Erratum to: Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR

Applied Microbiology and Biotechnology -     

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.     

Scanning Electron Microscopy of Pseudomonas syringae pv, morsprunorum on Sweet Cherry Leaves

Scanning electron microscopy indicated that sub-stomatal cavities on sweet cherry leaves are “protected sites” which shelter resident populations of Pseudomonas syringae pv. morsprunorum. Bacteria entered the stomata, multiplied in the cavities and emerged in a mass 6 days after inoculation. There were no visible symptoms, suggesting that the pathogen colonized the host in “sub-clinical” numbers to generate populations which were then released onto the leaf surface under suitable conditions.     

Structure of the sidechain of lipopolysaccharide from Pseudomonas syringae pv. morsprunorum C28

The sidechain of the lipopolysaccharide from the phytopathogen Pseudomonas syringae pv. morsprunorum C28 was shown to be composed of D-rhamnose. Using 1H and 13C-NMR spectroscopy, methylation analysis, Smith degradation and optical rotation data, the repeat unit was found to have the structure: ----3)-D-Rhap-(alpha 1----3)-D-Rhap-(alpha 1----2)-D-Rhap-(alpha 1---- and a degree of polymerization of approximately 70. Attention is drawn to the possible prevalence of D-6-deoxyhexoses in the lipopolysaccharides of plant pathogenic bacteria.     

Systemic Invasion of Immature Sweet Cherry Fruit by Pseudomonas syringae pv. morsprunorum Through Blossoms1

Cherry blossoms inoculated with a rifampicin-resistant strain of Pseudomonas syringae pv. morsprunorum died or gave rise to fruits containing necrotic spots at or near the blossom ends. Scanning electron microscopy of developing fruits indicated that the pathogen had invaded the entire pericarp, including the endocarp. Bacteria also spread to the fruit stalk and, to a lesser extent, to the spurs. Mesocarp cells below the lesion collapsed. Infected fruit, stalks, and spurs contained, respectively, ca. 10⁹, 10⁷, and 10² colony forming units of P. syringae pv. morsprunorum as determined by a dilution plate method on an agar medium supplemented with 50 μg/ml rifampicin. This is the first report of systemic spread of P. syringae from blossoms to developing fruit of a deciduous crop.     

DNA markers for identification of Pseudomonas syringae pv. actinidiae

The specific DNA fragment was screened by RAPD analysis of Pseudomonas syringae pv. actinidiae, as well as similar strains that were isolated from kiwifruits. The primer C24 detected a fragment that is specific in P. syringae pv. actinidiae. This fragment was cloned. The pathovar-specific fragment was detected from a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae using the cloned fragment as a probe. The sequence size of the cloned fragment was determined as 675 bp. A DNA Database search suggested that the fragment was a novel one. Approximately 9 kb of a single fragment was detected only in the P. syringae pv. actinidiae by a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae. Similar strains were also detected with the use of the cloned fragment as a probe. Since the genomic DNAs were digested with HindIII without a cleavage site, the result reveals that the cloned fragment exists on the genome of P. syringae pv. actinidiae as a single copy. A pair of primers that produced a 492 bp single fragment (only in the strains of P. syringae pv. actinidiae) were synthesized, based on the pathovar-specific sequences of the cloned fragment of P. syringae pv. actinidiae. The development of the primers and probe made it possible to diagnose the bacterial canker infection from leaves or trunks of kiwifruit trees before any symptom appeared on the tree.     

A simple and efficient PCR method for the specific detection of Pseudomonas syringae pv phaseolicola in bean seeds

Using Southern blot hybridizations, it was found that the gene encoding the phaseolotoxin-insensitive ornithyl carbamoyl transferase (argK) was specific for Pseudomonas syringae pv. phaseolicola, the causal agent of the halo-blight disease. Based on these findings, a PCR protocol was developed for the specific detection of P.syringae. pv. phaseolicola in water-extracts of soaked bean seed. For this PCR protocol, two oligonucleotide primers were designed, based on the sequence of argK, which allowed the detection of a specific 1kb fragment. The protocol is simple since PCR was directly applied to bacterial suspensions, thus avoiding DNA extraction. The sensitivity of detection was increased by allowing the bacteria present in seed extracts to multiply in semi-selective media for 18h prior to PCR amplification. The detection threshold by visual detection using ethidium bromide staining was one naturally infected seed in lots of 400 to 600 seeds.     

PCR Detection of Cyclic Lipodepsinonapeptide-Producing Pseudomonas syringae pv. syringae and Similarity of Strains

Many strains of Pseudomonas syringae pv. syringae produce one of four classes of small cyclic lipodepsinonapeptides: syringomycins, syringostatins, syringotoxins, or pseudomycins. These metabolites are phytotoxic and growth inhibitory against a broad spectrum of fungi. Their production is dependent upon the expression of conserved biosynthesis and export genes syrB and syrD, respectively. PCR and oligonucleotide primers specific for a 752-bp fragment of syrB were used to identify cyclic lipodepsinonapeptide-producing strains of P. syringae pv. syringae. In contrast, PCR amplification with primers based on syrD did not always correlate with possession of the syrD gene, as indicated by Southern blot analysis, or with cyclic lipodepsinonapeptide production. Sequence comparisons of 400 nucleotides from the syrB PCR-amplified fragments showed 94% plot similarity among 27 strains. In a sequence phenogram, syringostatin and syringotoxin producers were grouped apart from syringomycin-producing strain B301D, with sequences that differed by eight and nine conserved base substitutions, respectively. PCR amplification of the 752-bp syrB fragment offers rapid and accurate detection of cyclic lipodepsinonapeptide-producing strains, and its sequence provides some predictive capabilities for identifying syringotoxin and syringostatin producers.     

Effects of Pseudomonas syringae pv. morsprunorum Strain C28, Outer Membrane and LPS Preparations on Cherry Callus

Callus cultures were established from cherry (Prunus avium) cvs. Napoleon and Colt, respectively susceptible and resistant to race‐1 strains of Pseudomonas syringae pv. morsprunorum, by growth on Schenk–Hildebrandt medium. On Napoleon callus, necrosis began earlier and proceeded more rapidly when inoculated with the virulent race‐1 cherry isolate strain C28, than with mutants of diminished virulence derived from it, or with the virulent plum isolates D10 and D17. Colt tissue displayed poorer viability and showed susceptibility to strain C28 and the plum isolates. Callus from both sources was somewhat susceptible to the saprophytes P. aeruginosa NCIMB 8295 and P. fluorescens NCIMB 3756. Strain C28 grew on suspended Napoleon callus cells over a period of 3–4 days, causing leakage of UV‐absorbing compounds and K⁺, with a concomitant rise in extracellular pH. P. fluorescens NCIMB 3756 showed no growth on suspended callus for 6 days. EDTA‐extracted outer membrane (OM) from strain C28 caused leakage of UV‐absorbing material and K⁺, which was later reabsorbed, with little change in pH. The presence of OM suppressed the growth of a subsequent inoculum of strain C28, possibly due to complexation of the available Ca²⁺ and/or Mg²⁺ in the surrounding medium, by the component lipopolysaccharide (LPS). OM from the rough avirulent mutant strain C28‐2 induced leakage of K⁺, but not of UV‐absorbing material, and did not prevent the growth of subsequently inoculated strain C28. Smooth LPS from strain C28 did not cause leakage of K⁺ or of UV‐absorbing material and did not prevent growth of C28. The relevance of these findings is discussed in relation to disease.     

Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR

Speed is a key area in our development of PCR assays for Bacillus anthracis. We believe that the strand specific detection of amplicons within 10 min is a realistic goal and that this will be achieved through fluorescent in-tube assays. We have used the Idaho LightCycler to study and develop candidate assays for B. anthracis. New strand specific fluorescent methods have been developed and a number of formats have been studied for speed and sensitivity. Internal controls have been developed as a method of improving our assay confidence. In this communication we will introduce the field of rapid PCR whilst discussing previous work in the areas described above, the development of our own rapid assay and a novel internal control system for B. anthracis. This work used PCR assays and hardware that are either commercially available, or have been previously described in open literature publications.     

Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco

Isolates of Pseudomonas syringae pv. tabaci, including 21 strains of the wildfire pathogen and 2 strains of the angular leafspot pathogen, were isolated from 143 rhizosphere and soil samples collected from 11 tobacco fields in Wisconsin. These pathogens were isolated by inoculating rhizosphere and soil washings into tobacco leaves and isolating the bacteria from wildfire or angular leafspot lesions that developed on the leaves. The wildfire isolates were from the rhizospheres of tobacco and Panicum capillare and from soil. While the majority of these were from wildfire-diseased fields, one isolate was from a field without disease symptoms; both angular leafspot isolates were from fields without angular leafspot symptoms. The majority of wildfire isolates were race 1, but three were race 0, and one was a new race. In three fields multiple races of wildfire were found. Both angular leafspot isolates were race 1. Two wildfire and one angular leafspot isolates were from fields where the cultivars were resistant to the races isolated.     

Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IB using SCAR primers

AIMS: The aim of this study was to develop a specific and sensitive identification method for Rhizoctonia solani AG 1-IB isolates based on phylogenetic relationships of R. solani AG-1 subgroups using rDNA-internal transcribed spacer (rDNA-ITS) sequence analysis. METHODS AND RESULTS: A neighbour-joining tree analysis of 40 rDNA-ITS sequences demonstrated that R. solani AG-1 isolates cluster separately in six subgroups IA, IB, IC, ID, IE and IF. A molecular marker was generated from a random amplified polymorphic DNA fragment (RAPD). After conversion into a sequence-characterized amplified region (SCAR), a specific primer set for identification of subgroup AG 1-IB was designed for use in a polymerase chain reaction (PCR). The primer pair amplified a single DNA product of 324 bp. CONCLUSIONS: R. solani AG-1 subgroups were discriminated by sequence analysis of the ITS region. The designed SCAR primer pair allowed an unequivocal and rapid detection of R. solani AG 1-IB in plant and soil samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequence analysis of the rDNA-ITS region can be used for differentiation of subgroups within AG-1. The use of the developed SCAR primer set allowed a reliable and fast identification of R. solani AG 1-IB and provides a powerful tool for disease diagnosis.     

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from 1D and 2D 1H- and 13C-NMR spectra combined with extensive FAB-MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodepsinonapeptide. Its peptide moiety corresponds to Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acetylated by 3-hydroxytetradecanoic acid.     

Pseudomonas syringae pv. actinidiae detection in kiwifruit plant tissue and bleeding sap

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.     

Characterization of Pyoverdinpss, the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin_pss, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin_pss of approximately 50 μg/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of ≥ 10 μM. Pyoverdin_pss was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin_pss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin_pss was structurally unique since amino acid analysis of reductive hydrolysates yielded β-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin_pss exhibited a relatively high affinity constant for Fe(III), with values of 10²⁵ at pH 7.0 and 10³² at pH 10.0. Iron uptake assays with [⁵⁵Fe]pyoverdin_pss demonstrated rapid active uptake of ⁵⁵Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin_pss. The chemical and biological properties of pyoverdin_pss are discussed in relation to virulence and iron uptake during plant pathogenesis.     

Molecular and Physiological Characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola Strains That Produce the Phytotoxin Coronatine

The chlorosis-inducing phytotoxin coronatine is produced by several Pseudomonas syringae pathovars, including glycinea, morsprunorum, atropurpurea, and the closely related tomato and maculicola. To date, all coronatine-producing pv. glycinea, morsprunorum, and atropurpurea strains that have been examined carry the gene cluster that controls toxin production on a large plasmid. In the present study the genomic location of the coronatine gene cluster was determined for coronatine-producing strains of the pv. tomato-maculicola group by subjecting their genomic DNA to pulsed-field electrophoresis and Southern blot analysis with a hybridization probe from the coronatine gene cluster. The cluster was chromosomally borne in 10 of the 22 strains screened. These 10 strains infected both crucifers and tomatoes but could not use sorbitol as a sole source of carbon. The remaining 12 coronatine-producing strains had plasmid-borne toxin gene clusters and used sorbitol as a carbon source. Only one of these strains was pathogenic on both crucifers and tomatoes; the remainder infected just tomatoes. Restriction fragment length polymorphism analysis of the pv. tomato-maculicola coronatine gene clusters was performed with probes from P. syringae pv. tomato DC3000, a tomato and crucifer pathogen. Although the coronatine cluster appeared, in general, to be highly conserved across the pv. tomato-maculicola group, there were significant differences between plasmid-borne and chromosomally borne genes. The extensively studied coronatine cluster of pv. glycinea 4180 closely resembled the plasmid-borne clusters of the pv. tomato-maculicola group.     

Specific molecular detection of Phytophthora sojae using conventional and real-time PCR

Phytophthora rot, caused by Phytophthora sojae, is one of the most damaging diseases of soybean (Glycine max) worldwide. This disease can be difficult to diagnose and other Phytophthora species can infect soybean. Accurate diagnosis is important for management of Phytophthora rot. The objective of this study was to evaluate polymerase chain reaction (PCR) methods for rapid and specific detection of P. sojae and diagnosis of Phytophthora rot. PCR assays using two sets of primers (PS and PSOJ) that target the ITS region were evaluated for specificity and sensitivity to P. sojae. Genomic DNA extracted from 11 species of Phytophthora and 19 other species of fungal and oomycete pathogens were used to test the specificity of each primer set. The previously published PS primers amplified DNA from P.?sojae and from four other Phytophthora species using conventional PCR, indicating they are not specific for P. sojae. The new PSOJ primers amplified DNA only from P. sojae using conventional and real-time PCR and not from Phytophthora sansomeana, which has been found in soybean production areas, indicating that they are specific for P. sojae. The PSOJ primers were also used to detect P. sojae in diseased soybean tissue and infested soil. The PCR assays based on the PSOJ primers are specific, rapid, and sensitive tools for the detection of P. sojae.     

Pathogenicity and identification of the lilac pathogen, Pseudomonas syringae pv. syringae van Hall 1902

Comparative in planta studies with Pseudomonas syringae pv. syringae have established optimum conditions for disease expression in lilac in terms of inoculum concentration, host age and post-inoculation conditions (temperature and day-length). Reproducible disease reactions required an inoculum concentration exceeding the ED⁵⁰, 5 × 10⁶ cfu/ml, and a temperature for post-inoculation incubation not exceeding 19°C. A revised host range of P. syringae pv. syringae, proposed on the basis of confirmation of pathogenicity of strains to lilac, comprises 44 species from monocotyledonous and dicotyledonous plants. Nine new hosts Abelmoschus esculentus, Bromus willdenowii, Camellia sinensis, Centrosema pubescens, Citrullus lanatus, Cotoneaster sp., Cucumis melo, Populus×euramericana and Triticum aestivum, are recorded. A comparative laboratory study was made of strains of P. syringae pv. syringae using more than 30 selected biochemical and nutritional tests. The pathovar could be characterised on the basis of 11 of these which may prove to be useful determinative tests.