Progress 04/25/07 to 04/24/12
Outputs
Progress Report Objectives (from AD-416): Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. Approach (from AD-416): Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with BNYVV, the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. The Salinas lab maintains a collection of bipartite, whitefly-transmitted Criniviruses, and is recognized internationally as a leader in the study of this genus of viruses. The lab has identified and characterized most criniviruses currently known to infect sugarbeet and vegetable crops. Rapid-detection methods for nearly all viruses in this genus have been developed and published through this project and are in use throughout the world. We made significant strides toward understanding epidemiology and transmission parameters of this emerging genus, including Cucurbit yellow stunting disorder virus (CYSDV), an emergent crinivirus severely impacting cucurbit production in the US. The Salinas Virology Lab works closely with grower organizations and extension personnel, advising other scientists throughout the world and regulatory authorities in determining the extent of infection, educating growers and developing management tactics to minimize losses, working closely with the ARS melon breeding program in Salinas to advance development of resistance, and to identify important hosts serving as sources for virus transmission to melon. Research by the ARS Virology Lab in Salinas on Beet necrotic yellow vein virus (BNYVV) which causes rhizomania, and its vector, the soil-borne fungus, Polymyxa betae, has been critical to understanding this disease. The lab developed specific and sensitive diagnostic assays, and research led to taxonomic reclassification of some sugar beet viruses. The ARS Virology Lab in Salinas described a new strain of BNYVV that overcomes Rz1 gene resistance, and has linked the resistance-breaking trait to amino acid changes in RNA3. Research nearing completion is focused on clarifying how BNYVV infection and host resistance alter protein expression in sugarbeet, and utilizing this information to block virus infection and disease development. The Salinas Lab also recently described another soil-borne virus of sugarbeet, Beet oak leaf virus (BOLV) that alters expression of BNYVV symptoms during co-infection. Additional studies have genetically characterized soilborne viruses of lettuce associated with the diseases, lettuce dieback (two tombusviruses sequenced, taxonomic relationships characterize, developing methods for controlled environment resistance evaluations) and lettuce big vein (Mirafiori lettuce big vein virus; MLBVV), and examined epidemiological factors contributing to control and spread. Collaborative efforts with sugarbeet and vegetable breeding programs in Salinas have facilitated development of genetic resistance to all three soilborne viruses (BNYVV, Tombusviruses and MLBVV). Curly top, transmitted by the beet leafhopper (Circulifer tenellus) and caused by Beet curly top virus (BCTV) and related curtovirus species, has reduced yields of sugar beet and vegetables since the late 1800s. The Salinas Lab has identified crop and weed reservoirs, is examining factors that drive changes in curtovirus population structure in the western US, and is working toward release of novel methods for control of these viruses in sugarbeet and tomato. Significant Activities that Support Special Target Populations: Provided guest demonstration in Earth Science to fifth grade students at Los Padres Elementary School, Salinas, CA. (Oct. 25, 2011 and Dec. 13, 2011). Presented information on research and careers in agricultural science to students at North Salinas High School (Oct. 13, 2011) and Salinas High School (Feb. 3, 2012) in Salinas, CA, educating high school students on potential career options. Provided internships and training to two 9th grade honors biology students from Salinas High School (Salinas, CA), including a specific research project and hands-on experience with basic molecular biology methods including nucleic acid extraction, reverse transcription PCR, and agarose gel analysis. Accomplishments 01 Development of methods for lab-based evaluation of lettuce for resistanc to tombusviruses. Lettuce dieback disease, caused by the tombusviruses, Tomato bushy stunt virus (TBSV) and the closely related and Lettuce necrotic stunt virus (LNSV), results in necrosis, stunting and death of lettuce plants throughout all western lettuce production regions. Prior these studies no effective methods had been developed for greenhouse evaluation of lettuce germplasm for resistance to this disease since symptoms do not develop readily in greenhouses or growth chambers. Treatment of plants with long-days and high temperatures was found to induce disease symptoms on lettuce similar to those found under field conditions; there was, however, considerable variation in consistency, which suggested that an additional factor may be required for uniform symptom development. Research continues to optimize requirements for mor rapid symptom development of dieback-resistant lettuce. 02 Completed the sequencing of the tombusvirus, Lettuce necrotic stunt viru Lettuce necrotic stunt virus (LNSV) was first identified in the late 1990s by ARS researchers in the virology lab in Salinas, California, as novel virus and the causative agent of lettuce dieback disease, causing stunting and death of lettuce plants throughout all western lettuce production regions. Prior to current studies, only a small portion of th LNSV genome had been sequenced. ARS researchers in Salinas, California, determined the complete genome sequence of an isolate of LNSV obtained from lettuce in the Salinas Valley (California), and compared the sequen to related viruses in the genus Tombusvirus, family Tombusviridae. Resul demonstrated LNSV was clearly a unique virus, and clarified relationship among several members of the genus tombusvirus including discovery that the coat protein of LNSV was nearly identical to that of the older and poorly characterized Moroccan pepper virus. This information clarifies t relationship of LNSV to other members of the genus Tombusvirus, and provided more detailed information for use in virus detection. 03 Completed the sequencing of the tombusvirus, Moroccan pepper virus. Moroccan pepper virus (MPV) was first discovered in diseased pepper plan from Morocco in 1975, was serologically distinct from other viruses in t genus, Tombusvirus, but only the sequence of the MPV coat protein gene w known, and limited symptom information was available. Three isolates of MPV were obtained and sequenced in order to clarify the relationship between MPV and the recently sequenced Lettuce necrotic stunt virus (LNS which causes lettuce dieback. ARS scientists in Salinas, California, found that there was 97 percent common identity among the genomes of the three MPV isolates and LNSV, which confirmed that MPV and LNSV were the same virus species. A unique set of diagnostic hosts for MPV was determined based on comparative studies on the California isolate and other tombusviruses. This clarifies relationships within the genus Tombusvirus, demonstrates that MPV is present in North America, and provides molecular methods for identification of MPV isolates in other locations throughout the world. 04 Beet oak-leaf virus (BOLV) is transmitted by Polymyxa betae. It has man structural features resembling Beet necrotic yellow vein virus (BNYVV), yet BOLV produces only sporadic and mild symptoms on sugarbeet. Studies conducted by ARS in Salinas, California, have demonstrated that BOLV is serologically distinct from BNYVV, and may suppress BNYVV during mixed infections. Research is in progress to determine the nucleotide sequence of BOLV. The results may lead to future studies to identify protein interactions between BNYVV and BOLV responsible for suppression of BNYVV causal agent of rhizomania disease, a devastating pathogen of sugarbeet. 05 Protein changes associated with ability of Beet necrotic yellow vein vir (BNYVV) to overcome the sugarbeet Rz1 resistance gene. Rhizomania, caus by BNYVV, is widely distributed in most sugarbeet growing areas of the world and can result in complete loss of crop in the absence of resistan varieties. New variants of BNYVV overcome the first reported and widely used resistance rhizomania. ARS scientists in Salinas, California, demonstrated that a relatively small number of changes in sugarbeet protein expression were associated with traditional (Rz1 gene) resistanc to BNYVV infection. Proteins associated with infection by Rz1 resistance breaking variants have been identified, and further analysis is in progress. These studies are expected to lead to methods to prolong the longevity of Rz resistance sources by understanding the fundamental mechanisms that cause resistance to break down. 06 Identification of wild and cultivated reservoir hosts of Cucurbit yellow stunting disorder virus (CYSDV) in the desert region of southwest U.S. CYSDV is a recently emerged virus that adversely affects melon and watermelon production in the desert southwest of the U.S. (the inland deserts of California and southwestern Arizona), Florida and Texas. ARS scientists in Salinas, California, determined that CYSDV infects a much broader range of crop and weed plants than was previously known. It infects species in seven families in addition to cucurbits. The Salinas lab determined the distribution and prevalence of CYSDV and other viruse in crop and weed hosts in the desert southwest U.S. This information has been reported to melon and watermelon producers educating growers and us to develop management tactics to minimize losses to this devastating vir disease. 07 Curly top concentration, field incidence and transmission. Beet severe curly top virus (BSCTV) and Beet mild curly top virus (BMCTV) are the tw most abundant curtovirus species, but they have not always been the dominant forms, and in some areas of the southwestern U.S. new curtoviru species have been identified. ARS scientists in Salinas, California, compared virus accumulation, competition and transmission among common weed and crop curtovirus hosts in order to identify factors that drive t emergence of new virus species, as well as to determine what factors cau a variant to become dominant. Single and mixed infections of BSCTV and BMCTV were established in several weed and crop hosts, to determine efficiency of accumulation in each host plant species individually, as well as which virus dominates during mixed infections. Results indicated differential accumulation of each virus depending on host plant, and shifts in accumulation patterns during mixed infection. Transmission studies demonstrated variation in transmission efficiency of each virus among host plants. Results to date added to the knowledge of factors driving emergence and dominance among curtoviruses and contribute to overall knowledge of curtovirus epidemiology. 08 Primers for detection, differentiation, and quantification of BNYVV and other soil-borne viruses associated with rhizomania disease of sugar bee Four soil-borne viruses impact U.S. sugar beet production: Beet necroti yellow vein virus (BNYVV), Beet soil-borne mosaic virus (BSBMV), Beet so borne virus (BSBV), and Beet oak leaf virus (BOLV). ARS scientists in Salinas, California, as well as other labs have shown that mixed infecti influence virus accumulation in sugarbeet. Primers for the detection of all three soil-borne viruses affecting U.S. sugar beet production for which genome sequence information is available (BNYVV, BSBMV, BSBV) were designed and combined in specific ratios into a single reaction mix for identification of these viruses in single and mixed infections in field samples. Primers were developed to quantify levels of BNYVV during singl and mixed infections with BSBMV and BSBV using different fluorescent dye Early results demonstrated independent amplification of each of the thre viruses without cross-reactivity in, and will be useful to evaluate individual virus levels in mixed infections of sugar beet. 09 Vector transmission of Blackberry yellow vein associated virus (BYVaV). This virus belongs to subgroup 1 of the genus Crinivirus based on genomi sequence comparisons. All members of of this group are transmitted by th greenhouse whitefly, but the vector of BYVaV was unknown. ARS scientists in Salinas, California, evaluated two whitefly species, Trialeurodes vaporariorum and T. ablutilonea as potential vectors of BYVaV. Both T. vaporariorum and T. abutilonea were able to transmit BYVaV with about 30 transmission efficiency. Roses were also discovered to be a host of the virus, but it remains to be determined whether whiteflies can transmit t virus to rose. These studies provide important epidemiological informati toward management of BYVaV. 10 Genetic variability of Endornaviruses associated with melon germplasm. Endornaviruses are double-stranded RNA viruses that infect hosts in the kingdoms Plantae, Fungi and Chromista, and are transmitted through polle but do not induce visible symptoms. Preliminary studies indicated the presence of a melon endornavirus species in wild and cultivated cucurbit species. Studies were conducted to determine if virus variation is relat to co-evolution with melon as a result of breeding and host plant selection. Endornaviruses were amplified from melon accessions that represented genotypes from around the world. Sequence analysis of RT-PCR products by ARS scientists in Salinas, California, supported the hypothesis that crossing of melons influenced variability in the virus. Results provide documentation of virus genetic adaptation associated wit plant breeding.
Impacts
(N/A)
Publications
- Wintermantel, W.M., Anchieta, A.G. 2012. The genome sequence of lettuce necrotic stunt virus indicates a close relationship to moroccan pepper virus. Archives of Virology. doi:10.1007/s00705-012-1307-x.
- Wintermantel, W.M., Natwick, E.T. 2012. First report of Alfalfa mosaic virus infecting basil (Ocimum basilicum L.) in California. Plant Disease. 96(2):295.
- Robertson, N.L. 2012. First report of apple mosaic virus in Alaska. Plant Disease. 96(3):463.
- Robertson, N.L., Quito, D., Martin, R.R. 2012. Alaskan Ribes L. and Rubus L. Plant Species Surveyed for Viruses. Acta Horticulturae. 946:237-242.
- Barney, D.L., Hummer, K.E., Robertson, N.L., Gilmore, B.S. 2012. Ribes L. Gene Bank Management in the United States. Acta Horticulturae. 946:73-76.
- Liu, H., Koike, S.T., Xu, D., Li, R. 2012. First report of turnip mosaic virus in tomatillo (Physalis philadelphica) in California. Plant Disease. 96(2):296.
- Koike, S.T., Liu, H., Sears, J.L., Tian, T., Daugovish, O., Dara, S. 2012. Distribution, cultivar susceptibility, and epidemiology of Apium virus Y on celery in coastal California. Plant Disease. 96(5):612-617.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. Approach (from AD-416) Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with (BNYVV), the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. (IBC info pending). Replaces 5305-22000-010-00D (3/07). The Salinas lab maintains a collection of bipartite, whitefly-transmitted Criniviruses, and is recognized internationally as a leader in the study of this group of viruses. The lab has identified and characterized most of the criniviruses that are currently known to infect sugarbeet and vegetable crops, and has developed detection methods for viruses within the genus. New rapid-detection methods were developed through this project for rapid identification and differentiation of most known criniviruses from one another. Studies continue on Cucurbit yellow stunting disorder virus (CYSDV), an emergent crinivirus severely impacting cucurbit production in the US Desert Southwest. Our program has been actively assisting other laboratories in acquiring the tools to monitor for CYSDV as well as other crinivirus species throughout the world, working with grower organizations, extension personnel, company representatives and regulatory authorities, educating growers and developing methods to minimize losses, and determining the most important non-cucurbit hosts that harbor CYSDV. Beet necrotic yellow vein virus (BNYVV), which causes rhizomania and its vector, the soil-borne fungus, Polymyxa betae, were detected for the first time in the Western Hemisphere by the Salinas Virology Lab. Research on BNYVV and related viruses has been critical to understanding of the disease and facilitating development by the lab of highly specific and sensitive diagnostic assays. Results from studies of soil-borne sugarbeet viruses led to taxonomic reclassification of some viruses. The laboratory recently described a new strain of BNYVV that overcomes Rz1 gene resistance and differs from other resistance-breaking (RB) strains, and determined the RB trait linked to amino acid changes in RNA3. The Salinas lab has also described two other soilborne viruses of sugarbeet (Beet Soilborne Mosaic Virus (BSBMV) and beet oak-leaf virus (BOLV)) that interact with BNYVV under field conditions. Identified, in 2011, two sugarbeet breeding lines with resistance to the P. betae. Additional studies have genetically characterized soilborne viruses of lettuce associated with the diseases, lettuce dieback (tombusviruses) and lettuce big vein (Mirafiori lettuce big vein virus; MLBVV), and examined epidemiological factors contributing to control and spread. Collaborative efforts with sugarbeet and vegetable breeding programs in Salinas have facilitated development of genetic resistance to all three soilborne viruses (BNYVV, Tombusviruses and MLBVV) and continue to advance methods for disease management and development of resistance sources. The Salinas lab identified crop and weed reservoirs of curtoviruses, causative agents of curly top disease, is examining factors that drive changes in curtovirus population structure in the western US, and is working toward release of novel methods for their control on several crops. Additionally, studies on emerging viruses led to biological and molecular characterization of a wide array of viruses, and development of detection tools and management methods. Significant Activities that Support Special Target Populations Hosted Los Padres Elementary School fifth grade students, June 1, 2011 and discussed �Rhizomania disease of sugar beet�. Career Day Presentation at Washington Middle School, Salinas, CA, providing knowledge of career options to 7th and 8th grade students on Apr. 27, 2011. �What it�s like to be a plant pathologist and scientist with the USDA-ARS.� Provided internships and training to two science 8th grade students from Washington Middle School (Salinas, CA), including hands-on experience with basic molecular biology methods including nucleic acid extraction, reverse transcription PCR, and agarose gel analysis. Accomplishments 01 Resistance in sugar beet against Polymyxa betae, the causal agent of rhizomania disease. Rhizomania, caused by Beet necrotic yellow vein vir (BNYVV), is one of the most destructive diseases of sugar beet. BNYVV is exclusively transmitted by the soil-borne microorganism Polymyxa betae. genes for resistance to P. betae can be identified in sugar beet these m reduce BNYVV infection and lead to a decrease in incidence and potential for the virus to overcome other forms of BNYVV resistance. The ARS Virology Lab in Salinas, CA used quantitative (real-time) reverse transcription-polymerase chain reaction (qRT-PCR) analysis to determine levels of P. betae colonization of sugarbeet roots, identifying two suga beet breeding lines with reduced populations of P. betae. These lines ar potential candidates in the search for resistant gene (s) to P. betae. 02 Rapid identification and differentiation of both known and unknown crinivirus species. Criniviruses have emerged as a serious threat to vegetable and fruit production throughout the world but their diagnosis challenging. ARS scientists in Salinas, CA developed a method based on t polymerase chain reaction to identify and discriminate known criniviruse in single or mixed crinivirus infections. Unknown criniviruses can be detected, based on areas of sequence conservation among members of the genus. The method provides an easy, single reaction method for rapid identification of crinivirus infections. 03 Development of methods for lab-based evaluation of lettuce for resistanc to lettuce dieback disease. Lettuce dieback disease, caused by the tombusviruses, Tomato bushy stunt virus (TBSV) and the closely related a Lettuce necrotic stunt virus (LNSV), results in necrosis, stunting and death of lettuce plants throughout all western lettuce production region To date no effective methods have been developed for greenhouse evaluati of lettuce germplasm for resistance to this disease since symptoms do no develop readily in greenhouses or growth chambers. The ARS Virology Lab Salinas, CA found that treatment of lettuce with long-days and high temperatures was found to induce consistent disease symptoms. Efficient disease assays in the laboratory will lead to more rapid development of dieback-resistant lettuce. 04 First to identify a novel carmovirus - Honeysuckle ringspot virus - isolated from honeysuckle. A virus with spherical particles 29 to 31 nm in diameter was isolated from honeysuckle plants exhibiting purple ringspots, and has been tentatively named as Honeysuckle ringspot virus (HnRSV). Virus associated double stranded (ds)RNA was isolated from infected honeysuckle leaves, and consistently revealed one major band of about 4.0 kb and two subgenomic RNAs of approximately 1.7 and 1.3 kb, resembling a carmovirus. The complete genome sequence of HnRSV has been sequenced, and its organization, amino acid sequence and taxonomic analysis indicate HnRSV should be classified as a new species in the gen Carmovirus, family Tombusviridae. Knowledge of virus sequence and taxono facilitates detection and monitoring for this new virus in horticultural and vegetable crops to determine its impact on agriculture in California and other states. 05 Beet oak-leaf virus may suppress Beet necrotic yellow vein virus during mixed infections of sugarbeet. Beet oak-leaf virus (BOLV) is transmitte by the fungus Polymyxa betae, and has many structural features resemblin Beet necrotic yellow vein virus BNYVV, yet BOLV produces only sporadic a mild symptoms on sugarbeet, indicating it is much milder than BNYVV. ARS scientists in Salinas, CA have demonstrated that BOLV is serologically distinct from BNYVV, and can suppress BNYVV during mixed infections. Thi means that BOLV may be useful in either cross-protection or more likely engineered resistance as a means to suppress BNYVV and prevent symptoms rhizomania disease. 06 Biological characterization and complete genomic sequence of Celery mosa virus. Celery mosaic virus (CeMV), Apium virus Y (ApVY), and Carrot vir Y (CarVY) are important viral pathogens of celery, cilantro and carrot, respectively, but it was not clear whether these should be considered separate virus species or strains of a single species. Comparisons of co protein sequences available in the GenBank did not clearly separate CeMV from ApVY or CarVY. ARS researchers at Salinas, California reported the host range, aphid transmission, serological reactions, as well as the complete genomic sequence of CeMV. Their research provided conclusive evidence that CeMV is a distinct species in the genus Potyvirus from either ApVY or CarVY. This knowledge will facilitate improved detection technologies by providing sequence information necessary for development of molecular methods to differentiate these three closely related specie as well as control through knowledge of host and vector relationships. 07 Protein changes associated with ability of Beet necrotic yellow vein vir (BNYVV) to overcome the sugarbeet Rz1 resistance gene. Rhizomania, caus by BNYVV, is widely distributed in most sugarbeet growing areas of the world, and can result in complete loss of crop in the absence of resista varieties. New BNYVV variants overcome the most common source of resistance that is controlled by the Rz1 gene. BNYVV pathotype IV, a resistance-breaking pathotype from California�s Imperial Valley, overcom the Rz1 resistance gene, but is unable to infect and cause disease on beets containing the Rz2 resistance gene. The ARS Virology Lab in Salina CA identified proteins associated with infection by Rz1 resistance- breaking variants pathotype IV. These studies will lead to methods to prolong the longevity of Rz resistance sources by understanding the fundamental mechanisms that cause resistance to break down. 08 Molecular and biological characterization of Carrot thin leaf virus (CTL infecting cilantro plant. CTLV was isolated from a commercial cilantro field in California by ARS virologists in Salinas. The CTLV isolate was also shown to infect anise, carrot, celery, chervil, dill, and parsley. The complete genomic sequence of CTLV was determined to be 9,491 nucleotides, excluding the 3� poly (A) tail, and its genomic organizatio is typical of potyviruses. This is the first complete genomic sequence determined for CTLV, and comparisons with available genomic sequences of other potyviruses indicate that CTLV is genetically distinct from Carrot virus Y (CarVY) and several CarVY-related potyviruses. This information clarifies relationships among vegetable and herb-infecting potyviruses, and will facilitate correct virus identification in international commer 09 Diodia vein chlorosis virus is tranmitted by two whitefly species and ha a limited host range. Several whitefly-transmitted viruses in the genus Crinivirus, have emerged in agricultural crops in the past two decades, many of which infect small fruit crops, specifically strawberry and blackberry. Diodia vein chlorosis virus (DVCV) is an understudied crinivirus closely related to other criniviruses viruses known to infect blackberry as well as vegetable crops. The ARS Virology Lab in Salinas, evaluated host range among hosts known to harbor viruses closely related to DVCV, and confirmed transmission by a second whitefly species, Trialeurodes vaporariorum. Results demonstrated a very narrow host range for this virus and suggest it is not likely a serious pathogen of agricultural crops, but determined that like some other members of the Crinivirus genus, it can be transmitted with differing efficiency by mor than one whitefly species. 10 Wild and cultivated reservoir hosts of Cucurbit yellow stunting disorder virus (CYSDV) in US desert Southwest. CYSDV is a recently emerged virus affecting production in the US desert Southwest, Florida and Texas. The ARS Virology Lab in Salinas, CA recently determined that CYSDV infects a much broader range of crop and weed plants than was previously believed, infecting species in 7 families in addition to Cucurbitaceae. Scientists at the ARS virology lab continue to identify new hosts and are examining which of these new hosts are most significant agriculturally for transmission of virus to crops by its whitefly vector, Bemisia tabaci. Additionally, studies have examined the distribution and prevalence of CYSDV and other viruses in crop and weed hosts in the desert production region. The Virology Lab works closely with the melon breeding program a the USDA-ARS in Salinas, CA in evaluating a new source of resistance in melon, and is actively involved in educating growers and developing management tactics to minimize losses.
Impacts
(N/A)
Publications
- Wintermantel, W.M., Hladky, L.L. 2010. Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR. Journal of Virological Methods. 170(1-2):106- 114.
- Wintermantel, W.M. 2010. Transmission efficiency and epidemiology of criniviruses. Bemisia: Bionomics and Management of a Global Pest. DOI 10. 1007/978-90-481-2460-2_10.
- Gulati Sakhuja, A.N., Liu, H. 2011. Infectious full-length clones of Calibrachoa Mottle Virus (CbMV). Journal of Antivirals and Antiretrovirals. 3(1): 001-007.
- Xu, D., Liu, H., Li, F., Li, R. 2011. Complete genome sequence of Celery mosaic virus and its relationship to other members of the genus Potyvirus. Archives of Virology. 156:917-920.
- Xu, D., Liu, H., Koike, S.T., Li, F., Li, R. 2010. Biological characterization and complete genomic sequence of Apium virus Y infecting celery. Virus Research. 155:76-82.
- Wintermantel, W.M. 2011. A comparison of disinfectants to prevent spread of potyviruses in greenhouse tomato production. Plant Health Progress. DOI: 10.1094/PHP-2011-0221-01-RS.
- Tzanetakis, I.E., Wintermantel, W.M., Poudel, B., Zhou, J. 2011. Diodia vein chlorosis virus is a group-1 crinivirus. Archives of Virology. DOI:10. 1007/s00705-011-1055-3.
- Gulati Sakhuja, A.N., Rains, L., Tian, T., Liu, H. 2011. The complete nucleotide sequence and genome organization of a novel carmovirus - Honeysuckle ringspot virus isolated from honeysuckle. Archives of Virology. 156:1635-1640.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. Approach (from AD-416) Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with BNYVV, the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. Replaces 5305-22000-010-00D (3/07). The Salinas lab maintains a collection of bipartite, whitefly-transmitted Criniviruses, and is recognized internationally as a leader in the study of this genus of viruses. The lab has identified and characterized most criniviruses currently known to infect sugarbeet and vegetable crops. Rapid-detection methods have been developed through this project and submitted for publication this year for differentiation of most members of the genus. The method will provide diagnostic labs throughout the world and extension personnel with tools to rapidly differentiate viruses that may appear similar, and should identify related but uncharacterized viruses related to members of the genus. Research is continuing toward understanding epidemiology and transmission parameters for this emerging genus, including Cucurbit yellow stunting disorder virus (CYSDV), an emergent crinivirus severely impacting cucurbit production in the US. The Salinas Virology Lab works closely with grower organizations and extension personnel, advising other scientists throughout the world and regulatory authorities in determining the extent of infection, educating growers and developing management tactics to minimize losses, and to identify hosts serving as sources for virus transmission. Research by the ARS Virology Lab in Salinas on Beet necrotic yellow vein virus (BNYVV) which causes rhizomania, and its vector, the soil-borne fungus, Polymyxa betae, has been critical to understanding this disease. The lab developed specific and sensitive diagnostic assays, and research led to taxonomic reclassification of some sugar beet viruses. The lab recently described a new strain of BNYVV that overcomes Rz1 gene resistance, and has linked the resistance-breaking trait to amino acid changes in RNA3. Ongoing research is focused on clarifying how BNYVV infection and host resistance alter protein expression in sugarbeet, and utilizing this information to block virus infection and disease development. The Salinas Lab also recently described another soil-borne virus of sugarbeet, Beet oak leaf virus (BOLV) that may interact with BNYVV under field conditions. Additional studies have genetically characterized soilborne viruses of lettuce associated with the diseases, lettuce dieback (two tombusviruses, one described by ARS Salinas) and lettuce big vein (Mirafiori lettuce big vein virus; MLBVV), and examined epidemiological factors contributing to control and spread. Collaborative efforts with sugarbeet and vegetable breeding programs in Salinas have facilitated development of genetic resistance to all three soilborne viruses (BNYVV, Tombusviruses and MLBVV). Curly top, transmitted by the beet leafhopper (Circulifer tenellus) and caused by Beet curly top virus (BCTV) and related curtovirus species, has reduced yields of sugar beet and vegetables since the late 1800s. The Salinas Lab has identified crop and weed reservoirs, is examining factors that drive changes in curtovirus population structure in the western US, and is working toward release of novel methods for control across several crops. Accomplishments 01 Production of polyclonal antibodies against expressed coat proteins of Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TIC TICV and ToCV result in losses for tomato production in Florida, California, Mexico, the Mediterranean and other production regions throughout the world, but reliable, inexpensive serological detection methods have been lacking. TICV and ToCV coat proteins were expressed an purified using affinity columns, and used for production of polyclonal antiserum in rabbits for each virus. The antiserum developed by ARS researchers at Salinas, CA is effective for serological detection and differentiation of each virus using specific ELISA methods. Further development and refinement of detection with additional techniques will facilitate use of these antisera for detection of these viruses througho the world. 02 Infectious RNA transcripts derived from cloned cDNA of Calibrachoa Mottl Virus (CbMV). A new virus causing leaf mottling, chlorotic blotch and interveinal yellowing symptoms on Calibrachoa, an important new horticultural plant both in Europe and the United States was recently identified by ARS researchers at Salinas, California. The causal agent o this disease was named Calibrachoa mottle virus (CbMV). The ARS virolog lab at Salinas sequenced the entire virus genome. In order to confirm th CbMV sequence, an infectious full-length cDNA clone of CbMV was constructed and demonstrated to be infectious when RNA generated from th clone was inoculated to indicator plants. Calibrachoa is an important ne horticultural plant in Europe and in the United States. ARS researchers Salinas, California identified a new carmovirus, Calibrachoa mottle viru (CbMV), infecting Calibrachoa plants and the entire CbMV genome has been sequenced. In order to gain deeper insight into their gene organization and expression, the ARS virology lab at Salinas constructed the full- length of inifectious cDNA clone of CbMV. Successful construction of ful length infectious cDNA clone of CbMV makes it possible to develop molecular tools that can be used to understand the gene functions of thi virus. 03 Biological characterization and complete genomic sequence of Apium virus infecting celery. Apium virus Y (ApVY) was first identified in celery plants in California in 2007 by ARS researchers at Salinas, California. Comparisons of the coat proteins did not clearly separate ApVY from Cele mosaic virus (CeMV) and Carrot virus Y (CarVY) as distinct species. Thes three potyviruses are important viral pathogens of celery, cilantro and carrot. ARS researchers at Salinas, California reported the host range, aphid transmission, serological reactions and complete genomic sequence the celery isolate of ApVY. These results provide conclusive evidence th ApVY is a distinct species in the genus Potyvirus. 04 Emergence and epidemiology of Cucurbit yellow stunting disorder virus (CYSDV) in the US Desert Southwest. In the fall of 2006 CYSDV was first identified in California, Arizona and adjacent areas of Sonora, Mexico, significantly reducing yields and now fall planted acreage. The ARS virology lab in Salinas has monitored this virus over three seasons sinc emergence and mapped its movement and infection of alternate hosts, alon with whitefly population and dispersal patterns throughout the region, with cooperation from state and county personnel. Knowledge generated illustrates a virus that rapidly established and entrenched in this important melon production region. Valuable information is being relayed to production agriculture for improved disease management. 05 Identification of wild and cultivated reservoir hosts of Cucurbit yellow stunting disorder virus (CYSDV) in US Desert Southwest. CYSDV is a recently emerged virus affecting production in the US Desert SW, Florida and Texas, and the ARS Virology Lab in Salinas, CA has recently determin CYSDV infects a much broader range of crop and weed plants than was previously believed, infecting species in 7 families in addition to Cucurbitaceae. Studies at the ARS virology lab in Salinas have continue to identify new hosts and are examining which of these new hosts are mos significant agriculturally for transmission of virus to crops by its whitefly vector, Bemisia tabaci. The Virology Lab works closely with th melon breeding program at the USDA-ARS in Salinas, CA in evaluating a ne source of resistance in melon, and is actively involved in educating growers and developing management tactics to minimize losses. 06 First identified Tobacco rattle virus in spinach in California. In 2009, commercially grown spinach exhibited symptoms of a previously unrecogniz virus-like disease in California, with symptoms consisting of general chlorosis, bright yellow blotches and spots. Symptomatic plants were unmarketable and were not harvested. ARS researchers at Salinas, California using a series of diagnostic tools including electron microscopy, serological, and molecular analyses, identified the causal virus as Tobacco rattle virus (TRV). This is the first report of TRV in spinach in California. 07 First identification of Beet necrotic yellow vein virus infecting spinac in California: a possible new threat to spinach production in the state. In 2009, spinach plants from fields in California exhibited vein clearin mottling, interveinal yellowing and stunting symptoms. ARS researchers a Salinas, California using electron microscopy, serological and molecular analyses identified the causal agent as Beet necrotic yellow vein virus (BNYVV). BNYVV is transmitted by spores of a soil-inhabiting fungus, and is primarily known for causing rhizomania, one of the most devastating diseases of sugarbeet in the world. This fungal vector was widely distributed in the field. BNYVV could be a new threat to spinach production in the state.
Impacts
(N/A)
Publications
- Gulati Sakhuja, A.N., Liu, H. 2010. Complete Nucleotide Sequence and Genome Organization of Calibrachoa Mottle Virus (CbMV) - a new Species in the Genus Carmovirus, Family Tombusviridae. Virus Research. 147:216-223.
- Koike, S.T., Tian, T., Liu, H. 2010. First report of Tobacco rattle virus in spinach in California.. Plant Disease. 94-125.
- Gulati Sakhuja, A.N., Sears, J.L., Nunez, A., Liu, H. 2009. PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST PELARGONIUM ZONATE SPOT VIRUS COAT PROTEIN EXPRESSED IN ESCHERICHIA COLI AND APPLICATION FOR IMMUNODIAGNOSIS. Journal of Virological Methods. 160: 29-37.
- Wintermantel, W.M., Hladky, L.L., Gulati Sakhuja, A.N., Li, R., Liu, H., Tzanetakis, L.E. 2009. The complete nucleotide sequence and genome organization of Tomato infectious chlorosis virus: A distinct crinivirus most closely related to Lettuce infectious yellows virus. Archives of Virology. 154:1335-1341.
- Liu, H., Mou, B., Richardson, K.L., Koike, S.T. 2010. First report of Beet necrotic yellow vein virus infecting spinach in California. Plant Disease. 94:640.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. Approach (from AD-416) Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with BNYVV, the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. (IBC info pending). Replaces 5305-22000-010-00D (3/07). Significant Activities that Support Special Target Populations The Salinas lab maintains a collection of bipartite, whitefly-transmitted Criniviruses, and is recognized internationally as a leader in the study of this group of viruses. The lab has identified and characterized most of the criniviruses that are currently known to infect sugarbeet and vegetable crops, and has developed detection methods for most known criniviruses. Studies published this year have demonstrated that competition between viruses impacts the efficiency of crinivirus transmission differently depending on the host plant and is influenced by virus accumulation in the host plant. In the fall of 2006 a new crinivirus, Cucurbit yellow stunting disorder virus (CYSDV) emerged for the first time in the U.S. Desert Southwest. Our program has been actively assisting other laboratories in acquiring the tools to monitor for this virus, working with grower organizations, extension personnel, other scientists and regulatory authorities in determining the extent of infection, educating growers and developing management tactics to minimize losses. A manuscript published this year demonstrated that the host range of CYSDV is much broader than was previously believed, and several weed hosts can serve as reservoirs for transmission of the virus back to cucurbits. Beet necrotic yellow vein virus (BNYVV) which causes rhizomania, and its vector, the soil-borne fungus, Polymyxa betae, were detected for the first time in the Western Hemisphere by the Salinas Virology Lab. Research on BNYVV and related viruses has been critical to understanding of the disease and facilitating development by the lab of highly specific and sensitive diagnostic assays. Results from studies of soil-borne sugarbeet viruses led to taxonomic reclassification of some viruses. The laboratory recently described a new strain of BNYVV that overcomes Rz1 gene resistance, that differs from other resistance-breaking (RB) strains by having only the standard 4 RNAs, rather than a fifth as is present in RB strains from Europe and Asia. Recent work by the Salinas Lab has linked the RB trait to amino acid changes in RNA3. The Salinas lab has also described two other soilborne viruses of sugarbeet (BSBMV and BOLV) that compete and interact with BNYVV under field conditions. Additional studies have genetically characterized soilborne viruses of lettuce associated with the diseases, lettuce dieback (two tombusviruses, one described by ARS Salinas) and lettuce big vein (Mirafiori lettuce big vein virus; MLBVV), and examined epidemiological factors contributing to control and spread. Collaborative efforts with sugarbeet and vegetable breeding programs in Salinas have facilitated development of genetic resistance to all three soilborne viruses (BNYVV, Tombusviruses and MLBVV) . Technology Transfer Number of Active CRADAS: 1 Number of Other Technology Transfer: 4
Impacts
(N/A)
Publications
- Wintermantel, W.M., Cortez, A.A., Anchieta, A.G., Gulati Sakhuja, A.N., Hladky, L.L. 2008. Co-infection by two criniviruses alters accumulation of each virus in a host-specific manner and influences efficiency of virus transmission. Phytopathology 98: 1340-1345.
- Wintermantel, W.M., Hladky, L.L., Cortez, A.A., Natwick, E.T. 2009. A new expanded host range of Cucurbit yellow stunting disorder virus includes three agricultural crops. Plant Disease 93: 685-690.
- Liu, H., Sears, J.L., Mou, B. 2009. Spinach: A new natural host of Impatiens necrotic spot virus in California.. Plant Disease 93:673, 2009
- Ling, K., Wintermantel, W.M., Bledsoe, M. 2008. Genetic Composition of Pepino mosaic virus Population in North American Greenhouse Tomatoes. Plant Disease. 92:1683-1688.
- Strausbaugh, C.A., Wintermantel, W.M., Gillen, A.M., Eujayl, I.A. 2008. Curly top survey in the Western United States. Phytopathology. 98(11):1212- 1217.
- Liu, H. 2009. BEET CHLOROSIS. Compendium of the Beet Diseases and Pests, 2nd Edition, P. 49-50, APS Press, St. Paul, MN.
- Liu, H. 2009. BEET LEAF CURL. Compendium of the Beet Diseases and Pests, 2nd Edition, P 54-55 APS Press, St. Paul MN.
- Liu, H. 2009. BEET YELLOW NET. Compendium of the Beet Diseases and Pests, 2nd Edition, APS Press, St. Paul, MN. p. 57-58.
- Liu, H. 2009. BEET YELLOW STUNT. Compendium of the Beet Diseases and Pests. 2nd Edition, P 50-51, APS Press, St. Paul, MN.
- Liu, H. 2009. LETTUCE INFECTIOUS YELLOWS. Compendium of the Beet Diseases and Pests, 2nd Edition, P. 55-56, APS Press, St. Paul MN.
- Liu, H. 2009. BEET SAVOY. Compendium of the Beet Diseases and Pests, 2nd Edition, P. 55, APS Press, St. Paul, MN.
- Wintermantel, W.M. 2009. BEET CURLY TOP VIRUS. In: Compendium of Beet Diseases and Pests. 2nd Ed., R.M. Harveson and L.E. Hanson, eds., APS Press, St. Paul, MN. pp 51-53.
- Wintermantel, W.M. 2009. BEET YELLOWS VIRUS. In: Compendium of the Beet Diseases and Pests. 2nd Ed., R.M. Harveson, eds., APS Press, St. Paul, MN. pp 46-47.
- Wintermantel, W.M. 2009. BEET MILD YELLOWING VIRUS. In:Compendium of the Beet Diseases and Pests.2nd Ed., R.M. Harveson and L.E. Hanson, eds., APS Press, St. Paul, MN. p 50.
- Wintermantel, W.M. 2009. CUCUMBER MOSAIC VIRUS. In: Compendium of the Beet Diseases and Pests. 2nd Ed., R.M. Harveson and L.E. Hanson, eds.,APS Press, St. Paul, MN. p 53.
- Liu, H. 2009. BEET YELLOW VEIN. Compendium of the Beet Diseases and Pests, 2nd Edition, P. 57, APS Press, St. Paul, MN.
- Tian, T., Liu, H., Koike, S. 2008. First Report of Apium Virus Y on Cilantro, Celery, and Parsley in California. Plant Disease 92:1254.
- Wintermantel, W.M. 2009. BEET MOSAIC VIRUS. In: Compendium of the Beet Diseases and Pests. 2nd Ed., R.M. Harveson and L.E. Hanson, eds., APS Press St. Paul, MN. pp 53-54.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. Approach (from AD-416) Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with BNYVV, the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. (IBC info pending). Replaces 5305-22000-010-00D (3/07). Significant Activities that Support Special Target Populations The Salinas lab maintains a collection of bipartite, whitefly-transmitted Criniviruses, and is recognized internationally as a leader in the study of this group of viruses. The lab has identified and characterized and most of the criniviruses that are currently known to infect sugarbeet and vegetable crops, and has developed detection methods for most known criniviruses. Studies recently completed (in press) have demonstrated that competition between viruses impacts the efficiency of crinivirus transmission differently depending on the host plant. In the fall of 2006 a new crinivirus, Cucurbit yellow stunting disorder virus (CYSDV) emerged for the first time in the US Desert Southwest. Our program has been actively assisting other laboratories in acquiring the tools to monitor for this virus, working with grower organizations, extension personnel, other scientists and regulatory authorities in determining the extent of infection, educating growers and developing management tactics to minimize losses. Beet necrotic yellow vein virus (BNYVV) which causes rhizomania , and its vector, the soil-borne fungus, Polymyxa betae, were detected for the first time in the Western Hemisphere by the Salinas Virology Lab. Research on BNYVV and related viruses has been critical to understanding of the disease and facilitating development by the lab of highly specific and sensitive diagnostic assays. Results from studies of soil-borne sugarbeet viruses led to taxonomic reclassification of some viruses. The laboratory recently described a new strain of BNYVV that overcomes Rz1 gene resistance, that differs from other resistance-breaking (RB) strains by having only the standard 4 RNAs, rather than a fifth as is present in RB strains from Europe and Asia. Recent work by the Salinas Lab has linked the RB trait to amino acid changes in RNA3. The Salinas lab has also described two other soilborne viruses of sugarbeet (BSBMV and BOLV) that compete and interact with BNYVV under field conditions. Additional studies have genetically characterized soilborne viruses of lettuce associated with the diseases, lettuce dieback (two tombusviruses, one described by ARS Salinas) and lettuce big vein (Mirafiori lettuce big vein virus; MLBVV), and examined epidemiological factors contributing to control and spread. Collaborative efforts with sugarbeet and vegetable breeding programs in Salinas have facilitated development of genetic resistance to all three soilborne viruses (BNYVV, Tombusviruses and MLBVV) . Curly top, transmitted by the beet leafhopper (Circulifer tenellus) and caused by Beet curly top virus (BCTV) and related curtovirus members of the genus, Curtovirus, has impacted yields of sugar beet and vegetables since the late 1800s. The Salinas lab has identified crop and weed reservoirs, characterized changes in curtovirus population structure and determined this is likely influenced by environmental and cropping changes, and is developing novel methods for control. This relates to NP 303, Component 4. Technology Transfer Number of Active CRADAS: 1
Impacts
(N/A)
Publications
- Liu, H., Lewellen, R.T. 2008. Suppression of Resistance-breaking Beet Necrotic Yellow Vein Virus Isolates by Beet Oak-leaf Virus in Sugar Beet.. Plant Disease 92:1043-1047
- Polston, J.E., Hladky, L.L., Akad, F., Wintermantel, W.M. 2008. First Report of Cucurbit Yellow Stunting Disorder Virus in Cucurbits in Florida. Plant Disease. 92:1251
- Wintermantel, W.M., Hladky, L.L. 2008. Resistance to curly top viruses through virus induced gene silencing. Phytopathology. 92:S172
- Larson, R.L., Wintermantel, W.M., Hill, A.L., Fortis, L.L., Nunez, A. 2008. Proteome changes in sugar beet in response to Beet necrotic yellow vein virus. Physiological and Molecular Plant Pathology. doi:10.1016/j.pmpp. 2008.04.003
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. Approach (from AD-416) Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with BNYVV, the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. (IBC info pending). Replaces 5305-22000-010-00D (3/07). Significant Activities that Support Special Target Populations This project began in April 2007, information from the first 3 months will be presented in the 2008 Project Report when accomplishments are more complete. Accomplishments Identification and Management of Cucurbit Yellow Stunting Disorder Virus (CSYDV) in the American Desert Southwest: In the fall of 2006 a new crinivirus, Cucurbit yellow stunting disorder virus (CYSDV) was identified for the first time in the southwestern desert melon production areas of California and Arizona, by scientists from the USDA-ARS Virology Lab, as well as scientists from the University of California-Davis and the University Arizona. The USDA Virology lab has developed a broad array of detection methodologies to accurately identify and confirm infection, both advanced and early stage infections, that are being used by an increasing number of labs throughout the U.S. We are monitoring spring and fall crops for CYSDV incidence and associated incidence of the whitefly vector, B. tabaci. Our lab is working closely with state and county personnel to determine incidence (nearly 100% in fall 2006, but lower in the spring 2007 crop, as expected) and distribution. Our lab is conducting host range studies on regional crops and weeds, working closely with the melon breeding program at the USDA-ARS in Salinas, CA to evaluate a putative new source of resistance in melon, and is actively involved in educating growers and developing management tactics to minimize losses. All research described above is being conducted by the USDA-ARS Virology Lab in Salinas. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2C: Population Dynamics, Spread, and Epidemiology of Pathogens. Distribution and Molecular Analysis of Resistance-breaking Isolates of Beet Necrotic Yellow Vein Virus (BNYVV) in the United States: The economically devastating rhizomania disease of sugarbeet is caused by the soil-borne, Polymyxa betae-transmitted BNYVV, and can only be controlled effectively by the use of resistant cultivars. We determined that new resistance-breaking BNYVV isolates, first identified in 2003-2004 in the Imperial Valley of California, likely evolved from the existing A- pathotype based on single-strand conformation polymorphism and sequence analyses, and have identified amino acid changes unique to the resistance breaking isolates. Soil surveys recently completed indicated that the resistance-breaking isolates not only exist in the Imperial Valley, but also throughout many of the major U.S. sugarbeet production regions. The identification of resistance-breaking isolates from throughout the U.S. demonstrates that selection pressure resulting from widespread planting of Rz1 resistance is driving emergence of resistance-breaking BNYVV strains, and illustrates the urgency for developing new sources or methods of resistance. All work performed by the USDA-ARS Virology Lab in Salinas, CA. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Characterization of Lettuce Chlorosis Virus and Development of Efficient Molecular Detection: Lettuce chlorosis virus (LCV), family Closteroviridae, genus Crinivirus, emerged as a threat to lettuce and sugar beet production in the desert regions of the southwestern U.S. in the early 1990�s, producing interveinal yellowing, stunting, and brittleness of affected leaves. We (the USDA-ARS Virology Lab in Salinas, CA) developed molecular probes and LCV-specific RT-PCR primer pairs and demonstrated their efficacy for rapid identification of LCV infected lettuce and sugarbeet plants, and are continuing to characterize LCV on a molecular level. Results of our studies are providing industry with the diagnostic tools necessary for early identification, facilitating more effective disease control and decreasing losses. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen biology, Virulence Determinants, and genetics of the Pathogen. First Report of Pelargonium Zonate Spot Virus from Tomato in the United States: Pelargonium zonate spot virus (PZSV) was first isolated from tomato in southern Italy in 1982 and has since been identified in France and Spain. In June of 2006, more than 100 tomato (Lycopersicon esculentum Mill.) plants exhibiting symptoms of stunting, malformation, yellow rings and line patterns on the leaves, and concentric chlorotic ringspots on the stems, similar to those of PZSVwere observed in 7 acres of tomato in Yolo County, California. The causal agent was mechanically transmitted to several indicator species. Two field infected tomato plants and one each of the mechanically inoculated host plant were positive with the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using a commercial PZSV IdentiKit (Neogen Europe Ltd., Scotland, UK). Partially purified virions stained with 2% uranyl acetate contained spherical to ovate particles ranging between 25 to 35 nm. Published sequences of PZSV were used to design 3 sets of primer pairs specific for PZSV RNAs 1 through 3. Total nucleic acids were extracted from field infected tomato plants. Amplicons had 92%, 94%, and 96% nucleotide sequence identity to PZSV RNA1, RNA2 and RNA3, respectively. The symptomatology, serology, particle morphology, and nucleotide sequences confirm the presence of PZSV in a tomato field in California, and we have developed tools for the rapid detection of this virus in the United States. All work performed by the USDA-ARS Virology Lab in Salinas, CA. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Development of Novel Sources of Resistance to Beet Severe Curly Top Virus (BSCTV) in Sugarbeet and Tomato: BSCTV and related curtoviruses are responsible for severe losses in numerous crops each year, including tomato and sugarbeet. Current studies are focused on development of virus induced gene silencing (VIGS) to obtain complete resistance against BSCTV and other curtoviruses in tomato and sugarbeet, and have identified two types of constructs capable of significantly reducing virus concentrations in greenhouse experiments using two independent delivery systems. Additional studies are continuing in efforts to obtain complete resistance in both crops. The ability to elicit VIGS for control of curtoviruses will provide the vegetable and sugarbeet industries with alternative and potentially more effective control methods, reducing the need for excessive pesticide application. All work performed by the USDA- ARS Virology Lab in Salinas, CA. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Subtractive Proteomics to Characterize Resistance and Susceptibility of Sugarbeet to Beet Necrotic Yellow Vein Virus (BNYVV): New studies are using subtractive proteomics to identify proteins induced in resistant and susceptible sugarbeet (Beta vulgaris) as a result of infection with BNYVV. Studies conducted at the Sugarbeet Research Unit at the USDA-ARS in Ft. Collins, CO in collaboration with the Virology Lab at the USDA-ARS in Salinas, CA have identified differences in protein expression in susceptible sugarbeet among healthy sugarbeet, sugarbeet exposed to the virus-free vector of BNYVV, Polymyxa betae, and BNYVV infected sugarbeet. In vitro expression of BNYVV proteins is in progress for use in protein interaction arrays to identify functional interactions between virus and host plant. Results should elucidate physiological and biochemical changes that differ between healthy and BNYVV infected sugarbeet, leading to targeted methods to prevent BNYVV from eliciting symptoms of rhizomania disease. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2B: Plant-Microbe- Vector Interactions. Characterization of a U.S. Isolate of Beet Black Scorch Virus: The first reported U.S. isolate of Beet black scorch necrovirus (BBSV) was obtained and characterized by the USDA-ARS Virology Lab in Salinas, CA in collaboration with the USDA-ARS in Fargo, ND. The complete nucleotide sequence of the genomic RNA of the virus, designated BBSV-Co, exhibits 93% similarity to the genome of the �Ningxia� isolate of BBSV from China, with higher similarity for individual proteins (up to 97%), and it was determined that the U.S. isolate of BBSV may contain and additional gene absent from the Chinese isolate. Analysis of the coat protein by isoelectric focusing and by mass spectroscopy indicates the presence of phosphorylated resides, and it was determined that the American isolate lacked the two terminal adenosine nucleotides in the published sequences of BBSV from China. Rabbit anti-BBSV antiserum with high sensitivity was produced from a purified preparation of the virus. Results will benefit detection and monitoring for this virus in U.S. beet producing regions, and knowledge of its relationship to international BBSV isolates. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen biology, Virulence Determinants, and genetics of the Pathogen. Technology Transfer Number of Active CRADAS and MTAS: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 4 Number of Newspaper Articles,Presentations for NonScience Audiences: 2
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