Ophiostomatoid Fungi Research Articles

Chalaropsis pruni sp. nov. (Ceratocystidaceae), a new species from Prunus serrulata var. pubescens in South Korea

A routine survey in a natural forest located in the Gangwon Province of Korea was undertaken in 2022 to establish an inventory of potentially pathogenic fungi that might affect tree health in the country. An ophiostomatoid fungus was consistently isolated from naturally occurring wounds on Prunus serrulata var. pubescens. Isolates were subjected to morphological and DNA sequence comparisons. The results showed that the fungus resided in a distinct taxonomic lineage, and the novel species is described here as Chalaropsis pruni sp. nov.

Three Ophiostomatalean Fungi Associated with Bark Beetles from Pinus thunbergii Infested by Bursaphelenchus xylophilus in Laoshan Mountain (Shandong, China)

Pine wilt disease (PWD) is a devastating disease that occurs worldwide and affects conifers infested by the pine wood nematode (PWN, Bursaphelenchus xylophilus). PWD has caused serious economic and ecological losses in China. The mechanism of disease outbreak is complex, with the associated fungi, specifically ophiostomatoid fungi, thought to play an essential role. However, few ophiostomatoid fungal associates of PWD have been accurately identified. In the present study, we isolated fungi from bark beetles collected from Pinus thunbergii infested by the pine wood nematode on Laoshan Mountain, Shandong province. Three ophiostomatalean fungi were identified and assigned to Graphilbum and Ophiostoma based on phylogenetic analyses and comparison of morphological and cultural features, namely Gra. laoshanense sp. nov., Gra. translucens, and O. ips. This study increases the understanding of the diversity of ophiostomatoid fungi associated with PWD and provides resources for parsing this complex disease.

New Ophiostoma species associated with Dryocoetes himalayensis and decline of Juglans regia in Czechia

AbstractA new ophiostomatoid fungus involved in the decline of walnuts was discovered in Czechia. The fungus was isolated from necrotic wood surrounding the larval galleries of Dryocoetes himalayensis, the alien bark beetle attacking walnut trees. This insect species is rather unique within hardwood-associated bark beetle species for its aggressiveness and ability to attack apparently healthy trees. The isolated fungus, associated with an exotic insect vector is described as a new species, Ophiostoma juglandis sp. nov., based on its unique morphology and phylogenetic position. The pathogenicity tests confirmed the aggressiveness of the species to both Juglans regia and J. nigra plants, manifested by formation of extensive bleeding and necrotic lesions. The symbiotic relationship between O. juglandis and D. himalayensis is expected due to massive presence of the fungal spores on surface of adult beetles in pits forming the elytral striae. In the case of hardwood-associated ophiostomatoid fungal species, phytopathogenicity is very rarely observed. The described species is exceptional in this ability, which corresponds to the aggressive behaviour of its vector. The role of the fungus in walnut tree mortality caused by D. himalayensis is discussed.

Pathogenicity and induced resistance in Larix kaempferi and Larix olgensis inoculated with Endoconidiophora fujiensis.

With climate warming and economic globalization, insect-microbe assemblages are becoming increasingly responsible for various devastating forest diseases worldwide. Japanese larch (Larix kaempferi) is extensively cultivated in China because of its high survival rate, rapid maturation and robust mechanical properties. Endoconidiophora fujiensis, an ophiostomatoid fungus associated with Ips subelongatus, has been identified as a lethal pathogen of L. kaempferi in Japan. However, there is a dearth of research on the pathogenicity of E. fujiensis in larches in China. Therefore, we investigated the pathogenicity of E. fujiensis in introduced L. kaempferi and indigenous larch (Larix olgensis) trees and compared the induced resistance responses to the pathogen in both tree species in terms of physiology and gene expression. Five-year-old saplings and 25-year-old adult trees of L. olgensis and L. kaempferi were inoculated in parallel during the same growing season. Endoconidiophora fujiensis exhibited high pathogenicity in both larch species, but particularly in L. kaempferi compared with L. olgensis adult trees; adult L. olgensis was more resistant to E. fujiensis than adult L. kaempferi, which was reflected in higher accumulation of defensive monoterpenes, such as myrcene, 3-carene and limonene and the earlier induction of defense genes catalase (CAT) and pathogenesis-related protein 1 (PR1). This study contributes to our understanding of the interactions between bark beetle-associated ophiostomatoid fungi and host larches, from phenotypic responses to alterations in secondary metabolites via defense- and metabolism-related gene activation, providing a valuable foundation for the management of larch diseases and pests in the future.

Pathophysiology and transcriptomic responses of Pinus armandii defenses to ophiostomatoid fungi.

Pinus armandii Franch. is an ecologically and economically important evergreen tree species native to western China. Dendroctonus armandi Tsai and Li and pathogenic ophiostomatoid fungi pose substantial threats to P. armandii. With the interplay between species, the defense mechanisms of P. armandii have evolved to withstand external biotic stressors. However, the interactions between P. armandii and pathogenic ophiostomatoid fungal species/strains remain poorly understood. We aimed to analyze the pathophysiological and molecular changes in P. armandii following artificial inoculation with four ophiostomatoid species (Graphilbum parakesiyea, Leptographium qinlingense, Ophiostoma shennongense and Ophiostoma sp. 1). The study revealed that L. qinlingense produced the longest necrotic lesions, and G. parakesiyea produced the shortest. All strains induced monoterpenoid release, and monoterpene levels of P. armandii were positively correlated with fungal virulence (R2=0.93, P<0.01). Co-inoculation of two dominant highly (L. qinlingense) and weakly virulent (O. shennongense) pathogens reduced the pathogenicity of the highly virulent fungi. Transcriptomic analysis of P. armandii (LQ: L. qinlingense treatments, QS: co-inoculation treatments and OS: O. shennongense treatments) showed that the expression pattern of differentially expressed genes (DEGs) between QS and OS was similar, but different from that of LQ. The DEGs (LQ vs QS) involved in flavonoid biosynthesis and phenylpropanoid biosynthesis were downregulated. Notably, compared with LQ, QS significantly decreased the expression of host defense-related genes. This study provides a valuable theoretical basis for managing infestations of D. armandi and associated ophiostomatoid fungi.

Evidence that Ophiostomatoid Fungal Symbionts of Mountain Pine Beetle Do Not Play a Role in Overcoming Lodgepole Pine Defenses During Mass Attack.

Mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins) is a devastating forest insect pest that has killed millions of hectares of pines in western North America over the past two decades. Like other bark beetles, MPB vectors ophiostomatoid fungal species, some of which are pathogenic to host pine species. The phytopathogenicity of these fungal symbionts has sparked considerable debate regarding their role in facilitating MPB attack success. We tested the hypothesis that MPB ophiostomatoid fungal associates like Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield contribute to overwhelming host defenses during MPB mass attack. We compared responses of mature lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) trees growing in natural stands that were mass attacked by MPB with those inoculated with G. clavigera by examining host defense hormones, secondary metabolites, and gene expression profiles. The jasmonate and ethylene signatures of necrotrophic pathogen-triggered response were identified in G. clavigera-inoculated trees, but only the jasmonate signature of a herbivore-triggered response was measured in MPB-attacked trees. Several G. clavigera-induced changes in pine phenolic metabolite profiles and phenolic biosynthesis gene expression patterns were absent in MPB-attacked pines. These findings indicate that ophiostomatoid fungi like G. clavigera are not a major factor in overwhelming host defenses during MPB mass attack. Instead, fungal pathogenicity likely is more important in aiding MPB colonization and development within the host tree. Phenolics appear to play a larger role in the host response to G. clavigera than to MPB, although phenolics may also influence MPB feeding and behavior. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Field effects of oxygenated monoterpenes and estragole combined with pheromone on attraction of Ips typographus and its natural enemies

IntroductionCentral European Norway spruce monocultures face Ips typographus outbreaks due to decreasing resistance. These beetles use volatile compounds to communicate and select suitable host trees. Spruce trees, beetles, and their symbiotic ophiostomatoid fungi emit oxygenated monoterpenes, including 1,8-cineole, α-terpineol, camphor, carvone, terpinen-4-ol, isopinocamphone, and pinocamphone, and the phenylpropanoid estragole, particularly in the infestation phase. These compounds trigger strong responses in I. typographus antennae, motivating our field study.ObjectiveThis study aimed to assess how adding these compounds to the aggregation pheromone of Ips typographus modulates the attraction of this bark beetle and its natural enemies.MethodsIn combination with I. typographus pheromone, estragole, 1,8-cineole, (±)-camphor, (–)-carvone, alpha-terpineol, (–)-terpinen-4-ol, (+)-pinocamphone, and (+)-isopinocamphone at low, medium, and high doses were tested in pheromone traps at two sites in the Czech Republic.ResultsAll 1,8-cineole doses and the high estragole dose acted as anti-attractants for I. typographus, whereas all (+)-isopinocamphone doses enhanced their attraction to pheromone. Catches of natural enemies, the Staphylinidae and Pteromalidae, varied by location.Conclusion1,8-cineole, isopinocamphone, and estragole may play vital roles in tritrophic interactions among spruce trees, and I. typographus and its natural enemies, and these compounds may be developed into new or enhanced semiochemical-based pest control methods.

Ophiostomatatoid fungi (Ascomycota) associated with Ips acuminatus (Coleoptera) in eastern Poland

Ips acuminatus (Coleoptera, Scolytinae) is a pine-infesting bark beetle that occurs throughout Europe and Asia. Recently, the insect has killed numerous Scots pines (Pinus sylvestris) in eastern Poland. Several species of ophiostomatoid fungi are associated with I. acuminatus in Europe, but no research has been done on the fungi associated with this bark beetle in Central Europe specifically. The aim of this study was to identify the ophiostomatoid fungal associates of I. acuminatus in eastern Poland, where tree mortality caused by this beetle species has recently increased. Field surveys in Puławy and Mircze Forest Districts yielded a total of 2 269 fungal isolates from 237 beetles and 204 beetle galleries. Isolates were grouped based on morphology and representatives of each group were identified based on DNA sequences of the ITS, LSU, β-tubulin, calmodulin and elongation factor 1-α gene regions. A total of seven previously described species of ophiostomatoid fungi were identified. The dominant species were Graphilbum acuminatum and Sporothrix pseudoabietina. This study revealed that the community of ophiostomatoid fungi associated with I. acuminatus in Poland is different from those reported in other regions of Europe. In addition, molecular data suggest that S. pseudoabietina is a synonym of S. villosa in the Sporothrix gossypina &amp; S. stenoceras species complexes.

Features of Scots Pine Mortality Due to Incursion of Pine Bark Beetles in Symbiosis with Ophiostomatoid Fungi in the Forest-Steppe of Central Siberia

Forest decline is a significant issue affecting critical ecosystem processes worldwide. Here, we describe mortality in Pinus sylvestris L. monitored trees caused by the inhabitation of pine bark beetles (Tomicus minor Hart.) in symbiosis with ophiostomatoid fungi (Ophiostoma piceae (Munch) H. et P. Sydow) infection in the forest-steppe of central Siberia. Stem sap flow (Q) and stem diameter fluctuations (dRc) were monitored in eight pine trees during seven consecutive growing seasons (2015–2021). In addition, microcore sampling every ten days allowed the determination of stem wood formation in monitored trees in the 2021 growing season. During 2020 and 2021, two cases of Q termination were recorded among the monitored trees, with microcores revealing no cambium formation. Thus, the seasonal Q onset matches the beginning of the beetle dispersal period when they attack and inhabit tree stems. The decline of circumferential stem size began 10–12 days after Q onset, during the massive inhabitation of beetles into the stems. The disturbance of Q in trees occurred in 21–23 days, and total cessation of Q was observed 23–26 days after the Q onset at the beetle’s egg development phase. The timing of dRc disturbance and Q cessation observed directly coincides with the beetle life cycle. Thus, the phenology of pine trees and T. minor beetles is driven by seasonal weather conditions, particularly the cumulative air temperature (&gt;0 °C).

Filamentous fungal associates of the alder bark beetle, Alniphagus aspericollis, including an undescribed species of Neonectria.

Bark beetles (Coleoptera: Curculionidae; Scolytinae) are tree-infesting insects that consume subcortical tissues and fungi. Species capable of killing their host trees are most commonly associated with conifers, as very few bark beetle species infest and kill hardwood hosts directly. The alder bark beetle, Alniphagus aspericollis, is a hardwood-killing bark beetle that colonizes and kills red alder, Alnus rubra. Conifer-killing bark beetles have well-known associations with symbiotic ophiostomatoid fungi that facilitate their life histories, but it is unknown whether A. aspericollis has any fungal associates. This study was conducted to identify any consistent filamentous fungal associates of A. aspericollis and characterize the consistency of observed beetle-fungus relationships. Beetles and gallery phloem samples were collected from seven sites throughout the Greater Vancouver region in British Columbia, Canada. Filamentous fungi were isolated from these samples and identified by DNA barcoding using the internal transcribed spacer (ITS) region and other barcode regions for resolution to the species-level for the most dominant isolates. The most common fungal associate was a previously undescribed Neonectria major-like fungus, Neonectria sp. nov., which was isolated from ~67% of adult beetles, ~59% of phloem samples, and ~94% of the beetle-infested trees. Ophiostoma quercus was isolated from ~28% of adult beetles, ~9% of phloem samples, and ~56% of infested trees and deemed a casual associate of A. aspericollis, while a putatively novel species of Ophiostoma was more infrequently isolated from A. aspericollis and its galleries. Cadophora spadicis, a new record for red alder, was rarely isolated and is probably coincidentally carried by A. aspericollis. Overall, A. aspericollis was only loosely associated with ophiostomatoid fungi, suggesting that these fungi have little ecological significance in the beetle-tree interaction, while Neonectria sp. nov. may be a symbiote of A. aspericollis that is vectored by the beetle.

Ophiostomatální houby v porostech borovice lesní (Pinus sylvestris L.) postižených dlouhodobým suchem

In this study, presence of ophiostomatiod fungi (absence, presence) on wood cross-section samples (total 72) from freshly cut Scots pine (Pinus sylvestris L.) trees (total 24) at four sites (Valtice, Příšťpo, Brodce, Vrbová Lhota) affected by long-term drought was evaluated in relation to: sampling period (spring, summer, autumn), tree age (30–40, 50–90 years), wood sample origin (bottom trunk, middle trunk, twigs), tree defoliation (&gt;25–60%, &gt;60–99%), and presence of other taxa. Ophiostomatoid fungi were present on 33% wood cross-section samples from 50% trees. Presence of ophiostomatoid fungi was significantly different in relation to sampling period (i.e. the highest in autumn, the lowest in spring), and was directly proportional to both presence of mites and Nematocera larvae; it was insignificantly different in relation to site, tree age, wood sample origin, defoliation, and presence of other taxa. These results show: (1) frequent presence of ophiostomatoid fungi in wood tissues of Scots pine trees, (2) insignificant effect of ophiostomatoid fungi on health status of the trees, (3) relationship of presence of ophiostomatoid fungi with both presence of mites and Nematocera larvae.

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

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

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.

The occurrence and pathogenicity of fungi associated with Orthotomicus erosus on Pinus brutia in the Southern Marmara, Türkiye

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 &amp; 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.

Sušenje i umanjenje stabilnosti prestarelih stabala Populus × euramericana u urbanim sredinama - studija slučaja Železnički park, Novi Sad

Populus × euramericana is a species characterized by fast growth and simple vegetative propagation and is often planted in the parks of Novi Sad. During 2017, dieback of P. x euroamericana was observed in the Železnički Park in Novi Sad. The dieback continued in the following years, the leaning of the trees was noticed as well as mushrooms of the poplar rot fungus Cyclocybe aegerita growing in clusters at tree bases. Thus in October 2022, the static stability and internal structure of P. × euramericana wood was checked using semi-destructive micro-drilling method and IML PD 400 resistograph and the different stages of the structural erosion of the wood were observed. Ophiostomatoid fungi, fusarioid fungi and C. aegerita were isolated from symptomatic wood samples. Molecular phylogenetic analyzes confirmed that the fruiting bodies at tree bases belong to the species C. aegerita. Frequent climate extremes along with other stressful factors in urban areas make poplar trees sensitive to windstorms, windbreaks, and attack of opportunistic pathogens. Therefore, it is essential to conduct intensive monitoring of health and stability of poplar trees in urban areas and this should be done using resistance measurement devices for the detection of wood decay like resistograph. This instrument provides insight into the tree structure and the possibility of timely reaction to make city parks a safe and pleasant place for people to rest and relax.

Molecular characterization of a novel endornavirus isolated from Ophiostoma bicolor associated with bark beetles.

Ophiostoma bicolor is a pathogenic fungus associated with bark beetles that can cause serious damage to host plants. In this study, a novel fungal virus, "Ophiostoma bicolor endornavirus 1" (ObEV1), was obtained from O. bicolor, and its complete genome sequence was determined. ObEV1 has a single-stranded positive-sense (+ ss) RNA genome of 10,119 nucleotides. Sequence annotation and comparison showed that the viral genome has a single large open reading frame (ORF) encoding a polyprotein of 362.48 kDa. The polyprotein contains seven conserved domains: RNA-dependent RNA polymerase (RdRp), viral RNA helicase 1 (VHel1), viral methyltransferase (VMet), DEAD-like helicase (DEXDc), gliding-GltJ (G1), large tegument protein UL36 (PHA), and YlqF-related-GTPase (Y). Sequence comparisons and phylogenetic analysis showed that ObEV1 is a novel mycovirus belonging to the genus Betaendornavirus of the family Endornaviridae. This is the first report of a mycovirus in the ophiostomatoid fungus O. bicolor.

Ophiostomatoid fungi synergize attraction of the Eurasian spruce bark beetle, Ips typographus to its aggregation pheromone in field traps.

Eurasian spruce bark beetle, Ips typographus is a destructive pest of the Norway spruce (Picea abies). Recent outbreaks in Europe have been attributed to global warming and other anthropogenic impacts. Bark beetles are guided by multiple complex olfactory cues throughout their life cycle. Male-produced aggregation pheromones, comprising 2-methyl-3-buten-2-ol and cis-verbenol, have been identified as the most powerful attractants for dispersing conspecifics. In addition to host trees, bark beetles interact with multiple organisms, including symbiotic ophiostomatoid fungi, which may promote beetle colonization success and offspring development. Previously, in a short-distance laboratory assay, we demonstrated that I. typographus adults are attracted to the volatile organic compounds (VOCs) produced by three symbiotic fungi: Grosmannia penicillata, Endoconidiophora polonica, and Leptographium europhioides. Furthermore, the abundant fusel alcohols and their acetates were found to be the most attractive odorants in the fungal VOC profile. In this study, using a long-distance field-trapping experiment, we analyzed the role of fungal VOCs as attractants for dispersing I. typographus. Two types of fungal lures were tested in combination with pheromones in traps: (1) live cultures of fungi grown on potato dextrose agar (PDA) and (2) dispensers containing synthetic fusel alcohols and their acetates in equal proportions. Subsequently, the composition of VOCs emitted from live fungal lures were analyzed. We found that the symbiotic fungi synergistically increased the attraction of beetles to pheromones in field traps and the attractiveness of live fungal lures depended on the fungal load. While one Petri dish with E. polonica, when combined with pheromones synergistically increased trapping efficiency, three Petri dishes with L. europhioides were required to achieve the same. The synthetic mix of fungal fusel alcohols and acetates improved the catch efficiency of pheromones only at a low tested dose. VOC analysis of fungal cultures revealed that all the three fungi produced fusel alcohols and acetates but in variable composition and amounts. Collectively, the results of this study show that, in addition to pheromones, bark beetles might also use volatile cues from their symbiotic fungi to improve tree colonization and reproductive success in their breeding and feeding sites.

Symbiotic Fungi of an Ambrosia Beetle Alter the Volatile Bouquet of Cork Oak Seedlings.

In Portugal, fungal symbionts of the ambrosia beetle Platypus cylindrus affect tree vigor of cork oak (Quercus suber) and are linked with the cork oak decline process. Fungal symbionts play crucial roles in the life history of bark and ambrosia beetles and recent work indicates complex interactions on the fungal and plant metabolic level. Colonized trees may respond with an array of currently unknown volatile metabolites being indicative of such interactions, acting as infochemicals with their environment. In this study, we examined volatile organic compounds (VOCs) of cork oak seedlings wound inoculated with strains of three fungal associates of P. cylindrus (Raffaelea montetyi, R. quercina, and Ceratocystiopsis sp. nov.) over a 45-day period by means of thermodesorption gas chromatography-mass spectrometry techniques. Fungal strains induced largely quantitative but species-specific changes among the 58 VOCs characterized. Overall, monoterpenes-the major volatiles of cork oak foliage-were significantly reduced, possibly a result of fungal biotransformation. Acetophenone, sulcatone, and nonanal-volatiles known for mediating ambrosia beetle behavior-increased in response to fungal inoculation. Qualitative VOC profiles of excised tissue of wood lesions (21 VOCs) and pure fungal cultures (60 VOCs) showed little overlap with seedling VOCs, indicating their plant-derived but fungal-induced origin. This chemoecological study expands on the limited knowledge of VOCs as infochemicals emitted from oak trees threatened by oak decline in relation to beetle-vectored ophiostomatoid fungi. It opens new avenues of research to clarify mutualistic or pathogenic aspects of these complex symbiotic interactions and develop new control strategies for P. cylindrus, including its mycobiota.

Pathophysiology and transcriptomic analysis of Picea koraiensis inoculated by bark beetle-vectored fungus Ophiostoma bicolor.

Ophiostomatoid fungi exhibit a complex relationship with bark beetles; exhausting of host tree defenses is traditionally regarded as one of the key benefits provided to beetle vectors. Ophiostoma bicolor is one of the dominant species of the mycobiota associated with Ips genus bark beetles which infect the spruce trees across the Eurasian continent. Host spruce trees resist fungal invasion through structural and inducible defenses, but the underlying mechanisms at the molecular level, particularly with respect to the interaction between bark beetle-associated fungi and host trees, remain unclear. The aim of this study was to observe the pathological physiology and molecular changes in Picea koraiensis seedlings after artificial inoculation with O. bicolor strains (TS, BH, QH, MX, and LWQ). This study showed that O. bicolor was a weakly virulent pathogen of spruce, and that the virulent of the five O. bicolor strains showed differentiation. All O. bicolor strains could induce monoterpenoid release. A positive correlation between fungal virulence and release of monoterpenoids was observed. Furthermore, the release rate of monoterpenoids peaked at 4 days post-inoculation (dpi) and then decreased from 4 to 90 dpi. Transcriptomic analysis at 4 dpi showed that many plant-pathogen interaction processes and mitogen-activated protein kinase (MAPK) metabolic processes were activated. The expression of monoterpenoid precursor synthesis genes and diterpenoid synthesis genes was upregulated, indicating that gene expression regulated the release rate of monoterpenoids at 4 dpi. The enriched pathways may reveal the immune response mechanism of spruce to ophiostomatoid fungi. The dominant O. bicolor possibly induces the host defense rather than defense depletion, which is likely the pattern conducted by the pioneers of beetle-associated mycobiota, such as Endoconidiophora spp.. Overall, these results facilitate a better understanding of the interaction mechanism between the dominant association of beetles and the host at the molecular level.

Fungal Communities of the Pine Wilt Disease Complex: Studying the Interaction of Ophiostomatales With Bursaphelenchus xylophilus.

Considered one of the most devastating plant–parasitic nematodes worldwide, Bursaphelenchus xylophilus (commonly known as pinewood nematode, PWN) is the causal agent of the pine wilt disease in the Eurasian coniferous forests. This migratory parasitic nematode is carried by an insect vector (Monochamus spp.) into the host tree (Pinus species), where it can feed on parenchymal cells and reproduce massively, resulting in the tree wilting. In declining trees, PWN populations are strongly dependent on fungal communities colonizing the host (predominantly ophiostomatoid fungi known to cause sapwood blue-staining, the blue-stain fungi), which not only influence their development and life cycle but also the number of individuals carried by the insect vector into a new host. Our main aim is to understand if PWN-associated mycobiota plays a key role in the development of PWD, in interaction with the PWN and the insect vector, and to what extent it can be targeted to disrupt the disease cycle. For this purpose, we characterized the fungal communities of Pinus pinaster trees infected and non-infected with PWN in three collection sites in Continental Portugal with different PWD temporal incidences. Our results showed that non-infected P. pinaster mycoflora is more diverse (in terms of abundance and fungal richness) than PWN-infected pine trees in the most recent PWD foci, as opposed to the fungal communities of long-term PWD history sites. Then, due to their ecological importance for PWN survival, representatives of the main ophiostomatoid fungi isolated (Ophiostoma, Leptographium, and Graphilbum) were characterized for their adaptative response to temperature, competition in-between taxa, and as food source for PWN. Under the conditions studied, Leptographium isolates showed promising results for PWN control. They could outcompete the other species, especially O. ips, and significantly reduce the development of PWN populations when compared to Botrytis cinerea (routinely used for PWN lab culturing), suggesting this to be a natural antagonist not only for the other blue-stain species but also for the PWN.