Brian W. Bahder

Phylogeny of Geminivirus Coat Protein Sequences and Digital PCR Aid in Identifying Spissistilus festinus as a Vector of Grapevine red blotch-associated virus

Grapevine red blotch-associated virus (GRBaV) is a single-stranded DNA virus, proposed to be a member of the family Geminiviridae, and is associated with grapevines showing red blotch symptoms in North America. The existence of the virus was reported in 2012, and subsequently detected in grapevines in major grape production regions. We investigated if a vector exists that can transmit GRBaV in vineyards. Phylogenetic analysis of the predicted amino acid sequence of coat protein (CP) of GRBaV with the CP of 23 geminiviruses representing all seven genera of the family Geminiviridae revealed that GRBaV-CP was most similar to that of Tomato pseudo-curly top virus, a geminivirus known to be transmitted by a treehopper (Membracidae), a family that is closely related to leafhoppers (Cicadellidae). To identify vectors of GRBaV, hemipteran species within and nearby wine grape vineyards where virus spread was suspected were collected and transmission assays were conducted. Among the species tested, the three-cornered alfalfa hopper Spissistilus festinus (Hemiptera: Membracidae) was able to both acquire the virus from a grapevine infected with GRBaV and transmit the virus to healthy grapevines in the laboratory. In commercial vineyards, lateral shoots of grapevines girdled due to feeding injury by the adult three-cornered alfalfa hopper also tested positive for the virus using digital PCR. These findings represent an important step in understanding the biology of GRBaV and develop management guidelines.

Pheromone-Based Monitoring of Pseudococcus maritimus (Hemiptera: Pseudococcidae) Populations in Concord Grape Vineyards

ABSTRACT The grape mealybug, Pseudococcus maritimus (Ehrhorn), is the dominant mealybug in Washingtons Concord grape vineyards (Vitis labrusca L.). It is a direct pest of fruit clusters and a vector of grapevine leafroll-associated viruses. Using traps baited with the sex pheromone of Ps. maritimus, we determined the optimal trap density for monitoring Ps. maritimus, with the goal of providing a more rapid monitoring method for Ps. maritimus than visual surveys. Varying densities of pheromone-baited traps (one, four, and eight traps per 12.14 ha) were deployed in Concord vineyards to monitor Ps. maritimus seasonal phenology in 2010 and 2011. In both years, flights of adult males were detected in early May and captures peaked twice per season in mid-June and mid-August, indicating two generations each year. Trap data were analyzed using Taylors Power Law, Iwaos patchiness regression, and the K parameter of the negative binomial model to determine optimal sample size. The formula using the K parameter provided the lowest required sample size, showing that four to eight traps per 12.14 ha were needed to provide 30% sampling precision efficiency throughout the entire season. Fewer traps were needed during flight peaks when trap capture numbers were great. Only one pheromone-baited trap per 12.14 ha was sufficient to provide Ps. maritimus flight phenology data to make informed management decisions. Species-specific pheromone-baited traps deployed for Pianococcus ficus (Signoret), Pseudococcus longispinus (Targioni Tozzetti), and Pseudococcus viburni (Signoret) did not detect any of these species in the vineyards sampled.

Pseudococcus maritimus (Hemiptera: Pseudococcidae) and Parthenolecanium corni (Hemiptera: Coccidae) Are Capable of Transmitting Grapevine Leafroll-Associated Virus 3 Between Vitis x labruscana and Vitis vinifera

ABSTRACT The grape mealybug, Pseudococcus maritimus (Ehrhorn), and European fruit lecanium scale, Parthenolecanium corni (Bouché), are the predominant species of Coccoidea in Washington State vineyards. The grape mealybug has been established as a vector of Grapevine leafroll-associated virus 3 (GLRaV-3) between wine grape (Vitis vinifera L.) cultivars, elevating its pest status. The objective of this study was to determine if GLRaV-3 could be transmitted between Vitis x labruscana L. and V. vinifera by the grape mealybug and scale insects. Three transmission experiments were conducted with regard to direction; from V. vinifera to V. x labruscana L., from V. x labruscana L. to V. x labruscana L., and from V. x labruscana L. to V. vinifera. Each experiment was replicated 15 times for each vector species. Crawlers (first-instars) of each vector species were allowed 1-wk acquisition and inoculation access periods. The identities of viral and vector species were confirmed by reverse transcription-polymerase chain reaction, cloning, and sequencing of species-specificDNAfragments.GLRaV-3wassuccessfully transmittedbyboth species in all experiments, although Ps. maritimus was a more efficient vector under our experimental conditions.Tothe best of our knowledge, this study represents the firstdocumentedevidence of interspecific transmission of GLRaV-3 between two disparate Vitis species. It also highlights the potential role of V. x labruscana L. intheepidemiologyofgrapevineleafroll disease asasymptomlesssourceofGLRaV-3inoculum.

An Evaluation of the Flora Adjacent to Wine Grape Vineyards for the Presence of Alternative Host Plants of Grapevine red blotch-associated virus

Grapevine red blotch-associated virus (GRBaV) is a recently discovered virus of concern to wine grape production in North America. While the vector of this virus is unknown, other elements of virus epidemiology are essential to develop guidelines for the management of the virus as well as to assist in the search for its vector. The objective of this study was to evaluate vegetation within and surrounding GRBaV-infected vineyards to identify potential virus reservoirs that may serve as sources of inoculum. In this study, 13 plant species were sampled throughout the year and were tested for the presence of GRBaV. Of the 13 species tested, two species, Rubus armeniacus and wild grapes (Vitis californica × V. vinifera), tested positive by quantitative PCR. Of these two species, only wild grapes were determined to be a true host. This study documents the first time GRBaV has been confirmed in an alternative host or in a species outside of a commercial vineyard and suggests that a mechanism exists by which GRBaV moves between plant species that is not human-mediated. The precise role that wild grapes play in the epidemiology of GRBaV remains unknown.

Microsatellite Variation of two Pacific Coast Drosophila suzukii (Diptera: Drosophilidae) Populations

ABSTRACT The vinegar fly, Drosophila suzukii (Diptera: Drosophilidae), is a recent invader in North America that has become a serious threat to small fruit production. It was first detected in California in 2008 and in Washington state in 2009. In this study, D. suzukii populations from the area of the original detection on Californias central coast and from eastern Washington, the United States, were sampled over a 3-year period to determine genetic variation in both using microsatellite markers. Six different loci were successfully amplified and included in the analysis. These loci included nanos, elf1, antennapedia, mastermind, z600, and tenA. The population from eastern Washington was highly monomorphic with one locus, mastermind, having multiple alleles. There was greater genetic variation in the coastal California population with all loci having multiple alleles, with the exception of tenA. Owing to the relatively low levels of genetic variation in the eastern Washington population compared with the coastal California population, it appears that the D. suzukii population in the eastern Washington region has undergone a significant bottleneck.

Termites (Isoptera: Kalotermitidae, Rhinotermitidae, Termitidae) of Ecuador

Abstract Termites are an abundant and diverse group in the Neotropics with about 500 species representing 83 genera. The paucity of the termite fauna recorded from Ecuador is due, in part, to a lack of deliberate surveys. We revise the termite fauna of Ecuador and raise the number of species from 25 species to 72 based on our recent termite surveys. Of the 72 species, 18 could not be conclusively identified and are likely new species. Given the limited area that has been covered in surveys of the Ecuadorian termite fauna, there are undoubtedly many more species to be recorded for Ecuador, primarily in the eastern lowland areas, cloud forests on both the eastern and western slopes of the Andes, and the Amazonian lowland forests.

Multilamina teevani gen. et sp. nov., a microsporidian pathogen of the neotropical termite Uncitermes teevani.

A new genus and species of microsporidia is described from adults of the termite Uncitermes teevani (Emerson) (n. comb., formerly Armitermes teevani), collected in Ecuador. Masses of elongate, ovoid, uninucleate spores were localized to the coelomic cavity of adult workers and measured 6.29×3.33μm (fresh) and 5.83×3.00μm (fixed). These spores were individually contained within a multi-layered sporophorous vesicle and contained an isofilar polar filament with 24-28 coils. Blast-n analysis revealed that the small subunit ribosomal DNA (ssrDNA) sequence of this new species exhibited 85% identity with that of a Varimorpha species from the fire ant, Solenopsis richteri, and slightly less (78-85% identity) to a large clade of microsporidian parasites from mosquitoes and microcrustacea. The morphological and sequence data support the conclusion that Multilamina teevani gen. et sp. nov. is a novel microsporidium and distinct from any previously described genera or species.

Digital PCR technology for detection of palm infecting phytoplasmas belonging to group 16SrIV that occur in Florida

Phytoplasmas are an economically important group of plant pathogens that negatively impact a wide variety of plants in agricultural and natural ecosystems. In Florida, palm trees are essential elements in the nursery and landscaping industries that suffer from diseases caused by phytoplasmas that are related to each other but are classified in two different subgroups, 16SrIV-A and 16SrIV-D. In this study, a TaqMan assay was developed for digital polymerase chain reaction (dPCR) to detect both palm-infecting phytoplasmas found in Florida. When compared with real-time PCR assays and nested PCR assays, dPCR was capable of detecting the phytoplasmas at much lower concentrations than was possible by using real-time PCR and nested PCR. Additionally, the assay was capable of detecting 16SrIV-B phytoplasma as well as isolates representing the 16SrI and 16SrIII phytoplasma groups. Due to sequence identity of primer annealing regions across diverse phytoplasmas, the assay is likely to be successful for detection of a wide variety of phytoplasmas. The increased sensitivity of this dPCR assay over real-time PCR will allow for earlier detection of phytoplasma infection in palm trees, as well as for screening of salivary glands of candidate insect vector species. These advantages should aid timely management decisions to reduce disease spread and rapid determination of phytoplasma transmission by vectors.

Relative abundance and phenology of Drosophila Fallén, 1815 (Diptera: Drosophilidae) species in south-central Washington State

Abstract. A monitoring program for a recently introduced vinegar fly, Drosophila suzukii Matsumura, 1931, was conducted in south-central Washington State, U.S.A. from March 2011 to November 2013. Along with D. suzukii, a complex of nine additional Drosophila Fallén, 1815 species were captured in baited traps and identified to species. The Drosophila were captured in Nalgene® and Haviland traps baited with apple cider vinegar or a sugar yeast mixture that were distributed among seven different horticultural crops or unmanaged habitats. All flies captured were identified to species and quantified for each sampling period. The species identified and quantified included D. busckii Coquillett, 1901, D. funebris Fabricius, 1787, D. hydei Sturtevant,1921, D. immigrans Sturtevant, 1921, D. melanogaster Meigen, 1830, D. simulans Sturtevant, 1919, D. obscura Fallén, 1823, D. subobscura Collin, 1936, D. subquinaria Spencer, 1942 and D. suzukii. The predominant species in 2011 were the obscura group and D. hydei. In 2012, the predominant species were the melanogaster and obscura groups. The predominant species in 2013 were the melanogaster group and D. suzukii. Throughout the study, each species exhibited unique patterns in activity that varied from year to year. The results of this study reveal a greater diversity of Drosophila in the inland Pacific Northwest, U.S.A. Holarctic shrub-steppe environment than previously documented, highlighting the need for more in-depth research on any competition between D. suzukii and local Drosophila species.