2010 Annual Report
1a.Objectives (from AD-416)
Investigate pathogens and diseases of quarantine significance that occur in clonal plant germplasm that must enter the U.S. through federal quarantine programs. Characterize poorly described pathogens and diseases of quarantined crops that limit entry and distribution of plant germplasm. Enhance testing of quarantine crops for known pathogens by developing detection methods that represent improvements in sensitivity, speed, and reliability when compared to currently used procedures. Develop tools to effectively eliminate pathogens of quarantine significance to facilitate the availability of pathogen-free germplasm to stakeholders in a safe and timely fashion. Transfer research data, methods, and protocols to USDA-APHIS to support testing programs and science-based regulatory decisions.
1b.Approach (from AD-416)
Conduct research to identify new methodologies and protocols for diagnostic testing of quarantined plant germplasm, with an emphasis on molecular techniques, to shorten the duration material is held in quarantine and increase the reliability of indexing programs. Determine the etiology of poorly described quarantine diseases using a wide range of greenhouse and laboratory techniques. Conduct molecular characterization studies of quarantine pathogens and investigate their genetic diversity in order to refine testing methods. Develop protocols for the in vitro cultivation of prohibited genera germplasm and the therapeutic elimination of quarantine pathogens, thereby salvaging valuable and sometimes unique and endangered germplasm. Transfer research data, protocols, and products to USDA-APHIS for incorporation into testing and regulatory programs.
A project has been initiated to develop an improved bio-indicator for the USDA small fruit quarantine program. A new indicator for Gooseberry vein-banding associated virus (GVBaV) is needed based on past experiences and discussions with APHIS scientists. Reports have identified cv. Jonkheer van Tets as a superior bio-indicator to the currently used cv. Amos Black. Replicated trials with these two varieties have been inoculated with GVBaV and are being observed for symptom development. A more effective indicator for this virus will help the quarantine program detect and exclude foreign isolates of GVBaV from the United States.
A project has been initiated in collaboration with ARS scientists in Geneva, NY to investigate the effect of cryopreservation on virus elimination from cherry trees infected with several viruses. Plant material had been placed under liquid nitrogen storage conditions at the ARS laboratory in Ft. Collins, CO. Preliminary results showed Cherry Virus A and Prunus Necrotic Ringspot Virus were detected in the plantlets, but not Little Cherry Virus-2, suggesting the effect of the cryopreservation on the virus elimination depends on the virus species. We are currently testing young plants regenerated from in vitro cultures to confirm these results.
A PCR assay using a pair of consensus primers is being tested for the detection of Sugarcane bacilliform virus (SCBV). In collaboration with ARS scientists at Canal Point, FL, the assay was used to test the field samples collected in Florida. Results showed that the infection of the virus was common in sugarcane fields. However, it is not clear if the amplication was from virions or endogenus pararetrovirus and additional inverstigations are needed. SCBV is an important quarantine pathogen in the movement of sugarcane germplasm and improved detection techniques are needed.
Sweet potato chlorotic stunt virus (SPCSV) infects sweet potato and causes severe disease when mix infected with Sweet potato feathery mottle virus. The titer of the virus is low in infected plants. RT-PCR using different primer pairs (either previously published or newly designed) is being tested to detect the virus at different seasons. Our prelimary result confirmed that the best time to detect the virus is when plants are young. The RT-PCR assay will be useful for rapid and reliable detection and diagnosis of these viruses in quarantine, certification and virus survey programs.
To develop a virus eradication protocol, several species of virus-infected small fruits in the genera Ribes and Rubus infected were established in the greenhouse and as in vitro cultures. Media were optimized for Ribes and Rubus to obtain the best growth for a diverse range of species within each genus. Evaluation of pathogen elimination procedures (chemo-, thermo-, and meristem tip culture, singly and in combination) has begun. Plants generated through therapy treatments have tested negative as in vitro cultures and for up to 6 months of growth in the greenhouse. Additional viruses/plant species host combinations are being tested with different treatment routines to determine superior protocols for production of virus-free plants.
Development of a polyprobe to detect six viroids of pome and stone fruit trees. Six viroids have been reported to infect pome and stone fruit trees and all are important quarantine pathogens in the international movement of germplasm. NGRL scientists developed an assay using a non-radioactive probe (polyprobe) to detect all six viroids on a single membrane using dot blot htbridizations. The polyprobe was highly sensitive in detecting the viroids in trees with both single and mixed infections. The assay was validated by testing hundreds of field samples, including samples from the National Plant Germplasm System pome and stone fruit collections. The technology has been transferred to APHIS for possible inclusion in quarantine testing protocols and to a commercial diagnostic company to evaluate under a Material Transfer Agreement.
Virus survey of cherry trees in the national genetic resources. RT-PCR assays were developed or adapted for detecting for the following 12 viruses infecting stone fruits: American plum line pattern virus (APLPV), Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CRMV), Cherry necrotic rusty mottle virus (CNRMV), Cherry rasp leaf virus (CRLV), Cherry virus A (CVA), Little cherry virus 1 (LChV-1), Little cherry virus 2 (LChV-2), Prune dwarf virus (PDV), Plum bark necrotic stem associated virus (PBNSaV), Prunus necrotic ringspot virus (PNRSV) and Tomato ringspot virus (ToRSV). These assays were used in virus survey to evaluate samples collected from cherry trees in the NPGS repositories in Davis, CA and Geneva, NY, the National Mall of the National Park Service, and the U.S. National Arboretum (USNA) in Washington, D.C. Several viruses and different combinations and infection rates were detected in the four locations. Infections of CVA and LChV-2 were common in Geneva, the USNA, and the National Mall. Sequence analyses of the RT-PCR products of the two viruses indicated that the genetic variation of CVA was lower than that of LChV-2. This survey helped to establish the virus infection status of important cherry germplasm collections in the U.S. and to validate protocols that are useful to the APHIS quarantine program.
Complete nucleotide sequence of Gooseberry vein banding associated virus. Gooseberry vein banding associated virus (GVBaV) is an important viral pathogen of Ribes spp. Characterization of the virus is important for developing diagnostic tools for the APHIS quarantine program. In this study, complete genomic sequences of four isolates of GVBaV from different hosts and countries were determined. Analysis of the sequence data shows that this virus is indeed a distinct virus in genus Badnavirus and, unlike most other badnaviruses, the genetic variation among the four isolates is low. The genomic sequences will be useful information for taxonomic comparisons of the virus and in the development of reliable molecular detection methods.
Complete nucleotide sequence of Apium virus Y. Apium virus Y (ApVY) is a recently identified potyvirus infecting several apiaceous crops including celery and cilantro. The host range and vector transmission of ApVY were studied, and its complete genomic sequence was determined in collaboration with ARS scientists from Salinas, CA and the University of California-Davis. Results show that ApVY is a distinct virus closely related to Celery mosaic virus and Carrot virus Y. Biological characterization of the virus is important for developing management strategies and the genomic sequence will be useful information for taxonomic comparisons and in the development of reliable molecular detection methods.
Elimination of SCMV, SrMV, SCSMV, FDV, and SCYLV from sugarcane by meristem tip cultures from apical and axillary buds. Sugarcane mosaic virus, Sorghum mosaic virus, Sugarcane streak mosaic virus, Fiji disease virus, and Sugarcane yellow leaf virus are pathogens of significance to the federal quarantine program. NGRL scientists developed a protocol for elimination of these five viruses from infected sugarcane plants through the use of harvested growing points approximately 1 millimeter in size (meristems) using tissue culture techniques. A novel method of harvesting meristems from apical tips and axillary buds was developed to greatly increase the number of potentially virus-free meristems. Plants grown for up to 18 months in a greenhouse were tested by RT-PCR assays. Greater than 50% of the meristems originally placed in the treatment were recovered as virus-free plants. When integrated into the APHIS quarantine system, this virus eradication technique will allow valuable germplasm that is infected to be treated and distributed to genebanks, and to sugarcane breeders to facilitate the development of new and improved varieties for the U.S. sugarcane industry.
Trial Inoculations of Nicotiana sp. Indicator Plants for Detection of Solanum Viruses M, S, T, and V. The importation of potato (Solanum sp.) plants to the United States for curation and breeding new varieties carries with it the risk of importing potentially devastating foreign viruses. As part of the federal quarantine program, all new Solanum imports in quarantine are inoculated to a set of virus-sensitive herbaceous plants to test for foreign pathogens. NGRL scientists, in collaboration with scientists from the Animal and Plant Health Inspection Service (APHIS), completed research to identify superior bio-indicators plants from the genus Nicotiana for detection of viruses or strains of viruses of potato not present in the United States. This research provides greater reliability in the quarantine detection system for potato viruses M, T, and V.