ESTABLISHMENT OF MONOXENIC INOCULA FOR SCALING-UP IN-VITRO CULTURES OF THE ENTOMOPATHOGENIC NEMATODES STEINERNEMA SPP AND HETERORHABDITIS SPP
ESTABLISHMENT OF MONOXENIC INOCULA FOR SCALING-UP IN-VITRO CULTURES OF THE ENTOMOPATHOGENIC NEMATODES STEINERNEMA SPP AND HETERORHABDITIS SPP
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DOI：
10.1163/187529293x00330
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发表时间：
1993-07-01
期刊：
影响因子：
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通讯作者：
EHLERS, RU
中科院分区：
文献类型：
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作者：
LUNAU, S;STOESSEL, S;EHLERS, RU
昆虫病原线虫-细菌复合体（Steinernema spp./异小杆线虫属- 黄杆菌属）用于昆虫害虫的生物防治需要扩大真正的单菌培养物。通过将表面灭菌的dauer幼鱼添加到Xabridus bovienii的细菌草坪中来建立Steinernema feltiae的单菌培养物的尝试总是导致培养物的多异性。因此，开发了通过线虫卵的表面灭菌或通过妊娠雌性的碱裂解来获得无菌的第一阶段线虫幼虫的方法。所描述的程序允许在建立单菌培养物之前控制axenization的成功。所有检测的斯氏线虫属以及在无菌条件下在未处理的无菌大鼠肾上繁殖的菌株（18）。对S. carpocapsae在装有200 ml液体培养基的转瓶中达到超过18，000线虫/ml的密度。通过将无菌线虫添加到它们的特定细菌共生体的培养物中来获得斯氏线虫的单菌培养物。尝试启动异小杆线虫的繁殖。在无菌培养中，向孵化的幼鱼添加不同的生长因子是不成功的。因此，通过将无菌的第一阶段幼体与其特定的共生细菌组合来建立单菌培养物。它们对所有测试的H.细菌门。西北欧异小杆线虫菌株只能进行无菌化，然后在单菌条件下培养时，通过碱裂解获得它们的卵。异小杆线虫HSH和H.细菌属（菌株HD 01）不受所用液体培养基的影响，但取决于卵分离当天妊娠雌雄同体的发育状态。当介质的渗透压较低时，幼体的存活率显著降低。为了建立这两种菌株的单菌液体培养物，将不同生长培养基中的细菌悬浮液加入到无菌的第一阶段幼体中。菌株HSH仅在一种培养基中发育和繁殖，而HD 01在测试的4种培养基中的3种中发育和繁殖。在液体条件下异杆线虫的单菌培养物。只有当幼鱼发育成雌雄同体时才能成功。
Mass production of entomopathogenic nematode-bacterium complexes (Steinernema spp./Heterorhabditis spp. - Xenorhabdus spp.) for biological control of insect pests require scaling up of truly monoxenic cultures. Attempts to establish monoxenic cultures of Steinernema feltiae by addition of surface-sterilized dauer juveniles to bacterial lawns of Xenorhabdus bovienii always resulted in polyxenicity of the cultures. Therefore, methods were developed to obtain bacteria-free first stage nematode juveniles either by surface sterilization of nematode eggs or by alkaline lysis of gravid females. The procedures described allow control of the success of the axenization before monoxenic cultures are established. All tested Steinernema spp. and strains (18) reproduced under axenic conditions on untreated sterile rat kidney. The axenic culture of S. carpocapsae reached a density of over 18,000 nematodes/ml in a spinner flask filled with 200 ml liquid medium. Monoxenic cultures of steinernematids were obtained by addition of axenic nematodes to cultures of their specific bacterial symbiont. Attempts to initiate the reproduction 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-free first stage juveniles with their specific symbiotic bacterium. They were successful with all tested strains of H. bacteriophora. North-West European Heterorhabditis strains could only be axenized and subsequently cultured under monoxenic conditions when their eggs were obtained by alkaline lysis. Hatching of juveniles after alkaline lysis of Heterorhabditis sp. (strain HSH) and H. bacteriophora (strain HD01) was not influenced by the liquid media used but depended on the state of development of the gravid hermaphrodites on the day of egg isolation. The survival of the juveniles was significantly reduced when the osmolarity of the medium was low. To establish monoxenic liquid cultures of both strains, bacterial suspensions in different growth media were added to the axenic first stage juveniles. Strain HSH developed and reproduced 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.