Detection and identification of the crucifer pathogen, <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>, by PCR amplification of the conserved Hrp/type III secretion system gene <Emphasis Type="Italic">hrcC</Emphasis>

The development of a rapid detection method for Xanthomonas campestris pv. campestris (Xcc) in crucifer seeds and plants is essential for high-throughput certification purposes. Here we describe a diagnostic protocol for the identification/detection of Xcc by PCR amplification of fragments from the pathogenicity-associated gene hrcC. Under stringent conditions of amplification, a PCR product of 519 bp from hrcC was obtained from a collection of 46 isolates of Xcc, with the exception of two isolates from radish. No amplicons were obtained from 39 pure cultures of the phytopathogenic bacteria Xanthomonas campestris pv. cerealicola, X. campestris pv. juglandis, X. campestris pv. pelargonii, X. campestris pv. vitians, X. arboricola pv. pruni, X. axonopodis pv. phaseoli, X. axonopodis pv. vesicatoria, X. vesicatoria, Pseudomonas syringae pv. phaseolicola, P. syringae pv. syringae, P. syringae pv. tomato, P. fluorescens, P. marginalis, Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum. In addition, PCR reactions were negative for fifty unidentified environmental isolates purified from the surface of crucifers. The PCR fragment was obtained from four strains previously classified as X. campestris pv. aberrans, X. campestris pv. armorociae, X. campestris pv. barbarae and X. campestris pv. incanae using pathogenicity assays. Our PCR protocol specifically detected Xcc in inoculated leaves, seeds and naturally infected leaves of crucifers.     

Protein electrophoresis and DNA:DNA hybridizations of xanthomonads from grasses and cereals1

More than 120 Xanthomonas campestris strains pathogenic for grasses and cereals were compared by polyacrylamide gel electrophoresis (SDS-PAGE) of their whole-cell proteins. Genotypic relationships between representative strains of the electrophoretic groups were determined by DNA:DNA hybridizations. Two major groups of bacteria were delineated. The first included X. campestris pv. graminis, pv. arrhenatheri and some isolates from Bromus, which could be differentiated from each other by their protein fingerprints, and also the following pathovars which it was impossible to differentiate by SDS-PAGE: cerealis, hordei, poae, secalis, translucens and undulosa. DNA:DNA hybridizations indicated that significant degrees of DNA-binding (>60% D) exist between all these pathovars. In the second group, strains of X. campestris pv. holcicola, pv. vasculorum and pv. oryzae were related at 40–45% DNA-binding, while strains of pv. oryzae and pv. oryzicola were genotypically highly related (85% D). All the pathovars of this second group could be differentiated from each other by their protein electrophoretic fingerprints.     

Genetic relationships among strains of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis>pv. <Emphasis Type="Italic">campestris</Emphasis>revealed by novel rep-PCR primers

Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.     

The genetic characterization of Xanthomonas arboricola pv. juglandis,the causal agent of walnut blight in Poland

Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC‐, BOX‐PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I‐391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.     

Bacterial pathogens of Gramineae: systematic review and assessment of quarantine status for the EPPO region1

Bacterial pathogens of Gramineae principally belong to the genera Clavibacter, Erwinia, Pseudomonas and Xanthomonas, the last being the most important. A general survey of these pathogens is given, with details on nomenclature, symptoms, natural host range, geographical distribution and potential quarantine significance for the EPPO region. The status of Xanthomonas campestris pathovars with overlapping broad and narrow host ranges on Gramineae is discussed. It is proposed to adopt a broad concept of X. campestris pv. translucens and evaluate it as a potential quarantine hazard for the EPPO region.     

Identification of genomic regions associated with resistance to blackleg (Leptosphaeria maculans) in canola using genome wide association study

Black rot of crucifers is one of the most important diseases of wild rocket (Diplotaxis tenuifolia L. (D.C.)) caused by the seedborne pathogen Xanthomonas campestris pv. campestris. From 2005, it frequently affected this cultivation in the south of Italy, leading to heavy crop losses. In the present work, we aimed to describe the physiological and molecular characteristics of twenty X. campestris pv. campestris strains isolated from plants and seeds. Ten Xanthomonas spp. strains contaminating seeds were identified on the basis of molecular characterization and in vivo pathogenicity on a discriminating host range. Some of seed-borne isolates were ascribed to the species Xanthomonas campestris pv. raphani and X. campestris pv. incanae, indicating the occurrence of non-host pathogenic Xanthomonas on wild rocket seeds. As well as the presence of pathogenic bacteria, even non-pathogenic Xanthomonas spp. strains were detected on the seeds, underlying the importance of identifying them to evaluate the suitability of lots intended for sowing. A phylogeny using 69 Gyrase B (gyrB) sequences retrieved from the literature, was also carried out, highlighting species relatedness. Overall, this study provides a comprehensive framework for Xanthomonas species affecting wild rocket in Southern Italy.

     

Pathogenicity and virulence gene content of Xanthomonas strains infecting Araceae,formerly known as Xanthomonas axonopodis pv. dieffenbachiae

Bacterial leaf blight of aroids is caused by a heterogeneous group of xanthomonads listed as Xanthomonas axonopodis pv. dieffenbachiae (Xad) on the EPPO A2 quarantine list. Recently, Xad strains were shown not to belong to X. axonopodis but to the species X. citri, X. phaseoli and X. euvesicatoria. Here, to verify the pathovar designation, 11 representative strains were tested for pathogenicity on six aroid genera. They had overlapping host ranges and only the strain isolated from Syngonium showed host specificity. The X. citri strains, isolated from various hosts, showed dissimilarity in virulence to the tested aroid genera. The X. phaseoli strains, isolated from Anthurium and Syngonium, were generally more virulent and, additionally, induced systemic infections. The X. euvesicatoria strains, isolated from Philodendron, were scored as not pathogenic on the tested aroids. Four representative strains were genome sequenced and showed a variable virulence‐associated gene content. Pathogenicity to aroids was correlated with the presence of three specific T3 effector genes and with a T6SS gene sequence. Together, the phylogenetic and pathogenic differentiation among Xad strains justifies the installation of three pathovar epithets for the pathogens on aroids: X. phaseoli pv. dieffenbachiae comb. nov. for the strains isolated from Anthurium; X. phaseoli pv. syngonii comb. nov. for the strain isolated from Syngonium; and X. citri pv. aracearum comb. nov. for the strains isolated from Aglaonema, Xanthosoma and Dieffenbachia. It is proposed that phytosanitary regulations for xanthomonads on aroids are restricted to these three pathovars.     

Development of a specific molecular tool for detecting Xanthomonas campestris pv. musacearum

A specific and rapid diagnostic tool has been developed to detect Xanthomonas campestris pv. musacearum, the causal agent of bacterial wilt of banana. PCR primers were developed from intergenic regions of X. campestris pv. musacearum following its partial sequence. A total of 48 primers were tested for specificity to X. campestris pv. musacearum strains collected from various regions in Uganda. These were also tested for specificity against related Xanthomonas species from the vasicola group, Xanthomonas species pathogenic to other crops, and against those existing saprophytically on banana plants. Seven primer sets (Xcm12, Xcm35, Xcm36, Xcm38, Xcm44, Xcm47 and Xcm48) were found to be very specific to X. campestris pv. musacearum. These primer sets directed the amplification of the expected product for all 52 strains of X. campestris pv. musacearum collected from different locations in Uganda. No amplification products were obtained with unrelated phytopathogenic bacteria or endophytic/epiphytic bacteria from banana. A detection limit of 10³ CFU mL^?1 corresponding to about four cells per PCR reaction was observed when X. campestris pv. musacearum cells were used for all the seven primer sets. The DNA samples from symptomless plant tissues also tested positive with primer set Xcm38. The specific PCR method described here is a valuable diagnostic tool which can be used to detect the pathogen at early stages of infection and monitor disease.     

Differentiation of Xanthomonas campestris pv. Phaseoli from Xanthomonas campestris pv. Phaseoli var. Fuscans by PFGE and RFLP

Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, the causal agents of the common and fuscous bacterial blight of beans, appear to be phenotypically identical except that the latter can produce a melanin-like pigment in culture. Ten isolates of X. campestris pv. phaseoli and 12 isolates of X. campestris pv. phaseoli var. fuscans were examined using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP). The average genome sizes for X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were 3850.6±48.9 and 3584.3±68.1kb respectively. The genetic relatedness of the isolates was determined from macrorestriction patterns generated using XbaI. Cluster analysis indicated that the non-fuscous and fuscous strains are distinct. RFLP results, based on the highly conserved hrp genes and a pectate lyase gene from Xanthomonas, also indicated that the two bacteria are genetically different. The results obtained in this study suggest that this pathovar can be segregated into two subgroups under a recently proposed reclassification of the Xanthomonas genus.     

Characterization of isolates that cause black rot of crucifers in East Africa

A study was conducted in the East African countries of Kenya, Tanzania and Uganda in the months of July and August 2009 with the objectives of assessing the status of black rot and race structure of Xanthomonas campestris pv. campestris in the three countries. Samples infected with black rot were collected from farmers’ fields mainly from Brassica oleracea crops (broccoli, cabbage, cauliflower and kales). A total of 399 farms were surveyed of which 260 were from Kenya, 91 from Tanzania and 48 from Uganda. Following successful isolations, a total of 249 isolates of the causal agent, Xanthomonas campestris pv. campestris were recovered. Pathogenicity of all isolates was confirmed on B. oleracea susceptible cultivars Copenhagen Market F1 and Wirosa F1. Sixty of the 250 isolates were race-typed using a differential set Brassica spp. Only two races, 1 (Kenya and Tanzania) and 4 (Kenya, Tanzania and Uganda) were observed however, another race (5) was observed from one isolate recovered from a B. rapa sample obtained from Tanzania in 2003. Genomic fingerprinting with repetitive-PCR revealed clusters that did not depict significant correlations between isolates and geographical location, isolates and host adaptation or isolates and race. However, it did demonstrate existence of genetic differences within the East African X. campestris pv. campestris population indicating that it is not a similar clonal population of the same genetic background.     

Consequences of the taxonomic revision of Xanthomonas axonopodis pv. dieffenbachiae and of the analysis of its associated pathogenicity for the recommendation of regulation (EPPO A2 List)

Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of bacterial blight of Araceae (aroids), is a regulated pest in several countries and is included in the EPPO A2 List. Reference strains of Xanthomonas axonopodis pv. dieffenbachiae have recently been reclassified into the species Xanthomonas phaseoli, Xanthomonas citri and Xanthomonas euvesicatoria on the basis of different features, including multilocus sequence analysis, average nucleotide identity and homology in DNA–DNA hybridization analyses. Based on pathogenicity tests, Constantin et al. (2017) proposed naming the pathogens on aroids as X. phaseoli pv. dieffenbachiae, X. phaseoli pv. syngonii and X. citri pv. aracearum. Recommendations are made on how to deal with these changes for the group of pathogenic bacteria for Araceae. The name Xanthomonas axonopodis pv. dieffenbachiae on the EPPO List should be adjusted to the names proposed in the taxonomic study by Constantin et al. (2016). The current EPPO Diagnostic Standard is directed at strains pathogenic on Anthurium. They mainly belong to X. phaseoli pv. dieffenbachiae, but some also to X. citri pv. aracearum that are not detected by the EPPO Diagnostic Standard. Xanthomonas phaseoli pv. syngonii strains are also aggressive, but with a host range restricted to Syngonium. The pathogenicity specific to aroids of the bacterial isolates reclassified as Xanthomonas euvesicatoria was not confirmed and no pathovar epithet has been retained for these strains.     

油菜黑腐病病原细菌的鉴定

近年来,在湖北省油莱上发生一种病害,油菜抽薹后,主轴上产生暗绿色水渍状长条斑,病部溢出大量乳黄色粘稠物,后为黑褐色腐烂,其主轴萎缩卷曲,角果干秕、枯死。被害维管束变褐,髓部变黄。叶片上很少见病斑。从这种病株上分离到了致病细菌,经全面、系统鉴定,确定为黑腐病黄色单胞杆菌[Xanthomonas campestris(Pammel)Dowsom]。由黑腐病菌引起的上述症状,国内外未曾报道过。另外,对来自六种十字花科寄生的七个黑腐病菌株,作了致病力比较测定,结果差异显著,以油莱两个黑腐病菌株致病力最强,表明可能存在不同生理型。     

Investigation of Genomic Variability of Xanthomonas Arboricola Pv. juglandis by AFLP Analysis

Xanthomonas arboricola pv. juglandis is the causal agent of walnut blight, one of the most important and widespread diseases of Persian (English) walnut (Juglans regiaL.), causing severe damage to leaves, twigs and nuts. To investigate the genomic variability of X. arboricolapv. juglandis, 66 isolates obtained from different countries (England, France, Italy, The Netherlands, Romania, Spain, USA, and New Zealand) were analysed using the Amplified Fragment Length Polymorphism (AFLP) technique. EcoRI and MseI were used as restriction endonucleases. Primers with a core sequence including endonuclease recognition sites and a 3^prime-terminal cytosine selective base for MseI primer, or no selective base for EcoRI primer, were used. Data were analysed by means of a multiple correspondence analysis. A total of 76 amplified polymorphic DNA fragments were used to compute relationships among isolates. The AFLP profiles of X. arboricola pv. juglandis isolates appeared to be reliably distinguishable from X. arboricola pv. pruni and X. arboricola pv. corylina, and from other Xanthomonas species, i.e. X. campestris pv. campestris, X. fragariae, X. hortorum, X. axonopodis pv. vesicatoria. Though this pathogen is associated with one single host genus, a high level of genomic diversity was found. This diversity might be partly explained by the geographic origin. Nevertheless, isolates with different patterns were collected within one country, and similar molecular patterns were found in isolates collected at different sites. However, genetic diversity might be influenced by exchanging vegetative material from different countries. Mixing of X. arboricola pv. juglandis isolates might have partly concealed the influence of the geographic location from which the bacteria were isolated.     

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow-pigmented bacteria isolated from blight-affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re-emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.     

Genomics‐informed design of loop‐mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level

The objective of this study was to develop a rapid, sensitive detection assay for the quarantine pathogen Xanthomonas arboricola pv. pruni, causal agent of stone fruit bacterial spot, an economically important disease of Prunus spp. Unique targets were identified from X. arboricola pv. pruni genomes using a comparative genomics pipeline of other Xanthomonas species, subspecies and pathovars, and used to identify specific diagnostic markers. Loop‐mediated isothermal amplification (LAMP) was then applied to these markers to provide rapid, sensitive and specific detection. The method developed showed unrivalled specificity with the 79 tested strains and, in contrast to previously established techniques, distinguished between phylogenetically close subspecies such as X. arboricola pv. corylina. The sensitivity of this test is comparable to that of a previously reported TaqMan^? assay at 10³ CFU mL^?1, while the unrivalled speed of LAMP technology enables a positive result to be obtained in <15 min. The developed assay can be used with real‐time fluorescent detectors for quantitative results as well as with DNA‐staining dyes to function as a simplified strategy for on‐site pathogen detection.     

Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

Specificity of polycllonal and monoclonal antibodies for the identification of Xanthomonas campestris pv. campestris

Polyclonal and monoclonal antibodies (PCAs and MCAs), produced to whole cells and flagellar extracts ofXanthomonas campestris pv.campestris (Xcc), respectively, were tested for specificity. In immunofluorescence microscopy (IF) the three PCAs tested, reacted at low dilutions with all Xcc strains, some other xanthomonads and non-xanthomonads. At higher dilutions most cross-reactivity with non-xanthomonad strains disappeared. However, the cross-reactivity with strains ofX. c. pv.vesicatoria (Xcv),X. c. pv.amoraciae (Xca) andX. c. pv.phaseoli var. fuscans (Xcpf) remained.Six MCA-producing cell clones viz. 20H6, 2F4, 18G12, 10C5, 17C12 and 16B5 were selected for specificity tests with an enzyme immunoassay (EIA), IF and a dot-blot immunoassay (DBI). None of the MCAs reacted with all Xcc strains in IF and EIA. In DBI, only MCAs 17C12 and 16B5 reacted with all Xcc strains. All six MCAs tested, cross-reacted in one of either tests with other pathovars ofX. campestris, such as Xcv or Xca. The MCAs were also tested in immunoblotting experiments using total bacterial extracts, cell envelope and flagellar extracts. MCAs 20H6, 2F4, 18G12 and 10C5 reacted with the lipopolysaccharide (LPS) of Xcc. MCAs 16B5 and 17C12 reacted with a 39 kilodalton and a 29 kilodalton protein, respectively.It is concluded that the PCAs and MCAs discussed in this study may be used for routine identification and differentiation of (a group of) Xcc strains. The significance of the cross-reactions with other pathovars ofX. campestris needs to be determined by testing seed lots.     

PCR-mediated Detection of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>by Amplification of the 16S–23S rDNA Spacer Region Sequence

A detection method specific for Xanthomonas oryzae pv. oryzae, the pathogen responsible for bacterial blight of rice, was based on the polymerase chain reaction (PCR) and designed by amplifying the 16S–23S rDNA spacer region from this bacterium. The nucleotide sequence of the spacer region between the 16S and 23S rDNA, consisting of approximately 580-bp, from X. oryzae pv. oryzae, X. campestris pv. alfalfae, X. campestris pv. campestris, X. campestris pv. cannabis, X. campestris pv. citri, X. campestris pv. cucurbitae, X. campestris pv. pisi, X. campestris pv. pruni and X. campestris pv. vitians, was determined. The determined sequences had more than 95% identity. Therefore, a pair of primers, XOR-F (5′-GCATGACGTCATCGTCCTGT-3′) and XOR-R2 (5′-CTCGGAGCTATATGCCGTGC-3′) was designed and found to specifically amplify a 470-bp fragment from all strains of X. oryzae pv. oryzae isolated from diverse regions in Japan. No PCR product was amplified from X. campestris pathovars alfalfae, campestris, cannabis, carotae, cucurbitae, dieffenbachiae, glycines, pisi, pruni, vitians or zantedeschiae, except for pathovars citri, incanae and zinniae. The method could also detect the pathogen in infected rice leaves within 3 hr, at a detection limit of 4×10¹ cfu/ml. Received 17 December 1999/ Accepted in revised form 10 April 2000     

Characterization of the pathogenicity of strains of Pseudomonas syringae towards cherry and plum

Bacterial canker is a major disease of Prunus avium (cherry), Prunus domestica (plum) and other stone fruits. It is caused by pathovars within the Pseudomonas syringae species complex including P. syringae pv. morsprunorum (Psm) race 1 (R1), Psm race 2 (R2) and P. syringae pv. syringae (Pss). Psm R1 and Psm R2 were originally designated as the same pathovar; however, phylogenetic analysis revealed them to be distantly related, falling into phylogroups 3 and 1, respectively. This study characterized the pathogenicity of 18 newly genome‐sequenced P. syringae strains on cherry and plum, in the field and laboratory. The field experiment confirmed that the cherry cultivar Merton Glory exhibited a broad resistance to all clades. Psm R1 contained strains with differential specificity on cherry and plum. The ability of tractable laboratory‐based assays to reproduce assessments on whole trees was examined. Good correlations were achieved with assays using cut shoots or leaves, although only the cut shoot assay was able to reliably discriminate cultivar differences seen in the field. Measuring bacterial multiplication in detached leaves differentiated pathogens from nonpathogens and was therefore suitable for routine testing. In cherry leaves, symptom appearance discriminated Psm races from nonpathogens, which triggered a hypersensitive reaction. Pathogenic strains of Pss rapidly induced disease lesions in all tissues and exhibited a more necrotrophic lifestyle than hemibiotrophic Psm. This in‐depth study of pathogenic interactions, identification of host resistance and optimization of laboratory assays provides a framework for future genetic dissection of host–pathogen interactions in the canker disease.