Chromosomal and Plasmid Diversity of Agrobacterium Strains Isolated from Ficus benjamina Tumors

Agrobacteria were previously isolated from tumors developing on branches and aerial and hypogeous roots of weeping fig plants in Italy and in The Netherlands. A representative group of 48 strains was analyzed by PCR–RFLP of 16S and 16S + IGS ribosomal regions, PCR–RFLP of six Ti plasmid (pTi) regions and characterized for plasmid content. Two groups of agrobacteria were separated by cluster analysis of PCR–RFLP profiles of rrs gene: seventeen strains were similar to the new species Agrobacterium larrymoorei, while the remaining strains were included within the agrobacterium biovar 1 group. Sixteen different plasmid profiles from one to five plasmids were observed. In addition, 21 ribotypes and 20 pTi structures were arranged in many different combinations, showing that fig agrobacteria were characterized by a wide heterogeneity. A general lack of correlation between strain ribotypes and plasmid content was observed.     

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.     

Pathogenic and genetic variability of Ralstonia solanacearum strains from the Philippines

In the Philippines, bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting vegetables and banana. In this study, 89 strains of R. solanacearum isolated from various hosts were screened for their biovar, phylotype, pathogenicity, and genetic diversity. Foreign strains were included for comparison with these Philippine strains. Results of the biochemical and multiplex-PCR tests divided the Philippine strains into five biovars (1, 2, 3, 4, and N2) and three phylotypes (I, II, and IV). Three potato strains belonged to biovar N2/phylotype IV. Pathogenicity tests divided the strains into five pathogenicity types based on their virulence in tomato, potato, eggplant, sweet pepper, and tobacco. Strains classified as biovar N2 were weakly pathogenic to potato (pathogenicity type III) and almost all strains isolated from banana were not pathogenic to the test plants except potato (pathogenicity type V). The results of AFLP analysis divided the strains into four clusters. Cluster 1 was composed of strains isolated from solanaceous crops, ginger (Zingiber officinale), and Morus sp. from the Philippines and other Asian countries. Cluster 2 grouped the potato strains (biovar N2) from the Philippines and Japan and blood disease bacterium strains from Indonesia. Cluster 3 contained the local and foreign strains isolated from potato (biovar 2) and banana (biovar 1). Cluster 4 consisted only of the tomato strain from the USA.     

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.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Assessment of recent outbreaks of <Emphasis Type="Italic">Dickeya</Emphasis> sp. (syn. <Emphasis Type="Italic">Erwinia chrysanthemi</Emphasis>) slow wilt in potato crops in Israel

Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease incidence of 2–30% (10% in average). In addition to typical wilting symptoms on the foliage, in cases of severe infection, progeny tubers were rotten in the soil. Six strains were characterised by biochemical, serological and PCR-amplification. All tests verified the strains as Dickeya sp. The rep-PCR and the biochemical assays showed that the strains isolated from blackleg diseased plants in Israel were very similar, if not identical to strains isolated from Dutch seed potatoes, suggesting that the infection in Israel originated from the Dutch seed. The strains were distantly related to D. dianthicola strains, typically found in potatoes in Western Europe, and were similar to biovar 3 D. dadanti or D. zeae. This is the first time that the presence of biovar 3 strains in potato in the Netherlands is described. One of the strains was used for pathogenicity assays on potato cvs Nicola and Mondial. Symptoms appeared 2 to 3 days after stem inoculation, and 7 to 10 days after soil inoculation. The control plants treated with water, or plants inoculated with Pectobacterium carotovorum, did not develop any symptoms with either method of inoculation. The identity of Dickeya sp. and P. carotovorum re-isolated from inoculated plants was confirmed by PCR and ELISA.     

Biochemical and genetical analysis reveal a new clade of biovar 3 <Emphasis Type="Italic">Dickeya</Emphasis> spp. strains isolated from potato in Europe

Sixty-five potato strains of the soft rot-causing plant pathogenic bacterium Dickeya spp., and two strains from hyacinth, were characterised using biochemical assays, REP-PCR genomic finger printing, 16S rDNA and dnaX sequence analysis. These methods were compared with nineteen strains representing six Dickeya species which included the type strains. A group of twenty-two potato strains isolated between 2005-2007 in the Netherlands, Poland, Finland and Israel were characterised as belonging to biovar 3. They were 100% identical in REP-PCR, dnaX and 16S rDNA sequence analysis. In a polyphasic analysis they formed a new clade different from the six Dickeya species previously described, and may therefore constitute a new species. The strains were very similar to a Dutch strain from hyacinth. On the basis of dnaX sequences and biochemical assays, all other potato strains isolated in Europe between 1979 and 1994 were identified as D. dianthicola (biovar 1 and 7), with the exception of two German strains classified as D. dieffenbachia (biovar 2) and D. dadantii (biovar 3), respectively. Potato strains from Peru were classified as D. dadantii, from Australia as D. zeae and from Taiwan as D. chrysanthemi bv. parthenii, indicating that different Dickeya species are found in association with potato.     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.     

An Efficient Microtiter System to Determine Agrobacterium Biovar

A microtiter system for biovar characterization of Agrobacterium strains which simplifies the analysis of a large number of isolates is described. This method is based on incubation of bacterial strains in microplate wells previously amended with media specifically used by the different Agrobacterium biovars. More than 150 purified Agrobacterium strains isolated from the most common host plants were analysed by the microtiter system. It proved to be an excellent tool using less reagents, time and space for incubation in comparison with the traditional method.     

Eucalyptus occidentalis plantlets are naturally infected by pathogenic Agrobacterium tumefaciens

In the Sidi M’djahed nursery (Algeria), over 60,000 eucalyptus (Eucalyptus occidentalis) plantlets exhibited tumour-like growths localized at the crown of the plants that resembled crown galls caused by Agrobacterium tumefaciens. Bacteria colonizing the galls were isolated and purified. Most (22 out of 24) of the isolates had cultural and biochemical characteristics similar to those of strains of the biovar 1 of A. tumefaciens. Twenty out of 22 Agrobacterium isolates induced tumour formation on various test plants. In PCR experiments, DNA extracted from these virulent strains yielded an amplification signal corresponding to a 247-bp fragment located within the virulence region of nopaline type Ti plasmid. Consistent with this, the opine nopaline was detected in the tumours induced on test plants – but not on eucalyptus plants. Nopaline was degraded by the 20 pathogenic isolates that were also sensitive to agrocin 84, indicating the presence of a nopaline-type pTi in these strains. The chromosomal region encoding the 16S rRNA was analyzed in a sub-population of the pathogenic agrobacterial isolates. The analyzed strains were found to belong to the ribogroup of the reference strain B6. Interestingly, Eucalyptus camaldulensis and Eucalyptus cladocalyx grown in the same nursery and in the same soil substrate developed no galls.     

Crown gall of tobacco caused by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> biovar 1 in tobacco fields

Crown gall disease of tobacco was found in Iwate Prefecture, Japan in 1995. Ten bacterial isolates, obtained from the galls of tobacco, were identified as Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 biovar 1 based on their ability to induce galls on the 14 tested plants, including tobacco after needle-prick inoculation, and on 12 cultural, physiological, and biological characteristics. The growth of the causal organism was not inhibited in vitro by agrocin of A. radiobacter strain K84. This report is the first on the natural occurrence of crown gall caused by A. tumefaciens on tobacco plants.     

Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.

Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die‐back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.     

Differentiation ofVerticillium dahliae populations on the basis of vegetative compatibility and pathogenicity on cotton

Complementary auxotrophic nitrate-nonutilizing (nit) mutants were used to investigate vegetative compatibility within 27 strains ofVerticillium dahliae isolated from several hosts originating from Africa, Asia, Europe and the United States. Using about 500nit mutants generated from these strains, three vegetative compatibility groups, 1, 2 and 4, were identified. Simultaneously, virulence of each strain was assessed on cultivars ofGossypium hirsutum, G. barbadense andG. arboreum, based upon Foliar Alteration Index (FAI) and Browning Index (BI) estimation. The strains in VCG1 were of both the cotton-defoliating pathotype and race 3 (on cotton) but were non pathogenic on tomato; those in VCG2 and VCG4 were of the nondefoliating pathotype and belonged to different races on cotton and on tomato. Hyaline mutants deriving from parental wild-type strain showed differences in pathogenicity but were always assigned to the parental VCG. A relationship was established between VCGs and the taxonomic position of host plants. Data fromnit pairings indicated that the sub-populations ofV. dahliae (VCGs) may not be completely isolated genetically.     

Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.     

Pseudomonas syringae pv. avii (pv. nov.), the Causal Agent of Bacterial Canker of Wild Cherries (Prunus avium) in France

Bacterial strains isolated from cankers of wild cherry trees (Prunus avium) in France were characterized using numerical taxonomy of biochemical tests, DNA–DNA hybridization, repeat sequence primed-PCR (rep-PCR) based on REP, ERIC and BOX sequences, heteroduplex mobility assay (HMA) of internal transcribed spacer (ITS) as well as pathogenicity on wild cherry trees and other species of Prunus. They were compared to reference strains of Pseudomonas syringae pathovars isolated from wild and sweet cherry and various host plants. Wild cherry strains were closely related to P. syringae (sensu lato) in LOPAT group Ia (+ - - - +). Wild cherry strains were pathogenic to wild cherry trees and produced symptoms similar to those observed in orchards. They were pathogenic also, but at a lesser extent, to sweet cherry trees (cv. Napoléon). The wild cherry strains were collected from five different areas in France and appeared to constitute a very homogeneous group. They showed an homogenous profile of a biochemical and physiological characteristics. They were closely related by DNA–DNA hybridization and belonged to genomospecies 3 `tomato'. Rep-PCR showed that wild cherry strains constitute a tight group distinct from P. s. pv. morsprunorum races 1 and 2 and from other P. syringae pathovars. HMA profiles indicated that the ITS of all wild cherry strains were identical but different from P. s. pv. persicae strains since the two heteroduplex bands with reduced mobility were generated by hybridization with the P. s. pv. persicae pathotype strain CFBP 1573. The 8 genomospecies of Gardan et al. (1999) have not been converted into formal species as they cannot be differentiated by biochemical tests. Therefore, the pathovar system within P. syringae was currently used. P. syringae pv. avii is proposed for this bacterium causing a wild cherry bacterial canker and strain CFBP 3846 (NCPPB 4290, ICMP 14479) is designated as the pathotype.     

Characterization and PCR-based Typing of Xanthomonas campestris pv. vesicatoria from Peppers and Tomatoes in Serbia

During the last two decades bacterial strains associated with necrotic leaf spots of pepper and tomato fruit spots were collected in Serbia. Twenty-eight strains isolated from pepper and six from tomato were characterized. A study of their physiological and pathological characteristics, and fatty acid composition analysis revealed that all of the strains belong to Xanthomonas campestris pv. vesicatoria. Being non-amylolytic and non-pectolytic, pathogenic on pepper but not on tomato, containing lower amounts of fatty acid 15 : 0 ante–iso, the pepper strains were designated as members of the A group of X. campestris pv. vesicatoria. However, the tomato strains hydrolyzed starch and pectate, caused compatible reactions on tomato but not on pepper, had higher percent of 15 : 0 ante–iso fatty acid, and were classified into B phenotypic group and identified as X. vesicatoria. PCR primers were developed which amplified conserved DNA regions related to the hrp genes of different strains of X. campestris pv. vesicatoria associated with pepper and tomato. Restriction analysis of the PCR product resulted in different patterns and enabled grouping of the strains into four groups. When xanthomonads isolated from pepper and tomato in Serbia were analyzed, they clustered into two groups corresponding to the grouping based on their physiological and pathological characteristics. According to the reaction of pepper and tomato differential varieties, the strains from pepper belong to races P7 and P8 and tomato strains belong to the race T2. All strains were sensitive to copper and streptomycin. Advantages and disadvantages of various bacterial spot management practices are discussed.     

Specific Detection of Biovars of Ralstonia solanacearum in Plant Tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 10³cfu ml^–1, which corresponds to 1cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.     

Characterization of Pectobacterium species from Iran using biochemical and molecular methods

Characteristics of forty strains from macerated potato tubers and water-soaked lesions of some ornamental plants were studied in north parts of Iran. The causal organisms isolated from infected tissues were identified as Pectobacterium spp. based on their physiological and biochemical assays and confirmed by species and subspecies specific PCR and RFLP analysis of 16S–23S intergenic transcribed spacer region. Artificial inoculation of isolates to their related hosts generated the same symptoms on potato and ornamental plants, from which the same bacteria were isolated and identified. We detected two groups of atypical isolates in this study. The first group from potato classified as Pectobacterium carotovorum subsp. carotovorum by phenotypic tests but was unable to elicit HR on tobacco leaves, to grow at 37°C and to amplify the pel gene relevant to this subspecies. The second one from ornamental plants which was again characterized as Pectobacterium carotovorum subsp. carotovorum in biochemical assays, produced a unique ITS-RFLP profile different from all of known Pectobacterium species and subspecies. Our findings based on phylogenetic analysis using concatenated partial sequences of housekeeping genes mdh and gapA, indicated the occurrence of P. wasabiae as a novel species in potato storage in Iran. Furthermore we detected a distinct clade of Pectobacterium spp. from some ornamental plants including Schlumbergera bridgesii, Syngonium podophyllum and Iris spp.     

Detection of <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> by PCR using novel <Emphasis Type="Italic">virD2</Emphasis> gene-specific primers that discriminate two subgroups

Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.     

Analysis of genetic and biological characters of Japanese potato strains of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

We assessed the geographic distribution, biovar, phylotype, DNA fingerprints (rep-PCR), and/or endoglucanase sequence of potato bacterial wilt pathogen, Ralstonia solanacearum (Rs), in Japan. Rs has been isolated from potato fields in southwestern, warm, temperate regions. Of the 188 isolates, 74 belonged to biovar N2 (39%), 44 to biovar 3 (24%), and 70 to biovar 4 (37%). Biovars N2 and 4 strains were widely distributed, from northern (Hokkaido) to southern (Okinawa) Japan. Based on the results of multiplex-PCR analysis, every potato strains belonged to either phylotype I or IV. Phylotype I comprised both biovars 3 and 4 strains. On the other hand, phylotype IV included biovar N2 strains. None of the strains belonged to phylotype II or III or biovar 1 or 2. Phylogenetic analysis based on DNA fingerprints and endoglucanase gene sequences clarified the genetic diversity of the Japanese potato strains and the close genetic relationship between the Japanese strains and the Asian strains in phylotypes I and IV.