摘要
本研究旨在揭示解淀粉芽胞杆菌JK10防治蓝莓根癌病的生防机制。利用改良发酵培养基对解淀粉芽胞杆菌JK10菌株进行发酵,用盐酸沉淀、硫酸铵沉淀、乙酸乙酯萃取、大孔树脂吸附四种方法对JK10发酵液中的活性物质进行分离,利用MALDI-TOF-MS对活性最高的粗提物进行分析明确其种类;对JK10进行绿色荧光蛋白标记,明确其在蓝莓根际土及组织内的定殖能力;使用高通量测序技术对蓝莓健康根组织、根癌病根组织及JK10处理的根癌病根组织内生细菌群落结构进行分析。结果表明,四种方法得到的粗提物对根癌土壤杆菌均有良好的拮抗效果,其中大孔树脂吸附法得到的粗提物抑菌活性与产率均最高。JK10发酵液中能检测出bacillomycin D和fengycin两类脂肽类化合物、大环内酯类抗生素Divergolide D和聚酮类化合物Bacillaene;菌株JK10能够稳定定殖于蓝莓根际土、蓝莓根组织细胞间隙和蓝莓叶中,定殖数量在接种35 d后趋于稳定,在蓝莓根际土和蓝莓叶中分别为1×10^(6)、4.0×10^(5)cfu/g;蓝莓根癌病根组织在施入生防菌JK10菌株后,相比自然条件下蓝莓根癌病根组织内生细菌菌落结构发生改变,物种多样性增加,结构更加趋向于健康蓝莓根组织内生细菌。综上所述,解淀粉芽胞杆菌JK10通过产生对蓝莓根癌病菌有抑制活性的物质,稳定定殖在蓝莓根际土及蓝莓根、叶组织中,改变蓝莓根内生细菌的群落结构而达到防治蓝莓根癌病的目的。
Crown gall disease caused by Agrobacterium tumefaciens is a serious threat to blueberry production.In this study,we aimed to explore the biocontrol mechanism of Bacillus amyloliquefaciens JK10 to manage crown gall disease of blueberry via pot and field experiments.We extracted crude extract with the maximum antibacterial activity against A.tumefaciens from the fermentation medium of B.amyloliquefaciens JK10,using four methods:acid precipitation,ammonium sulfate precipitation,ethyl acetate extraction,and microporous resin adsorption.High-throughput sequencing technique was used to investigate the bacterial community structure associated with blueberry healthy root tissue,crown gall root tissue,and crown gall root tissue treated with B.amyloliquefaciens JK10.Furthermore,B.amyloliquefaciens JK10 was labeled with a green fluorescent protein (GFP) to confirm its colonization ability in the blueberry plant parts.Results of this study,demonstrated that the crude extract obtained by four methods have strong antagonistic effects on A.tumefaciens.Among all methods,the crude extract obtained by the macroporous resin adsorption method has maximum antibacterial activity.Two types of lipopeptide compounds bacillomycin D and fengycin,and the macrolide antibiotic Divergolide D and the polyketide Bacillaene,were detected in the crude extract of B.amyloliquefaciens JK10.Confocal microscopy micrographs revealed that B.amyloliquefaciens JK10 can successfully colonize the intercellular spaces of blueberry roots and emits fluorescence light.Whereas,plate culture methods were adopted to confirm the colonization ability in the blueberry leaf tissues,which showed that B.amyloliquefaciens JK10 also colonized in the blueberry leaf as fluorescence color was oberved under UV-light.The number of B.amyloliquefaciens JK10 stabilized in the rhizosphere soil of blueberry and the leaves of blueberry were 1×10^(6)cfu/g and 4.0×10^(5)cfu/g,respectively,after 35 days of post inoculation.Application of B.amyloliquefaciens JK10 significantly increased the alpha diversity (richness) and beta diversity(community structure) indices as compared with control (untreated diseased blueberry plants).The species diversity was increased and the structure tends to be healthier in blueberry diseased plants treated with B.amyloliquefaciens JK10 compared with control.In summary,we conclude that B.amyloliquefaciens JK10 mitigates the crown gall disease of blueberry through production of antibacterial substances that inhibit the growth of crown gall pathogen,niche exclusion (sharing space with pathogen),and by changing the bacterial community structure.Altogether,our study demonstrates the possible biocontrol mechanism of B.amyloliquefaciens JK10 to manage blueberry crown gall disease.
作者
姚博
张晋豪
何依璐
梁旭清
崔兴国
荣浪飞
周星海
魏方俊
何祖雷
姬广海
魏兰芳
YAO Bo;ZHANG Jinhao;HE Yilu;LIANG Xuqing;CUI Xingguo;RONG Langfei;ZHOU Xinghai;WEI Fangjun;HE Zulei;JI Guanghai;WEI Lanfang(State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,Yunnan Agricultural University,Kunming 650201,China;Agricultural Foundation Experiment Teaching Center,Yunnan Agricultural University,Kunming 650201,China;Qujing Jiawo Modern Agriculture Company,Qujing 655000,China)
出处
《中国生物防治学报》
CSCD
北大核心
2022年第3期613-625,共13页
Chinese Journal of Biological Control
基金
国家重点研发计划(2019YFD1002000)
云南省“万人计划”产业技术领军人才(YNWR-CYJS-2019-046)。
关键词
解淀粉芽胞杆菌
根癌土壤杆菌
生物防治
高通量测序
定殖
活性物质
Bacillus amyloliquefaciens
Agrobacterium tumefaciens
biocontrol
high-throughput sequencing
colonize
antibacterial substance
