Diversity of fungi associated with Monochamus alternatus larval habitats in Bursaphelenchus xylophilus-infected Pinus massoniana and identification of two new ophiostomatalean species (Ascomycota, Ophiostomatales)

Bursaphelenchus xylophilus, a pathogenic pine wood nematode (PWN), is responsible for pine wilt disease (PWD), which has caused significant economic and ecological damage worldwide, particularly in East Asia. Multiple biological factors, such as the beetle vector Monochamus, symbiotic bacteria and associated fungi, are involved in the disease infection cycle. This study isolated and identified the fungal communities of Monochamus alternatus larval galleries and pupal chambers from different instars through field investigation, morphological observation and multi-locus DNA sequence analyses in Zhejiang Province, China. A total of 255 and 454 fungal strains were isolated from M. alternatus galleries and pupal chambers infected with PWN, from the 2^nd–3^rd and 4^th–5^th instar larvae, respectively. A total of 18 species of fungi were identified, 14 species were isolated from the 2^nd–3^rd instar larval galleries and six species from the galleries and pupal chambers of the 4^th–5^th instar larvae. Amongst them were six species belonging to four genera of ophiostomatalean fungi, including two novel species, Graphilbum xianjuensis sp. nov. and Ophiostoma taizhouense sp. nov. and four known species, Ceratocystiopsis weihaiensis, Ophiostoma ips, Sporothrix zhejiangensis and S. macroconidia. The findings revealed that the fungal diversity and abundance of the 2^nd–3^rd instar larvae differed markedly from those of the 4^th–5^th instar larvae. This difference could be the result of fungal succession. This study provides a thorough understanding of the fungi associated with PWD and lays the groundwork for future research.