Distinguishing pathovars of Pseudomonas syringae on peas: nutritional, pathogenicity and serological tests

Of the published methods to distinguish Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. pisi , inoculation of susceptible cultivars was the most reliable. Results were confirmed by inoculation of lemon fruit.
A much more rapid and convenient serological method was developed to distinguish the two pathovars. Antisera against glutaraldehyde-fixed cells had a high level of specificity in Ouchterlony gel double-diffusion tests and, after cross-absorption with heterologous antigens, in indirect ELISA. Antiserum to P. syringae pv. pisi has considerable potential to detect pea seed infected with this pathogen.     

Testing for Pseudomonas syringae pv. atrofaciens and Xanthomonas campestris pathovars on cereals in Italy

During recent years, recurrent attacks of basal glume rot/leaf blight attributed to Pseudomonas syringae pv. atrofaciens have been observed on cultivars of durum wheat grown in the Po valley (IT). The main aim of this study was to test for this pathogen and Xanthomonas campestris pathovars in commercial seed lots of cereals produced in different regions of Italy, as well as in symptomatic plants collected in the field. None of the analyses led to the detection of xanthomonads. In testing for P.s. atrofaciens , representative bacterial isolates were selected and characterized; this was done by combining conventional identification tests and computerized densitometric analysis of electrophoretic patterns of cell proteins obtained using the SDS-PAGE technique. P.s. atrofaciens was detected in seed samples (barley, wheat and durum wheat) and symptomatic plants (durum wheat) grown in northern and central Italy. The possible pathogenic role of other unrelated fluorescent pseudomonads in this interaction was also examined. Difficulties in discriminating pvs syringae and atrofaciens of P. syringae are discussed and the uncertain taxonomic relationship between these two pathovars is emphasized.     

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.     

Specificity of polyclonal antibodies to different antigenic preparations of Pseudomonas syringae pv. pisi strain UQM551 and Pseudomonas syringae pv. syringae strain L

Polyclonal antibodies were produced against sonicated and heat-killed cells of Pseudomonas syringae pv. pisi strain UQM551 and Pseudomonas syringae pv. syringae strain L, and their specificities were compared. Evidence is presented that the serological specificity between these two pathovars lies in surface antigens. Of the surface antigens purified and tested, only flagella and lipopolysaccharide from the cell wall showed no cross-reactivity with heterologous antisera. Antisera to glutaraldehyde-fixed flagella of the two strains showed a high level of specificity. At a species or genus level, antisera prepared from heat-killed cells of P. syringae distinguished this species from all other bacterial species and genera tested, including strains of Pseudomonas fluorescens, Escherichia coli, Agrobacterium and Rhizobium.     

Identification of two serological flagellar types (H1 and H2) inPseudomonas syringae pathovars

Flagellar antigen specificity was studied for the speciesPseudomonas syringae, P. viridiflava andP. cichorii. After checking their motility, bacteria were reacted against six polyclonal antisera containing anti-O (LPS) and anti-H (flagellar) antibodies by indirect immunofluorescent staining. Two distinct flagellar serotypes (H1 and H2) were described. The distribution of H1 and H2 serotypes was then determined for a collection of 88 phytopathogenicPseudomonas strains. Serotype H1 was possessed byP. syringae pv.aptata (12 strains),P. s. pv.helianthi (2),P. s. pv.pisi (11), andP. s. pv.syringae (13). Serotype H2 was possessed byP. cichorii (2),P. s. pv.delphinii (1),P. s. pv.glycinea (4),P. s. pv.lacrymans (1),P. s. pv.mori (1),P. s. pv.morsprunorum (10),P. s. pv.persicae (1),P. s. pv.phaseolicola (8),P. s. pv.tabaci (10) andP. s. pv.tomato (1).P. viridiflava (5) revealed HI, H2 and untyped flagella. The following isolates were untypable by the H1/H2 system:P. corrugata (3),P. fluorescens (2),P. tolaasii (1). H1/H2 serotypes distribution is not linked toP. syringae O-serogroups. On the other hand, H1/H2 distribution seems remarkably linked to the new genospecies of theP. syringae group.Abbreviations CFBP French Collection of Phytopathogenic Bacteria, Angers, France - ICMP International Collection of Micro-organisms from Plants, Auckland, New-Zealand - NCPPB National Collection of Plant Pathogenic Bacteria, Harpenden, Great Britain     

Recent problems with Pseudomonas syringae pv. pisi in UK1

Bacterial blight of peas caused by Pseudomonas syringae pv. pisi was found for the first time in a UK field crop during 1985. The outbreak was confined to a seed crop of the compounding protein pea cv. Belinda. All stocks derived from the original imported basic seed lot were traced and the seed crops from some 180 farms were removed from the certification schemes and were not allowed to go for planting. The pathogen, belonging to race 2, was found in approximately two thirds of these stocks. Protein pea seed is produced in UK, whereas most seed for vining crops is imported. The UK climate with its cool, wet summers is considered ideal for the establishment of pea blight and we consider that the disease could be particularly damaging in susceptible cultivars. Statutory control methods will be reviewed in an effort to maintain freedom from the disease. Surveys of field crops will also be carried out.     

Comparison of ethylene-producing Pseudomonas syringae strains isolated from kudzu (Pueraria lobata) with Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. glycinea

The relationships among strains of Pseudomonas syringae pv. glycinea (Psg) and Pseudomonas syringae pv. phaseolicola (Psp) isolated from kudzu ( Pueraria lobata) and bean ( Phaseolus vulgaris) were investigated. All strains tested showed a close phenotypic similarity, with the exception of the utilization of inositol and mannitol as well as the production of toxins. On this basis the strains could be divided into three groups. Group 1 consists of all strains of pathovar glycinea, group 2 includes all Psp strains isolated from kudzu, and all Psp strains isolated from bean belong to group 3. This grouping was also reflected in the genetic fingerprints using the polymerase chain reaction (PCR) with primers that anneal to dispersed repetitive bacterial sequences (rep-PCR). The rep-PCR generated fingerprints were unique for each of the three groups. The strains of group 2, Psp strains isolated from kudzu, possess certain characteristics of group 1 (ethylene production) and group 2 (phaseolotoxin production). The Psp strains from kudzu can be clearly differentiated from Psp strains isolated from bean. They utilize mannitol, produce ethylene, and are strongly pathogenic to kudzu, bean, and soybean. The results obtained show that the Psp strains from kudzu should be separated from the pathovar phaseolicola and should represent their own pathovar.     

Patterns of interaction between isolates of three pathovars of Pseudomonas syringae and accessions of a range of host and nonhost legume species

Isolates of three pathovars of Pseudomonas syringae were tested against 10 legume species. Some isolates of all pathovars showed cultivar-specific interactions with at least one legume species outside the expected host range. Lablab purpureus and Phaseolus lunatus were found to be hosts to isolates of both P. syringae pv. glycinea and P. syringae pv. phaseolicola, while Lathyrus latifolius was host to isolates of P. syringae pv. pisi and P. syringae pv. glycinea . Lens culinaris showed patterns of interaction with isolates of all three pathovars. Gene models based on mathematical estimates of minimum gene numbers agreed with those previously published for the interactions of P. syringae pv. pisi with Pisum sativum and P. syringae pv. phaseolicola with Phaseolus vulgaris. Two different gene-for-gene models based on five resistance/avirulence gene pairs were proposed to explain observed interactions between Glycine max and P. syringae pv . glycinea . Pathogen isolates which contained no known avirulences defined on their respective host species were found to carry cryptic avirulences recognized by other plant species. Estimates of minimum gene numbers required to explain the interactions of a plant species with all pathogen isolates or to explain the interactions of the isolates of one pathovar with all plant accessions were consistently lower than the sum of the minimum gene numbers required to explain the interactions of each individual component.     

Antagonisme entre les Pseudomonas fluorescents et plusieurs pathovars de Pseudomonas syringae. Application comme méthode de lutte contre la moucheture bactérienne de la tomate1

Deux Pseudomonas du groupe fluorescens-putida,à forte activité inhibitrice vis-à-vis de bactéries phytopathogènes, ont été confrontéà une gamme de 23 pathovars de Pseudomonas syringae. lis ont confirmé leur activité antagoniste sur ces derniers germes. Deux essais réalisés dans des conditions normales de culture de plein champ ont démontré la potentialité de ces antagonistes comme moyen de lutte contre la moucheture bactérienne, due àP. syringae pv. tomato. Des travaux complémentaires ont montré que ?activité antagoniste des deux germes inhibiteurs n'est pas semblable, ?un des deux agissant essentiellement par la production ?un sidérophore. Le mécanisme ?action du second germe n'est pas déterminé.     

Induction of pear blossom blast caused by Pseudomonas syringae pv. syringae

Conditions were established for inducing pear blossom blast caused by Pseudomonas syringae pv. syringae on both attached and detached shoots. The incidence of blossom blast was proportional to the logarithm of the P.s. pv. syringae population under optimal temperature, moisture, and bloom developmental stage. Highest incidence of blossom infection followed occurrence of a major exotherm (an increase in temperature caused by the heat of fusion from ice formation within blossom tissue) in the presence of P. s. pv. syringae. The exotherm was detected inside ovary tissue at temperatures ranging from –1.8 to –3.5 C. Wetness duration following the thawing process was less important than wetness during and immediately after the freeze event. Blossoms inoculated, then air-dried or removed from low-temperature treatment prior to occurrence of an exotherm, had a low incidence of infection, The full bloom stage of blossom development was more susceptible to blossom blast than either the open cluster or tight cluster stages of development.     

Bacterial inflorescence rot of grapevine caused by Pseudomonas syringae pv. syringae

Molecular sequencing (rpoB) and standard pathological and microbiological methods identified Pseudomonas syringae pv. syringae (Pss) as the causal agent of bacterial inflorescence rot of grapevines (Vitis vinifera) in three vineyards in Tumbarumba, NSW, Australia in 2006 and 2007. Pss strains from shrivelled berries and necrotic inflorescences of diseased grapevines were used to inoculate leaves and inflorescences of potted cv. Semillon grapevines. Pss caused disease symptoms similar to those experienced in the field, including angular leaf lesions, longitudinal lesions in shoot tissues and rotting of inflorescences from before flowering until shortly after fruit set. High humidity promoted symptom severity. The necrotic bunch stem and leaf lesions were susceptible to the development of Botrytis cinerea infections. Cryo‐scanning electron microscopy (cryoSEM) indicated that Pss entered leaves and inflorescence tissues via distorted, open, raised stomata surrounded by folds of tissue that appeared as ‘star‐shaped’ callose‐rich complexes when viewed by UV light microscopy. In necrotic tissues, cryoSEM revealed Pss within petiole parenchyma cells and air‐filled rachis xylem vessels. This is the first report of inflorescence and hence fruit loss caused by Pss in grapevines. The disease is described as ‘bacterial inflorescence rot’ and regarded as one that expands the previously reported pathology of grapevines caused by P. syringae. This study also indicated that infection by Pss might promote destructive B. cinerea infections when the fungus is already present but latent, although further experimentation is needed to prove such an interaction.     

Pathogenicity and virulence factors of Pseudomonas syringae

In 1994, Oku reported that plant pathogens, mainly fungal pathogens, require three essential abilities to infect plants: to enter plants, to overcome host resistance, and to evoke disease. Because the infectious process of phytopathogenic bacteria differs from that of fungal pathogens, we have attempted to characterize pathogenicity, the ability of a pathogen to cause disease, using the phytopathogenic bacterium Pseudomonas syringae as a representative pathogen. To establish infection and incite disease development, bacteria first have to enter a plant. This process requires flagella- and type IV pili-mediated motility, and active taxis is probably necessary for effective infection. After bacteria enter a plant’s apoplastic spaces, they need to overcome host plant resistance. To do this, they secrete a wide variety of hypersensitive response and pathogenicity (Hrp) effector proteins into the plant cytoplasm to interfere with pathogen/microbe-associated molecular pattern- and effector-triggered immunity, produce phytohormones and/or phytotoxins to suppress plant defense responses and extracellular polysaccharides to prevent access by antibiotics and to chelate Ca²⁺, and activate the multidrug resistance efflux pump to extrude antimicrobial compounds for successful colonization. Furthermore, to evoke disease, bacteria produce toxins and Hrp effectors that compromise a plant’s homeostasis and injure plant cells. The expression of these virulence factors depends on the infection processes and environmental conditions. Thus, the expression and function of virulence factors interact with each other, creating complex networks in the regulation of bacterial virulence-related genes.     

Motility-mediated regulation of virulence in Pseudomonas syringae

We have investigated Pseudomonas syringae pv. tabaci–plant interactions using a large variety of virulence-related mutants. A flagellin-defective mutant, ΔfliC, lost flagellar motility and the ability to produce N-acyl homoserine lactones; it had reduced ability to cause disease symptoms, but the expression of genes encoding a multidrug efflux pump transporter, mexEFoprN, was activated. A type IV pili (T4P)-defective mutant, ΔpilA, lost swarming motility, had reduced expression of hrp-related genes and virulence toward the host tobacco plant, but expression of the genes encoding another multidrug efflux pump transporter, mexABoprM, was activated. These results suggest that the genes regulating flagella- and T4P-mediated motilities also regulate expression of other virulence-related genes.     

A study of populations of Pseudomonas syringae pv. syringae on stonefruits in Victoria

In a survey of the major stonefruit nurseries in Victoria during winter 1978 and 1979, Pseudomonas syringae pv. syringae , the causal organism of bacterial canker, was found to be present on most of the stonefruit material in all nurseries but was detected most frequently on apricot.
The epiphytic populations of P.s. pv. syringae on leaves, buds and shoots of apricot and cherry were assessed periodically between 1979 and 1983 by determining the proportion of trees bearing the bacterium or by counting numbers of bacteria. Populations consistently reached peak levels during spring and late autumn, with highest levels in spring. Populations were lowest during mid- to late summer. High proportions of tree contamination and high populations coincided with periods when maximum temperatures ranged from 19° to 25°C, and when rainfall was moderately high. The significance of these findings in the light of information from other studies on the seasonal variability of host susceptibility, and in relation to chemical control, is discussed.
There was no evidence of occurrence of P.s. pv. morsprunorum in Victoria.     

Identification of Pseudomonas syringae pv. syringae causing bacterial leaf blight of Miscanthus sinensis

Journal of Plant Diseases and Protection - In the late summer of 2015, severe leaf blight occurred on Miscanthus sinensis grown on natural riverside lands of the Han River in Seoul, South Korea....     

Evaluation of drench treatments with phosphonate derivatives against Pseudomonas syringae pv. syringae on pear under controlled environment conditions

Several phosphonate derivatives including theoomycetic antifungal agents phosphonate andtris-o-ethylphosphonate (fosetyl), theethylene-releasing compound 2-chloroethylphosphonate(ethephon), and the antibiotic2-epoxypropylphosphonate (phosphomycin) were evaluatedfor in vitro and in planta activityagainst Pseudomonas syringae pv. syringae.Inhibition of colony growth in CYE agar byphosphonate, fosetyl and etephon was very slight(minimal inhibitory concentrations MIC= 0.31–;0.62 gHP /l). Also, survival of P. syringae pv. syringae in aqueous solutions ofphosphonate or fosetyl was high. Only phosphomycinshowed significant antibacterial activity invitro (MIC=10-20 µg HP /ml) comparedto streptomycin (1-2 µg a.i./ml). Potted pearplants irrigated with these chemicals and inoculatedwith Pseudomonas syringae pv. syringae had significantly less disease than non-treatedcontrols ( P<0.001). Phosphomycin was the mostactive compound with a median effective dose(ED₅₀) of less than 0.62 g HP /l.Activities of the other phosphonates were weak butconsistent between experiments. The ED₅₀s on wholeplants were 2.1, 3.3, and 6.9 g HP /l for ethephon, phosphonate and fosetyl, respectively. TheED₅₀of P. syringae pv. syringaeincreased from 6.5 in non-treated controls to 7.7-8.8log₁₀ cfu/ml on plants treated with phosphonatesat 1.86 g HP /l. It was concluded thatdrench treatment with fosetyl is not a practicaloption for control of P. syringae pv. syringae on pear.