Bio-stimulation by seed priming with Bacillus subtilis for suppressing seed-borne fungal pathogens of vegetables in Bangladesh

Aqueous formulation of rhizopheric beneficial bacteria viz. Bacillus subtilis, an exotic strain collected from Russia was assayed for its performance to suppress the growth of various seed-borne fungi of vegetable. The performance of the bio-agent was appraised based on growth suppression by dual culture method and reduction of seed-borne fungi in the bio-agent treated seeds. Ten seed samples comprise of summer and winter vegetables were examined collected from different local farmers. A total of fourteen fungal species belongs to twelve genera viz. Fusarium moniliforme, Botrytis cinerea, Aspergillus flavus, F. oxysporum, Botryodiplodia theobromae, Macrophomina phaseolina, A.niger, Cercospora sp., Phoma exigua, Rhizopus sp., Colletotrichum sp., Phytophthora sp., Penicillium sp., and Curvularia sp. were recorded from different untreated seeds. Vegetables seeds were treated in a concentration (10) of aqueous formulation of B. subtilis for two hours followed by air drying for 30 minutes. In general, the incidences of all fungal species of all kinds of vegetables seeds were reduced. But, selectively the best performances were observed in tomato, brinjal, cucumber, wax gourd and okra where most of the fungal species were completely suppressed by seed treatment with Bacillus subtilis. The growth of seed-borne fungi was inhibited highest in wax gourd (100%). The growth inhibition was also at satisfactory level in cucumber (95%), tomato (95%), brinjal (92%) and okra (86%) seeds. The findings of this research indicate the possibility of utilizing B. subtilis as seed treating agents instead of chemical fungicides to control seed-borne diseases of vegetables.


Introduction
Eco-friendly sustainable disease management strategies are the prime concern for crop producers across the world where bio-agents collected from the natural ecosystem play a pivotal role.Stimulation of plants innate immunity by exploiting natural bio-agents has been utilized in many plant diseases.Induced systemic resistance activated by microbial components can resist a wide range of pathogens (Alizadeh et al. 2013).Bacillus subtilis produces a wide range of bioactive molecules among which lipopeptides; surfactin, iturin and fengycin families.In a recent study it is reported that surfactin, a bioactive compound derived from Bacillus subtilis showed elicitation of systemic resistance (ISR) in tomato against tomato mosaic virus (Hussein et al. 2016 andDevendra et al. 2009).Plant protection from various disease resulting from ISR elicited by Bacillus spp.(Van Loon, 2007).has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases (Turner andBackman, 1991 andAbo-Elnaga, 2006).Bio-priming is a new technique of seed treatment that integrate biological and physiological aspects of disease control has recently been used as alternative method for controlling many seeds and soil borne pathogens (Ferreira et al., 1991;El-Mohamedy, 2008).Coating seeds of vegetables with bio-control agent such as Bacillus subtilis is the most effective treatment for controlling many seed-borne pathogens.Seed priming with B. subtilis have since been shown to control various diseases in vegetables.The inhibitory effect of Bacillus subtilis on plant pathogenic fungi has been frequently reported in laboratory, greenhouse, and field studies.B. subtilis is able to synthesize more than 60 different types of antibiotics, mainly in polypeptides, many of which possess antifungal effects.B. subtilis produces antibiotics, including some called iturins, which help the bacteria compete with other microorganisms either by killing them or reducing their growth rate (Singh et al., 2016).When applied directly to seeds, B. subtilis colonizes the developing root system, competing with various disease organisms that attack root systems.According to the manufacturers, B. subtilis also inhibits plant pathogen spore germination and interferes with the attachment of the pathogen to the plant.When soil or seed-applied, it is claimed that B. subtilis feeds off plant root exudates, depriving pathogens of a food source.
Keeping these views in mind, the present investigation was designed to assay the potential of B. subtilis for seed priming for reducing the incidence of seed-borne fungi of several vegetable crops which can eventually improve the quality seedlings as well as increase the yield of crops and reduce indiscriminate use of chemical pesticides.

Materials and Methods
Seeds of ten vegetable crops (Bottle gourd, Sweet gourd, Snake gourd, Wax gourd, Cucumber, Tomato, Brinjal, Raddish, Bean and Okra) were collected from the farmers of Mymensingh district.These seeds were stored in zip-lock bags in refrigerator for further studies.In order to investigate the viability of the formulated product of Bacillus subtilis, colony forming unit (cfu) was observed and counted on Nutrient Agar (NA) medium.One mL of suspension from each dilution series was poured on NA medium and wiped with a sterile glass rod.The next day, colony forming unit (whitish colony of bacterium) was formed and their number was counted.To detect the seed-borne fungi Blotter method was followed according to ISTA rules for seed testing (ISTA, 2001).Three layers of blotting papers (Whatman no. 1) were soaked in sterilized water and placed at the bottom of 9 cm diameter plastic petridish and over which the seeds were plated.Four hundred seeds were examined per sample.The seeds were incubated in the incubation chamber at 20 ± 2 0 C under alternating cycles of 12 hours near ultraviolet (NUV) light and darkness for 7 days.Additional water was supplemented to moisten the blotter paper when necessary.In order to prepare and select the dilution series from stock solution of Bacillus subtilis 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 times dilution solutions were prepared with distilled water.Before, conducting any experiment the viability of the imported formulation was examined by making the series dilution and subsequent plating on Nutrient Agar (NA) medium and incubated them at room temperature for 2-3 days.Colony developed on NA medium were examined visually and counted.Seed priming was accomplished by dipping the vegetables seeds in 10 -3 dilution of stock solution of B. subtilis for 2 hrs at room condition (25±2 C).Afterwards, the solution was drained out and the seeds were allowed to dry.Then, the seeds were placed in blotting paper according to the ISTA rules for seed health testing (ISTA, 2001).Each individual incubated seed was observed under stereomicroscope at 16x and 25x magnifications in order to record the incidence of seed-borne fungi.Most of the associated fungi were detected by observing their growth characters on the incubated seeds on blotter paper.The fungal associations were presented as percent incidence for individual fungi.

Effect of seed treatment by Bacillus subtilis on the association of seed-borne fungi in summer vegetable seeds
A total of five summer vegetables viz.sweet gourd, wax gourd, snake gourd, cucumber and okra were considered for investigating the association of seed-borne fungi collected from the farmers.The most fungi belong to the genera Fusarium, Macrophomina, Colletotrichum, Aspergillus, Curvularia, Botrytis, Rhizopus, Phoma were predominantly associated at various intensity with most of the seed samples (Fig. 1-5).In sweet gourd, the highest (44%) prevalence was recorded in case of Aspergillus niger followed by Rhizopus sp.(35%), Fusarium oxysporum (34%), F. moniliforme (28%).The prevalence of M. phaseolina, Colletotrichum sp., A. niger was completely suppressed by seed priming by Bacillus subtilis suspension (Fig. 1).
Among all the seed-borne fungi of summer vegetables, seed treatment by B. subtilis showed the strong suppression ability, the best suppression ability was observed in case of okra and cucumber seeds where almost 100% suppression of seed-borne fungi was recorded.

Effect of seed treatment by Bacillus subtilis on the association of seed-borne fungi in winter vegetable seeds
A total of five winter vegetables viz.bottle gourd, bean, raddish, tomato and brinjal were considered for investigating the association of seed-borne fungi collected from the farmers.The most fungi belong to the genera Fusarium, Macrophomina, Colletotrichum, Aspergillus, Curvularia, Botrytis, Rhizopus, Phoma, Phytophthora, Penicillium, Cercospora were predominantly associated at various intensity with most of the seed samples (Fig. 6-10).In bottle gourd the highest (66%) prevalence was recorded in case of Botrytis cinerea followed by Fusarium oxysporum (24%), F. moniliforme (18%).The prevalence of M. phaseolina, Cercospora sp., Phoma exigua, and A. niger was completely suppressed by seed treatment by Bacillus subtilis suspension (Fig. 6).(Fig. 7).

Fig. 7 .
Fig. 7. Effect of Bacillus subtilis on percent prevalence of seed-borne fungi in Bean (Phaseolus vulgaris)

Fig. 9 .
Fig. 9. Effect of Bacillus subtilis on percent prevalence of seed-borne fungi in Tomato (Solanum lycopersicum)