Article(id=1276190679491547234, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276190518317023323, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2024.05.020, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1667145600000, receivedDateStr=2022-10-31, revisedDate=1675612800000, revisedDateStr=2023-02-06, acceptedDate=null, acceptedDateStr=null, onlineDate=1782197168467, onlineDateStr=2026-06-23, pubDate=1716566400000, pubDateStr=2024-05-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782197168467, onlineIssueDateStr=2026-06-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782197168467, creator=13701087609, updateTime=1782197168467, updator=13701087609, issue=Issue{id=1276190518317023323, tenantId=1146029695717560320, journalId=1235980609244409860, year='2024', volume='45', issue='5', pageStart='873', pageEnd='1093', issueExtLink='null', onlineDate='null', pubDate='1716566400000', pubDateStr='2024-05-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782197130040, creator='13701087609', updateTime=1782197317472, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276191304694493587, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276190518317023323, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276191304694493588, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276190518317023323, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1052, endPage=1060, ext={EN=ArticleExt(id=1276190679785148516, articleId=1276190679491547234, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Correlation Analysis Between Anthocyanins Content in Pennisetum purpureum Leaves and Phyllosphere Bacterial Community, columnId=1236318328365577171, journalTitle=Chinese Journal of Tropical Crops, columnName=Agricultural Ecology & Environmental Protection, runingTitle=null, highlight=null, articleAbstract=

Anthocyanins, also called anthocyans, are a group of plant secondary metabolites of flavonoid derivatives. Anthocyanins play an important role in mediating plant stress responses. However, whether anthocyanins are also involved in regulating phyllosphere bacterial communities remains unknown. To evaluate the differences of phyllosphere bacterial communities in plants with different anthocyanin contents, this study analyzed the phyllosphere bacterial communities of two cultivars of elephant grass with contrasting anthocyanins content, Pennisetum purpureum cv. Purple (Purple) with a high amount of anthocyanins and P. purpureum cv. Mott (Mott) with relatively low anthocyanins. The anthocyanins content determination results showed that the anthocyanins content in Purple leaves was nearly 20 times of that in Mott leaves. High throughput sequencing of 16S rRNA and analysis of the microbiome community indicated that though alpha diversity was not significantly changed, the bacterial communities of Purple was significantly different from that of Mott. In Purple, the relative abundance of endopphytic Proteobacteria and Bacteroidetes, and epiphytic Deinococus-Thermus were significantly increased, whereas epiphytic Bacteroidetes and Firmicutes were significantly decreased. The composition of microbiomes (ANCOM) at the OTU level, the relative abundance analysis at the genus level and correlation analysis between epiphytes and anthocyanins showed that the relative abundance of Methylobacterium, Unclassified Methylobacteriaceae and Deinococcus, which showed a significant positive correlation with anthocyanins content, were significantly increased in the episphere of Purple. In summary, the phyllosphere bacterial communities were significantly different between Purple and Mott. The findings from this work would provide new insights into the understanding of the superior forage value of P. purpureum cv. Purple, and lay a foundation to further investigate the mechanism of how plant secondary metabolism products regulate phylloshpere microorganisms.

, authors=null, authorsList=Junjie LIU, Yuanyuan HUANG, Xiao LEI, Liangfa GE, authorCompany=null, correspAuthors=Liangfa GE, 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, fund=null), CN=ArticleExt(id=1276190683098648690, articleId=1276190679491547234, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=象草叶片花青素含量与叶际细菌群落的相关性分析, columnId=1236292523270918153, journalTitle=热带作物学报, columnName=农业生态与环境保护, runingTitle=null, highlight=null, articleAbstract=

花青素又称为花色素,是植物黄酮类次生代谢产物。大量研究表明,花青素在调控植物的抗逆反应中起着重要作用,但花青素是否影响植物的叶际细菌仍未知。为探究叶片花青素与叶际细菌间的相关性,本研究以花青素含量高的紫色象草(Pennisetum purpureum cv. Purple,Purple)及花青素含量低的矮象草(P. purpureum cv. Mott,Mott)为研究材料,通过对叶片花青素含量进行定量分析,利用16S rRNA高通量测序对其叶际细菌群落结构及相对丰度进行分析,比较紫色象草和矮象草叶际细菌群落结构的差异性。结果表明:紫色象草叶片的花青素含量是矮象草的近20倍。紫色象草和矮象草的叶际细菌α多样性无显著差异,但是β多样性差异较大。操作分类单元(operational taxonomic unit,OTU)水平的相对丰度主成分分析结果表明,紫色象草和矮象草的叶际细菌群落组成呈显著差异。与矮象草相比,紫色象草叶内生变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)的相对丰度更高。在叶表附生微生物中,紫色象草中的异常球菌-栖热菌门(Deinococus-Thermus)相对丰度更高,而矮象草的拟杆菌门和厚壁菌门(Firmicutes)相对丰度更高。OTU水平的微生物群落组成分析(analysis of composition of microbiomes,ANCOM)、属水平的相对丰度差异分析及其与花青素含量的相关性分析结果显示,2种象草叶表面的甲基杆菌属(Methylobacterium)、Unclassified_ Methylobacteriacea)和异常球菌属(Deinococcus)的相对丰度与其花青素含量呈正相关,且紫色象草中的相对丰度显著高于矮象草。综上,紫色象草和矮象草叶表面附生细菌群落存在显著差异。本研究探索了不同花青素含量象草叶际细菌群落的差异性,为研究紫色象草的优异饲用价值提供新思路,为解析植物次生代谢物与叶际微生物的关系提供理论依据。

, authors=

刘俊杰(1997—),男,博士研究生,研究方向:草基因功能研究与育种

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* 葛良法(GE Liangfa),E-mail:
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刘俊杰(1997—),男,博士研究生,研究方向:草基因功能研究与育种

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刘俊杰(1997—),男,博士研究生,研究方向:草基因功能研究与育种

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Red wine polyphenols influence carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles of colonic mucosa in F344 rats[J]. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2005, 591(1/2): 237-246., articleTitle=Red wine polyphenols influence carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles of colonic mucosa in F344 rats, refAbstract=null), Reference(id=1277242069378794114, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, doi=null, pmid=null, pmcid=null, year=2017, volume=18, issue=11, pageStart=690, pageEnd=699, url=null, language=null, rfNumber=[45], rfOrder=49, authorNames=HALL A B, TOLONEN A C, XAVIER R J, journalName=Nature Reviews Genetics, refType=null, unstructuredReference=HALL A B, TOLONEN A C, XAVIER R J. Human genetic variation and the gut microbiome in disease[J]. 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A:叶片正面;B:叶片背面;左侧为矮象草,右侧为紫色象草。C:叶片花青素含量,**表示差异极显著(P<0.001)。

, figureFileSmall=cLoSL1pXdwf9Ar2KkHfuRQ==, figureFileBig=kUfaRUcihkvTD4MwEY3Rqw==, tableContent=null), ArticleFig(id=1277242064312074814, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Fig. 2, caption=Alpha diversity index of phyllosphere bacterial communities, figureFileSmall=9w8t0WiPeKEg+uqbJhfO0g==, figureFileBig=mzscuNeQwXbP3iOSYDQ5NQ==, tableContent=null), ArticleFig(id=1277242064387572287, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=图2, caption=叶际细菌群落的α多样性指数, figureFileSmall=9w8t0WiPeKEg+uqbJhfO0g==, figureFileBig=mzscuNeQwXbP3iOSYDQ5NQ==, tableContent=null), ArticleFig(id=1277242064454681152, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Fig. 3, caption=Beta diversity analysis of elephant grass phyllosphere bacteria, figureFileSmall=SEQxZC0fI6KfTlJLOCgHqQ==, figureFileBig=m6oIrJMVzpMNTGD19lBfgg==, tableContent=null), ArticleFig(id=1277242064534372929, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=图3, caption=象草叶际细菌β多样性分析

A:基于Bray-Curtis的主成分分析,圆圈表示样本95%置信区间;B:基于UPGMA的聚类分析。

, figureFileSmall=SEQxZC0fI6KfTlJLOCgHqQ==, figureFileBig=m6oIrJMVzpMNTGD19lBfgg==, tableContent=null), ArticleFig(id=1277242064593093186, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Fig. 4, caption=Relative abundance of phyllosphere bacteria of elephant grass at phyla level, figureFileSmall=se7kn1LCKkPzEOz/7Kc7+w==, figureFileBig=A2WR7RPLvla7sMPBHy1aKA==, tableContent=null), ArticleFig(id=1277242064651813443, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=图4, caption=2种象草叶际细菌在门水平的相对丰度, figureFileSmall=se7kn1LCKkPzEOz/7Kc7+w==, figureFileBig=A2WR7RPLvla7sMPBHy1aKA==, tableContent=null), ArticleFig(id=1277242064731505220, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Fig. 5, caption=Significantly different OTUs between two cultivars of elephant grass, figureFileSmall=X3MSG21WOTgvsIGR3BpkcA==, figureFileBig=RIVDiryyITFu6Q+FeqL6zg==, tableContent=null), ArticleFig(id=1277242064794419781, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=图5, caption=2种象草叶际微生物差异OTU

每个点代表独立的OTU,红色点表示差异显著的OTUs。

, figureFileSmall=X3MSG21WOTgvsIGR3BpkcA==, figureFileBig=RIVDiryyITFu6Q+FeqL6zg==, tableContent=null), ArticleFig(id=1277242064857334342, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Fig. 6, caption=Relative abundance of episphere bacteria communities at genus level between two cultivars of elephant grass, figureFileSmall=7D/7ItBpxhLIU18SKP7A2Q==, figureFileBig=JQgGxrF6WjBAraNICiGg/A==, tableContent=null), ArticleFig(id=1277242064924443207, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=图6, caption=2种象草叶表面附生细菌群落属水平的相对丰度

*表示差异显著(P<0.05);**表示差异极显著(P<0.01)。

, figureFileSmall=7D/7ItBpxhLIU18SKP7A2Q==, figureFileBig=JQgGxrF6WjBAraNICiGg/A==, tableContent=null), ArticleFig(id=1277242064974774856, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Tab. 1, caption=

Effects of anthocyanins on bacterial community structure assessed by PERMANOVA

, figureFileSmall=null, figureFileBig=null, tableContent=
区室Compartment配对比较Pairwise comparisonF决定系数R2P
叶内部Purple vs Mott2.422 4800.232 4280.011*
叶表面Purple vs Mott9.974 9270.475 5640.002**
), ArticleFig(id=1277242065050272329, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=表1, caption=

PERMANOVA评估花青素含量对细菌群落结构的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
区室Compartment配对比较Pairwise comparisonF决定系数R2P
叶内部Purple vs Mott2.422 4800.232 4280.011*
叶表面Purple vs Mott9.974 9270.475 5640.002**
), ArticleFig(id=1277242065129964106, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Tab. 2, caption=

Relative abundance of phyllosphere dominant bacteria at phyla level between two cultivars of elephant grass

, figureFileSmall=null, figureFileBig=null, tableContent=
门Phylum叶内部Endosphere叶表面Episphere
矮象草Mott紫色象草Purple矮象草Mott紫色象草Purple
变形菌门Proteobacteria19.8660.22*49.4368.21
拟杆菌门Bacteroidetes54.0726.27*20.102.91*
厚壁菌门Firmicutes21.8011.224.630.09*
放线菌门Actinobacteria0.720.5325.7212.50
异常球菌-栖热菌门Deinococus-Thermus00.050.1016.10*
), ArticleFig(id=1277242065201267275, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=表2, caption=

2种象草叶际优势菌门的相对丰度

, figureFileSmall=null, figureFileBig=null, tableContent=
门Phylum叶内部Endosphere叶表面Episphere
矮象草Mott紫色象草Purple矮象草Mott紫色象草Purple
变形菌门Proteobacteria19.8660.22*49.4368.21
拟杆菌门Bacteroidetes54.0726.27*20.102.91*
厚壁菌门Firmicutes21.8011.224.630.09*
放线菌门Actinobacteria0.720.5325.7212.50
异常球菌-栖热菌门Deinococus-Thermus00.050.1016.10*
), ArticleFig(id=1277242065280959052, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Tab. 3, caption=

Different OTUs in episphere bacteria communities between two cultivars of elephant grass

, figureFileSmall=null, figureFileBig=null, tableContent=
OTU名称OTU name属GenusWW Value相对丰度Relative abundance/%
矮象草Mott紫色象草Purple
OTU7甲基杆菌属Methylobacterium2170.1615.60***
OTU8异常球菌属Deinococcus2070.1015.22*
OTU69Unclassified__Methylobacteriaceae200201.40**
), ArticleFig(id=1277242065343873613, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=表3, caption=

2种象草叶表面附生细菌群落差异OTU

, figureFileSmall=null, figureFileBig=null, tableContent=
OTU名称OTU name属GenusWW Value相对丰度Relative abundance/%
矮象草Mott紫色象草Purple
OTU7甲基杆菌属Methylobacterium2170.1615.60***
OTU8异常球菌属Deinococcus2070.1015.22*
OTU69Unclassified__Methylobacteriaceae200201.40**
), ArticleFig(id=1277242065402593870, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=EN, label=Tab. 4, caption=

Correlation analysis between the relative abundance of episphere bacteria communities at the genus level and the content of anthocyanins

, figureFileSmall=null, figureFileBig=null, tableContent=
属GenusSperman相关系数Sperman correlation coefficientP
甲基杆菌属Methylobacterium0.63430.019 90
异常球菌属Deinococcus0.67700.011 04
Unclassified_ Methylobacteriaceae0.84400.000 29
), ArticleFig(id=1277242065482285647, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276190679491547234, language=CN, label=表4, caption=

2种象草叶表面附生细菌属水平的相对丰度与花青素含量的相关性分析

, figureFileSmall=null, figureFileBig=null, tableContent=
属GenusSperman相关系数Sperman correlation coefficientP
甲基杆菌属Methylobacterium0.63430.019 90
异常球菌属Deinococcus0.67700.011 04
Unclassified_ Methylobacteriaceae0.84400.000 29
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象草叶片花青素含量与叶际细菌群落的相关性分析
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刘俊杰 1 , 黄圆圆 1 , 雷虓 1 , 葛良法 1, 2, *
热带作物学报 | 农业生态与环境保护 2024,45(5): 1052-1060
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热带作物学报 |农业生态与环境保护 2024 , 45 (5) : 1052 -1060
象草叶片花青素含量与叶际细菌群落的相关性分析
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刘俊杰1, 黄圆圆1, 雷虓1, 葛良法1, 2, *
作者信息
  • 1.华南农业大学林学与风景园林学院草业科学教研室/广东省草业工程技术研究中心,广东广州 510642
  • 2.农业农村部国家大豆改良中心广东分中心,广东广州 510642
通讯作者:
* 葛良法(GE Liangfa),E-mail:
Correlation Analysis Between Anthocyanins Content in Pennisetum purpureum Leaves and Phyllosphere Bacterial Community
Junjie LIU1, Yuanyuan HUANG1, Xiao LEI1, Liangfa GE1, 2, *
Affiliations
  • 1.Department of Grassland Science, College of Forestry and Landscape Architecture, South China Agricultural University / Guangdong Engineering Research Center for Grassland Science, Guangzhou, Guangdong 510642, China
  • 2.Guangdong Subcenter of National Center for Soybean Improvement, Guangzhou, Guangdong 510642, China
出版时间: 2024-05-25 doi: 10.3969/j.issn.1000-2561.2024.05.020
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花青素又称为花色素,是植物黄酮类次生代谢产物。大量研究表明,花青素在调控植物的抗逆反应中起着重要作用,但花青素是否影响植物的叶际细菌仍未知。为探究叶片花青素与叶际细菌间的相关性,本研究以花青素含量高的紫色象草(Pennisetum purpureum cv. Purple,Purple)及花青素含量低的矮象草(P. purpureum cv. Mott,Mott)为研究材料,通过对叶片花青素含量进行定量分析,利用16S rRNA高通量测序对其叶际细菌群落结构及相对丰度进行分析,比较紫色象草和矮象草叶际细菌群落结构的差异性。结果表明:紫色象草叶片的花青素含量是矮象草的近20倍。紫色象草和矮象草的叶际细菌α多样性无显著差异,但是β多样性差异较大。操作分类单元(operational taxonomic unit,OTU)水平的相对丰度主成分分析结果表明,紫色象草和矮象草的叶际细菌群落组成呈显著差异。与矮象草相比,紫色象草叶内生变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)的相对丰度更高。在叶表附生微生物中,紫色象草中的异常球菌-栖热菌门(Deinococus-Thermus)相对丰度更高,而矮象草的拟杆菌门和厚壁菌门(Firmicutes)相对丰度更高。OTU水平的微生物群落组成分析(analysis of composition of microbiomes,ANCOM)、属水平的相对丰度差异分析及其与花青素含量的相关性分析结果显示,2种象草叶表面的甲基杆菌属(Methylobacterium)、Unclassified_ Methylobacteriacea)和异常球菌属(Deinococcus)的相对丰度与其花青素含量呈正相关,且紫色象草中的相对丰度显著高于矮象草。综上,紫色象草和矮象草叶表面附生细菌群落存在显著差异。本研究探索了不同花青素含量象草叶际细菌群落的差异性,为研究紫色象草的优异饲用价值提供新思路,为解析植物次生代谢物与叶际微生物的关系提供理论依据。

象草  /  花青素  /  16S rRNA  /  叶际微生物  /  细菌群落

Anthocyanins, also called anthocyans, are a group of plant secondary metabolites of flavonoid derivatives. Anthocyanins play an important role in mediating plant stress responses. However, whether anthocyanins are also involved in regulating phyllosphere bacterial communities remains unknown. To evaluate the differences of phyllosphere bacterial communities in plants with different anthocyanin contents, this study analyzed the phyllosphere bacterial communities of two cultivars of elephant grass with contrasting anthocyanins content, Pennisetum purpureum cv. Purple (Purple) with a high amount of anthocyanins and P. purpureum cv. Mott (Mott) with relatively low anthocyanins. The anthocyanins content determination results showed that the anthocyanins content in Purple leaves was nearly 20 times of that in Mott leaves. High throughput sequencing of 16S rRNA and analysis of the microbiome community indicated that though alpha diversity was not significantly changed, the bacterial communities of Purple was significantly different from that of Mott. In Purple, the relative abundance of endopphytic Proteobacteria and Bacteroidetes, and epiphytic Deinococus-Thermus were significantly increased, whereas epiphytic Bacteroidetes and Firmicutes were significantly decreased. The composition of microbiomes (ANCOM) at the OTU level, the relative abundance analysis at the genus level and correlation analysis between epiphytes and anthocyanins showed that the relative abundance of Methylobacterium, Unclassified Methylobacteriaceae and Deinococcus, which showed a significant positive correlation with anthocyanins content, were significantly increased in the episphere of Purple. In summary, the phyllosphere bacterial communities were significantly different between Purple and Mott. The findings from this work would provide new insights into the understanding of the superior forage value of P. purpureum cv. Purple, and lay a foundation to further investigate the mechanism of how plant secondary metabolism products regulate phylloshpere microorganisms.

Pennisetum purpureum  /  anthocyanins  /  16S rRNA  /  phyllosphere microorganisms  /  bacterial community
刘俊杰, 黄圆圆, 雷虓, 葛良法. 象草叶片花青素含量与叶际细菌群落的相关性分析. 热带作物学报, 2024 , 45 (5) : 1052 -1060 . DOI: 10.3969/j.issn.1000-2561.2024.05.020
Junjie LIU, Yuanyuan HUANG, Xiao LEI, Liangfa GE. Correlation Analysis Between Anthocyanins Content in Pennisetum purpureum Leaves and Phyllosphere Bacterial Community[J]. Chinese Journal of Tropical Crops, 2024 , 45 (5) : 1052 -1060 . DOI: 10.3969/j.issn.1000-2561.2024.05.020
定殖于叶际(phyllosphere,植物地上部分)的细菌和真菌等微生物被称为叶际微生物(phyllosphere microorganisms),其中以细菌的丰度最大[1-4]。依据定殖位置的不同,可将叶际微生物分为两类,一类是附生在植物表面的微生物,称为附生微生物(epiphytes);另一类是定殖于植物内部的微生物,称为内生微生物(endophytes)[5]。研究表明,附生微生物的丰度和丰富度远大于内生微生物,而内生微生物与植物细胞的关系较附生微生物更密切[6]。叶际微生物主要来源于土壤,部分来源于空气[7-9]。对于微生物来说,叶际并不是一个稳定的环境,其组成受环境因素的影响,这些因素包括气候、土壤类型、地理位置、温度、光照和湿度等[10-12]。此外,宿主植物的基因型、年龄以及宿主植物内部的次生代谢物也会对叶际微生物群有较大影响[13-15]
多项研究表明,植物叶片内部的次生代谢产物和有机挥发物会影响叶际微生物群落的构成[15-18]。黄酮类化合物——花青素是植物体内重要的水溶性次生代谢产物,影响植物果实、花朵和叶片的颜色[19]。花青素具有参与植物体内非生物胁迫和生物胁迫抗逆反应,减轻植物受环境、病害的伤害等功能[20-21]。此外,花青素还具有抗氧化、抗癌、软化血管和降低血糖等多种功效,具有极高的药用价值,已广泛应用于医药领域[22]。目前对花青素合成及调控的分子机制已有较为深入的研究[19,22],但植物中的花青素对植物叶际微生物群落是否产生影响有待进一步研究。
象草(Pennisetum purpureum)是狼尾草属多年生草本植物,广泛种植于热带亚热带地区[23]。象草具有产量高、生长速度快等特性,常用作饲料、青贮饲料、生物乙醇和纸张的原料以及生态修复等[23]。紫色象草(P. purpureum cv. Purple)叶片为紫色,其叶片较矮象草(P. purpureum cv. Mott)富含多种花青素[24]。因花青素的诸多优点,紫色象草被视为比绿色象草更佳的饲用品种[25-26]
本研究采用16S rRNA高通量测序技术对紫色象草和矮象草的叶际细菌群落进行比较分析,从细菌多样性、群落结构等方面对比紫色象草和矮象草叶际细菌群落,探究不同花青素含量的象草叶际细菌的差异性,为花青素与植物叶际微生物群落相互作用的研究和实际利用提供理论支持。
供试材料为摩特矮象草(P. purpureum cv. Mott,Mott)和紫色象草(P. purpureum cv. Purple,Purple),种植于广东省广州市华南农业大学草业科学系引种园(23°10'N,113°22'E),2种象草种植距离约为4 m。
用0.1%盐酸-甲醇溶液配制矢车菊素-3-O-葡萄糖苷(Sigma-Aldrich,#52976)的标准溶液,浓度分别为20.0、12.5、10.0、5.0、2.5 mg/L。使用分光光度计Nano-Photometer NP80(implen)测530 nm处吸光度,得出标准曲线。
称取1 g象草叶片用于花青素浓度测定。低温破碎叶片组织后,加入10 mL 0.1%盐酸-甲醇溶液,震荡后超声5 min,4 ℃避光保存过夜。5000 r/min离心10 min,取上清液,加入5 mL氯仿及5 mL ddH2O,摇匀后5000 r/min离心10 min,取上层红色液体,测530 nm处OD值,依据标准曲线计算样品花青素浓度。
于2022年6月在华南农业大学草业科学系引种园采集象草样本,选取长势一致的象草作为试验材料,每株象草作为一个独立生物学重复,每种材料设置5~7个生物学重复。将所取叶片组织浸没于无菌磷酸盐缓冲液(phosphate-buffered saline,PBS)中,室温震荡洗涤20 min,收集液体并重复1次。取出植物组织,加入无菌PBS,超声洗涤10 min,收集上述洗涤液,于0.22 μm滤膜过滤,滤膜保存于–80 ℃,用于提取叶表面附生微生物DNA。剩余植物组织使用5%次氯酸钠溶液浸泡5 min,无菌水漂洗3次后储存于–80 ℃,用于提取叶内生微生物总DNA。
选用FastDNA® SPIN Kit for soil(MP Biomedicals)试剂盒提取样本DNA。使用引物799F(5′-AACMGGATTAGATA CCCKG-3′)、1193R(5′-ACGTCATCCCCACCTT CC-3′)和1392R(5′-ACGGGCGGTGTGTRC-3′)对细菌16S rDNA V5~V7区域进行巢式扩增。第一轮PCR扩增使用引物799F和1392R,程序为:经95 ℃ 3 min预变性后,95 ℃变性30 s;55 ℃退火30 s,72 ℃延伸30 s进行27个循环扩增,,然后72 ℃稳定延伸10 min。第一轮扩增产物使用AxyPrep DNA Gel Extraction Kit(Axygen Biosciences)回收纯化后作为第二轮PCR反应的模板。第二轮PCR使用引物799F和1193R,除循环数变更为13外,其余程序同第一轮PCR。PCR反应体系为:5×TransStart FastPfu缓冲液4 μL,2.5 mmol/L dNTPs 2 μL,上游引物(5 μmol/L)0.8 μL,下游引物(5 μmol/L)0.8 μL,TransStart FastPfu DNA聚合酶0.4 μL,模板DNA 10 ng,用ddH2O补足至20 μL,每个样本3个重复。将同一样本的PCR产物混合后使用2%琼脂糖凝胶纯化回收PCR产物。纯化后的PCR产物使用2%琼脂糖凝胶电泳进行质量检测,并检测DNA浓度。PCR产物质量及浓度合格后,使用NEXTFLEX Rapid DNA-Seq Kit进行建库,利用Illumina公司的Miseq PE300平台进行测序。
测序所得原始数据使用QIIME2-2022.2平台进行质量过滤和分类学分析[27]。利用DADA2软件去除引物,对序列进行质量过滤和校正,鉴别出操作分类单元(operational taxonomic units,OTU)的代表序列,生成OTU表[28]。每个OTU的代表序列使用Greengenes(13_8)数据库进行分类学注释。依据OTU表,使用QIIME2软件计算出样本α多样性的Shannon多样性指数和Faith系统发育多样性指数;使用QIIME2软件构建样本间的Bray-Curtis距离矩阵,基于该矩阵进行主成分分析(PCoA),并使用非加权组平均法(UPGMA)对样本进行聚类分析。通过置换多元方差分析(permutational multivariate analysis of variance,PERMANOVA)计算微生物群落结构的差异以及微生物群落组成分析(analysis of composition of microbiomes,ANCOM)探究样本间OTU的差异[29-30]。利用OriginPro 2022b软件中的Welch’s t检验分析属水平相对丰度的差异,通过Spearman相关性分析检验属水平相对丰度与花青素含量的相关性,并绘制柱状图。
紫色象草与矮象草的叶型相似,但颜色差异很大。由于花青素的积累,紫色象草的叶片正面和背面均为紫色,而矮象草叶片整体为绿色(图1A图1B)。为进一步探究2种象草叶片花青素含量的差异,分别对2种象草叶片的花青素含量进行定量分析。结果表明,紫色象草叶片中的花青素含量为(150.15±3.41)μg/g,矮象草为(7.80±0.29)μg/g,2种象草叶片花青素含量呈极显著差异(P<0.001),紫色象草叶片中的花青素含量是矮象草的近20倍(图1C)。
采用16S rRNA测序技术对2种象草叶片表面附生细菌及叶片内生细菌群落进行分析,共得到2 994 242条reads,经分析得到26门、45纲、76目、120科、155属和881个OTU。Shannon指数是反映物种丰富度和均匀度的一个均匀指标;Faith系统发育多样性指数反映群落的丰富度。本研究利用这2个指数在OTU水平分别对两类样本进行α多样性分析。结果表明,紫色象草和矮象草同一区室的α多样性均无显著差异(图2)。
为进一步探究花青素对象草叶际细菌群落结构的影响,根据OTU水平的相对丰度,对紫色象草和矮象草的叶际细菌群落进行主成分分析(图3A)。主成分1解释度为40.48%,主成分2解释度为19.29%,总解释度为59.77%。其中,叶表面附生细菌样本和叶内生细菌样本在X轴(主成分1)出现分离。进一步分析2种象草的叶际细菌群落发现,2种象草叶片内部细菌样本距离较近,而叶片表面附生细菌样本在Y轴(主成分2)出现分离,紫色象草和矮象草样品明显分开。使用非加权组平均法(unweighted pair-group method with arithmetic means,UPGMA)对叶际细菌样本进行聚类分析也得出相似结果:所有样本可以聚类为3个分支,分支1包含所有叶片内部细菌样本,分支2为紫色象草表面细菌样本,分支3为矮象草表面细菌样本(图3B)。此外,通过置换多元方差分析(permutational multivariate analysis of variance,PERMANOVA)发现,2种象草叶片表面附生细菌样本的差异较叶片内生细菌更显著(表1)。以上结果显示,紫色象草和矮象草叶表细菌群落存在较大差异。
为解析象草叶际细菌组成,在细菌门水平上对不同样本进行相对丰度分析(图4表2)。结果显示,叶片内部细菌的优势菌门(占比≥1%)为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes);叶片表面细菌的优势菌门除上述3种外,还包括放线菌门(Actinobacteria)和异常球菌-栖热菌门(Deinococus-Thermus)。紫色象草叶片表面变形菌门和异常球菌-栖热菌门的相对丰度显著大于矮象草,而拟杆菌门相对丰度则显著小于矮象草。以上结果表明,紫色象草和矮象草叶际微生物优势菌门及其相对丰度存在差异。
对紫色象草和矮象草的OTU相对丰度进行组间差异分析(图5)。图中横轴clr统计量表示差异量的大小,当其为正值时,表示紫色象草中该OTU的相对丰度高于矮象草,负值则相反;纵轴W统计量表示显著度。在2种象草叶片内部细菌群落中未发现相对丰度差异显著的OTU,而在叶片表面细菌群落中发现3个相对丰度显著差异的OTU,紫色象草OTU7(甲基杆菌属Methylobacterium)、OTU8(异常球菌属Deinococcus)和OTU69(Unclassified_Methylobacteriaceae)的相对丰度均高于矮象草(表3)。在细菌属水平上,紫色象草表面细菌群落中的甲基杆菌属、异常球菌和Unclassified_Methylobacteriaceae的相对丰度显著高于矮象草。同时,Spearman相关性分析得出,象草表面甲基杆菌属、异常球菌和Unclassified_Methylobacteriaceae的相对丰度与花青素含量呈正相关(图6表4)。
花青素是植物的重要次生代谢物,是影响植物颜色的关键色素之一[22]。多项研究表明,花青素参与植物抗逆反应,能提高植物耐逆境胁迫能力[31-32]。目前,学界对于花青素的生物合成通路以及调控网络已有较为清晰的认识,已发现多种转录因子参与花青素合成的调控,如MYB家族、bHLH家族和WD蛋白等。多项研究解析了作物中影响花青素合成的关键调控因子[33-35],如蒺藜苜蓿中过表达LAP1基因可促进花青素在叶片中的积累[36];玉米中ZmR1基因的序列变异及表达水平影响了花青素在整个植株上的积累[37];白色葡萄中VvmybA1基因启动子区域存在反转座子插入,导致花色素苷合成基因转录受阻,影响了浆果着色,从而使果实呈现白色[38]
紫色象草是叶片高花青素含量的象草品种。本研究发现,紫色象草叶片中的花青素含量是矮象草叶片中花青素含量的近20倍。研究表明,2种象草花青素种类不同,紫色象草花青素多为天竺葵素和锦葵色素的衍生物,矮象草花青素多为矢车菊素的衍生物[24]。目前对于紫色象草叶片高花青素含量的遗传机理已有一定的解析。紫色象草叶片中花青素合成基因表达水平较绿色叶片的矮象草显著上调,多个调控花青素合成的转录因子的序列具有多态性,其表达水平与矮象草差异显著[24],这些重要基因的多态性及差异表达可能是导致紫色象草叶片花青素高积累的重要原因。
植物的次生代谢物可影响其叶际微生物群落结构[15-18]。为探究不同花青素含量植物叶际微生物群落的差异性,本研究选取2个不同花青素含量的象草品种(紫色象草和绿色叶片的矮象草),通过对2个品种叶际细菌进行α多样性分析、β多样性分析、OTU相对丰度分析以及细菌群落组成分析,比较紫色象草和矮象草叶际细菌结构。2种象草叶际细菌高通量测序结果表明,紫色象草与矮象草叶际细菌群落的优势菌门及其相对丰度均存在差异。PCoA分析、样本聚类分析以及PERMANOVA分析结果表明,紫色象草和矮象草表面的附生细菌群落出现显著分离,提示叶片表面附生细菌群落可能与花青素含量及种类相关。此外,花青素可改变叶片对光的吸收[39]。因此,推测紫色象草叶片中的花青素可能通过影响叶片中光线的透过率,改变叶片背部的光线强度,进而影响叶面附生细菌的群落结构。
本研究在叶片附生细菌中发现3个OTUs的相对丰度差异显著,这些OTUs分别来自甲基杆菌属(Methylobacterium)、异常球菌属(Deinococcus)和Unclassified_Methylobacteriaceae,其中甲基杆菌属从属于甲基杆菌科。研究发现,紫色象草叶表面甲基杆菌属的相对丰度较矮象草显著提升,进一步分析发现,甲基杆菌属的相对丰度与花青素含量呈正相关。研究表明,根际和叶际的甲基杆菌可以提高作物的产量、增强植物的抗逆性[40-41]。紫色象草适应性强,产草量高[26],其叶表面附生的甲基杆菌相对丰度的增加,可能促进紫色象草的生长,进一步提高紫色象草的产量和抗逆性,这也为植物与叶际微生物的相互作用提供一定的证据。异常球菌属具有抗辐射、抗氧化等特性[42]。已有研究报道,银杏叶片中异常球菌属的相对丰度与叶片中类黄酮物质的浓度具有正相关效应,体外培养异常球菌属细菌时添加黄酮类化合物可提高其增殖速度[17]。异常球菌属的细菌具有蔗糖-4-葡糖基转移酶(amylosucrase),以槲皮素(quercetin,一种黄酮类化合物)为底物,可以生成多种槲皮素糖苷(quercetin glucosides),提示异常球菌属细菌可有效利用黄酮类化合物[43]。本研究中,异常球菌属的相对丰度在富含花青素的紫色象草叶片表面上更高,表明其相对丰度与花青素浓度相关,为黄酮类化合物促进异常球菌属细菌增殖提供了新证据。
研究表明,与绿色象草相比,使用高花青素含量的紫色象草喂养动物可提高动物的产量[25]。此外,在饲料中添加红酒多酚物质可改变动物的肠道菌群结构[44],肠道内菌群结构会影响宿主的健康状况[45]。本研究发现,叶际细菌群落结构与叶片花青素含量存在联系,提示多酚物质可能对细菌产生影响,改变其群落结构,为解析紫色象草对动物生长的促进作用提供了新的研究方向。
研究表明,紫色象草叶际细菌群落结构与矮象草存在显著差异,表明象草叶片中的花青素含量和象草叶际细菌群落结构相关。在紫色象草叶片表面,能促进植物生长的甲基杆菌属以及抗辐射、抗氧化的异常球菌属的相对丰度更高,表明花青素含量的提升可能促进这类对宿主有积极作用的细菌的生长,进而辅助植物的抗逆反应。本研究从细菌菌落结构的角度解析了象草叶片中的花青素与叶际细菌菌落结构的相关性,为解析紫色象草的优良生产性能及饲用价值提供了新思路。
  • 广州市重点研发计划项目(202103000066)
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doi: 10.3969/j.issn.1000-2561.2024.05.020
  • 接收时间:2022-10-31
  • 首发时间:2026-06-23
  • 出版时间:2024-05-25
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  • 收稿日期:2022-10-31
  • 修回日期:2023-02-06
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广州市重点研发计划项目(202103000066)
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    1.华南农业大学林学与风景园林学院草业科学教研室/广东省草业工程技术研究中心,广东广州 510642
    2.农业农村部国家大豆改良中心广东分中心,广东广州 510642

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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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