Interaction Among Indigenous Plasmids Of Sinorhizobium Fredii Strains And Its Effect On Symbiotic Nitrogen Fixation

The indigenous plasmids of Sinorhizobium fredii HN01, YC4 and WWG18 were labelled with Tn5-Mob-sacB .Labelled plasmids were cured on TY medium containing 10% sucrose except the second larger plasmid of YC4 was identified the function of plasmids by pot plant experiment..The Labelled plasmids were transferred into Agrobacerium tumefaciens GMI9023 and S.fredii AB359 by tri-parental mating with the helper plasmid pRK2073 to identify the nodulation ability on soybean.The results indicated that the broad host characteristics of HN01 was determined by the symbiotic plasmid, pSymHNOl, since recepient strain AB359 recovered the ability to nodulate on Heinong33 by introduction of pSym HN01.The pSymHNOlb of HN01 was transferred into WWG18SR and C361SR. . The results of plasmid detection, curing and pot plant experiment revealed that the incompatibility was existed between pSymHNOlb and symbiotic plasmid of WWG18SR or the third plasmid of C361SR. But pSymHNOlb was compatible with the non-symbiotic plasmid of WWG18SR and symbiotic plasmid of C361SR. Using plasmid incompatibility, The symbiotic plasmid of WWG18SR and one nonsymbiotic plasmid of C361SR were eliminated successfully.A 270MD second larger plasmid named pSymYC4b of Sinorhizobium fredii YC4b, a spontaneous Nod~-_ mutant on soybean cultivar Heinong33, was mobilized into another S. fredii HN01SR which contained three indigenous large plasmids (pHN01a,pSymHN01b, and pHN01c).A new pattern of plasmid incompatibility was observed that two stable indigenous plasmids (pSymHNOlb and pHNOlc) of HN01SR were cured simultaneously by the introduction of pSymYC4b. Furthermore, an unstable plasmid named pHY4 whose molecule weight was larger than both pSymYC4b and pSymHN01b was detected together with pSymYC4b in one transconjuant.Two spontaneous nodulation mutants named YC4B and YSC3 were isolated from YC4.Mutant YSC3 formed ineffective nodules on all tested soybean cultivars and G.soja in the conditions of sand pot experiments and fewer bacteroid inside of nodules in comparison with the nodules formed by wild strain YC4. The results showed that the size of the second larger plasmid of YSC3 was larger than the pSymYC4b which indicated that pSymYC4b was amplified in YSC3. Mutant YC4B lost completely nodulation ability on all tested soybean cultivars and G.soja although its plasmid map was the same with YC4.YC4 producted the unique lipochitooligosaccharde Nod factors (LCOs) containing more hydrophobic substitution in comparison with LCOs from other four strains detected by Thin-layer chromatography (TLC). Mutant YSC3 producted more and different LCOs than that of its parental strain YC4. It was proved that incubation temperature has an important influence on the LCOs production of YSC3, LCOs production was greatly increased at 28℃ in comparison with at 23℃, but the LCOs production of YC4 and HN01 have no great change in two temperatures.WWG18SRN1 containing the deleted pSymHNOlb was obtained when pSymHN0lb was mobilized from HN01 into WWG18SR. It showed narrow host range on soybeancultivars comparing with WWG18SR, HN01 and another transconjugant WWG18SRN2 containing the complete pSymHN0lb. WWG18SRN1 formed only ineffective nodules on all G.soja and soybean cultivars tested.Reference strains were marked by luxAB genes and used for competitive nodulation tests in pot plant experiments. The results indicated that the competitive ability of strains tested was mainly determined by chormosomc genes. Both of symbiotic and non-symbiotic plasmids and soybean cultivars shown no significant influence on strain competitiveness.The effectiveness of mutants HN01 and WWG18 non-symbiotic plasmid cured showed no difference with their wild strains.The symbiotic plasmid of Rhizobium.leguminosarum bv.viciae 1229 was transferred into HN01SR and its pSym cured derivatives HND100. The transconjugant HND100(pSym1229) expressed the same LCOs as that of R.leguminosarum 1229 in free-living condition analysed by TLC method. The results of pot plant tests indicated that HND100(pSym1229...