Physiological, Biochemical and Molecular Analyses of an Italian Collection of Agrobacterium tumefaciens strains

Physiological, biochemical and molecular characteristics of Agrobacterium tumefaciens strains isolated in Italy from different host plants were analysed. Diseased plants were collected from several nurseries located in nine different regions. Out of 1293 strains isolated from 12 fruit tree and six ornamental plant species, a group of 120 strains was chosen as representative of the whole collection. The majority of the strains were biovar 2 (82.5%), agrocin 84 sensitive, and were isolated from stone fruit trees. Most of the strains identified as biovar 1 were isolated from ornamental plants and were insensitive to A. radiobacter antagonistic strain K84. Some strains that were isolated from Euonymus spp, Prunus GF 677 and Pyrus communis (pear) OHF tumours could not be allocated to any of the three Agrobacterium biovars. PCR-restriction fragment length polymorphism of the rrs gene plus the intergenic spacer was used for strain fingerprinting and characterisation. Results showed a wide genetic variability within the biovar 1 strains and homogeneity within the biovar 2 group. Biovar 2 strains from Sardinia were highly variable and differed from the biovar 2 strains isolated from the other regions of Italy.     

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.     

Detection of tumorigenic Agrobacterium strains from infected apple saplings by colony PCR with improved PCR primers

With the colony polymerase chain reaction (PCR), the specificity of newly prepared primer sets VCF2/VCR2, VCF3/VCR3, VCF4/VCR4, and VCF5/VCR5 to Ti or Ri plasmids in Agrobacterium strains were compared to that of the conventional set VCF/VCR. At first, control strains, which consisted of a nonpathogenic strain and phytopathogenic strains carrying Ti or Ri plasmid, were used. VCF3/VCR3 and VCF5/VCR5 were highly specific to all the phytopathogenic strains, whereas the others were not. These two primer sets were superior to VCF/VCR in their specificity with colony PCR to tumorigenic Agrobacterium strains isolated from apple saplings.     

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.     

Multiplex PCR for the identification of Agrobacterium biovar 3 strains

A biovar 3-specific primer set Ab3-F3/Ab3-R4 was designed based on the comparison of sequences of the 16S rDNA region of agrobacteria and related rhizobia for rapid identification of Agrobacterium biovar 3 strains. A 570-bp 16S rDNA fragment was amplified from cell lysates of Agrobacterium biovar 3 strains by polymerase chain reaction (PCR) using Ab3-F3/Ab3-R4 primers. Discrimination of Agrobacterium tumefaciens biovar 3 from Agrobacterium radiobacter biovar 3 and of Agrobacterium biovar 3 strains from other Agrobacterium strains was done simultaneously using multiplex PCR with a mixture of two primer sets (Ab3-F3/Ab3-R4 and VCF3/VCR3) previously designed for the virC region of Ti-plasmid and Ri-plasmid.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

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.     

Interaction of Root-knot Nematodes (RKN) and the Bacterium Agrobacterium Tumefaciens in Roots of Prunus Cerasifera: Evidence of the Protective Effect of the Ma RKN Resistance Genes Against Expression of Crown Gall Symptoms

Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode (RKN), M. hapla. Some clones of this plum carry single Ma resistance genes that control M. arenaria, M. incognita and M. javanica. The four above mentioned RKN and Myrobalan progenies segregating for Ma were used in experiments aimed at obtaining a better knowledge of the interaction between AT and RKN in relation to the RKN resistance genes. Prunus rooted cuttings, naturally infected with the bacterium were repotted, grown and inoculated individually with RKN. In a first experiment, Prunus plants were (i) either inoculated with 10,000 juveniles (J2s) of M. arenaria to provide a short inoculum pressure (SIP) or (ii) inoculated by association with one M. arenaria-galled tomato root system that produced a high and durable inoculum pressure of the same nematode species. Four months after RKN inoculation, plants were rated for nematode and bacterial root galling symptoms. RKN and AT galls were more numerous and more homogenous under DIP than under SIP. Nevertheless, for both inoculum regimes, AT galls were present in the RKN-susceptible clones (= carrying none of the Ma genes) and absent in the RKN-resistant clones. Subsequent experiments, conducted under DIP with M. arenaria, M. incognita, M. javanica and M. hapla, also showed, for the three first species, the presence of AT galls only in RKN-susceptible clones whereas Prunus plants inoculated with M. hapla and nematode-free controls were free of AT galls. Consequently RKN act as a wound agent in the AT infection process of Myrobalan plum only when the plant develops a compatible reaction (i.e. when it lacks the Ma resistance genes). Considering that J2s do penetrate the roots of resistant plants, the absence of crown gall symptoms on this material even under durable inoculum pressure strengthens the hypothesis that this nematode stage has a very weak effect on plant cells during the infection process. This is the first evidence of the protective effect of a RKN resistance gene against the expression of root crown gall consecutive to RKN infection. The protective effect of Ma and presumably of other RKN resistance genes against AT is a strong argument for their introgression into Prunus and other Rosaceae or bacterium-susceptible crops.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation as a tool for random mutagenesis of <Emphasis Type="Italic">Colletotrichum trifolii</Emphasis>

We transformed Colletotrichum trifolii, the causal agent of alfalfa anthracnose, using Agrobacterium tumefaciens as a new tool for random insertional mutagenesis. Fungal spores of C. trifolii were transformed with T-DNA including the hygromycin phosphotransferase gene (hph). Southern analysis showed that every randomly selected transformant had a unique hybridization pattern of T-DNA, suggesting that the T-DNA was randomly integrated into the fungal genome. More significantly, about 75% of transformants had a single copy of the T-DNA. The results demonstrate that insertional mutagenesis via A. tumefaciens is a useful tool for studying the function of C. trifolii genes.     

Pathogenic and Genetic Diversity of Soilborne Isolates of Cylindrocladium from Banana Cropping Systems

Pathogenicity and genetic variation were investigated within a collection of 104 banana-infecting isolates of Cylindrocladium (teleomorph Calonectria) originating from different countries and representing previously described morphological taxa or species. These root-rot fungi, along with endoparasitic nematodes, have been reported to be causal agents of necrotic lesions that induce root breakage and toppling of banana plants. Little is known about the individual pathogenic effects of the species involved or their genetic diversity. In the present study, among the five morphological taxa found in the banana rhizosphere, only isolates showing an atypical morphology relative to Cylindrocladium gracile (named Cy. gracile-like isolates) and Cy. spathiphylli isolates were pathogenic on banana cultivar Grande Naine. When comparing the latter isolates with others of the same species, but originating from different hosts, an analysis of rDNA spacer polymorphism partitioned isolates of Cy. spathiphylli by host into a banana – tea group and a Heliconia – Spathiphyllum group. Furthermore, isolates from Heliconia were not pathogenic on banana. A pathogenicity assessment of representative isolates from the Cy. gracile-like and the Cy. spathiphylli taxa on six different banana cultivars yielded no evidence of differential interactions between isolates and banana genotypes. Significant differences in susceptibility between banana genotypes were nevertheless detected that could potentially be exploited by breeders. Random amplified polymorphic DNA analysis revealed a genetic similarity ranging from 70% to 100% within Cy. spathiphylli isolates from bananas regardless of the geographic origin. Moreover, Cy. gracile-like isolates were highly similar but showed only 60% similarity relative to the Cy. gracile reference isolates, thus raising questions about their species status.     

Rapid and Specific Detection of Virulent Pseudomonas avellanae Strains by PCR Amplification

Specific oligonucleotides, based on hrpW (hypersensitive response and pathogenicity) gene sequences encoding harpin protein in phytopathogenic bacteria, were designed to detect and identify virulent strains of Pseudomonas avellanae by polymerase chain reaction (PCR). A population of virulent P. avellanae strains, isolated in central Italy (Viterbo region), was assessed with hrpW-derived primers, producing a specific band of about 350 base pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and from hazelnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Agrobacterium, Erwinia, Brenneria, Pseudomonas, Ralstonia, Xanthomonas or from hazelnut-associated bacteria, indicating the specificity of these primers. Moreover DNA from strain ISPaVe-MCB-596, isolated from north Italy (Piedmont region) and belonging to the less aggressive population of P. avellanae, did not amplify in PCR. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for virulent P. avellanae strains and a useful tool to evaluate the progress of sanitation of the area.     

中国及欧洲各国的苹果蠹蛾种群遗传多样性分析

为研究不同国家和地区苹果蠹蛾Cydia pomonella种群的遗传多样性,对采自我国新疆维吾尔自治区和黑龙江省,以及德国、法国和西班牙等欧洲国家的11个种群共60个苹果蠹蛾个体进行基因组重测序,有效测序样本数为56个,共获得8 455 843个群体单碱基突变（single nucleotide polymorphism,SNP）位点。利用SNP数据进行了苹果蠹蛾群体遗传进化树的构建、主成分分析以及遗传结构分析。结果显示,入侵我国黑龙江省的苹果蠹蛾与德国BD种群更为接近,我国新疆维吾尔自治区与黑龙江省苹果蠹蛾种群有一定的分化,前者种群遗传多样性更低。表明我国东北地区和西北地区的苹果蠹蛾种群具有不同的遗传多样性特征,入侵我国的苹果蠹蛾种群可能来源于欧洲。     

PCR primers for identification of opine types of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in Japan

The polymerase chain reaction (PCR) is a rapid, precise method for detecting and identifying pathogenic bacteria. In addition to the published primers for identification of Agrobacterium tumefaciens up to species level, two sets of primers were designed to identify the nopaline and octopine types of Agrobacterium tumefaciens. The RBF-RBR primer set designed based on the nopaline type T-DNA right border detected the nopaline type A208 and R225f strains, and the ocsF-ocsR primer set derived from the ocs gene of the octopine type A. tumefaciens detected the octopine type A348 strain. After polymerase Chain reaction (PCR) amplification by the RBF-RBR primers, the A208 and R225f strains could be differentiated from each other by restriction fragment length polymorphism digestion using the restriction enzymes DraI and XbaI. Multiple colonies can be screened at one time in a single PCR tube with satisfactory efficiency, thereby allowing rapid detection of pathogenic A. tumefaciens. Following a rough screening by classical biovar medium and -methyl-d-glucoside medium, the developed PCR system was introduced to identify isolates collected from soil and crown gall samples. Of 42 isolates determined to be A. tumefaciens, 7 were found to be octopine type; all the rest were R225f type.     

Identification and Characterisation of Xanthomonas campestris pv. campestris Strains from Tanzania by Pathogenicity Tests, Biolog, rep-PCR and Fatty Acid Methyl Ester Analysis

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a major disease constraint to cabbage production by smallholder farmers in Africa. Variability exists within the pathogen, and yet differentiation of Xcc strains from other closely-related xanthomonads attacking crucifers is often difficult. The Biolog system, fatty acid methyl ester analysis using microbial identification system (MIS), rep-PCR and pathogenicity tests were used to identify and characterise Xcc strains from Tanzania. Great diversity was observed among Xcc strains in their Biolog and rep-PCR profiles. Specific rep-PCR genomic fingerprints were linked to some geographical areas in the country. Most of the Xcc strains were clustered in two groups based on their fatty acid profiles and symptom expression in cabbage although some deviant strains were found. Each of the methods allowed a degree of identification from species, pathovar to the strain level. Biolog and MIS identified all Xcc strains at least to the genus level. Additionally, Biolog identified 47% of Xcc strains to the pathovar and 43% to strain level, whereas MIS identified 43% of the strains to pathovar level. In the absence of a database, the utility of rep-PCR for routine diagnosis of strains was limited, although the procedure was good for delineation of Xcc to the strain level. These findings indicate the existence of Xcc strains in Tanzania that are distinct from those included in Biolog and MIS databases. The limitations noticed warrant continued improvement of databases and inclusion of pathogenicity testing, using universally susceptible cultivars, as an integral part of strain identification.     

Biological control of grapevine crown gall by nonpathogenic <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent against crown gall of grapevine (Vitis vinifera L.). A mixture of the nonpathogenic strain VAR03-1 and a tumorigenic strain G-Ag-27 of A. vitis at cell ratios of 1 : 1, 3 : 1, 9 : 1, and 99 : 1 significantly inhibited gall formation and size on stems of tomato (Lycopersicon esculentum Mill.). Strain VAR03-1 also inhibited gall formation on stems of both tomato and grapevine at a 1 : 1 cell ratio with several tumorigenic A. vitis strains isolated from different fields of grapevine in Japan. In biological control tests, when roots of grapevine and tomato seedlings were soaked in a cell suspension of strain VAR03-1 for 24 h before a 1-h soaking in a cell suspension of the pathogen and subsequent planting in pots of infested soil, strain VAR03-1 significantly reduced the incidence of gall formation on both plants.     

Genetic and Pathogenic Diversity of Pseudomonas avellanae Strains Isolated from Corylus avellana Trees in North-west of Italy, and Comparison with Strains from Other Regions

Forty strains of Pseudomonas avellanae isolated from hazelnut (Corylus avellana L.) trees in the Langhe district of Italy were compared with 15 strains collected from various geographic areas. All strains were assessed by rep-PCR genomic fingerprinting using ERIC, REP and BOX primer sets. Cluster analysis was performed by means of UPGMA. To check the possible differential virulence of the strains, pathogenicity tests were carried out by inoculating leaf scars of hazelnut trees in early autumn. Cluster analysis indicated that, during a four-year study, at least five groups of strains were isolated from different hazelnut orchards located in the small district of Langhe. Two groups were isolated from the same twig. Such strains showed around 20% similarity with other P. avellanae strains collected from northern Greece and central Italy. The strains from Langhe were less aggressive to hazelnut than strains from northern Greece and central Italy. These results and previous genomic characterizations indicate a possible correlation between genomic profile type and regional geographic distribution of P. avellanae strains. In addition, the genetic variability found in the strains from Langhe indicates that such populations are older than the more homogenous P. avellanae populations from other regions.     

Population structure of the rice sheath blight pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-1 IA from India

The population structure of Rhizoctonia solani AG-1 IA causing rice sheath blight from India was evaluated for 96 isolates using seven RFLP loci. Nineteen of the isolates did not hybridise to R. solani AG-1 IA RFLP probes and rDNA analyses subsequently confirmed that they were either Ceratobasidium oryzae-sativae isolates or another Rhizoctonia sp. The population structure of the remaining 77 R. solani AG-1 IA Indian isolates was similar to that of a previously characterized Texas population. Clonal dispersal of R. solani AG-1 IA in India was moderate within fields and no clones were shared among field populations. Low levels of population subdivision and small genetic distances among populations were consistent with high levels of gene flow. Frequent sexual reproduction was indicated by the fact that most populations were in Hardy–Weinberg equilibrium (HWE). The two loci (R68 and R111) that deviated significantly from HWE showed an excess of heterozygosity. Although Texas and Indian populations were geographically very distant, they exhibited only moderate population subdivision, with an F_ST value of 0.193.     

基于DNA条形码技术的湖南省柑橘主产区实蝇幼虫分子鉴定

为明确湖南省柑橘主产区的实蝇入侵为害现状,从该省7个市(自治州)22个地点收集柑橘蛆果中的幼虫,利用DNA条形码技术对其进行分子鉴定,并以DNA条形码序列作为分子标记探究橘大实蝇Bactrocera minax的20个中国地理种群(湖南省6个组群共16个种群、其它3省市4个种群)以及1个印度地理种群间的亲缘地理关系,分析橘大实蝇在我国的遗传进化关系。结果表明,仅采集自邵阳市城步苗族自治县柑橘蛆果中的5头幼虫被鉴定为蜜柑大实蝇B. tsuneonis,其余21个地点采集的595头幼虫均被鉴定为橘大实蝇。21个橘大实蝇地理种群的平均单倍型多样性为0.75,核苷酸多样性为0.0032,核苷酸差异数为2.13,中国所有地理种群均具有较高的遗传多样性;单倍型网络进化图显示湖南、重庆、贵州种群共享的单倍型H3为原始单倍型,表明其为比较原始的种群;AMOVA分析结果显示种群内个体间遗传变异占总体变异的59.04%,是遗传变异的主要来源;遗传分化结果表明湖南省6个组群间均出现了中度至高度的遗传分化,FST在0.0521～0.7795之间。表明DNA条形码技术可用于蜜柑大实蝇和橘大实蝇幼虫的分子鉴定及其种群遗传进化分析。     

Diversity and detection of Korean Erwinia pyrifoliae strains as determined by plasmid profiling, phylogenetic analysis and PCR

Twenty-five strains of Erwinia pyrifoliae were investigated for their plasmid profiles and genetic relatedness. Four types of plasmid profile were observed for the first time, suggesting intraspecific plasmid profile diversity in E. pyrifoliae . Moreover, BOX-PCR and phylogenetic analysis based on the 16S-23S intergenic transcribed spacer (ITS) region showed genetic variations among E. pyrifoliae strains, although all strains were clustered in one group and separated from E. amylovora . On the other hand, ERIC-PCR and phylogenetic analysis based on partial groEL gene sequences revealed close genetic relatedness among the strains. Amplification with EpSPF and EpSPR primers of a fragment of approximately 0·65 kb from the genomic DNA of all E. pyrifoliae strains, but not from E. amylovora strains, suggested that this primer set is useful for identification of this pathogen.