Lettuce Big-vein Research Articles

Genome sequence of a novel phlebovirus associated with lettuce big vein disease infecting lettuce (Lactuca spp.).

Lettuce big vein disease is a disease complex involving at least two RNA viruses, both transmitted by the soilborne fungus Olpidium virulentus. Here, we present the genomic sequence of a novel unrelated third negative-stranded RNA virus, belonging to the family Phenuiviridae, recovered from infected lettuce plants.

Konya İli Marul Ekim Alanlarında Sorun Olan Virüs Hastalıklarının Belirlenmesi

Lettuce (Lactuca sativa L.) is a member of the family Compositae (Asteraceae). This plant, which is grown for its leaves, is grown in open field and greenhouse conditions almost everywhere in the world and in Turkey. With the present study virus diseases of lettuce and their prevalence in Konya province was revealed for the first time. For this purpose, leaf samples were taken from lettuce plants showing virus diseases symptoms with surveys carried out in Konya province lettuce planting areas from May to August in 2020. Then the collected lettuce leaf samples were tested in laboratory conditions by Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA) method to reveal infections of Cucumber mosaic virus (CMV), Miraflori lettuce big vein virus (MiLBVV), Lettuce mosaic virus (LMV), and Tomato spotted wilt virus (TSWV). According to the information obtained from the results of the study, it was determined that 40 out of 97 (41.23%) lettuce plant samples and all (6) weed samples were infected with at least one of the viruses. In lettuce leaf samples; TSWV (27.83%), LMV (12.37%), CMV (10.31%) and MiLBVV (5.15%) infections have been detected. In weed samples; infections of CMV (83.33%), LMV (66.66%), and TSWV (50%) have been revealed. The infections of TSWV, LMV, CMV, and MiLBVV on lettuce plants in Konya province were reported firstly with the study.

Characterization of an isolate of Lettuce big-vein associated virus (LBVaV) detected in naturally infected tomato (Solanum lycopersicum L.) in Slovakia

A tomato plant (Solanum lycopersicum Linnaeus, labelled KVE) displaying virus-like symptoms, tested negative for common tomato viruses, was subjected to high-throughput sequencing (HTS) on the Illumina MiSeq platform using ribosomal RNA-depleted total RNA as a template. The analysis has revealed the contigs mapping to Lettuce big-vein associated virus (LBVaV). The near complete LBVaV-KVE sequence of RNA1 and RNA2 revealed 95.0 and 94.9% identity with the reference sequence, the same length of translated products and a typical varicosavirus genome organisation. After initial long-term maintenance of LBVaV-KVE in the original plant, the virus could be detected by RT-PCR or nanoLC-ESI-Q-TOF in new plants generated from lateral shoot cuttings or inoculated by stem chips, although not uniformly. So far, LBVaV was reported to infect lettuce and related species. Our study expands the natural host range of the LBVaV to tomato.

Complete genome sequence of a novel varicosavirus infecting tall morning glory (Ipomoea purpurea).

A novel varicosa-like virus was identified in a tall morning glory (Ipomoea purpurea) plant by high-throughput sequencing and tentatively named "morning glory varicosavirus" (MGVV). The complete genome of MGVV contains two segments of negative-sense single-stranded RNA of 6409 (RNA1) and 5288 (RNA2) nucleotides. RNA1 encodes a 224.3-kDa large protein (224K), and RNA2 encodes four putative proteins of 48.6 kDa (49K), 46.4 kDa (46K), 35.7 kDa (36K), and 36.8 kDa (37K), respectively. The 224K and 49K proteins show amino acid sequence similarity to the large protein (39.4%) and the 49K protein (22.6%), respectively, of red clover-associated varicosavirus, and the 36K protein shares 19.6% amino acid sequence similarity with protein 3 of lettuce big-vein associated virus. The 46K and 37K proteins share no significant sequence similarity to known functional viral sequences. Phylogenetic analysis based on the large protein of MGVV and other rhabdoviruses showed that MGVV clustered with the varicosaviruses. These analyses indicate that MGVV is a novel member of the genus Varicosavirus in the family Rhabdoviridae.

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples.

Serological methods serve as a direct or indirect means of pathogen infection diagnosis in plant and animal species, including humans. Dot-ELISA (DE) is an inexpensive and sensitive, solid-state version of the microplate enzyme-linked immunosorbent assay, with a broad range of applications in epidemiology. Yet, its applicability is limited by uncertainties in the qualitative output of the assay due to overlapping dot colorations of positive and negative samples, stemming mainly from the inherent color discrimination thresholds of the human eye. Here, we report a novel approach for unambiguous DE output evaluation by applying machine learning-based pattern recognition of image pixels of the blot using an impartial predictive model rather than human judgment. Supervised machine learning was used to train a classifier algorithm through a built multivariate logistic regression model based on the RGB (“Red,” “Green,” “Blue”) pixel attributes of a scanned DE output of samples of known infection status to a model pathogen (Lettuce big-vein associated virus). Based on the trained and cross-validated algorithm, pixel probabilities of unknown samples could be predicted in scanned DE output images, which would then be reconstituted by pixels having probabilities above a cutoff. The cutoff may be selected at will to yield desirable false positive and false negative rates depending on the question at hand, thus allowing for proper dot classification of positive and negative samples and, hence, accurate diagnosis. Potential improvements and diagnostic applications of the proposed versatile method that translates unique pathogen antigens to the universal basic color language are discussed.

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.

First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece

HomePlant DiseaseVol. 104, No. 10First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Impatiens Necrotic Spot Virus Infecting Lettuce in GreeceD. Beris, I. Malandraki, O. Kektsidou, N. Vassilakos, and C. VarveriD. Beris†Corresponding author: D. Beris; E-mail Address: d.mperi@bpi.grhttp://orcid.org/0000-0003-3308-8742Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, I. Malandrakihttp://orcid.org/0000-0001-5623-5261Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, O. KektsidouLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, N. VassilakosLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, and C. VarveriLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this authorAffiliationsAuthors and Affiliations D. Beris † I. Malandraki O. Kektsidou N. Vassilakos C. Varveri Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, Greece Published Online:18 Aug 2020https://doi.org/10.1094/PDIS-11-19-2316-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat During the autumn of 2018 and the winter of 2019, virus-like disease symptoms were observed in four fields of lettuce (Lactuca sativa) in Kalamos, Marathonas, and Leonidio regions of central and south-eastern Greece. In all cases, the disease affected 30 to 40% of the plants. Symptoms consisted of brown ringspots, necrotic lesions, vein necrosis, and leaf distortion resembling those associated with tospovirus infections. Six symptomatic plants (four in 2018 and two in 2019) were collected from four different fields and were tested for the presence of tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), impatiens necrotic spot virus (INSV), groundnut ringspot virus (GRSV), and chrysanthemum stem necrosis virus (CSNV) with double antibody sandwich ELISA specific and generic polyclonal antisera, respectively (Loewe Biochemica, Germany). All samples were found negative for TSWV but positive for other Tospovirus infection with the generic ELISA test. Total RNA was purified from all six symptomatic samples and reverse transcription polymerase chain reaction (RT-PCR) was performed with the gM410/gM870c primer pair (Chen et al. 2012) targeting the conserved NSm genomic region of tospoviruses. Direct Sanger sequencing of the 500-bp PCR products obtained from all six samples revealed the presence of INSV Tospovirus, and showed 97.5 to 98.7% identity at nucleotide level with the respective region of the Chinese isolate HDL (GU112503). To further characterize our INSV isolate, total RNA isolated with NucleoZOL reagent (Macherey-Nagel, Germany) from one symptomatic lettuce was subjected to high-throughput sequencing (HTS) on a Next Seq platform (Illumina, U.S.A.) by using the TruSeq Stranded mRNA Library Prep kit (Illumina) for the construction of the poly-A selected HTS library. The analysis generated approximately 14,000,000 single-end reads of 75-bp length that were analyzed with Geneious bioinformatics software (Biomatters, New Zealand). After the de novo assembly of the unique reads (7,448,124 reads), contigs (in total 108,234 contigs ranging in size from 150 to 12,000 bp) were annotated with BLAST, and the presence of INSV was confirmed. Assembled nucleotide sequences of S (MN553562) and M (MN553561) segments exhibited 88 to 98% coverage and 99% identity at nucleotide level with an Italian isolate (DQ425096 and DQ425095, respectively). Nucleotide sequence of the L segment also showed 64% coverage with 88% identity with the Italian isolate (DQ425094). Furthermore, HTS analysis revealed the presence of lettuce big-vein associated virus (LBVaV, Rhabdoviridae, Varicosavirus genus). To isolate INSV, Nicotiana tabacum, an insusceptible host of LBVaV, was used. Infected N. tabacum tissues were used to reproduce symptoms in virus-tested-negative lettuce plants after mechanical inoculation. In the latter plants, INSV presence was confirmed with RT-PCR assays targeting specific regions of the three INSV segments by respective primer pairs designed based on the HTS analysis. Sequences of the three PCR products were 100% identical with the HTS-assembled INSV segments of the respective regions and confirmed the presence of all virus segments. INSV was first described in Impatiens spp. plants (Law and Moyer 1990) and is an important pathogen for several crops including ornamental plants, tomato, and lettuce (Kuo et al. 2014). The relatively high incidence of the virus in different regions of Greece suggests that INSV is an emerging pest for lettuce cultivation in Greece. To the best of our knowledge, this is the first report of INSV in Greece.The author(s) declare no conflict of interest.

Identification and characterization of lettuce big vein disease (LBVD) in lettuce (Lactuca sativa) crops in Adana and Mersin provinces in Turkey

This study was carried out for the purpose of determination of Lettuce big vein disease (LBVD), identification of the virus or virus complex (MiLBVV and/or LBVaV) causing disease and characterization of causal agent in the fields where the lettuce cultivation was made extensively in Adana and Mersin provinces during autumn and winter months between 2015 and 2017. A total of 160 samples were collected from lettuce (Lactuca sativa L.) plants suspected to be infected with LBVD simptomatologically in surveys. In the result of the ELISA tests, 52 samples were found to be infected with MiLBVV. RT-PCR studies were performed to identify the causative agents of LBVD by using specific primer pairs for MiLBVV (MiLBV-F; MiLBVV-R) and LBVaV (VP-248; VP-249) showed that LBVD is caused by MiLBVV or mixed infection of MiLBVV+LBVaV. LBVaV infection was not detected alone in lettuce plants with LBVD symptoms. Sequence analyses showed that Adana and Mersin isolates of MiLBVV formed in separate groups on phylogenetic tree. While Yakapınar (2-MiLBVV 1 E10), Yumurtalık (6-MiLBVV 1 G10) and Yüreğir (4-MiLBVV 1 F10) isolates from Adana province clustered with Argentina and Iran isolates, Yenice 1 (11-MiLBVV 2 H10) and Yenice 2 (12-MiLBVV 2 E11) isolates from Mersin province were in the same group with Netherlands and Egypt isolates in another group. In addition, the Yüreğir isolate of LBVaV (4-VP 248 A12) clustered with isolates from Saudi Arabia, United States, United Kingdom, Australia and Netherlands.

First report of lettuce big-vein associated varicosavirus infecting lettuce in China

First report of lettuce big-vein associated varicosavirus infecting lettuce in China

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.

Characterization of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus infecting lettuce in Saudi Arabia.

During 2014 and 2015, 97 lettuce plants that showed big-vein-disease-like symptoms and seven weed plants were collected from the Riyadh region. DAS-ELISA revealed that 25% and 9% of the lettuce plants were singly infected with LBVaV and MiLBVV, respectively, whereas 63% had a mixed infection with both viruses. The results were confirmed by multiplex reverse transcription polymerase chain reaction using primers specific for LBVaV and MiLBVV. LBVaV and MiLBVV were also detected in Sonchus oleraceus and Eruca sativa, respectively. The nucleotide sequence of LBVaV and MiLBVV Saudi isolates ranged from 94.3-100%, and their similarities to isolates with sequences in the GenBank database ranged from 93.9 to 99.6% and 93.8 to 99.3%, respectively. Olpidium sp. was present in the roots of lettuce plants with big-vein disease and it was shown to facilitate transmission of both viruses.

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.

Distribution of Lettuce Big-Vein Incidence Under Three Irrigation Systems.

Spatial patterns of lettuce big-vein (LBV) incidence under furrow, sprinkler, and subsurface drip irrigation systems were determined. Because LBV pathogen is a virus and is vectored by the soilborne chytrid Olpidium brassicae, different irrigation systems likely affect the movement of the vector and were hypothesized to result in different distribution patterns and levels of the disease. Lettuce plants were mapped by recording the location of each LBV-infected or healthy plant in arbitrarily selected plots of sizes 16 by 30, 20 by 30, and 18 by 50 m in Salinas, Gonzales, and Santa Maria in California. Data were arrayed into different quadrat sizes by rearrangement, and disease incidence was calculated for each quadrat. Frequency distribution analysis and spatial autocorrelation analyses were performed on this data. LBV incidence was aggregated in all furrow-irrigated fields, four of five subsurface drip-irrigated fields, and two of three sprinkler-irrigated fields. The remaining fields had a random distribution. As the quadrat size increased, index of aggregation decreased, and vice versa. In fields under sprinkler irrigation, regardless of whether the spatial pattern of LBV was random or aggregated, no directional orientation occurred. However, under furrow or subsurface drip irrigation, the aggregation mostly occurred across the rows. Although irrigation type influenced LBV distribution pattern and incidence in lettuce fields, the differential effects of irrigation type on vector O. brassicae could not be discerned in this study. The sprinkler irrigation practiced in lettuce production until thinning may influence the vector distribution and the subsequent irrigation methods adapted for the remainder of the season in individual fields may play a significant role in disease incidence.

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.

Control of Lettuce Big-vein Disease by Combined Use of Resistant Cultivars and Fungicides

Control of Lettuce Big-vein Disease by Combined Use of Resistant Cultivars and Fungicides

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 and Partial Characterization of Viral Agent of Lettuce Big Vein in Tehran Province

Background and Aims: The causal agents of viral lettuce big vein disease are two viruse, Lettuce big vein associated virus (Varicosavirus) and Mirafiori lettuce virus (Ophiovirus). These viruses have coat proteins of similar size but have different morphologies and serologically unrelated.The purpose of this study was to distinguish and detect LBVaV and MiLV in lettuce fields in Tehran Province. Patients and Methods: A total 344 samples with mosaic and big vein, head stunt, leaf deformation and motteling symptoms were collected from lettuce fields in Tehran Province.Using DAS – ELISA and specific antiserum for MiLV (DSMZ, AS-0798) and RTPCR for LBVaV. Positive samples in ELISA and RT-PCR were inoculated on index plants,including Chenopodium quinoa, Chenopodium amaranticolor, Lactuca sativa and Nicotiana occidentalis p1. Results: The results of ELISA and RT-PCR about MiLV showed that, virus is transmitted on C.quinoa and produced chlorotic local lesion but about LBVaV, RT-PCR showed that C.quinoa and C.amaranticolor were infected and the virus caused chlorotic local lesion. Extraction of total RNA with three methods using RNAWIZ buffer, Guanidium isothiosianat buffer and Qiagen kit showed that exteraction with RNeasy plant minikit (Qiagen company) is better for RT-PCR. RT-PCR with LBVaV and MiLV specific primer pairs (were designed with Navaro et al. 2004) were performed and the fragment length were amplified for LBVaV and MiLV respectively 296bp and 469bp. The sequence nucleotides of CP of LBVaV was determined and had high similarity with other isolates in gene bank. Conclusion: This is the first report of occurrence of these viral diseases in lettuce in Iran (Tehran Province).