A Study On Phosphate-solubilizing Bacteria Of Rhizosphere Soil In Walnut Plant Grown In Sichuan Province

Walnut plays an important role in agriculture economy and ecological environment of mountainous areas in Sichuan province, because of economic nuts and water and soil conservation. However, walnut plants often grow vigorously into 30-40 feet in height, and assimilate and consume lots of soil nutrients, especially for phosphorus. So to fertilize, including phosphorus is absolutely necessary to get high economic effect of walnut production. Whereas, the more closely the negative effects face to us, the more chemical fertilizer used in field, such as less unproducible phosphate and harder environmental pollution. Biological fertilizer is one of effective ways to absolve this puzzledom. Up to now, no reports have been found for phosphorus sollubilizing bacteria (PSB) in walnut field management.In this study, rhizosphere soil of 10-year-old walnut plants were sampled separately in the ten important production areas in Sichuan province. The soil microorganisms were isolated and the PSBs with high ability of phosphorus solubilizing were screened out from all the rhizosphere soil samples. These PSBs were identified effectively according to their biophysical and biochemical characteristics and 16S rDNA sequencing. The culture conditions were optimized through orthogonal experiments for the three PSBs. At last, this three PSBs with high ability of phosphorus sollubilizing were inoculate into one-year-old seedlings of pecan (Carya illinoensis) to observe the inoculation effects on such as plant growth, soil nutrients, microorganism and activities of soil enzymes.The main results according to this study are as follows:1. By using selective culture media, the phosphate solubilizing bacteria (PSB) in the walnut rhizosphere were isolated and identified with their community structure and genus studied. The results showed that:(1) The number of PSB that formed clear zones was among 3.71×105～35.21×105cfu/g dry soil (DS) across all the sites. (2) The altitude could not influence the PSB number. The maximum PSB number was recorded in cinnamon soil followed by yellow soil. The distribution of PSB was influenced by soil organic matter, soil available phosphorus, the content of alkali-hydrolysable nitrogen. The PSB number were found to be significantly positive correlated with organic matter or alkali-hydrolysable nitrogen of rhizosphere soil(r=0.639, r=0.724), but were significantly negative correlated to available phosphor content of soil(r=-0.896). (There were a strong negative correlation between the amount of PSB and available phosphorus content of soil. (r=-0.896). (3) The PSB isolated from all the rhizosphere soil were belonged to nine genera according to the colony morphogenesis, biochemical characterization, and Bacillus, Psedumonas and Arthrobacter were the dominant ones. (4) The diversity of PSB population could vary with different ecological environments. The diversity index of PSB was highest in Hanyuan site compared with the lowest index in Guangyuan site. Richness index changed as similar to the diversity index among all the sampling sites. The evenness index was highest in Guangyuan site and was the lowest in Nanjiang site. As for domination index, it was the highest in Xingwen site, whereas the lowest in Hanyuan site.2. Phosphate solubilizing activities of 37 strains of PSBs were tested on solid tricalcium and liquid phosphate medium. The soluble-P concentration ranged between 81.09 mg/L and 233.35 mg/L with variations among different strains. A strong negative correlation (r=-0.698) between pH and soluble P concentration was observed. Identification of 37 PSBs were carried out by 16S rDNA sequencing. Fifteen strains belonged to genus Pseudomonas, nine to genus Bacillus, three to genus Stenotrophomonas, two to genus Acinetobacter, one to genus Cupriavidus, one to genus Agrobacterium, one to genus Arthrobacter, one to genus Pantoe, one to genus Rhodococcus.3. The optimal culture media and phosphate solubilizing conditions of PSB W25, W9 and W12 were studied separately. We found that their growth and phosphate dissolving ability of these three bacteria strains were different under conditions of different carbon sources, nitrogen sources, initial pH, inoculums and liquid volumes. The growth ability of bacteria strains was positively correlated with their phosphorus dissolving abilities. The results of orthogonal experiments showed that:(1) for W25, the optimal carbon and nitrogen source were glucose at the concentration of 20.0 g/L, NH4C1 at the concentration of 1.0 g/L with pH7.5, inoculums of 4%, liquid volume of 75 mL. The importance of the four factors are ranked in decreasing order:carbon source> nitrogen source> liquid volume> the initial pH> inoculums. The phosphate solubilizing capacity of it was up to 204.56 mg/L under the optimized conditions of cultivation, being 16.28% higher than CK; (2) While for W9, the optimal carbon and nitrogen source were sucrose at the concentration of 30.0 g/L and NH4C1 at 1.0 g/L with pH7.0, inoculums of 6%, liquid volume of 100 mL. The importance of the four factors are ranked in decreasing order:carbon source> the initial pH> nitrogen source> inoculum> liquid volume. The phosphate solubilizing capacity of this bacteria strain was 192.25 mg/L under the optimized conditions of media, being 12.43% higher than CK; (3) For W12, the optimal carbon and nitrogen source were glucose at the concentration of 30.0 g/L, NH4NO3 at the concentration of 1.0 g/L with pH6.0, inoculums of 7%, liquid volume of 75 mL; The importance of the four factors are ranked in decreasing order: nitrogen source>liquid volume> carbon source> inoculum> the initial pH. The phosphate solubilizing activity of W12 was 192.12 mg/L under the optimized conditions of cultivation, being 18.44% higher than CK.4. The PSB strains W25, W9, W12 were marked with streptomycin, and the stable streptomycin resistance strains W25', W9'and W12'were obtained. Their colonization dynamics in walnut rhizosphere were studied under pot experiment condition by counting with selective medium. The results of potted experiment showed that:(1) the three strains could successfully colonize in the rhizosphere of walnut seedlings. (2) Both in sterilized and unsterilized soil, the colonization dynamics was similar for all the three bacteria strains. The highest colonization level of W25'and W12'were reached on 20 days after inoculation,1.28x108 cfu/g DS and 1.88x108 cfu/g DS,1.94x107 cfu/g DS and 2.09×107 cfu/g DS, respectively. But the survival number of W9'reached the peak value after inoculation of 10 days,2.02×107 cfu/g DS and 2.36×107 cfu/g DS under conditions of sterilized and unsterilized soil, respectively. And then, the population of the three strains decreased gradually and tended to be stable on 60 days after inoculation; (3) Colonization level in unsterilized soil was higher than sterilized soil.5. The effects of PSB strains on growth, photosynthesis characteristics, nutrient uptake and rhizosphere properties of walnut under pot experiment were studied. The results show that: (1) The plant height, biomass, net photosynthesis rate (Pn), total content of nitrogen and phosphorus of walnut seedlings were higher after inoculation with the PSB strains; (2) Application of the three PSB strains increased the content of available phosphorus and alkali-hydrolyzable nitrogen in the rhizosphere of walnut; (3) Amounts of bacteria and actinomyces of rhizosphere soil increased, but the amount of soil fungi decreased to some extent after inoculation with strains W25, W9 and W12. At the same time, activities of phosphatase and urease were improved effectively. The higher phosphorus solubilizing and growth promoting effects were observed for all the three PSBs in the low content of available phosphorus of potted soil. The maximum number of population of rhizosphere bacteria and actinomyces were measured at the shoot growth stage followed by defoliation and leaf expansion stage. Nevertheless, the amounts of rhizosphere fungi changed in opposite direction. For phosphatase and urease activities, both of them reached its maximum at the shoot growth stage followed by defoliation and leaf expansion stage of walnut seedlings; (4) The PSB strain could act with higher phosphate-solubilizing ability and growth promoting effect of plant under conditions of low available P in soil; (5) At the same phosphorus level of potted soil and bacteria strain, some important indexes including plant height, biomass, Pn value, total phosphorus and nitrogen content of plant, available nutrient of soil under conditions of unsterilized soil were higher than that under conditions of sterilized soil. (6) In conclusion, PSB strains could improve micro-ecological environment in rhizosphere soil of walnut plant, increase the enzyme activities which would promote effective absorption of N and P nutrition for walnut seedlings.