基于高通量测序的土壤可培养有机解磷菌多样性研究  

Diversity Assessment of Soil Culturable Organic Phosphate-solubilizing Bacteria Based on High-throughput Sequencing

作　　者：夏围围 邹萌萌 张泽霖 陈晓芬 王玉芳 孙祥鑫[3] 贾仲君[3,4] XIA Weiwei;ZOU Mengmeng;ZHANG Zelin;CHEN Xiaofen;WANG Yufang;SUN Xiangxin;JIA Zhongjun(Jiangsu Provincial University Key Laboratory of Agricultural and Ecological Meteorology,School of Ecology and Applied Meteorology,Nanjing University of Information Science and Technology,Nanjing 210044,China;Soil and Fertilizer&Resources and Environment Institute,Jiangxi Academy of Agricultural Sciences,Nanchang 330200,China;Key Laboratory of Soil and Sustainable Agriculture,Chinese Academy of Sciences,Nanjing 211135,China;Northeast Institute of Geography and Agroecology,Chinese Academy of Sciences,Changchun 130102,China)

机构地区：[1]南京信息工程大学生态与应用气象学院/农业与生态气象江苏省高校重点实验室,南京210044 [2]江西省农业科学院土壤肥料与资源环境研究所,南昌330200 [3]土壤与农业可持续发展重点实验室(中国科学院),南京211135 [4]中国科学院东北地理与农业生态研究所,长春130102

出　　处：《土壤》2024年第5期999-1006,共8页Soils

基　　金：国家自然科学基金项目(42177280)资助。

摘　　要：采用常规细菌培养基(BPM)、无机解磷菌培养基(IPM)和有机解磷菌培养基(OPM)分别对土壤解磷菌进行平板培养,经3次传代富集,获得可培养微生物菌落集合,连同原位土壤,以phoD基因为分子标靶进行高通量测序分析,评价土壤本底和可培养解磷菌多样性差异,及其在不同培养基中的富集规律。结果表明:基于phoD基因的高通量测序结果能够较大程度覆盖土壤本底和菌落富集物中phoD相关解磷菌种群,Goods coverage指数大于98.3%。土壤可培养解磷菌phoD基因的α多样性显著低于原位土壤,其中Shannon和Simpson指数在IPM中最低,而在BPM和OPM中无显著差异;Chao 1指数在不同培养基富集物中均无显著差异,仅为原位土壤的5.3%~8.4%。在微生物属水平对phoD基因序列进行分类注释发现,原位土壤共检测到34属可分类解磷菌,可培养解磷菌的比例约为26.5%~41.2%。原位土壤和不同培养基富集物之间的phoD相关解磷菌共有属仅为6个,多为可培养的优势属,包括Pseudomonas、Bradyrhizobium、Cupriavidus、Sinorhizobium、Xanthomonas和Actinoplanes。进一步通过LEf Se(LDA effect size)差异分析显示,共有属中有4个在不同培养基中存在显著富集差异,Cupriavidus在IPM和OPM中显著富集,而Pseudomonas、Bradyrhizobium和Xanthomonas在BPM和OPM中显著富集。此外,Burkholderia仅在OPM中被检测。最后,在属水平上还存在大量pho D基因序列目前无明确分类,在所测序列中占比为0.13%~59.6%。本研究通过将传统微生物培养技术与高通量测序技术相结合,定量评价了培养基成分对土壤可培养解磷菌pho D基因多样性的影响,揭示了pho D相关解磷菌的可培养特性和生理特点,为发掘和利用土壤解磷菌资源提供了参考。未来亟待培养技术创新,以定向分离和鉴定更多解磷菌,增强对环境解磷资源微生物的全面认识。By using conventional bacteria medium(BPM),inorganic phosphate-solubilizing bacteria(PSB)medium(IPM)and organic PSB medium(OPM),culturable microbial colonies were collected after third serial passage on agar plates.High-throughput sequencing analysis of phoD gene as molecular target were employed to evaluate the diversity differences of PSB in the background soil and colony enrichments from different media.The results showed that phoD gene sequences obtained from high-throughput sequencing could largely cover phoD-related PSB communities in the background soil and colony enrichments,and the Goods coverage index exceeded 98.3%.αdiversity of phoD gene in colony enrichments were significantly lower than the background soil,and Shannon and Simpson indices of phoD gene were the lowest in IPM,but had no significant difference between BPM and OPM.Chao 1 index of phoD gene showed no significant difference in different media,accounting for 5.3%–8.4%of the background soil.According to the taxonomic annotation of phoD gene,34 genera of PSB were detected in the background soil and the proportion of culturable PSB were only about 26.5%–41.2%.Among the background soil and the colony enrichments from different media,there were 6 common genera of PSB,which usually dominated in the culturable PSB communities,including Pseudomonas,Bradyrhizobium,Cupriavidus,Sinorhizobium,Xanthomonas and Actinoplanes,.Further LEfSe(LDA effect size)analysis showed that 4 genera above had significant differences among different media,Cupriavidus was significantly enriched in both IPM and OPM,while Pseudomonas,Bradyrhizobium and Xanthomonas were significantly enriched in both BPM and OPM.Moreover,Burkholderia was only detected in OPM.Finally,at the genus level,lots of phoD gene sequences couldn’t be clearly classified at present which accounted for 0.13%to 59.6%of the total phoD sequences.In conclusion,by combining traditional microbial culture technology with high-throughput sequencing technology,this study quantitatively analyzed the influenc

关 键 词：高通量测序 解磷菌 phoD基因 无机磷 有机磷 

分 类 号：X171[环境科学与工程—环境科学] S154.3[农业科学—土壤学]