| Acid soil widely distributes in our country and covers all over southern fifteen provinces with a total area up to 2.03×10~7hm~2. Soil acidity is a major factor that limits crops and agricultural productivity. With the expansion of Legume pasture cultivation, legume plant in southern has became the important developmental direction. However, soil acidity is a major limit factor of legume pasture growth, which reduces the establishment of rhizobia-legume symbiosis and inhibits the survival of rhizobia, and thus blocks nitrogen fixation. In order to enhance the output and quality of legume, it is necessary to select appropriate acid-tolerant rhizobia to inoculate on the legume pastures growing on new reclaimed acidic soil.Pueraria lobata, commonly known as the fastest growing plant in the world, is a perrennial leguminous vine that could flourish under severe soil conditions. Pueraria lobata is characterized by high stress resistance, excessive nitrogen fixation, perennial, rapid elongation rates and wide distribution. Therefore, Pueraria lobata is regarded as an ideal crop to develop modern agriculture with the potential of increasing production. The blooming rootstocks of Pueraria lobata growing deeply into soils are able to keep water and soil and reduce soil erosion. Under southern acid soil conditions, symbiosis between Pueraria lobata and rhizobium has laid a foundation for increasing soil fertility, holding soil and water, improving entironment, and playing an vital role in converting arable land to forestry as well as beautifing mountains and rivers. Thus, planting Pueraria lobata in southwest area has great practical significance in China.Acid-tolerant rhizobia resources are rare in our country. Pueraria lobata distributes widely and adapts to various soil conditions including neutral and acid soil. It is not difficult to isolate and select acid-tolerant rhizobia from acid soil. This paper studied the acid tolerance and acid-tolerant mechanism of rhizobia strains isolated. The results provided hints for examining acid-tolerant mechanism at molecular level and promoted the application of Pueraria lobata. 1 Rhizobia were isolated from Pueraria lobata growing on acidic and yellow soil (pH4.6) on Jinyun Mountain in Chongqing. Compared with plants noninoculated, fresh root weight and subterranean of Pueraria lobata plants inoculated with PR390 exhibited a 537% and 330% increase, respectively. It also showed that the nodulation rate reached 68.33%. Nodules weight and activities of enzymes involved innitrogen fixation were higher than other strains. PR390 was able to grow normally in agar plates with pH4.8 and had a higher acid tolerance. The acidic condition influenced the cell sizes and diameter of colonies dramatically.2 Growth curves of PR390 were examined under different acidity (pH7.0, 5.5, 5.0, 4.8, 4.6, 4.4). The results indicated that acidity inhibited the growth of rhizobia. The lower the acidity was, the slower the growth of rhizobia with the characteristics of a longer lag phase, a lagged log phase, a delayed stationary phase and a longer generation time were. PR390 possessed the strong acid tolerance and was able to grow under pH4.6. The growth of rhizobia affected the pH value of culture similarly: causing the pH value of culture to increase and approach to neutral in the end.3 PR390 grew best in neutral soil suspension. The number of rhizobia decreased with the pH value of soil reducing. The increasing and decreasing trends of rhizobia number were examined under the acid soil suspension at pH4.6. The rhizobia number was up to 5.43×10~7CFU/ml in acid soil suspension, which indicated that PR390 could grow in barrenly acid soil suspension and had strong acid tolerance.4 In order to study the acid-tolerant mechanism, the lethal pH was examined. The lower the acidity was, the less rhizobia survived. Growth of rhizobia was not examined under pH 3.5. This indicated that strong acid condition did completely lethal harm to strain cell. The external pH of PR390, never induced at pH5.0, went up to pH3.42 gradually under the shock of pH3.3. It showed that capability of containing H~+ was very strong. It was speculated that PR390 could keep neutral entironment by contradicting the entering protons. The proton flux assay proved that PR390, induced at pH5.0, had the best acid adaptive tolerant response and the external pH held the line. It indicated that PR390 induced at pH5.0 was more capable of preventing H~+ from entering cells, which survived better in acid condition with an increased survival rate of 46.39%. It was speculated that special structure of cells after acid-induction may assist preventing H~+ from entering cells.5 PR390 was inoculated into liquid culture medium with pH4.6 and pH 7.0, respectively. It showed that K~+ concentration quickly went up at beginning and remained stable generally later. Meanwhile, external K~+ concentration in acid condition was 633mg/L and 33mg/L higher than in neutral condition. It showed that K~+ played an important role in acid tolerance of rhizobia. Potassium has been shown to be important in pH homeostasis. Higher external levels of potassium may assist in the survival of rhizobiaat sublethal levels of protons by maintenance of internal pH through anion exchange.6 Wether adding chloramphenicol or not neutral culture medium, the both logCFU curves kept parallel. Rhizobia obtained conditions for its own growth under neutral condition and resistance to chloramphenicol to some degree. Even if chloramphenicol was added to culture medium, the growth of rhizobia was not affected. Adding chloramphenicol to acidic culture medium, the logCFU of the number of living cells decreased 2.62 and drop time advanced 120 minutes, which speeded up the progress of cells death. It was speculated that PR390 may activate specific proteins within cell, which were favourable to live longer in acidic environment. These proteins may be restrained by chloramphenicol in acidic environment.7 The meristems of stem tips of Pueraria lobata were used as explants invitro culture. The optimum medium for callus induction from the meristems was MS+2,4-D0.5 mg/L. Then the calli were transferred to MS+6-BA 0.5 mg/L+NAA 0.5 mg/L for subsequent induction. The differentiation rate of calli on MS+6-BAlmg/L was as high as 100%. The shoots rooted readily on MS+NAA 0.1 mg/L, with a rooting rate of 60%.
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