链格孢菌LD-2的改造与发酵
|
摘要
在对由黑龙江八一农垦大学农药研究室分离获得的链格孢菌(Alternaria)菌株LD2形态特征、生物学特性、流行病学及毒性的致病机理等一系列研究后,认为该菌可作为小蓟(Cephalanoplos segetum)生防潜力菌使用并具有进一步的研究潜质。但由于野生菌种本身生防能力不强,难以得到理想的防治效果,本研究通过物理(紫外线)和化学(亚硝基胍)复合诱变的方法对野生菌种进行改造,然后对改造后菌种生长的培养基配方进行优化以及发酵条件等进行研究,最后采取20 L发酵罐对其进行实验中试即放大培养。研究结果如下:
1.采用紫外线诱变链格孢菌LD2的孢子悬浮液(30min),筛选到突变株LD2-13。该菌株较原始菌株孢子产量提高了1.33×10~7个/mL,感病率提高了11.1%,死亡率提高了11.2%。通过传代稳定性验证试验处理,结果表明紫外线诱变得到的菌株LD2-13传代稳定性较好。采用NTG诱变出发菌株LD2-13的孢子悬浮液(75min),筛选到突变株LD2-13-4,该菌株较野生菌株LD2孢子产量提高了2.73×10~7个/mL,感病率提高了24.9%,死亡率提高了16.1%。将亚硝基胍诱变得到的菌株LD2-13-4进行传代稳定性验证试验处理以及对玉米、大豆的安全性试验,结果表明该菌株传代稳定性较好,而且对大豆、玉米均安全,无感病现象。
2.采用单因素实验和正交实验优化出的液体摇瓶发酵培养基为:蛋白胨5.0 g/L、蔗糖20.0 g/L、麸皮50.0 g/L、KH_2PO_4 3.0 g/L、MgSO_4·7H_2O 1.0 g/L、FeSO_4·7H_2O 0.1 g/L、无水CaCl_2 0.1 g/L。在上述培养基条件下采用单次单因素实验筛选出的液体摇瓶发酵培养条件为:250mL三角瓶装液量100 mL,接种量4%,接种种龄3 d,初始pH7.8,摇床转数180 r/ min,30℃振荡培养15d。在上述发酵工艺条件分生孢子产量可达到5.3×10~7个/mL,较该菌在固体(PDA)培养基上培养,分生孢子产量有所增加。
3.在20 L发酵罐中,发酵放罐的最佳时间为68 h,分生孢子产量达7.2×10~7个/mL,最终得发酵液总体积11.4 L,干重产量为76.3 g。
Heilongjiang Agricultrue University pesticide laboratory separated Alternaria that named fungi-LD2.After reserched its morphological characteristics, biology,epidemiology and the pathogenic mechanism of toxicity,the results showed that this fungi can be used to biocontrol Cephalanoplos segetum and further to reserch potential. However,Due to wild species Biocontrol ability itself is not strong,it is difficult to obtain the desired effect,Therefore,This study used physical (ultraviolet) and chemical (nitrosoguanidine) composite mutagenesis method to transform the wild strains.Then reserch growth medium and the fermentation conditions of modified fungi. Finally,cultured strains in 20L fermentor as second test to enlarge production.The results are as follows:
1. Through the use of spore suspension of ultraviolet mutagenesis LD2 Alternaria alternata (30min),screening mutant LD2-13.The strain of spore production increased 1.33×10~7 months / mL,infection rate inceased 11.1% and mortality increased 11.2% than the original strain. Through testing to verify genetic stability, the results showed that the UV-induced strain LD2-13 genetic stability was better. Through the use of spore suspension of NTG mutagenesis LD2-13 Alternaria alternata (75min),screening mutant LD2-13-4, The strain of spore production increased 2.73×10~7 months / mL,infection rate inceased 24.9% and mortality increased 16.1% than the original strain. The verification test of genetic stability and the security test of corn, soybean to mutant LD2-13-4, The results showed that the genetic stability of the strain was well, but also for soybean and corn were safe, non-flu patients phenomenon.
2. Single factor experiments and orthogonal experiment to optimize the liquid fermentation medium are as follows:peptone 5.0g/L、sucrose 20.0g/L.bran 50.0g/L、KH_2PO_4 3.0g/L、MgSO_4·7H_2O 1.0g/L、FeSO_4·7H_2O 0.1g/L、CaCl2 0.1g/L.In the medium under the conditions of the use of a single factor experiment screened liquid fermentation culture conditions are as follows:250mltriangle bottle capacity 100mL,inoculation quantity4%,Inoculation days3d,original pH7.8,shaker revolution180r/ min,30℃surge culture 15d. Fermentation process in the above-mentioned conditions for spore production could reach 5.3×10~7 months / mL,more fungi in the solid(PDA)culture medium,spore production increased。
3. In 20L fermentor,the best time to fermentation tank68h,spores output 7.2×10~7个/mL,The end,the total volume of fermentation 11.4 L,dry weight output 76.3g。
1.采用紫外线诱变链格孢菌LD2的孢子悬浮液(30min),筛选到突变株LD2-13。该菌株较原始菌株孢子产量提高了1.33×10~7个/mL,感病率提高了11.1%,死亡率提高了11.2%。通过传代稳定性验证试验处理,结果表明紫外线诱变得到的菌株LD2-13传代稳定性较好。采用NTG诱变出发菌株LD2-13的孢子悬浮液(75min),筛选到突变株LD2-13-4,该菌株较野生菌株LD2孢子产量提高了2.73×10~7个/mL,感病率提高了24.9%,死亡率提高了16.1%。将亚硝基胍诱变得到的菌株LD2-13-4进行传代稳定性验证试验处理以及对玉米、大豆的安全性试验,结果表明该菌株传代稳定性较好,而且对大豆、玉米均安全,无感病现象。
2.采用单因素实验和正交实验优化出的液体摇瓶发酵培养基为:蛋白胨5.0 g/L、蔗糖20.0 g/L、麸皮50.0 g/L、KH_2PO_4 3.0 g/L、MgSO_4·7H_2O 1.0 g/L、FeSO_4·7H_2O 0.1 g/L、无水CaCl_2 0.1 g/L。在上述培养基条件下采用单次单因素实验筛选出的液体摇瓶发酵培养条件为:250mL三角瓶装液量100 mL,接种量4%,接种种龄3 d,初始pH7.8,摇床转数180 r/ min,30℃振荡培养15d。在上述发酵工艺条件分生孢子产量可达到5.3×10~7个/mL,较该菌在固体(PDA)培养基上培养,分生孢子产量有所增加。
3.在20 L发酵罐中,发酵放罐的最佳时间为68 h,分生孢子产量达7.2×10~7个/mL,最终得发酵液总体积11.4 L,干重产量为76.3 g。
Heilongjiang Agricultrue University pesticide laboratory separated Alternaria that named fungi-LD2.After reserched its morphological characteristics, biology,epidemiology and the pathogenic mechanism of toxicity,the results showed that this fungi can be used to biocontrol Cephalanoplos segetum and further to reserch potential. However,Due to wild species Biocontrol ability itself is not strong,it is difficult to obtain the desired effect,Therefore,This study used physical (ultraviolet) and chemical (nitrosoguanidine) composite mutagenesis method to transform the wild strains.Then reserch growth medium and the fermentation conditions of modified fungi. Finally,cultured strains in 20L fermentor as second test to enlarge production.The results are as follows:
1. Through the use of spore suspension of ultraviolet mutagenesis LD2 Alternaria alternata (30min),screening mutant LD2-13.The strain of spore production increased 1.33×10~7 months / mL,infection rate inceased 11.1% and mortality increased 11.2% than the original strain. Through testing to verify genetic stability, the results showed that the UV-induced strain LD2-13 genetic stability was better. Through the use of spore suspension of NTG mutagenesis LD2-13 Alternaria alternata (75min),screening mutant LD2-13-4, The strain of spore production increased 2.73×10~7 months / mL,infection rate inceased 24.9% and mortality increased 16.1% than the original strain. The verification test of genetic stability and the security test of corn, soybean to mutant LD2-13-4, The results showed that the genetic stability of the strain was well, but also for soybean and corn were safe, non-flu patients phenomenon.
2. Single factor experiments and orthogonal experiment to optimize the liquid fermentation medium are as follows:peptone 5.0g/L、sucrose 20.0g/L.bran 50.0g/L、KH_2PO_4 3.0g/L、MgSO_4·7H_2O 1.0g/L、FeSO_4·7H_2O 0.1g/L、CaCl2 0.1g/L.In the medium under the conditions of the use of a single factor experiment screened liquid fermentation culture conditions are as follows:250mltriangle bottle capacity 100mL,inoculation quantity4%,Inoculation days3d,original pH7.8,shaker revolution180r/ min,30℃surge culture 15d. Fermentation process in the above-mentioned conditions for spore production could reach 5.3×10~7 months / mL,more fungi in the solid(PDA)culture medium,spore production increased。
3. In 20L fermentor,the best time to fermentation tank68h,spores output 7.2×10~7个/mL,The end,the total volume of fermentation 11.4 L,dry weight output 76.3g。
引文
[1]王韧.我国杂草生防的现状及若干问题的讨论[J].生物防治通报,1986,2(4):173～177
[2]胡萃,王韧.国外应用微生物防治杂一草的进展[J].生物防治通报,1987,3(1):35～38
[3]陈勇强.国外微生物除草剂的研究及应用现状[J].天津农业科学,1998,4(2):5～9
[4] Harris,P.,Zwolfer,H.Screening of pyhtophagous insects for biological control of weeds[J].Canada Entomology,1968,100:295～303
[5]丁建清.农田杂草的生物防治[J].中国生物防治,1995,11(3):129～133
[6]高昭远,干静娥.菟丝子的生物防除“鲁保一号”的研究进展[J].生物防治通报,1992,8(4):173～175
[7] Womack J q Eccleston G.M.,Burge M.N.A vegetable oil based emulsion for my coherbicide delivery[J].Biological control,1996,(6),23～28
[8] Julien,M.H.,et al.Biocontrol of weeds:A world catalogue of agents and their target weeds,second edition[J].UK,Wallingford,1987,144
[9] Gibbs,A.J.and Meischke,H.R.C.In pests and parasites as migrants[A]:Cambridge :Cambridge University Press, 1985,171～177
[10] Kenney D S.Devine TM the way it was developed-an industrialist’s view[J].Weed Science,1986,34:5～16
[11] Bowers R C.Commercialization of Collego an industrialist’s view[J].Weed Science,1986,34:24～25
[12] Kadir J B,Charudattan R,Stall W M,et al.Field efficacy of Dactylaria higginsii as a bioherbicide for the control ofpurple nutsedge(Cyperus rotundus) [J].Weed technology,2000,14(1):1～6
[13] De Jone,M.D.,Scheepens,P.C.,Zadoks,J.C.Risk analysis for biological control:a Dutch case study in biocontrol of Prunus serotina by the fungus Chondrostereum purpureum[J].Plant Dis.1990,74:189～194
[14] Becker E M,Hintz,et al.PCR-based genetic markers for detection and infection frequency analysis of the biocontrol fungus Chondrostereum purpureum on Sitka alder and trembling aspen[J].Biological Control.1999,15(1):71～80
[15] Prasad R,Kushwaha S.Ecological based Weed Management for the 21st century:Biological Control of Forest Weeds by Mycoherbicide Agent,Chondrostereum purpureum[C].18th Asian-Pacific Weed Science Society Conference.May 28-Jun 2,2001 Beijing.P.R.China.2001:348～352
[16] Paul Y.dela Bastide,Hong Zhu.et al.Chondrostereum purpureumAn Alternative to Chemical Herbicide Brush Control,The seventh International Sym posium on Environmental Concerns in Rights-of-Way Management Sep9-13.2000Calgary[C].Alberta.Canada,2002:120~124
[17] El-Sayed W,Kang Z,Hurle K.Infection process of Phomopsis convolvulus as a mycoherbicide for Convolvulus arvensis L.Zeitschrift for Pflanzenkrank heiten and Pflanzenaschutz, 2002,109(2):180～192
[18] Goto,M.The relationship between Emmalocera sp.and barnyardgrass and its potential as a biological control.Integrated management of paddy and aquatic weeds in Asia,1992,113～121
[19] Watanabe,H,A.Uchino,and M.Tachibana:Application period of Drechslera monoceras,plant pathogen for the mycoherbicide,to control Echinochloa oryzicola easing rice fields of Tohoku,Japan.The Proceedings I of the 18th Asian-Pacific Weed Science Society Conference,May 28-June2,2001,Beijing,416～421
[20]亦冰.生物除草剂Tasmart[J].世界农药.2004,26(6):46～47
[21] Saxena S,Pandey A K.Evaluation of an indigenous isolate of Alternaria alternata (LC-508) for use as a mycoherbicide for Lantana camara L[J].Crop Protection,2002,21(1):71～73
[22]李春光.除草微生物禾长蠕袍菌种诱变改良及分子鉴定.[D].北京,中国水稻研究所,2004
[23] Zhang W.M,And A.K.Watson.Characterization of Growth and Conidia Production of Exserohilum monoceras on Different Substrates[J].Biocontrol Science and Technology,1997,7:75～86
[24] Zhang W.M,M.Sulz,and T.Myketiak.Potential of using microbes and microbial natural products for control of weeds[C],The Proceedings I of the 18th Asian-Pacific Weed Science Society Conference,May 28-June2,2001,Beijing,358～365
[25] Zhang W.M.et al.Screening of adjuvants for bioherbicide formulation with Colletotrichum spp.and Phoma spp[J].Biological Contro1,2003,26:95～108
[26]王磊.杀禾黄杆菌(Camperico)——种用于灭杀一年生杂草早熟禾的活体生物除草剂.世界农药,2001,23(5)49～51
[27] Imaizumi,S.Biological control of annual bluegrass(Poa annua I.) [C].Abatract of 15 th Pan Pacific Weed Science Soc.Congress at Tsukuba,1995,B:693～697
[28] Imaizumi,S.,Honda,M.,Fujimori,T.Effect of temperature on the control of annual bluegrass(Poa annua L.)with Xanthomonas campestris pv.poae(JT-P482) [J].Biological control,1999,16:13～17
[29]张天宇.胶胞炭疽菌菟丝子专化型.真菌学报,1985,4(4):234～239
[30]向梅梅.莲子草假隔链格孢的寄主范围及对空心莲子草的控制作用[J].植物病理学报,2002,32(3):286～287
[31]刘焕禄,刘亦学,刘晓琳等.微生物除草剂研究概况与建议[J].天津农学院学报,2000,7(4)36～39
[32]陈勇,倪汉文.尖角突脐孢对稻田稗草的防除效果[J].植物保护学报,2001,28(1)73～76
[33]黄世文,余柳青,罗宽.稻田杂草生物防治研究现状、问题及展望[J].植物保护,2004,30(5):5～11
[34]曾青,强胜.低剂量化学除草剂对菌株Qz-97a侵染波斯婆婆纳的影响[J].植物保护学报,2002,29(4):361～365
[35]强胜.大批量生产百日草链格孢菌孢子的技术[J].中国生物防治,1997.13(4):169～172
[36]张青文,扬奇华,周明藏,等.细格孢氮杂酸防治蚜虫和红蜘蛛研究初报[J].植物保护,1990,(2):34～35
[37]向梅梅,刘任,曾永三.空心莲子草叶斑病菌研究初报[J].吉林农业大学学报,1998,20(增刊):124
[38] BARRET O R W,TORRES A N L.Nimbya alternantherae and Cercospora alternantherae:two new records of fungalpathogens on Alternanthera philoxeroides(alligatorweed) in Brazil [J].Australasian Plant Pathology,1999,28(2):103～107
[39]强胜.链格孢菌作为真菌除草剂防除紫茎泽兰潜力的研究[D].南京农业大学博士论文,1998
[40] QiangSheng,Brett A,Summerell.Pathogenicity of Alternariaal-ternata on crofton weed (Eupatorium adenophorum)[A].The Proc.of 17th Asian-Pacific Weed Science Society Conference [C].Thailand Plant Protection Society,1999:556～561.
[41]李荣金,强胜.百日草链格孢菌毒素对加拿大一枝黄花叶片伤害的生理生化研究[J].西北植物学报,2006,26(5):995～1000
[42]周俊初.微生物遗传学[M].北京:中国农业出版社,1994
[43]周德庆.微生物学教程[M].北京:高等教育出版社,1993.245～249
[44]贺小贤.生物工艺原理[M].北京:化学工业出版社,2003.21～22
[45]于洪梅.风味粟米酱米曲霉的分离筛选及诱变育种的研究[D].吉林大学,2007
[46]陈丽梅,汪旭,李启云,等.链霉菌诱变育种方法综述[J].吉林农业科学,2006,31(2):62～63
[47]北京光学学会激光医用专业委员会编译.激光医学基础与临床[M].北京:人民出版社,1989:261～268
[48]余增亮.离子束生物技术引论[M].合肥:安徽科学出版社,1998,247
[49]魏赛金,付学琴,程新,等. 60Coλ射线对南昌霉素生产菌的诱变选育[J].中国抗生素杂志,2002,27(10):580～581
[50]赵永亮.玉米化学诱变研究进展[J].华北农学报,1996,11(4):24～28
[51]王元东.诱变育种在创造玉米新种质中的应用[J].北京农业科学,1999,17(2):12～16
[52]马惠平,赵永亮,杨光宇.诱变技术在农作物育种中的应用[J].遗传,1998,20(6):41～43
[53]微生物诱变育种编写组.生物诱变[M].科学出版社,1973,46～47
[54]盛祖嘉.微生物遗传学[M].北京:科学出版社,1987
[55]许耀奎.作物诱变育种[M].上海:上海科学技术出版社,219～222
[56]张铭堂.诱变[J].科学农业,1996,44(1,2):37～52
[57]许建平.枯草芽抱杆菌B826的化学诱变及筛选研究[[J].浙江农业大学学报,1998,24(6):595～596
[58]贺筱蓉,李永泉,赵小立,等.原生质体诱变选育去甲基金霉素高产菌[J].浙江大学学报,1997,24(2):170～177
[59]吴振倡,王凤仙,陈守川,等.铜蒸汽激光及其与氯化锂复合选育龟裂链霉菌的研究[J].激光生物学,1997,6(2):1068～1070
[60]沈萍,范秀荣,李广武,等.微生物学实验[M].北京:高等教育出版社,1999,6(3):125～127
[61]罗大珍等.现代微生物发酵及技术教程[M].北京大学出版社,2006(1)
[62]熊宗贵,白秀峰,徐新民,等.发酵工艺原理[M].北京:中国医药出版社,1995,4～8
[63]熊宗贵.发酵工艺原理[M].北京:中国医药出版社,2003
[64]吴克,刘斌.木霉T6木聚糖酶液态发酵生产研究[J].食品与发酵工业,2001,27(3)9～12
[65] Duck H.Sub.Biotech.Bioeng,1978,32:821～825
[66] Xue GP,Johnson JS,Smyth DJ,et al.Temperature regulated expression of the tac.laclsystem for overproduction of a fungal xylanase in Escherichia coli.,Appl Microbiol Biotechnol.1996 Mar:45(1-2):120～126.
[67]曹军卫.微生物工程[M].北京:科学出版社,2002,11～12
[68]陈坚.发酵工程试验技术[M].化学工业出版社,2003(1)
[69]魏薇.小蓟叶片上链格孢菌的研究[J].黑龙江八一农垦大学学报,2008,20(1):40～42
[70]施巧琴,吴松刚.工业微生物育种学[M].福州:福建科学技术出版社,1991.
[71]章健,杨云华.定向选育衣康酸高产菌株的研究[J].工业微生物.2000,30(3):1～3
[72]蔡晶晶,李季伦.土曲霉(Aspergillus terreus)产生洛伐他汀(lovastatin)的研究[J].微生物学杂志.2000,20(4):1～4
[73]冮洁，刘晓兰，吴耘红，等．衣康酸生产菌种的定向选育和产酸条件的研究[J].微生物学通报.2003,30(2):20～24
[74]刘建军,姜鲁燕,李丕武,等.发酵法生产衣康酸技术的研究-衣康酸生产菌株的选育(Ⅰ) [J].食品与发酵工业.2003,29(2):17～21.
[75]沈萍.微生物学.北京:高等教育出版社,2000
[76]梁文平,征斐能.21世纪农药发展的趋势:绿色农药与绿色农药制剂[J].农药,1999,38(9):1～2
[77]蒋琳,马承铸.生物农药研究进展[J].上海农业学报,2000,16(增刊):73～77
[78]郝林华,蒋振波,陈靠山,等.枯草芽孢杆菌(Bacillus subtilis)液体发酵条件[J].上海交通大学学报(农业科学版),2006,24(4):380～385
[79] Cook R J.Making greateruseo fintroduced microor ganism for biological control of plant.Pathogens[J].Annular Review Phytopathology,1993,31:53～58
[80]肖怀秋,李玉珍,兰立新.复合诱变原生质体选育高产中性蛋白酶菌株[J].中国酿造,2008,(15):52～53
[81]赵凯,王颖,王旋,等.NTG和UV复合诱变选育紫杉醇高产菌株的研究[J].黑龙江大学自然科学学报,2008,25(1):74～77
[82]曾莹,王常高.NTG和UV复合诱变米曲霉原生质体选育米蛋白分解菌的研究[J].湖北农学院学报,1994,14(2):49～54
[83]赵达,刘伟成,裘季燕,等.枯草芽孢杆菌B03液体发酵条件的优化[J].华北农学报,2008,23(2):205～209
[84]杨亚珍,张建民,余欢.黑曲霉液体发酵产植酸酶条件的研究[J].试验研究,2007,28(5):27～28
[85]廖湘萍,易华荣,杨翠珍.苏云金芽孢杆菌液体发酵的研究[J].酿酒,2007,34(3):53～54
[2]胡萃,王韧.国外应用微生物防治杂一草的进展[J].生物防治通报,1987,3(1):35～38
[3]陈勇强.国外微生物除草剂的研究及应用现状[J].天津农业科学,1998,4(2):5～9
[4] Harris,P.,Zwolfer,H.Screening of pyhtophagous insects for biological control of weeds[J].Canada Entomology,1968,100:295～303
[5]丁建清.农田杂草的生物防治[J].中国生物防治,1995,11(3):129～133
[6]高昭远,干静娥.菟丝子的生物防除“鲁保一号”的研究进展[J].生物防治通报,1992,8(4):173～175
[7] Womack J q Eccleston G.M.,Burge M.N.A vegetable oil based emulsion for my coherbicide delivery[J].Biological control,1996,(6),23～28
[8] Julien,M.H.,et al.Biocontrol of weeds:A world catalogue of agents and their target weeds,second edition[J].UK,Wallingford,1987,144
[9] Gibbs,A.J.and Meischke,H.R.C.In pests and parasites as migrants[A]:Cambridge :Cambridge University Press, 1985,171～177
[10] Kenney D S.Devine TM the way it was developed-an industrialist’s view[J].Weed Science,1986,34:5～16
[11] Bowers R C.Commercialization of Collego an industrialist’s view[J].Weed Science,1986,34:24～25
[12] Kadir J B,Charudattan R,Stall W M,et al.Field efficacy of Dactylaria higginsii as a bioherbicide for the control ofpurple nutsedge(Cyperus rotundus) [J].Weed technology,2000,14(1):1～6
[13] De Jone,M.D.,Scheepens,P.C.,Zadoks,J.C.Risk analysis for biological control:a Dutch case study in biocontrol of Prunus serotina by the fungus Chondrostereum purpureum[J].Plant Dis.1990,74:189～194
[14] Becker E M,Hintz,et al.PCR-based genetic markers for detection and infection frequency analysis of the biocontrol fungus Chondrostereum purpureum on Sitka alder and trembling aspen[J].Biological Control.1999,15(1):71～80
[15] Prasad R,Kushwaha S.Ecological based Weed Management for the 21st century:Biological Control of Forest Weeds by Mycoherbicide Agent,Chondrostereum purpureum[C].18th Asian-Pacific Weed Science Society Conference.May 28-Jun 2,2001 Beijing.P.R.China.2001:348～352
[16] Paul Y.dela Bastide,Hong Zhu.et al.Chondrostereum purpureumAn Alternative to Chemical Herbicide Brush Control,The seventh International Sym posium on Environmental Concerns in Rights-of-Way Management Sep9-13.2000Calgary[C].Alberta.Canada,2002:120~124
[17] El-Sayed W,Kang Z,Hurle K.Infection process of Phomopsis convolvulus as a mycoherbicide for Convolvulus arvensis L.Zeitschrift for Pflanzenkrank heiten and Pflanzenaschutz, 2002,109(2):180～192
[18] Goto,M.The relationship between Emmalocera sp.and barnyardgrass and its potential as a biological control.Integrated management of paddy and aquatic weeds in Asia,1992,113～121
[19] Watanabe,H,A.Uchino,and M.Tachibana:Application period of Drechslera monoceras,plant pathogen for the mycoherbicide,to control Echinochloa oryzicola easing rice fields of Tohoku,Japan.The Proceedings I of the 18th Asian-Pacific Weed Science Society Conference,May 28-June2,2001,Beijing,416～421
[20]亦冰.生物除草剂Tasmart[J].世界农药.2004,26(6):46～47
[21] Saxena S,Pandey A K.Evaluation of an indigenous isolate of Alternaria alternata (LC-508) for use as a mycoherbicide for Lantana camara L[J].Crop Protection,2002,21(1):71～73
[22]李春光.除草微生物禾长蠕袍菌种诱变改良及分子鉴定.[D].北京,中国水稻研究所,2004
[23] Zhang W.M,And A.K.Watson.Characterization of Growth and Conidia Production of Exserohilum monoceras on Different Substrates[J].Biocontrol Science and Technology,1997,7:75～86
[24] Zhang W.M,M.Sulz,and T.Myketiak.Potential of using microbes and microbial natural products for control of weeds[C],The Proceedings I of the 18th Asian-Pacific Weed Science Society Conference,May 28-June2,2001,Beijing,358～365
[25] Zhang W.M.et al.Screening of adjuvants for bioherbicide formulation with Colletotrichum spp.and Phoma spp[J].Biological Contro1,2003,26:95～108
[26]王磊.杀禾黄杆菌(Camperico)——种用于灭杀一年生杂草早熟禾的活体生物除草剂.世界农药,2001,23(5)49～51
[27] Imaizumi,S.Biological control of annual bluegrass(Poa annua I.) [C].Abatract of 15 th Pan Pacific Weed Science Soc.Congress at Tsukuba,1995,B:693～697
[28] Imaizumi,S.,Honda,M.,Fujimori,T.Effect of temperature on the control of annual bluegrass(Poa annua L.)with Xanthomonas campestris pv.poae(JT-P482) [J].Biological control,1999,16:13～17
[29]张天宇.胶胞炭疽菌菟丝子专化型.真菌学报,1985,4(4):234～239
[30]向梅梅.莲子草假隔链格孢的寄主范围及对空心莲子草的控制作用[J].植物病理学报,2002,32(3):286～287
[31]刘焕禄,刘亦学,刘晓琳等.微生物除草剂研究概况与建议[J].天津农学院学报,2000,7(4)36～39
[32]陈勇,倪汉文.尖角突脐孢对稻田稗草的防除效果[J].植物保护学报,2001,28(1)73～76
[33]黄世文,余柳青,罗宽.稻田杂草生物防治研究现状、问题及展望[J].植物保护,2004,30(5):5～11
[34]曾青,强胜.低剂量化学除草剂对菌株Qz-97a侵染波斯婆婆纳的影响[J].植物保护学报,2002,29(4):361～365
[35]强胜.大批量生产百日草链格孢菌孢子的技术[J].中国生物防治,1997.13(4):169～172
[36]张青文,扬奇华,周明藏,等.细格孢氮杂酸防治蚜虫和红蜘蛛研究初报[J].植物保护,1990,(2):34～35
[37]向梅梅,刘任,曾永三.空心莲子草叶斑病菌研究初报[J].吉林农业大学学报,1998,20(增刊):124
[38] BARRET O R W,TORRES A N L.Nimbya alternantherae and Cercospora alternantherae:two new records of fungalpathogens on Alternanthera philoxeroides(alligatorweed) in Brazil [J].Australasian Plant Pathology,1999,28(2):103～107
[39]强胜.链格孢菌作为真菌除草剂防除紫茎泽兰潜力的研究[D].南京农业大学博士论文,1998
[40] QiangSheng,Brett A,Summerell.Pathogenicity of Alternariaal-ternata on crofton weed (Eupatorium adenophorum)[A].The Proc.of 17th Asian-Pacific Weed Science Society Conference [C].Thailand Plant Protection Society,1999:556～561.
[41]李荣金,强胜.百日草链格孢菌毒素对加拿大一枝黄花叶片伤害的生理生化研究[J].西北植物学报,2006,26(5):995～1000
[42]周俊初.微生物遗传学[M].北京:中国农业出版社,1994
[43]周德庆.微生物学教程[M].北京:高等教育出版社,1993.245～249
[44]贺小贤.生物工艺原理[M].北京:化学工业出版社,2003.21～22
[45]于洪梅.风味粟米酱米曲霉的分离筛选及诱变育种的研究[D].吉林大学,2007
[46]陈丽梅,汪旭,李启云,等.链霉菌诱变育种方法综述[J].吉林农业科学,2006,31(2):62～63
[47]北京光学学会激光医用专业委员会编译.激光医学基础与临床[M].北京:人民出版社,1989:261～268
[48]余增亮.离子束生物技术引论[M].合肥:安徽科学出版社,1998,247
[49]魏赛金,付学琴,程新,等. 60Coλ射线对南昌霉素生产菌的诱变选育[J].中国抗生素杂志,2002,27(10):580～581
[50]赵永亮.玉米化学诱变研究进展[J].华北农学报,1996,11(4):24～28
[51]王元东.诱变育种在创造玉米新种质中的应用[J].北京农业科学,1999,17(2):12～16
[52]马惠平,赵永亮,杨光宇.诱变技术在农作物育种中的应用[J].遗传,1998,20(6):41～43
[53]微生物诱变育种编写组.生物诱变[M].科学出版社,1973,46～47
[54]盛祖嘉.微生物遗传学[M].北京:科学出版社,1987
[55]许耀奎.作物诱变育种[M].上海:上海科学技术出版社,219～222
[56]张铭堂.诱变[J].科学农业,1996,44(1,2):37～52
[57]许建平.枯草芽抱杆菌B826的化学诱变及筛选研究[[J].浙江农业大学学报,1998,24(6):595～596
[58]贺筱蓉,李永泉,赵小立,等.原生质体诱变选育去甲基金霉素高产菌[J].浙江大学学报,1997,24(2):170～177
[59]吴振倡,王凤仙,陈守川,等.铜蒸汽激光及其与氯化锂复合选育龟裂链霉菌的研究[J].激光生物学,1997,6(2):1068～1070
[60]沈萍,范秀荣,李广武,等.微生物学实验[M].北京:高等教育出版社,1999,6(3):125～127
[61]罗大珍等.现代微生物发酵及技术教程[M].北京大学出版社,2006(1)
[62]熊宗贵,白秀峰,徐新民,等.发酵工艺原理[M].北京:中国医药出版社,1995,4～8
[63]熊宗贵.发酵工艺原理[M].北京:中国医药出版社,2003
[64]吴克,刘斌.木霉T6木聚糖酶液态发酵生产研究[J].食品与发酵工业,2001,27(3)9～12
[65] Duck H.Sub.Biotech.Bioeng,1978,32:821～825
[66] Xue GP,Johnson JS,Smyth DJ,et al.Temperature regulated expression of the tac.laclsystem for overproduction of a fungal xylanase in Escherichia coli.,Appl Microbiol Biotechnol.1996 Mar:45(1-2):120～126.
[67]曹军卫.微生物工程[M].北京:科学出版社,2002,11～12
[68]陈坚.发酵工程试验技术[M].化学工业出版社,2003(1)
[69]魏薇.小蓟叶片上链格孢菌的研究[J].黑龙江八一农垦大学学报,2008,20(1):40～42
[70]施巧琴,吴松刚.工业微生物育种学[M].福州:福建科学技术出版社,1991.
[71]章健,杨云华.定向选育衣康酸高产菌株的研究[J].工业微生物.2000,30(3):1～3
[72]蔡晶晶,李季伦.土曲霉(Aspergillus terreus)产生洛伐他汀(lovastatin)的研究[J].微生物学杂志.2000,20(4):1～4
[73]冮洁，刘晓兰，吴耘红，等．衣康酸生产菌种的定向选育和产酸条件的研究[J].微生物学通报.2003,30(2):20～24
[74]刘建军,姜鲁燕,李丕武,等.发酵法生产衣康酸技术的研究-衣康酸生产菌株的选育(Ⅰ) [J].食品与发酵工业.2003,29(2):17～21.
[75]沈萍.微生物学.北京:高等教育出版社,2000
[76]梁文平,征斐能.21世纪农药发展的趋势:绿色农药与绿色农药制剂[J].农药,1999,38(9):1～2
[77]蒋琳,马承铸.生物农药研究进展[J].上海农业学报,2000,16(增刊):73～77
[78]郝林华,蒋振波,陈靠山,等.枯草芽孢杆菌(Bacillus subtilis)液体发酵条件[J].上海交通大学学报(农业科学版),2006,24(4):380～385
[79] Cook R J.Making greateruseo fintroduced microor ganism for biological control of plant.Pathogens[J].Annular Review Phytopathology,1993,31:53～58
[80]肖怀秋,李玉珍,兰立新.复合诱变原生质体选育高产中性蛋白酶菌株[J].中国酿造,2008,(15):52～53
[81]赵凯,王颖,王旋,等.NTG和UV复合诱变选育紫杉醇高产菌株的研究[J].黑龙江大学自然科学学报,2008,25(1):74～77
[82]曾莹,王常高.NTG和UV复合诱变米曲霉原生质体选育米蛋白分解菌的研究[J].湖北农学院学报,1994,14(2):49～54
[83]赵达,刘伟成,裘季燕,等.枯草芽孢杆菌B03液体发酵条件的优化[J].华北农学报,2008,23(2):205～209
[84]杨亚珍,张建民,余欢.黑曲霉液体发酵产植酸酶条件的研究[J].试验研究,2007,28(5):27～28
[85]廖湘萍,易华荣,杨翠珍.苏云金芽孢杆菌液体发酵的研究[J].酿酒,2007,34(3):53～54