Isolation And Idntification Of Endophytic Bacteria In The Root Nodule Of Young Period Paulownia Fortunei

In order to explore the symbiotic nitrogen fixation system of non-leguminous plants Paulownia and microorganisms,a preliminary study was carried out on isolation and culture conditions and the phylogenetic position of endophy bacteria in root nodule of Paulowia.The paper is about the studies of the traditional physiological and biochemical characteristics,growth adaptability,back-test,and phylogenetic analysis of 16S rDNA complete sequence and nifA gene fragment sequence of endophytic bacteria were isolated and purified from the root nodules of Paulownia fortunei(Seem.)Hemsl collected from Qinzhou and Nanning of Guangxi province.The results are as follows:1.Twenty-one endophytic bacteria strains of Paulownia root nodules were isolated and purified.From the morphological characteristics of the bacteria,except for strain PG-7,the other strains were rod-shaped,Gram staining was negative,all strains had flagella and no spores,33% of the strains had capsule,the morphology of these strains has many similarities with rhizobium.From the identification of physiological and biochemical characteristics,there are obvious differences among different strains.There were negative reaction about use of 3-ketolactose,10% of the strains could hydrolyze starch,90% were able to use nitrate,33% of the strains could deoxidize the nitrate of medium into nitrite,most of the strains showed the positive reaction of the beef paste peptone,95% produced acid in BTB reaction.Most of the tested strains could utilize KNO3 and(NH4)2HPO4 nitrogen sources to varying degrees,95% of the strains could utilize nitrate and 90% could utilize ammonium salt.Most strains can grow well on medium containing carbon source.2.The optimum growth temperature of endophytic bacteria in paulownia nodules is about 30 ℃,and most strains can not tolerate high and low temperatures.The range of acid and alkali tolerance is wide,48% of the strains can grow under the condition of pH 4.0～9.0,with strong alkali resistance and relatively poor acid resistance.The salt tolerance was good,and 81% of the strains can grow at a concentration of 2.0% NaCl.Most of the tested strains had the strongest resistance to Sm and Cm,but no resistance to Tc and Gm,the resistance of different strains to antibiotics was significantly different.3.In the nodulation test,none of the test-tube seedlings inoculated with 9 strains of endophytic strains of had nodules,but the growth of Paulownia seedlings was significantly promoted in the pot experiment.The symbiosis of different endophytic bacteria and plants was significantly different,the seedling height and diameter growth of inoculated strain PG-9 were the highest.4.Through 16S rDNA complete sequence analysis,the twenty-one tested strains could be divided into six bacterial genera,namely Rhizobium,Agrobacterium,Herbaspirillum,Burkholderia,Mitsuaria and Sphingobium.Among them,67% of the strains were Rhizobium and Rhizobium was in a dominant position,indicating that the endophytic bacteria of Paulownia root nodules have rich genetic diversity.5.The nifA gene fragments of the tested strains were amplified and sequenced,the comparative analysis showed that,the nifA gene of strain PG-7 was 97% similar to that of Burkholderia,while the other strains were generally similar to the nifA gene of Rhizobium,and the similarity with reference strains was 95% or more,the highest being 99%.