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AIMS: Dickeya species are high consequence plant pathogenic bacteria; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, Dickeya dianthicola. METHODS AND RESULTS: A signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome-based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed a detection limit of 10 fg DNA. CONCLUSION: The developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding programme. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

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Aphid-transmitted papaya ringspot virus (PRSV) is the greatest disease threat to the commercial production of papaya worldwide. Specific ultrasensitive assays are important for the early detection of PRSV in the field. We have developed a single-tube nested PCR (STNP) assay to address this need. Two nested PCR primer sets were designed to target the P3 gene of PRSV. The annealing temperatures and concentrations of both primer pairs were optimized to reduce potential competition between primer sets in STNP. The assay is more sensitive than regular RT-PCR as determined by serial dilutions of cDNA and RNA templates and sample extracts from infected plants. STNP is capable of detecting PRSV in plants 7 days post-inoculation, whereas RT-PCR and ELISA are capable of detecting PRSV 14 to 21 days post-inoculation. This new assay can also detect PRSV from virus infected but asymptomatic plants. This system could assist epidemiological studies in the field and in quarantine protocols by enabling early detection of very low PRSV infection rates in the field and in imported plant samples. Keywords: early detection; quarantine protocols.

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The coconut rhinoceros beetle, Oryctes rhinoceros (L.), is a major pest of coconut and other palm trees. An incipient coconut rhinoceros beetle population was recently discovered on the island of Oahu, Hawaii and is currently the target of a large, mutiagency eradication program. Confounding this program is the widespread presence of another scarab beetle on Oahu, the oriental flower beetle, Protaetia orientalis (Gory and Percheron 1833). Eggs, early life stages, and fecal excrement of coconut rhinoceros beetle and oriental flower beetle are morphologically indistinguishable, thereby creating uncertainty when such specimens are discovered in the field. Here, we report the development of a multiplex PCR assay targeting cytochrome oxidase I of coconut rhinoceros beetle and oriental flower beetle that can rapidly detect and distinguish between these insects. This assay also features an internal positive control to ensure DNA of sufficient quantity and quality is used in the assay, increasing its reliability and reducing the chances of false negative results.

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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.

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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.

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An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), an important factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcription of RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested polymerase chain reaction (PCR) to amplify a segment of the coat protein gene of the PMWaV-2. The outer primers were designed to anneal at higher temperatures than the nested primers to prevent primer competition in consecutive amplification reactions. To reduce potential competition further, the outer primers were used at one-thousandth the concentration of the nested primers. The specificity and sensitivity of this assay are much greater than PCR using only a single primer-pair. A TaqMan(®) probe was also designed for use in quantitative PCR to detect and quantify the PCR amplification products directly in a single-tube assay. The advantages of the single-tube assays using both conventional and quantitative PCR are reduced handling time and prevention of cross contamination compared to regular nested PCR in which the reactions are carried out in two separate tubes.

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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.

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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.

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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). A405nm 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.

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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.

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A complex of Pineapple mealybug wilt-associated viruses (PMWaVs) that can infect pineapple (Ananas comosus) is correlated with reduced yields and mealybug wilt of pineapple. The incidences of PMWaV-1 and PMWaV-2 at planting, fruit harvest, the beginning of the ratoon crop, and ratoon fruit harvest were determined for end, side, and central regions of planting blocks in eight commercial fields. Differences in virus incidence for the three regions at ratoon harvest were highly significant (P = 0.0018). Central regions of planting blocks had lower virus incidences at the time of ratoon fruit harvest. Collection of propagation material from the central regions of planting blocks will help to minimize PMWaV incidence in fields planted with this material.

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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.

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Pineapple mealybug wilt-associated virus-1 (PMWaV-1; family Closteroviridae, genus Ampelovirus) belongs to a complex of mealybug-transmissible viruses found in pineapple worldwide. In this study, the complete genome of PMWaV-1 was sequenced and found to be 13.1 kb in length, making it the smallest in the family. The genome encoded seven open reading frames (ORFs) and was unusual for an ampelovirus due to the lack of an intergenic region between the RdRp and p6 ORFs, an ORF encoding a relatively small coat protein (CP), and the absence of an ORF encoding a coat protein duplicate (CPd). Phylogenetic analyses placed PMWaV-1, plum bark necrosis stem pitting-associated virus and some grapevine leafroll-associated viruses in a distinct clade within the genus Ampelovirus.

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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.

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The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3'-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5' to 3', potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3' terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family CLOSTEROVIRIDAE: the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.

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Restriction fragment length polymorphisms (RFLP) were examined in three isoforms of a gene family encoding subtilisin-like proteases (Pr1A, Pr1B, and Pr1C) in several isolates of the entomopathogenic fungus Metarhizium anisopliae. RFLP variation was not observed in any of the Pr1 genes from isolates within the same genetically related group. Between genetically related groups and between isolates from disparate geographical areas, the greatest variation in RFLP patterns was observed for Pr1A. When variation does occur at Pr1B and Pr1C, it was generally observed at an EcoRI site. Metarhizium anisopliae var. majus strain 473 and a M. flavoviride isolate were most dissimilar in RFLP patterns at all Pr1 genes when compared to the M. anisopliae strains. We suggest that Pr1 genes represent a gene family of subtilisin-like proteases and that the Pr1A gene encodes for the ancestral subtilisin-like protease which has subsequently duplicated and rearranged within the genome.

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