摘要
为了明确新疆棉田土壤中黄萎病菌的致病类型及微菌核密度,本研究经重组巢氏PCR建立了双重巢氏PCR,以落叶型黄萎病菌菌株V592和非落叶型黄萎病菌菌株I6的混合DNA为模板,对其检测特异性和灵敏度进行了分析,检测了新疆棉田208份土壤样品的黄萎病菌致病类型。基于实时荧光定量聚合酶链式反应(qPCR)建立Ct值与大丽轮枝菌拷贝数及Ct值与微菌核相关性的标准曲线,获得大丽轮枝菌拷贝数和菌核数的关系,定量测定了棉田土壤中黄萎病菌的种群密度。检测结果显示,双重巢氏PCR可同时检测棉花黄萎病菌的落叶型和非落叶型菌系,灵敏度比普通PCR至少提高了104倍。落叶型菌系占供试土样的98.6%,非落叶型菌系占供试土样的38.9%,且非落叶型菌系几乎都与落叶型菌系混合发生,表明落叶型菌系是棉田土壤中的优势致病类型。拷贝数(y)与微菌核(x)之间的线性关系为y=11.54x。不同地块、不同植棉区土壤带菌量均存在差异,阿克苏棉田土壤中微菌核密度最高,塔城的微菌核密度最低。
This research aimed to determine the pathotypes and microsclerotial density of Verticillium dahlia in cotton field soil in Xinjiang.Duplex nested PCR was established for detection of defoliating and non-defoliating strain of V.dahliae by recombining nested PCR for defoliating strain detection and nested PCR for non-defoliating strain detection.The specificity and sensitivity of duplex nested PCR were evaluated using the mixed DNA of defoliating strain V592 and non-defoliating strain I6 as template.The total of 208 soil samples collected from different cotton regions were detected by duplex nested PCR to determine the pathotypes of V.dahliae in soil.In order to quantify microsclerotial density of V.dahliae in the soil,the standard curve relating the Ct values to the log-transformed copy number of the inserted V.dahliae DNA fragment in plasmids and the standard curve relating the Ct values to known number of microsclerotia were established based on qPCR,respectively,to derive to relate DNA copy and microsclerotium numbers.The detection resuts showed that duplex nested PCR could be used to detect defoliating and non-defoliating strains of V.dahliae simultaneously,and the detection sensitivity was at least 104 times higher than that of ordinary PCR.The detection results of V.dahliae in cotton field soil showed that the defoliating and non-defoliating strains accounted for 98.6%and 38.9%,respectively,furthermore,almost all non-defoliating strains were mixed with defoliating strains,indicating that defoliating strains were the dominant pathotypes in cotton field soil.Based on the two standard curves,a simple linear model(y=11.54x)was derived to relate DNA copy(y)and microsclerotium numbers(x).Quantitative detection of microsclerotial density of V.dahliae in cotton field soil showed that there were significant differences not only in different field plots but also in different cotton planting areas,and the density of microsclerotia in Aksu cotton field was the highest,and the lowest in Tacheng cotton field.
作者
徐灿
刘启
蔡梦杭
高峰
黄家风
XU Can;LIU Qi;CAI Menghang;GAO Feng;HUANG Jiafeng(College of Agriculture/Key Laboratory of Oasis Agricultural Pest Management and Plant Protection Resources Utilization,Xinjiang Uygur Autonomous Region,Shihezi University,Shihezi,Xinjiang 832003,China)
出处
《石河子大学学报（自然科学版）》
CAS
北大核心
2019年第6期734-741,共8页
Journal of Shihezi University(Natural Science)
基金
国家公益性行业(农业)科研专项经费资助(201503109)
国家自然科学基金(31560494)
关键词
棉花黄萎病菌
棉田土壤
致病型
菌核密度
Verticillium dahliae from cotton
cotton field soil
pathotypes
microsclerotial density
