TaqMan Real-Time PCR Assay for Specific Detection and Differentiation of Xanthomonas translucens pv. undulosa from Other Pathovars Targeting a Recombination Mediator Gene, recF.

Bacterial leaf streak and black chaff diseases of wheat caused by Xanthomonas translucens pv. undulosa is becoming a major constraint to growers and trade since it is seedborne. Molecular tools for specific detection/differentiation of pv. undulosa are lacking. We report the development of a TaqMan real-time PCR for specific detection/identification of pv. undulosa targeting the recombination mediator gene (recF). Analysis of the complete recF (1,117 bp) sequences identified the gene as a reliable phylogenetic marker for identification of pv. undulosa, differentiating it from the other pathovars; recF-based sequence homology values among the 11 pathovars correlated well with genome-based DNA-DNA hybridization values. The discriminatory power of recF to differentiate pv. undulosa from the other pathovars is due to nucleotide polymorphic positions. We used these nucleotide polymorphisms to develop a TaqMan PCR for specific detection of pv. undulosa. The specificity of the assay was validated using 67 bacterial and fungal/oomycete strains. The selected primers and the double-quenched FAM-labeled TaqMan probe were specific for the detection of 11 pv. undulosa/secalis strains. The 56 strains of other X. translucens pathovars (n = 39) and non-Xanthomonas spp. (n = 17) did not exhibit any detectable fluorescence. Also, greenhouse-inoculated and naturally infected wheat leaf samples showed positive reactions for the presence of pv. undulosa DNA but not healthy control plants. The TaqMan assay reliably detected as low as 1-pg DNA amount and 10 colony forming units of the target pathogen per reaction. This TaqMan assay could be useful to regulatory agencies with economic benefits to wheat growers.

Pseudomonas quebecensis sp. nov., a bacterium isolated from root-zone soil of a native legume, Amphicarpaea bracteata (L.) Fernald, in Quebec, Canada.

Four bacterial strains (S1Bt3, S1Bt7, S1Bt30 and S1Bt42T) isolated from soil collected from the rhizosphere of a native legume, Amphicarpaea bracteata, were investigated using a polyphasic approach. Colonies were fluorescent, white-yellowish, circular and convex with regular margins on King's B medium. Cells were Gram-reaction-negative, aerobic, non-spore-forming rods. Oxidase- and catalase-positive. The optimal growth temperature of the strains was 37 °C. Phylogenetic analysis of the 16S rRNA gene sequences placed the strains within the genus Pseudomonas. Analysis of the 16S rRNA-rpoD-gyrB concatenated sequences clustered the strains and well separated from Pseudomonas rhodesiae CIP 104664T and Pseudomonas grimontii CFM 97-514T with the type strains of the closest species. Phylogenomic analysis of 92 up-to-date bacterial core gene and matrix-assisted laser desorption/ionization-time-of-flight MS biotyper data confirmed the distinct clustering pattern of these four strains. Digital DNA-DNA hybridization (41.7 %-31.2 %) and average nucleotide identity (91.1 %-87.0 %) values relative to closest validly published Pseudomonas species were below the species delineation thresholds of 70 and 96 %, respectively. Fatty acid composition results validated the taxonomic position of the novel strains in the genus Pseudomonas. Phenotypic characteristics from carbon utilization tests differentiated the novel strains from closely related Pseudomonas species. In silico prediction of secondary metabolite biosynthesis gene clusters in the whole-genome sequences of the four strains revealed the presence of 11 clusters involved in the production of siderophore, redox-cofactor, betalactone, terpene, arylpolyene and nonribosomal peptides. Based on phenotypic and genotypic data, strains S1Bt3, S1Bt7, S1Bt30 and S1Bt42T represent a novel species for which the name Pseudomonas quebecensis sp. nov. is proposed. The type strain is S1Bt42T (=DOAB 746T=LMG 32141T=CECT 30251T). The genomic DNA G+C content is 60.95 mol%.

The Mitochondrial Genome of the Sea Anemone Stichodactyla haddoni Reveals Catalytic Introns, Insertion-Like Element, and Unexpected Phylogeny.

A hallmark of sea anemone mitochondrial genomes (mitogenomes) is the presence of complex catalytic group I introns. Here, we report the complete mitogenome and corresponding transcriptome of the carpet sea anemone Stichodactyla haddoni (family Stichodactylidae). The mitogenome is vertebrate-like in size, organization, and gene content. Two mitochondrial genes encoding NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit I (COI) are interrupted with complex group I introns, and one of the introns (ND5-717) harbors two conventional mitochondrial genes (ND1 and ND3) within its sequence. All the mitochondrial genes, including the group I introns, are expressed at the RNA level. Nonconventional and optional mitochondrial genes are present in the mitogenome of S. haddoni. One of these gene codes for a COI-884 intron homing endonuclease and is organized in-frame with the upstream COI exon. The insertion-like orfA is expressed as RNA and translocated in the mitogenome as compared with other sea anemones. Phylogenetic analyses based on complete nucleotide and derived protein sequences indicate that S. haddoni is embedded within the family Actiniidae, a finding that challenges current taxonomy.

Analysis of Draft Genome Sequences of Two New Pantoea Strains Associated with Wheat Leaf Necrotic Tissues Caused by Xanthomonas translucens Reveals Distinct Species.

We report whole-genome sequences of two new Pantoea strains (DOAB1048 and DOAB1050) isolated from necrotic wheat leaves caused by Xanthomonas translucens The draft genome sequences of DOAB1048 and DOAB1050 consist of 52 and 57 scaffolds and have sizes of 4,795,525 bp and 4,962,883 bp with 4,418 and 4,517 coding sequences, respectively.