The development and evaluation of Baculovirus formulations for the biological control of the African cotton bollworm

Abstract

Wettable powder and suspension formulations of a Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) biopesticide were developed for the control of the African cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). Greenhouse trials of an unformulated suspension of HearSNPV were conducted on tomato plants artificially infested with H. armigera larvae to determine the efficacy of the virus without formulation additives. Two greenhouse trials were carried out. An application rate of 1.00 x 1013 occlusion bodies per hectare (OBs/ha) significantly reduced the number of larvae per plant compared to the control group in greenhouse trial 1, and application rates ranging between 4.80 x 1011 to 4.80 x 1012 OBs/ha significantly reduced the number of larvae compared to the control group in greenhouse trial 2. In terms of pest reduction, the HearSNPV treatments in greenhouse trial 2 were comparable to a commercially available biopesticide (Dipel, containing Bacillus thuringiensis). The effect of the purity, in terms of bacterial contamination, of the inoculum used to infect H. armigera insects was compared at three different storage temperatures (4, 25 and 37 °C) for three different storage periods (7, 30 and 90 days). No significant difference was found between the bacterial counts of the homogenates prior to storage. However, the total bacterial aerobic counts increased on storage and were highest for homogenates prepared from the crude inoculum which averaged 5.16 log cfu/mg, compared to 3.92 log cfu/mg and 2.90 log cfu/mg for the purified and control (sterile distilled water) inoculums respectively. The contaminating bacteria were identified using 16S rDNA sequence analysis, and found to be a Bacillus and Enterococcus species. This suggests that bacterial contamination should be minimized from the start of baculovirus production, since the microbial load can increase on storage. Additives, namely Instant Starch (IS) and Xanthan Gum (XG) were used to prepare HearSNPV suspensions and these were evaluated under accelerated storage conditions, corresponding to storage at room temperature for two years. The IS suspension completely lost its insecticidal activity after storage, while the XG and Unformulated (UF) suspensions were 4.8 and 3 times respectively, less insecticidal after storage. The microbial load decreased from an initial total aerobic count of 8.0 log cfu/ml each, to 3.24 log cfu/ml, 6.86 log cfu/ml and 4.26 log cfu/ml for the IS, UF and XG suspensions respectively. Two bacterial genera were isolated from these samples, namely Bacillus and Paenibacillus. The pH of the suspensions remained near neutral, with the exception of the IS suspension, which had a pH of approximately 3.5 after storage. The XG suspension displayed the best stability on storage, followed by the UF suspension. A spray-dryer was used to develop a wettable baculovirus powder and Response Surface methodology was used to optimize the process. Two carriers, namely Polysaccharide-MS and Polysaccharide-WM were used and inlet temperature, air speed and feed flow rate were used as the model factors. The response factors monitored were powder yield (mg/ml), active ingredient yield of the occlusion bodies (OBs/mg), and moisture content (%). The effect of spray-drying on the microbial load of the samples was also examined. The optimal conditions for Polysaccharide-WM were determined to be an inlet temperature of 130 °C, air speed dial setting of 45 and feed flow rate of 9 ml/minute. According to the predictive models obtained, this would give a powder yield of 337.5 mg, active ingredient yield of 8.0 x 105 OBs/mg and moisture content of 4.8 %. The optimal conditions for Polysaccharide-MS were determined to be an inlet temperature of 130 °C, air speed dial setting of 45 and feed flow rate of 9 ml/minute. According to the predictive models obtained, this would give a powder yield of 110.9 mg/ml, active ingredient yield of 1.0 x 106 OBs/mg and moisture content of 4 %. Spraydrying reduced the microbial load of the sample four-fold. Baculovirus biopesticides in the form of suspensions and wettable powders were developed and evaluated in this project and will form the foundation for the development of commercial biopesticides for the control of the African cotton bollworm.