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...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.展开更多
Marinobufagenin(MBG)is a bufadienolide compound belonging to the cardiac glycosides class.The bufadienolides are present in humans as well as in some plants and animals.But the major source for these compounds is loca...Marinobufagenin(MBG)is a bufadienolide compound belonging to the cardiac glycosides class.The bufadienolides are present in humans as well as in some plants and animals.But the major source for these compounds is located in the parotid and skin gland secretions of some toad species.MBG is acting as a human endogenous cardiac inotrope and is demonstrating展开更多
The predatory behavior of invasive species can affect their ecological impact, and offer opportunities for targeted control. In Australia, tadpoles of invasive cane toads （Rhinella marina） do not consume eggs of nat...The predatory behavior of invasive species can affect their ecological impact, and offer opportunities for targeted control. In Australia, tadpoles of invasive cane toads （Rhinella marina） do not consume eggs of native anurans, but are strongly attracted to （and consume） newly-laid eggs of conspecifics; chemical cues from such eggs （or adult secretions） thus can be used to attract toad tadpoles to traps. Do other invasive anurans show similar selectivity？ Our laboratory trials on a Chinese population of invasive American bullfrogs （Lithobates catesbeianus） revealed similar behaviors as exhibited by Australian cane toads. Bullfrog tadpoles rarely consumed the eggs of native anurarts, but were attracted to both bullfrog eggs and bullfrog skin secretions. Although the attraction response was less intense in bullfrogs than in cane toads, it might nonetheless enable selective removal of bullfrog tadpoles from invaded sites.展开更多
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...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.展开更多
基金financially supported by the Key Technologies Research and Development Program of China(2011BAE06B04-16)
文摘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.
文摘Marinobufagenin(MBG)is a bufadienolide compound belonging to the cardiac glycosides class.The bufadienolides are present in humans as well as in some plants and animals.But the major source for these compounds is located in the parotid and skin gland secretions of some toad species.MBG is acting as a human endogenous cardiac inotrope and is demonstrating
基金supported by grants from the National Natural Science Foundation of China(31370545)National Science Foundation of China (31572284)
文摘The predatory behavior of invasive species can affect their ecological impact, and offer opportunities for targeted control. In Australia, tadpoles of invasive cane toads （Rhinella marina） do not consume eggs of native anurans, but are strongly attracted to （and consume） newly-laid eggs of conspecifics; chemical cues from such eggs （or adult secretions） thus can be used to attract toad tadpoles to traps. Do other invasive anurans show similar selectivity？ Our laboratory trials on a Chinese population of invasive American bullfrogs （Lithobates catesbeianus） revealed similar behaviors as exhibited by Australian cane toads. Bullfrog tadpoles rarely consumed the eggs of native anurarts, but were attracted to both bullfrog eggs and bullfrog skin secretions. Although the attraction response was less intense in bullfrogs than in cane toads, it might nonetheless enable selective removal of bullfrog tadpoles from invaded sites.
基金supported by grants from the National Natural Science Foundation of China (31070072,31171201)the Program for New Century Excellent Talents in University (NCET-0900717)partially supported by the National Key Technologies Research and Development Program (2011BAE06B04)
文摘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.
