Selection of Insecticidal and Fungicidal Engineering Strain by Protoplasts Fusion of Inactivated Parental Strain Bacillus thuringiensis and Bacillus subtilis

doi:10.16409/j.cnki.2095-039x.2017.04.015

Abstract

Abstract: Fermentation broth of Bacillus thuringiensis B7 caused 85.06% mortality of Plutella xylostella 2nd instar larvae at 96 h after treatment. Bacillus subtilis TL2 showed antagonistic effects on pathogenic fungi Fusarium oxysporum and FPhytophthora capsici, which reduced more than 60% of mycelial growth of these fungi at 5 d after confronting incubation. The best condition for protoplast obtaining, complete inactivation, and protoplast fusion of these two bacterial strains were determined. The best protoplast formation rate and regeneration rate were 85.00% and 29.41%, respectively, for strain B7 under 1.0 mg/L of lysozyme for 70 min. The best protoplast formation rate and regeneration rate were 80.00% and 25.00%, respectively, for strain TL2 under 1.25 mg/L of lysozyme for 50 min. The best protoplasts inactivation condition for strain TL2 was water bath at 60℃ for 35 min, and for strain B7, ultraviolet radiation for 25min. The best protoplast fusion condition was water bath at 30℃ for 20 min. Then the mixture was spread on regeneration medium plates. Seventeen fusants were obtained after 10 times of subcultures, and the fusant TLB15 showed both high insecticidal and fungicidal activity. Fermentation broth of TLB15 resulted in 93.44% mortality of P. xylostella 2nd instar larvae at 96 h after treatment and inhibited 60.00% and 62.79% of mycelial growth of F. oxysporum and P. capsici, respectively (5 d of confrontation cultivation).

Key words: protoplast fusion, Bacillus subtilis, Bacillus thuringiensis

CLC Number:

S476.12

ZHAO Xiaoyan, ZHOU Fangyuan, ZHOU Hongzi, WU Xiaoqing, ZHANG Guangzhi, ZHANG Xinjian, LI Tianyuan, XIE Xueying. Selection of Insecticidal and Fungicidal Engineering Strain by Protoplasts Fusion of Inactivated Parental Strain Bacillus thuringiensis and Bacillus subtilis[J]. journal1, 2017, 33(4): 537-544.

References

[1] 孙冰冰, 李伟, 魏军, 等. 生防芽胞杆菌的研究进展[J]. 天津农业科学, 2015, 21(12):102-107.
[2] 刘国红, 刘波, 王阶平, 等. 芽胞杆菌分类与应用研究进展[J]. 微生物学通报, 2016, 9(1):1-11.
[3] 叶晶晶, 曹宁宁, 吴建梅, 等. 生防芽胞杆菌的应用研究进展[J]. 西北农林科技大学学报, 2014, 42(8):185-190.
[4] 梁小龙, 赵峰, 史荣久, 等. 原生质体融合构建高效产脂肽工程菌[J]. 中国生物工程杂志, 2014, 34(11):76-84.
[5] 葛菁萍, 安琦, 张玉环, 等. 灭活原生质体融合选育木糖、葡萄糖共发酵酿酒酵母工程菌[J]. 菌物学报, 2014, 33(1):87-96.
[6] 曾柏全, 李淼, 冯金儒. 双亲灭活青霉菌与枯草芽孢杆菌原生质体融合[J]. 中国食品学报, 2015, 15(6):45-49.
[7] 李佳玮, 张文琴, 林海, 等. 双亲灭活原生质体融合法选育阿维菌素高产菌株[J]. 沈阳药科大学学报, 2006, 23(5):320-323.
[8] Kannan R, Rajamanickam C, Annamalai T, et al. Biocontrol efficacy of protoplast fusants between Bacillus thuringiensis and Bacillus subtilis against Spodoptera litura Fabr[J]. Archives of Phytopathology and Plant Protection, 2014, 47(11):1365-1375.
[9] Liu Y Q, Wang Y P, Pan N S, et al. Studies on formation regeneration and interspecific fusion of protoplasts from Bacillus subtilis and Bacillus thuringiensis[J]. Acta Microbiologica Sinica, 1994, 34(1):76-80.
[10] Yan N L, Qiu S X, He H, et al. Construction of antagonistic insecticidaland endophytic strains of Bacillus spp. by protoplast fusion[J]. Chinese Journal of Agricultural Biotechnology, 2005, 2(2):131-135.
[11] 颜念龙, 邱思鑫, 何红, 等. 原生质体融合构建防病、杀虫和内生多功能工程菌[J]. 农业生物技术学报, 2004, 12(6):704-708.
[12] 刘伊强, 王雅平, 潘乃褪, 等. 芽抱杆菌原生质体的形成、再生及种间融合的研究[J]. 微生物学报, 1994, 34(1):76-80.
[13] 陈海荣, 刘前刚, 高书锋, 等. 原生质体融合构建水稻病害生防多功能工程菌株[J]. 湖南农业科学, 2010(1):13-15.
[14] 赵晓燕, 洪永聪, 郭凯, 等. TL2的鉴定及其对植物病原菌的抑制作用[J]. 福建农业学报, 2013, 28(4):353-356.
[15] 公维亮, 薛正莲, 周扬, 等. 原生质体诱变及融合选育那西肽高产菌株[J]. 中国抗生素杂志, 2016, 41(5):335-339.
[16] 郭建强, 柴秀娟, 庞学兵, 等. 芽胞杆菌高产纤维素酶菌株的诱变选育与培养基优化[J]. 核农学报, 2016, 31(2):263-270.
[17] 王金晶, 秦昶. 影印平板法方法的研究与改进[J]. 洛阳师范学院学报, 2005, 5:113-114.
[18] 桑利伟, 刘爱勤, 孙世伟, 等. 一株拮抗胡椒瘟病菌的生防菌S的鉴定及其生物学特性研究[J]. 中国生物防治学报, 2015, 31(2):291-296.
[19] 吴刚, 尤民生, 赵士熙, 等. 苏云金杆菌预处理小菜蛾对有机磷和氨基甲酸酯杀虫剂的增效作用[J]. 昆虫学报, 2001, 44(4):454-460.
[20] 薛正莲, 刘阳, 王洲, 等. 基因组改组选育产酯化酶地衣芽胞杆菌[J]. 中国生物工程杂志, 2013, 3(8):43-48.