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Sirindhorn thailandiensisAdamski & Malikul

Proceedings of International Conference on Biodiversity: IBD2019

on 22nd - 24th May 2019

at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand

Organized by:

Ministry of Higher Education, Science, Research and Innovation

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Proceedings of International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand ii

Proceedings of

International Conference on Biodiversity: IBD2019

Biodiversity for Sustainable Bioeconomy on 22nd - 24th May 2019

at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand

First Edition, September 2019

Copyright © 2019 by:

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Academic committee Asst. Prof. Dr. Apiradee Saelim Prince of Songkla University, Pattani campus

Prof. Dr. Benchamas Silayoi Kasetsart University

Assoc. Prof. Dr. Boonsatien Boonsoong Kasetsart University

Asst. Prof. Dr.Chitchol Phalaraksh Chiang Mai University

Assoc. Prof. Dr. Decha Wiwatwittaya Kasetsart University

Dr. Weerachai Nanakorn Queen Sirikit Park

Assoc. Prof. Dr. Mullica Jaroensutasinee Walailak University

Assoc. Prof. Dr. Narumon Sangpradub Khon Kaen University

Asst. Prof. Dr. Noppadon Kitana Chulalongkorn University

Assoc. Prof. Philip D. Round Mahidol University

Asst. Prof. Dr. Pratueng Jintasakul Khorat Fossil Museum

Dr. Putarak Chomnuti Mae Fah Luang University

Prof. Dr. Saisamorn Lumyong Chiang Mai University

Mr. Sakanan Plathong Prince of Songkla University

Asst. Prof. Dr. Santi Watthana Suranaree University of Technology

Prof. Dr. Savitree Limtong Kasetsart University

Dr.Somying Thunhikorn Department of National Parks, Wildlife and Plant Conservation

Dr. Tanit Changthavorn Biodiversity-Based Economy Development Office

Prof. Dr. Wanchai De-Eknamkul Chulalongkorn University

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Asst. Prof. Dr. Witsanu Attavanich Kasetsart University

All right reserved. No part of this book may be reproduced, stored in retrieval system or transmitted in any form or by any mean: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without permission from the publishers.

Proceedings of International Conference on Biodiversity: IBD2019 On 22nd - 24th May 2019

at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand iii

CONTENT

Discover of giant chili in Nan Province, Thailand 1 - 4

Vichai Puripunyavanich, Narisra Suwan, Taweepong Na Nan

and Penjan Sutthanukul

Participatory approaches for Volkameria inermis conservation through herbal bioproducts at

the Sirinart Rajini Mangrove Ecosystem Learning Center, Prachuap Khiri Khan Province

5 - 8

Nudchanard Rukklin, Jedsada Kongkasurichay, Panisa Rodpai,

Nidanuch Sungpia and Komson Hongpadharakiree

Endophytic fungi from wild plants and their antifungal and plant-growth promoting properties 9 - 15

Sakuntala Siri-Udom, Nattamon Somjai and Wanwipa wongchaiya

Diversity of entomopathogenic fungi in protected forest in the Eastern of Thailand 16 - 20 Winanda Himaman, Panrada Jangsantear, Baramee Sakolruk,

Kittima Duengkae, Suchada Mongkolsamrit, Wasana Noisripoom,

Janet Jennifer Luangsa-Ard and Isarapong Vorapab

Diversity of mushrooms at Mu Ko Chang National Park, Trat Province 21 - 32

Baramee Sakolrak, Panrada Jangsantear, Winanda Himaman,

Tiplada Tongtapao, Chanjira Ayawong and Kittima Duengkae

Health benefit screening on 45 Thai human probiotics (TISTR strains): their safety and

immunomodulatory activity in macrophage function

33 - 44

Bhusita Wannissorn, Weerasak Taengphan, Prapaipat Klungsupya,

Supatjaree Ruengsomwong and Thanchanok Muangman

Records of potential antimicrobial activity of soil actinomycetes isolated from

community forest, Ban Khoklam Sang Aram, Udon Thani Province

41 - 47

Metinee Wasoontharawat and Panthong Kulsantiwong

Screening on anti-inflammatory property of 45 Thai human probiotics from

Biodiversity Research Centre of TISTR

48 - 54

Prapaipat Klungsupya, Weerasak Taengphan, Bhusita Wannissorn

Supatjaree Ruengsomwong and Thanchanok Muangman

Effect of water temperature on growth, survival and health status

of East Asian Bullfrog (Hoplobatrachus rugulosus) Larvae

55 - 61

Sornsawan Liamtong, Kanokporn Saenphet , Monruedee Chaiyapo and Supap Saenphet

Antioxidant activity and hair growth promoting activity of flavonoid extracts

from Phyllodium pulchellum and Uvaria rufa Blume on cultured mouse vibrissa hair follicles

62 - 66

Patcharida Penpakkula, Kanokporn Saenphetb and Supap Saenphet

Chemical composition and antibacterial activities of essential oils of Lavandula × hybrid

‘boysemberry ruffles’, L. pedunculata ‘princess’ and L. × hybrid ‘high five purple’

67 - 72

Suchawadee Insawang and Patcharee Pripdeevech

Discovery of plant antimicrobial peptides and laboratory scale production 73 - 76

Parichart Burns, Jutatape Watcharachaiyakup, Patchima Sithisarn,

Pimpilai Saengmanee, Vinitchan Ruanjaichon and Sonthichai Chanpreme

Diversity of indigenous vegetables in Puparn Royal Development Study Centre,

Sakon Nakhon Province, Thailand

77 - 85

Hathairat Chokthaweepanich, Kwankamol Tawaitakam and Noppadol Kaewkumsai

Phytochemical screening and antimicrobial activity of Indian almond leaf extracts 86 - 88

Ratthayaporn Thansuwan, Jirapron Jitturongarpron, Jittima Buatib,

Thanyaporn Tangjaroenchai and Noppadon Muangsue

Proceedings of International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand iv

Study of chemical composition and antioxidant properties of Sangyod and

Tubtimchumpae rice bran oil

89 - 93

Pavinee Yampeng , Hathairat Rimkeeree and Supanida Winitchai

Checklist of bee genus Megachile Latreille, 1802 in Thailand (Hymenoptera:Megachilidae) 94 - 101

Nontawat Chatthanabun and Natapot Warrit

Diversity of caterpillars (Order Lepidoptera) in KhaoYai National Park,

Nakhon Ratchasima Province

102 - 115

Paradorn Dokchan, Nanthasak Pinkaew, Sunisa Sanguansub

and Sravut Klorvuttimontara

Proposal to reduce anthocyanin-deficient banana Musa siamensis to a M. rubra variety 116 - 121

Wandee Inta, Panida Kongsawadworakul, Unchera Viboonjun,

Paweena Chuenwarin, Paweena Traiperm and Sasivimon Chomchalow Swangpol

Species richness and distribution of marine spider crabs (Majoidea) in Thailand 122 - 128

Kamonchanok Wongissarakul, Pattanee Jantrarotai and Puntip Wisespongpand

The effectiveness of basic bait traps for collecting adult flies 129 - 135

Amornrat Ninon and Taeng On Prommi

An assessment of habitat connectivity for the endangered Malayan tapir in Thailand 136 - 142

Damisa Kaminsin and Naparat Suttidate

Satun UNESCO Global Geopark, the readiness for sustainable tourism 143 - 147

Suwimon Hengpatana, Phannipha Anuruksakornkul, Apinya Wanaset,

Taweesin Tangseng and Danuvas Suwanwong

Analysis of factors related to bamboo structure for preventing erosion and restoration of coastal

area on the Upper Gulf of Thailand

148 - 155

Patcharaporn Yaowasooth

Exploring the suitability map of wild banana (Musa serpentina Swangpol & Somana)

in Thailand using species distribution models with the limited occurrence data

156 - 162

Thanayut Changruenngam and Jantrararuk Tovaranonte

Stimulating the access to biodiversity and technologies to combat climate change 163 - 173

Fabrice Mattei

International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019

at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand 1

Proceedings of International Conference on Biodiversity: IBD2019 (2019); 1 - 4

Discover of giant chili in Nan Province, Thailand

Vichai Puripunyavanich1*, Narisra Suwan2, Taweepong Na Nan2 and Penjan Sutthanukul3

1Thailand Institute of Nuclear Technology, Ongkharak District, Nakhon Nayok, Thailand 2Nan Agricultural Research and Development Center, Muang District, Nan, Thailand

3Sukhothai Horticultural Research Center, Si Satchanalai District, Sukhothai, Thailand

*Corresponding author e-mail: [email protected]

Abstract: From the research project of anthracnose resistance breeding in chili, the native chili in northern and

north eastern region of Thailand were explored for its diversities of native wildtype. The result showed that most

of the chili cultivars that Thai farmer planting after rice harvesting season were commercial F1 hybrid. However,

some farmers still planted native wild types chilies. The objective of Thai chili farmer was to trade the product to

the middle merchants or directly sale to seasoning factory. Diversities of Thai native chili was rather narrow genetic

base because Thai farmers select only dark red colour and large size fruit that the factory is needed and discarded

the other characters. By incident, giant chili cultivar was noticed in remote valley of Tha Wangpha District, Nan

Province, Thailand. Some giant chili fruits are up to 25-30 cm in length and 3-5 cm in width with dark red colour.

This giant chili was an open pollinated cultivar which only few farmers in that area planted and collected it

continuously for more than ten years. The chili specialist at the Department of Agriculture also has never found

the giant chili at this size before. However, after the researcher group requested some seeds from a farmer and

brought to planted at Sukhothai Horticultural Research Center, most of the giant chili were died or became dwarf

plants. It is indicated that the giant chili cultivar may have highly specific to a certain area, thus, further

investigation and research study on the giant chili are needed.

Keywords: Nan, chili, fruit size, Capsicum annuum

Introduction

Chili pepper or Prik Chifah in Thai is a Thai local vegetable that become widely known among consumers

as it has hot taste and color right to the recipes. Chili is considered as one of the most important commercial spice

crops and is widely used as universal spice, named as wonder spice. Different varieties are cultivated for various

uses like vegetable, pickles, spice and condiments. Chili pepper has botanically known as Capsicum annuum L.

and belongs to the genus Capsicum, under the Solanaceae family which closed relatives used as vegetables: potato,

tomato, and eggplant. Chili originated in Latin America and was introduced to Asia by Spain and Portugal around

three hundred years ago. Most cultivated chili belonging to one of two major species groups.

1. Capsicum annuum includes chili pepper, sweet peppers and the pimiento (pimento), or Spanish pepper.

Sometime they have grown principally for use in commercial chili sauces.

2. Capsicum frutescens includes the "hot" chili and cayenne peppers; these have extreme pungency.

C. frutescens species may contain as much as 1 percent capsaicin and is one of the most important ingredients in

many different cuisines throughout the world as it adds pungency taste, flavor and color to the dishes (Ministry of

agriculture of India, 2009). The world production was reported by FAO in 2010 approximately 26.8 and 2.8 ton

for fresh and dried fruit. Usually, chili pepper is an annual plant but it can grow as a woody biennial in warm

climates. Thai farmers often started planting it in the nursery as seedling and then transplanted to the production

field. Chili pepper is a dicotyledonous plant. Flowers are insect pollinated and harvest green or red fruit. Fruit

color of chili peppers changes from mature green to full red color or yellow depending on its varieties. Their fruit

varies substantially in shape, pericarp thickness, color and pungency. The pungency of chili peppers is caused by

compound capsaicin. Chili peppers are range from 2,000 to 25,000 unit capsaicin whereas hot chilies are range

between 60,000 to 80,000 unit capsaicin. In Thailand, fresh edible chili peppers are usually harvested when red

while fresh chili peppers in commercial chili sauces are only harvested in red or dark red.

In Thailand, farmer usually grow chili pepper in November to April after harvesting rice. The area for

chili production was about 2-4 rais / family. The chili cultivar was determinate by the middle merchants or dealer

which was deal with the factory. Company F1 hybrid of Mae-ping 8000 was the introduced cultivar, following by

Kweawmanee 26, Sunkampang, Xian and Yoksiam. In some case, if there were contacted with collector, the farmer

may harvest only ripe chilies. Chili peppers fruit was usually sent to factory within 24 hours after harvesting. Thick

flesh and dark red color chili pepper were highly needed for the commercial chili sauces factory. The important

problem for the production is the pest infected by both insects and diseases. Major diseases of chili are anthracnose,

bacterial spot, and Phytophthora blight while major insects are broad mite, red spider mite, whitefly, etc. Most of

the Thai farmer buy chili seeds from a company. Usually F1 hybrid seeds are served for the farmers from the

company through a dealer. Very few Thai farmers developed open pollinated seed from natural germplasm by

http://edis.ifas.ufl.edu/PP104

http://www.ces.ncsu.edu/depts/pp/notes/Vegetable/vdin018/vdin018.htm

http://vegetablemdonline.ppath.cornell.edu/factsheets/Cucurbit_Phytoph.htm

International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand 2

themselves. On the beginning of this project, researchers tried to discover the anthracnose resistance in chilli which

may be hidden in natural resource conditions in north and north eastern region of Thailand. However, sometimes

the result did not correspond to the project target that had been set as some hybrid chili-pepper with vigour

characters hidden in the deep remote area of the country could be find.

Materials and Methods

1. Exploration of chili peppers samples were done in north and north eastern region of Thailand. Samples

of leaves, flowers, seeds and fruits were collected, observed, studied and took photographs in some areas at

Yasothon and Mukdahan, Chiang Mai, Sakhon Nakhon, Nan, Phrae, Roi Ed, Khon Kaen, Ubon Ratchathani,

Phitsanulok, Sukhothai, etc.

2. Around 40 sample of farmers were interviewed

- Farmer bought the seed from whom?

- Do farmers use native chili varieties?

- Are there contracts between farmers and company?

- Pest control process

- Period of planting

3. Morphology and taxonomy studies at Sukhothai Horticultural Research Center

4. Field plot test conducts at Sukhothai Horticultural Research Center for some interested cultivars.

Result & Discussion

From field exploration around the north and north eastern Thailand, it is founded that most of chili

cultivars which Thai farmer planting after rice field season were commercial F1 hybrid. Maeping 8000, Keaw-

manee 26 and Yok-siam which generally introduced by the company through the middle man in both north and

north eastern regions. Some farmers had contracted with the company whereas some farmers were no contracted.

Those contracted farmers are received price guarantee from the company. However, some farmers did not accept

the guarantee price and sale their products by themselves through independent middle merchants or directly to

chili sauces factory somewhere such as at Sukhothai, Sisaket, Ubon Ratchathani Province. There was setting up

the community enterprise or agricultural co-operative units but most of them were not strong. The price guarantee

from company was attracted the farmers to make decision to plant chili after their rice field season. Although the

objective of Thai chili farmer was mostly trade to the middle merchants or directly sent to seasoning factory. Only

thick flesh and dark red color chili pepper were needed for the commercial chili sauces factory and some products

which the factory did not accept, the farmer would sale it in the local market nearby their village. At that time, the

exploration of anthracnose disease indicated that it was widely spread in both chili pepper (Capsicum annuum)

and hot chili (Capsicum frutescens). But the farmers use only chemical disease control such as benomyl, mancozeb,

carbendazim or prochloraz to control this disease, so there was no anthracnose resistance in chili cultivars or native

cultivars in Thailand.

Although most of Thai farmers planted F1 hybrid chili which bought from the company, however, few of

them still planted native wild types. Diversities of Thai native chili was rather narrow genetic base because Thai

farmers select only dark red colour and large size fruit which factory needed and discard the other characters. The

exploration in Nan Province were conducted deep to the remote valley area of Tumbol Jomphra, Tha Wangpha

District. Some farmers planted the commercial F1 hybrid chili peppers cultivar under company contract. Some

farmers planted the commercial F1 hybrid chili peppers paralleled with their native inbred lines. All of their chili

pepper cultivars are open pollinated cultivars which different from F1 hybrid. The quality and quantity of the

cultivars look similar to the company F1 hybrid. It did not name after their native cultivars were breed, therefore,

all those cultivars were called by coding with Nan 1001, 1002, 1003, 1004…etc. Usually, the farmers sold only F1

hybrid chili pepper products to the company under contract through a dealer. But their native chili pepper products

were sold to freelance middle man or sold in local market. There was no farmer sold their chili pepper fruits

directly to chili sauce factory because there was no any factory nearby. In addition, the native ‘Sornthong Nan’

cultivar was investigated. Mr. Sawai Bhrammanee was the cultivar breeder which was not under contract from any

company. He explained ‘Sornthong Nan’ cultivar has very thick flesh and sweet. There was freelance dealer sold

his products to Nam Prik Num (Northern Thai Green Chilli Dip) factories because ‘Sornthong Nan’ has very thick

flesh and sweet fruit. By incident, giant chili cultivar was noticed. The land owner and the giant chili pepper

breeder is Mr. Kamsarn Sutthisan. He told the research group that the breeding of giant chili pepper was done for

more than ten years from native varieties crossed over with commercial varieties and did selection year by year

until receiving the right chili pepper line. However, this genetical line is still not so stable, there possesses a range

from giant chili pepper size to the normal chili pepper size in the same field. The giant chili pepper was sold in

the local market. There was no middle man from any company bought this giant chili pepper because it was not

under contract with any company. Some giant chili fruits are up to 25-30 cm in length and 3-5 cm in width with

dark red colour and thick flesh fruits. This giant chili was an open pollinated cultivar. After receive giant chili

Proceedings of International Conference on Biodiversity: IBD2019 On 22nd - 24th May 2019


pepper samples from the owner, it was brought to Sukhothai Horticulture Research Centre. The chili specialist of

the Department of Agriculture said that the chili with giant size has never been found before. Although the giant

chili has dark red colour and thick flesh fruit but it produces rather few seeds of only 40-50 seeds/fruit. The giant

chili pepper seed were planted in Sukhothai Horticultural Research Centre whereas nearby many chili seasoning

factories. Most of them were died or became dwarf plants and got a few normal size fruits. It is indicated that this

giant chili cultivar was highly specific to area base Nan Province or may be highly specific to area only in Tumbol

Jomphra, Tha Wangpha District.

To avoid patent conflict and to breed a new cultivar base on this giant chili pepper, mutation breeding

program by gamma irradiation was setting up. The research hope to discover a giant chili pepper that able to adapt

and survive at Sukhothai Horticulture Research Centre where there are chili seasoning factories in Sukhothai

Province, therefore, it is expected that the breeding of a mutant line will be accomplished to support the Sukhothai

area. Large size, dark red colour and thick flesh fruit characters will be selected in the research program for the

near future research.

Conclusion

Although the first target of anthracnose resistance in native chili exploration was not discovered in north

and north eastern region of Thailand. Most of chili cultivars were commercial F1 hybrid from a company. From

the exploration in Nan Province, some of farmers planted native wild types or inbred lines that they did breeding

it themselves. Diversities of Thai native chili was rather narrow genetic base. There were some open pollinated

chili pepper cultivars developed by several farmers. Quality and Quantity of their chili peppers fruit were similar

to F1 hybrid of any company. ‘Sornthong Nan’ and Giant chili pepper cultivar were discovered in Nan Province.

The native ‘Sornthong Nan’ is suitable to make Nam Prik Num (Northern Thai Green Chilli Dip). Some giant chili

pepper fruits were very big up to 25-30 cm in length, dark red colour and thick flesh fruits are suited for chili sauce

factory requirement. The giant chili pepper and native ‘Sornthong Nan’ were also open pollinated cultivars.

However, most of the giant chili pepper were died or became dwarf plants after they were planted in Sukhothai

Province.

Acknowledgement

We would like to thank AVRDC (Asian Vegetable Research and Development Centre), Kasetsart

University, Kamphaengsaen Campus, Nakhon Pathom) for their valuable suggestions and comments. We also

thank all authorities and their staff in every agricultural station and especially researchers from Department of

Agriculture for their technical supports.

References

Department of agriculture and co-operation, Ministry of agriculture of India. 2009. Postharvest profile of chilli. p.80

Kraikruan W., Supkweaw U., Sachati S. and Suthanigool P. 2014. Supply chain of processing chili production in

Sukhothai Province. The 53th Kasetsart University Annual Conference. Feb. 3-6, 2015. p. 279-286 (in Thai script).

Mahasuk P., Chinthaisong J. and Mongkolporn O. 2013. Differential resistances to anthracnose in Capsicum baccatum as responding to two Colletotrichum pathotypes and inoculation methods. Breed Science. 2013 Sep; 63(3): 333 – 338.

Mongkolporn O., Montri P., Supakaew T. and Taylor P.W.J. 2010. Differential reactions on mature green and

ripe chili fruit infected by three Colletotrichum spp. Plant Disease Journal. 94: 306–310.

Puripunyavanich V., Phadvibulya V. and Boonsirichai K. 2007. Effect of Gamma Irradiation on Chili Mutation.

The 10th Conference on Nuclear Science and Technology. Bangkok International Trade & Exhibition

Centre. June 16-17, 2007.

Ratanacherdchai K., Wang H. K., Lin F.C. and Soytong K. 2007. RAPD analysis of Colletotrichum species causing

chilli anthracnose disease in Thailand. Journal of Agricultural Technology. 2007; 3:211–219.

Than P.P., Prihastuti, H., Phoulivong S., Taylor P.W.J. and Hyde K.D. 2008. Chilli anthracnose disease caused

by Colletotrichum species. Journal of Zhejiang University Science B. 2008 Oct; 9(10): 764–778.

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International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand 4

Figure 1. Nan 1008, Nan 1009 and Nan 1015 chili pepper lines.

Figure 2. Nan 1014, Nan 1016 and Nan 1017 chili pepper lines.

Figure 3. Sorn Thing Nan, Xian F1 hybrid (Jia Tai) and Sunkampang F1 hybrid (Sorn Dang) chili pepper lines.

Figure 4. Giant chili pepper line.

Figure 5. Giant chili pepper line breeder and ‘Sorn Thong Nan’ breeder.

Proceedings of International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019



Participatory approaches for Volkameria inermis conservation through herbal

bioproducts at the Sirinart Rajini Mangrove Ecosystem Learning Center,

Prachuap Khiri Khan Province

Nudchanard Rukklin*, Jedsada Kongkasurichay, Panisa Rodpai, Nidanuch Sungpia,

and Komson Hongpadharakiree

Sirinart Rajini Mangrove Ecosystem Learning Center, Pranburi District, Prachuab Khiri Khan, Thailand


Abstract: The study aimed to examine participatory approach using commercial values of herbal bioproducts and

local attitudes to expand the plant habitats inside the Sirinart Rajini Mangrove Ecosystem Learning Center at Pak

Nam Pran Sub-district. Volkameria inermis was chosen among other mangrove plants for its values. Forty

respondents, twenty of them whose incomes were promoted from Volkameria inermis herbal products, while

another twenty respondents lived adjacent the center. Their attitudes were collected by questionnaire surveys and

analyzed by descriptive statistical analysis including percentage, mean, and One-way ANOVA with a significance

level (α) of 0.05. Incomes increased by added values of mangrove herbal products made from the plant were

analyzed. The study showed that respondents whose incomes were promoted through the herbal products had high

level of awareness at 61.77% on the plant value and the mangrove forest conservation while another group of

respondents showed their moderate awareness with 44.28%. The results showed significant influence (p-value =

0.024) of knowledge on the plant benefits and the higher incomes earned from the herbal products on the attitude

towards a plant and mangrove natural resource conservation. A total income obtained from selling the plant

products of center during January 2016 to September 2018 was totally 226,990 baht. Among the products, the

incomes from selling the soap and hot balm made from the plant were 222,535 baht and 4,455 baht respectively,

made by total fresh leaves of plant 109.34 kilograms or 2,076 baht per kilogram wet weights. It can be concluded

that a community income promotion using local mangrove forest resources was an approach to the community’s

values and awareness on the mangrove forest conservation, leading to community participation in tree planting,

caring, and restoring of targeted plant species as well as using the natural resources in sustainable ways.

Keywords: Participatory approach, conservation, Volkameria inermis, herbal bioproducts

Introduction

Mangrove forest conservation with participatory policy under the Sirinart Rajini Mangrove Ecosystem

Learning Center, at the Klong Kao – Klong Koy National Reserved Forest, Pak Nam Pran Sub-district, Pran Buri

District in Prachuap Khiri Khan Province has been ecological restored since 1981 with the area of 786 rai (126

hectares) (Paphavasit et al., 2014). The policy on community participation on the mangrove forest was applied to

the mangrove management following His Majesty King Rama IX’s speeches "The locals benefit from the

mangrove forest, so they must help to conserve it" on 16 November 2002 by His Majesty King Rama IX delivered

during the Public Company Limited’s 1 Million-Rai Reforestation Project in Commemoration of His Majesty the

King Rama IX’s 50th Anniversary of His Accession to the Throne. Following the Speech, becoming a policy and

actions, the restored mangrove forest tracts of Forest plantation target 29 and forest plantation target 29/3 provided

the forest ecosystem benefits with the building the participation of the community in the conservation of mangrove

planting to sustainability among communities at Pak Nam Pran Sub-district of Pran Buri District. To achieve the

target on community participation on the mangrove forest management, there were four strategic issues were

included to the forest operations which were knowledge, community and society, environment, and ecotourism.

Social entrepreneurship is an effective tool for promoting participatory resource-based management. Network of

villagers who earned benefits from herbal plants of mangrove forest was established under the Center as a group

of mangrove forest herbal bioproducts. The social entrepreneurship is known as a process of creating value by

combining resources in new concepts of social networking to seek for opportunities regarding social values and

needs. Management of herbal bioproduct values to promote more socio-incomes among the members and

strengthen the mangrove forest was anchored as foundation of capacity building conservation expanding around

the Center. The process of creating value of mangrove plant bioproducts regarding local needs such as hot balm

oils for the development of product values of mangrove plants and encourage the sustainable mangrove forest

management under the Center. Promotion of mangrove forest services for local communities in particular, various

mangrove plant species growing in the center’s area is for occupation promotion and socio-incomes of group

members. There are more than 20 plant species that could be utilized as herbal bioproducts with simple production

process and meet to the market needs of local consumption. Their medicinal characteristics were reported and

utilized by local wisdoms and scientific research studies. For example, boiled water from bark of Rhizophora

Proceedings of International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand 6

apiculata can heal diarrhea, nausea, vomiting, fever, or chronic wounds clean (Faculty of Science Chulalongkorn

University as cited in Paphavasit et al., 2014) as well as the boil water or blended bark of Xylocarpus granatum

can cure wounds and bruise. The boiled water of green leaves of Samma Nga or Volkameria inermis is used to

clean wounds, treat skin diseases, and bathing to reduce rash (Kung Krabaen Bay Royal Development Study

Center. 2006). Among those plant speices such as Rhizophora apiculata, Rhizophora mucronata, Xylocarpus

moluccensis, Xylocarpus granatum, Thespesia populnea, Avicenniaalba, Avicennia marina, Bruguiera cylindrical,

Acanthus ilicifolius, Volkameria inermis, and etc, the local plant named Samma Nga (Volkameria inermis) was

selected for participatory management model using commercial values to expand the area of plantation in the

Center area.

The plant contains flavone derivatives including hispidulin, acacetin, and diosmetin. The hispidulin is

found in Volkameria inermis reported as a potential medicinal plant species by Faculty of Pharmacy, Mahidol

University. Potential therapeutic role of hispidulin extracted from the plant leaves is volatile substance that

decongestive bronchial congestion. More substances of acacetin and diosmetin were found in the plant leaves and

exhibits several pharmacological and medicinal properties especially their roles in anti-inflammation of the skin,

rash, bruise and sprain.

From highly potential therapeutic roles of chemical substances found in Volkameria inermis, but in very

small quantity of leaves available at the Center, the plant will be promoted through commercial values and local

attitudes to expand the plant habitats inside the Center. Social mechanisms especially social entrepreneurship using

the potential of herbal bioproducts to generate the diversified socio-incomes and community participation in the

programs of Volkameria inermis planting is assessed. Assessment of effectiveness of conservation practices that

related to Sirinart Rajini Center programs including knowledge transfer and occupation promotion using benefits

from herbal bioproducts of Volkameria inermis. The results of assessment would be the management model for

sustainable forest management using participatory approach together with commercial values development of

herbal bioproducts (Figure. 1).


In order to find the participatory approaches using the social entrepreneurship operating the herbal

bioproduct values from Volkameria inermis available at the Sirinart Rajini Mangrove Ecosystem Learning Center,

Pak Nam Pran Sub-district, field survey was conducted using semi-structure questionnaires. The contents of

questionnaires are divided into three data sets.

A. Demographic information the 40 respondents.

B. Benefits of Volkameria inermis to promote the mangrove forest conservation.

C. Participatory approaches for Volkameria inermis planting in the Center area to promote the

mangrove forest conservation.

Attitudes survey, totally forty respondents was conducted by selecting the twenty respondents from

communities in the center and another twenty respondents from adjacent communities. The collected data sets

were analyzed using the Statistical Package for Social Sciences program for averages percentage and mean values.

One-way ANOVA at a significance level (α) of 0.05 was chosen to reveal the significant differences of two groups

of respondents on the studied data sets. Moreover, using the literature reviews and experts opinions for qualitative

data analysis, the records of socio-incomes obtained from the benefits of herbal bioproducts made from Volkameria

inermis in 2016 to 2018 was analyzed.

Result & Discussion

The forty respondents were in the middle ages older than 51 years old. The twenty respondents from the

observed group who earned benefits form the Volkameria inemis herbal bioproducts were mainly female at 65%

at 51-60 years old. Their education information showed that 30% of this group graduated in an elementary school

or lower and 50% of the respondents were graduated in high school. For the source of main incomes, there were

30% of respondents earning their incomes from fishery, and 30% of those had their total incomes around 5,000-

8,000 baht per month.

The other twenty respondents who lived adjacent mangrove forest of Sirinart Rajini Mangrove Ecosystem

Learning Center were found females 60% and age of 60 years old of respondents were found around 30%. There

were about 30% of respondents graduated in high school, and among the respondents they were found being a

merchant. There were 40% of them earned incomes 5,000-8,000 baht per month and another 60% of them earned

incomes more than 15,000 baht.



The results of interviews of all respondents showed that their attitudes on the benefits of herbal plant and

importance of mangrove forest, the respondents whose incomes were earned from the herbal bioproducts were

found at 61.77% while another group of respondents showed their moderate awareness with 44.28%.

The results showed significant attitudes (p-value = 0.024) on the plant benefits and the higher incomes

earned from the herbal bioproducts on the attitude towards a plant and mangrove natural resource conservation.

The demographic factors including gender, age, education, occupation and incomes per month did not significantly

affect (p-value = 0.534, 0.824, 0.636, 0.752, and 0.983, respectively) to the attitudes a plant and mangrove natural

resource conservation.

In addition, the results showed the respondents whose their incomes were from herbal bioproducts needed

training programs for the knowledge related to the benefits of Volkameria inermis and should developed other

herbal bioproducts to provide alternative choices for consumers. The Volkameria inermis may be reduced if

harvested continuously, so more areas inside the Center should be for Volkameria inermis plantation as well as the

determination of timing of restoring the Volkameria inermis back to the area.

Socio-incomes obtained from the benefits of herbal bioproducts made from Volkameria inermis January

2016 to September 2018 revealed that the plant provided the respondents more incomes. The center with the group

of mangrove forest herbal bioproducts produced the herbal bioproducts earned more incomes from selling the

herbal bioproducts totally 226,990 baht. Among the different types of bioproducts, the incomes from selling the

soap made from the plant were 31,975, 87,600 and 107,145 baht in 2016, 2017 and 2018 respectively. The soap

made from total fresh leaves of Volkameria inermis 109.34 kilograms or 2,076 baht per kilogram wet weights.

Conclusion

The results showed the significant attitudes on the benefits of herbal plant, Volkameria inermis

conservation through incomes promotion of selected stakeholders from the community at Pak Nam Pran Sub-

district, Pran Buri District in Prachuap Khiri Khan Province and from the Center adjacent communities. The

respondents whose incomes were promoted through the herbal bioproducts had high level of awareness at 61.77%

on the plant value and the mangrove forest conservation while another group of respondents showed their moderate

awareness with 44.28%. The results showed significant attitudes (p-value = 0.024) on the plant benefits and the

higher incomes earned from the herbal bioproducts on the attitude towards a plant and mangrove natural resource

conservation. Moreover, they needed knowledge related to the benefits of Volkameria inermis and other herbal

bioproducts to provide alternative choices for consumers. To increase the numbers of this herbal plant, more areas

inside the Center should be provided for plantation as well as the determination of timing of restoring the

Volkameria inermis back to the area.

The occupation promotion to community was an approach to the community’s values and awareness on

the mangrove forest conservation, leading to community participation in planting, caring, and restoring of targeted

plant species as well as generating the incomes and using the natural resources in sustainable ways.

Acknowledgement

The authors gratefully thank the respondents and officers of the Sirinart Rajini Mangrove Ecosystem

Learning Center for their information. Also, thank you very much to the PTT Public Company Limited for

supporting the research budget. Finally, we would like to thank Dr. Kallaya Suntornvongsagul, Environmental

Research Institute of Chulalongkorn University for useful comments and suggestions on the English language and

structure of our manuscript.

References

Faculty of Pharmacy Mahidol University. 2017. Herbal Plants: Volkameria inermis. Retrieved from

https://pharmacy.mahidol.ac.th/siri/index.php?page=search_detail&medicinal_id=38.

Forestry Kung Krabaen Bay Royal Development Study Center. 2006. Medicinal plants of beach forests and

mangrove forests in the Kung Krabaen Bay Royal Development Study Center area. Chanthaburi.

Paphavasit, N., Siriboon, S., Jaiperm, J. and Mookui, P. 2014. Sirinath Rajini Mangrove Ecosystem Learning

Center. From mangrove plantation to mangrove forest enhancing human development (1ed.).

Bangkok: PTT co., Ltd and Department of Science, Chulalongkorn University. Bangkok.

Paphavasit, N., Chawasiri, W., and Theerathanathor, V. 2012. Herbal plants in the mangrove forest Thung Tase

in Trang Province. Yves Rocher (France) and Yves Rocher (Thailand) Ltd. And Department of

Science, Chulalongkorn University. Pra Suk Chai Printing Part., Ltd. Bangkok.


Figure 1. Research framework diagram.

Sustainable Forest Management using Participatory Forest Conservation

Community Based Participatory Approach

Attitudes on Benefits of Forest

Conservation

Benefits for Socio-economics:

-Herbal medicines

-Food

Sustainable Management

Tools




Endophytic fungi from wild plants and their antifungal and

plant-growth promoting properties

Sakuntala Siri-Udom*, Nattamon Somjai and Wanwipa wongchaiya Department of Biology, Faculty of Science, Udon Thani Rajabhat University, Muang District,

Udon Thani, Thailand


Abstract: A total of 118 endophytic fungi were isolated from 21 wild plants in northeastern of Thailand. Most

endophyte were isolated from Maesa ramentacea (Roxb.) A. DC. (12.7%), followed by Baeckea frutescens

(11.9%) and Anneslea fragrans Wall. (6.8%), respectively. Endophytic fungi were more prevalent in the leaves

(59.3%) than the branches/stems and the flowers. Isolate Gu02 and Gu03 from flowers of Gluta usitata (Wall.)

Ding Hou exhibited board range of antifungal activity above 50% growth inhibition by dual culture technique

toward to Colletotrichum gloeosporioides, Phellinus noxius and Rigidoporus microporus. Isolate Gu03 inhibited

mycelial growth of the pathogenic fungi, P. noxius and R. microporus causing root rot disease of rubber tree with

74.1% and 88.1% inhibition, respectively. Isolate Gu03 produced plant growth hormone, indole acetic acid (IAA)

at the concentration of 331.5 µg/ml. Volatile metabolite-producing endophytic fungus, isolate Cs05 was isolated

from leaf tissues of Cycas siamensis Miq. The volatile organic compounds (VOCs) of isolate Cs05 were active

against P. noxius and R. microporus with 100% growth inhibition.

Keywords: Antifungal activity, endophytic fungi, indole acetic acid, wild plant

Introduction

Endophytic fungi live in symbiosis with plant tissues and play an important role in plant growth (Young-

Hyun et al., 2012). They have been identified in nearly 300,000 plant species (Strobel and Daisy, 2003) and dwell

in root, stem, leaf, flower, fruit and seed (Yu et al., 2018). Most endophytic fungi are members of the Ascomycota,

with only a few reports of basidiomycetous endophytes, these often being orchid mycorrhizas (Rungjindamai et

al., 2008). Natural select the evolution of beneficial endophyte strains and several endophytes are found to produce

bioactive compounds that protect plant from insect pests and pathogens (Saikkonen et al., 2004). They play an

important role in plant defense including the function as growth promoter and enable the host survival under

extreme conditions (Rosa et al., 2012). Although, chemical pesticides were used to protect plant disease, but

pesticide use may lead to environmental pollution and might threaten human health (Yu et al., 2018). Recently,

the development and spread of drug-resistant pathogens are still a global problem and there is a need to search for

new active agents with antimicrobial activity (Espinel et al., 2001). Thus, endophytic fungi are source of novel or

bioactive metabolites for pharmacological and agricultural applications (Idris et al., 2013; Deshmukh et al., 2018).

Natural products from fungal endophyte showed antagonistic activity to inhibit several pathogenetic

organism such as bacteria, fungi, viruses and protozoans. Furthermore, they could be plant growth regulators such

as indole acetic acid (IAA), one of the most physiologically active auxins. Fungi produce a wide variety of plant

hormone such as gibberellins (GAs), abscisic acid (ABA), and auxin (Young-Hyun et al., 2012; Khan et al., 2017).

IAA is a product of L-tryptophan metabolism by various microorganism including Plant growth promoting

rhizobacteria (PGPR) (Ahmad et al., 2005). The volatile metabolite-producing endophytic fungi have not been

commonly reported. But some endophytic fungi that belong to the families Diaporthaceae, Hypocreaceae,

Stachybotryaceae and Xylariaceae of the phylum Ascomycota are notable for their capacity to form volatile

metabolites with antimicrobial activity (Stinson et al., 2003; Banerjee et al., 2010; Suwannarach et al., 2013; Siri-

Udom et al., 2015). The mixture of fungal volatile compounds that have been identified were aldehydes, alcohols,

benzene derivatives, cyclohexanes, ketones, hydrocabons, heterocycles, phenol, thioalcohols, thioesters and their

derivatives (Mercier et al., 2007; Morath et al., 2012). In agriculture, fungal VOCs with antimicrobial activity may

be used as a mycofumigant to control plant disease.

In this study, we aimed to evaluate the diversity of endophytic fungi that live in association with wild

plants. Furthermore, to assessed the potential of the effective fungi that capable of producing bioactive agents,

plant growth promoting metabolites and volatile metabolites against fungal pathogens.


Preparation and surface sterilization of plant materials

Twenty-one healthy wild plant species were used as source for isolation of endophytic fungi. Eight plants

were collected from The Royal-initiated Lam Huai Bong Forest Area Development Project (16°54′23″N, 102°

24′47″E), located in, Nong Bua Lam Phu Province. Six plants were collected in Dong Kheng community forestry,


Nong Bua Lam Phu Province (16°56′49″N, 102°34′31″E). Seven plants were collected from Ban Thai Seri

community forestry, Bueng Kan Province (18°00 ′54″N, 103°57′49″E). Plant materials were cut randomly into

small segments (leaf and flower, 5 mm x 5 mm; stem/branch 5 mm long). All segments were sterilized by soaking

in 75% ethanol for 30 s, 2% sodium hypochlorite for 3 min, and 95% ethanol for 30s under a laminar flow hood

(Suwannarach et al., 2010).

Isolation of endophytic fungi

Each plant materials were placed on potato dextrose agar (PDA) containing Rose bengal (0.033 g/l) and

chloramphenicol (50 mg/l). Petri dishes were sealed with Parafilm® M (Bemis company, Inc., USA) and incubated

at room temperature (25±2 C) for 2 weeks. Emerging fungi were transferred to fresh PDA plates. The medium

was also be used for subculture and stock culture. Identification of endophytic fungi was carried out on the basis

of morphological characteristics (Huang et al., 2008). The fungal isolates were grown on PDA and incubated at

room temperature to observe morphological characteristic such as color, shape and size of spores. Spore production

of endophytic fungi was studied on different media, such as malt agar (MA), PDA and water agar (WA).

Antagonism against fungal pathogens by dual culture method

The antagonistic analysis was observed from the interaction between fungal endophytes and the pathogens

using dual culture technique. An agar plug (6 mm diameter) 4-day-old fungal endophytes growing on PDA was

inoculated on PDA part of the petri dish, then an agar plug of fungal pathogens was inoculated opposite side of

the Petri dish. The control plates were inoculated with either pathogens. All Petri dishes were wrapped with

Parafilm® M and incubated at room temperature (25±2 °C) for 7 days. The percentage of inhibition of fungal

growth after the dual culture test was calculated with the following equation: [(R1- R2)×100] ÷ R1 , where R 1

was the average colony radius of each tested fungi measured in the control plates (without the tested fungi), and R

2 was the average colony radius that calculated from the tested plates.

Antimicrobial assay of VOCs

The parallel-growth isolation technique was adapted for the antagonism test of fungal VOCs. An agar

plug of volatile-producing endophytic fungi was inoculated on the PDA part of a two-compartment Petri dish, and

allowed to grow at room temperature (25±2 °C) for 2-4 days. Then, an agar plug (6-mm diameter) 4-day-old fungal

pathogens growing on PDA was inoculated on PDA on the opposite side of the Petri dish. All Petri dishes were

wrapped with parafilm® M and incubated at room temperature (25±2°C) for 3-7 days. The percent inhibition of

fungal growth after the dual culture test was calculated. The viability of the test fungal pathogens was observed

by transferring them from the test plates and re-growing in fresh PDA (Strobel et al. 2001).

Indole acetic acid (IAA) production

All isolates of endophytic fungi were inoculated in 5 ml potato dextrose broth (PDB) with L-tryptophan

(2 mg/ml) and incubated in the dark at room temperature (25±2 °C) with shaking at 150 rpm on a shaker for 5

days. The broth cultures were filtrated by two layers of gauze cloth to separate the broth cultures and mycelia. A

modified method described by Ahmad et al., 2005 was used for screening IAA production. All filtrates (1 ml) were

mixed with 2 ml of Salkowski’s reagent (1 ml of 0.5 M FeCl3; 50 ml of 35% Perchloric acid (HClO4)) and

incubated in the dark for 30 min. The development of pink color indicated IAA production and the absorbance at

530 nm was measured. The level of IAA production was estimated by standard IAA graph.

Result & Discussion

Isolation of endophytic fungi

A total of 118 endophytic fungi were isolated from 21 plant species, the most of them were isolated from

Maesa ramentacea (Roxb.) A. DC. (12.7%), followed by Baeckea frutescens (11.9%) and Anneslea fragrans Wall.

(6.8%), respectively (Table 1). Most of them colonized in leaf (59.3%) and only 3 isolates colonized in flower of

plant sample (Figure 1. and Table 1). Furthermore, 6 volatile producing endophytic fungi were isolated from 5

wild plants, including M. ramentacea. Isolate Cs05 did not produce spore and belonged to mycelia sterilia group.

Endophytic fungi are presented in most of plant section, especially healthy leaf tissue (Kharwar et al., 2011;

Karunai and Balagengatharathilagam, 2014; Yu et al., 2018) according to this study, most endophytic fungi were

more prevalent in the leaves than the branches/stems and the flower. Although, Zheng et al. (2016) proposed that

the diversity of endophytic fungi is generally significantly higher in the stems than in the leaves.

Endophytic population varies from plants to plants and species to species (Nair and Padmavathy, 2014).

However, the endophytes population was affected by the environmental conditions under which the host is growing

and the endophyte profile may be is more diversified in tropical areas (Karunai and Balagengatharathilagam,

2014). The isolate numbers of endophytic fungi are closely correlated with the sampling range of the plant age,

such as increasing the collecting plant specimens of different ages, to enable the isolation of additional fungal

endophytes (Yu et al., 2018).



Wild plant in family Myrsinaceae can potentially be explored for estrogenic activity and for sources of

phytoestrogens (Jamal et al., 2012), genus Maesa was reported the fungal endophyte in genus Diaporthe lived in

fruit (Gomes et al., 2013). Coniochaeta ligniaria, an endophytic fungus with antifungal activity was isolated from

leaves of Baeckea frutescens (family Myrtaceae) obtained from the Phu Luang wildlife sanctuary in Thailand

(Kokaew et al., 2011). Fungal endophyte can live in various wild plant tissue, genus Fusarium was the most

common fungal isolate from root of wild banana (Musa acuminate, family Musaceae), which is native to Southeast

Asia and is an ancestor of the edible banana (Zakaria et al., 2016). Several endophytic fungi, such as Colletotrichum

gloeosporioide, Phomopsis sp., Phyllosticta capitalensis and Corynespora were consistently isolated from the

different tree in tropical forest of southern India. In dry season, endophytic diversity was greater in the dry thorn

forest than in the dry deciduous forest (Murali et al., 2007).

Antagonistic activity of endophytic fungi

Based on the results, 22 isolates of endophytic fungi could inhibit the mycelial growth of fungal pathogens

and only 16 isolates showed the percentage of inhibition above 50%. Isolate Gu03 showed the greatest percentage

of inhibition of mycelial growth with R. microporus (88.1%) and P. noxius (74.1%), respectively (Figure 2. and

Table 2). Isolate Cc12 showed the greatest percentage of inhibition of mycelial growth with C. gloeosporioides

(64.9%) by overgrowth of the tested pathogen (Table 2).

Root rot disease is the most serious problem of rubber tree plantation and present in many countries,

including Thailand. In this study, the antagonistic test showed that the causing pathogen, R. microporus and

P. noxius was against by isolate Gu02 and Gu03 with the highest percentage of inhibition. The antagonistic activity

of fungi to inhibit mycelial growth of the root rot pathogens was considered to control root rot disease in the field

according to the previous studies such as the use of Trichoderma hazianum (Jayasuriya and Thennakoon, 2007)

and Chaetomium cupreum to control R. microporus in vivo (Kaewchai and Soytong, 2010).

The bioactive compounds were produced by the plant but in a mutualistic association with the host plant,

endophytes may enhance bioactive metabolites that process bioactivity such as antibacterial and antifungal activity

in host plant (Radu and Kqueen, 2002; Tejesvi et al., 2007).

Antimicrobial assay of VOCs

The volatile metabolites produce by endophytic fungi were tested for their antimicrobial activity. Isolate

Cs05 showed the greatest antimicrobial activity with all test organisms (Table 3). It was isolated from leaf tissues

of Cycas siamensis (family Cycadaceae), which did not produce spores on several media, including MA, PDA and

WA, and had rope-like mycelium with coiled structures. Many endophytic fungi are known to produce the

bioactive compounds in the form of volatile metabolites with anti-microbial, anti-oxidant and anti-proliferative

activities, cytotoxicity, and fuel production (Naik, 2018). In the family Xylariaceae, Muscodor albus is the first

known fungal endophyte isolated from Cinnamomum zeylanicum, which produces bioactive volatile metabolites

(Strobel et al., 2001; Ezra et al., 2004). An endophytic fungus, Nodulisporium sp. produced the mixture of volatile

compounds were active against plant pathogens. The most abundant identified compound was 1, 8 cineole, 1-

butanol, 2-methyl, and phenyl ethanol alcohol and most importantly cyclohexane, propyl, which is a common

ingredient of diesel fuel (Hassan et al., 2013).

Indole acetic acid (IAA) production

Eighteen isolates of endophytic fungi showed their ability to produce IAA in preliminary test. The range

of IAA production was 11.3 µg/ml to 331.5 µg/ml (Table 2). Endophytic fungus, isolate Gu03 produced maximum

concentration of IAA, following by isolate Gu01 and Cc12, respectively (Table 2). This result was supported by

the previous studies that showed several endophytic fungi, which produced IAA and increased seed germination

and plant growth (Khan et al., 2012, 2015, 2017; Zhou et al., 2013; Kedar et al., 2014). Endophytic fungi enhance

plant growth by produce various secondary metabolites, including IAA, flavonoids and flavnols. These compounds

were determined in the culture filtrate of Aspergillus fumigatus, an endophyte in leaves of wild plant in family

Solanaceae, Withania somnifera (Mehmood et al., 2018). IAA is essential for crop growth and development

because it enhances root, flower development, and other processes (Reinhardt et al., 2000). However, there are

reports that IAA is important tool to against fungal pathogens, such as Colletotrichum spp. (Yue et al., 2000).

Conclusion

Endophytic fungi colonized in plant tissue, especially healthy leaf tissue. They had antimicrobial and

plant-growth promoting properties. Biological control by endophytic fungi to prevent disease and promote plant

growth offer an attractive alternative method for disease management without the negative impact of the chemical

control. Furthermore, volatile metabolite-producing endophytes may be an alternative biological approach as

biofumigation in control of plant diseases.


Acknowledgement

This study was supported by grants from Research and Development Institute Udon Thani Rajabhat

University.

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Figure 1. Colony of isolate Gu02 and Gu03 on PDA (A-B) and isolate Cs05, volatile-producing endophytic

fungus on PDA (C), scale bar = 2 cm.

Figure 2. Preliminary testing for antifungal activity of endophytic fungi (isolate El20 and Gu03) against plant

pathogenic fungi by dual culture technique (A-B). Antifungal activity of VOCs from isolate Mr35 and Cs05 after

the exposure of VOCs for 3 and 7 day, respectively (C-D), scale bar = 1.5 cm.

Table 1. Number of endophytic fungi isolated from wild plants.

Plant species Family Leaf Stem/Branch Flower Total

Vein Intervein

* The Royal-initiated Lam

Huai Bong Forest Area

Development Project

Cycas siamensis Cycadaceae 2 0 1 0 3

Dipterocarpus tuberculatus Dipterocarpaceae 0 2 2 0 4

Ficus microcarpa Moraceae 1 1 2 0 4

Gluta usitata Anacardiaceae 0 0 0 3 3

Hoya ovalifolia Asclepiadaceae 2 4 0 0 6

Melientha suavis Opiliaceae 1 2 2 0 5

Shorea obtusa Dipterocarpaceae 3 0 3 0 6

Shorea siamensis Dipterocarpaceae 5 0 1 0 6

* Dong Kheng community

forestry

Cratoxylum formosum Clusiaceae 1 0 4 0 5

Dialium cochinchinense Leguminosae -

Caesalpinioideae

2 0 0 0 2

Dioecrescis erythroclada Rubiaceae 0 0 1 0 1

Ellipanthus tomentosus Connaraceae 3 0 2 0 5

Erythrophleum succirubrum Leguminosae -

Caesalpinioideae

0 0 1 0 1

Streblus asper Moraceae 6 1 0 0 7

* Ban Thai Seri community

forestry

Anneslea fragrans Theaceae 1 2 5 0 8

Baeckea frutescens Myrtaceae 6 0 8 0 14

Catunaregam tomentosa Rubiaceae 2 4 0 0 6

Dipterocarpus obtusifolius Dipterocarpaceae 1 6 0 0 7



Plant species Family Leaf Stem/Branch Flower Total

Vein Intervein

Eurycoma longifolia Simaroubaceae 2 2 0 0 4

Maesa ramentacea Myrsinaceae 3 3 9 0 15

Syzygium gratum Myrtaceae 2 0 4 0 6

Table 2. Antifungal activity and IAA production of endophytic fungi from wild plants.

Isolate number Percentage of inhibition of microbial growth IAA

(µg/ml) C. gloeosporioides P. noxius R. microporus

Af01 64.1 0 0 74.4

Af03 51.3 0 0 84.4

Af08 37.7 0 0 70.1

Af10 51.3 0 0 79.0

Bf09 26.1 67.2 64.3 0

Cc12 64.9 23.5 5.9 150.8

Cs01 53.9 0 0 32.6

Ct01 54.0 0 0 0

Dc01 53.2 0 0 86.8

De03 63.7 23.5 29.4 76.8

El20 52.5 8.5 54.5 11.3

Et09 40.5 10.3 0 103.9

Fb08-1 54.1 0 0 65.2

Gu01 36.5 7.1 44.7 243.1

Gu02 50.2 65.5 38.0 50.0

Gu03 35.4 74.1 88.1 331.5

Mr28 43.1 31.0 57.1 71.3

Mr29 28.6 69.0 28.6 0

Ms12-2 20.0 0 0 86.3

Ss09 20.2 0 0 53.7

Ss35 12.3 0 0 69.1

Su21 52.2 0 0 0

Table 3. Antifungal activity of VOCs from endophytic fungi.

Plant sample Isolate number Percentage of inhibition of microbial growth

C. gloeosporioides P. noxius R. microporus

Baeckea frutescens Bf17 23.5 7.7 43.5

Cycas siamensis Cs05 100/D 100/D 100/D

Hoya ovalifolia Ho21 0 0 30.4

Maesa ramentacea Mr23 9.1 65.1 26.1

Mr35 60.9 1.4 58.8

Melientha suavis Ms01-2 0 0 21.7

*D = Dead



Diversity of entomopathogenic fungi in protected forest

in the Eastern of Thailand

Winanda Himaman1*, Panrada Jangsantear1, Baramee Sakolruk1,

Kittima Duengkae1, Suchada Mongkolsamrit2, Wasana Noisripoom2,

Janet Jennifer Luangsa-Ard2 and Isarapong Vorapab1

1Department of National Parks, Wildlife and Plant Conservation (DNP), Chatuchak District, Bangkok, Thailand 2National Center of Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology

Development Agency (NSTDA), Khlong Luang District, Pathum Thani, Thailand


Abstract: The entomopathogenic fungi or insect fungi are widespread in nature. They are a well-known as a rich

source of bioactive compounds which prove to useful in medicinal and agricultural applications. There are about

400 species recorded from natural forests in Thailand. However, our knowledge of entomopathogenic fungi

species diversity in the eastern of Thailand is limited. During June 2017-December 2018, we studied species

diversity of entomopathogenic fungi in Khao Soi Dao Wildlife Sanctuary, Chanthaburi Province and Mu Ko Chang

National Park, Trat Province. A total of 537 samples were classified to 3 families; Clavicipitaceae, Cordycipitaceae and

Ophiocordycipitaceae in the order Hypocreales. These fungi were revealed into 43 species based on morphological

character study. The infected insects were in 8 major orders including Coleoptera, Hemiptera, Hymenoptera,

Isoptera, Lepidoptera, Neuroptera, Orthoptera and spider in Araneae order of class Arachnida. The most infected

insects were in Hymenoptera order (44.87 percentage). The most abundant species was Ophiocordyceps unilateralis.

In addition, Ophiocordyceps species found on coleoptera larvae and orthoptera in Mu Ko Chang National Park will

be tentatively described as new species based on morphology and phylogenetic analysis.

Keywords: Entomopathogenic Fungi, Diversity, Taxonomy, Protected Forest, Eastern

Introduction

The term entomopathogenic refers to those microorganisms that are capable of attacking insects using

them as hosts to develop part of their life cycle (Delgado and Murcia, 2011). Entomopathogenic fungi are fungi

that parasitizes the wide range of insects and spiders. The entomopathogenic species are found in almost all

ecosystems. The largest numbers of fungal species that are pathogenic to insects belong to the order (Molnar et

al., 2010). For more than one thousand years, medicinal Ophiocordyceps sinensis has been known as a unique

Tibet's prized parasitic fungus in the Qinghai-Tibetan Plateau for its mysterious life history (Lo et al., 2013).

The genus Beauveria and Metarhizium (Cordycipitaceae) play an important role in controlling insect populations

and have been increasingly utilized as biological control agents of insect pests throughout the world. Nowadays,

the entomopathogenic fungi are becoming increasingly popular from medical and pharmacological researchers

because these fungi are abundant source of useful natural products with various biological activities (Das et al.,

2010). There are about 700 species of entomopathogenic fungi worldwide (Roy et al., 2006).

Thailand is rich in biodiversity located in the tropical areas (Luangsa-ard et al. 2010). At this present,

more than 400 species of entomopathogenic species were reported from Thailand (Luangsa-ard et al. 2010). The

fungus Cordyceps gentilis on a hornet from northern Thailand was the first species recorded by Petch (1932)

which was later considered as Cordyceps sphecocephala by Hywel-Jones (1995).

The diversity of entomopathogenic fungi of the eastern part is poorly known and few collections have

been made. The objectives of the present study were to explore diversity of entomopathogenic fungi in eastern

Thailand. Specifically, Department of National Parks, Wildlife and Plant Conservation (DNP) and BIOTEC have

contributed to the studying of diversity of entomopathogenic fungi in Khao Soi Dao Wildlife Sanctuary (KSD),

Chanthaburi Province and Mu Ko Chang National Park (MKC), Trat Province, Thailand.


Collecting area

Entomopathogenic fungi specimens were collected in Khao Soi Dao Wildlife Sanctuary and Mu Ko

Chang National Park during June 2017-December 2018.

Specimen collection and isolation

The undersides and upper sides of leaves, stems and leaf litter in the forest were examined for fungi

growing on dead insects. Collected specimens were placed in plastic boxes and returned to the laboratory for

further examination. Fresh materials were examined and isolated in pure culture from both the anamorph and

http://www.indexfungorum.org/names/families.asp?FamilyName=Cordycipitaceae



teleomorph using the technique described by Luangsa-ard et al. (2012). Strains were isolated from the infected

specimens on potato dextrose agar (PDA). Cultures were incubated at 25°C for up to 10 weeks with high moisture

provided by a moist cotton sheets. Slow growing isolates were daily checked. After incubation, fungal growth

was transferred with a sterile needle to new PDA plates. Fungal culture were deposited in the Forest and Plant

Conservation Research Office and the BIOTEC Culture Collection (BCC).

Morphological study Fruiting bodies were examined using free hand sections under a stereo-microscope (Olympus SZX16).

Water-mounted slides were prepared for a microscope study and photographed under a compound microscope

(Olympus CX41). Morphological characteristics of the fungus were examined using classical mycological

techniques based on growth rate and macroscopic and microscopic characteristics. All specimens were dried at

50oC using food dehydrator. The IF numbers refer to herbarium material and to cultures deposited at the Forest

herbarium (mushroom section), DNP and the BCC, Thailand. In this study, clearly distinguishable morphological

samples will be studied molecular taxonomy according to Luangsa-ard et al. (2018). However, the data of

molecular taxonomy will not be showed and discussed in this paper.

Result & Discussion

During this study, we have collected infected insects 397 and 140 samples from KSD and MKC,

respectively. A total of 537 entomopathogenic fungi were collected and then identification on the basis of morphological

characters at Forest Microbiology Laboratory, DNP. The specimens were mostly founded during rainy season

(May to July). All samples were identified 15 genera belonging to 3 families; Clavicipitaceae, Cordycipitaceae

and Ophiocordycipitaceae in the order Hypocreales. These fungi were revealed into 43 species as shown in table

1. Currently, the strains of entomopathogenic fungi are listed in the following orders: Hypocreales (various genera),

Onygenales (Ascosphaera genus), Entomophthorales, and Neozygitales (Entomophthoromycota) (Mora et al., 2017).

Fungal species were varied on collections made over different site. Among these species, some were reported as

biocontrol agents, some with medicinal properties. Ophiocordyceps unilateralis was the most abundant entomopathogenic

fungi species of affixed to the underside of a leaves. Kobmoo et al. (2015) mentioned that O. unilateralis is an

ubiquitous pathogen of ants and in addition the species had hidden phylogenetic diversity associated with host

specificity. The occurrence of B. bassiana and M. anisopliae were zero number in KSD comparing with both were

common in MKC.

The host insects were in 8 major orders including Coleoptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera,

Neuroptera, Orthoptera and spider in Araneae of class Arachnida. In fact, the hosts are spread among 20 of the 31

orders of insects, in all developmental stages: eggs, larvae, pupae, nymphs, and adults (Araújo and Hughes, 2016).

In this study, the highest frequency of infection insects were in Hymenoptera order (44.87 percentage). Some

fungal species were highly host specific, whereas others had broad host. The genera Aschersonia, Hypocrella and

Moelleriella were specific to scale insects. The Gibellula specimens were regularly collected on spider hosts that

indicate its specific presence. Ants were found to be infected by Ophiocordyceps irangiensis/myrmecophila on

the ground or underneath the leaf litter. A thin single yellow synnema of the O. irangiensis/ myrmecophila arising

from the host was produced. In this case, the hosts were found on the ground, or underneath the leaf litter. In this

study, we found that there were 19 species with hosts in the order Hemiptera. This has clearly shown that Hemiptera

is wide host range. Faria and Wraight (2007) reported approximately 60% of insect diseases are caused by pathogenic fungi.

The isolation of these fungal species in culture was successful in some fungal species. However, the

isolation of O. unilateralis was needed a complicated method and culture medium according by Wongsa et al. (2005).

Furthermore, two Ophiocordyceps species will be described as new species based on morphology and

phylogenetic analysis. The Ophiocordyceps sp. 1 resemble Ophiocordyceps bruneipunctata, buried in the forest

soil, were found parasitizing coleoptera larvae collected from dry evergreen forest at Mu Ko Chang National Park.

The fungus produced pale pink stroma on morphological character. The Ophiocordyceps sp. 2 was collected from

rotting log. Hosts were immature orthopteran. Their morphological character and phylogenetic study clearly

distinct from genera Ophiocordyceps. The surveys of entomopathogenic fungi in Thailand have a very long history

with BIOTEC staff. Their several field studies have reported the new species of entomopathogenic fungi from

natural forests (Luangsa-ard et al., 2017; Luangsa-ard et al., 2018; Khonsanit et al., 2019; Kuephadungphan et al.,

2019). These findings encourage further research on the fungus in protected forests including national parks and

wildlife sanctuaries in Thailand.

Conclusion

Entomopathogenic fungi were mainly found in order Hypocreales (Clavicipitaceae, Cordycipitaceae and

Ophiocordycipitaceae). Fungus diversity was 43 species from both protected forests in the eastern part of Thailand.

Fungal common to both study sites was Ophiocordyceps irangiensis/ myrmecophila. Two species of Ophiocordyceps

proved to be undescribed and will be proposed as new species after confirmed with molecular results. At this

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/developmental-stage

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/pupa

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/nymph


present, studying biodiversity need to explore the potential economic benefit from entomopathogenic fungi

concurrently. Thus, there is an important to contribute the collaboration with many research groups including

biotechnologists, pharmacologists, analytical chemists, medical scientists etc. (Shrestha et al., 2016).

Acknowledgement

We would like to thank the staff of Khao Soi Dao Wildlife Sanctuary and Head of Mu Ko Chang National

Parks for splendid hospitality whilst we were on field trips. We also would like to thank Forest and Plant

Conservation Research Office, Department of National Parks, Wildlife and Plant Conservation for the support of

the project "Diversity of invertebrate-pathogenic fungi and wild mushrooms in protected forest in eastern and

northeastern of Thailand".

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doi:10.1017/S0953756205003321.



Table 1. Diversity of entomopathogenic fungi in KSD, Khao Soi Dao Wildlife Sanctuary and MKC, Mu Ko

Chang National Park in eastern, Thailand (June 2017 to December 2018).

Genus/species Host/Insect Order KSD MKC

Family Clavicipitaceae

Aschersonia badia

Aschersonia confluens

Aschersonia luteola

Aschersonia marginata

Aschersonia placenta

Aschersonia sp.

Aschersonia oxystoma

Conoideocrella luteorostrata

Conoideocrella tenuis

Scale insect/Hemiptera









0

2

0

1

6

2

0

2

3

1

8

3

1

1

1

3

7

0

Hypocrella badia

Hypocrella calendulina

Hypocrella discoidea

Hypocrella sp.

Hypocrella luteola

Metarhizium anisopliae

Moelleriella mollii

Moelleriella raciborskii






Coleopteran/Coleoptera



1

5

2

1

0

0

3

8

0

0

6

0

1

1

7

4

Family Cordycipitaceae

Akanthomyces pistillariiformis

Akanthomyces sp.

Beauveria bassiana

Beauveria sp.

Cordyceps javanica

Cordyceps sp.

Cordyceps tenuipes

Cordyceps tuberculata

Engyodontium sp.

Gibellula pulcha

Gibellula sp.1

Gibellula sp.2

Isaria sp.

Torrubiella hemipterigenum

Moth/Lepidoptera

Moth/Lepidoptera



Spider/Araneae

Spider/Araneae

Lepidopteran pupae/Lepidoptera

Moth/Lepidoptera


Spider/Araneae

Spider/Araneae

Spider/Araneae

Spider/Araneae

Spider/Araneae

Leafhopper/ Hemiptera

0

0

0

0

1

1

1

0

0

0

1

1

8

0

1

1

1

21

5

0

0

2

3

1

1

2

3

0

1

3


Table 1. (continued)

Genus/species Host/Insect Order KSD MKC

Family Ophiocordycipitaceae

Ophiocordyceps irangiensis/myrmecophila

Ophiocordyceps longissima

Ophiocordyceps sporangifera

Ophiocordyceps sp. 1

Ophiocordyceps sp. 2

Ant/Hymenoptera

Cicada/Hemiptera

Elateridae larvae/Coleoptera

Coleopteran larvae/Coleoptera

Orthopteran/Orthoptera

52

3

0

0

0

29

7

1

28

7

Ophiocordyceps sphecocephala

Ophiocordyceps unilateralis

Ophiocordyceps cf. communis

Ophiocordyceps nutan

Ophiocordyceps houaynhangensis

Polycephalomyces nipponicus

Purpureocillium takamizusanensis

Wasp/Hymenoptera

Ant/Hymenoptera

Termite/Isoptera

Stink bug/Hemiptera

Lepidopteran/Lepidoptera

Neuroptera

Stink bug/Hemiptera

0

141

51

2

49

10

1

3

15

0

0

0

0

0

Total 397 140

Figure 1. a) Ophiocoryceps sp. 1 in habitat showing pale pink stroma emerging from coleopteran larva

underneath the forest ground b) Ophiocoryceps sp. 2 with mushroom-like head on orthopteran.

a b




Diversity of mushrooms at Mu Ko Chang National Park, Trat Province

Baramee Sakolrak*, Panrada Jangsantear, Winanda Himaman, Tiplada Tongtapao,

Chanjira Ayawong and Kittima Duengkae

Forest and Plant Conservation Research Office, Department of National Parks,

Wildlife and Plant Conservation, Chatuchak District, Bangkok, Thailand


Abstract: Diversity of mushrooms at Mu Ko Chang National Park was carried out by surveying the mushrooms

along natural trails inside the national park. During December 2017 to August 2018, a total of 246 samples were

classified to 2 phyla Fungi; Ascomycota and Basidiomycota. These mushrooms were revealed into 203 species

based on their morphological characteristic. They were classified into species level (78 species) , generic level

(103 species) and unidentified (22 species). All of them were divided into 4 groups according to their ecological

roles in the forest ecosystem, namely, saprophytic mushrooms 138 species (67.98%), ectomycorrhizal mushrooms

51 species (25.12%) , plant parasitic mushrooms 6 species (2.96%) termite mushroom 1 species (0.49%). Six

species (2.96%) were unknown ecological roles and 1 species as Boletellus emodensis (Berk.) Singer are both of

the ectomycorrhizal and plant parasitic mushroom. The edibility of these mushrooms were edible (29 species) ,

inedible (8 species) and unknown edibility (166 species). Eleven medicinal mushroom species were recorded in

this study. The most interesting result is Spongiforma thailandica Desjardin, et al. has been found, the first report

found after the first discovery in 2009 at Khao Yai National Park by E. Horak, et al.

Keywords: Species list, ecological roles, edibility, protected area, Spongiforma thailandica

Introduction

Mushroom is a group of fungi which has the reproductive part known as the fruit body or fruiting body

and develops to form and distribute the spores. The mushrooms are a very large class of organisms which in their

structure have some similarities to plants, by they lack chlorophyll and are thus unable to build up the carbon

compounds essential to life. Instead, they draw their sustenance ready-made from living or dead plants or even

animals, as animal do (Phillips, 2006). They can be epigeous or hypogeous and are divided into two distinct groups

according to sexual reproductive forms as Basidiomycetous and Ascomycetous mushrooms. The Basidiomycetous

mushrooms are a diverse group of fungi composed of agarics, boletes, tooth fungi, chanterelles, coral fungi,

polypores, puffballs, earth stars, stinkhorns, bird’s nest fungi and jelly fungi. The Ascomycetous mushrooms are

included the cup fungi, morels, truffles, earth tongues, and saddle fungi. There are approximately 1.5 million species

of fungi found on earth (Hawksworth, 2001; 2004) and approximately 41,000 species of mushrooms (Deshmukh,

2004) . An estimated 1,069 mushroom species have been reported as being used for food purposes (Boa, 2004) .

According to Sangwanit et al.(2013) the numbers of mushrooms recorded in Thailand were over 2,500 species.

Mu Ko Chang National Park is a big archipelago of over 40 islands in Trat Province, east of Thailand. It

became established in 1982 as 45th national park of Thailand. The total area is 650 square kilometers of which

two-thirds is marine area. Its landscape is mostly high mountains and stone cliffs. The highest mountain is Khao

Salak Phet (743 meters above mean sea level) . The forests are very abundant as tropical rain forest, mangrove

forest and beach forest. There are many waterfalls on the island such as Than Mayom Waterfall, Khlong Phlu

Waterfall, etc. There are many plants, including Dipterocarpus alatus, Anisoptera costala, Hopea odorata,

Terminalia catappa, Calophyllum inophyllum and Acanthus ebracteatus. The park’s wildlife includes 29 mammal

species, 74 types of birds and 42 kinds of reptiles such as Ko Chang Frog (Limnonectes kohchangae Smith) , the

endemic animal of Ko Chang (Department of National Parks, Wildlife and Plant Conservation, 2019) . In 1902,

Rostrup, the first recorder species of mushroom in Ko Chang, reported 94 species of fungi including 1 rust fungus,

2 Myxomycetes and 91 mushrooms species (Rostrup, 1902) . This research is the first mushroom survey and

reported after 117 years ago.


Study areas

The mushroom specimens were collected along the nature trails inside Ko Chang main islands showed

in Figure 1. such as Than Mayom Waterfall, Khlong Phlu Waterfall, Khlong Nonsi Waterfall, Salak Phet trail and

Ban Bang Bao trail in rainy and winter seasons of the years from 2017 to 2018 (December 2017 to August 2018).

Proceedings of International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand

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Mushroom identification All collected mushrooms were studied on macroscopic and microscopic characteristics. Macroscopic

characters required for identification such as size and color of the pileus and stipe, habit, habitat, substratum, odor,

presence or absence of veil and volva were noted from the fresh fruit bodies in the field and photographed in their

natural habitat (Largent, 1973). Microscopic characters required such as shape, size, color and ornamented spores,

basidia, cystidia and chemical reaction on Melzer’s solution (Largent et al. , 1977). All characteristics were used

to identify all mushrooms into species if possible by various identification keys and books (Corner and Bas, 1962;

Dring, 1964; Corner, 1966; 1972; 1981; Lowy, 1951; 1952; Moser, 1973; Pegler, 1986; Largent and Baroni, 1988;

Ruksawong and Flegel, 2001; Imazeki et al. , 2005; Phillips, 2006; Miller and Miller, 2006; Chandrasrikul et al. ,

2008 Sanoamuang, 2010; Sangwanit et al. , 2013; Bandara et al. , 2017) . Besides these keys and books, some

authentic websites were accessed such as www.mushroomexpert.com. After proper identification, the current name

of the identified mushrooms and their taxonomic details were accessed from the website: www.catalogueoflife.org and

www.indexfungorum.org (accessed on March 6, 2019). All dried specimens are deposited at the Forest Herbarium

(Mushrooms Section), under the supervision of Department of National Parks, Wildlife and Plant Conservation.

Result & Discussion

The 246 mushroom specimens were collected from various nature trails in Mu Ko Chang National Park

in main island. They were classified to 2 phyla; Ascomycota and Basidiomycota. These mushrooms were revealed

into 203 species based on their morphological characteristic only. They were classified into species level (78

species) , generic level ( 103 species) and unidentified ( 22 species) ( Table 1.) . Since this study used only

morphological characteristics for classification. If they were studied more by molecular techniques. The number

and species of collected mushrooms may be changed.

All of collected mushrooms were divided into 4 groups according to their ecological roles in the forest

ecosystem, namely, saprophytic mushrooms 138 species ( 67. 98% ) , ectomycorrhizal mushrooms 51 species

(25.12%), plant parasitic mushrooms 6 species (2.96%) termite mushroom 1 species (0.49%). Six species (2.96%)

were unknown ecological roles and 1 species as Boletellus emodensis (Berk. ) Singer are both of the ectomycorrhizal

and plant parasitic mushroom (Table 1. and Figure 2.).

The edibility of these mushrooms were edible (29 species) , inedible (8 species) and unknown edibility

(166 species). The eleven medicinal mushroom species were recorded (Table 1. and Figure 3.). Most of edible mushrooms

were in the genus Russula which were found at least five species such as R. alboareolata Hongo, R. cyanoxantha

(Schaeff.) Fr., R. delica Fr., etc. This genus was well known as one of the finest foods derived from the forest especially

in the north and northeast of Thailand and had a high price around 270 baht/kg (Sakolrak et al., 2018). According

to this study, none of Ko Chang villagers collected wild mushrooms for eating or selling. It is possible that the

economic system in this area tied to tourism.

In 1902, Rostrup, the first mushroom species recorder in Ko Chang, reported 94 species of fungi

including 1 rust fungus, 2 Myxomycetes and 91 mushrooms species. There were only four mushroom species that

were found in this study as Microporus xanthopus ( Fr. ) Kuntze, Pycnoporus sanguineus ( L. ) Murrill,

Schizophyllum commune Fr. and Stereum ostrea (Blume & T. Nees) Fr. So the summary numbers of mushroom

species are reported from Mu Ko Chang National Park are 290 species.

The most interesting result is the first report found of Spongiforma thailandica Desjardin, et al. outside

the Khao Yai National Park, the first discovery in 2009 by E. Horak, et al. (Desjardin et al., 2009). The specimens

were collected in December 2017. The research team has tried to track more specimens but still cannot collect

more specimens. The macroscopic and microscopic characters of S. thailandica, which found at Mu Ko Chang

National Park (Figure 4.), has quite similar to previous publication (Desjardin et al., 2009) except some chemical

reaction tests for example 10% potassium hydroxide (10% KOH) that it was not tested in this study. The fresh

fruit bodies that found at Kho Yai National Park were immediately deep purple to purplish black in 10% KOH

(Desjardin et al. , 2009) . In this occasion, the research team tried to extract DNA according to Desjardin et al.

(2009) method. The PCR reaction was performed for internal transcribed spacer ( ITS) regions using the primer

combinations ITS1-ITS4 and sequencing was done commercially (Marcrogen, Korea). The ITS gene sequencing

results were compared with the GenBank database (http: / /www.ncbi.nlm.nih.gov/ ) using BLAST search. The

resultant ITS gene sequences were aligned using CLUSTAL W software (Larkin et al. , 2007) in the program

BioEdit (Hall, 2011). The results showed 98.32% similar to Spongiforma thailandica BBH DED 7873. However,

98.32% similar is still not strong enough. If we study in more other genes regions. The difference may be found

and leading to being a new species in the future.

Conclusion

This research was the second time to survey diversity of mushrooms at Mu Ko Chang National Park after

117 years ago. Two hundred and three mushrooms species were collected inside Ko Chang island, the main island

of Mu Ko Chang National Park, from various nature trails. The consisted of saprophytic mushrooms, ectomycorrhizal

http://www.mushroomexpert.com/

http://www.catalogueoflife.org/

http://www.indexfungorum.org/

http://www.ncbi.nlm.nih.gov/



mushrooms, plant parasitic mushrooms, termite mushroom, unknown ecological roles and both of the ectomycorrhizal

and plant parasitic mushroom are 138, 51, 6, 6, 1 and 1 species respectively. The twenty nine species of edible

mushrooms were recorded in this survey. The most interesting result is that the first report found that Spongiforma

thailandica Desjardin, et al. were spread outside the Khao Yai National Park after the first discovery in 2009.

Acknowledgement

This research was funded by Department of National Parks, Wildlife and Plant Conservation (DNP) ,

Ministry of Natural Resources and Environment. We would like to thank Mr. Itsarapong Voraphab and Mr. Boonsong

Sriyotsombat for their kind helps on fieldwork and Mr. Kosit Nilrat, Head of Mu Ko Chang National Parks, and his

staffs for various facilities.

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Figure 1. Map of Mu Ko Chang National Park (DNP, 2019); 1 = Khlong Nonsi Waterfall, 2 = Khlong Phlu

Waterfall, 3 = Than Mayom Waterfall, 4 = Salak Phet trail, and 5 = Ban Bang Bao trail.

Figure 2. The ecological roles of mushrooms collected from Mu Ko Chang National Park.

Figure 3. The edibility information of mushrooms collected from Mu Ko Chang National Park.

1

2 3

4

5


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Figure 4. Spongiforma thailandica were found at Mu Ko Chang National Park; a. fruit bodies, b. basidiospores.

Table 1. Species list of mushrooms collected from Mu Ko Chang National Park, Trat Province, their edibility

information (EI) and ecological roles (ER).

Scientific Name EI ER

Phylum Ascomycota

Class Geoglossomycetes

Order Geoglossales

Family Geoglossaceae

Trichoglossum hirsutum (Pers.) Boud. Une Unk Class Leotiomycetes

Order Helotiales

Family Helotiaceae

Bisporella citrina (Batsch) Korf & S. E. Carp. Une Sap

Class Pezizomycetes

Order Pezizales

Family Pyronemataceae

Aleuria luteonitens (Berk. & Broome) Gillet Une Sap Family Sarcoscyphaceae

Cookeina sulcipes (Berk.) Kuntze Une Sap Cookeina tricholoma (Mont.) Kuntze Une Sap

Class Sordariomycetes

Order Xylariales

Family Xylariaceae

Xylaria allantoidea (Berk.) Fr. Une/Med Sap Xylaria polymorpha (Pers.) Grev. Une/Med Sap Xylaria sp.1 Une Sap Xylaria sp.2 Une Sap Xylaria sp.3 Une Sap

Phylum Basidiomycota

Class Agaricomycetes

Order Agaricales

Family Agaricaceae

Agaricus trisulphuratus Berk. Une Sap Agaricus sp.1 Une Sap Agaricus sp.2 Une Sap Calvatia gardneri (Berk.) Lloyd Edi Sap Coprinus sp.1 Une Sap Cyathus striatus (Huds.) Willd. Ine Sap

a. b.



Scientific Name EI ER Cyathus sp.1 Une Sap Lepiota sp.1 Une Sap Lepiota sp.2 Une Sap Leucocoprinus fragilissimus (Ravenel ex Berk. & M.A. Curtis) Pat. Une Sap Lycoperdon sp.1 Une Sap Lycoperdon sp.2 Une Sap Unidentified 1 Une Sap

Family Amanitaceae

Amanita javanica (Corner & Bas) T. Oda, C. Tanaka & Tsuda Edi Ect Amanita mira Corner & Bas Une Ect Amanita virgineoides Bas Edi Ect Amanita sp.1 Une Ect

Family Clavariaceae

Clavaria fumosa Pers. Une Sap Clavaria sp.1 Une Sap Clavaria sp.2 Une Sap Clavaria sp.3 Une Sap Clavulinopsis helvola (Pers.) Corner Une Sap

Family Entolomataceae

Entocybe nitida (Quél.) T.J. Baroni, Largent & V. Hofst. Une Sap Entoloma sp.1 Une Sap Entoloma sp.2 Une Sap Entoloma sp.3 Une Sap

Family Hygrophoraceae

Hygrocybe cantharellus (Fr.) Murrill Edi Sap Hygrocybe sp.1 Une Sap Hygrocybe sp.2 Une Sap Hygrocybe sp.3 Une Sap

Family Hymenogastraceae

Gymnopilus sp.1 Une Sap Naematoloma sp.1 Une Sap Psilocybe sp.1 Une Sap

Family Inocybaceae

Crepidotus sp.1 Une Sap Inocybe rimosa (Bull.) P. Kumm. Une Ect

Family Lyophyllaceae

Termitomyces sp.1 Edi Ter Family Marasmiaceae

Campanella junghuhnii (Mont.) Singer Une Sap Campanella sp.1 Une Sap Campanella sp.2 Une Sap Marasmius siccus (Schwein.) Fr. Une Sap Marasmius sp.1 Une Sap Tetrapyrgos nigripes (Fr.) E. Horak Une Sap Trogia infundibuliformis Berk. & Broome Une Sap

Family Mycenaceae

Mycena sp.1 Une Sap Mycena sp.2 Une Sap


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Scientific Name EI ER Mycena sp.3 Une Sap Mycena sp.4 Une Sap Mycena sp.5 Une Sap Panellus pusillus (Pers. ex Lév.) Burds. & O.K. Mill. Une Sap Xeromphalina tenuipes (Schwein.) A.H. Sm. Une Sap

Family Omphalotaceae

Gymnopus sp.1 Une Sap Family Physalacriaceae

Oudemansiella canarii (Jungh.) Höhn. Edi Sap

Family Pleurotaceae

Pleurotus sp.1 Une Sap Family Psathyrellaceae

Psathyrella candolleana (Fr.) Maire Edi Sap

Family Pterulaceae

Deflexula fascicularis (Bres. & Pat.) Corner Une Sap Family Schizophyllaceae

Schizophyllum commune Fr. Edi/Med Sap

Family Tricholomataceae

Clitocybe sp.1 Une Sap Collybia sp.1 Une Sap Collybia sp.2 Une Sap Collybia sp.3 Une Sap Collybia sp.4 Une Sap Collybia sp.5 Une Sap Resupinatus applicatus (Batsch) Gray Une Sap

Unidentified 2 Une Sap Unidentified 3 Une Unk Unidentified 4 Une Unk Unidentified 5 Une Sap

Order Auriculariales

Family Auriculariaceae

Auricularia nigricans (Sw.) Birkebak, Looney & Sánchez-García Edi/Med Sap Auricularia thailandica Bandara & K.D. Hyde Edi Sap Auricularia sp.1 Une Sap

Order Boletales

Family Boletaceae

Boletellus emodensis (Berk.) Singer Edi Ect/Sap Boletus sp.1 Une Ect Boletus sp.2 Une Ect Boletus sp.3 Une Ect Boletus sp.4 Une Ect Boletus sp.5 Une Ect Boletus sp.6 Une Ect Boletus sp.7 Une Ect Boletus sp.8 Une Ect Chalciporus piperatus (Bull.) Bataille Edi Ect Phylloporus bellus (Massee) Corner Edi Ect Phylloporus sp.1 Edi Ect



Scientific Name EI ER Spongiforma thailandica Desjardin, Manfr. Binder, Roekring & Flegel

Une Ect Strobilomyces strobilaceus (Scop.) Berk. Edi Ect

Family Calostomataceae

Calostoma japonicum Henn. Une Ect Family Sclerodermataceae

Scleroderma sinnamariense Mont. Une Ect Scleroderma sp.1 Une Ect Scleroderma sp.2 Une Ect Scleroderma sp.3 Une Ect

Order Cantharellales

Family Cantharellaceae

Cantharellus sp.1 Une Ect Craterellus aureus Berk. & M.A. Curtis Une Ect Craterellus odoratus (Schwein.) Fr. Une Ect

Family Hydnaceae

Hydnum rufescens Pers. Une Ect Order Geastrales

Family Geastraceae

Geastrum mirabile Mont. Une Sap Geastrum rufescens Pers. Une Sap Geastrum sp.1 Une Sap

Family Gomphaceae

Phaeoclavulina cyanocephala (Berk. & M.A. Curtis) Giachini Edi Ect Ramaria sp.1 Une Sap Ramaria sp.2 Une Sap Ramaria sp.3 Une Ect

Order Hymenochaetales

Family Hymenochaetaceae

Coltricia cinnamomea (Jacq.) Murrill Ine Sap Hymenochaete rubiginosa (Dicks.) Lév. Une Sap Hymenochaete sp.1 Une Sap Hymenochaete sp.2 Une Par Hymenochaete sp.3 Une Par Hymenochaete sp.4 Une Sap Hymenochaete sp.5 Une Sap Unidentified 6 Une Sap Unidentified 7 Une Par Unidentified 8 Une Par

Order Phallales

Family Phallaceae

Phallus indusiatus Vent. Edi Sap Unidentified 9 Une Sap

Order Polyporales

Family Fomitopsidaceae

Daedalea sp.1 Une Sap Daedalea sp.2 Une Sap


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Scientific Name EI ER Family Ganodermataceae

Amauroderma rude (Berk.) Torrend Edi/Med Sap Amauroderma rugosum (Blume & T. Nees) Torrend Edi Sap Ganoderma applanatum (Pers.) Pat. Edi Sap Ganoderma sp.1 Une Sap Ganoderma sp.2 Une Sap Ganoderma sp.3 Une Sap Ganoderma sp.4 Une Sap

Family Meruliaceae

Cymatoderma elegans Jungh. Ine Sap Podoscypha sp.1 Une Sap

Family Phanerochaetaceae

Byssomerulius corium (Pers.) Parmasto Une Sap Family Polyporaceae

Cerioporus mollis (Sommerf.) Zmitr. & Kovalenko Une Sap Hexagonia tenuis (Fr.) Fr. Une Sap Lentinus arcularius (Batsch) Zmitr. Une Sap Lentinus fasciatus Berk. Une Sap Lentinus squarrosulus Mont. Edi/Med Sap Microporus xanthopus (Fr.) Kuntze Ine Sap Nigrofomes sp.1 Ine Sap Picipes badius (Pers.) Zmitr. & Kovalenko Une Sap Polyporus grammocephalus Berk. Une Sap Polyporus retirugus (Bres.) Ryvarden Une Sap Polyporus sp.1 Une Sap Polyporus sp.2 Une Sap Polyporus sp.3 Une Sap Polyporus sp.4 Une Sap Polyporus sp.5 Une Sap Polyporus sp.6 Une Sap Pycnoporus sanguineus (L.) Murrill Ine Sap Trametes cingulata Berk. Ine Sap Trametes versicolor (L.) Lloyd Edi/Med Sap Trametes sp.1 Une Sap Trametes sp.2 Une Sap Trametes sp.3 Une Sap Trametes sp.4 Une Sap Trametes sp.5 Une Sap Unidentified 10 Une Sap

Unidentified 11 Une Sap Unidentified 12 Une Sap

Order Russulales

Family Peniophoraceae

Peniophora sp.1 Une Sap Peniophora sp.2 Une Sap

Family Russulaceae

Lactarius piperatus (L.) Pers. Edi/Med Ect Lactarius sp.1 Une Ect



Scientific Name EI ER Lactarius sp.2 Une Ect Lactarius sp.3 Une Ect Lactarius sp.4 Une Ect Russula alboareolata Hongo Edi/Med Ect Russula cyanoxantha (Schaeff.) Fr. Edi Ect Russula delica Fr. Edi/Med Ect Russula densifolia Secr. ex Gillet Edi Ect Russula nigricans Fr. Edi Ect Russula rosea Pers. Une Ect Russula violeipes Quél. Edi Ect Russula sp.1 Une Ect Russula sp.2 Une Ect Russula sp.3 Une Ect Russula sp.4 Une Ect Russula sp.5 Une Ect Russula sp.6 Une Ect Russula sp.7 Une Ect Russula sp.8 Une Ect Russula sp.9 Une Ect

Family Stereaceae

Stereum ostrea (Blume & T. Nees) Fr. Ine Sap Stereum sp.1 Une Sap

Order Stereopsidales

Family Stereopsidaceae

Stereopsis sp.1 Une Sap Order Thelephorales

Family Thelephoraceae

Thelephora vialis Schwein. Une Ect

Unidentified 13 Une Unk Unidentified 14 Une Sap Unidentified 15 Une Sap Unidentified 16 Une Sap Unidentified 17 Une Unk Unidentified 18 Une Sap Unidentified 19 Une Unk Unidentified 20 Une Par Unidentified 21 Une Par Unidentified 22 Une Sap

Class Dacrymycetes

Order Dacrymycetales

Family Dacrymycetaceae

Calocera viscosa (Pers.) Fr. Une Sap Calocera sp.1 Une Sap Dacrymyces minor Peck Une Sap

Class Tremellomycetes

Order Tremellales

Family Tremellaceae


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Scientific Name EI ER Tremella mesenterica Retz. Edi/Med Sap Tremella sp.1 Une Sap

Remark: Abbreviations of edibility information (EI): Edi = edible mushroom; Ine = inedible mushroom;

Med = medicinal mushroom and Une = unknown edibility and abbreviations of ecological roles (ER):

Ect = ectomycorrhizal mushroom; Sap = saprophytic mushroom; Ter = termite mushroom and

Unk = unknown ecological role




Health benefit screening on 45 Thai human probiotics (TISTR strains): their safety and

immunomodulatory activity in macrophage function

Bhusita Wannissorn1*, Weerasak Taengphan2, Prapaipat Klungsupya2, Supatjaree Ruengsomwong1

and Thanchanok Muangman2

1Biodiversity Research Centre (BRC), Thailand Institute of Scientific and Technological Research (TISTR),

Techno Polis, Khlong Luang District, Pathum Thani, Thailand 2Expert Centre of Innovative Herbal Products (InnoHerb), Thailand Institute of Scientific and Technological

Research (TISTR), Techno Polis, Khlong Luang District, Pathum Thani, Thailand


Abstract: A total of 45 human probiotic strains (namely Med 1 to Med 45) from the probiotic bank of Thailand

Institute of Scientific and Technological Research (TISTR) were analyzed for their cytotoxic and phagocytic

activities on macrophage RAW 264.7 cells. Their heat killed supernatants were prepared from 48 hr-culture

anaerobically grown in de Man Rogosa and Sharpe (MRS) broth supplemented with 0.05% L-cystiene and were

tested without dilutions (at 100% concentration). Results obtained from the water-soluble tetrazolium salt (WST-1)

assay suggested that 27 out of 45 strains were non-cytotoxic to macrophage RAW 264.7 cells. Among them, 10

strains clearly demonstrated a cell-proliferation promoting activity with cell survival rate greater than 100%

compared with the untreated cells. The phagocytic activity of all 45 TISTR probiotics were determined by their

capability on up-taking phenol red particles which acted as foreign bodies. The results revealed that 16 out of 45

TISTR probiotic strains possessed positive phagocytosis in macrophage RAW 264.7 cells at various degrees.

A significant increase in percentage of phagocytosis (% phagocytic activity) in RAW 264.7 cells induced by these

16 TISTR probiotic strains was found in a range of 2.58 to 39.89% in comparison to control (0% phagocytic

activity, untreated cells) and β-glucan (64.43% phagocytic activity, positive control). Among 45 TISTR probiotic

strains, Med 1, Med 31, Med 39 and Med 42 are considered to be the most active strains regarding to their safety

and phagocytic activities on macrophage RAW 264.7 cells when tested under conditions described in this study.

Keywords: Cytotoxicity, immunomodulation, macrophages RAW 264.7, phagocytosis, probiotics

Introduction

Due to their beneficial effects on health, probiotics, live microorganisms conferring health benefit to the

host when administered in sufficient amount (FAO and WHO, 2011), have been widely applied as ingredients in

functional foods and dietary supplements. The Markets and Markets reported that probiotics market is estimated

to grow at USD 49.4 billion in 2018 and is projected to grow at a compound annual growth rate (CAGR) of 7.0%

from 2018, to reach a value of USD 69.3 billion by 2023 due to highly rising health awareness among consumers

worldwide. Apart from the abilities in enhancement intestinal barrier, preventing of pathogens via acidification,

production of antimicrobial substances and competitive exclusion, stimulation of immune system is considered as

one of the probiotics’ mechanisms which in turn generating health benefits to the host (Kumar Bajaj et al., 2015).

However, these mechanisms are strain-specific (Collado et al., 2012; Lee et al., 2016), not all probiotics can show

equally effectiveness. Hence, screening of a large number of candidate probiotics in order to obtain the effective

strains are still required.

Regarding the Innovative Center for Production of Industrially Used Microorganisms (ICPIM), Thailand

Institute of Scientific and Technological Research (TISTR), we have set up the probiotic bank with the aim to

collect and conserve local probiotics for commercial uses. Extensive isolation of effective probiotics has been

done from various sample sources in Thailand. Recently, 45 strains have been isolated from flowers and fruits

locally grown in the northern part of Thailand. All isolates were selected for probiotic properties based on

notification of Ministry of Public Health (2011) such as non-hemolytic activity, sensitivity to antibiotic, tolerance

to gastric- and intestinal juices, adhesion to human epithelial cell lines and antimicrobial activity against certain

intestinal pathogens. In this study, we further characterized immunomodulatory effects of these 45 isolates on

cytokine profiles using macrophage RAW 264.7 cell line.


Probiotics and growth conditions

Forty five strains of TISTR human probiotics (namely Med 1 to Med 45) from the probiotic bank of

Culture Collection of Microorganism Centre, TISTR were anaerobically grown in de Man Rogosa and Sharpe

(MRS) broth supplemented with 0.05% L-cysteine for 48 hr. Each 48 hr grown culture was adjusted with normal

saline to obtain the final concentration of 108 CFU/ml. Then, cell pellets of each culture were collected by centrifugation


for 5 min at 8,500 rpm at 25°C. After 3 times washing with normal saline, the washed pellets were suspended in

Dulbecco's Modified Eagle Medium (DMEM) high glucose (Biowest, USA) without antibiotics before being

heated at 110 °C for 10 min. The 45 heat killed supernatants were tested without dilutions (at 100% concentration).

Macrophages culture

The mouse monocyte macrophage cell line, RAW 264.7 (ATCC TIB-71TM) was purchased from American

Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were grown as adherent in Dulbecco’s Modified

Eagle Medium (DMEM) (Biowest, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS)

(GIBCO®, USA) and 1% (v/v) penicillin-streptomycin (GIBCO®, USA) in humidified atmosphere incubator with

5% CO2 at 37°C. The cells were collected by centrifugation (Kobota, Japan) at 1500 rpm, 4°C for 5 min, aspirated

and fresh culture medium at similar volume was added before dispensing into new culture flasks. All steps of

culturing and maintaining of RAW 264.7 cells were conducted in aseptic condition using the Biological Safety

Cabinet Class II (SANYO, Japan). For experimentation, cells were seeded onto 96-well plates at a density of

2x104 cells/well and incubated for 24 hrs before treatments.

Cytotoxicity analysis by WST-1 assay

The cytotoxic property of 45 TISTR human probiotics (heat killed supernatants) on macrophage RAW

264.7 cells were determined using 4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate

(WST-1) assay. Its principle is based upon the conversion of tetrazolium salt to soluble formazan crystals by

succinate dehydrogenase in the mitochondria of metabolically active cells. Therefore, in the dead cells this reaction

will not be occurred. After incubation, the probiotic supernatants were removed and washed once with 10 µl of

PBS and then 100 µl of WST-1 solution (Sigma, USA) was added to each well. The plates were incubated in

darkness with 5% CO2 in a humidified atmosphere incubator at 37°C for 30 min. The cell viability was determined

by the optical density (OD) at the wavelength of 450 nm with microplate reader (Tecan Infinite 200 Pro, Austria).

Mitomycin C (MMC) solution (100 µl) at 20 µg/ml prepared in DMEM was used as positive control. The percentage

viability of cells related to control wells containing cell culture medium (DMEM) without samples was calculated

as following equation:

% Cell viability = (Absorbance of treated cells / Absorbance of treated cells) x 100

Phagocytic activity by neutral red uptake assay

The phagocytosis ability of macrophages was measured by colorimetric neutral red uptake assay. The assay

was performed to determine phagocytosis ability of macrophage RAW 264.7 cells when treated with heat killed

supernatants of 45 TISTR human probiotics. The principle of this assay is based upon membrane permeability

and lysosomal activity of viable cells in response to pharmaceuticals, chemicals and environmental compounds

as well as nutrients. In this study, RAW 264.7 cells were treated (at 200 µl total volume) with 100% concentration

of heat killed supernatants of 45 TISTR human probiotics or or β-glucan (Sigma, USA) at 100 µg/ml for 24 hr.

By the end of incubation time, the treatments were removed and cells were washed 3 times with PBS followed by

adding of 100 µl of 0.075% neutral red solution (Sigma, USA) to each well. The reactions were kept at 37C for

60 min. The supernatant was removed and 100 µg/ml cell lysis solution (ethanol and 0.01% acetic acid at the ratio

of 1:1) was added to lysed cells at 37C for 120 min. The quality of dye incorporated into RAW 264.7 cells in

measured by spectrophotometry at 540 nm wavelength. The percentage of phagocytosis activity was calculated

as following equation:

% Phagocytic activity = Absorbance of sample – Absorbance of control x 100

Absorbance of control

Statistical analysis

All experimental data were expressed as mean ± standard error (Mean ± SE) of triplicated determination.

The significance of difference was used to compare mean (p<0.05). All analyses were performed using One-way

ANOVA and Tukey’s Honestly Significant Difference test.

Result & Discussion

Effects of probiotics on macrophage cell viability

MMC is a chemotherapeutic drug that has been used in cancers treatment. It works by intercalating the DNA

together. Hence, DNA cannot come apart during cell division resulting in no cells division and apoptosis. Damage

to nuclear DNA is thought to be its primary mechanism of cell death in MMC action (Snodgrass et al., 2013).

In this study, MMC (20 µg/ml concentration) was used as a positive control for cell proliferation test using the

WST-1 assay. After RAW 264.7 cells were treated with heat killed supernatants probiotics at 100 % concentration

and assessed by WST-1 assay, all probiotics exhibited the % cell viability as illustrated in Table1. Morphologies

of cells following treatments were demonstrated in Figure 1. The results were expressed as % cell inhibition


on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand 35

compared with the control cells. It was found that most TISTR probiotic strains (27 out of 45 strains) showed no

significant cytotoxic activity against RAW 264.7 cells. Among them, 9 strains including Med1, Med2, Med3,

Med4, Med5, Med7, Med8, Med10 and Med11 clearly demonstrated a cell-proliferation promoting activity with

cell survival rate greater than 100% compared with the untreated cells. However, the highest cytotoxic effect

against RAW 264.7 cells was observed in 6 strains (Med23, Med27, Med34, Med35, Med44, Med45) with cell

growth inhibition greater than 90% (cell survival rate less than 10%). RAW 264.7 cells treated with MMC

exhibited a marked decrease in cell survival to 17.04 ± 0.43 % (Table 1.)

Phagocytic activity of probiotics in macrophage function

It has been well-known that probiotics promote various health benefits (Sanders, 2008). They can inhibit

overgrowth of pathogenic bacteria by antibacterial substance production and stimulating the immune system. They

have been demonstrated that the effects on the variety of immune cells such as phagocytes (macrophages and

neutrophils), lymphocytes and natural killer (NK) cells. The neutral red uptake assay is a method to detect cell

viability or lysosome activity assay based on the ability of viable cells to incorporate and bind neutral red within

lysosomes (Guan et al., 2011). It is generally performed on adherent cells. The quantity of dye incorporated into

cells is measured by spectrometry at 540 nm, and is directly proportional to the number of cells with an intact

membrane. In this study, stimulation of phagocytic function by 45 TISTR probiotics in macrophage RAW 264.7

cells was investigated by phenol red assay meaning the amount of neutral red internalized in phagosomes (Figure1).

β-glucans are naturally occurring polysaccharides that are produced by bacteria, yeast, fungi and many plants

especially mushrooms (Han et al., 2008). β-glucan is recognized by the dectin-1 and C3 receptors on macrophages

that triggers phagocytosis (Goodridge et al., 2009). We selected β-glucan (at 100 µg/ml concentration) and used

as a positive control in this study. As demonstrated in Table 2, the results from phenol red uptake assay revealed

that 16 out of 45 TISTR probiotic strains possessed positive phagocytosis in macrophage RAW 264.7 cells. An

increase in percentage of phagocytosis (% phagocytic activity) in RAW 264.7 cells induced by these 16 TISTR

probiotic strains (Med1, Med2, Med3, Med4, Med6, Med10, Med11, Med14, Med16, Med30, Med31, Med36,

Med39, Med40, Med41, Med42) was found in a range of 2.58 to 39.89% in comparison to control (0% phagocytic

activity, untreated cells) and β-glucan (64.43% phagocytic activity, positive control) (Table 2). Though we found

out that these TISTR probiotics were effective on activation of macrophages function (phagocytosis), their

mechanisms are still unknown. They may or may not function like β -glucans by binding to dectin-1 receptor on

macrophages and lead to activation of macrophages to engulf pathogens through phagocytosis and digestion them

with lysosomal enzymes. Further studies on this matter are required.

Conclusion

Among 45 heat-killed TISTR probiotic strains, Med 1, Med 31, Med 39 and Med 42 are considered to

be the most active strains regarding to their safety and phagocytic activities on macrophage RAW 264.7 cells.

These results suggest that administration of these four TISTR probiotic strains may result in an initiation of

immune reaction against foreign materials. Therefore, they can be employed as a natural resource for development

to healthy dietary supplement such as functional food products.

Acknowledgement

This work was performed by Thailand Institute of Scientific and Technological Research (TISTR) under

financial support by a grant from the Ministry of Science and Technological (MOST) during 2018-2020.

References

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Health and nutritional properties of probiotics in food including powder milk with live lactic acid

bacteria. FAO and WHO Joint and Expert Committee Report.

Goodridge, S., Underhill, D., and N. Touret. 2012. Mechanisms of fc receptor and dectin-1 activation for phagocytosis.

Traffic. 13 (8):1062-1071.

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form the roots of Actinidia kolomikta on macrophage. Int J Biol. 3 (2): 3-10.

Han, M., Han, Y., Hyun, S., and Shin, H. 2008.Solubilization of water-insoluble β-glucan isolated from

Ganoderma lucidum. J Env Biol. 29 (2): 234-242.

Kumar Bajaj, B., Claes, I., and Lebeer, S. 2015. Functional mechanisms of probiotics. J Microbiol Biotech Food

Sci. 4 (4): 321-327.


Lee, C., Van, G., Taverne, N., Meijerink, M., Bron, A., Spano, G., and Kleerebezem, M. 2016. Strain-specific

features of extracellular.

polysaccharides and their impact on Lactobacillus plantarum-host interactions. Appl Environ Microbiol 82

(13):3959–3970.

Sanders, M. 2008. Probiotics: Definition, Sources, Selection, and Uses. Clin Infec Dis. 46 (2): 58-61.

Snodgrass, G., Collier, A., Coon, A., and Pritsos, A. 2010. Mitomycin C inhibits ribosomal RNA: A novel

cytotoxic mechanism for bioreductive drug. Cold Spring Harb. Perspect. Biol.5:1-16.



Table 1. Cytotoxicity determination of 45 TISTR human probiotics against RAW 264.7 cells by WST-1 assay.

% Cell viability

range Probiotic stains

Cytotoxicity

(% cell viability)

80-100

Med1 102.60 ± 3.18a

Med2 109.92 ± 6.08a

Med3 110.85 ± 6.70a

Med4 108.98 ± 6.89a

Med5 103.98 ± 8.30a

Med6 99.00± 4.67a

Med7 104.59 ± 14.13a

Med8 111.58 ± 1.65a

Med10 117.74 ± 4.05a

Med11 108.34 ± 5.55a

Med14 87.40 ± 0.24a

Med16 91.48 ± 10.04a

Med30 84.90 ± 15.91a

Med31 89.94 ± 2.90a

Med39 86.07± 1.65a

Med41 82.62 ± 4.52a

Med42 85.73 ± 6.91a

60-79

Med9 79.11 ± 5.01a

Med17 68.13 ± 2.09c

Med19 65.15 ± 5.19c

Med21 62.13 ± 8.60c

Med36 77.21 ± 3.17a

Med38 69.50 ± 3.30c

Med40 73.26 ± 8.64c

40-59

Med18 49.30 ± 9.78d

Med24 51.66 ± 7.90d

Med26 48.39 ± 1.60d

Med28 58.66 ± 17.06c

20-39

Med13 28.62 ± 4.54c

Med22 39.73 ± 1.66d

Med25 30.40 ± 8.48d

Med32 30.06± 5.39d

0-19

Med12 10.41 ± 1.16b

Med15 10.40 ± 3.64b

Med20 14.06 ± 10.28b

Med23 8.18 ± 0.35b

Med27 9.95 ± 1.53b

Med29 16.09 ± 13.21b

Med33 11.90 ± 4.75b

Med34 9.14 ± 1.81b

Med35 9.00 ± 0.75b

Med37 15.59 ± 6.00b

Med43 11.90 ± 6.33b

Med44 9.33 ± 1.71b

Med45 9.82 ± 1.97b

Positive control MMC (20 µg/ml) 17.04 ± 0.43b

* Each value is mean ± SD (n=3). One way ANOVA and Tukey’s Honestly Significant Difference (P<0.05).

Difference later in the same row are significantly different.


Figure 1. Morphology of RAW 264.7 cells after treated with TISTR probiotic strains in WST-1 assay. (A) Control

(100 % cell viability); (B) Probiotics (80-100 % cell viability); (C) Probiotics (60-79 % cell viability); (D)

Probiotics (40-59 % cell viability); (E) Probiotics (20-39 % cell viability); (F) Probiotics (1-19 % cell viability).



Figure 2. Phagocytic activity of macrophage RAW 264.7 cells analyzed by neutral red uptake assay following

treatments with 45 TISTR probiotic strains; (A) Control; (B) Beta-glucan (high effect by 64% phagocytic activity);

(C) TISTR probiotics (moderate effect by 21- 40% phagocytic activity); (D) TISTR probiotics (mild effect or less

than 20% phagocytic activity); (E) TISTR probiotics (no effect in phagocytic activity).


Table 2. Phagocytic activity of 45 TISTR probiotic strains in RAW 264.7 cells by neutral red uptake assay.

% Phagocytosis range Probiotic strain % Phagocytic activity

21-40

Med1 39.89 ± 7.86a

Med4 32.07 ± 9.22a

Med31 29.89 ± 7.86a

Med39 39.39 ± 5.66a

Med42 32.13 ± 3.23a

1-20

Med2 16.58 ± 2.99b

Med3 4.94 ± 3.60b

Med6 2.58 ± 5.97b

Med10 9.15 ± 9.39b

Med11 18.09 ± 8.37b

Med14 13.27± 2.12b

Med16 15.71± 2.43b

Med30 8.68 ±5.09b

Med 36 2.58 ± 5.97b

Med40 4.94 ± 2.99b

Med41 16.58 ± 3.60b

No effects Med5, Med7, Med8, Med9, Med12, Med13, Med15, Med17, Med18,

Med19, Med20, Med21, Med22, Med23, Med24, Med25, Med26, Med27

Med28, Med29, Med32, Med33, Med34, Med35, Med37, Med38, Med43

Med44, Med45

Positive control β-glucan

(100 µg/ml) 64.43 ± 8.68

*Each value is mean ± SD (n=3). One way ANOVA and Tukey’s Honestly Significant Difference (P<0.05).

Difference later in the same row are significantly different.




Records of potential antimicrobial activity of soil actinomycetes isolated from

a community forest in Ban Khoklam Sang Aram, Udon Thani Province

Metinee Wasoontharawat 1* and Panthong Kulsantiwong 2 1Department of Biology, Faculty of Science, Udon Thani Rajabhat University, Muang District, Udon Thani, Thailand 2Department of Biology, Faculty of Science, Udon Thani Rajabhat University, Muang District, Udon Thani, Thailand


Abstract: The WHO (2015) released a report of an increasing number of infections due to the global antimicrobial

drug resistance crisis. Unexplored areas may be possible for sources of actinomycetes with potent antimicrobial

compounds. This study was directed towards isolation of actinomycetes that produce antimicrobial substances

from unexplored soil in a community forest in Ban Khoklam Sang Aram, Udon Thani Province. The survey

showed diversity of actinomycetes and rare actinomycetes in this area. Ten of 232 isolated actinomycetes showed

potent antimicrobial activity by growth inhibition of Bacillus subtillis, Candida albicans, Escherichia coli,

Pseudomonas aeroginosa and Staphylococcus spp. after testing using agar disc diffusion and agar well diffusion

tests. These isolates were identified as species in the genera Streptomyces (9 isolates) and Microbispora (1 isolate).

Keywords: Microbial diversity, actinomycetes, Antimicrobial activity, Soil microorganism, Pathogenic microorganism

Introduction

Actinomycetes are Gram-positive bacteria. They are unique in their formation of branching filaments with

fungi like hyphae and asexual spores. Their bacterial DNA has a high G+C content, 55-75% (Lechevalier and

Lechevalier, 1967; Embley and Stackebrandt, 1994). Actinomycetes is a major microbial population that is widely

distributed and inhabits the soil (Jaralla et al., 2014). They play role in soil biodegradation and humus formation

(McCathy and Williams, 1992; Agadagba, 2014). Moreover, many actinomycetes are pharmaceutically important

for production of secondary metabolites as antibiotics, vitamins and enzymes (Shahidi et al., 2004; Xu et al., 2005;

Arifuzzaman et al., 2010). Soil actinomycetes produce many of currently used antibiotics including Erythromycin,

Gentamycin, Rifamycin and Streptomycin (Jeffrey, 2008). Therefore actinomycetes are potentially important

strains for producing new bioactive compounds, especially antibiotics that have diverse clinical effects and have

activity against many pathogens (Atta and Ahamd, 2009; Usha et al., 2011).

This work was aimed to investigate diversity of an actinomycetes population in the soil of a community

forest in Ban Khoklam Sang Aram, Udon Thani Province and their antimicrobial activities, for the first time.

This forest has not yet been studied. Additionally, this area yields Streptomyces species and rare actinomycetes

strains with high antimicrobial activity.


Collection of soil samples

Seven soil samples were randomly collected from various sites in a community forest in Ban Khoklam

Sang Aram, Udon Thani Province. The seventh sites where the collected soil sample were takes were diverse.

They include 1) loam with a leaf covering, 2) loam with a decayed wood covering, 3) fine sand with no vegetation

and a leaf covering that served as drainage for rainfall and 4) sandy loam surrounding plant roots. The soil was

taken at a depth of 1-10 cm from the soil surface. Approximately 500 grams of each soil sample was collected

and was placed in sterile polyethylene bags and kept at room temperature. These were promptly transferred to a

laboratory for analysis of their actinomycetes population.

Preparation of soil samples

The soil samples were air-dried at room temperature until a constant weight was attained and were stored

at 4C prior to processing for actinomycetes isolation. They were pretreated to stimulate actinomycetes growth

and eliminate unwanted Gram-negative bacteria. Each soil sample divided into two parts for separate pretreatment

methods. One part was pretreated with wet heat and the other was pretreated with air dry plus calcium carbonate

(CaCO3) following the methodology of Fang et al. (2017). Then the samples were further examined for diversity

of actinomycetes colonies and actinomycetes isolates.


Enumeration isolation and purification of actinomycetes

Actinomycetes in each soil were isolated using a 10-fold serial dilution of soil and spread plate technique.

Diluted soil solution was spread on a humic acid (HA) medium (Hayakawa and Nonomura, 1987) and starch-casein

agar (SCA) (Okazaki et al., 1983) supplement with 1 mg/mL of nalidixic acid and 1 mg/mL of cycloheximide. All

spread plates were incubated at 37C for a period of 14 days.

A number of diverse morphological colonies appeared in each dilution on both the HA and SCA media on

day 14. The various colony morphologies were carefully counted and recorded. Then, the density of the actinomycetes

population was determined in units of CFU/g dried soil as the number colonies on a plate times the dilution factor.

Various colonies on HA and SCA media were then picked with sterile needles and transferred to an

International Streptomyces Project 2 (ISP2) medium. Each isolate was purified using a streak plate technique and

cultured at 37C for a period of 3-5 days until pure single colonies appeared.

Preliminary Screening for antimicrobial activity

Preliminary screening of the antimicrobial activity of purified isolates was done using a streak plate technique

following Lemons et al. (1985). One isolate was long single streak at the corner of ISP2 solid medium plate. Four

pathogenic strains were used to investigate the antagonistic activity of actinomycetes isolates. They were Bacillus subtilis,

Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. These pathogenic microorganisms were

suspended (0.5 McFarland) and streaked perpendicular to the actinomycetes strain using a sterilized loop. The plate was

then incubated at 37C for a period of 14 days. Clear zones, indicating microbial inhibition, and their widths were determined

in millimeters.

Secondary screening for antimicrobial activity

Each purified isolate expressing growth inhibition in the preliminary screening was cultured in 100 ml ISP2 broth

on a 250 ml rotary shaker (180 revolutions per minute, rpm) at 28+2°C for 4 days. They were then transferred to

a new YEME broth supplemented with 0.1% CaCO3, as a production medium. The cultures were incubated a rotary

shaker (180 rpm) at room temperature for 14 days. The culture broth was filtered through Whatman No.1 filter paper

to separate the mycelium. A clear supernatants were obtained that were extracted three times with ethyl acetate at a 1:1

(v/v) ratio with vigorous shaking for 1 hour according the method of Rao et al. (2017). The bioactive compounds were in

the ethyl acetate phase that separated from the aqueous phase. The ethyl acetate phase was evaporated under a reduced

vacuum at 80C. The obtained bioactive crude extract was used to test the antimicrobial activity of isolates using agar disc

diffusion and agar well diffusion tests.


The antimicrobial activity of purified isolates expressing growth inhibition in the preliminary screening were

confirmed by agar disc diffusion and agar well diffusion methods as described by Mohseni et al. (2013) and Omar et al.

(2000), respectively. Nystatin and nalidixic acid agents were used as controls for anti-Gram positive and anti-Gram

negative bacterial activity, respectively.

Morphological identification of actinomycetes

Preliminary identification of actinomycetes isolates grouped the bacterial as Streptomyces and non- Streptomyces,

(rare actinomycetes). It was performed by observing the growth rates of bacteria on an International Streptomyces

Project (ISP) medium (Shirling and Gottli, 1966). Streptomyces grew faster than non- Streptomyces, as was also

reported by Abdullah et al. (2016).

Isolates were identified at the genus level based on microscopic and macroscopic studies of actinomycetes

following Shirling and Gottileb (1966), Muthu et al. (2013) and Barka et al. (2015). Macroscopic examination

considered colony features such as size, shape, color, absence or presence of aerial mycelium and spore formation.

Microscopic characteristics observed were aerial and substrate mycelium, conidia, spore type, and especially spore chains.

Result & Discussion

Enumeration and isolation of actinomycetes

Actinomycetes in soil samples were isolated using HA medium and SCA medium supplement with

nalidixic acid (as antifungal agent) and cycloheximide (as antibacterial agent) at 37C for 7-14 days. The diversity

of the actinomycetes population was viewed from various types of colony morphology, size, color, elevation, surface

and pigmentation, as they grew on a solid medium.

The results indicated a high diversity of actinomycetes from soil samples collected from a community

forest in Ban Khoklam Sang Aram, Udon Thani Province, on both media. This data is shown in Figure 1.

The various soil samples had actinomycetes populatons in the range of 1.1 x 102 - 5.3 x 102 CFU/g dried soil on

HA plates and 2.1 x 102 - 9.4 x 102 CFU/g dried soil on SCA plates. The various colonies had obviously different

colony characteristic on the media. The highest numbers of colonies with different morphology were found on the

SCA medium as it supported significantly more growth than the HA medium. Most actinomycetes isolates grew



better when hydrolyzing starch and casein than the humid acid in the HA medium (Ravel et al., 2000). The types

of colonies grown on SCA medium are shown in Figure 2.

The most populated sample had 9.4 x 102 CFU/g dried soil on SCA medium, from the fourth site in the

forest. Its soil was a fine sand without vegetation and the soil was saturated with water. This may have been due

to a recent rainfall. Furthermore, the soil might contain soil nutrients, such as organic carbon, that originated from

other sites. According to Stackebrandt et al. (1991) and Baby et al. (2002), fertile soils or soil containing organic

carbon, nitrogen, and phosphorus is an important soil feature that can enhance the grow the rate and diversity of

an actinomycetes population. Alternatively, the highest number of diverse colonies on HA medium was 5.3 x 102

CFU/g dried soil from soil at the seventh site. This soil was a loam with a decayed wood covering. The colony

morphology from soil taken at this site was different than from other soil sources. So, it was of interest to

investigate these bacteria. Actinomycetes isolates have been found on decomposing wood since some actinomycetes

can produce hydrolytic enzymes to break down the lignocellulose of wood (Bontemps et al., 2013; Ding et al.,

2004). The lowest number of colonies on both media was 2.1 x 102 CFU/g dried soil on SCA medium from soil

at third site, as fine sand without vegetation. It was in an area where there was water flow due to rainwater runoff.

A total of 232 actinomycetes were isolated from seven soil samples collected at various locations within

this community forest.

Primary screening for antimicrobial activity All actinomycetes isolates underwent a preliminary screening for antimicrobial activity using an agar cross

streak method. It found that ten isolates possess inhibitory activity against pathogenic fungi and bacteria. These

were the 8.2.4HV, 8.2.5HV, 6.1HV-1, 20.2.6, 15.1, RH13-26, 11.1.2, 10. 4.1, 10.5, and RH12-1 isolates.


The ten isolates were further tested to confirm their antimicrobial activity using a crude ethyl acetate extract

with disc diffusion and well diffusion methods. Their antimicrobial activity was measured in terms of a clear zone

that formed. As shown in Table 1, many isolates were active against more than one of the tested pathogenic

microorganisms. Bacillus subtillis growth was inhibited by eight isolates. These were 6.1HV-1, 20.2.6, 15.1, 11.1.2,

10.5, 8.2.4HV, 10.4.1 and RH12-1, ranked from highest to lowest antimicrobial activity. Maximum and minimum

inhibition zones of B. subtillis of these isolates were 10 and 1 mm, respectively. Staphylococcus aureus growth

was inhibited by seven isolates viz., 15.1, 11.1.2, 10.5, 8.2.4HV, 8.2.5HV, 10.4.1, and RH12-1. The isolates

displayed maximum and minimum inhibition zones for S. aureus of 8 and 4 mm, respectively. Pseudomonas

aeruginosa growth was inhibited by eight isolates viz., 8.2.4HV, RH13-26, 15.1, 10.5, 8.2.5HV, 11.1.2, 10.4.1, and

20.2.6. The isolates displayed maximum and minimum inhibition zones for P. aeruginosa inhibition of 20 and

4 mm, respectively. Candida albicans growth was inhibited by three isolates viz., 8.2.4HV, 10.5, and 8.2.5HV.

The isolates displayed maximum and minimum inhibition zones for C. albicans of 10 and 6 mm, respectively. No

isolate showed growth inhibition of E. coli. Isolates 8.2.4HV and 10.5 showed inhibition against four pathogenic

microorganisms, but not against E. coli. Some isolates (10.4.1 and RH12-1) showed no inhibition of pathogenic

microorganisms. Actinomycetes isolates all produced secondary metabolites, such as bioactive antibiotics, to

inhibit growth of some pathogens. These results are similar to those of Thangapandian et al. (2007) and

Gebreyohannes et al. (2013) who studied antimicrobial actinomycetes isolated from rhizosphere soil, water, and

sediment.

The isolate 10.5 displayed antimicrobial activity against four pathogens including Bacillus subtilis, S.

aureus, P. aeruginosa, and C. albicans, comparing with other isolates. The results showed in Figure 3.

Identification of actinomycetes

A total of 232 microorganisms were preliminary identified as Streptomyces and non- Streptomyces (rare

actinomycetes), by observing their growth rate on ISP medium. It was found that most of the isolates (206) showed

the typical morphology of Streptomyces and few isolates (26) were found to be rare actinomycetes.

Only ten isolates expressed antimicrobial activity. They were identified at the genus level by examining

their morphological characteristics, i.e., presence of aerial mycelium, conidia, pigment per Barka et al. (2015) and

Shirling and Gottileb (1966). The results indicate that these isolates can be divided into two genera, Streptomyces

and Microbispore. Isolates 8.2.4HV, 8.2.5HV, 6.1HV-1, 20.2.6, 15.1, 11.1.2, 10.5, RH13-26, and RH12-1 are

Streptomyces. They have diverse types of spores including retinaculiapert, oligosporous, spira and spiral spores.

Only isolate, 10.4.1, was a Microbispore spp. and it showed disporus spores as depicted in Figure 4.

Conclusion

This work clearly showed that soil samples collected from a community forest in Ban Khoklam Sang

Aram, Udon Thani Province contain actinomycetes that possess antibacterial and antifungal activities. The data

demonstrates the diversity of actinomycetes population in the form of colony morphology. Additionally, these

isolates were effective in producing antimicrobial metabolites to against B. subtilis, S. aureus, P. aeruginosa and

C. albicans. It is revealed that this location is a good source of antimicrobial actinomycetes, both Streptomyces


and rare-actinomycetes. Further work will be performed to determine their potential application to control human

and plant pathogens.

Acknowledgement

The authors acknowledge Udon Thani Rajabhat University for support for research funding. We are

especially thankful to Prof. Dr. Somboon Tanasupawat, Department of Biochemistry and Microbiology, Faculty

of Pharmaceutical Sciences, Chulalongkorn University for advice and support with some agents and pathogens in

this work. We thank Mr. Prechawut Pimphakdee, Department of Biology, Faculty of Science, Udon Thani

Rajabhat University, Udon Thani for his technical assistance.

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Baby, U.I., Tensingh Baliah, N., Ponmurugan, P., and Premkumar, R. 2002. Population level of certain of certain

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Barka, E.A., Vatsa, P., Sanchez, L., Gaveau-Vaillant, N., Jacquard, C., Klenk, H.P., Clément, C., Ouhdouch, Y.

and van Wezel, G.P. 2 0 1 5 . Taxonomy, Physiology, and Natural Products of Actinobacteria. Microbiol

Mol Biol Rev 80(1): 1-43.

Bontemps, C., Toussaint, M., Revol, P.-V., Hotel, L., and Jeanbille, M. 2013. Taxonomic and functional diversity of

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Embley, T., and Stackebrandt, E. 1994. The molecular phylogeny and systematics of the actinomycetes. Annual

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Fang, B.-Z., Salam, N., Han, M.-X., Jiao, J.-Y., Cheng, J., Wei, D.-Q., Xiao, M., and Li, W.-J. 2017. Frontiers in

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producing actinomycetes from water and sediments of Lake Tana Ethiopia. Asian pacific journal of tropical

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production ability and antimicrobial activity of actinomycetes isolated from soil in Hillah/Iraq. IOSR

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Jeffrey, L.S. 2008. Isolation characterization and identification of actinomycetes from agriculture soils at

Semongok, Sarawak. African journal of biotechnology 7: 3697-3702.

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0

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Figure 1. Morphological diversity of actinomycetes colony in soil samples collected from a community forest

in Ban Khoklam Sang Aram, Udon Thani Province using HA and SCA media.

Figure 2. Various morphological colonies of actinomycetes growing on SCA medium supplemented with

nalidixic acid and cycloheximide of culture broth of actinomycetes isolates.

Table 1. Antimicrobial activity of actinomycetes isolates showed inhibition zone against the tested pathogenic

microorganisms.

Isolate Inhibition (mm)

B. subtilis S. aureus P. aeruginosa E.coli C. albicans

8.2.4HV 1 4 20 0 10

8.2.5HV 0 4 7 0 6

6.1HV-1 10 0 0 0 0

20.2.6 10 0 4 0 0

15.1 10 8 8 0 0

RH13-26 0 0 10 0 0

11.1.2 10 8 7 0 0

10.4.1 3 4 4 0 0

10.5 5 8 8 0 9

RH12-1 3 3 0 0 0

1 2 3 4 5 6 7



Figure 3. Antimicrobial activity of the 10.5 isolate against pathogens: A, Bacillus subtilis; B, S. aureus; C, P.

aeruginosa; D, C. albicans using an agar disc diffusion method and an agar well diffusion method; nystatin as control

for anti-gram positive bacteria; nalidixic acid as anti-Gram negative bacteria.

Figure 4. Morphology of colonies and spores of actinomycetes isolates: A, 8.2.4HV, retinaculiapert spore; B,

8.2.5HV, spirals spore; C, 6.1HV-1, spira spore; D, 15.1, oligosporous spore; F, RH12-1, spirals spore; G, 10.4.1,

disporus spore.

A C

D F G

A B C D

A B C D

B



Screening on anti-inflammatory property of 45 Thai human probiotics from

Biodiversity Research Centre of TISTR

Prapaipat Klungsupya1*, Weerasak Taengphan1, Bhusita Wannissorn2, Supatjaree Ruengsomwong2

and Thanchanok Muangman1

1Expert Centre of Innovative Herbal Products (InnoHerb), Thailand Institute of Scientific and Technological

Research (TISTR), Techno Polis, Khlong Luang Distric, Pathum Thani, Thailand 2Biodiversity Research Centre (BRC), Thailand Institute of Scientific and Technological Research (TISTR),

Techno Polis, Khlong Luang District, Pathum Thani, Thailand.


Abstract: Probiotics are defined as living microorganisms which can provide various health benefits. They can

inhibit overgrowth of pathogenic bacteria by antibacterial substance production and stimulating the health

function in gut system. Moreover, probiotics can promote the affect on innate immunity such as phagocytosis,

and anti-inflammatory activity. The anti-inflammatory effect of probiotics is attributed to the released cytokines

from immune cells such as lymphocytes, mast cells and macrophages. The aim of this study was to determine

the anti-inflammatory activity of 45 TISTR human probiotic strains on lipopolysaccharide-induced macrophage

RAW 264.7 cell line. Heat-killed supernatants of these 45 probiotic strains (Med1 to Med45) prepared and tested

at 100% concentration. Lipopolysaccharides (LPS, 10 µg/ml) of 100 µl was used to induce inflammation. Results

were collected based on the measurements of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6

(IL-6) productions using ELISA Kit assays. The results revealed that 20 out of 45 TISTR probiotics yielded various

degrees of their anti-inflammatory capacity with %NO inhibition ranging from 19.91±1.50 to 82.04±3.71% compared

with β-glucan (83.93±0.07%, positive control). Stimulation of RAW 264.7 macrophages with LPS induced a high

production of NO by 378.61 pg/ml. The reduction in TNF-α levels (% TNF-α inhibition) was seen in 16 TISTR

probiotics with the highest activity at 52.46% (Med 8 strain) and the lowest at 5.69% (Med 9 strain). Considering

on % IL-6 inhibition, among 45 TISTR probiotic strains, 18 strains expressed positive results by 5.79 to 68.79%.

The results suggest that many TISTR probiotic strains are potential of anti-inflammatory activity on macrophage

RAW 264.7 cells. These findings can support the utilization of TISTR probiotics in preventing and down-regulating

inflammation.

Keywords: Anti-inflammation, interleukin, macrophages, nitric oxide, probiotics, tumor necrosis factor-α

Introduction

Thailand Institute of Scientific and Technological Research (TISTR) by Biodiversity Research Centre

(BRC) is responsible for culture collection of bacteria, yeasts, molds and seaweeds which are beneficial to

industrial and environmental usage. At the moment, BRC has more than 500 human probiotics collected and

isolated from Thai population kept in the bank. Probiotics have been promoted the affecting on innate immunity

such as phagocytosis, bactericidal activity and anti-inflammatory activity. The anti-inflammatory effect of

probiotics is attributed to the released cytokines from immune cells such as lymphocytes, mast cells and

macrophages (Thomas and Versalovic, 2010). Macrophages express a variety of surface receptors that including

cytokine receptors (CRs), the interleukin-1 receptor (IL-1R) and complement receptor for pathogen associated

molecular pathways (PAMPs) such as lipopolysaccharide (LPS). The activated macrophages also produce and

secrete cytokines such as TNF-α, IL-1, IL-6, IL-8, IL-12 and IFN)-γ, that are important for inflammatory and

acquired immune response (Guan et al., 2011; Ariel et al., 2012). Rodes et al., (2013) reported that the pro-inflammatory

cytokine concentration of TNF-α, IL-1beta was reduced by probiotics Lactobasilus and Bifidobacterium but anti-

inflammatory cytokine was increased on lipopolysaccharide induced-RAW 264.7 macrophages. Therefore, macrophages

behave as critical play in the initiation, maintenance, and resolution of inflammation through the release of variety

of factors in response to an activating stimulus (Moro et al., 2012). The aim of the present study was to determine

the anti-inflammatory mediator effect of 45 TISTR human probiotic strains on lipopolysaccharide-induced macrophage

RAW 264.7 cell line.

To assort local probiotic strains for Thailand Institute of Scientific and Technological Research (TISTR)

probiotic bank, we randomly collected various sources of flowers and fruits grown in the northern part of Thailand

to isolate bacterial strains. All isolated bacterial strains were screened for probiotic properties according to a

notification of Thai Food and Drug Administration (FDA), Ministry of Public Health (MOPH) and World Health

Organization (WHO) starting from gram straining, catalase-oxidase activity, hemolytic activity, antimicrobial

activity against potentially pathogens, antibiotic susceptibility, gastric acidity resistance (pH 2 and 3), bile salt tolerance,

adherence to human epithelial cell line, and immunomodulatory activity in macrophage function. They were gram



positive, rod shape, catalase-negative, and negative hemolytic activity bacteria. All of them have antimicrobial

activity against pathogens such as Escherichia coli, Staphylococcus aureus. The antibiotic susceptibility, gastric acidity

and bile salt resistance and adherence to human epithelial cell lines of them were already proved. After probiotic

properties screening, we got 45 probiotic strains in total. Therefore, this study was carried out to evaluate anti-inflammation

activity of these 45 TISTR probiotics.


Probiotic strains and growth condition

A total of 45 TISTR human probiotic strains (namely Med 1 to Med 45) from the TISTR culture collection

bank of Biodiversity Research Centre (BRC) were cultivated in de Man Rogosa and Sharpe (MRS) broth (Merck,

Germany) with 0.05% L-cysteine (Merck, Germany) under anaerobic condition at 37C for 48 hr. T hey were

adjusted their turbidity to the concentration to match that of McFarland No. 0.5 (approximate 108 CFU/ml). Cell pellets

were obtained by centrifugation at 8,500 rpm for 5 min at 25°C and washed 3 times with 0.85% NaCl. After

washing, they were resuspended with 10 ml of Dulbecco's Modified Eagle Medium (DMEM) high glucose

(Biowest, USA) without antibiotics before heating at 110°C for 10 min. The supernatants of each probiotic strain

was kept at -20°C until use.

Macrophages culture

RAW 264.7 macrophage cell line (ATCC TIB-71TM) was purchased from American Type Culture Collection

(ATCC) (Manassas, VA, USA). The cells were cultured as adherent in Dulbecco’s Modified Eagle Medium

(DMEM) (Biowest, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (GIBCO®,

USA) and 1% (v/v) penicillin-streptomycin (GIBCO®, USA) in humidified atmosphere incubator with 5% CO2

at 37°C. The cells were collected by centrifugation (Kobota, Japan) at 1500 rpm, 4°C for 5 min, aspirated and

fresh culture medium was added before dispensing into new culture flasks. All steps of culturing and maintaining

of RAW 264.7 macrophage cells were conducted in aseptic condition using the Biological Safety Cabinet Class II

(SANYO, Japan).

Determination of the anti-inflammatory activity in RAW 264.7 macrophage cells.

Nitric oxide measurement by Griess reagent assay Griess reagent system is based on the chemical reaction which uses sulfanilamide and N-1-napthylethylenediamine

dihydrochloride (NEDD) under phosphoric acid conditions. When sulfanilamide is added, the nitrites (NO2–) form

a sulphonamide. Then, the NEDD was added, the sulphonamide was formed a pink colour of diazonium. To determine

the anti-inflammatory activity, 200 µl of RAW 264.7 macrophage cells in DMEM were seed onto 96-well plates

at a density of 2x104 cells/well and incubated for 24 hr at 37°C in humidified atmosphere incubator with 5% CO2.

The overnight culture media were removed and RAW 264.7 macrophage cells (2x106 cells/well) were separately

treated with 100 µl of probiotics (Med1-Med45) or β-glucan at 100 µg/ml (positive control) and incubated for 24

hr. Culture media were replaced by 100 µl (10 µg/ml concentration) of lipopolysaccharide (LPS) from Escherichia

coli (Sigma, USA) and incubated at 37°C in humidified atmosphere incubator with 5% CO2 for 24 hr. After

incubation, the culture media was analyzed for nitric oxide (NO) production using the Griess reaction. Briefly,

the culture media was mixed with 100 μl of Griess reagent (1% sulfanilamide, 5% phosphoric acid and 1%

Naphthyletylenediamine dihydrochloride) and incubated at room temperature for 10 min. Evaluation of the concentration

of NO in the supernatants of the RAW 264.7 cell culture was carried out with enzyme-linked immunosorbent

assay (ELISA) kit (Thermo, USA) according to the manufacturer’s instruction. Then, the absorbance at 540 nm

was measured using microplate reader (Tecan Infinite 200 Pro, Austria). The amount of nitrite presented in the

samples was measured with the sodium nitrite serial dilution standard curve.

Cytokines measurement

RAW 264.7 macrophage cells were prepared in 96-well plate as previously described in NO measurement

assay. Following removal of overnight culture medium, cells (2x106 cells/well) were treated with 100 µl of each TISTR

probiotics (Med1-Med45) or β-glucan (Sigma, USA) at 100 µg/ml which was served as a positive control. Cells were

incubated at 37°Cin humidified atmosphere incubator with 5% CO2 for 24 hr. Then, culture media were replaced

with 100 µl of LPS (Sigma, USA) at 10 µg/ml and incubated for 24 hr. The supernatants of treated-RAW 264.7

macrophage cells were collected by centrifugation at 1500 rpm, 4°C for 5 min and analyzed for two cytokine

measurements including interleukin-6 (IL-6) and tumor necrosis- alpha (TNF-α) by Human IL-6 ELISA kit

(Thermo, USA) and Human TNF-α ELISA kit (Thermo, USA), respectively. The secreted cytokine concentrations

were quantified on the basis of a linear dose-response standard curve.



All data were expressed as mean ± standard error (Mean ± SE) of triplicated determination. The

significance of difference was used to compare mean (p<0.05). All analyses were performed using One-way

ANOVA and Tukey’s Honestly Significant Difference test.

Result & Discussion

In this study, the anti-inflammatory capacity of 45 TISTR probiotic strains was determined on % inhibition

of NO, TNF-α and IL-6 productions. Following treatments, the morphology of RAW 264.7 macrophage cells was

examined under the inverted microscopy as demonstrated in Figure 1. It was observed that 25-26 TISTR probiotic

strains exhibited cytotoxic property (cell viability less than 50%) against RAW 264.7 macrophage cells when tested

at 100% concentration (undiluted heat-killed supernatants). This observation was confirmed by cell viability and

proliferation test using WST-1 assay whose principle is based upon a conversion of tetrazolium salts to colored

formazan compounds by mitochondrial succinate-tetrazolium reductase which exists in viable cells. The results

of WST-1 assay were analyzed by spectrophotometric quantification. However, results of WST-1 assay suggested

that at least 27 out of 45 TISTR probiotic strains were non-cytotoxic to RAW 264.7 macrophage cells. Among them,

10 strains demonstrated a cell-proliferation promoting activity with cell survival rate greater than 100% compared

with the untreated cells.

Macrophages are a type of phagocytic cells of the innate immune system. They play a crucial role and function

as one of the first line of host defense mechanism against invading pathogen, foreign materials and inflammation

(Flannagan et al., 2007; Ariel et al., 2012). Macrophages are activated in the inflammatory process including cytokines,

bacterial lipopolysaccharide (LPS), extracelluar matrix proteins, and other chemical mediators. Activated

macrophages release several different chemical mediators, including ROS, NO, IL-6, TNF-α, and PGE2, that

perpetuate the pro-inflammatory response (Shaikh 2011; Soromou et al., 2012). To determine the effect of TISTR

probiotics on NO and pro-inflammatory mediators (TNF-α, IL-6) productions, RAW 264.7 cells were treated with

45 heat-killed TISTR probiotic strains (Med1 - Med45) at 100 % concentration for 24 hr prior to stimulate with

LPS at a concentration of 10 µg/ml for 24 hr. Then, concentrations of NO, TNF-α and IL-6 mediators in medium

were determined in cell supernatants. Under the stimulus with LPS which was a pro-inflammatory molecule,

RAW 264.7 macrophage cells exhibited high concentration levels of NO (up to 126.17±5.33 µM), TNF-α (up to

378.61±0.52 pg/ml) and IL-6 (up to 149.73±1.52 pg/ml). Overall of 45 heat-killed TISTR probiotic strains, 19

strains were able to reduce the production of NO demonstrating as “% NO inhibition” (Figure 2). Overall, Med2,

Med6 and Med10 showed the best anti-inflammatory activity on RAW264.7 macrophage cells. Their anti-

inflammatory capacity was close to β-glucan (83.93±0.07%, positive control). The decrease of NO production

was not related to viability of cells, which was not affected by LPS stimulation (data not shown).

Anti-inflammatory activity of 45 heat-killed TISTR probiotic strains was confirmed by inhibition of

productions of pro-inflammatory cytokines focusing on TNF-α and IL-6. RAW264.7 cells were first pre-treated

with TISTR probiotic samples (Med1 - Med45) and then stimulated with LPS for verifying their protective anti-

inflammatory activity. The known anti-inflammatory compound, β-glucan at concentration of 100 µg/ml was used

as a positive control. β-glucans are naturally occurring polysaccharides that are produced by bacteria, yeast, fungi

and many plants (Han et al., 2008). Results on % inhibition of TNF-α and IL-6 productions were shown in Figures

3 and 4, respectively. It was found that 19 out of 45 TISTR probiotic strains yielded various degrees of their anti-

inflammatory capacity against LPS-induced TNF-α and IL-6 mediators in RAW264.7 macrophage cells. The

reduction in TNF-α levels (% TNF-α inhibition) was found in 18 TISTR probiotics with the highest activity by

52.46±2.98% (Med 8) and the lowest at 5.56±32.66% (Med 9) (Figure 3). Regarding % IL-6 inhibition, among

45 TISTR probiotic strains, 18 strains expressed positive results by 5.79±2.80% (Med40) to 68.53±1.63% (Med1)

- 68.79±2.79% (Med41) (Figure 4). However, the anti-inflammatory capacity was not found in 26 TISTR probiotic

strains tested including Med12, Med13, Med15, Med17, Med18, Med19, Med20, Med21, Med22, Med23, Med24,

Med25, Med26, Med27, Med28, Med29, Med32, Med33, Med34, Med35, Med36, Med37, Med38, Med43 and

Med45 strains. This might be due to their cytotoxic property when tested at 100% concentration as described in

WST-1 assay results.

Conclusion

Besides increasing inflammation and stimulating the immune system, macrophages play an important

anti-inflammatory role and can decrease immune reactions through the release of cytokines. The results generated

by this study suggest that many TISTR probiotic strains (positive results found in 18-20 strains among 45 strains

tested) potentially exhibit anti-inflammatory activity on RAW 264.7 macrophage cells. These findings can support

the utilization of TISTR probiotics in preventing and down-regulating inflammation. They can be utilized as

ingredients for functional foods and dietary supplement products.

https://en.wikipedia.org/wiki/Inflammation

https://en.wikipedia.org/wiki/Anti-inflammatory

https://en.wikipedia.org/wiki/Cytokines



Acknowledgement

This work was performed by Thailand Institute of Scientific and Technological Research (TISTR) under

financial support by a grant from the Ministry of Science and Technological (MOST) during 2018-2020.

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Figure 1. Morphology of RAW 264.7 macrophage cells after treated with TISTR probiotic strains. (A) Control

(100% cell viability), (B) Probiotics (80-100% cell viability), (C) Probiotics (60-79% cell viability) (D) Probiotics

(40-59% cell viability), (E) Probiotics (20-39% cell viability), (F) Probiotics (1-19% cell viability).



Figure 2. Inhibitory effect on LPS-induced nitric oxide (NO) production in RAW 264.7 macrophage cells by

heat-

killed TISTR probiotic strains at 100% concentration. Beta-glucan (100 µg/ml) was served as positive control.

Figure 3. Inhibitory effect on LPS-induced tumor necrosis-alpha (TNF-α) production in RAW 264.7 macrophage

cells by heat-killed TISTR probiotic strains at 100% concentration. Beta-glucan (100 µg/ml) was served as

positive control.

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Figure 4. Inhibitory effect on LPS-induced Interleukin-6 (IL-6) production in RAW 264.7 macrophage cells by

heat-killed TISTR probiotic strains at 100% concentration. Beta-glucan (100 µg/ml) was served as positive

control.

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Treatments




Effect of water temperature on growth, survival and health status

of East Asian Bullfrog (Hoplobatrachus rugulosus) Larvae

Sornsawan Liamtong1, Kanokporn Saenphet2, Monruedee Chaiyapo2 and Supap Saenphet2*

1Master's Degree Program in Biology, Faculty of Science, Chiang Mai University, Muang District, Chiang Mai, Thailand 2Department of Biology, Faculty of Science, Chiang Mai University, Muang District, Chiang Mai, Thailand


Abstract: East Asian Bullfrog (Hoplobatrachus rugulosus) is an economically and nutritionally important animal

in Thailand. However, many factors including UV radiation, infection and especially the fluctuation of temperature

adversely affect the East Asian Bullfrog culture. Thus, the aims of this study was to investigate the effects of water

temperature changes on the survival and growth as well as oxidative stress of the H. rugulosus tadpoles. The result

showed that cold shock and heat shock significantly decreased the survival rate of the tadpoles when compared to

those of control groups. Cold shock at 23°C showed the highest survival rates among the cold shocked groups and

the heat shock at 27°C showed the highest survival rates among the heat shocked groups. However, there was no

significant difference (p>0.05) of their body length at day 0 and day 7 among the experimental groups. Histological

examination showed blood congestion and a large number of melanomacrophage and Kupffer cell in the liver

tissue of tadpoles exposed to cold and heat shock when compared to those of the controls. Moreover, blood congestion

was found in tadpoles exposed to the cold shock (17°C). In addition, cold and heat shock groups revealed

significantly increased malondialdehyde (MDA) level in whole body of the tadpoles compared with the control

group. Moreover, the level of MDA in the cold shock group was significantly greater than that in the heat shock

group. The data obtained from this study will help to raise awareness of temperature shock in farm-raised frogs

and others wild amphibian species. Keywords: Growth, Hoplobatrachus rugulosus, Survival, Tadpole, Temperature.

Introduction

East Asian Bullfrog (Hoplobatrachus rugulosus, Wiegmann, 1835) is an anuran amphibian with a widespread

distribution from central China, Myanmar, Peninsular, Malaysia including Thailand. They are commonly found

in wetlands and paddy fields (Diesmos et al., 2004; Gratwicke et al., 2009). This species is economically important

in Thailand because it is used as food by the local people and they are a very good source of protein. Moreover,

frozen frog legs of farm-raised frogs are a part of the exports to many countries (Oza, 1990). However, they are

susceptible to changes in their natural environment brought on, for example, by pollution, pesticides, diseases,

especially the climate change.

Frogs are ectothermic animals, water temperature is one of the most important environmental factors affecting

the physiology of organisms because they do not have a thermoregulatory mechanism (Goldstein et al., 2017).

Earlier studies indicated that temperature fluctuation could influence their survival, growth and physiological functions

in the wild and farmed frogs (Wang et al., 2012; Bellakhal et al., 2014). Moreover, Sahoo and Kara (2014) demonstrated

that cold shock on Tiger frogs (H. rugulosus) caused acute mortality in the larvae. Thus, these phenomena were the main

causes of the mortality and the decline in frog populations. In addition, Kern et al. (2015) found that water temperature

fluctuation on Australian frogs (Limnodynastes peronii) inhibited growths and developments by significant

decrease in total length (TL), body mass (Mb) and body length (BL) when compared with those reared in constant

water temperature. Furthermore, temperature fluctuation can lead to oxidative stress in organisms by increased

production of reactive oxygen species (ROS), which cause lipid peroxidation (Sahin, 2004). Sahoo and Kara

(2014) reported that the exposure of short-term cold stress to common Indian toads (Bufo melanostictus)

significantly increased ROS that was indicated by the rising of lipid peroxidation in the liver, kidney and brain

tissues. Additionally, thermal stresses can also effectively induce histological alterations in the liver of other

ectotherms, such as fat droplets accumulation (Liu et al., 2018), mitochondrial swelling (Xu et al., 2018), hepatocyte

degeneration and necrosis (Ates et al., 2006). It has been reported that histological examination in the liver tissue

of giant spiny frog (Quasipaa spinosa) exposed to cold and heat shock, erythrocyte extravasation as hemorrhage

and congestion was observed. Inflammatory cell infiltration was conspicuous and enlargement of sinusoids was

also presented (Liu et al, 2018). Therefore, the present study aimed to investigate the effects of water temperature

changes on the survival and growth as well as oxidative stress of the H. rugulosus tadpoles.

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Animals and experimental design

Experiments were performed with H. rugulosus larvae. Tadpoles, Gosner stage 26, were obtained from

Thunpisit frog farm, Chiang Mai province. They were kept in dechlorinated water with laboratory conditions at

least 24 hours for acclimation. The tadpoles were fed twice daily with commercial tadpole diet. Experimental designs

were as followed:

Experiment 1 – The frog tadpoles were divided into 7 groups comprising of 30 tadpoles each. Group 1-3

were served as cold shock groups and groups 4-6 were served as heat shock groups. For cold shock groups, the initial

temperature was set at 25°C and then decreased to 23°C, 20°C and 17°C within 4 hours. For heat shock groups,

the initial temperature was set at 25°C and then increased to 27°C, 30°C and 33°C within 4 hours. The control

group was maintained at 25°C throughout the experimental period. Each treatment was conducted in triplicate.

Dead larvae, if present, were counted and removed daily. After 7 days of exposure, the survived tadpoles were

measured for their body length to determine the growth rate. The liver tissues of frog tadpoles were collected and

processed for histological investigation.

Experiment 2 – The frog tadpoles were divided into 3 groups comprising of 30 tadpoles each. Group 1 was

served as control group and the temperature was set at 25°C. Group 2 was set as cold shock group and the temperature

was started at 25°C and then decreased to 23°C (the temperature which was produced the highest viability of cold

shock-exposed larvae from the experiment I). Group 3 was heat shock group and the temperature was started at

25°C and then increased to 27°C (the temperature which was produced the highest viability of heat shock-exposed

larvae from the experiment I). Each treatment was conducted in triplicate. The tadpoles were exposed to heat and

cold temperatures until they reached the Gosner stage 42. They were then collected for determining oxidative

stress by evaluating the level of malondialdehyde (MDA).

Histopathological examinations The tadpoles at day 7 were collected and whole body were fixed in Bouin's solution. After fixation for

24-30 h, tissues were dehydrated through a graded series of ethanol, cleared in xylene, and then embedded in

paraffin. Six μm sections were made and stained with hematoxylin and eosin (H&E). The sample was analyzed

by assessing the histopathological alterations under a light microscope.

Estimation of lipid peroxidation in tadpoles tissue. Twenty milligrams of homogenated tadpoles tissue were mixed in 1 ml of 0.1 M Phosphate Buffer

Solution (PBS), pH 7.4 at -4 °C. The mixed sample was centrifuged at 1,155 x g for 10 minutes and the supernatant

was stored at -40°C for examination of MDA within 24 hours. The degree of lipid peroxidation was determined

colorimetrically using the method of Buege and Aust (1976). Briefly, 100 µl of sample was mixed with 450 µl of normal

saline, 200 µl of thiobarbituric acid (TBA) and 1,000 µl of trichloroacetic Acid (TCA). The mixture was heated

at 100°C for 30 minutes, cooled with running tap water and then added with 2,000 µl of distilled water, mixed

with vortex mixer and centrifuged at 1,155 x g for 10 minutes. The absorbance at 532 nm was then measured and

calculated for MDA concentration against the MDA standard curve.

Estimation of protein in tadpoles tissue.

The level of protein content from each tissue was estimated following the method described by Bradford

(1976) and the bovine serum albumin (BSA) was used as a standard protein. Five milligrams of Coomassie Blue G

were dissolved in 1 ml of 25% (v/v) methanol containing 42.5% (v/v) H3PO4. One volume of the prepared dye

was diluted with four volumes of distilled water, and 0.04 mL of each sample was mixed with 2 mL of diluted

dye. The absorbance was read at 590 nm and calculated for protein concentration against the protein standard curve.

Statistical Analyses All statistical analyses were managed with Statistical Package for Social Science (SPSS) software version

20.0 for Mac OSX (SPSS Inc., IBM). Values were verified for normality and homogeneity of variance by Shapiro-

Wilk test and Levene's tests, respectively. Mean comparisons were carried out by one way analysis of variance

(ANOVA) followed by Tukey's HSD test.

Result & Discussion

Survival and growth of tadpoles

As shown in Figure 1, cold shock and heat shock groups revealed significantly decreased survival rate of

the tadpoles when compared to those of the control groups (p<0.05). Moreover, the survival rate of tadpoles in

cold shock groups was not significantly different from that of heat shock groups. The highest survival rates in cold

and heat shock were found at 23°C and 27°C, respectively. However, there was no significant difference (p>0.05)

of their body length at day 7 among the experimental groups. (Figure 2) The highest survival rates obtained from

our study were slightly associated with the studies of Wang et al. (2012) which reported that the mortality rate of



Tiger frog (H. rugulosus) exposed to cold shock were 28.1% at 12°C, 87. 5% at 10°C and 100% at 8°C.

Additionally, Bellakhal et al. (2014) reported that African green frog (Pelophylax saharicus) survival was reduced

at temperatures above 28 °C. These inferred that the temperature fluctuation on the survival rate among species

may be determined by differences in their thermal optimum for survival. The larval tadpole H. rugulosus used by

Wang et al. and P. saharicus used by Bellakhal might have greater thermal optimum range than the older larvae

used in our study. In addition, the body length of tadpoles obtained from our study are contradiction with the

studies of Kern et al. (2015) which, reported that body length of L. tasmaniensis tadpoles raised in temperature

shock was significantly lower than those of tadpoles in the constant temperature. As a result, tadpoles obtained

from cold and heat shock had reduced survival when compared with tadpoles reared in the constant temperature

since the temperature has been well known as the most pervasive abiotic factor to influence physiological function

because of thermodynamic effects on biochemical reactions which underline survival and growth (Somero, 2002).

Environmental temperature determines body temperature in most ectotherms (Guderley, 2004; Seebacher and

Murray, 2007). Consequently, the temperature fluctuation could affect the physiology, survival and growth of

tadpole as seen in our study.

Histopathological changes in tadpole liver

Histopathological changes of liver were observed in all treated group (Figure 3). A larger number of

melanomacrophage and Kupffer cells were observed in the liver tissue of tadpoles exposed to cold and heat shock

Moreover, blood congestion was also found in the liver of the tadpoles exposed to the cold shock (17°C) (Figure 3D).

Our results were consistent with the studies of Corsaro et al. (1990) which reported that melanomacrophages

in R. esculenta increased during the winter (about 5–10°C) and decreased during the summer (about 20°C –25°C).

The liver has long been well known to be the main organ for detoxification that could suffer serious morphological

alterations in tadpoles exposed to temperature shock. Therefore, its histological changes could be used as biomarkers

for environmental stress (Sichel et al., 1987). The hematopoietic organs of ectothermic animals commonly display

pigmented cells with phagocytic activity called melanomacrophage (Barni et al., 1999). They are focal accumulations

of pigmented macrophages in the liver. These may contain four types of brown to black pigments, namely melanin,

lipofuscin, ceroid, and hemosiderin/ferritin, which absorbs and neutralizes free radicals, cations, and other potentially

toxic agents derived from the degradation of phagocytized cellular material. Thus, it appears that these cells are

sensitive to stimuli as a way to adapt to stress conditions (Sayed, et al., 2015). In addition, Kupffer cells are

specialized resident macrophages in the liver and the activated kupffer cells appears to modulate acute hepatocyte

injury and chronic liver responses. They constitute the macrophage population of the body to encounter the bacteria

which derived from the gastrointestinal tract and transported to the liver via the portal vein. Thus, the increase in

the number of Kupffer cells in the liver tissue might be the response of the defense system when animals are in

contact with environmental stress. Moreover, it has been reported that blood congestion was present in the liver

tissues of giant spiny frog (Q. spinosa) under cold and heat stresses (Liu et al., 2018) which was consistent with

our results. It could be attributed to the destruction of hepatic erythrocytes and newly produced erythrocytes

accumulated in the liver (Maekawa et al., 2012).

Lipid peroxidation in tadpole tissue

Biochemical analysis of tadpole tissue in the present experiments indicated that cold shock (23°C)

(0.079±0.009 mM/mg protein) and heat shock groups (27°C) (0.050±0.002 mM/mg protein) induced a significant

increase in MDA level compared with the control group. (0.021±0.006 mM/mg protein). Moreover, the level of MDA

in the cold shock group (0.079±0.009 mM/mg protein) was significantly greater than those in the heat shock group

(0.050±0.002 mM/mg protein) (Figure 4).

This is in agreement with the findings of previous studies of Sahoo and Kara (2014) which report that

MDA level in the liver, kidney and brain tissues of the common Indian toad (B. melanostictus) exposed in cold

and heat shock were higher than those of the tadpoles exposed in constant temperature. In addition, the level of MDA

of the liver of goldfish (Carassius auratus) obtained in heat shock was higher than that was obtained in the constant

temperature. As a result, temperature change induces a significant increase in the MDA level in tissue of ectotherm

animals. (Abele et al., 2002). It is commonly known that the MDA is one of the important biochemical compounds

used as an indicative of reactive oxygen species (ROS) being generated from lipid peroxidation. In general, ROS,

which can be neutralized by a variety of antioxidants, are generated by cellular metabolism (Lushchak and

Bagnyukova, 2006). However, excessive ROS would damage various chemical and biological membranes and it

has been suggested as a cause of toxicity in several organs. Furthermore, oxidative stress has also been shown to

affect organ dysfunction and tissue damage. The cold and heat stress, which induces elevated thermogenesis by

enhanced substrate combustion along with increased oxygen consumption, has been reported to produce ROS,

which cause lipid peroxidation and play an important role in tissue injury (De Quiroga et al., 1991).


Conclusion

In summary, we concluded that the temperature change caused decrease survival and growth of East

Asian Bullfrog (H. rugulosus). In addition, temperature shock caused oxidative stress that was demonstrated by

the increased MDA level and triggered the histopathological alterations in the livers of H. rugulosus tadpoles.

It could be suggested that the data obtained from this study would help to raise awareness of temperature shock in

farm-raised frogs and others wild amphibian species.

Acknowledgement

This work was supported by the Research and Researchers for Industries (RRi)(MSD6110063) and the

Graduate School, Chiang Mai University.

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Figure 1. Survival of H. rugulosus tadpoles exposed to cold shock (23°C, 20°C, 17°C) and heat shock (27°C,

30°C, 33°C) for 7 days as compared to control group (25°C) (Difference in labelling letter (a, b, c) represents

significant difference at P < 0.05).

Figure 2. Body length of H. rugulosus tadpoles exposed to cold shock (23°C, 20°C, 17°C) and heat shock (27°C,

30°C, 33°C) for 7 days as compared to control group (25°C) (Difference in labelling letter (a, b) represents

significant difference at P < 0.05)

c

c

b

a

b

b

bc

0.00

20.00

40.00

60.00

80.00

100.00

1 2 3 4 5 6 7

Su

rviv

al

(%)

Days of experiment

17°C 20°C 23°C 25°C

27°C 30°C 33°C

a a a a a a ab b b b b b b

.00

.20

.40

.60

.80

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1.20

17°C 20°C 23°C 25°C 27°C 30°C 33°C

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)

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Day 1 Day 7



Control group

Cold shock group

Heat shock group

Figure 3. Liver tissue of H. rugulosus tadpoles (A) control; (B) exposed to cold shock (23°C); (C) exposed to cold

shock (20°C); (D) exposed to cold shock (17°C); (E) exposed to heat shock (27°C); (F) exposed to heat shock

(30°C); (G) exposed to heat shock (33°C); white arrowhead : melanomacrophage; yellow arrow : Kupffer cell;

black arrow : blood congestion; H&E. x200.

Figure 4. The level of malondialdehyde (MDA) of H. rugulosus tadpoles exposed to cold shock (23°C) and heat

shock (27°C) for 7 days as compared to control group (25°C) (Difference in labelling letter (a, b, c) represents

significant difference at P<0.05.)

0.00

0.01

0.02

0.03

0.04

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25°C 23°C 27°C

MD

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M)/

mg p

rote

in

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b

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Antioxidant activity and hair growth promoting activity of flavonoid extracts

from Phyllodium pulchellum and Uvaria rufa Blume on cultured mouse vibrissa hair follicles

Patcharida Penpakkul1, Kanokporn Saenphet2* and Supap Saenphet3 1Master’s Degree Program in Biology, Faculty of Science, Chiang Mai University, Muang District, Chiang Mai, Thailand

2Department of Biology, Faculty of Science, Chiang Mai University, Muang District, Chiang Mai, Thailand


Abstract: This study aims to determine antioxidant activity and hair growth promoting activity of flavonoid extracts

from the aerial parts of Phyllodium pulchellum and the stems of Uvaria rufa Blume on cultured mouse vibrissa hair

follicles. Flavonoid extracts of both plants were tested for their antioxidant activities using total antioxidant assay,

2,2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric

oxide radical scavenging assays. To investigate hair growth promotion, vibrissa follicles of mouse were isolated and

cultured in 95 % CO2, 5 % O2 and 37⁰C. Cultured vibrissa follicles were treated with flavonoid extracts of P. pulchellum

(PPE) and U. rufa (URE) at concentrations of 0.01-20 µg/ml for three days. The length of hair follicles was

measured every 24 hours for three days. Total flavonoids contents of PPE and URE were 10.14 ± 0.20, 10.28 ±

0.60 mg quercetin per extract, respectively. We found that PPE and URE had total antioxidant activities at 10.53 ± 0.26

and 11.29 ± 0.04 mg ascorbic acid equivalent per gram extract, respectively. Moreover, PPE and URE markedly

exhibited antioxidant activities with the IC50 values at 1.67 mg/ml and 2.44 mg/ml for ABTS assay, and 1.34 mg/mL

and 2.45 mg/ml for DPPH assay, and 0.44 mg/ml and 0.34 mg/ml for nitric oxide radical scavenging assay. PPE

at 1 µg/ml significantly increased the length of hair follicles at 24 and 48 hours while URE at 0.01-1 µg/ ml

significantly elongated hair follicles at 48 and 72 hours when compared to those of controls. The results suggest that

the flavonoid extracts from P. pulchellum and U. rufa are the new sources of antioxidants and the potential promoting

hair growth agents.

Keywords: Antioxidant, hair growth, Phyllodium pulchellum, Uvaria rufa Blume

Introduction

Hair loss is a concern problem which can occur in both men and women. This problem brings loss of

self-esteem and mental health problems (Williamson et al., 2001). Although there are effective drugs for treating hair loss

which are finasteride and minoxidil, they contribute several adverse side effects. Finasteride caused male infertility

and gynecomastia while minoxidil can lead to skin irritation, itching and dryness and hypotension (Chilba et al., 2011;

Ramot et al., 2009; Rossi et al., 2012; Mehta et al., 1975). Due to the limited usage of synthetic drugs, natural product

has become attractive. Hair growth promoting activities of various plants; Centella asiatica Linn., Panax ginseng,

Avicennia marina and Thujae occidentalis were previously reported (Saansoomchai et al. , 2018; Matsuda et al. ,

2003; Jain et al., 2015; Park et al., 2003). The major phytochemical constituent in these plants is flavonoids, which

is well-known for its antioxidant properties. Since oxidative stress is related to hair loss, consumption of antioxidants

can prevent the miniaturization of hair. Over-production of free radicals in scalp skin can trigger dermal papilla cell

apoptosis, eventually leads to hair shedding (Trüeb, 2015). Therefore, natural antioxidant treatment for maintaining

healthy scalp is necessary.

Phyllodium pulchellum and Uvaria rufa Blume are distributed in Thailand and used in traditional

medicine as remedy for hair loss. P. pulchellum was reported to have hepatoprotective effect while U. rufa was

reported as effective treatment against benign prostatic hyperplasia in experimental animals (Fan et al. , 2018;

Buncharoen et al. , 2016) . However, there is no evidence for hair growth promoting activity of these two plants.

Thus, this study aims to investigate hair growth promoting activity as well as antioxidant of extracts from P.

Pulchellum and U. rufa to confirm their traditional usage.


Reagents

2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), 2,4-dinitrophenylhydrazine (DNPH), basal medium,

bovine calf serum, bovine pituitary extract, fibroblast growth factor, gallic acid and insulin were purchased from

Sigma Aldrich (MO, USA). Ammonium molybdate was obtained from Unilab (Sydney, Australia). Ascorbic acid

was purchased from Prolabo (Leuven, Belgium). Penicillin/streptomycin was obtained from Merck (Darmstadt, Germany).



Plant materials

The aerial parts of P. pulchellum were collected in Chiang Mai Province and the stems of U. rufa were

obtained from Buriram Province, Thailand.

Extraction

All plant materials were chopped, cleaned and dried. The dried sample was defatted with petroleum ether

using a Soxhlet extractor at 40-60⁰C. Then, the residue was extracted with ethyl acetate. The ethyl acetate extracts

were evaporated by a rotary evaporator at 50⁰C (Peleg et al., 1991) to obtain the flavonoid extracts of P. Pulchellum

(PPE) and U. rufa (URE). Total flavonoid contents were determined by using aluminium chloride solution at 420 nm

(Ordonez et al. , 2006) . The flavonoids in PPE and URE were expressed as mg quercetin equivalent per gram

extract (mgQE/g extract).

Measurement of antioxidant capacity

ABTS radical scavenging activity

The scavenging of ABTS radical was tested according to the method described by Re et al (1998). Briefly,

the working ABTS solution was prepared by mixing 7 mM ABTS solution with 2. 45 mM potassium persulfate.

The mixture was allowed to react in the dark at room temperature for 12 h. The ABTS•+ solution was then diluted

with deionized water to obtain an absorbance of 0.7 ± 0.02 at 734 nm. Different concentrations of each sample

were mixed with 1 ml of the ABTS+. The absorbance was measured at 734 nm at 0 (initial absorbance) and 1 (final

absorbance) minute of incubation. Gallic acid was used as the standard control. The inhibition of ABTS•+ was calculated

by using the following equation:

% inhibition of ABTS•+ = [Abs (initial)- Abs(final) / Abs (initial)] x 100

The results were expressed as median inhibitory concentration (IC50) value.

DPPH radical scavenging activity

The DPPH assay was done according to the method of Susanti et al. (2007). 100 µl of each sample was added

to 2 ml of 0.1 mM DPPH·solution. The mixture was vigorously shaken and the absorbance (initial absorbance) of the mixture

was recorded immediately at 517 nm. Then, it was incubated in the dark for 30 min at room temperature and the final

absorbance at 517 nm of the mixture was measured. Gallic acid was used as the standard control. The DPPH·scavenging

ability was calculated as:

% inhibition of DPPH· = [Abs (initial)- Abs(final) / Abs (initial)] x 100

The IC50 value of each sample as well as antioxidant standard were calculated. Each sample was performed in triplicate.

Nitric oxide scavenging activity

The nitric oxide scavenging activity assay was conducted according to the procedure described by

Marcocci et al. (1994). Sodium nitroprusside (10 mM) in phosphate buffer saline was prepared to produce nitric oxide

radicals, which was measured by using Griess reagent. 500 µl of sodium nitroprusside solution was then mixed

with 1 ml of various concentrations of each extract. The mixture was incubated at room temperature for 180 min.

After incubation, 500 µl of Griess reagent was added. Gallic acid was used as the standard positive control. The absorbance

of the mixture was taken at 546 nm.

Total antioxidant capacity

The phosphomolybdenum method was used to investigate the total antioxidant capacity in PPE and URE

extracts (Oyaizu, 1986. Phosphomolybdenum reagent was prepared by mixing 0.6 M sulfuric acid, 28 mM sodium

phosphate and 1% of ammonium molybdate. The reagent was then mixed with 500 µl of each sample. The mixtures

were incubated at 95⁰C for 10 min. The absorbance of the mixture was subsequently measured at 695 mm. The results

were expressed as mg ascorbic acid equivalent per gram extract (mgAAE/g extract)

Isolation and culture of mouse vibrissa follicles

For isolation experiment, five-week-old ICR mice were isolated as previously described ( Jindo et al. ,

1994). Briefly, the mystacial pads were removed from upper lip region. Intact vibrissa follicles were then carefully

disaggregated under a stereomicroscope using a scalpel and tweezers. Isolated vibrissa follicles were placed in

24-well plates containing 1 ml of basal medium supplemented with 0.002 ml/ml bovine pituitary extract, 0.5µg/ml

bovine insulin, 0.05 ml/ml calf serum, 0.5 ng/ml fibroblast growth factor, 50 U/ml penicillin and 50 µg/ ml

streptomycin ( Junlatat and Sripanidkulchai, 2014) . The follicles were cultured in an incubator with 5% CO2 at

37⁰C. For each assay, PPE and URE were diluted with DMSO and added to the medium to yield various

concentrations (0.001 - 10 µg/ml). DMSO (0.1%) was used as a control. The medium was discarded and replaced

every 24 h. During the period of culture (72 h), the cells were photographed at 24, 48 and 72 h using a stereo microscope.

The difference of the length of vibrissa hair follicles were calculated using ImageJ software and expressed as

mean ± S.D.



All values were expressed as mean ± S.D. of at least three samples. The significant differences among

experimental groups were determined by one-way analysis of variance (ANOVA), followed by Duncan’s test using

SPSS version 16. P-values less than 0.05 were regarded as significant.

Result & Discussion

Antioxidant activity of PPE and URE

Oxidative stress is the disturbance which leads to damaging of cellular structures and may be a factor of

hair loss (Trüeb, 2015) . The association between oxidative stress and hair loss has been reported by Bahta et al.

(2008) that the dermal papilla cells were susceptible to oxidative stress and resulting in miniaturization of hair

follicles. In addition, Beoy et al. (2010) have suggested that consuming the supplement containing antioxidants

can decrease oxidative stress in the hair scalp as well as increase the numbers of hairs.

The low IC50 values of all parameters obtained from our results indicated that PPE and URE extracts

exhibited the ability to scavenge free radicals. PPE and URE markedly performed antioxidant activities with the

IC50 values at 1.67 mg/ml and 2.44 mg/ml for ABTS assay, and 1.34 mg/mL and 2.45 mg/ml for DPPH assay,

respectively. Moreover, URE also exhibited excellent nitric oxide scavenging activity, especially URE which IC50

value was not much higher than that of gallic acid (0.34 mg/ml for URE and 0.23 mg/ml for gallic acid) . PPE,

however, exhibited lower capacity to scavenge nitric oxide than URE by showing IC50 value at 0.44 mg/ml (Table

1). Determination of total antioxidant capacity of PPE and URE expressed values with 10.53 ± 0.26 and 11.29 ±

0.04 mg AAE/g extract, respectively. Total flavonoid contents of PPE and URE were 10.14 ± 0.20, 10.28 ± 0.60

mgQE/g extract. The potent antioxidant activities of PPE and URE found in our study might contribute to their

flavonoid compounds.

As previous study reported that flavonoids are powerful antioxidants which can reduce oxidative stress

in the hair scalp (Pietta, 2000; Beoy et al., 2010). Thus, flavonoids in P. pulchellum and U. rufa may be the agents

that reduce the free radicals in the hair scalp. To ensure hair growth promoting activity of PPE and URE via

decreasing oxidative stress, determination of hair growth promoting activity in vivo needs to be further evaluated.

Elongation of vibrissa hair follicles

Flavonoids could enhance hair quality not only by decreasing free radicals in the hair scalp, but also promoting

elongation of hair follicles. The previous study reported that green tea extract containing flavonoid promoted hair

growth by inducing elongation of hair follicles and inhibiting apoptosis of dermal papilla cells (Kwon et al., 2007).

In addition, flavonoids contained in Panax ginseng performed significant stimulation on hair growth of vibrissa

follicles after 48 h of culture (Matsuda et al., 2003).

In this study, the vibrissa follicles treated with PPE at 1 µg/ml significantly increased the elongation of

hair follicles after 24 and 48 h of culture (128.02 ± 21.97 and 116.81 ± 28.05 µm, respectively) when compared

with the control (Figures 1, 3) . In the URE group, the follicles were significantly longer than those treated with

vehicle control after 48 and 72 h of culture (Figure 3). The follicles treated with URE at 0.01, 0.1 and 1 µg/ml for

24 h expressed elongation with values 123.76 ± 69.58, 136.47 ± 12.46 and 115.04 ± 26.20 µm, respectively. For

72 h of culture, the URE treatment at 0.01, 0.1 and 1 µg/ml enhanced the hair growth by 111.38 ± 17.82, 113.47

± 2.83 and 149.83 ± 25.59 µm, respectively (Figure 2) . Hence, these results suggested that PPE and URE have

potential hair growth promoting activity in vitro resulting from the presence of flavonoids as the major constituents.

Conclusion

In conclusion, the flavonoid extracts from P. pulchellum and U. rufa are potent antioxidants and able to

promote hair growth by inducing hair elongation. The antioxidant capacity of these two extracts is recommended

as the mechanism underlying their hair growth promoting activity.

Acknowledgement

This research was supported by the Research and Researchers for Industries, Thailand Research Fund

(MSD61I0046). We also gratefully acknowledge Graduate School, Chiang Mai University.



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Kwon, O. S., Han, J. H., Yoo, H. G., Chung, J. H., Cho, K. H., Eun, H. C., and Kim, K. H. 2007. Human hair growth

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Matsuda, H., Yamazaki, M., Asanuma, Y., and Kubo, M. 2003. Promotion of hair growth by Ginseng radix on cultured

mouse vibrissal hair follicles. Phytotherapy Research 17(7): 797-800.

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from the flowers of Melastoma malabathricum L. Food Chemistry 103(3): 710-716.

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European Academy of Dermatology and Venereology15(2): 137-139.


Figure 1. Hair growth promoting effect of PPE on cultured mouse vibrissa hair follicles for 24, 48 and 72 h.

Follicles were treated with 0.01, 0.1, 1, 10 and 20 µg/ml of PPE. Data are exhibited as mean ± S.D. (n = 3), *p <

0.05 compared to control.

Figure 2. Hair growth promoting effect of URE on cultured mouse vibrissa hair follicles for 24, 48 and 72 h.

Follicles were treated with 0.01, 0.1, 1, 10 and 20 µg/ml of URE. Data are exhibited as mean ± S.D. (n = 3), *p <

0.05 compared to control.

Figure 3. Elongation of cultured mouse vibrissa hair follicles treated with PPE or URE demonstrated a significant

increase in length of hair shaft when compared to the control at 24, 48 and 72 h.

Table 1. Antioxidant activity of PPE and URE and gallic acid.

Treatment IC50 values (mg/ml)

ABTS method DPPH method Nitric oxide inhibition

method

PPE 1.67 ± 0.20 1.34 ± 0.16 0.44 ± 1.20

URE 2.44 ± 0.29 2.45 ± 0.30 0.34 ± 0.48

Gallic acid 0.08 ± 0.18 0.002 ± 0.00 0.23 ± 0.76

Each value is expressed as mean ± S.D. from triplicate.

0

20

40

60

80

100

120

140

160

24Hr 48Hr 72Hr

The

incr

ease

d l

ength

(µ

m)

Control

0.01

0.1

1

10

20

0

20

40

60

80

100

120

140

160

180

24 Hr 48 Hr 72 Hr

The

incr

ease

d l

ength

(µ

m)

Control

0.01

0.1

1

10

20

**

* *

*

*

*

*

Control PPE URE

24 h

48 h

72 h




Chemical composition and antibacterial activities of essential oils of Lavandula × hybrid

‘boysemberry ruffles’, L. pedunculata ‘princess’ and L. × hybrid ‘high five purple’

Suchawadee Insawang1 and Patcharee Pripdeevech1,2* 1School of Science, Mae Fah Luang University, Muang District, Chiang Rai, Thailand

2Center of Chemical Innovation for Sustainability (CIS), Mae Fah Luang University, Muang District, Chiang Rai, Thailand


Abstract: Essential oils of Lavandula plants are used in perfumery and therapeutic applications. They are mainly

planted in Mediterranean area and several regions in Asia. According to its beneficial property, chemical composition

and bioactivity of various species of Lavandula plants have been studied. Thus, this study aimed to extract the essential

oils of three species from Lavandula plants grown in Thailand including L. × hybrid ‘boysemberry ruffles’, L. pedunculata

‘princess’ and L. × hybrid ‘high five purple’. The yield of essential oils obtained among these samples were 0.11-0.24 %w/w.

The chemical composition of all essential oils were investigated using gas chromatography- mass spectrometry.

A total of 89 volatile compounds were identified. Four main compounds including 1,8-cineole, fenchone, camphor

and α-pinene were detected in all samples with different contents. For the antibacterial activity assay, the experiment

was performed by agar disc diffusion method against four Gram-negative bacteria (Klebsiella pneumonia, Salmonella

typhimurium, Escherichia coli and Pseudomonas aeruginosa) and five Gram-positive bacteria (Staphylococcus

epidermidis, S. aureus, Bacillus subtilis, Enterococcus faecium and Streptococcus pyogens). The result showed that

the great antibacterial activity was obtained from the essential oil of L. pedunculata ‘princess’ compared to those

found in essential oils of L. × hybrid ‘boysemberry ruffles’ and L. × hybrid ‘high five purple’, respectively. Different

antibacterial activities among these samples could be correlated to major components such as 1,8-cineole, fenchone

and camphor. It was indicated that essential oil of L. pedunculata ‘princess’ may be used as natural antibacterial agent in

therapeutic applications.

Keywords: Antibacterial, chemical composition, essential oil, gas chromatography-mass spectrometry, Lavandula.

Introduction

Essential oils from plant species ‘Lavandula’ have been receiving many interests due to their applications

in many industries such as food, pharmaceutical, and cosmetic. In cosmetic industry, the oils can be used in the a production

of perfumes, colognes, body wash and other cosmetics (Cavanagh and Wilkinson, 2005; Lesage-Meessen et al., 2015).

The use of lavender essential oil might depend on their cultivar source due to the variety of composition. The volatiles

constituents in the extracted oil are feasibly depend on many factors such as cultivar and extraction method. It has

been found that linalool, linalyl acetate, 1,8-cineole, β-ocimene (both cis- and trans-), terpinen-4-ol and camphor are

the main constituents of lavender oil. (Cavanagh and Wilkinson, 2002; Costa et al., 2012). Lavandula essential oil

has the antimicrobial activity against various fungi and bacteria causing skin infection (Kunicka‐Styczyńska et al., 2009).

It was found that there is a relation between volatiles in the essential oil and antimicrobial strength. Linalool, a volatile

components in lavender essential oil, has a strong antimicrobial strength (Guo et al., 2018). In other studies, it is

reported that lavender oil is used for relieving symptoms of psoriasis, dermatitis, and eczema (Cavanagh and

Wilkinson, 2002; Matos et al., 2009). Another factor that affect quality and yield of the oil is an extraction method.

Hydrodistillation is chosen to extract essential oils in this study due to it is a popular method using less heat and

retaining most of the volatiles in the sample (Costa et al., 2012). Therefore, the aims of this study are to extract

essential oils from selected species, analyze volatile compounds using gas chromatography-mass spectrometry

(GC-MS) and screen their antibacterial activities.


Plant materials

The sample Lavandula plants including L. pedunculatas ‘princess’, L. x hybrid ‘high five purple’ and L.

x hybrid ‘boysemberry ruffles’ were collected from Angkhang Royal Agricultural Station, Chiang Mai, Thailand

on April 2018.

Extraction of essential oils

Fresh arial part of each sample (250 g) was subjected to hydrodistillation. The essential oils were extracted

for three h. The obtained essential oils were dried over anhydrous Na2SO4, and diluted with dichloromethane

(1:100 v/v) prior subjecting to GC-MS injection port.


Analysis of volatile compounds

The volatile components were also identified by GC-MS using a Hewlett–Packard 5973 MSD apparatus

(Agilent 5973 network mass selective detector) from Agilent Technologies, USA with fused-silica capillary columns.

The temperature program was started at 60°C and raised up to 220°C with a rate of 3°C/min. Helium was used as

carrier gas with a flow rate of 1 mL/min. Injector and detector temperatures were set at 250 °C and 280°C, respectively.

Electron impact ionization was employed with electron energy of 70 eV. The acquisition was performed in scanning

mode (mass range m/z 35-300 u). Identification of all volatile compounds was performed by comparing their mass

spectra to those obtained from NIST 05 and Wiley 7N libraries and literature (Angioni et al., 2006; Adams, 2017).

Moreover, linear retention indices correlating with C9-C18 n-alkanes were used to confirm identification.

Antibacterial activity assay

Antibacterial activity was performed using the paper disc diffusion method of Zaidan (2005). The bacteria

strains including Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, S. pyrogens DMST 17020,

Bacillus subtilis TISTR 008, Enterococcus faecium ATCC 29212, Escherichia coli ATCC 25922, Klebsiella

pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853 and Salmonella typhimurium ATCC 13311 were

obtained from the Department of Medical Science, Ministry of Health, Bangkok, Thailand. All strains were subcultured

in Müller-Hinton Broth (YM, Difco, USA) at 37°C for 24 h. All pathogenic bacterial was further spread in Müller-

Hinton agar. Different concentrations of essential oils were prepared by two-fold dilution method using hexane

as solvent until the final concentration was obtained as follows: 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.39 mg/mL.

Thirty microliters of each essential oil sample were dropped on a sterilized 6 mm diameter paper disc (WhatmanTM, USA),

and the Petri dish plates were placed in culture plates prior incubating at 37°C for 24 h. Hexane and chloramphenicol

antibiotic were used as negative and positive control, respectively. The result was reported as minimum inhibition

concentration (MIC) and zone inhibition diameter. Each experiment was carried out in triplicate.

Result & Discussion

Essential oils from three Lavandula species, L. pedunculatas ‘princess’ , L. x hybrid ‘high five purple’

and L. x hybrid ‘boysemberry ruffles’ were extracted by hydrodistillation method. All essential oils were clear

with pale yellow and strong piquant smell. All essential oils yielded ranging between 0.11-0.24 %w/w. The results

of volatile constituents in the essential oils from Lavandula species analyzed by GC-MS are listed in Table 1.

A total of eighty-nine volatile constituents were detected in the essential oils extracted from the selected Lavandula species.

Forty-five compounds were observed in the essential oil of L. x hybrid ‘boysemberry ruffles’ while the essential

oils from L. pedunculatas ‘princess’ and L. x hybrid ‘high five purple’ contained 42 and 38 volatile compounds,

respectively. It was also found that 1,8-cineole, fenchone, camphor and α-pinene were the main compounds in all

Lavandula species with different contents. This might be the result of their genetic diversity. Camphor found to be

the most abundant volatile in L. pedunculata ‘princess’ was similar to those found previously in the study of

Gonçalves and Romano (2013). Camphor is known as antimicrobial (Zuccarini, 2009), thus the essential oil with

the highest amount of camphor could be expected to provide the greatest antibacterial activity in this study. As show

in Table 2. the essential oil of L. pedunculatas ‘princess’ showed the best result in the microbial inhibitory

activities, considering from its ability to inhibit microbial growth by MIC and zone inhibition diameter. It was

able to inhibit most of the bacterial pathogens used in this study. S. aureus was evaluated as the most sensitive

bacteria with MIC and zone inhibition diameter of L. pedunculatas ‘princess’ essential oil being 0.39 mg/mL and

7.37 mm, respectively. On the other hand, K. pneumoniae and B. subtilis were considered to be less sensitive

when being tested with all essential oils. The greatest antibacterial activity of L. pedunculatas ‘princess’ might be

due to the presence of major volatile compounds, such as 1,8-cineole, fenchone, camphor (Vakilian et al., 2011).

Conclusion

This study reports the essential oil composition and antibacterial activities of three different Lavandula

species grown in Thailand. Each Lavandula species appeared to have unique volatile profiles and antibacterial

activities. The essential oil of L. pedunculata ‘princess’ showed a broad spectrum of antibacterial activities,

suggesting thatit may be used as antimicrobial agent in the food, cosmetic and therapeutic applications.

Acknowledgement

The authors would like to thank Angkhang Royal Agricultural Station for providing access to the lavender

samples.The author thanks Mae Fah Luang University for supporting of financial and GC-MS.



References

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Table 1. Volatile constituents of essential oils from three Lavandula species analysed by GC-MS.

No. Compound RI* RI**

% relative peak area

L. x hybrid

‘boysemberry

ruffles’

L. x hybrid

‘high five

purple’

L. pedunculatas

‘princess’

1 tricyclene 918 921 0.11±0.18 0.31±0.27

2 α-pinene 928 932 2.31±0.07 0.02±0.00 4.75±0.18

3 α-fenchene 940 945 1.60±0.07

4 camphene 941 946 1.97±0.08 4.78±0.14

5 sabinene 962 969 0.37±0.13 0.10±0.0.5 0.13±0.07

6 β-pinene 970 974 0.95±0.08 0.47±0.11 0.37±0.11

7 3-octanone 976 979 0.02±0.00

8 myrcene 984 988 0.33±0.13 0.34±0.10 0.32±0.04

9 3-octanol 985 988 0.06±0.10 0.11±0.03

10 δ-2-carene 998 1001 1.45±0.11

11 α-phellandrene 999 1002 0.11±0.05

12 δ-3-carene 1002 1008 1.87±0.07

13 α-terpinene 1010 1014 0.02±0.00 0.13±0.04

14 p-cymene 1015 1020 0.33±0.13 0.09±0.03

15 o-cymene 1018 1022 0.47±0.10

16 limonene 1020 1024 9.73±0.11

17 1,8-cineole 1022 1026 28.81±0.14 12.29±0.15 9.73±0.10

18 Z-β-ocimene 1029 1032 1.05±0.10 0.70±0.07

19 β-ocimene 1041 1044 0.23±0.13 0.02±0.00

20 pulegone 1048 1053 0.02±0.00

21 γ-terpinene 1050 1054 0.08±0.04 0.19±0.02

22 cis-linalool oxide 1060 1067 0.18±0.02

23 cis-vertocitral C 1070 1076 0.07±0.02

24 camphenilone 1072 1078 0.09±0.04 0.13±0.05

25 fenchone 1079 1083 5.50±0.13 21.91±0.14 23.73±0.13

26 isoterpinolene 1080 1085 2.09±0.13

29 trans-vertocitral C 1096 1105 0.02±0.00

30 camphor 1138 1141 33.62±0.17 46.65±2.13 30.30±1.24

31 isoborneol 1148 1155 0.56±0.17

32 δ-terpineol 1156 1162 1.52±0.11 0.58±0.14

33 lavandulol 1161 1164 0.50±0.04

34 terpinen-4-ol 1170 1174 0.87±0.08

35 santalone 1173 1177 0.71±0.13

36 p-methyl acetophenone 1173 1179 0.08±0.10

37 α-terpineol 1180 1186 1.21±0.13 0.74±0.10 0.47±0.13

38 trans-isocarveol 1183 1187 0.61±0.14

39 myrtenal 1190 1195 0.17±0.10

40 verbenone 1199 1204 0.20±0.17 0.12±0.16 0.18±0.14

41 endo-fenchyl acetate 1214 1218 0.20±0.11

42 carvone 1234 1239 0.09±0.02 0.18±0.08

43 iso-isopulegyl acetate 1275 1283 0.66±0.13 2.84±0.13

44 bornyl acetate 1283 1287 5.28±0.28

45 lavandulyl acetate 1284 1288 0.02±0.00

46 geranyl formate 1295 1298 1.06±0.14

47 cis-pinocarvyl acetate 1305 1311 0.22±0.13 1.79±0.12

48 neo-verbanol acetate 1315 1319 0.13±0.10

49 E-cis-jasmonol 1324 1328 1.07±0.14



No. Compound RI* RI**

% relative peak area

L. x hybrid

‘boysemberry

ruffles’

L. x hybrid

‘high five

purple’

L. pedunculatas

‘princess’

50 trans-carvyl acetate 1335 1339 3.16±0.28

51 α-ylangene 1370 1373 0.19±0.14

52 α-duprezianene 1383 1387 0.85±0.13

53 β-cubebene 1383 1387 0.02±0.00

54 iso-longifolene 1384 1389 0.13±0.07 0.05±0.02

55 sesquithujene 1400 1405 0.05±0.02

56 acora-3,7(14)-diene 1401 1407 0.20±0.13

57 α-barbatene 1403 1407 0.02±0.00

58 α-gurjunene 1406 1409 0.22±0.11 0.02±0.00

59 α-cedrene 1407 1410 0.15±0.05

60 aromadendrene 1433 1439 0.08±0.02

61 Z-β-farnesene 1435 1440 0.02±0.00

62 γ-muurolene 1472 1478 0.15±0.04 0.02±0.00

63 amorpha-4,7(11)-diene 1474 1479 0.05±0.02 0.10±0.07

64 β-vetispirene 1491 1493 0.18±0.04

65 valencene 1494 1496 0.06±0.02

66 epizonarene 1496 1501 0.05±0.02

67 α-chamigrene 1498 1503 0.07±0.02

68 premnaspirodiene 1499 1505 1.36±0.13

69 Z-α-bisabolene 1499 1506 0.11±0.04

70 δ-amorphene 1507 1511 0.09±0.02

71 γ-cadinene 1508 1513 0.32±0.04

72 sesquicineole 1509 1518 0.02±0.00

73 7-epi-α-selinene 1517 1521 0.05±0.02

74 10-epi-cubebol 1525 1533 0.11±0.04

75 E-β-ionol acetate 1531 1537 0.07±0.02

76 α-calacorene 1538 1542 0.19±0.13

77 trans-dauca-4(11),7-diene 1547 1556 0.22±0.11

78 E-nerolidol 1552 1562 0.10±0.03

79 longipinanol 1560 1567 0.40±0.07

80 zierone 1565 1574 0.37±0.13

81 santalenone 1567 1576 0.02±0.00 0.05±0.02

82 β-copaen-4α-ol 1581 1590 0.93±0.07 0.51±0.07 0.02±0.00

83 khusimone 1591 1604 0.18±0.14

84 β-humulene epoxide 1595 1614 0.06±0.02

85 epi-cedrol 1605 1618 0.37±0.15

86 β-acorenol 1619 1637 0.12±0.14

87 α-muurolol 1629 1644 0.65±0.08

88 himachalol 1648 1652 0.17±0.08

89 α-bisabolol oxide B 1649 1658 0.02±0.00

%yield (w/w) 0.24±0.13 0.11±0.32 0.22±0.18

Total of volatile components 45 38 42

RI*; calculated retention indices, RI**; retention indices from literature Adams (2017).


Table 2. Antibacterial activities of essential oils from three Lavandula species and chloramphenicol expressed as

inhibition zone diameter (mm) and MIC.

Bacterial strain

Essential oil MIC (mg/mL)

(diameter±SD)

Chloramphenicol MIC (µg/mL)

(diameter±SD) L. pedunculatas

‘princess’

L. x hybrid

‘boysemberry ruffles’

L. x hybrid

‘high five

purple’

Klebsiella pneumoniae 12.50

(7.67±0.06)

6.25

(7.40±0.17)

12.50

(8.17±0.21)

500.00

(9.30±2.50)

Salmonella typhimurium 1.56

(7.23±0.15)

6.25

(7.30±0.10)

500.00

(8.25±2.10)

Escherichia coli 0.78

(8.37±0.15)

12.50

(7.27±0.15)

12.50

(7.57±0.15)

7.81

(10.80±1.11)

Pseudomonas aeruginosa 0.78

(7.33±0.11)

12.50

(7.67±0.32)

Staphylococcus epidermidis 3.12

(7.37±0.15)

12.50

(7.83±0.15)

12.50

(7.33±0.23)

15.62

(9.60±2.20)

Staphylococcus aureus 0.39

(7.37±0.06)

15.62

(12.40±1.40)

Bacillus subtilis 12.50

(7.53±0.21)

125.00

(13.10±1.10)

Enterococcus faecium

12.50

(7.37±0.21)

500.00

(7.21±0.18)

Staphylococcus pyogens 3.12

(7.33±0.15)

1.56

(7.10±0.10)

15.62

(9.60±2.20)




Discovery of plant antimicrobial peptides and laboratory scale production

Parichart Burns1,2,3*, Jutatape Watcharachaiyakup2,3, Patchima Sithisarn4, Pimpilai Saengmanee2,

Vinitchan Ruanjaichon1 and Sonthichai Chanpreme5

1National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology

Development Agency, Khlong Luang District, Pathum Thani, Thailand

2Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen District, Nakhorn Pathom, Thailand 3Center of Excellence on Agricultural Biotechnology, Chatuchak Distance, Bangkok, Thailand

4Department of Veterinary Public health, Faculty of Veterinary Medicine, Kasetsart University Kamphaeng Saen District,,

Nakhorn Pathom, Thailand 5Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen District,

Nakhorn Pathom, Thailand *Corresponding author e-mail: [email protected]

Abstract: Plant antimicrobial peptides (PAMPs) are natural peptide molecules derived from plants with antimicrobial

properties. Their targets range from viruses, bacteria to fungi with the activities of replication or growth inhibition,

slow and abnormality growth induction, bactericides and fungicides. With the availability of genome, transcriptome

and metabolome databases, bioinformatics has been successfully identified these beneficial molecules and in silico

studied of their properties. Four peptides including P5, P9, P25 and P36 were selected from retrieved genomic

and transcriptome data of monocots including maize, sorghum, rice and sugarcane. Their properties were analysed

online tools such as the predicted protein server, Phyre2 and AMPA. The molecules had potential to be antimicrobial

peptides. In order to determine their activities, E. coli expression system was used to produce these peptides in

fusion form. The fusion proteins were selected, digested, purified and concentrated using affinity absorption and

size selection methods. Spectrophotometry indicated that the purified peptides had high concentration and can be

used for further study.

Keywords: defensin, bioinformatics, E. coli expression system, laboratory scale production, protein stability

Introduction

Antimicrobial peptides (AMPs) are found in invertebrates, vertebrates, plant and bacteria. Most AMPs

are cationic or amphiphilic. Hence their interactions are to anionic charge molecules. Modes of AMP functions

include 1) binding to negatively charged surfaces of gram negative bacterial outer membrane, 2) interact with

bacterial cell wall and 3) interact with bacterial cell membrane. These actions cause the restriction cell division,

causing cell wall remodeling and subsequently cell lysis. In some cases, AMPs interact with specific targets within

the bacterial cells causing the disruption of cellular functions such as protein synthesis (Bechinger and Gorr, 2018).

They are, therefore, valuable not only for plant pathogen protection but also medical and veterinary treatment.

Some plant AMPs reportedly had antifungal and antibacterial activities against both plant and human pathogens

(Sathoff et al., 2019). Although share modes of functions, plant microbial peptides have a distinct character of high

cysteine residues which form multiple disulfide bonds. Most of them are low and moderate in size (molecular weight).

These features provides advantages of chemical, thermal and proteolytic activities. There are seven major PAMP

families; thionins, defensin, hevein, knottin, hairpinin, lipid transfer proteins (LTPs) and snakins (Tam et al., 2015).

Recently, many plant genome and transcriptome databases are available in public domain. Therefore,

bioinformatics approach becomes a power tool in identifying and retrieving PAMPs data. Schmitt et al., (2016)

reported the link between variations of AMP amino acid sequences and AMP binding capacity to bacterial membranes.

Plant AMPs are generally accumulated from specific organs such as leaves, flower and seeds. Several purification

steps are required for purified AMPs (Tang et al., 2018). Alternatively, in silico approach combining with de novo

synthesis via chemical reaction has become an alternative approach to conventional method for production and

an improvement of AMP (Mikut et al., 2016). The aim of this study is to 1) identify target AMPs and retrieve

the genome/transcriptome data from monocots databases using in silico study 2) to amplify AMP genes from monocots

and 3) produce purified PAMPs in laboratory scale.


Plant databases

There are many plant genome and transcriptome databases available on line. However, only those with

monocots data (sugarcane, rice, maize and sorghum) were included (Table 1). Criteria such as keywords and

sequence similarity were used in BLAST and FASTA search. The outcome from various sources were compared

and analyzed. The PAMP genomic DNA and cDNA sequences were retrieved. They were aligned using MacVector 12.6.


In silico study on protein properties and antimicrobial activity

Protein secondary and tertiary structures and other important characters including solubility, transmembrane

probability and stability were investigated. The antimicrobial activities were determined using on line software (Table 1).

Primer design and PCR amplification

Primers were designed based on retrieved sequences using Primer3 (Untergasser et al., 2012). The target

genes were amplified using Reverse transcription PCR. The DNA were cloned and automate sequenced. The authenticity

were determined using BLASTX software (Altschul et al., 1997).

E. coli Expression system

DNA fragments were cloned into pMAL-c2 (New Englan Biolab, USA) and recombinant plasmids were

transformed into E. coli. The optimal temperature were investigated in small scale conditions. The fusion protein

was produced in laboratory scale under optimized conditions. It was purified and digested with factor Xa. PAMPs

was further purified and concentrated.

Result & Discussion

Four plant microbial peptides (P5, P9, P25 and P36) were successfully retrieved from plant databases.

They were identified as defensin and snakin. They displayed antimicrobial activities and relatively stable in

cytoplasm. The coding regions were cloned into pMALc2 E. coli expression vector. The work processes were

summarized in Figure. 1 E. coli expression system is widely used for heterologous protein expression because of

the cost effective and routine/standard protocol for E. coli production (Chen, 2012).The temperature, aeration and

duration for induction were tested to fine optimized condition for protein production (Papaneophytou and Kontopidis,

2014; Singha et al., 2017). The fusion proteins were produced in laboratory scale and maintained within bacterial

cells. They were released using sonication and purified from other bacterial proteins via specific binding. PAMPs

were digested out of fusion proteins, purified and concentrated using size exclusion chromatography. The concentration

and protein content were summarized in Table 2. The results indicated variation of protein recovery and yield.

Interestingly, the deviation results from the last steps of factor Xa digestion and protein recovery. This could be

specific protein tertiary structure that reduce availability of factor Xa site or the current process caused misfolding

of specific protein (Overton, 2013).

Conclusion

Plant antimicrobial peptides (PAMPs) were effectively produced in laboratory scale using bioinformatics

approaches combining with E. coli expression system. This method can be applied to other AMPs production.

Acknowledgement

This research is financially supported by Thailand Research Fund (TRF) through National Research

Council of Thailand (NRCT). National Center for Genetic Engineering and Biotechnology (BIOTE) and Center

for Agricultural Biotechnology (CAB) provided laboratory facilities and equipment.

References

Bechinger, B., and Gorr, S. U. 2017. Antimicrobial peptides: mechanisms of action and resistance. Journal of Dental

research 96(3): 254260.

Chen, R. 2012. Bacterial expression system for recombinant protein production: E. coli and beyond. Biotechnology

Advances 30: 11021107.

Mikut, R., Ruden, S., Reischl, M., Breitling, F., Volkmer, R., and Hilper, K. 2016. Improving short antimicrobial peptides

despite elusive rules for activity. Biochimica et Biophysica Acta (BBA)-Biomembranes 1858:10241033.

Overton, T. W. 2014. Recombinant protein production in bacterial hosts. Drug Discovery Today 19(5): 590601.

Papaneophytou, C.P. and Kontopidis, G. 2014. Statistical approaches to maximize recombinant protein expression

in Escherichia coli: A general view. Protein Expression and Purification 94: 2232.

Sathoff, A. E., Velivelli, S., Shah, D. M. and Samac, D. A. 2019. Plant defensing peptides have antifungal and

antibacterial activity against human and plant pathogens. Phytopathology 109: 402408.

Schmitt, P., Rosa, R. D. and Destoumieux-Garzón, D. 2016. An intimate link between antimicrobial peptide

sequence diversity and binding to essential components of bacterial membranes. Biochimica et Biophysica

Acta (BBA)-Biomembranes 1858: 958–970.

Singha, T. K., Gulati, P., Mohanty, A., Khasa, Y., Kapoor, R. K. and Kumar, S. 2017. Efficient genetic approaches

for improvement of plasmid based expression of recombinant protein in Escherichia coli: A review.

Process Biochemistry 55: 1731.



Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman D. J. 1997. Gapped BLAST

and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 25(17)

: 33893402.

Tam, J. P., Wang, S., Wong, K. H. and Tan, W. L. 2015. Antimicrobial peptides from plants. Pharmaceuticals 8(4): 711757.

Tang, S. S., Prodhan, Z H., Biswas, S. K., Le, C. F. and Sekaran, S. D. 2018. Antimicrobial peptides from different

plant sources: isolation, characterization and purification. Phytochemistry 154: 94105.

Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M. and Rozen, S.G. 2012. Primer3-

new capabilities and interfaces. Nucleic Acids Research 40(15): e115.


Figure 1. The overview of plant AMP protein production at laboratory scale.

Table 1. Websites used in this study for A) Plant genome and transcriptome databases and B) Protein analysis

and antimicrobial activity.

Name Location Tools

Ensembl Plants

Home http://plants.ensembl.org/species.html BLAST/BLAT

NCBI https://www.ncbi.nlm.nih.gov/ BLAST, keyword search

Phytozome12 https://phytozome.jgi.doe.gov/pz/portal.html BLAST, keyword search

PlantGDB http://www.plantgdb.org/prj/GenomeBrowser/ BLAST, pattern search

Sugarcane genome

hub http://sugarcane-genome.cirad.fr/

BLAST, keyword search, primer

design

PredyFlexy http://www.dsimb.inserm.fr/dsimb_tools/predyfl

exy/index.html

Flexibility and Local Structure

prediction from sequence

Pro Pi https://www.protpi.ch Protein tools, peptide tools

AntiBP2 http://crdd.osdd.net/raghava/antibp2/index.html Server for antibacterial peptide

prediction

Antimicrobial

Sequence Scanning

System (AMPA)

http://tcoffee.crg.cat/apps/ampa/do

A theoretical approach to spot

active regions in antimicrobial

proteins.

Table 2. Protein concentration and total protein content of four plant antimicrobial peptides. Bradford assay was

used for determination of protein concentration with bovine serum albumin (BSA) as a protein standard.

Step Protein concentration

(mg/mL)

Total protein (milligram)

Cell suspension 0.455 - 1.083 182-433.2

Cell disruption 7.65 -8.27 153.0-169.5

Fusion protein 4.15-7.19 4.15-7.19

Purified protein 0.014-0.765 0.0032-137.7

https://www.ncbi.nlm.nih.gov/




Diversity of indigenous vegetables in Puparn Royal Development

Study Centre, Sakon Nakhon Province, Thailand

Hathairat Chokthaweepanich1*, Kwankamol Tawaitakam1and Noppadol Kaewkumsai2 1Department of Agriculture and Resources, Faculty of Natural Resources and Agro-Industry,

Kasetsart University Chalermphrakiat Sakonnakhon province Campus, Muang District, Sakon Nakhon, Thailand 2The Puparn Royal Development Study Centre, Muang District, Sakon Nakhon, Thailand


Abstract: A study on diversity of indigenous vegetables in Puparn Royal Development Study Centre, Sakon

Nakhon province during June 2015 to May 2016 was purposed to collect and classify the indigenous vegetables

with revealing their potential as food resources, edible parts and utilization method. Sixty-three species, sixty

genera and forty-three families were identified. The four majorities of plant families were Zingiberaceae,

Fabaceae, Araceae, and Asteraceae, respectively. Additionally, identified indigenous vegetables could be

classified into six groups based on consumed part including underground stem (4 species), leaves and young shoots

(35 species), stem (2 species), flower (4 species), fruit (6 species) and whole plant (21 species). Most of utilization

methods were used as salad, steamed or boiled vegetables eaten with chili paste, minced meat, or seasoning in

food ingredients. Keywords: indigenous vegetables, diversity, plant utilization, Sakon Nakhon province

Introduction

The Puparn Royal Development Study Centre is established in Ban Na Nok Khao, Huai Yang sub district,

Muang district, Sakon Nakhon province on November 25, 1982. This centre is one of six development study

centres that is settled in northeastern region of Thailand. A total land area is approximately 2,128 hectares (13,300

rais) comprising about 368 hectares (2,300 rais) of an agricultural development zone and 1,760 hectares (11,000

rais) of peripheral areas earmarked for forestry development that is located within Pa Phu Lom Khao – Phu Peck

National Reserved Forests and Mai Kraya Loei Namphung Forest Project. Most of ecological systems in this

location are natural forest consisted of Dipterocarp forest (60%), dry evergreen forest (13%), mixed deciduous

forest (12%) and agricultural area (12%). Additionally, people who live in this area and nearby can get benefits

from plants in this forest [Office of the Royal Development Projects Board (ORDPB), 2018].

There are many species of indigenous vegetables in Thailand especially northeastern region. Deewiset

and Khumklang (1998) collected 139 species of indigenous vegetables in northeastern region. Khumklang et al.

(2000) reported that there were more than 220 species of indigenous vegetables in Thailand and majority of them

were found in the northeastern region. Indigenous vegetables can be consumed either wholly or in parts of plant.

The edible parts of vegetables may be root, stem, leave, flower, fruit, rhizome or bulb. Moreover, some mushroom

can also be consumed as vegetable (Shirai and Rambo, 2008; Nnamani et al., 2010; Chokthaweepanich et al.,

2016). These local vegetables are not only an excellent source of vitamins and minerals, but they can also be used

for several purposes such as food, medicinal plants, ornamental plants and household incomes (Omara-Achong et

al., 2012; Abukutsa-Onyango, 2014; Srisawat et al., 2016; Sseremba et al., 2017; Phumthum et al., 2018).

Indigenous vegetables can be prepared from locally available resources for food and nutritional security.

However, the local vegetables are known and take benefit in different patterns depend on traditional knowledge,

tasting culture, location, seasonal availability and social group (Lee et al., 2008; Omara-Achong et al., 2012;

Abukutsa-Onyango, 2014; Khuankaew et al., 2014). Although economic vegetables are widely eaten in Thailand

at the present, indigenous vegetables are still important to people who live in the rural area (Agoreyo et al., 2012;

Ayaz et al., 2015; Sseremba et al., 2017) because indigenous vegetables are cheap and substantial source of amino

acid, protein, vitamins and minerals (Okafor, 1983). Moreover, many researchers reported beneficial effects from

local vegetable consumption such as high antioxidant (Hong et al., 2004; Tangkanakul et al., 2006; Panpipat et

al., 2010; Chao et al., 2014; Baang et al., 2015; Kongkachuichai et al., 2015; Tinrat, 2016), flavonoid contents

(Baang et al., 2015; Kankanamge and Amarathunga, 2017), phenolic compound (Panpipat et al., 2010; Tinrat,

2016; Kankanamge and Amarathunga, 2017; Yakoh et al., 2018), preventing hypertension (Chuchote et al., 2015),

etc.

Unfortunately, traditional thai vegetables are risk to loss since the younger generations who prefer the

introduced species (e.g. broccoli, cabbage, cauliflower, lettuce, tomato, etc.), don’t know cooking techniques or

recipe using indigenous vegetables (Somnasang and Moreno-Black, 2000; Kolsuwat, 2002; Setalaphruk and Price,

2007). Homemade meals are substituting with fast food in Thailand (Kosulwat, 2002). Economic or introduced


vegetables are key ingredients in many dishes. Although many indigenous vegetables are sale in the local market,

some people may know their plant name, kinds, cooking process (Shirai and Rambo, 2008). Therefore, it will be

a worthwhile attempt to study on diversity and classification of indigenous vegetables covering their used parts

and utilization method in the Puparn Royal Development Study Centre.


Study Area

This study was conducted in the Puparn Royal Development Study Centre located in Sakon Nakhon

province, Thailand during June 2015 to May 2016. It is located between latitudes 170 04’ to 170 07’ north and

longitudes 1040 00’ to 1040 04’ East (Figure 1). The range of height is between 180-420 meters above moderate

sea level. The average temperature in this area is approximately 26.40C. The lowest temperature is around 12.10C

and highest is around 37.90C. The average precipitation is 362.1 mm per year and average annual relative humidity

is 72.9%. Soil condition in this study area is sandstone of conglomerate type and shale with lower layers of soil

comprised shale, sandstone and limestone (ORDPB, 2018).

Data Collection

Indigenous vegetables were collected and photographed from three forest areas including Dipterocarp

forest, dry evergreen forest and mixed deciduous forest. All samples were recorded plant ecology and

morphological characteristics especially disappeared traits in herbarium specimens or preserved plants in alcohol-

glycerine such as habit, latex, color, taste and flavor. Moreover, the local name, used part, and utilization methods

also were gathered from local people. The collected plants were identified in the fields and laboratory by

taxonomists or comparing with voucher specimens at Bangkok Herbarium Museum, Department of Agriculture,

Bangkok. Finally, all specimens were reconfirmed using literature on the flora of Thailand, Flora of China and

Thai plant names (Revised edition 2014) book (Smitinand, 2014).

Result & Discussion

Sixty-three indigenous vegetable species belong to 60 genera and 43 families (Table 1) were collected in

the Puparn Royal Development Study Centre. They could be classified into 4 main groups including

monocotyledonous plants 15 species, dicotyledonous plants 41 species, fern 2 species and fungi 5 species. The

most discovered plant families were 5 species (7.94%) in Zingiberaceae, 4 species (6.35%) in Fabaceae, 3 species

(4.76%) each in Araceae and Asteraceae. Moreover, They could be classified into 9 groups (Figure 2) depend on

plant habit (Smitinand, 2014) consisted of tree 9 species (14.29%), shrub 9 species (14.29%), herb 25 species

(39.68%), climbing 9 species (14.29%), scandent 1 species (1.59%), palm 1 species (1.59%), bamboo 2 species

(3.17%), fern 2 species (3.17%) and fungi 5 species (7.94%). All fungi were 5 species of mushrooms that grew in

the rainy season. Herbs were the dominant indigenous vegetables in this study because they contained less woody

tissue in all vegetative parts than tree and shrub, whereas tree and shrub took longer time for new branch, shoot

and fruit (Thongpukdee et al., 2014). Besides, the main features of surveyed areas were deciduous forest, open

canopy and abundance of herbs in the ground layer (Rundel and Boonpragob, 1995).

The majority species of indigenous plants were found in Dipterocarp forest (34 species), mixed deciduous

forest (30 species), wet areas (19 species) and dry evergreen forest (17 species), respectively. The Dipterocarp

forest contained more indigenous species than others because this forest type covered 60% of all areas (ORDPB,

2018), while the mixed deciduous forest was indicated higher plant diversity when species-area ratio was

examined (Figure 3). Dipterocarp forest was considered to be less diversity than evergreen forests or wet forests

due to the conditions of low moisture, high temperature and low annual rainfall averages (Murphy and Lugo, 1986;

Powers et al., 2018). Besides, most of trees in Dipterocarp forest are shedding their leaves in the dry season

(Janzsn, 1998; Himmapan and Kaitpraneet, 2008; Elliiott et al., 2013).

The plant parts used from local people both in single or multiple preparations (Figure 4 and Table 1) were

underground stem 4 species (6.35%), leave and young shoot 35 species (55.56%), stem 2 species (3.17%), flower

4 species (6.35%), fruit 6 species (9.52%) and whole plant 21 species (33.33%). The results revealed that the

greater vegetable consumption was leave and young shoot because this part was young and appreciated for

cooking. Additionally, leafy vegetables were quick to prepare and an admired source of food and medicine

(Turreira-García et al., 2017). The investigation of this study reported that most utilization methods were used in

cooked or fresh vegetables as salad, steamed or boiled vegetables eaten with chili paste, spicy minced meat, or

seasoning in food ingredient. Furthermore, they could be cooked in a variety of dishes; for example, soup, stir-

fried, curry or as a sweet dessert. Some lifestyle diet was similar to other regions in Thailand (Ngamsiri and

Thananoppakun, 2014; Turreira-García et al., 2017) and other countries such as in Kenya (Gido et al., 2017),

Nepal (Uperty et al., 2012), Nigeria (Nnamani et al., 2010; Arowosegbe et al., 2018).



Various indigenous species have been presented to be used several ways as well as vegetables, for

instance, fruit (e.g. Aegle marmelos, Antidesma ghaesembilla, Passiflora foetida, Garcinia cowa, Schleichera

oleosa); medicinal plants (e.g. Acmella oleracea, Alpinia sp., Limnophila geoffrayi, Phlogacanthus pulcherrimus,

Glinus oppositifolius); ornamental plants (e.g. Ardisia crenata, Curcuma sessilis, Curcuma singularis, Cratoxylum

formosum, Caryota mitis, Nymphaea pubescens); and sweet dessert (e.g. Cissampelos pareira, Dioscorea alata,

Dioscorea sp.). Some local vegetables should not be eaten raw because of highly toxic prussic acid and must be

cooked or fermented before consuming to neutralize the hydrocyanic acid (CSIR, 1950; Coursey, 1967) such as

wild yam (Dioscorea sp.), giant taro (Colocasia gigantea) and unicorn plant (Lasia spinosa). Some indigenous

vegetables in this study were reported as a good source of minerals and vitamins (Office of the Primary Health

Commission. 1997; Nutrition Division, 2001; Sirival, 2008; Kingkachuichai et al., 2015): phosphorus (e.g.

Dracaena angustifolia, Lobelia chinensis, Persicaria odorata); potassium (e.g. Blumea napifolia, Dracaena

angustifolia, Lobelia chinensis,); calcium (e.g. Blumea napifolia, Cissampelos pareira, Lobelia chinensis); iron

(e.g. Diplazium esculentum, Hydrocotyle umbellata); vitamin A (e.g. Careya sphaerica, Centella asiatica,

Cissampelos pareira, Hydrocotyle umbellata, Lasia spinosa); and vitamin C (e.g. Aegle marmelos, Careya

sphaerica, Cissus hastate, Melientha suavis, Passiflora foetida). Consequently, the local vegetables can help the

rural people to prevent malnutrition (Omara-Achong et al., 2012; Kongkachuichai et al., 2015; Phumthum et al.,

2018) and to get rid of common illness (Chotchoungchatchai et al., 2012; Khuankaew et al., 2014; Phumthum et

al., 2018).

Conclusion

The Puparn Royal Development Study Centre can be represented as a major source of plant diversity.

Forty-three families with sixty-three species of indigenous vegetables were encountered in different four

ecosystems. A number of vegetables were found in the Dipterocarp forest. Leave and young shoot of diverse

species were preferred to food preparation. There were alternative methods to make use of indigenous plants

except vegetables such as medicine, ornamental plant or dessert. These vegetables are a good option for contributes

to food and nutrition security.

Acknowledgement

We are grateful to Office of the Royal Development Projects Board (ORDPB) for providing research

funding. We thank the Puparn Royal Development Study Centre for transportation and assistance during

fieldwork. Finally, we are also thankful to Dr. Yingyong Paisooksantivattana and Dr. Sranya Vajrodaya (Kasetsart

University) for some plant identification

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Figure 1. Map of the Puparn Royal Development Study Centre, Sakon Nakhon, Thailand

Source: https://www.iucn.org/content/combining-science-local-knowledge-ecosystem-based-adaptation-kok-

klang-village-sakon-nakhon

Figure 2. Classification of indigenous vegetables in the Puparn Royal Development Study Centre based on plant

habit

Figure 3. A comparison of species number within each forest type in the Puparn Royal Development Study

Centre

3%

14%

3%

8%

40%

2%

2%

14%

14%bamboo

climbing

fern

fungi

herb

palm

scandent

shrub

tree

0% 20% 40% 60% 80% 100%

Dipterocarp forest

Mixed Deciduous forest

Dry Evergreen forest

Wet areas

Percentage of area size

Number of species

https://www.iucn.org/content/combining-science-local-knowledge-ecosystem-based-adaptation-kok-klang-village-sakon-nakhon

https://www.iucn.org/content/combining-science-local-knowledge-ecosystem-based-adaptation-kok-klang-village-sakon-nakhon



Figure 4. Edible part of indigenous vegetables in the Puparn Royal Development Study Centre

Table 1. Habit, ecosystem and edible parts of indigenous vegetables in Puparn Royal Development Study Centre

Family Scientific name Vernacular

name

Habit Ecosystem

*

Edible parts**

D

F

M

D

F

D

E

F

W

A

U

st

S

t

S

h

F

l

F

r

W

h

Acanthaceae Phlogacanthus

pulcherrimus T.

Anderson.

Di pla chon Shrub / /

Alismataceae Limnocharis flava (L.)

Buchenau

Phak phai Herb / / /

Amanitaceae Amanita sp. Het ra ngok Fungi /

Amaranthacea

e

Alternanthera sessilis (L.)

R. Br. ex DC.

Phak pet thai Herb / / /

Ancistroclada

ceae

Ancistrocladus tectorius

(Lour.) Merr. Hu kwang

Climb

ing

/

Apiaceae Centella asiatica (L.)

Urb.

Phak nok Herb / / /

Hydrocotyle umbellata L. Phak waen

kaeo Herb / / /

Apocynaceae Aganonerion

polymorphum Pierre ex

Spire

Som lom Climb

ing

/ / /

Amphineurion

marginatum (Roxb.) D.J.

Middleton

Khruea sai tan Climb

ing

/ / /

Araceae Amorphophallus sp. Buk Herb / / / / /

Colocasia gigantea

(Blume) Hook f. Bon Herb / / /

Lasia spinosa (L.)

Thwaites

Phak nam Herb / / /

Arecaceae Calamus sp. Wai Scand

ent

/ / /

Caryota mitis Lour. Tao rang Palm / /

Asparagaceae Dracaena angustifolia

(Medik.) Roxb. Khon Khan Shrub

/ /

Asteraceae Acmella oleracea (L.)

R.K. Jansen

Phak khrat hua

waen Herb / / /

Blumea napifolia DC. Phak hon han Herb / /

Crassocephalum

crepidioides (Benth.) S.

Moors.

Phak kho on Herb / / /

Athyriaceae Diplazium esculentum

(Retz.) Sw. Phak kut Fern / / /

Auriculariacea

e

Auricularia auricula-

judae (Bull.) J.Schröt.

Het hu nu Fungi

/

Campanulacea

e Lobelia chinensis Lour. Phak khi som Herb / / /

Clusiaceae Garcinia cowa Roxb. ex

Choisy Cha muang Shrub / /

0

5

10

15

20

25

30

35

underground

stem

leave & shoot stem flower fruit whole plant

Nu

mb

er o

f p

lan

t sp

ecie

s

Edible parts of indigenous vegetables



name

Habit Ecosystem

*

Edible parts**

D

F

M

D

F

D

E

F

W

A

U

st

S

t

S

h

F

l

F

r

W

h

Convolvulace

ae

Ipomoea aquatica Forssk. Phak bung Herb / / /

Costaceae Cheilocostus speciosus (J.

Koenig) C. D. Specht

Ueang mai na Herb / / / /

Dioscoreaceae Dioscorea alata L. Man Climb

ing

/ / /

Dioscorea sp. Kloi Climb

ing

/ / /

Diplocystacea

e

Astraeus hygrometricus

(Pers.) Morgan

Het phao Fungi

/

Ebenaceae Diospyros filipendula

Pierre ex Lecomte

Lam bit dong Tree / /

Fabaceae Adenanthera pavonina L. Ma klam ton Tree / / /

Caesalpinia mimosoides

Lam. Phak khaya Climb

ing

/ /

Crotalaria sp. Hing hai Herb / / / /

Droogmansia

godefroyana (Kuntze)

Schindl.

Chai hin Shrub / /

Hypericaceae Cratoxylum formosum

(Jacq.) Benth. & Hoof. f.

ex Dyer subsp. formosum

Tio khao Tree / / / / /

Lecythidaceae Careya aeborea Roxb. Kradon Tree / /

Lygodiaceae Lygodium polystachyum

Wall. ex Moore

Ya li phao Fern / / / /

Melastomatac

eae

Melastoma

malabathricum L. Khlong khleng Shrub / / / / /

Memecylon scutellatum

(Lour.) Hook. & Arn.

Mueat ae Tree / / / /

Menispermace

ae

Cissampelos pareira L.

var. hirsuta (Buch. ex

DC.) Forman

Khruea ma noi Climb

ing

/ / /

Menyanthacea

e

Nymphoides indica (L.)

Kuntze Bua ba Herb / /

Molluginacea

e

Glinus oppositifolius (L.)

A. DC.

Sadao din Herb / / /

Myrtaceae Syzygium antisepticum

(Blume) Merr. & L. M.

Phak mek Tree / / /

Nymphaeacea

e

Nymphaea pubescens

Willd Bua sai Herb / /

Barclaya sp. Phak lin fan Herb / /

Opiliaceae Melientha suavis Pierre Phak wan pa Shrub / / /

Passifloraceae Passiflora foetida L. Ka thok rok Climb

ing

/ /

Phyllanthacea

e

Antidesma ghaesembilla

Gaertn.

Mao khai pla Tree / / /

Plantaginacea

e

Limnophila geoffrayi

Bonati

Phak kha

yaeng Herb / / /

Poaceae Gigantochloa albociliata

(Munro) Kurz.

Phai rai Bamb

oo

/ /

Vietnamosasa ciliata (A.

Camus) T. Q. Nguyen

Phai chot Bamb

oo

/ /

Polygonaceae Persicaria odorata

(Lour.) Soják

Phak phai Herb / / /

Primulaceae Ardisia crenata Sims Ta kai bai

kwang Shrub / / /

Russulaceae Russula densfolia (Secr)

Gill.

Het than Fungi /

Russula emetica (Schaeff.

Ex Fr.) Pers. S.f. Gray

Het num mak Fungi

/

Rutaceae Aegle marmelos (L.)

Corrêa ex Roxb.

Ma tum Tree / / / /

Clausena guillauminii

Tanaka

Song fa Shrub / /

Sapindaceae Schleichera oleosa

(Lour.) Merr.

Mak kho Tree / / / / /

Symplocaceae Symplocos

cochinchinensis (Lour) S.

Moore

Chum cha Shrub / /

Vitaceae Cissus hastata Miq. Som sandan Climb

ing

/ /




name

Habit Ecosystem

*

Edible parts**

D

F

M

D

F

D

E

F

W

A

U

st

S

t

S

h

F

l

F

r

W

h

Zingiberaceae Alpinia sp. Kha Herb / / / /

Boesenbergia sp. Krachai pa Herb / /

Curcuma sessilis Gage Krachiao

daeng Herb / / /

Curcuma singularis

Gagnep. Krachio khao Herb / / /

Kaempferia galanga L. Pro Herb / / /

Note: *Ecosystem: DF = Dipterocarp Forest; MDF = Mixed Deciduous Forest; DEF = Dry Evergreen Forest; WA

= Wet Area

**Edible part: Ust = underground stem; St = stem; Sh = young shoot and leaves; Fl = flower; Fr = fruit;

Wh = whole plant



Phytochemical screening and antimicrobial activity of Indian almond leaf extracts

Ratthayaporn Thansuwan1*, Jirapron Jitturongarpron1, Jittima Buatib1,

Thanyaporn Tangjaroenchai1,2and Noppadon Muangsue3 1Demonstration School University of Phayao, Muang District, Phayao, Thailand

2School of Science, University of Phayao, Muang District, Phayao, Thailand 3School of Allied Health Sciences, University of Phayao, Muang District, Phayao, Thailand


Abstract: The antimicrobial activity of Indian almond leaf extracts, prepared by extraction using water, %70 ethyl

alcohol and 9 5% ethyl alcohol, were tested for their efficacy by paper disc diffusion method on Muller Hinton

Agar (MHA). The results showed that all three extracts could inhibit mainly the growth of Staphylococcus aureus

with the inhibition zones of 4.33 mm, 3.33 mm and 6.67 mm. respectively. No growth inhibition was observed with

other microbial cultures of Escherichia coli, Klebsiella sp., Citrobacter sp. and Enterobacter sp. Chemical analysis

by Gas Chromatography–Mass Spectrometry (GC-MS) revealed that there were 31 compounds that were matched with

the data in the library,of which, benzenethiol 1,2,3- benzenetriol or “Pyrogallol” was found to be the most abundance.

Keywords: Antimicrobial activity, Indian almond leaf extract, Phytochemical analysis, GC-MS

Introduction

Indian almond tree (Terminalia catappa) is a Combretaceous plant belonging to the tropical almond family.

It is widely distributed throughout all regions of Thailand. In aquaculture, the Indian almond leaves have been claimed

to have active compound(s) that promote wound healing. It has been used in Thailand for curing injured siamese

fighting fishs, presumably to increase thickness of keratin layer of the fish. In the literature, the leaf part of the plant

has been shown to have a potential to use as an alternative treatment for chemical substances and antibiotics [1].

The objectives of this study were to evaluate the antibacterial activity of the Indian almond leaf extracts in order to get

the most effective extracts that could inhibit the growth of microbial pathogens, and to evaluate the potential antimicrobial

compounds in the Indian almond leaf extracts by Gas Chromatography–Mass Spectrometry (GC-MS) analysis.


Extraction of Indian almond leaves

The Indian almond leaves, collected from School of Agriculture and Natural Resources, University of Phayao,

were washed with deionized water and immediately dried in an oven at 50 °C for 24 hours. The fried leaves were

then ground into powder by using motar and pestle. The leaf powder was then extracted separately with three

types of solvent, namely water, 70% ethanol and 95% ethanol for 3 days (100 g per 1000 ml). After filtration, the

extract was evaporated by using a rotary evaporator to dryness. The extracts were kept at 37°C until being used.

Antibacterial activity of Indian almond leaf extracts

Antibacterial tests were performed using disc agar diffusion method. The bacterial suspension of

Staphylococcus aureus, Escherichia coli, Klebsiella sp., Citrobacter sp. and Enterobacter sp. was each diluted to

1.5×108 CFU/ml and swabbed on the surface of MHA agar. Plates were incubated for 24 hours at room temperature.

Antimicrobial activity was detected by measuring the inhibition zone around the disc (in mm.). Tetracycline (TE 30 μg/disc)

was used as positive control.

Phytochemical analysis of Indian almond leaf extracts by gas chromatography – mass spectrometry (GC-MS)

The Indian almond leaf extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS)

analysis and compounds were matched with data in the library.

Result & Discussion

The efficacy test for antimicrobial activity of the leaf extracts was conducted by disc diffusion method

on Muller Hinton agar (MHA). The lowest concentrations of the three Indian almond leaf extracts prepared by using

water, 70% ethanol and 95% ethanol were 31.25, 31.25 and 15.63 mg/ml, respectively. The results obtained from

the disc diffusion method showed that Staphylococcus aureus the inhibition zone could only be observed with

Staphylococcus aureus with the diameters of 4.33 mm for the water extract, 3.33 mm for the 70% ethanol and

6.67 mm for the 95% ethanol (Table 1). No significant inhibition zones were observed with other microbial cultures,

including Escherichia coli, Klebsiella sp., Citrobacter sp. and Enterobacter sp. Therefore, it was suggested that

the antimicrobial activities of the extracts are relatively specific.

mailto:[email protected]



From the Gas chromatography – mass spectrometry (GC-MS) analysis, there were 31 compounds detected

in the 95% Indian almond leaf extract (Figure.1). Among these, only the top 10 constituents with the highest

abundance were listed (Table 2). The lowest concentrations of Indian almond leaves extracted by water, 70%

ethanol and 95% ethanol that could inhibit the growth of Staphylococcus aureus at were found to be 31.25, 31.25 and

15.63 mg/ml, respectively. GC-MS studies suggested that pyrogallol tannin might be the active compound which

is similar to the results reported previously Sundaram Poongulali (2016) and Muthuraman Sundararaman (2016).

Table 1. Inhibition zone of Indian Almond leaf extracts against pathogenic bacteria.

Solution

Concentration

of the extract

(mg/ml)

inhibition zone (mm)

Staphylococcus

aureus

Escherichia

coli

Klebsiella

sp.

Citrobactor

sp.

Enterobacter

sp.

Tetracycline (control) ≥19 29.67 - 20 20

95% eth

125 19.67 - - - -

62.5 15 - - - -

31.25 12.67 - - - -

15.63 6.67 - - - -

70% eth

125 20 - - - -

62.5 14.33 - - - -

31.25 3.33 - - - -

15.63 - - - - -

H2O

125 23 - - - -

62.5 17.33 - - - -

31.25 4.33 - - - -

15.63 - - - - -

Figure 1. A typical chromatogram of the chemical constituents present in 95% ethanolic crude extract of

Terminalia catappa.


Table 2. Major constituents of Indian almond leaves extracted by 95% ethanol based on by Gas chromatography – mass

spectrometry (GC-MS).

Conclusion

The antimicrobial efficacy of Indian Almond leaf extracts prepared by using three kinds of solvent water,70%

ethanol and 95% ethanol were conducted by testing the ability of the extracts to inhibit the growth of bacteria.

The results showed that the 95% ethanol extract are the best in inhibiting the growth of Staphylococcus aureus.

Water is lower ability to inhibit Staphylococcus aureus and 70% ethanol is the lowest. But it has not affect the inhibition

of Escherichia coli.

Acknowledgement

This project was supported by the Science Classroom in University Affiliated School (SCiUS) under the

Ministry of Science and Technology, jointed by Demonstration School, School of Science University of Phayao

and School of Allied Health Sciences, University of Phayao.

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No. R.T. min Compounds Molecular formula Molecular weight % peak area

1 7.644 Benzenetriol Benzene-1,2,3-

triol Pyrogallol C6H6O3 126.111 g/mol 100.00%

2 35.177 Stigmast-5-en-3-ol C29H50O 414.718 g/mol 74.31%

3 29.311 Tetracosahexaene C24H38 326.568 g/mol. 48.60%

4 32.773 Vitamin E C29H50O2 430.706 g/mol 42.40%

5 16.423 Hexadecanoic acid ethyl ester C18H36O2 284.48 g/mol 27.45%

6 7.896 1,3a-epoxy-3ah-indene - - 22.24%

7 36.519 Aristol-a-en-8-one C15H22O 218.34 g/mol 22.47%

8 15.9 Hexadecanoic acid

Hexadecanoate C16H32O2 256.4 g/mol 19.71%

9 19.366 Ethyl-9-octadecenoate C20H38O2 310.522 g/mol 10.23%

10 35.767 Olean-12-en-3-ol C32H52O2 468.766 g/mol 8.72%




Study of chemical composition and antioxidant properties of Sangyod and

Tubtimchumpae rice bran oil

Pavinee Yampeng1*, Hathairat Rimkeeree1 and Supanida Winitchai2

1Faculty of Agro-Industry, Kasetsart University, Chatuchak District, Bangkok, Thailand 2 Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University,

Chatuchak District, Bangkok, Thailand


Abstract: Sangyod rice bran oil is an oil extracted from a local rice strain grown in Phattalung Province,Thailand.

Tubtimchumpae rice bran oil is another kind oil obtained from Tubtimchumpae rice grown in Kamphaengphet Province,

Thailand. Both types of red rice containing high anthocyanin were prepared for the oils by cold-press extraction.

Since they do not pass through heat processing, they are rich in nutritional values. The aim of this research was to study

the chemical compositions and antioxidant properties of Sangyod and Tubtimchumpae rice bran oils. The results

showed that the chemical compositions of in terms of free fatty acids, acid value, peroxide value, unsaponifiable

matter and saponification of the two rice bran oils were significantly different (p≤0.05). Both rice bran oils consisted

of high unsaturated fatty acid contents (80.46 and 80.35%, respectively), with oleic acid and linoleic acid as main

components. However, Tubtimchumpae rice bran oil contained higher tocopherol than Sangyod rice bran oil

(11.88 and 9.13 mg/100g, respectively). Total phenolic contents were found to be 1.48±0.05 and 1.54±0.03 mg

gallic acid equipvalent/100 g oil, respectively. DPPH (IC50) values were 13.90 and 13.97 µ g/ml, ABTS (IC50)

were 15.99 and 16.09 µ g/ml, respectively. In addition, the two types of rice bran oil were found to possess

important nutritional values, such as unsaturated fatty acids, antioxidants and vitamin E (α-tocopherol). Therefore,

it was suggested that these oils can be used to increase health benefits in food products.

Keywords: Sangyod rice bran oil, Tubtimchumpae rice bran oil, chemical composition, antioxidant properties.

Introduction

Rice bran oil (RBO) is the oil extracted from the germ and inner husk of rice. RBO contains significant

unsaturated fatty acids, saturated fatty acids and micronutrients, such as tocopherol, tocotrienol and oryzanol.

Several studies have shown that RBO has an effect on metabolic activities, causing the decrease of bad cholesterol LDL

but without significant affecting the blood level of HDL cholesterol (Yoshino et al., 1989, Qureshi et al., 1991,

Hegsted et al., 1990, Kahlon et al., 1992, Hegsted and Windhauser 1993). RBO containing high antioxidant activities

is known to have health benefits and for improving stability of foods during the storage (Shahid et al., 2005). RBO

also contains high concentration of tocopherols compared with other seed oils (Kao and Luh, 2010). Previous

studies have also shown that the rice grains with dark red or purple color contain high amount of anthocynanins,

a group of natural pigments found in deep purple or reddish fruits and vegetables (Yawadio et al., 2013). On the other

hand, rice grains with red color consist high amount of polyphenols, phenolics and flavonoids (Gunaratne et al., 2013;

Sompong et al., 2011). Sangyod rice is a traditional rice variety grown in Phatthalung province for more than a hundred years.

Sangyod Muang Phatthalung rice is soft with approximately 14-15% of the amylose content. The analysis of its nutrient

volume has been shown that Sangyod rice has high nutritional value, iron, vitamin B and niacin (Department of Health, 2004).

For Tubtimchumphae rice, it is a new Thai rice varieties which is a hybrid between Jasmin rice and Sangyod rice.

It contains high amount of phenolic, flavonoid, Vitamin E (α-tocopherol and γ-oryzanol) (Rice Department, 2016).

The objective of this research work was to study the chemical composition and antioxidant properties of RBOs obtained

from the two rice varieties, in order to provide information for their uses in food and supplementary products.


Materials

Sangyod rice bran oil (SRBO) was obtained from Phattalung Province’s Community Enterprise and

Tubtimchumpae rice bran oil (TRBO) from Kamphaengphet Province’s Community Enterprise.

Physical parameters

The color of RBOs was measured by the CIE L*, a*, b* with standard illuminant D65 and standard observer 10°

by Spectrophotometer (CM-3500d, Minolta, Japan)

mailto:[email protected]*

https://www.sciencedirect.com/topics/medicine-and-dentistry/oilseed


Chemical parameters

Acid value (AV), free fatty acid (FFA), Peroxide value (PV), iodine value (IV), saponification value (SV) and

unsaponifiable matter were determined according to the methods described in AOAC (2000).

Determination of fatty acid composition

The fatty acid compositions were determined by GC-FID according to the methods described by Kasetsart

Agricultural and Agro-Industrial Product Improvement Institute, Thailand. Capillary GC (Agilent 6890 Serie) equipped

with a capillary column (BPX 70 SGE; 70% Cyanopropyl Polysilphenylene-siloxane; 30 m×0.25 mm–0.25 μm

film) was used. The GC conditions operated at the initial temperature of 160 °c for 0.5 min, then increased to 160-200 °c

at 10°c /min and the final temperature of 200 °c for 4.6 min with a total run time of 9.1 min. The flow rate of gas (Nitrogen)

was 1 ml/min. injection volume of 1 μl were used. The fatty acid composition was obtained by comparison of the peak

retention times with the respective fatty acids standards.

Determination of total phenolic content

The total phenolic content (TPC) of the crude RBO extracts was determined using Folin–Ciocalteu’sreagent

method as described by Singleton et al., (1999) Oil extracts (300 µL) were mixed with 1.5 ml of Folin-Ciocalteu’s

reagent, left for 6 min and subsequently 1.25 ml of 7% sodium carbonate solution was added. The mixture was diluted

to 3 ml with deionized water. The absorbance of the resulting mixture was read at 725 nm using a UV visible

spectrophotometer (UV 1601, Shimadzu, Japan) after leaving for 30 min at the room temperature (25 °C). The measurement

was compared to a standard curve of gallic acid concentrations, which were expressed mg gallic acid equivalents/ g sample.

Determination of α-tocopherol

The α-tocopherol content in RBO was determined by Institute of Food Research and Product Development,

Thailand, using an in-house method based on BS EN 12823-1:2000

Determination of DPPH radical scavenging capacity

DPPH radical-scavenging effects were determined by using the method modified from that reported previously

by Brand-Williams et al., (1995). A solution of DPPH was prepared in methanol, and the solution of RBO was dissolved

in DMSO to obtain the concentration between 5 and 30 µg/mL. Then, RBO and 1 ml of 0.1 mM DPPH solution

were mixed in test tube. The mixture was shaken and incubated for 30 min in the dark at room temperature. The absorbance

was determined at 517 nm by UV-VIS spectrophotometer (Shimadzu 160A, Japan). The radical scavenging activity

was expressed as IC50 value that denotes the concentration of the sample required to scavenge 50% of DPPH radicals.

Determination of ABTS radical scavenging capacity

ABTS assay was determined according to the method described by Re et al., (1999) ABTS radical was generated

by reacting 7 mM ABTS and 2.45 mM potassium persulfate at room temperature in dark for 16 h. The ABTS solution

was diluted with 80 % ethanol to an absorbance around 0.700 at 734 nm. 2 ml of ABTS solution was added to 0.1 ml

of the extracts and mixed thoroughly. The reaction mixture was kept at room temperature for 6 min, absorbance

was measured at 734 nm by UV-VIS spectrophotometer (Shimadzu 160A, Japan).

Result & Discussion

Physical parameters: The CIE Lab color measurement method was determined by its color coordinates: L* represent

the difference between light (L*= 100) and dark (L* = 0), a* represent the difference between green (–a*) and red

(+a*) and b* represent the difference between blue (-b*) and yellow (+b*). It was found that the color of SRBO

(L* = 56.48, a* = 15.60 and b* = 93.43) was darker, more green and yellow than the color of TRBO (L* = 43.64,

a* = 13.97 and b* = 74.11). Therefore, the color of two varieties rice bran oil were significantly different (p≤0.05).

based on the mentioned physical parameters.

Chemical parameters: The AV and FFA values in the samples of oil or fat were determined after the hydrolysis

of triglycerides by the enzyme lipase in which its activity was increased rapidly after the milling process. In terms

of lipid quality, the value of PV content is used as a parameter, and the values of IV is used to indicate the amount

of unsaturation in RBO. The results showed that the values of AV, FFA, PV and IV of SRBO were 13.36 mg KOH/g,

6.71％, 1.78 mg eq/kg oil and 96.86 g Iodine/100 g oil, respectively. While the values of TRBO were 7.12 mg

KOH/g oil, 3.58 ％, 1.91 mg eq/kg oil and 100.74 g Iodine/100 g oil, respectively. CODEX standard (1999) has

suggested that the maximum levels of AV for Crude rice bran oil is 4 mg KOH/g, the maximum level of PV is 10

mg eq/kg oil, and the recommended IV value is between 90–105 g Iodine/100 g oil. According to Tao et al.(1993),

the level of FFA value for RBO should be not over 5%. Our results showed that the RBO samples had the AV

and FFA contents higher than the recommendations. This could be affected by the raw material quality, as the

bran did not stabilize before being used. The bran quality deterioration may be due to the presence lipase activity.

For the PV and IV contents, the values of both rice bran oils were lower than those of the CODEX standard.

Saponification value is the reaction that free hydroxide breaks the ester bonds between the fatty acids and glycerol

of a triglyceride, resulting in free fatty acids and glycerol. Unsaponification value is the measure of non-lipids



constituents (sterols, pigments, and hydrocarbons) in the oil (Bodger et al. 1982). In this study, the saponification

value and unsaponifiable matter of SRBO were determined to be 196.48 mg KOH/g oil and 2.29%, and of TRBO

were 189.49 mg KOH/g oil and 3.49%, respectively. Statistical analysis showed that there were significant

differences (p≤0.05) in the saponification values and unsaponifiable matter of two varieties oils.

Total phenolic content, DPPH and ABTS radical scavenging capacity: The result of total phenolic content

(TPC) was expressed as gallic acid equivalent, showing that the phenolic compounds in RBO contributes to their

antioxidant properties as summarized in Table 2. The total phenolic contents of SRBO (1.48 mg GAE/100g) and

TRBO (1.54 mg GAE/100g) were not significantly different (p>0.05). For DPPH and ABTS radical scavenging

activities of the RBO extracts, their values were expressed as IC50 which is inversely proportional to the antioxidant

activity. Our results showed that the DPPH and ABTS radical scavenging activities of the two varieties RBO extracts

were not significantly different (p>0.05). On the other hand, the α-tocopherol contents were found to be higher in TRBO

(9.13 mg/100g oil) than in SRBO (11.88 mg/100g oil), which were significantly different in both varieties (p ≤ 0.05).

It is well known that tocopherols possesses high antioxidant properties and the greatest vitamin E potency (Chen

and Bergman, 2015). Therefore, DPPH, ABTS radical scavenging activity and Total phenolic content of TRBO

were found to be higher than those values in SRBO.

Fatty Acid Composition: The fatty acid compositions of SRBO and TRBO are showed in Table 3.

There were eight fatty acids identified including oleic acid (C18:1), linoleic acid (C18:2), palmitic acid (C16:0),

stearic acid (C18:0), linolenic acid (C18:3), arachidonic acid (C20:0), myristic acid (C14:0) and palmitoleic

(C16:1). The saturated fatty acids were found in both rice bran oils such as myristic, palmitic, stearic and arachidonic

acids with the concentrations of 0.34–0.45%, 15.80–16.35%, 2.23–2.40% and 0.97-0.99%, respectively. The unsaturated

fatty acids were found to be palmitoleic, oleic, linoleic and linolenic acids with the concentration of 0–0.14%,

41.74–42.04%, 36.22–36.89% and 0.97-0.99%, respectively. The main components of the fatty acids were oleic

acid, linoleic acid and palmitic acid. However, the fatty acid contents of SRBO and TRBO were not significantly

different (p ≥ 0.05). According to Mingyail. et al., (2017) the major fatty acids in the RBO samples are oleic acid,

linoleic acid and palmitic acid. The content of the unsaturated fatty acids was apparently higher than the saturated

fatty acids.

Conclusion

The results of this study showed that the fatty acid profiles of SRBO and TRBO contain a high percentage

of the unsaturated fatty acids (approximately 80%), with the main components of oleic acid and linoleic acid. The results

also suggested that, the two rice bran oils can be used as a raw material in the production of functional foods. They

provided different physicochemical and antioxidant properties, which are potentially beneficial for the nutrition and

health of human.

Acknowledgement

This wor was supported by Faculty of Agro-Industry, Kasetsart University, Kasetsart Agricultural and Agro

Industrial Product Improvement Institute (KAPI) and Kasetsart University Research and Development Institute (KURDI).

Referenes

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USA: AOAC International.

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Shanks, P., and Southwell, K. 1982. An investigation of the extraction, refining and composition of oil

from Winged bean (Psophocarpus tetragonolobus D.C.). J Am Oil Chem Soc. 59:523–530.

Brand-Williams, W., Cuvelier, M.E., and Berset, C. 1995. Use of a Free Radical Method to Evaluate Antioxidant

Activity. Lebensmittel Wissenschaft und Technologie, 28: 25–30.

Chen, M.H., and Bergman, C.J. 2005. A rapid procedure for analysing rice bran tocopherol, tocotrienol and

c-orizanol contents. J Food Anal 18:139–151.

CODEX STANDARD. 1999. CODEX standard for named vegetable oils. Department of Health. 2004. Nutrition

analysis of Sanyod Phatthalung rice. Department of Health, Ministry of Public Health, Thailand.

Gunaratne, A., Wu, K., Li, D., Bentota, A., Corke, H., and Cai, Y.Z. 2013. Antioxidant activity and nutritional

quality of traditional red-grained rice varieties containing proanthocyanidins. Food Chem 138: 1153-1161.

Hegsted, M., Windhauser, M., Lester, S., and Morris, S. 1990. Stabilized rice bran and germ product on hypercholersterolemia.

Food Chem. News 32(37).

Hegsted, M., and Windhauser, M. M. 1993. Reducing human heart disease risk with rice bran. La. Agric. 36(3):22-24.

Institute of food research and product development: Kasetsart University. Thailand: IFRPD [Online] Available

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444922/#CR5

http://ifrpd.ku.ac.th/en/about/index.php


Kahlon, T., Saunders, R., Sayre, R., Chow, F., Chin, M., and Betschart, A. 1992. Cholesterol-lowering effects

of rice bran and rice bran oil fraction in hypercholesterolemic hamsters. Cereal Chem. 69:485-489.

Kao, C., and Luh, B. S. 1991. in Rice Utilization. Vol. 2. B. S. Luh, ed. Avi: New York. Pages 295-311.

Qureshi, A. A., Qureshi, N., and Wright, J. J. K. 1991. Lowering of serum cholesterol in hypercholesterolemic

humans by tocotrienols (palmvitee). Am. Clin. Nutr. 53:s1021-s1026.

Mingyai1, S., Kettawan, A., Srikaeo, K., and Singanusong, Riantong. 2017. Physicochemical and Antioxidant

Properties of Rice Bran Oils Produced from Colored Rice Using Different Extraction Methods. J Oleo

Sci. 66, (6) 565-572.

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Activity Applying An Improved ABTS Radical Cation Decolorization Assay. Free Radical Biology and Medicine

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Thailand. Shahid, I., Bhanger, M.I., and Farooq, A. 2005. Antioxidant properties and components of

some commercially available varieties of rice bran in Pakistan. Food Chem 93: 265-272.

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Table 1. Physical-Chemical properties of SRBO and TRBO.

*mean significantly different (p ≤ 0.05)

Table 2. Total phenolic content, α-tocopherol, DPPH and ABTS radical scavenging of SRBO and TRBO.

*mean significantly different (p ≤ 0.05), ns mean significantly different (p > 0.05)

Table 3. The fatty acid compositions of Sangyod rice bran oil and Tubtimchumpae rice bran oil.

ns mean significantly different (p > 0.05)

ND : Not detected.

Properties SRBO TRBO

L* 56.48±0.15 43.64±0.14

a* 15.60±0.03 13.97±0.05

b* 93.43±0.29 74.11±0.27

Acid value* 13.36±0.32 7.12±0.40

Free fatty acid (as % oleic)* 6.71±0.16 3.58±0.20

Peroxide value (PV)* 1.48±0.24 1.91±0.10

Iodine value (IV)* 96.86±1.64 100.74±1.05

Saponification value* 196.48±3.19 189.49±2.23

Unsaponifiable matter* 2.29±0.85 3.49±0.29

Properties SRBO TRBO

Total phenolic content (mg GAE/100 g)ns 1.48±0.05 1.54±0.03

DPPH assay (IC50 , µg/ml)ns 13.97±1.07 13.90±1.64

ABTS assay (IC50 , µg/ml)ns 15.99±2.19 16.09±1.98

α-Tocopherol (mg/100 g)* 9.13±1.52 11.88±1.71

Fatty Acid Composition (%) SRBO TRBO

myristic acid (C14:0)ns 0.34±0.03 0.45±0.03

palmitic acid (C16:0) ns 16.35±0.12 15.80±0.09

palmitoleic (C16:1) ns 0.14±0.12 ND

stearic acid (C18:0) ns 2.23±0.06 2.40±0.02

oleic acid (C18:1) ns 42.04±0.24 41.74±0.09

linoleic acid (C18:2) ns 36.22±0.29 36.89±0.08

linolenic acid (C18: 3) ns 1.70±0.08 1.72±0.01

arachidonic acid (C20: 0) ns 0.97±0.05 0.99±0.04

Proceeding of International Conference on Biodiversity: IBD2019 on 22nd - 24th May 2019 at Centara Grand & Bangkok Convention Centre at CentralWorld, Bangkok, Thailand 94


Checklist of bee genus Megachile Latreille, 1802 in Thailand (Hymenoptera: Megachilidae)

Nontawat Chatthanabun and Natapot Warrit*

Department of Biology & Center of Excellence in Entomology, Faculty of Science, Chulalongkorn University,

Pathum Wan District, Bangkok, Thailand


Abstract: A checklist of Thai megachilid bees is reported for the first time. Species names listed were recorded

from specimens collected throughout Thailand and deposited at the Chulalongkorn University Natural History

Museum, Bangkok and through literature review. A total of 30 Thai Megachile species are listed with locality

information and notes on floral records, diagnostic characters, and nesting habitats (when appropriate). Due to

their abundance, we suggest that Megachile (Amegachile) bicolor, M. (Callomegachile) disjuncta, M. (Creightonella) fraterna,

and M. (Pseudomegachile) velutina are good candidates for being future crop pollinators.

Keywords: Diversity, Leafcutter bee, Pollination, Pollinator, Resin bee.

Introduction

Megachile Latreille, 1802 (Hymenoptera; Megachilidae) is one of the most diverse bee genera that include

leafcutter, resin, and mason bee. Megachile has a wide distribution and diverse habitats in most regions of the world

(Michener, 2007; Gonzalez et al., 2012). Presently, over 1,500 species are already named and described (Michener, 2007;

Ascher & Pickering, 2019). These bees include many important pollinators for natural ecosystems and agricultural

areas, e.g. Megachile rotundata, a well-known alfalfa crop pollinator (Delaplane & Mayer, 2000; Michener, 2007).

Historically, Southeast Asian megachilid bees are poorly known, underrepresented in collections, and inadequately

explored (Engel & Gonzalez, 2011; Gonzalez & Engel, 2012). Therefore, megachilid bees in Thailand are not well

recognized and protected from various anthropogenic threats such as habitat destruction and insecticide usages in

agriculture areas. This work attempts to contribute to the basic knowledge of Thai megachilid bee fauna. The checklist

comprises number of species and valid names, their distributions in Thailand, and notes on some species.


588 specimens (368♀, 220♂) collected throughout Thailand and deposited at the Chulalongkorn University

Natural History Museum (CUNHM) since 1962 were examined (see in Table 1). In addition, literature on Thai Megachile

taxonomic treatments were reviewed following Tadauchi and Tasen (2009), Engel and Gonzalez (2011), Gonzalez

& Engel, 2012, Ascher et al., (2016) and the Discoverlife Bee Species Guide and World Checklist (Ascher and

Pickering, 2019).

Result & Discussion

This checklist recorded 30 species of Megachile in Thailand through specimens examination and literature

review. For generic and subgeneric classification we follow Michener (2007) and Trunz et al. (2016), whereas for species

level identification, the specimens were verified with their original descriptions. In addition, notes on floral records,

diagnostic character, and nesting habitats are provided below. Species and collecting localities names are alphabetically

ordered.

Family: Megachilidae

Genus: Megachile Latreille, 1802

Subgenus: Aethomegachile Engel and Baker, 2006

Megachile (Aethomegachile) conjuncta Smith, 1853

Distribution: Chiang Mai.

Note: There is a sexual dimorphism between male and female bees.

Megachile (Aethomegachile) laticeps Smith, 1853

Distribution: Bangkok, Surat Thani.

Note: Superficially resembling to M. (Callomegachile) umbripennis but it can be easily

distinguished by mandible with an incomplete cutting edge in the second interspace and

hidden cutting edge in the third interspace.

Megachile (Aethomegachile) siamensis Cockerell, 1927

Distribution: Chiang Mai, Mae Hong Son.

Note: Only male was described.

Megachile (Aethomegachile) trichorhytisma Engel, 2006



Distribution: Chiang Rai, Loei

Note: Male can be easily recognized by the modification of its last flagellomere.

Subgenus: Amegachile Friese, 1909

Megachile (Amegachile) bicolor (Fabricius, 1781)

Distribution: Chiang Mai, Kanchanaburi, Mukdahan, Nakhon Nayok, Nakhon Pathom,

Nong Khai, Phayao, Ratchaburi, Saraburi, Tak.

Note: A widespread species in Oriental region, particularly India and southern China. This

species is commonly found in sunhemp field (Crotalaria juncea L.) and its nesting biology

was reported back from 1955 by Von Rudolf Altevogt.

Subgenus: Callomegachile Michener, 1962

Megachile (Callomegachile) atratiformis (Meade-Waldo, 1914)

Distribution: Uthai Thani.

Note: Engel and Gonzalez (2011) reported the species was collected on Dipterocarpus

obtusifolius Teijsman & Miquel.

Megachile (Callomegachile) disjuncta (Fabricius, 1781)

Distribution: Bangkok, Chiang Mai, Phayao, Surat Thani, Yala.

Note: This species forages on numerous types of plants, especially in Fabaceae. In addition,

Soh (2014) was first to report its nest construction using resins.

Megachile (Callomegachile) faceta (Bingham, 1897)

Distribution: No specific location provided.

Megachile (Callomegachile) facetula (Cockerell, 1918)


Note: This species is superficially similar to M. (Callomegachile) faceta though can be

distinguished by the presence of dense fulvous hairs on the vertex of the former species.

Megachile (Callomegachile) fulvipennis (Smith, 1879)

Distribution: Surat Thani.

Note: Soh (2014) reported the species using resin for nest construction and visited

Peltophorum pterocarpum (Fabaceae) and Memecylon sp. (Melastomataceae).

Megachile (Callomegachile) impressa (Friese, 1903)


Note: Only male was described.

Megachile (Callomegachile) monticola (Smith, 1858)

Distribution: No specific location provided

Note: This species has a wide distribution in south China.

Megachile (Callomegachile) odontophora Engel, 2011

Distribution: Nakhon Ratchasima

Note: Engel and Gonzalez (2011) reported the species was collected on Sindora siamensis

Teijsman & Miquel (Fabaceae).

Megachile (Callomegachile) ornata (Smith, 1853)

Distribution: Chiang Mai

Note: Tadauchi and Tasen (2009) identified only one specimen from a teak plantation.

Megachile (Callomegachile) tuberculata (Smith, 1858)

Distribution: No specific location provided

Note: Ascher et al. (2016) reported the species was collected on Grammatophyllum

speciosum Blume (Orchidaceae).

Megachile (Callomegachile) umbripennis (Smith, 1853)

Distribution: Bangkok, Phayao.

Note: This species has a wide distribution range in Southeast Asia and was reported to be

adventive in the US.

Subgenus: Carinula Michener, McGinley, and Danforth, 1944

Megachile (Carinula) fruhstorferi Friese, 1903

Distribution: Saraburi.

Note: This species is superficially similar to M. (Callomegachile) stulta though can be easily

distinguished by the presence of yellow felted hairs on the metasomal terga.

Subgenus: Chelostomoda Michener, 1962

Megachile (Chelostomoda) aureocincta Cockerell, 1927

Distribution: Nakhon Si Thammarat.

Megachile (Chelostomoda) nematocera Cockerell, 1929



Subgenus: Creightonella Cockerell, 1908

Megachile (Creightonella) fraterna Smith, 1853

Distribution: Ayutthaya, Chiang Mai, Kanchanaburi, Petchabun, Phayao, Ratchaburi,

Saraburi, Thonburi.

Note: This species usually forages on sunhemp flower (Crotalaria juncea L.) and constructs

its nest underground in the sun hemp field.

Megachile (Creightonella) atrata Smith, 1853

Distribution: Chumphon, Surat Thani, Yala.

Note: This species only occur in south Thailand. The bee forages on sunhemp flower

(Crotalaria juncea L.) and constructs its nests underground in the sun hemp field.

Subgenus: Eutricharaea Thomson, 1872

Megachile (Eutricharaea) gathela Cameron, 1908


Megachile (Eutricharaea) griseopicta Radoszkowski, 1882


Note: This species is superficially similar to M. (Callomegachile) faceta though can be

easily distinguished by the presence of the hidden cutting in the third interspace of the

mandible.

Megachile (Eutricharaea) hera Bingham, 1897

Distribution: Kamphaeng Phet, Mae Hong Son, Nakhon Pathom, Petchabun, Phetchaburi,

Phang Nga, Phayao, Sakon Nakhon, Samut Sakorn, Ubon Ratchathani, Udon Thani.

Note: This species has a wide distribution and commonly found in Thailand.

Subgenus: Lophanthedon Gonzalez and Engel, 2011

Megachile (Lophanthedon) amputata Smith, 1857


Note: This species is superficially similar to M. (Aethomegachile) laticeps though can be

easily distinguished by the presence of the outer ridge on the mandible.

Megachile (Lophanthedon) dimidiata Smith, 1853

Distribution: Lumpang.

Note: This species is superficially similar to M. (Pseudomegachile) velutina though can be

easily distinguished by the presence of the outer ridge on the mandible and absence of

dense long hairs on the inner adductor interspace of the mandible.

Megachile (Lophanthedon) erythropoda Cameron, 1901

Distribution: Songkhla.

Megachile (Lophanthedon) ferruginea Friese, 1903

Distribution: Nong Bua Lam Phu.

Note: This species is superficially similar to M. (Lophanthedon) dimidiata though can be

easily distinguished by the presence of fulvous hairs on the apical margin of the metasomal

terga.

Subgenus: Pseudomegachile Friese, 1898

Megachile (Pseudomegachile) lanata (Fabricius, 1775)


Note: A common species found in India.

Megachile (Pseudomegachile) velutina Smith, 1853

Distribution: Lumpang, Nakhon Si Thammarat, Pattani.

Note: Commonly found in sunhemp field (Crotalaria juncea L.).

This report listed 30 Megachile species recorded in Thailand, which consist of 18 species of mason and

resin bees from 4 subgenera: Callomegachile, Carinula, Lophanthedon, and Pseudomegachile. Leafcutter bees

are represented for 12 species from 5 subgenera: Aethomegachile, Amegachile, Chelostomoda, Creightonella, and

Eutricharaea. Callomegachile has the highest number of species for Thai megachilid bees, whereas others are

infrequently reported. Despite Megachile wide distribution in Thailand, historical records are sporadic and rare.

Here, we documented 27 provinces in Thailand for the occurrence of Megachile. Of these 8 provinces are new

records for Thailand: Ayutthaya, Chumphon, Kanchanaburi, Mukdahan, Nakhon Pathom, Nong Khai, Petchabun,

and Thonburi.

Remarkably, three of the most abundance in agricultural areas, the leafcutter bees Megachile (Amegachile)

bicolor (Figure 1a), Megachile (Creightonella) fraterna (Figure 2a) and mason bee Megachile (Pseudomegachile)

velutina (Figure 2e), live and nest in sunhemp field. According to above, we suggest that these bees are potential

candidates for future pollination study. Another candidate is Megachile (Callomegachile) disjuncta (Figure 1e),

usually foraging on fabaceae and easy adoption human artifacts.



Two close species of Thai Megachile, which have fulvous hairs on prosoma and mesosoma with the yellow

wings: the female of Megachile (Lophanthedon) dimidiata and Megachile (Pseudomegachile) velutina. These bees

have similar appearance, but differ in outer ridge on mandible, which are reaching to mandible base and reaching

only three-fourth mandible respectively. Female of Megachile (Aethomegachile) conjuncta and Megachile

(Callomegachile) disjuncta, which are black with white hairs on propodeum and first metasomal tergite, closely

resemble and they are sympatric species, but differ in scopal color, orange-red and black respectively. The male

of Megachile (Aethomegachile) conjuncta and Megachile (Aethomegachile) laticeps, black covered with fulvous

hairs, are very similar but differ in apical of gonostylus. Anonther interesting case is the Müllerian mimicry that

occurs to Megachile (Callomegachile) fulvipennis and Megachile (Callomegachile) tuberculata (Ascher et al., 2016).

Of the 5 species of Thai Megachile, include, Megachile (Aethomegachile) siamensis, Megachile (Aethomegachile)

trichorhytisma, Megachile (Callomegachile) monticola, Megachile (Callomegachile) tuberculata and Megachile

(Lophanthedon) dimidiate, are collected since past decades and there is no any recent appearance. For this reason,

we suggest that these bees may be extinct from Thailand. Nevertheless, these list may be not completely verifiable

because unclear distribution pattern, poor records and observations are difficult to determine and remain questionable.

In addition, anthropogenic activities such as deforestation, expansion of urban areas, and irresponsible tourism

are cause of habitat destruction and reduction of pollinators (Kjøhl et al., 2011).

Conclusion

30 species of Thai Megachile are recorded in 27 provinces. Here, we note some informations of Thai Megachile

such as diagnostic characters, food plants or nesting biology. Due to their high abundance and commonly found

in crop fields in Thailand, we suggest that M. (Amegachile) bicolor, M. (Callomegachile) disjuncta, M. (Creightonella)

fraterna, and M. (Pseudomegachile) velutina may be potential candidates for future pollination studies in Thailand

for food crops. In Addition, threats to the decline of Megachile species in Thailand should be of great concern since

they can be affected through anthropogenic activities.

Acknowledgement

The authors would like to thank the following institutions and persons as follow: Chawakorn Kunsete,

Pakorn Nalinrachatakan, Patsavee Utaipanon, Puttita Pasukdee, Sirat Lertjintanakit and Varat Sivayyapram, CU

Bee and Spider Research Lab, Chulalongkorn University, Thailand. We would like to express their gratitude towards

to The Scholarship from the Graduate School, Chulalongkorn University to commemorate the 72nd anniversary of

his Majesty King Bhumibol Adulyadej is gratefully acknowledged and The 90th Anniversary Chulalongkorn University

Fund (Ratchadaphiseksomphot Endowment Fund) (GCUGR1125604070M).

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Figure 1. Pictures of candidates for future pollination studies (a–d) Megachile (Amegachile) bicolor (a) dorsal

view (b) lateral view (c) frontal view of mandible (d) lateral view of scopa (e–h) Megachile (Callomegachile)

disjuncta (e) dorsal view (f) lateral view (g) frontal view of mandible (h) lateral view of scopa.


Figure 2. Pictures of candidates for future pollination studies (a–d) Megachile (Creightonella) fraterna (a) dorsal

view (b) lateral view (c) frontal view of mandible (d) lateral view of scopa (e–h) Megachile (Pseudomegachile)

velutina (e) dorsal view (f) lateral view (g) frontal view of mandible (h) lateral view of scopa.



Table 1. Examined specimens.

Subgenus sex

locality female male

Aethomegachile 55 43 Bangkok, Chiang Mai, Chiang Rai, Loei, Mae Hong Son,

Surat Thani

Amegachile 72 20 Chiang Mai, Kanchanaburi, Mukdahan, Nakhon Nayok,

Nakhon Pathom, Nong Khai, Phayao, Ratchaburi,

Saraburi, Tak

Callomegachile 120 62 Bangkok, Chiang Mai, Nakhon Ratchasima, Phayao,

Surat Thani, Uthai Thani, Yala

Creightonella 56 18 Ayutthaya, Chiang Mai, Chumphon Kanchanaburi,

Petchabun, Phayao, Ratchaburi, Saraburi, Thonburi.

Eutricharaea 24 70 Chiang Mai, Kamphaeng Phet, Mae Hong Son, Nakhon

Pathom, Petchabun, Phetchaburi, Phang Nga, Phayao,

Sakon Nakhon, Samut Sakorn, Ubon Ratchathani, Udon

Thani.

Lophanthedon 1 - Lumpang

Pseudomegachile 40 7 Lumpang, Nakhon Si Thammarat, Pattani



Diversity of Caterpillars (Order Lepidoptera) in KhaoYai National Park,

Nakhon Ratchasima Province

Paradorn Dokchan1,2*, Nanthasak Pinkaew1, Sunisa Sanguansub1 and Sravut Klorvuttimontara3 1Department of Entomology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University

KamphaengSaen Campus, Kamphaeng Saen Dictrict, Nakhon Pathom, Thailand 2Environmental Entomology Research and Development Centre, Faculty of Agriculture at KamphaengSaen,

Kasetsart University KamphaengSaen Campus, KamphaengSaen District, Nakhon Pathom, Thailand 3Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng Saen Campus, Kamphaeng Saen

District, Nakhon Pathom, Thailand *Corresponding author e-mail:[email protected]

Abstract: The study of caterpillars diversity was started by sampled caterpillars from 500 meters line transect every

100 meters above mean sea level from 100 meters above mean sea level thru 1,200 meters above sea level in

KhaoYai National Park. Caterpillars were sampled every month from January 2017 – June 2017. A total of 3,434 specimens

were identified to 86 species, 55 genera, and 19 families and 37 morphospecies. The most abundant species was

Euremablanda (n=1,280). The highest diversity was found in 500 meters above mean sea level (H'= 2.66) and the similarity

of caterpillar that occurred in different elevation was low. Keywords: caterpillars, elevation, diversity, KhaoYai National Park.

Introduction

Khao Yai National Park is a Thailand's first national park, it is the third largest national park in Thailand.

Situated mainly in Nakhon Ratchasima Province. Khao Yai is part of Dong Phayayen-Khao Yai Forest Complex,

a world heritage site declared by UNESCO. In at least five different forest type, Khao Yai National Park has complex

ecosystem with richness of plant and animal such as mammal bird reptile and insects. Butterflies and Moths are

familiar insects with people. There are recorded of 214 butterflies species in Dong Phayayen-Khao Yai Forest Complex

include Khao Yai National Park (Lapkratok and Suwanwaree, 2010). Considering their abundance, moths and

butterflies caterpillars are not well known. It’s just a few records of caterpillars and foodplant in Khao Yai National Park.

The main objectives of the research were to determine the species of caterpillars and food plants in Khao Yai

National Park and to investigate the caterpillar diversity in different seasons and effect by biotic factor and abiotic factor.


Caterpillars collecting and rearing

The line transects walk modified from Pollard (1977). In different elevation, total 12 line transect are setting

in every 100 meters above mean sea level (=MMSL) from 100 – 1,200 MMSL. Every line transects are 500 meters length

and 5 meters width each both side along the transect. The transect walked is starting monthly from January – June 2017.

Caterpillars are sampled from every line transect by visual searching from ground surface up to 2 meters

on the host plant. The individual caterpillars are put in the plastic box. The caterpillar food plants were cut and put in

the plastic bag. The food plants were used for feed caterpillar and another purpose was for plant identified.

After field surveys, caterpillars will be moved to a bigger container. Leaves are changed and clean the container

every day along the development from larva to pupa. After pupation, the pupa are transferred into other plastic boxes

separately for rearing until adult emerge.

Specimens preservation and identification

Specimens are pinned and preserved on the setting board. After that, the setting boards were moved to a

hot air oven with 50 o C for two weeks. The specimens are moved to the insect boxes with code number and

insect’s information label. Then all of the specimens were identified by All specimens are identified by Cerny and

Pinratana (2009), Ek-Amnuay (2012), Holloway (1983; 1985; 1986; 1987; 1988a; 1988b; 1989; 1993; 1996; 1997;

1999; 2003; 2005), Inoue et al. (1997), Khanal and Smith (1997), Kononenko and Pinratana (2005), Pinratana

(1981; 1983; 1985),Pinratana and Eliot (1992; 1996), Robinson et al. (1994),Schintlmeister and Pinratana (2007),Smith

(1997), Solovyev (2008) Tan and Khoon (2012) and Zolotuhin and Pinratana (2005)



Caterpillar diversity calculation

Caterpillar diversities are calculated by diversity indexes such as Shannon index, Simpson index, evenness

index and Similarity index as follows:

where H' = Shannon Index

pi = The proportion abundance of the i species

i = number of i species individual

ln = Natural logarithm

Simpson’s index

Simpson’s index = 1- pi2

where pi = The proportion abundance of the i species

i = number of i species individual

Shannon species evenness index

J' = H'/Hmax

where Hmax = ln S

J' = H'/ ln S

H' = Shannon Index

ln = Natural logarithm

S = Number of species at each site

Result & Discussion

Total 3,434 individuals were collected and divide by 19 Family, 55 genera, 86 species and 37 morphospecies of

caterpillars. The most diverse species were Noctuidae and Pyralidae (15 species). In each elevation, The highest

number of individual was found in 1,000 MMSL (n=1,280) (figure 1). The most abundance was found in 500 MMSL

(figure 1.) and the highest diversity was found in 500 meters above mean sea level (H'= 2.66) (table 1.) consist

with McCoy (1990) studied on The distribution of insects include butterflies and moths along elevational gradients.

He reported that the highest species richness does frequently occur among insect in mid-elevation. In 800, 900

and 1,000 MMSL, we found that elevations were very high number of individual but their Shannon index values

were quite low (H' = 0.842, 1.135 and 0.460 respectively) and considered with Shannon species evenness index

values were low too (J' = 0.311, 0.367 and 0.237 respectively) (table 1). The Shannon index and Shannon species

evenness index values can reveal that some dominat species can be found. The highest individual of caterpillar

species was Eurema blanda (figure). A total 1,280 individuals were found and 788 individual were found in 1000 MMSL.

The similarity of caterpillar that occurred in different elevation represented by similarity index value.

The similarity measures between each elevation and another elevation were quite low (similarity index > 0.5),

some were absolutely different (similarity index = 0). It was because of the hostplant that occurred in each

elevation were different (table 2.)

Conclusion

Khao Yai National Park has a richness and high diversity of caterpillar. A total of 3,434 individuals were

identified into 19 Family, 55 genera, 86 species and 37 morphospecies. The most diverse species were Noctuidae

and Pyralidae. The dominant species was Eurema blanda. The highest species diversity was found in 500 MMSL.

The similarity measures between each elevation and another elevation is quite low, some were absolutely different.

However, the study period was quite short for higher analysis. A longer term sampling and monitoring are needed

to continue and observe changes in diversity. The physical and biological factor that be influencing to diversity

of caterpillars in different area are the futher study.

Acknowledgement

We thank all our friends who involve with this research, especially Miss Sopita Muadsub for caterpillar

image edited. We also would like to thank all of Khao Yai National Park rangers and officer for kindly supported.


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Figure 1. The number of individual caterpillar fluctuation in different elevation from January - June 2017

in Khao Yai National Park.

Figure 2. The number of species and caterpillar family fluctuation in different elevation from January - June

2017 in Khao Yai National Park.

Figure 3. Eurema blanda A) caterpillar B) Adult.

A B


Table 1. A diversity indexes value in different elevation from January-June 2017 in Khao Yai National Park.

Table 2. The similarity indexes value between different elevation from January-June 2017

in Khao Yai National Park.



APPENDIX

Appendix figure 1. The Buterfly caterpilars: F. Danaidae A (Tirumala septentrionis, F. Hesperiidae)

B (Bibasis sena) C (Burara gomata) D (Capitha purreea) E (Choaspes furcate) F (Hasora schoenherr)

G ( Notocrypta paralysos) H (Tagiades vajuna)

A

D

B

G

E

C

H

F


Appendix figure 2. The Buterfly caterpilars: F. Lycaenidae A (Arhopala abseus) B (Cheritra freja)

C (Flos sp.) D (Heliophorus epicles) E (Horaga sp.) F (Nacaduba sp.) G (Spindasis lohita)

H (Surendar quercetorum)

A

D

B

G

E

C

H

F

A B



Appendix figure 3. Butterfly caterpilars: F. Nymphalidae A (Athyma nefte) B (Charaxes bernardus)

C (Coelites nothis) D (Cyrestis thyodamas) E (Discophora sondaica) F (Faunis canens) G (Herona marathus)

H (Lebadea Martha)

D

G

E

C

H

F

A

D

B

C


Appendix figure 4. Butterfly caterpilars: F. Nymphalidae (cont.) A (Moduza procris) B (Neptis hylas)

C (Parasarpa dudu) D (Prothoe franck) E (Rohana parisatis) F (Tanaecia julii)

G (Tanaecia lepidea) H (Zeuxidia amethystus)

G

E

H

F

A

D

B

E

C

F



Appendix figure 5. Butterfly caterpilars: F. Papilionidae A (Graphium Agamemnon) B (Graphium antiphates)

C (Graphium doson) D (Graphium megarus) E (Lamproptera megas)

F (Losaria coon) G (Papilio Memnon) H (Papilio paris)

Appendix figure 6. Butterfly caterpilars: F. Pieridae A (Eurema hecabe) B (Eurema simulatrix)

G H

A B

A

D

B

C


Appendix figure 7. Moth caterpilars: F. Bombycidae A (Prismosticta tiretta F. Drepanidae) B (Drepana sp. F.

Gemetridae) C (Dyaphania militaris) D (Genusa simplex) E (Hyposidra sp.) F (Pingasa sp. F. Lasiocampidae)

G (Gastropacha sp.) H (Kunugia divaricate)

G

E

H

F

A

D

B

E

C

F



Appendix figure 8. Moth caterpilars: F. Lasiocampidae A (Kunugia sp.) B (Trabala vishnu) C (Trabala sp. F. Limacodidae)

D (Darna sp.) E (Parasa sinica) F (Setora sp. F. Lymantriidae) G (Calliteara horsfieldii) H (Orgyia postica)

Appendix figure 9. Moth caterpilars: F. Notodontodae A (Phalera grotei) B (Stauropus sp. F. Pyralidae)

C (Parotis marginata) D (Pygospila tyres F. Saturniidae) E (Antheraea assamensis) F (Attacus atlas)

G H

A

D

B

C

E F


Appendix figure 10. Moth caterpilars: F. Saturniidae A (Actias maenas) B (Cricula trifenestrata F. Sphingidae)

C (Acherontia Lachesis) D (Acosmeryx anceus) E (Cephonodes hylas) F (Daphnis hypothous) G (Meganoton

nyctiphanes) H (Neogurelca himachala)

A

D

B

G

E

C

H

F

A B



Appendix figure 11. Moth caterpilars: F. Sphingidae (cont.) A (Pergesa acteus) B (Theretra clotho)

C (Theretra oldenlandiae F. Thyrididae) D (Calindoea atripunctalis) E (Glanycus tricolor F. Zygaenidae)

F (Cyclosia panthona) G (Cyclosia papilionaria) H (Prosopandrophila distincta)

D

G

E

C

H

F



Proposal to reduce anthocyanin-deficient banana Musa siamensis to a M. rubra variety

Wandee Inta, Panida Kongsawadworakul, Unchera Viboonjun, Paweena Chuenwarin,

Paweena Traiperm and Sasivimon Chomchalow Swangpol*

Department of Plant Science, Faculty of Science, Mahidol University, Ratchathewi District, Bangkok, Thailand


Abstract: Musa rubra Wall. ex Kurz and M. siamensis Häkkinen & Rich. H. Wallace are small wild banana species

with erect inflorescences of orange and yellow bracts, respectively. While M. rubra is commonly found in North-East

India, Myanmar to Western Thailand, M. siamensis, described from a specimen collected in Eastern Thailand

bordering Cambodia, is known only in cultivation. Striking characters shared by both M. rubra and M. siamensis

include extended rhizome, shape and texture of inflorescence bracts, morphological characters of flowers and

smooth-surface seeds. Bract coloration caused by changes in cellular anthocyanin contents and revealed by expression

levels of genes involved e.g. dihydroflavonol 4-reductase (DFR) is the lone recognized difference between the two

taxa. It is, therefore, proposed here to reduce this yellow-bract taxon to M. rubra var. siamensis. These and other

bananas with a wide range of colorful bracts are useful as proper models for genetics, biochemistry, cytology and

biotechnology in flavonoid biosynthesis pathway. Keywords: Chek Meas banana, Leucoanthocyanidin, Musa laterita Cheesman, Rhodochlamys, Thai Gold banana

Introduction

Ornamental banana species possess attractive bract colors such as orange, pink, bright red, purple, and also,

though less frequently found, yellow and green. Among these colorful taxa, we have suspected that two species

in the Rhodoclamys sections, Musa rubra Wall. ex Kurz with orange bracts and M. siamensis Häkkinen & Rich.

H. Wallace with yellow ones, are conspecific.

The Musa genus has been classified into sections by Cheesman in 1947. Two out of his four sections,

Musa and Rhodoclamys, both having basic chromosome number x=11, are proved later by the genetic evidences

to be very closely allied (Cheesman 1947, Wong et al. 2002). They possess less glaucous, rarely or never polished,

non- or less overlapping inflorescence bracts, often dorsiventrally compressed, sometimes subglobose to irregular

angulate seeds, though the two sections are distinguished by different rachis directions; Musa is with hanging to

horizontal inflorescences and Rhodoclamys with erect ones.

Musa rubra was first discovered by Wallich in Burma (Myanmar) and grown in Calcutta Botanical Gardens

at least since as earlier as 1845 (Voigt, 1845; Hooker, 1895). It was later described and published in 1867 by Kurz

from specimens he collected by himself. Afterward, Cheesman (1949) separately described a banana grown from

seeds also collected from that country as M. laterita, however this name is recently declared as a synonym of M. rubra

by Joe et al (2016).

In 2007, Häkkinen and Wallace described M. siamensis from a specimen collected in 2002 in Eastern

Thailand. It has been called “Chek Meas”and used as ornamental plants by the Cambodians for a long time and

later commercialized by Thai nursery industry under the name “Thai Gold.” This species is closely related to M. rubra

by having similar itinerant rhizomes, smooth-surface seeds, and molecular and cytogenetic evidences (Häkkinen

and Wallace 2007; Čížková et al. 2015).

Color variation of banana male inflorescence bracts is of aesthetic value for ornamental purposes and

undoubtedly intended to attract pollinators. Among other factors, anthocyanin components and combinations are

stated as the cause of this diversity (Kitdamrongsont et al. 2008, Kongsawadworakul et al. 2016). Several anthocyanin

pigments, genes and biosynthetic pathways have been known to pay roles. Though it is rare for a single species

to contain genes encoding the entire spectrum of flower colors (Ahmed et al. 2014), bract colors of related Musaceae

species widely range from orange to pink and deep purple and could be useful in the study of anthocyanin biosynthesis.

Identification of anthocyanins by high performance liquid chromatography (HPLC) , mass spectrometry (MS) ,

and tandem mass spectrometry (MS/MS) revealed six major derivatives in Musaceae, i.e. delphinidin-3-rutinoside,

cyanidin-3-rutinoside, petunidin-3-rutinoside, pelargonidin-3-rutinoside, peonidin-3-rutinoside and malvidin-3-

rutinoside. It was found that bracts of M. rubra contain only non-methylated anthocyanin, i.e., delphinidin-3-rutinoside,

and cyanidin-3-rutinoside. On the other hand, in M. acuminata with yellow bracts and Ensete glaucum (Musaceae)

with green bracts, no anthocyanin could be detected. It was suspected that anthocyanin biosynthetic pathway in these

anthocyanin-deficient bananas may have been obstructed at leucoanthocyanidin- to-anthocyanidin step or prior

( Kitdamrongsan et al. 2008, Figure. 1) . One of the enzymes involving in this pathway is dihydroflavonol 4-



reductase (DFR) which catalyzes the production of flavan-3,4-diols ( leucoanthocyanidins) via the reduction of

colorless dihydroflavonols (Tian et al. 2017).

Apart from collected morphological data and investigation of floral morphological characters, we studied

expression levels of DFR from specimens of the two taxa with two different bract colors, one of which is suspected

to be a mutant of the other. The results of this study would clarify taxonomic statuses of these bananas and will

be beneficial in genetic resource selection for breedings and future studies on morphology, molecular biology and

phylogenetics. These bananas with colorful bracts would be proper models in research involving flavonoid

biosynthesis pathway and evolution of bract color variation in plants.


Two accessions of rare M. siamensis Häkkinen & Rich. H. Wallace from cultivations were investigated

along with three accessions of M. Rubra Wall. ex Kurz collected from two natural populations and a market.Three

accessions of M. Acuminate Colla subsp. siamea N.W. Simmonds and two accessions of M. ornata Roxb. were

also examined for comparison (Table 1). Morphological features were investigated and photographed in the fields.

Bracts were collected and stored in ice box and refrigerator before transferring to deep freezer and kept at -8 0 ⁰C

until used. Mature flowers from male inflorescences were preserved in 70% ethanol for morphological study.

Anthocyanin extraction and analysis

Frozen bract samples were ground to a fine powder with a mortar and pestle. The powdered bracts (0.2 g)

were extracted with 5 ml of methanol:HCL (99:1, v/v) at 4°C in darkness overnight. The extracts were centrifuged

at 10,000 g for 15 min at 4°C to precipitate the debris. The clear supernatant was transferred to a new tube, and the absorption

of the extracts at 530 and 657 nm was determined. Total anthocyanin content was calculated according to the

method of Mehrtens et al. (2005) using the following formula modification: QAnthocyanins = A530-0.25xA657,

where QAnthocyanins is the amount of anthocyanins and A530 and A657 are the absorption values at the indicated

wavelengths. The obtained data were the mean of two independent replicates.

Expression analysis by quantitative real-time PCR (qRT-PCR)

The qRT-PCR analysis was performed with the ABI-7500 real-time PCR machine (Applied Biosystem).

Twenty µl of PCR reactions containing 1 µl of 5-fold cDNA dilution, 0.4 µM of each primer, 0.2 mM dNTP mix,

2 mM MgCl2, 0.8 U of Platinum Taq DNA polymerase (Invitrogen) and 1000-fold dilution of SYBR green I (Sigma)

in 1X PCR buffer. The Ct (cycle threshold) data were determined using default threshold settings. The relative

expression was calculated as 2-∆Ct where ∆Ct = (Ct of candidate gene – Ct of internal control actin). The analyses

were performed in triplicate.

Result & Discussion

Musa acuminata is quite different from other species in this study. It has pendent rachis and fruits in two

rows and was previously grouped in the Musa section (Cheesman 1947) apart from the Rhodoclamys with erect type

of inflorescence and fruits in one row, which M. ornata, M. rubra and M. siamensis belong to. Besides inflorescence

bract colors, the latter three species are distinct in several aspects. Similar to most banana species in the Musa genus,

M. ornata has suckers and angular rough seeds. On the other hand, M. rubra and M. siamensis possess short

pseudostem of 1-1.5 m with extended rhizomes and subglobose seeds with smooth seed surface. These characters

are unique and rarely found among the members of the Musa genus. Though M. itinerans Cheesman also has long

travelling rhizome, its pseudostems are vigorous at 4-6 m tall. Moreover, with close investigation of floral morphology,

it was found that the flowers of M. rubra and M. siamensis having median inner tepal with acuminate apex and wing

apex as long as apex or nearly so (Figure 3C and D, arrow), are quite similar comparing to M. ornata which have

median inner tepal with attenuate apex and no wing apex present in neither M. ornata nor M. acuminata (Figure

3A and B). These distinct characters were compiled in key to species as stated below.

Key to Species based on Morphology

Herbs small or large; perennial, monocarpic, monoecious. Underground stem a rhizome or corm, commonly

suckering. Pseudostems clump, erect, formed by closely clasping leaf sheaths. Leaves spirally arranged, leaf blade oblong

to lanceolate. Inflorescence erect to pendulous. Bracts spirally arranged, usually with bright color. Flowers basal

male sterile, terminal female sterile; perianth in 2 whorls with 3 outer tepals and 2 inner ones united into a compound

tepal, 5-lobed; adaxial inner tepal free. Stamens 5; Pistil 1; ovary inferior, 2 rows of ovules in each loculus. Stigma 3-lobed

to clavate, or capitate. Fruit a fleshy berry. Seed surface smooth or rough.

1. Pseudostems less than 1.5 m high, inflorescence erect, fruits in one row.

2. Rhizomatous, male bract orange red or yellow, median inner tepal apex

acuminate, seed surface smooth…………………..………………………….. M. rubra, M. siamensis


2. Suckering, male bract pink, median inner tepal apex attenuate, seed surface

rough…………………….…………………………………………………... M. ornata

1. Pseudostems more than 1.5 m high, inflorescence pendent, fruits in two rows…… M. acuminata

Anthocyanin content The analysis revealed the correlation of total anthocyanin contents and color variation of male inflorescence

bracts. The total anthocyanin level was highest in the red purple bract and, in parallel, sample of the yellow bracts

yielded low anthocyanin contents (Figure 4A).

Expression analysis of DFR gene by qRT-PCR Preliminary real-time RT-PCR analysis showed that the anthocyanin biosynthetic gene, DFR, expressed

differently in the banana bracts of distinct colors, i.e. low or undetectable in M. siamensis (Figure 4B). This low

or undetectable level of DFR gene in M. siamensis is consistent with the absence of DFR gene expression in white

pomegranate (Zhao et al. , 2015) . This result suggested that DFR may be one of the main factors responsible for

anthocyanin accumulation in the bract of banana species.

Previous researches on molecular biology of wild bananas which included M. rubra and M. siamensis

stated that they are closely related. Čížková et al. (2015) reported that the two species share the same number of

45S rDNA and 5S rDNA loci and were grouped in the same cluster. Later, Christelová et al. (2017) constructed a

dendrogram based on the results of SSR analysis and revealed that M. ornata formed a distinct separate cluster

distantly positioned from the other Rhodochlamys entries which M. rubra and M. siamensis belong to. In addition,

information on distribution areas of the two species hints their position in the classification. The fact that M. siamensis

is rare and has never been found in natural habitats by the authors, while M. rubra distributes widely from North-

East India, Myanmar to Western Thailand indicates that the former could be an unusual mutant of the latter. From

these findings on morphology and molecular biology, we propose that M. rubra and M. siamensis are conspecific

and reduce taxonomic status of M. siamensis to M. rubra Wall. ex Kurz var. siamensis (Häkkinen & Rich. H. Wallace)

Swangpol & Inta.

Conclusion

Several characters including pseudostem heights, inflorescence positions, and number of fruit row, can

be used to distinguish M. acuminata subsp. siamea, M. ornata and M. rubra. On the other hand, M. rubra and M.

siamensis are quite similar and only difference is their inflorescence bract colors. The data obtained from this

study indicated the relation of this color modification and the expression of gene involved in anthocyanin

biosynthetic pathway. Further investigations are underway to study the expressions of the other structural genes

and its regulators including in different banana species. This information will ease the understanding of molecular

genetic background of the color differences in the banana bracts and further studies on co-evolutionary aspects

with pollinators.

Acknowledgement

The projects led by S.C.S. has been supported by Mahidol University and the Thailand Research Fund

(TRF) (RSA56_80033), Biodiversity-based Economy Development Office (Public Organization- BEDO) and the

Faculty of Science, Mahidol University, Bangkok, Thailand.

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Table 1. List of banana accessions in this study.

1 Cultivated clones purchased from a plant market. 2 Specimens grown as ornamental plants in public parks.

Figure 1. Simplified scheme of the flavonoid biosynthesis pathway modified from that of petunia (Quattrocchio, 2006).

Figure 2. Male inflorescence of Musa L. (Musaceae) showing different bract colors; (A) purple, M. acuminata

Colla subsp. siamea N. W. Simmonds, (B) pink, M. ornata Roxb., (C) orange, M. rubra Wall. ex. Kurz, and (D)

yellow, M. siamensis Häkkinen & Rich. H. Wallace. Photos not to scale.

No. Acc. No.

SS & JS Species Source/Origin

1 001 Musa acuminata Colla subsp. siamea N.W. Simmonds Phetchabun

2 247 Musa acuminata Colla subsp. siamea N.W. Simmonds Kanchanaburi

3 300 Musa acuminata Colla subsp. siamea N.W. Simmonds Nakhon Nayok

4 449 Musa ornata Roxb. Bangkok 1

5 542 Musa ornata Roxb. Chiang Mai 2

6 352 Musa rubra Wall. ex Kurz Mae Hong Son

7 420 Musa rubra Wall. ex Kurz Mae Hong Son

8 516 Musa rubra Wall. ex Kurz Bangkok 1

9 524 Musa siamensis Häkkinen & Rich.H.Wallace Bangkok 1

10 624 Musa siamensis Häkkinen & Rich.H.Wallace Bangkok 2



Figure 3. Drawing of floral parts showing, from left to right, a compound tepal, a free tepal, five stamens and a

pistil of each taxa (A) M. acuminata subsp. siamea, (B) M. ornata, (C) M. rubra, (D) M. siamensis. Arrow

indicated wing apex. Scale bar = 0.5 cm.

Figure 4. Total anthocyanin content in banana bract (A) and the relative expression pattern of DFR genes in bracts

of M. siamensis, M. rubra and M. acuminata (B). Notice that relative expression of DFR in M. siamensis is low

or undetectable. Relative expression profiles (means of the normalized expression) were obtained by quantitative

real-time PCR analyses. Bars are the standard errors from the means.

A B



Species richness and distribution of marine spider crabs (Majoidea) in Thailand

Kamonchanok Wongissarakul1*, Pattanee Jantrarotai2 and Puntip Wisespongpand3 1Thailand Natural History Museum, National Science Museum, Khlong Luang District, Pathum Thani, Thailand 2Department of Zoology, Faculty of Science, Kasetsart University, Chatuchak District, Bangkok, Thailand 3Department of Marine Science, Faculty of Fisheries, Kasetsart University, Chatuchak District, Bangkok, Thailand


Abstract: The spider crabs are classified into Superfamily Majoidea. Species richness of spider crab in Thailand

were studied by collected specimens from local fishing boat along the coast of the Gulf of Thailand. Some specimens

were also examined from museums and institutes collection during September 2013 to December 2018. A total of 811

specimens were identified into 4 families, 21 genera and 40 species. Family Epialtidae were found the highest number

of spider crab species, comprising of 23 species (57.5%). The species richness of spider crab from the Andaman

Sea was higher than from the Gulf of Thailand at 35 and 19 species, respectively. There were two new records of Thailand:

Tiarinia angusta and Tylocarcinus styx. Furthermore, the results from this study indicated that the richness of marine

spider crab species was diversified due to habitat variation in marine ecosystem. Keywords: Majoidea, Marine environment, Species diversity, Spider crab, Thailand.

Introduction

Among the brachyuran superfamilies, the spider crabs (superfamily Majoidea) are one of the most diverse

group. Recently, the number of extant species of spider crabs comprised of over 800 species around the world (Ng

et al., 2008; Davie et al., 2015). Spider crabs distribute in all oceans especially in the Indo-West Pacific region.

Spider crabs are also called decorator crabs or masking crabs because they use various materials from their environment

such as pieces of algae, sponges, small pebbles and bits of shell decorate themselves with hook-setae for camouflage

and food reserve (Guinot et al., 1995; Guinot & Wicksten, 2015). Some spider crabs is popular for the live-seafood

trade such as snow crabs or queen crabs (Chionoecetes opilio) and Atlantic spider crabs (Maja brachydactyla)

(Ng, 1998; Marques et al., 2010). Most species of spider crabs play an important role as omnivorous, scavenger, consumer

and prey in marine ecosystem (Warner, 1977).

In the past, the identification key of spider crabs was complicated and confused because there were more than

ten subfamilies in family Majidae (Rathbun, 1925; Garth, 1958; Griffin & Tranter, 1986; Stevcic, 1994). Moreover,

there are widely different in morphological characters within family and subfamilies. Therefore, the genus of spider

crabs has been frequently transferred among families or subfamilies. As a result, it is a great practical problem

when using the identification keys. Later for clarifying the taxonomy of spider crabs, they were classified based

on their larval and more adult characters such as eyestalk and basal antenna characters. Finally, spider crabs are

classified in superfamily Majoidea with six families (Ng et al., 2008).

The previous studies of marine crabs in Thailand mostly focus on the commercial crabs such as Grapsid

crabs (Mutchacheep, 1973) and Portunid crabs (Naiyanetr, 1997 and Viruhpintu, 1989). For the spider crabs, there

are a few studies on spider crab diversity in Thailand with a great confusion in classification and numbers of species

because they are very diversified and the classification status have changed for a many time. The latest spider crabs

classification in Thailand (Naiyanetr, 2007), has been reported in only one family (16 genera, 32 species) which

is an outdating data and uncertainly about the species name. Therefore, the purpose of this study is to clarify and

updated classification and provide the distribution of marine spider crabs in Thailand.


Specimens collection

Specimens were collected from the gill nets, which fishing by local fishing boat of the fishery villages.

The sampling sites comprised of six stations along the Inner coast, Eastern coast and Upper coast of the Gulf of Thailand

(Figure 1, Table 1) during September 2013 to January 2015. All specimens were preserved in 70% ethanol and deposited

in the Crustacean Collection, Zoological Museum, Department of Zoology, Faculty of Science, Kasetsart University

(ZMKUcru).

The specimens in museum collection and institution collection were also examined in order to complete

overview of marine spider crabs in Thailand viz. follow; The Phuket Marine Biological Center (PMBC) where

specimens were collected in Andaman sea and from BIOSHELF project (Aungtonya et al, 2000), Marine crabs



laboratory in Marine Science Department, Faculty of Fisheries, Kasetsart University (MSCI), Thailand Natural

History Museum (THNHM) and Princess Maha Chakri Sirindhorn Natural History Museum (PSUZC).

Taxonomic identification

The spider crabs in this study were identified and described based on the mature specimens. The classification

of families and the status of species followed the checklist by Ng et al. (2008). The identification key to species were

followed Alcock (1895), Griffin (1974), Sakai (1938, 1965), Guinot & Forges (1985), Griffin & Tranter (1986),

Wagner (1986), Dai & Yang (1991), Poore (2004), Davie (2011) and Ng & Forges (2015).

Result & Discussion

Species richness of spider crabs

A total of 811 specimens of marine spider crab were identified in superfamily Majoidea with four families,

five subfamilies, 21 genera and 40 species, as present in Table 2. There were two species, Tiarinia angusta and

Tylocarcinus styx are supposed to new record in Thailand. From field collection, spider crabs were identified into

three families, six genera and 12 species; Schizophrys aspera, Prismatopus halimoides, P. aculeatus, Micippa

thalia, M. philyra, Hyastenus aries, H. hilgendorfi, H. diacanthus, Doclea armata, D. canalifera, D. rissoni and

Camposcia retusa. The spider crab species was consistent with many previous studies (Wisespongpand et al.,

2010: 2013: 2016; Kaewgunha & Tangkrock-olan, 2007; Singlaem et al., 2013 and Khanadee et al., 2014) because

we collected specimens from the same areas. However, the number of spider carb species in this study was higher than

the previous studies because we collected specimens from many areas and collected periods longer than those of studies.

The family with the highest number of spider crab species was Epialtidae which contained 23 species

(57.5% of total). Spider crabs in genus Doclea had the highest number of species which composed of six species.

We found two species, D. canalifera and D. canaliformis that have never been reported in the decapod checklist

of Thailand (Naiyanetr, 2007). In this study, D. canalifera was dominant species and its distribution was in the

Inner, Eastern and Upper coast of Gulf of Thailand, which was supported by several previous studies (Rathbun,

1910; Wagner, 1986; Griffin & Tranter, 1986; Dai & Yang, 1991). On the other hand, D. canaliformis was an

uncommon species and its distribution was only in the Gulf of Thailand (Wagner, 1986). In family Epialtidae, we

reclarified D. alcocki to D. muricata from the PMBC collection which previously were reported by Ng & Davie

(2002) and Naiyanetr (2007). D. ovis resembled to D. canaliformis in morphological characters. However, the character

of the first male gonopod and gonopore were practically used for species identification.

For the family Hymenosomatidae, we found only one genus and one species, E. magnum. This species

was known as the endemic species in the mangal area of Ranong province, Andaman Sea, which corresponded to

the report by Ng & Chuang (1996) and Naiyanetr (2007). Therefore, the management of this mangroves area is

seriously point for the long term protection and conservation of this species.

In this study, we did not found two specimens, Rochinia pulchra and Hoplophrys oatesii, which were

recorded by Promdam (2009) and Jaingam (2013) that these specimens were preserved in PMBC and MSCI,

respectively. Furthermore, we did not found Paratymolus pubescens and P. vannus (family Inachidae) which was

agreed with Singlaem et al (2013) and Khanadee et al. (2014) studies but differed from Rathbun (1910) who

recorded these two species from Chanthaburi coast, Eastern Gulf of Thailand. This may be because these two

species were restricly distributed in rocky bottom and coral reef (Griffin & Tranter 1986), where these habitats

were good refuge for spider crabs and it is the area that difficult to collect the specimens.

As result, there were the numbers of spider crab species more than Naiyanetr’s report in the decapod

checklist of Thailand (Naiyanetr, 2007). According to Naiyanetr (2007), he reported that there were 32 spider crab

species while this study found 40 species. The spider crab species that were not previously reported as follow;

Paramaja gibba, Tiarinia angusta, Micippa platipes, Tylocarcinus styx, Naxioides taurus, Hyastenus borradailei,

Phalangipus filiformis, Doclea canaliformis, D. canalifera, D. muricata, Huenia heraldica, Xenocarcinus

depressus, X. tuberculatus, X. conicus, Oncinopus araneus, Pleistacantha rubida, Platymaia alcocki and

Cystomaia suhmii. The increasing in number of spider crab species in this study was because we collected

specimens from several field areas especially in the Eastern Gulf of Thailand where there was less information

about spider crab species. Moreover, this study also reexamined spider crab specimens from several museum

collections. The MSCI, THNHM and PSUZC collections contain most of spider crab specimens collected from

various types of fishing gears throughtout the coast of the Gulf of Thailand and Andaman Sea, and including the

coral reefs. Those specimens were also collected by SCUBA diving, from the gill nets which vertically placed

near the coral reefs or rocky shores, and by the trawl nets, traps or crab dredges. On the other hand, the PMBC

collection contains most of the spider crab specimens came from the coast and the deep-water in Andaman Sea

by using traps (Aungtonya et al., 2000). Therefore, the specimens from this study came from different habitats

and from various types of fishing gears which lead to the discovery of many spider crab species including new

habitats and new localities of spider crab distribution.


Geographic distribution

Within the geographic area of this study, the number of marine spider crab species that occurrence in

Andaman Sea was higher those of the Gulf of Thailand, 35 species found in the Andaman Sea and 19 species in

the Gulf of Thailand. Among these, there were 21 species only in Andaman Sea, 5 species only in Gulf of Thailand

and 14 species in both areas (Table 2.). As a result, the number of spider crab species in Andaman Sea had a considerably

more than the Gulf of Thailand due to their geographical differences (Jaingam et al., 2007). Therefore, Andaman

Sea should be promoted as Biodiversity Important Area (BIA) for further conservation and protection of marine

crab species in Thailand.

In this study, the intertidal zone had the highest number of spider crab species (22 species) due to the

diversity of habitats that suitable for spider crabs which was to many studies. For example, Doclea spp. lived on

muddy bottom of the nearshore (Wagner, 1986 and Dai & Yang, 1991), Phalangipus spp. lived on sandy to

muddy-sandy bottom of the offshore (Griffin, 1973). While Xenocarcinus depressus, X. tuberculatus, X. conicus,

Huenia heraldica and Menaethius monoceros lived in the coral reef by associated with the other marine organisms

such as sponges, gorgonians, sea whips, corallines, and algae (Griffin & Tranter, 1986; Jaingam, 2013). Moreover,

there were some spider crab species such as S. aspera, Prismatopus spp., Micippa spp. and Camposcia retusa

lived by decorating themselves with a piece of sponges, coral, algae or pebbles for camouflage, defence, shelter

and/or food supply (Jaingam et al., 2008; Wisespongpand et al., 2012). In addition, spider crabs can be used as an

indicator species of coral reef ecosystem such as Xenocarcinus spp and Huenia heraldica. Therefore, the revised

and updated classification of spider crabs in Thailand are important for the further study of spider crab diversity

due to they are species assemblages in most marine ecosystems, resulting in the complexity of the food chain and

finally lead to the stable of marine ecosystems.

Conclusion

The species richness of marine spider crab was found in the Andaman Sea higher than in the Gulf of

Thailand especially in the intertidal zone where there were the variety of habitat environments such as muddy

bottom, sandy-muddy bottom and coral reef. The highest number species was belonging to family Epialtidae by

contain 23 species. In this family, spider crab genus Doclea was highest species richness, which composed of six

species. There are two species, Tiarinia angusta and Tylocarcinus styx are supposed to be new records in Thailand.

The habitat of most spider crab species, are in marine environment except the Hymenosomatid crab (Brackish-

water environment). In order to fulfill information of the crab biodiversity in Thailand, we should be studying in

Brackish-water and freshwater areas. This study provides an updated classification of marine spider crabs in Thailand

for further spider crab identification, which it is importance for measuring the diversity of marine crabs in the future.

Acknowledgement

This study supported by Faculty of Science Kasetsart University Postgraduate Studentship (ScKUPGS)

and funding source for graduate students of Zoology Department, Faculty of Science, Kasetsart University. We

would like to thank Mr. Rueangrit Promdam of the Princess Maha Chakri Sirindhorn Natural History Museum for

lending materials and give important spider crab data for this study. We sincere thanks also goes to Prof. Peter Ng

Kee Lin of Lee Kong Chian Natural History Museum at Singapore, for his valuable suggestion and information

of the spider crab identification. We would like to express my gratitude to all curators of Reference Collection at

Phuket Marine Biological Center for provided me of this opportunity and for helping in laboratory facilities during

the examination under their care.

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Figure 1. Map showing collection localities. (C = Chonburi; R = Rayong; CH = Chanthaburi; T = Trat;

P = Prachuapkhirikhan Province).

Table 1. Sampling sites for this study.

Sampling site Station Latitude Longitude

Region Province

Inner coast Chonburi North Pattaya 12º57’26N 100º53’02E

South Pattaya 12º55’30N 100º52’5E

Eastern coast Rayong Suan-son beach 12º37’58N 101º28’42E

Chanthaburi Chao-Lao beach 12º32’11N 101º55’43E

Trat Laem-Ngob 12º10’08N 102º23’55E

Upper coast Prachuapkhirikhan Bang-Kum 11º33’56N 99º39’43E

Laem-Kum beach 11º28’4N 99º37’20E

Table 2. Species richness and geographic distribution with habitat noted. (GT = Gulf of Thailand, AS = Andaman Sea).

Superfamily Majoidea (Samouelle, 1819) Geographic

distribution Habitat Noted

Family Subfamily Species GT AS

Hymenosomatidae Elamena magnum Ng & Chuang, 1996 - Mangal

Majidae Majinae Paramaja gibba (Alcock, 1895) - Deep-water

Schizophrys aspera (H. Milne Edwards, 1834) Coral reef

Prismatopus halimoides (Miers, 1879) Intertidal/Coral

reef

P. aculeatus (H. Milne Edwards, 1834) Intertidal/Coral

reef

P. longispinus (De Haan, 1839) - Offshore

Mithracinae Tiarinia angusta Dana, 1851 - Coral reef

Micippa thalia (Herbst, 1803) Intertidal/Coral

reef

M. platipes Rüppell, 1830 - Intertidal/Coral

reef

M. philyra (Herbst, 1803) - Intertidal/Coral

reef


Epialtidae Pisinae Tylocarcinus styx (Herbst, 1803) - Coral reef

Naxioides taurus (Pocock, 1890) - Offshore

Hyastenus planasius (Adams & White, 1848) - Intertidal

H. aries (Latreille, 1825) Intertidal/Coral

reef

H. borradailei (Rathbun, 1907) - Coral reef

H. hilgendorfi De Man, 1887 Intertidal/Coral

reef

H. diacanthus (De Haan, 1839) - Intertidal/Coral

reef

Phalangipus hystrix (Miers, 1886) - Offshore

P. indicus (Leach, 1815) - Offshore

P. filiformis Rathbun, 1916 Offshore

P. longipes (Linnaeus, 1758) - Offshore

Doclea armata De Haan, 1839 Intertidal

D. canaliformis Ow-Yang in Lovett, 1981 - Intertidal

D. canalifera Stimpson, 1857 - Intertidal

D. ovis (Fabricius, 1787) Intertidal

D. muricata (Herbst, 1788) - Intertidal

D. rissoni Leach, 1815 Intertidal

Tychinae Criocarcinus superciliosus (Linnaeus, 1758) - Intertidal/Coral

reef

Epialtinae Huenia heraldica (De Haan, 1837) - Coral reef

Menaethius monoceros (Latreille, 1825) Rocky

shore/Coral reef

Xenocarcinus depressus Miers, 1874 - Coral reef

X. tuberculatus White, 1847 - Coral reef

X. conicus (A. Milne-Edwards, 1865) - Coral reef

Inachidae Oncinopus aranea (De Haan, 1839) - Coral reef

Achaeus brevirostris (Haswell, 1879) Coral reef

A. lacertosus Stimpson, 1858 Coral reef

Camposcia retusa (Latreille, 1829) Coral reef

Pleistacantha rubida (Alcock, 1895) - Deep-water

Platymaia alcocki Rathbun, 1918 - Deep-water

Cystomaia suhmii Miers, 1885 - Deep-water




The effectiveness of basic bait traps for collecting adult flies

Amornrat Ninon and Taeng On Prommi* Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen District,

Nakhon Pathom, Thailand *Corresponding author e-mail: [email protected]

Abstract: The aim of the present study is to compare the effectiveness of pork liver bait traps (PLBTs) and fish

meat bait traps (FMBTs) for collecting adult flies. Bait traps used to collect these species can be damaged by anthropogenic

or environmental effects. In this study, PLBTs and FMBTs were hung from trees in the three areas (tree near the pig

farm, tree near the dormitories and tree near the fresh market) of the Kasetsart University, Kamphaeng Saen Campus,

Nakhon Pathom Province, Thailand. This activity was carried out on the first Monday of each week for two months.

During the study, 4,252 specimens were collected. Of all these species, 2903 (68.27%) were obtained from PMBTs,

while 1,349 (31.73%) were obtained from FMBTs. Calliphoridae, belonging to three species [Chrysomya

megacephala (Fabricius), C. rufifacies (Macquart) and C. nigripes (Aubertin)] were the most abundance in this study.

The number of species collected from PMBTs was the same found in the FMBTs, but the number of each individual

in each species was different. This study provides baseline information on the necrophilous fauna for estimating

postmortem interval in cases of human death in Thailand.

Keywords: Basic meat bait trap; biodiversity; Diptera, forensic entomology.

Introduction

Fauna studies were conducted to determine bioavailability in a region. It is extremely important to understand

and evaluate processes that appear in nature as a result of gradually increasing anthropogenic effects and resultant

climatic changes. Results of fauna studies are commonly used in applied sciences including entomology, veterinary

science, public health care, agriculture, forestry and ecology, and in the protection of the environment in addition

to basic sciences such as zoology, zoogeography, population genetics, systematics, microbiology and parasitology

(Braioni, 1994; Tamutis et al., 2011). Fauna studies are also useful in elimination of the order of Diptera,

Calliphoridae, Sarcophagidae, Oestridae and Muscidae in public health and veterinary studies, which lead to myiasis

by leaving their larvae and eggs on the wounds of humans and animals (Green et al. 2005; Sankari & Ramakrishnan

2010; Avula et al. 2011; Aggarwal et al. 2014; Jervis-Bardy et al. 2014). Another applied science benefiting from

the results of fauna studies is forensic entomology, which determines postmortem interval, cause of death and whether

corpses have been moved, which contribute to solving forensic cases.

In studies in basic and applied sciences, bait traps used to attract flies to determine the fauna of a region

depend on the sense of smell and types of nutrition of flies (Cavallari et al. 2014). The most well-known traps are

carrion-baited Martín-Vega et al. 2013; Martín-Vega & Baz 2013), followed by basic meat bait traps (BMBTs),

sticky traps, synthetic smell traps and chemical (insecticide) traps (Boonchu et al. 2003; Harvey et al. 2010; Nurita

& Abu 2010). BMBTs, usually prepared with beef and beef liver, are the first traps to be preferred in that they are

inexpensive and effective (Boonchu et al. 2003). The goal of the present study is to compare the effectiveness of pork

liver bait traps (PLBTs) and fish meat bait traps (FMBTs) the collection of flies.


Insect collection

Traps were prepared as shown in Figure 1. Pork liver bait traps (PLBTs) and fish meat bait traps (FMBTs)

to serve as attractant, each 200 g, were placed in the traps. As shown in Figure 1, the upper part of each trap, raincoats

and the ropes used in hanging the traps were secured on the same place on the pipes. The traps were hung on the pipe

under the tree near the fresh market (Figure 1A), the tree near the dormitories (Figure 1AB) and the tree near the pig

farm (Figure 1C) in the Kasetsart University, Kamphaeng Saen Campus, at 08.00 h on the first Monday of each month.

Then, traps were left in that place until 17.00 h on the Friday of that same week. The traps were operated during

February and March 2019.

On the first Friday of each week, live adults in the PLBT and FMBT were killed with chloroform. All

the adult, larvae and pupae in both the PLBT and FMBT were removed. Ambient temperatures and relative

humidity were obtained from Nakhon Pathom Meteorological Station which is located near the sampling sites.



In order to determine the diversity of species in PLBT and FMBT sample groups in our study, Shannon

Wiener and Simpson diversity and evenness indices were used. Diversity indices were calculated using PC-ORD ver.5.1

(McCune and Mefford, 2006).

To compare the mean temperature and relative humidity in each collection time, a t- test was used to

obtain a 95% confidence level. To evaluate the relationship between environmental factors and insect species, a

Pearson correlation coefficient was used. Statistical analyses were performed using SPSS software (version 16.0).

Result & Discussion

Average relative humidity and temperatures at the study sites are shown in Table 1. For the duration of

the experiment, the mean temperature ranged 24.99±1.21−29.33±1.73°C, whereas the relative humidity ranged

71.07±7.19−80.29±5.47. There was not a significant difference ambient temperatures measured and relative humidity

at the experimental site in each collection time (Table 1.).

A total of 4,252 specimens were collected during two months of the experiment. Of all these species,

2,903 (68.27%) were obtained from PMBTs, while 1,349 (31.73%) were obtained from FMBTs (Table 2.).

Calliphoridae, belonging to three species [Chrysomya megacephala (Fabricius) (Figure 2.), C. rufifacies (Macquart)

(Figure 3.) and C. nigripes (Aubertin) (Figure 4.)] were the most abundance in this study. The other insect group

found in these trap were Muscidae (Musca domestica), Sarcophagidae (Sarcophagida dux), Histeridae (Saprinus sp.)

and Dermestidae (Dermestes maculatus). The number of species collected from PMBTs was the same found in

the FMBTs, but the number of each individual in each species was different.

The purpose of the present study is to compare the effectiveness of pork liver bait traps (PLBTs) and fish

meat bait traps (FMBTs) in the collection of flies. Table 3 showing the diversity index results, similarities are seen

in terms of species composition in both samples. Thus, in terms of species composition of the traps, rates are

similar. The value of diversity index in both tarps was not high because of the area that expose to insect is limit.

The proper range of Shannon`s diversity index ranged 1−3 (Magurran, 1988).

According to the Pearson correlation test, the meteorological factors were associated with the Calliphoridae

species, Chrysomya megacephala and Chrysomya nigripes (Table 4.). This fact emphasizes that the bait traps

temperatures are important with respect to the rate of bait traps decomposition; relative humidity is also important

because it acts directly on the decomposition of the bait trap meat and promotes the emergence of an alternative

decomposition stage (Moura et al., 1997). Because temperature and relative humidity conditions throughout the study

exhibited was not variation, it was not possible to demonstrate the influence of these factors on the presence of

insects on the carcass or on the decomposition process itself (Rungsri et al., 2018).

Conclusion

This was a preliminary study. It is necessary to repeat and replicate it at different times of the year so as

to provide multiple sets of baseline succession data for Thailand that encompass all seasons. However, the information

obtained during this study could be useful for providing initial database information as no succession data was

previously available in central Thailand. Furthermore, these results could also possibly stimulate other entomologists

in Thailand and initiate future studies. The use of fresh meat traps can increase the number of flies collected. The fact

for this study, flies meat bait traps (FMBTs) was attached flies more than pork liver bait traps (PLBTs). Unfortunately,

fish meat bait traps was destroyed by some vertebrate in this area. Fauna studies have scientific and sociocultural

importance. Subsequently, they play an important role in raising public awareness in the determination of endemic

species, climatic change and natural history, and in the protection of nature.

Acknowledgement

This research was supported by Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng

Saen Campus.

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Figure 1. Design of liver of pork meat bait traps (PMBTs) and fish meat bait traps (FMBTs) operated under the

tree near the fresh market (A), the tree near the dormitories (B) and the tree near the pig farm (C) in the Kasetsart

University, Kamphaeng Saen Campus, Nakhon Pathom Province, Thailand.

Figure 2. Adult and larvae of Chrysomya megacephala.



Figure 3. Adult and larvae of Chrysomya rufifacies.

Figure 4. Adult and larvae of Chrysomya nigripes.


Table 1. Environmental variable in the study area. (Data from Nakhon Pathom meteorological Station).

Sampling date Air temperature (ºC) Humidity (%)

January 7, 2019 24.99 ±1.21a 75.86 ±4.53a

January 14, 2019 27.14 ±1.52a 80.29 ±5.47a

January 21, 2019 26.85 ±1.44a 78.64 ±5.59a

January 28, 2019 25.12 ±1.80a 72.14 ±7.40a

February 5, 2019 27.59 ±1.81a 73.93 ±6.53a

February 12, 2019 29.13 ±1.60a 76.43 ±5.96a

February 19, 2019 28.79 ±1.45a 75.50 ±6.11a

February 26, 2019 29.33 ±1.73a 71.07 ±7.19a

Remark: a, b, c = the relationship of environmental factors is similar in the sampling sites.

Table 2. Abundance of flies species collected in the pork liver bait traps (PLBTs) and fish meat bait traps

(FMBTs); L1 = first instar larval; L2 = second instar larval; L3 = third instar larval; P = Pupae; A = Adults)

Family Genus/Species Stage

Site 1 Site 2 Site 3

FMBT PLBT FMBT PLBT FMBT PLBT

Calliphoridae Chrysomya megacephala A 126 319 126 200 49 223 (Fabricius) L1 0 0 0 0 0 51 L2 22 37 3 63 0 110 L3 351 448 87 283 173 517 P 0 0 1 0 0 0 Total 499 804 217 546 222 901 % 74.48 22.37 55.93 70.45 76.29 77.81 Chrysomya rufifacies A 50 21 0 55 5 26 (Macquart) L2 0 0 0 0 0 123 L3 73 0 79 77 54 11 Total 123 21 79 132 59 160 % 18.36 2.16 20.36 17.03 20.27 13.82 Chrysomya nigripes A 6 25 14 21 0 11 (Aubertin) L3 31 112 78 72 9 79 Total 37 137 92 93 9 90

% 5.52 14.12 23.71 12 3.09 7.77

Muscidae Musca domestica A 0 4 0 0 0 5 Total 0 4 0 0 0 5

% 0 0.41 0 0 0 0.43

Sarcophagidae Sarcophagida dux A 0 4 0 0 0 2 Total 0 4 0 0 0 2

% 0 0.41 0 0 0.17

Histeridae Saprinus sp. A 6 0 0 3 0 0 Total 6 0 0 3 0 0

% 0.90 0 0 0.39 0 0

Dermestidae Dermestes maculatus A 5 0 0 1 1 0 Total 5 0 0 1 1 0

% 0.74 0 0 0.13 0.34 0 Total 670 970 388 775 291 1158

% 100 100 100 100 100 100



Table 3. Biodiversity indices.

Indices Pork liver bait traps (PLBTs) Fish meat bait traps (FMBTs)

Site 1 Site 2 Site 3 Site 1 Site 2 Site 3

Species number 5 5 5 5 3 4

Number of individuals 970 775 1158 670 388 291

Evenness 0.348 0.517 0.436 0.478 0.901 0.474

Shannon`s diversity index 0.560 0.833 0.702 0.769 0.990 0.657

Simpson`s diversity index 0.2925 0.4602 0.3695 0.4084 0.5895 0.3759

Table 4. The relationship between the environmental factors and insect species.

Taxon/factor Air Temperature (ºC) Humidity (%)

FMBTs PLBTs FMBTs PLBTs

r sig r sig r sig r sig

Chrysomya megacephala 0.359 0.552 0.958** 0.001 -0.889* 0.044 -0.435 0.465

Chrysomya rufifacies -

0.718 0.282 0.611 0.582 0.601 0.399 0.837 0.369

Chrysomya nigripes 0.440 0.459 -1.000** -0.880 * 0.049 -1.000**

Remark: * Correlation is significant at the 0.05 level (2-tailed).

** Correlation is significant at the 0.01 level (2-tailed).



An assessment of habitat connectivity for the endangered Malayan tapir in Thailand

Damisa Kaminsin and Naparat Suttidate* Walailak University, Department of Biology, School of Science, Thasala District, Nakhon Si Thammarat, Thailand

Corresponding author e-mail: [email protected]*

Abstract: The Malayan tapir (Tapirus indicus) is threatened by habitat loss and fragmentation, causing populations

to become small and isolated. An assessment of habitat connectivity patterns is crucial for the survival of the species

because connectivity facilitates species movement and gene flow. Our goal was to identify habitat connectivity

networks for the Malayan tapir in Thailand. We first defined suitable habitat for tapirs using satellite data: evergreen

and deciduous forest patches with the minimum patch size of 13 km2 (average home range size), elevation, slope,

and mean annual precipitation. We then applied circuit theory analysis to assess potential dispersal corridors using

Circuitscape software. We identified 38 suitable habitat forest patches and 13 potential dispersal corridors for tapirs

in Thailand. We found that Khlong Saeng-Khao Sok forest complex was well connected with average habitat patch

size of 2,247.78 km2. In contrast, Chumphon forest complex was the most fragmented forest complex with the highest

number of suitable habitat patches and potential dispersal corridors. In addition, we found ten potential dispersal

corridors within the forest complexes, while there were three potential dispersal corridors between Western –

Kaeng Kra Chan, Kaeng Kra Chan – Chumphon and Chumphon – Khlong Saeng – Khao Sok. The results provide

better understanding of the current pattern of habitat connectivity networks and a guideline for priority areas for

conservation planning. We highlight that an assessment of habitat connectivity pattern can be applied as a wildlife

management tool to assist conservation efforts for the Malayan tapir and other endangered mammals in the region.

Keywords: Circuit theory, Conservation, Corridor, Habitat connectivity, Mammal

Introduction

The Malayan tapir (Tapirus indicus) is a globally endangered species (IUCN, 2016). It is also listed as

an endangered species in the Wild Animal Reservation and Protection Act, B.E. 2535 of Thailand and as Appendix

I status in the Convention on International Trade in Endangered Species (CITES) appendices (CITES, 2017). The

Malayan tapirs is a frugivore and is one of the most important seed dispersal mammals in tropical forest ecosystems.

Malayan tapirs help to decrease plant competition, increasing survival rates of seeds, and decreasing the impacts

of disease. Therefore, the Malayan tapir plays a crucial role in plant communities and tropical biodiversity. Currently,

tapir populations have declined rapidly due to anthropogenic activities, such as habitat loss and fragmentation,

caught by chance, hunting, and road accidents (Corlett, 2007; Holden et al., 2003). Specifically, habitat loss and

fragmentation can fracture a population into at-risk sub-populations. A small, isolated sub-population within a habitat

patch is more likely to become locally extinct due to limited movement between suitable habitats and low genetic

variation among sub-populations (de la Torre, et al., 2017). Thus, the survival of Malayan tapirs in fragmented

landscapes depends upon maintaining connectivity between isolated populations (Fahrig & Merriam, 1985; Noss

et al., 1996; Taylor et al., 1993).

Habitat connectivity is the connectedness between patches of suitable habitats to facilitate movement for

a given species (Lindenmayer & Fischer, 2013). The importance of habitat connectivity is to maintain a specie’s

home range, enhance gene flow among sub-populations, increase the opportunity for adaptation in response to

environmental changes, and mitigate the risk of extinction (Brodie et al., 2015; McRae et al., 2008; Sulistyawan

et al., 2017). In addition, connectivity plays a crucial role in conservation planning where the goal is often to

preserve resilient habitat networks, and design linkages of high-quality habitat (i.e., dispersal corridors) between

remnant patches or protected areas (Soule & Terborgh, 1999). Habitat connectivity has been successfully applied

in conservation planning for species in many regions, such as deer (Odocoileus hemionus) in California and

Arizona (Beier et al., 2006), jaguars (Panthera onca) in Mexico (de la Torre et al., 2017), and pandas (Ailuropoda

melanoleuca) in China (Sulistyawan et al., 2017; Wang et al., 2014). Despite the advancements in theory and application,

the assessment of habitat connectivity for Malayan tapirs has not yet been studied in Thailand which hampers conservation

efforts (Lynam et al., 2012). Broad-scale conservation planning for Malayan tapirs cannot be successful without

identifying suitable habitat patches, dispersal corridors, and an effective assessment of a connectivity network that

can maintain the viability of tapir populations and facilitate gene flow among populations (Lynam et al., 2012).

The goal of our study was to identify habitat connectivity networks for the Malayan tapir in Thailand.

First, we defined suitable habitat patches for Malayan tapirs, and then evaluated the patterns of habitat connectivity

using circuit theory.




Study area

Our study area included 35 protected areas in seven forest complexes, Thailand where the Malayan tapir

populations occupy: (1) Western forest complex (Kamphaeng Phet, Tak, Nakhon Sawan, Kanchanaburi, Suphan

Buri, and Uthai Thani Provinces); (2) Kaeng Kra Chan forest complex (Prachuap Khiri Khan, Ratchaburi and

Phetchaburi Provinces); (3) Chumphon forest complex (Pratap Khiri Khan, Ranong and Chumphon Provinces);

(4) Khlong Saeng-Khao Sok forest complex (Surat Thani, Ranong, Phang Nga, Chumphon and Ranong

Provinces); (5) Khao Luang forest complex (Nakhon Si Thammarat Province); (6) Khao Banthad forest complex

(Phattalung, Trang, Satun and Songkhla Provinces); and (7) Hala Bala forest complex (Yala, Narathiwat, and

Pattani Provinces) (Kanchanasaka 2015). Preferred forest habitat areas for the Malayan tapir are evergreen and

deciduous forests with an approximate total area of 22,803 km2 (Figure 1).

Figure 1. Study area covered evergreen and deciduous forest patches with an average area greater than 13 km2

was shown in white with seven forest complex boundaries.

Identifying Malayan tapir’s habitat patches To identify habitat patches for Malayan tapirs in Thailand, we employed four habitat requirement variables

from satellite data: habitat types, elevation, slope, annual precipitation. we defined suitable habitat patches as the

following criteria:

(1) Evergreen forest and deciduous forest patches with a minimum habitat patch size of 13 km2 based on

average home range size for tapirs in Thailand (Lynam et al., 2012; Williams & Petrides, 1980). Evergreen forest

and deciduous forest data were obtained from the land use and land cover map of Thailand for the year 2016 derived

by the Land Development Department, Thailand.

(2) Suitable elevation for the Malayan tapir was < 1,500 meters. We derived elevation data from the

CGIAR - Consortium for Spatial Information (http://srtm.csi.cgiar.org)

(3) Suitable slope for the Malayan tapir was from 0° to 30°. We derived the slope data by transforming

the elevation data to degree slope using spatial analyst tool in ArcGIS version 10.2.1.

(4) Annual precipitation for the Malayan tapir differed among regions in Thailand. Accordingly, we

assigned mean annual precipitation in southern forest complexes (i.e., Chumphon FC, Khlong Saeng - Khao Sok

FC, Khao Luang FC, Khao Banthad FC, and Hala Bala FC) to a minimum of 3,500 mm. On the other hand, mean

annual precipitation in western forest complexes (Kaeng Kra Chan FC and Western FC) was assigned to a minimum


of 2,500 mm. Annual precipitation was obtained from WorldClim version 2 (http://worldclim.org/version2) (Fick

& Hijmans, 2017).

We prepared all habitat data in raster format with 1-km resolution because it is a proper resolution for

spatial analysis of mammals in Thailand (Lynam et al., 2012). We analyzed suitable habitat patches for the Malayan

tapir using ArcGIS program version 10.2.1.

Assessing habitat connectivity for Malayan tapirs

To evaluate potential dispersal corridors for the Malayan tapir, we used Circuitscape software version

4.0 (McRae et al., 2008). Circuitscape applies electrical circuit theory and random walk theory to calculate

pairwise resistances and create maps of current flowing between focal nodes (McRae et al., 2008). Circuitscape

requires two inputs: (1) a resistance map (i.e., land use and land cover with a degree of movement resistance for

Malayan tapirs); and (2) a focal node map (i.e., forest habitat patches). We prepared the resistance map by assigning

resistance values to each land use and land cover raster, ranking from 1 to 100. We assigned lowest resistance

values to forest habitat: evergreen and deciduous forest patches. Higher resistance values were assigned to other

natural habitats: swamp forest, rangeland, marsh and swamp, and small natural water body. Highest resistance

values were assigned to agricultural areas, such as forest plantation, and orchards (Table 1). For complete barriers,

we assigned them as no data areas (i.e., settlements, roads, perennial agricultural areas, and large water bodies).

Circuitscape created current maps between pairs of forest patches using pairwise analysis and iterated all pairs

into focal nodes (McRae, et al., 2013).

Table 1. Resistance values assigned to create the resistance map.

Category Resistance score

Evergreen forest 1

Deciduous forest 1

Swamp forest 20

Forest plantation 30

Rangeland 40

Marsh and swamp 50

Small natural water body 60

Agricultural areas 100

Result & Discussion

We identified 38 potential suitable forest patches in seven forest complexes with approximately an area

of 23,731 km2 and average patches size of 625 km2 (Table 2). Chumphon forest complex had the highest number

of forest habitat patches, while Khao Luang and Khlong Saeng - Khao Sok had the lowest number of forest habitat

patches. Additionally, we found large forest habitat patches (> 625 km2) in Western, Kaeng Kra Chan, Khao Luang

and Khlong Saeng-Khao Sok forest complexes. In contrast, we found many small forest habitat patches (< 625 km2)

in the Chumphon, Hala-Bala, and Khao Banthad forest complexes (Table 2). In addition, we found that Western

and Kaeng Kra Chan forest complexes had seven forest patches which included both evergreen and deciduous

forest habitat types. On the other hand, we found that suitable forest habitat patches in the south of Thailand were

only evergreen forests due to high rainfall and humidity (Kanchanasaka, 2015; Lynam et al., 2012).

We also identified 13 potential dispersal corridors: 10 dispersal corridors within the forest complexes

(Table 3), and three dispersal corridors between forest complexes: (A)Western forest complex – Kaeng Kra Chan

forest complex; (B) Kaeng Kra Chan forest complex – Chumphon forest complex; (C)Chumphon forest complex

– Khlong Saeng – Khao Sok forest complex (Figure 2).



Table 2. Suitable forest patches for the Malayan tapir in Thailand.

Forest complex Number of patches Mean patches size (km2)

Chumphon 9 169.07

Hala Bala 8 241.04

Western 7 998.70

Kaeng Kra Chan 6 915.41

Khao Banthad 3 504.34

Khao Luang

Khlong Saeng – Khao Sok

3

2

596.47

2,247.78

Total 38 625

Table 3. Potential dispersal corridors within forest complexes for the Malayan tapir in Thailand.

Forest complex Number of corridors

Chumphon

Namtok Ngao NP - Lam Nam Kraburi NP

Namtok Ngao NP - Tungraya-Nasak WS

Tungraya-Nasak WS - Prince Chumphon Park (South) WS

Prince Chumphon Park (South)WS-Prince Chumphon Park (North) WS

4

Kaeng Kra Chan

Kuiburi NP (Patch 1) - Kaeng Kra Chan NP

Kuiburi NP (Patch 1) - Kuiburi NP (Patch 2)

2

Khao Luang

Tai Rom Yen NP - Namtok Si khid NP

Namtok Yong NP - Khao Luang NP

2

Hala Bala

Budo-Sungai Padi NP (Patch 2) - Budo-Sungai Padi NP (Patch 3)

Hala Bala WS - Budo-Sungai Padi NP (Patch 1)

2

Khao Banthad 0

Western 0

Khlong Saeng – Khao Sok 0

Total 10

* NP: Natural Park, WS: Wildlife Sanctuary


Figure 2. The results showed pairwise current flow density (Amps/cell) between three forest complexes: (A)

Western forest complex (1) and Kaeng Kra Chan forest complex (2); (B) Kaeng Kra Chan forest complex (2) and

Chumphon forest complex (3); (C) Chumphon forest complex (3) and Khlong Saeng – Khao Sok forest complex

(4). The highest current density (shades of yellow) indicated areas of potential dispersal corridors. Areas, where

connectivity was most difficult were shown in purple. Evergreen and deciduous forest habitat patches with an

area greater than 13 km2 were shown in green.

Discussion

Our goal was to identify habitat connectivity networks for the Malayan tapir in Thailand. We assessed

patterns of potential forest habitat patches and dispersal corridors within and between forest complexes. Our

results provide important baseline information for Malayan tapir conservation planning. Especially, the Malayan

tapir populations are at risk of locally extinct primarily due to habitat loss and fragmentation (Lynam et al., 2012).

Recent estimate Malayan tapir abundance in Thailand is approximately 538 – 720 individuals, occupying seven

forest complexes: Western, Kaeng Kra Chan, Chumphon, Khlong Saeng-Khao Sok, Khao Luang, Khao Banthad

and Hala Bala forest complexes (Kanchanasaka 2015).

Our results demonstrated that Chumphon, Hala Bala, Khao Banthad, and Khao Luang forest complexes

were more fragmented than other forest complexes. These forest complexes experience changes in land use and

land cover from anthropogenic activities, such as perennial crop, field crop, orchard, built-up areas, and artificial

water bodies. Malayan tapir populations are more likely to be separated in those forest complexes, and occupy

small proportions of the available forest habitat patches (Lynam et al., 2012; Clements et al., 2012). Malayan

Tapirs avoid human-made landscape and anthropogenic activity because they are nocturnal and solitary, and also

prefer wetter places in denser forest habitat (Clements et al., 2012). Malayan tapirs may also avoid natural water

bodies, if such areas were used by human activities (Lynam et al., 2012; Kanchanasaka 2015). In addition,

Chumphon forest complex had the lowest mean patches size, indicating that forest habitat patches in this forest

complex become more fragmented and degraded. This might due to geographical location and topography where

Chumphon forest complex is located along the western border of Thailand and potentially vulnerable to human

disturbance (Lynam et al., 2012; Kanchanasaka 2015).

We found that Chumphon forest complex had the highest number of potential dispersal corridors,

indicating habitat fragmentation and degradation. However, there was no potential dispersal corridor in Western

forest complex, and Khlong Saeng – Khao Sok forest complex because such forest complexes are well connected

and most of protected areas in each forest complex are adjacent to each other (Lynam et al., 2012; Kanchanasaka 2015).

The current flows among adjacent forest patches were substantially higher than the current flows in nonadjacent

forest patches, indicating that short distance dispersal corridors can facilitate tapirs’ movement better than the



long-distance corridors. The analyses revealed that potential dispersal corridors with low resistance mostly were

evergreen forest and deciduous forest.

The results showed that there were more potential dispersal corridors within the forest complexes than

between the forest complexes. Also, corridors within the forest complexes were connected to larger forest habitat

patches. Similarly, the study of habitat connectivity for Pumas (Puma concolor) in the southwestern United States

(Arizona and New Mexico) demonstrates that maximum current flow corridors are more likely to be located within

close proximity to high quality habitat (Dickson et al., 2013). Furthermore, we found that forest patches in Hala

Bala forest complex were more isolated with the long-distance potential corridors than other forest complexes due

to conversion of natural habitat to perennial crops. We found no potential dispersal corridors in Khlong Saeng-

Khao Sok forest complex and Western forest complex because the complexes have the largest forest patch size

with adjacent patches. Conversely, we found that forest patches in Khao Banthad forest complex were threatened

by agricultural areas that resulted in no connectedness between patches.

We suggest that Western forest complex and Khlong Saeng-Khao Sok forest complex had the lowest

habitat fragmentation with high quality habitat for Malayan tapir populations. This forest complex could

potentially be an important source for habitat connectivity network.

Conclusions

An assessment of potential suitable habitat patches and dispersal corridors for Malayan tapirs in Thailand

provides preliminary information to assist conservation planning and management for the Malayan tapir in Thailand.

Specifically, less fragmented and well-connected evergreen and deciduous forest patches can potentially be an

important refuge for the endangered Malayan tapir in Thailand. Habitat connectivity is crucial to maintain the

viability of Malayan tapirs and can help conservation management for other endangered species in the regions.

Acknowledgement

We would like to thank Walailak University and the Development and promotion of science and technology

talents project (DPST) for funding. We are thankful to the Land Development Department (Thailand) for sharing

the land use and land cover map of Thailand for the year 2016.

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Satun UNESCO Global Geopark, the readiness for sustainable tourism

Suwimon Hengpatana*, Phannipha Anuruksakornkul, Apinya Wanaset,

Taweesin Tangseng and Danuvas Suwanwong

Faculty of Economics, Srinakharinwirot University, Wattana District, Bangkok, Thailand


Abstract: Satun Geopark is recently UNESCO Global Geopark creating pride for the people in the country. The area

covers four of seven districts of Satun Province namely Thungwa, Manang, La-Ngu, and Muang Satun. The Geopark

attraction points are categorized into four types. It consists of ecotourism and adventure attraction; sea – islands

attraction; attraction for learning; and intangible/ cultural heritage. The famous tourist attractions are Li-Pe Island,

Hin Ngam Island, Adang-Ra Wi Islands, Tarutao National Park, Petra National Park, Pak Bara Beach, Satun Karst

Zone includes Le- Stegodon Sea Cave, Chet Khot Cave, and Phu Pha Phet Cave. Being a global geopark can

attract eco-tourists from all over the world. Therefore, in the Satun geopark, there must be efficient and proper tourism

management not only to preserve the existing ecosystem but also impress the tourists. Logistics management is consequently

applied to sustainable tourism. Satun Geopark logistics management can be categorized into four components which

consist of geological resources and landscape; transportation; amenities; and environmental management as well as

creating the value of tourism products using three supporting factors: information and communication technology;

innovation; and sustainable management provide tourism value for the tourists in terms of flexible, convenient, fast,

and safe. The objectives of this paper are to discover the component of logistics management and to evaluate the logistics

management potential for sustainable tourism for Satun UNESCO Geopark. The results could be used for government

agencies, and the private sector involved such as Satun Provincial Administrative Organization, Sub-district Administrative

Organization in the area as well as travel service operators.

Keywords: Satun UNESCO Global Geopark, logistics management, sustainable tourism, geotourism

Introduction

In Thailand, tourism has become a pioneer sector and made a significant contribution to the country’s

gross domestic product (GDP). It generates income and creating employment for local people. During January to

August 2018, there are 25,886,325 foreign tourists. Compared to the same period of last year tourism expanded by 9.94%,

in line with the expansion of tourists in almost all regions and generated total revenue of 1,350,317.90 million

baht, expanding by 12.85% from the same period of last year. From the large amount of tourists, the management

of sustainable tourism is needed.

Thailand is a country with abundant of natural resources, including cultural and historical attractions.

Southern Thailand contains many islands and beautiful beaches. As UNESCO announced to Satun geopark is

UNESCO global geopark on April 17, 2018, creating pride for the people in the country. Being a global geological

park can attract eco-tourists from all over the world. Satun Geopark covered four districts of Satun Province

namely; Muang, La Ngu, Tungwa, and Manang located along the Coast of Andaman Sea. It possesses many

beautiful islands such as Li-Pe Island, Hin Ngam Island, Adang-Ra Wi Islands, and Dong Islands located in Tarutao

National Park. Moreover, Satun Geopark also features some attractions about culture and the local way of lives.

The Geopark attraction points can be categorized into four types; (1) ecotourism and adventure attraction such as

Le-Stagodon Cave, Puphapet Cave, Chet Chod Cave, Thanpliew Waterfall; (2) sea – islands attraction such as

Adang - Ra Wi Island, Li-Pe Island, Tarutao Island, Petra Island. There are also dunes called Dragon’s back at

Tan-Yong-Po and many diving sites such as Jabang diving site; (3) attraction for learning such as Satun Geopark

Museum, many fossil sites such as Khao Noi stone hierarchy, trilobite fossil at Tarutao Island, nautiloid at Khao

Dang. There are also many Karst in Thungwa and Manang Districts; (4) intangible/ cultural heritage such as Ma-Ni,

a native Nigrito at Manung District, floating boat tradition of Orang Laut, the indigenous sea people at Adang and

Li-Pe Island. Since there are many types of attractions in the area, and some area is famous for foreign tourists,

there must be attracted abundant of tourists both Thai and foreigner tourists. Therefore, in the Satun geopark, there

must be efficient and proper tourism management not only to preserve the existing ecosystem but also impress

the tourists. Logistics management is the way to manage sustainable tourism. By managing the tourism logistics,

it is the planning of the administration of tourism products and services before tourists travel until tourists come

to travel conveniently throughout the journey.

This article, therefore, is the studied the logistics management components for tourism and to evaluate

the logistics management potential for sustainable tourism of Satun UNESCO Global Geopark. The government


sector can apply the findings to prepare for the infrastructure, logistics system, and facilities to develop logistics

management for sustainable tourism to facilitate the tourists and to create a balance in the economy, society, and environment.


The area of the study is Satun Geopark (as known as Satun UNESCO Global Geopark) which composed of four

of seven desticts of Satun Province namely; Muang, Thungwa, La-Ngu, and Manang District. The area covers four types

of geopark attraction points; ecotourism and adventure attraction; sea – islands attraction; attraction for learning;

and intangible/ cultural heritage.

Geotourism is a form of tourism that sustains or enhances the geographical characters of a location such

as its geology, environment, culture, heritage, aesthetics, and the well-being of the communities. (Tourtellot, 2011).

There are several works of literature about geotourism management in different principles. Valiakos (2010)

mentioned five components of geological park management. It composes of 1) geology and landscape which consist

of three elements; territory; geography conservation; and natural and cultural heritage 2) management structure

3) interpretation and environmental study 4) geotourism 5) sustainable economic management in the area. Lewis

T.O. Cheung (2014) explained the factors that affect geopark. There are the external and the internal value of the park.

The external value of the park consists of five components. There are convenient public transportation, easy access

to the bathroom, visitor guides and useful maps, adequate leisure facilities, and clear road signs. Also, the internal

value of the park consists of four components which are outstanding scenery and landscape; beautiful mountain

and attractive area; coast, seashore, and beautiful beach; and interesting geology and ecology. He also analyzed

the three main components of motivation in tourism. It includes 1) travel for novelty which are five elements that

tourist have; enjoyment and fun; freedom; new experiences in different lifestyles; tried exotic foods; and seen as

much as can be seen during the free time. 2) Leisure travel which includes what the tourists want to have in travel;

body movements and participation in sports; positive attitude about the trip after returning home; and have been

changed from busy jobs. 3) Tourism for escape from something which is an escape from the need at home;

searching for excitement; meet people with similar interests. Moreover, Patrick (2014) described how to develop

ideas and activities in geological park. He mentions four categories. 1) Sustainable mobility: the concept of public

transport or bicycle access 2) Sustainable hotel business 3) Geothermal energy from Geological Park 4) Energy and

climate strategy.

Result & Discussion

The integrity of tourism resources in ecology, terrain, and nature, Satun Geopark create various types

of natural tourism activities. The activity has been classified into various types (and still counting). The tourism

sector today aims to cater to the needs and preferences of all types of tourists, and thus, seems to take into consideration

specific areas of their interest. In present, there are many tour operators provide variety of tourism activities for

the tourist. Their offices located near Pak Bara Port. The tourists are able to choose the activities or the trip that they prefer

from the tour operator in the area or book it from internet.

The tourism in Satun Geopark can be categorized as following:

1) Adventure Tourism (as known as adventure travel). This kind of tourism has been famous among adventure seeker

in Satun Geopark. The adventure tourists indulge in challenging activities such as water rafting, scuba diving, caving,

hiking, rock climbing, and so on. There are several adventure points in Satun Geopark such as Le-Stegodon Cave,

Chet Chod Cave, Wangsaithong Waterfall, and so on. The diving sites in the area are Jabang Pinnacle, Adang-

Rawi, Li-Pe Island, Tarutao Islands, and Petra Islands. The tourists can go diving by buying a package tour from tour

operator at Pak-Bara Port and nearby and speed boat rental is also available.

2) Cultural Tourism. The concept of cultural tourism encompasses things, such as history of a given region, the lifestyle

of people in a particular geographical locale, architecture, oral traditions, religions, festivals, cuisine, and so on. Tourists

can visit the floating boat which is the native fishery tradition.

3) Ecotourism. Ecotourism also encompasses the concepts of geotourism. It involves the traveling to primary

attractions which are natural beauty or settlements of indigenous communities. The tourists can learn the lifestyle

of Mani (the native Nigrito) in Thungwa, Manang, and La-Ngu Districts.

4) Educational Tourism. There are many fossil sites in Satun Geopark for geo-scientist, geologist, students, and

other people who interested in studying variety of fossil. The Satun Geopark terrain originated as a sea floor in the Cambrian

Period more than 500 million years ago. The oldest fossils of primitive sea life in Thailand were found in the red

sandstones on Tarutao Island.

As various types of attraction points and variety activities provided in Satun Geopark, the logistics

management is consequently applied to sustainable tourism. From the review literature, logistic management of sustainable

tourism of geopark can be categorized into four components and applied to Satun UNESCO Global Geopark as

in Table 1. The components of logistics management for sustainable tourism are 1) transportation 2) amenities 3)



geological resources and landscape, and 4) environmental management as well as creating value of tourism

products using three supporting factors: information and communication technology; innovation; and sustainable

management provide tourism value for the tourists in terms of flexible, convenient, fast, and safe.

The evaluation of the particular component of logistics management for sustainable tourism of Satun

Geopark is as following:

1) Transportation. There are three sub-component combining to Satun Geopark. A) The transportation from the

visitor’s origin to Satun Geopark is essential for the visitor to decide whether they travel to travel site or not. The transportation

information should inform the visitor how to go to the main attraction points such as the type of vehicle, the time

table of the train from the nearby airport or train station to Satun Geopark and back. B) The transportation network

among geo-point is significant. In the present, the visitor or the tourist would like to travel by public transportation.

The local transit in Satun Geopark has to be improved to support the coming visitors travel from site to site. There

is still a lack of local transportation. The best way for the visitor is to rent a car or a motorcycle, but there is an insufficient

service provider in the area. C) Transportation from Satun Geopark to Langkawi Island, Malaysia. Because it is

convenient for the visitor to travel from Langkawi Island to Satun Geopark, it takes about one hour by speed boat

from Langkawi Island to Li-Pe Island. Also, there is an international airport in Langkawi Island. The transportation

in the area is the obstacle for Satun Geopark tourism development, and it has to be improved shortly.

2) Amenities. It composes of accommodation, restaurant, primary healthcare and hospital, public service, and

travel service provider. The accommodation in Satun Geopark is hotel and homestay. There are many hotels and

resorts in Li-Pe Island and land, but there is still a lack of homestay in the area. For primary public healthcare and

hospital, there are emergency medical service ( EMS) system provided by every Sub-District Administrative

Organization (SAO) in Satun Geopark, but in some SAOs have to improve their services. There is also a marine

emergency service from Tarutao National Park located at Rawi Island. The public service in the area is security

checkpoints for the police. The travel service providers are private businesses mostly located near Pak Bara Port.

3) Geological Resources and Landscape. The sub-component of this part are a territory and natural beauty,

ecological value, nature, cultural heritage, and landscape design around the geopark site. There are abundant

resources of attraction in Satun Geopark. The landscape around geopark must have well design and fit the nature.

4) Environmental management. There is four sub-component consist of water supply, electricity supply, waste

disposal, and air pollution control. In the present, there are wastewater and waste disposal in some areas. This

problem has to be appropriately diminished. The Chief of SAOs and stakeholders in the area have to solve these

problems. The obstacle is political in the local areas.

Conclusions

Satun Geopark is recently UNESCO Global Geopark. Being a global geological park can attract eco-tourists

from all over the world. Therefore, in the Satun geopark, there must be efficient and proper tourism management

not only to preserve the existing ecosystem but also impress the tourists. Logistics management is consequently

applied to sustainable tourism. Satun Geopark logistics management can be categorized into four components. It

consists of geological resources and landscape; transportation; amenities; and environmental management as well

as creating the value of tourism products using three supporting factors: information and communication technology;

innovation; and sustainable management provide tourism value for the tourists in terms of flexible, convenient, fast, and safe.

The logistics management for sustainable tourism needs a lot of support from both local and central

government. The government has not only promoted and supported the tourism industry, but also requiring the

tourism industry to be a part of the country’s strategic plans such as National Economic and Social Development Plan,

National Tourism Development Strategy, and Service sector development strategy of Thailand. The strategies

focus on driving policies to promote, support and develop tourism, both products and service systems, to be able

to facilitate tourists for maximum efficiency along with sustainable development for tourism which will lead to

income generation in the country and increase the competitiveness of the country.

Acknowledgement

The authors acknowledge the financial support provided by The Thailand Research Fund (TRF) for

research project “Logistics Management for Sustainable Tourism of Satun UNESCO Global Geopark”.


References

Cheung, TOL; Fok, L. 2014. The motivations and environmental attitudes of nature-based visitors to protected

areas in Hong Kong. Available on: https://mailattachment.googleusercontent.com/attachment/u/0/?ui=2

&ik=8381694a82&view=att&th=164b059fb9885723&attid=0.5&disp=inline&safe=1&zw&saddbat=A

NGjdJ9 (16 July 2018)

Lewis T.O. Cheung, Lincoln Fok & Wei Wei Fang. 2014. Understanding geopark visitors' preferences and

willingness to pay for global geopark management and conservation. Available on:

https://www.tandfonline.com /doi/abs/10.1080/14724049.2014.941848 (16 July 2018)

Petrick. 2014. Genetics, neuroscience, and psychopathology: Clothing the emperor. Available on:

http://globalgeoparksnetwork.org/wp-content/uploads/2015/09/6th-International-UNESCO Conference-

on-Global-Geoparks_Sept_13.pdf (16 July 2018)

Piboonrungroj and Disney. 2012. Tourism supply chains: a conceptual framework. PhD Networking Conference.

Tourtellot Jonathan. 2011. UNESCO’s geoparks “Clarify” geotourism. Available on:

https://blog.nationalgeographic.org/2011/11/16/unescos-geoparks-clarify-geotourism/ (2 July 2019)

Zouros N.,Valiakos I. 2010. Geoparks management and assessment. Available on

https://ejournals.epublishing.ekt.gr/index.php/geosociety/article/view/11262 (16 July 2018)



Table 1. The Components of Logistics Management of Sustainable Tourism of Geopark of Satun UNESCO

Global Geopark.

Component Sub-Component Source

1. Transportation - Transportation from origin to Satun

Geopark

- Transportation network in Satun

Province and nearby

- Transportation from Satun Geopark to

Langawi Island, Malaysia

Zouros N.,Valiakos I.

(2010),Lewis T.O. Cheung

(2014), Piboonrungroj and

Disney ( 2012(

2. Amenities - Accommodation

- Restaurant

- Primary healthcare and hospital

- Public service

- Travel service provider


(2010 ,Lewis T.O. Cheung

(2014)

3. Geological Resources and

Landscape

- Territory and natural beauty and

ecological value

- Nature

- Cultural heritage

- Landscape design around geopark site


(2010), Petrick (2014)

4. Environmental management - Water supply

- Electricity supply

- Waste disposal

- Air pollution control


(2010), Petrick (2014)



Analysis of factors related to bamboo structure for preventing erosion and restoration

of coastal area on the Upper Gulf of Thailand

Patcharaporn Yaowasooth Marine and Coastal Resources Research and Development Center, the Upper Gulf of Thailand, Muang District,

Samut Sakhon, Thailand Corresponding author e-mail: [email protected]

Abstract: Coastal erosion caused by anthropogenic, as well as natural processes in the coastal region of Thailand

and other countries is becoming a big issue at present days, because coastal erosion have negative impact on

human and coastal ecosystem. To prevent coastal erosion in the Upper Gulf of Thailand, the Department of Marine

and Coastal Resources (DMCR) carried out a project for the installation of bamboo structures and planting of trees

in the coastal area. At the end of the project, it has been found that the restoration of coastal ecosystem and

prevention from further coastal erosion were succeeded in some areas, on the other hand different result shown in

other areas. Based on the DMCR project outcome, this study analyzed the factors related to prevention of erosion

and the restoration of the coastal zone structure, in the Upper Gulf of Thailand. This study found that the success

of the project that is the prevention of erosion and restoration of the coastal area related with people’s participation,

strong leadership, government support and specification of bamboo. From focus group discussion, it was found

that strong policies for the protection and maintenance of installed bamboo structures and thereby appropriate

revision of policy are also an important factor for sustainability of the project. Moreover, devoted local leader,

and active committee members are needed to maintain, and monitor the project. This study results are applicable

for other coastal regions of different countries, with similar soil characteristics, to prevent erosion and restoration

of coastal areas. Keywords: Coastal erosion, bamboo structure, Upper Gulf of Thailand

Introduction

Thailand has a coastline measuring of 3,148 km, divided into the Gulf of Thailand with a length of 2,055

km and rest 1,093 km with the Andaman Sea. Coastal erosion mainly occurs along the coast of the Gulf of Thailand.

In seventeen provinces of Thailand 830 km along the coastline has been eroded, where 730 km belong to the Gulf

of Thailand and rest only 100 km coastline belong to Andaman Sea (Suraswadi, 2016). It also observed that the path

of erosion goes from the east to west coast of the Gulf of Thailand. Considering the erosion rate, some areas are declared

as critical area, where the erosion rate is over 5 meters per year, in other areas rate of erosion is 1-5 meters per year.

The Upper Gulf of Thailand, which started from the Bang Pa Kong River mouth to the Tha Chin River mouth

with a coastline of about 41.8 km has suffered the most severe erosion. From 2009, the rate of mangrove forest

deforestation in the Upper Gulf of Thailand is high and there has been a shift of livelihood along the coast to shrimp

farms. This trend increased the shrimp farms from 64,336 hectares to 237,220 hectares within the year of 2009 to

2016, it also damaged mangrove forest higher than 64 % (DMCR, 2011). Besides that, growing salt farms,

industry, agricultural and infrastructure make the deforestation problem more critical. The effect of coastal

erosion, Thamma-apipon, (2012) found coastal erosion affected the local communities in three aspects; there was

an economic impact, social impact, and environmental impact. Coastal erosion has effects to ecosystems and natural

resources. Mangrove forests, coral reefs, sea grass and land will be affected directly, ultimately, it affects the ecological

balance in the marine and coastal area. Coastal erosion affects the environment including; loss of land, mangrove

deterioration, marine animal reduction, loss of coastal scenery, adaption of house structure and prevention of erosion.

These directly affect the coastal community. Coastal erosion affected the economic on fisheries, aquaculture,

housing, tourism, and industry. After the effect of an economic system, people lose their resources, land, jobs, and

income etc. Tourism is the main source of income for Thailand. Besides that, industry of fisheries can create jobs

and increase income for the community as well as the country. Almost one-fourth of Thai people live in the 22

coastal provinces. That coastal erosion has an effect on the economic sector by various ways. It reduces the value

of land, results in income loss, and increases the retraction of the house and coastal defence structure costs. Dean

(2009) found that the cost of different erosion control structures is especially pertinent given the current recession.

The division of coastal management, in all states, reduces its budget by 7% (Gregson, 2009) and this reduction in

budget made the impact on enforcement and permitting personnel for local erosion control structures. And coastal

erosion has the impact on the social life of the people who live in coastal areas, and the number of affected people

are more than 12 million. Communities affected by erosion, lose their land and property, must change lifestyle by

migration. It is also the cause of changing the folkway of humans in coastal areas. Coastal communities migrated

to other areas which resulted in loss of life and cultural traditions of the communities; left some communities



homeless and resulted in the unavailability of work which normally has effects to other sectors of the economy.

Anxiety in a new career that may affect the state of mind and family relationships. Moreover, it is the cause of declining

living standards or not as good as the original. (DMCR, 2011B)

Community leader Khok Kham and Phanthai Norasing, Samut Sakhon Province of Thailand organized

a meeting with the local people for finding the solution of coastal erosion by bringing the folk wisdom. Local

fishermen are using bamboo for make fish trap and stable for shell aquatic. Fishermen noticed that after installation

of bamboo for aquaculture mussels, sediment accumulated near the area of bamboo streak. Observing that, they

installed bamboo streak adjacent each other without keeping any space between the streaks, which generate more

sediment accumulation easily and reduced the wave action. Local people used this technology to prevent coastal

erosion and increase the sediment along the coastline, and also planted mangrove tree to restore mangrove forest.

Coastal erosion issues are recognized to be solved urgently. The technology of using bamboo streak to

prevent erosion in the coastal areas by community are now proven. DMCR with community in coastal areas started

DMCR project to prevent coastal erosion by installation of bamboo structure, which reduced the weave energy,

generate sediment accumulate and helps for reforestation. The project started in 2011 on the Upper Gulf of

Thailand and includes eight areas: Khok Kham Moo 3, Khok Kham Moo 8, Bang Ya Praek Moo 6, Phanthai

Norasing Moo 6, Bang Krachao Moo 1 Sub-district, Samut Sakhron Province, Bang Kaeo Moo 10 Sub-district,

Samut Song Kram Province, Laem Fa Pha Moo 11 Sub-district, Samut Prakan Province and Song Khlong Moo 9

Sub-district, Chachoengsao Province. Communities with DMCR improved the design of the bamboo structure.

The summary of the design was bamboo size at least 3 inches width with 5 meters long, installed in 5 rows, depth

of about 2 meters emerged into the ground and 3 meters kept above from the ground, installed row alternating

zigzag design with the bamboo stick and at least 35 stick per 1meter bamboo structure combined with 5 row. The

ways for boat is 10 meters long and had a bamboo streak to cover the boat ways about 10 meters (Figure 2). This

project is a good example for prevent erosion, restoration of coastal area and local people are willing to prevent

erosion to make their life stable. In 2013 Intatha studied about factors influencing the capacity of communities to

respond to coastal erosion in the Upper Gulf of Thailand. This study shows. In the past most common method

used to protect the coast was rock placements (hard structure). But later soft structure such as bamboo structure

used to protect high coastal erosion. Moreover, the government and other stakeholders had applied a combination

of protection and restoration by installation bamboo structure and planting trees. This combined method shows

better results.

DMCR project found some areas were succeeded to prevent coastal erosion and restoration of coastal

area by using bamboo structure, but some areas were failed. This experience make an interest to researchers to

find the factors related to success of bamboo structure to prevent erosion and restoration of coastal area on the

Upper Gulf of Thailand and to determine the findings to be incorporated in existing guidelines and policies for

review as well as successful implementation by using bamboo structures to prevent erosion and restoration of

coastal areas. Besides that, this study results will be useful for policy modification and implementation to prevent

erosion and restoration of coastal area.


Study area

The study focused on three areas of the Upper Gulf of Thailand where DMCR project implemented. The areas

are; Bang Ya Phraek Sub-district, Samut Sakhon Province, Bang Kaeo Sub-district, Samut Songkhram Province,

and Song Khlong Sub-district, Chachoengsao Province, Thailand.

Data collection and sampling methods

Primary and secondary data both are collected for this research which is particularly relevant to the study.

Primary data was collect from questionnaires (Quantitative survey) a sample population of 333 people from 1973

people (National statistical office Thailand, 2016) as well as from focus group discussion (Qualitative survey).

This research conducted in coastal areas of Bang Ya Phraek sub-district, Samut Sakhon province, Bang Kaeo sub-

district, Samut Songkhram province, and Song Khlong sub-district, Chachoengsao province, of Thailand. Secondary

data was collected from various sources such as articles published in journals and books etc.


Sample size: Theoretical aspects, formulas. Simplified formula for proportions (Taro Yamane, 1967)

𝑛 =𝑁

1+𝑁(𝑒)2

𝑛 =1973

1+1973(0.05)2

𝑛 = 333 people

n = the sample size

N = the population size

e= the acceptable sampling error

*95% confidence level and p = 0.5 are assume

Data analysis

Data analysis is one of the most sophisticated activities of the study to produce the correct results. In this

study, quantitative and qualitative data were used for analysis. One of the best software for analyzing statistical

data Statistical Package for Social Sciences (SPSS 15.0 for windows evaluation version) was used in this study.

Quantitative data analysis

The data collected through a personal interview by using pre-designed questionnaire and collected data

analyzed by SPSS. Inferential statistics and descriptive both types of data were collected for quantitative data

analysis. Collected data were coded, sorted, and categorized for analyzing by software. Analyzed data used to

prepare Multinominal logistic regression to analyze the factors related to a bamboo structure to prevent erosion

and restoration of coastal area on the Upper Gulf of Thailand.

Multinominal logistic regression

In logistic regression analysis, there is only one independent variable, the logistic regression model can

be written as

Pr𝑜𝑏 (𝑒𝑣𝑒𝑛𝑡) =𝑒𝛽0+𝛽1𝑋

1 + 𝑒−(𝛽0+𝛽1𝑋)

Or Pr𝑜𝑏 (𝑒𝑣𝑒𝑛𝑡) =1

1+𝑒−(𝛽0+𝛽1𝑋)

β0 and β1 Coefficients were estimated from the data

X is an independent variable

E is natural logarithm with a value of 2.718

From the above equation, we derive a new equation as

Pr𝑜𝑏 (𝑒𝑣𝑒𝑛𝑡) =𝑒𝑧

1 + 𝑒𝑧

Or Pr𝑜𝑏 (𝑒𝑣𝑒𝑛𝑡) =1𝑒𝑧

1 + 𝑒−𝑧

By Z= β0+β1X1+ β2X2+……………+ βpXp and the event not as incident from the equation.

Prob (no event) = 1-Prob (event)

Qualitative data analysis

Focus group discussion and interview with community leader was done to accumulate qualitative data.

In focus group, discussion representative from local dwellers, local government officer, and a representative from

different occupation of that area were confirmed.

Result & Discussion

Socio-economic characteristics of the respondents The number of population in the study areas were 1,973 people. For personal interview 333 respondents

were chosen randomly. Almost 90% of respondents were local dwellers and their life is dependent on marine and

coastal ecosystem for livelihoods. Most of the respondents age limit were in the range of 41-60 years. The main

occupation of the dwellers in the study area was fishery and their education level was primary 71%, Major part of

the respondents 66%, monthly income was in the class of $142- $425 (5,001 – 15,000 Baht) the result is similar

to Thai people’s average income 3,976-12,275 Baht (World Bank, 2016).



Awareness about coastal erosion issue of the respondents The study found that most of the respondents in the study area were aware of coastal erosion, and the value

was higher than 85%. The awareness of respondents about social (migration, change occupation etc.), economic

(loss of income, loss of land, spend money for protecting land and house etc.), and environmental (loss of mangrove

forest, coastal resources degraded etc.) effect due to erosion also analyzed in the study. The results of the study

showed that the respondents are firstly conscious about the social effect, then secondly the economic effect, and

thirdly the environmental effect.

Respondent’s opinion about the impact of bamboo structures About the impact of bamboo structures in preventing erosion and restoration of coastal areas as well as

in socio-economic factors a significant portion of the respondents had a positive view. 34% respondents thought

the impact of bamboo structures on social benefit is high, where 37% respondents thought the impact bamboo

structures of economic benefit is high, and highest 63% respondents thought bamboo structures have high positive

impact on environmental benefit. On the other hand, for social benefits perspective, 39% respondents thought the

impact of bamboo structure in social benefit is low, where 38% and 25% respondents thought the impact of bamboo

structure in economic and environmental benefit is low respectively.

Respondent’s opinion about factors of success Budget

Budget is the main driving force in success to prevent erosion and restoration of the coastal area.

Respondent’s first preference was the government fund collaboration with the community. Their second choice

was NGO fund collaboration with community and the third was only community funded project.

Participation of stakeholders For success of the project, participation from different stakeholders is another important factor. Among

all stakeholders, respondent's first priority was participation of leader, the second priority was participation of the

community, and third was government official participation.

Miscellaneous factors Comparing various factors together, the study results found shows that specification of bamboo (type, species,

age, and thickness etc.) was the first preference from the respondent. Environmental (area, wave, and wind etc.)

condition was in the second position and in the third position the species of tree used for restoration of the coastal area.

Respondent’s opinion about policy and regulations For management of coastal area "The strategies for erosion control" policy of Thailand was established

in 2012. However, the contents of the policy is not enough to describe the management of bamboo structure for

erosion control in coastal areas. This study found that most of the respondent’s suggestion is to modify the current

policy without delay for providing more emphasis on using bamboo structure to prevent erosion and restoration

of coastal area. Moreover, almost 65% respondents suggested that policy should be modified after discussion with

the community of the coastal area, as well as policy should be modified as per suggestion of the community. For

the success of the policy implementation, 43% respondents recommended that collaboration of government and

community will be the main factor.

Participation of the Respondents in the Project Considering the full study area, the study found that participation in the project activity of respondents

was significantly high. 75% of the respondents were present in clarification meeting about use of bamboo structure

to prevent erosion and restoration of the coastal area. On the other hand, considering individual study area

participation rate was different, and values were 92%, 65% and 46% in Bang Ya Phreak, Bang Kaeo and Song

Khlong sub-district respectively. Similar results found for installation of bamboo structures and planting mangrove trees. 56% respondents

participated in the installation of bamboo structures and planting mangrove trees. But for individual area,

participation rate was 79%, 45%, and 17% in Bang Ya Phreak, Bang Kaeo and Song Khlong Sub-district respectively. Participation for monitoring after installation and planting for full study area was 50%. Where, for individual

study area participation values were 63%, 49% and 27% in Bang Ya Phreak, Bang Kaeo and Song Khlong Sub-district

respectively. 77% of all respondents, recommended that the maintenance activity should be continued after installation

of bamboo structures, and planting mangrove trees to prevent erosion and to restore the coastal area. Besides that,

respondents demand to continue maintenance activities were recorded at 94%, 71%, and 49% in Bang Ya Phreak,

Bang Kaeo and Song Khlong Sub-district respectively. Data related to participation in project activity shows, in Khlong Sub-district the participation rate was

very low compared with Bang Ya Phreak and Bang Kaeo Sub-district.


Statistical analysis 33 factors were included in the questionnaire survey to find the most effective factors for using bamboo

structures to prevent coastal erosion and restoration of the study area. The collected data was analyzed by

Multinominal regression model of SPSS program. The output shows the factors that are significant with project success.

In this study, failed and partially successful area’s data was analyzed through comparing the data of successful area. Multinominal logistic analysis showed among the 33 factors 4 factors were highly significance for project

success and the significance results between project success and these four factors at 0.01 significance level (Table 1).

Four significant factors are leadership, participation in project activities (includes meeting, installation of bamboo

structures, planting trees, monitoring and maintenance), government support, and the specification of bamboo.

It indicates, to make a project success for prevent coastal erosion and restoration of coastal area strong leadership,

active participation of the beneficiary in project activity, government positive support, and maintain the quality of

bamboo by following the specification is essential.

Project leader’s opinion The researcher also carried out personal interviews with the project leader of the 3 study area. All leaders

came from the local community, but they have different views and different level of involvement in the project activity.

Bang Ya Phreak Leader’s opinion Leader of the Bang Ya Phreak was the initiator of using bamboo structures to protect coastal erosion in

his area. Bang Ya Phreak area facing the coastal erosion problem for a long period of time, and it destroyed their

land and property. Rock protection work, for controlling erosion, was not working properly in that area. Besides

that, rock protection created more erosion to the adjacent area after construction. Another problem was the very

clayey soil of Bang Ya Phreak area, which are not suitable for rock protection. At that time, he observed the

success of protecting coastal erosion in Khok Kham Sub-district by using bamboo structures. This observation

encouraged him to initiate bamboo structure installation in Bang Ya Phreak area to prevent erosion, and restores

more area by increasing sedimentation. To start that activity, he spend his personal income, as well as convinced

the community to organize a fund for installing bamboo structure to prevent erosion. He identified erosion as an important issue for coastal ecosystem. He address erosion issues in the order

of the economic effect, the social effect and the environmental effect. He has been highly involved with these

activities for more than 11 years. The people who were not capable or had no interest in bamboo installation

activity, he involved them in other supporting works like establishing a mangrove tree nursery by donating mangrove

trees for restoration of the coastal area. He also invited the children in the area and took them to learn folkways

and planting of mangrove trees. Moreover, he communicated with the universities, colleges, and schools to send

their students for participating in bamboo installation and mangrove tree planting. Among the factors related with the success of the prevention of erosion and restoration by using bamboo

structures, he prioritized community participation with self-willingness is the most important factor for success,

followed by the specification of bamboo and lastly, the involvement of stakeholders from outside of community

such as government, NGOs for support knowledge and budget. His opinion about the future of the project was

that, these activities should be continued to prevent erosion and restorations of the coastal area. Restore land and

mangrove forest will enhance the aquatic life as well as natural resources of the area. Proper and sustainable use

of those resources in the future, will enhance the community people's quality of life and help them to enjoy a

comfortable and happier life.

Bang Kaeo Leader’s opinion The project starting story was in Bang Kaeo area almost the same to Bang Ya Phreak area. Bang Kaeo

area also suffers coastal erosion for many years. The leader of Khok Kham project area known about the problem

of Bang Kaeo. The leader with people from his community visit Khok Kham. After visiting, Bang Kaeo leader

realized that, to prevent coastal erosion and restoration installation of bamboo structures should be start in Bang

Kaeo area. To implement the same project, he talked with the local community of Bang Kaeo area, but unfortunately

most of them were not agreed to do. At that stage, he decided to start the project by spending his own money for

set an example in front of local community with the help of agreed people. He was highly involved for more than 11 years with these projects as well as highly aware about coastal

erosion issue and the importance of the coastal ecosystem. He chose to address these issues in the following order:

social effect, economic effect and finally the environmental effect. He also communicated with some NGOs and

organizations that could provide funds. He found that, if they can show some success NGOs and other organizations

would support them because of their corporate social responsibility (CSR). He discussed all the findings with the

community and finally, the community agreed to start to install bamboo structures to prevent coastal erosion and

restoration of the area. The leader of Bang Kaeo had a view that, the factors related with the success to prevent erosion and

restoration by using bamboo structure are interconnected. He prioritized community participation are the most

important factor for success. Quality of bamboo and monitoring the repair work were his second priority. His third

priority was the approach of leader. He believes that a leader should maintain the approach of ‘not give up on a task



until you succeed’. His opinion about the future of the project was that, these activities should be continued for a better

future of the community. He believes that this project will be the best example for green technology to prevent

coastal erosion and restoration. Besides that, from the success of the project, future generations will get a better

quality life.

Song Khlong leader opinion The project opening story in Song Khlong area was different from the other two study areas. Before starting

the project activities, officers of the project organized a meeting with the leader and the community of Song Khlong

area. In the meeting the number of participants from the community was low. After that, officers arranged more

meetings with the leader and gave him instructions to ensure the participation of the community in the project activities.

However, later on, without ensuring community participation, project officers installed bamboo structures with

the help of laborers. Due to the lack of monitoring, after a few years they found all the bamboo structures were damaged. He was involved in 5 years in project activities, He considered economic effect is the first, social effect

is the second, and finally the environmental effect is the third important issue for erosion in the coastal ecosystem.

He believes that, in Song Khlong area, rock structures will be effective for preventing coastal erosion instead of

bamboo structure, but for restoration of that area planting mangrove trees will be the good solution. He also

suggested that before starting the project, the government should make some focused group discussions with the community

to check the willingness of the people to participate in the project. Among the various factors, the participation of the community was chosen by him as the most important

factor for successful prevention of coastal erosion and restoration of the coastal area by using bamboo structures.

The natural environment in the coastal area (strong winds and high sea waves, soil characteristics, etc.) was

selected by his as the next important parameter for success of the project. Beside these, he recognized specification

of bamboo, and policies of the government are also important factors for success of the project.

Focus group discussion In focus group discussion, the main topic was "Guidelines for policy modification on use of bamboo

structures to prevent coastal erosion and restoration of the coastal areas successfully by the community ". The guidelines

for policy modification and implementation formulated by the focused group discussions were community involvement

should be the first priority at the design stage of the project. Participation of local community should be ensured

from the beginning of the project and continue until the end. At the start, this includes looking for the project site or

area, planning for the project, analysis of the problem in the area, evaluation of the damaged resources. During

implementation stage, it includes installation of the structures and restoration activities, monitoring activities, and finally

evaluation of the project. Sustainability of the structure depends on regular monitoring and maintenance, for this reason, sufficient

budget have to insure for maintenance program. Knowledge should be shared with the community people as well

as the officers of the project. Besides this, training about implementation, monitoring, and maintenance activities,

as well as visits in success areas to get acquainted with their experiences should be organized. Ownership of the newly restored areas should be belongs to the government. Restored area should not

be sold or rented by the government. Moreover, the government must include instructions on the policy about the

use of restored lands for community as public goods with sustainable resource management. The policy should

emphasize for increase awareness about economic benefits, environmental benefits, and social benefits of the

community by using bamboo structures for prevention and restoration of the coastal area. Penalty impose methodology to a person or a group who destroy the bamboo structures or hampers project

activities should also be included in policy. To select the leader with high morality and awareness on environmental,

policy should provide a proper guideline through community discussions.

Conclusions

The Upper Gulf of Thailand facing severe coastal erosion because of natural process and human

activities. The DMCR has the project to prevent and restoration of the coastal area on the Upper Gulf of Thailand

by using bamboo structures. The bamboo structures suitable sediment characteristics in that areas. Sediment

characteristic on the Upper Gulf of Thailand is sandy clay loam (Sawangarreruks at all, 2013). After implementation,

DMCR found the bamboo structures can prevention of erosion were succeed in many areas, but in some areas it

failed. This study was done to find the factors related to the success of using a bamboo structure for preventing

erosion and restoration of coastal area in the Upper Gulf of Thailand. A pre-designed questionnaire survey, consisting of 33 factors was done to collect data from 333 respondents

about the success of the project using the bamboo structure for preventing erosion and restoration of coastal area.

The data was analyzed by Multinominal regression model of SPSS program and found that among the thirty-three

factors, four factors were highly significant with the significant value at 0.01 and had relation to the success of

DMCR project. Four significant factors for success of the project were people’s participation in the project

(installation of bamboo, planting trees, meetings, and maintenance), strong leadership, government support for various


activities, and the specification of bamboo. The result from questionnaire survey shows that 75% of total respondents

participated in the project activity, where the percentage of participation was higher than 90% in the successful

area. Strong leadership is essential for project success was chosen by 60% of the respondents. Where government

support on different activities is an essential factor for success was chosen by 84% of the respondents. Comparing with

various quality issues and environment factors, specification of the bamboo was chosen by 90% of the respondents. (Appendices) Three focus group discussions were carried out in the study area to get some guidelines for policy

modification and implementation about using bamboo structures to prevent erosion and restoration of coastal

areas. The findings from the focus group discussion were, policy should be more specific and details about using

bamboo structure and current policy should be modified without delay for successful implementation of the

project. Penalty impose methodology should be included in the policy for destroying the bamboo structures and

hampering the project activity. Moreover, leader with high morality should be chosen by the community.

Policy recommendations 1. Do not carry out activities or do any actions that may result in or cause problems in increasing coastal

erosion.

2. Establish rules for the construction process to prevent coastal erosion.

3. Continuing the project to prevent coastal erosion in the area.

Acknowledgement

Above all, I would like to profoundly thank the Korea Environment and Technology Institute (KEITI)

of the Government of the Republic of Korea for tuition and subsistence allowances. Sincere thanks to the Ministry

of Natural Resources and Environment (MNRE) of Thailand for granting me this opportunity to acquire more

knowledge in my field of interest and for their cooperation. I am grateful and very thankful to my advisor,

Professor Jin-Young Jung Ph.D., and Dusit Wechakit Ph.D. from suggestions, teachings, imparting of knowledge

and data to the unconditional contribution directives that greatly improved this paper. I would like to extend my

gratitude to Professor Yohan Lee Ph.D. and Ferdous Ahmed for their assistance to improve this paper, the support,

encouragement and care rendered to me.

References

Department of marine and coastal. 2011. “Prevention of coastal erosion in Thailand”. Strategic Management

Prevention of coastal erosion. Marine and Coastal Resources Journal, 20 December 2018. 70 – 78.

Department of marine and coastal. 2011. Monitoring and evaluation result of monitoring and evaluation the

restoration of coastal ecosystems. By planting bamboo slow wave Samut Prakan and Chachoengsao

province case study. Chulalongkorn University. Bangkok: GRT.

Department of marine and coastal. 2013. “Coastal areas of Thailand”. Accessed February 25. 2016.

http://marinegiscenter.dmcr.go.th/km/coastal-zone-management01 /#.V12 fs 7t97IU.

Department of marine and coastal 2015. Bamboo slow wave manual to prevent coastal erosion. DMCR, Bangkok: GRT.

Gregson, J. 2009. Executive Secretary notes. Paper presented at the NC Coastal Resources Committee Meeting

February 2009, Morehead City, NC.

Intatha, C. 2013. Factors influencing the capacity of communities to respond to coastal erosion in the Upper Gulf

of Thailand. Mahidol University, Thailand. National statistical office Thailand. 2016. “Number of

population”. Accessed May 8. 2016. http://service.nso.go.th/nso/nsopublish/districtList/page1.htm

Statistics Solutions. 2016. “Multinomial Logistic Regression.” Accessed December 23. 2016.

http://www.statisticssolutions.com/mlr/.

Suchart Sawangarreruks. S., lertkasetvittaya. N., Yaowasooth. P., luangkhamin. C., Passada and Nikomjit. S.

2013. “After effect of flooding on the Macro benthic community in the Upper Gulf of Thailand.” Key

challenge of marine Biodiversity Conservation in our Changing Climate 9-11 September 2013,

Department of Marine and Coastal Resources Research and Development Center Upper Gulf of Thailand.

Suraswadi Pinsak. 2016. “The new coastal resources law, Marine protected areas and nature-based solutions for

coastal erosion in Thailand.” Accessed December 20. 2018.

https://www.iucn.org/sites/dev/files/import/downloads/1431_1443_the_new_coastal_resources_law__

marine_protected.pdf

Thama-apipon Sanawaya. 2012. Studied the effects of the problems of coastal erosion on coastal communities.

Silpakorn University, Thailand.

World Bank. 2016. “Thai people average income.” Accessed December 20. 2018.

http://www.worldbank.org/th/news/press-release/2011/08/02/thailand-now-upper-middle-income-economy.

Yamane, Taro. 1967. Statistics: An Introductory Analysis, 2nd Ed., New York: Harper and Row.



Figure 1. Study areas on the Upper Gulf of Thailand. DMCR, 2013

Figure 2. DMCR bamboo stick structure. DMCR, 2015

Table 1. The results of multinominal regression model.

Project (a) B S.E. Wald Sig. Exp (B)

Failed Leadership 3.481 .817 18.171 .000 32.498

People’s participation 2.174 .389 31.180 .000 8.792

Government support 4.396 .786 31.273 .000 81.121

Specification of bamboo 1.339 .551 5.903 .015 3.815

Partially success Leadership .513 1.035 .246 .620 1.671

People’s participation 1.386 .305 20.598 .000 3.999

Government support 2.923 .780 14.047 .000 18.590

Specification of bamboo .068 .555 .015 .903 1.070

a. The reference category is: Success.

b. This parameter is set to zero because it is redundant

B = The regression coefficient of each independent variable that will be used to create the forecasting equation.

S.E. = Standard error.

Wald = Wald statistic, the way to find out if explanatory variables in a model are significant.

Sig. = Significant.

Exp (B) = Exponentiated of B.

https://www.statisticshowto.datasciencecentral.com/explanatory-variable/

https://www.statisticshowto.datasciencecentral.com/what-is-statistical-significance/



Exploring the suitability map of wild banana (Musa serpentina Swangpol & Somana)

in Thailand using species distribution models with the limited occurrence data

Thanayut Changruenngam and Jantrararuk Tovaranonte* School of Science, Mae Fah Luang University, Muang District, Chiang Rai, Thailand


Abstract: Species distribution model (SDMs) is one of the powerful tools to predict the suitability map to address

the ecology and conservation approaches. However, the limited number of occurrence data has been a problem in

model performance. Here, the three algorithms (MaxEnt, generalized linear model: GLM, and random Forest: RF)

were selected to project the suitable map with eight occurrences of Musa serpentina Swangpol & Somana wild

banana which endemic to the west of northern and central Thailand and eleven environmental variables. Due to a limitation

of occurrence data, the fuzzy logic had been applied to GLM and RF model to enhance the occurrence data.

MaxEnt is presence-only data, it could not use the fuzzy logic to create pseudo absence data. The results showed

that three climatic variables had affected all three models especially precipitation of warmest quarter (BIO8) and

precipitation of coldest quarter (BI019). All algorithms could predict the suitable map well with high AUC values

(> 0.9). GLM had the highest performance with AUC value of 0.991.

Keywords: MaxEnt, Random forest, GLM, Fuzzy logic, Musa serpentine

Introduction

Species distribution models (SDMs) play an important role for conservation of endangered species. It can

predict future results under climate change and generate species distribution models under geospatially explicit

layers of abiotic or biotic data which defines the ecological requirements of species under study (Franklin, 2010).

SDMs consist of 2 categories according to the characteristics of data. First, presence data-only algorithms such as

genetic algorithm for ruleset prediction (GARP) and maximum entropy (MaxEnt) (Phillips, 2008). MaxEnt is

commonly shown accurate prediction capabilities of these models. Second, binary presence/absence data

algorithms such as classical modelling approach: the generalized linear model (GLM) and modern method:

random forest (RF) and boosted regression trees (BRT). From previous studies, consideration of the actual values

of the predictions which emphasize more clearly found that MaxEnt, BRT and GLM performed well, followed by

RF then GARP (Elith & Graham, 2009). The applications of SDMs are widely available, such as predicting

distribution of disease vectors. (Khatchikian et al., 2011). The other applications include conservation of the

suitable habitat of species (Tovaranonte et al., 2015) and the evaluation of species vulnerability to climate change

(Trisurat, Shrestha, & Kjelgren, 2011).

Musa serpentina Swangpol & Somana (‘Nakkharat’ or ‘Naga’) is a rare wild banana in Musaceae. It is

endemic to the west of northern and central Thailand such as Tak, Mae Hong Son and Kanchanaburi Provinces.

It commonly grows in the open mixed deciduous forest by stream banks or low slopes by roadsides. The altitude

of habitat is approximately 240-570 meters above sea level. Due to a small seed set and drastic fragmentation, its

conservation status is considered as endangered species based on IUCN Red List Categories and Criteria: Version 3.1

(Criteria D in Section V; IUCN, 2001)(Swangpol & Somana, 2011).

SDMs should provide conservation practitioners with the estimated spatial distributions of species

requiring attention. These species are often rare and have a small sample size, posing challenges for creating

accurate species distribution models. The studies indicated that the number of known occurrences had greatly

affected the accuracy of SDMs whereas MaxEnt always had the highest accurate predictive ability (Hernandez,

Graham, Master, & Albert, 2006). Wisz et al. (2008) compared AUC form 14 SDMs and found MaxEnt and GLM

had higher performance. GLM gave the highest AUC compared to RF and MaxEnt in different species distribution

(Elith & Graham, 2009). However, RF algorithm can also generate better species predictive distributions for a

habitat prediction of small sample size. while MaxEnt had lower performance (Mi, Huettmann, Guo, Han, & Wen,

2017). Since the sample size of Musa serpentina is small. Hence, the suitability map of Musa serpentine should

be predicted by MaxEnt, RF and GLM algorithm.

GLM and RF are algorithms for binary presence/absence data. The methods of pseudo-absence selection

influence a performance of species distribution models which is binary presence/absence data (Mary S. Wisz &

Guisan, 2009). The pseudo-absence data was established by using opposite condition with environment of Musa

serpentina. Generally, the boundaries of suitable environment cannot be clearly defined. Therefore fuzzy logic is

a tool for increasing flexibility of boundaries of suitable environment. It is used to handle the concept of partial

truth where the true values may range between completely true and completely false that is these values of variables



are any real numbers in closed interval 0 to 1 (0 = false, 1= truth). These are a feature of the membership function

of fuzzy set.

Here, this study aimed to compare the predicted performance between GLM and RF which developed

by Fuzzy pseudo-absence data with MaxEnt for the small sample size condition. And there is establish the

suitability map for the conservation of the habitat of Musa serpentina.


Study area and the occurrence data

Thailand is located between 5°45′ and 20°30′N and 97°30′ and 105°45′E. The total area is 511,731 square

kilometers or around 200,000 square miles. There are eight localities of Musa serpentina were obtained from

published data (Swangpol & Somana, 2011), herbarium data and unpublished data

Figure 1. (a). Distribution map of Musa serpentina in the west side of northern and central regions of Thailand

(Swangpol & Somana, 2011) and (b). suitability map of Musa serpentina overlaid by the area of Band 4 and 5 of three models

Environmental variables

Twenty environmental variables were used at 30 seconds (1km2) resolution. Twenty climate variables

were downloaded from the WorldClim website (http://www.worldclim.org, 11th March 2017, Hijmans et al., 2005).

These variables are BIO1 (annual mean temperature), BIO2 (mean diurnal range), BIO3 (Isothermality), BIO4

(temperature seasonality), BIO5(Max temperature of warmest month), BIO6 (min temperature of coldest Month),

BIO7 (temperature annual range), BIO8 (mean temperature of wettest quarter), BIO9 (mean temperature of driest

quarter), BIO10 (mean temperature of warmest quarter), BIO11 (mean temperature of coldest quarter), BIO12

(annual precipitation) BIO13 (precipitation of wettest month), BIO14 (precipitation of driest month), BIO15

(precipitation seasonality), BIO16 (precipitation of wettest quarter), BIO17 (precipitation of driest quarter),

BIO18 (precipitation of warmest quarter), BIO19 (precipitation of coldest quarter) and Alt (altitude).

Fuzzy Logic

Fuzzy Logic is a computer system that works by fuzzy set invented by Zadeh (Zadeh, 1965). It is used

to handle the concept of partial truth where the truth values may range between completely true and completely

false (0 = false, 1= truth). These are a feature of the membership function of fuzzy set. It was created by the degree

of truth as an extension of valuation. The membership functions were used as input and output variables of the

rule-based system (IF-THEN rules) derived from knowledge. Fuzzy logic was used to create pseudo-absence data

for identifying suitable habitat of Musa serpentine. After that, areas with probability level of less 0.6 as area for

1,000 randomly sampled was used as pseudo-absence data or Fuzzy pseudo-absence data.

Fuzzy pseudo-absence data

A suitable habitat of Musa serpentina is in highly disturbed habitats of open mixed deciduous forest by

stream banks or low slopes by roadsides and altitude 240–570 meters (Swangpol & Somana, 2011). The open

mixed deciduous forest often found at height of 50-800 meters, rainfall range from 1,200-1,600 mm per year and

not found in the South of Thailand (Marod, Pinyo, Duengkae, & Hiroshi, 2010). The presence data and absence

data were defined as 1 and 0 respectively. A membership function of fuzzy logic was created with Triangular

function and the rule-based system as follows.

Input

Altitude (0 -1): low (less 300 m), appropriate (200 - 600 m), high (more than 500)

Rainfall (0-1): low (less 1300 mm), appropriate (1,100 - 1,700 mm), high (more than 1500 mm)

Legend

") serpentina

1

2

3

4

5

b a


Output

Minimum of the Fuzzy membership from the input (0 - 1)

Rule-based system 1. If altitude = low then 0

2. If altitude = High and then 0

3. If altitude = appropriate and rainfall = low then 1

4. If altitude = appropriate and rainfall = appropriate then 1

5. If altitude = appropriate and rainfall = high then 0

The map outputs were overlaid type "and" are shown in Figure 2. We used areas with a level of less 0.6

as an area for 1,000 randomly sampled to use as pseudo-absence data (Morgane, Frederic, Jiguet, Cecile Helene,

& Wilfried, 2013).

Figure 2. The fuzzy map in a fuzzy logic system to create pseudo-absence data from (a) altitude data, (b) rainfall

data and (c) The overlaid map

Modelling and evaluation methods

The program named MaxEnt version. 3.2.1 was used to project eight samples and 1000 background

samples were created randomly. The function random forest was used to build an ensemble of classification trees.

The model generated 500 trees from both binary presence data and Fuzzy pseudo-absence data. We used function

GLM to create all possible subsets of models with the options for each variable from presence data and Fuzzy

pseudo-absence data. Area Under the ROC Curve (AUC) was used to evaluate the performance of the models

(Liu, White, & Newell, 2011). The AUC is the current best practice for evaluating the success of a model for

binary presence/absence data (Rushton et.al, 2004). Therefore, the value of AUC is an efficient tool for testing

the performance of SDMs. For example, in 2013, the value of AUC was used to evaluate the distribution model

of the particle to find the most effective model (Tovaranonte et al., 2015). The criteria for the AUC values that

are frequently seen are 0.90-1.00: excellent, 0.80-0.90: good, 0.70-0.80: fair, 0.60-0.70: bad and 0.50-0.60: failed

(Swets, 2015). In addition, each model different in prediction which can be observed from the Pearson product-

moment correlation coefficient and percentage of duplicate area. Summarizing the modelling and evaluation

methods are shown in Figure 3.

a b c

Probability



Figure 3. Flow chart summarizing the modelling and evaluation methods.

Result & Discussion

The suitability map of Musa serpentine

The evaluation of habitat prediction from the sample areas revealed the generalizability of the model

predictions. This method was used to evaluate that occurrence data matched prophecy of the distribution area, as

a spatial assessment of model performance. It is a visual method to show the transferability of models from

sampled to interesting areas. The suitability map for Musa serpentina in Thailand was developed in three models

(MaxEnt, GLM and RF) as shown in Figure 4. Those maps were classified by the criteria for the probability values

are 0 - 0.20: Band 1, 0.21-0.40: Band 2, 0.41-0.60: Band 3, 0.61-0.80: Band 4, and 0.81 - 1: Band 5. Each Band

was shown in green according to the intensity of the shades. Hence, the dark green color indicated that the most

suitable locations were in the western part of Thailand.

The area predicted by MaxEnt model most appeared in Band 3 about 41% of Thailand. However, for

Band 4 and 5 where probability more than 0.6 had the sum area about 8%, spread in the west and the lower North

of Thailand. GLM model provided the most area predicted at Band 1 where 64% of Thailand after that the area

continued to decrease to 1% in Band 5. For RF model had no area of prediction in Band 1 but it had the most at

Band 2 (about 48%) and had the predicted area in Band 4 and 5 higher than the area predicted by MaxEnt and

GLM. Results of the predicted area of each model are shown in Table 1.

Band 4 and 5 of each model represented to the habitat of Musa serpentina and had a total area of 172,988 km2.

While the overlapping areas of three models had 30,942 km2 are shown in the green area (Figure 1b). The three

suitability maps predicted by MaxEnt, GLM and RF had high performance. However, the overlapping of these

maps represented the suitability map of Musa serpentina did likewise. Hence, the overlay maps cover all 8

samples are shown in Figure 1a. Therefore, this area is the suitability map that we recommend to be the

conservation of the habitat for Musa serpentina. Band 5 represented more specific residential of Musa serpentina

where the suitability maps from the three models had a total area of 46,281 km2. However, the area with the highest

probability presence was developed by overlapping of Band 5 in each model. It had 450 km2 spread in the North

and West of Thailand especially in Mae Hong Son Province (Figure 5d).

Fuzzy logic system

20 Environmental data

8 Occurrence data Knowledge base

Fuzzy pseudo

absence data Presence Data

MaxEnt GLM RF

Percentage of duplicate area

Pearson product-moment correlation coefficient

AUC test for a model performance


Figure 4. Suitability map of Musa serpentina in Thailand created from (a) MAXENT, (b) Random Forest and (c)

GLM.

Species distribution models MaxEnt, GLM and RF had high performance for creating the suitability map of Musa serpentina. Those

maps can provide conservation practitioners to estimate the accurate spatial distributions of Musa serpentina.

However, each algorithm had estimated in difference size of the area in the map. Therefore, the percentage of

duplicate area, Pearson product-moment correlation coefficient and AUC from each model were compared.

Figure 5. The same area predicted from MaxEnt, RF and GLM (a) Band 2, (b) Band 3, (c) Band 4, (d) Band 5

Percentage of the duplicate area represented the prediction area of each algorithm in the same band. However, the

predicted size of areas from three models were different. Band 1 had no duplicate area as the RF's map had a

probability of not less than 0.2. Range 0.21 - 0.40 (Band 2) had duplicated area for 52% which is close to GLM's

predicted area (Figure 5a) and Band 3 had 27% of duplicated area (Figure 5b). Band 4 and Band 5 were used to define

the suitability habitat because of the probability > 0.6. However, the predicted area of each model is different.

Therefore, predicted for the same area of Band 4 found 13 % (Figure 5c). Band 5 is the model's predicted area that

has the highest probability (>0.80), represented to the suitability map of Musa serpentina where all models shown

a duplicate area of 9 %.

Table 1. Percentage of predicting area by MAXENT, GLM, RF of Musa serpentine in Thailand.

Band Probability

Model’s predicted area of

Musa serpentine in Thailand (%) Percentage of duplicate

area (%) MAXENT GLM RF

1 0.00 – 0.20 28 64 0 0

2 0.21 – 0.40 24 24 48 52

3 0.41 – 0.60 41 6 25 27

4 0.61 – 0.80 7 4 21 13

5 0.81 – 1.00 0.06 1 7 9

The r-value of MaxEnt and GLM had a strong positive relationship (0.564) but the relationship between

RF with the remaining two models had a moderate positive relationship (Table 2). However, the reliability of

the correlation coefficient depends on hypothesis test of the significance of the correlation coefficient where H0:

ρ = 0, H1: ρ ≠ 0 represented no significant correlation exists and a significant correlation exists. Testing

the significance of the correlation coefficient of every pair of model's predicted maps had p-value ≤ 0.001 (Table 2).

Therefore, there is sufficient evidence to conclude that there is a significant linear positive relationship between

a

Legend

0-0.20

0.21-0.40

0.41-0.60

0.61-0.80

0.81-1

b c

Probability

b c d a



these prediction maps. In other words, MaxEnt GLM and RF established the suitability map of Musa serpentina

are in the same direction.

Table 2. the Correlation Coefficient of Model's predicted maps.

Model MAXENT GLM RF

r p-value r p-value r p-value

MAXENT 1

GLM 0.664 0.00* 1

RF 0.446 0.00* 0.478 0.00* 1

*P-Value < 0.05

The Area under ROC curve (AUC) was usually used to evaluate model accuracy. The prediction performance

of three models were similar. The three models had relatively high AUC values. Our results showed that the AUC

values from GLM were highest (0.991), followed by RF (0.989) and MaxEnt (0.931), respectively. Three variables included precipitation of warmest quarter, mean temperature of wettest quarter and precipitation of coldest quarter

had affected MaxEnt models especially precipitation and maximum temperature of warmest month, precipitation

of warmest quarter and precipitation seasonality which were the top three of the variables affected GLM model.

For RF model was the most affected from variables precipitation of wettest month, precipitation of coldest quarter

and precipitation seasonality. It can be seen that precipitation of warmest quarter (BIO18) and precipitation of

coldest quarter (BIO19) were the two variables that had affected all models. In addition, both variables represented

characteristic of the habitat of Musa serpentina with open mixed deciduous forest.

Conclusions

The prediction of the suitability map of Musa serpentina found that the most suitable locations were in

the North and West part of Thailand. However, the areas with the highest probability of presenting were in Mae

Hong Son Province cover the area of 450 square kilometers

MaxEnt is acceptable as a high-performance model for the prediction of the species distribution with

small sample size. Fuzzy pseudo-absence data can develop the algorithm of GLM and Random Forest as efficient

as MaxEnt at the small sample size. Although, the predicted area of each model had some differences whereas

PMCC test can represent that each algorithm established the suitability map of Musa serpentina in the same

direction. In conclusion, all three algorithms are highly efficient tools of species distribution models for limited

occurrence data

Acknowledgement

We would like to thank our colleagues at the Computational Science Department, School of Science,

Mae Fah Luang University for fruitful interaction and encouragement of the first author. We would like to thank

Chiang Rai Rajabhat University for supporting the ArcGIS software and Mae Fah Luang University for funding

this research.

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Stimulating the access to biodiversity and technologies to combat climate change

Fabrice Mattei

Rouse & Co. International (Thailand) Ltd, Klongtoey District, Bangkok, Thailand

Corresponding author e-mail: [email protected], [email protected]

Abstract: On 13 November 2017, in a joint statement issued by the Executive Secretaries at the UN Climate

Change Conference 2017 ("COP 23") taking place in Bonn, Germany1, the Executive Secretaries of respectively

the Convention on Biological Diversity ("CBD"), the United Nations Convention to Combat Desertification

("UNCCD") and the United Nations Framework Convention on Climate Change ("UNFCCC") called for

establishing a financial mechanism, called Facility, to secure finance for large projects that will help to address

common issues relating to climate change:“We are calling for the establishment of a new Project Preparation

Facility to bridge this gap and promote an integrated, coherent and multi-disciplinary approach to these related

issues while supporting the respective mandates of the three Rio Conventions.”2 This declaration officially

recognizes what was widely known; biodiversity and climate change are interconnected. Biodiversity is affected

by climate change, and equally the conservation of biodiversity contributes to mitigate climate change impacts3.

The promises made under the CBD and UNFCCC to conserve biodiversity and to combat climate change both rely

on "access". Facilitating the access to Genetic Resources ("GR") under the CBD while facilitating the access to

technologies under the UNFCCC and Paris Agreement ("PA"). This paper challenges the assumptions behind those

promises (1) before it evaluates the financial mechanisms which are supposed to stimulate the access to

biodiversity by developed countries and to climate change technologies by developing countries (2). It then

investigates what are the legal mechanisms which could further stimulate the access to biodiversity and climate

change technologies including empowering the Green Climate Fund ("GCF") to own and license climate change

technologies (3).

Keywords: Biodiversity, Technology Transfer, Climate Change, Green Climate Fund, Paris Agreement on Climate Change.

Introduction

On 13 November 2017, in a joint statement issued by the Executive Secretaries at the UN Climate Change

Conference 2017 ("COP 23") taking place in Bonn, Germany , the Executive Secretaries of respectively the

Convention on Biological Diversity ("CBD"), the United Nations Convention to Combat Desertification ("UNCCD") and

the United Nations Framework Convention on Climate Change ("UNFCCC") called for establishing a financial

mechanism, called Facility, to secure finance for large projects that will help to address common issues relating to

climate change:

“We are calling for the establishment of a new Project Preparation Facility to bridge this gap and

promote an integrated, coherent and multi-disciplinary approach to these related issues while supporting

the respective mandates of the three Rio Conventions.”

This declaration officially recognizes what was widely known; biodiversity and climate change are interconnected.

Biodiversity is affected by climate change, and equally the conservation of biodiversity contributes to mitigate

climate change impacts. The promises made under the CBD and UNFCCC to conserve biodiversity and to combat

climate change both rely on "access". Facilitating the access to Genetic Resources ("GR") under the CBD while

facilitating the access to technologies under the UNFCCC and Paris Agreement ("PA"). This paper challenges the

assumptions behind those promises (1) before it evaluates the financial mechanisms which are supposed to

stimulate the access to biodiversity by developed countries and to climate change technologies by developing

countries (2). It then investigates what are the legal mechanisms which could further stimulate the access to

biodiversity and climate change technologies including empowering the Green Climate Fund ("GCF") to own and

license climate change technologies (3).



1. Assumptions behind stimulating the access to biodiversity and technologies

1.1 Assumptions behind stimulating the access to biodiversity

Under the CBD, one way of ensuring the conservation of biodiversity is to facilitate its access and use for

commercial or research purposes by third parties through the Access and Benefit Sharing ("ABS")4. The promotion

of access to biodiversity is based on several assumptions, three of them. First, GR from biodiversity-rich countries

are ‘green gold’5. Second, limiting access to GR will inhibit the already failing bio-prospecting activity and any

financial and non financial benefits to share so that the conservation of biodiversity will be at risk6. Third, the

access to GR is mostly between resource-rich to biodiversity rich countries also known as North-South7. These

assumptions are challengeable. Studies reveal that access to GR by developed countries is less appealing than it

first appears, due in particular to the development of biotechnology and options for off-site access to GR. There is

no clear study confirming that access to biodiversity contributes to its conservation. There is a debate between

conservation and preservation of biodiversity. It contends that the access and sustainable use of biodiversity don't

necessarily ensure its preservation. Finally, the access and use of GR is not always north-south driven8. Since the

assumptions behind the access to biodiversity are not fully grounded, the CBD's access mechanism is weakened.

1.2 Assumptions behind stimulating the access to technologies

(i) Technologies' coverage

The access to technologies for mitigation of, and adaptation to, climate change has been much debated

especially at the Conferences of the Parties ("COP") of the UNFCCC. There is debate on what the term

“technology” covers and what are the critical technologies to give access to9? Chapter 24 of Agenda 21 under the

UNFCCC refers to "Environmentally Sound Technologies" (“ESTs”), which are intended to encompass the

following technologies: technologies protecting the environment, less polluting technologies, technologies using

resources in a more sustainable manner, technologies aiming at recycling of waste and products, and technologies

handling residual wastes.

The Intergovernmental Panel for Climate Change (“IPCC”) further distinguishes between “Climate

Change Mitigation Technology” ("CCMT") and “Climate Change Adaptation Technology” ("CCAT"). CCMT

covers technological change and substitution that reduce energy resource inputs and emissions, while CCAT

covers technologies intended to reduce the harmful effects arising from expected climate change. Finally, the PA

further specifies the stage of technology cycle which matter to give access to, it should be "early stages of the

technology cycle"10, rather than mature technologies.

To sum up, the term “technology” should cover environmentally friendly technologies at early stages of

the technology cycle that involve energy efficiency, alternatives to fossil fuel and carbon generation, pollution and

toxic remediation, water purification, recycling, safety and health concerns, renewable resources.

(ii) Access scope to technologies

Under the UNFCCC and the PA, the access to and transfer of technologies is an encompassing notion in

climate change adaptation and mitigation. The first assumption under the UNFCCC is that climate change

technologies are under developed countries’ ownerships and their transfer to developing countries is also under

their control:

Article 4, paragraph 5 of the UNFCCC reads: “The developed country Parties shall take all practicable

steps to promote, facilitate and finance, as appropriate, the transfer of, or access to, environmentally

sound technologies and know-how to other Parties, particularly developing country Parties, to enable

them to implement the provisions of the Convention.”

Chapter 34 of Agenda 21 of Transfer Of Environmentally Sound Technology, Cooperation And Capacity-

building11 further suggests that the access and transfer of “ESTs” should be promoted in a balanced manner taking

into account the interests of developed and developing countries:

"To promote, facilitate, and finance, as appropriate, the access to and the transfer of environmentally

sound technologies and corresponding know-how, in particular to developing countries, on favourable



terms, including on concessional and preferential terms, as mutually agreed, taking into account the need

to protect intellectual property rights as well as the special needs of developing countries for the

implementation of Agenda 21;"12

The Kyoto Protocol also promotes the access to climate change technology in its Article 10 by repeating

UNFCCC’s paradigm according to which developed countries shall “take all practical steps” to facilitate the

transfer of, or access to, transfer ETSs to developed countries:

“All Parties...shall: (c) Cooperate in the promotion of effective modalities for the development,

application and diffusion of, and take all practicable steps to promote, facilitate and finance, as

appropriate, the transfer of, or access to, environmentally sound technologies…in particular to

developing countries…”13

More recently, the PA sets the stage for urgently needed climate change technologies’ access,

development and transfer. Notably, countries anchored the Technology Mechanism (“TM”) within the PA and

decided that it would be a key element in implementing the PA. They strengthened the TM, requesting further

work on technology access, research, development and demonstration, as well as enhancing endogenous capacities

and technologies (e.g. locally created and developed technologies).

The assumptions under the UNFCCC and PA are challengeable. First, there is a growing number of

developing countries which are heavily engaged in climate change technologies, developing and patenting

inventions in this area. For example, the Thai company Siam Cement is heavily engaged in environmental friendly

paper and cement.

The Siam Cement Public Company Limited ("SCG") is a Thai company which was established over 100 years

ago in Thailand in 1913, following a Royal Decree of His Majesty King Rama VI in order to eliminate reliance

on imports of cement, as well as fully using own country natural resource14. The goal is to diversify and meet

the economic growing in Thailand.

SCG established and participated in new industries under SCG. SCG comprises over 100 companies under 5

business groups (Cement, paper, chemical, building material, distribution) employing approximately 40,000

employees, handling more than 80,000 product items and filing over 150 patents a year. In 2005, SCG established

a working committee dedicated specifically to innovation.

In 2017, SCG had nearly 1,700 staff members in the R&d department. This included over 100 PhD graduates

and it has a research budget of more than 424,240,40.00 USD per year, again resulting in over USD

4,250,400,250.40 of sales from high value-added products.

A wide range of environmental friendly products have been invented, produced and distributed worldwide by

SCG. For example, products that use minimal natural resources such as "Idea Green paper"15, which reduces the

use of trees by 30 %. Another innovative product is "EL Green plastic pellets"16 (used for plastic packaging);

this is biodegradable under certain temperatures and moisture levels. Another green invention is SCG Marine

Cement17. It is sea water resistant and the greenhouse gas emissions resulting from its production have been

reduced by at least 350 kilograms per ton of cement. These locally made and developed technologies effectively

reduces greenhouse gas emissions and contribute to mitigate climate change impacts18

Second, early stage technologies are not generally opened to access and transfer to developing countries, due to

their confidentiality, owners’ high return on investment expectations, and delays in patenting them especially in

developing countries. There is also a significant part of climate change technology which is mature technology.

One example is solar technology. China has engaged into this area becoming the world’s largest developer,

manufacturer and exporter of solar cells. This was due partially because the technology used was several

generations old and many patents were in public domain19.


Since the assumptions for facilitating access to technologies are not fully well grounded, the UNFCC and PA

access facilitation mechanisms are also weakened.

2. Financial mechanisms to stimulate the accesses to biodiversity and technologies

The financial mechanisms to stimulate the access to biodiversity and climate change technologies under

the CBD, UNFCCC and PA are of various kinds; public, private and hybrid.

2.1 Financial mechanisms to stimulate the accesses to biodiversity

The financial mechanisms to stimulate the access to biodiversity are dominantly (i) private and to some

extend (ii) hybrid between public and private financing sources. There is a consensus that the conservation of

biodiversity cannot be fully achieved through private financial stimulants. Although the participation of the private

sector in promoting the access to biodiversity through the Access and Benefit Sharing (“ABS”) scheme is

encouraged under the CBD and related COPs, its real impact on the conservation of biodiversity is negligible.

(i) Private financial mechanisms

The externalization of biodiversity conservation costs is possible with the help of payments from

industrialized nations' companies to developing countries for their access and use of GR and TK. Upon reviewing

the COPs decisions on financial resources, they are pushing the GEF to take into account the potential of private

sector involvement. However, the private sector has little or no incentive to collaborate with GEF’s activities. In

1996 and in 1999, the Secretariat of the CBD submitted two proposals for Council consideration aimed at

increasing the role and importance of the private sector in the GEF20. However, no measures have taken place

pursuant to these two proposals. The problem of biodiversity lies in the external uncompensated benefits that

diverse resources render to the global community.

There is a specific mechanism under the CBD to allow private financing of biodiversity conservation

known as the Access and Benefit Sharing (“ABS”) promoting the access to GR and TK and their use for

commercial or non commercial purposes.

Private law contracts regulating Prior Inform Consent ("PIC") and Material Transfer Agreement ("MTA")

are the core instruments of ABS. Conservation of biodiversity is supposed to be linked to monetary and non-

monetary benefits shared by the users with the providers of the GR. According to the literature there was over-

expectation on the capacity to extract value from GR especially from bio-prospecting for new industrial products,

such as pharmaceuticals. These activities are unlikely to generate significant funds to conserve biodiversity. One

reason for this is that a considerable percentage of the added value in bio-prospecting projects accrues outside the

country where it takes place. In addition, the assumption that private companies were prepared to subject

themselves to stringent conditions and complex procedures to get access to GR and associated TK has proven

wrong. Companies are also unwilling to incur significant up-front costs in bio-prospecting, especially if samples

of the same resources are available in other countries without or with less stringent regulations.

(ii) Hybrid financial mechanisms

Debt-for-nature swaps are financial transactions that reduce a developing country’s debt stock or service

in exchange for a commitment to protect nature from the debtor-¬government. It is a voluntary transaction whereby

the donor(s) cancels the debt owned by a developing country’s government. The savings from the reduced debt

service are invested in conservation projects. Several organizations such as the Conservation International, The

Nature Conservancy, and the World Wildlife Fund have participated in debt-for-nature swaps. The first debt-for-

nature-swap took place in 1987. It was between Conservation International and the Bolivian government according

to which Conservation International purchased the Bolivian debt having of an original value of USD 650,000 for

the amount of USD 100,000. In return, the Bolivian government agreed to expand an environmentally protected

area by 1.2 million hectares where 13 of the 18 threatened species in this country live. When looking at the

conservation needs, total debt of developing countries and rarity of debt-for-nature swaps agreements this financial

mechanism is of little significance to conserve biodiversity through accessing it.

2.2 Financial mechanism to stimulate the access to climate change technologies

The PA refers to "Financial Mechanism" of the UNFCCC to strengthen cooperative action on technology

transfers by facilitating the access to technologiwa, especially to "early stages of technology cycle". Article 10.5

of the PA reads as follows:

“Accelerating, encouraging and enabling innovation is critical for an effective, long-term global

response to climate change and promoting economic growth and sustainable development. Such effort

shall be, as appropriate, supported, including by the Technology Mechanism and, through financial

means, by the Financial Mechanism of the Convention, for collaborative approaches to research and

development, and facilitating access to technology, in particular for early stages of the technology cycle,

to developing country Parties.”



The financial support to accessing to technologies is provided by the GCF. It cooperates with the

Technology Mechanism that was first mentioned in the Copenhagen Accord in 2009 and was formally established

one year later during the COP 16 held in Cancun, Mexico and is reiterated in the PA. The GCF, together with the

GEF, are the two operating entities of the Financial Mechanism and as such represent the main channels through

which sources of international climate finance are flowing.

The Paris Decision 1/CP.2121 serving as guidance for the implementation of the PA and pre-2020 action,

"strongly urges developed country Parties to scale up their level of financial support, with a concrete roadmap to

achieve the goal of jointly providing USD 100 billion annually by 2020 for mitigation and adaptation"22 .

Furthermore, the Decision adds that prior to 2025 the COP shall set a new ‘collective quantified goal from a floor

of USD 100 billion per year’23.

Most climate change technologies relate to CCMT rather than CCAT24. Since its establishment in 1991,

the GEF has been funding projects on CCMT in developing countries. As at June 30, 2017, the GEF has supported

867 projects on CCMT with over USD 5.3 billion GEF funding in more than 165 countries25. Out of 867 projects

that were implemented in developing countries, 31.1 % in Asia, 25.3 % were in Africa, 18.5 % in LAC, and 17.2

% in Eastern Europe and Central Asia. In addition, there were 69 global and regional projects that account for

eight per cent of the overall CCM portfolio. The table below categorizes these 867 projects in the areas of low

carbon energy, energy efficiency, renewable energy, sustainable transport and urban systems. However out of the

867 projects it appears that only 67 relate to technology transfers which is approximately 7.7 %. It indicates that

access to technologies by developing countries is very limited.

3. Legal mechanisms to stimulate the access to biodiversity and climate change technologies

3.1 Legal stimulants for accessing to biodiversity

The CBD relies on a contractual model to grant access to genetic resources and ensure benefit sharing in

case of exploitation thereof. National policy makers determine criteria for ensuring benefits sharing. Bio-rich

countries' national access regimes to biodiversity were adopted in a context of concerns which are still valid about

the unauthorized access and misappropriation of GR and the lack of compensation for the benefits their commercial

exploitation could generate. Access is generally regarded as a threat rather than an opportunity. Consequently,

such regimes have been mostly defensive, aimed at controlling rather than facilitating the access and use of GR

for research and commercial purposes26. Those stringent regimes have had limited or no effects on conservation

since, as we mentioned earlier, ABS mechanism has not proven to contribute to the conservation of biodiversity.

The following are some examples.

South Africa’s ABS regulations27 require that the applicant for a permit be either a corporation registered

under South African law, or a natural person who is a citizen or a permanent resident of South Africa. If the

applicant is a foreign natural or corporation, they must apply jointly with a South African corporation or citizen or

permanent resident. Therefore, foreign individuals, corporations or institutions cannot apply for a permit alone.

In Costa Rica28, if the applicant lives in a foreign country, he or she must assign a legal representative

who is a resident of Costa Rica.

India also has a differential procedure to handle foreign applications, as does the Andean Community,

Brazil and others. Some countries like India have created a separate procedure depending on nationality, or if the

materials accessed or research results will be sent abroad29.

The Philippines has a relatively long history of regulating access to biodiversity and impose stringent

conditions against bio-prospectors which deter most access to biodiversity30.

(I) Legal stimulants for accessing to technologies

The challenge faced in climate change sector is to encourage innovations in this field while at the same

time promoting the access, diffusion and the transfer of these technologies to developing countries. Technology

transfer is crucial and is seen by developing countries as a prerequisite to adhere to treaties like the UNFCCC. For

example, at the 1992 Rio Earth Summit, China and India insisted that developed countries commit themselves to

technology transfer as a requirement for developing country support for the proposed agreements. Discussions in

general about technology transfer for climate change mitigation have been affected by deep divides between

developed and developing countries. Most developing countries see patents as a barrier to access to technologies


and their transfer, while developed countries see patents as a necessary mechanism to incentivize and license

climate change technologies. Consequently, like with the access regime to biodiversity under CBD, developing

countries' legislations on patent have been mostly defensive, aiming even at imposing compulsory licenses against

developed countries' inventions having the potential to reduce greenhouse gas emissions. The cleavage is strong

enough to explain why the PA is silent on intellectual property, including patent.

Stimulating access to technologies requires strong incentives. Most climate technologies are owned and

developed by commercial companies. Giving access to their technologies by developing countries implies risking

intellectual property rights and empowering existing or potential competitors. Some negotiations on climate

change technology transfer have not identified this problem. At the Second COPs to the UNFCCC in 1996, the

Chinese government published a booklet "The List of Chinese Government Needed Technologies" in which it

demanded climate change technology equipments including integrated gasification combined cycles, fuel cells,

and rice husk energy transfer instruments. However, the booklet remains silent on any compensation for

technology owners. The demands of China and other developing countries to access to technologies can be driven

by their desire to enhance their competitiveness explaining why such demands are usually resisted by developed

countries, creating impasses about how to stimulate the access to ESTs.

GCF's juridical personality and legal capacity

Due to the difficulties to incentivize companies to give access and transfer their technologies and know how to

developing countries, we should investigate whether the GCF could actually own and license technologies which

would allow it to invest into R&D projects and facilitate the implementation of climate change mitigating

approaches in developing countries where they are most needed. Legally speaking, there are no reasons why the

GCF could not own green technologies and patents. The GCF is not only a juristic person but it also has the legal

capacity to own and license patents. The Governing instrument for the GCF, decision 3/CP.17, 11 December 2011,

article 7 reads as follows:

“In order to operate effectively internationally, the Fund will possess juridical personality and will have

such legal capacity as is necessary for the exercise of its functions and the protection of its interests.”

GCF's mandate to own and license technologies

The next question is whether it is the GCF's mandate to own and license technologies? Under the PA, the mandate

of the GCF is to facilitate the access and transfer of technologies and R&D cooperation. Articles 10, 10.5 and

10.6 of the PA refer to the Technology Mechanism and Financial Mechanism as key instruments to allow the

access and transfer of technologies to developing countries.

The COPs Draft Decision no. 7/CP.21 gives to the GCF some specific tasks for its future actions including

"undertaking R&D activities":

"Also invites the Board of the Green Climate Fund, in line with paragraph 38 of the governing instrument

of the Green Climate Fund, to consider ways to provide support, pursuant to the modalities of the Green

Climate Fund, for facilitating access to environmentally sound technologies in developing country

Parties, and for undertaking collaborative research and development for enabling developing country

Parties to enhance their mitigation and adaptation action;"

The COPs decision 3/CP.17 at its paragraph 38 entrusts the GCF to "ensure adequate resources for […] technology

development and transfer […]".

The 2016 Green Climate Fund's publication entitled “Support for facilitating access to environmentally

sound technologies and for collaborative research and development” at paragraphs 23-28 refer to options for R&D

collaboration which the GCF could undertake.

Paragraph 24 reads:

"The benefits of research and development can help countries to address their climate and sustainable

development in the longer term. Even though research and development as an activity poses many risks,

it is only through this process that new technologies are developed and brought to the market."

The GCF solemnly declares the need to invest in R&D and is willing to devote investment, despite the risks that

are inherent.



Paragraph 25 is even more specific on the possibilities for GCF to finance R&D activities:

"GCF could provide financing for research and development activities and help countries tackle the he

challenges faced in the early stages of the technology development cycle […]"

It further mentions in its Paragraph 26 possible R&D activities which it could finance as follows:

"(a) Competitive GCF innovation funding to stimulate local/regional interest and investment through the

tailored request for proposals;

(b) Promoting micro-finance for research and development projects in developing countries;

(c) Working with governments to strengthen national innovation systems;

(d) Facilitating the establishment of research center networks on environmentally sound technologies;

and

(e) Catalyzing research partnerships with relevant stakeholders, including collaboration with the private

sector.

The Board may wish to consider the following options for providing such support for research and

development:

(a) Business incubation and financial support for viable, new technologies to be deployed in developing

countries; or

(b)Capacity-building programmes/request for proposals for developing countries to enhance endogenous

capacities related to climate technologies."

Paragraph 27 further explains:

"The first option [means a) above] would focus on bringing new, viable technologies to market through

business incubation, and early and growth stage financing. The Private Sector Facility could potentially

play an important role in this area, attracting venture capital and angel investors to deploy their capital

in developing countries."

The GCF plans so explicitly to finance R&D activities in order to bring technologies to market. Certainly, the GCF

is not mandated to fund basic research, which is wise, because the cycle to produce a workable technology is long

and uncertain. However, the phase of industrialization of technology is often a time that patents are filed. The issue

of industrial property will arise so necessarily for incubation projects in which the GCF would invest.

To summarize, the GCF has legal personality and capacity, as it may perform all legal operations that

allow it to achieve its missions. The GCF should be able to own technologies and license them. This would also

mean that owners of patented technologies could donate a license to GCF which may then manage a patent pool.

The GCF could use these licenses for its beneficiaries, mainly developing countries.

Result & Discussion

From the review above, key findings are that facilitating the access to climate change technologies and

the access to GR have shown little impacts on mitigating or adapting to climate change. This result ties well with

previous studies wherein access to biodiversity hasn’t necessarily resulted in improved biodiversity conservation.

One concern about the findings of access to biodiversity was that limited information is available on bio-

prospecting activities and how the access to biodiversity is usually retributed. Likewise, there is lack of information

on technology transfers of EST, especially between private entities.

Conclusion

The conservation of biodiversity by facilitating its access to developed countries has shown little results.

Most rich bio-resources countries have enacted defensive legislations which deter bio-prospectors from entering

into MTAs to access to GR and TK. Furthermore, the development of biotechnology offers viable alternatives to

GR. It can be concluded that continuing promoting the access to biodiversity under the CBD won't offset the fast

decline of GR. Emphasizes should be placed on other mechanisms that access, for example public financial

instruments. Promoting the access to climate change technologies is also challenging. However, the option for the

GCF to invest in green technologies including through incubator whose mission is to support, including financially,

the development of existing technologies is promising. R&D in the field of adaptation of existing technologies is

conducive to collaboration, because it requires double competencies, both technological and knowledge of the


conditions specific to each country or region. In addition, it is often considered that technology held by western

companies need to be adapted to the specific conditions of other countries. Integrating a licensing system into the

GCF could better ensure developing countries gain easier and faster access to climate change technologies. The

GCF and the Technology Mechanism are exciting new institutions offering the possibility of a global license

system that ensures that intellectual property, royalties, investment etc are not barriers to technology access, and

can become the missing link between innovating and giving accessing to technologies.

References

1 Joint Statement by the Executive Secretaries of the Rio Conventions calling for the establishment of a Project

Preparation Facility (PPF) to increase financing for large-scale, transformative projects which integrate

action on land degradation, biodiversity loss, and global warming. https://www.cbd.int/cooperation/joint-

statement-rio-convention-2017-en.pdf 2 Supra no. 2 3 The CBD Secretariat has published two technical series on biodiversity and climate change to support the

implementation of relevant adaptation activities. Technical Series No. 10 – Interlinkages between

Biological Diversity and Climate Change; https://www.cbd.int/doc/publications/cbd-ts-10.pdf

Technical Series No. 25 – Guidance for Promoting Synergy among Activities Addressing Biological

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https://www.cbd.int/doc/publications/cbd-ts-25.pdf 4Joint Statement by the Executive Secretaries of the Rio Conventions calling for the establishment of a Project

Preparation Facility (PPF) to increase financing for large-scale, transformative projects which integrate

action on land degradation, biodiversity loss, and global warming. https://www.cbd.int/cooperation/joint-

statement-rio-convention-2017-en.pdf 5Supra no. 2 6The CBD Secretariat has published two technical series on biodiversity and climate change to support the

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“However, the traditional view of north-south technology transfer (e.g., from resource-rich to

biodiversity-rich countries in the original context of the CBD) is being challenged increasingly because

south-south technology transfer is growing in importance”;

“It is likely that, in future, south-south transfer will require greater attention as more agreements are made

to foster these interactions”

“It is likely that technology transfer is more frequent among countries with economic, geographic,

cultural, or historic ties”. 12Heleen de Coninck and Shikha Bhasin, Meaningful technology development and transfer: A necessary condition

for a viable climate regime, Towards a Workable and Effective Climate Regime, Radboud University;

Energy research Centre of the Netherlands (ECN) pp. 451-464 13Article 10.5 of the Paris Agreement "Accelerating, encouraging and enabling innovation is critical for an

effective, long-term global response to climate change and promoting economic growth and sustainable

development. Such effort shall be, as appropriate, supported, including by the Technology Mechanism



and, through financial means, by the Financial Mechanism of the Convention, for collaborative

approaches to research and development, and facilitating access to technology, in particular for early

stages of the technology cycle, to developing country Parties." 14Transfer Of Environmentally Sound Technology, Cooperation And Capacity-building, http://www.un-

documents.net/a21-34.htm 15Paragraph 14 b) http://www.un-documents.net/a21-34.htm 16Kyoto Protocol to the United Nations Framework Convention on Climate Change of 1998

http://unfccc.int/resource/docs/convkp/kpeng.pdf 17http://www.scg.com/en/01corporate_profile/ 18http://www.scgpackaging.com/others/home-office-paper/idea-green/EN 19http://www.scg.com/id/05sustainability_development/eco-value.html 20http://www.scg.com/en/08news_release/01_news/detail.php?ContentId=2164 21Orose Leelakulthanit, Sustainability: The Case Of Siam Cement Group (SCG), Journal of Business Case Studies

– Fourth Quarter 2014 Volume 10, Number 4; Innovative IP development, Siam Cement Group

http://www.wipo.int/ipadvantage/en/articles/article_0054.html 22Cynthia Cannady, Access to Climate Change Technology by Developing Countries, ICTSD Global Platform on

Climate Change, Trade Policies and Sustainable Energy, Issue Paper 25, September 2009, pp. 1-44. 23Porter Gareth, Raymond Clemencon, Waafas Ofosu-Amaah, and Michael Philips, Communicative Structure and

Governance of the Global Environment

Through Policy Networks of International Aid Organizations, 1998. 24Shah Md. Mushfiqur Rahman, A critical examination of the Financial Mechanism as adopted in the Convention

on Biological Diversity (CBD) from a developing country’s perspective, The Northern University Journal

of Law, Volume I (2010). 25Elisa Morgera,The Evolution of Benefit Sharing: Linking Biodiversity and Community Livelihoods, 26Gordon C. Rausser University of California, Berkeley Arthur A. Small Columbia University, Valuing Research

Leads: Bioprospecting and the Conservation of Genetic Resources, Journal of Political Economy, 2000,

vol. 108, no. 1. Nicolas Pauchard, Access and Benefit Sharing under the Convention on Biological

Diversity and Its Protocol: What Can Some Numbers Tell Us about the Effectiveness of the Regulatory

Regime? Resources 2017, 6, 11; doi:103 390/resources6010011 pp.1-15, Barett, C.B.; Lybbert, T.J. Is

bioprospecting a viable strategy for conserving tropical ecosystems? Ecol. Econ. 2000, 34, 293–300,

Moretti, C.; Aubertin, C. Stratégies des firmes pharmaceutiques: La bioprospection en question. In Les

Marchés de la Biodiversité; Aubertin, C., Pinton, F., Boisvert, V., Eds.; Institut de Recherche Pour le

Développement (IRD): Paris, France, 2007., Morgera, E.; Tsioumani, E.; Buck, M. Unraveling the

Nagoya Protocol: A Commentary on the Nagoya Protocol on Access and Benefit-sharing to the

Convention on Biological Diversity.

http://booksandjournals.brillonline.com/content/books/9789004217188

“Key publications on the ABS regime implemented through the CBD and its protocol note its difficulties,

so far, in achieving its objectives, as very few ABS agreements have been concluded and consequently

very few benefits have been shared."

“However, to our knowledge, there is not any accurate counting of either access permits granted or ABS

agreements concluded since the entry into force of the CBD or the NP. I found piecemeal information

indicating that the number is small but no systematic inventory. I consider that providing reliable data

about access permits and ABS agreements is essential to analyze the concrete functioning of the regime.

This is the core aim of our contribution.” 27Cabrera, M.J. (2003) ‘Bioprospecting: policy, regulatory and market incentives’, paper presented at the

Megadiverse Countries Meeting on Environmental Legislation on Access to Genetic Resources,

Protection of Traditional Knowledge and Intellectual Property Rights, Cusco, 27–29 November, pp. 246-247 28Cox, P.A. 2001. Ensuring equitable benefits: The Falealupo Convenant and the isolation of anti-viral drug

prostatin from a Samoan Medicinal Plant. Pharmaceutical Biol. 39, 33-40. 29Debt for Nature Swaps, Financing Solutions for Sustainable Development (2017) 30James P. Resor, Debt-for-nature swaps: a decade of experience and new directions for the future,

http://www.fao.org/docrep/w3247e/w3247e06.ht 31http://unfccc.int/meetings/copenhagen_dec_2009/items/5262.php 32http://unfccc.int/resource/docs/2015/cop21/eng/10a01.pdf 33Paragraph 15 of the Paris Decision 34Paragraph 54 of the Paris Decision 35Antoine Dechezleprêtre, Matthieu Glachant, Ivan Hascic, Nick Johnstone, Yann Ménière, nvention and Transfer

of Climate Change Mitigation Technologies on a Global Scale: A Study Drawing on Patent Data (2009)

Mines paris Tech http://personal.lse.ac.uk/dechezle/Innovation_diffusion_climate_techs_AFD.pdf


United Nations Environment Programme (UNEP)/European Patent Office (EPO) study Patents and clean

energy technologies in Africa, 2013, www.epo.org/clean-energy-africa; 36Report of the Global Environment Facility to the Twenty-third Session of the Conference of the Parties to the

United Nations Framework Convention on Climate Change, August 2017

https://www.thegef.org/sites/default/files/documents/Final%20COP%2023%20Report%20August%203.pdf 37Carlos M. Correa, Do national access regimes promote the use of genetic resources and benefit sharing?, Int. J.

Environment and Sustainable Development, Vol. 4, No. 4, 2005, pp. 444-463. Jorge Cabrera Medaglia,

Frederic Perron-Welch And Freedom-Kai Phillips, Overview Of National And Regional Measures On

Access And Benefit Sharing (2004) Centre for International Sustainable Development Law (CISDL). 38Neil R. Crouch South Africa’s bioprospecting, access and benefit-sharing legislation: current realities, future

complications, and a proposed alternative South African Journal of Science 104, September/October

2008, South Africa’s legislative framework on bioprospecting, access and benefit sharing, 7th Pan-

African ABS Workshop Hans Merensky Hotel, Phalaborwa 25 February-1 March 2013,

http://www.abs-initiative.info/uploads/media/Lactitia_Tshitwamulomoni_-_DEA_-

_South_Africa_s_legislative_framework.pdf 39Carment Richerzhagen , The effectiveness of access and benefit sharing in Costa Rica: implications for national

and international regimes, Ecological Economics, Volume 53, Issue 4, 2005 pp.445-460 40Indian ABS guidelines http://nbaindia.org/uploaded/pdf/Gazette_Notification_of_ABS_Guidlines.pdf 41Carlos M. Correa, Do national access regimes promote the use of genetic resources and benefit sharing?, Int. J.

Environment and Sustainable Development, Vol. 4, No. 4, 2005, pp. 444-463. 42Tim Forsyth, Climate change investment and technology transfer in Southeast Asia,

http://personal.lse.ac.uk/FORSYTHT/Harr-ch13.pdf 43Legal Personality and the Green Climate Fund (2011) http://legalresponseinitiative.org/wp-

content/uploads/2013/09/BP38E-Briefing-Paper-Legal-Personality-of-the-Green-Climate-Fund-8-

December-2011.pdf 44"Accelerating, encouraging and enabling innovation is critical for an effective, long-term global response to

climate change and promoting economic growth and sustainable development. Such effort shall be, as

appropriate, supported, including by the Technology Mechanism and, through financial means, by the

Financial Mechanism of the Convention, for collaborative approaches to research and development, and

facilitating access to technology, in particular for early stages of the technology cycle, to developing country Parties." 45"Support, including financial support, shall be provided to developing country Parties for the implementation of

this Article, including for strengthening cooperative action on technology development and transfer at

different stages of the technology cycle, with a view to achieving a balance between support for mitigation

and adaptation. The global stocktake referred to in Article 14 shall take into account available information

on efforts related to support on technology development and transfer for developing country Parties." 46http://unfccc.int/resource/docs/2015/cop21/eng/l07.pdf 47Paragraph 22 of the COPs Decision no. 7/CP.21 48http://unfccc.int/resource/docs/2011/cop17/eng/09a01.pdf#page=55 "The Board shall also ensure adequate

resources for capacity-building and technology development and transfer. The Fund will also provide

resources for innovative and replicable approaches." 49GCF/B.14/02, 30 September 2016 https://www.greenclimate.fund/documents/20182/409835/GCF_B.14_02_-

_Support_for_facilitating_access_to_environmentally_sound_technologies_and_for_collaborative_rese

arch_and_development.pdf/410006c7-c3f6-4abc-96fe-cfa9280994e0 50Menno van der Veen Department of Biotechnology, Section Biotechnology and Society, Delft University of

Technology, Delft, The Netherlands. The Green Climate Fund as a Patent Pool for Innovations, Nature

Biotechnology (2012) 51Report Of The Global Environment Facility To The Twenty-Fourth Session Of The Conference Of The Parties

To The United Nations Framework Convention On Climate Change, 55th GEF Council Meeting

December 17 – 20, 2018 Washington, D.C, available at :

http://www.thegef.org/sites/default/files/council-meeting-

documents/EN_GEF.C.55.Inf_.XX_UNFCCC_CoP_Report.pdf



Table 1. - Summary of GEF projects on climate change mitigation by phase in USD million.

Phase

Technology

transfer/

Innovative

low-carbon

technologies

(LCTs)

Energy

efficiency

Renewable

energy Transport/Urban AFOLU SGP

Mixed

and

Other

Total

GEF

Pilot

(1991-

1994)

Number of

Projects 2 7 12 2 2 0 3 28

GEF Amount 10.1 33.3 94.5 9 4 - 46.7 197.6

Co-financing 0.1 341.2 1,848 2 0.1 - 145.9 2,337.20

GEF-1

(1994-

1998)

Number of

Projects 2 16 16 0 0 0 6 40

GEF Amount 8.2 134.4 146.9 - - - 27 316.4

Co-financing 6.2 447.5 809.7 - - - 94.5 1,357.80

GEF -2

(1998-

2002)

Number of Projects 6 32 44 6 1 0 6 95

GEF Amount 102.3 189.9 227.8 30 0.9 - 19.1 570.1

Co-financing 827.8 2,025.40 1,097.80 28.3 1 - 182.9 4,163.30

GEF-3

(2002-

2006)

Number of

Projects 4 29 53 13 0 0 14 113

GEF Amount 64.6 228.2 248.6 88.8 - - 76.3 706.5

Co-financing 309.2 1,310.10 1,462.30 886.1 - - 348.4 4,316.00

GEF-4

(2006-

2010)

Number of

Projects 9 83 48 20 25 3 15 203

GEF Amount 46.3 382.5 118.9 110.9 121.5 65.3 88.6 934

Co-financing 215.2 3,747.40 856.8 2,082.70 870.9 44.50 490.4 8,307.90

GEF-5

(2010-

2014)

Number of

Projects 38 38 56 27 69 10 17 255

GEF Amount 223.7 199.1 206.6 125.3 515.9 159 105.7 1,535.30

Co-financing 1,797.60 4,355.70 2,022.50 2,558.10 2,386.80 160.50 1,046.10 14,327.30

GEF-6 to

date

(2014-

2017)

Number of Projects 6 15 25 25 36 11 15 133.00

GEF Amount 16.9 119.2 143.1 203.9 485 53.3 45 1,066.50

Co-financing 82.4 825.9 2,928.60 2,894.00 3,021.20 80.80 303.80 10,136.70

Total

Number of

Projects 67 220 254 93 133 24 76 867.00

GEF

Amount 472.1 1,286.70 1,186.30 567.8 1,127.40 277.60 408.50 5,326.50

Co-financing 3,238.30 13,053.10 11,025.70 8,451.40 6,279.90 285.90 2,611.90 44,946.20

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