rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.     

Several pseudomonads, associated with the cultivated mushrooms Agaricus bisporus or Pleurotus sp., are hemolytic

Pseudomonas tolaasii, causing brown blotch disease on cultivated mushrooms, and yielding a white line precipitate towards P. “reactans”, has been shown to induce lysis of erythrocytes. Some Finnish strains isolated from diseased mushroom fruit bodies, although harboring the typical features of P. tolaasii, proved to be distinct, and have been allocated to a nov. sp. P. costantinii. We examined in these study whether all brown blotch causing agents were hemolytic. The induction of erythrocytes lysis seemed to be a rather common feature of mushroom associated-pseudomonads, especially for strains involved in the production of a white-line-in agar.     

Characteristics of stress response in a mushroom-pathogenic bacterium,Pseudomonas tolaasii, during the interaction withPleurotus ostreatus and carbon/nitrogen starvation in vitro

A brown blotch bacterium,Pseudomonas tolaasii strain PT814, expresses a high degree of cross-protection against generalized stress imposed by physical/chemical treatment, H₂O₂, UV, high temperature, ethanol and NaCl during the interaction withPleurotus ostreatus. Stress resistance was also noted in the bacterium in vitro under limited carbon and nitrogen sources. In addition, changes in cell morphology from a “metabolically active” rod to an “energy-saving” spherical shape were detected during starvation and the interaction. All the changes under stress were reversible. A homologue ofrpoS (σ ^S), a regulator that controls such physiological status during starvation in other bacteria, was identified inP. tolaasii strain PT814. Data suggest that the bacterium is able to withstand a complex stress environment for its survival through changes in its metabolic pattern.     

Actinomycins inhibit the production of the siderophore pyoverdines in the plant pathogen Pseudomonas cichorii SPC9018

ABSTRACT

Pyoverdines, a group of peptide siderophores produced by Pseudomonas species, function not only in iron acquisition, but also in their virulence in hosts. Thus, chemical inhibition of pyoverdine production may be an effective strategy to control Pseudomonas virulence. In the plant pathogen Pseudomonas cichorii SPC9018 (SPC9018), pyoverdine production is required for virulence on eggplant. We screened microbial culture extracts in a pyoverdine-production inhibition assay of SPC9018 and found Streptomyces sp. RM-32 as a candidate-producer. We isolated two active compounds from RM-32 cultures, and elucidated their structures to be actinomycins X₂ and D. Actinomycins X₂ and D inhibited pyoverdine production by SPC9018 with IC₅₀ values of 17.6 and 29.6 μM, respectively. Furthermore, pyoverdine production in other Pseudomonas bacteria, such as the mushroom pathogen P. tolaasii, was inhibited by the actinomycins. Therefore, these actinomycins may be useful as chemical tools to examine pyoverdine functions and as seed compounds for anti-Pseudomonas virulence agents.     

Isolation of a gram-positive bacterium effective in suppression of brown blotch disease of cultivated mushrooms,Pleurotus ostreatus andAgaricus bisporus, caused byPseudomonas tolaasii

A Gram-positive bacterium was isolated from a rottingPleurotus ostreatus fruiting body that markedly reduced the level of extracellular toxins (i.e., tolaasins) produced byPseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The isolated bacterium is saprophytic but not parasitic nor pathogenic toP. ostreatus. A low ratio, ca. 10⁻³ cells of the isolated bacterium for oneP. tolaasii cells, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease inP. ostreatus andAgaricus bisporus. The suppression of the disease development, however requires the initial cell density equivalent to ca. 10⁻¹ cells of the isolated bacterium for one cells of the pathogen. The effects is ascribed to the inactivation of tolaasin by the live, suppressive bacterial cells, and not to metabolites secreted from the organism into culture media. Examination by conventional bacteriological tests and with testing kits, i.e., MicroStation^TMSystem Release 3.5 (Biolog Inc., Hayward, CA), ATB Expression (bioMerieux Inc. Japan) and VITEK (bioMerieux Inc. Japan), failed to assign the organism to any defined bacterial genus. The suppressive bacterium may be useful in future for the development of biocontrol system and/or the construction of genetically modified edible fungi resistant to the disease caused byP. tolaasii.     

Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens

An anaerobic phthalate isomer-degrading strain (JT^T) that we previously isolated was characterized. In addition, a strictly anaerobic, mesophilic, syntrophic phthalate isomer-degrading bacterium, designated strain JI^T, was isolated and characterized in this study. Both were non-motile rods that formed spores. In both strains, the optimal growth was observed at temperatures around 37°C and neutral pH. In syntrophic co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, both strains could utilize two or three phthalate isomers for growth, and produce acetate and methane as end products. Strain JT^T was able to grow on isophthalate, terephthalate, and a number of low-molecular weight aromatic compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate, 2,5-dihydroxybenzoate, 3-phenylpropionate in co-culture with M. hungatei. It could also grow on crotonate, hydroquinone and 2,5-dihydroxybenzoate in pure culture. Strain JI^T utilized all of the three phthalate isomers as well as benzoate and 3-hydroxybenzoate for growth in co-culture with M. hungatei. No substrates were, however, found to support the axenic growth of strain JI^T. Neither strain JT^T nor strain JI^T could utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, Fe (III) or 4-hydroxybenzoate as electron acceptor. Phylogenetically, strains JT^T and JI^T were relatively close to the members of the genera Pelotomaculum and Cryptanaerobacter in ‘Desulfotomaculum lineage I’. Physiological and chemotaxonomic characteristics indicated that the two isolates should be classified into the genus Pelotomaculum, creating two novel species for them. Here, we propose Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov. for strain JT^T and strain JI^T, respectively. The type strains are strains JT^T (= DSM 16121^T = JCM 11824^T = NBRC 100523^T) and JI^T (= JCM 12282^T = BAA-1053^T) for P. terephthalicum and P. isophthalicum, respectively.Nucleotide sequence accession number: The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequences of strains JT^T and JI^T are AB091323 and AB232785, respectively     

The structure of a pyoverdine from Pseudomonas sp. CFML 96.188 and its relation to other pyoverdines with a cyclic C-terminus

From Pseudomonas sp. CFML 96.188 a pyoverdine was isolated and its primary structure was elucidated by spectroscopic methods and degradation reactions. This strain is of interest as it accepts the structurally different pyoverdines from several other Pseudomonas strains. They all have in common as a specific structural feature a C-terminal cyclic substructure, the importance of which for the recognition of a pyoverdine at the cell surface of a given strain will be discussed.     

Molecular genetic characterization of bacterial isolates causing brown blotch on cultivated mushrooms in Japan

The polymerase chain-reaction (PCR) was used to amplify 16S ribosomal DNA (16S rDNA) from bacteria, identified asPseudomonas tolaasii orP. fluorescens, causing brown blotch on cultivated mushrooms in Japan. PCR-amplified 16S rDNA was analyzed on the basis of nucleotide sequence and restriction fragment length polymorphisms (RFLP) to determine the specific identity of isolates. Banding patterns obtained through PCR using primers corresponding to repetitive extragenic palindromic sequences of enteric bacteria (REP-PCR) were used to determine the relatedness of conspecific isolates. AllP. tolaasii isolates and a mushroom pathogen identified asP. fluorescens had identical RFLP patterns and partial 16S sequences, and are considered conspecific. An isolate ofP. fluorescens from creamery wastes (IFO 3507) differed slightly from isolates ofP. tolaasii in both 16S sequence (0.8%) and RFLP patterns (d=0.08), and had almost entirely different REP-PCR bands (d=0.88–1.0). Phylogenetic analyses based on 16S sequences indicated thatP. tolaasii andP. fluorescens are close members ofPseudomonas sensu stricto. REP-PCR shows promise in characterizing isolates pathogenic on different mushroom crops. Two isolates ofP. tolaasii pathogenic onPleurotus ostreatus had identical banding patterns, but three isolates fromLentinula edodes showed the greatest diversity. Contribution No. 312 of the Tottori Mycological Institute, Totori, Japan.     

Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga

Eight bacterial strains identified as P1, P2, Y1, Y2, W1, W2, G, and R were isolated from a long-term laboratory culture of the green alga Chlorella ellipsoidea. Although it is unknown how these bacterial strains have been maintained with the C. ellipsoidea culture, all appeared to promote the growth of C. ellipsoidea. Co-inoculation of each bacterial strain with C. ellipsoidea resulted in 0.5–3 times greater algal growth than that of C. ellipsoidea alone. The most effective bacterium (i.e., strain P1) was selected and further characterized. Biochemical analysis and transmission electron microscopy revealed that strain P1 is closely related to the genus Brevundimonas. Sequence analysis of the 16S rRNA of strain P1 showed 99.9 and 99.4% nucleotide sequence identity to that of B. nasdae and B. vesicularis, respectively. In addition to the growth promotion of C. ellipsoidea by strain P1, the growth of strain P1 was also significantly enhanced by co-culturing with C. ellipsoidea, indicating a symbiotic relationship between the bacterium and alga. Scanning electron microscopy showed the direct adhesion of strain P1 cells to the surface of C. ellipsoidea cells, as well as the development of abundant crinkles on the surface of co-cultured C. ellipsoidea cells. Handling editor: J. Padisak     

Sexual fertility of fortyFusarium strains from the EuropeanFusarium sambucinum project

Fungus strains designated asFusarium sambucinum, F. torulosum, orFusarium sp. nov. were crossed withMAT1-1 andMAT1–2 tester strains ofGibberella pulicaris. Of the 40 field strains that were crossed with the tester strains, 13 strains produced fertile crosses and 27 strains did not produce fertile crosses. One strain designated asF. torulosum was fertile with a tester strain ofG. pulicaris, suggesting that this is an intraspecies cross and that the strain isG. pulicaris, and, consequently,F. sambucinum rather thanF. torulosum. The lack of fertile crosses between tester strains and 27 of the 40 field strains suggests that these strains are notG. pulicaris. Although the ability to form a fully fertile cross with a tester strain can determine the species of a fertile strain, it is more problematic to exclude a strain only because it is infertile.     

大蒜浸出液对平菇细菌性褐斑病病原菌托拉斯假单胞杆菌的抑菌作用

【背景】由托拉斯假单胞杆菌(Pseudomonas tolaasii)引起的平菇细菌性褐斑病在国内外大面积发生,导致产量降低,并有潜在的安全风险,寻找安全有效的抑菌剂对产业发展具有重要意义。【目的】通过5种不同溶剂提取得到大蒜浸出液,测定其对平菇细菌性褐斑病病原菌托拉斯假单胞杆菌的抑制作用,同时检测其对平菇菌丝生长的作用。【方法】利用抑菌圈法测定5种不同的大蒜浸出液对托拉斯假单胞杆菌的抑菌作用,利用平板扩散法筛选能促进平菇菌丝生长的药剂及适宜的浓度。【结果】5种大蒜浸出液原液对托拉斯假单胞杆菌均有显著的抑菌活性,其中大蒜山杏壳木醋液浸出原液抑菌效果最强。不同浓度的大蒜浸出液抑菌作用比较发现,浓度为10%的大蒜山杏壳木醋液浸出液具有较好的抑菌效果,其抑菌效果与0.33 mg/mL的链霉素相当,并对平菇菌丝生长有显著的促进作用,菌丝生长速度显著大于对照,并且菌丝浓密,边缘整齐。【结论】本研究为大蒜与山杏壳木醋液复配药剂防治平菇细菌性褐斑病奠定了实验基础。     

Reclassification of Pichia membranifaciens sensu Kurtzman

Forty strains which were stocked as Pichia membranifaciens sensu Kurtzman and 9 strains stocked as Candida valida, anamorphs of P. membranifaciens, in the Institute for Fermentation, Osaka (IFO) were reclassified based on the data of base composition of nuclear DNA, DNA/DNA hybridization, coenzyme Q system, electrophoretic karyotype, and base sequence of 18S rDNA. P. membranifaciens complex was assigned into 3 groups: (I) P. membranifaciens group, including 25 strains with high DNA homologies to the type strain of P. membranifaciens (72–98%); (II) P. manshurica group, including 18 strains with high DNA homology of 79–95% to the type strain of P. manshurica; and a group including the remaining 6 strains, which had low DNA homology to the above two species. GC content was 42.9–45.3 mol% for the P. membranifaciens group, 40.0–42.0 mol% for the P. manshurica group, and 27.2–44.7 mol% for the remaining group. All three groups had ubiquinone Co Q-7. Of the 6 anomalous strains, IFO 0162 was identified as Pichia deserticola, and IFO 0839 and IFO 0840 were identified as Issatchenkia occidentalis; but IFO 0842, IFO 0843, and IFO 1788 were thought to be unknown strains.     

Characterization of the pyoverdines ofAzotobacter vinelandii ATCC 12 837 with regard to heterogeneity

Summary Azotobacter vinelandii strain ATCC 12 837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows thatA. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to theAzotobacter-type family of pyoverdines.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

Application of Siderotyping for Characterization of Pseudomonas tolaasii and “Pseudomonas reactans” Isolates Associated with Brown Blotch Disease of Cultivated Mushrooms

Pyoverdine isoelectric focusing analysis and pyoverdine-mediated iron uptake were used as siderotyping methods to analyze a collection of 57 northern and central European isolates of P. tolaasii and “P. reactans.” The bacteria, isolated from cultivated Agaricus bisporus or Pleurotus ostreatus mushroom sporophores presenting brown blotch disease symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401–407, 1979) and their pathogenicity towards A. bisporus and were grouped into siderovars according to the type of pyoverdine they produced. Seventeen P. tolaasii isolates were recognized, which divided into two siderovars, with the first one containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 “P. reactans” isolates divided into eight siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake studies demonstrated an identity for some “P. reactans” isolates, with reference strains belonging to the P. fluorescens biovars II, III, or V. Thus, the easy and rapid methods of siderotyping proved to be reliable by supporting and strengthening previous taxonomical data. Moreover, two potentially novel pyoverdines characterizing one P. tolaasii siderovar and one “P. reactans” siderovar were found.     

Multi-gene analysis reveals previously unrecognized phylogenetic diversity in Aliivibrio

The “Vibrio fischeri species group” recently was reclassified as a new genus, Aliivibrio, comprising four species, Aliivibrio fischeri, Aliivibrio logei, Aliivibrio salmonicida, and Aliivibrio wodanis. Only limited phylogenetic analysis of strains within Aliivibrio has been carried out, however, and taxonomic ambiguity is evident within this group, especially for phenotypically unusual strains and certain strains isolated from bioluminescent symbioses. Therefore, to examine in depth the evolutionary relationships within Aliivibrio and redefine the host affiliations of symbiotic species, we examined several previously identified and newly isolated strains using phylogenetic analysis based on multiple independent loci, gapA, gyrB, pyrH, recA, rpoA, the luxABE region, and the 16S rRNA gene. The analysis resolved Aliivibrio as distinct from Vibrio, Photobacterium, and other genera of Vibrionaceae, and resolved A. fischeri, A. salmonicida, A. logei, and A. wodanis as distinct, well-supported clades. However, it also revealed that several previously reported strains are incorrectly identified and that substantial unrecognized diversity exists in this genus. Specifically, strain ATCC 33715 (Y-1) and several other strains having a yellow-shifted luminescence were not members of A. fischeri. Furthermore, no strain previously identified as A. logei grouped with the type strain (ATCC 29985^T), and no bona-fide strain of A. logei was identified as a bioluminescent symbiont. Several additional strains identified previously as A. logei group instead with the type strain of A. wodanis (ATCC BAA-104^T), or are members of a new clade. Two strongly supported clades were evident within A. fischeri, a phylogenetic structure that might reflect differences in the host species or differences in the ecological incidence of strains. The results of this study highlight the importance of basing taxonomic conclusions on examination of type strains.     

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity – comparable to that obtained with the common preservative calcium propionate (0.3% w/v) – of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.     

DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

绿针假单胞菌的研究进展及农业应用潜力

绿针假单胞菌(Pseudomonas chlororaphis)是目前研究较多的生防菌种之一.19世纪初被Miguela首次分离,将其鉴定为假单胞菌(Pseudomonas),并将机会性病原菌绿脓杆菌作为其模式菌株,而后Peix于2007年重新将其分类为绿针假单胞菌(P.chlororaphis).目前该菌种已报道有4...