Blake R. Bextine

Serratia marcescens, a phloem-colonizing, squash bug-transmitted bacterium: causal agent of cucurbit yellow vine disease.

Cucurbit yellow vine disease (CYVD), which can inflict heavy losses to watermelon, pumpkin, cantaloupe, and squash in U.S. production areas from the midwest to northeastern states, causes phloem discoloration, foliar yellowing, wilting, and plant decline. Bacteria were cultured from the phloem of crown sections of symptomatic plants of Citrullus lanatas and Cucurbita pepo. Those bacteria testing positive in CYVD-specific polymerase chain reaction (PCR) were all gram negative and appeared morphologically identical, producing creamy white, smooth, entire, convex colonies on Luria-Bertani or nutrient agar. Characterized cucurbit-derived strains of Serratia marcescens were introduced into greenhouse-grown squash plants by puncture inoculation and into field-grown squash plants by enclosure with S. marcescens-fed squash bugs, Anasa tristis. Up to 60% of the bacteria-inoculated plants in the greenhouse and up to 17% of field plants caged with inoculative squash bugs developed phloem discoloration and tested positive for S. marcescens by CYVD-specific PCR. None of the controls developed phloem discoloration or tested positive by PCR. Of the diseased field plants, 12% (2 of 35) also yellowed, wilted, and collapsed, exhibiting full symptom development of CYVD. However, neither plant collapse nor decline was observed in the greenhouse-grown, puncture-inoculated plants. The morphology, growth habit, and PCR reaction of bacteria cultured from crown tissue of a subset of plants in each experimental group were indistinguishable from those of the inoculum bacteria. Evidence presented from our studies confirms that the squash bug can transmit S. marcescens, the CYVD causal bacterium. The S. marcescens-A. tristis relationship described here is the first instance in which the squash bug has been identified as a vector of a plant pathogen. Our experiments represent a completion of the steps of Kochs postulates, demonstrating that S. marcescens is the causal agent of CYVD and that the squash bug, A. tristis, is a vector of the pathogen.

Advances in RNA interference: dsRNA Treatment in Trees and Grapevines for Insect Pest Suppression

RNA interference (RNAi) is a breakthrough technology that has significantly impacted contemporary approaches to control the damage caused by insect pests. The method permits functional genomics studies in many organisms that are difficult to study, moving science beyond model systems and laboratory studies. Reviews on applications of RNAi promise to improve human health and agricultural production (Castanotto and Rossi 2009, Siomi and Siomi 2009, Bellés 2010, Huvenne and Smagghe 2010). To move RNAi into real-world applications such as Citrus sp. and viticulture, we evaluated the movement and persistence of dsRNA in citrus trees and grapevines (Hunter et al. 2010ab). Most well-known RNAi studies continue to rely on injecting the dsRNA molecules directly into the organism; this approach is not suitable for use in the field. If host-delivered RNAi-based management approaches are to be implemented, plants must successfully uptake the dsRNA and retain it long enough for the target insects to ingest it through feeding (Hunter et al. 2010c, Huvenne and Smagghe 2010). We propose the development of a new class of environmentally-friendly, ‘Highly Specific Pest Control’ (HiSPeC) substances. HiSPeC’s are highly specific, with no adverse effects on non-target species. Equally important, these substances are environmentally friendly and safe to handle. This report entails current efforts of HiSPeC’s that use plant-systemic delivery of an RNAi agent. To facilitate a realworld implementation of a host-delivered RNAi strategy, robust intake of the dsRNA and system-wide spread of the silencing action must be achieved. In some invertebrates, there exists a systemic RNAi pathway (Jose et al. 2009); other invertebrates depend on receptor-mediated endocytosis for cell internalization of the dsRNA (Saleh et al. 2006, Jose and Hunter 2007). The dsRNA can have very low LD-50, in the pictograms; results showed specificity to silence arginine kinase, AKtranscripts in psyllids and leafhoppers with the corresponding dsRNA experiments in lab tests. RNAi strategies will demand mass-production of dsRNA, efficient delivery methods, and methods to validate its environmental stability (Hunter et al. 2010c). The longevity of the dsRNA in Citrus trees showed suitability to develop an area-wide pest suppression approach. This study reports the robust nature and persistence of the RNAi pathway metabolites in whole-plant systems targeting insects. Persistence of dsRNA in psyllids and leafhoppers was detectable for 5-8 days post ingestion from plants, while detection in treated Citrus was at least 57 days post treatment (Fig. 1). Small interfering RNA (siRNA) was detectable into the third month. ________________________

Characterization of the Asian Citrus Psyllid Transcriptome.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a vector for the causative agents of Huanglongbing, which threatens citrus production worldwide. This study reports and discusses the first D. citri transcriptomes, encompassing the three main life stages of D. citri, egg, nymph and adult. The transcriptomes were annotated using Gene Ontology (GO) and insecticide-related genes within each life stage were identified to aid the development of future D. citri insecticides. Transcriptome assemblies and other sequence data are available for download at the International Asian Citrus Psyllid Genome Consortium website [http://psyllid.org/download] and at NCBI [http://www.ncbi.nlm.nih.gov/bioproject/29447].

Using Quantitative Real Time PCR Melt Curve Analysis of Partial CO1 Sequence for Rapid Biotype Differentiation of Bactericera cockerelli (Hemiptera: Triozidae)

Abstract. The potato psyllid, Bactericera cockerelli (Sulc), is a native of North America and occurs from Central America to Canada. A newly emerging plant pathogen, Candidatus Liberibacter solanacearum, associated with potato psyllids in North America and New Zealand, has become a major concern in many solanaceous crops worldwide. In potato, Solanum tuberosum L., the resulting disease is called zebra chip, but similar foliar and vascular symptoms have been reported in tomato, Solanum lycopersicum L., and peppers, Capsicum annuum L. To date, it is known that potato psyllids transmit Candidatus Liberibacter solanacearum, and they are the primary mode of pathogen spread in potato, tomato, and pepper agroecosystems in North America and New Zealand. Potato psyllids have a rather wide range of hosts, including wild plants, from which the psyllids may spread to crop plants. Within the insect species, B. cockerelli, two biotypes are reported to exist. This differentiation was originally associated with hardiness to cold temperatures and can be monitored through genotypic variations in the cytochrome oxidase 1 (CO1) gene. In this study, two potato psyllid biotypes from four locations were delineated by melt temperature analysis following Sybr Green quantitative real-time PCR. In North Dakota, a northern extreme for the central range of potato psyllids, 100% of samples were “Central Biotype”; whereas, both biotypes (Central and Western) were found in Colorado where the two biotypes seem to overlap.

Comparative Analysis of Antennae Sensory Arrays in Asian Citrus Psyllid, Diaphorina citri, and Potato Psyllid, Bactericera cockerelli (Hemiptera)

Abstract. Scanning electron microscopy was used for comparison of the morphological basis for host detection and mating behaviors by olfactory reception in two psyllid species, the Asian citrus psyllid, Diaphorina citri Kuwayama, and potato/tomato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psyllidae). The two psyllids inhabit different plant niches. The Asian citrus psyllid is essentially monophagous, feeding primarily on citrus, but the potato psyllid feeds on a wide range of solanaceous plants. This study identified two antennal sensory arrays, with a more complex arrangement occurring in the Asian citrus psyllid than potato psyllid. The antennal length of the Asian citrus psyllid was 0.23 mm and contained 10 segments, while the antenna of the potato psyllid was 0.60 mm long with 10 segments. Both species had two multi-porous, single-walled bristles apical to the sensillus terminalis. These bristles were longer in potato psyllids. A few mechanosensory and chemosensory hairs were found on all antennal segments of both species, with more sensillae on distal segments. Asian citrus psyllid coevolved with its citrus host in tropical Asian countries; locating the strongly aromatic plants probably was less difficult. Asian citrus psyllid females would be constrained to a specific host plant, facilitating mate detection. Potato psyllid has fewer olfactory sensilla and feeds on a wider host range. A wide host range suggests that potato psyllid may have increased sensitivity to specific chemical cues required to locate mates that could be on different species of host plants.

Delivery system using sodium alginate virus loaded pellets to red imported fire ants (Solenopsis invicta, Hymenoptera: Formicidae).

ABSTRACT Microencapsulation as a delivery mechanism of SINV-1 and other molecules such as dsRNA, offers an approach to Solenopsis invicta Buren management that is target specific and fits current approaches to baiting ants with toxins and/or RNA-interference. The delivery method presented here targets ground dwelling, foraging ants with an ant-infecting virus which is specific to the genus, Solenopsis. Endemic ant-infecting viruses, like S. invicta viruses (SINV-1, SINV-2, and SINV-3) are being evaluated for efficacy in S. invicta population suppression. In this study, SINV-1 (TX5 strain) was extracted from S. invicta colonies and microencapsulated in sodium alginate pellets. Pellets containing extracted whole virions were offered to confirmed non-infected S. invicta colonies. Colonies were sampled every 5 d and tested by reverse transcription polymerase chain reaction (RT-PCR) for presence of viral RNA. The longevity of control and viral pellets were also evaluated. Within 30 d, post-feeding of virus, 35% of S. invicta colonies acquired SINV-1 infection (P = 0.03). Thus, microencapsulation as a delivery mechanism was successful to deliver SINV-1 to S. invicta colonies. Future incorporation of this economically affordable method can be implemented to deliver biological agents for specific ant species and to augment current approaches that bait ants. While a virus was used to demonstrate delivery, an adequate and affordable virus production system still needs to be developed before a viral strategy can be adopted as a tool for biological control of fire ants.

Seasonal Increase of Xylella fastidiosa in Hemiptera Collected from Central Texas Vineyards

ABSTRACT Yellow sticky traps were placed in six vineyards in central Texas from 2003 to 2006 and in locations outside the vineyards in 2004–2006. In total, 72 collections on 55 dates were examined. Xylem fluid-feeding insects were removed and identified to species and then analyzed by polymerase chain reaction to determine the presence or absence of Xylella fastidiosa Wells et al. Of the 1318 insects removed, 13 species were found, dominated by Homalodisca vitripennis (Germar), Clastoptera xanthocepahala Germar, and Graphocephala versuta (Say). Insects testing positive for X. fastidiosa were analyzed further using fluorescence resonance energy transfer probes to determine the genotype of the bacterium, which fell into four groups: subspecies fastidiosa, multiplex, sandyi, and unknown subspecies. Vineyards known to be affected by Pierces disease had more insects that were contaminated by the bacterium than those that were not as affected. X. fastidiosa subsp. fastidiosa, the causative agent of Pierces disease, was found more commonly in insects collected from vineyards than from insects collected outside the vineyards. Conversely, the subspecies multiplex and sandyi, which are not known to cause disease in grape, were more commonly found in insects collected outside the vineyard. The percentage of individuals contaminated with the bacterium increased over the course of the growing season, and the data suggest that vector insects acquired X. fastidiosa subsp. fastidiosa from infected grapevines, a necessary precursor for vine to vine transmission to occur. Management options, including the use of systemic insecticides and plant roguing, would be effective for this type of transmission model.

Cytochrome B Sequences of Potato Psyllids, Bactericera cockerelli (Sulc) 1 , from North and Central America

Potato psyllid, Bactericera cockerelli (Sulc), is an insect pest of major concern in North and Central America because it is the vector of Candidatus Liberibacter solanacearum (Clover), the causal agent of zebra chip disease of potato, Solanum tuberosum L. (Liefting et al. 2009). Zebra chip is characterized by chlorosis, leaf scorching, wilting, vascular discoloration, swollen nodes, twisted stems, aerial tubers, and build up of complex simple sugars along the medullary rays of the tuber (Secor et al. 2009). As a result, potato tubers become saturated with simple sugars that burn easily when fried, resulting in potato chips with an unappetizing appearance and flavor. The first confirmed incident of zebra chip occurred in Saltillo, Mexico, in 1994; by 2000, the pathogen had spread to the Rio Grande Valley of South Texas. The migratory patterns of the potato psyllid resulted in northward and southward expansion of outbreaks of zebra chip. To date, the pathogen has been confirmed as far north as Washington State (Munyaneza et al. 2009), and as far south as Nicaragua (Bextine 2012). Exportation of the vector has resulted in the spread of Ca. Liberibacter solanacearum (Clover) to New Zealand and is estimated to have caused millions of dollars in crop losses (Liefting et al. 2009). Populations of potato psyllids from different geographical areas are identifiable by genomic analysis. Using a single nucleotide polymorphism present within a 514-bp cytochrome oxidase I (COI) sequence, two biotypes were discernable (Liu et al. 2006, Jackson et al. 2009). The central biotype is predominantly found east of the Rocky Mountains, and the western biotype is found to the west (Munyaneza 2012). Population differentiation is critical for the study and management of potato psyllids. The western biotype is considered an invasive population and resistant to agrochemical treatments traditionally used to control hemipterans (Liu et al. 2006). This can also be a critical tool in tracking the source of anthropological importation, allowing for more directed action and response. Potato psyllids were collected from seven sites in North and Central America -Colorado, Texas, Washington, Chihuahua, Saltillo, Toluca, and Guatemala. A 3,025-bp region of the mitochondrion genome was amplified by using AmpliTaq Gold 360 Master Mix (Applied Biosystems, Carlsbad, CA), and primers -GCA GGA TCC GCG GCC WTG RGG HCA AAT ATC WTT TTG RGG DGCGCA GGT ACC TCG AGT ATG TAC AMA TYG CCC GTC AYT CTTfrom Thao and Baumann (2004). The resulting amplicons were sequenced by

Impacts of an Orange Oil Solvent and Stickem® on the Detection of Xylella fastidiosa DNA in Glassy-Winged Sharpshooters, Homalodisca vitripennis (Hemiptera: Cicadellidae)

ABSTRACT Xylella fastidiosa is a plant pathogenic bacterium that causes many economically important agricultural diseases and is transmitted by the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae). Efficient detection of X. fastidiosa in field collected H. vitripennis in an area-wide management program can contribute to risk assessment associated with insect presence in vineyards. Prior to conducting molecular assays for detection of X. fastidiosa in individual insects, H. vitripennis must be removed from yellow sticky traps with a solvent such as orange oil. In this study, we determined the effect of orange oil concentration on extraction of individual H. vitripennis following trap removal on detection of X. fastidiosa by qRT-PCR. In a ten-fold dilution series of orange oil, increasing amounts of orange oil caused decreasing levels of X. fastidiosa detection in standardized positive samples. Additionally, tests on the effects of Stickem® brand trap adhesive on qRT-PCR and development of methods which lowered the concentration of orange oil often present in field samples determined the point where detection of X. fastidiosa was negatively impacted. These results benefit the monitoring and screening for Xylella fastidiosa from leafhoppers collected on sticky cards used in regulatory area-wide management.

Comparison of Bacterial Communities of Flower Thrips (Frankliniella tritici1) and Potato Psyllid (Bactericera cockerelli2 )

Abstract. The potato psyllid, Bactericera cockerelli (Sulc), is the primary insect vector of the bacterium Candidatus Liberibacter solanacearum Leifting et al., the causal agent of zebra chip disease in solanaceous crops. Some phloem-feeding insects harbor bacterial communities determined by the food sources of the insects. Horizontal transmission of bacteria between individuals of the same species and insects of different species, with their host plants as reservoirs occurs in several plant insect systems. Ca. L. solanacearum is symbiotic in potato psyllid and can colonize plants before transmitted to its next insect host. Flower thrips, Frankliniella tritici (Fitch), that co-colonize plants have tested positive for Ca. L. solanacearum, although vector status is unknown. The bacterial community of thrips co-colonizing plants infected with Ca. L. solanacearum was analyzed and compared to that of potato psyllids on the same plants. Bacterial communities of samples were sequenced via the Illumina MiSeq platform and analyzed with macQIIME. Of the three thrips samples subjected to 16s bacterial community sequencing, the bacterium Ca. L. solanacearum comprised 6, 1, and 3% of their total bacterial community; of the three psyllid samples screened it comprised 1, 43, and 20% of the total bacterial community. This suggests that thrips can harbor the bacterium Ca. L. solanacearum.