CCD detection of lux-marked Pseudomonas syringae pv. phaseolicola L-forms associated with Chinese cabbage and the resulting disease protection against Xanthomonas campestris

R.N. WATERHOUSE, H. BUHARIWALLA, D. BOURN, E.J. RATTRAY AND L.A. GLOVER. 1996. CCD image-enhancement was used to detect lux -marked Pseudomonas syringae pv. phaseolicola L-form bacteria in association with Chinese cabbage. Bioluminescence from colonizing bacteria was maximal 8 d after association. Bioluminescing L-forms were reisolated from 22 d post-association tissue. L-forms but not parental, cell-walled forms, were effective at inducing protection against a heterologous pathogen ( Xanthomonas campestris ).     

Pseudomonas syringae pv. phaseolicola: from 'has bean' to supermodel

Pseudomonas syringae pv. phaseolicola causes halo blight of the common bean, Phaseolus vulgaris, worldwide and remains difficult to control. Races of the pathogen cause either disease symptoms or a resistant hypersensitive response on a series of differentially reacting bean cultivars. The molecular genetics of the interaction between P. syringae pv. phaseolicola and bean, and the evolution of bacterial virulence, have been investigated in depth and this research has led to important discoveries in the field of plant-microbe interactions. In this review, we discuss several of the areas of study that chart the rise of P. syringae pv. phaseolicola from a common pathogen of bean plants to a molecular plant-pathogen supermodel bacterium. TAXONOMY: Bacteria; Proteobacteria, gamma subdivision; order Pseudomonadales; family Pseudomonadaceae; genus Pseudomonas; species Pseudomonas syringae; Genomospecies 2; pathogenic variety phaseolicola. MICROBIOLOGICAL PROPERTIES: Gram-negative, aerobic, motile, rod-shaped, 1.5 μm long, 0.7-1.2 μm in diameter, at least one polar flagellum, optimal temperatures for growth of 25-30°C, oxidase negative, arginine dihydrolase negative, levan positive and elicits the hypersensitive response on tobacco. HOST RANGE: Major bacterial disease of common bean (Phaseolus vulgaris) in temperate regions and above medium altitudes in the tropics. Natural infections have been recorded on several other legume species, including all members of the tribe Phaseoleae with the exception of Desmodium spp. and Pisum sativum. DISEASE SYMPTOMS: Water-soaked lesions on leaves, pods, stems or petioles, that quickly develop greenish-yellow haloes on leaves at temperatures of less than 23°C. Infected seeds may be symptomless, or have wrinkled or buttery-yellow patches on the seed coat. Seedling infection is recognized by general chlorosis, stunting and distortion of growth. EPIDEMIOLOGY: Seed borne and disseminated from exudation by water-splash and wind occurring during rainfall. Bacteria invade through wounds and natural openings (notably stomata). Weedy and cultivated alternative hosts may also harbour the bacterium. DISEASE CONTROL: Some measure of control is achieved with copper formulations and streptomycin. Pathogen-free seed and resistant cultivars are recommended. USEFUL WEBSITES: Pseudomonas-plant interaction http://www.pseudomonas-syringae.org/; PseudoDB http://xbase.bham.ac.uk/pseudodb/; Plant Associated and Environmental Microbes Database (PAMDB) http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; PseudoMLSA Database http://www.uib.es/microbiologiaBD/Welcome.html.     

Production of monoclonal antibodies against Xanthomonas campestris pv. mangiferaeindicae and their use to investigate differences in virulence

G.M. SANDERS, J.A. VERSCHOOR, S. VAN WYNGAARD, L. KORSTEN AND J.M. KOTZE 1994. Four Xanthomonas campestris pv. mangiferaeindicae isolates from mango black spot lesions were grouped according to differences in virulence and used to raise monoclonal antibodies (mAbs). Two immunization approaches were followed. In the first, four groups of mice were immunized, each with a different isolate and the spleens from each group homogenized together for cell fusion. The second approach entailed immunization of a single group of mice with bacteria pooled from all four isolates. The resultant mAbs were characterized with regard to the antigen binding specificity and antibody class. A relationship between mAb binding specificity and virulence of the bacteria was shown by Western blot analysis.     

Gene-for-gene interactions between Pseudomonas syringae pv. phaseolicola and Phaseolus.

The gene for cultivar-specific avirulence to Phaseolus vulgaris cv. Tendergreen in races 3 and 4 of Pseudomonas syringae pv. phaseolicola was isolated and sequenced. Genomic clones from libraries of race 3 in pLAFR1 and race 4 in pLAFR3, which altered the phenotype of race 5 from virulent to avirulent in Tendergreen, were found to possess a common approximately 15-kb region of DNA that contained the determinant of avirulence. Subcloning and insertion mutagenesis with Tn1000 located an avirulence gene within a 1.4-kb BglII/HindIII DNA fragment in races 3 and 4. Comparison of the nucleotide sequences of regions of DNA that confer avirulence confirmed that both races have an identical gene for avirulence (designated avrPph3) comprising 801 base pairs (bp) and predicted to encode a cytoplasmic protein of 28,703 Da. A sequence, TGCAACCGAAT, 91% homologous to the motif found in promoter regions of avrB and avrD from P. s. pv. glycinea was located 89-99 bp upstream of the start of the open-reading frame 1. Hybridization experiments showed that avrPph3 was not plasmid-borne and was absent from isolates of P. s. pv. phaseolicola races 1, 2, 5, 6, 7, and 8, P. cichorii, P. s. pvs. coronafaciens, glycinea, maculicola, pisi, syringae, and tabaci. Cosegregation studies of crosses between cultivars resistant (Tendergreen) and susceptible (Canadian Wonder) to races 3 and 4 and transconjugants of race 5 confirmed that a gene-for-gene relationship controls specificity in the interaction between Tendergreen and races 3 and 4 of P. s. pv. phaseolicola.     

野生型油菜黄单胞菌的过氧化氢释放特性

采用外源过氧化氢和油菜组织处理油菜黄单胞菌野生型(XpW)和烷基过氧化物还原酶亚基C突变型(Xp1),检测了各体系中过氧化氢的释放情况,并测定了油菜黄单胞菌野生型的最大过氧化氢耐受浓度范围,以明确植物-病原菌互作过程中是否具有病原细菌源的过氧化氢产生.结果显示:(1)过氧化氢处理3.5 h后,对外源过氧化氢的清除率相对于0.5 h时XpW为100%,Xp1为-26%,说明野生型对培养体系中过氧化氢的清除率高于突变型;油菜组织处理后,体系中产生和积累的过氧化氢情况是:XpW为3.5 h时比0.5 h时高2.105倍,Xp1为3.5 h时比0.5 h时低25.2%,说明野生型培养体系中产生和积累的过氧化氢量高于突变型.(2)野生型的最大过氧化氢耐受浓度范围为1.715 08×105～2.450 11×105 μmol/L,远远高于油菜组织处理XpW菌液时体系中最大检测到的过氧化氢浓度,说明本实验中油菜组织处理XpW菌液过程中产生的过氧化氢的浓度不能够杀灭XpW.由此推测,油菜组织处理体系中野生型油菜黄单胞菌能够产生部分的过氧化氢,其过氧化氢的产生与烷基过氧化物还原酶亚基C相关,支持植物-病原细菌相互作用过程中可能有细菌源的过氧化氢产生的观点.     

Characterization of eight excision plasmids of Pseudomonas syringae pv. phaseolicola

Summary Pseudomonas syringae pv. phaseolicola strain LR719 contains a 150 kilobase pair (kb) plasmid pMC7105, stably integrated into its chromosome. Occasionally, single colony isolates of this strain contain an excision plasmid. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb; the largest contained approximately 130 kb of chromosomal DNA sequences. Restriction maps of pMC7105 were developed to deduce the site of integration and to identify the fragments in which recombination occurred to produce each excision plasmid. The eight excision plasmids were arranged into five classes based on the sites where excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of adjacent fragments, is present in all excision plasmids and thought to contain the origin of replication. The site of integration on pMC7105 maps within BamHI fragment 8. This fragment shows homology with seven other BamHI fragments of pMC7105 and with five chromosomal fragments identified among the excision plasmids. The data strongly suggest that the integration of pMC7105 may have occurred at a repetitive sequence present on the chromosome and on the plasmid.     

Ethylene Production by Cell-Free Extract of the Kudzu Strain of Pseudomonas syringae pv.phaseolicola

A cell-free ethylene-forming system of Pseudomonas syringaepv.phaseolicola (Kudzu strain) was characterized by its psychrophilictrait. Ethylene was most effectively produced from -ketoglutaricacid (-KG) at 0.5 mM followed by glutamate and then istidineat 5 to 10 mM. The presence of FeSO₄ was essential to the cell-freesystem. DTT and histidine greatly stimulated ethylene production;the latter could be substituted to some extent by its analogues.The optimum pH value and temperature for the ethylene-formingreactions were pH 7.0 and 25?C, respectively. Ethylene formationfrom -KG was inhibited in the presence of carbonates or organicacids of the TCA cycle, whereas that from glutamate was inhibitedin the presence of ammonium salts. Ethylene production from-keto--methylthiobutyric acid in the cell-free system was largelydependent on non-enzymical processes in the presence of DTTand FeSO₄. The ethylene-forming reactions were inhibited completelyby 1 mM n-propyl gallate and 1 mM p-chloromercuribenzoic acidand partly by coenzymes such as pyridoxal-1-phosphate, folicacid, and flavin mononucleotide at 5mM. The complete systemfor the highest ethylene production consisted of: 0.5 mM -KG,50 mM HEPES (pH 7.0), 5 mM DTT, 0.5 mM FeSO₄, and 10 mM histidine.The amount of ethylene produced in this system was equivalentto 40 to 50% of that produced by the living cells. (Received October 22, 1986; Accepted January 19, 1987)     

Multiplication of Pseudomonas syringae pv. phaseolicola"in planta"

In the compatible combination of the halo blight disease of bean Pseudomonas phaseolicola was able to colonize large areas of the intercellular space of leaves, such that these confluent water congested areas became visible as water-soaked spots. Most of the plant cell walls in the infected region maintained their normal shape, even when the cytoplasm had collapsed. Some inward bending of plant cell walls preceded their rather slow degradation and final replacement by bacterial masses. Neighbouring plant cells appeared to be metabolically active. In resistant leaves no indications of active bacterial attachment or encapsulation could be observed. However, bacteria appeared to be more densely packed in resistant leaves, and relatively more plant cells completely collapsed as compared with susceptible leaves. From 8—14 days after inoculation, the bacterial concentration did not change much in susceptible or resistant leaves, indicating the absence of bactericidal components. Even Pseudomonas pisi snowed some multiplication in bean leaves (immune reaction), but its growth stopped earlier than that of P. phaseolicola. in the resistant cultivars, probably due to a different mechanism of resistance. Although less bacteria were determined in the intercellular washing fluid (IF) compared with leaf homogenates, the high bacterial concentrations in the IF supported our observation that an effective encapsulation of bacteria in resistant leaves did not occur.     

Multiplication of Pseudomonas syringae pv. phaseolicola "in planta"

Multiplication of Pseudomonas phaseolicola was determined in 17 different bean cultivars ( Phaseolus vulgaris ) and 9 other plant species, and the effect of different inoculation methods and conditions was also studied.
In susceptible leaves, a generation time of 2.1 h was determined in the early phase (2 days after inoculation). Different multiplication rates in susceptible and resistant leaves were clearly observed 4 days after inoculation. At this time the first small water-soaked spots were visible in the susceptible cultivars. Bacteria multiplied up to the 7th day after inoculation with a maximum of 10⁹ cells per cm² leaf (equal to ca. 4 × 10¹⁰ bacterial cells/cm³). At the same time, the water-soaked spots had reached their maximum size in most cases. Thus, bacterial multiplication and development of water-soaked spots paralleled each other.
In resistant leaves, no water-soaked spots appeared, and the final bacterial concentration was 1/1000–1/100 of that in susceptible leaves. Gomparison of races 1 and 2 in several bean cultivars indicated the non-existence of a gene-for-gene relationship with this disease. Old leaves were less susceptible to infection. Some bacterial multiplication was also observed in non-host plants. There was a general correlation between bacterial multiplication in the non-host plants and their botanical relation to Phaseolus vulgaris .     

Harpin of Pseudomonas syringae pv. phaseolicola harbors a protein binding site

Harpin HrpZ of plant-pathogenic bacterium Pseudomonas syringae elicits a hypersensitive response (HR) in some nonhost plants, but its function in the pathogenesis process is still obscure. HrpZ-interacting proteins were identified by screening a phage-display library of random peptides. HrpZ of the bean pathogen P. syringae pv. phaseolicola (HrpZPph) shows affinity to peptides with a consensus amino acid motif W(L)ARWLL(G/L). To localize the peptide-binding site, the hrpZPph gene was mutagenized with randomly placed 15-bp insertions, and the mutant proteins were screened for the peptide-binding ability. Mutations that inhibited peptide-binding localized to the central region of hrpZPph, which is separate from the previously determined HR-inducing region. Antiserum raised against one of the hrpZPph-binding peptides recognized small proteins in bean, tomato, parsley, and Arabidopsis thaliana but none in tobacco. On native protein blots, hrpZPph bound to a bean protein with similar pI as the protein recognized by the peptide antiserum. The result suggests a protein-protein interaction between the harpin and a host plant protein, possibly involved in the bacterial pathogenesis.     

Ornithine carbamoyltransferase genes and phaseolotoxin immunity in Pseudomonas syringae pv. phaseolicola

Two different DNA fragments encoding ornithine carbamoyltransferase (OCTase) were cloned from Pseudomonas syringae pv. phaseolicola NPS3121. These fragments did not cross-hybridize and encoded OCTases which differed with respect to their sensitivity to purified phaseolotoxin, an OCTase inhibitor produced by this phytopathogenic bacterium. Recombinant plasmids carrying these DNA fragments complemented OCTase-deficient strains of Escherichia coli and Pseudomonas aeruginosa. Extracts of the complemented E. coli strain contained OCTase enzyme activities with similar degrees of sensitivity to purified phaseolotoxin as extracts of P.s.phaseolicola grown at either 20 or 30°C. The OCTase activity detectable in extracts of P.s.phaseolicola grown at 20°C is insensitive to phaseolotoxin while that detectable in extracts of cells grown at 30°C is sensitive to the toxin. E.coli HB101 harboring recombinant plasmids carrying the gene(s) encoding the phaseolotoxin-insensitive enzyme activity exhibited resistance to purified phaseolotoxin. The results of Tn5 mutagenesis and Southern blotting and the pattern of complementation of OCTase-deficient and Tox^- mutant strains suggest that the gene(s) encoding the phaseolotoxin-insensitive OCTase is part of a gene cluster involved in phaseolotoxin production.     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

In planta conditions induce genomic changes in Pseudomonas syringae pv. phaseolicola

The co-evolution of bacterial plant pathogens and their hosts is a complex and dynamic process. Plant resistance can impose stress on invading pathogens that can lead to, and select for, beneficial changes in the bacterial genome. The Pseudomonas syringae pv. phaseolicola (Pph) genomic island PPHGI-1 carries an effector gene, avrPphB (hopAR1), which triggers the hypersensitive reaction in bean plants carrying the R3 resistance gene. Interaction between avrPphB and R3 generates an antimicrobial environment within the plant, resulting in the excision of PPHGI-1 and its loss from the genome. The loss of PPHGI-1 leads to the generation of a Pph strain able to cause disease in the plant. In this study, we observed that lower bacterial densities inoculated into resistant bean (Phaseolus vulgaris) plants resulted in quicker PPHGI-1 loss from the population, and that loss of the island was strongly influenced by the type of plant resistance encountered by the bacteria. In addition, we found that a number of changes occurred in the bacterial genome during growth in the plant, whether or not PPHGI-1 was lost. We also present evidence that the circular PPHGI-1 episome is able to replicate autonomously when excised from the genome. These results shed more light onto the plasticity of the bacterial genome as it is influenced by in planta conditions.     

Cell Surface Properties of Pseudomonas syringae pv. phaseolicola Wild-type and hrp Mutants

The cell surface hydrophobicity and charge as well as surface polysaccharides of eight independent prototrophic hrp::-Tn5 mutants (Lindgren et al., J. Bacteriol. 168 , 512–522, 1986) were compared to the wild-type parent strain NPS3121 of Pseudomonas syringae pv. phaseolicola. No significant differences were found in cell surface charge, but mutant strain NPS4005 exhibited significantly lower cell surface hydrophobicity than the wild-type and the other mutant strains. The mutant strains all retained the ability to produce the exopolysaccharides (EPS) levan, a neutral fructan, and alginate, an acidic polymer. Relative amounts of EPS produced in vitro was dependent on culture conditions. Lipopolysaccharide (LPS) chemotypes were similar for all nine strains. Chemical as well as ¹³C-NMR analyses of the O-antigens from four wild-type strains of P. s. pv. phaseolicola representing two physiological races as well as the O-antigens of two strains of P. s. pv. syringae which belong to the same serogroup as P. s. pv. phaseolicola indicated that all of the O-antigens were very similar if not identical. LPS of three strains of P. s. pv. phaseolicola produced in vitro or in planta were also compared and no significant differences were detected. The altered phenotype of the Tn5 mutants of P. s. pv. phaseolicola does not appear to be due to changes in the ability to produce exopolysaccharides or to an altered composition of cell surface polysaccharides (LPS and EPS). However, a change in an unidentified cell surface component(s) leading to lowered cell surface hydrophobicity of mutant strain NPS4005 may be important.     

Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola.

Levansucrase (EC 2.4.1.10), an exoenzyme of Pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on TMAE-Fraktogel and butyl-Fraktogel. The enzyme has molecular masses of 45 kDa under denaturing conditions and 68 kDa during gel filtration of the native form. In isoelectric focusing, active bands appeared at pH 3.55 and 3.6. Maximum sucrose cleaving activities were measured at pH 5.8 to 6.6 and 60 degrees C. The enzyme was highly tolerant to denaturing agents, proteases, and repeated freezing and thawing. The molecular weight of the produced levan depended on temperature, salinity, and sucrose concentration. The enzyme had levan-degrading activity and did not accept raffinose as a substrate. Comparison of the N-terminal amino acid sequence with the predicted amino acid sequence of levansucrases from Erwinia amylovora and Zymomonas mobilis showed 88 and 69% similarity, respectively, in amino acids 5 to 20. No similarity could be detected to levansucrases of gram-positive bacteria in the first 20 amino acids. By comparison of all levansucrases which have been sequenced to date, the enzyme seems to be conserved in the gram-negative bacteria. The rheological behavior of the product levan prompted a new assessment of the enzyme's role in pathogenesis. Depending on formation conditions, levan solutions exclude other polymer solutions. This behavior supports the presumption that the levansucrase is important in the early phase of infection by creating a separating layer between bacteria and plant cell wall to prevent the pathogen from recognition.     

Layered basal defenses underlie non-host resistance of Arabidopsis to Pseudomonas syringae pv. phaseolicola

Arabidopsis is a non-host for Pseudomonas syringae pv. phaseolicola NPS3121 (Pph), a bacterial pathogen of bean. Pph does not induce a hypersensitive response in Arabidopsis. Here we show that Arabidopsis instead resists Pph with multi-layered basal defense. Our approach was: (i) to identify defense readouts induced by Pph; (ii) to determine whether mutations in known Arabidopsis defense genes disrupt Pph-induced defense signaling; (iii) to determine whether heterologous type III effectors from pathogens of Arabidopsis suppress Pph-induced defense signaling, and (iv) to ascertain how basal defenses contribute to resistance against Pph by individually or multiply disrupting defense signaling pathways with mutations and heterologous type III effectors. We demonstrate that Pph elicits a minimum of three basal defense-signaling pathways in Arabidopsis. These pathways have unique readouts, including PR-1 protein accumulation and morphologically distinct types of callose deposition. Further, they require distinct defense genes, including PMR4, RAR1, SID2, NPR1, and PAD4 . Finally, they are suppressed differentially by heterologous type III effectors, including AvrRpm1 and HopM1. Pph growth is enhanced only when multiple defense pathways are disrupted. For example, mutation of NPR1 or SID2 combined with the action of AvrRpm1 and HopM1 renders Arabidopsis highly susceptible to Pph. Thus, non-host resistance of Arabidopsis to Pph is based on multiple, individually effective layers of basal defense.     

PCR-based detection of Xanthomonas campestris pv. phaseoli var. fuscans in plant material and its differentiation from X. c. pv. phaseoli

A PCR-based method was developed for the specific detection of Xanthomonas campestris pv. phaseoli var. fuscans from plant material. Primers Xf1 and Xf2, based on a sequence conserved amplified region (SCAR) derived from RAPD PCR analysis of X. c. pv. phaseoli var. fuscans , amplified a DNA fragment of 450 bp from all such isolates. In contrast, no amplification product was obtained from any X. c. pv. phaseoli isolates, or from any other DNAs tested. As few as 10 cells of X. c . pv. phaseoli var. fuscans (equivalent to about 100 fg DNA) could be detected in vitro . In planta , following an initial inoculation of as little as one cell, an amplification product was generated after only 2 d of incubation, allowing highly sensitive detection 10 d before disease symptoms were observed. Moreover, the failure to amplify DNA from X. c . pv. phaseoli isolates shows that these primers provide a rapid, improved method to differentiate these two varieties using PCR.     

Methods for recovery and immunodetection of Xanthomonas campestris pv. phaseoli in navy bean seed

Non-selective enrichment procedures were evaluated for recovery of Xanthomonas campestris pv. phaseoli (fuscans strain) from artificially inoculated navy bean seed. A marked increase in recovery of the pathogen was obtained when the mixtures (bacterium plus bean seed) were suspended in Pseudomonas Agar F medium at 28°C for 48 h. Detection of this pathogen by indirect immunofluorescence microscopy (IF) and indirect enzyme-linked immunosorbent assay (ELISA) with a specific monoclonal antibody was compared. The IF system was not only more sensitive but also more reliable than ELISA for detection of the pathogen. The method is particularly useful for evaluation of the common bacterial blight status of seedlots before planting out.     

Water Stress in Leaves of Pbaseolus vulgaris Infected with Xanthomonas campestris pv. phaseoli

Infection of bean leaves ( Phaseolus vulgaris ) by Xanthomonas campestris pv. phaseoli in the field frequently resulted in the appearance of isolated flaccid areas in green leaf tissue adjacent to necrotic and chlorotic lesions. The flaccid leaf areas had significantly higher stomatal resistances compared to nearby turgid areas on the same leaf, and the turgid areas had stomatal resistances that were the same or only moderately elevated compared to those of healthy leaves. The flaccid tissues also had significantly lower relative water contents than turgid tissues on the same leaf demonstrating that pathogeninduced water stress was localized. The levels of free proline, another indicator of water stress, were directly correlated (r²= 0.556) with disease severity. The change in free proline content implied that water stress increased in direct proportion with the amount of tissue infected. Water stress may be due to the disruption of xylem elements by the invasion of X. c. phaseoli from nearby lesions. One result of xylem invasion could have been severe water deficits which were sufficient to cause stomatal closure and leaf flaccidity; however, this effect was highly localized and the remainder of the diseased leaf was either significantly less water stressed or not affected.     

Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola

Aims:  To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean ( Phaseolus vulgaris L.).
Methods and Results:  Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium , Erwinia and Pantoea .
Conclusion:  A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria .
Significance and Impact of the Study:  This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.