十字花科蔬菜黑斑病菌的PCR鉴定

 在对十字花科蔬菜黑斑病菌(Alternaria sp.)3个种及相近种的5.8SrDNA和其侧翼ITS区进行测序的基础上,分别设计合成了鉴定白菜黑斑病菌3个种的特异性引物。PCR扩增结果表明:Abre1和Abre2引物对能特异性扩增芸苔链格孢(A.brassicae)371bp的片段,Abra1和Abra2引物对能特异性扩增甘蓝链格孢(A.brassicicola)457bp的片段,Ajap1和Ajap2引物对能特异性扩增萝卜链格孢(A.japonica)411bp的片段,而且其它近源种未扩增出目标片段,说明这3个引物对可以作为十字花科蔬菜黑斑病菌3个种快速检测鉴定的分子特征标记。     

甘薯茎线虫rDNA-ITS1区的PCR扩增与序列分析

 利用PCR技术获得了甘薯茎线虫rDNA-ITS1区序列。序列分析表明,采自我国河北、山东、安徽的甘薯茎线虫16个地理种群的ITS1区序列分化为短型(S)和长型(L)2种基因型。山东费县芍药山乡4个地理种群为L型,ITS1区长度为466 bp;河北、安徽和山东费县新庄乡的12个地理种群为S型,ITS1区长度为288 bp。甘薯茎线虫与鳞球茎茎线虫(Ditylenchus dipsaci)的rDNA-ITS1序列同源性为52.0%~52.5%,与腐烂茎线虫(D.destructor)序列同源性为82.0%~85.4%;我国甘薯茎线虫不同地理种群间的序列同源性为96.6%~100.0%。     

香蕉穿孔线虫(Radopholus similis)特异性分子检测技术研究

 香蕉穿孔线虫(Radopholus similis)是一种国际公认的极具毁灭性的有害线虫,也是我国重要的进境检疫性有害生物。利用线虫通用引物对香蕉穿孔线虫7个不同群体的ITS进行PCR扩增、克隆及测序,获得ITS片段序列长度为706bp。经与国外香蕉穿孔线虫种群及近缘种的ITS序列进行比对及同源性分析,构建设计了1对香蕉穿孔线虫的特异性引物RsF1/RsR1,特异性扩增片段长度为271 bp;同时引入D2A/D3B作内标,研究出特异性检测香蕉穿孔线虫的一步双重PCR分子技术和方法,该项检测技术特异性好,耗时较短、操作简便、可靠。     

小麦苗枯病菌的ITS分析及PCR检测

 小麦苗枯病菌(Clavibacter fangii,Cf)是引起小麦细菌性苗枯病的病原,本研究用16S~23S rDNA间的内源转录间隔区(internally transcribed spacer,ITS)序列通用引物L1(5'-AGTCGTAACAAGGTAGCCGT-3')和L2(5'-GTGCCAAGGCATCCACC-3')扩增Cf和其它相关细菌的基因组DNA;并对其PCR产物进行回收、克隆和测序,将所获序列和其它已报道的细菌ITS序列进行多重比较后设计出Cf的特异性引物I1(5'-TGCCAAGTCACACTGAGACGA-3')和I2(5'-CAATGATCTACCACCCTCCGA-3')。此引物可以从Cf中扩增出351bp的特异性片段,而其余参试的21个细菌PCR反应结果均为阴性。该方法可以应用于小麦苗枯病菌的快速、可靠检测。此外,本研究对多种植物病原棒形杆菌的ITS序列进行比较研究,发现其具有一定的分类意义。     

马铃薯腐烂茎线虫特异性分子检测技术研究

 本研究利用通用引物(rDNA1/rDNA2)研究了21个国内甘薯茎线虫(Ditylenchus destructor)群体和1个韩国马铃薯茎线虫(D.destructor)群体的rDNA-ITS序列,从21个国内群体中扩增出2个大小不同的ITS片段,分别约为940bp和1100bp;经克隆、序列测定和分析比对发现其ITS区存在特异性差异,分别命名为A型和B型,其中18个群体DdTH、DdCL、DdJN、DdMY1、DdYX1、DdZZ、DdLN,DdDX1、DdFN,DdYX2、DDSX1、DdDX2、DdXY,DdLL、DdSX2、DdLY,DdMY2和DdPY的ITS扩增产物约为940bp,称之为A型马铃薯腐烂茎线虫(940bp),3个群体DdSH,DdTS,DdYS为B型马铃薯腐烂茎线虫(1100bp)。设计构建并筛选出A型和B型马铃薯腐烂茎线虫2对特异性引物DdS1/DdS2和DdL1/DdL2,分别扩增出A型马铃薯腐烂茎线虫、B型马铃薯腐烂茎线虫群体的特异片段252bp和485bp;引入D3A/D3B作为内标,设计出一步双重PCR检测技术;同时优化了检测体系和PCR反应程序。该技术具有较高的特异性和灵敏性,能快速、准确地检测出不同型的马铃薯腐烂茎线虫群体。     

河南郑州小麦禾谷孢囊线虫(Heterodera avenae)的核糖体基因ITS序列和RFLP分析

 采用PCR技术对河南郑州禾谷孢囊线虫群体的核糖体基因(ribosomal DNA,rDNA)内转录间隔区(Internal Transcribed Spacers,ITS)进行扩增,获得片段长度约为1040bp。利用UPGMA方法分析了河南郑州禾谷孢囊线虫群体与近缘种的系统发育关系,结果表明:中国Heterodera avenae群体,H.australis和H.pratensis亲缘关系很近。8种限制性内切酶(Restriction Enzyme,RE)酶切禾谷孢囊线虫ITS的扩增产物,其中HindⅢ、AvaⅠ不能酶切PCR产物;Alu Ⅰ酶切PCR产物,获得560bp和480bp2个片段;RsaⅠ和Hinf Ⅱ酶切后分别得到3个片段(700、320、20bp和820、180、40bp);CfoⅠ是3个酶切位点(740、150、110、40bp);HaeⅢ和MvaⅠ能分别清晰地观察到3个片段(420、350、180bp和400、340、280bp),但有微小片段无法清晰观察到。9个种群所得RFLP图谱一致,说明郑州禾谷孢囊线虫群体可能是同一种群且不同于欧洲群体(typeA)和印度群体(typeB)的C型。     

进口美国苹果上拟盘多毛孢菌的鉴定

从进口的美国华盛顿州红帅(reddelicious)苹果中分离到一种引起苹果果实腐烂的病原真菌,根据该真菌的菌落及分生孢子的形态特征,以及该真菌DNA的ITS序列,将其鉴定为拟盘多毛孢菌(Pestaloti-opsissp.)。该真菌菌丝生长和孢子萌发的最适温度是25℃。分生孢子长梭形直立或稍弯曲,15·6~22·2(18·8)μm×4·8~7·1(5·7)μm,5个细胞,4个隔膜均为真隔膜,中间3个细胞暗褐色,两端细胞淡色,3个有色细胞中第1、2个有色细胞颜色较深,茶褐色;第3个有色细胞淡褐色;分隔处稍缢缩;孢子顶端有2~3根不分支的附属丝,长7·0~19·5(11·9)μm,基细胞具短柄,长0·4~3·2(1·4)μm。分生孢子梗较长,呈淡色,不分支,无隔膜。用ITS区通用引物PM1/PM4对该真菌DNA进行PCR扩增和测序,获得了525bp的ITS区序列。     

进境美国大豆中大豆茎褐腐病菌的截获

 从进境的美国大豆豆秆样品中分离到2株疑似大豆茎褐腐病菌Phialophora gregata的分离物247-8和8300-5,2株分离物在PGM培养基上菌落圆形,边缘规则,白色至暗褐色,表面隆起,粗糙,轮纹状。分生孢子卵形至椭圆形,无色,平滑,单胞,平均大小4.3 μm×1.9 μm。分生孢子梗具有瓶梗状结构,无色,无隔膜或有隔膜,大小(5~16)μm×(2~3) μm,呈桶型或长颈瓶型。特异性引物BSRIGS1/2、BSR1/2和Pgl/4分别扩增分离物247-8的DNA得到预期1 022 bp、483 bp和499 bp的产物;分离物8300-5的DNA经PCR扩增分别得到834 bp、483 bp和499 bp的预期条带。2株分离物的ITS区序列完全一致,与GenBank中大豆茎褐腐病菌(登录号AB190396、DQ459387、DQ459386和AF132804)的序列相似性为100%。人工接种大豆幼嫩植株茎基部均产生大豆茎褐腐病菌的典型症状。根据分离物形态特征、PCR检测、序列分析以及致病性测试结果,将进境美国大豆样品中的分离物247-8和8300-5鉴定为大豆茎褐腐病菌P. gregata。     

车前草穗枯病菌分子鉴定

采用真核生物通用引物ITS1/ITS4,对来自江西省吉安县2个车前草穗枯病菌菌株进行rDNA-ITS区段的PCR扩增,对扩增产物进行测序,结果获得2个长度均为579 nt且序列完全一致的rDNA-ITS序列,该序列与GenBank中当归间座壳菌(Diaporthe angelicae)及其无性态拟茎点霉菌(Phomopsis subordinaria)对应序列的同源性高达99.0%,根据以rDNA-ITS序列同源性大小划分种类的标准,将车前草穗枯病菌鉴定为当归间座壳菌。     

小麦禾谷胞囊线虫(Heterodera avenae)的核糖体基因(rDNA)限制性片段长度多态性研究

 采用PCR技术扩增出中国和摩洛哥禾谷胞囊线虫群体的核糖体基因(rDNA)的内转录间隔区(ITS)片段的长度约为1 060 bp。用11种限制性内切酶(RE)酶切禾谷胞囊线虫ITS扩增产物,共产生27个酶切片段。用AluI和RsaI酶切ITS扩增产物证明中国禾谷胞囊线虫ITS属于"B型",而摩洛哥禾谷胞囊线虫ITS属于"A型"。用HinfI酶切后,7个中国禾谷胞囊线虫群体产生2个RFLP片段(860和200 bp),而摩洛哥群体产生3个RFLP片段(520、340和200 bp),HinfI揭示出中国与摩洛哥禾谷胞囊线虫ITS之间存在差异。AvaI和HindⅢ不能酶切禾谷胞囊线虫ITS。用CfoI、Bsh1236I、MsrFI、ScrFI、HaeⅢ和MvaI 6种RE酶切中国和摩洛哥禾谷胞囊线虫群体的rDNA-ITS,均分别得到相同类型的RFLP分布型,因此这6种RE不能揭示中国与摩洛哥群体的rDNA-ITS的差异。     

流动沙漠腹地柽柳叶片的生长规律

研究极端干旱区植物叶片生长特征对筛选防风固沙植物有重要意义。在塔克拉玛干沙漠腹地塔中植物园柽柳专类园区,选择8种不同柽柳,研究了生长季内叶片生长规律。结果显示:1)柽柳属不同种的单叶表面积增长模型较为类似,总体差异不大,但其增长的规律性极强,以y=ax2+bx-c二项式增长模型为主;2)方差分析发现,不同柽柳种间单叶面积差异极显著(F>F0.01);单叶鲜重和干重的差异亦明显;3)柽柳单叶表面积与其干重存在正相关关系,以y=axb的乘幂关系为主。说明塔中沙漠植物园不同柽柳叶面积增长特征有所不同,防护林和固沙树种选择过程中可优先考虑叶面积增长迅速,生物量积累快的柽柳-长穗柽柳和刚毛柽柳。     

瘿蚊科3种昆虫核糖体DNA的克隆及序列分析

瘿蚊科是双翅目昆虫中的重要类群?本研究对瘿蚊科3种昆虫—麦红吸浆虫?菊花瘿蚊和食蚜瘿蚊的核糖体DNA进行PCR扩增?克隆?测序及序列分析, 并对ITS-1在麦红吸浆虫4个地理种群中的遗传变异情况进行分析?结果表明, 从3种昆虫中获得的核糖体DNA序列包括:部分的18S rDNA(44 bp)?28S rDNA(37 bp), 全部的ITS-1(487~535 bp), 5.8S rDNA(121 bp)及ITS-2(336~352 bp)序列?3种昆虫ITS序列的碱基差异百分比在17.21%~29.59%之间, 共含有206个变异位点?ITS-1序列在麦红吸浆虫4个地理种群中比较保守, 只有4个变异位点, 单倍型多样性为0.311 7~0.796 5, 核苷酸多样性为0.000 6~0.002 2?本研究为今后瘿蚊科昆虫的分类鉴定?系统发育和遗传进化等相关研究提供了基础?     

Intraspecific Relationship of Plasmopara halstedii Isolates Differing in Pathogenicity and Geographic Origin Based on ITS Sequence Data

Sequence parts of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were analysed to screen for the intraspecific variability of a non-coding genomic region in 15 Plasmopara halstedii populations of different pathotype and geographic origin. Samples revealed uniformity in a ca. 790 Bp fragment comprising of the ITS-1, 5.8S and front parts of the ITS-2. In contrast, clear differences were found in a ca. 810 Bp fragment of the ITS-2 thus allowing differentiation between populations of pathotype 100, 310 and 330 and a group of populations representing pathotypes 700, 701, 703, 710 and 730. Samples of pathotypes 700 to730 originated from Slovakia, France, and Germany, but were uniform in both ITS sequence parts, thus indicating very recent origin of these highly aggressive physiological races. The potential use of ITS sequences for pathotype differentiation and phylogenetic studies in P. halstedii is discussed.     

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     

Internal Transcribed Spacer Regions 1 and 2 and Random Amplified Polymorphic DNA Analysis of Didymella bryoniae and Related Phoma Species Isolated from Cucurbits

ABSTRACT Didymella bryoniae (anamorph Phoma cucurbitacearum) is the causal agent of gummy stem blight, although other Phoma species are often isolated from cucurbit plants exhibiting symptoms of the disease. The molecular and phylogenetic relationships between D. bryoniae and these Phoma species are unknown. Isolates of D. bryoniae and Phoma obtained from cucurbits grown at various geographical locations in the United States were subjected to random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS) sequence analysis (ITS-1 and ITS-2) to determine the molecular and phylogenetic relationships within and between these fungi. Using RAPD fingerprinting, 59 isolates were placed into four phylogenetic groups, designated RAPD group (RG) I, RG II, RG III, and RG IV. D. bryoniae isolates clustered in either RG I (33 isolates), RG II (12 isolates), or RG IV (one isolate), whereas all 13 Phoma isolates clustered to RG III. There was greater than 99% sequence identity in the ITS-1 and ITS-2 regions between isolates in RG I and RG II, whereas isolates in RG III, P. medicaginis ATCC 64481, and P. exigua ATCC 14728 clustered separately. On muskmelon seedlings, a subset of RG I isolates were highly virulent (mean disease severity was 71%), RG II and RG IV isolates were slightly virulent (mean disease severity was 4%), and RG III isolates were nonpathogenic (disease severity was 0% for all isolates). The ITS sequences indicate that RG I and RG II are both D. bryoniae, but RAPD fingerprints and pathogenicity indicate that they represent two different molecular and virulence subgroups.     

柽柳属植物镜下器官特征描述及分类学意义

新疆柽柳属植物为新疆重要的固沙植物,有１８种１变种。面积大（５５３万ｈｍ＾２）,分布广,由于花器小不易观察,种间易杂交,使本属植物成为有花植物中分类最为困难的属之一。为精确分类,为该属植物分类提供解剖学依据,本实验将新疆的１４种柽柳植物的叶、花、种子等器官在解剖镜下放大二十倍,进行观察描述,并对关键特征拍照。总结出结构典型、特征稳定、专属性强的识别特征,且易掌握、好记忆,使种间性状的相识性明显区分     

中亚荒漠生态系统中的关键种——柽柳（Tamarix spp.）

生物多样性是一个综合的具有复杂相互关系的概念，研究内容极其多种多样。不同生态系统关键种（Ｋｅｙｓｔｏｎ ｓｐｅｃｉｅｓ）的确定及其生态作用与经济开发的研究是生物多样性研究的主要方向之一。本文从柽柳在荒漠生态系统中的生态学地位和对分布区人群经济生活中的重要作用角度，说明这类植物是干旱荒漠区生态系统中的关键种之一，在生物多样性保护中要给予更多的关注。     

Exceptional length of ITS in <Emphasis Type="Italic">Plasmopara halstedii</Emphasis> is due to multiple repetitions in the ITS-2 region

Plasmopara halstedii, cause of downy mildew of sunflower, is a pathogen of worldwide economic importance. Efforts to amplify the ITS-region from this organism revealed an unexpected fragment length of about 2600 bp, in contrast to about 900 bp, reported for other members of the Peronosporaceae. First attempts to obtain the complete sequence of the P. halstedii fragment were unsuccessful, due to repeated elements in ITS, which were uncovered later on. The presence of a single EcoRI-site allowed us to apply a restriction-ligation procedure to amplify parts of the ITS fragment separately. Sequencing of these fragments revealed the presence of four copies of a tandemly arranged repetitive element in the ITS-2 region. The complete sequence was obtained by using a sequencing primer which annealed shortly before the repetitions so covering the gap in the sequence around the restriction site. The ITS sequence in P. halstedii (AY773346) consisted of 2587 bp in total, with ITS-2 accounting for 2212 bp alone. This is the longest ITS-2 sequence reported so far for any examined species.     

Genetic Diversity Within Colletotrichum acutatum sensu Simmonds

ABSTRACT Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1-5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.