Termite sanitary behaviour and their associated bacteria protect against latent Xylaria and entomopathogenic Metarhizium

Fungus-growing termites harbour diverse microbial communities in their combs and guts. However, culture-based surveys, cross-colony and cross-regional comparisons and assessments of their antimicrobial activities against entomopathogens and fungal weeds remain limited. Through culture-dependent isolation, we surveyed the bacterial and fungal diversity associated with the combs and guts of termites in the genera Macrotermes and Odontotermes , recovering 600 non- Termitomyces fungal isolates representing 79 genera, as well as 59 bacterial isolates. Pathogenicity tests on Macrotermes gilvus revealed that a Metarhizium isolate obtained from a healthy comb was highly virulent to termites. Combs incubated in the boxes yielded 112 isolates of the weed Xylaria and multi-locus phylogenetic analyses revealed high species diversity. Together, these findings revealed two major microbial threats within healthy termite fungus combs. First, highly virulent entomopathogens that directly threaten termite survival. Second, a diverse assemblage of comb-specialised xylariaceous fungi that persist latently and compete for comb nutrients. Behavioural assays revealed that termites exhibited sharply differential hygienic responses to fungi co-occurring in soil plates, aggressively burying Metarhizium regardless of comb presence, but responded weakly to co-evolved Xylaria . Newly-hatched termites also displayed innate avoidance of food contaminated with Metarhizium conidia. Several comb- and gut-associated bacterial isolates, as well as termite-generated soil boluses, inhibited both Metarhizium and Xylaria in vitro . This study underscores the utility of culture-based approaches for elucidating microbial diversity and ecology in termite nests and highlights the roles of host behaviour and comb- and gut-associated bacteria in suppressing both fungal weed invasions and entomopathogen infections.