| Alfalfa (Medicago sativa L.), a kind of leguminous forage with high quality, is acknowledged as the "king of forge". Alfafa planting and acid-tolerant rhizobia inoculation can enhance crop yield and soil fertility. However, alfalfa can hardly survive in the southern area of China due to large area of soil with low PH and lack of acid-tolerant rhizobia. Under acidic condition, such nutrients as Ca, Mg, P, Mo etc which are necessary elements in soil may be deficient because of loss and Al will release in the form of ion, which can result in the poisoning of crops by Al and acid. Technologies of plant fertilization and microbial fertilization were combined together to discuss and verify effects of alfalfa planting and inoculation of beneficial microorganisms for rhizophere on improvement of soil fertility of newly-cultivated land. Alfalfa-rhizobia which is sensitive to acid was selected to investigate the effect of Al on organic acid secretion of alfalfa rhizoshpere, quorum-sensing of rhizobia and nitrogen fixation performance of the symbiotic system under neutral (PH 7.0) and acid (PH.5.0) condition. The experimental results indicate that:1. Alfalfa planting and inoculation of beneficial microorganisms for rhizophere can raise the amount of microorganism and fertilize the newly-explored soil (P＜0.05). The weight of nodule, the stem length, the weight of rootstock, weight of the overground part and total nitrogen content was increased by 198.18%,65.7%,220.30%,397.27% and 25.26% after inoculating PGPR. The amount of bacteria, fungi, actinomycetes, rhizobium, silicate bacteria and nitrogen-fixing bacteria was increased by 54.94%, 11.90%,18.26%,27.20%,30.01% and 40.49% respectively compared with those in unused land. The content of microbial biomass carbon was raised by 60.12% and microbial biomass nitrogen processed by inoculation of beneficial microorganisms for rhizophere was increased by 63.28%.2. In order to observe the colonization and migration of rhizobia in acidic soil, Rhizobia 91522 was marked by fluorescent protein. Through the electrophoresis analysis, pDsRed2 plasmid was channeled into rhizobia and 91522' was obtained. The comparison of the growth curve of the 91522 and 91522' suggested that their growth curves overlapped in whatever conditions and there was no remarkable difference between the number of nodule and the raw weight of nodule, which implied that inoculation by labeled bacteria had no effect on nodulation ability of alfalfa and it can be applied to observe the infection of rhizobia on leguminous crops. Compared with the neutral condition, the acidic condition with PH.5 would delay the growth of 91522 and 91522'for about 12hours and the average generation time was prolonged for about 10 hours. Quorum-sensing analysis of 91522'showed its horizontal curve was similar with growth curve. That was to say the phase in which quorum-sensing activity was gradually promoted was just the logarithmic phase of bacteria growth. As quorum-sensing stopped growing, the logarithmic phase of bacteria growth turned into stable phase. It demonstrated that quorum-sensing activity controlled cell population and its multiplication of 91 522'.3. The water culture experiments revealed the influence mechanism of Al on symbiotic nitrogen-fixing system under the acidic condition. In the acidic condition, Al affected the elongation of root. When the concentration of Al was raised from 20umol/L to 30umol/L, the elongation of root decreased from 47.31% to 32.63%. Activated Al had impact on root hair deformation under acidic condition. For example, the deformation rate of root hair with the Al concentration as 30umol/L was merely 4.9%. However, under neutral condition Al had no significant effect on root hair deformation, which implied that in acidic condition Al influenced the identification of alfalfa rhizobia and its host. Correspondingly, with the increasing of Al concentration, secretion malic acid and oxalic acid in rhizophere of symbiotic nitrogen-fixing system dramatically mounted up (p<0.05).4. Under neutral condition each alfalfa can bear 28-30 nodules on average. The number of nodules remarkably reduced with the increasing of Al concentration. When the Al concentration was 30umol/L each plant can bear 3-4 nodules on average. As far as nodulation rate was concerned there was no notable influence of Al concentration on alfalfa nodulation in neutral condition. But in acidic condition with the increase of Al concentration nodulation rate significantly decreased (p<0.05) within the range from 48.35% to 10.98%. The kinetics curve of alfalfa nodulation indicated that after 8 days of inoculation nodules appeared under neutral condition but in acidic condition nodules turned up after 14 days of inoculation. Besides, nodulation was significantly delayed with the rising of Al concentration.5. Alfalfa was planted in glass bead compartment incubators to further investigate the influence of acidic Al stress on agronomical characteristics of alfalfa-rhizophia, micro-zoological sytem of rhizophere and quorum-sensing. Results indicated that after Al treatment the content of exchangeable Al arised greaty. Furthermore, with the rising of Al concentration the content increased remarkably (p<0.05). While the Al concentration was 30umol/L, the exchangeable Al came to 1.1mg/kg. At the time, the growth of alfalfa was restrained and alfalfa yield dropped. Under acidic condition, Al with high concentration (30umol/L) could block alfalfa growth. The number of nodules, the raw weight of stem, the stem length and the length of underground part decreased by 70.7%,70.6%,59.5% and 95% compared to those without Al. Microbial biomass was obviously larger in neutral condition than that in the acidic condition (p<0.05). There was no noteworthy effect of Al concentration on distribution of microbial biomass under neutral condition. But things were different in acidic condition (p<0.05). With Al concentration as 30umol/L, the amount of bacteria, fungi, actinomycetes, rhizobium dropped by 95.7%,94.7%,91.4% and 69.2% compared with those without Al. Measurement of quorum-sensing showed that under neutral condition the activity ofβ-galactosidase was obviously higher than that in acidic condition. Along with the rising of Al concentration the activity ofβ-galactosidase dropped significantly under acidic condition (p<0.05).6. Therefore the harm mechanism of the acidic environment and Al stress on plant, especially the alfalfa-rhizobia system, in the aspect of microzoological level of rhizophere was concluded from findings mentioned above. For the micorecological nutrients of root:Acidic soil conditions can promote dissolution of Al in soil lattice and its accumulation in soil solution (i.e. exchangeable Al). The concentration of exchangeable Al increased linearly with the rising of the total Al concentration. Except for the content of organic matters, other indicators of soil nutrients decreased significantly with the increase of Al concentration (p<0.05). For the rhizospere microflora:the amount of bacteria, fungi, and actinomycetes in the microzoological environment of rhizophere under acidic condition dropped by 70%-90% becaused of Al stress compared with acidic conditions without Al. The influence mechanism of acidic Al on alfalfa-rhizobia can be demonstrated in the following three aspects in particular:The first one is its impact on plants and acidic Al can influence elongation of root hair and its number; the second is on the rhizobia. Acidic Al can reduce the quorum-sensing activity dramatically and further reduce the number of rhizobia in the rhizophere; the third is on the alfalfa-rhizobia system. It can affect generation and transmission of identifying signals between rhizobia and its host, and reduce the deformation rate of root hair. Thus macroscopically such agronomic characteristics as the number of nodules, the raw weight of stem, the length of stem and the length of underground part etc of the alfalfa-rhizobia were significantly reduced under the influence of acidic Al.7. Inoculating PGPR which including rhizobium can as one of the effective method by respairing the growth of alfalfa under acidity and aluminum stress condtion.
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