芽孢杆菌Ya-1的定殖特性及其对辣椒根际细菌多样性的影响  

Colonization characteristics of Bacillus sp.Ya-1 and its effects on rhizosphere bacterial diversity of pepper

作　　者：赵志祥[1] 李道敏 谭诗梦 严婉荣[1] 王宝 肖彤斌 ZHAO Zhi-xiang;LI Dao-min;TAN Shi-meng;YAN Wan-rong;WANG Bao;XIAO Tong-bin(Institute of Plant Protection,Hainan Academy of Agricultural Sciences/Research Center for the Quality Safety and Standards of Agricultural Products,Hainan Academy of Agricultural Sciences/Hainan Key Laboratory for Control of Plant Diseases and Insect Pests/Scientific Observation and Experiment Station of Crop Pests in Haikou,Ministry of Agriculture and Rural Affairs,Haikou,Hainan 571100,China)

机构地区：[1]海南省农业科学院植物保护研究所/海南省农业科学院农产品质量安全与标准研究中心/海南省植物病虫害防控重点实验室/农业农村部海口作物有害生物科学观测实验站,海南海口571100

出　　处：《南方农业学报》2024年第11期3346-3357,共12页Journal of Southern Agriculture

基　　金：海南省自然科学基金高层次人才项目(324RC537);海南省省属科研院所技术研究开发专项(FW20230002);海南省冬季瓜菜产业技术体系项目(HNARS-05-ZJ04);海南省农业科学院科技创新团队项目(HAAS2023TDYD12)。

摘　　要：【目的】研究芽孢杆菌Ya-1(简称Ya-1)在辣椒根际、根和叶中的定殖特性及其对辣椒根际细菌多样性的影响,为微生态调控防治辣椒枯萎病及芽孢杆菌的开发利用提供参考依据。【方法】以自然土+蛭石(Ns)和灭菌土+蛭石(Ss)2组基质盆栽辣椒为试验材料,采用利福平标记法,分别对2组基质设接种无菌水(A)、接种抗200μg/mL利福平的Ya-1突变株(Ya-1-200)发酵液(B)、接种辣椒枯萎病菌SMPLJLD-1孢子悬浮液(C)和接种Ya-1-200发酵液+辣椒枯萎病菌SMPLJLD-1孢子悬浮液(D)4个处理,接种后0、1、2、3、7、10和20 d,测定Ya-1在根际、根和叶片的定殖量的,并结合绝对定量PCR验证Ya-1的定殖规律。同时利用高通量测序技术对接种0、10、20 d根际细菌多样性及其群落结构差异进行比较分析。【结果】稀释分离法检测Ya-1在辣椒植株及根际中的定殖情况结果显示,接种3 d后,B、D处理下根际和叶中Ya-1的定殖量逐渐下降,20 d后趋于稳定,定殖量均大于1×10^(4) CFU/g。绝对定量PCR检测结果显示,B、D处理后,辣椒根际、根和叶中Ya-1单位样本拷贝数呈先增加后减少的变化趋势,接种3 d后达到峰值,并在20 d后稳定在1.0×10^(5) copies/g以上。基于高通量测序分析结果显示,Ns组接种10 d与接种20 d相比,B处理ACE指数和Chao1指数显著降低(P<0.05,下同),而C和D处理2个指数显著升高。辣椒根际细菌群落中相对丰度大于4%的优势菌门包括放线菌门、酸杆菌门、拟杆菌门、绿弯菌门、芽单胞菌门和变形菌门。接种10 d时,C处理辣椒根际细菌群落中芽单胞菌门相对丰度最低(4.07%),B处理该菌门的相对丰度最高(6.77%)。与A处理相比,B处理在接种10和20 d时贪铜菌属、Aquicella、Trinickia、Crenobacter、产碱杆菌属、戴氏菌属、假单胞菌属、Pseudogulbenkiania、芽孢杆菌属、罗河杆菌属和Vogesella均明显富集;接种10 d后,与A处理相比,B处理鞘氨醇单胞菌属�【Objective】To investigate the colonization characteristics of Bacillus sp.Ya-1 in rhizosphere,root and leaf of pepper and its effects on rhizosphere bacterial diversity,so as to provide reference for the control of pepper wilt disease by microecological regulation and the further development and utilization of Bacillus sp.【Method】Natural soil+vermicu‐lite(Ns)and sterilized soil+vermiculite(Ss)matrix potted pepper were used as experimental materials.Using rifampi‐cin labeling method,the 2 groups of matrix were inoculated with sterile water(A),inoculated with Ya-1 mutant(Ya-1-200)fermentation broth that was resistant to 200μg/mL rifampicin(B),inoculated with spore suspension of pepper fu‐sarium wilt bacteria SMPLJLD-1(C),and inoculated with YA-1-200 fermentation broth+spore suspension of pepper fu‐sarium wilt bacteria SMPLJLD-1(D).At 0,1,2,3,7,10 and 20 d after inoculation,the colonization amounts of Ya-1 in rhizosphere,root and leaf were determined,and the colonization rule of Ya-1 was verified by absolute quantitative PCR.At the same time,the diversity and community structure difference of rhizosphere bacteria at 0,10 and 20 d after ino-culation were compared and analyzed by high-throughput sequencing technology.【Result】The results of the detection of Ya-1 colonization in pepper plants and rhizosphere using rifampicin labeling method showed that after 3 d of inoculation,the colonization amount of Ya-1 in rhizosphere soil and leaves under treatments B and D gradually decreased,and tended to be stable after 20 d,and it was above the level of 1×10^(4) CFU/g after 20 d.The results of absolute quantitative PCR showed that after B and D treatment,the copy number of Ya-1 unit samples in rhizosphere,root and leaf of pepper in‐creased first and then decreased,reached the peak after 3 d of inoculation,and stabilized above 1.0×10^(5) copies/g after 20 d.Analysis based on high throughput sequencing showed that comparing with 10 and 20 d after inoculation in Ns group,ACE index and Chao1 index in B tre

关 键 词：芽孢杆菌 利福平标记 绝对定量PCR 定殖 根际细菌多样性 

分 类 号：S641.306[农业科学—蔬菜学]