河南省紫荆黄萎病菌的分离和鉴定

为明确河南省郑州市紫荆山公园紫荆植株枯死的病因,采集具有典型黄萎病症状的紫荆植株,采用组织分离法分离出2株病原菌Vcg1和Vcg4,并进行鉴定。该病原菌在PDA培养基中菌落菌丝灰白色,分生孢子梗轮生,后期产生微菌核。PCR扩增菌株Vcg1和Vcg4 r DNA-ITS区段并测序,序列比对结果表明其与大丽轮枝菌(V.dahliae Kleb.)同源性最高。通过分生孢子悬浮液伤根接种法测定目标菌株在紫荆幼苗上的致病性,结果发现该病原菌能引起紫荆发病,维管束变褐色,严重的导致紫荆植株死亡。以上结果表明引起郑州市紫荆山公园紫荆枯死的病原菌为大丽轮枝菌。     

糖化酶工业株的鉴定及糖化酶基因的克隆

对4种糖化酶工业株的rDNA片段进行克隆,在对比其rDNA-ITS1序列与GenBank数据库的基础上,结合形态学观察对菌株进行了鉴定。结果显示,4种菌株分别为黑曲霉、无花果曲霉、米曲霉和灰色小克银汉霉。通过Me-gAlign得到的进化树表明,前3种菌株亲缘关系较近,三者与灰色小克银汉霉的亲缘关系较远。进一步克隆了黑曲霉、无花果曲霉和米曲霉的糖化酶基因,并在毕赤酵母中实现了分泌表达。     

细口杯环线虫形态学和分子特征分析

为探讨细口杯环线虫(Cylicocyclus leptostomum)的分类地位和系统发育关系,本试验利用数码显微镜对细口杯环线虫进行了形态观察,运用PCR扩增其核糖体DNA内部转录间隔区(rDNA-ITS)序列,并从GenBank中下载12种圆线虫的ITS序列,以马圆形线虫(Strongylus equinus)为外群,运用最大似然法(maximum likelihood,ML)构建系统发育树。结果显示,细口杯环线虫中等大小,口囊呈圆柱形,宽度大于深度,口囊底部有小齿;口囊壁前端薄,后端基部有明显的环箍形增厚;外叶冠由20～24个小叶组成,内叶冠由50～60个小叶组成;食道漏斗较小;雄虫生殖锥较长,呈圆锥形;雌虫尾部直,尾尖呈指形;所测ITS序列总长度为837bp,其中ITS1长366bp,5.8S长153bp,ITS2长318bp;ITS1的GC含量(46.0%)明显高于ITS2(39.8%);经BLAST同源性比对分析,本研究线虫ITS序列与GenBank上登录的同种线虫序列(登录号:AJ004849、Y08587)同源性达99.85%,与阿氏杯环线虫的同源性达99.0%,与杯环属内其他线虫的同源性仅为93.33%～98.45%;ITS序列种间差异远大于种内差异;系统进化分析显示,细口杯环线虫与阿氏杯环线虫的亲缘关系较近,而与杯环属内其他线虫的亲缘关系相对较远。综上所述,ITS序列可作为鉴定寄生线虫的分子遗传标记,证实所采标本是细口杯环线虫,并首次在国内报道了细口杯环线虫的ITS序列,为该线虫的进一步研究奠定基础。     

大白菜褐腐病（茎基腐病）的病原菌鉴定

2011年在北京、河北、江苏等地的部分大白菜种植区,当地俗称的大白菜“茎基腐病”普遍严重发生,实际调查后共采集到大白菜“茎基腐病”病样12份,分离获得12个真菌分离物,经形态学鉴定,分离到的菌株均为立枯丝核菌Rhizoctonia solani;采用真菌通用引物ITS1/ITS4对病原菌rDNA-ITS进行PCR扩增,并将测序结果在GenBank中进行同源性比对分析,分子生物学鉴定结果与形态学鉴定结果一致。将分离到的菌株分别回接健康的大白菜植株,植株表现出与田间相似的发病症状,重新进行病原菌的分离,得到相同的病原物。基于形态学、分子生物学鉴定和致病性试验最终确定北京、河北、江苏等地大白菜 “茎基腐病” 病原菌为立枯丝核菌Rhizoctonia solani,大白菜“茎基腐病”与大白菜褐腐病、大白菜立枯病属同病异名。     

南昌市郊梨轮纹病病原菌鉴定

为了明确江西省南昌市郊区梨轮纹病菌的种类归属,本文从果园采集呈典型症状的梨轮纹病样本,采用组织分离法进行病菌分离和病菌纯化,获得5个梨轮纹病菌分离株,其培养性状相同且与已报道的葡萄座腔菌Botryosphaeria dothidea的培养性状一致。用菌饼对离体梨枝条进行接种,结果5个分离株均导致典型病斑,对该病斑进行再分离,又分离到与原接种菌一致的病原菌。任选其中的一个分离株NC-1进行rDNA-ITS序列扩增和序列测定,获得片段长为543bp的核苷酸序列,通过BLAST分析,发现该序列与GenBank中葡萄座腔菌的同源性高达100%。结果表明,南昌市郊区梨轮纹病菌属于葡萄座腔菌。     

红瑞木溃疡病病原菌的鉴定

为了明确近年来引起上海辰山植物园红瑞木上枝干溃疡病的病原菌种类,首先对病原菌分离并依据柯赫氏法则进行了致病性测定和病原菌验证,然后进行了形态学特征观察、rDNA-ITS序列分析和系统发育树构建。病原菌形态学特征与葡萄座腔菌(Botryosphaeria dothidea)一致,其rDNA-ITS序列(已提交GenBank,登录号:JN638454)与GenBank中葡萄座腔菌相似性达到99%,在系统发育树上与葡萄座腔菌处于同一分支。将辰山植物园红瑞木枝干溃疡病的病原菌鉴定为葡萄座腔菌(B.dothidea)。     

江西瑞昌山药炭疽病菌的形态及分子鉴定

为了明确江西瑞昌山药炭疽病病原菌种类归属,本文从当地采集呈典型症状的炭疽病叶片进行了病原菌分离鉴定.通过组织分离获得8个在培养性状和分生孢子形态大小均一致且均具有致病性的分离株,8个分离株在PDA平板上菌落初为白色,后变为灰色至深灰色,菌落中央产生橘红色黏质分生孢子团.分生孢子无色,长椭圆形至纺锤形,单胞,大小为(15.6~18.0)μm×(3.6~6.0)μm.对其中之一的分离株YRRC-1进行rDNA-ITS区段扩增和序列测定,获得长度为536 bp的rDNA-ITS序列,该序列与胶孢炭疽菌(Colletotrichum gloeosporioides)的对应序列同源性达100%.根据分离病菌的培养特征、形态大小和序列鉴定结果,认为瑞昌山药炭疽病菌属于胶孢炭疽菌(C. gloeosporioides).     

Comparison of sequences for the internal transcribed spacer region in <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG 1-ID and other subgroups of AG 1

The rDNA-ITS sequence of Rhizoctonia solani AG 1-ID was determined and compared to those of R. solani AG 1-IA, AG 1-IB, and AG 1-IC. The similarity of the isolates from each AG 1 subgroup was almost identical (99%–100%), whereas it was lower between subgroups (91%–95%) than within subgroups. Phylogenetic analysis indicated that isolates of AG 1-ID and other subgroups were separately clustered. Isolates of R. solani AG 1 were clearly separated from R. solani AG 2-1, AG 4, and binucleate Rhizoctonia AG-Bb and AG-K. These results showed that analysis of the rDNA-ITS sequence is an optimal criterion for differentiating R. solani AG 1-ID from other subgroups of R. solani AG 1.     

Comparison of rDNA-ITS Sequences between Potato and Tobacco Strains in <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-3

Genetic diversity among 51 isolates of Rhizoctonia solani AG-3, representing potato and tobacco populations, was inferred from the sequences of the internal transcribed spacer (ITS) and 5.8S ribosomal RNA (rRNA) gene. The 5.8S rDNA sequence was completely conserved not only in AG-3, but across all the AG isolates examined, whereas the rDNA-ITS sequence was found to be variable among the isolates. The nucleotide sequence similarity in the ITS 1 region was high (96-100%) for isolates within each of the two populations, but was 91-92% for isolates from different populations. The AG-3 isolates had 56 to 91% sequence similarities in the ITS 1 region with R. solani isolates of the other AGs. Phylogenetic analysis based on the ITS-5.8S rDNA sequence data indicated that the different populations in AG-3 are distantly related to each other. Genetic divergence between the two populations was also supported by the results of DNA-DNA hybridization studies. This study suggests that AG-3 consists of two genetically isolated groups corresponding to separate subgroups: AG-3 PT (potato type) and AG-3 TB (tobacco type). Specific primer sets for the detection of the two AG-3 subgroups were developed from the aligned rDNA-ITS sequences. Received 22 April 1999/ Accepted in revised form 2 July 1999     

栀子叶斑病及其病原的鉴定

首次报道在栀子(Gardenia jasminoides)上存在由拟盘多毛孢属真菌引致的叶斑病。从湖北省长阳县采集到栀子叶斑病病害标本,并利用柯赫氏法则证实该病由真菌GJ-1菌株所引致;根据GJ-1菌株的形态学特征和ITS序列的分析、比较,证实GJ-1菌株属于拟盘多毛孢属(PestalotiopsisSteyaert)真菌;GJ-1菌株的ITS序列与P.gracilis的ITS同源性达100%,因此推定它属于P.gracilis。