胶孢炭疽菌生防放线菌SD-29的鉴定及抗菌活性评价
|
摘要
【背景】胶孢炭疽菌是引起橡胶炭疽病的一种重要病原菌,可导致橡胶树产胶量下降。【目的】从山东青岛一农田土壤中分离出一株胶孢炭疽菌生防放线菌SD-29,并对其进行鉴定及抗菌活性评价。【方法】采用对峙生长法及菌丝生长速率法对菌株SD-29的拮抗活性进行鉴定;利用乙酸乙酯萃取法提取菌株SD-29发酵液粗提物并进行活性评价;根据菌株SD-29的形态特征、生理生化及16S rRNA基因序列进行鉴定。【结果】菌株SD-29对胶孢炭疽菌具有较强的抑制活性,皿内抑制活性达到82.6%。发酵液粗提物对菌丝生长的EC50为13.6μg/mL,100μg/mL的粗提物对胶孢炭疽菌孢子萌发抑制率达到63.16%,其对感炭疽病橡胶叶片的防治效果达到48.96%。根据该菌的形态特征、生理生化特征及16S rRNA基因序列分析鉴定菌株SD-29为Streptomyces yatensis。【结论】菌株SD-29对胶孢炭疽菌有较强的防治效果,具有潜在的应用价值。
[Background] Colletotrichum gloeosporioides can infect rubber tree, and decrease rubber production. [Objective] An antagonistic strain SD-29 against C. gloeosporioides was isolated from farmland soil in Qingdao, Shandong province. The strain was identified, and its antifungal activity was evaluated.[Methods] The antifungal activity of strain SD-29 was determined using the methods of confrontation growth and mycelium growth rate. The crude extract of the fermentation broth was obtained by ethyl acetate extraction, and its activity was also evaluated. Then, strain SD-29 was identified based on morphology,physiological, biochemical characteristics and 16S rRNA sequence. [Results] Strain SD-29 had a strong inhibitory activity to C. gloeosporioides with the inhibition rate of 82.6%. The EC50 of fermentation broth extractions was 13.6 μg/mL. Crude extract(100 μg/mL) inhibited conidial germination with the inhibition rate of 63.16%, and its control effect on the rubber leaves infected with C. gloeosporioides was up to 48.96%.According to the results of morphology, physiological, biochemical characteristics and 16S rRNA sequence,strain SD-29 was identified as Streptomyces yatensis. [Conclusion] Strain SD-29 has a strong control effect on C. gloeosporioides.
[Background] Colletotrichum gloeosporioides can infect rubber tree, and decrease rubber production. [Objective] An antagonistic strain SD-29 against C. gloeosporioides was isolated from farmland soil in Qingdao, Shandong province. The strain was identified, and its antifungal activity was evaluated.[Methods] The antifungal activity of strain SD-29 was determined using the methods of confrontation growth and mycelium growth rate. The crude extract of the fermentation broth was obtained by ethyl acetate extraction, and its activity was also evaluated. Then, strain SD-29 was identified based on morphology,physiological, biochemical characteristics and 16S rRNA sequence. [Results] Strain SD-29 had a strong inhibitory activity to C. gloeosporioides with the inhibition rate of 82.6%. The EC50 of fermentation broth extractions was 13.6 μg/mL. Crude extract(100 μg/mL) inhibited conidial germination with the inhibition rate of 63.16%, and its control effect on the rubber leaves infected with C. gloeosporioides was up to 48.96%.According to the results of morphology, physiological, biochemical characteristics and 16S rRNA sequence,strain SD-29 was identified as Streptomyces yatensis. [Conclusion] Strain SD-29 has a strong control effect on C. gloeosporioides.
引文
[1]Lan JQ.Study on the taxonomy of Colletotrichum Corda et al.in China and biological peculiarity of Colletotrichum gloeosporioides(Penz.)Sacc.in Mango[D].Yangling:Doctoral Dissertation of Northwest A&F University,2012(in Chinese)兰建强.我国炭疽菌属Colletotrichum部分种分类及芒果胶孢炭疽菌生物学特性研究[D].杨凌:西北农林科技大学博士学位论文,2012
[2]Zhang GZ,Zhang C,Jin BC,et al.Toxicity determination of fungicide to fungal pathogens causing Anthranenose and Gray Mold of strawberry[A]//Advances in Strawberry Research IV[C].Beijing:Chinese Society for Horticultural Science,2015:4(in Chinese)张国珍,张璨,靳宝川,等.几种杀菌剂对草莓炭疽菌和灰霉病菌的室内毒力测定[A]//草莓研究进展(IV)[C].北京:中国园艺学会,2015:4
[3]Cao XL,Liu HJ,Guo XJ,et al.Preliminary isolation and identification of the antagonistic bacterial strain 5A5-3 against common orchid diseases[J].Forest Research,2013,26(5):598-602(in Chinese)曹晓璐,刘慧娟,郭晓军,等.兰花炭疽病拮抗细菌5A5-3菌株的初步筛选和鉴定[J].林业科学研究,2013,26(5):598-602
[4]Luo SS.Isolation,screening and biocontrol efficacy assessment of antagonism yeast against postharvest mango anthracnose[D].Haikou:Master’s Thesis of Hainan University,2015(in Chinese)罗珊珊.采后芒果炭疽病拮抗酵母菌的筛选鉴定及生防效果评价[D].海口:海南大学硕士学位论文,2015
[5]Jiao JH.Yam anthracnose biocontrol 30702 biocontrol characteristics and identification[D].Haikou:Master’s Thesis of Hainan University,2016(in Chinese)焦敬华.山药炭疽病生防菌30702的生防特性及分类鉴定[D].海口:海南大学硕士学位论文,2016
[6]Hu YM,Yang L.Biological control of plant pathogens with microorganism[J].Chinese Journal of Biological Control,2006,22(S1):190-193(in Chinese)胡燕梅,杨龙.利用微生物防治植物病害的研究进展[J].中国生物防治,2006,22(S1):190-193
[7]Deng L,Liu JA,Zhou GY,et al.Antifungal activity of broth of Camellia anthracnose of antagonistic actinomycete CF17 and isolation and purification of its antifungal substance[J].Journal of Fujian Agriculture and Forestry University(Natural Science Edition),2017,46(1):15-20(in Chinese)邓雷,刘君昂,周国英,等.油茶炭疽病拮抗放线菌CF17发酵液的抑菌活性及活性成分的分离纯化[J].福建农林大学学报:自然科学版,2017,46(1):15-20
[8]Sun PP,Jia XH,Cui JC,et al.Selection,identification and characterization of actinomyces L-30 for the biocontrol of Pear Gray Mold[J].Acta Horticulturae Sinica,2016,43(12):2335-2346(in Chinese)孙平平,贾晓辉,崔建潮,等.梨灰霉病拮抗放线菌L-30的筛选、鉴定及作用机制研究[J].园艺学报,2016,43(12):2335-2346
[9]Shirling EB,Gottlieb D.Methods for characterization of Streptomyces species[J].International Journal of Systematic Bacteriology,1966,16(3):313-340
[10]Williams ST,Goodfellow M,Alderson G,et al.Numerical classification of Streptomyces and related genera[J].Journal of General Microbiology,1983,129(6):1743-1813
[11]Hu RM.Classification and Identification of Actinomycetes[M].Chengdu:Sichuan Institute of Antibiotic Industry,1987胡润茂.放线菌的分类与鉴定[M].成都:四川抗菌素工业研究所,1987
[12]Actinomycete Classification Group.Institute of Microbiology,Chinese Academy of Sciences.Handbook of Streptomyces Identification[M].Beijing:Science Press,1975(in Chinese)中国科学院微生物研究所放线菌分类组.链霉菌鉴定手册[M].北京:科学出版社,1975
[13]Zhang K,Wu ML,Gu LS,et al.Identification and preliminary evaluation of a biocontrol actinomycete strain gz-8 against Colletotrichum gloeosporioides[J].Chinese Journal of Pesticide Science,2017,19(6):708-715(in Chinese)张凯,吴曼莉,辜柳霜,等.胶孢炭疽菌生防放线菌gz-8的鉴定及生物活性初步评价[J].农药学学报,2017,19(6):708-715
[14]Ye JL,Yue CY,Pan H,et al.Screening of antagonistic actinomyces to Erwinia carotovora and study on its antibacterial activity[J].Journal of Anhui Agricultural Sciences,2010,38(30):16908-16910(in Chinese)叶晶龙,乐超银,潘虹,等.魔芋软腐病拮抗放线菌的筛选及其抑菌活性研究[J].安徽农业科学,2010,38(30):16908-16910
[15]Hou X,Zhang GQ,Hu X,et al.Screening and inhibitory effect of antagonistic endophytic bacteria in peach against Monilinia fructicola[J].Microbiology China,2017,44(8):1874-1881(in Chinese)侯旭,张国庆,胡晓,等.桃褐腐病菌拮抗性内生细菌的筛选及其抑病效果[J].微生物学通报,2017,44(8):1874-1881
[16]Wu Z,Zhang J,Sun L,et al.Identification of antagonistic strain1706 and its effect on strawberry powdery mildew[J].Chinese Journal of Biological Control,2013,29(3):401-405(in Chinese)武哲,张俊,孙蕾,等.放线菌菌株1706的鉴定及其对草莓白粉病的防治效果[J].中国生物防治学报,2013,29(3):401-405
[17]Liu YL.Primary studies on actinomycete M34 and separation of antimicrobial in gredients from the fermentation broth[D].Wuhan:Master’s Thesis of Huazhong Agricultural University,2008(in Chinese).刘艳玲.放线菌M34菌株及其发酵液抗菌活性物质的初步研究[D].武汉:华中农业大学硕士学位论文,2008
[18]Li XJ,Cheng LX,Wu YB,et al.New methods of testing fermentation liquid antagonistic effect of antagonistic bacteria and antimicrobial spectrum[J].Biotechnology,2007,17(1):55-58(in Chinese)李小俊,成丽霞,吴彦彬,等.拮抗菌抗菌谱及发酵液拮抗能力测定的新方法[J].生物技术,2007,17(1):55-58
[19]Himaman W,Thamchaipenet A,Pathom-aree W,et al.Actinomycetes from Eucalyptus and their biological activities for controlling Eucalyptus leaf and shoot blight[J].Microbiological Research,2016,188-189:42-52
[20]Subramani R,Aalbersberg W.Marine actinomycetes:an ongoing source of novel bioactive metabolites[J].Microbiological Research,2012,167(10):571-580
[21]Jia Y,Jia LY,Huang JX.The bio-control and application of actinomycetes against plant diseases[J].Journal of Xi’an University of Arts&Science(Natural Science Edition),2012,15(3):6-10(in Chinese)贾雨,贾丽苑,黄建新.放线菌对植物病害的防治作用及应用[J].西安文理学院学报:自然科学版,2012,15(3):6-10
[22]Chen SQ,Wang SR.Antagonism of actinomycete DX23 to Colletotrichum coccodes of potato and its identification[J].Journal of Gansu Agricultural University,2015,50(2):95-99(in Chinese)陈淑琴,王生荣.放线菌DX23对马铃薯炭疽病菌的拮抗作用及其鉴定[J].甘肃农业大学学报,2015,50(2):95-99
[23]Du YX,Shi NN,Ruan HC,et al.Screening and identification of actinomycetes antagonistic to Fusarium oxysporum and Phomopsis asparagi[J].Fujian Journal of Agricultural Sciences,2013,28(10):1027-1031(in Chinese)杜宜新,石妞妞,阮宏椿,等.香蕉枯萎病菌和芦笋茎枯病菌的拮抗放线菌筛选与鉴定[J].福建农业学报,2013,28(10):1027-1031
[24]Boukaew S,Prasertsan P.Suppression of rice sheath blight disease using a heat stable culture filtrate from Streptomyces philanthi RM-1-138[J].Crop Protection,2014,61:1-10
[25]Sadeghian M,Bonjar GHS,Sirchi GRS.Post harvest biological control of apple bitter rot by soil-borne actinomycetes and molecular identification of the active antagonist[J].Postharvest Biology and Technology,2016,112:46-54
[26]Zhang Y.Isolation of against some phytopathogenic fungi actinomyces and study of their metabolites with bioactivity[D].Harbin:Master’s Thesis of Northeast Agricultural University,2012(in Chinese)张阳.抗部分植物病原真菌放线菌的筛选及其活性代谢产物的研究[D].哈尔滨:东北农业大学硕士学位论文,2012
[2]Zhang GZ,Zhang C,Jin BC,et al.Toxicity determination of fungicide to fungal pathogens causing Anthranenose and Gray Mold of strawberry[A]//Advances in Strawberry Research IV[C].Beijing:Chinese Society for Horticultural Science,2015:4(in Chinese)张国珍,张璨,靳宝川,等.几种杀菌剂对草莓炭疽菌和灰霉病菌的室内毒力测定[A]//草莓研究进展(IV)[C].北京:中国园艺学会,2015:4
[3]Cao XL,Liu HJ,Guo XJ,et al.Preliminary isolation and identification of the antagonistic bacterial strain 5A5-3 against common orchid diseases[J].Forest Research,2013,26(5):598-602(in Chinese)曹晓璐,刘慧娟,郭晓军,等.兰花炭疽病拮抗细菌5A5-3菌株的初步筛选和鉴定[J].林业科学研究,2013,26(5):598-602
[4]Luo SS.Isolation,screening and biocontrol efficacy assessment of antagonism yeast against postharvest mango anthracnose[D].Haikou:Master’s Thesis of Hainan University,2015(in Chinese)罗珊珊.采后芒果炭疽病拮抗酵母菌的筛选鉴定及生防效果评价[D].海口:海南大学硕士学位论文,2015
[5]Jiao JH.Yam anthracnose biocontrol 30702 biocontrol characteristics and identification[D].Haikou:Master’s Thesis of Hainan University,2016(in Chinese)焦敬华.山药炭疽病生防菌30702的生防特性及分类鉴定[D].海口:海南大学硕士学位论文,2016
[6]Hu YM,Yang L.Biological control of plant pathogens with microorganism[J].Chinese Journal of Biological Control,2006,22(S1):190-193(in Chinese)胡燕梅,杨龙.利用微生物防治植物病害的研究进展[J].中国生物防治,2006,22(S1):190-193
[7]Deng L,Liu JA,Zhou GY,et al.Antifungal activity of broth of Camellia anthracnose of antagonistic actinomycete CF17 and isolation and purification of its antifungal substance[J].Journal of Fujian Agriculture and Forestry University(Natural Science Edition),2017,46(1):15-20(in Chinese)邓雷,刘君昂,周国英,等.油茶炭疽病拮抗放线菌CF17发酵液的抑菌活性及活性成分的分离纯化[J].福建农林大学学报:自然科学版,2017,46(1):15-20
[8]Sun PP,Jia XH,Cui JC,et al.Selection,identification and characterization of actinomyces L-30 for the biocontrol of Pear Gray Mold[J].Acta Horticulturae Sinica,2016,43(12):2335-2346(in Chinese)孙平平,贾晓辉,崔建潮,等.梨灰霉病拮抗放线菌L-30的筛选、鉴定及作用机制研究[J].园艺学报,2016,43(12):2335-2346
[9]Shirling EB,Gottlieb D.Methods for characterization of Streptomyces species[J].International Journal of Systematic Bacteriology,1966,16(3):313-340
[10]Williams ST,Goodfellow M,Alderson G,et al.Numerical classification of Streptomyces and related genera[J].Journal of General Microbiology,1983,129(6):1743-1813
[11]Hu RM.Classification and Identification of Actinomycetes[M].Chengdu:Sichuan Institute of Antibiotic Industry,1987胡润茂.放线菌的分类与鉴定[M].成都:四川抗菌素工业研究所,1987
[12]Actinomycete Classification Group.Institute of Microbiology,Chinese Academy of Sciences.Handbook of Streptomyces Identification[M].Beijing:Science Press,1975(in Chinese)中国科学院微生物研究所放线菌分类组.链霉菌鉴定手册[M].北京:科学出版社,1975
[13]Zhang K,Wu ML,Gu LS,et al.Identification and preliminary evaluation of a biocontrol actinomycete strain gz-8 against Colletotrichum gloeosporioides[J].Chinese Journal of Pesticide Science,2017,19(6):708-715(in Chinese)张凯,吴曼莉,辜柳霜,等.胶孢炭疽菌生防放线菌gz-8的鉴定及生物活性初步评价[J].农药学学报,2017,19(6):708-715
[14]Ye JL,Yue CY,Pan H,et al.Screening of antagonistic actinomyces to Erwinia carotovora and study on its antibacterial activity[J].Journal of Anhui Agricultural Sciences,2010,38(30):16908-16910(in Chinese)叶晶龙,乐超银,潘虹,等.魔芋软腐病拮抗放线菌的筛选及其抑菌活性研究[J].安徽农业科学,2010,38(30):16908-16910
[15]Hou X,Zhang GQ,Hu X,et al.Screening and inhibitory effect of antagonistic endophytic bacteria in peach against Monilinia fructicola[J].Microbiology China,2017,44(8):1874-1881(in Chinese)侯旭,张国庆,胡晓,等.桃褐腐病菌拮抗性内生细菌的筛选及其抑病效果[J].微生物学通报,2017,44(8):1874-1881
[16]Wu Z,Zhang J,Sun L,et al.Identification of antagonistic strain1706 and its effect on strawberry powdery mildew[J].Chinese Journal of Biological Control,2013,29(3):401-405(in Chinese)武哲,张俊,孙蕾,等.放线菌菌株1706的鉴定及其对草莓白粉病的防治效果[J].中国生物防治学报,2013,29(3):401-405
[17]Liu YL.Primary studies on actinomycete M34 and separation of antimicrobial in gredients from the fermentation broth[D].Wuhan:Master’s Thesis of Huazhong Agricultural University,2008(in Chinese).刘艳玲.放线菌M34菌株及其发酵液抗菌活性物质的初步研究[D].武汉:华中农业大学硕士学位论文,2008
[18]Li XJ,Cheng LX,Wu YB,et al.New methods of testing fermentation liquid antagonistic effect of antagonistic bacteria and antimicrobial spectrum[J].Biotechnology,2007,17(1):55-58(in Chinese)李小俊,成丽霞,吴彦彬,等.拮抗菌抗菌谱及发酵液拮抗能力测定的新方法[J].生物技术,2007,17(1):55-58
[19]Himaman W,Thamchaipenet A,Pathom-aree W,et al.Actinomycetes from Eucalyptus and their biological activities for controlling Eucalyptus leaf and shoot blight[J].Microbiological Research,2016,188-189:42-52
[20]Subramani R,Aalbersberg W.Marine actinomycetes:an ongoing source of novel bioactive metabolites[J].Microbiological Research,2012,167(10):571-580
[21]Jia Y,Jia LY,Huang JX.The bio-control and application of actinomycetes against plant diseases[J].Journal of Xi’an University of Arts&Science(Natural Science Edition),2012,15(3):6-10(in Chinese)贾雨,贾丽苑,黄建新.放线菌对植物病害的防治作用及应用[J].西安文理学院学报:自然科学版,2012,15(3):6-10
[22]Chen SQ,Wang SR.Antagonism of actinomycete DX23 to Colletotrichum coccodes of potato and its identification[J].Journal of Gansu Agricultural University,2015,50(2):95-99(in Chinese)陈淑琴,王生荣.放线菌DX23对马铃薯炭疽病菌的拮抗作用及其鉴定[J].甘肃农业大学学报,2015,50(2):95-99
[23]Du YX,Shi NN,Ruan HC,et al.Screening and identification of actinomycetes antagonistic to Fusarium oxysporum and Phomopsis asparagi[J].Fujian Journal of Agricultural Sciences,2013,28(10):1027-1031(in Chinese)杜宜新,石妞妞,阮宏椿,等.香蕉枯萎病菌和芦笋茎枯病菌的拮抗放线菌筛选与鉴定[J].福建农业学报,2013,28(10):1027-1031
[24]Boukaew S,Prasertsan P.Suppression of rice sheath blight disease using a heat stable culture filtrate from Streptomyces philanthi RM-1-138[J].Crop Protection,2014,61:1-10
[25]Sadeghian M,Bonjar GHS,Sirchi GRS.Post harvest biological control of apple bitter rot by soil-borne actinomycetes and molecular identification of the active antagonist[J].Postharvest Biology and Technology,2016,112:46-54
[26]Zhang Y.Isolation of against some phytopathogenic fungi actinomyces and study of their metabolites with bioactivity[D].Harbin:Master’s Thesis of Northeast Agricultural University,2012(in Chinese)张阳.抗部分植物病原真菌放线菌的筛选及其活性代谢产物的研究[D].哈尔滨:东北农业大学硕士学位论文,2012