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2025年8月13日 星期三
农业生物技术学报  2022, Vol. 30 Issue (6): 1210-1218    DOI: 10.3969/j.issn.1674-7968.2022.06.016
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
两种甘蔗梢腐病病原分子检测技术体系的建立及应用
李婕1, 李银煳1, 普家荣2, 张荣跃1, 单红丽1, 李文凤1, 王晓燕1, 普金安2, 黄应昆1,*
1 云南省农业科学院 甘蔗研究所/云南省甘蔗遗传改良重点实验室,开远 661699;
2 新平县经济作物工作站,新平 653400
Establishment and Application of Molecular Detection Technology System for Two Kinds Pathogens of Sugarcane (Saccharum officinarum) Pokkah boeng
LI Jie1, LI Yin-Hu1, PU Jia-Rong2, ZHANG Rong-Yue1, SHAN Hong-Li1, LI Wen-Feng1, WANG Xiao-Yan1, PU Jin-An2, HUANG Ying-Kun1,*
1 Sugarcane Research Institute, Yunnan Province Academy of Agricultural Science/Yunnan Key Laboratory of Sugarcane Genetic Improvement, Kaiyuan 661699, China;
2 Economic Crop Workstation in Xinping County, Xinping 653400, China
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摘要 甘蔗(Saccharum officinarum)是我国最重要的糖料作物,甘蔗梢腐病(Pokkah boeng)对我国蔗糖产业高质量发展带来严重威胁。本研究基于甘蔗梢腐病主要病原菌拟轮枝镰孢(Fusarium verticillioides)和层出镰孢(Fusarium proliferatum)的核糖体DNA内转录间隔区(nuclear ribosomal DNA internal transcribed spacer, rDNA-ITS)序列分别设计了特异性引物,并对采自云南省不同蔗区不同主栽品种上的117份典型甘蔗梢腐病样品进行了PCR分子检测,明确了该区域这2种甘蔗梢腐病主要病原菌的发生情况。结果表明,引物Fp-F3/Fp-R3可从拟轮枝镰孢菌及其侵染甘蔗中扩增出400 bp的特异性片段,而其他甘蔗病害病原菌均未扩增出此片段,该引物最低能检测到的拟轮枝镰孢菌DNA浓度为30 pg/µL;引物Fp-F4/Fp-R4可从层出镰孢菌及其侵染甘蔗中扩增出362 bp的特异性片段,而其他甘蔗病害病原菌均未扩增出此片段,该引物最低能检测到的层出镰孢菌DNA浓度为300 pg/µL。117份样品中有112份样品检出拟轮枝镰孢菌,阳性检出率为95.7%;有103份样品检出层出镰孢菌,阳性检出率为88%;有103份样品为拟轮枝镰孢菌和层出镰孢菌复合侵染,复合侵染率为88%;另有5份样品均未检测出这2种病原菌,可能为其他种引起的甘蔗梢腐病。分别选取不同蔗区不同主栽甘蔗品种上的23个拟轮枝镰孢菌和19个层出镰孢菌的PCR扩增产物进行测序,结果显示,拟轮枝镰孢菌和层出镰孢菌扩增产物序列分别与拟轮枝镰孢菌(GenBank No. KU508286)和层出镰孢菌(GenBank No. MK252904)序列相似性高达99.45%~100%和99.26%~100%。挑取部分序列构建系统发育树,系统发育分析显示测序序列分属于拟轮枝镰孢菌组和层出镰孢菌组。结果表明,本研究建立的检测体系能够准确、快速的检测拟轮枝镰孢菌和层出镰孢菌,云南不同蔗区不同主栽品种轮枝镰孢菌和层出镰孢菌检出率均较高,为云南甘蔗梢腐病重要病原,且复合侵染现象较普遍,其中普洱、临沧、红河和玉溪蔗区拟轮枝镰孢菌为优势种,但层出镰孢菌检出率也较高,且存在其他种。本研究为甘蔗梢腐病病害早期诊断和及时防控提供技术支撑和实践依据。
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李婕
李银煳
普家荣
张荣跃
单红丽
李文凤
王晓燕
普金安
黄应昆
关键词 甘蔗梢腐病拟轮枝镰孢层出镰孢系统进化分析    
Abstract:Sugarcane (Saccharum officinarum) is the most important sugar crop in China. Sugarcane Pokkah boeng disease poses a serious threat to the high-quality development of the sucrose industry in China. Therefore, specific primers were designed based on the nuclear ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences of 2 main pathogens of sugarcane Pokkah boeng Fusarium verticillioides and Fusarium proliferatum, respectively. The results showed that primers Fv-F3/Fv-R3 could amplify a specific amplification fragment of 400 bp from F. verticillioides and its infected sugarcane, but not other pathogens of sugarcane diseases. The detection sensitivity for genomic DNA of F. verticillioides was 30 pg/µL. Primers Fp-F4/Fp-R4 could amplify a specific amplification fragment of 362 bp from F. proliferarum and its infected sugarcane, but not other pathogens of sugarcane diseases. The detection sensitivity for genomic DNA of F. proliferarum was 30 pg/µL. A total of 117 samples with typical Pokkah boeng symptoms were collected from different main cultivated sugarcane varieties in different sugarcane areas of Yunnan Province were detected by PCR with these two pairs of specific primers. And the occurrence of the two main pathogens of sugarcane Pokkah boeng in this area was clarified. The results showed that 112 (95.7%) of the 117 samples were infected by F. verticillioides, 103 (88%) were infected by F. proliferatum. 103 (88%) were complex infected by F. verticillioides+F. proliferatum. The two pathogens were not detected in other 5 samples, which may be caused by other species. The PCR products of 23 F. verticillioides and 19 F. proliferatum from different main sugarcane varieties in different sugarcane regions were selected for sequencing and analysis. The results showed that the F. verticillioides and F. proliferatum amplification product sequences had 99.45%~100% similarity with F. verticillioides (KU508286) and 99.26%~100% similarity with F. proliferatum (MK252904), respectively. Some sequences were selected to construct phylogenetic tree, they were divided into F. verticillioides group and F. proliferatum group. The results of this study showed that the established detection system could accurately and quickly detect F. verticillioides and F. proliferatum. The detection rates of F. verticillioides and F. proliferatum were high from different main sugarcane varieties in different sugarcane areas of Yunnan Province. They were important pathogens of sugarcane Pokkah boeng in Yunnan province, and the compound infection was common. Among them, F. verticillioides was the dominant species in sugarcane areas such as Puer, Lincang, Honghe, and Yuxi sugarcane areas. However, the detection rate of F. proliferatum was also high in these sugarcane areas, and maybe there were other species of sugarcane Pokkah boeng pathogens might be present. This study provides technical support and practical basis for early diagnosis and timely prevention and control of these diseases.
Key wordsSugarcane Pokkah boeng    Fusarium verticillioides    Fusarium proliferatum    Phylogenetic analysis
收稿日期: 2021-07-23     
ZTFLH:  S432.1  
基金资助:财政部和农业农村部国家现代农业产业技术体系专项(CARS-170303); “云岭产业技术领军人才”培养项目(2018LJRC56); 云南省现代农业产业技术体系建设专项(YNGZTX-4-92)
通讯作者: * huangyk64@163.com   
引用本文:   
李婕, 李银煳, 普家荣, 张荣跃, 单红丽, 李文凤, 王晓燕, 普金安, 黄应昆. 两种甘蔗梢腐病病原分子检测技术体系的建立及应用[J]. 农业生物技术学报, 2022, 30(6): 1210-1218.
LI Jie, LI Yin-Hu, PU Jia-Rong, ZHANG Rong-Yue, SHAN Hong-Li, LI Wen-Feng, WANG Xiao-Yan, PU Jin-An, HUANG Ying-Kun. Establishment and Application of Molecular Detection Technology System for Two Kinds Pathogens of Sugarcane (Saccharum officinarum) Pokkah boeng. 农业生物技术学报, 2022, 30(6): 1210-1218.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.06.016     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I6/1210
 
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