Insecticide efficacy and emergence timing of the Douglas-fir twig weevil.

The Douglas-fir twig weevil (Cylindrocopturus furnissi Buchanan) (Coleoptera: Curculionidae) has recently emerged as a significant pest of Christmas trees grown in the Pacific Northwest United States. The larvae girdle and disfigure twigs, which adversely affects tree marketability. Trees produced for export are also routinely destroyed for phytosanitary reasons when C. furnissi is discovered at border crossings. Due to historically being a sporadic and benign pest on planted and natural Douglas-fir (Psuedotsuga menziesii), there is a lack of chemical management options. In laboratory experiments, we assessed the knockdown effects (ability to kill or incapacitate) of 4 insecticides commonly used on Christmas trees: one assay tested knockdown after direct contact for 24 h, and the other assay tested knockdown after being allowed to feed on treated twigs with 2 days, 7 days, and 14 days residuals. Concurrently, we monitored temperature and adult C. furnissi emergence at a noble fir bough farm for 2 years to estimate the ideal degree-day window for applying insecticides. Bifenthrin and esfenvalerate knocked down all weevils on contact within just 4 h, whereas chlorpyrifos and acephate failed to achieve 100% knockdown within 24 h. Only acephate failed to knock down more weevils than the control (water) after feeding on treated twigs, regardless of the insecticide residue age. Degree-day modeling revealed a variable emergence window between the 2 years but 50% of adult emergence occurred between approximately 1,000-1,100 degree days (1st January, 50 °F (10 °C), single sine). Future work should assess the resulting management recommendation: apply bifenthrin or esfenvalerate once annually just after 1,000 growing degree days for 2 or more years prior to harvest.

Physical characteristics of soil-biodegradable and nonbiodegradable plastic mulches impact conidial splash dispersal of Botrytis cinerea.

Botrytis cinerea causes gray mold disease of strawberry (Fragaria ×ananassa) and is a globally important pathogen that causes fruit rot both in the field and after harvest. Commercial strawberry production involves the use of plastic mulches made from non-degradable polyethylene (PE), with weedmat made from woven PE and soil-biodegradable plastic mulch (BDM) as emerging mulch technologies that may enhance sustainable production. Little is known regarding how these plastic mulches impact splash dispersal of B. cinerea conidia. The objective of this study was to investigate splash dispersal dynamics of B. cinerea when exposed to various plastic mulch surfaces. Mulch surface physical characteristics and conidial splash dispersal patterns were evaluated for the three mulches. Micrographs revealed different surface characteristics that have the potential to influence splash dispersal: PE had a flat, smooth surface, whereas weedmat had large ridges and BDM had an embossed surface. Both PE mulch and BDM were impermeable to water whereas weedmat was semi-permeable. Results generated using an enclosed rain simulator system showed that as the horizontal distance from the inoculum source increased, the number of splash dispersed B. cinerea conidia captured per plate decreased for all mulch treatments. More than 50% and approximately 80% of the total number of dispersed conidia were found on plates 10 and 16 cm away from the inoculum source across all treatments, respectively. A significant correlation between the total and germinated conidia on plates across all mulch treatments was detected (P<0.01). Irrespective of distance from the inoculum source, embossed BDM facilitated higher total and germinated splashed conidia (P<0.001) compared to PE mulch and weedmat (P = 0.43 and P = 0.23, respectively), indicating BDM's or embossed film's potential for enhancing B. cinerea inoculum availability in strawberry production under plasticulture. However, differences in conidial concentrations observed among treatments were low and may not be pathologically relevant.

Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris.

Gray bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen, Rhizoctonia tuliparum (Rtul). Sclerotia present in infected bulbs, as well as overwintering sclerotia in soil and field debris, are the primary sources of infection. A method for accurate and sensitive detection of Rtul from soil and infected bulbs, and estimation of inoculum threshold levels, is needed for the management of disease caused by this pathogen. We designed a unique set of primers targeting the ITS2 region of the Rtul genome and developed a highly sensitive quantitative PCR (qPCR)-based method for Rtul identification using these primers, where the threshold of detection was approximately 1 fg Rtul DNA. The assay was more sensitive with sclerotia collected from the field (natural) than with those grown in the lab, and more sensitive with natural-light than natural-dark sclerotia. Also, the detection method was more sensitive when sclerotia were extracted from soil than from bulb tissue. The qPCR method was highly specific, as no PCR amplification was detected when genomic DNA from 62 non-Rtul Rhizoctonia isolates from a wide range of anastomosis groups were tested. To understand the evolutionary relationships and genomic diversity of Rtul, we performed phylogenetics of the ITS1-5.8S-ITS2 region and ITS2-molecular morphometric characterization (MMC) of Rtul isolates. The three Rtul isolates whose ITS sequences were available in GenBank formed a distinct phylogenetic clade with Ceratobasidium anceps as the nearest relative. Furthermore, MMC analysis revealed genetic divergence among these three Rtul isolates.

First report of leaf blight caused by Phytophthora ramorum on periwinkle (Vinca minor) in Washington State, USA.

Phytophthora ramorum (Werres, De Cock & Man in't Veld) was recovered from symptomatic foliage of periwinkle at a botanical garden in WA in March 2015. Symptoms were tan colored lesions with a dark brown margin visible on both surfaces of the leaf and were found on wounds or around leaf margins. Periwinkle is native to Europe and is commonly used for ground cover in ornamental landscapes. It is known to be invasive in US forests near the urban/wildland interface. Potential spread of P. ramorum into WA forests is of regulatory concern, as well as long distance spread to other states via nursery stock (7 CFR §301.92-2). Phytophthora ramorum was isolated from symptomatic foliage by excising leaf pieces 4-6 mm in diameter and surface-sterilizing in 0.6% sodium hypochlorite followed by two rinses in sterile water. Leaf pieces were plated on PARP medium (Ferguson and Jeffers 1999) and after 2-3 days at 20°C, slow-growing dense colonies with coralloid hyphae were isolated onto V8 agar. Colony morphology and chlamydospore production were consistent with descriptions of P. ramorum (Werres et al. 2001), except that the isolate was slower growing and had irregular, non-wildtype morphology (Elliott et al. 2018) compared to other isolates of P. ramorum. ITS and COX1 regions of mycelial DNA was amplified and sequenced to confirm the identity of P. ramorum using primers ITS1/ITS4 (White et al. 1990) and COX1F1/COX1R1 (Van Poucke et al. 2012). Sequences were submitted to GenBank (accession nos. ITS MT031975, COX1 MT031974). BLAST results showed at least 98% similarity with sequences of P. ramorum (ITS, MN540640 [98%]; COX1, EU124920 [100%]), and belonged to the NA1 clonal lineage. Pathogenicity of P. ramorum to periwinkle was confirmed by completing Koch's Postulates. Inoculum was grown on V8 agar plates at 20°C for two weeks until sporangia were abundant. A zoospore suspension was produced by flooding plates with 7 ml sterile water, incubating for 2 hours at 5°C, then for an additional hour at 24°C. Zoospores were observed under the microscope and quantified with a hemocytometer, then diluted to 2 x 105 zoospores/ml. A 10 µl droplet of inoculum was placed at one wounded and one unwounded site on six leaves on each of four plants. In addition, a set of four plants was inoculated by dipping foliage on one branch per plant into the zoospore suspension for 30 seconds. A set of four control plants were mock inoculated in the same manner using sterile water. The trial was repeated once. Inoculated plant materials were incubated in a moist chamber for 3-5 days and free moisture was present on foliage upon removal. Plants were held in a biocontainment chamber (USDA-APHIS permit # 65857) at 20C and symptom development assessed after 7 days (Figure S1). . Symptoms developed on foliage inoculated using both methods in both trials. Phytophthora ramorum was isolated once from droplet inoculated foliage at a wounded site on one plant. Reisolation onto PARP and then V8 agar was conducted from surface-sterilized symptomatic tissue and the presence of P. ramorum confirmed by observation of colony morphology and chlamydospore production. The presence of P. ramorum was also confirmed with DNA extraction from symptomatic foliage from plants from each of the two trials followed by PCR and sequencing of the COX1 gene (EU124920, 100%) (Figure S2). None of the water-inoculated controls were positive for P. ramorum. Low isolation success could be attributed to reduced pathogenicity due to being a non-wildtype isolate. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1019284 and USDA APHIS Cooperative Agreement AP17PPQS&T00C070.

First report of leaf blight caused by Phytophthora ramorum on cherry laurel (Prunus laurocerasus) in Washington State, USA.

In April 2014, Phytophthora ramorum (Werres, De Cock & Man in't Veld) was recovered from symptomatic foliage of cherry laurel (Prunus laurocerasus) at an ornamental plant nursery in Washington State. Cherry laurel, also known as English laurel, is widely propagated in WA because it is commonly used in landscaping. It is invasive in forests near the urban/wildland interface in the western US and in Europe (Rusterholz et al. 2018). Given its popularity as an ornamental species, the potential of this host to spread P. ramorum is of regulatory concern due to possible long distance spread to other states via nursery stock. Foliar symptoms consisted of dark brown lesions near wounds or around leaf margins where water collected. Shot-hole symptoms characterized by abscission zones and dropping of infected tissues were also observed. Lesions expanded beyond the margin of the shot-hole in some cases (Figure S1A). Phytophthora was isolated from symptomatic foliage by surface-sterilizing leaf pieces in 0.6% sodium hypochlorite and 2 rinses in sterile water. They were plated on PARP medium (Ferguson and Jeffers 1999). After 2-3 days, a slow-growing dense colony with coralloid hyphae was isolated onto V8 agar. P. ramorum was identified by observing morphological features (Figure S1B). Colony and spore morphology matched that of P. ramorum (Werres et al. 2001). The isolate was confirmed as P. ramorum by PCR and sequencing of ITS and COX1 regions using primers ITS1/ITS4 (White et al. 1990) and COX1F1/COX1R1 (Van Poucke et al. 2012). Sequences were submitted to GenBank (accession nos. ITS MT031969, COX1 MT031968). BLAST results showed at least 99% similarity with sequences of P. ramorum (ITS, KJ755124 [100%]; COX1, EU124926 [99%]). Multilocus genotyping with microsatellite markers placed the isolate in the EU1 clonal lineage. Pathogenicity of P. ramorum on cherry laurel was confirmed by completing Koch's Postulates using the isolate taken from this host. Two trials were done in a biocontainment chamber (USDA-APHIS permit # 65857) since P. ramorum is a quarantine pathogen and greenhouse trials could not be conducted, using detached stems from mature, visibly healthy cherry laurel plants growing in a landscape. Phytophthora ramorum inoculum was grown on V8A plates at 20®C for 2 weeks until sporangia were abundant. A zoospore suspension was produced by flooding plates with 7 ml sterile water, incubating for 2 hours at 5®C, then 1 hour at 24®C. Zoospores were observed with light microscopy, quantified with a hemocytometer and diluted to 1 x 104 zoospores/ml. A 10 µl droplet was placed at 3 wounded and 3 unwounded sites on 4 leaves per branch. In addition, a set of samples was inoculated by dipping foliage into the zoospore suspension for 30 seconds. A set of controls was mock inoculated using sterile water. Four branches per inoculation treatment were used and the trial was repeated once. Inoculated plant materials were incubated in moist chambers for 3-5 days at 20®C. Free moisture was present on foliage upon removal. Symptom development was assessed after incubation in the biocontainment chamber at 20®C for 7 days (Figure S1C). Phytophthora ramorum was reisolated from symptomatic tissue and the recovered culture was verified morphologically and by PCR and sequencing. It was isolated more often from foliage dipped in zoospore suspension than droplet inoculated, and more from wounded than unwounded sites. None of the water-inoculated controls were positive for P. ramorum. The presence of P. ramorum was also confirmed with DNA extraction from surface-sterilized symptomatic foliage followed by PCR and sequencing of the COX1 gene (EU124926, 100%) (Figure S2). To our knowledge, this is the first report of P. ramorum naturally infecting cherry laurel in the United States. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1019284 and USDA APHIS Cooperative Agreement AP17PPQS&T00C070 Literature cited Ferguson and Jeffers, 1999. Plant Disease 83:1129-1136 Van Poucke, K. et al. 2012. Fungal Biology 116: 1178-1191. http://dx.doi.org/10.1016/j.funbio.2012.09.003 Werres, S. et al. 2001. Mycol. Res. 105:1155-1165. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

Genetic analysis reveals unprecedented diversity of a globally-important plant pathogenic genus.

Genus Botrytis contains approximately 35 species, many of which are economically-important and globally-distributed plant pathogens which collectively infect over 1,400 plant species. Recent efforts to genetically characterize genus Botrytis have revealed new species on diverse host crops around the world. In this study, surveys and subsequent genetic analysis of the glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis and ethylene-inducing proteins 1 and 2 (NEP1 and NEP2) genes indicated that Botrytis isolates collected from peony fields in the United States contained more species diversity than ever before reported on a single host, including up to 10 potentially novel species. Together, up to 16 different phylogenetic species were found in association with peonies in the Pacific Northwest, which is over a third of the total number of species that are currently named. Furthermore, species were found on peonies in Alaska that have been described on other host plants in different parts of the world, indicating a wider geographic and host distribution than previously thought. Lastly, some isolates found on peony share sequence similarity with unnamed species found living as endophytes in weedy hosts, suggesting that the isolates found on peony have flexible lifestyles as recently discovered in the genus. Selected pathogenicity, growth, and morphological characteristics of the putatively new Botrytis species were also assessed to provide a basis for future formal description of the isolates as new species.

A Novel Phagomyxid Parasite Produces Sporangia in Root Hair Galls of Eelgrass (Zostera marina).

The objective of this study was to identify the parasite causing the formation of root hair galls on eelgrass (Zostera marina) in Puget Sound, WA. Microscopic and molecular analyses revealed that a novel protist formed plasmodia that developed into sporangia in root hair tip galls and released biflagellate swimming zoospores. Root hair galls were also observed in the basal section of root hairs, and contained plasmodia or formed thick-walled structures filled with cells (resting spores). Phylogenetic analyses of 18S rDNA sequence data obtained from cells in sporangia indicated that the closest relative of the parasite with a known taxonomic identification was Plasmodiophora diplantherae (86.9% sequence similarity), a phagomyxid parasite that infects the seagrass Halodule spp. To determine the local geographic distribution of the parasite, root and soil samples were taken from four eelgrass populations in Puget Sound and analyzed for root hair galls and parasite DNA using a newly designed qPCR protocol. The percent of root hairs with galls and amount of parasite DNA in roots and sediment varied among the four eelgrass populations. Future studies are needed to establish the taxonomy of the parasite, its effects on Z. marina, and the factors that determine its distribution and abundance.

Interactions Between Root Rotting Phytophthora, Abies Christmas Trees, and Environment.

Defining host-pathogen interactions between species of root-rotting Phytophthora and Abies in Christmas tree production areas is important for tailoring management activities on a regional scale and for developing molecular tools for identifying resistant host species. Classifying Abies species as resistant or susceptible is complicated by regional variation in abundance and aggressiveness of Phytophthora species and the influence of environment on symptom expression and host vigor. Because previous studies performed to assess host response to Phytophthora root rot (PRR) have focused on one or a few species of either the host or pathogen, a multifactorial experiment was conducted to assess the responses of seven species of Abies challenged with three isolates each of four Phytophthora species under contrasting temperature conditions. Evaluation of mortality, root rot severity, and remaining root biomass after 16 weeks of exposure to the pathogen confirmed prior inferences regarding inherent variation in the resistance responses of various species of Abies and demonstrated evidence of variation in aggressiveness among species of Phytophthora as well as different isolates of the same Phytophthora species. The ambient temperatures at which studies were conducted had a conspicuous effect on host mortality, root rot severity, and radial growth of Phytophthora. Understanding how host responses differ under variable pathogen attack and ambient environment will improve efforts to control PRR using host species substitutions on infested ground.

Characterization of phenotypic variation and genome aberrations observed among Phytophthora ramorum isolates from diverse hosts.

BACKGROUND: Accumulating evidence suggests that genome plasticity allows filamentous plant pathogens to adapt to changing environments. Recently, the generalist plant pathogen Phytophthora ramorum has been documented to undergo irreversible phenotypic alterations accompanied by chromosomal aberrations when infecting trunks of mature oak trees (genus Quercus). In contrast, genomes and phenotypes of the pathogen derived from the foliage of California bay (Umbellularia californica) are usually stable. We define this phenomenon as host-induced phenotypic diversification (HIPD). P. ramorum also causes a severe foliar blight in some ornamental plants such as Rhododendron spp. and Viburnum spp., and isolates from these hosts occasionally show phenotypes resembling those from oak trunks that carry chromosomal aberrations. The aim of this study was to investigate variations in phenotypes and genomes of P. ramorum isolates from non-oak hosts and substrates to determine whether HIPD changes may be equivalent to those among isolates from oaks. RESULTS: We analyzed genomes of diverse non-oak isolates including those taken from foliage of Rhododendron and other ornamental plants, as well as from natural host species, soil, and water. Isolates recovered from artificially inoculated oak logs were also examined. We identified diverse chromosomal aberrations including copy neutral loss of heterozygosity (cnLOH) and aneuploidy in isolates from non-oak hosts. Most identified aberrations in non-oak hosts were also common among oak isolates; however, trisomy, a frequent type of chromosomal aberration in oak isolates was not observed in isolates from Rhododendron. CONCLUSION: This work cross-examined phenotypic variation and chromosomal aberrations in P. ramorum isolates from oak and non-oak hosts and substrates. The results suggest that HIPD comparable to that occurring in oak hosts occurs in non-oak environments such as in Rhododendron leaves. Rhododendron leaves are more easily available than mature oak stems and thus can potentially serve as a model host for the investigation of HIPD, the newly described plant-pathogen interaction.

Botrytis euroamericana, a new species from peony and grape in North America and Europe.

A novel species of Botrytis isolated from peony in Alaska, USA, and grape in Trento District, Italy, was identified based on morphology, pathogenicity, and sequence data. The grape and peony isolates share sequence homology in the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis- and ethylene-inducing protein 1 and 2 (NEP1 and NEP2) genes that place them in a distinct group closely related to B. aclada, a globally distributed pathogen of onions. Genetic results were corroborated with morphological and pathogenicity trials that included two isolates of B. cinerea and two isolates of B. paeoniae from peony in Alaska and one isolate of B. aclada. The authors observed differences in colony and conidia morphology and ability to cause lesions on different host tissues that suggest that the grape and peony isolates represent a distinct species. Most notably, the grape and peony isolates did not colonize onion bulbs, whereas B. aclada readily produced lesions and prolific sporulation on onion tissue. The new species Botrytis euroamericana is described herein.

Comparison of Five Detection and Quantification Methods for Phytophthora ramorum in Stream and Irrigation Water.

Propagules of Phytophthora ramorum, the causal agent of sudden oak death (SOD) and ramorum blight, can be recovered from infested stream and nursery irrigation runoff using baiting and filtration methods. Five detection methods, including pear and rhododendron leaf baits, Bottle O' Bait, filtration, and quantitative polymerase chain reaction (qPCR) performed on zoospores trapped on a filter were compared simultaneously in laboratory assays using lab or creek water spiked with known quantities of P. ramorum zoospores. The detection threshold for each method was determined and methods that could be used to quantify zoospore inoculum were identified. Filtration and qPCR were the most sensitive at detecting low levels of zoospores, followed by wounded rhododendron leaves, rhododendron leaf disks, and pear baits. Filtration, qPCR, and leaf disks were able to quantify P. ramorum zoospores ranging from 2 to 451 direct-plate CFU/liter while wounded leaves and pear baits appeared to be better at detection rather than quantification. The ability to detect and quantify P. ramorum inoculum in water will assist scientists, regulatory agencies, and nursery personnel in assessing the risk of spreading P. ramorum in nurseries and landscape sites where untreated infested water is used for irrigation.

Sydowia polyspora associated with current season needle necrosis (CSNN) on true fir (Abies spp.).

Current season needle necrosis (CSNN) has been a serious foliage disorder on true fir Christmas trees and bough material in Europe and North America for more than 25y. Approximately 2-4 weeks after bud break, needles develop chlorotic spots or bands that later turn necrotic. The symptoms have been observed on noble fir (Abies procera), Nordmann fir (A. nordmanniana) and grand fir (A. grandis) on both continents. CSNN was reported as a physiological disorder with unknown aetiology from USA, Denmark, and Ireland, but was associated with the fungus Kabatina abietis in Germany, Austria and Norway. In 2007, a fungus that morphologically resembled K. abietis was isolated from symptomatic needle samples from Nordmann fir from Austria, Denmark, Germany, Norway, and USA. Sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA of these cultures, plus a K. abietis reference culture from Germany (CBS 248.93), resulted in Hormonema dematioides, the imperfect stage of Sydowia polyspora, and thus the taxonomy is further discussed. Inoculation tests on Nordmann fir seedlings and transplants with isolates of S. polyspora from all five countries resulted in the development of CSNN symptoms. In 2009, S. polyspora was also isolated from symptomatic needles from Nordmann fir collected in Slovakia.

Population genetic analysis infers migration pathways of Phytophthora ramorum in US nurseries.

Recently introduced, exotic plant pathogens may exhibit low genetic diversity and be limited to clonal reproduction. However, rapidly mutating molecular markers such as microsatellites can reveal genetic variation within these populations and be used to model putative migration patterns. Phytophthora ramorum is the exotic pathogen, discovered in the late 1990s, that is responsible for sudden oak death in California forests and ramorum blight of common ornamentals. The nursery trade has moved this pathogen from source populations on the West Coast to locations across the United States, thus risking introduction to other native forests. We examined the genetic diversity of P. ramorum in United States nurseries by microsatellite genotyping 279 isolates collected from 19 states between 2004 and 2007. Of the three known P. ramorum clonal lineages, the most common and genetically diverse lineage in the sample was NA1. Two eastward migration pathways were revealed in the clustering of NA1 isolates into two groups, one containing isolates from Connecticut, Oregon, and Washington and the other isolates from California and the remaining states. This finding is consistent with trace forward analyses conducted by the US Department of Agriculture's Animal and Plant Health Inspection Service. At the same time, genetic diversities in several states equaled those observed in California, Oregon, and Washington and two-thirds of multilocus genotypes exhibited limited geographic distributions, indicating that mutation was common during or subsequent to migration. Together, these data suggest that migration, rapid mutation, and genetic drift all play a role in structuring the genetic diversity of P. ramorum in US nurseries. This work demonstrates that fast-evolving genetic markers can be used to examine the evolutionary processes acting on recently introduced pathogens and to infer their putative migration patterns, thus showing promise for the application of forensics to plant pathogens.

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum.

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

Use of shaking treatments and preharvest sprays of pyrethroid insecticides to reduce risk of yellowjackets and other insects on Christmas trees imported into Hawaii.

Insects are commonly found by Hawaii's quarantine inspectors on Christmas trees imported from the Pacific Northwest. To reduce the risk of importing yellowjacket (Vespula spp.) queens and other insects, an inspection and tree shaking certification program was begun in 1990. From 1993 to 2006, the annual percentage of shipped containers rated by Hawaii quarantine inspectors as moderately or highly infested with insects was significantly higher for manually shaken trees than for mechanically shaken trees. Between 1993 and 2001, 343 insect species in total were recovered from Christmas trees. Live western yellowjacket [Vespula pensylvanica (Saussure)] queens were intercepted both from containers certified as manually shaken and from containers certified as mechanically shaken. The standard manual shaking protocol removed about one-half of the queens from Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] trees that were naturally infested with western yellowjacket queens. We investigated the use of preharvest sprays of permethrin as a complement to shaking procedures used to control yellowjackets and other insects. Western yellowjacket queens and honey bees (surrogates for wasp pests) were exposed to Noble fir foliage that had been sprayed in the field with permethrin > 6 wk before harvest. Pesticide residues provided complete control (moribundity or mortality) in both species. The sprays did not affect needle retention or quality of Noble fir foliage. We conclude that preharvest sprays of pyrethroid insecticides could be used in combination with mechanical shaking to greatly reduce the quarantine risk of yellowjacket queens and other insects in exported Christmas trees.