Abstract
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.
Highlights
Lettuce big-vein is a damaging disease responsible for important quality and yield losses worldwide
Semi-quantitative RTPCR was used to validate the presence of the above viruses in RNA pools; Mirafiori lettuce big-vein virus (MiLBVV); and lettuce big-vein associated virus (LBVaV) were readily detected in all three pools, while neither ranunculus white mottle virus (RWMV) nor melon necrotic spot virus (MNSV) could be confirmed in spite of the use of two primer pairs for the former
Our analysis revealed that most virus-derived small RNAs (vsRNAs) had a 5′ end uridine for all viral genomes and in all three pools analyzed with the only exception of vsRNAs from RNA4 of MiLBVV in pool 3, which mostly had a cytosine in that position (Figure 3D)
Summary
Lettuce big-vein is a damaging disease responsible for important quality and yield losses worldwide. Relatively recent data in the literature still questioned the LBVD etiology, as plants diagnosed positive for LBVaV but negative for MiLBVV by ELISA were symptomatic at least in a field experiment in Italy (Roggero et al, 2003); differential sensitivity of the detection method used for each virus may underlie this conflicting observation. Both viruses are transmitted by zoospores of a chytridiomycete fungus, Olpidium virulentus, a soil-borne obligate root parasite (Hartwright et al, 2010; Maccarone et al, 2010a). Control strategies are difficult, as O. virulentus infects a wide range of weed species which can act as reservoirs (Campbell and Fry, 1966; Navarro et al, 2005). Maccarone (2009) has detected the presence of MiLBVV and LBVaV in extracts of seeds collected from plants infected by both viruses, so seed transmission of these viruses is possible (Maccarone, 2009)
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