First Report of Nerine Latent Virus and Amazon Lily Mosaic Virus Infecting Amaryllis in Indonesia

Amaryllis (Hippeastrum sp.; Amaryllidaceae) is widely cultivated in Indonesia as an ornamental plant. In Indonesia, plants from this family have never been found to be infected by potyviruses or carlaviruses which are genera with wide host ranges, including ornamental plants (Figure S1) Diseased plants of Hippeastrum sp. were collected from two different locations in Bogor (West Java, Indonesia) between October 2023 and March 2024. The samples exhibited uneven colour, malformation and necrosis on the leaves (Figure 1). Total nucleic acid was extracted from the leaves using cetyltrimethylammonium bromide buffer (Doyle and Doyle 1987) to serve as templates for PCR amplification. One-step RT-PCR was conducted using universal primer pairs to amplify the partial coat protein and nucleic acid binding protein regions for carlaviruses as well as the partial coat protein and 3′UTR for potyviruses. The primers used for carlavirus detection were AlcarF (5′-TGCTGCYTTTGATACYTTCGAT-3′)/Poty1 (5′-GGATCCCGGGTTTTTTTTTTTTTTTTTV-3′) with an expected amplification product of c. 715 bp, whereas primers U341 (5′-CCGGAATTCATGRTITGGTGYATIGAIAAYGG-3′)/Poty1, with an expected amplicon of c. 750 bp, were used for potyvirus detection (Cremer et al. 2021; Gibbs and MacKenzie 1997; Langeveld et al. 1991). The RT-PCR mix consisted of 12.5 µL 2 × Dream Taq Green Master Mix (Thermo-Fisher Scientific, USA), 2 µL of each primer (10 µM of working primer), 1 µL ribonuclease inhibitor (40U/µL), 0.5 µL (200U/µL) reverse transcriptase (NZYtech, Portugal), 2 µL total RNA, and nuclease-free water to a final volume of 25 µL. One-step RT-PCR was initiated with cDNA synthesis at 45°C for 60 min followed by pre-denaturation at 94°C for 1 min. The next 35 cycles consisted of denaturation at 94°C for 30 s, annealing at 56°C for 60 s, and elongation at 72°C for 60 s; the last step was one cycle of post-elongation at 72°C for 10 min. Positive amplicons were sent to First Base, Malaysia for Sanger sequencing in both directions. Pairwise alignment of sequences was done using Geneious Prime v. 2025.1.3 (Biomatters Ltd., New Zealand) and the BLAST suite of algorithms. A phylogenetic tree was generated using the RAxML v. 8 method in Geneious Prime. Nerine latent virus (NeLV; Carlavirus) was identified in H. reginae (GenBank Accession no. LC860497.1) and Hippeastrum sp. (LC860498.1) sharing the highest identity of 97.9% and 98.2% (646 nt) with the South Korean (ON228217.1) and Australian isolates (JQ395044.1), respectively. The phylogenetic tree showed that the NeLV isolate clustered together with other NeLV isolates from different host ranges (Figure 2). Amazon lily mosaic virus (ALiMV; Potyvirus) was detected only in Hippeastrum sp. (LC860647.1); the isolate shared 94.5% identity (651 nt) with an ALiMV isolate from the Netherlands (PV167214.1). The phylogenetic tree clustered Indonesian ALiMV isolates with isolates from Japan and the Netherlands in one group (Figure 3). Previous studies have reported the presence of NeLV in the United States, the United Kingdom, the Netherlands and South Korea (Heo and Choi 2021; Jordan et al. 2018; Monger and Eden 2019). The host range has been observed to include Crinum sp., Narcissus sp. Nerine bowdenii, H. hybridum, Haemanthus albiflos, and diverse species of Amaryllidaceae (Jordan et al. 2018; Monger and Eden 2019). ALiMV was firstly reported from Amazon lily (Eucharis grandiflora) in Japan (Terami et al. 1995). To the best of our knowledge, this study represents the first report of NeLV and ALiMV infecting amaryllis in Indonesia. This study was funded by the ACIAR project number SLAM/2018/145. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.