Chlorotic Dwarf-associated Virus Research Articles

Construction of an Infectious Clone of Citrus Chlorotic Dwarf-Associated Virus and Confirmation of Its Pathogenicity.

Citrus chlorotic dwarf disease (CCDD) seriously affects the citrus industry. Citrus chlorotic dwarf-associated virus (CCDaV) is speculated to be the causal agent of CCDD. However, this speculation has not been confirmed by fulfilling Koch's postulates. In this study, an infectious clone comprising a 1.6-fold tandem CCDaV genome in the binary vector pBinPLUS was constructed and agro-inoculated into 'Eureka' lemon (Citrus limon) seedlings through vacuum infiltration. At 60 days postinoculation, 25% of the 'Eureka' lemon seedlings developed symptoms of crinkling and curling that were the same as those associated with the wild-type virus. Western blotting and graft transmission assays confirmed that the infectious clone systemically infected 'Eureka' lemon seedlings. In addition, CCDaV can establish infection on three more Citrus species and one hybrid, although at different infection rates. These findings support that CCDaV is the primary causal agent of CCDD. The infectious CCDaV clone will allow further studies on the functions of viral proteins and molecular interactions of CCDaV with its hosts.

Identification of RNA silencing suppressor encoded by citrus chlorotic dwarf-associated virus

IntroductionCitrus chlorotic dwarf-associated virus (CCDaV) is an economically important citrus virus associated with leaf curling, deformation, and chlorosis found in China. Plants have evolved RNA silencing to defend against viral infections; however, the mechanism by which CCDaV suppresses RNA silencing in citrus remains unknown.MethodsSix proteins encoded by CCDaV were ectopically expressed in Nicotiana benthamiana 16c using the pCHF3 vector to identify RNA-silencing suppression activities.ResultsV2 protein encoded by CCDaV suppressed local RNA silencing and systemic RNA silencing triggered by GFP RNA, but did not impede short-distance movement of the RNA silencing signal in N. benthamiana 16c. GFP fluorescence observations showed that the ability of V2 protein to suppress RNA silencing was weaker than tomato bushy stunt virus P19. Deletion analysis showed that the putative nuclear localization signal (NLS, 25–54 aa) was involved in the RNA silencing suppression activity of V2 protein. Furthermore, V2 protein cannot block dsRNA-triggered RNA silencing. The subcellular localization assay suggested that V2 protein was localized to nucleus of N. benthamiana.ConclusionOverall, the results of this study demonstrate that CCDaV-V2 acts as an activity of silencing suppression. This is the first reported RNA-silencing suppressor encoded by Citlodavirus and will be valuable in revealing the molecular mechanism of CCDaV infection.

Regulation of Nicotiana benthamiana cell death induced by citrus chlorotic dwarf-associated virus-RepA protein by WRKY 1.

Citrus chlorotic dwarf-associated virus (CCDaV) is a Citlodavirus species in the Geminiviridae family that causes tremendous economic loss to the citrus industry in China. Some proteins encoded by geminiviruses are crucial for the interaction between the virus and its host plant. However, the exact functions of CCDaV-encoded proteins such as CCDaV-RepA have not been investigated. This study presents evidence that CCDaV-RepA elicits a hypersensitive response (HR)-like cell death in Nicotiana benthamiana that was accompanied by the production of H2O2 and ion leakage, which suggested that CCDaV-RepA is a potential recognition target for inducing host defense responses. Furthermore, the rolling-circle replication motifs of CCDaV-RepA are associated with triggering HR-like cell death in N. benthamiana. Confocal microscopy and deletion mutagenesis assays showed that CCDaV-RepA was located in the nucleus, while the first eight amino acids (aa) at the N terminus and two regions located between aa residues 122-263 and 220-264 of RepA were not associated with nuclear localization. Tobacco rattle virus-induced gene silencing of the key signaling cascade components revealed that HR-like cell death induced by RepA was inhibited in WRKY1-silenced N. benthamiana. Moreover, WRKY1 expression was upregulated in RepA-GFP infiltrated Overall, the results suggest that NbWRKY1 positively regulated CCDaV-RepA -induced cell death in N. benthamiana. These findings provide novel information for further research on the interactions between CCDaV and the host plant.

Complete genome sequence analysis of a novel citlodavirus isolated from the leaves of Myrica rubra in Yunnan.

Through high-throughput sequencing, a novel citlodavirus, tentatively named "Myrica rubra citlodavirus 1" (MRV1,accession no. OP374189), was isolated from the leaves of Myrica rubra in Yunnan exhibiting narrow deformity of leaf tips, shrinkage, and chlorosis along the veins. The complete genome sequence was determined and analyzed using cloning and Sanger sequencing. MRV1 is a single-stranded circular non-enveloped DNA virus with a genome size of 3775 nucleotides and contains six open reading frames (ORFs). The virion-sense genome strand encodes a coat protein (CP, nt 750-1,493, 247 aa), two hypothetical movement proteins (V3, nt 382-666, 94 aa; and V2, nt 461-895, 144 aa), and one movement protein (MP, nt 1,527-2,438, 303 aa). The complementary strand of the genome encodes two replication proteins (RepA, nt 3,712-2,834, 292 aa; Rep, nt 2,867-2,553, 104 aa). The MRV1 genome contains the stem-loop motif 5'-TAATATTAC-3', which is a highly conserved nonanucleotide motif found in the origin of virion-strand replication in geminiviruses. Genome sequence alignment analysis showed that citrus chlorotic dwarf associated virus (CCDaV, accession no. JQ920490) shared the highest nucleotide sequence similarity (66.10% identity) with MRV1. Phylogenetic analysis showed that CCDaV is the closest known relative of MRV1, and that these viruses clustered in a single branch within a clade consisting of citlodaviruses. These results indicate that MRV1 should be regarded as a new species of the genus Citlodavirus in the family Geminiviridae.

Fast detection of Citrus chlorotic dwarf-associated virus by loop-mediated isothermal amplification

Fast detection of Citrus chlorotic dwarf-associated virus by loop-mediated isothermal amplification

Citrus chlorotic dwarf associated virus. [Distribution map

Abstract A new distribution map is provided for Citrus chlorotic dwarf associated virus. Geminiviridae: Citlodavirus. Hosts: Citrus spp.

Citrus chlorotic dwarf associated virus. [Distribution map

Abstract A new distribution map is provided for Citrus chlorotic dwarf associated virus. Geminiviridae: Citlodavirus. Hosts: Citrus spp.

First Insight into Genetic Variation and Population Structure of The Emerging Citrus chlorotic dwarf-associated virus (CCDaV, genus Citlodavirus)

Citrus spp. is widely planted in tropical and subtropical regions, including in Turkey and other Mediterranean countries. Due to its widespread vector and climate change, Citrus chlorotic dwarf-associated virus (CCDaV), a member of the newly formed genus Citlodavirus, is one of the emerging viruses that can be a serious constraint to Citrus crops production in the coming years. Therefore, in-silico analysis on all available isolates in NCBI GenBank was performed to provide the first insight into the genetic population and evolution of CCDaV, which may contribute to its control. CCDaV phylogroups based on full genome, complete movement protein, and complete coat protein sequences were found to be not associated with isolate origins or host species, and all isolates also shared a high genetic identity among them. However, neutrality tests indicated that the current populations are expanding, driven by new mutations. Low Fixation index (FST) values (0.00000-0.36207) confirmed no genetic separation among different ORFs of isolates from three countries. The constructed TimeTree suggested that CCDaV emergence was very recent compared to the other three members of the genus Citlodavirus. Therefore, the obtained results of this study could also expand our knowledge on other even more obscure citladovirus and even other plant DNA viruses, which are still less studied than RNA viruses.

New geographic distribution and molecular diversity of Citrus chlorotic dwarf-associated virus in China

In 2009, an emerging citrus viral disease caused by Citrus chlorotic dwarf-associated virus (CCDaV) was discovered in Yunnan Province of China. However, the occurrence and spread of CCDaV in other citrus-growing provinces in China is unknown to date. To better understand the distribution and molecular diversity of CCDaV in China, a total of 1 772 citrus samples were collected from 11 major citrus-growing provinces and were tested for CCDaV by PCR. Among these, 134 citrus samples from Guangxi, Yunnan and Guangdong were tested positive for CCDaV, demonstrating that the occurrence and spread of CCDaV are increasing in China. The complete genome sequences of 17 CCDaV isolates from different provinces and hosts were sequenced. Comparisons of the whole-genome sequences of the 17 CCDaV isolates as well as the 15 isolates available in GenBank revealed that the sequence identity was about 99–100%, showing that the CCDaV isolates were highly conserved. Phylogenetic studies showed that the 32 CCDaV isolates belonged to four different groups based on geographical origins and host species, and that CCDaV isolates from China and Turkey were clustered into different groups. The results provide important information for clarifying the distribution and genetic diversity of CCDaV in China.

Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease.

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Geminiviruses: Taxonomic Structure and Diversity in Genomic Organization.

Geminiviridae is one of the best-characterized and hence, one of the largest plant-virus families with the highest economic importance. Its members characteristically have a circular ssDNA genome within the encapsidation of twinned quasi-icosaheadral virions (18-38 nm size-range). Construction of a narrative review on geminiviruses, to have a clearer picture of their genomic structure and taxonomic status. A thorough search was conducted for papers and patents regarding geminiviruses, where relevant information was used to study their genomic organization, diversity and taxonomic structure. Geminiviruses have been classified into nine genera (viz., genus Begomovirus, Mastrevirus, Curtovirus, Topocuvirus, Becurtovirus, Turncurtovirus, Capulavirus, Eragrovirus and Grablovirus) having distinct genomic organizations, host ranges and insect vectors. Genomic organization of all genera generally shows the presence of 4-6 ORFs encoding for various proteins. For now, Citrus chlorotic dwarf-associated virus (CCDaV), Camellia chlorotic dwarf-associated virus (CaCDaV) and few other geminiviruses are still unassigned to any genera. The monopartite begomoviruses (and few mastreviruses) have been found associated with aplhasatellites and betasatellites (viz., ~1.3 kb circular ssDNA satellites). Recent reports suggest that deltasatellites potentially reduce the accumulation of helper-Begomovirus species in host plants. Some patents have revealed the methods to generate transgenic plants resistant to geminiviruses. Geminiviruses rapidly evolve and are a highly diverse group of plant-viruses. However, research has shown new horizons in tackling the acute begomoviral diseases in plants by generating a novel bio-control methodology in which deltasatellites can be used as bio-control agents and generate transgenic plants resistant to geminiviruses.

First Report of Duranta leaf curl virus Infecting Ficus virens Showing Leaf Curl Symptoms in Pakistan

Ficus virens (family: Moraceae) is a deciduous tree found in the Indian subcontinent, some other Southeast Asian countries, and northern Australia. It is valued for its latex, wood, and medicinal and edible products. In December 2018, during a routine survey to record begomoviruses other than cotton infecting viruses in Pakistan, curling of young leaves, which is a typical symptom of infection by viruses of the genus Begomovirus, was observed in F. virens trees in a park in Lahore, central Punjab, Pakistan. The total DNA of three trees was extracted using the Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and used as a template in PCR amplification with begomovirus-specific primers: Beg-F (5′-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3′) and Beg-R (5′- CTGCCACAACCATGGATTCACGCACAGGG-3′). The amplified 1-kb DNA was cloned into the pGEM-3Zf (+) vector (Promega, Madison, WI) and sequenced by a commercial sequencing service (Macrogen, Seoul, Korea). NCBI BLAST results revealed a nucleotide (nt) sequence identity of 95, 93, and 92% with chilli leaf curl India virus, rose leaf curl virus, and papaya leaf crumple virus, respectively. Based on the sequence obtained, new primers, DCP-F (5′-AGCACATTGGTAAAGTCATGTGTG-3′) and DCP-R (5′-GGATATCATGTCTGGACTCAAATG-3′), were designed to obtain full-length DNA-A of the begomovirus. An approximately 2.8-kb product was amplified and cloned. The resulting sequence (GenBank accession no. MN537564) showed 97.43% nt identity with duranta leaf curl virus (DLCV; KT948069), a Begomovirus species reported in the ornamental shrub Duranta erecta in Pakistan by Iram et al. (2005) and by Anwar et al. (2017). In the former case, only DNA-A of DLCV was found without any associated molecules such as DNA-B or DNA β, whereas, in the latter case, tomato leaf curl New Delhi virus DNA-B was found in association with the DNA-A of DLCV, like in many other Old World begomoviruses. The attempt to amplify DNA-B through PCR using newly designed primers NDB-F (5′-AAACACAGGAGGGATCGGAG-3′) and NDB-R (5′-CACAGATTTCCTTACGCGTATATT-3′) based on the sequence KT948071 was unsuccessful. Similarly, efforts to detect alphasatellite and betasatellite molecules in infected F. virens using universal primers (Bull et al. 2003; Briddon et al. 2002) were unsuccessful. Furthermore, Southern blot hybridization was carried out using [³²P]-radiolabeled PCR product of 1 kb obtained with Beg-F/R primers as a probe. This produced dsDNA bands of different conformations as well as a ssDNA band, which confirms the presence of DLCV in F. virens. Members of the family Geminiviridae, to which begomoviruses belong, mainly infect herbaceous hosts, but they have also been reported in a few woody hosts, such as citrus chlorotic dwarf associated virus in citrus (Loconsole et al. 2012), grapevine red blotch virus in grape (Al Rwahnih et al. 2013), jatropha mosaic virus in Jatropha multifida (Polston et al. 2014), and apple geminivirus 1 in apple tree (Liang et al. 2015). To our knowledge, this is the first report of DLCV infecting F. virens in Pakistan. The results obtained here indicate that F. virens may serve as an alternative host of DLCV and a possible source of infection to the commonly grown D. erecta ornamental garden hedge.

First Report of Citrus Chlorotic Dwarf-Associated Virus on Pomelo in Nakhon, Thailand

Citrus chlorotic dwarf disease (CCDD) was first reported in Turkey in the mid-1990s (Korkmaz et al. 1995) and subsequently in China (Guo et al. 2015). Symptoms included various chlorotic patterns, leaf crinkling, and other types of leaf distortion. Currently, CCDD is the most serious citrus disease in Turkey, where severe losses on different species are reported (Loconsole et al. 2012). Citrus chlorotic dwarf-associated virus (CCDaV; family Geminiviridae) is the causal agent of CCDD (Loconsole et al. 2012). In spring 2019, a new disease was observed at some Ruby Green pomelo orchards in Nakhon, Thailand, with symptoms of severe leaf curling, distortion, and chlorosis on young flushes. Approximately 20% of Ruby Green pomelo trees in this area were symptomatic. The budwoods from four symptomatic Ruby Green pomelo plants were grafted onto 15 plants each of healthy Ruby Green pomelo and Sanhongmiyou pomelo (Citrus grandis) plants and covered by insect-proof net. Three months later, the young flushes of these graft-inoculated plants displayed typical symptoms of CCDD. Total DNA extracts (Plant Genomic DNA Extraction Kit; Bioer, Hangzhou, China) were obtained from these symptomatic plants and 45 additional Ruby Green pomelo plants (symptomatic = 16, asymptomatic = 29), which were collected from four orchards in Nakhon. PCR tests were performed with CCDaV-specific primers (sense, 5′-ACAAGACTATCATAGCACGAGACG-3′; and antisense, 5′- TTTGAACTGTTTAAGTCCATCCC-3′) designed based on the conserved region of movement protein (accession no. JQ920490). The expected amplicon (789 bp) was obtained from the symptomatic Ruby Green pomelo samples and identified by BLASTn analysis as specific to CCDaV. All CCDaV-infected plants showed severe leaf curling, distortion, and chlorosis. Furthermore, all the samples tested negative for citrus yellow vein clearing virus, citrus tristeza virus, citrus tatter leaf virus, satsuma dwarf virus, and citrus exocortis viroid by reverse transcription PCR. The complete CCDaV genome was amplified from three randomly collected field samples (Tha1-17, Tha1-19, and Tha30) using two pairs of previously described primers, 1409fw/341rev and 221fw/1542rev (Loconsole et al. 2012). The obtained sequences were deposited in GenBank under accession numbers MN509440 to MN509442. In pairwise comparisons, the three CCDaV isolates from Thailand shared 99.5 to 99.6% complete genome identities with each other and 99.1 to 99.7% identities with corresponding sequences of 12 CCDaV isolates (JQ920490, KF561253, KX840467 to KX840470, and MG566050 to MG566055) previously reported from Turkey and China. Phylogenetic analysis of these 15 genomic sequences showed that the CCDaV isolates from Thailand clustered into the same clade as isolates from Turkey and apart from isolates from China. To our knowledge, this is the first report of CCDaV in Thailand. Our finding emphasizes the need for CCDaV indexing in production and distribution of pathogen-free citrus plants in Thailand.

Geographic distribution and molecular characterization of Turkish isolates of the citrus chlorotic dwarf-associated virus

Citrus chlorotic dwarf-associated virus (CCDaV) was first reported in Turkey’s Eastern Mediterranean Region (EMR) in the late 1980s. No studies have been conducted on its presence in the other important citrus cultivation regions of Turkey until this publication. To investigate the potential spread of CCDaV we conducted surveys in commercial orchards, residential and hotel gardens, and recreational areas in four different citrus cultivation regions outside of the EMR. Plants were examined visually and a total of 141 samples showing symptoms similar to CCDaV were collected. Only one out of 141 samples tested positive for CCDaV. This sample was collected from garden grounds of a hotel in Antalya and, to the best of our knowledge, is the first report of CCDaV infection outside of the EMR in Turkey. CCDaV infection was not detected in any of the surveyed commercial citrus orchards. To determine the molecular characteristics and population structures of the CCDaV isolates, additional surveys were performed to obtain CCDaV isolates from different citrus species in EMR. Following sample collection and virus identification work, complete genome sequences were determined for CCDaV isolates from five different citrus species and a citrus hybrid cultivated commercially in Turkey. Analysis of these genomes showed sequence identities over 96% between themselves and with the Chinese CCDaV isolates. Phylogenetic studies showed that Turkish and Chinese samples clustered into different groups, based on their complete genomes and gene regions.

Passion Fruit Chlorotic Mottle Virus: Molecular Characterization of a New Divergent Geminivirus in Brazil.

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae: passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus. Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12–30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891–921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7–3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina, Arabidopsis thaliana, and Passiflora edulis.

Discovery of a novel geminivirus associated with camellia chlorotic dwarf disease

Next-generation sequencing of small RNAs and PCR amplification with two pairs of back-to-back primers, followed by cloning and sequencing, allowed identification of a novel geminivirus isolate provisionally named camellia chlorotic dwarf-associated virus (CaCDaV). The complete genome sequence of CaCDaV comprises 3,687 nucleotides (nts) and six open reading frames (ORFs). The genome and putative proteins of CaCDaV were most closely related to those of two unclassified geminiviruses: citrus chlorotic dwarf-associated virus (CCDaV, 3,640 nt) and mulberry mosaic dwarf-associated virus (MMDaV, 2,952 nt). Phylogenetic analysis revealed a distinct clade for CaCDaV, CCDaV and MMDaV, supporting their inclusion in a new genus within the family Geminiviridae. Because CaCDaV and CCDaV shared a high genome nucleotide sequence identity (54.8%), we propose that the CaCDaV should be included as a member in this new genus.

Distribution and molecular characterization of citrus chlorotic dwarf-associated virus in China

In 2009, a new citrus viral disease caused by Citrus chlorotic dwarf-associated virus (CCDaV) was discovered in China. To more effectively monitor the presence of CCDaV, a survey was conducted in 175 citrus orchards from 11 major citrus growing provinces in China from May 2014 to August 2016. 4 out of a total of 2, 432 citrus samples tested positive for CCDaV, demonstrating there were still sporadic CCDaV cases occurred in China, and the virus was restricted to a few orchards. In this study, the complete genome sequences of four CCDaV isolates from Eureka lemon were sequenced and characterized. Comparisons of the whole genome sequences of these four CCDaV isolates as well as two isolates previously reported from around the world revealed that sequence identity ranged from 98.1% to 99.8%, indicating that there is a very low level of sequence heterogeneity among CCDaV isolates of different geographic origins. To our knowledge, this is the first extensive survey conducted in China for CCDaV incidence.

Capulavirus and Grablovirus: two new genera in the family Geminiviridae

Geminiviruses are plant-infecting single-stranded DNA viruses that occur in most parts of the world. Currently, there are seven genera within the family Geminiviridae (Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus). The rate of discovery of new geminiviruses has increased significantly over the last decade as a result of new molecular tools and approaches (rolling-circle amplification and deep sequencing) that allow for high-throughput workflows. Here, we report the establishment of two new genera: Capulavirus, with four new species (Alfalfa leaf curl virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus and Plantago lanceolata latent virus), and Grablovirus, with one new species (Grapevine red blotch virus). The aphid species Aphis craccivora has been shown to be a vector for Alfalfa leaf curl virus, and the treehopper species Spissistilus festinus is the likely vector of Grapevine red blotch virus. In addition, two highly divergent groups of viruses found infecting citrus and mulberry plants have been assigned to the new species Citrus chlorotic dwarf associated virus and Mulberry mosaic dwarf associated virus, respectively. These species have been left unassigned to a genus by the ICTV because their particle morphology and insect vectors are unknown.

Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease

High-throughput sequencing of small RNAs allowed the identification of a novel DNA virus in a Chinese mulberry tree affected by a disease showing mosaic and dwarfing symptoms. Rolling-circle amplification and PCR with specific primers, followed by sequencing of eleven independent full-length clones, showed that this virus has a monopartite circular DNA genome (∼ 2.95 kb) containing ORFs in both polarity strands, as reported previously for geminiviruses. A field survey showed the close association of the virus with diseased mulberries, so we tentatively named the virus mulberry mosaic dwarf-associated virus (MMDaV). The MMDaV genome codes for five and two putative proteins in the virion-sense and in the complementary-sense strands, respectively. Although three MMDaV virion-sense putative proteins did not share sequence homology with any protein in the databases, functional domains [coiled-coil and transmembrane (TM) domains] were identified in two of them. In addition, the protein containing a TM domain was encoded by an ORF located in a similar genomic position in MMDaV and in several other geminiviruses. As reported for members of the genera Mastrevirus and Becurtovirus, MMDaV replication-associated proteins are expressed through the alternative splicing of an intron, which was shown to be functional in vivo. A similar intron was found in the genome of citrus chlorotic dwarf-associated virus (CCDaV), a divergent geminivirus found recently in citrus. On the basis of pairwise comparisons and phylogenetic analyses, CCDaV and MMDaV appear to be closely related to each other, thus supporting their inclusion in a putative novel genus in the family Geminiviridae.

DEEP SEQUENCING OF SMALL RNAS FROM CITRUS AFFECTED BY GRAFT-TRANSMISSIBLE DISEASES OF UNKNOWN AETIOLOGY LEADS TO DISCOVERY OF TWO NOVEL VIRUSES

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.