Certain aspects of resistance of plum trees to bacterial canker: Part III. The action of cell-free filtrates of Pseudomonas mors-prunorum Wormald and related phyto-pathogenic bacteria on plum trees

A technique for introducing 2-3 c.c. liquid into the bark of plum trees is described. Parallel injections with dyestuff throughout the spring failed to afford a reliable index of the invasive powers of the various test fluids introduced in this manner.
A susceptible variety of plum (Giant Prune) showed greater injury by cell-free filtrates of Ps. morsprunorum than did a resistant variety (Warwickshire Drooper), which showed negligible injury. The greatest injuries were caused by filtrates of cultures 5 weeks old, and over, especially when concentrated.
Of other bacteria tested Ps. prunicola, Ps. syringae strains, Syringa 3, Ap. No. 1, Ap. No. 2, and Bact. pruni , yielded damaging filtrates, whereas Ps. syringae , strains pear 6 and 7, Ps. tabaci, Ps. fluorescens, Ps. marginalis, Ps. phaseolicola, Ps. tumefaciens, Ps. pisi and Ps. cerasi failed to do so.
Some evidence is advanced for the view that the deleterious activity of Ps. mors-prunorum may be due in part to an endotoxin of protein nature obtainable from the dried bacterial cells by acetic acid extraction.     

Studies on the Bacteriophagy of Pseudomonas mors-prunorum, Ps. syringae and Related Organisms

S ummary : A group of 23 phages, mainly isolated with Pseudomonas mors-prunorum and Ps. syringae as the propagating strains, was tested against more than 200 pseudomonads including named plant pathogens and a variety of saprophytes. The majority of the phages had a wide host range, and did not distinguish between plant pathogens and saprophytes, thus confirming the close relationship between these two groups. The most reactive bacteria were 60 English isolates of Ps. mors-prunorum , 48 from cherry and 12 from plum, and 28 isolates of Ps. syringae from pear. Patterns of reaction within these 3 groups were relatively homogeneous and each group was distinct and differed from all other isolates tested. Ps. syringae isolates from other hosts were less uniform and occasionally shared reaction patterns with other species, e.g. Ps. cannabina and Ps. glycinea. Similar relationships were observed with phages at both high titre and at routine test dilution including the difference in phage sensitivity between the cherry and plum strains of Ps. mors-prunorum. On the basis of 7 biochemical tests the plum and cherry strains were indistinguishable but they differed from all Ps. syringae isolates tested by giving white growth in 5% sucrose broth and in failing to liquefy gelatin. Furthermore, unlike most Ps. syringae isolates they were also unable to hydrolyze aesculin and were tyrosinase positive. There was no clear evidence in this investigation of correlation between phage sensitivity and biochemical activity. Eleven isolates from various European countries, designated Ps. mors-prunorum , differed widely both in phage sensitivity and biochemical activity and some of them may be more appropriately called Ps. syringae. Others may be intermediate forms between these species. The relationship between Ps. mors-prunorum and Ps. syringae and the nomenclature of these organisms are discussed and a concept of ecotypes suggested as a substitute for species.     

Characterization of Pseudomonas pathovars isolated from rosaceous fruit trees in East Algeria

A survey of bacterial diseases due to Pseudomonas on rosaceous fruit trees was conducted. In forty two orchards located in the Constantine region ( East Algeria). Pseudomonas isolates were identified on the bases of their cultural and biochemical characteristics . A total of fifty nine phytopathogenic bacteria were isolated from diseased pome and stone fruit trees. Thirty one strains comparable to Pseudomonas syringae pv. syringae were isolated from cherry (Prunus avium L.), plum (P. domestica L.), apricot (P. armeniaca L.), almond (P. dulcis L.) and pear trees (Pirus communis L.); sixteen strains comparable to Pseudomonas syringae pv. morsprunorum were obtained from samples of cherry and plum. Twelve strains of Pseudomonas viridiflava were isolated from cherry, apricot and peach (Prunus persica L.).     

A Note: Serological study of four pathovars of Pseudomonas syringae: Ps. syr. aptata, Ps. syr. tabaci, Ps. syr. mors-prunorum and Ps. syr. phaseolicola

The antigenic reactions of 35 strains of four pathovars of Pseudomonas syringae (Ps. syr. aptata, Ps. syr. tabaci, Ps. syr. mors-prunorum and Ps. syr. phaseolicola ) were studied by double diffusion and indirect immunofluorescent staining, and anti-whole-cell and anti-LPS-extract sera. It had already been shown that the precipitating lines in Ouchterlony double-diffusion tests, due to bacterial LPS, were suitable for the distinction of O-serogroups. The investigation of serological cross-reactions between the 35 strains and 20 antisera revealed that three pathovars were serologically homogeneous: Ps. syr. aptata, Ps. syr. tabaci and Ps. syr. phaseolicola. They could fit into three O-serogroups formerly described: namely APTPIS, TAB and PHA. The O-serogroups APTPIS and TAB showed some common antigens. The 10 strains of Ps. syr. mors-prunorum studied were distributed into two O-serogroups (eight strains belonging to the O-serogroup MOP1, one strain to MOP2, and the last strain failed to react with any of the serogroups).     

Preliminary description of biocidal (syringomycin) activity in fluorescent plant pathogenic Pseudomonas species

Strains representing the fluorescent plant pathogenic Pseudomonas spp., Ps. agarici , Ps. asplenii , Ps. avellanae , Ps. beteli , Ps. caricapapayae , Ps. cichorii , Ps. corrugata , Ps. ficuserectae , Ps. flectens , Ps. fuscovaginae , Ps. marginalis , Ps. meliae , Ps. savastanoi , Ps. syringae , Ps. tolaasii and Ps. viridiflava were tested for biocidal activity using Aspergillus niger as assay organism. Inhibitory behaviour was found in strains of Ps. asplenii , Ps. blatchfordae , Ps. cichorii , Ps. corrugata , Ps. fuscovaginae , Ps. marginalis , Ps. marginalis pv. pastinacea , Ps. syringae pv. syringae , Ps. syringae pv. aptata , Ps. syringae pv. atrofaciens , Ps. syringae pv. lapsa , Ps. tolaasii , and strains of a Pseudomonas sp. pathogenic to Actinidia , in the Ps. savastanoi genomic sp. Antifungal activity could be identified with the production of members of the syringomycin family of toxins by strains in Ps. syringae , Ps. asplenii and Ps. fuscovaginae . These toxin reactions support suggestions made elsewhere of the synonymy of the latter two species. In a preliminary characterization using tests for stability to heat, protease, acid and alkaline treatments, unknown toxins consistent with syringomycin-like toxins the strains from Actinidia speciesColour RGB 0,0,128. The toxins from Ps. cichorii and from Ps. corrugata differed in their reactions from all other agents. Pseudomonas tolaasii produces the antifungal compound tolaasin. The white line reaction with ' Ps. reactans ', a test for tolaasin production by strains of Ps. tolaasii , was confirmed as specific for this compound. Some of these low molecular weight toxins may be produced by some of these plant pathogenic strains.     

Identification of Pseudomonas tolaasi: the White Line in Agar and Mushroom Tissue Block Rapid Pitting Tests

A sharply defined white line of precipitate forms in Pseudomonas Agar F (Difco) between the opaque white colonies of Pseudomonas tolaasi and translucent colonies of certain unidentified pseudomonads. This visible interaction has been utilized in a specific and reliable method for the identification of Ps. tolaasi. The white line test was positive when 113 isolates of Ps. tolaasi from five different countries were examined, whereas 154 isolates of pseudomonads other than Ps. tolaasi , including Ps. corrugata, Ps. delphinii, Ps. fluorescens, Ps. lachrymans, Ps. marginalis, Ps. pastinaceae, Ps. phaseolicola, Ps. aeruginosa, Ps. putida, Ps. syringae, Ps. mors-prunorum, Ps. cichorii, Ps. antirrhini, Ps. viridiflava, Ps. caryophylli, Ps. cepacia, Ps. mendocina, Ps. stutzeri, Ps. acidivorus and Ps. lemoignei did not give the white line reaction with a reacting translucent colony pseudomonad. Browning of mushrooms in host tests does not help in the identification of Ps. tolaasi , but a conspicuous pitting produced in less than 10 min at the cut surface of mushroom tissue is as specific as the white line test in detecting Ps. tolaasi in suspension in distilled water.     

Numerical systematics of bacteria of the genus Pseudomonas

In 290 strains of bacteria belonging to the genus Pseudomonas, 120 morphological and physiologo-biochemical characters were studied and the results obtained thereby were analyzed by the methods of numerical taxonomy using computers. The majority of strains were subdivided into 11 clusters: Ps. aeruginosa (1), Ps. putida (2), Ps. rathonis (5), Ps. syringae (8), Ps. pseudoalcaligenes (9), Ps. maltophilia (10), Ps. acidovorans (11), Ps. testosteroni (12), Ps. mendocina (13), Ps. cepacia (14), Ps. fluorescens (3). The latter cluster included also the strains identified earlier as Ps. aurantiaca, Ps. lemonnieri, Ps. fluoro-violaceus, and Ps. aureofaciens. Three clusters contained strains which could not be identified and probably should be regarded as distinct species. The characteristics have been selected useful for diagnostics of the above Pseudomonas bacteria and the subgroups of Ps. fluorescens.     

Pseudomonas syringae pv. japonica (Mukoo 1955) Dye et al. 1980 is a junior synonym of Ps. syringae pv. syringae van Hall 1902

An investigation of the biochemical, nutritional and pathogenic reactions of strains of Pseudomonas syringae pv. japonica and Ps. syringae pv. syringae showed them to be indistinguishable. Pseudomonas syringae pv. japonica is a junior synonym of Ps. syringae pv. syringae.     

Isozyme analysis for the identification of Pseudomonassyringae pathovar pisi strains

Distinction between Pseudomonas syringae pathovar (pv.) pisi (Ps. syr. pisi) , responsible for bacterial blight of pea ( Pisum sativum ), and pv. syringae (Ps. syr. syringae) , still requires strain inoculation onto peas. Patterns of enzymes including esterase (EST) and superoxide dismutase (SOD) were examined for diagnostic purposes. Profiles of 59 Ps. syr . pisi strains and 53 Ps. syr . syringae strains were compared. Pseudomonas syringae pisi was characterized by one unique zymotype for SOD and two slightly different zymotypes for EST. Pseudomonas syringae syringae zymotypes were very heterogeneous with 10 different zymotypes for SOD and 32 for EST. Twenty-four percent of the Ps. syr . syringae strains shared SOD zymotype 1 of Ps. syr . pisi , thus preventing the use of this enzymatic system for identification. In contrast, the two EST zymotypes of Ps. syr. pisi strains were specific to the pathovar and could be used for its identification. The two Ps. syr. pisi EST patterns were correlated to race structure of the pathovar, zymotype 1 corresponding to races 2, 3, 4 and 6, and zymotype 2 to races 1, 5 and 7. Esterase isozyme profiling was proposed as a new identification procedure for bacterial pea blight agent.     

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.     

Characteristics of English Isolates of Pseudomonas syringae van Hall from Pear

S ummary . The cultural and biochemical characters of 30 isolates of Pseudomonas syringae from pear are described and compared with isolates of Ps. syringae from other hosts and with a variety of Pseudomonas spp. from plant material.     

Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola

Aims:  To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean ( Phaseolus vulgaris L.).
Methods and Results:  Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium , Erwinia and Pantoea .
Conclusion:  A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria .
Significance and Impact of the Study:  This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.     

Detection of tabtoxin-producing strains of Pseudomonas syringae by PCR

AIMS: The present study describes a system based on PCR to distinguish tabtoxin-producing strains of Pseudomonas syringae from other Ps. syringae plant pathogens that produce chlorosis-inducing phytotoxins. METHODS AND RESULTS: Thirty-two strains of Ps. syringae and related species were examined. Two sets of PCR primers were developed to amplify genes (tblA and tabA) required for tabtoxin production. Only a PCR product of 829 bp or 1020 bp was produced in PCR reactions with the tblA or tabA primer sets, respectively, and cells from tabtoxin-producing pathovars of Pseudomonas syringae. All known non-tabtoxin producing bacterial species failed to produce an amplification product with either primer set. CONCLUSIONS: PCR of genes required for tabtoxin production is a simple, rapid and reliable method for identifying tabtoxin-producing strains of Ps. syringae. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol can effectively distinguish tabtoxin-producing strains of Ps. syringae from other Ps. syringae pathovars and Ps. syringae pv. tabaci strains from other tabtoxin-producing Ps. syringae pathovars.     

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.     

Serological and molecular size characterization of flagellins of Pseudomonas syringae pathovars and related bacteria

Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P. chlororaphis, P. cichorii, P. corrugata, P. fluorescens, P. fuscovaginae, P. stutzeri, P. viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting. Eighty-six strains of the P. syringae group pathovars, P. cichorii and P. viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa. Similarities between the P. syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out. The distribution of H1 and H2 antigens in the nine recently described genomospecies of P. syringae-P. viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads. The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.     

A numerical taxonomic study of the Pseudomonas flora isolated from poultry meat

Pseudomonas strains were isolated from both fresh and cold-stored broiler skin. Phenotypically-based numerical taxonomic techniques were used to characterize the isolates and 36 reference strains. For this purpose, Biolog GN Microplates, API 20NE and a number of other biochemical tests were used. Jaccard clustering revealed the predominance of four major Pseudomonas groups: Ps. fragi, Ps. lundensis, strains belonging to Ps. fluorescens biovars and an unidentified group of strains displaying a high degree of similarity to Ps. fluorescens biovars. Within Ps. fluorescens, biovar A was best represented. The marked proteolytic character of members of Ps. fluorescens biovars A, B and C, as well as of members of the unidentified cluster, supports their possible role in the origin of organoleptic defects. In the Ps. lundensis cluster, a distinct group of Ps. lundensis-like species was found. Further genotypic studies should be carried out to clarify the taxonomic status of the Ps. lundensis-like strains and that of the unidentified group resembling Ps. fluorescens biovars A and B.     

Comparison of Plant Pathogenic Pseudomonads from Fruit Trees

S ummary . The characteristics of 59 pathogenic pseudomonads isolated from stone fruit trees in south east England, 30 from cherry and 29 from plum, were compared with 41 isolates from pear trees in a wide range of biochemical, physiological and lesion tests. Many characteristics were common, but several consistent and stable differences were found between the stone fruit and pear groups, each of which exhibited a high level of homogeneity. The few atypical isolates in each group deviated from the majority in one or two respects only. Ten citrus and 3 lilac isolates were biochemically and physiologically indistinguishable from the pear isolates but had distinctive phage and bacteriocin sensitivities. The stone fruit isolates correspond to Pseudomonas morsprunorum and the pear isolates to Ps. syringae. The relationship between these two 'species'is discussed.     

A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water

M. GENNARI AND F. DRAGOTTO. 1992. Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

Comparison of ATPase-encoding type III secretion system hrcN genes in biocontrol fluorescent Pseudomonads and in phytopathogenic proteobacteria

Type III protein secretion systems play a key role in the virulence of many pathogenic proteobacteria, but they also occur in nonpathogenic, plant-associated bacteria. Certain type III protein secretion genes (e.g., hrcC) have been found in Pseudomonas sp. strain SBW25 (and other biocontrol pseudomonads), but other type III protein secretion genes, such as the ATPase-encoding gene hrcN, have not been found. Using both colony hybridization and a PCR approach, we show here that hrcN is nevertheless present in many biocontrol fluorescent pseudomonads. The phylogeny of biocontrol Pseudomonas strains based on partial hrcN sequences was largely congruent with the phylogenies derived from analyses of rrs (encoding 16S rRNA) and, to a lesser extent, biocontrol genes, such as phlD (for 2,4-diacetylphloroglucinol production) and hcnBC (for HCN production). Most biocontrol pseudomonads clustered separately from phytopathogenic proteobacteria, including pathogenic pseudomonads, in the hrcN tree. The exception was strain KD, which clustered with phytopathogenic pseudomonads, such as Pseudomonas syringae, suggesting that hrcN was acquired from the latter species. Indeed, strain KD (unlike strain SBW25) displayed the same organization of the hrpJ operon, which contains hrcN, as P. syringae. These results indicate that the occurrence of hrcN in most biocontrol pseudomonads is not the result of recent horizontal gene transfer from phytopathogenic bacteria, although such transfer might have occurred for a minority of biocontrol strains.