SCREENING AND IDENTIFICATION OF MICROBES WITH POTENTIAL ANTAGONIStIC ACTIVITY AGAINST

PHYTOPHTHORA CAPSICI

Tang LokIng

(39082)

QK Bachelor of Science with Honours

604.2 (Resource Biotechnology)

AS6 2015

TI64 2015

P sat Khidmat Maklumat Akademik UNI E ITI MALAYSIA SARAWAK

SCREENING AND IDENTIFICATION OF MICROBES WITH POTENTIAL ANTAGONISTIC ACTIVITY AGAINST PHYTOPHTHORA CAPSICI

TangLokIng (39082)

This project is submitted in partial fulfilment of the requirement for the Degree of Bachelor of Science with honours

(Resource Biotechnology)

Supervisor: Dr. Samuel Lihan Co-supervisor: Associate Prof. Dr. Awang Ahmad SaUehin Awang Husaini

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2015

Acknowledgement

First of all, I would like to express my appreciation to my supervisor, Dr. Samuel

Lihan and co-supervisor, Associate Professor Dr. Awang Ahmad Sallehin Awang Hussaini

for their guidance and supervision to me throughout the this research.

In addition, I would like to thank to all the postgraduate students from the virology

laboratory and the lab assistant ofVirology laboratory for their precious assistance, advices

and concerns to me.

Next, I would like to show appreciation for my lab-mates and friends, Chai Shin

Nei, Lee Sai Yeng and Soh Khar Mun for their supports and concerns throughout my

research progress.

Last but not least, I would like to express my thankfulness to my family members

especially my mother for their continuous fmancial and emotional support throughout my

research.

I

I

I

(

UNIVERSITI MALAYSIA SARA WAK

Grade:

" Please tick (..J) Final Year Project Report o Masters o PhD o

DECLARATION OF ORIGINAL WORK

This declaration is made on the ... . ~~ ... ..... day of... }~.~~ .. .... 2015.

Student's Declaration:

I TANG LOK lNG, 39082, FACULTY OF RESOURCE SCIENCE AND TECHNOLOGY hereby declare that the work entitled SCREENING AND IDENTIFICATION OF MICROBES WITH POTENTIAL ANTAGONISTIC ACTIVITY AGAINST PHYTOPHTHORA CAPSICI is my original work. I have not copied from any other students' work or from any other sources except where due reference or acknowledgement is made explicitly in the text, nor has any part been written for me by another person.

Date submitted Tang Lok Ing (39082)

Supervisor's Declaration:

I DR. SAMUEL LIHAN hereby certifies that the work entitled SCREENING AND IDENTIFICATION OF MICROBES WITH POTENTIAL ANTAGONISTIC ACTIVITY AGAINST PHYTOPHTHORA CAPSICI was prepared by the above named student, and was submitted to the "FACULTY" as a * partial/full fulfillment for the conferment of DEGREE OF BACHELOR OF SCIENCE WITH HONOURS (RESOURCE BIOTECHNOLOGy), and the aforementioned work, to the best of my knowledge, is the said student's work.

Received for examination by: Dr. Samuel Lihan

DR. SAMUEL LlHAN nPensyarah «an.a Kul

Jabalan Biologl M~leknologi sumber !=akulti Sain5 dan. e aUwaK Unl-Jersi\l MalaYSia Shan SarawaK 91300 \~oti\ :samara .

Date:__'2_3t-f6_'\_5~__

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(Date) \lll lhll~\~ e n Kal1el1 Pens ara . i Molekul

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III

PII a KI idmat Maklumal Akademik UNIVERSITJ MALA 'SIA SARAWAK

Table of Contents

Acknow ledgement

Declaration

Table ofContents

List ofAbbreviations

List ofTables

List of Figures

Abstract

1.0 Introduction

2.0 Literature Review

2.1 Black pepper

2.2 Phytophthora disease

2.3 Chemical agents against Phytophthora capsid

2.4 Biological control agents (BCAs)

3.0 Materials and Method

3.1 Recovering of bacteria from glycerol stock

3.2 Isolation ofthe bacteria

3.3 Preparation of fungus

3.4 Antagonistie activities against Phytophthora capsid

3.4.1 Spore gennination test

3.4.2 Double plate assay

3.5 Molecular characterization

3.5.1 Preparation for DNA extraction

3.5.2 Boiled-Cell Method for DNA Extraction

IV

I

II

IV

VI

VIII

IX

2

5

6

7

8

10

10

10

11

11

12

12

3.5.3 Polymerase Chain Reaction (PCR) Amplification

3.5.4 Agarose Gel Electrophoresis

3.6 16S rRNA Gene Sequencing

3.6.1 DNA Purification from Gel using QIAquick Gel Exraction Kit

3.6.2 Gene Sequencing

4.0 Results

5.0 Discussion

6.0 Conclusion

7.0 References

.'

12

14

15

16

17

28

32

33

v

Iii

°C

cm

III

BLAST

ddH20

DNA

h

ml

rnA

MgCh

mm

mt

NA

NB

NCB!

PCR

PDA

List of Abbreviations

Degree Celcius

Centimeters

Microliters

Basic Local Alignment Search Tool

Double Distilled Water

Deoxyribonucleic acid

Hours

Milliliters

Milli ampere

Magnesium Chloride

Minutes

Metric Ton

. Nutrient Agar

Nutrient Broth

National Center for Biotechnology Information

Polymerase Chain Reaction

Potato Dextrose Agar

VI

PDB Potato Dextrose Broth

PIDG Percentage ofInhibition ofDiameter Growth

rpm Rotation per minute

TBE Tris-borate-EDTA

TSA Tryptic Soy Agar

UV Ultraviolet

v Volt

VII

List of Tables

Tables

Table 3.1

Table 3.2

Table 3.3

Table 4.1

Table 4.2

Table 4.3 I,',

Descriptions Pages

The sequences of the peR primers. 13

The master mix used for peR amplification. 13

The step cycle, temperature and duration for peR 14

amplification.

The calculated average of radii of inhibition zone of 19

iso lated bacteria.

The table showed the average diameter growth and PIDG 23

value of the antifungal activities of the isolated bacteria.

The closest match of the sequences ofbacterial isolate with 27

the database

VIII

List of Figures

Figures Descriptions Pages

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Results for isolation of the bacteria by streaking for a 17

few times to obtain s single colony.

Radii of inhibition zone in the spore germination test. 21

Spore germination test after 72 h. 22

Double plate assay. 24

Graph of isolated bacteria against percentage 25

inhibition ofdiameter growth.

Agarose gel showed the DNA extracted from the 26

bacteria.

IX

II ,

Screening and identification of microbes with potential antagonistic activity against Phytophthora capsid

Tang Lok Ing

Resource Biotechnology Programme Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Phytophthora capsid infection on the black pepper can affect the production of black pepper. This infection can be managed using fungicides such as metalaxyl, copper oxychloride and phosphonate. However, the use of these fungicides against the infection can cause damage to the environment. Thus, it is important to look for a new approach to control P. capsid. The aims of this study are to isolate and characterize the bacteria that are antagonism against P. capsid. In this study, the bacteria were screened for their antagonistic activity through spore germination test and double plate assay. Hence, KDKBB83 and SB (2)6 that showed high antagonistic activity was selected for molecular characterization. The mo lecular characterization was done by using 16S rRNA sequence analysis. Analysis of 16S rRNA sequencing suggested the closest identities of the bacterial isolates as Serratia marcescens and Pseudomonas aeruginosa by using Basic Local Alignment Search Tool (BLAST) via National Center for Biotechnology Information (NCBI).

Keywords: Phytophthora capsici, antagonistic activity, 16S rRNA sequencing, spore germination test, double plate assay

ABSTRAK

Jangkitan yang disebabkan oleh Phytophthora capsici dalam lada hilam akan menjejaskan pengeluaran lada hitam. Jangkilan ini boleh diuruskan dengan menggunakan racun kulat seperti metalaxvl. oksiklorid tembaga dan phosphonate. Tetapi, penggwwan raclin kulat in; akan menyebabkan kerosakan terhadap alam sekitar. Olell ilu. ia adalah sangat penting untuk mencari kaedah baru untuk menga».'al jangkitan ini. Tlijuan kajian ini ada/ah untuk mengasingkan dan mengetahlli identiti bakteria yang boleh mengQ\-1/a1 tumbuhan kulat. 'Dalam kajian ini, bakteria telah disaring melailli spora lIjian percambahan dan plat penglljian berganda. J~di, KDKBB83 dan SB (2)6 yang mellu'!iukkan aktiviti permusuhan nyata terhadap P. capsid telah dipilih lIntlik pencirian molekul. Pencirian molekul telah dtialankan dengan menggunakan 16S rRNA analisis IIrutan. Analisis tersebut telah mencadangkan identiti terdekat isolat bakteria sebagai Serrtlfia marcescens and Pseudomonas aeruginosa dengan menggunakan Basic Local Alignment Search Tool (BLAST) melalui National Center {or Biotechnolog;:, In{ormation (NCB!).

Kata kunci: Phytophthora capsici, aktiviti permusuhan, 16S rRNA al1alisis urulan, Spora I{jian perclImhahan, PIal pengl{jian berganda

1.0 Introduction

Black pepper is one of the important economic crops in the Sarawak. The scientific

name of the black pepper is Piper nigrum. This plant belongs to the family Piperaceae and

it is well known as the 'king of spices' (Saha et al., 20l3). The black pepper is grown since

millennia and it is still remained as one of the valuable spices in the world (Yogesh &

Mokshapathy, 2013). The fruits of the black pepper are the part that is used as the spice

and seasoning. Currently, black pepper is widely cultivated in India, Brazil, Indonesia,

Malaysia, Sri Lanka, Vietnam, and China (Sivaraman et al., 1999). Yogesh and

Mokshapathy (2013) stated that the spiceness of the black pepper is because of the

presence of chemical piperine. Moreover, black pepper is also used in the cosmetic

industry and medical products (Rosli et al., 2013). Black pepper is best grown in the hot

hwnid tropics which require conditions such as high rainfall, uniform temperature and high

relative humidity (Krishnamurthy et al., 2010).

Phytophthora capsici infection leads to the foot rot of the black pepper. The foot

rot is also referred to as 'sudden wilt' (Tran et al., 2008). This infection affects the

production ofblack pepper and it causes the economic loss. P. capsici infection can spread

fro m the roots to the all parts ofthe pepper plant and the infection primarily arises from the

roots where there is a water soaked area (Hausbeck & Lamour, 2004). According to

Manohara (2007), the temperature and light can affect the formation of the oospore of P.

capsici. It is well grown in the humid environment of25-30 °C and pH of5.5 to 6.0 (Lee &

Lwn, 2004). In addition, this infection also can spread through wind dispersal and rain

splash ofspores (Wong, 2004).

2

The symptoms of the P. capsici infection are distinctive black lesions fonned on

the stem, grayish brown lesions fonned on the leaves and fruit covered with the white

sporangia (Ristaino & Johnston, 1999). This infection may end with the dropping of

berries, shedding of leaves and breaking off of branches (Wong, 2004). Drenth and Guest

(2004) mentioned the factors that contributed to the effectiveness ofPhytophthora as plant

pathogens. The factors are capability of producing various types of spores, rapid

sporulation on host tissue within 3-5 days of infection, capability to live in or outside the

host tissue and production of sporangia (Drenth & Guest, 2004). According to Drenth and

Guest (2004), the pathogens also contain different biochemical pathways as compared to

the true fungi. Besides, the pathogens increase under humid and wet environments and this

leads to the difficulty of applying of the protectant fungicides (Drenth & Guest, 2004).

Hence, the fungicides are least efficient under these environments.

The strategies that have been taken for the management of Phytophthora infection

are cultural practices, organic amendments, chemical control and biological control

(Anandaraj & Sanna, 1995). For the chemical control, the Bordeauc mixture, metalaxyl,

copper oxychloride and phosphonate have been used (Dinu et aI., 2007). However, the use

of the fungicides is not environmentally-friendly. Thus, there is a need to look for an

alternative way that is safe to be used in the management ofthe disease.

Recently, the "biological control using microorganisms is an alternative method to

control Phytophthora infection. According to Weller (2007), Pseudomonas spp. can be

found in the agricultural soils and contain many traits that make them can be act as the

biological control agent. One of the traits is the production of wide spectrum of bioactive

metabolites such as antibiotics, siderophores and volatiles (Weller, 2007). Thus, this

research is conducted to look for other potential biological agents against P. capsici.

3

The objectives ofthe study were to:

I. To isolate the bacteria that is antagonistic against the P. capsici.

2. To characterize the bacteria that is antagonism to the P. capsici.

4

J

P I SM Khi ma Maklllmal Akadema· . ,' "I'. TI M. LAY IA SARAW K

2.0 Literature Review

2.1 Black pepper

Black pepper belongs to the family Piperaceae and it is originated from the state of

Kerala in south-western India (Drenth & Sendall, 2004). It is suitable to be planted in the

hot humid tropics (Krishnamurthy et aI., 2010). Therefore, Malaysia is one of countries

that export black pepper. In Malaysia, Sarawak is the main state to harvest black pepper.

The main harvesting areas in Sarawak are Kuching, Samarahan, Sri Aman, Sarikei and

Sibu (Krishnamurthy et ai., 2010).

Lee and Lum (2004) stated that there are about 60,000 fann families involved in

the cultivation of the black pepper in Sarawak. International Pepper Community (n.d.)

stated that the production of black pepper in Malaysia for year 2012 was 26,000 mt and

10,454 mt of the production was exported from Malaysia. The exported pepper comprised

of 8,404 mt of black pepper and 2,050 mt of white pepper (International Pepper

Community, n.d.). There is an agent which is known as Malaysian Pepper Board (MPB)

that is held responsible for marketing and relevant services of the black pepper (Rosli et ai.,

2013),

Drenth and Sendall (2004) stated that the black pepper is propagated vegetative and

the fruits start to produce within two years of planting. The vines of the black pepper able

to harvest fruit for 12 to 15 years. There are different types of pepper occurring in the

market and this is resulting from the berries harvested by P. nigrum (Drenth & Sendall,

2004). Black pepper is the result of mature dried berries. Black pepper is also known as

"Black gold" because ofthe durability and value (Yogesh & Mokshapathy, 2013).

5

,..

2.2 Phytophthora disease

Manohara (2007) stated that Phytophthora capsid is a heterothallic fungus. The

sexual reproduction requires two compatible mating types which are designated Al and A2

for oospore fonnation. The oospore fonnation can be affected by light, temperature,

oospore maturity, enzyme treatment and chemical treatment. According to Manohara

(2007), the light can inhibit the oospores fonnation. The oospores of the fungus can live in

extreme environmental conditions and they are able to survive outside the host tissue for

long periods. For the asexual spore fonnation, there are three fonns available which are

sporangia, zoospores and chlamydospores (Drenth & Guest, 2004).

Drenth & Guest (2004) stated that there are different species of Phytophthora and

these species have different degree of host specificity. For examples, P. fragariae var. rubi

can only infect a single species whereas P. cinnamomi can infect over 1000 diverse species

ofthe host plant. P.capsici can attack ove! 40 different host plant species.

P. capsici is a soilborne pathogen and the Phytophthora infection usually occurs

during the wet weather (Lee & Lum, 2004). Lee and Lum (2004) stated that this infection

can affects the whole parts of the plants such as the leaves, berries, spikes, branches, stems

and roots. The infection causes the foot rot of black pepper and the foot rot can be spread

by rain splash and root contact (Lee & Lum, 2004). Their study also stated that the fungus

can live in the soil without the presence of a host for at least 18 months.

According to Drenth and Guest (2004), there are various factors contributing to the

effectiveness of the Phytophthora pathogens. The fIrst factor is that the pathogens belong

to the Kingdom Stramenopiles and their biochemical pathways are different as compared

to the true fungi. Thus, the fungicides are not able function effectively in the management

6

of the diseases. The second factor is they are able to live without a host for a long period.

Lastly, the pathogens can survive under humid and wet environment. This contributes to

the difficulty in applying ofthe fungicides (Drenth & Guest, 2004).

2.3 Chemical agents against Phytophthora capsici

The fungicides are used to control the Phytophthora infection. Drenth and Guest

(2004) stated that the fungicides used can be divided into two types which are protectant

and systemic. Bordeaux mixture is one of the protect ant fungicides. Bordeaux mixture

have been used to control the diseases that caused by different species of Phytophthora.

However, this fungicide contains several disadvantages. There is copper occurs in the

ingredients of the Bordeaux mixture. Copper is toxic to some plants and non-target

organisms. Preparation and applying of this fungicide requires extensive labour. Besides,

the fungicide can be washed off easily since there is high rainfall in the tropical areas

(Drenth & Guest, 2004).

Metalaxyl is one ofthe most widely used systemic fungicides. According to Drenth

and Guest (2004), metalaxyl fungicide is transported in the xylem with an upward

movement in the plants. If this fungicide is applied as a foliar spray, there will be not effect

occurs due to the upward movement in the plants and fungicide does not transports to the

roots. Hence, this fungicide is normally applied as a soil drench and it can attack over all

species ofPhytophthora.

Drenth and Guest (2004) also stated that the metalaxyl fungicide is effective against

the fungus by inhibit RNA synthesis, inhibit sporangium formation and reduces

chlamydospore and oospore formation. However, this fungicide also contains several

disadvantages such as the drainage of chemicals into the soil and water systems and

7

developed resistance among Phytophthora (Drenth & Guest, 2004). The drainage of

chemicals into the soil and water systems can be destructive to the environment and human

health.

2.4 Biological control agents (BeAs)

According to Pliego et al. (2011), the tenn of ''biological control" in the field of

plant pathology is referred to the application of the microbial that shows antagonistic

activity to suppress diseases. The biological control agent is considered as a safer

management option as compared to the used of the chemicals. There are different modes of

action shown by bacterial biocontrol agents such as hyperparasitism, lytic enzymes,

antibiotics, waste products, physical or chemical interference, competition and induction of

host resistance (Pliego et al., 2011). For example, the waste product, hydrogen cyanide

produced by Pseudomonas fluorescens can be used against Phytophthora infestans in the

tomato. In addition, Pseudomonas sp. can .produce antibiotics, cyclic lipopeptides to

suppress Phytophthora infestans in the tomato (Pliego et aI., 2011).

Akgiil and Mirik (2008) stated that Bacillus megaterium are effective in

suppressing P. capsici. Pseudomonas aeruginosa, Pseudomonas putida and Bacillus

megaterium also have been discovered as effective biological control agents against

Phytophthora capsici in the black pepper (Aravind et aI., 2009). Besides, Bacillus

megaterium, Bacillus cereus, Enterobacter cloacae and Bacillus thuringiensis also showed

effective antifungal activities against Fusarium oxysporum in the black pepper (Edward et

al., 2013). Yap (2012) also stated that Bacillus amyloliqueJaciens, Bacillus atrophaeus,

Bacillus subtilis and Bacillus vallismortis are capable to be used as potential biological

control agents against phytopathogenic fungi. According to Rahman et al. (2007) stated

8

that Burkholderia cepacia showed high antagonistic activity against Colletotrichum

gloeosporioides. Sivanantham et al. (2013) also stated that Pseudomonas maltophila and

Bacillus circulans had strong inhibitory effect against Botryodiplodia theobromae,

Alternaria porri, Fusarium oxysporum and Sclerotium rolfsii. The Burkholderia sp. H-6

inhibits the growth of the fungi, P. capsid, Fusarium graminearumt and Sclerotinia

libertiana (Wang et al., 2010).

Rajan et al. (2002) also identified that Trichoderma harzianum and Trichoderma

virens have the ability to control the P. capsd infection in the black pepper. Besides, T.

hamatum also can effectively against foot rot disease. But, the combination of these

bacteria showed no decreasing of the foot rot disease in the black pepper. This may due to

the competition between these isolates and the effect of single isolates might be cancelled

(Rajan et al., 2002). Anith (2003) also mentioned that Trichoderma viride have been used

as biocontrol agent against plant pathogenic fungus, P. capsici in the black pepper.

9

3.0 Materials and methods

The materials used are listed in Appendix 1.

3.1 Recovering of bacteria from glycerol stock

Twenty three (n= 23) bacterial isolates obtained from virology laboratory of UNIMAS

with labeling SB (1)1, KDKS549, KRS624, KRBRI5, KDKBB83, KRS684, KDKS550,

KRS625, HR (1)7, HB (1)8, HR (1)9, HB (2)6, HR (1)8, HB (1)9, BR (1)6, BR (1)10, SB

(1)10, HR (2)6, DB (2)9, DB (2)7, SB (2)6, HB (2)7 and DB (2)8 were subculture into

nutrient broth by pipetting 100 III from glycerol stock. The bacterial solutions were shaken

(120 rpm) for 2 days.

3.2 Isolation of the bacteria

About 20 J,ll of the bacterial solutions w~ spread on the TSA plates. All the plates were

incubated at 28°C for 2 days. In order to obtain the pure colony, the bacterial colony were

selected from each agar plate and purified by streaking on the TSA plates. The plates were

incubated at 28°C and the results were observed.

3.3 Preparation of fungus

The plant pathogen fungus, Phytophthora capsici was obtained from the stock culture and

cultured onto malt extract agar (MEA) plates at 28°C for 7 days. The results were

observed.

10

3.4 Antagonistic activities against Phytophthora capsici

3.4.1 Spore germination test

The Phytophthora capsid suspension from the seven days POB culture was spread over

the PDA plates by using the sterile swab and then allowed to dry for 10 min. The sterile

paper discs containing 30 III of nutrient broth (NB) supernatant of bacterial cultures were

placed on the agar. The disc with uninoculated NB was used as control and triplicate was

done for this experiment. The plates were incubated for 24 h, 48 hand 72 h at 28°C and

the radii of the clearing zones around the discs were measured.

3.4.2 Double plate assay

The bacteria and P. capsid were cultured on the NA and POA plates for 2 and 5 days. The

cover lids ofNA and POA plates were removed. The POA plate was inverted over the NA

plate and two plates were sealed together with parafilm. The plates were then incubated at

28°C for 3 days. The NA plate remained uninoculated for the control and duplicate was

done. The growth inhibition of bacteria towards P. capsid was measured based on the

percentage inhibition ofdiameter growth as follows (Edward et aI., 2013).

PIDG (%) =01-02 X 100 % 01

01: Diameter growth of P. capsid in control plat~ and D2: Diameter growth of P.capsici

in the double plate.

11

3.5 Molecular characterization

3.5.1 Preparation for DNA extraction

Two bacteria were selected based on the results of spore germination test and spore

germination test for DNA extraction. A pure colony ofeach bacteria culture was inoculated

from NA into the NB. The bacterial suspensions were shaken (120 rpm) at room

temperature for 24 h.

3.5.2 Boiled-CeU Method for DNA Extraction

Approximately 2.0 ml of each culture suspension was pipetted into 2.0 ml of centrifuge

tube and centrifuged at 10,000 rpm for 5 min. The supernatant was discarded and extra 2.0

ml of the bacterial suspension was pipetted into the centrifuge tube and centrifuged again

at 10,000 rpm for 5 min. The supernatant was discarded again and the pellet was

suspended with 500 ).11 of sterile distilled water. After that, the mixture was homogenized

using vortex until there was no clump being observed. The centrifuge tubes were

transferred into a boiling bath at 100 De for 10 min. Then, the centrifuge tubes were

immediately cooled in ice at -20 De for 5 min. Finally, the centrifuge tubes were

centrifuged at 10,000 rpm for 10 min. The supernatant was transferred into new sterile 0.6

ml tubes. The tubes were kept at 4 De for polymerase chain reaction.

3.5.3 Polymerase Chain Reaction (PCR) Amplification

The peR amplification was done using universal primers 27F (forward) and 519R

(reverse), The primer sequences used were listed in the Table 3.1. The peR master mix

used for the peR amplification was shown in the Table 3.2. The temperature, step cycle

and duration ofthe peR amplification were listed in the Table 3.3.

12

Table 3.1: The sequences of the PCR primers.

Primers Sequences (From 5' to 3')

27F AGAGTTTGATC(M)TGGCTCAG

51 9R G(W)ATTACCGCGGC(K)GCTG

M= CIA; W=AlT and K=T/G

Table 3.2: The master mix used for PCR amplification.

Components Volume (,.d)

PCRbuffer 10.0

MgCh 6.0

dNTPs 3.0

27F forward primer 1.0

519R reverse primer 1.0

8.0

DNA 20.0

Taq DNA polymerase 1.0 ..

Total 50.0

13