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.

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

Marine-derived Bacillus strains have been proved to be a very promising source for natural product leads.However,transformation of environmental strains is much more difficult than that of domesticated strains.Here,we report the development of an efficient and robust electroporation-based transformation system for marine-derived Bacillus marinus B-9987,which is a macrolactin antibiotics producer and a very promising biological control agent against fungal plant diseases.The transformation efficiency was greatly enhanced 103-fold by using unmethylated plasmid to bypass modification-restriction barrier,and using glycine betaine to protect cells from electrical damages during electroporation.Addition of HEPES and 2 mmol L?1MgCl2 further improved the efficiency by additional 2-fold,with a maximum value of 7.1×104 cfu/μg pHT3101.To demonstrate the feasibility and efficiency of the protocol,a green fluorescent protein reporter system was constructed;furthermore,phosphopantetheinyl transferase gene sfp,which is essential to the biosynthesis of polyketides and nonribosomal peptides,was overexpressed in B-9987,leading to increased production of macrolactin A by about 1.6-fold.In addition,this protocol is also applicable to marine-derived Bacillus licheniforms EI-34-6,indicating it could be a reference for other undomesticated Bacillus strains.To our knowledge,this is the first report regarding the transformation of marine-derived Bacillus strain.