New species of pathogenic Pseudomonas isolated from citrus in Tunisia: Proposal of Pseudomonas kairouanensis sp. nov. and Pseudomonas nabeulensis sp. nov.

A collection of Pseudomonas strains was isolated in different regions of Tunisia in the period 2016–2017 from the fruits and leaves of Citrus sinensis cv. ‘Valencia Late’ and Citrus limon cv. ‘Eureka’ plants with symptoms of blast and black pit disease. A phylogenetic analysis of the housekeeping gene rpoD was used for strain identification at the species level. The results demonstrated the affiliation of these strains with the genus Pseudomonas and revealed the presence of 11 strains representing two putative new species in two monophyletic branches. These strains were analyzed morphologically and genotypically by multilocus sequence analyses of the rpoD, gyrB and 16S rRNA (rrs) gene sequences, and their phenotypic characteristics by API 20NE and Biolog GEN III. Plant pathogenic properties were confirmed on fruits and detached leaves of C. limon cv. ‘Eureka’. Fatty acids and WC MALDI-TOF MS major protein profiles were determined. The genomes of both representatives were sequenced. The average nucleotide index and genome-to-genome distance from KC12^T and E10B^T are below the cut-off established for a described species. These results support the conclusion that the strains KC12^T, KC17, KC20, KC22, KC24A, KC25 and KC26 represent a novel species of Pseudomonas, for which the name of Pseudomonas kairouanensis is proposed. The type strain is KC12^T (=CECT9766 and CFBP 8662). The strains E10B^T, E10AB, E10CB1 and Iy3BA represent another novel species of Pseudomonas for which the name of Pseudomonas nabeulensis is proposed; the type strain is E10B^T (=CECT9765 and CFBP 8661).     

Pseudomonas gallaeciensis sp. nov., isolated from crude-oil-contaminated intertidal sand samples after the Prestige oil spill

Strains V113^T, V92 and V120 have been isolated from sand samples taken at the Atlantic intertidal shore in Galicia, Spain, after the Prestige oil spill. A preliminary analysis of the 16S rRNA and the partial rpoD gene sequences indicated that these strains belonged to the Pseudomonas genus, but they were distinct from any known Pseudomonas species. They were extensively characterized by a polyphasic taxonomic approach and phylogenetic data that confirmed that these strains belonged to the Pseudomonas pertucinogena group. Phylogenetic analysis of 16S rRNA, gyrB and rpoD gene sequences showed that the three strains were 99% similar and were closely related to members of the P. pertucinogena group, with less than 94% similarity to strains of established species; Pseudomonas pachastrellae was the closest relative. The Average Nucleotide Index based on blast values was 89.0% between V113^T and the P. pachastrellae type strain, below the accepted species level (95%). The predominant cellular fatty acid contents and whole cell protein profiles determined by MALDI-TOF mass spectrometry also differentiated the studied strains from known Pseudomonas species. We therefore conclude that strains V113^T, V92 and V120 represent a novel species of Pseudomonas, for which the name Pseudomonas gallaeciensis is proposed; the type strain is V113^T (= CCUG 67583^T = LMG 29038^T).     

Pseudomonas aestusnigri sp. nov., isolated from crude oil-contaminated intertidal sand samples after the Prestige oil spill

Strains VGXO14^T and Vi1 were isolated from the Atlantic intertidal shore from Galicia, Spain, after the Prestige oil spill. Both strains were Gram-negative rod-shaped bacteria with one polar inserted flagellum, strictly aerobic, and able to grow at 18–37 °C, pH 6–10 and 2–10% NaCl. A preliminary analysis of the 16S rRNA and the partial rpoD gene sequences indicated that these strains belonged to the Pseudomonas genus but were distinct from any known Pseudomonas species. A polyphasic taxonomic approach including phylogenetic, chemotaxonomic, phenotypic and genotypic data confirmed that the strains belonged to the Pseudomonas pertucinogena group. In a multilocus sequence analysis, the similarity of VGXO14^T and Vi1 to the closest type strain of the group, Pseudomonas pachastrellae, was 90.4%, which was lower than the threshold of 97% established to discriminate species in the Pseudomonas genus. The DNA–DNA hybridisation similarity between strains VGXO14^T and Vi1 was 79.6%, but below 70% with the type strains in the P. pertucinogena group. Therefore, the strains should be classified within the genus Pseudomonas as a novel species, for which the name Pseudomonas aestusnigri is proposed. The type strain is VGXO14^T (=CCUG 64165^T = CECT 8317^T).     

Concordance between whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and multilocus sequence analysis approaches in species discrimination within the genus Pseudomonas

Multilocus sequence analysis (MLSA) is one of the most accepted methods for the phylogenetic assignation of Pseudomonas strains to their corresponding species. Furthermore, updated databases are essential for correct bacterial identification and the number of Pseudomonas species is increasing continuously. Currently, 127 species are validly described in Euzéby's List of Species with Standing in Nomenclature, and 29 novel species have been described since the publication of the last comprehensive MLSA phylogenetic study based on the sequences of the 16S rDNA, gyrB, rpoB and rpoD genes. Therefore, an update of the sequence database is presented, together with the analysis of the phylogeny of the genus Pseudomonas. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight (WC-MALDI-TOF) mass spectrometry (MS) analysis has been applied very recently to the identification of bacteria and is considered to be a fast and reliable method. A total of 133 type strains of the recognized species and subspecies in the genus Pseudomonas, together with other representative strains, were analyzed using this new technique, and the congruence between the WC-MALDI-TOF MS and MLSA techniques was assessed for the discrimination and correct species identification of the strains. The utility of both methods in the identification of environmental and clinical strains is discussed.     

Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin

Fluorescent Pseudomonas strains producing the antimicrobial secondary metabolite 2,4-diacetylphloroglucinol (Phl) play a prominent role in the biocontrol of plant diseases. A subset of Phl-producing fluorescent Pseudomonas strains, which can additionally synthesize the antimicrobial compound pyoluteorin (Plt), appears to cluster separately from other fluorescent Pseudomonas spp. based on 16S rRNA gene analysis and shares at most 98.4% 16S rRNA gene sequence identity with any other Pseudomonas species. In this study, a polyphasic approach based on molecular and phenotypic methods was used to clarify the taxonomy of representative Phl⁺ Plt⁺ strains isolated from tobacco, cotton or wheat on different continents. Phl⁺ Plt⁺ strains clustered separately from their nearest phylogenetic neighbors (i.e. species from the ‘P. syringae’, ‘P. fluorescens’ and ‘P. chlororaphis’ species complexes) based on rpoB, rpoD or gyrB phylogenies. DNA-DNA hybridization experiments clarified that Phl⁺ Plt⁺ strains formed a tight genomospecies that was distinct from P. syringae, P. fluorescens, or P. chlororaphis type strains. Within Phl⁺ strains, the Phl⁺ Plt⁺ strains were differentiated from other biocontrol fluorescent Pseudomonas strains that produced Phl but not Plt, based on phenotypic and molecular data. Discriminative phenotypic characters were also identified by numerical taxonomic analysis and siderotyping. Altogether, this polyphasic approach supported the conclusion that Phl⁺ Plt⁺ fluorescent Pseudomonas strains belonged to a novel species for which the name Pseudomonas protegens is proposed, with CHA0^T (=CFBP 6595^T, =DSM 19095^T) as the type strain.     

Dehydrogenase activity of pseudomonas species

Single-strain cultures of Pseudomonas fragi, P. fluorescens, P. putrefaciens, and strains of two marine species, Pseudomonas type I and Pseudomonas type II, were found to be capable of reducing added acetaldehyde, proprionaldehyde, and butyraldehyde to the corresponding alcohols at 21 C. All species studied reduced propionaldehyde at 6 C. P. fragi, Pseudomonas type I, and Pseudomonas type II reduced butanone at 6 and 21 C. P. fragi and Pseudomonas type II reduced acetone at 21 C. Dehydrogenase activity was found in some cultures in which growth was not evident. Under aerobic conditions, a strain of P. fragi reduced added propionaldehyde to n-propanol quantitatively within 36 hr at 21 C.     

New Pseudomonas spp. Are Pathogenic to Citrus

Five putative novel Pseudomonas species shown to be pathogenic to citrus have been characterized in a screening of 126 Pseudomonas strains isolated from diseased citrus leaves and stems in northern Iran. The 126 strains were studied using a polyphasic approach that included phenotypic characterizations and phylogenetic multilocus sequence analysis. The pathogenicity of these strains against 3 cultivars of citrus is demonstrated in greenhouse and field studies. The strains were initially grouped phenotypically and by their partial rpoD gene sequences into 11 coherent groups in the Pseudomonas fluorescens phylogenetic lineage. Fifty-three strains that are representatives of the 11 groups were selected and analyzed by partial sequencing of their 16S rRNA and gyrB genes. The individual and concatenated partial sequences of the three genes were used to construct the corresponding phylogenetic trees. The majority of the strains were identified at the species level: P. lurida (5 strains), P. monteilii (2 strains), P. moraviensis (1 strain), P. orientalis (16 strains), P. simiae (7 strains), P. syringae (46 strains, distributed phylogenetically in at least 5 pathovars), and P. viridiflava (2 strains). This is the first report of pathogenicity on citrus of P. orientalis, P. simiae, P. lurida, P. moraviensis and P. monteilii strains. The remaining 47 strains that could not be identified at the species level are considered representatives of at least 5 putative novel Pseudomonas species that are not yet described.     

Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology

Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.     

Sensitivity to vinyl phenol derivatives produced by phenolic acid decarboxylase activity in Escherichia coli and several food-borne Gram-negative species

Ferulic, p-coumaric, and caffeic acids are phenolic acids present in soil, food, and gut, which have antimicrobial effects. Some Gram (+) bacteria metabolize these phenolic acids into vinyl derivatives due to phenolic acid decarboxylase activity (PAD) involved in the phenolic acid stress response (PASR). In this study, the antimicrobial activity of phenolic acids and their vinyl derivatives was tested on a panel of desirable and undesirable food-borne bacteria, especially Gram (?) species of Salmonella, Enterobacter, Klebsiella, and Pseudomonas, most of them without PAD activity. Native and engineered Escherichia coli strains either expressing or not PAD activity were included. Gram (?) bacteria of the panel were not significantly inhibited by phenolic acids at 3 mM, but were dramatically inhibited by the corresponding vinyl derivatives. On the contrary, Gram (+) bacteria displaying the PASR face the toxicity of phenolic acids by PAD activity and are not inhibited by vinyl phenols. In E. coli, the genes aaeB and marA, encoding efflux pumps for antimicrobial compounds, are upregulated by the addition of p-coumaric acid, but not by its derivative 4-vinyl phenol (p-hydroxystyrene). These results suggest that phenolic acids and their vinyl phenol derivatives produced by PAD (+) species could have a significant impact on undesirable or pathogenic food-borne Gram (?) bacteria in complex microbial ecosystems.     

Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.     

Pseudomonas versuta sp. nov., isolated from Antarctic soil

In this study we analysed three bacterial strains coded L10.10^T, A4R1.5 and A4R1.12, isolated in the course of a study of quorum-quenching bacteria occurring in Antarctic soil. The 16S rRNA gene sequence was identical in the three strains and showed 99.7% pairwise similarity with respect to the closest related species Pseudomonas weihenstephanensis WS4993^T. Therefore, the three strains were classified within the genus Pseudomonas. Analysis of housekeeping genes (rpoB, rpoD and gyrB) sequences showed similarities of 84-95% with respect to the closest related species of Pseudomonas, confirming its phylogenetic affiliation. The ANI values were less than 86% to the closest related species type strains. The respiratory quinone is Q9. The major fatty acids are C16:0, C16:1 ω7c/ C16:1 ω6c in summed feature 3 and C18:1 ω7c / C18:1 ω6c in summed feature 8. The strains are oxidase- and catalase-positive. Growth occurs at 4–30 °C, and at pH 4.0–10. The DNA G+C content is 58.2–58.3 mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains L10.10^T, A4R1.5 and A4R1.12 into a novel species of Pseudomonas, for which the name P. versuta sp. nov. is proposed. The type strain is L10.10^T (LMG 29628^T, DSM 101070^T).     

rpoD Gene Pyrosequencing for the Assessment of Pseudomonas Diversity in a Water Sample from the Woluwe River

A water sample from a noncontaminated site at the source of the Woluwe River (Belgium) was analyzed by culture-dependent and -independent methods. Pseudomonas isolates were identified by sequencing and analysis of the rpoD gene. Culture-independent methods consisted of cloning and pyrosequencing of a Pseudomonas rpoD amplicon from total DNA extracted from the same sample and amplified with selective rpoD gene primers. Among a total of 14,540 reads, 6,228 corresponded to Pseudomonas rpoD gene sequences by a BLAST analysis in the NCBI database. The selection criteria for the reads were sequences longer than 400 bp, an average Q₄₀ value greater than 25, and >85% identity with a Pseudomonas species. Of the 6,228 Pseudomonas rpoD sequences, 5,345 sequences met the established criteria for selection. Sequences were clustered by phylogenetic analysis and by use of the QIIME software package. Representative sequences of each cluster were assigned by BLAST analysis to a known Pseudomonas species when the identity with the type strain was greater than or equal to 96%. Twenty-six species distributed among 12 phylogenetic groups or subgroups within the genus were detected by pyrosequencing. Pseudomonas stutzeri, P. moraviensis, and P. simiae were the only cultured species not detected by pyrosequencing. The predominant phylogenetic group within the Pseudomonas genus was the P. fluorescens group, as determined by culture-dependent and -independent analyses. In all analyses, a high number of putative novel phylospecies was found: 10 were identified in the cultured strains and 246 were detected by pyrosequencing, indicating that the diversity of Pseudomonas species has not been fully described.     

High-Performance Liquid Chromatography Analyses of Pyoverdin Siderophores Differentiate among Phytopathogenic Fluorescent Pseudomonas Species

The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P. syringae. Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P. syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two β-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.     

Pseudomonas caspiana sp. nov., a citrus pathogen in the Pseudomonas syringae phylogenetic group

In a screening by multilocus sequence analysis of Pseudomonas strains isolated from diverse origins, 4 phylogenetically closely related strains (FBF58, FBF102^T, FBF103, and FBF122) formed a well-defined cluster in the Pseudomonas syringae phylogenetic group. The strains were isolated from citrus orchards in northern Iran with disease symptoms in the leaves and stems and its pathogenicity against citrus plants was demonstrated. The whole genome of the type strain of the proposed new species (FBF102^T = CECT 9164^T = CCUG 69273^T) was sequenced and characterized. Comparative genomics with the 14 known Pseudomonas species type strains of the P. syringae phylogenetic group demonstrated that this strain belonged to a new genomic species, different from the species described thus far. Genome analysis detected genes predicted to be involved in pathogenesis, such as an atypical type 3 secretion system and two type 6 secretion systems, together with effectors and virulence factors. A polyphasic taxonomic characterization demonstrated that the 4 plant pathogenic strains represented a new species, for which the name Pseudomonas caspiana sp. nov. is proposed.     

Immunological cross reactions among strains of Hydrogenomonas,Pseudomonas and Alcaligenes

Immunodiffusion tests were used to determine the serological interrelationships of extracts of Hydrogenomonas eutropha and H. facilis with antisera to 19 strains of Pseudomonas, 2 Alcaligenes species and the 2 homologous antisera. The existence of 3 antigens common to H. facilis and H. eutropha was demonstrated. The 3 serologically identical antigens were also shared among some pseudomonads and Alcaligenes species. We conclude that strains within the genera Hydrogenomonas, Pseudomonas and Alcaligenes represent a continuous series of serologically interrelated organisms.     

Application of the profiles of amino acid utilization as the sole carbon and nitrogen sources for pseudomonad taxonomy

Profiles of utilization of 20 amino acids were determined for 218 strains of 10 Pseudomonas species (P. aeruginosa, P. putida, P. fluorescens, P. stutzeri, P. alcaligenes, P. pseudoalcaligenes, P. luteola, P. oryzihabitans, P. mendocina, and P. chlororaphis), including the type strains of these species. Amino acid utilization was determined on minimal salt agar with an amino acid as the sole source of nitrogen and carbon. All the investigated pseudomonad species had species-specific profiles of amino acid utilization. For the type strains of all species, Jaccard’s coefficients of community were different (Sj = 0.31–0.82), in accordance with the interspecies differences. The similarity between the intraspecies variants of the profiles and that of the type strain was high; for 98% of P. aeruginosa strains, Sj = 0.85−1.0; for 100% of P. putida, P. stutzeri, and P. alcaligenes strains, Sj was 0.87–1.0, 0.90, and 0.86–1.0, respectively. Only for P. fluorescens and P. pseudoalcaligenes were low Sj of the intraspecies profiles revealed, in accordance with the known phenotypic heterogeneity of these species. These results agree with the known pseudomonad classification, and the method is therefore valid for identification of known species and for determination of the new members of the genus Pseudomonas.     

Genetic and functional diversity among root-associated psychrotrophic Pseudomonad’s isolated from the Himalayan plants

Out of 534 psychrotrophic bacteria, 12 bacteria were selected on the basis of plant growth promoting activities at 4 °C and identified as Pseudomonas genus. These strains showed high level of genetic polymorphisms based on RAPD and rep-PCR fingerprinting. This genetic variability revealed that isolates belonging to same species were as high as the variability among different species. Further inoculation of these Pseudomonas strains significantly improves root/shoot biomass and nutrients uptake of lentil plant as compared to non-bacterized control after 40 days of seed showing. Agglomerative hierarchical clustering analysis of pot assay results revealed that genetically diverse strains showing the same prototype in functional parameter and representing diverse blueprint of plant growth promoting attributes. Results of present findings explain the huge beneficial microbial resources from root zone of hilly crops of Himalayan region that could be effectively exploited as bio-inoculums for cold climatic condition.     

Roles of Microorganisms in the Deterioration of Rockfish

Seven species of microorganisms indigenous to fish were inoculated into sterile fish muscle, obtained from Sebastodes alutus and Sebastalobus alascanus, and the production of total volatile acids (TVA) and total volatile bases (TVB) was determined during storage at 1 C. No production of TVA and TVB was noted in sterile fish, fish inoculated with Pseudomonas type III species (PIII-985), or fish inoculated with Flavobacterium (F-1070). The inactivity of PIII-985 was due to its low maximal growth level, whereas the inactivity of F-1070 was due to its slow growth rate. Pseudomonas type I species (PI-950), Pseudomonas type II species (PII-952), and two Achromobacter (Acinetobacter) species (A-981 and A-70) grew and actively produced TVA and TVB. TVB levels increased steadily even after the microbial number reached a maximum, whereas TVA levels declined toward the end of the storage period. It appeared that a minimal microbial growth level of 10⁸ cells/g was needed before detectable levels of TVA or TVB could be produced. Regardless of genera, microorganisms which grew well in fish were able to produce TVA and TVB.     

Polyphosphate Accumulation by Pseudomonas putida CA-3 and Other Medium-Chain-Length Polyhydroxyalkanoate-Accumulating Bacteria under Aerobic Growth Conditions

Pseudomonas putida CA-3 accumulates polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) concurrently under nitrogen limitation. Five other mclPHA-accumulating Pseudomonas strains are capable of simultaneous polyP and mclPHA biosynthesis. It appears that polyP is not the rate-limiting step for mclPHA accumulation in these Pseudomonas strains.