hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria.

The majority of bacterial plant diseases are caused by members of three bacterial genera, Pseudomonas, Xanthomonas, and Erwinia. The identification and characterization of mutants that have lost the abilities to provoke disease symptoms on a compatible host and to induce a defensive hypersensitive reaction (HR) on an incompatible host have led to the discovery of clusters of hrp genes (hypersensitive reaction and pathogenicity) in phytopathogenic bacteria from each of these genera. Here, we report that predicted protein sequences of three hrp genes from Pseudomonas solanacearum show remarkable sequence similarity to key virulence determinants of animal pathogenic bacteria of the genus Yersinia. We also demonstrate DNA homologies between P. solanacearum hrp genes and hrp gene clusters of P. syringae pv. phaseolicola, Xanthomonas campestris pv. campestris, and Erwinia amylovora. By comparing the role of the Yersinia determinants in the control of the extracellular production of proteins required for pathogenicity, we propose that hrp genes code for an export system that might be conserved among many diverse bacterial pathogens of plants and animals but that is distinct from the general export pathway.     

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.     

Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora

Involvement of an oxidative burst, usually related to incompatible plant/pathogen interactions leading to hypersensitive reactions, was investigated with Erwinia amylovora, the causal agent of fire blight of Maloideae subfamily of Rosaceae, in interaction with pear (Pyrus communis; compatible situation) and tobacco (Nicotiana tabacum; incompatible situation). As expected, this necrogenic bacterium induced in tobacco a sustained production of superoxide anion, lipid peroxidation, electrolyte leakage, and concomitant increases of several antioxidative enzymes (ascorbate peroxidases, glutathion reductases, glutathion-S-transferases, and peroxidases), in contrast to the compatible pathogen Pseudomonas syringae pv tabaci, which did not cause such reactions. In pear leaves, however, inoculations with both the disease- and the hypersensitive reaction-inducing bacteria (E. amylovora and P. syringae pv tabaci, respectively) resulted in superoxide accumulation, lipid peroxidation, electrolyte leakage, and enzyme induction at similar rates and according to equivalent time courses. The unexpected ability of E. amylovora to generate an oxidative stress even in compatible situation was linked to its functional hrp (for hypersensitive reaction and pathogenicity) cluster because an Hrp secretion mutant of the bacteria did not induce any plant response. It is suggested that E. amylovora uses the production of reactive oxygen species as a tool to provoke host cell death during pathogenesis to invade plant tissues. The bacterial exopolysaccharide could protect this pathogen against the toxic effects of oxygen species since a non-capsular mutant of E. amylovora induced locally the same responses than the wild type but was unable to further colonize the plant.     

The presence of diverse IS elements and an avrPphD homologue that acts as a virulence factor on the pathogenicity plasmid of Erwinia herbicola pv. gypsophilae

The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) and Erwinia herbicola pv. betae (Ehb) is dependent on a native plasmid (pPATH(Ehg) or pPATH(Ehb)) that harbors the hrp gene cluster, genes encoding type III effectors, phytohormones, biosynthetic genes, and several copies of IS1327. Sequence analysis of the hrp-flanking region in pPATH(Ehg) (cosmid pLA150) revealed a cluster of four additional IS elements designated as ISEhel, ISEhe2, ISEhe3, and ISEhe4. Two copies of another IS element (ISEhe5) were identified on the upstream region of the indole-3-acetic acid operon located on the same cosmid. Based on homology of amino acids and genetic organization, ISEhe1 belongs to the IS630 family, ISEhe2 to the IS5 family, ISEhe3 and ISEhe4 to different groups of the IS3 family, and ISEhe5 to the IS1 family. With the exception of ISEhe4, one to three copies of all the other IS elements were identified only in pathogenic strains of Erwinia herbicola pv. gypsophilae and Erwinia herbicola pv. betae whereas ISEhe4 was present in both pathogenic and nonpathogenic strains. An open reading frame that exhibited high identity (89% in amino acids) to AvrPphD of Pseudomonas syringae pv. phaseolicola was present within the cluster of IS elements. An insertional mutation in the AvrPphDEh, reduced gall size in gypsophila by approximately 85%. In addition, remnants of known genes from four different bacteria were detected on the same cosmid.     

Pathogenicity, Non-Host Hypersensitivity and Host Defence Non-Permissibility Regulatory Gene hrpX is Highly Conserved in Xanthomonas Pathovars

Xanthomonas campestris pv. campestris and X. oryzae pv, oryzae contain the 1428 base pair hrpX gene whose product is involved in the regulation oi hrp genes required for pathogericity, non-host hypersensitivity and non-permissibility of compatible host defence responses. Previous Southern blot hybridization studies have suggested that hrpX is conserved in several X. campestris pathovars and X. oryzae. strains. We have confirmed and extended these findings using hrpX gene amplification by polymerase chain reaction, coupled with Southern blot hybridization analyses. Sixteen distinct pathovars of X. campestris and 12 strains of X. oryzae pv, oryzae were shown to contain homologs of hrpX which were not apparent in heterologous bacteria such as Agrobacterium tumefaciens, A. rhizogenes, Erwinia carolovora ssp. carotovora, Pseudomonas syringae pv, glycinea. P. syringae pv, labaci , and Escherichia coli. The hrpX gene is therefore highly conserved among Xanthomonas species and its gene product strongly resembles positive regulatory proteins of the AraC protein family,     

Phenotypic expression of the Pseudomonas syringae pv. syringae 61 hrp/hrm gene cluster in Escherichia coli MC4100 requires a functional porin.

Plants, in general, appear to be able to detect the presence of incompatible Pseudomonas syringae strains by a hypothetical cell-cell recognition process to initiate inducible defense mechanisms that contribute to disease resistance. A 25-kb hrp/hrm gene cluster isolated from P. syringae pv. syringae 61(pHIR11) enables Escherichia coli to elicit a hypersensitive response (HR), a plant response generally considered to be a manifestation of recognition and resistance. To identify the nature of the HR-eliciting signal produced by E. coli cells carrying pHIR11, bacterial surface features were surveyed by immunological and biochemical procedures. No immunoreactive epitopes or outer membrane proteins were detected that were associated with expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100. Phenotypic expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100, however, was found to be dependent upon ompC and ompF, which control outer membrane permeability to hydrophilic solutes. The results suggest that deployment of the HR-eliciting signal occurs via outer membrane porins and imply that a low-molecular-weight, hydrophilic factor mediates signal exchange between the bacterium and the responding plant cell.     

Analysis of the role of the Pseudomonas syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally non-polar hrpZ deletion mutations, truncated HrpZ fragments, and hrmA mutations

Pseudomonas syringae pv. syringae , like many plant pathogenic bacteria, secretes a 'harpin' protein that can elicit the hypersensitive response (HR), a defensive cellular suicide, in non-host plants. The harpin-encoding hrpZ gene is located in an operon that also encodes Hrp secretion pathway components and is part of the functional cluster of hrp genes carried on cosmid pHIR11 that enables saprophytic bacteria like Escherichia coli and Pseudomonas fluorescens to elicit the HR in tobacco leaves. We have constructed functionally non-polar hrpZ deletion mutations, revealing that HrpZ is necessary for saprophytic bacteria carrying pHIR11 to elicit a typical HR, whereas it only enhances the elicitation activity of P. s. syringae . Partial deletion mutations revealed that the N-terminal 153 amino acids of HrpZ can enable E. coli MC4100-(pHIR11) to elicit a strong HR. hrpZ subclone products comprising the N-terminal 109 amino acids and C-terminal 216 amino acids, respectively, of the 341 amino acid protein were isolated and found to elicit the HR. P. fluorescens (pHIR11 hrmA  ::Tn phoA ) mutants do not elicit the HR, but cell fractionation and immunoblot analysis revealed that they produce and secrete wild-type levels of HrpZ. Therefore, elicitor activity resides in multiple regions of HrpZ, P. syringae produces elicitor(s) in addition to HrpZ, and HrpZ is essential but not sufficient for HR elicitation by saprophytic bacteria carrying pHIR11.     

Interaction between Pseudomonas syringae pv. Pisi and Isolated Pea Mesophyll Protoplasts

The interaction between five races of Pseudomonas syringae pv. pisi (PSP) and isolated mesophyll protoplasts obtained from five pea cultivars was studied. There was no trend in the attachment of bacterial cells to surfaces of compatible and incompatible host protoplasts. The viability of protoplasts from compatible and incompatible host-pathogen interactions did not differ significantly; however, changes in the viability of cultivar Kelvedon Wonder protoplasts, compatible with all five races, was relatively more stable following inoculation with bacteria than those of cultivar Fortune, incompatible with all of the five races. Protoplast cell wall regeneration did not take place until 24–48 h after isolation. It is concluded that the use of pea protoplasts as a model system for studying the pea-PSP interaction appears to have considerable potential for the future, but more basic research is required.     

Phosphatidylinositol, a phospholipid of ice-nucleating bacteria.

The nature of the phospholipids of the various bacteria that have ice nucleation activity in supercooled water has been determined. The seven bacteria studied included Pseudomonas syringae, Erwinia herbicola, three Escherichia coli K-12 strains that are phenotypically Ice+ because they contain plasmids with different amounts of either P. syringae or E. herbicola cloned DNA, and two E. coli K-12 strains without cloned ice gene DNA. All five Ice+ bacterial strains contained small amounts (0.1 to 1.0% of the total phospholipids) of phosphatidylinositol (PI), a phospholipid not previously detected in E. coli, Pseudomonas, or Erwinia species. The Ice- E. coli strains also contained trace level of PI that amounted to 2 to 30% of the level found in the Ice+ E. coli strains. Extracts of Ice+ strains contained low but measurable activities of PI synthase, while the activities in Ice- strains amounted to only 8 to 12% or less of that found in extracts of Ice+ bacteria. The functioning of the ice gene apparently increased both the PI synthase activity and the PI content of Ice+ strains from low endogenous levels. The relative ice nucleation activity at -4 degrees C or above (class A nucleation activity) of all Ice+ strains was found to be proportional to their PI content. The addition of myo-inositol (5 x 10(-4) M) to synthetic culture media increased the class A nucleation activity of both Ice+ E. coli strains and P. syringae up to sevenfold but had no stimulating effect on ice nucleation at lower temperatures (class B and class C nucleation activities). If these cells after fusion with PI vesicles were incubated with an energy source, the class A nucleation activity increased 70-fold over that present before fusion. These results indicate that PI plays an important role in ice nucleation at warm temperatures and is a likely precursor or component of the class A structure.     

Role of Extracellular Polysaccharide (EPS) Slime of Plant Pathogenic Bacteria in Protecting Cells to Reactive Oxygen Species

Pseudomonas syringae pv. phaseolicola , a phytopathogenic bacterium, seemed very sensitive in planta to the adverse action of reactive oxygen species (ROS) produced by two chemical systems. The disease symptoms in host plants were also suppressed by ROS. Several other plant pathogenic bacteria ( P. syringae pv. pisi, Erwinia amylovora, Xanthomonas campestris pv. pelargonii ) as well as P. fluorescens were also sensitive in vitro to the inhibiting or killinig action of ROS. It was shown that O₂ and H₂O₂ were produced in our two chemical systems and were involved in the killing action. OH'however was not involved in the adverse action on bacteria of the ROS. Superoxide dismutase and catalase were able to reverse the killing action of ROS. When the EPS slime around bacteria was removed by washing and centrifuging the cells, bacteria were more sensitive to ROS. However, when the cells of EPS- mutants were washed and centrifuged, their sensitivity to the killing action of ROS did not change because the lack of slime around the mutant cells.
The EPS^- Tn5 mutants of P. syringae pv. phaseolicola and the natural EPS^- mutant of E. amylovora were more sensitive to ROS than the wild type strains. These results support the idea that the EPS slime protects bacteria from ROS (O^-₂ and H₂O₂).     

The Selective Activity of a Benzoxazole Brightener against Phytopathogenic Bacteria

A cationic benzoxazole compound used commercially as an optical brightener was found to have a selective bactericidal effect at low concentrations on a wide range of bacterial phytopathogens; many strains of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas were tested. Known phytopathogenic species of Corynebacterium, Pseudomonas and Xanthomonas were rapidly killed, whereas saprophytic strains of Corynebacterium and Pseudomonas were resistant to 500 parts/10⁶. The phytopathogenic Erwinia spp. were inhibited only by the higher concentrations of AN, and some saprophytic E. herbicola var. herbicola strains were slightly sensitive. The extent and nature of this selective bactericidal property is examined and discussed. Resistant mutant colonies were very rarely encountered. The results are of significance in that the recognition of such phytopathogens under laboratory conditions is made easier. The resistance of Ps. aeruginosa to the compound and its almost unique ability to utilize it as a sole carbon source offer a means of isolating this organism.     

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.     

Antimicrobial activity of glycosidase inhibitory protein isolated from Cyphomandra betacea Sendt. fruit

Broad-spectrum antimicrobial activity of an invertase inhibitory protein (IIP) isolated from Cyphomandra betacea ripe fruits is documented. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. This IIP inhibited the growth of xylophagous and phytopatogenic fungi (Ganoderma applanatum, Schizophyllum commune, Lenzites elegans, Pycnoporus sanguineous, Penicillium notatum, Aspergillus niger, Phomopsis sojae and Fusarium mango) and phytopathogenic bacteria (Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae pv. syringae and Erwinia carotovora var carotovora). The IIP concentration required to completely inhibit the growth of all studied fungi ranged from 7.8 to 62.5 microg/ml. Phytopatogenic bacteria were the most sensitive, with MIC values between 7.8 and 31.25 microg/ml. Antifungal and antibacterial activities can be associated with their ability to inhibit hydrolytic enzymes. Our results indicate the possible participation of IIP in the plant defense mechanism and its potential application as a biocontrol agent against phytopathogenic fungi and bacteria.     

Effect of solar radiation and predacious microorganisms on survival of fecal and other bacteria.

The effect of solar radiation and predacious microorganisms on the survival of bacteria of fecal and plant origin was studied. The decline in the numbers of Escherichia coli cells in estuarine water samples was found to be significantly greater in the presence of both naturally occurring microbial predators and solar radiation than when each of these factors was acting independently. The effect of solar radiation on microbial predators was negligible, whereas the susceptibility of bacteria to light-induced decay varied from one organism to another, as follows: Klebsiella pneumoniae greater than E. coli greater than Salmonella typhimurium, Streptococcus faecium, Enterobacter aerogenes, Erwinia herbicola.     

Detached leaf enrichment: a method for detecting small numbers of Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria in seed and symptomless leaves of tomato and pepper

A method for detecting 10¹-10² cells of phytopathogenic bacteria ( Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria ) in either tomato or pepper seed was developed. The method is based on the enrichment of the compatible pathogen inside a detached leaf of its host when placed on a water agar medium. It was found to be superior to the diagnostic growth media method commonly used and to permit the detection of the pathogens in symptomless plants.     

Antagonistic Activities of Epiphytic Bacteria from Soybean Leaves against Pseudomonas syringae pv. glycinea in vitro and in planta

Abstract Over two growing seasons, 273 bacterial strains were isolated from soybean leaves without and with bacterial blight symptoms caused by Pseudomonas syringae pv. glycinea (Psg). The majority of the isolates from leaves with symptoms were identified as Psg (43%), followed by Erwinia herbicola (21%), and Enterobacter/Erwinia (19%). The isolates from leaves without symptoms included mainly a group of unidentified Gram-negative bacteria (22%), Psg (21%), and E. herbicola (18%). Psg colonized the soybean leaves prior to saprophytic bacteria, and remained dominant during both seasons on healthy, as well as infected, leaves. Eighty-two saprophytic isolates were tested in vitro for their antagonistic activities against Psg, using an agar-diffusion assay. For the in planta assay, Psg and each isolate were simultaneously inoculated into wounds of pin-pricked leaves of greenhouse-grown soybean plants. Twenty-nine isolates were antagonistic in vitro. Nineteen isolates were able to suppress the growth of Psg and prevented the formation of leaf spots in planta when mixtures of isolate and pathogen were inoculated at ratios >1. Only 9 of the 82 isolates inhibited Psg in vitro as well as in planta. Most antagonists detected belonged to the genera Pseudomonas and the species Erwinia herbicola. The in planta assay should be a reliable predictor of field performance for screening of biological control agents. Received: 8 April 1996; Accepted: 22 October 1996