A high-throughput plate method for nucleic acid extraction from beet leafhopper (Hemiptera: Cicadellidae) and potato psyllid (Hemiptera: Triozidae) for pathogen detection.

Plant pathogens that are transmitted by insect vectors cause considerable damage to crops when pests or pathogens are not detected early in the season and populations are not controlled. Knowledge of pathogen prevalence in insect pest populations can aid growers in their insect pest management decisions but requires the timely dissemination of results. This process requires that specimen capture, identification, nucleic acid extraction, and molecular detection of a pathogen(s) occur alongside a platform for sharing results. The potato psyllid (Bactericera cockerelli, Sulc; Hemiptera: Triozidae) and beet leafhopper (Circulifer tenellus, Baker; Hemiptera: Cicadellidae) transmit pathogens to potato and other vegetable or seed crops each season in the northwestern United States. While the potato psyllid has been tested for pathogen occurrence for the past decade, testing of the beet leafhopper is a new endeavor and substantially increases the specimen number that must be tested by our laboratories each season. To aid in the rapid processing of individual insect specimens, we optimized and validated a new high-throughput 96-well plate nucleic acid extraction method for use in place of a standard 1.5-ml single-tube extraction method. Processing efficiency, in terms of total specimens processed over a 2-day period, improved 2.5-fold, and the cost associated with processing a single sample was nearly cut in half with this newly developed plate nucleic acid extraction method. Overall, this method has proven to be an excellent tool for the rapid testing of large numbers of small, individual insect vectors to enable timely dissemination of data on pathogen prevalence to growers.

New Assays for Rapid Detection of Beet Leafhopper-Associated Plant Pathogens, 'Candidatus Phytoplasma trifolii', Beet Curly Top Virus, and Spiroplasma citri.

The beet leafhopper Circulifer tenellus is an important pest of agricultural crops in the United States, where it transmits beet curly top virus, beet leafhopper-transmitted virescence agent phytoplasma, and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects beet curly top virus and Spiroplasma citri, and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast and accurate results to growers for implementation in their insect pest monitoring programs.

A robust SNP-haplotype assay for Bct gene region conferring resistance to beet curly top virus in common bean (Phaseolus vulgaris L.).

Beet curly top virus (BCTV), which is synonymous with curly top virus (CTV), causes significant yield loss in common bean (snap and dry beans) cultivars and several other important crops. Common bean cultivars have been found to be resistant to CTV, but screening for resistance is challenging due to the cyclical nature of epidemics and spotty feeding by the leafhopper that vectors the virus. We used an SNP dataset for the Snap Bean Association Panel (SnAP) agro-inoculated with CTV-Logan (CA/Logan) strain to locate the Bct gene region to a 1.7-Mb interval on chromosome Pv07 using genome-wide association study (GWAS) analysis. Recombinant lines from the SnAP were used to further narrow the Bct region to a 58.0-kb interval. A missense SNP (S07_2970381) in candidate gene Phvul.007G036300 Exonuclease V (EXO5) was identified as the most likely causal mutation, and it was the most significant SNP detected by GWAS in a dry bean population (DBP) naturally infected by the CTV-Worland (Wor) strain. Tm-shift assay markers developed for SNP S07_2970381 and two linked SNPs, S07_2970276 and S07_2966197, were useful for tracking different origins of the Bct EXO5 candidate gene resistance to CTV in common bean. The three SNPs identified four haplotypes, with haplotype 3-1 (Haplo3-1) of Middle American origin associated with the highest levels of CTV resistance. This SNP-haplotype assay will enable breeders to track resistance sources and to develop cultivars with better CTV resistance.

Bacterial Endosymbionts Identified From Leafhopper (Hemiptera: Cicadellidae) Vectors of Phytoplasmas.

Insects often harbor bacterial endosymbionts that provide them with nutritional benefit or with protection against natural enemies, plant defenses, insecticides, and abiotic stresses. Certain endosymbionts may also alter acquisition and transmission of plant pathogens by insect vectors. We identified bacterial endosymbionts from four leafhopper vectors (Hemiptera: Cicadellidae) of 'Candidatus Phytoplasma' species by direct sequencing 16S rDNA and confirmed endosymbiont presence and identity by species-specific conventional PCR. We examined three vectors of Ca. Phytoplasma pruni, causal agent of cherry X-disease [Colladonus geminatus (Van Duzee), Colladonus montanus reductus (Van Duzee), Euscelidius variegatus (Kirschbaum)] - and a vector of Ca. Phytoplasma trifolii, the causal agent of potato purple top disease [Circulifer tenellus (Baker)]. Direct sequencing of 16S identified the two obligate endosymbionts of leafhoppers, 'Ca. Sulcia' and 'Ca. Nasuia', which are known to produce essential amino acids lacking in the leafhoppers' phloem sap diet. About 57% of C. geminatus also harbored endosymbiotic Rickettsia. We identified 'Ca. Yamatotoia cicadellidicola' in Euscelidius variegatus, providing just the second host record for this endosymbiont. Circulifer tenellus harbored the facultative endosymbiont Wolbachia, although the average infection rate was only 13% and all males were Wolbachia-uninfected. A significantly greater percentage of Wolbachia-infected Ci. tenellus adults than uninfected adults carried Ca. P. trifolii, suggesting that Wolbachia may increase this insect's ability to tolerate or acquire this pathogen. Results of our study provide a foundation for continued work on interactions between leafhoppers, bacterial endosymbionts, and phytoplasma.

Acquisition and Transmission of 'Candidatus Liberibacter solanacearum' Differs Among Wolbachia-Infected and -Uninfected Haplotypes of Bactericera cockerelli.

'Candidatus Liberibacter solanacearum' (Lso) causes disease symptoms and economic losses in potato, tomato, and other solanaceous crops in North America. Lso is transmitted to plants by the potato psyllid, Bactericera cockerelli, which occurs as distinct haplotypes named western, central, and northwestern that differ in the presence or absence of the bacterial endosymbiont, Wolbachia. Previous work showed that all three vector haplotypes can transmit Lso, but it was not clear whether acquisition and transmission rates of Lso were equal among the haplotypes. The goal of our study was to compare Lso infection rates among psyllids of the western, central, and northwestern haplotypes. Using data collected from several years of periodic testing of Lso infection of laboratory-reared potato psyllid colonies, we showed that psyllids of the western and central haplotypes are more likely to harbor Lso than are psyllids of the northwestern haplotype. We then used greenhouse assays to demonstrate that psyllids of the northwestern haplotype are less likely to acquire and transmit Lso than those of the western haplotype. Lso infection rates corresponded with Wolbachia infection among the three psyllid haplotypes. The Wolbachia-infected central and western haplotypes were more likely to harbor and transmit Lso than the Wolbachia-free northwestern haplotype. Results demonstrate that potato psyllids of the western and central haplotypes pose a greater risk for spread of Lso in crops and suggest a pattern between infection with Lso and Wolbachia in potato psyllid.

Development of a Loop-Mediated Isothermal Amplification (LAMP) Method to Detect the Potato Zebra Chip Pathogen 'Candidatus Liberibacter solanacearum' (Lso) and Differentiate Haplotypes A and B.

'Candidatus Liberibacter solanacearum' (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signals from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 nontarget plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately, and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.

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.

Comparative Phylogenomic Analysis Reveals Evolutionary Genomic Changes and Novel Toxin Families in Endophytic Liberibacter Pathogens.

Liberibacter pathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato zebra chip. These bacteria are endophytic and nonculturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding about Liberibacter pathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of the Liberibacter pathogens and their free-living, nonpathogenic, ancestral species, aiming to identify major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. Using gene neighborhood analysis and phylogenetic classification, we systematically uncovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages of Liberibacter and only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including new toxins homologous to the EspG/VirA effectors, a YdjM phospholipase toxin, and a secreted endonuclease/exonuclease/phosphatase (EEP) protein. Our results substantially extend the knowledge of the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenic Liberibacter species, which will facilitate the design of functional experiments and the development of new methods for detection and blockage of these pathogens. IMPORTANCELiberibacter pathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover the genes/proteins important to Liberibacter pathogenesis. Further, earlier bioinformatics studies failed to identify key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis of Liberibacter pathogens along with their ancestral nonpathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new light on the disease mechanism of Liberibacter pathogens and will facilitate the development of new detection and blockage methods targeting the toxins.

First Report of Curly Top of Coriandrum sativum Caused by Beet curly top virus in the Columbia Basin of Washington State.

Two fields of coriander (Coriandrum sativum L.) seed crops of proprietary cultivars were observed in the Columbia Basin of Washington in July 2020 with 40 and 90% incidence of plants showing stunting and leaf and stem discoloration, sometimes with mild leaf curl. Foliar discoloration ranged from yellow to red and purple. Sweep-netting along the field edges collected one beet leafhopper (Circulifer tenellus Baker; BLH), the known vector of Beet curly top virus (BCTV), Beet leafhopper transmitted virescence agent (BLTVA) phytoplasma, and Spiroplasma citri, all of which affect Solanaceae and Apiaceae crops in Washington (Crosslin et al. 2006; Johnson and Martin 1998; Lee et al. 2006). Nucleic acids extracted from leaves and petioles of 12 coriander plants (8 from Field 1 and 4 from Field 2) using the Dellaporta method, and from the BLH using the CTAB method (Crosslin et al. 2006) were subjected to PCR assays to detect the BLH-transmitted pathogens which cause yellow and purple discoloration in potato (Solanum tuberosum L.) and carrot (Daucus carota subsp. sativus (Hoffm.) Arc.) in this region. BLTVA was targeted using a species-specific nested PCR assay with primers P1 and P7, followed by primers FU5 and BLTVA-int (Crosslin et al. 2006); S. citri was targeted using primers P89-F and P89-R (Yokomi et al. 2008); and BCTV was targeted using curtovirus primers BCTV2-F and BCTV2-R (Strausbaugh et al. 2008). BLTVA and S. citri were not detected in the plants, but curtovirus was detected in 10 of the 12 plants. All three pathogens were detected from the single BLH. A 519 bp region of the curtovirus capsid protein gene was amplified from seven plants (5 from Field 1 and 2 from Field 2) and the BLH, and cloned into TOP10 Escherichia coli cells using the pCR-2.1 TOPO vector (Invitrogen, Carlsbad, CA). Three clones were sequenced from each sample. For each of six plant samples and the BLH, the three clones were identical and consensus sequences were generated (GenBank Accessions MW234419 to MW234425). For the seventh plant, two clones were identical in sequence (MW234426) and the third contained 12 single nucleotide polymorphisms (MW234427). All sequences were subjected to an NCBI BLASTn analysis and showed 98.3 to 99.8% identity with BCTV sequences. Additional PCR assays with primers BMCTV-C1 2213F and BMCTV-C1 2609R (Strausbaugh et al. 2008), targeting the C1 gene of the Worland strain of BCTV, detected BCTV-Worland-like strains in all plants and the BLH, confirming that BCTV was present and indicating that the strain-specific primer pair was more sensitive than the universal curtovirus primers. Yield losses in the two fields were approximately 60%, with reduced seed size but not seed quality. BCTV infections in coriander crops have been observed in the Columbia Basin in 2002, 2005, 2008, and 2013, with yield losses ranging from 10 to 100% per field, though official reports were not made following the diagnoses (Crosslin, du Toit, and Frost, unpublished data). BCTV has caused millions of dollars of losses in the U.S. in crops such as sugar beet (Beta vulgaris subsp. vulgaris L.), tomato (S. lycopersicum L.), and pepper (S. annuum L.) (Johnson and Martin 1998). This is the first publication of BCTV affecting seed production of the specialty crop C. sativum. The observation of 90% incidence of symptoms in one field suggests that resistant cultivars and/or insect pest management practices are needed to prevent significant impacts of BCTV on coriander seed production in this semi-arid region.

KASP Markers Reveal Established and Novel Sources of Resistance to Pea Seedborne Mosaic Virus in Pea Genetic Resources.

Pea seed-borne mosaic virus (PSbMV) is both seedborne and aphid-transmitted and can cause economic losses for pea (Pisum sativum L.) production by reducing yield through decreased seed weight and number. The P1 pathotype is especially virulent, affecting this important vegetable crop across the United States and internationally in regions of West Asia, North Africa, Europe, and Australia. Previously, two kompetitive allele-specific PCR (KASP) genotyping markers (eIF4E resistant 1 and 2) were developed and validated on P. sativum accessions identifying two PSbMV pathotype P1 resistance alleles in the eukaryotic translation initiation factor gene, eIF4E. The current study utilized these novel markers to rapidly evaluate 318 genetic resource accessions maintained as part of the United States Department of Agriculture National Plant Germplasm System's Pea Single Plant Collection (PSPC). The evaluations also included 58 commercial and other plant introduction (PI) lines that were assessed for the two eIF4E resistance alleles. All genotyping results were validated in greenhouse assays by confirmation of observable disease symptoms after inoculations and by enzyme-linked immunosorbent assays. The eIF4E resistant 1 and 2 alleles were found in 18 accessions from the PSPC, five commercial lines, and 14 other PI accessions. A single PSPC accession showed resistance to PSbMV pathotype P1 that is believed to be a novel source of resistance based on sequencing analysis of eIF4E. Sources of resistance were identified in the PSPC and in commercial cultivars that can be introgressed into breeding lines using traditional techniques to reduce the time and cost required to generate germplasm with superior disease-resistant traits.

Seasonal Population Dynamics of Potato Psyllid (Hemiptera: Triozidae) in the Columbia River Basin.

Understanding factors that affect the population dynamics of insect pest species is key for developing integrated pest management strategies in agroecosystems. Most insect pest populations are strongly regulated by abiotic factors such as temperature and precipitation, and assessing relationships between abiotic conditions and pest dynamics can aid decision-making. However, many pests are also managed with insecticides, which can confound relationships between abiotic factors and pest dynamics. Here we used data from a regional monitoring network in the Pacific Northwest United States to explore effects of abiotic factors on populations of an intensively managed potato pest, the potato psyllid (Bactericera cockerelli Sulc), which can vector Candidatus Liberibacter psyllaurus, a bacterial pathogen of potatoes. We assessed effects of temperature on psyllid populations, and show psyllid population growth followed predictable patterns within each year, but there was considerable variation across years in psyllid abundance. Examination of seasonal weather patterns suggested that in 2017, when psyllid populations were less abundant by several orders of magnitude than other years, a particularly long and cold period of winter weather may have harmed overwintering populations and limited population growth. The rate of degree-day accumulation over time, as well as total degree-day accumulation also affected trap catch abundance, likely by mediating the number of psyllid generations per season. Our findings indicate that growers can reliably infer the potential magnitude of risk from potato psyllids using monitoring data, date of first detection, seasonal weather patterns, and population size early in the growing season.

Development and Validation of KASP Markers for the Identification of Pea seedborne mosaic virus Pathotype P1 Resistance in Pisum sativum.

As pesticides have become heavily relied on for management of insect pests vectoring economically important pathogens of vegetable crops, development of pathogen-resistant germplasm remains a promising alternative to reduce or eliminate costly and timely chemical inputs. Molecular markers can be used to rapidly identify resistant genotypes to aid breeders in advancing germplasm. This study developed two kompetitive allele-specific PCR (KASP) genotyping markers for rapid screening of Pisum sativum genotypes for resistance to Pea seedborne mosaic virus pathotype P1 (PSbMV-P1), the most economically devastating strain worldwide. The KASP markers differentiate two eIF4E PSbMV-P1-resistant allelic variants from susceptible eIF4E variants. A single nucleotide polymorphism (Resistant 1) and a 3-basepair deletion (Resistant 2) present in either of the two resistant alleles were used for marker design. Forty-four P. sativum lines previously characterized for resistance to PSbMV were inoculated with PSbMV-P1 in a greenhouse, observed for visual symptoms, assayed for virus susceptibility by enzyme-linked immunosorbent assay (ELISA), and genotyped by KASP marker analysis. The KASP markers were 100% accurate in characterizing PSbMV-P1-susceptible and PSbMV-P1-resistant genotypes when correlated with the ELISA results. The Resistant 1 marker also correlated with resistance to PSbMV pathotypes P2 and P4 completely, making this marker a new advanced tool for P. sativum breeding programs.

Growth and Yield Performance of Solanum tuberosum Grown from Seed Potatoes Infected with 'Candidatus Liberibacter solanacearum' Haplotypes A and B.

Zebra chip (ZC) disease of potato (Solanum tuberosum) is associated with infection by 'Candidatus Liberibacter solanacearum' (Lso). Two haplotypes of Lso-A and B-occur in the United States. Lso haplotype B is more virulent than haplotype A, causing greater disease incidence in tubers, more severe symptoms, and greater loss in tuber yield. This study assessed whether tubers from infected plants generate new infected plants the following year. The effects of both Lso haplotypes A and B on tuber resprout were examined on five potato cultivars. When compared with noninfected tubers, overall plant emergence rate from Lso A- or B-infected tubers was lower, plants emerged slower, and plants generated lower daughter tuber yields in weight and quantity. Plants generally emerged poorly from Lso B-infected tubers and produced lower daughter tuber yields than Lso A-infected tubers. Regardless of Lso treatment, all daughter tubers were asymptomatic, and only 0.3% tested positive for Lso in experiments conducted over 2 years. This suggests that plants generated from Lso A- and Lso B-infected seed potatoes with severe ZC symptoms are likely not a significant source of Lso in potato fields.