【作者】 魏巍；

【导师】 许艳丽；

【作者基本信息】 中国科学院研究生院(东北地理与农业生态研究所) ， 生态学， 2012， 博士

【摘要】 根腐病作为大豆连作后主要发生的根部病害，是东北黑土区大豆连作障碍的主要原因之一。然而近年发现经过长期连作，大豆根腐病发病情况得到明显的控制，因此提出了长期连作可能形成根腐病抑制性土壤的假设。为了验证这一假设，应用中国科学院海伦农业生态试验站大豆连作定位试验区，以大豆长期连作—大豆根腐病病原微生物—病原抑制性微生物三者关系为研究对象，结合传统分离计数、形态学鉴定、致病性检测、核酸序列分析、实时荧光定量PCR（Real-time PCR）和变性梯度凝胶电泳（DGGE）等方法，研究了大豆连作方式下根腐病原菌种类，以及其在3年连作、20年连作和20年轮作土壤中的种群密度、结构及致病性分化。同时，通过微生物群落的解析明确长期连作土壤中根腐病原菌抑制性微生物种类、抑制能力以及其在3种轮作方式中的种群密度和结构，以期证实上述假设并为更深入、系统地研究大豆连作障碍生物机制提供方法指导。通过苗期大豆生长发育情况的调查结果表明，与大豆3年连作种植相比较，大豆20年连作种植显著地改善了大豆的根长、地上和地下鲜重生长发育情况，仅对株高的影响不显著。同时，大豆根腐病病情指数亦较3年连作显著降低。大豆连作根部位定殖真菌共分离到分属7个属的84个菌株。致病性测定结果显示仅镰孢菌属Fusarium spp.的部分菌株具有大豆根腐病致病能力。结合转录延长因子序列（EF-1α）的系统发育分析和致病性检测结果，确定尖镰孢F. oxysproum、禾谷镰孢F. graminearum和燕麦镰孢F.avenaceum为病原镰孢菌，木贼镰孢F. equiseti、腐皮镰孢F. solani及拟轮枝镰孢F.verticillioides为非致病性镰孢菌。大豆根部镰孢菌属DGGE检测表明尖镰孢菌F.oxysproum为最优势种，因此，大豆连作方式下大豆根腐病原菌以尖镰孢为主，联合禾谷镰孢和燕麦镰孢复合侵染所引起。结合稀释平板法和Real-time PCR方法，检测了大豆20年连作方式下土壤镰孢菌种群的基因组DNA质量和CFU数量，分别为4.5ng/g干土和2.7×10~4个/g干土，均显著低于3年连作的16.0ng/g干土和6.0×10~4个/g干土。传统分离鉴定和DGGE结果显示，3年连作方式土壤镰孢菌属中以尖镰孢菌为优势菌，且其在20年连作方式土壤中的优势度明显下降。由镰孢菌属各个种的分离频率可知，20年连作方式镰孢菌种群结构多样性、均匀度和优势度指数均较3年连作方式有显著的改善。而对DGGE结果的主成分分析和聚类分析结果表明，大豆20年连作方式通过降低尖镰孢菌和禾谷镰孢菌的密度，从而改变土壤镰孢菌属种群结构。研究至此确定了大豆长期连作可以形成根腐病抑制性土壤。结合传统分离计数、Real-time PCR以及DGGE方法研究土壤中的微生物群落结果表明，大豆20年连作方式对土壤细菌群落的密度和结构没有产生明显的影响，仅应用DGGE方法挑选出优势细菌类群，即荧光假单胞菌Pseudomonas fluorescens。但长期连作可以降低土壤真菌群落的CFU数量，增加真菌群落多样性水平，并改变群落结构构成。确定了与20年连作方式相关的特异性真菌类群，即厚垣轮枝菌Verticiliumchlamydosporium和木霉菌属真菌Trichoderma spp.。同时对土壤细菌与真菌群落密度的比较，大豆3年连作方式下CFU和基因组DNA比率分别为88.9（±22.3）和1.5（±0.8），均低于大豆20年连作的111.7（±14.5）和2.2（±2.8）。分离的44株荧光假单胞菌P. fluorescens和8株厚垣轮枝菌V. chlamydosporium中未发现具有抑制性作用的菌株，而在44株木霉菌株Trichoderma中得到具有抑制性的菌株32株。其中24株分离自大豆3年连作土壤，且23株具有病原镰孢菌的抑制能力；18株分离自大豆20年连作土壤，8株具有抑制能力。3年连作土壤中以哈茨木霉菌T.harzianum为主，抑制方式主要为重寄生作用；20年连作方式主要以绿木霉T. viren为主，抑制方式主要为次生代谢产物。盆栽结果显示，3年连作分离的木霉菌具有更高的大豆根腐病发病抑制效果，但20年连作分离的木霉菌在可以抑制根腐病的同时，还具有对大豆生长发育的促生作用。最终确定木霉菌Trichoderma spp.中的哈茨木霉T.harzianum和绿木霉T. virens是大豆长期连作过程中产生的大豆根腐病抑制性微生物之一。综上所述，东北黑土区大豆长期连作方式可以形成根腐病抑制性土壤，减轻大豆根腐病发病程度，抑制病原镰孢菌的生长。而木霉菌属真菌可以作为抑制性微生物控制根腐病病原种群密度，从而达到对根腐病的控制作用。更多还原

【Abstract】 As the primary root disease in the continuous cropping field of soybean, soybean root rotis one of the principal factors that causing the continuous cropping obstacles in NortheastChina. However, the situation of soybean root rot disease is amended significantly after thelong-tern continuous cropping, which means that the disease-suppressive soil has formed bythis management. For sustaining this hypothesis, from the relationship among continuouscropping of soybean, pathogenic microbes and suppressive microbes, the pathogenicmicroorganism and their population abundance, diversity and pathogenic differentiation werestudied using the traditional isolating, counting and morphological identification methodscombined with sequencing analysis, real-time PCR and denaturing gradient gel electrophoresis(DGGE) techniques. Meanwhile, according to the analysis of soil microbial population in long-tern continuous cropping, the suppressive microbes and their population abundance anddiversity in three rotation systems were explored.Compared with3years continuous cropping, the situation of root length, ground freshweight and underground fresh weight were improved significantly by20years measure(P<0.05), but not for plant height (P>0.05). The attack of soybean root rot was also milder than3years treatment. There were84strains isolated from the suffering root of soybean classifiedinto7genuses. However, only some strains from Fusarium genus could cause the soybean rootrot. Using the sequencing analysis of EF-1α and DGGE method, the Fusarium speciescolonizing on the soybean roots were confirmed, including F. avenaceum, F. culmorum, F.equiseti, F. graminearum, F. solani, F. oxysproum, F. verticillioides and a relative specie with F.oxysproum. Through the test of pathogenicity, the pathogenic species were confirmed,including F. avenaceum, F. graminearum, F. oxysproum, and F. equiseti, F. solani and F.verticillioides were the nonpathogenic ones.Combined dilution plate method and real-time PCR, the genomic DNA quality and theCFU of soil Fusarium population in20years continuous cropping treatment were4.5ng pergram of soil and2.7×10~4per gram of soil, which significantly lower than that in3yearstreatment. Meanwhile, the result of identification and DGGE showed that the dominance of F.oxysproum species in20years treatment had decreased from the level of3years treatment.According to the isolation frequency of Fusarium spp., the diversity, evenness and dominanceof Fusarium population in20years continuous cropping had been improved than3years one.From the result of principal component analysis (PCA) and cluster analysis, the Fusariumpopulation in20years treatment had been altered by decreasing the abundance of certain dominant species.Using all above method to analyze the microbial population in long-tern continuouscropping field, it was shown that the abundance and structure of bacterial population was notinfluenced. There were not any specific microbes to select, and just pick the dominant speciesPseudomonas fluorescens. However, the CFU of fungal population was decreased, the diversitywas increased and the structure was altered thoroughly. Using the DGGE method, the specificmicrobes had been selected, which were Verticilium chlamydosporium and Trichoderma spp..Meanwhile, the bacterial to fungal ratio of CFU and genomic DNA quality in3years treatmentwere88.9（±22.3）and1.5（±0.8）respectively, both of which lower than that in20yearstreatment.According to the test of suppression capability, there were not any suppressive strains in44P. fluorescens and8V. chlamydosporium isolates. However, there were32suppressiveisolates in all44Trichoderma strains. Except one T. viride strain,23strains isolated from3years treatment owned capability that inhibited the pathogenic Fusarium, but there were just8strains among all18ones from20years treatment represented suppressive ability. T.harzianum was the dominant species in3years treatment, which act the superparasitism as thesuppressive mechanism. T. virens was the dominant species in20years treatment, which usingthe secondary metabolic production as the suppressive mechanism. Meanwhile, all strains from3years treatment were slightly better than that from20years treatment as a biological controlagent for soybean root rot disease, but all Trichoderma strains from20years treatment alsopresented growth promotion for soybean plants. The linear relation between Trichoderma spp.and Fusarium spp. was established by the result of CFU and genomic DNA quality. T.harzianum and T. virens were the dominant species in3years treatment, but this dominance ofT. harzianum had decreased after20years continuous cropping.更多还原