A rapid bioassay screen for quantifying nucleopolyhedroviruses (Baculoviridae) in the environment

A quantitative bioassay method for the detection of Helicoverpa armigera (Lepidoptera: Noctuidae) singly encapsulated nucleopolyhedroviruses (HaSNPVs) in soil is described. Calibration curves used for estimating soil virus titres in environmental samples were generated by incorporating a sterilised soil into a semi-synthetic insect diet then inoculating known concentrations of an HaSNPV into the soil-diet mixture. Calibration curves were constructed for soil diets containing varying proportions of soil: 0, 1, 5, 10 and 25% soil (w/v). Their accuracy was assessed in a series of blind tests in which the actual soil virus concentration fell within the estimated mean 95% confidence region for each of three samples. The five soil-diet incorporation rates were compared in terms of larval survivorship and growth rate. There was no significant difference in larval survivorship after 10 days (i.e. for the duration of the bioassay period). The stage structure of bioassay larvae at 10 days and pupal weight at 20 days was significantly different for individuals reared on 25% soil-diet in terms of both a slower growth rate and a lower mean pupal weight compared to individuals reared on 10, 5 and 1% soil diets. This did not, however, appear to lead to greater variability in bioassay response at the high soil rate at 10 days. The level of sensitivity of virus detection achieved using this method was extremely good with the LC10 value for mid-first instar H. armigera larvae reared on the 25% soil-diet estimated at 26 polyhedral inclusion bodies (PIBs) per gram of soil. The suitability of using this approach for quantifying Helicoverpa NPVs in Australian soils was assessed by comparing percent bioassay infection across a range of five isolates known to be present in Australia. The effect of soil pH and soil management (cultivated versus non-cultivated) on percent bioassay infection was also examined. In both cases, no significant differences were observed. Finally, percent idopathic mortality, percent NPV infection and estimates of Helicoverpa SNPV concentration in a selection of samples from the Australian environment are presented.