Use Red Fluorescence Protein Mark To Monitor Symbiosis Process Between Alfalfa And Its Rhizobium Under Acidity Stress

. Rhizobium-legume symbiotic is one of the most efficient nitrogen fixation system in nature. It takes an important role in ecological and economic value. Alfalfa and Rhizobium are very sensitive to acidic environment. A large area of southern acid soils severely hampered alfalfa planting and its industrialization in southern China. So researching rhizobia identification, colonization and migration with alfalfa plants in acidic soils are of great significance. It has been studied to screen acid tolerant alfalfa rhizobium stain and researching rhizobium-alfalfa symbiotic nitrogen system. In the study, we used red fluorescent protein marked acid tolerant alfalfa rhizobium strain91512and91532. Their competitive nodulation abilities and migration with alfalfa plants were evaluated by the method of rfp. The results of this study are showed as follows:1、Using the electricity transformation method, the labeled S.meliloti91512and91532with rfp gene separately, denoted as E91512and E91532has been successfully obtained. E91512and E91532in pure culture and symbiotic conditions could all stably express red fluorescent protein. Growth curve and dynamic curves of nodulation of labeled strain E91512and E91532have no significant difference with S.meliloti91512and91532. Inoculating rhizobium significantly promotes the growth of alfalfa compared to the control treatment.2、This paper studied the effect of acid stress on labeled strains E91512and E91532in alfalfa infection, colonization, and migration using test tube hydroponics. The results showed that the pH5.2treatment significantly reduced the rhizobium’s migration rate from the roots upward to stem and leaves compared to the pH5.8and pH7.0treatments. In pH7.0and pH5.8hydroponic fluid treatment, labeled strains E91532started migration in10to15days after innoculation, from the root upward to the stem leaves, while E91532was founded in stem and leaves15to20days after inoculation. The dynamic migration process of these two strains, E91512and E91532from root upward to leaves was in pH5.8hydroponic fluid. There is no significant difference between pH7.0and pH5.8regarding migration rate for marked strains E91532, while the migration rate of E91512under pH5.8was significantly lower than it under pH7.0. Obviously, acidity affected more to E91512than to E91532.3、Marked strain E91512and E91532colonized in root, stem and leaf of alfalfa plants under pH7.0, pH5.8and pH5.2in water medium cultivation with reduced density from root to leaves. pH5.2in water medium was significantly reduced rhizobium population density in alfalfa roots, stems than pH5.8and pH7.0treatments, which was similar to acid yellow soil, sandy loam soil and purplish soil mediums. Acidic stress not only affects the survival of rhizobium, but also reduces the colonization density and migration rates of rhizobium in alfalfa plants.4、This paper studies the competitive nodulation ability of different acid tolerant strains under different acidic soils and different inoculum in situ with the red fluorescent protein markers. The results showed that the competitive nodulation ability of the strain related to their genetic characteristics ability and acid tolerance. In all of the three pH soils, pH5.2,5.8and7.0, the competitive nodulation ability of rhizobia MA2inoculation plants are all higher than rhizobia91512. Under the volume ratio E91532+91532of100:1and1:1, the nodule rate of91532is only accounted for7.78%and52.15%in acid yellow soil. This result is significantly lower than the nodule rate E91532:MA2of acid tolerant strain MA2, which accounted for20.71%and68.89%of nodule rate. But in the sandy loam soil and purplish soil, their nodule rates are almost the same. Therefore the competitive nodulation ability of strains associates with soil types.Furthermore, the competitive nodulation ability of strains associates with the inoculum quantity. The nodule rate of different volume ratio of E91532to MA2were all in order of100:1＞1:1＞1:100. Appropriately increasing the quantity of inoculum can improve the nodule rate.5、Inoculation rhizobium can improve soil nutrients in different soil types. For example, inoculation rhizobium treatments, E91532:91532, E91532:91512and E91532:MA2significantly increases the soil alkaline hydrolysis nitrogen (AHN) content comparing with the control in the acid yellow soil, increased by21.45%,20.28%,26.67%. Nitrogen content (TN or AHN) of treatment E91532+MA2was significantly improved by16.06%from the control in acidic soil. The number of rhizobia, microbial activity and rhizobium quorum sensing level were affected by soil type, i.e. they were significantly lower in acid yellow soil (pH4.73) than in sandy loam soil (pH6.27) and purplish soil (pH7.51). Rhizobium quorum sensing level was53.14Miller Units/50g in acid yellow soil, significantly lower than it was in sandy loam soil and purplish soil. The treatments with E91532+MA2ratio of1:1and1:100had higher rhizobia number, microbial activity, quorum sensing level and nodulation rate. Correlation analysis showed that rhizobium quorum sensing level was significantly positively related to nodule number on alfalfa, nodule rate and plant biomass in sandy loam and purplish soil. Quorum sensing plays an important role in Rhizobium-Alfalfa symbiotic nitrogen-fixing system.