The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as ‘High risk plants, plant products and other objects’. This Scientific Opinion covers plant health risks posed by graftwood, whips, bare root plants and potted plants of Castanea sativa imported from the United Kingdom. The assessment was performed considering the available scientific information, including the technical information provided by the UK. All pests associated with the commodities were evaluated against specific criteria. Three EU regulated pests (Cryphonectria parasitica, Dryocosmus kuriphilus, Phytophthora ramorum), present in the UK and associated with the commodity, were considered relevant for this opinion. One pest that is not regulated in the EU (Phytophthora kernoviae) fulfilled all relevant criteria and was selected for further evaluation. For the selected pest, the risk mitigation measures described in the submitted technical dossier were evaluated. An expert judgement was given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The degree of pest freedom varied among the commodities evaluated, with P. kernoviae being most frequently expected on the imported bare root and potted plants. The Expert Knowledge Elicitation indicated with 95% certainty that 9060 or more units per 10,000 will be free from P. kernoviae.

Integrating factors affecting the success of invasive insect pests into dynamical models can help assessing their invasion risks and control. Here, we model the spread of a gall-forming hymenopteran parasite of chestnut trees, Dryocosmus kuriphilus, and its control agent, Torymus sinensis, across 23 natural forest sites located in the French Eastern Pyrenees. The integration of field estimates of the levels of bottom-up (frequency, density and genetic susceptibility of chestnut trees) and top-down (hyperparasitism by native insects and fungi) control of the pest in a Nicholson-Bailey model allowed to identify source and sink sites for the invasive species and its control agent. Comparisons with the observed levels of hyperparasitism by T. sinensis showed that it was found in 7/23 sink sites. The extension of our modelling into a two-site model showed that dispersal rates as low as 1‰ can be responsible for the persistence of T. sinensis in sinks, regardless of the precise dynamical regime of D. kuriphilus-T. sinensis coexistence in the source. Although dispersal promotes the persistence of the control agent and tends to homogenize its effectiveness in both sites, it was also shown to reduce the global biological control effectiveness at high rates of coupling.

The basic helix-loop-helix (bHLH) transcription factor family plays crucial roles in plant growth, development, and responses to environmental stresses. However, a systematic characterization of bHLH genes in Castanea mollissima has been lacking. We performed a genome-wide identification of bHLH genes in C. mollissima, followed by phylogenetic, structural, motif, chromosomal distribution, and cis-element analyses. Co-expression network analysis and transcriptomic profiling under Dryocosmus kuriphilus infestation were conducted, and representative genes were validated using qRT-PCR across seed developmental stages. A total of 124 non-redundant bHLH genes (CmbHLHs) were identified and classified into eight subfamilies, consistent with conserved gene structures and motif compositions, particularly motif-1 and motif-2 of the canonical bHLH domain. Chromosomal mapping revealed non-random distribution and tandem duplication events. Promoter analysis indicated enrichment of cis-elements responsive to stress, hormone signaling, and developmental regulation. Co-expression analysis highlighted hub genes, including CmbHLH82 and CmbHLH57, with potential regulatory functions. Transcriptomic data showed that most CmbHLHs were downregulated under D. kuriphilus infestation, notably CmbHLH63 and CmbHLH51. qRT-PCR confirmed the dynamic expression of five selected genes during seed development. These findings provide the first comprehensive overview of the bHLH gene family in Chinese chestnut. The results highlight candidate genes potentially involved in development and insect resistance, thereby laying a foundation for future functional and breeding studies.

Asian chestnut gall wasp (ACGW) (Dryocosmus kuriphilus Yasumatsu), native to China, is an invasive pest that causes significant economic losses in Castanea species. While some cultivars show full resistance by inhibiting insect development in buds, the underlying defense mechanisms remain unclear. In this study, the accumulation and distribution of hydrogen peroxide (H2O2) were investigated in dormant buds of chestnut cultivars that are resistant and susceptible to D. kuriphilus by using the 3,3′-diaminobenzidine (DAB) staining method. Buds were examined under a stereomicroscope during key stages of pest development, including oviposition, transition from egg to larva, gall induction, and gall development. Baseline levels of H2O2 were detected in all buds; however, these levels varied among cultivars, with resistant cultivars exhibiting lower basal levels. The degree of H2O2 accumulation was found to vary depending on plant–insect interaction, physiological processes, and cultivar-specific traits. Histochemical staining revealed that brown spots indicative of H2O2 accumulation were concentrated in the vascular bundles of leaf primordia and in the apical regions. In resistant hybrid cultivars, the defense response was activated at an earlier stage, while in resistant Castanea sativa Mill. cultivars, the response was delayed but more robust. Although consistently high levels of H2O2 were observed throughout the pest interaction in susceptible cultivars, gall development was not inhibited. During the onset of physiological bud break, increased H2O2 accumulation was observed across all cultivars. This increase was associated with endodormancy in susceptible cultivars and with both defense mechanisms and endodormancy processes in resistant cultivars. These findings highlight the significant role of H2O2 in plant defense responses, while also supporting its function as a multifunctional signaling molecule involved in gall development and the regulation of physiological processes.

The chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu) is an invasive pest that attacks species of the genus Castanea, inducing gall formation on buds and leaves, which can significantly reduce tree growth, fruiting, and overall chestnut production. Native to China, D. kuriphilus has become a serious threat to chestnut orchards worldwide. Torymus sinensis Kamijo, a parasitoid also originating from China, is highly specific to D. kuriphilus and is currently considered the most effective biological control agent against this pest. This study aimed to evaluate the establishment of T. sinensis as well as its effectiveness in controlling D. kuriphilus at release sites between 2020 and 2023. Releases of T. sinensis were conducted in the municipality of Bragança with a sex ratio of 120 females to 70 males. The parasitoids were randomly released across three chestnut trees with infestation levels ranging from moderate (26–50% of the canopy affected by galls) to very severe (>80% of the canopy affected). At each release site, 250 galls were collected annually, and 10% of these galls were dissected to calculate parasitism rates by T. sinensis. Results revealed a positive correlation between the monitoring year and the parasitism rate. Following the releases, parasitism rates increased gradually, reaching values between 15% and 40%. T. sinensis successfully established itself in chestnut orchards and parasitized D. kuriphilus, despite normal population fluctuations being observed across years and orchards.

The phosphatidylethanolamine binding protein (PEBP) family plays an important part in growth and development of plants. Castanea mollissima is an economic plant with significant financial value and has become an important food source in the Northern Hemisphere. However, the PEBP genes in C. mollissima have not been studied yet. In this study, six PEBP genes (CmPEBP1 ∼ CmPEBP6) were identified in C. mollissima and comprehensively analyzed in terms of physicochemical properties, phylogeny, gene structures, cis-regulatory elements (CREs), transcription factor interaction, and expression profiles. The six CmPEBP genes were categorized into three subfamilies according to the phylogeny analysis, and all of them share extremely similar gene and protein structures. A total of 136 CREs were identified in the promoter regions of the CmPEBP genes, mainly related to growth and development, environmental stress, hormone response, and light response. Comparative genomic analysis indicated that the expansion of the CmPEBP genes was mainly driven by dispersed duplication, and the CmPEBP3/CmPEBP5 derived from eudicot common hexaploidization (ECH) events retained orthologous genes in all species studied. A total of 259 transcription factors (TFs) belonging to 39 families were predicted to be regulators of CmPEBP genes, and CmPEBP4 was predicted to interact with the most TFs. The RNA-seq data analysis indicated the potential roles of CmPEBP genes in the ovule, bud, and flower development of C. mollissima, as well as in the response to temperature stress, drought stress, and the gall wasp Dryocosmus kuriphilus (GWDK) infestation. Additionally, the expression of CmPEBP genes in C. mollissima seed kernel development and their response to temperature stress were confirmed by RT-qPCR assays. This study gives references and directions for future in-depth studies of PEBP genes.

The complexity of mycobiota associated with chestnut galls induced by Dryocosmus kuriphilus in Galicia (Northwestern Spain)

One of the most prominent problems related to biological invasions is the variation of local species composition, which often leads to ex novo interspecific interactions. Here, we explored and analysed the native species composition of gall inducers and their associated parasitoids and inquilines in Spanish areas invaded by Dryocosmus kuriphilus Yasumatsu 1951 (Hymenoptera: Cynipidae), an invasive pest of chestnut trees. After a quantitative description of these species' assemblages, we analysed through bipartite networks the level of the trophic specialisation of parasitoids and inquilines when considering either the host taxonomic identity, the host plant species or the host gall morphological type. We sampled galls of D. kuriphilus and native species of Cynipidae in different Spanish areas, including those where the exotic parasitoid Torymus sinensis Kamijo 1982 (Hymenoptera: Torymidae) had been released for D. kuriphilus biological control. The results indicate that the native parasitoids recruited by D. kuriphilus come almost exclusively from native communities on Quercus galls, except for one species from Rosa. Galls of D. kuriphilus had the second most diverse species composition; despite this species assemblage arose ex novo in less than a decade. The bipartite networks resulted more specialised when considering host plant taxa than when gall types and the host taxa were accounted. In such trophic webs, there were few parasitoid/inquiline specialist and many generalist species, which agrees with the rapid recruitment by D. kuriphilus. Higher parasitoid species richness in D. kuriphilus galls is likely due to their being a largely unexploited available resource for the native natural enemies of cynipid wasps.

ABSTRACTIn this work, we investigate the native parasitoid community colonising galls by an invasive pest: the oriental chestnut gall wasp (OCGW), Dryocosmus kuriphilus (Hymenoptera; Cynipidae). We tried to understand how native parasitoids react after the arrival of the pest (two‐year sampling period) in the province of Zamora (Castilla & León region, N‐W Spain). For this purpose, we collected both, chestnut galls from six stands and oak galls from surrounding oak groves. Our results highlight the response to D. kuriphilus provided by the oak gall wasps that naturally inhabit Quercus pyrenaica. The parasitoid community showed a core assemblage composed of Eurytoma brunniventris, Torymus flavipes and T. sinensis in OCGW galls, and Baryscapus diaphantus, Bootanomyia dorsalis, Eupelmus urozonus, E. brunniventris, Mesopolobus lichtensteini, T. affinis, and T. flavipes in oak galls. We identified the torymid T. flavipes as one of the most common parasitoids of OCGW, in addition to the exotic parasitoid T. sinensis, never officially released in the region but present in the study area. In addition, we intend to know its possible incidence on non‐target cynipids. We have found an intense connection in the food web with some chalcids that establish interactions with the gall wasp, both in adult instar (emerging from reared galls) and larval (presence of larvae in OCGW gall chambers, identified by dissection). We also describe the flight phenology of D. kuriphilus in our study area (as far as we know, the first one focused in Castilla & León region) and evaluate the overlap between OCGW and native and exotic parasitoids showing a promising role of native guild as biocontrollers.

Dryocosmus kuriphilus, commonly known as the chestnut gall wasp, belongs to the family Cynipidae and is native to China. It is a highly invasive insect species causing serious damage to chestnut trees and has rapidly spread to various continents, including Europe, North America, and Oceania. The D. kuriphilus has become one of the important pests of chestnut plants in the world and is listed as a quarantine object by the European and Mediterranean Plant Protection Organization (EPPO). In this study, we used PacBio long reads, Illumina short reads, and Hi-C sequencing data to construct a chromosome-level assembly of the D. kuriphilus genome. The assembled genome includes 14,729 contigs with a total length of 2.28 Gb and a contig N50 of 0.8 Mb. With Hi-C technology, 2.17 Gb (95.02%) of contigs were anchored and oriented into the 10 pseudochromosomes with the scaffold N50 of 198.8 Mb and the scaffold N90 of 158.8 Mb. In total, 24,086 protein-coding genes were predicted in the assembled D. kuriphilus genome as the reference gene set. A total of 1.82 Gb repeats (occupying 79.7% of the genome), including 1.42 Gb of transposable elements and 0.40 Gb of tandem repeats, were identified in D. kuriphilus genome. In the evaluation of completeness, the BUSCO analysis determined a level of 98.1% completeness for the assembled genome sequences based on the Insecta database (OrthoDB version 10). The high-quality genome assembly of D. kuriphilus will not only provide a valuable reference for the study of its evolutionary history and genetic structure but also facilitate the research of host-pest interactions and invasiveness. Moreover, this genome assembly will promote in the development of effective management strategies to mitigate the economic and ecological impacts of this invasive pest on chestnut trees and ecosystems.

In recent years, the Asian chestnut gall wasp (ACGW) Dryocosmus kuriphilus has been reported to have a high incidence in Italy and other Mediterranean basin countries. In 2021-2022, a study was undertaken in the Basilicata Region (Southern Italy) to investigate the relationship between the galls produced by ACGW on sweet chestnut (Castanea sativa Mill.) and fungal pathogens. In particular, the fungal diversity from green and necrotic galls collected from two important sweet chestnut sites (Melfi and Rionero in Vulture) was investigated. Nineteen fungal taxa were identified based on their morphological and molecular traits. In both localities, the most frequent species isolated from green and necrotic galls were Gnomoniopsis castaneae, Colletotrichum acutatum, and Pestalotiopsis sp. It is essential to understand the role played by the galls as an inoculum source for sweet chestnut fungal pathogens, particularly for G. castaneae, an emerging pathogen of which biology is still poorly understood. Findings from the present study stressed that the complex relationship between host-insect-microbial community needs to be elucidated to be able to control the pathogenic fungi and consequently maintain sweet chestnut trees' health as they play a key role in the local agriculture (horticulture, forestry) and subsidiary economy

The cross-hybridization of American chestnut (Castanea dentata (Marsh.) Borkh.) with Chinese chestnut (Castanea mollissima Bl.) is a promising strategy for restoring a blight-resistant strain of this keystone species to the Appalachian mountains. To assess the ecological impacts of hybridization on invertebrate communities, we conducted a study across chestnut plots with varying degrees of hybridization (75%, 94%, or 100% American chestnut). Our findings indicate American chestnut hybridization impacted invertebrate communities above- and belowground. Aboveground insect community composition, insect herbivory, gall infestation, and belowground invertebrate diversity were all altered. While some of these differences could be explained by different growth habits or environmental differences, stark differences in Asian chestnut gall wasp infestation (Dryocosmus kuriphilus Yasumatsu.) suggest a genetic component. These results suggest that chestnut hybridization, and particularly expanded restoration efforts using chestnut hybrids, could impact invertebrate communities above- and belowground in addition to pest dynamics. Understanding these effects is crucial for successful chestnut restoration and ecosystem management.

This study delved into the larval development and the morphological and anatomical transformations that occur in the galls of chestnut trees (Castanea mollissima BL.) and are induced by the chestnut gall wasp Dryocosmus kuriphilus Yasumatsu (GWDK) across various stages: initial, growth, differentiation, maturity, and lignification. Chestnut galls in the five development stages were collected. Gall structural characteristics were observed with an anatomical stereomicroscope, and anatomical changes in galls were analyzed with staining and scanning electron microscope techniques. The chestnut gall wasp laid its eggs on young leaves and buds. Chestnut gall wasp parasitism caused plant tissues to form a gall chamber, with parenchyma, protective, and epidermal layers. The development of the gall structure caused by the infestation of the GWDK gall led to the weakening of the reactive oxygen species (ROS) elimination ability of the host. The accumulation of ROS led to cell wall peroxidation, resulting in structural damage and diminished host resistance, and the parenchyma layer exhibited significant nutrient supply and thickening. The thickness of the protective and epidermal layers varied notably across different growth stages. The oviposition of the chestnut gall wasp induced modifications in the original plant tissues, with gall formation being most favorable in young tissues, correlating with the maturity level of the host plant tissues. Variances in the internal structures of the galls primarily stemmed from nutrient supplementation, while those in the external structure were attributed to defensive characteristics. This research contributes a foundational understanding of gall development induced by the chestnut gall wasp in Chinese chestnut, offering valuable insights into the intricate interplay between insect infestation and plant physiology.

Abstract The chestnut gall wasp (CGW) Dryocosmus kuriphilus is a serious pest of chestnuts (Castanea sativa) in many chestnut growing areas in Turkey. Out of 200 galls randomly collected from four different areas of Yalova and Giresun provinces, 116 showed necrosis, while 84 were asymptomatic. Fungi associated with the necrotic and asymptomatic galls were determined based on morphology and DNA sequencing. Gnomoniopsis smithogilvyi, Colletotrichum acutatum, Penicillium glabrum, Botrysphaeria dothidea, Trichoderma atroviridea, and Botrytis cinerea were found on 49, 11, 10, 8, 7, and 1 necrotic galls, respectively. On the other hand, G. smithogilvyi, Fusarium proliferatum, Aureobasidium sp., C. godetiae, Rhizopus stolonifer, P. glabrum, C. acutatum, Cladosporium sp., Alternaria spp., and Aspergillus sp. were hosted by 37, 35, 15, 14, 12, 8, 6, 3, 1, and 1 asymptomatic galls, respectively. Seven fungi, G. smithogilvyi, C. acutatum, F. proliferatum, B. dothidea, Cryphonectria parasitica, Diplodina castanea, and Penicillium sp. were isolated from 31, 6, 4, 1, 1, 1, and 1 of the dead larvae of D. kuriphilus, respectively. Two fungi, F. proliferatum and Penicillium sp., were isolated from the adults of Torymus sinensis, a parasitoid of CGW. Pathogenicity of G. smithogilvyi, C. acutatum, and F. proliferatum detected from dead larvae of D. kuriphilus and galls was tested on young chestnut saplings: the former two produced necrosis while the third one did not.

Abstract Invasive alien species pose a challenge to ecosystem stability and crop production, so understanding their interactions with the natural communities they invade is crucial to mitigate negative effects. The oriental chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), is an invasive insect that attacks chestnut trees. It was first recorded in Spain in 2012 and is now presumably present in the majority of chestnut stands in the country. We assessed variation in the abundance of D. kuriphilus galls and the composition of their associated parasitoid community (Hymenoptera: Chalcidoidea) during a 4‐year period (2014–2017) in 12 sampling sites in Catalonia (Spain, northeastern Iberian Peninsula), and identified which species contributed most significantly to the control of D. kuriphilus . The results show that the abundance of individual parasitoid species varied during the study period and that the number of species increased year after year. There was also a transition in the parasitoid community composition across the years. Our data suggest that the joint action of the non‐native parasitoid Torymus sinensis Kamijo and the native T . notatus (Walker) (Hymenoptera: Chalcidoidea: Torymidae) led to the decline of D . kuriphilus until it was no longer considered a pest. Also, the observed pool of parasitoid species ended up being relatively homogeneous across sites over the years, which may be due to the high natural dispersal of these parasitoids (both by active flight and passive wind dispersal).

The Asian chestnut gall wasp (ACGW) (Hymenoptera Dryocosmus kuriphilus Yasumatsu) is a severe pest of sweet chestnut (Castanea sativa Mill.) with a strong impact on growth and nut production. A comparative field trial in Central Italy, including provenances from Spain, Italy, and Greece, was screened for ACGW infestation over consecutive years. The Greek provenance Hortiatis expressed a high proportion of immune plants and was used to perform a genome-wide association study based on DNA pool sequencing (Pool-GWAS) by comparing two DNA pools from 25 susceptible and 25 resistant plants. DNA pools were sequenced with 50X coverage depth. Sequence reads were aligned to a C. mollissima reference genome and the pools were compared to identify SNPs associated with resistance. Twenty-one significant SNPs were identified and highlighted a small genomic region on pseudochromosome 3 (Chr 3), containing 12 candidate genes of three gene families: Cytochrome P450, UDP-glycosyltransferase, and Rac-like GTP-binding protein. Functional analyses revealed a putative metabolic gene cluster related to saccharide biosynthesis in the genomic regions associated with resistance that could be involved in the production of a toxic metabolite against parasites. The comparison with previous genetic studies confirmed the involvement of Chr 3 in the control of resistance to ACGW.

Quantifying Asian chestnut gall wasp (Dryokosmus kuriphilus Yasumatsu) impact on fruit yield and on tree growth using terrestrial LiDAR

A retrospective examination of early pest and disease outbreaks, such as ink disease, offers new insights into their impact on ecosystems and landscapes. Ink disease, caused by Phytophthora spp., is one of the most destructive diseases affecting Castanea sativa Mill. It first appeared in Europe in the early 18th century, with the initial recorded case in Italy (Piedmont) dating back to 1845. However, its spread instilled significant concerns in several chestnut-growing regions primarily devoted to fruit production. In 1938, a comprehensive assessment of Phytophthora xcambivora outbreaks was conducted in the Bolognese Apennines (Italy), providing a detailed overview of chestnut cultivation status. Thirty-six disease foci were documented, and laboratory tests confirmed the presence of ink disease. To understand the disease’s impact on chestnut ecosystem and landscape over the past 85 years, the 1938 sites were revisited to assess chestnut persistence and phytosanitary status, with Phytophthora species detected using isolation and molecular techniques. Monitoring data revealed that C. sativa still thrives in all but one site, with its wood seemingly able to coexist in dynamic equilibrium with the disease. While P. xcambivora was still detected in several foci, the extent of damage appeared limited. The potential for natural control, likely influenced by the complexity of soil biota, along with the natural spread of hypovirulence in chestnut blight and biological control of the Asian chestnut gall wasp, could explain the continued presence of chestnut in the investigated area and potentially throughout the Italian chestnut range, despite nearly two centuries of ink disease prevalence. Nevertheless, ongoing monitoring is essential to dynamically comprehend the factors at play and their efficacy, particularly in the context of climate change and the possible spread of other Phytophthora species. The survival of chestnut ecosystems amidst ink disease underscores the preservation of the economic, ecological, and landscape values associated with these woodlands.

Chinese chestnut (Castanea mollissima BL.) is a well-known fruit tree that has been cultivated in East Asia for millennia. Leaves and buds of the plant can become seriously infested by the gall wasp Dryocosmus kuriphilus (GWDK), which results in gall formation and associated significant losses in fruit production. Herbivore-induced terpenes have been reported to play an important role in plant-herbivory interactions, and in this study, we show that upon herbivory by GWDK, four terpene-related compounds were significantly induced, while the concentrations of these four compounds in intact buds were relatively low. Among these compounds, (E)-nerolidol and (E, E)-α-farnesene have frequently been reported to be involved in plant herbivory defenses, which suggests direct and/or indirect functions in chestnut GWDK defenses. Candidate terpene synthase (TPS) genes that may account for (E)-nerolidol and (E, E)-α-farnesene terpene biosynthesis were characterized by transcriptomics and phylogenetic approaches, which revealed altered transcript levels for two TPSs: CmAFS, a TPS-g subfamily member, and CmNES/AFS, a TPS-b clade member. Both genes were dramatically upregulated in gene expression upon GWDK infestation. Furthermore, Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana showed that CmAFS catalyzed the formation of (E, E)-α-farnesene, while CmNES/AFS showed dual (E)-nerolidol and (E, E)-α-farnesene synthase activity. Biochemical assays of the recombinant CmAFS and CmNES/AFS proteins confirmed their catalytic activity in vitro, and the enzymatic products were consistent with two of the major volatile compounds released upon GWDK-infested chestnut buds. Subcellular localization demonstrated that CmAFS and CmNES/AFS were both localized in the cytoplasm, the primary compartment for sesquiterpene synthesis. In summary, we show that two novel sesquiterpene synthase genes CmAFS and CmNES/AFS are inducible by herbivory and can account for the elevated accumulation of (E, E)-α-farnesene and (E)-nerolidol upon GWDK infestation and may be implicated in chestnut defense against GWDK herbivores.

Morphological characterization of the antenna of Torymus sinensis (Hymenoptera: Torymidae) and a comparison within the superfamily Chalcidoidea