| Rhizobia can develop symbiotic relationships with legume plants,often resulting in the fixation of atmospheric nitrogen,which hold both economic and ecological benefits.Inoculating rhizobia becomes a common measure in promoting plants growth and increasing productivity in farming systems.The rhizobia in seeds and plants are endophytic bacteria,and rhizobia can also be found in the seeds of alfalfa,which have advantage over resident rhizobia on nodulation.The benefit is that endophytic rhizobia can interact with the elongating hypocotyl before seeds encounter resident soil rhizobia,which allows the endophytic rhizobia in seeds to efficiently nodulate.But fewer studies have been conducted on the source and formation process of endophytic rhizobia in seeds,on the migration channel and the colonization dynamics of the rhizobia by the interference factors.In this study,we inoculated Medicago sativa L.Gannong No.5 alfalfa with two fluorescent tagged rhizobia,Ensifer meliloti LZgn5f?isolated from an Medicago sativa L.Gannong No.5 seed,an endogenous rhizobium gn5f?and Ensifer meliloti 12531f?a standard alfalfa rhizobia species,obtained from the China Microbiological Culture Collection Center,and was originally isolated from Melilotus Suaveolens L.,an exogenous rhizobium 12531f?,to add to alfalfa seedlings with boron,gibberellin and matrine exogenous interference materials,and select the appropriate interference material level to study the effect of inoculation methods and exogenous interference materials on the migration pathway,colonization position and the dynamic processes of both endogenous and exogenous rhizobia in alfalfa plants,the results showed that:?1?Inoculated alfalfa seedlings using fluorescent tagged rhizobia with the addition of 0,0.01,0.5,1,10 and 100 mg L-11 boron and found optimum boron level could enhance the colonization ability of the two rhizobia in alfalfa seedlings.Under 1mg L-11 and 100 mg L-11 boron levels,12531f and gn5f grew better than at other levels,respectively.The two rhizobia mainly colonized roots,with a density of 2184.99 cfu g-11 and 58307.11 cfu g-11 by adding 100 mg L-11 and 0.5mg L-11 boron into 12531f and gn5f respectively;They could migrate to the aerial tissues,by adding 1 mg L-11 boron into 12531f could promote it migrate to and well colonize lower stem and lower leaf with the densities of 62.74 cfu g-11 and 23.38 cfu g-1?P<0.05?,respectively;by adding100 mg L-11 boron into gn5f could promote it migrate to and well colonize lower stem with the density of 57.55 cfu g-1?P<0.05?.No fluorescent tagged rhizobia could be detected in the control treatment.The individual plant nodule number,individual plant nodule weight,aboveground fresh weight,aboveground dry weight,root fresh weight and root dry weight were increased highest after adding 1 mg L-11 and 100 mg L-1boron to 12531f and gn5f,respectively.?2?Inoculated alfalfa seedlings using fluorescent tagged rhizobia with the addition of 0,0.5,1,10 and 100 mg L-11 gibberellin and found optimum gibberellin level could enhance the colonization ability of the two rhizobia in alfalfa seedlings.With the addition of 10 mg L-11 gibberellin into 12531f could promote it migrate to and well colonize lower stem,upper stem and upper leaf with the densities of 54.72 cfu g-1,16.72 cfu g-11 and 95.91 cfu g-1?P<0.05?,respectively;adding 1 mg L-11 gibberellin into gn5f could promote it migrate to and well colonize lower stem and lower leaf with the densities of 52.08 cfu g-11 and 321.40 cfu g-1?P<0.05?,respectively.No fluorescent tagged rhizobia could be detected in the control?sterile distilled water?treatment.The individual plant nodule number,individual plant nodule weight,aboveground fresh weight,aboveground dry weight,root fresh weight and root dry weight were increased highest after adding 10 mg L-11 and 1 mg L-11 gibberellin to12531f and gn5f,respectively.?3?Inoculated alfalfa seedlings using fluorescent tagged rhizobia with the addition of 0,100,200,300 and 400 mg L-11 matrine and found optimum matrine level could enhance the colonization ability of the two rhizobia in alfalfa seedlings.Optimum matrine level enhanced the colonization of both fluorescent tagged rhizobia in alfalfa roots and migrate to the aerial tissues and most colonize lower stem and upper stem through the application of adding 300 mg L-11 matrine into 12531f,with the densities of 1.18×104 cfu g-11 and 3.03×102 cfu g-1?P<0.05?,respectively;with the addition of 100 mg L-11 matrine into gn5f could promote it migrate to and well colonize lower stem with the densities of 9.00×103 cfu g-1?P<0.05?.No fluorescent tagged rhizobia were detected in the control treatment.Alfalfa seedling growth parameters like individual plant nodule number,individual plant nodule weight,aboveground fresh weight,aboveground dry weight,root fresh weight and root dry weight were increased highest when 300 mg L-11 matrine adding 12531f and 100 mg L-11 matrine added to gn5f treatments.?4?Different methods?root drench,taproot slightly damaging and drench,flower spray and root drench with the addition of matrine?were used to inoculate reproductive stage alfalfa.The migration channel,distance and colonization position were almost same for the two fluorescent tagged rhizobia,that they mainly colonized fibrous roots,and the colonization quantity in lateral root cortices and lateral root steles was less than 5%that in fibrous roots.No FTR were detected in the control samples.In aerial tissues,when alfalfa plants were inoculated during the bud stage,both rhizobia strains mainly colonized the leaf and stem;during the flower stage,the rhizobia mainly colonized the flower;during the pod stage,the rhizobia mainly colonized pod skin.When combined exogenous materials with inoculation methods,the results were effectively——During bud stage,root drench 12531f with the addition of matrine enhanced it migrated to and colonized stem,while root drench gn5f enhanced it migrated to and colonized stem and leaf.During flower stage,a spray inoculation treatment resulted in more 12531f colonizing reproductive tissues,root drench gn5f with the addition of matrine enhanced it migrated to and flower tissues and pod.During pod stage,more rhizobial strains 12531f colonized seeds using the root drench treatment,and more gn5f colonized seeds using the root drench with matrine treatment.?5?When root and flower inoculation methods were used at flower and pod stages alfalfa with the addition of different exogenous materials?boron,gibberellin and matrine?,we found when flower spray 12531f with the addition of 1 mg L-11 boron resulted in more colonizing seeds,with the density of 5.33 cfu grain-1?P<0.05?,while for gn5f,flower spray without adding material resulted in more colonizing pod skin and seed with the densities of 2.67 cfu grain-11 and 1.33 cfu grain-1,respectively.During pod stage,pod skin spray 12531f with the addition of 1 mg L-11 boron resulted in more migrated to and colonized pod skin and seed with the densities of 25.33 cfu grain-1and 7.01 cfu grain-1,respectively;more gn5f colonized pod skin and seed with the addition of 100 mg L-11 boron,the densities to be 36.67 cfu grain-11 and 22.00 cfu grain-1,respectively.To enhance the colonization of the reproductive stage alfalfa tissues,especially in seeds,the pod stage is the ideal stage for the two fluorescent tagged rhizobia inoculations.Pod skin spray gn5f and 12531f with the addition of 100mg L-11 and 1 mg L-11 boron inoculation treatment worked best,respectively.?6?Medicago sativa L.Gannnong No.5 seeds were used as the materials which were obtained with three inoculation methods?taproot slightly damaging and drench,root drench and root drench with matrine to inoculate pod stage alfalfa with two rhizobia,Ensifer meliloti LZgn5f?gn5f?and Ensifer meliloti 12531f?12531f?and root drench with sterile water?.Envelope,envelope+cloth bag and aluminum foil storage packing materials were used under 25?,25?dried,-4?and 4?,respectively,to store the seeds for six months.Then the colonization quantity of endogenous rhizobia in seeds was detected.We found that the endogenous rhizobia in seeds obtained by inoculating gn5f was significantly higher?P<0.05?when stored under-4?;the endogenous rhizobia in seeds obtained by inoculating 12531f was significantly higher?P<0.05?when stored under 25?dried condition.The endogenous rhizobia colonization number of 25?dried and-4?were higher than 25?and 4?when seeds were obtained from inoculation the same rhizobia. |
PDF Full Text Request