摘要
【目的】从二化螟个体中分离获得昆虫病原线虫共生菌,从中筛选对水稻纹枯病菌具有高抑菌活性的菌株,以进一步丰富昆虫病原线虫共生菌资源,为昆虫病原线虫共生菌应用于二化螟和水稻纹枯病的防治提供理论基础。【方法】采用血淋巴法从斯氏线虫侵染致死的二化螟个体中分离昆虫病原线虫共生菌,利用NBTA培养形态结合16SrRNA扩增测序鉴定昆虫病原线虫共生菌种类;利用平板培养法测定昆虫病原线虫共生菌对水稻纹枯病菌的抑菌率,筛选获得高抑菌活性菌株;对高抑菌活性菌株进行全基因组测序;通过液体培养获得高抑菌活性菌株生长曲线;利用单因素试验和正交试验优化高抑菌活性菌株液体发酵培养基的碳源、氮源和无机盐,筛选菌株液体发酵的装液量、菌种接种量、发酵温度和转速最佳条件。【结果】共分离获得5株昆虫病原线虫共生菌,鉴定为嗜线虫致病杆菌(Xenorhabdus nematophila)。NBTA培养特性表明5株昆虫病原线虫共生菌中1株为次生菌,其余4株为初生菌;其中初生菌株HN04对水稻纹枯病菌的抑菌率最高,为73.62%,次生菌株HN01的抑菌率最低,为55.29%,菌株HN01与4株初生菌的抑菌率差异显著(P<0.05),选择抑菌率最高的菌株HN04进行后续研究。全基因组测序结果显示,菌株HN04由1条环状染色体组成,大小为7998302bp,平均GC含量为44.03%;菌株HN04对数生长期为4~28h,稳定期为28~52 h,52 h后进入衰亡期。单因素和正交试验优化菌株HN04液体发酵条件,获得培养基组合为2.0%麦芽糖、2.0%蛋白胨、1.0%NaCl,各因素对发酵液中菌体浓度的影响顺序为氮源因子>无机盐因子>碳源因子;最佳液体发酵条件为三角瓶装液量30%~40%、菌种接种量4%~12%、发酵温度28~32°C、转速180 r/min。【结论】从二化螟个体中分离获得嗜线虫致病杆菌HN04,该菌株对水稻纹枯病菌具有较高的抑菌活性。优化的液体发酵方案可用于快速、大批量培养菌株HN04菌悬液。菌株HN04具有开发为防治二化螟和水稻病害生防菌的潜力。
【Objective】The strains of symbiotic bacteria from entomopathogenic nematodes were isolated from Chilo suppressalis individuals,the strain with high antibacterial activity against rice sheath blight was screened to further enrich the resources of entomopathogenic nematode symbiotic bacteria,which could provide theoretical basis for the application of entomopathogenic nematode symbiotic bacteria in the control of Chilo suppressalis and rice sheath blight.【Method】Strains of symbiotic bacteria from entomopathogenic nematodes were isolated by hemolymph separation method from the dead Chilo suppressalis individuals infected with Steinernema.NBTA culture morphology combined with 16S rRNA am‐plification sequencing was used to identify the symbiotic species of entomopathogenic nematodes.Plate culture method was used to determine the inhibitory rate of symbiotic bacteria from entomopathogenic nematodes against rice sheath blight strain,and strain with high antibacterial activity against rice sheath blight was screened.The whole genome of strain with high antibacterial activity was sequenced,and the growth curve of strain with high antibacterial activity was obtained by liquid culture.The carbon source,nitrogen source and inorganic salt of the strain liquid fermentation medium of strain with high antibacterial activity were optimized by single factor test and orthogonal test,and the optimal condi‐tions for liquid fermentation,inoculation amount,fermentation temperature and rotation speed were screened.【Result】Five strains of symbiotic bacteria from entomopathogenic nematodes were isolated and identified as Xenorhabdus nema‐tophila strains.NBTA culture characteristics showed that 1 strain of the 5 entomopathogenic nematode symbiotic bacteria was secondary bacteria,and the other 4 strains were primary bacteria.The primary strain HN04 had the highest inhibitory rate against rice sheath blight(73.62%),and the secondary strain HN01 had the lowest inhibitory rate(55.29%),strain HN01 was significantly different from that of the 4 primary strains(P<0.05).The strain HN04 with the highest inhibitory rate was selected for follow-up study.The results of whole genome sequencing showed that strain HN04 consisted of a ring chromosome with a size of 7998302 bp and an average GC content of 44.03%.The logarithmic growth period of strain HN04 was 4-28 h,the stable period was 28-52 h,and it entered the decline period after 52 h.Single factor and or‐thogonal experiment optimized the liquid fermentation conditions of strain HN04,and the medium combination was 2.0%maltose,2.0%peptone,1.0%NaCl.The main order of influence of each factor on the concentration of bacteria in the fer‐mentation solution was nitrogen source factor>inorganic salt factor>carbon source factor.The optimal liquid fermenta‐tion conditions were 30%-40%of liquid in triangular bottle,4%-12%of inoculum,fermentation temperature 28-32°C and rotation speed 180 r/min.【Conclusion】Xenorhabdus nematophila strains are isolated from Chilo suppressalis.The pri‐mary Xenorhabdus nematophila bacteria HN04 has the highest antibacterial activity against rice sheath blight.The opti‐mized fermentation scheme can be used to rapidly culture HN04 strain suspensions in large quantities.HN04 has potential application value in controlling Chilo suppressalis and rice sheath blight.
作者
邵晨霞
朱华珺
刘絮宁
杨祎
唐少军
任锐
颜莲莲
鲁湘
许隽
刘洋
SHAO Chen-xia;ZHU Hua-jun;LIU Xu-ning;YANG Yi;TANG Shao-jun;REN Rui;YAN Lian-lian;LU Xiang;XU Jun;LIU Yang(Hunan Institute of Microbiology/Yuelushan Laboratory,Changsha,Hunan 410009,China;Longping Huangpu Research Institute of Agricultural Science and Technology,Guangzhou,Guangdong 510715,China;Hunan Hybrid Rice Research Center,Changsha,Hunan 410001,China)
出处
《南方农业学报》
北大核心
2025年第4期1005-1016,共12页
Journal of Southern Agriculture
基金
国家重点研发计划项目(2021YFD1401100)
湖南省重点研发计划项目(2023NK2016)
湖南省科技创新计划项目(2024RC8167)。
关键词
昆虫病原线虫共生菌
嗜线虫致病杆菌
二化螟
水稻纹枯病
抑菌活性
液体发酵条件优化
symbiotic bacteria from entomopathogenic nematodes
Xenorhabdus nematophila
Chilo suppressalis
rice sheath blight
antibacterial activity
liquid fermentation condition optimization
作者简介
第一作者:邵晨霞(1980-),https://orcid.org/0009-0000-2116-5128,高级农艺师,主要从事农业微生物菌种的资源收集、功能挖掘、菌剂研发与应用工作。主持或作为主要成员参与国家重点研发计划项目“稻飞虱灾变机制与可持续防控技术研究”、国家级平台“国家植保微生物种质资源库建设”、中央引导地方科技发展资金项目“药用桑黄高效栽培技术及产品加工的研究与产业化示范”、湖南省重点研发计划项目“湖南优势种植业功能微生物种质资源挖掘选育与应用”等项目20余项。获长沙市农业标准制修订项目奖1项,获授权国家发明专利6项;制订省地方标准13项、企业标准3项;出版著作2部,在《Frontiers in Microbiology》《Journal of Ethnopharmacology》《南方农业学报》《农业生物技术学报》等国内外期刊上发表论文30余篇,E-mail:ashaochenxia@outlook.com;通信作者:许隽(1984-),https://orcid.org/0000-0002-9774-1811,博士,研究员,主要从事微生物功能研究及资源开发工作,E-mail:33022366@qq.com;通信作者:刘洋(1984-),https://orcid.org/0009-0007-9026-119X,博士,副研究员,主要从事农作物虫害绿色防控研究工作,E-mail:52073200@qq.com。
