ANOVA, analysis of variance. *P<0.05. **P<0.01. A two-factor interaction analysis showed that both the pH and CaCl2 concentration had significant effects on the bud lengths and root lengths of D. glomerata, L. perenne, andM. sativa except for CaCl2 on L. perenne. The treatments of pH and CaCl2 all had significant interaction on the bud lengths and root lengths of D. glomerataand M. sativa (Table 5).
Discussions
4.1 Effect of pH value on seed germination of herbageThe soil pH depends on the location, bedrock, climate and vegetation type [16]. The response of seed germination to pH change has been a concern for a long time [17]. The effect of pH value on seed germination has become an important indicator to assess whether a species can adapt to changes in soil pH [18,19]. Different species vary in their pH values and thresholds for germination; some species have a wider pH range and a higher germination rate [20]. Other species only germinate at certain pH values. The response of M. sativa to soil acidity during germination has also been studied. It can grow well and produce large yields even in highly acidic soils (pH < 4.0) [21]. The results showed that the germination rates of M. sativa, D. glomerata and L. perenne were significantly inhibited at pH 4.55 and 7.03 to 9.18. Low and high hydrogen (H+) concentrations inhibit the utilization of other metal ions by herbages [22]. However, slightly acidic conditions tend to be favorable. Studies have shown that slightly acidic conditions are beneficial to the germination of several types of feed crops[23]. Seeds placed on filter paper in petri dishes germinated at pH 3 to 8 without any statistically significant differences. This is also the reason why the buds and young roots in this experiment can promote their growth at weakly acidic conditions. In addition, higher pH values can affect germination by inhibiting proteolytic enzymes involved in the metabolism of seed storage compounds [24]. Hydroxyl ions (OH-) may also interfere with the uptake of essential anions [25]. Therefore, the osmotic pressure of the membrane is affected, leading to the inhibition of enzyme activity, and, in turn, the change in membrane potential inhibits seed germination, buds and root cell elongation.4.2 Effect of CaCl2 concentration on the seed germination of herbageKarst areas in the Guizhou Province are rich in calcium; the CaO weighted average content is 25.27%-55.63% [26]. Such high levels of calcium have become an important limiting factor for the germination of forage seeds. Salt stress can lead to ionic stress, osmotic stress, and secondary stresses, particularly oxidative stress, in plants [27]. The stress of salt on seed germination primarily includes osmotic stress, toxic effects, ion absorption imbalances and alkaline stress [28], However, as an important element for plant growth and development, calcium controls a series of important physiological processes and enzyme activities related to seed germination [29,30]. In this study, the germination of M. sativa seeds did not respond significantly to the increase in CaCl2 concentration, while the D. glomerata and L. perenne seeds germinated significantly more poorly under these conditions. Overall, there was no apparent effect of CaCl2 on the buds and young roots at low concentrations, while the seeds were significantly inhibited at high concentrations. These results indicate that D. glomerata andL. perenne seeds are likely to be more sensitive to CaCl2 than M. sativa seeds under salt-stressed or non-stressed conditions. They also showed that the M. sativaseeds had a higher tolerance to salt during germination than those of the forage crops red clover (Trifolium pratense L.) and white clover (T. repens L.) in comparison with their salt tolerance [31]. Although plant seedlings can grow in solutions with low salt concentrations, high salinity may substantially inhibit root elongation, particularly in the young roots of M. sativa [32]. High salinity is commonly owing to high concentrations of Na+ and Cl- in the soil solution, resulting in hyperosmotic and hyperionic conditions, respectively, which impede the plant from absorbing water and nutrients from the soil [33]. Ca is a vital element for cell division, the maintenance of structure and building cell walls, and it plays an important role in imbibition during seed germination. Ca2+ can mitigate the adverse effect of salinity during seed germination [2], which confirms the observed result of CaCl2 treatment in salt-stressed seeds. Higher salinity results in a greater reduction in the germination rate and increases the time to germination [34].4.3 Effects of different pH values and CaCl2concentrations on the germination of forage seedsIn this study, through a two-factor variance analysis of seed germination, pH and CaCl2 concentration, we found that the pH had a more significant effect on the seed germination rate ofM. sativa, D. glomerata and L. perenne compared with CaCl2, while the combined treatment of pH and CaCl2 significantly reduced the seed germination ofD. glomerata and L. perenne. Studies have shown that, owing to the high pH, a low concentration of alkali stress also strongly inhibited seedling growth, and the detrimental effect was much more marked than that of salt stress [35]. Alternatively, this study found that under the interaction of calcium salt stress and acid-base stress, the pH requirement of L. perenne decreased, and the CaCl2 increased, indicating that both are involved in the regulation of seed germination. Thus, the results also indicate that seed germination and seedling stages have different physiological responses to the salt and alkali stresses. The specific molecular mechanisms merit further research. To manage salt stress, plants have developed a series of strategies that are regulated by changes in gene and protein expression, which change in specific metabolic and signaling pathways. Our results clearly showed that the acid, alkali and salt tolerance of the three herbage seeds had a substantial influence on each other. When the salt stress concentration began to change, the herbage seeds could adapt to salt stress at a suitable pH condition. pH stress and calcium salt stress are actually two different types of stress, and the salt-alkali tolerance of the three herbage seeds is largely affected by the interaction of salt and pH.
Acknowledgements
We acknowledge the laboratory of the Department of Grassland Science, Guizhou University. We thank Xin Liu, Guiying Liu, Yini Wang and Xiaolong Tian, for their valuable suggestions and help with the laboratory analysis. This work was funded by the Science and Technology Department of Guizhou Province (Grant No. Qian Ke He Zhicheng [2020]1Y076 and [2021] Yiban503).Author Contributions:Conceptualization, Z.W., S.T., X.Z. and B.J.; methodology, Z.W., S.T., and X.Z.; software, Z.W., S.T., and X.Z.; validation, Z.W., B.J., X.Z., and H.S.; investigation, Z.W., X.Z., B.J., H.S., Y.Z. and Y.W.; writing—original draft preparation, Z.W., S.T., B.J. and X.Z. All authors have read and agreed to the published version of the manuscript.Finding: This work was supported by the Science and Technology Department of Guizhou Province (Grant No. Qian Ke He Zhicheng [2020]1Y076 and [2021]Yiban503).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Not applicable.Conflicts of Interest: The authors declare no conflict of interest.