Alleviation of salt stress in rapeseed (Brassica napus L.) plants by biochar-based rhizobacteria: new insights into the mechanisms regulating nutrient uptake, antioxidant activity, root growth and productivity

ABSTRACT Amendment of saline soils with biochar and rhizobacteria is a new technique to diminish the negative impacts of salt stress on plants. Hence, an original pot experiment was conducted in a glass greenhouse at the University of Tabriz to investigate the performance of salt-stressed (0, 6 and 12 dS m−1 NaCl) rapeseed (Brassica napus L.) plants in response to different biochar-related treatments (non-biochar, biochar, biochar-based Pseudomonas putida RS-198, biochar-based Azotobacter chroococcum RS-106 and both bacteria). The biochar-related treatments reduced sodium content, generation of reactive oxygen species, lipid peroxidation and enzymatic antioxidants in plant tissues while enhanced nutrient content, non-enzymatic antioxidants, main and lateral roots lengths and weights, main/lateral root length ratio, specific root length, root diameter, shoot length and weight, leaf area, chlorophyll content and seed and oil yields under saline conditions. The biochar-based Pseudomonas + Azotobacter was the superior treatment for reducing the harmful impacts of salt stress on rapeseed plants, which resulted in 83–109% and 69–115% improvements in seed and oil yields of this crop under moderate and high salinities, respectively. Therefore, inoculation of biochar with both rhizobacteria might be a novel approach for improving salt tolerance and productivity of rapeseed plants.


Introduction
Rapeseed (Brassica napus L.) is a polyploid plant and one of the most important oil seed crops with high dietary value. Its cultivation is increasingly growing worldwide because of its high protein content (Gacek et al. 2018) and healthy fatty acid composition including high oleic acid and low linolenic acid contents (Spasibionek et al. 2020) in seeds. The preserved fatty acid profile and bioactive compounds of rapeseed oil have the health benefits such as regulating blood lipid, insulin sensitivity, glycemic control and antioxidant and cytotoxic activity (Chew 2020). Rapeseed is categorized as moderately salt tolerant (Ashraf and McNeilly 2004). However, the productivity of this plant may be limited at high salinity levels. Salt stress triggers inhibition in plant growth and development due to high absorption of sodium ions (Na + ). The increment of Na + accumulation in plant tissues reduces the uptake of some essential nutrients including potassium (K + ), calcium (Ca 2+ ), magnesium (Mg 2+ ), nitrogen (N) and phosphorus (P) (Ghassemi-Golezani and Abdoli 2021), leading to a decline in K + /Na + and Ca 2+ /Na + ratios and thus ionic toxicity and nutrient imbalance of saltstressed plants (Forni et al. 2017). The Na + toxicity could also lead to oxidative damages in plant cells. Generation of reactive oxygen species (ROS) in plant tissues under salt stress may cause massive depletion of cytosolic K + in plant roots and endanger cells survival. Some plants may remove a certain amount of ROS generated by moderate salt stress, but under high salinity, there is often an imbalance between generation and scavenging of ROS due to excess production of ROS, which causes peroxidation of lipids and membrane damage. The enzymatic and non-enzymatic components of an antioxidant defense system protect plant cells against ROS and decrease oxidative damage caused by stress (Abdoli et al. 2020;Abdoli and Ghassemi-Golezani 2021). In addition, ionic and oxidative stresses induced by salinity disrupt different growth aspects of plants, particularly structure and anatomy of roots, leading to a reduction in biomass and seed yield .
Various organic amendments such as biochar have been established to decrease the toxic effects of salinity in plants ( . The waste woods are pyrolyzed under anaerobic conditions to produce a carbon-rich material, which is commonly referred as biochar (Busch et al. 2015). This useful material has a great effect on crop production and remediation of toxic ions ). Amendment of soil by biochar may be a promising method to enhance soil C storage and concurrently to increase crop productivity by altering the physical, chemical, and biological properties of soil (Farhangi-Abriz and Ghassemi-Golezani 2021). Biochar can be produced by a range of organic compounds through biotic and abiotic processes (Dong et al. 2017) that affect the bioavailability of nutrients (Budai et al. 2016). This is a nutrient source, which directly controls plant root nutrient acquisition and indirectly alters soil nutrient content (Farhangi-Abriz and Ghassemi-Golezani 2021). According to Akhtar et al. (2015), the addition of 5% (w/w) biochar to a soil reduced Na + but enhanced K + in wheat (Triticum aestivum) leaves, thus lowering the ratio of Na + /K + . A biochar with appropriate physicochemical properties can be used to alter soil pH, organic matter, cation exchange capacity (CEC), and fertility . The root growth of maize plants was also remarkably improved by biochar amendments (Liu et al. 2021).
Inoculation of plant growth promoting rhizobacteria (PGPRs) is another alternative to organic amendments for the alleviation of salt stress in crops (Chu et al. 2019). The useful salt-tolerant rhizobacteria have been reported to increase salt tolerance in plants through improving soil structure, altering root architecture, reducing sodium uptake, enhancing water and nutrient absorption, producing hormones, lowering adverse impacts of ethylene, and stimulating the expression of genes involved in defense system (Joseph et al. 2012;Etesami and Noori 2019). Some PGPRs can reduce ethylene production under stressful conditions (Zafar-Ul-Hye et al. 2018). The PGPRs inoculated salt-stressed plants can produce indole-3-acetic acid (IAA), which helps to increase root growth and water uptake (Forni et al. 2017;Tahir et al. 2019). Ion regulation by PGPRs through modulating the expression of HKT1 (K + transporter 1) enables plants to alleviate some of the detrimental impacts of salt stress (Zhang et al. 2008). The inoculation with ACC deaminase producing PGPRs improves the uptake of some nutrients that can induce the synthesis of chlorophyll (Zafar-Ul-Hye et al. 2018).
The Pseudomonas bacterium, as an important biofertilizer and PGPRs, can be applied to enhance organic matter decomposition and phosphate solubilization in soils, which promote plant defense against salt stress (Chu et al. 2019). Pseudomonas can solubilize insoluble phosphate (Otieno et al. 2015), which increases the availability of other micro-and macro-nutrients for plants. This bacterium inhibits stress-induced ethylene synthesis via activating the ACC deaminase and metabolizing 1-aminocyclopropane-1-carboxylic acid (Washington et al. 2016). Azotobacter is another aerobic rhizobacteria that fixes nitrogen and stimulates plant growth by nutrient supplementation and production of hormones such as auxins, gibberellins and cytokinins (Ahmad et al. 2008;Joseph et al. 2012). Two salt-tolerant strains of this bacterium mitigated salt stress by reducing Na + and enhancing K + uptake by maize plants (Rojas-Tapias et al. 2012).
The biochar can be applied with PGPRs to develop a sustainable system that enhances available nutrients to the growing plants as well as increases tolerance to environmental stresses. Addition of biochar-based rhizobacteria to the soil might be an effective new method to promote the efficiency of low-input systems under abiotic stresses such as salinity. Therefore, this research was performed for the first time to evaluate the possible roles of biochar-based Pseudomonas putida and Azotobacter chroococcum RS-106 on nutrient uptake, antioxidant capacity, root growth and productivity of salt subjected rapeseed plants.

Soil and biochar characteristics
The biochar was produced by pyrolysis of peach (Prunus persica L. Batsch) residues under the anoxic conditions at about 560°C (Qian et al. 2013). The soil and biochar samples were analyzed according to Carter and Gregorich (2008). The carbon, hydrogen, nitrogen and oxygen contents of soil and biochar were quantified by an elemental analyzer (Elementar group, Hanau, Germany), and other nutrients were measured by a flame photometer (Corning flame photometer, 410). A pH meter (Model: HI 99121, Hanna Instrument, USA) was used to determine the pH of soil and biochar, and the ammonium acetate method was applied to measure the CEC (Chapman 1965). The properties of soil and biochar are listed in Table 1.

The experimental design and treatments
A pot experiment was performed in a glass greenhouse (146 Wm −2 light intensity, about 13 h photoperiod, and day and night temperatures of 28°C and 23°C, respectively) of university of Tabriz, using a factorial arrangement with randomized complete block design in three replications. The bacterial strains (10 8 cfu mL −1 ) were provided by the Soil and Water Research Institute, Karaj, Iran. Initially, salt tolerant strains of rhizobacteria were mixed with biochar (100 mL Kg −1 ). The soil treatments were non-biochar (NB), biochar (B), biochar-based Pseudomonas putida , biochar-based Azotobacter chroococcum RS-106 (BA) and biochar enriched with both bacteria (BPA). The pots for non-biochar treatment were filled with 3 kg soil, and the pots for biochar-related treatments were filled with a mixture of 3 kg soil and 90 g biochar (30 g biochar per 1 Kg soil), using 45 pots for sowing and 5 unsown pots in general. Specific amounts of sodium chloride were dissolved in tap water (pH of 7.2) to provide different saline solutions (6 and 12 dS m −1 NaCl). Seeds of Brassica napus (10 seeds) were sown in each pot (45 pots) and immediately irrigated with tap water and saline solutions (0, 6 and 12 dS m −1 NaCl; as non-saline, moderate and high salinities, respectively) up to 100% field capacity (FC) according to the treatments. The water loss of substrates was regularly measured in unsown pots, and this loss was compensated by tap water in all pots during plant growth and development.
After establishment, seedlings were thinned to keep four plants per pot. Then, 10 g of the NPK fertilizer (Master 20-20-20-Valagro-Italy) was dissolved in a liter of tap water and 500 mL of that solution was added to each pot. The images of rapeseed plants at 58 days after sowing under high salinity for different biochar-related treatments are shown in a supplementary file. At early flowering, two plants from each pot were removed to measure physiological parameters. The remaining plants in each pot (two plants) were harvested at maturity, and seeds, shoots and roots were separated for laboratory measurements.

Ions analysis
The samples of dried roots and leaves were separately burned in an electric furnace at 560°C for 7 h and then digested in 10 mL of 1 N HCl at 25°C for 24 h. The sodium, potassium, calcium, and magnesium contents (mg g −1 dry weight) in the digested samples were measured by a flame photometer (Corning flame photometer, 410). The nitrogen and phosphorus contents were determined by Kjeldahl and yellow (Fan et al. 2016) methods, respectively.

O 2 •− , H 2 O 2 , and Malondialdehyde
The O 2 •− generation was measured according to Bai et al. (2015). The H 2 O 2 concentration was measured by following the method of Vijayaraghavareddy et al. (2017). The fresh root and leaf samples were separately homogenized in trichloroacetic acid (TCA) and subsequently were centrifuged at 12,000 × g for 15 min. 500 μl of the supernatant was then mixed with 500 μl of potassium phosphate buffer and 1 mL of potassium iodide. The absorbance was read at 390 nm, using a spectrophotometer (Dynamica, Halo DB-20 UV-Visible, the United Kingdom).
The method of Qin et al. (2018) was applied to determine the malondialdehyde (MDA) content (mmol g −1 FW). Initially, 0.5 g of root and leaf samples were homogenized in 5 mL of trichloroacetic acid (5%) and centrifuged at 1800 g at 25°C for 10 min. The upper layer was added to 2-thiobarbituric acid (TBA) in a tube and heated at 98°C for 10 min, and then it was cooled at 25°C. After centrifugation, the absorbance was recorded at 532 nm.

Enzymatic antioxidants
Antioxidant enzyme activities in plant tissues were assayed spectrophotometrically according to the method described by Qin et al. (2022). The homogenate samples of plant tissues (root and leaf) in sodium phosphate buffer were centrifuged at 12,000 × g for 10 min at 4°C. Then, the upper layer was collected for enzyme extraction. The nitro blue tetrazolium method was used to assay the activity of superoxide dismutase (SOD) as Ug −1 FW. The catalase (CAT) activity was estimated by recording the absorbance at 240 nm and expressed as Ug −1 FW. The peroxidase (POD) activity was measured by changes in absorbance at 470 nm for 210 s.

Non-enzymatic antioxidants
The method of Leng et al. (2017) was followed to determine total phenolics in rapeseed organs. The root and leaf samples were separately homogenized with 5 ml 85% methanol at about 25°C and centrifuged at 12,000 × g for 10 min. 100 μl of supernatant was diluted by adding 150 μl of distilled water and 1.25 ml of freshly prepared 50% Folin-Ciocalteu reagent. The extract was kept in darkness for 4 minutes at room temperature. 1 ml of Na 2 CO 3 solution was added, and the absorbance was read at 760 nm. The standard curve of Gallic acid was used to calculate total phenolics (as mg of Gallic acid equivalent per g leaf fresh weight). The aluminum chloride colorimetric method (Sembiring et al. 2018) was applied to measure flavonoids using the standard curve of quercetin.

Leaf area and chlorophyll content
A portable area meter (model ADC-AM 300 UK) was used to measure leaf area (LA) of a random plant from each pot as cm 2 at pod formation stage. The total chlorophyll content of the leaves was measured spectrophotometrically according to the method described by Arnon (1949).

Root and shoot growth and seed yield
At maturity, two remaining plants from each pot were removed, and the roots and shoots were separated by cutting from crown. The GiA Roots software (Benfey laboratory, Duke University, Durham, North Carolina, USA) was used to determine the root length and diameter at the final harvest (Galkovskyi et al. 2012). All the digital images were taken using a Canon EOS 750D digital camera with 18-135 mm IS STM (Canon, Japan). Eventually, the root samples were dried at 75°C for 48 h and weighed. The lengths of the shoots were also measured, and subsequently, they were dried at 75°C for 48 h and weighed. The seeds of the plants from each pot were then separated, and seed yield per plant was calculated.

Oil extraction
A sample of 0.5 g seed was ground, and oil percentage was determined using the Soxhlet apparatus and petroleum ether for 4 hours. After that, oil yield per plant was calculated as:

Statistical analysis
The data were analyzed by the MSTAT-C software (two-way ANOVA) after testing normality by Kolmogorov-Smirnov test. The means of data were compared by Duncan multiple range test at p≤ 0.05. All figures were drawn by Excel 2019. The correlations between traits were assessed by principal component analysis (PCA).

Soil pH and cation exchange capacity
Significant interaction between salinity and biochar-based rhizobacteria treatments was found for pH and cation exchange capacity of soil after plant harvesting. Increasing salt toxicity considerably decreased these parameters of soil. Application of biochar and biochar-based rhizobacteria, especially biochar-based Pseudomonas + Azotobacter, increased the soil pH and CEC under saline and non-saline conditions. This combined treatment in comparison with non-biochar treatment enhanced soil pH by about 4%, 6% and 7% and CEC by about 25%, 32% and 24% under 0, 6 and 12 dS m −1 NaCl, respectively. Differences between BP and BPA on soil CEC under 6 and 12 dS m −1 salinities were not statistically significant. However, the biochar + bacteria-related treatments were resulted in statistically similar soil pH under 12 dS m −1 NaCl ( Figure 1).

Ion contents of plants
The interaction of salinity × biochar-based rhizobacteria was significant for the ion contents of roots and leaves of rapeseed plants ( Table 2). The Na content in roots and leaves was generally enhanced, but K, Ca, Mg, N and P contents were reduced as a result of salinity increment. Biochar + bacteria treatments had no significant effects on root and leaf Na, Ca, Mg, root K and leaf N under non-saline condition. However, leaf K, root N and root and leaf P were increased in plants grown in biochar treated soils under non-saline condition. The biochar-based bacteria, particularly both bacteria, significantly reduced the Na content of roots and leaves and enhanced essential nutrients under saline conditions. The BPA treatment resulted in a decrement of root Na by about 27% and leaf Na by about 50% under high salinity. Nevertheless, the improvement of K, Ca, Mg, N and P in plant tissues by this treatment under moderate salinity was about 75%, 37%, 34%, 72% and 78%, respectively. However, the BPA treatment enhanced K, Ca, Mg, N and P in plant tissues by about 73%, 85%, 68%, 76% and 130% under moderate salinity, respectively. Differences between root and leaf nutrients (except leaf N) in BP and BPA treatments under high salinity were statistically similar. The BA treatment in comparison with BP treatment significantly increased root and leaf N under moderate salinity (Table 2).

ROS generation and lipid peroxidation
The interaction of salinity × biochar enriched bacteria was significant for ROS generation and lipid peroxidation in roots and leaves of rapeseed. Increasing salinity led to an increment in O 2 •− and H 2 O 2 generation and lipid peroxidation. The biochar-inoculated bacteria did not show a significant effect on ROS generation and lipid peroxidation in roots and leaves under non-saline condition. Nevertheless, ROS generation and lipid peroxidation were considerably reduced by biochar-related treatments under moderate and high salinities (   Figure 2).

Enzymatic and non-enzymatic antioxidants
Significant interaction between salinity and biochar-based rhizobacteria treatments was found for the CAT, SOD, POX activities, and phenolic and flavonoid contents in rapeseed roots and leaves (p ≤ 0.01). Rising salinity noticeably enhanced antioxidant activity of roots and leaves. The biocharand bacteria-related treatments had no significant effects on enzymatic and non-enzymatic antioxidants in rapeseed roots and leaves under non-saline condition. Biochar and biochar-inoculated rhizobacteria reduced enzymatic and enhanced non-enzymatic antioxidant activities of plants. The plants grown in biochar inoculated by Pseudomonas + Azotobacter had the highest non-enzymatic antioxidant activities under saline conditions. The BPA in comparison with NB increased nonenzymatic antioxidants by 53.5% in roots and 45.5% in leaves of rapeseed plants. The phenolic contents were enhanced by biochar inoculated with both bacteria by about 26% and 79% under moderate and high salinities, respectively. However, the increment of flavonoid contents in plants grown under this treatment was about 22% and 72% under 6 and 12 dS m −1 NaCl, respectively (Table 3).

Root growth
The interaction of salinity × biochar-related treatments was significant for the length and weight of main and lateral roots, main/lateral root length ratio, specific root length and root diameter (Table 4). These parameters were decreased with increment of salinity. Application of different biochar and biochar-based bacterial treatments increased root parameters (except main/lateral root length ratio of unstressed plants) under all salinity levels. The biochar-based rhizobacteria treatments enhanced main/lateral root length ratio under saline conditions, particularly under high salinity (Table 4; Supplemental Figure). The superiority of biochar-based Pseudomonas + Azotobacter on growth parameters was more than the individual bacterial treatments, especially under saline conditions. The main root length, main and lateral root weights, specific root length and root diameter under moderate salinity and the main/lateral root length ratio under high salinity were similarly affected by bacteria related treatments. Differences between BPA and BP treatments in main and lateral root lengths and weights, specific root length and root diameter under moderate and high salinities were not statistically significant (Table 4).
The biochar-based Pseudomonas + Azotobacter enhanced the length of the main root by 5%, 53% and 134% and the length of the lateral roots by 18%, 44% and 88% under non-salinity, and 6 and 12 dS m −1 NaCl salinities, respectively. The BPA treatment increased the main root weight by 21% and 49% under moderate and high salinities, respectively, and the lateral root weight by 15%, 22% and 59% under non-saline, 6 and 12 dS m −1 NaCl, respectively. The improvement of main/lateral root length ratio, specific root length and root diameter due to BPA treatment under high salinity was about 20%, 35% and 38%, respectively.

Principal component analysis (PCA)
The PCA was used to assess the relations among nutrients, antioxidants, root and shoot lengths and weights and yield parameters of rapeseed plants. The PCAs were contributed up to 92% of variance in leaf and root traits (Figure 3). The Na content, enzymatic and non-enzymatic antioxidants and ROS were inversely related, but the K, Ca, Mg, N and P contents, soil pH and CEC were directly related to root, shoot and yield parameters. A strong positive correlation between phenolics and flavonoids was found. The antioxidants were also highly related to Na, ROS and MDA. The soil pH and CEC were more effective in enhancing lateral root growth than the main root. However, the main root length, weight and main/lateral root lengths had stronger positive correlation with the K, Ca, Mg, N and P than soil properties. Increasing nutrient contents and improving soil properties stimulated the growth and yield of plants ( Figure 3).

Discussion
Increasing Na toxicity and decreasing mineral nutrient uptake (Table 2) were the result of reduction in cation exchange capacity and pH of saline soils (Figure 1), which disrupted membrane permeability of root cells, leading to nutrient imbalance. Excessive Na content in the plant tissues can potentially enhance the generation of ROS including O 2 •− and H 2 O 2 . This causes lipid peroxidation (Figures 2 and 3) and induces the SOD, CAT and POX activities and phenolic and flavonoid syntheses (  (Ghorbanpour 2015). Flavonoids can protect plants from salt-induced oxidative stress by inhibition of lipoxygenase enzyme, which converts polyunsaturated fatty acids into oxygen-containing derivatives (Nijveldt et al. 2001). Ionic and oxidative stresses due to salinity reduced leaf growth and total chlorophyll content (Table 5), leading to a reduction in root parameters (Table 4), and seed and oil yields ( Table 5). Declining of CEC and pH of soil ( Figure 1) and nutrients, especially Ca, Mg and P contents of plants (Table 2), under saline condition negatively affected root growth (Supplemental Figure). It was reported (Koch et al. 2020) that interruption of the Mg translocation may result in an impaired photo assimilate partitioning and finally less root growth. The P deficiency may induce abscisic acid synthesis that can mediate the changes in root architecture (Vysotskaya et al. 2020). This reduction can also be caused by elevation of ethylene synthesis under salt stress (Qin et al. 2019).
Our results clearly proved the hypothesis that the addition of biochar with rhizobacteria to the soil improves salt tolerance, root and shoot morphology and physiological performance of rapeseed plants. Increasing nutrient uptake and translocation by the plants under biochar and biochar-based rhizobacteria treatments, especially under biochar inoculated with Pseudomonas + Azotobacter led to a reduction in Na content of plant tissues (Table 2). Biochar and particularly biochar-based rhizobacteria decreased sodium but increased essential nutrient availability to plants via enhancing cation exchange capacity ( Figure 1) and nutrient bioavailability in soil ( Table 2). The major role of biochar in ameliorating plant toxicity is related to its high adsorption capacity, which limits the uptake of toxic ions by the plants (Medyńska-Juraszek et al. 2020. Similarly, Ghassemi-Golezani and Farhangi-Abriz (2021) stated that modified biochar significantly enhances cation exchange capacity but reduces exchangeable sodium percentage of soil. Pseudomonas and Azotobacter can enrich soil with P and N by solubilizing insoluble phosphate (Otieno et al. 2015) and producing some hormones (Joseph et al. 2012), respectively. The exopolysaccharides produced by Pseudomonas sp. AK-1 were able to bind free sodium from the soil, making Na inaccessible to plants and maintaining normal plant growth up to 200 mM NaCl (Kasotia et al. 2016).
The augmentation of antioxidant capacity (enzymatic and non-enzymatic) is a key mechanism for scavenging reactive oxygen species under salt toxicity. This can be stimulated by biochar-based Pseudomonas + Azotobacter (Table 3, Figure 2) via reducing Na and enhancing K, N and P uptakes (Table 2) and increasing non-enzymatic antioxidants such as phenolics and flavonoids (Table 3). However, reduced enzymatic antioxidants in inoculated plants (Table 3) could be attributed to the transcriptional changes. Inoculation of plants with Pseudomonas PS01 up-regulated LOX2 and downregulated APX2 and GLYI7 genes under salt stress (Chu et al. 2019). It was also reported that salt- tolerant PGPR strain IG3 modulates the expression profile of rbcL (codes for the Rubisco) and WRKY1 (a transcription factor involved in plant response to biotic stress) genes in salt subjected oat plants (Sapre et al. 2018).
The positive effects of biochar-related treatments and particularly biochar inoculated with Pseudomonas + Azotobacter on shoot growth under salinity were mainly attributed to augmented main and lateral root elongation, lateral root distribution (Table 4, Supplemental Figure), nutrient bioavailability (particularly magnesium, nitrogen and phosphorus contents) ( Table 2) and nonenzymatic antioxidants (Table 3). The results showed that the enhanced plant productivity was achieved through the expansion of root system by biochar + bacteria related treatments (Supplemental Figure, Table 4, Figure 4). Enhancing the main and lateral root length, main/lateral root length ratio and specific root length by these treatments (Table 4) led to an increment in the amplitude of nutrient absorption in rhizosphere by root cells (Table 2). Improving the main/lateral root length of all plants in biochar + bacteria -related treatments under saline conditions, especially under high salinity (Table 4, Supplemental Figure), indicates the critical roles of main roots in plant survival under high salinity. This change in root architecture (Supplemental Figure) can enhance nutrient uptake (Table 2) and crop performance under saline conditions. Production of ACC deaminase, an enzyme involved in decreasing the stress-induced ethylene synthesis in the root tissues, by rhizobacteria (Glick 2014) could also improve root growth. The biochar + bacteria-related treatments enhanced leaf area and chlorophyll content by improving nutrient uptake (Table 2) and antioxidant capacity (Table 3). Similarly,  found that addition of biochar and biochar-based nanocomposite to the soil reduced sodium uptake and enhanced chlorophyll content index and biomass of safflower plants under salinity. The increment of root and leaf growth and chlorophyll content by biochar-based Pseudomonas + Azotobacter resulted in a considerable improvement of shoot growth and seed and oil yields of rapeseed (Table 5, Figure 3).

Conclusions
Increased Na content, ROS generation and lipid peroxidation, and decreased mineral nutrients, leaf growth and chlorophyll content reduced growth and productivity of salt-stressed rapeseed plants. However, biochar and particularly biochar inoculated with rhizobacteria improved root and shoot growth, total chlorophyll content, seed production and oil yield of plants via alleviation of ionic and oxidative stresses by decreasing Na uptake, O 2 •− , H 2 O 2 and MDA contents and increasing root and leaf nutrients, and stimulating oxidative defense system by non-enzymatic antioxidants. These beneficial effects were more pronounced in plants grown under biochar-based Pseudomonas + Azotobacter. The increment of nutrient absorption under biochar + bacteria related treatments was strongly attributed to the enhanced root elongation and lateral root distribution. The root growth and distribution showed a strong correlation with pH and CEC of the soil and nutrient contents of plants based on principal component analysis. These results suggest that incorporation of biochar with rhizobacteria, especially with Pseudomonas + Azotobacter, can improve crop productivity under salinity via mitigating ionic and oxidative stresses and enhancing nutrients, antioxidant capacity, root and shoot growth and chlorophyll content.