解磷芽孢杆菌的筛选鉴定及其对玉米促生机理的研究

发布时间：2018-11-09 16:38

【摘要】：磷是作物必需的大量养分元素,土壤中磷大部分为难溶态,难以被植物吸收利用。为此,本研究采用磷酸钙液体培养基富集培养方法,从土壤中分离具有高效溶解难溶性磷的菌株L1和L2;采用玉米盆栽和田间试验,研究了各菌株对土壤磷有效性、植株磷含量和植株生物量的提高效果;采用绿色荧光蛋白基因标记法,研究了菌株L1在玉米根表定殖的时间和位置。主要结果如下:(1)经筛选得到8株解磷菌,在1%的磷酸钙液体培养基,30 oC培养72 h时,培养液中水溶性磷含量为73.72~392.56 mg·L-1,其中菌株L1、L2、L7的溶磷效果相对较高,其水溶性磷含量分别为392.56 mg·L-1、296.77 mg·L-1、353.59 mg·L-1,为此,进一步分析了这两个菌株的菌落形态、生理生化和16S rDNA测序特征,初步鉴定菌株L1为巨大芽孢杆菌(Bacillus megaterium),菌株L2为阿氏芽孢杆菌(Bacillus aryabhattai),菌株L7为巨大芽孢杆菌(Bacillus megaterium)。(2)2015年6月的玉米盆栽结果,在接种菌株后25天,菌株L1和L2处理土壤的有效磷含量比CK土壤分别提高了30%和21%。2015年9月的盆栽数据显示,在第15天及第25天,各菌株显著提高了土壤磷有效性,尤其是第25天,L1处理组土壤有效磷含量较CK提高了46%,较L2提高了32%。(3)两种菌剂均能够显著提高玉米植株的磷累积量。第一次玉米盆栽试验,与CK相比,L1处理组的磷累积量显著提高了22.41%。在第二次盆栽实验中,L1和L2处理组的磷累积量分别提高了34.04%,27.71%。盆栽条件下L1处理组较L2处理组更有效地促进了植物对磷的吸收。玉米田间实验两组处理的累积量则分别是55.95%,57.63%。(4)菌株L1比L2显著提高植株茎叶干重、根干重和玉米果实产量。L1和L2处理组相较于CK,盆栽玉米的茎叶干重增加了18.78%、18.62%,根干重增加14.84%,、4.69%。在玉米田间实验中,菌处理的植株茎叶干重增加了75.28%,、60.75%,根干重增加了100.70%、99.82%,产量增加了9.56%、8.09%。以上结果说明,菌株L1的溶磷效果高于L2,L1处理土壤磷的有效性、植株磷吸收和植株生物量均高于L2处理。为此采用绿色荧光蛋白(GFP)标记方法,通过分子克隆方法,构建了荧光强度高,稳定好的GFP标记菌株L1,荧光亮度较强,每24h转接一代连续7代后,GFP质粒稳定性依旧高达87%,说明构建菌株可用于研究菌株L1在玉米根系的定殖机制。在玉米发芽后2天,再接种GFP标记菌株L1,24小时后,观察玉米根表表面和内部均有GFP菌株L1,由此确证了L1菌株已成功定殖在玉米根毛内部细胞。综上,这些结果显示了菌株L1在改善土壤磷有效性和提高玉米磷养分的潜力,具有巨大的生物肥料潜质并且有望在农业可持续发展中发挥促进作用。同时在实验室条件下,确证了L1成功定殖在玉米根毛表面和内部细胞,为下一步菌株L1在原位土壤和植物根际定殖机制研究打下基础。
[Abstract]:Phosphorus is a necessary nutrient element in crops. Most of the phosphorus in soil is difficult to be absorbed by plants. Therefore, the strain L1 and L2 with insoluble phosphorus were isolated from soil by using calcium phosphate liquid culture medium. Pot and field experiments were carried out to study the effect of each strain on soil phosphorus availability, phosphorus content and biomass, and the time and position of strain L1 colonization in maize root surface were studied by green fluorescent protein gene marker method. The main results are as follows: (1) 8 strains of phosphorus releasing bacteria were selected and cultured on 1% calcium phosphate liquid medium for 72 h for 30 oC. The water-soluble phosphorus content in the culture medium was 73.72 ~ 392.56 mg / L ~ (-1), in which strain L _ (1) and L _ (2) were obtained. The water-soluble phosphorus content of L7 was 392.56 mg L-1296.77 mg L-1353.59 mg L-1, respectively. The colony morphology, physiological and biochemical characteristics and 16s rDNA sequencing characteristics of the two strains were further analyzed. The results of corn pot culture of Bacillus megaculis (Bacillus megaterium), strain L2, (Bacillus aryabhattai), strain L7 and Bacillus megaterium (Bacillus megaterium). (2 in June 2015 were preliminarily identified, and 25 days after inoculation. The available phosphorus content of soil treated with strain L1 and L2 was 30% higher than that of CK soil, respectively, and the pot culture data of September 21.2015 showed that, on the 15th and 25th days, the soil phosphorus availability was significantly increased by the strains, especially on the 25th day. Compared with CK and L2, the soil available phosphorus content of L1 treatment increased by 46% and 32% respectively. (3) the accumulation of phosphorus in maize plants was significantly increased by two kinds of fungicides. In the first maize pot experiment, phosphorus accumulation in L1 treatment group was significantly higher than that in CK treatment group (22.41%). In the second pot experiment, the accumulation of phosphorus in L1 and L2 groups was increased by 34.04 and 27.71, respectively. Under pot condition, L1 treatment group was more effective than L2 treatment group in promoting plant phosphorus absorption. (4) strain L1 significantly increased stem and leaf dry weight, root dry weight and corn fruit yield compared with L2 treatment. L1 and L2 treatments were significantly higher than CK,. The stem and leaf dry weight of potted corn increased 18.78% and the root dry weight increased 14.84% and 4.69% respectively. In maize field experiment, the dry weight of stem and leaf increased by 75.2880.75, the dry weight of root increased by 100.70and 99.82, and the yield increased by 9.56and 8.09. The results showed that the phosphorus solubilization effect of strain L1 was higher than that of L2N 1 treatment, and the phosphorus uptake and biomass of plant were higher than that of L2 treatment. Using green fluorescent protein (GFP) labeling method and molecular cloning method, a stable GFP labeled strain L1 with high fluorescence intensity was constructed. The stability of GFP plasmid was still up to 87 indicating that the constructed strain could be used to study the colonization mechanism of strain L1 in maize root system. After 2 days of germination and 24 hours after inoculation of GFP labeled strain L1, GFP strain L1 was observed on the surface and inside of maize root surface. It was confirmed that L1 strain had been successfully colonized in the inner cells of maize root hair. In conclusion, these results indicate that strain L1 has great potential in improving soil phosphorus availability and maize phosphorus nutrient, and is expected to play a promoting role in the sustainable development of agriculture. At the same time, it was confirmed that L1 was successfully colonized on the surface and inner cells of maize root hair under laboratory conditions, which laid a foundation for the further study of colonization mechanism of L1 in situ soil and plant rhizosphere.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S154.3;S513

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