Multi-Loci Sequence Typing (MLST) for Two Lacto-Acid Bacteria (LAB) Species: Pediococcusparvulus and P.damnosus

The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P.parvulus when using either conventional PCR or Real Time PC

Pythium sterilum sp. nov. isolated from Poland, Spain and France: its morphology and molecular phylogenetic position

In a survey of Phytophthora species associated with forest decline in Spain, Poland and France, we found three Pythium isolates, which have been characterized with internal transcribed spacer rRNA gene sequences and with classical morphological descriptors for Pythium spp. These isolates showed unique internal transcribed spacer sequences, different enough from those of any described species to justify new species status. These three distinct isolates failed to produce any sex organs with an entirely asexual reproduction and were found to represent a new species for which the name Pythium sterilum is proposed. This paper describes and illustrates the morphology of P. sterilum and presents its taxonomic position and relationships with other, related Pythium species belonging to clade

A new species of Pythium with ornamented oogonia: morphology, taxonomy, internal transcribed spacer region of its ribosomal RNA, and its comparison with related species

Pythium spiculum sp. nov. was isolated from soil samples taken in a vineyard in the Burgundian region of France and from different locations in Spain and Portugal. The oomycete has spiny oogonia and does not sporulate readily. It resembles Pythium mamillatum Meurs, but has its own distinguishing characteristics. It also exhibits sickle-shaped as well as spherical appressoria which at times are associated with sex organs like those found in Pythium abappressorium Paulitz and Pythium contiguanum Paul. Sequencing of the internal transcribed spacer region of its nuclear ribosomal DNA and a close look at its morphological characters have now enabled us to describe it as a new species. The internal transcribed spacer region of its rRNA gene sequence is comprised of 945 bases. This oomycete is closely related to the members that form ornamented or spiny oogonia like Pythiummamillatum, Pythium spinosum and Pythium irregulare but also with those producing smooth-walled oogonia like Pythium paroecandrum, Pythium sylvaticum and Pythium cylindrosporum. Taxonomic description of this new species, its comparison with related oomycetes, the sequence of the internal transcribed spacer region of its rRNA gene and the phylogenetic tree, are given her

An unknown trees die back caused by Pseudomonas species in Switzerland

A model for tree pathogen diagnosis – Prunus domestica L. has been studied against pathogenic bacteria. An orchard of 110 trees of P. domestica showed dying back symptoms in May 2009 and nineteen of these trees were eradicated and burnt for prophylaxis. No symptoms correlated with those caused by pathogens previously observed in stone fruit die back in Europe or elsewhere (Pseudomonas syringae pv syringae van Hall, Pseudomonas syringae pv morsprunorum Lazarowtz, Phytophthora sp., Diaporthe perniciosa Marchal., phytoplasma or viruses) were not found. Interestingly, cutting the trunk in transversal sections allowed the observation of stem heart necrosis which was mostly important at the grafting point. Isolations from necrotic stem heart allowed to identify anot yet described Pseudomonas species not related to P. syringae. The method described in the paper for isolation of pathogenic bacteria and their quick an reliable identification can be also applied for detection of pathogens in forest tree plantations.Przemysław Szmi

Phytophthora polonica, a new species isolated from declining Alnus glutinosa stands in Poland

In a survey of Phytophthora associated with alder decline in Poland, several isolates of a homothallic Phytophthora spet al, which could not be assigned to other taxa including Phytophthora alni subspecies, were consistently recovered from rhizosphere soil samples. Their morphology and pathogenicity, as well as sequence data for three nuclear regions (internal transcribed spacer rDNA, elongation factor-1α and β-tubulin) and a coding mitochondrial DNA region (nadh1), were examined. The new Phytophthora species is characterized by the moderate to slow growth rate of its colony in carrot agar at 20°C, high optimal (c. 30°C) and maximum (c. 38°C) growth temperatures, formation of catenulate, often lateral, hyphal swellings, large chlamydospores in agar media and in soil extract, persistent, ovoid to ellipsoid nonpapillate sporangia and large oogonia with paragynous and sometimes amphigynous antheridia. Phytophthora polonica was slightly pathogenic to alder twigs and not pathogenic to trunks of several tree species. In a phylogenetic analysis using either Bayesian inference or maximum likelihood methods, P. polonica falls in clade 8 ‘sensu Kroon (2004)' of Phytophthor

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1α and β-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and β-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthor

Evolution of the cutinase gene family: Evidence for lateral gene transfer of a candidate Phytophthora virulence factor

Lateral gene transfer (LGT) can facilitate the acquisition of new functions in recipient lineages, which may enable them to colonize new environments. Several recent publications have shown that gene transfer between prokaryotes and eukaryotes occurs with appreciable frequency. Here we present a study of interdomain gene transfer of cutinases – well documented virulence factors in fungi – between eukaryotic plant pathogens Phytophthora species and prokaryotic bacterial lineages. Two putative cutinase genes were cloned from Phytophthora brassicae and Northern blotting experiments showed that these genes are expressed early during the infection of the host Arabidopsis thaliana and induced during cyst germination of the pathogen. Analysis of the gene organisation of this gene family in Phytophthora ramorum and P. sojae showed three and ten copies in tight succession within a region of 5 and 25 kb, respectively, probably indicating a recent expansion in Phytophthora lineages by gene duplications. Bioinformatic analyses identified orthologues only in three genera of Actinobacteria, and in two distantly related eukaryotic groups: oomycetes and fungi. Together with phylogenetic analyses this limited distribution of the gene in the tree of life strongly support a scenario where cutinase genes originated after the origin of land plants in a microbial lineage living in proximity of plants and subsequently were transferred between distantly related plant-degrading microbes. More precisely, a cutinase gene was likely acquired by an ancestor of P. brassicae, P. sojae, P. infestans and P. ramorum, possibly from an actinobacterial source, suggesting that gene transfer might be an important mechanism in the evolution of their virulence. These findings could indeed provide an interesting model system to study acquisition of virulence factors in these important plant pathogens

Comment contrer biologiquement cylindrocladium buxicola et volutella buxi

Après un premier article sur la lutte contre la pyrale du buis, nous traitons ici d'essais de lutte biologique contre les champignons cylindrocladium buxicola et volutella buxi

Phylogenetic analysis and Real Time PCR detection of a presumbably undescribed <i>Peronospora </i>species on sweet basil and sage

Downy mildew of sweet basil (Ocimum basilicum) has become a serious disease issue for the producers of sweet basil in Switzerland since it was first recorded in 2001. Reported in Africa in Uganda as early as 1933, major outbreaks of this disease in Europe were first noted in Italy in 1999 and in the USA from 1993. Previous reports have named the pathogen as Peronospora lamii. Its preferential hosts belong to the Lamiaceae family including basils (Ocimum spp.), mints (Menta spp.), sages (Salvia spp.) and other aromatics. This study investigated the taxonomic status of the downy mildew pathogen, using both morphological characters and molecular analysis of the ITS region of the rDNA. The inherent variability of conidial dimensions made species differentiation difficult. Sequence homology and phylogenetic analysis of nine collections of the Peronospora on sweet basil showed unique ITS sequences distinct from those of P. lamii and any other sequenced Peronospora species. This paper describes and illustrates the morphology of this presumably undescribed species of Peronospora. Its taxonomic position and relationships with other related species in the same genus are presented and discussed. In addition to this work, PCR primers for real time PCR analysis have been developed for the specific detection of this downy mildew pathogen from infected tissues or seeds. It is shown that these primers can also be used in classic PCR