| The symbiotic azotification between Rhizobia and leguminous forages plays an importantrole in ameliorating soil fertilization, enhancing the productivity of forage grass and crop, as wellas improving ecological environment. Previous work indicated that endogenous Rhizobia widelyexisted in the seeds and plants of leguminous forages, while no direct proof was found to provethe migration and pathway of Rhizobia between roots and seeds. In order to make clear themigrational pathway and rules of endogenous rhizobia in alfalfa plants, as well as the factors thatinfluence the migration of endogenous rhizobia in host plants, triparental mating technology wasused to transfer cfp fluorescent protein into Sinorhizobium meliloti.12531(standard alfalfa rhizobia)and Rhizobium meliloti. GN5(isolated from the endogenous rhizobia in seeds of Medicago sativaGannong NO.5) to obtain exogenous fluorescence marked rhizobia S.12531f and endogenousfluorescence marked rhizobia R.GNf. Inoculation methods, inoculation positions and the effect ofexogenous interfering substances on the migration and transferring rules of endogenous rhizobiaand exogenous invasive rhizobia in plants were tested based on the bud stage, seedling stage andreviving Gannong NO.5(1a after cultivation) that cultivated in the open fields. The results showedthat:1. Both similarity and heterogeneity were found for endogenous R.GNf and exogenousS.12531f in terms of host seedlings infection and transportational process. For the similarity, boththe two marked bacteria could infect the root of seedling and enter the stem. For the heterogeneity,endogenous bacteria had relatively smaller differnece in bacterial density in stems and roots, andcould enter the cotyledon of seedling, while exogenous bacteria could not enter the cotyledon ofseeding within24h, and the bacterial densities in roots and stems of the host plants was relativelygreater, indicating that in the seedlings, a selective barrier exists in the transportational process ofrhizobia from root to stem and cotyledon, the1stselective barried occurred between root and stem,which could possibly decrease the quantity of exogenous bacteria while play no effect onendogenous, and the2ndselective barrier occurred between stem and cotyledon, which could cutoff the migration of exogenous bacteria to cotyledon.2. After infecting into seedlings and field plants, the marked bacteria could be migrated toroot and stem, but could not be migrated upward from inoculation position, or colonized in thepathway between the inoculation position and stem base, and therefore formed a discontinuousdistribution of marked bacteria in the plant tissues. The enter speed of exogenous bacteria from the scar to root was found faster than that from the medulla to root, whereas endogenous bacteria hadreversed situation. To field plants, the scar on the stem was good for the migration of exogenousbacteria, while the pathway between medulla and root was found more appropriate for themigration of endogenous (injected treatments). The daub tests conducted on young leaves ofdifferent parts indicate that both stem-scratching inoculation and leaf-daub inoculation could makethe marked bacteria migrated downward but in different pathways; the marked bacteria thatdaubed on different parts of leaves could move downward to stem and root, but could neither enterthe leaves in other positions nor move upward. In10-12d seedlings, the rhizobia migrationalpathway from euphylla to root hadn't been formed, and the bacteria daubed onto the scratchedleaves could not infect the plants.3. In all stages of growth, the infected marked bacteria had the pathway to be migratedupward to the aboveground part. After the roots of bud seedling, young seedling and field plantsbeing treated with bacterial solution, the detected marked bacteria in different positions werefound with significant difference, but both the two marked bacteria could enter the root of alfalfaand been migrated to stem. Endogenous marked bacteria R.GNf could enter cotyledon of buds,lower leaves of seedlings and upper leaves of field plants, whereas exogenous S.12531f could onlymove upward to stems when exogenous substances were absent. When the root (for bud seedlings)or the original nodules (for field plants) been cut off, the injured roots gave more ways to rhizobiainfection, which significantly increased the quantity of marked bacteria in root tissues, andpromoted the migration of the two bacteria in bud seedlings and endogenous bacteria in plants tomake more endogenous bacteria moved to cotyledon of seedlings, but which also inhibited theupward movement of endogenous bacteria in field plants.4. Low concentraion LaCl3, IAA and alien extracellular polysaccharides could inordinatelyincrease the distribution density of marked bacteria that existed in the root of host bud seedlingsand young seedlings, while LaCl3significantly decreased the distribution density of S.12531inhost stem, and also inhibited the migration of endogenous bacteria to stem and cotyledon. IAAcould increase the quantity of exogenous bacteria that existed in stems, but decreased the bacterialdensity of endogenous bacteria that existed in stem and cotyledon. Exogenous substances withdifferent mechanisms like root damage, the change or damage of cell wall conformation, playdifferent effects on the infection and migration of marked bacteria, but could only urge the markedbacteria to enter the root, while could not play an positive role in the further transportation of the marked bacteria to stem and cotyledon. The usage of extracelluar polysaccharides of endogenousbacteria could decrease the selective pressure from root-stem barrier in bud seedlings, but couldnot going through the selective barrier between leaf and stem by using exogenous bacteria;exogenous extracellular polysaccharides could increase the distributional density of endogenousbacteria in stems of bud seedlings and young seedlings.5. On condition of normal temperature, rhizobia could infected the young seedlings fromoutside and be migrated from root to stem, few endogenous marked bacteria could move to thelower leaves. The stems of young seedlings were selective to bacteria stains, exogenous bacteriacould exist in stem, but the detected quantity takes only a very low proportion in the totalendogenous bacteria, which was significantly lower than other endogenous bacteria existed in thesame part. Low temperature at4℃could inhibit the infection and migration of rhizobia, and theinhibitory effect of which on exogenous rhizobia was found stronger than that on endogenousrhizobia. Balanced nutrition supply could promote rhizobia infecting plant roots, while themicro-environment which is nitrogen deficient was beneficial for the migration of marked rhizobiato seedlings stem.6. Both endogenous and exogenous marked rhizobia could keep the continuity of migratingto stems after the infection into roots, and arrived at lower stem2days later,5days later,endogenous bacteria could come into upper leaves, while exogenous bacteria could only arrievedat upper stem, at this time, bacterial density in plant tissues except in root were found the highest,on6-7thday, the density decreased progressively without change in distributional position.7. For plants that grown for40d after root-soaked inoculation treatment and that were in theprimary stages of vegetative growth in the fields, the marked bacteria of which could still bedetected move upward to the aboveground part, but only endogenous bacteria could enter upperleaves of plants, indicating that the reviving plants which were in the primary stages of vegetativegrowth had the upward pathway for rhizobia migration, while the distribution of marked bacteriawas discontinued, some pathway and tissue were selective to marked bacteria and could makethem colonized indefinitely. The exogenous bacteria could exist about40d in the stems and rootsof host plants with approximately the same density of endogenous bacteria, but could not enter theupper leaves like endogenous, indicating that selective barriers for rhizobia migration are exist inalfalfa plants.
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