Développement des marqueurs moléculaires spécifiques pour l'identification et la quantification de deux espèces de champignons mycorhiziens arbusculaires en utilisant la PCR en temps réel
Arbuscular Mycorrhizal Fungi (AMF) are able to form symbiosis with approximately 80% of plant species, including most important corps. This symbiosis is known as Arbuscular Mycorrhiza which has been largely used in agriculture to promote plant growth by enhancing minerals uptake and protecting plants against biotic and abiotic stresses. Despite the ecological and economical importance of this symbiosis, specific markers for spore quantification of commercial AMF strains by quantitative real-time PCR (qPCR) are extremely limited and none has been rigorously validated for quality control of manufactured products such as biofertilizers. Most of the existing AMF markers developed either for AMF identification or for AMF quantification purposes are based on either morphological characters, on Fatty Acids Methyl Esters (FAME) profiles, on specific isozymes or antibodies, or on nuclear DNA. It has been shown that mitochondrial (mt) genomes are conserved among AMF species. This allowed us to use mt genomes in order to develop efficient isolate-specfic markers. Using the alignments of 11 complete AMF mt genomes, a qPCR assay using a hydrolysis probes designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of the model R. irregularis isolate DAOM197198 and another probe was designed in nad1-cox2 intergene specific to Glomus cerebriforme. The specificity tests performed, using standard PCR and qPCR, clearly showed that the primers specifically amplified the isolate DAOM-197198 or Glomus cerebriforme DAOM220722. Quantification assays were successfully undertaken on unknown samples in liquid suspensions, commercial solid formulations and soil samples to show the accuracy and reproducibility of the assays. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate Glomus cerebriforme DAOM220722. The latter could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.
Identification PCR quantitative Sol Glomus cerebriforme DAOM220722 Glomus irregulare DAOM197198 Mycorrhiza Quantitative PCR Specific TaqMan markers Soil