Location: Crop Improvement and Protection Research2009 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 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).
1. Expanded host range of Cucurbit yellow stunting disorder virus (CYSD). The CYSD virus was first identified in epidemic proportions in California, Arizona, and adjacent areas of Sonora, Mexico in fall 2006 when it caused severe yield and fruit quality losses to melon crops. ARS scientists in Salinas, California with university collaborators and industry funding found that Cucurbit yellow stunting disorder virus infected a broader range of crops and weeds than was previously known. The virus infects species in seven families in addition to cucurbits. The expanded host range enables the virus to infect Spring-melon crops, which makes it a serious threat to both Spring and Fall melon crops in these areas. This information has been disseminated to growers for development of management tactics to minimize losses.
2. Protein interactions of sugar beet with rhizomania disease. Rhizomania is a major disease of sugar beet worldwide that is caused by the soilborne Beet necrotic yellow vein virus. ARS researchers at Salinas, California, and Ft. Collins, Colorado, expressed proteins of this virus in a model bacterial system. This accomplishment enables examination of functional interactions between virus and host plant proteins involved in virus infection and disease development. Results should identify protein interactions that lead to disease development and that are altered in host resistance. This research will lead to specific and sophisticated methods to prevent rhizomania disease.
3. Specific point mutations affect expression of rhizomania disease of sugarbeets. Rhizomania is a major disease of sugarbeet worldwide that is caused by the soilborne Beet necrotic yellow vein virus. Genetic resistance is available in sugarbeet, but certain strains of the virus overcome the resistance. ARS researchers at Salinas, California in collaboration with researchers in France studied the effect of mutations in RNA3 at amino acids 67 and 68 in Beet necrotic yellow vein virus RNA3 induced different symptoms on two host plant species. DNA sequence variations that enable strains of the virus to overcome or break resistance in sugarbeet produced symptoms distinctly different from those produced by non-resistance breaking strains of the virus. These results demonstrate the significance of point mutations in development of Beet necrotic yellow vein virus symptoms, indicating a potentially important role in the interaction of Beet necrotic yellow vein virus with multiple host plants.
4. Point mutation enables virus to overcome resistant sugarbeets. Rhizomania is a major disease of sugarbeet worldwide that is caused by the soilborne Beet necrotic yellow vein virus. Genetic resistance is available in sugarbeet, but certain strains of the virus overcome the resistance. ARS researchers at Salinas, California, confirmed that strains of Beet necrotic yellow vein virus with specific point mutations in RNA3 overcome both resistant genes in sugarbeet whether or not they were alone or together. All genetic variants of the virus that contained the P25 point mutations were able to infect resistant varieties. This test confirmed the role of these amino acid changes in the ability of Beet necrotic yellow vein virus to overcome rhizomania resistance in sugarbeet.
5. Complete genome sequence of the Crinivirus, Tomato infectious chlorosis virus. Tomato infectious chlorosis virus, transmitted by the greenhouse whitefly is an important disease affecting tomato production in California, Mexico, many parts of Europe and some parts of Asia. ARS researchers at Salinas, California, have determined the complete nucleotide sequence of this virus, and compared it with other members of the genus Crinivirus. Results show this virus is most closely related to Lettuce infectious yellows virus, an older member of the genus genetically distinct from other members. There are thus three distinct crinivirus groups with Tomato infectious chlorosis virus and Lettuce infectious yellow virus classified together in group 3, and the other members of the genus remaining in the previously recognised groups 1 and 2. These results changed the taxonomic structure of the genus crinivirus and will lead to new understanding of host-vector-virus interactions.
6. Transiently-induced silencing of a sugarbeet and tomato virus. Beet Severe Curly Top virus (BSCT) and related curtoviruses are responsible for severe losses in numerous crops each year, including tomato and sugarbeet. ARS researchers at Salinas, California, used virus-induced gene silencing to obtain high-level resistance against BSCT virus and a related curtovirus in tomato and a related tobacco species. This induced resistance to BSCT virus provided disease control well in tomato and a relative of tobacco but there was limited control in sugarbeet. The ability to elicit induced silencing of curtoviruses will provide the vegetable and sugarbeet industries with alternative and potentially more effective disease control methods while reducing pesticide usage.
7. Crinivirus protein expression constructs for protein binding studies. The two criniviruses Tomato chlorosis virus and Tomato infectious chlorosis virus, are transmitted by the greenhouse whitefly and have several host plants in common. Tomato chlorosis virus is, however, also transmitted by other whitefly species. ARS researchers at Salinas, California, cloned genes for selected proteins of these two viruses and expressed them in a model bacterial system for use in whitefly protein binding studies as a method to identify whitefly proteins involved in virus transmission. This accomplishment will lead to new knowledge about vector specificities of criniviruses, an emerging group of plant viruses worldwide. It will also facilitate gaining knowledge of their movement into new areas, their subsequent development, and a means for their control.
8. DNA primers for quantitative studies of curtoviruses. Curly top disease, which is caused by a group of viruses designated curtoviruses, has been a problem on sugarbeet and vegetable crops throughout the western U.S. for many years. ARS researchers at Salinas, California, and Kimberly, Idaho, developed the DNA primers for individual, quantitative Polymerase Chain Reaction (PCR) detection of three curtovirus species. This enabled them to confirm that two curtoviruses are the primary agents responsible for causing curly top disease. Furthermore, quantitative analysis using DNA primers will facilitate studies on the relative competitiveness of each curtovirus within weed and crop hosts, which will lead to knowledge of factors that change curtovirus population structure over time and thus play a role in approaches to prevent viral susceptibility.
9. Widespread emergence of a blackberry virus. Blackberry yellow vein associated virus is a recently recognized crinivirus that poses a threat to blackberry production throughout the U.S. ARS researchers at Salinas, California, characterized the genome of this virus at the molecular level and used this information to determine that it is present in a significant percentage of blackberry varieties. This information enables transmission studies to identify the vector responsible for transmission of this virus and further characterization of factors contributing to its establishment and persistence in the field. It will also help determine minimum duration of vector feeding necessary for transmission and virus host range. This information will help improve management leading to a reduction in virus transmission in the field, as well as knowledge of important reservoir and other host plants that contribute to the domestic and international movement of Blackberry yellow vein associated virus.
10. Polyclonal antiserum for detection and diagnosis of Pelargonium zonate spot virus (PZSV). PZSV is difficult to diagnose but was recently identified in tomato plants in the United States, and found to cause stunted, malformed leaves and stems. ARS researchers at Salinas, California, developed the first polyclonal antiserum for serological detection and diagnosis of this virus. When used in Western blot analysis, the new antiserum detected viral coat protein and whole (intact) virus in infected plant samples, and was effective for indirect ELISA-based virus detection at dilutions of up to 1:16000 without cross reactivity which demonstrated that it is a highly reliable antiserum. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of the virus using serological methods. This research will be of benefit in monitoring the virus in tomato and other hosts.
11. First report of Apium virus Y on cilantro, celery, and parsley in California. Over the past six years, cilantro, celery, and parsley in California have exhibited virus-like symptoms that reduce yield. ARS researchers at Salinas, California, confirmed the presence of a virus in affected samples of these crops. Electron microscopy studies confirmed the presence of flexuous rod-shaped particles in symptomatic tissues. Molecular diagnosis identified Apium virus Y in diseased plants. Apium virus Y was previously identified in celery and weeds in Australia and New Zealand, but to our knowledge this is the first report of this virus on cilantro, celery, and parsley in California.
12. Spinach is a new, natural host of Impatiens necrotic spot virus in California. Monterey County, California ,is a major producer of spinach for fresh consumption. Discovery of severely stunted spinach plants in October 2008 with yellowed, thickened and deformed leaves was considered a possible new threat to spinach production. ARS researchers at Salinas, California, determined the plants were infected with Impatiens necrotic spot virus. The plants were not infected with the closely related, Tomato spotted wilt virus, Cucumber mosaic virus, or Tobacco mosaic virus. This is the first report of natural occurence of Impatiens necortic spot virus infection of spinach in California. Increased prevalence of the vector could provide the means for Impatiens necrotic spot virus to become a yield limiting disease of spinach in California and other parts of the United States. This information will enable growers and extension personnel to be more vigilant of this virus problemm.
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.