Seeds play a vital role for the healthy production of

Sci. & Cult. 77 (7 8) 308-312 (2011) Some Aspects of Stored Wheat Seeds with Respect to Seedborne Species of Alternaria, Aspergillus, Fusarium and Penicillium at Gorakhpur ABSTRACT : The present paper deals with the seedborne species of Alternaria, Aspergillus, Fusarium and Penicillium associated with deterioration of wheat seeds during storage. Forty seven (47) species belonging to four genera have been isolated by using standard methods. Highest number of species are those of Aspergillus and least of Alternaria. Most of the species confirm their pathogenic nature. Weight loss has been recorded to be highest for Aspergillus flavus and least by Fusarium chlamydosporum. Per cent loss of carbohydrate, protein and phosphorus has been found to be in between 0.60-4.80, 0.52-1.82 and 2-8 respectively by different species of these four genera. Key words : Seedborne mycoflora, Triticum aestivum, Nutritional value Seeds play a vital role for the healthy production of crop. They are known to carry pathogens which cause heavy yield losses. Besides this, the pathogens present either within the seed or over the surface decrease their market value largely due to losses in nutritional components and secretion of secondary metabolites which are harmful for the human beings and animals as well as because of bad odour and texture. Wheat is the most important cereal crop of India and is grown nearly in each part of the country. From the available literature it is revealed that much work has been done on the deterioration of wheat by fungi during storage before 1990 (Christensen 1, Christensen and Kaufmann 2, Panasenko 3, Agarwal et al. 4, Mehrotra and Basu 5, Sejiny et al. 6, Duthie et al. 7, Prasad and Pathak 8, Sauer 9 and Kunwar 10 ). During 1990 onwards the work which has been done on seed health is basically related with mycotoxins, seed abnormalities and control measures. Also onward 1990 no much work has been done on the stored wheat seeds with respect to seedborne species of four genera i.e. Alternaria, Aspergillus, Fusarium and Penicillium. Therefore, a comparative study of seedborne species of four genera Alternaria, Aspergillus, Fusarium and Penicillium on some aspects of stored wheat seeds has been carried out. Materials and Methods : Samples of seeds of Triticum aestivum Linn. (wheat) were collected from whole salers or retail shops of Gorakhpur from June 2005 to 2006 in pre-sterilized gunny bags and brought to the laboratory. The samples were stored at 24 + 2 0 C. The samples were studied for the symptoms and associated mycoflora twice in a month for two years. The detection of associated seed borne fungi was carried out by Standard Blotter Method and Agar Plate Method as recommended by the International Seed Testing Association (1966). In each method seeds were dipped with 0.1 per cent solution of mercuric chloride for ten minutes then washed several times by sterilized water. Data were recorded for the presence of species of Alternaria, Aspergillus, Fusarium and Penicillium per seed from June 2005 to May 2007. The species of these four genera found to be associated with wheat seeds were identified on the basis of their morphology and cultural characteristics as recommended by Gilman 11, Ellis 12, Subramanium 13, Domsch and Gams 14, Raper and Thom 15 and Mukerjee and Juneja 16. Pathogenicity test were conducted to confirm the pathogenic nature of isolated fungi on the wheat seeds. The seeds were surface sterilized with 0.1 per cent solution of mercuric chloride by dipping them for ten minutes and then washed several times with sterilized water. Sterilized seeds were dried in an oven at 60 0 C for one hour. Sterilized and dried seeds were kept in presterilized flask and were seperately inoculated with seven day old mycelial culture of species of Alternaria, Aspergillus, Fusarium and Penicillium. Species were aseptically inoculated by picking up a disc of 5 mm diameter with the help of sterilized cork borer. The flask containing infected seeds were thoroughly shaken mechanically for uniform distribution of inoculum, plugged with sterilized cotton plugs and then incubated at 24 + 2 0 C. The pathogenicity of the organism was considered established only when Koch s postulates were fully satisfied. For each species five replicats were maintained. Weight loss : To observe change in the weight of seeds due to infection caused by pathogenic fungi fresh and healthy seeds were surface sterilized and inoculated with respective species of fungi as described above. Control sets were maintained in which the pathogenic fungi was not inoculated. Weight loss was noted after incubation 308 SCIENCE AND CULTURE, JULY-AUGUST, 2011

for one week at 24 + 2 0 C. Loss in weight was determined by following formula : Where, W w Weight loss = x 100 W W = weight of the infested seed before incubation w = weight of the infested seed after incubation For the estimation of chemical constituents i.e. carbohydrate, protein and phosphorus of infected seeds of wheat by pathogenic speices of Alternaria, Aspergillus, Fusarium and Penicillium methods of Dreywood 17, Lowry et al 18. and Fiske and Subba Rao 19 respectively were followed. All the seeds were surface sterilized by dipping them in 0.1 per cent solution of mercuric chloride for ten minutes and then washed several times with sterilized water and dried in an oven at 60 0 C for one hour. Samples of 100 gm of sterilized and dried seeds were kept in pre-sterilized flasks. Then the seeds were seperately inoculated by seven-day old cultures of pathogenic species i.e. Alternaria, Aspergillus, Fusarium and Penicillium. The inoculated seeds were kept at 24 + 2 0 C for one month in presterilized gunny bags for the estimation of carbohydrate, protein and phosphorus. Observation and Result : A total of 47 species belonging to Alternaria, Aspergillus, Fusarium and Penicillium were found associated with the seeds of wheat when plated on moist blotter and agar medium. Highest number of species i.e. 25 were those of Aspergillus followed by 12 of Fusarium and 9 of Penicillium. Their was only one species of Alternaria. Results given in Table 1 revealed that per cent concentration of Aspergillus was highest (66.14) followed TABLE 1: Concentration of Allternaria, Aspergillus, Fusarium and Penicillium in storage of wheat seeds from June 2005 to May 2007 Fungal genera Total number Total number Per cent of colonies of colonies concentration of Genera of all genera of particular genera 1 2 3 4 Alternaria 40 1270 3.14 Aspergillus 840 1270 66.14 Fusarium 175 1270 13.77 Penicillium 215 1270 16.92 by Penicillium (16.92),Fusarium (13.77) and Alternaria (3.14). Out of 25 species of Aspergillus per cent concentration was highest for A. flavus (19.88) followed by A. niger (15.20). It was least in A. repens (0.11). Out of 12 species of Fusarium highest per cent concentration was that of F. solani (22.85) followed by F. semitectum (21.14) and it was least in F. poa (0.57). Highest per cent concentration was 26.51 followed by 19.06 for P. oxalicum and P. citrinum respectively. It was least i.e., 0.93 for P. capsulatum (Table 2). TABLE 2: Showing per cent concentration of species of fungi isolated from wheat seeds during storage Fungi Per cent concentration Alternaria A. alternata (Fr.) Keissler 1.00 Aspergillus A. amestelodamii (Mang.) Thom and Church 7.14 A. aureus Thom and Raper 0.83 A. awamori Nakazawa 2.38 A. candidus Link ex Fries 1.78 A. chevalieri (Mangin) Thom and Church 3.69 A. clavatus Desmazieres 2.85 A. flavipes (B & S) Thom and Church 2.26 A. flavus Link 19.88 A. foetidus Thom and Raper 0.95 A. fumigatus Fresenius 10.11 A. giganteus Wehmer 0.71 A. nidulans (Eidam) Wint 3.33 A. niger Van Tieghem 15.20 A. ochraceus Wilhelm 5.71 A. oryzae (Ahlb.) Cohn 5.95 A. parasiticus Speare 0.59 A. repens (Corda) de Bary 0.11 A. ruber (K, S and B) Thom and Church 0.95 A. tamarii Kita 1.66 A. sydowi (B and S) Thom and Church 0.47 A. terreus Thom 7.38 A. triticus Sp. nov. 0.59 A. ustus (Bain) Thom and Church 3.21 A. variecolor Thom and Raper 0.59 A. versicolor(vuill.) Tirabaschi 1.54 VOL. 77, NOS. 7 8 309

Fusarium F. chlamydosporum Wr. and Rg. 6.28 F. culmorum (W.G. Smitch) Sacc. 6.28 F. equiseti (Corda) Sacc. 6.28 F. graminearum Schwabe 3.42 F. moniliforme Sheldon 6.28 F. nivale (Fries) Cesati 5.71 F. oxysporum SchlechtVar. redolens 13.71 F. poa (Peck) Wollenw 0.57 F. roseum Wr. 2.85 F. semitectum Berkeley and Ravenel 21.14 F. solani (Mart.) Sacc. 22.85 F. sporotrichoides Sherb 4.57 Pencillium P. capsulatum Raper and Fennel 0.93 P. citrinum Thom 19.06 P. frequentans Westling 9.30 P. funiculosum Thom 3.25 P. notatum Westling 13.48 P. oxalicum Curric and Thom 26.51 P. urticae Bainier 6.97 P. chrysogenum Thom 17.20 P. expansum Link ex Fries 11.62 Results of pathogenicity test and weight loss has been given in Table 3. About 19 species of Aspergillus were pathogenic because they confirmed Koch s postulates remaining 6 species i.e., A. aureus, A. flavipes, A. giganteus, A. repens, A. ruber and A. variecolor did not confirm Koch s Postulates. Average weight loss by all the pathogenic fungi was 2.80 per cent. Individually loss in weight was highest in A. flavus (6.20 per cent) and least in A. triticus (0.40 per cent) treatments. All the species of Fusarium except F. roseum were pathogenic and average loss in weight by all the species of Fusarium was 2.78 per cent. Individually loss in weight was highest in Fusarium nivale (4 per cent) and least in F. chlamydosporum (0.40 per cent) treatments. Their were only 5 species of Penicillium viz., P. citrinum, P. notatum, P. oxalicum, P. chrysogenum and P. expansum which were found to be pathogenic and average loss in weight was slightly higher than those of Aspergillus and Fusarium. Individually loss in weight was highest in P. citrinum and least in P. expansum, treatments. The only species of Alternaria i.e. A. alternata was pathogenic and loss in weight was 3.20 per cent. Out of 36 pathogenic species of Alternaria, Aspergillus, Fusarium and Penicillium loss in weight was highest in A. flavus(6.20 per cent) and least in A. triticus (0.40 per cent) and F. sporotrichoides (0.40 per cent), treatments. Results given in Table 3 revealed the effect of pathogenic species of Alternaria, Aspergillus, Fusarium and Penicillium on carbohydrate, protein and phosphorus contents of wheat seeds during storage. Average loss in per cent of carbohydrate and phosphorus was highest in the species of Aspergillus (1.51 and 1.36 respectively) infected seeds. Followed by Penicillium (1.56 and 1.60 respectively) infected seeds. However, average loss in protein was maximum in Alternaria (1.30 per cent) followed by Penicillium (1.07 per cent) infected seeds. Alternaria did not affect phosphorus contents of wheat. Out of 36 pathogenic species loss in carbohydrate content was highest in A. terreus (4.80 per cent) and protein in P. citrinum (2.12 per cent) infected seeds. Phospohrus content of wheat seeds was not affected by most of the pathogenic species. However it was maximally affected in A. flavus and P. citrinum (8 per cent) infected seeds. Individually loss of carbohydrate was maximum in A. terreus (4.80 per cent), F. oxysporum (1.80 per cent) and P. chrysogenum (3.60 per cent) infected seeds. Protein content was maximally affected in A. flavus, A. terreus, F. oxysporum i.e. 1.82 per cent and P. citrinum 2.12 per cent. Individually the phosphorus content was maximally decreased in A. flavus and P. citrinum (8 per cent) infected seeds. Discussion : The seeds after harvest are stored in bulk usually for a long period (more than two years) with high moisture content. This certainly increases all the hazards that accompany storage including those caused by fungi. Deterioration in stored grain is manifested by decrease in germination, processing quality, sick or germ damaged grain, heating and mustiness. Therefore, deterioration affect germination, nutritional contents, trade and grading of seeds which results economic loss to the growers and health hazards to consumers. The occurrence of different genera and species in varying concentrations may be attributed to their moisture requirement, temperature, humidity and time of the storage period. Among the Aspergillus, A. flavus, A. niger, A. terreus and A. amestelodamii have been frequently observed in wheat grains. Similar findings have been reported by 310 SCIENCE AND CULTURE, JULY-AUGUST, 2011

TABLE 3 : Showing pathogenic species of Alternaria, Aspergillus, Fusarium and Penicillium and their effect on weight, carbohydrate, protein and phosphorus content of wheat seeds during storage Parameters Nutritional constituents Fungi Pathogenicity Weight loss Carbohydrate Protein Phosphorus (%) (%) (%) (%) 1 2 3 4 5 6 Alternaria A. alternata + 3.20 1.80 1.30 0 Aspergillus A. amestelodamii + 3.00 0 0 0 A. aureus - - - - - A. awamori + 2.80 0 0 0 A. candidus + 2.00 0 0 0 A. chevalieri + 1.40 0 0 0 A. clavatus + 2.00 0 0 0 A. flavipes - - - - - A. flavus + 6.20 3.60 1.82 8.00 A. foetidus + 2.60 0 0 0 A. fumigatus + 6.00 3.00 0.78 2.00 A. giganteus - - - - - A. nidulans + 3.20 3.60 1.56 4.00 A. niger + 3.60 4.20 0.78 6.00 A. ochlraceus + 3.40 3.60 0.52 0 A. oryzae + 2.80 2.40 0.52 0 A. parasiticus + 1.40 1.80 0.52 0 A. repens - - - - - A. ruber - - - - - A. tamarii + 3.40 1.20 0 0 A. sydowi + 0.80 0 0 0 A. terreus + 5.00 4.80 1.82 6.00 A. triticus + 0.40 0 0 0 A. ustus + 1.00 0.60 0 0 A. variecolor - - - - - A. versicolor + 2.00 0 0 0 Average Loss 2.78 1.51 0.43 1.36 Fusarium F. chlamydosporum + 0.40 0 0 0 F. culmorum + 3.80 0 0 0 F. equiseti + 1.80 0 0 0 F. graminearum + 2.20 0 0 0 F. moniliforme + 3.20 1.20 0 0 Flannigan 20, Mehrotra and Basu 5, Abdel Hafez 21, and Prasad and Pathak 8. Out of 9 species of Penicillium, four viz., P. oxalicum, P. citrinum, P. chrysogenum and P. notatum have been frequently observed in wheat grains in this study. This is in confirmation with other workers Mehrotra and Basu 5, and Abdel Hafez 21. A total of 13 species of Fusarium have been found to be associated with the seeds of wheat. F. solani, F. semitectum and F. oxysporum are most prevalent. Similar results have been reported by other workers Abdel Hafez 21, Mehrotra and Basu 5, Prasad and Pathak 8, and Gonzalez et al 22. However, in the present study Aspergillus aureus, A. foetidus, A. giganteus, F. roseum and P. expansum are the new records for the wheat grains. The results of pathogenicity test reveal that 35 species were pathogenic of which 19, 11, 4 and 1 are of Aspergillus, Fusarium, Penicillium and Alternaria respectively. From the literature available it has been noticed that for the wheat grains this type of work is probably being reported for the first time. From the list of species it is clear that the presence in grains of these fungi is significant from the point of view of decrease in weight loss, their texture (appearance) and especially in food values. Genus Aspergillus was most conducive because it decreased carbohydrate, protein and phosphorus to the maximum extent followed by Penicillium, Fusarium and Alternaria. VOL. 77, NOS. 7 8 311

F. nivale + 4.00 0 0 0 F. oxysporum + 3.80 1.80 1.82 2.00 F. poa + 3.20 0 0 0 F. roseum - - - - - F. semitectum + 4.20 0.60 0.52 0 F. solani + 3.60 0.60 0 0 F. sporotrichoides + 0.40 0 0 0 Average Loss 2.78 0.38 0.21 0.18 Penicillium P. capsulatum - - - - - P. citrinum + 4.50 2.40 2.12 8.00 P. decumbens - - - - - P. frequentans - - - - - P. notatum + 1.60 0 0 0 P. oxalicum + 3.00 1.20 1.20 0 P. urticae - - - - - P. chrysogenum + 4.00 3.60 1.04 0 P. expansum + 1.00 0.60 0 0 Average Loss 2.82 1.56 1.07 1.60 + : Present ; -: Absent Received : 19 March, 2009 Revised 25 February, 2011 1 C.M. Christensen. Botanical Review. 23 : 108-131, (1957). 2 C.M. Christensen and H.H. Kaufman. Annual review of Phytopathology, 3 : 69-83, (1965). 3 V.T. Panasenko. Botanical Review. 33 : 189-215, (1967). 4 V.K. Agarwal, S.B. Mathur and P. Neergaard. Indian Phytopathology. 25 : 91-100, (1972) 5 B.S. Mehrotra and Monica Basu. Indian J. Mycol. Pl. Pathol. 6 : 43-50, (1976). 6 M.J. Sejiny, K.A. Tawfik and M.K. EL-Shaieb. Rev. Pl. Pathol. 64 : 2249, (1984). 7 J.A. Duthie, R. Hall, A.V. Asselin, Canadian Journal of Plant Pathology. 8 (3) : 282-288, (1986). 8 T. Prasad and S.S. Pathak. Indian Phytopathology. 40 (1) : 39-46, (1987). 9 D.B. Sauer. International Journal As it is well known that wheat grains are important for their carbohydrate content which is found to be most affected in the present study by several species of all the four genera. Similar results have been given by Dube et al 23. Effect of these four genera on phosphorus content of wheat grain has not been reported earlier and therefore it is also a new finding. The fungi which have been found to be associated beside lowering downs the nutritional value also create health hazards because of their capacity to produce toxins by some of these species. Acknowledgement Authors are thankful to UGC, New Delhi for providing financial support and Head of the Department of Botany, D.D.U. Gorakhpur University for providing necessary facilities. Department of Botany, D.D.U. Gorakhpur University Gorakhpur, Bihar 273009,U.P., India *email : vardha1979@rediffmail.com ASHA SWAMI, REKHA SRIVASTAVA* AND N. MISRA of Food Microbiology. 7(3) : 267-275, (1988). 10 I.K. Kunwar, Plant Science, 99 : 437-443, (1989). 11 J.C. Gilman. A Manual of Soil Fungi. (1967) (Oxford and IBH Publishing Co., Calcutta, Bombay, New Delhi). 12 M.B. Ellis, Dematiaceous Hyphomycetes, Principal Mycologist (1971). (Common Wealth Mycological Institute). Kew. Survey, England. 608. 13 C.V. Subramanian. Hypomycetes : An account of Indian species cercosporae. 930, (1971). (Indian Council of Agricultural Research, New Delhi). 14 K.H. Domsch and W. Gams. Fungi in Agricultural Soils. (1972) (Printed in Great Britain by T. and Constable Ltd. Edinburg). 15 K.B. Raper and C. Thom. A Manual of Penicillia (1949). (The Williams and Wilikans Co. Baltimore). 16 K.G. Mukerjee and R.C. Juneja, Fungi of India (1962-72), Emkay Publications. Delhi. 17 R. Dreywood. 1946. Methods in Microbiology Vol. 58, Norris, J.R. and Ribbons, D.W. (Ed.) Academic Press, London. 1971 : 265-272, (1946). 18 O.H. Lowry, N.J. Rosenbrongh, A.L. Farr and R.J. Randull. J. Biol.Chem. 193 : 265-275, (1951). 19 C.H. Fiske and Y. Subba Rao. J. Biol. Chem. 66 : 375, (1925). 20 B. Flannigan. Tr. Br. Mycol. Soc. 71(1) : 37-42, (1978). 21 S.I.I. Abdel-Hafez. Mycopathalogia. 85 : 53-57, (1984). 22 H.H.L. Gonzalez, G.A. Molto, A. Pacin, S.L. Resnik, M.J. Zelaya, M. Masana and E.J. Martinez. Mycopathologia. 165(2) : 105-114, (2008). 23 S. Dube, H.S. Shukla, S.C. Tripathi. Indian Phytopathology. 41(4) : 633-635, (1988). 312 SCIENCE AND CULTURE, JULY-AUGUST, 2011