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Abstract
Bacillus amyloliquefaciens YN201732 is an endophytic bacteria with high biocontrol efficiency and broad-spectrum antimicrobial activities. In order to clarify the main active ingredients and their antifungal mechanisms against powdery mildew of tobacco, this study is focused on lipopeptide obtained through acid precipitation and organic solvent extraction. HPLC and LCMS-IT-TOF were used to separate and identify antimicrobial lipopeptides. Findings revealed that bacillomycin D plays an important role against surrogate fungal pathogen Fusarium solani. Synthetic pathways of sfp, bacillomycin D, and fengycin were separately disrupted. The sfp gene knockout mutant B. amyloliquefaciens YN201732M1 only showed minor antagonistic activity against F. solani. While Erysiphe cichoracearum spore germination was inhibited and pot experiments displayed a significant decrease in tobacco powdery mildew. The spore inhibition rate of YN201732M1 was only 30.29%, and the pot experiment control effect was less than 37.39%, which was significantly lower than that of the wild type. The inhibitory effect of mutant YN201732M2 (deficient in the production of bacillomycin D) and mutant YN201732M3 (deficient in the production of fengycin) on the spore germination of E. cichoracearum were 50.22% and 53.06%, respectively, suggesting that both fengycin and bacillomycin D had potential effects on spore germination of powdery mildew. Interestingly, in a greenhouse assay, both B. amyloliquefaciens YN201732M2 and YN201732M3 mutants displayed less of a control effect on tobacco powdery mildew than wild type. The results from in vitro, spore germination, and greenhouse-pot studies demonstrated that antimicrobial lipopeptides especially bacillomycin D and fengycin may contribute to the prevention and control of tobacco powdery mildew. In addition, gene mutation related to lipopeptide synthesis can also affect the biofilm formation of strains.
Highlights
Erysiphe cichoracearum is the primary cause of powdery mildew in tobacco plants, and it results in substantial reduction of yields and quality of tobacco in China (Xing et al, 2015)
The results show that the n-butanol extract had obvious antifungal activity against F. solani, and the average zone of inhibition diameter was 2.63 ± 0.17 cm
The inhibition zone of mutant YN201732M3 was 3.97 ± 0.06 mm, and the antifungal effect was slightly lower than that of the wild-type strain. These results show that antimicrobial lipopeptides except for fengycin could inhibit the pathogen of F. solani
Summary
Erysiphe cichoracearum is the primary cause of powdery mildew in tobacco plants, and it results in substantial reduction of yields and quality of tobacco in China (Xing et al, 2015). Bacillus as an ideal biocontrol bacterium has been used for biological control of crop diseases and insect pests, especially endophytic B. amyloliquefaciens, which are ubiquitously found to be safe microorganisms with proven excellent in planta colonization aptitudes (Cao et al, 2011; Liu et al, 2017) and versatility in effectively protecting plants from pathogens (Ongena and Jacques, 2008). They can compete with pathogens for nutrients, secretes antimicrobial substances, and induce the plant’s defense system to resist the invasion of pathogens (Wang et al, 2017). Antimicrobial peptides are powerful weapons of biocontrol strains against other pathogenic microorganisms, and their various structural types can help Bacillus effectively avoid the development of pathogen resistance and maintain the antagonistic advantage of biocontrol agents (Raaijmakers et al, 2010)
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