长期施肥对土壤中大豆根瘤菌多样性及结瘤特性的影响

There is interaction effect between soil microorganisms and environment. Soil microorganisms community are affected by environmental factor, such as long term fertilization, changed soil attribute and, in turn, the environment is affected by the microorganisms, as decomposers, are critical to nutrient recycling in ecosystems. That is because long term fertilization can influence the soil microbial community by changing soil attribute. The soybean rhizobia, one kind of soil microorganisms, can response quickly to the changed soil environmental conditions, especially after various fertilizer were applied to the field. Soybean rhizobia-one kind of nitrogen-fixing bacteria is more sensitive to long term fertilization regimes than the other soil microorganisms. Therefore, three groups of long-term fertilizer field experiments were chosen to study the evolutionary process of genetic diversity, nodulation and nitrogen fixation characters of soybean rhizobia. The molecular biotechnology were used to evaluate the changes of genetic diversity of soybean rhizobia isolated from the soil with different physical and chemical properties, which are affected by long term fertilization regimes. Then, compared the evolutionary process of genetic diversity of soybean rhizobia isolated from the before and after long term fertilization regimes. Besides, the 15N natural abundance technology was used to estimate the ability of symbiotic N2 fixation. And the influence mechanism was also revealed with long term different fertilization regimes in this study. The nitrogen fixation efficiency was determined under different soil attribute and fertilization regimes, and the inter correlation was also studied in those complex correlation among fertilization, soil attribute and the efficiency of nitrogen fixation. The evolutionary characters of genetic diversity of soybean rhizobia ware specified with different long term fertilization regimes. Meanwhile, the factors for highly efficient ability of symbiotic N2 fixation were analyzed, and the major and related impact factors that affected the evolutionary process of soybean rhizobia were clarified as well. Besides, the highly efficient soybean rhizobia were screened in related experiments of our study for popularization of fertilization regimes. The study can provide scientific evidences for improve the ability of symbiotic N2 fixation with fertilizer application and then enrich in symbiotic fixation theory.

土壤中微生物和环境之间具有相互作用，长期施肥会改变土壤的属性，进而引起土壤中微生物变化。土壤中大豆根瘤菌和土壤中其它微生物一样，会对不同的施肥制度产生响应，由于大豆根瘤菌是固氮微生物，对长期施肥尤其敏感。利用3组长期定位田间不同施肥试验，采用15N自然丰度技术和分子生物学方法，研究长期施肥引起土壤理化性状的变化过程中大豆根瘤菌的演化特征；长期不同施肥制度对大豆根瘤菌结瘤固氮的影响机制；及施肥和土壤属性变化与大豆根瘤菌固氮效率变化的内在关系。以期阐明在春大豆主产区各种施肥制度下，土壤中大豆根瘤菌的演化特征；明确影响大豆根瘤菌高效固氮的因素；阐明土壤中大豆根瘤菌变化过程及其相关的土壤主控因子，筛选出的适合当地施肥制度的高效大豆根瘤菌，为生产上通过施肥和培肥土壤提高大豆根瘤菌固氮效率提供科学依据，为丰富大豆共生固氮理论增添新的内容。

通过试验得出本研究试验区分离到的大豆根瘤菌多样性较低且均为慢生根瘤菌，主要分为5个类群，分别为Bradyrhizobium sp. I，Bradyrhizobium sp. II，Bradyrhizobium sp. III，B. japonicum USDA 6T和B. diazoefficiens USDA 110T。不同施肥管理措施相比，施用有机肥（NPOM）有利于根瘤菌丰度、H’指数、D指数和J指数的增加。NPOM处理下的大豆根瘤干重、数量和固氮酶活性的变化较CK1处理分别增加了84.39%，128.57%，13.0%。不同土地利用方式相比，草地（GL）处理的根瘤菌丰度、H’指数、D指数和J指数值均最高。GL处理中分离到了Bradyrhizobium sp. I，Bradyrhizobium sp. II，Bradyrhizobium sp. III和B. japonicum USDA 6T，其中Bradyrhizobium sp. II只在GL处理中分离到。农田（CL）处理的Bradyrhizobium sp. I为优势种（50.0%）。GL处理中Bradyrhizobium sp. I和Bradyrhizobium sp. III占总菌株数的比例相当，分别为42.5%和35.0%。土壤有机碳（OC）含量、pH和速效磷（AP）含量对B. japonicum USDA 6T，Bradyrhizobium sp. I和Bradyrhizobium sp. II的分布具有正相关影响，Bradyrhizobium sp. III的分布具有负相关性影响。土壤容重（BD）对B. japonicum sp. III的分布具有正相关性影响。因此在黑龙江大豆主产区要想达到土壤中根瘤菌丰度、多样性指数高，大豆结瘤固氮及产量高的多重目的，施肥管理措施和土地利用方式需要进行适当的调整。