Development of a new entomopathogenic nematode species, steinernema innovation : biological characterization and mass production.
Entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis are employed as environmental friendly biopesticides to control key pests in high value agricultural crops. Their successful application in developed countries has proved their effectiveness. The production of EPN for large scale commercial application has been restricted to developed countries because high capital cost of setting up liquid fermentation, and the high running costs of such units. If EPNs are to be commercialized in developing countries, then this will probably be achieved using in vivo culturing in insect larvae, or by using in vitro solid culture on a small to medium scale. The Republic of South Africa bans the importation of exotic EPN products by Amendment 18 of Act 36 of 1983. Therefore EPN products in South Africa will need to be developed from indigenous species. This study focused on optimizing the solid culture production and biological characterization of an indigenous nematode, Steinernema innovationi, as a biocontrol agent. A good production system must achieve high yield, maintain high virulence and the product must have a reasonable shelf-life. Optimum production temperature was determined using in vivo culturing of S. innovationi inside Galleria mellonella larvae. Performance was measured by quantifying the first day of emergence of infective juveniles (IJs) from cadavers, yield of IJs and IJ length, at five temperatures ranging from 18°C to 25°C. Optimum temperature was selected as 22°C based on high yield and slow emergence. There was a yield of 92,756 ± 28,089 IJs/larva, and 84,056 ± 27,832 IJs/larva, at 22°C and 25°C, respectively, which were not significant different. However, IJ emergence was significantly slower at 22°C, which provided more time for nutrient uptake and therefore these IJs had a greater nutrient reserve. There was no correlation observed between IJ length vs. temperature and IJ length vs. yield. A medium containing a puree of the larvae of the common house fly (Musca domestica) and 3% canola oil produced the highest yield of IJs (781,678 ± 221 IJs/5g), the highest level of live IJs (>84%) and the lowest level of adults (<10%) remaining in the medium at the time of harvest, compared to five other media formulations. All media were subjected to in vitro mass production at 22°C. A liquid inoculum of the EPN gave higher yields than a solid culture inoculum, irrespective of concentration. The length of IJ did not affect virulence against last instar larvae of G. mellonella, which was >90% in all experiments. A study characterizing optimum storage temperature of S. innovationi was carried out at five temperatures ranging from 5°C to 25°C in aqueous suspension over a period of 84 days. Survival was highest and most stable at 15°C, ranging from 84% to 88% after 84 days storage. Storage of the EPN in a sponge at a concentration of 2.5 million IJs in 15ml 0.1% formalin solution was successful, with an improvement of 6% compared to aqueous storage at 15°C. Furthermore, storage of the EPN in a sponge at 25°C, after a period of low temperature (15°C) storage for 84 days, did not have a detrimental impact on IJ survival and infectivity (87% and 95%, respectively). The new EPN (S. innovationi) was characterized by studying traits related to its infectivity (infectivity under a range of temperatures [10°C to 35°C], foraging behaviour & persistence under field conditions). Time till death was shortest at 25 and 30°C (average 1 day). The highest number of established IJs was recovered at 25°C (mean = 27°C). The nematode infected Galleria mellonella larvae at all depths and was capable of covering a distance of up to 15cm in 24 hours. There was no statistical difference between in vitro (40%) and in vivo (27%) cultured IJ persistence 4 weeks post application. Furthermore, there was no statistical difference in nematode recovery after 1 and 4 weeks post application of in vitro (33% & 40%, respectively) and in vivo (60% & 27%, respectively) produced IJs. Thermal activity was optimal at 25°C, the new species was classified as a cruiser and proved to survive under field conditions for at least 4 weeks. Susceptibility of larvae and/or pupae of Eldana saccharina (Walker), Sesamia calamistis (Hampson), Chilo partellus (Swinhoe), Tenebrio molitor (Linnaeus), Galleria mellonella (Linnaeus), Cydia pomonella (Linnaeus), Plutella xylostella (Linnaeus), and Gryllidae acheta (Linnaeus) representing three orders (Coleoptera, Lepidoptera & Orthoptera) was tested at a low and high concentration of 50 and 500 IJs, respectively. The hosts G. acheta, C. partellus and P. xylostella showed least susceptibility with maximum mortalities at the 500 IJs concentration of 28%, 45% and 92%, respectively. All other hosts suffered 100% mortality. Pupal mortality ranged from 47% to 68%. An LC50 and LC70 of 3 IJs/larva and 31 IJs/larva, respectively, was calculated for the black cutworm, Agrotis ipsilon (Hufnagel). These results provided a broad guideline on the relative pathogenicity of this new species against different hosts. A cost analysis was calculated for in vitro solid culture of the EPN, including the cost of rearing an insect-based nutrient component. An estimated retail price was then compared to the costs of market products around the globe. An estimated retail price (R90.61) for S. innovationi was considerably lower than the market price for other Steinernema species, which ranged from R271.50 to R458.55, in South African rands. The production system developed in this study for S. innovationi offers a highly competitive small to medium scale production method to produce EPN products without having to invest in large scale liquid fermentation equipment, by using relatively cheap production media ingredients, and simple solid culture growing conditions.