First report of zucchini yellow mosaic virus (ZYMV) infecting Cucurbita maxima in Peru

Zucchini yellow mosaic virus (ZYMV), a member of the family Potyviridae, was first reported in Italy in 1973 (Desbiez & Lecoq, 1997). Since then, it has been reported in various regions worldwide, including Asia, Europe, Africa, and the Americas. This virus has caused severe economic epidemics in cucurbit crops globally, causing symptoms such as mosaic, foliar mottling, and fruit deformities (Berbati et al., 2023; Ali; Damayanti et al., 2021; Clarke, 2020). In Peru, this virus is causing severe damage to cucurbit crops. This study reports the detection of ZYMV in a Macre squash (Cucurbita maxima) fields through high-throughput sequencing (HTS) and Sanger sequencing. In September 2022, C. maxima cv. Macre plants exhibiting symptoms of foliar mosaic and fruit deformation were observed in fields located in La Molina, Lima, Peru. To assess the virus distribution in Peru, a total of 270 C. maxima samples comprising both symptomatic and asymptomatic plants were randomly collected between June and August 2023 from various regions: Chiclayo (25 samples), Lima (152), Ica (47), and Arequipa (46). ZYMV detection was performed using DAS-ELISA (DSMZ, QC-SOP-0087), revealing a prevalence of 60–71%. From these, a symptomatic young leaf was collected from each of the five plants (M1, M2, M3, M4, and M5) to extract total RNA using the SV Total RNA Isolation System kit (Promega) and lyophilize them. RNA from the M1 sample was used to construct a ribosomal RNA-depleted library and sequenced on the NextSeq 1000 platform, yielding 30 million 150 bp paired-end reads. Data were processed via the Galaxy platform following Mendoza et al. (2022). Trimmomatic v0.32 was used to trim low-quality reads, resulting in 59,262,180 reads, which were assembled into 77,583 contigs with SPAdes v3.12.0. Contigs were analyzed by tBLASTx against the RefSeq2022 viral database using Geneious vR11 (Biomatters, NZ) A total of 140 contigs exclusively corresponding to ZYMV were identified and assembled de novo, generating a consensus sequence of 9,484 nt with 98.9% identity to a ZYMV sequence from the United Kingdom by (Cucurbita pepo; GenBank OM471983) and 91.6 % identity to one from Brazil (Citrullus lanatus; GenBank MN364667). Mapping of the trimmed reads revealed 4,017,426 aligned reads with 65,544x coverage, confirming ZYMV as the sole causal agent of the observed disease. To validate the HTS-derived sequence, specific primers ZYMV4274_F/ZYMV4663_R and ZYMV8362_F/ZYMV9402_R were designed to amplify 412 bp and 1,063 bp fragments corresponding to the CI (GenBank PV599321) and the NIb C-terminal regions extending to the initial 3’UTR (GenBank PV599322), respectively, via RT-PCR. The amplified sequences from the M1 sample were confirmed through Sanger sequencing, showing 99.9% identity to the HTS-derived sequence. Additionally, ZYMV was reactivated from the lyophilized M1 sample by inoculating C. pepo var. grey zucchini, which was subsequently used as the inoculum source for mechanical inoculation on 21 plant species from the families Cucurbitaceae, Solanaceae, Lamiaceae, Fabaceae and Amaranthaceae. Symptomatic infection was observed seven days after inoculation in cucurbit species, including C. moschata, C. maxima, C. pepo, C. ficifolia, Cucumis sativus, C. melo and Citrullus lanatus, while Luffa spp. and Cyclanthera pedata showed no signs of infection. Meanwhile, Chenopodium quinoa and Ch. amaranticolor exhibited local chlorotic lesions on the inoculated leaves. All inoculated plants were confirmed by DAS-ELISA. Observed symptoms varied depending on species and cultivar, ranging from mild to severe mosaic, vein banding, leaf deformation, and vein clearing. To date, in South America, ZYMV has only been reported in Argentina, Brazil, Chile, Colombia, Guyana, Paraguay, and Venezuela (Spadotti et al., 2015). Therefore, this study represents the first official report of ZYMV in Peru with the isolate designated as ZYMV_Lim (GenBank OQ181225). The virus is widespread across major key squash-producing regions along the Peruvian coast.