Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

Molecular characterization of a novel cytorhabdovirus associated with paper mulberry mosaic disease.

A novel cytorhabdovirus, tentatively named "paper mulberry mosaic-associated virus" (PMuMaV), was discovered and identified by transcriptome sequencing (RNA-seq), small RNA sequencing (sRNA-seq), and RT-PCR amplification. The whole-genome sequence of PMuMaV is 13,736 nucleotides (nt) in length and contains six open reading frames (ORFs) encoding a nucleocapsid protein (N), a phosphoprotein (P), a putative movement protein (P3), a matrix protein (M), a glycoprotein (G), and an RNA-dependent RNA polymerase (L). The coding sequences are flanked by a 194-nt leader and a 370-nt trailer sequence at the 3' terminus and 5' terminus, respectively. Pairwise sequence comparisons showed that PMuMaV is related to northern cereal mosaic virus (NCMV, 38.97%), barley yellow striate mosaic virus (BYSMV, 38.86%), and maize yellow striate virus (MYSV, 38.76%), and phylogenetic analysis also placed these viruses together into the same branch, thus suggesting that PMuMaV is a member of a new species in the genus Cytorhabdovirus.

Identification of the Potential Virulence Factors and RNA Silencing Suppressors of Mulberry Mosaic Dwarf-Associated Geminivirus.

Plant viruses encode virulence factors or RNA silencing suppressors to reprogram plant cellular processes or to fine-tune host RNA silencing-mediated defense responses. In a previous study, Mulberry mosaic dwarf-associated virus (MMDaV), a novel, highly divergent geminivirus, has been identified from a Chinese mulberry tree showing mosaic and dwarfing symptoms, but the functions of its encoded proteins are unknown. In this study, all seven proteins encoded by MMDaV were screened for potential virulence and RNA silencing suppressor activities. We found that V2, RepA, and Rep affect the pathogenicity of a heterologous potato virus X. We showed that V2 could inhibit local RNA silencing and long-distance movement of the RNA silencing signal, but not short-range spread of the green fluorescent protein (GFP) silencing signal in Nicotiana benthamiana 16c plants. In addition, V2 localized to both subnuclear foci and the cytoplasm. Deletion mutagenesis of V2 showed that the basic motif from amino acids 61 to 76 was crucial for V2 to form subnuclear foci and for suppression of RNA silencing. Although the V2 protein encoded by begomoviruses or a curtovirus has been shown to have silencing suppressor activity, this is the first identification of an RNA silencing suppressor from a woody plant-infecting geminivirus.

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.

Unusual genomic features of a badnavirus infecting mulberry.

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95 % sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.

Complete Genome Sequence of Mulberry Vein Banding Associated Virus, a New Tospovirus Infecting Mulberry.

Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5'-/3'-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.

Complete genome sequence of a novel monopartite geminivirus identified in mulberry (Morus alba L.).

The genome sequence of a novel geminivirus from mulberry samples exhibiting crinkle leaf symptoms is reported. The sequence consisted of 2952 nt, containing four open reading frames (ORFs) in the viral-sense strand and two ORFs in the complementary-sense strand. The size of the genome and the conserved origin of replication are similar to those of members of the family Geminiviridae, but the genomic organization, number of ORFs, and especially five contiguous GAAAAA repeats positioned upstream of ORF1 distinguish it from other geminiviruses. Phylogenetic analysis coupled with ORF analysis suggests that this is a novel virus that does not fit into the established seven genera of the family Geminiviridae. The virus, found in Zhenjiang, Jiangsu province, China, is tentatively named mulberry crinkle leaf virus isolate Jiangsu (MCLV-js).

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.

A new nepovirus identified in mulberry (Morus alba L.) in China.

An isometric virus was identified in mulberry leaves showing symptoms of mulberry mosaic leaf roll (MMLR) disease. Its genome consists of two (+)ssRNAs. RNA1 and RNA2 have 7183 and 3742 nucleotides, excluding the 3'-terminal poly(A) tail. Based on phylogenetic analysis of the RNA1-encoded polyprotein and CP amino acid sequences, the properties of the the 3'-UTR of RNA1 and RNA2, and <75 % identity in the CP amino acid sequence, this virus is proposed to be a new member of the genus Nepovirus, subgroup A. Since a causal relationship between this virus and MMLR has not been established, it is tentatively referred to as MMLR-associated virus.

A new report of a mosaic dwarf viroid-like disease on mulberry trees in China.

A viroid-like disease causing mosaic leaves and dwarfism was found on mulberry plants in Zhejiang, China. Grafting of stems from infected plants onto healthy plants resulted in the same symptoms on the healthy plants. A circular small RNA (Mmd-v RNA1) was isolated from the infected plant leaves and caused identical symptoms after more than two years. Nucleotide sequencing indicated that the Mmd-v RNA1 contains 356 nt (GenBank accession no. NC_011362) without viroid characteristic regions, only sharing 30 nt sequence identity with that of the Cherry small circular viroid-like RNA 1 (isolate cscRNA1.150, GenBank accession no. DQ357628). This description is the first of viroid-like RNA infection of mulberry trees.