Evaluation of selected free-living diazotrophic bacteria for plant growth promotion and biological control of damping-off fungi.

Abstract

Inoculation with free-living diazotrophic bacteria is well documented to enhance vegetative growth and yield increases of various crops coupled with suppression of sublethal pathogens. The use of microbial inoculants has been identified as an alternative or supplement to use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. The search for effective free-living diazotrophic bacterial strains for formulation as biofertilizers has been on going since the 1970’s and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 250 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within KwaZulu-Natal, province, Republic of South Africa. These were evaluated for plant growth-promotion and biological control of damping-off fungi initially by in vitro screening. The growth promotion traits tested included, phosphate-solubilization, production of indole-3-acetic acid, production of ammonia and acetylene reduction. Biocontrol traits evaluated included siderophore-production, antibiosis, and production of hydrogen cyanide (HCN). Biochemical and molecular bioassay tests were conducted to identify the twenty most promising isolates selected in the in vitro study. The twenty isolates were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of maize (Zea mays L.) and wheat (Triticum aestivum L.) under greenhouse conditions. The five most promising isolates identified for growth promotion under greenhouse conditions for each crop were assessed for their effects on the germination of maize in vitro and growth and yields of two maize and two wheat cultivars, when combined with a low dose of nitrogenous fertilizer in field trials. The five Bacillus subtilis (Ehrenberg) Cohn isolates that suppressed the growth of a wide range of pathogenic fungi in vitro were tested for their efficacy against damping-off of wheat caused by Rhizoctonia solani Kühn in the greenhouse. These isolates were further studied for their modes of action against R. solani in vitro. The modes of action tested included antibiosis, production of siderophores, extracellular enzymes, production of hydrogen cyanide (HCN) and antibiotic resistance. The twenty most promising bacterial isolates identified from the in vitro screening reduced acetylene to ethylene, produced indole-3-acetic acid and siderophores, one isolate solubilized phosphate, and 9 inhibited the growth of R. solani. These twenty isolates enhanced growth of maize and wheat above the Uninoculated Control under greenhouse conditions. The growth enhancements varied with bacterial isolate x crop species interactions, which identified five different isolates for each of the two crops. Relative to the Uninoculated Control, the best five isolates significantly (P = 0.001) enhanced the growth of maize and wheat at all fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry biomass of maize and wheat, and enhanced root development of maize in the greenhouse. Inoculation of maize and wheat with the two most promising isolates identified from the field trial for each crop, in combination with 65% and 50% of the recommended amount of nitrogenous fertilizer for maize and wheat, respectively, caused the same increases in shoot biomass as the Fully Fertilized Control. Application of a combination of the best bacterial isolates and 35% nitrogenous fertilizer resulted in the same or greater shoot dry biomass and yields of both maize and wheat under field conditions. Shoot dry biomass of wheat increased by 75% above the Uninoculated Control and 30% above the Fully Fertilized Control. The wheat yield increased by 95% above the Uninoculated Control and 43% above the Fully Fertilized Control. Seed inoculation with the best isolates combined with 35% N increased yields of maize by 41% above the Uninoculated Control and 15% above the Fully Fertilized Control. The best isolates significantly (P < 0.001) increased plant height, chlorophyll levels and shoot biomass of maize relative to the Uninoculated Control. There was a positive correlation between chlorophyll level and yield, chlorophyll level and shoot dry biomass, height and shoot dry biomass and height and yield of maize at P = 0.01 with r values of 0.87, 0.77, 0.92 and 0.81, respectively. The isolates that exhibited multiple plant-growth promoting traits in vitro, increased shoot biomass of both maize and wheat in the greenhouse and field, and caused yield increases in the two crops under field conditions. Five B. subtilis isolates inhibited the growth of some of the pathogenic fungi tested in vitro up to 95%. Seed inoculation with the same isolates significantly (P = 0.001) suppressed R. solani damping-off of wheat under greenhouse conditions and exhibited multiple mechanisms of disease control in vitro. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising the yields. The B. subtilis isolates obtained in this study can effectively control R. solani damping-off of wheat, fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture. Key words: Free-living bacteria; plant growth-promotion; diazotrophs; biological nitrogen fixation; phosphate-solubilization; siderophores; indole-3-acetic acid; biocontrol; damping-off