Transcriptomic analysis of the fungus Graphilbum sp. in response to the pine wood nematode.

Graphilbum species are important blue stain fungi associated with pine trees and are widely distributed throughout Asia, Australia, and North Africa. Pine wood nematode (PWN) primarily feed on ophiostomatoid fungi such as Graphilbum sp. in wood, the population of PWNs was increased, and incomplete organelle structures were observed in Graphilbum sp. hyphal cells following exposure to PWNs. In this study, we showed that Rho and Ras were involved in the MAPK pathway, SNARE binding and small GTPase-mediated signal transduction, and their expression was upregulated in the treatment group. However, the expression of the Rab7 involved in MAPK and small GTPase-mediated signal pathway was downregulated in the treatment group. Thus, further research is needed to study the MAPK pathway and related Ras and Rho genes in Graphilbum sp. associated with the PWN population. Overall, transcriptomic analysis clarified the basic mechanisms of mycelial growth in Graphilbum sp. fungus used as a food source by PWNs.

Identification of ophiostomatalean fungi associated with Tomicus pilifer infesting Pinus koraiensis in Northeastern China.

Ophiostomatalean fungi usually facilitate bark beetles to infest tree hosts and seriously endanger the health of coniferous forests. Tomicus pilifer Spessivtsev is a common endemic bark beetle in Asia and primarily threatens Pinus koraiensis. Tomicus species have similar morphology; however, they can be differentiated by their genetic characteristics through phylogenetic analyses. To date, the 28S rDNA sequence of T. pilifer and the diversity of ophiostomatalean fungi associated with T. pilifer have not been reported. In this study, we aimed to clarify the taxonomic status of T. pilifer and identify ophiostomatalean fungi associated with T. pilifer infesting P. koraiensis in northeastern China. In total, 315 ophiostomatalean fungal strains were isolated from 62 adults of T. pilifer and 220 tissue samples from T. pilifer galleries in Jilin Province. Thirty-five representative strains were further identified by comparing their morphological and physiological characteristics and conducting the phylogenetic analysis of ITS, ITS2-LSU, TUB2, and TEF1-α. We identified nine species of ophiostomatalean fungi belonging to four genera, which included six novel species (Ceratocystiopsis changbaiensis sp. nov., Leptographium linjiangense sp. nov., Leptographium qieshaoense sp. nov., Ophiostoma piliferi sp. nov., Ophiostoma tonghuaense sp. nov., and Ophiostoma yaluense sp. nov.), two previously described species (Graphilbum interstitiale and Ophiostoma fuscum), and one undefined specie (Ceratocystiopsis sp. 1). To the best of our knowledge, this is the first report of G. interstitiale and O. fuscum in China and the fungal diversity of ophiostomatalean in T. pilifer. The dominant species were O. piliferi and L. qieshaoense, representing 39.37% and 35.87% of the isolates, respectively. The results of this study provide valuable information on the symbiotic relationship between bark beetles and ophiostomatalean fungi.

Diversity of Ophiostomatoid Fungi Associated with Dendroctonus armandi Infesting Pinus armandii in Western China.

Pinus armandii (P. armandii) is extensively abundant in western China and, as a pioneer tree, and prominently influences local ecology. However, pine forests in this region have been significantly damaged by Dendroctonus armandi (D. armandi) infestations, in close association with ophiostomatoid fungi. This study aimed to identify the diversity of ophiostomatoid fungi associated with D. armandi infesting P. armandii in western China. A total of 695 ophiostomatoid fungal strains were isolated from 1040 tissue pieces from D. armandi galleries and 89 adult beetles at four sites. In this study, based on multiloci DNA sequence data, as well as morphological and physiological characteristics, seven species belonging to five genera were identified including three known species, Esteyea vermicola, Graphium pseudormiticum and L. wushanense, two novel taxa, Graphilbum parakesiyea and Ophiostoma shennongense, and an unidentified Ophiostoma sp. 1. A neotype of Leptographium qinlingense. Ophiostoma shennongense was the dominant taxon (78.99%) in the ophiostomatoid community. This study provides a valuable scientific theoretical basis for the occurrence and management of D. armandi in the future.

Diversity of fungi associated with Monochamusalternatus larval habitats in Bursaphelenchusxylophilus-infected Pinusmassoniana and identification of two new ophiostomatalean species (Ascomycota, Ophiostomatales).

Bursaphelenchusxylophilus, 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 Monochamusalternatus 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 2nd-3rd and 4th-5th instar larvae, respectively. A total of 18 species of fungi were identified, 14 species were isolated from the 2nd-3rd instar larval galleries and six species from the galleries and pupal chambers of the 4th-5th instar larvae. Amongst them were six species belonging to four genera of ophiostomatalean fungi, including two novel species, Graphilbumxianjuensis sp. nov. and Ophiostomataizhouense sp. nov. and four known species, Ceratocystiopsisweihaiensis, Ophiostomaips, Sporothrixzhejiangensis and S.macroconidia. The findings revealed that the fungal diversity and abundance of the 2nd-3rd instar larvae differed markedly from those of the 4th-5th 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.

Ophiostomatoid species associated with pine trees (Pinus spp.) infested by Cryphaluspiceae from eastern China, including five new species.

Cryphaluspiceae attacks various economically important conifers. Similar to other bark beetles, Cr.piceae plays a role as a vector for an assortment of fungi and nematodes. Previously, several ophiostomatoid fungi were isolated from Cr.piceae in Poland and Japan. In the present study, we explored the diversity of ophiostomatoid fungi associated with Cr.piceae infesting pines in the Shandong Province of China. We isolated ophiostomatoid fungi from both galleries and beetles collected from our study sites. These fungal isolates were identified using both molecular and morphological data. In this study, we recovered 175 isolates of ophiostomatoid fungi representing seven species. Ophiostomaips was the most frequently isolated species. Molecular and morphological data indicated that five ophiostomatoid fungal species recovered were previously undescribed. Thus, we proposed these five novel species as Ceratocystiopsisyantaiensis, C.weihaiensis, Graphilbumtranslucens, Gr.niveum, and Sporothrixvillosa. These new ophiostomatoid fungi add to the increasing number of fungi known from China, and this evidence suggests that numerous novel taxa are awaiting discovery in other forests of China.

Ophiostomatoid fungi associated with pine bark beetles and infested pines in south-eastern Australia, including Graphilbum ipis-grandicollis sp. nov.

The ophiostomatoid fungi are an assemblage of ascomycetes which are arguably best-known for their associations with bark and ambrosia beetles (Curculonidae) and blue stain (sap stain) of many economically important tree species. These fungi are considered a significant threat to coniferous forests, which has resulted in numerous studies characterising the diversity of bark beetles and their ophiostomatoid associates globally. The diversity of ophiostomatoid fungi present in Australian pine plantations, however, remains largely undetermined. The aims of this study were therefore to reconsider the diversity of ophiostomatoid fungi associated with Pinus in Australia, and to establish the baseline of expected taxa found within these plantation ecosystems. To achieve this, we reviewed Australian plant pathogen reference collections, and analysed samples collected during forest health surveillance programs from the major pine growing regions in south-eastern Australia. In total, 135 ophiostomatoid isolates (15 from reference collections and 120 collected during the current study) were assessed using morphological identification and ITS screening which putatively distinguished 15 taxonomic groups. Whole genome sequencing (WGS) of representative isolates from each taxon was performed to obtain high-quality sequence data for multi-locus phylogenetic analysis. Our results revealed a greater than expected diversity, expanding the status of ophiostomatoid fungi associated with Pinus in Australia to include 14 species from six genera in the Ophiostomatales and a single species residing in the Microascales. While most of these were already known to science, our study includes seven first records for Australia and the description of one new species, Graphilbum ipis-grandicollis sp. nov.. This study also provides an early example of whole genome sequencing (WGS) approaches replacing traditional PCR-based methods for taxonomic surveys. This not only allowed for robust multi-locus sequence extraction during taxonomic assessment, but also permitted the rapid establishment of a curated genomic database for ophiostomatoid fungi which will continue to aid in the development of improved diagnostic resources and capabilities for Australian biosecurity.

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 Pinusyunnanensis 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 ß-tubulin (TUB2), elongation factor 1α (TEF1-α) 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, Esteyavermicola, Leptographiumyunnanense, Ophiostomabrevipilosi, O.canum, O.minus and O.tingens and four novel taxa, described as Graphilbumanningense, O.aggregatum, Sporothrixpseudoabietina 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; Ophiostomacanum 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.