Differential patterns of ophiostomatoid fungal communities associated with three sympatric Tomicus species infesting pines in south-western China, with a description of four new species

Bark beetles and their associated fungi, which cause forest decline and sometimes high mortality in large areas around the world, are of increasing concern in terms of forest health. Three Tomicus spp. (T. brevipilosus, T. minor and T. yunnanensis) infect branches and trunks of Pinus yunnanensis and P. kesiya in Yunnan Province, in south-western China. Tomicus spp. are well known as vectors of ophiostomatoid fungi and their co-occurrence could result in serious ecological and economic impact on local forest ecosystems. Nonetheless, knowledge about their diversity, ecology, including pathogenicity and potential economic importance is still quite rudimentary. Therefore, an extensive survey of ophiostomatoid fungi associated with these Tomicus species infesting P. yunnanensis and P. kesiya was carried out in Yunnan. Seven hundred and seventy-two strains of ophiostomatoid fungi were isolated from the adult beetles and their galleries. The strains were identified based on comparisons of multiple DNA sequences, including the nuclear ribosomal large subunit (LSU) region, the internal transcribed spacer regions 1 and 2, together with the intervening 5.8S gene (ITS) and the partial genes of beta-tubulin (TUB2), elongation factor 1 alpha (TEF1-alpha) and calmodulin (CAL). Phylogenetic analyses were performed using maximum parsimony ( MP) as well as maximum likelihood (ML). Combinations of culture features, morphological characters and temperature-dependent growth rates were also employed for species identification. Eleven species belonging to five genera were identified. These included six known species, Esteya vermicola, Leptographium yunnanense, Ophiostoma brevipilosi, O. canum, O. minus and O. tingens and four novel taxa, described as Graphilbum anningense, O. aggregatum, Sporothrix pseudoabietina and S. macroconidia. A residual strain was left unidentified as Ophiostoma sp. 1. The overall ophiostomatoid community was by far dominated by three species, representing 87.3% of the total isolates; in decreasing order, these were O. canum, O. brevipilosi and O. minus. Furthermore, the ophiostomatoid community of each beetle, although harbouring a diversity of ophiostomatoid species, was differentially dominated by a single fungal species; Ophiostoma canum was preferentially associated with and dominated the ophiostomatoid community of T. minor, whereas O. brevipilosi and O. minus were exclusively associated with and dominated the ophiostomatoid communities of T. brevipilosus and T. yunnanensis, respectively. Eight additional species, representing the remaining 12.7% of the total isolates, were marginal or sporadic. These results suggested that sympatric Tomicus populations are dominated by distinct species showing some level of specificity or even exclusivity.