不同核盘菌菌株及其近缘种的RAPD分析

 本文利用随机引物扩增多态性DNA (RAPD)技术分析了7个生物学性状差异较大核盘菌(Sclerotinia sclerotiorum)的遗传多样性,并同三叶草核盘菌(S.trifoliorum)、小核盘菌(S.minor)的代表性菌株和莴苣上的一种产菌核病原菌的代表菌株Let-19进行了比较。结果表明40个引物中8个引物能稳定地从供试菌株中扩增出多态性DNA片段。通过分析这些多态性片段可以看出7个供试核盘菌菌株之间的遗传相似系数变化幅度为0.505 2~0.793 1,而核盘菌、三叶草核盘菌,小核盘菌和Let-19之间的遗传相似系数的变幅则为0.194 2~0.385 3。莴苣上的菌株Let-19的RA PD图谱同供试其它种的菌株既存在明显差异的DNA片段电泳带,又显示出一些位置一致的DNA片段电泳带。因而Let-19同核盘菌属真菌的亲缘关系较近。供试的引物中OPL14既能介导从供试的7个核盘菌菌株和3个近缘种的3个菌株的DNA样品中扩增出相同的DNA片段,又能扩增出种或菌株特异性DNA片段。因而RAPD技术适于研究核盘菌的遗传多样性及分析核盘菌属真菌的亲缘关系。     

Phylogenetic relationship and genetic diversity of <Emphasis Type="Italic">Agrobacterium</Emphasis> spp. isolated in Poland based on <Emphasis Type="Italic">gyrB</Emphasis> gene sequence analysis and RAPD

The genetic diversity of 47 strains of Agrobacterium originating from different host plants and geographical locations in Poland, together with 12 strains from other countries was investigated. It was analyzed using RFLP of DNA fragment amplified with primers UP-1 and UP-2r flanking part of gyrB and parE genes, gyrB sequencing and randomly amplified polymorphic DNA (RAPD) technique. On the basis of obtained results, we found the majority of agrobacteria isolated in Poland belong to biovar 2. However, among others, three strains distinct from type strains of all the known Agrobacterium species, were discovered. All three methods showed no correlation between genetic diversity and geographical origin or the host plant of all studied strains but they revealed high diversity of the tested agrobacteria. The highest diversity was observed within strains of biovar 1, whereas those of biovar 2 were found to be the more homogenous group. The topology of the constructed gyrB tree corresponds to topologies of 16S and 23S rDNA trees obtained in this and other studies, but the gyrB tree had deeper branching. In the case of RAPD, it was possible to find a unique DNA fingerprint for almost each strain tested. The gyrB gene appeared to be a good phylogenetic marker with high discrimination power allowing better differentiation between species and strains, whereas the RAPD technique can serve as a tool for single strain typing.     

河南省小麦黑胚病优势病原菌链格孢菌(Alternaria spp.)遗传多样性分析

 采用RAPD技术对分离自河南省各地的43株小麦黑胚病优势病原菌(Alternaria spp.)菌株进行了遗传多样性分析。17个随机引物共扩增出151条清晰的DNA条带,所扩增出的DNA条带均为多态带,说明河南省小麦黑胚病菌存在着丰富的遗传多样性。利用NTSYS软件进行了病原菌的聚类分析,结果表明,河南省小麦黑胚病菌主要有2个种,即Alternaria alternata和A. tenuissima,种间的遗传相似系数的变化幅度为0.62~0.92。来自同一个地区的小麦黑胚病菌菌株基本上聚在了一起,表现出很近的亲缘关系,来自不同地区之间的菌株也可以交叉聚类。病原菌遗传多样性分析进一步验证了形态学的鉴定结果。     

Phylogenetic Relationships Among Global Populations of Pseudomonas syringae pv. actinidiae

ABSTRACT Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit (Actinidia spp.) vines, was first detected in Japan in 1984, followed by detections in Korea and Italy in the early 1990s. Isolates causing more severe disease symptoms have recently been detected in several countries with a wide global distribution, including Italy, New Zealand, and China. In order to characterize P. syringae pv. actinidiae populations globally, a representative set of 40 isolates from New Zealand, Italy, Japan, South Korea, Australia, and Chile were selected for extensive genetic analysis. Multilocus sequence analysis (MLSA) of housekeeping, type III effector and phytotoxin genes was used to elucidate the phylogenetic relationships between P. syringae pv. actinidiae isolates worldwide. Four additional isolates, including one from China, for which shotgun sequence of the whole genome was available, were included in phylogenetic analyses. It is shown that at least four P. syringae pv. actinidiae MLSA groups are present globally, and that marker sets with differing evolutionary trajectories (conserved housekeeping and rapidly evolving effector genes) readily differentiate all four groups. The MLSA group designated here as Psa3 is the strain causing secondary symptoms such as formation of cankers, production of exudates, and cane and shoot dieback on some kiwifruit orchards in Italy and New Zealand. It is shown that isolates from Chile also belong to this MLSA group. MLSA group Psa4, detected in isolates collected in New Zealand and Australia, has not been previously described. P. syringae pv. actinidiae has an extensive global distribution yet the isolates causing widespread losses to the kiwifruit industry can all be traced to a single MLSA group, Psa3.     

Molecular Variability Within Diaporthe/Phomopsis helianthi from France

ABSTRACT Diaporthe/Phomopsis helianthi causes brown stem canker of sunflower (Helianthus annuus) and is responsible for considerable yield loss. This species shows considerable variation for morphological characters, growth, and pathogenicity. Molecular variability of two sample groups was assessed with amplified fragment length polymorphism (AFLP) markers. Isolates of the first sample were collected from infected sunflower tissues from the main regions in France where the crop is grown, whereas isolates from the second sample came from stems within a single field of sunflower. A soybean strain was taken as an outgroup for AFLP analyses. Within sample one, the greatest genetic distance among isolates was 0.97, whereas it was 0.44 within sample two isolates. For the whole of France, the average genetic distance was 0.68, whereas in the one field it was 0.12. Nei's genetic diversity indices were 0.20 and 0.06 for France and for one field, respectively. The greatest genetic distance was found between isolates from the most northern crops. The greatest genetic distance between D. helianthi isolates and the strain isolated from soybean was similar to that observed for D. helianthi isolates from different geographical areas. The problems in defining the genus Phomopsis are discussed. It is shown that internal transcribed spacer sequencing could be a useful criteria for Diaporthe/Phomopsis species determination. The considerable genetic variability of the pathogen could lead to the occurrence of new strains that could be more aggressive or more resistant to chemical control.     

基于全基因序列的中国北方3省(区)马铃薯Y病毒遗传多样性分析

本研究以马铃薯Y病毒(PVY)全基因组为基础,分析吉林、黑龙江和内蒙古3省(区)PVY群体遗传多样性和群体分化,并评估突变、重组、选择等遗传力所起的作用。根据已报道的PVY全基因序列保守区设计4对引物,采用片段重叠法对来自内蒙古和吉林的24个PVY分离物全基因序列进行测定,并联合NCBI中已登录的9个黑龙江分离物全基因组序列进行遗传多样性参数评估、群体分化检验和分子变异等分析。结果显示,我国北方3省(区)PVY群体遗传多样性高,其中内蒙古和黑龙江PVY群体遗传多样性高于吉林群体,并且3个群体之间呈现一定程度的遗传分化。分子变异分析发现在PVY基因组中存在1 786个变异位点,表明我国北方3省(区)PVY群体变异程度较高,并且这种高变异度有85.54%来自各个马铃薯种植区内PVY个体的遗传变异。重组分析和系统发育分析发现,我国北方3省(区)PVY群体中重组株系占比高达90.3%,并具有明显的株系多样性,表明PVY重组株系已成为我国北方3省(区)马铃薯种植区的流行株系。选择压力分析显示,使用FEL和IFEL法分别检测出501个和315个净化压力选择位点,这表明3省(区)PVY群体受净化选择压力为主。以上结果表明,中国北方3省(区)PVY群体遗传多样性高,突变、重组和自然选择都对遗传多样性和群体分化存在一定影响。     

Evaluation of the Genetic Diversity of Xylella fastidiosa Strains from Citrus and Coffee Hosts by Single-Nucleotide Polymorphism Markers

ABSTRACT The aim of this study was to obtain information about genetic diversity and make some inferences about the relationship of 27 strains of Xylella fastidiosa from different hosts and distinct geographical areas. Single-nucleotide polymorphism (SNP) molecular markers were identified in DNA sequences from 16 distinct regions of the genome of 24 strains of X. fastidiosa from coffee and citrus plants. Among the Brazilian strains, coffee-dependent strains have a greater number of SNPs (10 to 24 SNPs) than the citrus-based strains (2 to 12 SNPs); all the strains were compared with the sequenced strain 9a5c. The identified SNP markers were able to distinguish, for the first time, strains from citrus plants and coffee and showed that strains from coffee present higher genetic diversity than the others. These markers also have proven to be efficient for discriminating strains from the same host obtained from different geographic regions. X. fastidiosa, the causal agent of citrus variegated chlorosis, possesses genetic diversity, and the SNP markers were highly efficient for discriminating genetically close organisms.     

Use of random amplified polymorphic DNA (RAPD) to identify races 1, 2, 4 and 8 of Fusarium oxysporum f. sp. dianthi in Italy

The RAPD fingerprinting procedure was used in combination with pathogenicity assays on differential cultivars to characterize a representative collection of 72 Fusarium spp. isolates of different geographic origin collected from diseased carnation. In F. oxysporum f. sp. dianthi, isolates were grouped according to the physiologic race: group 1 included isolates of race 4; group 2 was formed by isolates of race 2 and single representatives of races 5 and 6; group 3 included isolates of races 1 and 8. No correlation was found between RAPD data and geographic origin of the isolates tested: representatives of race 2 isolated in Italy, Israel and Japan had the same amplification profile. Three isolates which showed a low level of pathogenicity on all carnation cultivars tested shared an identical amplification pattern and are probably saprophytic F. oxysporum. Finally, two F. redolens isolates from Japan and seven non-pathogenic isolates of F. proliferatum collected from diseased carnation in Italy, Israel and The Netherlands were clearly distinguishable according to their RAPD fingerprint. The results are discussed in relation to previous studies on the genetic diversity of F. oxysporum f. sp. dianthi and to the development of forma specialis- and pathotype-specific diagnostic tools.     

玉米新月弯孢菌(Curvularia lunata)的RAPD分析

 对分离自玉米弯孢菌叶斑病标样中的77株新月弯孢菌和1株来自水稻的新月弯孢菌进行RAPD分析表明,菌株间具有丰富的遗传多样性,在相似系数约0.60处,所有菌株被聚为3个组,但88.0%的菌株聚入第Ⅰ组内,其余菌株被聚入另外2个组内。第Ⅰ组内共有69个菌株,包含来自不同区域的致病性较强的菌株,是玉米弯孢菌叶斑病的优势类群,其余2个类群主要是一些致病能力较弱或不致病的菌株。结果表明,新月弯孢菌种内菌株的遗传多样性与致病性相关,但与菌株地理来源无明显的直接关系。     

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。     

广东果树上17种拟茎点霉的RAPD分析

 自320个随机引物中筛选出适于拟茎点霉属真菌种间亲缘关系分析的15个随机引物,并优化了RAPD分析的扩增体系,在此基础上,对广东果树上17种拟茎点霉进行了RAPD分析。各菌株间的Nei相似系数UPGMA法聚类结果表明:来源于不同地区的2个Phomopsis mangiferae Ahmad菌株和2个P.macadami Z.D.Jiang et P.K.Chi菌株都分别以0.636和0.589的相似系数两两首先聚在一起,而不同的种则只在小于0.54的相似系数范围内聚类,体现了种间及种内的亲缘关系差异程度;聚类群与寄主植物不具相关性,同种植物上的不同拟茎点霉,即使是分自相同寄生部位也不能聚在一类;支持形态学上将生于柑桔枝和黄皮茎、沙梨叶和果、杨梅叶和枝以及同是生于龙眼叶的共8个拟茎点霉分别鉴定为不同的种,而不支持将P. cytosporella Penz.et Sacc.与P. mangiferae合并为一个种的观点;RAPD技术可作为拟茎点霉属真菌种间的亲缘关系分析的重要手段。     

Genetic Structure of Mycosphaerella populorum (Anamorph Septoria musiva) Populations in North-Central and Northeastern North America

ABSTRACT In order to characterize the genetic variation of the poplar pathogen Mycosphaerella populorum (anamorph Septoria musiva), we have studied seven North American populations using the polymerase chain reaction random amplified polymorphic DNA (RAPD) technique. The fungal populations were sampled in 2001 and 2002 by obtaining 352 isolates from cankers and leaf spots in hybrid poplar plantations and adjacent eastern cottonwood (Populus deltoides). A total of 21 polymorphic RAPD markers were obtained with the six RAPD primers used. A fine-level scale analysis of the genetic structure within the populations revealed that subpopulations sampled on P. deltoides and on hybrid trees were not significantly differentiated. In contrast, analyses performed on the entire data set showed high levels of haplotypic diversity and moderate to high genetic differentiation, with 20% of the expected genetic diversity found at the interpopulation level. Moreover, a high and significant correlation between genetic and geographic distances among populations was found, suggesting isolation by distance of the sampled populations. Although the occurrence of the sexual stage of this fungus remained unclear in field populations, five of the six populations were at gametic equilibrium for RAPD loci, suggesting the occurrence of recombination episodes in Septoria musiva populations. Overall, S. musiva appears to consist of differentiated subpopulations, with both asexual and sexual recombination contributing to the local level of genetic structure.     

Comparison of RAPD and AFLP Marker Analysis as a Means to Study the Genetic Structure of Botrytis cinerea Populations

To assess the genetic relationships of Botrytis cinerea populations in Almería (Spain), 44 isolates of B. cinerea, collected from six commercial greenhouses (subpopulations), were analysed by Random Amplified Polymorphic DNA (RAPD) and amplified-fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per primer with AFLP than with RAPD (16 compared to 4). However, RAPD detected polymorphisms more frequently per loci than AFLP (56% compared to 32%). The analysis of population structure revealed that the genetic diversity within subpopulations (H_S) accounted for 96% of the total genetic diversity (H_T) , while genetic diversity among subpopulations represented only 4% of the total diversity, independently of whether they were analysed with RAPD or AFLP markers. The relative magnitude of gene differentiation between subpopulations (G_ST) and the estimate of the number of migrants per generation (N_m) averaged similar values when estimated with RAPD or AFLP markers (0.039 and 0.036, or 12.32 and 13.39, respectively). The results obtained in dendrograms were in accordance with the gene diversity analysis. However, the diversity of B. cinerea was higher when analysed by RAPD than with AFLP. In these cases, the isolates could not be grouped by greenhouse or fungicide resistance (except those sensitive to carbendazim and resistant to procymidone). Both the RAPD and AFLP technologies are suitable for studies of genetic structure of B. cinerea populations, although RAPD generated more polymorphisms per loci than AFLP, and provided a better explanation of the genetic relationships between isolates.     

Biological and molecular characterization of a new cucurbit-infecting tobamovirus

ABSTRACT An uncharacterized virus was isolated from greenhouse-grown cucumber plants. Biological and serological data described in the present study indicated that the virus belonged in the genus Tobamovirus. The host range of the virus included several plant species within the family Cucurbitaceae. The virus designated Cucumber fruit mottle mosaic virus (CFMMV) causes severe mottling or mosaic on cucumber fruits, and its fast spread within greenhouses could lead to significant economic losses in cucumber crops. The genome of CFMMV has been completely sequenced and its genome organization was typical of a Tobamovirus. However, its sequence was distinct from other described viruses within the group of cucurbit-infecting Tobamoviruses. Comparisons of sequences and phylogenetic analysis suggested that the cucurbit-infecting Tobamoviruses be separated into two subgroups: subgroup I comprising the strains and isolates referred to in the literature as Cucumber green mottle mosaic virus (CGMMV) (CV3, CV4, CGMMV-W, CGMMV-SH, and CGMMV-Is) and subgroup II comprising CFMMV, Kyuri green mottle mosaic virus (KGMMV), and the Yodo strain of CGMMV, which is closely related to KGMMV and may be considered a strain of it.     

Genetic Diversity and Pathogenic Variation of Colletotrichum lindemuthianum in the Three Centers of Diversity of Its Host, Phaseolus vulgaris

ABSTRACT Population subdivision of Colletotrichum lindemuthianum, the causal agent of anthracnose, was studied in three regions located in three centers of diversity of its host, Phaseolus vulgaris. Random amplified polymorphic DNA (RAPD) markers, restriction endonuclease analysis of the amplified ribosomal internal transcribed spacer region, and virulence on a set of 12 cultivars were used to assess the genetic diversity of C. lindemuthianum strains isolated in Mexican, Ecuadorian, and Argentinean wild common bean populations. The three regions were significantly differentiated for molecular markers. For these markers, Mexico was the most polymorphic and the most distant from Ecuador and Argentina. The majority of the RAPD alleles present in Ecuador and Argentina were found in Mexico, suggesting that Andean populations have been derived from the Mesoamerican center. Pathogenicity tests on a set of 12 cultivars showed that all but one of the Mexican strains were virulent exclusively on Mesoamerican cultivars. Argentinean strains were virulent preferentially on southern Andes cultivars, and the Ecuadorian strains, except for one strain, were avirulent on all cultivars. These results suggest an adaptation of strains on cultivars of the same geographic origin. Thus, based on molecular and virulence markers, C. lindemuthianum strains isolated from wild common bean populations were divided into three groups corresponding to host gene pools.     

Genetic fingerprinting of Iranian Xanthomonas campestris pv. campestris strains inducing black rot disease of crucifers

Northern Iran has one of the largest and most diverse populations of cultivated crucifers in Iran. Symptoms of black rot disease were observed in 40 % of fields. To assess the genetic diversity of Xanthomonas campestris pv. campestris (Xcc) strains, associated with black rot disease, 40 strains were isolated from infected samples of crucifers such as cabbage, radish, cauliflower, turnip and kohlrabi, and were collected from different geographic regions of northern Iran including West and East Azarbayjan and Ardabil provinces. Bacterial strains were characterized by their morphological, biochemical and physiological features and pathogenicity tests. Four races were found in northern Iran (1, 4, 5 and 6) and the majority of the tested strains belonged to either race 4 (45 %) or race 6 (20 %). To examine the distribution of dispersed repetitive DNA, Enterobacterial Repetitive Intergenic Consensus (ERIC), BOX, Repetitive Extragenic Palindromic (REP) and random amplified polymorphic DNA (RAPD) sequences in the genome of Xcc using conserved primers. The different markers produced characteristic banding patterns and the similarity matrices from binary banding data was derived with the similarity for qualitative data program (SIMQUAL). On the basis of the fingerprint patterns generated by the combination data set of both rep-PCR and RAPD, the Xcc strains were differentiated into seven clusters (A–G) at 76 % similarity level. The geographical origin of the Iranian strains does not seem to be correlated with the RAPD and rep-PCR clusters. The clusters seem to be more related to the race of the strains. This is the first study on genetic diversity of Xcc strains inducing black rot disease of crucifers in Iran.     

Phylogenetic analysis of Trypanosomatina (Protozoa: Kinetoplastida) based on minicircle conserved regions

Phylogenetic relationships within the suborder Trypanosomatina were inferred from the kinetoplast DNA minicircle conserved region sequences. Trees built using distance-matrix (Neighbor-Joining) and maximum parsimony methods showed that the minicircle conserved regions (CRs) provide a sensitive and specific molecular marker suitable for phylogenetic analyses of subspecies and strains of trypanosomatid flagellates, as testified by the subdivision of the genus Leishmania into the subgenera Leishmania. Viannia and Sauroleishmania. However, since Phytomonas and monogenetic parasites of insects represent the earliest diverging groups, the CRs do not seem to be useful for inference of relationships among major lineages of the order Kinetoplastida.     

我国南方地区主要根结线虫DNA变异的RAPD分析

 本试验用5组随机引物对来自我国南方地区的30个根结线虫种群进行RAPD分析,并从中筛选出多态性较好的引物12个。共扩增出179条DNA多态带,各供试种群间存在着丰富的遗传多态性。扩增结果表现出种间差异大于种内差异的共同趋势,这表明上述12个引物能够较客观地反映种群间亲缘关系的远近。北方根结线虫与另外3种线虫(南方根结线虫、爪哇根结线虫、花生根结线虫)的亲缘关系最远;在3种主要根结线虫中,爪哇根结线虫与南方根结线虫的亲缘关系相对较近。基于种群间的相似系数分析和应用UPGMA法构建的聚类树状图,显示出不同的根结线虫在较低的相似性系数范围聚类,而绝大多数种内的不同种群均以较高的相似性系数聚在一起,这与形态分类基本一致,反映了形态学分类的分子遗传本质,同时也表明了应用RAPD技术进行根结线虫亲缘关系分析和种类鉴定具有合理性和可行性。本文还对RAPD方法对南方根结线虫小种鉴定的可能性进行了初步探讨。