European Hazelnut Research Articles

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.

Desiccation sensitivity and successful cryopreservation of oil seeds of European hazelnut (Corylus avellana)

AbstractThe sensitivity of a Polish provenance of dormant Corylus avellana seeds to extreme desiccation and cryopreservation in liquid nitrogen (LN, −196°C) was investigated. No sensitivity to desiccation was observed as seeds readily germinated and exhibited seedling emergence, even when the critical water content (WC) of seeds (nuts devoid of pericarp) was reduced to 0.027 g H2O/g dry mass, g g−1 by drying. Results of germination and seedling emergence tests indicated that seeds tolerated cooling in LN when desiccated to a WC in the range of 0.05–0.10 and 0.08–0.10 g g−1, respectively. The results of this study demonstrate the feasibility of long‐term cryopreservation of European hazelnut seeds. As the seeds of this species have been classified in several different categories (orthodox, suborthodox and recalcitrant), based on their response to desiccation and low temperature, the assignment of the seeds of hazelnut to a specific category is provided and discussed.

European Hazelnut and Almond Genetic Resources: Safeguard and Traditional Uses

The extensive worldwide interest in plant genetic resources regarding the opportunities offered by their use is clearly described by the objectives set out in both the Convention on Biological Biodiversity (CDB) and the FAO International Treaty on Plant Genetic Resources for Food and Agriculture (PGRFA). This article presents the European AGRI GEN RES SAFENUT project, including its methods and results, as an example of a resourceful strategy for reorganizing and sharing hazelnut and almond genetic resources. The project emphasizes how crucial it is to preserve not only genetic resources per se, but also the unique cultural value of the traditional and historical uses of hazelnut and almond genetic resources, which people have conserved and, in some cases, enhanced.

A Real-Time PCR Assay for Early Detection of Eastern Filbert Blight.

Eastern filbert blight (EFB) is a devastating disease of European hazelnut, Corylus avellana, which causes economic losses in Oregon, where 99% of the U.S. crop is produced. The causal fungus, Anisogramma anomala, is native to eastern North America, where it is found associated with the American hazelnut (C. americana). Although C. americana is tolerant, EFB causes cankers, branch dieback, and death of C. avellana. Detection and identification of A. anomala is time consuming using conventional methods because the fungus can only be cultured from sporulating perithecia and the disease symptoms and signs only show 12 to 16 months after infection. In this study, a TaqMan real-time polymerase chain reaction (PCR) assay based on a ribosomal DNA internal transcribed spacer was developed for A. anomala. The assay was validated with multiple isolates of A. anomala, closely related species, common environmental microorganisms, and over 100 C. avellana samples. The real-time PCR assay detected as low as 0.12 pg of A. anomala genomic DNA, and positively diagnosed EFB on 82% of asymptomatic plants as early as 15 weeks from infection. The real-time PCR assay is more sensitive and faster than traditional diagnostic methods. It can facilitate hazelnut breeding and disease management by early and accurate diagnosis of EFB.

Microwave Irradiation for Dry-Roasting of Hazelnuts and Evaluation of Microwave Treatment on Hazelnuts Peeling and Fatty Acid Oxidation

<p>European hazelnut is an important nut crop in Italy, where about 121,750 tons of in-shell nuts are produced every year. Roasting is the most important practice for hazelnut preservation and commonly is carried out in commercial electrical ovens at 120-160°C for 10-20 min. This needful practice is time and energy expensive, so the development of new processing methods is required to reduce processing costs and to obtain top quality roasted nuts. The aim of this study was to develop a simple microwave treatment for hazelnuts peeling and roasting.</p> <p>With this aim, some physical (colour, temperature, moisture) and chemical (taste, lipoxygenase activity, fatty acids, vitamins, sensory attributes) features of inshell nuts and kernels of three Italian hazelnut varieties (Tonda di Giffoni, Tonda Romana and Nocchione) after conventional oven or microwave roasting were evaluated.</p> <p>Results showed that microwave roasting of kernels for 450 s gave a higher peeling score than the conventional oven treatment. This paralleled with better colour and taste scores for microwaved roasted kernels. Furthermore, a 360-450 s microwave roasting was able to inactivate almost completely lipoxygenases, avoiding adverse effects on fatty acids hydroperoxides and PUFA content. A shorter microwave treatment (360 s) was enough to obtain good peeling and sensory scores of inshell hazelnuts.</p> Taken together our results indicated that microwave technology can be successfully applied to both kernels and inshell hazelnuts to obtain suitable peeling and high quality roasted nuts.

De novo sequencing of hazelnut bacterial artificial chromosomes (BACs) using multiplex Illumina sequencing and targeted marker development for eastern filbert blight resistance

Bacterial artificial chromosome (BAC) libraries are widely used in map-based cloning of plant genes. Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Muller, is a devastating disease of European hazelnut (Corylus avellana L.) in the Pacific Northwest. A dominant allele at a single locus from the obsolete pollenizer “Gasaway” confers complete resistance. Our map-based cloning efforts use a BAC library for “Jefferson” hazelnut, which is heterozygous for resistance. Screening the library with primer pairs designed from RAPD markers closely linked to the EFB resistance locus identified 38 BACs. We sequenced 28 of these BACs using Illumina technology, by multiplexing with barcoded adapters. De novo sequence assembly using the programs Velvet and SOPRA and further alignment using CodonCode Aligner generated contigs whose length ranged from 393 to 108,194 bp. The number of contigs per BAC ranged from 1 to 19, and estimated coverage of assembled BACs ranged from 64 % to 100 %. Preliminary analysis of the sequences identified 779 simple sequence repeats (SSRs), from which we developed 23 markers. Of these, 17 were assigned to linkage group 6 adjacent to the disease resistance locus, five were placed on other linkage groups, and one could not be assigned to a linkage group. The BAC sequences and new SSR markers will be useful for our efforts at map-based cloning of the disease resistance gene.

Eastern Filbert Blight-resistant Hazelnuts from Russia, Ukraine, and Poland

Stable genetic resistance to the fungal disease eastern filbert blight (EFB), caused by Anisogramma anomala, is vital for sustainable production of European hazelnut (Corylus avellana) in eastern North America. In this study, new hazelnut germplasm from the Russian Federation, Ukraine, and Poland (a total of 1844 trees from 66 seed lots) was subjected to A. anomala under field conditions over at least five years in New Jersey. Plants were then rated for the presence of EFB using an index of 0 (no disease) through 5 (all stems containing cankers). Nuts of the resistant trees were evaluated to identify plants with improved kernel characteristics. Genomic DNA of these trees was also screened with sequence-characterized amplified region (SCAR) markers generated by the primers BE-03, BE-33, and BE-68, which are closely linked to the single dominant R-gene of ‘Gasaway’, to assess the resistant seedlings for the presence of this well-known source of resistance. At final evaluation, 76 trees remained free of disease with nine expressing only minor symptoms (rating 1 or 2). The resistant trees spanned 24 different seed lots representing all three countries. The remaining trees ranged from moderately to severely infected with 81% of the total collection rating 5. Several of the resistant trees were found to produce commercial-sized (≈12 mm diameter), round kernels that blanched well. Although the results of the ‘Gasaway’ SCAR primers were inconclusive, the diverse collection origins and disease phenotypes provide evidence that novel sources of resistance were likely identified in this study. These new plants should broaden the genetic base of EFB-resistant C. avellana hazelnut germplasm available for breeding.

Cytogenetic characterization by in situ hybridization techniques and molecular analysis of 5S rRNA genes of the European hazelnut (Corylus avellana)

The European hazelnut (Corylus avellana L.) is widespread in Europe, where it has been cultivated for centuries. Despite progress in genetics, most of the cytogenetic aspects of this species have been overlooked. The aim of this study was to fill in this gap and obtain basic information on the chromosome structure of this species. Karyomorphological analysis confirmed the chromosome number 2n = 22 and showed that, despite their apparent uniformity, the chromosomes could be separated into three groups of different size: large (L), medium (M), and small (S). As a first step towards the physical mapping of the hazelnut chromosomes, we applied FISH to localize the position of rRNA genes (rDNA). The sites of 45S and 5S rDNA enabled us to identify two chromosome pairs belonging, respectively, to the L and S groups. The self-GISH procedure revealed that repetitive DNA is concentrated in the pericentromeric regions of the chromosomes, as with other species with rather small genomes. The analysis of 5S rDNA repeats offered additional information on the hazelnut genome by obtaining the whole sequence of the transcribed region so far unpublished. The overall results constitute a substantial advance in hazelnut cytogenetics. Further investigation of other species of Corylus could be an effective approach to understanding the phylogenesis of the genus and resolving taxonomic problems.

A Quick and Simple Method to Evaluate Anisogramma anomala Ascospore Viability

Viability of ascospores of Anisogramma anomala, cause of eastern filbert blight on European hazelnut, was assessed using the vital stain trypan blue (working solution of 0.05% in lactoglycerol). Viable ascospores only had faint blue staining around their cell walls while non-viable ascospores absorbed the stain and turned dark blue. The number of viable (non-stained) ascospores as determined by trypan blue was similar to the proportion of ascospores germinating on culture media. Viability of field collected ascospores from rainwater spore traps ranged from 41 to 68%. Disease incidence of hazelnut seedlings was more closely related to differences in ascospore abundance than to differences in ascospore viability. Accepted for publication 3 January 2013. Published 9 May 2013.

First Report of Shoot Blight and Canker Caused by Diplodia coryli in Hazelnut Trees in Chile.

Shoot blight, twig and branch cankers with grayish discoloration, roughened tissue, and dieback were observed in 5-year-old European hazelnut trees (Corylus avellana L.) cv. Barcelona at two commercial plantations in the Allipén locality, Region de La Araucanía (38° 59' 24.76″ S, 72° 29' 35.58″ W), Chile, during the 2011 to 2012 growing season. In addition, discoloration of the wood was observed in cross-sections. In order to isolate the causal agent, entire twigs and shoot fragments with cankers were sterilized in 0.5% sodium hypochlorite for 2 minutes, followed by two rinses with sterile distilled water. The tissues were then stored in a humid chamber. Sub-epidermal black pycnidia with sporulation were detected on the symptomatic tissue. Conidia were transferred to potato dextrose agar (PDA) (Difco, Lawrence, KS) and incubated at 25°C in the dark. The mycelia were black, creeping, and compact in appearance. The mature conidia were dark brown with a single septum, slightly constricted at the septum, and ovoid with a broadly rounded apex; some had a truncated base. Conidia had the following measurements: (20.0-) 23.1 ± 1.9 (-28.0) × (10-) 11.9 ± 1.2 (-15) μm with an average length/width ratio of 1.95 ± 0.17 (n = 50). These morphometric characteristics correspond to those of Diplodia coryli Fuckel (1870), teleomorph: Botryosphaeria sensu lato. The identity of the fungus was confirmed using internal transcribed spacer (ITS) rDNA sequencing completed at CABI, United Kingdom. The sequencing report indicated that the isolate (IMI-501235a) had 100% homology with a reference strain (CBS 242.51) in the CBS collection. The obtained sequence was deposited in GenBank (Accession No. JX163116). The anamorphs of Botryosphaeria have been divided into up to 18 genera (1), many of which are not clearly defined. Diplodia (3), including D. coryli (CBS 242.51) and D. juglandis (CBS 188.87), have been included within the genus Dothiorella (2), but the taxon names have not been formally changed. A pathogenicity test was conducted with one isolate (IMI-501235a) and four 1-year-old plants of hazelnut cultivar Barcelona. Plants were maintained in individual bags in greenhouse conditions (14/10 hours dark/light, 20°C; 70% RH). Prior to inoculation, plant tissues were externally disinfected with sodium hypochlorite (2%) and rinsed with sterile distilled water. Each plant was inoculated at fresh wound sites on two shoots and three twigs around each vegetative bud. The inoculum consisted of an agar plug with mycelia (5 mm) from the margin of an actively growing colony cultured on PDA media for 7 days. Each wound was covered with moistened cotton and sealed with Parafilm; a control plant was inoculated in the same way with agar only. After 3 months, fragments of necrotic and discolored vascular system tissues from inoculated shoots were removed and incubated on PDA. D. coryli was consistently recovered from these tissues, satisfying Koch's postulates. The control plant showed no symptoms of the disease. D. coryli has been reported to cause symptoms of dieback (dead branches) in Italy and Spain. To our knowledge, this is the first report of D. coryli on C. avellana cv. Barcelona in Chile. European hazelnut is an emerging crop in Chile, grown mainly for export, and management strategies for this disease will need to be developed.

Assembly and Characterization of the European Hazelnut ‘Jefferson’ Transcriptome

ABSTRACTEuropean hazelnut (Corylus avellana L.) is of worldwide agricultural significance, with breeding efforts focused on combining high nut yield and nut quality with resistance to diseases such as eastern filbert blight (EFB), a cause of severe crop loss in much of the United States. Oregon State University recently released a resistant cultivar, ‘Jefferson’ (OSU 703.007), that was chosen for transcriptome sequencing to establish further genomic resources for C. avellana L. We used Illumina ribonucleic acid sequencing (RNA‐seq) to characterize complementary DNA (cDNA) libraries from four hazelnut tissues, including leaves, catkins, bark, and whole seedlings. The 6.8 Gb of hazelnut transcriptome data was assembled de novo into 28,255 contigs with an average length of 532 bp and an N50 (the minimum contig length necessary such that all contigs of equal or greater length will equal half of the bases of the assembly) of 961 bp. Sequence comparisons using BLASTX and gene ontology (GO) classifications were used to generate automated descriptive function annotations. High similarity of the predicted proteins to sequences in related plants demonstrates the validity of the transcript contigs, with 80.8% having similarity to grape (Vitis vinifera L.), poplar (Populus trichocarpa Torr. & A. Gray), and castor bean (Ricinus communis L.) sequences in the public domain. A survey of GO terms enriched among tissue‐specific transcripts further validates the assembly. A basic local alignment search tool (BLAST) portal and web resources (http://hazelnut.cgrb.oregonstate.edu [accessed 8 Jan. 2010]) are available and will be of importance to breeders for marker‐assisted breeding efforts.

Eastern Filbert Blight Susceptibility of American × European Hazelnut Progenies

Eastern filbert blight (EFB), caused by Anisogramma anomala, is a devastating disease of Corylus avellana, the European hazelnut of commerce, and is considered the primary limiting factor of production in eastern North America. Conversely, C. americana, the wild American hazelnut, is generally highly tolerant of EFB, although it lacks many horticultural attributes necessary for commercial nut production. Hybrids of C. americana and C. avellana combine the EFB resistance of the wild species with the improved nut quality of the European species. However, inheritance of EFB resistance from C. americana remains unclear with existing hybrids derived from a very limited selection of parents. To investigate this topic, C. americana and advanced-generation C. americana × C. avellana hybrids were crossed with susceptible C. avellana and the resulting seedlings exposed to EFB through field inoculations and natural disease spread. In the winter after their fifth growing season, plants were rated for the presence of EFB using an index of 0 (no disease) through 5 (all stems containing cankers). The three progeny related to C. americana ‘Rush’ segregated for resistance in a ratio of one resistant to one susceptible, suggesting the presence of a single dominant R gene. A wide array of disease responses was observed for the other progenies with some expressing little EFB resistance or tolerance and others showing a distribution of disease phenotypes typical of control by multiple genes. Overall, the results indicate that both qualitative and quantitative resistance is present in C. americana. They also suggest that the choice of C. americana parent as well as the C. avellana parent will play a significant role in obtaining useful levels of EFB resistance in hybrid offspring, although the degree of disease expression in the parents may not be a useful predictor of progeny performance. Thus, more research is needed to understand inheritance of resistance, especially in advanced-generation backcrosses to susceptible C. avellana.

Pheromone detection of the introduced forest pest Megaplatypus mutatus (=Platypus mutatus) (Chapuis) (Platypodinae, Curculionidae) in Italy

Megaplatypus mutatus (Chapuis) (=Platypus mutatus), an ambrosia beetle native to South America, attacks standing live trees of a wide range of forest and fruit tree species, and it is particularly damaging to commercial poplar plantations. In 2000, M. mutatus was observed for the first time in Italy, in the province of Caserta, near Naples. The development of a pheromone-based monitoring system, for detecting the spread and for managing M. mutatus infestations, is an important goal for both European and South American control and surveillance programs. Using a three component pheromone blend developed in Argentina into commercial funnel traps we were able to asses the level of dispersion of this pest in the Italian Campania region. Insects were captured in all the plantations suspected of being infested based on the presence of active parental and larval galleries. We also provide the first report of the attack followed by completion of the life cycle of M. mutatus in European hazelnut, Corylus avellana L. (Betulaceae), an important nut species native to Europe and Western Asia.

Identification and Mapping of DNA Markers Linked to Eastern Filbert Blight Resistance from OSU 408.040 Hazelnut

Eastern filbert blight (EFB) of European hazelnut (Corylus avellana L.), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a major disease problem and production constraint in Oregon’s Willamette Valley. Host genetic resistance is viewed as the most economical means of controlling this disease. Marker-assisted selection has been extensively used for ‘Gasaway’ resistance in the hazelnut breeding program at Oregon State University (OSU). Concern over potential breakdown of this single resistance gene prompted a search for new sources of resistance. Selection OSU 408.040 showed no signs or symptoms of the fungus after a series of disease inoculations, and resistance was transmitted to half of its offspring, indicating control by a dominant allele at a single locus. In this study, we identified six random amplified polymorphic DNA (RAPD) and 11 simple sequence repeat (SSR) markers linked to EFB resistance from OSU 408.040. The new markers supplement the previously identified amplified fragment length polymorphism (AFLP) markers. A linkage map constructed in the progeny OSU 245.098 × OSU 408.040 spanned a distance of 19.5 cM with the resistance locus cosegregating with AFLP marker A8-150 and located between SSR markers LG675 and LG682. Using SSR markers as anchor loci, OSU 408.040 resistance was assigned to linkage group 6 (LG6). Comparison with the previously mapped ‘Gasaway’ resistance locus showed that resistance from OSU 408.040 maps to the same location.

A bacterial artificial chromosome library for ‘Jefferson’ hazelnut and identification of clones associated with eastern filbert blight resistance and pollen–stigma incompatibility

European hazelnut (Corylus avellana L.) is the only economically important nut crop in the family Betulaceae. Because of its small genome size (~385 Mb / 1C), relatively short life cycle, availability of a dense linkage map, and amenability to transformation by Agrobacterium, the European hazelnut could serve as a model plant for the Betulaceae. Here we report the construction of a bacterial artificial chromosome (BAC) library for 'Jefferson' hazelnut using the cloning enzyme MboI and the vector pECBAC1 (BamHI site). The library consists of 39,936 clones arrayed in 104,384-well microtitre plates with a mean insert size of 117 kb. The genomic coverage of the library is estimated to be about 12 genome equivalents. This library provides a valuable resource for the map-based cloning of two important genes, the resistance gene from 'Gasaway' that confers resistance to eastern filbert blight caused by the fungus Anisogramma anomala (Peck) E. Müller and the S locus that controls pollen-stigma incompatibility. Fine-resolution mapping near the two loci was carried out using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. Fine mapping at the disease resistance locus showed that markers W07-375 and X01-825 flanked the resistance locus at distances of 0.06 and 0.05 cM, respectively. The S locus is flanked by markers 204-950 and KG819-200 at distances of 0.14 and 0.24 cM, respectively. Assuming that 1 cM corresponds to a physical distance of 430 kb, it will take approximately two to three chromosome walks to assemble BAC contigs that span both loci.

DNA Markers Linked to Eastern Filbert Blight Resistance from a Hazelnut Selection from the Republic of Georgia

The hundred-year history of the european hazelnut (Corylus avellana L.) industry in the Pacific northwestern United States is threatened by eastern filbert blight (EFB) caused by the fungus Anisogramma anomala (Peck) E. Müller. Marker-assisted selection has been extensively used for ‘Gasaway’ resistance in the hazelnut breeding program at Oregon State University. Concern over possible breakdown of this single resistance gene provides an incentive to look for new sources of resistance. OSU 759.010, a selection from the Republic of Georgia, has remained free of EFB after inoculations over several years. Random amplified polymorphic DNA (RAPD) markers linked to resistance were identified by screening primers against three resistant seedlings, three susceptible seedlings, and the parents of a segregating seedling population. For the progeny OSU 759.010 × OSU 653.068, 13 linked markers were identified. The markers most closely linked to resistance were 695-1800 on the proximal side and H12-640, 373-700, 349-450, and F08-700 on the distal side. Four of the five markers also segregated in the progeny OSU 759.010 × OSU 665.076, whereas H12-640 was monomorphic. Segregation for disease response in the first population showed a surplus of resistant seedlings, approaching a 3:1 ratio, with closely linked RAPD markers showing similar ratios. In the second population, the observed segregation for disease response and associated markers did not deviate from the expected 1:1 ratio. Based on cosegregation with simple sequence repeat (SSR) markers, resistance from OSU 759.010 was assigned to linkage group 2. Resistance to EFB from ‘Gasaway’ and ‘Ratoli’ was previously mapped to linkage groups 6 and 7, respectively. Therefore, OSU 759.010 provides a novel source of EFB resistance and markers 695-1800, 373-700, 349-450, and F08-700 have potential for use in marker-assisted selection to pyramid EFB resistance alleles.

Characterization of American hazelnut (Corylus americana) accessions and Corylus americana × Corylus avellana hybrids using microsatellite markers

The American hazelnut (Corylus americana Marshall) is native to the eastern United States and Southern Canada. In the early decades of the last century, breeding work by several individuals attempted to combine the cold hardiness and disease resistance of the American hazelnut with the larger nut size of the European hazelnut (C. avellana L.). Many hybrid selections grown today trace back to these early efforts. Over the past three decades, representatives of C. americana were collected and are preserved in Corvallis, Oregon. Seeds were collected along roadsides, in hedgerows, in woodland clearings, and in gardens. The seeds were germinated and the best of the resulting seedlings were preserved for later use. In this study, genetic diversity was studied in 87 American and 67 hybrid hazelnut selections using 21 microsatellite loci. The total number of alleles was 266 in the 162 accessions examined, of which 229 were present in C. americana accessions, but only 168 in the Arbor Day Farm hybrids, and 116 in the hybrids of C. americana ‘Rush’. In the American accessions, a high level of genetic diversity was observed (H e = 0.74, H o = 0.68). A genetic similarity matrix of the American and hybrid accessions, and a few European cultivars, was constructed and the resulting dendrogram revealed seven major groups: European cultivars and ‘Rush’ hybrids, two groups of Arbor Day Farm hybrids, three groups of C. americana accessions, and a mixed group of hybrid and American accessions. Analysis confirmed the reported parentage of 10 hybrids. The genetic diversity among the American hazelnut accessions, untapped to date, will be useful in breeding hybrids that will allow expansion of hazelnut production into eastern North America.

First Report of Eastern Filbert Blight on Corylus avellana 'Gasaway' and 'VR20-11' Caused by Anisogramma anomala in New Jersey.

Eastern filbert blight (EFB) is a serious disease of European hazelnut, Corylus avellana L., which causes economic losses in Oregon (OR) where 99% of the U.S. crop is produced. The causal organism, Anisogramma anomala (Peck) E. Müller, is native east of the Rocky Mountains where it is found in association with C. americana Marshall. While C. americana is tolerant, EFB causes cankering, branch dieback, and death of C. avellana (3). EFB was first discovered in Washington State in the late 1960s (1). Since then, it has spread throughout the Willamette Valley of OR. In OR, 'Gasaway', an obsolete pollinizer, shows complete resistance to EFB, conferred by a dominant allele at a single locus (4). 'Gasaway' has been widely used in breeding at Oregon State University (OSU) to develop resistant cultivars that are used in most new orchards. In January 2008, cankers containing rows of dark brown elliptical stroma, characteristic of EFB, were first observed on more than 25 trees of 'Gasaway' growing at the Rutgers University research farms in Adelphia and North Brunswick, NJ. At that time, cankers were also found on 18 trees of 'VR20-11' growing on the research farms. 'VR20-11,' an offspring of 'Gasaway' that carries the same resistance gene, was released by OSU for use as a pollinizer for 'Barcelona', an EFB-susceptible but widely grown cultivar in OR. Additional cankers were observed on the New Jersey trees in January 2009 and 2010. To our knowledge, this is the first report of EFB on either cultivar under field conditions. The cankers are smaller than those on susceptible cultivars. Of 61 cankers on 10 trees of 'Gasaway', the average length was 11 cm with a range of 4 to 42 cm. Canker lengths on susceptible trees are typically 20 to 100 cm. The cankers appear otherwise alike with stromata, 2 to 4 × 2 mm, up to 2 mm high; perithecia upright, in the lower part of stroma; asci ellipsoid, 35 to 45 × 9 to 12 μm; and ascospores 8 to 11 × 4 to 5.5 μm, hyaline, smooth, ellipsoid, 2-celled, with the lower cell very short (1 to 1.5 μm long and wide). Genomic DNA was isolated from ascospores excised from cankers of 'Gasaway' and 'VR20-11'. ITS1F and ITS2 primers were used to amplify and sequence the internal transcribed spacer 1 region (ITS1) of the rRNA genes (GenBank Accession Nos. HM565133 and HM565132). BLAST analysis of the 238-bp segments showed 99% homology with a sequence of A. anomala (EU683064). Phylogenetic analysis also confirmed that the two isolates are A. anomala. To test viability, 'Gasaway' cankers were excised and ascospore suspensions (1 × 106 spores ml-1) were applied to 15 trees of susceptible 'Barcelona' in March 2008 following the protocol of Johnson et al. (2). In December 2009, 12 of 15 inoculated trees expressed EFB. 'Gasaway' has shown no signs or symptoms of infection by A. anomala over several decades of exposure in OR, which is believed to have a limited diversity of the fungus due to a single-point introduction. Our findings suggest quarantine efforts must be bolstered to prevent further introductions of A. anomala into the Pacific Northwest to protect the viability of the U.S hazelnut industry.

DNA markers linked to eastern filbert blight resistance in “Ratoli” hazelnut (Corylus avellana L.)

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Muller, is a major disease problem and production constraint in orchards of European hazelnut (Corylus avellana L.) in Oregon’s Willamette Valley. Host genetic resistance is viewed as the most economical means of controlling this disease. A dominant resistance gene from “Gasaway” has been used extensively in the hazelnut breeding program at Oregon State University, but concern about the durability of a single resistance gene stimulated a search for new sources of resistance. “Ratoli,” a minor cultivar from Spain, showed no signs or symptoms of the fungus following a series of inoculations. The objective of this study was to study segregation for disease response in two progenies from crosses of Ratoli with susceptible selections and identify linked DNA markers. About half of the seedlings were resistant, suggesting control by a dominant allele at a single locus. A total of 900 random amplified polymorphic DNA (RAPD) primers and 64 amplified fragment length polymorphism (AFLP) primer combinations were screened. Four RAPD markers and two ALFP markers were identified and a linkage map constructed. On this map, disease resistance was flanked by AFLP marker C4-255 and RAPD marker G17-800 at distances of 0.4 cM and 2.8 cM, respectively. Based on co-segregation with SSR markers, Ratoli resistance was assigned to linkage group 7 while Gasaway resistance is on linkage group 6. Ratoli provides a novel source of EFB resistance, and robust RAPD marker G17-800 is useful for marker-assisted selection.

Response of Hazelnut Accessions to Greenhouse Inoculation with Anisogramma anomala

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a devastating disease of European hazelnut (Corylus avellana L.) in the Pacific Northwest. Host genetic resistance from ‘Gasaway’ has been used extensively for breeding hazelnuts at Oregon State University. Concern over the durability of this single-gene resistance prompted a search for new sources of resistance. In this study, 86 accessions from 11 countries were evaluated for their response to greenhouse inoculation with the pathogen. Nine accessions showed complete resistance, including one from Chile (‘Amarillo Tardio’), two from Serbia (‘Crvenje’ and ‘Uebov’), one from southern Russia (OSU 495.072) and five from Moscow, Russia. These new sources of EFB resistance have geographically diverse origins and will broaden the genetic base of EFB-resistant hazelnut germplasm. The previously reported resistance of ‘Grand Traverse’ from Michigan and the susceptibility of ‘Closca Molla’ from Spain were confirmed.