高效固氮芽孢杆菌的选育及其效应研究
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摘要
本研究从105个不同生态环境下的植物根际土中筛选得到11株固氮酶活性高的固氮芽孢杆菌菌株,并对其形态特征、生理生化特征、16S rDNA序列、对环境的适应性及其对植物的促生效果和植物根际土壤细菌群落特征作了系统研究,结果如下:
固氮芽孢杆菌的筛选与鉴定:首先利用芽孢杆菌中芽孢的抗热性将土壤溶液在100℃沸水中煮10-15分钟,然后用选择性无氮培养基进行初筛,得到347株菌落形态不同的菌株;接着用乙炔还原法测定固氮酶活性,结果表明170株能测出固氮酶活性,其中12株比参比菌株圆褐固氮菌(Azotobocter chroococcum)固氮酶活性高,这12株菌的编号依次为:Y1、C2、B3、D4、E5、Y6、S7、S8、S9、N10、Y11和Y12。结合形态学特征、生理生化特征和16S rDNA序列分析对这12株进行了初步鉴定,结果表明其中菌株Y12属于微杆菌属(Miclvbacterium sp.),菌株Y1、S9、N10属于类芽孢杆菌属(Paenibacillus sp.),其余8株属于芽孢杆菌属(Bacillus sp.)。
对小白菜的接种效应:筛选的11株固氮酶活性高的芽孢杆菌在小白菜上进行接种试验,试验结果表明,接种固氮芽孢杆菌菌液对小白菜的生长起到促进作用,而且植株的鲜重、干重都有明显的提高,其中鲜重增幅38.2%～69.8%,干重增幅25.9%～69%。同时,植株的全氮量也有明显的提高,其中10个接种菌液处理分别达到0.05显著水平。测定接种B3、S9菌株根际土壤的全氮、有效磷、速效钾含量发现,根际土壤的全氮、有效磷、速效钾含量都有一定程度的提高。
对根际土壤细菌多样性的影响:采用PCR-DGGE方法分析Y1、B3、D4、S7、S8、S9、N10、Y11等8株固氮芽孢杆菌接种小白菜根际后,对根际土壤细菌多样性的影响。结果表明,与不接种菌液的对照土壤相比,接种菌液后的土壤细菌多样性提高,而且不同接种菌的处理对土壤细菌多样性的影响也不同。
环境因子的适应性:无氮培养条件下,测定温度、酸碱度、铵盐对B3、S9菌株固氮能力的影响,结果表明,在15～40℃下均能生长并表达固氮酶活性;在偏酸和偏碱的条件下(pH4～9)均能保持较高的固氮酶活性;能耐受环境中的一定浓度的铵,B3和S9分别在0.5mmol/L和1mmol/LNH_4~+时有最高固氮酶活性。由此可知,B3和S9菌株对环境具有较广的适应性。
In this thesis, eleven strains of nitrogen-fixing bacillus with high nitrogenase activity were selected from 105 samples of rhizosphere soil. At the same time, the further studies for them were conducted. The results obtained are as follows:
The selection and identification of nitrogen-fixing bacillus: A total 347 isolates were selectively obtained, based on their growth on nitrogen-free medium and their resistance to 100℃for 10-15 min. These isolates were tested for their nitrogenase activity using acetylene reduction assay (ARA). The result suggested that 170 isolates showed nitrogenase activity. The nitrogenase activities of 12 isolates were higher than the reference strain (Azotobocter chroococcum). These 12 isolates were Y1, C2, B3, D4, E5, Y6, S7, S8, S9, N10, Y11 and Y12. They were identified based on configuration, physiological and biochemical characterization, 16S rDNA sequence. Isolates Y12 was identified as Microbacterium sp. Isolates Y1, S9 and N10 were identified as Paenibacillus sp. Isolates C2, B3, D4, E5, Y6, S7, S8 and Y11 were identified as Bacillus sp.
The effect of inoculation on the pakchoi: The pakchoi was inoculated with 11 bacillus strains of high nitrogenase activity respectively. The results indicated that the treatments with nitrogen-fixing bacillus had positive effect on pakchoi's seedling growth and led to the significant increase in fresh, dry plant weights of pakchoi. The plant fresh weight was increased by 38.2%～69.8% and the plant dry weight was increased by 25.9%～69%. Meanwhile, the total nitrogen content of pakchoi seeding was also increased considerablely, reaching significant level for 10 strains of them respectively. In addition, the experiment showed that both B3 and S9 strains had slight influence on total nitrogen, available phosphorus and quick-acting potassium contents of the rhizosphere soil.
The impact of bacterial diversity in rhizosphere soil: With PCR-DGGE method, it was detected the fluctuation of bacterial community diversity in pakchoi rhizosphere soil, which was influenced by nitrogen-fixing bacillus. The results showed that the bacterial community diversity increased as the treatment with nitrogen-fixing bacillus, and the different treatments had different impact of the diversity.
The applicability to diverse environmental factors: Effects of diverse environmental factors on nitrogenase activity (ARA) of both B3 and S9 strains in nitrogen-free culture were investigated in our experiments. The results implied that these two strains could easily adapt to different cultural conditions: it could propagate quickly and fix nitrogen at a temperature range of 15℃to 40℃and at a pH range of 4 to 9. Moreover, low NH_4~+ concentration had little effect on its nitrogenase activity. When the NH_4~+ concentration was about 0.5mmol/L and 1mmol/L respectively, their highest nitrogenase activities could be detected.
固氮芽孢杆菌的筛选与鉴定:首先利用芽孢杆菌中芽孢的抗热性将土壤溶液在100℃沸水中煮10-15分钟,然后用选择性无氮培养基进行初筛,得到347株菌落形态不同的菌株;接着用乙炔还原法测定固氮酶活性,结果表明170株能测出固氮酶活性,其中12株比参比菌株圆褐固氮菌(Azotobocter chroococcum)固氮酶活性高,这12株菌的编号依次为:Y1、C2、B3、D4、E5、Y6、S7、S8、S9、N10、Y11和Y12。结合形态学特征、生理生化特征和16S rDNA序列分析对这12株进行了初步鉴定,结果表明其中菌株Y12属于微杆菌属(Miclvbacterium sp.),菌株Y1、S9、N10属于类芽孢杆菌属(Paenibacillus sp.),其余8株属于芽孢杆菌属(Bacillus sp.)。
对小白菜的接种效应:筛选的11株固氮酶活性高的芽孢杆菌在小白菜上进行接种试验,试验结果表明,接种固氮芽孢杆菌菌液对小白菜的生长起到促进作用,而且植株的鲜重、干重都有明显的提高,其中鲜重增幅38.2%～69.8%,干重增幅25.9%～69%。同时,植株的全氮量也有明显的提高,其中10个接种菌液处理分别达到0.05显著水平。测定接种B3、S9菌株根际土壤的全氮、有效磷、速效钾含量发现,根际土壤的全氮、有效磷、速效钾含量都有一定程度的提高。
对根际土壤细菌多样性的影响:采用PCR-DGGE方法分析Y1、B3、D4、S7、S8、S9、N10、Y11等8株固氮芽孢杆菌接种小白菜根际后,对根际土壤细菌多样性的影响。结果表明,与不接种菌液的对照土壤相比,接种菌液后的土壤细菌多样性提高,而且不同接种菌的处理对土壤细菌多样性的影响也不同。
环境因子的适应性:无氮培养条件下,测定温度、酸碱度、铵盐对B3、S9菌株固氮能力的影响,结果表明,在15～40℃下均能生长并表达固氮酶活性;在偏酸和偏碱的条件下(pH4～9)均能保持较高的固氮酶活性;能耐受环境中的一定浓度的铵,B3和S9分别在0.5mmol/L和1mmol/LNH_4~+时有最高固氮酶活性。由此可知,B3和S9菌株对环境具有较广的适应性。
In this thesis, eleven strains of nitrogen-fixing bacillus with high nitrogenase activity were selected from 105 samples of rhizosphere soil. At the same time, the further studies for them were conducted. The results obtained are as follows:
The selection and identification of nitrogen-fixing bacillus: A total 347 isolates were selectively obtained, based on their growth on nitrogen-free medium and their resistance to 100℃for 10-15 min. These isolates were tested for their nitrogenase activity using acetylene reduction assay (ARA). The result suggested that 170 isolates showed nitrogenase activity. The nitrogenase activities of 12 isolates were higher than the reference strain (Azotobocter chroococcum). These 12 isolates were Y1, C2, B3, D4, E5, Y6, S7, S8, S9, N10, Y11 and Y12. They were identified based on configuration, physiological and biochemical characterization, 16S rDNA sequence. Isolates Y12 was identified as Microbacterium sp. Isolates Y1, S9 and N10 were identified as Paenibacillus sp. Isolates C2, B3, D4, E5, Y6, S7, S8 and Y11 were identified as Bacillus sp.
The effect of inoculation on the pakchoi: The pakchoi was inoculated with 11 bacillus strains of high nitrogenase activity respectively. The results indicated that the treatments with nitrogen-fixing bacillus had positive effect on pakchoi's seedling growth and led to the significant increase in fresh, dry plant weights of pakchoi. The plant fresh weight was increased by 38.2%～69.8% and the plant dry weight was increased by 25.9%～69%. Meanwhile, the total nitrogen content of pakchoi seeding was also increased considerablely, reaching significant level for 10 strains of them respectively. In addition, the experiment showed that both B3 and S9 strains had slight influence on total nitrogen, available phosphorus and quick-acting potassium contents of the rhizosphere soil.
The impact of bacterial diversity in rhizosphere soil: With PCR-DGGE method, it was detected the fluctuation of bacterial community diversity in pakchoi rhizosphere soil, which was influenced by nitrogen-fixing bacillus. The results showed that the bacterial community diversity increased as the treatment with nitrogen-fixing bacillus, and the different treatments had different impact of the diversity.
The applicability to diverse environmental factors: Effects of diverse environmental factors on nitrogenase activity (ARA) of both B3 and S9 strains in nitrogen-free culture were investigated in our experiments. The results implied that these two strains could easily adapt to different cultural conditions: it could propagate quickly and fix nitrogen at a temperature range of 15℃to 40℃and at a pH range of 4 to 9. Moreover, low NH_4~+ concentration had little effect on its nitrogenase activity. When the NH_4~+ concentration was about 0.5mmol/L and 1mmol/L respectively, their highest nitrogenase activities could be detected.
引文
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