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.     

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     

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.     

Functional analysis of the 3′ end of avrBs3/pthA genes from two Xanthomonas species

The phytopathogens Xanthomonas oryzae pathovar (pv.) oryzae and Xanthomonas axonopodis pv. citri each contain several avrBs3/pthA family genes. Structural features of these genes important for avirulence and/or virulence functions include a central region of multiple direct repeats and three nuclear localization signals (NLSs) and an acidic activation domain (AAD) at the 3′ end. To identify other regions critical to function in the 3′ ends of these genes, we constructed several chimeras using apl1 and apl2 from X. axonopodis pv. citri and avrXa10 and avrXa7 from X. oryzae pv. oryzae and evaluated their functions by inoculation to citrus and rice. The apl1 and avrXa7 genes are major virulence determinants in citrus and rice, respectively, while the contributions of apl2 and avrXa10 to virulence are negligible or not measurable. Constructs that contained a 417 bp HincII-SphI fragment from the 3′ end of apl1 in combination with the repeats from avrXa7, avrXa10, and apl1 caused a canker phenotype on citrus. Interchange of the HincII-SphI fragment between avrXa7 and avrXa10 abolishes avrXa7 avirulence function and reduces its virulence but it does not affect avrXa10 avirulence function in rice. avrXa7 caused a hypersensitive response (HR) in citrus and replacement of it's 3′ end with that of apl1 resulted in loss of canker and induction of HR. Thus, the HincII-SphI fragment of the avrBs3/pthA gene family is important for avirulence and virulence functions in two different plant species, Oryza sativa and Citrus natsudaidai HAYATA.     

Selection forMeloidogyne incognita virulence against resistance genes from tomato and pepper and specificity of the virulence/resistance determinants

Experiments were designed to analyze the relationships between the root-knot nematodeMeloidogyne incognita and resistant tomato and pepper genotypes. From a natural avirulent isolate, near-isogenic nematode lineages were selected with virulence either against the tomatoMi resistance gene or the pepperMe3 resistance gene. Despite the drastic selection pressure used, nematodes appeared unable to overcome the pepperMe1 gene, therefore suggesting some differences in the resistance conferred byMe1 andMe3 in this species. Nematodes virulent onMi-resistant tomatoes were not able to reproduce onMe1-resistant nor onMe3-resistant peppers, and nematodes virulent onMe3-resistant peppers were not able to reproduce onMi-resistant tomatoes nor onMe1-resistant peppers. These results clearly demonstrate the specificity ofM. incognita virulence against resistance genes from both tomato and pepper, and indirectly suggest that gene-for-gene relationships could occur between these two solanaceous crops and the nematode.     

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.     

Polyphosphate kinase is essential for swarming motility, tolerance to environmental stresses, and virulence in Pseudomonas syringae pv. tabaci 6605

Polyphosphate kinase (PPK), encoded by the ppk gene, is a principal enzyme responsible for synthesis of inorganic polyphosphate (poly P) from ATP in many Gram-negative bacteria. In order to elucidate the functions of poly P in Pseudomonas syringae pv. tabaci 6605, an in-frame deletion mutant of the ppk gene (ppk) was constructed. The ppk mutant did not accumulate poly P, whereas the wild-type strain accumulated a large quantity. The mutant had reduced swarming motility, even though it retains swimming motility like the parental strain. The mutant exhibited increased sensitivity to prolonged incubation and environmental stresses, such as heat shock and oxidative stress and reduced exopolysaccharide (EPS) production compared to the wild-type. Northern blot analysis revealed that expression of the rpoS gene, encoding the stationary phase sigma factor RpoS, was reduced in ppk in the logarithmic phase, indicating that rpoS is regulated by the ppk gene. The poly P deficient mutant had significantly reduced ability to cause disease in its host tobacco plant and in planta growth of the mutant was also significantly reduced in host tobacco leaves as compared to the wild-type strain. Thus, our results suggest that poly P plays an important role in the virulence of P. syringae pv. tabaci 6605.     

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.     

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.     

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.     

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.     

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     

Age-related resistance to Pseudomonas syringae pv. tomato is associated with the transition to flowering in Arabidopsis and is effective against Peronospora parasitica

As plants mature it has been observed that some become more resistant to normally virulent pathogens. The ability to manifest the Age-Related Resistance (ARR) response in Arabidopsis to Pseudomonas syringae pathovars tomato (Pst) coincided with the transition to flowering in plants both delayed and accelerated in the transition to flowering. ARR was also associated with a change in PR-1 gene expression, such that young plants expressed PR-1 abundantly at 3 days post inoculation (dpi) while mature plants expressed much less. The Arabidopsis ARR response requires SA accumulation via isochorismate synthase (ICS1) [24]. ICS1 was expressed one dpi with virulent and avirulent Pst in both young and mature plants. The ARR response was also effective versus avirulent Pst providing an additional 4-fold limitation in bacterial growth. Arabidopsis ARR was found to be ineffective against two necrotrophs, Erwinia carotovora subspecies carotovora (bacterium) and Botrytis cinerea (fungus) and one obligate biotroph, Erysiphe cichoracearum (fungus). However, mature wild type, SA-deficient sid2 and NahG plants supported little growth of the obligate biotrophic oomycete, Peronospora parasitica. Therefore ARR to P. parasitica appears to be SA-independent, however the level of ARR resistance was somewhat reduced in these mutants in some experiments. Thus, there may be numerous defence pathways that contribute to adult plant resistance in Arabidopsis.     

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.     

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.     

Molecular characterization and in planta detection of Fusarium moniliforme endopolygalacturonase isoforms

The activation of Fusarium moniliforme endopolygalacturonase (endoPG) was studied during infection of maize plants. EndoPG is a plant cell wall degrading enzyme that cleaves the pectin component causing cell death. The authors generated several hybridoma cell lines producing endoPG specific monoclonal antibodies. One monoclonal antibody was selected and successfully used in Western blotting analysis to detect F. moniliforme endoPG secretion in vitro and in planta. Two F. moniliforme strains (FC-l0 and 62264) were used for the studies. Both strains revealed the expression of a single endoPG in vitro as in planta. EndoPG from strain FC-10 presented four isoforms whereas only two isoforms were revealed in the endoPG from strain 62264. Differences were also found in the sequences of the two endoPG genes indicating the presence of endoPG variability among F. moniliforme strains.     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.