First Report of a New Potexvirus Isolate from Citrus in the United States

In March 2015, virus-disease like symptoms were observed on greenhouse-grown lemon seedlings at USDA-ARS in Fort Pierce, FL. Symptoms appeared first on newly matured leaves, characterized by chlorotic flecking, mosaic, and vein necrosis on the abaxial side (Supplemental Figure 1), followed by leaf drop. The disease was traced to symptomatic lemon (‘Bakh’ and ‘Fourny’) trees in a variety collection at the USDA-ARS farm in Fort Pierce. All 44 seedlings from the infected tree showed typical symptoms three months after planting in the greenhouse. The virus was successfully transmitted to citrus and Chenopodium quinoa by grafting. Due to low virus titer in lemon, efforts to identify it using routine high-throughput sequencing were not successful. Consequently, we concentrated the viral RNA from total RNA isolated from symptomatic leaves using a RNeasy Plant Mini Kit (Qiagen, Germany) by depleting host cytoplasmic, mitochondrial and chloroplast rRNA (QIAseq FastSelect Plant, Qiagen) and using RNAClean XP beads (Beckman Coulter, U. S. ). We made a random primed cDNA library (Liefting et al. , 2021) for next generation sequencing via a ligation sequencing kit (SQK-LSK114, Oxford Nanopore Technologies, UK) followed by high-throughput sequencing using a MinION Mk1D (Oxford Nanopore Technologies). A total of 25, 486, 706 reads with a mean length of 347 bases was generated. Geneious (2023. 2. 1, Biomatters) was used for de novo viral genome assembly, producing a 6, 602 nucleic acid (nt) contig. To maximize coverage, all reads were subsequently aligned to the assembled contig using Bowtie2 (Langmead and Salzberg, 2012), which mapped 1, 356 reads with a mean length and coverage of 480 nt and 75. 4X, respectively. Rapid amplification of cDNA ends (RACE) was used to characterize the 5’ and 3’ terminal sequences (Takara Bio USA, Inc. , U. S. ), and the full-length cDNA clones were sequenced via Oxford Nanopore Technology (Plasmidsaurus, US), which showed 99. 7% nt identity to our assembled contig. The complete genome consisting of 6, 673 nts was deposited in GenBank as Accession No. PX389693. A BLASTn of the genome sequence resulted in highest coverage (75%) and identity (69. 6%) to the potexvirus, papaya mosaic virus at the genome level (GenBank Accession No. MN203139. 1). However, compared with the incomplete genome sequence of Lagenaria mild mosaic virus (Accession No. NC₀43079. 1), it showed 72. 5% nt identity with 57% coverage. All the reads were analyzed against the NCBI virus and viroid genome reference database, which revealed no additional viruses or viroids. . We propose citrus fleck mosaic (CFM) as a name for this disease. To detect CFM virus, we designed coat protein-specific primers CFMV-CPv (5’-ACAAAGTACGCTGCCTTCGA-3’), CFMV-CPvc (5’-TGTGGGTGAGCGAATGAGTC-3’), and TaqMan probe CFMV-CPp (5’-TGGAGAACCCGGCAGCCATGCA-3’). Citrus plants growing in the USDA-ARS greenhouses and farm in Fort Pierce were assayed using the Luna® Universal Probe One-Step RT-qPCR Kit (New England Biolabs, Ipswich, MA). We detected CFMV with Ct values of 31. 2 to 35. 5 in 7 of 15 greenhouse samples and 9 of 40 field samples, including lemon, lime, and grapefruit plants. To the best of our knowledge, this is the first report of a new potexvirus isolate infecting citrus in the U. S. , adding a new member to three other citrus viruses within the family Alphaflexiviridae. Further investigation into the transmission, host range, molecular host-virus interactions, and virus management is warranted for CFM.