烟草黑胫病拮抗真菌的筛选及活性评价
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摘要
为获得更多防治烟草黑胫病的拮抗真菌,从烟草黑胫病发病烟株根际土壤分离20株真菌,通过平板对峙法筛选,发现菌株ZP2-1、ZP2-3、ZP2-4、GZ3、GZ7对烟草黑胫病菌具有较好的抑制效果。形态学和分子生物学分析表明,ZP2-1为里氏木霉(Trichoderma reesei)、ZP2-3为拟康宁木霉(Trichoderma koningiopsis)、ZP2-4为黑曲霉(Aspergillus niger)、GZ3为烟曲霉(Aspergillus fumigatus)、GZ7为红棕肉座菌(Hypocre rufa)。ZP2-4具有较强分泌纤维素酶和蛋白酶的能力且固体发酵产孢能力显著(p<0.01)高于其余4株菌。ZP2-1、GZ3、ZP2-4固态发酵产物纤维素酶活性相当且显著(p<0.01)高于ZP2-3和GZ7。田间试验结果显示,ZP2-1、GZ3、ZP2-4对烟草黑胫病田间防治效果分别为59.50%、61.82%、85.00%。
In order to obtain more antagonistic fungi for prevention and control of tobacco black shank, 20 fungal strains were isolated from tobacco rhizosphere soil, among which ZP2-1, ZP2-3, ZP2-4, GZ3, and GZ7 had antagonistic effects on Phytophthora nicotianae. Molecular and morphological analysis showed that these strains were identified as Trichoderma reesei ZP2-1, Trichoderma koningiopsis ZP2-3, Aspergillus niger ZP2-4, Aspergillus fumigatus GZ3 and Hypocre rufa GZ7. ZP2-4 has a strong ability to secrete cellulase and protease and the ability of sporulation in solid fermentation was higher than that of the other 4 strains(p < 0.01). The cellulase activities of the solid fermentation products of ZP2-1, GZ3 and ZP2-4 were fairly high and significantly higher than that of ZP2-3 and GZ7(p < 0.01). ZP2-1, GZ3 and ZP2-4 with high spore producing ability and cellulase activity in solid fermentation were selected for field trial. The results showed that the control efficiency against tobacco black shank of ZP2-1, GZ3 and ZP2-4 were 59.50%, 61.82% and 85.00%, respectively.
In order to obtain more antagonistic fungi for prevention and control of tobacco black shank, 20 fungal strains were isolated from tobacco rhizosphere soil, among which ZP2-1, ZP2-3, ZP2-4, GZ3, and GZ7 had antagonistic effects on Phytophthora nicotianae. Molecular and morphological analysis showed that these strains were identified as Trichoderma reesei ZP2-1, Trichoderma koningiopsis ZP2-3, Aspergillus niger ZP2-4, Aspergillus fumigatus GZ3 and Hypocre rufa GZ7. ZP2-4 has a strong ability to secrete cellulase and protease and the ability of sporulation in solid fermentation was higher than that of the other 4 strains(p < 0.01). The cellulase activities of the solid fermentation products of ZP2-1, GZ3 and ZP2-4 were fairly high and significantly higher than that of ZP2-3 and GZ7(p < 0.01). ZP2-1, GZ3 and ZP2-4 with high spore producing ability and cellulase activity in solid fermentation were selected for field trial. The results showed that the control efficiency against tobacco black shank of ZP2-1, GZ3 and ZP2-4 were 59.50%, 61.82% and 85.00%, respectively.
引文
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[21]蔡勇.烟草黑胫病高效生防木霉菌的筛选及其拮抗机制的研究[D].长沙:湖南农业大学,2010.CAI Y.Resarch on the screening and antagonistic mechanism of the high effective biocontrol Trichodermaagainst Phytophthora parasitica var.nicotianae[D].Changsha:Hunan Agricultural University,2010.
[22]周晓见,査鑫垚,董夏伟,等.烟草青枯病拮抗真菌烟曲霉发酵条件的优化[J].河南农业科学,2011,40(12):93-97.ZHOU X J,ZHA X Y,DONG X W,et al.Optimization of fermentation conditions of fungus inhibiting Ralstonia solanacearum by orthogonal test[J].Journal of Henan Agricultural Sciences,2011,40(12):93-97.
[23]郭夏丽,李红丽,王岩,等.一种烟曲霉及其应用:CN102533566A[P].2012-07-04.GUO X L,LI H L,WANG Y,et al.A kind of Aspergillus fumigatus and its application:CN102533566A[P].2012-07-04.
[24]王岩,李红丽,郭夏丽,等.一种黑曲霉菌及其应用:CN102732430A[P].2012-10-17.WANG Y,LI H L,GUO X L,et al.A kind of Aspergillus niger and its application:CN102732430A[P].2012-10-17.
[25]高雪丽,吴坚平,徐刚,等.侧钩木霉的分离、鉴定及产孢条件优化[J].中国生物工程杂志,2014,34(2):84-92.GAO X L,WU J P,XU G,et al.Isolation,identification of Trichoderma ghanense and optimization of spores production[J].China Biotechnology,2014,34(2):84-92.
[26]Kang S W,Park Y S,Lee J S,et al.Production of cellulases and hemicellulases by Aspergillus niger KK2from lignocellulosic biomass[J].Bioresource Technology,2004,91(2):153-156.
[27]袁康培,郑春丽,冯明光.黑曲霉HU53菌株产酸性蛋白酶的条件和酶学性质[J].食品科学,2003,24(8):46-49.YUAN K P,ZHENG C L,FENG M G.Conditions and enzymology properties of acid protease produced by Aspergillus niger HU53[J].Food Science,2003,24(8):46-49.
[28]徐同,柳良好.木霉几丁质酶及其对植物病原真菌的拮抗作用[J].植物病理学报,2002,32(2):97-102.XU T,LIU L H.Chitinases from Trichoderma spp.and their antagonism against phytopathogenic fungi[J].Acta Phytopathologica Sinica,2002,32(2):97-102.
[2]胡燕,王开运,许学明,等.烯酰吗啉对我国烟草黑胫病菌的毒力研究[J].农药学学报,2006,8(4):339-343.HU Y,WANG K Y,XU X M,et al.Inhibitory effect of dmiethomorph on Phytophthora parasitica var.nicotianaein China[J].Chinese Journal of Pesticide Science,2006,8(4):339-342.
[3]Banaay C G B.Trichoderma ghanense promotes plant growth and controls disease caused by Pythium arrhenomanes in seedlings of aerobic rice variety Apo[J].Philippine Agricultural Scientist,2012,95(2):175-184
[4]宋晓妍,孙彩云,陈秀兰,等.木霉生防作用机制的研究进展[J].中国农业科技导报,2006,8(6):20-25.SONG X Y,SUN C Y,CHEN X L,et al.Research advances on mechanism of Trichoderma in biological control[J].Review of China Agricultural Science and Technology,2006,8(6):20-25.
[5]Harman G E.Myths and dogmas of biocontrol changes in perceptions derived from research on Trichoderma harzinum T-22[J].Plant Disease,2007,84(4):377-393.
[6]Schuster E,Dunncoleman N,Frisvad J C,et al.On the safety of Aspergillus niger-a review[J].Applied Microbiology&Biotechnology,2002,59(4-5):426.
[7]高秋芬,倪辉,李利君,等.黑曲霉胞外酶对柑橘果汁的脱苦作用[J].中国食品学报,2014,14(4):156-163.GAO Q F,NI H,LI L J,et al.Characterization of extracellular enzymes from Aspergillus niger for debittering citrus juice[J].Journal of Chinese Institute of Food Science and Technology,2014,14(4):156-163
[8]李静.黑曲霉发酵制备香蕉皮可溶性膳食纤维研究[J].中国食品添加剂,2015(5):137-141.LI J.Study on extraction of soluble dietary fiber from banana peel with Aspergillus niger[J].China Food Additives,2014(5):137-141.
[9]胥海东,王永泽,王金华.津巴布韦烟叶中淀粉酶和蛋白酶产生菌的分离及鉴定[J].生物技术通讯,2011,22(4):536-539.XU H D,WANG Y Z,WANG J H.Isolation and identification of protease and amylase producing bacteria strains from the Zimbabwe tobacco leaves[J].Letters in Biotechnology,2011,22(4):536-539.
[10]张建强,李亚澜,李勇.纤维素降解菌的分离鉴定及固态发酵条件[J].西南交通大学学报,2006,41(4):442-446.ZHANG J Q,LI Y L,LI Y.Investigation into screening identifying and solid-state fermentation of cellulolytic strains[J].Journal of Southwest Jiaotong University,2006,41(4):442-446.
[11]农业部微生物肥料质量监督检验测试中心,中国农业科学院土壤肥料研究所.农用微生物菌剂:GB20287—2006[S].北京:中国标准出版社,2006:1-14.Quality Supervision and Testing Center for Microbial Fertilizer of Ministry of Agriculture,Soil and Fertilizer Institute,Chinese Academy of Agricultural Sciences.Agricultural microbial inoculum:GB 20287—2006[S].Beijing:China Standard Press,2006:1-14.
[12]NIEMANN H,L?SEKANN T,BEER D D,et al.Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink[J].Nature,2006,443(7113):854.
[13]兰楠,祁高富,喻子牛,等.油菜内生真菌的分离鉴定及抑菌作用[J].华中农业大学学报,2011,30(3):270-275.LAN N,QI G F,YU Z N,et al.Isolation,identification and anti-fungal action of endophytic fungi of rapeseed[J].Journal of Huazhong Agricultural University,2011,30(3):270-275.
[14]尚博菲.土壤真菌多样性研究及塑料降解真菌菌株的筛选[D].长春:吉林大学,2013.SHANG B F.The research of soil fungal diversity and screening of degradable plastics fungal strains[D].Changchun:Jilin University,2013.
[15]BORNEMAN J,HARTIN R J.PCR primers that amplify fungal r RNA genes from environmental siamples[J].Applied&Environmental Microbiology,2000,66(10):4356-4360.
[16]中国烟草总公司青州烟草研究所.烟草病虫害分级及调查方法GB/T 23222—2008[S].北京:中国标准出版社,2008:1-6.Qingzhou Tobacco Research Institute of China tobacco company.Grade and investigation method of tobacco diseases and insect pests GB/T 23222—2008[S].Beijing:China Standard Press,2008:1-6.
[17]EL_KOMY M H,SALEH A A,ERANTHODI A,et al.Characterization of novel Trichoderma asperellumisolates to select effective biocontrol agents against tomato Fusarium wilt[J].Plant Pathology Journal,2015,31(1):50-60.
[18]VALENZUELA N L,ANGEL D N,ORTIZ D T,et al.Biological control of anthracnose by postharvest application of Trichoderma.spp.on maradol papaya fruit[J].Biological Control,2015,91:88-93.
[19]INNOCENTI G,ROBERTI R,PIATTONI F.Biocontrol ability of Trichoderma harzianum strain T22 against Fusarium wilt disease on water-stressed lettuce plants[J].Biological Control,2015,60(4):1-9.
[20]陈方新,齐永霞,戴庆怀,等.哈茨木霉对几种植物病原真菌的拮抗作用及其抗药性测定[J].中国农学通报,2005,21(11):314-317.CHEN F X,QI Y X,DAI Q H,et al.In-Lab determination of antagonism against severl plant pathogenic fungi and resistance to fungieides of Trihoderma harzianum[J].Chinese Agriculture Science Bulletin,2005,21(11):314-317.
[21]蔡勇.烟草黑胫病高效生防木霉菌的筛选及其拮抗机制的研究[D].长沙:湖南农业大学,2010.CAI Y.Resarch on the screening and antagonistic mechanism of the high effective biocontrol Trichodermaagainst Phytophthora parasitica var.nicotianae[D].Changsha:Hunan Agricultural University,2010.
[22]周晓见,査鑫垚,董夏伟,等.烟草青枯病拮抗真菌烟曲霉发酵条件的优化[J].河南农业科学,2011,40(12):93-97.ZHOU X J,ZHA X Y,DONG X W,et al.Optimization of fermentation conditions of fungus inhibiting Ralstonia solanacearum by orthogonal test[J].Journal of Henan Agricultural Sciences,2011,40(12):93-97.
[23]郭夏丽,李红丽,王岩,等.一种烟曲霉及其应用:CN102533566A[P].2012-07-04.GUO X L,LI H L,WANG Y,et al.A kind of Aspergillus fumigatus and its application:CN102533566A[P].2012-07-04.
[24]王岩,李红丽,郭夏丽,等.一种黑曲霉菌及其应用:CN102732430A[P].2012-10-17.WANG Y,LI H L,GUO X L,et al.A kind of Aspergillus niger and its application:CN102732430A[P].2012-10-17.
[25]高雪丽,吴坚平,徐刚,等.侧钩木霉的分离、鉴定及产孢条件优化[J].中国生物工程杂志,2014,34(2):84-92.GAO X L,WU J P,XU G,et al.Isolation,identification of Trichoderma ghanense and optimization of spores production[J].China Biotechnology,2014,34(2):84-92.
[26]Kang S W,Park Y S,Lee J S,et al.Production of cellulases and hemicellulases by Aspergillus niger KK2from lignocellulosic biomass[J].Bioresource Technology,2004,91(2):153-156.
[27]袁康培,郑春丽,冯明光.黑曲霉HU53菌株产酸性蛋白酶的条件和酶学性质[J].食品科学,2003,24(8):46-49.YUAN K P,ZHENG C L,FENG M G.Conditions and enzymology properties of acid protease produced by Aspergillus niger HU53[J].Food Science,2003,24(8):46-49.
[28]徐同,柳良好.木霉几丁质酶及其对植物病原真菌的拮抗作用[J].植物病理学报,2002,32(2):97-102.XU T,LIU L H.Chitinases from Trichoderma spp.and their antagonism against phytopathogenic fungi[J].Acta Phytopathologica Sinica,2002,32(2):97-102.