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First Report of Avocado Sunblotch Viroid (ASBVd) Naturally Infecting Avocado (Persea americana) in Greece

Lotos, L., Kavroulakis, N., Navarro, B., Di Serio, F., Olmos, A., Ruiz-Garcia, A. B., Katis, N. I., Maliogka, V. I.
Plant disease 2018 v.102 no.7 pp. 1470
Avocado sunblotch viroid, Northern blotting, Persea americana, RNA, avocados, clones, complementary DNA, crop yield, cultivation area, fruits, gels, genome, growing season, leaves, nucleotides, nylon, orchards, polyacrylamide gel electrophoresis, pulp, reverse transcriptase polymerase chain reaction, shoots, tissues, tree diseases, trees, Crete, Greece
Avocado (Persea americana) is an important fruit crop for the island of Crete, on which almost the entire yield of avocado fruit in Greece is produced. The total cultivation area is estimated at more than 400 ha ( During the 2016 growing season, fruits exhibiting depressed, greenish, yellow-white craters, which are typical symptoms of avocado sunblotch disease, were spotted on a single tree in an orchard located in the province of Chania, Crete. No symptoms were observed on the leaves or shoots of the tree nor on any other tree in the orchard. The causal agent of sunblotch disease is avocado sunblotch viroid (ASBVd, family Avsunviroidae). In order to determine if the viroid was present, total RNA preparations from leaves, fruit skin, and pulp from the symptomatic tree were extracted (Maliogka et al. 2015) and tested by a two-step RT-PCR (Schnell et al. 1997), which amplifies the cDNA of the complete ASBVd genome. Leaves from nine nonsymptomatic trees from the same field were also analyzed. The RT-PCR performed on all the tissues of the symptomatic sample yielded the expected unit-length amplicon, as well as the dimeric and polymeric forms of the viroid, whereas all the nonsymptomatic samples were found negative. The unit-length amplicon was directly sequenced, whereas the dimeric amplicon was excised from the gel, purified, and cloned into a pGEM-T Easy vector (Promega) and subsequently sequenced (VBC-Biotech). The whole genome sequence of ASBVd (249 nucleotides) was determined from the monomeric RT-PCR product and from two dimeric clones and was deposited in the European Nucleotide Archive (ENA). Two different variants were identified from the symptomatic fruit, AvoCr0-A and B (accession nos. LT966069 and LT966070, respectively). BLASTn results showed that the Greek variants of ASBVd (AvoCr0-A and B) are 95 to 99% identical to the already characterized isolates of ASBVd. The presence of the viroid in the symptomatic tree was also confirmed by northern blot analysis (Torchetti et al. 2012). For this aim, total RNAs were separated by 5% PAGE electrophoresis in denaturing conditions, electro-transferred to a nylon membrane, and hybridized with a DIG-labeled full-length RNA probe specific for the plus strand of ASBVd (provided by Dr. Ricardo Flores, UPV-CSIC, Spain). Circular and linear forms of ASBVd RNAs comigrating with those of the positive control (also provided by Dr. R. Flores) were observed. The presence of additional infected trees in the same field is not excluded, but further investigation is needed to obtain conclusive data in this respect. To our knowledge, this is the first report of ASBVd in Greece. Given the importance of the sunblotch disease, future studies should be performed to determine the prevalence and the significance of this viroid in Greek orchards.