Isolation,identification and growth-promoting characteristics of endophytic nitrogen-fixing bacteria in upland rice roots
[Objective]Upland rice was an important germplasm resource.The present study aimed to obtain strains with good growth-promo-ting effects on upland rice through isolation,screening and identification of endophytic nitrogen-fixing bacteria in the roots of mature upland rice.[Method]The combined nitrogen fixation solid culture medium was used for cultivation.Isolation of endophytic nitrogen-fixing bacteria from the roots of mature upland rice,molecular identification and phylogenetic analysis were conducted by amplifying the nitrogenase nifH gene and 16S rRNA gene,measuring biochemical indicators,and identifying their morphology.The capacities of phosphorus dissolution and i-ron carriers production were verified by using the transparent circle method.Using nitrogen fixing bacteria Azotobacter chroococcum as a control(CK),the activities of nitrogenase and 1-aminocyclopropane-1-carboxylic acid(ACC)deaminase secreted by the strains were measured u-sing an ELISA assay kit.Determination of auxin(IAA)content using Salkowski colorimetric method and its growth characteristics were stud-ied.The upland rice seeds were soaked in separated nitrogen-fixing strains at a concentration of 1％(about 8 x 107CFU/mL)to promote ger-mination and study their growth-promoting effects.[Result]The isolated strain URR45 was a nitrogen-fixing bacterium,belonging to a ther-mophilic type of microorganism.The optimal growth temperature was 32-50 ℃,the optimal pH was 6.0-6.5,and it was not salt tolerant.It secreted a nitrogenase content of 220.15 U/L,an ACC content of 898.69 ng/mL,and an auxin content of 14.91 mg/L.The nitrogenase and ACC content were significantly higher than those of the control.The strain could utilize carbon sources such as glucose and maltose and belonged to the fermentation-type bacteria.It had the characteristics of hydrolyzing gelatin,dissolving phosphorus,and producing iron carri-ers.After 16S rRNA gene sequencing and NCBI BLAST comparison,the strain was identified as Herbaspirillum seropedicae belonging to the Pseudomonadota phylum,Betaproteobacteria class,Burkholderiales order,Oxalobacteraceae family,Herbaspirillum genus,and Gram's stain was negative.The results of the growth-promotion experiments showed that the average length of the main root,the average length of the lateral root and the average number of hair roots were significantly higher than the control group.[Conclusion]The strain has a certain promoting effect on upland rice seedlings and can be developed and utilized as a functional endophytic nitrogen-fixing bacterium in agriculture.
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