Article(id=1276530007312830922, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276529901037548535, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2025.06.019, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1735660800000, receivedDateStr=2025-01-01, revisedDate=null, revisedDateStr=null, acceptedDate=1740672000000, acceptedDateStr=2025-02-28, onlineDate=1782278070523, onlineDateStr=2026-06-24, pubDate=1750780800000, pubDateStr=2025-06-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782278070523, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782278070523, creator=13701087609, updateTime=1782278070523, updator=13701087609, issue=Issue{id=1276529901037548535, tenantId=1146029695717560320, journalId=1235980609244409860, year='2025', volume='46', issue='6', pageStart='1279', pageEnd='1532', issueExtLink='null', onlineDate='null', pubDate='1750780800000', pubDateStr='2025-06-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782278045186, creator='13701087609', updateTime=1782298980105, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276617708544328532, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276529901037548535, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276617708544328533, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276529901037548535, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1472, endPage=1479, ext={EN=ArticleExt(id=1276530007614820813, articleId=1276530007312830922, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Biocontrol of Banana Bacterial Soft Rot by Quorum Quenching Bacterium GXMZU-5 and Its Mechanism Study, columnId=1236292524264968282, journalTitle=Chinese Journal of Tropical Crops, columnName=Plant Protection & Bio-safety, runingTitle=null, highlight=null, articleAbstract=

In order to investigate the biocontrol ability and mechanism of Burkholderia GXMZU-5 with quorum quenching activity, the biocontrol effect was verified through experiments on bacterial soft rot of banana fruits and potted seedlings (Dickeya zeae GR-1), and the biocontrol mechanism was verified by measuring the content of biofilm, the ability to remove extracellular polysaccharides, the ability to inhibit the metabolic activity of biofilm, and PCR amplification of QQ enzyme homologous genes. The results showed that GXMZU-5 could effectively degrade acylhomoserine lactone (AHLs) signal molecules and had AHLs quenching ability. In the biocontrol experiment on banana fruits, the surface of the mixed bacteria experimental group was healthier than that of the control group, with no obvious black spots or soft rot signs. The cross-section and longitudinal section results of banana fruits showed that the lesion area of the treatment group was significantly reduced. In the potted plant control experiment, there were no obvious signs of rot at the injection site of the mixed bacteria group, and the natural plant height recovered by 56% compared with the pathogen group, indicating that GXMZU-5 had a significant inhibitory effect on the disease caused by Dickeya zeae GR-1. The results for exploring of the biocontrol mechanism showed that the total content of biofilm and the content of extracellular polysaccharides in the mixed bacteria system were lower than those of the two bacteria cultured separately. The MTT method for measuring biofilm metabolism indicated that GXMZU-5 could effectively reduce the biofilm metabolic activity of Dickeya zeae GR-1 and occupied a dominant position in the mixed bacteria system. The exploration of the biocontrol mechanism indicated that GXMZU-5 could reduce the biomass and vitality of Dickeya zeae GR-1 biofilm through quenching effect, thereby obtaining biocontrol ability. The amplification of QQ enzyme homologous genes showed that GXMZU-5 had the aiiA lactonase gene and could quench AHLs signal molecules. This study verified the biocontrol ability of GXMZU-5 and preliminarily explored its biocontrol mechanism, providing a certain reference basis for the use of quenching bacteria to control plant bacterial diseases.

, authors=null, authorsList=Tianhan QIN, Jinxin YANG, Lu WANG, Yingze DUAN, Jing WEI, Jiahui LI, Yingzhi ZHU, Zhanbiao LI, authorCompany=null, correspAuthors=Yingzhi ZHU, Zhanbiao LI, 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=1276530011783959016, articleId=1276530007312830922, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=群体感应淬灭活性生防菌GXMZU-5对香蕉细菌性软腐病的生物防治及其机制研究, columnId=1236292524520820846, journalTitle=热带作物学报, columnName=植物保护与生物安全, runingTitle=null, highlight=null, articleAbstract=

为探究具有群体感应淬灭活性的伯克霍尔德菌(Burkholdria)GXMZU-5生物防治的能力及其机制,通过对香蕉果实及盆栽苗细菌性软腐病病菌Dickeya zeae GR-1的防治试验验证其生物防治效果,通过测定生物膜含量、胞外多糖清除能力、生物膜代谢活性抑制能力及QQ酶同源基因PCR扩增验证其生物防治机制。结果表明:GXMZU-5可有效降解酰基高丝氨酸内酯信号分子(acyl-homoserine lactones,AHLs),具有AHLs淬灭能力;在香蕉果实上进行的生物防治试验中,GXMZU-5的混菌处理组果实表面相较于对照组更健康,未出现明显黑斑和软腐迹象,香蕉果实横切和纵切结果表明,处理组的病斑扩散面积大幅度减少;盆栽防治试验中,混菌处理组的香蕉假茎注射处无明显腐烂迹象,自然株高相比病原菌组恢复了56%,表明GXMZU-5具有显著抑制病原菌发病的作用。对其生物防治机制探究表明,混菌体系下的生物膜总含量、胞外多糖含量小于2种菌单独培养;MTT法测定生物膜代谢表明,GXMZU-5可有效地降低GR-1的生物膜代谢活动,且在混菌体系内占主要优势位置。说明GXMZU-5可通过淬灭效应降低GR-1生物膜生物量及活力,从而获得生物防治能力;对QQ酶同源基因进行扩增表明,GXMZU-5具有aiiA内脂酶基因,可淬灭AHLs信号分子。本研究验证了GXMZU-5的生物防治能力,并初步探究了其防治机制,为淬灭菌防治植物细菌性病害提供一定的参考依据。

, authors=

覃天涵(1999—),男,硕士研究生,研究方向:微生物与植物互作。

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* 朱英芝(ZHU Yingzhi),E-mail:
李战彪(LI Zhanbiao),E-mail:
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覃天涵(1999—),男,硕士研究生,研究方向:微生物与植物互作。

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(in Chinese), articleTitle=Solation, properties and structural characterization of VFM, a new quorum sensing signal of Dickeya dadantii, refAbstract=null), Reference(id=1276530038312931963, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530007312830922, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[24], rfOrder=40, authorNames=陈少华, 李绮婷, 范兴辉, 叶田, 李欣, 梁梓侨, journalName=null, refType=null, unstructuredReference=陈少华, 李绮婷, 范兴辉, 叶田, 李欣, 梁梓侨. 溶蛋白芽孢杆菌在防治依赖群体感应信号分子的致病菌及病害方面的应用: 201910843482[P]. 2024-11-20., articleTitle=溶蛋白芽孢杆菌在防治依赖群体感应信号分子的致病菌及病害方面的应用, refAbstract=null), Reference(id=1276530038388429436, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530007312830922, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[24], rfOrder=41, authorNames=CHEN S H, LI Q T, FAN X H, YE T, LI X, LIANG Z Q, journalName=null, refType=null, unstructuredReference=CHEN S H, LI Q T, FAN X H, YE T, LI X, LIANG Z Q. Application of proteolytic bacillus in the control of pathogens and diseases dependent on quorum sensing signaling molecules: 201910843482[P]. 2024-11-20. 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companyName=null, departmentName=null, remark=5.农业农村部华南果蔬绿色防控重点实验室,广西南宁 530007)])], figs=[ArticleFig(id=1276530029303566906, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530007312830922, language=EN, label=Fig. 1, caption=Ability of strain CV026 to utilize CHL-6, figureFileSmall=GfpOXSEq3muevnkPySsOEw==, figureFileBig=GZzL8VseIOAB/mXoKu5Owg==, tableContent=null), ArticleFig(id=1276530029672665659, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530007312830922, language=CN, label=图1, caption=CV026验证菌株利用CHL-6的能力试验

A: MSM+CHL-6; B: E. coli +CHL-6; C: GXMZU-5+CHL-6.

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A:外观;B:横切;C:纵切。a:LB;b:GR-1;c:GXMZU-5+GR-1;d:GXMZU-5。

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A: LB; B: GR-1; C: GXMZU-5+GR-1.

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A: LB; B: GR-1; C: GXMZU-5+GR-1.

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M: DL2000 DNA marker.

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A: GXMZU-5; B: GR-1; C: GXMZU-5+GR-1.

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群体感应淬灭活性生防菌GXMZU-5对香蕉细菌性软腐病的生物防治及其机制研究
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覃天涵 1, 3 , 杨金新 1, 3 , 王璐 1 , 段莹泽 1 , 韦婧 1 , 李佳惠 1 , 朱英芝 1, 2, 3, * , 李战彪 2, 4, 5, *
热带作物学报 | 植物保护与生物安全 2025,46(6): 1472-1479
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热带作物学报 |植物保护与生物安全 2025 , 46 (6) : 1472 -1479
群体感应淬灭活性生防菌GXMZU-5对香蕉细菌性软腐病的生物防治及其机制研究
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覃天涵1, 3, 杨金新1, 3, 王璐1, 段莹泽1, 韦婧1, 李佳惠1, 朱英芝1, 2, 3, * , 李战彪2, 4, 5, *
作者信息
  • 1.广西民族大学海洋与生物技术学院/广西多糖材料与改性重点实验室,广西南宁 530008
  • 2.广西作物病虫害生物学重点实验室,广西南宁 530007
  • 3.海洋生物资源开发与利用国际合作重点实验室,广西南宁 530008
  • 4.广西农业科学院植物保护研究所,广西南宁 530007
  • 5.农业农村部华南果蔬绿色防控重点实验室,广西南宁 530007
通讯作者:
* 朱英芝(ZHU Yingzhi),E-mail:
李战彪(LI Zhanbiao),E-mail:
Biocontrol of Banana Bacterial Soft Rot by Quorum Quenching Bacterium GXMZU-5 and Its Mechanism Study
Tianhan QIN1, 3, Jinxin YANG1, 3, Lu WANG1, Yingze DUAN1, Jing WEI1, Jiahui LI1, Yingzhi ZHU1, 2, 3, * , Zhanbiao LI2, 4, 5, *
Affiliations
  • 1.School of Marine and Biotechnology, Guangxi Minzu University / Guangxi Key Laboratory of Polysaccharide Materials and Modification, Nanning, Guangxi 530008, China
  • 2.Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Nanning, Guangxi 530007, China
  • 3.Key Laboratory of International Cooperation for Exploitation and Utilization of Marine Bio-resources, Nanning, Guangxi 530008, China
  • 4.Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530007, China
  • 5.Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China, Ministry of Agriculture and Rural Affairs, Nanning, Guangxi 530007, China
出版时间: 2025-06-25 doi: 10.3969/j.issn.1000-2561.2025.06.019
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为探究具有群体感应淬灭活性的伯克霍尔德菌(Burkholdria)GXMZU-5生物防治的能力及其机制,通过对香蕉果实及盆栽苗细菌性软腐病病菌Dickeya zeae GR-1的防治试验验证其生物防治效果,通过测定生物膜含量、胞外多糖清除能力、生物膜代谢活性抑制能力及QQ酶同源基因PCR扩增验证其生物防治机制。结果表明:GXMZU-5可有效降解酰基高丝氨酸内酯信号分子(acyl-homoserine lactones,AHLs),具有AHLs淬灭能力;在香蕉果实上进行的生物防治试验中,GXMZU-5的混菌处理组果实表面相较于对照组更健康,未出现明显黑斑和软腐迹象,香蕉果实横切和纵切结果表明,处理组的病斑扩散面积大幅度减少;盆栽防治试验中,混菌处理组的香蕉假茎注射处无明显腐烂迹象,自然株高相比病原菌组恢复了56%,表明GXMZU-5具有显著抑制病原菌发病的作用。对其生物防治机制探究表明,混菌体系下的生物膜总含量、胞外多糖含量小于2种菌单独培养;MTT法测定生物膜代谢表明,GXMZU-5可有效地降低GR-1的生物膜代谢活动,且在混菌体系内占主要优势位置。说明GXMZU-5可通过淬灭效应降低GR-1生物膜生物量及活力,从而获得生物防治能力;对QQ酶同源基因进行扩增表明,GXMZU-5具有aiiA内脂酶基因,可淬灭AHLs信号分子。本研究验证了GXMZU-5的生物防治能力,并初步探究了其防治机制,为淬灭菌防治植物细菌性病害提供一定的参考依据。

群体感应淬灭  /  伯克霍尔德菌  /  香蕉细菌性软腐病  /  生物防治

In order to investigate the biocontrol ability and mechanism of Burkholderia GXMZU-5 with quorum quenching activity, the biocontrol effect was verified through experiments on bacterial soft rot of banana fruits and potted seedlings (Dickeya zeae GR-1), and the biocontrol mechanism was verified by measuring the content of biofilm, the ability to remove extracellular polysaccharides, the ability to inhibit the metabolic activity of biofilm, and PCR amplification of QQ enzyme homologous genes. The results showed that GXMZU-5 could effectively degrade acylhomoserine lactone (AHLs) signal molecules and had AHLs quenching ability. In the biocontrol experiment on banana fruits, the surface of the mixed bacteria experimental group was healthier than that of the control group, with no obvious black spots or soft rot signs. The cross-section and longitudinal section results of banana fruits showed that the lesion area of the treatment group was significantly reduced. In the potted plant control experiment, there were no obvious signs of rot at the injection site of the mixed bacteria group, and the natural plant height recovered by 56% compared with the pathogen group, indicating that GXMZU-5 had a significant inhibitory effect on the disease caused by Dickeya zeae GR-1. The results for exploring of the biocontrol mechanism showed that the total content of biofilm and the content of extracellular polysaccharides in the mixed bacteria system were lower than those of the two bacteria cultured separately. The MTT method for measuring biofilm metabolism indicated that GXMZU-5 could effectively reduce the biofilm metabolic activity of Dickeya zeae GR-1 and occupied a dominant position in the mixed bacteria system. The exploration of the biocontrol mechanism indicated that GXMZU-5 could reduce the biomass and vitality of Dickeya zeae GR-1 biofilm through quenching effect, thereby obtaining biocontrol ability. The amplification of QQ enzyme homologous genes showed that GXMZU-5 had the aiiA lactonase gene and could quench AHLs signal molecules. This study verified the biocontrol ability of GXMZU-5 and preliminarily explored its biocontrol mechanism, providing a certain reference basis for the use of quenching bacteria to control plant bacterial diseases.

quorum quenching  /  Burkholderia  /  banana bacterial soft rot  /  biological control
覃天涵, 杨金新, 王璐, 段莹泽, 韦婧, 李佳惠, 朱英芝, 李战彪. 群体感应淬灭活性生防菌GXMZU-5对香蕉细菌性软腐病的生物防治及其机制研究. 热带作物学报, 2025 , 46 (6) : 1472 -1479 . DOI: 10.3969/j.issn.1000-2561.2025.06.019
Tianhan QIN, Jinxin YANG, Lu WANG, Yingze DUAN, Jing WEI, Jiahui LI, Yingzhi ZHU, Zhanbiao LI. Biocontrol of Banana Bacterial Soft Rot by Quorum Quenching Bacterium GXMZU-5 and Its Mechanism Study[J]. Chinese Journal of Tropical Crops, 2025 , 46 (6) : 1472 -1479 . DOI: 10.3969/j.issn.1000-2561.2025.06.019
群体感应(quorum sensing,QS)是一种微生物间进行通信的机制,细菌通过自发产生并释放信号分子到环境中[1],种群随后根据信号分子的浓度来调控诸如生物发光、生物膜形成、酶的产生等生理活动[2]。QS信号分子有很多种类,如酰基高丝氨酸内酯(acyl-homoserine lactones,AHLs)[3]、顺式-11-甲基-2-十二烯酸(diffusible signaling factor,DSF)[4]、烷基喹啉分子(alkyl-quinolones,AQ)[5]等,经研究发现,部分植物病原菌也通过利用群体感应完成侵染活动,例如胡萝卜软腐果胶杆菌(Pectobacterium carotovorum subsp. carotovorum,Pcc)通过感知AHLs,调控产生多聚半乳糖醛酸酶和果胶酸盐裂解酶,从而破坏植物细胞壁完成侵染[6],野油菜黄单胞菌(Xanthomonas campestris pv. campestris,Xcc)依赖DSF调控各种胞外多糖的产生及各类致病因子的产生[7]
细菌性病害是植物主要病害之一,常引起巨大的经济损失[8],目前主要通过化学农药和微生物源农药进行防治,但过度使用农药可导致病原菌产生耐药性[9-11]。群体感应淬灭(quorum quenching,QQ)是切断微生物种群间交流的一种机制,通过形成信号分子类似物竞争性地与受体结合[12];或形成可特异性降解信号分子的酶[13]等形式切断病原微生物的群体交流,使病原菌的毒力因子表达水平下降从而达到生物防治的效果[14]。KALIA等[15]研究发现二酮哌嗪(diketopiperazine,DKP)作为一种竞争类似物,可通过竞争与AHLs受体结合,从而引起群体感应水平下降并减弱病原菌致病能力。ZHANG等[16]发现Acinetobacter sp.对Pcc具有明显的防治效果,GC-MS分析表明该菌可有效降解AHLs,从而干扰细菌的群体感应系统,有效抑制病原菌的致病性。
香蕉细菌性软腐病是我国香蕉主要病害之一,病原为Dickeya zeae,主要引起香蕉叶片枯黄,球茎或球茎与假茎交界处出现黑褐色斑点,随后迅速扩散至整株植株,并开始腐烂,直至植株死亡[17],我国香蕉每年均遭受细菌性软腐病的大面积侵害,且侵害范围不断扩大,导致严重的经济损失。本研究以香蕉细菌性软腐病菌为防治对象,探究一株具有群体感应淬灭活性的伯克霍尔德菌(Burkholdria)GXMZU-5对香蕉细菌性软腐病的生物防治效果,以期为防治香蕉细菌性软腐病提供一种新型防治方式,也为伯克霍尔德菌GXMZU-5在植物细菌性病害防治中的合理应用提供理论基础。
AHLs指示菌紫色杆菌CV026、伯克霍尔德菌(Burkholdria)GXMZU-5、大肠杆菌(Escherichia coli)均由本课题组保存,Dickeya zeae GR-1(GR-1)由广西农业科学院付岗老师课题组馈赠。
N-酰基高丝氨酸内酯信号分子(acyl-homoserine lactones,AHLs)CHL-6、5-(4,6-二氯三嗪基)氨基荧光素(5-DTAF)均购自上海麦克林生化科技股份有限公司。
香蕉购自广西海吉星农产品国际物流水果批发市场,香蕉幼苗(威廉斯品种)采自广西农业科学院。
LB(Luria-Bertani)培养基、NB(nutrinet broth)培养基、TSB(trypticase soy broth)培养基均购自青岛海博生物科技有限公司。MSM(minimal salt medium)基础盐培养基:硫酸铵2 g,七水合硫酸镁0.2 g,二水合氯化钙0.01 g,七水合硫酸亚铁0.001 g,十二水合磷酸氢二钠1.5 g,磷酸二氢钾1.5 g,蒸馏水1000 mL,pH 6.8。T100-PCR仪为美国伯乐公司生产,正置荧光显微镜BX51TRF为奥林巴斯公司产品,Eproch酶标仪为美国伯腾公司生产,其余均为常规试剂。
取菌株GXMZU-5、CV026于液体LB培养基中活化,在30 ℃、200 r/min条件下培养24 h,培养结束后以此为种子液,按10%接种量将GXMZU-5接种至1 mL MSM培养基中,并添加10 μL的CHL-6为唯一碳源。CV026接种至LB培养基中,在30 ℃、200 r/min条件下培养24 h,培养结束后取1 mL CV026均匀涂布于LB平板,晾干后取无菌2.5 cm圆形模具制作圆饼,再使用无菌200 μL枪头倒扣在圆饼中央挖洞。取发酵GXMZU-5 MSM培养液,利用0.22 μm过滤器进行除菌过滤,取滤液进行点样。设立空白MSM培养液+CHL-6,以及大肠杆菌+CHL-6为对照,每组均点样10 μL,在30 ℃条件下培养24 h,观察平板内CV026菌株产生的紫色色素情况。
菌株GXMXU-5和GR-1在NB平板上进行活化,然后分别挑取单菌落接种至NB培养基中,在30 ℃、200 r/min条件下培养24 h,调节其OD600值至1.0。然后将GR-1均匀涂布于整个平板,待其晾干后,于平板中央放置牛津杯,并向牛津杯中加入200 μL GXMZU-5菌液,观察平板上的拮抗情况。
将培养24 h的GXMXU-5和GR-1菌液的OD600调节至1.0,然后选取健康、表皮无瑕疵且状态一致的香蕉果实,使用1 mL无菌注射器穿透果肉表皮进行注射,试验组注射1 mL GXMZU-5+病原菌(1∶1等体积混合)混合液,对照注射1 mL空白培养液、病原菌,每个处理设3次重复。于28 ℃保湿培养3 d,观察果实发病情况。
将培养24 h的GXMXU-5、GR-1菌液的OD600调节至1.0,选取长势一致的健康香蕉苗,使用无菌注射器于假茎处进行注射,试验组注射1 mL GXMZU-5+病原菌(1∶1等体积混合)混合液,对照注射1 mL无菌培养液、病原菌,每个处理设3次重复,接种后的香蕉苗于网室内自然条件下培养。持续10 d观察植株发病情况,并在10 d后测量植株的自然株高。
参考KHALID等[18]的方法,合成AHLs内脂酶(aiiA)基因的扩增引物,引物序列为F(5ʹ-3ʹ):GATGGCCTGGAGAATGAC;R(5ʹ-3ʹ):GCGTGTAGGGTATGAGCC,扩增片段大小为257 bp。扩增条件:95 ℃预变性5 min,95 ℃变性30 s,59.9 ℃退火40 s,72 ℃延伸1 min,30次循环,然后在72 ℃下延伸10 min。采用1%琼脂糖凝胶电泳验证条带是否存在,同时判断PCR产物大小。
将GR-1与GXMZU-5设置单独、混菌培养,接种至5 mL 5% NB培养基中,于30 ℃下培养5 d,结束后弃培养基,加入无菌水清洗2遍后风干15 min,风干后滴加甲醇固定10 min,风干,后向管内加入0.1%结晶紫染色20 min,然后用无菌水漂洗,风干10 min,加入33%冰醋酸没过管壁溶解,于96孔板孔中加入200 μL溶解液,测定其OD595,以OD595表示生物膜含量。每组设置3个重复。
取培养24 h的菌液作为种子液,按1%接种量将GXMZU-5、GR-1接种至无菌NB培养基中,设置单一菌接种对照组、1∶1混菌试验组,于30 ℃下培养30 h,培养结束后在5000 r/min条件下离心20 min,收集上清液,加入3倍无水乙醇于4 ℃过夜,收集胞外多糖(EPS),收集结束后在8000 r/min条件下离心10 min,弃上清液后,置于烘箱中完全烘干后称重,设置3次重复。
参照齐龙升等[19]的方法,取培养24 h的菌液调整至对数期,然后按10%接种量接种至无菌5% NB培养基中,充分混匀后转接至96孔板内,设置GXMZU-5、GR-1单一培养组以及1∶1混菌试验组,每孔200 μL。将96孔板置于25 ℃下培养,分别于培养1、2、3、4、5 d后去除孔内悬浮细胞,用无菌磷酸缓冲液洗涤生物被膜2次,清除孔内的浮游菌体,随后采用MTT方法进行染色,利用酶标仪测定其OD595,设置3次重复。
参照齐龙升等[19]的方法进行制片,并利用正置荧光显微镜观察,在6孔细胞培养板板内放置无菌盖玻片(14 mm×14 mm),然后将过夜培养的2种菌液稀释至其OD600为0.5,每孔加入2 mL菌液,混菌组按1∶1进行混合,置于30 ℃下培养48 h。吸去孔中悬浮菌液,用无菌生理盐水冲洗玻片3次,避光条件下用4%多聚甲醛固定30 min,用无菌磷酸缓冲液冲洗3次,然后向玻片滴加5-(4,6-二氯三嗪基)氨基荧光素(5-DTAF)直至没过玻片,避光下孵育2 h,然后用无菌磷酸缓冲液重复冲洗玻片,用锡纸将玻片包裹,防止玻片受光线照射,于正置荧光显微镜下观察玻片。
CV026是一株人工突变菌株,主要用途为检测环境内是否存在AHLs分子,CV026自身不能产生AHLs,但当检测到外源的AHLs时会启动相关基因产生紫色色素。本研究中,经添加外源CHL-6与GXMZU-5培养24 h后,菌板无紫色色素产生,对照则出现明显的紫色,结果表明,GXMZU-5具有降解AHLs的能力(图1)。
经24 h培养后,GXMZU-5与GR-1混合培养后无明显抑菌圈,证明GXMZU-5不是通过拮抗作用对GR-1产生抑制作用,可以用于群体感应淬灭的后续研究(图2)。
香蕉经保湿培养后,混菌处理的病斑面积比病原菌单一处理的大幅度减少(图3A)。沿注射处横切后,淬灭菌本身未引起香蕉产生腐烂症状,而混菌处理后可明显减少果肉腐烂面积(图3B)。将果肉沿注射处纵切后,混菌处理后发病扩散面积显著减弱(图3C)。结果表明,GXMZU-5对GR-1有较好的生物防治作用。
香蕉盆栽经持续浇水后,对叶片状态、假茎注射处进行观察,病原菌对照组出现叶片明显发黄变黑、植株较矮,且注射处有明显腐烂黑斑症状(图4);混菌处理的发病症状明显减弱,且自然株高高于病原菌单一处理(图5);假茎注射处无明显黑斑(图6)。与2.3生物防治试验结果吻合,表明GXMZU-5对GR-1在盆栽试验中也呈现一定的生物防治效果。
内脂酶是群体感应淬灭环节中一类重要的酶类,本研究选定aiiA酶基因作为研究目标,结果表明,在257 bp左右出现明显且单一条带(图7),将单一条带进行切胶回收并送至奥克生物科技有限公司进行测序,将测序结果与NCBI进行比对后,发现与aiiA基因高度近似,证实GXMZU-5代谢产物中至少包含内脂酶,可以有效阻止病原菌的群体感应进程。
生物膜是微生物的一种天然屏障,通过生物膜的形式,病原菌可以在生物膜内更为稳定地增殖并提高抗逆性从而更有利地侵染植物,生物膜的生成受群体感应的调控。本研究将GXMZU-5与GR-1单独、混合培养5 d后,用结晶紫法对对照、混菌组的生物膜含量进行提取并测定其OD595,结果表明,混菌组的生物膜含量较两菌单独培养组低(图8),表明GXMZU-5不仅具有清除病原菌生物膜的能力,还会降低其自身生物膜的产生水平。
胞外多糖是生物膜组成的重要物质,为印证2.6中清除生物膜的结论,通过三倍醇沉法获得胞外多糖进行称重,GXMZU-5单独处理的胞外多糖含量为0.0034 g,GR-1单独处理的胞外多糖含量为0.0016 g,混菌处理的的胞外多糖含量为0.0018 g,总体低于两菌单独产量之和。证实在混菌体系下胞外多糖的产生量总体下降。此结论与2.6中生物膜总含量减少的结果一致,证明GXMZU-5对GR-1胞外多糖的产生有抑制作用。
为反映混菌体系下2种菌的优势种群及其活性,本研究通过使用96孔板进行MTT染色。结果显示在5% NB培养基中,GXMZU-5在培养1 d时活性最好,其OD595值为1.436,其生物膜代谢活性较高,混菌体系的OD595值几乎接近单独培养,而GR-1的代谢活性极低,其OD595仅为0.143,表明GXMZU-5在混菌初期为优势菌群;培养2 d时,GXMZU-5的生物膜代谢能力下降,但其活性仍高于GR-1;培养3~4 d时,GXMZU-5的生物膜代谢活性进一步降低,推测此时GXMZU-5的生物膜代谢暂时到达饱和,呈下降趋势,但与混菌组OD595值相差不大,仍然处于代谢优势状态;培养5 d时,GXMZU-5的生物膜代谢重新开始,始终高于GR-1的生物膜代谢活性,而混菌组的生物膜代谢活性与第4天的相近,第4天的OD595值为1.017,第5天的OD595值为1.0275,而GR-1的生物膜代谢活性持续增加(图9),证实GXMZU-5具有显著的抑制生物膜代谢的能力,符合2.6中生物膜清除、2.7中胞外多糖代谢的结论。
使用正置荧光显微镜观察GXMZU-5与GR-1在混合培养下的生物膜形成情况,由图10可以看出,GXMZU-5与GR-1单独培养时,均有明显、大片的荧光,能观察到大量的生物膜生成。而在混菌状态下,产生的荧光强度、生物膜面积均比单独培养时明显减少,进一步验证GXMZU-5对自身及GR-1的生物膜产生具有一定影响。
香蕉是我国重要的热带水果之一,深受大众喜爱。广西是我国香蕉主要种植区,其地表温度高、高温多雨的环境易生产优质果,但相应地也促进各类病原菌发病。近年来,由Dickeya zeae导致的香蕉细菌性软腐病使广西香蕉产量大幅下降,且发病面积逐年扩大,亟需寻找一种高效、绿色可持续发展的防治方式。研究发现,部分化学药剂可有效防治香蕉细菌性软腐病,如王铜和噻菌铜在防治香蕉细菌性软腐病方面表现出较好效果[20],但过度使用化学农药易导致土壤结构遭受破坏。已有针对Dickeya zeae导致的香蕉细菌性软腐病开展了一定的防治研究,如番华彩等[21]通过筛选,将春雷霉素等药剂应用于田间,防治效果达到90.1%,在多种作物上表现出显著的防治效果。周佳暖等[22]针对Dickeya zeae导致的香蕉细菌性软腐病致病机理进行了详细探究,表明致病性主要与胞外多糖、细胞壁降解酶等密切相关。群体感应淬灭作为一种新型防治方法,其毒性和危险性相对较低,但针对Dickeya zeae的群体感应淬灭研究极少。因此,本研究筛选到一株具有强淬灭AHLs信号分子能力的菌株GXMZU-5,在香蕉软腐病防治中取得明显的生物防治效果,为使用群体感应淬灭菌应用于香蕉细菌性软腐病的防治提供了一定的思路与研究基础。
本研究证实GXMZU-5对采用AHLs信号分子的病原细菌具有显著的群体感应淬灭效应,并对由AHLs调控的生物膜相关的内容进行了研究。结果表明,GXMZU-5可有效抑制Dickeya zeae GR-1的生物膜形成,此外胞外多糖的产量也大幅度下降。在混菌体系中,GXMZU-5的细胞活性占主导地位,表明该菌可能通过影响Dickeya zeae GR-1的生物膜代谢进程导致病原菌在自然环境中抗逆性减弱进而导致毒力下降。目前针对Dickeya zeae的研究,还发现其具有一种控制菌体发挥毒力的新型信号通路Vfm[23]。本研究已证实GXMZU-5可有效降解群体感应信号分子AHLs,并在防治试验中证实GXMZU-5可有效防治香蕉细菌性软腐病,但GXMZU-5对Dickeya zeae的Vfm信号分子是否具有淬灭作用的研究还在进行中,已有学者研究表明淬灭菌可能同时具备淬灭多种信号分子的能力,如陈少华等[24]发现了一株溶蛋白芽孢杆菌具有同时淬灭DSF、AHLs两种信号分子的能力。因此,推测GXMZU-5也可能通过同时淬灭多种信号分子,总体降低病原菌的毒力,从而达到有效防治香蕉细菌性软腐病的作用。今后将对GXMZU-5的生物防治机理进行深入探究,或对其淬灭信号分子的通路进一步拓宽探索,以期为群体感应淬灭应用于生物防治提供新的思路。
  • 广西自然科学基金项目(2023GXNSFAA026116)
  • 广西作物病虫害生物学重点实验室项目(22-035-31-23KF04)
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排序方式:
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2025年第46卷第6期
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doi: 10.3969/j.issn.1000-2561.2025.06.019
  • 接收时间:2025-01-01
  • 首发时间:2026-06-24
  • 出版时间:2025-06-25
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  • 收稿日期:2025-01-01
  • 录用日期:2025-02-28
基金
广西自然科学基金项目(2023GXNSFAA026116)
广西作物病虫害生物学重点实验室项目(22-035-31-23KF04)
作者信息
    1.广西民族大学海洋与生物技术学院/广西多糖材料与改性重点实验室,广西南宁 530008
    2.广西作物病虫害生物学重点实验室,广西南宁 530007
    3.海洋生物资源开发与利用国际合作重点实验室,广西南宁 530008
    4.广西农业科学院植物保护研究所,广西南宁 530007
    5.农业农村部华南果蔬绿色防控重点实验室,广西南宁 530007

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* 朱英芝(ZHU Yingzhi),E-mail:
李战彪(LI Zhanbiao),E-mail:
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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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