Screening Of Extracellular Protease Mutants Of Bacillus Subtilis And Their Antagonistic Function

Bacillus species, which are both resistant to adverse environment and usually against bacterial and fungal pathogens, are among the dominant microorganisms in the soil and plant microecological systems. The biocontrol mechanisms of Bacillus include antagonism, competition and induced plant systemic resistance. The action of fungal cell-wall degrading enzymes such as protease, chitinase, andβ-1.3-glucanase were considered as one of the main mechanisms involved in the antagonistic process. Present study showed that the role of chitinase andβ-1.3-glucanase in biocontrol was closely related to the cell-wall components, which are complicatedly composed of chitin,β-1.3-glucan and several kinds of proteins. Extracellular protease may contribute to the antagonism of fungal pathogens cooperated with chitinase andβ-1.3-glucanase.Therefore, in order to clarify the status and function of extracellular protease in bicontrol and to provide supporting evidence for application and genetic improvement of Bacillus isolates with great potency, the objectives of this study were: (1) to determine the cell-wall proteins content of seven fungal pathogens which caused severe damage to crops in Shandong Province, (2) to analyze the correlation between the inhibitory effect of seven Bacillus strains and the activity of three cell-wall degrading enzymes, (3) to obtain protease mutants by means of UV-irradiation, DES and Microwave treatments and to investigate the activity of cell-wall degrading enzymes of mutants and the wild type, (4) to study the antagonistic activity of protease mutants and to test, in vitro, the effect of cell-free culture filtrate of mutants on hyphae and conidia germination of F. oxysporum f.sp. vasinfectum, (5) to identify the genetic stability of protease mutants. The main conclusions of this paper are as follows:1. According to the result of Bradford's method, the covalently bound proteins content was higher than that of the non-covalently bound proteins and the content of total cell-wall proteins in seven fungal pathogens differed greatly from each other. The highest content of 7.411 mg/g was observed in F. oxysporum f.sp. vasinfectum while the lowest content of 2.626 mg/g in B. cinerea. F. oxysporum f.sp. niveum, F. oxysporum f.sp. cucumerinum and R. cerealis shared the similar results which were respectivly 4.927 mg/g, 4.550 mg/g, 4.275 mg/g, while the content of F. graminearum was 3.898 mg/g.2. The correlation analysis of Bacillus strains between the inhibitory effect and the activity of cell-wall degrading enzymes is an elementary research for strain screening and improvement of biocontrol activity. According to the diverse results of different Bacillus strains in control of five plant pathogenic fungi, three biological indexes: protease,β-1.3-glucanase and chitinase were introduced. And then correlation coefficient was calculated for each of the above. The result showed that the protease acted an important part in process of antagonism against pathogenic fungi whileβ-1.3-glucanase and chitinase showed certain correlation. The correlation coefficient of protease amounted to 0.915, and followed byβ-1.3-glucanase to 0.811, chitinase to 0.572 when F. oxysporum f.sp. vasinfectum with highest cell-wall proteins content used as the target. The same conclusions were drawn from the investigation of F. oxysporum f.sp. cucumerinum, F. graminearum and A. alternata whereas different conditions existed in F. oxysporum f.sp. niveum with chitinase amounting to 0.824, protease to 0.623 andβ-1.3-glucanase to 0.497.3. Bacillus subtilis T2 was a potential biocontrol agent against a broad-spectrum plant pathogenic fungi with the ability to produce extracellular protease. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of extracellular protease derivates. Two protease mutants with genetic stability were obtained by means of UV-irradiation, DES and Microwave treatments. It was revealed that extracellular protease activity of the positive mutant T2-18 was elevated by 108.5% and the negative mutant T2-2 decreased by 81.1% after 48 h cultivation, whereas the activity of chitinase andβ-1.3-glucanase did not change significantly compared to the wild type T2.4. In vitro bioassays showed that the positive mutant T2-18 proved to be better antagonistis against F. oxysporum f.sp. vasinfectum. Its cell-free culture filtrate showed great inhibition to mycelial growth and conidia germination of the pathogen, which the swelling, distortion hyphae and shorten germination tubes were observed under light microscope. On the contrary, the inhibitory effect of the negative mutant T2-2 decreased markedly than the wild type T2, and there was no obvious effect on morphology of the hyphae and conidia. In experiments conducted on cucumber infected by F. oxysporum f.sp. cucumerinum, significant difference in inhibition was observed between wild type and the protease mutants. The mortality rate of cucumber seedlings treated with the positive mutant T2-18 was merely 40.0% and the negative mutant T2-2 was 79.6%, while the wild type T2 was 54.3% and the control was 80.1%. This study suggests the possibility of using mutants with secretion of extracellular protease against plant pathogenic fungi.5. It is important for the mutants with genetic stability to function normally in various environment. Two protease mutants were taken to inherit from generation to generation. The result showed that the capacity of enzyme production and the antagonists did not change significantly compared to the wild type. So, they can be applied into practice stably.