W. B. Borth

Pineapple bacilliform CO virus: Diversity, Detection, Distribution, and Transmission

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.

First report of Tomato yellow leaf curl virus in Hawaii.

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 states

A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2).

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.

Spatial and temporal incidences of Pineapple mealybug wilt-associated viruses in pineapple planting blocks.

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.

Genome characterization and distribution of Taro bacilliform CH virus on taro in Hawaii, USA

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.

Transgene-mediated resistance to Papaya ringspot virus: challenges and solutions

Taro bacilliform CH virus (TaBCHV) is the second badnavirus found infecting taro (Colocasia esculenta), after Taro bacilliform virus (TaBV). We determined the complete genome of TaBCHV Hawaii Strain (KY359389) from taro in Hawaii and examined its occurrence and distribution. Alignment analysis showed the TaBCHV Hawaii Strain has 93% and 94% sequence identity to China strains TaBCHV-1 and TaBCHV-2 respectively. Phylogenetic analysis basing on the whole genome sequence of 21 selected badnaviruses, showed that the TaBCHV Hawaii Strain clustered within the same branch as the two China strains. All sequences from available TaBCHV strains are most closely related to Piper yellow mottle virus (PYMoV; NC022365) and are most distant from TaBV (NC004450). We used RT-PCR to determine the occurrence and distribution of TaBCHV on taro in Hawaii. The positive rates of the virus on the five islands were 72% on Oahu, 53% on Hawaii, 89% on Maui, 73% on Molokai, and 78% on Kauai, with a mean positive rate of 73%. This is the first report of the complete genome sequence and the first report of the occurrence of this virus in the United States.

Correction to: Genome characterization and distribution of Taro bacilliform CH virus on taro in Hawaii, USA

Global papaya production is severely affected by papaya ringspot disease caused by Papaya ringspot virus (PRSV). Management of this potyvirus is challenging, due to 1) its non-persistent transmission by numerous aphid species and 2) the diversity of PRSV strains that exists within a country or between different geographical regions. Papaya cultivars with transgenic resistance have reduced the impact of the disease. There are no effective alternatives to transgenic resistance available in areas where disease pressure is high. In Hawaii, transgenic papayas such as “SunUp” and “Rainbow” have remained resistant to PRSV more than two decades saving the commercial papaya industry. Following the success in Hawaii, researchers from other countries have focused on developing PRSV-resistant transgenic papaya. These transgenic cultivars often demonstrated an initial transitory resistance that was ultimately overcome by the virus. For other cases, resistance was inconsistent. That is, some transgenic lines were resistant while others were not. Transgenic cultivars are now losing PRSV-resistance for various reasons in China and Taiwan. In this review, we present an update on work with transgenic papaya with resistance to PRSV. The focus is on factors affecting transgenic resistance in papaya and our attempt to explain why the Hawaiian scenario of complete and durable resistance has not been replicated in other regions. The utilization of more recent technologies to the development of virus resistance in papaya is also discussed.

First Report of Bean common mosaic virus Infecting Lima Bean in Hawaii

Due to some error captions for Fig. 1 and Fig. 2 have somehow been switched and needs to be read as: Fig. 1: Genome organization of Taro bacilliform CH virus (TaBCHV) Hawaii Srain.

First Report of Banana bunchy top virus in Heliconia spp. on Hawaii

Bean common mosaic virus (BCMV; genus Potyvirus, family Potyviridae) is a single-stranded RNA virus that infects leguminous crops worldwide, and is transmitted in a nonpersistent manner by aphids. In July 2016, several plants exhibiting mosaic, mottling, chlorotic leaf symptoms, and stunting were found in a community garden in Honolulu, HI. Leaves from symptomatic Phaseolus lunatus (Lima bean), Araceae spp., Colocasia esculenta, Cordyline fruticosa, Crinium spp., Heliconia spp., Passiflora spp., Polyscias spp., and Syngonium spp. were collected from the community garden and tested for potyvirus infection using a Potyvirus-specific ELISA (Agdia, Elkhart, IN). P. lunatus and Passiflora spp. leaf tissue tested positive in ELISA. Total RNA was isolated from P. lunatus and Passiflora spp. leaf tissues using an RNeasy Plant Mini Kit (Qiagen, Redwood City, CA). Reverse transcription (RT)-PCR assays were conducted using a universal primer set designed to amplify a partial sequence of the nuclear inclusion B (NIb) gene of the potyviral genome: NIb2F (5′GTITGYGTIGAYGAYTTYAAYAA-3′) and NIb3R (5′-TCIACIACIGTIGAIGGYTGNCC-3′) (Zheng et al. 2010). The expected 350-bp products were amplified from the P. lunatus and Passiflora spp. samples, cloned into the pGEM-T Easy vector (Promega, Madison, WI), and sequenced. The sequence of the products from Passiflora spp. had an expected high similarity to Watermelon mosaic virus (WMV), which was previously reported infecting Passiflora spp. in Hawaii (Watanabe et al. 2016). The products from P. lunatus (accession no. KY473075) had Quick Links Add to favorites

Engineering Resistance to Viruses

Banana bunchy top virus (BBTV) infects Musa spp. and causes banana bunchy top disease (BBTD), one of the most significant constraints to banana (Musa spp.) production worldwide (Xie and Hu 1995). The virus is transmitted by banana aphids (Pentalonia spp.), which can also feed on heliconia (Heliconia spp.) (Watanabe et al. 2013). Heliconias, native to the tropical Americas, are common landscape plants in Hawaii and have naturalized in some disturbed areas and forests. If heliconias are hosts of BBTV, they may potentially pose a significant threat to banana production in Hawaii. In July 2016, heliconia (Heliconia aurantiaca) plants with chlorotic leaves and green flecking of the veins were observed in a community garden in Honolulu, HI (21.2851°N, 157.8280°W). Fifteen symptomatic samples were tested for BBTV by PCR assays using virus-specific primers BBTV-RT1F (5′ACCAGCCGACTACATGTCTG-3′) and BBTV-RT2R (5′-TCCTCAACAC GGTTGTCTTC-3′). The expected 155-bp fragment of the partial capsid protein (CP) gene was amplified from eight of the 15 samples. These samples also tested positive in triple-antibody sandwich (TAS)-ELISA for BBTV (Agdia, Elkhart, IN), confirming the presence of the virus. Thirty-two additional heliconia samples with similar symptoms were collected from the same location and 22 tested positive in seven PCR tests using two different sets of BBTV-specific primer pairs: Repgene-specific primers BBTV-RepF1 (5′-CCATCAACAATCCCACAACA-3′) and BBTV-RepR1 (5′ACAGTATGACCGCGCGCTTCTT-3′); and CP-specific primers BBTV-HAF1 (5′TCCGAAGAATCCATCAAGA-3′) and BBTV-HAR1 (5′-ACACGGTTGTCTTCCTCAAA-3′). The Quick Links Add to favorites