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Use of a RT-qPCR Method to Estimate Mycorrhization Intensity and Symbiosis Vitality in Grapevine Plants Inoculated
Morgane Duret1, Xi Zhan1, Lorène Belval1
1Laboratoire Vigne, Biotechnologies et Environnement, Université de Haute Alsace, Université de Strasbourg, E.A. 3991, 33 rue de Herrlisheim, BP 50568, CEDEX 008, 68000 Colmar, France.
Abstract:
Assessing the mycorrhization level in plant roots is essential to study the effect of arbuscular mycorrhizal fungi (AMF) on plant physiological responses. Common methods used to quantify the mycorrhization of roots are based on microscopic visualization of stained fungal structures within the cortical cells. While this method is readily accessible, it remains time-consuming and does not allow checking of the symbiosis vitality. The aim of this work is thus to develop an efficient method for assessing the intensity and vitality of mycorrhiza associated with grapevine through gene expression analyses by RT-qPCR. To this end, grapevine plants were inoculated with the AMF Rhizophagus irregularis (Ri). The relationship between mycorrhization level, assessed by microscopy, and expression of several fungus and grapevine genes involved in the symbiosis was investigated. In AMF-inoculated plants, transcript amounts of fungal constitutively-expressed genes Ri18S, RiTEF1α and RiαTub were significantly correlated to mycorrhization intensity, particularly Ri18S. Grapevine (VvPht1.1 and VvPht1.2) and AMF (GintPT, Ri14-3-3 and RiCRN1) genes, known to be specifically expressed during the mycorrhizal process, were significantly correlated to arbuscular level in the whole root system determined by microscopy. The best correlations were obtained with GintPT on the fungal side and VvPht1.2 on the plant side. Despite some minor discrepancies between microscopic and molecular techniques, the monitoring of Ri18S, GintPT and VvPht1.2 gene expression could be a rapid, robust and reliable method to evaluate the level of mycorrhization and to assess the vitality of AMF. It appears particularly useful to identify AMF-inoculated plants with very low colonization level, or with non-active fungal structures. Moreover, it can be implemented simultaneously with the expression analysis of other genes of interest, saving time compared to microscopic analyses.
