Pt SorRes 30(1) 65-71 2014 satus of Seed Mycoflora of Lucerne (Medicago sativa L.) SC Jain and Rekha Kumawat siagurwal087@gmail.com wither were also seen Seed M sample *E’ lant Pathology, S.K.N. College of Agriculture, Jobner-303329 (Jaipury Ravastine mples were collected from different sources, showing deformed (shrivelled and gait ned), discoloured (brown) and damaged (insects and mechanical) seeds. Impurities of one all the six samples. Six different kinds of fungal spores were detected by shing Test on the seed testa of all the seed samples tested. The spore load was maximum in while it was minimum in sample ‘F’. A total of eight seed borne fungi were obtained in or Motter and Agar Plate Methods and these were Allernaria alternata, Aspergillus flavus, Aspergillus ricer, Cladosporium cladosparioides, Drechslera australiensis, Fusarium moniliforme, Fusarium sporwn and Rhizopus 5 1 Sollowed by “AY, “BY, 4 Scovmrds: Lucerne, Aspergillus, Ger YPRODUCTION vig or altalla (Medicago sativa L.) vernacularly | Rika” is one of the important fodder crop of ic and genus Medicago grown on ros scule in USA, Argentina, Australia, India, New ul, Frans, ttaly and USSR Rahusflian, iis grown as fodder erop almost in all ‘cept partially in one or two district like ual chittor where it also grown for seed 11s (Anonymous, 2009-10). One of the ypovtaut factors which limit the production of the crop 1» foulder or a5 seeds in Rajasthan or in other seed reas is the use of contaminated/ contaminated seeds purchased by the farmers from «al seed selling shopkeepers or rarely seeds saved \ hom. which culminating into heavy losses atall the 20> ol the crop growth, specially at germination by nce mortality (seed rots). post vice moriality (wilt, damping off, seedling rot citing various fungal diseases at »p whose pathogens also reported te through such seeds. ! PERIALS AND METHODS lection of seed samples inpies nf lucerne seeds were collected. Out of Pout thur samples were collected from local seed Highest count of mycoflora was abserved hy Agar Plate Method in sample “D’ and ‘F" in comparison to Blotter Method. ration, Seed mycoflora, Agar Plate Method. selling shops situated in nearby areas of vegetable grain mandies of Bagru, Phulera, Renwal. Chomu towns which belongs tb nearby areas Jobner vicinity (District-Jaipur, Rajasthan) and the fifth samples was collected from Bilada a town belongs to Jodhpur district ‘where some farmers cultivated the lucerne crop both as fodder and seed production purpose (Table 1). At each mandi /town level 5-15 samples were collected from 5-15 shops/farmers depending upon the number of shops/farmers and availability of seeds on shops or in farmers houses situated in respective mandi/town respectively. Ateach mandi/town level all the collected samples mixed together to represent a composite sample of that particular mandi / town. Sixth sample was obtained from office of Assistant Agriculture Officer, Department of Agriculture Govt. of Rajasthan, Jobner (Jaipur, Rajasthan) who distributes the improved seeds of lucerne (developed/collected by them from registered seed producing agencies where seed production done by usingimproved cultivation /storave practices) to the farmers belong to different viliazes of unit area (lobner / town and adjoining viit grow green fodder for their animals. Sample collected 2-3 weeks before sowing from the she town, kept in fresh cotton cloth bags brought laboratory and stored in refrigerator fort Sampling was done by the methods suggested 1976 and Vishunawat. 2007Ccece C Pankaj Agar'.al, SC Jain and Rekha Kumawat lable t: Place of samples collection in Jobner vicinity Distriet-Jaipury and Bilada (District Jodhpur) Rajasthan, ‘ner code number Place of collection Code No. 2 | chome Phulera Renwal alolalml> Bilada® 6 | Depn or Agreatture, L Govt, of Rajasthan F * Local seed producing village belong to Jadhpur district Examii ation of Dry Seeds The method suggested by Agarwal and Sinclair (1987) vith slight modification was followed. Twenty grams seeids from each sample were taken at random and divided into 4 fractions (5.0 gm). Each fraction was spreaded on bottom of a Petridish and examined with the help of a hand-lens or if required under stereobinocular microscope. The inspected material was categorized as follows A. Discoloured seeds (i) Brown % Deformed sceds () Shrivelled 1) Gall formed, Damaged seeds (i By insects D. Impurities (i) Other crop seeds (ii) Stone or sand/ Ash partials ii) Plant debries E. Apparently healthy seeds Seeds and impurities of each category were pooled separately and weighted on modern electric balance and per cent content by weight was calculated Incidence of mycoflora, in particular category was also recorded by using standard incubation methods. Seed Washing Test The Seed Washing Test suggested by Agarwal and Sinclair (1987) was followed with slight modification, oh Tine Toaraal of Plan Sevonce Res From each sample 600 seeds (approximately 2 am by weight) were selected at random and divided into two ‘groups of 300 seeds and were immersed in 20 ml double 2alass distilled and sterlized water in 100ml conical flask and shaken for 15 minutes on a mechanical shaker. The seeds were discarded and the liquid was ‘centrifuged at 2500 rpm for 15 minutes. The supematant was discarded and sediment was resuspended in 2 ml of lactophenol. A drop of suspension was placed at the centre of the haemacyrometer and spores of the funy were counted in 10 large squares, chosen at randam under stereobinocular microscope. Spore load per seed was calculated by using following formula :- Nxv Spore load per seed = <—— Where, N = Total number of spores counted/ number of squares X = Volume of mounting between thecover giass and above the square covered (area of large square x depth of chamber = 10* cm?) Volume of the mounting fluid added to the sediment’and Number of seeds washed ve ne Incubation Method Mycoflora associated with seed samples cotfevied post storage (just before next sowing) stage were isolated using two incubation methods 1.¢. Blotter Method and Agar Plate Method (ISTA, 1976 and Agarwal and Sinclair, 1987) and incubated at 22 + 1 °C with 12 hours of light altering with 12 hrs of darkness. ‘The seeds were examined alternatively on 3° to 7 day of incubation. RESULTS AND DISCUSSION Examination of dry seed samples revealed the presence of deformed (shrivelled and gall formed seeds), discoloured (brown) and damaged (mechanically and insect) seeds. In addition to inert material (impurities and apparently healthy seeds (Table 2). It is likely that different types of seed mycoflora during storage might have caused such deformation / discolouration in seedsStatus of Seu malities and impurities in different lucerne seed samples J Myroflora of Lucerne (Medicago sativa 1.) vie oe Tern ony 3 rin ine mop fe ed 5 zi home See eerie trrso tarot Eteos ] Detormed ove Beene esa [Saves ue | 3s | te | oe | te | is ae nies Beet eeep a |e oc) mn alle cso 1h ei ae | a8 | ae eds | ee 17 impurities ' [PRPS eter srp sw | 2m | as | 20 | am | ive elon |S | | te | Ley os ‘sows | ora ro | 150 | 200 | 10 | 2m | ow | eamasaang asia testers imLomsia noe a Talons sees enn Se Te Bltenel F=Biatt P= Dept of gules Con 1 Bact vocnce of such deformation and discolouration — sp,, Fusarium sp. and Rhizopus sp. Total “nga! siongw ith impurities have also been reported during Joad was observed to be less in sample F . Table vopeotion » examination of dry seeds of gram, black seam, cowpea, mothbean and sesame by Ahmed and Reidy, 1993; Khatik, 1998; Goyal, 1996; Cheema, 1997; ali 2001 and Singh and Singh, 1983, respectively. Sirangely, six fungal species could be detected by the ‘Sowd Washing Test and these fungi were Alternaria 1». Aspergillus sp. Cladosporium sp. Drechslera ‘comparison to sample collected from different regio" ‘A possible reason for the occurrence of fungi on see coat might be the contamination during harvest: follo -.." by the prevailing favourable weather condi during si.sage for the survival and multiplication of furw Non-adoption of improved cultural practices by ‘>: farmers might be another reason also. Presence oi fuble 3: Fungal spore load in different lucerne seeds samples detected by Seed Washing Test Seat Tovar Fone 1 Teal oe ie Ime oi Alt. Asp. cid Fus. Rh. a iso _ 228 1.50, ans $.20 050 h 125 Lis 050 2.00 4.00 1.00, ve! ms Lo 1.00, o4s 1.50 2.70 1.20 : a5 425 1.00 0.75 228 04s | 7 3.00 20 3.00 6.00 075, re "he as 03S 1.00 150) 0.25 ‘P04 (300 » 2) seeds reennia sp. Asp = Aspergillus sp.. Ck wi= Chom, C=Phulera = Renwal ‘adosporium sp, Dre = Drechslera sp. Fas, = Fusarium gh E=Diladat —F = Deptt. of Agriculture, Gos The Journal of Plani Sve“8 Pankaj Agarwal, 5 C Jain and Rekha Kumawat Table 4: Per cent incidence of mycoflora of lucerne seeds isolated by Blotter Method | Mycomtors T Per cent incidence [sample TI ‘ x ® ¢ D z Fo] uw j_Mernaria alternata 4.50 225 “350 3.00 6350 | 100 1 peril fas 700 250 300 soo | i020 | 200 iipergi niger 350 450 7.00 x50 | ers | aa Cindospartan cadosporiores | 318 200 30 sot lasso a oop le Drechalera australiensis 7.00) 550 325 3.00 1050 [208 | sz Tasoriim moniliforme 335 750 as | a0 | aso | a0 | om Fusarium epeporam 7200 | 1050 900 | 750 | 1850 | a0 Ricophas se 300 325 250 | 200 | 330 | 100 Tol 4 mpeaora sao | 3700 | soo | 2030 | avs] aay * Seed tested 400 seeds / samples ~ ~ A= Bagru B = Chom ula -D~Renwal_—E=Dilads* = Deptt. of Ait, Govt. of Raa different fungal spore on seed coat of clusterbean, fenugreek. cowpea, mothbean and soybean, have been also reported by Jaiman, 2003; Singh, 2001; Cheema, 1997; Ramnath ef ai., 1970 and Gujar, 2011. This ‘method provides quick detection of fungi which adhere to the seed surface in the form of easily indentifiable spores. 1m the present investigation, Blotter Method, Agar Plate Method and Seedling Symptom Test were employed for the isolation of seed mycoflora of lucerne. “otal eight fungi ie, Alternaria alternata, Aspergillus nig? Aspergillus flavus, Cladosporium cladosporivite Drechslera australiensis, Fusarium moniliform. Fusarium oxysporum and Rhizopus sp. were isolated from the samples of lucerne by Blotter Method and ‘Agar Plate Method (Table 4 & 5 and Fig. 1) In general variation was observed in Blotter Metliod and Agar Plate Method. Higher per cent counts of fun table S: Per cent incidence of mycoffora of lucerne seeds isolated by Agar Plate Method Mycotora _ Per cent incidence / sample a a ¢ D E ierariaaliernata 650 325 a5 a0 | 850 [sports Raves 50 550 450 00 | wso[ 250) 9% [cece 375 5.00 430 350 | soo | am) sit a (Cladosporium cladosporioides | 450 350 200 230 | 430 [ 130) sun] Drecstera austratensis 330 600 350 as | nso[ sof oa | Fusarium moniliforme 7050 | _#25 700 550 | woo [ 250) | Fsartum oxsporam 335 1150 950 sas [oof asof vm [Rhizoma sp 00 3.50 3.00 250 | 4.00 2.00 1 Ton % myeatora aso [4630 ars | 3500 | 7400 | 2150 * Seed tested = 200 seeds / samples A» Bagra B= Chomu, C~Phulera D = Renwal E = Bilada F = Deptt, of Agriculture. Govt. of Rajasthan i-Iournal of Plant Science ResearchStas of Seed Mvcoflora of Lucerne /Medivage sativa L Altemaria altemata Aspergillus niger Aspergillus flavus B A B Cladosporium cladosporides ‘vcutial yrowth on seed B = Conidia witgh conidiophore Lncome (Medicago satva L.) seed showing growth of seed byre mycator The Town= 70 Pankay Agurwal, S.C Juin and Rekha Kumawat Drechslera australiensis = Fusarium moniliforme Fusarium oxysporum A Rhizopus sp. oe A = Mycelial growth on seed 18 = Conidia with und ithStatus of Seed Mycoflora of Lucerne (Medicago sativa Ly 7) sece observed in Agar’ Plate Method (46.70%) as ‘compared to Blotter Method (38.76%). This variation zhi be due to the reasons that some of the weak and ‘ow ing flngi could not grow in Blotter Method #» comparisons to fast growing saprophytic fungi, pre sterilization of seeds and different substratum the method employed (de-Tempe. 1961 *canve and Sadd, 1962 and Jain, 1990), Neergaard s8 Saude (1962) also observed that Blotter Method Plate Method are equally valuable and supplementary to each other. Deterioration of seeds Sarious raps in storage has also réported by Christensen (1973). REFERENCES \carval V K and Sinelait B 1987 Prineiplesof Seed Pathology Vol. 1 (l= 157) and Vol.« I (PI-153) CRC Press. Ine Foca Raten Florida USA, Anined KM and Reddy CR 1993 A pictorial guide to the ‘dentifieation of seed borne fungi of sorghum, peal, ‘inger mille. chickpea, pigeonpes and groundnut. Inf Bulletin No. 34 ICRISAT, Patacheru, A.P. - 02324, pp. 192 \somymous 2009-10 Agriculture Statisties of Rajasthan rectorate of Economics and Statistics, Rajasthan, Yoana Bhawan. 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