禾本科植物内生真菌研究3:产生子座而无有性孢子的Neotyphodium属内生真菌-拂子茅属植物新共生体

2006年5月在南京市南郊发现了着生内生真菌子座的禾本科植物。子座长47.5~165.0mm,白色,包裹其宿主植物的旗叶叶鞘;于4月下旬形成,7月中旬完全崩溃;在自然条件下、人工交配试验中,子座上均未形成有性生殖结构。宿主植物直立,高90~120cm,有丰富的根状茎;连续2个花期均无抽穗。根据植物外部特征、叶表皮微形态特征以及rDNA-ITS序列的分析结果,将这种不抽穗的禾本科植物鉴定为拂子茅属植物(Calamagrostis sp.)。从29株着生子座的植株和87株不着生子座的植株中全部检出了内生真菌,从中分离得到了20个菌株。根据分生孢子、分生孢子梗的形态特征,分离菌株被鉴定为Neotyphodium属内生真菌。宿主植物的特殊性和菌株的子座形成能力显示了这是一个禾本科植物-内生真菌的新组合。这个组合是产生子座而不形成有性世代的第3例,并且这3例组合的宿主都是翦股颖族植物。     

rDNA-ITS sequence analysis of pathogens of cucumber downy mildew and cucumber powdery mildew

To determine the pathogens of cucumber downy mildew and cucumber powdery mildew by molecular marker, we amplified and sequenced the rDNA-ITS region of the pathogens of cucumber downy mildew and cucumber powdery mildew collected from the Shanghai region. The intra-/interspecific sequence difference was analyzed by rDNA-ITS sequence. The results show that the length of rDNA-ITS1 and rDNA-ITS2 of cucumber downy mildew’s pathogen was 141 bp and 406 bp, respectively, with GC contents of 41.13% in ITS1 and 46.8% (Minhang and Jinshan District, sm1 and sm2) or 46.55% (Pudong District, sm3) in ITS2. The rDNA-ITS sequence was intraspecific conservation. The interspecific difference was related with their kin relationship. The pathogen of cucumber downy mildew was identified as Pseudoperonospora cubensis by molecular marker. The length of rDNA-ITS1 and rDNA-ITS2 of cucumber powdery mildew’s pathogen was 136 bp and 89 bp, respectively, with GC contents being 59.56% and 66.29%, and rDNA-ITS sequence being highly conservative in this study that was the same as Sphaerotheca cucurbitae. But the sequence difference between the strains in the Shanghai region in this study with S. fuliginea was 4.5%, which was identified by morphology. It is suggested that the pathogen of cucumber powdery mildew should be further clarified and determined. __________ Translated from Journal of Northwest A & F University (Nat. Sci. Ed.), 2007, 35(10): 155–158 [译自: 西北农林科技大学学报(自然科学版)]     

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。     

蚕豆赤斑病病原菌分离鉴定及生物学特性研究

采用组织分离法进行病原物的分离、柯赫氏法则进行致病性测定,并利用真菌通用引物ITS1/ITS4和蚕豆葡萄孢特异性引物G3PDHfor/G3PDHrev进行基因组DNA扩增与序列分析,对甘肃省渭源县蚕豆赤斑病的致病菌种类进行研究。结果表明：该病原菌为蚕豆葡萄孢（Botrytis fabae）,系统发育树分析表明该病原菌与GenBank已报道的蚕豆葡萄孢属聚为一类。生物学特性测定表明,该病原菌菌丝生长最适培养基为PDA培养基,最适碳源和氮源分别为乳糖和酵母膏,最适温度为20 ℃,最适pH为8,对光照不敏感。     

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。     

马铃薯腐烂茎线虫RPA-LFD可视化快速检测方法的建立

 马铃薯腐烂茎线虫是为害我国甘薯和马铃薯的一种重要植物病原线虫,也是我国重要的检疫性有害生物。为实现对该线虫的准确、快速且可视化的检测,本研究以马铃薯腐烂茎线虫rDNA-ITS序列为靶标构建了重组酶聚合酶结合侧流层析试纸条(RPA-LFD)的可视化快速检测体系。该体系可在39 ℃条件下15 min内特异性地完成对马铃薯腐烂茎线虫的检测,对A型(甘薯种群)和B型(马铃薯种群)单头线虫(J4)的检出底限均为3 125^-1头线虫,可以直接对土壤和甘薯茎中的马铃薯腐烂茎线虫进行检测,灵敏度可达1头(J4)/10 g土壤和1头(J4)/2 g甘薯茎组织。该体系操作简便、成本低廉、结果可视,可为马铃薯腐烂茎线虫的早期预警和口岸检疫提供技术支撑。     

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.     

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.     

新疆巩留县杏果实斑点病病原菌鉴定

[目的]鉴定引起新疆巩留县杏树果实斑点病病原菌,研究造成杏树果实病害的原因,为当地杏树防治工作提供依据。[方法]采用常规组织分离法分离得到罹病杏真菌菌株,利用传统形态学观察和分子系统学分析相结合的方式对所分离出菌株进行分类鉴定及其致病性检测。[结果]杏果病斑处的病原菌在显微镜下观察到分生孢子形态与经PDA培养基培养后观察到分生孢子形态均与Thyrostroma carpophilum（Lév.） B.Sutton所产生分生孢子一致;将分离获得的3株真菌的rDNA-ITS片段测序后与NCBI参考序列进行多重序列比对的结果显示,其序列与T.carpophilum一致性为100%;在基于ITS基因序列构建的系统发育树中,3株菌与T.carpophilum聚在同一分支。在接种了T.carpophilum后,杏果实和叶片均产生明显病斑并且从其所产生的病斑上再次分离到所接菌,满足柯赫氏法则。[结论]从新疆巩留县杏果实病斑处分离获得的3株真菌,经鉴定为引起杏穿孔病的病原菌T.carpophilum。这是该菌首次在该地区发现并报道。