烟草黑胫病菌、青枯病菌拮抗放线菌的筛选及控病研究
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摘要
烟草是我国重要的经济作物之一,近几年来,重庆烟区以烟草黑胫病、青枯病发病最为严重,造成大量经济损失。在烟草病害的多种防治方法中,生物防治对烟叶生产的可持续发展具有较好的应用前景。放线菌是生防微生物的重要来源,在植物病害的生物防治中占有重要地位。本试验从采自我国几大烟区的烟草根围土壤中分离筛选出对烟草黑胫病、青枯病病原菌有拮抗效果的放线菌,进行温室控病研究及菌株的鉴定,并进一步探讨其生防控病机理。具体结果如下:
1、从重庆、贵州、云南、河南、陕西、山东这几个我国主要的烟草生产地区共采集烟草根围土壤样品57份。在高氏一号培养基上采用平板稀释法分离获得415株放线菌。
2、将分离获得的415株放线菌分别采用平板对峙法和平板喷雾法对烟草黑胫病菌、烟草青枯病菌进行初筛,得到有拮抗活性的菌株178株,占分离总数的42.9%。其中对烟草黑胫病菌有拮抗活性的菌株134株,占分离总数的32.3%。对烟草青枯病菌有拮抗活性的菌株77株,占分离总数的18.6%。而对两种病害均有拮抗作用的活性菌株为33株,占分离总数的7.95%。
3、对初筛拮抗半径在10mm以上的烟草黑胫病菌拮抗菌以及初筛拮抗半径在5mm以上的烟草青枯病菌拮抗放线菌菌株进行复筛,测定单个菌株的平板抑菌效果。对烟草黑胫病菌抑菌带宽在15～35mm拮抗菌共有11株菌株,其中菌株HLL-4、BX 2-5-4抑菌带宽平均值为33.3mm和32mm;对烟草青枯病菌的抑菌圈直径在35～55mm的活性菌株有7株,菌株GAS 1-9和YYD 3-32抑菌圈直径均在50mm以上。
4、温室盆栽试验结果显示,参试拮抗菌株发酵原液对两种病害均有良好的防治效果。对烟草黑胫病的防治效果在38.13%～88.89%;相对防效50%以上的菌株8株,占总数的61.5%;防效在80%以上的菌株2株,菌株BX2-5-4的防治效果最好,达88.89%;其次为BX1-1,防效为88.13%。拮抗菌对烟草青枯病的防治效果在37.89%～90.06%;防效在60%以上的菌株有6个,占总数的85.7%。相对防效最高的菌株为SXL 1-7,防效90.06%;其次为菌株GAS1-9,相对防效为85.72%。
5、通过形态和培养特征的观察、生理生化的测定对平板抑菌和温室控病筛选出的13株拮抗菌进行分类鉴定,初步认为13株拮抗菌均为放线菌,其中链霉菌属(Streptromyces)6株,包括白孢类群、绿色类群、黄色类群和粉红孢类群弗氏亚群;链轮丝菌属(Streptroverticillium)3株;糖单孢菌属(Saccharomonospora)1株;鞘孢囊菌属(Elytrosporangium)1株;孢囊放线菌属(Actinosporangium)1株。另外对5株温室防效较好的菌株更深入的进行了16S rDNA序列及系统发育分析,结合其形态学及生理生化特性,确定菌株的分类地位。结果表明:菌株GAS1-9为娄彻氏链霉菌(Streptromyces rochei);BX1-1为变异链霉菌(Streptromycesvariabilis);BX2-5-4为吸水链霉菌(Streptromyces hygroscopicus);SYZ2-6为放线细菌(Actinomycetales bacterium);SXL1-7与皱褶链霉菌(Streptromycesplicatus)最相近。
6、对5株温室防效较好的菌株进行拮抗代谢产物分析,结果表明,参试菌株都具有良好的生防潜力。其中SYZ2-6和BX1-1均能产生几丁质酶、纤维素酶、蛋白酶以及嗜铁素等生防活性物质。
Tobacco black shank caused by Phytophthora parasitica,tobacco bacterial wilt caused by Ralstonia solannacearum,are the important diseases with serious loss in the tobacco area of Chongqing.Biocontrol is an promising measure of methods in tobacco disease control.Actinomycetes is important source with potential biocontrol effect.In this study,some actinomyces strains with antagonistic Phytophthora parasitica & Ralstonia solannacearum were isolated from tobacco rhizosphere soil in main tobacco areas of China,the control efficacy at greenhouse experiment,taxonomic identification,and the biocontrol mechanism by analyzed.
1.57 soil samples of tobacco fields were collected from Chonging,Guizhou, Yunnan,Henan,Shannxi and Shandong provinces,the main tobacco planting areas of China.A total number of 415 isolates of actinomycetes were isolated by dilution cultured on Gause's Synthetic Agar plate.
2.The all of actinomycetes isolates by tested with the Phytophthora parasitica by pairing culture on plate method,and with the Ralstonia solannacearum by plate spraying method.And 178(42.9%of the all isolates) were found to be antagonistic effect against the pathogen.134(32.3%of the all isolates) against Phytophthora parasitica,which i.e.were 77 isolates(18.6%) agianst Ralstonia solannacearum and the 33 isolates(7.95%) can antagonistic the both pathogens.
3.Farther screening were carried out by each strain with plate inhibition effect against Phytophthora parasitica over 10 mm inhibition radius and against Ralstonia solannacearum over 5 mm inhabition radius.The results showed that 11 strains had good antagonistic effects on Phytophthora parasitica,and the inhibition zone diameters ranged from 15 to 35 mm,in which the inhibition zone of strain HLL3-4 and BX2-5-4 were 33.3 mm and 32mm,respectively.7 strains which against Ralstonia solannacearum were found the inhibition zone diameters ranged from 35 to 55mm,the inhibition diameters of strain GAS1-9 and YYD3-32 were over than 50mm.
4.The results showed that the zymolysis liquid of all the actinomycetes had good biocontrol effects against the two diseases at the greenhouse experiments.The biocontrol effect against Phytophthora parasitica ranged from 38.13%to 88.89%.In which have 8(61.5%of the all strains)strains with relative efficiency over 50%,2 strains with relative efficiency over 80%were BX 2-5-4 strain and BX 1-1 strain, which the control effect reach to 88.89%and 88.13%,respectively..The biocontrol effect of actinomycetes against Ralstonia solannacearum ranged from 37.89%to 90.06 %.In which have 6(85.7%of the all strains) with relative efficiency over 60%,the strains SXL1-7 had the best control effect of 90.06%,followed the strain GAS1-9 with the control effect of 85.72%.
5.Based on the characteristics of morphology and physiology-biochemistry,plate screening and greenhouse experiment,13 antagonsists were primarily identified as actinomycetes.In which 6 strains belong to Streptromyces including Albosporus, Viridis,Flavus,Roseosporus Fradiae,and 3 strains belong to Streptroverticillium,other 3 stains belong to Saccharomonospora,Elytrosporangium and Actinosporangium respectively.Furthermore,5 strains had good biocontrol effect at greenhouse were analyzed with morphology,physiological and biochemical properties,16S rDNA phylogenetic tree,the results showed that the GAS1-9 was identified as Streptromyces rochei,BX1-1 was Streptromyces variabilis,BX2-5-4 was Streptromyces hygroscopicus,SYZ2-6 was Actinomycetales bacterium,SXL1-7 was Streptromyces plicatus.
6.Analysis of the metabolic producetions of the five strains,the results indicated all of the strains have good biocontrol effects,and the strains SYZ2-6 and BX1-1 could produce active substance for biocontrol,such as chitinase,cellulases,proteases, siderophores and so on.
1、从重庆、贵州、云南、河南、陕西、山东这几个我国主要的烟草生产地区共采集烟草根围土壤样品57份。在高氏一号培养基上采用平板稀释法分离获得415株放线菌。
2、将分离获得的415株放线菌分别采用平板对峙法和平板喷雾法对烟草黑胫病菌、烟草青枯病菌进行初筛,得到有拮抗活性的菌株178株,占分离总数的42.9%。其中对烟草黑胫病菌有拮抗活性的菌株134株,占分离总数的32.3%。对烟草青枯病菌有拮抗活性的菌株77株,占分离总数的18.6%。而对两种病害均有拮抗作用的活性菌株为33株,占分离总数的7.95%。
3、对初筛拮抗半径在10mm以上的烟草黑胫病菌拮抗菌以及初筛拮抗半径在5mm以上的烟草青枯病菌拮抗放线菌菌株进行复筛,测定单个菌株的平板抑菌效果。对烟草黑胫病菌抑菌带宽在15～35mm拮抗菌共有11株菌株,其中菌株HLL-4、BX 2-5-4抑菌带宽平均值为33.3mm和32mm;对烟草青枯病菌的抑菌圈直径在35～55mm的活性菌株有7株,菌株GAS 1-9和YYD 3-32抑菌圈直径均在50mm以上。
4、温室盆栽试验结果显示,参试拮抗菌株发酵原液对两种病害均有良好的防治效果。对烟草黑胫病的防治效果在38.13%～88.89%;相对防效50%以上的菌株8株,占总数的61.5%;防效在80%以上的菌株2株,菌株BX2-5-4的防治效果最好,达88.89%;其次为BX1-1,防效为88.13%。拮抗菌对烟草青枯病的防治效果在37.89%～90.06%;防效在60%以上的菌株有6个,占总数的85.7%。相对防效最高的菌株为SXL 1-7,防效90.06%;其次为菌株GAS1-9,相对防效为85.72%。
5、通过形态和培养特征的观察、生理生化的测定对平板抑菌和温室控病筛选出的13株拮抗菌进行分类鉴定,初步认为13株拮抗菌均为放线菌,其中链霉菌属(Streptromyces)6株,包括白孢类群、绿色类群、黄色类群和粉红孢类群弗氏亚群;链轮丝菌属(Streptroverticillium)3株;糖单孢菌属(Saccharomonospora)1株;鞘孢囊菌属(Elytrosporangium)1株;孢囊放线菌属(Actinosporangium)1株。另外对5株温室防效较好的菌株更深入的进行了16S rDNA序列及系统发育分析,结合其形态学及生理生化特性,确定菌株的分类地位。结果表明:菌株GAS1-9为娄彻氏链霉菌(Streptromyces rochei);BX1-1为变异链霉菌(Streptromycesvariabilis);BX2-5-4为吸水链霉菌(Streptromyces hygroscopicus);SYZ2-6为放线细菌(Actinomycetales bacterium);SXL1-7与皱褶链霉菌(Streptromycesplicatus)最相近。
6、对5株温室防效较好的菌株进行拮抗代谢产物分析,结果表明,参试菌株都具有良好的生防潜力。其中SYZ2-6和BX1-1均能产生几丁质酶、纤维素酶、蛋白酶以及嗜铁素等生防活性物质。
Tobacco black shank caused by Phytophthora parasitica,tobacco bacterial wilt caused by Ralstonia solannacearum,are the important diseases with serious loss in the tobacco area of Chongqing.Biocontrol is an promising measure of methods in tobacco disease control.Actinomycetes is important source with potential biocontrol effect.In this study,some actinomyces strains with antagonistic Phytophthora parasitica & Ralstonia solannacearum were isolated from tobacco rhizosphere soil in main tobacco areas of China,the control efficacy at greenhouse experiment,taxonomic identification,and the biocontrol mechanism by analyzed.
1.57 soil samples of tobacco fields were collected from Chonging,Guizhou, Yunnan,Henan,Shannxi and Shandong provinces,the main tobacco planting areas of China.A total number of 415 isolates of actinomycetes were isolated by dilution cultured on Gause's Synthetic Agar plate.
2.The all of actinomycetes isolates by tested with the Phytophthora parasitica by pairing culture on plate method,and with the Ralstonia solannacearum by plate spraying method.And 178(42.9%of the all isolates) were found to be antagonistic effect against the pathogen.134(32.3%of the all isolates) against Phytophthora parasitica,which i.e.were 77 isolates(18.6%) agianst Ralstonia solannacearum and the 33 isolates(7.95%) can antagonistic the both pathogens.
3.Farther screening were carried out by each strain with plate inhibition effect against Phytophthora parasitica over 10 mm inhibition radius and against Ralstonia solannacearum over 5 mm inhabition radius.The results showed that 11 strains had good antagonistic effects on Phytophthora parasitica,and the inhibition zone diameters ranged from 15 to 35 mm,in which the inhibition zone of strain HLL3-4 and BX2-5-4 were 33.3 mm and 32mm,respectively.7 strains which against Ralstonia solannacearum were found the inhibition zone diameters ranged from 35 to 55mm,the inhibition diameters of strain GAS1-9 and YYD3-32 were over than 50mm.
4.The results showed that the zymolysis liquid of all the actinomycetes had good biocontrol effects against the two diseases at the greenhouse experiments.The biocontrol effect against Phytophthora parasitica ranged from 38.13%to 88.89%.In which have 8(61.5%of the all strains)strains with relative efficiency over 50%,2 strains with relative efficiency over 80%were BX 2-5-4 strain and BX 1-1 strain, which the control effect reach to 88.89%and 88.13%,respectively..The biocontrol effect of actinomycetes against Ralstonia solannacearum ranged from 37.89%to 90.06 %.In which have 6(85.7%of the all strains) with relative efficiency over 60%,the strains SXL1-7 had the best control effect of 90.06%,followed the strain GAS1-9 with the control effect of 85.72%.
5.Based on the characteristics of morphology and physiology-biochemistry,plate screening and greenhouse experiment,13 antagonsists were primarily identified as actinomycetes.In which 6 strains belong to Streptromyces including Albosporus, Viridis,Flavus,Roseosporus Fradiae,and 3 strains belong to Streptroverticillium,other 3 stains belong to Saccharomonospora,Elytrosporangium and Actinosporangium respectively.Furthermore,5 strains had good biocontrol effect at greenhouse were analyzed with morphology,physiological and biochemical properties,16S rDNA phylogenetic tree,the results showed that the GAS1-9 was identified as Streptromyces rochei,BX1-1 was Streptromyces variabilis,BX2-5-4 was Streptromyces hygroscopicus,SYZ2-6 was Actinomycetales bacterium,SXL1-7 was Streptromyces plicatus.
6.Analysis of the metabolic producetions of the five strains,the results indicated all of the strains have good biocontrol effects,and the strains SYZ2-6 and BX1-1 could produce active substance for biocontrol,such as chitinase,cellulases,proteases, siderophores and so on.
引文
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