Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids

Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.     

High-resolution melting analysis as a powerful tool to discriminate and genotype Pseudomonas savastanoi pathovars and strains

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants.     

Detection of Pseudomonas savastanoi pv. savastanoi in olive plants by enrichment and PCR

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.     

Genetic characterization of spoilage pseudomonads isolated from retail-displayed beef

Aim:  This study genetically characterized Pseudomonas isolated from beef using the random amplification of polymorphic DNA (RAPD) method and correlate predominant genotypes with spoilage changes.
Methods and Results:  Pseudomonads were recovered from beef loins and steaks on days 0, 2, 4, 6, 8 and 10. A total of 309 pseudomonads were grouped into 50 RAPD types (>85% similarity). One major RAPD type contained 45% of the isolates comprising 71%, 45%, 31%, 35%, 50% and 37% of isolates from days 0, 2, 4, 6, 8 and 10, respectively, from steaks and 48% of the isolates recovered from beef loins. Nineteen RAPD types consisted of isolates that were shared between more than two sampling times, whereas the remaining 31 types were unique to one particular time.
Conclusions:  A genetically diverse Pseudomonas population was present on the loins and steaks at each sampling time. Although pseudomonads associated with beef loins were transferred to the steaks prepared from it, a genetically diverse Pseudomonas population emerged during the retail display.
Significance and Impact of the Study:  Information about the heterogeneous nature of Pseudomonas recovered from meat would help understanding the spoilage owing to predominant strains. The meat industry can use the knowledge to develop control strategies for prevalent spoilage strains.     

Comparison of strains of Pseudomonas syringae pv. savastanoi from olive leaves and knots

A collection of strains of Pseudomonas syringae pv. savastanoi was subjected to numeric phenetic analysis of 60 characters using unweighted average linkage on the simple matching coefficient. Most strains recovered by washing random leaves in April and October shared lower similarity values between themselves than with the majority of those isolated from 6-month-old knots in October and April, respectively.     

Highly sensitive detection of Pseudomonas savastanoi pv. savastanoi in asymptomatic olive plants by nested-PCR in a single closed tube

A nested-polymerase chain reaction (PCR) has been set up to be performed in a single closed tube for the detection of Pseudomonas savastanoi pv. savastanoi. Nested-PCR coupled with dot-blot hybridization was able to detect up to one cell of the target per ml of olive extract, showing the greatest sensitivity compared with all previously reported detection assays. Validation of the developed procedure for diagnosis and epidemiological purposes was achieved by testing ca. 240 asymptomatic plant samples from olive trees. When performing the other previously reported techniques (bacterial isolation and single PCR), P. savastanoi was detected in 50 of the analyzed samples, while with the new developed nested-PCR assay, the bacterium was detected in 82 samples.     

Genome sequence analyses of Pseudomonas savastanoi pv. glycinea and subtractive hybridization-based comparative genomics with nine pseudomonads

Bacterial blight, caused by Pseudomonas savastanoi pv. glycinea (Psg), is a common disease of soybean. In an effort to compare a current field isolate with one isolated in the early 1960s, the genomes of two Psg strains, race 4 and B076, were sequenced using 454 pyrosequencing. The genomes of both Psg strains share more than 4,900 highly conserved genes, indicating very low genetic diversity between Psg genomes. Though conserved, genome rearrangements and recombination events occur commonly within the two Psg genomes. When compared to each other, 437 and 163 specific genes were identified in B076 and race 4, respectively. Most specific genes are plasmid-borne, indicating that acquisition and maintenance of plasmids may represent a major mechanism to change the genetic composition of the genome and even acquire new virulence factors. Type three secretion gene clusters of Psg strains are near identical with that of P. savastanoi pv. phaseolicola (Pph) strain 1448A and they shared 20 common effector genes. Furthermore, the coronatine biosynthetic cluster is present on a large plasmid in strain B076, but not in race 4. In silico subtractive hybridization-based comparative genomic analyses with nine sequenced phytopathogenic pseudomonads identified dozens of specific islands (SIs), and revealed that the genomes of Psg strains are more similar to those belonging to the same genomospecies such as Pph 1448A than to other phytopathogenic pseudomonads. The number of highly conserved genes (core genome) among them decreased dramatically when more genomes were included in the subtraction, suggesting the diversification of pseudomonads, and further indicating the genome heterogeneity among pseudomonads. However, the number of specific genes did not change significantly, suggesting these genes are indeed specific in Psg genomes. These results reinforce the idea of a species complex of P. syringae and support the reclassification of P. syringae into different species.     

Characterization of Aeromonas encheleia strains isolated from aquatic environments in the Czech Republic

Aims:  Characterization and identification of Aeromonas strains isolated from surface and underground waters using phenotypic and genotyping methods.
Methods and Results:  Biotyping using the ENTEROtest 24 kit and conventional biochemical and physiological tests assigned four strains to Aeromonas encheleia , whereas three isolates were identified as ambiguous Aeromonas bestiarum/Aeromonas caviae and one strain as Aeromonas eucrenophila/Aeromonas encheleia . Further characterization grouped the analysed strains together with Aer. encheleia CCM 4582^T and assigned the analysed group as members of Aer. encheleia species using ribotyping, whole-cell protein analysis and ERIC-PCR fingerprinting. The results obtained were verified by DNA gyrase A subunit gene sequencing. All analysed isolates showed unique molecular patterns, except for isolates P 1769 and CCM 7407, which revealed the same Eco RI ribotype profile and proved to be identical strains.
Conclusions:  Our results imply that Aer. encheleia strains occur in unpolluted surface as well as in underground waters and demonstrate applied methods as suitable for their identification.
Significance and Impact of the Study:  To our best knowledge, this is the first report of the isolation and identification of Aer. encheleia in the Czech Republic.     

Study of the coumarate decarboxylase and vinylphenol reductase activities of Dekkera bruxellensis (anamorph Brettanomyces bruxellensis) isolates

Aim:  To evaluate the coumarate descarboxylase (CD) and vinylphenol reductase (VR) activities in Dekkera bruxellensis isolates and study their relationship to the growth rate, protein profile and random amplified polymorphic DNA (RAPD) molecular pattern.
Methods and Results:  CD and VR activities were quantified, as well, the growth rate, intracellular protein profile and molecular analysis (RAPD) were determined in 12 isolates of D. bruxellensis . All the isolates studied showed CD activity, but only some showed VR activity. Those isolates with the greatest growth rate did not present a different protein profile from the others. The FASC showed a relationship between RAPD molecular patterns and VR activity.
Conclusion:  CD activity is common to all of the D. bruxellensis isolates. This was not the case with VR activity, which was detected at a low percentage in the analysed micro-organisms. A correlation was observed between VR activity and the RAPD patterns.
Significance and Impact of the Study:  This is the first study that quantifies the CD and VR enzyme activities in D. bruxellensis, demonstrating that these activities are not present in all isolates of this yeast.     

Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis

Aims:  The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method.
Methods and Results:  Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis, using arithmetic means (UPGMA). Isolates grouped according to host, with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus -specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2), and an internal amplification control was developed, to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi.
Conclusions:  RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus , with a detection limit of 10 fg.
Significance and Impact of the Study:  Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems.     

Regulation of 3-indoleacetic acid production in Pseudomonas syringae pv. savastanoi. Purification and properties of tryptophan 2-monooxygenase

The oxidative decarboxylation of L-tryptophan to yield 3-indoleacetamide, catalyzed by tryptophan 2-monooxygenase, represents a controlling reaction in the synthesis of indoleacetic acid by Pseudomonas savastanoi (Pseudomonas syringae pv. savastanoi), a gall-forming pathogen of olive (Olea europea L.) and oleander (Nerium oleander L.). Production of indoleacetic acid is essential for virulence of the bacterium in its hosts. Tryptophan 2-monooxygenase was characterized to determine its role in indoleacetic acid metabolism in the bacterium. The enzyme was purified to apparent homogeneity from Escherichia coli cells containing the genetic locus for this enzyme obtained from P. savastanoi. The preparation contained a single polypeptide with a mass of 62,000 that cross-reacted immunologically with a homologous protein in P. savastanoi. The holoenzyme contained one FAD moiety/subunit with properties consistent with a catalytic function. The enzyme preparation catalyzed an L-tryptophan-dependent O2 uptake and yielded 3-indoleacetamide as a product. Enzyme activity fit simple Michaelis Menten kinetics with a Km for L-tryptophan of 50 microM. 3-Indoleacetamide and 3-indoleacetic acid were identified as regulatory effectors. The apparent Ki for 3-indoleacetamide was 7 microM; that for indoleacetic acid was 225 microM. At Km concentrations of tryptophan, enzyme activity was inhibited 50% by 25 microM 3-indoleacetamide. In contrast, 230 microM indoleacetic acid was required to effect a similar inhibition. Phenylalanine and tyrosine were ineffective as regulatory metabolites. These results indicate that IAA synthesis in P. savastanoi is regulated by limiting tryptophan and by feedback inhibition from indoleacetamide and indoleacetic acid.     

Recovery of Pseudomonas savastanoi pv. savastanoi from symptomless shoots of naturally infected olive trees.

Seasonal dynamics of Pseudomonas savastanoi pv. savastanoi (Psv) on stems and leaves from symptomless shoots of naturally infected olive trees was monitored in Spanish olive orchards. Data inferred from the comparison between washing of leaves and dilution-plating versus leaf printing of individual leaves suggested that Psv population sizes varied by over several orders of magnitude, among leaves sampled concurrently from the same shoot. We did not find significant differences between leaves and stems, in respect to the number of samples where Psv was isolated or detected by PCR, showing that Psv colonizes both leaves and stems. The frequencies of Psv isolation and average populations were highly variable among field plots. No correlation between Psv populations and those of non-Psv bacteria in any plant material or field plot was observed. However, where both Psv and yellow Pantoea agglomerans colonies were isolated a positive correlation was found. In a selected field plot, dynamics of Psv over three years showed significant differences between summer and the rest of seasons. The highest Psv population occurred in warm, rainy months, while low numbers were generally found in hot and dry months.     

Nucleotide sequence and expression of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology with Agrobacterium tumefaciens tmr and tzs loci.

The nucleotide sequence of a Pseudomonas trans-zeatin producing gene (ptz) from the pCK1 plasmid of Pseudomonas syringae pv. savastanoi strain 1006 has been determined. This gene confers upon E. coli the ability to synthesize and secrete several cytokinins including trans-zeatin, iso-pentenyladenine and their respective N9-ribosyl derivatives. Sequence analysis indicates an open reading frame encoding a protein of 234 amino acids with a molecular weight of 26,816. Significant sequence homology is found between ptz and both the tzs and tmr genes from Agrobacterium tumefaciens. The results suggest a close relationship between the cytokinin biosynthetic pathways in P. savastanoi and A. tumefaciens.     

Genotyping of Edwardsiella ictaluri isolates in Japan using amplified-fragment length polymorphism analysis

Aim:  The major objective of the present study was to clarify genetic relationship of isolates of Edwardsiella ictaluri in Japan, which was first found from ayu Plecoglossus altivelis in Japanese rivers in 2007.
Methods and Results:  Ten isolates of Edw. ictaluri in 2007–2008 from ayu and the 1 isolate from bagrid catfish Pelteobagrus nudiceps in Japan were subjected to amplified-fragment length polymorphism (AFLP) analysis. The strains isolated from catfish in United States (ATCC strains) or Indonesia were used as reference strains. The AFLP profiles were all the same among the isolates from Japan, while the polymorphic DNA bands were observed among the strains from United States or Indonesia. The isolates from Japan and Indonesia constituted a genogroup different from the ATCC strains on a dendrogram constructed from the AFLP profiles.
Conclusion:  No DNA polymorphisms were found among Japanese Edw. ictaluri isolates.
Significance and Impact of the Study:  A single clonality of the Edw. ictaluri isolates in Japan suggests the single source of the organism, and the infection in ayu is in the early stage of epidemics.     

Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.     

Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.