Screening, identification and biochemical cultivation of nitrogen-fixing bacteria in tobacco rhizosphere in a biochemical incubator

Nitrogen is one of the most important nutrients in the growth process of flue-cured tobacco. Through low-temperature cultivation and research in a biochemical incubator, it was found that more than 60% of the nitrogen nutrients absorbed by flue-cured tobacco come from the soil, and fertilizer nitrogen only accounts for about 35% of the total nitrogen. Therefore, increasing the nitrogen supply in the soil has important practical significance for high yields of flue-cured tobacco. Autotrophic nitrogen-fixing bacteria are a type of rhizosphere bacteria that live in soil organic matter and promote plant growth. The nitrogen-fixing bacteria screened in this experiment are all autotrophic nitrogen-fixing bacteria, all of which are Bacillus. Bacillus thuringiensis, Bacillus subtilis, methylotrophic Bacillus, and Bacillus megaterium are all bacteria with nitrogen-fixing functions.

The 16S sequence of N7 has a similarity of 98% with Bacillus megaterium, which can be inferred that this strain is Bacillus megaterium. Bacillus megaterium is a probiotic. In the 1940s, Bacillus megaterium had been experimentally studied. Scholars Sundar, Molla, and Ellott found that a mixed bacterial agent made with Bacillus megaterium as the core can degrade insoluble phosphorus compounds. In 1958, Hin first isolated a strain of Bacillus megaterium with high nitrogen fixation activity in Japan. Compared with biological fertilizers, Bacillus megaterium has its own unique and excellent characteristics, and its survival and adaptability are strong. This type of strain will play a very important role in the supply of non-artificial nitrogen. Bacillus megaterium not only has the function of efficiently degrading nitrite in tobacco, but also studies have shown that the microbial preparation made of Bacillus megaterium sprayed on tobacco leaves can effectively improve the aroma of the leaves and reduce the protein content, which greatly improves the quality of tobacco. In addition, the combined action of Bacillus megaterium and Arthrobacter can effectively improve the comprehensive utilization of waste tobacco leaves and the quality of tobacco sheets, which has great economic significance for tobacco companies.

The 16S sequences of N2, N5, N6, N9, and N10 have a similarity of 98% with Bacillus subtilis, from which it can be inferred that these strains are Bacillus subtilis. Bacillus subtilis is a probiotic that can not only improve the germination rate of plant seeds, but also has nitrogen fixation activity. At present, studies have shown that Bacillus subtilis is an endophytic nitrogen-fixing bacterium that colonizes in the host plant and directly secretes nitrogen-fixing products for the plant to absorb. It is not easily disturbed by external environmental conditions, and is more conducive to fully exerting the nitrogen-fixing efficiency and showing higher nitrogen-fixing efficiency. In the study of endophytic nitrogen-fixing bacteria in sugarcane, the efficient nitrogen-fixing activity of Bacillus subtilis and Bacillus megaterium was confirmed. Studies by English et al. showed that tobacco leaves can quickly produce a pleasant aroma after being inoculated with Bacillus subtilis and Bacillus circulans separately or in combination. And studies have shown that Bacillus subtilis can also be used to effectively prevent and control tobacco bacterial wilt. The 16S sequence of N1 is 99% similar to that of Bacillus thuringiensis, from which it can be inferred that the bacterium is Bacillus thuringiensis. Bacillus thuringiensis is not only commonly used as a microbial insecticide, but also has efficient nitrogen-fixing function. The 16S sequences of N4, N8, and N12 are 99% similar to those of Bacillusmethylotrophicus, from which it can be inferred that these bacteria are methylotrophic Bacillus with efficient nitrogen-fixing function. However, there are relatively few studies on the nitrogen fixation function of Bacillus thuringiensis and Bacillus methylotrophus, and the nitrogen fixation activity of these two species needs further study. The analysis results of ARDRA are roughly consistent, which confirms the correctness of our classification.

This experiment studied the suitable culture conditions and optimal pH value of Bacillus megaterium. Sucrose was used as the optimal carbon source for N7 for the next step of research. Compared with sucrose, glucose is a monosaccharide and is generally easier to be used by bacteria, but it produces acid faster during its metabolism, which causes the pH value of the culture medium to decrease rapidly, which may be the main reason for its lower growth rate than sucrose. The optimal initial pH value for the rapid growth of N7 is 7.50, and the strain grows well under neutral or alkaline conditions, which is consistent with the suitable culture conditions for plant growth-promoting bacteria reported in the literature. In low-temperature culture, the optimal growth temperature of N7 is 30℃. This may be because the lower temperature (25℃) causes the metabolism of the bacteria to be slower, which affects the growth and division rate of the bacteria. The higher temperature (40℃) exceeds the optimal temperature of the enzyme in the bacteria, which reduces its catalytic efficiency and affects the growth of the bacteria.