Article(id=1204800729945977819, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1204800727341310425, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250392, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1747324800000, receivedDateStr=2025-05-16, revisedDate=null, revisedDateStr=null, acceptedDate=1748534400000, acceptedDateStr=2025-05-30, onlineDate=1765176478134, onlineDateStr=2025-12-08, pubDate=1764777600000, pubDateStr=2025-12-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1765176478134, onlineIssueDateStr=2025-12-08, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1765176478134, creator=13701087609, updateTime=1765176478134, updator=13701087609, issue=Issue{id=1204800727341310425, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='12', pageStart='5191', pageEnd='5649', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1765176477513, creator=13701087609, updateTime=1765176611928, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1204801291189986067, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1204800727341310425, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1204801291189986068, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1204800727341310425, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=5452, endPage=5468, ext={EN=ArticleExt(id=1204800730206024675, articleId=1204800729945977819, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Effect of inoculating associative nitrogen-fixing bacteria on the root-associated bacterial community of sugarcane, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the effect of inoculating the associative nitrogen-fixing bacteria strain Paraburkholderia RBCS-17 on the root-associated bacterial community of sugarcane. Methods We employed 16S rRNA gene high-throughput sequencing combined with QIIME 2-based bioinformatics analysis to investigate the effects of inoculating Paraburkholderia RBCS-17 on the alpha diversity, beta diversity, composition, and co-occurrence network of the root-associated bacterial community of sugarcane. Results Inoculating Paraburkholderia RBCS-17 did not significantly affect the diversity but significantly changed the structure of the root-associated bacterial community. Further studies showed that the inoculation significantly increased the relative abundance of Burkholderia, Dyella, and Pseudomonas, while reducing the relative abundance of certain potentially detrimental bacteria such as Ralstonia. In addition, the inoculation altered the key module composition of the bacterial co-occurrence network in sugarcane roots, which suggested that inoculation might influence potential bacterial interactions. Conclusion Inoculating the associative nitrogen-fixing bacterial strain Paraburkholderia RBCS-17 modified the root-associated bacterial community structure of sugarcane, promoting the enrichment of potentially beneficial bacteria while suppressing potentially harmful ones. These findings provide new insights into the intricate interactions among associative nitrogen-fixing bacteria, host plants, and indigenous bacteria.

, correspAuthors=Yongjia ZHONG, authorNote=null, correspAuthorsNote=
*E-mail:
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目的 研究接种联合固氮菌副伯克霍尔德氏菌(Paraburkholderia) RBCS-17对甘蔗根系相关细菌群落的影响。 方法 采用16S rRNA基因高通量测序技术,结合基于QIIME 2的生物信息分析,探究接种联合固氮菌Paraburkholderia RBCS-17对甘蔗根系相关细菌群落的α多样性、β多样性、细菌组成及细菌共现网络的影响。 结果 接种联合固氮菌Paraburkholderia RBCS-17对甘蔗根系细菌群落多样性的影响不显著,但显著改变了根系细菌群落的结构。深入研究表明,接种固氮菌显著提高了伯克霍尔德氏菌属(Burkholderia)、戴氏菌属(Dyella)和假单胞菌属(Pseudomonas)的相对丰度,同时降低了某些潜在有害细菌如罗尔斯通氏菌属(Ralstonia)的相对丰度。此外,接种处理还改变了甘蔗根系细菌共现网络的关键模块组成,表明接种联合固氮菌可能影响了细菌之间潜在的相互作用。 结论 接种联合固氮菌Paraburkholderia RBCS-17能够通过改变甘蔗根系细菌群落结构,促进潜在有益细菌的富集,抑制潜在有害细菌,为联合固氮菌-植物-土著细菌复杂的相互作用提供了新的见解。

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A: Box plot of sugarcane root-associated bacterial alpha diversity based on Shannon index; B: Box plot of sugarcane root-associated bacterial alpha diversity based on Simpson index; C: Box plot of sugarcane root-associated bacterial alpha diversity based on ACE index; D: Box plot of sugarcane root-associated bacterial alpha diversity based on Chao1 index. ns: No significant difference., figureFileSmall=tJfP+5QaOS8jZ4F/c1t1oA==, figureFileBig=L5EoJg/wNi2E35giAjDkug==, tableContent=null), ArticleFig(id=1217784597849493706, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=CN, label=图1, caption=接种联合固氮菌甘蔗根系相关细菌的α多样性分析。A:基于Shannon指数的甘蔗根系相关细菌的α多样性箱线图;B:基于Simpson指数的甘蔗根系相关细菌的α多样性箱线图;C:基于丰度的覆盖估计值(abundance-based coverage estimator, ACE)指数的甘蔗根系相关细菌α多样性箱线图;D:基于Chao1指数的甘蔗根系相关细菌的α多样性箱线图。ns:无显著差异。, figureFileSmall=tJfP+5QaOS8jZ4F/c1t1oA==, figureFileBig=L5EoJg/wNi2E35giAjDkug==, tableContent=null), ArticleFig(id=1217784598134706395, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=EN, label=Figure 2, caption=PCoA, MDS, and ANOSIM analyses of sugarcane root-associated bacteria inoculated with combined nitrogen-fixing bacteria. A: PCoA plot based on Bray-Curtis distance matrix; B: PCoA plot based on Jaccard similarity index; C: PCoA plot based on Unweighted UniFrac distance matrix; D: PCoA plot based on Weighted UniFrac distance matrix (85% confidence interval ellipses shown for each treatment); E: Scatter plot derived from MDS analysis; F: Boxplot of ANOSIM analysis. The R-value ranges from -1 to 1. When R>0, differences between groups are greater than within groups (P<0.05 indicates statistical significance)., figureFileSmall=QjP2B/siK/bffg3cg2oF+A==, figureFileBig=VPVkpVRUD+xRcfbw6oYNSw==, tableContent=null), ArticleFig(id=1217784598239564008, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=CN, label=图2, caption=接种联合固氮菌甘蔗根系相关细菌的PCoA分析、MDS分析及ANOSIM分析。A:基于Bray-Curtis距离矩阵绘制的PCoA图;B:基于Jarcard相似度绘制的PCoA图;C:基于Unweighted UniFrac距离矩阵绘制PCoA图;D:基于Weighted UniFrac距离矩阵绘制的PCoA图(每个处理使用圆圈表示85%置信区间范围);E:基于MDS分析绘制的散点图;F:ANOSIM分析绘制的箱图[R-value介于(-1,1)之间,当R>0时说明组间差异大于组内差异,P<0.05表示统计具有显著性]。, figureFileSmall=QjP2B/siK/bffg3cg2oF+A==, figureFileBig=VPVkpVRUD+xRcfbw6oYNSw==, tableContent=null), ArticleFig(id=1217784598344421617, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=EN, label=Figure 3, caption=Composition analysis of sugarcane root-associated bacteria at the order and genus levels under different treatments. A: Stacked bar plot showing order-level taxonomic composition of sugarcane root-associated bacteria across treatments; B: Stacked bar plot showing genus-level taxonomic composition of sugarcane root-associated bacteria across treatments., figureFileSmall=a8vxUDLbUtY5Q/4WgQXOIQ==, figureFileBig=TEUs8RhIh/h6iVD3H1hUfg==, tableContent=null), ArticleFig(id=1217784598491222273, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=CN, label=图3, caption=不同处理下甘蔗根系相关细菌在目及属分类水平下的组成分析。A:不同处理下甘蔗根系相关细菌目水平物种组成堆叠柱状图;B:不同处理下甘蔗根系相关细菌属水平物种组成堆叠柱状图。, figureFileSmall=a8vxUDLbUtY5Q/4WgQXOIQ==, figureFileBig=TEUs8RhIh/h6iVD3H1hUfg==, tableContent=null), ArticleFig(id=1217784598612857098, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=EN, label=Figure 4, caption=LEfSe analysis of the LDA distribution bar diagram and the evolutionary branch diagram. A: LDA score distribution histogram from LEfSe analysis; B: Cladogram of differentially abundant taxa from LEfSe analysis., figureFileSmall=lfUVLgqsv+vpBPXXASRL2g==, figureFileBig=M8hjzHIBMDwDTw38XFF66A==, tableContent=null), ArticleFig(id=1217784598738686227, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=CN, label=图4, caption=LEfSe分析的LDA分布柱状图与进化分支图。A:LEfSe分析LDA分布柱状图;B:LEfSe分析进化分支图。, figureFileSmall=lfUVLgqsv+vpBPXXASRL2g==, figureFileBig=M8hjzHIBMDwDTw38XFF66A==, tableContent=null), ArticleFig(id=1217784598830960925, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=EN, label=Figure 5, caption=Analysis of species abundance differences in STAMP software at family and genus levels. A: Family-level taxonomic bar plot showing treatment-specific differences in sugarcane root-associated bacteria; B: Genus-level taxonomic bar plot illustrating treatment-group variations in sugarcane root-associated bacteria., figureFileSmall=ihoZ/r/ywgPssLR3fM+VCw==, figureFileBig=4t+k0rMy1YDSEseOJUG3Uw==, tableContent=null), ArticleFig(id=1217784599153922347, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=CN, label=图5, caption=STAMP软件分析科水平和属水平下的差异物种。A:甘蔗根系相关细菌科水平的组间差异分析;B:甘蔗根系相关细菌属水平的组间差异分析。, figureFileSmall=ihoZ/r/ywgPssLR3fM+VCw==, figureFileBig=4t+k0rMy1YDSEseOJUG3Uw==, tableContent=null), ArticleFig(id=1217784599254585649, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=EN, label=Figure 6, caption=Co-occurrence network of root-associated bacterial under different treatments. A: Co-occurrence network of sugarcane root-associated bacteria at the genus level in the control group; B: Co-occurrence network of sugarcane root-associated bacteria at the genus level in the inoculated group., figureFileSmall=U5IKZItS7D2KKOGcs1ukhQ==, figureFileBig=RqdFSyQbcx59zcxS79o8rg==, tableContent=null), ArticleFig(id=1217784599334277436, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=CN, label=图6, caption=不同处理的根系相关细菌共现网络。A:对照组甘蔗根系相关细菌属水平的共现网络图;B:接种组甘蔗根系相关细菌属水平的共现网络图。, figureFileSmall=U5IKZItS7D2KKOGcs1ukhQ==, figureFileBig=RqdFSyQbcx59zcxS79o8rg==, tableContent=null), ArticleFig(id=1217784599455912263, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=EN, label=Table 1, caption=

Co-occurrence network topology parameters of root-associated bacterial under different treatments

, figureFileSmall=null, figureFileBig=null, tableContent=

处理

Treatment

节点数

Nodes number

边数

Edges number

节点平均度

Node average degrees

聚类系数

Clustering coefficient

模块化

Modularity

Control821313.1950.9230.918
Inoculation811243.0620.9010.713
), ArticleFig(id=1217784599564964173, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1204800729945977819, language=CN, label=表1, caption=

不同处理的根系相关细菌共现网络拓扑参数

, figureFileSmall=null, figureFileBig=null, tableContent=

处理

Treatment

节点数

Nodes number

边数

Edges number

节点平均度

Node average degrees

聚类系数

Clustering coefficient

模块化

Modularity

Control821313.1950.9230.918
Inoculation811243.0620.9010.713
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接种联合固氮菌对甘蔗根系相关细菌群落的影响
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李瑞瑞 , 刘冉 , 廖红 , 钟永嘉 *
微生物学报 | 研究报告 2025,65(12): 5452-5468
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微生物学报 | 研究报告 2025, 65(12): 5452-5468
接种联合固氮菌对甘蔗根系相关细菌群落的影响
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李瑞瑞, 刘冉, 廖红, 钟永嘉*
作者信息
  • 福建农林大学 资源与环境学院,根系生物学研究中心,福建 福州
Effect of inoculating associative nitrogen-fixing bacteria on the root-associated bacterial community of sugarcane
Ruirui LI, Ran LIU, Hong LIAO, Yongjia ZHONG*
Affiliations
  • Root Biology Center, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China
出版时间: 2025-12-04 doi: 10.13343/j.cnki.wsxb.20250392
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目的 研究接种联合固氮菌副伯克霍尔德氏菌(Paraburkholderia) RBCS-17对甘蔗根系相关细菌群落的影响。 方法 采用16S rRNA基因高通量测序技术,结合基于QIIME 2的生物信息分析,探究接种联合固氮菌Paraburkholderia RBCS-17对甘蔗根系相关细菌群落的α多样性、β多样性、细菌组成及细菌共现网络的影响。 结果 接种联合固氮菌Paraburkholderia RBCS-17对甘蔗根系细菌群落多样性的影响不显著,但显著改变了根系细菌群落的结构。深入研究表明,接种固氮菌显著提高了伯克霍尔德氏菌属(Burkholderia)、戴氏菌属(Dyella)和假单胞菌属(Pseudomonas)的相对丰度,同时降低了某些潜在有害细菌如罗尔斯通氏菌属(Ralstonia)的相对丰度。此外,接种处理还改变了甘蔗根系细菌共现网络的关键模块组成,表明接种联合固氮菌可能影响了细菌之间潜在的相互作用。 结论 接种联合固氮菌Paraburkholderia RBCS-17能够通过改变甘蔗根系细菌群落结构,促进潜在有益细菌的富集,抑制潜在有害细菌,为联合固氮菌-植物-土著细菌复杂的相互作用提供了新的见解。

副伯克霍尔德氏菌RBCS-17  /  细菌群落结构  /  细菌多样性  /  细菌组成  /  伯克霍尔德氏菌属

Objective To investigate the effect of inoculating the associative nitrogen-fixing bacteria strain Paraburkholderia RBCS-17 on the root-associated bacterial community of sugarcane. Methods We employed 16S rRNA gene high-throughput sequencing combined with QIIME 2-based bioinformatics analysis to investigate the effects of inoculating Paraburkholderia RBCS-17 on the alpha diversity, beta diversity, composition, and co-occurrence network of the root-associated bacterial community of sugarcane. Results Inoculating Paraburkholderia RBCS-17 did not significantly affect the diversity but significantly changed the structure of the root-associated bacterial community. Further studies showed that the inoculation significantly increased the relative abundance of Burkholderia, Dyella, and Pseudomonas, while reducing the relative abundance of certain potentially detrimental bacteria such as Ralstonia. In addition, the inoculation altered the key module composition of the bacterial co-occurrence network in sugarcane roots, which suggested that inoculation might influence potential bacterial interactions. Conclusion Inoculating the associative nitrogen-fixing bacterial strain Paraburkholderia RBCS-17 modified the root-associated bacterial community structure of sugarcane, promoting the enrichment of potentially beneficial bacteria while suppressing potentially harmful ones. These findings provide new insights into the intricate interactions among associative nitrogen-fixing bacteria, host plants, and indigenous bacteria.

Paraburkholderia RBCS-17  /  bacterial community structure  /  bacterial diversity  /  bacterial community composition  /  Burkholderia
李瑞瑞, 刘冉, 廖红, 钟永嘉. 接种联合固氮菌对甘蔗根系相关细菌群落的影响. 微生物学报, 2025 , 65 (12) : 5452 -5468 . DOI: 10.13343/j.cnki.wsxb.20250392
Ruirui LI, Ran LIU, Hong LIAO, Yongjia ZHONG. Effect of inoculating associative nitrogen-fixing bacteria on the root-associated bacterial community of sugarcane[J]. Acta Microbiologica Sinica, 2025 , 65 (12) : 5452 -5468 . DOI: 10.13343/j.cnki.wsxb.20250392
自然界中的植物并非单独存在,其体内及周围栖息着数以万计的微生物(包括细菌、真菌等),这些微生物群体被统称为植物微生物组[1]。根系作为植物与土壤环境相互作用的关键界面,不仅是吸收矿质养分的主要器官,也是植物与土壤微生物进行信号交流和物质交换的关键部位[2]。与植物根系相关的微生物群落被称为根系微生物群,主要包括共生菌(如固氮菌、菌根真菌)、病原体和共栖菌[3]。根据受宿主植物影响和调控的程度,根系微生物可划分为3类,即根际微生物(根周围约2 mm范围内)、根表微生物以及根内微生物[4]。研究表明根系微生物通过多种途径影响宿主植物的生长和健康,包括促进养分吸收利用、调控宿主植物免疫系统及对非生物逆境的适应等[5]。由于根系微生物在植物养分吸收利用和生物防治等方面起着关键作用,定向调控根系微生物的有益功能已被视为推动农业绿色发展的关键策略[6]
甘蔗(Saccharum officinarum L.)作为全球重要的糖料和生物能源作物,具有极高的经济价值[7]。作为一种高光合效率的C4作物,甘蔗的固碳效率高于C3植物。为维持体内碳氮平衡,甘蔗需要吸收大量氮素以满足正常生长和发育的需求[8-9]。目前,在农业生产中氮肥施用仍是保障甘蔗产量和糖分积累的主要措施。然而,氮肥利用率普遍较低,仅有15%-50%的氮素能被甘蔗有效吸收利用[10]。氮肥过量或不合理施用引发了一系列问题:一方面,过量施用氮肥可能抑制作物生长,甚至导致减产;另一方面,未被植物吸收的氮通过淋溶或气体排放进入环境,造成水体富营养化、温室气体排放等环境问题[11]。面对这一现状,如何提高氮肥利用效率并减少施用量已成为当前甘蔗种植中亟待解决的重要课题,这不仅关系到农业生产的经济效益,也是实现农业可持续发展的重要方向。
近年来,作物相关的固氮微生物因具有降低化学氮肥使用潜力的特性备受科研界关注[12-17]。据报道,接种内生固氮菌Y-1a可通过增加甘蔗的株高、茎径、平均节间长度和单茎重来促进甘蔗生长[18]。定殖于野生水稻叶片的菌株草螺菌属(Herbaspirillum sp.) B501能够通过联合固氮作用显著提高水稻植株的15N浓度[19]。此外,天然存在于玉米根际的洋葱伯克霍尔德菌除可促进玉米生长外,部分菌株还能有效抑制有害根际微生物的生长[12]。在越南低肥力的酸性土壤条件下接种越南伯克霍尔德菌(Burkholderiavietnamiensis) TVV75菌株可使水稻产量显著增加[20]。研究还指出,伯克霍尔德氏菌/副伯克霍尔德氏菌(Burkholderia/Paraburkholderia)可能是甘蔗核心微生物群落的重要组分,是对甘蔗有益的内生细菌[21]。例如,定殖在甘蔗根部的澳大利亚伯克霍尔德氏菌(Burkholderia australis sp.) 11能在氮素含量低的条件下,通过生物固氮作用固定氮素,进而促进甘蔗生长[16]。本课题组最新研究表明,在低氮条件下甘蔗通过根系分泌黏液以及黏液中的苯甲酸特异性招募具有固氮能力的Burkholderia/Paraburkholderia,进一步发现gus [β-葡萄糖醛酸酶基因(β-glucuronidase gene)]标记的Paraburkholderia RBCS-17在甘蔗根系显著定殖,并通过固氮作用促进甘蔗生长[17]。尽管禾本科作物联合固氮的研究已取得一定进展,但接种固氮微生物如何影响作物根系相关的微生物群落仍不清楚。
本研究以课题组前期分离的甘蔗联合固氮菌Paraburkholderia RBCS-17为供试菌株,利用16S rRNA基因测序技术及生物信息学分析研究接种联合固氮菌对甘蔗根系细菌群落的影响,以期为理解外来菌对土著细菌群落的影响,以及固氮菌-宿主植物-根系细菌复杂的互作关系提供新的见解,也为农业生产中应用有益微生物提供理论指导。
本研究所用的甘蔗品种为‘ROC22’。甘蔗组培苗由福建农林大学国家甘蔗工程技术研究中心提供,在本实验室进行扩繁、壮苗及生根培养。炼苗完成后,将组培苗分成单株并去除植株的分蘖。挑选长势一致的单株,用二级水洗去根部残余的培养基,用海绵夹住植株,塞入打孔的黑色泡沫板中。在面包箱中加入营养液,使甘蔗根部浸没其中,置于人工气候室中培养14 d,待新根长出约5 cm后用于开展以下试验。
试验所用固氮菌株为Paraburkholderia RBCS-17,分离自低氮栽培条件下的甘蔗根系[17]。试验所用土壤采集自福建农林大学国家甘蔗工程技术研究中心的甘蔗试验地(119°14′E,26°5′N)的田间土壤,使用前用孔径4 mm的筛子过筛以去除石子、植物碎屑等杂质。
将菌株Paraburkholderia RBCS-17从-80 ℃取出解冻,在LB平板上划线活化,挑取单克隆至LB液体培养基中,于28 ℃、220 r/min的恒温摇床中培养24 h (培养至平台期,OD600为2.0)。将培养结束的菌液6 000 r/min离心20 min收集菌体,并进一步用低氮营养液(530 μmol/L N)洗去残留的培养基,再用低氮营养液重悬菌体,调节OD600至0.2备用;准备甘蔗幼苗,使用蛭石:珍珠岩=2:1的生长基质种植甘蔗幼苗,通过在甘蔗根基部浇灌20 mL OD600为0.2的菌液,使菌液与基质混合均匀(以接种等量的无菌水作为对照组),然后置于植物生长间共培养1周。甘蔗生长过程中所用营养液为改良的霍格兰营养液[22],氮水平设为530 μmol/L N。之后将共培养的甘蔗连同生长基质移植到大田条件下,每个处理均设置4次重复。小区面积为7 m2 (5.0 m×1.4 m),每个重复设2垄,垄长为5 m株距1 m,试验小区采用随机区组排列。待甘蔗生长至伸长期,采集分析接种联合固氮菌对甘蔗根系细菌群落的影响。
在大田条件下生长至伸长期的甘蔗,利用铁锹挖取甘蔗根系,轻轻抖去根系上松散的土壤,收集甘蔗根系。将3株独立的甘蔗根系混合为一个生物学重复,每个处理收集5份生物学重复。因此,分别收集5份对照处理和5份接种联合固氮菌Paraburkholderia RBCS-17的甘蔗根系,并立即用干冰将甘蔗根系速冻,带回实验室进一步处理。将收集的根系样品放入三角瓶中,加入无菌PBS溶液没过根样,在220 r/min摇床中振荡以洗去甘蔗根系表面的土壤,重复多次直至振荡后无明显土壤洗下[23]。将甘蔗根系样品取出,用吸水纸吸去表面水分,然后用液氮将甘蔗根系研磨至粉末状。接着取0.5 g的组织粉末,利用PowerSoil DNA提取试剂盒(Qiagen公司)提取组织总DNA,DNA的提取按照试剂盒说明书进行操作。为避免植株组织DNA的影响,以DNA为模板,使用引物799F (5′-AACMGGATTAGATACCCKG-3′)和1193R (5′-ACGTCATCCCCACCTTCC-3′)扩增细菌16S rRNA基因的V5-V7区[24-25],PCR产物利用QIAquick胶提取试剂盒(Qiagen公司)进行纯化,产物提交到上海美吉生物医药科技有限公司,利用NovaSeq平台进行扩增子测序分析(Illumina公司)。
Illumina NovaSeq 6000平台下机数据使用QIIME 2平台进行数据处理[26]。原始数据使用q2-demux导入,使用cutadapt插件去除序列引物和linker。双端测序序列使用vsearch进行合并,保留q-score>25质量的序列进行下一步分析[27]。使用deblur插件对序列进行去噪,矫正测序错误碱基,保留序列长度以保证有99.50%以上的序列达到要求。使用去噪后生成的特征表进行微生物数据挖掘,并使用SILVA数据库按照97%相似度注释OTUs[28]。微生物群落结构多样性使用QIIME 2的diversity插件进行计算,生成α多样性并使用Kruskal-Wallis test进行非参数检验。β多样性的距离矩阵生成后,在R语言(version 4.0.3)中使用vegan包和ggplot2包绘制主坐标分析(principal coordinates analysis, PCoA)图,并使用vegan包对各个距离矩阵进行组间差异分析(analysis of similarities, ANOSIM)。使用QIIME 2的taxa插件生成物种堆叠柱状图,对不同分类水平的微生物种类进行统计,并使用R语言的ggplot2绘图。差异微生物分析使用在线线性判别分析(linear discriminant analysis effect size, LEfSe)程序和STAMP软件对不同处理的biomarker进行分析[29]
α多样性分析显示,Shannon (图1A)、Simpson (图1B)、ACE (图1C)和Chao1 (图1D)等指数在甘蔗对照组和实验组之间均无显著性差异(P>0.05),表明接种联合固氮菌Paraburkholderia RBCS-17未显著改变甘蔗根系细菌群落的物种丰富度和均匀度(图1)。通过β多样性分析发现,接种处理显著影响了甘蔗根系细菌群落结构。PCoA分析结果表明,对照组和实验组的样品组间存在明显的区别,组内呈现明显聚类趋势(图2A-2D)。此外,实验组和对照组在多维尺度分析(multidimensional scaling, MDS)中也呈现出类似趋势(图2A)。上述结果说明接种联合固氮菌对细菌群落结构有一定影响。进一步通过ANOSIM分析结合置换多元方差分析(permutational multivariate analysis of variance, PERMANOVA)检验,对组间细菌群落结构的差异进行统计分析。结果显示,接菌与不接菌处理下的组间差异大于组内差异(R=0.968, P=0.01),即接种联合固氮菌显著改变了甘蔗根系相关的细菌群落结构(P<0.05) (图2B)。综上所述,研究表明接种Paraburkholderia RBCS-17对甘蔗根系相关的细菌群落多样性影响不显著,而对根系相关细菌群落的结构影响显著。
为明确接种联合固氮菌Paraburkholderia RBCS-17对甘蔗根系相关细菌群落组成的影响,分别从目和属分类水平对细菌组成进行比较分析。首先,目水平的物种堆积柱状图分析结果显示(图3A),对照组在目水平上相对丰度大于1.0%的优势细菌[30]分别为β-变形菌目(Betaproteobacteriales, 63.7%)、棒杆菌目(Corynebacteriales, 12.7%)、根瘤菌目(Rhizobiales, 11.6%)、假单胞菌目(Pseudomonadales, 4.5%)、黄单胞菌目(Xanthomonadales, 2.8%)、芽孢杆菌目(Bacillales, 1.3%)。接菌处理在目水平上的优势细菌分别为β-变形菌目(Betaproteobacteriales, 45.0%)、棒杆菌目(Corynebacteriales, 18.6%)、根瘤菌目(Rhizobiales, 13.9%)、黄单胞菌目(Xanthomonadales, 9.0%)、假单胞菌目(Pseudomonadales, 8.7%)、芽孢杆菌目(Bacillales, 1.7%)。进一步对属水平的物种堆积柱状图进行分析(图3B),结果显示,对照组的优势细菌分别为罗尔斯通氏菌属(Ralstonia, 29.3%)、红球菌属(Rhodococcus, 12.6%)、伯克霍尔德氏菌属(Burkholderia, 12.1%)、草螺菌属(Herbaspirillum, 11.5%)、苍白杆菌属(Ochrobactrum, 7.9%)、假单胞菌属(Pseudomonas, 4.5%)。接菌处理的优势细菌分别为伯克霍尔德氏菌属(Burkholderia, 39.3%)、红球菌属(Rhodococcus, 18.6%)、苍白杆菌属(Ochrobactrum, 11.9%)、假单胞菌属(Pseudomonas, 8.7%)、戴氏菌属(Dyella, 8.7%)。
本研究还进一步使用LEfSe分析对不同处理条件下的甘蔗根系的生物标记物进行分析。LEfSe分析结果显示:接种Paraburkholderia RBCS-17后根系菌群组成发生显著改变(图4A4B),主要富集的是β-变形菌纲(Betaproteobacteriales)的伯克霍尔德氏菌属(Burkholderia),γ-变形菌纲(Gammaproteobacteria)的戴氏菌属(Dyella)、假单胞菌属(Pseudomonas),α-变形菌纲(Alphaproteobacteria)的苍白杆菌属(Ochrobactrum)以及δ-变形菌纲(Deltaproteobacteria)的蛭弧菌属(Bdellovibrio)。此外,细菌组成分析结果显示(图3):在目水平下,接菌处理的黄单胞菌目(Xanthomonadales)的相对丰度与对照组相比增加了6.2%,且差异极显著(P<0.01)。相比之下,β-变形菌目(Betaproteobacteriales)的相对丰度则显著降低(P<0.05),相对丰度下降了18.7%。在属分类水平下,接菌处理的甘蔗根系戴氏菌属(Dyella)的相对丰度与对照组相比增加了6.0%,且差异极显著(P<0.01),伯克霍尔德氏菌属(Burkholderia)相对丰度增加了27.3% (P<0.05)。
为进一步分析接种Paraburkholderia RBCS-17后甘蔗根系显著变化的细菌,利用STAMP软件分别对在科和属分类水平下显著变化的细菌进行分析。科分类水平的分析结果显示(图5A),接种Paraburkholderia RBCS-17后罗河杆菌科(Rhodanobacteraceae)和假单胞菌科(Pseudomonadaceae)相对丰度显著升高(P<0.05),而伯克霍尔德氏菌科(Burkholderiaceae)相对丰度显著降低(P<0.05)。属分类水平的分组差异图结果表明(图5B),接种Paraburkholderia RBCS-17处理伯克霍尔德氏菌属(Burkholderia)、戴氏菌属(Dyella)和假单胞菌属(Pseudomonas)的相对丰度显著增加(P<0.05),而草螺菌属(Herbaspirillum)、马赛菌属(Massilia)、根瘤菌属(Rhizobium)、罗尔斯通氏菌属(Ralstonia)、诺卡氏菌属(Nocardioides)的相对丰度显著降低(P<0.05)。上述结果表明,接种联合固氮菌显著影响甘蔗根系的部分细菌的相对丰度。
微生物共现网络可揭示微生物间复杂的相互作用关系,能反映潜在的生态联系和生态过程。进一步基于微生物群落在属分类水平的相关性,对接种Paraburkholderia RBCS-17处理和对照组的根系相关细菌共现网络进行构建和解析。如表1所示,接种Paraburkholderia RBCS-17处理细菌的共现网络由81个节点和124条边组成,对照组的细菌共现网络由82个节点和131条边组成。此外,接菌处理和对照组的细菌共现网络的模块化程度分别为0.713和0.918,表明两者的细菌群落的网络模块化均较高(模块化指数大于0.400),而对照组细菌群落的网络模块化高于接菌处理,表明对照组细菌群落的共现网络模块的连接更复杂。此外,接菌处理和对照组的细菌群落聚类系数为0.901和0.923,节点平均度分别为3.062和3.195,表明接菌处理下细菌群落的稳定性小于对照组。同时,接种Paraburkholderia RBCS-17对甘蔗根系相关细菌网络核心模块的细菌群落产生了一定的影响(图6A)。对照组的核心模块细菌(紫色模块)由假双头斧形菌(Pseudolabrys)、甲基杆菌(Methylobacterium)、微枝形杆菌(Microvirga)、鞘氨醇杆菌(Sphingobium)以及间孢囊菌(Intrasporangium)属的细菌组成(图6B),接种Paraburkholderia RBCS-17的核心模块细菌(紫色模块)分别由伯克霍尔德氏菌/副伯克霍尔德氏菌(Burkholderia/Paraburkholderia)、草螺菌属(Herbaspirillum)、鞘氨醇单胞菌(Sphingomonas)、嗜酸链球菌(Streptacidiphilus)、寡养单胞菌(Stenotrophomonas)、慢生根瘤菌(Bradyrhizobium)和代夫特菌(Delftia)属的细菌组成,表明接种联合固氮菌改变了重要模块的细菌组成。
根系微生物与植物的养分吸收、应对逆境胁迫以及健康等密切相系[4]。根系微生物群落的组成和动态变化受多种因素调控,主要包括土壤理化性质、气候条件、宿主植物基因型以及微生物互作等[31]。在本研究中,接种联合固氮菌Paraburkholderia RBCS-17显著改变了甘蔗根系相关的细菌群落结构(图2),但细菌群落多样性(Shannon、Simpson、ACE和Chao1等指数)并未发生明显变化(图1)。这表明外源微生物添加显著影响了甘蔗根系相关细菌群落的塑造,该研究结果与前人研究一致,说明外源微生物可显著影响作物根系相关的微生物群落结构[23,32-34]。进一步对其组成分析发现,接种联合固氮菌Paraburkholderia RBCS-17显著提高了伯克霍尔德氏菌属(Burkholderia)、戴氏菌属(Dyella)和假单胞菌属(Pseudomonas)的相对丰度(图3)。值得注意的是,随着基因组研究的深入,于2017年基于系统发育分析对Burkholderia属进行了拆分,其中具有植物促生和环境适应性的菌种被重新归类为Paraburkholderia[35]。鉴于ParaburkholderiaBurkholderia存在密切的系统发育关系,分析中检测到的Burkholderia属细菌信号可能包含了目标接种的Paraburkholderia菌株,这一结果也印证了接种处理的有效性。
Paraburkholderia RBCS-17可能通过2种途径影响根系微生物组:一是与土著细菌直接相互作用;二是通过与宿主植物互作间接调控细菌群落。这些富集的细菌具有重要的生态功能:据报道,伯克霍尔德氏菌属(Burkholderia)中的一些菌种具备固氮能力,能够直接将大气中的氮气转化为植物可利用的铵态氮,从而减少化学氮肥的使用[17]。假单胞菌属(Pseudomonas)常因其产生植物激素(如生长素、细胞分裂素)的能力而受到关注,这些激素对刺激植物细胞分裂、促进根系发育及提高植物抗逆性具有重要作用[36]。虽然有关戴氏菌属(Dyella)的研究相对较少,但近年来也发现其可能具有促进植物生长及生物防治病害的潜力,能通过竞争抑制有害病原菌的生长维护植物健康[37]
在根系相关环境中,菌与菌之间可能形成复杂的相互作用网络。本研究显著富集的伯克霍尔德氏菌属、戴氏菌属和假单胞菌属之间可能会竞争根系分泌物中的营养物质,如糖类、氨基酸等。由于伯克霍尔德氏菌属和假单胞菌属本身具有较强的代谢能力和产生抗菌物质的能力[36,38],可能在竞争中占据一定优势。此外,伯克霍尔德氏菌属或假单胞菌属产生的植物激素、有机酸等也可能为戴氏菌属提供有利的生长条件,而戴氏菌属可能通过其他方式,如改善土壤结构,间接促进伯克霍尔德氏菌属和假单胞菌属的生长[36-38]。总体而言,接种Paraburkholderia RBCS-17通过改变甘蔗根系细菌群落组成促进了甘蔗根系潜在有益细菌的富集,这些细菌之间竞争与协同并存的生态关系共同维持了群落的动态平衡,对植物健康生长和土壤生态系统功能具有重要作用。这一发现为理解微生物-植物-环境互作机制提供了新的见解。
生物固氮是土壤氮素循环的重要组成部分,它为农业生态系统贡献了约一半的总氮输入[39-40]。这一过程不仅显著降低了农业生产对化学氮肥的依赖,更在维持生态系统氮平衡和促进可持续发展方面发挥着不可替代的作用。近年来,联合固氮已成为研究热点,受到广泛关注。其固氮效率虽低于典型的根瘤菌-豆科植物共生体系,但具有更广泛的宿主适应性,特别是在禾本科作物(如甘蔗、玉米、水稻等)生产中显示出巨大的应用潜力[12-17]
本课题组最新研究成果表明,在低氮环境条件下接种联合固氮菌Paraburkholderia RBCS-17可有效促进甘蔗和玉米对氮素的获取,提高植株的氮含量[16]。此外也有相关报道,接种联合固氮菌对甘蔗的氮素吸收效率具有显著的促进作用,同时甘蔗植株的干重显著增加[34,41-42]。本研究结果显示,接种Paraburkholderia RBCS-17菌株能显著提升伯克霍尔德氏菌(Burkholderia)的相对含量(图3图4)。同时发现接种联合固氮菌Paraburkholderia RBCS-17后甘蔗根系相关细菌网络核心模块(紫色模块)出现了Burkholderia/Paraburkholderia属的细菌,表明接种联合固氮菌改变了甘蔗根系相关细菌重要模块的组成(图6)。据报道,伯克霍尔德氏菌属的部分成员已被证明具有固氮能力[16-17]。接种联合固氮菌Paraburkholderia RBCS-17可能通过调控甘蔗根系相关细菌群落,特别是通过增加有益菌伯克霍尔德氏菌的丰度来改善甘蔗根系的微生态环境。
对照组罗尔斯通氏菌(Ralstonia)的相对丰度较高,而接种后的甘蔗根系相关细菌中其相对丰度显著降低,伯克霍尔德氏菌(Burkholderia)则成为了最大的优势菌(图3图4)。目前的研究表明,罗尔斯通氏属(Ralstonia)包含一些重要的植物病原菌,其中青枯雷氏菌(Ralstonia solanacearum)作为最常见的植物病原细菌之一,能引起多种农作物产生细菌性萎蔫病害,造成巨大的经济损失[43]。这些结果表明,接种Paraburkholderia RBCS-17能够降低甘蔗根系潜在有害细菌的相对丰度,从而减少甘蔗病害的发生。这有助于减少农药的使用量,降低农业生产成本,同时也有助于保护生态环境和人体健康。通过生物防治的方式控制甘蔗病害是实现农业可持续发展的重要途径之一。
本研究发现,接种Paraburkholderia RBCS-17显著改变了甘蔗根系相关细菌群落结构,提高了伯克霍尔德氏菌属(Burkholderia)、戴氏菌属(Dyella)和假单胞菌属(Pseudomonas)等潜在有益细菌的相对丰度,降低了潜在有害细菌的丰度。接种处理还改变了细菌共现网络的关键模块,增强了细菌间互作。这些发现为开发微生物肥料、促进甘蔗生长及减少氮肥依赖提供了科学依据,对甘蔗生产的可持续发展具有重要意义。
李瑞瑞:数据分析,论文撰写及修改;刘冉:研究构思和设计,数据收集与处理;廖红:研究构思和设计,论文审阅;钟永嘉:研究构思与设计,基金获取,论文撰写和修改。
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
  • 福建省自然科学基金(2022J01617)
  • 福建农林大学杰出青年科研人才计划(xjq202120)
  • 国家重点研发计划(2021YFF1000500)
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2025年第65卷第12期
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doi: 10.13343/j.cnki.wsxb.20250392
  • 接收时间:2025-05-16
  • 首发时间:2025-12-08
  • 出版时间:2025-12-04
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  • 收稿日期:2025-05-16
  • 录用日期:2025-05-30
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Natural Science Foundation of Fujian Province(2022J01617)
福建省自然科学基金(2022J01617)
Distinguished Young Scientific Research Talent Program of Fujian Agriculture and Forestry University(xjq202120)
福建农林大学杰出青年科研人才计划(xjq202120)
National Key Research and Development Program of China(2021YFF1000500)
国家重点研发计划(2021YFF1000500)
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    福建农林大学 资源与环境学院,根系生物学研究中心,福建 福州

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2种不同金属材料的力学参数

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

Genus
种数
Number of
species
占总种数比例
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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