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.

Impact of metal salts on the survival, development, and oviposition behavior of coconut rhinoceros beetle (Coleoptera: Scarabaeidae).

Oryctes rhinoceros (Coleoptera: Scarabaeidae) is an invasive pest of palms in the Pacific Region, including Hawaii, for which limited management options are available. O. rhinoceros larvae develop in materials rich in organic materials such as green waste and animal manure. Biosolid waste within Hawaii's infestation zone, however, was determined to inhospitable to O. rhinoceros. To determine if the elevated metal salts present in the biosolid waste was responsible for this observation, O. rhinoceros life stages were acutely and chronically exposed to several metal salts at increasing concentrations to determine the impact of these salts on survival, development, and oviposition behavior. Acute (7 days) exposure to mulch rehydrated in solutions of CaCl2, KCl, MgCl2, or NaCl increasing in concentration from 0 to 0.7 M resulted in increased mortality, with concentrations > 0.5 M generally being 100% lethal to both first and second-instar larvae. A similar trend in survival was observed in subsequent experiments using a horticultural grade of Epsom salt (MgSO4) at 1×, 2×, and 5× the recommended application rate. Chronic exposure (eggs reared on mulch through pupation) to Epsom salt at these same rates resulted in significantly delayed instar development and reduced adult biometrics at both 1× and 2× levels. Similar to the acute exposure, eggs exposed to 5× levels did not hatch, or the first instar died soon after emergence. In choice experiments, gravid females showed no oviposition preference for media hydrated with water or 2× Epsom salt but did avoid ovipositing in mulch rehydrated in 5× Epsom salt. These trials demonstrate a potentially novel approach to managing pest populations of O. rhinoceros.

Genetic Diversity of Viral Populations Associated with Ananas Germplasm and Improvement of Virus Diagnostic Protocols.

Pineapple (Ananas comosus L. [Merr.]) accessions from the U.S. Tropical Plant Genetic Resources and Disease Research (TPGRDR) in Hilo, Hawaii were subjected to RNA-sequencing to study the occurrence of viral populations associated with this vegetatively propagated crop. Analysis of high-throughput sequencing data obtained from 24 germplasm accessions and public domain transcriptome shotgun assembly (TSA) data identified two novel sadwaviruses, putatively named "pineapple secovirus C" (PSV-C) and "pineapple secovirus D" (PSV-D). They shared low amino acid sequence identity (from 34.8 to 41.3%) compared with their homologs in the Pro-pol region of the previously reported PSV-A and PSV-B. The complete genome (7485 bp) corresponding to a previously reported partial sequence of the badnavirus, pineapple bacilliform ER virus (PBERV), was retrieved from one of the datasets. Overall, we discovered a total of 69 viral sequences representing ten members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the pineapple mealybug wilt-associated virus (PMWaV) complex as well as PSVs. PMWaV-1, -3, and -6 presented recombination events across the quintuple gene block, while no recombination events were found for PMWaV-2. High recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for the specific identification and detection of viruses infecting pineapple based on the diverse viral populations characterized in this study. Given the high occurrence of recombination events, diversity, and discovery of viruses found in Ananas germplasm, the reported and validated RT-PCR assays represent an important advance for surveillance of viral infections of pineapple.

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.

First Detection and Genome Characterization of a New RNA Virus, Hibiscus Betacarmovirus, and a New DNA Virus, Hibiscus Soymovirus, Naturally Infecting Hibiscus spp. in Hawaii.

Hibiscus (Hibiscus spp., family Malvaceae) leaves exhibiting symptoms of mosaic, ringspot, and chlorotic spots were collected in 2020 on Oahu, HI. High-throughput sequencing analysis was conducted on ribosomal RNA-depleted composite RNA samples extracted from symptomatic leaves. About 77 million paired-end reads and 161,970 contigs were generated after quality control, trimming, and de novo assembly. Contig annotation with BLASTX/BLASTN searches revealed a sequence (contig 1) resembling the RNA virus, hibiscus chlorotic ringspot virus (genus Betacarmovirus), and one (contig 2) resembling the DNA virus, peanut chlorotic streak virus (genus Soymovirus). Further bioinformatic analyses of the complete viral genome sequences indicated that these viruses, with proposed names of hibiscus betacarmovirus and hibiscus soymovirus, putatively represent new species in the genera Betacarmovirus and Soymovirus, respectively. RT-PCR using specific primers, designed based on the retrieved contigs, coupled with Sanger sequencing, further confirmed the presence of these viruses. An additional 54 hibiscus leaf samples from other locations on Oahu were examined to determine the incidence and distribution of these viruses.

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.

A Virus Infecting Hibiscus rosa-sinensis Represents an Evolutionary Link Between Cileviruses and Higreviruses.

Hibiscus (Hibiscus spp.) are popular ornamental and landscape plants in Hawaii which are susceptible to foliar diseases caused by viruses belonging to the genera Cilevirus and Higrevirus (family Kitaviridae). In this study, a virus infecting H. rosa-sinensis plants displaying foliar symptoms consistent with infection by a kitavirus, including yellow chlorotic blotches with a green perimeter, was characterized. The genome consisted of two RNAs 8.4 and 4.4 kb in length, and was organized most similarly to cileviruses, but with important distinctions. These included the location of the p29 homolog as the 3'-terminal open reading frame (ORF) of RNA2 instead of its typical locus at the 3'-end of RNA1; the absence of a p15 homolog on RNA2 and the adjacent intergenic region which also harbors small putative ORFs of unknown function; and the presence of an ORF encoding a 10 kDa protein at the 3'-terminal end of RNA1 that was also found to be present in the hibiscus green spot virus 2 genome. Spherical particles approximately 55-65 nm in diameter were observed in infected leaf tissue, and viral RNA was detected by reverse-transcription PCR in individual mites collected from symptomatic plants tentatively identified as Brevipalpus yothersi. Although phylogenetic analyses placed this virus between the higrevirus and cilevirus clades, we propose the tentative taxonomic placement of this virus, designated hibiscus yellow blotch virus (HYBV), within the genus Cilevirus.

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.

ICTV Virus Taxonomy Profile: Closteroviridae.

Viruses in the family Closteroviridae have a mono-, bi- or tripartite positive-sense RNA genome of 13-19 kb, and non-enveloped, filamentous particles 650-2200 nm long and 12 nm in diameter. They infect plants, mainly dicots, many of which are fruit crops. This is a summary of the ICTV Report on the family Closteroviridae, which is available at ictv.global/report/closteroviridae.

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.

Air potato (Dioscorea bulbifera) plants displaying virus-like symptoms are co-infected with a novel potyvirus and a novel ampelovirus.

Air potato (Dioscorea bulbifera) plants being grown at the Florida Department of Agriculture and Consumer Services Division of Plant Industry Biological Control Laboratory II in Alachua County, Florida were observed exhibiting foliar mosaic symptoms characteristic of virus infection. A double-stranded RNA library generated from a symptomatic plant underwent high-throughput sequencing to determine if viral pathogens were present. Sequence data revealed the presence of two viral genomes, one with properties congruent with members of the genus Potyvirus (family Potyviridae), and the other with members of the genus Ampelovirus (family Closteroviridae). Sequence comparisons and phylogenetic placement indicate that both viruses represent novel species. The names "dioscorea mosaic virus" and "air potato virus 1" are proposed for the potyvirus and ampelovirus, respectively.

Oviposition Preferences and Behavior of Wild-Caught and Laboratory-Reared Coconut Rhinoceros Beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), in Relation to Substrate Particle Size.

The coconut rhinoceros beetle (CRB), Oryctes rhinoceros (L.) (Coleoptera: Scarabaeidae), has become one of the most important coconut and oil palm pests. This species was detected attacking coconut palms on O'ahu, Hawai'i in December 2013, and an eradication program was initiated. One of the major challenges for eradication has been the identification of new breeding sites. Information on the factors influencing oviposition is needed to aid in finding sites likely to host the immature stages of this insect. In this study, a series of choice tests were conducted to assess the oviposition preferences of both laboratory-reared and wild-caught CRB. Mated females, of both lab-reared and wild-caught beetles, were offered for oviposition a choice between sand and two mulch substrates, one with small and one with large particle sizes. Both types of CRB laid eggs preferentially in substrate of small particle size rather than large and none laid eggs in sand. Lab-reared and wild-caught CRB differed in their oviposition behavior and size. These results can be used to aid in the identification of breeding sites for management programs and eradication efforts.

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.

Complete Nucleotide Sequence of a Novel Hibiscus-Infecting Cilevirus from Florida and Its Relationship with Closely Associated Cileviruses.

The complete nucleotide sequence of a recently discovered Florida (FL) isolate of hibiscus-infecting cilevirus (HiCV) was determined by Sanger sequencing. The movement and coat protein gene sequences of the HiCV-FL isolate are more divergent than other genes of the previously sequenced HiCV-HI (Hawaii) isolate.

Potato virus Y (PVY) Isolates from Physalis peruviana are Unable to Systemically Infect Potato or Pepper and Form a Distinct New Lineage Within the PVYC Strain Group.

Poha, or cape gooseberry (Physalis peruviana L.), is a plant species cultivated in Hawaii for fresh fruit production. In 2015, an outbreak of virus symptoms occurred on poha farms in the South Kohala District of the island of Hawaii. The plants displayed mosaic, stunting, and leaf deformation, and produced poor fruit. Initial testing found the problem associated with Potato virus Y (PVY) infection. Six individual PVY isolates, named Poha1 to Poha6, were collected from field-grown poha plants and subjected to biological and molecular characterization. All six isolates induced mosaic and vein clearing in tobacco, and three of them exhibited O-serotype while the other three reacted only with polyclonal antibodies and had no identifiable serotype. Until now, PVY isolates have been broadly divided into pepper or potato adapted; however, these six PVY isolates from poha were unable to establish systemic infection in pepper and in four tested potato cultivars. Whole-genome sequences for the six isolates were determined, and no evidence of recombination was found in any of them. Phylogenetic analysis placed poha PVY isolates in a distinct, monophyletic "Poha" clade within the PVYC lineage, suggesting that they represented a novel, biologically and evolutionarily unique group. The genetic diversity within this poha PVYC clade was unusually high, suggesting a long association of PVYC with this solanaceous host or a prolonged geographical separation of PVYC in poha in Hawaii.

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.