Commercial use of entomopathogenic nematodes against key insect pests of deciduous fruit, grapes and citrus in South Africa requires massive numbers of nematodes for inundative field application. High-technology in vitro liquid culture requires development to mass culture and to formulate these nematodes for commercial purposes. Heterorhabditis zealandica was identified as a species with potential as a biological control agent against a wide range of key insect pests. The first step towards the development of in vitro mass culture of H. zealandica was to establish monoxenic cultures of both the nematode and its Photorhabdus sp. symbiont, using in vitro liquid culture technology. The body length of various H. zealandica life stages during in vitro development was measured to determine the growth characteristics of H. zealandica in liquid culture. The growth curve of the symbiotic bacteria during the process time was measured to determine when the stationary phase was reached, as this would indicate the optimum time required for inoculating infective juveniles (IJs) and for aiding in maximum IJ recovery. On day 15, the IJs reached a maximum density of 41100/ml, while the hermaphrodites and females reached their highest density on day 16 at 9800/ml and 7700/ml, respectively, after which the experiment was terminated. Bioassays using Galleria mellonella were performed to compare the virulence between in vitro- and in vivo-produced nematodes, which indicated in vitro-produced nematodes to be significantly less virulent. This study illustrates that H. zealandica and its Photorhabdus sp. symbiont can be successfully cultured in liquid. However, two generations occurred during the process time, instead of the desirable one generation. Future research goals would include developing methods to increase the percentage recovery in the liquid culture, as doing so would increase the number of nematodes produced per ml and it would also reduce the processing time significantly.
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