A method evaluating ectomycorrhizal fungal colonization in vitro by using relative quantitative PCR

Ectomycorrhiza (ECM) significantly influences the establishment and stability of forest ecosystems. While evaluating ECM colonization is important for ECM symbiosis research, conventional methods using microscopes prove both time-intensive and subjective. To overcome this, we developed a high throughput and objective method for evaluating ECM colonization in a target tree under laboratory conditions by using quantitative PCR (qPCR). Primers specific for <i>Populus tomentosa</i>, <i>Cenococcum geophilum</i> (Cg), and <i>Laccaria japonica</i> (Lj) were designed and validated. After separately inoculating Cg and Lj, as well as co-inoculating Cg and Lj to <i>P. tomentosa</i>, the root samples were harvested at three different time points; 10, 20, and 40 days post-inoculation. ECM colonization was assessed by conventional microscopic method and qPCR with the designed primers. The designed primers showed a high specificity and quantitativity for each species, indicating their practical use in quantifying individual ECM species in tripartite conditions. Both ectomycorrhizal species showed a positive correlation between the conventional method and the qPCR method: Cg (Spearman correlation = 0.917, <i>p</i> &lt; 0.001), Lj (Spearman correlation = 0.690, <i>p</i> &lt; 0.01). Therefore, by using highly species-specific primers, we established a method to evaluate ECM colonization in vitro by qPCR, which can be an alternative and solution to the conventional method.