Beet Leafhopper-transmitted Virescence Agent Research Articles

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.

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.

Detection of Pathogens Associated with Psyllids and Leafhoppers in Capsicum annuum L. in the Mexican States of Durango, Zacatecas, and Michoacán

In fall 2014, 5 to 75% percent of chili and bell pepper (Capsicum annuum L.) in commercial fields located in the Mexican states of Durango, Zacatecas, and Michoacán had symptoms of deformed, small, mosaic, curled, and chlorotic leaves; shortened internodes; plant dwarfing; or phyllody and rosetting leaf tips. At the same time, leafhoppers and psyllids were observed in the fields, and more than 50 beet leafhoppers (Circulifer tenellus) and nearly 300 potato psyllids (Bactericera cockerelli) were collected from the pepper plants and adjacent weeds. Based on the insect pressure and observed symptoms, nearly 400 pepper samples were collected across this region of Mexico and tested for the presence of leafhopper- and psyllid-associated pathogens. In all, 76% of the pepper samples were found to be infected with 'Candidatus Liberibacter solanacearum', beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma, a strain of a curtovirus, or a combination of any two or three of these pathogens. Additionally, 77% of the collected leafhoppers and 40% of the psyllids were infected with one or more of these pathogens, in addition to Spiroplasma citri. Specifically, the leafhoppers were infected with BLTVA phytoplasma, S. citri, or a strain of curtovirus. Of particular interest, potato psyllids were not only infected with 'Ca. L. solanacearum' but also with phytoplasmas that belong to the groups 16SrVI subgroup A and 16SrI subgroup A. The presence of mixed infections in pepper plants and the insect vectors highlights the need for growers to effectively control both leafhoppers and potato psyllids from solanaceous crops in this region of Mexico in order to prevent the spread of these bacterial and viral pathogens.

First Report of Beet Leafhopper Transmitted Virescence Agent Phytoplasma in Capsicum annuum and Circulifer tenellus in Mexico

First Report of Beet Leafhopper Transmitted Virescence Agent Phytoplasma in <i>Capsicum annuum</i> and <i>Circulifer tenellus</i> in Mexico

Monitoring and Controlling the Beet Leafhopper Circulifer tenellus in the Columbia Basin

The beet leafhopper (BLH, Circulifer tenellus Baker) is a frequent pest of potatoes in the Columbia Basin of Oregon and Washington. Beet leafhoppers vector the causal agent of potato Purple top disease, known as Beet Leafhopper Transmitted Virescence Agent (BLTVA). Over 70 % of BLH in the region can be infected with BLVTA. Current control methods include making regular insecticide applications beginning in May; however it is unclear if those applications are needed. Thus, are insecticide applications necessary to control BLH and reduce BLTVA through the entire season? To answer that question, a field study was conducted in 2008 and 2009 to assess season-long control of BLH. There were 14 treatments, including the control, that were based on gradually eliminating insecticide applications weekly. Plots were sampled weekly for BLH and surveyed for Purple Top disease. Beet leafhoppers were sampled using sweep nets and an inverted leaf blower. Natural BLH populations were low in 2008 and 2009, but differed significantly among treatments in 2009 for inverted leaf blower and sweep net samples. Yields were assessed at the end of each season and no significant differences were found. Trends in the yield, BLH populations and BLTVA incidence support early-season insecticide application when BLH populations are low. The practice of using a calendar spray regime was not supported as yields were similar regardless of insecticide applications. As BLH populations were low, the results emphasized the importance of monitoring before making insecticide applications.

Characterisation of the tolerance to the beet leafhopper transmitted virescence agent phytoplasma in the PI128655 accession of Solanum peruvianum

AbstractIn 1992, Thomas and Hassan reported an apparent immunity and tolerance to the beet leafhopper transmitted virescence agent (BLTVA) phytoplasma in the PI128655 accession of Solanum peruvianum (Plant Disease 76:139). We revisited this interaction in an effort to better characterize the bases of plant resistance and tolerance to phytoplasmas. After challenge with a BLTVA strain, plants of the PI128655 accession fell into two distinct symptom‐based groups, termed class I and class II. Class II plants displayed symptoms on the vegetative parts and phloem hyperplasia, whereas class I plants showed symptoms only on the inflorescence parts, and no phloem hyperplasia. Plants of both classes hosted similar titres of BLTVA, demonstrating that the lower symptom severity in class I plants was due to tolerance rather than resistance. However, this tolerance was not effective against all phytoplasmas, as both classes of plants were equally susceptible to a stolbur phytoplasma strain. A genetic analysis suggested that the BLTVA tolerance of class I plants was monogenic. Finally, a reciprocal graft experiment showed that the virulence of the BLTVA strain seemed to increase after propagation through class II plants. These results provide valuable insights on the tolerance of plants to phytoplasma diseases, and highlight the usefulness of the S. peruvianum/BLTVA experimental pathosystem.

Evaluation of Beet Leafhopper Transmitted Virescence Agent Damage in the Columbia Basin

Potato purple top disease is caused by a phytoplas- ma known as Beet Leafhopper Transmitted Virescence Agent (BLTVA), which is vectored by the beet leafhopper (BLH, Circulifer tenellus Baker). Previous studies determined that BLTVA can cause significant reductions in yield and tuber quality; however, quantifying the damage caused by BLTVA and the insect vector has been challenging. In 2009-2011, potato plants at different growth stages were exposed to vary- ing densities of BLH in a screen house located at the Hermiston Agricultural Research and Extension Center in Hermiston, OR. The densities of potentially infective BLH were one BLH per plant (low), two BLH per plant (medium), and five BLH per plant (high). Releases occurred at the following growth stages: vegetative, tuber initiation, tuber bulking, and maturation. The treatments were arranged in a randomized complete block design with three replications per treatment. Disease incidence was monitored weekly and yield was assessed. When all 3 years were combined, we found that increasing rates of disease incidence correlated with decreas- ing yields. We also found that greater yield losses were ob- served with later BLH release times. With both correlations, differences between years were a strong contributing factor. There was a mean decrease in yield of 0-12 % at a density of oneBLH per plant,6-19% attwoBLH per plant,and 6-20% for five BLH per plant. These general trends in yield loss suggest that economically relevant damage may occur at levelsas low as one or two potentially infective BLH per plant in the Columbia Basin.

Population Dynamics of the Beet Leafhopper (Hemiptera: Cicadellidae) in the Columbia Basin as Influenced by Abiotic Variables

ABSTRACT Beet leafhoppers (Circulifer tenellus Baker) have been identified as the vector for a plant-pathenogenic phytoplasma known as beet leafhopper-transmitted virescence agent. Beet leaf-hopper-transmitted virescence agent causes purple top disease in potatoes, which can reduce yields and tuber quality. A trapping network, composed of ≈100 sites, monitors leafhoppers in the Columbia Basin of Oregon and Washington through a collaborative effort of regional researchers and stakeholders. Yellow sticky cards were used to determine the timing and spatial distribution of beet leafhoppers in the Columbia Basin; insects were counted weekly from early April through late October in 2006, 2007, and 2008. Weather data collected from a network of weather stations in Oregon and Washington were used in a nonparametric multiplicative regression analysis to determine which abiotic environmental variables might influence beet leafhopper populations. Weather conditions (mean temperature, dew point, precipitation, and wi...

Population Dynamics of the Beet Leafhopper in Northeastern Oregon and Incidence of the Beet Leafhopper-Transmitted Virescence Agent Phytoplasma

Beet leafhoppers were collected weekly on yellow sticky traps placed at 36 locations in Morrow and Umatilla Counties in northeastern Oregon in April through November 2007, 2008, and 2009. Insects were counted, collected, and a subset of the insects was tested for the presence of the beet leafhopper-transmitted virescence agent phytoplasma, the causal agent of potato purple top disease in this region. Beet leafhoppers were present throughout the sampling period and the number of insects collected peaked in June of each year with smaller peaks in July and October. Of the 804 insects tested for phytoplasma in 2007, 2008, and 2009, 21, 18, and 22% tested positive for phytoplasma, respectively. Most of the phytoplasma-positive insects were collected from mid-June through July. Positive insects, however, were collected as late as 13 November in 2007 and 2008. These data indicate that a relatively high proportion of the beet leafhoppers in this area are harboring the phytoplasma. Therefore, the potential for development of purple top disease of potatoes from migrating beet leafhoppers in this important potato producing region is quite high and measures to control this pest throughout the growing season are probably necessary in order to reduce disease pressure.

Incidence of the beet leafhopper-transmitted virescence agent phytoplasma in local populations of the beet leafhopper, Circulifer tenellus, in Washington State.

Phytoplasma diseases are increasingly becoming important in vegetable crops in the Pacific Northwest. Recently, growers in the Columbia Basin and Yakima Valley experienced serious outbreaks of potato purple top disease that caused significant yield loss and a reduction in tuber processing quality. It was determined that the beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was the causal agent of the disease in the area and that this pathogen was transmitted by the beet leafhopper, Circulifer tenellus Baker (Hemiptera: Cicadellidae). To provide the most effective management of phytoplasmas, timing of insecticide applications targeted against insects vectoring these pathogens should be correlated with both insect abundance and infectivity. Beet leafhoppers were collected from a potato field and nearby weeds in Washington throughout the 2005, 2006, and 2007 growing seasons and tested for BLTVA by PCR to determine the incidence of this phytoplasma in the insects. In addition, overwintering beet leafhoppers were collected throughout Columbia Basin and Yakima Valley and tested for BLTVA to investigate if these insects might constitute a source of inoculum for this phytoplasma from one season to the next. Results showed that 29.6% of overwintering leafhoppers collected near potato fields carried the phytoplasma. BLTVA-infected leafhoppers were also found in both potatoes and nearby weedy habitats throughout the growing season. PCR testing indicated that a large proportion of beet leafhoppers invading potatoes were infected with the phytoplasma, with an average of 20.8, 34.8, and 9.2% in 2005, 2006, and 2007, respectively. Similarly, BLTVA infection rate in leafhoppers collected from weeds in the vicinity of potatoes averaged 28.3, 24.5, and 5.6% in 2005, 2006, and 2007, respectively. Information from this study will help develop action thresholds for beet leafhopper control to reduce incidence of purple top disease in potatoes.

Characterization of a Phytoplasma Associated with Cabbage Yellows in Iran.

In 2001, a disease tentatively named Iranian cabbage yellows (ICY) was observed in cabbage fields of Zarghan (Fars Province, Iran). The major symptoms of the disease were yellowing, little leaves, plant stunting, opening of the head, and proliferation of the buds at the base of the stem into a witches'-broom. Among leafhoppers collected in cabbage fields, only Circulifer haematoceps transmitted the ICY agent. The disease agent was transmitted by the leafhopper from cabbage to cabbage, cauliflower, rape, and periwinkle, causing phytoplasma-type symptoms in these plants. Polymerase chain reaction (PCR) using phytoplasma-specific primer pair P1/P7 and nested PCR using P1/P7 and R16F2n/R16R2 primer pairs amplified products of expected size (1.8 and 1.2 kb, respectively) from symptomatic cabbage plants. Both restriction fragment length polymorphism (RFLP) of nested PCR products (1.2 kb) and phylogenetic analyses of 16S-23S rDNA spacer region sequence indicated that the ICY phytoplasma had the closest relationship to subgroup A members of the clover proliferation group, including beet leafhopper-transmitted virescence agent, 'Candidatus Phytoplasma trifolii', Columbia Basin potato purple top phytoplasma, and vinca virescence phytoplasma. Cabbage is reported as a new natural host to the 16SrVI group of phytoplasmas.

Development of a Real-Time, Quantitative PCR for Detection of the Columbia Basin Potato Purple Top Phytoplasma in Plants and Beet Leafhoppers.

A quantitative, real-time "TaqMan" polymerase chain reaction assay (real-time PCR) was developed which was capable of detecting and quantifying a group 16SrVI phytoplasma in DNA extracts prepared from infected tomatoes, potatoes, and beet leafhoppers (Circulifer tenellus). Primers and probe were designed from the 16S rRNA gene of the Columbia Basin potato purple top phytoplasma, which is closely related to the beet leafhopper transmitted virescence agent. The detection limit in phytoplasma-infected tomato DNA was approximately 50 pg. The concentration of phytoplasma varied considerably among potato plants showing symptoms of purple top. The pathogen was readily detected in extracts from single or groups of five beet leafhoppers. As with infected potatoes, the concentration of phytoplasma in individual leafhoppers was variable. The assay also detected aster yellows (group 16SrI) and pigeon pea witches'-broom (group 16SrIX) phytoplasmas in infected periwinkle plants. The real-time PCR was at least as sensitive as the commonly used and more labor-intensive nested PCR for detection of the pathogen.

Comparative analysis of the plasmids from two isolates of “ Candidatus Phytoplasma australiense”

Two plasmids from the plant-pathogenic mollicute “ Candidatus Phytoplasma australiense” were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607 bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635 bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of “ Ca. P. australiense.” The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.

Beet Leafhopper (Hemiptera: Cicadellidae) Transmits the Columbia Basin Potato Purple Top Phytoplasma to Potatoes, Beets, and Weeds

Experiments were conducted to determine whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits the purple top phytoplasma to potato, Solanum tuberosum L.; beets, Beta vulgaris L.; and selected weed hosts. The beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was identified as the causal agent of the potato purple top disease outbreaks that recently occurred in the Columbia Basin of Washington and Oregon. The phytoplasma previously was found to be associated almost exclusively with the beet leafhopper, suggesting that this insect is the probable vector of BLTVA in this important potato-growing region. Eight potato cultivars, including 'Russet Burbank', 'Ranger Russet', 'Shepody', 'Umatilla Russet', 'Atlantic', 'FL-1879', 'FL-1867', and 'FL-1833', were exposed for a week to BLTVA-infected beet leafhoppers. After exposure, the plants were maintained outdoors in large cages and then tested for BLTVA by using polymerase chain reaction after 6 to 7 wk. The leafhoppers transmitted BLTVA to seven of the eight exposed potato cultivars. Sixty-four percent of the exposed plants tested positive for the phytoplasma. In addition, 81% of the BLTVA-infected potato plants developed distinct potato purple top disease symptoms. Beet leafhoppers also transmitted BLTVA to beets and several weeds, including groundsel, Senecio vulgaris L.; shepherd's purse, Capsella bursa-pastoris (L.) Medik); kochia, Kochia scoparia (L.) Schrad; and Russian thistle, Salsola kali L. This is the first report of transmission of BLTVA to potatoes, beets, and the above-mentioned four weed species. Results of the current study prove that the beet leafhopper is a vector of the potato purple top disease.

Beet Leafhopper (Hemiptera: Cicadellidae) Transmits the Columbia Basin Potato Purple Top Phytoplasma to Potatoes, Beets, and Weeds

Experiments were conducted to determine whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits the purple top phytoplasma to potato, Solanum tuberosum L.; beets, Beta vulgaris L.; and selected weed hosts. The beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was identified as the causal agent of the potato purple top disease outbreaks that recently occurred in the Columbia Basin of Washington and Oregon. The phytoplasma previously was found to be associated almost exclusively with the beet leafhopper, suggesting that this insect is the probable vector of BLTVA in this important potato-growing region. Eight potato cultivars, including ‘Russet Burbank’, ‘Ranger Russet’, ‘Shepody’, ‘Umatilla Russet’, ‘Atlantic’, ‘FL-1879’, ‘FL-1867’, and ‘FL-1833’, were exposed for a week to BLTVA-infected beet leafhoppers. After exposure, the plants were maintained outdoors in large cages and then tested for BLTVA by using polymerase chain reaction after 6 to 7 wk. The leafhoppers transmitted BLTVA to seven of the eight exposed potato cultivars. Sixty-four percent of the exposed plants tested positive for the phytoplasma. In addition, 81% of the BLTVA-infected potato plants developed distinct potato purple top disease symptoms. Beet leafhoppers also transmitted BLTVA to beets and several weeds, including groundsel, Senecio vulgaris L.; shepherd’s purse, Capsella bursa-pastoris (L.) Medik); kochia, Kochia scoparia (L.) Schrad; and Russian thistle, Salsola kali L. This is the first report of transmission of BLTVA to potatoes, beets, and the above-mentioned four weed species. Results of the current study prove that the beet leafhopper is a vector of the potato purple top disease.

Beet Leafhopper (Hemiptera: Cicadellidae) Settling Behavior, Survival, and Reproduction on Selected Host Plants

Experiments were conducted to determine the settling behavior, survival, and reproduction of the beet leafhopper, Circulifer tenellus (Baker), when maintained on selected host plants. This leafhopper was recently identified in the Columbia Basin of Washington and Oregon as the probable vector of the beet leafhopper-transmitted virescence agent phytoplasma, causal agent of several vegetable crop diseases, including potato purple top. Plants selected for study were sugar beet, Beta vulgaris L.; radish, Raphanus sativus L.; dry bean, Phaseolus vulgaris L.; potato, Solanum tuberosum L.; carrot, Daucus carota L.; and tomato, Lycopersicon esculentum Mill. Leafhopper adults were confined on caged plants, and settling behavior was observed during a 72-h period and survival was monitored for 40 d. Also, oviposition and nymphal production were investigated by maintaining leafhoppers for approximately 90 d on each of the selected plants. Sixty to 100% of leafhoppers settled on all studied plants during the first 5 h, but settling on bean and tomato declined sharply thereafter. Leafhopper mortality was very high on bean and tomato, with 95 and 65% of the leafhoppers, respectively, dying in about a week. In contrast, 77, 90, and 95% of leafhoppers maintained on potato, sugar beet, and radish, respectively, survived until the end of the 40-d experimental period. Beet leafhopper oviposition and nymphal production and development only occurred on sugar beet, radish, and potato; reproduction was lower on potato.

Beet Leafhopper (Hemiptera: Cicadellidae) Settling Behavior, Survival, and Reproduction on Selected Host Plants

Experiments were conducted to determine the settling behavior, survival, and reproduction of the beet leafhopper, Circulifer tenellus (Baker), when maintained on selected host plants. This leafhopper was recently identified in the Columbia Basin of Washington and Oregon as the probable vector of the beet leafhopper-transmitted virescence agent phytoplasma, causal agent of several vegetable crop diseases, including potato purple top. Plants selected for study were sugar beet, Beta vulgaris L.; radish, Raphanus sativus L.; dry bean, Phaseolus vulgaris L.; potato, Solanum tuberosum L.; carrot, Daucus carota L.; and tomato, Lycopersicon esculentum Mill. Leafhopper adults were confined on caged plants, and settling behavior was observed during a 72-h period and survival was monitored for 40 d. Also, oviposition and nymphal production were investigated by maintaining leafhoppers for ≈90 d on each of the selected plants. Sixty to 100% of leafhoppers settled on all studied plants during the first 5 h, but settling on bean and tomato declined sharply thereafter. Leafhopper mortality was very high on bean and tomato, with 95 and 65% of the leafhoppers, respectively, dying in about a week. In contrast, 77, 90, and 95% of leafhoppers maintained on potato, sugar beet, and radish, respectively, survived until the end of the 40-d experimental period. Beet leafhopper oviposition and nymphal production and development only occurred on sugar beet, radish, and potato; reproduction was lower on potato.

Association of Beet Leafhopper (Hemiptera: Cicadellidae) with a Clover Proliferation Group Phytoplasma in Columbia Basin of Washington and Oregon

At least 16 taxa of cicadellids and delphacids were tested by polymerase chain reaction (PCR) for the presence of a phytoplasma in the clover proliferation group, designated 16SrVI. Nucleic acid extracts from individual insects or groups of 5-10 were tested using PCR primers designed from the DNA sequence of 16S-23S rRNA or ribosomal protein genes of the pathogen. The beet leafhopper, Circulifer tenellus (Baker), was most often associated with the phytoplasma, with approximately 16% of the insects testing positive. The phytoplasma was occasionally found associated with Ceratagallia spp. Leafhopper species that were not associated with the phytoplasma included Macrosteles spp., Dikraneura spp., Colladonus montanus (Van Duzee), Circulifer geminatus (Van Duzee), Ballana spp., Amplysellus spp., Paraphlepsius spp., Texananus spp., Balclutha spp., Latalus spp., Erythroneura spp., Exitianus exitiosus (Uhler), and unidentified delphacids. The detected phytoplasma was similar to, or synonymous with, the beet leafhopper-transmitted virescence agent that is associated with the potato purple top disease in the Columbia Basin region of Washington and Oregon. This is in contrast to the phytoplasma associated with potato purple top disease in Mexico that is related to aster yellows (group 16SrI). The association of the group 16SrVI phytoplasma almost exclusively with the beet leafhopper suggests that this insect is the major vector of the phytoplasma in this region.

Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent.

The complete nucleotide sequences of the two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent (BLTVA) have been determined. The larger plasmid, pBLTVA-1, was 10 785 nt in length and contained 11 putative ORFs, almost all of which were duplicated or triplicated on the plasmid due to the presence of large repeated regions. The sequence contained a series of tandem repeats, the largest of which was 338 nt long. The sequences of ORFs 4 and 11 showed homology with the replication genes of plasmids from other phytoplasmas and from geminiviruses. ORF9, the only ORF present as a single copy, showed homology with DNA primase genes from bacterial chromosomes and contained the conserved zinc finger and topoisomerase/primase domains. None of the other eight ORFs showed homology with known sequences in the GenBank database. pBLTVA-2 was 2587 nt in length, and all of its sequence was nearly identical to sequences from pBLTVA-1, most of which spanned ORFs 10 and 11, including the 338 nt tandem repeat. Analysis of 30 strains of BLTVA showed that most of the 11 putative ORFs were present, but the size of the plasmids varied in these strains.

Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Potato Purple Top Disease in Washington and Oregon.

An epidemic of purple top disease of potato (Solanum tuberosum) occurred in the Columbia Basin Region of Washington and Oregon in 2002 and 2003, causing great economic loss in the potato industry (1). Symptoms of potato purple top (PPT) were characterized by upright terminal shoots, upward leaf rolling, chlorosis, red or purplish discoloration of new leaves, proliferation of axillary shoots with basal swelling, and the formation of aerial tubers. Preliminary studies on PPT disease suggested phytoplasma as a possible cause (1). In this study, 78 potato samples (including five asymptomatic) were collected from five fields throughout the region. A nested polymerase chain reaction (PCR) with primer pair P1/P7 in the first amplification followed with primer pair R16F2n/R16R2 was performed to detect the presence of phytoplasmas in infected plants (2). Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Eighty-four percent (63% in the first amplification) of the symptomatic samples and 60% (0% in the first amplification) of the asymptomatic samples tested positive. Low phytoplasma titers and the presence of PCR inhibitors accounts for the low detection rate in the first PCR amplifications. RFLP analyses of 16S rDNA with enzymes MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma associated with PPT belonged to the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) (2). 16SrVI-A currently consists of three members, CP (GenBank Accession No. AY500130), potato witches'-broom (GenBank Accession No. AY500818), and vinca virescence (VR) (GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (2). The taxonomic affiliation of PPT phytoplasma was confirmed by phylogenetic analysis of cloned 16S rDNA (GenBank Accession Nos. PPT4, AY496004; PPT8, AY496005). The 16S rDNA sequences of the PPT strains were closely related to VR with 99.7% sequence homology compared with 99.2% with CP. A high correlation between the symptoms and the presence of 16SrVI-A phytoplasmas in the potato plants suggests that these phytoplasmas play an etiological role in PPT disease. To gain further evidence, a modified test of Koch's postulates was conducted. Infected tissues from four phytoplasma-positive potato samples (including PPT4 and PPT8) were grafted onto healthy potato seedlings. Within 60 days after grafting, the potato seedlings developed symptoms similar to those in the original diseased samples. The newly infected plants were maintained through cuttings. RFLP analysis of 16S rDNA indicated that the phytoplasmas detected in each of the seedlings and cuttings were identical to those in the scions. These results confirmed the probable etiological role of CP group, subgroup 16SrVI-A phytoplasma strains in PPT disease in Washington and Oregon. There are two other confirmed cases of phytoplasmas (BLTVA and aster yellows phytoplasma) associated with PPT disease in Utah (4) and Mexico (3).