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Seed transmission is an important factor in the epidemiology of plant pathogens. Geminiviruses are serious pests spread in tropical and subtropical regions. They are transmitted by hemipteran insects, but a few cases of transmission through seeds were recently reported. Here, we investigated the tomato seed transmissibility of the begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), one of the agents inducing the tomato yellow leaf curl disease, heavily affecting tomato crops in the Mediterranean area. None of the 180 seedlings originating from TYLCSV-infected plants showed any phenotypic alteration typical of virus infection. Moreover, whole viral genomic molecules could not be detected in their cotyledons and true leaves, neither by membrane hybridization nor by rolling-circle amplification followed by PCR, indicating that TYLCSV is not a seed-transmissible pathogen for tomato. Examining the localization of TYLCSV DNA in progenitor plants, we detected the virus genome by PCR in all vegetative and reproductive tissues, but viral genomic and replicative forms were found only in leaves, flowers and fruit flesh, not in seeds and embryos. Closer investigations allowed us to discover for the first time that these embryos were superficially contaminated by TYLCSV DNA but whole genomic molecules were not detectable. Therefore, the inability of TYLCSV genomic molecules to colonize tomato embryos during infection justifies the lack of seed transmissibility observed in this host.

Chickpea chlorotic dwarf virus (CpCDV) a polyphagous mastrevirus, family Geminiviridae, is reported to infect several economically important hosts, among which tomato, in numerous world areas. NGS studies have recently linked the "hard fruit syndrome" of watermelon to CpCDV infection. In this study, we surveyed for CpCDV infection tomato fields in several Tunisian areas and we report this virus for the first time in tomato in this Mediterranean country. We detected CpCDV by PCR with specific primers and obtained a full-length sequence of a tomato-derived isolate, resulting identical to the Tunisian watermelon-derived isolate, and classifiable as CpCDV strain A. Following tomato agro-inoculation of the CpCDV infectious clone under controlled conditions, we checked infection by stem squash, dot blotting and PCR, and demonstrated the ability of CpCDV to replicate in tomato tissues. The alterations observed in fruits of CpCDV-infected tomatoes were similar to those previously described for watermelon, providing hints about the perturbation of fruit development mediated by this virus.

The soil-borne cereal mosaic virus (SBCMV) is a furovirus that can infect durum (Triticum turgidum ssp. durum) and common (T. aestivum L.) wheat. SBCMV is transmitted to the roots of the host plants by Polymyxa graminis Led., a plasmodiophorid protist that persist in soil up to 20 years. SBCMV is widespread in European countries, and in Italy is present in the northern and central regions, where it causes yield losses on susceptible wheat cultivars often above 50%. The impact of all viruses in wheat may be greater in Europe due to climate change. Notably, a spontaneous mutation of a new variant of SBCMV, that infects plants at high temperatures, was isolated in Japan. In the future, the occurrence of similar mutations may increase due to global temperature increase. Little information is available on genetic determinants for SBCMV resistance and the identification of new sources of resistance is needed. Thus, we are performing a genome wide association study to identify genomic regions related to resistance to SBCMV in durum wheat. Since wild genotypes are an important source of crop genetic diversity, we selected a sub-collection of about 200 landraces from the Global Durum Wheat Panel, already genotyped with Illumina iSelect 90K SNP array technology. Population structure was performed to define the genetic diversity of the sub-collection. The selected genotypes will be grown in greenhouse using a soil infected by SBCMV. Infection will be evaluated by symptoms and by qRT-PCR. The association between genotypic and phenotypic data will be allow us to identify genomic regions and related candidate genes for resistance to SBCMV. This work is supported by Regione Lombardia, d.d.s. n. 4403 28/03/2018, grant n° 42 - SURF.

Tomato plants can establish symbiotic interactions with arbuscular mycorrhizal fungi (AMF) able to promote plant nutrition and prime systemic plant defenses against pathogens attack, the mechanism involved is known as mycorrhiza-induced resistance (MIR). However, studies on the effect of AMF on viral infection, still limited and not conclusive, indicate that AMF colonization may have a detrimental effect on plant defenses against viruses, so that the term &ldquo, mycorrhiza-induced susceptibility&rdquo, (MIS) has been proposed for these cases. To expand the case studies to a not yet tested viral family, that is, Bromoviridae, we investigated the effect of the colonization by the AMF Funneliformis mosseae on cucumber mosaic virus (CMV) infection in tomato by phenotypic, physiological, biochemical, and transcriptional analyses. Our results showed that the establishment of a functional AM symbiosis is able to limit symptoms development. Physiological and transcriptomic data highlighted that AMF mitigates the drastic downregulation of photosynthesis-related genes and the reduction of photosynthetic CO2 assimilation rate caused by CMV infection. In parallel, an increase of salicylic acid level and a modulation of reactive oxygen species (ROS)-related genes, toward a limitation of ROS accumulation, was specifically observed in CMV-infected mycorrhizal plants. Overall, our data indicate that the AM symbiosis influences the development of CMV infection in tomato plants and exerts a priming effect able to enhance tolerance to viral infection.

Background Small RNAs (sRNAs) are short non-coding RNA molecules (20–30 nt) that regulate gene expression at transcriptional or post-transcriptional levels in many eukaryotic organisms, through a mechanism known as RNA interference (RNAi). Recent studies have highlighted that they are also involved in cross-kingdom communication: sRNAs can move across the contact surfaces from “donor” to “receiver” organisms and, once in the host cells of the receiver, they can target specific mRNAs, leading to a modulation of host metabolic pathways and defense responses. Very little is known about RNAi mechanism and sRNAs occurrence in Arbuscular Mycorrhizal Fungi (AMF), an important component of the plant root microbiota that provide several benefits to host plants, such as improved mineral uptake and tolerance to biotic and abiotic stress. Results Taking advantage of the available genomic resources for the AMF Rhizophagus irregularis we described its putative RNAi machinery, which is characterized by a single Dicer-like (DCL) gene and an unusual expansion of Argonaute-like (AGO-like) and RNA-dependent RNA polymerase (RdRp) gene families. In silico investigations of previously published transcriptomic data and experimental assays carried out in this work provided evidence of gene expression for most of the identified sequences. Focusing on the symbiosis between R. irregularis and the model plant Medicago truncatula, we characterized the fungal sRNA population, highlighting the occurrence of an active sRNA-generating pathway and the presence of microRNA-like sequences. In silico analyses, supported by host plant degradome data, revealed that several fungal sRNAs have the potential to target M. truncatula transcripts, including some specific mRNA already shown to be modulated in roots upon AMF colonization. Conclusions The identification of RNAi-related genes, together with the characterization of the sRNAs population, suggest that R. irregularis is equipped with a functional sRNA-generating pathway. Moreover, the in silico analysis predicted 237 plant transcripts as putative targets of specific fungal sRNAs suggesting that cross-kingdom post-transcriptional gene silencing may occur during AMF colonization. Electronic supplementary material The online version of this article (10.1186/s12864-019-5561-0) contains supplementary material, which is available to authorized users.

The nucleocapsid protein (N) gene of tomato spotted wilt virus (TSWV) was inserted into Osteospermum ecklonis via Agrobacterium tumefaciens leaf strips co-cultivation. Sixteen primary transformant clones of two O. ecklonis genotypes were analysed. Southern blots of restricted genomic DNA demonstrated integration of the transgene and indicated the number of integrated copies. Expression of the transgene was estimated by DAS-ELISA and Western and Northern blotting. Plants were challenged with TSWV inoculation, either mechanically or by the thrips Frankliniella occidentalis; they were then monitored for symptom appearance and tested by TAS-ELISA for infection. Inoculation of the transgenic clones via the natural TSWV vector was more efficient and led to the identification of 1 clone, characterised by multiple transgene integration and no transgene expression, with improved resistance to TSWV.

The potential of Arundo donax to grow in degraded soils, characterized by excess of salinity (Na+), and phosphorus deficiency (-P) or excess (+P) also coupled with salinity (+NaP), was investigated by combining in vivo plant phenotyping, quantification of metabolites and ultrastructural imaging of leaves with a transcriptome-wide screening. Photosynthesis and growth were impaired by + Na, -P and + NaP. While + Na caused stomatal closure, enhanced biosynthesis of carotenoids, sucrose and isoprene and impaired anatomy of cell walls, +P negatively affected starch production and isoprene emission, and damaged chloroplasts. Finally, +NaP largely inhibited photosynthesis due to stomatal limitations, increased sugar content, induced/repressed a number of genes 10 time higher with respect to + P and + Na, and caused appearance of numerous and large plastoglobules and starch granules in chloroplasts. Our results show that A. donax is sensitive to unbalances of soil ion content, despite activation of defensive mechanisms that enhance plant resilience, growth and biomass production of A. donax under these conditions.

Arundo donax L. is an invasive species recently employed for biomass production that emits large amounts of isoprene, a volatile compound having important defensive role. Here, the potential of A. donax to grow in degraded soils, characterized by poor fertility, eutrophication and/or salinization, has been evaluated at morphological, biochemical and transcriptional level. Our results highlight sensitivity of A. donax to P deficiency. Moreover, we show that A. donax response to salt stress (high sodium, Na+), which impaired plant performance causing detrimental effects on leaf cells ultrastructure, is characterized by enhanced biosynthesis of antioxidant carotenoids and sucrose. Differently from Na+, high phosphorous (P) supply did not hamper photosynthesis although it affected carbon metabolism through reduction of starch content and by lowering isoprene emission. In particular, we revealed on salt-stress leaves that high P enhanced the expression of genes involved in abiotic stress tolerance, but further increased diffusive limitations to photosynthesis and slowed-down sugar turnover without modifying isoprene emission. Therefore, despite limiting productivity, high P improved A. donax tolerance to salinity by favouring the accumulation of carbohydrates that protect cells and increase osmotic potential, and by stimulating the synthesis of antioxidants that improves photo-protection and avoids excessive accumulation of reactive oxygen species.HighlightsArundo donax is sensitive to elevated salinity. High phosphorous supply to salt-stressed A. donax enhances transcriptomic changesthat induce the onset of physiological mechanisms of stress tolerance but limits productivity.

The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species., Viruses are potential vectors for horizontal gene transfer. Here, studying viral infection of sugar beet plants, the authors report the generation of virus-host circular DNA hybrids and provide a picture of the initial steps in virus-mediated horizontal transfer of chromosomal DNA between plant species.

Cicer arietinum L. (chickpea) is the world's fourth most widely grown pulse. Chickpea seeds are a primary source of dietary protein for humans, and chickpea cultivation contributes to biological nitrogen fixation in the soil, given its symbiotic relationship with rhizobia. Therefore, chickpea cultivation plays a pivotal role in innovative sustainable models of agro-ecosystems inserted in crop rotation in arid and semi-arid environments for soil improvement and the reduction of chemical inputs. Indeed, the arid and semi-arid tropical zones of Africa and Asia have been primary areas of cultivation and diversification. Yet, nowadays, chickpea is gaining prominence in Canada, Australia, and South America where it constitutes a main ingredient in vegetarian and vegan diets. Viruses and plant parasitic nematodes (PPNs) have been considered to be of minor and local impact in primary areas of cultivation. However, the introduction of chickpea in new environments exposes the crop to these biotic stresses, compromising its yields. The adoption of high-throughput genomic technologies, including genome and transcriptome sequencing projects by the chickpea research community, has provided major insights into genome evolution as well as genomic architecture and domestication. This review summarizes the major viruses and PPNs that affect chickpea cultivation worldwide. We also present an overview of the current state of chickpea genomics. Accordingly, we explore the opportunities that genomics, post-genomics and novel editing biotechnologies are offering in order to understand chickpea diseases and stress tolerance and to design innovative control strategies.

During 2016, a survey was carried out to investigate the presence and distribution of Tomato leaf curl New Delhi virus (ToLCNDV) in Campania region (Southern Italy). In three fields in the Castel Volturno and Giugliano in Campania municipalities (Caserta and Napoli provinces respectively), an unusual disease was noted affecting pumpkin (Cucurbita moschata) plants of the variety “Lunga di Napoli”, a traditional local ecotype. Symptoms consisted of stunting and severe foliar yellow mosaic, which, in some cases, resulted in completely yellow leaves. The affected plants produced less flowers and fruits. The incidence of the disease ranged from 80 to 100% of the plants. The insect vector of ToLCNDV, Bemisia tabaci, was found in each sampled field and the genotype of twenty specimens for each population was identified as previously described (Parrella et al., 2012 and 2014). The Q1 and Q2 variants of the Mediterranean species were identified with a percentage ranging from 11-14% and 86-89% respectively. Four sy...

Cicer arietinum L. (chickpea) is the world's fourth most widely grown pulse. Chickpea seeds are a primary source of dietary protein for humans, and chickpea cultivation contributes to biological nitrogen fixation in the soil, given its symbiotic relationship with rhizobia. Therefore, chickpea cultivation plays a pivotal role in innovative sustainable models of agro-ecosystems inserted in crop rotation in arid and semi-arid environments for soil improvement and the reduction of chemical inputs. Indeed, the arid and semi-arid tropical zones of Africa and Asia have been primary areas of cultivation and diversification. Yet, nowadays, chickpea is gaining prominence in Canada, Australia, and South America where it constitutes a main ingredient in vegetarian and vegan diets. Viruses and plant parasitic nematodes (PPNs) have been considered to be of minor and local impact in primary areas of cultivation. However, the introduction of chickpea in new environments exposes the crop to these biotic stresses, compromising its yields. The adoption of high-throughput genomic technologies, including genome and transcriptome sequencing projects by the chickpea research community, has provided major insights into genome evolution as well as genomic architecture and domestication. This review summarizes the major viruses and PPNs that affect chickpea cultivation worldwide. We also present an overview of the current state of chickpea genomics. Accordingly, we explore the opportunities that genomics, post-genomics and novel editing biotechnologies are offering in order to understand chickpea diseases and stress tolerance and to design innovative control strategies.

Tomato leaf curl disease is one of the main constraints in tomato production worldwide. It is induced by viruses including Tomato leaf curl virus (ToLCV). Plant viruses can both induce and be themselves targeted by gene silencing machinery. Certain animal viruses were found to be targeted by host miRNAs but, there is no similar experimental report for plant viruses. In this study, we investigated if tomato-encoded miRNAs target ToLCV using an in silico analysis. Results showed that ToLCV can be targeted by three tomato miRNAs, named miR156, miR159, and miR403. Following virus infection of susceptible tomato plants cv Moneymaker, miR159 and miR403 were upregulated, while miR156 was unchanged. In the plants of the resistant tomato variety PS550, that accumulated 50-fold less viral DNA and did not show disease symptoms, miR156 was upregulated, while the two others remained unchanged. In addition, the expression level of three genes known as targets of these miRNAs, i.e. SPL6b, MYB33 and AGO2a, was measured in both susceptible and resistant tomato varieties by qRT-PCR. The possible role of the predicted miRNAs and regulation of their target genes in symptom development by ToLCV is discussed.

Tomato yellow leaf curl disease is a major constraint for tomato production worldwide and availability of new resistant materials is of great importance for breeding programmes. A phenotypic survey was undertaken to evaluate the level of resistance to the main tomato yellow leaf curl disease-inducing viruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus, in several commercial tomato cultivars, never characterised before. Seven weeks post inoculation, two cultivars resulted in high resistant phenotypes to both begomoviruses, and four were tolerant to at least one of them. In the two highly resistant hybrids (SJ12, RFT112), symptoms were completely absent and viral DNA was from 102 to 105 fold lower than in susceptible plants. Molecular marker analysis revealed that these genotypes harbour the resistant genes Ty-1/Ty-3 and Ty-2. Given their high resistance, they can be considered good candidates for cultivation and breeding in areas where incidence of TYLCD is very elevated.

In Tunisia, watermelon (Citrullus lanatus) is one of the most common and popular summer fruit crops. The crop is widely cultivated in the central and southern parts of the country with a yearly production of about 400,000 tons. Hard fruit syndrome of watermelon is a well-known syndrome, reported in Tunisia since 1994, when up to 70% incidence was reported in some areas; in recent years, the incidence ranged between 10 and 40%, leading growers to switch to alternative crops. The etiology of the hard fruit syndrome of watermelon is uncertain up to now, but two begomoviruses (Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus) have been recently found associated with this syndrome (Mnari-Hattab et al. 2014), suggesting a viral etiology for symptoms initially attributed to physiological and/or nutritional disorders. Between 2005 and 2015, watermelon fields were surveyed in different areas of Tunisia (Kairouan, Zaghouan, Gabes, Gafsa, and Beja). Samples were collected from unmarketable fruits exhibiting chlorotic mottling on rind and white hard portions inside the flesh. Total DNA from 11 samples were subjected to rolling circle amplification using ?29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, U.K.). A single DNA fragment of about 2,500 bp was obtained following SpeI digestion from three samples. The SpeI-cut DNA derived from a sample collected in Zaghouan Province in 2015 was cloned in pBluescript KS(+) and fully sequenced in both directions. The sequence was 2,571 nt long and exhibited the genomic organization typical of mastreviruses. Alignment analysis against 63 mastrevirus genome sequences using STD program (Muhire et al. 2013) showed high nucleotide sequence identity values with Chickpea chlorotic dwarf virus (CpCDV) genome isolates (about 86%), with the maximum identity score of 98% with an isolate from chickpea collected in Syria (FR687959); identity percentage toward other mastreviruses was less than 70%. The new CpCDV sequence was submitted to GenBank under accession number KX580024. A primer pair CpCDV-CP-F (5?-GCAGAATCAAGGGCGAAGAG-3?) and CpCDV-CP-R (5?-CGGACCGGGACCATAGTAAG-3?) was used to check for CpCDV infection in field watermelon samples; 10 of 11 samples were positive in PCR amplifying a 501-bp diagnostic fragment in the coat protein gene. PCR analyses were also conducted for 39 additional watermelon samples collected in Kairouan (21 samples), Gafsa (4 samples), and Zaghouan (14 samples), the main growing areas in Tunisia, during 2005, 2014, and 2015. Thirty-two of them tested positive for CpCDV. A 1.8-mer construct was engineered in the binary plasmid pBin19, used to transform Agrobacterium tumefaciens LBA4404, and then used to challenge Nicotiana benthamiana plants in infectivity assays. Symptoms of downward leaf curling were observed at 7 days post inoculation in upper uninoculated leaves that later became crumpled, with the plant exhibiting marked chlorosis and dwarfing. CpCDV infection was confirmed by tissue print hybridization using a digoxigenin-labeled probe. CpCDV infecting several species has been reported from many tropical and subtropical countries and was recently found in Tunisia on chickpea (Kumari et al. 2015). To our knowledge, this is the first report of CpCDV infecting watermelon. The finding of CpCDV in samples with the hard fruit syndrome adds an unexpected piece to the complex puzzle of its etiology issue.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) which infects species in the families Cucurbitaceae and Solanaceae (Padidam et al., 1995; Mizutani et al., 2011). Begomoviruses are transmitted by the whitefly Bemisia tabaci in a persistent manner (Rosen et al., 2015). In October 2015, severe symptoms not previously reported by growers in the horticultural area of the Province of Trapani (Sicily, Italy) were observed on zucchini squash (Cucurbita pepo) in open fields. The symptoms included yellow mosaic, severe leaf curling, swelling of veins of young leaves, shortening of internodes, roughness of the skin of fruit and reduced fruit size; the symptoms were reminiscent of those caused by begomoviruses. Total DNA was extracted from young leaves of 22 plants by phenol/chloroform extraction and ethanol precipitation. PCR was performed with the A1F/A1R primer pair (Mizutani et al., 2011) for the DNA-A component and the pair described by Ruiz et al. (2015) for the DNA-B component to amplify a ~1200-bp fragment of DNA-A and a ~890 bp fragment of DNA-B, respectively. All 10 samples were positive by PCR with both primer pairs. No amplification products were obtained using primers specific for the monopartite begomoviruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus (Davino et al., 2008). DAS-ELISA analysis for Cucumber mosaic virus, Papaya ring spot virus and Zucchini yellow mosaic virus (Loewe Phytodiagnostica, Germany) yielded negative results. The amplification products of DNA-A and DNA-B components from three plants were purified using the UltraClean® PCR Clean-Up kit (Mo-Bio, USA) and the nucleotide sequences were determined in both orientations using an ABI 3130XL Genetic Analyzer (Life Technologies, USA). The sequences obtained from the three samples showed 99% identity. A Basic Local Alignment Search Tool (BLAST) analysis showed an identity >99% with sequences of DNA-A and DNA-B of Tomato leaf curl New Delhi virus (ToLCNDV) isolates recently reported in Spain on zucchini (GenBank Accession Nos. KF749224 and KF749227) and tomato (KM977733 and KM977734). The DNA-A and DNA-B sequences of one sample were deposited in GenBank (KU145141 and KU145142). Sicily is an important region for horticulture in the Mediterranean basin and Europe in general. With the possible opening of a free trade area in countries bordering the Mediterranean basin, Sicily may become a transit centre for flow of plant materials, potentially carrying new pathogens, between Europe and North Africa. To our knowledge, this is the first report of ToLCNDV in Italy, as well as the first identification of a bipartite begomovirus in Italian crops.

The measurement of the intensity of light reflected by interfaces with extremely low reflectivity in water enables the label-free, multiplex detection of bio-molecular interactions. The binding of targets in solution to surface-immobilized probes (e.g. antibodies) can be quantified using a simple instrumental set-up, comprising a LED source and a camera. The condition of low reflectivity can be achieved by using a perfluorinated plastic substrate with refractive index very close to that of water. Here we show that, despite its simplicity, the method enables label-free detection in complex samples with high absorbance and turbidity. Diagnostic markers of Tomato spotted wilt virus are revealed in crude plant extracts of Datura stramonium leaves at early stage of infection. The measuring procedure requires only the addition of the sample into a cuvette and the acquisition of a series of images for a few minutes. The light reflectivity of the spots of specific antibodies increases more rapidly if the sample is prepared with infected plants.

Beet curly top Iran virus (BCTIRV) is a geminivirus with unusual genomic organisation, recently reported in Iran, infecting sugarbeet and a few other plant species. Although three BCTIRV sequences have been reported, demonstration that BCTIRV DNA is the causal agent of the disease was missing. A full-length genomic DNA was obtained from symptomatic leaves of sugarbeet collected in the Sivand area of Iran, and its nucleotide sequence was determined (BCTIRV-Siv, 2845 nt). To satisfy Koch's postulates, an infectivity assay was developed by inserting a 1.4-mer of BCTIRV-Siv DNA in Agrobacterium tumefaciens and using it in agroinoculation experiments. The cloned viral DNA was capable of infecting sugarbeets, reproducing the leaf curling and vein enations observed in the field. These results demonstrate that the single DNA component of BCTIRV is sufficient for infectivity. Host range studies indicated that some economically important crops can be affected, such as spinach, tomato and sweet pepper, as well as important laboratory plants including Nicotiana benthamiana, Arabidopsis thaliana and Jimson weed. Circulifer haematoceps, the dominant leafhopper species present in sugarbeet fields in Iran, was successfully used to transmit the disease. The availability of an infectious clone will facilitate extended host range studies, to determine the potential risks to other crops, as well as genetic studies on this unusual member of the family Geminiviridae.

Mixed viral infections can induce different changes in symptom development, genome accumulation and tissue tropism. These issues were investigated for two phloem-limited begomoviruses, Abutilon mosaic virus (AbMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) in Nicotiana benthamiana plants doubly infected by either the potyvirus Cowpea aphid-borne mosaic virus (CABMV) or the tombusvirus Artichoke mottled crinkle virus (AMCV). Both RNA viruses induced an increase of the amount of AbMV, led to its occasional egress from the phloem and induced symptom aggravation, while the amount and tissue tropism of TYLCSV were almost unaffected. In transgenic plants expressing the silencing suppressors of CABMV (HC-Pro) or AMCV (P19), AbMV was supported to a much lesser extent than in the mixed infections, with the effect of CABMV HC-Pro being superior to that of AMCV P19. Neither of the silencing suppressors influenced TYLCSV accumulation. These results demonstrate that begomoviruses differentially respond to the invasion of other viruses and to silencing suppression.

Tomato hybrids obtained from homozygous progeny of line 30-4, engineered for Tomato spotted wilt virus (TSWV) resistance, were tested under field conditions in two locations with their corresponding nontransgenic hybrids. No transgenic hybrid became infected, but 33 to 50% of plants of each nontransgenic hybrid became infected with a severe reduction of marketable fruit production. The transgenic hybrids conformed to the standard agronomic characteristics of the corresponding nontransgenic ones. Fruit were collected from the nontransgenic plots included in the experimental field and from border rows, and seed were used to estimate the flow of the transgene via pollen. No transgene flow was detected in the protected crops; however, in the open field experiment, 0.32% of tomato seedlings were found to contain the genetic modification. Immunity to TSWV infection in 30-4 hybrids was confirmed in laboratory conditions using mechanical inoculation and grafting. Thrips inoculation in leaf discs of line 30-4 demonstrated that TSWV replication was inhibited at the primary infection site but not in leaf discs of a commercial hybrid containing the naturally occurring resistance gene Sw-5. Due to the high economic value of tomato crops worldwide and the importance of TSWV, the engineered resistance described here is of practical value for breeding into cultivars of commercial interest, because it could be combined with naturally occurring resistance, thus greatly reducing the ability of the virus to develop resistance-breaking strains.

The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had ≥50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of γ-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic γ-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.

The sequence of the single-stranded RNA genome of Indian citrus ringspot virus (ICRSV) consists of 7560 nucleotides. It contains six open reading frames (ORFs) which encode putative proteins of 187.3, 25, 12, 6.4, 34 and 23 kDa respectively. ORF1 encodes a polypeptide that contains all the elements of a replicase; ORFs 2, 3 and 4 compose a triple-gene block; ORF5 encodes the capsid protein; the function of ORF6 is unknown. Phylogenetic analysis of the complete genome and each ORF separately, and database searches indicate that ICRSV, though showing some similarities to potexviruses, is significantly different, as in the presence of ORF6, the genome and CP sizes, and particle morphology. These differences favour its inclusion in a new virus genus.

Beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV) are known as the causal agents of curly top disease in beet and several other dicotyledonous plants in Iran. These viruses are transmitted by Circulifer species, and until now, there has been no confirmed report of their seed transmission. A percentage (38.2–78.0%) of the seedlings developed from the seeds of a petunia local cultivar under insect-free conditions showed stunting, interveinal chlorosis, leaf curling, and vein swelling symptoms, and were infected by BCTV when tested by PCR. Presence of BCTV in seed extracts of petunia local cultivar was confirmed by PCR and IC-PCR, followed by sequencing. Agroinoculation of curly top free petunia plants with a BCTV infectious clone resulted in BCTV infection of plants and their developed seeds. These results show the seed infection and transmission of BCTV in a local cultivar of petunia. Similar experiments performed with BCTIV showed that this virus is also seed transmissible in the same cultivar of petunia, although with a lower rate (8.8–18.5%). Seed transmission of curly top viruses may have significant implications in the epidemiology of these viruses.

Big-vein is a widespread and damaging disease of lettuce, transmitted through soil by the chytrid fungus Olpidium brassicae, and generally supposed to be caused by Lettuce big-vein virus (LBVV; genus Varicosavirus). This virus is reported to have rigid rod-shaped particles, a divided double-stranded RNA genome, and one capsid protein of 48 kD, but has not been isolated or rigorously shown to cause the disease. We provide evidence that a totally different virus, here named Mirafiori lettuce virus (MiLV), is also very frequently associated with lettuce showing big-vein symptoms. MiLV was mechanically transmissible from lettuce to Chenopodium quinoa and to several other herbaceous test plants. The virus was partially purified, and an antiserum prepared, which did not react with LBVV particles in decoration tests. As reported for LBVV, MiLV was labile, soil-transmitted and had a single capsid protein of 48 kD, but the particles morphologically resembled those of ophioviruses, and like these, MiLV had a genome of three RNA segments approximately 8.5, 1.9 and 1.7 kb in size. MiLV preparations reacted strongly in Western blots and in ISEM with antiserum to Tulip mild mottle mosaic virus, an ophiovirus from Japan also apparently Olpidium-transmitted. They reacted weakly but clearly in Western blots with antiserum to Ranunculus white mottle virus, another ophiovirus. When lettuce seedlings were mechanically inoculated with crude or partially purified extracts from MiLV-infected test plants, many became systemically infected with MiLV and some developed big-vein symptoms. Such plants did not react in ELISA using an LBVV antiserum or an antiserum to tobacco stunt virus, and varicosavirus-like particles were never seen in them in the EM after negative staining. We conclude that MiLV is a hitherto undescribed virus assignable to the genus Ophiovirus. The cause or causes of lettuce big-vein disease and the properties of LBVV may need to be re-evaluated in light of our results.

Tomato yellow leaf curl disease is spreading in southern Europe, where it has quickly become a serious problem. In recent years, several virus isolates have been characterised. Although with some genetic variability, all isolates found in Europe belong to one of two species Tomato yellow leaf curl-Sardinia (TYLCV-Sar) or Tomato yellow leaf curl-Israel (TYLCV-Is). Several methods were tested to identify and type TYLCV isolates from field samples: (1) RFLP of a DNA fragment amplified from the coat protein gene; (2) PAGE of a fragment amplified from the C2 gene; (3) dot-blot hybridisation. All methods enabled the detection of the TYLCVs and provided good indications for attributing them to one species or the other. However, for typing purposes, the RFLP method was the most reliable, due to the easily recognisable pattern produced by the two virus species present in Europe. Dot-blot hybridisation is less expensive for identifying TYLCVs in large numbers of samples, particularly when a mixture of two probes is used. PAGE of the C2 fragment is the fastest of the methods tested.

Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a post in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes.

The arbuscular mycorrhizal (AM) symbiosis is considered a natural instrument to improve plant health and productivity since mycorrhizal plants often show higher tolerance to abiotic and biotic stresses. However, the impact of the AM symbiosis on infection by viral pathogens is still largely uncertain and little explored. In the present study, tomato plants were grown under controlled conditions and inoculated with the AM fungus Funneliformis mosseae. Once the mycorrhizal colonization had developed, plants were inoculated with the Tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus causing one of the most serious viral diseases of tomatoes in Mediterranean areas. Biological conditions consisted of control plants (C), TYLCSV-infected plants (V), mycorrhizal plants (M), and TYLCSV-infected mycorrhizal plants (MV). At the time of analysis, the level of mycorrhiza development and the expression profiles of mycorrhiza-responsive selected genes were not significantly modified by virus infection, thus indicating that the AM symbiosis was unaffected by the presence and spread of the virus. Viral symptoms were milder, and both shoot and root concentrations of viral DNA were lower in MV plants than in V plants. Overall F. mosseae colonization appears to exert a beneficial effect on tomato plants in attenuating the disease caused by TYLCSV.

Tomato line 30.4 was obtained engineering the nucleocapsid (N) gene of tomato spotted wilt virus into plant genome, and immunity to tomato spotted wilt virus infection of its self-pollinated homozygous progeny was observed. Despite the presence of a high amount of transgenic transcripts, transgenic proteins have not been detected, suggesting a mechanism of resistance mediated by RNA. In the present study, we identify post-transcriptional gene silencing as the main mechanism of resistance, which is able to spread systemically through grafting, and show that the line 30.4 resistant plants produce both 24 and 21-22 nt N-gene specific siRNA classes. The transgenic locus in chromosome 4 shows complex multiple insertions of four T-DNA copies in various orientations, all with 3′ end deletions in the terminator and part of the N gene. However, for three of them, polyadenylated transcripts are produced, due to flanking tomato genome sequences acting as alternative terminators. Interestingly, starting at the fifth generation after the transformation event, some individual plants show a tomato spotted wilt virus-susceptible phenotype. The change is associated with the disappearance of transgene-specific transcripts and siRNAs, and with hyper-methylation of the transgene, which proceeds gradually through the generations. Once it reaches a critical threshold, the shift from post-transcriptional gene silencing to transcriptional silencing of the transgene eliminates the previously well established virus resistance. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

A polyclonal rabbit antiserum was obtained to the nucleoprotein of tomato spotted wilt tospovirus expressed as a recombinant fusion protein in E. coli . In indirect plate trapping ELISA, the antiserum gave similar titres against purified TSWV nucleocapsids in native form, the fusion protein and the carrier protein. The crude antiserum was also tested by Western blotting, indirect plate trapping ELISA and immunogold electron microscopy of thin sections; purified immunoglobulins were tested by DAS-ELISA. In all cases, with both glasshouse and field material, the antibodies had good detectability and specificity. By ELISA and Western blots against other tospoviruses, impatiens necrotic spot and groundnut bud necrosis viruses did not react but there was a reaction with groundnut ringspot virus, reflecting the nucleoprotein amino acid sequence similarity. These antibodies combine specificity to the target protein and versatility with regard to all the more important serological techniques. There were no undesired reactions resulting from immunization using a complex virus purified from infected host material.