Phenotypic variability in different strains of Pseudomonas syringae subsp. savastanoi isolated from different hosts

One hundred and sixty strains of Pseudomonas syringae subsp. savastanoi from Olea europaea, Olea europaea var. sylvestris, Nerium oleander, Fraxinus angustifolia and Retama sphaerocarpa, and four type strains of other pathovars were studied, investigating 102 phenotypic traits, among which we include biochemical characteristics, assimilation of different carbon sources, sensitivity or resistance to antibiotics and indoleacetic acid (IAA) production. Results were analysed with an affinity dendrogram via the Jaccard coefficient. They indicate an influence of environmental factors on the formation of the 15 phenons obtained, since isolated (knot) strains from the same species but different geographical areas are segregated. Segregation, also detected in strains from different hosts within the same area, added to the pathogenicity test helps to characterise these strains as different pathovars.     

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.     

Occurrence of unusual strains of Pseudomonas syringae subsp. savastanoi on olive in central Italy1

Bacteria forming levan colonies and not producing fluorescent pigments have been isolated from olive knots and the olive phylloplane in central Italy. By their pathogenicity to olive and their morphological, biochemical and physiological features, they clearly belong to Pseudomonas syringae subsp. savastanoi.     

Knot Formation Caused by Pseudomonas syringae subsp. savastanoi on Olive Plants Is hrp-Dependent

ABSTRACT The virulence of Pseudomonas syringae subsp. savastanoi, which causes hyperplastic symptoms (knots) on olive plants, is associated with secreted phytohormones. We identified a Tn5-induced mutant of P. syringae subsp. savastanoi that did not cause disease symptoms on olive plants although it was still able to produce phytohormones. In addition, the mutant failed to elicit a hypersensitive response in a nonhost plant. Molecular characterization of the mutant revealed that a single Tn5 insertion occurred within an open reading frame encoding a protein 92% identical to the HrcC protein of P. syringae pv. syringae. Moreover, sequence analysis revealed that the gene encoding the HrcC protein in P. syringae subsp. savastanoi was part of an operon that included five genes arranged as in other phytopathogenic bacteria. These results imply that hrp/hrc genes are functional in P. syringae subsp. savastanoi and that they play a key role in the pathogenicity of this plant pathogen.     

Systemic spread of Pseudomonas savastanoi pv. savastanoi in olive explants

The manner in which the bacterium Pseudomonas savastanoi pv. savastanoi ( Pss ), the causal agent of knot disease, infects olive plants is erratic and has not been fully documented. To investigate the process of Pss invasion, olive explants were inoculated in vitro and examined visually and by light microscopy at 2-weekly intervals for 10 weeks. In all host genotypes tested, interaction with the pathogen resulted in: (i) a progressive collapse of the stem, originating at the inoculation site at the apex of the explant, and proceeding downwards; and (ii), localized outgrowths on the stem located at various distances from the inoculation site. Histological analysis revealed that the anatomy of the outgrowths closely resembled that of knots formed in vivo ; they showed that Ps. savastanoi also diffused within the olive explants through the xylem vessels, and that the olive host reacted to pathogen invasion, possibly by producing substances of polysaccharidic and/or phenolic nature.     

Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.

Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die‐back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.     

Interaction between Pseudomonas savastanoi pv. savastanoi and Pantoea agglomerans in olive knots

Results of a survey of olive knot disease in central Italy in 2002 and 2003 showed that Pantoea agglomerans was found associated with the pathogen Pseudomonas savastanoi pv. savastanoi ( Ps. savastanoi ) in 70% of the olive knots examined. Pathogenicity tests in which these two bacteria were co-inoculated on the stems of 1-year-old olive plants at ratios of 1:1, 1:100 and 100:1 showed that the growth of P. agglomerans was apparently aided by the presence of an actively growing population of Ps. savastanoi . At the same time, however, a dominant population of P. agglomerans at the inoculation site tended to depress the growth of Ps. savastanoi , probably because of competition for space and nutrients between these bacteria and by means of antibiotic production by P. agglomerans. In some cases the association of P. agglomerans, which in culture was found to produce indole-3-acetic acid but not cytokinins, with Ps. savastanoi resulted in an increase in the size of knots. This boosting effect of P. agglomerans on proliferation was probably due to the release of IAA by this bacterium at the inoculation sites.     

Genetic variability of Iranian strains of Pseudomonas syringae pv. syringae causing bacterial canker disease of stone fruits

Strains of Pseudomonas syringae pv. syringae (Pss) were isolated from healthy and diseased stone fruits tissues sampled from 38 stone fruits orchard sites in Iran in 2010 and 2011. These strains were tested for pathogenicity and the presence of the syrB gene and were genetically characterized by using ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and IS50 (insertion sequences) primers and PCR. All 78 strains of Pss tested were moderately to highly pathogenic on Loring peach seedlings. A total of 78 isolates of the Pss amplified a 752-bp fragment with the syrB primers. To assess genetic diversity among the strains, genomic DNA was extracted from strains and used in rep-PCR and IS50-PCR analysis. Cluster analysis was performed using UPGMA. The strains of Pss were separated into nine distinguishable genotypic groups by the combination data set of both rep-PCR and IS50-PCR at 73 % similarity level. There was no significant correlation between genetic diversity and geographical origin of the isolates. These results indicate that a combination of rep-PCR and IS50-PCR fingerprinting can be used as a high resolution genomic fingerprinting method for elucidating intrapathovar diversity among strains of Pss. The results of this study demonstrated the existence of a considerable genetic diversity among Pss strains causing canker of stone fruit trees in Iran. In this study, genetic variability in Iranian strains of Pss were established, which will be of immense use in the development of resistant genotypes against this bacterial pathogen.     

Genetic,biochemical and pathogenic diversity of Pseudomonas syringae pv. pisi strains

Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep‐PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep‐PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.     

Dissemination of Pseudomonas savastanoi pv. savastanoi populations and subsequent appearance of olive knot disease

Pseudomonas savastanoi pv. savastanoi (Psv) is the causal agent of olive knot disease. The bacterium survives epiphytically and gains ingress through new wounds where infections and colonization result in knot formation. The natural spread of the bacterium and the subsequent appearance of the disease in olive orchards is poorly understood. The aim of this study was to monitor Psv epiphytic populations in inoculated plants with knots versus non‐inoculated healthy trees within the same orchard over four years. Additionally, disease severity was measured in both inoculated and non‐inoculated control trees. Epiphytic Psv populations moved from inoculated to non‐inoculated trees, although average Psv populations were higher in inoculated trees. Olive knot severity increased over the course of the study in all treatments and cultivars, with all plants reaching a high level of disease by the end of the study. However, the delay in the onset of disease was longer in non‐inoculated than in inoculated trees. Molecular typing of Psv isolates recovered from non‐inoculated control trees confirmed that they were similar to the inoculated strain. These data demonstrate that Psv can move over short distances in olive orchards through dissemination of epiphytic bacteria and suggest a relationship between the presence of epiphytic Psv and the number of knots on trees.     

Relationship of genetic structure of Pseudomonas savastanoi pv. savastanoi populations from Italian olive trees and patterns of host genetic diversity

A total of 360 Pseudomonas savastanoi pv. savastanoi isolates obtained from 11 Italian olive ( Olea europaea ) cultivars grown in different provinces were assessed with repetitive PCR using short interspersed elements of the bacterial genome as primers (ERIC, BOX and REP primer sets). The population structure of the isolates was determined by using three different hierarchical clustering algorithms: UPGMA, single-link and complete-link methods. REP primers were the most discriminatory. The various fingerprints obtained from the same cultivar and locality persisted over 2 years of knot sampling. Repetitive PCR and UPGMA analysis, using the three data sets combined, revealed 20 patterns with an overall similarity of 81%, with no grouping of the isolates. The resulting dendrogram shows a bush-like topology. Similar results were obtained with the other two clustering methods. In contrast, data obtained from the literature showed that the genetic structure of olive is characterized by bifurcated dendrograms and clear grouping of cultivars. Therefore it appears that the host plant and its pathogen did not cospeciate. The strict adaptation of the bacterium to olive would represent a case of association by colonization.     

Genetic and phenotypic diversity of Mediterranean populations of the olive knot pathogen,Pseudomonas savastanoi pv. savastanoi

Pseudomonas savastanoi pv. savastanoi (Psav) is a member of P. syringae sensu lato, and causes olive knot disease, a disease first reported over 2000 years ago. Analysing 124 isolates of Psav from 15 countries by rep‐PCR, the population genetic structure of Psav was investigated. A total of 113 distinct fingerprints were detected. Cluster analysis revealed the existence of two clusters and four subclusters. These clusters were associated with the geographic origin of isolates, which in turn correspond to historic human migration events and trade routes across the Mediterranean Sea. In contrast, multilocus sequence typing (MLST) of 2788 bp of the gapA, gltA, gyrB and rpoD genes found only one variable site among 77 representative isolates. Virulence variation was observed within the Psav population, with the most virulent strains generating knots that had a weight that was 10‐fold greater than those generated by the least virulent strains. Taken together, these data suggest that today's Psav population is the result of clonal expansion of a single strain, that moderate migration of the pathogen occurred between countries, and that changes in virulence arose during its evolution.     

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

Genetic diversity, presence of the syrB gene, host preference and virulence of Pseudomonas syringae pv. syringae strains from woody and herbaceous host plants

A total of 101 Pseudomonas syringae pv. syringae strains, obtained from international culture collections or isolated from diseased tissues of herbaceous and woody plant species, were assessed by repetitive PCR using the BOX primer, and for the presence of the syrB gene. Representative strains were also tested for pathogenicity to lilac, pear, peach, corn and bean, as well as for virulence to lemon and zucchini fruits. The unweighted pair-group method using arithmethic averages analysis (UPGMA) of genomic fingerprints revealed 17 different patterns which grouped into three major clusters, A, B and C. Most of the strains (52·4%) were included in patterns 1–4 of group A. These patterns comprised strains obtained from either herbaceous or woody species, and showed four fragments of similar mobility. Genetic variability was ascertained for strains isolated from apple, pear, apricot, Citrus spp. and cereals. No clear relationship was observed between host plant and bacterial genomic fingerprint. Variability was also observed in pathogenicity and virulence tests. The inoculation of pear leaves discriminated strains isolated from pear as well as the very aggressive strains, whereas inoculation of lilac, peach and corn did not discriminate the host plant from which the strains were originally isolated. Lemon fruit inoculation proved very effective for P. syringae pv. syringae virulence assessment. The syrB gene was present in almost all strains.     

In vitro analysis of the interaction of Pseudomonas savastanoi pvs. savastanoi and nerii with micropropagated olive plants

This study assessed the use of in vitro olive plants to evaluate the virulence of Pseudomonas savastanoi pv. savastanoi strains isolated from olive and P. savastanoi pv. nerii strains isolated from oleander knots. First, different olive isolates were inoculated into stem wounds and differences in knot formation and weight of overgrowths were observed for the selected strains. Tissue proliferation was clearly visible in all inoculated plants 30 days after inoculation. Virulence of P. savastanoi pv. nerii mutants with defects in regard to biosynthesis of indole-3-acetic acid and/or cytokinins was tested using this system. In agreement with data previously reported, all mutant strains multiplied in olive but induced attenuated symptoms. To analyze the virulence of P. savastanoi pv. savastanoi affected in their ability to grow in olive tissue, a trpE tryptophan auxotroph mutant was generated using a collection of signature tagged mutagenesis transposons. Virulence of this mutant was clearly reduced as evidenced by swelling of the olive tissue that evolved into attenuated knots. Furthermore, mixed infections with its parental strain revealed that the wild-type strain completely out-competed the trpE mutant. Results shown here demonstrate the usefulness of in vitro olive plants for the analysis of P. savastanoi pvs. savastanoi and nerii virulence. In addition, this system offers the possibility of quantifying virulence differences as weight of overgrowths. Moreover, we established the basis for the use of mixed infections in combination with signature tagged mutagenesis for high-throughput functional genomic analysis of this bacterial pathogen.