ESTABLISHMENT OF MONOXENIC INOCULA FOR SCALING-UP IN-VITRO CULTURES OF THE ENTOMOPATHOGENIC NEMATODES STEINERNEMA SPP AND HETERORHABDITIS SPP

Mass production of entomopathogenic nematode-bacterium complexes (Stei nernema spp./Heterorhabditis spp. - Xenorhabdus spp.) for biological c ontrol of insect pests require scaling up of truly monoxenic cultures. Attempts to establish monoxenic cultures of Steinernema feltiae by ad dition of surface-sterilized dauer juveniles to bacterial lawns of Xen orhabdus bovienii always resulted in polyxenicity of the cultures. The refore, methods were developed to obtain bacteria-free first stage nem atode juveniles either by surface sterilization of nematode eggs or by alkaline lysis of gravid females. The procedures described allow cont rol of the success of the axenization before monoxenic cultures are es tablished. All tested Steinernema spp. and strains (18) reproduced und er axenic conditions on untreated sterile rat kidney. The axenic cultu re of S. carpocapsae reached a density of over 18,000 nematodes/ml in a spinner flask filled with 200 ml liquid medium. Monoxenic cultures o f steinernematids were obtained by addition of axenic nematodes to cul tures of their specific bacterial symbiont. Attempts to initiate the r eproduction of Heterorhabditis spp. in axenic cultures by addition of different growth factors to hatched juveniles were unsuccessful. Hence , monoxenic cultures were established by the combination of bacteria-f ree first stage juveniles with their specific symbiotic bacterium. The y were successful with all tested strains of H. bacteriophora. North-W est European Heterorhabditis strains could only be axenized and subseq uently cultured under monoxenic conditions when their eggs were obtain ed by alkaline lysis. Hatching of juveniles after alkaline lysis of He terorhabditis sp. (strain HSH) and H. bacteriophora (strain HD01) was not influenced by the liquid media used but depended on the state of d evelopment of the gravid hermaphrodites on the day of egg isolation. T he survival of the juveniles was significantly reduced when the osmola rity of the medium was low. To establish monoxenic liquid cultures of both strains, bacterial suspensions in different growth media were add ed to the axenic first stage juveniles. Strain HSH developed and repro duced in only one medium and HD01 in 3 of 4 media tested. Under liquid conditions monoxenic cultures of Heterorhabditis spp. were successful only when the juveniles developed to hermaphrodites.