3.1 Effects of different treatments on the infection rate and growth index of switchgrass
Under saline-alkali stress, Ri established an excellent symbiotic relationship with switchgrass. Apparent vesicles and mycelial structures were observed in the inoculated roots (Fig. S1a). According to the determination of mycorrhizal infection rate, compared with the Ri treatment group, the addition of biochar significantly increased the infection rate of Ri to the root system by 13.1% (P < 0.05) (Fig. S1b). In addition, no mycorrhizal infection was found in CK and BC treatments, suggesting that sterilization eliminated the effects of indigenous AMF.
As shown in Fig. 1a, 1b and 1d, the plant height of switchgrass under Ri, BC and Ri + BC treatment increased by 82.8%, 69.9% and 101.8%, respectively, compared with CK treatment. Compared with CK, Ri, BC and Ri + BC, the aboveground biomass increased by 67.19%, 64.98% and 135.4%, respectively, and the underground biomass increased by 82.4%, 68.6% and 121.4%, respectively. The plant height and biomass of the Ri + BC treatment were significantly higher than those of the Ri and BC treatment. These results indicated that Ri combined with biochar promoted the growth of switchgrass. Tillering is an important agronomic trait affecting plant biomass. In this study, Ri and Ri + BC treatment significantly increased tillering number of switchgrass (Fig. 1c), indicating that Ri may be a key factor affecting tillering of switchgrass. The results of two-factor ANOVA showed that Ri inoculation and biochar addition had significant effects on plant height, tiller number and dry weight and had significant interaction between plant height and root dry weight (P < 0.05).
3.2 Effects of different treatments on malondialdehyde, osmotic regulatory substances and antioxidant enzymes of switchgrass
Biochar and Ri alone or combined significantly reduced MDA content in leaves and roots (Fig. 3a). Compared with CK treatment, MDA content in BC, Ri and Ri + BC treated roots decreased by 27.4%, 35.7% and 41.2%, respectively. MDA content in leaves decreased by 29.5%, 33.3% and 45.9%, respectively. Ri combined with biochar significantly affected soluble sugar in the roots and leaves of switchgrass (Fig. 3b). Salt and alkali stress can reduce the soluble sugar content of plants, while Ri and biochar alone or in combination can significantly increase the soluble sugar content of switchgrass. The soluble sugar content of roots and leaves under Ri + BC treatment is the highest, 1.72 and 1.48 times that under CK treatment, respectively.
As shown in Fig. 3c, compared with CK treatment, BC, Ri, and Ri + BC treatment significantly increased the proline content in the roots and leaves of switchgrass. There was no significant difference in proline content between BC and Ri treatment groups (P > 0.05), while Ri + BC treatment had a significant difference with other treatments (P < 0.05). The changing trend of sucrose content in the roots and leaves of switchgrass was consistent with that of proline. Among them, the sucrose content in the roots and leaves of Ri + BC treatment was the highest, 49.3% and 59.6% higher than that of CK treatment, respectively (Fig. 3d). The results of two-factor ANOVA also showed that Ri and biochar significantly affected the osmotic regulatory substances of switchgrass and had significant interactions with MDA, proline in leaves and MDA, proline and soluble sugar in roots (P < 0.05).
Saline-alkali stress usually induces oxidative stress in plants, causing them to break the homeostasis of reactive oxygen species elimination and production. Ri and biochar had significant effects on the antioxidant enzymes of switchgrass leaves and roots but only had significant interaction on root SOD and leaf POD (P < 0.05). As shown in Fig. 3e-h. Compared with CK treatment, BC, Ri and Ri + BC treatment significantly increased SOD, POD, APX and CAT contents in roots and leaves. It is worth noting that SOD, POD, APX and CAT in the root of the Ri + BC treatment group were increased by 76.9%, 71.1%, 200.2% and 52.5%, respectively, compared with CK treatment. SOD, POD, APX and CAT in leaves increased by 47.3%, 138.2%, 169.3% and 104.1%, respectively, compared with CK treatment.
3.3 Effects of different treatments on endogenous hormones of switchgrass
Ri and biochar significantly affected the contents of CTK, IAA, MT and SLs in switchgrass and only had significant interaction between leaf SLs and root MT and CTK (P < 0.05). As shown in Fig. 4a-c, compared with CK treatment, BC, Ri, and Ri + BC treatment significantly increased the contents of CTK, IAA and MT in switchgrass. Among them, CTK, IAA and MT in leaves treated with Ri + BC were 2.1, 2.1 and 1.2 times that of those treated with CK treatment, respectively. CTK, IAA and MT in root were 2.2, 2.4 and 1.2 2.8 times of CK treatment, respectively. In contrast to the former, BC, Ri and Ri + BC all reduced SLs content in leaves and roots, among which Ri + BC had the most significant effect on SLs in leaves and roots, decreasing by 6.76ng/g and 5.88ng/g, respectively, compared with CK treatment (Fig. 4d).
3.4 Effects of different treatments on ion concentration and ion homeostasis
The salt ions' content in switchgrass's roots and leaves was measured (Fig. 4a,b,c,d). The results showed that the accumulation of Na+ in roots was more significant than in leaves under saline-alkali stress. Compared with CK treatment, BC, Ri, and Ri + BC treatments significantly reduced Na+ content in switchgrass leaves and roots. At the same time, there was no significant difference in Na+ content in the leaves and roots of BC and Ri treatments, but there were significant differences in Na+ content between BC and Ri treatment and Ri + BC treatment (P < 0.05) (Fig. 4a). Salt and alkali stress can inhibit the absorption of K+ by plants. However, this study found that biochar and Ri alone or in combination increased the K+ content in plant leaves and roots, among which the K+ content in leaves and roots treated with Ri + BC was 1.61g/kg and 1.85/kg higher than that in CK treatment group, respectively (Fig. 4b). The accumulation of K+ in leaves was higher than that in roots. In contrast to the distribution of Na+ and K+, switchgrass leaves accumulated more Ca2+ and Mg2+. Under saline-alkali stress, compared with BC, Ri, and Ri + BC treatments, Ca2+ and Mg2+ contents in switchgrass leaves and roots in the CK treatment group were the lowest, and there were significant differences between them and other treatments (Fig. 4c, d). The two-factor analysis of variance showed that Ri and biochar only had significant interaction on Na + content in switchgrass leaves and roots (P < 0.05).
Ri and biochar had significant effects on K+/Na+, Ca2+/Na+, Mg2+/Na+ and Ca2+/Na+ ratios between leaves and roots of switchgrass and had significant interactions on K+/Na+ and Ca2+/Na+ in roots (P < 0.05). As shown in Figure. 4e, f, and g, the root ion ratio of switchgrass under BC and Ri treatment was higher than that under CK treatment, but the difference between them was insignificant. The K+/Na+, Ca2+/Na+ and Mg2+/Na+ ratios of switchgrass leaves and roots in the Ri + BC treatment group were significantly increased (P < 0.05), which were 2.51–2.49 times, 2.85–2.53 times and 3.63–2.89 times of those in CK treatment group, respectively. As shown in Fig. 4h, Ca2+/Mg2+ in plant leaves and roots was significantly reduced in the Ri + BC treatment group (P < 0.05).
Compared with CK treatment, leaf and root K+, Ca2+, Mg2+, K+/Na+, Ca2+/Na+ and Mg2+/Na+ in Ri treatment group increased by 12%~20%, 23%~32%, 25%~38%, 78%~79%, 95%~98% and 97%~107%, respectively. Na+ and Ca2+/Mg2+ decreased by 37%~33% and 3%~5%, respectively. It was also found that compared with BC treatment, the K+, Ca2+, Mg2+, K+/Na+, Ca2+/Na+ and Mg2+/Na+ of Ri + BC treatment increased by 18%~16%, 21%~25%, 23%~62%, 62%~68%, 69%~91% and 75%~163%, respectively. Na+ was reduced by 7–9% (Figure S2a, b). The data showed that the changes in ion content and ion ratio in the Ri + BC treatment were greater than in the Ri treatment group. The results showed that the absorption of K+, Ca2+ and Mg2+ ions was improved by reducing the content of sodium ions in BC, Ri and Ri + BC treatments.
3.5 Influence of relative expression of saline-alkali tolerance genes in switchgrass
In this experiment, the leaves and roots of switchgrass under different treatments were analysed by QRT-PCR. Stress response genes (PvNAC1, PvP5CS1, PvDREB2A, PvDREB1B), active oxygen scavenging genes (PvPOD, PvCAT), ion transport genes (PvSOS1, PvNHX1, PvHKT) and cellulose biosynthesis genes (PvCesA4) were screened. QRT-PCR verification on leaves and roots found that the relative expression levels of related genes in BC, Ri and Ri + BC treatment groups were significantly higher than those in CK treatment (P < 0.05). In Ri + BC treatment, the relative expression levels of leaf and root saline-alkali resistance genes (PvNAC1, PvP5CS1, PvDREB2A, PvDREB1B, PvPOD, PvCAT, PvSOS1, PvNHX1, PvHKT) and cellulose biosynthesis genes (PvCesA4) were CK, respectively Processed (7.18–4.13 times, 21.40–7.09 times, 5.23–4.12 times, 6.24–4.79 times, 17.23–6.86 times, 15.19–7.33 times, 19.70–9.58 times, 6.37–4.78 times, 20.37–2.65 times) and (8.58–5.07 times). The results also showed that the relative expression levels of PvP5CS1, PvNAC1, PvCAT and PvCesA4 genes in leaves treated with BC, Ri and Ri + BC were higher than in roots.
3.6 Effects of different treatments on the energy quality of switchgrass
Cellulose and hemicelluloses are the main components of plant cell walls, and they play an essential role in the field of energy and materials, which can be used to produce flammable gases (such as methane) or liquid fuels (such as biodiesel, ethanol, etc.). Calorific value is an important index to measure the energy content of energy plants. In this study, BC, Ri and Ri + BC treatments significantly increased cellulose content, hemicellulose content and calorific value of switchgrass and decreased lignin and ash content (Table 1). Compared with CK treatment, Ri + BC treatment increased cellulose, semi-fiber and calorific value by 2.3%, 2.0% and 4.8%, respectively, while lignin and ash decreased by 0.8% and 0.9%, respectively. The results showed that Ri combined with biochar under saline-alkali stress had the best effect on increasing cellulose, hemicellulose and caloric value of switchgrass. The results of the two-factor variance showed that BC and Ri had significant effects on cellulose, hemicellulose, lignin and ash, and Ri + BC had significant interaction with hemicellulose (P < 0.05).
Table 1
Contents of cellulose, hemicellulose, lignin, GCV and ash in switchgrass under different treatments
Treatment
|
Cellulose (%)
|
Hemicellulose (%)
|
Lgnin
(%)
|
GCV (MJ/kg)
|
Ash
(%)
|
CK
|
26.9 ± 0.7c
|
27.1 ± 0.2c
|
5.3 ± 0.3a
|
16.8 ± 0.1b
|
6.0 ± 0.3a
|
BC
|
28.7 ± 0.4ab
|
28.4 ± 0.3b
|
4.8 ± 0.2b
|
17.5 ± 0.1a
|
5.6 ± 0.3b
|
Ri
|
28.0 ± 0.3b
|
28.1 ± 0.3b
|
5.0 ± 0.2b
|
17.4 ± 0.4a
|
5.5 ± 0.3b
|
Ri + BC
|
29.2 ± 0.6a
|
29.1 ± 0.4a
|
4.6 ± 0.2b
|
17.6 ± 0.3a
|
5.1 ± 0.2b
|
F(BC)
|
*
|
***
|
*
|
NS
|
**
|
F(Ri)
|
***
|
***
|
**
|
*
|
*
|
F(Ri*BC)
|
NS
|
*
|
NS
|
NS
|
NS
|
Note: Results are six replicates of mean ± standard deviation. Different letters indicated that the difference between treatments was statistically significant (P < 0.05). *P < 0.05, **P < 0.01, ***P < 0.001, NS. no significant. |
3.7 Principal component analysis and correlation analysis
Principal component analysis was performed on biomass, ion absorption, osmotic regulatory substances, antioxidant enzymes, plant hormones and other indicators of switchgrass (Fig. 6a). The results showed that the total PCA interpretation rate was about 94.61%, and there were apparent differences among different treatments, among which the difference between Ri + BC treatment and CK treatment was the most significant. Mantel test results showed cellulose and hemicellulose were mainly related to Na+, osmoregulatory substances, antioxidant enzymes and plant hormones in leaves and roots. In contrast, lignin, GCV and ash were primarily associated with plant mineral element ions (Fig. 6b, c). Cellulose and hemicellulose were more closely correlated with the contents of osmotic regulatory substances, antioxidant enzyme activities and plant hormones in leaves and roots (the correlations were higher in leaves than in roots). The contents of Na+ in leaves were positively correlated with Ca2+/Mg2+, MDA and SLs and negatively correlated with other indexes. The same results were also obtained in roots. Random forest prediction analysis showed that the ratio of leaf SLS and root Mg2+ / Na+ had the highest interpretation of cellulose. Leaf CTK and root MT explained 113.69% and 93.29% of hemicellulose, respectively. The effects of leaf CTK and root K+ on lignin were the largest, respectively. Leaf CTK and root Ss had the highest interpretation rate of GCV. Leaf MT and root SLs significantly affected ash content (Fig. 3d-m).