Visualising confirmation of the endophytic relationship of Metarhizium anisopliae with maize roots using molecular tools and fluorescent labelling

Metarhizium anisopliae, an entomopathogenic fungus, is found to contribute to plant nutrient uptake and condidered as a plant endophyte. However, there is very little visual evidence that demonstratrates the endophytic relationship. Since the incidence of Metarhizium within host plants may be ephemeral or of limited distribution within host tissue, visualisation is reliant on both presence and the ability to detect the fungus even when present at low levels. We inoculated maize seeds with an egfp-labelled isolate of M. anisopliae, and then detected the egfp fragment by qualitative and quantitative PCR, and examined fluorescence of mycelia in the inoculated roots by laser scanning confocal microscopy (LSCM). The optimised PCR qualitatively identified the presence of egfp-labelled M. anisopliae at a 1:3000 M. anisopliae-maize DNA ratio. Root samplings of inoculated plants at intervals over 24 days were discovered egfp-labelled fragments with 14-d roots having the highest proportion with amplification. The egfp-label concentrations ranged very low in 2.976 × 10^{−7.42∼−7.02} ng/μL, with a rise process in 4–11 days after M. anisopliae treatment. Under LSCM, fluorescent mycelia in roots were identified, and the clear images were captured to show hyphal development extending along a longitudinal line of root cells and even forming a hyphal mass in the intercellular space. There were many hyphae at the branch junctions of the lateral roots. The methodology used to detect the fungus will be important for exploring the multi-trophic function of M. anisopliae and co-evolution in plants.