Seed Biostimulant MGW9 (SB-MGW9) Biopriming Improves Salt Tolerance during Maize Seed Germination


 Crop performance is seriously affected by high salt concentrations in soils. To develop more new seed pre-sowing treatment technologies it is crucial to improve the salt tolerance of seed germination. Here we isolated and identified the strain Bacillus sp. MGW9 and developed the seed biostimulant MGW9 (SB-MGW9) by the strain. Effect of seed biopriming with SB-MGW9 in maize (Zea mays L.) under saline condition were studied. The results showed that the Bacillus sp. MGW9 has the characteristics of salt tolerance, nitrogen fixation, phosphorus dissolution, indole-3-acetic acid production and the like. Seed biopriming with SB-MGW9 enhanced the performance of maize during seed germination under salinity stress to improve the germination energy, germination percentage, shoot/seedling length, primary root length, shoot/seedling fresh weight, shoot/seedling dry weight, root fresh weight and root dry weight. SB-MGW9 biopriming also alleviates the salinity damage to maize by improving relative water content, chlorophyll content, proline content, soluble sugar content, root activity, activities of superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, decreasing the malondialdehyde content. Especially, the field seedling emergence of maize seeds in saline-alkali soil can be improved by SB-MGW9 biopriming. Therefore, maize seed biopriming with SB-MGW9 can be an effective approach to resist the inhibitory effects of salinity stress and promote seed germination and seedling growth.


Introduction
Soil salinization is an increasingly serious agricultural problem in the world. It seriously affects the growth and development of crops, resulting in serious loss of productivity. As a result of poor irrigation water, over-fertilization and deserti cation processes, cultivated soils around the world have become more saline and alkaline. More than 800 million hectares of land worldwide are currently affected by salt stress (Ramadoss et al. 2013). Maize (Zea mays L.) is an important global cereal crop whose production needs to be increased to meet the food needs of a growing world population (Tilman et al. 2011).
Nevertheless, the growth of maize and grain quality can be severely affected by salinity, drought, high temperature and other adverse environmental conditions (Sabagh et al. 2020). Salt stress is emerging as a particular constraint to global crop production, and it is estimated that it will affect about 20% of the world's irrigated land and will lead to a loss of up to 50% of the land by the middle of the twenty-rst century (Mahajan and Tuteja 2005;Zhu 2001). Sodium chloride (NaCl), as the main form of soil salinity, can lead to crop yield reduction or even death by making root water uptake more di cult, and lead to plant poisoning by accumulating high concentrations of Na + and Cl − in plants (Deinlein et  Various methodologies are in vogue to develop stress-tolerant varieties, either through conventional breeding or through transgenic technology. Alternatively, however, simpler and more economical practices are competing to solve this problem. Seed priming is a farmer-friendly technique recommended by many researchers for better establishment and growth even under adverse conditions (Filippou et al. 2013a; dos Santos Araújo 2021). It is well known that different environmental stresses often activate similar cell signaling pathways and cellular responses, and seed priming can activate these signaling pathways early in growth and lead to faster plant defense responses. Different seed priming methods employed to mitigate stress tolerance as reported by many researchers are main as follows: hydropriming, halo-and osmopriming, matrix priming, thermopriming, biopriming, drum priming, priming using growth regulators, nutrient priming and redox priming.
In recent years, microorganism and its engineering technology play an important role in helping plants to resist abiotic stress and improving crop yield and quality, which has become an effective way to alleviate plant growth stress and has broad application prospects (de Vries et al. 2020; Mahanty et al. 2016; Venkateswarlu et al. 2008). In particular, the use of some microbial agents such as Azospirillum, Bacillus, Gliocladium, Pseudomonas, Rhizobium, Trichoderma, and other biopriming treatment on seeds to improve seed viability or vigour is very worthy of further study. Some progress has been made in seed biopriming, and the growth-promoting ability of microorganisms may be highly speci c to certain plant species, cultivars and genotypes (Bashan 1998; Moeinzadeh et al. 2011; dos Santos Araújo 2021). In order to reduce the toxic effects of high salt on plant growth, some plant growth promoting bacterias (PGPBs) have been developed to improve the salt tolerance of plants (Ali et al. 2014). PGPBs are considered to be microorganisms that can grow in, on or around plant tissues, stimulating plant growth by a variety of mechanisms, such as synthesis of phytohormones, xation of non-symbiotic nitrogen, dissolution of inorganic phosphate and mineralization of organic phosphate and/or other nutrients, and antagonism against phytopathogenic microorganisms (Esitken et al. 2010).
Shahid et al. (2011) observed that treatment of seeds with T. viride improved germination and vigour of chickpea. Under salt stress, Bacillus subtilis and Pseudomonas uorescens could signi cantly increase the fresh weight, dry weight, photosynthetic pigments, proline, total free amino acids and crude protein content of radish roots and leaves (Mohamed and Gomaa 2012). Under 320mM NaCl stress, the root elongation and dry weight of Hallobacillus sp. SL3 and Bacillus halodenitri cans PU62 were increased by more than 90% and 17.4%, respectively, compared with those of uninoculated wheat seedlings (Ramadoss et al. 2013). Many research ndings suggest that that seed biopriming with different bene cial microorganisms can not only improve seed quality, but also improve seedling vigour and resistance to abiotic and biotic stresses, thus providing an innovative crop protection tool for sustainable improvement of crop yields.
In recent years, a kind of agricultural product called plant biological stimulants can help crops resist abiotic stress, which has attracted much attention (Akhtar et al. 2008;Hamel and Plenchette 2007;Harrier and Watson 2004;van der Heijden et al. 2004). By de nition, a plant biostimulant is any substance or microorganism applied to plants with the aim to enhance nutrition e ciency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content (du Jardin 2015). There is very little research using seed biostimulant to improve the germination and emergence ability of crop seeds under abiotic and biotic stresses. Our purpose was to isolate and identi cate the salt-tolerant bene cial strains for the development of seed biostimulant, and to study the effects of bio-priming on seed germination and seedling emergence of different maize varieties under salt stress by pre-sowing treatment with the probiotics as bio-initiators, so as to provide a basis for the research on improving seed quality.

Materials And Methods
Isolation and cultivation of strains Bacterial strains were isolated from the extremely arid soil samples near the Great Wall of Ming Dynasty in Shandan County of Gansu Province (100.88E, 38.84N). Soil samples were taken and were placed in sterile sealed bags and stored at -20 ℃ in August 2017. The soil sample is diluted by 10-fold gradient dilution method. Weigh 20 g of soil sample, mix it well and grind it, pour it into a triangular ask containing 80mL of sterile water, shake it well and mix it well, put it into a triangular ask, add 80mL of sterile water, take the supernatant and dilute it into soil suspension with the concentration of 10 −3 , 10 −4 , 10 −5 and 10 −6 . The strain was isolated by dilution-spreading plate method, and 200µL of diluent was spread on beef extract peptone agar medium containing 10 different concentrations of NaCl (5,7,8,9,10,11,12,13,14 and 15% (w/v)) (agar 15-25 g per litre, peptone 10.0 g, sodium chloride 5.0g, beef extract 3.0g, pH 7.4-7.6) and six replicates per concentration. After 6 to 7 days of culture at 28 ℃, the colonies with different morphological characteristics were selected on the plate, and the single colony was puri ed on the beef extract peptone agar plate by plate streaking method. The candidate bacterial strains were numbered and maintained in strain preservation tube with 25% (V/V) glycerol and stored at -80 ℃. In this work, we selected candidate bacterial strain MGW9 for further study as follows.

Screening for salt-tolerance level and growth promoting characteristics of strain MGW9
To a ask containing 50 mL of nutrient broth, NaCl was added to give a nal salt concentration of 5,7,8,9,10,11,12,13,14 and 15% (w/V). The strain MGW9 of active growth were then added to each ask and incubated on a rotating shaker at 30 ℃ with 180 rpm. Bacterial growth was determined as OD 600nm to determine salt tolerance again.
The strain MGW9 was streaked and inoculated into a nitrogen-free culture medium containing 0-15% of NaCl, cultured in a dark incubator at 30 °C for 4-6 days, the growth condition of the strain MGW9 was observed, and whether the strain had the nitrogen-xing capacity was detected according to the presence or absence of colonies on a plate, and each NaCl concentration is repeated for three times.
The strain MGW9 was placed on bacterial inorganic and organic phosphorus media containing 0-15% NaCl and incubated at 30 ℃ for 7 days, respectively, and the strain was observed for the presence of transparent circles, i.e phosphate-solubilizing circles, with three replicates per NaCl concentration. If a clear area appears around the colony indicating that it has the property of dissolving phosphate. The diameter of phosphate-solubilizing zone (D) and colony diameter (d) were measured, and the phosphatesolubilizing ability of strain MGW9 was qualitatively tested by D/d. According to the method described by Li et al. (2019b), the phosphate solubilization index (PSI) = (colony diameter + halo zone diameter)/colony diameter.
According to the method of indole-3-acetic acid (IAA) production test described by Li and Jiang (2017), King's B medium containing 100 mg ml -1 L-tryptophan and 0-15% NaCl was used to screen for IAA production. The culture supernatant of the candidate strain was mixed with Salkowski reagent at a ratio of 1:1 (V: V -1 ). The pink mixture indicates the generation of IAA and its density was recorded at OD 530nm .
The concentration of IAA produced was estimated from a standard curve of IAA in the range of 0-100 μg mL −1 .
Note: according to the salt-tolerance level of strain MGW9, we choose the maximum salt concentration to study its growth-promoting characteristics.

Identi cation of strain MGW9
The strain MGW9 was cultured on beef extract peptone agar medium at 28 ℃ for 48 h with 200 rmp with three replicates, and then its morphological characteristics were observed by microscope. Genomic DNA of the strain MGW9 was extracted by bacterial genomic DNA rapid isolation kit (Sangon Biotech (Shanghai) Co., Ltd., China), and identi ed according to the complete 16s rDNA sequence. The 16s rDNA was amplify by PCR using a forward 27F primer (5'-AGAGTTTGATCCTGGCTCAG-3') and the reverse 1492R primer (5'-GGTTACCTTGTTACGACTT-3'). The sequencing of PCR reaction products was completed by Qingdao Pacino Gene Biotechnology Co., Ltd. Sequence homology of nucleotides was compared using the blast search program. The tightly related sequences were aligned by Clustalx using MEGA version 5.1 software package, and the phylogenetic tree was constructed by Neighbor Joining (NJ) method. The bootstrap replications (1000) were used as statistical support for nodes in the phylogenetic tree.
The 16S rRNA gene sequence of strain MGW9 was deposited in the NCBI GenBank under accession number MW663489.
Seed priming using the seed biostimulant MGW9 (SB-MGW9) Seeds of hybrid maize 'Zhongdi 175' (ZD175), 'Zhengdan 958' (ZD958) and 'Denghai 605' (DH605) were used. Pure maize seeds were randomly selected from each sample for the following experiments. i) Thousand-seed weight (TSW) test: the TSW was measured using 500 seeds in each of the three replicates and then converted to thousand seed weight (Li et al. 2019a).
ii) Seed moisture content (SMC) test: the seeds were ground and dried at 130 ± 0.5 ℃ for 4 hours (h) and the moisture content basis was calculated from the fresh weight (ISTA 2007).
iii) Seed water absorption test: 100 maize seeds of the three maize varieties were measured the initial weight, then soaked in sterile water, taken out every 2 h, wiping off oating water on the surfaces of the seeds, measuring the weight and calculating the water absorption of the seeds at different time points. And the water absorption characteristic equation of maize seed was obtained by curve tting analysis of the average water absorption of three maize seed samples at each time point. iv) Preparing SB-MGW9: the strain MGW9 was inoculated into a beef extract peptone liquid culture medium and cultured in a fermentation tank at the stirring speed of 150 r/minute (min), the culture temperature of 28 ℃ and the ventilation rate of 1.5 L/min for 48-60 h, and the number of the MGW9 is adjusted to be 1.0×10 8 -1.5×10 8 cfu/mL, and the pH value of the bacterial liquid is adjusted to be 7.0-8.0. v) Seed priming with SB-MGW9: two-factor randomized block design was used in the experiment.
Soaking time for the factor A, set two different time: 3 and 6 h. Moisturizing time was factor B, which was divided into two different time: 12 and 24 h. After treatment, the primed seeds were air dried at 25 ℃ to near their original moisture contents. Factors A and B were randomly divided into 4 treatments, and the untreated group (no priming) was used as control (C). Treatment 1 (T1) means the seeds soak for 3 h, and moisturize for 12 h; Treatment 2 (T2) means the seeds soak for 3 h, and moisturize for 24 h; Treatment 3 (T3) means the seeds soak for 6 h, and moisturize for 12 h; Treatment 4 (T4) means the seeds soak for 6 h, and moisturize for 24 h.
Germination and seedling growth test The pure seeds are randomly selected for standard germination test. The seed surface was sterilized with 1% NaClO (w/v, Beijing Chemical Reagent Company, Beijing, China) for 10 minutes, then washed three times with distilled water and air dried for use. The seeds are germinated by adopting a rolling paper germination. Firstly, two pieces of germinating paper (Anchor Paper Co., St Paul, MN, USA) are stacked and moistened by 100 mmol/L NaCl solution, and the redundant water on the paper is removed by a towel; Secondly, the primed seeds are alternately placed on a germination paper bed, the directions of the seed holes are consistent, the paper bed is rolled up and placed into a self-sealing bag, the seed hole ends are vertically placed into a Versatile Environmental Test Chamber (MGC-350HP, Shanghai Yiheng Technology Instrument Co., Ltd., Shanghai, China) with three replicates of 100 seeds and incubated at 25 ± 0.5 ℃ and on an illumination cycle of 12 hours of light and 12 hours of darkness. Each treatment was repeated 3 times, with 100 seeds per repetition (Jiang et al. 2016). The no priming seeds were used as control. The germination energy (GE) and germination percentage (GP) were measured on the 4th and 7th day after the experiment was established. GP is the normal seedling number on the 7th day after seed planting (Li et al. 2019a). While counting the GP, 10 seedlings with uniform size were randomly selected to measure six indices, including shoot/seedling length (SL), primary root length (PRL), shoot/seedling fresh weight (SFW), shoot/seedling dry weight (SDW), root fresh weight (RFW) and root dry weight (RDW). For SDW and RDW, the plant tissue (shoot/seedling or root) were dried at 105 ± 0.5 ℃ for 8 h.

Assay for biochemical index
After passing through a 2 mm sieve, the sand was sterilized and placed in plastic pots (volume 150 ml) of 100 g of sterilized sand per pot. The content of deionized water in the sterilized sand is 10% (V: W).
Salt treatment was carried out by supplementing deionized water with NaCl at a nal concentration of 100 mM. After the primed seeds are placed in the sand bed, the pot is sealed with transparent preservative lm. Seeds not primed were used as control. There were three replications for each treatment and 15 seedlings per replication. The relative water content (RWC) of the leaf samples was determined, expressed as a percentage, referring to the method of Ghahfarokhi et al. (2015). The chlorophyll content was measured by SPAD502 Plus meter. The level of lipid peroxidation was determined by the content of malondialdehyde (MDA), the content of proline was determined by extraction with 3% 5-sulfosalicylic acid at room temperature, and the content of soluble sugar was measured by anthrone-sulfuric acid method (Zhu et al. 2010). The root activity was determined by triphenyltetrazolium chloride (TTC) method (Li et al. 2015). Approximately 500 mg of a fresh leaf sample was homogenize in 10 ml of a 0. Field seedling emergence test For field seedling emergence (FSE), the samples were sown at the saline-alkali land in Binzhou (soil salt content is 0.61%, pH is 7.72), Dongying (soil salt content is 0.54%, pH is 7.63), and Weifang (soil salt content is 0.56%, pH is 7.57) experimental base, Shandong, China, in 2020. In this study, the row spacing, row spacing and line length were 0.06, 0.06 and 0.60 m, respectively. The seeds were sown using the single seed sowing method with 10 seeds per row and 10 rows per repetition (continuous row, 100 seeds) in three repetitions. The arrangement of seeds was designed by the method of partition comparison. In June, the eld emergence test was completed. FSE was measured at three-leaf stage of maize.

Statistical analysis
Data were analyzed by a one-way analysis of variance (ANOVA) using the SAS statistical software package (SAS Institute, 1999), followed by the calculation of the lowest significant differences (LSD).
The data was used to make a comparison and statistical analysis by SPSS 11.0, and Excel. The work was completed in the Seed Science and Engineering Laboratory of Qingdao Agricultural University from March to September 2020.

Isolation, identi cation and characteristics of strain MGW9
According to the morphological characteristics, 19 strains of salt-tolerant bacteria were isolated from soil samples. Among all the isolated strains, the strain MGW9 not only has the salt tolerance of 12% NaCl, but also has good nitrogen xation, phosphorus dissolution and indole-3-acetic acid (IAA) production performance. The characteristics of the strain MGW9 cultured under the condition of 12% NaCl mainly include the following three aspects: i ) Nitrogen xation: the strain MGW9 was streaked on nitrogen-free medium and cultured in dark incubator at 30 ℃ for 4-6 days, and MGW9 colonies were found on the plate; ii) Phosphorus dissolution: the transparent circle of MGW9 could be observed on the 3rd day after inoculation in organic phosphate and inorganic phosphate medium, and the size of transparent circle tended to be stable until the 7th day. When the transparent zone was stable, the ratio of the diameter of dissolving phosphorus zone (D) to the diameter of bacterial colony (d) was 1.7 in organic phosphorus medium, and 1.95 in inorganic phosphorus medium; iii) IAA production: the standard curve equation of IAA concentration and absorbance change was Y = 0.025X + 0.001 (R 2 = 0.985, Y represents absorbance, X represents IAA concentration). IAA production of the strain MGW9 in the king's B medium was 19.24 mg/L.
Blast search and phylogenetic analysis at the National Center for Biotechnology Information (NCBI) of the United States showed that the strain MGW9 had 99.0% sequence homology with Bacteria WSB-1 (KJ950500.1). Based on its morphology including gram-positive staining, the cells are rod-shaped and 16s rDNA genetic sequence (1424bp), the strain was identi ed as Bacillus sp. MGW9. The strain MGW9 has been preserved in the China General Microbiological Culture Collection Center on November 6, 2019; CGMCC No. 18690; The test result of the collection center is that the strain MGW9 is alive and is recommended to be classi ed as Bacillus sp. (Fig. 1). The thousand seed weight (TSW) and seed moisture content (SMC) of seed samples from three maize varieties were 342.7-361.3g and 11.3-11.8% (lower than the safe water content 13%), respectively. The water absorption curve equation of maize seed was Y = K(6.519X − 0.224X 2 + 2.879), K is coe cient of variation. In the seed imbibition stage, the water absorption rate of the three varieties of seed samples showed a trend of rst fast and then slow change, and the change became stable after 12 hours (Fig. 2).

Figure 2 here
Effects of seed biopriming with SB-MGW9 on maize seed germination under normal and saline conditions Compared to normal condition, the germination energy (GE) and germination percentage (GP) of three maize varieties sample seeds were signi cantly decreased under salt stress, the GE of ZD175, ZD958 and DH605 decreased by 5%, 20.5% and 14.5%, respectively, and the GP of ZD175, ZD958 and DH605 decreased by 10%, 11.6% and 8.5%, respectively. Compared with the control, the GE and GP of seeds after priming treatments were higher than the control, and the priming effect was different under different germination environment. In the normal germination environment, except ZD958, the GE and GP of ZD175 and DH605 sample seeds after biopriming were not signi cantly different from the control (P < 0.05). And there was no signi cant difference in the GE among different priming treatments of the same variety, and the GP was the same (Fig. 3a, b). This may be related to the fact that the priming effect was not obvious when the initial level of seed vigour of sample was high. Under salinity stress, the GE and GP of seeds after priming treatments were signi cantly higher than that of the control (P < 0.05) except the GP of ZD175-T1, and the priming effects of different priming treatments were different (Fig. 3c, d).
Comprehensively analyzing the GE and GP of different priming treatments, the results showed that T3 had the best seed biopriming effect (Fig. 3).

Figure 3 here
Effects of seed biopriming with SB-MGW9 on maize seedling growth under salinity stress condition Under salinity stress, the six seedling growth indices after seed biopriming were higher than the control. The suitable seed biopriming treatment with SB-MGW9 was different for different varieties. The suitable treatments of ZD175 were T2 and T3, and the suitable treatments of ZD958 were T3 and T4, and DH605 were T2 and T3 (Table 1). Comprehensive consideration of the six indices of seedling growth, the most suitable treatment for the three maize varieties was T3. Compared with the control, the shoot/seedling length (SL), primary root length (PRL), shoot/seedling fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW), root dry weight (RDW) of ZD175 increased by 49  According to Duncan's multiple range test, different letters in the same column indicate signi cant differences between treatments at the 0.05 level. SL = shoot/seedling length; PRL = primary root length, SFW = shoot/seedling fresh weight, RFW = root fresh weight, SDW = shoot dry weight; RDW = root dry weight; S = seedling (s).
Effects of seed biopriming with SB-MGW9 on the RWC, Chl content, MDA content, proline content, soluble sugar content and root activity of maize seedlings under salinity stress Under salinity stress, compared with the control, the relative water content (RWC), chlorophyll (Chl) content, proline content, soluble sugar content and root activity of the seedlings of the three maize varieties after seed biopriming were signi cantly increased except for the malondialdehyde (MDA) content (P < 0.05). By comparing and analyzing the data of six biochemical indices, it can be seen that different seed biopriming treatments have different seed biopriming effects. According to the content of MDA, the suitable seed biopriming treatments were T3 and T4, but there was no signi cant difference between T3 and T4. And from the other ve indices, the suitable seed biopriming treatments for the three maize varieties were T2 and T3, and there was also no signi cant difference between T2 and T3. Comprehensive consideration of the six biochemical indices data, the suitable seed biopriming treatment for the three maize varieties was T3. Compared with the control, the RWC, chlorophyll, proline, soluble sugar content and root activity of ZD175-T3 increased by 9.1%, 12.3%, 49.1%, 37.2% and 25.0% respectively, and those of ZD958-T3 increased by 7.3%, 9.3%, 59.9%, 29.4% and 15.9% respectively, and those of DH605-T3 increased by 5.0%, 12.3%, 56.9%, 24.0% and 14.3% respectively. In addition, the MDA content in seedling of ZD175-T3, ZD958-T3 and DH605-T3 decreased by 32.2%, 24.3% and 29.4%, respectively (Fig. 4).

Figure 4 here
Effects of seed biopriming with SB-MGW9 on the SOD, CAT, POD and APX activities of maize seedlings under salinity stress Compared with the control, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) in maize seedlings increased signi cantly after seed biopriming with SB-MGW9 (P < 0.05). Comprehensive analysis of the four enzyme activity data, different seed biopriming treatments had different priming effects. The suitable seed biopriming treatments for the three maize varieties were T2 and T3, and there were no signi cant differences in the four enzyme activity indices between T2 and T3. The most suitable treatments for different maize varieties were T3 for ZD175 and DH605, and T2 for ZD958. Compared with the control, the SOD, CAT, POD and APX activities of ZD175-T3 were increased by 42.1%, 23.4%, 36.1% and 63.9% respectively; the SOD, CAT, POD and APX activities of ZD958-T2 were increased by 26.8%, 19.6%, 43.9% and 94.9% respectively; the SOD, CAT, POD and APX activities of DH605-T3 were increased by 47.0%, 20.7%, 33.5% and 27.2% respectively (Fig. 5).   (Fig. 6). A recent trend in sustainable development is the use of bene cial microorganisms to increase the nutrient use e ciency of eld crops without compromising soil health (Meena et al. 2017). Biopriming is an emerging and promising seed and/or seedling treatment tool for inducing systemic resistance to abiotic and biotic stresses in treated crop. It is a process of biological treatment of seeds refers to the process of combining seed hydration and inoculation with bene cial organisms to protect seeds (Rakshit et al. 2015). In most cases, microbial inoculants such as rhizospheric or endophytic microorganisms (bacteria Here, the objective of this study was to investigate the effects of SB-MGW9 biopriming on seed germination and seedling growth of maize under salt stress. Related reports show that some microorganisms can improve the growth performance of plants under stress environment by providing plant hormones, soluble phosphate, xed nitrogen, and other substances (Hayat et al. 2010;Ji et al. 2014), and the characteristics of the strain are similar to Bacillus sp. MGW9. Some researchers began to pay attention to the application of microorganisms in seed pre-sowing treatment because of the ability of bene cial microorganisms to inhibit diseases, better crop germination ability and vitality. We set up four seed biopriming treatments according to the water absorption characteristics of maize seeds (Fig. 2), including seed soaking time and moisturizing time. The results of germination test showed that the germination energy (GE) and germination percentage (GP) of three maize varieties under normal and salt stress conditions were increased after seed biopriming treatment (Fig. 3), but the GE and GP of ZD175 (T1, T2, T3 and T4) and the GP of DH605 (T1, T2, T3 and T4) were not signi cantly different from the control under normal condition (Fig. 3a). However, under saline condition, the GE and GP of T2, T3 and T4 of ZD175 and the four seed biopriming treatments of ZD958 and DH605 were signi cantly higher than that of the control (P < 0.05) (Fig. 3b) (Timperio et al. 2007). However, compared with non-bioprimed seeds, it was observed that suitable SB-MGW9 biopriming treatment could signi cantly increase the chlorophyll content of maize seedlings under salt stress (Fig. 4b).
Salt stress often induces the increase of reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2− ) and hydroxyl radical (·OH) in plants, resulting in oxidative damage to plants (Hyodo et al., 2017). Malondialdehyde (MDA) is a kind of lipid peroxidation product, which is considered to be one of the important indices of oxidative damage to cell membrane caused by ROS (Parida and Das 2005). Our results suggest that salt stress induces an increase in MDA content in maize seedlings, suggesting that the presence of salt stress may enhance membrane lipid peroxidation, leading to increased membrane permeability, electrolyte extravasation, and ultimately damage to the cell membrane system. However, ZD175-T3, ZD958-T4 and DH605-T3 signi cantly decreased by 32.2%, 27.1% and 29.4%, respectively, compared to non-bioprimed seeds (Fig. 4c). This indicated that maize seedlings had stronger tolerance to oxidative stress after seed biopriming with SB-MGW9. This is similar to the result that the content of MDA in mycorrhizal inoculated maize plants is lower than that in non-mycorrhizal plants under temperature stress (Zhu et al. 2010). In addition, proline is a good osmotic agent and radical scavenger to stabilize subcellular structure, which can quench single O 2− or directly react with OH (Filippou et al. 2013b). Under stress conditions, proline accumulation may be due to increased synthesis and decreased degradation, which helps to maintain cell water status and protect cell membranes and proteins (Kishor and Sreenivasulu 2014). Bano and Fatima (2009) showed that microorganisms (Rhizobium and Pseudomonas) introduced in the rhizosphere can improve water use e ciency of maize plants, induce the synthesis of osmotic regulators such as proline, and help maintain the integrity of cell membranes. This is consistent with the results of this study, under salt stress, the proline content of biopriming maize seedlings was signi cantly higher than that of non-biopriming seedlings (Fig. 4d).
In plants, carbohydrate metabolism is involved in key processes in response to abiotic stresses, with key roles in carbon storage, osmotic homeostasis, osmoprotectants and free radical scavenging (Gangola and Ramadoss 2018 activity is a general indicator of the ability of roots to absorb water and nutrients. The decrease of root activity is harmful to the growth and development of maize seedlings. The increase of root activity was bene cial to absorb more water and nutrients in the process of maize seed germination and seedling formation. In our study, an increase in root activity was observed by seed biopriming with SB-MGW9 (Fig. 4f).
Abiotic stresses, including salinity, are often interrelated, either individually or in combination. They lead to excessive production of reactive oxygen species (ROS) in plants, such as superoxide anion radical . In our study, compared with non-bioprimed seeds, the activities of SOD, POD, CAT and APX in maize seedlings were signi cantly increased after biopriming treatment (P < 0.05), indicating that SB-MGW9 biopriming may improve the antioxidant defense capacity of maize seedlings under salt stress (Fig. 5).
In addition, the eld seedling emergence (FSE) of seeds after bio-priming was measured in saline and alkaline soil of Binzhou, Dongying and Weifang, respectively in this work. From the results of FSE determination, although the biopriming effects of different treatments on seed samples of three maize varieties were different, on the whole, compared with the seeds without biopriming treatment, the FSE of priming seeds was signi cantly improved (Fig. 6). The results further indicated that SB-MGW9 biopriming could effectively improve the salt stress resistance of maize at the stage of seed germination and seedling.
In this study, we've isolated and identi ed the strain Bacillus sp. MGW9 (CGMCC No. 18690) with the characteristics of salt tolerance, nitrogen xation, phosphorus solubilization, IAA production and so on.
We've also developed the SB-MGW9, and demonstrated that maize seeds biopriming with SB-MGW9 can resist the inhibitory effects of NaCl stress and promote seed germination and seedling growth. According to our current research results, we argue that the use of SB-MGW9 biopriming to improve salt stress resistance of maize seed germination may be to improve the antioxidant capacity of plants, increase the RWC, the content of chlorophyll, proline, soluble sugar, root activity and other aspects of the comprehensive role to promote plant growth.
The results indicated that the suitable biopriming treatments of SB-MGW9 for the three maize varieties were T2 (the seeds soak for 3 h, and moisturize for 24 h) and T3 (the seeds soak for 6 h, and moisturize for 12 h), and SB-MGW9 may be the promising technique to decrease the deleterious effects of salt stress for maize seed germination and seedling. In addition, based on the current research results, the molecular regulation mechanism of SB-MGW9 biopriming to improve maize seed vigour and whether spraying SB-MGW9 on maize seedlings can improve the effect of salt resistance etc. need to be further studied.

Declarations
Declaration of competing interest All authors declare that no potential con ict of interest.