Use of a RT-qPCR Method to Estimate Mycorrhization Intensity and Symbiosis Vitality in Grapevine Plants Inoculated with Rhizophagus irregularis

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.

Exogenous myristate promotes the colonization of arbuscular mycorrhizal fungi in tomato

“…This is due to errors between microscopy results and qRT-PCR results (Bodenhausen et al, 2021). In Ri2, RiEF1a expression was not detected in either treatment, since the M% was too low to detect the expression of RiEF1a (Duret et al, 2022).…”

Positioning absorptive root respiration in the root economics space across woody and herbaceous species

“…It is worth noting that in this study we estimated AM fungal colonisation by quantifying the ratio of DNA copy numbers between arbuscular mycorrhizal fungi and roots. This molecular quantification method of AM fungal colonization has been used in an increasing number of studies (Arruda et al., 2022; Bodenhausen et al., 2021; Duret et al., 2022) as it may require less expertise than conventional microscopy. As a support to this methodology, we found a significant (but not very strong) correlation between molecular and microscopic quantification of AM fungal colonisation among a subset of our species ( r = 0.46, p = 0.04, Figure S4).…”

“…Across AM plant species, the abundance of AM fungi in absorptive roots was estimated by quantifying the DNA copy number of AM fungi relative to the DNA copy number of root tissue (Arruda et al, 2022;Bodenhausen et al, 2021;Duret et al, 2022). We used primers AMG1F (ATAGG GAT AGT TGG GGGCAT) and AM1 (GTTTC CCG TAA GGC GCCGAA) (Hewins et al, 2015) to quantify AM fungi, and primers trnL-g (GGGCA ATC CTG AGC CAA) and trnL-h (CCATT GAG TCT CTG CAC CTATC) to quantify the roots (Taberlet et al, 2007).…”

Efficiently recording and processing data from arbuscular mycorrhizal colonization assays using AMScorer and AMReader

“…• Correlations between the microscopic assessment and multiple fungal gene expressions have been assessed in grapevine (Duret et al, 2022).…”

“…( Peŕez-Tienda et al, 2014;Choi et al, 2020;Evangelisti et al, 2021;Duret et al, 2022;Shi et al, 2022) Plant RNA qRT-PCR • AM symbiosis-specific gene expression.…”