BIOCONTROL OF SEEDLING BLIGHT BY CO- INOCULATION WITH MICROBIAL CONSORTIA

Abstract

Compost has been used in agriculture since ancient time. It is useful to improve the fertility of the soil but its role in growth promotion and disease suppression is due to compost inhabiting bacteria (CIB). Plant growth promoting rhizobacteria (PGPR) regulate plant growth and act as biocontrol agents. Use of fungicide is common to control fungus, with detrimental residual effects. Biocontrol agents can be used to reduce the harmful effect of pesticides. Hypothesizing that PGPR and CIB can play role in controlling seedling blight in maize this prospect was studied with a consortium of PGPR and CIB. A number of PGPR and CIB strains were isolated from maize rhizosphere and compost respectively. After preliminary tests, twenty five isolates each of PGPR (Mb1 to Mb25) and CIB (Cb1 to Cb25) were selected and maintained for further experimentation. Fungal isolates were isolated from the maize plants, suffering from seedling blight. After in-vitro studies for germination test, twelve strains each of PGPR and CIB were selected, characterized and screened individually for growth promotion and disease suppression through jar experiments under axenic conditions. Synergism of isolates was also tested through in-vitro studies. The four best performing isolates of PGPR (Mb4, Mb5, Mb6 and Mb7) and CIB (Cb1, Cb4, Cb8 and Cb9) were selected to prepare consortium and test their compatibility through jar experiment under controlled conditions. Results showed that best performing consortium were Mb4 + Cb4, Mb4 + Cb9, Mb7 + Cb4 and Mb7 + Cb9. The potential of consortium for growth promotion and disease suppression was tested by trials in pots, under semi-controlled conditions. Disease severity decreased from 82% in the control to 11% in consortium inoculation. Intercellular CO2 concentration (Ci) was higher in the control (357 μmol mol-1 air) and decreased to 116 μmol mol-1 air in consortium inoculation. Higher values for stomatal conductance (165 mMm-2S-1), vapor pressure deficit (4 k-Pascal), evapotranspiration (5.7mM water m-2 S-1), assimilation of CO2 (19 μmol CO2 m-2s-1), water use efficiency (3.4%), chlorophyll a (2 mg g-1), chlorophyll b (3 mg g-1) and relative water contents (97%) were observed by single and consortium inoculation while membrane permeability (87.7%) was higher in control. Proline contents (5 μmol/g), total soluble sugars (11 mg g-1 FW), catalase activity (4.3 mM H2O2/min/g) and ascorbate peroxidase activity (0.63 μM H2O2/min/mg protein) were also significantly higher with inoculated treatment. Overall each set of the consortium suppressed disease and promoted the growth and yield of test crop. The bacterial metabolites of strains Mb4, Mb7, Cb4 and Cb9, as extracted by methanol were analyzed by HPLC system.The chromatograms of allisolates, exhibited different patterns and many peaks were detected. Based on comparison with standards, phenols in the metabolites were identified. Chlorogenic acid and caffeic acid were present in the metabolites of all four strains, while ferulic acid was present in smaller quantity in all isolates. Higher amount of ascorbic acid were detected in Mb7 and Cb9 while quercetin, gallic acid, p-caumaric acid, m- caumaric acid, vanillic acid and cinnamic acid were also present in all the isolates in smaller quantities. Root exudates were analyzed by HPLC against standards of pyruvic acid, citric acid, malleic acid, chlorogenic acid, oxalic acid, caffeic acid, fumaric acid, cinnamic acid and salicylic acid. Cinnamic acid and malleic acid were present in exudates of undiseased control while a diseased control contained chlorogenic acid, caffeic acid and salicylic acid. Exudates, collected from inoculated treatments containedchlorogenic acid, malleic acid and salicylic acid. Inoculation with PGPR isolates (Mb4, Mb7) induced the secretion of caffeic acid while that of Cb9 cause the induction of benzoic acid by roots under diseased conditions. The efficacy of different consortia was proved. Characterization of the selected bacterial isolates revealed that all isolates had more than one mechanism of action to control disease. Root exudates from soil ofundiseased plants could more effectively stimulate conidial germination than that from the diseased plants. Under undiseased condition root exudates had stimulatory effect on conidial germination (95% conidial growth) and showed highest germination, followed by control in diseased condition with 76% conidial growth. While lowest growth (39%) was observed by the addition of root exudates, collcted from consortium treatment in diseased soil. Compared with the root exudates from single inoculated plants, the root exudates from co-inoculated plants have significant effect to decrease the growth of conidia. The effect of the root exudates on mortality of spores ranges from 5 to 50% in undiseased soil and 24 to 61% in diseased soil. Root exudates, collected from single and consortium treatments from diseased soil showed decreasing trends of conidial growth compared to treatments with no exudates. Root exudates suppressed the growth of conidia by 43% (single inoculation) to 100% (by consortium) compared to treatments without exudates. The number of conidia varied from 3.2 to 3.5 × 105 CFU mL-1 by root exudates while the number ranges from 5 to 5.6 × 105 CFU mL-1 without exudates. Consortium showed 2.7 × 105 CFU mL-1 with exudates while treatment with no exudates showed 5.4 × 105 CFU mL-1. Root exudates supported the growth of bacteria in both, undiseased and diseased soil. All treatments with exudates enhanced the growth of bacteria compared to treatments with no exudates. Moreover growth with exudates from undiseased soil increased the growth more than from diseased soil. Consortium dominated supporting the growth of bacteria in both undiseased and diseased soil. Inoculation with the consortium of isolates exhibited percent increase in fresh cob (up to 53%) and dry cob (41%) yield, cob length (51%), grain yield (up to 55%), 1000-grain weight (up to 37%), K contents in grains and straw (1.8 and 0.8), %N in grain and straw (up to 2.7 and 1.0% respectively) and %P in grains and straw (up to 1.8 and 0.8% respectively) of maize compared to un-inoculated control. So the growth promotion caused by these selected consortia could be a function of multiple mechanisms. Co-inoculation overcome the adverse effects of pathogen and enhances the growth and yield of maize. Selected bacterial isolates could be used as biocontrol agent against Fusarium oxysporum in maize. However, the use of the bacterial isolates as biocontrol agents should be avoided without knowledge of the particular information about the compatibility of the isolates toward specific crop varieties, fungal isolates, soil and environmental conditions for which it could be used. In short, isolates of PGPR and CIB, recognized for the growth, yield promotion and diseases suppression could be used efficiently and effectively against specific host under specific set of soil and environmental conditions for maximum benefits.