不同种植年限宁夏枸杞根际微生物多样性变化

Microbial Biodiversity in Rhizosphere of Lycium bararum L. Relative to Cultivation History

为了解长期人工种植枸杞根际土壤微生物的种群结构变化特征,利用Illumina Mi Seq测序平台分别对种植5 a、10 a和15 a宁夏枸杞根际土壤微生物基因组总DNA中16S和18S r DNA基因的部分区域进行测序,经UPARSE pipeline和RDP classifier软件进行聚类分析和物种注释。结果表明,长期种植宁夏枸杞不会改变其根际土壤p H,但会导致根际土壤全磷、有效磷、全盐含量和电导率升高。测序结果表明,不同种植年限枸杞根际土壤细菌群落α多样性无显著变化,但真菌群落多样性在种植10 a枸杞中较种植5 a时显著降低(p<0.05),表明根际细菌群落多样性受种植年限影响较小。从门的分类水平看,酸杆菌门、放线菌门、拟杆菌门、厚壁菌门、绿弯菌门、泉古菌门、蓝菌门、芽单胞菌门、变形菌门以及真菌子囊菌门、担子菌门、接合菌门的比例在不同种植年限的枸杞根际土壤中显著改变(p<0.05)。属水平的分析也表明,共有27个细菌属和16个真菌属的比例发生改变(p<0.05),这些结果表明枸杞根际土壤微生物群落组成受种植年限的影响更大。相关性分析结果表明,种植年限、土壤全磷及有效磷含量是影响枸杞根际微生物群落结构的主要因子。

The rhizosphere is a critical interface where exchange of substance takes place between plants roots and their surrounding soil. In the rhizosphere,interactions between the plant and soil microbes,though affected by a series of factors,such as physic-chemical properties of the rhizospheric soil,genotype of their host plant,can be beneficial to growth of the plant,the microbes or both. However,it is still not very clear how long-term cultivation of Lycium bararum L. would affect soil microbial community structure in the rhizosphere of the plant. Therefore,rhizospheric soil samples were collected from Lycium bararum L. fields different in cultivation history（5 a,10 a and 15 a）in a farm of Nanliang,Ningxia,China,for analysis of physic-chemical properties,such as p H,electrical conductivity,SOM,total salt,total and readily available N,P and K,etc. Results showed that p H remained unchanged in all the fields,while total salt content,total and readily available phosphorus and electrical conductivity in the soil increased significantly with the age of cultivation. Total genomic DNA was isolated from the rhizosphere soil using a Power Soil DNA Isolation Kit for sequence analysis of V4 sections of 16 S r DNA as indicator of bacterial diversity and ITS2 sections of 18 S r DNA gene as indicator of fungal diversity,with the aid of the Illumina Mi Seq system. Results of the sequencing were assembled and clustered with the FLASH,QIIME,and UPARSE pipeline software packages. In the end,is was found that the α diversity of the rhizosphere bacteria community did not vary much between the fields different in cultivation history,but fungal diversity did,and decreased with the cultivation going on from 5 a to 10 a（p〈0.05）. For analysis of changes in microbial community structure at the phyla and genus levels,the software of RDA Classifier to denote each OUT by species. It was found that Proteobacteria（22.2%）,Crenarchaeota（15.1%）,Bacteroidetes（13.9%）,A c i d o b a c t e r i a（12.4%）, C h l o ro f l e x i（10.3%）, G e m m a t i m o n a d e t e s（4.8%）, A c t i n o b a c t e r i a（4.7%）,Planctomycetes（4.0%）and Verrucomicrobia（2.0%）were the dominate bacterial groups and Ascomycota（29.5%）and Basidiomycota（11.7%）were the dominant fungal group in the rhizosphere of the plant. However,about 55.8% of the fungi found in the rhizosphere were still unknown in the taxonomy. Besides,the sequencing further demonstrated that soil microbial community structure in the rhizosphere varied sharply between the fields different in cultivation history and stood out uniquely in each field from the others. The variation between fields different in cultivation history was particularly significant in terms of the ratio of Acidobacteria,Actinobacteria,Bacteroidetes,Fimicutes,Chloroflexi,Crenarchaeota,Cyanobacteria,Gemmatimonadetes,Proteobacteria,Ascomycota,Basidiomycota and Zygomycota in the rhizospheric microbial community（p〈0.05）. The protion of unknown fungal groupsin taxonomy was much higher in the 10 a（76.6%）and 15 a（61.4%）Lycium bararum L. fields than in the 5 a（32.5%）fields（p〈0.05）. The analysis at the genera level also shows that the ratios of 27 genera of bacteria and 16 genera of fungi changed with the cultivation going on（p〈0.05）. In order to further analyze relationships of soil community,with soil physic-chemical properties,hundred-grain weight and cultivation history,SPSS 16.0 software was used to work out pearson’s correlation coefficients between the data,which show that cultural history is significantly related to richness of Firmicutes,Proteobacteria,Verrucomicrobia,Basidiomycota and Zygomycota（p〈0.05）,content of total phosphorus in the rhizosphere soil is to richness of Firmicutes,Gemmatimonadetes,Proteobacteria,Basidiomycota,Zygomycota（p〈0.05）and content of readily available phosphorus is to richness of Acidobacteria,Gemmatimonadetes,Ascomycot,Basidiomycota and Zygomycota（p〈0.05）. The findings further demonstrated that the three factors,cultural year,total phosphorus and readily available phosphorus,are the key factors affecting soil microbial composition in the rhizosphere of Lycium bararum L. To sum up,cultivation history affects soil microbial community structure more than soil microbial diversity in the rhizosphere soil of Lycium bararum L.,which may be related to soil phosphorus metabolism in the rhizosphere.