Psyllids in Natural Habitats as Alternative Resources for Key Natural Enemies of the Pear Psyllids (Hemiptera: Psylloidea).

The pear psyllids (Cacopsylla spp.; Psylloidea) comprise ~24 species of sap-feeding insects distributed in Europe, temperate Asia, and (as introductions) in the Americas. These pear-specialized insects are among the most damaging and difficult to control pests in orchards. Biological control increasingly is being used to replace or partially replace insecticidal management of pear psyllids. Many key natural enemies of pear psyllids regularly occur in non-orchard habitats on native plants. The presence of beneficial species both in orchard and non-orchard habitats (here referred to as "spillover") has prompted suggestions that native plants and their associated psyllids should be conserved as alternative resources for natural enemies of pear psyllids. The expectation is that the natural enemies will move from those habitats into psyllid-infested orchards. This review shows that psyllids in native habitats are important resources for several key predators and parasitoids of pear psyllids. These resources are critical enough that some beneficials exhibit an almost nomadic existence as they move between plant species, tracking the seasonal appearance and disappearance of psyllid species. In contrast, other natural enemies show minimal or no spillover between orchard and non-orchard habitats, which likely is evidence that they exhibit limited movement at best between orchard and non-orchard habitats. To show conclusively that spillover also indicates that a beneficial species disperses between native habitats and orchards requires difficult research on insect movement. This review concludes with a brief discussion of these difficulties and possible solutions.

Seasonal activity of Trechnites insidiosus (Hymenoptera: Encyrtidae) and its host Cacopsylla pyricola (Hemiptera: Psyllidae) in pear.

Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae) is the most expensive and challenging insect pest of commercial pear trees in the Pacific Northwest. Integrated pest management (IPM) programs are working toward relying more heavily on natural enemies to reduce insecticide use. Trechnites insidiosus (Crawford) (Hymenoptera: Encyrtidae) is the main parasitoid of C. pyricola, but little is known about its biology in the region. Developing sampling tools is important for the deployment of IPM programs, including monitoring of natural enemies. In this study, we examined 2 conventional monitoring methods: beat trays and yellow sticky cards, in addition to screened sticky cards and 3D-printed cylinder traps. Additionally, we tested an overwintering trap for the collection of parasitized C. pyricola. The trapping methods were tested in orchards in Oregon and Washington. Unscreened cards caught the most T. insidiosus and C. pyricola, followed by screened cards, cylinder traps, and then beat trays. Beat trays sometimes failed to catch any T. insidiosus, even when it was found in abundance via other methods. Screened cards and cylinder traps reduced bycatch and increased ease of identifying T. insidiosus. Specimens from the cylinder traps were also more suitable for use in molecular analysis. The overwintering traps were effective at capturing parasitized C. pyricola, but were highly variable year to year. The ideal trapping method will vary based on research needs (e.g., DNA preservation, reducing bycatch, catching higher numbers), but both screened sticky cards and cylinder traps were viable methods for monitoring T. insidiosus and its host.

Association of Two Bactericera Species (Hemiptera: Triozidae) With Native Lycium spp. (Solanales: Solanaceae) in the Potato Growing Regions of the Rio Grande Valley of Texas.

Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) is a vector of 'Candidatus Liberibacter solanacearum' (Lso), the pathogen that causes potato zebra chip. Zebra chip incidence varies regionally, perhaps because of geographic differences in species of noncrop hosts available to the vector and in susceptibility of those hosts to Lso. Native and introduced species of Lycium (Solanales: Solanaceae) are important noncrop hosts of B. cockerelli in some regions of North America. Susceptibility of native Lycium species to Lso is uncertain. We investigated the use of two native species of Lycium by B. cockerelli in South Texas and tested whether they are susceptible to Lso. Bactericera cockerelli adults and nymphs were collected frequently from L. berlandieri Dunal and L. carolinianum Walter. Greenhouse assays confirmed that B. cockerelli develops on both species and showed that Lso infects L. carolinianum. Molecular gut content analysis provided evidence that B. cockerelli adults disperse between potato and Lycium. These results demonstrate that L. berlandieri and L. carolinianum are likely noncrop sources of potato-colonizing B. cockerelli in South Texas and that L. carolinianum is a potential source of Lso-infected psyllids. We also routinely collected the congeneric psyllid, Bactericera dorsalis (Crawford), from both Lycium species. These records are the first for this psyllid in Texas. Bactericera dorsalis completed development on both native Lycium species, albeit with high rates of mortality on L. berlandieri. B. dorsalis acquired and transmitted Lso on L. carolinianum under greenhouse conditions but did not transmit Lso to potato. These results document a previously unknown vector of Lso.

Identification of three new 'Candidatus Liberibacter solanacearum' haplotypes in four psyllid species (Hemiptera: Psylloidea).

Eleven haplotypes of the bacterium, 'Candidatus Liberibacter solanacearum', have been identified worldwide, several of which infect important agricultural crops. In the United States, haplotypes A and B are associated with yield and quality losses in potato, tomato, and other crops of the Solanaceae. Both haplotypes are vectored by potato psyllid, Bactericera cockerelli. Recently, a third haplotype, designated F, was identified in southern Oregon potato fields. To identify the vector of this haplotype, psyllids of multiple species were collected from yellow sticky cards placed near potato fields during two growing seasons. Over 2700 specimens were tested for 'Ca. L. solanacearum' by polymerase chain reaction. Forty-seven psyllids harbored the bacterium. The infected specimens comprised four psyllid species in two families, Aphalaridae and Triozidae (Hemiptera: Psylloidea). Nucleic acid and/or amino acid sequence analysis of the 'Ca. L. solanacearum' 16S ribosomal RNA, 50S ribosomal proteins L10/L12, and outer membrane protein identified three new haplotypes of the bacterium, designated as Aph1, Aph2 and Aph3, including two variants of Aph2 (Aph2a and Aph2b). The impact of these new haplotypes on solanaceous or other crops is not known. The vector of 'Ca. L. solanacearum' haplotype F was not detected in this study.

Bacterial Endosymbionts of Bactericera maculipennis and Three Mitochondrial Haplotypes of B. cockerelli (Hemiptera: Psylloidea: Triozidae).

Insects harbor bacterial endosymbionts that provide their hosts with nutritional benefit or with protection against natural enemies, plant defenses, insecticides, or abiotic stresses. We used directed sequencing of 16S rDNA to identify and compare endosymbionts of Bactericera maculipennis (Crawford) and the western, central, and northwestern haplotypes of B. cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae). Both species are native to North America, are known to harbor the plant pathogen 'Candidatus Liberibacter solanacearum' and develop on shared host plants within the Convolvulaceae. The Old-World species Heterotrioza chenopodii (Reuter) (Psylloidea: Triozidae), now found in North America, was included as an outgroup. 16S sequencing confirmed that both Bactericera species harbor 'Candidatus Liberibacter solanacearum' and revealed that both species harbor unique strains of Wolbachia and Sodalis. However, the presence of Wolbachia and Sodalis varied among haplotypes of B. cockerelli. The central and western haplotypes harbored the same strains of Wolbachia, which was confirmed by Sanger sequencing of the wsp and ftsZ genes. Wolbachia was also detected in very low abundance from the northwestern haplotype by high-throughput sequencing of 16S but was not detected from this haplotype by PCR screening. The northwestern and central haplotypes also harbored Sodalis, which was not detected in the western haplotype. Heterotrioza chenopodii harbored an entirely different community of potential endosymbionts compared with the Bactericera spp. that included Rickettsia and an unidentified bacterium in the Enterobacteriaceae. Results of this study provide a foundation for further research on the interactions between psyllids and their bacterial endosymbionts.

Crude Extracts and Alkaloids Derived from Ipomoea-Periglandula Symbiotic Association Cause Mortality of Asian Citrus Psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae).

Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is an important economic pest of citrus crops because it vectors the causal pathogen of huanglongbing (HLB; aka citrus greening). Population suppression of D. citri with insecticides has been disproportionally relied on for HLB management and a greater diversity of more sustainable tools is needed. Periglandula spp. is a fungal endosymbiont (family Clavicipitaceae) that forms a mutualistic relationship with members of plants in family Convolvulaceae. This association results in the production of ergot alkaloids that were previously documented as having psyllicidal properties. We investigated the mortality and behavior of D. citri exposed to crude extracts from morning glories in the plant family Convolvulaceae, as well as synthetic ergot alkaloids. Nymphs and adults were exposed to the crude plant extracts from Periglandula positive species of Convolvulaceae, as well as five synthetic ergot alkaloids. Treatments were prepared by exposing clippings of citrus to 100 ng/µL of crude extract from Periglandula-positive species of Ipomoea (I. imperati, I. leptophylla, I. pandurata and I. tricolor), and Turbina corymbosa, and from one Periglandula-negative species (I. alba) (100 ng/µL). Mortality of adult and nymphal D. citri was significantly higher than the control after exposure to extracts from I. tricolor and I. imperati. The synthetic ergot alkaloids, lysergol (10-100 ng/µL), ergonovine maleate (100 ng/µL), agroclavine (10-100 ng/µL), and ergosine (10-100 ng/µL) increased mortality of D. citri nymphs, while ergosine (100 ng/µL) and agroclavine (100 ng/µL) increased mortality of adults compared to water controls. Fewer D. citri adults settled on plants treated with crude extracts or synthetic ergot alkaloids than on water controls at 48 h after release. D. citri that fed on citrus leaves treated with 10 ng/µL solution of crude extract from the Periglandula-positive species Ipomoea (I. imperati, I. leptophylla, I. pandurata, I. tricolor), and Turbina corymbosa excreted significantly less honeydew compared with a negative water control and extract from Periglandula-negative species (I. alba). Our results indicate that crude extracts and ergot alkaloids exhibit toxic and sub-lethal effects on D. citri that could be useful for management of this pest.

Stylet Probing Behavior of Two Bactericera (Hemiptera: Psylloidea: Triozidae) Species on Host and Nonhost Plants.

Understanding host use by psyllids (Hemiptera: Psylloidea) benefits from comparative studies of behavior on host and nonhost plant species. While most psyllid species develop on one or a few closely related plant species, some species are generalized enough to develop on species across plant families. We used electropenetography (EPG) technology to compare probing activities of an oligophagous psyllid (Bactericera cockerelli (Sulc)) and a host-specialized psyllid (Bactericera maculipennis) on two species of Solanaceae (potato, Solanum tuberosum L. and matrimony vine, Lycium barbarum L.) and two species of Convolvulaceae (field bindweed, Convolvulus arvensis L. and sweet potato, Ipomoea batatas). Bactericera cockerelli develops on all four species, albeit with longer development times on Convolvulaceae. Bactericera maculipennis develops only on Convolvulaceae. Bactericera cockerelli fed readily from phloem of all four species, but the likelihood of entering phloem and duration of time in phloem was reduced on suboptimal hosts (Convolvulaceae) relative to behavior on Solanaceae. We observed instances of cycling between bouts of phloem salivation and ingestion in assays of optimal (Solanaceae) hosts not observed on Convolvulaceae. The Convolvulaceae-specialized B. maculipennis (Crawford) failed to feed from phloem of nonhosts (Solanaceae). Both psyllid species readily ingested from xylem of all plant species, irrespective of host status. Our finding that phloem feeding by B. maculipennis did not occur on potato has implications for understanding epidemiology of phloem-limited psyllid-vectored plant pathogens. Our results also showed that EPG assays detect subtle variation in probing activities that assist in understanding host use by psyllids.

Host plants and Wolbachia shape the population genetics of sympatric herbivore populations.

Changing climate and land-use practices have the potential to bring previously isolated populations of pest insects into new sympatry. This heightens the need to better understand how differing patterns of host-plant association, and unique endosymbionts, serve to promote genetic isolation or integration. We addressed these factors in populations of potato psyllid, Bactericera cockerelli (Sulc), a generalist herbivore that vectors a bacterial pathogen (Candidatus Liberibacter solanacearum, causal pathogen of zebra chip disease) of potato (Solanum tuberosum L.). Genome-wide SNP data revealed two major genetic clusters-psyllids collected from potato crops were genetically similar to psyllids found on a common weed, Lycium spp., but dissimilar from those found on another common non-crop host, Solanum dulcamara L. Most psyllids found on Lycium spp. and potato represented a single mitochondrial cytochrome oxidase I (COI) haplotype that has been suggested to not be native to the region, and whose arrival may have been concurrent with zebra chip disease first emerging. The putatively introduced COI haplotype usually co-occurred with endosymbiotic Wolbachia, while the putatively resident COI haplotype generally did not. Genetic intermediates between the two genetic populations of insects were rare, consistent with recent sympatry or reproductive isolation, although admixture patterns of apparent hybrids were consistent with introgression of genes from introduced into resident populations. Our results suggest that both host-plant associations and endosymbionts are shaping the population genetic structure of sympatric psyllid populations associated with different non-crop hosts. It is of future interest to explicitly examine vectorial capacity of the two populations and their potential hybrids, as population structure and hybridization might alter regional vector capacity and disease outbreaks.

Susceptibility of Physalis longifolia (Solanales: Solanaceae) to Bactericera cockerelli (Hemiptera: Triozidae) and 'Candidatus Liberibacter solanacearum'.

The potato psyllid, Bactericera cockerelli (Sulc), is a major pest of potato (Solanum tuberosum L.; Solanales: Solanaceae) as a vector of 'Candidatus Liberibacter solanacearum', the pathogen that causes zebra chip. Management of zebra chip is challenging in part because the noncrop sources of Liberibacter-infected psyllids arriving in potato remain unknown. Adding to this challenge is the occurrence of distinct genetic haplotypes of both potato psyllid and Liberibacter that differ in host range. Longleaf groundcherry (Physalis longifolia Nutt.) has been substantially overlooked in prior research as a potential noncrop source of Liberibacter-infected B. cockerelli colonizing fields of potato. The objective of this study was to assess the suitability of P. longifolia to the three common haplotypes of B. cockerelli (central, western, and northwestern haplotypes), and to two haplotypes of 'Ca. L. solanacearum' (Liberibacter A and B haplotypes). Greenhouse bioassays indicated that B. cockerelli of all three haplotypes produced more offspring on P. longifolia than on potato and preferred P. longifolia over potato during settling and egg-laying activities. Greenhouse and field trials showed that P. longifolia was also highly susceptible to Liberibacter. Additionally, we discovered that infected rhizomes survived winter and produced infected plants in late spring that could then be available for psyllid colonization and pathogen acquisition. Results show that P. longifolia is susceptible to both B. cockerelli and 'Ca. L. solanacearum' and must be considered as a potentially important source of infective B. cockerelli colonizing potato fields in the western United States.

Mortality of Potato Psyllid (Hemiptera: Triozidae) on Host Clippings Inoculated With Ergot Alkaloids.

Our previous study provided correlative evidence that morning glory species harboring endophytic fungi (Periglandula) are resistant to potato psyllid [Bactericera cockerelli (Sulc)], whereas species free of fungi often allowed psyllid development. In this study, we manipulated levels of ergot alkaloids in host tissues by inoculating clippings from potato plants with extracts from morning glories that harbor Periglandula [Ipomoea leptophylla Torrey, Ipomoea imperati (Vahl) Grisebach, Ipomoea tricolor Cavanilles, Ipomoea pandurata (L.) G. F. Meyer, and Turbina corymbosa (L.)] and one species (Ipomoea alba L.) that does not harbor the endophyte. Ergot alkaloids (clavines, lysergic acid amides, and ergopeptines) were detected in potato clippings, thus confirming that leaves had taken up compounds from solutions of crude extracts. Psyllid mortality rates on inoculated clippings ranged between 53 and 93% in treatments producing biochemically detectable levels of alkaloids, when compared with 15% mortality in water controls or the alkaloid-free I. alba. We then tested synthetic analogs from each of the three alkaloid classes that had been detected in the crude extracts. Each compound was assayed by inoculating clippings of two host species (potato and tomato) at increasing concentrations (0, 1, 10, and 100 µg/ml in solution). Psyllids exhibited a large and significant increase in mortality rate beginning at the lowest two concentrations, indicating that even very small quantities of these chemicals led to mortality. Feeding by nymphs on artificial diets containing synthetic compounds resulted in 100% mortality within 48 h, irrespective of compound. Further testing of ergot alkaloids to characterize the mode of action that leads to psyllid mortality is warranted.

The Artificial Sweetener, Erythritol, Has Insecticidal Properties Against Pear Psylla (Hemiptera: Psyllidae).

Erythritol is a dietary sweetener that is used for low-calorie or diabetic diets. Although safe for human consumption, erythritol is lethal to certain Dipteran pests, but insecticidal effects of erythritol on phloem-feeding insects have yet to be examined. Our goal was to determine whether erythritol has insecticidal activity against pear psylla, Cacopsylla pyricola (Foerster) (Hemiptera: Psyllidae). We first demonstrated that ingestion of erythritol solutions compared with water by pear psylla caused reduced feeding, impaired motor functions, and reduced survival time of adults. We then tested whether foliar treatment of pear leaves with erythritol was also lethal to pear psylla. Foliar treatment of erythritol led to reduced 3-d survival of pear psylla nymphs and adults, and reduced rates of oviposition by pear psylla adults. Psylla adults also preferred to settle on untreated leaves than on erythritol-treated leaves in preference assays. Finally, we conducted field experiments to test whether applications of erythritol provided pear trees with protection against pear psylla under natural field conditions. Those experiments showed a reduction in pear psylla nymphs on erythritol-treated trees compared with untreated trees, but only if the erythritol was completely dissolved into solution by heating. Laboratory trials confirmed the importance of heating. Results of our experiments demonstrate that erythritol is insecticidal to pear psylla nymphs and adults and provide the first report that erythritol is lethal to a phloem-feeding insect. These findings suggest that erythritol may provide a new safe and effective tool for the management of pear psylla.

Host and Non-host 'Whistle Stops' for Psyllids: Molecular Gut Content Analysis by High-Throughput Sequencing Reveals Landscape-Level Movements of Psylloidea (Hemiptera).

Psyllids (Hemiptera: Psylloidea) are phloem-feeding insects that tend to be highly specific in their host plants. Some species are well-known agricultural pests, often as vectors of plant pathogens. Many pest psyllids colonize agricultural fields from non-crop reproductive hosts or from non-host transitory and winter shelter plants. Uncertainty about which non-crop species serve as sources of psyllids hinders efforts to predict which fields or orchards are at greater risk of being colonized by psyllids. High-throughput sequencing of trnL, trnF, and ITS was used to examine the dietary histories of three pest and two non-pest psyllid species encompassing a diversity of lifecycles: Cacopsylla pyricola (Förster) (Psyllidae), Bactericera cockerelli (Sulc) (Triozidae), Diaphorina citri Kuwayama (Liviidae), Aphalara loca Caldwell (Aphalaridae), and a Cacopsylla species complex associated with Salix (Malphighiales: Salicaceae). Results revealed an unexpectedly high level of feeding on non-host species by all five psyllid species. The identification of the dietary history of the psyllids allowed us to infer their landscape-scale movements prior to capture. Our study demonstrates a novel use for gut content analysis-to provide insight into landscape-scale movements of psyllids-thus providing a means to pinpoint the non-crop sources of pest psyllids colonizing agricultural crops. We observed previously unknown psyllid behaviors during our efforts to develop this method and discuss new research directions for the study of psyllid ecology.

Survival and development of potato psyllid (Hemiptera: Triozidae) on Convolvulaceae: Effects of a plant-fungus symbiosis (Periglandula).

Plant species in the family Solanaceae are the usual hosts of potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae). However, the psyllid has also been shown to develop on some species of Convolvulaceae (bindweeds and morning glories). Developmental success on Convolvulaceae is surprising given the rarity of psyllid species worldwide associated with this plant family. We assayed 14 species of Convolvulaceae across four genera (Convolvulus, Calystegia, Ipomoea, Turbina) to identify species that allow development of potato psyllid. Two populations of psyllids were assayed (Texas, Washington). The Texas population overlaps extensively with native Convolvulaceae, whereas Washington State is noticeably lacking in Convolvulaceae. Results of assays were overlain on a phylogenetic analysis of plant species to examine whether Convolvulaceae distantly related to the typical host (potato) were less likely to allow development than species of Convolvulaceae more closely related. Survival was independent of psyllid population and location of the plant species on our phylogenetic tree. We then examined whether presence of a fungal symbiont of Convolvulaceae (Periglandula spp.) affected psyllid survival. These fungi associate with Convolvulaceae and produce a class of mycotoxins (ergot alkaloids) that may confer protection against plant-feeding arthropods. Periglandula was found in 11 of our 14 species, including in two genera (Convolvulus, Calystegia) not previously known to host the symbiont. Of these 11 species, leaf tissues from five contained large quantities of two classes of ergot alkaloids (clavines, amides of lysergic acid) when evaluated by LC-MS/MS. All five species also harbored Periglandula. No ergot alkaloids were detected in species free of the fungal symbiont. Potato psyllid rapidly died on the five species that harbored Periglandula and contained ergot alkaloids, but survived to adulthood on seven of the nine species in which ergot alkaloids were not detected. These results support the hypothesis that a plant-fungus symbiotic relationship affects the suitability of certain Convolvulaceae to potato psyllid.

Elicitors of Host Plant Defenses Partially Suppress Cacopsylla pyricola (Hemiptera: Psyllidae) Populations Under Field Conditions.

Defense elicitors are products that activate acquired defense responses in plants, thus rendering the plants less susceptible to attack by a broad range of pests. We demonstrated previously under laboratory conditions that foliar applications of the defense elicitors Actigard (acibenzolar-S-methyl), Employ (harpin protein), or ODC (chitosan) to potted pear trees (Pyrus communis L.) each caused an increase in mortality of Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae) nymphs and altered the settling and oviposition behavior of the adults. In this study, we monitored C. pyricola populations over a 3-yr period on orchard-grown trees treated with water (untreated control), Actigard, Employ, or ODC. Fewer nymphs were observed on trees treated with elicitors compared with untreated trees in both 2014 and 2016. A similar but statistically nonsignificant pattern was observed in 2015 when nearly 30% fewer nymphs were observed on trees treated with elicitors versus untreated controls. Observed reductions in psyllid numbers by defense elicitors were modest and do not warrant the use of these products alone for managing C. pyricola. However, these products are often used for management of fire blight, and our observations that elicitors also reduce C. pyricola populations may be useful for system-wide integrated pest management approaches.

Bacterial Endosymbionts of the Psyllid Cacopsylla pyricola (Hemiptera: Psyllidae) in the Pacific Northwestern United States.

Insects often have facultative associations with bacterial endosymbionts, which can alter the insects' susceptibility to parasitism, pathogens, plant defenses, and certain classes of insecticides. We collected pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae), from pear orchards in Washington and Oregon, and surveyed them for the presence of bacterial endosymbionts. Adult psyllids were collected on multiple dates to allow us to assay specimens of both the summer ("summerform") and the overwintering ("winterform") morphotypes. Two endosymbionts, Arsenophonus and Phytoplasma pyri, were detected in psyllids of both morphotypes in both states. A separate survey revealed similar associations present in psyllids collected in 1987. Arsenophonus was present in 80-100% of psyllids in all growing regions. A slightly lower proportion of summerform than winterform psyllids harbored the bacterium. Arsenophonus was present in the bacteriomes and developing oocytes of most psyllids, indicating that this endosymbiont is transovarially transmitted. This bacterium was also observed in the salivary glands and midguts of some psyllids. Phytoplasma pyri was present in a greater proportion of pear psylla from orchards near Yakima, WA, than from other regions, and was present in a higher proportion of winterforms than summerforms. We did not detect Wolbachia, Profftella, or Liberibacter europaeus, which are associated with other psyllid pests, including other species of Cacopsylla. Our study is the first to survey North American populations of C. pyricola for endosymbionts, and provides a foundation for further research on how bacterial associations may influence the ecology and management of this pest.

"Candidatus Liberibacter solanacearum" Associated With the Psyllid, Bactericera maculipennis (Hemiptera: Triozidae).

The psyllid Bactericera maculipennis (Crawford) (Hemiptera: Triozidae) often cohabits field bindweed (Convolvulus arvensis, Solanales: Convolvulaceae) and other plants with the congeneric psyllid, Bactericera cockerelli (Sulc), in the Pacific Northwestern United States. Bactericera cockerelli is a vector of "Candidatus Liberibacter solanacearum," the pathogen associated with zebra chip disease of potato (Solanales: Solanaceae). Because B. maculipennis and B. cockerelli both naturally occur on certain plants, we surveyed B. maculipennis adults collected from Washington and Idaho for presence of "Ca. L. solanacearum" to determine whether this psyllid also harbors this pathogen. Liberibacter was present in 30% of field-collected B. maculipennis and in 100% of colony-reared psyllids. Sequences of 16S rDNA and microsatellite markers revealed that "Ca. L. solanacearum" from B. maculipennis was closely related to Liberibacter haplotype B from B. cockerelli. Results of laboratory assays demonstrated that Liberibacter can be transmitted between B. cockerelli and B. maculipennis on plants within the Convolvulaceae. Potato plants challenged with Liberibacter-infected B. maculipennis did not become infected, apparently because potato is not a suitable host for the psyllid. We therefore conclude that B. maculipennis is not a direct threat to potato production, despite its association with Liberibacter. We are the first to report that "Ca. L. solanacearum" is associated with a psyllid other than B. cockerelli in North America. Results of our study demonstrate the importance of understanding the complete ecology of psyllids-including interactions with other psyllids on non-crop hosts-in predicting what crops or regions are potentially susceptible to the spread of Liberibacter.

Assessing the Likelihood of Transmission of Candidatus Liberibacter solanacearum to Carrot by Potato Psyllid, Bactericera cockerelli (Hemiptera: Triozidae).

'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited bacterium that severely affects important Solanaceae and Apiaceae crops, including potato, tomato, pepper, tobacco, carrot and celery. This bacterium is transmitted to solanaceous species by potato psyllid, Bactericera cockerelli, and to Apiaceae by carrot psyllids, including Trioza apicalis and Bactericera trigonica. Five haplotypes of Lso have so far been described, two are associated with solanaceous species and potato psyllids, whereas the other three are associated with carrot and celery crops and carrot psyllids. Little is known about cross-transmission of Lso to carrot by potato psyllids or to potato by carrot psyllids. Thus, the present study assessed whether potato psyllid can transmit Lso to carrot and whether Lso haplotypes infecting solanaceous species can also infect carrot and lead to disease symptom development. In addition, the stylet probing behavior of potato psyllid on carrot was assessed using electropenetrography (EPG) technology to further elucidate potential Lso transmission to Apiaceae by this potato insect pest. Results showed that, while potato psyllids survived on carrot for several weeks when confined on the plants under controlled laboratory and field conditions, the insects generally failed to infect carrot plants with Lso. Only three of the 200 carrot plants assayed became infected with Lso and developed characteristic disease symptoms. Lso infection in the symptomatic carrot plants was confirmed by polymerase chain reaction assay and Lso in the carrots was determined to be of the haplotype B, which is associated with solanaceous species. EPG results further revealed that potato psyllids readily feed on carrot xylem but rarely probe into the phloem tissue, explaining why little to no Lso infection occurred during the controlled laboratory and field cage transmission trials. Results of our laboratory and field transmission studies, combined with our EPG results, suggest that the risk of Lso infection and spread between psyllid-infested solanaceous and Apiaceae crops is likely to be negligible under normal field conditions.

Gut Content Analysis of a Phloem-Feeding Insect, Bactericera cockerelli (Hemiptera: Triozidae).

Potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a key pest of potato (Solanum tuberosum L., Solanales: Solanaceae) and a vector of "Candidatus Liberibacter solanacearum," the pathogen associated with zebra chip disease. In addition to its presence on cultivated crops, the psyllid regularly occurs on numerous uncultivated annual and perennial species within the Solanaceae. A better understanding of landscape-level ecology of B. cockerelli would substantially improve our ability to predict which potato fields are most likely to be colonized by infected psyllids. We developed three PCR-based methods of gut content analysis to identify what plant species B. cockerelli had previously fed upon. These methods included-1) sequencing PCR amplicons of regions of plant-derived internal transcribed spacer (ITS) or the chloroplast trnL gene from psyllids, 2) high-resolution melting analysis of ITS or trnL real-time PCR products, and 3) restriction enzyme digestion of trnL PCR product. Each method was used to test whether we could identify psyllids that had been reared continuously on potato versus psyllids reared continuously on the perennial nightshade, Solanum dulcamara. All three methods of gut content analysis correctly identified psyllids from potato and psyllids from S. dulcamara Our study is the first to demonstrate that plant DNA can be detected in a phloem-feeding insect. Gut content analysis, in combination with other landscape ecology approaches, could help elucidate patterns in landscape-level movements and host plant associations of B. cockerelli.

Horizontal Transmission of "Candidatus Liberibacter solanacearum" by Bactericera cockerelli (Hemiptera: Triozidae) on Convolvulus and Ipomoea (Solanales: Convolvulaceae).

"Candidatus Liberibacter solanacearum" (Proteobacteria) is an important pathogen of solanaceous crops (Solanales: Solanaceae) in North America and New Zealand, and is the putative causal agent of zebra chip disease of potato. This phloem-limited pathogen is transmitted to potato and other solanaceous plants by the potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae). While some plants in the Convolvulaceae (Solanales) are also known hosts for B. cockerelli, previous efforts to detect Liberibacter in Convolvulaceae have been unsuccessful. Moreover, studies to determine whether Liberibacter can be acquired from these plants by B. cockerelli are lacking. The goal of this study was to determine whether horizontal transmission of Liberibacter occurs among potato psyllids on two species of Convolvulaceae, sweet potato (Ipomoea batatas) and field bindweed (Convolvulus arvensis), which grows abundantly in potato growing regions of the United States. Results indicated that uninfected psyllids acquired Liberibacter from both I. batatas and C. arvensis if infected psyllids were present on plants concurrently with the uninfected psyllids. Uninfected psyllids did not acquire Liberibacter from plants if the infected psyllids were removed from the plants before the uninfected psyllids were allowed access. In contrast with previous reports, PCR did detect the presence of Liberibacter DNA in some plants. However, visible amplicons were faint and did not correspond with acquisition of the pathogen by uninfected psyllids. None of the plants exhibited disease symptoms. Results indicate that horizontal transmission of Liberibacter among potato psyllids can occur on Convolvulaceae, and that the association between Liberibacter and Convolvulaceae merits additional attention.

Use of Electrical Penetration Graph Technology to Examine Transmission of 'Candidatus Liberibacter solanacearum' to Potato by Three Haplotypes of Potato Psyllid (Bactericera cockerelli; Hemiptera: Triozidae).

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium 'Candidatus Liberibacter solanacearum' (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid (Central, Western, Northwestern). All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato ("inoculation access period", or IAP) to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions of vector efficiency.