The C. longepaniculatum was collected from the C. longepaniculatum base of Hongyan Mountain in Yibin, and an endophytic fungus 2J1 (Penicillium commune) was isolated from the C. longepaniculatum plant and identified in the early stage. It was preserved in PDA medium [16] .

40 mg/L of endophytic fungus 2J1 crude elicitor was added to the C. longepaniculatum suspension cell culture until the 7th day, and an equal amount of PDA medium was added as a control. The effects of endophytic fungus 2J1 crude elicitor on Ca²⁺ concentration and essential oil content in C. longepaniculatum suspension cells within 24 h were shown in Figures 1-3, respectively. The results shown in the figure are the average of 3 independent experiments.

It can be seen from Figure 1 that the C. longepaniculatum suspension cells treated by the 2J1 crude elicitor can produce a change in Ca²⁺ concentration. The maximum intracellular Ca²⁺ concentration is 21 h after the elicitor treatment, and the Ca²⁺ concentration in the treated group at this time is about 1.66 times that of the control group. As shown in Figure 2 and Figure 3, the amount of essential oil produced by the C. longepaniculatum cell suspension cells after the inducer treatment is significantly different from that of the control group, and the content of the cells grows with the growth of the cells, and the cells grow for 21 days, (14 d after treatment), the yield peaked. At this time, the cells treated with the inducer 2J1 produced 1,8-eucalyptus oil in an amount of 11.725 mg/L, and the produced α-terpineol was 4.577 Mg/L. The amount of volatile oil that is subsequently produced gradually decreases.

After 7 days of culture of suspension culture of C. longepaniculatum, 2J1 crude elicitors of endophytic fungi with different concentrations (0, 20, 40, 60, 80 mg/L) were added to detect the change of Ca²⁺ concentration and accumulation of volatile oil in suspension cells of C. longepaniculatum. The amount is shown in Figures 4-6. The results shown in the figure are the average of 3 experiments.

Figure 4 shows that the change in Ca²⁺ concentration in the suspension cells of C. longepaniculatum is dependent on the concentration of endophytic 2J1 elicitor. When the concentration of elicitor was 0 - 40 mg/L, the concentration of Ca²⁺ in the suspension cells of C. longepaniculatum increased gradually. When the concentration of inducer was 40 mg/L, the concentration of Ca²⁺ in the suspension cells of C. longepaniculatum peaked. When the concentration is greater than 40 mg/L, the intracellular Ca²⁺ concentration gradually decreases. This phenomenon is similar to the dependence of the accumulation of volatile oil in the C. longepaniculatum suspension cells on the endophytic fungal 2J1 elicitor concentration as shown in Figure 5 and Figure 6. This suggests that the Ca²⁺ signaling pathway may be one of the signaling pathways that mediate endophytic fungal elicitors to promote the accumulation of volatile oil in C. longepaniculatum suspension cells.

In order to further study the role of Ca²⁺ in the endophytic fungus 2J1 crude elicitor in the synthesis of volatile oil from the suspension of C. longepaniculatum, the concentration of Ca²⁺ in the suspension of C. longepaniculatum in 5 groups was detected after 7 days of culture. The situation (Figure 7) and the synthesis of volatile oil (Figure 8), wherein the endophytic fungi 2J1 crude elicitor, exogenous Ca²⁺, LaCl₃ were added at a concentration of 40 mg/L, 10 mmol/L, 20 mmol/L.

图3-8 5个组中挥发油的合成情况

As can be seen from Figure 7 and Figure 8, in the control group without the inducer added, the concentration of Ca²⁺ in the suspension cells of the C. longepaniculatum was low, and the amount of 1,8-eucalyptus oil that can be detected was only 4.43 mg/L. Adding the endophytic fungus 2J1 crude elicitor can increase the concentration of Ca²⁺ in the suspension cells of C. longepaniculatum (compared with A and B in Figure 7); it can also promote the accumulation of volatile oil, and the 1,8-eucalyptol under the action of 2J1 elicitor, the amount of synthetic oil was 13.41 mg/L, which was 3.03 times that of the control group (compared with A and B in Figure 8). The addition of exogenous Ca²⁺ promoted the increase of Ca²⁺ concentration in the suspension of C. longepaniculatum (compared with A and C in Figure 7); it also promoted the accumulation of volatile oil (compared with A and C in Figure 8), but The promotion effect was inferior to the addition of the endophytic fungus 2J1 crude elicitor experimental group (comparison of A, B and C in Figure 8). Adding endophytic fungus 2J1 crude elicitor and exogenous Ca²⁺ promoted the increase of Ca²⁺ concentration in the suspension of C. longepaniculatum, and the Ca²⁺ concentration reached the maximum (compared with A and D in Figure 7), and also promoted the synthesis of volatile oil (compared with A and D in Figure 8). The concentration of Ca²⁺ in the suspension cells was increased after add exogenous Ca²⁺ and LaCl₃ compared with the control group, but the content of volatile oil decreased slightly (compared with A and E in Figure 7, A and in Figure 8). This indicates that LaCl₃ can inhibit the promotion of volatile oil synthesis by endophytic fungal elicitors and exogenous Ca²⁺.

图3-8 5个组中挥发油的合成情况

It is generally believed that the signal transduction process of endophytic fungal inducer synthesis of secondary metabolites in plant cells is: endophytic fungal elicitor as an extracellular stimulator first recognizes and binds to specific receptors on plant cell membranes, thereby promoting the cells produce specific intracellular messenger substances and regulate the expression of related genes in the nucleus through corresponding signal transduction pathways [19] , ultimately activating the defensive secondary metabolic system in the cells to promote secondary metabolite synthesis. Therefore, according to this theory, it can be speculated that the change of Ca²⁺ concentration in cells after 2J1 crude elicitor treatment may be the pre-reaction, and calcium as a common second messenger in plant cells, after a series of process reactions, eventually leading to the accumulation of volatile oil. Under stress conditions such as elicitors, plant cells can sense and transduce external stress signals through a variety of signaling molecules and signaling pathways [19] . Studies have shown that the calcium signaling pathway is located upstream of the NO signaling molecule [13] . The calcium signaling pathway is thought to be an important pathway for fungal elicitors to induce plant cell defense responses [12] . The results of this study indicated that the concentration of Ca²⁺ reached the peak at 21 h after culture of the endophytic fungus 2J1 crude elicitor. At the same time, the endophytic fungus 2J1 crude elicitor was used to treat the suspension of C. longepaniculatum. After data analysis, it was found that the concentration of Ca²⁺ and the accumulation of volatile oil in the suspension of C. longepaniculatum showed a similar trend with the change of concentration; further study the role of Ca²⁺ on the synthesis of volatile oil in the suspension of C. longepaniculatum by 2J1 crude elicitor. The C. longepaniculatum suspension cells may exist a signal pathway regulating the synthesis of volatile oil: calcium signaling pathway. And this pathway has different effects in regulating the different components of synthetic volatile oil. Adding endophytic fungus 2J1 crude elicitor can promote the synthesis of α-terpineol to 3.31 times of the control group, while 1,8-eucalyptol can only increase by 1.90 times; addition of exogenous Ca²⁺ can also promote the increase of volatile oil, but the effect is not as good as after the addition of inducer. Moreover, the three experimental groups added with LaCl₃ showed that the synthesis of volatile oil was significantly inhibited.

Although this study has further studied the regulation mechanism of Ca²⁺-mediated endophytic fungal elicitors on the synthesis of oil volatile oil, there are two aspects that can be further studied: the endophytic fungus crude inducer is used in this study. The filtrate of the mycelium is inactivated, and the endophytic fungal inducers are divided into four categories: oligosaccharides, glycoproteins, proteins, and unsaturated fatty acids. The components are complex, and it is presumed to be oligosaccharides in this experiment. Oligosaccharides are one of the more common inducers [20] . However, in order to fully exert the inducer effect, it is necessary to further isolate and purify the endophytic fungus 2J1 crude elicitor, and explore a better method for preparing elicitors [21] . The addition of LaCl₃ could not completely inhibit the accumulation of volatile oil from the endophytic fungus 2J1 crude elicitor on the suspension of C. longepaniculatum, indicating that endophytic fungal elicitors can also cause accumulation of volatile oil by other means, or it may be calcium signal. With multiple calcium signaling pathways, LaCl₃ may only inhibit one of the signaling pathways. Therefore, the synthetic pathway of plant secondary metabolites is very complicated, especially the involvement of calcium signaling pathways in secondary metabolic regulation is particularly complicated, and further research is needed.