基于PCR检测的4种土传病害病原菌侵染棉花的动态分析
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摘要
【目的】立枯病、红腐病、枯萎病和黄萎病是新疆棉花上的4种主要土传病害,研究4种病害的病原菌在新疆棉花上的侵染动态,分析各自的侵染始期和最佳防治时期,为病害高效防控提供依据。【方法】针对4种主要病害的病原菌,对混合接菌后分期采集的棉株进行特异性引物PCR检测,依据检测结果,进行各病原菌侵染动态分析及混合侵染分析。【结果】4种病原菌从棉花子叶期即能侵染;立枯丝核菌侵染相对较早、较快,拟轮枝镰孢霉次之;2种病原菌均在棉苗前期有较高的侵染株率,3叶期达到高峰,此后侵染株率逐渐降低;而尖孢镰孢霉和大丽轮枝菌,虽从子叶期开始即可侵染,但侵染株率较低,直到蕾铃期侵染株率呈渐增趋势;病原菌之间的混合侵染普遍存在,以两菌混合侵染居多。【结论】子叶期为4种病原菌的侵染初期;立枯丝核菌和拟轮枝镰孢霉以苗期侵染为特点;尖孢镰孢霉和大丽轮枝菌以子叶期到蕾铃期均可侵染为特点;病原菌之间常有混合侵染发生。
【Objective】 Rhizoctonia damping off, Fusarium red rot, Fusarium wilt and Verticillium wilt are four major soil-borne diseases on cotton in Xinjiang. This project aims to clarify the infection dynamics of the four diseases of cotton in Xinjiang and analyze the initial infection period and the best control period of each disease in the hope of providing a basis for the high efficiency prevention and control of the disease. 【Methods】In this study, specific primer PCR was used to detect the pathogen of four main diseases, and the dynamic analysis and mixed infection analysis of each pathogen were carried out according to the detection results.【Results】The results indicated that the four pathogens could be infested from the cotyledon stage of cotton; the infection of Rhizoctonia solani was detected relatively earlier and faster, followed by Fusarium verticillioides, the infection rate of the two pathogens was high in the early stage of cotton seedling and reached the peak at the three-leaf stage, and the detected rate decreased gradually after that. Although the Fusarium oxysporum and Verticillium dahliae could also be infected from the cotyledon stage, but the rate of detection in the cotyledon to the one-leaf stage was low, Until the time of the boll stage invasion plant rate was gradually increasing. The mixed infection between the pathogens was widely spread, and the mixed infection of the two species pathogens was the most common.【Conclusions】Cotyledon stage is the infection start period of four species pathogens. R. solani and F. verticillioides is characterized by seedling invasion, while F.oxysporum and V.dahliae is characterized by invasion from cotyledon stage to boll stage; There are often mixed infections among pathogens.
【Objective】 Rhizoctonia damping off, Fusarium red rot, Fusarium wilt and Verticillium wilt are four major soil-borne diseases on cotton in Xinjiang. This project aims to clarify the infection dynamics of the four diseases of cotton in Xinjiang and analyze the initial infection period and the best control period of each disease in the hope of providing a basis for the high efficiency prevention and control of the disease. 【Methods】In this study, specific primer PCR was used to detect the pathogen of four main diseases, and the dynamic analysis and mixed infection analysis of each pathogen were carried out according to the detection results.【Results】The results indicated that the four pathogens could be infested from the cotyledon stage of cotton; the infection of Rhizoctonia solani was detected relatively earlier and faster, followed by Fusarium verticillioides, the infection rate of the two pathogens was high in the early stage of cotton seedling and reached the peak at the three-leaf stage, and the detected rate decreased gradually after that. Although the Fusarium oxysporum and Verticillium dahliae could also be infected from the cotyledon stage, but the rate of detection in the cotyledon to the one-leaf stage was low, Until the time of the boll stage invasion plant rate was gradually increasing. The mixed infection between the pathogens was widely spread, and the mixed infection of the two species pathogens was the most common.【Conclusions】Cotyledon stage is the infection start period of four species pathogens. R. solani and F. verticillioides is characterized by seedling invasion, while F.oxysporum and V.dahliae is characterized by invasion from cotyledon stage to boll stage; There are often mixed infections among pathogens.
引文
[1] 新疆维吾尔自治区统计局.新疆统计年鉴[M].北京:中国统计出版社,2018:343-345.Xinjiang Uygur Autonomous Region Statistics Bureau.(2018).Xinjiang Statistical Yearbook [M].Beijing:China Statistics Press:343-345.(in Chinese)
[2] 林玲.棉花黄萎病研究进展[J].棉花学报,2014,26(3):260-267.LIN Ling.(2014).Research Progress in Cotton Verticillium Wilt [J].Cotton Science,26(3):260-267.(in Chinese)
[3] 荆宝健.控制新疆棉花苗期病害的有效途径[J].新疆农垦科技,2009,(1):31-32.JING Bao-jian.(2009).The Effective Measure to Control the Seedling Disease of Cotton in Xinjiang [J].Xinjiang Farm Research of Science and Technology,(1) :31-32.(in Chinese)
[4] 王芙蓉,乔清华,张景霞,等.棉花根际真菌群落结构及其动态变化[M],中国农学会棉花分会2017年年会暨第九次会员代表大会2017:中国河南郑州.WANG Fu-rong,QIAO Qing-hua,ZHANG Jing-xia,et al.(2017).Community structure and dynamic changes of cotton rhizosphere fungi [M].China Agriculture Society Cotton Branch 2017 Annual Meeting and Ninth Member Congress:Zhengzhou,Henan,China.(in Chinese)
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[6] 姜玉英,陆宴辉,李晶,等.新疆棉花病虫害演变动态及其影响因子分析[J].中国植保导刊,2015,35(11):43-48.JIANG Yu-ying,LU Yan-hui,LI Jing,et al.(2015).Evolution dynamics of cotton pests and diseases in Xinjiang and their influencing factors [J].China Plant Protection,35(11):43-48.(in Chinese)
[7] 李焕宇,付婷婷,张云,等.5种方法提取真菌基因组DNA作为PCR模板效果的比较[J].中国农学通报,2017,33(16):28-35.LI Huan-yu,FU Ting-ting,ZHANG Yun,et al.(2017).Comparison of Five Methods for Extracting Fungal Genomic DNA as a PCR Template [J].Chinese Agricultural Science Bulletin,33(16):28-35.(in Chinese)
[8] 赵永坡,初雷霞,岳开华.等.太子参根际尖孢镰刀菌绝对荧光定量检测方法的建立及其应用[J].江西农业大学学报,2014,36(5):1 127-1 131.ZHAO Yong-po,CHU Lei-xia,YUE Kai-hua,et al.(2014).Establishment and Application of Absolute Fluorescence Quantitative Detection of Fusarium oxysporum in Rhizosphere of Radix Pseudostellariae [J].Journal of Jiangxi Agricultural University,36(5):1,127-1,131.(in Chinese)
[9] 黄怀冬,肖姬玲,张屹,等.一种改进的尖孢镰刀菌的实时荧光定量PCR检测方法[J].植物保护,2017,43(4):129-133.HUANG Huai-dong,XIAO Ji-ling,ZHANG Yi,et al.(2017).An improved real-time quantitative PCR assay for Fusarium oxysporum [J].Plant Protection,43(4):129-133.(in Chinese)
[10] 张吉祥.甘蓝枯萎病菌1、2号生理小种特异基因鉴定及应用[D].北京:中国农业科学院,2014.ZHANG Ji-xiang.(2014).Identification and application of physiological race specific genes of Fusarium oxysporum f.1 and No.2 [D].PhD Dissertation.Chinese Academy of Agricultural Sciences,Beijing.(in Chinese)
[11] 伍恩宇.茄科蔬菜立枯丝核菌的遗传分化及分子检测[D].泰安:山东农业大学植物保护学院,2008.WU En-yu.(2008).Genetic Differentiation and Molecular Detection of Rhizoctonia solani in Solanaceae [D].Master Dissertation.Shandong Agricultural University,Tai'an.(in Chinese)
[12] 李瑞琴,刘星,邱慧珍,等.发生马铃薯立枯病土壤中立枯丝核菌的英冠定量PCR快速检测[J].草业学报,2013,22(5):136-144.LI Rui-qin,LIU Xing,QIU Hui-zhen,et al.(2013).Quantitative PCR detection of the negative crown of Rhizoctonia solani in the potato blight [J].Acta Pratacultura Sinica,22(5):136-144.(in Chinese)
[13] 刘萍花,方敦煌,吴祖建.应用多重PCR检测烟草黑胫病菌、根黑腐病菌、猝倒病菌及立枯病菌[J].福建农林大学学报(自然科学版),2015,44(4):345-349.LIU Ping-hua,FANG Dun-huang,WU Zu-jian.(2015).Detection of Tobacco Black rot fungus,root rot pathogen,sputum pathogen and Phytophthora infestans by multiplex PCR [J].Journal of Fujian Agricultural and Forestry University,44(4):345 -349.(in Chinese)
[14] K.A.Abd-Elsalam,A.Asran-Amal,F.Schnieder,et al.Molecular detection of Fusarium oxysporum f.sp.vasinfectum in cotton roots by PCR and real-time PCR assay [J].Journal of Plant Diseases and Protection,2006,113(1):14-19.
[15] 庞莉,李梅,孙青,等.棉花黄萎病病原菌大丽轮枝菌的快速分子检测[J].植物保护学报,2016,43(6):892-899.PANG Li,LI Mei,SUN Qing,et al.(2016).Rapid molecular detection of Verticillium dahliae,a pathogen of cotton Verticillium wilt [J].Chinese Journal of Plant Protection,43(6):892-899.(in Chinese)
[16] Murillo,I.,Cavallarin,L.,& Segundo,B.S.(1998).The development of a rapid pcr assay for detection of fusarium moniliforme.European Journal of Plant Pathology,104(3):301-311.
[2] 林玲.棉花黄萎病研究进展[J].棉花学报,2014,26(3):260-267.LIN Ling.(2014).Research Progress in Cotton Verticillium Wilt [J].Cotton Science,26(3):260-267.(in Chinese)
[3] 荆宝健.控制新疆棉花苗期病害的有效途径[J].新疆农垦科技,2009,(1):31-32.JING Bao-jian.(2009).The Effective Measure to Control the Seedling Disease of Cotton in Xinjiang [J].Xinjiang Farm Research of Science and Technology,(1) :31-32.(in Chinese)
[4] 王芙蓉,乔清华,张景霞,等.棉花根际真菌群落结构及其动态变化[M],中国农学会棉花分会2017年年会暨第九次会员代表大会2017:中国河南郑州.WANG Fu-rong,QIAO Qing-hua,ZHANG Jing-xia,et al.(2017).Community structure and dynamic changes of cotton rhizosphere fungi [M].China Agriculture Society Cotton Branch 2017 Annual Meeting and Ninth Member Congress:Zhengzhou,Henan,China.(in Chinese)
[5] 李国英.新疆棉花病虫害及防治[M].北京:中国农业出版社,2017.LI Guo-ying.(2017).Cotton pests and diseases in Xinjiang [M].Beijing:China Agriculture Press.(in Chinese)
[6] 姜玉英,陆宴辉,李晶,等.新疆棉花病虫害演变动态及其影响因子分析[J].中国植保导刊,2015,35(11):43-48.JIANG Yu-ying,LU Yan-hui,LI Jing,et al.(2015).Evolution dynamics of cotton pests and diseases in Xinjiang and their influencing factors [J].China Plant Protection,35(11):43-48.(in Chinese)
[7] 李焕宇,付婷婷,张云,等.5种方法提取真菌基因组DNA作为PCR模板效果的比较[J].中国农学通报,2017,33(16):28-35.LI Huan-yu,FU Ting-ting,ZHANG Yun,et al.(2017).Comparison of Five Methods for Extracting Fungal Genomic DNA as a PCR Template [J].Chinese Agricultural Science Bulletin,33(16):28-35.(in Chinese)
[8] 赵永坡,初雷霞,岳开华.等.太子参根际尖孢镰刀菌绝对荧光定量检测方法的建立及其应用[J].江西农业大学学报,2014,36(5):1 127-1 131.ZHAO Yong-po,CHU Lei-xia,YUE Kai-hua,et al.(2014).Establishment and Application of Absolute Fluorescence Quantitative Detection of Fusarium oxysporum in Rhizosphere of Radix Pseudostellariae [J].Journal of Jiangxi Agricultural University,36(5):1,127-1,131.(in Chinese)
[9] 黄怀冬,肖姬玲,张屹,等.一种改进的尖孢镰刀菌的实时荧光定量PCR检测方法[J].植物保护,2017,43(4):129-133.HUANG Huai-dong,XIAO Ji-ling,ZHANG Yi,et al.(2017).An improved real-time quantitative PCR assay for Fusarium oxysporum [J].Plant Protection,43(4):129-133.(in Chinese)
[10] 张吉祥.甘蓝枯萎病菌1、2号生理小种特异基因鉴定及应用[D].北京:中国农业科学院,2014.ZHANG Ji-xiang.(2014).Identification and application of physiological race specific genes of Fusarium oxysporum f.1 and No.2 [D].PhD Dissertation.Chinese Academy of Agricultural Sciences,Beijing.(in Chinese)
[11] 伍恩宇.茄科蔬菜立枯丝核菌的遗传分化及分子检测[D].泰安:山东农业大学植物保护学院,2008.WU En-yu.(2008).Genetic Differentiation and Molecular Detection of Rhizoctonia solani in Solanaceae [D].Master Dissertation.Shandong Agricultural University,Tai'an.(in Chinese)
[12] 李瑞琴,刘星,邱慧珍,等.发生马铃薯立枯病土壤中立枯丝核菌的英冠定量PCR快速检测[J].草业学报,2013,22(5):136-144.LI Rui-qin,LIU Xing,QIU Hui-zhen,et al.(2013).Quantitative PCR detection of the negative crown of Rhizoctonia solani in the potato blight [J].Acta Pratacultura Sinica,22(5):136-144.(in Chinese)
[13] 刘萍花,方敦煌,吴祖建.应用多重PCR检测烟草黑胫病菌、根黑腐病菌、猝倒病菌及立枯病菌[J].福建农林大学学报(自然科学版),2015,44(4):345-349.LIU Ping-hua,FANG Dun-huang,WU Zu-jian.(2015).Detection of Tobacco Black rot fungus,root rot pathogen,sputum pathogen and Phytophthora infestans by multiplex PCR [J].Journal of Fujian Agricultural and Forestry University,44(4):345 -349.(in Chinese)
[14] K.A.Abd-Elsalam,A.Asran-Amal,F.Schnieder,et al.Molecular detection of Fusarium oxysporum f.sp.vasinfectum in cotton roots by PCR and real-time PCR assay [J].Journal of Plant Diseases and Protection,2006,113(1):14-19.
[15] 庞莉,李梅,孙青,等.棉花黄萎病病原菌大丽轮枝菌的快速分子检测[J].植物保护学报,2016,43(6):892-899.PANG Li,LI Mei,SUN Qing,et al.(2016).Rapid molecular detection of Verticillium dahliae,a pathogen of cotton Verticillium wilt [J].Chinese Journal of Plant Protection,43(6):892-899.(in Chinese)
[16] Murillo,I.,Cavallarin,L.,& Segundo,B.S.(1998).The development of a rapid pcr assay for detection of fusarium moniliforme.European Journal of Plant Pathology,104(3):301-311.