取食洋白蜡和旱柳光肩星天牛...异及关键纤维素降解细菌筛选_贾若峰.pdf

doi：10.11707/j.1001-7488.LYKX20210919取食洋白蜡和旱柳光肩星天牛幼虫肠道中的细菌多样性差异及关键纤维素降解细菌筛选*贾若峰1谷奇1孙一鸣1陆鹏飞1俱世博2乔海莉2（1.北京林业大学省部共建森林培育与保护教育部重点实验室北京 100083；2.中国医学科学院北京协和医学院药用植物研究所北京 100193）摘要：【目的】从肠道细菌多样性角度探究光肩星天牛幼虫对传统抗性树种洋白蜡的适应性原因。【方法】基于16S rDNA 高通量测序技术，分别对用洋白蜡、旱柳饲喂的光肩星天牛幼虫肠道细菌进行了分子鉴定及多样性分析；然后通过富集培养，从未经饲喂的幼虫中筛选出了 31 株纤维素降解细菌，以透明水解圈直径（D）/菌落直径（d）为标准初步测定了各菌株纤维素降解能力；对降解能力最强的 4 个菌株进行了滤纸复筛试验。【结果】使用洋白蜡饲喂后，厚壁菌门、芽孢杆菌纲、乳杆菌目、链球菌科、乳球菌属为优势类群；使用旱柳饲喂后，厚壁菌门、芽孢杆菌纲、乳杆菌目、肠球菌科、肠球菌属为优势类群；变形菌门、-变形菌纲、肠杆菌目、肠杆菌科、拉乌尔菌属在两者中都为优势类群。两者肠道细菌群落最显著的差异在于，取食洋白蜡幼虫链球菌科、乳球菌属相对丰度显著高于取食旱柳的幼虫，而肠球菌科、肠球菌属相对丰度显著低于取食旱柳的幼虫。直接取食洋白蜡枝干的幼虫肠道纤维素降解细菌种类丰富，隶属于 4 门、5 纲、5 目、7 科、7 属，15 种；优势属包括微杆菌属、纤维单胞菌属、假单胞菌属；其中纤维素降解能力最强的 4个菌株分别为 2 株假单胞菌和 2 株产黄纤维单胞菌；二者在滤纸复筛试验中显著地分解了滤纸，进一步验证了其良好的纤维素降解能力。【结论】在取食洋白蜡光肩星天牛幼虫肠道中，链球菌科、乳球菌属为优势类群，可能发挥重要功能但不直接降解纤维素；放线菌门下的微杆菌属、纤维单胞菌属和变形菌门下的假单胞菌属非优势类群但纤维素降解能力强，可能发挥降解纤维素的功能；上述菌群共存于光肩星天牛肠道，参与其适应洋白蜡的过程。关键词：16S rDNA；光肩星天牛；洋白蜡；旱柳；肠道细菌；纤维素降解细菌中图分类号：S763.38文献标识码：A文章编号：10017488(2023)04011715Differences in Bacterial Diversity and Key Cellulose-Degrading Bacteria in the Intestinal Tractof Anoplophora glabripennis(Coleoptera:Cerambycidae)Larvae Feeding onFraxinus pennsylvanica and Salix matsudanaJia Ruofeng1Gu Qi1Sun Yiming1Lu Pengfei1Ju Shibo2Qiao Haili2（1.Key Laboratory for Silviculture and Conservation of Ministry of Education,Beijing Forestry UniversityBeijing 100083；2.Institute of Medicinal PlantDevelopment,Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100193）Abstract：【Objective】This study aims to explore the adaptive reasons of Anoplophora glabripennis larvae to Fraxinuspennsylvanica,a traditional resistant species,in terms of intestinal bacteria diversity.【Method】The A.glabripennis larvae werefed with F.pennsylvanica and Salix matsudana,respectively ,and the high-throughput sequencing technology was used tosequence 16S rDNA of the larvae intestinal bacteria,with which the molecular identification and diversity analysis of intestinalbacteria were conducted.Subsequently,through accumulation culture,31 strains of cellulose-degrading bacteria were screened outfrom unfed larvae.The cellulose-degrading ability of each strain was determined by the transparent circle diameter(D)/colonydiameter(D)as the standard,and the filter paper re-screening test was conducted on the four strains with the strongest cellulose-degrading ability.【Result】The results showed that Phylum Firmicutes,Class Bacilli,Order Lactobacillales,FamilyStreptococcaceae and the genus Lactococcus were predominant in the larvae intestinal tract after being fed with F.pennsylvanica.Whereas Phylum Firmicutes,Class Bacillus,Order Lactobacillales,Family Enterococcaceae and the genus Enterococcus were 收稿日期：20211223；修回日期：20230306。基金项目：国家重点研发计划课题(2017YFD0600103)；国家自然科学基金面上项目（31570643）；国家级大学生创新创业训练项目（G201910022008）。*陆鹏飞为通讯作者。第 59 卷 第 4 期林业科学 Vol.59，No.42 0 2 3 年 4 月SCIENTIA SILVAE SINICAEApr.，2 0 2 3predominant in the larvae intestinal tract after being fed with S.matsudana.Phylum Proteobacteria,Class Gammaproteobacteria,Order Enterobacteriales,Family Enterobacteriaceae and the genus Raoultella were predominant in the both feedings.The mostsignificant difference between the two groups was that the relative abundance of Family Streptococcaceae and genus Lactococcusin the larvae feeding on F.pennsylvanica was significantly higher than that in the larvae feeding on S.matsudana,while therelative abundance of Family Enterococcaceae and genus Enterococcus was significantly lower than that in the larvae feeding onS.matsudana.In the intestinal bacterial community of larvae feeding directly on the branch of F.pennsylvanica,there wereabundant species of cellulose-degrading bacteria,belonging to 4 phyla,5 classes,5 orders,7 families,7 genera and 15 species.Atthe genus level,Microbacterium,Cellulomonas and Pseudomonas were predominant.Among them,two strains of Pseudomonassp.and two strains of C.flavigena had the strongest cellulose-degrading ability.The filter paper was decomposed significantly inthe re-screening test,which further verified that the strains had strong cellulose-degrading ability.【Conclusion】FamilyStreptococcaceae and genus Lactococcus are the predominant bacteria in the intestinal tract of A.glabripennis larvae feeding onF.pennsylvanica,which may play an important role but do not degrade cellulose directly.Genera Microbacterium andCellulomonas in Phylum Actinobacteria and genus Pseudomonas in Phylum Proteobacteria are not the predominant group ofbacteria,but they have strong cellulose degradation ability and may play a role in cellulose degradation.These bacteria co-exist inthe intestinal tract of A.glabripennis and are involved in its adaptation process to the F.pennsylvanica.Key words：16S rDNA；Anoplophora glabripennis；Fraxinus pennsylvanica；Salix matsudana；intestinal bacteria；cellulose-degrading bacteria 昆虫抵御植物次生物质的能力和它们的肠道微生物群落相关，这些微生物可以影响昆虫的寄主范围(Hammer et al.,2015)。其中，肠道内共生细菌由于世代周期短、适应性强，在植物昆虫相互作用的协同进化中具有突出的重要性(Spiteller et al.,2000)。例如椴六点楔天牛(Saperda vestita)肠道内含有的纤维素降解菌可以帮助其消