Roots of F1 seedlings from a cross between Actinidia chinensis and Actinidia eriantha were collected from orchard of Anhui agricultural university in Anhui province, China. They were washed with sterile water to remove the rhizosphere soil on the surface. Then the roots were sterilized with 75% (w/v) alcohol and 8% (w/v) sodium hypochlorite for 2 mins respectively and washed using sterilized deionized water four times. Solid NFB medium supplemented with 0.01g/L KNO3 were inoculated with serial dilutions of crushed roots. The composition of the NFB medium is as follows (L-1): malate (5.0 g); K2HPO4 (0.5 g); MgSO4·7H2O (0.2 g); NaCl (0.1 g); CaCl2·2H2O (0.02 g); bromothymol blue 0.5% in 0.2 M KOH (2 ml); sterile, filtered vitamin solution (1 ml); sterile, filtered micronutrient solution (2 ml); 1.64% FeEDTA solution (4 ml); KOH (4.5 g). The pH was adjusted to 6.8 and 1.75 g/L agar was added. The vitamin solution contains, in 100 ml, biotin (10 mg) and pyridoxol-HCl (20 mg), dissolved at 100 °C in a water bath. The micronutrient solution consists of the following (L-1): CuSO4·5H2O (40 mg); ZnSO4·7H2O (0.12 g); H3BO3 (1.4 g); Na2MoO4·2H2O (1.0 g); MnSO4·H2O (1.175 g). After 24h incubation at 37 °C, one loop of pellicle-forming culture was transferred into fresh broth medium. Further purification was done on NFB agar plates. The purified strain was preserved at -80℃ as a glycerol suspensions.
To establish the phylogenetic position of strain CCTCC AB 2021101T, the 16S rRNA gene sequence was determined in this study and subjected to comparative analysis. Genomic DNA from cells was extracted using a commercial genomic DNA extraction kit (Aidlab Biotechnologie). The primer pair 27F (5’-AGAGTTTGATCCT GGCTCAG-3’) and 1492R (5’- GGTTACCTTGTTACGACTT-3’) was used for amplification of the 16S rRNA gene. The PCR product was gel purified using Gel Extraction kit D2500-01 (Omega Biotek) and then cloned into a plasmid vector using a TA cloning kit (TaKaRa). The 16S rRNA gene cloned in the plasmid vector was sequenced by Sangon (Shanghai, China) using a downstream vector primer M13R (5’- CAGGAAACAGCTATGACC-3’) and an upstream vector primer M13F 5’-GTAAAACGACGGCCAGT-3’) as the sequencing primers. Identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon-e server (http://eztaxon-e.ezbiocl oud.net/; (Kim et al., 2012)). Multiple alignments were performed using the CLUSTAL X program (Thompson, 1997). Phylogenetic trees with 1200 bootstrap replications were reconstructed using the MEGA 6.0 program with the maximum composite likelihood model (Tamura et al., 2013). Clustering was performed with the neighbourjoining method (Saitou&Nei, 1987).
Morphological examination and the optimum growth condition
Cell morphology was studied by scanning electron microscope (S-4800, Hitachi) after bacterial were immersed in 2.5% (v/v) glutaraldehyde fixative solution at 4℃ for 12 hours and by transmission electron microscopy (JEM-1400; JEOL) after staining with 0.2 % uranyl acetate as well as by light microscopy (model A3000; Zeiss). Gram-staining was performed as described by Beveridge et al. (2009). Growth was tested using nutrient broth of NMS, NFB, TY and LB at 30-45℃ (5℃ increments) and pH 5–9 (1 pH unit increments).
Measurement of cellular fatty acid and G+C content
Cellular fatty acid profiles of isolate A. actinidiae CCTCC AB 2021101T, A. humicireducens SgZ-5T, A. lipoferum ATCC 29707T and A. oryzae COC8T were determined with a gas chromatograph, using the Sherlock Microbial Identification System (MIDI), according to a standard protocol (Garcia et al., 1993). For the analysis of DNA G+C content, DNA samples were prepared and degraded enzymically into nucleosides as described by Mesbah et al. (1989).
nifH PCR amplification
To estimate nitrogen-fixing ability of the isolate, a 320-base fragment of the nifH gene (enconding dinitrogenase reductase) was amplified from extracted DNA using two pairs of primers, FGPH19 (5’-TACGGCAARGGTGGNATHG-3’) plus POLR (5’-ATSGCCATCATYTCRCCGGA-3’) and AQER (5’-GACGATGTAGATYTCC TG-3’) plus POLF (5’-TGCGAYCCSAARGCBGACTC-3’). The PCR condition was described by Poly et al. and this product was purified and sequenced (Poly et al., 2001). The resulting sequence was compared with nifH sequences retrieved from NCBI using BLAST.
To further confirm the nitrogen-fixing capability of the isolate, the acetylene-reduction assay was performed using the described procedure (Hardy et al., 1973). 50 ml vials containing 15 ml NFB medium were inoculated with strain CCTCC AB 2021101T, sealed with rubber septa and incubated at 30℃ in the dark. When the OD was 0.8, 10 % (v/v) of the air phase was replaced with acetylene (Koch&Evans, 1966) and the vials were reincubated. The amount of ethylene was measured over 48h by using a gas chromatograph system (7820A, Agilent Technology).