【作者】 王进闯；

【导师】 王敬国；

【作者基本信息】 中国农业大学 ， 植物营养学， 2014， 博士

【摘要】 为了加深理解大豆连作的生物学障碍机理,本文采用高通量测序和末端限制性片段多态性分析(T-RFLP)、聚丙烯酰胺梯度凝胶电泳(DGGE)和定量(qPCR)方法,研究了连作13年的大豆根际细菌、真菌和线虫,以及镰刀菌、大豆疫霉菌的群落结构和丰度的动态。同时,通过与环境因子进行相关性分析,阐明驱动根际生物类群动态变化的关键因子。另外,通过建立微宇宙系统,利用DGGE和qPCR的方法,研究了大豆异黄酮对镰刀菌和大豆疫霉菌的影响,探讨了大豆异黄酮对主要病原菌的生态功能。结果如下：(1)连作1-3年根际真菌群落的丰度显著高于连作6-13年,而真菌的香农指数和均匀度在6-13年显著高于1-3年。在连作2-3年内,疾病真菌(Boeremia和Clavariopsis)的相对丰度更高,而有益菌(Bionectria, Melanospora和Hypocrea)的相对丰度较低。另外,根的分泌物如类黄酮、染料木因和pH是调节真菌群落的重要因子。细菌丰度从1-3年迅速减少,之后保持相对稳定。细菌群落组分受可利用性磷(AP)、硝态氮(N03-)和pH的影响。这些结果说明从起初的健康土壤到致病土壤的转化可能主要是由于致病真菌的相对丰度较高,而且真菌群落与细菌比例增加有关。之后,从致病土壤到抑病土壤的转化和土壤抑制特性的可持续性可能与土壤中有益菌的比例提高有关。根系分泌物和土壤性质是驱动微生物群落变化的因子。(2)在1到13年,镰刀菌的群落结构和丰度都没有清楚的时间趋势。土壤的异黄酮物质和镰刀菌群落的变化具有明显相关性。另外,大豆疫霉菌的丰度从第1到第3年增加,之后开始减少。大豆疫霉菌与类黄酮的浓度呈负相关。物种少的镰刀菌群落是物种丰富的镰刀菌群落的子集,说明在种库中连续的演替是巢氏和物种的丧失。镰刀菌群落可能由随机和环境过程影响,镰刀菌群落可能不是导致大豆产量降低的主要因子,根际中大豆疫霉菌的密度或许更重要。(3)线虫群落结构在连作期间发生了明显变化。线虫的物种丰富度随着连作年限的增加呈逐渐降低的趋势。第1年物种丰富度最高,第3年的丰富度显著低于第1年,之后逐渐降低。大豆根际土中共检测到16个线虫群落T-RF(末端限制性片段),且大多数T-RF能从克隆文库中鉴定。在大豆根际土中,食细菌线虫(Acrobeloides)是最丰富的有机体。在连作2-3年,植物寄生线虫相对丰度增加,而在连作后期,植物寄生线虫相对丰度减少。NMDS分析显示,第1年线虫群落与其余年限分开,而第2和第3年聚集较近,后面9、11和13年聚集较近。另外,线虫群落结构与pH、土壤有机质(SOM)、速效磷(AP)、细菌数量和真菌数量相关。线虫丰度呈先增后降的趋势,最高值出现在第6年。线虫的基因拷贝数与土壤NH4+和染料木因(genisten)浓度呈显著正相关,而与N03-和细菌的基因拷贝数呈显著负相关。说明多个因素驱动着连作过程中线虫群落变化,不仅受到土壤性质的影响,而且受到微生物群落影响。线虫群落的变化,特别是植食性线虫的变化,可能是导致连作障碍的因素之一。(4)外源添加黄酮物质改变了镰刀菌群落结构。培养前期(第3天),黄酮溶液对镰刀菌丰度影响最大,随着培养时间的延长,黄酮溶液对镰刀菌丰度的影响较小。可是,在培养第3天,黄酮溶液没有改变大豆疫霉菌的丰度,当培养7天后,高浓度的大豆苷元和染料木因以及混合液对大豆疫霉菌产生了显著的抑制作用。培养14天,只有最高浓度的混合液促进了大豆疫霉菌的数量。大豆苷元和染料木因对镰刀菌和大豆疫霉菌能产生促进或抑制作用,主要取决于培养时间和黄酮物质的浓度。通过上述研究得出以下结论(1)长期大豆连作使土壤细菌、真菌、线虫和镰刀菌群落的物种丰富度降低；(2)连作使土壤生物群落的基因拷贝数减少。更多还原

【Abstract】 Continuous crop monoculture results in a transformation from a conducive to suppressive soil, but we lack a comprehensive understanding of how soil microbiota develop in the process over time. The aim of this study was to investigate whether the dynamic processes in the composition and abundance of rhizosphere bacterial, fungal, nematode, as well as fusarium communities and Phytophthora sojae in the long-term soybean monoculture (up to13years) are linked to the transformation and which the main factor drive shifts in these soil microbial taxa. To determine abundance of bacterial, fungal, nematode, fusarium communities and P. sojae, we used quantitative PCR (qPCR), and to study the structure and diversity of bacterial, fungal and fusarium communities, we used a454-pyrosequencing technique. Furthermore, we used T-RFLP to test the compositions and abundances of nematode community in soils. In addition, to understand the ecological function of root exudates, we established a microcosm system to evaluate the effects of genstein and daidzin as well as their mixture on fusarium communities and P. sojae. The results are as following:(1) The abundances of fungal communities were significantly higher from1to3years than those from6to13years. The Shannon index and evenness of fungal communities are higher from6to13years than those from1to3years. Evenness is negative related to fungal abundance. The relative abundance of pathogenic fungi such as Boeremia and Clavariopsis were higher in the second and third cycle, whereas the relative abundance of beneficial fungi such as Bionectria, Melanospora, and Hypocrea were decreased. However, after6-year soybean monoculture, fungal communities were characterized by several beneficial species, while the abundances of pathogenic fungi were lower than year2and3. In addition, the special root exudates such as isoflavones, genstein and soil chemical factors, primarily pH, had a regulatory effect on the fungal communities. Bacterial abundance were rapidly reduced from1to3years and remained constant until the end of experiment. Bacterial community composition was affected by Olsen P, NO3-and pH. Our results suggest that the transformation from initial healthy to conducive soil may be attributed to not only high proportion of fungal pathogen taxa, but also a large fraction of fungal community and a small fraction of bacterial community. Then, the transformation from conducive to suppressive soil and the sustainment of soil suppressive may be due to the high proportion of beneficial taxa in soils.(2) No clearly temporal compositional trends in Fusarium community composition and abundance were observed from1to13year. Isoflavonoids concentrations in soils were significantly correlated to Fusarium community shifts. In addition, P. sojae abundance increased from1to3years, whereas, the abundance of P. sojae was negatively correlated to isoflavonoids concentrations. The strong response to isoflavonoids of these two rhizosphere fungi populations highlights that soil microbes depend more on plant physiology than soil chemical parameters. Fusarium communities from species-poor fields were found to be subsets of those in richer fields, indicating nestedness and a progressive ’loss’ from the species pool. Our results suggest that Fusarium community is influenced by the combination of deterministic and stochastic processes.that the Fusarium community present in soybean fields might not be the main factor in yield shift, but P. sojae population density might was of an importance.(3) The species richness of nematode communities was decreased with the increase of continuous monoculture. Acrobeloides was prevalent in the soil. The relative abundances of plant parasite nematode are higher from2to3years than those from6to13years. NMDS indicated that the nematode communities in rhizophere soil collected from the9,11, and13years closely clustered together, outside of the first three years. Furthermore, nematode community composition was affected by available P, soil organic matter (SOM), pH, bacterial and fungal abundance. The abundance of nematode community was increased from1to6years, and then it was decreased from9to13years. In addition, the abundance of nematode community was positively correlated to NH4and genisten and was negatively correlated to NO3-and bacterial abundance. Our results suggest that nematode community is influenced by soil microbiology and many soil factors, and that nematode community present in soybean fields might be an important factor in yield shift.(4) Exogenous daidzin and genistein as well as their mixture resulted in the changes of Fusarium community. The number of the intense bands assayed by DGGE that may represent numerically dominant fusarium species were increased from the third day to the seventh day and then decreased at the forteenth day. Cluster analyses indicated that samples from different treatements could be distinguished from each other. Daidzin, genistein and their mixture have strongly impacted on the abundance of Fusarium community in incubated the third day, but have only weakly affected on it with the increase of incubated days. However, these solutions have no effect on the abundance of P. sojae in incubated the third day, while the high concentrations of these solutions have an inhibitory effect on it in incubated the seventh day. In incubated the forteen days, only the highest concentrations of these solutions increased the abundance of P. sojae. The inhibitory or sitimulatory effects of isoflavones on these two pathogenic fungi depended on incubated time and their concentrations.In conclusion, we found that long-term monoculture soybean led to decreases of richness in soil bacterial, fungal and nematode communities and that soil biotic community sizes were decreased by monoculture soybean.更多还原