François Lefort

Phylogenetic analysis and Real Time PCR detection of a presumbably undescribed Peronospora 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.

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 1 alpha and beta-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 beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.

Gnomoniopsis smithogilvyi causes chestnut canker symptoms in Castanea sativa shoots in Switzerland.

A screening of Castanea sativa scions for grafting for the presence of endophytes showed that the opportunistic fungal pathogen Gnomoniopsis smithogilvyi was the most abundant member of the endophytic flora. This fungus is known as a pathogen affecting chestnut fruits in Italy and Australia. Here, we present evidence that it causes cankers very similar to the ones due to Cryphonectria parasitica infection on twigs and scions of chestnut trees. We found natural infections of G. smithogilvyi in healthy grafted plants as well as in scions from chestnut trees. The identity of the fungus isolated from asymptomatic tissues was verified by applying Kochs postulates and corroborated by DNA sequencing of four different gene regions. In contrast to C. parasitica that appears on the bark as yellow to orange pycnidia, stromata and slimy twisted tendrils, G. smithogilvyi forms orange to red and black pycnidia, gray stromata and cream-colored to beige slimy twisted tendrils on the bark. These Swiss strains are closely related to G. smithogilvyi strains from Australia and from New Zealand, Gnomoniopsis sp. and Gnomoniopsis castanea from New Zealand, Italy, France and Switzerland. While the strains from Ticino are genetically very close to G. smithogilvyi and G. castanea from Italy, the differences between the strains from Ticino and Geneva suggest two different origins. The present study supports the hypothesis that a single species named G. smithogilvyi, which is known to be the agent of chestnut rot, also causes wood cankers on chestnut.

Green Fluorescent Protein (GFP) as a Reporter Gene for the Plant Pathogenic Oomycete Phytophthora ramorum

ABSTRACT. Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2‐based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron, one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non‐labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum. Limitations of the technology are discussed.

Direct PCR for DNA Barcoding in the Genera Phytophopthora and Pythium

ABSTRACT A protocol for direct polymerase chain reaction (DPCR) amplification of commonly used phylogenetic markers of the genera Pythium and Phytophthora from mycelia was developed. This has proven useful for direct PCR amplification from hypha, zoospores and infected plant material, which is quicker and simpler, than other proposed attempts. Optimal reaction conditions are described allowing amplification of commonly used phylogenetic markers with a size of up to 1 kbp. This approach proved successful for amplification of selected markers from hundreds of Phytophthora, Pythium and fungal isolates and has been so far used in routine genotyping identification of oomycota and fungi in our laboratory.

Whole-Genome Shotgun Sequence of Arthrospira platensis Strain Paraca, a Cultivated and Edible Cyanobacterium

ABSTRACT Here we report the whole-genome shotgun sequence of a Peruvian strain of Arthrospira platensis (Paraca), a cultivated and edible haloalkaliphilic cyanobacterium of great scientific, technical, and economic potential.

SELECTION AND MASS PRODUCTION OF SANTALUM ALBUM L. CALLI FOR INDUCTION OF SESQUITERPENES

ABSTRACT Young hypocotyl segments obtained from aseptically germinated seeds of Santalum album L. (5 weeks old) were used to induce callus formation. Growth of different calli types was induced on hypocotyls segments under different conditions. Resulting green calli were maintained on MS medium and Gamborg basal medium supplemented with 2,4D (2,4-Dichlorophenoxyacetic acid) (0.5 μM) and Kin (Kinetin) (10 μM). Numerous concentrations of 2,4D and Kin were tested in combination with inducers of the sesquiterpene metabolism and one promising callus type was selected for mass production. This interesting type of callus has been used for searching functional genes in the biosynthetic pathway leading to sesquiterpenes molecules.

Whole-Genome Shotgun Sequence of Bacillus amyloliquefaciens Strain UASWS BA1, a Bacterium Antagonistic to Plant Pathogenic Fungi

ABSTRACT We report here the whole-genome shotgun sequence of Bacillus amyloliquefaciens strain UASWS BA1, isolated from inner wood tissues of a decaying Platanus × acerifolia tree. This strain proved to be antagonistic to several plant pathogenic fungi and oomycetes and can be developed as a biological control agent in agriculture.

Whole-Genome Shotgun Sequence of Pseudomonas viridiflava, a Bacterium Species Pathogenic to Ararabidopsis thaliana.

ABSTRACT We report here the first whole-genome shotgun sequence of Pseudomonas viridiflava strain UASWS38, a bacterium species pathogenic to the biological model plant Arabidopsis thaliana but also usable as a biological control agent and thus of great scientific interest for understanding the genetics of plant-microbe interactions.

Whole-Genome Sequences of Seven Strains of Bacillus cereus Isolated from Foodstuff or Poisoning Incidents.

ABSTRACT We present here the whole shotgun genome sequences of seven strains of Bacillus cereus isolated from foodstuff samples or food poisoning incidents.