International of Phytopathology POTENTIALITY OF BIOAGENTS ON SEED QUALITY ENHANCEMENT IN CHICKPEA

Planting healthy seeds and other inputs have an impact on seed quality, fungal invasion deteriorates the seed quality in soil and storage. Studies on chickpea carried out at Seed Research and Technology Center, Rajendranagar, Hyderabad during spring 2010-11 & 2011-12 revealed that benomyl was found effective followed by Pseudomonas fluorescens in the inhibition of mycelial growth of Fusarium solani . Maximum germination percentage was recorded with Benomyl (Benomyl 500 WP) @ 2 g/kg seed (treated check) and Tebuconazole @ 1 ml/ kg seed (95 per cent) followed by treatment with P. fluorescens @ 10 g/kg seed along with soil application of P. fluorescens @ 3 kg/acre (94 per cent). Maximization of growth parameters like root length, shoot length and total seedling length were observed with Benomyl @ 2 g/ kg seed as 17.0 cm, 10.3 cm and 27.3 cm, respectively. Considering seedling vigour index as an important seed quality character, P. fluorescens and Benomyl @ 2 g/kg seed recorded high seedling vigor index. The per cent recovery of infested seeds was found to be low with treated seeds when compared to the control.


INTRODUCTION
Chickpea is the most nutritive pulse among different food legumes being extensively used as protein adjunct to starchy diet. Of the various diseases, fungal diseases especially, the wilt caused by species of Fusarium remains to be a challenging task in terms of management since it is soil-borne in nature (Agrios, 2000;Singh et al., 1986). Planting high quality seeds, one of the cheapest input in crop production, is the key to agricultural progress. Response of other inputs in crop production depends on seed material used. Seed quality of chickpea was affected by incidence of seed borne diseases like wilt and root rot. Earlier workers reported influence of cultural, regulatory, physical, chemical and biological methods on the control of the seed and soil borne pathogens. In modern agriculture, agrochemicals are unavoidable, but bio-agents are important components in integrated pest management programme and sustainable agriculture (Chaudhary et al., 2007). The combined use of biocontrol agents and chemical pesticides has attracted much attention as a way to obtain synergistic or additive effects in the control of soil borne pathogens (Maurya et al., 2008). Among these, chemical methods offer a good choice to farmer to combat the diseases (Wise et al., 2009). But owing to so many problems in view of ecosystem and environment, a rapid shift has been made from synthetic products to bio products, which are eco friendly and beneficial. Keeping these aspects in view, the present study was carried out based on preliminary studies to find out the effect of various bio-agents on seed quality by minimizing the incidence of seed borne diseases and also increasing soil micro flora.

MATERIAL AND METHODS
Preliminary studies on seed mycoflora of chickpea were carried out under laboratory conditions and their inhibition was tested with four fungicides viz., Azoxystrobin, Tebuconazole, Carbendazim and benomyl and three bio-agents ie., Trichoderma viride, Bacillus subtilis and Pseudomonas fluorescence by rolled towel and seed inoculation technique. Fungicides (0.2 and 0.3%) and bio-agents (0.8 and 1%) were tested in two concentrations against Fusarium oxysporium seed pathogenic (root rot and wilt) fungi. Per cent inhibition of mycelia growth was carried out by the seed inoculation and seed rolled technique (Pankaj Sharma, 2010). Seed Inoculation Technique: Healthy surface sterilized seeds (1:1000 mercuric chloride) of cultivar Annegiri were artificially inoculated with 7 days old culture of fungal species separately. Inoculated seeds were treated with fungicides. The treated seeds were placed on the moist blotter paper in plates @ 5 seeds/plate. Inoculated untreated seeds served as control. Observations were recorded after 10 days of incubation. In-vitro Evaluation of Fungicides by Seed Rolled Towel Method: Seeds were treated with fungicides by following wet seed treatment with 0.2 per cent and 0.3 per cent fungicidal solution. Seeds were soaked in the fungicidal solutions for 2 hrs. Then the seeds were dried under shade. Three replications of 50 seeds per treatment were tested in moist paper towel (rolled towel) method. The plates were incubated in BOD incubator at 20±2° C under 12 hrs of light and 12 hrs dark. The untreated samples served as control. The per cent germination and per cent infection were recorded. (Pankaj Sharma, 2010).

In-vitro Evaluation of Bio-agents by Rolled Paper Towel Method:
The powder formulation of antagonist's viz., Trichoderma viride, Bacillus subtilis and Pseudomonas fluorescence were used for seed treatment to test their efficacy in overcoming seed borne fungal infection of chickpea under in-vitro conditions by rolled towel method. Seeds of moderately infected chickpea were treated with different bio-agents at the rate 10 6 cfu. The seeds were shaken in shaker for 20 min for the coating of bio-agents formulation and then stored in separate boxes for 24 hrs. The treated seeds were tested in 5 replications of 50 seeds per replication. Seeds without treatment served as control. These paper towels were incubated at 20±2° C for seven days under 12 hrs light and 12 hrs darkness. After 7 days of incubation, per cent germination, per cent infection and seedling vigor were calculated as germination x seedling length. Based on the above findings of in-vitro conditions, the field study was taken up at Seed Research and Technology Centre, Rajendranagar, Hyderabad during spring, 2010-2012. Treatments comprising of bioagents, fungicides and their combinations viz., Trichoderma viride @ 10 g/kg seed, soil application of T. viride @ 2 kg/acre, Bacillus subtilis @ 5 g/kg seed, Benomyl (Benomyl 500 WP) @ 2 g/kg seed, Pseudomonas fluorescens @ 10 g/kg seed, combination of Trichoderma viride @ 10 g/kg seed + soil application of T. viride @ 2 kg/acre, combination of B. subtilis @ 5 g/kg seed + Benomyl @ 2 g/kg seed (treated check), AMISTAR (23% w/w azoxystrobin) @ 1 g/kg seed, Tebuconazole (100% Raxil T @ 25 g/l = Tebuconazole 4 g/l + Triflunuron) @ 1 ml/kg seed, P. fluorescens @ 10 g/kg seed + foliar spray of P. fluorescens @ 6-10 g/l, P. fluorescens @ 10 g/kg seed + soil application of P. fluorescens @ 3 kg/acre along with untreated check were imposed. The experiment was laid out in a randomized block design in three replications with all the recommended agronomic practices. Observations on various disease parameters such as crop stand, per cent disease incidence of wilt and root rot was assessed. Yield attributing characters viz., plant height, branches per plant, pods per plant, seeds per plot, seed yield per plot (g), seed yield per ha (q) and 100 seed weight (g) were recorded for 10 randomly selected plants from each treatment. Various seed quality parameters like germination percentage, root length (cm), shoot length (cm), total seedling length and seedling vigor index I were studied under laboratory conditions as per the ISTA rules (1985) and expressed in percentage. Ten normal seedlings were selected at random in each replication for recording seedling length in centimeters (cm) and oven dried at 80 o C for 17 h and weighed (g) for seedling dry weight. Seed vigor index I was calculated as product of germination and seedling length (Abdul-Baki and Anderson, 1973). Average values were computed and the data was subjected to statistical analysis (Panse and Sukhatme, 1985).

RESULTS AND DISCUSSION
All the fungicides and bio-agents significantly reduced mycelia growth as compared to control. Minimum per cent mycelia growth was observed in Tebuconazole tested against Aspergillus niger. The per cent inhibition in mycelia growth was in the range of 24.9 to 96 per cent in different tested fungicides (Table 1). Among the fungicides tested, benomyl was more effective in maximum germination and minimum seed borne infestation (92 and 16) followed by Pseudomonas ( 84 and 18). These results are in agreement in the inhibition of mycelial growth of Fusarium solani. with those of Singh and Jha (2003). At higher concentration, all fungicides were found effective in controlling seed borne mycoflora. These results are in agreement with the findings of Pankaj Sharma (2010) where the seed borne mycoflora was effectively controlled by fungicides. At higher concentration, all the metabolic activities of all fungi could be arrested which in turn results into complete destroy of the internal parts of the seed (Ibiam et al., 2006).
The bio-agents were tested for their efficacy against seed borne fungal infection of chickpea by rolled paper towel method. Among the three (Table 2)  Seed treatment with strains of Pseudomonas increased emergence and yield of pigeon pea planted in Fusarium infested soil and were equivalent to fungicidal seed treatment (Gupta et al., 2011). Seed quality is the one of the most important basic input characterized in terms of longer viability and good vigor. Besides quantity of seed realized and their effectiveness in reducing disease incidence, quality of seed also plays an important role in adjudging the performance of bio-agents for chickpea crop. Seed treatment with P. fluorescens @ 10 g/kg seed recorded minimum germination of 83.0 per cent (Table 3). While, maximum germination was recorded with Binomil seed treatment @ 2 ml/kg seed and Tebuconazole @ 1 ml/ kg seed (95 per cent) followed by treatment with P. fluorescens @ 10 g/kg seed along with soil application of P. fluorescens @ 3 kg/acre (94 per cent). Similar results of plant growth promotion and reduced wilt incidence by P. fluorescens in pigeonpea was reported by Gupta et al. (2011). Similarly, root length, shoot length and total seedling length were highest in case of seed treatment with Benomyl @ 2 ml/ kg seed (17.0 cm, 10.3 cm and 27.3 cm respectively). Considering seedling vigor index as an important seed quality character, Benomyl @ 2g/ kg seed recorded high seedling vigor index (2588) followed by bioagents. Further, the per cent disease infested seeds were also found to be low with P. fluorescens seed treatment. Hence seed quality can be enhanced by P. fluorescens instead of chemicals with reduced wilt and root rot incidence.