ARI REVIEW FOR 2016-2017

Production of healthy local citrus propagating material For several commercial varieties, it is possible to reintroduce presently healthy material from overseas Citrus Production Centres. However, this always includes the risk of the introduction of new unknown diseases. In addition, for local varieties there is not such possibility. For these reasons it was decided to employ contemporary techniques, as micrografting in vitro to free from virus and other disease problems valuable local citrus varieties and/or clones, including ‘Lapithou’ and ‘Polyphori’ lemon, ‘Arakapa’ mandarin, local Jaffa orange and others. The micrografting in vitro technique was used in Cyprus since 2000 to free from virus and other pathogens valuable local citrus varieties and/or clones. A survey in commercial groves was conducted for selection of elite mother plants of citrus, including ‘Polyphori’ and ‘Lapithou’ lemon ( Citrus limon Burm f.), Jaffa, Siekeriko and Aematoysiki orange ( C. sinensis L.), ‘Arakapas’ mandarin ( C. reticulata Blanco ), Frappa ( C. grandis (L.) Osbeck ), Bergamot ( C. bergamia Risso & Poit. ), Coumantantas ( C. comandatore ) and Pummelo ( C. maxima ). All mother plants were tested for viruses and viroids by biological indexing, by ELISA for CTV, and by RT-PCR for viroids. The standard procedure of shoot-tip grafting technique was used for elimination of viroids and CPsV in the selected citrus isolates. Micrografted plants, re-grafted on sour orange seedlings in vivo , and successfully established and transferred to the glasshouse, were tested 6-9 months later for the viruses and viroids present in meristem donor mother plants. ( T. Kapari-Isaia, L.C. Papayiannis ) 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 ) Detection of citrus viroids in Cyprus Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In recent years, advanced molecular diagnostic tools, such as real-time PCR, have been used widely for the rapid detection of nucleic acid targets. The 5- -nuclease fluorogenic assay, also known as TaqMan real-time PCR, is a powerful and rapid technique providing increased sensitivity and specificity and thus alleviating the need for post PCR manipulations, such as gel electrophoresis. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimised for the simultaneous detection of CEVd and HSVd. The assay’s analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity and specificity was 100%. In direct comparison, the developed assay presented 1000-fold more analytical sensitivity than conventional. The increased assay sensitivity has allowed the use of alternative template preparation methods such as crude extracts spotted on nylon membranes, offering users a cost effective alternative template preparation method. The developed assay, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and citrus certification schemes. ( L.C. Papayiannis ) Production, maintenance and distribution of healthy stone fruit material Virus-free material is maintained in a pre-basic plantation under insect-proof screen at the Zygi and Saittas Experimental Stations. Currently, 52 stone fruit accessions are included in either plantation, kept in sanitary condition by application of strict measures and regular indexing in conjunction with optical observation for either fungal problems or genetic aberrations. The plantations provide with propagating material for the basic or mother plantations of the Department of Agriculture and for the experimental glasshouses of the ARI. ( T. Kapari-Isaia, L.C. Papayiannis ) 44 Plant Protection