Bacillus amyloliquefaciens 11B91 inoculation enhances the growth of quinoa (Chenopodium quinoa Willd.) under salt stress

Laboratory materials

Bacillus amyloliquefaciens 11B91 was obtained from the Northwest Institute of Eco-Environmental and Resources, Quinoa seed (Longli No. 1) was obtained from the Animal Husbandry, Pasture and Green Agriculture Institute (Gansu, China).

Addition of 11B91 under normal conditions and salt treatment

The seeds were disinfected by soaking quinoa seed in 0.5% KMnO₄ for 10 min and rinsing in sterile water until it was colorless. For the seed soaking treatment, Luria-Bertani (LB) liquid medium was first prepared by mixing of distilled water (1 L) with tryptone (10 g), yeast extract (5 g), and sodium chloride (10 g) according to the recipe at pH 7.2 ± 0.2, and then sterilizing it at 121 °C for 20 min; the LB liquid medium was inoculated with strain Bacillus amyloliquefaciens 11B91 and incubated at 28 °C and 180 r/min to facilitate rapid culture(the OD₆₀₀ value was approximately 1) (Jira et al., 2018). Quinoa seeds were soaked in LB liquid medium (blank control group) and LB liquid medium with Bacillus amyloliquefaciens 11B91 (treatment group) for 5 min, ensuring that the seeds were completely submerged (He et al., 2018).

After seed soaking, the seeds were cultured in a petri dish with sterile filter artical. After 3 days of germination, the seeds were transferred to a plate containing sterilized vermiculite and cultured with half-strength Hoagland’s nutrient solution. (containing 2 mM KNO₃, 0.5 mM NH₄H₂PO₄, 0.5 mM Ca(NO₃)₂, 0.5 mM MgSO₄, 0.5 mM Fe-citrate, 92 µM H₃BO₃, 18 µM MnCl₂·4H₂O, 1.6 µM ZnSO₄·7H₂O, 0.6 µM CuSO₄·5H₂O, and 0.7 µM (NH₄)₆Mo₇O₂₄·4H₂O) (2 L). Relevant physiological data were measured at 2, 3 and 4 weeks of quinoa growth (Zhao et al., 2016).

After 4 weeks of quinoa growth, salt treatments were carried out by adding a half-strength Hoagland nutrient solution with salt concentrations of 0, 150 and 300 mmol/L NaCl. Three replicates were set up for each treatment. Relevant physiological data were measured at the first, second, and third weeks of the salt treatment (Zhao et al., 2016).

Determination of biomass

The seedlings of each treatment group were cleaned. An analytical balance was used to determine the fresh weight of the shoots and roots. After being weighed, the samples were oven-dried at 60 °C for 72 h, and after cooling for 48 h, the dry weight was obtained. Each parameter was measured in six replicates.

Determination of chlorophyll content

Fresh leaves (0.05 g) were weighed and recorded by a specific weight (G). After crushing, they were placed in 10 mL centrifuge tubes, and 10 mL ethanol acetone (1:1) mixed solution was added. Samples were stored in the light for 48 h, and the absorbance value was determined with UV-spectrophotometer (UV-1800; Shimadzu, Suzhou, China) at 663 and 645 nm wavelengths (using solution as a blank control) (Zhang et al., 2019). The contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) were calculated as follows:

$$\mathrm{C}\mathrm{h}\mathrm{l}a={\displaystyle \frac{\left(12.72A_{663}-2.59A_{645}\right)V}{1000W}}$$

$$\mathrm{C}\mathrm{h}\mathrm{l}b={\displaystyle \frac{\left(22.88A_{645}-4.67A_{663}\right)V}{1000W}}$$

Note: W: veight of fresh leaves; V: volume of ethanol acetone (1:1) mixed solution; A663 and A645: the absorbance value was determined with a spectrophotometer at 663 and 645 nm wavelengths, respectively.

Root activity

The triphenyl tetrazolium chloride (TTC) colorimetric method was used to determine root activity in quinoa. Quinoa roots (0.5 g) were mixed with 10 mL of 0.4% TTC solution, succinic acid and phosphoric acid buffer in equal amounts. A TTC standard curve was obtained, and the TTC reduction was calculated against the standard curve (Muhammad et al., 2019).

$$Rootactivity={\displaystyle \frac{Ch}{w}}$$

Note: C: TTC (μg), w: root weight (g), h: time (h).

Total phosphorus content

For the determination of the total phosphorus content, a method described by Tisarum et al. (2020) was used. Data were collected as previously described (Cai et al., 2021). Specifically, masses of dried and ground quinoa plants, including the roots, were weighed, decocted with hydrogen peroxide (H₂O₂) and concentrated H₂SO₄, allowed to cool and then fixed in ultrapure water. Then, the total phosphorus content of the quinoa was determined using the molybdenum blue colorimetric method. Total P (mg·g⁻¹ DW) was measured at 420 nm with a UV-spectrophotometer using potassium dihydrogen phosphate as the calibration standard.

Statistical analyses

Excel 2010 was used to collect and process the original data. SPSS version 21.0 (IBM Corporation, Chicago, IL, USA) was used to identify differences between means at a significance level of p ≤ 0.05. The Duncan multi-interval test was used for one-way analysis of variance (ANOVA). The results of the growth and physiological data are presented as the mean ± standard error. The data were plotted using ORIGIN software (Zhao et al., 2016).

11B91 treatment affected the biomass of quinoa seedlings under normal conditions

In the 11B91 treatment, the fresh weight of the aboveground parts at the second and third weeks after inoculation and the fresh weight of the roots at the third week were significantly higher than that in the blank group (p < 0.05). In the second and third weeks of the 11B91 treatment, the fresh weight of the aboveground parts was 59.54% and 73.95% higher than that of the blank group (Fig. 1A), respectively. There was no significant difference in the aboveground fresh weight of 11B91-treated seedlings in the first week. In the third week of 11B91 treatment, the fresh weight of the roots was 75.36% higher than that of the blank group (Fig. 1B). There was no significant difference in fresh root weight (p < 0.05) between the first and second weeks of 11B91-treated seedlings, and the fresh root weight of 11B91—treated seedlings was 49.66% higher than the blank group in the third week (Fig. 1B).

There was no significant difference in aboveground dry weight of 11B91-treated seedlings in the first, second and third weeks (Fig. 1C). In the first and second weeks, the dry weight of the 11B91-treated seedlings increased by 27.46% and 16.83%, respectively. In the second week, the dry weight of the roots of the 11B91-treated seedlings was significantly higher than that of the blank group (p < 0.05). The dry root weight of the 11B91-treated seedlings was 1.36 times higher than that of the blank group. In the first and third weeks, there was no significant difference in root dry weight of 11B91-treated seedlings (p < 0.05). At the third week of 11B91 treatment, the dry root weight was 49.56% higher than that of the blank group (Fig. 1D).

11B91 treatment affected the chlorophyll content of quinoa seedlings under normal conditions

The content of chlorophyll a in the first and third weeks of quinoa seedlings treated with B. amyloliquefaciens 11B91 was significantly higher than that of the blank group (p < 0.05) (Fig. 2A). The chlorophyll a content in the first and third weeks of the 11B91 treatment was 65.32% and 23.48% higher than that in the blank group. There was no significant difference in chlorophyll a content in the second week of 11B91-treated seedlings (p < 0.05), and the content of chlorophyll a in seedlings treated with 11B91 was 11.23% higher than in the blank group (Fig. 2A).

The content of chlorophyll b in the first week of quinoa seedlings treated with B. amyloliquefaciens 11B91 was significantly higher than that in the blank group (p < 0.05); the content of chlorophyll b in the first week of seedlings treated with 11B91 was 58.50% higher than that in the blank group (Fig. 2B). There was no significant difference in chlorophyll b content in the second and third weeks of 11B91-treated seedlings (p > 0.05); the content of chlorophyll b in 11B91-treated quinoa seedlings was 45.42% higher than that in the blank group in the third week (Fig. 2B).

Effects of 11B91 on the root activity of quinoa seedlings under normal conditions

The root activity was significantly higher in 11B91-treated seedlings than in the blank group in the first and second weeks (p < 0.05; Fig. 3) and was 54.44% and 5.45% higher, respectively.

Effects of 11B91 on the total phosphorus content of quinoa seedlings

The total phosphorus content of quinoa seedlings treated with 11B91 was significantly higher (16.66%; p < 0.05) than that of the blank group in the first week (Fig. 4).

Effect of 11B91 on quinoa growth under salt treatment

Under B. amyloliquefaciens 11B91 treatment, in the first and second weeks of 0 mM salt treatment, in the first, second and third weeks of 150 mM salt treatment, and in the first and third weeks of 300 mM salt treatment, the fresh weight was significantly higher than that without 11B91 treatment (p < 0.05). The fresh weight of quinoa treated with 11B91 was 63.99% and 83.55% higher than that of the blank group in the first and second weeks of 0 mM salt treatment (Figs. 5A and 5C). The fresh weight in the 150 mM salt treatment was 57.59%, 88.18% and 42.89% higher than that in the blank group at 1, 2 and 3 weeks, respectively (Figs. 5A, 5C and 5E). Under 300 mM salt treatment, the fresh weight was 66.98% and 1.07 times higher than that of the blank group in the first and third weeks (Figs. 5A, 5E). There was no significant difference in the fresh weight between the blank group and 11B91 treatment in the 0 mM salt treatment in the third week and the second week under 300 mM (p < 0.05). The fresh weight in the 11B91 treatment was 23.98% higher than that of the blank group in the third week of 0 mM salt treatment and 54.99% higher in the second week of 300 mM salt treatment than in the blank group (Figs. 5C and 5E).

The dry weight of 11B91-treated quinoa seedlings treated with 150 mM salt in the second week and 300 mM salt in the first week was significantly higher than that of the blank group (p < 0.05) (Figs. 5B and 5D). The dry weight of the 150 mM salt treatment was 64.86% higher than that of the blank group in the second week of 11B91 treatment; the dry weight was 1.33 times, 75.99% and 45.03% higher than in the blank group in the 300 mM salt treatment. Under the B. amyloliquefaciens 11B91 treatment, in the first, second and third weeks of 0 mM salt treatment, in the first and third weeks of 150 mM salt treatment, and in the second and third weeks of 300 mM salt treatment, the dry weight showed no significant difference compared to the treatment without 11B91 (p < 0.05). No significant difference was found in dry weight in the 0 mM treatment at three weeks or the treatment during the first and third weeks of salt treatment.

The dry weight in the 11B91 treatment was 21.07% and 11.26% higher than that of the blank group in the 0 mM salt treatment in the first and third weeks, respectively. In the 150 mM salt treatment in the first and third weeks, the dry weight was 20.54% and 10.85% higher than that in the blank group (Figs. 5B and 5F).

Effect of 11B91 on the chlorophyll content of quinoa under salt treatment

The content of chlorophyll a in quinoa treated with 11B91 was significantly higher than that in the blank group (p < 0.05) in the 0 mM salt treatment at 2 and 3 weeks, 150 mM salt treatment in the first week, and 300 mM salt treatment in the first, second and third weeks (Figs. 5A, 6C and 6E). The chlorophyll a content in the second and third weeks in the 0 mM salt treatment was 23.48% and 57.79% higher than that in the blank group, respectively. The chlorophyll a content in the first week with 150 mM salt treatment was 91.36% higher than that in the blank group, and under 300 mM salt treatment, it was 95.07%, 162 % and 34.76% higher than that in the blank group at 1 and 2 weeks, respectively. No significant difference was found in the chlorophyll a content between seedlings in the 0 mM salt treatment in the first week, 150 mM salt treatment in weeks 2 and 3(p < 0.05). The chlorophyll a content in the first week with 11B91 in the 0 mM salt treatment was 11.23% higher than that in the blank group (Figs. 6A and 6E). The chlorophyll b content in the second week of 0 mM salt treatment and in the second week of 300 mM salt treatment was significantly higher than that in the blank group (p < 0.05) (Fig. 6D). The chlorophyll b content in the second week of 11B91 and 0 mM salt treatment was 45.42% higher than that in the blank group, and in the 300 mM salt treatment, it was 76.37% higher in the second week than in the blank group. The chlorophyll b content between seedlings in the 0 mM salt treatment in the first and third weeks, 150 mM salt treatment in the first, second and third weeks, and 300 mM salt treatment in the first and third weeks was not significantly different (p < 0.05). The chlorophyll b content in the third week of the 0 mM salt treatment was 24.82% higher than that in the blank group, and in the first and third weeks of the 300 mM salt treatment, it was 109% and 20.53% higher than that in the blank group, respectively (Fig. 6F).

Effect of 11B91 on quinoa root activity under salt treatment

Under 11B91 treatment, in the first week with 150 mM salt treatment the root activity was significantly lower than in the blank group. Under 11B91 treatment, in the second and third weeks with 150 mM salt treatment and in the third week with 300 mM salt treatment, the root activity was significantly higher than in the blank group (Figs. 7A–7C). The root activity under 11B91 treatment in the second and third weeks of 150 mM salt treatment was 7.17% and 33.07% higher than that in the blank group, respectively, and 26.87% higher in the third week after 300 mM salt treatment than in the blank group (Fig. 7C). There was no significant difference in root activity between the 11B91 and blank groups in the first, second and third weeks of 0 mM salt treatment and in the first and second weeks of 300 mM salt treatment (p < 0.05). The root activity of the 11B91-treated seedlings was 5.45% higher than that of the blank group in the first week of 0 mM salt treatment (Fig. 7A).

Effect of 11B91 on the total P content of quinoa under salt treatment

The total phosphorus content of plants in all LB treatments showed a decreasing trend with increasing salt concentrations at different salt treatment times (Figs. 8A–8C). The inoculation of 11B91 maintained a relatively stable P content in plants under salt stress. In the second week, the total phosphorus content under 150 and 300 mM salt treatment in seedlings treated with 11B91 was significantly higher than that of the blank group (p < 0.05). In the second week, the total phosphorus content of the 11B91 treatment was 7.40% higher under 150 mM salt treatment than that in the blank group and 42.73% higher under 300 mM salt treatment than that in the blank group (Fig. 8B). There was no significant difference in total phosphorus content between the 11B91 and blank groups in the first and third weeks of 150 and 300 mM salt treatment (Figs. 8A and 8C).