Pseudomonas Syringae Pathovars Research Articles

Asymmetric total syntheses of (+)-coronafacic acid and (+)-coronatine, phytotoxins isolated from Pseudomonas syringae pathovars

Abstract Asymmetric total synthesis of (+)-coronafacic acid (2), was accomplished via intramolecular 1, 6-conjugate addition as the key step. The chiral ester (+)-7 was prepared via two approaches: starting from (R)-(+)-4-acetoxy-2-cyclopenten-1-one (12), and using catalytic asymmetric Michael reactions promoted by heterobimetallic BINOL complexes. Coupling between (+)-2 and the protected coronamic acid 8 and subsequent deprotection by hydrogenolysis provided (+)-coronatine (1). This is the first asymmetric total synthesis of (+)-1.

Ethylene Production by Pseudomonas syringae Pathovars In Vitro and In Planta

Significant amounts of ethylene were produced by Pseudomonas syringae pv. glycinea, pv. phaseolicola (which had been isolated from viny weed Pueraria lobata [Willd.] Ohwi [common name, kudzu]), and pv. pisi in synthetic medium. On the other hand, the bean strains of P. syringae pv. phaseolicola and strains of 17 other pathovars did not produce ethylene. P. syringae pv. glycinea and P. syringae pv. phaseolicola produced nearly identical levels of ethylene (about 5 x 10(sup-7) nl h(sup-1) cell(sup-1)), which were about 10 times higher than the ethylene level of P. syringae pv. pisi. Two 22-bp oligonucleotide primers derived from the ethylene-forming enzyme (efe) gene of P. syringae pv. phaseolicola PK2 were investigated for their ability to detect ethylene-producing P. syringae strains by PCR analysis. PCR amplification with this primer set resulted in a specific 0.99-kb fragment in all ethylene-producing strains with the exception of the P. syringae pv. pisi strains. Therefore, P. syringae pv. pisi may use a different biosynthetic pathway for ethylene production or the sequence of the efe gene is less conserved in this bacterium. P. syringae pv. phaseolicola isolated from kudzu and P. syringae pv. glycinea also produced ethylene in planta. It could be shown that the enhanced ethylene production in diseased tissue was due to the production of ethylene by the inoculated bacteria. Ethylene production in vitro and in planta was strictly growth associated.

Stimulation of exopolysaccharide production by fluorescent pseudomonads in sucrose media due to dehydration and increased osmolarity

Exopolysaccharides produced by plant pathogenic bacteria are thought to play an important role in both the general ecology and the virulence of the producing organism. The environmental factors affecting exopolysaccharide production in planta by Pseudomonas syringae pathovars are not known. We tested the effect of increased medium osmolarity and dehydration on exopolysaccharide production in a sucrose-containing medium by three P. syringae pathovars, one ( P. syringae pv. phaseolicola) capable of levan and alginate production and two ( P. syringae pv. papulans and pv. savastanoi) capable of only alginate production. Addition of NaCl and ethanol to the medium led to increased accumulation of alginate by all three pathovars as well as increased levan production by P. syringae pv. phaseolicola. Culture fluids of the two non-levan producers also contained increased amounts of neutral carbohydrate which was not levan. Based on sugar compostion this material may have originated from outer membrane lipopolysaccharide. In addition, the ratio of neutral material (levan or not) to alginate varied dependent on culture conditions.

Stimulation of exopolysaccharide production by fluorescent pseudomonads in sucrose media due to dehydration and increased osmolarity

Exopolysaccharides produced by plant pathogenic bacteria are thought to play an important role in both the general ecology and the virulence of the producing organism. The environmental factors affecting exopolysaccharide production in planta by Pseudomonas syringae pathovars are not known. We tested the effect of increased medium osmolarity and dehydration on exopolysaccharide production in a sucrose-containing medium by three P. syringae pathovars, one (P. syringae pv. phaseolicola) capable of levan and alginate production and two (P. syringae pv. papulans and pv. savastanoi) capable of only alginate production. Addition of NaCl and ethanol to the medium led to increased accumulation of alginate by all three pathovars as well as increased levan production by P. syringae pv. phaseolicola. Culture fluids of the two non-levan producers also contained increased amounts of neutral carbohydrate which was not levan. Based on sugar compostion this material may have originated from outer membrane lipopolysaccharide. In addition, the ratio of neutral material (levan or not) to alginate varied dependent on culture conditions.

The HrpZ proteins of Pseudomonas syringae pvs. syringae, glycinea, and tomato are encoded by an operon containing Yersinia ysc homologs and elicit the hypersensitive response in tomato but not soybean.

The Pseudomonas syringae pathovars are composed of host-specific plant pathogens that characteristically elicit the defense-associated hypersensitive response (HR) in nonhost plants. P. s. pv. syringae 61 secretes an HR elicitor, harpinPss (HrpZPss), in a hrp-dependent manner. An internal fragment of the P. s. pv. syringae 61 hrpZ gene was used to clone the hrpZ locus from P. s. pv. glycinea race 4 (bacterial blight of soybean) and P. s. pv. tomato DC3000 (bacterial speck of tomato). DNA sequence analysis revealed that hrpZ is the second ORF in a polycistronic operon. The amino acid sequence identities of HrpZPss/HrpZPsg and HrpZPss/HrpZPst were 79 and 63%, respectively. Although none of the HrpZ proteins showed significant overall sequence similarity with other known proteins, HrpZPst contained a 24-amino acid sequence that is homologous with a region of the PopA1 elicitor protein of the tomato pathogen, Pseudomonas solanacearum GMI1000. hrpA, the upstream ORF, was highly divergent: The amino acid sequence identities of HrpAPss/HrpAPsg and HrpAPss/HrpAPst were 91 and 28%, respectively, and no HrpA sequence showed similarity to known proteins. In contrast, the predicted products of the downstream ORFs in P. s. pv. syringae and P. s. pv. tomato, hrpB, hrpC, hrpD, and hrpE showed varying levels of similarity to those of yscI, yscJ, yscK, and yscL. These are colinearly arranged genes in the virC locus of Yersinia spp., which are involved in the secretion of the Yop virulence proteins via the type III pathway. The similarity of the Ysc proteins was generally stronger in comparisons with the P. s. pv. tomato Hrp proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products

Production of the chlorosis-inducing phytotoxin coronatine in the Pseudomonas syringae pathovars atropurpurea, glycinea, maculicola, morsprunorum, and tomato has been previously reported. DNA hybridization studies previously indicated that the coronatine biosynthetic gene cluster is highly conserved among P. syringae strains which produce the toxin. In the present study, two 17-bp oligonucleotide primers derived from the coronatine biosynthetic gene cluster of P. syringae pv. glycinea PG4180 were investigated for their ability to detect coronatine-producing P. syringae strains by PCR analysis. The primer set amplified diagnostic 0.65-kb PCR products from genomic DNAs of five different coronatine-producing pathovars of P. syringae. The 0.65-kb products were not detected when PCR experiments utilized nucleic acids of nonproducers of coronatine or those of bacteria not previously investigated for coronatine production. When the 0.65-kb PCR products were digested with ClaI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P. syringae. A restriction fragment length polymorphism was detected in the amplified region of P. syringae pv. atropurpurea, since this pathovar lacked a conserved PvuI site which was detected in the PCR products of the other four pathovars. The 0.65-kb PCR products from six strains comprising five different pathovars of P. syringae were cloned and sequenced. The PCR products from two different P. syringae pv. glycinea strains contained identical DNA sequences, and these showed relatedness to the sequence obtained for the pathovar morsprunorum. The PCR products obtained from the pathovars maculicola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)

Recognition responses in pathogen/non-host and race/cultivar interactions involving soybean ( Glycine max) and Pseudomonas syringae pathovars

Previous work with tobacco and bacteria which cause a hypersensitive response (HR) indicated that the K +/H + response (increase in extracellular pH and K +) and active oxygen production are associated with early events leading to a HR. This study demonstrates that these early responses also occur in soybean upon recognition of pathogen of other host species as well as incompatible races of Pseudomonas syringae pv. glycinea, a pathogen of soybean. Pathogens of other host species stimulated the K +/H + response in leaf disc and cell suspension assays in all soybean cultivars tested. These pathogens also stimulated the active oxygen response in cell suspension assays as observed using luminol-dependent chemiluminescence. Races of P. syringae pv. glycinea stimulated these responses in incompatible interactions which lead to early cell death as monitored by Evans Blue. However, the race/cultivar compatibility under the leaf disc or cell suspension assay conditions differed in some cases from that observed in intact leaves. In all cases, the K +/H + and active oxygen responses preceded hypersensitive cell death by several hours. This study extends our previous findings from tobacco studies in that in soybean these early responses occur upon recognition of bacteria which induce an HR not only in pathogen/non-host interactions hut also race/cultivar interactions.

Evaluation of a commercial microbial identification system based on fatty acid profiles for rapid, accurate identification of plant pathogenic bacteria

D.E. STEAD, J.E. SELLWOOD, J. WILSON AND I. VINEY, 1992. Fatty acid profiles of 773 strains representing 25 taxa of plant pathogenic and related saprophytic bacteria were compared with two commercially available broad‐spectrum libraries and one self‐generated library based primarily on cultures from the National Collection of Plant Pathogenic Bacteria. The accuracy of identification at specific level was often 100%, although for some closely related species and infraspecific taxa accuracy was sometimes significantly less than this. The accuracy of identification of Xanthomonas campestris pathovars was much better than for Pseudomonas syringae pathovars. Almost all identifications were made within24–48 h. Standardization of cultural conditions was essential. Hydroxy fatty acids were of great taxonomic value in classification of Gram‐negative bacteria. Improved library development and standardization of cultural and analytical techniques will further increase the accuracy of identification. Fatty acid profiling offers a valuable rapid, accurate method for identification of many bacteria.

Bioassay of Toxins as a Diagnostic Method for Pseudomonas syringae Pathovars

Abstract Six pathovars of Pseudomonas syringae were tested for bioassay of toxins. P. syringae pv. syringae and pv. maculicola showed an inhibitory effect on the growth of Escherichia coli in mineral medium. The inhibition for P. syringae pv. syringae was reversed by L-arginine, not by L-glutamine and L-citrulline. It appears that the biochemical target of the toxin(s) from P. syringae pv. syringae is involved in the metabolism of citrulline to arginine in the urea cycle. Under the experimental conditions, the toxin activity was not detected with the isolate of P. syringae pv. phaseolicola in vitro, but the isolate retained it's pathogenicity in a susceptible variety of bean. Zusammenfassung Bioassay von Toxinen als Methode zur Diagnostizierung von Pseudomonas syrrngae-Pathovaren Die Toxinaktivität von sechs Pathovaren von Pseudomonas syringae wurde in einem Biotest geprüft. P. syringae pv. syringae und pv. maculicola zeigten eine hemmende Wirkung auf das Wachstum von Escherichia coli in Mineralmedium. Die Hemmung von P. syringae pv. syringae wurde durch L-Arginin aufgehoben, jedoch nicht durch L-Glutamin und L-Citrullin. Es scheint, daß die biochemische Erkennungsstelle (Target) des Toxins von P. syringae pv. syringae in Verbindung steht mit dem Metabolismus von Citrullin zu Arginin im Harnstoffzyklus. Unter den Versuchsbedingungen konnte in vitro keine Toxizität bei P. syringae pv. phaseolicola gefunden werden, jedoch behielt das Isolat seine Pathogenität gegenüber einer empfindlichen Bohnenart.

The relationship between the growth rate of Pseudomonas syringae pathovars and the hypersensitive reaction in tobacco

Pseudomonas syringae pathovars were starved to deplete amino acids in the absence (starved) or in the presence of chloramphenicol (starved + Cm). When amino acids were replaced (after 3, 30 and 60 min of starvation) the starved culture resumed growth in vitro immediately, whereas the starved + Cm culture started to grow only after a lag period. This phenomenon was exploited to study the possible relationship between the induction of the hypersensitive reaction (HR) in plant leaves and the growth rate of the bacteria. It was found that in an incompatible host-parasite system the induction of HR was delayed, in the case of starved + Cm treated bacteria, by the same period of time as the length of the lag period found in vitro, and that for an efficient HR induction the bacterial growth rate must exceed a certain threshold value. In a compatible host-parasite system the induction of the HR-type necrosis induced by the starved + Cm treated bacteria was delayed beyond the length of the lag period found in vitro. From these results we can conclude that a correlation exists between the capability of the bacteria for growth and the kinetics of HR induction.

Bacterial growth and phytoalexin elicitation in soybean cell suspension cultures inoculated with Pseudomonas syringae pathovars

Two commonly used media for the culturing of numerous plant tissues, 1-B5 and 1-B5C, were tested for their ability to support bacterial growth. No significant growth of strains representing four of the six major genera of phytopathogenic bacteria occurred in 1-B5 liquid medium, in contrast to 1-B5C, making 1-B5 highly suitable for studies of most phytopathogenic bacteria—plant tissue culture interactions. Both compatible and incompatible strains of the soybean bacterial leaf pathogen Pseudomonas syringae pv. glycinea plus strains of the bean pathogens P. syringae pv. phaseolicola and pv. syringae , induced production of the phytoalexin glyceollin in soybean cv. Mandarin cell suspension cultures. Glyceollin production was elicited without a concomitant hypersensitive response (HR). Induced glyceollin production by cell suspension cultures declined as the number of culture transfers increased. There was no expression of resistance to incompatible strains of pv. glycinea , pv. phaseolicola or pv. syringae by the suspension cultured soybean cells. This was indicated by: (1) highest glyceollin production by soybean cell line Sb-2 after inoculation with a compatible pv. glycinea strain, (2) greater growth of pv. syringae strains than pv. glycinea and pv. phaseolicola and (3) an incompatible pv. glycinea strain grew as well as compatible pv. glycinea strains. Of several bacterial strains which elicited an HR on soybean leaves in vivo , only pv. syringae strain Y30 caused a rapid decline in viability of suspension cultured soybean cells. Various bioassays were used in an attempt to determine if production of the pv. syringae Phytotoxin syringomycin was responsible.

Toxin production as a distinguishing character for some Pseudomonas syringae pathovars : P. syringae pv. glycinea versus P. syringae pv. phaseolicola

Three strains of Pseudomonas syringae pv. glycinea from Glycine wightii (previously Glycine javanica) were found to have toxin-producing characteristics suggesting the presence of phaseolotoxin, which was confirmed by a chemical examination of the toxin. With the use of pathogenicity tests, serology tests and toxin characterizations, these 3 strains were compared with 5 strains of P. syringae pv. glycinea from Glycine max and 4 strains of P. syringae pv. phaseolicola. The results showed that the 3 strains from G. wightii were P. syringae pv. phaseolicola rather than pv. glycinea as previously recorded. Pathogenicity tests with a variety of P. syringae pv. phaseolicola strains demonstrated that G. wightii is a host for this organism.