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.     

Characterization of <Emphasis Type="Italic">Dickeya</Emphasis> strains isolated from potato and river water samples in Finland

Plant pathogenic enterobacteria in the genera Pectobacterium and Dickeya (formerly classified as Erwinia) were isolated from diseased potato stems and tubers. The isolated bacteria were identified as P. atrosepticum, P. carotovorum and pathogens in the genus Dickeya with PCR tests. Furthermore, Dickeya strains were isolated from river water samples throughout the country. Phylogenetic analysis with 16S-23S rDNA intergenic spacer sequences suggested that the Dickeya strains could be divided into three groups, two of which were isolated from potato samples. Phylogenetic analysis with 16S rDNA sequences and growth at 39°C suggested that one of the groups corresponds to D. dianthicola, a quarantine pathogen in greenhouse cultivation of ornamentals, while two of the groups did not clearly resemble any of the previously characterised Dickeya species. Field trials with the strains indicated that D. dianthicola-like strains isolated from river samples caused the highest incidence of rotting and necrosis of potato stems, but some of the Dickeya strains isolated from potato samples also caused symptoms. The results showed that although P. atrosepticum is still the major cause of blackleg in Finland, virulent Dickeya strains were commonly present in potato stocks and rivers. This is the first report suggesting that Dickeya, originally known as a pathogen in tropical and warm climates, may cause diseases in potato in northern Europe.     

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.     

Two new effective semiselective crystal violet pectate media for isolation of Pectobacterium and Dickeya

Pectolytic bacteria, including Pectobacterium spp. and Dickeya spp., are best isolated on crystal violet pectate (CVP), a semiselective medium containing pectin. The source of pectin is essential, because pectolytic bacteria are not able to degrade all of them. The aims of this study were to identify a new pectin source and to perfect formulations of semiselective CVP media to isolate the pectolytic bacteria Pectobacterium spp. and Dickeya spp. from different environmental compartments (plants, soil and water). The AG366 pectin, selected after screening six different formulations, was incorporated into single‐layer (SL‐CVP_AG366) and double‐layer (DL‐CVP_AG366) CVP media. Both media were compared with those based on Bulmer, Sigma‐Aldrich and Slendid‐Burger pectins, using 39 Pectobacterium and Dickeya strains. All strains formed deep cavities on AG366‐CVPs, whereas nine did not produce cavities on Bulmer or Sigma‐Aldrich media. Recovery rates were similar on DL‐CVP_AG366, Sigma‐Aldrich and Bulmer CVPs for a given taxon, and did not differ significantly between SL‐ and DL‐CVP_AG366. Pectolytic bacteria were successfully isolated on both media from field samples of diseased potatoes, carrots, tobacco, onions, radishes and ornamentals. AG366 is thus a high‐performance pectin source for the elaboration of CVP media suitable to isolate Dickeya and Pectobacterium. It is also efficient for enrichment purposes in liquid medium. The validation of AG366 as an improved source of pectin to recover the polyphagous Pectobacterium and Dickeya in different environmental compartments is essential given the current worldwide emergence and recrudescence of these bacteria.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> can cause potato blackleg in temperate climates

It is well established that the pectinolytic bacteria Pectobacterium atrosepticum (Pca) and Dickeya spp. are causal organisms of blackleg in potato. In temperate climates, the role of Pectobacterium carotovorum subsp. carotovorum (Pcc) in potato blackleg, however, is unclear. In different western and central European countries plants are frequently found with blackleg from which only Pcc can be isolated, but not Pca or Dickeya spp. Nevertheless, tubers vacuum-infiltrated with Pcc strains have so far never yielded blackleg-diseased plants in field experiments in temperate climates. In this study, it is shown that potato tubers, vacuum-infiltrated with a subgroup of Pcc strains isolated in Europe, and planted in two different soil types, can result in up to 50% blackleg diseased plants.     

Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences

This study used a novel computational pipeline to exploit draft bacterial genome sequences in order to predict, automatically and rapidly, PCR primer sets for Dickeya spp. that were unbiased in terms of diagnostic gene choice. This pipeline was applied to 16 draft and four complete Dickeya genome sequences to generate >700 primer sets predicted to discriminate between Dickeya at the species level. Predicted diagnostic primer sets for both D. dianthicola (DIA‐A and DIA‐B) and ‘D. solani’ (SOL‐C and SOL‐D) were validated against a panel of 70 Dickeya reference strains, representative of the known diversity of this genus, to confirm primer specificity. The classification of the four previously sequenced strains was re‐examined and evidence of possible misclassification of three of these strains is presented.     

Genes responsible for coronatine synthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> present in the genome of soft rot bacteria

Primers for the PCR amplification of homologous genes encoding polyketide coronafacic acid and coronafacic ligase in the cells of Pectobacterium atrosepticum SCRI1043 (BX950851) were developed to study the presence of these genes in the genome of Pectobacterium sp. and Dickeya sp. Coronafacic ligase catalyses the formation of coronatine from polyketide coronafacic acid and coronamic acid. Coronatine is a toxin produced by Pseudomonas syringae and is one of the major virulence factors in this bacterium. This study using several strains of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. isolated in different countries, indicated that all strains of P. atrosepticum possess genes coding coronafacic acid (cfa gene cluster) and coronafacic ligase (cfl). However, these genes were present only in the genome of five out of 50 tested P. carotovorum subsp. carotovorum strains and two out of 34 strains of Dickeya sp. tested. The PCR products homologous to the sequence of cfa7 and cfl gene fragments were sequenced in order to check the level of homology between genes of P. atrosepticum, P. carotovorum subsp. carotovorum and Dickeya sp. The sequences of the gene fragments amplified from all P. atrosepticum strains were almost identical (100% and 99.97%, respectively). The homology of the sequences obtained for P. atrosepticum and sequences of five P. carotovorum subsp. carotovorum and two Dickeya sp. was lower, between 89.69% to 95.00% for the cfl gene fragment, and about 94% for the cfa7 gene fragment.     

Distribution of <Emphasis Type="Italic">Dickeya</Emphasis> spp. and <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.     

Symptoms and yield reduction caused by Dickeya spp. strains isolated from potato and river water in Finland

Biochemical characterisation of Dickeya strains isolated from potato plants and river water samples in Finland showed that the majority of the strains were biovar 3. They thus resembled the strains recently isolated from potato in the Netherlands, Poland and Israel and form a new clade within the Dickeya genus. About half of the Finnish isolates resembling strains within this new clade were virulent and caused wilting, necrotic lesions and rotting of leaves and stems. Similar symptoms were caused by D. dianthicola strains isolated from one potato sample and from several river water samples. Frequently, the rotting caused by the Dickeya strains was visible in the upper parts of the stem, while the stem base was necrotic from the pith but hard and green on the outside, resulting in symptoms quite different from the blackleg caused by Pectobacterium atrosepticum. The presence of Dickeya in the symptomatic plants in the field assay was verified with a conventional PCR and with a real-time PCR test developed for the purpose. The virulent Dickeya strains reduced the yield of individual plants by up to 50% and caused rotting of the daughter tubers in the field and in storage. Management of Dickeya spp. in the potato production chain requires awareness of the symptoms and extensive knowledge about the epidemiology of the disease.     

Simultaneous and selective detection of two major soft rot pathogens of potato: Pectobacterium atrosepticum (Erwinia carotovora subsp. atrosepticum) and Dickeya spp. (Erwinia chrysanthemi)

Dickeya spp. and Pectobacterium atrosepticum are major pathogens of potato. Current methods to detect these soft-rotting bacteria require separate identification steps. Here we describe a simple method allowing simultaneous detection of both pathogens based on multiplex PCR. The sensitivity of the primer sets was first examined on purified genomic DNA of the type strains Dickeya chrysanthemi 2048^T and P. atrosepticum 1526^T. The specificity and detection limits of the primer sets were successfully tested on 61 strains belonging to various Dickeya and Pectobacterium species, on artificially inoculated and on naturally contaminated potato plants. This new method provides a gain in time and materials, the main advantages for large-scale processes such as pathogen-free seed certification.     

Spatial analysis of blackleg‐affected seed potato crops in Scotland

Potato blackleg, caused by Pectobacterium and Dickeya species, is one of the most significant bacterial diseases affecting potato production globally. Although it is generally accepted to be a seedborne disease, the processes underlying the spread of disease largely remain unknown. Spatial point pattern analysis was applied to blackleg occurrence in seed potato crops in Scotland during the period of 2010–2013 (approximately 8000 blackleg‐affected crops), to assess whether its distribution was random, regular or aggregated, and the spatial scales at which these patterns occurred. Blackleg‐affected crops derived from mother stocks with symptoms were omitted from the analyses in order to examine the statistical evidence for horizontal transmission of blackleg. The pair correlation function was used to test for global spatial autocorrelation, and results indicated significant (P < 0·05) clustering of incidence at a wide range of spatial scales. Strength of clustering (degree of aggregation) among blackleg‐affected crops was notably larger at spatial scales of 25 km or less. A hot‐ and coldspot analysis was performed to test for local spatial autocorrelation, and statistically significant clusters of high and low values of disease were found across the country. These analyses provide the first quantitative evidence of localized and large‐scale spatial clustering of potato blackleg. Understanding the mode(s) of inoculum dispersal will be important for developing new management strategies that minimize host–pathogen contacts in potato and numerous other crops affected by pathogenic Pectobacterium and Dickeya species.     

Pathogenicity and aggressiveness in populations of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> from Belgian fruit orchards

The pathogenicity of 99 Belgian Pseudomonas syringae strains representative of the genetic diversity encountered in Belgian fruit orchards was evaluated by using 17 pathogenicity tests conducted on pear, cherry, plum, lilac, sugar beet and wheat. The P. syringae pv. morsprunorum strains were pathogenic to stone fruit species but the race 1 strains possessing the cfl gene involved in coronatine production were pathogenic in more tests than those lacking the gene. Also, sweet cherry twigs were a better material to detect pathogenic strains of race 1 and sour cherry twigs of race 2, which accorded with race 2 presence in sour cherry orchards in Belgium. Three groups were defined in the pv. syringae based on pathogenicity. One group pathogenic in 71.1% of the tests and to lilac included toxic lipodesipeptide-producing (TLP+) strains. The second group pathogenic in 26.8% of the tests and non-pathogenic to lilac included TLP+ strains. The thirth group pathogenic in 9.1% of the tests and almost specifically pathogenic to pear included TLP− strains. The three groups were genetically heterogeneous. Although strain-host relationships were noted within the pv. syringae, aptata and atrofaciens when considering the strain origins, such relationships were not found in the pathogenicity tests, suggesting that pathogenicity tests could probably not reproduce all the aspects of the host-pathogen interactions. None of the pathogenicity tests was able to provide all the information provided by the complete study. A test on pear buds indicated that strains different from the pv. syringae were pathogenic to pear.     

Studies on the interaction between the biocontrol agent,Serratia plymuthica A30, and blackleg‐causing Dickeya sp. (biovar 3) in potato (Solanum tuberosum)

Interactions between Serratia plymuthica A30 and a blackleg‐causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co‐inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antagonist and of the pathogen in planta were studied by dilution plating and confocal laser scanning microscopy (CLSM) using fluorescent protein‐tagged strains. Pathogen‐free minitubers were vacuum‐infiltrated with DsRed‐tagged Dickeya sp. IPO2222 and superficially treated during planting with a water suspension containing GFP‐tagged S. plymuthica A30. A30 reduced the blackleg incidence from 55% to 0%. Both the pathogen and the antagonist colonized the seed potato tubers internally within 1 day post‐inoculation (dpi). Between 1 and 7 dpi, the population of A30 in tubers increased from 10¹ to c. 10³ CFU g^?1 and subsequently remained stable until the end of the experiment (28 dpi). Populations of A30 in stems and roots increased from c. 10² to c. 10⁴ CFU g^?1 between 7 and 28 dpi. Dilution plating and CLSM studies showed that A30 decreased the density of Dickeya sp. populations in plants. Dilution plating combined with microscopy allowed the enumeration of strain A30 and its visualization in the vascular tissues of stem and roots and in the pith of roots, as well as its adherence to and colonization of the root surface. The implications of these finding for the use of S. plymuthica A30 as a biocontrol agent are discussed.     

Characterization of Dickeya strains isolated from potato grown under hot‐climate conditions

Dickeya strains isolated in Israel in 2006–2010 were characterized by dnaX sequence analysis, pulsed‐field gel electrophoresis (PFGE), biochemical assays and pectolytic activity, and found to be homogeneous: most of them could be classified as ‘Dickeya solani’. Of the 34 strains isolated from imported seed tubers or potato plants grown from imported seed, 32 were typed as ‘D. solani’ and only two were characterized as Dickeya dianthicola. Biovar typing indicated that all ‘D. solani’ strains were biovar 3. ‘Dickeya solani’ strains were most closely related to Dickeya dadantii subsp. dieffenbachiae according to PFGE and dnaX analyses and both species exhibited high pectolytic activity. Expression levels of two putative virulence genes, pelL (encoding a pectic enzyme) and dspE (encoding a type III effector) were significantly induced in ‘D. solani’ strains isolated from potato plants or tubers grown in hot climates such as the Negev region in Israel, compared to those isolated from seed tubers imported from the Netherlands, France or Germany. Results of this study support the hypothesis that ‘D. solani’ strains isolated in Israel are also clonal; however, they appear to be more virulent than strains isolated in Europe.     

Phylogenetic study of Japanese Dickeya spp. and development of new rapid identification methods using PCR–RFLP

Forty-one representative Japanese Dickeya spp. (Erwinia chrysanthemi) strains isolated from 24 plants in Japan were investigated using multilocus sequence analysis of recA, dnaX, rpoD, gyrB and 16S rDNA; PCR–RFLP (restriction fragment length polymorphism) of recA, rpoD and gyrB genes; PCR genomic fingerprinting; and biochemical tests. Based on the recA, dnaX, rpoD, gyrB and 16S rDNA sequences and PCR genomic fingerprinting, the strains were essentially divided into six groups (I–VI). Group I corresponded to D. chrysanthemi, group II corresponded to D. dadantii, group III to D. dianthicola and group IV to D. zeae. Meanwhile, group V and group VI could not be assigned to any existing Dickeya species, and they were deduced to be two putative new species. The PCR–RFLP analysis of gyrB, rpoD and recA clearly differentiated the six groups of Dickeya strains. From the results of the biochemical tests, the strains were assigned to biovars 1, 3, 5, 8 and 9; only one strain (SUPP 2525) was not assignable to the existing biovars. We also showed that the PCR–RFLP analysis of rpoD, gyrB and recA can be used as a rapid technique to identify Japanese Dickeya strains.     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.     

Characterization of bacterial isolates from rotting potato tuber tissue showing antagonism to Dickeya sp. biovar 3 in vitro and in planta

Possibilities for biocontrol of biovar 3 Dickeya sp. in potato were investigated, using bacteria from rotting potato tissue isolated by dilution plating on nonselective agar media. In a plate assay, 649 isolates were screened for antibiosis against Dickeya sp. IPO2222 and for the production of siderophores. Forty‐one isolates (6·4%) produced antibiotics and 112 isolates (17·3%) produced siderophores. A selection of 41 antibiotic‐producing isolates and 41 siderophore‐producing isolates were tested in a potato slice assay for control of the Dickeya sp. Isolates able to reduce rotting of potato tuber tissue by at least 50% of the control were selected. Isolates were characterized by 16S rDNA analysis as Bacillus, Pseudomonas, Rhodococcus, Serratia, Obesumbacterium and Lysinibacillus genera. Twenty‐three isolates belonging to different species and genera, 13 producing antibiotics and 10 producing siderophores, were further characterized by testing acyl‐homoserine lactone (AHL) production, quorum quenching, motility, biosurfactant production, growth at low (4·0) and high (10·0) pH, growth at 10°C under aerobic and anaerobic conditions and auxin production. In replicated greenhouse experiments, four selected antagonists based on the in vitro tests were tested in planta using wounded or intact minitubers of cv. Kondor subsequently inoculated by vacuum infiltration with an antagonist and a GFP (green fluorescent protein)‐tagged biovar 3 Dickeya sp. strain. A potato endophyte A30, characterized as S. plymuthica, protected potato plants by reducing blackleg development by 100% and colonization of stems by Dickeya sp. by 97%. The potential use of S. plymuthica A30 for the biocontrol of Dickeya sp. is discussed.     

Strain-specific alfalfa water stress induced by <Emphasis Type="Italic">Xylella fastidiosa</Emphasis>

Differences in the virulence of a pathogen among host species can occur because hosts differ in their resistance or tolerance to infection or because of underlying genetic variation in the pathogen. The xylem-limited bacterium Xylella fastidiosa is pathogenic to dozens of plant species throughout the Americas, and is structured into genetically and biologically distinct strains. In some plants X. fastidiosa causes striking leaf scorch symptoms and in others, such as alfalfa, stunting is the primary symptom. The mechanism by which these symptoms occur has been debated. We tested the hypothesis that symptoms result from X. fastidiosa-induced water stress, and that the magnitude of water stress is strain-dependent. We mechanically inoculated alfalfa plants with one of 14 isolates (5 identified genetically as “almond” and 9 as “grape” isolates), and compared stable carbon isotope ratios among isolates. Infected plants showed significant isotopic shifts (up to 2% on average) relative to healthy plants that were consistent with water stress. Moreover, there were significant differences in water stress among isolates, with a tendency for grape isolates to cause more severe water stress than almond isolates. There was also a positive relationship between plant infection level and isotopic shift (slope ± SE = 0.273 ± 0.068), which supports the hypothesis that X. fastidiosa symptoms result from bacterial multiplication and vessel occlusion. Unexpectedly, however, water stress was not correlated with measures of alfalfa stunting. These results suggest X. fastidiosa induces strain-specific levels of water stress, but factors other than water stress alone are responsible for stunting.     

Taxonomic Position of the Causal Pathogen of Bacterial Shoot Blight of Pear

Bacteriological properties and DNA-DNA homology values were compared among the pathogen causing bacterial shoot blight of pear (BSBP) isolated in 1994–1996, Erwinia amylovora isolated outside of Japan, and other Amylovora group bacteria. Bacteriological properties of BSBP strains were identical to those of E. amylovora in the majority of tests, but differed distinctively in several tests, including hydrolysis of esculin and acid production from salicin, etc. BSBP strains differed from the others in the Amylovora group in many other tests. DNA homology among the strains of BSBP ranged from 85 to 103% and from 83 to 110% among strains of E. amylovora. In contrast, the values between BSBP strains and E. amylovora strains were 55 to 81%, while those between BSBP strains and other Amylovora group strains were 42% or less. We consider, therefore, that the BSBP pathogen may well be included in E. amylovora at the species level. E. amylovora, including BSBP strains, however, can be classified into four biovars based on differences in nine tests such as growth factor requirements and crater formation on high sucrose medium. Namely, there are two biovars from Maloideae sources, one from Rubus idaeus, and one from the source of BSBP in Hokkaido. The presence of these biovars suggests a correlation with geographical, serological, and pathogenic differentiations in the species of E. amylovora. The BSBP pathogen in Hokkaido was identified as E. amylovora bv. 4 which is distinct from E. amylovora bv. 1, 2 and 3 isolated in countries outside of Japan. Received 29 July 1999/ Accepted in revised form 12 October 1999