| Legume seeds are the product of long-term natural selction and coevolution of rhizobia and seeds.They are more competitive than the native rhizobium in constructing symbiotic nitrogen-fixing system with host plants.However,the paths and influencing factors of rhizobium invasion into and migration in plants,and colonization in seeds are not clear.And few studies have been conducted on the establishment of target rhizobia and improved seeds symbiont through the invasion,migration,and colonization of target rhizobia in seeds.In this study,two M.sativa cultivars,WL343HQ(M.sativa cv.WL343HQ,W),and Gannon 5(M.sativa cv.Gannong No.5,G),and three cyan fluorescent protein(CFP)-labeled rhizobium,Rhizobium LH3436f(3436f),Ensifer meliloti 12531f(12531f),Rhizobium GN5f(gn5f)were used as materials.The effects of potassium dihydrogen phosphate(KH2PO4)and fulvic acid(FA)on the migration and colonization of rhizobia strains in M.sativa seedlings were explored.Different concentrations of KH2PO4 and FA were added to three kinds of rhizobium suspension to screen the concentrations suitable for rhizobium growth.M.sativa seedling roots were then inoculated with the corresponding rhizobial strains added with the KH2PO4 and FA of selected concentrations.And the dynamic changes in the migration and colonization of rhizobia strains in M.sativa seedlings after they intruded into M.sativa root,along with their effects on M.sativa seedlings growth were determined.The excellent combination of M.sativa cultivars,marker rhizobium,and KH2PO4 and FA concentrations were screened out as well.Finally,to study the effects of KH2PO4 and FA on the migration and colonization of three labeled rhizobium in M.sativa at the reproductive stage,flowers and pods were also inoculated with marker rhizobium added with KH2PO4 or FA at the flower and pod stage.Results showed that:(1)After inoculation of labeled rhizobium added with different concentrations of KH2PO4 and FA on M.sativa seedlings,we found that optimum concentrations of KH2PO4 and FA levels could enhance the migration and colonization of the three marker rhizobium in M.sativa root,stem and leaves.For WL343HQ,50 mg/L KH2PO4 could promote large-scale colonization of 3436f in M.sativa root system,with the colonization density reaching 27075.12 cfu/g,which was significantly higher than other treatments(P<0.05).And 3436f could quickly migrated to and colonize the upper stems and upper leaves under 50 mg/L KH2PO4,where the densities reached 19.20 cfu/g and 9.94 cfu/g,respectively;0.01%FA could promote the rapid migration of 3436f to the roots,lower leaves and upper stems.In terms of Gannon 5,50 mg/L KH2PO4 promoted the migration and colonization of gn5f in roots,lower stems,and lower leaves,with the quantity reached 31750.31 cfu/g,1381.32 cfu/g,and 49.99 cfu/g,respectively;0.02%FA could promote the migration of gn5f from root to leaves,and colonizion the leaves,with the densities reached 43640.90 cfu/g(roots)and 313.24 cfu/g(leaves)(2)The labeled rhizobium were added with KH2PO4 and FA,and inoculated on M.sativa at the reproductive stage.For WL343HQ,0.01%FA could promote the colonization of 3436f in roots,stems,leaves,and pistils;0.08%FA make gn5f migrate to and colonize stems,pistils and seeds.For Gannong 5,200 mg/L KH2PO4 could promote the migration and colonization of 3436f in roots(8888.27 cfu/g),and increase their colonization density in leaves;0.06%FA allowed 1253 1f to colonize leaves,petals,stamens,and pistils,with the densitis reached 1550.74 cfu/g,85.67 cfu/g,7.67 cfu/g,and 60 cfu/g,respectively(3)For the colonization of labeled rhizobium in M.sativa seedlings,the addition of KH2PO4 had a better promoting effect on the colonization number,whereas FA had a better promoting effect on the colonization time and stability(4)The inoculate treatments that could promote the colonization of labeled rhizobium in M.sativa seeds were:for M.sativa cv.WL343HQ,the pods smeared with 3436f added with 0.01%FA,which had a colonization density of 23.08 cfu/seed at 6 months after harvested;the flower smeared with 1253 1f added with 50 mg/L KH2PO4 and gn5f added with 0.08%FA,which gave colonization densities of 22.69 cfu/seed and 10.77 cfu/seed at 6 months after harvested.For M.sativa cv.Gannong No.5,the flowers smeared with 3436f added with 200 mg/L KH2PO4 and gn5f added with 0.02%FA,which had colonization densities of 27.69 cfu/seed and 21.04 cfu/seed at 6 months after harvested. |
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