用宏基因组学方法研究绿肥对水稻根际微生物磷循环功能基因的影响

  【目的】  绿肥对土壤微生物磷循环的影响受到研究者们的广泛关注，但绿肥影响土壤磷循环的微生物机理尚不明确。利用宏基因组学方法剖析长期绿肥还田对土壤磷循环功能微生物的影响，旨在阐明绿肥对土壤磷循环影响的微生物机理，为绿肥的科学利用提供依据。  【方法】  以中国湖南祁阳绿肥及稻草还田定位试验的水稻根际土为材料，利用宏基因组学方法，对土壤微生物DNA进行PE150 (双端读长150 bp) 宏基因组测序，使用MetaWRAP软件中的read_qc模块进行质控，用assembly模块megahit方法进行组装；基于组装的较长序列 (> 1000 bp) 使用Diamond软件的BLASTX和UniProtKB/SWISS-PROT数据库进行序列比对，根据磷活化 (磷酸酯矿化、膦酸酯矿化、无机磷溶解)、磷吸收 (膦酸酯运输、磷酸酯运输和无机磷酸盐运输) 和缺磷诱导响应调控三大类主要磷循环功能相关基因 (重点关注的64个磷循环功能基因) 进行筛选；再利用R语言进一步分析水稻根际土壤中磷循环功能基因相对丰度及其与土壤理化指标之间的关系，阐述绿肥对土壤磷循环功能微生物的影响。  【结果】  土壤pH等9个常规指标在有、无绿肥处理之间均无显著差异。无稻草还田时，绿肥处理对64个磷循环功能基因中的11个相对丰度影响显著，这11个基因广泛分布在除磷酸酯运输及无机磷溶解外的5个功能组中，其中phnA、phnN、phnV等3个基因相对丰度上升，phnI、phnL、glpB、glpO、pitA、phoA、phoP、phoU等8个基因相对丰度下降。而在稻草还田时，绿肥处理仅phnPP基因相对丰度显著降低。相关性分析显示，土壤pH与磷酸酶活性、无机磷溶解基因pqqB、pqqC 、pqqCD、pqq E、pqqF相对丰度呈显著负相关 (P < 0.05)；无机磷溶解pqqB、pqqC 、pqqCD、pqq E等4个基因相对丰度分别与有效磷含量、磷酸酶活性以及有机氮含量呈显著正相关 (P < 0.05)。冗余分析 (RDA) 结果表明，绿肥处理通过pH、AP以及磷酸酶活性影响磷循环功能基因。  【结论】  绿肥翻压下水稻根际微生物磷功能基因相对丰度深受稻草还田的影响。与单绿肥或单稻草还田相比，绿肥翻压基础上增加稻草还田对磷循环功能基因影响效果不明显，秸秆甚至减弱了绿肥对磷循环功能基因的影响，其原因可能是绿肥和稻草还田下不同微生物及不同磷循环功能基因之间存在激烈竞争。

Impact of green manure on microbial phosphorus cycling genes in rice rhizosphere as investigated by metagenomics

  【Objectives】  The effect of green manure on soil phosphorus-cycle(P-cycle) has gained great attention, but the involved microbial mechanism is largely unknown. In order to clarify the microbial mechanism of how green manure impacts on soil P-cycle, we comprehensively analyzed the effects of long-term green manure on soil microbial P-cycle genes by Metagenomics.  【Methods】  The study was based on a long-term green manure field experiment in Qiyang, Hunan, China. The rice rhizosphere soil was collected in the four treatments, including with or without green manure returning in combination with or without rice straw returning. The shortgun metagenomic sequencing was performed on an Illumina NovaSeq platform with a PE150 strategy. The read_qc module in MetaWRAP was used for quality control, and the assembly module megahit method was used for assembly. The assembled long sequences (> 1000 bp) were aligned with SWISS-PROT databases using Diamond BLASTX, and then screened according to 64 P-cycle genes seven functional groups of the three major categories including P activation (phosphate mineralization, phosphonate mineralization, inorganic P solubilization), P uptake (phosphonate transport, organic phosphate transport, inorganic P transport) and P-starvation inducible response and regulation. Then, the R software was used to analyze the relative abundance of P-cycle genes, and to analyze the relationship between the relative abundance of genes and soil properties.  【Results】  Nine soil physicochemical and biological indexes including pH showed no significant differences between the treatments of green manure and non-green manure under both conditions of straw and non-straw amendment. Among the 64 functional genes related to P cycle, the relative abundances of 11 genes were significantly affected by green manure returning under condition of without straw amendment. These 11 genes were widely distributed in five groups except organic phosphate transport and inorganic phosphate solubilizing genes, and the relative abundance of 3 genes, i.e. phnA, phnN, phnV, were increased, and those of the other 8 genes such as phnI, phnL, glpB, glpO, pitA, phoA, phoP and phoU, were decreased. However, only the relative abundance of phnPP gene was significantly decreased by green manure in straw-amended plots. Correlation analysis showed that soil pH was significantly and negatively correlated with acid phosphatase activity and relative abundance of inorganic phosphate-soluble genes pqqB, pqqC, pqqCD, pqqE and pqqF significantly (P < 0.05). The relative abundance of four genes pqqB, pqqC, pqqCD and pqqE in the functional group of inorganic P solubilization were positively correlated with available P content, phosphatase activity and organic N content (P < 0.05). Redundancy analysis (RDA) showed that pH, available P content and phosphatase activity were the three important factors significantly correlated with the effect of green manure treatment on P-cycle genes.  【Conclusions】  Green manure could impact multiple processes of the P cycling in rice rhizosphere under straw amendment. Straw amendment under green manure returning does not have that significant effect like without straw amendment on the P-cycle related genes, or even weaken the effect possibly due to competition among microorganisms and P-functional genes involved in straw and green manure decomposition.