Location: Crop Improvement and Protection Research2011 Annual Report
1a. 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.
1b. 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).
3. Progress Report
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.
1. Resistance in sugar beet against Polymyxa betae, the causal agent of rhizomania disease. Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is one of the most destructive diseases of sugar beet. BNYVV is exclusively transmitted by the soil-borne microorganism Polymyxa betae. If genes for resistance to P. betae can be identified in sugar beet these may 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 sugar beet breeding lines with reduced populations of P. betae. These lines are potential candidates in the search for resistant gene (s) to P. betae.
2. 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 is challenging. ARS scientists in Salinas, CA developed a method based on the polymerase chain reaction to identify and discriminate known criniviruses 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.
3. Development of methods for lab-based evaluation of lettuce for resistance to lettuce dieback disease. 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. To date no effective methods have been developed for greenhouse evaluation of lettuce germplasm for resistance to this disease since symptoms do not develop readily in greenhouses or growth chambers. The ARS Virology Lab in 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.
4. 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 genus Carmovirus, family Tombusviridae. Knowledge of virus sequence and taxonomy facilitates detection and monitoring for this new virus in horticultural and vegetable crops to determine its impact on agriculture in California and other states.
5. Beet oak-leaf virus may suppress Beet necrotic yellow vein virus during mixed infections of sugarbeet. Beet oak-leaf virus (BOLV) is transmitted by the fungus Polymyxa betae, and has many structural features resembling Beet necrotic yellow vein virus BNYVV, yet BOLV produces only sporadic and 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. This means that BOLV may be useful in either cross-protection or more likely engineered resistance as a means to suppress BNYVV and prevent symptoms of rhizomania disease.
6. Biological characterization and complete genomic sequence of Celery mosaic virus. Celery mosaic virus (CeMV), Apium virus Y (ApVY), and Carrot virus 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 coat 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 species, as well as control through knowledge of host and vector relationships.
7. Protein changes associated with ability of Beet necrotic yellow vein virus (BNYVV) to overcome the sugarbeet Rz1 resistance gene. Rhizomania, caused 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 resistant 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, overcomes the Rz1 resistance gene, but is unable to infect and cause disease on beets containing the Rz2 resistance gene. The ARS Virology Lab in Salinas, 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.
8. Molecular and biological characterization of Carrot thin leaf virus (CTLV) 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 organization 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 commerce.
9. Diodia vein chlorosis virus is tranmitted by two whitefly species and has 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, CA 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 more 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 at 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.
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.