rDNA-ITS在植物病原真菌分子检测中的应用

文章综述了rDNA序列结构特点及ITS序列在植物病原真菌的分类鉴定、分子检测、病害诊断及土壤中病原真菌的检测方面的应用,并对其应用前景进行了评述。     

借助ITS序列用GFP标记巴西橡胶树棒孢霉落叶病病原菌

由多主棒孢菌(Corynespora cassiicola)侵染引起的巴西橡胶树棒孢霉落叶病是影响橡胶树产胶量的主要病害之一。为了开展该病原菌在活体叶片中的病理学研究,本实验将绿色荧光蛋白gfp基因表达盒插入了多主棒孢菌经定点突变后的ITS序列中,构建了重组质粒p ITGH,并经PEG介导转化了原生质体,获得了GFP标记的多主棒孢菌转化子,所获得的转化子经过连续7次转接培养后仍能在分生孢子、分生孢子梗、芽管和菌丝中稳定地发出强烈的绿色荧光,其生长特性和致病性与野生菌株无明显差异。     

贡柑黑腐病病原菌的分子鉴定

贡柑黑腐病是近年来在贡柑上发生的一种新型病害,该病害可导致嫩梢快速枯死,大量嫩叶脱落,严重影响树势和产量。本研究从病区采集发病枝叶,对贡柑黑腐病病原菌进行分离、纯化,并采用rDNA-ITS序列分析技术对其进行分子鉴定。结果表明,贡柑黑腐病病原菌为链格孢属Alternaria Nees真菌,rDNA-ITS序列与GenBank中芸薹链格孢菌A.brassicae(Berk.)Sacc.和柑桔链格孢菌A.citri Ellis et Everhart的ITS序列的同源性为99%。结合形态学特征,推断该病原菌为柑桔链格孢菌。     

柱花草炭疽病菌的准确快速鉴定

采用常规组织分离法分离得到2个柱花草炭疽病菌致病性菌株,利用ITS通用引物ITS1／ITS4对炭疽菌rDNA—ITS进行扩增,电泳检测得到约500bp大小的目的条带,经过生物信息学分析2个菌株均为刺盘孢属胶胞炭疽病菌,且两者之间同源性达到99％,对其侵染能力进行检测,5个柱花草品系的抗病性有所不同。     

Identification of Powdery Mildew Pathogen and Ribosomal DNA-ITS Sequence Analysis on Melon

Identification of powdery mildew pathogens on melon（Cucumis melo） is important for melon breeding and diseaseresistant germplasm selection. In this study, a powdery mildew pathogen that infected melon plants in Heilongjiang Province, China, was investigated in terms of host identification, morphological characteristics and phylogenetic relationships. The morphological characteristics of the pathogen were observed at five phases in the life cycle： germinating conidia, primary germ tube, hyphae, conidiophores, and colonization. The conidia were elliptical, colorless, catenulate, and the average length was 29.07 μm and average width was 17.82 μm. One ascus and eight ascospores were produced. DNA was extracted from 0.01 g conidiophores from a strain of powdery mildew pathogen that infected melon. ITS ribosomal DNA region（524 bp） was amplified with the universal ITS1 and ITS4 primers. The nucleotide sequence showed 100% similarity with ITS sequences for three Podosphaera fusca strains obtained from the GenBank database. The identity of the pathogen was confirmed as Sphaerotheca fuliginea. International standard differential hosts were used to identify S. fuliginea strain as 2F race. These results supported the notion that Podosphaera fusca was a synonym of S. fuliginea.     

镰刀菌引起的北京市草莓根腐病病原鉴定

对北京市昌平区采集的35个草莓根腐病病样进行病原鉴定,结果分离获得13个菌株,根据培养特征和形态学鉴定,确定分离到的菌株均为尖孢镰刀菌（Fusarium oxysporum）|选取真菌通用引物ITS1/ITS4扩增菌株的rDNA-ITS序列并进行测序,通过与GenBank数据库序列比对,分子鉴定结果与形态学一致。将分离到的菌株回接到健康草莓幼苗上,植株表现出与田间发病相似的症状,重新分离能够得到相同的病原物。基于形态学、分子生物学鉴定及致病性检测,最终确定北京市昌平区草莓根腐病的病原菌为Fusarium oxysporum。     

松材线虫rDNA-ITS1区分子检测与鉴定

利用两对松材线虫特异性引物,分别对来自日本、美国和葡萄牙的松材线虫虫样进行PCR检测,成功扩增出330 bp和220 bp rDNA-ITS1区基因片段,该方法对松材线虫成虫或幼虫均能作出准确鉴定.     

茄子绒菌斑病病原菌鉴定及生物学特性研究

本文针对辽宁省近年茄子设施栽培生产上趋重发生的叶部病害——绒菌斑病,对该病的病原菌进行了鉴定及其生物学特性研究。通过病原菌形态学鉴定,柯赫氏法则证病,并对病菌经rDNA-ITS测序,鉴定该病致病菌为真菌灰毛茄钉孢［Passalora nattrassii?(Deighton)U. Braun & Crous］。病菌生物学特性测定结果表明：病菌菌丝生长以PDA+茄叶煎汁培养基为最适;适宜菌丝生长的碳源和氮源分别为可溶性淀粉和胰蛋白胨;适宜温度为25 ℃;最适pH为6;黑暗条件下菌丝生长较好;菌丝致死温度为59 ℃,10min。病菌分生孢子萌发试验结果表明,茄叶汁能明显促进其萌发,碳源以麦芽糖、氮源以硫酸铵最适于孢子萌发,孢子萌发的适宜温度为30 ℃;最适pH为5;光暗交替条件下萌发情况较好;分生孢子致死温度为57 ℃,10min。     

苦瓜枯萎病菌的鉴定及其遗传多样性分析

 对分离获得的32株苦瓜枯萎病菌菌株进行形态学特征和寄主专化型测定, 结果表明, 测试的苦瓜枯萎病菌株均为尖孢镰刀菌苦瓜专化型 (Fusarium oxysporum f. sp. momordicae), 这些菌株可以侵染苦瓜和瓠瓜幼苗, 但不侵染其他葫芦科瓜类作物。对苦瓜枯萎病菌菌株的rDNA-ITS区 (ITS1、5.8S和ITS2)序列进行扩增测序, 结果显示其序列长度均为456 bp;聚类分析表明测序菌株与镰刀菌属中尖孢镰刀菌不同专化型的菌株聚为一群。利用RAPD标记技术分析苦瓜枯萎病菌的遗传多样性, 结果显示苦瓜枯萎病菌株与其他葫芦科瓜类作物枯萎病菌株间的遗传相似系数范围为0.59~0.99, 当遗传相似系数为0.85时, 供试的48个菌株分成10个类群 (G_1~10)。在RAPD聚类树中所有苦瓜枯萎病菌株聚在一个分支上 (G₁群), 菌株间的遗传相似系数范围为0.92~1.00, 具有较高的遗传相似性, 且菌株的聚群与地理来源存在一定的相关性。     

采后胡萝卜腐烂病病原菌鉴定

In October 2012, infected post harvest carrot root were found with decay symptom in several farmer markets around Kunming city of Yunnan Province, China. The symptom was grey white initially, then became brown to black with visible perithecia. According pathogenicity, morphology and sequence of the rDNA internal transcribed spacer region ( ITSI-5.8S-ITS2 ), the pathogen was identified as Ceratocystis fimbriata. The disease has been reported only in Brazil before, this is the first report on C. fimbriata as the pathogen of carrot(Daucus carota) decay post harvest in China .