Safe antifungal lipopeptides derived from Bacillus marinus B-9987 against grey mold caused by Botrytis cinerea

Agricultural application studies, including field experiments and acute toxicity tests, were conducted for lipopeptides secreted by marine-derived Bacillus marinus B-9987. Benefiting from commercially available scaled-up lipopeptide purification, the sample of impurities(isolated from target lipopeptides), raw extracted sample(purity: 9.08%), partially purified sample(purity: 20.86%), and highly purified sample(purity: 87.51%) were prepared from B. marinus B-9987 fermentation broth, and used in lab-scale antagonism tests, field experiments, swarming motility tests, and acute toxicity tests. Operations and conditions in field experiments were consistent with the Pesticide-Guidelines for the Field Efficacy Trials(GB/T 17980.28-2000), and acute toxicity tests were executed according to Toxicological Test Methods of Pesticides for Registration(GB 15670-1995). In agar diffusion tests in vitro and pot tests in vivo, all lipopeptide samples with different purities significantly inhibited Botrytis cinerea; meanwhile the sample of impurities isolated from target lipopeptides were not effective against B. cinerea. Results of lab-scale tests showed that the target lipopeptides were effective substances against B. cinerea. Thus, partially purified and raw extracted samples were used in field experiments instead of the highly purified sample for cost saving. In the field experiments against rose grey mold, biological control efficacy of 500 mg L–1 lipopeptides reached 67.53%, slightly lower than 74.05% reached by the agrochemical pyrimethanil. However, pyrimethanil severely suppressed B. marinus B-9987, whereas the lipopeptides promoted swarming motility and biocontrol efficacy of Bacillus biomass. Lipopeptides at 87.51% purity were tested for systemic acute toxicity and confirmed as low-toxicity substances. In conclusion, low-toxicity lipopeptides were potential alternatives to agrochemicals, and they also performed good promotion when combined with homologous biological control microorganism. There were 2 breakthroughs in this research:(1) marine-derived bacterial lipopeptides inhibited grey mold caused by B. cinerea in field experiments; and(2) purified bacterial lipopeptides(sample purity: >87.51%) were determined to be low-toxicity substances by systemic acute toxicity tests, satisfying the strict requirement of pesticide registration in China(required purity: >85%). This study provides support for using extracellularBacillus-derived lipopeptides commercially similar to Bacillus-based biological control agents.