| Ormosia henryi Prain belongs to a plant of Papilionaceae Ormosia,which is a precious material and landscaping tree,with high economic value.It can be symbiotic with rhizobium to form a nodule for nitrogen fixation.It is still unclear which rhizobium can be symbiotic with O.henryi? what are its characteristics? whether they have phenotypic diversity and genetic diversity? what inoculation effects do they have after artificial inoculation of rhizobium? The solution of these problems is of great significance for the investigation of the symbiotic nitrogen fixation system between O.henryi and rhizobium,the enrichment of the gene resources of rhizobium in China,and the artificial inoculation of high-efficiency strains to improve the quality of seedlings.In response to these problems,the research team extensively collected the root nodules of O.henryi in its distribution area,carried out wild distribution characteristics and habitat survey,analyzed the coupling relationship between the diversity of O.henryi-rhizobium and environment,explored the composition and distribution of nodule bacteria using traditional culture methods and high throughput sequencing technology,isolated and purified rhizobium of O.henryi,re-inoculated and tested the function of rhizobium,verified the phenotypic traits and genetic diversity of rhizobium by physiological and biochemical determination and molecular biological methods,make clear the phylogenetic status of the rhizobium,and appraised drought resistance after artificial inoculation of rhizobium for screening excellent growth-promoting and drought-resistant strains,the main conclusions are as follows:(1)The root nodules of O.henryi are mainly concentrated in the soil layer of 0-30 cm.The number and weight of the nodules in the 10-20 cm soil layer are the largest.The nodules are mainly born on the lateral roots,and the shape is spherical,elliptical,long strip,rod-shaped bifurcation,ginger-like and coral-like,the little root nodules are only 1-2mm,and the large ones can reach more than 20 mm.The color of the nodules are light yellow,yellowish brown,light brown,dark brown,etc.,belonging to the infinite nodule type.The growth period of Rhizobium colony is 2-7 days,the diameter is 1.5-6mm,creamy or translucent,the edge is neat,the surface is convex,the mucus is produced,the luster is not absorbed,the Congo red is not absorbed,and the Gram stain is negative.The shapes of Rhizobium are rod-shaped,wooden stick shaped,partially X and T-shaped,with an average size of 3.12±0.70?m×0.69±0.08?m.(2)Soil pH has the greatest influence on the size of O.henryi nodules.In the range of pH 3.76-6.89,the short diameter of diameter increases with the increase of pH.The nodule weight is most related to the content of hydrolyzed nitrogen in soil,the higher the content of hydrolyzed nitrogen in soil,the heavier the single nodule.In the distribution of bacteria in O.henryi nodules nodules,the soil factor has a greater influence than the geographical factor.the diversity of bacteria in O.henryi nodules increases with the increase of available potassium content in soil.(3)The bacteria in root nodule of O.henryi is of high diversity,and can be annotated to 6 phylum,11 classes,20 orders,34 families,and 61 genera,including nodulation symbiotic bacteria and non-nodulation commensal bacteria.The 6 phylums are as follows,Proteobacteria,Firmicutes,Actinobacteria,Bacteroidetes,Spirochaetes and Melainabacteria.And Proteobacteria contains most of the nodulation symbiotic bacteria,constituting the dominant phylum of the root nodule bacteria with Firmicutes.(4)The phenotypic diversity of O.henryi rhizobium is abundant,and most of the strains have a wide range of carbohydrate utilization ability,and a few strains have poor utilization of carbon sources.All test strains could utilize L-aspartic acid,L-cysteine,L-tyrosine,L-leucine,L-glutamic acid as the sole nitrogen source,they were the least resistant to cephalosporins and most resistant to lysins.The test strains generally have strong acid resistance and poor alkali resistance;they have a wide adaptability to temperature,and most of them could grow at 4 ?-40 ?,and a small number of strains could growing at 60 ° C.Most of the strains grew better in the absence of NaCl,1% NaCl,and 2% NaCl.With the increase of NaCl concentration,the growth of O.henryi rhizobium was worse.68.4% of the strains produced acid in BTB,and 31.6% of the strains produced alkali in BTB.All tested strains did not produce oxidase,98.2% of the strains produced catalase,and 96.5% of the strains could not grow in the broth.57 strains tested could be divided into 5 supervised groups at 82% similarity level.(5)The 16S rRNA amplification products of O.henryi rhizobium were digested with four restriction enzymes(Hae III,Hinf I,MspI and RsaI)to obtain 22,21,21 and 15 restriction endonucleases,respectively.Cut-spectrum types and 50 combinations of types showed high genetic diversity.The 16S r RNA PCR-RFLP and BOX-PCR fingerprints of the tested strains were divided into 6 categories at 75% and 70% similarity levels.The distribution of these genetic maps had no obvious regional characteristics.16S rRNA,PCR-RFLP,BOX-PCR fingerprint and full sequence of 16S rRNA gene three clustering dendrograms show good developmental consistency.57 strains of nodulation strains were located in Rhizobium,Azorhizobiu,Mesorhizobium,Bradyrhizobium,Sinorhizobium,Burkholderia 6 genera.(6)The photosynthesis rate and transpiration rate of the leaves of the seedlings of O.henryi were higher than that of the control.The inoculating rhizobium could significantly increase the chlorophyll fluorescence parameters of the seedlings of the flowering wood,and promote the aboveground and underground growth and total biomass accumulation of the seedlings of O.henryi.It indicates that the regeneration of O.henryi was closely related to the symbiosis of rhizobium.There were significant differences in the growth-promoting effects of different strains.According to the comprehensive evaluation of promotion,the top five strains were No.45,No.46,No.32,No.51,No.47,which could be used as an excellent growth-promoting strain.(7)Inoculation with Rhizobium could significantly improve the drought resistance of the seedlings of O.henryi and the physiological responses of the different strains inoculated to the moderate drought stress were significant.Inoculation with rhizobium could reduce the effect of drought stress on the relative permeability of the plasma membrane of leaves,increase the content of osmotic substances such as proline and soluble sugar in the plant,and increase the activity of active oxygen and free radical scavenging enzyme SOD,thereby reducing the dehydration damage caused to cells,and could effectively reduce the accumulation of membrane lipid peroxidation product MDA,improve the drought tolerance of flowering palm seedlings.The drought resistance productivity and the water use efficiency of the seedlings were significantly increased after inoculation.The light energy conversion efficiency and the potential activity of PSII in the PSII reaction center were improved,and the adaptability to drought stress was enhanced.The comprehensive evaluation results of drought resistance showed that strains No.34,No.35,No.32,No.43,No.28 and No.37 were beneficial to improve the drought resistance of the seedlings and could be used as an excellent drought-resistant strain.Comprehensive promotion effect and drought resistance index,the first five strains with the best combined effect of promoting growth and drought resistance were obtained from strains No.38,No.35,No.42,No.47 and No.32. |
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