The evaluation of the toxicity of bacillus thuringiensis cry proteins against reference and field populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), in South Africa

Chen, Chia-yu
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Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a major Western Hemisphere pest that has established and damaged maize crops in South Africa since 2016. The cultivation of Bacillus thuringiensis (Bt) maize that expresses insecticidal Bt Cry proteins is a control method of FAW in the Western Hemisphere and is also a control method prevalent in South Africa. However, the toxicity of purified Bt Cry proteins expressed by Bt maize for the control of FAW in South Africa has not been evaluated. Thus, the main aim of the study was to evaluate the toxicity of agronomically important Bt Cry proteins against a reference and field populations of FAW in South Africa. FAW is characterised by two genetically distinct strains which are the rice strain (RS) and the corn strain (CS). The two strains have been reported to exhibit strain-specific responses to Bt Cry proteins. Thus, all populations of FAW in the study were first genetically characterised to assess whether the susceptibilities of FAW populations to Bt Cry proteins are strain-related. The majority of FAW specimens (73%), characterised by strain-diagnostic single nucleotide polymorphisms (SNPs) in the downstream segment of the mitochondrial cytochrome oxidase subunit I gene (COIB) and the triosephosphate isomerase (Tpi) exon and intron regions, were found to be hybrids of CS and RS FAW, with variations in the Tpi exon and intron regions. To evaluate the toxicity of Bt Cry proteins (Cry1Ac, Cry1Ab, Cry2Ab2.820 and Cry1A.105) to first and second instar FAW larvae, the mortality responses, and growth-inhibitory responses of first and second instar FAW larvae were evaluated by droplet-feeding and diet-overlay bioassays. The toxicity of the Bt Cry proteins against a reference FAW population (V1) was compared with five field populations that were sampled 3 years later. Variation in the susceptibilities of FAW larvae to Bt Cry proteins was observed for all six populations, however, the V1 population was the least susceptible to all the Bt Cry proteins when compared to the field populations. The degree of toxicity of Bt Cry proteins also differed between the different FAW populations. The variability in the susceptibilities of FAW to Bt Cry proteins may be due to the fitness costs associated with resistance alleles, regional selection pressures due to geographical location differences, or strain-related genetic differences. Despite the variation in the susceptibilities between the FAW populations, Cry2Ab2.820 and Cry1A.105 were the most toxic to all the populations in terms of both mortality and growth inhibitory responses. These two most toxic proteins in the present study are expressed together in the MON89034 event in Bt maize. Thus, the toxicity of Cry2Ab2.820 and Cry1A.105 was further evaluated by assessing the interaction between Cry2Ab2.820 and Cry1A.105 at different ratios, using v isobolographic analysis and the calculation of the synergy factor (SF). At five different ratios of Cry2Ab2.820:Cry1A.105, four ratios (1:1, 1:2, 1:3 and 2:1) were synergistic and one ratio (3:1) was antagonistic. Thus, when expressed in Bt maize at ratios that enable synergistic interactions, the Cry2Ab2.820 and Cry1A.105 proteins would be more toxic to FAW larvae than each of the proteins alone. Overall, the toxicity of Bt Cry proteins to reference and field populations from three different provinces in South Africa revealed that FAW populations can be expected to be mildly susceptible to Cry1Ab and highly susceptible to Cry2Ab2.820 and Cry1A.105. The study sets a baseline of the toxicity of Bt Cry proteins to FAW in South Africa and provides an important foundation for future resistance screening programmes in South Africa.
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2022