In vitro and in vivo experiments
Antifungal effects of surfactin on B. cinerea in vitro
The rates of inhibition of mycelial growth of B. cinerea under surfactin stress are shown in Figure. 1 The results showed that the inhibition was concentration dependent over 5 days. The surfactin treatment groups (10 mg/L, 20 mg/L, 30 mg/L) showed the highest inhibition rates at 2 d, 1 d and 3 d, with 9.6%, 15.66% and 20.98% inhibition, respectively; at the end of the experiment (5 d), the inhibition rates decreased to 4.61%, 5.92% and 15.79%, respectively. The inhibition rates in the surfactin treatment groups (40 mg/L, 50 mg/L) increased significantly within 5 days. At 5 d, the inhibition rates in the 40 mg/L and 50 mg/L surfactin treatment groups were 44.08% and 53.95%, respectively, showing a significant concentration dependence (P < 0.05). The EC50 was 46.42 mg/L for 5 d.
Surfactin significantly reduced the spore germination rate of B. cinerea (Figure. 2). The inhibition rate was dependent on the surfactin concentration. The rate of inhibition in the 10 mg/L surfactin treatment group was low, only 13.63%. There was no significant difference between the 20 mg/L and 30 mg/L surfactin groups (P > 0.05). When the concentration of surfactin increased to 40 mg/L, the rate of inhibition increased to 33.71%, which was 2.47 times that of the 10 mg/L surfactin treatment group. The rate of inhibition in the 50 mg/L surfactin treatment group was 42.67%, which was 3.13 times that in the 10 mg/L group. The surfactin treatment groups (40 mg/L, 50 mg/L) showed a significant concentration dependence for the rate of inhibition (P < 0.05).
Inhibitory effects of surfactin on B. cinerea in vivo
As shown in Figure. 3, surfactin reduced the fruit disease incidence and spot diameter with artificial B. cinerea inoculation. The incidence in the surfactin treatment group was significantly lower than that in the control group (P < 0.05, Figure. 4a). At 2 d, the disease incidence was 61.9% in the control group and 19.05% and 9.52% in the 2 EC50 and 4 EC50 surfactin treatment groups, respectively. The disease incidence in the 8 EC50 surfactin treatment group was 0%. After 3 days of storage, the disease incidence in the 8 EC50 surfactin treatment group increased to 33.33%, and there was no significant difference between the 8 EC50 and the 4 EC50 surfactin treatment group (P > 0.05). At 4 d, the disease incidence rate with surfactin treatment at 2 EC50 was the lowest (52.38%). The disease incidence was 57.14% in the 4 EC50 and 8 EC50 surfactin treatment groups, showing no significant difference.
At 2 d, the lesion diameters in the 2 EC50, 4 EC50 and 8 EC50 surfactin treatment groups were 3.69, 1.31 and 0.83 mm, respectively, showing a significant difference (P < 0.05, Figure. 4b). At 3 d, the lesion diameters in the 2 EC50, 4 EC50 and 8 EC50 surfactin treatment groups were 1.86, 1.38 and 1.29 mm, respectively, which were 22.41%, 11.67% and 15.52% of those in the control group, respectively. At 4 d, the surface diameters in the 2 EC50, 4 EC50 and 8 EC50 treatment groups were 22.60%, 17.36% and 15.37% of that in the control group, while the surface diameters in the three surfactin treatment groups were 2.98, 2.29 and 2.02 mm, respectively, showing a significant difference compared with the control group (P < 0.05). The surfactin concentration was positively correlated with the diameter of the disease spots.
Antifungal mechanisms of surfactin
Effects of surfactin on the mycelial morphology of B. cinerea
Through scanning electron microscopy, we observed that the mycelia in the control group were complete, smooth and normal without fracture (Figure. 5a, red arrow). The mycelia in the 4 EC50 surfactin treatment group were completely distorted and wrinkled (Figure. 5b, red arrow), with no intact mycelium observed.
Effects of surfactin on the membrane integrity of B. cinerea
PI is a common nucleic acid dye that can penetrate the membrane of dead cells and stain the nucleus to appear red in a fluorescent environment. The effects of different concentrations of surfactin on the cell integrity of B. cinerea are shown in Fig. 6. There was a small amount of red fluorescence in the control group (Figure. 6a), while the red fluorescence in the surfactin-treated group covered almost the entire visual field (Figure. 6b). Among the spores, only a very small number were stained by PI in the control group (Figure. 6c), while the number of spores stained by PI in the surfactin-treated group was significantly higher than that in the control group (Figure. 6d).
Effects of surfactin on the total lipid and ergosterol levels of B. cinerea
Surfactin significantly reduced the total lipid and ergosterol levels of B. cinerea (Figure. 7). The total fat content in the control group was 145.62 g/kg and that in the 4 EC50 surfactin treatment groups was 122.36 g/kg, showing a significant difference (P < 0.05). The ergosterol content in the control group was 3.07%, and that in the 4 EC50 surfactin treatment groups was decreased to 2.07%, showing a significant difference (P < 0.05).
Effects of surfactin on the ROS content of B. cinerea
The fluorescent probe DCFH-DA can be hydrolyzed to DCFH by esterase across the cell membrane, which can be oxidized by intracellular ROS to generate DCF with green fluorescence. The effects of surfactin on the ROS content of B. cinerea are shown in Fig. 8. In mycelia, there was a small amount of intermittent green fluorescence in the visual field of the control group (Figure. 8a), while in the 4 EC50 surfactin-treated group, there was a large amount of green fluorescence with strong continuity (Figure. 8b). In spores, the amount of green fluorescence in the 4 EC50 surfactin treatment groups was significantly higher than that in the control group (Figure. 8c, d).
Changes in fatty acid composition in winter jujube as affected by gray mold and surfactin treatment
In total, 12 fatty acids were identified in different winter jujube groups, including 5 saturated fatty acids (SFAs), 5 monounsaturated fatty acids (MUFAs) and 2 polyunsaturated fatty acids (PUFAs) (Table 1). SFAs accounted for the highest proportion (48.31–54.87%) in each group. The dominant SFAs were palmitic acid (C16:0) and stearic acid (18:0), accounting for 32.00-36.06% and 14.66–17.37%, respectively. The SFA content in the mechanical injury group was the highest, and those of C16:0 and C18:0 were 35.87% and 17.37%, respectively. The SFA content in the inoculated control group was the lowest, and those of C16:0 and C18:0 were 32.00% and 14.66%, respectively. Compared with the nonmechanical injury group, the increase in C18:0 in the mechanical injury group was statistically significant (P < 0.05). Compared with the mechanical injury group, the SFA content in the 2EC50 group was statistically similar (p > 0.05), while in the 8EC50 group, it decreased significantly (P < 0.05).
Overall, MUFAs were significantly higher than PUFAs (P < 0.05). These two groups of unsaturated fatty acids (UFAs) accounted for 30.27%-33.85% and 13.24%-18.24%, respectively (Table 1). The dominant UFAs were palmitoleic acid (C16:1), oleic acid (C18:1), linoleic acid (C18:2) and α-linolenic acid (C18:3). UFAs in the mechanical injury group were the lowest, among which the percentages of C16:1, C18:1, C18:2 and C18:3 were 14.8%, 14.94%, 8.74% and 4.50%, respectively. UFAs in the inoculated control group were the highest, among which the percentages of C16:1, C18:1, C18:2 and C18:3 were 14.63%, 17.96%, 13.49% and 4.35%, respectively. Compared with the mechanical injury group, UFAs in the 2EC50 group were statistically the same (p > 0.05), while those in the 8EC50 group were significantly higher (P < 0.05) and were similar to those in the nonmechanical injury group (P < 0.05).
Table 1
Fatty acid composition (%) of winter jujube in different treatment groups. (lowercase letters show the statistical significance of the difference between groups)
Fatty acids
|
nonmechanical injury group(A)
|
mechanical injury group(B)
|
inoculated control group(C)
|
2EC50 Surfactin(D)
|
8EC50 Surfactin(E)
|
C14:0
|
1.39 ± 0.28a
|
1.47 ± 0.14a
|
1.05 ± 0.11a
|
1.20 ± 0.24a
|
1.28 ± 0.25a
|
C14:1(n-3)
|
0.45 ± 0.02b
|
0.77 ± 0.01a
|
0.43 ± 0.00b
|
0.26 ± 0.16b
|
0.41 ± 0.01b
|
C16:0
|
32.59 ± 0.23ab
|
35.87 ± 0.23ab
|
32.00 ± 0.46b
|
36.06 ± 1.92a
|
32.37 ± 0.58ab
|
C16:1(n-7)
|
12.57 ± 0.18c
|
14.8 ± 0.08a
|
14.63 ± 0.55a
|
14.10 ± 0.06ab
|
13.28 ± 0.28bc
|
C17:0
|
0.53 ± 0.01a
|
0.16 ± 0.20ab
|
0.13 ± 0.16ab
|
-b
|
0.55 ± 0.03a
|
C18:0
|
15.23 ± 0.34bc
|
17.37 ± 0.19a
|
14.66 ± 0.25c
|
15.91 ± 0.45b
|
14.78 ± 0.45c
|
C18:1(n-9)
|
7.50 ± 0.09c
|
7.12 ± 0.07c
|
10.92 ± 0.48a
|
9.04 ± 0.21b
|
8.86 ± 0.24b
|
C18:1(n-7)
|
8.54 ± 0.12b
|
7.82 ± 0.07c
|
7.04 ± 0.05d
|
8.40 ± 0.19bc
|
9.40 ± 0.26a
|
C18:2(n-6)
|
12.18 ± 0.09b
|
8.74 ± 0.14d
|
13.49 ± 0.28a
|
11.74 ± 0.13bc
|
11.49 ± 0.04c
|
C18:3(n-3)
|
6.06 ± 0.20a
|
4.50 ± 0.25ab
|
4.35 ± 0.13ab
|
2.86 ± 1.75b
|
4.99 ± 0.26ab
|
C22:0
|
1.75 ± 0.03a
|
-c
|
0.47 ± 0.58bc
|
-c
|
1.13 ± 0.70ab
|
C22:1(n-9)
|
1.21 ± 0.19ab
|
1.37 ± 0.08a
|
0.83 ± 0.53ab
|
0.41 ± 0.50b
|
1.47 ± 0.12a
|
SFA
|
51.49 ± 0.28bc
|
54.87 ± 0.27a
|
48.31 ± 0.32d
|
53.18 ± 2.13ab
|
50.11 ± 0.45cd
|
MUFA
|
30.27 ± 0.56c
|
31.89 ± 0.03b
|
33.85 ± 0.5a
|
32.21 ± 0.82b
|
33.42 ± 0.65ab
|
PUFA
|
18.24 ± 0.29a
|
13.24 ± 0.29c
|
17.85 ± 0.18a
|
14.61 ± 1.7bc
|
16.47 ± 0.3ab
|
UFA
|
48.51 ± 0.85bc
|
45.13 ± 0.31c
|
51.69 ± 0.69a
|
46.82 ± 2.52cd
|
48.89 ± 0.95ab
|
Principal component analysis (PCA) was conducted on the composition of fatty acids in different groups (Fig. 9). The contribution rate of the first component was 36.9%, that of the second component was 30.4%, and the total contribution rate of the two components was 67.3% (Fig. 9). The mechanical damage group (B) was further different from the other groups. The inoculated control group (C) and the two surfactin treatment groups (D, E) were discrete. 8 EC50 surfactin treatment group (E) and the nonmechanical injury group (A) partially overlapped, showing no significant difference.