Big-vein Disease 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.

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.

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.

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.

Relationship between physical and chemical properties of soil and lettuce big-vein disease severity in Kagawa prefecture

Relationship between physical and chemical properties of soil and lettuce big-vein disease severity in Kagawa prefecture

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.

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.

PH降下型肥料の施用がレタスビッグベイン病の発生とレタスの生育に及ぼす影響

pH降下型肥料の施用がレタスビッグベイン病の発生とレタスの生育に及ぼす影響

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.

DAS-ELISA quantification of resting spores of Olpidium virulentus in roots and correlation between resting spore density in soil and severity of lettuce big-vein disease

A method to quantify resting spores of Olpidium virulentus, fungal vector of Mirafiori lettuce big-vein virus, which causes lettuce big-vein disease, in lettuce roots was developed using a DAS-ELISA with a polyclonal antibody against the resting spores and used to evaluate the relationship between the resting spore density in soil and disease severity. When lettuce seedlings were grown on soils containing a powder made from diseased roots with a known number of resting spores, symptoms developed from ca. 102 spores/g of soil or more. This simple method can be used in soil diagnosis and risk assessment of the disease.

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.

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.

Preparation and characterization of polyclonal antibody against resting spores of Olpidium virulentus, fungal vector of lettuce big-vein disease

Research on Olpidium virulentus, the vector of the serious lettuce big-vein disease, is difficult because its resting spores persist in soil and retain virus for years and the fungus is not culturable. We developed protocols to obtain purified, viable resting spores from infected roots using an enzymatic treatment and two-step density gradient centrifugation and to generate a polyclonal antibody against these spores. The antibody was species-specific in a direct immunostaining assay and in western blots produced two specific bands (ca. 30.5, 29.0 kDa) against resting spores. The detection limit for resting spores in an indirect ELISA was 200 spores/100 μL.

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.

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).

The relationship of Olpidium brassicae (Wor.) Dang. to the big-vein disease of lettuce

Summary Inoculation of lettuce with tobacco-necrosis virus isolated from big-vein plants did not cause big-vein symptoms. Olpidium brassicae was always found in roots of lettuce grown in big-vein-infected soil but not in two gardens where big vein did not occur. Olpidium resting spores were sedimented from big-vein root sap by low-speed centrifuging. Inoculation of lettuce with resuspended sediment produced big-vein symptoms while inoculation with the supernatant liquid did not. Veronica tournefortii Gmel., Stellaria media (L) Vill., and Spergula arvensis L. were found to be hosts of O. brassicae. Inoculation of lettuce with root sap of these species gave rise to big-vein symptoms. Soil treatment with captan, phygon, copper oxychloride, and thiram, all reduced incidence of big vein. These results suggest that O. brassicae is the cause of big-vein disease of lettuce.

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).