Pichia anomala 0732-1代谢产物乙酸在生防哈密瓜细菌性果斑病中的作用机制研究

Bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli is a new devastating disease of hami melon in Xinjiang. It has been rank as an importance internal and external quarantine plant disease in china. Because the pathogen can spread rapidly in a field, often causes fruit rot, induce hami melon production reduction and there has no effective management, the disease is responsible for high depreciation and significant economic losses every year. The disease has been a significant threat to china and global cucurbit production. Effective management for control BFB will require to research. Based on the results of some prophase research works, this project will adopt the classical plant pathology research methods and combine with the theories and technologies of biochemistry or molecular biology. The objectives of this work are: (1) to systematic study some functions of acetate acid produced by Pichia anomala 0732-1 in the interaction among yeast, bacteria and host. Effects of acetate acid produced by P. anomala 0732-1 on physiological-biochemical characteristics, the acyl-homoserine lactone (AHL)-type quorum sensing system and biofilms formation in Acidovorax avenae subsp. citrulli are emphases investigation in this work; (2) to illuminate the antibiosis mechanism of action of acetate acid produced by P. anomala 0732-1 on Aac; (3)to establish optimization of medial components and cultural conditions for producing acetate acid by P.anomala 0732-1; (4) to verify whether or not the mechanism of inducing resistance in melon by acetate acid. The results of project research will fully reveal the mechanism of P. anomala 0732-1 against Aac and provide a safe and effective biological control approach for BFB. At the same time, it has important theoretical and practical significance for promoting the famous local hami melon production.

哈密瓜细菌性果斑病是哈密瓜生产中一种毁灭性病害。是我国对内对外检疫性重大植物病害。其具有发病快、防治难、为害重的特点，常造成果实腐烂，导致减产，给瓜农造成很大经济损失，成为瓜生产中巨大的威胁。有效防治该病倍受人们关注。本项目在已有研究的基础上，综合运用经典植物病理学研究技术，结合生物化学和分子生物学等仪器分析的理论和技术，系统研究Pichia anomala 0732-1代谢产物乙酸在酵母菌、哈密瓜细菌性果斑病菌(Aac)和寄主间互作关系中的作用，重点研究P. anomala 0732-1代谢产物乙酸对Aac生理生化特性、群体感应、生物膜形成的影响；阐明乙酸对Aac抗生作用机制；明确P. anomala 0732-1产乙酸最佳发酵条件；进一步验证乙酸是否具有诱导哈密瓜植株抗性作用。以期全面揭示P. anomala 0732-1的抑细菌机理，为安全有效防治哈密瓜果斑病提供一条生物防治途径。

通过本项目的研究，明确了P .anomala 0732-1代谢产物乙酸对Acidovorax avenae subsp. Citrulli（Aac）具有显著抗生作用，随着乙酸浓度的增加（pH降低），Aac菌体电导率值升高，胞内ATPase活性降低，且胞内外pH值梯度增加，细胞正常功能受阻，Aac菌体畸形，细胞壁残缺。pH值小于3时，Aac失去泳动能力且无法生长。对构建的Aac成膜体系中加入乙酸，乙酸对Aac成膜初期及成熟期均有抑制作用，pH值越低，导致形成的生物膜结构疏松，菌体形态严重受损、变形，甚至无法形成生物膜。当pH值为5、4.5时，Aac显著受到抑制作用，对数生长期明显滞后于CK。当pH为3.7时，Aac生长浓度随着培养时间的延长而增加，β-乳糖苷酶的活性与Aac生长浓度呈正相关。当pH值小于5时，胞外多糖的产生呈下降趋势。由此表明乙酸对Aac群体感应信号分子的产生具有一定的影响，进而进一步阐明P .anomala 0732-1代谢产物乙酸或由乙酸营造的酸性环境是直接导致Aac无法存活的直接原因，也是P. anomala 0732-1防治哈密瓜细菌性果斑病的重要生防机制。对于酵母菌产酸体系优化，获得最优发酵条件为培养液初始pH 9.4、培养温度25 ℃、装样量61 mL（150 mL三角瓶），较优化前提高了32.6%。另外研究表明，温度为28℃，湿度大于65%易于定殖，且酵母种群数量随着接种时间的延长而增加，并趋于平稳。不同浓度酵母菌悬液接种哈密瓜叶片，均能造成叶表pH值的下降。利用乙酸喷布盆栽哈密瓜幼苗叶片，叶片POD、SOD、PAL和CAT酶活性均有不同程度的升高，证明了乙酸对寄主具诱导抗性的作用。利用乙酸溶液处理后的哈密瓜带菌种子，幼苗的发病率明显降低。当乙酸溶液pH为2时，发病率为0。表明乙酸对哈密瓜带菌种子有明显防效作用。在田间防效测定中，利用pH为3的乙酸溶液防治哈密瓜细菌性果斑病的防治效果可达35.76%。乙酸在果蔬保鲜（真菌病害）方面研究较多，但在细菌病害方面国内外鲜有报多，综上研究，为揭示酵母菌防治细菌性病害的本质，拓宽了酵母菌防治植物病害的范围提供理论依据。