First report of citrus yellow vein clearing virus (CYVCV) on citrus in Italy.

In March 2024, symptoms of vein clearing (Fig. S1) were observed on leaves of six lemon (Citrus limon (L.) Burm. f.) trees grown in a private garden in Palma Campania (Campania region, Italy). Symptoms strongly resembled those of yellow vein clearing disease, caused by Potexvirus citriflavivenae (citrus yellow vein clearing virus, CYVCV), genus Potexvirus, sub-genus Mandarivirus (Loconsole et al., 2012; Cui et al., 2018). CYVCV was reported in Pakistan, India, China, Iran, Turkey, Korea and USA (Abrahamian et al., 2024; Catara et al., 1993; EPPO, 2024; Sun and Yokomi, 2024; Jin et al., 2024). Since 2022, considering the potential impact of this emerging virus, CYVCV has been included in the EPPO alert list and it has not been reported in any European Union (EU) Member State where the virus is not regulated. To ascertain possible infection with CYVCV, leaf samples were collected and tested by reverse transcription polymerase chain reaction (RT-PCR). Total nucleic acids (TNAs) from leaves of the symptomatic lemon and non-symptomatic lemon and sweet orange (Citrus sinensis (L.) Osbeck) trees grown in the same garden, were extracted using the silica-capture extraction method (Foissac et al. 2001). TNAs were tested by RT-PCR using CYVCV specific primers (Table S1) designed by Chen et al., (2014) targeting the coat protein gene. All symptomatic trees and an asymptomatic sweet orange from the same garden tested positive generating an amplicon of the expected size (612 bp). Direct Sanger sequencing of amplicons obtained from two symptomatic lemons and one non-symptomatic sweet orange (An PP842725-PP842727) followed by BLAST search showed 97.9-98.08% sequence identity (query coverage 100%) with CYVCV isolates previously reported from a mandarin (An OQ418501) and a lemon (An OQ418493) in California. In contrast, no amplicons were detected in samples from non-symptomatic lemon trees. Virus identity was confirmed by RT-PCR with different primers (Table S1) targeting the RNA-dependent RNA polymerase gene of CYVCV. Amplicons of the expected size (832 bp) were obtained only from samples previously tested positive to the virus and their sequences determined by Sanger sequence (An PQ284953-PQ284955) showed 97.98-98.11% identity (query coverage 100%) with CYVCV sequence previously reported from a mandarin (An OR251443) in New Delhi. Since CYVCV is transmitted by insects (Önelge et al., 2011a, 2011b, Zhang et al., 2018, 2019), TNAs (Foissac et al., 2001) from Aphis aurantii infesting symptomatic lemons, and of A. spiraecola and Dialeurodes citri infesting symptomatic lemon and non-symptomatic sweet orange trees were assayed by RT-PCR with primer pairs designed by Chen et al., (2014). Three samples of 15 specimens from three different trees were tested for each insect. In contrast to D. citri, which tested negative, an amplicon of 612 bp was detected from A. aurantii and A. spiraecola and Direct sequencing of amplicons (An PP842721-PP842724) confirmed the nature of the virus and showed that insect-derived viral sequences shared nucleotide identity of 99.3-100% with those from the infected trees. Altogether, these data support that CYVCV is present in Southern Italy. Likely, it has been introduced illegally through infected citrus propagation material. Given the large A. aurantii and A. spiraecola populations in the areas where CYVCV has been detected, a fast spread of the virus to other areas of Campania region can be expected. Even if the impact of CYVCV on citrus production in Italy remains to be assessed, prompt and extensive surveys may be helpful to establish the actual distribution of the virus. This would allow for a better evaluation of appropriate measures to be adopted to achieve sustainable citrus production. At the best of our knowledge, this is the first report of CYVCV in Italy and in the EU territory.