Genetic diversity of Melon necrotic spot virus and Olpidium isolates from different origins

The geographic incidence, genetic diversity and phylogenetic relationships of Melon necrotic spot virus (MNSV) and Olpidium isolates were studied in three cucurbit species from several Latin American and European countries on different collecting dates. Of the 112 cucurbit samples analysed, 69 from Guatemala, Honduras, Mexico, Panama and Spain were DAS‐ELISA‐positive for MNSV. Olpidium bornovanus and O. virulentus infections, and MNSV infections mixed with these Olpidium species, were observed for all these countries. Twenty‐nine MNSV isolates from all the origins where the virus was detected were selected and amplified by RT‐PCR. The resulting RT‐PCR of the p29, p89, p7A, p7B and p42 proteins was used to estimate the genetic diversity and the phylogenetic relationships of the MNSV population. The sequences obtained in this study were compared with the MNSV sequences of the NCBI database, and three groups were recovered by nucleotide composition according to geographical origins: the EU‐LA genotype group (with two subgroups: EU and LA, European and Latin American isolates, respectively), the JP melon genotype group (Japanese melon reference isolates) and the JP watermelon genotype group (Japanese watermelon reference isolates). The genetic diversity in the entire p7A and p7B proteins of MNSV suggests that these coding regions are under strong selective pressure. Additionally, the rDNA‐ITS region was analysed in 40 O. bornovanus and O. virulentus isolates associated with each geographical location and host examined. Phylogenetic analysis showed two groups for each Olpidium species, and these groupings were related to the host from which they were originally isolated.     

Virus transmission by host-specific strains ofOlpidium bornovanus andOlpidium brassicae

Zoospores of 12 isolatesO. bornovanus from geographically diverse sites and representing the three host specific cucurbit strains were tested as vectors for seven viruses using watermelon bait plants and the in vitro acquisition method. All isolates of the cucumber, melon, and squash strains transmitted melon necrotic spot carmovirus (MNSV) and cucumber necrosis tombusvirus (CNV) but none transmitted petunia asteroid mosaic tombusvirus (PAMV) or tobacco necrosis necrovirus (TNV). The isolates varied as vectors of three other carmoviruses: cucumber leaf spot virus (CLSV); cucumber soil borne virus (CSBV); and squash necrosis virus (SqNV). All cucumber isolates transmitted CLSV and SqNV but not CSBV. Some of the melon isolates transmitted CLSV and SqNV but none transmitted CSBV. Two squash isolates transmitted CSBV and SqNV but not CLSV. Two isolates ofO. brassicae transmitted only TNV and a third did not transmit any of the viruses. The species of bait plant sometimes affected transmission. The most efficient vector strains ofO. bornovanus, as determined by reducing zoospores and virus in the inoculum, were the cucumber strain for CLSV; the cucumber strain for CNV if cucumber was the bait plant or melon strain if watermelon was the bait plant; and the squash strain for SqNV. The plurivorous strain ofO. brassicae was the most efficient vector of TNV.Olpidium bornovanus is the first vector reported for CSBV and is confirmed as a vector of SqNV. It is proposed that all carmoviruses may have fungal vectors.Ligniera sp. did not transmit any of the viruses in one attempt.Abbreviations CLSV cucumber leaf spot virus - CNV cucumber necrosis virus - CSBV cucumber soil borne virus - MNSV melon necrotic spot virus - PAMV petunia asteroid mosaic virus - SqNV squash necrosis virus - TNV tobacco necrosis virus - TBSV tomato bushy stunt virus     

Amino acid substitution in the coat protein of Melon necrotic spot virus causes loss of binding to the surface of Olpidium bornovanus zoospores

Melon necrotic spot virus (MNSV) is transmitted by the fungus Olpidium bornovanus. In this study, we used immunofluorescence microscopy to detect MNSV particles over the entire surface of the O. bornovanus zoospore; MNSV particles were not detected on the related fungus O. virulentus, which cannot transmit MNSV. The amino acid substitution Ile → Phe at position 300 in the MNSV coat protein resulted in loss of both specific binding and fungal transmission, while virion assembly and biological aspects were unaffected. Taken together, these results suggest that the MNSV coat protein acts as a ligand to the O. bornovanus zoospore as part of a fungal-vector transmission system.     

Multigene phylogenetic analysis of inter- and intraspecific relationships in <Emphasis Type="Italic">Venturia nashicola</Emphasis> and <Emphasis Type="Italic">V. pirina</Emphasis>

Isolates of Venturia species isolated from pear in Japan, China, Taiwan and Israel were used in this study to analyze their molecular phylogenetic relationship. The nucleotides of rDNA-ITS, partial β-tubulin and elongation factor 1α genes were sequenced directly after PCR. Based on these sequence data two phylogenetic groups could be distinguished. Isolates collected from Asian pears such as Japanese and Chinese pears formed a distinct evolutionary lineage from those derived from European and Syrian pears. This result corroborated the early taxonomic separation of V. nashicola from V. pirina. In addition, trees from single-locus data sets and the combined data set showed that all isolates of V. nashicola were included in a monophyletic group and representative isolates of five pathological races originating from different locations and cultivars formed a single lineage. In contrast, two distinct evolutionary lineages were revealed in V. pirina and isolates of five races were scattered in two lineages. Israeli isolates of race 2 as well as two Japanese isolates of V. pirina formed a distinct lineage from other isolates of this species, while other Israeli isolates belonging to races 1, 3, 4, and 5 were closely related to each other and formed another lineage. It was indicated that the evolution of pathological races in V. nashicola might have occurred relatively recently as compared with V. pirina.     

Molecular Phylogenetic Analysis of Ribosomal DNA Internal Transcribed Spacer Regions and Comparison of Fertility in <Emphasis Type="Italic">Phomopsis </Emphasis>Isolates from Fruit Trees

Sequences of the ribosomal DNA (rDNA) internal transcribed spacer (ITS) regions were examined to infer a molecular phylogeny of small-spored Phomopsis isolates, designated W-type (mainly white colony, weakly virulent, bearing both alpha and beta conidia at 25°C on PDA) and G-type (mainly gray colony, highly virulent, bearing only alpha conidia at 25°C on PDA), and P. amygdali from fruit trees. Phomopsis G-type and P. amygdali were a monophyletic group distinct from the W-type. The W-type isolates were divided into two monophyletic groups. Diaporthe citri, D. tanakae, P. asparagi, P. viticola, P. vitimegaspora and D. nomurai, which are morphologically distinguishable from W- and G-types, differed from the W- and G-types in molecular phylogenetic analyses. PCR-RFLP analysis of rDNA ITS regions was useful to distinguish each of the Phomopsis species and groups using three restriction enzymes. In mating tests, W-type isolates from fruit trees were heterothallic and inter-fertile even between isolates belonging to the different monophyletic groups. Isolates of the G-type and P. amygdali collected in Japan were cross-fertile. Some isolates from Lunaria annua, Ulmus glabra and Juglans regia belonged to one of the two monophyletic groups of the W-type and were cross-fertile with W-type isolates from Rosaceous fruit trees. Received 27 September 1999/ Accepted in revised form 27 January 2000     

Genetic diversity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan based on phylogenetic analyses of rDNA-IGS and <Emphasis Type="Italic">MAT1</Emphasis> sequences

Twenty-eight isolates of Fusarium oxysporum f. sp. spinaciae (FOS; the causal agent of spinach wilt) collected from Japan were assessed for mating type and subjected to phylogenetic analysis. Mating type analysis revealed all isolates to be MAT1-2, suggesting that there is no sexual recombination within the population. Phylogenetic analyses based on nucleotide sequences of the ribosomal DNA intergenic spacer (IGS) and the mating type locus (MAT1) suggested that FOS is polyphyletic. The cluster analysis based on IGS showed four phylogenetic groups (S1–S4) among the isolates. Two distinct lineages, S1 and S3, included FOS isolates both of the vegetative compatibility group (VCG) types, 0330 and 0331, demonstrating that VCG differentiation in FOS may not necessarily reflect the phylogenetic relationships based on IGS and MAT1-2-1.     

Effect of soil matric water potentials on germination of ascospores of Monosporascus cannonballus and colonization of melon roots by zoospores of Olpidium bornovanus

Results document, for the first time, the role of soil moisture on a unique, tripartite, host-specific rhizosphere interaction (i.e., Cucumis melo-Monosporascus cannonballus-Olpidium bornovanus). Specifically, colonization of cantaloupe roots by zoospores of O. bornovanus and germination of ascospores of M. cannonballus were highest at a soil matric potential of ?0.001 MPa but significantly inhibited at a matric potential of only ?0.01 MPa. Matric water potentials of ?0.01 MPa or drier are characteristically inhibitory to the motility of zoosporic microbes but not hyphal growth of filamentous fungi like M. cannonballus. These results support our previous conclusion that germination of ascospores of M. cannonballus, a destructive root pathogen of cantaloupe is mediated by O. bornovanus, an obligate, zoosporic fungus.     

Phylogenetic analyses of plant-growth-promoting rhizobacteria isolated from tomato,lettuce, and Japanese pepper plants in Hyogo Prefecture,Japan

Approximately 30,000 fluorescent bacterial strains isolated from tomato, lettuce, eggplant, Chinese cabbage, and Japanese pepper plants at seven different locations in Hyogo Prefecture, were screened for plant-growth-promoting (PGP) activity to induce disease resistance against bacterial wilt in tomato. The 37 strains that had higher PGP activity were subjected to molecular phylogenetic analyses using the sequences of the 16S rRNA, gyrB and rpoD genes. Most of the strains were identified as Pseudomonas fluorescens or its close relative, P. putida, while a few strains were grouped with more distantly related bacterial species such as Enterobacter and Stenotrophomonas. The phylogenetic relationships among tomato and lettuce isolates mostly coincided with the source locality and host plants, with a few exceptions. In contrast, isolates from Japanese pepper plants did not form their own cluster but represented several different bacterial species.     

Molecular analysis of Spanish populations of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">dianthi</Emphasis> demonstrates a high genetic diversity and identifies virulence groups in races 1 and 2 of the pathogen

Fusarium wilt, caused by Fusarium oxysporum f. sp. dianthi (Fod), is the most important carnation disease worldwide. The knowledge of the diversity of the soil population of the pathogen is essential for the choice of suitable resistant cultivars. We examined the genetic diversity of Fod isolates collected during the period 1998–2008, originating from soils and carnation plants in the most important growing areas in Spain. Additionally, we have included some Fod isolates from Italy as a reference. Random amplified polymorphic DNA (RAPD) fragments generated by single-primer PCR were used to compare the relationship between isolates. UPGMA analysis of the RAPD data separated Fod isolates into three clusters (A, B, and C), and this distribution was more related to aggressiveness than to the race of the isolates. The results obtained in PCR amplifications using specific primers for race 1 and race 2, and SCAR primers developed in this work, correlated with the molecular groups previously determined from the RAPD analysis, and provided new molecular markers for the precise identification of the isolates. Results from successive pathogenicity tests showed that molecular differences between isolates of the same race corresponded with differences in aggressiveness. Isolates of races 1 and 2 in cluster A (R1I and R2I isolates) and cluster C (R1-type isolates) were all highly aggressive, whereas isolates of races 1 and 2 in cluster B (R1II and R2II isolates) showed a low aggressiveness profile. The usefulness of the molecular markers described in this study has been proved in double-blind tests with Fod isolates collected in 2008. Results from this work indicate a change in the composition of the Spanish Fod population over time, and this temporal variation could be related to the continuous change in the commercial carnation cultivars used by growers. This is the first report of genetic diversity among Fod isolates in the same race.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

Phylogenetic relationships and genome organisation of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> causing anthracnose in strawberry

Colletotrichum acutatum is a major plant pathogen which infects a broad range of host plants. Extensive research has been carried out on C. acutatum populations affecting various hosts in different geographical locations, showing a considerable genotypic and phenotypic diversity. Anthracnose, caused by Colletotrichum spp., is the major disease of cultivated strawberry, Fragaria x ananassa. In the present study, the phylogenetic relationships within a worldwide sample of fifty-two C. acutatum isolates collected from different strawberry cultivars have been established, by using ITS sequence analyses. Twenty-nine isolates clustered in the molecular group A2, in which seventeen out of eighteen Spanish isolates were included; this may indicate that the group A2 is the key group in Spain. The molecular polymorphism among C. acutatum isolates was determined by southern-blot hybridisation using a telomeric DNA probe. Results indicated that the minimum number of estimated chromosomes ranges between six and nine. The molecular characterisation of C. acutatum isolates was completed using the Pulsed-Field Gel Electrophoresis (PFGE) technique that resolved from six to nine chromosomal bands, this number being coincident with the number of chromosomes obtained by telomeric fingerprinting. The minimum total genome size was estimated to range from 29 to 36 Mb. Comparison of karyotypes patterns and southern-blot analysis demonstrated a high level of molecular polymorphism among C. acutatum isolates from different origins.     

山东甘薯主要病毒的鉴定及多样性分析

为明确山东省甘薯病毒病发生现状,在重病区调查采样,通过鉴别寄主、电镜和分子检测技术明确主要病毒种类;并克隆病毒外壳蛋白基因序列,利用Mega 5.0构建系统进化树进行遗传分析。结果显示,巴西牵牛嫁接甘薯染病枝条后叶片黄化、褪绿及皱缩;病样组织中存在大量600~900 nm的线状病毒粒子和柱状内含体。24份病样中检测到甘薯羽状斑驳病毒、甘薯潜隐病毒、甘薯G病毒、甘薯曲叶病毒和甘薯褪绿矮化病毒5种病毒,其中23份为复合侵染,存在11种侵染类型。遗传分析显示山东省甘薯羽状花叶病毒主要为EA、O和C株系,甘薯潜隐病毒与周边省份分离物相近,甘薯G病毒与中国海南和美国分离物相近,甘薯曲叶病毒分属3个株系。表明山东地区甘薯病毒种类繁多,侵染模式复杂,病毒遗传结构具有多样性。     

来源于湖北省枣疯病植原体分离物16S rDNA和rp基因的序列分析

以田间采集的来源于我国湖北省枣树产业主产区随州市随县种植的表现为"枣疯病"症状的枣树分离株为试材,对其16S rDNA和核糖体蛋白(ribosomal protein,rp)基因采用Nested-PCR进行扩增以及序列分析。结果表明,湖北JWB-Hubei植原体分离物16S rDNA基因的核苷酸序列与我国山东、河南等地的分离株一致率均为99%以上,在进化树中位于同一亚组的不同进化分支;虚拟RFLP图谱分析表明,JWB-Hubei属于16SrV-B亚组一个成员,与其进化树分组结果一致。JWBHubei分离株rp基因的核苷酸序列也与我国山东、陕西等地区的分离株一致率均为99%以上,在进化树中聚为同一亚组,与报道的基于RFLP分类属于rpV-C亚组的中国枣疯病分离物(JWB)聚集于同一亚组不同分支。该研究结果明确了湖北省枣疯病植原体的分类地位以及与来源于我国不同地区枣疯病分离株之间的遗传进化关系,为进一步研究植原体的株系划分、基因遗传变异研究提供了理论基础。     

Molecular analysis and virus transmission tests place Olpidium virulentus, a vector of Mirafiori lettuce big-vein virus and tobacco stunt virus, as a distinct species rather than a strain of Olpidium brassicae

The rDNA-ITS sequences of ten single-sporangium isolates of Olpidium virulentus (a noncrucifer strain of Olpidium brassicae), which transmits Mirafiori lettuce big-vein virus (MLBVV) and tobacco stunt virus (TStV), were compared with those of six single-sporangium isolates of O. brassicae. The sequence similarity within isolates of O. virulentus or O. brassicae was almost identical (98.5%–100.0%), but was low between the two species (79.7%–81.8%). In a phylogenetic analysis of the rDNA-ITS region, O. virulentus and O. brassicae fell into two distinct clusters, indicating that O. virulentus, a vector of MLBVV and TStV, is a distinct species rather than a strain of O. brassicae.     

Molecular genetic variability of Italian binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. isolates from strawberry

Fifty-eight binucleate Rhizoctonia isolates were collected over six years from strawberry plants displaying symptoms of black root rot in Italy. Almost all isolates were able to produce necrosis on strawberry roots, most of them also showed this ability on faba bean and, with lower frequency, on a crucifer and a cereal crop used in rotation with strawberry in Italy. The sequence alignment of Internal Transcribed Spacer (ITS) regions of 51 binucleate Rhizoctonia were analyzed and compared with a set of eight sequences representative of Rhizoctonia isolate Anastomosis Groups (AG) already found to be pathogenic on strawberry (AG-A, AG-G, AG-I and AG-F). The neighbour-joining tree, based on ITS region sequences, divided Italian strawberry Rhizoctonia isolates into two main clusters corresponding to AG-A and AG-G. The results were confirmed by hyphal anastomosis tests. The clustering obtained with the phylogenetic tree was also confirmed using PCR-Restriction Fragment Length Polymorphism of 28S rDNA to compare some isolates, defined as AG-A and AG-G on the basis of ITS region sequence analysis, with representative AG isolates pathogenic on strawberry. The AG-A and AG-G Rhizoctonia spp. were widespread in Italian strawberry-growing areas, although with different relative frequencies: AG-G was most frequent in northern (latitude 44°N) and AG-A in southern (latitude 39–40°N) Italy. Analysis of MOlecular VAriance, based on geographic location, showed that Rhizoctonia molecular variations between northern and southern Italy accounted for 36.6% of the total, but most of the variations (61%) occurred within each of the four geographical regions from where the isolates originated.     

Genetic Variability of Central European Isolates of the <Emphasis Type="Italic">Fusarium graminearum</Emphasis> Species Complex

The main causative agents of Fusarium head blight in central Europe are Fusarium graminearum and F. culmorum. We examined the mycotoxin producing ability, aggressiveness and molecular variability of F. graminearum isolates. Altogether twenty-six Hungarian, three Austrian isolates and representatives of eight species identified in the F. graminearum species complex were involved in this study. Mycotoxin producing abilities of the isolates were tested by GC-MS and HPLC. The central European isolates were found to belong to chemotype I (producing deoxynivalenol). Most isolates produced more 15-acetyl-deoxynivalenol than 3-acetyl-deoxynivalenol suggesting that they belong to chemotype Ib. All F. graminearum isolates were found to be highly pathogenic in in vitro aggressiveness tests. Phylogenetic analysis of random amplified polymorphic DNA profiles, and restriction profiles of the intergenic spacer region of the ribosomal RNA gene cluster of the isolates allowed clustering of the central European isolates into 17 and 16 haplotypes, respectively. When RAPD and IGS-RFLP data were combined, almost every single central European F. graminearum isolate could be differentiated (27/29 haplotypes). Sequence analysis of a putative reductase gene of some isolates was also performed. Based on molecular data, the majority of the central European isolates belonged to F. graminearum sensu stricto characteristic to the northern hemisphere, with the exception of one Hungarian isolate, which was not related to any known species of the F. graminearum species complex based on sequence data. The taxonomic assignment of two other Hungarian isolates, previously suggested as belonging to F. boothii based on mitochondrial DNA restriction profiles, was supported by sequence analysis.     

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。     

Genetic diversity and pathogenicity of Sphaceloma rosarum (teleomorph Elsinoë rosarum) causing spot anthracnose on roses

Roses produced or grown in the field, as well as pot‐grown and cut roses, are attacked by different fungal pathogens causing leaf spot diseases. The incorrect identification and scoring of these pathogens and the lack of information about their genetic and pathotype diversity hamper resistance breeding. This is especially true for the hemibiotrophic ascomycete Sphaceloma rosarum, which is often confused with other fungi. Here for the first time, the genetic variability between isolates at both the molecular and morphological level is analysed. Eighty leaf spot samples were collected from different rose genotypes at five different locations, and 15 single conidial isolates established. All of the samples showed high morphological similarities to the reference isolate CBS 213.33 that was obtained from a public repository. By sequencing a part of the large subunit (LSU) of the 28S ribosomal RNA and phylogenetic analysis, high sequence similarities were shown to other Sphaceloma species for 13 of the isolates and the CBS reference. One of the isolates clustered with Septoria species and another clustered with Seimatosporium species. UPGMA clustering with 145 polymorphic AFLP markers resulted in five distinct groups in the majority rule consensus tree for the 14 S. rosarum isolates, including the CBS reference. Jaccard similarities ranged from 0·31 to 0·91. A detached leaf assay using a differential set of five rose genotypes led to the classification of the five tested isolates as five distinct pathotypes. Therefore, grouping depending on the avirulence gene diversity was clearly different from clustering using selectively neutral AFLP markers that were evenly distributed throughout the genome.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">brasiliensis</Emphasis> causing blackleg on potatoes in South Africa

In South Africa during the 2006/2007 potato growing season, outbreaks of blackleg occurred, causing severe economic losses in commercial potato production fields. Symptoms were initially observed on only one stem per plant, on which the top leaves rolled upwards, wilted and became necrotic. As symptoms progressed to the lower leaves with subsequent leaf desiccation, a light to dark brown discolouration of the vascular system at the stem base developed, followed by external darkening. Under prevailing wet and humid conditions stems became slimy and pale. In the stems, the pith became necrotic and hollow. These symptoms were similar to those described in Brazil, where the causal agent was identified as a new subspecies, Pectobacterium carotovorum subsp. brasiliensis (Pbcb). Isolations from plants showing typical blackleg symptoms were made on CVP medium. Sequences and phylogenetic analysis of the partial 16S–23S rDNA intergenic spacer region indicated that the isolates were Pbcb. Comparison of PCR-RFLP patterns of the 16S–23S rDNA of isolates to reference cultures confirmed the identity of the South African blackleg strains as Pbcb, identical to strain 8 isolated in Brazil. This is the first report of Pbcb in South Africa and it appears to be the most important causal agent of blackleg in South Africa. The disease poses a major potential threat to the South African potato industry especially in terms of seed exports, tuber quality and yield.     

Molecular characterization of <Emphasis Type="Italic">Pythium</Emphasis> group F isolates by ribosomal-and intermicrosatellite-DNA regions analysis

Pythium group F is a ubiquitous, though minor, pathogen in several soilless and soil cultures; investigations were carried out to analyze different regions of the DNA and better understand the nature of this group. Fourty-two isolates were obtained from a variety of plants (cucumber, lettuce, tomato) grown in soil or soilless cultures collected in various countries (Canada, Denmark, France, Norway, Sweden and United Kingdom). All Pythium group F isolates displayed amplified ITS1-5,8S-ITS2 ribosomal DNA region (rDNA) of similar length, whereas polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) revealed that, among the seven enzymes used, polymorphism was only identified with Hin6I. After cloning of ITS1-5,8S-ITS2 rDNA region from Pythium group F isolates that displayed restriction polymorphism patterns with Hin6I, comparisons of sequence and restriction mapping data showed a slight variation consisting in a single base change. Inter Simple Sequence Repeat (ISSR)-PCR method was also used to obtain data related to the entire genome and not only to a single DNA region. It identified repeated motifs in the genome of Pythium group F isolates. Two primers (CAC)₅ and (CCA)₅ detected polymorphism, and isolates were classified among 11 molecular clusters. The genetic diversity of this group was not correlated with the geographical locations or the host plants from which the isolates originated. Polymorphism of Pythium group F isolates pointed out by ISSR is discussed