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.

Strain-Specific Resistance to Potato virus Y (PVY) in Potato and Its Effect on the Relative Abundance of PVY Strains in Commercial Potato Fields.

Potato virus Y (PVY) is a serious threat to potato production due to effects on tuber yield and quality, in particular, due to induction of potato tuber necrotic ringspot disease (PTNRD), typically associated with recombinant strains of PVY. These recombinant strains have been spreading in the United States for the past several years, although the reasons for this continuing spread remained unclear. To document and assess this spread between 2011 and 2015, strain composition of PVY isolates circulating in the Columbia Basin potato production area was determined from hundreds of seed lots of various cultivars. The proportion of nonrecombinant PVYO isolates circulating in Columbia Basin potato dropped ninefold during this period, from 63% of all PVY-positive plants in 2011 to less than 7% in 2015. This drop in PVYO was concomitant with the rise of the recombinant PVYN-Wi strain incidence, from less than 27% of all PVY-positive plants in 2011 to 53% in 2015. The proportion of the PVYNTN recombinant strain, associated with PTNRD symptoms in susceptible cultivars, increased from 7% in 2011 to approximately 24% in 2015. To further address the shift in strain abundance, screenhouse experiments were conducted and revealed that three of the four most popular potato cultivars grown in the Columbia Basin exhibited strain-specific resistance against PVYO. Reduced levels of systemic movement of PVYO in such cultivars would favor spread of recombinant strains in the field. The negative selection against the nonrecombinant PVYO strain is likely caused by the presence of the Nytbr gene identified in potato cultivars in laboratory experiments. Presence of strain-specific resistance genes in potato cultivars may represent the driving force changing PVY strain composition to predominantly recombinant strains in potato production areas.

RNA-Seq analysis of resistant and susceptible potato varieties during the early stages of potato virus Y infection.

BACKGROUND: Potato virus Y (PVY) is one of the most important plant viruses affecting potato production. The interactions between potato and PVY are complex and the outcome of the interactions depends on the potato genotype, the PVY strain, and the environmental conditions. A potato cultivar can induce resistance to a specific PVY strain, yet be susceptible to another. How a single potato cultivar responds to PVY in both compatible and incompatible interactions is not clear. RESULTS: In this study, we used RNA-sequencing (RNA-Seq) to investigate and compare the transcriptional changes in leaves of potato upon inoculation with PVY. We used two potato varieties: Premier Russet, which is resistant to the PVY strain O (PVY(O)) but susceptible to the strain NTN (PVY(NTN)), and Russet Burbank, which is susceptible to all PVY strains that have been tested. Leaves were inoculated with PVY(O) or PVY(NTN), and samples were collected 4 and 10 h post inoculation (hpi). A larger number of differentially expressed (DE) genes were found in the compatible reactions compared to the incompatible reaction. For all treatments, the majority of DE genes were down-regulated at 4 hpi and up-regulated at 10 hpi. Gene Ontology enrichment analysis showed enrichment of the biological process GO term "Photosynthesis, light harvesting" specifically in PVY(O)-inoculated Premier Russet leaves, while the GO term "nucleosome assembly" was largely overrepresented in PVY(NTN)-inoculated Premier Russet leaves and PVY(O)-inoculated Russet Burbank leaves but not in PVY(O)-inoculated Premier Russet leaves. Fewer genes were DE over 4-fold in the incompatible reaction compared to the compatible reactions. Amongst these, five genes were DE only in PVY(O)-inoculated Premier Russet leaves, and all five were down-regulated. These genes are predicted to encode for a putative ABC transporter, a MYC2 transcription factor, a VQ-motif containing protein, a non-specific lipid-transfer protein, and a xyloglucan endotransglucosylase-hydroxylase. CONCLUSIONS: Our results show that the incompatible and compatible reactions in Premier Russet shared more similarities, in particular during the initial response, than the compatible reactions in the two different hosts. Our results identify potential key processes and genes that determine the fate of the reaction, compatible or incompatible, between PVY and its host.

Diversity and evolution of potato mop-top virus.

Nearly complete sequences of RNA-CP and 3'-proximal RNA-TGB were determined for 43 samples of potato mop-top virus (PMTV) originating from potato tubers and field soil from Sweden, Denmark and the USA. The results showed limited diversity and no strict geographical grouping, suggesting only a few original introductions of PMTV from the Andes. Two distinguishable types of RNA-CP and RNA-TGB were found in the samples, but no specific combination of them correlated with spraing symptoms in tubers. Lack of positive selection in the coding sequences indicates that there is no specific molecular adaptation of PMTV to new vectors or hosts.

Identification of a fourth haplotype of Bactericera cockerelli (Hemiptera: Triozidae) in the United States.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a pest of potato and other solanaceous crops in North and Central America and New Zealand. Previous genotyping studies have demonstrated the presence of three different haplotypes of B. cockerelli in the United States corresponding to three geographical regions: Central, Western, and Northwestern. These studies utilized psyllids collected in the western and central United States between 1998 and 2011. In an effort to further genotype potato psyllids collected in the 2012 growing season, a fourth B. cockerelli haplotype was discovered corresponding to the Southwestern United States geographical region. High-resolution melting analyses identified this new haplotype using an amplicon generated from a portion of the B. cockerelli mitochondrial cytochrome c oxidase subunit I gene. Sequencing of this gene, as well as use of a restriction enzyme assay, confirmed the identification of the novel B. cockerelli haplotype in the United States.

A global comparison of Bactericera cockerelli (Hemiptera: Triozidae) microbial communities.

The potato psyllid (Bactericera cockerelli Sulc) is an economically important insect pest of solanaceous crops such as potato, tomato, pepper, and tobacco. Historically, the potato psyllid's range included central United States, Mexico, and California; more recently, populations of this insect have been reported in Central America, the Pacific Northwest, and New Zealand. Like most phytophagous insects, potato psyllids require symbiotic bacteria to compensate for nutritional deficiencies in their diet. Potato psyllids harbor the primary symbiont, Candidatus Carsonella ruddii, and may also harbor many secondary symbionts such as Wolbachia sp., Sodalis sp., Pseudomonas sp., and others. These secondary symbionts can have an effect on reproduction, nutrition, immune response, and resistances to heat or pesticides. To identify regional differences in potato psyllid bacterial symbionts, 454 pyrosequencing was performed using generic 16S rRNA gene primers. Analysis was performed using the Qiime 1.6.0 software suite, ARB Silva, and R. Operational taxonomic units were then grouped at 97% identity. Representative sequences were classified to genus using the ARB SILVA database. Potato psyllids collected in California contained a less diverse microbial community than those collected in the central United States and Central America. The crop variety, collection year, and haplotype did not seem to affect the microbial community in potato psyllids. The primary difference between psyllids in different regions was the presence and overall bacterial community composition of Candidatus Carsonella ruddii and Wolbachia.

Low-level detection of candidatus Liberibacter solanacearum in Bactericera cockerelli (Hemiptera: Triozidae) by 16s rRNA Pyrosequencing.

Accurate detection and quantification of Candidatus Liberibacter solanacearum (CLs), the putative causal agent of zebra chip disease of potato (Solanum tuberosum L.), in the potato psyllid, Bactericera cockerelli (Sulc), has become necessary to better understand the biology of the disease cycle. Studies on the transmission efficiency of potato psyllids have shown inconsistencies with field surveys. There have also been reports of laboratory colonies inexplicably losing and regaining CLs infection as detected by polymerase chain reaction (PCR). Until now, DNA primers were used to detect CLs in potato psyllid tissue using conventional polymerase chain reaction (PCR) and gel electrophoresis or by real-time quantitative PCR. In this study, CLs was detected using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) at levels identifiable by PCR, and low levels, including samples with only one cell of CLs. Potato psyllids with <300 pyrosequencing reads did not show positive using conventional PCR. These results indicate that the currently accepted PCR diagnostic technique produces false negatives due to detection limits higher than what is generally present in field collected psyllids, and also provides an explanation as to why laboratory colonies seem to lose and regain CLs infection.

Temporal and spatial analysis of potato psyllid haplotypes in the United States.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is an economically important pest of potato (Solanum tuberosum L.) crops across the western and central United States, as it is known to cause psyllid yellows disease and to transmit the bacterium that causes zebra chip disease. Recent genotyping of B. cockerelli collected during the 2011 potato growing season identified three psyllid haplotypes within the western and central United States according to their geographical regions: northwestern, western, and central. To understand potato psyllid population dynamics before the year 2011, high resolution melting analysis of the B. cockerelli mitochondrial cytochrome oxidase I-like gene was used to identify the haplotypes of over 450 archived psyllids collected in the western and central United States between the years 1998 and 2010. Results show that the northwestern haplotype was present in Washington State as early as 1998 and has persisted in this region since that time. Likewise, psyllids of the western haplotype have also been present in Washington and Oregon before 2011.

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains.

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.

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.

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes.

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20-25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3' terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.

Carrot Purple Leaf: A New Spiroplasmal Disease Associated with Carrots in Washington State.

During the growing seasons of 2003 and 2004, a disease occurred in several carrot crops in south central Washington with symptoms suggestive of infection by phytopathogenic mollicutes (phytoplasmas and spiroplasmas). In the fall, many affected carrot plants exhibited extensive purple or yellow-purple leaf discoloration, general stunting of shoots and taproots, and formation of bunchy, fibrous secondary roots. For detection of the putative causal agents, polymerase chain reaction (PCR) assays were performed using primers specific to phytoplasmas as well as primers specific to plant-pathogenic spiroplasmas. Restriction fragment length polymorphism (RFLP) analyses of PCR-amplified 16S rDNA sequences revealed that about 81% of affected plants showing dark purple or yellow-purple leaf symptoms tested positive for Spiroplasma citri. Of affected plants showing mild purple discoloration of leaf margins, 18% tested positive for a phytoplasma strain belonging to the clover proliferation group (16SrVI), subgroup 16SrVI-A, and 11% for another phytoplasma strain belonging to the aster yellows group (16SrI), subgroup 16SrI-A. Nucleotide sequence analysis of cloned 16S rDNA confirmed the phytoplasma group affiliations. Some symptomatic plants were co-infected with S. citri and either aster yellows phytoplasma or clover proliferation group phytoplasma. To our knowledge, this is the first documentation of spiroplasma infection of carrot in the United States.