Use of biocides to minimize microbial contamination in Spodoptera exigua multiple nucleopolyhedrovirus preparations

The presence of contaminant microbes in baculovirus-based insecticides is regulated by phytosanitary product registration authorities. We aimed to determine whether the abundance of microbes in suspensions of Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies (OBs) could be reduced by treatment with a range of biocidal compounds. The diversity of contaminant bacteria was determined by next-generation sequencing of the 16S rRNA gene. Overall, 97.9% of sequences detected were Gammaproteobacteria (mostly Pseudomonas spp. and Enterobacteriaceae) and 2.1% were Firmicutes (mostly Enterococcus spp.). Colloidal silver, benzalkonium chloride and chlorhexidine digluconate were identified as highly effective biocides. Incubation of OB suspensions with high concentrations of colloidal silver (450 mg/l) or benzalkonium chloride (6000 mg/l) resulted in marked reductions in colony forming unit counts over a 180 day period at 4° or 25 °C. Benzalkonium chloride and colloidal silver treatments, at either 4 or 25 °C, did not affect the insecticidal activity of OBs over an 80 day period. However, OB activity decreased following 180 days of treatment by benzalkonium chloride at either 4 or 25 °C, or by colloidal silver at 25 °C, but not at 4 °C. Counts of OBs revealed a significant decrease in OB numbers in benzalkonium chloride-treated suspensions after 180 days at both temperatures, whereas colloidal silver-treated OBs were not affected. Benzalkonium chloride also caused aggregation of OBs at the concentration tested. We conclude that biocidal compounds can markedly reduce the abundance of contaminant microorganisms in OB suspensions, and can be accompanied by reductions in OB infectivity and OB numbers in some circumstances. Future studies should focus on lower concentrations of biocides that do not affect OBs in long-term storage.