Azospirillum Actinidiae sp. nov., a Nitrogen-Fixing Bacterium Isolated From The Roots of Kiwifruit Plants

A novel diazotrophic bacterium, designated CCTCC AB 2021101 T , was isolated from fresh roots of kiwifruit. Cells of strain CCTCC AB 2021101 T were Gram-negative, aerobic and rod-shaped, with motility provided by peritrichous agella. The 16S rRNA analysis showed that strain CCTCC AB 2021101 T belongs to the genus Azospirillum and is closely related to Azospirillum melinis (98.32%), Azospirillum oryzae (97.73%), Azospirillum lipoferum (96.98%), Azospirillum humicireducens (96.49%) and Azospirillum largimobile (96.01%) and lower sequence similarity (<96.0 %) to all other species of the genus Azospirillum. Strain CCTCC AB 2021101 T was able to grow well at 35–40 ℃ and pH 6.0–7.0, and tolerated up to 3.0 % (w/v) NaCl. The major saturated fatty acids are C 14:0 , C 16:0 and C 18:0 . C 18:1 ω7c and C 16:0 3-OH were the major unsaturated and hydroxylated fatty acid. The G+C content was 67.8 mol%. Strain CCTCC AB 2021101 T gave positive amplication for dinitrogen reductase (nifH gene). Highest nifH gene sequence similarities were obtained with Azospirillum brasilense AWB14 T (95.9%), Azospirillum zeae Gr24 T (95.56%), Azospirillum picis DSM 19922 T (96.79%), Azospirillum lipoferum B22 T (94.88%) and Azospirillum oryzae COC8 T (94.88%). The activity of the nitrogenase of the strain was further conrmed by acetylene-reduction assay, which was recorded as 81 nmol ethylene h -1 . Based on these data, strain CCTCC AB 2021101 T is considered to represent a novel endophytic diazotrophs species in the genus Azospirillum, for which the name Azospirillum actinidiae sp. nov. is proposed. The type strain is CCTCC AB 2021101 T .


Introduction
Plant Growth-Promoting Bacteria (PGPB) were a large number of probiotic communities widely distributed in soil (Glick, 2012). They were free-living around the roots of plants or colonized some or a portion of a plant's interior tissues to form speci c symbiotic relationships with plants (e.g., Rhizobia spp. and Frankia spp.) (Gtari et al., 2020;Liu et al., 2019). PGPB promote plant growth directly usually by facilitating resource acquisition or modulating plant hormone levels, or indirectly by decreasing the inhibitory effects of various biotic and abiotic stresses (Glick, 1995). For instance, the diazotrophic bacteria are able to x nitrogen and provide it to plants (Fox et al., 2016;Geddes et al., 2015;Ren et al., 2019). Some soil bacteria were described to promote solubilization and bioavailability of inorganic or organic phosphorus by synthesizing low molecular weight organic acids or various phosphatases (Zeng et al., 2017;Rodrıǵuez&Fraga, 1999;Rodriguez et al., 2004). And some other bacteria were shown to synthesize low-molecular mass siderophores, molecules with an exceptionally high a nity for Fe 3+ to facilitate iron uptake by plants (Jin et al., 2006;Sharma et al., 2003;Siebner-Freibach et al., 2003). Even bacterial volatile was produced to protect plants from drought stress (Song et al., 2008).
Although the nitrogen xation in planta has not yet been shown unequivocally in Azospirillum, the contribution of Azospirillum to plant growth is signi cant. It is generally accepted that members of the genus Azospirillum can enhance the growth of plants by the production of phytohormones, which are signal molecules that interfere with plant metabolism (Bashan&Holguin, 1997). For example, IAA was shown to be biosynthesized by Azospirillum brasilense to modify root morphology of wheat (Dobbelaere et al., 1999).
Kiwifruit is often referred as the king of fruits owing to its remarkably high vitamin C content and . Probiotics in the roots or rhizosphere soil of kiwifruit plants has not yet been reported. In the present study, a novel diazotrophic bacterium belonging to genus Azospirillum was isolated from the roots of kiwifruit plants, for which the name Azospirillum actinidiae was proposed. And the phylogenetic position and physiological properties were investigated. The nitrogen-xing capability was determined by acetylene-reduction assay using a gas chromatograph system.

Isolation
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 KNO 3 were inoculated with serial dilutions of crushed roots. The composition of the NFB medium is as follows (L -1 ): malate (5.0 g); K 2 HPO 4 (0.5 g); MgSO 4 ·7H 2 O (0.2 g); NaCl (0.1 g); CaCl 2 ·2H 2 O (0.02 g); bromothymol blue 0.5% in 0.2 M KOH (2 ml); sterile, ltered vitamin solution (1 ml); sterile, ltered 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 ( forming culture was transferred into fresh broth medium. Further puri cation was done on NFB agar plates. The puri ed strain was preserved at -80℃ as a glycerol suspensions.

Phylogenetic analysis
To establish the phylogenetic position of strain CCTCC AB 2021101 T , 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 ampli cation of the 16S rRNA gene. The PCR product was gel puri ed 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. Identi cation 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 xative 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 pro les of isolate A. actinidiae CCTCC AB 2021101 T , A. humicireducens SgZ-5 T , A.
lipoferum ATCC 29707 T and A. oryzae COC8 T were determined with a gas chromatograph, using the Sherlock Microbial Identi cation 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).

Acetylene-reduction assay
To further con rm the nitrogen-xing 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 2021101 T , 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).

Phylogenetic relationship
The 16S rRNA sequence analysis revealed that our isolated strain is a member of the genus Azospirillum, clustering with other Azospirillum species (Fig. 1) Cells are Gram-negative, aerobic, rod-shaped (3.1-3.3 µm in length and 1.1-1.3 µm in width), motile with peritrichous agella and mesophilic ( Fig. 2 and 3). Colonies are white, circular and raised after 48 hours of incubation on NFB at 30℃. Colony size is about 1-2 mm in diameter. Four mediums (NMS, NFB, TY and LB) were tested for optimum growth. The bacterial strain can grow well in NFB and TY medium (Fig.   4). The optimum temperature for growth was tested using 30, 35, 40 and 45°C, respectively. The best growth was observed at 35 and 40°C and no growth occurred at 45°C (Fig.5). Of the range of pH values tested (5.0-9.0), the best growth occurred at pH 6.0-7.0 (Fig.6). Growth did not occur in the presence of 3% NaCl (Fig.7). The major saturated fatty acids were C 14:0 , C 16:0 and C 18:0. C 18:1 ω7c and C 16:0 3-OH were the major unsaturated and hydroxylated fatty acids ( Table 1). The G+C content of the genomic DNA of strain CCTCC AB 2021101 T was determined to be 67.8±0.2 mol%, which is within the range for similarity with the genus Azospirillum.

Nitrogen-xing ability
Nitrogenase gene nifH involved in activation of the Fe protein, iron molybdenum cofactor biosynthesis and electron donation was required for nitrogen xation. To verify nitrogen-xing ability of strain CCTCC AB 2021101 T , the nifH gene was ampli ed and the expected 320 bp ampli cation product was obtained (Fig. 8). This PCR product was puri ed and sequenced. The sequence was deposited in GenBank (OL958547). Comparison of the results through an NCBI BLAST search revealed that highest sequence similarities with the nifH gene of CCTCC AB 2021101 T were A. brasilense AWB14 T (95.9%), A. zeae Gr24 T (95.56%), A. picis DSM 19922 T (96.79%), A. lipoferum B22 T (94.88%) and A. oryzae COC8 T (94.88%).
To con rm the activity of the nitrogenase, acetylene-reduction assay was performed by a gas chromatograph system in NFB medium. The acetylene and ethylene standard gas had a strong signal at the retention time of 4.708 min and 4.219 min, respectively ( Fig.9 a and b). In the culture medium without inoculation, there were a CO 2 signal and an acetylene peak at the retention time of 2.909 min and 4.651 min, respectively (Fig.9 c). The ethylene peaks were detected at the retention time of 4.222 min and 4.217 min in the culture medium inoculated by positive control Sinorhizobium meliloti (Rm1021) and Azospirillum actinidiae (Fig. 9 d and

Competing interests
The authors have no relevant nancial or non-nancial interests to disclose.     The growth response (a) and curve (b) of strain CCTCC AB 2021101 T to four culture mediums, NMS, NFB supplemented with 0.01g/L KNO 3 , TY and LB.