First detection and complete genome sequence of a new tobamovirus naturally infecting Hibiscus rosa-sinensis in Hawaii.

High-throughput sequencing was used to analyze Hibiscus rosa-sinensis (family Malvaceae) plants with virus-like symptoms in Hawaii. Bioinformatic and phylogenetic analysis revealed the presence of two tobamoviruses, hibiscus latent Fort Pierce virus (HLFPV) and a new tobamovirus with the proposed name "hibiscus latent Hawaii virus" (HLHV). This is the first report of the complete sequence, genome organization, and phylogenetic characterization of a tobamovirus infecting hibiscus in Hawaii. RT-PCR with virus-specific primers and Sanger sequencing further confirmed the presence of these viruses. Inoculation experiments showed that HLFPV could be mechanically transmitted to Nicotiana benthamiana and N. tabacum, while HLHV could only be mechanically transmitted to N. benthamiana.

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.

First report of plumeria mosaic virus infecting Plumeria spp. in the United States.

Plumeria (Plumeria spp.) is a small ornamental tree grown in subtropical and tropical areas, providing shade and attractive and fragrant flowers in public and private landscapes. In Hawaii, plumeria is also commercially grown, with flowers harvested for cut flower and lei production. In 2019, a total of 10 cut flower plumeria farms in Hawaii sold 7,356,702 blossoms, valued at $395,791 (USDA/NASS). In July 2021, a commercial plumeria farm in Maui County, Hawaii reported trees with flowers exhibiting color breaking, and leaves with mosaic and vein banding. To determine if a viral pathogen was present, leaf tissue from a symptomatic plant was processed using a Double-RNA Viral dsRNA Extraction Kit for plant tissue (iNtRON Biotechnology, Korea), followed by random amplification (Melzer et al. 2010). The resulting products underwent paired-end sequencing on a MiSeq platform (Illumina, CA) at the Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory at the University of Hawaii. Of the 2,913,913 paired-end 300-bp reads generated, Geneious Prime (Biomatters, New Zealand) mapped 350,686 to plumeria mosaic virus (PluMV; KJ395757.1) and 293,911 to frangipani mosaic virus (FrMV; JN555602.2), with mean coverage depths of 10,318 and 7,348 respectively. A 6,648 nt contig representing the near full-length genome of PluMV-HI (OP342599) was found to be >97% identical to PluMV isolates PluMV-DR_TW (KX881422.2) and PluMV-Plu-Ind-1 (KJ395757.1). Similarly, a 6,631 nt contig representing the near full-length genome of FrMV-HI (OP342600) was found to be >98% identical to FrMV isolates FrMV-P (Lim et al. 2010; HM026454.1) and FrMV-Fr-Ind-1 (Kumar et al. 2015; JN555602.2). Bioinformatic analyses (Olmedo-Velarde et al. 2019) of the reads that did not map to PluMV and FrMV suggested the presence of a novel ampelovirus and reovirus in the tissue. To confirm the presence of PluMV and FrMV in Hawaii, leaves were collected in June 2022 from 5 plumeria trees on the Maui County farm and 16 ornamental plumeria trees from Honolulu County. Total RNA extracted as described by Li et al. (2008) as well as non-template (water) and positive (HTS tissue sample) controls underwent RT-PCR using random primers for cDNA synthesis, followed by virus-specific primers for PluMV (1133: 5'-TGGGCAAATAATCCGGCTATAC-3'/1134: 5'CCGGAGAGAGCATCAAACAA-3') and FrMV (1129: 5'-TGAGTTTAGGTCGCAGTTGATAG-3'/1130: 5-'AAAGACCAGAACCTCCAGAAAG-3') in singleplex reactions. The results indicated that all 5 plumeria samples from Maui County were positive for both PluMV and FrMV, whereas 6 out of 16 plumeria samples collected from Honolulu County tested positive for PluMV only. Both PluMV and FrMV are tobamoviruses (Virgaviridae) that can be transmitted mechanically and spread through the introduction and/or movement of cuttings, the primary method of plumeria propagation. Both PluMV and FrMV cause leaf mosaic (Kumar et al. 2015; Lim et al. 2010; Lin et al. 2020) and FrMV may also cause color break or "splash" in flowers, which was observed at the Maui County farm. This represents the first report of PluMV in the USA. FrMV was detected in Florida (Dey et al. 2020), but this is the first report of the virus in Hawaii. Additional surveys are required to determine the incidence of both viruses in other counties of Hawaii. The putative discovery of a novel ampelovirus and reovirus in plumeria warrants further research to characterize these agents and determine their distribution and impact on plumeria health.

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.

Genome characterization of fig umbra-like virus.

The complete genome of a new umbra-like virus from edible fig (Ficus carica) was identified by high-throughput sequencing. Based on its similarity to umbra-like virus genome sequences available in GenBank, the proposed name of this new virus is "fig umbra-like virus" (FULV). The genome of full-length FULV-1 consists of 3049 nucleotides organized into three open reading frames (ORFs). Pairwise comparisons showed that the complete nucleotide sequence of the virus had the highest identity (71.3%) to citrus yellow vein-associated virus (CYVaV). In addition, phylogenetic trees based on whole-genome nucleotide sequences and amino acid sequences of the RNA-dependent RNA polymerase showed that FULV forms a monophyletic lineage with CYVaV and other umbra-like viruses. Based on the demarcation criteria of the genus Umbravirus, and lack of two umbravirus ORFs, we propose that FULV is a putative new member of the umbra-like virus clade within the family Tombusviridae.

Examination of the Virome of Taro Plants Affected by a Lethal Disease, the Alomae-Bobone Virus Complex, in Papua New Guinea.

Alomae-bobone virus complex (ABVC) is a lethal but still understudied disease that is limited to the Solomon Islands and Papua New Guinea. The only virus clearly associated to ABVC is Colocasia bobone disease-associated virus (CBDaV). Taro (Colocasia esculenta) plants with and without symptoms of ABVC disease were sampled from two locations in Papua New Guinea and examined for viruses using high-throughput sequencing (HTS). Similar to previous reports, isolates of CBDaV were present only in symptomatic plants, further supporting its role in the disease. The only other viruses consistently present in symptomatic plants were badnaviruses: taro bacilliform virus (TaBV) and/or taro bacilliform CH virus (TaBCHV). If ABVC requires co-infection by multiple viruses, CBDaV and badnavirus infection appears to be the most likely combination. The complete genomes of two isolates of CBDaV and TaBCHV, and single isolates of TaBV and dasheen mosaic virus, were obtained in this study, furthering our knowledge of the genetic diversity of these relatively understudied taro viruses. HTS data also provided evidence for an agent similar to umbra-like viruses that we are tentatively designating it as Colocasia umbra-like virus (CULV).

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.