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松材线虫生防放线菌的筛选、鉴定及其毒性因子初步研究

黄冰纷 陈俊梅 李文鹏 宋希磊 王博文 张成 赵素雅 牛秋红

黄冰纷, 陈俊梅, 李文鹏, 宋希磊, 王博文, 张成, 赵素雅, 牛秋红. 松材线虫生防放线菌的筛选、鉴定及其毒性因子初步研究[J]. 北京林业大学学报, 2019, 41(4): 99-106. doi: 10.13332/j.1000-1522.20180302
引用本文: 黄冰纷, 陈俊梅, 李文鹏, 宋希磊, 王博文, 张成, 赵素雅, 牛秋红. 松材线虫生防放线菌的筛选、鉴定及其毒性因子初步研究[J]. 北京林业大学学报, 2019, 41(4): 99-106. doi: 10.13332/j.1000-1522.20180302
Huang Bingfen, Chen Junmei, Li Wenpeng, Song Xilei, Wang Bowen, Zhang Cheng, Zhao Suya, Niu Qiuhong. Screening and identification of actinomycetes for biological control of Bursaphelenchus xylophilus and preliminary study on their toxicity factors[J]. Journal of Beijing Forestry University, 2019, 41(4): 99-106. doi: 10.13332/j.1000-1522.20180302
Citation: Huang Bingfen, Chen Junmei, Li Wenpeng, Song Xilei, Wang Bowen, Zhang Cheng, Zhao Suya, Niu Qiuhong. Screening and identification of actinomycetes for biological control of Bursaphelenchus xylophilus and preliminary study on their toxicity factors[J]. Journal of Beijing Forestry University, 2019, 41(4): 99-106. doi: 10.13332/j.1000-1522.20180302

松材线虫生防放线菌的筛选、鉴定及其毒性因子初步研究

doi: 10.13332/j.1000-1522.20180302
基金项目: 河南省高校科技创新人才支持计划项目(17HASTIT041),研究生创新基金项目(2017cx008、2018cx010)
详细信息
    作者简介:

    黄冰纷。主要研究方向:线虫生物防治。Email:1365914254@qq.com 地址:473000 河南省南阳市卧龙区卧龙路1638号南阳师范学院

    责任作者:

    牛秋红,博士,教授。主要研究方向:微生物学与分子生物学。Email:qiuhongniu723@163.com 地址:同上

  • 中图分类号: S763.306.4

Screening and identification of actinomycetes for biological control of Bursaphelenchus xylophilus and preliminary study on their toxicity factors

  • 摘要: 目的从土壤中筛选、鉴定出对松材线虫有较高杀灭活性的放线菌,并确定其毒力因子。方法以藤黄八叠球菌、枯草杆菌、大肠杆菌3个敏感菌株为抑菌检测对象,从南阳师范学院校园内、白河边、宝天曼、卧龙岗等地采集的土壤样品中筛选有抑菌活性的放线菌,采用形态学观察以及基于16S rRNA序列分析等方法对抑菌活性最高的菌株进行分类鉴定,将该菌株进行杀松材线虫活性测试,并分离纯化出杀线活性物质。结果筛选出4株有抑菌活性的放线菌菌株:C611、C612、C614、C619,其中C611菌株对藤黄八叠球菌、枯草杆菌和大肠杆菌均有较高抑制活性。结合该菌株形态学、生理学特征以及16S rRNA序列分析等结果,将其初步确定为链霉菌属。将C611发酵液处理松材线虫7 d后,线虫死亡率高达85%,空白对照组死亡率为0,对C611发酵液进行分离纯化后,初步确定活性成分为呋喃它酮。使用含量为0.1%呋喃它酮纯溶液测试杀线虫活性,作用于线虫5 d后其死亡率高达96%。结论本研究筛选到的生防放线菌C611,鉴定出其活性物质为呋喃它酮,丰富了松材线虫生防菌的种类,本研究首次发现呋喃它酮对线虫有较强毒杀作用,为松材线虫生物防治奠定了一定的理论基础。

     

  • 图  1  C611对3个敏感菌株的抑制结果

    A. C611对大肠杆菌的抑制结果;B. C611对枯草芽孢杆菌的抑制结果;C. C611对藤黄八叠球菌的抑制结果。A, inhibitory effect on E. coli of C611; B, inhibitory effect on B. subtilis of C611; C, inhibitory effect of C611 on S. lutea.

    Figure  1.  Inhibitory effects of C611 on three sensitive strains

    图  2  4个菌株菌落形态

    Figure  2.  Colony morphology of 4 strains

    图  3  C611菌丝及孢子丝

           A. C611菌丝形态;B. C611孢子丝形态。A, hyphae morphology of C611; B, spore silk morphology of C611.

    Figure  3.  Hyphae and spore filaments of C611

    图  4  放线菌分离株C611的系统发育学分析

    发育树节点的数字表示Bootstrap值;括号中的数字为序列登录号。The numbers at each branch points indicate Bootstrap value; numbers in paretheses are accession No. of sequence in GenBank.

    Figure  4.  Phylogenetic analysis of C611 based on 16S rRNA sequence

    图  5  C611发酵液及空白处理后的松材线虫形态

    A. 无菌水对照处理7 d后的松材线虫形态;B. C611发酵液处理7 d后的松材线虫形态。A, the morphology of Bursaphelenchus xylophilus after 7 days treated by C611 fermentation liquor; B, the morphology of Bursaphelenchus xylophilus after 7 days treated by aseptic water.

    Figure  5.  Morphology of Bursaphelenchus xylophilus after treated by C611 fermentation broth and blank control

    图  6  C611杀线虫活性物质色谱分析图

    Figure  6.  Chromatographic analysis of active substances of C611

    图  7  0.1%呋喃它酮及空白处理后的松材线虫形态

    A. 无菌水处理4 h后线虫形态;B. 0.1%呋喃它酮处理4 h处理后线虫形态;C. 无菌水处理20 h后线虫形态;D. 0.1%呋喃它酮处理20 h处理后线虫形态。A, the morphology of Bursaphelenchus xylophilus after 4 hours treated by aseptic water;B, the morphology of B. xylophilus after 4 hours treated by 0.1% furaltadone;C, the morphology of B. xylophilus after 20 hours treated by aseptic water; D, the morphology of B. xylophilus after 20 hours treated by 0.1% furaltadone.

    Figure  7.  Morphology of Bursaphelenchus xylophilus after treated by 0.1% furaltadone and blank control

    表  1  抑菌测试结果对比

    Table  1.   Comparison of the inhibitory effect of actinomycetes on different test strains

    菌株编号
    Strain No.
    藤黄八叠球菌
    Sarcina lutea
    枯草杆菌
    Bacillus subtilis
    大肠杆菌
    Escherichia coli
    C611 +++ ++ +++
    C612 + +++ ++
    C614 +
    C619 ++ +
    注:“+”表示抑菌效果,“−”表示无抑菌效果,“+”越多代表抑菌效果越好。Notes: “+” means bacteriostatic effect, “−” means no bacteriostatic effect, more “+” means better antibacterial effects.
    下载: 导出CSV

    表  2  C611发酵液杀线虫活性

    Table  2.   Activity of killing nematode of C611 fermentation broth %

    组别 Group 死亡率 Mortality
    3 d 4 d 5 d 6 d 7 d
    C611 30 40 50 60 85
    空白对照 Blank contrast 0 0 0 0 0
    下载: 导出CSV

    表  3  0.1%呋喃它酮杀线虫活性

    Table  3.   Nematode activity of 0.1% furazolidone %

    时间 Time 死亡率 Mortality
    0.1%呋喃它酮
    0.1% furazolidone
    空白对照
    Blank contrast
    4 h 10 (0) 0 (0)
    1 d 30.33 (0.58) 0 (0)
    2 d 41.67 (2.89) 0 (0)
    3 d 71.67 (2.89) 0 (0)
    4 d 91 (1.73) 1 (0)
    5 d 96 (1.73) 1.33 (0.58)
    注:括号内数据为标准差。Note: data in paretheses are standard deviation.
    下载: 导出CSV
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  • 收稿日期:  2018-09-27
  • 修回日期:  2018-11-23
  • 刊出日期:  2019-04-01

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