草酸在生防菌盾壳霉与核盘菌互作中的作用及其机制解析

Oxalic acid is a host-non-specific phytotoxin produced by Sclerotinia sclerotiorum. Numerous studies have been conducted to determine the phytotoxicity and the mechanisms of oxalic acid. Results showed that oxalic acid acts both as a phytotoxin and as a signal molecule in pathoigenesis of S. sclerotiorum. Coniothyrium minitans is a mycoparasite and an important biocontrol agent of S.sclerotiorum. Our previous studies showed that oxalic acid can inhibit both conidial germination and mycelial growth of C.minitans and reduce mycoparasitism of C.minitans on S.sclerotiorum. On the other hand, C.minitans can produce antifungal substances under acidic ambient pH created by oxalic acid from S. sclerotiorum and finally C.minitans can degrade oxalic acid. These findings indicate that oxalic acid might play an important role in the interaction between C.minitans and S.sclerotiorum. Under the circumstances, we proposed to study the effect of the antifungal substances produced by C.minitans on biosynthesis of oxalic acid in S.sclerotiorum, to clone oxalate decarboxylase genes in C.minitans, to analyze the function and the expression pattern of the oxalate decarboxylase gene in C.minitans, and to detect oxalic acid induction of programmed cell death (PCD) in Cm and the anti-PCD mechanisms in C.minitans.The results from the project will ascertain the role of the oxalate decarboxylase genes responsible for degradation of oxalic acid in C.minitans, will boraden our underatnding of the induction effect of oxalic acid on generation of programmed cell death in C.minitans and of the effect of anti-PCD on mycoparastism of C.minitans against S.sclerotiorum, and will lay a solid foundation for control of S.sclerotiorum using the oxalate decarboxylase genes and for genetic modification of C.minitans.

草酸（OA）是核盘菌（Ss）产生的一种寄主非专化毒素。前人对OA的生物活性及机制进行了许多研究。结果表明OA在Ss致病过程中扮演着毒素和信号分子角色。盾壳霉（Cm）是Ss的一种重寄生菌和生防菌。我们前期研究结果表明：OA可抑制Cm分生孢子萌发及菌丝生长，并导致其重寄生效率下降。与此同时，Cm可巧妙地利用OA营造的酸性环境产生抗真菌物质（AFS），并最终降解OA。这些结果为揭示Cm/Ss互作机制提供了重要线索。鉴此，本项目提出研究Cm产生AFS对Ss合成OA的抑制效果，克隆Cm草酸脱羧酶基因（cmoxdc基因）；研究cmoxdc基因功能、表达模式及生防作用；研究OA对Cm细胞程序化死亡（PCD）的诱导作用、Cm抗PCD的分子机制及生防作用。项目的完成将明确cmoxdc基因功能，明确OA诱导Cm发生PCD的效果及Cm抗PCD的生防意义。为利用cmoxdc基因控制油菜菌核病及改良Cm奠定基础。

本项目旨在从遗传角度证明盾壳霉降解草酸毒素的生防作用，并揭示盾壳霉感受pH环境的分子机制。从盾壳霉基因组中克隆得到两个草酸脱羧酶基因，分别命名为Cmoxdc1和Cmoxdc2。Cmoxdc1表达受酸性pH环境和草酸诱导，而Cmoxdc2表达则不受受酸性pH环境和草酸诱导。对这两个基因进行了敲除，对其中敲除突变体进行了互补。通过比较野生型和敲除突变体降解草酸能力、重寄生能力和抗真菌活性，证明盾壳霉降解草酸对其寄生核盘菌菌丝至关重要。在感受pH环境分子机制方面，从盾壳霉基因组中克隆到CmpacC基因及6个pal基因。对其分别进行了敲除和互补。结果表明：CmpacC正向调控盾壳霉重寄生作用，负向调控盾壳霉降解草酸和抗真菌活性。上述结果为揭示盾壳霉与核盘菌之间互作的分子机理提供了新的分子证据。对于改良盾壳霉野生菌株，提供其生防效果奠定了理论基础。在《Environmental Microbiology》上发表研究论文2篇。