
First Report of Nectarine Virus M in Grapefruit (Citrus × paradisi Macfad.) in Association with Citrus Chlorotic Blotch Disease in Texas, U.S.A.
- Avijit Roy1 †
- Ashrafou Ouro-Djobo2
- Sam Grinstead1
- John Hammond3
- Mamoudou Sétamou4
- Olufemi J. Alabi2
- 1United States Department of Agriculture (USDA)-Agricultural Research Service, Molecular Plant Pathology Laboratory, BARC-West, Beltsville, MD 20705
- 2Department of Plant Pathology & Microbiology, Texas A&M AgriLife Research and Extension Center, Weslaco, TX 78596
- 3USDA-ARS, Floral and Nursery Plants Research Unit, USNA, Beltsville, MD 20705
- 4Texas A&M University-Kingsville Citrus Center, Weslaco, TX 78599
During November 2019, four leaf samples (TX1 to TX4) with citrus leprosis-like symptoms in ‘Rio Red’ grapefruit trees were collected from La Feria, Cameron County, Texas, U.S.A., and sent to USDA-Animal and Plant Health Inspection Service–Plant Protection Quarantine, Plant Pathogen Confirmatory Diagnostics Laboratory at Laurel, Maryland, for pathogen identification and confirmatory testing. Ribo-depleted libraries for all four samples were prepared for high-throughput sequencing (HTS) analysis, using the RNA extracts of individual grapefruit samples. HTS yielded 13.6 to 22.8 million 75-bp paired-end raw reads per sample library but no potential virus-like agent was identified at the time. Recent advances in bioinformatic tools (Roy et al. 2024) prompted a revisit of the archived HTS data, and several virus contigs were identified. The assembled contigs covered approximately 82% of the nectarine marafivirus M (NeVM) genome (GenBank accession KT273413) with read depths of 4.72 to 9.96 per nucleotide (nt). In addition, a few Caulimoviridae and Retroviridae contigs were also identified in all four libraries. NeVM was previously discovered from budwoods of nectarine trees from California using HTS and shown to infect peach (Villamor et al. 2016), but no other biological or serological data were reported. Foliar chlorotic blotch symptoms, reminiscent of the 2019 findings, were observed in adjacent Rio Red grapefruit blocks during September 2023. To investigate the association of chlorotic blotch symptoms with NeVM, 12 symptomatic and 4 nonsymptomatic grapefruit samples were collected for testing. A conventional RT-PCR primer pair, Marafi Gen-1F (5′-AACATGAAGAACGGSTTCGACG-3′) and NeVM-1R (5′-TTCATGGTGTGCATGGCRTTYTG-3′), was designed using HTS-derived NeVM contigs and utilized for the development of a detection assay. The results of the ∼671-bp amplicon sequencing showed that 13 (12 + 1) of the 16 grapefruit plants (81.25%) were positive for NeVM and shared 87.63 to 92.25% nt sequence identities with the nectarine isolates of NeVM (KT273411-13) and 78% with the Canadian Prunus isolate 13TF170 (MZ291915). To confirm the first report of NeVM in grapefruit trees, the archived leaf tissue sample (TX4) and 2023 tested samples (LF1 and LF2) from La Feria, TX, were selected for genetic analysis. The primer pair Marafi Gen-1F and NeVM-1R targeting the helicase domain of NeVM successfully amplified the expected fragment size of 671 bp. The amplicon sequence of the TX4 isolate shared 97.76 and 89.87% nt identities with the isolates LF1 and LF2, respectively, while LF1 shared a 90.76% nt identity with LF2. Sequence variation was observed for a 1,906-bp overlapping amplicon obtained with the primer pairs NeVM-2F (5′-CTGTTCGCCGAATGCATCAAYCT-3′)/Marafi Gen-1R (5′-AGTAGTACCCGCAGAAGGTGG-3′) and Marafi Gen-2F (5′-CCACCTTCTGCGGGTACTACT-3′)/Marafi Gen-2R (5′-CTGGAGGTGTTTTCCTTCACCTG-3′) spanning the catalytic domain and tymovirus coat protein region of NeVM. The analysis showed that the 1,906-bp amplicon sequence of the isolate TX4 shared 94 and 95% nt identities with the isolates LF2 and LF1, respectively, but only a 91% nt identity between them. Overall, the 1,906-bp amplicon of all three Texas grapefruit isolates shared 91.08 to 92.29% nt identities with American prunus isolates (KT273411-13) and a 75% nt identity with the Canadian isolate (MZ291915). Three sequences of 671-bp and 1,906-bp amplicons were deposited in GenBank under accession numbers PP767656 to PP767661. From the regulatory point of view, NeVM fails to satisfy the criteria to be considered as a potential quarantine pest for the European Union because of the absence of information on its biology, distribution, and economic impact (Bragard et al. 2019). However, this report expands the natural host range of NeVM to include grapefruit. From an epidemiological standpoint, more data on the host range, varietal susceptibility, and genetic variability among citrus and prunus isolates are needed to confirm the association of NeVM infection with symptom development.
The author(s) declare no conflict of interest.
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Funding: Funding was provided by USDA-ARS (NP303-8042-22000- 319-000-D).
The author(s) declare no conflict of interest.