817 Deep Sequencing of Small RNAs from Citrus Affected by Graft- Transmissible Diseases of Unknown Aetiology Leads to Discovery of Two Novel Viruses Giuliana Loconsole 1 , Annalisa Giampetruzzi 2 , Pasquale Saldarelli 2 , Nuket Onelge 3 , Raymond K. Yokomi 4 and Maria Saponari 2,* 1 Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università di Bari “Aldo Moro”, Bari, Italy 2 Istituto di Virologia Vegetale, Consiglio Nazionale delle Ricerche, UOS Bari, Italy 3 Çukurova University, Agriculture Faculty, Plant Protection Department, Adana, Turkey 4 United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Parlier, CA, USA *Corresponding author: m.saponari@ba.ivv.cnr.it Abstract Diseases of unknown aetiology remain difficult or impossible to diagnose. Illumina next generation sequencing (NGS) technology was used to generate sequence datasets from citrus affected by two such diseases: citrus chlorotic dwarf (CCDD) (whitefly-transmitted) and yellow vein clearing disease (YVCD) (aphid- transmitted). Contigs from small RNAs were assembled and used to screen sequence homologies against the virus database in GenBank. DNA fragments from CCDD- affected plants were used to re-construct a circular single-stranded DNA viral genome with homologies to geminiviruses. The genome size and organization of the provisionally named Citrus chlorotic dwarf-associated virus (CCDaV) was shown to be a highly divergent member of the family Geminiviridae. Similarly, the whole genome sequence of a putative filamentous virus associated with CYVCD-affected lemon plants was reconstructed. The genome structure was found to be typical of flexiviruses and sequence data suggested that CYVCV is a new species in the genus Mandarivirus. PCR-based assays for CCDaV and CYVCV were developed that will be an important diagnostic tool for citrus disease management programs in Turkey and other citrus-producing regions. Keywords: citrus chlorotic dwarf, yellow vein clearing, geminivirus, Mandarivirus, Parabemisia myricae, Aphis craccivora, Aphis spiraecola INTRODUCTION Citrus is an economically important fruit crop in Turkey that is threatened by invasive and graft-transmissible pathogens. These pathogens, if detectable, can be controlled by exclusion, use of pathogen-free plants, vector control or resistant cultivars. Two emerging disorders, Citrus chlorotic dwarf disease (CCDD) and Yellow vein- clearing disease (YVCD), are presently restricted to Turkey and/or neighbor countries and both cause severe damage to infected trees. Both disorders were of unknown etiology and, as such, examined in this report. CCDD is graft-transmissible and was first observed in the mid-1980s in the south Mediterranean region of Turkey which accounts for ~85% of the country’s citrus production (Kersting et al., 1996). Records indicate that CCDD incidence has increased up to 60-70% in recent years (European Food Safety Authority, 2008). CCDD is considered to be the most serious disease of citrus in Turkey where severe losses were due to the reduction in number and size of fruits on different cultivars. Disease symptoms include crinkling, curling, inverted cupping and distortion of the leaves with occasional chlorotic patterns and variegation, and reduced size. Shoots of affected young trees have short internodes which results in a bushy, stunted tree. The putative CCDD agent was shown to be transmitted by the bayberry whitefly, Parabemisia myricae Kuwana (Hemiptera: Aleyrodidae) in a persistent or semi-persistent manner (Korkmaz and Garnsey, 2000). Ultrastructural images of thin citrus leaf sections did not reveal the Proc. XII th Intl. Citrus Congress Eds.: B. Sabater-Muñoz et al. Acta Hort. 1065, ISHS 2015