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Duddingtonia flagrans冻干制剂厚壁孢子优化培养及其杀线虫幼虫剂量研究
陈林军,刘省段,杨晓野,等
作者单位
陈林军 内蒙古农业大学 兽医学院农业部动物疾病临床诊疗技术重点实验室 
刘省段 内蒙古农业大学 兽医学院农业部动物疾病临床诊疗技术重点实验室 
杨晓野,等 内蒙古农业大学 兽医学院农业部动物疾病临床诊疗技术重点实验室 
摘要:
【目的】探究捕食线虫性真菌Duddingtonia flagrans冻干制剂厚壁孢子的批量培养方法及其对线虫幼虫的毒杀剂量。【方法】通过玉米粒或大麦粒培养基的单步培养法,以及先在含有0.05%琼脂粉的沙氏葡萄糖肉汤培养基中培养7 d后继而转接到玉米粒或大麦粒培养基的双步培养法,以培养3~5周后洗脱的每克固体培养基中的厚壁孢子数为依据,对D.flagrans的最优培养方法进行筛选;然后使用最优培养法培养D.flagrans厚壁孢子并将其冻干后用于体外杀线虫幼虫试验,研究D.flagrans冻干制剂的杀线虫幼虫剂量。【结果】培养基质不同时,D.flagrans的菌落颜色有一定差异,玉米粒培养基中的菌落颜色呈微黄色,而大麦粒培养基中的菌落呈白色;D.flagrans的适宜培养时间为3周。采用单步培养法时,玉米粒培养基培养的厚壁孢子数量为2.4×105个/g,大麦粒培养基培养的厚壁孢子数量为3.2×105个/g;而采用双步培养法时,玉米粒培养基培养的厚壁孢子数量为3.0×105个/g,大麦粒培养基培养的厚壁孢子数量为3.5×105个/g。D.flagrans冻干制剂杀线虫幼虫剂量的阈值为每克粪便4×105D.flagrans厚壁孢子,相应对线虫幼虫的杀虫率为95.2%。【结论】采用双步培养法培养厚壁孢子并将其制备成冻干制剂,在D.flagrans生物防治寄生性线虫病方面有较好的应用前景。
关键词:  Duddingtonia flagrans  厚壁孢子  培养优化  线虫幼虫  毒杀剂量
DOI:
分类号:
基金项目:国家公益性行业(农业)科研专项(201303037);内蒙古自然科学基金项目(2015MS0308);家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2015KFKT013)
Studies on culture optimization of preparation freeze-drying Duddingtonia flagrans chlamydospores and its dose for killing nematode larvae
CHEN Linjun,LIU Xingduan,YANG Xiaoye,et al
Abstract:
【Objective】In order to study the batch culture of nematode-trapping fungus Duddingtonia flagrans chlamydospores for freeze-drying and its dose of killing nematode larvae.【Method】D.flagrans chlamydospores were produced by using two methods:one was that the mycelial inoculum was only cultivated in corn or barley medium;while another was firstly cultivated in sabouraud dextrose broth medium containing 0.05% of agar,then transferred to the corn or barley medium.Finally,one optimal method was screened for batch culturing nematode trapping fungus D.flagrans chlamydospores based on the number of chlamydospores that was eluted from the medium in a period of 3-5 weeks.To obtain the dose of killing nematode larvae of freeze-drying D.flagrans in vitro,the optimal cultivation method was used to get the D.flagranschlamydospores and then it was freeze-dryed and the experiment of killing nematode larvae in sheep faeces was finished.【Result】The color of D.flagrans colony had certain difference when cultured in different medium,its color was yellowish that cultured in corn medium,the other was white that cultured in barley medium. The appropriate cultivating time was 3 weeks.Cultured it by one-phase,2.4×105 of chlamydospores per gram were obtained that cultured it only in corn medium;3.2×105 of chlamydospores per gram were obtained that cultured it only in barley medium.Cultured it by two-phase,3.0×105 chlamydospores per gram were collected in corn medium and 3.5×105 chlamydospores per gram were collected in barley medium.The maximum insecticidal rate of nematode larvae was 95.2% when 4×105 lyophilized D.flagrans chlamydospores were mixed with per gram of sheep faeces.【Conclusion】The results showed that the best method for cultivating nematode trapping fungus D.flagrans chlamydospores was two phase culture,and make its spore suspension to do lyophilized preparation,it can achieve better application prospects in biological control of livestock gastrointestinal nematodes.
Key words:  Duddingtonia flagrans  chlamydospores  optimization of cultivation  nematode larvae  dose of killing