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.

Tobacco leaf mildew caused by Rhizopus oryzae (Mucorales, Zygomycota) is an important and devastating post-harvest disease during flue-cured tobacco period, and also is known to cause diseases of fruits and vegetables. In this study, assessment of several candidate biological control agents were first tested in vitro to determine their antifungal activities and potential strains were further applied to tobacco leaves to prevent pathogen colonization during the tobacco-curing process. In vitro screening of 36 bacteria and the isolates of one fungus were performed for their antifungal activities against R. oryzae using dual culture method. Potential five isolates viz. Bacillus amyloliquefaciens B9601-Y2 (Y2), B. amyloliquefaciens YN201728 (YN28), Pseudomonas sp. (Pb), and B. amyloliquefaciens YN201732 (YN32) and T. harzianum B (Th-B) from total screened isolates have shown remarkable results for controlling the mycelial growth of R. oryzae. Finally, out of these five isolates, B. amyloliquefaciens B9601-Y2 potentially reduced the mycelial growth of fungal pathogen with great inhibitory effect. In order to get a better understanding of the biocontrol effect of B9601-Y2 in a flue-curing barn, various suspension density tests with two application methods involving spraying and soaking were examined. Two application methods of B. amyloliquefaciens B9601-Y2 had 98.60 and 98.15% control effects, respectively. In curing barn, the incidence in the treatment group was significantly reduced and tobacco leaves did not get mildew. Altogether, the study demonstrated that candidate bacterial strain B. amyloliquefaciens B9601-Y2 is a potential antagonist for the management of tobacco leaf mildew during flue-curing.

Introduction: Tobacco root rot caused by Fusarium spp. is a soil-borne vascular disease that severely affects tobacco production worldwide. To date, the community composition and functional shifts of the rhizosphere microbiome in tobacco plants infected with Fusarium root rot remain poorly understood.Methods: In this study, we analyzed the differences in the compositions and functions of the bacterial and fungal communities in the rhizosphere and root endosphere of healthy tobacco plants and tobacco with Fusarium root rot using amplicon sequencing and metagenomic sequencing.Results and discussion: Our results showed that Fusarium root rot disrupted the stability of bacteria–fungi interkingdom networks and reduced the network complexity. Compared to healthy tobacco plants, the Chao1 index of bacterial communities in the rhizosphere soil of diseased plants increased by 4.09% (P < 0.05), while the Shannon and Chao1 indices of fungal communities decreased by 13.87 and 8.17%, respectively (P < 0.05). In the root tissues of diseased plants, the Shannon index of bacterial and fungal communities decreased by 17.71–27.05% (P < 0.05). Additionally, we observed that the rhizosphere microbial community of diseased tobacco plants shifted toward a pathological combination, with a significant increase in the relative abundance of harmful microbes such as Alternaria, Fusarium, and Filobasidium (89.46–921.29%) and a notable decrease in the relative abundance of beneficial microbes such as Lysobacter, Streptomyces, Mortierella, and Penicillium (48.48–81.56%). Metagenomic analysis further revealed that the tobacco rhizosphere microbial communities of diseased plants played a significant role in basic biological metabolism, energy production and conversion, signal transduction, and N metabolism, but their functions involved in C metabolism were significantly weakened. Our findings provide new insights into the changes in and interactions within the rhizosphere and root endosphere microbiomes of tobacco plants under the stress of Fusarium soil-borne fungal pathogens, while laying the foundation for the exploration, development, and utilization of beneficial microbial resources in healthy tobacco plants in the future.