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

2021 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. Additional resources in the merged project will strengthen the research in the current Objective 1: Objective 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]

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
Under Objective 1a: We examined samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples included: a) plants of Lindera benzoin from a Maryland nursery, showing a strong foliar mosaic and distortion, which was determined by reverse transcriptase-polymerase chain reaction assay to be infected with an emaravirus recently sequenced by a collaborating ARS scientist at Beltsville, Maryland, in samples with similar symptoms from Indiana and Maryland, and also detected in Kentucky, Missouri, Ohio, and Virginia; the virus has been tentatively named Lindera severe mosaic virus, and in collaboration with an ARS mite expert, the putative eriophyid mite vector found on infected plants was identified as Phyllocoptes linderafolius. b) plants of Callicarpa americana showing foliar mosaic were shown to be infected with Constricta yellow dwarf virus (previously known as Potato yellow dwarf virus-CYDV). This is new host for this virus, which mainly infects solanaceous plants and a few herbaceous hosts. Under Objective 1d: In collaboration with other ARS colleagues at Beltsville, Maryland, several samples were subjected to high-throughput sequencing, including: a) a composite sample of Liriope muscari showing chlorotic or whitish foliar streaking, in which long flexuous virus-like particles were previously revealed by electron microscopy, yielded near complete sequences of a novel allexivirus, and of tobacco streak virus; b) a sample originating from clover, and subsequently propagated in Nicotiana benthamiana. This was previously determined to be infected by a potexvirus, but high throughput sequencing revealed the presence of a complex mixed infection of two distinct isolates of clover yellow mosaic virus and also two distinct isolates of white clover mosaic virus; c) samples of Magnolia tripetala from Massachusetts were similar to nearly complete sequences of either apple mosaic virus (previously detected in a sample from Pennsylvania) or a carlavirus closely related to isolates previously detected in a sample from Virginia, and to the full sequence previously identified by a colleague from the University of Mississippi in a sample from North Carolina; d) a plant of Rehmannia angulata showing an obvious mosaic was found to be infected with an isolate of cucumber mosaic virus; e) samples from other Callicarpa americana plants showing foliar mosaic were not found to be infected with either of two novel emaraviruses already under study, but were shown to be infected with a novel badna virus; and, f) in partnership with Korean collaborators, three new virus:crop combinations were detected for the first time in Korea - Passiflora latent virus was identified infecting persimmon (Diospyros kaki), Tomato mosaic virus in chili pepper (Capsicum annuum), and Cucurbit chlorotic yellows virus in muskmelon and Oriental melon (Cucumis melo). Under Objective 2a: In partnership with Korean collaborators, full length infectious clones of three isolates of radish mosaic virus differing in host responses were developed and pseudo-recombinants generated by inoculating groups of plants with RNA1 of each isolate separately with RNA2 of each of the three isolates. By this means it was shown that induction of systemic necrosis in Nicotiana benthamiana is determined by RNA1 of isolates A or G, whereas severe mosaic, necrotic ringspots, and veinal necrosis in radish was determined by RNA1 of isolate B. Thus isolates A or G and showed reciprocal results to isolate B for symptom severity and systemic necrosis between N. benthamiana and radish. Japanese scientists had previously implicated an amphipathic helix in the helicase protein encoded by RNA1 as the inducer of necrosis in N. benthamiana, but the amino acid sequence of this helix is identical in the helicase proteins of the three Korean isolates, suggesting that a differential interaction with host proteins of N. benthamiana and radish determined by another region of the helicase is responsible for the reciprocal symptom difference in these two hosts.

Accomplishments
1. Development of a high-throughput virulence screening method for Ralstonia solanacearum species complex strains. R. solanacearum species complex strains are the causative agents for wilting diseases of many plants, including the economically important brown rot of potato. ARS scientists at Beltsville, Maryland, developed a high-throughput virulence screen implemented in 96-well microtiter plates using Nicotiana glutinosa grown in soft water agar which saves space, time, and resources. This assay can be used to rapidly screen many strains, isolates, or mutants for disease symptoms under both cool (20C) and warm (28C) temperature conditions to narrow down isolates for more detailed testing. This assay provides a valuable tool for future work in understanding genetics of virulence of R. solanacearum species complex strains, especially the cool virulence of the select agent race 3 biovar 2 group of R. solanacearum, leading toward development of effective control strategies.

2. A sensitive and specific tool to detect and identify strains of Xylella fastidiosa infecting American mulberry and Italian olive. The bacterium X. fastidiosa causes serious plant diseases in many important agricultural crops and landscape trees. In Italy, it was identified for the first time in 2013 in olive trees affected by a devastating disease denoted ‘olive quick decline syndrome’. Recently, it has also been found in other European countries including Germany, France, Spain, and Portugal. An ARS scientist in Beltsville, Maryland, collaborated with scientists in Italy to develop TaqMan-based qPCR, Loop-mediated isothermal amplification (LAMP), and Fluorescence of TaqMan Probe upon Dequenching - Loop-mediated Isothermal Amplification (FTP-LAMP) assays based on a unique gene identified by the ARS scientist. The new FTP-LAMP assay is highly sensitive, detecting down to 100 fg genomic DNA of X. fastidiosa, and highly specific (98.7% vs 89% for other LAMP-based assays). Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP assay represents a useful tool not only for the specific detection of the olive-associated strain in Italy, but also in differentiating the Italian De Donno strain from other strains of X. fastidiosa already reported in Italy, Germany, France, Spain, and Portugal.

3. First complete genome sequence of the genus Carlavirus type species, Carnation latent virus. The International Committee on Taxonomy of Viruses (ICTV) is charged with the task of developing, refining, and maintaining a universal virus taxonomy. Carlavirus is a genus of plant viruses that includes 53 species recognized by ICTV. Molecular sequences of many of these species are well-represented in the NCBI GenBank database. However, surprisingly, the type species for this genus, Carnation latent virus (CLV), had no full-length genomic sequences in the database. ARS scientists in Beltsville, Maryland, determined the full-length genome sequence of CLV using high-throughput sequencing. The complete sequence was determined to be 8,513 nucleotides and was deposited in GenBank. The sequence data and phylogenetic analysis confirm that carnation latent virus is distinct from all other recognized carlaviruses. This full genome sequence of Carnation latent virus will now serve as the Genome Reference Sequence to which all other carlaviruses are compared.

4. Discovery of a Streptomyces scabies-infecting bacteriophage from Egypt with promising biocontrol traits. Potato common scab caused by Streptomyces scabies is one of the most economically important diseases infecting potato. Biological control using bacteriophages is a promising strategy for controlling this disease. An ARS scientist in Beltsville, Maryland, in collaboration with scientists in Egypt, discovered and characterized a novel bacteriophage (designated SscP1EGY) isolated from a potato field in Egypt that showed high lytic efficacy against seven tested S. scabies strains, but showed no lytic activity against three beneficial Streptomyces species, other beneficial bacterial species, and non-target plant pathogenic bacteria. In greenhouse experiments, treatment of S. scabies-inoculated potato tubers with phage SscP1EGY resulted in superficial rather than pitted scab lesions, and reductions in the number of lesions and overall severity of scab, compared to inoculated tubers without phage treatment. These results suggest that SscP1EGY has potential as a biological control agent for this economically important pathogen.

5. Identification and characterization of a global regulator, AclR, in Acidovorax citrulli. LuxR-type transcriptional regulators are essential for many physiological processes in bacteria, including pathogenesis. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. However, the LuxR-type transcriptional factors in A. citrulli have not been well studied, except the previously reported LuxR-type regulatory protein, AcrR, involved in regulating virulence and motility. Scientists in China, in collaboration with an ARS scientist in Beltsville, Maryland, characterized a second LuxR-type regulator, AclR, in the group II strain Aac-5 of A. citrulli by mutagenesis and transcriptomic analysis. They found that AclR plays a global role in transcriptional regulation in A. citrulli influencing motility, biofilm formation, and virulence, and provides a new perspective regarding the regulatory network of biological functions in A. citrulli. Increased understanding of the molecular mechanisms behind the regulation of A. citrulli by AclR will facilitate the development of effective control strategies to combat this important bacterial pathogen.

6. Genome sequences of two distinct carlaviruses from a mixed infection in veronica. Mixed infections may result in either more severe or attenuated symptoms compared to single infections. A Veronica hybrid showing foliar mosaic and distortion was previously found by ARS researchers in Beltsville, Maryland, to be infected by a mixture of two distinct carlaviruses. Near-complete sequences of both butterbur mosaic virus (ButMV) and helenium virus S (HelVS) revealed that two distinct isolates of ButMV were present, one of which was defective, having a deletion of almost the complete sequence of a gene normally considered essential; this is the first example of a defective isolate of a carlavirus being maintained in the viral population through complementation by the fully functional ButMV isolate. The sequence of HelVS is the first near-complete genome of any HelVS isolate. This work expands the known occurrence of viruses in this perennial flowering ornamental; both viruses are transmitted by aphids and have restricted natural host ranges; ButMV has previously been reported only from Japanese butterbur (Petasites japonica) in Japan and South Korea, while HelVS has previously been reported only from Helenium amarum and Impatiens holstii in Europe and the USA. Testing of stock plants of veronica prior to mass propagation is suggested to minimize the distribution of these viruses in nursery-grown plants.

U.S. DEPARTMENT OF AGRICULTURE

Floral and Nursery Plants Research: Washington, DC