高效溶磷木霉菌株复合诱变选育及促生作用
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摘要
为获得高效溶磷菌株并探讨其促生效果,本研究以木霉T6为出发菌株,分别采用微波、硫酸二乙酯(DES)、紫外(UV)单一和微波-DES-UV复合诱变的方法对其进行处理,并测定了诱变菌株对番茄幼苗的促生效果。结果表明,单一最佳诱变条件为:900 W微波辐照70 s,2%DES处理40 min,20 W UV照射90 s。利用3种单一最佳诱变条件组合进行复合诱变获得了一株具有高效溶磷能力的突变株T6-MDU45,该突变株的溶磷量为353.46μg·mL~(-1),较出发菌株提高了259.10%,其培养9 d的生长速率和产孢量分别为38.58 mm·d~(-1)和8.02×10~9 CFU·mL~(-1),与出发菌株无显著差异。多次传代试验表明,该突变株遗传稳定性较好。对番茄幼苗的促生试验结果表明,突变菌株T6-MDU45灌根处理后,番茄幼苗的株高、根长、茎粗、地上部干质量、地下部干质量、植株地上部和地下部磷较CK分别提高了29.29%、41.36%、27.45%、44.87%、108.33%、36.55%和109.92%,且能显著提高光合效率,具有明显的促生作用。综上,通过复合诱变可明显提高木霉菌株的溶磷促生能力,获得的突变菌株为土壤改良及多功能木霉制剂、微生物肥料的开发提供了优良的菌种资源。
In this study, T6 strain of Trichoderma sp. with low ability of dissolving phosphorus was mutated by microwave, DES, UV and compound mutation in order to obtain the strain with highly efficient in dissolving phosphorus. In addition, the growth-promoting effect on tomato of the mutant strain was tested. The results showed that, the proper irradiation for microwave was radiation with 900 W for 70 s, and for 2% DES and 20 W UV, the irradiation time was 40 min and 90 s, respectively. Combined these three mutations, T6-MDU45 mutant with highly efficient in dissolving phosphorus was obtained and its′ dissolved phosphorus content reached up to 353.46 μg·mL~(-1), and was 259.10% higher than that of the original strain. The growth rate and sporulation quantity of mutant were 38.58 mm·d~(-1) and 8.02×10~9 CFU·mL~(-1) respectively, no significant differences from those of initial strains. Meanwhile, the passage experiment showed that the mutant strain was stable heritable. The results of growth-promoting effect on tomato showed that conidial suspension of T6-MDU45 could significantly promote the growth of tomato. Compared with the control, the plant height, root length, stem diameter, dry weight of shoots, dry weight of roots, shoots P and roots P of tomato seedlings treated with T6-MDU45 conidial suspension were significantly increased by 29.29%,41.36%,27.45%,44.87%,108.33%,36.55% and 109.92%, respectively, photosynthetic efficiency was distinctly improved and growth was promoted obviously. Therefore, compound mutation could enhance the ability of T6 strain in dissolving phosphorus. The obtained mutant strain provided excellent strain resources for soil improvement and development of multi-function microbial fertilizer.
In this study, T6 strain of Trichoderma sp. with low ability of dissolving phosphorus was mutated by microwave, DES, UV and compound mutation in order to obtain the strain with highly efficient in dissolving phosphorus. In addition, the growth-promoting effect on tomato of the mutant strain was tested. The results showed that, the proper irradiation for microwave was radiation with 900 W for 70 s, and for 2% DES and 20 W UV, the irradiation time was 40 min and 90 s, respectively. Combined these three mutations, T6-MDU45 mutant with highly efficient in dissolving phosphorus was obtained and its′ dissolved phosphorus content reached up to 353.46 μg·mL~(-1), and was 259.10% higher than that of the original strain. The growth rate and sporulation quantity of mutant were 38.58 mm·d~(-1) and 8.02×10~9 CFU·mL~(-1) respectively, no significant differences from those of initial strains. Meanwhile, the passage experiment showed that the mutant strain was stable heritable. The results of growth-promoting effect on tomato showed that conidial suspension of T6-MDU45 could significantly promote the growth of tomato. Compared with the control, the plant height, root length, stem diameter, dry weight of shoots, dry weight of roots, shoots P and roots P of tomato seedlings treated with T6-MDU45 conidial suspension were significantly increased by 29.29%,41.36%,27.45%,44.87%,108.33%,36.55% and 109.92%, respectively, photosynthetic efficiency was distinctly improved and growth was promoted obviously. Therefore, compound mutation could enhance the ability of T6 strain in dissolving phosphorus. The obtained mutant strain provided excellent strain resources for soil improvement and development of multi-function microbial fertilizer.
引文
[1] 柯春亮,陈宇丰,周登博,黄绵佳,张锡炎,高祝芬. 香蕉根际土壤解磷细菌的筛选、鉴定及解磷能力[J]. 微生物学通报,2015,42(6):1032-1042
[2] 杨顺,杨婷,林斌,刘杏忠,向梅春. 两株溶磷真菌的筛选、鉴定及溶磷效果的评价[J]. 微生物学报,2018,58(2): 264-273
[3] Gong M B,Du P,Liu X,Zhu C X. Transformation of inorganic P fractions of soil and plant growth promotion by phosphate-solubilizing ability of Penicillium oxalicum Ⅱ[J]. Journal of Microbiology,2014,52(12):1012-1019
[4] 赵小军,李志洪,刘龙,崔婷婷. 种还分离模式下玉米秸秆还田对土壤磷有效性及其有机磷形态的影响[J]. 水土保持学报,2017,31(1):243-247
[5] 章明清,李娟,孔庆波,姚宝全,陈燕花. 菜-稻轮作对菜田氮、磷利用特性和富集状况的影响[J]. 植物营养与肥料学报, 2013,19(1):117-126
[6] Saravanakumar K,Shanmuga Ⅴ A,Kathiresan K. Effect of Trichoderma on soil phosphate solubilization and growth improvement of Avicennia marina[J]. Aquatic Botany,2013,104:101-105
[7] 乔欢,吴小芹,王早. 一株嗜松青霉JP-NJ4 的解磷特性[J]. 微生物学报,2014,41(9):1741-1748
[8] Zaidi A, Khan M S, Ahemad M, Oves M, Wani P A. Recent advances in plant growth promotion by phosphate-solubilizing microbes[M]//Khan M S,Zaidi A,Musarrat J. Microbial Strategies for Crop Improvement. Berlin: Springer,2009: 23-50
[9] Mehta P,Walia A,Chauhan A,Shirkot C K. Plant growth promoting traits of phosphate-solubilizing rhizobacteria isolated from apple trees in trans Himalayan region of Himachal Pradesh [J]. Archives of Microbiology,2013,195(5):357-369
[10] 吴鹏飞,张冬明,郝丽虹,漆智平. 解磷微生物研究现状及展望[J]. 中国农业科技导报,2008,10(3): 40-46
[11] Fernondez L A,Zalba P,Gomez M A,Sagardoy M A. Phosphate solubilization activity of bacterial strains in soil and their effect on soybean growth under greenhouse conditions[J]. Biology and Fertility of Soils,2007,43(6):805-809
[12] 张硕成. 木霉菌生态学及其在生防中的应用[J]. 应用生态学报,1991,2(1):85-88
[13] 张树武,徐秉良,薛应钰,古丽君. 长枝木霉对小麦禾谷孢囊线虫的致死作用[J]. 应用生态学报,2014,25(7):2093-2098
[14] 张树武,刘佳,徐秉良,古丽君,薛应钰. 长枝木霉对禾谷胞囊线虫寄生和致死作用的显微观察及测定[J]. 应用生态学报,2013,24(10):2055-2960
[15] Elad Y. Trichoderma harzianum T39 preparation for biocontrol of plant disease-control of Botrytis cinerea,Sclerotinia sclerotiorum and Cladosporum fulvum[J]. Biocontrol Science and Technology,2000,10(4):499-507
[16] Altomare C,Norvell W A,Bjorkman T,Harman G E. Solubilization of phosphates and micronutrients by the plant growth promoting and biocontrol fungus Trichoderma harzianum Rifai[J]. Environmental Microbiology,1999,65(7):2926-2933
[17] Azarmi R,Hajieghrari B,Giglou A. Effect of Trichoderma isolates on tomato seedling growth response and nutrient uptake[J]. African Journal of Biotechnology,2011,10(31):5850-5855
[18] 薛应钰,叶巍,张树武,刘佳,徐秉良. 微波诱变选育高效溶磷木霉菌株的研究[J]. 干旱地区农业研究,2016,34(4):231-236
[19] 虞龙,张宇,龚文静,安肖,徐翔. 氯化锂-紫外-离子束复合诱变红霉素高产菌株研究[J]. 原子能科学技术,2011, 45(7):780-784
[20] 李蕤,骆军,虞磊,鲁润龙,潘仁瑞. 复合诱变筛选产细胞壁水解酶木霉菌株及产酶条件的优化[J]. 微生物学通报,2002,29(5):47-52
[21] 韩立亚,冯培勇,张玉香,张丽. 复合诱变选育高产纤维素酶绿色木霉菌株[J]. 安徽农业科学,2009,37(9):3869-3870,3883
[22] 赵丹萍. 产几丁质酶木霉的诱变选育及对玉米纹枯病的生物防治[D]. 雅安:四川农业大学,2010
[23] 尹婷,徐秉良,梁巧兰,古丽君,李荣峰. 耐药性木霉T2菌株的筛选、紫外诱变与药剂驯化[J]. 草业学报,2013,22(2):117-122
[24] 李剑峰,张淑卿,师尚礼,霍平慧. 微波诱变选育耐药高效溶磷苜蓿根瘤菌[J]. 原子能科技技术,2009,43(12):1071-1076
[25] 岳斌,王宝维,张名爱,荆丽珍,张倩. 鹅源草酸青霉溶磷效果及对鹅磷代谢的影响[J]. 青岛农业大学学报(自然科学版),2008,25(1):34-37
[26] 王爱萍,刘立君,梁秀丽,姚旭霞,周彤. 钒钼黄分光光度法测定有机化合物中的微量磷[J]. 化学分析计量,2007,16(2):49-50
[27] 鲍士旦. 土壤农化分析[M]. 北京:中国农业出版社,2000
[28] 高俊凤. 植物生理学实验指导[M]. 北京:高等教育出版社,2006
[29] 葛菁萍,蔡柏岩,宋刚,孙宗祥,平文祥. 土壤中功能真菌的分离及其解磷能力的初步研究[J]. 中国土壤与肥料,2009,6:84-86
[30] Zhan S W,Gan Y T,Xu B L,Xue Y Y. The parasitic and lethal effects of Trichoderma longibrachiatum against Heterodera avenae[J]. Biological Control,2014,72:1-8
[31] 王玉霞,胡基华,刘宇帅,姜威,孟利强,李晶,曹旭,陈静宇,张淑梅. 低温生防菌株的诱变选育及生防效果[J]. 核农学报,2017,31(10): 1873-1880
[32] 张曼曼,邓春生,马金奉,燕荣,耿兵,李顺鹏. 多功能木霉的筛选及鉴定[J]. 农业环境科学学报,2012,31(8):1571-1575
[33] 杨合同,于雪云,魏艳丽,周红姿,宋莉璐,扈进冬,李纪顺 . 多功能植病生防木霉的分离筛选与鉴定[J]. 山东科学,2009,22(5):211-214
[34] 薛应钰,叶巍,张树武,徐秉良,杨百昶. 紫外诱变选育木霉高效解磷菌株[J]. 核农学报,2015,29(8):1509-1516
[35] 刘俊梅,王庆,王丹,杨盼盼,丁伟,朴春华,于寒松. 紫外线和硫酸二乙酯复合诱变选育PHB高产菌株[J]. 微生物学通报,2018,43(10):2242-2248
[36] 覃玲灵. 里氏木霉的紫外诱变和高活性纤维素酶突变株的选育[D]. 长沙:中南林业科技大学,2011
[37] 李瑞芳,赵玉峰,杨世清,伊艳杰,田泱源. 枯草芽胞杆菌BS501a复合诱变菌株及抗菌谱研究[J]. 植物保护,2011,37(5):86-91
[2] 杨顺,杨婷,林斌,刘杏忠,向梅春. 两株溶磷真菌的筛选、鉴定及溶磷效果的评价[J]. 微生物学报,2018,58(2): 264-273
[3] Gong M B,Du P,Liu X,Zhu C X. Transformation of inorganic P fractions of soil and plant growth promotion by phosphate-solubilizing ability of Penicillium oxalicum Ⅱ[J]. Journal of Microbiology,2014,52(12):1012-1019
[4] 赵小军,李志洪,刘龙,崔婷婷. 种还分离模式下玉米秸秆还田对土壤磷有效性及其有机磷形态的影响[J]. 水土保持学报,2017,31(1):243-247
[5] 章明清,李娟,孔庆波,姚宝全,陈燕花. 菜-稻轮作对菜田氮、磷利用特性和富集状况的影响[J]. 植物营养与肥料学报, 2013,19(1):117-126
[6] Saravanakumar K,Shanmuga Ⅴ A,Kathiresan K. Effect of Trichoderma on soil phosphate solubilization and growth improvement of Avicennia marina[J]. Aquatic Botany,2013,104:101-105
[7] 乔欢,吴小芹,王早. 一株嗜松青霉JP-NJ4 的解磷特性[J]. 微生物学报,2014,41(9):1741-1748
[8] Zaidi A, Khan M S, Ahemad M, Oves M, Wani P A. Recent advances in plant growth promotion by phosphate-solubilizing microbes[M]//Khan M S,Zaidi A,Musarrat J. Microbial Strategies for Crop Improvement. Berlin: Springer,2009: 23-50
[9] Mehta P,Walia A,Chauhan A,Shirkot C K. Plant growth promoting traits of phosphate-solubilizing rhizobacteria isolated from apple trees in trans Himalayan region of Himachal Pradesh [J]. Archives of Microbiology,2013,195(5):357-369
[10] 吴鹏飞,张冬明,郝丽虹,漆智平. 解磷微生物研究现状及展望[J]. 中国农业科技导报,2008,10(3): 40-46
[11] Fernondez L A,Zalba P,Gomez M A,Sagardoy M A. Phosphate solubilization activity of bacterial strains in soil and their effect on soybean growth under greenhouse conditions[J]. Biology and Fertility of Soils,2007,43(6):805-809
[12] 张硕成. 木霉菌生态学及其在生防中的应用[J]. 应用生态学报,1991,2(1):85-88
[13] 张树武,徐秉良,薛应钰,古丽君. 长枝木霉对小麦禾谷孢囊线虫的致死作用[J]. 应用生态学报,2014,25(7):2093-2098
[14] 张树武,刘佳,徐秉良,古丽君,薛应钰. 长枝木霉对禾谷胞囊线虫寄生和致死作用的显微观察及测定[J]. 应用生态学报,2013,24(10):2055-2960
[15] Elad Y. Trichoderma harzianum T39 preparation for biocontrol of plant disease-control of Botrytis cinerea,Sclerotinia sclerotiorum and Cladosporum fulvum[J]. Biocontrol Science and Technology,2000,10(4):499-507
[16] Altomare C,Norvell W A,Bjorkman T,Harman G E. Solubilization of phosphates and micronutrients by the plant growth promoting and biocontrol fungus Trichoderma harzianum Rifai[J]. Environmental Microbiology,1999,65(7):2926-2933
[17] Azarmi R,Hajieghrari B,Giglou A. Effect of Trichoderma isolates on tomato seedling growth response and nutrient uptake[J]. African Journal of Biotechnology,2011,10(31):5850-5855
[18] 薛应钰,叶巍,张树武,刘佳,徐秉良. 微波诱变选育高效溶磷木霉菌株的研究[J]. 干旱地区农业研究,2016,34(4):231-236
[19] 虞龙,张宇,龚文静,安肖,徐翔. 氯化锂-紫外-离子束复合诱变红霉素高产菌株研究[J]. 原子能科学技术,2011, 45(7):780-784
[20] 李蕤,骆军,虞磊,鲁润龙,潘仁瑞. 复合诱变筛选产细胞壁水解酶木霉菌株及产酶条件的优化[J]. 微生物学通报,2002,29(5):47-52
[21] 韩立亚,冯培勇,张玉香,张丽. 复合诱变选育高产纤维素酶绿色木霉菌株[J]. 安徽农业科学,2009,37(9):3869-3870,3883
[22] 赵丹萍. 产几丁质酶木霉的诱变选育及对玉米纹枯病的生物防治[D]. 雅安:四川农业大学,2010
[23] 尹婷,徐秉良,梁巧兰,古丽君,李荣峰. 耐药性木霉T2菌株的筛选、紫外诱变与药剂驯化[J]. 草业学报,2013,22(2):117-122
[24] 李剑峰,张淑卿,师尚礼,霍平慧. 微波诱变选育耐药高效溶磷苜蓿根瘤菌[J]. 原子能科技技术,2009,43(12):1071-1076
[25] 岳斌,王宝维,张名爱,荆丽珍,张倩. 鹅源草酸青霉溶磷效果及对鹅磷代谢的影响[J]. 青岛农业大学学报(自然科学版),2008,25(1):34-37
[26] 王爱萍,刘立君,梁秀丽,姚旭霞,周彤. 钒钼黄分光光度法测定有机化合物中的微量磷[J]. 化学分析计量,2007,16(2):49-50
[27] 鲍士旦. 土壤农化分析[M]. 北京:中国农业出版社,2000
[28] 高俊凤. 植物生理学实验指导[M]. 北京:高等教育出版社,2006
[29] 葛菁萍,蔡柏岩,宋刚,孙宗祥,平文祥. 土壤中功能真菌的分离及其解磷能力的初步研究[J]. 中国土壤与肥料,2009,6:84-86
[30] Zhan S W,Gan Y T,Xu B L,Xue Y Y. The parasitic and lethal effects of Trichoderma longibrachiatum against Heterodera avenae[J]. Biological Control,2014,72:1-8
[31] 王玉霞,胡基华,刘宇帅,姜威,孟利强,李晶,曹旭,陈静宇,张淑梅. 低温生防菌株的诱变选育及生防效果[J]. 核农学报,2017,31(10): 1873-1880
[32] 张曼曼,邓春生,马金奉,燕荣,耿兵,李顺鹏. 多功能木霉的筛选及鉴定[J]. 农业环境科学学报,2012,31(8):1571-1575
[33] 杨合同,于雪云,魏艳丽,周红姿,宋莉璐,扈进冬,李纪顺 . 多功能植病生防木霉的分离筛选与鉴定[J]. 山东科学,2009,22(5):211-214
[34] 薛应钰,叶巍,张树武,徐秉良,杨百昶. 紫外诱变选育木霉高效解磷菌株[J]. 核农学报,2015,29(8):1509-1516
[35] 刘俊梅,王庆,王丹,杨盼盼,丁伟,朴春华,于寒松. 紫外线和硫酸二乙酯复合诱变选育PHB高产菌株[J]. 微生物学通报,2018,43(10):2242-2248
[36] 覃玲灵. 里氏木霉的紫外诱变和高活性纤维素酶突变株的选育[D]. 长沙:中南林业科技大学,2011
[37] 李瑞芳,赵玉峰,杨世清,伊艳杰,田泱源. 枯草芽胞杆菌BS501a复合诱变菌株及抗菌谱研究[J]. 植物保护,2011,37(5):86-91