In Silico Characterization and Gene Expression Analysis of Toll Signaling Pathway-Related Genes in Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri is the main vector of citrus greening disease, also known as Huanglongbing (HLB). Currently, mitigating HLB depends on the control of D. citri using insecticides. To design innovative control strategies, we should investigate various biological aspects of D. citri at the molecular level. Herein we explored the Toll signaling system-related proteins in D. citri using in silico analyzes. Additionally, the transcripts of the identified genes were determined in all life stages from eggs to adults. Our findings reveal that D. citri genome possesses Toll signaling pathway-related genes similar to the insect model, Drosophila melanogaster, with slight differences. These genes include cact, TI, Myd88, Dif/DI, pll, tub, and spz encoding Cactus, Toll, Myeloid differentiation factor 88, Dorsal related immunity factor/Dorsal, Pelle, Tube, and Spaetzle, respectively. Unlike D. melanogaster, in D. citri Dorsal, immunity factor and Dorsal are the same protein. In addition, in D. citri, Pelle protein possesses a kinase domain, which is absent in Pelle of D. melanogaster. Gene expression analysis showed the transcript for cact, TI, Myd88, pll, tub, and spz are maximum in adults, suggesting the immunity increases with maturity. Instead, Dif/DI transcripts were maximal in eggs and adults and minimal in nymphal stages, indicating its role in embryonic development. The overall findings will help in designing pioneering control strategies of D. citri based on repressing its immunity by RNAi or CRISPR and combining that with biological control.

Diaphorina citri flavi-like virus localization, transmission, and association with Candidatus Liberibacter asiaticus in its psyllid host.

Huanglongbing is caused by Candidatus Liberibacter asiaticus (CLas) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with CLas are unknown. DcFLV was found in various organs of D. citri, including the midgut and salivary glands, where it co-localized with CLas. CLas-infected nymphs had the highest DcFLV titers compared to the infected adults and CLas-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and CLas were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with CLas in the vector. These results provide new insights on the interactions between DcFLV, the D. citri, and CLas.

Phytoene desaturase-silenced citrus as a trap crop with multiple cues to attract Diaphorina citri, the vector of Huanglongbing.

Huanglongbing (HLB) is one of the most destructive diseases in citrus worldwide. Unfortunately, HLB has no cure and management relies on insecticides to reduce populations of the vector, Diaphorina citri Kuwayama (Hemiptera: Liviidae). We propose an attract-and-kill strategy using a trap crop as an alternative to vector control to reduce transmission of the pathogen, 'Candidatus Liberibacter asiaticus'. We evaluated vector response to phytoene desaturase-silenced citrus trees using virus-induced gene silencing technology. Citrus tristeza virus (CTV) was used to produce a phytoene desaturase-silenced citrus (CTV-tPDS) that expresses visual, olfactory, and gustatory cues to attract D. citri. We found that D. citri were more attracted to CTV-tPDS plants with noticeably better fecundity and overall population fitness than on control plants. Moreover, rearing D. citri on CTV-tPDS plants significantly increased their survival probability compared with those reared on control plants. CTV-tPDS plants possessed reduced content of both carotenoid and chlorophyll pigments resulting in a consistent photobleached phenotype on citrus leaves which provided a sufficient close-range visual attractant to stimulate D. citri landing. Additionally, CTV-tPDS plants exhibited an enriched profile of volatile organic compounds (VOCs), which offered adequate olfactory cues to attract psyllid from long-range. Finally, CTV-tPDS plants exhibited an enriched metabolite content of phloem sap and leaves which offered appropriate gustatory cues that influenced probing/feeding behavior. We believe that introducing CTV-tPDS plants (as a trap crop) to D. citri-infested orchards will attract and congregate psyllids to facilitate their removal from the target crop with insecticides or by other means. This new strategy could be deployed relatively quickly and economically to HLB-impacted citrus industries. Moreover, it is an eco-friendly strategy because it should partially reduce the input of chemical insecticides ameliorating the indirect cost of HLB infection.

Grassy Weeds and Corn as Potential Sources of Barley yellow dwarf virus Spread Into Winter Wheat.

Barley yellow dwarf virus (BYDV) is an important vector-borne pathogen of cereals. Although many species of grasses are known to host BYDV, knowledge of their role in virus spread in regional agroecosystems remains limited. Between 2012 and 2016, Idaho winter wheat production was affected by BYDV. BYDV-PAV and the bird cherry-oat aphid (BCOA) (Rhopalosiphum padi L.) vector were commonly present in the affected areas. A series of greenhouse bioassays were performed to examine whether two types of corn (Zea mays L.), dent and sweet, and three commonly found grassy weeds, downy brome (Bromus tectorum L.), green foxtail (Setaria viridis L.), and foxtail barley (Hordeum jubatum L.), can be inoculated with BYDV (species BYDV-PAV) by BCOA and also act as sources of the virus in winter wheat. BCOA successfully transmitted BYDV-PAV to both corn types and all weed species. Virus titers differed between the weed species (P = 0.032) and between corn types (P = 0.001). In transmission bioassays, aphids were able to survive on these host plants during the 5-day acquisition access period and later successfully transmitted BYDV-PAV to winter wheat (var. SY Ovation). Transmission success was positively correlated with the virus titer of the source plant (P < 0.001) and influenced by weed species (P = 0.028) but not corn type. Overall, the results of our inoculation and transmission assays showed that the examined weed species and corn types can be inoculated with BYDV-PAV by BCOA and subsequently act as sources of infections in winter wheat.

Effect of the level of "Candidatus Liberibacter solanacearum" infection on the development of zebra chip disease in different potato genotypes at harvest and post storage.

Potato psyllid (Bactericera cockerelli Sulc)-transmitted "Candidatus Liberibacter solanacearum" (Lso) has been negatively impacting the potato industry in the United States as well as other potato-producing countries. Lso has been linked to a condition known as zebra chip (ZC) that affects yield and quality of potato tubers. Efforts to find sources of resistance to ZC have primarily focused on greenhouse evaluations based on a single inoculation time prior to harvest. Plant response to infection, however, could be influenced by the developmental stage of the host plant, and ZC may continue to develop after harvest. The objectives of this study were to quantify Lso inoculation success, Lso titer, ZC severity and Lso development during storage in eight potato genotypes. These evaluations were conducted on plants infested with Lso-positive psyllids at 77, 12, and 4 days before vine removal (DBVR). The evaluated genotypes were categorized according to their relative resistance to Lso and tolerance to ZC symptoms. Lso inoculation success in the genotype family A07781, derived from Solanum chacoense, was lower than that of the susceptible control ('Russet Burbank'). A07781-4LB and A07781-3LB genotypes were characterized relatively resistant to the pathogen and highly tolerant to ZC symptoms, while A07781-10LB was categorized as susceptible to Lso but relatively tolerant to symptom expression. In stored potatoes, increase in Lso concentrations was observed for all infestation times. However, significantly higher Lso titer was detected in tubers infested 12 DBVR and the effect was similar across genotypes. Overall, the A07781 family can be considered as a promising source of resistance or tolerance to ZC.

Immunofluorescence Assay to Study Early Events of Vector Salivary Gland Colonization by Phytoplasmas.

To visualize phytoplasmas at early stages of vector infection, an immunofluorescence assay was developed. The chapter provides experimental details on dissection of salivary glands, incubation of the dissected organs with phytoplasma suspension, fixation, embedding, sectioning, labeling, and final visualization with confocal microscopy. All the procedure will be described for the leafhopper Euscelidius variegatus, natural vector of "Candidatus phytoplasma asteris" and laboratory vector of Flavescence dorée phytoplasma.

Postharvest Development of 'Candidatus Liberibacter solanacearum' in Late-Season Infected Potato Tubers under Commercial Storage Conditions.

Zebra chip (ZC) disease of potato is associated with the putative pathogen 'Candidatus Liberibacter solanacearum', which is transmitted by the potato psyllid Bactericera cockerelli (Hem., Triozidae). The present study was initiated to investigate 'Ca. L. solanacearum' development during and following typical commercial storage practices. Using bacteriliferous psyllids, Russet Norkotah potato tubers were infested in field cages 14, 10, and 4 days before harvest. Changes in 'Ca. L. solanacearum' detection rate, 'Ca. L. solanacearum' titer, and concentrations of phenolic compounds were documented throughout storage. 'Ca. L. solanacearum' titer continued to increase during storage. Although significant increases in the frequency of 'Ca. L. solanacearum' detection were observed in all infestation treatments, the impact of 'Ca. L. solanacearum' infection on tuber quality remained comparatively low in plants infected 4 days before harvest, because the majority of the tubers remained asymptomatic. Minimizing storage and retail chain movement durations would help to limit 'Ca. L. solanacearum' impact on tuber quality in tubers infected 14 and 10 days before harvest. This study also demonstrated that 'Ca. L. solanacearum' can relocate from a newly infected leaf to a tuber in as little as 4 days. Psyllid management is recommended until at least 4 days before green harvest, when psyllid pressure is high in fields in which tubers are destined for commercial storage.

Characterization of host plant resistance to zebra chip disease from species-derived potato genotypes and the identification of new sources of zebra chip resistance.

'Candidatus Liberibacter solanacearum' (Lso), an uncultivable phloem-limited phytopathogenic bacteria, is known to be associated with Zebra Chip disease (ZC), which represents a major threat to potato production in the US and elsewhere. This pathogen is transmitted by the phloem-feeding potato psyllid, Bactericera cockerelli Sulc (Hem. Triozidae). Currently, there are no reports of resistance to ZC in cultivated potatoes. This greenhouse study was conducted to evaluate Lso transmission success and the susceptibility of 11 potato breeding clones, representing diverge genetic background, to ZC, in relation to a susceptible commercial cultivar, Russet Burbank. Individual plants were exposed to two Lso-positive potato psyllids for 48 hours. The percentage of successful Lso transmission varied across the evaluated genotypes ranging between 7 and 57%. Freshly-cut and fried tubers showed significant variation in ZC symptom severity among the breeding clones, with several genotypes expressing relative tolerance when compared to Russet Burbank. None of the evaluated clones showed statistically lower Lso titers than Russet Burbank with the exception of one genotype in the second year of the study. However, the presence of a non-significant relationship between average symptom severity and Lso titer indicated variations in phenotypic responses (i.e. tolerance) to Lso existed among evaluated breeding lines. Breeding clones A07781-3LB, A07781-4LB and A07781-10LB had relatively lower Lso titer (low susceptibility) and tuber symptom expression (high tolerance) among the tested genotypes. As these three clones represent full siblings, the observed effects could be indicative of the presence of a genetic basis for resistance/tolerance to ZC. Findings provide a better understanding of resistance/tolerance to ZC, and contribute to continued efforts in breeding for resistance to this disease.

Full Mitochondrial Genome Sequence of the Sugar Beet Wireworm Limonius californicus (Coleoptera: Elateridae), a Common Agricultural Pest.

We report here the full mitochondrial genome sequence of Limonius californicus, a species of click beetle that is an agricultural pest in its larval form. The circular genome is 16.5 kb and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes.

Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

BACKGROUND: Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization. METHODS: Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells. RESULTS: Artificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process. CONCLUSION: Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.