棉花黄萎病生防菌的筛选及挥发性抑菌物质检测
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摘要
旨在筛选具有高效抑制棉花黄萎菌的芽孢杆菌并对其挥发性抑菌物质进行检测。采用稀释涂布平板法从棉花、玉米、向日葵根际以及苦豆子植株内分离得到菌株共65株,通过初筛、复筛共筛选到1株高效根际拮抗菌X4和内生菌N4,它们对棉花黄萎菌有较强抑制作用;通过 16S rDNA基因序列分析进行分子鉴定,平板对峙法检测2株生防菌对棉花黄萎菌的抑菌活性,生防菌发酵滤液处理法检测对棉花黄萎菌菌丝生长、孢子萌发、微菌核萌发、毒力蛋白产量的影响,GC-MS法对2株生防菌挥发性抑菌物质进行检测。结果表明:经分子检测2株高效拮抗菌均为芽孢杆菌;经2株1生防菌发酵滤液处理的棉花黄萎菌的菌丝生长受到抑制,菌株N4的抑制率为65.15%、菌株X4的抑制率为58.82%,且菌丝出现严重形变;经处理的黄萎菌孢子萌发率、微菌核萌发率、毒力蛋白产量均显著下降;研究发现2株拮抗菌的挥发性物质具有抑菌作用,GC-MS法检测2株菌发挥性物质主要是2,3-丁二醇、3-甲基丁酸、2-甲基丁酸和异丁酸,纯品物质检验发现2-甲基丁酸、3-甲基丁酸和异丁酸对棉花黄萎菌均有抑菌活性,2,3-丁二醇没有抑菌活性。可见:2个菌株对棉花黄萎菌具有潜在生防能力,可为生防菌剂的研发提供优良菌种。
To screen the Bacillus with high inhibition to cotton Verticillium and to detect the volatile antimicrobial substance,65 strains were isolated from cotton, maizeand sunflower rhizosphere and Sophora alopecuroides plant by plate dilution method. After primary and secondary screening, two strains of Bacillus with strong inhibitory effect on cotton Verticillium were obtained,that was X4 isolated from rhizosphere and N4 isolated from plant.Molecular identification was done to analyze gene sequence of 16 S rDNA. Plate confrontation method was used to detect biocontrol effects on cotton Verticillium;the aseptic fermentation of the biocontrol Bacillus was used toevaluate the influence on mycelial growth, spore germination of microsclerotia, germination and virulence protein of cotton Verticillium were detected; the antimicrobial substance was detected by GC-MS method.The results showed that these two antagonistic strains were identified as Bacillus by molecular detection; the growth of cotton Verticillium mycelium was strongly inhibited by sterile fermentation liquid treatment of biocontrol Bacillus,with the inhibition rate, 65.15% and 58.82%, of strain N4 and X4 respectively,accompanied by severe deformation of mycelium;under the treatments of antagonistic bacteria, the spore germination rate, microsclerotium germination rate, and the virulence protein yield of Verticillium were significantly decreased;the dominant volatile substances of two strains were 2-methyl butyric acid, 3-methybutyric acidisobutyric acid and 2,3-butanediol detected by GC-MS. The inspection with pure substance showed that,with the exception of 2,3-butanediol,2-methyl butyric acidand 3-methyl butyric acid and isobutyric acid had antibacterial ability tocotton Verticillium. In conclusion,Bacillus N4 and X4 have strong antibacterial ability on Verticillium, and the volatile substances, 2-methyl butyric acid, 3-methyl butyric acid and isobutyric acid have strong inhibitory effect, which indicated that bacillus N4 and X4 are good potential biocontrol resources, andused to develop excellent biocontrolstrains.
To screen the Bacillus with high inhibition to cotton Verticillium and to detect the volatile antimicrobial substance,65 strains were isolated from cotton, maizeand sunflower rhizosphere and Sophora alopecuroides plant by plate dilution method. After primary and secondary screening, two strains of Bacillus with strong inhibitory effect on cotton Verticillium were obtained,that was X4 isolated from rhizosphere and N4 isolated from plant.Molecular identification was done to analyze gene sequence of 16 S rDNA. Plate confrontation method was used to detect biocontrol effects on cotton Verticillium;the aseptic fermentation of the biocontrol Bacillus was used toevaluate the influence on mycelial growth, spore germination of microsclerotia, germination and virulence protein of cotton Verticillium were detected; the antimicrobial substance was detected by GC-MS method.The results showed that these two antagonistic strains were identified as Bacillus by molecular detection; the growth of cotton Verticillium mycelium was strongly inhibited by sterile fermentation liquid treatment of biocontrol Bacillus,with the inhibition rate, 65.15% and 58.82%, of strain N4 and X4 respectively,accompanied by severe deformation of mycelium;under the treatments of antagonistic bacteria, the spore germination rate, microsclerotium germination rate, and the virulence protein yield of Verticillium were significantly decreased;the dominant volatile substances of two strains were 2-methyl butyric acid, 3-methybutyric acidisobutyric acid and 2,3-butanediol detected by GC-MS. The inspection with pure substance showed that,with the exception of 2,3-butanediol,2-methyl butyric acidand 3-methyl butyric acid and isobutyric acid had antibacterial ability tocotton Verticillium. In conclusion,Bacillus N4 and X4 have strong antibacterial ability on Verticillium, and the volatile substances, 2-methyl butyric acid, 3-methyl butyric acid and isobutyric acid have strong inhibitory effect, which indicated that bacillus N4 and X4 are good potential biocontrol resources, andused to develop excellent biocontrolstrains.
引文
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[6]喻梦花.棉花黄萎病防治[J].农业与技术,2013(1):111.YU M H.Prevention and control of cotton Verticillium wilt[J].Agriculture&Technology,2013(1):111.
[7]MAZZOLA M,MANICA L M.Apple replant disease:roleof microbial ecology in cause and control[J].Annual Review of Phytopathology,2012,50(1):45.
[8]杨金霞.柑橘木虱内生细菌种类多样性[D].福州:福建农林大学,2013.YANG J X.Species diversity of endophytic bacteria in Citrus sinensis[D].Fuzhou:Fujian Agriculture and Forestry University,2013.
[9]彭研,陈相艳,裘纪莹,等.生防芽孢杆菌的研究进展[J].山东农业科学,2013,45(7):138-140.PENG Y,CHEN X Y,QIU J Y,et al.Research progress of Bacillus subtilis[J].Shandong Agricultural Sciences,2013,45(7):138-140.
[10]曹君,高智谋,潘月敏,等.枯草芽孢杆菌BS菌株和哈茨木霉TH-1菌株对棉花枯黄萎病菌的拮抗作用[J].植物病理学报,2005(S1):170-172.CAO J,GAO ZH M,PAN Y M,et al.Antagonistic effects of Bacillus subtilis strain BS and Trichoderma harzianum TH-1strain on Verticillium dahliae[J].Chinese Journal of Plant Pathology,2005(S1):170-172.
[11]李宝庆,鹿秀云,郭庆港,等.枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定[J].中国农业科学,2010,43(17):3547-3554.LI B Q,LU X Y,GUO Q G,et al.Separation and identification of lipopeptides and volatile substances from Bacillus subtilis BAB-1[J].Scientia Agricultura Sinica,2010,43(17):3547-3554.
[12]FERNANDO W G D,RAMAARATHNAM R,KRISH-NAMOORTHY A S,et al.Identification and use of potential bacterial organic antifungal volatiles in biocontrol[J].Soil Biology&Biochemistry,2005,37(5):955-964.
[13]车建美,郭慧慧,刘波,等.产挥发性物质芽胞杆菌对枇杷炭疽病菌的抑制作用及其鉴定[J].福建农业学报,2014(5):469-474.CHE J M,GUO H H,LIU B,et al.Inhibition and identification of Bacillus sp.producing Bacillus anthracis[J].Fujian Journal of Agricultural Sciences,2014(5):469-474.
[14]汪远.海洋细菌AiL3和Kc-38菌株防治芒果炭疽病研究[D].广东湛江:广东海洋大学,2010.WANG Y.Study on the control of mango anthracnose by marine bacteria AiL3and Kc-38[D].Zhanjiang Guangdong:Guangdong Ocean University,2010.
[15]张金凤,韩琴,王晓楠,等.解淀粉芽孢杆菌41B-1R对棉花黄萎病的防效研究[J].核农学报,2016,30(3):468-475.ZHANG J F,HAN Q,WANG X N,et al.Study on the control effect of Bacillus amyloliquefaciens 41B-1R on cotton Verticillium wilt[J].Journal of Nuclear Agricultural Sciences,2016,30(3):468-475.
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[17]郎剑锋,杨蕊,赵荣艳.对玉米茎基腐病菌有拮抗活性木霉菌株的筛选[J].安徽农业科学,2010,38(5):2419-2420.LANG J F,YANG R,ZHAO R Y.Screening of Trichoderma strain against antagonistic activity of maize stem-based rot pathogen[J].Anhui Agricultural Sciences,2010,38(5):2419-2420.
[18]曹明慧,冉炜,杨兴明,等.烟草黑胫病拮抗菌的筛选及其生物效应[J].土壤学报,2011,48(1):151-159.CAO M H,RAN W,YANG X M,et al.Screening of antagonistic bacteria against tobacco black shank and its biological effects[J].Journal of Soil Science,2011,48(1):151-159.
[19]张霞,武志芳,张胜潮,等.贵州浓香型白酒大曲中霉菌的18SrDNA系统发育分析[J].应用与环境生物学报,2011(3):334-337.ZHANG X,WU ZH F,ZHANG SH CH,et al.Development of 18SrDNA system of mold in Guizhou Luzhou-flavor liquor[J].Journal of Applied and Environmental Biology,2011(3):334-337.
[20]尹敬芳,张文华,李健强,等.辣椒疫病生防菌的筛选及其抑菌机制初探[J].植物病理学报,2007,37(1):88-94.YIN J F,ZHANG W H,LI J Q,et al.Screening of biocontrol bacteria against pepper blight and its antibacterial mechanism[J].Chinese Journal of Plant Pathology,2007,37(1):88-94.
[21]田绍仁,聂太礼,王梦亮,等.拮抗细菌C-02防治棉花黄萎病的机理研究[J].棉花学报,2012,24(5):420-426.TIAN SH R,NIE T L,WANG M L,et al.Mechanism of antagonistic bacterium C-02against cotton Verticillium wilt[J].Cotton Science,2012,24(5):420-426.
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