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Article(id=1217471087013712128, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1217471079325549522, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250599, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1753977600000, receivedDateStr=2025-08-01, revisedDate=null, revisedDateStr=null, acceptedDate=1757433600000, acceptedDateStr=2025-09-10, onlineDate=1768197326663, onlineDateStr=2026-01-12, pubDate=1767456000000, pubDateStr=2026-01-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768197326663, onlineIssueDateStr=2026-01-12, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768197326663, creator=13701087609, updateTime=1768197326663, updator=13701087609, issue=Issue{id=1217471079325549522, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='1', pageStart='1', pageEnd='475', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768197324830, creator=13701087609, updateTime=1768198886678, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217477630291530315, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1217471079325549522, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217477630291530316, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1217471079325549522, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=443, endPage=455, ext={EN=ArticleExt(id=1217471087240204552, articleId=1217471087013712128, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Isolation and plant growth-promoting function evaluation of bacteria from rhizosphere soil and phyllosphere of Citrus reticulata cv. Gonggan in Deqing, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Citrus reticulata cv. Gonggan in Deqing is a national geographical indication product of China, and its quality formation is intricately linked to microbial ecology. In-depth exploration of the microbial resources associated with Gonggan is of great significance for enhancing the quality and increasing the yield of this fruit. [Objective] To isolate and culture bacteria from the rhizosphere soil and phyllosphere surface of Gonggan in Deqing and systematically evaluate their plant growth-promoting functions, thus providing applicable bacterial resources for the eco-friendly cultivation of Gonggan in Deqing. [Methods] Samples of rhizosphere soil and leaves of Gonggan in Deqing were collected. Bacteria within the samples were isolated via the dilution plating method. Bacterial strains were identified based on the homology of their 16S rRNA gene sequences. The functional media for phosphate solubilization, potassium release, nitrogen fixation, and siderophore production were used to evaluate the plant growth-promoting ability of bacterial strains, and the strains with strong plant growth-promoting functions were screened out. Ultimately, pot experiments were conducted to validate the plant growth-promoting functions of the selected strains. [Results] A total of 240 bacterial isolates were obtained, including 96 strains from the rhizosphere soil and 144 strains from the phyllosphere. These strains belonged to 51 genera, 29 families of 4 phyla, where Pseudomonadota was the dominant phylum and Bacillaceae was the dominant family. Functional characterization revealed that 230 (95.83%) strains exhibited at least one plant growth-promoting function, while 123 strains (51.25%) possessed three or more such functions. Given the extended growth cycle of Gonggan in Deqing, we utilized tomato as a model plant to evaluate the plant growth-promoting functions of eight bacterial strains possessing at least two functions through pot experiments. The results demonstrated that the tested strains significantly increased both plant height and fresh weight of tomato plants. [Conclusion] The rhizosphere soil and phyllosphere of Gonggan in Deqing harbor abundant functional microbial resources. This study successfully screened multiple strains of plant growth-promoting bacteria, and pot experiments demonstrated their significant ability to promote tomato growth. These findings provide a solid theoretical foundation for exploiting microbial resources of Gonggan in Deqing and advancing eco-friendly cultivation practices.

, correspAuthors=Honghui ZHU, authorNote=null, correspAuthorsNote=
*Tel: +86-20-87685699, E-mail:
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#These authors contributed equally to this work.

, authorsList=Honghong DONG, Qian YANG, Mingrong DENG, Yunfeng LI, Honghui ZHU), CN=ArticleExt(id=1217471090566287722, articleId=1217471087013712128, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=德庆贡柑根围土壤及叶际细菌的分离与植物促生功能, columnId=1192149544164012138, journalTitle=微生物学报, columnName=研究报告, runingTitle=null, highlight=null, articleAbstract=

德庆贡柑(Citrus reticulata cv. Gonggan in Deqing)作为中国国家地理标志产品，其品质形成与微生物生态密切相关。深入探究贡柑相关微生物资源对提升贡柑品质和产量意义重大。 【目的】 分离培养德庆贡柑根围土壤及叶际表面的细菌，系统评价其促植物生长功能，为德庆贡柑的绿色栽培提供可用的促生菌资源。 【方法】 分别采集德庆贡柑根围土壤及叶片样本，采用稀释涂布法分离样本中的细菌，根据16S rRNA基因序列同源性鉴定菌株。利用溶磷、解钾、固氮和产铁载体的功能培养基评价菌株的促生能力，筛选高活性菌株。通过盆栽试验验证筛选出的菌株的促生功能。 【结果】 本研究共分离得到240株细菌，其中从贡柑根围土壤分离出96株，从叶际分离出144株。这些菌株隶属于4门29科51属，其中假单胞菌门(Pseudomonadota)为优势门，芽孢杆菌科(Bacillaceae)为优势菌科。240株细菌中，230株菌具有溶磷、解钾、固氮、产铁载体和产吲哚-3-乙酸(indole-3-acetic acid, IAA)中的一种能力，占比达95.83%；123株菌具有3种以上促生能力，占比为51.25%。鉴于德庆贡柑的生长周期较长，本研究以番茄作为模式植物，通过盆栽试验评价8株具有2种及以上功能的菌株的促生功能。供试菌株显著增加了番茄的株高和鲜重。 【结论】 德庆贡柑根围土壤和叶际蕴含着丰富的功能微生物资源。本研究成功筛选出多株促生细菌菌株，盆栽试验证明它们能显著促进番茄生长。研究结果为德庆贡柑微生物资源的开发利用以及绿色栽培技术的发展提供了坚实的理论依据。

, correspAuthors=朱红惠, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=zINKyDUij5QQeowJeCvDfw==, magXml=hfcjMpU7kRxe/cB4/ecJPA==, pdfUrl=null, pdf=4H9jcSNO5F9od3QjAWZG6Q==, pdfFileSize=3477818, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=H9ov+CAPY0XEf0JBFndY0A==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=WvwM8a9zJqA0yv1T9WZcaw==, mapNumber=null, authorCompany=null, fund=null, authors=

作者贡献声明

董红红：研究构思和设计、论文撰写和修改；杨茜：细菌分离鉴定、数据收集和处理、参与论文撰写；邓名荣：项目管理、监督管理；李云锋：监督管理；朱红惠：获取基金、试验设计和论文修订。

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Gonggan in Deqing at the phylum level (A) and genus level (B)., figureFileSmall=WbtBmepLaoGVmaVNgsissA==, figureFileBig=T5D5sj19kE9A/tXl3P0bmQ==, tableContent=null), ArticleFig(id=1226557140043740016, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=CN, label=图1, caption=德庆贡柑根围土壤及叶际细菌在门水平(A)和属水平(B)的分类分布情况, figureFileSmall=WbtBmepLaoGVmaVNgsissA==, figureFileBig=T5D5sj19kE9A/tXl3P0bmQ==, tableContent=null), ArticleFig(id=1226557140211512182, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=EN, label=Figure 2, caption=Distribution of plant growth-promoting bacteria in rhizosphere soil and phyllosphere of Citrus reticulata cv. Gonggan in Deqing at genus level. A: The distribution of 210 IAA-producing bacteria at the genus level; B: The distribution of 199 siderophore-producing bacteria at the genus level; C: The distribution of 109 dissolved organic phosphorus-dissolving bacteria at the genus level; D: The distribution of 89 nitrogen-fixing bacteria at the genus level; E: The distribution of 62 dissolved inorganic phosphorus-dissolving bacteria at the genus level; F: The distribution of 45 potassium-dissolving bacteria at the genus level., figureFileSmall=J1d2gT8HrdvrOZ4feVhNQQ==, figureFileBig=0RNw1IT5kY4kHkfDp6lGlA==, tableContent=null), ArticleFig(id=1226557140349924226, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=CN, label=图2, caption=德庆贡柑根围土壤及叶际促生功能细菌在属水平的分布。A：210株产IAA细菌在属水平的分布；B：199株产铁载体细菌在属水平的分布；C：109株溶解有机磷细菌在属水平的分布；D：89株固氮细菌在属水平的分布；E：62株溶解无机磷细菌在属水平的分布；F：45株解钾细菌在属水平的分布。, figureFileSmall=J1d2gT8HrdvrOZ4feVhNQQ==, figureFileBig=0RNw1IT5kY4kHkfDp6lGlA==, tableContent=null), ArticleFig(id=1226557140467364745, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=EN, label=Figure 3, caption=Functional evaluation results of plate assays for highly active plant growth-promoting strains. A: Organic phosphate solubilization; B: Inorganic phosphate solubilization; C: Nitrogen fixation (nitrogen-free medium); D: Nitrogen fixation (Ashby’s medium); E: Potassium solubilization; F: Siderophore production., figureFileSmall=Qu/2ds/Oxkt0CanwsGh7sA==, figureFileBig=yueAFqF4tDoIeDhUa8IpWw==, tableContent=null), ArticleFig(id=1226557140597388176, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=CN, label=图3, caption=部分高活性促生菌株的平板功能评价结果。A：溶有机磷；B：溶无机磷；C：固氮(无氮培养基)；D：固氮(阿须贝氏培养基)；E：解钾；F：产铁载体。, figureFileSmall=Qu/2ds/Oxkt0CanwsGh7sA==, figureFileBig=yueAFqF4tDoIeDhUa8IpWw==, tableContent=null), ArticleFig(id=1226557140739994521, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=EN, label=Figure 4, caption=Analysis of proportions of bacteria with different plant growth-promoting functions in the rhizosphere soil and phyllosphere of Citrus reticulata cv. Gonggan in Deqing. A: IAA production; B: Siderophore production; C: Organic phosphorus solubilization; D: Nitrogen fixation; E: Inorganic phosphorus solubilization; F: Potassium solubilization., figureFileSmall=obNTUAVMwxf4lZRPJAlH2A==, figureFileBig=Uxc6FIDFLpkcjT3bhTuf2g==, tableContent=null), ArticleFig(id=1226557140853240735, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=CN, label=图4, caption=德庆贡柑根围土壤及叶际不同促生功能的细菌占比分析。A：产IAA；B：产铁载体；C：溶有机磷；D：固氮；E：溶无机磷；F：解钾。, figureFileSmall=obNTUAVMwxf4lZRPJAlH2A==, figureFileBig=Uxc6FIDFLpkcjT3bhTuf2g==, tableContent=null), ArticleFig(id=1226557140966486952, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=EN, label=Figure 5, caption=Pot experiment of functional strains on tomato growth promotion., figureFileSmall=0JmFE9g1WjRh4XQPtyEq0w==, figureFileBig=IiMS6pO6BEV3aeL9nNvB6w==, tableContent=null), ArticleFig(id=1226557141033595822, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=CN, label=图5, caption=功能菌株对番茄促生的盆栽试验, figureFileSmall=0JmFE9g1WjRh4XQPtyEq0w==, figureFileBig=IiMS6pO6BEV3aeL9nNvB6w==, tableContent=null), ArticleFig(id=1226557141159424950, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=EN, label=Figure 6, caption=The plant height (A) and fresh weight (B) of tomatoes after inoculation with functional strains. ns: No significant difference; **: The difference was significant (P<0.01); ***: The difference was the most significant (P<0.001); ****: The difference was extremely significant (P<0.000 1)., figureFileSmall=DZPCPcprQNT8upRYAn8akA==, figureFileBig=l/LGTAO9PP+UEFzadIX1Kw==, tableContent=null), ArticleFig(id=1226557141285254077, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471087013712128, language=CN, label=图6, caption=功能菌株接种处理后番茄植株的株高(A)和鲜重(B), figureFileSmall=DZPCPcprQNT8upRYAn8akA==, 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德庆贡柑根围土壤及叶际细菌的分离与植物促生功能
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董红红 1 , 杨茜 1, 2 , 邓名荣 1 , 李云锋 2 , 朱红惠 1, *
微生物学报 | 研究报告 2026,66(1): 443-455
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微生物学报 | 研究报告 2026, 66(1): 443-455
德庆贡柑根围土壤及叶际细菌的分离与植物促生功能
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董红红1, 杨茜1, 2, 邓名荣1, 李云锋2, 朱红惠1, *
作者信息
  • 1.广东省科学院微生物研究所，华南应用微生物国家重点实验室，农业农村部农业微生物组学与精准应用重点实验室，农业农村部农业微生物组学重点实验室，广东省菌种保藏与应用重点实验室，广东 广州
  • 2.华南农业大学 植物保护学院，广东 广州
Isolation and plant growth-promoting function evaluation of bacteria from rhizosphere soil and phyllosphere of Citrus reticulata cv. Gonggan in Deqing
Honghong DONG1, Qian YANG1, 2, Mingrong DENG1, Yunfeng LI2, Honghui ZHU1, *
Affiliations
  • 1.State Key Laboratory of Applied Microbiology Southern China, Key Laboratory of Agricultural Microbiomics and Precision Application (MARA), Key Laboratory of Agricultural Microbiome (MARA), Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China
  • 2.College of Plant Protection, South China Agricultural University, Guangzhou, Guangdong, China
出版时间: 2026-01-04 doi: 10.13343/j.cnki.wsxb.20250599
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摘要
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德庆贡柑(Citrus reticulata cv. Gonggan in Deqing)作为中国国家地理标志产品，其品质形成与微生物生态密切相关。深入探究贡柑相关微生物资源对提升贡柑品质和产量意义重大。 【目的】 分离培养德庆贡柑根围土壤及叶际表面的细菌，系统评价其促植物生长功能，为德庆贡柑的绿色栽培提供可用的促生菌资源。 【方法】 分别采集德庆贡柑根围土壤及叶片样本，采用稀释涂布法分离样本中的细菌，根据16S rRNA基因序列同源性鉴定菌株。利用溶磷、解钾、固氮和产铁载体的功能培养基评价菌株的促生能力，筛选高活性菌株。通过盆栽试验验证筛选出的菌株的促生功能。 【结果】 本研究共分离得到240株细菌，其中从贡柑根围土壤分离出96株，从叶际分离出144株。这些菌株隶属于4门29科51属，其中假单胞菌门(Pseudomonadota)为优势门，芽孢杆菌科(Bacillaceae)为优势菌科。240株细菌中，230株菌具有溶磷、解钾、固氮、产铁载体和产吲哚-3-乙酸(indole-3-acetic acid, IAA)中的一种能力，占比达95.83%；123株菌具有3种以上促生能力，占比为51.25%。鉴于德庆贡柑的生长周期较长，本研究以番茄作为模式植物，通过盆栽试验评价8株具有2种及以上功能的菌株的促生功能。供试菌株显著增加了番茄的株高和鲜重。 【结论】 德庆贡柑根围土壤和叶际蕴含着丰富的功能微生物资源。本研究成功筛选出多株促生细菌菌株，盆栽试验证明它们能显著促进番茄生长。研究结果为德庆贡柑微生物资源的开发利用以及绿色栽培技术的发展提供了坚实的理论依据。

德庆贡柑  /  根围土壤  /  叶际  /  促生细菌

Citrus reticulata cv. Gonggan in Deqing is a national geographical indication product of China, and its quality formation is intricately linked to microbial ecology. In-depth exploration of the microbial resources associated with Gonggan is of great significance for enhancing the quality and increasing the yield of this fruit. [Objective] To isolate and culture bacteria from the rhizosphere soil and phyllosphere surface of Gonggan in Deqing and systematically evaluate their plant growth-promoting functions, thus providing applicable bacterial resources for the eco-friendly cultivation of Gonggan in Deqing. [Methods] Samples of rhizosphere soil and leaves of Gonggan in Deqing were collected. Bacteria within the samples were isolated via the dilution plating method. Bacterial strains were identified based on the homology of their 16S rRNA gene sequences. The functional media for phosphate solubilization, potassium release, nitrogen fixation, and siderophore production were used to evaluate the plant growth-promoting ability of bacterial strains, and the strains with strong plant growth-promoting functions were screened out. Ultimately, pot experiments were conducted to validate the plant growth-promoting functions of the selected strains. [Results] A total of 240 bacterial isolates were obtained, including 96 strains from the rhizosphere soil and 144 strains from the phyllosphere. These strains belonged to 51 genera, 29 families of 4 phyla, where Pseudomonadota was the dominant phylum and Bacillaceae was the dominant family. Functional characterization revealed that 230 (95.83%) strains exhibited at least one plant growth-promoting function, while 123 strains (51.25%) possessed three or more such functions. Given the extended growth cycle of Gonggan in Deqing, we utilized tomato as a model plant to evaluate the plant growth-promoting functions of eight bacterial strains possessing at least two functions through pot experiments. The results demonstrated that the tested strains significantly increased both plant height and fresh weight of tomato plants. [Conclusion] The rhizosphere soil and phyllosphere of Gonggan in Deqing harbor abundant functional microbial resources. This study successfully screened multiple strains of plant growth-promoting bacteria, and pot experiments demonstrated their significant ability to promote tomato growth. These findings provide a solid theoretical foundation for exploiting microbial resources of Gonggan in Deqing and advancing eco-friendly cultivation practices.

Citrus reticulata cv. Gonggan in Deqing  /  rhizosphere soil  /  phyllosphere  /  plant growth-promoting bacteria
董红红, 杨茜, 邓名荣, 李云锋, 朱红惠. 德庆贡柑根围土壤及叶际细菌的分离与植物促生功能. 微生物学报, 2026 , 66 (1) : 443 -455 . DOI: 10.13343/j.cnki.wsxb.20250599
Honghong DONG, Qian YANG, Mingrong DENG, Yunfeng LI, Honghui ZHU. Isolation and plant growth-promoting function evaluation of bacteria from rhizosphere soil and phyllosphere of Citrus reticulata cv. Gonggan in Deqing[J]. Acta Microbiologica Sinica, 2026 , 66 (1) : 443 -455 . DOI: 10.13343/j.cnki.wsxb.20250599
正文
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德庆贡柑(Citrus reticulata cv. Gonggan in Deqing)作为我国特有的柑橘地方品种，其产业化开发模式在广东省取得了显著成效。肇庆市德庆县凭借独特的自然禀赋，成功培育出被誉为“中华柑王”的优质贡柑品种，该品种先后获得国家原产地域产品认证(2008年)、全国名特优新农产品名录(2020年)及国家地理标志保护农产品(2021年)等权威认证，现已成为区域农业经济的核心支柱产业[1]。这一产业的成功不仅彰显了地方特色农业的经济价值，更凸显了农业生态系统中微生物资源开发的战略意义。
在农业生态系统的多维作用框架下，植物-微生物互作网络对作物产量与品质的调控具有决定性影响。根际与叶际作为植物的特异性生态位，富集了具有促生功能的关键生态功能群[2-3]。这些微生物通过多重机制协同作用促进植物生长。例如，植物根际促生菌(plant growth-promoting rhizobacteria, PGPR)分泌的吲哚-3-乙酸(indole-3-acetic acid, IAA)类植物生长调节物质可优化根系发育[4]；根际细菌产生铁载体的能力赋予了内生细菌在植物根系定殖和排除来自同一环境的其他微生物的各种优势[5]；固氮菌可通过生物固氮、酶促反应和反硝化等作用使环境中的氮为植物所利用[6]；溶磷菌通过分泌低分子有机酸和合成磷酸酶的方式溶磷和矿化有机磷，从而改良土壤肥力[6]；解钾菌通过矿化难溶性钾素提升养分生物有效性[4,6]；而叶际生防菌则通过抗菌肽等次生代谢物构建病原菌抑制屏障[4,7-9]。这些功能微生物的开发利用已成为提升作物抗逆性、实现农业可持续发展的重要生物技术路径。
尽管研究人员已对广西壮族自治区钟山县及广东省仁化县等产区的贡柑开展了果园土壤中解磷菌的筛选[10]及叶际柑橘溃疡病拮抗菌资源的挖掘[11]等研究，但针对德庆贡柑核心产区的系统性微生物资源调查仍属空白。为填补这一空白，本研究聚焦德庆贡柑产区，结合传统分离培养技术与现代分子鉴定技术系统解析其根围土壤及叶际表面可培养细菌的多样性与种类组成。在此基础上，结合促生功能评价体系筛选优势功能菌株，以期为开发具有地域特色的微生物菌剂提供理论依据，对提升贡柑品质、优化农业生态环境及推动乡村振兴均具有重要实践价值。
1 材料与方法
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1.1 材料
1.1.1 样品采集
细菌分离试验所用样品为德庆贡柑(Citrus reticulata cv. Gonggan in Deqing)根围土壤及叶片，于2024年11月7日采集自广东省肇庆市德庆县的贡柑果园(23°17′21.41′′N，112°12′32.40′′E)。
1.1.2 主要试剂和仪器
三羟甲基氨基甲烷(Tris)、乙二胺四乙酸(ethylenediaminetetraacetic acid, EDTA)、无水乙醇、75%乙醇、次氯酸钠、丙三醇、铬天青S、Salkowski比色液等试剂均为国产分析纯；PCR所用Buffer、dNTP、Taq酶均购自南京诺唯赞生物科技股份有限公司。
PCR仪，赛默飞世尔科技(中国)有限公司；超净工作台，苏州安泰空气技术有限公司；生化培养箱，天津市泰斯特仪器有限公司。
1.1.3 培养基和主要试剂配方
R2A培养基、无机磷培养基、有机磷细菌培养基、无氮培养基、固氮菌用阿须贝氏培养基(HB8540)、M1997培养基和CAS检测培养基，Hopebio公司；NA培养基(022010)，广东环凯微生物科技有限公司。
Salkowski比色液：50 mL 35% HClO4，1 mL 0.5 mol/L FeCl3；PBS缓冲液：KH2PO4 0.24 g/L，Na2HPO4 1.44 g/L，NaCl 8 g/L，KCl 0.2 g/L，Tween-20 500 μL/L，pH 7.4；碱性裂解液：25 mmol/L NaOH，0.2 mmol/L Na2-EDTA， pH 12.0；中和缓冲液：40 mmol/L Tris-HCl，pH 7.5。
1.2 贡柑根围土壤及叶际细菌的分离与纯化
从广东省肇庆市德庆县的2处果园采集贡柑根围土壤和叶片样品，将同一样地来源的健康根围土壤和叶片进行均等并样，每个样地通过五点法采集5份健康样品，每个样品分别称取土壤和叶片2 g，合并后共10 g。将样品放入500 mL三角瓶，土壤加100 mL无菌水，叶片加200 mL无菌PBS缓冲液，28 ℃、200 r/min振荡30 min，叶片样品超声(40 Hz)洗脱5 min；分别吸取1 mL土壤悬液和超声后的叶片洗脱液到装有9 mL无菌水的试管中进行梯度稀释，其中叶片样品依次稀释为10-2、10-3、10-4，土壤样品增加稀释梯度10-5和10-6，稀释好的土壤和叶片样品分别取100 µL和200 µL均匀涂布到R2A培养基上，28 ℃培养2-7 d，选择合适稀释梯度挑取单菌落，通过反复划线进行纯化培养。
1.3 贡柑根围土壤及叶际分离细菌的鉴定
从平板上刮取纯培养的菌体，利用碱性裂解法提取细菌DNA。细菌16S rRNA基因序列采用通用引物27F (5ʹ-AGAGTTTGATCCTGGC TCAG-3ʹ)和1492R (5ʹ-GGTTACCTTGTTACGA CTT-3ʹ)进行PCR扩增。PCR反应体系(25 µL)：GreenTaq Mix (p131) 12.5 µL，上、下游引物(10 µmol/L)各1 µL，DNA模板2 µL，ddH2O 8.5 µL。PCR反应条件：95 °C预变性3 min；95 °C变性15 s，55 °C退火15 s，72 °C延伸90 s，共35个循环；72 °C终延伸5 min。PCR产物经琼脂糖凝胶电泳检测后送至苏州金唯智生物科技有限公司进行Sanger测序。测序得到27F和1492R的两端序列，用DNAMAN软件进行序列拼接，得到16S rRNA基因的全长序列，通过EzBioCloud (https://www. ezbiocloud.net/)数据库在线比对[12]，得到相应菌株的分类信息。鉴定菌株使用25%甘油于-80 ℃保藏。
1.4 菌株促生功能评价
菌株的功能评价参照文献[13]的方法进行：利用铬天青S培养基[14]对菌株产铁载体能力进行定性测定。菌株产铁载体定量测定：等量混合(各100 μL)各菌株的无细胞上清液和铬天青S检测液，静置1 h后用酶标仪测定630 nm波长的吸光值As，以加ddH2O为对照调0，另取等体积无菌培养基与铬天青S检测液混合，测定的吸光值为参比值Ar。铁载体量根据公式(1)计算[15]。通过Salkowski比色法检测菌株产IAA的能力[16]，并以IAA标准品梯度稀释绘制标准曲线，定量测定菌株产IAA的量；利用溶磷、解钾和固氮筛选培养基对菌株的溶磷功能(包括有机磷和无机磷)、解钾及固氮功能进行定性测定，通过观察筛选培养基平板的特异性透明圈大小评价菌株功能[17-18]。
铁载体量=(Ar-As)/Ar×100%
1.5 植物促生试验
以‘中蔬四号’番茄为试验材料，对筛选获得的多功能菌株进行盆栽促生实验。分别挑取菌株单菌落于100 mL NA液体培养基中，30 ℃、200 r/min培养2 d；4 ℃、8 000 r/min离心5 min收集菌体，使用无菌水重悬菌体，再次离心，洗去残留培养基，收集菌体再次用无菌水重悬并调整菌悬液OD600=1.0。挑选长势相近的番茄幼苗45盆。处理组(OD600=1.0的菌悬液)将配制好的菌液均匀地浇灌到幼苗根系附近，每盆浇灌5 mL，对照组(CK)每盆浇灌无菌水5 mL，每组8盆重复。每天浇水1-2次，置于广东省科学院微生物研究所温室培养，培养条件：28 ℃，12 h (光)/12 h (暗)，湿度为65%。处理15 d时对每个处理的8株幼苗测量株高和鲜重，并观察番茄幼苗的叶片生长情况，以此考察菌株的促生功能。
1.6 数据处理
采用Excel及GraphPad Prism 8进行数据处理和图片绘制[19]，使用IBM SPSS Statistics 26进行数据分析，利用ANOVA进行单因素方差分析(P<0.05)。
2 结果与分析
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2.1 德庆贡柑根围土壤及叶际细菌的分离鉴定
本研究共分离到240株细菌，其中贡柑根围土壤96株，叶际144株。对所有菌株的分类地位进行统计分析后发现，所分离到的240株细菌隶属于4门5纲20目29科51属(图1)。其中，100株为假单胞菌门(Pseudomonadota)菌株，69株为放线菌门(Actinomycetota)菌株，66株为芽孢杆菌门(Bacillota)菌株，拟杆菌门(Bacteroidota)最少，仅有5株(图1A)；在科水平上，微杆菌科(Microbacteriaceae)中的短小杆菌属(Curtobacterium)、叶居菌属(Frondihabitans)与胺杆菌属(Amnibacterium)分离数量最多，共32株，占比13.3%，其次为甲基杆菌科(Methylobacteriaceae)和芽孢杆菌科(Bacillaceae)，占比均为9.1%；在属水平上，优势属为短小杆菌属(Curtobacterium)，共分离出33株，占比13.8%，其次为假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)，占比分别为9.6%和7.9% (图1B)。
2.2 德庆贡柑根围土壤及叶际分离细菌的功能评价
对所分离出的240株贡柑根围土壤及叶际细菌进行了IAA、产铁载体、溶解有机磷、溶解无机磷、解钾和固氮等功能的评价，结果发现230株菌具有至少一种促生功能，占比95.83% (图2)。
在这些促生功能菌株中有210株能够产IAA (图2A)，占比91.30%。其中，柠檬色短小杆菌(Curtobacterium citreum) CYT1802产IAA能力最强，达到68.99 mg/L；其次为植物内生戈特弗里德氏菌(Gottfriedia endophyticus) CR15和天蓝黄链霉菌(Streptomyces coelicoflavus) CR78，产IAA的量分别为55.39 mg/L和54.36 mg/L。有199株细菌能够产铁载体(图2B)，占比86.52%。定量测定结果显示，阿氏普里斯特氏菌(Priestia aryabhattai) XXL03的产铁载体能力最强，为37.80%；其次是暹罗芽孢杆菌(Bacillus siamensis) CR99和海泥短小杆菌(Curtobacterium oceanosedimentum) XXL08，产铁载体的量分别为34.03%和29.93%。定性测定结果显示，霍氏假单胞菌(Pseudomonas rhodesiae) CLY10产铁载体能力最强，透明圈直径达4.60 cm；其次是神秘伯克霍尔德氏菌(Burkholderia aenigmatica) HIP6和新洋葱伯克霍尔德氏菌(Burkholderia cenocepacia) YOP2。有109株细菌能够溶解有机磷(图2C)，占比47.39%，其中新洋葱伯克霍尔德氏菌(Burkholderia cenocepacia) YOP2溶解有机磷能力最强，透明圈直径达4.69 cm；其次是霍氏假单胞菌(Pseudomonas rhodesiae) CLY10和奶色副白色单胞菌(Paracandidimonas lactea) CR48 (图3)。有89株细菌具有固氮能力(图2D)，占比38.70%，其中藤黄短小杆菌(Curtobacterium luteum) CLX11固氮能力最强，透明圈直径达3.00 cm；其次是白色短小杆菌(Curtobacterium albidum) CLX03和海泥短小杆菌(Curtobacterium oceanosedimentum) CLX23 (图3)。有62株菌具有溶解无机磷的功能(图2E)，占比26.96%，其中韩中大鞘氨醇单胞菌(Sphingomonas chungangi) YFL15溶无机磷能力最强，透明圈直径达2.61 cm；其次是海泥短小杆菌(Curtobacterium oceanosedimentum) CXT37和霍氏假单胞菌(Pseudomonas rhodesiae) CLY19 (图3)。有45株细菌具有解钾功能(图2F)，占比19.57%，其中霍氏假单胞菌(Pseudomonas rhodesiae) CLY10解钾能力最强，透明圈直径达1.78 cm；其次是新洋葱伯克霍尔德氏菌(Burkholderia cenocepacia) YOP2和德雷氏泛菌(Pantoea deleyi) CXT1101 (图3)。
对这些功能菌株进一步分析发现，除解钾功能外，分离到的产IAA、产铁载体、溶解无机磷、固氮及溶解有机磷的功能菌株中短小杆菌属(Curtobacterium)的菌株占比最多，其次是假单胞菌属(Pseudomonas)、芽孢杆菌属(Bacillus)和肠杆菌属(Enterobacter) (图2)。此外，还发现肠杆菌属(Enterobacter)和泛菌属(Pantoea)的细菌相比其他属菌株具有更强的解钾能力(图2F)。
通过UpSet图和韦恩图对230株贡柑促生细菌的功能进行分析发现，有123株细菌同时具备3种以上的促生功能，其中包含33株同时具有3种功能的细菌，隶属于18属26种。其中鞘氨醇单胞菌属(Sphingomonas)和芽孢杆菌属(Bacillus)数量最多，均有4株，占比均为12.1%；其次为假单胞菌属(Pseudomonas)、甲基杆菌属(Methylobacterium)和短小杆菌属(Curtobacterium)，占比均为9.1%。有39株同时具有4种功能的细菌，隶属于17属24种，其中短小杆菌属(Curtobacterium)数量最多，有12株，占比30.8%，其次为芽孢杆菌属(Bacillus)、普里斯特氏菌属(Priestia)和肠杆菌属(Enterobacter)，占比均为10.3%。有37株同时具有5种功能的细菌，隶属于12属19种，其中短小杆菌属(Curtobacterium)数量最多，有13株，占比35.1%，其次为肠杆菌属(Enterobacter)和泛菌属(Pantoea)，占比分别为21.6%和10.8%。有14株同时具有6种功能的细菌，隶属于6属10种，其中泛菌属(Pantoea)数量最多，有6株，占比42.9%；其次为短小杆菌属(Curtobacterium)、微杆菌属(Microbacterium)和肠杆菌属(Enterobacter)，占比均为14.3% (图4)。
2.3 功能菌株的盆栽促生效果验证
以功能为导向，本研究随机选取8株具有不同功能特性的促生菌株，通过盆栽试验对其功能进行验证。所选取的8株菌株中，1株假单胞菌新物种(Pseudomonas sp.) CR1201具有产铁载体和分泌IAA 2种功能，1株韩中大鞘氨醇单胞菌(Sphingomonas chungangi) YFL15具有溶无机磷、产铁载体和分泌IAA 3种功能；1株白色短小杆菌(Curtobacterium albidum) CLX03具有溶有机磷、固氮、产铁载体和分泌IAA 4种功能；1株柠檬色短小杆菌(Curtobacterium citreum) CYT1802、1株喜花泛菌(Pantoea anthophila) CYT0101和1株神户肠杆菌(Enterobacter kobei) CXT52同时具有5种促生功能；1株藤黄短小杆菌(Curtobacterium luteum) CXT01和1株假罗根坎普氏肠杆菌(Enterobacter pseudoroggenkampii) CXT46同时具有6种促生能力。接种这些功能菌株能够促进盆栽番茄的生长，与对照相比，番茄植株的株高更高，根系更加发达(图5)。统计分析结果显示，供试的8株促生菌株中除了柠檬色短小杆菌CYT1802外，其余菌株均能显著提高番茄的株高(图6A)。植株鲜重测定结果统计分析显示，仅柠檬色短小杆菌CYT1802、喜花泛菌CYT0101和神户肠杆菌CXT52接种处理后番茄鲜重与对照无显著差异，其余5株菌接种处理后番茄鲜重均显著高于对照(图6B)。
3 讨论与结论
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广东省肇庆市德庆县农业部门最新统计数据显示，2024年德庆贡柑产业发展再上新台阶：种植规模持续扩大，种植面积达0.29万hm2；截至目前，德庆贡柑已畅销至全国各地，销售量达1.148亿斤；预计总产量将攀升至1.64亿斤，实现总产值8.2亿元，较以往增长30.15%。这些数据不仅彰显了德庆贡柑作为“国家地理标志产品”的市场认可度，更折射出特色农业在乡村振兴战略中的核心价值。在农业农村部大力推进特色水果产业高质量发展的政策框架下，德庆贡柑产业正迎来转型升级的关键期。目前，推动产业向“绿色化、高效化、现代化”迈进已成为核心任务。
研究指出植物-微生物互作在调控土壤理化性质、提升植物产量与抗逆性方面发挥着重要作用[20]。在柑橘类植物的微生物研究中，众多成果已证实假单胞菌门(Pseudomonadota)是根际与叶际可培养细菌中的优势菌群。本研究聚焦于广东省肇庆市德庆县的主要经济柑橘品种——德庆贡柑，从其根际土壤和叶际样品中分离出共240株细菌。经鉴定，这些分离菌株主要隶属于假单胞菌门、放线菌门和芽孢杆菌门。在科分类水平上，优势类群为芽孢杆菌科，其次是微杆菌科和假单胞菌科。这一组成特征与其他柑橘类植物的相关研究结果基本一致，表明上述细菌类群在柑橘类植物根际及叶际环境中普遍存在。
基于分离获得的细菌，本研究进一步筛选了具有特定功能的菌株(如有机/无机磷溶解、固氮、解钾、产IAA和铁载体合成)，从德庆贡柑中共筛选出230株分属于芽孢杆菌科、微杆菌科和假单胞菌科等的促生细菌。结果表明，德庆贡柑蕴藏着丰富多样的促生细菌资源，其中部分菌株在溶磷、固氮、解钾及促进植物生长等方面表现卓越。为验证其实际促生效果，从中随机选取了8株具备2种以上上述功能的代表性菌株(包括泛菌属、鞘氨醇单胞菌属、肠杆菌属、短小杆菌属及假单胞菌属等)进行番茄盆栽促生实验，发现这些菌株能够显著促进番茄生长。这些发现与文献报道相符，如阴沟肠杆菌(Enterobacter cloacae) DJ在盐碱条件下能够促进黄瓜幼苗的生长[21]；鞘氨醇单胞菌(Sphingomonas sp.) CX7产生的胞外多糖对大麦生长具有促生作用[22]；泛菌(Pantoea sp.) FBS135不仅能够促进马尾松幼苗的生长，还能提高其抗逆性[23]；水稻内生成团泛菌(Pantoea agglomerans) HAUM1在提高水稻的株高、鲜重及干重方面优势显著[24]。本研究结果进一步证实了这些菌属在植物促生方面的巨大潜力。
综上所述，本研究筛选获得的功能菌株为后续开发贡柑高效复合微生物菌剂提供了宝贵的资源。未来的研究应结合宏基因组学技术与田间试验，深入挖掘这些促生细菌资源的产业化潜力，为德庆贡柑绿色、可持续生产提供坚实的科技支撑，推动产业持续健康发展。
基金
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  • 省级乡村振兴战略专项资金种业振兴项目(2022-440000-43010104-9463)
  • 广东省自然科学基金(2024A1515011067)
  • 广东特支计划(2021JC06N628)
文献
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参考文献 引证文献
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2026年第66卷第1期
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doi: 10.13343/j.cnki.wsxb.20250599
  • 接收时间：2025-08-01
  • 首发时间：2026-01-12
  • 出版时间：2026-01-04
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  • 收稿日期：2025-08-01
  • 录用日期：2025-09-10
基金
Guangdong Strategic Special Fund for Rural Revitalization(2022-440000-43010104-9463)
省级乡村振兴战略专项资金种业振兴项目(2022-440000-43010104-9463)
Natural Science Foundation of Guangdong Province(2024A1515011067)
广东省自然科学基金(2024A1515011067)
Guangdong Special Support Program(2021JC06N628)
广东特支计划(2021JC06N628)
作者信息
    1.广东省科学院微生物研究所，华南应用微生物国家重点实验室，农业农村部农业微生物组学与精准应用重点实验室，农业农村部农业微生物组学重点实验室，广东省菌种保藏与应用重点实验室，广东 广州
    2.华南农业大学 植物保护学院，广东 广州

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
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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