| This study selected 7 selected representative plots according to different vegetation types in alpine meadow, such as azalea shrub (Rhododendron fruticosa shrub land), alpine willow shrubs (Salix cupularis fruticosa shrub land), Potentilla fruticosa alpine shrub (Dasiphoru fruticosa shrub land ), Polygonum grass (Polygonum grassland), grass steppe (Grass grassland), marsh grass (Swamp grassland) and Kobresia (Kobresia grassland). Isolated bacteria on surface soil (0 20cm) and discovered the dissolving capacity and dissolving mechanism by using microbiological methods and molecular biology methods, as well as selected some of superior dissolving strains to explored rock bottle fermentation in conditions and so on. And phylogenetic study has been studied by sequencing the 16SrDNA of some better dissolving strains. The results are as follows:1. Screen and Preliminary study of phosphorus dissolving bacteria and their capacity in soil of Alpine vegetation36 strains of bacteria were isolated based on chromo and the feature, including Y5, Y4 and Y15 are same in two area, so final is 33. After 20 generations of subculture in beef extract peptone medium, 20 Phosphate solubilizing bacteria decreased the 60.61% of total in phosphate solubilizing capacity. Determined D/d value of 13 remaining bacteria on Ca3(PO4)2, results showed that 13 solubilizing bacteria on Ca3 (PO4)2 are dissolution, after 8d D/d values were between 1.214.01. But the large difference was in dissolution results. Studies on the 13 strains, there is one excellent dissolving strain (D/d value≥4), accounting for 7.69%; dissolving effect of good (2≤D/d<4), 8 strains are accounting for 61.54%; effect of normal (1.5≤D/d<2), two strains are accounting for 15.38%; solubilizing effect of poor (D/d<1.5), there is only one accounting for 7.69%. Solubility of 13 solubilizing bacteria were tested by shake bottle qualitative determination of solubilizing Ca3(PO4)2, the results showed that the solubilizing capacity of those strains are quite different in the 1% significance level ,Y2 and other solubilizing ability of bacteria are very significant in differences, which dissolving the best;Y5,Y6,Y11 solubilizing effect have weak solubilizing effect, the significant level were not reached, but their dissolving capacity compared to the others were reached significant differences level; there was no significant difference between the solubilizing capacity of the four Y20, Y21, Y28 andY15.2. 4 Determination of capcity of excel strains of phosphate solubilizing bacteria to three types of phosphate ground phosphate rockEffect from 13 stable dissolving dissolving dissolving bacteria was better to select the four measured on three rock phosphate (rock phosphateⅢ, phosphate rockⅣ, rock phosphateⅣⅢ) dissolving capacity. In the same strain under different temperature conditions on the dissolution of phosphate rock capacity varies, the same rock phosphate under the same strain at different temperatures are not the same amount of its dissolution. Dissolving the best of which is at 10℃, strain Y2Ⅳon phosphate rock dissolution, the dissolving rate of 4.737 percent. Various strains of 30℃for the dissolution of phosphate rock on the effects of both began to decline, 35℃and reached a minimum, the basic effect is no longer dissolving. The strain on the effect of rock phosphate dissolutionⅣcommon good, under the same conditions, higher than the phosphate rockⅢ03 percentage points, while the phosphate rockⅣⅢthan 07 percentage points higher.Sand culture method, the sand culture of each strain culture in turn direct extraction, fumigation, digestion, determination of four rock phosphate solubilizing bacteria on a dissolution case, the results show: The culture of each strain fumigation digestion increased after the dissolving Liang Jun, indicating that phosphate solubilizing bacteria can be transformed not only the direct release of phosphorus utilization of plants for the environment, can also be part of the phosphorus stored in the body as their source of phosphorus, in addition to some was on assimilation for their growth needs. But the strain on the release of phosphate rock, storage, assimilation vary. The phosphate rock and phosphate rockⅢⅣ, 4 ones solubilizing bacteria during its dissolution, in addition to Y3 dissolve rock phosphate rock phosphate dissolution Y1ⅢandⅣ, the remaining, more than 50% of the total amount of dissolving P for direct release into the environment and going. For phosphate rockⅣⅢ, only strain Y4 to dissolve the direct release of phosphorus of 51.20%, the remaining three strains were the most of the store or assimilation of phosphorus dissolved. 4 rock phosphate solubilizing bacteria on the storage capacity of phosphorus are, but generally speaking, stocks are not large, only in strains Y2ⅣⅢon phosphate rock dissolution process, the dissolved phosphorus 73.12% stored in the cells. In assimilation of phosphorus, the various rock phosphate have no obvious pattern, all strains assimilate varied greatly.3. Dynamics and mechanism of phosphate solubilizing bacteria Y1 and screen of flask culture conditionsSelecting the best combination of the strain Y1 and the determination of rock phosphate solubilizing compositionⅣdynamic and dissolving mechanism were tested. When strain Y1 in 8d, D/d valued of 3.94 in maximum, followed by D/d value is not changed greatly with dissolving effect stabilized. Quantitative determination of the solubilizing dynamic, the strain Y1 in maximum 96h after the dissolving effect, flask in available phosphorus content of up to 3.995mg; however, Y1 keep its capcity in 24168 hours . Y1 strain of Phosphate Rock Phosphate Solubilization mechanism ofⅣresults: bacteria after 36h culture, the control of phosphorus content of 5.171mg, content of available phosphorus in acid 7.547mg, enzyme treatment available phosphorus content of 8.666mg, phosphorus acid and enzyme content of the common role of 7.316mg. Shows that enzyme treatment on the dissolution of phosphate rock and most obvious, followed by acid, acid and enzyme interaction rather minimal.Solubilizing bacteria on Y1 flask culture conditions were the best initial screening, the results show that: strain Y1 flask optimal conditions for fermentation temperature: 15℃30℃; medium ingredients: corn meal 1%, 1% buckwheat noodles ; pH value 7.5; 250ml flask of liquid volume are installed with 50ml.4. Identification of 16SrDNA in phosphate solubilizing bacteria13 isolates with Gram-negative bacteria for the majority, a total of 10, accounting for 76.92%, relatively small multi-cell, but the morphological diversity, there are rod-shaped, short rod-shaped, spherical, oval, almost spherical. Colony color to white majority, most translucent, a small number of bacteria in vivo with Bacillus, mostly round. For selection four strains of phosphate solubilizing bacteria in significant effect,16SrDNA genes of Y2, Y3 and Y4 are phylogenetically belonged to the the genus Erwinia billingiae (Gene Bank: AM055711.1), Pseudomonas reactans(Gene Bank: DQ257418.1) and Serratia plymuthica(Gene Bank: EF064206.1), with the significance 99%. According to the analysis, Y2, Y3 and Y4 are Erwinia billingiae, Pseudomonas reactans and Serratia plymuthica, which can be preliminarily determinated.However, 16SrDNA gene of Y1 has not been sequenced, and expecting further determination. |
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