Molecular and Biological Characterization of a Novel Tobamovirus Infecting Sunn Hemp (Crotalaria juncea) in Hawaii.

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.

Preserving plant samples from remote locations for detection of RNA and DNA viruses.

Viral diseases in plants have a significant impact on agricultural productivity. Effective detection is needed to facilitate accurate diagnosis and characterization of virus infections essential for crop protection and disease management. For sensitive polymerase chain reaction (PCR)-based methods, it is important to preserve the integrity of nucleic acids in plant tissue samples. This is especially critical when samples are collected from isolated areas, regions distant from a laboratory, or in developing countries that lack appropriate facilities or equipment for diagnostic analyses. RNAlater ® provides effective, reliable sample storage by stabilizing both RNA and DNA in plant tissue samples. Our work indicated that total RNA or DNA extracted from virus-infected leaf samples preserved in RNAlater ® was suitable for reverse transcription polymerase chain reaction (RT-PCR), PCR, Sanger sequencing, high-throughput sequencing (HTS), and enzyme-linked immunosorbent assay (ELISA)-based diagnostic analyses. We demonstrated the effectiveness of this technology using leaf tissue samples from plants with virus symptoms grown in farmers' fields in Bangladesh. The results revealed that RNAlater ® technology was effective for detection and characterization of viruses from samples collected from remote areas and stored for extended periods. Adoption of this technology by developing countries with limited laboratory facilities could greatly increase their capacity to detect and diagnose viral infections in crop plants using modern analytical techniques.

Development of an ultra-sensitive single-tube nested PCR assay for rapid detection of Campylobacter jejuni in ground chicken.

Traditional culture-based detection methods for Campylobacteri jejuni, a leading cause of human bacterial gastroenteritis worldwide, are time-consuming, cumbersome, and lacking in reliability. While polymerase chain reaction (PCR) has been frequently used for pathogen testing, it might generate false-negative results due to inadequate sensitivity. This study was the first to explore novel single-tube nested PCR (STN-PCR) to detect pathogens in food. Two pairs of nested PCR primers were designed based on the hippuricase gene of C. jejuni. The annealing temperatures and concentrations of nested primers were optimized to ensure the sequential use of outer and inner pairs of primers during amplification. Efficacy of the developed STN-PCR assay was compared with standard culture-based methods and conventional PCR in artificially contaminated ground chicken homogenate. Limit of detection of the STN-PCR assay was determined to be 3.6 × 101 CFU/ml of C. jejuni in the homogenate without enrichment, which was 100 times lower than conventional PCR. Moreover, 0.1 CFU/g of C. jejuni in ground chicken homogenate was identified by STN-real time PCR (rtPCR) after 24 h of enrichment, while a 48-h enrichment was required for culture-based methods and conventional rtPCR. This developed assay provides a powerful tool for rapid, highly specific, and ultra-sensitive detection of C. jejuni and may potentially be used to identify contaminated chicken and other foods.

Characterization of taro reovirus and its status in taro (Colocasia esculenta) germplasm from the Pacific.

Taro reovirus (TaRV) has been reported infecting taro (Colocasia esculenta) in the South Pacific, but information on the virus is limited. Here, we report the genome sequence of a reovirus infecting taro in Papua New Guinea that had 10 genomic segments ranging from 1.1 to 3.9 kilobase pairs (kbp) in length with a total genome length of 26.3 kbp. TaRV was most closely related to rice ragged stunt virus (RRSV) but did not cross-react with RRSV polyclonal antisera. TaRV was not detected in 82 germplasm accessions of taro in Hawaii, or samples collected in American Samoa, Fiji, Guam, Palau, or Vanuatu.

Novel Fig-Associated Viroid-Like RNAs Containing Hammerhead Ribozymes in Both Polarity Strands Identified by High-Throughput Sequencing.

Based on high-throughput sequencing (HTS) data, the existence of viroid-like RNAs (Vd-LRNAs) associated with fig trees grown in the Hawaiian Islands has been predicted. One of these RNAs has been characterized as a circular RNA ranging in size from 357 to 360 nucleotides. Structural and biochemical features of this RNA, tentatively named fig hammerhead viroid-like RNA (FHVd-LR), markedly resemble those previously reported for several viroids and viroid-like satellite RNAs (Vd-LsatRNAs), which are non-protein-coding RNAs infecting their hosts autonomously and in combination with a helper virus, respectively. The full-length sequence of FHVd-LR variants was determined by RT-PCR, cloning, and sequencing. Despite a low global sequence identity with known viroids and Vd-LsatRNAs, FHVd-LR contains a hammerhead ribozyme (HRz) in each polarity strand. Northern blot hybridization assays identified the circular and linear forms of both polarity strands of FHVd-LR and showed that one strand, assigned the (+) polarity, accumulates at higher levels than the (-) polarity strand in vivo. The (+) polarity RNA assumes a rod-like secondary structure of minimal free energy with the conserved domains of the HRzs located in opposition to each other, a feature typical of several viroids and Vd-LRNAs. The HRzs of both FHVd-LR polarity strands were shown to be active in vitro during transcription, self-cleaving the RNAs at the predicted sites. These data, together with the sequence variability observed in the cloned and sequenced full-length variants, indicate that FHVd-LR is a novel viroid or Vd-LsatRNA. According to HTS data, the coexistence of FHVd-LR of different sizes in the same host cannot be excluded. The relationships of FHVd-LR with previously reported viroids and Vd-LsatRNAs, and the need to perform bioassays to conclusively clarify the biological nature of this circular RNA, are discussed.

Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus).

The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.

Papaya Ringspot Virus Isolates From Papaya in Bangladesh: Detection, Characterization, and Distribution.

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.

Characterization of Ti Ringspot-Associated Virus, a Novel Emaravirus Associated with an Emerging Ringspot Disease of Cordyline fruticosa.

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.

Occurrence of tomato leaf curl Bangladesh virus and associated subviral DNA molecules in papaya in Bangladesh: molecular detection and characterization.

Forty-five papaya samples showing severe leaf curl symptoms were tested by PCR with a degenerate primer set for virus species in the genus Begomovirus. Of these, 29 were positive for tomato leaf curl Bangladesh virus (ToLCBV). The complete genome sequences of ToLCBV (GenBank accession no. MH380003) and its associated tomato leaf curl betasatellite (ToLCB) (MH397223) from papaya isolate Gaz17-Pap were determined and characterized. Defective betasatellites were found in ToLCBV-positive papaya isolates Gaz19-Pap, Gaz20-Pap and Gaz21-Pap. This study confirmed that papaya is a host of ToLCBV, ToLCB, and other defective and recombinant DNA satellites in Bangladesh.

A New Strain of Bean Common Mosaic Virus From Lima Bean (Phaseolus lunatus): Biological and Molecular Characterization.

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.

Detection of Jasmine virus H and characterization of a second pelarspovirus infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants displaying virus-like symptoms.

Star jasmine (Jasminum multiflorum) plants growing in Hawaii expressing a diverse array of virus-like foliar symptoms were examined for the presence of a causal agent. Symptomatic tissues collected from three locations on the island of Oahu, Hawaii consistently harbored double-stranded (ds)RNAs approximately 4.2 and 1.7 kbp in size. Sanger and high-throughput sequencing approaches revealed these dsRNAs were from two distinct virus species co-infecting the same host plant. One of these two viruses was the recently characterized Jasmine virus H (JaVH), and the second we designated as Jasmine mosaic-associated virus (JMaV). Both viruses were subsequently found, by high-throughput sequencing, in a single angelwing jasmine (J. nitidum) plant exhibiting similar ringspot symptoms and growing at the U.S. National Arboretum in Washington, DC. Phylogenetic placement, genome organization, and sequence comparisons indicate these two viruses are classifiable as members of the genus Pelarspovirus (family Tombusviridae). To determine if either of these viruses were associated with the observed symptoms, a PCR-based detection assay was developed to detect and distinguish these two viruses in several Hawaii-grown plants. All 32 samples collected from four Oahu locations displayed symptoms. All 32 samples were positive for JaVH, and 16 were positive for JMaV. An asymptomatic star jasmine plant from the island of Hawaii was negative for both JaVH and JMaV. Both viruses were also found in a symptomatic J. sambac sample from Maryland while only JMaV was detected in a symptomatic Jasminum sp. sample from California.

Characterization of Canna yellow mottle virus in a New Host, Alpinia purpurata, in Hawaii.

Canna yellow mottle virus (CaYMV) is an important badnavirus infecting Canna spp. worldwide. This is the first report of CaYMV in flowering ginger (Alpinia purpurata) in Hawaii, where it is associated with yellow mottling and necrosis of leaves, vein streaking, and stunted plants. We have sequenced CaYMV in A. purpurata (CaYMV-Ap) using a combination of next-generation sequencing and traditional Sanger sequencing techniques. The complete genome of CaYMV-Ap was 7,120 bp with an organization typical of other Badnavirus species. Our results indicated that CaYMV-Ap was present in the episomal form in infected flowering ginger. We determined that this virus disease is prevalent in Hawaii and could potentially have significant economic impact on the marketing of A. purpurata as cut flowers. There is a potential concern that the host range of CaYMV-Ap may expand to include other important tropical plants.

Molecular Characterization and Distribution of Two Strains of Dasheen mosaic virus on Taro in Hawaii.

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.

Deep sequencing of banana bract mosaic virus from flowering ginger (Alpinia purpurata) and development of an immunocapture RT-LAMP detection assay.

Banana bract mosaic virus (BBrMV) has never been reported in banana plants in Hawaii. In 2010, however, it was detected in a new host, flowering ginger (Alpinia purpurata). In this study, we characterize the A. purpurata isolate and study its spread in flowering ginger in Hawaii. A laboratory study demonstrated that BBrMV could be transmitted from flowering ginger to its natural host, banana, therefore raising a serious concern about the potential risk to the rapidly growing banana industry of Hawaii. To quickly monitor this virus in the field, we developed a robust immunocapture reverse transcription loop-mediated isothermal amplification (IC-RT-LAMP) assay. Deep sequencing of the BBrMV isolate from A. purpurata revealed a single-stranded RNA virus with a genome of 9,713 nt potentially encoding a polyprotein of 3,124 aa, and another predicted protein, PIPO, in the +2 reading-frame shift. Most of the functional motifs in the Hawaiian isolate were conserved among the genomes of isolates from one found in the Philippines and India. However, the A. purpurata isolate had an amino acid deletion in the Pl protein that was most similar to the Philippine isolate. Phylogenetic analysis of an eastern Pacific subpopulation that included A. purpurata was closest in genetic distance to a Southeast Asian subpopulation, suggesting frequent gene flow and supporting the hypothesis that the A. purpurata isolate arrived in Hawaii from Southeast Asia.

Analysis of pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors.

Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.

A cilevirus infects ornamental hibiscus in Hawaii.

The complete nucleotide sequence of a virus infecting ornamental hibiscus (Hibiscus sp.) in Hawaii with symptoms of green ringspots on senescing leaves was determined from double-stranded RNA isolated from symptomatic tissue. Excluding polyadenylated regions at the 3' termini, the bipartite RNA genome was 8748 and 5019 nt in length for RNA1 and RNA2, respectively. The genome organization was typical of a cilevirus: RNA1 encoded a large replication-associated protein with methyltransferase, protease, helicase and RNA-dependent RNA polymerase domains as well as a 29-kDa protein of unknown function. RNA2 possessed five open reading frames that potentially encoded proteins with molecular masses of 15, 7, 62, 32, and 24 kDa. The 32-kDa protein is homologous to 3A movement proteins of RNA viruses; the other proteins are of unknown function. A proteome comparison revealed that this virus was 92 % identical to citrus leprosis virus cytoplasmic type 2 (CiLV-C2), a recently characterized cilevirus infecting citrus with leprosis-like symptoms in Colombia. The high sequence similarity suggests that the virus described in this study could be a strain of CiLV-C2, but since the new genus Cilevirus does not have species demarcation criteria established at present, the classification of this virus infecting hibiscus is open to interpretation. This study represents the first documented case of a cilevirus established in the United States and provides insight into the diversity within the genus Cilevirus.

Molecular characterization of closteroviruses infecting Cordyline fruticosa L. in Hawaii.

In Hawaii, common green ti plants (Cordyline fruticosa L.) have been shown to harbor Cordyline virus 1 (CoV-1) which, along with Little cherry virus 1 (LChV-1), and Grapevine leafroll-associated virus 7 (GLRaV-7), form a distinct clade within the family Closteroviridae. Preliminary work has indicated that, aside from CoV-1, three additional closteroviruses may infect common green ti plants in Hawaii. In this study, pyrosequencing was used to characterize the genomes of closteroviruses infecting a single common green ti plant. The sequence data confirmed the presence of CoV-1 as well as three additional closteroviruses. Although all four viruses had the same general genome organization, the sequence divergence between the RNA-dependent RNA polymerase, heat shock protein 70 homolog, and coat protein ranged from 22 to 61%, indicating these represent four distinct closterovirus species. The names CoV-2, CoV-3, and CoV-4 are proposed for the three new viruses. Phylogenetic analyses placed CoV-2, CoV-3, and CoV-4 in the same clade as CoV-1, LChV-1, and GLRaV-7.

Characterization of a virus infecting Citrus volkameriana with citrus leprosis-like symptoms.

A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. 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 ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 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. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus 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. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.

An assemblage of closteroviruses infects Hawaiian ti (Cordyline fruticosa L.).

The ti plant (Cordyline fruticosa L.) is culturally important throughout most of Polynesia and has considerable economic importance in Hawai'i where the foliage is commonly used in cultural ceremonies as well as food and ornamental industries. In Hawai'i, ringspot symptoms were recently observed on leaves of the common green variety of ti growing in Kahalu'u on the island of O'ahu, and Wailuku and Hana on the island of Maui. High molecular weight double-stranded (ds)RNAs were isolated from the leaves of symptomatic plants as well as plants without symptoms. A cDNA library derived from the dsRNAs present in symptomatic plants was generated and sequenced. These sequences indicated at least four distinct clostero-like viruses were present in the plants, and phylogenetic analyses suggested they were most closely related to Little cherry virus 1, an unassigned member of the family Closteroviridae. The 16,883 nucleotide genome of one of these viruses was determined and predicted to contain ten open reading frames with an organization typical of closteroviruses. Reverse-transcription PCR revealed this virus was present in both symptomatic and asymptomatic ti plants, making it unlikely to be responsible for the observed ringspot symptoms. We propose the name Cordyline virus 1 (CoV-1) for this virus and include it as a new, unassigned member of the family Closteroviridae.

Genetic diversity and evidence for recent modular recombination in Hawaiian Citrus tristeza virus.

The Hawaiian Islands are home to a widespread and diverse population of Citrus tristeza virus (CTV), an economically important pathogen of citrus. In this study, we quantified the genetic diversity of two CTV genes and determined the complete genomic sequence for two strains of Hawaiian CTV. The nucleotide diversity was estimated to be 0.0565 + or - 0.0022 for the coat protein (CP) gene (n = 137) and 0.0822 + or - 0.0033 for the p23 gene (n = 30). The genome size and organization of CTV strains HA18-9 and HA16-5 were similar to other fully sequenced strains of CTV. The 3'-terminal halves of their genomes were nearly identical (98.5% nucleotide identity), whereas the 5'-terminal halves were more distantly related (72.3% nucleotide identity), suggesting a possible recombination event. Closer examination of strain HA16-5 indicated that it arose through recent recombination between the movement module of an HA18-9 genotype, and the replication module of an undescribed CTV genotype.