The Influence Of Long Term Rice-rice-green Manure Rotationon Rice Bulk Soil And Root-associated Bacteria

Green manure is one of effective measures to maintain and improve soil fertility and quality. It also has the superiorities of crop rotation and the reduction of fertilizer pollution. Nowadays, environmental problems have become increasingly prominent. So, the application of green manure is significant for the protection of ecological environment, and the improvement of soil health. At present, the fundamental research related to green manure rotation is relatively weak, which has become one of the limiting factors in the application and promotion of green manure. The related research was not systematic due to the limitation of methods and technology. Especially, the study of the impact of green manure on rhizosphere microorganisms of afterreap crop are limited so far. This subject would intend to explore the influence of different green manure crop rotation on paddy soil, rice rhizosphere and rice root endogenous microbial compositions. Samples were taken from HuNan QiYang farmland system national field scientific observation station which has 30-years history of long-term rice- rice- green manure(GM) rotation, including Milk Vetch(MV), Ryegrass(RG) and Rape(RP) and rice-rice-winter fallow(WF), based on 16 S rRNA Illumina Miseq sequencing, Q- PCR and DGGE methods combined with the biochemical indexes of the soil.(1) The Rice root-associated microbiomes include the paddy bulk soil microbe, rice rhizosphere soil microbe and rice root endogenous microbes. The results showed that there were significant differences of the communities and composition among the three parts of the bacteria, in which the microbial diversity of paddy bulk soil was significantly higher than rhizosphere and rice root, and the application of green manure increased the disparity. In the taxonomic level of the genus, the microbial composition of the paddy soil was antagonistic to the rice root samples. Such as some bacterial populations in the paddy bulk soil were detected with higher percentage like Gemmatimonas, Janthinobacterium and Acidobacteria etc, which were lower in the rice roots;in contrast,there are also some populations, such as Anaeromyxobacter, Pantoea、Burkholderia etc, which are widely distributed in the rice roots, but with lower precentage in paddy bulk soil. And the rhizosphere soil is the obvious enrichment of Pseudomonas, Clostridium and Acinetobacter.(2) The application of green manure in winter obviously changed the structure of bacteria in rice rhizosphere. Microbial diversity was reduced, but the numbers of microorganisms were increased. The bacterial composition had also changed distinctly, The highest percentage of RHWF(Rhizosphere of rice-rice-winter follow) was Firmicutes, but the predominant population in Green manure treatments was Proteobacteria. However, there was no significantly difference among the three kinds of green manure treatments, and the rice rhizosphere effects might still account for the dominant factor. CCA results showed that bacterial communities in the green manure treatment group were significantly different in winter fallow treatment group, but the kinds of green manure had little effect. In addition the green manure treatment group of bacteria community has a good correlation with soil organic matter, available phosphorus contents and nitrogenase activity.(3) Different green manure rotations had different effects on rice root endophytes, but they had no significant difference on diversity of endophyte. The Q-PCR of 16 S rDNA results showed that the application of green manure could increase the number of rice roots endophytic bacteria, especially milk vetch and ryegrass. Rice root in milk vetch showed a strong enrichment effcet on the genera of Burkholderia and Pseudomonas.(4) Green manure improved the soil pH to some extent and increased the organic matter content and nitrogenase activity of the soil. But there were little difference of impacts among different green manure on soil physical and chemical properties. Although green manure has been verified some effects on the composition of soil microbe, it has little effect on the community structure. In addition, we found that the percentange of Pseudomonadales and Burkholderiales were obviously lower in paddy bulk soil of the milk vetch treatment than in other treatments, but the content of which was significantly higher in the root endophytes than in other treatments.(5) The results of quantitative PCR of nifH gene showed that the copy number of rice root endophytes was significantly lower than that in paddy bulk soil and rice rhizosphere soil. Application of manure could increase the copy number of nifH genes in rice rhizosphere soil, but in paddy soil and rice root, only milk vetch and ryegrass treatments increased. On the contrary, the nifH gene numbers in rape group decreased. DGGE electrophoresis patterns demonstrated that maximum number of bands was in the ryegrass, followed by milk vetch,winter follow and rape. The effects of different green manure on rice root-associated nitrogen fixing bacteria are different. In the rice root, Burkholderia kururiensis and Rhodopseudomonas spp. coexisted together in all treatments, and Agromonas oligotrophica only existed within rice roots of the green manure groups.