Improved salt tolerance of Chenopodium quinoa Willd. contributed by Pseudomonas sp. strain M30-35

Background Plant-growth-promoting rhizobacteria (PGPR) can promote plant growth and enhance plant tolerance to salt stress. Pseudomonas sp. strain M30-35 might confer abiotic stress tolerance to its host plants. We evaluated the effects of M30-35 inoculation on the growth and metabolite accumulation of Chenopodium quinoa Willd. during salt stress growth conditions. Methods The effects of M30-35 on the growth of C. quinoa seedlings were tested under salt stress. Seedling growth parameters measured included chlorophyll content, root activity, levels of plant- phosphorus (P), and saponin content. Results M30-35 increased biomass production and root activity compared to non-inoculated plants fertilized with rhizobia and plants grown under severe salt stress conditions. The photosynthetic pigment content of chlorophyll a and b were higher in M30-35-inoculated C. quinoa seedlings under high salt stress conditions compared to non-inoculated seedlings. The stability of P content was also maintained. The content of saponin, an important secondary metabolite in C. quinoa, was increased by the inoculation of M30-35 under 300 mM NaCl conditions. Conclusion Inoculation of M30-35 rescues the growth diminution of C. quinoa seedlings under salt stress.


INTRODUCTION
Salinity is a major threat to crop production, especially in arid and semi-arid areas (Flowers & Colmer, 2008;Schleiff, 2008). Salt stress has a negative effect on plant growth, and development (Yu et al., 2018), and it is challenging to determine the efficient use of saline soilsfor food production. Two primary strategies, modified agricultural practices and the development of new varieties adapted to saline soils, are being used for growing crops in saline soils. These strategies have produced useful, results but they are still in the immersed in LB liquid medium (blank control) and Pseudomonas sp. M30-35 bacterial solution (treatment group) for 20 min. Then, the C. quinoa seeds were germinated on moist filter paper in Petri dishes and kept for 48 h at 25 • C in darkness (Gururani & Upadhyaya, 2013). After germination, the plantlets were transferred to plastic pots (upper diameter, 48 mm; bottom, 23 mm; depth, 50 mm). The pots contained vermiculite, which was previously heated at 121 • C for 20 min for sterilization. Each pot was inoculated with 1 mL of M30-35 suspension culture or 1 mL of liquid LB medium. Each treatment was irrigated with half-strength Hoagland solution (2 mM KNO 3 , 0.5 mM NH 4 H 2 PO 4 , 0.5 mM Ca(NO 3 ) 2 , 0.5 mM MgSO 4 , 0.5 mM Fe-citrate, 92 µM H 3 BO 3 , 18 µM MnCl 2 · 4H 2 O, 1.6 µM ZnSO 4 · 7H 2 O, 0.6 µM CuSO 4 · 5H 2 O, and 0.7 µM (NH 4 ) 6 Mo 7 O 24 · 4H 2 O) (2 L). The plants were grown in a greenhouse with a 16:8 h (L:D) photoperiod, 26 • C day and 22 • C night temperature, and a relative humidity of 65 ± 10% (He et al., 2018).
Plants aged 14 d were subjected to salt stress by treatment with 0, 150, or 300 mM of NaCl solutions. Treated seedlings were harvested at 7 d and 21 d after salt treatment to record the plant biomass and physiological measurements (Su et al., 2017).

Determination of the seedling biomass
Plants were removed from the pots, and the roots were washed with sterilized water to remove attached soil. Then, the seedlings were divided into shoot and root portions. The samples were dried in an oven at 70 • C for 4 d, and the dry weight was measured (Elesawi et al., 2018).

Determination of chlorophyll contents
The relative chlorophyll content was measured using the ethanol-acetone method, according to the method previously reported (Han et al., 2014). Chlorophyll was extracted from the third compound leaf (50 mg) with an 80% (v/v) cold acetone and 95% ethyl alcohol mix (1:1). A spectrophotometer (Shimadzu, UV-1800, Suzhou, China) was used to read the absorbance at 663 nm for chlorophyll a and 645 nm for chlorophyll b contents. Six samples were measured in each treatment. Each measurement was conducted at 10:00 a.m.

Determination of root activity
Root activity was determined using the triphenyltetrazolium chloride (TTC) colorimetric method (Muhammad et al., 2019). C. quinoa seedling roots were washed, and the root tip samples were excised. Then, the root tips were dried with filter paper. The reaction mixture consisted of 0.2 g root samples, 5 mL of PBS (pH 7.0), and 5 mL of 0.4% TTC in a tube, with the root tips fully immersed in the solution for 2 h at 37 • C. A 2 mL amount of 1 M of sulfuric acid was added to stop the reaction. The roots were removed from the reaction solution, blotted, and homogenized using a mortar and pestle. The red extraction was collected by ethyl acetate to determine the final volume of 10 mL. The absorbance was determined at 485 nm against a blank of ethyl acetate. The standard curve reduction value was TTC(y): y = 0.0021 × −0.0113, R = 0.9945, where y represents the absorbance reading. The root vigor = TTC(x)/W × time reduction value, where W is the root fresh weight, and the time was 2 h.

Determination of total P content
Phosphorus content was determined by the molybdenum blue spectrophotometry method (Tisarum et al., 2020). Dried shoot and root samples (100 mg) were added to 8 mL of H 2 SO 4 and soaked overnight. Then, this was digested at 300 • C for 90 min until fumes were produced. After slight cooling, 0.5 mL of H 2 O 2 was added to the tubes, and heated at 300 • C until the solution was colorless or clear. The volume of extract was maintained by adding distilled water up to 100 mL in the volumetric flask. Then, 10 mL of the previous digestion liquid was transferred to a 50 mL volumetric flask, and dinitrophenol indicator was added. Afterwards, 5 mL of molybdenum antimony anti-coloring agent was added, shaken well, and added with up to 50 mL of ultrapure water. Total P (mg g −1 DW) was measured at 420 nm by a spectrophotometer using KH 2 PO 4 as the calibration standard.

Saponin quantification
The content of total saponin in the different salt treatments of C. quinoa was determined by colorimetry (Liao et al., 2017) using oleanolic acid as the standard. C. quinoa saponins were retrieved by microwave heating. The dried shoot and root samples (500 mg) were extracted with 10 mL of 75% alcohol placed in a ultrasonic bath for 30 min at 60 • C. The extract solution was then evaporated to dryness in 60 • C water. Next, both the 200 µL 5% vanillin acetic acid (vanillin: glacial acetic acid was 5:100, W/V) and 800 µL perchloric acid were added to different test tubes. The liquid was boiled for 15 min, and up to 5 mL of glacial acetic acid was added in a volumetric flask after cooling. The absorbance was determined at 546 nm using a spectrophotometer against the reagent blank.

Data analysis
Excel 2010 was used to collect and process the original data. All data were analyzed by one-way analysis of variance (ANOVA). Duncan's post-hoc multiple comparison tests were used to identify differences between means at a significance level of P ≤ 0.05 using SPSS version 21.0 (IBM Corporation) statistical software. Results of the growth and physiological data are presented as means ± standard error. Data were plotted using the ORIGIN software.

M30-35 effects on C. quinoa growth
With increased salinity for 7 d and 21 d, the dry weight of shoots decreased in the control and the M30-35 medium. For the 7 d treatment, different NaCl concentrations had no effect on the shoot dry weight of the control, while 150 mM of NaCl increased the shoot dry weight of seedlings inoculated with M30-35 (Fig. 1A). For the 21 d treatment, M30-35 and the control had similar levels of reduced growth. In contrast, M30-35 reduced the decline of shoot dry weight (Figs. 1A and 1B). M30-35 slightly improved shoot growth, compared to the control, under NaCl treatment.
We compared the effect of adding Luria broth (LB) to plants grown in the presence or absence of M30-35. The roots maintained normal growth after 7 d in the NaCl treatment. However, roots were reduced by exposure to NaCl for 21 d. Compared to the control,

Influence of M30-35 on the chlorophyll content of C. quinoa
After a prolonged time period, both chlorophyll a and chlorophyll b in C. quinoa seedlings showed similar changes under the NaCl treatments (Fig. 2). After NaCl treatment for 7d, the chlorophyll a and chlorophyll b levels in the controls significantly decreased with the increase in salt concentrations. However, the levels of chlorophyll a and b in the M30-35 treatment maintained relative stability ( Figs. 2A and 2C). Under 300 mM NaCl conditions the chlorophyll a content of the M30-35 treatment significantly increased by 40.48%, compared to the controls (P < 0.05). In contrast, the content of chlorophyll a and b in the control or M30-35 medium in the NaCl treatment at 21 d remained stable except for a

Impact of M30-35 on the root activity of C. quinoa
The activity of root system is related to the survival rate of plant, which is an important index to reflect the quality of plant seedlings and evaluate the response of plants to stress (Zhang et al., 2019). With increasing NaCl concentration, the C. quinoa root activity in both the control and M30-35 medium improved at 150 mM, but subsequently declined by 300 mM, at both 7 d and 21 d (Fig. 3). Compared to the control group, the root activity of seedlings inoculated with M30-35 improved by 11.88% (P < 0.05) at 150 mM NaCl for 7 d, and improved by 27.12% (P < 0.05) under 300 mM of NaCl for 21 d.

The effect of M30-35 on the total P contents of C. quinoa
The P content of all LB-treated plants were reduced after exposure to salt stress (Fig. 4). The inoculation of M30-35 maintained a relatively stable P content in plants under salt stress. The P content of the M30-35 treatment was significantly higher than that in the controls under 150 mM of NaCl for 7 d (Fig. 4A).

Effect of M30-35 on the saponin content in C. quinoa seedlings
Similar to the change pattern of root activity, the total saponin content initially increased and then decreased. In the 300 mM NaCl treatment, M30-35 significantly increased the saponin content by 17.40% (P < 0.05). However, M30-35 had no significant effect on the saponin content in the 150 mM NaCl treatment (Fig. 5).  et al., 2017;Han et al., 2014). Under salt stress, PGPR can increase the root length, root surface area, and number of root tips, thereby enhancing the uptake of plant nutrition, and ultimately improving plant growth and development under stress (Egamberdieva & Kucharova, 2009;Trivedi et al., 2020). Roots colonized by the M30-35 strain enhanced the tolerance of the whole plant to salt stress, and the enhancement was reflected by increasing plant root growth and nutritional status (He et al., 2018). Halotolerant strains of bacteria (Enterobacter sp.
[MN54]) can promote the plant health and performance of C. quinoa (Yang et al., 2016). The present results also demonstrated that M30-35 significantly increased the dry weight of roots under saline conditions and also increased the dry weight of the shoots. Serratia liquefaciens KM4 promoted maize (Zea mays L.) salt tolerance by inducing the accumulation of the biomass yield (Elesawi et al., 2018). M30-35 might improve the salt tolerance of C. quinoa in a similar manner. However, this hypothesis requires further verification.
Photosynthesis is the basis for increasing in plant biomass and provides the energy needed for metabolism. Changes in chlorophyll levels can be considered to be a biochemical marker of salt tolerance, which reflects the physiological state of a plant's response to salt (Bernal-Vicente et al., 2018;Singh & Gautam, 2013). Therefore, chlorophyll plays key roles in plant development and stress resistance processes, and its concentration has been shown to decline less rapidly in strong stress-resistant plants, when compared to stress-sensitive plants (Liu & Jiang, 2010). In the present study, the application of M30-35 reduced the decline of chlorophyll a and b in the leaves of C. quinoa seedlings after 7 d of NaCl treatment, indicating the potential role of M30-35 in protection against salt stress. In the 300 mM NaCl treatment, seedlings inoculated with M30-35 had significantly increased chlorophyll a content after the 7 d treatment, and chlorophyll b content in the 7 d treatment, and increased chlorophyll b levels after the 21 d treatment. These results suggest that M30-35 could maintain the C. quinoa chlorophyll content, especially when seedlings were under severe salt stress. These results were similar to observations made on white clover and ryegrass (Han et al., 2014;He et al., 2018). The plant root system is among the most important components of plants, and is sensitive to salt stress (Liu et al., 2019). Salt stress can reduce root vigor which was defined as the capacity for deoxidization, and affect absorption of water and nutrition (He et al., 2018). However, increased antioxidant substance accumulation and increased antioxidant enzyme activities can protect plants against the oxidative injury caused by salt stress, thereby giving the plants increased resistance to salt stress (Liu et al., 2019;Wang et al., 2014). Enzymes such as superoxide dismutase in the root of Kandelia candel under varying salt stresses exhibited a trend from ascent to descent (Wang et al., 2014). The root activity of C. quinoa was significantly greater under the 150 mM NaCl treatment, compared to the control. The higher accumulation of antioxidant substances was likely due to PGPR-induced salt stress response strategy in plants. This observation suggests that the 150 mM NaCl concentration is a moderate salt treatment for C. quinoa. Significantly elevated root activity in the M30-35 treatment was also observed under 150 and 300 mM of NaCl. In a similar study, Trichoderma longibrachiatum T6 significantly increased the root activity of wheat seedlings under salt stress (Zhang et al., 2019). According to the recent research results reported by He et al. (2018), M30-35 promoted the growth of ryegrass, by enhancing root activity under salt stress. Hence, the growth promotion in C. quinoa roots might be correlated to an increase in root activity under salt stress.
Salinity interferes with the absorption of macro-elements (K, Ca, Mg, P, and S) and micro-elements (Zn, Fe, Mn, Cu, and B) by plants, leading to nutrient deficiency and metabolic disorders (Egamberdieva et al., 2017;Kim et al., 2017;Munns & Tester, 2008). P is a key nutrient element in plants and plays an important role in the process of plant growth and development. The solubilization of bound inorganic P and mobilization of organic P by PGPR are mechanisms that have a positive effect on plant growth (Liu et al., 2014;Zeng, Wu & Wen, 2016). Under high salt conditions, eggplant seedlings inoculated with Achromobacter increased the absorption of P (Mayak, Tirosh & Glick, 2004), and inoculated wheat seedlings with Bacillus increased the levels of P in seedlings (Upadhyay & Singh, 2015). Our results showed that the P content of non-inoculated C. quinoa seedlings under salt stress was significantly lower than seedlings not exposed to salt stress. This indicated that salt stress can influence P absorption. The results of the present study indicate that inoculation of M30-35 can maintain (P) homeostasis and mitigate the salt stress impact on C. quinoa.
C. quinoa is rich in saponins (belonging to the triterpenoid saponins) mainly concentrated in the pericarp (Kuljanabhagavad et al., 2008). The saponins contents vary in different C. quinoa varieties and are in the range of 47.11-136.98 mg/100g, which are often influenced by biotic and abiotic factors (Szakiel, Paczkowski & Henry, 2011). Under 200 mM of salt stress, the C. quinoa seedlings accumulated higher levels of saponins than control (Gomezcaravaca et al., 2012). Furthermore, Yang et al. (2018) demonstrated that saponins might be a biostimulant for germination in C. quinoa, especially under salt stress. Thus, saponin might be involved in the response of C. quinoa to salt stress. Our results showed that 150 mM of NaCl induced the synthesis of saponins in C. quinoa seedlings. Besides, the C. quinoa plants inoculated with M30-35 exhibited higher saponins levels compared to un-inoculated plants under 300 mM NaCl salt stress. M30-35 might enhance the salt tolerance of C.quinoa by partially increasing their saponins accumulation.

CONCLUSIONS
The effects of Pseudomonas sp. strain M30-35 on the growth and physiology of C. quinoa seedlings were evaluated. Under salt stress, M30-35 promoted the growth of C. quinoa, and increased the root and total biomass of C. quinoa seedlings. The chlorophyll a and chlorophyll b content in the 300 mM NaCl treatment was increased by inoculation of M30-35. M30-35 improved the root activity and accumulation of total saponins and alleviated the decline in P content under salinity. These data provide evidence that M30-35 inoculation of C. quinoa seedlings can improve their salt tolerance.