First report of citrus yellow vein-associated virus in Aeonium arboreum in Europe

In April 2024, virus-like symptoms were observed in two potted plants of Aeonium cv. "black head" (Aeonium arboreum var. atropurpureum; Family Crassulaceae) growing in a private garden located in the Pontecagnano Faiano municipality (Salerno province, south Italy). This variety is highly appreciated for its dark purple leaves, whose colour becomes more intense in summer and in full light. Symptoms consisted of green spots of 2-6 mm on leaves, sometimes confluent to form slightly larger and more evident areas on the dark purple background colour. To assess the virome of symptomatic Aeonium plants, total RNAs were extracted from symptomatic leaves of one plant using the SNC50 kit (Sigma, USA) and, following ribodepletion, subjected to RNASeq high-throughput sequencing (HTS) on an Illumina NovaSeq platform (2x150 nt paired reads). The raw reads (24.953.652 total) were trimmed based on quality and ambiguity, using CLC Genomics Workbench 21 (CLC-GW-21; Qiagen, USA) default setting and resulting trimmed reads (average length 148.7 nt) were assembled de novo into contigs using the same programme. Viral contigs were identified through BlastN and BlastX analysis against default GenBank database (https://www.ncbi.nlm.nih.gov/blast), in particular, the Core nucleotide database (Core_nt) and ClusteredNR database (nr_cluster_seq). Contigs were assembled with CLC-GW-21 default settings and a minimum contig size of 250 nt, using a conservative e-6 e-value cut-off to identify viral contigs. Two contigs of respectively 2303 nt (39.331 reads, 2449x average coverage; Accession no. PZ289316) and 2154 nt (58.855 reads, 3900x average coverage; Accession no. PZ289317) were identified, showing very high percentages of nucleotide identity of respectively 92.9% (96% coverage) and 93.1% (90% coverage) with the Hadhar sunflower isolate of citrus yellow vein-associated virus (CYVaV; unclassified virus) from Iraq (PX308628). The two identified contigs showed 90% of nucleotide identity among them. No other contigs belonging to other viruses were identified in the HTS library. To confirm the presence of CYVaV, the RNA used for the HTS analysis and the RNA obtained from a second symptomatic plant were tested by RT-PCR using the specific primers AeoF (5’-GAGTACGGAAGSGAGTGGTTCG-3’) and AeoR (5’-AGAGTTGCTACCCCCAGATACAATCCC-3’) designed on the longest contig. Amplicons of the expected size (609 bp) were obtained from RNAs of both symptomatic plants, and in direct Sanger sequencing they showed 100% sequence identity with the same genomic portion of the original contig. No amplicons were obtained from an asymptomatic Aeonium plant. CYVaV was previously reported, causing citrus yellow-vein disease (CYVD), in a wild citrus (Citrus macroptera) in the USA (JX101610; Kwon et al., 2021), in hemp (Cannabis sativa) (OQ068388; Jarugula et al., 2023) in the USA, and in a pool of five cucurbit species in USA (PX904363; direct submission). This is the first report of CYVaV in Aeonium plants and in Europe. Further investigations would be necessary to understand if Aeonium could be a potential reservoir and source of CYVaV inoculum for vector-mediated spread to primary host-citrus.