Establishment of RNA Interference Genetic Transformation System and Functional Analysis of FlbA Gene in Leptographium qinlingensis.

Leptographium qinlingensis is a pathogenic fungus of Pinus armandii that is epidemic in the Qinling Mountains. However, an effective gene interference strategy is needed to characterize the pathogenic genes in this fungus on a functional level. Using the RNA silencing vector pSilent-1 as a template, we established an RNA interference genetic transformation system mediated by Agrobacterium tumefaciens GV3101, which is suitable for the gene study for Leptographium qinlingensis by homologous recombination and strain interference system screening. The LqFlbA gene was silenced using the RNA interference approach described above, and the resulting transformants displayed various levels of silencing with a gene silencing effectiveness ranging from 41.8% to 91.4%. The LqFlbA-RNAi mutant displayed altered colony morphology, sluggish mycelium growth, and diminished pathogenicity toward the host P. armandii in comparison to the wild type. The results indicate that this method provides a useful reverse genetic system for studying the gene function of L. qinlingensis, and that LqFlbA plays a crucial role in the growth, development, and pathogenicity of L. qinlingensis.

Ascarosides Promote the Prevalence of Ophiostomatoid Fungi and an Invasive Pathogenic Nematode, Bursaphelenchus xylophilus.

Understanding the coevolution of pathogens and their associated mycoflora depend upon a proper elucidation of the basis of their chemical communication. In the case of pine wilt disease, the mutual interactions between cerambycid beetles, invasive pathogenic nematodes, (Bursaphelenchus xylophilus) and their symbiotic ophiostomatoid fungi provide a unique opportunity to understand the role of small molecules in mediating their chemical communication. Nematodes produce ascarosides, a highly conserved family of small molecules that serve essential functions in nematode biology and ecology. Here we demonstrated that the associated fungi, one of the key natural food resources of pine wood nematodes, can detect and respond to these ascarosides. We found that ascarosides significantly increase the growth of L. pini-densiflorae and Sporothrix sp. 1, which are native fungal species in China that form a symbiotic relationship with pinewood nematodes. Hyphal mass of L. pini-densiflorae increased when treated with asc-C5 compared to other ophiostomatoid species. Field results demonstrated that in forests where higher numbers of PWN were isolated from beetle galleries, L. pini-densiflorae had been prevalent; the same results were confirmed in laboratory studies. Furthermore, when treated with asc-C5, L. pini-densiflorae responded by increasing its production of spores, which leads to a higher likelihood of dispersal by insect vectors, hence explaining the dominance of L. pini-densiflorae over S. sp. 1 in the Tianwang and Nanlu Mountains within the Northern Forestry Centre of China. These findings provide an emphatic representation of coevolution of pine wood nematode and its associated fungi. Our results lay a broader foundation for a better understanding of inter-kingdom mutualisms and the chemical signals that mediate their establishment.

Three new Leptographium spp. (Ophiostomatales) infecting hardwood trees in Norway and Poland.

Species of Leptographium are characterized by mononematous or synnematous conidiophores and are commonly associated with different arthropods. Some of them also produce a sexual state characterised by globose ascomata with elongated necks. Compared to investigations on coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various hardwood tree species in Norway and Poland, three unusual species, which fit in the broader morphological description of Leptographium spp., were found in association with Trypodendron domesticum, Trypodendron signatum and Dryocoetes alni, and from wounds on a variety of hardwoods. Phylogenetic analyses of sequence data for six different loci (ITS1-5.8 S-ITS2, ITS2-LSU, ACT, ß-tubulin, CAL, and TEF-1α) showed that these Leptographium species are phylogenetically closely related to the species of the Grosmannia olivacea complex. The first species forms a well-supported lineage that includes Ophiostoma brevicolle, while the two other new taxa resided in a separate lineage; possibly affiliated with Grosmannia francke-grosmanniae. All the new species produce perithecia with necks terminating in ostiolar hyphae and orange-section shaped ascospores with cucullate, gelatinous sheaths. These species also produce dark olivaceous mononematous asexual states in culture. In addition, two of the newly described species have a second type of conidiophore with a short and non-pigmented stipe. The new Leptographium species can be easily distinguished from each other by their appearance and growth in culture. Based on novel morphological characters and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium tardum sp. nov., Leptographium vulnerum sp. nov., and Leptographium flavum sp. nov. are provided.

Genetic and genomic evidence of niche partitioning and adaptive radiation in mountain pine beetle fungal symbionts.

Bark beetles form multipartite symbiotic associations with blue stain fungi (Ophiostomatales, Ascomycota). These fungal symbionts play an important role during the beetle's life cycle by providing nutritional supplementation, overcoming tree defences and modifying host tissues to favour brood development. The maintenance of stable multipartite symbioses with seemingly less competitive symbionts in similar habitats is of fundamental interest to ecology and evolution. We tested the hypothesis that the coexistence of three fungal species associated with the mountain pine beetle is the result of niche partitioning and adaptive radiation using SNP genotyping coupled with genotype-environment association analysis and phenotypic characterization of growth rate under different temperatures. We found that genetic variation and population structure within each species is best explained by distinct spatial and environmental variables. We observed both common (temperature seasonality and the host species) and distinct (drought, cold stress, precipitation) environmental and spatial factors that shaped the genomes of these fungi resulting in contrasting outcomes. Phenotypic intraspecific variations in Grosmannia clavigera and Leptographium longiclavatum, together with high heritability, suggest potential for adaptive selection in these species. By contrast, Ophiostoma montium displayed narrower intraspecific variation but greater tolerance to extreme high temperatures. Our study highlights unique phenotypic and genotypic characteristics in these symbionts that are consistent with our hypothesis. By maintaining this multipartite relationship, the bark beetles have a greater likelihood of obtaining the benefits afforded by the fungi and reduce the risk of being left aposymbiotic. Complementarity among species could facilitate colonization of new habitats and survival under adverse conditions.

Two new Leptographium spp. reveal an emerging complex of hardwood-infecting species in the Ophiostomatales.

Species of Leptographium are generally characterized by mononematous conidiophores and are commonly associated with bark beetles and weevils. These species are responsible for sapstain and in some cases serious diseases on a range of primarily coniferous trees. In comparison with coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various tree species in Norway and Poland, three unusual species, which fit the broader morphological description of Leptographium spp., were found in association with Scolytus ratzeburgi, Dryocoetes alni and Trypodendron domesticum on a variety of hardwoods, and from wounds on Tilia cordata. Phylogenetic analyses of sequence data for three gene regions (ITS2-LSU, ß-tubulin, and TEF1-α) showed that these Leptographium species are phylogenetically closely related to each other and form a well-supported lineage that included Grosmannia grandifoliae and Leptographium pruni. The first species could be distinguished from the other Leptographium species based on conidiophores arising from spiral hyphae, chlamydospore-like structures and a hyalorhinocladiella-like synanamorph in culture. The second species differs from the previous one by having distinctly shorter conidiophores and smaller conidia. This species also produces a well-developed sporothrix-like synanamorph with denticulate conidiogenous cells. Based on these unusual morphological characteristics and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium trypodendri sp. nov. and L. betulae sp. nov. are provided. The third group of isolates belonged to Grosmannia grandifoliae, representing the first report of this species outside of the USA. The newly defined G. grandifoliae complex is the first species complex in Leptographium s.l. consisting of only hardwood-infecting species.

The CYP51F1 Gene of Leptographium qinlingensis: Sequence Characteristic, Phylogeny and Transcript Levels.

Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

Modeling the pest-pathogen threats in a warming world for the red turpentine beetle (Dendroctonus valens) and its symbiotic fungus (Leptographium procerum).

BACKGROUND: Dendroctonus valens along with its symbiotic fungi have caused unprecedented damage to pines in China. Leptographium procerum, its primary symbiotic fungus, facilitates the invasion and colonization of the pest, thereby aggravating ecological threats. Assessing shifts in the niches and ranges of D. valens and its symbiotic fungus could provide a valuable basis for pest control. Here, we conducted niche comparisons between native and invasive populations of D. valens. Then, we employed standard ecological niche models and ensembles of small models to predict the potential distributions of D. valens and L. procerum under climate change conditions and to estimate areas of overlap. RESULTS: The niche of invasive population of D. valens in Chinese mainland only occupied a limited portion of the niche of native population in North America, leaving a substantial native niche unfilled and without any niche expansion. The suitable regions for D. valens are predicted in central and southern North America and central and northeastern Chinese mainland. The overlap with the suitable regions of L. procerum included eastern North America and the central and northeastern Chinese mainland under historical climatic scenarios. The regions susceptible to their symbiotic damage will shift northward in response to future climate change. CONCLUSIONS: Projected distributions of D. valens and its symbiotic fungus, along with areas vulnerable to their symbiotic damage, provide essential insights for devising strategies against this association. Additionally, our study contributes to comprehending how biogeographic approaches aid in estimating potential risks of pest-pathogen interactions in forests within a warming world. © 2024 Society of Chemical Industry.

Safety evaluation of the food enzyme phosphodiesterase I from the non-genetically modified Leptographium procerum strain FDA.

The food enzyme phosphodiesterase I (oligonucleotide 5'-nucleotidohydrolase; EC 3.1.4.1) is produced with the non-genetically modified Leptographium procerum strain FDA by DSM Food Specialties B.V. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.171 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 5848. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The mitogenomes of Leptographium aureum, Leptographium sp., and Grosmannia fruticeta: expansion by introns.

Introduction: Many members of the Ophiostomatales are of economic importance as they are bark-beetle associates and causative agents for blue stain on timber and in some instances contribute towards tree mortality. The taxonomy of these fungi has been challenging due to the convergent evolution of many traits associated with insect dispersal and a limited number of morphological characters that happen to be highly pleomorphic. This study examines the mitochondrial genomes for three members of Leptographium sensu lato [Leptographium aureum (also known as Grosmannia aurea), Grosmannia fruticeta (also known as Leptographium fruticetum), and Leptographium sp. WIN(M)1376)]. Methods: Illumina sequencing combined with gene and intron annotations and phylogenetic analysis were performed. Results: Sequence analysis showed that gene content and gene synteny are conserved but mitochondrial genome sizes were variable: G. fruticeta at 63,821 bp, Leptographium sp. WIN(M)1376 at 81,823 bp and L. aureum at 104,547 bp. The variation in size is due to the number of introns and intron-associated open reading frames. Phylogenetic analysis of currently available mitochondrial genomes for members of the Ophiostomatales supports currently accepted generic arrangements within this order and specifically supports the separation of members with Leptographium-like conidiophores into two genera, with L. aureum grouping with Leptographium and G. fruticeta aligning with Grosmannia. Discussion: Mitochondrial genomes are promising sequences for resolving evolutionary relationships within the Ophiostomatales.

Phylogenetic analyses allow species-level recognition of Leptographium wageneri varieties that cause black stain root disease of conifers in western North America.

Leptographium wageneri is a native fungal pathogen in western North America that causes black stain root disease (BSRD) of conifers. Three host-specialized varieties of this pathogen were previously described: L. wageneri var. wageneri on pinyon pines (Pinus monophylla and P. edulis); L. wageneri var. ponderosum, primarily on hard pines (e.g., P. ponderosa, P. jeffreyi); and L. wageneri var. pseudotsugae on Douglas-fir (Pseudotsuga menziesii). Morphological, physiological, and ecological differences among the three pathogen varieties have been previously determined; however, DNA-based characterization and analyses are needed to determine the genetic relationships among these varieties. The objective of this study was to use DNA sequences of 10 gene regions to assess phylogenetic relationships among L. wageneri isolates collected from different hosts. The multigene phylogenetic analyses, based on maximum likelihood and Bayesian inference, strongly supported species-level separation of the three L. wageneri varieties. These results, in conjunction with previously established phenotypic differences, support the elevation of L. wageneri var. ponderosum and L. wageneri var. pseudotsugae to the species level as L. ponderosum comb. nov. and L. pseudotsugae comb. nov., respectively, while maintaining L. wageneri var. wageneri as Leptographium wageneri. Characterization of the three Leptographium species, each with distinct host ranges, provides a baseline to further understand the ecological interactions and evolutionary relationships of these forest pathogens, which informs management of black stain root disease.

Bark Beetles Utilize Ophiostomatoid Fungi to Circumvent Host Tree Defenses.

Bark beetles maintain symbiotic associations with a diversity of microbial organisms, including ophiostomatoid fungi. Studies have frequently reported the role of ophiostomatoid fungi in bark beetle biology, but how fungal symbionts interact with host chemical defenses over time is needed. We first investigated how inoculations by three fungal symbionts of mountain pine beetle affect the terpene chemistry of live lodgepole pine trees. We then conducted a complimentary laboratory experiment specifically measuring the host metabolite degradation by fungi and collected the fungal organic volatiles following inoculations with the same fungal species on lodgepole pine logs. In both experiments, we analyzed the infected tissues for their terpene chemistry. Additionally, we conducted an olfactometer assay to determine whether adult beetles respond to the volatile organic chemicals emitted from each of the three fungal species. We found that all fungi upregulated terpenes as early as two weeks after inoculations. Similarly, oxygenated monoterpene concentrations also increased by several folds (only in logs). A large majority of beetles tested showed a strong attraction to two fungal species, whereas the other fungus repelled the beetles. Together this study shows that fungal symbionts can alter host defense chemistry, assist beetles in overcoming metabolite toxicity, and provide possible chemical cues for bark beetle attraction.

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.

Molecular Mechanism of Overcoming Host Resistance by the Target of Rapamycin Gene in Leptographium qinlingensis.

Leptographium qinlingensis is a fungal symbiont of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandii) that must overcome the terpenoid oleoresin defenses of host trees to invade and colonize. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include the decomposing and use of these compounds as a nitrogen source. Target of Rapamycin (TOR) is an evolutionarily conserved protein kinase that plays a central role in both plants and animals through integration of nutrients, energies, hormones, growth factors and environmental inputs to control proliferation, growth and metabolism in diverse multicellular organisms. In this study, in order to explore the relationship between TOR gene and carbon sources, nitrogen sources, host nutrients and host volatiles (monoterpenoids) in L. qinlingensis, we set up eight carbon source treatments, ten nitrogen source treatments, two host nutrients and six monoterpenoids (5%, 10% and 20%) treatments, and prepared different media conditions. By measuring the biomass and growth rate of mycelium, the results revealed that, on the whole, the response of L. qinlingensis to nitrogen sources was better than carbon sources, and the fungus grew well in maltose (carbon source), (NH4)2C2O4 (inorganic nitrogen source), asparagine (organic nitrogen source) and P. armandii (host nutrient) versus other treatments. Then, by analyzing the relationship between TOR expression and different nutrients, the data showed that: (i) TOR expression exhibited negative regulation in response to carbon sources and host nutrition. (ii) The treatments of nitrogen sources and terpenoids had positively regulatory effects on TOR gene; moreover, the fungus was most sensitive to ß-pinene and 3-carene. In conclusion, our findings reveal that TOR in L. qinlingensis plays a key role in the utilization of host volatiles as nutrient intake, overcoming the physical and chemical host resistances and successful colonization.

Phylogeny of Regulators of G-Protein Signaling Genes in Leptographium qinlingensis and Expression Levels of Three RGSs in Response to Different Terpenoids.

Leptographium qinlingensis is a bark beetle-vectored pine pathogen in the Chinese white pine beetle (Dendroctonus armandi) epidemic in Northwest China. L. qinlingensis colonizes pines despite the trees' massive oleoresin terpenoid defenses. Regulators of G-protein signaling (RGS) proteins modulate heterotrimeric G-protein signaling negatively and play multiple roles in the growth, asexual development, and pathogenicity of fungi. In this study, we have identified three L. qinlingensis RGS genes, and the phylogenetic analysis shows the highest homology with the regulators of G-protein signaling proteins sequence from Ophiostoma piceae and Grosmannia clavigera. The expression profiles of three RGSs in the mycelium of L. qinlingensis treated with six different terpenoids were detected, as well as their growth rates. Under six terpenoid treatments, the growth and reproduction in L. qinlingensis were significantly inhibited, and the growth inflection day was delayed from 8 days to 12-13 days. By analyzing the expression level of three RGS genes of L. qinlingensis with different treatments, results indicate that LqFlbA plays a crucial role in controlling fungal growth, and both LqRax1 and LqRgsA are involved in overcoming the host chemical resistances and successful colonization.

Abundance and Diversity of Ophiostomatoid Fungi Associated With the Great Spruce Bark Beetle (Dendroctonus micans) in the Northeastern Qinghai-Tibet Plateau.

The role of several virulent tree pathogens in host death has been overlooked because of the aggressiveness of their associated bark beetles. The great spruce bark beetle (Dendroctonus micans) is a widely distributed beetle that infests coniferous plants in Eurasia; however, its associated fungi have been poorly studied. Therefore, in this study, we elucidated the diversity of ophiostomatoid fungi associated with D. micans in the northeastern Qinghai-Tibet Plateau through field investigation, laboratory isolation, and culture analyses. A total of 220 strains of ophiostomatoid fungi were isolated from adults and tunnel galleries of D. micans infesting Picea crassifolia. We identified that the isolated strains belonged to eight ophiostomatoid species, including five new species (Ophiostoma huangnanense sp. nov., Ophiostoma maixiuense sp. nov., Ophiostoma sanum sp. nov., Leptographium sanjiangyuanense sp. nov., and Leptographium zekuense sp. nov.), one undefined species (Ophiostoma sp. 1), and two known species (Ophiostoma bicolor and Endoconidiophora laricicola), using phylogenetic analysis of multigene DNA sequences and morphological characteristics. This is the first time that E. laricicola, a pioneer invader and virulent pathogen, has been reported in China. We found that E. laricicola was the dominant species, accounting for 40.91% of the total number of ophiostomatoid communities. This study enriched the knowledge of the fungal associates of D. micans and elucidated that it carried the virulent pathogen E. laricicola at a surprisingly high frequency. Our findings show increased species association between D. micans and ophiostomatoid fungi and provide a basis for understanding the occurrence of forest diseases and pests.

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.

Interspecific variation in spruce constitutive and induced defenses in response to a bark beetle-fungal symbiont provides insight into traits associated with resistance.

Differences in defensive traits of tree species may predict why some conifers are susceptible to bark beetle-fungal complexes and others are not. A symbiotic fungus (Leptographium abietinum (Peck) M.J. Wingf.) associated with the tree-killing bark beetle (Dendroctonus rufipennis Kirby) is phytopathogenic to host trees and may hasten tree decline during colonization by beetles, but defense responses of mature trees to the fungus have not been experimentally examined. To test the hypothesis that interspecific variation in spruce resistance is explained by defense traits we compared constitutive (bark thickness and constitutive resin ducts) and induced defenses (resin flow, monoterpene composition, concentration, phloem lesion formation and traumatic resin ducts) between two sympatric spruces: Engelmann spruce (Picea engelmannii Parry ex Engelm.-a susceptible host) and blue spruce (Picea pungens Engelm.-a resistant host) in response to fungal inoculation. Four central findings emerged: (i) blue spruce has thicker outer bark and thinner phloem than Engelmann spruce, which may restrict fungal access to phloem and result in less beetle-available resource overall; (ii) both spruce species induce monoterpenes in response to inoculation but blue spruce has higher constitutive monoterpene levels, induces monoterpenes more rapidly, and induces higher concentrations over a period of time consistent with spruce beetle attack duration; (iii) Engelmann and blue spruce differed in the monoterpenes they upregulated in response to fungal inoculation: blue spruce upregulated α-pinene, terpinolene and γ-terpinene, but Engelmann spruce upregulated 3-carene and linalool; and (iv) blue spruce has a higher frequency of constitutive resin ducts and produces more traumatic resin ducts in annual growth increments than Engelmann spruce, though Engelmann spruce produces more resin following aseptic wounding or fungal inoculation. These findings suggest that higher constitutive resin duct densities and monoterpene concentrations, as well as the ability to rapidly induce specific monoterpenes in response to L. abietinum inoculation, are phenotypic traits associated with hosts resistant to spruce beetle colonization.

Fungal Interactions and Host Tree Preferences in the Spruce Bark Beetle Ips typographus.

The spruce bark beetle Ips typographus is the most damaging pest in European spruce forests and has caused great ecological and economic disturbances in recent years. Although native to Eurasia, I. typographus has been intercepted more than 200 times in North America and could establish there as an exotic pest if it can find suitable host trees. Using in vitro bioassays, we compared the preference of I. typographus for its coevolved historical host Norway spruce (Picea abies) and two non-coevolved (naïve) North American hosts: black spruce (Picea mariana) and white spruce (Picea glauca). Additionally, we tested how I. typographus responded to its own fungal associates (conspecific fungi) and to fungi vectored by the North American spruce beetle Dendroctonus rufipennis (allospecific fungi). All tested fungi were grown on both historical and naïve host bark media. In a four-choice Petri dish bioassay, I. typographus readily tunneled into bark medium from each of the three spruce species and showed no preference for the historical host over the naïve hosts. Additionally, the beetles showed a clear preference for bark media colonized by fungi and made longer tunnels in fungus-colonized media compared to fungus-free media. The preference for fungus-colonized media did not depend on whether the medium was colonized by conspecific or allospecific fungi. Furthermore, olfactometer bioassays demonstrated that beetles were strongly attracted toward volatiles emitted by both con- and allospecific fungi. Collectively, these results suggest that I. typographus could thrive in evolutionary naïve spruce hosts if it becomes established in North America. Also, I. typographus could probably form and maintain new associations with local allospecific fungi that might increase beetle fitness in naïve host trees.

Ophiostomatoid fungi associated with Ips subelongatus, including eight new species from northeastern China.

Ips subelongatus is a major pest that infects larch plantations over large areas of northern and northeastern China. Ips species are closely associated with ophiostomatoid fungi that are morphologically well-adapted for dispersal by beetles. These associations result in important threat for coniferous forests worldwide. The aim of this study was to characterize the ophiostomatoid communities associated with I. subelongatus infesting Larix species and sympatric Pinus sylvestris var. mongolica in northeastern China forests. Morphological and multilocus phylogenetic approaches (based on six markers: ITS, LSU, 60S, ß-tubulin, EF-1α, and CAL gene regions) allowed identifying 14 species of four genera (Ceratocystiopsis, Endoconidiophora, Leptographium and Ophiostoma). Eight species are showed to be new to science. Most strains resided in two Ophiostoma species complexes, viz. the O. clavatum and the O. ips complexes, all together accounting for 76.8% of all isolates. Ophiostoma hongxingense sp. nov., O. peniculi sp. nov., and O. subelongati sp. nov. (O. clavatum complex) and O. pseudobicolor sp. nov. (O. ips complex) were the four dominant species. The ophiostomatoid communities associated with larch bark beetles, I. cembrae and I. subelongatus, in Europe and Asia, China and Japan, also were compared. These comparisons showed distinct, specific assemblage patterns.