Morphological and Phylogenetic Analyses Reveal a New Species of Ceratocystiopsis (Ophiostomataceae, Ophiostomatales) Associated with Ips subelongatus in Inner Mongolia (China) with Weak Host Pathogenicity

Ophiostomatoid fungi are known for their associations with bark beetles, and some species are important sources of tree diseases. Ceratocystiopsis is a genus of the ophiostomatoid fungi in order Ophiostomatales. The shortage of DNA barcodes for many species in this genus has resulted in the presence of many unnamed cryptic species. In this study, Ceratocystiopsis subelongati sp. nov. associated with Ips subelongatus infesting Pinus sylvestris var. mongolica in Inner Mongolia, China, was identified and described based on phylogenetic inference of multi-gene DNA sequences and morphological characteristics. The species is characterized by a hyalorhinocladiella- to sporothrix-like asexual state and an optimal growth temperature of 30 °C. Artificial inoculation tests in the field showed that it is mildly pathogenic to five-year-old larch trees, the main host of I. subelongatus. It is also the first described Ceratocystiopsis species associated with I. subelongatus in China. This discovery should provide new avenues for studying the symbiosis between bark beetles and ophiostomatoid fungi.

Fungal Communities Vectored by Ips sexdentatus in Declining Pinus sylvestris in Ukraine: Focus on Occurrence and Pathogenicity of Ophiostomatoid Species

Drought-induced stress and attacks by bark beetle Ips sexdentatus currently result in a massive dieback of Pinus sylvestris in eastern Ukraine. Limited and fragmented knowledge is available on fungi vectored by the beetle and their roles in tree dieback. The aim was to investigate the fungal community vectored by I. sexdentatus and to test the pathogenicity of potentially aggressive species to P. sylvestris. Analysis of the fungal community was accomplished by combining different methods using insect, plant, and fungal material. The material consisted of 576 beetles and 96 infested wood samples collected from six sample plots within a 300 km radius in eastern Ukraine and subjected to fungal isolations and (beetles only) direct sequencing of ITS rDNA. Pathogenicity tests were undertaken by artificially inoculating three-to-four-year-old pine saplings with fungi. For the vector test, pine logs were exposed to pre-inoculated beetles. In all, 56 fungal taxa were detected, 8 exclusively by isolation, and 13 exclusively by direct sequencing. Those included nine ophiostomatoids, five of which are newly reported as I. sexdentatus associates. Two ophiostomatoid fungi, which exhibited the highest pathogenicity, causing 100% dieback and mortality, represented genera Graphium and Leptographium. Exposure of logs to beetles resulted in ophiostomatoid infections. In conclusion, the study revealed numerous I. sexdentatus-vectored fungi, several of which include aggressive tree pathogens.

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

Cryphalus piceae 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. Ophiostoma ips 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 Ceratocystiopsis yantaiensis, C. weihaiensis, Graphilbum translucens, Gr. niveum, and Sporothrix villosa. 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.

Filamentous Fungi and Yeasts Associated with Mites Phoretic on Ips typographus in Eastern Finland

The European spruce bark beetle (Ips typographus) has become a major forest pest in Finland in recent years. The beetle is a well-known vector of mainly ophiostomatoid fungi causing blue-stain of timber and pathogens that have the ability to amplify the insect damage. It also vectors other associated organisms, such as phoretic mites. The ecology of these mites remains poorly understood, including their associations with fungi. In this study, we considered filamentous fungi and yeasts associated with mites phoretic on I. typographus. Fungal identifications were based on DNA sequences and phylogenetic analyses of the ITS and/or partial β-tubulin gene regions. Fifteen fungal species were detected, including eight yeasts and seven filamentous fungi. Eleven percent of the beetles carried mites and of these 74% carried at least one fungal species. An average of two fungal species were carried per mite. The most commonly found filamentous fungi were Grosmannia penicillata (25%), Ophiostoma bicolor (19%), O. ainoae (12%) and O. brunneolum (12%). Of the yeast species, the most commonly found was Wickerhamomyces bisporus (47%). This study is the first to report yeasts associated with I. typographus and its phoretic mites in Finland. Majority of the filamentous fungal species found are those previously reported in association with I. typographus. The results also confirmed that many of the fungal species commonly found on I. typographus are also associated with its phoretic mites. However, the nature of the symbiosis between the mites, beetles and fungal associates remains to be understood.

Ophiostomatoid Fungi Associated with Cryphalus Piceae in Shandong province in eastern China

Cryphalus piceae parasitizes various economically important conifers. Similar to other bark beetles, C. picea vectors an assortment of fungi and nematodes. Previously, several ophiostomatoid fungi were isolated from C. piceae in Poland and Japan. In the present study, we explored the diversity of ophiostomatoid fungi associated with C. 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. Through this study, we recovered 176 isolates of ophiostomatoid fungi representing at least seven species. Ophiostoma ips was the most frequently isolated species. Analyses of molecular and morphological data indicated four of the ophiostomatoid fungal species recovered in this study were previously undescribed. Hereby, we described these species as Ceratocystiopsis yantaiensis sp. nov., C. weihaiensis sp. nov., Graphilbum translucens sp. nov. and Sporothrix villosa sp. nov. A majority of the ophiostomatoid fungi recovered in this study were novel species. This suggests that the forests in China harbour an assortment of undescribed ophiostomatoid fungi yet to be discovered.

Ophiostomatoid fungi including a new species associated with Asian larch bark beetle Ips subelongatus, in Heilongjiang (Northeast China)

Ips subelongatus (Coleoptera, Scolytinae) is an important bark beetle species that infests Larix spp. in Asia. Individuals of this beetle are vectors of ophiostomatoid fungi, on their exoskeletons, that are transmitted to infested trees. In this study, the symbiotic assemblage of ophiostomatoid fungi associated with I. subelongatus in Northeast China was studied. Fungal isolates were identified based on their morphological characters and sequences of ITS, beta-tubulin, elongation factor 1-alpha and calmodulin gene regions. In total, 48 isolates were collected and identified, residing in six taxa. These included a novel species, described here as Ophiostoma gmelinii sp. nov.

Ophiostomatoid fungi vectored by bark beetles and colonizing trees of Pinus sylvestris in Sumy region (Ukraine)

K. Davydenko[1], D. Baturkin[2] Intensive mortality of Pinus sylvestris trees has recently been observed in the Sumy region in eastern Ukraine. There are two pine bark beetle species (Ips acuminatus and Ips sexdentatus), which spread resulted in considerable forest damage in Ukraine. The study of ophiostomatoid fungi vectored by bark beetles is very important to assess total harm of these insects. Therefore, the aim of our research was i) to identify ophiostomatoid fungi associated with weakened and dying Scots pine trees infested by bark beetles in the Sumy region; ii) to test the pathogenicity of these ophiostomatoid fungi to evaluate their potential threat to Scots pine. The fungi were isolated from bark beetle galleries and identified based on morphological properties and DNA sequences. In total, eight ophiostomatoid fungi (Graphium sp., Grosmannia sp.1, Ophiostoma bicolor, O. ips, O. canum, O. piceae, O. minus, Ophiostoma sp.1) were isolated from Scots pine trees infested by bark beetles. Scots pine seedlings were inoculated with eight fungi and sterile medium (control) to evaluate their pathogenicity. The inoculated seedlings were examined finally in 6 month after inoculation. Inoculation with O. minus produced significantly largest lesions and only this fungus caused mortality of pine seedlings. In total, all eight fungal species inoculated caused resin exudation and staining the bark around inoculations in Scots pine seedlings and five fungi caused different rate of seedlings decline. The size of stained sapwood was also greater following O. minus inoculations than other fungi or the control. All ophiostomatoid fungi caused significantly longer necrotic lesions and more occlusions in the sapwood than the controls. Therefore, based on the ability of various ophiostomatoid fungi to weaken and kill pine seedlings and stain sapwood, O. minus was the most dangerous species for Scots pine trees, followed by Graphium sp. and Ophiostoma sp.1. The occurrence of ophiostmatoid fungi in the sapwood of Scots pine is consistent with the concept of their primary role in the colonization of the fresh sapwood of trees in the succession of microorganisms during wood decay.