DCSI Vol. 09 No.

2: 257 - 261 (2013)

Research Article DCSI 9: No.2; 257 261 (2013)

Sanjay R. Maske and Umesh P. Mogle

Received: 17 November, 2013 Revised: 30 November, 2013 Accepted: 22 December, 2013 Online: www.jdcsi.in/

Determination of seed borne mycoflora of Cowpea (Vigna unguiculata (L.) Walp.)

Sanjay R. Maske and Umesh P. Mogle1 Department of Botany, K. S. K. College Beed (M. S.)IndIa 1 Department of Botany, J. E. S. College, Jalna (M. S.) India sanjaymaske11@gmail.com

Abstract:

Seed born mycoflora of cowpea was determined by using blotter and agar plate method; Five cowpea seed samples were collected from local markets of Beed district (MS) in India. A total of 25 fungal species in 12 genera isolated. The isolated fungal species were Fusarium oxysporum, F. equisti, F. solani F. Monliforme, F. semitectum, F. sporotrichioides, F. anthophilum, F. graminearum, Penicillium notatum, Cladosporiumherbarum, Mucorhiemalis, Rhizopusoryzae, Aspergillus niger, A. flavus, A. glaucus, A. Fumigatus, Colletotrichum lindemuthianum. Alternaria longissima, Cephalosporium acromonium, Phytopthera sp. sclerotium sp. Macrophomina phaseolina. Rhizoctonia solani, R. bataticola. R. nigricans. The most frequently isolated fungal species were Aspergillus Sp. Fusrium Sp. Penicillum, Mucoretc. The untreated seeds were found to be associated with highest percent incidence of mycoflora than treated seeds.About 65% of the samples were infested with Fussarium oxysporum, Aspergillus nigar, Rhizopus oryzae, Penicillium notatum sp. Of the two methods, The agar plate method was better than the blotter method in isolating seed born fungi as compared to blotter.The blotter method was the best for the detection of Aspergillus flavus, Alternaria longissima, Fusrium sp. Penicillum, while agar plate method was suitable for the detection of, A. Fumigatus, colletotrichum lindemuthianum. Alternaria longissima, sclerotium sp. Macrophomina phaseolina. Rhizoctonia solani, R. nigricans. Key words: Cowpea seed, seed mycoflora, standard blotter, agar plate method.
adapted and highly variable crop, cultivated

Introduction Cowpea (Vigna unguiculata (L.) Walp) is one of the most ancient human food source and has probable been used as a crop plant since Neolithic times (Chevalier, 1964). It is now a broadly

around the world primarily for seed, but also as a vegetable, a cover crop and for fodder. Cowpea is one of the important food legume crop grown by many small and large-scale farmers in tropical and

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subtropical regions (Gitaitis and Nilakhe, 1982; Buaet et al., 1998). The people obtain a good supply of proteins, Carbohydrates and vitamins from all the plant parts used for food, especially from seed (Quin, 1997; Sing et al., 2002). Such a nutritive crop infected from pathogens like fungi, bacteria, nematodes etc. Seeds have been shown to harbor number of fungi. Many of them are known to cause important diseases. growth and finally affect quantity and quality of crops and discolourations decrease germinibility, mycotoxin production and total decay. Quenton et al., 2003. Infected seeds with pathogens often fail to germinate, transmit disease from seed to seedling and from seedling to growing plants, thus seedborne diseases cause enormous losses to the crops (Fakir et al., 2002). Some of the fungi produce mycotoxins, which can have adverse effects on the health of both farm animals and humans. Fungi that establish on the developing seed in the field may be broadly of two kinds, saprophytic field fungi and pathogenic fungi (Wright et al., 1995). According to Champion (1997), many fungi considered to be saprophytes, pathogens or others that lost their ability to cause disease, can survive in latency inside of the seeds, becoming active when these seed germinate. Seed born fungi may results in loss in germination, discoloration and shriveling (Nutsuga et al., 2004; Castilloet al., 2004). The seed-borne fungi of Solanum xanthocorpum were detected by moist blotter (B) and agar (A) plate methods as recommended by ISTA (1956), De Tempe (1970), Neergaard (1973) and Agarwal et al., 1973. The detection of seed borne fungi can be easily done using the blotter test which permits 1979; mycelium Maude, growth and formation of fruiting bodies on the seed surface (Neergaard, 1996), becoming possible the identification of fungal species by morphology. The objectives of this study is to paper These fungi cause severe loses to stored seeds, seedlings & later stages of plant

Sanjay R. Maske and Umesh P. Mogle

isolate and identify fungi contaminating cowpea seeds in Beed District (MS) in India Materials and Methods: Collection of Seed sample The method described by Neergaard (1973) has been used for the collection of seed sample. Five (500 kg. of each) seed sample of Cowpea were collected from local market of Beed in (M.S.) in India and preserve in cloth bag at laboratory condition during the studies. Detection of seed mycoflora The external and internal seed mycoflora was detected by using standard moist blotter method and by agar plate method seed as recommended international testing

association (ISTA, 1996) and Neergaard (1973). a) Standard Blotter paper Method For detecting external seed mycoflora 100 healthy seeds from each sample were taken and composite sample was made. Untreated 10 seeds from the samples were taken and placed at equal distance on three layers of moistened blotter paper in pre-sterilized petriplates of 9 cm diameter. For detecting internal seed mycoflora 100 healthy seeds from each sample were taken and composite sample was made and seeds were treated with 0.1% HgCl2. Solution for five minute and then washed thoroughly with sterile distilled water. Treated 10 seeds from the samples were taken and placed at equal distance on three layers of moistened blotter paper in pre sterilized petriplates of 9 cm diameter. All the petriplates (Treated and untreated) with three replicates were used per treatment. The petriplates were arranged in a randomized block design. All the petriplates were incubated at 25 + 2 conditions for 7 days. b) Agar plate method In this method presterilize petriplates were poured with 15 ml. of autoclaved Potato dextrose agar medium (PDA). On cooling of the medium treated and untreated seeds per plate of to be studied were equally spaced aseptically. A
0

C under diurnal

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set of 10 seeds of samples of cowpea with three replicates design. were PDA used was to per treatment. with The 1% petriplates were arranged in randomized block used amended suppress streptomycinesulphate
0

Sanjay R. Maske and Umesh P. Mogle

hyphae or spores aseptically into sterile media with alcohol flamed inoculating needle in an inoculating chamber. All the plates were incubated at 25 +0C under diurnal conditions for 7 days. Microscopic observed structure a of fungal isolates was for under compound microscope

bacterial
th

growth. All the petridishes were incubated at 25 + 2 C under diurnal conditions for 7 days. After 8 days all these petriplates were examined directly under a stereoscopic binocular microscope. The treated seeds were nicked with a pointed needle to find out the actual location of mycoflora associated with different seed components (Seed coats, Tegmen, Embryo).The population of the fungal isolate was determined by direct counting of the colonies on the plates. The colonies were first designated on the basis of certain colony morphological characteristics especially

proper identification at 40X magnification. Results and Discussion: Isolation of Seed mycoflora from cowpea was done critically and results are given in the fom of table (Table- 1 and Fig.1). In order to study the total association of seed born fungi, Cowpea seeds were plated on blotter paper and agar plate The seeds pretreated with 0.1% HgCl2 Solutions were placed on blotter paper and agar plate for the isolation of internal seed born fungi. It is clear from the obtained results that 25 fungal species in 12 genera isolated from the untreated and treated cowpea seed used in the study. The fungi appeared clearly on the culture plate between 5-7 days of incubation. The difference of the fungi on blotter paper was different from that of Fussarium equisti agar (17.00) F.solani plate. (15.00)

color, sporulation type, shape of spores, and mycelium up to species level with the help of compound microscope. Pure cultures were prepared from each of the designated fungal colonies observed on blotter paper seeds and agar plates seeds by transferring Observation was apparently due to the fact that PDA supplied nutrients for the growth of fastidious organisms. Table 1 indicates that Cowpea seed was associated with 25 fungi such as, Fusarium oxysporum, F. equisti, F. solani F. Monliforme, F. semitectum, F. F. sporotrichioides, F. anthophilum, Penicilliumnotatum, Mucorhiemalis, A. flavus, A. A. Fumigatus, Phytoptherasp. Sclerotiumsp graminearum,

Aspergillus glaucus(18.00) Alternaria longissima (14.58) Cladosposporium herbarum(14.00) and Macrophomina oryzae(11.00) semitectum, phaseolina, Penicillium F. (12.00) notatum, Rhizopus (10.00). F.

Sclerotium sp (9.45). Phytopthera sp. (9.00). graminearum nigricans F. sporotrichioides, Fussarium to least. anthophilum,

Rhizoctonia. (9-5%),

Cladosporiumherbarum, Rhizopusoryzae, glaucus, Alternarialongissima, Cephalosporium acromonium, Aspergillusniger,

R. bataticola etc Mucorhiemalis

were intermediate within the range of

colletotrichumlindemuthianum.

Cephalosporium acromonium (3.25%), were found In treated Cowpea seeds only 10 fungi were found. Aspergillus niger shows maximum percentage Fussarium incidence oxysporium (5.00) (3.79) followed and by Aspergillus

Macrophomin aphaseolina. Rhizoctoniasolani, R. bataticola, R. nigricans. In case of standard blotter the percent incidence of untreated and treated seed is as follows. On the untreated (37.50) seeds gave of Cowpea % Aspergillusflavus highest

flavus, A. fumigatus, Fussarium solani, Rhizopus oryzae cladosporium herbarum, F. anthophilum, F. semitectum, Rhizoctonia solani. and Rhizoctonia nigrian (0.60%) were found to least In case of

incidence followed by A. niger (33.00) Fussarium oxysporum (25.00) Aspergillus fumigates (22.25)

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Agar plate the percent incidence of untreated and treated seed is as follows. On the untreated seeds of Cowpea Aspergillus flavus(42.00) gave highest percent incidence followed by A. niger (39.75) Fussariumo xysporum(33.00) Aspergillus fumigatus (29.00) A. glaucus (27.25) Fussarium equisti (26.25) F. solani (23.60) Alternaria herbarum longissima (20.00) (21.47) Rhizopus (16.50%) Cladosposporium colletotrichum

Sanjay R. Maske and Umesh P. Mogle

fungi was higher in the untreated seeds than in the treated seeds. Acknowledgement: The authors express their gratitude to the Dr. S. M. Talekar, Head, Dept. of Botany K. S. K. college Beed, for the laboratory facilities and thankful to Principal Dr. S.D. Naikwade Principal K.S.K. College K.S.K. Beed and Nakhate Beed for A.B. Vice principal College necessary

oryzae (19.00) Macrophomina phaseolina, (17.60) lindemuthianum, Phytopthera sp. (16.25). Penicillium notatum, (15.75) F. semitectum, (15.25)Rhizoctonia solani, (14.00) bataticola. were F. Monliforme,(13.25%)R. F. sporotrichioides, within range of nigricans Fussarium (12-8%) (12.00.) and Sclerotium sp. F. graminearum, R. anthophilum, Cephalosporium acromonium, etc. intermediate Mucorhiemalis (7.00%), were found to least. In treated Cowpea seeds only 7 fungi were found. Aspergillusglaucus incidence niger F. shows maximum by Rhizocto (2.00%) notatum, percentage Aspergillus (4.42%)followed (3.00%) Monliforme, Penicillium

facilities and inspiration during the course of investigation. References: Agrawal PK, 1976. Identification of suitable seed storage places in India on the basis of temperature and relative humidity condition. Seed Res. 4(1):6-11. Bua B, Adioala E, Apio F, 1998. Screening cowpea germplasm for resistance to bacterial blight in Uganda.InternationalJournal of Pest Management,44: 185-189. Castillo, MD, HHL Gonzulez, EJ Martinez, AM Pacin and SL Resnik, 2004.Mycoflora and potential production Academic for mycotoxin production of freshly area. Publishers Mycopathologia. Dorderecht, Kluwer

nianigrian(2.75%) oxysporium

harvested black bean from the Argentinean main Netherlands,

Cladosporium herbarum, (2.00%) and Fussarium (1.58%) (1.54%) were found to least. In comparing the three isolation procedures, it was found that more isolates were obtained on the PDA method followed by RBA and CDA. This is to be expected since seeds which were placed on PDA had been subjected to surface sterilization and PDA is more sensitive in detecting even traces of infection (Neergaard, 1977). The result of the present investigation emphasize that the percentage incidence of Aspergillus flavus was significantly high on seeds of cowpea followed by Aspergillus niger and Fusarium oxysporium. It was proving that more fungi were isolated with agar plate method than blotter paper method because agar plate favours the growth of fungi due to potato dextrose agar content. It was also prove that the percentage of

158: 107- 112. Chevalier A, 1964.Cowpea in Africa .Revue de Botonique Appliquce Agriculture Tropicale,24:128 Champion R,1997. Identifier les champignons transmis par les semences. Paris. INRA. Fakir GA, Hossain I, Ahmed MU, Asad-udDoullah M, Alam M, 2002.Quality of farmers.Boro and T. Aman rice seeds collected before sowing from Bogra, Rajshahi and Rangpur districts of Bangladesh. A paper presented in the review and planning meeting of the Rice Seed Health Improvement (SHIP), PETRRA project held on 17-18 April, at BRRI, Gazipur, Bangladesh. Gitaitis RD, Nilakhe SS, 1982. Detection of Xanthomonas campestrispv. Vignicola in Southern pea seed. Plant Disease,66: 20-22. ISTA 1966.International rules for seed testing. Proc. Int. Seed Asso.32:565-589.

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Maude RB, 1996.Seedborne diseases and their control: Principles and practice. Cambridge.CAB International. Neergard, Paul 1973.Detection of seed borne pathogens by culture tests.Seed Sci. and Technol.,1: 217-254. Nutsuga SK, Vibeke L, Atokple IDK and Ayensu FK, 2004. Seed-borne Mycoflora of Major Food Crops in Ghana. Journal of Science and Technology, 24:20. Quenton K, AS Theresa, and and FO Walter, PR Johon,VDW 2003.Mycoflora Liana SS Gardon, Mycotoxins

Sanjay R. Maske and Umesh P. Mogle

Singh BB, Ehlers JD, Sharma B, Filho FR, 2002. Recent progress in Cowpea breeding, In: Fatokun CA Tarawali SA, Singh BB, Kormawa PM, Tamo M (eds) Challenges and enhancing sustainable opportunities for production. Cowpea

Proceeding of the world Cowpea conference III held at the International institute of Tropical Agriculture (IITA), Ibadan, Nigeria, 4-8 september 2000. IITA, Ibadan Nigeria, pp.22-40. ISBN 978131-190-8 Wright M, Delimini L, Luhanga J, Mushi C and Tsin, H, 1995. The quality of farmer saved seed in Ghana, Malawi and Tanzania. NRI Research Report.

fumonisin

associated with cowpea (Vigna unguiculata (L.) Walp). Seeds. J. Agric. Food Chem. 51:21882192.

Table 1: Percentof incidence of mycoflora isolated from Cowpea seeds SR. No 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21 22) 23) 24) 25) Name of fungus Aspergillus A. flavus, A,niger A. Fumigatus A. glaucus Alternaria longissima, Rhizoctonia solani R. bataticola. R.nigrian Fusarium equisti F. semitectum, F. Monliforme, F. sporotrichioides F.anthophilum F.graminearum, Fusariumoxysporum F. solani Phytopthera sp. Cladosporium herbarum, Mucorhiemalis, Rhizopus oryzae, Colletotrichum lindemuthianum Cephalosporium acromonium Macrophomina phaseolina sclerotium sp. Penicillium notatum, Blotter Method UT 37.50 33.00 22.25 18.00 14.58 7.00 6.50 8.00 17.00 7.16 4.75 7.00 5.00 7.42 25.00 15.00 9.00 14.00 5.50 11.00 7.00 3.25 12.00 9.45 10.00 TT- Treated seed TT 5.00 2.60 2.00 --1.00 --- 0.60- --1.37 --1.57 --3.79 2.00---1.72 --1.72 0.67 --Agar method UT 42.00 39.75 29.00 27.50 21.47 14.00 10.75 12.00 26.25 15.25 13.25 9.00 8.45 11.50 33.00 23.60 16.25 20.00 7.00 19.00 16.50 8.00 17.60 12.00 15.75 1.54 2.75 2.00 1.58 2.00 3.00 --4.42 TT

UT- Untreated seed

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