Increased sensitivity of detection of plant viruses obtained by using a fluorogenic substrate in enzyme‐linked immunosorbent assay

The fluorogenic substrate 4‐methylumbelliferyl phosphate (MUP) of alkaline phosphatase was compared with the chromogenic substrate p‐nitrophenyl phosphate (NPP) in tests for plant viruses by enzyme‐linked immunosorbent assay (ELISA). In tests on leaf extracts of squash infected with prune dwarf virus, Chenopodium quinoa and apple infected with apple mosaic virus (ApMV), and potato infected with potato leafroll virus (PLRV), MUP increased sensitivity 2–16 times, the smallest and greatest increases being obtained with ApMV (in apple) and PLRV respectively. In similar tests on 21 dormant PLRV‐infected potato tubers, sensitivity was increased 2–4 times with 13 tubers, but the two substrates gave the same detection end‐points with eight tubers. When individual seeds of potato plants infected with the Andean potato calico strain of tobacco ringspot virus were tested, the virus was detected in virtually all seeds by MUP‐ELISA, but detection by NPP‐ELISA was inefficient unless absorbance values were measured after overnight incubation at 4 °C, instead of after 2 h at room temperature. In tests on Myzus persicae carrying PLRV and Sitobion avenae carrying barley yellow dwarf virus (BYDV), both viruses were consistently detected in a greater proportion of individual aphids by MUP‐ELISA than NPP‐ELISA irrespective of whether incubation was for 2 h at room temperature or overnight at 4 °C. The effeciency of detection of virus in single viruliferous aphids by MUP‐ELISA was not decreased by grouping with one or four non‐viruliferous aphids but was decreased (PLRV) or greatly decreased (BYDV) by grouping with nine. MUP‐ELISA and transmission tests to Physalis floridana seedlings (2–3 day inoculation access periods) both detected PLRV in most individual M. persicae, but the results obtained with the two methods did not correlate completely. In similar tests for BYDV in individual S. avenae, virtually all aphids transmitted BYDV to oat seedlings during a 3‐day inoculation access period but it was subsequently detected by MUP‐ELISA in less than half of them. By contrast, MUP‐ELISA detected PLRV in most viruliferous M. persicae even after they had fed for 3 days on Chinese cabbage, a non‐host for this virus.