Role of Thrips palmi and Parthenium hysterophorus pollen in active spread of tobacco streak virus in the cotton ecosystem.

Tobacco streak virus incidence in the cotton field, cv.CO14 at Department of Cotton, Tamil Nadu Agricultural University (TNAU), Coimbatore, India was nearly 36.50 %. Cotton plants infected with TSV exhibits different types of symptoms, including necrotic spots, lesions, mosaic, purplish necrotic rings, square drying, veinal necrosis and drying of terminal shoots. The highly prevalent thrips species in this cotton ecosystem was established as Thrips palmi (60.00 %) by morphological (ESEM) and molecular methods (RT-PCR using mtCOI primers). The density of the alternate weed host, Parthenium hysterophorus, was 15.05 plants per m2 in these fields. Association of Thrips palmi with Parthenium was confirmed, when observed under environmental scanning electron microscope (ESEM), Parthenium pollen grains (i.e., average size @ 15000X =12.94 µm) were found adhering to its body. Molecular studies through RT-PCR confirmed the presence of TSV in the leaves and pollen grains of symptomatic and symptom-free Parthenium plants collected from the cotton field (cv. CO14). Therefore, the combined role of Thrips palmi and the Parthenium pollen grains in the transmission of TSV was examined; acquiring of TSV and its presence in the body of Thrips palmi instars and adults after 72 h of AAP was convincingly demonstrated using RT-PCR, NASH and qPCR. However virus acquired thrips could not transmit the virus. Pollen from TSV infected Parthenium plants when dusted on cotton (ANKUR 2110) seedlings along with virus acquired or non-acquired thrips led to symptom development 22 days after sowing. From the study it is evident that thrips only facilitate the movement of TSV borne pollen grains, and thereby contributing to active spread of the virus.

Molecular characterization of a new begomovirus infecting Synedrella nodiflora in South India.

Begomoviruses (family Geminiviridae) cause important diseases in many crops. In addition, many wild plants are reservoirs of begomoviruses, which are a potential menace for nearby crops. A novel begomovirus was isolated from a weed of the species Synedrella nodiflora (Compositae) exhibiting yellow vein symptoms in South India. This virus had a typical monopartite Old World begomovirus genome and was accompanied by a betasatellite. Sequence comparison revealed that this virus represents a new species in the genus Begomovirus. Recombination analysis showed that the novel begomovirus originated through recombination between the begomoviruses ageratum yellow vein Sri Lanka virus and tomato leaf curl Sri Lanka virus.

Two distinct begomoviruses associated with an alphasatellite coinfecting Emilia sonchifolia in Thailand.

Emilia sonchifolia is a traditionally used medicinal plant that is widespread in tropical and subtropical regions of the world. Yellow vein symptoms were observed in E. sonchifolia plants in fields in the county of Koh Samui, Surat Thani Province, Thailand, in August 2015. Two distinct begomoviruses, designated TH4872-6 and TH4872-9, and an associated alphasatellite were obtained from an E. sonchifolia leaf sample (TH4872). Sequence analysis showed that the full-length sequence of TH4872-6 was most closely related to that of ageratum yellow vein China virus (AYVCNV), with 85.7% identity, suggesting that it is a novel begomovirus, while the TH4872-9 sequence closely resembled cotton leaf curl Multan virus (CLCuMuV) with 99.1% identity. The alphasatellite sequence showed the highest nucleotide sequence identity (92.8%) to an isolate of tobacco curly shoot alphasatellite (TbCSA) originating from China. Recombination analysis revealed that the isolate TH4872-6 is a potential recombinant begomovirus, derived from ageratum yellow vein virus (AYVV) and tobacco leaf curl Thailand virus (TbLCTHV). This study represents the first report of begomoviruses identified in E. sonchifolia in Thailand.

Complete genome analysis of a novel umbravirus-polerovirus combination isolated from Ixeridium dentatum.

Two novel viruses, isolated in Bonghwa, Republic of Korea, from an Ixeridium dentatum plant with yellowing mottle symptoms, have been provisionally named Ixeridium yellow mottle-associated virus 1 (IxYMaV-1) and Ixeridium yellow mottle-associated virus 2 (IxYMaV-2). IxYMaV-1 has a genome of 6,017 nucleotides sharing a 56.4% sequence identity with that of cucurbit aphid-borne yellows virus (genus Polerovirus). The IxYMaV-2 genome of 4,196 nucleotides has a sequence identity of less than 48.3% with e other species classified within the genus Umbravirus. Genome properties and phylogenetic analysis suggested that IxYMaV-1 and -2 are representative isolates of new species classifiable within the genus Polerovirus and Umbravirus, respectively.

Complete genome sequence of a novel potyvirus, callistephus mottle virus, identified in Callistephus chinensis.

The complete genomic sequence of a novel putative member of the genus Potyvirus was detected from Callistephus chinensis (China aster) in South Korea. The genomic RNA consists of 9,859 nucleotides (excluding the 3' poly(A) tail) and contains the typical open reading frame of potyviruses, encoding a putative large polyprotein of 3,154 amino acids. The Callistephus virus is most closely related to plum pox virus and members of the ApVY subgroup which showed 50-52 % polyprotein amino acid sequence identity. These results suggest that the Callistephus virus is a novel member of the genus Potyvirus, tentatively named "callistephus mottle virus" (CalMV).

Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.

Salivary gland morphology, tissue tropism and the progression of tospovirus infection in Frankliniella occidentalis.

Tomato spotted wilt virus (TSWV) is transmitted by thrips in a propagative manner; however, progression of virus infection in the insect is not fully understood. The goal of this work was to study the morphology and infection of thrips salivary glands. The primary salivary glands (PSG) are complex, with three distinct regions that may have unique functions. Analysis of TSWV progression in thrips revealed the presence of viral proteins in the foregut, midgut, ligaments, tubular salivary glands (TSG), and efferent duct and filament structures connecting the TSG and PSG of first and second instar larvae. The primary site of virus infection shifted from the midgut and TSG in the larvae to the PSG in adults, suggesting that tissue tropism changes with insect development. TSG infection was detected in advance of PSG infection. These findings support the hypothesis that the TSG are involved in trafficking of TSWV to the PSG.

Complete genome sequence of yacon necrotic mottle virus, a novel putative member of the genus Badnavirus.

The complete genome sequence of a previously undescribed virus isolated from a yacon plant exhibiting necrotic mottle, chlorosis, stunting, and leaf malformation symptoms in Gyeongju, Korea, was determined. The genome of this virus consists of one circular double-stranded DNA of 7661 bp in size. The genome contained four open reading frames (ORFs 1 to 4) on the plus strand that potentially encode proteins of 26, 32, 234, and 25 kDa. Protein BLAST analysis showed that ORF3, which is the largest ORF, has 45 % amino acid sequence identity (with 89 % coverage) to the ORF3 of fig badnavirus 1 (FBV-1), a recently identified badnavirus. Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Badnavirus. The name yacon necrotic mottle virus (YNMoV) is proposed for this new virus.

Virus-induced gene silencing for Asteraceae--a reverse genetics approach for functional genomics in Gerbera hybrida.

Virus-induced gene silencing (VIGS) is a natural defence mechanism in plants which leads to sequence-specific degradation of viral RNA. For identifying gene functions, Tobacco rattle virus (TRV)-based VIGS has been applied for silencing of endogenous genes in many plant species. Gerbera hybrida (Asteraceae) has emerged as a novel model for studies in flower development and secondary metabolism. For this highly heterozygous species, functional studies have been conducted through reverse genetic methods by producing stable transgenic lines, which, however, is labour-intensive and time-consuming. For the development of TRV-based VIGS system for gerbera, and for the first time for an Asteraceaeous species, we screened several gerbera cultivars and optimized the agroinfiltration methods for efficient silencing. Gene fragments for gerbera phytoene desaturase (GPDS) and Mg-chelatase subunits (GChl-H and GChl-I), expressed from a TRV vector, induced silencing phenotypes in leaves, scapes, and involucral bracts indicating their feasibility as markers for green tissues. In addition, robust silencing symptoms were achieved in gerbera floral tissues by silencing the anthocyanin pathway gene for chalcone synthase (GCHS1) and a gerbera B-type MADS-box gene globosa (GGLO1), confirming the phenotypes previously observed in stable transgenic lines. Unexpectedly, photobleaching induced by GPDS and GChl-H or GChl-I silencing, or by the herbicide norflurazon, resulted in silencing of the polyketide synthase gene G2PS1, which has no apparent connections to carotenoid or chlorophyll biosynthesis. We have shown feasibility of VIGS for functional studies in gerbera, but our results also show that selection of the marker gene for silencing must be critically evaluated.

Effect of host species on the distribution of mutational fitness effects for an RNA virus.

Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multi-host parasites and thus for the emergence of new pathogens. Here we characterize the DMFE for a collection of twenty single-nucleotide substitution mutants of Tobacco etch potyvirus (TEV) across a set of eight host environments. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas the other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail. Within the Solanaceae hosts, the distribution contained an excess of deleterious mutations, whereas for the non-Solanaceae the fraction of beneficial mutations was significantly larger. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host.

Complete nucleotide sequence and genome organization of butterbur mosaic virus.

Butterbur mosaic virus (ButMV), a member of the genus Carlavirus, was isolated from Japanese butterbur. Here we report the complete nucleotide sequence and genome organization of ButMV. The genome of ButMV consists of 8,662 nucleotides in length and is predicted to contain six ORFs. The ButMV replicase and CP genes share 46.4-54.9 and 43.2-62.1% nucleotide and 38.6-46.6 and 31.3-65.0% amino acid sequence identities, respectively, with those of other carlaviruses. Based on phylogenetic analysis, we suggested that ButMV replicase and CP is most closely related to coleus vein necrosis virus and carnation latent virus, respectively. Together, our results demonstrate that ButMV was a distinct species of the genus Carlavirus.

Six novel begomoviruses infecting tomato and associated weeds in Southeastern Brazil.

The incidence of tomato-infecting begomoviruses has sharply increased in Brazil following the introduction of the B biotype of the whitefly vector in the early 1990s. Five definitive species and six tentative species have been described since then. Here, we report the detection of members of an additional six novel species, three in tomato and three infecting weeds that are commonly associated with tomato fields: Blainvillea rhomboidea, Sida rhombifolia and Sida micrantha. Tomato and weed samples were collected in two major tomato-growing regions of southeastern Brazil in 2005 and 2007. Two of the novel viruses were present in tomato plants collected in Paty do Alferes, Rio de Janeiro state. Three novel viruses were present in weed samples collected in Coimbra, Minas Gerais state. One virus was present in tomato samples collected at both locations. Genome features indicate that all six species are typical New World, bipartite begomoviruses. However, the viruses belonging to two of the novel species did not cluster with the Brazilian viruses in a phylogenetic tree. These species could represent a distinct lineage of New World begomoviruses, found in Brazil for the first time.

Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families.

Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21-24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one of the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery.

Molecular characterization of CEVd strains that induce different phenotypes in Gynura aurantiaca: structure-pathogenicity relationships.

Two Citrus exocortis viroid isolates (CEVd-s and CEVd-129) that induce severe and mild symptoms in Gynura aurantiaca, respectively, have been characterized. They present nucleotide sequences in the pathogenicity motifs P(L), C and P(R) similar to those of "Class A" and "Class B". Infectivity and symptom expression in G. aurantica and tomato were evaluated with a selection of sequence variants recovered from both isolates. As expected, the two variants selected from CEVd-s induced severe symptoms. The variants selected from CEVd-129 induced mild symptoms, except one of them, named MJ, that presented an unusual genotype and induced severe symptoms in G. aurantiaca. The biological properties of MJ show that the two nucleotide changes of the C domain normally associated with the P(L) and P(R) motifs of "Class B" strains are not implicated in symptom expression. The relationship between "Class A" and "Class B" strains with the symptoms induced in clementine trees grafted on trifoliate orange is discussed.

Siegesbeckia yellow vein virus is a distinct begomovirus associated with a satellite DNA molecule.

Leaf samples of Siegesbeckia glabrescens showing yellow vein, enation, and stunting symptoms were collected in Guangdong province, China. A specific 500-bp product was consistently detected in total DNA extracts, amplified with universal primers specific for members of the genus Begomovirus. Comparison of partial DNA sequences revealed that these virus isolates were identical, and therefore isolates GD13, GD24 and GD27 were selected for further sequence analysis. The complete nucleotide sequences of GD13, GD24 and GD27 were all found to be 2768 nucleotides (nts) long, with two open reading frames (ORFs) in the virion-sense strand and four ORFs in the complementary-sense strand, typical of the Old World begomoviruses. Sequence identities among the three isolates ranged from 99.7 to 99.8%. When compared with other reported sequences of begomoviruses, GD13 was most closely related to papaya leaf curl China virus (AJ876548), with a sequence identity of 76.8%. In addition, all isolates were found to be associated with DNAbeta molecules. The complete DNAbeta sequences of isolates GD13, GD24 and GD27 were determined. Sequence analysis showed that they had highest sequence identity with DNAbeta of Eupatorium yellow vein virus (AJ438938) (44.0, 43.9 and 45.6% identity). GD13, GD24 and GD27 are considered to be isolates of a distinct begomovirus species for which the name Siegesbeckia yellow vein virus (SgYVV) is proposed.

Molecular characterization of a new begomovirus infecting Senecio scandens in China.

Three begomovirus isolates, G46, G83 and G84 from Senecio scandens showing yellow mosaic symptoms were collected from Guangxi Province, P.R. China. The isolates were detected by PCR using universal primers for begomoviruses. Comparison of partial DNA-A sequences (approximately 500 bp) of the isolates revealed their 98.7-99.1% identity. The isolate G46, chosen for complete DNA-A sequencing, consisted of 2746 nt and had a typical genomic organization of begomoviruses. The G46 DNA-A had the highest sequence identity (72.4%) with that of Ageratum leaf curl virus among begomoviruses. The molecular data suggest that the isolate G46 is a new begomovirus (species), for which the name Senecio yellow mosaic virus (Senecio yellow mosaic virus) is proposed.

A kissing-loop interaction in a hammerhead viroid RNA critical for its in vitro folding and in vivo viability.

Chrysanthemum chlorotic mottle viroid (CChMVd) RNA (398-401 nucleotides) can form hammerhead ribozymes that play a functional role in its replication through a rolling-circle mechanism. In contrast to most other viroids, which adopt rod-like or quasi-rod-like secondary structures of minimal free energy, the computer-predicted conformations of CChMVd and Peach latent mosaic viroid (PLMVd) RNAs are branched. Moreover, the covariations found in a number of natural CChMVd variants support that the same or a closely related conformation exists in vivo. Here we report that the CChMVd natural variability also supports that the branched conformation is additionally stabilized by a kissing-loop interaction resembling another one proposed in PLMVd from in vitro assays. Moreover, site-directed mutagenesis combined with bioassays and progeny analysis showed that: (1) single CChMVd mutants affecting the kissing loops had low or no infectivity at all, whereas infectivity was recovered in double mutants restoring the interaction; (2) mutations affecting the structure of the regions adjacent to the kissing loops reverted to wild type or led to rearranged stems, also supporting their interaction; and (3) the interchange between 4 nucleotides of each of the two kissing loops generated a viable CChMVd variant with eight mutations. PAGE analysis under denaturing and nondenaturing conditions revealed that the kissing-loop interaction determines proper in vitro folding of CChMVd RNA. Preservation of a similar kissing-loop interaction in two hammerhead viroids with an overall low sequence similarity suggests that it facilitates in vivo the adoption and stabilization of a compact folding critical for viroid viability.

Viral genetic determinants for thrips transmission of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) is transmitted exclusively by thrips in nature. A reassortment-based viral genetic system was used to map transmissibility by thrips to the medium (M) RNA of TSWV. To locate determinants of thrips transmission in the M RNA, 30 single-lesion isolates (SLIs) were generated from a single TSWV isolate that was inefficiently transmitted by thrips. Three of the 30 SLIs were transmitted by thrips, and 27 were not. Sequence analysis of the M RNA, thrips transmissibility assays, G(C) protein analysis, and transmission electron microscopic studies revealed that a specific nonsynonymous mutation (C1375A) in the G(N)/G(C) ORF of the M RNA resulted in the loss of thrips transmissibility without inhibition of virion assembly. This was in contrast to other nontransmissible SLIs, which had frameshift and/or nonsense mutations in the G(N)/G(C) ORF but were defective in virion assembly. The G(C) glycoprotein was detectable in the C1375A mutants but not in the frameshift or nonsense mutants. We report a specific viral determinant associated with virus transmission by thrips. In addition, the loss of transmissibility was associated with the accumulation of defective haplotypes in the population, which are not transmissible by thrips, rather than with the presence of a dominant haplotype that is inefficiently transmitted by thrips. These results also indicate that the glycoproteins may not be required for TSWV infection of plant hosts but are required for transmissibility by thrips.

The complete genome sequence of a new necrovirus isolated from Olea europaea L.

The complete nucleotide sequence of a virus isolated from Olea europaea L. (GP isolate), previously identified as an isolate of Tobacco necrosis virus D (TNV-D) based on its coat protein sequence, was determined. The viral RNA genome consists of 3683 nucleotides and contains five open reading frames. The putative RNA-dependent RNA polymerase shows 91.2% amino acid identity with that of an isolate of Olive latent virus 1 (OLV-1) and the coat protein reveals highest sequence identity with that of TNV-D. Based on the deduced genome organization and phylogenetic analysis of predicted functional translation products with that of other necroviruses, the GP isolate genome appears to represent an example of a new virus arisen by gene exchange and is proposed to be a new necrovirus, provisionally named Olive mild mosaic virus.

A DNAbeta associated with Tomato yellow leaf curl China virus is required for symptom induction.

We report here that all 25 isolates of Tomato yellow leaf curl China virus (TYLCCNV) collected from tobacco, tomato, or Siegesbeckia orientalis plants in different regions of Yunnan Province, China, were associated with DNAbeta molecules. To investigate the biological role of DNAbeta, full-length infectious clones of viral DNA and DNAbeta of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into Nicotiana benthamiana, Nicotiana glutinosa, Nicotiana. tabacum Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAbeta was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAbeta of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the betaC1 gene of TYLCCNV-Y10 DNAbeta was mutated, the mutants failed to induce leaf curl symptoms in N. benthamiana when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAbeta molecules were replicated. When N. benthamiana and N. tabacum plants were transformed with a construct containing the betaC1 gene under the control of the Cauliflower mosaic virus 35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of betaC1 transcripts, while transgenic plants transformed with the betaC1 gene containing a stop codon after the start codon remained symptomless. Thus, expression of a betaC1 gene is adequate for induction of symptoms of viral infection in the absence of virus.