Development, application and evaluation of three novel TaqMan qPCR assays for phosphine resistance monitoring in major stored product pests Tribolium castaneum and Rhyzopertha dominica.

Abstract

Stored product protection from insect pests relies heavily on the use of phosphine. The most serious drawback of phosphine is the development of resistance in major stored product insects worldwide, including the red flour beetle, Tribolium castaneum (Herbst) and the lesser grain borer, Rhyzopertha dominica (F.). Two genetic loci are responsible for phosphine resistance: the rph1 (S349G mutation in the cyt-b5-r homolog) in T. castaneum and the rph2 (P45/49S mutation in the dihydrolipoamide dehydrogenase (dld) gene) in T. castaneum and R. dominica. In this study, we have developed and applied high-throughput, practical and specific molecular diagnostics (TaqMan qPCR) for monitoring mutations S349G, P45S and P49S. In our pilot monitoring application, we have included phosphine-resistant and susceptible populations from different parts of the world (USA, Australia, Brazil) and European strains from Greece and Serbia. Our results for the resistant T. castaneum showed a P45S mutant allele frequency (MAF) of 100% and 75.0% in the populations from Serbia and Brazil, respectively. Regarding the susceptible T. castaneum, P45S was detected in Greece (MAF = 62.5%) and was absent in Australia (MAF = 0.0%). Additionally, the S349G mutation was found to be fixed in all resistant populations, while it was also detected in susceptible ones (frequencies: 65.0% and 100.0%). The only case where both mutations were fixed (100%) was a resistant population from Serbia. In R. dominica, the P49S mutation was found only in the two resistant R. dominica populations from Serbia and Greece (50.0% and 100%) and was absent from the susceptible one from Greece; thus, P49S seems to be a satisfactory indicator for monitoring phosphine resistance. Our P49S detection assay in R. dominica seems to be a viable option in this direction, yet its utilization needs additional large-scale confirmatory work. The identification of additional resistance markers also should be prioritized. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.