Eradication of Citrus tristeza virus from cultivar Zorica Rana (Citrus unshiu Marc.)

Varieties of Satsuma mandarin are commercially the most important citrus crop in Croatia due to their relative frost tolerance. Citrus tristeza virus (CTV) is the causal agent of tristeza, the most devastating viral disease of citrus. CTV has been detected in most of the Satsuma mandarin (Citrus unshiu Marc.) varieties including different and even the newest clones of cv. Zorica Rana. Raising virus-free plants is an important aspect of citrus cultivation in Croatia. Two-year old Satsuma mandarin plants cv. Zorica Rana from collection orchard that were previously shown to be CTV positive (ELISA-test and IC/RT-PCR) were selected for virus elimination. After three months of thermotherapy plants of three clones were used as a source of explants for shoot-tip grafting (micrografting) on Troyer citrange rootstock germinated in vitro following the method essentially described by Navarro et al. (1975). The original procedure was modified in performing grafting procedure (“ L” incision) and nutrition media composition. Clones: 3/21-01, 5/19-01, 9/12-01 and 5/23-01 were also subjected to CTV biological detection on two-year old seedlings of Mexican lime (Citrus aurantifolia L.), CRC Grapefruit (Citrus paradisi Macf.), Sour Orange (Citrus aurantium L.) and Madame Vinous Sweet Orange [Citrus sinensis (L.) Osbeck]. Micrografted plants were tested for the virus by DAS-ELISA using commercial antisera according to the manufacturers protocols and IC/RT-PCR using primers for the CTV coat protein gene. The same diagnostic methods were utilized for testing indicator plants. Observation of morphological changes and appearance of viral disease symptoms on citrus indicator hosts in comparison with positive and negative control plants had confirmed the presence of CTV in all tested clones of cv. Zorica Rana. Leaf cupping, chlorosis and vein enation were observed in Mexican lime seedlings only two months after grafting. Symptoms of stem-pitting (SP) started to appear on Mexican lime after seven months and, in the case of clone 5/23-01, in CRC Grapefruit seedlings, too. Lime reaction produced by CTV isolate from the mandarin clone 5/23-01 was much stronger in comparison with the other tested clones (growth retardation). No symptoms that could be attribuated to CTV infection were observed in Sweet and Sour Orange indicators which suggests the presence of mild to moderate SP-CTV strains in 'Zorica Rana' clone 5/23-01. Biotest results imply biological diversity among detected CTV isolates. CTV-free plants of three ‘ Zorica Rana’ clones were obtained by thermotherapy in combination with shoot-tip grafting in vitro from infected plants. Following this procedure, 20% of plants were tristeza-negative. The highest rate of successful grafts was obtained with 2-week old Troyer citrange seedlings. After shoot-tip grafting, successfully cultured plants were established in vivo, this resulted in ninety percent survival and excellent subsequent growth.