Combined effect of corn in the barrier crop and plant extracts against Cowpea mild mottle virus infecting soybean [Glycine max (L. ) Merr. ] in the field

Cowpea mild mottle virus (CpMMV) is one of the problems that can decrease soybean production. The research was conducted on the combined effects of corn in the barrier crop with plant extracts against CpMMV infecting soybean in the field. The field data was conducted using a Completely Randomized Design. The mean of disease incidence and disease severity is measured from total plants in each replicate plot on each treatment. Planting one or two of corn lines were grown at the edge four weeks before planting soybeans. Cashew nut shell (CNS), pagoda leaf, and rhizome of ginger extracts were applied using the sprayer and applied at 24 h before virus acquisition and transmission by whiteflies. The result showed that the virus incubation period ranged from 9−38 days after transmission longer than the untreated control. Planting two corn lines at the edge with CNS extract as bioactivator on soybean was the most extended incubation period of the virus and the lowest absorbance value DAS-ELISA of 0.20. There was a 73.11 % increase in the relative inhibition level of the virus. Planting corn at the edge with CNS extract proved to be more effective than soybean monoculture with CNS extract. However, soybean monoculture with CNS extract provides a better relative inhibition level of the virus (64.32 %) than planting two rows of corn on the edge combined with ginger of rhizome extract and planting two rows of corn on the edge with pagoda leaf extract as bioactivator on a soybean plant.

Transmission of Grapevine leafroll-associated virus-1 (Ampelovirus) and Grapevine virus A (Vitivirus) by the Cottony Grape Scale, Pulvinaria vitis (Hemiptera: Coccidae).

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]-1, -3, and -4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV-1 and GVA. This is the first report of GLRaV-1 and GVA transmission from grapevine to grapevine by this species.

Molecular characterization of a novel wheat-infecting virus of the family Betaflexiviridae.

Wheat plants showing yellowing and mosaic in leaves and stunting were collected from wheat fields in Henan Province, China. Analysis of these plants by transmission electron microscopy showed that they contained two types of filamentous virus-like particles with a length of 200-500 nm and 1000-1300 nm, respectively. RNA-seq revealed a coinfection with wheat yellow mosaic virus (WYMV) and an unknown wheat-infecting virus. The genome of the unknown virus is 8,410 nucleotides long, excluding its 3' poly(A) tail. It has six open reading frames (ORFs). ORF1 encodes a putative viral replication-associated protein (Rep), and ORFs 2, 3, and 4 encode the triple gene block (TGB) proteins. ORFs 5 and 6 encode the capsid protein (CP) and a protein with unknown function, respectively. Phylogenetic analysis showed that this novel virus is evolutionarily related to members of the subfamily Quinvirinae, family Betaflexiviridae. It is, however, distinct from the viruses in the currently established genera. Based on the species and genus demarcation criteria set by the International Committee on Taxonomy of Viruses (ICTV), we tentatively name this novel virus "wheat yellow stunt-associated betaflexivirus" (WYSaBV), and we propose it to be a member of a new genus in the family Betaflexiviridae.

Virus Pathogens in Australian Vineyards with an Emphasis on Shiraz Disease.

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on "Shiraz Disease" (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.

First complete genome sequence of garlic virus X infecting Allium sativum- G282 from India.

The present report communicates the first full genome sequencing of the Garlic virus X from northern India. The total genome size of Garlic virus X (MK503771) reported in this study is 8458 bp ssRNA. The full genome sequence analysis showed the close relationship of Garlic virus X from India to that of from China, Korea, Australia and Spain. The full genome sequence based study of Indian Garlic virus X reveals the geographical relationship of this virus in India and global origin which may assists in development of control strategy for this virus.

Characterization of the virome of shallots affected by the shallot mild yellow stripe disease in France.

To elucidate the etiology of a new disease of shallot in France, double-stranded RNAs from asymptomatic and symptomatic shallot plants were analyzed using high-throughput sequencing (HTS). Annotation of contigs, molecular characterization and phylogenetic analyses revealed the presence in symptomatic plants of a virus complex consisting of shallot virus X (ShVX, Allexivirus), shallot latent virus (SLV, Carlavirus) and two novel viruses belonging to the genera Carlavirus and Potyvirus, for which the names of shallot virus S (ShVS) and shallot mild yellow stripe associated virus (SMYSaV), are proposed. Complete or near complete genomic sequences were obtained for all these agents, revealing divergent isolates of ShVX and SLV. Trials to fulfill Koch's postulates were pursued but failed to reproduce the symptoms on inoculated shallots, even though the plants were proved to be infected by the four viruses detected by HTS. Replanting of bulbs from SMYSaV-inoculated shallot plants resulted in infected plants, showing that the virus can perpetuate the infection over seasons. A survey analyzing 351 shallot samples over a four years period strongly suggests an association of SMYSaV with the disease symptoms. An analysis of SMYSaV diversity indicates the existence of two clusters of isolates, one of which is largely predominant in the field over years.

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis.

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.

p15 encoded by Garlic virus X is a pathogenicity factor and RNA silencing suppressor.

Garlic virus X (GarVX) encodes a 15 kDa cysteine-rich protein (CRP). To investigate the function(s) of p15, its subcellular localization, role as a symptom determinant and capacity to act as a viral suppressor of RNA silencing (VSR) were analysed. Results showed that GFP-tagged p15 was distributed in the cytoplasm, nucleus and nucleolus. Expression of p15 from PVX caused additional systemic foliar malformation and led to increased accumulation of PVX, showing that p15 is a virulence factor for reconstructed PVX-p15. Moreover, using a transient agro-infiltration patch assay and a Turnip crinkle virus (TCV) movement complementation assay, it was demonstrated that p15 possesses weak RNA silencing suppressor activity. Removal of an amino acid motif resembling a nuclear localization signal (NLS) prevented p15 from accumulating in the nucleus but did not abolish its silencing suppression activity. This study provides the first insights into the multiple functions of the GarVX p15 protein.

Allexiviruses may have acquired inserted sequences between the CP and CRP genes to change the translation reinitiation strategy of CRP.

Allexiviruses are economically important garlic viruses that are involved in garlic mosaic diseases. In this study, we characterized the allexivirus cysteine-rich protein (CRP) gene located just downstream of the coat protein (CP) gene in the viral genome. We determined the nucleotide sequences of the CP and CRP genes from numerous allexivirus isolates and performed a phylogenetic analysis. According to the resulting phylogenetic tree, we found that allexiviruses were clearly divided into two major groups (group I and group II) based on the sequences of the CP and CRP genes. In addition, the allexiviruses in group II had distinct sequences just before the CRP gene, while group I isolates did not. The inserted sequence between the CP and CRP genes was partially complementary to garlic 18S rRNA. Using a potato virus X vector, we showed that the CRPs affected viral accumulation and symptom induction in Nicotiana benthamiana, suggesting that the allexivirus CRP is a pathogenicity determinant. We assume that the inserted sequences before the CRP gene may have been generated during viral evolution to alter the termination-reinitiation mechanism for coupled translation of CP and CRP.

Cryotherapy by encapsulation-dehydration is effective for in vitro eradication of latent viruses from 'Marubakaido' apple rootstock.

Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) are several major viral pathogens of apple trees, responsible for substantial damage to the world's apple industry. This study aimed to evaluate the effectiveness of the encapsulation-dehydration cryopreservation technique to eradicate these viral pathogens from in vitro shoot tips excised from 'Marubakaido' apple rootstock cultures. Axillary shoot tips were excised from in vitro cultures, encapsulated in alginate beads, precultured in MS salts, dehydrated in a laminar flow hood, immersed in liquid nitrogen, then warmed and recovered on medium. After LN exposure, in vitro rooting and acclimatization, recovered 'Marubakaido' plants exhibited 52% survival and 35% regrowth without callus formation. After 8 months of regrowth, PCR analyses revealed that all the plants were free of ACLSV and ASPV, but 2 out of 20 recovered plants were still infected with ASGV. This is the first report in Brazil of the application of cryotherapy to eradicate viral complexes in Malus. Cryotherapy can facilitate the production of virus-free plants by producing high quality plant material.

[Persistent Shallot virus X infection correlates with transcriptional repression of plant cell RNA-dependent RNA polymerase and DCL proteins in plant roots].

Shallot virus X is a typical representative of Allexiviruses. The transcription levels of principal genes involved in the RNA silencing in healthy and shallot virus X-infected plants have been quantified by real-time polymerase chain reaction. There is a negative correlation between the reproduction rates of RNA virus and the levels of RNA-dependent RNA polymerase and DCL proteins in roots and leaves of infected plants. These observations indicate that Shallot X virus employs noncanonical ways of overcoming the antiviral defense of the plant by systemic RNA silencing.

The unfolded protein response and programmed cell death are induced by expression of Garlic virus X p11 in Nicotiana benthamiana.

Garlic virus X (GarVX) ORF3 encodes a p11 protein, which contributes to virus cell-to-cell movement and forms granules on the endoplasmic reticulum (ER) in Nicotiana benthamiana. Expression of p11 either from a binary vector, PVX or TMV induced ER stress and the unfolded protein response (UPR), as demonstrated by an increase in transcription of the ER luminal binding protein (BiP) and bZIP60 genes. UPR-related programmed cell death (PCD) was elicited by PVX : p11 or TMV : p11 in systemic infected leaves. Examination of p11 mutants with deletions of two transmembrane domains (TM) revealed that both were required for generating granules and for inducing necrosis. TRV-based VIGS was used to investigate the correlation between bZIP60 expression and p11-induced UPR-related PCD. Less necrosis was observed on local and systemic leaves of bZIP60 knockdown plants when infected with PVXp11, suggesting that bZIP60 plays an important role in the UPR-related PCD response to p11 in N. benthamiana.

Integrated transcriptome catalogue and organ-specific profiling of gene expression in fertile garlic (Allium sativum L.).

BACKGROUND: Garlic is cultivated and consumed worldwide as a popular condiment and green vegetable with medicinal and neutraceutical properties. Garlic cultivars do not produce seeds, and therefore, this plant has not been the subject of either classical breeding or genetic studies. However, recent achievements in fertility restoration in a number of genotypes have led to flowering and seed production, thus enabling genetic studies and breeding in garlic. RESULTS: A transcriptome catalogue of fertile garlic was produced from multiplexed gene libraries, using RNA collected from various plant organs, including inflorescences and flowers. Over 32 million 250-bp paired-end reads were assembled into an extensive transcriptome of 240,000 contigs. An abundant transcriptome assembled separately from 102,000 highly expressed contigs was annotated and analyzed for gene ontology and metabolic pathways. Organ-specific analysis showed significant variation of gene expression between plant organs, with the highest number of specific reads in inflorescences and flowers. Analysis of the enriched biological processes and molecular functions revealed characteristic patterns for stress response, flower development and photosynthetic activity. Orthologues of key flowering genes were differentially expressed, not only in reproductive tissues, but also in leaves and bulbs, suggesting their role in flower-signal transduction and the bulbing process. More than 100 variants and isoforms of enzymes involved in organosulfur metabolism were differentially expressed and had organ-specific patterns. In addition to plant genes, viral RNA of at least four garlic viruses was detected, mostly in the roots and cloves, whereas only 1-4% of the reads were found in the foliage leaves. CONCLUSIONS: The de novo transcriptome of fertile garlic represents a new resource for research and breeding of this important crop, as well as for the development of effective molecular markers for useful traits, including fertility and seed production, resistance to pests and neutraceutical characteristics.

Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses.

Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples.

Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus.

Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

Complete sequence of an Apple stem grooving virus (ASGV) isolate from China.

The complete genome sequence of a Chinese isolate of Apple stem grooving virus (ASGV) was determined to be 6,495 nucleotides long, single-stranded, plus-sense RNA. The viral RNA has two overlapping open reading frames (ORFs): ORF1 and ORF2. Compared with the genome sequences of ASGV isolates available in GenBank, the nucleotide identities ranged from 80.1 to 86.3 %. The amino acid identities of proteins encoded by ORF1 and ORF2 ranged from 79.5 to 86.1 % and 82.0 to 85.9 %, respectively.

Cydonia japonica, Pyrus calleryana and P. amygdaliformis: three new ornamental or wild hosts of Apple stem pitting virus.

Japanese quince, ornamental and wild pear symptomless samples were infected with Apple stem pitting virus (ASPV). Identification of ASPV was achieved by different PCR assays that amplified either the RNA polymerase or coat protein gene regions. For further confirmation, 312 bp amplicons within the polymerase gene were sequenced and compared with previously published ASPV sequences and additional sequences of isolates from ancient Italian cultivars. Comparison of the partial sequences isolated from wild/ornamental hosts and from cultivated species revealed significant divergence levels. Among the wild/ornamental isolates, the PCT88 isolate from Pyrus calleryana was the most divergent, having an amino acid deletion and incorporating a unique stretch of amino acids not present in any other isolate. Further to this preliminary partial sequence data, statistical analysis demonstrated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. These results represent the first report of natural ASPV infection in these novel ornamental and wild Rosaceae hosts.

Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study.

BACKGROUND: Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. RESULTS: Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. CONCLUSIONS: The fast and efficient strategies described here should have broad applications, in particular for the study of "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents.

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed.