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作 者 ：任争光，林敏，姜文君，倪兴雅，梅桂英，韩升才，张力群

期 刊 ：植物病理学报 2012年 42卷 6期 页码：608-619

关键词：群体感应；西瓜食酸菌；致病力；甜瓜；

Keywords:quorum sensing, Acidovorax citrulli, virulence, melon,

摘 要 ：以N-乙酰高丝氨酸内酯（N-acyl-homoserine lactone, AHL）为信号分子的群体感应（quorum-sensing, QS）系统是很多病原细菌的重要致病性调控因子。本文自甜瓜果斑病菌——西瓜食酸菌（Acidovorax citrulli）菌株MH21中克隆到AHL信号合成基因luxI_MH21，并构建了其缺失突变体MΔluxI_MH21及转化有AHL信号降解酶编码基因aiiA和aidH的工程菌株MAiiA和MAidH。信号检测结果显示MΔluxI_MH21、MAiiA和MAidH菌株均无AHL信号产生，同时细菌的游动能力及在基本培养基中的生长速率均显著下降，但对细菌生物膜形成和在非寄主植物烟草上诱导过敏性坏死反应的能力没有影响。盆栽条件下，经低浓度（10⁴ CFU/mL）MΔluxI_MH21、MAiiA和MAidH菌株处理的甜瓜种子萌发后幼苗死亡率显著低于野生型MH21和luxI_MH21基因互补菌株MΔluxI_MH21HB的处理；而高浓度细菌（10⁸ CFU/mL）处理种子后，除MAidH菌株处理引起的死苗率明显低于野生型MH21处理，其他菌株与MH21没有显著差异。子叶注射试验也得到相似的结果，以低浓度细菌（10⁴ CFU/mL）注射甜瓜子叶后发现MΔluxI_MH21、 MAiiA和MAidH菌株甜瓜子叶中的繁殖速率及对子叶的致病力与野生型MH21相比均显著下降；而高浓度细菌（10⁸ CFU/mL）处理子叶时，MΔluxI_MH21和MAiiA菌株与野生菌MH21相比致病力无显著差异，仅有MAidH菌株的致病力明显下降。说明QS系统影响菌株MH21在低细菌浓度下对甜瓜幼苗的致病力，这种作用可能与影响细菌生长有关。

Abstract:The N-acyl-homoserine lactone(AHL)-depended quorum sensing(QS) is an important regulatory system for virulence expression in various bacterial pathogens. Acidovorax citrulli causing fruit blotch on melon contains the typical LuxI/LuxR-type QS system. In order to explore the effect of the QS on the pathogenicity of A. citrulli strain MH21, the luxI_MH21 gene encoding the AHL biosynthetic protein was cloned and the luxI_MH21 gene in-frame deletion mutant MΔluxI_MH21 was constructed. Meanwhile, AHL lactonase genes aiiA and aidH were introduced into wild-type MH21 and resulted in engineered strains MAiiA and MAidH, respectively. Compared to the wild type MH21, mutants MΔluxI_MH21, MAiiA and MAidH failed to produce AHL signals, and exhibited impaired swimming motility and reduced growth in minimal medium. However, the biofilm formation and the hypersensitive response-inducing capacity of non-host tobacco were not affected. In the pot experiments, seed treatments with the mutants MΔluxI_MH21, MAiiA and MAidH in a low concentration(10⁴ CFU/mL) caused reduced seedling mortalities compared to the treatments with wild-type MH21 and the complemented mutant MΔluxI_MH21HB, but this virulence defect was not observed when treated with high-concentration(10⁸ CFU/mL) mutants MΔluxI_MH21 and MAiiA. Similar results were found in melon cotyledons, where the mutants MΔluxI_MH21, MAiiA and MAidH were less virulent and multiplied poorly when infiltrated in low-concentration(10⁴ CFU/mL). But under high bacterial concentrations(10⁸ CFU/mL), both the mutants MΔluxI_MH21 and MAiiA produced severe necrotic lesions on melon cotyledons similar to that caused by the wild-type MH21. It should be noted that, unlike MΔluxI_MH21 and MAiiA, the engineered strain MAidH containing the aidH gene exhibited reduced virulence on the melon seedlings when applied in a high concentration. Our results suggested that the QS system was required for the full virulence of A. citrulli in low bacterial concentrations, and this effect probably mediated through its impact on bacterial growth.

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