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Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome

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Abstract

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study 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.

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Acknowledgements

This work is a part of a research project organized and coordinated by Premier Tech. The authors are grateful for financial support from NSERC Cooperative Research and Development (grant number CRDJP 468828-14), Premier Tech, and CRIBIQ. We would like to thank Dr. Serge Gagné, Dr. Younes Machrafi, Nicolas Bertrand, Éric Dion, François Gobeil, and Dr Alain Bélanger for recommendations and comments. We also thank Dr. David Morse and Dr. Karen Fisher-Favret for English editing and Dr D. Janos and two anonymous reviewers for their helpful comments.

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    Authors and Affiliations

    1. Centre de recherche sur les biotechnologies marines, 2e Rue Est, Rimouski, QC, G5L 9H3, Canada

      Amine Badri

    2. Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, 4101 Rue Sherbrooke Est, Montréal, QC, H1X 2B2, Canada

      Franck O. P. Stefani, Denis Beaudet & Mohamed Hijri

    3. Premier Tech, 1 avenue Premier, Campus Premier Tech, Rivière-du-Loup, QC, G5R 6C1, Canada

      Geneviève Lachance & Line Roy-Arcand

    4. Biopterre—Centre de développement des bioproduits, 1642, Rue de la Ferme, La Pocatière, Québec, G0R 1Z0, Canada

      Agathe Vialle

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    Correspondence to Mohamed Hijri.

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    Amine Badri and Franck O. P. Stefani contributed equally to this work.

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    Badri, A., Stefani, F.O.P., Lachance, G. et al. Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome. Mycorrhiza 26, 721–733 (2016). https://doi.org/10.1007/s00572-016-0708-1

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