Multi-locus Phylogeny and Cuticle-Degrading Enzyme Activities of Entomopathogenic Fungi from Forest Ecosystems in Northern Thailand.

The escalating demand for sustainable pest management strategies has intensified research interest in entomopathogenic fungi (EPF), particularly regarding their taxonomic diversity and biocontrol potential in tropical ecosystems. However, only a limited number of comprehensive surveys that combine molecular phylogenetics with functional characterization have been conducted in Asia. In the present study, we collected mycosed insect cadavers from forest areas in Chiang Mai Province, northern Thailand. Morphological characteristics and multilocus phylogenetic analysis were conducted to facilitate proper species identification. Fungal isolates were further screened for proteolytic, chitinolytic, and lipolytic activities using a preliminary whole-plate assay. Further, a larval mortality bioassay using the mealworm beetle, Tenebrio molitor, was performed to confirm pathogenicity and assess the insecticidal potential of the fungal isolate under controlled laboratory conditions. In the field, seven insect cadavers infected with six different EPF species were collected. The collected fungal isolations were identified as Beauveria asiatica, B. bassiana, Clonostachys rogersoniana, Cordyceps blackwelliae, two strains of C. tenuipes, and Purpureocillium takamizusanense. For the enzymatic assay, C. tenuipes (MFLUCC 25-0373) has the highest chitinolytic activity, while P. takamizusanense (MFLUCC 25-0376) showed the highest proteolytic activity. In addition, Cl. Rogersoniana (MFLUCC 25-0379), C. tenuipes (MFLUCC 25-0373), and P. takamizusanense (MFLUCC 25-0376) showed higher and similar lipolytic activity. Notably, no single isolate in this study exhibited detectable activity for all three enzymes. Except for B. bassiana (MFLUCC 25-0375) and C. blackwelliae (MFLUCC 25-0377), which showed chitinolytic and proteolytic activity, respectively, all other isolates demonstrated activity for at least two of the three key enzymes. Most fungal isolates induced initial larval mortality by the third day post-exposure; however, virulence levels varied over time. Notably, no clear correlation was observed between enzyme activity and larval virulence, suggesting that fungal pathogenicity is a multifactorial process not merely dependent on extracellular enzymatic activity. Therefore, here, cumulative larval mortality was considered the primary criterion for candidate selection for future biocontrol formulation development, under which B. bassiana (MFLUCC 25-0375) was identified as the most suitable candidate, exhibiting the highest virulence.