First report of Pepo aphid‐borne yellows virus on watermelon plants in Spain

Abstract

In 2021, fifty-two watermelon (Citrullus lanatus) samples exhibiting yellowing virus-like symptoms (Figures 1, 2) were collected from thirteen open-field plots in Murcia (southeastern Spain). Total RNA extraction and dot-blot molecular hybridization were performed to detect Cucumber vein yellowing virus, Cucurbit aphid-borne yellows virus (CABYV), Cucurbit chlorotic yellows virus, Cucurbit yellow stunting disorder virus and Zucchini yellow mosaic virus (Rabadán et al., 2023), but none of these viruses were detected. Leaf yellowing symptoms may also be confused with plant nutrient deficiency. However, considering the large aphid populations in the affected crops and the inconspicuous symptoms that poleroviruses often cause in affected cucurbits, we sought to check for the possibility of polerovirus infection. Five random samples were subjected to RT-PCR using the polerovirus primers PolGenUp2 and PolGen RT3 (Lotos et al., 2014). For three samples, a 593 bp amplicon was produced, encompassing part of the polerovirus RNA-dependent RNA polymerase (RdRp) and sequenced (STAB VIDA, Portugal). The sequencing results from the three amplicons confirmed the presence of a polerovirus and identified the virus as Pepo aphid-borne yellows virus (PABYV), with 96.7% nucleic acid identity to GenBank Accession No. LN865082, reported from Greece. PABYV is a phloem-restricted RNA plant viruses that is transmitted by aphids and has been detected in West Africa and the Mediterranean, including Greece, Italy and Syria (Lotos et al., 2016; Parrella et al., 2023). To understand the extent and distribution of PABYV in these watermelon production areas, we amplified the coat protein (CP) from a Spanish PABYV isolate as described by Masika et al. (2022). The CP was cloned into the pGEMT-easy vector to facilitate the synthesis of a specific RNA-probe labeled with digoxigenin. All the watermelon samples collected in 2021, and a further 68 symptomatic samples collected during the 2022 and 2023 seasons, were tested for PABYV by dot-blot molecular hybridization, as described by De Moya-Ruiz et al. (2021). The analysis revealed that the occurrence of PABYV ranged from 75 to 100% in watermelon crops during the three seasons. Dot-blot hybridization was also used to detect the other cucurbit viruses listed above. Only PABYV was detected in 2021 and 2022, while PABYV and CABYV were found in mixed infections in all samples from 2023. Three PABYV isolates per year were selected randomly, and the CP gene was amplified, sequenced and deposited in GenBank (OR253793-OR253801). Phylogenetic analysis was done using the maximum likelihood method in MEGA X. The nine PABYV Spanish isolates clustered in a group along with the Greek (LN865082), Italian (OP973153) and Syrian (KC960436) isolates (Figure 3). This would suggest a common origin for the PABYV isolates spreading in the Mediterranean area. This is the first report of PABYV in Spain. The emergence of this aphid-borne virus in a new geographic area represents further evidence of either recent spread to this area or that it has been present over a longer term but previously unreported and/or possibly unnoticed in mixed infections with other viruses. It is likely that PABYV infection can reduce cucurbit production. Therefore, it is crucial to conduct systematic and extensive monitoring of PABYV in Spain to ascertain its distribution and prevalence in cucurbit crops. Additionally, further research on the ecological aspects and biological traits of PABYV are necessary to understand its epidemiology in the Mediterranean basin. This study was conducted as part of the AGROALNEXT programme (PRTR-C17.l1), supported by MCIN with funding from NextGenerationEU and Fundación Séneca with funding from CARM. CDMR was supported by Fundación Séneca within a PhD programme (SENECA 21417/FPI/20).