In this chapter, the transmission, symptoms, ecology and epidemiology of the most economically important viruses that infect lentil are discussed. Sensitive assays available for detection (immunological protein-based methods and molecular nucleic acid-based methods), and appropriate measures (breeding for resistance, and cultural, chemical and integrated control methods) for the management of these viruses are briefly described.
Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM. About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.
A Syrian chickpea isolate of Chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus, family Geminiviridae) was purified and yielded 0.6–0.8 mg of purified virus per kg of infected chickpea tissue. The purified preparations were injected into a rabbit and an antiserum of good quality was obtained and used to evaluate different serological tests for the detection of CpCDV in infected chickpea leaf tissue and extracts. CpCDV was detected in sap dilutions of 1/640 by double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) and dot‐blot ELISA, and in sap dilutions of 1/1280 by direct antigen‐coating (DAC)‐ELISA using CpCDV immunoglobulin G (IgG) at 0.5 μg/ml. The antiserum was also able to detect the capsid protein of CpCDV by Western blot using raw antiserum at a dilution of 1/2000. The CpCDV raw antiserum (third bleeding) produced had a titre of 1/320 000 when determined by tissue‐blot immunoassay (TBIA); whereas, coating ELISA plates with CpCDV IgG at a concentration of 0.004 μg/ml was enough to detect the virus by DAS‐ELISA in a sap dilution of 1/20 using an enzyme conjugate at a dilution of 1/2000.
Australian pea varieties were evaluated for virus resistance using spreader plots sown with Pea seed-borne mosaic virus (PSbMV)-infected seed and aphid inoculations with Bean leafroll virus (BLRV). Natural infections by Bean yellow mosaic virus (BYMV) and Soybean dwarf virus (SbDV) allowed also screening for these viruses. Complete PSbMV resistance was limited to a few varieties, but BYMV resistance was relatively frequent. No consistent ranking of partial PSbMV resistance or PSbMV seed transmission rates were found. Selection for partial resistance to PSbMV is therefore not a practical breeding objective, while incorporating complete resistance is relatively easy. A wide range of resistance was found for BLRV and confirmed in field and greenhouse tests against local BLRV strains in Syria, but infection levels varied over years and no complete resistance was identified. Genotypes with a high level of partial resistance to BLRV also appeared to show resistance to the closely related SbDV.
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