National Clonal Germplasm Repository Research Articles

Characterization of Genetic Diversity in the Capsid Protein Gene of Grapevine Fleck Virus and Development of a New Real-Time RT-PCR Assay.

The grapevine fleck virus (GFkV) is a ubiquitous grapevine-infecting virus found worldwide, is associated with the grapevine fleck complex, and is often found in mixed infections with viruses of the grapevine leafroll complex and/or vitiviruses. Although GFkV has been studied for a long time, limited sequence information is available in the public databases. In this study, the GFkV sequence data available in GenBank and data generated at the Foundation Plant Services, University of California, Davis, were used to perform nucleotide sequence comparisons, construct a phylogenetic tree, and develop a new RT-qPCR assay. Sequence comparisons showed high genetic diversity among the GFkV isolates, and the phylogenetic analyses revealed a new group comprised of GFkV isolates identified in the present study. A new assay, referred to as GFkV-CP, was designed and validated using an existing GFkV positive control together with 11 samples known to be infected with combinations of different marafiviruses and maculaviruses but not GFkV. In addition, the newly designed assay was used in a field survey to screen grapevines from diverse geographical locations that are maintained at the United States Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR) in Winters, CA.

Vulnerability of pear (Pyrus) genetic resources in the U.S.

AbstractPears (Pyrus) represent an important part of consumer diets, and have the fourth highest production of non-citrus fruits, measured by fresh weight, in the U.S. They are maintained clonally and grown as composite plants, consisting of a scion (fruit bearing) cultivar grafted onto a rootstock cultivar. Up to 98% of existing production relies on only a few scion and rootstock cultivars, leaving the standing crop vulnerable to threats. Pears are faced with a wide range of biotic and abiotic threats and production vulnerabilities, some of which can be limited by integrating resistance and horticultural traits from wild and cultivated materials from around the world. The National Clonal Germplasm Repository (NCGR Corvallis), part of the USDA-ARS National Plant Germplasm System, maintains a large Pyrus collection from across the globe, consisting of 2793 Pyrus accessions from 37 species. The collection represents an important resource for preservation, research, and breeding efforts for pears. The crop vulnerability status of pears in the U.S. is currently moderate to high, with increasing threats and challenges. Breeding and preservation efforts, along with genetic, crop protection and production research are, however, actively targeting these needs.

Genetic Characterization of European Plum (Prunus domestica L.) Accessions from Norway Using ECPGR-Selected SSR Markers

In order to ensure the long-term sustainability of the conservation process of Norwegian plum germplasm, as well as to enhance the possibility of its utilization, a central plum heritage cultivar collection was established in 2020. In this study, 40 plum accessions maintained at the Ullensvang plum heritage cultivar collection were genetically characterized using a set of nine microsatellite markers recently approved by the ECPGR Prunus working group. The obtained molecular data were used to investigate the genetic identity, diversity, and structure among the analyzed accessions. No redundancies were detected among the plum accessions, which is in stark contrast to the previous molecular study on plum samples collected through an on-farm inventory of Southern Norway. Furthermore, the obtained data indicate that the Ullensvang collection contains a significant genetic diversity of Norwegian plum germplasm, previously held in decentralized sites. With that in mind, this collection can certainly be considered for the role of the National Clonal Plum Germplasm Repository. The nine microsatellite markers, recommended by ECPGR, revealed a genetic structure not entirely tied to previously proposed pomological groups, possibly indicating a history of hybridization among members of the various groups.

Complete genome sequence of vaccinium-associated virus C, a new member of the family Totiviridae from Vaccinium floribundum.

Blueberries (Vaccinium sp.) are a major crop grown in the Pacific Northwest region. Currently, there are at least 17 known viruses that infect blueberry plants, and some of them cause a wide range of symptoms and economic losses. A new virus, vaccinium-associated virus C (VaVC) (family Totiviridae, genus Totivirus) was identified in an imported blueberry accession from the USDA-ARS National Clonal Germplasm Repository in Corvallis, Oregon. The complete genomic sequence of VaVC was determined, but the biological significance of VaVC is unknown and requires further study. Additional Vacciniumsp. accessions should be screened to investigate the incidence of this new virus.

A Field Survey of Grapevine Germplasm Susceptible to Eutypa Dieback

Eutypa dieback of grapevine is a trunk disease that impacts vineyard productivity worldwide. Grape germplasm is typically evaluated for resistance to Eutypa dieback through controlled inoculations in the greenhouse, although the high level of replication required of this approach (40 plants per genotype) can limit the total number of genotypes evaluated. An alternative approach is to evaluate naturally infected genotypes in the field. We rated the incidence and severity of vines with the diagnostic leaf symptoms of Eutypa dieback and the incidence of mortality among such vines of 973 Vitis vinifera accessions (planted in duplicate) at the US Department of Agriculture, National Clonal Germplasm Repository in Davis, CA, USA, which is maintained as a living collection for grape research. Across 3 years and spanning a total of 5 years (2011, 2013, and 2015), 120 accessions had leaf symptoms in one or more years (“susceptible accessions”). Courbu blanc [Davis Vitis identification tag (DVIT) 2313], Frankenthal blanc (DVIT 2115), and Pinot gris (DVIT 0907) were the only accessions with leaf symptoms each year. Accessions with the most severe leaf symptoms (a rating of 5 points) were Chasselas Napoleon (DVIT 0375) and Queen of the Vineyard (DVIT 0496). We identified susceptible accessions—namely, those related to ‘Chasselas’ and ‘Muscat’—with a shared genetic background, based on a previous single nucleotide polymorphism genotyping effort of the collection. Especially for grapevine, a long-lived perennial that is meant to produce a crop for decades, knowledge of susceptible accessions and their pedigrees can help inform breeding programs and studies on the host response to infection.

Impact of laurel wilt on the avocado germplasm collection at the United States Department of Agriculture, Agricultural Research Service, Subtropical Horticulture Research Station

Abstract In late 2020, laurel wilt impacted the avocado collection at the United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Subtropical Horticulture Research Station in Miami, Florida. Monitoring of the ambrosia beetle (Coleoptera: Curculionidae) community over the next 18 months suggested that the primary vector, Xyleborus glabratus Eichhoff, was likely not responsible for spreading the fungal pathogen (Harringtonia lauricola [T.C. Harr., Fraerich & Aghayeva] Z.W. de Beer & M. Procter; Ophiostomatales). Rather, eight secondary vectors were detected in greater numbers, with Xyleborinus saxesenii (Ratzeburg) being the most abundant. After detection of laurel wilt, beetle numbers increased for 2–4 months, then gradually declined over the next year. This pest suppression was likely due to several countermeasures, including the prompt removal of infected trees and prophylactic macroinfusions with propiconazole fungicides. In addition, to preserve the genetic diversity of the Miami avocado germplasm, the Subtropical Horticulture Research Station is collaborating with other Agricultural Research Service facilities to establish backup collections at alternative locations free of the laurel wilt pathogen and its beetle vectors.

A Novel, Divergent Member of the Rhabdoviridae Family Infects Strawberry.

Strawberry (Fragaria × ananassa) is the most important berry crop worldwide and viruses pose a constant threat to the industry. In this communication, we describe a novel virus in the family Rhabdoviridae referred to as strawberry virus 3 (StrV-3). The virus does not show significant homology when compared with recognized rhabdoviruses and, therefore, the establishment of a new genus should be considered. A triplex reverse-transcription PCR test was developed and successfully employed in a survey of the National Clonal Germplasm Repository Fragaria collection. A CRISPR-Cas-based protocol was also developed and shown to detect the virus in as little as 1 fg of total RNA, a protocol to be used in the detection of the virus in candidate G1 plants. The strawberry aphid (Chaetosiphon fragaefolii) was evaluated-alas, unsuccessfully-as a potential vector of the virus. This work broadens our understanding of the family Rhabdoviridae and assists in the quest of releasing plant material free of viruses.

Identification, Characterization, and Detection of a Novel Strawberry Cytorhabdovirus.

In 2020, a novel agent was discovered in strawberry, a rhabdovirus closely related to lettuce necrotic yellows virus. The new virus, named strawberry virus 2 (StrV-2), was discovered in an accession of the Fragaria virus collection of the National Clonal Germplasm Repository (NCGR), and for this reason, it was studied in-depth. The complete StrV-2 genome was obtained and investigated in silico. Transmission was assessed using two aphid species whereas a multiplex RT-PCR test targeting plant and virus genes was developed and used to screen the NCGR Fragaria virus collection.

Phenotypic characterization of a strawberry (Fragaria ×ananassa Duchesne ex Rosier) diversity collection

Societal Impact statementTo secure a stable, safe food supply, the future of humanity depends on the conservation and application of cultivated and wild plant diversity. Strawberries are globally important. They are widely produced and provide significant human health benefits. In 2019, annual world production was 14.5 MT (FAOSTAT, 2022). This report characterizes agricultural traits of diverse heritage strawberries. The gene pool of breeding programs has traditionally included a reliable but limited set of cultivars. The traits of lesser known heritage strawberry cultivars and selections examined by our study could be of value in expanding gene pools by tapping into existing cultivated genetic diversity.Summary Strawberries are a berry crop of global significance. We present a comprehensive evaluation of strawberry diversity through phenotyping 284 strawberry cultivars from the U.S. Department of Agriculture, National Clonal Germplasm Repository Corvallis, Oregon, USA. We assessed 284 cultivars in 2019 and 2020 for quantified variation across 16 traits, including phenology, plant morphology, and fruit quality traits at full ripeness. Through cluster analysis, we determined groups of accessions that had desirable traits, including quality fruits, earliest fruit ripening, or high numbers of daughter plant production. We observed that this strawberry diversity panel displayed a wide range of phenotypic variation that could prove useful for future strawberry improvement. ORUS 2427‐1, NW 90054‐37, and “Independence,” each developed by the Oregon USDA breeding program, displayed a combination of the highest fruit quality traits. “Independence” also had the longest runner length and the highest number of daughter plants. Two California cultivars, Oso Grande and Cuesta, and the Oregon cultivar Pinnacle had a short ripening time, on average 32 days between flowering and harvest, with the range for the study group of 25–41 days. Our data provide insight for breeders to expand gene pools, producers who choose cultivars for green‐wall production, or geneticists who need data to prepare genome‐wide association studies.

Genetic Identity and Diversity of Apple Accessions within a Candidate Collection for the Norwegian National Clonal Germplasm Repository

In order to best conserve, as well as utilize, traditional apple germplasm in Norway, an apple heritage cultivar collection was established in Ullensvang, western Norway, which aims to become the National Clonal Germplasm Repository. The establishment of the apple heritage cultivar collection was preceded by a molecular study that aimed to genotype a large number of apple accessions maintained in various ex situ sites in western and south-eastern Norway, using a rather small set of eight SSR markers. However limited, the marker set managed to identify synonyms, homonyms, and duplicates within and among the investigated collections. In this study, 171 apple accessions from the Ullensvang apple heritage cultivar collection were genotyped using a set of 20 different SSR markers. Approximately half of the accessions have been previously genotyped using eight SSR markers, enabling an assessment of whether the use of a larger marker set would yield a more accurate characterization. Based on the obtained molecular data, the apple heritage cultivar collection was determined to hold a key part of the overall genetic diversity of the Norwegian apple germplasm. Furthermore, the twelve additional SSR markers were able to differentiate several accessions groups originally thought to be synonyms, as well as to provide a more detailed insight into the genetic structure of this germplasm.

Two fingerprinting sets for Humulus lupulus based on KASP and microsatellite markers.

Verification of clonal identity of hop (Humulus lupulus L.) cultivars within breeding programs and germplasm collections is vital to conserving genetic resources. Accurate and economic DNA-based tools are needed in dioecious hop to confirm identity and parentage, neither of which can be reliably determined from morphological observations. In this study, we developed two fingerprinting sets for hop: a 9-SSR fingerprinting set containing high-core repeats that can be run in a single PCR reaction and a kompetitive allele specific PCR (KASP) assay of 25 single nucleotide polymorphisms (SNPs). The SSR set contains a sex-linked primer pair, HI-AGA7, that was used to genotype 629 hop accessions from the US Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR), the USDA Forage Seed and Cereal Research (FSCR), and the University of Nebraska-Lincoln (UNL) collections. The SSR set identified unique genotypes except for 89 sets of synonymous samples. These synonyms included: cultivars with different designations, the same cultivars from different sources, heat-treated clones, and clonal variants. Population structure analysis clustered accessions into wild North American (WNA) and cultivated groups. Diversity was slightly higher in the cultivated samples due to larger sample size. Parentage and sib-ship analyses were used to identify true-to-type cultivars. The HI-AGA7 marker generated two male- and nine female-specific alleles among the cultivated and WNA samples. The SSR and KASP fingerprinting sets were compared in 190 samples consisting of cultivated and WNA accession for their ability to confirm identity and assess diversity and population structure. The SSR fingerprinting set distinguished cultivars, selections and WNA accessions while the KASP assays were unable to distinguish the WNA samples and had lower diversity estimates than the SSR set. Both fingerprinting sets are valuable tools for identity confirmation and parentage analysis in hop for different purposes. The 9-SSR assay is cost efficient when genotyping a small number of wild and cultivated hop samples (<96) while the KASP assay is easy to interpret and cost efficient for genotyping a large number of cultivated samples (multiples of 96).

Nuclear DNA contents and ploidy levels of North American Vaccinium species and interspecific hybrids

Breeding strategies for improving blueberry (Vaccinium corymbosum and V. virgatum) cultivars often include introgressing regionally adapted species into the cultivated gene pools through interspecific hybridization. However, these approaches are complicated by variation in ploidy, triploid blocks and infertility, production of unreduced gametes, and aneuploidy. The objective of this study was to use flow cytometry, k-mer distribution analysis, and known pedigree information to evaluate genome sizes (2C nuclear and 1Cx monoploid), and ploidy of diverse accessions from Vaccinium sections and species. A total of 369 accessions, including a diversity panel (DP) of 251 inter- and intra-specific hybrid Vaccinium accessions, as well as 118 non-hybrid Vaccinium species across multiple sections, were sampled from the North Carolina State University blueberry breeding program and the National Clonal Germplasm Repository. The nuclear DNA content was analyzed via flow cytometry. The mean (range) DNA content of diploid, tetraploid, and hexaploid reference species were 1.20 pg (0.99 pg in V. crassifolium ‘Well's Delight’ to 1.41 pg in V. caesariense NC79–24), 2.37 pg (2.11 pg in V. corymbosum ‘Concord’ to 3.01 pg in V. corymbosum DE599), and 3.64 pg (3.24 in V. constablaei NC83–21–2 to 3.80 in V. virgatum ‘Premier’ and NC4790), respectively. Of the 369 unique accessions analyzed for ploidy, 259 were tetraploid, 46 were diploid, one was triploid, 51 were pentaploid or aneuploid with 2C values between tetraploid and hexaploid values, and 12 were hexaploid. Tetraploid hybrid pedigrees, which involved hexaploid crosses within three prior generations, had a 2C value range between 2.22 pg and 2.59 pg. Interspecific pentaploid and aneuploid progeny 2C DNA content ranged from 2.61 pg to 3.15 pg. We speculate some of these progeny to be near tetraploids with extra chromosomes from hexaploid progenitors. Further karyotyping of these individuals is necessary to ascertain aneuploidy anomalies. This research provides an expanded knowledge base of genome sizes, ploidy, and reproductive pathways for diverse species and hybrids to enhance future breeding, improvement, and the genomic study of blueberry.

Exploring the diversity and genetic structure of the U.S. National Cultivated Strawberry Collection.

The cultivated strawberry (Fragaria ×ananassa) arose through a hybridization of two wild American octoploid strawberry species in a French garden in the 1750s. Since then, breeders have developed improved cultivars adapted to different growing regions. Diverse germplasm is crucial to meet the challenges strawberry breeders will continue to address. The USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon maintains the U.S. strawberry collection. Recent developments in high-throughput genotyping for strawberry can provide new insights about the diversity and structure of the collection, germplasm management, and future breeding strategies. Genotyping was conducted on 539 F. ×ananassa accessions using either the iStraw35 or FanaSNP 50 K Axiom array. Data for markers shared by the two arrays were curated for call quality, missing data, and minor allele frequency resulting in 4033 markers for structure assessment, diversity analysis, pedigree confirmation, core collection development, and the identification of haplotypes associated with desirable traits. The F. ×ananassa collection was equally diverse across the different geographic regions represented. K-means clustering, sNMF, and UPGMA hierarchal clustering revealed seven to nine sub-populations associated with different geographic breeding centers. Two 100 accession core collections were created. Pedigree linkages within the collection were confirmed. Finally, accessions containing disease resistance-associated haplotypes for FaRCa1, FaRCg1, FaRMp1, and FaRPc2 were identified. These new core collections will allow breeders and researchers to more efficiently utilize the F. ×ananassa collection. The core collections and other accessions of interest can be requested for research from the USDA-ARS NCGR via the Germplasm Resources Information Network (https://www.ars-grin.gov/).

Olea europaea geminivirus is present in a germplasm repository and in California and Texas olive (Olea europaea L.) groves.

Olea europaea geminivirus (OEGV) from olive accessions in Italy was characterized recently. OEGV was also detected during routine high-throughput sequencing screening of olive (cv. Leccino) material, and its complete bipartite genome segments were sequenced and shown to be 100% identical to those of the isolate from Italy. Using two pairs of newly designed primers targeting the AV1 and BV1 genes, OEGV was detected in randomly sampled olive trees from the U.S. Department of Agriculture National Clonal Germplasm Repository (USDA-NCGR) (21.4% or 6/28), commercial and residential settings in California (47.6% or 10/21), and an orchard in Texas (60% or 30/50). The cuttings for the USDA-NCGR-positive trees originated from the former Serbia and Montenegro, Spain, Italy, and Greece. Comparative analysis of the directly sequenced gene fragments from randomly selected samples showed that OEGV isolates from the different sources were 100% identical to each other. The results indicate that OEGV spread was likely facilitated by inadvertent movement of contaminated olive germplasm.

Sequencing a Strawberry Germplasm Collection Reveals New Viral Genetic Diversity and the Basis for New RT-qPCR Assays.

Viruses are considered of major importance in strawberry (Fragaria × ananassa Duchesne) production given their negative impact on plant vigor and growth. Strawberry accessions from the National Clonal Germplasm Repository were screened for viruses using high throughput sequencing (HTS). Analyses of sequence information from 45 plants identified multiple variants of 14 known viruses, comprising strawberry mottle virus (SMoV), beet pseudo yellows virus (BPYV), strawberry pallidosis-associated virus (SPaV), tomato ringspot virus (ToRSV), strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV), strawberry crinkle virus (SCV), strawberry polerovirus 1 (SPV-1), apple mosaic virus (ApMV), strawberry chlorotic fleck virus (SCFaV), strawberry crinivirus 4 (SCrV-4), strawberry crinivirus 3 (SCrV-3), Fragaria chiloensis latent virus (FClLV) and Fragaria chiloensis cryptic virus (FCCV). Genetic diversity of sequenced virus isolates was investigated via sequence homology analysis, and partial-genome sequences were deposited into GenBank. To confirm the HTS results and expand the detection of strawberry viruses, new reverse transcription quantitative PCR (RT-qPCR) assays were designed for the above-listed viruses. Further in silico and in vitro validation of the new diagnostic assays indicated high efficiency and reliability. Thus, the occurrence of different viruses, including divergent variants, among the strawberries was verified. This is the first viral metagenomic survey in strawberry, additionally, this study describes the design and validation of multiple RT-qPCR assays for strawberry viruses, which represent important detection tools for clean plant programs.

Reconstruction of the Largest Pedigree Network for Pear Cultivars and Evaluation of the Genetic Diversity of the USDA-ARS National Pyrus Collection.

The USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, maintains one of the world’s largest and most diverse living Pyrus collection. A thorough genetic characterization of this germplasm will provide relevant information to optimize the conservation strategy of pear biodiversity, support the use of this germplasm in breeding, and increase our knowledge of Pyrus taxonomy, evolution, and domestication. In the last two decades simple sequence repeat (SSR) markers have been used at the NCGR for cultivar identification and small population structure analysis. However, the recent development of the Applied Biosystems Axiom Pear 70K Genotyping Array has allowed high-density single nucleotide polymorphism (SNP)-based genotyping of almost the entire collection. In this study, we have analyzed this rich dataset to discover new synonyms and mutants, identify putative labeling errors in the collection, reconstruct the largest pear cultivar pedigree and further elucidate the genetic diversity of Pyrus.

Crop Wild Relatives as Germplasm Resource for Cultivar Improvement in Mint (Mentha L.).

Mentha is a strongly scented herb of the Lamiaceae (formerly Labiatae) and includes about 30 species and hybrid species that are distributed or introduced throughout the globe. These fragrant plants have been selected throughout millennia for use by humans as herbs, spices, and pharmaceutical needs. The distilling of essential oils from mint began in Japan and England but has become a significant industrial product for the US, China, India, and other countries. The US Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR) maintains a mint genebank in Corvallis, Oregon. This facility preserves and distributes about 450 clones representing 34 taxa, hybrid species, advanced breeder selections, and F1 hybrids. Mint crop wild relatives are included in this unique resource. The majority of mint accessions and hybrids in this collection were initially donated in the 1970s by the A.M. Todd Company, located in Kalamazoo, Michigan. Other representatives of diverse mint taxa and crop wild relatives have since been obtained from collaborators in Australia, New Zealand, Europe, and Vietnam. These mints have been evaluated for cytology, oil components, verticillium wilt resistance, and key morphological characters. Pressed voucher specimens have been prepared for morphological identity verification. An initial set of microsatellite markers has been developed to determine clonal identity and assess genetic diversity. Plant breeders at private and public institutions are using molecular analysis to determine identity and diversity of the USDA mint collection. Evaluation and characterization includes essential oil content, disease resistance, male sterility, and other traits for potential breeding use. These accessions can be a source for parental genes for enhancement efforts to produce hybrids, or for breeding new cultivars for agricultural production. Propagules of Mentha are available for distribution to international researchers as stem cuttings, rhizome cuttings, or seed, which can be requested through the GRIN-Global database of the US National Plant Germplasm System, subject to international treaty and quarantine regulations.

Field evaluation of olive (Olea europaea) genotypes for resistance to Pseudomonas savastanoi pv. savastanoi

Current commercial olive (Olea europaea) cultivars are susceptible to olive knot disease caused by Pseudomonas savastanoi pv. savastanoi. Here we report on the examination of the olive tree germplasm collection maintained at the USDA-ARS National Clonal Germplasm Repository (NCGR) in Winters, California for the incidence of olive knot disease. Over a two year period we evaluated olive knot disease incidence on 506 trees representing 144 unique accessions consisting of 104 named cultivars originating from the Mediterranean Basin, South America, and California, including several hybrids, and related subspecies e.g. Olea europaea ssp. cuspidata. All genotypes were replicated in four blocks with trees in these blocks ranging in age from 6 to 35 year old. The disease index was higher in 2014 (43.5%) than in 2013 (29.4%). The percentage of infection varied significantly (P < 0.05) between the different blocks in the study orchard. Eleven genotypes exhibited greater than 30% disease severity with Tragolea (Greece), Barnea (Israel) and Arbusana (Spain) being the most susceptible to olive knot disease. Eighty-six genotypes exhibited less than 10% disease severity with 17 genotypes of this group showing zero olive knot incidence over the two year observation period.

Recent acquisitions of Rubus L. at the USDA National Clonal Germplasm Repository, Corvallis, Oregon: profiles of four species

Recent acquisitions of <i>Rubus</i> L. at the USDA National Clonal Germplasm Repository, Corvallis, Oregon: profiles of four species

Host selection behavior mediated by differential landing rates of the walnut twig beetle, Pityophthorus juglandis, and associated subcortical insect species, on two western North American walnut species, Juglans californica and J. major

AbstractThe walnut twig beetle, Pityophthorus juglandis Blackman (Coleoptera: Scolytidae), vectors a phytopathogenic fungus, Geosmithia morbida Kolařík et al. (Hypocreales), which causes thousand cankers disease (TCD) in walnut (Juglans sp.) and wingnut (Pterocarya sp., both Juglandaceae) trees. We investigated an early point in disease inception in two walnut species – Juglans californica S. Wats. and Juglans major (Torr. ex Sitsgr.) Heller – native to riparian forests of the western USA by comparing P. juglandis flight and landing responses to small‐diameter branch sections. Twenty unbaited branch sections (10 each of J. californica and J. major) were presented in a completely randomized design to populations of P. juglandis at the USDA Agricultural Research Service, National Clonal Germplasm Repository (NCGR) Juglans collection located at Wolfskill Experimental Orchards (Winters, CA, USA) and at the California State University, Chico, Agricultural Teaching and Research Center (ATRC, Chico, CA). These assays were carried out within a 4‐ to 6‐year period when weekly flight surveys with aggregation pheromone‐baited multiple funnel traps revealed that P. juglandis flight activity–abundance was higher at the NCGR than at the ATRC. For the landing rate assays, adhesive‐coated acetate sheets were wrapped around the branch sections and exchanged weekly. Three assays were completed at the NCGR (assays 1–3), whereas one assay was completed at the ATRC (assay 4). Landing rates on these traps were compared between J. californica and J. major. Two additional assays (5 and 6) were completed at the NCGR to compare responses to branch sections of J. californica and to similarly sized cardboard tubes (negative control). All six assays were completed over a 4‐year span during the 4‐ to 6‐year weekly flight survey period. Pooled landing rates of male and female P. juglandis (assays 1–4) demonstrated a preference by both sexes for J. californica over J. major. In assay 5 there was no preference by males or females for J. californica over the negative control, perhaps due to the low flight activity–abundance of P. juglandis during the assay. When repeated at a time of higher flight activity–abundance (assay 6), male and female landing rates on J. californica exceeded those on the negative control. Females of the invasive fruit‐tree pinhole borer (an ambrosia beetle), Xyleborinus saxeseni (Ratzeburg), and an invasive bark beetle, Hypothenemus eruditus Westwood (both Coleoptera: Scolytidae), showed relatively higher flight responses than either sex of P. juglandis during most assays, suggesting higher population densities of these two other invasive species at the two orchards or a greater sensitivity to host volatiles. Xyleborinus saxeseni and H. eruditus preferred to land on J. major over J. californica and on J. californica over the negative control. Similarly, an invasive longhorned beetle, Nathrius brevipennis (Mulsant) (Coleoptera: Cerambycidae), showed a significant preference for J. major over J. californica, but not for J. californica over the negative control. More male N. brevipennis were trapped than females at both study sites [sex ratio ranged from 5:1 (assay 6) to 39:1 (assay 4)], and flight occurred only in the spring and early summer months. Another ambrosia beetle trapped at the NCGR and ATRC, Xyleborus affinis Eichhoff, represented the first records of this species from western North America. In summary, flight responses recorded in some of our assays for P. juglandis and several other subcortical insects on Juglans indicate that host preference by these insects may be determined by long‐range olfactory cues that do not involve pheromones.