Early detection protocols for the oak wilt fungal pathogen (Bretziella fagacearum) are crucial for forest health practitioners on the boundaries of the growing disease front. Established protocols use oak wilt-specific primers and gel electrophoresis to amplify and detect oak wilt among DNA extracted from nitidulid vectors. However, these protocols are prone to inconclusive results due to the presence of off-target amplification products similar in size to positive control bands. Using sequence-adapted oak wilt primers, we employed a metabarcoding amplicon sequencing approach that resolved inconclusive results and validated true oak wilt positives. We found that these off-target amplification products are co-occurring taxa from natural forest and beetle microbiomes, further necessitating a sequencing approach for early surveillance of oak wilt.

Oak wilt is a fungal related disease that is caused by the oak wilt fungus (Bretziella fagacearum [Bretz] Z.W. De Beer, S. Marincowitz, T.A. Duong, and M.J. Wingfield) which is an introduced vascular wilt fungus that mainly infects oak species (Quercus spp.). The purpose of this review paper is to provide an update on the environmental challenges that are contributing to the spread of oak wilt as well as providing an overview of the forest management options that are available to reduce its spread. This disease has killed millions of oaks in the Midwest and Texas and impacts forest health in the Eastern United States as well. As climate change shifts ranges northward, the distribution of oak wilt is predicted to spread north to Canada. Forest managers, landscapers, and researchers use various management techniques to mitigate the impacts the disease causes to economy, biodiversity, and aesthetics. Current management emphasizes removal of trees near or in the infection center to limit the overland mode of spread. To contain the underground mode of spread, root graft connections between oak trees needs to be severed. Monitoring is key to limiting the spread of oak wilt and will require a combination of remote sensing based technologies and nventory plot network for effective ground-truthing.

Entomopathogenic fungi are a group of fungi that infect and kill insects to obtain nutrients, thereby contributing to the natural regulation of insect populations. In recent years, they have been increasingly utilized as biological control agents, particularly in response to the rising prevalence of pesticide-resistant pests in agricultural systems. Representative examples include Beauveria bassiana and Metarhizium anisopliae, which are regarded as natural enemies of pests in agroecosystems. Since the first report of Korean oak wilt disease in 2004, the disease has continuously spread across the country and causes severe damage to deciduous oak species, especially Quercus mongolica. Although many efforts have been made to effectively control the disease, including chemical treatments, the control efficacy was shown to be low, and given the environmental side effects arising from the use of insecticides, there has been a demand for alternative control strategies. Integrated Pest Management in forests promotes ecological sustainability by reducing chemical pesticide use, conserving biodiversity, and enhancing long-term forest health. In this study, to mitigate issues with disease management strategies, assessments were made on three entomopathogenic fungi, B. bassiana, M. anisopliae, and Purpureocillium lilacinum, as potential biological control agents against oak wilt disease and its insect vector, Platypus koryoensis. In this regard, we investigated the insecticidal efficacy and LT50 of each entomopathogenic fungus, and the results showed that all three entomopathogenic fungal strains exhibited fast insecticidal effects against the insect vector, P. koryoensis, with M. anisopliae showing the fastest action, recording a lethal time to 50% mortality (LT50) of 58.7 h. The spores of M. anisopliae were found to be sensitive to high temperatures, while demonstrating a relatively high germination rate under UV exposure and strong initial germination ability at low temperatures.

Oak wilt, caused by the fungal pathogen Bretziella fagacearum, spreads via root grafts and insect vectors, threating oaks (Quercus spp.) and chestnuts (Castanea spp.) in the United States. Detection and management of B. fagacearum are crucial, as oak wilt can devastate forested and urban ecosystems. However, diagnosing oak wilt presents challenges and requires laboratory confirmation due to symptom similarities with other stressors. Common detection methods also have limitations. In this study, we optimized and validated an existing TaqMan real-time PCR assay, comparing it with a culture-based method and using nested PCR as the gold standard. We also developed a novel nondestructive sampling technique. Our optimized real-time PCR assay demonstrated a consistent 100% detection rate and accuracy across all branch sapwood samples. In contrast, the culture-based method varied significantly, achieving a 100% detection rate and accuracy only for fresh samples displaying sapwood discoloration. In the absence of sapwood discoloration, the culture detection rate and accuracy were 80 and 90%, respectively. For dry samples, these rates decreased significantly to 22 and 52%. The novel nondestructive sampling method used leaf petioles of fallen leaves to detect B. fagacearum from two tree hosts, using both optimized real-time PCR and culture-based methods. Our real-time PCR consistently outperformed the culture-based method, regardless of symptom severity in leaf samples. The real-time PCR offers improved efficiency, specificity, sensitivity, and turnaround time compared with nested PCR and culture-based methods. Our findings highlight the potential of detecting vascular-inhabiting pathogens from leaf petiole samples, particularly in scenarios requiring nondestructive sampling and high-throughput screening.

Korean oak wilt disease associated with Dryadomyces quercus-mongolicae recently emerged as a major tree disease in South Korea. A comprehensive transcriptome analysis is presented for D. quercus-mongolicae grown in vitro on three different culture media, identifying nearly 7,000 expressed transcripts. Most transcripts are associated with proteins essential for fungal survival and growth. The 40S ribosomal protein S25, ceramide very long chain fatty acid hydroxylase, Epl1 protein, and ADP/ATP translocase are particularly important due to their critical roles in the metabolism and environmental adaptation of fungi. Gene ontology analyses revealed that 39.4%, 61.2%, and 43.3% of transcripts were successfully annotated to biological process, molecular functions, and cellular component aspects, respectively. Furthermore, key metabolic pathways were elucidated, including sphingolipid metabolism, L-tryptophan biosynthesis, and glycolysis, which provide important information on physiological functioning of D. quercus-mongolicae. Overall, these findings provide key information on fundamental biological mechanisms of D. quercus-mongolicae.

Diseases are major natural disturbances to forest ecosystems that cause changes to overstory tree structure and influence seedling dynamics by altering environmental conditions via functional traits. Thus, identifying factors affecting seedling dynamics would improve broader understanding of the seedling regeneration process after disease disturbance.We investigated 13,010 current year seedlings from 59 woody species, 44 genera, and 21 families in a Japanese warm‐temperate secondary forest, following Japanese oak wilt disease (JOW) in 2009–2013 and in 2018–2020. We also quantified temporal changes in canopy openness and soil moisture. Structural equation model (SEM) was used to explore the effects of environmental conditions and seedling functional traits on current year seedling density in autumn for 13 dominant species.Canopy openness gradually increased, whereas soil water content decreased throughout the study site. After disruption by JOW, recruitment of current year seedlings increased through improvements in light conditions. The functional traits of current year seedlings were likely affected by soil nutrients in the late JOW period (2011–2013), whereas litterfall production influenced functional traits of current year seedlings in the post‐JOW period (2018–2020). Factors affecting seedling density varied as environmental conditions changed across the JOW periods. Seedling establishment was enhanced by increased canopy openness in the late JOW period and by low litterfall, high soil nutrient content, and high soil moisture in the post‐JOW period.Our study provides evidence that temporal changes in environmental conditions and seedling densities occurring after disruption by JOW differ from the changes in forests impacted by other natural disturbances.

Traditional methods for diagnosing bacterial or fungal infections, such as cell culture, are comprehensive but time-consuming and subjective. Microbial volatile organic compound (VOC) analysis offers a faster alternative, though challenges such as low concentrations and chemical heterogeneity persist. Gas chromatography-mass spectrometry (GC-MS), while highly sensitive, requires lengthy sample preparation. This study presents a novel approach using proton transfer-reaction mass spectrometry (PTR-MS) for direct headspace analysis of fungal cultures, eliminating the need for preconcentration steps. By culturing microbes in wide-mouth glass jars with septum caps, VOC profiles were obtained in under 30s for samples which were incubated for just 1day, thus significantly reducing the diagnosis time. Using Bretziella fagacearum, a model organism known for its distinctive fruity odor linked to oak wilt disease, this method demonstrated enhanced accuracy and speed in detecting characteristic VOCs. The high sensitivity and rapid turnaround of this technique offer a promising alternative to traditional cell culture and GC-MS methods, providing faster, more reliable diagnostics and reducing both the complexity and time required for pathogen identification.

Abstract In 2022, EFSA was mandated by the European Commission's Directorate‐General for Health and Food Safety (M‐2022‐00070) to provide technical assistance on the list of Union quarantine pests qualifying as priority pests, as specified in Article 6(2) of Regulation (EU) 2016/2031 on protective measures against plant pests. As part of Task C, EFSA conducted comprehensive expert knowledge elicitations for candidate priority pests on the lag period, rate of expansion and impact on production (yield and quality losses) and the environment. This report provides the rationale for the dataset on Bretziella fagacearum, delivered to the European Commission's Joint Research Centre, to feed the Impact Indicator for Priority Pest (I2P2) model and complete the pest prioritisation ranking exercise.

Summary A new rhabditid nematode was isolated from dead wood of Quercus crispula with Japanese oak wilt. The nematode is typologically close to Chylorhabditis epuraeae, and considered to be congeneric to this species. Phylogenetic analysis based on ribosomal DNA small subunit sequences corroborated this close relationship. Typologically, the new species is characterised by its relatively large body (1255-1518 μm in the male and 1610-1943 μm in the female), arrangement of bursal rays (4 + 1 + 1 + 3), dorsally directed first, fifth, and seventh rays, very long bipartite spicule, and some other minor traits. According to these morphological characters and molecular phylogenetic status, the new species is described and illustrated as Chylorhabditis crispulae n. sp. Genus Chylorhabditis was once synonymised with Buetschlinema, but resurrection of the genus is proposed and the generic characters of Chylorhabditis are emended/updated.

Characteristics of Damaged Trees in the First Record of Japanese Oak Wilt in Hokkaido, Northern Japan

Bretziella fagacearum is a destructive vascular wilt fungal pathogen affecting oaks in the United States and Canada. The epidemiology of oak wilt varies across different geographical locations, indicating the need to investigate the population dynamics of B. fagacearum to discern potential differences in its genotypes using genomic tools. A good-quality genome of B. fagacearum is crucial as a reference for population genetics studies. Here, we report a high-quality genome of B. fagacearum isolate C519. The genome assembly consists of nine scaffolds, corresponding to the nine chromosomes, totaling 27,072,536 bp with a GC content of 47.29%. It is predicted to encode 7,554 proteins, which are annotated using RNA sequencing data from the same isolate. The circular mitochondrial genome consists of a chromosome of 174,403 bp with a GC content of 28.59% and contains 54 open reading frames, including 14 core genes, 28 tRNAs, 4 rRNAs, and 8 hypothetical proteins. The reference genome can enhance the understanding of molecular epidemiology and biology of B. fagacearum, aiding in identifying genetic variations and pathogen–host interactions and developing diagnostic tools and disease management strategies. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2025.

The ambrosia beetle Platypus quercivorus (Murayama) is a vector of Japanese oak wilt, a fungal pathogen that causes mass mortality of Fagaceae trees. The male locates and lands on the host tree and releases an aggregation pheromone that attracts both males and females. Thus, aggregation pheromones and host tree volatiles are important chemicals for host location. However, the antennal responses to these volatiles have not yet been examined. To obtain physiological property of P. quercivorus antennae, we electrophysiologically recorded the antennal responses of this beetle to candidate volatiles, including isoprene and other leaf volatiles (1-hexanol, 2-ethyl-1-hexanol, trans-2-hexenal, decanal, and benzaldehyde) of the host trees, as well as quercivorol (aggregation pheromone) and ethanol (widely used for trapping this insect in the field). Electroantennograms (EAGs) exhibited clear dose-dependent responses to the tested volatiles, but antennae did not respond to decanal. Sexual differences in responses were not observed for almost all volatiles. However, the EAGs suggested that the quercivorol and trans-2-hexenal sensitivity of males was higher than that of females. Ethanol elicited a large EAG amplitude in both sexes, but the sensitivity of males or females to ethanol was lower than that to the other volatiles. EAGs showed a very weak response to isoprene only in males, although isoprene is considered a possible attractant for host location. Our study will facilitate to understand the mechanism of the host location ecologically and neurophysiologically.

Abstract Tree-afflicting pests, such as insects and pathogens, could change forests in ways promoting invasions by non-native plants. After tree death associated with the fungal pathogen oak wilt (Bretziella fagacearum) and its attempted containment (severing root connectivity and sanitation removal of infected trees), we examined change in cover of the non-native liana Oriental bittersweet (Celastrus orbiculatus Thunb.; hereafter Celastrus) at 28 sites in temperate black oak (Quercus velutina Lam.) forests, Ohio, USA. During our 5-yr study spanning 2020 to 2024, Celastrus cover increased significantly (P < 0.05) through time at oak wilt sites but not in untreated reference forest sites without evidence of oak wilt. Celastrus cover increased by an order of magnitude, up to an average of 32 times among oak wilt treatments up to 10 yr old. By 2024, Celastrus cover ranged from 6% to 22% on average in 5- to 10-yr-old oak wilt treatments, compared with 1% cover in reference forest. Results indicate that non-native plant invasion accelerated following disturbance associated with a fungal pathogen and its attempted containment and, more generally, suggest that tree-afflicting pests can promote invasive plants in forests. Co-management of tree-afflicting pests and non-native plants may become increasingly important to ensure forests recovering from tree mortality are dominated by native plants.

Oak wilt causes severe dieback of Quercus serrata, a dominant tree species in the lowlands across Japan. This study evaluated the effects of oak wilt on the wood-inhabiting fungal community and the decay rate of deadwood using a field monitoring experiment. We analysed the fungal metabarcoding community from 1200 wood samples obtained from 120 experimental logs from three forest sites at five different time points during the initial 1.5 years of the experiment. Death due to wilt significantly influenced the fungal community composition and reduced fungal diversity, likely due to the dominance of a limited number of species. The operational taxonomic unit richness, occurrence frequency, and DNA copy number of white rot fungi were also enhanced on the logs killed by wilt, depending on the sites. Structural equation modelling suggested that the wilt-initiated changes in the fungal community reduced the decay rate of oak logs. Temperature and wood moisture also affected the fungal community and log decomposition. These results suggest that, in addition to the direct effect of climate, oak wilt indirectly affects log decomposition by structuring the fungal community. Continuous monitoring is essential to evaluate the longer-term effects of oak wilt on the fungal decomposition of wood.

Nitidulid beetles (Coleoptera: Nitidulidae), the overland vectors of the Bretziella fagacearum fungus that causes oak wilt, were monitored in infection centers in Quercus rubra stands in northern Michigan, USA using baited, wind-oriented traps for 2 years. First nitidulid captures, accounting for <1.5% of total annual captures, occurred in late April in both years (<50 cumulative degree days [DDs]; base 10°C). A total of 1,188 and 735 beetles representing 19 and 18 species were captured in 2018 (18 traps; 3 sites) and 2019 (16 traps; 4 sites), respectively. Ground traps (1.5 m high) captured more beetles and species than traps on canopy branches of red oaks. Most nitidulids (81-86%) were captured in May and June, but frequent precipitation and cool spring temperatures extended activity into early July in 2019. In 2018, 336 beetles representing 12 species were screened for B. fagacearum spores, but only 20 beetles from 4 species bore viable spores. Mycelial mats on red oaks killed were 4-fold more abundant in 2019 than in 2018. Of the 225 beetles screened in 2019, 56 beetles representing 6 species had viable spores. Nearly all (96%) spore-bearing beetles in both years were captured in late May or June. In bimonthly xylem samples collected from healthy trees, large earlywood vessels, presumably more vulnerable to infection than latewood, were present from May to June in 2018 and until early July in 2019. Results are consistent with mid-May to mid-July high-risk periods designated in current state and regional guidelines for oak wilt management.

Abstract In recent years, Japanese oak wilt causing mass mortality of oak trees has exhibited a significant geographical expansion. Understanding the formation history of current local populations of Platypus quercivorus Murayama, a vector of Japanese oak wilt, is important foundational knowledge for effectively preventing forest damage. In this study, we used genome-wide single nucleotide polymorphism data to infer the population genetic structure and historical demography of P. quercivorus populations in eastern Japan, where Japanese oak wilt is rapidly expanding. We found at least 3 distinct genetic groups, each of which caused Japanese oak wilt in different areas. The supported scenario suggests that 1 of 2 genetic groups in the northeast first diverged from the ancestral population, then the other group diverged from the southwestern population, followed by gene flow between the 2 groups. Recent admixtures of the 2 northeastern groups were identified at certain sites. The geographic genetic structure of the populations suggested that each genetic group experienced recent range expansion. Although genetically independent populations had caused oak tree mass mortality in their original distribution range, our results support the hypothesis that the recent geographic expansion of Japanese oak wilt outbreaks is attributed to the range expansion of these P. quercivorus groups.

ABSTRACTOak wilt disease, caused by the fungus Bretziella fagacearum, can kill mature red oaks within months of infection, severely affecting biodiversity, landscapes, and industries. The disease, originally only present in the United States, was officially reported for the first time in Canada in June 2023. The aim of this study was to suggest a standardized assay and sample processing method to optimize oak wilt detection both in infection centers and ahead of the disease front. Two previously published molecular assays, a Nested PCR and a TaqMan qPCR, were compared to detect B. fagacearum in a variety of samples in a ring trial across five laboratories. Sample types investigated included eDNA from trapped insect vectors (sorted insects and bulk content from traps), infested and healthy oak wood chips, and B. fagacearum conidia dilutions. Results demonstrated that both Nested and TaqMan assays can be used for molecular confirmation of oak wilt, and results are reproducible across different labs. There is a general agreement between both detection assays when testing true‐positive and true‐negative samples. Both methods demonstrated overall good accuracy. The TaqMan assay was more sensitive and detected lower amounts of DNA target. Both tests were 100% specific to oak wood samples, which was the best sample type to use for detection. In general, samples with high Cts were more prompted to yield false negative Nested results. Detecting oak wilt from bulk insect samples was by far more rapid than sorted sap beetles, but resulted in lower detection signals, especially with the Nested assay. The time‐period when the insect traps were set up also had considerable influence on detection results. We hope this study helps to formulate guidelines in oak wilt detection and biosurveillance management.

Influence of cutting <i>Pleioblastus chino</i> on Japanese oak wilt in abandoned secondary forest.

BackgroundOak wilt disease, caused by Bretziella fagacearum is a significant threat to oak (Quercus spp.) tree health in the United States and Eastern Canada. The disease may cause dramatic damage to natural and urban ecosystems without management. Early and accurate diagnosis followed by timely treatment increases the level of disease control success.ResultsA rapid assay based on loop mediated isothermal amplification (LAMP) was first developed with fluorescence detection of B. fagacearum after 30-minute reaction time. Six different primers were designed to specifically bind and amplify the pathogen’s DNA. To simplify the use of this assay in the field, gold nanoparticles (AuNPs) were designed to bind to the DNA amplicon obtained from the LAMP reaction. Upon inducing precipitation, the AuNP-amplicons settle as a red pellet visible to the naked eye, indicative of pathogen presence. Both infected and healthy red oak samples were tested using this visualization method. The assay was found to have high diagnostic sensitivity and specificity for the B. fagacearum isolate studied. Moreover, the developed assay was able to detect the pathogen in crude DNA extracts of diseased oak wood samples, which further reduced the time required to process samples.ConclusionsIn summary, the LAMP assay coupled with oligonucleotide-conjugated gold nanoparticle visualization is a promising method for accurate and rapid molecular-based diagnosis of B. fagacearum in field settings. The new method can be adapted to other forest and plant diseases by simply designing new primers.

ABSTRACT The purpose of this study was to assess the effects of Japanese Oak Wilt Disease on mortality in a tree population. The study site was the permanent plot of 0.26 ha established in the secondary forest dominated by Abies firma, Tsuga sieboldii and Quercus serrata at the University of Tokyo Chiba Forest in the southeastern part of the Boso peninsula, Japan. We set the two observation periods of 1999–2008 and 2009–2021, and Japanese Oak Wilt Disease invaded the study site in the middle of the second period. We collected tree age data for both alive and dead trees in the two periods based on semi-nondestructive drilling measurements for trees using a RESISTOGRAPH. Survival analyses were conducted on the second-period data, and the demographic metrics such as mortality ratios and mean lifetimes were compared between the two periods. The results showed that: the mortality ratio of the tree population at old ages, where oaks (Quercus serrata) concentrated in their age distribution, significantly increased; the effect of the oak group was significant on mortality in the tree population; the hazard for the oak group was 24 times the hazard for the other tree species group; the survival curve reflected the mass mortality in oaks at old ages, which led to the decrease of mean lifetime less than half. Consequently, it was implied that the effects of Japanese Oak Wilt Disease impacted the demographic metrics of oak trees.