不同表面灭菌对棉花茎秆内黄萎病菌分离效果的比较
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摘要
【目的】筛选一套标准的表面灭菌分离获得棉秆内黄萎病病原菌方法。【方法】研究4种抑制材料内细菌生长的PDA选择性培养基中最低抗生素含量,评价四种不同方法对材料表面灭菌(75%乙醇、5%次氯酸钠、0.1%升汞和75%乙醇+5%次氯酸钠)效果。【结果】含混合抗生素(链霉素:氯霉素:青霉素m/m,1∶1∶1)的PDA培养基抑制细菌最低浓度为500 mg/L;新鲜棉秆材料和轻度腐败棉秆表面灭菌的最佳条件为75%乙醇处理4 min,重度腐败棉秆表面灭菌条件为75%乙醇+5%NaClO处理5 min。【结论】即新鲜棉秆材料和轻度腐败棉秆材料表面灭菌的最佳条件为75%乙醇处理4 min,重度腐败棉秆表面灭菌条件为75%乙醇+5%NaClO处理5 min。选择合适的灭菌材料和时间是分离棉花茎杆内黄萎病菌的关键。
【Objective】 To screen a standard method of surface sterilization separation in the cotton Verticillium wilt pathogen. 【Method】 The lowest antibiotic content in PDA selective medium for inhibiting bacterial growth in four test materials was studied in this experiment, and four different methods were used to evaluate the effect of sterilization of the material surface(75% ethanol, 5% NaClO, 0.1% HgCl and 75% ethanol+5% NaClO). 【Results】 The results showed that the lowest concentration of mixed antibiotic(streptomycin: penicillin: chloramphenicol m/m, 1∶1∶1) in PDA medium that could restrain bacteria growth was 500 mg/L; The best sterilization condition for the surface sterilization of fresh material or mild spoilage cotton stalk was 75% ethanol treatment for 4 min, and the surface sterilization condition of severe spoilage cotton stalk was 75% ethanol+5%NaClO for 5 min. 【Conclusion】 Selection of suitable sterilizing material and time is the key to isolate cotton Verticillium wilt to a large extent.
【Objective】 To screen a standard method of surface sterilization separation in the cotton Verticillium wilt pathogen. 【Method】 The lowest antibiotic content in PDA selective medium for inhibiting bacterial growth in four test materials was studied in this experiment, and four different methods were used to evaluate the effect of sterilization of the material surface(75% ethanol, 5% NaClO, 0.1% HgCl and 75% ethanol+5% NaClO). 【Results】 The results showed that the lowest concentration of mixed antibiotic(streptomycin: penicillin: chloramphenicol m/m, 1∶1∶1) in PDA medium that could restrain bacteria growth was 500 mg/L; The best sterilization condition for the surface sterilization of fresh material or mild spoilage cotton stalk was 75% ethanol treatment for 4 min, and the surface sterilization condition of severe spoilage cotton stalk was 75% ethanol+5%NaClO for 5 min. 【Conclusion】 Selection of suitable sterilizing material and time is the key to isolate cotton Verticillium wilt to a large extent.
引文
[1]朱荷琴,李志芳,冯自力,等.我国棉花黄萎病研究十年回顾及展望[J].棉花学报,2017,29(增刊):37-50.ZHU He-qin,LI Zhi-fang,FENG Zi-li,et al.(2017).Overview of cotton verticillium wilt research over the past decade in China and its prospect in future [J].Cotton Science,29(S):37-50.(in Chinese)
[2] Carpenter C W.The Verticillium wilt problem [J].Phytopathology,1914,4:393.
[3]刘海洋,王伟,张仁福,等.新疆主要棉区棉花黄萎病发生概况[J].植物保护,2015,41(3):138-142.LIU Hai-yang,WANG Wei,ZHANG Ren-fu,et al.(2015).Occurrence overviews of cotton verticillium wilt in Xinjiang [J].Plant Protection,41(3):138-142.(in Chinese)
[4]王玲飞.棉花内生真菌多样性分析及对黄萎病生防潜力评估[D].北京:中国农业科学院硕士论文,2014.WANG Ling-fei.(2014).Diversity Analysis of endophytic fungi in cotton and evaluation of control potential of verticillium wilt [D].Master Dissertation.Chinese Academy of Agricultural Sciences,Beijing.(in Chinese)
[5] Júlia Kuklinsky-Sobral,Welington Luiz Araújo,Mendes,R.,Isaias Olívio Geraldi,Pizzirani-Kleiner,A.A.,& Lúcio Azevedo.(2004).Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion.Environmental microbiology,6(12):1,244-1,251.
[6] Welington L,Araújo,Joelma,M.,Walter,M.,Jan Dirk,V.E.,Vuurde,J.W.L.V.,et al.(2002).Diversity of endophytic bacterial populations and their interaction with xylella fastidiosa in citrus plants.Applied & Environmental Microbiology,68(10):4,906-4,914.
[7] Stéphane Compant,Mitter,B.,Colli-Mull,J.G..Gangl,H.,& Sessitsch,A.(2011).Endophytes of grapevine flowers,berries,and seeds:identification of cultivable bacteria,comparison with other plant parts,and visualization of niches of colonization.Microbial Ecology,62(1):188-197.
[8]张冬冬,李术娜,郭晓军,等.一株棉花黄萎病拮抗芽孢细菌的分离鉴定及活性检测[J].棉花学报,2012,24(4):358-362.ZHANG Dong-dong,LI Shu-na,GUO Xiao-jun,et al.(2012).Isolation,Identification and Activity of an Antagonistic Spore Bacterium against Cotton Verticillium Wilt [J].Cotton Science,24(4):358-362.(in Chinese)
[9] Zhu He-qin,Feng Zi-li,Li Zhi-fang,et al.(2013).Characterization of two fungal isolates from cotton and evaluation of their potential for biocontrol of verticillium wilt of cotton.Journal of Phytopathology,161(2):70-77.
[10] Wei,F.,Fan,R.,Dong,H.,Shang,W.,Xu,X.,& Zhu,H.,et al.(2015).Threshold microsclerotial inoculum for cotton verticillium wilt determined through wet-sieving and real-time quantitative PCR.Phytopathology,105(2):220-229.
[11]付慧娟,刘方,黎绍惠,等.半野生棉抗黄萎病筛选及根系分泌物对黄萎病菌的影响[J].棉花学报,2011,23(5):387-393.FU Hui-juan,LIU Fang,LI Shao-hui,et al.(2011).Screening of verticillium wilt resistance on gossypium hirsutum races and their root exudate effects on the wilt pathogen [J].Cotton Science,23(5):387-393.(in Chinese)
[12]张芸,冯自力,冯鸿杰,等.内生球毛壳属真菌CEF-082对棉花黄萎病的控制作用[J].植物病理学报,2016,46(5):697-706.ZHANG Yun,FENG Zi-li,FENG Hong-jie,et al.(2016).Control effect of endophytic fungus chaetomiun globosum CEF-082 against verticillium wilt in gossypium hirsutum[J].Acta Phytopathologica Sinica,46(5):697-706.(in Chinese)
[13]孔德真,黎玉顺,裴娟,等.新疆乌苏地区棉花黄萎病菌分离鉴定和致病力分析[J].中国农学通报,2015,31(20):61-66.KONG De-zhen,LI Yu-shun,PEI Juan,et al.(2015).Identification and pathogenicity analysis of cotton verticillium dahlia strains from Wusu of Xinjiang [J].Chinese Agricultural Science Bulletin,31(20):61-66.(in Chinese)
[14]朱荷琴,冯自力,李志芳,等.分离自棉花的轮枝菌"种"的鉴定[J].中国农业科学,2013,46(10):2,032-2,040.ZHU He-qin,FENG Zi-li,LI Zhi-fang,et al.(2013).Identification of isolates of verticillium species from cotton [J].Scientia Agricultura Sinica,46(10):2,032-2,040.(in Chinese)
[2] Carpenter C W.The Verticillium wilt problem [J].Phytopathology,1914,4:393.
[3]刘海洋,王伟,张仁福,等.新疆主要棉区棉花黄萎病发生概况[J].植物保护,2015,41(3):138-142.LIU Hai-yang,WANG Wei,ZHANG Ren-fu,et al.(2015).Occurrence overviews of cotton verticillium wilt in Xinjiang [J].Plant Protection,41(3):138-142.(in Chinese)
[4]王玲飞.棉花内生真菌多样性分析及对黄萎病生防潜力评估[D].北京:中国农业科学院硕士论文,2014.WANG Ling-fei.(2014).Diversity Analysis of endophytic fungi in cotton and evaluation of control potential of verticillium wilt [D].Master Dissertation.Chinese Academy of Agricultural Sciences,Beijing.(in Chinese)
[5] Júlia Kuklinsky-Sobral,Welington Luiz Araújo,Mendes,R.,Isaias Olívio Geraldi,Pizzirani-Kleiner,A.A.,& Lúcio Azevedo.(2004).Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion.Environmental microbiology,6(12):1,244-1,251.
[6] Welington L,Araújo,Joelma,M.,Walter,M.,Jan Dirk,V.E.,Vuurde,J.W.L.V.,et al.(2002).Diversity of endophytic bacterial populations and their interaction with xylella fastidiosa in citrus plants.Applied & Environmental Microbiology,68(10):4,906-4,914.
[7] Stéphane Compant,Mitter,B.,Colli-Mull,J.G..Gangl,H.,& Sessitsch,A.(2011).Endophytes of grapevine flowers,berries,and seeds:identification of cultivable bacteria,comparison with other plant parts,and visualization of niches of colonization.Microbial Ecology,62(1):188-197.
[8]张冬冬,李术娜,郭晓军,等.一株棉花黄萎病拮抗芽孢细菌的分离鉴定及活性检测[J].棉花学报,2012,24(4):358-362.ZHANG Dong-dong,LI Shu-na,GUO Xiao-jun,et al.(2012).Isolation,Identification and Activity of an Antagonistic Spore Bacterium against Cotton Verticillium Wilt [J].Cotton Science,24(4):358-362.(in Chinese)
[9] Zhu He-qin,Feng Zi-li,Li Zhi-fang,et al.(2013).Characterization of two fungal isolates from cotton and evaluation of their potential for biocontrol of verticillium wilt of cotton.Journal of Phytopathology,161(2):70-77.
[10] Wei,F.,Fan,R.,Dong,H.,Shang,W.,Xu,X.,& Zhu,H.,et al.(2015).Threshold microsclerotial inoculum for cotton verticillium wilt determined through wet-sieving and real-time quantitative PCR.Phytopathology,105(2):220-229.
[11]付慧娟,刘方,黎绍惠,等.半野生棉抗黄萎病筛选及根系分泌物对黄萎病菌的影响[J].棉花学报,2011,23(5):387-393.FU Hui-juan,LIU Fang,LI Shao-hui,et al.(2011).Screening of verticillium wilt resistance on gossypium hirsutum races and their root exudate effects on the wilt pathogen [J].Cotton Science,23(5):387-393.(in Chinese)
[12]张芸,冯自力,冯鸿杰,等.内生球毛壳属真菌CEF-082对棉花黄萎病的控制作用[J].植物病理学报,2016,46(5):697-706.ZHANG Yun,FENG Zi-li,FENG Hong-jie,et al.(2016).Control effect of endophytic fungus chaetomiun globosum CEF-082 against verticillium wilt in gossypium hirsutum[J].Acta Phytopathologica Sinica,46(5):697-706.(in Chinese)
[13]孔德真,黎玉顺,裴娟,等.新疆乌苏地区棉花黄萎病菌分离鉴定和致病力分析[J].中国农学通报,2015,31(20):61-66.KONG De-zhen,LI Yu-shun,PEI Juan,et al.(2015).Identification and pathogenicity analysis of cotton verticillium dahlia strains from Wusu of Xinjiang [J].Chinese Agricultural Science Bulletin,31(20):61-66.(in Chinese)
[14]朱荷琴,冯自力,李志芳,等.分离自棉花的轮枝菌"种"的鉴定[J].中国农业科学,2013,46(10):2,032-2,040.ZHU He-qin,FENG Zi-li,LI Zhi-fang,et al.(2013).Identification of isolates of verticillium species from cotton [J].Scientia Agricultura Sinica,46(10):2,032-2,040.(in Chinese)