Pectobacterium parvum sp. nov., having a Salmonella SPI-1-like Type III secretion system and low virulence.

Pectobacterium strains isolated from potato stems in Finland, Poland and the Netherlands were subjected to polyphasic analyses to characterize their genomic and phenotypic features. Phylogenetic analysis based on 382 core proteins showed that the isolates clustered closest to Pectobacterium polaris but could be divided into two clades. Average nucleotide identity (ANI) analysis revealed that the isolates in one of the clades included the P. polaris type strain, whereas the second clade was at the border of the species P. polaris with a 96 % ANI value. In silico genome-to-genome comparisons between the isolates revealed values below 70%, patristic distances based on 1294 core proteins were at the level observed between closely related Pectobacterium species, and the two groups of bacteria differed in genome size, G+C content and results of amplified fragment length polymorphism and Biolog analyses. Comparisons between the genomes revealed that the isolates of the atypical group contained SPI-1-type Type III secretion island and genes coding for proteins known for toxic effects on nematodes or insects, and lacked many genes coding for previously characterized virulence determinants affecting rotting of plant tissue by soft rot bacteria. Furthermore, the atypical isolates could be differentiated from P. polaris by their low virulence, production of antibacterial metabolites and a citrate-negative phenotype. Based on the results of a polyphasic approach including genome-to-genome comparisons, biochemical and virulence assays, presented in this report, we propose delineation of the atypical isolates as a novel species Pectobacterium parvum, for which the isolate s0421T (CFBP 8630T=LMG 30828T) is suggested as a type strain.

Expression patterns and phylogenetic analysis of two xylanase genes (htxyl1 and htxyl2) from Helminthosporium turcicum, the cause of northern leaf blight of maize.

A xylanase gene (htxyl2) was cloned from Helminthosporium turcicum, the cause of northern leaf blight of maize by screening the genomic library from the fungus using a approximately 500 bp PCR fragment of the gene as a probe. The gene is a second xylanase gene cloned from the fungus and it is different from the previously cloned xylanase gene (htxyl1). The two genes are grouped into separate clades in a phylogenetic tree based on DNA sequences. The patterns of htxyl1 and htxyl2 transcript accumulation were studied in vitro with different carbon and nitrogen sources and during infection of maize by the pathogen. In culture high htxyl1 transcript was detected in mycelium obtained from medium containing birchwood xylan and birchwood xylan plus xylose as sole carbon sources. On the other hand htxyl2 transcript was detected in a medium containing birchwood xylan plus xylose but not in the medium where only birchwood xylan was used as the sole carbon source. Addition of glucose to the basal inducing media had inhibitory effect to the expression of both genes. The htxyl1 transcript accumulation declined sharply with an increase in concentration of ammonium sulphate and glutamic acid, while increased htxyl2 transcript was observed with an increase in concentrations of the two nitrogen sources. Abundant htxyl2 transcript was detected at early and late stages of infection of maize by Helminthosporium turcicum. On the other hand no detectable htxyl1 transcript was found in northern blot under the test conditions. This study demonstrated that in H. turcicum xylanolytic system htxyl1 and htxyl2 are differentially expressed and might play important roles in the saprophytic and pathogenic phases of the fungus, respectively.

Agrobacterium- tumefaciens-mediated transformation of Helminthosporium turcicum, the maize leaf-blight fungus.

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium- tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase ( hph) and the enhanced green fluorescent protein ( EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium- tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium- tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.