摘要
为建立荧光定量PCR检测杨树枯萎病的方法和明确前期分离的拮抗菌N6-34对杨树枯萎病致病菌尖孢镰刀菌的作用,本试验采用常规PCR法扩增尖孢镰刀菌的特异性片段,将此片段与载体连接构建质粒,提取质粒DNA在实时荧光定量PCR仪上采用两步法进行扩增并绘制实时荧光定量PCR标准曲线,同时对杨树进行不同灌根处理(T1:1×10~6 cfu/mL的尖孢镰刀菌孢子悬液和未接N6-34菌株的发酵培养液各20 mL;T2:1×10~6 cfu/mL的尖孢镰刀菌孢子悬液和灭活的N6-34菌株发酵培养液各20 mL;T3:1×10~6 cfu/mL的尖孢镰刀菌孢子悬液和N6-34菌株的发酵培养液各20 mL),并于10、20、30 d取样检测杨树根际尖孢镰刀菌数量。结果表明:本研究建立的方法能应用于土传尖孢镰刀菌导致的杨树枯萎病的检测;T1处理的尖孢镰刀菌的数量随处理时间的延长呈先下降后上升的趋势,T2和T3中的尖孢镰刀菌数量则呈下降趋势,表明N6-34能够抑制尖孢镰刀菌。本研究对指导杨树种植和病害预报具有极为重要的应用价值,能够为杨树病害管理提供科学依据。
This study was aimed to construct a real-time fluorescent quantitative PCR method for detection of poplar wilt pathogen and make clear of the effect of antagonistic bacterium N6-34 isolated earlier on Fusarium oxysporum. The specific fragment amplified by routine PCR was lingated with the vectors to constrct the plasmid, and then the plasmid DNA was extracted and amplified by two-step method on a real-time fluorescent quantitative PCR instrument. The stantard curve of real-time fluorescent quantitative PCR was also obtained. In addition, an experiment was conducted by irrigating root of poplar with different solutions. Three treatments were designed as T1 (20 mL of 1×10 6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with no N6-34), T2 (20 mL of 1×10 6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with inactivated N6-34) and T3 (20 mL of 1×10 6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with activated N6-34). The quantity of Fusarium oxysporum at the rhizosphere of poplar was detected after treated for 10, 20 and 30 days. The results showed that the real-time fluorescent quantitative PCR method established in this study could be used for the detection of poplar wilt caused by soil-borne Fusarium oxysporum. The quantity of Fusarium oxysporum decreased first and then increased under the T1 treatment, and decreased under the T2 and T3 treatments, which indicated that the antagonistic bacterium N6-34 could inhibit Fusarium oxysporum. This study would have very important application value for guiding poplar planting and disease prediction, and could provide scientific bases for poplar disease management.
作者
洒荣波
晁强
王晓辉
隋君康
刘训理
Sa Rongbo;Chao Qiang;Wang Xiaohui;Sui Junkang;Liu Xunli(College of Life Sciences, Taishan Medical University, Taian 271016, China;Forestry College of Shandong Agricultural University, Taian 271018, China;College of Life Sciences of Shandong Agricultural University, Taian 271018, China)
出处
《山东农业科学》
2019年第2期131-135,共5页
Shandong Agricultural Sciences
基金
国家林业公益性行业科研专项(201304212)
关键词
荧光定量PCR
杨树枯萎病
尖孢镰刀菌
定量检测
Fluorescent quantitative PCR
Poplar wilt
Fusarium oxysporum
Quantitative detection
