Biological control of the Asian chestnut gall wasp in Portugal: Insights from a mathematical model.

In recent years, the Asian gall wasp Dryocosmus kuriphilus has invaded chestnut trees and significantly affected the Portuguese chestnut production. Studies in other countries, such as Japan or Italy, have shown that the parasitoid Torymus sinensis can successfully achieve biological control of D. kuriphilus. Mathematical models help us to understand the dynamics of the interaction between the pest D. kuriphilus and its parasitoid T. sinensis and, consequently, they can help to implement measures that enhance crop pest management. In this work, the evolution of the density of D. kuriphilus and T. sinensis across time and space is studied through the numerical solution of models that include parameters based on observations made in Portugal. Simultaneous releases of the parasitoid are simulated at various locations and at different times. The results indicate that, in the case of a small and homogeneous orchard, biological control can be effective, but, in the case of extensive domains, the pest control is much more difficult to achieve. In order for biological control to be efficient, it is necessary to implement, in each chestnut-producing region, a collective strategy based on the annual monitoring of infestation levels.

Climate as a possible driver of gall morphology in the chestnut pest Dryocosmus kuriphilus across Spanish invaded areas.

The alien cynipid wasp Dryocosmus kuriphilus Yasumatsu, 1951 is a serious pest of chestnuts (Castanea spp.) in Japan, North America and Europe, causing fruit losses while inducing galls in buds. While D. kuriphilus galls have a recognizable and roughly invariable globular shape, their size varies, reaching up to 4 cm in diameter. Among other factors, such variation may depend on different climatic conditions in different attacked areas. Here, we sampled and measured 375 D. kuriphilus galls from 25 localities throughout the Iberian Peninsula, including both cold and rainy northern (Eurosiberian) areas and warm and dry central-southern (Mediterranean) areas, to test the effects of climate and geographical location on gall morphology. The analyses indicate that gall mass and volume follow a pattern that can be associated with a climatic cline. In particular, the Eurosiberian galls were smaller than the Mediterranean galls according to differences in climatic conditions. In the southern areas, the greater insolation regime does not allow the chestnut trees to be distributed at lower altitudes, but the high rainfall and humidity regime of the mountain enclaves allow their presence. These conditions of insolation and precipitation seem to influence the morphological characteristics of the galls of D. kuriphilus.

Transcriptome characterization and expression profiling in chestnut cultivars resistant or susceptible to the gall wasp Dryocosmus kuriphilus.

The oriental gall wasp Dryocosmus kuriphilus represents a limiting pest for the European Chestnut (Castanea sativa, Fagaceae) as it creates severe yield losses. The European Chestnut is a deciduous tree, having major social, economic and environmental importance in Southern Europe, covering an area of 2.53 million hectares, including 75,000 ha devoted to fruit production. Cultivars show different susceptibility and very few are resistant to gall wasp. To deeply investigate the plant response and understand which factors can lead the plant to develop or not the gall, the study of transcriptome is basic (fundamental). To date, little transcriptomic information are available for C. sativa species. Hence, we present a de novo assembly of the chestnut transcriptome of the resistant Euro-Japanese hybrid 'Bouche de Bétizac' (BB) and the susceptible cultivar 'Madonna' (M), collecting RNA from buds at different stages of budburst. The two transcriptomes were assembled into 34,081 (BB) and 30,605 (M) unigenes, respectively. The former was used as a reference sequence for further characterization analyses, highlighting the presence of 1444 putative resistance gene analogs (RGAs) and about 1135 unigenes, as putative MiRNA targets. A global quantitative transcriptome profiling comparing the resistant and the susceptible cultivars, in the presence or not of the gall wasp, revealed some GO enrichments as "response to stimulus" (GO:0050896), and "developmental processes" (e.g., post-embryonic development, GO:0009791). Many up-regulated genes appeared to be transcription factors (e.g., RAV1, AP2/ERF, WRKY33) or protein regulators (e.g., RAPTOR1B) and storage proteins (e.g., LEA D29) involved in "post-embryonic development". Our analysis was able to provide a large amount of information, including 7k simple sequence repeat (SSR) and 335k single-nucleotide polymorphism (SNP)/INDEL markers, and generated the first reference unigene catalog for the European Chestnut. The transcriptome data for C. sativa will contribute to understand the genetic basis of the resistance to gall wasp and will provide useful information for next molecular genetic studies of this species and its relatives.

Fungal community associated with adults of the chestnut gall wasp Dryocosmus kuriphilus after emergence from galls: Taxonomy and functional ecology.

The diversity of the fungal community associated with adults of Dryocosmus kuriphilus following emergence was examined using HTS analysis. Ascomycota dominated the fungal core-biome community. The functional guilds of the 90 taxa forming the core-biome were assessed, demonstrating three main groups: saprotrophs, plant pathogens and entomopathogens. Twenty-nine OTUs out of 90 were resolved to species level identifying 26 different fungal species. Among these species, many were cosmopolitan or previously recorded in Europe. Ten taxa were previously recorded on chestnut, including some recognized plant pathogens associated with foliage and green tissues such as Epicoccum nigrum, Gnomoniopsis castanea, Colletotrichum acutatum, Stromatoseptoria castaneicola, Ramularia endophylla. Beauveria bassiana; within the core microbiome, Fusarium larvarum represented the most abundant entomopathogenic species. Some of these species are known to impact directly or indirectly the vitality of the insects in the galls. The chestnut blight pathogen, Cryphonectria parasitica, was never found associated with D. kuriphilus. Based on the present study, an active role for D. kuriphilus as a vector of chestnut fungal endophyte/pathogens cannot be demonstrated but neither ruled out.

Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi.

The soil-borne oomycete Phytophthora cinnamomi is a highly destructive Phytophthora species associated with the decline of forest. This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768 bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24 h; at 36 h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichia pastoris and assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein.

Resistance to Phytophthora cinnamomi in American Chestnut (Castanea dentata) Backcross Populations that Descended from Two Chinese Chestnut (Castanea mollissima) Sources of Resistance.

Restoration of American chestnut (Castanea dentata) depends on combining resistance to both the chestnut blight fungus (Cryphonectria parasitica) and Phytophthora cinnamomi, which causes Phytophthora root rot, in a diverse population of C. dentata. Over a 14-year period (2004 to 2017), survival and root health of American chestnut backcross seedlings after inoculation with P. cinnamomi were compared among 28 BC3, 66 BC4, and 389 BC3F3 families that descended from two BC1 trees (Clapper and Graves) with different Chinese chestnut grandparents. The 5% most resistant Graves BC3F3 families survived P. cinnamomi infection at rates of 75 to 100% but had mean root health scores that were intermediate between resistant Chinese chestnut and susceptible American chestnut families. Within Graves BC3F3 families, seedling survival was greater than survival of Graves BC3 and BC4 families and was not genetically correlated with chestnut blight canker severity. Only low to intermediate resistance to P. cinnamomi was detected among backcross descendants from the Clapper tree. Results suggest that major-effect resistance alleles were inherited by descendants from the Graves tree, that intercrossing backcross trees enhances progeny resistance to P. cinnamomi, and that alleles for resistance to P. cinnamomi and C. parasitica are not linked. To combine resistance to both C. parasitica and P. cinnamomi, a diverse Graves backcross population will be screened for resistance to P. cinnamomi, survivors bred with trees selected for resistance to C. parasitica, and progeny selected for resistance to both pathogens will be intercrossed.

Transcriptome Analysis of Chinese Chestnut (Castanea mollissima Blume) in Response to Dryocosmus kuriphilus Yasumatsu Infestation.

Chinese chestnut (Castanea mollissima Blume) can be infested by Dryocosmus kuriphilus Yasumatsu, resulting in gall formation and yield losses. Research on the control of gall wasps using genomics approaches is rarely reported. We used RNA-seq to investigate the dynamic changes in the genes of a chestnut species (C. mollissima B.) during four gall-formation stages caused by D. kuriphilus. A total of 21,306 genes were annotated by BLAST in databases. Transcriptome comparison between different gall-formation stages revealed many genes that were differentially expressed compared to the control. Among these, 2410, 7373, 6294, and 9412 genes were differentially expressed in four gall-formation stages: initiation stage (A), early growth stage (B), late growth stage (C), and maturation stage (D), respectively. Annotation analysis indicated that many metabolic processes (e.g., phenylpropanoid biosynthesis, secondary metabolism, plant⁻pathogen interaction) were affected. Interesting genes encoding putative components of signal transduction, stress response, and transcription factors were also differentially regulated. These genes might play important roles in response to D. kuriphilus gall formation. These new data on the mechanism by which D. kuriphilus infests chestnuts could help improve chestnut resistance.

Tracking the origin and dispersal of the Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu (Hymenoptera, Cynipidae) in Europe with molecular markers.

The Asian chestnut gall wasp, Dryocosmus kuriphilus, is an invasive pest causing significant damage to chestnut trees (Castanea spp., Fagaceae). Originating from China, it has recently invaded a wide range of regions in Europe and North America. Understanding the population genetic structure of important invasive pests is very useful for improving the knowledge concerning routes of expansion and colonizing capacity. Despite its economic importance, limited attention has been given to D. kuriphilus origin and spread, or to its genetic structure. In this study, D. kuriphilus populations sampled in eight European countries were screened using both mitochondrial (cytochrome c oxidase subunit 1; COI) and nuclear (internal transcribed spacer 2; ITS2) sequences, and Amplified Fragment Length Polymorphism (AFLP) markers. The molecular markers COI and ITS2 highlighted the presence of a single haplotype in all the studied populations. The recorded mitochondrial haplotype was identical to one of the most widespread haplotypes occurring in the native area (China). AFLP results indicated that D. kuriphilus individuals belong to two genetically distinct clusters without any further geographic clustering. These results suggest that D. kuriphilus populations in Europe could be the result of a single introduction of a Chinese founder population characterized by two genetically distinct lineages that subsequently spread rapidly across Europe. However, the possibility that populations originated from multiple introductions of the same Chinese mitochondrial haplotype cannot be excluded. The reported results provide useful information concerning this invasive species, potentially facilitating integrated pest management.

Looking for variable molecular markers in the chestnut gall wasp Dryocosmus kuriphilus: first comparison across genes.

The quick spread of the chestnut gall wasp Dryocosmus kuriphilus in Europe constitutes an outstanding example of recent human-aided biological invasion with dramatic economic losses. We screened for the first time a set of five nuclear and mitochondrial genes from D. kuriphilus collected in the Iberian Peninsula, and compared the sequences with those available from the native and invasive range of the species. We found no genetic variability in Iberia in none of the five genes, moreover, the three genes compared with other European samples showed no variability either. We recorded four cytochrome b haplotypes in Europe; one was genuine mitochondrial DNA and the rest nuclear copies of mitDNA (numts), what stresses the need of careful in silico analyses. The numts formed a separate cluster in the gene tree and at least two of them might be orthologous, what suggests that the invasion might have started with more than one individual. Our results point at a low initial population size in Europe followed by a quick population growth. Future studies assessing the expansion of this pest should include a large number of sampling sites and use powerful nuclear markers (e. g. Single Nucleotide Polymorphisms) to detect genetic variability.

First interspecific genetic linkage map for Castanea sativa x Castanea crenata revealed QTLs for resistance to Phytophthora cinnamomi.

The Japanese chestnut (Castanea crenata) carries resistance to Phytophthora cinnamomi, the destructive and widespread oomycete causing ink disease. The European chestnut (Castanea sativa), carrying little to no disease resistance, is currently threatened by the presence of the oomycete pathogen in forests, orchards and nurseries. Determining the genetic basis of P. cinnamomi resistance, for further selection of molecular markers and candidate genes, is a prominent issue for implementation of marker assisted selection in the breeding programs for resistance. In this study, the first interspecific genetic linkage map of C. sativa x C. crenata allowed the detection of QTLs for P. cinnamomi resistance. The genetic map was constructed using two independent, control-cross mapping populations. Chestnut populations were genotyped using 452 microsatellite and single nucleotide polymorphism molecular markers derived from the available chestnut transcriptomes. The consensus genetic map spans 498,9 cM and contains 217 markers mapped with an average interval of 2.3 cM. For QTL analyses, the progression rate of P. cinnamomi lesions in excised shoots inoculated was used as the phenotypic metric. Using non-parametric and composite interval mapping approaches, two QTLs were identified for ink disease resistance, distributed in two linkage groups: E and K. The presence of QTLs located in linkage group E regarding P. cinnamomi resistance is consistent with a previous preliminary study developed in American x Chinese chestnut populations, suggesting the presence of common P. cinnamomi defense mechanisms across species. Results presented here extend the genomic resources of Castanea genus providing potential tools to assist the ongoing and future chestnut breeding programs.

Does Gnomoniopsis castanea contribute to the natural biological control of chestnut gall wasp?

Gnomoniopsis castanea has been reported as the causal agent of necrosis of chestnut wasp (Dryocosmus kuriphilus) galls. The fungus is frequently observed on galls in chestnut stands infested by the insect in Italy. In the present study the impact of gall necrosis and the dynamic of its development have been studied in mature and young Castanea sativa stands in Central Italy during spring and early summer, before the D. kuriphilus adult flies. Results suggest that gall necrosis develops from resident endophytic inoculum of G. castanea. During the 2 y of monitoring, no differences were found in incidence and severity of the disease. Gall necrosis increased exponentially during the season, reaching 75,4% of galls totally necrotized in the investigated site in mid July. Gall necrosis was shown to have a severe impact on D. kuriphilus vitality, mostly impacting the adults inside the galls. Gall necrosis by G. castanea appears to efficiently control gall wasp in chestnut stands, although the high virulence of the fungus to chestnut fruits precludes its use as biocontrol agent in biological control strategies.

Host Preference and Performance of the Yellow Peach Moth (Conogethes punctiferalis) on Chestnut Cultivars.

Suitability of plant tissues as food for insects varies from plant to plant. In lepidopteran insects, fitness is largely dependent on the host-finding ability of the females. Existing studies have suggested that polyphagous lepidopterans preferentially select certain host plant species for oviposition. However, the mechanisms for host recognition and selection have not been fully elucidated. For the polyphagous yellow peach moth Conogethes punctiferalis, we explored the effect of chestnut cultivar on the performance and fitness and addressed the mechanisms of plant-volatile-mediated host recognition. By carrying out laboratory experiments and field investigation on four chestnut Castanea mollissima cultivars (Huaihuang, Huaijiu, Yanhong, and Shisheng), we found that C. punctiferalis females preferentially select Huaijiu for oviposition and infestation, and caterpillars fed on Huaijiu achieved slightly greater fitness than those fed on the other three chestnut cultivars, indicating that Huaijiu was a better suitable host for C. punctiferalis. Plant volatiles played important roles in host recognition by C. punctiferalis. All seven chestnut volatile compounds, α-pinene, camphene, ß-thujene, ß-pinene, eucalyptol, 3-carene, and nonanal, could trigger EAG responses in C. punctiferalis. The ubiquitous plant terpenoids, α-pinene, camphene and ß-pinene, and their specific combination at concentrations and proportions similar to the emissions from the four chestnut cultivars, was sufficient to elicit host recognition behavior of female C. punctiferalis. Nonanal and a mixture containing nonanal, that mimicked the emission of C. punctiferalis infested chestnut fruits, caused avoidance response. The outcome demonstrates the effects of chestnut cultivars on the performance of C. punctiferalis and reveals the preference-performance relationship between C. punctiferalis adults and their offspring. The observed olfactory plasticity in the plant-volatile-mediated host recognition may be important for the forming of the relationship between yellow peach moth and chestnuts since it allows the polyphagous herbivores to adjust to variation in volatile emission from their host plants.

Abundance and Frequency of the Asiatic Oak Weevil (Coleoptera: Curculionidae) and Defoliation on American, Chinese, and Hybrid Chestnut (Castanea).

The Asiatic oak weevil, Cyrtepistomus castaneus Roelofs (Coleoptera: Curculionidae), is a nonnative defoliator of trees in the Fagaceae family in the United States but has not been studied on Castanea species in the southern Appalachian Mountains. Planted trees of Castanea dentata (Marsh.) Borkh. (Fagales: Fagaceae), Castanea mollissima Blume (Fagales: Fagaceae), and four hybrid breeding generations were evaluated in 2012 for insect defoliation and C. castaneus abundance and frequency. Defoliation was visually assessed throughout the growing season at two sites in the southern Appalachian Mountains (western North Carolina and eastern Tennessee). C. castaneus abundance and frequency were monitored on trees using beat sheets and emergence was recorded from ground traps. Asiatic oak weevils were more abundant and more frequently collected on American chestnut (Ca. dentata) and its most closely related BC3F3 hybrid generation than on the Asian species Ca. mollissima. In most months, C. castaneus colonization of hybrid generations was not significantly different than colonization of parental species. Frequency data for C. castaneus suggested that adults were distributed relatively evenly throughout the study sites rather than in dense clusters. Emergence of C. castaneus was significantly higher under a canopy dominated by Quercus species than under non-Quercus species or open sky. C. castaneus emergence began in May and peaked in late June and early July. These results may be useful for resource managers trying to restore blight-resistant chestnut to the Southern Appalachians while minimizing herbivory by insect pests.

New gall wasp species attacking chestnut trees: Dryocosmus zhuili n. sp. (Hymenoptera: Cynipidae) on Castanea henryi from southeastern China.

A new gall wasp species, Dryocosmus zhuili Liu et Zhu, is herein described from the southeastern Fujian province of China. The new species induces galls on trees of Henry's chestnut, Castanea henryi, which is also a native host for the notorious Oriental chestnut gall wasp (OCGW, Dryocosmus kuriphilus Yasumatsu). D. zhuili overlaps with OCGW in emergence time and induces galls morphologically similar to that of OCGW on similar plant parts. In a previous study, we reported considerable divergence between mtDNA CO1 (mitochondrial DNA Cytochrome c oxidase subunit I) sequences of these wasps and the true OCGW wasps and suggested the existence of a cryptic species. Herein, we confirm the identity of the new species based on morphological and biological differences and provide a formal description. Although the new species is relatively easily separated from OCGW on basis of morphology, field identification involving the two species can still be problematic because of their small body size, highly similar gall morphology, and other life history traits. We further discussed the potential of the new species to be a pest for the chestnut industry and the consequences of accidental introduction of this species into nonnative areas, especially with regard to the bisexual reproduction mode of the new species in contrast to the parthenogenetic reproduction mode of OCGW.

Resistance of Castanea mollissima Shuhe-WYL strain to Dryocosmus kuriphilus and its molecular mechanism.

The resistance of Castanea mollissima Shuhe-WYL strain to Dryocosmus kuriphilus and its molecular mechanism were examined. The larvae of D. kuriphilus were inoculated on the Shuhe-WYL and Qingzha strains, and mortality was observed and compared; the relative mRNA content of the OsCDPK2, receptor-like protein, OsNAC6 protein, KH domain protein, RNA-binding protein, and the bHLH genes was detected using real-time polymerase chain reaction, and then compared between the Shuhe-WYL and Qingzha strains. Phenylalanine ammonia-lyase content was detected by western blotting and compared between the inoculated Shuhe-WYL, non-inoculated Shuhe-WYL, and inoculated Qingzha strains. The mortalities of larvae inoculated on the bud, bracteal leaf, and cardiac lobe were lower in Shuhe-WYL than Qingzha at 48 and 96 h after inoculation; the contents of OsCDPK2, receptor-like protein, OsNAC6 protein, and bHLH in the cardiac lobe were higher in Shuhe-WYL than in Qingzha at 96 h after inoculation, but KH domain protein and RNA-binding protein were not significantly different. The content of phenylalanine ammonia-lyase in the cardiac lobe was higher in inoculated and non-inoculated Shuhe-WYL compared to inoculated Qingzha at 15, 30, 45, and 60 days, and higher in inoculated Shuhe-WYL than in non-inoculated Shuhe-WYL at 15, 30, 45, and 60 days. The content of phenylalanine ammonia-lyase in the cardiac lobe of inoculated Shuhe-WYL had no significant difference between at 60 and at 45 days; and was higher at 60 and 45 days than at 30 and 15 days; and was higher at 30 days than at 15 days (60≈45˃30˃15 days). The C. mollissima Shuhe-WYL strain was resistant to D. kuriphilus; high expression of OsCDPK2, receptor-like protein, OsNAC6 protein, and bHLH and phenylalanine ammonia-lyase may explain the mechanism.

A global phylogeny of leafmining Ectoedemia moths (Lepidoptera: Nepticulidae): exploring host plant family shifts and allopatry as drivers of speciation.

BACKGROUND: Host association patterns in Ectoedemia (Lepidoptera: Nepticulidae) are also encountered in other insect groups with intimate plant relationships, including a high degree of monophagy, a preference for ecologically dominant plant families (e.g. Fagaceae, Rosaceae, Salicaceae, and Betulaceae) and a tendency for related insect species to feed on related host plant species. The evolutionary processes underlying these patterns are only partly understood, we therefore assessed the role of allopatry and host plant family shifts in speciation within Ectoedemia. METHODOLOGY: Six nuclear and mitochondrial DNA markers with a total aligned length of 3692 base pairs were used to infer phylogenetic relationships among 92 species belonging to the subgenus Ectoedemia of the genus Ectoedemia, representing a thorough taxon sampling with a global coverage. The results support monophyletic species groups that are congruent with published findings based on morphology. We used the obtained phylogeny to explore host plant family association and geographical distribution to investigate if host shifts and allopatry have been instrumental in the speciation of these leafmining insects. SIGNIFICANCE: We found that, even though most species within species groups commonly feed on plants from one family, shifts to a distantly related host family have occasionally occurred throughout the phylogeny and such shifts are most commonly observed towards Betulaceae. The largest radiations have occurred within species groups that feed on Fagaceae, Rosaceae, and Salicaceae. Most species are restricted to one of the seven global biogeographic regions, but within species groups representatives are commonly found in different biogeographic regions. Although we find general patterns with regard to host use and biogeography, there are differences between clades that suggest that different drivers of speciation, and perhaps drivers that we did not examine, have shaped diversity patterns in different clades.

The evolution of bet hedging in response to local ecological conditions.

Genotypes that hedge their bets can be favored by selection in an unpredictably varying environment. Bet hedging can be achieved by systematically expressing several phenotypes, such as one that readily attempts to reproduce and one that procrastinates in a dormant stage. But how much of each phenotype should a genotype express? Theory predicts that evolving bet-hedging strategies depend on local environmental variation, on how the population is regulated, and on exchanges with neighboring populations. Empirically, however, it remains unknown whether bet hedging can evolve to cope with the ecological conditions experienced by populations. Here we study the evolution of bet-hedging dormancy frequencies in two neighboring populations of the chestnut weevil, Curculio elephas. We estimate the temporal distribution of demographic parameters together with the form of the relationship between fecundity and population density and use both to parameterize models that predict the bet-hedging dormancy frequency expected to evolve in each population. Strikingly, the observed dormancy frequencies closely match predictions in their respective localities. We also found that dormancy frequencies vary randomly across generations, likely due to environmental perturbations of the underlying physiological mechanism. Using a model that includes these constraints, we predict the whole distribution of dormancy frequencies whose mean and shape agree with our observed data. Overall, our results suggest that dormancy frequencies have evolved according to local ecological conditions and physiological constraints.

Detection of a hypersensitive reaction in the chestnut hybrid 'Bouche de Bétizac' infested by Dryocosmus kuriphilus Yasumatsu.

The purpose of the study was the identification of the mechanisms of resistance to Dryocosmus kuriphilus Yasumatsu in the hybrid-resistant cultivar 'Bouche de Bétizac' (Castanea sativa × Castanea crenata). Larvae and eggs of the insect are found in the buds of this cultivar at the end of winter, but there is no gall development after budburst. The hypothesis of the presence of a hypersensitive reaction (HR) in the buds was tested using diaminobenzidine (DAB) to detect H(2)O(2) and by Real Time PCR (RT-PCR) to evaluate the expression of a germin-like protein gene. HR in plants is elicited by the production of reactive oxygen compounds, such as H(2)O(2), and results in the programmed cell death. The DAB test was applied to buds of 'Bouche de Bétizac' and of the susceptible cultivar 'Madonna' (C. sativa) at different stages of budburst. The DAB staining produced brown areas in the swelling buds of 'Bouche de Bétizac', indicating the presence of H(2)O(2). On the contrary, all uninfested buds, as well as the infested buds of 'Madonna', appeared whitish. Papers report that germin and germin-like proteins (GLP) with oxalate oxidase activity are discrete markers of stress-responsive gene products. A strong expression of the chestnut GLP gene was detected by RT-PCR at bud swelling in infested 'Bouche de Bétizac' buds but not in 'Madonna' ones. The results support the hypothesis of the occurrence of an HR in 'Bouche de Bétizac' as response to the cynipid infestation, resulting in cell and larvae death.

Quick detection of Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) in chestnut dormant buds by nested PCR.

Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) develops in chestnut buds that remain asymptomatic from oviposition (June-July) until budburst; it is, thus, easily spread by plant material used in propagation. Therefore, it is particularly interesting to identify infested plant batches before their movement. Unfortunately, a non-destructive method for checking buds has not yet been developed, and the only technique available is the screening of a bud sample. The visual investigation is long and requires highly skilled and trained staff. The purpose of this work was to set up an effective and fast method able to identify the presence of first instar larvae of D. kuriphilus in a large number of chestnut buds by PCR. Four primer pairs were designed on nuclear and mitochondrial sequences of a set of seven gall wasp taxa and tested on five different cynipid's DNA. Nested diagnostic PCR was carried out on DNA extracted from samples of 2 g buds simulating four levels of infestation (larvae were added to uninfested buds); 320 bp amplicon of 28S sequence was chosen as a marker to detect one larva out of 2 g buds. The method showed a potential efficiency of 5000 to 15,000 buds per week, depending on bud size.