灰枣红点软腐病病原鉴定及其防治研究
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摘要
灰枣红点软腐病是2005年至今在河南新郑灰枣产区大面积发生的一种严重危害灰枣的新病害,这种病害不仅影响果实的正常生长,降低了果实的商品价值,而且所有病果完全失去食用价值。为了降低灰枣红点软腐病对河南省灰枣的危害,本文系统研究了灰枣红点软腐病的病原,并进行了化学药剂及生防菌的筛选,主要结果如下:
1本文对灰枣红点软腐病在河南省新郑枣产区的危害和发病特点进行了调查与研究,以形态学特征为基础并结合核糖体DNA-ITS序列对病原菌进行分析鉴定。结果表明:菌落初期气生菌丝为白色,5～6 d后变为灰色,两周后变为黑色,菌丝发达,绒毛状;分生孢子器球形,长196.9μm,宽213.3μm;分生孢子纺锤形,单胞,无色,长15～20μm,宽4.5～6.5μm;分生孢子梗帚状。分离菌株NXK核糖体DNA-ITS序列与葡萄座腔菌的同源性达到99.87 %,仅有2个碱基的差异,从而将灰枣红点软腐病的病原鉴定为葡萄座腔菌(Botryosphaeria dothidea)。
2采用多种促进葡萄座腔菌产孢的方法,对其产孢速度及产孢量进行研究。结果表明,B. dothidea在25℃下,CMA培养基上产孢速度最快,产孢量最大。
3采用生长速率法观测14种杀菌剂的抑菌效果,并对抑制菌丝生长效果较好的药剂采用梯度稀释法进行稀释,进行菌丝生长抑制试验,利用POLO软件计算其毒力指标,求出EC50。结果表明:炭疽福美、多菌灵、代森锰锌对菌丝抑制效果最好,与其他药剂间均达到极显著差异,抑菌率都在99%以上,EC50分别为6.633 mg/L,0.326 mg/L和27.978 mg/L。
4采用孢子萌发法对10种菌丝生长抑制率在90%以上的杀菌剂进行筛选,得到2种抑制效果好的药剂。并采用梯度稀释法对2种药剂进行毒力测定,利用POLO软件计算其毒力指标,求出EC50。结果表明:25%除菌通WP、25%酷乐咪鲜胺EC对孢子萌发抑制作用最强。对孢子萌发的抑制率均为100%,EC50分别为15.305 mg/L和50.158 mg/L。
5田间试验表明,5种自配方复合药剂都有一定的防治效果,与CK都达到了极显著差异,其中喷施3号配方枣果的发病率与喷施其他配方的枣果发病率之间都达到了极显著差异,发病率最低,为35.5%,其次是4号配方,发病率为44.5%,其余几个配方发病率都在70%左右,防治效果较差。
6以灰枣红点软腐病菌(B.dothidea)为指示菌,采用平板对峙法筛选对灰枣红点软腐病菌有拮抗效果的生防菌株,结果表明龟裂链霉菌(CN124)拮抗效果最好,抑菌率达97.52%。并且以龟裂链霉菌菌株CN124为试材,采用单因子试验与均匀试验相结合的方法,通过均匀设计回归分析,筛选出菌株CN124最佳发酵培养基配方。其最佳配方为:淀粉11.67%,玉米粉2.69%,(NH4)2SO4 0.22%,CaCO3 0%,NaCl 0.03%。
Fruit soft rot disease of Ziziphus jujuba was a new disease which had widely distributed and caused great damage in Xin zheng, Henan Province since 2005. It affected the normal growth of fruit and reduced the commercial value. Furthermore, all the infected fruits were not edible at all. To reduce the loss caused by soft rot disease of Ziziphus jujuba, the pathogen was isolated and identified. Several kinds of fungicides and antagonistic bacteria were also screened in this study. The main results were summarized as follows:
1 The occurrence, symptom characteristics and pathogen identification of soft rot disease of Ziziphus jujuba were studied, which widely occurred in xinzheng jujube areas of Henan province. Pathogens were identified by traditional morphological characteristics and ribosome DNA-ITS sequence analysis. The results showed that the villous aerial mycelium was white in the early germination, turned gray after 5 ~ 6 d and black after two weeks, which grew luxuriantly; Pycnidium was flask-shaped, 196.9×213.3μm; Conidium was spindle and colorless with a single spore, 15~20×4.5~6.5μm; Conidiophore was fastigiate. The homology of ribosome DNA-ITS sequence between strain NXK and Botryosphaeria dothidea reached to 99.87 %, with only 2 bp difference, and the pathogen of soft rot disease of Ziziphus jujuba was identified as B. dothidea.
2 To promote the sporulation speed and quantity of B. dothidea, some facilitative methods were used in this research. The results showed that B. dothidea had the fastest and largest amount of conidia on the CMA sporulation medium, and the optimum temperature was at 25℃.
3 The bacteriostatic effects of 14 fungicides were observed using the growth rate method. The fungicides which have good inhibitory effect were diluted by gradient dilution for carrying out the inhibitory experiments on the growth of mycelium. Toxicity index and EC50 were calculated by POLO software. The results showed that the inhibitory effects of anthrax fukumi, carbendazim and mancozeb were the best,and the correlation between the three fungicides and others was significant. The rate of the three bacteriostasis was more than 99%, and EC50 were 6.633 mg/L, 0.326 mg/L and 27.978 mg/L, respectivly.
4 Screening of 10 fungicides whose inhibition rate was up to 90% by spore germination method was conducted and 2 effective fungicides were obtained. Toxicity of the 2 fungicides was tested by gradient dilution. The toxicity index and EC50 was calculated with POLO software. The results showed that the inhibitory effect of 25% sterilization Tong WP, 25% Prochloraz EC were the best,and the correlation between the three fungicides and others were significant. The inhibition rates of the three bacteriostasis were 100%, and EC50 were 15.305 mg/L, and 50.158 mg/L, respectively.
5 The results of field test showed that 5 kinds of self-prescription pharmaceutical compound had a certain effect on soft rot disease of Ziziphus jujuba, which had reached a very significant difference compared with the control. The incidence rate of soft rot disease of Ziziphus jujuba treated by the 3rd formula was 35.5%, the lowest one which reached a significant difference compared with other treatments. Followed by the 3rd formula was the 4th, whose incidence rate was 44.5%, and the rest were less effective with incidence rate at about 70%.
6 The antagonistic microbes were selected by plates assay, using B. dothidea as an indicator strain. The results indicated that the antagonistic strain CN124, belonging to Streptomyces rimosus, had the best biocontrol effect, and the bacteriostasis rate reached to 97.52%. The fermentation medium of the highest rates of S.rimosus CN124 for B. dothidea was selected using single-factor test combined with the evenness test through the regression analysis. The results showed that the components of optimum medium was starch 11.67%, corn flour 2.69%, (NH4)2SO4 0.22%, CaCO3 0%, and NaCl 0.03%.
1本文对灰枣红点软腐病在河南省新郑枣产区的危害和发病特点进行了调查与研究,以形态学特征为基础并结合核糖体DNA-ITS序列对病原菌进行分析鉴定。结果表明:菌落初期气生菌丝为白色,5～6 d后变为灰色,两周后变为黑色,菌丝发达,绒毛状;分生孢子器球形,长196.9μm,宽213.3μm;分生孢子纺锤形,单胞,无色,长15～20μm,宽4.5～6.5μm;分生孢子梗帚状。分离菌株NXK核糖体DNA-ITS序列与葡萄座腔菌的同源性达到99.87 %,仅有2个碱基的差异,从而将灰枣红点软腐病的病原鉴定为葡萄座腔菌(Botryosphaeria dothidea)。
2采用多种促进葡萄座腔菌产孢的方法,对其产孢速度及产孢量进行研究。结果表明,B. dothidea在25℃下,CMA培养基上产孢速度最快,产孢量最大。
3采用生长速率法观测14种杀菌剂的抑菌效果,并对抑制菌丝生长效果较好的药剂采用梯度稀释法进行稀释,进行菌丝生长抑制试验,利用POLO软件计算其毒力指标,求出EC50。结果表明:炭疽福美、多菌灵、代森锰锌对菌丝抑制效果最好,与其他药剂间均达到极显著差异,抑菌率都在99%以上,EC50分别为6.633 mg/L,0.326 mg/L和27.978 mg/L。
4采用孢子萌发法对10种菌丝生长抑制率在90%以上的杀菌剂进行筛选,得到2种抑制效果好的药剂。并采用梯度稀释法对2种药剂进行毒力测定,利用POLO软件计算其毒力指标,求出EC50。结果表明:25%除菌通WP、25%酷乐咪鲜胺EC对孢子萌发抑制作用最强。对孢子萌发的抑制率均为100%,EC50分别为15.305 mg/L和50.158 mg/L。
5田间试验表明,5种自配方复合药剂都有一定的防治效果,与CK都达到了极显著差异,其中喷施3号配方枣果的发病率与喷施其他配方的枣果发病率之间都达到了极显著差异,发病率最低,为35.5%,其次是4号配方,发病率为44.5%,其余几个配方发病率都在70%左右,防治效果较差。
6以灰枣红点软腐病菌(B.dothidea)为指示菌,采用平板对峙法筛选对灰枣红点软腐病菌有拮抗效果的生防菌株,结果表明龟裂链霉菌(CN124)拮抗效果最好,抑菌率达97.52%。并且以龟裂链霉菌菌株CN124为试材,采用单因子试验与均匀试验相结合的方法,通过均匀设计回归分析,筛选出菌株CN124最佳发酵培养基配方。其最佳配方为:淀粉11.67%,玉米粉2.69%,(NH4)2SO4 0.22%,CaCO3 0%,NaCl 0.03%。
Fruit soft rot disease of Ziziphus jujuba was a new disease which had widely distributed and caused great damage in Xin zheng, Henan Province since 2005. It affected the normal growth of fruit and reduced the commercial value. Furthermore, all the infected fruits were not edible at all. To reduce the loss caused by soft rot disease of Ziziphus jujuba, the pathogen was isolated and identified. Several kinds of fungicides and antagonistic bacteria were also screened in this study. The main results were summarized as follows:
1 The occurrence, symptom characteristics and pathogen identification of soft rot disease of Ziziphus jujuba were studied, which widely occurred in xinzheng jujube areas of Henan province. Pathogens were identified by traditional morphological characteristics and ribosome DNA-ITS sequence analysis. The results showed that the villous aerial mycelium was white in the early germination, turned gray after 5 ~ 6 d and black after two weeks, which grew luxuriantly; Pycnidium was flask-shaped, 196.9×213.3μm; Conidium was spindle and colorless with a single spore, 15~20×4.5~6.5μm; Conidiophore was fastigiate. The homology of ribosome DNA-ITS sequence between strain NXK and Botryosphaeria dothidea reached to 99.87 %, with only 2 bp difference, and the pathogen of soft rot disease of Ziziphus jujuba was identified as B. dothidea.
2 To promote the sporulation speed and quantity of B. dothidea, some facilitative methods were used in this research. The results showed that B. dothidea had the fastest and largest amount of conidia on the CMA sporulation medium, and the optimum temperature was at 25℃.
3 The bacteriostatic effects of 14 fungicides were observed using the growth rate method. The fungicides which have good inhibitory effect were diluted by gradient dilution for carrying out the inhibitory experiments on the growth of mycelium. Toxicity index and EC50 were calculated by POLO software. The results showed that the inhibitory effects of anthrax fukumi, carbendazim and mancozeb were the best,and the correlation between the three fungicides and others was significant. The rate of the three bacteriostasis was more than 99%, and EC50 were 6.633 mg/L, 0.326 mg/L and 27.978 mg/L, respectivly.
4 Screening of 10 fungicides whose inhibition rate was up to 90% by spore germination method was conducted and 2 effective fungicides were obtained. Toxicity of the 2 fungicides was tested by gradient dilution. The toxicity index and EC50 was calculated with POLO software. The results showed that the inhibitory effect of 25% sterilization Tong WP, 25% Prochloraz EC were the best,and the correlation between the three fungicides and others were significant. The inhibition rates of the three bacteriostasis were 100%, and EC50 were 15.305 mg/L, and 50.158 mg/L, respectively.
5 The results of field test showed that 5 kinds of self-prescription pharmaceutical compound had a certain effect on soft rot disease of Ziziphus jujuba, which had reached a very significant difference compared with the control. The incidence rate of soft rot disease of Ziziphus jujuba treated by the 3rd formula was 35.5%, the lowest one which reached a significant difference compared with other treatments. Followed by the 3rd formula was the 4th, whose incidence rate was 44.5%, and the rest were less effective with incidence rate at about 70%.
6 The antagonistic microbes were selected by plates assay, using B. dothidea as an indicator strain. The results indicated that the antagonistic strain CN124, belonging to Streptomyces rimosus, had the best biocontrol effect, and the bacteriostasis rate reached to 97.52%. The fermentation medium of the highest rates of S.rimosus CN124 for B. dothidea was selected using single-factor test combined with the evenness test through the regression analysis. The results showed that the components of optimum medium was starch 11.67%, corn flour 2.69%, (NH4)2SO4 0.22%, CaCO3 0%, and NaCl 0.03%.
引文
[1]曲泽洲,王永蕙.中国果树志—枣卷[M].北京:中国林业出版社,1993.
[2]王淮洲,陈志远,李志媛,等.鲜枣中抗坏血酸的含量及其利用[J].营养学报,1986(1):15-23.
[3]王慧瑜,张晓申.灰枣的组织培养[J].植物生理学通讯.2002(6):595.
[4]杨丰年.枣树栽培与病虫害防治[M].中国农业出版社,1996.
[5]任国兰.枣树病虫害防治[M].北京:金盾出版社,1995,5.
[6]陈福礼.鲜食大枣炭疽病的综合防治[J].西北园艺,2002(5).43.
[7]刘惠珍.中国主要树种病原名录[M].北京:中国林科院林业研究所,1982.
[8]陈贻金,朱林元.枣缩果病及其防治技术的研究[J].林业科技通讯,1989 (8):1-4.
[9]曲俭绪,沈瑞祥,李志清,等.枣褐腐病病原研究[J].森林病虫通讯,1992 (2):1-4.
[10]徐樱,郑晓莲,赵光耀,等.枣缩果病病原研究[J].华北农学报,1995,10(增刊):139-143.
[11]康绍兰,邸垫平,李兴红,等.枣铁皮病病原鉴定[J].植物病理学报,1998,28(2):165-171.
[12]陈漠林,马元忠,李占林,等.枣缩果病发病过程及流行规律[J].河南科技,1996(4):11-12.
[13]郑晓莲,赵光耀,齐秋锁,等.枣干腰缩果病症状类型及侵染规律研究[J].植物保护,1998(4):17-19.
[14]郑晓莲.枣缩果病初侵染来源的初步研究[J].河北农业大学学报,1998,18(4):59-63.
[15]焦荣斌.枣缩果病的发生规律及综合防治[J].河北果树,2000(3):45-46.
[16]任士福,张彦婷.枣缩果病病原发病规律及防治[J].林业科技开发,2001,15(2):23-25.
[17]常聚普.枣轮纹病发病规律及综合防治技术[J].中国果树,2004(4):29-30.
[18]张立震,刘春琴,孙玉英,等.金丝小枣果实病害病原菌研究[J].林业科学,2004,40(6):190-193.
[19]林忠敏,赵晓军,赵子俊,等.红枣果实黑斑病的病原分离和鉴定[J].山西农业科学,2001,29(1):74-77.
[20]袁高庆,赖传雅,黄丽华.毛叶枣黑斑病的病原鉴定及其生物学特性研究[J].中国农学通报,2003,19(1):44-47.
[21]匡治州,许杨.核糖体rDNA ITS序列在真菌学研究中的应用[J].生命的化学,2004,24(2):120-122.
[22]杨佩文,李家瑞,杨勤忠,等.根肿病菌核糖体基因ITS区段的克隆测序及其在检测中的应用[J].云南农业大学学报,2003,18(3):228-233.
[23]张薇,魏海雷,张力群,等.紫花首楷菌核病病原鉴定及主要生物学特性[J].草业学报,2005,14(2):69-75.
[24]张竟宇,张正光,王源超.小麦印度腥黑穗病菌的分r检测[J].高技术通讯,2004(1):31-36.
[25]朱有勇,王云月,Lyon B R.尖孢镰刀菌核糖体基因ITS区段的克隆和测序[C].云南省植物病理重点实验室论文集.云南科技出版社.1997.
[26]朱有勇,王云月,Lyon B R.棉花黄萎病PCR检测[J].云南农业大学学报,1998,13(1):161-163.
[27]肖长坤,李健强,师迎春,等.卜多花科蔬菜黑斑病菌的PCR鉴定[J].植物病理学报,2005,35(3):278-282. [28 ] Carling D E.Hyphal anastomosis reaction,rDNA- internal transcribed spacer,and virulence levels among subsets of Rhizoctonia solani anastomosis group-2(AG-2) and AG-BI[J].Phytopathology,2002,92:43-50.
[29]王立安,张文利,王源超,等.人豆疫霉的ITS分子检测[J].南京农业大学学报,2004,27(3):38-41.
[30]谢勇,王云月,陈建斌,等.烟草黑胫病分子检测[J].云南农业大学学报,2000,15(2):176.
[31]赵杰. ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学,2004(4): 35-37.
[32]Henrion B. Tacon F.le. Martin F. Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes[J]. New Phytologist,1992(122):289-298.
[33] Raffle VL,Anderson NA,Furnier GR,et al. Variation in mating competence and random amplified polymorphic DNA in Laccaria bicolor(Agaricales) associated with three tree host species[J].Canadian Journal of Botany,1995(3):84-90.
[34] Karen O,Hogberg N,Dahiberg A,et al. Inter- and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis[J]. New Phytologist,1997(136):313-325.
[35] Pritsch K,boyle H,Munch JC,et al.Characterization and identification of black alder ectomycorrhiz as by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)[J]. New Phytologist,1997(137):57-369.
[36]黄学顺,付艳平,秦乐业,等.栀子叶斑病及其病原的鉴定[J].华中农业大学学报,2006, 25(1):21-25.
[37]赵嘉平,梁军,吕全,等.葡萄座腔菌属(Botryosphaeria)系统分类评述[J].林业科学研究,2007,20 (3):415-422.
[38]陈策.苹果果实轮纹病研究进展[ J].植物病理学报.1999,29(3):193-198.
[39] Smith H,Wingfield M J,Dewet J,et al. Genotypic diversity of Sphaeropsis sapinea from South Africa and Northerm Sumatra[J].Plant Disease.2000(84):139-142.
[40] Smith H,Crous P W,Wingfield M J,et al. Botryosphaeria eucalyptorum sp.nov.,a new species in the B.dothidea-complex on Eucalyptus in South Africa[J].Mycologia,2001,93( 2):277-285.
[41]张星耀,骆有庆.中国重大生物灾害[M].北京:中国林业出版社,2003.
[42] Zhonghua MA,Michailides T J. Characterization of Botryosphaeria dothidea isolates collected from pistachio and other plant hosts in California[J].Phytopathology,2002(92):519-526.
[43] Phillips A J L..Botryosphaeria species associated with diseases of grapevines in Portugal[J].Phytopathologia Medliterranea,2002(41):3-18.
[44]吴小芹,何月秋,刘忠华.葡萄座腔菌属所致树木溃疡病发生与研究进展[J].南京林业大学学报,2001,25 (1):61-66.
[45]王金利,秦国夫,贺伟,等.葡萄座腔菌属及其相关真菌的系统学研究进展[J].中国森林病虫,2003 (3):32-36.
[46]韩熹莱.中国农业百科全书—农药卷[M].北京:农业出版社,1993.
[47]刘勤生.化学农药的使用对农产品质量和环境污染的影响及防范对策[J].现代农业科技,2008(12):152-153.
[48]周明国.中国植物病害化学防治研究—第一卷[M].北京:中国农业科技出版社,1998.
[49]王万能,全学军,周跃钢,等.关于植物病害生物防治[J].重庆工学院学报,2004,18(2): 50-52.
[50] Leuchtmann A. Systematics,distribution,and host specificity of endophytes[J]. Nat Toxins,1992,1(3):150-162.
[51]蒋细良,朱昌雄,姬军红等.中生菌素对水稻白叶枯病的防治机制[J].中国生物防治,2003,19(2):69-72.
[52]蒋细良,谢德龄,倪楚芳等.中生菌素对真菌作用机理的研究[J].中国生物防治.1997,13 (2):69-71.
[53]张穗.井冈霉素A诱导水稻防御纹枯病反应机理研究[D].北京:中国农业大学图馆,2001.
[54]韩巨才,刘慧平,高计青等.农抗120对西瓜枯萎病作用方式的研究[J].西北农业大学学报,1995,15(2):122-125.
[55] Trejo-Estarda S R,Paszezynski A,Carwofrd D L.Antibioties and enzymes porduced by the biocontrol agent Streptomyces violacleusnigerYCED-9[J].Journal of Industrial Microbiology and Bioteehnology,1998,21:81-90.
[56] Eekwall E C,Sehottel J L.Isolation and characterization produced by the scab disease-suppressive Streptomyces distatochromogenes strain PonSSH[J].Journal of Industrial Microbiology and Bioteehnolog,1997,19(3):220-225.
[57]薛德林,胡江春,马成新等.海洋放线菌MB-97生物制剂在克服大豆连作障碍中的应用[J].现代化农业,2003,(12):19-21.
[58]沈寅初.新农药研究与开发[M].北京:化学工业出版社,1995.308-320.
[59] Knight S C,Anthony V M,Brady A M,el a1.Rationale and Perspectives on the development of fungicides [J].Annu. Rev.Phytopnthol.1997,35:349-372.
[60]洪华珠,杨红.杀虫微生物纲要[M].武昌:华中师范大学出版社,1997.
[61]丁雪.两株生防链霉菌的发酵条件优化及发酵液理化性质的研究[D].2008西北农林科技大学.
[62]刘秋,吴元华,于基成.龟裂链霉菌发酵液中抗真菌活性组分的分离纯化[J].植物保护学报,2004,31(4):353-358.
[63]刘秋,吴元华,于基成.东北地区保护地土壤拮抗放线菌的筛选[J].土壤,2004,36(5):573-575.
[64]陈坚,堵国成,李寅等.发酵工程实验技术[M].北京:化学工业出版社,2003.
[65]姚汝华.微生物工程工艺原理[M].广州:华南理工大学出社,1996.
[66]熊宗贵.发酵工艺原理[M].北京:中国医药出版社,2000.
[67]刘红宇,赵仪英.中生菌素高产菌的选育及发酵条件的研究[J].中国抗生素杂志.加02.27(4):246-248.
[68]任颧翔,戴玲妹,刘德华.使用大米粉液化糖化液发酵井冈霉素A[J].Pesticides, 2003,42(15 ):12-13.
[69]朱昌雄,宋培国,蒋细良等.中生菌素高产菌株发酵条件的研究中国生物防治.1997,13(3):114-117.
[70] Aharonowitz Y.,Demain A. L.Carbon catabolite regulation of Cephalosporin production in Streptomyces clavuligerus[J].Antimicrob Agents Chemother,1978,14(2):159-164.
[71] Aharonowitz Y.Nitrogen Metabolite Regulation of Antibiotic Biosynthesis[J].Annual Review of Microbiology,1980,34:209-233.
[72] Janice L.Doull,L eo C.Vining.Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2)suppressive efects of nitrogen and phosphate[J].Applied Microbiology and Biotechnology,1990,(32):449-454.
[73] Merrick M. J.,Edwards R.A. Nitrogen Control in Bacteria[J].Microbiological Review,1995, 59(4): 604-622.
[74] Coisne S.,Bechet M.,Blondeau R.Actinorhodin production by Streptomycesc coelicolor A3(2)in iron-restricted media[J].Leters in Applied Microbiology,1999,28:199-202.
[75]曹军卫,马辉文.微生物工程[M],北京:科学出版社,2002.
[76]吴献忠,李凤玲,工东昌,等.植病防治研究进展[J].莱阳农学院学报,1994,11(1):50-53.
[77]刘传德,吴桂本,宫本义,等.小麦主要病害化学防治研究进展[J].农药,2001,40(9):4-6.
[78] EI Tarabily KA,Hardy GE St J,Sivasithamparam-K,et al.The potential for the biological control of cavity-spot disease of carrots,caused by Pythium coloratum,by streptomycete and non-streptomycete actinomycetes[J].New-Phytologist.1997,137(3):495-507.
[79] E1 Abyard M S,EL saved M A,EL shanshoury A R,et al.Towards the biological control of fungal and bacterial deseases of tomato using antagonistic Streptomyces spp.[J].Plant and Soil.1993,149:185-195.
[80]谢德龄,倪楚芳,朱昌雄,等.中生菌素(农抗751)防治白菜软腐病的效果试验初报[J].生物防治通报,1990,6(2):74-75.
[81]宗兆锋,康振生.植物病理学原理[M].北京:中国农业出版社,2002,7.
[82] Paulo SR Coelho,Anuj Kumar Miehael Snyder.Genome-wide mutant collections:toolboxes for functional genemies[M].Curr.Opin.Microbiol.2000,3:309-315.
[83] Chaube H.S,Singh U.S.Plant Disease Mangement: Principles and Practices[M].1991,182-197.
[84] Cook R.J. The Nature and Practice of Biological Control of Plant Pathogens[M].American Phyto pathological Society,St.Paul,M N.1983.
[85] Cook R.J.Advances in Plant health management in the 20th century[J]. Ann.Rev.Phytopathol. 2000,38:95-116.
[86] Tsao P,Ann.Rev.Selective medium for isolation of Pathogenic fungi[J].Phytopa theology, 1970,8:157-186.
[87] David F.Farr,Marianne Elliott,Amy Y.Rossman1.Fusicoccum arbuti spp. nov.causing cankers on Pacific madrone in western north America with notes on Fusicoccum dimidiatum,the correct name for Scytalidium dimidiatum and Nattrassia mangiferae[J].Mycologia,2005,97(3):730-741.
[88]方中达.植病研究方法[M].1998,北京:中国农业出版社.
[89]薛峰.暗色丝孢真菌细基格孢属(Ulocladiμm)、匍柄霉属(Stemphyliμm)及链格孢属(Alternaria)的分类和分子系统学研究[D].2005,山东农业大学生命科学学院.
[90] Bernard Slippers,Pedro W.Crous,Sandra Denman et al.Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea[J].Mycologia,2004,96(1):83-101.
[91] Guo Zheng Qin,Shi Ping Tian.Biocontrol of postharvest diseases of jujube fruit by Cryptococcus laurentii combined with a low dosage of fungicides under different storage conditions[J].ProQuest Agriculture Journals.May 2004,(88):497-501.
[92]戴芳澜.真菌的形态和分类[M].1984,北京:科学出版社.
[93]陆家云.植物病原真菌学[M].2000,北京:中国农业出版社.
[94]赵嘉平,梁军,吕全等.葡萄座腔菌属(Botryosphaeria)系统分类评述[J].林业科学研究,2007,20(3):415-422.
[95]吴小芹,何月秋,刘忠华.2001.葡萄座腔菌属所致树木溃疡病发生与研究进展[J].南京林业大学学报,25(1):61-66.
[96] Hou L B,He W,Liu X Y,et al.Sequence analysis of ITS region in relationships of Chinese isolates of Cronartium( in Chinese)[J].Journal of Beijing Forestry University,2002, 24(5/6):175-182.
[97]张岳峰,吴小芹,王玲.马蹄金叶斑病发生与室内药剂毒力测定[J].林业科技开发, 2007,21(1):43-45.
[98]黄彰欣.植物化学保护实验指导[M].中国农业出版社,53-54.
[99]慕立义.植物化学保护研究法[M].中国农业出版社,73-74.
[100]苗建材.林木化学保护[M].东北林业大学出版社,1990.261-268.
[101]刘秋,闫建芳,艾勇等.菌株MY02发酵培养基的优化设计[J].吉林农业大学学报,2006(4):361-364.
[102]方开泰,马长兴.正交与均匀试验设计[M].北京:科学出版社,2001:3-25.
[103]杨华,纪明山,张广仁.苹果轮纹病拮抗细菌R8发酵条件的初步研究[J].北方园艺, 2007(12):219-220.
[2]王淮洲,陈志远,李志媛,等.鲜枣中抗坏血酸的含量及其利用[J].营养学报,1986(1):15-23.
[3]王慧瑜,张晓申.灰枣的组织培养[J].植物生理学通讯.2002(6):595.
[4]杨丰年.枣树栽培与病虫害防治[M].中国农业出版社,1996.
[5]任国兰.枣树病虫害防治[M].北京:金盾出版社,1995,5.
[6]陈福礼.鲜食大枣炭疽病的综合防治[J].西北园艺,2002(5).43.
[7]刘惠珍.中国主要树种病原名录[M].北京:中国林科院林业研究所,1982.
[8]陈贻金,朱林元.枣缩果病及其防治技术的研究[J].林业科技通讯,1989 (8):1-4.
[9]曲俭绪,沈瑞祥,李志清,等.枣褐腐病病原研究[J].森林病虫通讯,1992 (2):1-4.
[10]徐樱,郑晓莲,赵光耀,等.枣缩果病病原研究[J].华北农学报,1995,10(增刊):139-143.
[11]康绍兰,邸垫平,李兴红,等.枣铁皮病病原鉴定[J].植物病理学报,1998,28(2):165-171.
[12]陈漠林,马元忠,李占林,等.枣缩果病发病过程及流行规律[J].河南科技,1996(4):11-12.
[13]郑晓莲,赵光耀,齐秋锁,等.枣干腰缩果病症状类型及侵染规律研究[J].植物保护,1998(4):17-19.
[14]郑晓莲.枣缩果病初侵染来源的初步研究[J].河北农业大学学报,1998,18(4):59-63.
[15]焦荣斌.枣缩果病的发生规律及综合防治[J].河北果树,2000(3):45-46.
[16]任士福,张彦婷.枣缩果病病原发病规律及防治[J].林业科技开发,2001,15(2):23-25.
[17]常聚普.枣轮纹病发病规律及综合防治技术[J].中国果树,2004(4):29-30.
[18]张立震,刘春琴,孙玉英,等.金丝小枣果实病害病原菌研究[J].林业科学,2004,40(6):190-193.
[19]林忠敏,赵晓军,赵子俊,等.红枣果实黑斑病的病原分离和鉴定[J].山西农业科学,2001,29(1):74-77.
[20]袁高庆,赖传雅,黄丽华.毛叶枣黑斑病的病原鉴定及其生物学特性研究[J].中国农学通报,2003,19(1):44-47.
[21]匡治州,许杨.核糖体rDNA ITS序列在真菌学研究中的应用[J].生命的化学,2004,24(2):120-122.
[22]杨佩文,李家瑞,杨勤忠,等.根肿病菌核糖体基因ITS区段的克隆测序及其在检测中的应用[J].云南农业大学学报,2003,18(3):228-233.
[23]张薇,魏海雷,张力群,等.紫花首楷菌核病病原鉴定及主要生物学特性[J].草业学报,2005,14(2):69-75.
[24]张竟宇,张正光,王源超.小麦印度腥黑穗病菌的分r检测[J].高技术通讯,2004(1):31-36.
[25]朱有勇,王云月,Lyon B R.尖孢镰刀菌核糖体基因ITS区段的克隆和测序[C].云南省植物病理重点实验室论文集.云南科技出版社.1997.
[26]朱有勇,王云月,Lyon B R.棉花黄萎病PCR检测[J].云南农业大学学报,1998,13(1):161-163.
[27]肖长坤,李健强,师迎春,等.卜多花科蔬菜黑斑病菌的PCR鉴定[J].植物病理学报,2005,35(3):278-282. [28 ] Carling D E.Hyphal anastomosis reaction,rDNA- internal transcribed spacer,and virulence levels among subsets of Rhizoctonia solani anastomosis group-2(AG-2) and AG-BI[J].Phytopathology,2002,92:43-50.
[29]王立安,张文利,王源超,等.人豆疫霉的ITS分子检测[J].南京农业大学学报,2004,27(3):38-41.
[30]谢勇,王云月,陈建斌,等.烟草黑胫病分子检测[J].云南农业大学学报,2000,15(2):176.
[31]赵杰. ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学,2004(4): 35-37.
[32]Henrion B. Tacon F.le. Martin F. Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes[J]. New Phytologist,1992(122):289-298.
[33] Raffle VL,Anderson NA,Furnier GR,et al. Variation in mating competence and random amplified polymorphic DNA in Laccaria bicolor(Agaricales) associated with three tree host species[J].Canadian Journal of Botany,1995(3):84-90.
[34] Karen O,Hogberg N,Dahiberg A,et al. Inter- and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis[J]. New Phytologist,1997(136):313-325.
[35] Pritsch K,boyle H,Munch JC,et al.Characterization and identification of black alder ectomycorrhiz as by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)[J]. New Phytologist,1997(137):57-369.
[36]黄学顺,付艳平,秦乐业,等.栀子叶斑病及其病原的鉴定[J].华中农业大学学报,2006, 25(1):21-25.
[37]赵嘉平,梁军,吕全,等.葡萄座腔菌属(Botryosphaeria)系统分类评述[J].林业科学研究,2007,20 (3):415-422.
[38]陈策.苹果果实轮纹病研究进展[ J].植物病理学报.1999,29(3):193-198.
[39] Smith H,Wingfield M J,Dewet J,et al. Genotypic diversity of Sphaeropsis sapinea from South Africa and Northerm Sumatra[J].Plant Disease.2000(84):139-142.
[40] Smith H,Crous P W,Wingfield M J,et al. Botryosphaeria eucalyptorum sp.nov.,a new species in the B.dothidea-complex on Eucalyptus in South Africa[J].Mycologia,2001,93( 2):277-285.
[41]张星耀,骆有庆.中国重大生物灾害[M].北京:中国林业出版社,2003.
[42] Zhonghua MA,Michailides T J. Characterization of Botryosphaeria dothidea isolates collected from pistachio and other plant hosts in California[J].Phytopathology,2002(92):519-526.
[43] Phillips A J L..Botryosphaeria species associated with diseases of grapevines in Portugal[J].Phytopathologia Medliterranea,2002(41):3-18.
[44]吴小芹,何月秋,刘忠华.葡萄座腔菌属所致树木溃疡病发生与研究进展[J].南京林业大学学报,2001,25 (1):61-66.
[45]王金利,秦国夫,贺伟,等.葡萄座腔菌属及其相关真菌的系统学研究进展[J].中国森林病虫,2003 (3):32-36.
[46]韩熹莱.中国农业百科全书—农药卷[M].北京:农业出版社,1993.
[47]刘勤生.化学农药的使用对农产品质量和环境污染的影响及防范对策[J].现代农业科技,2008(12):152-153.
[48]周明国.中国植物病害化学防治研究—第一卷[M].北京:中国农业科技出版社,1998.
[49]王万能,全学军,周跃钢,等.关于植物病害生物防治[J].重庆工学院学报,2004,18(2): 50-52.
[50] Leuchtmann A. Systematics,distribution,and host specificity of endophytes[J]. Nat Toxins,1992,1(3):150-162.
[51]蒋细良,朱昌雄,姬军红等.中生菌素对水稻白叶枯病的防治机制[J].中国生物防治,2003,19(2):69-72.
[52]蒋细良,谢德龄,倪楚芳等.中生菌素对真菌作用机理的研究[J].中国生物防治.1997,13 (2):69-71.
[53]张穗.井冈霉素A诱导水稻防御纹枯病反应机理研究[D].北京:中国农业大学图馆,2001.
[54]韩巨才,刘慧平,高计青等.农抗120对西瓜枯萎病作用方式的研究[J].西北农业大学学报,1995,15(2):122-125.
[55] Trejo-Estarda S R,Paszezynski A,Carwofrd D L.Antibioties and enzymes porduced by the biocontrol agent Streptomyces violacleusnigerYCED-9[J].Journal of Industrial Microbiology and Bioteehnology,1998,21:81-90.
[56] Eekwall E C,Sehottel J L.Isolation and characterization produced by the scab disease-suppressive Streptomyces distatochromogenes strain PonSSH[J].Journal of Industrial Microbiology and Bioteehnolog,1997,19(3):220-225.
[57]薛德林,胡江春,马成新等.海洋放线菌MB-97生物制剂在克服大豆连作障碍中的应用[J].现代化农业,2003,(12):19-21.
[58]沈寅初.新农药研究与开发[M].北京:化学工业出版社,1995.308-320.
[59] Knight S C,Anthony V M,Brady A M,el a1.Rationale and Perspectives on the development of fungicides [J].Annu. Rev.Phytopnthol.1997,35:349-372.
[60]洪华珠,杨红.杀虫微生物纲要[M].武昌:华中师范大学出版社,1997.
[61]丁雪.两株生防链霉菌的发酵条件优化及发酵液理化性质的研究[D].2008西北农林科技大学.
[62]刘秋,吴元华,于基成.龟裂链霉菌发酵液中抗真菌活性组分的分离纯化[J].植物保护学报,2004,31(4):353-358.
[63]刘秋,吴元华,于基成.东北地区保护地土壤拮抗放线菌的筛选[J].土壤,2004,36(5):573-575.
[64]陈坚,堵国成,李寅等.发酵工程实验技术[M].北京:化学工业出版社,2003.
[65]姚汝华.微生物工程工艺原理[M].广州:华南理工大学出社,1996.
[66]熊宗贵.发酵工艺原理[M].北京:中国医药出版社,2000.
[67]刘红宇,赵仪英.中生菌素高产菌的选育及发酵条件的研究[J].中国抗生素杂志.加02.27(4):246-248.
[68]任颧翔,戴玲妹,刘德华.使用大米粉液化糖化液发酵井冈霉素A[J].Pesticides, 2003,42(15 ):12-13.
[69]朱昌雄,宋培国,蒋细良等.中生菌素高产菌株发酵条件的研究中国生物防治.1997,13(3):114-117.
[70] Aharonowitz Y.,Demain A. L.Carbon catabolite regulation of Cephalosporin production in Streptomyces clavuligerus[J].Antimicrob Agents Chemother,1978,14(2):159-164.
[71] Aharonowitz Y.Nitrogen Metabolite Regulation of Antibiotic Biosynthesis[J].Annual Review of Microbiology,1980,34:209-233.
[72] Janice L.Doull,L eo C.Vining.Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2)suppressive efects of nitrogen and phosphate[J].Applied Microbiology and Biotechnology,1990,(32):449-454.
[73] Merrick M. J.,Edwards R.A. Nitrogen Control in Bacteria[J].Microbiological Review,1995, 59(4): 604-622.
[74] Coisne S.,Bechet M.,Blondeau R.Actinorhodin production by Streptomycesc coelicolor A3(2)in iron-restricted media[J].Leters in Applied Microbiology,1999,28:199-202.
[75]曹军卫,马辉文.微生物工程[M],北京:科学出版社,2002.
[76]吴献忠,李凤玲,工东昌,等.植病防治研究进展[J].莱阳农学院学报,1994,11(1):50-53.
[77]刘传德,吴桂本,宫本义,等.小麦主要病害化学防治研究进展[J].农药,2001,40(9):4-6.
[78] EI Tarabily KA,Hardy GE St J,Sivasithamparam-K,et al.The potential for the biological control of cavity-spot disease of carrots,caused by Pythium coloratum,by streptomycete and non-streptomycete actinomycetes[J].New-Phytologist.1997,137(3):495-507.
[79] E1 Abyard M S,EL saved M A,EL shanshoury A R,et al.Towards the biological control of fungal and bacterial deseases of tomato using antagonistic Streptomyces spp.[J].Plant and Soil.1993,149:185-195.
[80]谢德龄,倪楚芳,朱昌雄,等.中生菌素(农抗751)防治白菜软腐病的效果试验初报[J].生物防治通报,1990,6(2):74-75.
[81]宗兆锋,康振生.植物病理学原理[M].北京:中国农业出版社,2002,7.
[82] Paulo SR Coelho,Anuj Kumar Miehael Snyder.Genome-wide mutant collections:toolboxes for functional genemies[M].Curr.Opin.Microbiol.2000,3:309-315.
[83] Chaube H.S,Singh U.S.Plant Disease Mangement: Principles and Practices[M].1991,182-197.
[84] Cook R.J. The Nature and Practice of Biological Control of Plant Pathogens[M].American Phyto pathological Society,St.Paul,M N.1983.
[85] Cook R.J.Advances in Plant health management in the 20th century[J]. Ann.Rev.Phytopathol. 2000,38:95-116.
[86] Tsao P,Ann.Rev.Selective medium for isolation of Pathogenic fungi[J].Phytopa theology, 1970,8:157-186.
[87] David F.Farr,Marianne Elliott,Amy Y.Rossman1.Fusicoccum arbuti spp. nov.causing cankers on Pacific madrone in western north America with notes on Fusicoccum dimidiatum,the correct name for Scytalidium dimidiatum and Nattrassia mangiferae[J].Mycologia,2005,97(3):730-741.
[88]方中达.植病研究方法[M].1998,北京:中国农业出版社.
[89]薛峰.暗色丝孢真菌细基格孢属(Ulocladiμm)、匍柄霉属(Stemphyliμm)及链格孢属(Alternaria)的分类和分子系统学研究[D].2005,山东农业大学生命科学学院.
[90] Bernard Slippers,Pedro W.Crous,Sandra Denman et al.Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea[J].Mycologia,2004,96(1):83-101.
[91] Guo Zheng Qin,Shi Ping Tian.Biocontrol of postharvest diseases of jujube fruit by Cryptococcus laurentii combined with a low dosage of fungicides under different storage conditions[J].ProQuest Agriculture Journals.May 2004,(88):497-501.
[92]戴芳澜.真菌的形态和分类[M].1984,北京:科学出版社.
[93]陆家云.植物病原真菌学[M].2000,北京:中国农业出版社.
[94]赵嘉平,梁军,吕全等.葡萄座腔菌属(Botryosphaeria)系统分类评述[J].林业科学研究,2007,20(3):415-422.
[95]吴小芹,何月秋,刘忠华.2001.葡萄座腔菌属所致树木溃疡病发生与研究进展[J].南京林业大学学报,25(1):61-66.
[96] Hou L B,He W,Liu X Y,et al.Sequence analysis of ITS region in relationships of Chinese isolates of Cronartium( in Chinese)[J].Journal of Beijing Forestry University,2002, 24(5/6):175-182.
[97]张岳峰,吴小芹,王玲.马蹄金叶斑病发生与室内药剂毒力测定[J].林业科技开发, 2007,21(1):43-45.
[98]黄彰欣.植物化学保护实验指导[M].中国农业出版社,53-54.
[99]慕立义.植物化学保护研究法[M].中国农业出版社,73-74.
[100]苗建材.林木化学保护[M].东北林业大学出版社,1990.261-268.
[101]刘秋,闫建芳,艾勇等.菌株MY02发酵培养基的优化设计[J].吉林农业大学学报,2006(4):361-364.
[102]方开泰,马长兴.正交与均匀试验设计[M].北京:科学出版社,2001:3-25.
[103]杨华,纪明山,张广仁.苹果轮纹病拮抗细菌R8发酵条件的初步研究[J].北方园艺, 2007(12):219-220.