| 50% of the world's arable land and potential arable land is the acid soil. It covers 15 provinces in the south throughout China with a total area of 2.03x107hm2, which occupies 21% of the total land area. However the proportion has been expanding due to such factors as climate and long-term application of fertilizers and so on. Growth of many leguminous plants grow in acidic soils is restrained and the survival of the rhizobia is also influenced by the acid soil. Thus it is crucial to investigate the impact of aciduric rhizobia on nodule and nitrogen fixation of alfalfa as well as on growth of leguminous plants under the acidic condition, and effecting acid envrionment with related stresses on symbiotic nitrogen-fixing system of rhizobium leguminosarum and biological yield.This paper focuses on alfalfa and acidduric alfalfa rhizobia and investigates effect of application of calcium, phosphorus, and innoculation amount of aciduric alfalfa rhizobia on alfalfa growth under the acidic condition. Their impact on nodule and nitrogen fixation of alfalfa and quorum-sensing of rhizobia is also explored. Relationship between different calcium, phosphorus level and inoculation amount, and rhizobium growth with rhizobia quorum-sensing level is discussed. The results are as follows:(1) Red fluorescent protein grains, pDsRed2-1, are able to express in large quantities in e. coli DH5a and emit stable red fluorescent. pDsRed2-1 grains extracted from e.coli DH5a are successfully imported into rhizobia by electrotransformation and the grains reproduce continuously, which equip the study of rhizobia growth and nodular performance with handy, accurate but efficient testing method.(2) In acidic environment, environmental pH is the main factor affecting alfalfa production and quality. Calcium and phosphorus application, and inoculation of aciduric alfalfa with different concentrations can reduce negative effects of environmental pH on alfalfa growth to some extent. Calcium is the main factors that affected root micorecological environment. Phosphorus is on the basis of calcium playing a better promoting function, high concentrations calcium within the inhibition. In addition, when calcium concentration is 5mmol/L, and phosphorus concentration is 4μmol/L, the best effect can be achieved. Calcium and phosphorus application significantly increased the level of rhizobium quorum-sensing, rhizobia's number, root hair deformation, rate of inoculation and nitrogenase activity, by 116.7%,50.1%,320%,163.2% and 78.6%, respectively, over the treatment of no calcium and phosphorus application. The treatment of incubation rhizobium also significantly increased the level of rhizobium quorum-sensing, rhizobia quantity, root hair deformation, rate of inoculation and nitrogenase activity, by 1452.7%,170%,336.6%,499% and 277.4%, respectively, over the treatment of no inoculation rhizobia. In a limited space, calcium, phosphorus, acid rhizobium can improve root micorecological environment, the level of rhizobium quorum-sensing, AHL secretion increased, rhizobium breeding, and the increase of rhizobium, advancing alfalfa nodule, increasing nodule fresh weight, which indicates in under the acid condition calcium and phosphorus application, inoculation amount of aciduric alfalfa can enhance deformation rate of root hair of alfalfa and improve nodular rate and nitrogenase activity.(3) Under the acidic condition, there is significance influence of application levels of calcium phosphorus and inoculation of aciduric alfalfa on such agronimic traits as weight of nodule of alfalfa plants; Calcium, phosphorus improve microecological environment, survival rhizobia postively, and nitrogenase activity of alfalfa nodule, enhancing the symbiotic nitrogen-fixing performance and nodular system to promote the growth alfalfa and improve alfalfa agronomic characters. Calcium and phosphorus application significantly increased the nodule weight, plant height, plant weight, fresh root weight, and plant's total nitrogen by 667.7%,80.2%,82.9%,196.2% and 26.2%, respectively, over the treatment of no calcium and phosphorus application. Application levels of calcium and phosphorus can remarkably affect raw weight of alfalfa nodule, plant height and concentration of total nitrogen (p<0.01); Their effect on raw weight of alfalfa plants with raw weight of roots is noteworthy (p<0.05). Furthermore the best effect is obtained with calcium concentration of 5mmol/L, and nitrogen concentration of 4μmol/L. Incubation rhizobium significantly increased the nodule weight, plant height, plant weight, fresh root weight, and plant's total nitrogen by 1800%,451%,315.4%,361.5%,864.2% and 180.9% respectively, over the treatment of no incubation rhizobium. Impact of inoculation of aciduric alfalfa with different concentrations on agronimic traits of alfalfa can reach a fairly high level (p<0.01). Incubation acid rhizobia directly improve alfalfa rhizobia quorum-sensing level and nitrogenase activity of alfalfa nodule to improve alfalfa agronomic characters.(4) By the correlation analysis with the help of SPSS, the nodule weight is correlated significantly with plant height, raw weight of roots, raw weight of the overground part as well as total nitrogen of plant, which implies application of calcium and phosphorus with appropriate concentrations, inoculation of aciduric rhizobia with certain amount, and nodular performance with fair symbiotic system can improve production and quality of alfalfa in acid soils. (5) In the acidic environment, application levels of calcium and phosphorus prominently affect secretion of oxalic acid and malic axie of alfalfa root (p<0.01). Calcium and phosphorus application significantly decreased oxalate and malate acid secretion by 26.1% and 72.1%, respectively, over the treatment of no calcium and phosphorus application. Calcium and phosphorus cause root oxalate and malic acid secretion capacity reduction, Calcium is the main factors that affected oxalate and malic acid secretion. Phosphorus is on the basis of calcium playing a better promoting function on oxalate acid secretion, play a better inhibition function on malic acid secretion; Calcium can resist acid stress and improve the roots environment leading to root which need not secrete more oxalate and malic acid to withstand acid environment. Incubation rhizobium significantly decreased oxalate and malate acid secretion by 34.7% and 39.2%, respectively, over the treatment of no incubation rhizobium. Inoculation amount of rhizobia reaching 105 CFU/mL can inhibit secretion of oxalic acid and malic acid of alfalfa root. However, while the amount of inoculation is excessive 108 CFU/mL, it has no impact on secretion of oxalic acid and malic acid.(6) In the acidic environment, there is noteworthy influence of application of calcium and phosphorus, and inoculation of aciduric rhizobia with different concentrations on amount of rhizobia (p<0.01), which indicates that application of calcium, phosphorus and rhizobia is beneficial to survival of rhizobia, nodule and nitrogen fixation of alfalfa rhizobia. Under the acidic condition application of calcium and phosphorus, and inoculation of aciduric rhizobia with different concentrations can remarkably influence quorum-sensing of rhizobia (p<0.01), indicating that application levels of calcium and phosphorus as well as inoculation of rhizobia with certain concentration can improve quorum-sensing level of rhizobia. In addition, as the concentration of calcium, phosphorus and rhizobia inoculation is 5mmol/L,4μmol/L and 109 CFU/mL respectively, quorum-sensing level is the strongest. By the correlation analysis with the help of SPSS, quorum-sensing level of rhizobia is positively correlated with the amount of rhizobia. The greater the amount of rhizobia, the stronger quorum-sensing level of rhizobia gets and vice versa. Besides, strong level of quorum-sensing of rhizobia can also indicate its large quantity and it is true on the contrary.
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