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United States Department of Agriculture

Agricultural Research Service

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Location: Crop Improvement and Protection Research

2010 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. 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 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.

4. Accomplishments
1. Production of polyclonal antibodies against expressed coat proteins of Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV). 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 and 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 throughout the world.

2. Infectious RNA transcripts derived from cloned cDNA of Calibrachoa Mottle 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 of this disease was named Calibrachoa mottle virus (CbMV). The ARS virology lab at Salinas sequenced the entire virus genome. In order to confirm the CbMV sequence, an infectious full-length cDNA clone of CbMV was constructed and demonstrated to be infectious when RNA generated from the clone was inoculated to indicator plants. Calibrachoa is an important new horticultural plant in Europe and in the United States. ARS researchers at Salinas, California identified a new carmovirus, Calibrachoa mottle virus (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 full length infectious cDNA clone of CbMV makes it possible to develop molecular tools that can be used to understand the gene functions of this virus.

3. Biological characterization and complete genomic sequence of Apium virus Y 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 Celery mosaic virus (CeMV) and Carrot virus Y (CarVY) as distinct species. These 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 of the celery isolate of ApVY. These results provide conclusive evidence that ApVY is a distinct species in the genus Potyvirus.

4. 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 since emergence and mapped its movement and infection of alternate hosts, along 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.

5. 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 determined 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 continued 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. 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.

6. First identified Tobacco rattle virus in spinach in California. In 2009, commercially grown spinach exhibited symptoms of a previously unrecognized 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.

7. First identification of Beet necrotic yellow vein virus infecting spinach in California: a possible new threat to spinach production in the state. In 2009, spinach plants from fields in California exhibited vein clearing, mottling, interveinal yellowing and stunting symptoms. ARS researchers at 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.

Review 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.


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.

Last Modified: 2/23/2016
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