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Abstract In order to study the fungistasis and antifungal mechanisms of biocontrol strain against Setosphaeria turcica, one endophytic bacterium strain YY1 was isolated from leaves of healthy maize(Zea mays) plants and was identified as Bacillus subtilis based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis in this study. Meanwhile, antifungal substances from the fermentation liquor of strain YY1 were deposited by the method of ammonium sulfate and exhibited a high antifungal activity as precipitated by 50% ammonium sulphate. These results implied that antifungal substances produced by strain YY1 may be proteinous material. It was shown on PDA plates that many plant pathogens such as Fusarium graminearum, Botryosphaeria dothidea, Botrytis cinerea and Curvularia lunata were inhibited effectively by both the fermentation liquor and the crude protein extracts. After treated by crude protein extracts, the substrate hyphal morphology had an aberration from filiform to bead-like, however the inhibited aerial hyphae were the same with that of the control. The conidial germination would start to be inhibited at the concentration of 0.35 μg/μL and could be inhibited completely at the concentration of 0.78 μg/μL, the IC50 was 0.46 μg/μL, but the crude protein extracts could not split the conidia, even the concentration was up to 17.36 μg/μL. Through the crude protein extracts incubation, the protoplast membrane was broken and the intracellular materials were dying out gradually at the concentration of 0.78 μg/μL. It was suggested that the crude protein extracts may change the structure or permeability of the plasma membrane. The semiquantitative RT-PCR results indicated that in the inhibited conidia of S. turcica, the expression of G protein γ subunit-encoding gene (Stgg-1) was inhibited completely, while other genes encoding three different subtypes of G protein α subunit (Stga-1, Stga-2 and Stga-3) and G protein β subunit (Stgb-1) were not expressed in both CK group and treatment group. The expression of protein kinase A regulated subunit gene(StPKA-r) and protein kinase A catalytic subunit gene(StPKA-c), which encoded the catalytic subunit and regulatory subunit of cAMP-dependent protein kinase A, decreased significantly, but adenylate cyclase encoding-gene (StAC) had slightly increased transcription level. The transcription level of mitogen-activated protein kinase kinase kinase gene(Stk1k) involved in mitogen-activated protein kinase (MAPK) signaling pathway and protein kinase C gene(StPKC) in Ca2+ signaling pathway displayed no difference during the process of treated or untreated conidial germination. The inhibitory ratios of crude protein extracts on RNA interference transformants of Stgg-1, StPKA-c and StPKA-r knockout mutant were decreased markedly, while the hyphal growth of Stk1k-silenced transformant had no distinction compared with that of the wild-type strain during face-to-face-culturing with those extracts on PDA media. So it was inferred that the molecular mechanisms of the crude protein extracts against S. turcica may mainly be mediated by cyclic adenosine monophosphate (cAMP) signaling pathway. This work will lay a foundation for finding new control techniques against Setosphaeria turcica
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Received: 27 February 2012
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