Salt stress is a widespread environmental problem, although great effort has been made on this problem  . It is well known that salinity affects almost every aspect of plant physiology and biochemistry. Salt stress decreases seed germination, retards plant development and reduces crop yield  . Excessive salt ions are absorbed by the plant, breaking the ion homeostasis of plant cells, causing osmotic stress, affecting the activity of the enzyme, and accumulating a large amount of toxic substances such as active oxygen in the cells. And several studies indicate that under salt stress, increased activity of antioxidant enzymes such as CAT, POD and SOD can significantly increase plant resistance.
Wheat (Triticum aestivum L.) is one of the most important crops in the world. Seed germination is a crucial stage in the life history of wheat. Under salt stress, seeds are very sensitive to salinity during germination and seedling stage. The salinization of the soil and other abiotic factors severely restrict the enhancement of wheat production. Therefore, research breakthroughs in this area are of great significance for improving wheat yield and quality.
A few studies have focused on alleviation of salt stress of plant, while the studies on the mitigate effect of bacteria on plant growth under salt stress are limited. B. subtilis is a non-pathogenic bacterium that is widely found in nature and has strong resistance to stress. B. subtilis QM3 is an antagonistic strain with biological control functions. It is isolated from the feces of Qinghai yak  . Previous studies have shown that it can promote the germination of wheat seeds, but it has not been studied whether it can improve the salt tolerance of seed germination.
The aim of this study was to explore the influence of B. subtilis QM3 on germination (GP, GT, GI) and antioxidant enzymes activities (SOD, POD and CAT) of wheat growing under different concentrations of salinity stress. The research results will provide some theoretical and practical bases to increase crop production in salt lick.
2. Materials and Methods
The commonly used media in this investigation were the beef extract peptone containing 1.0% peptone, 0.3% beef extract, 0.5% sodium chloride, 1.5% - 2.0% agar, pH 7.4 - 7.6, 1000 ml distilled water 121˚C, 20 min sterilization.
The concentrations of salt were 0, 50, 100, 150, 200 mmol/L and were prepared freshly as NaCl.
2.2. Bacteria Materials
B. subtilis QM3 used in the present study come from the Microbiological Lab, the School of Life Science, Shanxi Normal University. A culture of B. subtilis QM3 was obtained by transferring acolony from the activated culture plate into a 250 mL flask containing 100 mL beef extract-peptone medium and shaking in an orbital shaker at 150 rpm at 37˚C for 3 d  . Bacterial suspensions for the treatment of seeds were prepared as described: the bacteria are harvested by centrifugation at 3000× g for 10 min at 4˚C. The supernatant is reserved, and the cells are washed by sterile water twice and resuspended in sterile water adjust to OD600 = 0.872. There were 108 microbial cells per 1 ml solution. The concentration used in the experiment was 106 microbial cells per 1 ml solution.
2.3. Seed Materials
Seeds of the Lin feng No.3 (Triticum aestivum L.) came from Shanxi academy of agricultural sciences. Healthy seeds of similar size and mass were selected about more than 4000 seeds. Prior to germination, seeds were surface sterilized with 0.1% mercuric chloride for 5 - 10 minute to avoid fungus attack, and subsequently rinsed with sterile water  .
2.4. Germination Assay
Seeds were soaked with sterile water (Q), bacterium suspension (Y) and supernatant liquid (L) for 12hat 25˚C. Immediately after soaking 50 seeds were sown in each petri dishes. A filterpaper was dipped into petri dishes (9 cm diameter) with 20 ml NaCl solution of 0 mg/L, 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, respectively. The seeds were laid on filter papers uniformly with the embryo upturned and stored into the illumination plant box. The cultivated condition was set as follows: illumination lasting 12 h, light intensity is 1500 lux, temperature at 25˚C (daytime) or 18˚C (night), and moist condition  . To avoid changes in salinity, the original solution in each petri dish was removed daily and 10 ml of new solution was added and removed again as completely as possible  . All experiments described here were repeated three times. Seeds per petri dish were harvested 7 d after sowing.
Radicle protrusion was used as the criterion of germination, and the numbers of germinated seeds on the day after initiation were measured germination percentage throughout the test period. Seeds showing a normal epicotyl were considered as being alive. In the germination experiment, follow the method described by Lina Zhou et al.  . The number of germinated seeds was recorded each day. GP, GT and GI were calculated following the equations:
where, Gt means number of germination at tth day, T means selected number of seeds, N3d means number of germinated seeds in 3rd day, Dt means number of days up to tth day.
2.5.2. Antioxidant Enzymes Assays
SOD was assayed by the photochemical method described by Giannopolitis and Ries  . POD activity was assayed following the method of Xu and Ye with minor modifications  . For this,
2.6. Statistics Analysis
All data are the average of three repetitions. The obtained results were analyzed statistically by SPSS (version 11.0) statistical software. Data were analyzed by the analysis of variance and treatment mean comparison by using least significance difference (LSD; p = 0.05).
3.1. Effect on Wheat Seed Germination Soaked by B. subtilis QM3 under Salt Stress
Q, L and Y respectively represent the control group (treated with sterile water), supernatant liquid and bacterium suspension, GP, GT, and GI in the table respectively represent the germination percentage, the germination potential and the germination index. Data in the table is average ± standard error. The uppercase letters in the same vertical column of the table represent significant differences (p < 0.05) between sterile water treatments under different NaCl stress. Different lowercase letters represent a significant difference between the B. subtilis QM3 treatment and the Q under the same NaCl stress (p < 0.05).
In this experiment, seeds germinated for 3 days were selected to determine their germination indexes. The results are shown in Table 1. Compared with the control group (Q), B. subtilis QM3 (L, Y) significantly increased the level of wheat seed germination (50, 100, 150, 200 mmol/L). When without NaCl stress, L and Y treatments compared with Q, the GP was significantly increased by 4.44% and 5.22%; the GT increased by 6.58% and 15.79%; the GI increased by 6.30% and 22.05%. This result indicated that B. subtilis QM3 had an obvious promotion effect on CP, GT, GI. When without B. subtilis QM3 treatment, Treatment of wheat seeds with 100 mmol/L and higher concentrations of the NaCl caused inhibition of morphogenesis of wheat seedlings germination. When NaCl concentration were 100 mmol/L, L and Y treatments compared with Q, the GP was significantly increased by 11.11% and 11.6%; the GT increased by 23.33%
Table 1. Effect on wheat seed germination soaked by B. subtilis QM3 under salt stress.
and 25%; the GI increased by 27.5% and 26.25%; when NaCl concentration were 150 mmol/L, L and Y treatments compared with Q, the GP was significantly increased by 12.99% and 12.6%; the GT increased by 33.33% and 41.18%; the GI increased by 37.31% and 38.81%; when NaCl concentration were 200 mmol/L, L and Y treatments compared with Q, the GP was significantly increased by10.34% and 12.07%; the GT increased by 17.07% and 21.95%; the GI increased by 9.83% and 8.2%. The above data shows, when NaCl concentration was 100, 150 and 200 mmol/L, Y and L was higher than that of Q. When NaCl concentration were 150 mmol/L, B. subtilis QM3 had the best treatment effect. In a certain degree, B. subtilis QM3 had an alleviation effect on the germination of the wheat seeds under NaCl stress.
3.2. B. subtilis QM3 Soaking Effect on Activities of Antioxidant Enzymes under Salt Stress
3.2.1. SOD Activity
B. subtilis QM3 soaking effect on SOD activity under salt stress (Figure 1). With the increase of salt concentration, the SOD activity of wheat seeds increased and then decreased. Basically, under the salt concentration of 50 mmol/L, the SOD activity reached a maximum level, compared with Q, L and Y respectively increased by 21.35% and 40.84%; under the NaCl concentration were 50, 100, 150 and 200 mmol/L, the SOD activity of L and Y treatment was higher than control treatment (Q). When the salt concentration was 150 mmol/L, the increase was the largest compared with Q, and L and Y increased by 35.81% and 34.42%, respectively. At a salt concentration of 200 mmol/L, L and Y increased by 11.9% and 12.91%, respectively, compared to Q. Under the salt stress (50, 100, 150, 200 mg/L), B. subtilis QM3 could improve SOD activity.
3.2.2. POD Activity
NaCl treatment caused POD activity of L and Y treatments for wheat seed and the Q showed a tendency that POD activity were increased and then decreased with the increase of NaCl concentration (Figure 2). Under the salt concentration of 50 mmol/L, the POD activity reached a maximum level. Compared with Q, L and Y respectively increased by 20.27% and 31.17%, under the NaCl concentration were 50, 100, 150 and 200 mmol/L, the POD activity of L and Y treatment
Figure 1. B. subtilis QM3 soaking effect on SOD activity under salt stress. Q, L, and Y respectively represent the control group (treated with sterile water), supernatant liquid and bacterium suspension. The uppercase letters in the figure represent significant differences (p < 0.05) between sterile water treatments under different NaCl stress. Different lowercase letters represent a significant difference between the B. subtilis QM3 treatment and the Q under the same NaCl stress (p < 0.05).
Figure 2. B. subtilis QM3 soaking effect on POD activity under salt stress. Q, L, and Y respectively represent the control group (treated with sterile water), supernatant liquid and bacterium suspension. The uppercase letters represent significant differences (p < 0.05) between sterile water treatments under different NaCl stress. Different lowercase letters represent a significant difference between the B. subtilis QM3 treatment and the Q under the same NaCl stress (p < 0.05).
were higher than that of control treatment (Q). When the salt concentration was 150 mmol/L, the increase was the largest compared with Q, and L and Y increased by 29.45% and 36.57%, respectively. Under the salt stress (50, 100, 150, 200 mg/L), B. subtilis QM3 could improve POD activity.
3.2.3. CAT Activity
With the increase of salt concentration, the CAT activity of wheat seeds increased and then decreased (Figure 3). On the whole, under the NaCl concentration were 50, 100, 150 and 200 mmol/L, the CAT activity of L and Y treatment were higher than that of control treatment (Q). However, when NaCl concentrations were 50 mol/L, B. subtilis QM3 treatment was increased obviously than any other treatment, and L and Y increased by 77.68% and 87.25%, respectively. And when NaCl concentrations were 200 mmol/L, B. subtilis QM3 treatment
Figure 3. B. subtilis QM3 soaking effect on CAT activity under salt stress. Q, L, and Y respectively represent the control group (treated with sterile water), supernatant liquid and bacterium suspension. The uppercase letters in the figure represent significant differences (p < 0.05) between sterile water treatments under different NaCl stress. Different lowercase letters represent a significant difference between the B. subtilis QM3 treatment and the Q under the same NaCl stress (p < 0.05).
was the lowest at all. Under the salt stress (50, 100, 150, 200 mg/L), B. subtilis QM3 could improve CAT activity.
Excess salt stress can reduce seed germination, delay plant development, break the ion homeostasis of plant cells, affect the activity of enzymes, and accumulate a large amount of toxic substances such as active oxygen in cells. Salt stress can reduce seed germination, delay plant development, break the ion homeostasis of plant cells, affect the activity of enzymes, and accumulate a large amount of toxic substances such as active oxygen in cells.
Compared with the late stage, the early growth stage of the plant is more sensitive to salt, so it is particularly important to inoculate bacteria with reduced salt stress to make it more salt-tolerant  . Our result was carried out to evaluate exogenous application of B. subtilis QM3 enhancing NaCl tolerant of wheat through assessing on seed germination and antioxidant enzyme activity. The results show that excess salinity adversely (100, 150, 200 mmol/L) affected the germination of wheat, regardless of the B. subtilis QM3 treatment and the salt-stress level. And with increasing salt concentration, germination was progressively inhibited in the absence of bacteria. However, when the seeds were inoculated with B. subtilis QM3 the extent of seeds suppression was decreased and these treated seeds had greater GP, GT and GI than untreated seeds. On the other hand, GP, GT and GI signiﬁcantly decreased with an increase in NaCl concentration from 100 to 200 mmol/L. When NaCl concentration were 150 mmol/L, B. subtilis QM3 had the best treatment effect. This clearly demonstrates that the selected bacterium can alleviate some of the debilitating effects of salt stress, and bacterial suspension has the best treatment effect.
Under the condition of salt treatment, crops accumulate excessive reactive oxygen species (ROS), which cause peroxidation damage to crops. Plant cells have armed with a distinct antioxidant defense system that keeps ROS at minimal level by employing various enzymes like SOD, POD and CAT  . These enzymes resistance is positively correlated with enzyme activity.
SOD modulates relative amounts of O2− and decreases the risk of −OH radical formation.
CAT is the main scavenger of strong oxidant H2O2 in peroxisomes and it converts H2O2 to water and molecular oxygen. Generally, the increases of CAT activity are a strategy for improving the salt tolerance  .
Like CAT, POD plays also a vital role in plant defense against oxidative stress by scavenging H2O2 in chloroplast, cytosol, mitochondria and peroxisome of plant cells  .
In our study, with the increase of salt concentration, SOD activity POD activity and CAT activity of all treatments for wheat seeds showed a tendency that SOD activity and POD activity were increased and then decreased. Under the NaCl concentration of 50 mmol/L, the wheat antioxidant enzyme activity was promoted, it may due to the dual role of NaCl in plant. When with B. subtilis QM3 treatment, enzyme activity of wheat seeds under the treatment of L and Y were higher than that of Q. When the NaCl concentration was 50 mol/L, CAT activity was the greatest. Under salt stress (150 mmol/L), B. subtilis QM3 treatment maximized the relative growth rate of SOD activity and POD activity. And Y treatment was enhanced obviously than any other treatments. Based on these ﬁndings, B. subtilis QM3, was effective with respect to alleviating the negative effect of salinity on the growth of Wheat.
Overall, our results implied that salt stress caused severe damage on morphological indicators and antioxidant enzyme activity during the germination period of wheat seeds. And B. subtilis QM3 was effective with respect to alleviating the negative effect of salinity on the growth of Wheat.
To conclude, inoculating seeds with B. subtilis QM3 increased the germination percentages, GT and GI of wheat exposed to salt stress. When the plants exposed to NaCl, the bacterium depressed NaCl induced oxidative stress by improving antioxidant enzymes (SOD, POD, CAT), and promoting relative plant growth, resulting in increased plant tolerance to NaCl. The results of this study demonstrate that excess salinity is detrimental to wheat growth and B. subtilis QM3 inoculated have a positive effect on growth of wheat under salt stress. Selection of suitable inoculation methods is more conducive to promote wheat production, thus increasing crop yield. This phenomenon may be related to the combination of bacteria and wheat seeds, but the deeper reasons need to be further studied.
This work was financially supported by the Natural Science Foundation Project of Shanxi Province (201801D121206) and the Natural Science Foundation of Modern College of Humanities and Sciences of Shanxi Normal University (2018JCYJC01).
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