Fungi associated with fir beetles (Pityokteines vorontzovi) and their behavioural attraction

Fir beetles (Pityokteines vorontzovi, P. spinidens, and P. curvidens ) are among the most aggressive bark beetles in Europe. In most bark beetles symbiotic associates, in particular fungal ectosymbionts, are crucial for a successful colonization of trees due to their tree virulence, direct nutrition and detoxifying abilities of plant-defensive compounds. Despite the economic importance of fir beetles, fungal associates of fir beetles are poorly known and their potential functions remain enigmatic. Here we studied fungi from two fir beetle species (P. vorontzovi and P. curvidens) at three distinct locations in the black forest using culture-dependent and metabarcoding techniques. Fifty strains of 35 fungi were identified. The five core symbiotic fungi (Geosmithia sp, Ophiostoma piceae, Cladosporium cladosporioides, Penicillium brevicompactum and Graphilbum fragrans) were selected for further investigations. Growth tests at different temperatures reveaeld differences in fungal temperature preferences and potential niche differentiation. A novel behavioral bioassay was applied to investigate the beetles’ responses to fungal volatiles and gustatatory cues. Neither P. vorontzovi, nor P. curvidens showed significant preferences for any fungus based on its volatiles, however, phloem media colonized by three of the fungal species (Geosmithia sp, Ophiostoma piceae and Cladosporium cladosporioides) were significantly preferred by beetles for boring their tunnels. Such decisions could be the result of beetles’ recognition of specific fungal nutrients and/or detoxification products of plant-defensive compounds. Future studies are required to test the functions these fungi have for beetle nutrition and whether this preference for fungus-colonized substrate is also present during host-colonization in nature.