First Report of Lettuce chlorosis virus Infecting Bean in Spain

Abstract

In September 2011, symptoms typically associated with Bean yellow disorder virus (BYDV) such as intervenal mottling and yellowing on middle and lower leaves combined with brittleness were observed in green bean (Phaseolus vulgaris L.) produced in commercial greenhouses from Granada and Almeria provinces, Spain. The affected plants were all observed in greenhouses infested with Bemisia tabaci. However, collected samples tested negative for BYDV using a specific RT-PCR test (4). Electrophoretic double stranded (ds) RNA analysis from symptomatic plants revealed the presence of a slightly diffused high molecular weight dsRNA band of ~8.5 kb, similar to that produced by the crinivirus Lettuce cholorosis virus (LCV) (3). The dsRNA was purified and used for cDNA synthesis and PCR by uneven PCR (1) using primers derived from LCV genome sequences (GenBank FJ380118 and FJ380119). Amplified DNA fragments were cloned in pGEM-T Easy vector (Promega, Madison, WI) and sequenced. Two different sequences were obtained and the nucleotide and amino acid sequences were analysed using BLAST. Both showed the highest identity with different regions of the LCV genome. The sequence of the first product had 92% nucleotide and 98% amino acid sequence identity with the polyprotein (Orf1a) homologue from RNA1 of LCV (KC602376). The sequence from the second product (KC602375) revealed the highest nucleotide and amino acid identity with the heat shock protein 70 homologue from LCV (90% and 99%, respectively). Based on these sequences, two sets of specific primers were designed (LCVSP 3-forward 5′-AGTGACACAAGTTGGAGCCGAC-3′, LCVSP 4-low 5′-CAGTGTTTGTTGGATATCTGGGG-3′) and (LCVSP 1-forward 5′-TGTTGGAAGGTGGTGAGGTC-3′, LCVSP 2-low 5′-CAGAGACGAGTCATACGTACC-3′) and each produced amplicons of the expected size (463 and 434 nt, respectively) when used in RT-PCR from the collected field samples. Subsequent field surveys from 2012 to 2013 in commercial bean greenhouses confirmed the presence of LCV that apparently had replaced BYDV. Groups of 15 to 20 adults of B. tabaci introduced in clip cages were fed for 24 h on 12 green bean plants infected with LCV and later transferred to six seedlings of bean and six of lettuce (Lactuca sativa L.). After 2 and 4 weeks, total RNA from the lettuce and bean plants was extracted using Plant RNA Reagent (Invitrogen) and subjected to RT-PCR analysis with the LCV-SP 1-2 and LCVSP 3-4 primer sets. All six plants of bean and none of lettuce showed positive for LCV-SP and a repeat experiment revealed identical results. We also seeded and produced lettuce plants within a bean greenhouse that was naturally infected with the virus and infested with B tabaci whiteflies. Under these conditions, we observed that whiteflies migrated freely from the infected bean plants to lettuce. After 4 and 6 weeks, lettuce plants neither produced symptoms nor tested positive for LCV by RT-PCR. This result confirms the existence of a new putative strain of LCV, Lettuce chlorosis virus-SP, unable to infect lettuce plants. To date, natural infections of LCV have not been reported outside California, where the virus failed to infect P. vulgaris (2). This is also the first report of LCV in Spain that infects members of the family Leguminosae. Green bean in southeast Spain was produced in ~9,000 ha of greenhouses until the introduction of BYDV a decade ago, causing considerable economic damage. The recent finding of LCV-SP has urged the local phytosanitary inspections to include this virus in lab tests and to emphasize disease management strategies based on whitefly control. References: (1) X. Chen and R. Wu. Gene 185:195, 1997. (2) J. Duffus et al. Eur. J. Plant Pathol. 102:591, 1996. (3) N. M. Salem et al. Virology 390:45, 2009. (4) E. Segundo et al. Plant Pathol. 53:517, 2004.