液氨熏蒸下黄瓜连作土壤微生物区系特征及其定向调控

In this project, the scientific issues were proposed based on the phenomenon of the pre-application under field condition. On the strength of the environment-friendly soil fumigation methods, “ammonia fumigation”, we employ the MiSeq sequencing, real-time PCR and other detection technology combined with modern meta-analysis to analyze the impact of liquid ammonia fumigation on soil microbial structure and function and the effect of ammonia fumigation on specific features of soil microbial population. After the soil fumigation by ammonia, we will manipulate the soil microflora by organic fertilizer or bio-organic fertilizers, which contain a large number of beneficial microbes. Adopting modern determination and analysis methods, we will detect and analyze the effect of bio-organic fertilizer on interfering or recovering the function of soil microbial communities after soil fumigation, evaluate the effect of "ammonia fumigation" + "bio-organic fertilizer" processing mode on crop health and growth-promotion, and decipher the factors on impacting soil microflora manipulation. Through implementation of the project, we will reveal how related functional microbes respond to ammonia fumigation and how the bio-organic fertilizer manipulate soil microbial after the fumigation, clarify the microbial and ecological mechanism of this new concept with "environmentally friendly fumigation" + "microbial control" to control of cucumber wilt, and provide a theoretical basis for further strengthening biocontrol efficiency by regulating rhizosphere microorganisms.

本项目以前期的田间应用效果为基础提出科学问题，从液氨熏蒸这一环境友好型土壤消毒处理方法入手，利用MiSeq测序、real-time PCR等检测技术辅以现代宏分析方法，分析液氨熏蒸对土壤微生物结构和功能的影响，液氨熏蒸对土壤特定功能微生物数量的影响；同时以期利用含有大量有益微生物的有机肥或生物有机肥配施来定向培育熏蒸土壤的微生物区系，并利用现代检测和分析手段分析生物有机肥对土壤熏蒸干扰后土壤微生物群落与功能的培育效果，评价“液氨熏蒸”+“生物有机肥”处理土壤模式对作物的生长效应及其影响因素。以期通过项目实施，重点揭示相关功能微生物或优势菌对土壤液氨熏蒸的响应规律，以及熏蒸后生物有机肥对土壤微生物的构建机制，以此阐明用“环境友好型熏蒸”+“微生物调控”这种新理念来防控黄瓜枯萎病的微生物生态学机制，为防效的进一步强化和根际微生物调控提供理论依据。

黄瓜枯萎病是由尖孢镰刀菌黄瓜专化型（FOC, Fusarium oxysporum f. sp. Cucumerinum）侵染引起的土传病害，如何有效防控是一大难题。本项目通过田间和盆栽模拟试验结合土壤理化性状分析、土壤相关酶活性测定和MiSeq高通量测序技术，获得了液氨熏蒸配施生物有机肥防控黄瓜土传枯萎病的效果，探索了对土壤微生物结构与功能的影响，以及对健康高产土壤微生物区系构建的生态学机制。.获得的研究结果：（1）液氨熏蒸配施生物有机肥对黄瓜枯萎病发病率的防控效果达89.1-89.8%；黄瓜产量比对照增加64.5 - 155.39%。同时，我们还发现，液氨熏蒸配施生物有机肥对芹菜和黄瓜的根结线虫发病率防效达100%，芹菜的产量比对照增产31.8%；（2）熏蒸后土壤pH值、全氮量、铵态氮含量和硝态氮含量增加，表明熏蒸措施具有一定改善土壤酸化和提高部分土壤速效养分的作用。（3）通过对病原菌尖孢镰刀菌平板计数结果发现，液氨熏蒸处理很难分离到病原菌，并且至少能维持3个月不见病原菌增长，显著优于对照处理，表明液氨熏蒸能在作物生长期内较好的控制病原菌的数量。（4）通过高通量测序对土壤微生物群落分析发现，液氨熏蒸配施生物有机肥显著降低土壤中病原菌的相对丰度，提高某些土壤有益真菌的相对丰度（如：Penicillium），表明熏蒸联合生物有机肥不仅能控制病原菌的相对丰度，而且能改善土壤有益微生物，从而起到定向调控土壤微生物群落的作用。（5）通过对土壤相关酶活性测定发现，液氨熏蒸配施生物有机肥显著提高土壤硝酸还原酶、土壤过氧化氢酶等的活性，表明熏蒸联合生物有机肥还能促进土壤功能发挥，并有利于改善植株生长状况而增强防病效果。.综上所述，液氨熏蒸配施生物有机肥显著抑制芹菜、黄瓜枯萎病和根结线虫病的发生，并显著增加芹菜和黄瓜的产量，改善土壤理化性状与土壤微生物群落结构，降低土壤病原菌的相对丰度，从而达到防控黄瓜土传枯萎病的目标，有利于健康高产抑病土壤的培育。