Association of Verticillium dahliae and Verticillium longisporum with Chinese cabbage yellows and their distribution in the main production areas of Japan

Chinese cabbage yellows caused by Verticillium dahliae and V. longisporum is one of the most economically important diseases in Japan. Verticillium isolates from infected Chinese cabbage in Japan were identified as V. dahliae and V. longisporum based on morphological characteristics and newly developed molecular genotyping methods using group I intron of 18S rDNA, mitochondrial-SSU rDNA and cob gene. Although the two species were equally virulent to Chinese cabbage in the greenhouse, V. longisporum was more virulent in the field compared with V. dahliae. Among the 67 Verticillium isolates from Chinese cabbage, 53 were V. longisporum and considered to be the major pathogen. The isolation frequency of V. longisporum was 98?% in Ibaraki and eastern Gunma where Chinese cabbage was cropped from autumn to winter. In contrast, the frequency was 48?% in Nagano and north to west Gunma where the plant was cropped in cool summer and highland conditions and the rests were V. dahliae. This is the first report to show the different geographical distribution of V. longisporum and V. dahliae in Chinese cabbage production area of Japan.     

Retrotransposon-like elements in the genome of Verticillium dahliae may be used as DNA markers for fungal species and pathotypes

Using differential hybridization, two DNA fragments, VDf35 and VDf90, specific to Verticillium dahliae, were isolated. These fragments contained truncated open reading frames (ORFs) homologous to the gypsy-type retrotransposon. The ORFs of VDf35 and VDf90 were pol and gag homologs, respectively. In addition, VDf90 had a pol homolog without an ORF sequence. The pol homologs in VDf35 and VDf90 were similar to each other, and these two DNA fragments had completely identical sequences. Genomic Southern analysis revealed that numerous copies of these homologs existed in V. dahliae, suggesting that V. dahliae carries a gypsy-like retroelement. Genomic Southern and polymerase chain reaction (PCR) analysis also indicated that a large number of these homologs exist in V. longisporum as well as in V. dahliae, but only a few were present in V. albo-atrum. No homolog was found in either V. nigrescens or V. tricorpus. The uneven distribution of these homologs of the retroposon-like elements among Verticillium species suggested a close genetic kinship between V. dahliae and V. longisporum. PCR primers designed from VDf35 showed species- or pathotype-specific amplification. Therefore, this sequence may be useful as a DNA marker to identify species and pathotypes of V. dahliae. This is the first report on a retrotransposon-like sequence in the genome of phytopathogenic Verticillium species.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB095264 (VDf90) and AB095265 (VDf35)     

Genetic variability and virulence among Verticillium albo-atrum isolates from hop

Verticillium wilt, caused by Verticillium albo-atrum or V. dahliae, is an important disease of many worldwide crop species. In Europe, V. albo-atrum isolates infecting hop express different levels of virulence, inducing mild or lethal disease syndromes, and it is therefore an attractive model for studying the virulence of this pathogen. In this work, eleven amplified fragment length polymorphism (AFLP) primer combinations were used to analyze genetic variability among 55 V. albo-atrum hop isolates from four European hop growing regions, as well as isolates from other hosts and V. dahliae isolates. Cluster analysis divided V. albo-atrum and V. dahliae isolates into two well-separated groups. Within the V. dahliae cluster, isolates were separated without host specific grouping, although no host adapted isolates were included. In V. albo-atrum, the alfalfa isolates were distinct from isolates of other hosts, where a high association with virulence was observed in hop and tomato isolates. All lethal hop isolates were genetically different from mild hop isolates. The lethal hop isolates from England and Slovenia expressed the same virulence phenotype, although they showed a different AFLP pattern. The mild hop isolates formed two subgroups, to which isolates clustered irrespective of geographical location. These data suggest multiple origins of V. albo-atrum hop isolates, and the possible appearance of new virulent isolates in the future in other hop growing regions.     

Genetic structure of Verticillium dahliae isolates infecting olive trees in Tunisia using AFLP,pathogenicity and PCR markers

Since 2006, verticillium wilt of olive induced by Verticillium dahliae has caused considerable economic losses in olive orchards in Tunisia. The genetic structure of V. dahliae isolates collected from different olive growing regions was investigated using virulence tests, vegetative compatibility grouping (VCG) and amplified fragment length polymorphism (AFLP) analyses. In total, 42 isolates of V. dahliae from diseased olive trees were tested. Cluster analysis and principal coordinate analysis revealed that geographic origin was the main factor determining the genetic structure of V. dahliae populations and both methods indicated a genetic separation between the central and coastal isolates. Isolates were divided into two major groups: the AFLP‐I group included all isolates from Sidi Bouzid, Kairouan, Kasserine and Sfax (centre of the country) and the AFLP‐II group included isolates from Monastir, Zaghouane, Sousse, Mahdia (coastal region), and two isolates from Sfax. Analysis of the molecular variance (amova ) indicated a significant level of genetic differentiation among (76%) and within (23%) the two populations. Analyses of both the defoliating (D) and non‐defoliating (ND) pathotypes and VCG markers indicated that most of the isolates belong to VCG 2A and 4B/ND pathotype. The disease severity was highly variable among the isolates tested (P < 0·05) with no evidence of association between aggressiveness and geographical origin of the isolates. Overall, results of this study revealed a clear association between the genetic diversity of the isolates and their geographic origin, but not between genetic diversity and virulence patterns.     

Molecular and Pathotype Analysis of the Rice Blast Fungus in North Vietnam

Rice blast caused by Pyricularia oryzae is a devastating disease worldwide. In Vietnam, rice blast is especially severe in the Red River Delta in the North. The genetic diversity of 114 P. oryzae isolates collected from rice in 2001 in the Red River Delta and nine additional Vietnamese P. oryzae isolates was analysed using Amplified Fragment Length Polymorphism (AFLP). DNA similarity and cluster analysis based on 160 polymorphic AFLP markers showed twelve different AFLP genetic groups among the 123 field isolates. Isolates collected from japonica hosts clustered separately from indica host isolates with at least 60% dissimilarity with little evidence for gene flow between the two populations. In the 2001 population originating from indica hosts, three genetic groups were predominant and represented 99% of the isolates sampled. One predominant clonal lineage represented 59% of the 2001 indica host population and was found in eleven provinces in the Red River Delta of North Vietnam. Significant genotype flow could be demonstrated between the indica population south of Red river and the indica population north of Red river. There was significant linkage disequilibrium between the AFLP loci within the indica population, indicating that this is not a random mating population. Pathogenicity tests of 25 isolates selected from the 12 AFLP groups on a set of 29 differential rice lines revealed two avirulent isolates and 23 pathotypes. Different combinations of known resistance genes were found to have potential for blast resistance breeding for North Vietnam. First two authors contributed equally     

VCG and AFLP analyses identify the same groups in the causal agents of mango malformation in Brazil

The causal agents of mango malformation disease in Brazil are a new Fusarium lineage in the Gibberella fujikuroi species complex and Fusarium sterilihyphosum; however information on the genetic and geographical diversity of these pathogens in Brazil is missing. Vegetative compatibility group (VCG) and amplified fragment length polymorphism (AFLP) analyses were used to measure the genetic diversity within these populations. Both techniques identified the same genetic groups. Six VCG and AFLP groups were identified amongst isolates of the new lineage from Brazil. FB-VCG 1/AFLP I was the most widespread group, found in seven of the 13 sites sampled. The second most frequent group was recovered from three sites. The remaining four groups were recovered from single-sites. We think that this lineage represents a genetically and geographically diverse indigenous population that reproduces clonally. In F. sterilihyphosum, group FS-VCG 1/AFLP VII was found at three sites in the southeast region of Brazil. FS-VCG 2/AFLP VIII contained isolates from South Africa but not from Brazil. Fusarium mangiferae isolates from India and South Africa formed one group, while isolates from Egypt and the USA formed a second group. F. sterilihyphosum at present is represented by a small population that might have been introduced only once into a restricted area. The clonal nature of the observed populations suggests that these fungi either occur naturally on indigenous hosts and have jumped to the introduced mango host (introduced in Brazil) or that they originated with mango and went through a severe population bottleneck when they were introduced to Brazil from India or Southeast Asia.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Assessment of latent infection with <Emphasis Type="Italic">Verticillium longisporum</Emphasis> in field-grown oilseed rape by qPCR

Improvement of cultivar resistance is the key strategy to control the host-specialized pathogen Verticillium longisporum in oilseed rape (OSR). A special feature of this pathogen is its systemic, non-homogenous and delayed colonization of the plant xylem resulting in an extended symptomless period of latency. As a result, severity of infection in the field is difficult to score as it becomes apparent only at crop maturity stages when it may be confused with natural senescence. Assessment of Verticillium disease severity in OSR by visual scoring of microsclerotia on harvested stubbles unsatisfactorily reflects genotypic resistance as it is strongly affected by the ripening stage of the plant. To overcome these limitations, we developed a qPCR method, which unambiguously differentiates levels of quantitative resistance to V. longisporum in OSR genotypes under field conditions. The specificity and sensitivity of two primer pairs targeting ITS or tubulin loci in the V. longisporum genome were tested. While tubulin primers showed a high specificity to V. longisporum isolates, ITS primers exhibited a significantly higher sensitivity in detecting fungal DNA in stem tissue (limit of quantification =0.56 fg DNA) of field-grown pre-symptomatic plants. The best discrimination of resistant and susceptible OSR cultivars based on fungal DNA analysis in stem tissue was achieved at growth stage 80, at the transition of fungal vascular growth in viable plants to saprotrophic colonization of senescent stem tissues. Field screening data obtained with qPCR at growth stage 80 confirmed results from greenhouse testing thus corroborating the relevance and reliability of seedling assays for determining cultivar responses to V. longisporum in the field, as a useful tool for breeders in first selection of elite OSR genotypes with improved resistance to Verticillium.     

Molecular,physiological and pathogenic variability of <Emphasis Type="Italic">Pyrenochaeta lycopersici</Emphasis> associated with corky rot disease of tomato plants in Turkey

Isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot on tomato plants, were assessed for physiological and genetic characteristics using conventional and molecular techniques. All isolates were able to produce necrosis on tomato roots and classified into temperature group according to the optimal growth temperatures. Specific-PCR assays and DNA sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region confirmed the existence of both types (Type 1 and Type 2) of the pathogen among the isolates tested. All isolates were identified as Type 2 except for isolate Pl-4, which was classified as Type 1. Restriction fragment length polymorphism (RFLP) analysis with six enzymes resulted in three distinct banding patterns among the isolates depending on the length and restriction profiles of the rDNA intergenic spacer region. Inter-simple sequence-repeat analysis revealed a high level of genetic diversity among the isolates in agreement with the data of RFLP analysis. These results indicated that there were three different intraspecific groups among Turkish isolates of P. lycopersici. The presented study is the first attempting to characterize Turkish isolates of P. lycopersici. The results obtained will be useful in screening of tomato seedlings for resistance to P. lycopersici.     

河南省西部山区小麦白粉菌群体遗传多样性分析

为揭示河南省小麦白粉菌群体遗传结构、起源及进化关系,采用简单重复序列区间(inter-simple sequence repeats,ISSR)和扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分子标记技术对河南省西部山区4个小麦产区的35个小麦白粉菌单孢分离菌株进行了群体遗传多样性分析。结果显示:ISSR和AFLP分析均将35个菌株分为3个组,组Ⅰ包括来自卢氏和灵宝的大部分菌株;组Ⅱ包括来自栾川、卢氏和巩义的菌株;组Ⅲ由4个地区的个别菌株组成,同时包含1个闭囊壳释放子囊孢子获得的菌株。ISSR分析出菌株遗传距离分布在0.0139～0.6592之间,扩增多态性比率为64.83%,各菌株间的Shannon指数为0.2749;而AFLP分析所得的各菌株遗传距离变化幅度在0.1257～0.9322之间,扩增多态性比率为82.68%,各菌株间的Shannon指数为0.5100。可见,河南省小麦白粉菌具有丰富的遗传多样性,研究所用的2种方法均可用于遗传多样性分析,其中AFLP分析小麦白粉菌群体表现出更为丰富的遗传多样性。     

AFLP analysis of Russian <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> populations from wheat kernels and other hosts

Alternaria tenuissima is a common pathogen on a number of plants described in several geographic regions of the world. Genetic variation within and between Russian Far East, North West and Caucasus populations of A. tenuissima from wheat was examined. In addition, genetic differences between isolates from various hosts were estimated. In total, 101 isolates of A. tenuissima were studied using amplified fragment length polymorphism (AFLP) with four primer combinations. Wright’s fixation index (F _st), gene flow (N _m) and gene diversity (H _s) were calculated. AFLP banding patterns indicated significant genetic distance and at the same time a low level of gene flow between the Far East and the two other groups of isolates originating from the European part of country. The degree of similarity between the North West and Caucasus populations was very high, as was the migration rate. Isolates analysed by UPGMA-based cluster analysis were grouped according to location of origin but irrespective of plant host. Based on the F _st value, the group of isolates originating from wheat and barley were not found to differ significantly from each other.     

Plant Host Range of Verticillium longisporum and Microsclerotia Density in Swedish Soils

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, <5% of the samples had formed microsclerotia; for scentless mayweed, 5–10%; for oat, 10–20%; and for charlock and oilseed rape >80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g⁻¹ soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g⁻¹ soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.     

Pathogenicity,vegetative compatibility and amplified fragment length polymorphism (AFLP) analysis of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">radicis-cucumerinum</Emphasis> isolates from Turkish greenhouses

The objective of the current study was to characterize Fusarium oxysporum f. sp. radicis-cucumerinum isolates from cucumbers in Turkey in terms of pathogenicity, vegetative compatibility and amplified fragment length polymorphism (AFLP) variation. In the 2007 and 2008 greenhouse cucumber-growing seasons, surveys were conducted in Adana, Antalya, Hatay and Mersin provinces of the Mediterranean region of Turkey. Forty-seven fungal isolates of F. oxysporum were recovered from diseased cucumber plants. The pathogenicity of each isolate was tested on cucumber seedlings at the one-true-leaf stage. Forty of the 47 isolates of F. oxysporum were virulent on cucumber seedlings. Based on disease symptoms, the differential effect of temperatures of 17°C and 29°C on disease development, and the virulence on cucumber seedlings, these 40 isolates were identified as F. oxysporum f. sp. radicis-cucumerinum. Nitrate non-utilizing mutants were generated on minimal medium containing 1.5% KClO₃ and their phenotypes were determined. Mutants in different phenotypic classes were paired on minimal medium; of 40 F. oxysporum f. sp. radicis-cucumerinum isolates, thirty-eight were placed into VCG 0260. Remaining two strains were assigned to VCG 0261. The AFLP primers produced a total of 180 fragments between 200 and 500 bp in length for the 30 isolates tested. At a genetic similarity of 0.71, the UPGMA analysis separated the isolates into two distinct clusters. The first cluster, AFLP I, included 28 isolates, of which all belonged to VCG 0260. Two strains in the second AFLP cluster both belonged to VCG 0261.     

Diversity among isolates ofVerticillium lecanii as expressed by DNA polymorphism and virulence towardsBemisia tabaci

Thirty-six isolates ofVerticillium lecanii andVerticillium sp. were taken from different hosts (both insects and rusts) and geographical locations. The isolates were analyzed for genomic variability, as expressed by random amplified polymorphic DNA (RAPD), in relation to virulence onBemisia tabaci. Virulence on larvae ofB. tabaci within these isolates ranged from 0% to 83%. RAPD analysis was performed employing two different arbitrary decamer primers and the calculated similarity coefficients were subjected to cluster analysis using the unweighted average linkage (UPGMA) algorithm. The dendrograms obtained with each of the two primers were identical. Eight cluster groups and three unclustered isolates were obtained by selecting a similarity level of 80%. The amplification pattern of DNA obtained by RAPD for the various isolates suggested thatV. lecanii is a highly diverse species. No correlation could be established between virulence and either RAPD polymorphism of the fungal isolates or the insect host from which they were isolated. Generally, no correlation could be established between the clustering ofV lecanii strain and geographical location although a limited number of strains obtained from Russia and Georgia were assembled in the same cluster and those from Kazakhstan were clonal.     

Population studies on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> on potatoes and tomatoes in southern Germany

Fifty-seven isolates of Phytophthora infestans from blighted potato foliage were collected in 1995 in southern Germany and analysed for mating type and sensitivity to metalaxyl. Fifty-six of them were characterised as A1 and one as A2 mating types. Resistance to metalaxyl was observed frequently: 53 isolates were resistant, three were partially sensitive, and one was sensitive. In a subsequent field study in 1999, 84 isolates collected from blighted potato and tomato foliage were analysed for mating type. Seventy-two were characterised as A1 and twelve as A2 mating types. The response of 76 isolates to metalaxyl and to propamocarb was tested. The majority (42) of the 76 isolates was classified as resistant to metalaxyl; 31 were partially sensitive and only three isolates were sensitive. The results with propamocarb were less discrete; 10 isolates were classified as resistant and three were clearly sensitive. AFLP fingerprinting was used to examine the genetic structure of the southern German P. infestans population collected in 1999 and indicated that the tested population can be sub-divided into a tomato group, a potato group and a mixed group containing isolates collected from both crops. The presence of Ia and IIa mitochondrial DNA haplotypes indicates that the German P. infestans isolates belong to the new pathogen population that has also been reported in neighbouring regions of Europe. The present study indicates that at the beginning of the season only a few genotypes were present, and the population became genetically more variable at the end of the growing season.     

Verticillium diseases of vegetable crops in Brazil: Host range,microsclerotia production,molecular haplotype network,and pathogen species determination

Recent outbreaks of Verticillium wilt diseases in various vegetable crops have been reported in Brazil. This fact was our initial stimulus to carry out a nationwide survey aiming to determine their causal agent(s). Thus far, Verticillium dahliae has been reported as the predominant species based solely on morphological traits. As other Verticillium species can be associated with wilt diseases, we characterized a collection of 80 isolates, collected across 10 agricultural Brazilian regions, by combining morphological, biological, and molecular traits. A multilocus approach was employed for identification of Verticillium species with information from three genomic regions (ribosomal internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and actin genes). Only 21 out of the 80 isolates were unable to produce microsclerotia in culture. The analyses of all genomic regions indicated V. dahliae as the sole species associated with vascular wilt of distinct hosts, including major solanaceous vegetables and other hosts such as strawberry, okra, and cacao. Pathogenicity tests confirmed the infection by the V. dahliae isolates and the development of typical disease symptoms on their original hosts. This is the first nationwide characterization of Verticillium isolates associated with major vegetable crops in Neotropical areas. This provides valuable information to design sound management strategies for these diseases, mainly for establishing efficient rotation systems and for the development of resistant cultivars.     

A contribution to the knowledge of nematophagous species of Verticillium

Nine nematophagous species and two varieties ofVerticillium (Verticillium sect.Prostrata), occurring either as parasites of free-living nematodes or as parasites of cysts and eggs, are revised and keyed out.V. catenulatum is reduced to a variety ofV. chlamydosporium A similar fungus is distinguished asV. suchlasporium n. sp. (including a var.catenatum). Spicaria coccospora Drechsler is transferred toVerticillium. All species have been studied in pure culture. Ecological data are reviewed for each species.Samenvatting Negen nematofage soorten en twee variëteiten vanVerticillium (Verticillium sectieProstrata), die óf als parasieten van vrijlevende aaltjes óf als parasieten van cysten en eieren optreden, worden gereviseerd en er wordt een determinatiesleutel van gepresenteerd.V. catenulatum wordt gereduceerd tot een variëteit vanV chlamydosporium. Een hierop lijkende schimmel wordt onderscheiden als.V. suchlasporium n.sp. (incl. een variëteitcatenatum. Spicaria coccospora Drechsler wordt naarVerticillium overgebracht. Alle soorten werden in reincultuur onderzocht. Voor iedere soort worden oecologische gegevens vermeld.     

Virulence and Molecular Diversity in Cochliobolus sativus

ABSTRACT Spot blotch, caused by the fungal pathogen Cochliobolus sativus, is an important disease of barley in many production areas of the world. To assess genetic diversity in this pathogen, a worldwide collection of C. sativus isolates was evaluated for virulence on barley and DNA polymorphism. Three pathotypes (0, 1, and 2) were identified among the 22 isolates tested in this study and the 36 isolates characterized previously on three barley differentials (ND5883, Bowman, and NDB112) that differ in their resistance to C. sativus. Pathotype 2, which exhibits high virulence on cv. Bowman, was only found in North Dakota, whereas the other two pathotypes occurred in many other regions of the world. Genetic diversity of the 58 C. sativus isolates, together with isolates of three related pathogenic Cochliobolus spp. (C. heterostrophus, C. carbonum, and C. victoriae) was analyzed using amplified fragment length polymorphism (AFLP) markers. A total of 577 polymorphic AFLP markers were recorded among the 70 isolates of the four Cochliobolus spp. using eight primer combinations. Cluster analysis revealed distinct groups corresponding to the four different species, except in one case where race 0 of C. carbonum was placed in an outgroup that may belong to a different species. In C. sativus, 95 polymorphic AFLP markers were detected with the eight primer pairs used, and each isolate exhibited a unique AFLP pattern. Allelic diversity in the pathotype 2 group was lower (0.10) than in the pathotype 0 (0.23) and pathotype 1 (0.15) groups, indicating that pathotype 2 may have arisen more recently. Cluster analysis did not reveal a close correlation between pathotypes and AFLP groups, although two AFLP markers unique to pathotype 2 isolates were identified. This low correlation suggests that genetic exchange may have occurred through parasexual recombination in the fungal population. Some isolates collected from different regions of the world were clustered into the same AFLP group, suggesting that migration of the fungal pathogen around these regions has occurred.     

Genetic analyses of Pseudomonas syringae isolates from Belgian fruit orchards reveal genetic variability and isolate-host relationships within the pathovar syringae, and help identify both races of the pathovar morsprunorum

A collection of Pseudomonas syringae and viridiflava isolates was established between 1993 and 2002 from diseased organs sampled from 36 pear, plum and cherry orchards in Belgium. Among the 356 isolates investigated in this study, phytotoxin, siderophore and classical microbiology tests, as well as the genetical methods REP-, ERIC- and BOX- (collectively, rep-) and IS50-PCR, enabled identification to be made of 280 isolates as P. syringae pv. syringae (Pss), 41 isolates as P. syringae pv. morsprunorum (Psm) race 1, 12 isolates as Psm race 2, three isolates as P. viridiflava and 20 isolates as unclassified P. syringae. The rep-PCR methods, particularly BOX-PCR, proved to be useful for identifying the Psm race 1 and Psm race 2 isolates. The latter race was frequent on sour cherry in Belgium. Combined genetic results confirmed homogeneities in the pvs avii, and morsprunorum race 1 and race 2 and high diversity in the pv. syringae. In the pv. syringae, homogeneous genetic groups consistently found on the same hosts (pear, cherry or plum) were observed. Pathogenicity on lilac was sometimes variable among Pss isolates from the same genetic group; also, some Psm race 2 and unclassified P. syringae isolates were pathogenic to lilac. In the BOX analyses, four patterns included 100% of the toxic lipodepsipeptide (TLP)-producing Pss isolates pathogenic to lilac. Many TLP-producing Pss isolates non-pathogenic to lilac and the TLP-non-producing Pss isolates were classified differently. Pseudomonas syringae isolates that differed from known fruit pathogens were observed in pear, sour cherry and plum orchards in Belgium.     

Genetic diversity and pathogenicity of Pseudomonas syringae pv. aptata isolated from sugar beet

Pseudomonas syringae pv. aptata is the causal agent of bacterial leaf spot disease of sugar beet (Beta vulgaris). During 2013, 250 samples were collected from leaf lesions with typical symptoms of bacterial leaf spot in commercial fields of sugar beet in Serbia, and 104 isolates of P. syringae pv. aptata were obtained. Identification and characterization was performed using biochemical, molecular and pathogenicity tests. Identification included LOPAT tests and positive reactions using primers Papt2F and Papt1R specific for P. syringae pv. aptata. Repetitive (rep) sequence‐based PCR typing with ERIC, REP and BOX primers revealed high genetic variability among isolates and distinguished 25 groups of different fingerprinting profiles. Pulse‐field gel electrophoresis (PFGE) and multilocus sequence analysis (MLSA) of representative isolates showed higher genetic variability than in rep‐PCR analysis and distinguished three and four major genetic clusters, respectively. A pathogenicity test performed with 25 representative isolates on four cultivars of sugar beet confirmed the occurrence of leaf spot disease and showed correlation between the most aggressive isolates and the genetic clusters obtained in MLSA. All these findings point to the existence of several lines of P. syringae pv. aptata infection in Serbia that are genetically and pathologically different.