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

<i>Metarhizium anisopliae</i>, 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 <i>Metarhizium</i> 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 <i>egfp</i>-labelled isolate of <i>M. anisopliae</i>, and then detected the <i>egfp</i> 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 <i>egfp</i>-labelled <i>M. anisopliae</i> at a 1:3000 <i>M. anisopliae</i>-maize DNA ratio. Root samplings of inoculated plants at intervals over 24 days were discovered <i>egfp</i>-labelled fragments with 14-d roots having the highest proportion with amplification. The <i>egfp</i>-label concentrations ranged very low in 2.976 × 10^{−7.42∼−7.02} ng/μL, with a rise process in 4–11 days after <i>M. anisopliae</i> 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 <i>M. anisopliae</i> and co-evolution in plants.金龟子绿僵菌（Metarhizium anisopliae）是一种昆虫病原真菌，研究发现其可促进植物养分吸收，同时被认定为植物内生真菌。然而，能够直接证明其与植物建立内生共生关系的可视化证据却十分匮乏。由于寄主植物体内的金龟子绿僵菌可能仅短暂存在，或在宿主组织中分布有限，因此即使真菌含量极低，可视化检测仍需同时满足真菌存在且可被检出的条件。本研究采用携带增强绿色荧光蛋白基因（egfp）的金龟子绿僵菌菌株接种玉米种子，通过定性与定量PCR技术检测egfp片段，并利用激光扫描共聚焦显微镜（LSCM）观察接种根系中菌丝的荧光信号。经优化的PCR反应可在金龟子绿僵菌与玉米DNA比例为1:3000时，定性检出携带egfp标记的金龟子绿僵菌。对接种后24天内不同时段采集的根系样本进行检测，均检出egfp标记片段，其中接种后14天的根系样本扩增比例最高。egfp标记的浓度极低，范围为2.976×10^−7.42~−7.02 ng/μL，且在金龟子绿僵菌接种后的4~11天内呈上升趋势。在激光扫描共聚焦显微镜下，可观察到根系内发出荧光的菌丝，清晰成像显示菌丝沿根细胞纵向延伸生长，甚至在细胞间隙形成菌丝团；侧根的分支连接处也分布有大量菌丝。本研究建立的真菌检测方法，对深入探究金龟子绿僵菌的多营养级功能及其与植物的协同进化关系具有重要意义。