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BACKGROUND: Oak 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. RESULTS: A 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. CONCLUSIONS: In 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.

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Tree mortality due to global change-including range expansion of invasive pests and pathogens-is a paramount threat to forest ecosystems. Oak forests are among the most prevalent and valuable ecosystems both ecologically and economically in the United States. There is increasing interest in monitoring oak decline and death due to both drought and the oak wilt pathogen (Bretziella fagacearum). We combined anatomical and ecophysiological measurements with spectroscopy at leaf, canopy, and airborne levels to enable differentiation of oak wilt and drought, and detection prior to visible symptom appearance. We performed an outdoor potted experiment with Quercus rubra saplings subjected to drought stress and/or artificially inoculated with the pathogen. Models developed from spectral reflectance accurately predicted ecophysiological indicators of oak wilt and drought decline in both potted and field experiments with naturally grown saplings. Both oak wilt and drought resulted in blocked water transport through xylem conduits. However, oak wilt impaired conduits in localized regions of the xylem due to formation of tyloses instead of emboli. The localized tylose formation resulted in more variable canopy photosynthesis and water content in diseased trees than drought-stressed ones. Reflectance signatures of plant photosynthesis, water content, and cellular damage detected oak wilt and drought 12 d before visual symptoms appeared. Our results show that leaf spectral reflectance models predict ecophysiological processes relevant to detection and differentiation of disease and drought. Coupling spectral models that detect physiological change with spatial information enhances capacity to differentiate plant stress types such as oak wilt and drought.

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In the summer of 2021, a 20-year-old 'Colossal' (Castanea sativa × C. crenata hybrid) tree in a commercial chestnut orchard in northwest Michigan suddenly declined. Until 2023, an additional 26 adjacent trees declined, suggesting the occurrence of root-graft transmission of the pathogen. The initial wilting of leaves progressed to complete tree death in about 10 days. Symptoms included wilting, and bronzing, followed by tanning starting at leaf apex and margins, with significant defoliation. Sometimes black-to-brown streaks of discoloration appear in the sapwood, with no signs of mycelial mat production on dead trees. Branches from symptomatic trees in two different areas of the orchard were submitted to Plant and Pest Diagnostics at Michigan State University. Bretziella fagacearum (Bretz) Z.W. de Beer, Marinc., T.A. Duong & M.J. Wingf. was detected in both samples using nested PCR (Wu et al. 2011) and qPCR (Bourgault et al. 2022). The products of the nested PCR were sequenced (GenBank accession nos. OR522695-OR522696) and BLASTn search results showed 100% identity to an ex-type strain of B. fagacearum (MH865866). Surface-sterilized discolored sapwood chips were plated on acidified potato dextrose agar (aPDA). Bretziella fagacearum was consistently recovered; colony and endoconidia morphology aligned with the description of the pathogen (De Beer et al. 2017). A pure culture (BF277) was obtained for inoculation experiments. To confirm pathogenicity, 10 'Colossal' chestnut seedlings (average stem diameter of 9 mm) were inoculated in the greenhouse with a 14-day old culture of BF277. Using a conical drill bit, two 0.4 mm diameter holes were drilled, one was 5 cm above the soil line at a 45° angle and the other was on the opposite side of the stem at least 10 cm above the soil line. A 50-µl conidial suspension (1 × 107 conidia per ml) was applied and the holes were sealed with Parafilm. Five 'Colossal' seedlings were inoculated with sterile water. Leaf epinasty with bent petioles was observed 14 days later. Leaf wilting and necrosis similar to natural infection in the orchard were observed at 24 and 34 days after inoculation, respectively. Water-inoculated control plants showed no symptoms. Bretziella fagacearum was reisolated from symptomatic plants by surface sterilizing leaf petioles with 75% ethanol (30 s), followed by 10% (v/v) bleach (1 min), and two rinses with sterile deionized water (>1 min). Petiole pieces (~1 cm) were plated on aPDA. The pathogen was reisolated from six symptomatic plants and detected using qPCR in the remaining four seedlings. Bretziella fagacearum was not detected in control plants. The identity of the recovered fungus was confirmed following the amplification of the internal transcribed spacer (ITS) from extracted genomic DNA, as described in Chahal et al. 2022. The resulting PCR product was sequenced and assembled into a consensus sequence using Geneious Prime. The consensus sequence (accession no. OR515809) revealed 100% identity to the ex-type of B. fagacearum (KU042044). This is the first record of B. fagacearum infecting chestnut trees in Michigan. Previously, B. fagacearum has been reported infecting Chinese chestnut (C. mollissima) in Missouri (Bretz and Long, 1950). Oak wilt is widely distributed in Michigan and is the predominant disease afflicting red oaks in the Midwestern U.S. Consequently, constant vigilance and monitoring are essential in chestnut orchards to promptly detect and effectively manage potential infections.

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Bretziella fagacearum, the ascomycete fungus oak wilt, is considered a virulent threat to North American oaks, but the influence of the physical environment on this pathosystem remains unclear, particularly at the forest scale. This study explored the influence of terrain and soil factors on B. fagacearum infections, applying discrete and continuous spatial models to investigate the question, besides proximity to other infections, which environmental factors influenced B. fagacearum incidence? Locations of infections were recorded from 586 confirmed B. fagacearum sites, identified from 2004 through 2021 in a 76 km2 area of deep, sandy glacial outwash in Chequamegon-Nicolet National Forest, northern Wisconsin. Public datasets derived from remote sensing were incorporated as covariates, describing terrain elevation (USGS 10-m DEM), soil physical and chemical properties (POLARIS), and forest composition (WiscLand2). Spatial models included generalized additive models (GAMs) and Neyman-Scott cluster process models. The results indicated that spatial dependence and the distribution of oak forests were the most important drivers of B. fagacearum distribution in this area, with more minor influence from elevation, hill shade, and drainage patterns. Comparison between modeling approaches indicated that-at this scale and in this area-the most accurate models were those that included host distribution, spatial dependence, and quantitative terrain and soil descriptions. However, a close approximation could be attained using nonlinear models (GAMs) that incorporated only host distribution and spatial dependence.

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Nitidulidae trapping performed from 2018 to 2021 to characterize flight behaviors of potential vectors of the oak wilt pathogen yielded three new species records for Canada, six new species records for Ontario, and three new species records for Manitoba. The new records for Canada include Carpophilus (Ecnomorphus) corticinus reported from Ontario, C. (Myothorax) nepos reported from Ontario and Manitoba, and Glischrochilus (Librodor) obtusus reported from Ontario. In addition, the following species are first recorded in Ontario: Carpophilus (Ecnomorphus) antiquus, C. (Megacarpolus) sayi, Stelidotacoenosa; and also in Manitoba: Carpophilus (Megacarpolus) lugubris and Cychramusadustus. Collection data is provided for the two provinces and national records.

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Two new compounds, a new lactone, quercilactone A (1), and (17R)-hydroxynafuredin (5), as well as five known compounds, scytalone (2), 3S,4R-hydoxy-scytalone (3), nafuredin (4), (+)-(3R,5R)-3-hydroxy-5-decanolide (6) and 3-ethyl-4-hydroxy-6-methyl-2H-pyran-2-one (7), were isolated from Raffaelea quercivora, a fungus that causes Japanese oak wilt disease. The structures of these compounds were determined by 1D and 2D NMR spectroscopic analyses. The absolute configuration at C-17 of 5 was determined to be R by the modified Mosher's method. Compounds 1, 2, and 7 exhibited weak phytotoxic activity in lettuce seedlings at a concentration of 100 µg mL-1.

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Oak wilt disease caused by Raffaelea quercus-mongolicae has emerged obviously in Korea. We selected antifungal isolates against R. quercus-mongolicae among 368 endophytic fungal isolates from different parts of oak and pine trees. The experiment was conducted in the primary and secondary screenings by dual culture test. The antifungal activity of the selected isolates was assessed in culture filtrate test based on the inhibition rates in mycelial growth, sporulation, and spore germination of oak wilt fungus. Five isolates, E089, E199, E282, E409 and E415, showed strong antifungal activity in culture filtrate test, and their antifungal activity decreased on the culture media supplemented with heated culture filtrate. Higher mycelial growth inhibitions on the unheated media were recorded in E409 (Colletotrichum acutatum), E089 (Daldinia childiae), E415 (Alternaria alternata) and E199 (Daldinia childiae) with the inhibition rates of 79.0%, 70.1%, 68.9% and 64.5%, respectively. These isolates also had the higher sporulation inhibitions on unheated media with the rates of 96.8%, 84.2%, 82.8% and 80.5%, respectively. The spore germination of the oak wilt fungus was completely inhibited by E282 (Nectria balsamea) on both unheated and heated media. These results showed that a higher number of potent antifungal isolates against oak wilt fungus was isolated from the petiole compared to the other parts. This study could contribute to the development of biological control approaches for the management of oak wilt disease caused by R. quercus-mongolicae.

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Oak wilt is slowly expanding in the northeastern United States. Several nitidulid beetle species are known vectors of the fungus [Bretziella fagacearum (Bretz) Z. W. De Beer, Marinc., T. A. Duong, and M. J. Wingf (Microascales: Ceratocystidaceae)] that causes this disease, acquiring spores from fungal mats on infected trees and transmitting them to uninfected trees. Survey and fungal isolation from captured nitidulid beetles could be an important tool for detecting the presence of this disease in a geographic area not previously known to have oak wilt. In preparation for monitoring activities in such areas, two trapping studies were conducted in the northeastern United States: 1) trap test comparing the efficacy of wind-oriented pipe, multiple-funnel, and modified pitfall traps for nitidulids and 2) wet and dry collection cup comparison. Lures were a combination of nitidulid pheromones and fermenting liquid. Results support the use of multiple-funnel traps over the other two trap types, for both targeted species-specific surveys and community sampling. More total nitidulids, Colopterus truncatus (Randall), and Glischrochilus fasciatus (Olivier) were captured in wet collection cups compared with dry cups. Twenty-seven fungal species were isolated, none of which were B. fagacearum. Many fungi isolated from beetles were plant pathogens, indicating that in addition to the oak wilt fungus, sap beetles may contribute to the spread of other plant diseases.

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Korean oak wilt (KOW) is vectored by the beetle Platypus koryoensis (Murayama) (Coleoptera: Curculionidae), a native species of Korea, whose dispersal distance is a key factor determining the spread of damage by KOW. To estimate dispersal distance at stand level, we conducted a mark-release-recapture (MRR) experiment and validated its results using an independent data. Sticky traps were attached to the trunks of oak trees up to 48.8 m from the release point. Beetles were marked with different three fluorescent powders by date and released, and the number of recaptured beetles was counted 90 min after release. To validate the flight distance, annual mean dispersal distance of P. koryoensis population was analyzed using GPS coordinates of oak trees with the symptom of KOW recorded in the field from 2012 to 2014 in independent oak stands that have been damaged by KOW since 2012. The beetles were recaptured only on the day they were released, suggesting that the beetles only make one flight. The percentage of recaptured beetles was 6.0 ± 1.6%. The mean dispersal distance was 18.0 ± 1.3 m, and more than 85% of recaptured beetles were caught within 25 m. Annual movement distances in infested stands were 24.1 and 19.9 m from 2012 to 2013 and 2013 to 2014, respectively, similar to the dispersal distance obtained from our MRR experiment. Our results showed that the dispersal distance of P. koryoensis estimated by MRR is a useful process for predicting the spread of areas damaged by KOW.

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Following a request from the European Commission, the EFSA Plant Health (PLH) Panel performed a pest categorisation of Bretziella fagacearum, a well-defined and distinguishable fungal species of the family Ceratocystidaceae. The species was moved from the genus Ceratocystis to a new genus Bretziella following phylogenetic analysis of the species and its close relatives. The former species name Ceratocystis fagacearum is used in the Council Directive 2000/29/EC. The pathogen is regulated in Annex IAI as a harmful organism whose introduction into the EU is banned. B. fagacearum is only reported from the USA, where it causes a wilt disease on Quercus spp. Other hosts are reported based on inoculation trials, although Chinese chestnut (Castanea mollissima) is reported to be naturally infected. No North American oak species has been found to be immune to the disease. The European oak species Quercus robur, Quercus petraea and Quercus pubescens were found to be susceptible in inoculation experiments. The pest could enter the EU via wood (with and without bark, including wood packaging material), plants for planting and cut branches. Hosts and favourable climatic conditions are common in the EU, thus facilitating establishment. The pest would be able to spread following establishment by means of root grafts, insect vectors and movement of wood, plants for planting and other means. The pest introduction would have impacts in woodland and plantations, as oak wilt disease is often lethal in a short period of time. Wood treatment (debarking, kiln drying, fumigation), prompt removal of affected trees and creating root-free zones between affected and healthy stands are available control measures. The main knowledge gaps concern (i) the survival of the fungus in wood during transport and the association with propagation material, (ii) the presence of suitable vectors in Europe and (iii) the relative susceptibility of the oak species native to Europe under natural conditions. The criteria assessed by the Panel for consideration as a potential quarantine pest are met. For regulated non-quarantine pests, the criterion on the pest presence in the EU is not met.

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Raffaelea (Ophiostomatales) is a genus of more than 20 ophiostomatoid fungi commonly occurring in symbioses with wood-boring ambrosia beetles. We examined ambrosia beetles and plant hosts in the USA and Taiwan for the presence of these mycosymbionts and found 22 isolates representing known and undescribed lineages in Raffaelea. From 28S rDNA and ß-tubulin sequences, we generated a molecular phylogeny of Ophiostomatales and observed morphological features of seven cultures representing undescribed lineages in Raffaelea s. lat. From these analyses, we describe five new species in Raffaelea s. lat.: R. aguacate, R. campbellii, R. crossotarsa, R. cyclorhipidia, and R. xyleborina spp. nov. Our analyses also identified two plant-pathogenic species of Raffaelea associated with previously undocumented beetle hosts: (1) R. quercivora, the causative agent of Japanese oak wilt, from Cyclorhipidion ohnoi and Crossotarsus emancipatus in Taiwan, and (2) R. lauricola, the pathogen responsible for laurel wilt, from Ambrosiodmus lecontei in Florida. The results of this study show that Raffaelea and associated ophiostomatoid fungi have been poorly sampled and that future investigations on ambrosia beetle mycosymbionts should reveal a substantially increased diversity.

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The epidemic of mass mortality of oak trees by Japanese oak wilt has affected secondary deciduous broadleaved forests that have been used as coppices in Japan. The dieback of oak trees formed gaps in the crown that would be expected to enhance the regeneration of shade-intolerant pioneer species. However, foraging by sika deer Cervus nippon has also affected forest vegetation, and the compound effects of both on forest regeneration should be considered when they simultaneously occur. A field study was conducted in Kyôto City, Japan to investigate how these compound effects affected the vegetation of the understory layer of these forests. The presence/absence of seedlings and saplings was observed for 200 quadrats sized 5 m ×5 m for each species in 1992, before the mass mortality and deer encroachment, and in 2014 after these effects. A hierarchical Bayesian model was constructed to explain the occurrence, survival, and colonization of each species with their responses to the gaps that were created, expanded, or affected by the mass mortality of Quercus serrata trees. The species that occurred most frequently in 1992, Eurya japonica, Quercus glauca, and Cleyera japonica, also had the highest survival probabilities. Deer-unpalatable species such as Symplocos prunifolia and Triadica sebifera had higher colonization rates in the gaps, while the deer-palatable species Aucuba japonica had the smallest survival probability. The gaps thus promoted the colonization of deer-unpalatable plant species such as Symplocos prunifolia and Triadica sebifera. In the future, such deer-unpalatable species may dominate gaps that were created, expanded, or affected by the mass mortality of oak trees.

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Japanese oak wilt (Raffaelea quercivora) is a vector-borne disease transmitted by the flying ambrosia beetle, Platypus quercivorus, and causes mass mortality in the fagaceous species of Japan. The data described in this article are available in Mendeley Data, DOI: 10.17632/xwj98nb39r.1 [1] and include the mortality status of 1089 Quercus crispula and 846 Quercus serrata trees and surrounding forest conditions. The findings using this dataset were published in M. Oguro, S. Imahiro, S. Saito, T. Nakashizuka, Relative importance of multiple scale factors to oak tree mortality due to Japanese oak wilt disease, For. Ecol. Manag. (2015) doi:10.1016/j.foreco.2015.07.016 [2].

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The ambrosia beetle, Platypus koryoensis, is an economically important pest affecting oak trees in Korea. Candida kashinagacola was isolated from galleries of the beetle in oak wood and identified by analyses of morphology, physiological properties, and nucleotide sequence of the large subunit ribosomal DNA. This is the first report on Candida species associated with oak wilt disease vectored by the ambrosia beetle, Platypus koryoensis, in Korea.

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The ambrosia beetle, Platypus koryoensis, is an economically important pest affecting oak trees in Korea. Candida kashinagacola was isolated from galleries of the beetle in oak wood and identified by analyses of morphology, physiological properties, and nucleotide sequence of the large subunit ribosomal DNA. This is the first report on Candida species associated with oak wilt disease vectored by the ambrosia beetle, Platypus koryoensis, in Korea.

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The ambrosia beetle, Platypus koryoensis, is a serious pest of oak trees in Korea. In this study we investigated filamentous fungi present in the body of the beetle. Fourteen genera of filamentous fungi belonging to Ascomycota and Basidiomycota were isolated. Among the isolated fungi, some were able to produce wood degrading enzymes. This is first report of fungi associated with P. koryoensis.

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The ambrosia beetle, Platypus koryoensis, is a serious pest of oak trees in Korea. In this study we investigated filamentous fungi present in the body of the beetle. Fourteen genera of filamentous fungi belonging to Ascomycota and Basidiomycota were isolated. Among the isolated fungi, some were able to produce wood degrading enzymes. This is first report of fungi associated with P. koryoensis.

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