CHARACTERIZATION AND MANAGEMENT OF EMERGING PLANT VIRUS DISEASES IN HAWAII

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016982
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822

Performing Department
Plant & Environmental Protection Sciences

Non Technical Summary
In Hawaii, various severe virus diseases seriously challenge agricultural stakeholder's livelihoods throughout the state. This proposal addresses topics of immediate concern and impact in Hawaii for which extramural funds cannot be rapidly or readily obtained. The emergence of new virus diseases of flowering ginger (Alpinia purpurata) associated with Canna yellow mottle virus (CaYMV) and Banana bract mosaic virus (BBrMV) have become a cause for concern for growers in the cut flower industry. If not addressed, the cut flower industry might suffer the same consequences we now see in the threatened banana industry. This proposal encompasses multistate activities by sharing our findings and any resources available through collaboration with programs of other land-grant institutions located in states threatened with viral diseases. This proposal is a collaborative project, encompassing both research and extension activities. The goal of this Hatch project is to develop virus detection assays and to characterize the important virus diseases of major crops in Hawaii in order to develop effective control strategies. The goal will be approached by working toward the following four specific objectives: (1) develop sensitive assays to detect plant viruses; (2) characterize the genome and molecular diversity of the viruses; (3) examine CRISPR-Cas technologies for development of virus-resistant banana plants; and (4) share the information with others.This Hatch project will focus on the first and the third areas of the CTAHR/USDA Program "Sustain, Protect, and Manage Hawai'i's Natural Resources and Environment" and "Invasive Species Education and Management". This is an integrated Hatch project.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1099	1101	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1099 - Tropical/subtropical fruit, general/other;

Field Of Science
1101 - Virology;

Keywords

plant

viruses

Goals / Objectives
In Hawaii, various severe virus diseases seriously challenge agricultural stakeholders' livelihoods throughout the state. This proposal addresses topics of immediate concern and impact in Hawaii for which extramural funds cannot be rapidly or readily obtained. Recently, Hawaii's largest banana (Musa spp.) producer Hamakua Springs shut down its operations, citing Banana bunchy top virus (BBTV) as the primary reason for ceasing production. The emergence of new virus diseases of flowering ginger (Alpinia purpurata) associated with Canna yellow mottle virus (CaYMV) and Banana bract mosaic virus (BBrMV) have become a cause for concern for growers in the cut flower industry. If not addressed, the cut flower industry might suffer the same consequences we now see in the threatened banana industry. Papaya leaf curl disease caused by several geminiviruses has been reported in China, India, and other countries. Recently, Tomato yellow leaf curl virus (TYLCV; a Geminivirus) was found in diseased papaya plants in Texas, USA. TYLCV and its whitefly vectors have been reported in Hawaii; therefore, TYLCV could be a potential problem for the papaya industry in Hawaii. This proposal encompasses multistate activities by sharing our findings and any resources available through collaboration with programs of other land-grant institutions located in states threatened with viral diseases. This proposal is a collaborative project, encompassing both research and extension activities.The goals of this Hatch project are to develop virus detection assays and to characterize the important plant virus diseases of major crops in Hawaii in order to develop effective control strategies. The goals will be approached by working towards the following four specific objectives: (1) develop sensitive and reliable assays to detect plant viruses; (2) characterize the genome and molecular diversity of the viruses; (3) explore the use of CRISPR-Cas technologies for development of virus-resistant banana plants; and (4) share all new information with others.

Project Methods
Objective 1. Develop sensitive and specific assays for detection of the new virusesWe have optimized conditions and strategies to isolate nucleic acid templates from plant samples that allow sensitive amplification of plant virus sequences by PCR or RT-PCR. We will design PCR or RT-PCR primers that can amplify the viruses recently identified. Using sequence information available in GenBank, and from our own preliminary research, primers for other emerging viruses will be prepared and conditions for PCR detection of these viruses using these primers will be optimized.Enzyme-linked immunosorbent assays (ELISA) systems have been established for detection of many viruses in our lab. Large numbers of plant samples can be rapidly processed and screened for virus infections using ELISA. Monoclonal and polyclonal antibodies to emerging viruses will be produced and used in ELISA to test large numbers of samples.These useful assays will be developed for any new viruses identified in this project.Objective 2: Characterize the genome and molecular diversity of the virusesWe will use next generation sequencing (NGS) technologies to identify new viruses and to characterize the genome organization and gene expression strategies of the viruses. For example, nucleic acids will be isolated from symptomatic plant tissues and libraries will be constructed from the total RNA or dsRNA templates. Suitable libraries will undergo the manipulations required for emulsion PCR and will then be deep sequenced with a GS FLX Titanium sequencer at the University of Hawaii's Advanced Studies of Genomics, Proteomics, and Bioinformatics laboratory (http://asgpb.mhpcc.hawaii.edu). Sequence data will be assembled using GS De Novo assembly software (Roche). Contigs and single reads will be compared with datasets at the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov). From these comparisons, we can then identify potential viruses associated with specific diseases. The sequence information will be used to develop PCR assays that can be used to detect virus in plant samples.Objective 3: Examine CRISPR-Cas technologies for development of BBTV-resistant banana plants.Design sgRNA constructs targeting BBTV: Banana bunchy-top virus (BBTV) is the most serious virus disease that afflicts banana. It has a multipartite genome consisting of at least six separate circular ssDNA components, each about 1kb in size that are packaged into small (18-20nm diameter) isometric virions (Burns et al., 1995). Each of BBTV's genomic components contains only one large ORF, except DNA-1 that encodes 2 proteins (Beetham 1999; Randles et al., 2000). All six of BBTV's genomic components share several highly conserved regions, including a stem-loop, a TATA box, a major conserved motif, and a poly-A coding motif (Randles et al., 2000). The putative replication-associated protein (Rep) is encoded by DNA-1 and has been shown to both initiate and direct BBTV replication (Horser et al., 2001). We have designed specific sgRNAs that target the Rep ORF, the highly conserved region, and the stem loop region of DNA-1 and will synthesize expression cassettes in the vector pUC119-gRNA.Transient assay system using the CRISPR-Cas technology in banana: A transient system to inject BBTV gene-silencing constructs into infected banana plants was reported recently (Elayabalan, 2017). Results from these experiments show that typical BBTV symptoms could be suppressed in new leaves of BBTV-infected banana plants by this approach. This system apparently needs to be optimized but it potentially offers a new and rapid approach to screen gene constructs that could be used to develop stable transgenic banana plants with BBTV resistance. This method will allow us to screen a large number of BBTV gene constructs with different gRNAs targeted to various regions and components of BBTV genomic DNA. BBTV-free banana tissue culture seedlings will be propagated. The banana plants will be injected with various gene constructs with different gRNAs and then challenged with BBTV using viruliferous aphids. The injected and inoculated banana plants will be tested by PCR for BBTV infections and evaluated for virus resistance based on symptom expression.Development of transgenic banana plants using the best constructs: We have been working on the development of transgenic banana plants using gene-silencing technologies for years and we have established a reliable system to produce transgenic banana plants. This transformation and regeneration system will be used to introduce the optimal gRNAs constructs selected from this study to produce transgenic banana plants. BBTV-resistant transgenic banana plants will be selected as described previously (Borth et al. 2011). Objective 4. Share the new information with others. All research results will be presented at meetings of grower and other stakeholders, and short communications describing the technology will be prepared by CTAHR staff and disseminated out to growers and other stakeholders. Workshops will be organized to demonstrate the techniques and procedures used to produce resistant plants. "Field day" demonstrations will be organized to educate growers on recognizing symptoms, reporting outbreaks, and preventing spread of this disease.Based on our results from Objectives 1 and 2, we will develop management strategies to mitigate the spread of virus diseases between the Hawaiian Islands and between alternate host plants. The management strategies will be disseminated through flyers and downloadable PDF formats. Information about the viruses including the symptoms produced in plants, transmission by vectors, and control strategies will also be made available online via specially designed websites. Oral presentations at grower, nursery, and floriculture meetings will be made to improve recognition and appropriate responses.Stakeholders will be provided with disease recognition, spread, and management information so that proactive disease management can mitigate the spread of these diseases within the Hawaiian Islands. Public access to our developed detection assays will provide growers with the tools necessary to identify infected material and make appropriate management decisions. The sequences of any newly characterized viruses in crops in Hawaii will be deposited in GenBank to provide international access and contribute to a better understanding of virus diversity.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Considering the ICTV species demarcation criteria for secovirids (<80% identity in the Pro-Pol region or <75% identity in the CP) [16], PVSA should be considered a new sadwavirus subclassified within the proposed subgenus "Cholivirus". These results support the placement of PSVA as a putative member of the genus Sadwavirus, family Secoviridae. Further research is needed to identify the biologicalvector of PSVA, investigate whether PSVA is related to the two isometric viruses infecting pineapple in Australia, and determine if this putative new pineapple virus is involved in the etiology of MWP. How have the results been disseminated to communities of interest?Published in refereed journals. What do you plan to do during the next reporting period to accomplish the goals?Complete the objectives.

Impacts
What was accomplished under these goals? The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Olmedo-Velarde, A., Navarro, B., Hu, J.S., Melzer, M.J., and Di Serio, R. 2020. Novel Fig-Associated Viroid-Like RNAs Containing Hammerhead Ribozymes in Both Polarity Strands Identi?ed by High-Throughput Sequencing Frontiers in Microbiology doi: 10.3389/fmicb.2020.01903
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hamim, I., Borth, W.B., Suzuki, J.Y., Melzer, M.J., Wall, M.M., and Hu, J.S. 2020. Molecular characterization of tomato leaf curl Joydebpur virus and tomato leaf curl New Delhi virus associated with severe leaf curl symptoms of papaya in Bangladesh Eur J Plant Pathol (2020) 158:457472
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Larrea-Sarmiento, A., Olmedo-Velarde, A., Green, J.C., Al Rwahnih, M., Wang, X., Li, Y.-H., Wu, W., Zhang, J., Matsumoto Brower, T., Wall, M., and Hu, J.S. 2020. Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus) Achives of Virology https://doi.org/10.1007/s00705-020-04592-9
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Green, J.C., Rwahnih, M.A., Olmedo-Velarde, A., Melzer, M.J., Hamim, I., Borth, W.B., Brower, T.M., Wall, M. and Hu, J.S. 2020. Further genomic characterization of pineapple mealybug wilt-associated viruses using high-throughput sequencing. Tropical Plant Pathology 45:64-72.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Arlinghaus, R., Aas, �., Al�s, J., Arismendi, I., Bower, S., Carle, S., Czarkowski, T., Freire, K.M.F., Hu, J., Hunt, L.M., Lyach, R., Kapusta, A., Salmi, P., Schwab, A., Tsuboi, J.-I., Trella, M., McPhee, D., Potts, W., Wo?os, A., and Yang, Z.-J. 2020. Global Participation in and Public Attitudes Toward Recreational Fishing: International Perspectives and Developments, Reviews in Fisheries Science & Aquaculture, DOI: 10.1080/23308249.2020.1782340

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Bean common mosaic virus is a species of the genus Potyvirus in the family Potyviridae. Bean common mosaic virus (BCMV) is transmitted mechanically and in a non-persistent manner by several species of aphids. Flowering ginger (Alpinia purpurata) is an important ornamental crop in Hawaii that has been previously shown to harbor single and mixed infections by the potyvirus banana bract mosaic virus (BBrMV) (Wang et al. 2010) and the badnavirus canna yellow mottle virus (CaYMV) (Zhang et al. 2017). In March 2019, flowering ginger plants with virus-like symptoms that were distinct from the mosaic and streaking symptoms produced by dual infections of BBrMV and CaYMV were observed on Oahu, Hawaii. Our results indicated the presence of BCMV in the symptomatic flowering ginger plants. To our knowledge, this is the first report of BCMV naturally infecting flowering ginger. It is unclear if its presence in ginger is contributing to the current serious die-back disease of flowering ginger in Hawaii (Green et al. 2018). Additional research is needed to determine the potential cause of the threat to the ginger industry in Hawaii. How have the results been disseminated to communities of interest?Gave a talkin the 14th International Plant Virus Epidemiology Symposium 2019 What do you plan to do during the next reporting period to accomplish the goals?Complete the objectives.

Impacts
What was accomplished under these goals? In March 2019, flowering ginger plants with virus-like symptoms that were distinct from the mosaic and streaking symptoms produced by dual infections of BBrMV and CaYMV were observed on Oahu, Hawaii. The symptoms included green mosaic patterns along leaf veins, plant stuntedness, and chlorosis. Symptomatic leaf samples were collected and total nucleic acids (TNAs) were extracted for testing by PCR. Using degenerate primers, the samples were assayed for the presence of potyviral and badnaviral infection (Zheng et al. 2008; Yang et al. 2003). All symptomatic samples were found to be infected by CaYMV, but BBrMV was not detected. Direct sanger sequencing and analysis of the resultant PCR products generated using the potyviral primers indicated the presence of BCMV. BLASTN search showed that the potyviral sequence (MN073501) shared 93.8% identity to BCMV strain A1 (MK282414), a new strain of BCMV characterized from Lima Bean in Hawaii (Feng et al. 2019). To detect BCMV infection, a BCMV-specific primer set (BCMV-CIg-F 5′-AGCTCGCCACATAAACAAGC-3′ and BCMV-CIg-R 5′-CTCAGAATGCGCGGRTTGAGC-3′) was designed that targeted sequences that targeted the cylindrical inclusion (CI) protein. The expected 350-bp PCR products were obtained and the resulting sequences (MN043985) displayed a high similarity with the corresponding CI protein gene from a BCMV isolate obtained from a Sesamum indicum plant growing in China (MK282414), sharing 96.56% and 95.28% identity at the nucleotide and amino acids levels, respectively. Subsequently, the samples also tested positive using a potyvirus group-specific ELISA and a BCMV-specific ELISA following the manufacturer's directions (Agdia, Elkhart, IN). In May 2019, four additional samples were collected from the same location. The BCMV primer-specific set was used in RT-PCR to verify infection by BCMV based on the amplification of PCR fragments of the expected size. The results of these assays further confirmed the presence of BCMV in flowering ginger.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Olmedo-Velarde, A., Park, A.C., Sugano, J., Uchida, J.Y., Kawate, M., Borth, W.B., Hu, J.S., and Melzer, M.J. 2019. Characterization of Ti ringspot-associated virus, a novel emaravirus associated with an emerging ringspot disease of Cordyline fruticosa (L.) Plant Disease https://doi.org/10.1094/PDIS-09-18-1513-RE
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hamim, I., Al Rwahnih, M., Borth, W.B, Suzuki, J.Y., Melzer, M.J., Wall, M.M., Green, J.C., and Hu, J.S. 2019. Papaya ringspot virus isolates from papaya in Bangladesh: detection, characterization and distribution. Plant Disease 103:2920-2924.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hamim, I., Borth, W.B., Melzer, M.J., Suzuki, J.Y., Wall, M.M., and Hu, J.S. 2019. Occurrence of tomato leaf curl Bangladesh virus and associated subviral DNA molecules in papaya in Bangladesh: molecular detection and characterization. Archives of Virology 164:1661-1665
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Feng, X., Orellana, G., Green, J., Melzer, M.J., Hu, J.S., and Karasev, A.V. 2019. A new strain of Bean common mosaic virus from lima bean (Phaseolus lunatus): biological and molecular characterization. Plant Disease https://doi.org/10.1094/PDIS-08-18-1307-RE