Mirafiori Lettuce Big-vein Virus Research Articles

Population genetic analysis of lettuce big-vein disease viruses and their vector fungi Olpidium virulentus in Ankara province, Turkey

Lettuce big-vein disease (LBVD) is caused by either Mirafiori lettuce big-vein virus (MiLBVV) alone, or co-infected with Lettuce big-vein associated virus (LBVaV). Both viruses are transmitted by the fungal vector Olpidium spp. In the present study, population genetics of these two viruses and their fungal vector, O. virulentus, were analyzed, using the nucleotide sequences of the virus coat protein (CP) partial genes and the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of the vector fungi. The analysis was conducted along with all previously reported LBVD viruses and Olpidium spp. populations. We used conventional PCR techniques to identify the viruses in lettuce samples and their fungal vector in soil samples collected from the root zone of field-grown lettuce plants in the Ankara province of Turkey. Phylogenetic analyses showed that LBVD viruses had at least two evolutionary lineages, and Ankara LBVD isolates were more closely related to the European populations. Also, we found that there is a strong negative selection pressure in the partial CP genes of LBVD viruses isolated from Ankara, due to the effective terrestrial climate. The negative selection pressure indicates balancing selection within Turkish isolates and other populations. The population of O. virulentus isolates from Ankara and other geographic regions were branched into five sub-clades, and there was no geographical separation in these isolates. Our analysis also showed Olpidium spp. to be monophyletic, and showed speciation among the isolates.

Occurrence and partial genetic characterisation of Lettuce big‐vein associated virus and Mirafiori lettuce big‐vein virus infecting lettuce in Jordan

AbstractLettuce big‐vein disease (LBVD) is an economically damaging disease that occurs in most lettuce production areas worldwide. Several studies have associated LBVD with a complex of two viruses, Lettuce big‐vein associated virus (LBVaV) and Mirafiori lettuce big‐vein virus (MiLBVV). Both viruses are transmitted through soil by the zoospores of the chytridiomycete fungus Olpidium virulentus. During surveys carried out in two growing seasons from 2015 to 2016 in the main vegetable growing areas in Jordan (Jordan Valley, Jerash, and Madaba), 179 lettuce plants, 46 lettuce seedlings, and 56 weed plants were collected. RNA extraction was conducted from leaves of all samples. Associated viruses were identified using reverse transcription‐polymerase chain reaction (RT‐PCR) with specific primer pairs targeting part of the coat protein (CP) genes of LBVaV and MiLBVV. RT‐PCR results revealed that, out of 225 lettuce samples tested, 32 (14%) and 44 (20%) were singly infected with LBVaV and MiLBVV, respectively, and 73 (32%) samples were dually infected with LBVaV and MiLBVV. Overall, MiLBVV was detected in more lettuce samples (117) compared with LBVaV which was detected in 105 samples. Moreover, one sample of each weed species of Chenopodium murale and Sonchus oleraceus tested positive for MiLBVV and LBVaV, respectively. The partial nucleotide sequence of the CP genes of LBVaV and MiLBVV Jordanian isolates showed similarities to other isolates available in the NCBI database ranging between 93.9–100% and 88.3–100%, respectively. Nucleotide sequence comparison of the Jordanian isolates and inference from phylogenetic trees with strong support revealed well‐defined sub‐groups with little or no correlation with planting area or geographic origin. The occurrence of both viruses in lettuce plants was associated with the presence of resting spores and zoospores of Olpidium spp. in lettuce plants grown under field and growth chamber conditions. Lettuce plants inoculated with Olpidium spp. zoospores isolated from roots of symptomatic lettuce plants, developed LBVD‐symptoms. At 60 days post‐transplantation, both viruses were detected by RT‐PCR in most of the plants analysed. Biological and molecular assays together with the light microscopy observations of resting spores and zoospores of Olpidium spp., revealed the presence of LBVaV, MiLBVV, and O. virulentus in big‐vein diseased lettuce plants.

Control of lettuce big‐vein disease by application of fungicides and crop covers

One of the economically important diseases of lettuce is lettuce big‐vein disease (LBVD), which leads to severe yield losses. LBVD is associated with a complex of two viruses, Lettuce big‐vein associated virus (LBVaV) and Mirafiori lettuce big‐vein virus (MLBVV). These viruses are transmitted by motile zoospores of Olpidium spp. fungi, which persist in the soil for decades through resting spores. In greenhouse and field experiments, this study tested whether changing plant and soil temperatures together with fungicide application would have a significant effect on controlling LBVD in lettuce. Soil fumigation with metam sodium was not effective at controlling the disease, as opposed to treatment with chloropicrin and methyl bromide. Moreover, the fungicides carbendazim and fluazinam were effective in reducing the incidence of Olpidium virulentus. Nevertheless, control of the fungal vector did not seem to be sufficient to control the disease due to the transition ability of the virus under low vector abundance. Crop covers, which affect the favourable environmental conditions for the viruses by lowering soil temperature and raising air temperature, reduced the disease symptoms. Combining fungicides with crop cover had a synergistic effect on reducing disease symptoms, thus providing a sustainable solution for LBVD.

Small RNA-Seq to Characterize Viruses Responsible of Lettuce Big Vein Disease in Spain.

The emerging lettuce big-vein disease (LBVD) is causing losses in lettuce production ranging from 30 to 70% worldwide. Several studies have associated this disease with Mirafiori lettuce big-vein virus (MiLBVV) alone or in mixed infection with lettuce big-vein associated virus (LBVaV). We used Illumina small RNA sequencing (sRNA-seq) to identify viruses present in symptomatic lettuce plants from commercial fields in Southern Spain. Data analysis using the VirusDetect tool showed the consistent presence of MiLBVV and LBVaV in diseased plants. Populations of MiLBVV and LBVaV viral small RNAs (sRNAs) were characterized, showing features essentially similar to those of other viruses, with the peculiarity of an uneven asymmetric distribution of MiLBVV virus-derived small RNAs (vsRNAs) for the different polarities of genomic RNA4 vs. RNAs1 to 3. Sanger sequencing of coat protein genes was used to study MiLBVV and LBVaV phylogenetic relationships and population genetics. The Spanish MiLBVV population was composed of isolates from three well-differentiated lineages and reflected almost all of the diversity reported for the MiLBVV species, whereas the LBVaV population showed very little genetic differentiation at the regional scale but lineage differentiation at a global geographical scale. Universal primers were used to detect and quantify the accumulation of MiLBVV and LBVaV in field samples; both symptomatic and asymptomatic plants from affected fields carried equal viral loads, with LBVaV accumulating at higher levels than MiLBVV.

Acidic soil conditions suppress zoospore release from zoosporangia in Olpidium virulentus

Lettuce big-vein disease, caused by Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus, is suppressed when the pH of field soil becomes acidic. Therefore, we evaluated the effect of soil pH on the activities of Olpidium virulentus, the vector of the viruses. We found that acidic soil, pH less than 6.0, significantly reduced O. virulentus infection of the root and influenced the detection rate of zoospores released in the surrounding water. We concluded that acidic soil suppresses zoospore release from zoosporangia.

Development of a colorimetric reverse transcription loop-mediated isothermal amplification assay for the detection of Mirafiori lettuce big-vein virus.

An RT-LAMP assay was developed to detect Mirafiori lettuce big vein virus (MiLBVV) and was compared with DAS-ELISA and RT-PCR. All primers were designed on the basis of the coat protein gene of the virus. In addition, a novel immunocapture (IC) RT-LAMP assay for rapid and easy detection of MiLBVV was developed, and factors such as safety, simplicity, cost, user-friendliness and safety were compared with those of DAS-ELISA, RT-PCR and RT-LAMP assays. Compared with DAS-ELISA and RT-PCR, RT-LAMP and IC-RT-LAMP had higher sensitivity (100-fold) but similar specificity, with the advantage of a shorter assay time and no need for RNA extraction (in IC-RT-LAMP). As RT-LAMP requires only very basic instruments and the results can be obtained by visual inspection (using GeneFinder™ dye), this technique provides a simple and reliable tool for laboratory research.

Bioinformatic and mutational analysis of ophiovirus movement proteins, belonging to the 30K superfamily

Ophioviridae is a family of segmented, negative-sense, single-stranded RNA plant viruses. We showed that their cell-to-cell movement protein (MP) is an isolated member of the 30K MP superfamily with a unique structural organization. All 30K MPs share a core domain that contains a nearly-invariant signature aspartate. We examined its role in the MP of Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV). Alanine substitution of this aspartate prevented plasmodesmata accumulation of MPMiLBVV, while MPCPsV was not affected. The capacity of ophiovirus MPs to increase the plasmodesmata size exclusion limit and non-cell autonomous protein feature was abolished in both mutants. To investigate the role of the signature aspartate in cell-to-cell movement, we constructed a new movement-deficient Tobacco mosaic virus vector used for trans-complementation assays. We showed that both ophiovirus MP mutants lack the cell-to-cell movement capacity, confirming that this signature aspartate is essential for viral cell-to-cell movement.

Development of marker-free transgenic lettuce resistant to Mirafiori lettuce big-vein virus.

Lettuce big-vein disease caused by Mirafiori lettuce big-vein virus (MLBVV) is found in major lettuce production areas worldwide, but highly resistant cultivars have not yet been developed. To produce MLBVV-resistant marker-free transgenic lettuce that would have a transgene with a promoter and terminator of lettuce origin, we constructed a two T-DNA binary vector, in which the first T-DNA contained the selectable marker gene neomycin phosphotransferase II, and the second T-DNA contained the lettuce ubiquitin gene promoter and terminator and inverted repeats of the coat protein (CP) gene of MLBVV. This vector was introduced into lettuce cultivars 'Watson' and 'Fuyuhikari' by Agrobacterium tumefaciens-mediated transformation. Regenerated plants (T0 generation) that were CP gene-positive by PCR analysis were self-pollinated, and 312 T1 lines were analyzed for resistance to MLBVV. Virus-negative plants were checked for the CP gene and the marker gene, and nine lines were obtained which were marker-free and resistant to MLBVV. Southern blot analysis showed that three of the nine lines had two copies of the CP gene, whereas six lines had a single copy and were used for further analysis. Small interfering RNAs, which are indicative of RNA silencing, were detected in all six lines. MLBVV infection was inhibited in all six lines in resistance tests performed in a growth chamber and a greenhouse, resulting in a high degree of resistance to lettuce big-vein disease. Transgenic lettuce lines produced in this study could be used as resistant cultivars or parental lines for breeding.

Quantification ofMirafiori lettuce big-vein virusand its vector,Olpidium virulentus, from soil using real-time PCR

Big vein disease of lettuce (Lactuca sativa) is an economically important disease transmitted through soil by Olpidium virulentus, and has occurred in most production areas worldwide. The disease is assumed to be caused by Mirafiori lettuce big-vein virus (MiLBVV). To understand the dynamics of the virus and its vector, MiLBVV and O. virulentus were directly detected in soil. DNA and RNA were extracted from 5 g soil using a bead beating method, followed by purification using adsorption to a column. Detection and quantification were performed using real-time PCR and a TaqMan probe that was prepared based on the CP region of MiLBVV and the rDNA-ITS region of O. virulentus, respectively. Furthermore, using a visual assessment of the incidence rate of big vein disease on lettuce in agricultural fields, the Ct values of MiLBVV and O. virulentus from soil were also determined using real-time PCR. The results showed that MiLBVV concentrations in the soil were high in the field, as also determined by a visual assessment of the incidence rate of big vein disease on lettuce. However, the amount of O. virulentus in soil was not directly correlated with the incidence of MiLBVV. From these results, it is suggested that the risk of lettuce crops developing big vein disease can be estimated using an index of the amount of MiLBVV in the soil.

First Report of Lettuce big-vein associated virus (Varicosavirus) Infecting Lettuce in Mexico

Lettuce (Lactuca sativa) is a common consumed vegetable and a major source of income and nutrition for small farmers in Mexico. This crop is infected with at least nine viruses: Mirafiori lettuce big-vein virus (MiLBVV), Lettuce big-vein associated virus (LBVaV), both transmitted by the soil-borne fungus Olpidium brassicae; Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV), Lettuce mottle virus (LMoV), Cucumber mosaic virus (CMV), Bidens mosaic virus (BiMV), and Lettuce mosaic virus (LMV) (1). From March to May 2012, a disease on lettuce was observed in the south region of Mexico City displaying mild to severe mosaic, leaf deformation, reduced growth, slight thickening of the main vein, and plant death. At the beginning of the epidemic there were just a few plants with visible symptoms and 7 days later the entire crop was affected, causing a loss of 93% of the plants. It was estimated by counting the number of severely affected or dead plants in three plots. No thrips, aphids, or whiteflies were observed in the crop during this time. Twenty plants with similar symptoms were collected and tested by RT-PCR using the primers LBVaVF 5'-AACACTATGGGCATCCACAT-3' and LBVaVR 5'-GCATGTCAGCAATCAGAGGA-3' specific for the coat protein gene of LBVaV, amplifying a 322-bp fragment. Primers CP829F 5'-CCWACTTCATCAGTTGAGCGCTG-3' and CP1418R 5'-TATCAGCTCCCTACACTATCCTCGC-3' were used to detect MiLBVV (2). No amplification was obtained for MiLBVaV in any plants tested. PCR products of approximately 300 bp were obtained from four out of 20 symptomatic lettuce samples tested for LBVaV, but not from healthy plant and water controls. These results suggest the presence of another virus in symptomatic lettuce plants. Amplicons were gel-purified and sequenced using LBVaVF and LBVaVR primers. A consensus sequence was generated using the Bioedit v. 5 program. Both sequences of these Mexican lettuce isolates were 100% identical (Accession Nos. KC776266.1 and KC776267.1) and had identities between 94 and 99% to all sequences of LBVaV available in GenBank. Additionally, when alignments were made using ClustalW, these sequences showed identities of 99.7% to Almeria-Spanish isolate (Accession No. AY581686.1); 99.4% to Granada-Spanish isolate (AY581689.1); 99.1% to Dutch isolate (JN710441.1), Iranian isolate (JN400921.1), Australian isolate (GU220725.1), Brazilian isolate (DQ530354.1), England isolate (AY581690.1), and American isolate (AY496053.1); 96.2% to Australian isolate (GU220722.1); 96.3% to Japanese isolate (AB190527.1); and 92.8% to Murcia-Spanish isolate (AY581691.1). Twenty lettuce plants were mechanically inoculated with leaf tissue taken from the four plants collected in the field and tested positive for LBVaV by RT-PCR; 12 days after inoculation, mosaic symptoms were observed in all inoculated plants and six of them were analyzed individually by RT-PCR obtaining a fragment of the expected size. To our knowledge, this is the first report of LBVaV infecting lettuce in Mexico. Further surveys and monitoring of LBVaV incidence and distribution in the region, vector competence of olpidium species, and impact on the crop quality are in progress.

Relationships Between the Pathogen Olpidium virulentus and Viruses Associated with Lettuce Big-Vein Disease.

Lettuce big-vein disease (LBVD) is a widespread problem in commercial lettuce producing areas. It was first reported in 1934 in California. In the United States in 2009, it was estimated that 100% of all commercial lettuce production was concentrated in California (in early spring) and Arizona (in winter). The disease also occurs worldwide, particularly in regions in Australia, Europe, Brazil, and New Zealand, and at high altitudes in subtropical regions. LBVD often results in decreased yield from infected plants, mainly in winter growing seasons. Symptoms of this disease are the obvious chlorophyll clearing, which causes the big-vein leaf symptoms from which the disease gets its name, along with crinkled leaves and a reduced head size, which is often oblong in shape. LBVD is associated with a complex of two viruses, Lettuce big-vein associated virus (LBVaV) and Mirafiori lettuce big-vein virus (MLBVV). Motile zoospores of Olpidium virulentus vector LBVaV and MLBVV to the roots of healthy plants and, in the absence of susceptible hosts, its resting spores retain the ability to harbor these viruses for decades in infested soil and in dry roots for 39 months. The ability of viruliferous resting spores to persist in production areas for such extended periods of time, and also the motility of viruliferous zoospores, pose significant difficulties for the management and/or control of the disease.

Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein

Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV), members of the Ophioviridae family, have segmented negative-sense single-stranded RNA genomes. To date no reports have described how ophioviruses spread within host plants and/or the proteins involved in this process. Here we show that the 54K protein of CPsV is encoded by RNA 2 and describe its subcellular distribution. Upon transient expression in Nicotiana benthamiana epidermal cells the 54K protein, and also its 54K counterpart protein of MiLBVV, localize to plasmodesmata and enhance GFP cell-to-cell diffusion between cells. Both proteins, but not the coat proteins (CP) of the respective viruses, functionally trans-complement cell-to-cell movement-defective Potato virus X (PVX) and Tobacco mosaic virus (TMV) mutants. The 54K and 54K proteins interact with the virus-specific CP in the cytoplasm, suggesting a potential role of CP in ophiovirus movement. This is the first study characterizing the movement proteins (MP) of ophioviruses.

Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus.

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

Occurrence and Partial Characterization of Lettuce big vein associated virus and Mirafiori lettuce big vein virus in Lettuce in Iran.

Mirafiori lettuce big vein virus (MiLBVV) and lettuce big vein associated virus (LBVaV) were found in association with big vein disease of lettuce in Iran. Analysis of part of the coat protein (CP) gene of Iranian isolates of LBVaV showed 97.1-100% nucleotide sequence identity with other LBVaV isolates. Iranian isolates of MiLBVV belonged to subgroup A and showed 88.6-98.8% nucleotide sequence identity with other isolates of this virus when amplified by PCR primer pair MiLV VP. The occurrence of both viruses in lettuce crop was associated with the presence of resting spores and zoosporangia of the fungus Olpidium brassicae in lettuce roots under field and greenhouse conditions. Two months after sowing lettuce seed in soil collected from a lettuce field with big vein affected plants, all seedlings were positive for LBVaV and MiLBVV, indicating soil transmission of both viruses.

Citrus psorosis and Mirafiori lettuce big-vein ophiovirus coat proteins localize to the cytoplasm and self interact in vivo

Citrus psorosis (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV) belong to the family Ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. To gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (CP) was studied. Immunoblot analysis of fractionated extracts derived from natural and experimental infected hosts indicated that the CP of CPsV occurs in the soluble cytoplasmic fraction. The cytoplasmic localization of this protein was confirmed by confocal microscopy of fluorescent protein (FP)-tagged CP following its expression in either CPsV-infected and healthy Citrus sinensis plants or in Nicotiana benthamiana plants. The same localization was observed for FP-tagged CP of MiLBVV. The CPs of CPsV and MiLBBV can undergo homologous and heterologous interactions as revealed by fluorescent lifetime imaging microscopy and co-immunoprecipitation analysis. A putative leucine zipper motif that is conserved among ophiovirus CP sequences may account for these interactions.

Symptom severity and viral protein or RNA accumulation in lettuce affected by big-vein disease

Big-vein disease (BVD) is a widespread and economically damaging disease in lettuce (Lactuca sativa L.). Typical symptoms are chlorotic clearing around leaf veins, leaf deformations, and impaired head development. In this research, we studied the relationship between symptom intensity and protein and viral RNA accumulation in infected plants. Naturally infected lettuce plants, from the field or greenhouse, were classified according to their symptomatology: mild, moderate, severe, and symptomless. Coat protein accumulation was evaluated by a double antibody sandwich/enzyme-linked immunosorbent assay (DAS-ELISA), and RNA levels were studied by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Virus coat protein accumulation did not differ for the two viruses associated with this disease among lettuce plants showing different symptom severity. Similarly, abundance of Mirafiori lettuce big-vein virus (MLBVV)-RNA3 or Lettuce big-vein associated virus (LBVaV)-RNA-2 were not different (P > 0.05) for diverse big vein disease severity rating scales. This suggests that symptom severity expressed by big-vein diseased lettuce plants did not necessarily reflect the accumulation of viruses associated with this disease in the host. Therefore, lettuce plants showing mild symptoms of BVD do not necessarily present lower virus levels than plants showing more severe symptomatology.

Identification of Mirafiori lettuce big‐vein virus and Lettuce big‐vein associated virus infecting Lactuca sativa with symptoms of lettuce big‐vein disease in Argentina

Lettuce big-vein disease (BVD) affects all major lettuce-producing areas of the world. The causal agent is Mirafiori lettuce big-vein virus (MLBVV), an ophiovirus transmitted by the soil-borne fungus Olpidium brassicae (Lot et al., 2002). MLBVV has been detected in many different areas of the world but never in Argentina. La Plata has about 700 ha of lettuce with a production of about 13 000 tonnes, and with about 70% of the total production from Buenos Aires Province. BVD has been detected in different areas in the north and west of the La Plata horticultural green belt. Many of the plants with BVD symptoms had leaf distortions of moderate severity, which affected their commercial value. Over the winters of 2007, 2008 and 2009, forty samples of lettuce with BVD symptoms, representing most of the commercial varieties, were taken from fields near La Plata. Samples tested positive by RT-PCR with universal primers for the genus Ophiovirus (Vaira et al., 2003) and/or by DAS-ELISA using a polyclonal antiserum against the viral coat protein of MLBVV (kindly provided by Dr Y. Kawazu, National Institute of Vegetable and Tea Science, Japan). The identity of MLBVV was verified by cloning and sequencing amplicons (GenBank Accession Nos. FJ555204-05), which shared 97% identity with both Italian (AY2046741) and Brazilian (DQ8548131) MLBVV isolates. Specific primers, Cps1 (5′-CTCATGACAAAAGAAGAGAAGC) and Cpas1 (5′-CACATCAAATATGAAGTTGTGCTC) were designed from MLBVV-RNA 3, to allow specific MLBVV detection in RNA extracts from collected samples. The primers were tested using samples with symptoms that were positive by RT-PCR and DAS-ELISA. An amplicon of the expected 450 bp was amplified from all infected samples. Sequence analysis (GU295451) demonstrated high identity (98%) with sequences deposited in the public databases. A block sampling method was employed to collect random field samples and estimate the incidence of the disease. These were scored for symptoms and tested for infection by RT-PCR (primers Cps1/Cpas1). In the three lots tested, the incidence of the disease reached 60% of plants inoculated. Additionally, soil transmission experiments were conducted using healthy lettuce seedlings planted into contaminated soil or by watering with rinse water from the roots of diseased field plants. After 5 weeks in a greenhouse up to 70% of the 15 seedlings inoculated with five field isolates showed big-vein symptoms and tested positive by DAS-ELISA and RT-PCR. Three of the plants used for transmission were also positive for Lettuce big-vein associated virus (LBVaV), detected by RT-PCR using specific primers (VP248 and VP249; Navarro et al., 2004). This is the first report confirming the presence of MLBVV and LBVaV infecting lettuce plants in Argentina. The authors would like to thank Dr. L.W. Timmer for helpful discussion and manuscript revision.

Identification of Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus Associated with Lettuce Big-Vein Disease in Slovenia.

Lettuce big-vein disease (LBVD) is an important disease of lettuce (Lactuca sativa L.) worldwide. Two viruses are reported to be associated with the disease, Lettuce big-vein associated virus (LBVaV) and Mirafiori lettuce big-vein virus (MLBVV). The majority of publications shows that MLBVV is the causal agent of LBVD, but two plants with big-vein symptoms and infected only with LBVaV were found by Roggero et al. (2). Lettuce plants with big-vein symptoms were observed in a greenhouse in Ljubljana and in a private garden near Sežana. Chlorotic vein banding with deformations of leaves were observed and the plants were generally smaller than those without symptoms. The seedlings at the first location were grown in sterilized soil and showed no such symptoms. Six symptomatic plants and two seedlings without symptoms from the first location and three symptomatic plants from the second location were analyzed for the presence of LBVaV and MLBVV. Total RNA was isolated from 50 mg of lettuce leaf tissue with a RNeasy plant mini kit (Qiagen, Chatsworth, CA). Multiplex reverse transcription-PCR with primers VP248 (5'-CGCCAGGATCTTTGATCCATCTG-3') and VP249 (5'-TTGCGACATGTTCCTCCTCATCG-3') for LBVaV, VP286 (5'-TATCAGCTCACATACTCCCTATCG-3') and VP287 (5'-CAACTAGCTCAGAATACATGCAG-3') for MLBVV, and VP383 and VP389 for internal control (1), was performed. Mixed infections with MLBVV and LBVaV were detected in four plants and only MLBVV was detected in two plants from the first location. None of the viruses was detected in the two seedlings without symptoms. Mixed infections with MLBVV and LBVaV were detected in all analyzed plants from the second location. According to these results, only MLBVV could be correlated with observation of disease symptoms. To our knowledge, this is the first reported association of MLBVV and LBVaV with big-vein disease in Slovenia. MLBVV was the only virus found in all samples with big-vein symptoms.

Genetic variation for big-vein symptom expression and resistance to Mirafiori lettuce big-vein virus in Lactuca virosa L., a wild relative of cultivated lettuce

Lactuca virosa L. is a wild relative of lettuce that is potentially an important source of resistance to big-vein disease, an economically damaging disease of lettuce. Identification of L. virosa accessions with resistance to Mirafiori lettuce big-vein virus (MLBVV), the disease causing agent, may be useful for lettuce breeding. The objectives of this research were to determine the genetic variation for big-vein symptom expression and MLBVV accumulation in diverse L. virosa accessions. Greenhouse testing was conducted to characterize variation for symptom expression 90–100 days after planting (DAP) with 70 L. virosa accessions in unreplicated experiments in 2001 and 2003, and with 10 accessions in an experiment with 3 replications conducted in 2004. In 2005, six replications of seven accessions were evaluated for the percentage of symptomatic plants 120 DAP and 180 DAP in a growth chamber experiment. Reverse transcription-polymerase chain reaction or nucleic acid spot hybridization was used to determine MLBVV presence or absence at each reading date. Genetic variation for symptom expression was confirmed among the L. virosa accessions, although the majority of tested accessions did not express big-vein symptoms. Symptomless infections were discovered, although accumulation of MLBVV to detectable levels appeared to be a slow process in L. virosa. Genetic variation for the incidence of MLBVV positive plants was identified within symptomless accessions, and suggests that symptom expression and MLBVV resistance may be independent factors contributing to big-vein resistance. Regardless, symptomless accessions with low MLBVV incidence were identified, and should be useful for breeding new big-vein resistant cultivars.

Sequence diversity in the coat protein gene of Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus infecting lettuce in Brazil

Lettuce big vein associated virus (LBVaV) and Mirafiori lettuce big vein virus (MLBVV) have been found in mixed infection in Brazil causing the lettuce big vein disease. Analysis of part of the coat protein (CP) gene of Brazilian isolates of LBVaV collected from lettuce, showed at least 93% amino acid sequence identity with other LBVaV isolates. Genetic diversity among MLBVV CP sequences was higher when compared to LBVaV CP sequences, with amino acid sequence identity ranging between 91% to 100%. Brazilian isolates of MLBVV belong to subgroup A, with one RsaI restriction site on the coat protein gene. There is no indication for a possible geografical origin for the Brazilian isolates of LBVaV and MLBVV.