Pseudomonas Syringae Pathovars Research Articles

Strategies for the biocontrol Pseudomonas infections pre-fruit harvest.

The efficiency of global crop production is under threat from microbial pathogens which is likely to be worsened by climate change. Major contributors to plant disease are Pseudomonas syringae (P. syringae) pathovars which affect a variety of important crops. This opinion piece focuses on P. syringae pathovars actinidiae and syringae, which affect kiwifruit and stone fruits, respectively. We discuss some of the current control strategies for these pathogens and highlight recent research developments in combined biocontrol agents such as bacteriophages and combinations of bacteriophages with known anti-microbials such as antibiotics and bacteriocins.

Potential applications of biosurfactants produced by different Pseudomonas syringae: A comparative study on interfacial activity, antimicrobial efficacy, and cytotoxicity

Finding novel biocompatible compounds with adequate interfacial activity and low toxicity still represents a great challenge for the scientific community. In this context, this work aims to compare for the first time the potential applications of biosurfactants produced by three Pseudomonas syringae pathovars: P. syringae pv tabaci (TA), P. syringae pv tomato (TO) and P. syringae pv syringae (SY). Isolated lipopeptides proved excellent emulsifying activity against a great variety of hydrocarbons and vegetable oils. The lowest minimum inhibitory concentrations against the pathogenic strain Staphylococcus aureus were observed for the biosurfactants produced by TA and SY (0.375 mg/mL). Non-cytotoxic effects were observed for concentrations of up to 10 mg/mL for these BS, and they proved differential cytotoxicity against cancer cell lines. These results open perspectives for the application of these surfactants as low-toxic bioemulsifiers and biotherapeutics.

Structural analysis and molecular substrate recognition properties of Arabidopsis thaliana ornithine transcarbamylase, the molecular target of phaseolotoxin produced by Pseudomonas syringae.

Halo blight is a plant disease that leads to a significant decrease in the yield of common bean crops and kiwi fruits. The infection is caused by Pseudomonas syringae pathovars that produce phaseolotoxin, an antimetabolite which targets arginine metabolism, particularly by inhibition of ornithine transcarbamylase (OTC). OTC is responsible for production of citrulline from ornithine and carbamoyl phosphate. Here we present the first crystal structures of the plant OTC from Arabidopsis thaliana (AtOTC). Structural analysis of AtOTC complexed with ornithine and carbamoyl phosphate reveals that OTC undergoes a significant structural transition when ornithine enters the active site, from the opened to the closed state. In this study we discuss the mode of OTC inhibition by phaseolotoxin, which seems to be able to act only on the fully opened active site. Once the toxin is proteolytically cleaved, it mimics the reaction transition state analogue to fit inside the fully closed active site of OTC. Additionally, we indicate the differences around the gate loop region which rationally explain the resistance of some bacterial OTCs to phaseolotoxin.

Antimicrobial Multiresistant Phenotypes of Genetically Diverse Pseudomonas spp. Isolates Associated with Tomato Plants in Chilean Orchards

Tomatoes are susceptible to bacterial diseases, mainly related to some Pseudomonas syringae pathovars. Many Pseudomonas species are considered innocuous, but some have shown the ability to opportunistically infect tomato plants. Antimicrobial compounds have been used to control pathogenic organisms, and this can lead to environmental selection of phenotypically resistant bacteria. We assessed the diversity of Pseudomonas species associated with tomato plants from Chilean orchards and analyzed antimicrobial resistance among the isolated strains. A total of 64 Pseudomonas isolates (P. syringae, P. viridiflava, P. fluorescens, P. koreensis, P. gessardii, and P. azotoformans) were evaluated for their phenotypic resistance to seven antimicrobial compounds, including copper, streptomycin, and five other antibiotics typically not used in agriculture. The results showed that 95%, 86%, 70%, 53%, 45%, and 1.6% of the isolates were resistant to rifampin, ampicillin, copper, chloramphenicol, streptomycin, and tetracycline, respectively, with no isolates being resistant to gentamicin. A total of 96.9% of Pseudomonas isolates exhibited a multiresistant phenotype to at least two of the antimicrobials tested. The most frequent multiresistance phenotype was Cu-Str-Amp-Cm-Rif (23.4%). The presence of Pseudomonas strains tolerant to conventional bactericides, metals, and other antimicrobials makes these bacteria an emerging threat to the agriculture industry and to human health.

Evaluation of bacteriophages in the biocontrol of Pseudomonas syringae pv. syringae isolated from cankers on sweet cherry (Prunus avium L.) in Turkey

BackgroundBacterial canker and subsequent gummosis are caused by multiple pathogens and lead to significant yield and productivity losses in sweet cherry cultivation in Turkey. This study identified that Pseudomonas syringae pathovars were responsible for bacterial canker on sweet cherry orchards by using classical and molecular methods and evaluated the biocontrol effects of bacteriophages against P. syringae pv. syringae.ResultsPathogenic bacteria were isolated from samples taken from plants showing symptoms of bacterial canker in cherry orchards located in İzmir and Manisa provinces. Specific pathogens were identified using pathogenicity, phenotypic tests, and simplex PCR. Bacteriophages effective against P. syringae strains were isolated from soil contaminated with pathogens identified in the diseased orchards using an optimized isolation protocol. The biocontrol activity of bacteriophage isolates against P. syringae pv. syringae was tested in vitro and in vivo. The results of pathogenicity tests on immature sweet cherry fruits and micropropagated cherry plantlets revealed 10 pathogenic bacteria isolates from 44 plant samples taken from sweet cherry orchards showing symptoms of bacterial canker.ConclusionsTen isolates were identified as Pseudomonas syringae pv. syringae. Nine different pure bacteriophage isolates were effective. The results indicated that bacteriophage isolates may demonstrate variable reactivity against P. syringae pathovars.

Isolation, Characterization, and Pathogenicity of Two Pseudomonas syringae Pathovars from Populus trichocarpa Seeds.

Pseudomonas syringae is a ubiquitous plant pathogen, infecting both woody and herbaceous plants and resulting in devastating agricultural crop losses. Characterized by a remarkable specificity for plant hosts, P. syringae pathovars utilize a number of virulence factors including the type III secretion system and effector proteins to elicit disease in a particular host species. Here, two Pseudomonas syringae strains were isolated from diseased Populus trichocarpa seeds. The pathovars were capable of inhibiting poplar seed germination and were selective for the Populus genus. Sequencing of the newly described organisms revealed similarity to phylogroup II pathogens and genomic regions associated with woody host-associated plant pathogens, as well as genes for specific virulence factors. The host response to infection, as revealed through metabolomics, is the induction of the stress response through the accumulation of higher-order salicylates. Combined with necrosis on leaf surfaces, the plant appears to quickly respond by isolating infected tissues and mounting an anti-inflammatory defense. This study improves our understanding of the initial host response to epiphytic pathogens in Populus and provides a new model system for studying the effects of a bacterial pathogen on a woody host plant in which both organisms are fully genetically sequenced.

The possible mechanisms of copper resistance in the pathogen Pseudomonas syringae pathovars in stone fruit trees

One of the most common diseases affecting Stone-fruits is Bacterial Canker, caused by Pseudomonas syringae pv. syringae (Pss) and morsprunorum (Psm). In this study, Pss and Psm were isolated from stone-fruit trees from the Western Aegean region (WA) and Lake Van Basin (LVB) of Turkey, where pesticide usage is high and low, respectively. We aimed to determine copper resistance levels, the involved resistance mechanisms and the reactions to copper pesticides of these isolates. The minimum inhibitory concentration (MIC) and lethal dose 50 (LD50) of the isolates were detected using CuSO4 and other Cu-pesticides. To determine the mechanisms of copper resistance, the presence of copA and cusA genes was investigated. The modified Fe/Cu Blue-CAS Agar media were used to investigate the relationship between the isolates’ copper resistance and each isolate’s ability to produce siderophores. The highest MIC value was 2 mM in CuSO4. The tolerance levels of the isolates from the WA and LVB regions were 29% and 47% susceptible, 29% and 47% low resistance and 42% and 6% resistant, respectively. The most successful pesticide was (CuSO4 + Ca (OH)2) + mancozeb. While none of the isolates had the cusA gene, four isolates had the copA gene, which was proven to be plasmid-borne. Differences between copA gene sequences were detected and were determined to be not related to the pathovars. The amount of siderophore produced against copper in Ps pathovars affected seems to be related with the resistance level. Also, Ps pathovars were able to tolerate copper at doses as high as 1.1 mM by producing siderophores, and at doses of 1.7 mM and above through the copA gene.

Quorum-dependent expression of rsmX and rsmY, small non-coding RNAs, in Pseudomonas syringae

Pseudomonas syringae pathovars are known to produce N-acyl-homoserine lactones (AHL) as quorum-sensing molecules. However, many isolates, including P. syringae pv. tomato DC3000 (PtoDC3000), do not produce them. In P. syringae, psyI, which encodes an AHL synthase, and psyR, which encodes the transcription factor PsyR required for activation of psyI, are convergently transcribed. In P. amygdali pv. tabaci 6605 (Pta6605), there is one nucleotide between the stop codons of both psyI and psyR. However, the canonical stop codon for psyI in PtoDC3000 was converted to the cysteine codon by one nucleotide deletion, and 23 additional amino acids extended it to a C-terminal end. This resulted in overlapping of the open reading frame (ORF) for psyI and psyR. On the other hand, stop codons in the psyR ORF of P. syringae 7 isolates, including pv. phaseolicola and pv. glycinea, were found. These results indicate that many pathovars of P. syringae have genetically lost AHL production ability by the mutation of their responsible genes. To examine whether PtoDC3000 modulates the gene expression profile in a population-dependent manner, we carried out microarray analysis using RNAs prepared from low- and high-density cells. We found the expressions of rsmX and rsmY remarkably activated in high-density cells. The activated expressions of rsmX and rsmY were confirmed by Northern blot hybridization, but these expressions were abolished in a ΔgacA mutant of Pta6605. These results indicate that regardless of the ability to produce AHL, P. syringae regulates expression of the small noncoding RNAs rsmX/Y by currently unknown quorum-sensing molecules.

Genomic Features and Lytic Activity of the Bacteriophage PPPL-1 Effective against Pseudomonas syringae pv. actinidiae, a Cause of Bacterial Canker in Kiwifruit.

Bacterial canker in kiwifruit is caused by Pseudomonas syringae pv. actinidiae (Psa). In this study, the bacteriophage PPPL-1 effective against Psa was characterized. Belonging to the Podoviridae family, PPPL-1 was effective against most Psa strains as well as most Pseudomonas syringae pathovars. PPPL-1 carries a 41,149-bp genome with 49 protein coding sequences and is homologous to the previously reported phiPSA2 bacteriophage. The lytic activity of PPPL-1 was stable up to 40°C, within a range of pH 3-11 and under 365 nm UV light. These results indicate that the bacteriophage PPPL-1 might be useful to control Psa in the kiwifruit field.

Essential oil composition and biological/pharmacological properties of Salmea scandens (L.) DC

Salmea scandens (L.) DC is an indigenous edible plant whose stem bark is traditionally used as food by people of Oaxaca, México. Proximate analysis of the edible stem bark revealed abundant amounts of fiber (43.67%) and protein (9.27%). GC and GC-MS analyses demonstrated that the essential oil from leaves contained high levels of germacrene D (47.1%) and elemol (15.3%), whereas that of the stem bark contained the alkylamides N-Isobutyl-(2E,4E,8Z,10E/Z)-dodecatetraenamide isomers (39.7%). Levels of these compounds in the essential oil from both organs were in similar concentrations in all seasons except winter. The HPLC purified N-Isobutyl-(2E,4E,8Z,10E/Z)-dodecatetraenamide isomers produced a non-competitive inhibition on porcine pancreatic lipase. The enzymatic assays with these compounds revealed a modification on Vmax (0.0431–0.0533 mM min−1) whereas the Km value (0.880–0.881 mM) was not significantly changed. Essential oil from the stem bark showed a high anti-microbial activity against some phytopathogenic microorganisms. The MIC's in μg mL−1 for Pseudomonas syringae pathovars were tabaci 56.1, tomato 91.2 and phaseolitica 196.4, for Clavibacter michiganensis 35.8 and Erwinia carotovora 48.1. The fungi Fusarium oxysporum and Phytophthora infestans had MIC's in μg mL−1 of 3.3 and 2.4 respectively. Same essential oil was highly effective against larvae from Aedes aegypti (LC50 = 0.3 μg mL−1) and Anopheles albimanis (LC50 = 2.5 μg mL−1).

Induction of a viable but not culturable (VBNC) state in some Pseudomonas syringae pathovars upon exposure to oxidation of an apoplastic phenolic, acetosyringone

Acetosyringone is a phenolic metabolite often found in plant apoplasts. Its oxidation by hydrogen peroxide and peroxidase results in a prolonged increase in the redox potential of the reaction mixture, similar to redox increases observed in tobacco suspension cells upon treatment with incompatible bacteria. Since high redox potentials, being oxidative, are generally detrimental to bacteria, the effect of acetosyringone oxidation on bacterial viability was examined. Pseudomonas syringae pv. syringae was added to reaction mixtures containing acetosyringone, hydrogen peroxide and peroxidase and samples were removed to determine viability by dilution plating. Initial studies were done with low bacterial concentrations, 105 CFU ml−1, to ensure that scavenging of H2O2 was negligible and did not interfere with the reaction mixture. No colonies were formed by bacteria that had been added to reaction mixtures with acetosyringone ranging from 25 to 100 μΜ. Examination of the bacteria by microscopy and flow cytometry, using fluorescent stains that indicate bacterial membrane integrity, suggested that these bacteria had maintained their membrane integrity. In addition they were able to respire based on oxygen uptake. When bacteria were added to on-going reaction mixtures at a time point after the prolonged redox response, the CFU ml−1 increased indicating that a stable reaction product was not responsible for the non-culturability bioactive effect. Other bacterial isolates, P. s. pv. tabaci and Pseudomonas fluorescens, were less susceptible to the bioactive effect of the acetosyringone oxidation. Other phenolics were tested and had lesser degrees of bioactivity and in some cases reduced the bioactivity of acetosyringone oxidation. The ‘viable but non-culturable’ (VBNC) state of the bacteria in this study is compared to that described for other medical and plant pathogens.

Determination of the Self-Association Residues within a Homomeric and a Heteromeric AAA + Enhancer Binding Protein

The σ54-dependent transcription in bacteria requires specific activator proteins, bacterial enhancer binding protein (bEBP), members of the AAA+ (ATPases Associated with various cellular Activities) protein family. The bEBPs usually form oligomers in order to hydrolyze ATP and make open promoter complexes. The bEBP formed by HrpR and HrpS activates transcription from the σ54-dependent hrpL promoter responsible for triggering the Type Three Secretion System in Pseudomonas syringae pathovars. Unlike other bEBPs that usually act as homohexamers, HrpR and HrpS operate as a highly co-dependent heterohexameric complex. To understand the organization of the HrpRS complex and the HrpR and HrpS strict co-dependence, we have analyzed the interface between subunits using the random and directed mutagenesis and available crystal structures of several closely related bEBPs. We identified key residues required for the self-association of HrpR (D32, E202 and K235) with HrpS (D32, E200 and K233), showed that the HrpR D32 and HrpS K233 residues form interacting pairs directly involved in an HrpR–HrpS association and that the change in side-chain length at position 233 in HrpS affects self-association and interaction with the HrpR and demonstrated that the HrpS D32, E200 and K233 are not involved in negative regulation imposed by HrpV. We established that the equivalent residues K30, E200 and E234 in a homo-oligomeric bEBP, PspF, are required for the subunit communication and formation of an oligomeric lock that cooperates with the ATP γ-phosphate sensing PspF residue R227, providing insights into their roles in the heteromeric HrpRS co-complex.

PCR amplification of the hrcV gene through specific primers for detecting Pseudomonas syringae pathovars

Pseudomonas syringae pathovars are important pathogens among phytopathogenic bacteria causing a variety of diseases in plants. These pathogens can rapidly disseminate in a large area leading to infection and destruction of plants. To prevent the incidence of the bacteria, appropriate detection methods should be employed. Routinely serological tests, being time-consuming and costly, are exploited to detect these pathogens in plants, soil, water and other resources. Over the recent years, DNA-based detection approaches which are stable, rapid, specific and reliable have been developed and sequence analysis of various genes are widely utilized to identify different strains of P. syringe. However, the greatest limitation of these genes is inability to detect numerous pathovars of P. syringae. Herein, by using bioinformatic analysis, we found the hrcV gene located at pathogenicity islands of bacterial genome with the potential of being used as a new marker for phylogenetic detection of numerous pathovars of P. syringae. Following design of specific primers to hrcV, we amplified a 440 bp fragment. Of 13 assayed pathovars, 11 were detected. Also, through experimental procedures and bioinformatic analysis it was revealed that the designed primers have the capacity to detect 19 pathovars. Our findings suggest that hrcV could be used as a gene with the merit of detecting more pathovars of P. syringae in comparison with other genes used frequently for detection purposes.

Top 10 plant pathogenic bacteria in molecular plant pathology.

Many plant bacteriologists, if not all, feel that their particular microbe should appear in any list of the most important bacterial plant pathogens. However, to our knowledge, no such list exists. The aim of this review was to survey all bacterial pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate the bacterial pathogens they would place in a 'Top 10' based on scientific/economic importance. The survey generated 458 votes from the international community, and allowed the construction of a Top 10 bacterial plant pathogen list. The list includes, in rank order: (1) Pseudomonas syringae pathovars; (2) Ralstonia solanacearum; (3) Agrobacterium tumefaciens; (4) Xanthomonas oryzae pv. oryzae; (5) Xanthomonas campestris pathovars; (6) Xanthomonas axonopodis pathovars; (7) Erwinia amylovora; (8) Xylella fastidiosa; (9) Dickeya (dadantii and solani); (10) Pectobacterium carotovorum (and Pectobacterium atrosepticum). Bacteria garnering honourable mentions for just missing out on the Top 10 include Clavibacter michiganensis (michiganensis and sepedonicus), Pseudomonas savastanoi and Candidatus Liberibacter asiaticus. This review article presents a short section on each bacterium in the Top 10 list and its importance, with the intention of initiating discussion and debate amongst the plant bacteriology community, as well as laying down a benchmark. It will be interesting to see, in future years, how perceptions change and which bacterial pathogens enter and leave the Top 10.

Choline Promotes Growth and Tabtoxin Production in a <i>Pseudomonas syringae</i> Strain

Some Pseudomonas syringae pathovars secrete tabtoxin, a monocyclic β-lactam antibiotic, responsible for chlorosis, the principal halo blight symptom in susceptible plants as oats, rye, barley, wheat and sorghum, among other. Here, we demonstrated that the production of tabtoxin in a P. syringae strain increased at least 150%, when choline, betaine or dimethylglycine were used as nitrogen source, or when choline was added as osmoprotectant in hyperosmolar culture media. Besides, we investigated the induction of phosphorylcholine phosphatase (PchP) activity when choline or its metabolites were used as nitrogen sources. PchP is an enzyme involved in Pseudomonas aeruginosa pathogenesis through its contribution to the breakdown of choline-containing compounds of the host cells. Considering these results and that the success of a pathogenic microorganism depends on its ability to survive and proliferate in its target tissue, we propose that choline is one of the plant signals that contribute to establishment of the infection by tabtoxin-producing strains of P. syringae.

Genes Involved in the Production of Antimetabolite Toxins by Pseudomonas syringae Pathovars

Pseudomonas syringae is pathogenic in a wide variety of plants, causing diseases with economic impacts. Pseudomonas syringae pathovars produce several toxins that can function as virulence factors and contribute to disease symptoms. These virulence factors include antimetabolite toxins, such as tabtoxin, phaseolotoxin and mangotoxin, which target enzymes in the pathways of amino acid metabolism. The antimetabolite toxins are generally located in gene clusters present in the flexible genomes of specific strains. These gene clusters are typically present in blocks of genes that appear to be integrated into specific sites in the P. syringae core genome. A general overview of the genetic organization and biosynthetic and regulatory functions of these genetic traits of the antimetabolite toxins will be given in the present work.

Chemical and metabolic aspects of antimetabolite toxins produced by Pseudomonas syringae pathovars.

Pseudomonas syringae is a phytopathogenic bacterium present in a wide variety of host plants where it causes diseases with economic impact. The symptoms produced by Pseudomonas syringae include chlorosis and necrosis of plant tissues, which are caused, in part, by antimetabolite toxins. This category of toxins, which includes tabtoxin, phaseolotoxin and mangotoxin, is produced by different pathovars of Pseudomonas syringae. These toxins are small peptidic molecules that target enzymes of amino acids’ biosynthetic pathways, inhibiting their activity and interfering in the general nitrogen metabolism. A general overview of the toxins’ chemistry, biosynthesis, activity, virulence and potential applications will be reviewed in this work.

In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.

Annual Meeting, Winnipeg, Manitoba, 2009

Annual Meeting, Winnipeg, Manitoba, 2009

Identification of Pseudomonas syringae pv. actinidiae as Causal Agent of Bacterial Canker of Yellow Kiwifruit (Actinidia chinensis Planchon) in Central Italy

AbstractAngular, necrotic leaf spot, longitudinal cracks along the petiole, oozing and wilting of branches were observed during summer 2008 on Actinidia chinensis (yellow kiwifruit) cultivar Hort16A, cultivated in different orchards located the province of Latina (central Italy). Symptoms closely resembled those incited by Pseudomonas syringae pv. actinidiae on kiwifruit A. deliciosa. Isolates obtained from typical lesions were assessed by means of biochemical, pathogenicity and host range tests and compared with some Pseudomonas syringae pathovars by enterobacterial repetitive intergenic consensus (ERIC‐PCR) analysis. The isolates belong to Pseudomonas LOPAT group Ia, incited the death of pot‐cultivated A. chinensis cv. Hort 16A and A. deliciosa cv. Hayward plants in few days, but did not cause any symptoms to the other inoculated plant species. Upon ERIC‐PCR analysis, all the isolates showed similarity with P. syringae pv. actinidiae NCPPB 3739, type‐strain of the pathovar. This is the first report of this pathogen on A. chinensis in Italy and, as far as we currently know, in the world.