First report of a new Potexvirus isolate from citrus in the United States

Authors: Duan, Yong Ping; Lopez Jr, Salvador; Bock, Clive; Adkins, Scott

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/5/2025
Publication Date: 1/4/2026
Citation: Duan, Y., Lopez Jr, S., Bock, C.H., Adkins, S.T. First report of a new Potexvirus isolate from citrus in the United States. Plant Disease. 2026. https://doi.org/10.1094/PDIS-09-25-1990-PDN.
DOI: https://doi.org/10.1094/PDIS-09-25-1990-PDN

Interpretive Summary: This is the first report of a new Potexvirus isolate, citrus fleck mosaic virus (CFMV) from citrus in the United States. Descriptions of the symptoms caused, and diagnostic methods used for CFMV are included. This report provides new insight of CFMV in Citrus and its potential association with HLB for growers, extension personnel, and local and Federal regulatory and research scientists.

Technical Abstract: 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 prominent chlorotic flecking, mosaic, and vein necrosis on the abaxial side (Supplemental Figure 1), followed by extensive leaf drop. The disease was traced to symptomatic lemon trees in a variety collection at the USDA-ARS farm in Fort Pierce. More seeds were collected from fruit of symptomatic plants and grown in a greenhouse. All 44 seedlings 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) followed by high-throughput sequencing using a MinION Mk1D (Oxford Nanopore Technologies, UK). A total of 25,486,706 reads with a mean length of 347 bases were generated. Geneious (2023.2.1, Biomatters) was used for de novo viral genome assembly, producing a contig of 6,602 nucleic acid (nt) long. 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., USA). 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). Comparing capsid protein gene and deduced amino acid sequences with Lagenaria mild mosaic virus (Accession No. YP_009664728), it showed 75.1% (nt) and 89.8% (aa) identity, respectively. All sequencing reads were analyzed through a local BLASTn against the NCBI virus and viroid genome reference database, which revealed no additional viruses or viroids in the data set. Viral sequences were further confirmed by reverse transcription-qPCR (RT-qPCR) amplification and Sanger sequencing with 99.7% nt identity. We propose citrus fleck mosaic virus (CFMV) as a name for this new potexvirus isolate. To detect CFMV, we designed coat protein-specific primers CFMV-CPv (5’-ACAAAGTACGCTGCCTTCGA-3’), CFMV-CPvc (5’-TGTGGGTGAGCGAATGAGTC-3’), and TaqMan probe CFMV-CPp (5’-TGGAGAACCCGGCAGCCATGCA). 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, grapefruit and pumelo plants with and without typical symptoms. 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 CFMV.