S. Lunau et al., ESTABLISHMENT OF MONOXENIC INOCULA FOR SCALING-UP IN-VITRO CULTURES OF THE ENTOMOPATHOGENIC NEMATODES STEINERNEMA SPP AND HETERORHABDITIS SPP, Nematologica, 39(3), 1993, pp. 385-399
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.