Evaluation of Properties of Extracellular Antifungal Metabolites Secreted by Four Antagonistic Burkholderia Strains for the Control of Colletotrichum truncatum

Abstract

In the replete of biological disease management strategies, microbial antagonism is becoming a promising disease control tool used by scientists. Among various mechanisms extended by antagonists, production of extracellular antifungal substances is of greater interest. Properties of cell-free culture supernatants of four antagonistic Burkholderia sp. against chilli anthracnose causal agent, Colletotrichum truncatum was assessed in this study. These antagonists were found to produce diffusible antifungal substances for antibiosis against C. truncatum. Cell-free culture supernatants of six days old nutrient broth cultures, which were centrifuged twice at 12000 rpm for 20 minutes and filtered through a 0.22 μm millipore membrane were used for the study. The effect of each of the extracts on C. truncatum mycelial growth and spore germination was evaluated and their heat stability, durability at both 4 º C and room temperature and their minimum inhibitory concentrations were also studied. All the selected antagonists were capable of producing and accumulating extra cellular antifungal compounds inhibiting the growth of C. truncatum within a five-day incubation period. Cell free culture supernatants contained 11.0 mg/ml total soluble solids. Culture filtrates of Burkholderia multivorans and B. gladioli isolates showed a remarkable thermostability at 120 ºC, while B. arboris and B. rinojensis culture filtrates were thermally stable up to 100 ºC. Cell-free culture supernatants of the antagonists retained their antifungal properties up to a maximum of 2 months at room temperature and up to 3 months at 4 ºC. Minimum inhibitory concentrations of culture filtrates of B. multivorans, B. arboris, B. gladioli and B. rinojensis were 4.85 mg/ml, 1.62 mg/ml, 7.42 mg/ml and 6.59 mg/ml respectively. Antifungal metabolites biosynthesized by Burkholderia sp. were mostly organic solvent soluble molecules, which could be extracted into ethyl acetate. The ethyl acetate fraction of the culture supernatant showed a minimum inhibitory concentration of 2 mg/ml on C. truncatum mycelial growth suppression. These properties of cell-free culture supernatants produced by the antagonistic Burkholderia strains provide an excellent opportunity for chilli anthracnose disease management.