Evaluating And Identifying Of Plant Growth Promoting Rhizobacteria Of Dominant Alpine Meadows

Alpine meadows have extremely important ecological effects and economic values, the growing environment of them have large temperature difference and changeable climate which made them have strong cold and drought tolerance and the rhizosphere environment of them are very bad. Therefore, if excellent plant growth promoting rhizobacteria could screened from rhizosphere of them, providing the high-quality sources of bacteria to the development of subsequent microbial fertilizers. This study choiced typical dominant alpine meadows Kobresia myosuroides and Polygonum viviparum as the research objects, by molybdenum blues colorimetry, acetylene reduction assay and solid phase extraction-high performance liquid chromatography measured, analysed and evaluated the phosphate solubilization, nitrogen fixation and plant hormones secretion of the rhizosphere growth promoting bacteria, and through observation of morphological characteristics, determination of physiological and biochemical characteristics and 16 S r DNA gene sequence analysis identified the excellent promoting strains. Got the results as follows:(1) At first, 68 strains were isolated from the rhizosphere of two dominant kinds of alpine meadows. After the second time of screening, 43 strains were isolated them. Statistics of the distribution of strains in different parts of the rhizosphere of the plants, found that the surface of roots significantly more than the interior of roots and soil adhering to roots.(2) There were 17 dissolved inorganic phosphorus strains and 23 dissolved organic phosphorus strains screened from 43 rhizosphere bacteria strains. By molybdenum blue colorimetric quantitatively determined the phosphate solubilization capacity of the strains. The results showed that, the soluble phosphorus content of dissolved inorganic phosphorus strains between 9.39 to 94.79 μg·m L-1, the dissolved phosphorus maximum strain was ZNRS2 which isolated from the rhizosphere of Kobresia myosuroides, and the next was SNRP2, the difference of dissolved inorganic phosphorus between each strains was significant(P<0.05); the soluble phosphorus content of dissolved organic phosphorus strains between 10.37 to 72.82 μg·m L-1, the dissolved phosphorus maximum strain is SKHP3 which isolated from the rhizosphere of Polygonum viviparum, and the next was SKRP2, the difference of dissolved organic phosphorus between each strains was significant(P<0.05).(3) There were 30 nitrogen fixation strains screened from 43 rhizosphere bacteria strains. Nitrogenase activity was determined quantitatively by acetylene reduction assay. The results showed that the nitrogenase activity of nitrogen fixing strains from 3.79 to 3193.07 nmol(C2H4)·h-1·m L-1, the nitrogenase activity is highest is strain SKRP2, there are 3 strains which nitrogenase activity were more than 100.00 nmol(C2H4)·h-1·m L-1, included strains SKRP1, SNHP1 and ZNRS3. There were 27 strains which nitrogenase activity were less than 100.00 nmol(C2H4)·h-1·m L-1, accounting for 90% of the total amount of nitrogen fixing strains.(4) Established a solid phase extraction-high performance liquid chromatography which could simultaneously separated and determined three plant hormones(indole-3-acetic acid, gibberellic acid and cytokinin), and used this method successfully detected the 3 kinds of plant hormones from fermentation products of 43 rhizosphere strain. The results showed that the plant hormones were very small in the fermentation products of the strains, and the orders of magnitude were all at 102 μg m L-1. These 43 bacterial strains had the ability to secrete plant hormone, and 37.21% of the strains can produced 3 kinds of plant hormones at the same time, 44.19% of the strains can produced 2 kinds of plant hormones at the same time. The ability of secreting plant hormones of the growth promoting strains were: cytokinin>gibberellic acid>indole-3-acetic acid. Statistics of the distribution of the plant growth promoting rhizobacteria which could secreted plant hormones of 2 plants showed that the surface of roots significantly more than the interior of roots and soil adhering to roots.(5) Strains ZNRS2, SNRP2, ZKHP3 and ZKRP1 can be used as a source of high quality phosphate dissolving bacteria; strains SKRP2, SNHP1 and ZNRS3 can be used as a source of high quality nitrogen fixing bacteria; strains SKHP3, ZNHP2, ZKRS2, ZKRP1 and ZKRP2 can be used as a source of high quality plant hormones; comprehensive analysis of the determination results of the characteristics of 43 strains of dissolved phosphorus, nitrogen fixation and secretion of plant hormone, 15 strains were screened out for excellent growth promoting strains, among them there were 7 strains secreted from Kobresia myosuroides: SNRS1, SNRS2, SNRP2, SKRS1, SKRP2, SKRP3, SKHP3, among them there were 8 strains secreted from Polygonum viviparum: ZNRS3, ZNRP3, ZNRP4, ZNHP2, ZKRS1, ZKRS2, ZKRP1, ZKRP2.(6) Through analysis on the observation of morphological characteristics, determination of physiological and biochemical characteristics and 16 S r DNA gene sequence analysis, identified 15 excellent plant growth promoting rhizobacteria. The results showed that the 15 strains belong to Bacillus, contains three different kinds, namely: Bacillus pumilus, Bacillus cereus and Bacillus subtilis.