| Acidic soils distribute widely throughout the world, and become much worse and worse, in which active aluminum ions free to come out from silicate minerals and cause aluminum toxicity in plants and microbiology. For leguminous plants, acid aluminum toxicity not only affect the host’s root growth and survival of legume rhizobia, but also affect the rhizobium and plant symbiotic nodulation process. And at the same time, because the effect of acid aluminum toxicity is so fast that plant apical cell is damaged and microorganisms individuals die within a few hours, thus it is crucial to investigate the repair/restoration effect on legume plant-rhizobium after sufferring from acid aluminum toxicity. In previous studies, we find calcium or phosphorus both can alleviate acid aluminum toxicity efficiently, but few of researches use them together. In present study, we used alfalfa and acid tolerate alfalfa rhizobium stain, Sinorhizobium meliloti91522, as materials in water and soil culture experiments to study the remediation effects of calcium and phosphorus on alfalfa-rhizobia after acid aluminum stress. The results of our research show as follow:(1) S.meliloti91522growth curve has been extremely limited, logarithmic phase and stable phase have been postponed under acid aluminum stress. Calcium and phosphorus application can reduce negative effects of acid aluminum stress on S.meliloti91522to some extent, calcium is the main factors that speed up the growth rate and take logarithmic period and stable period in advance, Phosphorus is on the basis of calcium playing a better promoting function, under different concentrations calcium including4μmol·L-1P, logarithmic period of S.meliloti91522has arrived4-6hours earlier compared with non Ca^P treatment. Because of the serious acid aluminum stress, it is difficult for survival of rhizobium. In the YMA medium whose pH is5.0and Al3+concentration is30μmol·L-1, S.meliloti91522mortality rate reached75.9%after12hours. A small amount of supplemental P (4μmol·L-1) increase the survival rate of S.meliloti91522to27.9%, and under5/10mmol·L-1Ca2+treatment, the survival rate can be increased to35.3%and37.1%respectively. Interaction of10mmol·L-1Ca2+and4umol·L-1P increase the survival rate to38.6%. Rhizobium quorum-sensing was limited by acid aluminum stress and considerably below (<70Miller Units), in a limited space, calcium and phosphorus can improve the level of rhizobium quorum-sensing, and cause AHL secretion increased, which was able to rise up to120Miller Units in the10mmol·L-1Ca2+and4μmol·L-1P treatment. The rising of QS level could be basis of restoration for information exchange between rhizobium and host plants.(2) We research the restoration effect of calcium and phosphorus on alfalfa primary root developing after12hours of acid aluminum stress and find out that, alfalfa primary root elongation is only3.3mm one day in this environmental stress, at the same time, calcium and phosphorus application had a good effect on restoration,5or10mmol·L-1Ca2+both can increase alfalfa primary root elongation significantly, the elongation could be up to19.7mm and14.3mm respectively, interaction of5mmol·L-1Ca2+and4μmol·L-1P achieve the best effect. So we can see proper concentration of calcium can effectively alleviate the inhibition of acid aluminum toxicity on primary root elongation, at the same time, a little phosphorus application can play a better promoting function.(3) Calcium and phosphorus application had a good effect on restoration for symbiotic process of alfalfa-rhizobium after1week of acid aluminum stress.5、10mmol·L-1Ca2+could repair and improve the QS level and nitrogenase activity significantly after acid aluminum stress, base on this,4μmol·L-1P application can play a better promoting function. Comparing with the control treatment, which is no Ca2+and P treatment, under the conditions of10mmol·L-1Ca2+and4umol·L-1P, the nodulation time was14days earlier, nodular number had increased by247.09%, the nodular ratio achieved to90%, hair deformation rate of M. sativa increased from3.33%to28.89%, the restoration effect of calcium and phosphorus on nodular kinetics curves was significant and begin with scaffolding. When Ca2+and P were added, the nodular number, tumor fresh weight, nodule appearing time and agronomic traits were recovered well. By combining5mmol·L-1Ca2+and4umol·L-1P, the nitrogenase activity improved significantly, and Ca2+could enhance the QS of5. meliloti significantly as well, hence promoting alfalfa to perceive the nodular signal. Preliminary tests proved that Ca2+and P could recover the symbiosis nodular process and nitrogenase activity after alfalfa suffered from acidity and Al toxicity.(4) In present study, the split pot device with two compartments was used to inoculate rhizobium secondhand and research the migration and colonization of strain S.meliloti91522under acid aluminum stress with calcium and phosphorus application, at the same time, analyzing the dynamic changes of QS in the soil. The S.meliloti91522migration rate is faster under5、10mmol·L-1Ca2+treatment than that in non-calcium treatment, base on this,30μmol·L-1P application can get the influence more significantly. In this experiment, we add aluminum into acid soil to create stress condition, by combining proper concentration of calcium and phosphorus (5mmol·L-1Ca2+and30μmol·L-1P) could improve survival rate and migration ability, and then, causing nodular efficiency of alfalfa enhanced in the acid aluminum soil. From the perspective of quorum sensing that is a chemical behavior in rhizobium, we find that calcium and phosphorus application could improve the quorum sensing of rhizobium significantly. Under5mmol·L-1Ca2+and30umol·L-1P treatment, the quorum sensing level stayed higher compared with other treatments in all whole cultivation process, providing a good basis for nodular.Otherwise, in a short period of time, calcium and phosphorus application caused some change in soil chemical property, such as exchangeable calcium content, effective phosphorus content, base ion saturation, cation exchange capacity and soil acid-buffer ability and so on. Through correlation analysis this study found that there was a significantly positive correlation between the exchangeable calcium content and inside/outside rhizobia quantity, the coefficient of association is0.316(p=0.009) and0.363(p=0.004) respectively. In the whole cultivation process there was a significantly positive correlation between the rhizobia quantity and quorum sensing, the coefficient of association is0.335(p=0.007). The coefficient of association between inside and outside rhizobia quantity is0.765(p=0.001), between inside and outside quorum sensing is0.919(p=0.001).(5) In this experiment, we used the acidic yellow soils which contain extra aluminum ion, the alfalfa and rhizobium have been growing in this soil for lweek, and then applied different concentration calcium and phosphorus, we found out that5mmol·L-1Ca2+and30μmol·L-1P combined can restore the negative effect of acid aluminum stress on alfalfa-rhizobium symbiosis. During this, the number of nodular, biomass and agronomic trait of alfalfa was improved significantly. Compared with control treatment,5mmol·L-1calcium application could improve the number and quorum sensing of rhizobium in the soil, and rise up the microbial biomass carbon and nitrogen, so we can say the microbial activity has been improved by calcium application. At the same time, P application can play a better promoting function. Through correlation analysis this study found that there was a significantly positive correlation between the number and quorum sensing of rhizobium in the soil with biomass of alfalfa, the coefficient of association is0.916(p=0.000) and0.847(p=0.001) respectively. It is an important reason for restoration effect functions of calcium and phosphorus on alfalfa-rhizobium symbiosis was that, the ability of acid aluminum tolerance of rhizobium was raised up. It’s worth noting that10mmol·L-1Ca2+has inhibited effect on every factors of alfalfa-rhizobium symbiosis except nodular number. It is possible that high calcium improve nodular but couldn’t improve the activity of every nodular, that not only dissipate much root space, but also dissipate fertilizer resources. Some mechanisms of restoration on alfalfa-rhizobium symbiosis under acid aluminum stress need much further quantitative research. |
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