短密木霉与白术根腐病菌的空间互作及相关蛋白的克隆与功能研究

The famous authentic herbs Atractylides macrocephala is disturbed by theroot rot，which is one of the important factors affecting the yield and quality of A. macrocephala. It has been confirmed that Trichoderma could prevent A. macrocephala root rot. In our previous experiments, a highly effective biocontrol fungi, Trichoderma brevicompactum, was screened and found to have good bacteriostatic and growth-promoting effects and to effectively prevent root decay. However, the antagonistic mechanism and molecular mechanism of interaction in the root system of T. brevicompactum and Fusarium oxysporum is not clear. Accordingly, we propose that in the micro-ecosystem of plant-microbe-soil, T. brevicompactum may alter the rhizosphere microbial flora, form a bacterial membrane at the root surface to prevent pathogen invasion and induce plant-derived antimicrobial activity through Microbe-associated molecular pattern (MAMPs). Based on the above hypothesis, some techniques including qPCR and MiSeq will be used to study the effect of T. brevicompactum on rhizosphere microbial flora and fluorescent labeling technology will be used to make interaction of T. brevicompactum and F. oxysporum visualization. The hydrophobic proteins and the plants interacting protein related with colonization of T. brevicompactum will be screened by Knockout, RNAi and RNA-seq. In this way, the molecular pathways of the plant immunity induced by T. brevicompactum was revealed systematically, and a more effective and theoretical support were established for the sustainable development of A. macrocephala and the exploitation of microbial fertilizer.

道地药材白术根腐病严重，目前主要靠农药和轮作，但收效一般。课题组通过前期研究，从白术根部筛选到短密木霉菌Trichoderma brevicompactum，实验发现其有良好的抑菌及促生作用，但对致病菌拮抗机制、互作模式以及分子机理尚不明确。显然，木霉在诱导白术抗病过程中，定会分泌一些疏水蛋白小分子作为MAMPs效应分子与白术互作。据此我们提出，在植物-微生物-土壤的微生态系统中，短密木霉会改变根际微生物区系，在根表大量定殖以防病菌入侵，并通过微生物相关分子模式诱导植株产生抗病性。因此，本项目拟用qPCR和MiSeq研究木霉对根际微生物区系的影响；同时构建木霉和镰刀菌的GFP/RFP穿梭载体，利用激光共聚焦明确其定殖及互作细节；并用基因敲除、瞬时干扰、RNA-seq等克隆其控制定殖的疏水蛋白及植株互作蛋白，研究其功能，揭示诱导植株免疫发生的分子途径，为白术可持续发展及菌肥开发奠定理论基础。

根际微生物和内生真菌在药材生长及抗病中扮演着重要的角色，从白术根部筛选到的短密木霉菌，实验发现其有良好的抑菌及促生作用，但对致病菌拮抗机制、互作模式以及分子机理尚不明确。我们的研究发现：（1）在盆栽实验中短密木霉的添加在提高白术存活率的同时，也能够提高白术的质量；通过对白术抗病性的检测，发现添加短密木霉的白术的过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、多酚氧化酶(PPO)的含量都有所增加，这说明生防菌的添加通过调节白术抗氧化酶体系来增强白术植株的防御酶活性并促进白术的生长。（2）添加短密木霉的白术根际真菌多样性动态检测结果表明，白术根际青霉菌属、木霉属、产紫蓝状菌属等促生菌的丰度增加，同时镰刀属的含量降低；白术根际细菌多样性动态检测结果表明：短密木霉的添加使生物多样性指数都有所增加，说明根际细菌的多样性增加，其中固氮菌(根瘤菌属等)、益生细菌如芽孢杆菌属、丁酸梭菌属等的丰度增加，具有生物修复功能的地杆菌属和能够合成有机物的红螺菌属的丰度也有所增加。表明短密木霉菌对白术促生作用除了自身作用以外，也通过调节根际菌群的丰度起作用。（3）尖孢镰刀菌的空间侵染结果表明，接种GFP-尖孢镰刀菌后，首先在白术根毛区发现孢子，然后出现在主根木质部，随后菌丝在木质部逐步定殖，表明致病菌在白术是从根毛侵入木质部定殖从而侵染植株全身，这可能对我们探索短密木霉与尖孢镰刀菌的空间互作模式提供了一种方向。（4）短密木霉菌疏水蛋白基因的克隆及原核表达获得了专属疏水蛋白，通过添加外源性疏水蛋白发现白术植株H2O2含量和超氧阴离子含量均减小；而白术植株的过氧化氢酶、超氧化物歧化酶、过氧化物酶的含量都有所增加。这表明，疏水蛋白的添加会增强白术抗病活性，极大程度地降低了植物氧损伤，短密木霉疏水蛋白有着对白术植株抗病害和促生的作用，值得进一步开发利用。