Using comparative genomics to develop a molecular diagnostic for the identification of an emerging pest Drosophila suzukii.

Drosophila suzukii (Spotted Wing Drosophila) has recently become a serious invasive pest of fruit crops in the USA, Canada, and Europe, leading to substantial economic losses. D. suzukii is a direct pest, ovipositing directly into ripe or ripening fruits; in contrast, other Drosophilids utilize decaying or blemished fruits and are nuisance pests at worst. Immature stages of D. suzukii are difficult to differentiate from other Drosophilids, posing problems for research and for meeting quarantine restrictions designed to prevent the spread of this pest in fruit exports. Here we used a combined phylogenetic and bioinformatic approach to discover genetic markers suitable for a species diagnostic protocol of this agricultural pest. We describe a molecular diagnostic for rapid identification of single D. suzukii larva using multiplex polymerase chain reaction. Our molecular diagnostic was validated using nine different species of Drosophila for specificity and 19 populations of D. suzukii from different geographical regions to ensure utility within species.

Overwintering hosts for the exotic leafroller parasitoid, Colpoclypeus florus: implications for habitat manipulation to augment biological control of leafrollers in pome fruits.

Thirty sites of managed and native habitats were surveyed for leafrollers (Lepidoptera: Tortricidae) in the apple producing region of central Washington State and northern Oregon from September through November 1997-2000 to discover species that supported overwintering by the parasitoid Colpoclypeus florus (Walker) (Hymenoptera: Eulophidae). C. florus, a species introduced from Europe, requires medium to large host larvae late in autumn on which to overwinter, and few leafroller species display this biology. Over the four years, five potential C. florus hosts were collected, including: Ancylis comptana (Froelich), Xenotemna pallorana (Robinson), and Syndemis sp. (Tortricidae), Filatima sp. (Gelechiidae), and Caloptilia burgessiellia (Zeller) (Gracillariidae). Of these, A. comptana, Syndemis sp., and Filatima sp. have been confirmed as overwintering hosts for C. florus. During the four years, the Syndemis sp. was rare and observed at only one location feeding on redosier dogwood, Cornus sericea L. (Cornales: Cornaceae) although, at this location, many of the larvae collected were parasitized by C. florus. Filatima sp. was common in the Yakima valley feeding on balsam poplar, Populus balsamifera L. ssp. trichocarpa (Torr. & Gray ex Hook) Brayshaw (Malpighiales: Salicaceae) but was rarely parasitized. A. comptana, however, was collected at many locations in central Washington and was frequently found as an overwintering host for C. florus. A. comptana was found feeding on two Rosaceae: Wood's rose, Rosa woodsii Lindl., and strawberry, Fragaria ananassa Duchesne (Rosales: Rosaceae). Based on the number of host larvae collected, A. comptana appears to be the primary overwintering host for C. florus in Washington. Introduction of A. comptana populations to near-orchard habitats may facilitate biological control of leafrollers that are orchard pests.

Occurrence of Two Little Cherry Viruses in Sweet Cherry in Washington State.

Little cherry disease, one of the major viral diseases of sweet cherry (Prunus avium) worldwide, is associated with either of two closteroviruses, Little cherry virus 1 (LChV-1) and Little cherry virus 2 (LChV-2). Two sets of primers corresponding to a portion of the replicase gene of LChV-1 and LChV-2 were used in one-tube reverse-transcription polymerase chain reactions to detect these viruses in total RNA extracts of field-collected sweet cherry tissues. LChV-1 and LChV-2 were detected both alone and in combination in five sweet cherry orchards in Washington State. Sequence analysis of a 240-nucleotide (nt) fragment of the replicase open reading frame (ORF)1b and a 232-nt fragment from a portion of ORF8 and the 3' untranslated region (UTR) of LChV-1 indicated that North American (NA) isolates shared 90 to 99% nucleotide identity in both genome segments analyzed. In contrast, comparisons of NA isolates to two Eurasian isolates of LChV-1 indicated shared nucleotide identities of 79 to 82% in the replicase fragment and 89 to 90% in the ORF8/3'UTR fragment. Sequence variation in the replicase region did not affect detection of LChV-1 in 12 isolates using the replicase-specific primers reported here. This article represents the first report of LChV-1 and LChV-2 in sweet cherry in Washington.

DNA diagnostics to identify internal feeders (Lepidoptera: Tortricidae) of pome fruits of quarantine importance.

A diagnostic polymerase chain reaction (PCR) method is presented for differentiating among the North American internal apple-feeding pests codling moth, Cydia pomonella (L.); oriental fruit moth, Grapholita molesta (Busck); lesser appleworm, Grapholita prunivora (Walsh); and cherry fruitworm, Grapholita packardi Zeller. An approximately 470-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in three to six specimens of each species. Consistent and diagnostic differences were observed among the species in two regions of COI from which forward and reverse primers were designed to amplify a 112-116-bp segment of the gene. The primer sets were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. Protocols were adapted for conventional and quantitative PCR, the latter being substantially faster. The method was validated as a decision-making tool for quarantine identifications for Mexico by representatives of their phytosanitary agency (Sanidad Vegetal). The method can facilitate identification of intercepted internal feeding Lepidoptera in apple and pear for many other importing nations.

Particle films for suppression of the codling moth (Lepidoptera: Tortricidae) in apple and pear orchards.

Studies were conducted in 1997 and 1998 to evaluate the effects of three particle film formulations consisting of kaolin and adjuvants on neonate larvae, ovipositing adult females, and eggs of the codling moth, Cydia pomonella (L.). Neonate larval walking speed, fruit discovery rate, and fruit penetration rate on apple host plants coated with particle films were significantly lower than on host plants without particle films in laboratory assays. Females oviposited less on host plants covered with a particle film residue than on untreated plants in laboratory choice and no-choice tests. Hatch rate of codling moth neonate larvae was unaffected by particle films sprayed on host plants either before or after oviposition. Fruit infestation rates were significantly reduced on particle film-treated trees compared with untreated trees for both first- and second-generation codling moth in field trials in both apple and pear orchards. Particle films appear to be a promising supplemental control approach for codling moth in orchards where moth density is high, and may represent a stand-alone method where moth densities are lower.

Effects of a kaolin-based particle film on obliquebanded leafroller (Lepidoptera: Tortricidae).

Studies were conducted in 1997 to evaluate the effects of the kaolin-based particle film formulation M96-018 on adults, eggs, and larvae of the obliquebanded leafroller, Choristoneura rosaceana (Harris). Particle film treatments significantly reduced female longevity, mating success, and number of egg masses oviposited compared with moths on untreated apple leaves in sleeve-cage and screen-cage tests. No differences in mating success or oviposition were caused by the application rates and coverage density of M96-018 on foliage. Females avoided ovipositing on particle film-treated leaves in choice tests. Larval hatch was not affected by topical application or residual exposure to M96-018. Larval weight gain and pupal weight were significantly reduced and larval mortality increased in no-choice feeding tests with M96-018. In choice tests, larvae preferred to feed on untreated leaf surfaces. The negative effects on larval development and survivorship on M96-018-treated foliage did not differ across a fourfold difference in spray application rate. A significant reduction in the number of infested shoots was found in orchard trials when M96-018 was applied before bud break in late March compared with untreated trees. No reductions in larval densities were found compared with an untreated control following prebloom and postbloom applications.

Detecting Cacopsylla pyricola (Hemiptera: Psyllidae) in predator guts using COI mitochondrial markers.

Cacopsylla pyricola (Förster) is one of the most important pests of pear in North America, where several native predators have been considered for integrated pest management (IPM) programmes. Two molecular markers of 271 and 188 bp were developed from C. pyricola cytochrome oxidase I (COI) fragments, in order to study the detection of this species in the gut of arthropod predators. Primer sensitivity and the detection period for pear psylla remains in the guts of Anthocoris tomentosus Pericart were determined. The sensitivity threshold was defined at 10-5 dilution of a C. pyricola fifth-instar nymph in all samples. Predator adults were evaluated immediately after ingestion of one to five C. pyricola nymphs (t = 0) and after 2, 4, 6, 8, 16, 24 and 32 h. Detection of the presence of C. pyricola DNA always lasted longer using the shorter fragment and was observed after 32 h of digestion using both markers. The primers amplifying the 188 bp fragment amplified all four psyllid species tested, whereas the primers designed to amplify the 271 bp fragment did so exclusively for C. pyricola and its close relative, Cacopsylla pyri (Linnaeus). Both primers failed to amplify DNA from representative species of the Coccinellidae, Chrysopidae, Hemerobiidae, Anthocoridae, Miridae, Salticidae, Aphididae, Tetranychidae and the Tortricidae, suggesting their suitability for general trophic studies.