Article(id=1226296959393247294, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226296952975966478, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20240592, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1727280000000, receivedDateStr=2024-09-26, revisedDate=null, revisedDateStr=null, acceptedDate=1732118400000, acceptedDateStr=2024-11-21, onlineDate=1770301578614, onlineDateStr=2026-02-05, pubDate=1738598400000, pubDateStr=2025-02-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770301578614, onlineIssueDateStr=2026-02-05, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770301578614, creator=13701087609, updateTime=1770301578614, updator=13701087609, issue=Issue{id=1226296952975966478, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='2', pageStart='421', pageEnd='861', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770301577085, creator=13701087609, updateTime=1770353593135, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226515124169650204, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226296952975966478, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226515124173844509, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226296952975966478, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=758, endPage=770, ext={EN=ArticleExt(id=1226296962341843162, articleId=1226296959393247294, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Screening and genomic analysis of biocontrol bacteria against cobweb disease of Oudemansiella raphanipes, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Oudemansiella raphanipes is a rare edible and medicinal mushroom, while it is highly susceptible to cobweb disease caused by Cladobotryum varium. [Objective] To obtain bacteria capable of controlling cobweb disease and promoting the mycelial growth of O. raphanipes from casing soil. [Methods] Bacteria were isolated from the casing soil of O. raphanipes, and their antagonistic activity against the mycelial growth of both O. raphanipes and C. varium was evaluated. The strains with disease-preventing and growth-promoting properties were screened out, and their functioning mechanisms were studied based on genome data. [Results] A total of 90 bacterial strains were isolated from the casing soil, of which 38 strains showed significant inhibitory activity against the mycelial growth of C. varium and no inhibitory activity against that of O. raphanipes. The supernatants of two strains significantly inhibited the mycelial growth of C. varium and promoted that of O. raphanipes. Based on the genome data, the above two strains were identified as Peribacillus simplex and Ochrobactrum rhizosphaerae, respectively, and they carried multiple functional genes and biosynthetic gene clusters of secondary metabolites. [Conclusion] Two bacterial strains capable of controlling the disease and promoting the growth of edible fungi were obtained, providing valuable microbial resources for the future biocontrol of cobweb disease.

, correspAuthors=Wentao QIN, authorNote=null, correspAuthorsNote=
*E-mail:
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卵孢小奥德蘑(Oudemansiella raphanipes)是一种食药兼用的珍稀食用菌,但其易受到异形枝葡霉(Cladobotryum varium)引起的蛛网病侵害。 【目的】 获得能够生物防控蛛网病且促进卵孢小奥德蘑生长的覆土细菌。 【方法】 从卵孢小奥德蘑覆土中分离细菌,检测其对卵孢小奥德蘑及蛛网病菌的拮抗活性,筛选出具有防病促生的多功能菌株,并基于基因组数据分析明确其潜在的生物机制。 【结果】 从覆土中共分离获得90株细菌,其中38株细菌对异形枝葡霉具有明显的抑制活性,且对卵孢小奥德蘑无抑制活性。进一步筛选发现,2株细菌的上清液不仅显著促进卵孢小奥德蘑菌丝的生长,还能显著抑制异形枝葡霉菌丝的生长。基于基因组数据分析,鉴定出上述2株细菌分别为简单近芽孢杆菌(Peribacillus simplex)与根际苍白杆菌(Ochrobactrum rhizosphaerae),且它们含有多种功能基因与次级代谢产物基因簇。 【结论】 本研究获得2株具有食用菌防病促生功能的细菌,为未来蛛网病的生态防控储备了有益微生物资源。

, correspAuthors=秦文韬, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=QYSEvHDEq0V/8gbSgIL0qg==, magXml=yZzOki3Nx3YFYGagcvYOiw==, pdfUrl=null, pdf=4SGj/KgifeMvslTRRgFKpQ==, pdfFileSize=6949815, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=L4bqC/6CJ35mWczApiwojQ==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=I0sMG+goFG9loL28eUH0bw==, mapNumber=null, authorCompany=null, fund=null, authors=

作者贡献声明

刘亚勇:数据收集与处理,论文撰写;张涛涛:微生物分离培养;高琳:微生物分离及鉴定;赵娟:课题讨论,论文修改;孟盼盼:微生物基因组测序;李莹菲:微生物分离培养;秦文韬:课题设计,实验指导,论文修改。

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A: Effect of supernatant on the mycelial growth of O. raphanipes (*: P<0.05; ***: P<0.001; ****: P<0.000 1); B: Effect of supernatant on the mycelial growth of C. varium (*: P<0.05; **: P<0.01); C: Effect of supernatant of strains 6-22 and 27-12 on the mycelial growth of O. raphanipes and C. varium., figureFileSmall=yCfDDC0O3c4RkTMnhjQ6zg==, figureFileBig=qjqTVO8a9NB62ER2WdLtlw==, tableContent=null), ArticleFig(id=1226514044014735981, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226296959393247294, language=CN, label=图4, caption=覆土细菌上清液对卵孢小奥德蘑和异形枝葡霉菌丝生长的影响。A:上清液对卵孢小奥德蘑菌丝生长的影响(*: P<0.05; ***: P<0.001; ****: P<0.000 1);B:上清液对异形枝葡霉菌丝生长的影响(*: P<0.05; **: P<0.01);C:菌株6-22和27-12的上清液对卵孢小奥德蘑和异形枝葡霉菌丝生长的影响。, figureFileSmall=yCfDDC0O3c4RkTMnhjQ6zg==, figureFileBig=qjqTVO8a9NB62ER2WdLtlw==, tableContent=null), ArticleFig(id=1226514044136370801, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226296959393247294, language=EN, label=Figure 5, caption=Phylogenetic tree and genome analysis of strain 6-22. 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防治卵孢小奥德蘑蛛网病生防菌筛选及其基因组分析
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刘亚勇 1 , 张涛涛 1 , 高琳 1 , 赵娟 1 , 孟盼盼 1, 2 , 李莹菲 1 , 秦文韬 1, *
微生物学报 | 研究报告 2025,65(2): 758-770
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微生物学报 | 研究报告 2025, 65(2): 758-770
防治卵孢小奥德蘑蛛网病生防菌筛选及其基因组分析
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刘亚勇1, 张涛涛1, 高琳1, 赵娟1, 孟盼盼1, 2, 李莹菲1, 秦文韬1, *
作者信息
  • 1 北京市农林科学院植物保护研究所,农业农村部北方果蔬有害生物绿色防控重点实验室(部省共建),北京
  • 2 河北农业大学 植物保护学院,河北 保定
Screening and genomic analysis of biocontrol bacteria against cobweb disease of Oudemansiella raphanipes
Yayong LIU1, Taotao ZHANG1, Lin GAO1, Juan ZHAO1, Panpan MENG1, 2, Yingfei LI1, Wentao QIN1, *
Affiliations
  • 1 Key Laboratory of Environment Friendly Management on Fruit and Vegetable Pests in North China (Co-construction by Ministry and Province), Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China
  • 2 College of Plant Protection, Hebei Agricultural University, Baoding, Hebei, China
出版时间: 2025-02-04 doi: 10.13343/j.cnki.wsxb.20240592
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卵孢小奥德蘑(Oudemansiella raphanipes)是一种食药兼用的珍稀食用菌,但其易受到异形枝葡霉(Cladobotryum varium)引起的蛛网病侵害。 【目的】 获得能够生物防控蛛网病且促进卵孢小奥德蘑生长的覆土细菌。 【方法】 从卵孢小奥德蘑覆土中分离细菌,检测其对卵孢小奥德蘑及蛛网病菌的拮抗活性,筛选出具有防病促生的多功能菌株,并基于基因组数据分析明确其潜在的生物机制。 【结果】 从覆土中共分离获得90株细菌,其中38株细菌对异形枝葡霉具有明显的抑制活性,且对卵孢小奥德蘑无抑制活性。进一步筛选发现,2株细菌的上清液不仅显著促进卵孢小奥德蘑菌丝的生长,还能显著抑制异形枝葡霉菌丝的生长。基于基因组数据分析,鉴定出上述2株细菌分别为简单近芽孢杆菌(Peribacillus simplex)与根际苍白杆菌(Ochrobactrum rhizosphaerae),且它们含有多种功能基因与次级代谢产物基因簇。 【结论】 本研究获得2株具有食用菌防病促生功能的细菌,为未来蛛网病的生态防控储备了有益微生物资源。

卵孢小奥德蘑  /  覆土  /  蛛网病  /  异形枝葡霉  /  生物防控

Oudemansiella raphanipes is a rare edible and medicinal mushroom, while it is highly susceptible to cobweb disease caused by Cladobotryum varium. [Objective] To obtain bacteria capable of controlling cobweb disease and promoting the mycelial growth of O. raphanipes from casing soil. [Methods] Bacteria were isolated from the casing soil of O. raphanipes, and their antagonistic activity against the mycelial growth of both O. raphanipes and C. varium was evaluated. The strains with disease-preventing and growth-promoting properties were screened out, and their functioning mechanisms were studied based on genome data. [Results] A total of 90 bacterial strains were isolated from the casing soil, of which 38 strains showed significant inhibitory activity against the mycelial growth of C. varium and no inhibitory activity against that of O. raphanipes. The supernatants of two strains significantly inhibited the mycelial growth of C. varium and promoted that of O. raphanipes. Based on the genome data, the above two strains were identified as Peribacillus simplex and Ochrobactrum rhizosphaerae, respectively, and they carried multiple functional genes and biosynthetic gene clusters of secondary metabolites. [Conclusion] Two bacterial strains capable of controlling the disease and promoting the growth of edible fungi were obtained, providing valuable microbial resources for the future biocontrol of cobweb disease.

Oudemansiella raphanipes  /  casing soil  /  cobweb disease  /  Cladobotryum varium  /  biocontrol
刘亚勇, 张涛涛, 高琳, 赵娟, 孟盼盼, 李莹菲, 秦文韬. 防治卵孢小奥德蘑蛛网病生防菌筛选及其基因组分析. 微生物学报, 2025 , 65 (2) : 758 -770 . DOI: 10.13343/j.cnki.wsxb.20240592
Yayong LIU, Taotao ZHANG, Lin GAO, Juan ZHAO, Panpan MENG, Yingfei LI, Wentao QIN. Screening and genomic analysis of biocontrol bacteria against cobweb disease of Oudemansiella raphanipes[J]. Acta Microbiologica Sinica, 2025 , 65 (2) : 758 -770 . DOI: 10.13343/j.cnki.wsxb.20240592
卵孢小奥德蘑(Oudemansiella raphanipes),俗称黑皮鸡枞,在全球热带和亚热带地区广泛分布,我国主要分布于云、贵、川等地[1]。卵孢小奥德蘑以其鲜嫩醇香的口感和独特的风味而闻名,富含多种氨基酸、多糖及微量元素[2-3],其子实体和发酵液中含有多糖、酶类、邻苯二酚、麦角甾醇、三萜类等活性化合物,在抗氧化、抗肿瘤、免疫调节和肝脏保护等方面发挥积极作用[4-5]。近年来,随着大健康理论、大食物观的提出,卵孢小奥德蘑作为食药兼用的高档保健珍稀菌类,在国内外市场上产品供不应求,发展前景广阔。
蛛网病是影响食用菌产业健康发展的重要病害之一,其病原菌为枝葡霉属(Cladobotryum)真菌[6]。不同种类枝葡霉会导致双孢菇、香菇、金针菇、卵孢小奥德蘑等食用菌发生蛛网病,侵染初期菌柄基部出现白色稀疏菌丝并逐渐蔓延,后期白色絮状菌丝覆盖菇体,导致其腐烂或萎缩[7-8]。课题组前期在北京地区发现了卵孢小奥德蘑蛛网病,并明确其病原菌为异形枝葡霉(C. varium),其发病率高达25%,给种植户造成了巨大的经济损失[9-10]。目前,该病害的致病机理尚不明确,国内外主要通过棚室消毒预防,发病后则多依赖苯菌酮、咪鲜胺等化学药剂[11-12],但缺乏选择性强、安全高效的防治措施。卵孢小奥德蘑作为绿色、健康食品,其独特的生产方式及较短的生长周期决定了不宜在子实体生长阶段施用化学药剂。近年来,生物防治以其绿色、环保、无残留等特点备受瞩目,其可有效抑制多种植物和食用菌的病原菌。例如,安全高效的生防菌贝莱斯芽孢杆菌(Bacillus velezensis)已被证实可有效抑制双孢菇蛛网病菌嗜菌枝葡霉(C. mycophilum)[13]。然而,关于卵孢小奥德蘑蛛网病的生物防治研究尚未见报道。
覆土是卵孢小奥德蘑栽培中的关键环节,其覆土中蕴含着丰富的有益细菌,构成了潜在的生防菌资源库[14-15]。许多微生物具有溶磷、解钾、固氮或产生生长激素、嗜铁素等生物活性物质的功能,能通过自身生理生化代谢活动参与营养物质和能量的循环转化,进而影响卵孢小奥德蘑原基形成和子实体分化[16]。研究表明,食用菌覆土中的有益菌可以通过分泌抗菌物质抑制病原菌的生长、分解土壤中的有害物质,或与特定的病原菌竞争营养和空间等,从而减少食用菌病原菌的数量,并诱导其子实体的形成,进而提高产量[17]。然而,目前尚不清楚覆土中的微生物是否具备拮抗卵孢小奥德蘑蛛网病菌的活性。
本研究旨在从卵孢小奥德蘑覆土中筛选、鉴定具有拮抗蛛网病菌菌丝生长,同时能促进其自身菌丝生长的细菌,以储备覆土有益微生物资源,探究覆土微生物的挖掘潜力,为卵孢小奥德蘑蛛网病的科学绿色防控奠定基础。
供试卵孢小奥德蘑菌株HPJZ和异形枝葡霉菌株JZBQA1均为课题组前期分离保存[7]。覆土采集于北京通州的卵孢小奥德蘑种植基地,为出菇期覆土。
牛肉膏蛋白胨培养基(g/L):牛肉膏3.0,蛋白胨10.0,NaCl 5.0,琼脂15.0。
LB固体培养基(g/L):胰蛋白胨10.0,酵母提取物5.0,NaCl 10.0,琼脂15.0。
LB液体培养基:LB粉末39.0 g/L,去离子水定容至1 000 mL。
PDA综合培养基:去皮土豆200 g/L煮熟取滤液,葡萄糖20 g/L,琼脂20 g/L,磷酸二氢钾3 g/L,无水硫酸镁1.5 g/L,蛋白胨5 g/L,维生素B1 10 mg/L,去离子水定容至1 000 mL。
取3 g土壤加45 mL无菌水制成土壤悬浮液,置于25 ℃、160 r/min培养过夜,将土壤悬浮液稀释至10-3,并取200 μL均匀涂布于牛肉膏蛋白胨培养基上,每份土壤悬浮液稀释液设置6个重复,根据不同菌落形态使用无菌牙签挑取单菌落至新的LB固体培养基上进行划线分离培养,获得细菌的纯培养菌株后,使用无菌枪头蘸取纯培养菌株置于装有3 mL LB液体培养基的无菌10 mL离心管中,25 ℃、160 r/m培养至OD600为1.5,保存于灭菌的30%甘油内。
将500 μL细菌菌液进行沸水浴5 min灭活,并以此作为DNA模板,使用细菌16S rRNA基因通用引物27F (5′-AGAGTTTGATCCTGGCTCAG-3′)和1492R (5′-CTACGGCTACCTTGTTACGA-3′)进行PCR扩增。PCR反应体系(50 μL):DNA模板4 μL,2×PCR Mix 25 μL,上、下游引物(10 μmol/L)各2 μL,ddH2O 17 μL。PCR反应条件:95 ℃预变性5 min;95 ℃变性30 s,55 ℃退火30 s,72 ℃延伸2 min,35个循环;72 ℃延伸7 min。取5 μL PCR产物经1%琼脂糖凝胶电泳检测后,将合格PCR产物送至北京诺赛基因组研究中心测序。测序结果在GenBank数据库中进行BLAST比对。
利用打孔器制作直径为10 mm的卵孢小奥德蘑/蛛网病菌接种块,将其接种至PDA平板中央(每个平板含有15 mL PDA培养基),25 ℃恒温培养。取500 μL细菌菌液接种到3 mL LB液体培养基的离心管中,25 ℃、160 r/min培养,定期测量OD600至其达到1.5,将菌液转至1.5 mL离心管中备用。待卵孢小奥德蘑/蛛网病菌菌落生长至直径3 cm时,使用无菌枪头蘸取细菌菌液在平板直径左右两端距菌落1.5 cm的位置划两条直线。每株细菌设置3个重复。以同等条件下蘸取LB液体培养基划线作为对照组,以同等条件下不做处理的平板作为空白对照。观察菌落生长情况,记录卵孢小奥德蘑/蛛网病菌菌落直径,计算抑菌率。抑菌率的计算如公式(1)所示。
X=(A0-A1)/A0×100%
式中:X为抑菌率;A0为对照菌落直径(mm);A1为处理菌落直径(mm)。
取3 mL细菌菌液(OD600为1.5)加入装有50 mL LB液体培养基的250 mL锥形瓶内,25 ℃、160 r/min培养2 d,之后8 000 r/min离心10 min,然后用0.22 μm除菌过滤器获得无菌上清液。取4 mL滤液加入到16 mL PDA培养基中。用直径为10 mm的打孔器获得相同接种量的卵孢小奥德蘑/蛛网病菌菌丝块,接种至平板中央。每隔24 h划线并记录卵孢小奥德蘑及其蛛网病菌菌丝的生长速度,直至菌丝长满平板。菌丝生长速度(V,mm/d)的计算如公式(2)所示。
V=[(D2-D1)/d]
式中:D1为第1次测量的直径(mm);D2为最后一次测量的直径(mm);d为测量天数。
同时,挑取平板上卵孢小奥德蘑及其蛛网病菌的菌丝,制成切片后通过ECLIPSE LV100ND显微镜(Nikon公司)观察,采用NIS-Elements显微成像软件拍照。
覆土细菌菌株以平板划线法接种到LB固体培养基上,25 ℃培养48 h后,观察并拍照记录菌落形态。挑取菌体利用革兰氏染色试剂盒(北京索莱宝科技有限公司)进行革兰氏染色,并通过显微镜观察拍照。将菌液与Salkowski试剂(由0.5 mol/L FeCl3和35%高氯酸组成)混合,在黑暗处放置一段时间后观察溶液颜色变化,分析细菌产生植物激素吲哚乙酸(indole-3-acetic acid, IAA)的情况。
采用细菌DNA提取试剂盒(TaKaRa公司)对细菌DNA进行提取,构建细菌基因组文库,利用Illumina测序平台进行测序,并采用从头测序组装(de novo测序组装)。基因组数据已保存于国家微生物科学数据中心(NMDC, http://nmdc.cn),数据编号分别为NMDC60197572和NMDC60197573。对于细菌基因组,利用MAFFT进行序列比对,同时采用最大似然法(maximum-likelihood),利用autoMLST网站(https://automlst.ziemertlab.com)根据基因组中的60个核心基因构建系统发育树,且设置重复检验次数为1 000次循环,以计算自展值(bootstrap value, BP)。利用antiSMASH分析细菌基因组含有的次级代谢产物基因簇,采用生物信息软件Anvi'o v8进行细菌泛基因组分析。
分别使用GraphPad Prism 8.0和OriginPro 2021软件分析数据和绘图。采用单因素方差分析(one-way ANOVA)检验组间差异。显著性差异的定义为P<0.05。
从覆土中分离、筛选并保藏不同菌落形态的细菌,共计90株细菌。通过16S rRNA基因序列的初步鉴定,它们分别来自于芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、微杆菌属(Microbacterium)、节杆菌属(Arthrobacter)等(图1),其中芽孢杆菌属(Bacillus)和假单胞菌属(Pseudomonas)占比较多(45%)。
通过平板对峙培养法检测了90株细菌对卵孢小奥德蘑菌丝生长的影响,结果显示其中 65株细菌对卵孢小奥德蘑菌丝生长无明显抑制活性,25株细菌对卵孢小奥德蘑菌丝生长具有不同程度的抑制作用。卵孢小奥德蘑的菌落直径为36.0-69.2 mm,抑菌率为23.1%-60.0%。如图2所示,Bacillus sp. 11-36和Pseudomonas sp. 55-10对卵孢小奥德蘑菌丝生长的抑菌率分别达到60.0%和50.0%,而金黄杆菌属(Chryseobacterium)菌株26-1对卵孢小奥德蘑生长的抑菌率仅为2.9%,但对异形枝葡霉有很强的抑制活性,抑菌率为51.4%。
为获得抑制蛛网病菌生长但不影响卵孢小奥德蘑生长的功能细菌,以对卵孢小奥德蘑无明显抑制作用的65株细菌为研究对象,检测其对异形枝葡霉菌丝生长的活性。对峙培养发现,其中的38株细菌抑制异形枝葡霉菌丝的生长,菌落直径范围为34.2-62.0 mm,抑菌率为31.1%-62.0% (图3)。38株细菌对异形枝葡霉菌的抑制率大于31.0%,这些潜在功能细菌种类丰富,分别属于假单胞菌属(7株)、芽孢杆菌属(6株)、赖氨酸芽孢杆菌属(Lysinibacillus) (2株)等21个属。
在上述研究的基础上,为进一步筛选覆土内促进卵孢小奥德蘑菌丝生长的菌株,检测了上述38株细菌的发酵上清液对卵孢小奥德蘑菌丝生长的影响。结果显示,5株细菌具有显著促进卵孢小奥德蘑菌丝生长的活性(图4A),分别为菌株4-7、52-32、10-18、27-12和6-22。同时,检测了38株细菌的上清液对异形枝葡霉菌丝生长的影响,结果发现26株细菌具有显著抑制异形枝葡霉的活性(图4B),这些菌株可成为卵孢小奥德蘑蛛网病的重要生防菌株资源。其中,2株细菌(27-12和6-22)对卵孢小奥德蘑菌丝生长具有显著的促生长作用,同时对异形枝葡霉菌丝生长具有显著的抑制作用。菌丝显微形态观察显示,2株菌对卵孢小奥德蘑菌丝无明显影响,但菌株6-22的上清液导致异形枝葡霉菌丝变形、破裂,形成明显的空泡(图4C);而菌株27-12的发酵上清液则能抑制异形枝葡霉菌丝的生长,并使其菌丝表面呈现凹凸不平的现象(图4C)。
菌株6-22的菌落近圆形,乳白色,边缘整齐,不透明,表面光滑呈油性,可产生IAA,革兰氏染色显示为革兰氏阳性菌。菌株6-22基因组与简单近芽孢杆菌(Peribacillus simplex) BA2H3基因组平均核苷酸同一性(average nucleotide identity, ANI)相似性达98.4%,系统发育树分析显示,二者也聚类在一起[maximum likelihood bootstrap (MLBP)=100%],综合形态学特征、生理生化特性和系统发育分析结果,鉴定菌株6-22为简单近芽孢杆菌。进一步基因组antiSMASH分析结果显示,菌株6-22含有10个生物合成基因簇(biosynthetic gene clusters, BGCs),这些基因簇包括非核糖体肽(non-ribosomal peptides, NRPs)、III型聚酮合酶(type III polyketide synthases, T3PKS)、萜烯类(terpene)、套索肽(lasso peptide)、铁载体(NI-siderophore)、含唑线性肽[linear azol(in)e-containing peptide, LAP]、β-内酯(betalactone)。通过序列比对分析,结果显示菌株6-22中的套索肽基因簇BGC-1.2与已知的paeninodin基因簇具有100%的相似性,铁载体基因簇BGC-4.1与schizokinen基因簇具有75%的相似性,非核糖体肽基因簇BGC-5.1与koranimine基因簇具有75%的相似性。其余BGCs则未找到与已知次级代谢产物的相似性,这表明菌株6-22可能含有7个新的次级代谢产物基因簇。泛基因组分析显示,菌株6-22含有8个IAA产生相关基因,9个溶磷功能相关基因,7个固氮相关基因以及铁载体基因(图5)。
菌株27-12的菌落近圆形,乳白色,边缘整齐,不透明,表面光滑呈油性,微弱的IAA产生能力,为革兰氏阴性菌。菌株27-12基因组与根际苍白杆菌(Ochrobactrum rhizosphaerae) SJY1基因组ANI相似性达97.0%,且系统发育树上与该菌株聚类在一个分支(MLBP=100%),综合形态学特征、生理生化特性和系统发育分析结果鉴定菌株27-12为根际苍白杆菌。进一步基因组antiSMASH分析结果显示,菌株27-12含有9个BGCs,包括萜烯类、β-内酯、氰化氢(hydrogen-cyanide)、芳基多烯类化合物(arylpolyene)等。序列比对发现9个BGCs均未找到序列相似的已知次级代谢产物,因而初步判定菌株27-12含有的9个BGCs为新的次级代谢产物基因簇。泛基因组分析显示,菌株27-12含有9个IAA产生相关基因,6个溶磷功能相关基因,3个固氮相关基因(图6)。
覆土是卵孢小奥德蘑栽培过程中至关重要的步骤,覆土中栖居着的微生物种类繁多,功能复杂,对食用菌菌丝的生长、子实体的分化和发育具有重要作用,影响其产量和出菇整齐度等特性[18]。本研究团队前期发现,卵孢小奥德蘑覆土中微生物极其丰富,主要分布于放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、绿弯菌门(Chloroflexi)、厚壁菌门(Firmicutes)等,相对丰度大于1%的属超过37个[15],包含了本研究分离到的芽孢杆菌属、假单胞菌属、微杆菌属、节杆菌属等多个属,这也进一步表明了卵孢小奥德蘑覆土中含有丰富的微生物资源尚待挖掘。
蛛网病作为食用菌常见的病害之一,目前仍缺乏绿色、安全且具有针对性的生物防控方法。蛛网病生防菌株应具备既不影响食用菌菌丝的正常生长,又能对病原菌表现出特异性的抑制活性。因此,那些具有广谱且强抑菌活性的细菌可能并不适宜合用于食用菌真菌病害的防控。例如,本研究发现Bacillus sp. 11-36和Pseudomonas sp. 55-10对卵孢小奥德蘑和蛛网病菌均具有较强的抑制活性,但它们可能对卵孢小奥德蘑菌丝的生长阶段产生不利影响,因此不适合用于防治卵孢小奥德蘑蛛网病。Büchner等研究表明,贝莱斯芽孢杆菌SZMC 25431具有防控双孢菇绿霉病的应用潜力[19],但该研究并未考虑生防菌对食用菌菌丝生长可能产生的影响。此外,覆土中可能存在能够促进食用菌生长的有益细菌。例如,平菇(Pleurotus ostreatus)覆土中假单胞菌种群的增加可促进子实体的形成及提高产量[20]。同样,研究发现卵孢小奥德蘑种植后,覆土中链霉菌(Streptomyces)和类诺卡氏菌(Nocardioides)的相对丰度增加利于卵孢小奥德蘑菌丝的生长[15,21]。本研究从覆土中筛选获得了2株具有双重功能的细菌,它们不仅能够拮抗蛛网病菌,还能促进卵孢小奥德蘑菌丝的生长。
基因组分析发现,潜在生防菌简单近芽孢杆菌6-22和根际苍白杆菌27-12可产生丰富的次级代谢产物。近芽孢杆菌属(Peribacillus)是2020年从芽孢杆菌属中划分出的新属,其特性与芽孢杆菌非常相近[22]。芽孢杆菌可产生丰富的抗菌活性物质,包括表面活性素(surfactin)、伊枯菌素(iturin)、芬芥素(fengycin)和杆菌溶素(bacilysin)等,能有效抑制多种病原真菌的生长[23]。AntiSMASH分析结果显示,菌株6-22的基因组中未预测到常见的抗真菌活性基因簇,如伊枯菌素和芬芥素,但发现了新的β-内酯、NRPs、PKS等次级代谢产物基因簇。这些新的次级代谢产物可能具有抗真菌活性,如β-内酯类物质alligamycin A可抑制曲霉(Aspergillus)[24],大肠杆菌产生的铁载体可抑制构巢曲霉(Aspergillus nidulans)的生长[25],NRPs类活性产物在芽孢杆菌与类芽孢杆菌(Paenibacillus)抗真菌活性中发挥重要作用[26]。由此,推断菌株6-22对异形枝葡霉的抑制作用可能源于β-内酯、铁载体及非核糖体肽等多种活性物质的协同作用。Allioui等研究表明,简单近芽孢杆菌具有抑制多种真菌的活性,如简单近芽孢杆菌Alg.24B2可抑制小麦叶枯病菌(Zymoseptoria tritici)菌丝的生长[27],简单近芽孢杆菌PHYB1和PHYB9两株菌能够抑制弯角镰孢菌(F. camptoceras),有效地防治黑种草根腐病[28]。然而,其抗真菌活性的代谢产物仍需进一步研究。目前,根际苍白杆菌具有抗真菌活性的报道极少,本研究中根际苍白杆菌27-12含有多个新的BGCs,包括β-内酯、酰基氨基酸(acyl amino acids)。Fait等[29]研究表明,酰基氨基酸类似表面活性剂具有抗真菌活性,根据菌株27-12所含的BGCs推断,可能是由β-内酯、酰基氨基酸等次级代谢产物发挥抗真菌活性。上述2株细菌的次级代谢产物仍有待进一步研究。一些芽孢杆菌可促进食用菌的生长,如蜡状芽孢杆菌(B. cereus)、苏云金芽孢杆菌(B. thuringiensis)等[30-31]。苍白杆菌也是一类重要的植物促生长功能菌,具有溶磷、产生IAA等活性[32-33]。本研究发现,简单芽孢杆菌菌株6-22可产生IAA,且基因组数据显示菌株6-22和27-12的基因组中含有丰富的溶磷、固氮相关功能基因,此结果与2株菌可促进卵孢小奥德蘑菌丝生长一致。
本研究成功筛选出2株针对卵孢小奥德蘑蛛网病的潜在生防菌,这些菌株不仅能有效抑制蛛网病菌菌丝的生长,同时还能促进卵孢小奥德蘑菌丝的生长。因此,它们具有巨大的应用潜力,可作为卵孢小奥德蘑蛛网病的生物防治菌剂进行开发。本研究的结果为未来蛛网病的生态防控提供了宝贵的微生物资源。
  • 北京市自然科学基金(6232009)
  • 北京市科技计划(Z231100003723001)
  • 北京市农林科学院创新能力建设专项(KJCX20230115)
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2025年第65卷第2期
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doi: 10.13343/j.cnki.wsxb.20240592
  • 接收时间:2024-09-26
  • 首发时间:2026-02-05
  • 出版时间:2025-02-04
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  • 收稿日期:2024-09-26
  • 录用日期:2024-11-21
基金
Beijing Natural Science Foundation(6232009)
北京市自然科学基金(6232009)
Beijing Science and Technology Program(Z231100003723001)
北京市科技计划(Z231100003723001)
Beijing Academy of Agricultural and Forestry Sciences Innovation Capacity Building Special Funding Project(KJCX20230115)
北京市农林科学院创新能力建设专项(KJCX20230115)
作者信息
    1 北京市农林科学院植物保护研究所,农业农村部北方果蔬有害生物绿色防控重点实验室(部省共建),北京
    2 河北农业大学 植物保护学院,河北 保定

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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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