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Sunn hemp ( Crotalaria juncea L.) cultivar Tropic Sun plants, stunted and displaying mottle and mosaic symptoms on foliage, were observed at a seed farm in Maui County, Hawaii. Lateral flow assays indicated the presence of either tobacco mosaic virus or a serologically related virus. High-throughput sequencing results coupled with real-time PCR experiments recovered the 6,455-nucleotide genome of a virus with an organization typical of tobamoviruses. Nucleotide and amino acid sequence comparisons and phylogenetic analyses indicated that this virus was most closely related to sunn-hemp mosaic virus but represents a distinct species. Sunn-hemp mottle virus (SHMoV) is being proposed as the common name of this virus. Transmission electron microscopy of virus extracts purified from symptomatic leaves revealed rod-shaped particles approximately 320 by 22 nm in size. In inoculation studies, the experimental host range of SHMoV appeared limited to members of the plant families Fabaceae and Solanaceae. Greenhouse experiments demonstrated plant-to-plant transmission of SHMoV that increased with ambient wind speed. Seeds from SHMoV-infected Tropic Sun were collected and were either surface disinfested or directly planted. A total of 924 seedlings germinated; 2 were positive for the virus, resulting in a seed transmission rate of 0.2%. Both infected plants came from the surface disinfestation treatment, suggesting that the virus might be unaffected by the treatment., Competing Interests: The author(s) declare no conflict of interest.

Papaya ringspot virus (PRSV) is the major constraint to papaya ( Carica papaya ) production in Bangladesh. Disease symptoms occurred in 90 to 100% of the plants surveyed. Full-length genomes of PRSV strains from severely infected papaya plants were determined using the Illumina NextSeq 500 platform, followed by Sanger DNA sequencing of viral genomes obtained by reverse-transcription PCR(RT-PCR). The genome sequences of two distinct PRSV strains, PRSV BD-1 (10,300 bp) and PRSV BD-2 (10,325 bp) were 74 and 83% identical to each other, respectively, at the nucleotide and amino acid levels. PRSV BD-1 and PRSV BD-2 were 74 to 75% and 79 to 88% identical, respectively, to other full-length PRSV sequences at the nucleotide level. Based on phylogenetic analysis, PRSV BD-2 was most closely related to PRSV-Meghalaya (MF356497) from papaya in India. PRSV BD-1 formed a branch distinct from the other PRSV sequences based on nucleotide and amino acid sequence comparisons. Comparisons of the genome sequences of these two strains with other sequenced PRSV genomes indicated two putative recombination events in PRSV BD-2. One recombinant event contained a 2,766-nucleotide fragment highly identical to PRSV-Meghalaya (MF356497). The other recombinant event contained a 5,105-nucleotide fragment highly identical to PRSV-China (KY933061). The occurrence rates of PRSV BD-1 and PRSV BD-2 in the sampled areas of Bangladesh were approximately 19 and 69%, respectively. Plants infected with both strains (11%) exhibited more severe symptoms than plants infected with either strain alone. The full-length genome sequences of these new PRSV strains and their distribution provide important information regarding the dynamics of papaya ringspot virus infections in papaya in Bangladesh.

Ti ringspot is an emerging foliar disease of the ti plant ( Cordyline fruticosa ) in Hawaii that is quickly spreading throughout the islands. Symptoms include small chlorotic ringspots on leaves that often coalesce to form larger lesions. Although several virus species have been discovered in symptomatic plants, none have been associated with these symptoms. Here, we report and characterize a novel virus closely associated with ti ringspot symptoms in Hawaii. The presence of double membrane bodies approximately 85 nm in diameter in symptomatic cells and sequence analyses of five genomic RNA segments obtained by high-throughput sequencing indicate that this virus is most closely related to members of the plant virus genus Emaravirus . Phylogenetic and sequence homology analyses place this virus on a distinct clade within the Emaravirus genus along with High Plains wheat mosaic emaravirus , blue palo verde broom virus, and Raspberry leaf blotch emaravirus . Sequence identity values with taxonomically relevant proteins indicate that this represents a new virus species, which we are tentatively naming ti ringspot-associated virus (TiRSaV). TiRSaV-specific reverse transcription PCR assays detected the virus in several experimental herbaceous host species following mechanical inoculation. TiRSaV was also detected in eriophyid mites collected from symptomatic ti plants, which may represent a putative arthropod vector of the virus.

Lima bean ( Phaseolus lunatus ) is a popular cultivated legume vegetable grown in the United States for dry bean or canned bean production. In 2017, two symptomatic P. lunatus plants exhibiting mosaic, vein banding, and growth retardation were collected in a public garden in Honolulu, HI. Both samples contained bean common mosaic virus (BCMV), and the two BCMV isolates were subjected to biological characterization on a panel of 11 differential cultivars of common bean ( P. vulgaris ), and to molecular characterization through whole genome sequencing. Both samples contained nearly identical BCMV sequences, named BCMV-A1, which, in turn, were 93% identical to the peanut stripe virus strain of BCMV. BCMV-A1 induced an unusually severe systemic necrosis in cultivar 'Dubbele Witte', and pronounced necrotic or chlorotic reaction in inoculated leaves of five other bean differentials. BCMV-A1 was able to partially overcome resistance alleles bc-1 and bc-2 expressed singly in common bean, inducing no systemic symptoms. Phylogenetic analysis of the BCMV-A1 sequence, and distinct biological reactions in common bean differentials suggested that BCMV-A1 represented a new lima bean strain of BCMV. In 2017, two BCMV isolates were collected in Idaho from common bean, and based on partial genome sequences were found 99% identical to the BCMV-A1 sequence. The data suggest that the lima bean strain of BCMV may have a wider circulation, including common bean as a host. This new strain of BCMV may thus pose a significant threat to common bean production.

Dasheen mosaic virus (DsMV) is one of the major viruses affecting taro (Colocasia esculenta) production worldwide. Whole genome sequences were determined for two DsMV strains, Hawaii Strain I (KY242358) and Hawaii Strain II (KY242359), from taro in Hawaii. They represent the first full-length coding sequences of DsMV reported from the United States. Hawaii Strains I and II were 77 and 85% identical, respectively, with other completely sequenced DsMV isolates. Hawaii Strain I was most closely related to vanilla mosaic virus (VanMV) (KX505964.1), a strain of DsMV infecting vanilla in the southern Pacific Islands. Hawaii Strain II was most closely related to a taro DsMV isolate CTCRI-II-14 (KT026108.1) from India. Phylogenetic analysis of all available DsMV isolates based on amino acid sequences of their coat protein showed some correlation between host plant and genetic diversity. Analyses of DsMV genome sequences detected three recombinants from China and India among the six isolates with known complete genome sequences. The DsMV strain NC003537.1 from China is a recombinant of KJ786965.1 from India and Hawaii Strain II. Another DsMV strain KT026108.1 is a recombinant of Hawaii Strain II and NC003537.1 from China. The third DsMV strain KJ786965.1 from India is a recombinant of Hawaii Strain II and NC003537.1 from China. To our knowledge, this is the first report of recombination events in DsMV. Both Hawaii Strains I and II of DsMV were found widespread throughout the Hawaiian islands.

In March 2013, taro plants (Colocasia esculenta [L.] Schott cv. Iliuaua) with leaves displaying veinal chlorosis and necrosis were observed on the island of Molokai. These symptoms were similar to those of taro vein chlorosis, a disease of taro caused by Taro vein chlorosis virus (TaVCV; family Rhabdoviridae, genus Nucleorhabdovirus). To explore this possibility, RNA was isolated from both symptomatic and asymptomatic taro leaves using the NucleoSpin RNA II extraction kit (Macherey-Nagel, Bethlehem, PA) according to the provided protocol, except that RLT Buffer (Qiagen Inc., Valencia, CA) was used as the initial extraction buffer. The RNAs were converted to cDNA using random primers and MMLV-RT reverse transcriptase (Promega, Madison, WI). The cDNA underwent PCR assays using primer sets Pol2A1/Pol2A2 and Cap2A/Cap2B which target the RNA-dependent RNA polymerase (RdRp) and putative nucleocapsid genes of TaVCV, respectively (1). Amplification products of the correct size were obtained for both primer sets, and these underwent molecular cloning using pGEM-T Easy (Promega). Three clones were selected and their sequences determined by dye-terminator sequencing. After primer sequence removal, the Pol2A1/Pol2A2 product (952 bp; GenBank Accession No. KF921085) and Cap2A/Cap2B product (1,050 bp; KF921086) were found to be 79 and 84% identical to a Fijian strain of TaVCV (AY674964), respectively. Samples from 328 plants with and without taro vein chlorosis symptoms were collected from 35 sites on five of the Hawaiian islands and assayed for TaVCV using the Pol2A1/Pol2A2 primer set as described above. The incidence of TaVCV in these samples was 21.6%, with positive samples coming from each island. Although a very strong association between symptoms and the presence of TaVCV was observed, eight asymptomatic plants were also positive, suggesting the detection assay was able to detect the virus before the onset of symptoms. Conversely, three symptomatic plants were found to be negative, suggesting the Pol2A1/Pol2A2 PCR assay might not detect all strains of TaVCV in Hawaii. A digoxygenin-labeled probe (Roche Applied Science, Indianapolis, IN) derived from the Pol2A1/Pol2A2 amplification product of one sample hybridized with the cDNA of only four of nine TaVCV-infected samples collected from three different islands in a dot-blot hybridization assay performed at high stringency. This probe did not hybridize with the cDNA of five TaVCV-negative samples. TaVCV exhibits a great deal of genetic diversity in the South Pacific nations where it is found; nucleotide divergence of up to 27% in regions of the RdRp gene has been reported (1). The high genetic divergence between the TaVCV isolate characterized in Hawaii and the TaVCV accession in GenBank, as well as the dot blot hybridization assay results support this observation. The widespread distribution of TaVCV in Hawaii suggests it is not a recent introduction. However, the common practice of farmers sharing taro propagules has likely accelerated its spread. An arthropod vector of TaVCV has yet to be identified, so it is unknown whether natural spread is also occurring in Hawaii. Taro has both economic and cultural importance to Hawaii. These findings, representing the first detection of TaVCV in Hawaii and the United States, illustrate the need to develop virus-free germplasm for local, national, and international distribution of this important staple crop. Reference: (1) P. Revill et al. J. Gen Virol. 86:491, 2005.

In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3'T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States. References: (1) F.-H. Chu et al. Phytopathology 91:361, 2001. (2) M. J. Melzer et al. Virus Genes 40:111, 2010. (3) M. Okuda and K. Hanada. J. Virol. Methods 96:149, 2001.

Members of the genus Badnavirus (family Caulimovirdae) have been identified in dicots and monocots worldwide. The genome of a pineapple badnavirus, designated Pineapple bacilliform CO virus-HI1 (PBCOV-HI1), and nine genomic variants (A through H) were isolated and sequenced from pineapple, Ananas comosus, in Hawaii. The 7,451-nucleotide genome of PBCOV-HI1 possesses three open reading frames (ORFs) encoding putative proteins of 20 (ORF1), 15 (ORF2), and 211 (ORF3) kDa. ORF3 encodes a polyprotein that includes a putative movement protein and viral aspartyl proteinase, reverse transcriptase, and RNase H regions. Three distinct groups of putative endogenous pineapple pararetroviral sequences and Metaviridae-like retrotransposons encoding long terminal repeat, reverse-transcriptase, RNase H, and integrase regions were also identified from the pineapple genome. Detection assays were developed to distinguish PBCOV-HI1 and genomic variants, putative endogenous pararetrovirus sequences, and Ananas Metaviridae sequences also identified in pineapple. PBCOV-HI1 incidences in two commercially grown pineapple hybrids, PRI 73-114 and PRI 73-50, was 34 to 68%. PBCOV-HI1 was transmitted by gray pineapple mealybugs, Dysmicoccus neobrevipes, to pineapple.

In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial displayed mottling symptoms similar to that caused by Tomato spotted wilt virus (TSWV) or other tospoviruses. The foliage from affected plants, however, appeared symptomless. Fruit and leaf tissue from affected plants were negative for TSWV analyzed by double antibody sandwich (DAS)-ELISA and/or TSWV ImmunoStrips (Agdia, Elkhart, IN) when performed following the manufacturer's instructions. 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 manufacturer's instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers 900 and Sprimer, which amplify sequences encoding the partial inclusion body protein (NIb), coat protein, and 3' untranslated region of potyviruses (1). A ~1,700-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 1,747-bp consensus sequence of the three clones was deposited in GenBank (Accession No. JQ429788) and, following primer sequence trimming, found to be 97% identical to positions 7,934 through 9,640 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) accessions from Korea (isolate '217' from tomato; EU586126) and California (isolate 'C' from pepper; M96425). To determine the incidence of PepMoV in the field trial, all 292 plants representing 14 tomato cultivars were assayed for the virus 17 weeks after planting using a PepMoV-specific DAS-ELISA (Agdia) following the manufacturer's directions. Plants were considered positive if their mean absorbance at 405 nm 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%. Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants was unsaleable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Guatemala (3) and South Korea (2). To our knowledge, this is the first report of this virus in Hawaii and the first report of this virus naturally infecting tomato in the United States. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) M.-K. Kim et al. Plant Pathol. J. 24:152, 2008. (3) J. Th. J. Verhoeven et al. Plant Dis. 86:186, 2002.

Onion (Allium spp.) production in Hawaii is mostly comprised of green onion and the locally prized sweet bulb onions (Allium cepa L.) that include short- and medium-day cultivars. 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 United States and the world (3). In June 2010, straw-colored, diamond-shaped lesions with occasional green islands were observed on leaves of sweet onion '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 to be positive in indirect ELISA with IYSV-specific polyclonal antisera (2). A 405nm readings after 1 h ranged from 0.263 to 2.067 for positive samples and 0.055 to 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 (4). In July 2010, symptomatic sweet onion from a commercial farm in upper Kula, Maui at the 1,060 to 1,220 m (3,500 to 4,000 foot) elevation tested positive for IYSV by ELISA. Green onion samples collected from a commercial farm in Omaopio, Maui, located approximately 0.8 km (0.5 mile) 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 onions 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 (1). Amplicons approximately 1.1 kb long 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 Accession Nos. 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 that the highest nucleotide identities of 97 to 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 most closely related to isolates from the western United States, Texas, and New Zealand. To date, to our knowledge, 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. References: (1) H. R. Pappu et al. Arch Virol. 151:1015, 2006. (2) H. R. Pappu et al. Plant Dis. 92:588, 2008. (3) H. R. Pappu et al. Virus Res. 141:219, 2009. (4) T. N. Smith et al. Plant Dis. 90:729, 2006.

Flowering ginger, Alpinia purpurata (Vieill.) K. Schum., is a popular cut flower and tropical landscape plant in Hawaii. In Hawaii, ginger flowers, including red and pink cultivars, are grown as field crops with an estimated annual sales of more than $1.6 million (USD) in 2006 (2). In June 2009, a commercial ginger flower grower from Waimanalo, Oahu, Hawaii reported plants with symptoms that included severe mosaic and stripes on the leaves. Flowers showed significant cupping and browning and growers report a reduction in size and shelf life. Symptomatic ginger was also identified at the Lyon Arboretum in Honolulu. Double-stranded RNAs (dsRNAs) were isolated from pooled leaf samples collected from 42 symptomatic plants at two locations on the island of Oahu to further characterize the pathogen associated with the symptomatic ginger. dsRNAs of approximately 0.7, 1.1, 1.8, 2.2, and 12 kb were present in the extractions from symptomatic plants but not in extractions from asymptomatic plants. Partial cloning and sequence analysis of the dsRNA revealed 95 to 98% nucleotide identity to sequences of P1, HC-Pro, C1, 6K2, VpG, NIb, and CP genes and the 3' untranslated region (total approximately 6 kb) of Banana bract mosaic virus (BBrMV). Total RNAs were also isolated from the symptomatic and asymptomatic plants from the Waimanalo farm and Lyon Arboretum. These RNA isolations were used in reverse transcription (RT)-PCR with primers Bract N1: 5'-GGRACATCACCAAATTTRAATGG-3' and Bract NR: 5'-GTGTGCYTCTCTAGCCCTGTT-3' (1), to amplify a 279-bp conserved region of the coat protein of BBrMV. Amplicons of the appropriate size were obtained from 38 of the symptomatic plants, whereas none were obtained from asymptomatic controls. RT-PCR amplicons of arbitrarily selected samples were cloned into pGEM-T Easy, sequenced, and found to be 99% identical to corresponding sequences of BBrMV. Furthermore, using double-antibody sandwich-ELISA assay and antibodies (3), we developed a system that can specifically detect BBrMV in infected flowering ginger plants and not in healthy appearing ginger. To our knowledge, this is the first report of BBrMV in flowering ginger in Hawaii. Further research is needed to determine if BBrMV infecting ginger poses a threat to banana, edible ginger, and other closely related ornamentals in Hawaii. References: (1) M. L. Iskra-Caruana et al. J. Virol. Methods 153:223, 2008. (2) Statistics of Hawaii Agriculture (2006). HDOA/USDA (NASS). 96, 2008. (3) J. E. Thomas et al. Phytopathology 87:698, 1997.

Tomato yellow leaf curl disease, caused by the begomovirus Tomato yellow leaf curl virus (TYLCV; family Geminiviridae), is an economically important disease of tomato (Solanum lycopersicum L.) that can be very destructive in tropical and subtropical regions (1). In October 2009, tomato plants showing stunted new growth, interveinal chlorosis, and upward curling of leaf margins were reported by a residential gardener in Wailuku, on the island of Maui. Similar symptoms were observed in approximately 200 tomato plants at a University of Hawaii research farm in Poamoho, on the island of Oahu in November 2009. The similarity between these symptoms and those of tomato yellow leaf curl disease and the presence of whiteflies (Bemisia spp.), the vector of TYLCV, suggested the causal agent was a geminivirus such as TYLCV. Total nucleic acids were extracted from a tomato plant sample from Wailuku and Poamoho and used in a PCR assay with degenerate primers PAR1c715 and PAL1v1978 for geminivirus detection (4). The ~1.5-kbp amplicon expected to be produced from a geminivirus template was generated from the symptomatic tomato plant samples but not from a greenhouse-grown control tomato plant. The amplicons were cloned by the pGEM-T Easy vector (Promega, Madison, WI). Three clones from each sample were sequenced, revealing 97 to 99% nucleotide identity to TYLCV sequences in GenBank and a 98.9% nucleotide identity between the Wailuku (Accession No. GU322424) and Poamoho (Accession No. GU322423) isolates. A multiplex PCR assay for the detection and discrimination between the IL and Mld clades of TYLCV was also performed on these isolates (2). A ~0.8-kbp amplicon was generated from both isolates confirming the presence of TYLCV and their inclusion into the TYLCV-IL clade (2). Seven symptomatic and three asymptomatic tomato plant samples from Poamoho were tested for TYLCV using a squash-blot hybridization assay (3) utilizing a digoxigenin-labeled probe derived from the ~1.5-kbp PCR amplicon. All symptomatic tomato plants and one asymptomatic tomato plant were found to be infected with TYLCV. How the virus entered Hawaii and how long it has been present is unknown. The most plausible route is through infected plant material such as an asymptomatic alternative host rather than viruliferous whiteflies. It appears TYLCV is not a recent introduction into Hawaii since the Wailuku gardener observed similar disease symptoms for a few years before submitting samples for testing. In January 2010, TYLCV was also detected in two commercial tomato farms on Oahu, posing a serious threat to the state's $10 million annual tomato crop. References: (1) H. Czosnek and H. Laterrot. Arch. Virol. 142:1392, 1997. (2) P. Lefeuvre et al. J. Virol. Methods 144:165, 2007. (3) N. Navot et al. Phytopathology 79:562, 1989. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple (Ananas comosus) worldwide. At least one Ampelovirus species, Pineapple mealybug wilt associated virus-2 (PMWaV-2), and mealybug feeding are involved in the etiology of MWP. A previously undescribed Ampelovirus sharing highest homology with PMWaV-1 and a putative deletion mutant sharing highest homology with PMWaV-2 were detected with reverse transcription-polymerase chain reaction (RT-PCR) assays using degenerate primers. Results were verified with additional sequence information and by immunosorbent electron microscopy. Sequence homology between the virus tentatively designated PMWaV-3, and PMWaV-1 and PMWaV-2, decreases toward the N-terminal across the HSP70 homolog, small hydrophobic protein, and RNA-dependent RNA polymerase open reading frames (ORF). Putative PMWaV-3 could not be detected with four different monoclonal antibodies specific for PMWaV-1 and PMWaV-2. The potential deletion mutant spanning the N-terminal of the HSP70 region was obtained from a pineapple accession from Zaire maintained at the USDA-ARS National Clonal Germplasm Repository in Hawaii. Putative PMWaV-3, like PMWaV-1 and PMWaV-2, is transmissible separately or in combination with other PMWaVs by Dysmicoccus brevipes and D. neobrevipes mealybugs. Plants infected with PMWaV-3 that were continuously exposed to mealybugs did not develop symptoms of MWP in the absence of PMWaV-2. Specific RT-PCR assays were developed for detection of putative PMWaV-3 and the deletion mutant.

Hibiscus rosa-sinensis Linn., family Malvaceae, is an attractive horticultural plant originating from China. Five viruses infecting H. rosa-sinensis that have been characterized previously are Hibiscus chlorotic ringspot virus (HCRSV, genus Carmovirus), Hibiscus latent ringspot virus (HLRSV, genus Nepovirus), Hibiscus yellow mosaic virus (genus Tobamovirus), Eggplant mottled dwarf virus (EMDV, genus Nucleorhabdovirus), and Okra mosaic virus (OkMV, genus Tymovirus) (2). Recently, two novel tobamoviruses infecting H. rosa-sinensis were characterized in Singapore and Florida (1). In this study, viral symptoms were observed on H. rosa-sinensis in Nanyang City in Henan Province, China. The systemic symptoms included dark and light green mosaic in young leaves, leaf puckering and malformation on older leaves, and significant stunting. Rod-shaped virus particles were isolated from H. rosa-sinensis expressing systemic symptoms. The virus was transmitted mechanically to 10 species from three families. Symptoms expressed on these plants included systemic leaf chlorosis and distortion on Lycopersicum esculentum, systemic mosaic on Capsicum annuum, Nicotiana tabacum, and Physalis floridana, and systemic chlorosis on Glycine max. N. tabacum-Xanthi nc and Datura stramonium were asymptomatic. The virus also produced chlorotic and necrotic local lesions on Chenopodium quinoa, C. amaranticolor, and C. murale. The virus was propagated in L. esculentum, N. tabacum, and P. floridana. Virions purified from systemically infected N. tabacum contained a single-stranded RNA of approximately 6.4 kb and a coat protein (CP) of approximately 17.6 kDa. The double-stranded RNA profile revealed a single band of approximately 6.4 kb. Sap extracted from virus-infected plants reacted positive with an antiserum prepared against Tobacco mosaic virus (TMV) using an antigen-coated plate enzyme-linked immunosorbent assay. The CP gene was amplified by reverse transcription-polymerase chain reaction with primers specific to Tomato mosaic virus (ToMV) and sequence data obtained from the resulting amplification product. The CP gene consisting of 159 amino acids (GenBank Accession No. AY313136) shared 99.37% identity with the ToMV Queensland isolate (GenBank Accession No. AF332868). On the basis of biology, serology, properties of virions, and the sequence of the CP gene, we conclude that the virus isolated from H. rosa-sinensis in China is Tomato mosaic virus(ToMV). References: (1) S. Adkins et al. Plant Dis. 87:1190, 2003. (2) M. H. V. van Regenmortel et al., eds. Virus Taxonomy. 7th Report of the ICTV, Academic Press, NY, 2000.

The individual and combined effects of Pineapple mealybug wilt associated virus-1 (PMWaV-1) infection in pineapple, Ananas comosus, and Rotylenchus reniformis on pineapple growth were evaluated under greenhouse and field conditions. Under greenhouse conditions, no effect of PMWaV-1 infection on pineapple growth or nematode reproduction was observed. Under field conditions, the interaction of PMWaV-1 and nematodes was evaluated in plant and ratoon crops. In the plant crop, pineapple in plots treated with the nematicide 1,3-dichloropropane showed increased vegetative growth, whereas virus infection had no effect on vegetative growth. Nematodes reduced the average fruit weight (P = 0.01), whereas PMWaV-1 infection did not (P > 0.14). However, more fruit in the largest size classes (sizes 7 and 8) were produced in PMWaV-1-free plots than in PMWaV-1-infected plots (P = 0.03). The average fruit weight decreased in the presence of virus or nematodes. The smallest fruits were from PMWaV-1-infected plants infested with R. reniformis. More early-ripening fruit (30%) were produced by plants infected with PMWaV-1 than by PMWaV-free plants (P < 0.05). PMWaV-1 infection may be one of the reasons for asynchronous fruit ripening, which is a top limiting factor for pineapple production in Hawaii. In the ratoon crop, PMWaV-1 infection reduced fruit weight by 9% (P < 0.01), whereas nematode effects were similar across treatments (P > 0.10). More fruit in the three largest size categories were produced in PMWaV-1-free plots than in PMWaV-1-infected plots (P < 0.01). PMWaV-1 infection reduced the number of fruit produced in the ratoon crop (P < 0.02). An interaction (P < 0.03) between R. reniformis and PMWaV-1 infection status was detected in the ratoon crop. The fewest fruit were produced in plots with PMWaV-1-infected plants that were nematode infected.

The impact of mealybug feeding and Pineapple mealybug wilt associated virus-1 (PMWaV-1) and PMWaV-2 infection on pineapple fruit yield, and the spread of PMWaV-1 and mealybug wilt of pineapple (MWP) were evaluated under field conditions with a randomized complete block design. Plots of PMWaV-1-free or infected plants were maintained mealybug-free or inoculated with mealybugs (Dysmicoccus spp.) at monthly intervals. Plants infected with PMWaV-2, an integral part of MWP etiology, were nested within plots that were maintained free of mealybugs, and in the plots of PMWaV-1 infected plants exposed to mealybugs. MWP, which only developed in PMWaV-2 infected plants exposed to mealybugs, resulted in a 35% reduction in yield when compared to PMWaV-free plants. Yield reductions were dependent on time of MWP symptom development; the earlier the expression of symptoms the greater the impact on fruit yields. An interaction effect between PMWaV infection, inclusive of both PMWaV-1 and PMWaV-2 infected plants, and mealybug exposure was detected in the plant crop (P < 0.02) but not in the ratoon crop (P > 0.59). This could be explained by the presence of MWP symptom expression during the plant crop and subsequent plant recovery in the ratoon crop. Virus infection, inclusive of PMWaV-1 and PMWaV-2, suppressed yield (P < 0.01) in the ratoon crop. The commercially desirable fruit sizes were most frequently obtained from PMWaV-free plants. Spatial analysis of PMWaV-2 spread, and MWP symptom expression in mealybug inoculated plots showed patterns of aggregation within rows and within beds but not between beds over the course of the study. Initial occurrence of MWP symptom expression in mealybug-inoculated plots was underdispersed indicating random occurrence of PMWaV-2 plants. After 6 months of mealybug exposure, patterns of both PMWaV-2 incidence and MWP were overdispersed. Management strategies are discussed.

Symptoms of leaf yellowing, reduced leaf size, and witches'-brooms have recently been observed affecting watercress (Nasturtium microphyllum Boen. × Rcbh.) in Hawaii. These symptoms are followed by the collapse of affected plants. This condition has led to 80 to 90% losses for one of the largest watercress farms on Oahu and is now affecting other watercress farms in the area. Nutritional deficiencies or toxicities, water salinity, and insect or mite feeding damage were investigated but could not be implicated in the etiology of this syndrome. Eighteen watercress plants with early yellowing or advanced symptoms and nine symptomless plants were analyzed for phytoplasma infection using polymerase chain reaction (PCR) assays with primer pairs P1/Tint or P1/P7 (4). Amplicons of the expected sizes were produced from all symptomatic plants, whereas no products were amplified from symptomless plants. Sequence analysis of the cloned PCR products confirmed their phytoplasma origin and indicated that the watercress was infected with a phytoplasma most similar to SAY (2), a severe strain of western aster yellows phytoplasma previously classified as a 16SrI-B group member (3). Leafhoppers collected from an affected watercress planting have been identified as the aster leafhopper (Macrosteles quadrilineatus Fbs.) This species is the most efficient vector of the aster yellows phytoplasma and had not been previously recorded in Hawaii. The only other phytoplasma disease known in Hawaii prior to this report is Dodonaea yellows (1), which affects one of the most common native plants (Dodonaea viscosa (L.) Jacq.) in dry upland forests on all the islands. Dodonaea yellows, however, has been attributed to an X-disease (16SrIII) group phytoplasma. The occurrence of an aster yellows group phytoplasma in watercress, a previously unrecorded host, and the presence of a very efficient vector, M. quadrilineatus, poses a serious threat to the production of other vegetable and floral crops in Hawaii. References: (1) W. Borth et al. Plant Dis. 79:1094, 1995. (2) C. Kuske and B. Kirkpatrick. Int. J. Syst. Bacteriol. 42:226, 1992. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) C. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Surveys for Pineapple mealybug wilt-associated virus-1 (PMWaV-1) and PMWaV-2 were conducted on pineapple samples from Hawaii and around the world. Tissue blot immunoassays (TBIAs) with two different monoclonal antibodies (MAb) specific to either PMWaV-1 or PMWaV-2 indicated that both closteroviruses are widely distributed throughout the pineapple-growing areas of the world. In the worldwide survey, PMWaV-1 was found in 80% of the mea-lybug wilt of pineapple (MWP)-symptomatic and 78% of the asymptomatic pineapple plants tested. A subset of plants was tested for PMWaV-2; 100% of the symptomatic plants and 12% of the asymptomatic plants were positive for this virus. A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to differentiate between PMWaV-1 and PMWaV-2. Oligonucleotide primers were designed using distinct regions of the HSP 70 homolog genes of the two viruses. PMWaV-specific RT-PCR assays and TBIAs were used to screen the pineapple accessions maintained at the United States Department of Agriculture-Agricultural Research Service National Clonal Germplasm Repository for PMWaV infection; 73% of the accessions were found infected with at least one PMWaV. Pineapple accessions found PMWaV-free were challenged with viruliferous mealybugs to test for immunity to PMWaV-1. No immune germ plasm was identified. Potential alternative virus hosts were screened for infection with virus-specific RT-PCR assays and TBIAs and were also challenged with viruliferous mealybugs. No alternate hosts of PMWaV-1 or PMWaV-2 were identified. PMWaV-1 infection was eliminated through axillary and apical bud propagation from infected crowns. Strategies to manage MWP are discussed.

Specific monoclonal antibodies made to a pineapple closterovirus (PCV) were used in a tissue blotting immunoassay (TBIA) for the detection of PCV in pineapple. More than 2,000 samples were tested in 5 days by one person using this rapid and reliable assay. A survey was conducted using this assay to test more than 20,000 Hawaiian pineapple samples for the presence of PCV. PCV was detected in symptomless pineapple plants in the field and in the USDA pineapple germ plasm collection. Studies of the association of PCV with mealybug wilt of pineapple (MWP) suggest that PCV may be involved in MWP.