Interaction Among Indigenous Plasmids Of Mesorhizobium Huakuii Strains And Its Effect On Symbiotic Nitrogen Fixation

Sixty two strains of M huakuii were isolated from nodules of Astragalus sinicus collected from rice-growing field of Huazhong Agriculture University. 57 typical strains of M huakuii were identified by cultural characteristics, microscopic observation and plant nodulation tests. The nodule appearence times of strains tested were different from 8d to 30d and for most strains varied from 10d to 22d. 44 strains were chosen to detect indigenous plasmid profile and the results showed that 13 strains harbored 3 plasmids, 21 strains harbored 2 plasmids, 7 strains only harbored 1 plasmids, 3 strains harbored no plasmid. The size of indigenous plasmids tested was from 150kb to 428kb.M huakuii HN3015 containing three mega-plasmids were labeled by Tn5-mob-sacB insertion and plasmid labeled mutants were obtained. Plasmid curing experiment was carried out by using the sacB positive selection method and plasmid cured or deleted derivatives were obtained. Same cultural characteristics were identified among HN3015 and its derivatives. The mutant HN3015-1 cured with its largest plasmid pMhHN3015c formed only white small nodules and lost nitrogen fixation ability in plant nodulation tests. The mutant HN3015-2 cured with its second largest plasmid pMhHN3015b lost nodulation ability. Only the mutant HN3015-3 or HN3015-6 cured or deleted with its smallest plasmid pMhHN3015a, could form pink effective nodules. Furthermore, curing of pMhHN3015a could enhance competitive nodulation ability and symbiotic efficiency of HN3015-3. The results from acidity and alkali tolerance assays indicated that three indigenous plasmids of HN3015 played positive control effect on its acidity tolerance, but played negative control effect on alkali tolerance. Surprisingly, all indigenous plasmids showed also negative control effect on its growth rate of HN3015.When plasmid pMhHN3015c labeled by Tn5-mob-sacB was transferred from HN3015T18 into HN308SR by tri-parental mating, the first largest plasmid of HN308SR was cured. The results implied that plasmids of pMhHN3015c and pMhHN308c were incompatible and might be ascribed to the same incompatible group. However, a new co-elimination phenomenon of pMhHN3015c:: Tn5-mob-sacB and pMhHN308a was detected when the transconjugant HN308N18 (harboring pMhHN3015c::Tn5-mob-sacB, pMhHN308b and pMhHN308a) was used to do plasmid curing tests. The plasmid cured derivative of HN308 harboring only pMhHN308b was obtained. Results of pot plant nodulation tests showed that both of HN308SRN18 and HN308SRN18D could form only white small nodules. Our results indicated that the introduction of pMhHN3015c could not replace the effect of pMhHN308c and failed to restore the nitrogen fixation ability of HN308SRN18. The results also indicated that pMhHN308c was relevant to nitrogen fixation ability and pMhHN308b relevant to nodulation ability.The second largest plasmid pMhHN308b of HN308SR was eliminated when pMhHN3015a::Tn5-mob-sacB was introduced into HN308SR. Results demonstrated that both of pMhHN3015a and pMhHN308b should be belonged to the same incompatible group and pMhHN3015a, pMhHN308a and pMhHN308c ascribed to the different incompatible groups. But plasmid cured derivatives of transconjugant HN308SRN29 failed to be obtained on TY plate containing 7% sucrose. Since the sizes of pMhHN3015a and pMhHN308a were almost the same and their positions on agarose gel were difficult to distinguished, so that two plasmids might be recombined. Results of pot plant nodulation tests showed that pMhHN308a was relevant to nodulation ability and transconjugant HN308SRN29 harboring pMhHN3015a, pMhHN308a and pMhHN308c lost nodulation ability. These results also implied that pMhHN3015a and pMhHN308a might be recombined and transposon Tn5 transferred into the chromosome which resulted in the lose of nodulation ability.When the two labeled plasmid pMhHN3015a::Tn5-mob-sacB and pMhHN3015c:: Tn5-mob-sacB were respectively transferred into 7653R-1SR harboring pMh7653Ra by tri-parental mating, the result showed that the two plasmids could respectively coexist with pMh7653Ra. Certainly, they were belonged to different incompatible groups. Results from plasmid curing test of 7653R-1SRN29 showed that one plasmid cured derivative 7653R-1SRN29D-B lost its plasmid pMhHN3015a and a new plasmid p76H4 appeared. But p76H4 could not be observed in another plasmid cured derivative 7653R-1SRN29D-A. Results from plant pot nodulation tests showed that the introduction of pMhHN3015c::Tn5-mob-sacB in transconjugant 7653R-1SRN18 failed to restore nitrogen fixation ability. Similar to 7653R-1SR, it could only form a few small white. Although Transconjugant 7653R-1SRN29 harboring pMhHN3015a::Tn5-mob-sacB and pMh7653Ra could still form null nodules, but its nodule number was more than that of wild strain 7653RSR. It was demonstrated that pMhHN3015a could inhibit symbiotic efficiency of HN3015, but pMhHN3015a could enhance the nodulation ability of 7653R-1SRN29. Plasmid cured mutant 7653R-1SRN29D-A could only form null nodules. But mutant 7653R-1SRN29D-B with a novel plasmid p76H4 and cured of pMhHN3015a::Tn5-mob-sacB lost its nodulation ability.When symbiotic plasmid pMh7653Rb labeled by Tn5-mob-sacB was transferred from 7653RT14 into HN308SR by tri-parental mating method, two stable indigenous plasmids of pMhHN308c and pMhHN308b of HN308SR was co-eliminated. Results of plant pot nodulation tests showed that pMh7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR, but failed to replace nitrogen fixation effect of pMhHN308b and pMhHN308c. Plasmid cured mutant HN308SRN14D harboring only pMhHN308a could also form nodules that demonstrated pMhHN308a was relevant to nodulation ability.When symbiotic plasmid pMh7653Rb::Tn5-mob-sacB was also transferred into HN3015SR by tri-parental mating method, its second largest pMhHN3015b was eliminated. The mutant HN3015SRN14D harboring pMhHN3015a and pMhHN3015c was obtained by plasmid curing tests from transconjugant HN3015SRN14. Results of plant pot nodulation tests showed that HN3015SRN14 formed only null nodule and HN3015SRN14D lost the nodulation ability.Primers of RC1 and RC3 were used to amplify the repC-like sequences from the above six trans-conjugants and their plasmid cured mutants, repC-like sequences were obtained from strains tested except of 7653R-1SRN18D and 7653R-1SRN29D. The sizes of the PCR products were about 750 bp. The repC sequences of ten strains tested showed 99% sequence similarity, but were obviously different from that of other rhizobia strains.