Source: UNIV OF HAWAII submitted to NRP
VEGETATIVELY-PROPAGATED CROPS OF IMPORTANCE TO HAWAII: PATHOGEN IDENTIFICATION, DETECTION, AND DEVELOPMENT OF CLEAN GERMPLASM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1004851
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Dec 17, 2014
Project End Date
Sep 30, 2019
Grant Year
(N/A)
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
Crops primarily propagated by vegetative means are prone to the accumulation of systemic pathogens. Taro (Colocasia esculenta) and sweet potato (Ipomea batatas) represent two such crops that have accumulated systemic pathogens during their centuries of cultivation in Hawaii. Not only can these pathogens be detrimental to crop production, but their presence may impact the ability to export these crops to out-of-state markets in the future. Many states and nations are developing agricultural security programs that will restrict the importation of propagative materials that are not certified to be free of known pathogens. As such, the overall goal of this project is to develop pathogen-free germplasm of vegetatively-propagated crops important to Hawaii. To achieve this, we will use modern sequencing technologies to identify pathogens that are infecting taro, sweet potato, and other vegetatively-propagated crops important to Hawaii. With this information, assays for their detection will be developed and shared with diagnosticians and other researchers. Cryotherapy and shoot-tip culture will be used to eliminate pathogens from select varieties of taro and sweet potato, and the detection assays will be used to evaluate their pathogen status. The horticultural characteristics of regenerated varieties will then be evaluated and demonstrated to growers and other stakeholders. The cultivation of clean taro and sweet potato germplasm has the potential to dramatically increase the production per unit area of these crops. In addition, establishing detection assays and pathogen elimination protocols will increase Hawaii's agricultural security by identifying and eliminating exotic pathogens from incoming germplasm.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2021454116020%
2124030110180%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 202 - Plant Genetic Resources;

Subject Of Investigation
4030 - Viruses; 1454 - Taro;

Field Of Science
1101 - Virology; 1160 - Pathology;
Goals / Objectives
1) Identify systemic pathogens in vegetatively-propagated crops of importance to Hawaii2) Develop diagnostic assays for the detection of exotic and novel pathogens3) Generate pathogen-free germplasm for select taro and sweet potato varieties4) Evaluate the performance of pathogen-free taro and sweet potato germplasm
Project Methods
Objective 1) Identify systemic pathogens in vegetatively-propagated crops of importance to HawaiiA procedure modified from Kreuze et al. (2009) and Roy et al. (2013) will be used to accomplish this objective. The PI has RNA samples from 328 taro plants and an expected ~300 sweet potato plants collected from across Hawaii. For the taro samples, a digital image was taken of each leaf depicting expressed symptoms. A subset of these RNA samples will be aggregated by crop and the small-interfering RNAs (siRNAs), representing viral sequences exposed to the host post-transcriptional gene silencing mechanism, will be isolated. Adaptors will be ligated to the siRNAs and the resulting ligation products will undergo amplification followed by massively parallel sequencing using either Illumina (Illumina Inc.) IonTorrent (Life Technologies), or SMRT (Pacific Biosciences) platforms. The resulting datasets will be assembled and annotated using Geneious 5.6.5 (Biomatters Ltd.), or similar software. Contiguous sequences will be compared to accessions in GenBank for identification. This process, in whole or in part, may be outsourced to a commercial laboratory specialized for these procedures. The resulting dataset will allow the identification of any viruses or sub-viral agents present in these plant samples. This will include both novel and known pathogens of taro and sweet potato. Although systemic pathogens of taro and sweet potato will be the main focus of this objective, this protocol will be able to rapidly identify systemic pathogens in other vegetatively-propagated crops if the need arises.Objective 2) Develop diagnostic assays for the detection of exotic and novel pathogensTaro vein chlorosis virus (TaVCV) was detected in Hawaii for the first time in 2013 (Long et al. in press). Standard PCR-based detection assays for this virus developed elsewhere (Revill et al. 2005) did not detect TaVCV in some Hawaii-grown plants expressing typical symptoms of taro vein chlorosis (Long et al. in press), perhaps due to the high genetic diversity of this virus (Revill et al. 2005, Long et al. in press). We have the RNA of 71 isolates of TaVCV collected from across Hawaii. We will sequence the nucleocapsid and polymerase genes of these isolates to establish the genetic diversity of this virus in Hawaii. From these data, we will develop real-time PCR primers and probe for the universal detection of these strains. In addition, we determine if this real-time PCR assay can detect TaVCV in plant samples that had typical symptoms of taro vein chlorosis, but were negative using existing TaVCV PCR-based assays (Revill et al. 2005). Similar real-time PCR-based assays will be developed using this procedure for additional novel pathogens that are detected in Objective 1. All real-time PCR assays will include an internal positive control targeting a host gene such as cytochrome oxidase I or ribulose-1,5-biphosphate carboxylase/oxygenase.Objective 3) Generate pathogen-free germplasm for select taro and sweet potato varietiesWe will select 4-6 taro varieties for the generation of pathogen-free germplasm. These varieties will include Lehua Maoli, Maui Lehua, and Bun Long, which have commercial importance to Hawaii. The other varieties selected will be based on stakeholder input. We will use thermal therapy in a combination of heat therapy to reduce virus titer, and cryotherapy, a procedure incorporating liquid nitrogen that kills mature, virus-infected cells but not meristematic cells that are typically virus-free (Wang et al. 2009, Wang and Valkonen 2009). Although multiple protocols have been developed for eliminating pathogens from taro (Hartman 1974; Li et al. 2002), we will initially use the cryotherapy protocol of Sant (2009). Briefly, taro explants containing basal corm and shoot meristematic tissue will be excised from plants grown in tissue culture without growth regulators at 34-36 oC to reduce virus titer. These explants will be cultured on MS medium with a high sucrose concentration for one month, also at 34-36 oC. A 1 mm3 shoot tip containing the meristematic dome and two leaf primordia will then be excised and immersed into osmoprotective solution, followed by a second cryoprotective solution. The shoot tips will then be placed in a cryovial containing the second cryoprotective solution and immersed in liquid nitrogen for at least 60 min. Following the liquid nitrogen treatment, the shoot tips will be rinsed and placed on MS medium in the dark for one week, followed by low light conditions for two weeks, then normal tissue culture conditions. The survival of the shoot tips will be recorded after two months. Shoot tips that regenerate into plants will be evaluated at 6 and 12 months for the presence of systemic pathogens using diagnostic assays developed in Objective 2. Pathogen-free plants will be propagated as independent lines for further evaluation.We will similarly select 4-6 Hawaiian sweet potato varieties for the generation of pathogen-free germplasm. Protocols for pathogen elimination from sweet potato are well established (Wang and Valkonen 2008a, b). We will follow these established protocols for this project. The general protocol is similar to that described above for taro, except that sweet potato can be grafted onto established, virus-free rootstock to hasten regeneration times.4) Evaluate the performance of pathogen-free taro and sweet potato germplasmRegenerated taro and sweet potato lines emerging from shoot tip culture and cryotherapy will be evaluated against the parental germplasm in greenhouse and field settings. Rooted tissue culture plants will be transferred to soil-less medium and hardened for 7-14 in the laboratory under high humidity and low light. To minimize any residual effects of tissue culture cultivation and associated growth regulators, the plants will be allowed to go through one crop cycle in a foundation block greenhouse. At least twenty propagules of these mature plants will be grown alongside similar propagules from an isogenic plant of the same variety that has not undergone the cryotherapy procedures outline in Objective 3 (controls). These side-by-side trials will take place in both the greenhouse and field. For sweet potato, the number and size (weight) of the tubers will be measured at the end of the crop cycle. For taro varieties typically grown for the corm, its size and weight will be measured at the end of the crop cycle. For taro varieties typically grown for leaves, the number and area of the harvested leaves will be measured throughout the crop cycle. Plants will also be assessed for morphological differences between the cryotherapy and control treatments. These morphological differences may include pigmentation, leaf shape, and leaf size.

Progress 12/17/14 to 09/30/19

Outputs
Target Audience:The target audiences reached during the project include fellow scientists, Cooperative Extension personnel, state department of agriculture personnel from Hawaii, Colorado, Michigan, Minnesota, Nebraska, New York, and Wisconsin, USDA APHIS personnel, and stakeholders in the fruit tree, root crop, and ornamental industries. These stakeholders were located in Hawaii, Guam, American Samoa, Samoa, Palau, and Vanuatu. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?For this project, we have trainedof sixundergraduate students, fivegraduate students, a post-doctoral researcher, and a laboratory technician in pathogen discovery and detection using approaches that include serology, polymerase chain reaction, and high-throughput sequencing. We have also trained over 15 staff in sample collection and high-throughput diagnostics for seed potato certification programs. Most of these students and staff who have left our program are now working in the agricultural or biosecurity industry at either private companies or government agencies. How have the results been disseminated to communities of interest?Results of this project have been disseminated to stakeholders by publication in peer-reviewed journals, extension series articles, grower meetings, and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goal 1)Identify systemic pathogens in vegetatively-propagated crops of importance to Hawaii Taro: During this project we have identified and sequenced the complete genome of taro vein chlorosis virus, taro bacilliform virus, and taro bacilliform China virus in Hawaiian taro plants, with each representing the first time these viruses were detected in the United States. Additionally, we identified taro vein chlorosis virus in American Samoa, Guam, Palau, Samoa and Vanuatu. Aside from Vanuatu, this virus has not been detected in any of these locations. We sequenced the complete genome of this virus from all of these locations which has helped us to i) develop more effective detection assays for this virus and ii) determine potential movement pathways in the Pacific region. We have sequenced the nearly complete genomes of taro reovirus and colocasia bobone disease associated virus from samples collected in Papua New Guinea. This represents the first genetic information on either of these viruses from this location. We have also characterized a new virus in taro coming from a sample from Samoa and have tentatively designated this virus as taro totivirus L. Banana: We completed a state-wide survey of banana pathogens in Hawaii consisting of 31 locations. Survey targets included two viruses not known to occur in the USA/Hawaii, banana virus X and banana mild mosaic virus, and Fusarium oxysporum cubense Tropical Race 4. All 226 plants samples were negative for these pathogens. Citrus: During a statewide survey of citrus pathogens, we identified apple stem grooving virus, citrus dwarf viroid, citrus exocortis viroid, citrus vein enation virus, and hop stunt viroid, infecting citrus in the state of Hawaii. The presence of these pathogens have been confirmed by sequencing either their partial or complete genomes. These are the first reports of these citrus pathogens in Hawaii. Ti: We characterized a novel viral pathogen of ti plants designated ti ringspot emaravirus. This virus has become widespread in Hawaii's ti plants, causing a disease that reduces their value as an ornamental crop. This virus is likely transmitted by eriophyid mites and we have submitted voucher specimens to both state and federal identifiers to assist in identification. Sweetpotato: We have identified four viruses for the first time in Hawaii's sweetpotato plants: sweet potato feathery mottle virus, sweet potato virus G, sweet potato virus 2, and sweet potato leaf curl virus. These are all targeted pathogens in the National Clean Plant Network's Sweetpotato Clean Plant Network and may be impacting sweetpotato production in Hawaii. Other crops: We have characterized two tombusviruses that infect star jasmine (Jasminum multiflorum) in Hawaii. One of these viruses is an isolate of Jasmine virus H, a pelarspovirus recently described in China, but has not been previously reported in Hawaii or the USA. The second virus is new to science and we have proposed the name jasmine mosaic associated virus. These two viruses are responsible for leaf mosaic and mottling of star jasmine plants across Oahu. We have also identified Tomato chlorotic dwarf viroid for the first time in Hawaii, with assistance from USDA-APHIS-PPQ. This pathogen was severely impacting a large greenhouse tomato operation on the Island of Hawaii.We have been working on a novel viral disease of sunn hemp plants grown at a commercial farm on the island of Molokai. This disease has greatly reduced the production of sunn hemp seeds destined for export from the state. From these samples we identified two viral pathogens. The first, Tobacco streak virus (TSV), is a first report for Hawaii. The presence of TSV, however, was not strongly associated with the observed symptoms. We have sequenced the genome a novel tobamovirus that we are tentatively naming Sunn hemp mottle virus. Based on mechanical inoculation studies, this virus appears to be responsible for the observed symptoms. Goal 2)Develop diagnostic assays for the detection of exotic and novel pathogens Taro vein chlorosis virus (TaVCV): We have expressed a tagged partial nucleocapsid protein of TaVCV in E. coli, and have purified two protein products. One appears to be the entire partial protein (approximately 40 kDa), and the second appears to be a cleaved version of this protein (approximately 20 kDa). We immunized mice and produced of monoclonal antibodies for the purpose of serological detection of this virus. Unfortunately, none of the hybridoma lines appeared viable for ELISA, although several worked in immunocapture RT-PCR assays. Based on the complete genome sequences we have generated for TaVCV isolates collected from across the Pacific, we have develop what appear to be robust RT-PCR primers targeting the polymerase gene of this virus. Ti ringspot associated virus: We have developed conventional RT-PCR assays for the detection of specific detection of each TiRaV genomic segment. These assays can be multiplexed with an internal control primer set targeting the rbcL gene of the host plant, Cordyline fruticosa, to ensure the input material (RNA/cDNA) is of suitable quality. We have similarly developed a qRT-PCR assay for the detection of TiRaV. Sunn hemp mottle virus: This virus appears to cross-react with commercial antisera used for the detection of Tobacco mosaic virus and related tobamoviruses. We have developed an RT-PCR assay for the specific detection of this virus and have recently developed a TaqMan-based qRT-PCR assay for its detection that is highly specific and sensitive. RT-PCR-based diagnostic assays have been developed for the following viruses: jasmine mosaic associated virus 1, jasmine virus H, taro bacilliform China virus, taro reovirus, and tomato chlorotic dwarf viroid. With our collaborators, we have developed an immunocapture Reverse-Transcription Loop Mediated Amplification (RTLAMP) assay for banana bract mosaic virus, and a multiplex RT-LAMP assay for three important banana pathogens: banana bunchy top virus, banana streak virus, and cucumber mosaic virus. Goal 3)Generate pathogen-free germplasm for select taro and sweet potato varieties We have performed thermal therapy followed by meristem excision and regeneration to produce plantlets of six taro varieties that are free of targeted viral pathogens. These target pathogens currently include taro reovirus, colocasia bobone disease-associated virus, cucumber mosaic virus, dasheen mosaic virus, and taro vein chlorosis virus. These varieties are currently maintained in tissue culture and two are also maintained in an insect-proof greenhouse. Through the process of thermal therapy and meristem isolation, we have generated eight commercial and heirloom sweetpotato varieties that are free of viruses targeted by the National Clean Plant Network's Sweetpotato Clean Plant Network: sweet potato feathery mottle virus, sweet potato virus C, sweet potato virus 2, sweet potato leaf curl virus, and sweet potato chlorotic stunt virus. These varieties are currently maintained in tissue culture and four are also maintained in an insect-proof greenhouse. Goal 4)Evaluate the performance of pathogen-free taro and sweet potato germplasm Co-PI Dr. Susan Miyasaka has conductedfield evaluation of sweetpotato germplasm in Kula, Maui. Tissue-cultured Okinawan sweet potato germplasm free of targeted viruses (TC) was compared with cuttings collected from commercial fields in Hawaii (CC). The yield from TC material was almost twice that of the CC in her trials, indicating the importance of using such materials. A similartrial being conducted on Kauai was initiated with cuttings from three of our sweetpotato varieties. No field trials involving target pathogen-free taro have been conducted due to insufficient starting material.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, D., Hamim, I., Borth, W.B., Melzer, M.J., Suzuki, J.Y., Wall, M.M., Matsumoto, T., Sun, G.F., and Hu, J.S. 2019. First report of apple of Peru (Nicandra physalodes) infected with pepper mottle virus in Hawaii. Plant Disease https://doi.org/10.1094/PDIS-06-18-1061-PDN
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, D., Ocenar, J., Hamim, I., Borth, W.B., Fukuda, M.T., Melzer, M.J., Suzuki, J.Y., Wall, M.M., Matsumoto, T., Sun, G.F., Ko, M., and Hu, J.S. 2019. First report of Bean yellow mosaic virus infecting nasturtium (Tropaeolum majus) in Hawaii. Plant Disease doi.org/10.1094/PDIS-06-18-1082-PDN
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, D., Boluk, G., Quinto, E.A., Hamim, I., Borth, W.B., Melzer, M.J., Green, J., Suzuki, J.Y., Wall, M.M., Matsumoto, T., Sun, G.F., and Hu, J.S. 2019. First report of Zucchini tigre mosaic virus infecting bitter melon (Momordica charantia) in Hawaii. Plant Disease https://doi.org/10.1094/PDIS-08-18-1391-PDN
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Olmedo-Velarde, A., Roy, A., Belanger, C.A., Watanabe, S., Hamasaki, R.T., Mavrodieva, V.A., Nakhla, M.K., and Melzer, M.J. 2019. First report of tomato chlorotic dwarf viroid infecting greenhouse tomato in Hawaii. Plant Disease https://doi.org/10.1094/PDIS-08-18-1401-PDN
  • 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., 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
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Dey, K.K., Sugikawa, J., Kerr, C., and Melzer, M. J. 2019. Air potato (Dioscorea bulbifera) plants displaying virus-lilke symptoms are co-infected with a novel potyvirus and a novel ampelovirus. Virus Genes 55:117-121
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Miyasaka, S.C., Motomura-Wages, S., Pulakkatu-Thodi, I., Melzer, M.J., Clark, C.A., LaBonte, D.R., and Villordon, A.Q. 2018. Field performance of tissue-cultured, virus-tested Okinawan sweetpotato and comparison with some promising cultivars in Hawaii. HortTechnology 28:676-683
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, D, Hamim, I., Borth, W.B., Melzer, M., Sun, G., Hu, J. 2018. First report of Dasheen mosaic virus infecting taro (Colocasia esculenta) in Bangladesh. Plant Disease doi.org/10.1094/PDIS-03-18-0442-PDN
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kong, A., Long, M., Arakaki, A., Melzer, M. 2018. First report of Tobacco streak virus infecting sunn hemp (Crotalaria juncea) in Hawaii. Plant Disease doi.org/10.1094/PDIS-04-18-0697-PDN
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Manley, M, Melzer, M., and Spafford, H. 2018. Oviposition preferences and behaviors of wild caught and lab reared coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera:Scarabaeidae), in relation to substrate particle size. Insects doi: 10.3390/insects9040141


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audiences reached during the reporting period include fellow scientists, Cooperative Extension personnel, state department of agriculture personnel from Hawaii, Michigan, Minnesota, Nebraska, New York, and Wisconsin, USDA APHIS personnel, and stakeholders in the fruit tree, root crop, and ornamental industries. These stakeholders were located in Hawaii, Guam, American Samoa, and Samoa. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?For this project period, we have continued the training oftwo undergraduate students, four graduate students, a post-doctoral researcher, and a laboratory technician in pathogen discovery and detection using approaches that include serology, polymerase chain reaction, and high-throughput sequencing. How have the results been disseminated to communities of interest?Results of this project have been disseminated to stakeholders by publication in peer-reviewed journals, extension series articles, grower meetings, and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on our four stated objectives as outlined in our work narrative and project timeline. Of primary importance is to continue to evaluate the performance of taro and sweet potato germplasm developed in our program that is free of targeted systemic pathogens.

Impacts
What was accomplished under these goals? 1) Identify systemic pathogens in vegetatively-propagated crops of importance to Hawaii Taro: We have sequenced the genomes of Taro vein chlorosis virus (TaVCV) isolates from Hawaii and Samoa. These represent only the second and third fully sequenced genomes of this virus. These genomes differ considerably from the TaVCV isolate from Fiji that has been previously sequenced. With these new data, better molecular diagnostic assays can now be developed. We have also confirmed the presence of TaVCV in both American Samoa and Samoa. These results diffused a taro trade embargo between these two neighboring Pacific Island Communities. We have also confirmed the presence and sequenced the genome of a Taro bacilliform CH virus (TaBCHV) isolate for the first time in Hawaii and the USA. A survey revealed this virus is widespread in Hawaii's taro germplasm, and has likely been present in the state for many years. Fortunately, we suspect its impact as a single infection is minor. We are, however, concerned that more severe symptoms may develop if plants with TaBCHV are co-infected with other taro viruses. We have performed high-throughput sequencing on symptomatic taro samples from Hawaii and Samoa. In addition to the viruses described above, we have also identified sequences that represent novel virus-like agents in these samples. We are currently characterizing these agents to determine their taxonomic placement and will determine if they represent potential taro pathogens of economic importance. Banana: We completed a state-wide survey of banana pathogens. This included two viruses not known to occur in the USA/Hawaii and Fusarium oxysporum cubense (Foc) TR4. All samples have been negative for Banana mild mosaic virus, Banana virus X, and Foc TR4. We did not observe any viral symptoms in these samples, although additional analyses are needed to determine if there are any sub-lethal or cryptic infections in these tissues. Other crops: We have been working on a novel viral disease of sunn hemp plants grown at a commercial farm on the island of Molokai. This disease has greatly reduced the production of sunn hemp seeds destined for export from the state. From these samples we identified two viral pathogens. The first, Tobacco streak virus (TSV), is a first report for Hawaii. The presence of TSV, however, was not strongly associated with the observed symptoms. We have sequenced the genome a novel tobamovirus that we are tentatively naming Sunn hemp mottle virus. Based on mechanical inoculation studies, this virus appears to be responsible for the observed symptoms. We are currently examining the rate of seed transmission of this new virus to determine if a seed certification program is needed for sunn hemp grown in Hawaii. We have identified Apple stem grooving virus,Citrus dwarf viroid,Citrus exocortis viroid, Citrus vein enation virus, andHop stunt viroid, infectingcitrus inthe state of Hawaii. Thepresence of these pathogens have been confirmed by sequencing either their partial or completegenomes. Thesearethe first reports of thesecitrus pathogens in Hawaii. We recently finished sequencing the genome of a novel emaravirus, tentatively named ti ringspot associated virus (TiRaV), which is associated with ti ringspot disease in Hawaii. We have completed the characterization of two tombusviruses that infect star jasmine (Jasminum multiflorum) in Hawaii. One of these viruses is an isolate of Jasmine virus H, a pelarspovirus recently described in China, but has not been previously reported in Hawaii or the USA. The second virus is new to science and we have proposed the name jasmine mosaic associated virus. These two viruses are responsible for leaf mosaic and mottling of star jasmine plants across Oahu. We have also identified Tomato chlorotic dwarf viroid for the first time in Hawaii, with assistance from USDA-APHIS-PPQ. This pathogen was severely impactinga large greenhouse tomato operation on the Island of Hawaii. 2) Develop diagnostic assays for the detection of exotic and novel pathogens Sunn hemp mottlevirus:This virus appears to cross-react with commercial antisera used for the detection ofTobacco mosaic virus and related tobamoviruses.We have developed an RT-PCR assay for the specific detection of this virus. Taro vein chlorosis virus (TaVCV): We have expressed a tagged partial nucleocapsid protein of TaVCV in E. coli, and have purified two protein products. One appears to be the entire partial protein (approximately 40 kDa), and the second appears to be a cleaved version of this protein (approximately 20 kDa). We have immunized mice for the production of monoclonal antibodies for the purpose of serological detection of TaVCV. A robust serological test such as enzyme-linked immunosorbernt assay (ELISA) would be an important tool for the detection of TaVCV, particularly in Pacific Island Communities where molecular diagnostic equipment and reagents may not be available. 3) Generate pathogen-free germplasm for select taro andsweet potato varieties Taro:We have six taro varieties ofcommercial and cultural importance to Hawaii in tissue culture. We have performed thermal therapy followed by meristem excision and regeneration to produce plantlets free of target viral pathogens. These target pathogens currently include Cucumber mosaic virus, Dasheen mosaic virus, and Taro vein chlorosis virus. Sweet potato:We have nine sweet potato varieties of commercial and cultural importance to Hawaii in tissue culture. We have performed thermal therapy followed by meristem excision and regeneration to produce plantlets free of target viral pathogens. These target pathogens include potyviruses (Sweet potato feathery mottle virus, Sweet potato virus C, Sweet potato virus G, and Sweet potato virus 2),a geminivirus (Sweet potato leaf curl virus), and a crinivirus (Sweet potato chlorotic stunt virus).We are currently increasing plants in a foundation greenhouse for goal 4). 4) Evaluate the performance of pathogen-free taro and sweet potato germplasm Co-PI Dr. Susan Miyasaka has initiated field evaluation of sweet potato germplasm in Kula, Maui. Tissue-cultured Okinawan sweet potato germplasm free of targeted viruses (TC) was compared with cuttings collected from commercial fields in Hawaii (CC). The yield from TC material was almost twice that of the CC in her trials, indicating the importance of using such materials. Similar trials with other sweet potato varieties and taro will be conducted in the near future.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, Y., Borth, W., Green, J., Hamim, I., Cao, K., Hu, J., and Melzer, M. 2017. Genome characterization and distribution of Taro bacilliform CH virus on taro in Hawaii, USA. European Journal of Plant Pathology https://doi.org/10.1007/s10658-017-1353-z
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Roy, A., Stone, A.L., Melzer, M.J., Shao, J., Hartung, J.S., Mavrodieva, V., Nakhla, M.K., Brlansky, R.H., and Schneider, W.L. 2018. Complete nucleotide sequence of a hibiscus infecting cilevirus Florida isolate and its relationship with closely associated cileviruses. Genome Announcements doi: 10.1128/genomeA.01521-17
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dey, K.K., Leite, M., Hu, J.S., Jordan, R., and Melzer, M.J. 2018. Detection of Jasmine virus H and characterization of a second pelarspovirus infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants displaying virus-like symptoms. Archives of Virology DOI :10.1007/s00705-018-3947-y
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dey, K.K., Melzer, M.J., Xiaoan, S., and Adkins, S. 2018. First report of Tuberose mild mottle virus infecting tuberose (Polianthes tuberosa) in the USA. Plant Disease 102:461
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Roy, A., Stone, A.L., Melzer, M.J., Hartung, J.S., Mavrodieva, V., Nakhla, M.K., Brlansky, R.H., and Schneider, W.L. 2018. First report of Cilevirus associated with green ringspot on senescent hibiscus leaves in Tampa, Florida. Plant Disease 102:1181
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Atibalentja, N., Fiafia, S.T., Gosai, R.C., and Melzer, M.J. 2018. First report of taro vein chlorosis virus on taro (Colocasia esculenta) in the U.S. Territory of American Samoa. Plant Disease 102:828
  • Type: Other Status: Published Year Published: 2017 Citation: Olmedo-Velarde, A., Hamasaki, R.T., Bushe, B., and Melzer, M.J. 2017. Tomato chlorotic dwarf viroid. College of Tropical Agriculture and Human Resources PD-113
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Feng, X., Orellana, G., Green, J., Melzer, M.J., and Karasev, A.V. 2018. Characterization of a Bean common mosaic virus isolate from lima bean (Phaseolus lunatus). International Congress of Plant Pathology, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fatdal. L., Sipes, B., and Melzer, M. 2017. Bioforensic studies in Rotylenchulus reniformis ⿿ sources and origin. Society of Nematologists Annual Meeting, Colonial Williamsburg, VA
  • Type: Other Status: Published Year Published: 2017 Citation: Melzer, M.J., Sether, D.M., Hu, J.S., and Alvarez, A.M. 2017. Citrus huanglongbing. College of Tropical Agriculture and Human Resources PD-112
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Olmedo-Velarde, A. and Melzer, M.J. 2018. Viroid diseases in Hawaii. International Congress of Plant Pathology, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Olmedo-Velarde, A., Watanabe, S., Hamasaki, R.T., and Melzer, M.J. 2018. First report of Tomato chlorotic dwarf viroid and Southern tomato virus infecting greenhouse tomato in Hawaii. International Congress of Plant Pathology, Boston, MA


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audiences reached during the reporting period include fellow scientists, Cooperative Extension personnel, state department of agriculture personnel from Hawaii, Michigan, Minnesota, Nebraska, North Dakota, and Wisconsin, USDA-APHIS personnel, and stakeholders in the fruit tree, root crop, and ornamental industries. These stakeholders were located in Hawaii, Guam, and American Samoa. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?For this project period, we have trained two undergraduate students, four graduate students, a post-doctoral researcher, and a laboratory technician in pathogen discovery and detection using approaches that include serology, polymerase chain reaction, and high-throughput sequencing. How have the results been disseminated to communities of interest?Results of this project have been disseminated to stakeholders by publication in peer-reviewed journals, extension series articles, grower meetings, and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on our four stated objectives as outlined in our work narrative and project timeline.

Impacts
What was accomplished under these goals? We have identified two viruses in taro using high-throughput sequencing that have not been previously reported in the USA. Taro bacilliform virus (TaBV) and Taro bacilliform China virus (TaBCHV). We have also sequenced the genomes of two divergent strains of Dasheen mosaic virus (DsMV), which also represent the first sequenced genomes of DsMV strains from the USA. Working with collaborators in American Samoa, we are performing confirmatory testing for Taro vein chlorosis virus (TaVCV) using assays developed in our laboratory. This will be the first report of TaVCV in the territory of American Samoa. We are currently conducting a state-wide survey of banana pathogens. This includes two viruses not known to occur in the USA/Hawaii and Fusarium oxysporum cubense (Foc) TR4. To date all samples have been negative for Banana mild mosaic virus, Banana virus X, and Foc TR4. At the end of this survey project (12/31/2017), the collected tissues from across the state will undergo dsRNA isolation and high-throughput sequencing to determine if any novel viruses or systemic pathogens are present in these samples. We have not observed any viral symptoms in these samples, however the high-throughput sequencing will identify if there are any sub-lethal or cryptic infections in these tissues. We have identified a novel viral disease of sunn hemp plants grown at a commercial seed farm on the island of Molokai. This disease has greatly reduced the production of sunn hemp seeds. We have identified a novel tobamovirus in this crop and are currently characterizing its genomic and biological properties. We have identified Citrus dwarf viroid infecting a tangelo tree located on the island of Hawaii. We are in the process of confirming this diagnosis using a second diagnostic approach. This is the first report of this pathogen in Hawaii. We are continuing to characterize the genome of an emaravirus, tentatively named ti ringspot associated virus (TiRaV), which is associated with ti ringspot disease in Hawaii. We have partially sequenced the RNA-dependent RNA polymerase and nucleocapsid segments of this virus. This sequencing is essential to confirm the taxonomic placement of this virus, and propose it as a new species in the genus Emaravirus to the International Committee on Virus Taxonomy. We have completed sequencing on the genomes of two novel tombusviruses that infect star jasmine (Jasminum multiflorum) in Hawaii. These viruses are tentatively named Jasmine mosaic associated virus 1 and Jasmine mosaic associated virus 2. We are working with collaborators at USDA in Beltsville, MD to perform serological relationships with closely related tobamoviruses in other ornamental crops. The current reverse-transcription (RT)-PCR assay for TaVCV that was developed by an Australian group was unable to detect strains of TaVCV present in Hawaii. We sequenced the polymerase gene of over 40 TaVCV isolates and identified an improved target region for an RT-PCR assay. This assay has proven to be much more robust at detecting TaVCV isolates from Hawaii. As similar situation occurred in American Samoa - TaVCV was not detected by researchers there using the previous assay, but when we shared our improved assay, they were able to detect this virus for the first time in American Samoa. We also contracted the development of 3 polyclonal antibodies to TaVCV. None appeared to be effective in an ELISA format, but one of the three antibodies was successfully incorporated into an immunocapture RT-PCR assay for TaVCV, which greatly simplifies the testing procedure and eliminates the need for an RNA extraction step using costly kits. We have developed a conventional RT-PCR assay for the specific detection of TiRaV. This assay can be multiplexed with an internal control primer set targeting the rbcL gene of the host plant, Cordyline fruticosa, to ensure the input material (RNA/cDNA) is of suitable quality. RT-PCR-based diagnostic assays have been developed for the following viruses: Jasmine mosaic associated virus 1, Jasmine mosaic associated virus 2, Taro badnavirus China virus, and the novel tobamovirus found in sunn hemp.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhang, J., Dey, K., Lin, B., Borth, W.B., Melzer, M.J., Sether, D., Wang, Y., Wang, I.-C., Shen, H., Pu, X., Sun, D, and Hu, J.S. 2017. Characterization of Canna yellow mottle virus in a new host, Alpinia purpurata, in Hawaii. Phytopathology 107:791
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, Y.N., Hu, J.S., Borth, W.B., Hamim, I., Green, J.C., and Melzer, M.J. 2017. First report of taro bacilliform CH virus (TaBCHV) on taro (Colocasia esculenta) in Hawaii, USA. Plant Disease 101:1334
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Dey, K., Melzer, M., Sun, X., and Adkins, S. 2017. Tomato chlorotic spot virus identified in Marsdenia floribunda in Florida. Plant Health Progress 18:144-145
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Green, K.J., Chikh-Ali, M., Hamasaki, R, Melzer, M.J., and Karasev, A.V. 2017. Potato virus Y (PVY) isolates from Physalis peruviana are unable to systemically infect potato or pepper and form a distinct new lineage within the PVYC strain group. Phytopathology 1433-1439
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, Y., Wu, B., Borth, W., Hamim, I., Green, J., Melzer, M., Hu, J. 2017. Molecular characterization and distribution of two strains of Dasheen mosaic virus on taro in Hawaii, USA. Plant Disease 1980-1989
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Dey, K., Melzer, M., and Hu, J. 2017. Virus Induced Gene Silencing in Plant Biotechnology Vol. 2: Transgenics, Stress Management, and Biosafety Issues, Sahni, S., Deo Prasad, B., and Kumar, P. (eds.). Apple Academic Press ISBN 9781771885812
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dey, K.K., Melzer, M.J., Kerr, C., Xiaoan, S., and Adkins, S. 2017. A new potyvirus found in Dioscorea bulbifera in Florida. Annual Meeting of the American Phytopathological Society, San Antonio, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Roy, A., Stone, A.L., Martinez, G.L., Otero-Colina, G., Melzer, M.J., Hartung, J.S., Wei, G., Mavrodieva, V.A, Brlansky, R.H., Schneider, W., and Nakhla, M.K. 2017. Development of two multiplex RT-PCRs for simultaneous detection of five cytoplasmic and three nuclear viruses associated with citrus leprosis complex. Annual Meeting of the American Phytopathological Society, San Antonio, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Melzer, M. 2017. Agrosecurity in Hawaii. 1st Annual Hawaii Invasive Pest Symposium, Kapaa, HI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Spafford, H., Melzer, M., Atwood, J.P., and Kaniaupio-Crozier, P. 2016. Biosecurity: Nurturing Health & Quality of Life. Hawaii Public Health Conference, Honolulu, HI


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences reached during the reporting period include fellow scientists, Cooperative Extension personnel, state department of agriculture personnel from Hawaii, Michigan, Minnesota, and Wisconsin,USDA-APHIS personnel, and stakeholders in the fruit tree, root crop,and ornamental industries. These stakeholders werelocated in Hawaii, Guam, and American Samoa. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?For this project, we have trained fourgraduate students (three Master; one PhD), a post-doctoral researcher, and a laboratory technician, all of whom are associated with the PI's laboratory during the current reporting period. We have also trained a visiting undergraduatestudent from Brazil who worked in our laboratory for 2months Potato virus Y and Watermelon mosaic virus in passionfruit and orchids. How have the results been disseminated to communities of interest?Results of this project have been disseminated to stakeholders by publication in peer-reviewed journals, extension series articles, grower meetings, and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on our four stated objectives as outlined in our work narrative and project timeline.

Impacts
What was accomplished under these goals? We have collected tissue from symptomatic taro (Colocasia esculenta) and ti (Cordyline fruticosa) plants from across Hawaii. Double-stranded RNAs were isolated from these samples and genetic libraries were generated. Two librariesfor taro and one library for ti underwent illumina sequencing using a HiSeq platform. We are currently performing bioinformatics analyses on this sequence data. Our preliminary efforts indicate several novel viral pathogens may be infecting these two crops. For taro, this may include novel badnavirus(es), caulimovirus(es), and potyvirus(es). For ti, this may include novel badnavirus(es), emaravirus(es), and closterovirus(es). In both crops, viruses with similarities to fungal viruses were also tentatively identified. We are currently developing PCR-based detection assays for these putative viruses to determine if they are indeed infecting these plants, or if they are artefacts or contamination associated with the illumina sequencing. In July 2016 we started collecting banana germplasm from across Hawaii. From these materials, similar libraries will be developed and examined by massively parallel sequencing. Taro vein chlorosis virus (TaVCV): The current reverse-transcription (RT)-PCR assay for TaVCV developed by an Australian group was unable to detect strains of TaVCV present in Hawaii. We sequenced the POL gene of over 40 TaVCV isolates and identified an improved target region for an RT-PCR assay. This assay has proven to be much more robust at detecting TaVCV isolates from Hawaii. We are currently evaluating its ability to detect TaVCV isolates from elsewhere in the Pacific. We also had three polyclonal antibodies raised against TaVCV antigens synthesized at GenScript (Piscataway, NJ). These antibodies are currently being evaluated for their utility in serological-based detection methods such as enzyme-linked immunosorbent assay (ELISA) and tissue blot immunoassay (TBIA). We have developed a TaqMan® probe-based quantitative (q)RT-PCR for the detection of a novel virus infecting ti we have tentatively named Ti ringspot associated virus (TiRaV). This multiplex assay also targets a host gene as an internal control for the assay. We are currently evaluating the assay to determine is ability to reliably detect different strains of the virus from across Hawaii. With our collaborators, we have developed an immunocapture Reverse-Transcription Loop Mediated Amplification (RT-LAMP) assay for Banana bract mosaic virus, and a multiplex RT-LAMP assay for three important banana pathogens: Banana bunchy top virus, Banana streak virus, and Cucumber mosaic virus. With our collaborators, we are developing a multiplex RT-PCR based assay for detecting and distinguishing the viruses associated with citrus leprosis disease. We have also collaborated with PathSensors, Inc., assisting in the quality control testing of their Cellular Analysis and Notification of Antigen Risk and Yields (CANARY)-based assay for Citrus leprosis virus C. We have worked to bring fifteen sweet potato accessions from our culture collection into tissue culture. At present, we have been successful in establishing eleven of these accessions. The four remaining accessions are in tissue culture, but have not yet grown due to either dormancy or death of the explant. For the eleven accessions that are growing, one has been sufficiently propagated so that attempts at virus therapy could be conducted. Twenty propagules of this variety (KeoKeo) have undergone thermal therapy for four weeks, followed by meristem isolation. Additional varieties need to be further propagated before virus therapy can be attempted.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Watanabe, S., Ruschel, R., Marrero, G., Sether, D., Borth, W., Hu, J., and Melzer, M. 2016. A distinct lineage of Watermelon mosaic virus naturally infects honohono orchid (Dendrobium anosmum) and passionfruit (Passiflora edulis) in Hawaii. New Disease Reports 34:13
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Chikh-Ali, M., Vander Pol, D., Nikolaeva, O.V., Melzer, M.J., and Karasev, A.V. 2016. Biological and molecular characterization of a tomato isolate of Potato virus Y (PVY) from the PVYC lineage. Archives of Virology 161:3561
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang, J., Borth, W.B., Lin, B., Dey, K.K., Melzer, M.J., Shen, H., Pu, X., Sun, D., and Hu, J.S. 2016. Deep sequencing of banana bract mosaic virus from flowering ginger (Alpinia purpurata) and development of an immunocapture RT-LAMP detection assay. Archives of Virology 161:1783-1795
  • Type: Other Status: Published Year Published: 2016 Citation: Long, M.H., Gosai, R.C., and Melzer, M.J. 2016. Taro vein chlorosis. College of Tropical Agriculture and Human Resources PD-111
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Marrero, G., Yasuhara-Bell, J., Melzer, M., and Alvarez, A. 2016. Loop-mediated isothermal amplification for the detection of soft rot causing Dickeya spp. Annual Meeting of the American Phytopathological Society, Tampa, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Melzer, M., Dey, K., Chan Borges, A., Long, M., Borth, W., Wichitrnithed, N., Hu, J., and Li, R. 2016. First report and characterization of Taro bacilliform virus in the USA. Annual Meeting of the American Phytopathological Society, Tampa, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Park, A. and Melzer, M. 2016. Molecular detection of Ti ringspot associated virus, a novel emaravirus associated with ti ringspot disease of Cordyline fruticosa (L.) in Hawaii. Annual Meeting of the American Phytopathological Society, Tampa, FL.


Progress 12/17/14 to 09/30/15

Outputs
Target Audience:The primary target audience of this project is the farmers who grow vegetatively propagated crops in Hawaii such as taro, sweet potato, and citrus. Large-scale processors such as HPC Foods Ltd./Taro Brand, as well as the small scale processors who purchase directly from growers and co-ops are also a local target audience. The Secretariat of the Pacific Community has interest in exchanging taro germplasm, as has the Center for Environment Research, Education and Development which signed an MOU with CTAHR in 2013 for the development of crops such as taro in Vietnam. Scientists and diagnosticians also represent target audiences with respect to the characterization of novel systemic pathogens and the development of diagnostic assays for their detection. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?For this project, we have trained three graduate students (two Master; one PhD), a post-doctoral researcher, and a laboratory technician, all of whom are associated with the PI's laboratory. We have also trained a visiting high school student from Thailand who worked in our laboratory for two months on Taro bacilliform virus, and a visiting PhD student who worked in our laboratory for 8 months on Star jasmine mosaic virus 1 and 2. How have the results been disseminated to communities of interest?Results of this project have been disseminated to stakeholders by publication in peer-reviewed journals, extension series articles, grower meetings, and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on our four stated objectives as outlined in our work narrative and project timeline.

Impacts
What was accomplished under these goals? Vegetatively-propagated crops are prone to infection by systemic pathogens such as viruses, viroids, and fastidious bacteria. The pathogens affecting vegetatively-propagated crops in the tropics are understudied. With this project, we have discovered several new pathogens infecting edibleand ornamental crops, as well as known pathogens that have not been previously reported in Hawaii or the USA. Some of these pathogens, such as Taro vein chlorosis virus, and Canna yellow mottle virus, have very recently become a serioushindrance to growers in Hawaii. We have identified these new, or previously unreported pathogens, thereby allowing the development of diagnostics for their routine detection, as well as the development of management strategies that can aid in their control. We are also in the process of producing pathogen-free germplasm for select crops (currently taro and sweet potato), and are currently distributing pathogen-free citrus germplasm to local growers and nurseries. With this germplasm that is free of targeted pathogens, we can ensure that growers have access to the highest quality planting materials. Objective 1. Identify systemic pathogens in vegetatively-propagated crops of importance to Hawaii We have identified Taro vein chlorosis virus andTaro bacilliform virus in taroand Canna yellow mottle virus in ornamental ginger. These are known viruses, but have not been previously reported in Hawaii. We have also recently identified novel viral pathogens in several vegetatively-propagated plants includingornamental ti plants (Ti ringspot associated virus, Cordyline virus 1, Cordyline virus 2, Cordyline virus 3, and Cordyline virus 4), ornamental star jasmine (Star jasmine mosaic virus 1 and Star jasmine mosaic virus 2), and a novel emara-like virus in coffee. Objective 2. Develop diagnostic assays for the detection of exotic and novel pathogens We have developed an improved RT-PCRassay for the detection of Taro vein chlorosis virus, that can detect more strains of this virus in Hawaii than the currently published detection protocol. We have also developed an RT-PCR assay for the detection of Ti ringspot associated virus, and are currently optimizing a multiplexquantitative RT-PCR assay for the detection of this virus, that includes an endogenous positive control. We have developed a LAMP-based assay for the detection of Canna yellow mottle virus. Objective 3. Generate pathogen-free germplasm for select taro and sweet potato varieties We have currently brought 15 sweet potato accessions from a UH field collection into tissue culture. These plants are currently being propagated to undergo pathogen elimination therapy by thermotherapy followed by meristem excision. One variety has undergone these therapies and the meristem explants are being regenerated. Once at sufficient size, they will be tested for targeted pathogens to determine if therapy was successful. We have currently brought 5 Hawaiian taro varieties into tissue culture. As with the sweet potato, these plants are being propagated to undergo pathogen therapy. All of these plants harbor Taro vein chlorosis virus, and some also harbor Dasheen mosaic virus. Two of these taro varieties have undergone therapy and are currently regenerating. Once at sufficient size, they will be tested to determine if the virus elimination therapy was successful. Objective 4. Evaluate the performance of pathogen-free taro and sweet potato germplasm No work on this objective was conducted during this reporting period.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Roy, A., Hartung, J.S., Schneider, W.L., Shao, J., Leon, M.G., Melzer, M.J., Beard, J.J., Otero-Colina, G., Bauchan, G.R., Ochoa, R., and Brlansky, R.H. 2015. Role bending: complex relationships between viruses, hosts and vectors related to citrus leprosis, an emerging disease. Phytopathology 105:1013-1025
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Dey, K.K., Borth, W.B., Melzer, M.J., and Hu, J.S. 2015. Application of circular polymerase extension cloning to generate infectious clones of a plant virus. Journal of Applied Biotechnology 3:34-44
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Zhang, J., Borth, W.B., Lin, B., Melzer, M.J., Shen, H., Pu, X., Nelson, S., and Hu, J.S. 2016. Multiplex detection of three banana viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP). Journal of Virological Methods.
  • Type: Other Status: Published Year Published: 2015 Citation: Hamasaki, R.T., Motomura, S.A., Melzer, M.J., and Bushe, B.C. 2015. Potato virus Y: A pathogen associated with an emerging disease of poha in Hawaii. College of Tropical Agriculture and Human Resources. PD-109
  • Type: Other Status: Under Review Year Published: 2016 Citation: Melzer, M.J., Sether, D.M., Hu, J.S., and Alvarez, A.M. 2016. Citrus huanglongbing. College of Tropical Agriculture and Human Resources.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Leite de Oliviera, M., Borth, W., Carrillo, J., Hu, J., Neupane, K., Stubblefield, S., and Melzer, M. 2015. Star jasmine (Jasminum multiflorum) plants in Hawaii are infected with multiple tombusviruses. 2015 Annual Meeting of the American Phytopathological Society, Pasadena, CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Chikh-Ali, M., Vander Pol, D., Nikolaeva, O.V., Melzer, M.J., and Karasev, A.V. 2015. A novel strain of Potato virus Y from tomato. 2015 Annual Meeting of the American Phytopathological Society, Pasadena, CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Zhang, J., Borth, W.B., Lin, B., Melzer, M.J., Shen, H., Pu, X, and Hu, J. 2015. Multiple detection of four banana viruses by reverse transcription loop-mediated isothermal amplication. 2015 Annual Meeting of the American Phytopathological Society, Pasadena, CA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Roy, A, Hartung, J.S., Shao, J., Leon, G., Melzer, M.J., Beard, J.J., Otera-Colina, G., Bauchan, G.R., Ochoa, R., Brlansky, R.H., and Schneider, W.L. 2015. Identification of Brevipalpus yothersi Baker as a vector and possible primary host of cytoplasmic citrus leprosis viruses. 2015 Annual Meeting of the American Phytopathological Society, Pasadena, CA
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Melzer, M.J. 2015. Metagenomics applied to virology. 48th Brazilian Congress of Phytopathology.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Melzer, M., Shimabuku, R., and Hu, J. 2015. New hosts for Hibiscus green spot virus 2. WERA-20 Annual Meeting 2015, Greenbelt, MD.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Melzer, M. 2015. The Clean Plant Network in Hawaii. Kauai Fruit Conference, Lihue, HI.