短密木霉菌RT-PCR检测体系的建立及其在南瓜根际的动态分析

doi:10.16409/j.cnki.2095-039x.2018.04.012

中国生物防治学报 ›› 2018, Vol. 34 ›› Issue (4): 574-581.DOI: 10.16409/j.cnki.2095-039x.2018.04.012

短密木霉菌RT-PCR检测体系的建立及其在南瓜根际的动态分析

谢学文¹, 程颖超¹, 王锟¹, 张红杰², 杨杰³, 石延霞¹, 柴阿丽¹, 李宝聚¹

1. 中国农业科学院蔬菜花卉研究所, 北京 100081;
2. 河北北方学院农林科技学院, 张家口 075000;
3. 西藏自治区农牧科学院蔬菜研究所, 拉萨 850000

收稿日期:2017-12-25 出版日期:2018-08-08 发布日期:2018-07-30
通讯作者: 李宝聚,博士,研究员,Email:Libaoju@caas.cn。
作者简介:谢学文,博士,助理研究员,Email:xiexuewen@caas.cn;程颖超,硕士研究生,Email:761017740@qq.com
基金资助:
国家重点研发计划（2016YFD0201010、2017YFD0201102）；中国农业科学院科技创新工程（CAAS-ASTIP-IVFCAAS）；农业部园艺作物生物学与种质创制重点实验室项目

Establishment of Real-time Fluorescent Quantitative PCR for Trichoderma brevicompactum and Its Dynamic Change in Pumpkin Rhizosphere

XIE Xuewen¹, CHENG Yingchao¹, WANG Kun¹, ZHANG Hongjie², YANG Jie³, SHI Yanxia¹, CHAI Ali¹, LI Baoju¹

1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
2. School of Agriculture and Forestry Science and Technology, Hebei North University, Zhangjiakou 075000, China;
3. Institute of Vegetable, Tibet Academy of Agriculture and Animal Husbandry Sciences, Lasa 850000, China

Received:2017-12-25 Online:2018-08-08 Published:2018-07-30

摘要/Abstract

摘要： 根据短密木霉菌31636 ITS基因序列设计特异性引物TB51F/51R，建立其荧光定量PCR （RT-PCR）检测体系。通过盆栽试验明确短密木霉菌31636对南瓜疫病的防治效果，并采用RT-PCR检测技术监测短密木霉菌31636与辣椒疫霉菌LJ12010805在南瓜根际土中的动态变化。试验结果表明，利用该引物建立的RT-PCR检测体系线性关系良好，灵敏度为0.16 pg/μL，是普通PCR的100倍。采用拌土法接种短密木霉菌31636菌悬液10 mL育苗对南瓜疫病的防效最佳，定植后第15、30 d时防治效果分别为75.63%和46.82%。RT-PCR检测结果显示基质土中短密木霉菌31636呈减-增-减的动态变化规律，15 d时菌量最大，拷贝数为3.44×10⁶copys/μL；于定植后第3、15 d辣椒疫霉菌LJ12010805菌量分别达到最大、最小值，拷贝数分别为2.42×10⁶copys/μL和5.76×10²copys/μL。而对照的辣椒疫霉菌LJ12010805菌量持续增加，监测的第30 d拷贝数最大，为8.42×10⁷copys/μL。

关键词: 短密木霉菌, 南瓜疫病, 荧光定量PCR, 动态变化, 防效

Abstract: A real-time fluorescent quantitative PCR detection assay of Trichoderma brevicompactum 31636 was developed based on a specific primer pair TB51F/51R which were designed based on the ITS gene. The pot experiment was conducted to determine the control effect of T. brevicompactum 31636 on pumpkin Phytophthora blight, and the dynamic changes of population of T. brevicompactum 31636 and Phytophthora capsici Leonian LJ12010805 in pumpkin rhizosphere soil were monitored by RT-PCR. The result showed that the assay has a good liner relationship with a high sensitivity of 0.16 pg/μL DNA, which is 100 times more than that of normal PCR. Inoculation with 10 mL T. brevicompactum 31636 bacterial suspension at sowing stage had a best control effect on pumpkin Phytophthora blight, with the efficacies of 73.11% and 46.82% on the 15th and 30th day, respectively. RT-PCR results showed that dynamic change of T. brevicompactum 31636 in matrix after planting was appeared three distinct phases that were decrease, increase and decrease. The amount of bacteria was the largest on 15th day, with the copy number of 3.44×10⁶ copys/μL. On the 3rd after planting, the amount of P. capsici LJ12010805 reached the maximum, then decreased gradually and reached the lowest on 15th day, with the copy number of 2.42×10⁶ copys/μL and 5.76×10²copys/μL, respectively. However, P. capsici LJ12010805 of the control showed an increasing trend, with the highest copy number of 8.42×10⁷copys/μL on the 30th day.

Key words: Trichoderma brevicompactum, pumpkin Phytophthora blight, real-time fluorescent quantitative PCR, dynamic change, control effect

中图分类号:

S476

谢学文, 程颖超, 王锟, 张红杰, 杨杰, 石延霞, 柴阿丽, 李宝聚. 短密木霉菌RT-PCR检测体系的建立及其在南瓜根际的动态分析[J]. 中国生物防治学报, 2018, 34(4): 574-581.

XIE Xuewen, CHENG Yingchao, WANG Kun, ZHANG Hongjie, YANG Jie, SHI Yanxia, CHAI Ali, LI Baoju. Establishment of Real-time Fluorescent Quantitative PCR for Trichoderma brevicompactum and Its Dynamic Change in Pumpkin Rhizosphere[J]. journal1, 2018, 34(4): 574-581.

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短密木霉菌RT-PCR检测体系的建立及其在南瓜根际的动态分析

Establishment of Real-time Fluorescent Quantitative PCR for Trichoderma brevicompactum and Its Dynamic Change in Pumpkin Rhizosphere

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