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).
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.
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.