Isolation And Identification Of Nitrogen-fixing Bacteria From Kiwifruit Roots

The genus Azospirillum is a group of plant growth-promoting bacteria widely distributed in roots and rhizosphere soil,it has been isolated from the roots of rice,corn,Miscanthus sinensis,and Leptochloa fusca.But there have been no reports of the bacteria being found in roots of kiwifruit.In this study,a strain of nitrogen-fixing helicobacter was isolated from the root of kiwifruit using NFB medium.The strain morphology was observed by scanning and transmission electron microscopy,and the nitrogenase activity of the strain was determined by acetylene reduction method.The growth promoting characteristics of the strain were studied by colorimetric method and phosphorus-dissolving ring method.In order to optimize the best culture conditions of the isolated strain,the growth of the strain was measured from the aspects of culture medium,temperature and p H.The following main results are now obtained:（1）16S r RNA sequence analysis showed that the isolated strain NFB2-8 belonged to the genus Azospirillum,and was closely related to Azospirillum melinis（98.32%）,Azospirillum oryzae（97.73%）,Azospirillum lipoferum（96.98%）,Azospirillum humicireducens（96.49%）and Azospirillum largimobile（96.01%）.（2）The nitrogenase coding gene nif H was amplified by PCR,and the activity of nitrogenase was determined to be 81 nmol ethylene h^-1 by acetylene reduction method.The resμLts proved that the isolated strain had the function of nitrogen fixation.（3）Gram staining test showed that NFB2-8 was a gram-negative bacterium.Scanning electron microscopy and transmission electron microscopy showed that cells of strain were rod-shaped,with motility provided with peritrichous flagella.The major saturated fatty acids of the stain are C_14:0,C_16:0 and C_18:0.C_18:1ω7c and C_16:0 3-OH were the major unsaturated and hydroxylated fatty acid.（4）The growth promotion test showed that the strain could secrete indole acetic acid （IAA）,and its IAA production capacity was determined to be 113.6 mg/L,and it also had a certain phosphorus solubility.（5）Experimental results showed that the strain grew well on NFB and TY medium.The optimum temperature range for the strain growth was 35℃～40℃,the optimum p H was 6.0～7.0,and the maximum salinity tolerance was 3%Na Cl.The above studies showed that strain NFB2-8 was a valuable probiotics with high potential in promoting the growth of kiwifruit,and could be developed as a reserve strain of kiwifruit microbial fertilizer.