Nickel mine soil is a potential source for soybean plant growth promoting and heavy metal tolerant rhizobia

Sampling and soil analysis

Soil samples were collected from a nickel mine (26°46′54.2″N, 102°06′53.2″E) in Xichang, Sichuan Province, China. Three sampling sites spaced 50 to 100 m apart were randomly selected within the area. From the sampling points, five topsoil (0–20 cm) subsamples spaced at least 5 m apart were collected and mixed to make one composite sample per site. The composite samples were quartered and stored on ice before being taken to the laboratory. The soil samples were ground, passed through a 2 mm nylon sieve, and air-dried. Soil water content in the fresh soil was determined by measuring the weight difference after soil samples had been dried at 105 °C for eight hours. Soil pH was measured using a PHS-3C pH meter (Shanghai Yoke, Shanghai, China) in a 2.5:1 water-soil slurry which had been left to settle overnight. Soil organic carbon content was determined using the K₂Cr₂O₇-H₂SO₄ method (Schumacher, 2002). Total nitrogen, phosphorus and potassium contents in the soil samples were determined using Kjeldahl method (Kjeltec 8400, FOSS, Sweden), Mo-Sb colorimetric method (WFJ2100, UNICO, China) and flame spectrophotometry (FP6410, Shanghai Precision & Scientific, China), respectively (Murphy & Riley, 1962; Page, Miller & Keeney, 1982; Yu et al., 2021). Available nitrogen content was determined using the alkali N-proliferation method; soil available phosphorus and potassium were extracted with sodium bicarbonate solution and NH₄AC solution, respectively, and measured using the previously described method (Wu et al., 2021). The contents of heavy metals were determined after digestion using mixed acid (HNO₃: HCLO₄ = 3: 1) using inductively coupled plasma optical emission spectrometry (ICP-OES; IRIS Intrepid II; Thermo Electron Corporation, Waltham, MA, USA).

Trapping and isolation of rhizobia

The indigenous rhizobial strains in the nickel mine soil were trapped using soybean cultivar Nandou No.12 bred by Nanchong Institution of Agricultural Sciences, Sichuan, China. The soybean seeds were sterilized by dipping into 95% alcohol for 3 min and 1% HgCl₂ for 5 min, followed by rinsing with sterile water (Yu et al., 2017). The soybean seeds were germinated in a pot filled with sterilized moist vermiculite in the dark for 24 h, and transplanted into pots filled with soil collected from the nickel mine site (S_N). The soybean plants were harvested after 90 days. Three root nodules per soybean plant were selected and sterilized using the above-mentioned methods. The nodules were incubated on beef extract peptone agar at 28 °C, and the surface sterilization was considered successful when no colonies appeared in 24 h. After that, nodules were crushed in plastic tubes and inoculated on yeast-extract mannitol agar (yeast extract 1.5 g L⁻¹, mannitol 1.0 g L⁻¹, K₂HPO₄ 0.5 g L⁻¹, MgSO₄ 7H₂O 0.2 g L⁻¹, NaCl 0.1 g L⁻¹, Congo red 0.04 g L⁻¹, agar 20 g L⁻¹). The plates were maintained at 28 °C for 7 to 10 days. During the incubation period, single colonies were selected based on colony morphology and purified by repeated streaking (Yu et al., 2017). Purified isolates with round, plump, milky white, mucilaginous and smooth-edged colonies were examined using light microscopy after Gram staining. Gram-negative isolates with rod-shaped cells were preserved in 20% (w/v) glycerol at −80 °C.

Phylogenetic diversity analysis

The isolates were grown in YM liquid medium (yeast extract 1.5 g L⁻¹, mannitol 1.0 g L⁻¹, K₂HPO₄ 0.5 g L⁻¹, MgSO₄ 7H₂O 0.2 g L⁻¹, NaCl 0.1 g L⁻¹, Congo red 0.04 g L⁻¹) for 24 h at 28 °C in a shaking incubator, and DNA was extracted using TIANamp Bacteria DNA Kit (TIANGEN, China). The genetic diversity of the isolates was assessed using BOX-A1R PCR fingerprinting with the primer 5′-CCTCGGCAAGGACGCTGACG-3′ (Chen et al., 2014). Amplification was done in a 10 µL volume system containing 5 µL of 2 × PCR mix, 0.2 µL of the primer (10 µmol L⁻¹), 1 µL of template DNA (50 ng mL⁻¹), and 3.8 µL double distilled water (ddH₂O). The BOX-A1R PCR thermal profile included initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min and extension at 65 °C for 8 min, and a final extension at 65 °C for 16 min. The amplified fragments (8 µL) from the amplification mixture and the 200 bp DNA ladder were separated in 2% (w/v) agarose gel with ethidium bromide at 80 V for 2.5 h, and were visualized under UV light with the patterns recorded. Based on the patterns, a BOX-A1R PCR cluster tree diagram was created using the unweighted pair group method with arithmetic averages (UPGMA) in NTSYSpc 2.1 (Yu et al., 2014).

Plant growth promotion ability test

Based on the BOX-A1R PCR fingerprints, eight representative rhizobial isolates were selected for the test of plant growth promotion ability using Leonard jars. Leonard jar and nitrogen-free nutrient solution were prepared as previously described (Kang et al., 2018; Yu et al., 2017). Soybean seeds were surface-sterilized and germinated as described above. Three sterilized seeds were transferred into the topsoil of a Leonard jar, which was then covered with a layer of approximately 3 cm moist vermiculite. When the soybean seedlings were 2–3 cm tall, the rhizobia culture in exponential phase (OD_600 nm 0.6∼0.8) was inoculated around the roots, and then the topsoil was covered with a layer of 1 cm autoclaved quartz sand. The non-inoculated soybean supplemented with nitrogen-free nutrient solution was set as the negative control, while the N⁺ treatment with 1 g L⁻¹KNO₃ as the nitrogen source in the nutrient solution was the positive control. Each treatment and control included three replicates. The soybean plants were kept in a greenhouse with artificial light-dark cycles: 17 h light, temperature 25 °C and humidity 80% (to simulate daytime); 7 h dark, temperature 17 °C and humidity 85% (to simulate nighttime). The jars were replenished with nutrient solution when needed. The soybean plants were harvested after 55 days. The number of nodules, root length, and plant height and weight were measured. The plant samples were dried at 105 °C for 30 min to deactivate enzymes, and then at 55 °C until constant weight. The nitrogen, phosphorus, and potassium contents of the dried roots and shoots were measured.

Sequence analysis

The isolates that nodulated soybean plants were further characterized using sequence analyses. The almost full length 16S rRNA gene was amplified using the primer pair 27F/1492R (Yu et al., 2017), the housekeeping genes atpD, recA, glnII, and rpoB using primer pairs atpD 255F/ atpD 782R, recA 63F/recA 555R, glnII 12F/glnII tsR, and rpoB 454F/ rpoB 1364R, respectively (Tang et al., 2012; Zhao et al., 2014), and the nitrogen fixation gene nifH using the primer pair nifHF/nifHI (Laguerre et al., 2001). All of the primers we used in this study were listed in Table S2. Amplification was done in a 30 µL volume system containing 15 µL of 2 × PCR mix, 0.15 µL of each primer, one µL of template DNA (50 ng mL⁻¹), and 13.7 µL ddH₂O. For the amplification of 16S rRNA, atpD, recA, glnII and rpoB genes, the thermal cycling conditions included an initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. For the amplification of nifH, the thermal cycling conditions included an initial denaturation at 95 °C for 3 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 59 °C for 1 min, extension at 72 °C for 5 min, and a final extension at 72 °C for 6 min. The amplified products were sequenced by Sangon Biotech (Shanghai, China). The fragments of atpD (274 bp), recA (245 bp), glnII (413 bp) and rpoB (534 bp) were concatenated for multilocus sequence analysis (MLSA). The sequences were matched against reference sequences of rhizobia in the GenBank of NCBI database using BLAST. The reference sequences of different rhizobia species we used for the phylogenetic analysis of the three genes (16SrRNA, housekeeping genes, and nifH) were separately listed in Tables S3, S4, and S5. The sequences of the isolates and the reference sequences were subjected to phylogenetic analysis using neighbor joining method in MEGA7.0. The phylogenetic trees were bootstrapped with 1,000 replications for each sequence to evaluate the reliability of the tree topologies (Saitou & Nei, 1987).

Heavy metal resistance test

For assessing the heavy metal-resistance ability of the isolates, 10 g L⁻¹ stock solutions of Cd²⁺ (CdCl₂ 2.5H₂O), Cr⁶⁺ (K₂Cr₂O₇), Cu²⁺ (CuCl₂ 2H₂O), Ni²⁺ (NiSO₄), and Zn²⁺ (ZnSO₄ 7H₂O) were prepared by dissolving the salts in ultrapure water. The isolates were grown in 5 mL YM liquid medium with 0, 4, 8, 12, 16, 20, 40, 60, 80, 100 mg L⁻¹ of each metal in an orbital shaker at 28 °C for 72 h, after which the optical density (OD_600 nm) of the cultures were measured using a spectrophotometer (UV-3300; Shanghai MAPADA, Shanghai, China) (Abd-Alla et al., 2012).

The minimum inhibitory concentrations (MIC) were determined by comparing the OD_600 nm value of the cultures spiked with metals to those without metals. MIC was defined as the lowest metal concentration resulting in a visually observable decrease in the growth curve of the isolates. Minimum lethal concentration (MLC) was defined as the lowest metal concentration resulting in an OD_600 nm value lower than 0.1.

Statistical analysis

Statistical differences in soil properties and soybean growth parameters were tested using one-way ANOVA and Fisher’s protected LSD (least significant difference) test at P ≤ 0.05 in IBM SPSS statistics 22.0. The differences in MIC and MLC values were not tested due to their zero variance.

Isolation of rhizobia from nickel mine soil

The soil in the nickel mining area was slightly acidic (pH 6.35), and the organic matter content was low (1.07%). The contents of total N, P and K were 261.35, 479.85, and 5869.73 mg kg⁻¹, respectively, and 15.08%, 2.93%, and 0.38% of them were available fragments (Table 1). Both nickel and lead contents were approximately 20 mg kg⁻¹, and cadmium, chromium, copper, iron, manganese and zinc were also detected (Table 1).

As a result of trapping rhizobial strains from the nickel mine soil using soybean as the host plant, 21 isolates were preliminarily identified as rhizobia based on colony morphology, microscopic examination of cell shape, and Gram staining. The BOXA1R-PCR fingerprinting (Fig. 1) of the isolates showed that sixteen distinct fingerprint patterns were found, indicating that these isolates from nickel mine soil were genetically diverse. The isolates were clustered into 8 groups at 87% similarity level.

Plant growth promotion ability of selected isolates

Based on the fingerprinting, we selected 8 isolates (YN1, YN5, YN8, YN10, YN11, YN13, YN27, YN30) for the plant growth promotion ability test. Only four isolates (YN5, YN8, YN10, YN11) nodulated soybean plants, with nodule numbers ranging from 50 to 54 per plant (mean value of six plants). The biomass of soybeans inoculated with the four symbiotic isolates (YN5, YN8, YN10, YN11) and isolate YN13 was significantly higher than that in the N⁻ treatment (P = 0.00) (Fig. 2A). The biomass of soybeans inoculated with isolate YN8 was on the same level as in the N⁺ treatment. The shoots of soybeans inoculated with the 4 symbiotic isolates (YN5, YN8, YN10, YN11) and isolate YN1 were significantly longer than those in the N⁻ treatment (P = 0.00, 0.01, 0.00, 0.00, 0.00). The roots of soybeans inoculated with the symbiotic isolates YN8, YN10, and YN11 were significantly longer than those in the N⁻ treatment (P = 0.00) (Fig. 2B). The shoot N content of soybean plants inoculated with the symbiotic strains was at the same level as in the N⁺ treatment, and the shoot N content of soybeans inoculated with the symbiotic strains of YN5, YN8, YN10, and YN11 as well as the isolate YN1 was significantly higher than that in the N⁻ treatment (P = 0.00) (Fig. 3A). The root N content of inoculated soybean plants was higher than that in the N⁻ treatment (Fig. 3A). The root P content of soybeans inoculated with the symbiotic isolates and isolate YN30 was lower than that in the N⁻ treatment (Fig. 3B). The shoot K content of soybeans inoculated with the isolate YN10 was higher than that in the N⁻ treatment (Fig. 3C). The root K content of soybeans inoculated with the isolates YN5, YN8, YN10, YN13, YN27 and YN30 was higher than that in the N⁻ treatment (Fig. 3C).

Sequence analyses of symbiotic isolates

As a result of the sequence analysis of the almost full length 16S rRNA gene, the symbiotic isolates were assigned to the genera Ensifer (formerly designated as Sinorhizobium) and Rhizobium (Fig. 4). The isolates YN5, YN8, and YN10 were respectively grouped together with the type strains E. fredii USDA205, E. americanum CFNEI156 and E. xinjiangense CCBAU110, and isolate YN11 with R. radiobacter ICMP 5856 (formerly Agrobacterium tumefaciens ICMP 5856). Based on the multilocus sequence analysis (MLSA) of the concatenated fragments of atpD (274 bp), recA (245 bp), glnII (413 bp) and rpoB (534 bp), the isolates YN5, YN8, and YN10 were identified as E. xinjiangense strains, and YN11 as Rhizobium radiobacter strain (Fig. 5). The nifH sequences from YN5, YN8, and YN10 were 100% similar with those from E. fredii CCBAU23314 and E. xinjiangense CCBAU110, and the nifH sequence from YN11 with that from R. radiobacter (Fig. 6).

Heavy metal resistance ability of symbiotic isolates

In general, E. xinjiangense YN5 and R. radiobacter YN11 tolerated higher levels of heavy metals compared to E. xinjiangense YN8 and YN10 (Fig. 7, Table S1). For Cd²⁺, the MIC and MLC values of E. xinjiangense YN5 and R. radiobacter YN11 were the highest. For Cr⁶⁺, the MLC of all the strains was 16 mg L⁻¹. For Cu²⁺, the MIC and MLC values of E. xinjiangense YN8 were the lowest. For Ni²⁺, the MLC values of YN5 and YN11 were higher than those of YN8 and YN10. For Zn²⁺, the MLC value of E. xinjiangense YN5 was 300 mg L⁻¹, i.e., over three times higher than that of R. radiobacter YN11 and over 18 to 75 times higher than those of YN11 and YN8, respectively.