Julien Levy

Diversity of Endosymbionts in the Potato Psyllid, Bactericera cockerelli (Hemiptera: Triozidae), Vector of Zebra Chip Disease of Potato

Zebra chip disease is an emerging, serious disease of solanaceous crops and the causal agent is a bacterium “Candidatus Liberibacter solanacearum” (CLs), also known as “Candidatus Liberibacter psyllaurous”, which is transmitted by the potato psyllid, Bactericera cockerelli (Šulc). We performed bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) of the 16S rDNA genes to determine the bacterial microbiota in adult insects from CLs-uninfected and CLs-infected strains of B. cockerelli and potato leaf samples. We obtained sequences from five bacterial species among the two psyllid strains, including “Candidatus Carsonella ruddii”, Wolbachia, CLs, and two transient bacteria, Acinetobacter and Methylibium. We did not detect any common bacteria between psyllids and potato leaf samples using pyrosequencing. We performed PCR analysis using species-specific 16S rDNA primers to confirm pyrosequencing results in individual psyllids including eggs, early-instars, late-instars, and adults of both sexes from both CLs-uninfected and CLs-infected psyllid strains. The primary endosymbiont, “Candidatus Carsonella ruddii” and Wolbachia were detected in all life-stages and sexes of both strains using PCR analyses. The percentage of CLs-infected individuals increased from early-instar (0%), late-instar (40%) until adulthood (60%) in the CLs-infected strain. We believe that CLs levels in early-instars are probably too low to be detected by standard PCR. Using PCR analyses, we confirmed the presence of Acinetobacter in CLs-uninfected and CLs-infected adults (75 and 25%, respectively) but not Methylibium. Further, we detected Acinetobacter in potato leaves using PCR indicating that the psyllids may have acquired this bacterium via feeding on the host plant.

Interactions "Candidatus Liberibacter solanacearum"-Bactericera cockerelli: Haplotype Effect on Vector Fitness and Gene Expression Analyses.

“Candidatus Liberibacter solanacearum” (Lso) has emerged as a serious threat world-wide. Five Lso haplotypes have been identified so far. Haplotypes A and B are present in the Americas and/or New Zealand, where they are vectored to solanaceous plants by the potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae). The fastidious nature of these pathogens has hindered the study of the interactions with their eukaryotic hosts (vector and plant). To understand the strategies used by these pathogens to infect their vector, the effects of each Lso haplotype (A or B) on psyllid fitness was investigated, and genome-wide transcriptomic and RT-qPCR analyses were performed to evaluate Lso gene expression in association with its vector. Results showed that psyllids infected with haplotype B had significantly lower percentage of nymphal survival compared to psyllids infected with haplotype A. Although overall gene expression across Lso genome was similar between the two Lso haplotypes, differences in the expression of key candidate genes were found. Among the 16 putative type IV effector genes tested, four of them were differentially expressed between Lso haplotypes, while no differences in gene expression were measured by qPCR or transcriptomic analysis for the rest of the genes. This study provides new information regarding the pathogenesis of Lso haplotypes in their insect vector.

Transcriptome Analysis of “Candidatus Liberibacter solanacearum” in Its Psyllid Vector, Bactericera cockerelli

“Candidatus Liberibacter solanacearum” (Lso) is an emergent pathogen of carrots in Europe and solanaceous plants in North and Central America and New Zealand. This bacterium is closely related to other pathogenic Candidatus Liberibacter spp., all vectored by psyllids. In order to understand the molecular interaction of this pathogen and its psyllid vector, Bactericera cockerelli, Illumina sequencing of psyllid harboring Lso was performed to determine if this approach could be used to assess the bacterial transcriptome in this association. Prior to sequencing, psyllid RNA was purified and insect and bacterial rRNA were removed. Mapping of reads to Lso genome revealed that over 92% of the bacterial genes were expressed in the vector, and that the COG categories Translation and Post-translational modification, protein turnover, chaperone functions were the most expressed functional categories. Expression levels of selected Lso genes were confirmed by RT-qPCR. The transcriptomic analysis also helped correct Lso genome annotation by identifying the expression of genes that were not predicted in the genome sequencing effort.

Solanum habrochaites, a Potential Source of Resistance Against Bactericera cockerelli (Hemiptera: Triozidae) and “Candidatus Liberibacter solanacearum”

ABSTRACT The potato psyllid, Bactericera cockerelli Sulc, also known as tomato psyllid, is a serious pest of solanaceous plants. Its host selection criteria are poorly understood. In this study, we tested whether the Solanum habrochaites (PI127826), a wild solanaceous plant known for its property to repel whiteflies, was repellent to potato psyllids. Using a combination of nonchoice assays and choice assays on different psyllid stages, we demonstrated that S. habrochaites is both repelling and toxic to potato psyllids compared with Solanum lycopersicum. However, those properties were not sufficient to avoid transmission of the plant bacterial pathogen “Candidatus Liberibacter solanacearum” vectored by potato psyllids, the causative agent of potato zebra chip disease. However, a lower bacterial transmission rate to S. habrochaites was observed compared with S. lycopersicum.

Methods for Rapid and Effective PCR-Based Detection of 'Candidatus Liberibacter solanacearum' From the Insect Vector Bactericera cockerelli: Streamlining the DNA Extraction/Purification Process

ABSTRACT This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of ‘Candidatus (Ca.) Liberibacter solanacearum,’ the bacterial causal agent of potato zebra chip disease and eventually for ‘Ca. Liberibacter asiaticus’ the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (>97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected ‘Ca. Liberibacter’ in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average ≈5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of ‘Ca. Liberibacter’ pathogens in psyllids.

Investigations on Putative Zebra Chip Tolerant Potato Selections

Abstract‘Candidatus Liberibacter solanacearum’ (Lso) has emerged as a devastating pathogen of solanaceous and other vegetable crops. In potato, Lso is the causative agent of zebra chip (ZC) disease, which threatens production in North and Central America and New Zealand. Lso has caused significant economic losses to the potato industry in Texas since the emergence of the disease in 2000. Presently, disease control relies on pesticide applications, but efforts are under way to identify plant resistance. The objective of this work was to validate the most promising cultivars identified as potentially resistant or tolerant to ZC from previous field trials in different Texas locations and in multiple years. An important component of the study was the development of protocols to effectively evaluate resistance or tolerance. Results show that, while none of the tested cultivars displayed resistance against ZC, differences in susceptibility among them were confirmed. Results also demonstrated the importance of performing cage trials in which vector and disease pressure can be controlled.Resumen“Candidatus Liberibacter solanacearum” (Lso) ha emergido como un patógeno devastador en cultivos de solanáceas y otras hortalizas. En papa, Lso es el agente causal de la zebra chip (ZC) o papa rayada, que amenaza la producción en Norte y Centroamérica, y Nueva Zelanda. Lso ha causado pérdidas económicas significativas a la industria de la papa en Texas desde que surgió la enfermedad en 2000. En la actualidad, el control de la enfermedad se respalda en aplicaciones de plaguicidas, pero hay esfuerzos en proceso para identificar resistencia en la planta. El objetivo de este trabajo fue validar las variedades más prometedoras, identificadas como potencialmente resistentes o tolerantes a ZC, de ensayos de campo previos en diferentes localidades texanas y en múltiples años. Un componente importante del estudio fue el desarrollo de protocolos para evaluar efectivamente la resistencia o tolerancia. Los resultados muestran que, mientras que ninguna de las variedades probadas mostró resistencia contra ZC, se confirmaron diferencias en la susceptibilidad entre ellas. Los resultados también demostraron la importancia de hacer ensayos en jaulas en los que se puede controlar la presión del vector y de la enfermedad.

The influence of bacteria on multi-trophic interactions among plants, psyllids, and pathogen

The recent emergence of several plant diseases caused by psyllid‐borne bacterial pathogens worldwide (Candidatus Liberibacter spp.) has created renewed interest on the interaction between psyllids and bacteria. In spite of these efforts to understand psyllid association with bacteria, many aspects of their interactions remain poorly understood. As more organisms are studied, subtleties on the molecular interactions as well as on the effects of the bacteria on the psyllid host are being uncovered. Additionally, psyllid‐borne bacterial phytopathogens can also affect the host plant, which in turn can impact psyllid physiology and behavior. Here, we review the current literature on different aspects of the influence of bacteria on multitrophic interactions among plants, psyllids, and pathogens. We then highlight gaps that need to be addressed to advance this field, which can have significant implications for controlling these newly emergent and other plant diseases.

Global gene expression in two potato cultivars in response to ‘Candidatus Liberibacter solanacearum’ infection

BackgroundTranscriptomic analyses were performed to compare the molecular responses of two potato varieties previously shown to differ in the severity of disease symptoms due to infection by “Candidatus Liberibacter solanacearum” (Lso), the causative agent of Zebra Chip in potato. A factorial design utilizing the two varieties and psyllids either harboring Lso or without bacteria was used to discriminate varietal responses to pathogen infection versus psyllid feeding. Plant response was determined from leaf samples 3 weeks after infection.ResultsIn response to Lso infection, 397 genes were differentially expressed in the variety Atlantic (most susceptible) as compared to 1027 genes in Waneta. Over 80% of the transcriptionally-changed genes were down-regulated in both varieties, including genes involved in photosynthesis or primary and secondary metabolism. Many of the Lso-responsive genes involved in stress responses or hormonal pathways were regulated differently in the two potato varieties.ConclusionsThis study focused on the time point just prior to the onset of symptom development and provides valuable insight into the mechanisms of Liberibacter pathogenicity, especially the widespread suppression of plant gene expression, including genes involved in plant defenses.

Transcriptome of Russet Norkotah and its clonal selection, TXNS278

ObjectivesPotato has a large genetic diversity. This diversity is in part due to somaclonal variability that appears within potato selections for which tubers are used as seeds. However, the potato tetraploid genome, as well as the use of tubers for crop propagation, does not allow for easy genetic studies. The objective is to gain knowledge at the genomic level from standard Russet Norkotah and a subclonal Russet Norkotah selection TXNS278.Data descriptionIn this report, we used RNA-seq, which allows genome-wide gene expression analysis to sequence the transcriptomes of the subclonal Russet Norkotah selection TXNS278 with standard Russet Norkotah grown in commercial fields. Among the selections, TXNS278 appeared in a multi-year analysis in Texas as a top No 1 yielding variety. Russet Norkotah and TXNS278 leaf and root transcriptomes were sequenced at two time points during growing season.