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In this study, we measured the contents of P in various forms in the topsoil (0~10cm) of 8representative pine (Pinus massoniana) forests in the main urban area of Chongqing. We used high-throughput sequencing technology to investigate the community characteristics of the phoD-harboring bacteria. The results showed that the surface soils of Pinus massoniana forests in the study area had relatively low phosphorus levels, with average total phosphorus (TP) contents of 192.787mg/kg in winter and 169.512mg/kg in summer. Among the inorganic phosphorus fractions, the content distribution followed the order: occluded phosphorus (O-P) > iron-bound phosphorus (Fe-P) > aluminum-bound phosphorus (Al-P) > calcium-bound phosphorus (Ca-P) > exchangeable phosphorus (Ex-P), showing a seasonal pattern of higher levels in winter and lower levels in summer. The dominant phyla of phoD-harboring bacteria in the soil were Proteobacteria, Actinobacteria, and Planctomycetes, collectively accounting for 96% of the average relative abundance. Correlation analysis showed a significant negative correlation (P<0.05) between the diversity of phoD-harboring bacteria and Al-P, as well as a significant positive correlation (P<0.05) with soil pH. Redundancy analysis indicated that Al-P and pH were the most important factors influencing the community structure of phoD-harboring bacteria. In conclusion, the topsoil of pine forests in the main urban area of Chongqing is generally P-deficient, and the P content is significantly influenced by season. The forms of P occurrence affect the community structure and diversity of phoD-harboring bacteria.

, correspAuthors=Dun-mei LIN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Shao-long LI, Hao-yang TU, Yan-ping RUAN, Liang ZHAO, Dun-mei LIN), CN=ArticleExt(id=1234106398167069006, articleId=1234106389396779815, tenantId=1146029695717560320, journalId=1234093305789726721, language=CN, title=马尾松林土壤磷赋存形态及其对含phoD基因解磷细菌群落的影响, columnId=1234106394572550190, journalTitle=中国环境科学, columnName=土壤污染与控制, runingTitle=null, highlight=null, articleAbstract=

本研究测定了重庆主城区内8个代表性马尾松林样地表层土壤(0~10cm)不同赋存形态磷元素含量,并基于高通量测序技术研究了含碱性磷酸酶(phoD)基因的细菌群落特征.结果表明,研究区马尾松林表层土磷含量较低,冬季和夏季土壤总磷(TP)含量均值分别为192.787,169.512mg/kg.其中无机磷形态含量分布为闭蓄态磷(O-P)>铁结合磷(Fe-P)>铝结合磷(Al-P)>钙结合磷(Ca-P)>可交换态磷(Ex-P),并呈现冬高夏低的季节变化格局.含phoD基因细菌群落优势门为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes),平均相对丰度达到96%.相关分析表明含phoD基因细菌多样性与Al-P之间存在显著负相关关系(P<0.05),与土壤pH值存在显著正相关关系(P<0.05).冗余分析表明Al-P和pH值是影响含phoD基因细菌群落结构的最主要因素.综上所述,重庆主城区马尾松林表层土普遍缺磷且磷含量受到季节变化的显著影响,磷赋存形态影响含phoD基因细菌群落结构及多样性.

, correspAuthors=林敦梅, authorNote=null, correspAuthorsNote=
* 责任作者,副教授,
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李绍隆(2000-),男,青海西宁人,重庆大学硕士研究生,主要从事土壤微生物群落相关研究..

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李绍隆(2000-),男,青海西宁人,重庆大学硕士研究生,主要从事土壤微生物群落相关研究..

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李绍隆(2000-),男,青海西宁人,重庆大学硕士研究生,主要从事土壤微生物群落相关研究..

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Nature2024630(8017):660-665., articleTitle=Microbial competition for phosphorus limits the CO2 response of a mature forest, refAbstract=null)], funds=[Fund(id=1234106407537143843, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, awardId=S202110611406, language=CN, fundingSource=重庆市大学生创新训练项目(S202110611406), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1234106398724911501, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, xref=null, ext=[AuthorCompanyExt(id=1234106398733300110, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, companyId=1234106398724911501, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400044, China)), AuthorCompanyExt(id=1234106398741688720, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, companyId=1234106398724911501, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=重庆大学,三峡库区生态环境教育部重点实验室,重庆 400044)])], figs=[ArticleFig(id=1234106403867128662, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=EN, label=Fig.1, caption=Contents of various phosphorus forms and their differences between winter and summer in topsoil of Pinus massoniana forests in main urban area of Chongqing, figureFileSmall=U7apzPxIeg3mIBd+Tikb6Q==, figureFileBig=scIh9yuHBUR2jnYbGS41LA==, tableContent=null), ArticleFig(id=1234106403980374880, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=CN, label=图1, caption=重庆主城区马尾松林表层土壤各种赋存形态磷含量及其冬夏季差异

(a)总磷(TP)、有机磷(OrP)和无机磷(IP)含量,(b)不同赋存形态无机磷含量.数据为均值±标准误(n=8).Wilcoxon检验.*表示存在显著差异,*P <0.05,** P <0.01

, figureFileSmall=U7apzPxIeg3mIBd+Tikb6Q==, figureFileBig=scIh9yuHBUR2jnYbGS41LA==, tableContent=null), ArticleFig(id=1234106404261393280, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=EN, label=Fig.2, caption=Relative abundance of the phoD-harboring bacteria at the phylum and genus levels in soil of Pinus massoniana forests, figureFileSmall=pVXoo6YElLVSwIMI8+yqYA==, figureFileBig=808/D0BMidZYQPetc0ZR3A==, tableContent=null), ArticleFig(id=1234106404433359760, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=CN, label=图2, caption=马尾松林土壤含phoD基因细菌在门水平和属水平的相对丰度, figureFileSmall=pVXoo6YElLVSwIMI8+yqYA==, figureFileBig=808/D0BMidZYQPetc0ZR3A==, tableContent=null), ArticleFig(id=1234106404613714848, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=EN, label=Fig.3, caption=Correlation between α-diversity indices of the phoD-harboring bacteria and various phosphorus forms and soil pH, figureFileSmall=AxUxRLSmFjA9ssDoxscJQQ==, figureFileBig=q01bfF+7Jyc+9mB92zS3QA==, tableContent=null), ArticleFig(id=1234106406085915567, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=CN, label=图3, caption=phoD基因细菌α多样性指数与各赋存形态磷及土壤pH值之间的相关性

图中圆圈面积及颜色深度代表相关系数大小.*,P < 0.05,**,P < 0.01

, figureFileSmall=AxUxRLSmFjA9ssDoxscJQQ==, figureFileBig=q01bfF+7Jyc+9mB92zS3QA==, tableContent=null), ArticleFig(id=1234106406257882043, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=EN, label=Fig.4, caption=Redundancy analysis results of phoD-harboring bacteria communities and various forms of phosphorus and soil pH in soil samples, figureFileSmall=COXmik1ZAfKaB+5VR9xh4A==, figureFileBig=s3+ZAvef5ngNEvAeT6PKgQ==, tableContent=null), ArticleFig(id=1234106406404682700, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=CN, label=图4, caption=phoD细菌与土壤磷赋存形态及土壤pH值的冗余分析结果

Bradyrhizobium慢生根瘤菌属;Streptomyces链霉菌属;Ramlibacter拉姆利杆菌属;Pseudomonas假单胞菌属

, figureFileSmall=COXmik1ZAfKaB+5VR9xh4A==, figureFileBig=s3+ZAvef5ngNEvAeT6PKgQ==, tableContent=null), ArticleFig(id=1234106406618592218, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=EN, label=Fig.5, caption=The relative importance of soil pH, organic phosphorus and various inorganic phosphorus forms on the bacterial community structure (genus level) containing the phoD gene, figureFileSmall=OuYoWd+8Y3x7XAfrV9DMhQ==, figureFileBig=tta95rAta9C21SDz7WG9WA==, tableContent=null), ArticleFig(id=1234106406731838437, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=CN, label=图5, caption=土壤pH值、有机磷(OrP)、及各无机磷形态对含phoD基因细菌群落结构(属水平)影响的相对重要性

在右侧点阵图中,每行对应一个环境因子.对于每一列,孤立黑点表示各环境因子的边际效应,多点间连线表示这些环境因子间的共同效应,各组分解释的变差百分比(来自变差分解)展示在上方柱形图中.左侧柱形图为各环境因子的单独效应(来自层次分割),其值等同于该环境因子的边际效应加上与其他环境因子的共同效应的平均分配值.共同效应影响较小的组合结果未展示.*,P < 0.05

, figureFileSmall=OuYoWd+8Y3x7XAfrV9DMhQ==, figureFileBig=tta95rAta9C21SDz7WG9WA==, tableContent=null), ArticleFig(id=1234106406861861875, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=EN, label=Table 1, caption=

Summary of Pinus massoniana forest plots in main urban area of Chongqing

, figureFileSmall=null, figureFileBig=null, tableContent=
样点编号地理位置经纬度样地海拔(m)林龄(a)土壤pH值
P1南岸区茶园106°36′29″E,29°29′38″N427374.40
P2南岸区夏家坪106°39′7″E,29°35′29″N446454.02
P3璧山区杨秀口106°18′29″E,29°46′19″N382274.22
P4南岸区大坪子106°45′11″E,29°31′37″N410464.00
P5南岸区小龙洞106°46′1″E,29°33′21″N393473.88
P6巴南区二温泉106°52′22″E,29°29′9″N364413.95
P7巴南区圣灯山106°41′9″E,29°12′21″N770494.16
P8江北区五宝镇106°49′8″E,29°37′26″N375413.84
), ArticleFig(id=1234106407029634045, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=CN, label=表1, caption=

重庆主城区马尾松林样地基本情况

, figureFileSmall=null, figureFileBig=null, tableContent=
样点编号地理位置经纬度样地海拔(m)林龄(a)土壤pH值
P1南岸区茶园106°36′29″E,29°29′38″N427374.40
P2南岸区夏家坪106°39′7″E,29°35′29″N446454.02
P3璧山区杨秀口106°18′29″E,29°46′19″N382274.22
P4南岸区大坪子106°45′11″E,29°31′37″N410464.00
P5南岸区小龙洞106°46′1″E,29°33′21″N393473.88
P6巴南区二温泉106°52′22″E,29°29′9″N364413.95
P7巴南区圣灯山106°41′9″E,29°12′21″N770494.16
P8江北区五宝镇106°49′8″E,29°37′26″N375413.84
), ArticleFig(id=1234106407168045059, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=EN, label=Table 2, caption=

Diversity index of the phoD-harboring bacteria in soil of Pinus massoniana forests

, figureFileSmall=null, figureFileBig=null, tableContent=
多样性指数最大值最小值平均值标准差变异系数(%)
Chao1丰富度指数866.32142.11403.85206.3851.10
Shannon多样性指数5.413.424.5110.58112.87
Pielou均匀度指数0.820.700.7110.0405.20
PD谱系多样性指数88.8628.2164.3018.6228.9
), ArticleFig(id=1234106407340011536, tenantId=1146029695717560320, journalId=1234093305789726721, articleId=1234106389396779815, language=CN, label=表2, caption=

马尾松林土壤含phoD基因细菌多样性指数

, figureFileSmall=null, figureFileBig=null, tableContent=
多样性指数最大值最小值平均值标准差变异系数(%)
Chao1丰富度指数866.32142.11403.85206.3851.10
Shannon多样性指数5.413.424.5110.58112.87
Pielou均匀度指数0.820.700.7110.0405.20
PD谱系多样性指数88.8628.2164.3018.6228.9
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马尾松林土壤磷赋存形态及其对含phoD基因解磷细菌群落的影响
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李绍隆 , 屠豪阳 , 阮艳萍 , 赵亮 , 林敦梅 *
中国环境科学 | 土壤污染与控制 2025,45(6): 3190-3198
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中国环境科学 | 土壤污染与控制 2025, 45(6): 3190-3198
马尾松林土壤磷赋存形态及其对含phoD基因解磷细菌群落的影响
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李绍隆 , 屠豪阳, 阮艳萍, 赵亮, 林敦梅*
作者信息
  • 重庆大学,三峡库区生态环境教育部重点实验室,重庆 400044
  • 李绍隆(2000-),男,青海西宁人,重庆大学硕士研究生,主要从事土壤微生物群落相关研究..

通讯作者:

* 责任作者,副教授,
The phosphorus forms in the soil of Pinus massoniana forest and their impact on the community of phosphorus-decomposing bacteria harboring phoD genes
Shao-long LI , Hao-yang TU, Yan-ping RUAN, Liang ZHAO, Dun-mei LIN*
Affiliations
  • Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400044, China)
出版时间: 2025-06-20
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本研究测定了重庆主城区内8个代表性马尾松林样地表层土壤(0~10cm)不同赋存形态磷元素含量,并基于高通量测序技术研究了含碱性磷酸酶(phoD)基因的细菌群落特征.结果表明,研究区马尾松林表层土磷含量较低,冬季和夏季土壤总磷(TP)含量均值分别为192.787,169.512mg/kg.其中无机磷形态含量分布为闭蓄态磷(O-P)>铁结合磷(Fe-P)>铝结合磷(Al-P)>钙结合磷(Ca-P)>可交换态磷(Ex-P),并呈现冬高夏低的季节变化格局.含phoD基因细菌群落优势门为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes),平均相对丰度达到96%.相关分析表明含phoD基因细菌多样性与Al-P之间存在显著负相关关系(P<0.05),与土壤pH值存在显著正相关关系(P<0.05).冗余分析表明Al-P和pH值是影响含phoD基因细菌群落结构的最主要因素.综上所述,重庆主城区马尾松林表层土普遍缺磷且磷含量受到季节变化的显著影响,磷赋存形态影响含phoD基因细菌群落结构及多样性.

马尾松林  /  磷赋存形态  /  phoD基因  /  解磷细菌多样性

In this study, we measured the contents of P in various forms in the topsoil (0~10cm) of 8representative pine (Pinus massoniana) forests in the main urban area of Chongqing. We used high-throughput sequencing technology to investigate the community characteristics of the phoD-harboring bacteria. The results showed that the surface soils of Pinus massoniana forests in the study area had relatively low phosphorus levels, with average total phosphorus (TP) contents of 192.787mg/kg in winter and 169.512mg/kg in summer. Among the inorganic phosphorus fractions, the content distribution followed the order: occluded phosphorus (O-P) > iron-bound phosphorus (Fe-P) > aluminum-bound phosphorus (Al-P) > calcium-bound phosphorus (Ca-P) > exchangeable phosphorus (Ex-P), showing a seasonal pattern of higher levels in winter and lower levels in summer. The dominant phyla of phoD-harboring bacteria in the soil were Proteobacteria, Actinobacteria, and Planctomycetes, collectively accounting for 96% of the average relative abundance. Correlation analysis showed a significant negative correlation (P<0.05) between the diversity of phoD-harboring bacteria and Al-P, as well as a significant positive correlation (P<0.05) with soil pH. Redundancy analysis indicated that Al-P and pH were the most important factors influencing the community structure of phoD-harboring bacteria. In conclusion, the topsoil of pine forests in the main urban area of Chongqing is generally P-deficient, and the P content is significantly influenced by season. The forms of P occurrence affect the community structure and diversity of phoD-harboring bacteria.

Pinus massoniana forests  /  phosphorus forms  /  phoD gene  /  diversity of phosphorus-solubilizing bacteria
李绍隆, 屠豪阳, 阮艳萍, 赵亮, 林敦梅. 马尾松林土壤磷赋存形态及其对含phoD基因解磷细菌群落的影响. 中国环境科学, 2025 , 45 (6) : 3190 -3198 .
Shao-long LI, Hao-yang TU, Yan-ping RUAN, Liang ZHAO, Dun-mei LIN. The phosphorus forms in the soil of Pinus massoniana forest and their impact on the community of phosphorus-decomposing bacteria harboring phoD genes[J]. China Environmental Science, 2025 , 45 (6) : 3190 -3198 .
土壤中的磷可分为有机磷和无机磷两大类,无机磷根据其活性或溶解性的不同可再划分为可交换态的磷(Ex-P)、铝结合磷(Al-P)、铁结合磷(Fe-P)、闭蓄态磷(O-P)、钙结合磷(Ca-P)及残渣态磷[1];其中,Ex-P可以被植物吸收利用,Al-P,Fe-P和Ca-P不能被植物直接吸收,但在一定条件下可以转化为植物可吸收利用的形态,而O-P是以水化氧化铁胶膜包被的磷酸盐,其溶解度很小,难以被生物利用.由于不同赋存形态的磷在生物有效性方面存在很大的差异[2],解析土壤磷的赋存形态是深入认识生态系统磷供应状态的重要基础.
有机磷是土壤总磷的重要组成部分,有机磷的矿化是生物获取磷元素的重要途径.在土壤中有机磷的降解主要受胞外酸性磷酸酶和碱性磷酸酶的催化[3].过去的研究表明,陆地生态系统中酸性磷酸酶主要由植物根系分泌,而碱性磷酸酶主要来自土壤微生物,特别是细菌[3-5].受到在酸性条件下酸性磷酸酶活性更强这一观点的影响[6],传统上一般认为酸性土壤的有机磷矿化主要受酸性磷酸酶的影响,因此在对酸性土壤开展的研究中较少有研究者关注碱性磷酸酶及其相关的微生物群落[7-8].然而,近年来一些研究表明,即使在酸性土壤中,影响土壤有机磷矿化的因素也可能是碱性磷酸酶.例如,在酸性土壤中也发现存在大量编码碱性磷酸酶的基因[9];在贡嘎山,酸性土壤中的有效磷主要受碱性磷酸酶的影响而不是酸性磷酸酶活性的影响[10].这些结果表明分泌碱性磷酸酶的细菌在酸性土壤的磷循环过程中也扮演了重要角色,揭示其群落结构及其影响因素对于深入认识土壤磷循环具有重要意义.细菌中编码碱性磷酸酶的基因主要包括phoA,phoDphoX[11-12].但在陆地生态系统中含phoD基因的细菌更为常见[11],而且有研究发现phoD基因丰度与碱性磷酸酶的活性存在正相关关系[3,13].因此,很多研究采用含phoD基因的细菌群落来表征具备合成碱性磷酸酶的土壤微生物群落[7,14-16].不过已有研究主要以农田生态系统为研究对象,而对森林生态系统中含phoD基因的细菌群落结构特征及其影响因素的认识还相对缺乏[15].
马尾松(Pinus massoniana)广布于我国南方地区,马尾松林总面积达到1001万hm2[17].由于具备优良的抗旱、耐贫瘠等特性,马尾松是长江上游低山丘陵区退耕还林最常用的树种[18].三峡库区是我国重点敏感生态区和长江中下游的生态屏障,分布有大面积马尾松林[18-19],同时马尾松林占重庆市森林总面积的近一半[17].重庆主城区“四山”(缙云山、中梁山、铜锣山和明月山)地处三峡库区腹地,也是重庆主城区的重要生态屏障,马尾松林是该区域最主要的植被类型.本研究在重庆主城区“四山”范围内选取了代表性马尾松林样地,解析了马尾松林表层土壤不同形态磷元素的含量水平,同时基于高通量测序技术研究了含碱性磷酸酶phoD基因的细菌群落特征,揭示该区域马尾松林土壤磷供应现状和参与磷循环的微生物群落结构特征和影响因素,以期为区域森林管理提供理论参考.
本研究在重庆主城区四山范围内开展.该区域位于三峡库区腹地,气候类型为亚热带季风性湿润气候,年平均气温16~18℃,年平均降水量在1000~1611mm之间,日平均气温大于10℃的积温约为6500℃.其中夏季(6~8月)月平均温度在26~29℃之间,降水约占年总降水的70%,积温约为3360℃;冬季(12~次年2月)月平均气温为4~8℃,降水明显减少,积温约为747℃.区域土壤类型以黄壤和黄棕壤为主.该区域整体植被覆盖率高,但残存的天然常绿阔叶林很少,以马尾松纯林和马尾松针阔混交林为主.
基于前期在该区域建设的马尾松林长期监测样地网络[20].考虑空间上的分布,共计选择8个马尾松林样地进行本研究,样地基本信息见表1.分别于2020年12月(冬季)及2021年7月(夏季)进行两次野外土壤样品采集.在每个20m×20m样地内,用内径为32mm土钻按S形曲线随机钻取12钻深10cm的表层土壤置于无菌自封袋内充分混合为一份土壤样品,放入装有冰块的冷藏箱中尽快带回实验室.带回的样品用无菌勺随机挖取约10g土壤置于无菌自封袋中,保存于-80℃冰箱用于后续微生物的测定.余下土样分为两部分,一部份土壤样品自然风干保存,去除杂物,研磨过100目筛装袋用于不同赋存形态磷含量的测定,另一部分土壤样品保存于-20℃冰箱中冷冻保存,用于土壤pH值的测定.
土壤pH值采用电位法测定,土水比为1:2.5(w/V).
土壤总磷(TP)经H2SO4-HClO4消解后用磷钼蓝比色法测定,有机磷(OrP)采用灼烧法测定,无机磷(IP)总量为总磷减去有机磷.
无机磷赋存形态分级采用Chang等[21]提出的C-J化学连续提取法进行,简要步骤如下:(1)用NH4Cl溶液浸提得到交换态磷(Ex-P)提取液;(2)上一步残渣使用NH4F溶液(用NH3·H2O调节pH值至8.2)浸提得到铝结合态磷(Al-P)提取液;(3)上一步残渣使用NaOH溶液浸提得到铁结合态磷(Fe-P)提取液;(4)在上一步残渣中加入Na3C6H5O7·2H2O缓冲液,振荡后加入Na2S2O4,90℃热水浴并加入NaOH搅拌,体系由灰白色逐渐变为浅黄绿色时冷却并离心,上清液使用三酸混合液(H2SO4-HNO3-HClO4)消解得到闭蓄态磷(O-P)提取液;(5)上一步残渣使用H2SO4浸提得到钙结合态磷(Ca-P)提取液;以上步骤获得的提取液均使用磷钼蓝比色法测定其中的磷元素含量.每份土壤样品均设置3个重复.
将-80℃冷冻保存的土壤样品置于冷冻干燥机进行干燥,用干冰保温寄至上海美吉生物医药有限公司进行细菌群落的测试分析.土壤样品使用FastDNA®SPIN Kit for Soil试剂盒抽提土壤微生物总DNA.在DNA质量检验合格后,使用phoD-733F(5′-TGGGAYGATCAYGARGT-3′)和phoD-1083R(5′-CTGSGCSAKSACRTTCCA-3′)引物对细菌碱性磷酸酶基因(phoD)进行PCR扩增,每份样品同时做3个重复.PCR在ABI GeneAmp® 9700型PCR仪中进行.PCR反应采用Pro Taq,20μL反应体系,包括10 μL 2×Pro Taq,正反向引物各0.8μL,10ng/μL DNA模板,最后用超纯水(ddH2O)补足至20μL.PCR扩增程序设定为:95℃预变性3min;95℃变性30s;55℃退火30s;72℃延伸40s,该步骤进行36个循环;然后在72℃延伸10min,最后降温至10℃保持直到关机.同一样本的PCR产物混合后用2%琼脂糖凝胶电泳进行质检,然后建库,最后在Illumina Novaseq PE2500平台上进行测序.获得的序列数据在QIIME2(v2020.2)平台上进行分析,使用DADA2插件对序列数据进行降噪处理,获得扩增子序列变体(amplicon sequence variants,ASVs).然后通过数据库(nt_v20200604)比对进行分类学注释.最后,按最小样本的序列数对所有样品进行抽平处理,该数据用于后续的分析.
使用配对样本Wilcoxon秩和检验分析土壤各赋存形态磷元素在夏、冬季之间是否存在显著差异.经Shapiro-Wilk检验数据服从正态性后,使用Pearson相关分析检验微生物群落α多样性指数与各赋存形态磷含量(经对数变换)以及土壤pH值之间的相关关系[22],显著性水平设定为P<0.05.其中,α多样性指数包括Chao1丰富度指数、PD谱系多样性指数、Shannon多样性指数和Pielou均匀度指数;以属水平下含phoD基因菌群落(经Hellinger转化以消除极端值的影响并减少高丰度物种的权重)为响应变量、各磷元素赋存形态(经对数变换)为解释变量进行冗余分析(RDA),将方差膨胀因子大于10的变量从模型中删除;之后使用变差分解(variation partitioning)和层次分割(hierarchical partitioning)解析不同解释变量及其组合的相对重要性[23].数据分析及绘图均在R(v4.4.1)软件中进行.
图1所示,不同样地土壤TP在冬季和夏季的变幅分别为162.107~273.671mg/kg, 136.587~255.099mg/kg,其均值分别为192.787,169.512mg/kg;无论哪个季节,土壤IP含量均高于OrP含量,占比变幅分别为41.3%~68.4%和31.6%~ 58.7%(图1(a)).
图1(b)所示,整体上重庆主城区马尾松林表层土壤的IP形态分布稳定,各样地在冬季和夏季均呈现出O-P> Fe-P> Al-P> Ca-P> Ex-P的分布格局,且并没有表现出很大的变异性.其中,植物可直接吸收利用的Ex-P(0.6%~2.1%)及释磷潜力较大的Fe-P(15.2%~34.1%)以及Al-P(7.9%~16.7%)的总占比低于50%.
马尾松林表层土壤中磷含量表现出明显的夏冬季差异,普遍呈现出冬高夏低的分布格局,特别是TP、O-P、Fe-P和Al-P在冬夏季之间有显著的差异(图1, P<0.05).
基于夏季土壤的高通量测序分析,共得到730450条有效序列(最大序列数100859,最小序列数67947).以最小序列数抽平后,以100%相似度聚类共得到1295个ASVs,注释结果分属于11门21纲46目71科134属.
整体来看,重庆主城区马尾松林土壤含phoD基因细菌在门水平下以变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、浮霉菌门(Planctomycetes)为优势门,其中变形菌门相对丰度最高,占比达62%(图2(a)).在属水平下各样地优势属均为拉姆利杆菌属(Ramlibacter)、链霉菌属(Streptomyces)、慢生根瘤菌属(Bradyrhizobium)和假单胞菌属(Pseudomonas),这四种属的总平均相对丰度占55%以上(图2(b)).但具体到各样地之中,链霉菌属(1.6%~42.2%)与慢生根瘤菌属(30.6%~2.2%)的相对丰度在不同样地间表现出此消彼长的格局(图2(b)).
phoD基因细菌α多样性指数见表2.研究区含phoD基因细菌具有较高的α多样性,各样地之间部分指数存在较大的差异.其中Chao1丰富度指数介于142.11~866.32之间,变异系数达到51.10%,Shannon多样性指数介于3.42~5.41之间.
对土壤磷素形态及pH值和含phoD基因细菌Alpha多样性指数进行Pearson相关分析.含phoD基因细菌群落PD谱系多样性指数、Shannon多样性指数,Pielou均匀度指数均与Al-P呈显著的负相关关系(图3P<0.05),Shannon多样性指数,Pielou均匀度指数和Chao1指数与土壤pH值呈显著的正相关关系(图3P<0.05),PD谱系多样性指数与Ex-P呈显著的负相关关系(图3P<0.05).
属水平下含phoD基因细菌群落与不同磷素形态的冗余分析结果见图4.前两个排序轴共解释了数据60.1%的变异.pH值及各土壤磷赋存形态均对第一主轴存在较大贡献,其中pH值与Al-P是影响含phoD基因细菌群落最强烈的两个因素.在属水平下丰度排名靠前的链霉菌属与Al-P、Fe-P、OrP、O-P之间存在一定的正相关关系、拉姆利杆菌与Ca-P之间存在正相关关系,而慢生根瘤菌属与Al-P则表现出负相关关系.
进一步进行变差分解和层次分割分析(图5),结果显示土壤pH值、有机磷、及各无机磷形态共解释了群落丰度总变差的38.3%,其中单独效应相对重要的因素依次为Al-P(16.57%,P<0.05)、pH值(15.19%,P<0.05)和OrP(4.69%).同时观察到所有6个因素的单独效应均小于其边际效应,而O-P不论是单独效应(-1.91%)还是共同效应(-1.11%)均表现为极小的负效应.
我国土壤总磷(TP)含量很低,介于170~1090mg/kg之间[24],而一般总磷量小于800~1000mg/kg以下时对于植物来说土壤便会出现磷素供应不足的情况[5].本研究中的土壤总磷含量较低(图1),这与我国南方地区普遍缺磷的认识一致[25-26],也表明磷元素缺乏是限制重庆主城区马尾松生长的重要因素.本研究中无机磷元素赋存形态整体上表现出Fe-P、Al-P较多,而Ca-P较少的分布格局,与酸性土壤中富铝化程度高、铝氧化物络合能力强[27]而钙磷溶解度大等因素相关.
与在西南亚高山与[28]中丘陵区[25]等地的研究结果中TP、Ex-P等磷元素赋存形态多表现夏高冬低(或秋低)型不同,本研究发现重庆主城区马尾松表层土壤磷含量除Al-P外都呈现出冬高夏低的季节分布格局.这可能是因为一方面重庆地区夏季降水集中,降雨量较大,磷元素可能通过淋溶作用流失.另一方面,温暖的夏季土壤条件适宜植物及微生物的生长,土壤中的磷元素被快速的消耗,但由于马尾松松针在化学元素组成上属于较难分解的凋落物[29],磷元素由植物体归还到土壤中的过程相对较慢,导致土壤磷库的补充相对困难.Yu[30]等的研究结果也表明人工林缺乏相关的磷保留机制来应对磷循环过程中磷的快速消耗过程.同时,夏季土壤温度和微生物活性的增加也增强了土壤磷的矿化能力[31],而这部分矿化生成的磷会迅速的被土壤中大量的铝氧化物络合固定,这可能是本文结果中夏季Al-P含量高于冬季的原因.
本研究与相关研究比较可发现在不同地区、不同林分[32-34]土壤中含phoD基因细菌中变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)为主要优势类群(图2(a)).值得注意的是,在海洋、耕地等研究地点的研究结果中蓝细菌门(Cyanobacteria)细菌同时具有较高的相对丰度,这和本研究的结果不同.Bergkemper等[35]在一项强酸性(pH=3.52)森林土壤的研究中的结果与本研究相似,这可能是因为相较于海洋、耕地等研究地点,发育成熟的森林生态系统郁闭度高,而土壤蓝细菌普遍是自养微生物,没有充分的光能维持自身生长需要,而较低的土壤盐度水平及强酸性的土壤pH值也会强烈的威胁蓝细菌的生存[36].进一步分析属水平的相对丰度结果显示(图2(b)),链霉菌属与慢生根瘤菌属之间呈现出“此消彼长”的格局,推测这两种微生物之间可能存在较强的竞争关系.有研究表明链霉菌属中的一些细菌能够通过产生抗生素和其他抗微生物物质来抑制其他细菌的生长,同时链霉菌属与慢生根瘤菌属之间还存在“邻里防御性互惠”的复杂相互作用使其可以通过分泌去铁胺(Desferrioxamine)限制慢生根瘤菌的元素竞争能力从而限制其生长[37].慢生根瘤菌属的细菌则通常与植物根系结合形成根瘤,在解除自身氮限制的同时,能通过共生体系内的磷循环或直接分解土壤中较易分解的IP组分[31]满足自身需要从而加强竞争优势.这一结果表明含phoD基因细菌的种间关系也是影响群落结构的重要原因.值得注意的是除根瘤菌外许多相对丰度优势的含phoD基因细菌同时也参与到土壤氮循环的过程中,例如假单胞菌属中的细菌在土壤反硝化过程中起到重要作用[30,38].这说明土壤氮循环与磷循环之间密切相关,而土壤含phoD基因细菌可能从中起到“桥梁”作用.
一般认为,pH值是影响微生物群落结构最主要的环境因素[39-40].但在本文的结果中,含phoD基因细菌群落α多样性指数与Al-P的相关性比pH值更强(图3),更进一步的分析也显示不论是单独效应还是共同效应,Al-P对土壤含phoD基因细菌群落结构的影响均高于土壤pH值(图5).与本文的结果相同,在许多研究中都观察到低磷含量土壤中phoD基因的丰度显著高于高磷含量土壤[35].因此,磷限制对土壤含phoD基因细菌的影响要强于pH值.一方面,过去的研究已经证实phoD基因受Pho调节子的控制[41],土壤的磷限制会上调微生物对碱性磷酸酶相关基因的表达,特别是适应更广泛底物的phoD基因[42-43].另一方面,本研究所处西南地区酸性土有机质含量低、酸性强以及活性铝含量高,土壤中大量的活性铝离子会和土壤中的生物“争抢”Ex-P,这一环境行为直接影响了土壤磷的生物可利用性.Al-P成为土壤中无机磷含量最多的部分.与此同时,随着Al-P含量增加,铝毒害[44]也可能成为导致微生物多样性下降的重要原因之一.pH值一般通过影响酶活性来影响土壤微生物生存,也能通过影响土壤磷赋存形态结构间接影响土壤含phoD基因细菌的群落结构.而微生物可以主动改变土壤环境,在酸性的环境中创造pH值中性的微位点[45-46],削弱了土壤pH值对含phoD基因细菌群落结构的直接影响.这同时解释了为什么尽管碱性磷酸酶活性主要在中性和碱性环境中占主导地位[47],但在本文的结果里强酸性的土壤中含phoD基因细菌的丰富度和多样性都较高.
自然演替理论认为,随着演替的进行,自然界中土壤磷素的有效性在时间尺度上逐渐下降,大量活性磷被集中固定到周转缓慢的土壤有机质和植物、微生物残体等有机磷库之中,导致磷循环过程中活跃的磷组分不断减少[48].在本文的结果中,作为碱性磷酸酶反应底物的OrP含量在土壤TP中占比较高,却并没有表现出对土壤含phoD基因细菌的群落结构存在显著影响(图5).这可能是因为开展本研究的马尾松人工纯林属于演替后期的成熟林,但土壤中磷循环路径较简单,土壤有机磷库的稳定性较差但是恢复力较强[35].有研究表明土壤磷匮乏时微生物会与植物竞争土壤中矿化产生的Ex-P并将其保持在微生物磷库当中[49],这可能导致虽然土壤OrP的矿化过程活跃,但整体上具有活性的磷只是在OrP范围内周转.
而土壤中大量的活性铝离子的络合固定影响了土壤磷的生物可利用性,表现为Al-P对含phoD基因细菌群落具有强烈的影响.O-P虽然是含量最高(图1)的无机磷组分,但由于其极稳定的物理化学性质导致它在土壤中不参与磷循环过程,在本文的结果中对于群落丰度变差的解释也几乎没有贡献.整体来看,各影响因素的共同效应与单独效应的比较结果表明各磷形态及pH值在综合环境下对含phoD基因细菌的群落结构影响更大,即各磷形态之间的赋存关系会加强其对微生物群落的影响.
4.1 重庆四山地区土壤普遍缺磷,冬季和夏季土壤总磷(TP)含量均值分别为192.787mg/kg,169.512mg/kg.总磷(TP)、无机磷(IP)含量受到季节变化的显著影响,呈现冬高夏低的变化趋势.其中各无机磷形态含量分布较为稳定,呈现出闭蓄态磷(O-P)>铁结合磷(Fe-P)>铝结合磷(Al-P)>钙结合磷(Ca-P)>可交换态磷(Ex-P)的分布格局.
4.2 马尾松林土壤含phoD基因细菌群落主要由变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes)组成,平均相对丰度达到96%.土壤含phoD基因细菌多样性与Al-P之间存在显著负相关关系(P<0.05),与pH值之间存在显著正相关关系(P<0.05). Al-P和pH值是影响含phoD基因细菌群落结构特征的最主要因素.
  • 重庆市大学生创新训练项目(S202110611406)
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重庆市大学生创新训练项目(S202110611406)
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2种不同金属材料的力学参数

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genus
种数
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species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
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Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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