拮抗草坪蘑菇圈生防菌剂的关键发酵工艺及优化
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摘要
为获得草坪蘑菇圈病原菌拮抗生防菌剂的优化参数,本研究以拮抗蘑菇圈病原-肉褐麟小伞菌(Lepiota brunneo-incarnata)的黄曲霉(Aspergillus flavus)CP01为目的菌,对其分别进行液、固态发酵条件的优化。通过单因素试验优化发酵时间、培养温度、培养基类型及初始pH等条件,对最佳液态发酵条件进行筛选;并通过单因素试验优化培养物料、外源养分、发酵时间和温度等条件,对最佳固态发酵条件进行筛选。结果表明:液体发酵条件下,马铃薯葡萄糖液体培养基(PDB)中菌丝体干重含量最高,而在青秸秆粉浸提液培养基(CSE)中有效菌数最高,且该发酵液对蘑菇圈病原的抑菌效果最佳,培养基最适初始pH为5、最佳发酵条件为30℃、5d;在固态发酵条件下,最适发酵培养物料为经浸提法去营养化的青秸秆粉,外源添加养分为料水比1∶50的青秸秆粉浸提液,最适发酵条件为33℃、9d。以上优化参数为草坪蘑菇圈生防菌剂的研发奠定了基础。
The aim of this study was to optimize the fermentation conditions for obtaining the biological inoculant of an antagonist of the fairy ring mushroomLepiota brunneoincarnata.Aspergillus flavus CP01 is a pathogen of the fairy ring mushroom,and we optimized its liquid and solid fermentation conditions.We screened for the best liquid fermentation conditions of the antagonist in terms of medium type,fermentation time,temperature,and initial pH using the single-factor method.We also screened for the optimal solid fermentation conditions of the target host fungus,viz.,L.brunneoincarnata,in terms of training material and additional nutrition type,fermentation time,and temperature using the single-factor method.Results showed that the optimal growth medium of A.flavus was potato dextrose broth(PDB),which achieved the maximum dry weight of mycelial bead,while the medium type ensuring the maximum number of effective bacteria and the best antibacterial effect was corn stalk leach liquor(CSE)with an optimal initial pH of 5.The optimal liquid fermentation conditions were identified as 30 ℃ and 5 days.The optimal medium type was counteract-eutrophication straw powder by extraction as training material and corn stalk leach liquor at 1∶50 as additional nutrition type for maximum spore yield.The optimal solid fermentation conditions were identified as 33℃and 9 days.These optimized parameters establish a technical basis for the development of an antibiological inoculant of fairy rings.
The aim of this study was to optimize the fermentation conditions for obtaining the biological inoculant of an antagonist of the fairy ring mushroomLepiota brunneoincarnata.Aspergillus flavus CP01 is a pathogen of the fairy ring mushroom,and we optimized its liquid and solid fermentation conditions.We screened for the best liquid fermentation conditions of the antagonist in terms of medium type,fermentation time,temperature,and initial pH using the single-factor method.We also screened for the optimal solid fermentation conditions of the target host fungus,viz.,L.brunneoincarnata,in terms of training material and additional nutrition type,fermentation time,and temperature using the single-factor method.Results showed that the optimal growth medium of A.flavus was potato dextrose broth(PDB),which achieved the maximum dry weight of mycelial bead,while the medium type ensuring the maximum number of effective bacteria and the best antibacterial effect was corn stalk leach liquor(CSE)with an optimal initial pH of 5.The optimal liquid fermentation conditions were identified as 30 ℃ and 5 days.The optimal medium type was counteract-eutrophication straw powder by extraction as training material and corn stalk leach liquor at 1∶50 as additional nutrition type for maximum spore yield.The optimal solid fermentation conditions were identified as 33℃and 9 days.These optimized parameters establish a technical basis for the development of an antibiological inoculant of fairy rings.
引文
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[2]张兆松.草坪仙女环的发生与防治.世界高尔夫,2013(6):170.Zhang Z S.The occurrence and control of lawn fairy ring.Golf World,2013(6):170.(in Chinese)
[3]李雄军,郭国梓,韩惠兰,慕康国,崔建宇,胡林.高尔夫草坪病害与防治简介.中国高尔夫管理,2007(4):40-43.Li X J,Guo G Z,Han H L,Mu K G,Cui J Y,Hu L.Brief introduction of golf turf diseases and prevention.China Golf Management,2007(4):40-43.(in Chinese)
[4]刘星,张书乐,刘国锋,邱慧珍,王蒂,张俊莲,沈其荣.土壤熏蒸-微生物有机肥联用对连作马铃薯生长和土壤生化性质的影响.草业学报,2015,24(3):122-133.Liu X,Zhang S L,Liu G F,Qiu H Z,Wang D,Zhang J L,Shen Q R.Soil fumigation and bio-organic fertilizer application promotes potato growth and affects soil bio-chemical properties in a continuous cropping system.Acta Prataculturae Sinica,2015,24(3):122-133.(in Chinese)
[5]毛连纲,颜冬冬,吴篆芳,马涛涛,王秋霞,李园,郭美霞,欧阳灿彬,曹坳程,郑建秋.土壤化学熏蒸效果的影响因素述评.农药,2013,52(8):547-551.Mao L G,Yan D D,Wu Z F,Ma T T,Wang Q X,Li Y,Guo M X,Ouyang C B,Cao N C,Zheng J Q.Review of affecting factors of soil fumigation.Agrochemicals,2013,52(8):547-551.(in Chinese)
[6]张成霞,南志标,李春杰,王彦荣.杀菌剂拌种防治草坪草病害的研究进展.草业学报,2005,14(6):14-22.Zhang C X,Nan Z B,Li C J,Wang Y R.Studies progress of fungicide seed treatments on the control of turfgrass diseases.Acta Prataculturae Sinica,2005,14(6):14-22.(in Chinese)
[7]Wong P T W,Siviour H.Control of ophiobiolus patch in agrostis turf using avirulent fungi and take-all suppessive soils in pot experiments.Annals of Applied Biology,1979,92:191-197.
[8]何秋,刘建秀.草坪草真菌病害的研究进展.草业科学,2006,23(4):95-104.He Q,Liu J X.Advances in fungi diseases research on turfgrasses.Patacultural Science,2006,23(4):95-104.(in Chinese)
[9]Papavizas G C.Trzchocerma and Glzoclaczum:Biology ecology and potential for biocontrol.Annual Review of Phytopathology,1985,23:23-54.
[10]方俊松.农业生物防治的特点及方法.现代农业科技,2014(7):170.Fang J S.Characteristics and methods of agricultural biological control.Modern Agricultural Science and Technology,2014(7):170.(in Chinese)
[11]蒋家珍,赵美琦,薄怀霞,肖杰,李学锋.生物农药在草坪病害防治中的应用研究.草业科学,2004,21(11):93-95Jiang J Z,Zhao M Q,Bo H X,Xiao J,Li X F.Study on application of biological fungicide to several turf diseases.Pratacultural Science,2004,21(11):93-95.(in Chinese)
[12]杨合同,唐文华,Ryder M.木霉菌与植物病害的生物防治.山东科学,1999,12(4):7-15.Yang H T,Tang W H,Ryder M.Trichoderma and biological control of plant diseases.Shangdong Science,1999,12(4):7-15.(in Chinese)
[13]姬承东,李剑峰.高尔夫草坪蘑菇圈真菌拮抗木霉菌株的筛选及其抑菌作用.草原与草坪,2017,37(3):81-85.Ji C D,Li J F.Isolation and screening of antagonistic Trichoderma strains against fairy ring on golf turf.Grassland and Turf,2017,37(3):81-85.(in Chinese)
[14]王晖,孙晓东,吕国忠.钩状木霉Th12菌株液体发酵条件的研究.安徽农业科学,2011,39(13):7568-7571.Wang H,Sun X D,Lyu G Z,Study on the liquid fermentation condition of Trichoderma hamatum Th12strain.Journal of Anhui Agricultural Sciences,2011,39(13):7568-7571.(in Chinese)
[15]校逸,胡潇涵,陈建帮,弓爱君.Bt生物农药固态发酵研究进展.金属世界,2009(1):99-102.Xiao Y,Hu X H,Chen J B,Gong A J.Review on Bio-pesticide solid-state fermentation of bacillus thuringiensis.Metal World,2009(1):99-102.(in Chinese)
[16]徐福建,陈洪章,李佐虎.固态发酵工程研究进展.生物工程进展,2002,22(1):44-48.Xu F J,Chen H Z,Li Z H.New developments in engineering aspects of solid-state fermentation.Progress in Biotechnology,2002,22(1):44-48.(in Chinese)
[17]韩永超.盾壳霉产生真菌物质的调控及其分子机理研究.湖北:华中农业大学博士学位论文,2010.Han Y C.Studies on regulation and the molecular mechanism of production of anti-fungal substances by the mycoparasite Coniothyrium minitans.PhD Thesis.Hubei:Huazhong Agricultural University,2010.(in Chinese)
[18]蔡信之,黄君红.微生物学实验.北京:科学出版社,2010:109-111.Cai X Z,Huang J H.Experiment of Microbiology.Beijing:Science Press,2010:109-111.(in Chinese)
[19]李剑峰,师尚礼,曹文侠,霍平慧,陈力玉,张淑卿.一种微生物发酵培养基的制备方法:中国专利,CN102277299.ZL201110146217.x.2013-08-21.
[20]高雪丽.侧钩木霉Y01的分离、鉴定与固态发酵产孢研究.杭州:浙江大学硕士学位论文,2014.Gao X L.The separation,identification and spore production by solid state fermentation of Trichoderma ghanense Y01.Master Thesis.Hangzhou:Zhejiang University,2014.(in Chinese)
[21]李剑峰,师尚礼,曹文侠,霍平慧,陈力玉,张淑卿.以青秸杆制备植物促生菌剂载体基质的方法:中国专利,CN102428914A.201110123931.7.2012-05-02.
[22]李进一,井印,雷琛琛.食用菌废料(菌糠)木霉发酵物对黄瓜土传病害防治及促生作用的初步研究.食用菌,2010(4):67-68.Li J Y,Jing Y,Lei C C.Preliminary study on the treatment of cucumber soil borne diseases and the growth promoting effects of Trichoderma spp..Edible Fungi,2010(4):67-68.(in Chinese)
[23]武为平,陈捷,李雅乾,陈立杰,胡荣荣,段玉玺.棘孢木霉产厚垣孢子的液体发酵条件研究.上海交通大学学报(农业科学版),2013,31(6):19-23.Wu W P,Chen J,Li Y Q,Chen L J,Hu R R,Duan Y X.Studies on the liquid fermentation conditions for producing Chlamydospores in Trichoderma asperellum.Journal of Shanghai Jiaotong University(Agricultural Science),2013,31(6):19-23.(in Chinese)
[24]蒋继志,王兴哲,王会仙.2种致病疫霉拮抗真菌混合发酵及抗菌物质稳定性研究.河北大学学报(自然科学版),2011,31(5):515-521.Jiang J Z,Wang X Z,Wang H X.Mixed fermentation of two antagonistic fungi against phytophthora infestans and stability of anti-fungi substances.Journal of Hebei University(Natural Science Edition),2011,31(5):515-521.(in Chinese)
[25]方中达.植物病理研究方法.北京:北京科学出版社,1979:82-86.Fang Z D.Method of Plant Pathology.Beijing:Beijing Science Press,1979:82-86.(in Chinese)
[26]杨军然.害虫的生物防治简况.贵州林业科技,1975(1):20-21.Yang J R.Biological control of pests.Forestry Science and Technology in Guizhou,1975(1):20-21.(in Chinese)
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