Isolation and identification of the antagonistic strain DM-54 of Bacillus amyloliquefacien against Verticillium dahliae, and optimization of antifungal protein producing conditions

Abstract

The strains capable of resistance against Verticillium dahliae Kleb were isolated and screened from the soils of cotton fields from several different provinces in China. A strain, coded DM-54, with a rather high antagonistic activity was obtained. Its morphological characteristics, physiological and biochemical properties and a 16 S rDNA sequence of this strain were further studied. The DM-54 strain was finally identified as a kind of Bacillusamyloliquefacien. Through a single factor experiment and an orthogonal experiment, the optimal shaking flask fermentation condition of strain DM-54 was found to be: media composed of 5% dextrin, 3% soy peptone, 0.02% MgSO₄, 0.01% CaCl₂, initial pH 7.0 and 10% inoculum volume, media volume 30/250 (mL/mL), fermentation temperature at 32ºC, rotating speed 200 r∙min^-1, fermentation time of 48 h. Its antagonistic activity was distinguished to be elevated, at about 39.9%. Our research offers an effective means for the massive production of antagonistic proteins.

Key words： cotton verticilliumwilt; antagonistic bacterium; isolation and identification; Bacillus amyloliquefacien; fermentation

Acknowledgements

This project was supported by the Hebei Natural Science Fund Projects, China (No. 398152).

References

1 Buchanan R E, Gibbons N E (1984). Bergey’s Manual of Determinative Bacteriology. Beijing: Science Press, 729–797

2 Cheng L J, Xue Q H, Lai H X (2000). Experimental Technology of Microbiology. Xi'an: World Publishing Corporation (in Chinese)

3 Dong X Z, Cai M Y (2001). Manual of systematic and Determinative Bacteriology. Beijing: Science Press, 349–388 (in Chinese)

4 Hu M (2004). Isolation, purification and characterization of an antifungal protein to Verticillium dahliae of cotton. Dissertation for the Master Degree. Baoding: University of Hebei, 14–18 (in Chinese)

5 Jiang Y, Huang L L, Chen C Q, Qiao H P, Kang Z S (2007). Screen, identification and optimized fermentation condition of an actinomycete strain against pathogenic fungus Fulvia fulva. Acta Microbiologica Sinica, 47(7): 622–627 (in Chinese)

6 Lane D J (1991). 16S/23S rRNA Sequencing. In: Stackebrandt E, Goodfellow, eds. Nucleic Acid Techniques in Bacteria Systematics. Chichester: John Wiley & Sons, 115–147 (in Chinese)

7 Li S Z, Ma P, Liu X Z, Huang H C, Chen X H (2001). Biological control of cotton verticillium wilt by antagonistic bacteria. Journal of Huazhong Agricultural University, 20(5): 42–425 (in Chinese)

8 Liang Q M, Qi D M, Jia J, Hui M, Niu T G (2005). Isolation of antagonistic Bacillus and purification of antifungal protein B₁₁₀-a. Plant Protection, 8(5): 31–35 (in Chinese)

9 Quan C S, Wang J H, Xu H T, Fan S D (2006). Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity. Acta Microbiologica Sinica, 46(1): 7–12 (in Chinese)

10 Rainey F A, Ward-Rainey N, Kroppenstedt R M, Stackebrandt E (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage; proposal of Nocardiopsaceae fam. nov. International Journal of Systematic Bacteriology, 46: 1088–1092

11 Saitou N, Nei M (1987). The Neighbour-joining Method: a New Method for Reconstructing Phylogenetic Trees. Mol Biol Evol, 406–425

12 Sambong K, Jung Y, Hongik K (1995). A phylogenetic analysis of the genus Saccharomonospora conducted with 16 S rRNA gene sequences. International Journal of Systematic Bacteriology, 45(2): 351–356

13 Shen P, Fan X R, Li G W (1999). Microbiology Experiment. Beijing: Higher Education Press (in Chinese)

14 Xia Z J, Gu B K, Wu A M (1996). A study on the antagonistic activity of cotton to Verticillium dahliae induced by the endophytic bacteria in the ectohiaosphere and endorhizosphere of soil. Chinese Journal of Biological Control, 12(1): 7–10 (in Chinese)

15 Yang H, Cai L W, Pan Q B, Chen J P, Shi Q H, Zhang E, Wang H Y, Chen L M, Yang M F, Huo J Q (2006). Research advance of cotton verticillium wilt. Jiangxi Cotton, 28(6): 3–6 (in Chinese)

16 Zhou Y F, Du H F, Yuan H S, Zhang Y L, Zhu B C (2007). Isolation and purification of antifungal protein from Paenibacillus to Verticillium dahliae. Cotton Science, 19(2): 98–101 (in Chinese)

17 Zhou Y F, Zhao X Y, Zhang Y, Feng S L, Wang R Y (2006). Expression of Vitreoscilla hemoglobin gene in Bacillus thuringiensis. Journal of Hebei University (Natural Science Edition), 26(1): 33–37 (in Chinese)