Characterisation of nematode symbiotic bacteria and the in vitro liquid ulture of Heterorhabditis zealandica and Steinernema yirgalemense

Entomopathogenic nematodes (EPNs), in combination with their associated bacterial symbionts, have proven to be effective against numerous insect pests. In this project, the associated symbiotic bacteria of three EPN species were isolated, and the potential of two nematode species to be mass cultured in liquid media was evaluated. Bacteria species from Heterorhabditis noenieputensis, Steinernema khoisanae and Heterorhabditis zealandica, which were found to be new species, have been described as such. Using in vitro mass culture techniques, it was illustrated that H. zealandica and its Photorhabdus symbiont, as well as Steinernema yirgalemense and its Xenorhabdus symbiont, can be successfully cultured in a liquid medium. However, the number of nematodes per ml of medium was found to be much higher for S. yirgalemense, with 77 000 IJs/ml (13 days), in comparison to the 41 000 IJs/ml (15 days) obtained for H. zealandica. The final aim of the project was to determine when Xenorhabdus reached the stationary phase, when grown in a 20-L fermenter, as this would be the time most suited for adding the infective juveniles (IJs) of S. yirgalemense for mass culture. The step concerned would also be the first taken toward the liquid mass culture of S. yirgalemense in industrial-size fermenters. Results from this experiment indicated the optimum amount of time required for adding nematodes to the bacterial culture in the fermenter and for ensuring the optimum recovery of IJs, as well as a subsequent high yield of nematodes within a minimum amount of processing time. This is the first report of its kind to investigate comprehensively the successful liquid culture of two South African EPN species for the sole purpose of evaluating potential commercialisation. Future research goals should be to increase the percentage IJ recovery directly after inoculation into liquid culture, which would increase the number of nematodes produced per ml medium, which would, therefore, significantly reduce the amount of processing time required.