Project : USDA ARS

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Research Project: Detection, Identification, and Characterization of New and Emerging Viral and Bacterial Diseases of Ornamental Plants

Location: Floral and Nursery Plants Research

2019 Annual Report

Objectives
The three 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. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. 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 (tested for absence of specific pathogens) 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
The overall approach is to develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively-propagated annuals and perennials. 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. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, new virus-specific and broad spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have the potential to identify any virus present and identify all components of mixed infections, and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST-pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods.

Progress Report
We examined samples of various ornamentals from plant disease clinics, nurseries, or individuals, including: i) a plant of Veronica found to be infected by Helenium virus S as with prior plants of three cultivars; ii) plants of Phlox maculata x Phlox glabberima, and Phlox carolina showing necrotic streaking and etching of leaves - electron microscopy revealed pinwheel cytoplasmic inclusions typical of potyviruses; iii) plants of Coreopsis showing brown leaf streaking - a putative potyvirus was detected by PCR using generic potyvirus primers. In collaboration with scientists from the Systematic Entomology Laboratory and the Electron and Confocal Microscopy Unit at Beltsville, Maryland, we continue to examine interactions between eriophyid mites and different rose species or genotypes received from collaborators under a USDA-NIFA-SCRI project to examine the viral, vector, and host factors affecting spread of Rose rosette virus and the associated disease in roses. Collaboration was initiated with scientists from USDA-APHIS and PathSensors, Inc. (Baltimore, Maryland) to develop improved protocols for detection of potyviruses using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) biosensor system. Several years ago, the USDA-ARS Beltsville Lab developed a hybridoma cell line that secretes a broad-spectrum reacting PTY-1 monoclonal antibody (McAb). This hybridoma and McAb, which detects almost all known aphid-transmitted potyviruses, was patented and licensed to Agdia, Inc., and forms the basis of Agdia’s “POTY Group Test” reagents, ELISA kits, and ImmunoStrip Test. CANARY is a fast, sensitive, and easy-to-use biosensor-based technology developed at MIT that has been successfully utilized for the detection of bacteria, viruses, and toxins. Several PTY-1 hybridoma-derived POTY CANARY biosensor clones were created at MIT-LL, initially tested in the APHIS-CPHST Beltsville Lab, and later evaluated collaboratively in the USDA-ARS Beltsville and PathSensors labs, for assay feasibility, bead capture efficacy, specificity and sensitivity of the reaction to detect target potyviruses. We have optimized the POTY biosensor assay and shown that it can detect diverse purified potyviruses down to less than 5 ng/ml and can detect multiple potyviruses in naturally infected sources, such as various isolates of potato virus Y in potato leaves and tubers. We continue to utilize broad-spectrum PCR primers for analysis of a wide range of suspected virus-infected plants, allowing detection of previously reported viruses in new hosts, or obtaining products for sequence analysis of newly-discovered viruses allowing their comparison to previously characterized viruses. Using generic primers capable of amplifying products from multiple potyviruses, or both potexviruses and carlaviruses, products were obtained from samples including: Coreopsis (an unidentified potyvirus), Veronica (Helenium virus S, a carlavirus), and bamboo (Bamboo mosaic virus, a potexvirus). In our ongoing research on the development of broad-spectrum reacting antibodies to important plant virus species, additional potexvirus and carlavirus sequences have been cloned for bacterial expression of the coat protein sequences to aid in the analysis of cross-reactive rabbit polyclonal (PcAb) and mouse monoclonal antibodies (McAbs) recently generated to synthetic peptide immunogens containing highly conserved regions of carlavirus or potexvirus coat proteins. Carlavirus peptide-specific PcAbs were screened against the immunogen peptides, virions and cloned CPs from more than 20 diverse carlaviruses and were shown to react with multiple carlaviruses. Eighty newly developed hybridoma cell lines secreting McAbs reactive to these key carlavirus epitopes were also developed and initially assayed; 10 cell lines were selected for further study. Potexvirus-specific PcAbs were also evaluated for broad-spectrum reactivity to peptides, virions and cloned CPs from about 20 diverse potexviruses and were shown to react with most of these potexviruses, as well as many carlaviruses. High throughput sequencing of an isolate of Tulip virus X (potexvirus) from lemon balm showing mosaic symptoms has been validated by amplification, cloning and conventional sequencing of RT-PCR products together representing most of the viral genome, including the 5’ and 3’ terminal regions. The genome sequence obtained differs from characterized isolates from tulip (Japan) or lily (Korea), likely reflecting adaptation to lemon balm, which is a dicotyledonous host in contrast to the prior monocotyledonous hosts. The genome of Helenium virus S (carlavirus) from Veronica has been partially validated by sequencing of RT-PCR products made with primers designed from the genome sequence from high throughput sequencing. In collaboration with scientists of the Central Science Laboratory, Food and Environment Research Agency, U.K. plant materials infected with Plantain virus X (PlVX) were identified, and the first ever sequence of this virus was obtained, along with a full genome sequence of two isolates by high throughput sequencing, and partial sequences of nine other isolates from the UK. A scientist from the Netherlands Food and Consumer Product Safety Authority/National Plant Protection Organization collaborated to identify and obtain partial sequences of two Dutch PlVX isolates from Plantago lanceolata. PlVX was most closely related to Actinidia virus X, reported in 2011 from Actinidia chinensis (Kiwifruit) imported to New Zealand from China. Sequence relatedness (89-100%) of the respective replicase and coat protein genes indicates that Actinidia virus X and Plantain virus X are synonymous, with the name Plantain virus X having precedence. Whereas PlVX was previously only reported from the U.K., it is now known to be present in the Netherlands in P. lanceolata, in New Zealand (and presumably China) in A. chinensis, and (as Actinidia virus X) from Canada in Ribes nigrum (Black currant). The experimental host range of PlVX includes species in at least five plant families in addition to the reported natural hosts, suggesting possible future host expansion. In partnership with Korean collaborators, infectious clones of two isolates of Tomato mosaic virus (ToMV) were produced and shown to induce symptoms of differing severity in Nicotiana benthamiana. Only two amino acid residues differentiate the proteins of each isolate, with one difference in each of the non-conserved and helicase domains of the replicase gene. Single amino acid substitutions between mild and severe symptom variants showed that the symptom differences were dependent on the residue change in the helicase domain. In partnership with Korean collaborators, infectious clones of five Turnip mosaic virus (a potyvirus) isolates from Jeju Island were produced. The viral determinants of stunting in Nicotiana benthamiana were examined by sequence comparisons and gene exchange to identify the genomic regions associated with symptom types. Four of five isolates caused mild symptoms; the fifth induced a hypersensitive response but differed by only five amino acid residues from one mild isolate. Exchange of genome fragments between these two isolates identified an amino acid residue in the HC-Pro gene that was required, but not sufficient, for induction of severe symptoms; this residue must also interact with a differential residue in the NIb gene and possibly another in the P3 gene. Variation in the P3 gene is known to influence symptoms in other potyviruses. In partnership with Korean collaborators, sequence differences between isolates of Tomato yellow leaf curl virus (TYLCV) collected in different regions of Korea in 2017 and 2018 were most closely related to previously characterized isolates belonging to either of two out of three phylogenetic clades of TYLCV, the ‘Japan’ cluster or the ‘China’ cluster, while some earlier-reported Korean isolates belong to a ‘Japan+China’ cluster. This suggests at least three separate introductions of TYLCV into Korea, where TYLCV was first detected in 2008, and that isolates from the ‘Japan’ cluster are becoming more established; only isolates of this group were detected in 2018. In partnership with Korean collaborators, interactions between the P3 and P3N-PIPO frameshift proteins of Turnip mosaic virus were examined by confocal laser scanning microscopy. Prior studies identified the P3 gene region as containing determinants of pathogenicity, but there is another essential function expressed as a frameshift protein, P3N-PIPO; differential amino acids occur in both the P3 and P3N-PIPO gene products. Fluorescently-labeled fusion proteins were expressed in Nicotiana benthamiana by agroinfiltration, and differences in subcellular localization observed between P3 and P3N-PIPO. The genomes of tree strains of Xylella fastidiosa were purified and characterized. The unique open reading frames (ORFs) of the tree, pauca and Italian olive strains were analyzed and used to develop specific qPCR tools for the detection and differentiation of pauca and Italian olive strains of X. fastidiosa (in collaboration with USDA-APHIS scientists). We isolated the first R. solanacearum-infecting phage from soil in the United States. Biological and molecular characterization of the phage were also determined. We also sequenced and characterized the first R. solanacearum-infecting phage isolated from Indonesia. We mutated five regions identified previously that contain Ralstonia solanacearum race 3 biovar 2 (r3b2)-unique DNA regions. One of the mutated regions was found to be associated with the cool virulence of r3b2, and this region was used for specific detection of r3b2 strains of R. solanacearum.

Accomplishments
1. Detection of select agent strains of Ralstonia solanacearum through a DNA region associated with cool virulence. Ralstonia solanacearum r3b2 strains cause a devastating brown rot disease of potato. These bacteria are regulated select agent pathogens in the U.S. because they are capable of surviving and infecting potatoes under cool temperatures (‘cool virulence’) in areas that produce seed potatoes, thereby potentially threatening U.S. agriculture. The DNA regions responsible for the cool virulence trait of R. solanacearum r3b2, however, are largely unknown and need to be identified for the accurate definition and specific detection of these select agents. ARS scientists in Beltsville, Maryland, found that when one specific DNA region of the r3b2 genome was mutated, the mutant bacterium’s ability to cause disease under cool, but not warm, temperature conditions was significantly reduced - suggesting the association of this region with the cool virulence of R. solanacearum r3b2. Using this information, we developed the first DNA-based detection method to target the DNA region associated with cool virulence of r3b2 for specific detection and eventual control of this important group of R. solanacearum pathogens.

2. Sequence variations affect symptoms and pathogenicity of Turnip mosaic virus. Turnip mosaic virus (TuMV) affects many food and ornamental crops; plant resistance to this virus is useful to control disease in some crops, but new isolates capable of breaking resistance often emerge. An ARS scientist in Beltsville, Maryland, collaborated with Korean scientists to generate infectious cDNA clones from 17 new radish isolates of TuMV, and differential pathogenicity and infectivity were demonstrated in radish, Chinese cabbage, and the experimental host Nicotiana benthamiana. Three classes of isolates producing only mild symptoms in N. benthamiana were identified, whereas all remaining isolates induced systemic necrosis; differential susceptibility or resistances were identified in two cultivars each of radish and Chinese cabbage, with one Chinese cabbage cultivar not infected by any isolate. Gene exchanges between a mild and a severe isolate identified a novel pathogenicity determinant allowing infection of the Chinese cabbage cultivar resistant to all of the original isolates. These results may allow breeders to identify potential new resistance genes in germplasm and their incorporation into new crop varieties. New resistance genes may be combined with other types of resistance to yield broader, longer-lasting resistance to multiple viral pathotypes.

3. Two determinants of Turnip mosaic virus are required to induce stem necrosis in Nicotiana benthamiana. Introduction of novel viral pathotypes into a new area may cause breakdown of crop resistance. An ARS scientist in Beltsville, Maryland, collaborated with Korean scientists to determine whether sequence differences between four radish isolates of Turnip mosaic virus from China were associated with the degree of symptom severity. Only two isolates induced obvious symptoms in the natural host (radish), whereas symptoms in an experimental host (Nicotiana benthamiana) varied from mild to severe mosaic with associated stem necrosis. A domain containing three of four differential amino acid residues distinguishing two isolates conferred most of the more severe symptom characteristics, but not stem necrosis, in Nicotiana benthamiana. An interaction between a particular differential residue in this domain and the fourth differential residue in a different domain is most likely required to induce stem necrosis, as neither alone was sufficient. These isolates induced differential symptom severity in two radish cultivars, indicating that the radish genotype itself affects symptom severity. Similarities between the Chinese isolates and previously characterized Korean isolates suggests genetic interchange between Chinese and Korean isolates through trade, or as a result of long-distance transmission by aphid vectors carried by prevailing winds.

4. Molecular and biological characterization of the first Ralstonia phage, RsoM1USA, isolated from soil in the United States. Ralstonia bacteriophages are viruses that specifically infect the bacterium Ralstonia solanacearum (Rs), a causal agent of a destructive bacterial wilt disease in tropical, subtropical and warm temperate regions of the world. They have recently been isolated from soils in Japan, Thailand, Korea and Egypt. In an effort to better understand their relationships with Rs and to determine their potential as biocontrol agents for Rs, ARS scientists in Beltsville, Maryland, purified and characterized RsoM1USA, the first Ralstonia-infecting phage isolated from soil in the United States. Infection of Ralstonia by phage RsoM1USA resulted in significantly reduced in vitro growth of the infected bacterium, but not disease symptoms caused by the bacterium in tomato plants. Our goal is to determine if RsoM1USA offers a competitive advantage to the infected bacterial strain for persistence of the bacterium in the environment.

5. Detection and first report of beet ringspot virus in ornamental Oxalis in the United States. Ornamental Oxalis triangularis, commonly known as False Shamrock because of its triangular leaves, is grown as a potted plant in the United States, especially for marketing in the spring around St. Patrick’s Day. ARS scientists in Beltsville, Maryland, discovered a previously unreported virus in Oxalis plants from Wisconsin that were showing chlorotic ringspot symptoms. Full genome sequence analysis revealed that this virus was a strain of beet ringspot virus (BRSV), a soil-borne virus originally thought to be synonymous with the quarantine-regulated nepovirus tobacco blackring virus. BRSV infects a wide range of plant species including potato, sugar beet, strawberry, turnip, wheat, oat, peach, begonia and Euonymus. This is the first report of BRSV in ornamental Oxalis. The information and detection tools developed in this work will be useful to ornamental nurseries, public and private plant disease diagnostic clinics, and other scientists who study ornamental viruses, as well as to state and federal regulatory officials to help them make timely and appropriate recommendations in safeguarding the movement of horticultural products into and throughout the U.S.

U.S. DEPARTMENT OF AGRICULTURE

Floral and Nursery Plants Research: Washington, DC