摘要
【目的】明确柑橘砂皮病不同发病等级及喷施生防菌枯草芽孢杆菌M-23后柑橘叶际细菌群落结构的变化,揭示生防菌防治柑橘砂皮病的叶际微生物学机制,为进一步研究柑橘砂皮病的生物防治提供理论基础。【方法】试验设对照区和喷施生防菌区2个区域,喷施生防菌区分别在2019年3月中旬柑橘春梢长2~3 cm、4月中旬、谢花2/3、果实蚕豆大小和果实乒乓球大小时喷施M-23菌株发酵液,喷药液量3 L/株;同时对照区喷施等量的灭菌NB液体培养基,在最后一次喷施生防菌15 d后进行防效调查。采集喷施生防菌区柑橘叶片(R4处理,病情等级0级)和未喷施生防菌区不同发病等级的柑橘叶片(R1、R2和R3处理,病情等级分别为0、5和9级),使用FastDNA^ⓇSpin Kit for Soil试剂盒提取柑橘叶片叶际微生物总DNA,通过Illumina Miseq^TM高通量平台测序分析微生物群落多样性。【结果】生防菌M-23对柑橘砂皮病的防治效果达74.02%。高通量测序共获得571091条序列,单一样品序列数17833~96147条,在97%的相似水平下进行生物信息统计分析聚类后获得2932个OTUs。4个处理的OTUs数量分别为1944、1545、1205和607个,表现为R1>R2>R3>R4。在门水平的组成上主要由变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、浮霉菌门(Planctomycetes)、梭杆菌门(Fusobacteria)、装甲菌门(Armatimonadetes)、酸杆菌门(Acidobacteria)和疣微菌门(Verrucomicrobia)组成。变形菌门是优势菌门,在R1、R2、R3和R4处理中的相对丰度分别为81.84%、87.93%、86.80%和68.82%。对照区的叶际微生物多样性与发病等级成反比;喷施生防菌后柑橘叶片叶际细菌群落多样性降低,在属水平上芽孢杆菌属(Bacillus)和假单胞菌属(Pseudomonas)的相对丰度得到明显增加,芽孢杆菌属的相对丰度达33.41%。【结论】喷施生防菌M-23能有效防治柑橘砂皮病。M-23菌株能在柑橘叶片上定殖和增殖,改善叶际细菌群落结构,提升有益菌的比例,使病情指数降低,具有一定的应用潜力。
【Objective】Focusing on different severities of Diaporthe citri and the shifts of bacterial community diversity in citrus phyllosphere after spraying with Bacillus subtilis M-23,this study was to reveal the phyllospheric microbiological mechanism of controlling D.citri with biocontrol bacteria and provide theoretical basis for further studies on biological control of D.citri.【Method】The experiment was conducted in two areas—the control area and the treatment area.In the treatment area,biocontrol bacteria M-23 was sprayed in the middle of March,2019,when the citrus spring tip grew 2-3 cm,in the middle of April,2/3 of flowers fell,the citrus size was like broad bean and the citrus size was like ping-pong ball,evenly onto citrus leaves with an inoculum of 3 L/plant.At the same time,the control area was sprayed with the same amount of sterilized NB medium.The control effect was investigated 15 d after the last application of biocontrol agents.Collecting leaves with different incidence grades in the sprayed areas(R4 treatment,incidence grade 0)and unsprayed areas(R1,R2 and R3 treatments,incidence grades 0,5 and 9 respectively).FastDNA^ⓇSpin Kit for Soil kit was used to extract the total DNA of phyllosphere microorganisms,and the diversity of microbial community was analyzed by Illumina Miseq^TM high throughput platform sequencing method.【Result】The result showed that the control effect of biocontrol bacterium M-23 on D.citri was 74.02%.A total of 571091 high quality sequences were obtained by high throughput sequencing,the number of single sample sequences ranged from 17833 to 96147,and 2932 OTUs were obtained by bioinformatics statistical analysis clustering at a similar level of 97%.The number of OTUs of the four treatments were 1944,1545,1205 and 607,respectively,in the following order:R1>R2>R3>R4.The horizontal composition of the phylum was mainly composed of Proteobacteria,Firmicutes,Bacteroidetes,Actinomycetes,Fusobacteria,Armatimonadetes,Acidobacteria and Verruciformis.Proteobacteria were dominant,and the relative abundances in R1,R2,R3 and R4 treatments were 81.84%,87.93%,86.80%and 68.82%,respectively.In the control area,phyllosphere microbial diversity was inversely proportional to the incidence level.After spraying the biocontrol agent,the diversity of the bacterial community in the phyllosphere decreased,and the relative abundance of Bacillus and Pseudomonas increased obviously at the genus level,the relative abundance of Bacillus reached 33.41%.【Conclusion】To sum up,spraying M-23 can effectively prevent the occurrence of D.citri.M-23 can colonize and proliferate on citrus leaves,which improves the community structure of phyllosphere bacteria,increases the proportion of beneficial bacteria,and reduces the disease index.Therefore,B.subtilis M-23 has certain application potential.
作者
李审微
洪艳云
李新文
何可佳
戴良英
卢晓鹏
宋娜
易图永
LI Shen-wei;HONG Yan-yun;LI Xin-wei;HE Ke-jia;DAI Liang-ying;LU Xiao-peng;SONG Na;YI Tu-yong(College of Plant Protection,Hunan Agricultural University,Changsha 410128,China;Hunan Provincial Key Laboratory of Plant Diseases and Pests,Changsha 410128,China;Hunan Plant Protection and Quarantine Station,Changsha 410005,China;College of Horticulture,Hunan Agricultural University,Changsha 410128,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2020年第7期1699-1705,共7页
Journal of Southern Agriculture
基金
现代农业(柑橘)产业技术体系岗位科学家项目(CARS-26)
湖南省水果产业技术体系柑橘植保岗位专家项目(湘农发〔2015〕137号)
湖南农业大学双一流建设子项目(SYL2019027)
湖南农业大学青年科学基金项目(19QN30)。
关键词
柑橘砂皮病
叶际细菌
生防菌
高通量测序
多样性
Diaporthe citri
phyllosphere bacteria
biocontrol bacteria
high-throughput sequencing
diversity
