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ARS Home » Northeast Area » Washington, D.C. » National Arboretum » Floral and Nursery Plants Research » Research » Research Project #423161

Research Project: NEW AND EMERGING VIRAL AND BACTERIAL DISEASES OF ORNAMENTAL PLANTS: DETECTION, IDENTIFICATION, AND CHARACTERIZATION

Location: Floral and Nursery Plants Research

2017 Annual Report


Objectives
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
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 Report
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
1. 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.

2. 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.

3. 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.


Review Publications
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. 98(6):1526-1536. https://doi.org/10.1099/jgv.0.000771.
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.
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.
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. ICTV virus taxonomy profile: Ophioviridae. Journal of General Virology. 98(6):1161-1162. https://doi.org/10.1099/jgv.0.000836.