Evaluating the Effects of Formulation Substrates on the Storage Duration of Entomopathogenic Nematodes

Abstract

Although there has been satisfactory success attained by utilizing entomopathogenic nematodes (EPNs) as bio-predators, cultivation of formulations prolonging lifespan for at least a year, whilst warranting distribution of high-grade nematodes, have not been accomplished on the international commercial front. Hence, this study was aimed at evaluating the effects of various formulation substrates [Diatomaceous earth (DE), coarse silica sand, and the insect-cadaver-approach], on the storage duration of formulated nematodes, particularly Oscheius sp. OGB-2022 (OR879788) and Cruznema sp. NTM-2021 (OQ408141) which were isolated from the moderately dry climatic conditions of Johannesburg, South Africa, and the dry winter climate of Leribe, Lesotho using soil baiting and White trap setup technique. These isolates demonstrated high pathogenicity towards the last Instar larvae of Tenebrio molitor L. (Coleoptera: Tenebrionidae) at room temperatures ranging from 25-35℃ after 4-7 days, and were revealed by the NCBI BLASTn bioinformatics search parameters of the 18S rDNA nucleotide sequence data that they belonged to and are closely related to other nematode species in the Oscheius and Cruznema genera of the Nematoda phylum. Moreover, bacterial isolates successfully extracted from the gut of 0.1% Sodium hypochlorite (NaClO) surface sterilized infective juveniles (IJs) of Oscheius sp. OGB-2022 and Cruznema sp. NTM-2021 were revealed by the NCBI BLASTn to have high affinity to species of genera Alcaligenes, Pseudomonas, and Citrobacter, respectively. The study found no substantial statistical differences in the survival and virulence of formulated IJs across various substrates and incubation temperatures. However, Oscheius sp. OGB- 2022 IJs were more virulent after incubation at 30℃, while Cruznema sp. NTM-2021 appeared more virulent after incubation at 25℃. The insect cadaver formulation, DE, and coarse silica sand showed the highest survival and virulence (>85%), suggesting potential for agricultural field applications. Based on observations made from the results obtained in this study, the insect cadaver and DE formulation are the substrates best suited for field application with standard agronomic equipment. The Oscheius sp. OGB-2021 and Cruznema sp. NTM-2021 IJs showed high survivability in insect cadaver formulations (>85%) and DE (>85%) when stored at 25℃ and 30℃ incubation temperatures for 2, -7, -14, and 21-day incubation durations. This suggests that future commercialization of these nematode isolates will have to be undertaken at these optimum temperatures in optimized formulation substrates.