Identification of Citrus yellow vein clearing virus in Mazandaran province

Citrus yellow vein clearing virus (CYVCV), a new member of the genus Mandarivirus (family Alfafelxiviridae), has been recently reported from Iran after being recognized in Pakistan, India, Turkey and China. Typical symptoms of infection on the leaves of sour orange, lemon and Persian lime, as local sensitive hosts, consist of yellowing lateral veins with corresponding water-soaked appearance on the lower surface of the leaves. Biological and molecular detection, some modes of transmission and phylogenetic analysis of CYVCV isolates of Mazandaran province have been investigated in the present study.

Typical induced symptoms by CYVCV infection; namely yellowing and water soaking of the lateral veins, was considered as the initial criterion for diagnosis of the disease within seedlings and trees of sour orange, lemon and Persian lime cultivars in nurseries and orchards in Mazandaran province.  Three isolates, out of 53 samples, were selected for more analysis from the region collected during the spring of 2016, 2017, and 2018. Inoculation of these isolates into bean plants, sour oranges and lemons were performed using mechanical and grafting methods under greenhouse conditions with controlled temperatures. In order to confirm the infection, total RNA of the collected samples and the inoculated plants were extracted by the SDS-Potassium acetate method. A two-step Reverse Transcription Polymerase chain reaction (RT-PCR) was carried out using a specific primer pair of the virus coat protein (CP) gene.

Characteristic symptoms of the disease were expressed after top-work grafting of the susceptible virus-free orchard cultivars on the trunks of diseased trees. Using specific primer pair in RT-PCR, an expected 614 bp fragment was amplified in symptomatic samples. The consensus sequences, obtained from direct sequencing the PCR products of the studied isolates based on forward and reverse specific primes, were similar to the sequences of other CYVCV isolates of China, India, Pakistan and Turkey at the level of 95.75 to 98.37%. A phylogenetic tree of this part of the genome, grouped Iranian isolates with two isolates from India and Turkey and two Pakistani isolates.

The present study confirmed the presence of Citrus yellow vein clearing virus in the north of the country. Lemon and Persian lime are the two most important susceptible hosts of the virus. These citrus varieties are also valuable cultivars in the south of the country and are considered tolerant genotypes to Witches' broom disease, which is prevalent in these areas. Preventing the spread of the yellow vein clearingdisease to the new infection foci by continuous monitoring of the citrus nurseries should be seriously considered.