Properties Of Physics, Genetics And Symbiotics Of Acid-tolerant Strains Of Rhizobium Meliloti Under Acid Stress

Alfalfa (Medicago sativa L) ,a perennial forage grass of legumina, which grows rapidly, leading to high yield rich in protein content accounting for the favorite food of livestock and is named "king of the foragegrass" eventually. Alfalfa grows well in neuter or slight alkaline soil in northern China with the optimal pH range 6 to 8, and extremely sensitive to acid soil, on the other hand. In recent years, in answer of call of developing western China, removing furrow to forest of grass progressing, industry structure regulating, and the growth area of alfalfa extending as well, planting alfalfa in south of China, where acid soils mainly attribute to, has become more and more important. Isolating acid-tolerant rhizobium that can create nodules in Alfalfa root attracts attention from researchers who are aiming at improving the Alfalfa condition in acid soil.We studied the growth of three rhizobia that can growth normal on YMA culture medium in pH=4.8, and the effect of nodulation with alfalfa; form a numerical value class tree fig according to the 16SrDNA sequence, we can confirm the status of the strains in the develop system; Using the cell scanning electron microscope photographes to analyze and research the physiologic characteristics when they tolerate acid. Then plot experiment was taken to verificate the symbiotic characteristics with alfalfa. The results are listed as follow:1. Inoculated the three rhizobia back in Fahraeus N-free solutions , every rhizobia inoculated six pH of tube (7.0, 6.0, 5.5, 5.2, 5.0, 4.8),three seedlings in every tube, three repeats. Cultivate 30 days, statistic the number of nodulation, plant height and fresh weight upground. The results indicated, whether in the neuter or acidity condition, inoculating rhizobium affected plant's growth action significantly. Compared with treats which inoculated different strains, it is distinctness when pH value was around 5.5-7.0, the number of nodules, plant height and fresh weight upground were higher when inoculated strain 91532 than that when inoculated strain 91512 and strain91522.2. After 16rDNA sequencing of amplified fragment on every strain, strain 91532 have already set out, and formed a numerical value class tree fig according to the sequence. Strain 91532 had a 99.5% comparability with Sinorhizobium meliloti EF201802. So we can predicate that strain91532 was a strain of Sinorhizobium meliloti. 3. Elizabeth J and Watkina W. 's results of researching the acid-tolerant R. meliloti: observing from cell scanning electron microscope photographes, the cells of acid-tolerant strains were turn to shorter and smaller in the acid liquid, but acid sensitivity strains had little change. Analyzed the strain 91532 which was acid-tolerant comparably higher and strain 96489 which was topic alkaline-tolerant strain according to the cell scanning electron microscope photographes. The results showed that: in the neuter condition the cells of 91532 average length was 2.110um, width was0.657um; in the acid condition, the average length of cells was 1.1887um, width was 0.6267um. However, the average length of cells in strain 96489 was 1.539um, average width was 0.613um; in the pH5.5 value condition, the average length of cells wasl.539um, width was 0.616um, which were very similar with those in neuter condition. In acid condition, the length of strain91532 was about half of that in neuter condition, and the width didn't change much.4. In order to study the mechanism of acid tolerance in strains, we did the proton flux experiment. The results indicated that: whether through induced with acid or not, the pH of acid sensitive strain 96489 and the three acid-tolerant strain was going up in 16 Min, but pH of strain96489 changed more after inducing than other three acid-tolerant strains. The survival rate of three acid-tolerant strains was up of 75% whether inducing or not; but the survival rate of inducing acid sensitivity strain 96489 rose to 63%, significant 37% higher than that which wasn't induced. Acid inducing made 96489 have an ATR action, it made the cell membrane ban H~+ in effeciently, which increased survival rate of strains in the high acid condition. The cell membrane of the 3 acid-tolerant S. meliloti had an ability of banning H~+ into cells, which is a stable genetic trait formed in the acid soil for the long time, that's why it isn't effect to acid induce for them.5. Plot experiment for planting alfalfa on Jinyun Mountain acid soils by inoculating the three acid-tolerant, one acid sensitivity S. meliloti and control three repeats, 15 plots in all. After harvest plants and analyzed, the results showed: inoculating rhizobium(acid-tolerant and acid sensitivity) affected the plant significantly. Plant height was very significantly correlated with nodule number, alfalfa fresh weight up-ground, and more significantly correlated with plant's total nitrogen for inoculating acid-tolerant strains than acid-sensitivity. Measure the number of microbial in every plot(Bacteria, fungi, actinomyces and rhizobium), the analysis showed that, planting alfalfa in the acid soil significantly enhanced the colonization of microbial in the soil; it was different notable of the colonization of microbial in the soil among the treats of inoculating acid-tolerant, acid sensitivity and control.