有效轮作体系构建香蕉健康根际土壤微生物区系机制研究

Continuous cropping biological obstacle of banana is reported to be one of the major limiting factors for banana production in China. The main cause of the wilt is the unbalanced microflora in the continuous cultivation system. Previous studies have shown that effective crop rotation could prevent Fusarium wilt occurrence. However, the mechanism of effective rotation crops decreasing the number of Fusarium oxysporum by interaction of rhizosphere and pathogen is still unknown. What kind of specific microbial groups and their number in the soil influced by effective rotation system to prevent Fusarium wilt occurrence are also still unknown. In this project, 454 sequencing technique will be used to analyze the specific microbial groups, Real-Time PCR technique will be used to detect the number of microbes, and in vitro dual-culture on plates technology will be used to analyze the number and diversity of functional microbes which could antagonize the pathogen, then influence of effective rotation crop systems on the number and diversity of rhizosphere soil microbes will be analized. Combined with influence of root exudates and plant residues on the growth and spore germination of the pathogen and influence of root exudates on the the number and diversity of functional microbes, we are trying to reveal the mechanism of building healthy microbial community in the banana rhizosphere soil to prevent the continuous cropping obstacle by effective rotation systems. This project will provide an important theoretical basis for controlling continuous cropping obstacle of banana.

香蕉连作生物障碍已经成为我国香蕉产业可持续发展的限制因子，连作后土壤微生物区系失衡是引起这种连作生物障碍的主要原因。前期研究表明，通过部分作物轮作可以恢复连作土壤微生物区系，克服连作生物障碍，然而不同轮作作物如何通过根际与病原菌互作降低根际病原菌的数量任未探明，影响了土壤中哪些具体微生物类群的存在且保证一定的数量，从而构建出健康的微生物区系，避免下茬香蕉植株发病迄今未知。本项目拟利用454测序技术分析土壤微生物具体类群，Real-Time PCR技术测定微生物数量，平板对峙技术分析具拮抗能力微生物的数量和多样性，分析有效轮作体系对下茬香蕉根际土壤微生物数量和多样性的影响，结合不同作物根系分泌物与残体对尖孢镰刀菌生长及孢子萌发的影响与分泌物对功能微生物数量和多样性的影响，阐明有效轮作体系构建连作香蕉健康根际土壤微生物区系，防控香蕉连作生物障碍的机制，最终为香蕉连作生物障碍的防控提供理论依据。

香蕉连作生物障碍是影响香蕉产业可持续发展最的瓶颈，科学的轮作是缓解香蕉连作生物障碍的有效措施，本研究在田间调研的基础上，选择菠萝、玉米、辣椒、茄子、番茄和香蕉轮作，研究轮作对土壤（根际和土体）理化性质、土壤微生物群落结构的影响，同时，收集轮作作物根系分泌物、残体，研究根系分泌物、残体对香蕉土传枯萎病致病菌病原菌孢子萌发和菌丝生长的影响。研究结果表明，菠萝、辣椒、茄子根系分泌物及残体抑制香蕉土传枯萎病病菌孢子的萌发和菌丝的生长的能力比玉米和番茄强，而田间土壤微生物群落的分析表明，利用热图（Heatmap）和维恩图（Venn）对菠萝、玉米轮作两个处理中相对丰度较高的OTU进行分析显示，两者之间真菌微生物群落结构差异较大，PCOA的分析结果显示，轮作与连作处理微生物群落结构区分为两个不同的方向；菠萝、辣椒、茄子轮作后均能显著降低土壤中尖孢镰刀菌菌的丰度；将轮作与施用生物有机肥两个措施联合应用在田间实验中，轮作结束后再植香蕉的结果显示，香蕉土传枯萎病的发病率从高到低依次为香蕉连作处理平均为70%、轮作后施普通有机肥处理菠萝为33%、辣椒为36.7%、茄子为32%，轮作后施生物有机肥处理菠萝为12%，辣椒轮为25.0%、茄子为19.5%，采用MiSeq高通量测序技术各处理中土壤微生物群落结构进行分析：香蕉连作处理中病原菌的相对丰度显著高于轮作（菠萝、茄子）+普通有机肥和轮作+生物有机肥处理（菠萝、茄子），虽然在香蕉根际中辣椒轮作处理+生物有机肥的镰刀菌属的丰度与连作香蕉之间差异不显著，但是经TA克隆分析显示，致病菌尖孢刀菌占整个尖孢镰刀菌属的比例最低；采用随机矩阵理论（Network）构建土壤微生物群落生态网络分析不同处理中高丰度OTU的网络构成、模块性和稳定性，结果显示，轮作联合生物有机肥处理的土壤微生物网络模块性更好，更为稳定。利用方差分解分析（VPA）来计算轮作方式、施肥方式和土壤理化三个因素对土壤细菌和真菌微生物群落构建的贡献率，结果显示，轮作方式对细菌和真菌群落构建的解释量是最高的，分别为4.19%和5.12%，其次为施肥种类。