Antifungal Activity Substance of Endogeny Bacillus in Maize(Zea mays) and Mechanisms against Setosphaeria turcica | 玉米内生芽胞杆菌的抗菌活性物质及其拮抗玉米大斑病菌机理的初步研究

Chinese. 为了研究生防菌株对玉米大斑病菌的抑菌作用，深化对生防菌抗菌机制的认识，本研究从玉米(Zea mays)植株体内分离拮抗玉米大斑病菌(Setosphaeria turcica)的内生细菌，对其抗菌物质及其抑菌机理进行初步研究。结果表明，所分离的内生菌株YY1经形态学观察、生理生化测定及16S rDNA序列分析，鉴定为枯草芽胞杆菌(Bacillus subtilis)。菌株YY1发酵液的硫酸铵沉淀物具有抑菌活性，且在硫酸铵50%饱和度时抑菌活性最强，说明YY1菌株产生的抗菌活性物质可能是蛋白类物质。该菌株及其蛋白粗提液均对禾谷镰刀菌(Fusarium graminearum)、苹果轮纹病菌(Botryosphaeria dothidea)、灰霉病菌(Botrytis cinerea)、玉米弯孢霉叶斑病菌(Curvularia lunata)等7种植物病原真菌有较强的拮抗作用。用蛋白粗提液处理菌丝、分生孢子、原生质体后经显微观察发现，大斑病菌的基内菌丝由丝状畸变为串珠状，当蛋白粗提液浓度为0.78 μg/μL时，可完全抑制分生孢子萌发，并导致原生质体裂解。通过抑制孢子萌发过程中信号途径相关基因的半定量RT-PCR分析和玉米大斑病菌不同信号途径相关基因突变体的抑制率统计，初步判定该抑菌过程主要通过cAMP信号转导途径发挥作用。本研究为寻找玉米大斑病菌新的防治方法和途径提供基础资料。

English. 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.