Effect of insect cuticular compounds on appressorium formation and metabolic activity in Metarhizium anisopliae

The rate of appressoria formation following conidial germination in Metarhizium anisopliae is closely associated with its pathogenicity. This study investigated the effects of insect cuticular compounds on the formation and metabolism in M. anisopliae by adding insect cuticle analogues. The results showed that amino acid compounds L-aspartic acid and glycyl-L-phenylalanine, amine-class compound L-sorbitol, aromatic compounds and fenofibric acid, and organic acid 4-(aminomethyl) benzoic acid promoted spore germination and appressorium formation (p ≤ 0.05). Among these, L-aspartic acid significantly promoted spore germination at all tested concentrations (p ≤ 0.05), but showed no significant effect on appressorium formation at low concentrations (p > 0.05). In contrast, high concentrations significantly enhanced appressorium formation (p ≤ 0.05). Moreover, the promotive effect on appressorium formation increased with concentration, and at 1 mg/mL both the spore germination rate and appressorium formation rates peaked across all time points, being significantly higher than those of the control (p ≤ 0.05). Both epigallocatechin gallate and glycerophosphocholine promoted spore germination and appressorium formation at low concentrations, whereas high concentrations significantly inhibited both processes (p ≤ 0.05). At higher concentrations, the organic acid compound mitifos inhibited conidial germination and appressorium formation (p ≤ 0.05). For the aromatic compound 2,6-dihydroxybenzoic acid, the inhibitory effect on conidial germination and appressorium formation increased with concentration. Analysis of the effects of L-aspartic acid on metabolism during appressorium formation in Metarhizium anisopliae using LC-MS metabolomics showed that the amino acid compound L-aspartic acid broadly perturbed the secondary metabolism of M. anisopliae. After appressorium formation, a total of 367 unique differential metabolites were identified. Prior to appressorium formation, L-aspartic acid supports the energy demands of cellular assembly and proliferation by promoting the activation of M. anisopliae including D-amino acid metabolism, biosynthesis of cofactors and amino acid biosynthesis, thereby strengthening stress defense and signal transduction. After appressorium formation, L-aspartic acid activates pathways in M. anisopliae including tryptophan metabolism, the two-component system, β-alanine metabolism and folate metabolism, providing signaling cues to drive hyphal growth and accelerating toxin production and secondary metabolite biosynthesis. Thus, L-aspartic acid provides substances and energy for spore germination, appressorium cell wall thickening and stress early warning of M. anisopliae, and enhances fungal defense capacity. After appressorium formation, L-aspartic acid further promotes fungal cuticle penetration, accelerates in-host colonization, and facilitates toxin synthesis to kill the host, thereby improving the infectivity of M. anisopliae in multiple ways. This study provides a theoretical basis for in-depth investigations into the infection mechanism of M. anisopliae and the mechanistic role of appressorium during infection.