Bacillus lipopeptides for a novel postharvest disease control technology

Postharvest diseases caused by fungal pathogens account for about 44% of global fruit and vegetable losses. Biocontrol strategies for postharvest disease are particularly attractive as they are environmentally benign and non-toxic. Current biocontrol strategies employ microorganisms as the biocontrol agents, with application under controlled environmental conditions mandatory for cell viability. Here, a novel biocontrol technology has been developed which uses microbial antifungal lipopeptides, namely fengycin and iturin. These do not require environmental control, and have the potential to be manipulated into bespoke products with efficacy against specific phytopathogens. Antifungal lipopeptides were produced by Bacillus amyloliquefaciens DSM 23117 and quantified by reverse phase high performance liquid chromatography (RP-HPLC). Using discrete ratios of ammonium to nitrate, the nitrogen source was shown to exhibit a significant influence on the production kinetics. The antifungal concentration in the culture supernatant exhibited a general downward trend with decreasing amounts of nitrate with a 2.6-fold difference in the maximum and minimum lipopeptide concentrations in the cultures containing only nitrate or ammonium respectively. A similar trend was observed in cell concentration which exhibited a 3.9-fold reduction, and a corresponding 1.5-fold increase in antifungal yield, over the same range. Purification of the culture supernatant was effected by acid precipitation to pH 1, 2, 3 and 4. recipitates were recovered by centrifugation and drying and the lipopeptides quantified by RP-HPLC. High recoveries (70-78% fengycin and 62-67% iturin) were attained at pH 1-3, with optimal purity (67% antifungal) at pH 3. Thin layer chromatography confirmed the low purity at pH 4 was due to contaminating proteins. Antifungal efficacy of a 10-fold concentrate was confirmed for Alternaria brassicicola, Botrytis cinerea, Rhizopus stolonifer, Monilinia fructigena and Penicillium expansum in zone clearing assays, demonstrating the successful production of a concentrated and partially purified biofungicide effective against postharvest phytopathogens.