线虫抑制性土壤中植物内生真菌的多样性研究

The pathogenic nematode causes a serious damage to agriculture in our country and it will gradually get great attention to find the safe and stable biological control measure. Some facultative endophytic fungi of plants in nematode suppressive soil can inhabit nematodes and might be one of the key functional population playing a role in suppressive soil. Due to its kinds of advantages, it will be a unique and economically effective way to develop the facultative endophytic fungi into nematode biological control. On the basis of systemically investigating three culture plots more infected and identifying biology suppressive soil, the project will focus on the nematodes affecting the different kinds of vegetables worse and lay a foundation for screening biological control fungi with high efficient inhabitation to nematodes and establishing a stable symbiotic relationship with hosts as well as possessing broad spectrum genotype by the way of isolating and identifing endophytic fungi, evaluating the activity of inhabiting nematodes of superior species , analyzing the genetic diversity of superior species associating with three ITS、TUB、RPB fragments and exploring the colonization ability of dominant fungi within roots by RT-PCR method. Based on the endophytic fungi, the project will finally explore the ecological mechanism of nematode suppressive soil through analyzing the fungi in roots and eggs by the oligonucleotide fingerprinting of rRNA genes (OFRG) and evaluating the contribution of the endophytic fungi in suppressive soil according to characteristics of the dominant endophytic fungi cultivated easily.

作物病原线虫在我国危害严重，寻找安全稳定的生物防治措施渐成为关注的热点。线虫抑制性土壤中植物部分兼性内生真菌对线虫有抑制活性，可能是抑制性土壤发挥作用的功能群之一，因其种种优势，将其开发成线虫生防制剂是一条独特和经济有效的途径。本项目瞄准对各种蔬菜影响影响较为严重的根结线虫，系统调查三种易感植物栽培区，鉴定生物抑制性土壤的基础上，分离鉴定可培养内生真菌，评估优势种的抑制线虫活性，联合ITS、TUB、RPB三个片段对优势种进行遗传多样性分析，用RT-PCR对其根内定殖能力进行研究，为筛选到既能高效抑制线虫又能与宿主建立稳定共生关系和具广谱基因型的生防菌株奠定基础。通过rRNA基因的寡核苷酸指纹图谱法分析根内和卵上的真菌，结合可培养优势内生真菌各方面特性评估内生真菌在抑制性土壤中的贡献，从内生真菌的角度探索线虫抑制性土壤发挥作用的生态机制。

线虫抑制性土壤中植物部分兼性内生真菌的杀线虫活性，为线虫生防制剂开发提供了一条独特和经济有效的途径。本项目以抑制性土壤及其栽培区内易感作物内生真菌和根际真菌为研究对象，评估了其抑制线虫能力，并对抑制性土壤抑制线虫机制进行了探索。研究结果表明抑制性土壤对根结线虫的抑制作用与土壤根结线虫初始虫口密度之间存在密切的负相关性。抑制性土壤中优势真菌属主要有：Fusarium，Trichoderma，Aspergillus，Chaetomium，Mucor，Absidia，Paecilomyces。对其中的300个菌株杀线虫活性分析，发现大部分菌株具有杀线虫活性，其中Aspergillus niger H1-1, F25, E34, D16等14株真菌的培养物24小时能杀死60%的根结线虫。我们推测这些菌株是抑制性土壤能够抑制线虫的关键因子。在非抑制性土壤中，这些真菌出现的频率较低或出现后不具备杀线虫活性。对杀线虫活性较强的三个代表菌株Aspergillus niger F25、H1-1、D16进行了发酵条件的优化并明确了对应的最佳发酵工艺，在最优条件下，F25、D16和H1-1发酵液8小时对靶标线虫的致死作用分别达到100%、99.2%和99.7%。分析了这三株菌株的代谢活性产物：D16菌株的活性产物为草酸钙配位化合物，初步确定了另外两株菌株代谢产物的基本化学性质。最后在剂型研究的基础上，通过温室和田间实验评价了两菌株对根结线虫的防治效果及在根部的定殖能力。最终筛选到两株既能高效抑制线虫又能与宿主建立稳定共生关系的生防菌株，同期防治效果高于噻唑膦和阿魏菌素，有很好的应用前景。