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Summary Drought predisposes forest trees to bark beetle‐induced mortality, but the physiological mechanisms remain unclear. While drought‐induced water and carbon limitations have been implicated in defensive failure and tree susceptibility, evidence demonstrating how these factors interact is scarce. We withheld water from mature, potted Pinus edulis and subsequently applied a double‐stem girdle to inhibit carbohydrate transport from the crown and roots. Within this isolated segment we then elicited a defense response by inoculating trees with a bark beetle‐fungal symbiont (Ophiostoma sp.). We quantified local mono‐ and sesquiterpenes (MST), nonstructural carbohydrates (NSC), and pressure potential of the inner bark. Both drought‐stressed and watered trees had similar NSC concentrations just before inoculation and depleted NSC similarly following inoculation, yet MST induction (i.e. increased concentration and altered composition) was constrained only in drought‐stressed trees. Thus, NSC consumption was largely unrelated to de novo MST synthesis. Instead, stoichiometric calculations show that induction originated largely from stored resin. Watered trees experiencing higher pressure potentials consistently induced higher MST concentrations. We demonstrate the importance of preformed resin toward an induced MST response in a semi‐arid conifer where drought‐constraints on defense occurred through biophysical limitations (i.e. reduced turgor hindering resin transport) rather than through substrate limitation. [ABSTRACT FROM AUTHOR]

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ABSTRACTAmerican elm (Ulmus americana), highly prized for its ornamental value, has suffered two successive outbreaks of Dutch elm disease (DED) caused by ascomycete fungi belonging to the genus Ophiostoma. To identify the genes linked to the pathogenicity of different species and lineages of Ophiostoma, we inoculated 2-year-old U. americana saplings with six strains representing three species of DED fungi, and one strain of the saprotroph Ophiostoma quercus. Differential expression analyses were performed following RNA sequencing of fungal transcripts recovered at 3- and 10-days post-infection. Based on a total of 8,640 Ophiostoma genes, we observed a difference in fungal gene expression depending on the strain inoculated and the time of incubation in host tissue. Some genes overexpressed in the more virulent strains of Ophiostoma encode hydrolases that possibly act synergistically. A mutant of Ophiostoma novo-ulmi in which the gene encoding the ogf1 transcription factor had been deleted did not produce transcripts for the gene encoding the hydrophobin cerato-ulmin and was less virulent. Weighted gene correlation network analyses identified several candidate pathogenicity genes distributed among 13 modules of interconnected genes.IMPORTANCEOphiostoma is a genus of cosmopolitan fungi that belongs to the family Ophiostomataceae and includes the pathogens responsible for two devastating pandemics of Dutch elm disease (DED). As the mechanisms of action of DED agents remain unclear, we carried out the first comparative transcriptomic study including representative strains of the three Ophiostoma species causing DED, along with the phylogenetically close saprotrophic species Ophiostoma quercus. Statistical analyses of the fungal transcriptomes recovered at 3 and 10 days following infection of Ulmus americana saplings highlighted several candidate genes associated with virulence and host-pathogen interactions wherein each strain showed a distinct transcriptome. The results of this research underscore the importance of investigating the transcriptional behavior of different fungal taxa to understand their pathogenicity and virulence in relation to the timeline of infection.

Fungal pathogens associated with bark beetles constitute one of the most significant problems to forest health. The Turkish pine (Pinus brutia Ten.) is a native species in the forests of Türkiye and occurs in the Mediterranean-type climate. The Southern Marmara is a natural occurrence area of Turkish pine in the Marmara Region. In the present study, trap logs were set up in pure Pinus brutia forests to investigate fungi associated with Orthotomicus erosus (Wollaston) (Mediterranean pine beetle) throughout Southern Marmara. Orthotomicus erosus adults, larvae, and their galleries were sampled and individually cultured on a 1% CSMA (cycloheximide–streptomycin malt agar) medium. Three ophiostomatoid fungi were identified using morphological characteristics and molecular genetic analyses: Ceratocystis (syn. Ophiostoma) ips (Rumbold) C. Moreau, Graphilbum sp. H.P. Upadhyay & W.B. Kendr., and Leptographium wingfieldii M. Morelet. All three species were new in records of the fungal flora of Türkiye. The most dominant of these species, Ceratocystis ips was isolated 69%. Unexpectedly, L. wingfieldii had a high-frequency association with O. erosus (27%). The pathogenicity tests showed that all three species could cause lesions on branches of Turkish pine but were non-pathogenic or weak pathogenic.

Vascular wilts are important diseases caused by plant pathogenic fungi that result in the rapid death of their plant hosts. This is due to a systemic defense mechanism whereby the plant induces the compartmentalization of the infected vascular system in order to reduce the propagation of the fungus. The ascomycete class Sordariomycetes contains several species that cause vascular wilts in diverse plant hosts, and they can be classified into four taxonomic orders. The genetic mechanisms of pathogenesis have already been investigated in Fusarium and Verticillium species, but they have not yet been compared with other well-known wilt-causing species, especially fungi causing oak wilt or Dutch elm disease (DED). Here we analyzed 20 whole genome assemblies of wilt-causing fungi together with 56 other species using phylogenetic approaches to trace expansions and contractions of orthologous gene families and gene classes related to pathogenicity. We found that the wilt-causing pathogens evolved seven times, experiencing the largest fold changes in different classes of genes almost every time. However, some similarities exist across groups of wilt pathogens, particularly in Microascales and Ophiostomatales, and these include the common gains and losses of genes that make up secondary metabolite clusters (SMC). DED pathogens do not experience large-scale gene expansions, with most of the gene classes, except for some SMC families, reducing in number. We also found that gene family expansions in the most recent common ancestors of wilt pathogen groups are enriched for carbohydrate metabolic processes. Our study shows that wilt-causing species evolve primarily through distinct changes in their repertoires of pathogenicity-related genes and that there is the potential importance of carbohydrate metabolism genes for regulating osmosis in those pathogens that penetrate the plant vascular system. [ABSTRACT FROM AUTHOR]

In the past 25 to 30 years, a marked increase has occurred in numbers of invasive alien pests and pathogens recorded, damaging agriculture, horticulture and forest environments. The reasons for this upsurge are clear: all involve human-driven factors, with, arguably, global trade as the most important component in the complex. Climate change is another major factor. Each plant transported through trade could carry cryptic pests or pathogens, which may be introduced into previously naïve environments and lead to major health issues on previously unaffected plants. Global trade in plants is a complex system, driven by a desire to use large plants in landscapes and demand for varied species for gardens. Examples of invasive alien pathogens causing wide-spread problems on a global scale currently include many Phytophthora species, Dothistroma needle blight of pines, and Xylella fastidiosa on a wide range of trees. A striking example recently was the spread of the Dutch elm disease pathogen, Ophiostoma novo-ulmi, in North America and Western Europe. Many invasive pathogens cause problems in localized regions, including Ceratocystis platani, killing Platanus in some European countries. Other examples include the threat of pine wilt nematode (Bursaphelenchus xylophilus), damage to Cupressus in Mediterranean landscapes caused by Seiridium cardinale, dieback of sweet chestnut (Castanea) caused by the canker pathogen Cryphonectria parasitica, and many others. In addition to global trade, climate change appears to be exacerbating the problems, with major influences on establishment and spread of alien invasive species. Hope lies in the enormous genetic ‘power’ of plants: humans could establish selection and breeding programmes that will ultimately enable us to plant trees with greater tolerance of changing climate and pests and pathogens, whether native or invasive.

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-a. 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. [ABSTRACT FROM AUTHOR]

Abstract 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.

Resistance to the antibiotic Cycloheximide has been reported for a number of fungal taxa. In particular, some yeasts are known to be highly resistant to this antibiotic. Early research showed that this resulted from a transition mutation in one of the 60S ribosomal protein genes. In addition to the yeasts, most genera and species in the Ophiostomatales are highly resistant to this antibiotic, which is widely used to selectively isolate these fungi. Whole-genome sequences are now available for numerous members of the Ophiostomatales providing an opportunity to determine whether the mechanism of resistance in these fungi is the same as that reported for yeast genera such as Kluyveromyces. We examined all the available genomes for the Ophiostomatales and discovered that a transition mutation in the gene coding for ribosomal protein eL42, which results in the substitution of the amino acid Proline to Glutamine, likely confers resistance to this antibiotic. This change across all genera in the Ophiostomatales suggests that the mutation arose early in the evolution of these fungi. [ABSTRACT FROM AUTHOR]

Oak decline presenting symptoms of tracheomycosis have been observed globally over long periods of time. Since the 1990s, oak decline has been considered to be a multifactorial process where abiotic predisposing factors play a significant role. Nevertheless, biotic factors, such as subcortical insects and fungal pathogens, may influence the decline process, as some insect species transmit spores of tracheomycotic pathogens. We investigated biotic agents (subcortical insects, Ophiostoma spp. and Phytophthora spp.) in six declining oak stands located within mesophytic and thermophytic zones in the Czech Republic, where five dry years occurred from 2015 to 2019. The spectrum of insect pests of oak stands was investigated using two methods: reared from logs from each stand and window traps. The presence of Ophiostoma fungi was assessed from cultivated woodblocks; Phytophthora pathogens were isolated from soil samples. In total, 2931 subcortical insect individuals were captured during the 2021 vegetation period. The most species-rich group of the subcortical insect complex involved in oak decline consisted of the families Cerambycidae: Xylotrechus antilope; Curculionidae: Scolytus intricatus, Xyleborus monographus; and Buprestidae: Agrilus biguttatus, A. sulcicollis. The presence of Ophiostoma was detected in all stands with different intensities between tree parts. Several important oak pests were positively correlated with the occurrence of Ophiostoma. On the contrary, from all soil samples, Phytophthora plurivora was found at only one studied oak stand. Based on the results, a new modified model of biotic and abiotic factors involved in the oak decline is proposed. [ABSTRACT FROM AUTHOR]

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 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, 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 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. [ABSTRACT FROM AUTHOR]

Simple Summary: The presence of the oak pinhole borer, the insect Platypus cylindrus, in Portuguese cork oak stands has drastically increased in the past few decades. This beetle excavates long galleries in the trunk while inoculating fungi (called ambrosia fungi) transported in special organs (mycangia) that will serve as food source for its offspring. The combined action of extensive boring into the heartwood and the inoculation of fungi leads to an increase in tree mortality. A new ambrosial fungus, Ceratocystiopsis quercina, was isolated from the insects' mycangia and from wilting trees, namely from the staining patches it causes on wood. Platypus cylindrus is the most common ambrosia beetle in stands of Quercus suber in Portugal. This insect farms specialized fungi in sapwood galleries, using its mycangia to carry and store these organisms. Some ectosymbiotic fungi carried by P. cylindrus are phytopathogenic and cause extensive tree mortality and severe economic losses. To understand the role of P. cylindrus fungal symbionts in stands of Q. suber we examined beetle galleries present in declining and/or dying cork oak trees during field surveys. Logs with active galleries were obtained in situ and from captured emerging beetles. Insects were aseptically dissected, and their mycangia and intestine were retrieved. Morphological and molecular profiles of fungal isolates obtained from cultured insect parts were carried out to accurately characterize and identify isolated fungi. Molecular characterizations were performed with DNA sequence data from four loci, i.e., LSU, SSU, 5.8S-ITS2-28S, and TUB. Morphological results consistently showed a collection of Ophiostoma-like fungal axenic isolates, while phylogenies inferred that this collection constitutes an undescribed taxon reported herein for the first time in association with P. cylindrus in Portuguese cork oak stands. The novel species was erected as Ceratocystiopsis quercina sp. nov. and constitutes a new phytopathogenic fungal species associated with symptoms of vegetative cork oak decline. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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, β-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.

Vascular wilts are important diseases caused by plant pathogenic fungi that result in the rapid death of their plant hosts. This is due to a systemic defense mechanism whereby the plant induces the compartmentalization of the infected vascular system in order to reduce the propagation of the fungus. The ascomycete class Sordariomycetes contains several species that cause vascular wilts in diverse plant hosts, and they can be classified into four taxonomic orders. The genetic mechanisms of pathogenesis have already been investigated in Fusarium and Verticillium species, but they have not yet been compared with other well-known wilt-causing species, especially fungi causing oak wilt or Dutch elm disease (DED). Here we analyzed 20 whole genome assemblies of wilt-causing fungi together with 56 other species using phylogenetic approaches to trace expansions and contractions of orthologous gene families and gene classes related to pathogenicity. We found that the wilt-causing pathogens evolved seven times, experiencing the largest fold changes in different classes of genes almost every time. However, some similarities exist across groups of wilt pathogens, particularly in Microascales and Ophiostomatales, and these include the common gains and losses of genes that make up secondary metabolite clusters (SMC). DED pathogens do not experience large-scale gene expansions, with most of the gene classes, except for some SMC families, reducing in number. We also found that gene family expansions in the most recent common ancestors of wilt pathogen groups are enriched for carbohydrate metabolic processes. Our study shows that wilt-causing species evolve primarily through distinct changes in their repertoires of pathogenicity-related genes and that there is the potential importance of carbohydrate metabolism genes for regulating osmosis in those pathogens that penetrate the plant vascular system.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Some fungal endophytes of forest trees are recognized as beneficial symbionts against stresses. In previous works, two elm endophytes from the classes Cystobasidiomycetes and Eurotiomycetes promoted host resistance to abiotic stress, and another elm endophyte from Dothideomycetes enhanced host resistance to Dutch elm disease (DED). Here, we hypothesize that the combined effect of these endophytes activate the plant immune and/or antioxidant system, leading to a defense priming and/or increased oxidative protection when exposed to the DED pathogen Ophiostoma novo-ulmi. To test this hypothesis, the short-term defense gene activation and antioxidant response were evaluated in DED-susceptible (MDV1) and DED-resistant (VAD2 and MDV2.3) Ulmus minor genotypes inoculated with O. novo-ulmi, as well as two weeks earlier with a mixture of the above-mentioned endophytes. Endophyte inoculation induced a generalized transient defense activation mediated primarily by salicylic acid (SA). Subsequent pathogen inoculation resulted in a primed defense response of variable intensity among genotypes. Genotypes MDV1 and VAD2 displayed a defense priming driven by SA, jasmonic acid (JA), and ethylene (ET), causing a reduced pathogen spread in MDV1. Meanwhile, the genotype MDV2.3 showed lower defense priming but a stronger and earlier antioxidant response. The defense priming stimulated by elm fungal endophytes broadens our current knowledge of the ecological functions of endophytic fungi in forest trees and opens new prospects for their use in the biocontrol of plant diseases. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

In the 1970s, clones of the two subspecies of Ophiostoma novo-ulmi, subsp. americana (SSAM) and subsp. novo-ulmi (SSNU) began to overlap in Europe, resulting in hybrid swarms. By 1983–1986, hybrids with high, SSAM-like growth and pathogenic fitness comprised ~75% of popula-tions at Limburg, Netherlands and Orvieto, Italy. We resampled these populations in 2008 to examine trends in hybrid fitness traits. Since preliminary sampling in 1979–1980, MAT-1 locus frequency had increased from ~0% to ~32% at Orvieto and 5% to ~43% at Limburg, and vegeta-tive incompatibility type frequency had changed from near clonal to extremely diverse at both sites. This represents an enormous increase in outcrossing and recombination potential, due in part to selective acquisition (under virus pressure) of MAT-1 and vic loci from the resident O. ulmi and in part to SSAM × SSNU hybridisation. Overt virus infection in the 2008 samples was low (~4%), diagnostic SSAM and SSNU cu and col1 loci were recombinant, and no isolates exhib-ited a parental SSAM or SSNU colony pattern. At both sites, mean growth rate and mean patho-genicity to 3–5 m clonal elm were high SSAM-like, indicating sustained directional selection for these characters, though at Orvieto growth rate was slower. The once frequent SSNU-specific up-mut colony dimorphism was largely eliminated at both sites. Perithecia formed by Limburg isolates were mainly an extreme, long-necked SSNU-like form, consistent with transgressive segregation resulting from mismatch of SSAM and SSNU developmental loci. Orvieto isolates produced more parental-like perithecia, suggesting the extreme phenotypes may have been se-lected against. The novel phenotypes in the swarms are remodelling O. novo-ulmi in Europe. Locally adapted genotypes may emerge. [ABSTRACT FROM AUTHOR]

Pine bark beetles are typically associated with complexes of fungi that could reveal different functional interaction. Thus, previously nonaggressive bark beetle Ips acuminatus is considering now to be among the most serious pests of pine forest in Ukraine and other European countries and vectored fungal community is very important to assess total harm of this bark beetle. The aim of this study was to reveal the vectored fungal community associated with the pine engraver beetle, I. acuminatus with special emphasis on pathogenic fungi for further evaluation of harm bark-beetle - fungi association for Ukrainian forest. In total, 288 adult beetles were collected from Scots pine trees at six different sites through Ukraine. DNA sequencing as fungal culturing from all beetles resulted in 1681 isolates and amplicons representing 42 fungal taxa. NCBI BLAST search revealed that the overall fungal community was composed of 94 species, of which 80.85% were Ascomycota, followed by Basidiomycota and unidentified fungal group, which accounted for 10.6% and 8.5 % of the total sequences, respectively. Among these, the most commonly detected fungi for pooling dataset were Sphaeropsis sapinea (23.6%), Cladosporium pini-ponderosae (19.44%), Ophiostoma ips (19.1%), Ophiostoma canum (19.1%) and Cladobotryum mycophilum (18.06%). In the pooled dataset of isolates and amplicons for each site, Shannon diversity indices ranged between 1.9 and 2.9 while Simpson diversity index varied between 0.69 and 0.89 indicating rich species diversity. In total twelve ophiostomatoid species were detected. All ophiostomatoid fungi were showing varying degrees of virulence and O. minus was the most aggressive fungus in previous studies. It is concluded that I. acuminatus vectors a species-rich fungal community including pathogens such as ophiostomatoid fungi, Sphaeropsis sapinea, different needle pathogens and wood decay fungi that seems to be very important for the assessment of threat of I. acuminatus to the pine forest in Ukraine.

Oak decline presenting symptoms of tracheomycosis have been observed globally over long periods of time. Since the 1990s, oak decline has been considered to be a multifactorial process where abiotic predisposing factors play a significant role. Nevertheless, biotic factors, such as subcortical insects and fungal pathogens, may influence the decline process, as some insect species transmit spores of tracheomycotic pathogens. We investigated biotic agents (subcortical insects, Ophiostoma spp. and Phytophthora spp.) in six declining oak stands located within mesophytic and thermophytic zones in the Czech Republic, where five dry years occurred from 2015 to 2019. The spectrum of insect pests of oak stands was investigated using two methods: reared from logs from each stand and window traps. The presence of Ophiostoma fungi was assessed from cultivated woodblocks; Phytophthora pathogens were isolated from soil samples. In total, 2931 subcortical insect individuals were captured during the 2021 vegetation period. The most species-rich group of the subcortical insect complex involved in oak decline consisted of the families Cerambycidae: Xylotrechus antilope; Curculionidae: Scolytus intricatus, Xyleborus monographus; and Buprestidae: Agrilus biguttatus, A. sulcicollis. The presence of Ophiostoma was detected in all stands with different intensities between tree parts. Several important oak pests were positively correlated with the occurrence of Ophiostoma. On the contrary, from all soil samples, Phytophthora plurivora was found at only one studied oak stand. Based on the results, a new modified model of biotic and abiotic factors involved in the oak decline is proposed.

Fungi assigned to the Ophiostomatales are of economic concern as many are blue-stain fungi and some are plant pathogens. The mitogenomes of two blue-stain fungi, Ophiostoma minus and Ophiostoma piliferum, were sequenced and compared with currently available mitogenomes for other members of the Ophiostomatales. Species representing various genera within the Ophiostomatales have been examined for gene content, gene order, phylogenetic relationships, and the distribution of mobile elements. Gene synteny is conserved among the Ophiostomatales but some members were missing the atp9 gene. A genome wide intron landscape has been prepared to demonstrate the distribution of the mobile genetic elements (group I and II introns and homing endonucleases) and to provide insight into the evolutionary dynamics of introns among members of this group of fungi. Examples of complex introns or nested introns composed of two or three intron modules have been observed in some species. The size variation among the mitogenomes (from 23.7 kb to about 150 kb) is mostly due to the presence and absence of introns. Members of the genus Sporothrix sensu stricto appear to have the smallest mitogenomes due to loss of introns. The taxonomy of the Ophiostomatales has recently undergone considerable revisions; however, some lineages remain unresolved. The data showed that genera such as Raffaelea appear to be polyphyletic and the separation of Sporothrix sensu stricto from Ophiostoma is justified.

Platypus cylindrus is the most common ambrosia beetle in stands of Quercus suber in Portugal. This insect farms specialized fungi in sapwood galleries, using its mycangia to carry and store these organisms. Some ectosymbiotic fungi carried by P. cylindrus are phytopathogenic and cause extensive tree mortality and severe economic losses. To understand the role of P. cylindrus fungal symbionts in stands of Q. suber we examined beetle galleries present in declining and/or dying cork oak trees during field surveys. Logs with active galleries were obtained in situ and from captured emerging beetles. Insects were aseptically dissected, and their mycangia and intestine were retrieved. Morphological and molecular profiles of fungal isolates obtained from cultured insect parts were carried out to accurately characterize and identify isolated fungi. Molecular characterizations were performed with DNA sequence data from four loci, i.e., LSU, SSU, 5.8S-ITS2-28S, and TUB. Morphological results consistently showed a collection of Ophiostoma-like fungal axenic isolates, while phylogenies inferred that this collection constitutes an undescribed taxon reported herein for the first time in association with P. cylindrus in Portuguese cork oak stands. The novel species was erected as Ceratocystiopsis quercina sp. nov. and constitutes a new phytopathogenic fungal species associated with symptoms of vegetative cork oak decline.

Fungi assigned to the Ophiostomatales are of economic concern as many are blue-stain fungi and some are plant pathogens. The mitogenomes of two blue-stain fungi, Ophiostoma minus and Ophiostoma piliferum , were sequenced and compared with currently available mitogenomes for other members of the Ophiostomatales. Species representing various genera within the Ophiostomatales have been examined for gene content, gene order, phylogenetic relationships, and the distribution of mobile elements. Gene synteny is conserved among the Ophiostomatales but some members were missing the atp9 gene. A genome wide intron landscape has been prepared to demonstrate the distribution of the mobile genetic elements (group I and II introns and homing endonucleases) and to provide insight into the evolutionary dynamics of introns among members of this group of fungi. Examples of complex introns or nested introns composed of two or three intron modules have been observed in some species. The size variation among the mitogenomes (from 23.7 kb to about 150 kb) is mostly due to the presence and absence of introns. Members of the genus Sporothrix sensu stricto appear to have the smallest mitogenomes due to loss of introns. The taxonomy of the Ophiostomatales has recently undergone considerable revisions; however, some lineages remain unresolved. The data showed that genera such as Raffaelea appear to be polyphyletic and the separation of Sporothrix sensu stricto from Ophiostoma is justified. [ABSTRACT FROM AUTHOR]

Dutch elm is a fungal infection affecting elm trees. This plant disease is caused by two types of related fungus, namely “Ophiostoma novo-ulmi and Ophiostoma ulmi”. This disease is mainly transmitted through elm bark beetles and root grafts. Dutch elm affects the water transportation of plants and eventually leads to plant death. This bibliometric analysis had been conducted to understand the active authors, organizations, journals, and countries involved in the research domain of “Dutch elm”. All published articles related to “leaf blister” from “Scopus”, were analyzed using the VOS viewer to develop analysis tables and visualization maps. This article had set the objective to consolidate the literature regarding the “Dutch elm” and also to find out the trends related to the same. The most active journals related to this plant disease are Mycological Research, Mycopathologia, and Nature. The highly active countries in this research domain are the USA, the United Kingdom, and India. The top organizations focusing on the Dutch elm disease are the Forest Research Station of the United Kingdom and Department of Botany and Plant Pathology of Michigan State University. The most active author who had made valuable contributions related to Dutch elm disease are Brasier C.M., Davelos A.L, Jarosz A.M, and Gil L. [ABSTRACT FROM AUTHOR]

Bacterial leaf spot creates a dark spot on leaves and can even kill the leaves. However, this disease can affect the stems, seeds, and fruits. This disease is caused by the pathogen variants “Xanthomonas“ and “Pseudomonas”. Wet and cool conditions; overcrowded plants poor soil drainage are favorable conditions for the spread of this disease. This bibliometric analysis had been conducted to understand the active authors, organizations, journals, and countries involved in the research domain of “Bacterial leaf spot”. All published articles related to “Bacterial leaf spot” from “Scopus”, were analyzed using the VOS viewer to develop analysis tables and visualization maps. This article had set the objective to consolidate the literature regarding the “Bacterial leaf spot” and also to find out the trends related to the same. The most active journals related to this plant disease are ISME Journal and Plant Disease. The USA is the country where active research is going on regarding Bacterial leaf spot, followed by Spain. The top organizations focusing on Bacterial leaf spot are the Department of Plant Pathology of the University of Florida, the Department of Plant Sciences, and the Department of Plant Pathology of the University of California. The most active author who had made valuable contributions related to Bacterial leaf spot are Jones J.B., Coaker G.L., Leveau J.H.J., Rastogi G., Sbodio A., Suslow T.V., Tech J.J. [ABSTRACT FROM AUTHOR]

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.

1. All bark beetles are in symbiosis with fungi. Although obligate mutualisms with fungi are common with tree‐killing bark beetles (primaries), fungi associated with non‐tree‐killing bark beetles (secondaries) are usually dismissed as commensals. 2. Using an ecological stoichiometric approach, we show secondaries are also involved in nutrition‐based mutualisms, some of which appear obligate, and that differences in symbiont provisioning efficiency have a potent effect on beetle carbon (C): nitrogen (N): phosphorus (P) ratios. 3. Some secondary beetles have high P contents and require efficient P provisioning via fungi, while others have low P contents that may allow them to exploit less efficient fungi or a broader range of species with variable efficiencies. A co‐occurring scavenger that feeds on nutrient‐poor bark beetle frass (excrement/boring residues) exhibited the lowest phosphorus content yet recorded for an invertebrate. 4. Our results generally support the growth‐rate hypothesis that posits differences in C:P and N:P ratios in consumers are due to differential allocation of P to P‐rich RNA to support growth. However, while the beetle species that accumulated the most biomass was considerably enriched in P and that with the least biomass was P‐poor, one beetle species that was P‐rich was also small possibly due to limitation by an element other than P. 5. Our results indicate that fungi are important to a broader range of bark beetles than previously recognised. Additional research is needed to describe how these various symbioses influence forest ecosystems via differential effects of fungi on host beetle fitness. [ABSTRACT FROM AUTHOR]

Conifer bark beetles are well known to be associated with fungal complexes, which consist of pathogenic ophiostomatoid fungi as well as obligate saprotroph species. However, there is little information on fungi associated with Ips acuminatus in central and eastern Europe. The aim of the study was to investigate the composition of the fungal communities associated with the pine engraver beetle, I. acuminatus, in the forest-steppe zone in Ukraine and to evaluate the pathogenicity of six associated ophiostomatoid species by inoculating three-year-old Scots pine seedlings with these fungi. In total, 384 adult beetles were collected from under the bark of declining and dead Scots pine trees at two different sites. Fungal culturing from 192 beetles resulted in 447 cultures and direct sequencing of ITS rRNA from 192 beetles in 496 high-quality sequences. Identification of the above revealed that the overall fungal community was composed of 60 species. Among these, the most common were Entomocorticium sp. (24.5%), Diplodia pinea (24.0%), Ophiostoma ips (16.7%), Sydowia polyspora (15.1%), Graphilbum cf rectangulosporium (15.1%), Ophiostoma minus (13.8%) and Cladosporium pini-ponderosae (13.0%). Pathogenicity tests were done using six species of ophiostomatoid fungi, which were inoculated into Scots pine seedlings. All ophiostomatoid fungi tested successfully infected seedlings of Scots pine with varying degrees of virulence. Ophiostoma minus was the only fungus that caused dieback in inoculated seedlings. It is concluded that I. acuminatus vectors a species-rich fungal community including pathogens such as D. pinea and O. minus. The fungal community reported in the present study is different from that reported in other regions of Europe. Pathogenicity tests showed that O. minus was the most virulent causing dieback in seedlings of Scots pine, while other fungi tested appeared to be only slightly pathogenic or completely non-pathogenic.

American elm ( Ulmus americana ), highly prized for its ornamental value, has suffered two successive outbreaks of Dutch elm disease (DED) caused by ascomycete fungi belonging to the genus Ophiostoma . To identify the genes linked to the pathogenicity of different species and lineages of Ophiostoma , we inoculated 2-year-old U. americana saplings with six strains representing three species of DED fungi, and one strain of the saprotroph Ophiostoma quercus . Differential expression analyses were performed following RNA sequencing of fungal transcripts recovered at 3- and 10-days post-infection. Based on a total of 8,640 Ophiostoma genes, we observed a difference in fungal gene expression depending on the strain inoculated and the time of incubation in host tissue. Some genes overexpressed in the more virulent strains of Ophiostoma encode hydrolases that possibly act synergistically. A mutant of Ophiostoma novo-ulmi in which the gene encoding the ogf1 transcription factor had been deleted did not produce transcripts for the gene encoding the hydrophobin cerato-ulmin and was less virulent. Weighted gene correlation network analyses identified several candidate pathogenicity genes distributed among 13 modules of interconnected genes.IMPORTANCE Ophiostoma is a genus of cosmopolitan fungi that belongs to the family Ophiostomataceae and includes the pathogens responsible for two devastating pandemics of Dutch elm disease (DED). As the mechanisms of action of DED agents remain unclear, we carried out the first comparative transcriptomic study including representative strains of the three Ophiostoma species causing DED, along with the phylogenetically close saprotrophic species Ophiostoma quercus . Statistical analyses of the fungal transcriptomes recovered at 3 and 10 days following infection of Ulmus americana saplings highlighted several candidate genes associated with virulence and host-pathogen interactions wherein each strain showed a distinct transcriptome. The results of this research underscore the importance of investigating the transcriptional behavior of different fungal taxa to understand their pathogenicity and virulence in relation to the timeline of infection., Competing Interests: The authors declare no conflict of interest.

Wounds on trees provide entrance for many members of the Ophiostomatales (Ascomycota), including economically damaging tree pathogens. Because very little is known regarding wound‐associated fungi on hardwoods in Europe or their potential nitidulid beetle vectors (Coleoptera), the aim of this survey was to explore the diversity of these fungi in Poland. Wound samples associated with 26 tree species were collected from 18 stands in Poland. Fungi belonging to the Ophiostomatales were isolated and identified based on morphology and DNA sequence comparisons of five gene regions, combined with phylogenetic analyses. In total, 32 taxa were identified, including 20 known and 12 currently undescribed species. In addition, Grosmannia cainii, Ophiostoma cf. ponderosae, O. sparsiannulatum and Sporothrix aurorae were reported for the first time outside of the USA. The wound‐associated members of the Ophiostomatales recovered from hardwoods showed high diversity and specificity. Among the species recovered, 16 belonged to Sporothrix, 11 represented Ophiostomas.l., five could be assigned to Leptographiums.l., and one Graphilbum species was detected. Among the studied tree species, wounds on Quercus robur appeared to be the most vulnerable to infection by members of the Ophiostomatales. All the nitidulid beetle species were found in association with species assignable to Ophiostoma and Sporothrix. This study detected a significant association between the occurrence of species belonging to the Ophiostomatales and the health status of the wounded trees. Some of the isolated species were host specific although Ophiostoma quercus, the most frequently encountered species, had a broad host range. [ABSTRACT FROM AUTHOR]

The first pandemic of Dutch elm disease (DED) in Europe and North America caused by the introduced Ophiostoma ulmi began in the early 1900s but declined unexpectedly in Europe from the 1930s onwards after killing 30–40% of the elms. Later a second pandemic caused by the much more aggressive Ophiostoma novo‐ulmi spread across the same areas and by 1990 most of Britain's c. 30 million mature elms had died. During the second pandemic, O. novo‐ulmi acquired debilitating viruses and changed from being largely clonal to highly heterogeneous through horizontal transfer of novel, sometimes deleterious, genes from O. ulmi. In the post‐epidemic period a new disease dynamic has emerged, with millions of small recruitment elms repeatedly attacked by sequential cycles of disease. This study investigated the possibility of pathogenic attenuation in O. novo‐ulmi as the epidemic has progressed. Isolates collected from the three original outbreak areas in Britain in 1982–3 (early post‐epidemic period) and 2011 (advanced post‐epidemic) were compared for two fitness components: pathogenic aggressiveness and in vitro growth rate. Mean aggressiveness of the 2011 isolates to clonal English elm (Ulmus procera) proved not to differ significantly from that of the 1982 isolates, either overall or within each outbreak area. Similarly, the mean growth rates of the 1982 and 2011 samples showed no differences. The implications of these findings are discussed in relation to the future of the current DED pandemic in Europe, the decline of the first pandemic, and the difficulty of drawing parallels between different tree–pathogen systems. [ABSTRACT FROM AUTHOR]

Fungal volatile compounds can mediate fungal-insect interactions. Whether fungi can emit insect pheromones and how volatile chemicals change in response to chemicals the fungi naturally encounter is poorly understood. We analyzed volatiles emitted by Grosmannia clavigera (symbiont of the mountain pine beetle) and Ophiostoma ips (symbiont of the pine engraver beetle) growing in liquid media amended with compounds that the fungi naturally encounter: (−)-α-pinene, (+)-α-pinene, (−)- trans -verbenol, verbenone, or ipsdienol. Nine volatile compounds were identified among the fungal and amendment treatments. Volatiles qualitatively and quantitatively differed between fungal species and among amendment treatments. The bark beetle anti-aggregation pheromone (−)-verbenone was detected from both fungi growing in (−)- trans -verbenol-amended media. G. clavigera and O. ips can emit beetle pheromones and other beetle semiochemicals, suggesting that ophiostomatoid fungi could contribute to the chemical ecology of bark beetles. However, such contributions could be modulated by the presence of other environmental chemicals. [ABSTRACT FROM AUTHOR]

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 ß-tubulin (TUB2), elongation factor 1a (TEF1-a) 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. [ABSTRACT FROM AUTHOR]

Highlights • Raffaelea lauricola collected from Taiwan was compared to R. lauricola (RL570) and 7 other species. • R. lauricola & R. aguacate had a larger number of ABC transporters, P450s and proteases. • Three distinct clusters formed: Rl - Ra ; Rq - m - Rq - Gc ; and Ophiostma. • Most R. lauricola proteins were orthologous to R. aguacate , but non-orthologous were also found. • A diversity of secondary metabolite synthesis clusters was found and some were species specific. Abstract Raffaelea lauricola is an invasive fungal pathogen and symbiont of the redbay ambrosia beetle (Xyleborus glabratus) that has caused widespread mortality to redbay (Persea borbonia) and other Lauraceae species in the southeastern USA. We compare two genomes of R. lauricola (C2646 and RL570) to seven other related Ophiostomatales species including R. aguacate (nonpathogenic close relative of R. lauricola), R. quercus-mongolicae (associated with mortality of oaks in Korea), R. quercivora (associated with mortality of oaks in Japan), Grosmannia clavigera (cause of blue stain in conifers) , Ophiostoma novo-ulmi (extremely virulent causal agent of Dutch elm disease), O. ulmi (moderately virulent pathogen that cause of Dutch elm disease), and O. piceae (blue-stain saprophyte of conifer logs and lumber). Structural and functional annotations were performed to determine genes that are potentially associated with disease development. Raffaelea lauricola and R. aguacate had the largest genomes, along with the largest number of protein-coding genes, genes encoding secreted proteins, small-secreted proteins, ABC transporters, cytochrome P450 enzymes, CAZYmes, and proteases. Our results indicate that this large genome size was not related to pathogenicity but was likely lineage specific, as the other pathogens in Raffaelea (R. quercus-mongolicae and R. quercivora) had similar genome characteristics to the Ophiostoma species. A diverse repertoire of wood-decaying enzymes were identified in each of the genomes, likely used for toxin neutralization rather than wood degradation. Lastly, a larger number of species-specific, secondary metabolite, synthesis clusters were identified in R. lauricola suggesting that it is well equipped as a pathogen, which could explain its success as a pathogen of a wide range of lauraceous hosts. [ABSTRACT FROM AUTHOR]

Abstract The bark beetle Ips typographus carries numerous fungi that could be assisting the beetle in colonizing live Norway spruce (Picea abies) trees. Phenolic defenses in spruce phloem are degraded by the beetle's major tree-killing fungus Endoconidiophora polonica , but it is unknown if other beetle associates can also catabolize these compounds. We compared the ability of five fungi commonly associated with I. typographus to degrade phenolic compounds in Norway spruce phloem. Grosmannia penicillata and Grosmannia europhioides were able to degrade stilbenes and flavonoids faster than E. polonica and grow on minimal growth medium with spruce bark constituents as the only nutrients. Furthermore, beetles avoided medium amended with phenolics but marginally preferred medium colonized by fungi. Taken together our results show that different bark beetle-associated fungi have complementary roles in degrading host metabolites and thus might improve this insect's persistence in well defended host tissues. Highlights • Ips typographus -associated fungi have the ability to degrade conifer phenolics. • Grosmannia penicillata degrades major spruce phenolics most rapidly. • Efficiency of phenolic degradation varies directly with fungal virulence. • Bark beetles avoid phenolics but prefer fungus-colonized medium. [ABSTRACT FROM AUTHOR]

Bark beetle outbreaks are increasing in frequency and intensity, generating massive inventories of dead trees globally. During attacks, trees are pre-inoculated with ophiostomatoid fungi via bark beetles, which has been shown to increase termite presence and feeding. These events may, in turn, alter biogeochemical cycles during decomposition. We examined these relationships by experimentally inoculating dead wood with bluestain fungi in a temperate pine forest. Across ten replicate plots, eight 0.5 m-long logs were inoculated with Ophiostoma minus and eight with distilled water. Half of the logs from each inoculation treatment were covered from above with a mesh cage barrier to exclude aboveground beetles while permitting access by belowground decomposers. After 1 year, significant increases in mass (34%) and decreases in moisture content (− 17%) were observed across all treatments, but no consistent changes in density were evident. C concentrations were 12% greater in bark when barriers were present and 17% greater in sapwood when barriers and inoculation fungi were absent. N concentrations were 16% greater in bark for fungal-inoculated logs and 27% greater when barriers were present. C:N ratios in A horizon soils under fungal-inoculated logs were 12% greater. Furthermore, termites were present fourfold more in fungal-inoculated logs than controls and the presence of termites was associated with 6% less C in sapwood and 11% more N in both sapwood and heartwood. Together these results suggest dead wood generated via bark beetle attacks has different biogeochemical responses during initial decomposition phases, which could have implications for the C status in forests following bark beetle outbreaks. [ABSTRACT FROM AUTHOR]

Abstract Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark and ambrosia beetles (Curculionidae: Scolytinae, Platypodinae). Fungi in the Ophiostomatales include serious tree pathogens as well as agents of timber blue-stain. Although these fungi have been extensively studied in the northern hemisphere, very little is known regarding their occurrence on hardwoods in Europe. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with bark and ambrosia beetles infesting hardwoods in Norway and Poland, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Norwegian and Polish isolates, using multigene phylogenetic analyses. Results obtained from five gene regions (ITS, LSU, β-tubulin, calmodulin, translation elongation factor 1-α) revealed four new Ophiostoma spp. These include Ophiostoma hylesinum sp. nov., O. signatum sp. nov., and O. villosum sp. nov. that phylogenetically are positioned within the Ophiostoma ulmi complex. The other new species, Ophiostoma pseudokarelicum sp. nov. reside along with Ophiostoma karelicum in a discrete, well-supported phylogenetic group in Ophiostoma s. stricto. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on hardwoods in Europe is poorly understood and that further studies are required to enrich our knowledge about these fungi. Graphical abstract Image 1 Highlights • Four new Ophiostoma species were isolated from hardwoods in Norway and Poland. • New species were described based on morphological and molecular characters. • Ophiostoma hylesinum , O. signatum , and O. villosum are part of the Ophiostoma ulmi complex. • Ophiostoma pseudokarelicum resides in a separate lineage in Ophiostoma s. stricto. • Many undescribed ophiostomatoid fungi could be associated with hardwoods in Europe. [ABSTRACT FROM AUTHOR]

During 2017, Dutch elm disease was investigated on 67 Ulmus glabra and 29 Ulmus laevis samples from 28 sites in Latvia. The presence of Ophiostoma novo‐ulmi was detected in 61.2% of the samples from U. glabra and in 34.5% of the samples from U. laevis. In 11% of cases, hybrids between subsp. novo‐ulmi and subsp. americana were identified, particularly in the sites in the southern part of Latvia. This study presents the first verified record of Ophiostoma novo‐ulmi and its subspecies in Latvia. [ABSTRACT FROM AUTHOR]

Excessive mortality of Scots pine (Pinus sylvestris) trees has recently been observed in the Záhorská nížina lowland in western Slovakia, Central Europe. Continuous drought, bark beetles and blue-stain fungi are likely to play a crucial role in this dieback of pines in a warm sandy area. Two blue-stain fungi, Ophiostoma ips and Ophiostoma minus, were found here on Scots pine trunks infested by bark beetles. These fungi were isolated and identified based on morphological properties and DNA sequences (ITS, LSU, β-tubulin). They colonized the inner bark, phloem and blue-stained sapwood, and were recorded in the galleries of Orthotomicus longicollis and other species of this genus. The occurrence of O. minus was massive; the fungus was found to produce perithecia abundantly in dying pines. Detailed descriptions, illustrations and global distribution of the two fungal species on host pine species are provided. The records of O. ips and those of O. minus are new for the mycoflora of Slovakia. Undetermined mycophagous nematodes were found inside the perithecia of O. ips. The role of bark beetles as real and potential vectors (Ips spp., Orthotomicus spp., Tomicus spp., Hylurgus ligniperda, Hylastes sp.) of blue-stain fungi in a declining pine forest is discussed. [ABSTRACT FROM AUTHOR]

The activity of the pine wood nematode Bursaphelenchus xylophilus leads to extremely serious economic, ecological and social losses in East Asia. The nematode causes pine wilt disease, which is currently regarded as the most important forest disease in China. The pathogenic nematode feeds on dendrocola fungi to complete its cycle of infection. As the vector of the nematode, the Japanese pine sawyer (Monochamus alternatus) also carries dendrocola fungi. Pine woods, infected by B. xylophilus and tunnelled by M. alternatus, are also inhabited by ophiostomatoid fungi. These fungi are well known for their association with many bark and ambrosia beetles. They can cause sapstain and other serious tree diseases. The aims of our study were to investigate and identify the ophiostomatoid communities associated with the epidemic pine wood nematode and the pine sawyer in Pinus massoniana and P. thunbergii forests, which are the main hosts of the pine wood nematode in China. Two hundred and forty strains of ophiostomatoid fungi were isolated from nematode and sawyer-infected trees in the coastal Shandong and Zhejiang Provinces, representing newly and historically infected areas, respectively. Six ophiostomatoid species were identified on the basis of morphological, physiological and molecular data. For the latter, DNA sequences of the internal transcribed spacer (ITS1-5.8S-ITS2) region and partial b-tubulin gene were examined. The ophiostomatoid species included one known species, Ophiostoma ips, three novel species, viz. Ophiostoma album sp. nov., Ophiostoma massoniana sp. nov. and Sporothrix zhejiangensis sp. nov. and two species whose identities are still uncertain, Ophiostoma cf. deltoideosporum and Graphilbum cf. rectangulosporium, due to the paucity of the materials obtained. The ophiostomatoid community was dominated by O. ips. This study revealed that a relatively high species diversity of ophiostomatoid fungi are associated with pine infected by B. xylophilus and M. alternatus in China. [ABSTRACT FROM AUTHOR]

Ophiostoma quercus ( Ascomycota , Ophiostomatales ) is a globally widespread, insect-vectored fungus that colonizes a wide diversity of hardwood and conifer hosts. Although the fungus is considered to be non-pathogenic, it is closely related to the fungi that cause Dutch elm disease. We examined the global diversity of O. quercus based on a ribosomal RNA marker and three unlinked gene regions. The fungus exhibited substantial morphological diversity. In addition, O. quercus had high genetic diversity in every continent from which it was collected, although the fungus was most diverse in Eurasia. There was no evidence of geographical clustering of haplotypes based on phylogenetic and network analyses. In addition, the phylogenetic trees generated based on the different markers were non-congruent. These results suggest that O. quercus has been repeatedly moved around the globe, because of trade in wood products, and that the fungal species most likely outcrosses regularly. The high genetic diversity of the fungus, as well as its ability to utilize a wide variety of arthropod vectors and colonize a tremendous diversity of tree host species makes O. quercus truly unique among ophiostomatoid fungi. [ABSTRACT FROM AUTHOR]

Ophiostoma novo-ulmi, a member of the Ophiostomatales (Ascomycota), is the causal agent of the current Dutch elm disease pandemic in Europe and North America. The complete mitochondrial genome (mtDNA) of Ophiostoma novo-ulmi subsp. novo-ulmi, the European component of O. novo-ulmi, has been sequenced and annotated. Gene order (synteny) among the currently available members of the Ophiostomatales was examined and appears to be conserved, and mtDNA size variability among the Ophiostomatales is due in part to the presence of introns and their encoded open reading frames. Phylogenetic analysis of concatenated mitochondrial protein-coding genes yielded phylogenetic estimates for various members of the Ophiostomatales, with strong statistical support showing that mtDNA analysis may provide valuable insights into the evolution of the Ophiostomatales. [ABSTRACT FROM AUTHOR]

Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of pine species in western North America. Mountain pine beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Ophiostoma montium (Rumbold) von Arx, Grosmannia aurea (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Leptographium longiclavatum (Lee, Kim, and Breuil) and Leptographium terebrantis (Barras and Perry). These fungi are vectored by MPB to new pine hosts, where the fungi overcome host defenses to grow into the sapwood. A tree's relative susceptibility to these fungi is conventionally assessed by measuring lesions that develop in response to fungal inoculation. However, these lesions represent a symptom of infection, representing both fungal growth and tree defense capacity. In order to more objectively assess fungal virulence and host tree susceptibility in studies of host-pathogen interactions, a reliable, consistent, sensitive method is required to accurately identify and quantify MPB-associated fungal symbionts in planta. We have adapted RNase H2- dependent PCR, a technique originally designed for rare allele discrimination, to develop a novel RNase H2-dependent quantitative PCR (rh-qPCR) assay that shows greater specificity and sensitivity than previously published PCR-based methods to quantify MPB fungal symbionts in pine xylem and MPB whole beetles. Two sets of assay probes were designed: one that amplifies a broad range of ophiostomoid species, and a second that amplifies G. clavigera but not other MPB-associated ophiostomoid species. Using these primers to quantify G. clavigera in pine stems, we provide evidence that lesion length does not accurately reflect the extent of fungal colonization along the stem nor the quantity of fungal growth within this colonized portion of stem. The sensitivity, specificity, reproducibility, cost effectiveness and high-throughput potential of the rh-qPCR assay makes the technology suitable for identification and quantification of a wide array of pathogenic and beneficial microbes that form associations with plants and other organisms, even when the microbial partner is present in low abundance [ABSTRACT FROM AUTHOR]

Summary: Pine plantations in Argentinian Patagonia cover ca. 95,000 ha in Chubut, Río Negro and Neuquén provinces. Exotic bark beetles (Orthotomicus laricis, Hylastes ater and Hylurgus ligniperda) commonly occur in freshly cut logs, stumps and slash. These beetles are vectors of “ophiostomatoid” fungi which include primary tree pathogens as well as important agents of blue stain. The aim of this study was to identify these beetle‐associated fungi. Sawing mills and pine plantations were surveyed three consecutive years. Fungal isolates from stained logs, processed wood and insect galleries were identified based on morphological and DNA sequence comparisons of ITS and β‐tubulin gene regions. Two Grosmannia, one Graphilbum and three Ophiostoma species were identified. Ophiostoma piliferum and O. peregrinum sp. nov. were the most frequently isolated taxa. O. peregrinum occurred in all provinces, colonizing different conifer species and, interestingly, also the native broadleaved species Nothofagus dombeyi. Pine plantation forestry in southern South America includes Argentina, Brazil, Chile, Paraguay and Uruguay. Emerging data from Argentina, Chile and Uruguay revealed some coincidences between these countries, but also several differences, probably, as a result of multiple introduction events. [ABSTRACT FROM AUTHOR]

Wilt diseases caused by vascular pathogens include some of the most damaging stresses affecting trees. Dutch elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, destroyed most of North American and European elm populations in the 20th century. The highly susceptible English elm, also known as Atinian clone, suffered the highest mortality rates during the last pandemic event, probably due to its lack of genetic diversity. To study the DED pathosystem, we inoculated English elm ramets with O. novo-ulmi and evaluated xylem anatomy, molecular response, and disease symptoms. The high DED susceptibility of the clone was linked to xylem structure. The transcript levels changed significantly for 1,696 genes during O. novo-ulmi invasion. Genes covering different steps of the plant immune system were identified, many of which showed homology with Arabidopsis thaliana genes involved in systemic acquired resistance. Induction of several pathogenesis-related proteins and repression of fasciclin-like arabinogalactan proteins and other cell wall biosynthesis pathways evidence unbalanced costs between growth and defence mechanisms far from the inoculation point. This study sheds light on elm molecular defence mechanisms against DED. [ABSTRACT FROM AUTHOR]

Five cultivars and two populations of wild-type seedlings of American elm ( Ulmus americana), 3 and 4 years old, were examined for differences in their abilities to compartmentalize and resist infection by artificially inoculating with Ophiostoma novo-ulmi. Morphological characteristics of tree defence, often referred to as the compartmentalization of decay in trees model, were used as a conceptual framework, with particular emphasis on the limiting of tangential spread of infection within the xylem and barriers that limit spread outwards to cells formed after infection. To investigate the change in functional xylem over time, 3-year-old trees were assessed at multiple time points following inoculation for hydraulic conductivity. Three and four-year-old cut trees were placed in 0.1% w/v safranin O for 18 to 24 hr to indicate functional xylem. Transverse sections of the stained stems were used to calculate per cent of sap-conducting xylem area and the per cent of circumference conducting of first formed cells and later formed cells. At each collection time, trees were assessed for disease severity on a 1-12 scale, based on the percentage of permanent wilt in the crown. There was considerable variation between cultivars in disease severity and their capacity to localize and resist infection. 'Prairie Expedition,' which had the lowest disease severity rating in 2015 and the second lowest in 2016, consistently limited the spread of infection into newly formed xylem and had functional xylem around the entire circumference of the stem at 90 days post-inoculation. 'Valley Forge' in 2016 had the lowest overall disease severity rating and was the only cultivar to consistently limit the tangential spread of infection within extant xylem. This research identifies key characteristics that some cultivars have to resist and limit infection and provides new information that can be used in disease screening programmes to evaluate other cultivars and older plant material. [ABSTRACT FROM AUTHOR]

Bark beetles (Coleoptera: Curculionidae, Scolytinae) are commonly recognised as important agents of tree mortality in coniferous forests of the Western Carpathians. They, together with weevils, are consistently associated with ophiostomatoid fungi. Information regarding conifer beetle-associated fungi in the Western Carpathians remains incomplete and unreliable, particularly with respect to fir-infesting bark beetles. This study aims to clarify associations between fungi in the genera Graphilbum, Leptographium, Ophiostoma and Sporothrix (Ophiostomatales) and their beetle vectors in Norway spruce ( Picea abies), European larch ( Larix decidua) and silver fir ( Abies alba). Samples associated with 20 bark beetle species and weevils were collected from nine stands in Poland and the Czech Republic. Fungi were isolated from adult beetles and galleries. Isolates were identified based on morphology, DNA sequence comparisons for four gene regions (ITS, LSU, ß-tubulin, TEF 1-α) and phylogenetic analyses. In total, 46 distinct taxa were identified, including 25 known and 21 currently unknown species. Several associations between fungi and subcortical insects were recorded for the first time. In addition, O. borealis and O. quercus were detected from A. alba for the first time. The composition of the fungal communities varied among the studied tree species and to a lesser degree among the beetle species. The spruce-infesting bark beetles were commonly associated with species of Leptographium s. l. and Ophiostoma s. str.; the larch-infesting bark beetles were often associated with Ophiostoma s. str. and Sporothrix, while the fir-infesting bark beetles were commonly associated with Ophiostoma s. str. and Graphilbum. The most commonly encountered fungal associates of the examined insects were (a) Grosmannia cucullata, G. piceiperda, Grosmannia sp. 1, Ophiostoma macroclavatum and O. piceae with the spruce-infesting bark beetles; (b) O. pseudocatenulatum and Sporothrix sp. 1 with the larch-infesting bark beetles; and (c) O. piceae, Ophiostoma sp. 2 and Graphilbum sp. 2 with the fir-infesting bark beetles. The differences in fungal associates among the bark beetle species occurring on P. abies, L. decidua and A. alba could be linked to the different habitats that these beetles occupy. [ABSTRACT FROM AUTHOR]