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

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

Identification and Discrimination of Pseudomonas syringae Isolates from Wild Cherry in England

A survey of wild cherry (Prunus avium) woodland plantations and nurseries was carried out in 2000/01. Trees with symptoms of bacterial canker were found in 20 of the 24 plantations visited and in three of seven nurseries. Fifty-four Pseudomonas syringae isolates from wild cherry together with 22 representative isolates from sweet cherry and 13 isolates from other Prunus spp., pear and lilac were characterised by physiological, biochemical, serological and pathogenicity tests. Isolates from wild cherry were predominantly P. syringae pv. syringae (Pss), but P. syringae pv. morsprunorum (Psm) races 1 and 2 were also found. Physiological and biochemical tests discriminated Psm races 1 and 2 from other P. syringae isolates. Agglutination and indirect-enzyme-linked immunosorbent assay tests with three different antisera showed that Psm race 1 and race 2 were very uniform and indicated high variability amongst other P. syringae isolates. However, pathogenic Pss isolates could not be distinguished from non-pathogenic isolates of P. syringae on the basis of physiological, biochemical or serological tests. Pathogenicity tests on rooted lilac plants and on micropropagated plantlets of lilac and two wild cherry clones differentiated Pss and Psm isolates and demonstrated a range of aggressiveness amongst Pss isolates. Serological tests could be used as an alternative to the classical physiological and biochemical tests to increase the speed of detection and discrimination of isolates, but pathogenicity tests are still necessary to discriminate the pathogenic Pss isolates.     

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.     

The use of PCR melting profile for typing of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> isolates from stone fruit trees

Of thirty fluorescent Pseudomonas isolates originating from symptomatic tissues of sweet (Prunus avium) and sour cherry (Prunus cerasus), plum (Prunus domestica), peach (Prunus persica) and apricot (Prunus armeniaca), 23 were identified as P. syringae using LOPAT tests. Further characterization of those isolates by GATTa and L-lactate utilization tests showed that 10 of them belonged to race 1, six to race 2 of P. syringae pv. morsprunorum (Psm) and six other isolates were identified as pathovar syringae (Pss). One isolate (791) was determined as atypical. Phenotypic determination and genetic analysis of studied isolates for toxin production revealed that isolates of Pss produced syringomycin, 3 Psm race 1 produced coronatine and 6 Psm race 2 produced yersiniabactin. Genetic diversity of all isolates was evaluated with the PCR melting profile (PCR MP) method. A dendrogram constructed with PCR MP patterns showed positive correlation with phenotypically distinguished pathovars. Isolates of Psm races 1 and 2 formed distinct, tight clusters, whereas Pss isolates were more heterogeneous. Isolate 791 was placed within Pss isolates. Bacteria identified as Pss caused more severe symptoms on immature cherry fruits compared to Psm, which corresponded to determined pathovars and races.     

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.     

Identification of pathovars and races of <Emphasis Type="Italic">Pseudomonas syringae,</Emphasis> the main causal agent of bacterial disease in pea in North-Central Spain,and the search for disease resistance

A total of 298 bacterial isolates were collected from pea cultivars, landraces and breeding lines in North-Central Spain over several years. On the basis of biochemical-physiological characteristics and molecular markers, 225 of the isolates were identified as Pseudomonas syringae, either pv. pisi (110 isolates) or pv. syringae (112), indicating that pv. syringae is as frequent as pv. pisi as causal agent of bacterial diseases in pea. Most strains (222) were pathogenic on pea. Further race analyses of P. syringae pv. pisi strains identified race 4 (59.1% of the isolates of this pathovar), race 2 (20.0%), race 6 (11.8%), race 5 (3.6%) and race 3 (0.9%). Five isolates (4.6%) showed a not-previously described response pattern on tester pea genotypes, which suggests that an additional race 8 could be present in P. syringae pv. pisi. All the isolates of P. syringae pv. syringae were highly pathogenic when inoculated in the tester pea genotypes, and no significant pathogenic differences were observed. Simultaneous infections with P. syringae pv. pisi and pv. syringae in the same fields were observed, suggesting the importance of resistance to both pathovars in future commercial cultivars. The search for resistance among pea genotypes suitable for production in this part of Spain or as breeding material identified the presence of resistance genes for all P. syringae pv. pisi races except for race 6. The pea cultivars Kelvendon Wonder, Cherokee, Isard, Iceberg, Messire and Attika were found suitable sources of resistance to P. syringae pv. syringae.     

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.     

Molecular characterization of Pseudomonas syringae isolates from fruit trees and raspberry in Serbia

Infection of fruit trees by Pseudomonas syringae is a potentially serious problem that may limit the establishment and sustained productivity of pome and stone fruit orchards in Serbia. To estimate possible diversity of Pseudomonas syringae fruit trees strains, we collected a set of strains in several areas of Serbia. The samples were taken from infected orchards with raspberry, plum, cherry, sour cherry, peach, pear and apple trees. Genetic diversity of P. syringae strains isolated from fruit trees was determined by using SpeI macrorestriction analysis of genomic DNAs by pulsed-field gel electrophoresis (PFGE) and REP-PCR. Molecular analysis showed that most of isolates had unique profiles, with the exception of isolates from plum and cherry that displayed profiles identical to each other and similar to P. syringae pv. morsprunorum. The study presented here clearly demonstrates the discriminative power of molecular techniques in enabling a detailed analysis of the genetic variations between strains of P. syringae from different pome and stone fruit hosts in Serbia.     

Identification of two serological flagellar types (H1 and H2) inPseudomonas syringae pathovars

Flagellar antigen specificity was studied for the speciesPseudomonas syringae, P. viridiflava andP. cichorii. After checking their motility, bacteria were reacted against six polyclonal antisera containing anti-O (LPS) and anti-H (flagellar) antibodies by indirect immunofluorescent staining. Two distinct flagellar serotypes (H1 and H2) were described. The distribution of H1 and H2 serotypes was then determined for a collection of 88 phytopathogenicPseudomonas strains. Serotype H1 was possessed byP. syringae pv.aptata (12 strains),P. s. pv.helianthi (2),P. s. pv.pisi (11), andP. s. pv.syringae (13). Serotype H2 was possessed byP. cichorii (2),P. s. pv.delphinii (1),P. s. pv.glycinea (4),P. s. pv.lacrymans (1),P. s. pv.mori (1),P. s. pv.morsprunorum (10),P. s. pv.persicae (1),P. s. pv.phaseolicola (8),P. s. pv.tabaci (10) andP. s. pv.tomato (1).P. viridiflava (5) revealed HI, H2 and untyped flagella. The following isolates were untypable by the H1/H2 system:P. corrugata (3),P. fluorescens (2),P. tolaasii (1). H1/H2 serotypes distribution is not linked toP. syringae O-serogroups. On the other hand, H1/H2 distribution seems remarkably linked to the new genospecies of theP. syringae group.Abbreviations CFBP French Collection of Phytopathogenic Bacteria, Angers, France - ICMP International Collection of Micro-organisms from Plants, Auckland, New-Zealand - NCPPB National Collection of Plant Pathogenic Bacteria, Harpenden, Great Britain     

Discrimination of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> races among strains from northwestern Spain by <Emphasis Type="Italic">Brassica</Emphasis> spp. genotypes and rep-PCR

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a severe seedborne disease of Brassica crops around the world. Nine races are recognized, being races 1 and 4 the most aggressive and widespread. The identification of Xcc races affecting Brassica crops in a target area is necessary to establish adequate control measures and breeding strategies. The objectives of this study were to isolate and identify Xcc strains from northwestern Spain by using semi-selective medium and pathogenicity tests, determine the existing races of Xcc in this area by differential series of Brassica spp., and evaluate the use of repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) to differentiate among the nine existing Xcc races. Seventy five isolates recovered from infected fields were identified as Xcc. Race-typing tests determined the presence of the following seven pathogen races: 1, 4, 5, 6, 7, 8 and 9. Race 4 was the most frequent in Brassica oleracea and race 6 in Brassica rapa crops, therefore breeding should be focussed in obtaining resistant varieties to both races. Cluster analysis derived from the combined fingerprints showed four groups, but no clear relationship to race, crop or geographical origin was found. Rep-PCR analysis was found not to be a reliable method to discriminate among Xcc races, therefore race typing of Xcc isolates should be done by using the differential series of Brassica spp. genotypes or another alternative approach.     

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.     

<Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">cerasicola</Emphasis>, pv. nov., the Causal Agent of Bacterial Gall of Cherry Tree

Bacterial gall on trunks and twigs of cherry trees (Prunus × yedoens, Someiyoshino) was found in Miyazaki and Saga prefectures, Japan. The surface of young galls are relatively smooth and light brown, but they become rough and dark brown. Characteristics of the bacterium isolated from galls on trunks or twigs are similar to those of Pseudomonas syringae pathovars, i.e., pv. actinidiae, pv. daphniphylli, pv. dendropanacis, pv. Morsprunorum, pv. myricae, pv. rhaphiolepidis, pv. syringae and pv. tremae. This bacterium produced galls on cherry and apricot, but not on 66 other species of plants belonging to 39 families. From these results, this bacterium was classified as a new pathovar of Pseudomonas syringae, and the name Pseudomonas syringae pv. cerasicola, pv. nov., is proposed. Strain M9501(ICMP 13926) was designated as the pathotype strain. Received 10 September 1999/ Accepted in revised form 24 December 1999     

Nucleotide Sequence and Organization of Copper Resistance Genes from Pseudomonas syringae pv. actinidiae

Copper-containing bactericides have been used to control bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae. However, the efficacy of copper has been reduced by the occurrence of copper-resistant strains. Analysis of the DNA sequence of a cluster region containing the copper-resistance genes from P. syringae pv. actinidiae suggested the presence of three possible different systems for copper resistance: copper-trapping, copper-efflux and copper-transport systems. Transposon insertional inactivation analysis indicated that the copper-trapping system was essential for copper resistance.     

A strain ofPseudomonas syringae which does not belong to pathovarphaseolicola produces phaseolotoxin

A bacterial strain, CFBP 3388, isolated from Vetch (Vicia sativa, L.) was identified asP. s. pv.syringae on the basis of nutritional and biochemical patterns which were obtained with classical tests and the Biolog system. It caused necrotic symptoms typical ofP. s. pv.syringae on bean leaves and pods after artificial inoculation. However, the isolate caused a citrulline-reversible inhibition ofE. coli in phaseolotoxin bioassay. Furthermore, with CFBP 3388 DNA as template a 1900 bp DNA fragment, specific for the phaseolotoxin DNA cluster ofP. s. pv.phaseolicola, was amplified by PCR. This is the first demonstration that an isolate ofP. syringae that is not pv.phaseolicola can produce phaseolotoxinAbbreviations bp base pair - kb kilobase - OCT Ornithine Carbamoyl Transferase     

Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst

A rapid detection method based on PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primer design was based on the P. syringae DC3000 hrpZ_Pst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island.A 532 bp product corresponding to an internal fragment of hrpZ_Pst was amplified from 50 isolates of P. syringae pv. tomato belonging to a geographically representative collection. The amplification product was also obtained from three coronatine-deficient strains of P. syringae pv. tomato.On the other hand, PCR did not produce any such products from 100 pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and 75 unidentified bacterial saprophytes isolated from tomato plants. The method was tested using leaf and fruit spots from naturally-infected tomato plants and asymptomatic nursery plants and artificially contaminated tomato seeds. The results confirmed the high specificity observed using pure cultures.     

Multiphasic Approach for the Identification of the Different Classification Levels Of Pseudomonas savastanoi Pv. Phaseolicola

The relationships between strains of Pseudomonas savastanoi pv. phaseolicola (P. sav. phaseolicola), P. syringae pv. tabaci (P. syr. tabaci) and P. syr. syringae which all cause disease on bean; the related species P. sav. glycinea and P. syr. actinidiae, and reference bacteria, were evaluated by studying the phenotypic and genetic diversity of a collection of 62 strains. All the P. sav. phaseolicola strains tested produced characteristic watersoaked lesions on bean pods. Other pathovars produced varying combinations of symptoms including necrotic lesions, with or without watersoaked centres and sunken tissue collapse of the lesion (P. syr. tabaci) and necrotic lesions with or without sunken collapse (P. syr. syringae). At the genomospecies level, all the strains of P. sav. phaseolicola, P. sav. glycinea and P. syr. tabaci, belonging to genomospecies 2, could be separated from P. syr. syringae strains (genomospecies 1) and P. syr. actinidiae strains (unknown genomospecies) by BOX-PCR and DNA/DNA hybridisation. To distinguish P. sav. phaseolicola, within genomospecies 2, from P. sav. glycinea and P. syr. tabaci, it was necessary to perform nutritional characterisations myo-inositol negative and p-hydroxy benzoate positive for P. sav. phaseolicola strains), PCR with specific primers designed from the tox region (positive for all of the P. sav. phaseolicola strains) and serotyping, as 71% of the P. sav. phaseolicola strains reacted as O-serogroup PHA1. Important intrapathovar variation was seen by genomic fingerprinting with REP and ERIC primers, as well as with RAPD primers (AE7 and AE10) and esterase profilings. While RAPD fingerprinting detected variability correlated with two race-associated evolutionary lines, REP, ERIC and esterase profiles revealed intrapathovar variation linked to some host origins, that separated the kudzu isolates, and the mungbean isolates, from the other P. sav. phaseolicola strains.     

An ultrastructural study, including cytochemistry and quantitative analyses, of the interactions between pseudomonads and leaves of Phaseolus vulgaris L.

Infection of Phaseolus vulgaris cultivars Red Mexican and Tendergreen with Pseudomonas fluorescens, P. syringae pv. coronafaciens, P.s. pv. phaseolicola races 1 and 3, and a mutant of race 3 (race 3 M1) with altered cultivar specific virulence was examined. In addition to qualitative observations of the development of colonies of bacteria and the responses in adjacent plant cells, quantitative analyses were made of the numbers of bacteria within sections of colonies, contact between bacteria and the plant cell wall, the accumulation of fibrillar acidic polysaccharides (which stained with ruthenium red) around bacteria, convolution of the plant cell membrane adjacent to bacteria, deposition of paramural papillae, rupture of the tonoplast and the occurrence of cytoplasmic disorganization. The presence at infection sites of material staining with the periodic acid, thiocarbohydrazide silver proteinate (PATAg) procedure to localize vicinal glycols was also quantified.Cells of P. fluorescens failed to multiply in the plant and seemed tightly bound at junctions between mesophyll cells. The pathovars of P. syringae all multiplied at similar rates during the first 12 h after inoculation and were not closely attached to the plant cell wall. Fibrillar, ruthenium red staining material, considered to be bacterial extracellular polysaccharides, accumulated around cells of the pathovars of P. syringae irrespective of the compatibility of their interaction with cultivars. Amorphous PATAg positive deposits formed around cells of the saprophyte and as condensed aggregates in colonies of P. syringae pathovars in tissue undergoing the hypersensitive reaction (HR) but not during compatible interactions. The PATAg positive material may be involved in the elicitation of responses by plants during the HR. The first response of plant cells to adjacent bacteria was the localized convolution of the plasma membrane; this response was neither race nor species specific. Deposition of paramural papillae was also found to be a non-specific response occurring during compatible and incompatible interactions. Some components of papillae were PATAg positive. Plasmolytic studies demonstrated that, during the HR, irreversible damage to the plasma membrane first occurred 5 and 8 h after initial convolution of the membrane following inoculation of cv. Tendergreen with P.s. pv. coronafaciens and P.s. pv. phaseolicola race 3 respectively. Tonoplast dysfunction appeared to follow irreversible damage to the plasma membrane and preceded loss of compartmentation and cytoplasmic collapse. The ultrastructural study showed that many plant responses were non-specific and therefore could be separated from irreversible damage to the plasma membrane which was the irrevocable event during the HR.     

Resistance to Pseudomonas syringae in a collection of pea germplasm under field and controlled conditions

A total of 242 Pisum accessions were screened for resistance to Pseudomonas syringae pv. pisi under controlled conditions. Resistance was found to all races, including race 6 and the recently described race 8. Fifty‐eight accessions were further tested for resistance to P. syringae pv. syringae under controlled conditions, with some highly resistant accessions identified. Finally, a set of 41 accessions were evaluated for resistance to P. syringae pv. pisi and pv. syringae under spring‐ and winter‐sowing field conditions. R2, R3 and R4 race‐specific resistance genes to P. syringae pv. pisi protected pea plants in the field. Resistance sources to race 6 identified under controlled conditions were ineffective in the field. Frost effects were also evaluated in relation to disease response. Results strongly suggest that frost tolerance is effective in lowering the disease effects caused by P. syringae pv. pisi and pv. syringae under frost‐stress conditions, even in the absence of disease resistance genes, although the highest degree of this protection is reached when frost tolerance and disease‐resistance genes are combined in the same genetic background.     

Differential Responses to Pea Bacterial Blight in Stems, Leaves and Pods Under Glasshouse and Field Conditions

Resistance to pea bacterial blight (Pseudomonas syringae pv. pisi) in different plant parts was assessed in 19 Pisum sativum cultivars and landraces, carrying race-specific resistance genes (R-genes) and two Pisum abyssinicum accessions carrying race-nonspecific resistance. Stems, leaves and pods were inoculated with seven races of P. s. pv. pisi under glasshouse conditions. For both race-specific and nonspecific resistance, a resistant response in the stem was not always associated with resistance in leaf and pod. Race-specific genes conferred stem resistance consistently, however, there was variability in the responses of leaves and pods which depended on the matching R-gene and A-gene (avirulence gene in the pathogen) combination. R2 generally conferred resistance in all plant parts. R3 or R4 singly did not confer complete resistance in leaf and pod, however, R3 in combination with R2 or R4 enhanced leaf and pod resistance. Race-nonspecific resistance conferred stem resistance to all races, leaf and pod resistance to races 2, 5 and 7 and variable reactions in leaves and pods to races 1, 3, 4 and 6.Disease expression was also studied in the field under autumn/winter conditions. P. sativum cultivar, Kelvedon Wonder (with no R genes), and two P. abyssinicum accessions, were inoculated with the most frequent races in Europe under field conditions (2, 4 and 6). Kelvedon Wonder was very susceptible to all three races, whereas P. abyssinicum was much less affected. The combination of disease resistance with frost tolerance in P. abyssinicum enabled plants to survive through the winter. A breeding strategy combining race-nonspecific resistance derived from P. abyssinicum with race-specific R-genes should provide durable resistance under severe disease pressure.     

A functional gene-for-gene interaction is required for the production of an oxidative burst in response to infection with avirulent Pseudomonas syringae pv. tomato in Arabidopsis thaliana

An early event correlated with the gene-for-gene hypersensitive response (HR) is the accumulation of active oxygen species (AOS), also known as the oxidative burst. We present data that genetically demonstrates that the oxidative burst is a downstream component of the RPS2- avrRpt2gene-for-gene signal cascade. An in planta AOS assay using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was modified for use with the Arabidopsis thaliana / Pseudomonas syringae pv.tomato (P. syringae pv. tomato) model system. An oxidative burst occurred between 8 and 15 hpi with avirulent P. syringae pv. tomato(avrRpt2), but not with virulent P. syringae pv. tomato. This burst preceded cell death and was not observed in the RPS2 Arabidopsis mutantsrps2-101C and rps2-201 inoculated with avirulent P. syringae pv. tomato. An HR-like response has been observed when plants undergoing a systemic acquired resistance (SAR) response are challenged with a normally virulent pathogen (manifestation stage of SAR), however an HR-like oxidative burst was not detected by the in planta AOS assay during this stage of SAR.