Amy Anchieta

Co-Infection by Two Criniviruses Alters Accumulation of Each Virus in a Host-Specific Manner and Influences Efficiency of Virus Transmission

Tomato chlorosis virus (ToCV), and Tomato infectious chlorosis virus (TICV), family Closteroviridae, genus Crinivirus, cause interveinal chlorosis, leaf brittleness, and limited necrotic flecking or bronzing on tomato leaves. Both viruses cause a decline in plant vigor and reduce fruit yield, and are emerging as serious production problems for field and greenhouse tomato growers in many parts of the world. The viruses have been found together in tomato, indicating that infection by one Crinivirus sp. does not prevent infection by a second. Transmission efficiency and virus persistence in the vector varies significantly among the four different whitefly vectors of ToCV; Bemisia tabaci biotypes A and B, Trialeurodes abutilonea, and T. vaporariorum. Only T. vaporariorum can transmit TICV. In order to elucidate the effects of co-infection on Crinivirus sp. accumulation and transmission efficiency, we established Physalis wrightii and Nicotiana benthamiana source plants, containing either TICV or ToCV alone or both viruses together. Vectors were allowed to feed separately on all virus sources, as well as virus-free plants, then were transferred to young plants of both host species. Plants were tested by quantitative reverse-transcription polymerase chain reaction, and results indicated host-specific differences in accumulation by TICV and ToCV and alteration of accumulation patterns during co-infection compared with single infection. In N. benthamiana, TICV titers increased during co-infection compared with levels in single infection, while ToCV titers decreased. However, in P. wrightii, titers of both TICV and ToCV decreased during mixed infection compared with single infection, although to different degrees. Vector transmission efficiency of both viruses corresponded with virus concentration in the host in both single and mixed infections. This illustrates that Crinivirus epidemiology is impacted not only by vector transmission specificity and incidence of hosts but also by interactions between viruses and efficiency of accumulation in host plants.

The complete nucleotide sequence and genome organization of tomato chlorosis virus

Summary.The crinivirus tomato chlorosis virus (ToCV) was discovered initially in diseased tomato and has since been identified as a serious problem for tomato production in many parts of the world, particularly in the United States, Europe and Southeast Asia. The complete nucleotide sequence of ToCV was determined and compared with related crinivirus species. RNA 1 is organized into four open reading frames (ORFs), and encodes proteins involved in replication, based on homology to other viral replication factors. RNA 2 is composed of nine ORFs including genes that encode a HSP70 homolog and two proteins involved in encapsidation of viral RNA, referred to as the coat protein and minor coat protein. Sequence homology between ToCV and other criniviruses varies throughout the viral genome. The minor coat protein (CPm) of ToCV, which forms part of the “rattlesnake tail” of virions and may be involved in determining the unique, broad vector transmissibility of ToCV, is larger than the CPm of lettuce infectious yellows virus (LIYV) by 217 amino acids. Among sequenced criniviruses, considerable variability exists in the size of some viral proteins. Analysis of these differences with respect to biological function may provide insight into the role crinivirus proteins play in virus infection and transmission.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii)

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

Colonization of Spinach by Verticillium dahliae and Effects of Pathogen Localization on the Efficacy of Seed Treatments

Verticillium wilt on spinach (Spinacia oleracea) is caused by the soilborne fungus Verticillium dahliae. The pathogen is seedborne and transmission through seed is a major concern because of the dispersal of the pathogen to areas where fresh and processing spinach crops are grown in rotation with susceptible crops. Reduction in seedborne inoculum minimizes pathogen spread; therefore, knowledge of pathogen localization in seed is critical to develop methods to reduce seedborne inoculum. Spinach seedlings were inoculated with conidial suspensions of a green fluorescent protein-tagged strain of V. dahliae and colonization events were followed through seed production by confocal laser-scanning microscopy. Between 24 to 96 h postinoculation (PI), conidia germinated and formed hyphal colonies on root tips and in root elongation zones. Hyphae colonized root cortical tissues both intra and intercellularly by 2 weeks, and colonized the taproot xylem with abundant mycelia and conidia that led to vascular discoloration coincident with foliar symptom expression by 8 weeks PI. At 10 weeks PI, the xylem of the upper stem, inflorescence, and spinach seed parts, including the pericarp, seed coat, cotyledons, and radicle, had been colonized by the pathogen but not the perisperm (the diploid maternal tissue). Maximum concentration of the fungus was in the seed coat, the outermost layer of the vasculature. Infection of V. dahliae in spinach seed was systemic and transmissible to developing seedlings. Additional analyses indicated that fungicide and steam seed treatments reduced detectable levels of the pathogen but did not eliminate the pathogen from the seed. This information will assist in the development of seed treatments that will reduce the seedborne inoculum transmission to crop production fields.

Season-Long Dynamics of Spinach Downy Mildew Determined by Spore Trapping and Disease Incidence

Peronospora effusa is an obligate oomycete that causes downy mildew of spinach. Downy mildew threatens sustainable production of fresh market organic spinach in California, and routine fungicide sprays are often necessary for conventional production. In this study, airborne P. effusa spores were collected using rotating arm impaction spore trap samplers at four sites in the Salinas Valley between late January and early June in 2013 and 2014. Levels of P. effusa DNA were determined by a species-specific quantitative polymerase chain reaction assay. Peronospora effusa was detected prior to and during the growing season in both years. Nonlinear time series analyses on the data suggested that the within-season dynamics of P. effusa airborne inoculum are characterized by a mixture of chaotic, deterministic, and stochastic features, with successive data points somewhat predictable from the previous values in the series. Analyses of concentrations of airborne P. effusa suggest both an exponential increase in concentration over the course of the season and oscillations around the increasing average value that had season-specific periodicity around 30, 45, and 75 days, values that are close to whole multiples of the combined pathogen latent and infectious periods. Each unit increase in temperature was correlated with 1.7 to 6% increased odds of an increase in DNA copy numbers, while each unit decrease in wind speed was correlated with 4 to 12.7% increased odds of an increase in DNA copy numbers. Disease incidence was correlated with airborne P. effusa levels and weather variables, and a receiver operating characteristic curve analysis suggested that P. effusa DNA copy numbers determined from the spore traps nine days prior to disease rating could predict disease incidence.

Detection and Quantification of Bremia lactucae by Spore Trapping and Quantitative PCR.

Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (Cq) value of 32, and Cq values >35 were considered to be nonspecific. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.

Plasmolysis and Vital Staining Reveal Viable Oospores of Peronospora effusa in Spinach Seed Lots

Production of oospores by Peronospora effusa, the causal agent of downy mildew on spinach (Spinacia oleracea), was reported on spinach seed over three decades ago. In view of the rapid proliferation of new races of P. effusa worldwide, seedborne transmission of this pathogen has been suspected but methods to test the viability of seedborne oospores have not been available. Eighty-two seed lots of contemporary spinach cultivars were evaluated for the presence of P. effusa using a seed-wash method and the sediment was examined by microscopy. Of the analyzed seed lots, 16% were positive for oospores and an additional 6% for sporangiophores characteristic of P. effusa. Application of a P. effusa-specific quantitative polymerase chain reaction assay showed that 95% of the 59 tested seed lots were positive for P. effusa. The viability of oospores from five seed lots that were proven to carry the pathogen from the above tests was tested using two independent methods, one involving plasmolysis and the other trypan blue staining. The oospores plasmolyzed in 4 M sodium chloride and were deplasmolyzed in water, demonstrating an active and viable cell membrane. Similarly, viable oospores failed to take up the trypan blue stain. Overall, 59% of the oospores were viable in the plasmolysis test and 45% with the trypan blue test. These results indicate the presence of P. effusa oospores in contemporary spinach seed lots, and suggest that the transmission of viable oospores of P. effusa in spinach seed does occur. Elimination of the pathogen on seed, in addition to other management approaches, will be useful in reducing the extent and severity of downy mildew on spinach crops and diminishing pathogen spread through seed.

Spatiotemporal Patterns in the Airborne Dispersal of Spinach Downy Mildew

Downy mildew is the most devastating disease threatening sustainable spinach production, particularly in the organic sector. The disease is caused by the biotrophic oomycete pathogen Peronospora effusa, and the disease results in yellow lesions that render the crop unmarketable. In this study, the levels of DNA from airborne spores of P. effusa were assessed near a field of susceptible plants in Salinas, CA during the winter months of 2013-14 and 2014/15 using rotating-arm impaction spore-trap samplers that were assessed with a species-specific quantitative polymerase chain reaction (qPCR) assay. Low levels of P. effusa DNA were detectable from December through February in both winters but increased during January in both years, in correlation with observed disease incidence; sharp peaks in P. effusa DNA detection were associated with the onset of disease incidence. The incidence of downy mildew in the susceptible field displayed logistic-like dynamics but with considerable interseason variation. Analysis of the area under the disease progress curves suggested that the 2013-14 epidemic was significantly more severe than the 2014-15 epidemic. Spatial analyses indicated that disease incidence was dependent within an average range of 5.6 m, approximately equivalent to the width of three planted beds in a typical production field. The spatial distribution of spores captured during an active epidemic most closely fit a power-law distribution but could also be fit with an exponential distribution. These studies revealed two important results in the epidemiology of spinach downy mildew in California. First, they demonstrated the potential of impaction spore-trap samplers linked with a qPCR assay for indicating periods of high disease risk, as well as the detection of long-distance dispersal of P. effusa spores. Second, at the scale of individual crops, a high degree of spatial aggregation in disease incidence was revealed.

The genome sequence of lettuce necrotic stunt virus indicates a close relationship to Moroccan pepper virus

Lettuce necrotic stunt virus (LNSV) causes severe losses to lettuce production in the western United States, which results in stunting, necrosis and death on all non-crisphead lettuces, as well as flower abortion and yield losses in greenhouse tomato production. The genome of LNSV was sequenced and has an organization typical of viruses of the genus Tombusvirus. Sequence comparisons indicated that much of the genome is relatively closely related to tomato bushy stunt virus; however, the coat protein is very closely related to that of isolates of Moroccan pepper virus (MPV).

Genetic variation among Beet curly top virus isolates infecting weed and crop hosts in California

Curly top disease is caused by Beet curly top virus (BCTV) and related curtovirus species, and is transmitted by the beet leafhopper (Circulifer tenellus). The disease occurs in several large, but geographically separate regions of western North America. BCTV re-emerged in 2001 as a serious threat to agriculture in the San Joaquin Valley of California and has continued to exert pressure on agriculture in this region. BCTV infects a broad range of crop hosts including sugar beet, pepper, and tomato, as well as numerous native weeds. Prior molecular characterization of a limited number of curtoviruses from broad areas of the western United States suggested that two distinct curtovirus species, Beet severe curly top virus (BSCTV or CFH strain) and Beet mild curly top virus (BMCTV or Worland strain) were responsible for most crop disease, but little information existed on curtovirus species distribution among weed hosts or species prevalence in the California sugarbeet crop. The aim of this study was to clarify the genetic variability among curtovirus isolates in California, and to determine if specific weed hosts might be reservoirs for exceptionally severe virus species, such as BSCTV. Data collected over 2 years focused on molecular characterization of large numbers of BCTV isolates from weed and crop hosts of the beet leafhopper in the San Joaquin Valley. Total nucleic acid was isolated from individual plants, and both universal and specific primers were used to amplify viral DNA. PCR amplification coupled with sequence analysis identified the prevalence of both BSCTV and BMCTV as the predominant curtovirus species in California, infecting both weeds and crops. The Logan strain of BCTV, historically associated with California, was not identified among over 200 isolates characterized. SUMMARY Beet curly top virus (BCTV) and related viruses (collectively known as curtoviruses) transmitted by the beet leafhopper Circulifer tenellus (Baker) have caused significant problems to irrigated agriculture in the western US since 1899 (Carsner and Stahl, 1924). BCTV is known to infect a broad range of crop and weed hosts in many plant families (Bennett, 1971). Crop hosts for which natural BCTV infection has been reported include sugarbeet, tomato, pepper, bean, spinach, and cucurbits. The leafhopper vector also feeds and breeds on an extensive range of plant hosts from different families (Cook, 1967). C. tenellus transmits curtoviruses most efficiently after a 48-hour acquisition-access feed on an infected source plant, but shorter feeding times (220 min.) also result in a low frequency of transmission. Curtovirus transmission by the vector requires a 4 hour latent period following ingestion, and leafhoppers can inoculate healthy plants by feeding for as little as a 1 min inoculation access period. Symptoms generally develop in plants within two weeks, depending on the host and age at infection. Leafhopper vectors retain the ability to transmit BCTV for days to weeks. BCTV is a monopartite geminivirus and the type member of the genus Curtovirus within the family Geminiviridae (Fauquet et al., 2003). Viruses within this group are characterized by circular ssDNA genomes of approximately 3.0 kilobases encapsidated within twin spherical particles. Many strains (up to 14) of BCTV were initially distinguished on the basis of differential symptomatology in sugarbeet (reviewed in Klein, 1992). The disease occurs in several large, but geographically separate regions of western North America. Curly top re-emerged in 2001 as a serious threat to agriculture in the San Joaquin Valley of California and has continued to exert pressure on agriculture in this region (Wintermantel and Kaffka, 2006). BCTV infects a broad range of crop hosts including sugarbeet, pepper, and tomato, as well as numerous native weeds. Prior molecular characterization of a limited number of curtoviruses from broad areas of the western United States suggested that two distinct curtovirus species, Beet severe curly top virus (BSCTV or CFH strain) and Beet mild curly top virus (BMCTV or Worland strain) were responsible for most crop disease (Stenger and McMahon, 1997), but little information existed on curtovirus species distribution among weed hosts or species prevalence in the California sugarbeet crop. The aim of this study was to clarify the genetic variability among curtovirus isolates in California, and to determine if specific weed hosts might be reservoirs for exceptionally severe virus species, such as BSCTV. Data collected over 2 years focused on molecular characterization of large numbers of curly top isolates from weed and crop hosts of the beet leafhopper in the San Joaquin Valley. Using the extensive host range information available for curly top, reported weed and crop hosts of the virus were collected from throughout California. The majority of beet leafhopper flights are reported to be shorter than 100 miles, and the spring breeding grounds of the leafhopper, the foothills of western San Joaquin Valley, are well documented in California. Weed samples for this study were collected primarily from this area, with some samples originating from the southern portion of the Salinas Valley, as well. Collection locations were made using global positioning systems (GPS) in order to map the locations where curtoviruses were detected. Crop samples, consisting of sugarbeet, tomato, and pepper were also collected from the San Joaquin Valley. Sample collection was conducted from May through September over a three year period from 2002-2004. Samples were scored as positive or negative for curtoviruses using PCRbased virus detection methods described below. Based on this information, some areas were clearly “hot-spots” for the presence of curly top virus species, although no strainspecific hot-spots were identified. Polymerase chain reaction (PCR)-based detection methods and DNA sequencing were used to confirm curtovirus infection and to identify different curtovirus species. This method involved using short strands of DNA (primers) that bind to complementary DNA sequences present in all curtovirus species (formerly known as different BCTV strains). After primer binding, an enzyme was used to extend the primers to make multiple copies of the original strand. The end result of this process is known as a PCR product. Samples that did not contain BCTV or related curtoviruses did not produce PCR products. The resulting PCR product was then directly sequenced. Sequencing results were compared with known sequences of curtovirus species to determine which species the isolate in question was most closely related to. Results indicated that the highest incidence of infection was in sugarbeet (78%) and wild mustard (73%), with somewhat lower incidence in Russian thistle (57%), tomato (55%), and London rocket (46%). Other weed and crop hosts had considerably lower incidence of curly top, as confirmed by detection of curtoviruses in plant tissue. Overall, 200 of 562 (36%) samples tested positive for BSCTV (formerly known as CFH strain) or BMCTV (formerly known as Worland strain). No traditional BCTV (formerly California/Logan strain) was found, although small pieces of DNA corresponding to the traditional BCTV (California/Logan) sequence were occasionally found interspersed among BSCTV or BMCTV sequences. Some recombinant curtoviruses were also identified. These involved sections of both BSCTV and BMCTV, suggesting recombination (exchange of viral genetic material) may readily occur between the different species within the region sequenced. The abundance of BSCTV and BMCTV, along with the lack of BCTV indicated a clear transition between curtovirus species prevalent in California during the mid 1900s and those present today, suggesting evolution and emergence of new curly top (curtovirus) species. Studies also addressed whether specific curtovirus species were associated with specific weed or crop hosts. Results demonstrated that all species were equally capable of infecting the different host species examined in this study, and that there appears to be little difference in host range between the different curtovirus species. ACKNOWLEDGEMENT Thanks to Rod Clark and Kelly Brannigan of the California Department of Food and Agriculture–Curly Top Virus Control Board for collection and GPS mapping of weed samples. This research was supported in part by funding provided by the California Beet Growers Association Industry Research Committee. LITERATURE CITED Bennett, C. W. 1971. The curly top disease of sugarbeet and other plants. The Am. Phytopathol. Soc. Monogr. No. 7. Carsner, E. and Stahl, C. F. 1924. Studies on curly-top disease of the sugarbeet. J. Agr. Res. 28:297-320. Cook, W. C. 1967. Life history, host plants, and migrations of the beet leafhopper in the western United States. U.S.D.A. Tech. Bull. 1365. 122 p. Fauquet, C.M., Bisaro, D.M., Briddon, R.W, Brown, J.K., Harrison, B.D., Rybicki, E.P., Stenger, D.C. and Stanley, J. 2003. Revision of taxonomic criteria for species demarcation in the family Geminiviridae, and an updated list of begomovirus species. Arch. Virol. 148: 405-421. Klein, M. 1992. Role of Circulifer / Neoaliturus in the transmission of plant pathogens. Pages 152-193 in: Advances in Disease Vector Research, Vol. 9. Springer-Verlag, New York, NY. Stenger, D. C. and McMahon, C. L. 1997. Genotypic diversity of beet curly top virus populations in the western United States. Phytopathology 87:737-744. Wintermantel, W.M. and Kaffka, S.R. 2006. Sugarbeet performance with curly top is related to virus accumulation and age at infection. Plant Disease 90: 000-000 (in press).