Identification of the biosynthetic gene cluster for 3-methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93.

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K(m), 7 mM; k(cat), 85 min(-1)). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

A polyphasic approach assigns the pathogenic Erwinia strains from diseased pear trees in Japan to Erwinia pyrifoliae.

AIMS: Bacterial shoot blight of pear in Japan (BSBP) is caused by Erwinia strains which were formerly associated with the species Erwinia amylovora, the causative agent of fire blight. The description of Erwinia pyrifoliae as a pear pathogen in Korea renewed a possible connection of the pear pathogens in both countries. METHODS AND RESULTS: Nucleotide sequence analysis of the 16S rRNA, the house keeping genes gpd and recA, as well as DNA-DNA hybridization kinetics and microbiological assays place the pear pathogens from Japan into the species E. pyrifoliae described as the causative agent of Asian pear blight in Korea. CONCLUSIONS: Erwinia pyrifoliae strains from Korea and the pear pathogenic Erwinia strains from Japan belong taxonomically into the same species, but show slight divergences in nucleotide sequences used for classification. The allocation is not only supported by microbiological properties, but also by a host range restricted to pear observed before by others. SIGNIFICANCE AND IMPACT OF THE STUDY: The data suggest that the BSBP disease observed at the island of Hokkaido was not fire blight and unify BSBP in Japan with the pear pathogenic species E. pyrifoliae from Korea.

Biological Control of Pseudomonas syringae pv. glycinea by Epiphytic Bacteria under Field Conditions.

The efficacy of a bacterial strain as a biocontrol agent in the field may be related to the ecological similarity between the biocontrol agent and the target pathogen. Therefore, a number of different Pseudomonas syringae strains were evaluated for their antagonistic activities in vitro (agar-diffusion assay) and in planta (greenhouse assay) against the target pathogen, Pseudomonas syringae pv. glycinea. Six strains of five different pathovars were found to be antagonistic in vitro as well as in planta. The epiphytic fitness of the antagonistic Pseudomonas syringae strain 22d/93 and its two antibiotic-resistant mutants were examined on soybean plants in the fields. After adaptation the parental strain and its mutants had the ability to establish and maintain large epiphytic populations (about 106 cfu/g FW) over the whole growing season after a single spray inoculation. The epiphytic behaviors of the mutants and the parent were not significantly different. The introduced bacteria did not influence the total bacterial population size. When the antagonist was coinoculated with the pathogen, the development of the pathogen was significantly reduced during the whole growing season. When the antagonistic strain was inoculated 4 weeks in advance of the pathogen, this antagonistic effect could be markedly enhanced. The final population size of the pathogen reached just 104 cfu/g FW and was significantly reduced to 0.12% compared to the pathogen alone. This study demonstrates that biological control of foliar pathogens through colonization of the host plants with near isogenic or ecologically similar antagonistical strains seems to be a realistic goal.

The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola.

ABSTRACT The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum.

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.

Toxin production by pathovars of Pseudomonas syringae and their antagonistic activities against epiphytic microorganisms.

75 strains of 21 various Pseudomonas syringae (P.) pathovars were investigated in different tests for their toxin production. Data from literature about the production of the known phytotoxins phaseolotoxin (pv. phaseolicola), tabtoxin (pv. coronafaciens, pv. tabaci), coronatine (pv. atropurpurea, pv. glycinea, pv. maculicola, pv. morsprunorum, pv. tomato), and toxins of the lipodepsipeptide group (pv. aptata, pv. atrofaciens, pv. syringae) could be confirmed. Besides, a production of the phytohormone ethylene was detected for P. phaseolicola isolates from kudzu (Pueraria lobata) and for all tested P. glycinea and P. cannabina strains. Strains of P. apii, P. aptata, P. atrofaciens, and P. tomato produced antimetabolic toxins which could be detected with an agar diffusion assay with Escherichia coli as indicator strain. These antimetabolites inhibit a step in the arginine/ornithine biosynthesis. P. maculicola strains caused inhibition zones in this assay which could not be reversed by the tested amino acids. All strains with inhibitory effect against E. coli and Geotrichum candidum were also proved for their antagonistic activity against a selection of typical phyllosphere microorganisms. Most of the phytotoxins possess antimicrobial activity with different spectrum and efficiency. Only the lipodepsipeptide-producers showed antifungal activities. Our results show that the production of toxins is a widespread property among Pseudomonas syringae pathovars, and that some pathovars can produce more than one toxin. This characteristic and the antimicrobial activity of most toxins could be of advantage for the toxin-producing bacteria to adapt to different habitats.

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.

Characterization of two epiphytic bacteria from soybean leaves with antagonistic activities against Pseudomonas syringae pv. glycinea.

The strains 48b/90 and 22d/93 are naturally occurring ephiphytes which were isolated from soybean leaves. On the basis of pheno- and genotypic characteristics 48b/90 was identified as Erwinia herbicola and 22d/93 as Pseudomonas syringae. These two isolates produced biological active substances against different indicator organisms. The E. herbicola strain showed clear antagonistic properties against Escherichia coli and Pseudomonas syringae pv. glycinea, but not against Geotrichum candidum. 22d/93 was active against P. glycinea and G. candidum, but not against E. coli. Strain 48b/90 produced at least two different inhibitors: an antibiotic substance and an inhibitor of the alginate synthesis. Strain 22d/93 produced at least three different compounds inhibitory to P. glycinea and one to G. candidum. Their activities against the bacterial blight pathogen, P. glycinea, can be observed in planta, too. Under the influences of the antagonists the pathogen multiplied at lower rates and to lower stationary phase population levels. The development of bacterial blight symptoms was suppressed.

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)

Molecular characterization of field isolates of Pseudomonas syringae pv. glycinea differing in coronatine production.

Coronatine-producing and non-producing strains of Pseudomonas syringae pv. glycinea have been examined. We found a connection between copper resistance and synthesis of coronatine. Published data implied that these properties may be encoded on different plasmids. Production of coronatine and copper resistance were also found to be correlated for pv. glycinea in 19 field-isolates from leaf spots of plants in a soybean field and in 28 strains of a bacterial culture collection. Genomic diversity within pv. glycinea was investigated by plasmid profiling, DNA hybridization studies and PCR analysis. All strains unable to produce coronatine (cor-) were sensitive to copper ions and showed no homology to DNA from plasmid pSAY1, which carries a gene cluster for steps in coronatine production. In addition, cor- strains could be distinguished from coronatine-producing strains by a single unique band when amplified by random primer PCR. Plasmid profiles of strains isolated from field-populations during 1983, 1985 and 1990 showed that coronatine-producing and non-producing strains were present. The plasmid patterns also varied in 28 strains examined from a culture collection. No correlation between plasmid patterns and race specificity was observed. Cosmid pSAY1 proved to be an effective probe for detection of the coronatine synthesis genes and also revealed polymorphisms in coronatine producing strains of pv. glycinea.

Occurrence of antimicrobial activities of bacteria from soybean leaf spots.

Bacteria were isolated from leaf spots of field grown soybeans during two growing seasons. The leaf spots yielded up to 4 different species and a total population size of about 10(7)-10(8) bacteria/cm2. The majority of the 192 isolates belonged to the species Pseudomonas syringae pv. glycinea (55%), causing leaf spots of bacterial blight on soybeans, and Erwinia herbicola (22%). The remaining isolates included bacteria from other genera, but occurred occasionally. The determination of biological activity of the isolates demonstrated that a high percentage of strains from the group Erwinia/Enterobacter produced biological active substances against Escherichia coli (69%) and against Chlorella pyrenoidosa (88%). The majority of P. syringae pv. glycinea strains failed to do so. None of the isolates affected the growth of Geotrichum candidum. The E. herbicola strains showed clear antagonistic properties against a wide range of isolated bacteria. Four E. herbicola strains inhibited the growth of nearly all other E. herbicola isolates and 6 other strains were active against most of the P. syringae pv. glycinea isolates. However, antagonistic interactions among strains isolated from a distinct leaf spot were very rarely.

Identification and population dynamics of bacteria in leaf spots of soybean.

The qualitative and quantitative composition of bacterial flora occurring inside the leaf spots of field grown soybeans was studied during the growing seasons (June to October) of 1989 and 1990. As a rule these leaf spots (necrotic lesions with chlorotic haloes) were caused by Pseudomonas syringae pv. glycinea. This pathogenic bacterium was predominantly found during the whole season in the symptomatic leaf tissue. Other species, mainly Erwinia herbicola, were also found in the same habitat. The population sizes of P. s. pv. glycinea increased from the beginning of symptom occurrence until July, stabilized until September, and then decreased a little. In general, the size of saprophytic populations was orders of magnitude lower than that of the pathogenic populations. The number of different bacterial genera per sample increased up to four genera per leaf spot by the end of the season. No significant influence of the occurring saprophytes on the population dynamics of the pathogen in planta could be observed.

[Studies on the variability of the phaseolotoxin production by Pseudomonas syringae pv. phaseolicola]. / Untersuchungen zur Variabilität der Phaseolotoxinbildung bei Pseudomonas syringae pv. phaseolicola.

Isolation of bacteria from a field of bean plants (Phaseolus vulgaris L.) with conspicuous symptoms of halo blight disease resulted in 123 bacterial strains from which 57 were identified as Pseudomonas syringae pv. phaseolicola . At 18 degrees C the phaseolotoxin production of the isolated strains differs widely in submerse culture. Only few strains produce high amounts of phaseolotoxin being comparable with those of the reference strain 1321, while most of the strains show a low capability of phaseolotoxin production. Furthermore, we proved the stability of phaseolotoxin production of 29 strains. After one year about 50% of the strains were extremely reduced in their capability of phaseolotoxin production. We found that the reason for this reduction of toxin production is the appearance of Tox- segregants within a Tox+ clone. At 24 degrees C all strains (with one exception) show considerable lower amounts of toxin production or none at all: maximally 30% of the toxin amounts synthesized at 18 degrees C were produced. At 28 degrees C none of the isolated strains produce phaseolotoxin . The present data allow the conclusion to be drawn that in natural environments there exists a wide spread regarding the amount and stability of the phaseolotoxin production.