| 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. Bacillus spp. is one of the important sources of phytase applied in industry. However, little is known about the role of phytase in the alleviation of the effect of salt stress to plant produced by Bacillus strains.Therefore, in order to evaluate whether the phytase secreted by Bacillus spp. can enhance the saline tolerance of plant, Bacillus strains, which were potential biocontrol agent against a broad spectrum of plant pathogenic fungi with the ability to produce extracellular phytase, were isolated. The objectives of this study were:(1) to reveal whether Bacillus strains can enhance the saline tolerance of plant in edaphic culturing experiment,(2) to obtain phytase mutants by means of DES and Microwave treatments,(3) to further investigate the activity of increasing resistance of wheat to salt stress of mutants and the wild type,(4) to identify the genetic stability of phytase mutants.The main conclusions of this paper are as follows:1. To reveal whether Bacillus strains T2 with phytase activity can alleviate the salt stress effect of NaCl to wheat seedlings in soil, physiological and biochemical activities of wheat under NaCl stress were studied by edaphic culture. The results showed that Bacillus subtilis had positive effect on wheat growth under free salt stress and salt stress. Compared to free Bacillus subtilis treatment, Bacillus subtilis accelerated the increase of plant height, plant fresh weight, total leaf chlorophyll content, soluble sugar content, praline content and enhanced the activities of protective enzymes, such as SOD, POD and CAT, but decreased the content of MDA under free NaCl stress, 180 mmol·L-1 NaCl, 360 mmol·L-1 NaCl and 540 mmol·L-1 NaCl stress. Significant increases in total leaf chlorophyll content were observed under free NaCl stress and 180 mmol·L-1 NaCl stress. Simultaneously, increase in SOD activities and decrease in content of MDA were observed under 180 mmol·L-1 NaCl, 360 mmol·L-1 NaCl and 540 mmol·L-1 NaCl stress. These may provide strong evidence that B. subtilis with phytase activity is effective for the increasing resistance of wheat to salt stress in soil.2. Strain Bacillus subtilis T2 was a potential biocontrol agent against a broad-spectrum plant pathogenic fungi with the ability to produce extracellular phytase.To improve its saline stress alleviating capacity, mutagenetic program was undertaken for the construction of extracellular phytase derivates. Two phytase mutants with genetic stability were obtained by means of DES and Microwave treatments. It was revealed that extracellular phytase activity of the positive mutant M15 was elevated by 31.7% and the negative mutant M3 decreased by 93.3% after 72 h cultivation compared to the wild type T2.3. To further evaluate whether the phytase secreted by T2 can enhance the saline tolerance of plant, hydroponic culturing experiment was undertaken. A phytase minus mutant M3 with genetic stability was obtained by means of DES treatment. The results of biochemical experiments revealed that the culture filtrates of wild-type strain T2 with phytase activity and purified phytase stimulated growth of wheat seedlings under phosphate limitation in the presence of 150 mmol·L-1 NaCl and phytate. Significant increases in plant height, fresh weight, total leaf chlorophyll content and TTC reducing power and decrease in content of MDA were observed in response to addition of phytase and culture filtrates of T2 to culture liquids. However, culture filtrates obtained from phytase-negative mutant strain M3 did not stimulate wheat seedlings growth under salt stress. These may provide strong evidence that phytase activity of B. subtilis is important for the increasing resistance of wheat to salt stress under phosphate limitation.4. It is important for the mutants with genetic stability to function normally in various environments. Two phytase mutants were taken to inherit from generation to generation. The result showed that the capacity of enzyme production and the salt stress alleviation effect did not change significantly compared to the wild type. So, they can be applied into practice stably. |
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