Dryocosmus Kuriphilus Research Articles

Native Parasitoids Recruitment as Potential Controllers of Dryocosmus kuriphilus (Hymenoptera; Cynipidae) in Recently Colonised Areas

Native Parasitoids Recruitment as Potential Controllers of <i>Dryocosmus kuriphilus</i> (Hymenoptera; Cynipidae) in Recently Colonised Areas

Chromosome-level genome assembly of Oriental chestnut gall wasp (Dryocosmus kuriphilus)

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.

Fungal diversity in chestnut galls induced by Dryocosmus kuriphilus from Basilicata Region (Southern Italy)

Fungal diversity in chestnut galls induced by Dryocosmus kuriphilus from Basilicata Region (Southern Italy)

Ecological Impact of American Chestnut Hybrid Restoration on Invertebrate Communities Above- and Belowground

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.

Anatomical Changes during Chestnut (Castanea mollissima BL.) Gall Development Stages Induced by the Gall Wasp Dryocosmus kuriphilus (Hymenoptera: Cynipidae).

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.

Fungi associated with necrotic and asymptomatic galls of Dryocosmus kuriphilus (Hymenoptera: Cynipidae) and the adults of its parasitoid Torymus sinensis in chestnut growing areas of Turkey

Fungi associated with necrotic and asymptomatic galls of <i>Dryocosmus kuriphilus</i> (Hymenoptera: Cynipidae) and the adults of its parasitoid <i>Torymus sinensis</i> in chestnut growing areas of Turkey

Temporal changes in the composition of parasitoid assemblages associated with the invasive chestnut gall wasp

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).

Identification of a Unique Genomic Region in Sweet Chestnut (Castanea sativa Mill.) That Controls Resistance to Asian Chestnut Gall Wasp Dryocosmus kuriphilus Yasumatsu.

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

The Asian chestnut gall wasp is an invasive pest that causes ecological and economic losses in the management of the sweet chestnut (Castanea sativa Mill.). The pest therefore needs to be monitored in order to assess its impact on tree growth and fruit yield. As part of this work, we propose an infestation level classification based on the number of galls per branch. For two sweet chestnut orchards infested by the wasp, terrestrial light detection and ranging (LiDAR) data were used to calculate tree growth and fruit yield was quantified in terms of nuts and burrs. The tree growth and fruit yield variables were statistically analysed to determine the impact of different infestation levels on these variables, and differences between pairs of infestation levels were tested for significance. Negative correlations were found for the fruit yield variables with infestation indicators, while positive correlations were found for the tree growth variables. Significant differences were observed in fruit yield and tree growth variables associated with different infestation levels.

85 years counteracting an invasion: chestnut ecosystems and landscapes survival against ink disease

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.

Terpene Synthase Gene Family in Chinese Chestnut (Castanea mollissima BL.) Harbors Two Sesquiterpene Synthase Genes Implicated in Defense against Gall Wasp Dryocosmus kuriphilus.

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

The parasitoid Torymus sinensis (Hymenoptera: Torymidae) has been successfully used in Italy since 2005 for biological control of the invasive cynipid Dryocosmus kuriphilus (Hymenoptera: Cynipidae), highly destructive for the economically relevant Castanea sativa (Fagales: Fagaceae). In order to investigate the morphological aspects related to sensorial behavior, a fine morphology study of the antennae and their sensilla was conducted by scanning electron microscopy on both sexes of T. sinensis. The antennae, composed of a scape, a pedicel and a flagellum with ten flagellomeres, had chaetic sensilla of six subtypes, placoid sensilla of three subtypes, trichoid sensilla, sensilla with a roundish grooved tip, and coeloconic sensilla. The chaetic sensilla of the first three subtypes were found in the scape and in the pedicel, and those of the last three subtypes, together with trichoid, roundish grooved tip and coeloconic sensilla, were found only on flagellomeres. Sexual dimorphism was detected in the morphology of the proper pedicel and the flagellum, and in the presence and distribution of the sensilla and their subtypes. The morphological aspects of the antenna of T. sinensis and of its sensilla were compared with those found in the family Torymidae and in other families of the extremely diverse superfamily Chalcidoidea.

Lifecycle of Dryocosmus kuriphilus Yasumatsu and Diversity and Importance of the Native Parasitoid Community Recruited in the Northern Region of Portugal.

In this work, the objective was to learn the life cycle of D. kuriphilus and the diversity of native parasitoids in the northern region of Portugal between 2017 and 2019. The places studied belonged to the regions of Entre-Douro-e-Minho, Beira Interior, and Trás-os-Montes. To achieve the proposed objectives, buds were collected for egg and larva observation, galls were collected for larva, pupa, and adult observation and monitoring, and emergency boxes were used to identify the fauna present in the galls. In this study, 92% of D. kuriphilus adults emerged between June and July, with emergences occurring until September. We also obtained adults in winter, so it is important to study, in future works, the hypothesis of this pest performing diapause. Regarding the study of native parasitoids, compared to other countries, the same families emerged, with good rates of natural parasitism, although with fluctuations over the years. In the three municipalities under study, 11 species were identified (Eupelmus azureus Ratzeburg, Eupelmus urozonus Dalman, Eurytoma brunniventris Ratzeburg, Megastigmus dorsalis (Fabricius), Ormyrus pomaceus (Geoffroy), Sycophila iracemae Nieves Aldrey, Sycophila variegata (Curtis), Sycophila biguttata (Swederus), Torymus flavipes (Walker), Torymus auratus (Mueller), and Torymus notatus (Walker)). The average parasitism rates varied between 1.92% and 10.68%.

Monitoring of the health status of Castanea sativa in the Belasitsa mountain, southwest Bulgaria

In the period 2017-2023, a survey for the assessment of the phytosanitary condition of sweet chestnut (Castanea sativa) was conducted in a permanent sample plot (PSP) Belasitsa, which is part of the European large-scale network for monitoring the health status of forest ecosystems under the International Co-operative Program &amp;lsquo;Forests&amp;rsquo;. The PSP is set in a natural chestnut stand in Belasitsa mountain at an altitude of 643 m. Data collected from the first years of the monitoring determined a slight deterioration in the health status of the chestnut trees caused by an infection with the fungal pathogen Cryphonectria parasitica. An improvement in trees&amp;rsquo; vitality and lack of active necrosis were observed in 2022-2023. On the wounds, initiation of callus along the wounded edges was reported. Currently, the introduction of new and dangerous invasive insect pests Dryocosmus kuriphilus, Corythucha arcuata, etc., has not been detected. Attacks of both pests could further deteriorate the health status of the sweet chestnut in Belasitsa.

ABOVE-GROUND STORAGE OF PARASITIZED DRYOCOSMUS KURYPHILUS CHESTNUT GALLS INCREASES SURVIVAL OF OVERWINTERING TORYMUS SINENSIS IMMATURES

The gall-inducing Asian Chestnut Gall Wasp (ACGW), Dryocosmus kuriphilus is one of the most harmful pests of Castanea species in the world. In 2005, Torymus sinensis, a species-specific parasitoid of the ACGW, native to China, was released in Italy as a biological control agent. After more than ten years from the first T. sinensis release, ACGW only sporadically affects chestnut production. However, some areas of Tuscany such as Villore (Tuscany, Italy) still remain active ACGW hotspots and even though its distribution is much more jeopardized than in the past, several recurrent outbreaks have been recently reported. In the present work, we evaluated the possibility of storing leaf galls in the field, during the cold season, in order to protect T. sinensis immatures overwintering within them. In particular, the hypothesis was tested that above-ground storage of leaf galls could increase parasitoid survival in a Castanea sativa orchard near Villore (Tuscany, Italy). Results showed that T. sinensis survival in leaf galls stored above ground was higher than that recorded on galls stored on the ground, proving that this method was effective in protecting overwintering immatures from adverse environmental conditions. Our study provided chestnut growers with a new tool aimed at enhancing T. sinensis activity in C. sativa orchards. Key Words: Castanea sativa, natural enemies, parasitoids survival, galls storings methods

SOME BIOTIC LIMITING FACTORS OF DRYOCOSMUS KURIPHILUS (HYMENOPTERA CYNIPIDAE): A SURVEY IN CHESTNUT ORCHARDS NEAR FLORENCE (ITALY)

The biological control of the Asian Chinese gall wasp (ACGW) Dryocosmus kuriphilus, is influenced by several factors. In 2013, a survey was carried out in three chestnut orchards near Florence (Italy) to investigate ACGW parasitization by native parasitoids and the introduced Torymus sinensis. The following two methods were adopted: the first based on the dissection of cynipid galls with the aim of inspecting cells within each gall and the second based on monitoring adult emergence from galls. Parasitization patterns due to gall position related to plant height were also evaluated. Native parasitoids were more abundant in the upper part of tree crowns in June, while in July and August their activity was more evident in the middle and lower parts of chestnut trees. Conversely, parasitization of T. sinensis, was detected at all plant heights. Plant position within stands also affected the performance of both native parasitoids and T. sinensis, with parasitization that progressively decreasead moving toward the interior of the orchards. Gall necrosis was equally evaluated as a limiting factor of ACGW survival. Among the isolated fungal species, Gnomoniopsis castaneae (Tamietti), an endophyte fungus harmful to fruits, colonized both healthy and necrotic galls together with ubiquitous components of fungal communities such as Alternaria sp., Aspergillus sp., Fusarium sp. and Colletotrichum fioriniae. Our results evidenced that T. sinensis was able control the ACGW, while native parasitoids played only a secondary, though non-negligible, role. Conversely, gall necroses had a minor impact on pest control, as their efficacy was limited to the early stages of gall development. Key words: biodiversity, gall-inducing insects, native parasitoids, fungal species

Climate Change and Invasive Insects in Forest Ecosystems - Recent Examples from Türkiye

Recent climate changes -in contrast to changes in the past- are faster, and humans play a significant role in speeding up the process. Fauna usually responds strikingly to the changing climate; however, effects on animal species varies greatly. Indirect effects on insects through their host plants like the shifted shooting time, and the changed chemical composition and texture of the plant are significant. Direct effects like changes of the climatic elements (temperature, humidity) may cause a wide range of reactions, like change of the distribution area (e.g., northward shift) and/or change in the insects’ biology (e.g., swarming time, generation cycle). The presence of invasive insects is usually not associated with changing climatic factors. Their arrival is mainly human activity driven and the changing climatic factors may promote their establishment only. Introductions are accidental in most cases, but there are also plenty of examples of intentionally introduced species. In this paper, we demonstrate the effect of invasive insect species on the forest ecosystems in Türkiye through the examples of the western conifer seed bug (Leptoglossus occidentalis), the box tree moth (Cydalima perspectalis), the Asian longhorn beetles (Anoplophora glabripennis &amp; A. chinensis), the chestnut gall wasp (Dryocosmus kuriphilus), the oak lace bug (Corythucha arcuata) and the black stem borer (Xylosandrus germanus). The importance of invasive forest insect monitoring is also discussed.

Torymus sinensis and its close relatives in Europe: a multilocus phylogeny, detailed morphological analysis, and identification key

The introduction of the biological control agent Torymus sinensis Kamijo (Hymenoptera, Chalcidoidea, Torymidae) to control the populations of the chestnut gall wasp Dryocosmus kuriphilus Yasumatsu (Hymenoptera, Cynipidae) is considered one of the successful programs in biological control. The species was involved in interspecific hybridisation in Japan and the specimens imported into Europe were derived from this hybrid lineage, showing signs of introgression. The discovery of mitochondrial haplotypes or possible Enolase haplotypes from T. beneficus or of specimens with shorter ovipositor does not necessarily imply that T. beneficus is present in Europe, only that the European specimens are of hybrid origin. Of the native European Torymus species associated with D. kuriphilus, the molecular and morphometric results indicate Torymus notatus (Walker) as the closest species to T. sinensis. The two are part of the same species-group (cyaneus group), are nested together in the multivariate ratio analysis and are the closest genetically based on all three nuclear markers: Enolase (1.5% divergence), Wingless (2%) and ITS2 (13%). However, on the mitochondrial marker COI the closest species is Torymus rubi (Schrank) at 9.9% divergence. As such, T. notatus is the most likely candidate for accidental interspecific hybridisation if this is to happen in Europe. We provide an illustrated identification key for the European species of Torymus associated with D. kuriphilus, an important but lacking tool for biological control programs.

Brown Rot Caused by Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) at the Level of the Chestnut Tree (Castanea sativa Mill.)

The European chestnut tree (Castanea sativa Mill.) has great economic importance, mostly due to the recognized nutritional value of its fruit. Thus, the development and improvement of the techniques of the production, preservation, and control of the diseases/pests of chestnut trees is a topic of great interest to producers, companies, researchers, and consumers to ensure the quality of this exceptional fruit. Recently, an emerging rot in chestnuts caused by the fungus Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae Tamietti) (Gnomoniaceae, Diaporthales) was reported both in Australia and Europe. Since then, the number of records of this pathogen in several countries of the world (Europe and Asia) where Castanea spp. is cultivated has been increasing. This disease, called “brown rot”, has been causing significant production losses, raising serious concerns for producers and the chestnut industry. This review describes the world distribution and life cycle of the causal agent of brown rot. The life cycle of G. smithogilvyi can involve primary infection, caused by ascospores, and secondary infection, related to the asexual phase of the fungus (conidia). Then, the analytical methods used to detect G. smithogilvyi are described. Furthermore, the incidences of the disease caused by G. smithogilvyi are presented, ranging from 5 to 94%, with high infection rates causing significant economic losses. The damages caused by G. smithogilvyi are discussed. In fact, it can act as an endophyte or as a pathogenic fungus, causing fruit rot, canker in several plant tissues, and necrosis in leaves, as well as in galls caused by the gall wasp Dryocosmus kuriphilus Yasumatsu. Possible pre- and post-harvest methods to mitigate the damage caused by moulds, and in particular G. smithogilvyi, are presented, including biocontrol agents and chemicals. Finally, some challenges and future prospects for a number of uncertainties related to the epidemiology, geographic distribution, spread, detection, and management of this disease are discussed.

Leaf Trait Hyperspectral Characterization of Castanea sativa Miller Affected by Dryocosmus kuriphilus Yasumatsu

While populations of the Asian chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu), an invasive pest affecting the European chestnut (Castanea sativa Miller), have started to be controlled biologically, this pest still conditions chestnut tree development. With the aim of assessing plant health status as a means of monitoring gall wasp infestation, we used a field spectroradiometer to collect data from leaves taken from 83 trees in two chestnut orchards. We calculated characteristic spectral signatures for pest infestation, and after training and validation, developed classifiers to distinguish between different infestation levels. Several partial least square discriminant analysis (PLS-DA) and random forest (RF) models were fitted with reflectance and transformed values to obtain characteristic curves reflecting infestation. Four wavelengths (560 nm, 680 nm, 1400 nm, and 1935 nm) were identified as showing the greatest differences between curves. The best overall accuracy (69.23%) was achieved by an RF model fitted with reflectance transformed values. Lower overall accuracy (26.92%) was achieved in distinguishing between infestation levels. In conclusion, while more specific differences in infestation levels were not detectable, our method successfully discriminated between gall absence and presence.