Molecular characterization ofPseudomonas syringae pv.tomato isolates from Tanzania

Bacterial speck caused byPseudomonas syringae pv.tomato is an emerging disease of tomato in Tanzania. Following reports of outbreaks of the disease in many locations in Tanzania, 56 isolates ofP. syringae pv.tomato were collected from four tomato- producing areas and characterized using pathogenicity assays on tomato, carbon source utilization by the Biolog Microplate system, polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis. All theP. syringae pv.tomato isolates produced bacterial speck symptoms on susceptible tomato (cv. ‘Tanya’) seedlings. Metabolic fingerprinting profiles revealed diversity among the isolates, forming several clusters. Some geographic differentiation was observed in principal component analysis, with isolates from Arusha region being more diverse than those from Iringa and Morogoro regions. The Biolog system was efficient in the identification of the isolates to the species level, as 53 of the 56 (94.6%) isolates ofP. syringae pv.tomato were identified asPseudomonas syringae. However, only 23 isolates out of the 56 (41.1%) were identified asPseudomonas syringae pv.tomato. The results of this work indicate the existence ofP. syringae pv.tomato isolates in Tanzania that differ significantly from those used to create the Biolog database. RFLP analysis showed that the isolates were highly conserved in theirhrpZ gene. The low level of genomic diversity within the pathogen in Tanzania shows that there is a possibility to use resistant tomato varieties as part of an effective integrated bacterial speck management plan. http://www.phytoparasitica.org posting August 8, 2008.     

The tomato Pto gene confers resistance to Pseudomonas floridensis,an emergent plant pathogen with just nine type III effectors

A newly discovered bacterial species, Pseudomonas floridensis, has emerged as a pathogen of tomato in Florida. This study compares the virulence and other attributes of P. floridensis to Pseudomonas syringae pv. tomato, which causes bacterial speck disease of tomato. Pseudomonas floridensis reached lower population levels in leaves of tomato as compared to the P. syringae pv. tomato strains DC3000 and NYT1. Analysis of the genome sequence of the P. floridensis type strain GEV388 revealed that it has just nine type III effectors including AvrPtoB_GEV388, which is 66% identical to AvrPtoB in DC3000. Five of these effectors have been previously reported to be members of a ‘minimal effector repertoire’ required for full DC3000 virulence on Nicotiana benthamiana; however, GEV388 grew poorly on leaves of this plant species compared to the DC3000 minimal effector strain. The tomato Pto gene recognizes AvrPtoB in race 0 P. syringae pv. tomato strains, thereby conferring resistance to bacterial speck disease. Pto was also found to confer resistance to P. floridensis, indicating this gene will be useful in the protection of tomato against this newly emerged pathogen.     

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.     

Reduction of Bacterial Speck (Pseudomonas syringae pv. tomato) of Tomato by Combined Treatments of Plant Growth-promoting Bacterium, Azospirillum brasilense, Streptomycin Sulfate, and Chemo-thermal Seed Treatment

Inoculation of tomato seeds with the plant growth-promoting bacterium Azospirillum brasilense, or spraying tomato foliage with A. brasilense, streptomycin sulfate, or commercial copper bactericides, separately, before or after inoculation with Pseudomonas syringae pv. tomato, the casual agent of bacterial speck of tomato, had no lasting effect on disease severity or on plant height and dry weight. Seed inoculation with A. brasilense combined with a single streptomycin foliar treatment and two foliar bactericide applications at 5-day intervals (a third or less of the recommended commercial dose) reduced disease severity in tomato seedlings by over 90% after 4 weeks, and significantly slowed disease development under mist conditions. A. brasilense did not induce significant systemic resistance against the pathogen although the level of salicylic acid increased in inoculated plants. Treatment of tomato seeds that were artificially inoculated with P. syringae pv. tomato, with a combination of mild chemo-thermal treatment, A. brasilense seed inoculation, and later, a single foliar application of a copper bactericide, nearly eliminated bacterial leaf speck even when the plants were grown under mist for 6 weeks. This study shows that a combination of otherwise ineffective disease management tactics, when applied in concert, can reduce bacterial speck intensity in tomatoes under mist conditions.     

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.     

实时荧光定量PCR法检测十字花科细菌性黑斑病菌

为有效防控我国的检疫性有害生物十字花科细菌性黑斑病菌Pseudomonas syringae pv.maculicola在国内的传播与蔓延,通过设计1对特异性引物3539,利用132株靶标和非靶标菌为模板进行PCR扩增,建立了实时荧光定量PCR法,并进行了模拟种子带菌试验。结果显示,引物3539为只针对十字花科细菌性黑斑病菌扩增出的特异性产物;在模拟种子带菌检测中,常规PCR对菌悬液的检测限为10~5CFU/m L,实时荧光定量PCR的检测限为10~3CFU/m L,其中10~8CFU/m L菌液的Ct值最低,为22.90,10~3CFU/m L菌液的Ct值最高,为35.73,且不同浓度菌液间的Ct值均有显著差异;不同带菌率模拟种子的检测结果表明,常规PCR和实时荧光定量PCR能检测到的带菌率分别为0.5%和0.1%。研究表明,实时荧光定量PCR法不仅可用于病种的检测,也可用于病害的早期诊断。     

PCR–RFLP identifies differences in hrpZ sequences to distinguish two genetic groups of Pseudomonas syringae pv. syringae strains from barley and wheat with bacterial black nod

Differences in hrpZ sequences determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) were used to investigate the molecular epidemiology of Pseudomonas syringae pv. syringae (PSS) strains that were isolated from diseased barley and wheat plants in Okayama, Japan. PCR–RFLP using HhaI separated PSS strains into two groups (A and B). Although specific PCR–RFLP groups of PSS strains were not always isolated from specific cultivars or seeds produced in a specific area, many strains isolated from barley and wheat belonged to PCR–RFLP groups A and B, respectively.     

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.     

Discrimination of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> isolates from sweet and wild cherry using rep-PCR

Bacterial canker is one of the most important diseases of cherry (Prunus avium). This disease can be caused by two pathovars of Pseudomonas syringae: pv. morsprunorum and pv. syringae. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) was investigated as a method to distinguish pathovars, races and isolates of P. syringae from sweet and wild cherry. After amplification of total genomic DNA from 87 isolates using the REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX primers, followed by agarose gel electrophoresis, groups of isolates showed specific patterns of PCR products. Pseudomonas syringae pv. syringae isolates were highly variable. The differences amongst the fingerprints of P. syringae pv. morsprunorum race 1 isolates were small. The patterns of P. syringae pv. morsprunorum race 2 isolates were also very uniform, with one exception, and distinct from the race 1 isolates. rep-PCR is a rapid and simple method to identify isolates of the two races of P. syringae pv. morsprunorum; this method can also assist in the identification of P. syringae pv. syringae isolates, although it cannot replace inoculation on susceptible hosts such as cherry and lilac.     

Effects of para-aminobenzoic acid on bacterial speck symptom development and Pseudomonas syringae pv. tomato populations in tomato leaves

Para-aminobenzoic (PABA) is reported to induce resistance against a range of plant pathogens in different crops in a salicylic acid-dependent manner. However, factors affecting its efficacy are not well understood. Foliar PABA applications on tomato seedlings reduced lesion incidence caused by Pseudomonas syringae pv. tomato (Pst) in a dose-dependent manner in distal leaves up to 18 mM under controlled environment conditions, but only three out of six commercial processing tomato cultivars tested showed a response to PABA. Leaves in direct contact with 9 and 18 mM PABA of both PABA-responsive and PABA-nonresponsive cultivars showed phytotoxicity. In a PABA-responsive cultivar, one, two and three PABA applications were equally effective at reducing lesion incidence in distal leaves, but the duration of control only persisted for approximately 7 days. Although PABA application reduced lesion incidence in distal leaves, the Pst population in leaves was unaffected. Lesions on PABA-treated plants were larger than nontreated plants, and thus the proportion of leaf surface area with lesions was unaffected by PABA treatment. In in vitro assays, 18 and 72 mM PABA produced zones of inhibition against Pst 15 and 50% larger than the ethanol control, demonstrating direct antimicrobial effects of PABA. PABA application did not affect symptom development in a mixed infection of Pst or Xanthomonas spp. in one field experiment with a PABA-responsive cultivar. Further research is needed to understand why PABA was unsuccessful in the field before it is to be used as a practical disease management tool for foliar bacterial diseases of tomato.

     

The hrpZ and hrpA genes are variable, and useful for grouping Pseudomonas syringae bacteria

The hrpS to hrpB regions from strains of Pseudomonas syringae were amplified by polymerase chain reaction (PCR) and the DNA sequence determined. The order of hrpS, hrpA, hrpZ, and hrpB was consistent among P. syringae strains. The sequence of hrpS was highly conserved. In a cluster analysis with the hrpS sequence, P. syringae strains were divided into four groups (I, II, III, and IV) and one undetermined strain, in agreement with previous studies. In contrast, the hrpZ sequences contained insertions, deletions, and base substitutions followed by changes in amino acids. Based on cluster analysis of hrpA, hrpZ, and hrpB, P. syringae strains could be divided into five groups. One of the four groups (group I) in the cluster analysis of hrpS could be further divided into two subgroups (groups IA and IB). Groups II, III, and IV were the same in the two analyses. Group-specific primers were designed, based on the DNA sequences of hrpZ, that could differentiate the groups of P. syringae strains. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB112552 to AB112581     

Detection of Pseudomonas syringae pv. actinidiae using polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

Several published polymerase chain reaction (PCR) primers to identify Pseudomonas syringae pv. actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains of P. syringae pv. actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen, P. syringae pv. theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections as P. syringae pv. actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB, acnB, rpoD, pgi and cts) showed that none of these six strains was phylogenetically similar to P. syringae pv. actinidiae. In contrast to the P. syringae pv. actinidiae type strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified as P. syringae pv. actinidiae. It was not possible to distinguish P. syringae pv. actinidiae from the phylogenetically similar P. syringae pv. theae using the ITS, gyrB, acnB, rpoD, pgi or cts gene regions to design PCR primers. Because P. syringae pv. theae is unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.     

Systemic resistance to bacterial leaf speck pathogen in Arabidopsis thaliana induced by the culture filtrate of a plant growth-promoting fungus (PGPF) Phoma sp. GS8-1

The culture filtrate (CF) from the plant growth-promoting fungus Phoma sp. GS8-1 was found to induce systemic resistance in Arabidopsis thaliana against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), and the underlying mechanism was studied. Roots of A. thaliana were treated with CF from GS8-1, and plants expressed a clear resistance to subsequent Pst infection; disease severity was reduced, and proliferation of pathogen was suppressed. Various mutants of A. thaliana were used to test whether the CF induced resistance through one of the known signaling pathways: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The CF was fully protective against Pst in Arabidopsis mutants jar1 and ein2 similar to wild-type plants. However, its efficacy was reduced in plants containing transgene NahG. Examination of systemic gene expression revealed that CF modulates the expression of SA-inducible PR-1, PR-2 and PR-5 genes, the JA/ET-inducible ChitB gene, and the ET-inducible Hel gene. Moreover, the expression of these genes was further enhanced upon subsequent stimulation after attack by Pst. Our data suggest that in addition to a partial requirement for SA, the signals JA and ET may also play a role in defense signaling in Arabidopsis.     

Differentiation of some pathovars of Pseudomonas syringae with monoclonal antibodies1

Two stable hybridoma clones secreting antibodies specific for three pathovars of Pseudomonas syringae were obtained from a fusion of murine myeloma cells with spleen cells of BALB/c mouse immunized with P. syringae pv. savastanoi. Undiluted hybridoma culture medium reacted strongly in indirect ELISA tests with 20 strains of pv. savastanoi, 10 strains of pv. tomato, and 3 strains of pv. papulans. There were no reactions with 23 (of 24) strains of pv. glycinea, three strains each of pvs pisi and tabaci, two strains of pv. tagetis and one each of pvs lachrymans and aptata. Hybridomas also reacted positively with six of 16 strains of pv. syringae and with one of three strains of pv. phaseolicola.     

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.     

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.     

Plant amphipathic proteins delay the hypersensitive response caused by harpinPssandPseudomonas syringaepv.syringae

Harpin_Pssfrom the plant pathogenPseudomonas syringaepv.syringaeis a proteinaceous elicitor that induces a hypersensitive response (HR) in non-host plants. The plant products which recognize harpin_Pssin the triggering of the HR are not yet known. According to the elicitor-receptor model, we hypothesize that an exogenous cell membrane receptor infiltrated into the intercellular space will interfere with the interaction between harpin_Pssand the putative receptor. We demonstrate a plant amphipathic protein (AP1) which can postpone the HR induced by harpin_Pssas well asP. syringaepv.syringae.AP1 was extracted by solubilizing proteins from healthy leaves in the non-polar n-octanol buffer followed by a polar Tris buffer. The amphipathic extracts were then further separated by gel filtration and anion exchange chromatography to obtain highly purified AP1. Similar proteins can be extracted from cotton, tomato, and sweet pepper. The N-terminal amino acid sequence of AP1 is conserved among cotton, tomato, and sweet pepper. The postponement of the harpin_Pss-mediated HR was characterized as a competitive dosage-dependent pattern of AP1. An analysis of the bacterial population development indicates that the effect of AP1 on the postponement of bacteria-mediated HR was attributed to the suppression of bacterial growth during the early stages of the HR. The time course analysis of the infiltration indicates that the postponement of HR resulted from the co-interaction between AP1 and the bacteria. Based on these results, we suggest that the postponement of bacteria-mediated HR is due to the interference of the interaction between harpin_Pssand the putative receptor in the plant. Our research provides a new approach to elucidating the role that plants may play in the nonhost response caused by pathogens.     

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

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

Cellular reactions in Arabidopsis following challenge by strains of Pseudomonas syringae: From basal resistance to compatibility

Reactions to strains of the bean halo-blight pathogen Pseudomonas syringae pv. phaseolicola (Pph) strain 1448A and the Arabidopsis and tomato pathogen P.s. pv. tomato (Pst) strain DC3000 were examined by transmission electron microscopy. Wild-type and hrpL mutant strains of Pph failed to multiply in the accession Columbia (Col)-5, but did not cause a hypersensitive reaction (HR). Symptomless non-host resistance to 1448A and the hrpL mutant was associated with the progressive alteration of the plant cell wall adjacent to bacteria, following the accumulation of membrane bound vesicles within the cytoplasm at reaction sites. Large papillae containing callose accumulated within challenged plant cells. Papillae also formed in the pmr4–1 mutant of Col-0 which lacks an inducible callose synthase but immunocytochemical labelling demonstrated that they contained very little β-1, 3 glucan. Some papillae formed in Col-5 in response to the virulent pathogen DC3000, but they dispersed during cell collapse and lesion formation. Transconjugants of Pph expressing the avirulence genes avrPpiA and avrPphB matching the RPM1 and RPS5 resistance genes, caused rapid and slow HR development, respectively. Although corpse morphology was observed our observations suggest that in Arabidopsis, plant cell death during the HR is programmed but represents a variant of necrosis rather than apoptosis. Cerium chloride staining revealed the accumulation of H₂O₂ at reaction sites. The strongest H₂O₂ response was found during the HR activated by avrPpiA but localised generation of peroxide was also found at sites of papilla deposition next to 1448A or the hrpL mutant. Accumulation of H₂O₂ during the HR, but not during wall alterations, was strongly suppressed by inhibition of NADPH oxidase. The differential effect of the inhibitor suggests an alternative source of H₂O₂ to modify the plant wall. Extension of peroxide-driven cross-linking reactions to bacterial cell walls may contribute to the restriction of bacterial multiplication. The lowest level of H₂O₂ occurred during the compatible reaction to DC3000. Characterisation of the cellular co-ordination of basal (non-host) resistance has revealed several potential targets for bacterial effector proteins.