Screening and identification of actinomycetes for biological control of Bursaphelenchus xylophilus and preliminary study on their toxicity factors
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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。Abstract:ObjectiveThis paper aims to screen and identify the actinomycetes with high killing activity against pine wood nematodes Bursaphelenchus xylophilus from soil, and determine their virulent factors.MethodUsing Sarcina lutea, Bacillus subtilis and Escherichia coli as testing strains to determine the antibacterial activities, the actimomycetes with biocontrol activities to B. xylophilus were screened from the soil samples of Nanyang Normal University campus, Baihe Edge, Baotianman and other fields. Morphological observation and molecular identifications based on 16S rRNA sequence analysis were used to classify and identify the strain with the highest activities. The activity of the strain against pine wood nematode was tested, and the virulent substance was isolated and purified.ResultFour actinomycete strains with antibacterial activity were screened: C611, C612, C614 and C619. The strain C611 showed the highest inhibitory activity against S. lutea, B. subtilis and E. coli among the four strains. The strain was identified as Streptomyces sp. according to the morphological and phylogenetic analysis. After 7 days of treatment of pine wood nematode with C611 fermentation broth, the mortality rate of nematode reached 85%, and that of blank control group was 0. The active ingredient was determined to be furan ketone (furaltadone) after separation and purification of C611 fermentation broth.The nematode mortality reached 96% after 5 days treated with 0.1% pure furatanone solution.ConclusionThe actinomycete C611 was isolated and identified furaltadone as a key virulence factor in this study, which enriched the species of biological control microbe against pine wood nematodes. It was first reported that furantone had strong toxicity to nematodes and laid a theoretical foundation for biological control of pine wood nematode.
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Keywords:
- actinomyces /
- antibacterial activity /
- pine wood nematode /
- death rate /
- active substance
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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
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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
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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
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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
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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 coliC611 +++ ++ +++ 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. 
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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
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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 contrast4 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.
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