Progress 04/09/12 to 04/08/17
Outputs
Progress Report Objectives (from AD-416): The objectives of this project are 1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops and develop corresponding diagnostic testing methods; 2) Determine the genome organization of selected important ornamental viruses and utilize full- length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity; and 3) identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. The overall approach is to develop knowledge, tools, and reagents that will aid U.S. floriculture companies to establish effective virus testing protocols and improve clean stock production for new vegetatively propagated annuals and perennials. Based on the knowledge and tools developed while identifying and characterizing new viruses, new virus- specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols will be developed. A Universal Plant Virus Microarray, currently being developed cooperatively under an NRI grant, will add new capabilities in identifying newly emerging viruses to the appropriate family and genus, thus significantly aiding selection of appropriate methods for further characterization. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two- hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized. Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR. This is the final report for Project 8020-22000-032-00D, which ended on 04/08/2017 and was replaced by 8020-22000-042-00D. Significant progress was made on all three objectives, which fall under NP303. Under Objective 1a: We examined samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples include a plant of Seemannia nematanthodes, from which a tobamovirus was isolated and purified; Celosia plants infected by an isolate of Alternanthera mosaic virus; leaves of Camellia japonica received from the Plant Diagnostic Center at the University of Florida, in which a novel member of the Betaflexiviridae, Camellia chlorotic ringspot-associated virus, was identified (in collaboration with colleagues in the National Germplasm Resources Laboratory); Sedum and hop samples with putative carlaviruses identified; Tomato chlorosis virus was identified in a tomato sample from a hydroponic greenhouse operation (collaboration with the Molecular Plant Pathology Laboratory); samples of cut-flower Alstroemeria infected with a potyvirus identified (collaboration with the National Germplasm Resources Laboratory); and two cultivars of Veronica with apparent flexuous virions awaiting further identification. A rhabdovirus detected from pepper plants from a plant breeder�s field was identified as Potato yellow dwarf virus, and was also detected in plants of nightshade (a potential over-wintering host) growing in the field margins (collaboration with the Genetic Improvement of Fruits and Vegetables Laboratory, and a scientist from the University of Kentucky). Two new species of pelarspoviruses infecting jasmine have been detected in Hawaii, California, Maryland and DC (collaboration with scientists at University of Hawaii and with colleagues in the National Germplasm Resources Laboratory). The host ranges of Alternanthera mosaic virus (AltMV) and Plantago asiatica mosaic virus (PlAMV) were further examined by mechanical inoculation of ornamental and other species from 20 plant families; an additional 10 species from seven plant families were identified as susceptible to AltMV, and 20 additional species from 13 plant families were identified as susceptible to PlAMV. These results show the potential for both viruses to cause disease in additional ornamental or other crops. In collaboration with scientists from the Systematic Entomology Laboratory and the Electron and Confocal Microscopy Unit at Beltsville, Maryland, we have continued to examine interactions between eriophyid mites and different rose species and genotypes. In addition to the known vector (Phyllocoptes fructiphilus) of Rose rosette virus (RRV) observed on most rose samples examined, another eriophyid mite (Callyntrotus schlechtendali) was identified on a local planting of landscape roses, in the absence of the known vector P. fructiphilus. Further studies are being carried out to determine whether C. schlechtendali is a possible vector of RRV, and whether the two mite species are mutually incompatible for colonization on the same plant. Samples of P. fructiphilus have been studied by laser-scanning confocal microscopy to generate a detailed three-dimensional model of the mite, and techniques are being examined to allow fixation of individual mites for imaging of the internal organs, and ultimately to allow localization of RRV within the mite vector to aid in understanding of RRV transmission. Under Objective 1b: We continued to utilize existing broad-spectrum PCR primers to obtain partial nucleotide sequence to allow identification of previously characterized viruses, or to identify newly-discovered viruses as distinct from those previously characterized. In addition, new primer sets for detection and identification of specific viruses were developed as needed. For example, new generic and virus-specific primer sets for detection and differentiation of previously characterized and newly discovered pelarspoviruses were developed and used for their detection in new crops. Additional primer sets for detection and differentiation of specific carlaviruses have been developed, as well as new primer sets to amplify portions of the Plantago asiatica mosaic virus (PlAMV) genome. Partial genome sequences of additional isolates of Alternanthera mosaic virus (AltMV) and PlAMV, and of several additional viruses have been generated. In collaborative research (a USDA-NIFA-SCRI Project) to develop efficient serological diagnostic tools to enable the rapid, user-friendly and accurate detection of the virus that causes rose rosette disease (RRV) , the nucleocapsid protein (NP) of RRV was cloned into a bacterial expression vector and the expressed protein was used as an immunogen for production of polyclonal and monoclonal antibodies (McAbs). Synthetic peptides from highly conserved regions of the NP have also been developed as a selecting antigen and for evaluation of antibody epitope specificity. All of these antibodies are currently being tested with healthy and RRV- infected rose samples. Under Objective 1d: Crinum is a large genus of herbaceous perennial flowering bulbs in the family Amaryllidaceae and most species have large, showy, fragrant flowers. Total RNA from leaves of individual Crinum plants growing at the US National Arboretum exhibiting mosaic symptoms were purified and cDNA libraries prepared for next-generation sequencing. Complete genome sequences were obtained for the two potyviruses CriMV and NeYSV and the carlavirus Nerine latent virus (NeLV) from a single plant. To our knowledge, this is the first report of NeYSV in the US and first report of NeLV in Crinum. The full genome sequences of the new pelarspoviruses, Jasmine mosaic associated virus 1 and 2, were also determined by Illumina MiSeq next- generation sequencing. Both of these viruses were found in mixed infections in symptomatic Jasminium nitidum plants growing at the U.S. National Arboretum. The full genome sequence of an isolate of Impatiens necrotic spot virus infecting a field sample of tomatillo (Physalis philadelphica) exhibiting mosaic symptoms was also determined by Illumina MiSeq next-generation sequencing (collaboration with scientists at Purdue University). Under Objective 2a: Although a full-length infectious clone of PlAMV was previously generated in collaboration with Italian and Dutch colleagues, neither of two alternative methods of inserting a fluorescent marker protein resulted in successful systemic infections of the model plant Nicotiana benthamiana. A third variant of the virus carrying the fluorescent marker protein as a translational fusion to the C-terminus of the viral coat protein has been produced to determine whether this will be able to spread systemically and allow visualization of the progression of infection by fluorescence imaging and confocal microscopy. In partnership with Korean collaborators, full-length infectious clones of three distinct isolates of Cucumber green mottle mosaic virus (CGMMV) were produced, and shown to induce distinct types of symptoms in Nicotiana benthamiana, varying from mild mosaic, mosaic without obvious leaf distortion, or severe mosaic with significant leaf distortion. These infectious clones will be used to prepare chimeric constructs in order to examine the contribution of the amino acid differences to symptom expression and pathogenicity. Two infectious clones of Pepper mild mottle virus (PMMoV) were also generated with the same Korean collaborators, which differed by only three amino acids residues in the 126 kDa replication protein, and four residues in the movement protein; no obvious symptom differences were observed in either N. benthamiana or pepper (Capsicum annuum). Although four amino acids differed between the respective movement proteins, no apparent differences in movement protein localization were observed, nor was any difference observed in efficiency of systemic movement between the isolates. Infectious clones of 17 isolates of Turnip mosaic virus (TuMV) were also produced in partnership with Korean collaborators, and showed variations in both sequence and symptom severity. Four of the cloned isolates, plus one previously sequenced infectious clone, produced only mild symptoms in Nicotiana benthamiana, whereas all other isolates induced systemic necrosis in the same host. Two mild and two severe isolates were used to screen 60 lines of radish to evaluate differences in resistance and susceptibility, with the ultimate goal of identifying new resistance genes. Under Objective 2b: In partnership with Korean collaborators, infectious clones were produced from two Korean isolates (S-47; J-76) of Pepper mild mottle virus (PMMoV), and both were shown to induce severe symptoms in Nicotiana benthamiana, and mild symptoms in Capsicum annuum. Isolate J-76 consistently induced a hypersensitive response in inoculated leaves, whereas isolate S-47 did not. The isolates differed by three amino acid residues in the 126 kDa replication protein, and by four residues in the movement protein (MP). GFP fusion proteins of the 126 kDa replicase protein showed distinct differences which suggest that there are differential interactions with a host protein as a result of the amino acid differences between the 126 kDa proteins of the two isolates, resulting in a hypersensitive response to J-76, but not to S-47. The residues responsible will be identified by creation of chimeric constructs or site-directed mutagenesis. Under Objective 3: We propagated and verified one tree strain of X. fastidiosa obtained from the American Type Culture Collection. Genomic DNAs were sequenced and compared to sequences from other landscape tree strains of X. fastidiosa for conservation of unique genes and phylogenetic relationships. Accomplishments 01 Development of a specific and sensitive assay for the detection of Plantago asiatica mosaic virus in ornamental lily. Lilies are a valuable internationally-traded ornamental crop, with cut flower production in California alone worth over $56 million wholesale. Plantago asiatica mosaic virus (PlAMV) was recently detected in lilies imported to the U.S. from the Netherlands, where losses of up to 80% have been reported in cut-flower lily production, indicating the potential for significant losses to U.S. producers. ARS scientists in Beltsville, Maryland, developed a specific and sensitive assay to detect the presence of PlAMV in asymptomatic lily plants. This assay is based on a sensitive ELISA test that produces no background reaction and also does not react with healthy plant tissue. Furthermore, the assay shows accurate results from two hours to several days after incubation, so is convenient to use in any laboratory environment. This assay has been used in host range tests and has been validated by the Plant Disease Clinic of the University of Maryland, so will be a valuable new tool for identification and control of this important plant virus. 02 Development of improved assays for detection and differentiation of Ralstonia solanacearum strains. R. solanacearum is a bacterial species that causes millions of dollars of crop losses in a wide range of plant species worldwide. One strain in particular, the r3b2 subgroup, is such a threat to U.S. agriculture that is has been designated as a select agent, and regulations require that all strains of R. solanacearum be designated as select agents until proven to be non-r3b2. Detection methods are needed that are sensitive, specific, accurate, and efficient in order to prevent the r3b2 strain from entering the U.S., and also to avoid unnecessary exclusion of non-r3b2 strains. ARS and APHIS scientists in Beltsville, Maryland, collaborated to develop improved molecular assays that can detect R. solanacearum (Rs16S primers) and specifically the r3b2 strain (RsSA3 primers). Using a rapid and sensitive methodology (TaqMan RT qPCR), these new primers proved to be more sensitive than previously developed primers, and the RsSA3 primer effectively and accurately differentiated the r3b2 select- agent strain. These primers will be used by federal and state diagnostic laboratories to improve detection and control of this important plant pathogen. 03 Detection of plant quarantine pathogen Ralstonia solanacearum r3b2 with portable POCKIT� and BLItz� systems. R. solanacearum (Rs) is a bacterial species that causes millions of dollars of crop losses in a wide range of plant species worldwide. One strain in particular, the r3b2 subgroup, is such a threat to U.S. agriculture that is has been designated a select agent, and regulations require that all strains of Rs be designated as select agents until proven to be non-r3b2. ARS and APHIS scientists in Beltsville, Maryland, collaborated with scientists at Rutgers University to develop two portable platforms for Rs r3b2 detection - the POCKIT� and the BLItz� - that significantly increase the sensitivity, speed, specificity, accuracy and portability of diagnostic assays for Rs in order to prevent the Rs r3b2 strain from entering the U.S., and also to avoid unnecessary exclusion of non-r3b2 Rs strains. The palm-sized POCKIT� can be used with previously published primers to detect as few as 10 bacteria in 32 minutes, including heat-inactivated samples and in plant tissue, and the BLItz� instrument has comparable speed and sensitivity to the commercial ImmunoStrip� with the advantage of higher r3b2 specificity. These two independent, portable systems can facilitate R. solanacearum r3b2 detection at the ports of entry and in field settings.
Impacts
(N/A)
Publications
- Stulberg, M.J., Rascoe, J., Li, W.N., Yan, Z., Nakhla, M.K., Huang, Q. 2016. Development and comparison of TaqMan-based real-time PCR assays for detection and differentiation of Ralstonia solanacearum strains. Current Microbiology. doi:10.1007/s00284-016109-z.
- Di, R., Huang, Q., Stulberg, M.J., Zhao, L., Levy, L. 2016. Detection of plant quarantine pathogen Ralstonia solanacearum r3b2 with portable POCKIT� and BLItz� systems. Journal of Plant Health. 1(1):103.
- Wang, T., Guan, W., Huang, Q., Yang, Y., Yan, W., Sun, B., Zhao, T. 2016. Quorum-sensing contributes to virulence, twitching motility, seed attachment and biofilm formation in the wild type strain Aac-5 of Acidovorax citrulli. Microbial Pathogenesis. 100:133-140.
- Wang, J., Wang, Y., Dai, P., Chen, D., Zhao, T., Li, X., Huang, Q. 2017. Pectobacterium carotovorum. subsp. brasiliense is a causal agent of bacterial leaf rot of tobacco in China. Plant Disease. 101(5):830.
- Cho, I., Park, M., Kwon, S., Choi, G., Hammond, J., Lim, H. 2016. First report of Persimmon cryptic virus and Persimmon virus A in Korea. Journal of Plant Pathology. 98(3):694.
- Cho, I., Igori, D., Lim, S., Choi, G., Hammond, J., Lim, H., Moon, J. 2016. Deep sequencing analysis of apple infecting viruses in Korea. Plant Pathology Journal . 32(5):441-451.
- Jang, C., Wang, R., Wells, J., Leon, F., Farman, M., Hammond, J., Goodin, M. 2017. Genome sequence variation in the constricta strain dramatically alters the protein interaction and localization map of Potato yellow dwarf virus. Journal of General Virology. doi:10.1099/igv.0.000771PMID:28635588.
- Seo, E., Lim, S., Hammond, J., Moon, J., Lim, H. 2016. Complete genome sequence of a novel potyvirus, Callistephus mottle virus identified in Callistephus chinensis. Archives of Virology. 161:3281-3283.
- Hammond, J., Kim, I., Lim, H. 2017. Alternanthera mosaic virus � and alternative �model� potexvirus of broad relevance. Korean Journal of Agricultural Science. 44:145-180.
- Han, S., Park, J., Han, J., Gong, J., Park, C., Kim, J., Seo, E., Domier, L.L., Hammond, J., Lim, H. 2017. New Korean isolates of Pepper mild mottle virus (PMMoV) differ in symptom severity, seed yield in pepper, and subcellular localization of the 126 kDa protein. Virus Genes. 53:434-445.
- Park, C., Ju, H., Han, J., Park, J., Kim, I., Seo, E., Kim, J., Hammond, J. , Lim, H. 2016. Complete nucleotide sequences and construction of full- length infectious cDNA clones of Cucumber green mottle virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters. Virus Genes. 53:286-299.
- Garcia, M., Dal Bo, E., Da Graca, J., Gago-Zachert, S., Hammond, J., Moreno, P., Natsuaki, T., Pallas, V., Navarro, J., Reyes, C., Robles Luna, G., Sasaya, T., Tzanetakis, I., Vaira, A., Verbeek, M. 2017. Family Ophioviridae: classification and features. Journal of General Virology. doi:10.1099/jgv.0.000836PMID:28635587.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): The objectives of this project are 1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops and develop corresponding diagnostic testing methods; 2) Determine the genome organization of selected important ornamental viruses and utilize full- length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity; and 3) identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. The overall approach is to develop knowledge, tools, and reagents that will aid U.S. floriculture companies to establish effective virus testing protocols and improve clean stock production for new vegetatively propagated annuals and perennials. Based on the knowledge and tools developed while identifying and characterizing new viruses, new virus- specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols will be developed. A Universal Plant Virus Microarray, currently being developed cooperatively under an NRI grant, will add new capabilities in identifying newly emerging viruses to the appropriate family and genus, thus significantly aiding selection of appropriate methods for further characterization. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two- hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized. Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR. Progress was made on all three objectives, which fall under NP303. Objective 1a. We have detected and identified variants of Gloriosa stripe mosaic virus (GSMV) in Gloriosa superba var. rothschildiana, G. superba �Himalayan�, and G. lutea (collaboration with a scientist in the National Germplasm Resources Laboratory); this virus has not previously been reported in the United States. We have also detected GSMV in a lily plant (Lilium leichtlinii) being grown in the same greenhouse � presumably resulting from aphid transmission from the infected gloriosa lilies. To the best of our knowledge, GSMV has not previously been reported from Lilium anywhere in the world. We had previously identified Narcissus yellow stripe virus (NYSV) in an ornamental Allium species, A. carinatum. We have now identified what appears to be a distinct potyvirus in a second plant of A. carinatum; the new potyvirus isolate is most closely related to NYSV, but has a level of coat protein amino acid sequence identity with NYSV that is below the level recognized as the boundary between potyvirus species. Other portions of the genome which have been sequenced show a similar level of divergence, indicating that the new isolate from A. carinatum is either a novel potyvirus species, or possibly a recombinant between NYSV and an as yet unidentified potyvirus. Amino acid sequence alignments of the coat protein gene and the region immediately upstream show that the new isolate has multiple amino acid residues distinguishing the new isolate from all available NYSV sequences, especially in the N-terminal region of the coat protein which differs most between potyvirus species, and also in the putative proteolytic cleavage site between the upstream NIb gene and the coat protein gene. Two new pelarspoviruses, tentatively designated Jasmine mosaic associated virus 1 and 2, were detected in Jasminium multiflorum, J. nitidum and J. sambac and partially sequenced. These were found singly or in mixed infections in symptomatic plants from California, District of Columbia, Hawaii and Maryland (in collaboration with the University of Hawaii and the National Germplasm Resources Laboratory). The complete genome of another new pelarspovirus infecting Clematis, Clematis chlorotic mottle virus, was determined (in collaboration with scientists at National Germplasm Resources Laboratory and University of Minnesota). Two distinct viruses, a new potyvirus (Shamrock chlorotic ringspot virus; SCRV) and a nepovirus, Beet ringspot virus, found in a mixed infection of ornamental Oxalis exhibiting chlorotic ringspot symptoms, were studied further (in collaboration with scientists at National Germplasm Resources Laboratory and University of Minnesota). The full length genomes of both viruses were determined. Preliminary results indicate that the symptoms present in dually-infected plants are caused solely by SCRV. A new potyvirus, Crinum mosaic virus, was detected in ornamental flowering bulb Crinum plants growing at the U.S. National Arboretum and was partially sequenced. Leaves of Phalaenopsis orchid received through USDA-APHIS from a greenhouse in New Jersey showed obvious chlorotic and necrotic ringspots similar to those previously reported for tospovirus infection. Electron microscopy of thin sections revealed tospovirus-like particles in many cells, and multiple osmophilic globules in chloroplasts; bioassay plants were inoculated from the infected Phalaenopsis leaves. Tests are currently underway to identify the particular tospovirus by RT-PCR with tospovirus-generic and species-specific primers. In collaboration with scientists from the Systematic Entomology Laboratory and the Electron and Confocal Microscopy Unit in Beltsville, Maryland we have examined interactions between the eriophyid mite vector (Phyllocoptes fructiphilus) of Rose rosette virus and different rose genotypes and species by scanning electron microscopy. Other eriophyid mites were also identified on rose leaves and flower buds. The presence and localization of trichomes and glandular hairs were observed to vary significantly between different rose species and genotypes, and eriophyid mites were observed to utilize densely packed trichomes and glandular hairs as apparent protection against predators, with many mite feeding sites and eggs close to the base of glandular hairs. On other leaves, multiple mite feeding sites were observed in close proximity to each other, frequently in epidermal cells which had a �deflated� appearance. In partnership with Korean collaborators, we identified a novel potyvirus from mottled plants of Callistephus chinensis (Chinese aster) in Korea, which has been fully sequenced and tentatively named Callistephus mottle virus (CalMV). CalMV was found to be most closely related to Plum pox virus, Celery mosaic virus, and Apium virus Y, at levels of sequence identity below the criteria for species differentiation (i.e., CalMV is a distinct potyvirus species), and to have distinct polyprotein cleavage sites between the mature proteins processed from the viral polyprotein. In addition, the efficiency of RNA silencing suppression and subcellular localization and aggregation of the CalMV helper component-protease (HC-Pro) protein were compared to those of other potyviruses, showing that CalMV HC-Pro had relatively weak RNA silencing suppression activity similar to that of a mild isolate of Turnip mosaic virus. Objective 1b. In collaborative research (a USDA-NIFA-SCRI project) to develop efficient serological diagnostic tools to enable the rapid, user- friendly and accurate detection of the virus that causes rose rosette disease (RRV), the nucleocapsid protein (NP) of RRV was cloned into a bacterial expression vector and the expressed protein was used as an immunogen for production of polyclonal and monoclonal antibodies (McAbs). Synthetic peptides from highly conserved regions of the NP have also been developed as a selecting antigen and for evaluation of antibody epitope specificity. Ten McAb-secreting hybridoma cells were initially selected for analysis. The McAbs all reacted to antigen in ACP-ELISA. Five were selected for further study: three that exhibited strong reactivity in both ACP- and TAS-ELISA and two that exhibited strong reactivity in ACP- and weak to zero reaction in TAS-ELISA. All of these antibodies are currently being tested with healthy and RRV-infected rose samples. Virus-specific and broad-spectrum PCR primers continue to be designed and evaluated for the detection of known and recently sequenced new or emerging viruses, especially, Carla-, Potex- and Potyviruses. Progress in the development of broad-spectrum reacting polyclonal, monoclonal and/or scFv antibodies for the detection of carlaviruses or potexviruses continues. Diverse isolates from these two genera are being collected and purified, and synthetic peptides have been designed and produced. Objective 2a. A full length infectious clone of Plantago asiatica mosaic virus (PlAMV) has been generated in collaboration with Italian and Dutch collaborators, and a fluorescent protein gene has been introduced into the genome by two separate strategies � as a fusion protein at the N- terminal end of the coat protein, and as an added gene inserted into a multiple cloning site. The wild-type full length clone readily induces systemic infection following inoculation of test plants by agroinfiltration, but the clones modified with the fluorescent marker protein have so far shown very limited ability to infect plants systemically. Experiments are in progress using laser-scanning confocal microscopy to examine local and systemic movement of the infectious clones expressing the fluorescent marker protein. Polyclonal and monoclonal antibodies to movement protein 1 (MP-1) of various pelarspoviruses developed previously were further evaluated. These are either specific to one species or cross-reactive to many or all species. These antibodies are being used in ongoing experiments confirming the gene exchange of the MP-1 gene between infectious clones of these distinct viruses, as well as in experiments to determine the expression, location, and functionality of the MP-1 protein in viral pathogenesis. Objective 3. Analysis of the genome sequences of the mulberry- and sycamore-infecting strains of X. fastidiosa revealed a unique sequence conserved in the two strains, which we used to design primers for detection. In addition, using the primer pair that recognizes both the American mulberry and Italian olive strains of X. fastidiosa, we developed a duplex-PCR for the specific detection of the American mulberry strains, and differentiation of the Italian olive strains from the American mulberry strains. Accomplishments 01 Prevalence and variability of Plantago asiatic mosaic virus in imported lily bulbs. Lilies are a valuable internationally-traded ornamental crop, with cut flower production in California alone worth over $56 million wholesale. Plantago asiatica mosaic virus (PlAMV) was recently detected in lilies imported to the U.S. from the Netherlands, where losses of up to 80% have been reported in cut-flower lily production, indicating the potential for significant losses to U.S. producers. However, neither the prevalence of PlAMV in different imported lily types, nor the degree of variability within the viral population, were known. Stocks of different imported lily types were tested by an ARS researcher in Beltsville, Maryland, in collaboration with a University of Maryland colleague, for the presence of PlAMV; large differences were found in infection rate (from zero to one hundred percent in different stocks). Sequencing of the PlAMV coat protein gene from different isolates revealed little variation in sequence, with all U.S. samples closely related to �European�-type isolates from Europe, Korea, and India, but distinct from Japanese and Russian isolates; this suggests a single source for the �European�-type isolates and distribution through international trade. Purified PlAMV was used to generate a diagnostic antiserum to detect PlAMV infection. This research provides new information and diagnostic capabilities to USDA- APHIS for a previously undetected virus in this highly valuable crop. 02 Use of differential pathogenicity of Turnip mosaic virus isolates to improve breeding for virus-resistant plants in Korea. Breeding and selection for plants with tolerance or resistance to virus infection is complicated by the variability between virus isolates. Identification of diverse virus isolates varying in pathogenicity is needed to aid scientists in screening for viral resistance to improve crop productivity. RNA silencing suppression is a means by which plant viruses counter the innate host plant defenses, affecting levels of virus accumulation and symptom severity in infected plants. An ARS scientist in Beltsville, Maryland, working in collaboration with scientists in Korea, identified differences in RNA silencing suppression efficiency of Turnip mosaic virus (TuMV) isolates from Korea. These TuMV isolates also differ in pathogenicity in both Chinese cabbage and radish; the isolates were used to screen more than 150 cultivars or breeding lines of cabbage and radish in Korea to identify lines with differential susceptibility or resistance. Eight cabbage lines and three radish lines with differential resistance were identified for use in further breeding. One gene of the virus was identified as the major determinant of the host response, providing a potential tool for plant breeders to select new resistant genotypes. 03 Development of a rapid and sensitive assay to identify select agent strains of R. solanacearum. R. solanacearum is a bacterial species that causes millions of dollars of crop losses in a wide range of plant species worldwide. One strain in particular, the r3b2 subgroup, is such a threat to U.S. agriculture that is has been designated a select agent, and new regulations require that all strains of R. solanacearum be designated as select agents until proven to be non-r3b2. Detection methods are needed that are sensitive, specific, accurate, and efficient in order to prevent the r3b2 strain from entering the U.S., and also to avoid unnecessary exclusion of non-r3b2 strains. ARS researchers in Beltsville, Maryland developed a multiplex qPCR assay that not only improves the specificity of r3b2 detection, but also simultaneously detects the pathogen at the newly proposed R. solanacearum species level for added confidence. This new assay includes an internal control for infected plant samples to ensure that the assay is working correctly, and takes advantage of a simple extraction method that they developed using an improved extraction buffer. The multiplex qPCR assay, especially when coupled with the improved extraction buffer method, allows for quick, easy and reliable detection and differentiation of the r3b2 strains of R. solanacearum. 04 Fast and easy typing of a partial endoglucanase gene sequence into phylotypes and select agent strains of R. solanacearum. An especially devasting strain of the bacterium R. solanacearum, designated as select agent strain r3b2, causes destructive brown rot of potato and is capable of surviving and infecting at lower temperatures than other isolates. Current select agent regulations mandate that all strains of R. solanacearum be considered select agents until further testing can exclude them as r3b2 strains. ARS researchers in Beltsville, Maryland developed PCR primers and wrote a new computer algorithm that allows direct amplification of a gene sequence from R. solanacearum from infected plant samples and then classifies the sequence into one of the three genospecies in the R. solanacearum species complex; the algorighm also identifies the highly regulated r3b2 strains of R. solanacearum belonging to phylotype IIB, sequevars 1&2. This program does not require specific expertise to use, gives results in seconds, and provides data interpretation for the user. The program and primers allow users to quickly identify an unknown sample of R. solanacearum to type and determine whether it is a r3b2 strain. The program can also serve as confirmation, since it is the only method that can easily and directly determine whether the strain in question is a sequevar 1 or 2 strain. 05 The ferric uptake regulator gene fur affects key functions in Xanthomonas vesicatoria. Iron is essential for the growth and survival of many organisms including plant pathogenic bacteria. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the iron uptake system in many bacteria, but its role in the bacterial spot pathogen Xanthomonas vesicatoria (Xv) is unknown. An ARS scientist in Beltsville, Maryland, in collaboration with scientists in China, investigated the function of the fur gene in Xv by generating a fur mutant strain. We found that the Fur gene negatively controls siderophore production, but positively regulates extracellular polysaccharide production, biofilm formation, swimming ability, quorum sensing, and virulence in tomato leaves. This study is important as it increases our understanding of bacterial pathogenesis and could lead to eventual control of this plant pathogen.
Impacts
(N/A)
Publications
- Stulberg, M.J., Huang, Q. 2015. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum. PLoS One. doi: 10.1371/journal.pone. 0139637.
- Bampi, D., Mituti, T., Paven, M.A., Hammond, J., Krause-Sakate, R. 2015. Leek yellow stripe virus isolates from Brazil form a distant clade based on the P1 gene. Journal of Plant Pathology. 97:457-463.
- Han, J., Kim, J., Cheong, J., Seo, E., Park, C., Ju, H., Cho, I., Gotoh, T. , Moon, J., Hammond, J., Lim, H. 2015. Survey of apple chlorotic leaf spot virus and apple stem grooving virus occurrence in Korea and frequency of mixed infections in apple. Journal of Faculty of Agriculture. 60:323-329.
- Li, M., Seo, E., Cho, S., Kim, J., Chung, J., Lim, H., Gotoh, T., Hammond, J., Lim, H. 2015. A 2014 nationwide survey of the distribution of Soybean mosaic virus (SMV), Soybean yellow mottle mosaic virus (SYMMV) and Soybean yellow common mosaic virus (SYCMV) major viruses in South Korean soybean fields, and changes. Journal of Faculty of Agriculture. 60:339-347.
- Seo, E., Kim, H., Kim, J., Gotoh, T., Hammond, J., Lim, H. 2015. Utilization of a tobacco rattle virus vector to clone an Nicotiana benthamiana cDNA library for VIGS. Journal of Faculty of Agriculture. 60:331-337.
- Chung, J., Kim, J., Ju, H., Han, J., Seo, E., Hammond, J., Lim, H. 2015. 2014 nationwide survey revealed Turnip mosaic virus, Radish mosaic virus and Cucumber mosaic virus as the major viruses in Korean Radish Fields. Research in Plant Disease. 21:235-242.
- Han, J., Chung, J., Kim, J., Seo, E., Kilcrease, J.P., Bauchan, G.R., Lim, S., Hammond, J., Lim, H. 2016. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus. Virus Genes. 52:592-596.
- Lim, H., Seo, E., Kim, H., Kim, J., Park, C., Gong, J., Him, I., Han, A., Kilcrease, J.P., Tsuchiya, K., Hammond, J., Lim, H. 2016. 2015 nationwide survey revealed Barley stripe mosaic virus in Korean barley fields. Journal of Faculty of Agriculture. 61:71-77.
- Han, J., Park, C., Seo, E., Kim, J., Hammond, J., Lim, H. 2016. Occurrence of Apple stem grooving virus in commercial apple seedlings and analysis of its coat protein sequence. CNU Journal of Agricultural Science. 43:21-27.
- Liu, H., Dong, C., Zhao, T., Han, J., Wang, T., Wen, X., Yang, G., Huang, Q. 2016. Functional analysis of the ferric uptake requlator gene, fur, in Xanthomonas vesicatoria. PLoS One. 11(2):e0149280. doi: 10.1371/journal. pone.0149280.
- Kamo, K.K., Lakshman, D.K., Pandey, R., Guaragna, M.A., Okubara, P.A., Rajasekaran, K., Cary, J.W., Jordan, R.L. 2015. Resistance to Fusarium oxysporum f. sp. gladioli in transgenic Gladiolus plants expressing either a bacterial chloroperoxidase or fungal chitinase genes. Plant Cell Tissue And Organ Culture. 124:541.
- Stalberg, M., Huang, Q. 2016. A computer program for fast and easy typing of partial endoglucanase gene sequence into phylotypes and sequevars 1&2 (select agents) of Ralstonia solanacearum. Journal of Microbiological Methods. 123:101-107.
- Yuan, Q., Jordan, R.L., Brlansky, R.H., Minenkova, O., Hartung, J.S. 2015. Development of single chain variable fragment (scFv) antibodies against surface proteins of �Ca. Liberibacter asiaticus�. Journal of Microbiological Methods. 122:1-7.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): The objectives of this project are 1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops and develop corresponding diagnostic testing methods; 2) Determine the genome organization of selected important ornamental viruses and utilize full- length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity; and 3) identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. The overall approach is to develop knowledge, tools, and reagents that will aid U.S. floriculture companies to establish effective virus testing protocols and improve clean stock production for new vegetatively propagated annuals and perennials. Based on the knowledge and tools developed while identifying and characterizing new viruses, new virus- specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols will be developed. A Universal Plant Virus Microarray, currently being developed cooperatively under an NRI grant, will add new capabilities in identifying newly emerging viruses to the appropriate family and genus, thus significantly aiding selection of appropriate methods for further characterization. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two- hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized. Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR. Under Objective 1a: Additional imported Asiatic and Oriental lily bulbs were found to be infected with Plantago asiatica mosaic virus (PlAMV); in 2015 every single bulb (four bulbs each of four cultivars of Asiatic lilies, and four bulbs each of three Oriental lily cultivars) were found to be infected by PlAMV. The virus was also detected in the packing material around the bulbs, suggesting exudation of PlAMV from the developing roots of the bulbs. A method using agarose to stabilize the root:growing media matrix prior to fixation for electron microscopy of root tissue of lilies and Nicotiana benthamiana infected with PlAMV was developed to examine the possible presence of exuded virus at the root surface. Virus-infected root cells were identified, but no virus has been detected external to the root so far. A survey of viruses present in ornamental Allium species (collaboration with a graduate student from Brazil) identified Leek yellow stripe virus, Onion yellow dwarf virus, and Narcissus yellow stripe virus (NYSV), all potyviruses. NYSV has not previously been reported from any Allium species. Also detected were: Shallot latent virus and Garlic common latent virus (carlaviruses), the allexiviruses Garlic virus A, Garlic virus B, Garlic virus C, Garlic virus D, Garlic virus E, the Garlic mite- borne filamentous virus, and Shallot virus X. Identification of all viruses was by PCR with genus-generic and virus-specific primers; and, for several viruses, was also confirmed by ELISA with virus-specific antibodies, or by sequencing of cloned PCR products. Two distinct viruses, a new potyvirus (Shamrock chlorotic ringspot virus; SCRV) and a nepovirus, Beet ringspot virus (BRV), found in a mixed infection of ornamental Oxalis exhibiting chlorotic ringspot symptoms, were studied further. The full length genomes of both viruses were determined. Preliminary results indicate that the symptoms present in dually-infected plants are caused solely by SCRV. The full length genome of the United States isolate of Catharanthus mosaic virus was also determined. (Collaboration with National Germplasm Resources Laboratory and Michigan Department of Agriculture personnel). A mixed infection of Veronica with two distinct isolates of Butterbur mosaic virus (ButMV) and an isolate of Helenium virus S (HelVS) has been examined in greater detail. The two ButMV isolates have distinct sequences across the regions that have been sequenced to date (>3 kb from the 3' end). One of these ButMV isolates has been determined to be defective, having a major deletion in one gene; the defective isolate is thus presumed to be dependent on the other ButMV isolate for functions encoded by the deleted gene. Approximately 3.1 kb of the 3' end of the HelVS genome has also been determined, and shows significant levels of identity to partial sequences of isolates from Helenium and Impatiens (collaboration with National Germplasm Resources Laboratory). Under Objective 1b: Garden roses, which form the cornerstone of the multi-billion dollar landscape industry, annually generate wholesale U.S. domestic production valued at ~ $400 million. Over the past few decades Rose Rosette Disease has become very serious and threatens to decimate the United States Rose industry. The causal agent, Rose rosette virus (RRV), is transmitted by wind-blown eriophyid mites, and can kill a rose within 2-3 years of infection. The nucleoprotein (NP) from several Maryland isolates of RRV have been cloned and sequenced and shown to have high sequence homology with 22 other U.S. isolates. In collaborative research (a USDA-NIFA-SCRI Project) to develop efficient serological diagnostic tools to enable the rapid, user-friendly and accurate detection of the virus, the NP of RRV has also been cloned into a bacterial expression vector. Purified expressed protein will be used as an immunogen for production of polyclonal, monoclonal and/or single-chain antibodies. Synthetic peptides from highly conserved regions of the NP have also been developed to be used as immunogen and selecting antigen. Specific primers were developed for the detection and identification of several viruses, including: Plantago asiatica mosaic virus, Helenium virus S, Butterbur mosaic virus, Shamrock chlorotic ringspot virus, Catharanthus mosaic virus, and Beet ringspot virus. Under Objective 2a: The complete sequence of an isolate of Plantago asiatica mosaic virus (PlAMV) was determined, and two infectious clones of PlAMV differing in symptom severity were generated (collaboration with Italian and Dutch scientists). A major portion of the genome of two distinct isolates of NYSV from ornamental Allium has been determined (collaboration with a graduate student from Brazil). Partial sequences of additional isolates of Alternanthera mosaic virus were determined. The P1 genes of two distinct (N- and S-type) isolates of Leek yellow stripe virus were separately expressed in Nicotiana benthamiana as an additional gene from an Alternanthera mosaic virus protein expression vector. The P1 proteins were also expressed in Nicotiana benthamiana as GFP fusion proteins by agro-infiltration to examine sub-cellular localization. Some increase in symptom severity was observed when the P1 protein of the S-type isolate was expressed from AltMV (collaboration with a graduate student from Brazil). Infectious clones of Alternanthera mosaic virus and Soybean yellow common mosaic virus have been adapted for use as virus-induced gene silencing vectors and applied to screen whitefly cDNA sequences for RNAi against whiteflies, and for identification of soybean gene functions, respectively (collaboration with Korean scientists). Polyclonal and monoclonal antibodies to movement protein 1 (MP-1) of various pelarspoviruses (using synthetic peptides containing species unique or genus conserved amino acid sequences) that are either specific to one species or cross-reactive to all species, respectively, were developed. These antibodies are being used in ongoing experiments confirming the gene exchange of the MP-1 gene between infectious clones of these distinct viruses, as well as in experiments to determine the expression, location, and functionality of the MP-1 protein in viral pathogenesis. Under Objective 2b: A yeast two-hybrid cDNA library of Nicotiana benthamiana was generated for examination of further host proteins interacting with the proteins of Alternanthera mosaic virus, Lolium latent virus, Plantago asiatica mosaic virus, and other viruses of interest. The sub-cellular localization of three proteins of several isolates of Turnip mosaic virus has been examined (as fusions to Green fluorescent protein) by confocal microscopy, with differences in localization associated with some amino acid variants between the equivalent proteins of different isolates. Sub-cellular localization of two proteins of each of two isolates of Barley stripe mosaic virus was also examined (collaboration with Korean scientists and graduate students). Under Objective 3.1: Several remaining �gaps� in the genome of the sycamore strain of X. fastidiosa were determined. Some open reading frames were manually annotated and the draft genome of this sycamore strain was published. Based on our previously published genome sequence of the mulberry- infecting strain of X. fastidiosa, we identified its unique open reading frames. One of the unique gene sequences was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. Primers designed based on this unique sequence were successfully tested in both the U.S. and Italy against targeted and non-targeted X. fastidiosa strains in cultures and in naturally infected plant samples to ensure their specificity. Under Objective 3.2: A multiplex PCR assay was developed and tested that includes 1) a plant primer pair as an internal control; 2) R. solanacearum species complex; and 3) r3b2-specific primer pairs against 95 target and non-target R. solanacearum, as well as out-group bacterial strains. The assay was also tested against different plants including potato, geranium, tobacco and tomato artificially inoculated with r3b2 or non-r3b2 strains of R. solanacearum. Based on the unique r3b2 and plant mitochondrial cytochrome oxidase subunit 1 (cox1) sequences previously identified by us, we designed r3b2- and plant-specific primers and probes. We also designed phylotype II or the newly proposed R. solanacearum species-specific primers and a probe based on previously published phylotype II primers and their amplicon sequence. We tested the multiplex qPCR assay in vitro and in planta. We also developed a simple extraction method using an extraction buffer we designed to reduce PCR inhibition from geranium tissues for easy and fast processing of infected plant samples. Accomplishments 01 A multiplex PCR assay to detect and differentiate select agent strains of Ralstonia solanacearum. The fungal pathogen R. solanacearum race 3 biovar 2 causes destructive brown rot of potato and is capable of surviving and infecting at lower temperatures. Current select agent regulations list all strains of R. solanacearum as select agents unless further testing can exclude them from race 3 biovar 2. ARS researchers at Beltsville, Maryland, developed a molecular assay that can identify whether a strain of R. solanacearum is race 3 biovar 2, and also exclude false negatives caused by unsuccessful DNA extraction or PCR inhibition. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States. 02 Specific detection and identification of American mulberry-infecting and Italian olive-associated strains of Xylella fastidiosa by polymerase chain reaction. X. fastidiosa causes bacterial leaf scorch in many important landscape trees and was also recently identified for the first time in Italy in olive trees affected by a devastating disease named �Olive Quick Decline Syndrome�. ARS researchers at Beltsville, Maryland, in collaboration with an Italian scientist have developed a PCR assay that can specifically detect and identify both the mulberry-infecting strains of X. fastidiosa strains in the U.S. and the olive-associated strains in Italy. The PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. It will also be useful for detection and identification of the new Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution, and biology of this new group. 03 Determination of the genome sequence of a Xylella fastidiosa strain causing sycamore leaf scorch disease in Virginia. X. fastidiosa causes bacterial leaf scorch in many important landscape trees, including sycamore. The disease is spread by insect vectors, with different strains of the bacteria infecting different plant species. ARS researchers at Beltsville, Maryland, determined the draft genome sequence of the sycamore strain �Sy-VA� in order to gain a better understanding of the molecular basis of strain divergence, host specificity, nutrient requirements, and pathogenicity. This information will also be used to develop genome-based specific detection methods for this important plant pathogen. 04 Determination and recognition of a new genus, Pelarspovirus, in the plant virus family Tombusviridae. Plant viruses are responsible for millions of dollars of losses to agronomic crops, vegetables, fruits, and ornamentals. Understanding the relationships and identity of viruses is critical to pathogen detection, diagnosis, and control. By studying viruses in geranium, an ARS scientist in Beltsville, Maryland, and cooperators proposed that six virus species are distinct enough to be given their own genus, named Pelarspovirus, within the family Tombusviridae. This discovery was made by studying biochemical and genomic features that differentiate these species from other related species. Characterization and classification of these and other plant viruses will aid in the detection and identification of new and emerging viruses infecting ornamentals. This knowledge will be used by state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into and through the United States.
Impacts
(N/A)
Publications
- Hammond, J., Bampi, D., Reinsel, M.D. 2015. First report of Plantago asiatica mosaic virus in imported Asiatic and Oriental lilies (Lilium hybrids) in the United States. Plant Disease. 99:292.
- Mollov, D.S., Guaragna, M., Lockhart, B., Rezende, J., Jordan, R. 2014. First report of Catharanthus mosaic virus in Mandevilla in the United States. Plant Disease. 99:165.
- Seo, E., Cho, S., Moon, J.S., Gotoh, T., Kim, H., Domier, L.L., Hammond, J. , Lim, H., Song, K. 2015. A high throughput soybean gene identification system developed using soybean yellow common mosaic virus (SYCMV). Journal of Faculty of Agriculture. 60:127-138.
- Ko, N., Kim, H., Kim, J., Cho, S., Seo, E., Kwon, H., Yu, Y., Gotoh, T., Hammond, J., Youn, Y., Lim, H. 2015. Developing an Alternanthera mosaic virus vector for efficient clonging of Whitefly cDNA RNAi to screen gene function. Journal of Faculty of Agriculture. 60:139-149.
- Guan, W., Shao, J.Y., Davis, R.E., Zhao, T., Huang, Q. 2015. Specific detection and identification of mulberry-infecting strains of Xylella fastidiosa by polymerase chain reaction. PLoS One. DOI: 10.1371/journal. pone.0129330.
- Scheets, K., Jordan, R.L., White, A., Hernandez, C. 2015. Pelarspovirus, a proposed new genus in the family Tombusviridae. Archives of Virology. 160:2385-2393.
- Scheets, K., Hernandez, C., Jordan, R.L., White, A. 2014. Create four new unassigned species in the family Tombusviridae. Archives of Virology. DOI:10.1007/s00705-015-2500-5.
- Guan, W., Shao, J.Y., Davis, R.E., Zhao, T., Huang, Q. 2014. Genome sequence of a Xylella fastidiosa strain causing sycamore leaf scorch disease in Virginia. Genome Announcements. 2(4):e00773-14.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): The objectives of this project are 1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops and develop corresponding diagnostic testing methods; 2) Determine the genome organization of selected important ornamental viruses and utilize full- length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity; and 3) identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. The overall approach is to develop knowledge, tools, and reagents that will aid U.S. floriculture companies to establish effective virus testing protocols and improve clean stock production for new vegetatively propagated annuals and perennials. Based on the knowledge and tools developed while identifying and characterizing new viruses, new virus- specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols will be developed. A Universal Plant Virus Microarray, currently being developed cooperatively under an NRI grant, will add new capabilities in identifying newly emerging viruses to the appropriate family and genus, thus significantly aiding selection of appropriate methods for further characterization. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two- hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized. Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR. Progress was made on all three objectives, which fall under NP303. Under Objective 1a: Additional isolates of Alternanthera mosaic virus (AltMV) were detected in Phlox stolonifera and P. divaricata, and in Mazus reptans; detection in M. reptans constitutes the first report for this host, and in mixed infection with Ligustrum necrotic ringspot virus. Plantago asiatica mosaic virus (PlAMV-MD) was detected for the first time in the United States in lily bulbs of several cultivars, all imported from the Netherlands. Although Nandina mosaic isolates of PlAMV have been known in the U.S. since 1978, Nandina mosaic isolates have never been reported to infect lilies. As PlAMV is known to cause significant losses in cut-flower lilies in both Europe and Asia, there is potential for economic loss to lily growers in the U.S. This work complements a collaboration with Dutch and Italian scientists to generate an infectious clone (Objective 2) of PlAMV to investigate transmission and movement of PlAMV within lilies. Thirteen species of ornamental Allium are being investigated for the presence of virus in collaborative research with a graduate student from Brazil. At least five viruses have been identified to date, including one potyvirus not previously reported to infect Allium species, as well as one carlavirus and three allexiviruses previously known to infect garlic, onion, or leeks. Two distinct sequences of Butterbur mosaic virus and one sequence of Helenium virus S (both carlaviruses) were obtained from a single plant of veronica showing mosaic symptoms (collaboration with National Germplasm Resources Laboratory). A new potyvirus, Shamrock chlorotic ringspot virus, was detected in ornamental Oxalis. At least one of these plants was also infected with a nepovirus, Beet ringspot virus; this is the first report of this virus in Oxalis. A U.S. isolate of Catharanthus mosaic virus was detected in Mandevilla; this is the first report of this virus in this host. All three of these viruses have been partially to fully sequenced. (Collaboration with National Germplasm Resources Laboratory and Michigan Dept. of Agriculture). Kalanchoe latent virus (carlavirus) and Kalanchoe mosaic virus (potyvirus) were detected in a Sedum plant with ringspots (collaboration with Michigan Dept. of Agriculture). Under Objective 1b: Additional members of the Alphaflexiviridae and Betaflexiviridae were successfully amplified using generic primers, and sequences obtained. Virus-specific primers were developed and tested for several species, including Ligustrum necrotic ringspot virus, Narcissus yellow stripe virus, Shamrock chlorotic ringspot virus, Catharanthus mosaic virus, and Beet ringspot virus. Several viruses detected under Objectives 1a and 1b were further characterized and partially sequenced. Under Objective 1c: Additional viruses, including some mixed infections, were successfully detected with the Universal Plant Virus Microarray (UPVM). In collaboration with scientists at the Danforth Center and the University of Utah, more than 100 additional UPVM slides were hybridized with test samples obtained from the DSMZ (Germany); the International Potato center (Peru); the International Center for Tropical Agriculture, Colombia; Washington State University, Pullman and Prosser, WA; from a USDA-ARS lab in Wooster, OH; and from our own local collections, and used to further validate the UPVM. Including these samples, the UPVM was validated for several additional viruses of different taxonomic groups, expanding the number of genera from which viral species have been correctly detected and identified to at least 44 genera and recognized taxonomic groups representing at least 15 families (with two genera unassigned to families). Two previously unassigned viruses were correctly identified to appropriate taxonomic groups. In addition, two recently-described viruses not represented by species-specific probes were identified to the correct genus. Nucleic acid extracts of several symptomatic samples (including �unknown� field samples from the International Potato Center), and previously characterized samples either treated by subtractive hybridization or untreated, were submitted for next generation sequencing (NGS) as well as being hybridized to the UPVM. The raw NGS results have been received, but not yet analyzed. Comparison of NGS results to UPVM results may reveal viruses not detected by the UPVM, due to either low titer (potentially including secondary components of mixed infections) or possibly novel viruses without representation among the 9556 virus- specific probes of the UPVM. Comparison of virus detection and identification in parallel samples either treated by subtractive hybridization, or not treated, will aid in determination of the cost- effectiveness of this treatment for both the UPVM and NGS approaches. Under Objective 2.1: Polyclonal and monoclonal antibodies to movement protein 1, MP-1 of various pelarspoviruses (using synthetic peptides containing species unique or genus conserved amino acid sequences) that are either specific to one species or cross-reactive to all species, respectively were developed. These antibodies will be useful in confirming the gene exchange of the MP-1 gene between infectious clones of these viruses as well as in experiments to determine the expression, localition, and functionality of the protein in viral pathogenesis. Under Objective 3.1: Sequence gaps that were lacking in our draft genome sequence of the mulberry strain of X. fastidiosa have been closed. Some open reading frames were manually annotated and the draft genome of this mulberry strain published. This mulberry strain was compared with other strains of X. fastidiosa, its unique open reading frames and designed primers identified. Those primers are now being tested for specific detection of this particular strain. The genes used in MLST in the mulberry strain were identified and used in establishing phylogenetic relationships between the mulberry strain and other X. fastidiosa strains. Under Objective 3.2: A multiplex PCR assay that includes using a host plant primer pair as an internal control was developed and tested, as well as R. solanacearum species- and r3b2-specific primer pairs, on 90 target and non-target R. solanacearum strains, including samples from soil and from different artificially infected plants including potato, geranium, and tomato to insure its specificity. Using the unique sequences that were previously identified, R. solanacearum species- and r3b2-specific, as well as plant-specific primers and probes were designed in order to develop a multiplex qPCR assay for R. solanacearum. Accomplishments 01 Determination of the complete genome sequence of a Xylella fastidiosa strain causing mulberry leaf scorch disease in Maryland. Xylella fastidiosa is a Gram-negative, nutritionally fastidious, xylem- inhabiting and insect-transmitted bacterium affecting over 30 plant families and causes bacterial leaf scorch and decline in many important woody ornamentals, including mulberry. ARS researchers in Beltsville, Maryland determined the draft genome of the mulberry strain (Mul-MD) in order to gain a better understanding of the molecular basis of strain divergence, host specificity, nutrient requirements, and pathogenicity, as well as to develop genome-based specific detection methods for this important plant pathogen. Understanding bacterial genome structures and functions, and the mechanisms of bacterial pathogenicity, will lead to a better understanding of host-pathogen interactions and mechanisms of plant disease resistance. This, coupled with more sensitive pathogen detection methods, will lead to the development of better, more environmentally friendly, disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer.
Impacts
(N/A)
Publications
- Seo, E., Nam, J., Kim, H., Lakshman, D.K., Bae, H., Hammond, J., Lim, H. 2014. Selective interaction between Chloroplast B ATPase and TGB1 retards severe symptoms caused by Alternanthera mosaic virus infection. Plant Pathology Journal. 11736-11743.
- Bobev, S.G., Taphradjiiski, O.I., Hammond, J., Vaira, A. 2013. First report of Freesia sneak virus associated with foliar necrosis of Freesia refracto in Bulgaria. Plant Disease. 97:1514.
- Nam, J., Nam, M., Bae, H., Lee, C., Lee, B., Hammond, J., Lim, H. 2013. AltMV TGB1 nucleolar localization requires homologous interaction and correlates with cell wall localization associated with cell-to-cell movement. Plant Pathology Journal . 29(4):454-459.
- Cho, S., Kim, J., Li, M., Seo, E., Lim, S., Moon, J., Hammond, J., Lim, H. 2013. Occurrance in Korea of three major soybean viruses, Soybean mosaic virus (SMV), Soybean yellow mottle mosaic virus (SYCMV), and Soybean yellow common mosaic virus (SYCMV) revealed by a nationwide survey of soybean fields. Research in Plant Disease. 19(4):313-325.
- Li, M., Kim, J., Seo, E., Hwang, E., Domier, L.L., Hammond, J., Youn, Y., Lim, H. 2014. Sequence variability in HC-Pro genes of Korean Soybean mosaic virus isolates is associated with differences in gene silencing suppression. Archives of Virology. 159(6):1373-1383.
- Lim, H.S., Vaira, A., Bae, H., Jang, C., Nam, J., Lee, C., Lee, Z., Hwang, J., Kim, H., Roh, M.S., Hammond, J. 2014. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein. Virology.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): The objectives of this project are 1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops and develop corresponding diagnostic testing methods; 2) Determine the genome organization of selected important ornamental viruses and utilize full- length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity; and 3) identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. The overall approach is to develop knowledge, tools, and reagents that will aid U.S. floriculture companies to establish effective virus testing protocols and improve clean stock production for new vegetatively propagated annuals and perennials. Based on the knowledge and tools developed while identifying and characterizing new viruses, new virus- specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols will be developed. A Universal Plant Virus Microarray, currently being developed cooperatively under an NRI grant, will add new capabilities in identifying newly emerging viruses to the appropriate family and genus, thus significantly aiding selection of appropriate methods for further characterization. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two- hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized. Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR. Progress was made on all three objectives, which fall under NP303. Under Objective 1a: Additional isolates of Alternanthera mosaic virus (AltMV) were detected in Phlox stolonifera and P. divaricata, and in Celosia hybrids from different sources. Several viruses were detected for the first time in new hosts (Ornithogalum mosaic virus in Ixia and Crocosmia; Triteleia mosaic virus in Brodaiea; Ligustrum necrotic ringspot virus in P. divaricata). Potentially �new� potyviruses were detected in Aconitum and Dichlostemma, and Aconitum latent virus carlavirus was detected in Aconitum (a new report for the U.S.). Parts of this work were carried out in collaboration with scientists at Agdia, Inc.; Michigan Dept. of Agriculture; and the Univ. Maryland. Under Objective 1b: Additional members of the Alphaflexiviridae and Betaflexiviridae were successfully amplified using generic primers, and sequences obtained. Under Objective 1c: Additional viruses, including some mixed infections, were successfully detected with the Universal Plant Virus Microarray (UPVM). Methods for normalizing amplification of viral and host RNAs were improved, allowing increased sensitivity of detection of low-titered. Several viruses detected under Objective 1a were further characterized and partially sequenced. Under Objective 2a: Interactions of several natural and mutant sequence variants of AltMV TGB1 were examined with a number of host proteins using Bimolecular Fluorescence Complementation (BiFC), in collaboration with a scientist at Chungnam National University (Korea) and at Beltsville using laser scanning confocal microscopy (LSCM). Under Objective 2b: Interactions of AltMV CP and TGB3 with plant host proteins were examined by LSCM, BiFC, and yeast two-hybrid analysis. Monoclonal antibodies were developed to several unique and conserved regions of the predicted movement protein of several pelarspoviruses. Under Objective 3.1: We obtained 20x draft genomes of four landscape strains of X. fastidiosa and are almost done with a fifth strain. We examined published MLST primers in the sequenced strains of X. fastidiosa. Under Objective 3.2: By using in silico genome subtraction, we identified 76 non-phage related fragments unique only to the r3b2 select agent strains of R. solanacearum. Similarly, we also identified sequences conserved among strains belonging to Ralstonia species complex, but not in other closely related species, as well as other plant pathogenic bacteria including Xanthomonas campestris and Pseudomonas syringii. With this information we designed and tested primers that are specific to R. solanacearum species complex, and to r3b2, respectively. We also developed a multiplex PCR assay by combining the species-, r3b2-, and plant-specific primers in a single reaction to allow fast and reliable detection and differentiation of r3b2 strains in plant extracts. Summaries to document research conducted under nine Specific Cooperative Agreements or Reimbursable Agreements between the Floral and Nursery Plants Research Unit and others can be found in those specific annual reports. Accomplishments 01 Improvement of current detection and identification methods for select agent strains of Ralstonia solanacearum via a multiplex PCR assay. R. solanacearum is a species complex that is pathogenic to a wide range of plant species in many regions throughout the world. Specifically detecting one subgroup of strains, the race 3 biovar 2 (r3b2) strains, is of utmost importance, since they are adapted to temperate climates and are listed as select agents due to their potential threat to US agriculture. ARS scientists at Beltsville, MD have designed R. solanacearum species- and r3b2-specific primers in a multiplex PCR assay, allowing for simultaneous detection and differentiation of r3b2 from non-r3b2 strains of R. solanacearum in a single reaction. An internal plant DNA control primer pair was also developed and included in the multiplex assays to improve the confidence and reliability of r3b2 detection in plant extracts by validating the plant extracts and excluding false negative results. Rapid, accurate, sensitive, and reliable detection and identification of r3b2 are critical for state and government officials to make timely and appropriate recommendations in safeguarding the movement of agricultural and horticultural products into the United States. 02 Critical interactions between viral proteins and plant host proteins. Plant viruses of the genus Potexvirus cause disease in a wide variety of crops; among these viruses, Alternanthera mosaic virus (AltMV) infects many taxonomically diverse ornamental crops. ARS scientists in Beltsville, MD, in collaboration with a scientist from Chungnam National University, Korea, have identified protein interactions between AltMV and the chloroplast of host plants that are critical in disease development. Mutation and gene expression studies revealed the role that these proteins may play in viral replication efficiency and movement, as well as the importance of chloroplast interactions in diseases caused by AltMV. This discovery will potentially allow disruption of the virus infection process through selection of plants that have protein variants that do not allow successful virus interactions, thereby leading to the development of virus-resistant plants. 03 First Report of Nerine yellow stripe virus in Amaryllis in the United States. Ornamental flower bulbs, including the well-known amaryllis cultivars, are increasingly important floriculture crops. A home gardener in California observed mosaic symptoms on the leaves of an Amaryllis belladonna plant growing in her garden. ARS Researchers in Beltsville, MD tested these leaves using PCR and electron microscopy and found that they contained a type of Potyvirus. Further analysis of DNA sequences and serological testing revealed the virus to be a unique US isolate of Nerine yellow stripe virus (NeYSV) that we designated NeYSV-US. NeYSV has previously been reported in the United Kingdom, the Netherlands, Australia, and New Zealand, but to our knowledge, this is the first report of Nerine yellow stripe virus in the United States.
Impacts
(N/A)
Publications
- Damsteegt, V.D., Stone, A.L., Smith, O.P., Mcdaniel, L., Sherman, D.J., Dardick, C.D., Hammond, J., Jordan, R.L., Schneider, W.L. 2013. A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia. Archives of Virology. 158:1235-1244.
- Li, W., Teixeira, D.C., Hartung, J.S., Huang, Q., Chen, J., Lin, H. 2012. Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis. Journal of Microbiological Methods. 92:79-89.
- Jang, C., Seo, E., Nam, J., Bae, H., Gim, Y., Cho, I., Lee, Z., Bauchan, G. R., Hammond, J., Lim, H. 2013. Insights into Alternanthera mosaic virus TGB3 functions: Interactions with Nicotiana benthamiana PsbO correlate with chloroplast vesiculation and veinal necrosis caused by TGB3 overexpression. Frontiers in Plant Science.
- Lim, H., Lee, M., Jang, C., Nam, J., Bae, H., Ju, H., Lee, M., Yu, Y., Hammond, J., Jackson, A.O. 2013. Interactions with the actin cytoskeleton are required for cell wall localization of barley stripe mosaic virus TGB proteins. Plant Pathology Journal . 29:17-30.
- Guan, W., Shao, J.Y., Singh, R., Davis, R.E., Huang, Q. 2012. A TaqMan- based real time PCR assay for specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander. Current Microbiology. 92:108-112.
- Solovyev, A.G., Makarova, S.S., Remizowa, M.V., Lim, H., Hammond, J., Owens, R.A., Kopertech, L., Schiemann, J., Morozov, S.Y. 2013. Possible role of the Nt-4/1 protein in macromolecular transport in vascular tissue. Plant Signaling and Behavior. 8(10):e25784.
- Guaragna, M.A., Lamborn, J., Hammond, J., Schadewijk, T., Jordan, R.L. 2013. First report of Nerine yellow stripe virus in Amaryllis in the United States. Plant Disease. 97(10):1389.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The objectives of this project are 1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops and develop corresponding diagnostic testing methods; 2) Determine the genome organization of selected important ornamental viruses and utilize full- length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity; and 3) identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. The overall approach is to develop knowledge, tools, and reagents that will aid U.S. floriculture companies to establish effective virus testing protocols and improve clean stock production for new vegetatively propagated annuals and perennials. Based on the knowledge and tools developed while identifying and characterizing new viruses, new virus- specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols will be developed. A Universal Plant Virus Microarray, currently being developed cooperatively under an NRI grant, will add new capabilities in identifying newly emerging viruses to the appropriate family and genus, thus significantly aiding selection of appropriate methods for further characterization. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two- hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized. Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR. This report documents progress for Project Number 1230-22000-032-00D, which started in May 2012 and continues research from Project Number 1230- 22000-022-00D, entitled �Detection, identification and characterization of new and emerging viral and bacterial diseases of ornamental plants" which terminated in April 2012. Ligustrum necrotic ringspot virus was detected for the first time in plants of Mazus radicans, in addition to further plants of Mazus reptans; this virus has previously been reported only in Ligustrum (privet), apart from our first identification and reports in New Guinea Impatiens, Phlox stolonifera and Mazus reptans. Nerine yellow stripe virus (NeYSV) was detected for the first time in Amaryllis in California. NeYSV has previously only been reported in the United Kingdom, the Netherlands, Australia, and New Zealand. A putative carlavirus has been identified by electron microscopy and a generic polymerase chain reaction (PCR) assay in Magnolia tripetala showing significant mosaic and ringspot symptoms from two locations; cloning and sequencing of the PCR product is in progress to further identify the virus. In collaboration with a plant breeder, we have previously identified the causal agent of a mosaic disease of peppers and tomatoes as a rhabdovirus. Partial sequence data indicates that the virus is most closely related to, but distinct from, Potato yellow dwarf virus. We have now sequenced approximately 25% of the viral genome by PCR extension from previously determined sequences. Samples of infected tissue are currently being analyzed by high-throughput �deep� sequencing. In collaboration with a graduate student at the University of Maryland, a hemi-nested PCR assay was developed to increase the sensitivity of detection of Cymbidium mosaic virus (CymMV). The new method yielded an essentially saturated signal, even for samples of the initial PCR product diluted 1:312,500. This assay is capable of detecting most, if not all, known isolates of CymMV, including recently-identified isolates that do not react with some CymMV-specific antisera. Several plant virus genes (coat protein genes of Nerine yellow stripe virus and Pelargonium chlorotic ringspot virus) and one modified Bacillus thuringiensis Cry6A protein gene (demonstrated to confer plant resistance to an endoparasitic nematode) were cloned into bacterial expression vectors. The expressed proteins were isolated, purified and used as immunogens for the production of antibodies that will be useful in detecting the respective proteins in infected or transgenic plants. Three landscape strains of Xylella fastidiosa (Xf) were isolated, verified, and propagated. One tree strain of Xf was obtained from ATCC and propagated and verified. Genomic DNAs for all four landscape tree strains were prepared for large-scale sequencing. GenBank sequences and contigs from NCBI�s whole genome and shotgun reads database for Ralstonia solanacearum have been downloaded and compared. Accomplishments 01 A PCR assay developed for the specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander. Oleander leaf scorch caused by X. fastidiosa has become a serious proble in recent years throughout the American west and southwest including California, Arizona and Texas. A sensitive and specific detection and quantification of oleander strains of X. fastidiosa is greatly needed in order to facilitate epidemiological and etiological studies and control this disease. In collaborative research with scientists from Louisiana State University and the Chinese Academy of Agricultural Sciences, ARS scientists at Beltsville, MD developed a new PCR assay that allows specific and sensitive detection and quantification of X. fastidiosa strains causing oleander leaf scorch disease, and differentiation of oleander strains from other strains of X. fastidiosa. This method will b useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shru
Impacts
(N/A)
Publications
- Huang, Q., Yan, X., Wang, J. 2011. Improved biovar test for Ralstonia solanacearum. Journal of Microbiological Methods. 88:271-274.
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