NSave Nature to Survive

ISSN: 0974-0376 NSave Nature to Survive : Special issue, Vol. VI: 181-185: 2014 AN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES www.theecoscan.in DETERMINATION OF SEED GERMINATION PERCENT AND EFFECT OF TRICHODERMA HARZIANUM RAFAI ON FRESH AND STORED SEED BY DIFFERENT METHODS Shakshi Singh et al., KEYWORDS Seed pathogens Trichoderma harzianum Percent germination Blotter method Multi pot method Paper towel method Proceedings of National Conference on Harmony with Nature in Context of Environmental Issues and Challenges of the 21 st Century (HORMONY - 2014) November 28-30, 2014, Udaipur, organized by Department of Environmental Sciences, Faculty of Earth Sciences M. L. Shukhadia University, Udaipur - 313 001 (Rajasthan) in association with National Environmentalists Association, India www.neaindia.org 181

NSave Nature to Survive QUARTERLY SHAKSHI SINGH*, ASHA SINHA, SHRVAN KUMAR AND SANWAR MAL YADAV Department of Mycology and Plant Pathology, IAS, Banaras Hindu University, Varanasi - 221 005 (U.P.), INDIA e-mail: singh.bhu683@gmail.com ABSTRACT The seed pathogen may adhere to the seed surface either in form of spores or mycelium or may be deep seated internally. Present study was undertaken to screen for experiment determination of the germination percentage of wheat, paddy, pigeonpea, chickpea were observed in fresh and stored seed by different method such as blotter method, Plastic pot method, Multi pot method and Rolled paper towel method. After 10 days observation were made in which the result is found that all fresh seed give the better germination than stored seed. Germination percent of fresh seed of wheat (93%), paddy (90%), pigeonpea (88%), chickpea (88%) by Blotter method. And effect of seed treatment by Trichoderma spore on germination were observed in stored and fresh seeds of wheat, paddy, pigeon pea and chick pea by plastic pot method and by multiport method. The Trichoderma treated seed show higher germination percentage (>85%) than control. The growth and development of crop plants were better in treated seed than untreated seed. *Corresponding author INTRODUCTION Fungal pathogens constitute the major portion of the pathogenic agents in crops. These eukaryotic organisms have been exerting pressure over crop cultivation since time immemorial. Although, they affect the crop by infecting it in any of its growth stages yet the picture is worse, when they get associated with the seed. Seed infection of crops by mycoflora has its own importance. Firstly, the seed is the only mode of propagation for many crops. When fungal pathogens establish themselves with the seed, they have the whole plant in their grip. Infected seeds may also serve as source of inoculums for the areas or fields, where the pathogen was previously unknown. Seed mycoflora are chiefly responsible for the deterioration of seed in storage and reduce the viability of seeds. Seed germination is also influenced by seed borne fungi (Suryanarayana and Bhombe, 1961 and Rai et al., 1997). Srivastava and Jaiswal (2000) concluded that Alternaria brassicae may be the cause of reduction in germination of the cauliflower. Ammara (2000) studied the seed borne pathogen and their role in poor germination. Tekrony (2003) reviewed prescription is an essential component seed vigour testing. Merinda et al. (2006) reported the predominant fungi were Alternaria, Fusarium, Aspergillus, effect on germination. Das and Das (2005) studied the efficiency of different antagonists in reducing the Sclerotinia rot disease of French bean in amended soil. Seed treated with the spore suspensions of Trichoderma harzianum, Gliocladium virens and Aspergillus flavus. For controlling the disease the most effective treatment was seed treatment with T. harzianum + soil treatment with FYM. The treatment not only increased plant growth and yield but also resulted in low soil ph and high organic matter content. Nayaka et al. (2008) studied on seed biopriming with Trichoderma harzianum for control of toxigenic Fusarium verticillioides and fumonisin in maize, they treat the seed with aconidial suspension of T. harzianum at the rate of 1 10 8 spore/ml. Harman (2011) studied on effect of Trichoderma harzianum against biotic, abiotic and physiological stresses in germinating seeds and seedlings when applied as seed treatment they find that seed treatment reduced the accumulation of lipid peroxidise in seedling under osmotic stress or in aged seed, they observed that application of T. harzianum increases the seedling vigour and ameliorates stress by inducing physiological protection in plants against oxidative damage. Mahamune and Kakde (2011), studied the incidence of seed borne mycoflora on French bean and its antagonistic activity against Trichoderma harzianum. Sultana et al. (2012) studied the integrated approach of mitigating root rot of chillie. In this study effective isolates of Trichoderma harzianum, Chaetomium globosum, fungicides and plant extract were screened. Trichoderma seed treatment + Chaetomium + garlic + vitavax gave best result. Chavan (2014), studied the importance of seed priming on field performance and find that priming generally improve most parameters of soybean variety like plant height, branches, number of pods etc. Ganesh Patel (2014) also studied on frequency and assessment of fungi at different grain formation stages and discolored seeds of sorghum. Seed biopriming improve the germination and growth of the wheat, paddy, pigeon pea and chick pea because Trichoderma harzianum produce secondary metabolites and colonize the seed surface. T. 182

FRESH AND STORED SEED BY DIFFERENT METHODS harzianum produce antagonistic and competitive effect with other seed borne pathogens. MATERIALS AND METHODS Determination of seed germination (%) of fresh and stored seed In this method plastic pots were used to test for determination of percent germination of rice, wheat, gram, pigeon pea seeds in glass house. The plastic pot filled with the pre-sterilized (95 0 C for 30 min. in autoclave) uniform soil mixture containing 4 parts peat with essential amount of fertilizer and seeds sown in them. The germination was counted 8 to 10 days after sowing. Multi pot tray method (Khare, 1996) Multi-pot trays were used to test the germination of rice, wheat, gram and pigeon pea seeds in glasshouse. The small pots were filled with the pre-sterilization (95 C for 30 min. in autoclave) uniform soil mixture containing 4 parts peat with essential amount of fertilizer and seeds were sown in them. The germination was counted 8 to 10 days after sowing. Blotter method (de Tempe, 1953) Three pieces of blotting paper were placed in fold in each Petri-dish 9 cm diameter. Sterilized water is used for moisten of the blotter paper. Seeds place in each Petri-dish and incubated at 25 ± 1 C. Three replicates were prepared. The percent germination of fresh and stored seeds were calculated after one week. Rolled paper towel method (Agrawal, 1994) The rolled paper towel method was used for the germination of the rice, wheat, pigeon pea, gram. Good quality towel paper (46 29 cm) free from toxic substance was taken for experiment. Sterilized water is used for moisten of the towel and one towel was placed over the seed. The towel paper was rolled in such a manner that seeds remain in place. The towel is then wrapped in a sheet of polyethylene to check surface the rolled paper was then kept in upright position and incubated at 25 ± 1 C. Determination of seed germination (%) of fresh and stored seed treated with biocontrol agent Trichoderma harzianum Rafai The seeds of rice, wheat, pigeon pea, gram was treated with Trichoderma harzianum spores suspension. For the preparation of spore suspension, 10 mm mycelial disc of 7 days old culture of Trichoderma harzianum was placed on the surface of 100 ml potato dextrose broth and incubated 25 ± 1 C for 7 days. After the incubation period, 30 ml double distilled water was added to conical flask containing Trichoderma harzianum spore and shaken on a rotary shaker at 100 rpm for 30 minutes. The concentration of Trichoderma harzianum spore in double distilled water was counted using haemocytometer and was adjusted to 1 10 6 spores per ml. The seed of French bean was soaked in water for 4 hours them coated with 7% acacia gum for obtaining the sticky seed surface. Seeds were dried in shade for 1 hour. The seeds soaked in spore suspension for 30 minutes while the control seed were immersed in sterile distilled water. In this method plastic pots were used to test for determination of percent germination of wheat, rice, pigeon pea, gram seeds in glass house. The plastic pot filled with the pre-sterilized (95 0 C for 30 min. in autoclave) uniform soil mixture containing 4 parts peat with essential amount of fertilizer and treated seeds sown in them. The germination was counted 8 to 10 days after sowing. Multipot tray method (Khare, 1996) Multi-pot trays were used to test the germination of rice, wheat, gram and pigeon pea seeds in glasshouse. The small pots were filled with the pre-sterilization (95 C for 30 min. in autoclave) uniform soil mixture containing 4 parts peat with essential amount of fertilizer and treated seeds were sown in them. The germination was counted 8 to 10 days after sowing. RESULTS AND DISCUSSION Determination of seeds germination of fresh and stored seeds In this experiment determination of the germination of fresh and stored seeds of rice, wheat, gram, pigeon pea were done by plastic pot method, multi-pot tray method, Blotter method, rolled paper towel method. The data of seed germination presented in Table 1. In plastic pots, the percent germination of fresh seeds of rice, wheat, pigeon pea, gram were observed more than stored seeds of rice, wheat, pigeon pea, gram. Multi pot tray method (Khare, 1996) In multi-pot trays, fresh seeds of rice, wheat, pigeon pea, gram shown more germination than stored seeds of rice, wheat, pigeon pea, gram (Table 1). Blotter method (de Tempe, 1953) The data of seed germination presented in Table 1 and Plate 1. In Blotter method, the percent germination of fresh seeds of rice, wheat, pigeon pea, gram were observed more than stored seeds of rice, wheat, pigeon pea, gram (Table 1). Rolled paper towel method (Agrawal, 1994) The data of seed germination presented in Table 1 & Plate 2. In rolled paper towel method, the percentage germination of fresh seeds of rice, wheat, pigeon pea, gram were observed more than stored seeds of rice, wheat, pigeon pea, gram (Table 1). Seed mycoflora are chiefly responsible for the deterioration of seed in storage and reduce the viability of seeds. Seed germination is also influenced by seed borne fungi (Suryanarayana and Bhombe, 1961; Rai et al. 1997; Badoni et al., 2009; Singh et al., 2011 and Ganesh and Patel, 2014). Determination of seed germination (%) of fresh and stored seeds treated with biocontrol agent Trichoderma harzianum Rafai 183

SHAKSHI SINGH et al., Table 1: Germination test of fresh and stored seeds by different methods Crop seed Blotter method Multi pot Plastic pot Rolled paper towel method Fresh Stored Fresh Stored Fresh Stored Fresh Stored Wheat 93.00(9.69) 82.00(9.11) 92.00(9.64) 81.00(9.05) 90.00(9.53) 82.00(9.10) 95.00(9.79) 89.00(9.48) Paddy 90.00(9.53) 80.00 (8.99) 89.00(9.48) 80.00(8.99) 88.00(9.43) 79.00(8.94) 93.00(9.69) 87.00(9.38) Pegeonpea 88.00(9.43) 75.00(8.71) 82.00(9.10) 72.00(8.54) 85.00(9.27) 72.00(8.54) 85.00(9.27) 80.00(8.99) Gram 88.00(9.43 77.008.83 82.00(9.10) 70.00(8.42) 81.00(9.05) 72.00(8.54) 90.00(9.53) 85.00(9.27) CD (5%) 0.19 0.26 0.29 0.16 0.28 0.19 0.29 0.30 Table 2: Effect of Trichoderma harzianum seed treatment on germination of fresh seeds by plastic pot method and multi pot method Crop Seed Multi pot method Fresh Control Stored Control Fresh Control Stored Control Wheat 95.00(9.79) 90.00(9.53) 87.00(9.38) 82.00(9.10) 96.00(9.84) 92.00(9.64) 86.00(9.32) 81.00(9.05) Paddy 92.00(9.64) 88.00(9.43) 85.00(9.27) 79.00(8.94) 94.00(9.74) 89.00(9.48) 86.00(9.32) 80.00(8.99) Pegeonpea 90.00(9.53) 85.00(9.27) 83.00(9.16) 72.00(8.54) 89.00(9.48) 82.00(9.10) 75.00(8.71) 72.00(8.54) Gram 89.00(9.48) 81.00(9.05) 79.00(8.94) 72.00(8.54) 88.00(9.43) 82.00(9.11) 74.00(8.65) 70.00(8.42) CD (5%) NS 0.23 0.17 0.29 0.25 0.21 0.20 0.36 A Fresh wheat seeds Stored wheat seeds B Fresh paddy seeds Stored paddy seeds C Stored pigeonpea seeds Fresh pigeonpea seeds D Fresh gram seeds Stored gram seeds Figure 1: Germination test of fresh and stored seed by blotter method Fresh wheat seed Stored wheat seed Fresh rice seed Stored rice seed Fresh pigeonpea seed Stored pigeonpea seed Fresh gram seed Stored gram seed Plate 2: Germination test of fresh and stored seed by rolled paper towel method In this experiment determination of the germination of fresh and stored seeds of rice, wheat, gram, pigeon pea were done by plastic pot method, multi-pot tray method. The data of seed germination presented in Table 2. In plastic pots, the percent germination of treated fresh and stored seeds of rice, wheat, pigeon pea, gram were observed more than untreated fresh and stored seeds of rice, wheat, pigeon pea, gram (Table 2). Multi-pot tray method (Khare, 1996) 184

FRESH AND STORED SEED BY DIFFERENT METHODS The data of seed germination presented in Table 2. In plastic pots, the percent germination of fresh and stored seeds of rice, wheat, pigeon pea, gram were observed more than fresh and stored seeds of rice, wheat, pigeon pea, gram (Table 2). The seed treatment with biocontrol agent were reported by Harman et al. (1989); Mukhopadhyay et al. (1989); Das and Das, (2005); Mohamedy and El-Baky, (2008); Harman (2011); Mahamune and Kakde, (2011), Sultana et al. (2012) and Chavan et al. (2014) enhance the seed germination and seedling vigour. CONCLUSION The germination percentage of Wheat, paddy, Pigeon-pea, Chick pea were observed in fresh and stored seed by different method such as blotter method,, Multi pot method and Rolled paper towel method. After 10 days observation were made in which the result is found that all fresh seed give the better germination than stored seed. Plastic pot method was best method for evaluating the germination percentage of any crop seeds. Effect of seed treatment by Trichoderma spore on germination were observed in stored and fresh seeds of wheat, paddy, pigeon pea and chick pea by plastic pot method and by multiport method. The Trichoderma treated seed show higher germination percentage than control. The growth and development of crop plants were better in treated seed than untreated seed. REFERENCES Agarwal, P. K. 1994. Principles of seed technology. Indian council of Agri. Research, New Delhi. Ammara, I., Anwar, S. A. and Khan, A. R. 2000. A Seed-borne pathogens associated with wheat seed and their role in poor germination. Pakistan Journal of Phytopathology. 13(2): 102-106. Badoni, A., Bisht, C. and Chauhan, J. S. 2009. Seed Age Effect on germinability in seeds of Rheum emodi Wall. ex Meissn: An endangered medicinal plant of Garhwal Himalaya. New York Science Journal. 2(4): 1554-0200. Chavan, N. G., Bhujbal, G. 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