Complete genome sequence of Japanese erwinia strain ejp617, a bacterial shoot blight pathogen of pear.

The Japanese Erwinia strain Ejp617 is a plant pathogen that causes bacterial shoot blight of pear in Japan. Here, we report the complete genome sequence of strain Ejp617 isolated from Nashi pears in Japan to provide further valuable insight among related Erwinia species.

Mutations in γ-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence.

Pseudomonas syringae pv. tomato DC3000 is a bacterial pathogen of Arabidopsis and tomato that grows in the apoplast. The non-protein amino acid γ-amino butyric acid (GABA) is produced by Arabidopsis and tomato and is the most abundant amino acid in the apoplastic fluid of tomato. The DC3000 genome harbors three genes annotated as gabT GABA transaminases. A DC3000 mutant lacking all three gabT genes was constructed and found to be unable to utilize GABA as a sole carbon and nitrogen source. In complete minimal media supplemented with GABA, the mutant grew less well than wild-type DC3000 and showed strongly reduced expression of hrpL and avrPto, which encode an alternative sigma factor and effector, respectively, associated with the type III secretion system. The growth of the gabT triple mutant was weakly reduced in Arabidopsis ecotype Landberg erecta (Ler) and strongly reduced in the Ler pop2-1 GABA transaminase-deficient mutant that accumulates higher levels of GABA. Much of the ability to grow on GABA-amended minimal media or in Arabidopsis pop2-1 leaves could be restored to the gabT triple mutant by expression in trans of just gabT2. The ability of DC3000 to elicit the hypersensitive response (HR) in tobacco leaves is dependent upon deployment of the type III secretion system, and the gabT triple mutant was less able than wild-type DC3000 to elicit this HR when bacteria were infiltrated along with GABA at levels of 1 mm or more. GABA may have multiple effects on P. syringae-plant interactions, with elevated levels increasing disease resistance.

Sensitive and Specific Detection of Xanthomonas oryzae pv. oryzae by Real-Time Bio-PCR Using Pathovar-Specific Primers Based on an rhs Family Gene.

The present study describes bio-polymerase chain reaction (PCR) assays to detect bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae in rice. Successful control of X. oryzae. pv. oryzae requires a specific and reliable diagnostic tool. However, other X. oryzae pathovars are detected by currently available molecular and serological methods. In this study, SYBR Green real-time and conventional PCR primer sets were designed based on an rhs family gene of X. oryzae pv. oryzae KACC10331 because these genes are structurally diverse. The specificity of the primers was evaluated using purified DNA from 11 isolates of two X. oryzae pathovars, 21 other Xanthomonas species, and 4 other reference phytopathogenic bacteria and fungi. The assay was also able to detect at least two genome equivalents of cloned amplified target DNA using purified DNA. Thus, the SYBR Green real-time PCR-based method can be used for the rapid and specific detection of X. oryzae pv. oryzae and will potentially simplify and facilitate diagnosis and monitoring of this pathogen and guide plant disease management.

Sensitive and specific detection of Xanthomonas campestris pv. vesicatoria by PCR using pathovar-specific primers based on rhs family gene sequences.

The present study describes PCR assay to detect bacterial spot caused by Xanthomonas campestris pv. vesicatoria in pepper and tomato. One set of PCR primer was developed to amplify gene required for an rhs family gene homologous to rhsA, cell envelope biogenesis, outer membrane. Only a PCR product of a 517bp was produced in PCR reaction with the Xanthomonas campestris pv. vesicatoria (XCVF/XCVR) primer set. A specific, and highly sensitive and rapid PCR assay for the detection of X. campestris pv. vesicatoria was achieved. The protocol can be used as a reliable diagnostic tool for specific detection of X. campestris pv. vesicatoria in pepper or tomato.

Genetic organization of the hrp genes cluster in Erwinia pyrifoliae and characterization of HR active domains in HrpNEp protein by mutational analysis.

The disease-specific (dsp) region and the hypersensitive response and pathogenicity (hrp) genes, including the hrpW, hrpNEp, and hrpC operons have previously been sequenced in Erwinia pyrifoliae WT3 [Shrestha et al. (2005a)]. In this study, the remaining hrp genes, including the hrpC, hrpA, hrpS, hrpXY, hrpL and hrpJ operons, were determined. The hrp genes cluster (ca. 38 kb) was comprised of eight transcriptional units and contained nine hrc (hrp conserved) genes. The genetic organization of the hrp/hrc genes and their orientation for the transcriptions were also similar to and collinear with those of E. amylovora, showing > or = 80% homologies. However, ORFU1 and ORFU2 of unknown functions, present between the hrpA and hrpS operons of E. amylovora, were absent in E. pyrifoliae. To determine the HR active domains, several proteins were prepared from truncated fragments of the N-terminal and the C-terminal regions of HrpN(Ep) protein of E. pyrifoliae. The proteins prepared from the N-terminal region elicited HR, but not from those of the C-terminal region indicating that HR active domains are located in only N-terminal region of the HrpN(Ep) protein. Two synthetic oligopeptides produced HR on tobacco confirming presence of two HR active domains in the HrpN(Ep). The HR positive N-terminal fragment (HN delta C187) was further narrowed down by deleting C-terminal amino acids and internal amino acids to investigate whether amino acid insertion region have role in faster and stronger HR activity in HrpN(Ep) than HrpN(Ea). The HrpN(Ep) mutant proteins HN delta C187 (D1AIR), HN delta C187 (D2AIR) and HN delta C187 (DM41) retained similar HR activation to that of wild-type HrpN(Ep). However, the HrpN(Ep) mutant protein HN delta C187 (D3AIR) lacking third amino acid insertion region (102 to 113 aa) reduced HR when compared to that of wild-type HrpN(Ep). Reduction in HR elicitation could not be observed when single amino acids at different positions were substituted at third amino acids insertion region. But, substitution of amino acids at L103R, L106K and L110R showed reduction in HR activity on tobacco suggesting their importance in activation of HR faster in the HrpN(Ep) although it requires further detailed analysis.

Antiviral activity of the exopolysaccharide produced by Serratia sp. strain Gsm01 against Cucumber mosaic virus.

The potential of the exopolysaccharide (EPS) from a Serratia sp. strain Gsm01 as an antiviral agent against a yellow strain of Cucumber mosaic virus (CMV-Y) was evaluated in tobacco plants (Nicotiana tabacum cv. Xanthi-nc). The spray treatment of plants using an EPS preparation, 72 before CMV-Y inoculation, protected them against symptom appearance. Fifteen days after challenge inoculation with CMVY, 33.33% of plants showed mosaic symptoms in EPS-treated plants compared with 100% in the control plants. The EPS-treated plants, which showed mosaic symptoms, appeared three days later than the controls. The enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of the leaves of the protected plants revealed that the EPS treatment affected virus accumulation in those plants. Analysis of phenylalanine ammonia lyase, peroxidase, and phenols in protected plants revealed enhanced accumulation of these substances. The pathogenesis-related (PR) genes expression represented by PR-1b was increased in EPS-treated plants. This is the first report of a systemic induction of protection triggered by EPS produced by Serratia sp. against CMV-Y.

WITHDRAWN: Erratum to "Tomato plants overexpressing CaKR1 enhanced tolerance to salt and oxidative stress" [Biochem. Biophys. Res. Commun. 363 (2007) 983-988].

This article has been withdrawn at the request of the author. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

WITHDRAWN: Erratum to "Tomato plants overexpressing CaKR1 enhanced tolerance to salt and oxidative stress" [Biochem. Biophys. Res. Commun. 363 (2007) 983-988].

This article has been withdrawn at the request of the author. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Tomato plants overexpressing CaKR1 enhanced tolerance to salt and oxidative stress.

CaKR1 from pepper leaves encodes an ankyrin repeat domain zinc finger that is thought to be involved in transcriptional regulation in response to pathogens and abiotic stresses. Transgenic tomato plants expressing CaKR1 show enhanced resistance to Phytophthora infestans. In this study, we further characterized this CaKR1-overexpressing transgenic tomato line. Morphologically, the leaves of the transgenic plants were thicker than those of control plants. Overexpressed transgenic plants also produced lower levels of free oxygen radicals, such as superoxide (O2-) and hydrogen peroxide (H2O2), and showed enhanced resistance to salinity and oxidative stress. In particular, transgenic plants produced higher levels of transcripts encoding the pathogenesis-related (PR) proteins LePR1, LePR2, and LePR3, as well as oxidative stress response proteins, such as superoxide dismutase (LeSOD2) and ascorbate peroxidase (LeAPX2 and LeAPX3). These results suggest that CaKR1 is a key signaling molecule regulating plant antioxidant metabolism and defense responses.

Transgenic tobacco expressing the hrpN(EP) gene from Erwinia pyrifoliae triggers defense responses against botrytis cinerea.

HrpN(EP), from the gram-negative pathogen, Erwinia pyrifoliae, is a member of the harpin group of proteins, inducing pathogen resistance and hypersensitive cell death in plants. When the hrpN(EP) gene driven by the OsCc1 promoter was introduced into tobacco plants via Agrobacterium-mediated transformation, their resistance to the necrotrophic fungal pathogen, Botrytis cinerea, increased. Resistance to B. cinerea was correlated with enhanced induction of SA-dependent genes such as PR-1a, PR2, PR3 and Chia5, of JA-dependent genes such as PR-1b, and of genes related to ethylene production, such as NT-EFE26, NT-1A1C, DS321, NT-ACS1 and NT-ACS2. However the expression of NPR1, which is thought to be essential for multiple-resistance, did not increase. Since the pattern of expression of defense-related genes in hrpN(EP)-expressing tobacco differed from that in plants expressing hpaG(Xoo) from Xanthomonas oryzae pv. Oryzae, these results suggest that different harpins can affect the expression of different defense-related genes, as well as resistance to different plant pathogens.

Characterization of Streptomycetes Causing Potato Common Scab in Korea.

Six representative Korean strains of streptomycetes (S33, S27, S71, S63, S77, and S78) that were pathogenic to potato were characterized based on phenotypic properties, analysis of 16S rRNA genes, production of thaxtomin A, and presence of nec1 and ORFtnp gene homologs. Strains S33 and S27 had typical characteristics of Streptomyces scabies and S. turgidiscabies, respectively, producing thaxtomin A and hybridizing to genes of nec1 and ORFtnp. Strain S71 produced thaxtomin A and had phenotypic and phylogenetic properties similar to those of S. acidiscabies, except having a greater minimum growth pH (4.5), production of a melanoid pigment on tyrosine agar, and failure to hybridize with nec1 and ORFtnp gene probes. In contrast, strains S63, S77, and S78 were phenotypically different from described scab pathogens. Spore colors of strains S63 and S77 were yellow-white or pale orange, respectively, with rectiflexuous chains. Strain S78 had thin and compact spores unlike typical S. acidiscabies (ATCC 49003). Phylogenetic analysis of strains S63, S77, and S78 based on 16S rRNA gene sequences showed low homology to that of described scab pathogens (less than 97.3, 96.0, and 96.3%, respectively). Strain S78 produced thaxtomin A, but did not have homologous sequences to nec1 and ORFtnp genes. Production of thaxtomin A and gene homologs of nec1 and ORFtnp were not detected in strains S63 and S77. All three strains grow at low pH, with minimal growth at pH 3.5 (S77 and S78) or 4.5 (S63). Streptomyces strains S63, S77, and S78 are novel pathogenic streptomycetes adapted to acidic soil conditions in Korea.

Regulations of marker genes involved in biotic and abiotic stress by overexpression of the AtNDPK2 gene in rice.

AtNDPK2 is involved in transcriptional regulation in response to pathogen and abiotic stresses. AtNDPK2-expressing transgenic rice plants showed regulation of the marker genes for chilling and oxidative stresses. In the present study, we produced AtNDPK2-overexpressing transgenic rice lines using the co-transformation method. Morphologically, the transgenic plants, compared with the control plants, were growth retarded. We investigated how AtNDPK2 overexpression influences the response of rice plants to marker genes related to chilling and ROS stress. The accumulation of transcripts of pBC442 and pBC601, related to chilling stress, was induced in AtNDPK2-overexpressed rice plants. On further investigation, we found that OsAPX1-, OsAPX2-, and OsSodB-scavenging free-oxygen radicals, such as superoxide (O2-) and hydrogen peroxide (H(2)O(2)), could be induced in AtNDPK2-overexpressed rice plants. In particular, transcripts encoding pathogenesis-related (PR) proteins OsPR2 and OsPR4, as well as oxidative stress response proteins, were confirmed to change the gene expression in the transgenic rice plants. Together, these results suggest that AtNDPK2 plays a regulatory role in chilling and antioxidant signaling in plants.

Phylogenetic analysis of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium on the basis of 16S rRNA gene and internally transcribed spacer region sequences.

A total of 128 strains was isolated from more than 23 legume hosts in Korea. Phylogenetic relationships between these Korean isolates and reference strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were analysed using their 16S rRNA gene and internally transcribed spacer (ITS) region sequences. Among the Bradyrhizobium strains, dendrograms based on both the 16S rRNA gene and ITS region sequences produced two main groups. The ITS tree yielded at least two new clusters that were discernable from the seven previously delineated genospecies. Large discrepancies were revealed between phylogenetic dendrograms based on 16S rRNA gene and ITS region sequences for members of the genus Rhizobium, reflecting their taxonomic heterogeneity. The amalgamation of Rhizobium and former members of Agrobacterium was confirmed using the 16S rRNA tree. Phylogenetic analysis of ITS region sequences showed that the Rhizobium giardinii clade (group II) and the Rhizobium radiobacter/Rhizobium rubi clade (group III) could be tentatively recognized as groups that are separable from the core group (group I), which includes Rhizobium leguminosarum. Dendrograms based on the 16S rRNA gene and ITS region sequences of Mesorhizobium strains were highly conflicting due to the poor taxonomic resolution of the 16S rRNA gene sequences and the low confidence in the ITS dendrogram. Several Korean isolates within the genus Mesorhizobium are thought to represent novel taxa when considering their relatively low ITS region sequence similarities (<80 %) to the reference strains.

Pseudomonas koreensis sp. nov., Pseudomonas umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov., novel species from farm soils in Korea.

Among Pseudomonas strains isolated from Korean agricultural soils, four strains (Ps 9-14 group: Ps 1-2, Ps 1-10, Ps 5-5 and Ps 9-14T) from the Suwon, Goesan and Samchok regions, three strains (Ps 3-10 group: Ps 2-22, Ps 3-1 and Ps 3-10T) from Umsong Region and four strains (Pss 26 group: Pss 14, Pss 25, Pss 26T and Pss 27) from Jinju Region were identified as three independent groups on the basis of 16S rDNA sequence analysis. While, on the basis of 16S rDNA sequence analysis, Ps 9-14T and Ps 3-10T form a phyletic line with Pseudomonas jessenii CIP 105274T, 'Pseudomonas pavonaceae' IAM 1155 and Pseudomonas graminis DSM 11363T, Pss 26T is grouped with Pseudomonas citronellolis ATCC 13674T and Pseudomonas nitroreducens IAM 1439T. According to DNA-DNA hybridization studies, strain Ps 9-14T shows high DNA relatedness to strain Ps 3-10T (52%) and Pseudomonas migulae CIP 105470T (49%) and strain Ps 3-10T reveals high relatedness to strain Ps 9-14T (48%) and P. jessenii CIP 105274T (45%). Strain Pss 26T shows high relatedness to P. citronellolis LMG 18378T (54%), P. nitroreducens ATCC 33634T (48%) and Pseudomonas aeruginosa LMG 1242T (48%). On the basis of phenotypic and genotypic analyses, three novel species of the genus Pseudomonas are proposed: Pseudomonas koreensis sp. nov. (type strain Ps 9-14T =LMG 21318T =KACC 10848T) for the Ps 9-14 group, Pseudomonas umsongensis sp. nov. (type strain Ps 3-10T =LMG 21317T =KACC 10847T) for the Ps 3-10 group and Pseudomonas jinjuensis sp. nov. (type strain Pss 26T =LMG 21316T =KACC 10760T) for the Pss 26 group.

Streptomyces luridiscabiei sp. nov., Streptomyces puniciscabiei sp. nov. and Streptomyces niveiscabiei sp. nov., which cause potato common scab disease in Korea.

Three plant-pathogenic isolates of Streptomyces spp., isolated from potatoes with common scab disease lesions in Korea, are described as novel species. Morphological and physiological properties of these isolates were distinct from those of previously described Streptomyces species. Strain S63(T) has yellow-white, smooth, cylindrical spores that are borne in monoverticillus flexuous spore-chains. Strain S77(T) has purple-red, spiny spores that are borne in simple rectus flexuous spore-chains. Strain S78(T) has white, smooth, cylindrical spores that are borne in simple rectus flexuous spore-chains. These three isolates differed from known pathogenic strains by analysis of 16S rRNA gene sequences in a previous study. Furthermore, genetic uniqueness of our isolates was confirmed by sequencing of the 16S-23S internal transcribed spacer (ITS) region, which indicated that isolates S63(T) and S78(T) belong to the genus Streptomyces and have low homology to other Streptomyces species (less than 71.2 and 75.7 %, respectively). The 16S-23S ITS region of strain S77(T) was not amplified by these primer sets. DNA-DNA hybridization results for all three isolates show distant relationships to previously described Streptomyces species; therefore, on the basis of polyphasic evidence, the names Streptomyces luridiscabiei sp. nov. for strain S63(T) (=LMG 21390(T)=KACC 20252(T)), Streptomyces puniciscabiei sp. nov. for strain S77(T) (=LMG 21391(T)=KACC 20253(T)) and Streptomyces niveiscabiei sp. nov. for strain S78(T) (=LMG 21392(T)=KACC 20254(T)) are proposed.