First Report of Hoya tobamovirus 2 in <i>Hoya</i> Species in Australia

Hoya tobamovirus 2 (HoTV-2; syn. Hoya necrotic spot virus, genus Tobamovirus, family Virgaviridae) infects ornamental Hoya plants and is recorded from Germany and South Korea (Gaafar et al. 2020; Kim and Jeong 2024). In 2022, one plant each of the Hoya hybrid cv. Sunrise and Hoya carnosa cv. Krinkle, with chlorosis and necrotic patches on their leaves and infected with impatiens necrotic spot virus (INSV; species Orthotospovirus impatiensnecromaculae, genus Othotospovirus, family Tospoviridae), were collected from a commercial nursery in Victoria, Australia. Total RNA was extracted from each sample using an RNeasy Plant Mini kit (Qiagen, Australia). Libraries were prepared with the NEBNext UltraII RNA Library Prep Kit (New England BioLabs, USA). Sequencing on a Novaseq 6000 instrument (Illumina, USA) resulted in 3,238,966 (cv. Sunrise) and 5,148,862 (cv. Krinkle) raw reads and trimming and filtering using fastp version 0.20.0 (Chen et al. 2018) resulted in 2,181,218 and 3,284,930 reads, respectively. The trimmed reads were de novo assembled using SPAdes version 3.14.1 (Prjibelski et al. 2020). Comparison of the assembled contigs to sequences in GenBank indicated the presence of HoTV-2 and INSV in both samples. The longest HoTV-2 contig (6409 nt) from Hoya hybrid cv. Sunrise had > 99% nucleotide identity with other HoTV-2 isolates (GenBank Accession Nos. OR876400.1, LC807720.1, MN961200.1 and PP701469.1). A total of 1437.8 reads re-mapped to the 6409 nt HoTV-2 contig from Hoya hybrid cv. Sunrise with a sequence depth of 1437.8X. PrimalScheme (Quick et al. 2017) was used to design 12 pairs of primers (Table 1), based on the sequence of isolate JKI_19-461-466 (OR876400.1) then RT-PCR and Sanger sequencing of overlapping genome regions confirmed the detection and genome arrangement of HoTV-2 in Hoya hybrid cv. Sunrise (HoTV-2 Vic; PV929820). HoTV-2 Vic had 99.92% and 99.84% nucleotide identity to isolate JKI_19-461-466 and the 5700 nt contig from H. carnosa cv. Krinkle, respectively. Samples from 50 asymptomatic plants of ornamental and native Hoya spp. were collected from three Australian states (New South Wales, n = 6; Queensland, n = 40; Victoria, n = 4). HoTV-2 was detected in 30/50 samples, all from Queensland, by RT-PCR using primers HoTV-2 F9 and HoTV-2 R9 (Table 1). The sequenced amplicons of nine samples had > 99.6% nucleotide similarity to HoTV-2 VIC. The positive samples represented 2 Australian native Hoya species, H. australis and H. macgillivrayi, and 15 non-native species, H. arnottiana, H. carnosa, H. cinnamomifolia, H. compacta, H. cumingiana, H. finlaysonii, H. fungi, H. fuscomarginata, H. kerii, H. latifolia, H. mindorensis, H. multiflora, H. obovata, H. obscura and H. shepherdii. RT-PCR confirmed transmission by mechanical inoculation of HoTV-2 Vic to two uninfected H. globulosa plants, which developed mild chlorotic mottling on older leaves compared to uninoculated plants, and two plants of H. coronaria, which remained symptomless (Figure 1). INSV was not detected in any inoculated or uninoculated plant by RT-PCR (Mumford et al. 1996). Further work is required to determine if HoTV-2 was the cause of chlorotic mottling in H. globulosa and if it has the potential to cause symptoms in other Hoya species and hybrids. This is the first report of HoTV-2 in Australia. We thank Stuart Worboys, James Cook University and Lynne Jones, Department of Agriculture, Forestry and Fisheries, for providing the Queensland Hoya samples.