First Report of rattail cactus necrosis-associated virus Infecting Prickly pear () in the United States.

Black-spined prickly pear (Opuntia macrocentra Engelmann; Cactaceae) is a cactus native to Arizona, New Mexico, Texas, and northwest Mexico. The plant is often grown for ornamental purposes in the United States. In February 2023, virus-like symptoms such as concentric ringspots and chlorotic spots were observed on O. macrocentra plants grown at the vicinity of Maricopa County Cooperative extension, University of Arizona, Phoenix, AZ (33°24'24.6"N, 111°59'15.3"W). Total RNA was extracted from two samples (YPHC-60-A and YPHC-60-B), following the protocol by Tzanetakis et al. (2007). Reverse transcription polymerase chain reaction (RT-PCR) was performed with degenerate tobamovirus, TobamodF/TobamodR (Li et al. 2018) and potexvirus, 1RC, Potex 2RC, and Potex 5 (van der Vlugt and Berendsen 2002) primers. An expected amplicon of 880 bp was obtained from both samples using TobamodF/TobamodR primers, while no amplification was observed with potexvirus primers. Further, RT-PCR was carried out using species-specific primers to detect cacti related tobamoviruses: cactus mild mottle virus (CMMoV), rattail cactus necrosis-associated virus (RCNaV) (Park et al. 2018) and Opuntia virus 2 (Salgado-Ortiz et al. 2020). Amplicons of ~540 bp were amplified from both samples using RCNaV specific primers, whereas no amplification was obtained using CMMoV and Opuntia virus 2 specific primers. Then, the amplicons from both YPHC-60 (A-B) isolates (540 bp) were Sanger sequenced and shared 99.22% nucleotide identity to each other. A BLAST search revealed 93% nucleotide identity with RCNaV CP sequences (KY581586.1, JF729471, and MT130378.1). The sequences were submitted in the GenBank (accessions no. OQ914798 and OR828526). Furthermore, complete RCNaV- RNA dependent RNA polymerase (RdRP) gene was amplified using primers 3490-s-5'GTAGGTGGTACCGCATAGCA-3'; 3490as 5'AAACGCAAGTCMRYGACYGA-3' (designed in this study from accession no. JF729471.1, position 3490-3509 and 4905-4925). The expected amplicons of ~1,500 bp were obtained from both YPHC-60 (A-B) samples and sequenced (GenBank: OQ914799 and OR823954) showing 87.5 % identity with RCNaV sequences (JF729471.1 and NC_016442.1). The maximum-likelihood phylogenetic tree clustered YPHC-60 (A-B) isolates in a single clade with other RCNaV isolates. RCNaV virus particles were isolated from YPHC-60 (A-B) and submitted for RNA extraction, testing positive for RCNaV by RT-PCR. Sap extract of YPHC-60 (A-B) prepared in 0.01 M phosphate buffer (pH =7.0) was used to mechanically inoculate 3 indicator plant species (n=10): Phaseolus vulgaris, Medicago sativa, and Cucumis melo. Also, infected tissue was used to graft Opuntia sp. plants. Symptoms such as local lesions were observed on M. sativa and vein thickening on P. vulgaris 14 days post-inoculation, while Opuntia sp. showed chlorosis 30 days after grafting. RCNaV infection in mechanically inoculated P. vulgaris, M. sativa, and Opuntia sp. was also confirmed through RT-PCR. C. melo and non-inoculated control plants did not show any symptoms, nor tested positive through RT-PCR. RCNaV has been reported earlier to infect cactus species in South Korea (Park et al. 2018) and O. albicarpa in Mexico (De La Torre-Almaráz et al. 2016), where it was found in several orchards. To the best of our knowledge, this is the first report of RCNaV infecting O. macrocentra in the United States. This study highlights that RCNaV is easily transmitted mechanically or by grafting, which could impact the nursery industry as most cacti are clonally propagated.