ARI Biennial Review for 2018-2019

Citrus viroids research Citrus viroids are listed among the most important pathogens that negatively affect the citrus industry worldwide. They are highly transmitted by grafting and other mechanical means. An extensive survey was conducted in Cyprus during 2015-2018 in order to identify the presence and incidence of citrus viroids using molecular based techniques, and, in selected samples, biological indexing. TaqMan assays were developed, evaluated and used for the detection of five citrus viroids. Results showed that Citrus exocortis viroid (CEVd) was the most widespread viroid with an incidence of 82%, followed by Hop stunt viroid (HSVd), which was detected in 55% of the 1,280 samples tested. Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd) and Citrus bark cracking viroid (CBCVd) were also identified at lower rates of 14, 10 and 8%, respectively. CEVd+HSVd was the most common viroid combination (70%), while CEVd+CBLVd, CEVd+CDVd and CEVd+CBCVd were detected at 11, 10 and 9.5% of the double viroid mixtures (25%). Triple viroid combinations were also recorded at 10% of the tested samples with CEVd and HSVd identified in all mixtures. Citrus viroid V was not detected in Cyprus. Sequencing analysis showed that all HSVd shared 100% nucleotide identity and were closely related to other isolates from the Mediterranean Basin, while CEVd isolates shared 97-100% homology to exocortis isolates from neighbouring countries, including Africa and Asia, suggesting multiple intoductions through contaminated budwood. Only 7.5% of the samples tested were negative to viroid infection, indicating the need for dissemination of virus/viroid-free propagating material. (L.C. Papayiannis, T. Kapari-Isaia) Evaluation of five new sour orange hybrid rootstocks for tolerance to citrus viroids Five citrus hybrid rootstocks are evaluated at Zygi Experimental Station for their tolerance to citrus viroids. The five citrus hybrids are the following: (1) Sour orange × Orange (Citrus aurantium L. × Citrus sinensis, (L.) Osbeck), (2) Sour orange × Satsouma (Citrus aurantium L. Citrus reticulata blanco), (3) Sour orange × Citrumelo 1452 × Lemon (Citrus aurantium L. × P. trifoliata (L.) Raf × Citrus paradisi Macf. × Citrus limon), (4) Sour orange × Volkameriana (Citrus aurantium L. × Citrus volkameriana) and (5) Sour orange × Carrizo (Citrus aurantium L ×Citrus sinensis (L.) × Poncirus trifoliata (L.) Raf ). (T. Kapari-Isaia, L.C. Papayiannis) In vitro techniques for elimination of pathogens in infected Citrus In vitro micrografting, chemotherapy and cold therapy techniques have been used for sanitation of citrus species and varieties infected by Citrus tristeza virus (CTV) and/or citrus viroids. Several citrus tree varieties, including the lemon varieties ‘Adamopoulou’, ‘Verna’, ‘Polyphori’ and ‘Lapithou’, the orange varieties ‘Navelina’, ‘Washington navel’, ‘Jaffa’, ‘Siekeriko’ and ‘Aematousiki’, the mandarin varieties ‘Clasuelina’, ‘Page’ and ‘Arakapas’, ‘Frappa’ and ‘Bergamot’ were selected. Four Mexican lime (ML) trees infected by severe or mild CTV isolates from Cyprus were used. All trees were tested for viruses and viroids by biological indexing, by ELISA for CTV and CPsV, and by RT- PCR for viroids. They were all found free of CPsV, CVV, concave gum and impietratura. Thirteen trees were infected by CEVd and/or other viroids and one was infected by both CTV and viroids. The micrografting technique in vitro was used for elimination of viroids and CTV in the selected citrus isolates. Chemotherapy in vitro was tested in ML trees infected by CTV. Coldtherapy in vitro was tested in Lapithou lemon tree infected by CEVd and HSVd. Micrografted plants were re-grafted on sour orange seedlings in vivo , or were potted, and those which were successfully established were transferred to the glasshouse. They were tested 6-9 months later for CTV and viroids present in meristem donor mother plants. One or more plants produced by micrografting from all mother plants were found free of CTV and/or viroids. Elimination of CTV on apical meristems was achieved by chemotherapy in vitro using 30, 40 and 50 mg/lt ribavirin or 30, 40 and 50 mg/lt methotrexate. Elimination of viroids by coldtherapy at 10 C for 5 weeks was not possible, but was successful by coldtherapy at 4 C for 5 weeks. (T. Kapari-Isaia, L.C. Papayiannis) 43 PLANT PROTECTION