Bacterial identification and phylogenetic analysis
The bacterial strain SPSB2 (OQ380690) was identified, and its phylogenetic position was inferred by using BLAST search analysis (http://www.ncbi.nlm.nih.gov/) of the 16S ribosomal RNA sequences of the isolates against the sequences in the NCBI database. A greater degree of 16S sequence identity was found between SPSB2 and Nitratireductor aquimarinus. Parameters for gap opening penalty and gap extension penalty in both pairwise and multiple sequence alignment were set at 15.00 and 6.66, respectively. After sequence alignment with Clustal W (version 7.222) the phylogenetic tree of the aligned 16S rRNA sequences was constructed by the neighbor-joining method of the MEGA 11 software, with 1,000 times bootstrap replicates (BS) (Figure S1).
Effect of SPSB2 and mannitol treatment on plant biomasses of tomato plant under NaCl and As stress
Our results revealed that the bacterial inoculation significantly induced tomato plants' shoot and root length by 17% and 53%, respectively, compared to control plants under normal conditions (Figs. 1 and 2). The results indicate that the length of shoot and root decreased significantly under NaCl stress compared to control plants. Our finding showed that the SPSB2 inoculation has a significant positive effect on tomato plant growth and mitigates the negative impact of NaCl stress, resulting in a considerable enhancement of both shoot and root length by 84.8% and 152.5%, respectively, compared to their non-inoculated counterparts. Mannitol treatment also significantly promoted shoot length by 13.8%, and surprisingly, the same mannitol treatment led to a significant decrease in root length, reducing it by about 45% under NaCl stress conditions. The combined treated plants with SPSB2 and mannitol exhibited significantly increased root length (94.1%) but unexpectedly reduced shoot length by 8.3% compared to plants treated with NaCl. Similarly, SPSB2 inoculated plants showed a considerable increase in fresh weight of shoot and root, i.e. 82.1% and 105.7%, respectively, compared to the plants in the control condition (Fig. 2C and D). Similar trends were observed in the plants under NaCl stress conditions. Combined treated plants with SPSB2 and mannitol showed enhanced fresh root weight (31.6%) when compared to control plants. Conversely, there was a significant decrease in fresh shoot weight, with a reduction of 14% compared to untreated plants. Similarly, under As stress condition, inoculation with plants with SPSB2 bacteria led to a significant increase in the shoot length by 104.8% and in the root length by 36.1%. The results showed that the combined treatment of SPSB2 and mannitol had a positive effect on the shoot and root length of tomato plants compared to untreated plants, as the combined treatment resulted in a notable increase of 66.4% in shoot length and 5.3% in root length when compared to the control and mannitol-treated plants. The results revealed that under As stress conditions, a significant increase occurred in the fresh weight of the tomato plant's shoot and root by 63.1% and 45.5%, respectively, through inoculation with SPSB2. Additionally, the combined application of SPSB2 and mannitol also influenced the fresh weight of both shoot and root compared to the control.
Effect of SPSB2 and mannitol treatment on chlorophyll and relative water contents of tomato plant under NaCl and As stress
The results in Figs. 3 illustrated that SPSB2-treated plants significantly increased the photosynthetic contents in the control conditions compared to untreated plants. The combined treatment of SPSB2 and mannitol has a consistent positive influence on both chlorophyll-a and carotenoid and significantly increased their contents under both NaCl (76.3% and 123.6%) and As (43.3%, and 50%) stress conditions respectively (Fig. 3A and 3C). Moreover, the combined treatment of SPSB2 and mannitol resulted in a significant increase (78%) in chlorophyll-b content in plants. Interestingly, plants treated with As dramatically increased Chlorophyll-b content, while SPSB2 inoculation significantly decreased (Fig. 3B). Similarly, SPSB2-treated plants significantly decreased their carotenoid content by 35% compared to NaCl stress plants. The combined treated plants with SPSB2 and mannitol exhibited increased carotenoid concentration in NaCl and As stress conditions. The RWC of the tomato leaves was significantly affected by SPSB2 inoculation under control conditions. Similarly, all treatments, including SPSB2, mannitol, and a combination of both, led to a significant increase in RWC in plants under NaCl stress, i.e., 71.1%, 56.5% and 59.9%, respectively. Furthermore, under As stress, the application of mannitol significantly improved (36.1%) RWC, while SPSB2 and combined treatments showed a 19.4% and 37.1% decrease, respectively, when compared to As stressed plants (Fig. 3D).
Effect of SPSB2 and mannitol treatment on catalase and protein contents of tomato plant under NaCl and As stress
Our results in Fig. 4A showed that the catalase contents of tomato plants increased in SPSB2-treated plants, while with mannitol treatment, a reduction of 3% occurred in the control plants. The catalase content in plants treated with SPSB2 and mannitol was similar to that of plants treated with 400 mM NaCl alone. In comparison, mannitol-treated plants showed a slight increase of 2.5% in catalase content compared to SPSB2-treated plants. Catalase contents were analyzed in inoculated bacteria and mannitol under the As stress condition. The results revealed that SPSB2 treatments significantly increased the catalase content by 3.4% in tomato plants. Similar catalase contents were observed in tomato plants treated with the combination of SPSB2 and mannitol. In Fig. 4B, a reduction in protein content was recorded in plants treated with mannitol. Meanwhile, in control conditions, a significant increase of 14.5% in protein content was observed in plants inoculated with SPSB2. A decrease in protein content was observed when the tomato plants were treated with NaCl and As stress conditions. However, SPSB2-and mannitol-treated plants showed increased protein content by 45.1% and 18.3% under 400 mM stress conditions, respectively. The protein content in plants treated with SPSB2, mannitol individually, and a combination of both was higher compared to As-stressed plants. Mannitol treatment notably induced a significant increase in protein content, with a boost of 54.3% compared to As stressed plants.
Effect of SPSB2 and mannitol treatment on sugar, starch and flavonols contents of tomato plant under NaCl and As stress
In Figs. 5, results indicated that mannitol treatments significantly increased sugar and starch contents by 90.3% and 68.9%, respectively, under control conditions. Under the NaCl stress condition, sugar and starch levels rose 36.6% and 32.1%, respectively. Under the As stress, the corresponding increases were 39.8% for sugar and 37.1% for starch. The sugar contents significantly increased in plants treated with mannitol compared to the NaCl stress plants. However, they were lower than SPSB2-treated plants under NaCl and As stress conditions. At the same time, the sugar and starch content of combined treatment plants showed similar results to those treated with NaCl stress, but their concentration significantly decreased under As stress conditions. The results presented in Fig. 5C indicate a significant effect of the inoculated SPSB2 on the flavonols contents compared to the control condition. Furthermore, inoculated SPSB2 and mannitol treatments led to a considerable 44.9% and 68.4% increase in the flavonol contents of the plants under 400 mM NaCl. However, we observed that combined treatment of SPSB2 and mannitol significantly inhibited flavonol contents, e.g., a 5.9% decrease compared to 400 mM NaCl stress condition. Similarly, in the case of As stress, the SPSB2 and mannitol significantly increase the flavonol contents.
Effect of SPSB2 and mannitol treatment on reduced glutathione and total polyphenol contents of tomato plant under NaCl and As stress
Reduced glutathione contents were observed to be decreased by 5.6% and 9.4% with SPSB2 and mannitol treatments, respectively, compared to non-treated plants under the control conditions. Furthermore, NaCl-treated plants exhibited significantly enhanced glutathione contents and reduced 15.7%, 33.1%, and 25.4% in glutathione contents by applying inoculated SPSB2, mannitol and combined treatments. The results of glutathione contents in As stress were opposite in plants treated with NaCl stress (Fig. 6A). The combined stress of NaCl and As significantly increased the glutathione content by 25.2%, and an almost 12.4% increase was observed in SPSB2-inoculated plants compared to As-stressed plants. The PPO contents decreased by 18.1% in plants receiving the mannitol treatment and increased by 19% in plants treated with bacteria under control conditions (Fig. 6B). The PPO contents in tomato plants dramatically decreased under salinity stress; however, the application of inoculated bacteria improved the PPO contents by 85.0%. Interestingly, both the individual and combined treatment of mannitol and SPSB2 exhibited almost similar effects on PPO contents as the individual treatment with mannitol resulted in a 35.9% increase, while the combined treatment showed a slightly lower increase of 25.9% under NaCl stress condition. The results revealed that PPO contents were almost similar in NaCl and As-treated plants; however, the mannitol and SPSB2 treatment significantly increased the content by 54.7% and 76.1% under As stress conditions.
PCA and correlation trait of tomato plants under NaCl and As stress
Principal Component Analysis (PCA) results explored the variability in morpho-physiological, biochemical, and antioxidative traits of tomato plants under control, SPSB2 inoculated, and stress conditions of NaCl and As stress (Fig. 7). The data was collected from untreated control, SPSB2 inoculated, and mannitol-treated plants under control and stress conditions of NaCl and As. SPSB2 bacterial strain alleviated the salinity and As stress conditions in tomato plants. Our results showed that PC1 has greater values than PC2 in all three plots (Fig. 7A, B, and C). The eigenvalue increased and decreased one by one, but the cumulative value was induced in stress conditions. In control, 400 mM NaCl and 0.5 mM As stress conditions were recognized in biplots as 73.12%, 54.75%, and 55.27% cumulative values (Table 1). The PCA analysis showed that plants treated with SPSB2 were prominently represented in the PC1, and the SPSB2 treatment significantly impacted the plants' physiological, biochemical, and antioxidant contents under control conditions. Catalase was negatively correlated with plant morphological traits with combined treatments. Moreover, Pearson correlation analysis was conducted to determine the extent of the relationship among the characteristics. The results significantly correlated the morphological, biochemical, and antioxidant traits under control and stress conditions of NaCl and As in both inoculated and non-inoculated plants. Surprisingly, shoot, root length, FWS, FWR, and protein contents were positively correlated with other traits. On the other hand, RWC has a significantly negative correlation with other features shown in (Fig. 7D). Overall, the results showed insight correlation interaction among different growth parameters in tomato plants under salt and arsenic stress and highlighted the potential of using SPSB2-treated plants to improve growth and stress tolerance.
Table 1
Eigenvalue, variance, and cumulative of Control, NaCl, and As stress.
| Eigenvalue | Percentage of Variance | Cumulative |
Control | 10.96779 | 73.12% | 73.12% |
4.03221 | 26.88% | 100.00% |
0 | 0.00% | 100.00% |
8.21311 | 54.75% | 54.75% |
NaCl 400 mM | 4.51075 | 30.07% | 84.83% |
2.27615 | 15.17% | 100.00% |
0 | 0.00% | 100.00% |
8.29107 | 55.27% | 55.27% |
As 0.5 mM | 4.94274 | 32.95% | 88.23% |
1.76619 | 11.77% | 100.00% |
0 | 0.00% | 100.00% |
Effect of SPSB2 and mannitol treatment on stomata of tomato plant under NaCl and As stress
Generally, abiotic stresses elevate the ROS levels and induce osmotic stress in plant cells. Abscisic acids (ABA) play a vital role in closing stomatal cells, thereby preventing the dehydration of transpiration pathways. Stomatal morphology was screened in the microscope to check the effect of inoculated SPSB2 bacterial strain and mannitol under salt and heavy metal stress conditions. As shown in Fig. 8, under NaCl stress, a higher stomatal density accompanied by a decrease in size was observed compared to SPSB2-inoculated plants. The stomatal density of the plants treated with NaCl alone and those treated with NaCl + B was observed, and the number of stomata increased in the NaCl + B group. Similar trends were observed in the NaCl + M and SPSB2 + mannitol-treated groups, indicating that bacterial (B) and mannitol treatments reduced the number of stomata. Moreover, plants treated with As stress exhibited smaller and open stomata compared to control and SPSB2-treated plants. On the other hand, in SPSB2 inoculated plants, larger and open stomata were observed, and mannitol treatment increased the size and openness of stomata under As stress.