|Yokomi, Raymond - Ray|
Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/2013
Publication Date: 1/20/2015
Citation: Loconsole, G., Giampetruzzi, A., Saldarelli, P., Onelge, N., Yokomi, R.K., Saponari, M. 2015. Deep sequencing of small RNAs from citrus affected by graft-transmissible diseases of unknown etiology leads to discovery of two novel viruses. Acta Horticulturae. 1065(2):817-824. Interpretive Summary: Graft-transmissible citrus pathogens which have eluded characterization are diagnosed by in planta indexing and resultant symptomotology. These tests require a climate-controlled, insect-free greenhouse and 6 to 12 months or longer to complete. New technologies are now available for the diagnosis and identification of viruses. Plants and animals possess a conserved (not changed over millennia) mechanism known as RNA interference which regulate gene expression. In plants, virus infection produces double-stranded (ds) RNA during replication. Host recognition of these long dsRNAs trigger endonucleases, known specifically as Dicer, to chop dsRNA into small interfering (si) RNAs which are incorporated into RNA-induced silencing complex (RISC) to guide specific gene silencing by an Argonaute protein (AGO) present in the complex. Analysis of the small RNAs (18-28 nucleotides in length) from virus-infected plants obtained by Next Generation Sequencing (NGS) can be used to identify viral siRNAs and overlapping viral siRNAs assembled into longer fragments (contigs) corresponding to the partial or complete viral genome by computational algorithms. These methods were used to identify two previously unknown citrus virus diseases from Turkey: citrus chlorotic dwarf (CCDD) and yellow vein clearing disease (YVCD). Illumina NGS was used to generate siRNA sequence data sets from CCDD- and YVCD-affected citrus. DNA fragments from CCDD-affected plants were used to re-construct a circular single-stranded DNA viral genome with sequence similarities to geminiviruses. Genome assembly indicated the putative CCDD-associated virus is a highly divergent member of the family Geminiviridae and was provisionally named Citrus chlorotic dwarf-associated virus (CCDaV). Using the same procedures, the whole genome of a putative filamentous virus associated with CYVCD-affected lemon was reconstructed. The putative CYVCD viral genome structure placed the newly discovered virus in the Flexiviridae. New serological, biological and molecular investigations were conducted on CYVCD and data suggested that the CYVCD virus represents a new species in the genus Mandarivirus and was provisionally named Citrus yellow vein clearing virus (CYVCV). PCR-based assays for CCDaV and CYVCV were developed and now serve as new diagnostic tools for rapid detection for use in citrus disease management and citrus budwood certification programs.
Technical Abstract: Citrus is susceptible to graft-transmissible pathogens; some of which are arthropod-transmitted. Citrus cultivars vary in susceptibility or tolerance to these pathogens and may remain symptomless when infected. Established tests for known disease agents are used in certification and disease management programs. However, diseases with unknown etiology 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 of unknown etiology: citrus chlorotic dwarf (CCDD) and yellow vein clearing disease (YVCD). CCDD is a serious citrus disease only known to occur in Turkey; whereas YVCD was first observed in Pakistan and India, and later in Turkey (2000) and China (2010). Contigs from small interfering RNAs were assembled and used to identify sequence homology found in the virus database of GenBank. Sequences from CCDD-affected plants were used to re-construct a circular single-stranded DNA viral genome with homology to geminiviruses. The genome size and organization indicated the virus associated with CCDD was a highly divergent member of the family Geminiviridae and was provisionally named Citrus chlorotic dwarf-associated virus (CCDaV). Similarly, the whole genome of a putative filamentous virus associated with CYVCD-affected lemon was reconstructed. The genome structure of the virus associated with CYVCD was determined to be that of a flexivirus. New serological, biological and molecular investigations was initiated on CYVCD which resulted in the pathogen named provisionally Citrus yellow vein clearing virus (CYVCV) and that CYVCV it is a new species in the genus Mandarivirus. PCR-based assays for CCDaV and CYVCV were developed and now serve as important diagnostic tools for citrus disease management programs in Turkey and in citrus budwood certification programs.