Project : USDA ARS

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Research Project: Detection, Biology, and Genomics of New and Emerging Viral and Bacterial Diseases of Ornamental Plants

Location: Floral and Nursery Plants Research

2023 Annual Report

Objectives
Objective 1: Identify and characterize new, emerging, and re-emerging viruses of major significance to ornamental and nursery crops, develop new technologies and specific reagents for their detection, and examine the virus-vector biology associated with selected key viral diseases. (NP303, C1, PS1A, PS1B; C2, PS2B, PS2D) Sub-objective 1A: Identification, characterization, distribution, and detection of emerging or previously unknown viruses. [Non-hypothesis research]. Sub-objective 1B: Development of broad-spectrum and virus-specific reagents. [Non-hypothesis research] Sub-objective 1C: Determine and characterize complete viral genome sequences and virus diversity using high-throughput sequencing. [Non-hypothesis research] Objective 2: Determine and analyze the viral genome sequences and organization of selected high-impact ornamental and nursery crop plant-infecting viruses, including the utilization of new tools to evaluate the role of viral genes and gene products in transmission, pathogenicity, and disease development. (NP303, C2, PS2A) Sub-objective 2A: Identify viral determinants of host specificity, pathogenicity, and vector transmissibility. [Hypothesis 1: Site-directed mutagenesis or exchange of genes or genome segments between infectious clones of virus isolates having different host ranges or symptom responses will allow attribution of host-specificity, symptom, or vector transmission determinants to specific gene/regions.] Sub-objective 2B: Identify essential virus-host protein interactions as potential targets for disruption of viral infection [Non-hypothesis 2: Identification of crucial interactions between viral proteins and host proteins will allow determination of those interactions necessary for the establishment of infection, or for cell-to-cell and/or long-distance movement within the plant, and elucidate future potential targets for disrupting virus infection or systemic movement by gene-editing of appropriate host proteins.] Sub-objective 2C: Identify the interactions between emaraviral particles and the eriophyid mite vector which confer transmission specificity. [Non-hypothesis research]. Objective 3: Characterize genomes and strains and bacteriophages of bacteria of major significance to ornamental and nursery crops for culture collection and develop accurate pathogen detection tools and potential effective control methods, including those for select agent strains of Ralstonia solanacearum (Rs). Sub-objective 3.1a: Identify important genetic components contributing to cool-virulence of Rs strains and develop a rapid purification-free plant DNA extraction method for detection of R. solanacearum species complex (Rssc), including the select agent r3b2 IIB-1&2 strains of Rs. [Non-hypothesis] Sub-objective 3.1b: Determine the role of bacteriophages isolated from Rssc-infested soil or directly from a tropical Rssc strain in virulence and competitive fitness of Rssc strains. [Non- hypothesis] Sub-objective 3.2: Determine genomes, identify unique ORFs and develop highly specific diagnostic markers to detect and differentiate landscape tree strains from other Xf strains. [Non- hypothesis].

Approach
Approach 1. 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 "new" uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Develop 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. High-throughput sequencing (HTS) of nucleic acids from plants infected with unknown viruses will yield information about the genomes of previously uncharacterized viruses. HTS has 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). HTS will also be used to examine virus diversity of selected viruses, including rose rosette virus. Approach 2. Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. 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. Develop and modify infectious clones of selected viruses by gene exchange and site-directed mutagenesis. Examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST-pull down assays. Virus-Induced Gene Silencing (VIGS) and/or protein over-expression will also be utilized. Identify and examine mechanisms and specificity of the interactions between rose rosette virus particles and the eriophyid mite vector, in addition to determining the minimal complement of viral RNAs necessary for mite transmission. Approach 3. Characterize genomes and strains and bacteriophages of bacteria of major significance to ornamental and nursery crops. Develop pathogen detection tools and effective control methods, including those for select agent strains of Ralstonia solanacearum (Rs). Identify genetic elements and bacteriophages contributing to cool-virulence and competitive fitness traits of Ralstonia solanacearum species complex (Rscc) strains for accurate detection and effective control of Rs. Determine the complete genomes of additional ornamental strains of Xylella fastidiosa (Xf) using HTS. Identify unique ORFs and develop highly specific diagnostic markers to detect and differentiate landscape tree strains from other Xf strains.

Progress Report
Under Objective 1A: RNA was extracted from samples of Callicarpa americana obtained from Alabama, Florida, South Carolina, Maryland, and Washington, DC, and compared to the original sample from Alabama, whose full genome was sequenced in 2017. Whereas the Alabama sample was previously found to be infected with two distinct emaraviruses, tentatively designated as Callicarpa mosaic-associated viruses 1 and 2 (CaMaV-1 and CaMaV-2), only CaMaV-2 was detected by nucleic acid amplification using isolate-specific primer sets from Florida, South Carolina, and Washington, DC, samples, and neither emaravirus was detected in the Maryland sample. The nucleoprotein gene of the Florida isolate was shown to be more diverse than the others. Additional symptomatic C. americana samples were recently obtained from two locations in Texas, and RNA was prepared for further testing. Emaraviruses for which the vector is known are all transmitted by various species of eriophyid mites. Only low numbers of eriophyid mites were observed on samples from Florida and Washington, DC, which were tentatively identified to the genus Aceria by a collaborating ARS mite expert. However, it is not currently known if this mite species is able to transmit CaMaV-1 or CaMaV-2, as at least six species of eriophyid mites have been reported from Callicarpa species in various countries. We have previously reported the detection of an emaravirus causing a severe foliar mosaic and distortion on spicebush (Lindera benzoin) in Maryland and named Lindera severe mosaic-associated virus (LSMaV), and possible seed transmission. In the summer of 2022, the presumed eriophyid mite vector of LSMV, Phyllocoptes linderafolius, was observed on the immature fruit of spicebush (in collaboration with an ARS mite expert). In the fall of 2022, seeds of spicebush were collected separately from seven apparently healthy plants and from five obviously symptomatic plants to further examine the possibility of seed transmission. A few seed from an obviously symptomatic plant were set aside for virus testing and microscopic examination, while the remainder of the seeds were cleaned and germinated separately from non-symptomatic and obviously symptomatic to observe for possible virus transmission to the emerging seedlings. When the seed collected from symptomatic plants were observed at high magnification under a dissecting microscope, and subsequently with a table-top scanning electron microscope, eriophyid mites (P. linderafolius) and their eggs were observed on the fruit, under the peduncle, which appears to offer a refuge from predatory mites. Interestingly, when seedlings emerged in spring, and were transplanted into trays, only four of about 485 seedlings (~320 from non-symptomatic plants, and ~165 from symptomatic plants) showed possible virus-like symptoms, only one of which continued to show symptoms on later-developing leaves; that single plant originated from a non-symptomatic tree. Results from both 2022 and 2023 suggest a seed transmission rate of less than 1%, as occurs with some other types of plant viruses. The nearly complete genome of Lindera severe mosaic-associated virus (LSMaV) was previously obtained, consisting of four RNA segments encoding the RNA-dependent RNA polymerase, the glycoprotein precursor, the nucleoprotein, and the presumed movement protein. Phylogenetic analysis of the amino acid sequences of these four proteins, and the equivalent proteins of previously described emaraviruses, confirmed that LSMaV is distinct from other emaraviruses but is most closely related to the viruses previously assigned to clade C, including raspberry leaf blotch, Ti ringspot-associated, High plains wheat mosaic, and common oak ringspot-associated viruses. We examined samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These included symptomatic material of Alocasia, Asclepias, blackberry, bur oak, Callicarpa, Freesia, Hamamelis, Hosta, Lilium, Magnolia, Norway maple, Petunia, Phlox, Sambucus, Viburnum, and white oak. Freesia samples with necrotic spots and streaks similar to the symptoms induced by Freesia sneak ophovirus (FreSV) were not found to be infected by FreSV, but some samples were shown to be infected by Freesia mosaic potyvirus (typically associated with mild mosaic symptoms), and three samples yielded positive results for a putative member of family Qinviridae. This virus was detected using polymerase chain reaction primers developed by Italian collaborators at the Institute for Sustainable Plant Protection of the National Research Council of Italy. Under Objective 1B: Reverse-transcription polymerase chain reaction (RT-PCR) analysis of samples of Hosta, Easter lily, Magnolia, and additional samples of Phlox and clover from different locations with broad-spectrum potexviruses or carlaviruses, allowing subsequent identification of the viruses by direct sequencing of the PCR products and/or selection of appropriate virus-specific primers to identify the particular viruses present in the samples. Under Objective 1C: Ornamental Oxalis, commonly known as the Shamrock plant, is grown as a potted plant in the United States, especially for marketing in the spring around St. Patrick’s Day. ARS researchers at Beltsville, Maryland, in collaboration with Dutch colleagues from Naktuinbouw and the Netherlands NPPO, determined the complete genome sequences of two isolates of a novel potyvirus, Shamrock chlorotic ringspot virus. A total RNA extract from partially purified virion preparations from Oxalis triangularis plants from WI (2012-2013) exhibiting chlorotic ringspot symptoms tissue was used as the template for a random PCR to produce a cDNA library. The near complete nucleotide sequence of SCRV-WI was obtained (in 2019) from multiple overlapping cDNA clones, coupled with 5’ and 3’ RACE cloning, and determined to be 10,120 nucleotides. In 2021, oxalis plants in the in vivo collection at the Plant Protection Organization of The Netherlands (NPPO-NL) exhibiting similar symptoms tested positive for potyvirus using generic potyviral primers. More recently, RNAseq analysis of a cDNA library sequenced on the Illumina NovaSeq 6000 platform revealed the presence of a 10,136 nt genome sequence which showed 98.5% identity with the SCMV-WI isolate. Based on the predicted polyprotein cleavage sites, the number and size of the predicted processed mature peptides, and phylogenetic relatedness to members of the family Potyviridae, SCRV appears to be a new species in the genus Potyvirus. Under Objective 2A: In collaboration with colleagues from Chungnam National University (Korea), two closely-related isolates of turnip mosaic virus with differential infectivity to Chinese cabbage (Brassica napus) were found to differ by only eight amino acid residues over the 3164-residue viral polyprotein. A single amino acid difference in the cytoplasmic inclusion protein was shown to be responsible for breaking viral resistance in two otherwise resistant Chinese cabbage cultivars. In contrast, another single amino difference in the viral genome-linked protein VPg was required for systemic infection of Chinese cabbage, indicating the presence of two distinct genes for resistance to turnip mosaic virus in the two resistant cultivars. These findings, and the associated virus isolates, will be useful for screening breeding lines of Chinese cabbage for virus resistance. Under Objective 3.2: In collaboration with colleagues from the Chinese Academy of Agricultural Sciences in Beijing, China, we determined the draft genome of a Xylella fastidiosa strain causing bacterial leaf scorch of American elm in Washington, DC. X. fastidiosa causes bacterial leaf scorch in many species of landscape trees, including American elm. This new genetic information will help scientists gain a better understanding of the molecular basis of strain divergence, host specificity, nutrient requirements, and pathogenicity, as well as develop genome-based specific detection methods for this important plant pathogen.

Accomplishments
1. New tools to differentiate cool-virulent strains in Ralstonia solanacearum species complex. R. solanacearum race 3 biovar 2 (r3b2) strains can cause a devastating brown rot disease of potato under cool temperature conditions, a phenotype termed cool virulence. As a result, they are listed as select agent pathogens in the U.S. and are subject to strict government quarantine and security responses. Currently only one PCR assay, developed previously by ARS researchers at Beltsville, Maryland, is available to identify the critically important cool virulence region for specific and accurate detection of the r3b2 strains. These scientists recently identified a gene, ripS1, that plays a role in the cool-virulence phenotype of r3b2 strains. They used this gene to develop an assay that can separate cool virulence strains, including r3b2, from non-cool virulence strains of R. solanacearum. Once a suspected sample tests positive for r3b2 by the original PCR assay, regulatory agencies can use this new test to confirm the presence of the cool-virulence-related ripS1 region in the samples to increase the confidence of detecting the cool-virulent select agent pathogen.

2. Identifying genetic diversity among rose rosette virus isolates - a critical first step in determining gene function and pathogenicity. Roses are one of the most widely cultivated ornamental plants in the world. Rose Rosette Disease has caused significant economic losses in the United States over the last several decades, spreading from wild roses and the invasive multiflora rose into cultivated roses. Rose rosette virus (RRV) is the causal agent, and very few commercial rose varieties are resistant to RRV infection. ARS researchers at Beltsville, Maryland, in collaboration with scientists at Texas A&M University, conducted a genomic study to better understand RRV genetic diversity, population structures, and the nature of the genetic changes among the complete genomes of 95 sequenced RRV isolates (each with 7 RNA segments). They found differences within each genome segment among isolates and evidence of genome segment reassortment. Several viral proteins also appeared to evolve independently, suggesting that diversity may be important for adapting to new host genotypes and plant resistance genes. These results suggest greater diversity among RRV isolates than previously recognized and suggest new avenues for disease management, including breeding for long-lasting RRV resistance in roses.

U.S. DEPARTMENT OF AGRICULTURE

Floral and Nursery Plants Research: Washington, DC