Project description:In this work, 454 pyrosequencing was used to build up a 3’ cDNA fragment library from a normalized library constructed from pooled RNA samples extracted at different stages of A. quisqualis mycoparasitization process (recognition, early and late parasitization). An extensive catalogue of unique transcripts was compiled and used to develop a microarray for large-scale analysis of genes involved in this mycoparasitism. We examined the transcriptomic changes that occur during the first stage of mycoparasitism (conidial germination). Our results showed that 1,776 transcripts are regulated during germination in the presence of powdery mildew. A striking feature of the gene catalogue was the presence of a number of genes predicted to encode proteins involved in the production of, glucanases, chitinases and extracellular proteases. This suggests that A. quisqualis causes the degradation of powdery mildew macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing of the mycelium.
Project description:In this work, 454 pyrosequencing was used to build up a 3M-bM-^@M-^Y cDNA fragment library from a normalized library constructed from pooled RNA samples extracted at different stages of A. quisqualis mycoparasitization process (recognition, early and late parasitization). An extensive catalogue of unique transcripts was compiled and used to develop a microarray for large-scale analysis of genes involved in this mycoparasitism. We examined the transcriptomic changes that occur during the first stage of mycoparasitism (conidial germination). Our results showed that 1,776 transcripts are regulated during germination in the presence of powdery mildew. A striking feature of the gene catalogue was the presence of a number of genes predicted to encode proteins involved in the production of, glucanases, chitinases and extracellular proteases. This suggests that A. quisqualis causes the degradation of powdery mildew macromolecular constituents to provide the carbon skeletons and energy for the synthesis of proteins and other components destined for the developing of the mycelium. Microarray oligo probes were designed based on 454 sequencing of 3'-ends of transcripts of a sample constituted by pooling RNAs extracted at different stages of A. quisqualis mycoparasitization process (recognition, early and late parasitization)
Project description:The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, France, and the co-rapporteur Member State, Germany, for the pesticide active substance Ampelomyces quisqualis strain AQ10 are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of Ampelomyces quisqualis strain AQ10 as a fungicide by spraying against powdery mildew in grapes and spray applications in tomato, pepper and aubergine grown under protection or in the open field. The reliable end points, appropriate for use in regulatory risk assessment are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.
Project description:Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes.