polyphasic taxonomy of fusarium causing wilt in cut flower...

1 | P a g e THE INTERNATIONAL JOURNAL OF BIOLOGICAL RESEARCH (TIJOBR) ISSN Online: 2618-1444 Vol. 3(3) July-Sep. 2020., 01-18; 2020 http://www.rndjournals.com

Research Article

THE INTERNATIONAL JOURNAL OF BIOLOGICAL RESEARCH (TIJOBR) ISSN Online: 2618-1444 Vol. 3(3) July-Sep. 2020., 01-18; 2020 http://www.rndjournals.com

Polyphasic Taxonomy of Fusarium causing wilt in cut flower crops (Chrysanthemum and

Marigold) and its chemical management Umar Muaz1*, Arooba Nawaz2, Akasha Mansoor2, Amar Ahmad Khan1, Zulnoon Haider1, Kamran Ahmad2

1Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan 2Department of Botany, University of Agriculture, Faisalabad, Pakistan.

*Corresponding author email: [email protected] ________________________________________________________________________________________________

Abstract Marigold (Tagetes erecta L.) and Chrysanthemum (Chrysanthemum L.) are important cut flower crops which are facing threat by wilting disease in Pakistan. Survey of important ornamental plant local nurseries, public parks and gardens of districts of Punjab Faisalabad, Lahore, Kasur, Vehari, and Islamabad were done. Samples of soil, root, shoot and leave from healthy as well as wilted portion of both crops were collected. Isolation was done to find the Fusarium species associated with diseased samples. Fusarium spp. was characterized using morphological characters. Cultural characters of Fusarium spp. on potato dextrose agar medium (color, texture and growth pattern) were studied. Microscopic characters of Fusarium equiseti on different magnification (Mycelial structure, conidia shape and size) were observed. Molecular characterization of morphologically identified Fusarium equiseti was done and submitted to Gene bank with accession no. MN135748 and MN135746. Characterized Fusarium equiseti was preserved on agar slants and dry filters papers in FMB-CC-UAF with Accession No. FMB0151, FMB0152. Pathogenicity was confirmed following by Koch’s postulates. Chemical control is one of the best management strategies that is used commonly to control the diseases. Different fungicides Score, Limin Lingx, Notyl and Myco Guard were used at different concentration 100, 200, 300 ppm in in-vitro conditions against pathogenic Fusarium equiseti. Percent inhibition of Limin Lingx was observed 89%. Most effective fungicide Limin Lingx was applied on diseased plants and after four weeks disease severity was reduced up to 60%. Key words: cut flower, Chrysanthemum, Marigold, chemical management ________________________________________________________________________________________ Introduction In Pakistan industry of Floriculture is grooming itself annually at the rate of 25% in last a few decades. This industry comprises the marketing of flower oils, nursery plants, bulbs, seeds, potted plants and micro propagated plants (Zilda et al., 2008). At international level business of cut-flowers is increasing at a rate of 25 percent annually. The international trading figures of floriculture are US$ 11 billion and share of cut-flowers in floriculture sector is 60% of the world trade. The global exports increased ten times from 0.5 billion to 5.1 billion in duration of 1995- 2005, which is being expected that it will be double up to 2025 (Singh et al., 2010). Many of the flowers cultivating countries of the world are earning a valuable foreign exchange by exporting the flowers. Among all those exporting countries, Netherlands is top ranked in the production and marketing of flowers. In the different zones of the world area under the flower production was 305,105 ha, of which the

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total area in European zone was 44,444 ha, Asia and Pacific 215,386 ha, North America 22,388 ha, the Middle East and Africa 2,282 ha and central and South Africa 17,605 ha. Total flowers production under greenhouses in various regions of world was 46,008 ha. The maximum area of India under ornamental crops was (88,600 ha) followed by China (59,527 ha), Japan (21,218 ha), Indonesia (34,000 ha), Brazil (10285 ha), USA (16400 ha), The Netherlands (8,017 ha), Taiwan (9.661 ha), Italy (7.654 ha), Germany (6,621 ha), the United Kingdom (6,804 ha), and Colombia (4,757 ha). The worldwide more than 145 countries are keenly interested in growing of ornamental crops and the area under these ornamentals is increasing gradually (Usman, 2013). Marigold (Tagetes erecta L.) is one of the important ornamental crop of family Asteraceae. It is herbaceous in nature. The leaves of Marigold plant are pinnate. It is one of the significant crop that is cultivated on commercial level as an ornamental crop. Marigold is aromatic and it produces the large size flowers having different colors and their color shade ranges from yellow to orange (Dole and Wilkins, 2005). Marigold has a wide importance in different business sectors like pharmaceutical and cosmetic industries. Extracts of marigold are utilized in field of medicine for the treatment of fungal diseases and different infections. It is also important in recovery of patients fighting with mammary tumor, cancer disease, ear arch and for the healing of burned and boiled skin (Bosma et al., 2003; Asif, 2008). Naturally some worthy chemicals are found in marigold leaves and flowers, like flavonoids, saponins, carotenoids, and useful oils etc. (Bilia et al., 2002). Chrysanthemum, one of important cut-flower crops of Asteraceae family has more than 30 species. Species of Chrysanthemum are wild in nature mostly. Chrysanthemum is native to Asia and north-eastern Europe (Nelson and Horst, 1997). Chrysanthemum is cultivated as annual crop throughout the globe and is used significantly for both indoor and outdoor purposes. Italy is one of the major growers. It has a cultivated area of 1180 ha and it has production of 437 million pieces (Garrett, 1970). Many of the viral, fungal and bacterial pathogens infect the Chrysanthemum seriously at its different stages. (Bhattacharjee and De, 2003). A variety of pathogens infect the cut-flowers but among those Fusarium oxysporium is one of the destructive pathogen which disturbs the plant from start to end stages (Garrett, 1970). The different pathogens have different adaptation behavior but Fusarium oxysporum is more destructive in warm climatic conditions (Locke et al., 1985). The genus of F. oxysporum comprises the ascomycete fungus especially filamentous in shape (Sordariomycetes, Hypocreales and Nectriaceae) and has several toxin producing pathogens of plants. Till this time round about 1000 species of Fusarium have been diagnosed causing many pathological problems like rots, wilts, blights and cankers in a variety of field, horticultural, ornamental, forest crops. Meanwhile this specie Fusarium, which has been defined by the both Snyder and Hansen, is being broadly acknowledged from more than 50 plus years (Harrington, 1999). When the pathogen of Fusarium oxysporum causes infection in the plant then it shows the yellowing, leaf fall and loss of turgidity of leaves. Plant growth is abrupt and processes of bud’s formation and florescence are disturbed harshly. Discoloration of plant leaves occurs. Photosynthetic mechanism of plant is affected badly. In case of attack of pathogen on vascular tissues, root and lower stem area of plant show the symptoms of discoloration. Regular availability of nutrients to plant is blocked and in most of the cases plant dies. In later stages of disease cream color whitish growth of mycelium can be observed in collar region (Ghosh and Singh 1982). As the wilt problem is one of the major problems in Pakistan. So for limited or no work has been done to identify the pathogen of Fusarium. Due to this reason the work was done for the detection, identification and characterization. The purpose of this study was also to study the etiology of the pathogen and its management. Preliminary research activities were conducted in Fungal Molecular Biology Lab, Department of Plant Pathology under the research project HEC Project # 6451. Keeping in view research was conducted for suitable management of this disease. Plan of work was set keeping in mind following objectives:



To confirm/find out the actual Fusarium species causing wilt in cut flower crops (Chrysanthemum and Marigold) in Pakistan.

To find the effective chemical control of Fusarium Wilt of cut flowers (Chrysanthemum and Marigold).

Materials and methods The research was conducted with exclusive goal to learn about wilting problem in cut-flower crops Marigold and Chrysanthemum caused by Fusarium equiesti. Further the methods and materials used to characterize the pathogen of Fusarium equiesti at morphological and molecular basis are these: (A) Materials Soil Soil samples of the pots of grown Chrysanthemum and Marigold were taken. Soil samples were taken from different areas and nurseries of Faisalabad, Lahore, Kasur, Vehari and Islamabad districts of Punjab, Pakistan. Diseased and healthy seeds, plants, plant parts were taken from nurseries and field growing areas. Mostly collected infected portions were of Desi variety. They were having the wilting symptoms. A variety of pathogens like Fusarium equiesti and many other saprophytic pathogens were isolated from diseased portions of plants. The fungicides used in doing the in vitro treatments were generally Mancozeb, Thiram, Captan etc. obtained from local markets from around Faisalabad. Disease incidence To evaluate the disease incidence a certain number of plants were observed and data was recorded on the data sheet. This calculation was done by formula

𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 =𝑁𝑜. 𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑒𝑑 𝑝𝑙𝑎𝑛𝑡𝑠

𝑁𝑜. 𝑜𝑓 𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑝𝑙𝑎𝑛𝑡𝑠× 100

Nurseries Visited to collect the samples of Chrysanthemum

City Nursery Contact no.

Faisalabad Neelam Nursery 03217676912

Faisalabad Evergreen Nursery 03006636815

Faisalabad New Ashraf Nursery 03006940041

Lahore Aziz Nursery Farm 03487559812

Lahore Faizan Nursery Farm 03464131798

Lahore Abbas Nursery Farm 03334222731

Islamabad Islamabad Nursery Farm 03005316735

Islamabad Fateh Muhammad Nursery 0514442877

Islamabad Pak Fiori Nursery 03465132920

Kasur Kmaal Nurseries 03144001314

Kasur Sardar Nursery Farm 03008007567

Kasur Green Valley Nursery 03018444375

Vehari Allah Bakhsh Nursery Farm 03038681870

Vehari Chohan Nursery Farm 03027737656

Vehari Awami Nursery Farm 03007725295

Nurseries Visited to Collect the Samples of Marigold



City Nursery Contact no.

Faisalabad Neelam Nursery 03217676912

Faisalabad Evergreen Nursery 03006636815

Faisalabad New Ashraf Nursery 03006940041

Lahore Aziz Nursery Farm 03487559812

Lahore Faizan Nursery Farm 03464131798

Lahore Abbas Nursery Farm 03334222731

Islamabad Islamabad Nursery Farm 03005316735

Islamabad Fateh Muhammad Nursery 0514442877

Islamabad Pak Fiori Nursery 03465132920

Kasur Kmaal Nurseries 03144001314

Kasur Sardar Nursery Farm 03008007567

Kasur Green Valley Nursery 03018444375

Vehari Allah Bakhsh Nursery Farm 03038681870

Vehari Chohan Nursery Farm 03027737656

Vehari Awami Nursery Farm 03007725295

Isolation of Pathogen. After collecting the infected samples, they were brought to FMB Lab. These sampled parts of plants i.e. seed, leaves and stem were cut down into 1cm pieces. Sampled portions of plants were cut into bits on the basis of visual symptoms. After the preparation of media (PDA) that was poured into the petri plates. After that those bits of sampled portions were placed on PDA with the help of disinfected forceps. After that the petri plates were wrapped with paraffin film and then they were incubated in culture room at 25C±2 for the period of 72 hours to check the colony growth. Identification and Purification. Morphologically identification of Fusarium was done by the help of keys and morphological explanations by Massey (1926) and McCulloch (1944). Picking of mycelium was done in laminar flow chamber in disease free environment. During this total procedure ethanol was used as disinfectant. For the purification of respective pathogen i.e. Fusarium was done by single hyphal technique under the microscopic observation. Moreover, the spores were observed critically under the different powers of microscope. Confirmation of spores was done. Plates having surely Fusarium pathogen were saved at the temperature of -40°C.

Fig.1 Identification and Purification of Respective Pathogen

Molecular Characterization Isolated respective pathogen from Marigold and Chrysanthemum was characterized on molecular basis.



Preservation of cultures The fungal pathogen when was isolated and confirmed then it was preserved. It was preserved at -40C. Dry filter technique. It is also known as Blotter paper Technique. In this firstly the rounded cuttings of Filter paper were autoclaved to prevent the contamination. The blotter paper is shifted to PDA plate. After that mycelia of the Fusarium was cultured on this paper. Then it was incubated in culture room. After a few days when there was a proper growth of cultured fungus, it was transferred to new plate. It was then put to be dry. When it was fully dry then it was cut into small bits. Then it was saved in -860C for future. Agar Slants preparation This activity was done to preserve the culture for long term. For this procedure the glass tubes were filled with PDA. They were kept to be solidified. This was done in laminar flow chamber. Then Fusarium was cultured in glass tube with the help of sterilized needle. Then they were plugged with cotton. They were wrapped to ensure zero contamination. These slants were saved in 40C temperature.

Fig.2 Preservation of Pure Cultures by Agar Slant Technique

Pathogenicity Test: Soil Sterilization. For the sterilization of soil, the Formalin chemical was used. For that purpose, it was mixed in water and shake it properly. After this the bag of a big size was filled with soil. Then the Formalin mixed with water was applied to soil. After this that bag of soil was covered for next 48 hours. Then that Formalin applied bag of soil was kept open for the next 24 hours. This was done in the research area of Plant Pathology. Seed Collection. For the pathogenicity test the seeds of different varieties of Marigold and Chrysanthemum were collected from the nurseries of Faisalabad. Different varieties seeds of both cut flowers are these:

Marigold Varieties Chrysanthemum Varieties

Marigold Inca Orange Tricolor Daisy

Marigold Inca Gold Crown Daisy

Marigold Inca Yellow Shasta Daisy

French Marigold Bonenza Bolero Corn Daisy

French Marigold Bonenza Orange Alecost

Desi



Nursery Raising and Transplantation. Nursery raising is an intensive care demanding activity. To raise the nursery of these cut flowers the artificial media like coco peat and peat moss were used. For nursery raising the cups were filled with soil and then the peat moss was mixed in it. Seeds of these flowers were dropped in to cups in the depth of roundabout one inch below the upper surface. After that the coco peat was spread on the upper surface of the soil. Watering was done at the proper interval of time to keep the soil moist. After one week the seeds were germinated. After the interval of about 20 days the nursery was ready. Then the nursery was transplanted into pots. Inoculum Preparation. For the preparation of inoculums first of all the PDB was prepared. PDB was autoclaved to avoid the any contamination. After cooling that PDB was brought to Laminar Flow Chamber. After that from the culture of Fusarium the mat was taken by the help of needle and was cultured into PDB flasks. Those flasks were incubated at 25±2 °C. After 3-4 days the mat had covered the total PDB. The growth of fungus was very lush. Inoculum was ready at that time. Inoculation. For inoculation process the plants were grouped into two groups. One was that which was inoculated before transplanting, and other was that which was inoculated after the transplantation at the time of flowering. Inoculum was mixed directly into the soil of pots. In vitro evaluation of fungicides against pathogenic Fusarium equisti. Fungicides at different concentration were evaluated against pathogenic Fusarium spp. using poisoned food technique. Fungicides were mixed in PDA at lukewarm stage, disc of Fusarium culture placed in center of poisoned medium containing plates. Control plates were maintained using only PDA medium. Plates were incubated and data were recorded. Percentage inhibition was calculated using formula:

𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 =𝐶 − 𝑇

𝐶× 100

Where, C = Pathogen growth in control plate. T = Pathogen growth in Dual Culture plate. To evaluate fungicides against Fusarium spp. on cut flowers crops (Marigold and Chrysanthemum) in green house

From lab experiment the most effective fungicide was used in inoculated Marigold and Chrysanthemum plants in green house. Disease severity was recorded. Data Recording. After the inoculation the plants were observed with regular interval of time. After the one and half month of inoculation symptoms were appeared. Data was recorded in research book on the basis of disease severity. In the same way in vitro the effect of different fungicides with different concentrations was also calculated.

Results and discussion Disease Documentation of Chrysanthemum Table 1: Disease Documentation of Chrysanthemum in Faisalabad District

City Nursery Contact no. Observed Plants Infected Plants Disease Incidence

Faisalabad Neelam Nursery

03217676912 20 08 40%

Faisalabad Evergreen Nursery 03006636815 17 10 58%

Faisalabad New Ashraf Nursery 03006940041 16 12 75%

During the survey of Faisalabad area three nurseries were visited. Among these nurseries the disease incidence was highest in New Ashraf Nursery and that was 75% while lowest incidence was in Neelam Nursery and that was 40%. Table 2: Disease Documentation of Chrysanthemum in Lahore District




Lahore Aziz Nursery Farm 03487559812 22 06 27%

Lahore Faizan Nursery

Farm 03464131798 18 09 50%

Lahore Abbas Nursery

Farm 03334222731 14 06 42%

From the city of Lahore 3 nurseries were surveyed to collect the samples of Chrysanthemum. Among these nurseries the disease incidence was 50% highest and 27% lowest. Highest incidence was in Faizan Nursery Farm and lowest incidence among surveyed nurseries was in Aziz Nursery Farm. Table 3: Disease Documentation of Chrysanthemum in Islamabad District


Islamabad Islamabad Nursery Farm

03005316735 10 02 20%

Islamabad Fateh Muhammad Nursery

0514442877 12 03 25%

Islamabad Pak Fiori Nursery 03465132920 11 03 27%

In Islamabad three nurseries were visited to examine the plants of Chrysanthemum. Among those three nurseries high incidence of disease was in Pak Fiori Nursery and that was 27% and the low incidence of disease was in Islamabad Nursery Farm. Table 4: Disease Documentation of Chrysanthemum in Kasur District


Kasur Kmaal Nurseries 03144001314 17 05 29%

Kasur Sardar Nursery Farm

03008007567 15 08 53%

Kasur Green Valley Nursery

03018444375 13 05 38%

In Kasur three nurseries were also visited to check the condition of affected Chrysanthemum plants. High disease incidence was in Sardar Nursery Farm and that was 53% and low incidence was Kamal Nurseries and that was 38%. Table 5: Disease Documentation of Chrysanthemum in Vehari District


Vehari Allah Bakhsh Nursery Farm

03038681870 19 04 21%

Vehari Chohan Nursery Farm

03027737656 21 10 47%

Vehari Awami Nursery Farm

03007725295 16 07 43%

In district Vehari for sampling of Chrysanthemum among visited 3 nursery farms the disease incidence was high in Chohan Nursery Farm and the incidence was 47% and lowest incidence was in Allah Bakhsh Nursery and that was 21%. Disease Documentation of Marigold in Various Districts of Punjab Table 6: Disease Documentation of Marigold in Faisalabad




Faisalabad Neelam Nursery

03217676912 18 03 16%

Faisalabad Evergreen Nursery 03006636815 12 02 16%

Faisalabad New Ashraf Nursery

03006940041 20 03 15%

For the sampling of Marigold three nurseries were visited to check the infection by the Fusarium pathogen. Incidence of disease in Marigold was high in 2 nurseries Neelam Nursery and Evergreen Nursery. Both of the nurseries were having incidence was 16% and third had 15%. Table 7: Disease Documentation of Marigold in Lahore


Lahore Aziz Nursery Farm 03487559812 17 01 06%

Lahore Faizan Nursery Farm

03464131798 22 01 04%

Lahore Abbas Nursery Farm

03334222731 19 02 10%

To check the incidence of disease on Marigold three nurseries were checked. In this respective incidence in Lahore was high in Abbas Nursery and lowest incidence was in Faizan Nursery. Highest incidence was 10% and lowest was 04% Table 8: Disease Documentation of Marigold in Islamabad.


Islamabad Islamabad Nursery Farm

03005316735 17 01 05%

Islamabad Fateh Muhammad Nursery

0514442877 20 01 05%

Islamabad Pak Fiori Nursery 03465132920 08 01 12%

In Islamabad city total three nurseries we sampled. Highest incidence of disease in Marigold was in Pak Fiori Nursery and that was 12%, while other both of nurseries were having the disease incidence of 05%. Table 9: Disease Documentation of Marigold in Kasur


Kasur Kmaal Nurseries 03144001314 10 01 10%

Kasur Sardar Nursery Farm

03008007567 20 01 05%

Kasur Green Valley Nursery

03018444375 19 02 11%

In visited nurseries of Kasur the sampling of Marigold was done. Incidence of disease was high in Green Valley Nursery and that was 11% and lowest was Sardar Nursery and that was 05%. Table 10: Disease Documentation of Marigold in Vehari


Vehari Allah Bakhsh Nursery Farm

03038681870 19 01 06%

Vehari Chohan Nursery Farm

03027737656 21 02 09%

Vehari Awami 03007725295 16 02 13%



Nursery Farm

In city of Vehari the disease incidence on Marigold was not so high. But among visited sites the highest incidence was 13% and that was reported in Awami Nursery Farm. Lowest incidence was recorded in Allah Bakhsh Nursery and that was 06%. Table 11: Frequency Percentage of Fusarium in Chrysanthemum (Dist. Faisalabad)

Region Fungus Frequency %age

Faisalabad Fusarium spp. 75%

Other (Saprophytic spp.) 25%

Table 12: Frequency Percentage of Fusarium in Chrysanthemum (Dist. Kasur)


Kasur Fusarium spp. 65%


Table 13: Frequency Percentage of Fusarium in Chrysanthemum (Dist. Vehari)


Vehari Fusarium spp. 85%


Table 14: Frequency Percentage of Fusarium in Chrysanthemum (Dist. Lahore)


Lahore Fusarium spp. 70%


Table 15: Frequency Percentage of Fusarium in Chrysanthemum (Dist. Islamabad)


Islamabad Fusarium spp. 62%


In the survey of five districts of Punjab in view of Chrysanthemum crop the frequency percentage of Fusarium was highest in Vehari and lowest frequency was in Islamabad region. Table 16: Frequency Percentage of Fusarium in Marigold (Dist. Lahore)


Lahore Fusarium spp. 45%


Table 17: Frequency Percentage of Fusarium in Marigold (Dist. Vehari)


Vehari Fusarium spp. 55%


Table 18: Frequency Percentage of Fusarium in Marigold (Dist. Faisalabad)


Faisalabad Fusarium spp. 60%


Table 19: Frequency Percentage of Fusarium in Marigold (Dist. Islamabad)


Islamabad Fusarium spp. 42%


Table 20: Frequency Percentage of Fusarium in Marigold (Dist. Kasur)




Kasur Fusarium spp. 30%


In the surveys of 5 districts of Punjab for checking the disease frequency of Fusarium in crop of Marigold various nurseries were visited and it was concluded that lowest disease frequency was in District Kasur and highest frequency was in Faisalabad. Identification of Fusarium spp. Morphological Characters of identified Fusarium equisti On PDA the culture of Fusarium equisti was having the white mycelia. It became pigmented with the passage of time. In a few cultures it was observed that sporodochia was at the center of culture. Macroconidia of this fungal pathogen were thick walled having the strong dorsi-ventral curvature and quite wide structure at its center. Spores of this culture were observed short in size and long macro conidia. Apical cells of this pathogen were elongated and were tapering to form a needle like structure. Apical cells were having the foot shape. Number of septa was five. Micro conidia were absent. Chlamydospores of Fusarium equisti were produced fastly in the duration of Limin Lingxiu-4 weeks. They were formed in the form of chains and clusters, and they pigmented with the passage of time. Molecular Characterization of Fusarium equisti

Fig.3 Molecular Characterization of Fusarium equisti

CBS 414.97 Fusarium pseudoanthophilum

CBS 748.97 Fusarium napiforme

CBS 449.97 Fusarium pseudocircinatum

CBS 409.97 Fusarium guttiforme

CBS 100057 Fusarium bactridioides

CBS 177.35 Fusarium bactridioides

CBS 405.97 Fusarium circinatum

CBS 407.97 Fusarium denticulatum

CBS 110286 Fusarium foetens

CBS 417.97 Fusarium pseudonygamai

CBS 125178 Fusarium ficicrescens

CBS 452.97 Fusarium begoniae

CBS 418.97 Fusarium ramigenum

CBS 456.97 Fusarium nisikadoi

CBS 183.34 Fusarium concolor

CBS 961.87 Fusarium polyphialidicum

FMB 0151 FMB-F4B-CO

FMB 0152 FMB-S1F5-Dg

NRRL 26419 Fusarium equiseti

CBS 458.93 Fusarium venenatum

CBS 637.76 Fusarium robustum

CBS 113234 Fusarium langsethiae

CBS 127525 Fusarium louisianense

CBS 636.76 Fusarium lunulosporum

CBS 127669 Fusarium nepalense

CBS 122858 Fusarium aethiopicum

CBS 122710 Fusarium sinensis

CBS 143231 Fusarium petersiae

CBS 393.96 Fusarium nurragi

CBS 397.96 Fusarium babinda

CBS 189.34 Fusarium sublunatum

CBS 100160 Fusarium beomiforme

CBS 749.97 Fusarium nygamai

CBS 450.97 Fusarium concentricum

CBS 258.54 Fusarium annulatum

CBS 738.97 Fusarium dlaminii

CBS 739.97 Fusarium acutatum

CBS 402.97 Fusarium acutatum

CBS 221.76 Fusarium fujikuroi

CBS 110311 Fusarium biseptatum

CBS 317.34 Fusarium penzigii

CBS 120718 Fusarium delphinoides

CBS 434.34 Fusarium domesticum

CBS 111.93 Fusarium zealandicum

CBS 502.94 Fusarium staphyleae

CBS 125727 Fusarium rectiphorum

CBS 125729 Fusarium pseudensiforme

CBS 140079 Fusarium solani



Fig. 4 PCR Amplification of Putative Phytopathogen

Preservation. The characterized specie of Fusarium “Fusarium equisti” was preserved in Agar Slants at 4 °C and dry filter paper at -86 °C. In Result of Pathogenicity Symptoms Observed. The inoculums prepared of identified specie Fusarium equisti was applied to soil for pathogenicity test. After this both of the crops were observed with proper gap of time. Chrysanthemum Symptoms. In pathogenicity test the plants were observed regularly on the gap of five days. After 25 days of the inoculation the symptoms of respective pathogen were observed. In these symptoms the leaves of plants of one side were having the symptoms of browning and yellowing. Flowers production was retarded. When stem was cut it had the browning symptoms. Marigold Symptoms. In test of pathogenicity the plants were examined time to time. In first month after inoculation there were no symptoms. At last after 45 days of inoculation symptoms of pathogen were appeared. In the wilting problem light and dark green mottling was observed and especially young leaves were wilted. Affected leaflets were distorted and were smaller than normal size. Flowering was disturbed. Flowers had the symptoms of wilting. In vitro evaluation of fungicides against pathogen of Fusarium equisti. Total four fungicides of commercial standard were used to evaluate them against the pathogen of Fusarium f sp. Marigold and Chrysanthemum on PDA added with fungicides at different concentrations 100, 200, 300ppm.The fungicides used in experiment will be these.

Score(Difenocotazol) (F1)

Limin Lingx (Azoxystrobin, Difenoconazole) (F2)

Notyl (Mancozeb, Cymoxonil) (F3) Myco Guard(Azoxystrobin) (F4)

They were preserved at 25 °C. The fungicides were evaluated on the basis of growth of Fusarium against them. All the fungicides were effective to manage the disease but fungicide Limin Lingxui was the most effective fungicide. Mcculloch (1944) for the first time suggested the chemicals to manage the Fusarium issues. He also reported that Chloropicrin is very effective for the management of Fusarium in soil. He did this in field conditions. Cow Perth waite and Magie (1954) suggested that Nebham and ZnSO4 applied to soil weekly decreases the dry condition of soil and plant is prevented from Fusarium issues. Table 21: Analysis of Variance Table After 3 days data of Fungicides

Source DF SS MS F P

Replication 2 0.0235 0.01173

Fungicide 4 17.7595 4.43986 3358.69 0.0000

Conc 2 0.2253 0.11267 85.23 0.0000

Fungicide*conc 8 0.0847 0.01058 8.01 0.0000



Grand Mean 0.7553 CV 4.81 The above ANOVA results showed clearly that P factor value of Fungicides have the highly significant results. In ANOVA figures the results of concentrations is also highly significant. The results of interaction of Fungicides and their concentrations is also highly significant. Table 22: LSD All-Pairwise Comparisons Test of Data for Fungicide after 3 days

Fungicide Mean

Control 2.0056 A

Myco Guard 0.5222 B

Score 0.4767 C

Notyl 0.4389 D

Limin Lingxiu 0.3333 E

LSD= 0.0351 Mean table data after 3 days showed that among all fungicides Limin Lingxiu fungicide was the most effective fungicide to stop the growth of mycelium. Table 23: LSD All-Pairwise Comparisons Test of Data for concentration

Concentration (ppm) Mean

100 0.8420 A

200 0.7553 B

300 0.6687 C

LSD= 0.0272 Among three concentrations comparison it was concluded that the concentration of 300 ppm showed the maximum control over growth of mycelium. These results showed clearly the increase of concentration decreases the growth of Fusarium spp. Table 24: LSD All-Pairwise Comparisons Test of Data for Fungicide*conc

Fungicide

Concentration Mean

Control 200 2.0167 A

Myco Guard 100 0.6500 B

Score 100 0.5767 C

Notyl 100 0.5500 C

Myco Guard 200 0.5167 CD

Score 200 0.4767 DE

Limin Lingxiu 100 0.4500 EF

Notyl 200 0.4500 EF

Myco Guard 300 0.4000 FG

Score 300 0.3767 GH

Limin Lingxiu 200 0.3167 H

Notyl 300 0.3167 H

Limin Lingxiu 300 0.2333 I

LSD= 0.0608 The interaction of fungicide Limin Lingxiu and its concentration 300ppm gave a maximum control over growth of

Error 28 0.0370 0.00132

Total 44 18.1299



Fusarium. Other treatments also showed the significant results and also showed inhibition against fungus growth but Limin Lingxiu fungicide with concentration of 300 ppm was the most effective.

Fig.5 Inhibition after 3 days Score= F1, Limin Lingx=F2, Notyl=F3, Myco Guard=F4.Data collected after 3 days it was observed that Limin Lingxiu fungicide showed the maximum inhibition against the mycelium of Fusarium on concentration of 300ppm. Other tested three fungicides were also effective but Limin Lingxiu fungicide was the top effective one in its efficacy. On this same concentration inhibition of Notyl fungicide was on the second position. Table 25: Analysis of Variance after 5 days data

Source DF SS MS F P

Replication 2 0.0231 0.01155

Fungicide 4 24.8750 6.21875 2966.02 0.0000

Conc 2 0.2396 0.11979 57.13 0.0000

Fungicide*conc 8 0.0665 0.00831 3.97 0.0030

Error 28 0.0587 0.00210

Total 44 25.2629

Grand Mean 0.8627 CV 5.31 The above ANOVA results showed clearly that after 5 days the factors the fungicides and their concentrations showed the significant results. The results of interaction of Fungicides and their concentrations is also highly significant. Table 26: LSD All-Pairwise Comparisons Test of Data for Fungicide after 5 days

Fungicide Mean

Control 2.3444 A

Myco Guard 0.5722 B

Score 0.5244 C

Notyl 0.4889 C

Limin Lingxiu 0.3833 D

LSD= 0.0442 Data collected after 5 days the Limin Lingxiu fungicide among all fungicides controlled the growth of fungus up to maximum. Table 27: LSD All-Pairwise Comparisons Test of Data for conc Data after 5 days

Concentration(ppm) Mean

100 0.9507 A

200 0.8653 B

300 0.7720 C



LSD= 0.0342 In data collected after 5 days the results showed that among 3 concentrations the 300 ppm controlled the growth of fungus. Table 28: LSD All-Pairwise Comparisons Test of Data for Fungicide*conc Data after 5 days

Fungicide Concentrations Mean





Score 100 0.6200 C

Notyl 100 0.6000 CD

Myco Guard 200 0.5667 CDE

Score 200 0.5267 DE

Limin Lingxiu 100 0.5000 EF

Notyl 200 0.5000 EF

Myco Guard 300 0.4500 F

Score 300 0.4267 FG

Limin Lingxiu 200 0.3667 G

Notyl 300 0.3667 G

Limin Lingxiu 300 0.2833 H

LSD= 0.0766 The interaction of fungicides and concentration showed that the fungicide Limin Lingxiu and its concentration 300 ppm maximumly controlled the growth of fungus.

Fig 6 Inhibition after 5 days Score= F1, Limin Lingx=F2, Notyl=F3, Myco Guard=F4 In data noted after five days inhibition percentage was calculated. In this respect once again the fungicide Limin Lingxiu was on top with the inhibition percentage of 89% on concentration of 300ppm. Except Limin Lingxiu others were also very effective against Fusarium. Table 29: Analysis of Variance Table for after 7 days Data

Source DF SS MS F P

Replicate 2 0.0036 0.00180

Fungicide 4 35.4127 8.85319 4743.18 0.0000



Conc 2 0.2406 0.12028 64.44 0.0000

Fungicide*conc 8 0.0655 0.00819 4.39 0.0016

Error 28 0.0523 0.00187

Total 44 35.7747

Grand Mean 0.9858 CV 4.38 ANOVA table results after 7 days data showed that P value of both factors fungicides and concentrations is significant. The results of interaction of both factors is also significant. Table 30: LSD All-Pairwise Comparisons Test of Data for Fungicide after 7 days

Fungicide Mean

Control 2.7556 A

Myco Guard 0.6222 B

Score 0.5789 C

Notyl 0.5389 C

Limin Lingxiu 0.4333 D

LSD= 0.0417 In results collected after 7 days all the fungicides controlled the growth of mycelium. Among these fungicides Limin Lingxiu fungicide was the most effective to control the growth of Fusarium pathogen. Table 31: LSD All-Pairwise Comparisons Test of Data for conc Data after 7 days

Conc Mean

100 1.0787 A

200 0.9787 B

300 0.9000 C

LSD=0.0323 In results after 7 days all of the used concentrations of fungicides controlled the pathogen growth. But among used concentrations 100, 200, 300ppm the best concentration was 300 ppm to control the mycelium growth up to maximum. table 32: LSD All-Pairwise Comparisons Test of Data for Fungicide*conc Data after 7 days

Fungicide Conc. Mean



Score 100 0.6767 C

Notyl 100 0.6500 C

Myco Guard 200 0.6167 CD

Score 200 0.5767 D

Limin Lingxiu 100 0.5500 DE

Notyl 200 0.5500 DE

Myco Guard 300 0.5000 E

Score 300 0.4833 EF

Limin Lingxiu 200 0.4167 F

Notyl 300 0.4167 F

Limin Lingxiu 300 0.3333 G

LSD= 0.0723 In results collected after 7 days after checking the interaction of fungicides and their concentrations showed



that Limin Lingxiu Fungicide with concentration of 300ppm gave maximum control against the growth of Fusarium.

Fig 7 Inhibition after 7 days

Score= F1, Limin Lingx=F2, Notyl=F3, Myco Guard=F4 In data collected after duration of 7 days it was noted that fungicide Limin Lingxiu once again showed the maximum inhibition against Fusarium. That inhibition was 89%. After the Limin Lingxiu, NOTYL was also effective. It had also a good response against Fusarium. Evaluation of Fungicides against Fusarium equisti on Marigold and Chrysanthemum in Green House In lab experiment among evaluated fungicides the most effective fungicide was applied to inoculated Marigold and Chrysanthemum plants in research field area. Disease severity was recorded. For this purpose, two groups of plants were prepared. Each group had 4 plants. One group was sprayed with Limin Lingxiu.Then disease severity was recorded on the week basis visit. Comparison was done between control and treated plants. Table 33: Analysis of Variance Table for Field trial data

Source DF SS MS F P

Replication 3 32.12 10.71

Fungicide 1 1326.12 1326.12 581.69 0.0000

Weeks 3 905.12 301.71 132.34 0.0000

Fungicide*Weeks 3 102.62 34.21 15.01 0.0000

Error 21 47.87 2.28

Total 31 2413.87

Grand Mean 29.438 CV 5.13 The ANOVA table data of Field trials showed that the P value of Fungicides was significant. In the same way the P value of weeks and their interaction with fungicides value was also highly significant. Table 34: LSD All-Pairwise Comparisons Test of Data for weeks

Weeks Mean

W4 36.75A

W3 31.375B

W2 27.375C

W1 22.250D

LSD= 1.5700 Among the data collected in four weeks back to back it was calculated that severity of disease was highest in week 4.



Table 35: LSD All-Pairwise Comparisons Test of Data for fungicide*weeks

Fungicide Weeks Mean

C W4 45.000A

C W3 38.750B

C W2 34.000C

C W1 25.750E

Limin Lingxiu W4 28.500D

Limin Lingxiu W3 24.000E

Limin Lingxiu W2 20.750F

Limin Lingxiu W1 18.750F

LSD= 2.2203 In comparison if we observe then we concluded that with the weeks increasing the severity was increased in control and fungicides applied group plants. But on the other hand if we compare the treated plants group and control plants group then severity was less in Limin Lingxiu fungicide treated plants. Discussion In vitro experiment four fungicides were evaluated against Fusarium spp. The different concentrations 100, 200, 300 ppm of each fungicide were used to check the effect of fungicides against the mycelial growth of Fusarium. Among different concentrations and different fungicides used in the experiment the most effective fungicide was Limin Lingxiu fungicide. The best and effective concentration was 300ppm to control the growth of Fusarium. Each fungicide showed the significant results at 300 ppm concentration. In the green house field conditions one of the most effective chemical among four chemicals was applied to inoculated diseased plants. Disease was increased with the weeks increase. Severity was higher in control than Limin Lingxiu fungicide treated plants. Systemic and non-systemic fungicides viz., carbendazim, myclobutanil, bitertanol, hexaconazole, mancozeb, captan and zineb were evaluated for their efficacy on mycelial growth of Fusarium oxysporum by food poisoning technique (Falck, 1907). It was concluded by results of experiments that fungicides which work systemically, they stopped the growth of Fusarium mycelial growth. They also inhibited the growth at different concentrations. It was suggested that the concentration of 1000 ppm of hexaconazole controlled the Fusarium growth maximumly. That growth was stopped on the 8.80 mm. On the second position carbendazim showed the great efficacy and controlled the growth of Fusarium up to 9.40 mm. On the third number was bitertanol (18.60 mm) and on fourth position was myclobutanil (20 mm). All the chemicals had the same concentrations. By this experimental work it was concluded that among the non-systemic fungicides, mancozeb was the most effective which stopped growth on 14. 20mm.On the other hand captan reduced growth and stopped it on 20.00 mm. Zineb fungicide was also effective and it stopped growth on 22 mm. Similar results were also formulate by Sommer (1982); Singh et al. (1997); Daradhiyar (1980); Kalra and Sokhi (1985); Banyal et al. (2008) and Patel et al. (2005) against other pathogenic fungi. Moreover, the fungicides have been reported to control the mycelia growth ap to maximum in Richard medium (Khan et al., 1997; Sharma, 2006). References.

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polyphasic taxonomy of fusarium causing wilt in cut flower...

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