Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial evaluating varietal resistance to Tomato spotted wilt virus (TSWV) and Tomato yellow leaf curl virus (TYLCV) displayed mottling symptoms similar to that caused by TSWV or other tospoviruses. The foliage from affected plants, however, did not display typical symptoms caused by TSWV and was negative for TSWV using a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and TSWV ImmunoStrips (AgDia, Elkhart, IN) when performed following the manufacturers instructions. A reverse-transcription PCR assay using universal tospovirus primers TsMCR2 and 3T12 amplified a ~550 bp product from the total RNA isolated from symptomatic plants but not healthy plants. This product was larger than the expected product size (350-450 bp) for a tospovirus. Surprisingly, following primer sequence trimming, the 517 bp amplification product was found to be 92 to 99% identical to nucleotide positions 4363 to 4879 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) sequence accessions in GenBank. To confirm the presence of PepMoV in these samples, total RNA from a symptomatic and an asymptomatic plant was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using Invitrogen SuperScript III reverse transcriptase (Life Technologies, Grand Island, NY) and primer 900 [5-CACTCCCTATTATCCAGG(T)16-3] following the enzyme manufacturers instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers. A ~1700 bp product was amplified from the cDNA of the symptomatic plant but not the asymptomatic plant. This product was cloned using pGEM-T Easy (Promega, Madison, WI) and three clones were sequenced at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. The consensus sequence of the three clones was deposited in GenBank (accession JQ429788) and found to be 96 to 97% identical to positions 7917 through 9640 of other PepMoV accessions in the database. To determine the incidence of PepMoV in the field, nearly 292 plants representing 14 tomato varieties were assayed for the virus using a PepMoV-specific DAS-ELISA (AgDia) following the manufacturers directions 17 weeks after planting. Plants were considered positive if their mean absorbance at 405nm was greater than the mean absorbance + 3 standard deviations + 10% of the negative control samples. The virus incidence ranged from 4.8 to 47.6% for the different varieties, with an overall incidence of 19.9%. PARTICIPANTS: Researchers at CATAR, UHM (including Wayne Borth, Michael Melzer, Jari Sugano, Theodore Radovich, Steve Fukuda, and Susan Migita) and at USDA-ARS-PBARC (including Dennis Gonsalves, Tracie Matsumoto) were involved in the research. TARGET AUDIENCES: Plant pathologists, plant virologists, plant breeders, extension agents, growers, etc. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants were unsalable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Honduras and South Korea. This is the first report of this virus in Hawaii, and to the best of our knowledge, the first report of this virus naturally infecting tomato in the USA.
Publications
- Sugano, J., Melzer, M., Pant, A., Radovich, T., Fukuda, S., Migita, S., and Ueda, J. 2011. Field Evaluations of Tomato Yellow Leaf Curl Virus Resistant Varieties for Commercial Production. College of Tropical Agriculture and Human Resources Publication, Plant Disease, PD-78.
- Melzer, M.J., Tripathi, S., Matsumoto, T., Keith, L., Sugano, J., Borth, W.B., Wieczorek, A., Gonsalves, D., and Hu, J.S. 2012. Tomato spotted wilt. College of Tropical Agriculture and Human Resources Publication, Plant Disease, PD-81.
- Sether, D.M., Melzer, M. J., Borth, W.B., and Hu, J.S. 2012. Characterization, Diversity, Distribution, and Transmission of a Pineapple Badnavirus in Commercial Pineapple Hybrids and Pineapple Germplasm Accessions in Hawaii. Plant Disease, 96(12): 1798-1804.
- Martelli, G.P., N. Abou Ghanem-Sabanadzovic, A.A. Agranovsky, M. Al Rwahnih, V.V. Dolja, C.I. Dovas, M. Fuchs, P. Gugerli, J.S. Hu, W. Jelkmann, N.I. Katis, V.I. Maliogka, M.J. Melzer, Saldarelli, P. 2012. Taxonomic revision of the family closteroviridae with special reference to the grapevine leafroll-associated members of the genus ampelovirus and the putative species unassigned to the family. Journal of Plant Pathology, 94(1): 7-19.
- Melzer, M. J., Sugano, J.S., Cabanas, D., Dey, K., Kandouh, B., Mauro, D., Rushanaedy, I., Srivastava, S., Watanabe, S., Borth, W.B., Tripathi, S., Mastsumoto, T., Keith, L., Gonsalves, D., and Hu, J.S. 2012 First report of Pepper mottle virus on Tomato in Hawaii. Plant Disease, 96(6):917.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Hibiscus green spot virus (HGSV) A Citrus volkameriana (Ten. & Pasq.) tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawai'i. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves, but not in healthy controls. Double-stranded (ds) RNAs approximately 8 and 3 kilobase pairs (kbp) in size were present in symptomatic leaf tissue, but not in healthy controls. Excluding poly (A) tails, the largest molecule, RNA1, was 8354 bp in length. The approximately 3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3169 and 3113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. An RT-PCR assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus (A. Gray) plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay, however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. Ti Ringspot We have collected common green ti plants with ringspot symptoms from a commercial farm on Oahu. These plants were found to be infected with four new, distinct species of closteroviruses which we have named Cordyline virus 1 (CoV-1), CoV-2, CoV-3, and CoV-4. The entire genome of CoV-1 was sequenced and partial genomic sequence was obtained for CoV-2, CoV-3, and CoV-4. Using this sequence information, a reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed that can detect and distinguish these four viruses in ti plants. We have currently processed 39 samples (3 from Oahu, 3 from Maui, and 33 from the Big Island) using the RT-PCR assay and have found that all four of these viruses are widespread in Hawaii's ti and can be found in both healthy and symptomatic plants. We are currently creating ti plants that are not infected with these closteroviruses. Closteroviruses cannot be transmitted by seed, but since the common green ti does not produce seed in Hawaii, this method of virus exclusion is unavailable. Instead, we have successfully taken common green ti into tissue culture and exposed them to cryotherapy. These meristematic cells regenerate, producing a virus-free plant. Approximately ~100 ti plantlets underwent cryotherapy, and 12 have survived the procedure. These plants are currently 8-10 cm in height and will soon be assayed to confirm they are closterovirus-free. They will then be planted in a field with ringspot disease. When they develop ringspot symptoms, presumably they will be infected only with the causal virus, and standard methods to characterize the virus will not be hindered by the presence of the closteroviruses. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Worked with HDOA, USDA, and CTAHR personnel to determine the distribution of viruses in Hawaii. Communicated with individual growers on the symptoms and management of virus diseases. Field days (2) allowed stakeholders to learn about virus diseases and to observe/evaluate resistant varieties from field trials. The overall impact of our research is to protect the fragile food security and the economy of Hawaii by providing the basic information necessary to control the spread of emerging plant virus diseases in the state. The common green ti plant was brought to the Hawaiian Islands by early Polynesians and has considerable cultural and commercial importance in Hawai'i. From 2004 to 2008, annual commercial sales of cut ti leaves averaged $541,000. In 2009, ti plant farmers in Kahalu'u, O'ahu reported ringspot symptoms on their common green ti plants. Identical ringspot symptoms were also observed on common green ti plants in Wailuku and Hana on the island of Maui in November 2009. These symptoms make the leaf unmarketable, and have greatly impacted the production of ti across Hawaii.
Publications
- Melzer et al. 2011. Characterization of a virus infecting Citrus volkameriana (Ten. & Pasq.) with citrus leprosis-like symptoms. Phytopathology (in press)
- Melzer et al. 2011. An assemblage of closteroviruses infects Hawaiian ti (Cordyline fruticosa L.). Virus Genes 42:254-260
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) is an important constraint to bulb and seed onion production in many onion growing regions of the continental U.S. and the world. In June, 2010, straw-colored, diamond-shaped lesions with occasional green islands were observed on leaves of the sweet onion variety Linda Vista in an insecticide trial on Maui for onion thrips (Thrips tabaci) control. Collapse and lodging occurred when lesions on leaves were severe. Seven bulbs with green leaves exhibiting lesions were collected from this onion field in the Pulehu region of the lower Kula district on Maui. Leaf samples that included a lesion or were within 1 cm of a lesion were found positive in indirect ELISA with IYSV-specific polyclonal antisera. A405nm readings after one hour ranged from 0.263 to 2.067 for positive samples and 0.055-0.073 for healthy onion controls. Four samples that were prepared from leaf tissue several centimeters away from a lesion tested negative in ELISA. Such uneven virus distribution in the plants has been previously reported. In July 2010, symptomatic sweet onion from a commercial farm in upper Kula, Maui at the 1060-1220 m elevation tested positive for IYSV using ELISA. Green onion samples collected from a commercial farm in Omaopio, Maui, located approximately 0.1 km north of Pulehu, have tested negative, suggesting distribution may be limited at this time. RNA was isolated from leaf tissue from the seven Linda Vista sweet onion collected from the Maui insecticide trial. Reverse transcription (RT)-PCR with forward and complementary primers 5-CTCTTAAACACATTTAACAAGCAC-3 and 5 -TAAAACAAACATTCAAACAA-3 flanking the nucleocapsid (N) gene encoded by the small RNA of IYSV was conducted as previously described. Amplicons approximately 1.1 kb in length were obtained from all seven symptomatic onion samples but not from healthy samples or water controls. Sequencing of selected amplicons confirmed IYSV infection. Three sequence variants (GenBank accessions HM776014 - HM776016) were identified from two RT-PCR reactions. Phylogenetic analyses of the three sequence variants with the neighbor joining procedure available through NCBI-BLASTn Tree View showed the highest nucleotide identities of 97-98% were shared with IYSV isolates from New Zealand (EU477515), Nevada (FJ713699), and northern California (FJ713700). Phylogenetic analyses with the N-gene showed the sequences from Hawaii are mostly closely related to isolates from the western U.S., Texas, and New Zealand. To date, IYSV has not been detected on the islands of Kauai, Oahu, Molokai, or Hawaii. The distribution and economic consequences of this disease to Hawaii's onion production are under investigation. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Onion (Allium cepa) is an economically important crop in the United States and is also an important component of diversified agriculture in Hawaii where both green and dry onions are cultivated. In 2006 nearly 3 million pounds of both types of onion with a farm value of ~$3.5M were produced on 270 acres of land in Hawaii (HDOA Statististics of Hawaii Agriculture, 2007). In 1989 a new disease termed scape blight was reported on onions grown in Idaho (USA) (Mohan and Wilson 1989). Within 10 years this disease had spread throughout onion growing regions of the western United States and since then has also been reported in many other countries including Brazil (1994), Israel (1998), Japan (1999), Italy (2003), Australia (2003), India (2006), and Peru (2006). The spread of this disease can be quite rapid once it is established. For example, an epidemic of this disease in Colorado (USA) increased from 6% incidence to more than 70% incidence from 2001 to 2003, with estimated losses of more than $50M in farm receipts. The causal agent of this disease was eventually shown to be a new tospovirus, distinct from the type-member Tomato spotted wilt virus (TSWV) and from Impatiens necrotic spot virus (INSV), and was named Iris yellow spot virus (IYSV). To date, at least 47 plant species have been reported to be infected by IYSV under field conditions including Allium spp., peppers, potato, and many weedy species. IYSV is efficiently vectored by the onion thrips (Thrips tabaci) but not by other thrips species that are known vectors of other tospoviruses. Like other tospoviruses, IYSV is thought to be acquired by larvae of T. tabaci, but can only be transmitted by second-stage larvae or adults after the virus multiplies and spreads within the vector. Infective adults remain viable vectors of IYSV throughout their lifetimes. We reported earlier this year that Iris yellow spot virus (IYSV) is now present in onion (Allium spp.) from Maui. This is the first report of IYSV in Hawaii. As in other areas of the world where IYSV occurs, this pathogen has the potential to severely impact onion production in Hawaii. Control of this emerging plant virus in onion crops focuses on limiting the spread of the vector most commonly by pesticide applications, cultural practices such as crop rotation, isolation, and sanitation, and the use of a limited number of resistant cultivars. We currently have molecular tools including RT-PCR and ELISA that enable sensitive and reliable detection and identification of IYSV. Such tools will allow us to conduct thorough surveys of onion farms that are urgently needed to determine the extent of IYSV infections in onion and other plant species in Hawaii so that effective control strategies can be designed and implemented before the industry suffers significant damage. Without such information, the onion industry in Hawaii faces a serious threat to their production.
Publications
- Melzer, M.J., Ogata, D., Fukuda, S.K., Shimabuku, R, Borth, W.B., Sether, D. M., and Hu, J.S. 2010. First report of Tomato yellow leaf curl virus in Hawaii. Plant Dis. 94:641.
- Sether, D. M., Borth, W. B., Melzer, M. J., and Hu, J. S. 2010. Spatial and temporal incidences of Pineapple mealybug wilt associated viruses in pineapple planting blocks. Plant Dis. 94:196-200
- Wang, I.C., Sether, D.M., Melzer, M.J., Borth, W.B., and Hu, J.S. 2010. First Report of Banana bract mosaic virus in Flowering Ginger, Alpinia purpurata, in Hawaii. Plant Dis. 94:921.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Citrus Tristeza Virus: We have transformed Mexican lime with this construct and have regenerated approximately 70 shoots, most of which have undergone in vitro micrografting to promote rapid growth. These shoots, each representing an individual line, will soon undergo molecular analyses to confirm their transgenic genotype. A second transformation construct, identical to the one described above, is also currently being developed in which the CaMV 35S promoter is being replaced with the sucrose synthase promoter of Arabidopsis thaliana. This sucrose synthase promoter is expressed only in the phloem tissues, and may provide an effective means of targeting only the cells where CTV, a phloem-associated virus, is found. We will install the field trial to evaluate the transgenic plants we have developed, or have been developed elsewhere. We will also characterize the new transgenic lines by molecular techniques to determine their genetic background. Citrus Blight: We have continued our survey for CB and found that the disease is present on the islands of Kauai, Oahu, Maui, and the Big Island. We have not yet determined whether trees afflicted with CB are present on Molokai. We have been growing the various rootstocks for the field trial in the greenhouse, but due to the slow growth of some varieties, such as Cleopatra mandarin, not all rootstocks have attained a suitable size for grafting. We plan to install the field trial and complete the CB survey and laboratory component of this project. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Citrus tristeza virus (CTV) and its most effective vector, the brown citrus aphid (BrCA) have been present in Hawaii for at least 50 years. No serious control strategies have ever been employed to control CTV or the BrCA, resulting in high incidence and diversity of the virus. As such, Hawaii is the ideal location to test plants putatively resistant to CTV that have been developed in Hawaii, the US mainland, or elsewhere. The development of citrus resistant to Hawaiian CTV strains would be beneficial to not only Hawaii, which has not been self-sufficient in citrus for decades, but also other citrus-producing regions in the US and abroad. Many of the strains that are found in these regions are also present in Hawaii, making plants resistant in Hawaii potentially resistant elsewhere. This project also addresses citrus blight (CB), one of the major hindrances of citrus production in Hawaii. Since CB can resemble another unrelated disease of citrus (tristeza), we are conducting a survey to determine the distribution of CB in Hawaii. CB can be managed through proper rootstock selection; different rootstocks have varying tolerance to the disease. Hawaii has many local rootstocks in use whose tolerance to CB is unknown. Also, many other rootstocks that are used elsewhere due to their tolerance to CB are currently not being used in Hawaii. Therefore we believe a field trial evaluating these various rootstocks will be greatly beneficial to Hawaii citrus growers. These funds will be used for salaries and supplies to continue this research. Citrus blight is not only a major problem in Hawaii, but also in Florida and other humid regions where citrus is grown. Advances in CB research in Hawaii can have direct applications to citrus production in Florida and elsewhere. Citrus has the potential to be an important and productive crop in Hawaii. We have determined that CB is one of the main factors inhibiting production in Hawaii. Identifying where CB is prevalent in Hawaii, and evaluating citrus rootstocks for performance against CB in Hawaii's soils/climate represents a simple, effective, and environmentally friendly method for the management of this disease.
Publications
- Melzer, M.J., Borth, W.B., Sether, D.M., Ferreira, S., Gonsaives, D. and Hu, J.S. 2009. Genetic diversity and evidence for recent modular recombination in Hawaiian Citrus tristeza virus. Virus Gene (in press)
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The nucleotide sequence of Pineapple mealybug wilt associated virus-3 (PMWaV-3) (Closteroviridae: Ampelovirus), spanning seven open reading frames (ORFs) and the untranslatable region of the 3' end was determined. Based on the amino acid identities with orthologous ORFs of PMWaV-1 (54-73%) and PMWaV-2 (13-35%), we propose PMWaV-3 is a new species in the PMWaV complex. PMWaV-3 lacks an intergenic region between ORF1b and ORF2, encodes a relatively small, 28.8 kDa, coat protein, and lacks a coat protein duplicate. Phylogenetic analyses were used to analyze seven different domains and ORFs from members of the family Closteroviridae. Two distinct clades within the recognized genus Ampelovirus were observed; one that includes PMWaV-3 and PMWaV-1 and several GLRaVs and another that includes PMWaV-2 and GLRaV-3, the type member of the genus Ampelovirus. Reverse transcription-polymerase chain reaction assays (RT-PCR) for five known PMWaVs were developed and used to screen important commercial pineapple hybrids in Hawaii. The hybrids imported from Costa Rica and Philippine Islands have PMWaV-3 incidences that vary from 7-69% and occur in single or mixed infections with PMWaV-1, PMWaV-2, and PMWaV-4. PMWaV-4 was initially identified in a pineapple accession maintained at the USDA-ARS National Clonal Germplasm Repository in Hilo, HI. Recently, we detected PMWaV-4 in a commercially important pineapple hybrid in Hawaii. PMWaV-5, initially identified in Australia, has not been detected in Hawaiian grown pineapple in our preliminary screenings. We also conducted field trials to evaluate virus spread with and without ant control. Ant control during the plant crop significantly reduced the spread of PMWaVs when grey mealybugs, Dysmicoccus neobrevipes, were the only vector detected. The efficiency of the ant control achieved through two different delivery systems was significantly impacted by the age and crop stage of the pineapple crop. Several temporal transmission characteristics of PMWaV-2 by the grey pineapple mealybugs (Dysmicoccus neobrevipes) such as acquisition access period (AAP), persistence and retention of infectivity of the virus were evaluated. Results obtained showed that PMWaV-2 is transmitted by the grey pineapple mealybug in a semi-persistent manner. Transmission experiments for the longtailed mealybug (Pseudococcus longispinus), showed that this mealybug is also a vector of PMWaV-2. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Four Pineapple mealybug wilt associated viruses (PMWaV-1, PMWaV-2, PMWaV-3, and PMWaV-4) have been identified in pineapple in Hawaii. A fifth ampelovirus, PMWaV-5 , has not yet been found in Hawaii. PMWaV-1, PMWaV-3, and PMWaV-4, and PMWaV-5 share similar genomic organization and are distinct from PMWaV-2. PMWaV-2 is correlated with mealybug wilt of pineapple (MWP) whereas PMWaV-1 is not associated with MWP, but is correlated with reduced yield. Knowledge of the genomic organization and relationships between the PMWaVs, MWP, and yield reduction, as well as their incidences and distributions in the hybrids of Hawaii and germplasm are critical factors in selecting propagation material and developing management strategies to minimize yield-loss or disease problems in the future. The application of diazinon, an organophophate insecticide, during the growth stage of the pineapple plant crop can, in some cases, be eliminated without increase in virus incidence if ants are controlled. The in-field use of Amdro Pro ant bait in pineapple fields correlated with reductions in the spread of Pineapple mealybug wilt associated viruses (PMWaVs) when grey pineapple mealybugs were present in the plant and ratoon crop cycles of pineapple. However, the ant control methodology used differs depending on stage of the crop. This knowledge has lead to the development of IPM strategies that incorporate several ant control methodologies depending on crop stage as a component for disease and virus management in pineapple by the plantations in Hawaii. Because pineapple is grown worldwide and is often imported into the US from other countries, the adoption of lower risk or lower impact strategies can provide global benefits. Thus, the elimination of diazinon applications early in the pineapple crop cycle when ant control is used reduces organophosphate use both locally and globally. Previous studies have shown that D. brevipes and D. neobrevipes are vectors of PMWaV-2. The information that PMWaV-2 is transmitted by D. neobrevipes in a semi-persistent mode is not only helpful in the type of control approaches to implement but also give an idea on the rate and distance of virus spread in the field by these vectors. Furthermore, the identification of longtailed mealybugs as vector of PMWaV necessitates the inclusion of this insect in the vector control program for PMWaV management.
Publications
- Hu, J. S., Sether, D. M., Melzer, M.J., Subere, C.V., Borth, W.B., and Karasev, A. 2008. The nucleotide sequence and genome organization of Pineapple mealybug wilt associated virus-1 and PMWaV-3 support a new genus in the family Closteroviridae. Amer. Virol. Conf. Proc. Cornell, NY.
- Subere, C.V.Q., Borth, W.B., Melzer, M.J., Sether, D.M., and J.S. Hu. 2008. Development of Real-time TaqMan RT-PCR assay for detection and quantification of an ampelovirus, Pineapple mealybug wilt associated virus-2 in pineapple plants. CTAHR Research Symposium Abstract 80. Melzer, M.J., Sether, D. M., Karasev, A.V., and Hu, J.S. 2008 Complete nucleotide sequence and genome organization of Pineapple mealybug wilt-associated virus-1. Archives of Virology 153:707-714.
- Sether, D. M., Melzer, M.J., Borth, W.B., and Hu, J.S. 2008. Genome organization and phylogenetic relationship of Pineapple mealybug wilt associated virus-3 with other family Closteroviridae members. Virus Genes ( in press)
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