Lavender (Lavandula × intermedia) plants showing severe decline were observed in fields in Rincón de Ademuz, Valencia, Spain, in November 2023 and July 2024. Symptoms included spike deformation, stem necrosis, collapse of the main axis, and eventual death. Symptomatic plants occurred in circular patches in plots. Two representative symptomatic plants were selected for high-throughput sequencing (HTS). Total RNA was extracted from leaves and stems using Plant/Fungi RNA Isolation Kit (Norgen Biotek, Canada), treated with DNase I, quantified with a DeNovix DS-11 spectrophotometer, and stored at -80°C. Library preparation and sequencing were performed using the TruSeq Stranded Total RNA Ribo-Zero Plant Kit (Illumina) and a NovaSeq X platform (2 × 150 bp). The first sample (IVIA188.1) yielded 57,676,035 high-quality reads (mean length 149.9 nt). De novo assembly with CLC Genomics Workbench v10.1.1 generated 291,713 contigs (mean 431 nt). BLASTn/x analysis identified 133 contigs (sizes ranging 201-5,717 nt, mean: 395.9 nt) with significant similarity (E-value ≤ 10⁻⁵⁰) to phlox virus M (PhVM; genus Carlavirus, species Carlavirus miphlocis). Mapping reads and de novo contigs allowed reconstruction of the complete PhVM genome (8,656 nt) (PX512721), supported by 3,561,806 mapped reads with mean coverage of 150.3×. The nucleotide sequence showed 97.7% similarity with the only available full-length sequence of this virus (OQ674100). The second sample (IVIA188.13) produced 45,528,387 high-quality reads (mean length 149.5 nt) and 147,016 contigs. Of these, 203 contigs (sizes ranging 203-5,405 nt, mean: 386.3 nt) matched PhVM sequences (E-value ≤ 10⁻⁵⁰). The complete genome of 8,635 nt (PX512722) was reconstructed with 3,655,865 reads mapping to it, providing a mean coverage of 476,959×. The nucleotide similarity with OQ674100 was 90.78% and between both Spanish isolates described here was 90.8%. Additional contigs (220-6,943 nt; mean 1,495 nt) showed distant similarity to caulimovirus-like sequences but were incomplete and non-informative. No other plant viruses or viroids were identified in either sample by the BLASTn/x analysis. In addition, other viruses previously reported in lavender (raspberry ringspot virus, alfalfa mosaic virus, cucumber mosaic virus, tobacco mosaic virus, tomato spotted wilt virus) as well as phytoplasmas were not detected by specific PCR based methods. PhVM infection was confirmed by RT-PCR in the 2 HTS-analized samples using specific primers amplifying the CP gene: PhlVM-CPF (5’-CCGTCTATTGACGAGTTGAGCA-3’) and PhlVM-CPR (5’-CACACCACACAACTTATTAAACAC-3’) (Tang et al., 2024). Sanger-sequenced confirmed 100% the bioinformatically reconstructed sequence in this genomic region. A survey of 40 plants from two affected and two unaffected fields in the same area detected 7 PhVM-positive samples: 5 symptomatic and 2 asymptomatic. Interestingly all positive samples were detected in the affected plots, whereas all samples from non-affected plots tested were negative for PhVM. These results suggest that the presence of PhVM could be involved in lavender decline, however more studies are required to clarify its role on the disease, either in single infection or in combination with other pathogens. To our knowledge, this is the first report of phlox virus M infecting lavender in Spain and the first potential association of this virus with severe decline symptoms in the crop.
Morán et al. (Sat,) studied this question.