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

Location: Floral and Nursery Plants Research

2016 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
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
1. 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.

2. 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.

3. 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.

4. 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.

5. 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.


None.


Review 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.