The use of surfactin in inhibiting Botrytis cinerea and in protecting winter jujube from the gray mold

DOI: https://doi.org/10.21203/rs.3.rs-2272644/v1

Abstract

Surfactin has the potential to be used as a food preservative. However, efficiency and action mechanism in various applications need more assessments and research. In this study, the antifungal effects and the mechanism of action of surfactin on the fungus Botrytis cinerea were investigated. The effects of applying surfactin for the removal of gray mold on the quality of winter jujube were investigated based on the changes in fruit fatty acids. The results showed that (1) surfactin significantly inhibited the growth of B. cinerea, the EC50 at 5 d was 46.42 mg/L. (2) Surfactin significantly reduced the disease incidence and diameter of gray mold-inoculated winter jujube in a concentration-dependent manner. For that treated with surfactin at the EC50, the incidence decreased by 38.89%. (3) For B. cinerea under surfactin treatment, the mycelial morphology changed, the levels of total lipids and ergosterol decreased, the reactive oxygen species levels increased, and the cell integrity was completely damaged. (4) For winter jujube inoculated by B. cinerea, the contents of saturated fatty acids decreased and unsaturated fatty acids increased. For those under the surfactin treatments, winter jujube maintained the fatty acid composition at the level of non-inoculated groups. Mechanical injury significantly changed the fatty acid composition of winter jujube; however, Surfactin not only was able to inhibit the growth of gray mold but also mitigated the adverse effects from mechanical injury. The present study demonstrated the potential applications of Surfactin in the preservation of postharvest fruit quality.

Introduction

The airborne fungus Botrytis cinerea is a common pathogenic fungus that causes postharvest rot of fruits and vegetables (Dean et al. 2012), leading to rot of tomato, strawberry, grape, eggplant, cucumber and various flowers and other plants (An et al. 2016; Keller et al. 2003; Ren et al. 2018; Williamson et al. 2007. Qin et al. 2004). Due to its characteristics of rapid reproduction, strong adaptability and wide transmission channels (Staats et al. 2005). B. cinerea has gradually become one of the important pathogenic fungi affecting the fruit and vegetable industry. On the one hand, it causes serious economic losses; on the other hand, rotten fruits and vegetables carrying pathogens and even producing some secondary toxic metabolites are harmful to human and animal health (Weiberg et al. 2013; Yang et al. 2014; Fernandez-Cruz et al. 2010; Enikova et al. 2020). Winter jujube is favored by global consumers for its rich nutrition and crisp taste (Gao et al. 2013; Wang et al. 2016; Pareek et al. 2013; Li et al. 2007). However, winter jujube is not resistant to storage. The storage period was only 2–5 days at room temperature. After harvest, it is easily infected by fungi such as B. cinerea, Alternaria spp., and Colletotrichum gloeosporioides. (Mirzaee et al. 2014). At present, methods for the prevention and control of postharvest gray mold of fruits and vegetables mainly rely on chemical drugs for sterilization. Although chemical fungicides can effectively inhibit the growth of fungi or kill them, the challenges posed by the residues and toxicity of chemical reagents, as well as drug resistance resulting from the continuous use of single chemical fungicides, make it difficult to use these methods on a large scale (Leroux et al. 2010; Janisiewicz et al. 2002; Jamalizadeh et al. 2011). Therefore, safer and more effective substitutes for chemical fungicides are being sought by researchers. Many previous studies have shown that microorganisms or metabolites with antibacterial activities can inhibit the generation of nutrients needed for the growth and development of pathogens and improve the resistance of plants to diseases (Dik et al. 1999; Zahavi et al. 2000; Zhang et al. 2005; Yu et al. 2008). Among them, Bacillus species have attracted much attention as biological control agents and play an important role in the control of B. cinerea (Akutsu et al. 1994; Hmidet et al. 2019).

Surfactin is a kind of lipopeptide biosurfactant mainly produced by the secondary metabolism of Bacillus (Arima et al. 1968; Park et al. 2019). Surfactin is a bacterial cyclic lipid peptide that contains seven amino acids and has an LLDLLDL chiral sequence linked by lactone bonds to β-hydroxy fatty acids with 13–15 carbon atoms (Abdelmawgoud et al. 2008; Peypoux et al. 1999). It represents a class of biosurfactants with the highest surface activity and has been widely applied thus far in oil recovery (Hadia et al. 2019; Yang et al. 2020), food (Gomes et al. 2012), medicine (Ahire et al. 2017; Liu et al. 2019) and agriculture (Desmyttere et al. 2019; Hazarika et al. 2019) application prospects and has been reported to have good antibacterial activity against a variety of bacteria and fungi, such as Gramella and mold, as well as viruses and mycoplasma (Gomaa et al. 2013; Horng et al. 2019; Płaza et al. 2013). The antibacterial activity of surfactin is thought to be closely related to its physicochemical properties (Patel et al. 2014; Buchoux et al. 2008); however, the underlying mechanism remains unclear. It is not known whether fruit quality is affected when applying surfactin for the control of gray mold during fruit preservation.

In this paper, the antifungal effects of surfactin on B. cinerea were studied. By observing and measuring the mycelial morphology, cell membrane system and accumulation of reactive oxygen species (ROS) in B. cinerea, the mechanism underlying the inhibitory effect of surfactin on B. cinerea was proposed. Fatty acid composition affects the fluidity of the cell membrane, which is closely related to fruit and vegetable texture. To evaluate the effects of surfactin on the control of B. cinerea, the changes in the composition of fatty acids in winter jujube were comparatively studied. The purpose of the present study was to (1) evaluate the inhibitory effect of surfactin on B. cinerea in vitro and in vivo; (2) analyze the cell rupture mechanism involved in (1); and (3) preliminarily evaluate the protective effect of surfactin on winter jujube from gray mold.

Materials And Methods

Biosurfactant and fruit

Surfactin was purchased from MedChemExpress LLC (Biological Company, Shanghai, China) (product number HY-129555), and the product purity was > 98%. The powdered surfactin reagent was dissolved in dimethyl sulfoxide (DMSO) to prepare the stock solution beforehand for use in the experiment.

Winter jujube was purchased from Jirong Picking Garden (Fruit Orchard, Binzhou, China). The fruit selected in the experiment were uniform in size and free from diseases, insect pests and mechanical injuries, and the brick red area of fruit skin was over 1/3 of the total area.

Antifungal effects of surfactin on B. cinerea in vitro and in vivo

In vivo experiments

Freshly prepared surfactin was added to sterilized PDA to prepare media with final surfactin concentrations of 10, 20, 30, 40, and 50 mg/L. Medium supplemented with an equal volume of DMSO was used as the control. The above medium was poured into a glass petri dish, and a cake of B. cinerea (ACCC 36028, purchased from the Agricultural Culture Collection of China) (9 mm) was inoculated into the center of the PDA petri dish. Each experimental group was set up with three replicates. The Petri dishes were cultured at 25°C for 5 days, and the colony diameter was measured regularly every day.

The inhibition rate was calculated as follows: IR(%) = [(D0-Dt)-9]/(D0-9)×100%, where Dt (mm) is the colony diameter of surfactin added to the experimental group. D0 (mm) is the colony diameter of the control group, and 9 (mm) is the diameter of the B. cinerea cake.

Five milliliters of sterile water was added to the petri dish after 7 days of culture, and the conidia were eluted with a sterilized coating rod to prepare a spore suspension. The number of spores was adjusted to 1×105 cfu/mL, and 2 ml of spore suspension was added to PDB medium. Freshly prepared surfactin was added to the medium to prepare experimental groups with final surfactin concentrations of 10, 20, 30, 40, and 50 mg/L, while DMSO was added to the control group. Three repetition groups were set for each concentration. Shock cultures at 120 r/min at 25°C for 6 h were observed under an optical microscope. Approximately 200 spores were selected to calculate the spore germination rate.

The rate of inhibition of spore germination was calculated as follows: IR(%) = (C-T)/C×100%, where T(%) is the spore germination rate of the experimental group with surfactin added, and C (%) is the spore germination rate of the control group.

In vitro experiment

The purchased fresh winter jujubes were disinfected with ethanol (75%) and air-dried for 1 h. A sterilized stainless steel rod (3 mm in diameter) was used to introduce a wound (3 mm in depth) to one side of the fruit. Each wound was inoculated with 10 µL of spore suspension (1×105 cfu/mL), and the fruit were stored at 25°C for 1 h. The inoculated jujubes were randomly divided into four groups. A total of 10 µL of freshly prepared surfactin at concentrations of 2 EC50, 4 EC50, and 8 EC50 was inoculated into the wounds. Winter jujubes without surfactin and with equal volumes of DMSO were used as the control group. Five repetitions were set for each group. Each replicate group contained 5 pieces of the fruits. The treated fruits were removed from the container and dried at room temperature. After storage at 20°C for 5 days, the disease incidence in winter jujube and the diameters of the disease spots (mm) were recorded regularly every day.

Inhibitory mechanism of surfactin on B. cinerea

Culture and collection of mycelia and spores

One milliliter of spore suspension (1×106 cfu/mL) was added to 100 mL of PDB medium. Shock culture was performed at 25°C and 120 rpm for 69 h. Freshly prepared surfactin solution was added to the medium at a working concentration of 4 EC50, and spore suspension with an equal volume of DMSO was used as the control group. The spore suspension was incubated at 25°C at 120 rpm for 3 h. After the culture, the mycelium was centrifuged at 4500 rpm for 5 min and washed with PBS solution (pH 7.8) three times.

Freshly prepared surfactin at a working concentration of 4 EC50 was added to 10 mL of PDB medium with 100 µL of spore suspension (1×106 cfu/mL). Cells were cultured at 25°C and 120 rpm for 3 h. After centrifugation at 5500 rpm for 5 min, spores were collected and washed with PBS solution (pH 7.8) three times.

Effects of surfactin on mycelial morphology

The collected mycelia were fixed with 2.5% glutaraldehyde for 24 h. After washing three times with PBS solution (pH 7.8), dehydration was carried out with anhydrous ethanol. Then, the mycelia were freeze-dried for 48 h and sprayed with gold coating for 1 min by ion sputtering. Mycelial morphology was observed by scanning electron microscopy (Hitachi, Tokyo, Japan).

Effects of surfactin on the plasma membrane integrity of B. cinerea

Mycelia and spores after culture collection were stained with 10 µM propidium iodide (PI) and incubated in darkness at 37°C for 30 min. The samples were centrifuged at 4500 rpm for 5 min and washed with PBS (pH 7.8) three times. The cells were observed under a laser scanning confocal microscope (Zeiss, Germany).

Effects of surfactin on total lipid and ergosterol levels in mycelia

Cultured and collected mycelia were freeze-dried for 48 h. Dried mycelia (0.05 g) were ground to powder with liquid nitrogen, and 4.0 mL of a methanol:chloroform:water mixture was added (2:1:0.8). The supernatant was centrifuged after shaking for 10 min. Then, 0.2 mL of 0.9% NaCl was added. Then, 0.5 mL of 90% H2SO4 was added to the collected precipitate and heated in a water bath at 100°C for 10 min. Then, 3 mL of vanillin phosphate solution was added, and the solution was mixed well and allowed to stand for 15 min. The absorbance value at 520 nm was determined, and the total fat content was calculated using a cholesterol standard curve.

Dried mycelia (0.1 g) were homogenized with liquid nitrogen, and 3 mL of 25% KOH ethanol (W/V) solution was added. After 20 min of ultrasonication, the sample was placed in a water bath at 80°C for 2 hours. One milliliter of sterile water and 3 mL of n-heptane were mixed for 5 min and then allowed to stand and stratify. The absorbance of the upper organic phase was measured at 281 nm and 230 nm.

The formula for calculating the ergosterol content was as follows: C(%) = [(A281/290)-(A230/518)]/W, A281 and A230 were the absorbance of ergosterol and 24 (28) dehydroergosterol, respectively, and W is the net dry weight (g) of the thallus (Johnston et al. 1984).

Changes in the ROS levels of B. cinerea under surfactin stress

Mycelia and spores were cultured and collected for later use. ROS detection kits (Solarbio Biology Engineering Institute, Beijing, China) were used to determine the ROS levels of mycelia and spores treated with surfactin in accordance with the operating manual.

Changes in the fatty acid composition of winter jujube as affected by gray mold and surfactin treatment

The purchased winter jujube was disinfected with ethanol (75%) and air-dried for 1 h. A sterilized stainless steel rod (diameter: 3 mm) was used to introduce a wound (depth: 3 mm) on one side of the fruit. These punched fruits were divided into four groups. Three groups were inoculated with 10 µL of spore suspension (1×105 cfu/mL). The inoculated fruit were stored at 25°C for 1 h. Then, two groups of inoculated fruits were injected with 10 µL of surfactin solutions with working concentrations of 2 EC50 and 8 EC50 into the wound surface. Another contaminate group was treated with the same volume of DMSO and was set as the inoculated control group. The wounded fruit that were not treated with spore suspension or surfactin were defined as the mechanical injury group. Five replicates of each group were set, and five fruits in each replicate contained five fruits. After drying at room temperature, all the groups were stored at 20°C for 5 days.

Fatty acid analysis was conducted according to the method of Metcalfe (Metcalfe et al. 1996). To sample the fruit tissue for fatty acid analysis, a fruit digger was used to extract pulp from around the wound. The same amount of pulp was sampled from the fruit part that was not punched or inoculated. These samples were grouped into nonmechanical injury groups. 2 g of the samples from each group was freeze dried. 100 mL of the frozen dried sample was ground to powder, and 2 mL of chloroform:methanol solution (V/V = 1:1) was added. After ultrasonic crushing for 10 min, centrifugation was performed at 3000 r·min− 1 for 10 min. The supernatant was collected and dried in a rotary evaporator (Great Wall Technology & Trade Co., LTD., Zhengzhou, China), and this process was repeated twice. Then, 100 µL of 1 µg·µL− 1 C19:0 fatty acid internal standard and 2 mL of 5%-6% potassium hydroxide methanol solution (V/V = 4:1) were added, and saponification was performed in a water bath at 60°C for 2 h. After cooling, 6 mL of chloroform n-hexane mixture (V/V = 1:4) was added for extraction, and the mixture was centrifuged at 3000 r·min− 1 for 10 min; the supernatant was collected and evaporated in a rotary evaporator. Then, 0.5 mL of boron trifluoride methanol solution was added to the water bath at 60°C for 1 h. After cooling, 6 mL of analytical grade n-hexane was used for extraction. After collecting the supernatant, 3 g of anhydrous sodium sulfate was added, and the solution was allowed to stand for 3 h. The supernatant was cyclically steamed again and analyzed by GC-MS (NYSE: A, Palo Alto, America) with 1 mL of chromatographic n-hexane at constant volume.

Based on the mass spectrometry analysis of each component, the molecular weight was determined. The mass spectra of the ion fragments were combined and searched against the NIST library and WILEY library. The percentage of fatty acids was calculated by the area normalization method. Based on the initial concentration and peak area of the internal standard C19:0 fatty acid and the peak area of each component to be measured, the absolute content of fatty acid components was calculated, and the response factor was calculated as 1.

Data analysis

Statistical analysis was carried out using the social science statistical software package (SPSS). The EC50 was obtained by linear regression between the surfactin concentration and rate of inhibition of B. cinerea using SPSS. One-way ANOVA (LSD) was used to determine the differences between the surfactin treatment group and the control group. Principal component analysis (PCA) was used to analyze the differences in fruit fatty acids among the different treatment groups. The significance level was set as P < 0.05. Origin software was used for plotting the data.

Results

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.

Discussion

In the present study, surfactin had a significant inhibitory effect on Botrytis cinerea. The higher the concentration was, the stronger the inhibition. Bacillus species produce a variety of antibacterial compounds, among which cyclolipopeptides (LPs) are a class of antibacterial substances with surfactant properties, including mainly iturin, fengycin and surfactin. LPs have bacteriostasis, growth promotion and defense enzyme activity induction effects (Vanittanakom et al. 1986; Ongena et al. 2008; Liu et al. 2010; Zhang et al. 2010; Goussous et al. 2017). Płaza et al. (2013) isolated surfactin from Bacillus subtilis cultured with molasses and explored its antibacterial activity in vitro. The results showed that it had a mild inhibitory effect on several common plant pathogens, such as B. cinerea A258, Nuclear discomycetes K2291 and Colletotrichum gloeosporioides A259.

Many studies have shown that the cell membrane is the most important target of active bacteriostatic substances of Bacillus (Zouari et al. 2016; Senna et al. 2017; Zhang et al. 2013; Guo et al. 2014). In the present study, surfactin caused deformation of the mycelia of B. cinerea, which usually indicates increased cell permeability and provides channels for leakage of small molecules and ions within cells. Jin et al. (2018) found that bacillomycin D, a secondary metabolite of Bacillus velezensis HN-2, caused irreversible damage to the cell wall and cell membrane of hyphae and spores of anthrax, leading to exudation of cell contents. Wang et al. (2020) found that surfactin and fengycin B obtained from Bacillus pumilus W-7 could inhibit Phytophthora filaria by inducing mycelial deformation. Xin et al. (2019) isolated the alkaloid antofine from Cynanchum atratum and found that it significantly damaged the membrane integrity of Penicillium digitatum after staining with PI. In this study, we found that the fluorescence intensity of the PI-stained cells was significantly increased in the surfactin-treated groups, indicating that surfactin treatment damaged the integrity of the cell membrane of B. cinerea (Setiawati et al. 2017; Zhang et al. 2019), which inhibited mycelial growth and spore germination.

Lipids are the main component of the cell membrane, and the reduction in lipid content usually hinders the transport of lipid soluble substances, resulting in the loss of selective permeability of the cell membrane (Wei et al. 2010). Helal et al. (2010) found that citronella significantly reduced the total lipid content of Aspergillus niger, leading to functional damage of cell membranes. Ergosterol is a unique component of the fungal cell membrane and plays an important role in protecting cell membrane integrity, fluidity and material transport (Verma et al. 2002; Li et al. 2012). Siahmoshteh et al. (2018) showed that the culture filtrate of Bacillus subtilis and Bacillus amyloliquefaciens affected ergosterol synthesis of parasitic Aspergillus NRRL2999, resulting in impaired cell membrane integrity. In this study, we found that surfactin significantly reduced the total lipid and ergosterol contents of B. cinerea, suggesting that cell membranes are important targets for surfactin.

ROS play an important role in cell viability, and excessive ROS production can cause oxidative damage to compounds in cells, inhibit enzymes, destroy cell membranes, and lead to cell dysfunction or death (Pi et al. 2010; Shi et al. 2012; Marchi et al. 2011). Massawe et al. (2018) showed that bacteriostatic substances produced by Bacillus velezensis VM11 induced the accumulation of ROS in the mycelial cells of Sclerotinia sclerotiorum, leading to cell damage. In this study, we found that surfactin can cause high accumulation of ROS in B. cinerea cells, suggesting that lipids, DNA and proteins in mycelia and spore cells may have undergone severe oxidative damage, which inhibited spore germination and mycelial growth.

Fruits are susceptible to fungal infection and mechanical injury during the postharvest or storage periods. From the results of the present study, surfactin had good antifungal activity in vivo, at least in inhibiting the growth of gray mold. Changes in fatty acids are related to cell membrane stability, fluidity and cell wall structure, which can affect fruit and vegetable quality (Meyer et al. 2010; Zhang et al. 2018). Chen et al. (2011) found that the lipoxygenase (LOX) activity in longan peel increased after Phomopsis longanae Chi infection, while the relative UFA content decreased and the relative SFA content increased. From the results of our study, the compositions of fatty acids were significantly changed by different treatments. Mechanical injury resulted in varied compositions of fatty acids that were significantly different from the nonmechanical injury groups and the gray mold-inoculated groups (both the control and the surfactin-treated groups). However, surfactin treatment, especially with the higher concentration (8EC50), helped the fruit maintain the fatty acid composition similar to that of the nonmechanical injury groups. The surfactin treatment group was mechanically injured before being inoculated with mold and surfactin. This phenomenon demonstrated that surfactin not only inhibited the growth of gray mold but also maintained the fatty acid quality of the fruit when it was mechanically injured. The fatty acid composition could be changed by physical treatment, such as a lower temperature. Cao et al. (2009) showed that methyl jasmonate can reduce the degree of chilling injury in loquat by reducing the fatty acid composition of the cell membrane. Gray mold combination significantly decreased the composition of SFA, while in the surfactin treatment groups, SFA contents increased back to the level of the non-inoculated groups. The contents of UFAs, such as C18:1 and C18:2, were also significantly affected by surfactin treatments, showing that lipid metabolism related to these fatty acids in the interaction of gray mold and mechanically injured fruit was altered by surfactin. These results suggest that the metabolic target for further studies in the mechanism of gray mold combination.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Acknowledgments

We thank the National Science and Technology Basic Resources Investigation Program of China. 

Authors’ contributions 

C.Z and P.Z designed the experiments and managed the project. P. X, X.T and Y.X performed the experiments and the data analysis. P. X, X.T and C.Z mainly wrote the manuscript. All authors contributed to manuscript writing and reviewing and approved the final version for submission. 

Funding

This work was supported by the National Science and Technology Basic Resources Investigation Program of China (2018FY100206). 

Availability of data and materials

All data generated and analyzed during this study are included in this published article.

References

  1. Abdel-Mawgoud AM, Aboulwafa MM, Hassouna AH (2008) Characterization of surfactin produced by Bacillus subtilis isolate BS5. Appl Biochem Biotechnol 150, 289-303. https://doi.org/10.1007/s12010-008-8153-z.
  2. Ahire JJ, Robertson DD, Van Reenen AJ, Dicks LMT (2017) Surfactin-loaded polyvinyl alcohol (PVA) nanofibers alters adhesion of Listeria monocytogenes to polystyrene. Mater Sci Eng C Mater Biol Appl 77, 27-33. https://doi.org/10.1016/j.msec.2017.03.248.
  3. Akutsu K, Hirata A, Yamamoto M, Hirayae K, Hibi T (1993) Growth inhibition of Botrytis spp. by Serratia marcescens B2 isolated from Tomato Phylloplane. Jpn J Phytopathol 59, 18-25. https://doi.org/10.3186/jjphytopath.59.18.
  4. An B, Li B, Li H, Zhang Z, Qin G, Tian S (2016) Aquaporin8 regulates cellular development and reactive oxygen species production, a critical component of virulence in Botrytis cinerea. New Phytol 209, 1668-1680. https://doi.org/10.1111/nph.13721.
  5. Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation,characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31, 488-494. https://doi.org/10.1016/0006-291X(68)90503-2.
  6. Buchoux S, Lai-Kee-Him J, Garnier M, Tsan P, Besson F, Brisson A, Dufourc EJ (2008) Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism. Biophys J 95, 3840-3849. https://doi.org/10.1529/biophysj.107.128322.
  7. Cao S, Zheng Y, Wang K, Jin P, Rui H (2009) Methyl jasmonate reduces chilling injury and enhances antioxidant enzyme activity in postharvest loquat fruit. Food Chem 115, 1458-1463. https://doi.org/10.1016/j.foodchem.2009.01.082.
  8. Chen YH, Lin HT, Lin YF, Zhao YF, Zhang JN (2011) Effects of Phomopsis longanae Chi infection on lipoxygenase activity and fatty acid constituents of membrane lipids in pericarp of harvested longan fruits. J Trop Subtrop Bot 19, 260-266. https://doi.org/10.3969/j.issn.1005-3395.2011.03.011.
  9. Dean R, van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J (2012) The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13, 414-430. https://doi.org/10.1111/j.1364-3703.2011.00783.x.
  10. Desmyttere H, Deweer C, Muchembled J, Sahmer K, Jacquin J, Coutte F, Jacques P (2019) Antifungal Activities of Bacillus subtilis Lipopeptides to two Venturia inaequalis strains possessing different tebuconazole sensitivity. Front Microbiol 10, 2327. https://doi.org/10.3389/fmicb.2019.02327.
  11. Dik AJ, Koning G, Köhl J (1999) Evaluation of microbial antagonists for biological control of Botrytis cinerea stem infection in cucumber and tomato. Eur J Plant Pathol 105, 115-122. https://doi.org/10.1023/A:1008623210258.
  12. Enikova RK, Stoynovska MR, Karcheva MD (2020) Mycotoxins in fruits and vegetables. J IMAB-Annu Proc (Scientific Papers). 26, 3139-3143. https://doi.org/10.5272/jimab.2020262.3139.
  13. Fernández-Cruz ML, Mansilla ML, Tadeo JL (2010) Mycotoxins in fruits and their processed products: analysis, occurrence and health implications. J Adv Res 1, 113-122. https://doi.org/10.1016/j.jare.2010.03.002.
  14. Gao QH, Wu CS, Wang M (2013) The jujube (Ziziphus jujuba Mill.) fruit: A review of current knowledge of fruit composition and health benefits. J Agric Food Chem 61, 3351-3363. https://doi.org/10.1021/jf4007032.
  15. Guo Q, Dong W, Li S, Lu X, Wang P (2014) Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease. Microbiol Res 169, 533-540. https://doi.org/10.1016/j.micres.2013.12.001.
  16. Goussous SA, Casford MTL, Murphy AC, Salmond GPC, Leeper FJ, Davies PB (2017) Structure of the fundamental lipopeptide surfactin at the air/water interface investigated by sum frequency generation spectroscopy. J Phys Chem B 121, 5072-5077. https://doi.org/10.1021/acs.jpcb.7b03476.
  17. Gomaa EZ (2013) Antimicrobial activity of a biosurfactant produced by Bacillus licheniformis strain M104 grown on whey. Braz Arch Biol Technol 56, 4396-4403. https://doi.org/10.1590/S1516-89132013000200011.
  18. Gomes MZ, Nitschke M (2012) Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria. Food Control 25, 441-447. https://doi.org/10.1016/j.foodcont.2011.11.025.
  19. Hadia NJ, Ottenheim C, Li S, Hua NQ, Stubbs LP, Lau HC (2019) Experimental investigation of biosurfactant mixtures of surfactin produced by Bacillus Subtilis for EOR application. Fuel 251, 789-799. https://doi.org/10.1016/j.fuel.2019.03.111.
  20. Hazarika DJ, Goswami G, Gautom T, Parveen A, Das P, Barooah M, Boro RC (2019) Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens. BMC Microbiol 19, 71-81. https://doi.org/10.1186/s12866-019-1440-8.
  21. Helal GA, Sarhan MM, Abu Shahla AN, Abou El-Khair EK (2006) Effects of Cymbopogon citratus L. essential oil on the growth, lipid content and morphogenesis of Aspergillus niger ML2-strain. J Basic Microbiol 46, 456-469. https://doi.org/10.1002/jobm.200510106.
  22. Hmidet N, Jemil N, Nasri M (2019) Simultaneous production of alkaline amylase and biosurfactant by Bacillus methylotrophicus DCS1: application as detergent additive. Biodegrad 30, 247-258. https://doi.org/10.1007/s10532-018-9847-8.
  23. Horng YB, Yu YH, Dybus A, Hsiao SH, Cheng YH (2019) Antibacterial activity of Bacillus species-derived surfactin on Brachyspira hyodysenteriae and Clostridium perfringens. AMB Express 9, 188-197. https://doi.org/10.1186/s13568-019-0914-2.
  24. Jamalizadeh M, Etebarian HR, Aminian H, Alizadeh A (2011) A review of mechanisms of action of biological control organisms against post-harvest fruit spoilage. EPPO Bull 41, 65-71. https://doi.org/10.1111/j.1365-2338.2011.02438.x.
  25. Janisiewicz WJ, Korsten L (2002) Biological control of postharvest diseases of fruits. Annu Rev Phytopathol 40, 411-441. https://doi.org/10.1146/annurev.phyto.40.120401.130158.
  26. Jin P, Wang H, Liu W, Fan Y, Miao W (2018) A new cyclic lipopeptide isolated from Bacillus amyloliquefaciens HAB-2 and safety evaluation. Pestic Biochem Physiol 147, 40-45. https://doi.org/10.1016/j.pestbp.2017.08.015.
  27. Johnston PA, Coddington A (1984) Drug resistance in the fission yeast Schizosaccharomyces pombe: pleiotropic mutations affecting the oleic acid and sterol composition of cell membranes. Curr Genet 8, 37-43. https://doi.org/10.1007/BF00405430.
  28. Keller M, Viret O, Cole FM (2003) Botrytis cinerea infection in grape flowers: defense reaction, latency, and disease expression. Phytopathol 93, 316-322. https://doi.org/10.1094/PHYTO.2003.93.3.316.
  29. Leroux P, Fritz R, Debieu D, Albertini C, Lanen C, Bach J, Gredt M, Chapeland F (2010) Mechanisms of resistance to fungicides in field strains of Botrytis cinerea. Pest Manage Sci 58, 876-888. https://doi.org/10.1002/ps.566.
  30. Li JW, Fan LP, Ding SD, Ding XL (2007) Nutritional composition of five cultivars of chinese jujube. Food Chem 103, 454-460. https://doi.org/10.1016/j.foodchem.2006.08.016.
  31. Li Y, Zhang W, Niu J, Chen Y (2012) Mechanism of photogenerated reactive oxygen species and correlation with the antibacterial properties of engineered metal-oxide nanoparticles. Acs Nano 6, 5164-5173. https://doi.org/10.1021/nn300934k.
  32. Liu J, Li W, Zhu X, Zhao H, Lu Y, Zhang C, Lu Z (2019) Surfactin effectively inhibits Staphylococcus aureus adhesion and biofilm formation on surfaces. Appl Microbiol Biotechnol 103, 4565-4574. https://doi.org/10.1007/s00253-019-09808-w.
  33. Liu X, Ren B, Chen M, Wang H, Kokare CR, Zhou X, Wang J, Dai H, Song F, Mei L (2010) Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3. Appl Microbiol Biotechnol 87, 1881-1893. https://doi.org/10.1007/s00253-010-2653-9.
  34. Marchi S, Giorgi C, Suski JM, Agnoletto C, Bononi A, Bonora M, Marchi ED, Missiroli S, Patergnani S, Poletti F, Rimessi A, Duszynski J, Wieckowski MR, Pinton P (2011) Mitochondria-ros crosstalk in the control of cell death and aging. J Signal Transduct 2012, 329635. https://doi.org/10.1155/2012/329635.
  35. Massawe VC, Rao AH, Farzand A, Mburu DK, Ochola SO, Wu L, Tahir HAS, Gu Q, Wu H, Gao X (2018) Volatile organic compounds of endophytic Bacillus spp. have biocontrol activity against Sclerotinia sclerotiorum. Phytopathol 108, 1373-1385. https://doi.org/10.1094/PHYTO-04-18-0118-R.
  36. Metcalfe LD, Schmitz AA, Pelka JR (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal Chem. 38, 524-535. https://doi.org/10.1021/ac60251a031.
  37. Meyer MD, Terry LA (2010) Fatty acid and sugar composition of avocado, cv. Hass, in response to treatment with an ethylene scavenger or 1-methylcyclopropene to extend storage life. Food Chem 121, 1203-1210. https://doi.org/10.1016/j.foodchem.2010.02.005.
  38. Mirzaee MR (2014) An overview of jujube (Zizyphus jujuba) diseases. Arch. Phytopathol. Plant Prot 47, 82-89. https://doi.org/10.1080/03235408.2013.804238.
  39. Mora I, Cabrefiga J, Montesinos E (2015) Cyclic lipopeptide biosynthetic genes and products, and inhibitory activity of plant-associated Bacillus against phytopathogenic bacteria. Plos One 10, 0127738. https://doi.org/10.1371/journal.pone.0127738.
  40. Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16, 115-125. https://doi.org/10.1016/j.tim.2007.12.009.
  41. Pareek S (2013) Nutritional composition of jujube fruit. Emir J Food Agric 25, 463-470. https://doi.org/10.9755/ejfa.v25i6.15552.
  42. Park G, Nam J, Kim J, Song J, Kim PI, Min HJ, Lee CW (2019) Structure and mechanism of surfactin peptide from Bacillus velezensis antagonistic to fungi plant pathogens. Bull Korean Chem Soc 40, 704-709. https://doi.org/10.1002/bkcs.11757.
  43. Patel H, Huynh Q, Bärlehner D, Heerklotz H (2014) Additive and synergistic membrane permeabilization by antimicrobial (lipo)peptides and detergents. Biophys J 106, 2115-2125. https://doi.org/10.1016/j.bpj.2014.04.006.
  44. Peypoux F, Bonmatin JM, Wallach J (1999) Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 51, 553-563. https://doi.org/10.1007/s002530051432.
  45. Pi J, Zhang Q, Fu J, Woods CG, Hou Y, Corkey BE (2010) Ros signaling, oxidative stress and Nrf2 in pancreatic beta-cell function. Toxicol Appl Pharmacol 244, 77-83. https://doi.org/10.1016/j.taap.2009.05.025.
  46. Płaza GA, Turek A, Kroacute IE, Szczygłowska R (2013) Antifungal and antibacterial properties of surfactin isolated from Bacillus subtilis growing on molasses. Afr J Microbiol Res 7, 3165-3170. https://doi.org/10.5897/AJMR2013.5565.
  47. Qin GZ, Tian SP (2004) Biocontrol of postharvest diseases of jujube fruit by Cryptococcus laurentii combined with a low dosage of fungicides under different storage conditions. Plant Dis 88, 497-501. https://doi.org/10.1094/PDIS.2004.88.5.497.
  48. Ren X, Song D, Liang Z, Kong Q (2018) Effect of mint oil against Botrytis cinerea on table grapes and its possible mechanism of action. Eur J Plant Pathol 151, 321-328. https://doi.org/10.1007/s10658-017-1375-6.
  49. Senna AD, Lathrop A (2017) Antifungal screening of bioprotective isolates against Botrytis cinerea, Fusarium pallidoroseum and Fusarium moniliforme. Ferment 3, 53. https://doi.org/10.3390/fermentation3040053.
  50. Setiawati S, Nuryastuti T, Ngatidjan N, Mustofa M, Jumina J, Fitriastuti D (2017) In vitro antifungal activity of (1)-N-2-methoxybenzyl-1,10-phenanthrolinium bromide against Candida albicans and its effects on membrane integrity. Mycobiology 45, 25-30. https://doi.org/10.5941/MYCO.2017.45.1.25.
  51. Shi X, Li B, Qin G, Tian S (2012) Mechanism of antifungal action of borate against Colletotrichum gloeosporioides related to mitochondrial degradation in spores. Postharvest Biol Technol 67, 138-143. https://doi.org/10.1016/j.postharvbio.2012.01.003.
  52. Siahmoshteh F, Hamidi-Esfahani Z, Spadaro D, Shams-Ghahfarokhi M, Razzaghi-Abyaneh M (2018) Unraveling the mode of antifungal action of Bacillus subtilis and Bacillus amyloliquefaciens as potential biocontrol agents against aflatoxigenic Aspergillus parasiticus. Food Control 89, 300-307. https://doi.org/10.1016/j.foodcont.2017.11.010.
  53. Staats M, van Baarlen P, van Kan JA (2005) Molecular phylogeny of the plant pathogenic genus Botrytis and the evolution of host specificity. Mol Biol Evol 22, 333-346. https://doi.org/10.1093/molbev/msi020.
  54. Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin--a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot 39, 888-901. https://doi.org/10.7164/antibiotics.39.888.
  55. Verma B, Robarts RD, Headley JV, Peru KM (2002) Extraction efficiencies and determination of ergosterol in a variety of environmental matrices. Commun Soil Sci Plan 33, 3261-3275. https://doi.org/10.1081/CSS-120014521.
  56. Wang B, Huang Q, Venkitasamy C, Chai H, Gao H, Cheng N, Cao W, Lv X, Pan Z (2016) Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus jujuba Miller) during three edible maturity stages. LWT-Food Sci Technol 66, 56-62. https://doi.org/10.1016/j.lwt.2015.10.005.
  57. Wang Y, Zhang C, Liang J, Wang L, Chang R (2020) Surfactin and fengycin b extracted from Bacillus pumilus W-7 provide protection against potato late blight via distinct and synergistic mechanisms. Appl Microbiol Biotechnol 104:7467-7481. https://doi.org/10.1007/s00253-020-10773-y.
  58. Weiberg A, Wang M, Lin FM, Zhao H, Zhang Z, Kaloshian I, Huang HD, Jin H (2013) Fungal small rnas suppress plant immunity by hijacking host rna interference pathways. Science 342, 118-123. https://doi.org/10.1126/science.1239705.
  59. Wei YH, Wang LC, Chen WC, Chen SY (2010) Production and characterization of fengycin by indigenous Bacillus subtilis F29-3 originating from a potato farm. Int J Mol Sci 11, 4526-38. https://doi.org/10.3390/ijms11114526.
  60. Williamson B, Tudzynski B, Tudzynski P, van Kan JA (2007) Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol 8, 561-580. https://doi.org/10.1111/j.1364-3703.2007.00417.x.
  61. Xin Z, Ouyang Q, Wan C, Che J, Tao N (2019) Isolation of antofine from Cynanchum atratum BUNGE (Asclepiadaceae) and its antifungal activity against Penicillium digitatum. Postharvest Biol Technol 157, 110961. https://doi.org/10.1016/j.postharvbio.2019.110961.
  62. Yang J, Li J, Jiang Y, Duan X, Qu H, Yang B, Chen F, Sivakumar D (2014) Natural occurrence, analysis, and prevention of mycotoxins in fruits and their processed products. Crit Rev Food Sci Nutr 54, 64-83. https://doi.org/10.1080/10408398.2011.569860.
  63. Yang Z, Zu Y, Zhu J, Jin M, Cui T, Long X (2020) Application of biosurfactant surfactin as a pH-switchable biodemulsifier for efficient oil recovery from waste crude oil. Chemosphere 240, 1-8. https://doi.org/10.1016/j.chemosphere.2019.124946.
  64. Yu T, Zhang HY, Li XL, Zheng X (2008) Biocontrol of Botrytis cinerea in apple fruit by Cryptococcus laurentii and indole-3-acetic acid. Biol Control 46, 171-177. https://doi.org/10.1016/j.biocontrol.2008.04.008.
  65. Zahavi T, Cohen L, Weiss B, Schena L, Daus A, Kaplunov T, Zutkhi J, Ben-Arie R, Droby S (2000) Biological control of Botrytis, Aspergillus and Rhizopus rots on table and wine grapes in Israel. Postharvest Biol Technol 20, 115-124. https://doi.org/10.1016/S0925-5214(00)00118-6.
  66. Zhang B, Dong C, Shang Q, Han, Y, Li P (2013) New insights into membrane-active action in plasma membrane of fungal hyphae by the lipopeptide antibiotic bacillomycin L. Biochim Biophys Acta 1828, 2230-2237. https://doi.org/10.1016/j.bbamem.2013.05.033.
  67. Zhang DJ, Liu RF, Li YG, Tao L, Tian L (2010) Two new antifungal cyclic lipopeptides from Bacillus marinus B-9987. Chem Pharm Bull 58(12), 1630-1634. https://doi.org/10.1248/cpb.58.1630.
  68. Zhang H, Zheng X, Fu C, Xi Y (2005) Postharvest biological control of gray mold rot of pear with Cryptococcus laurentii. Postharvest Biol Technol 35, 79-86. https://doi.org/10.1016/j.postharvbio.2004.03.011.
  69. Zouari I, Jlaiel L, Tounsi S, Trigui M (2016) Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds. Biol Control 100, 54-62. https://doi.org/10.1016/j.biocontrol.2016.05.012.
  70. Zhang S, Lin Y, Lin H, Lin Y, Chen H, Wang Y, Shi J, Lin Y (2018) Lasiodiplodia theobromae (Pat.) Griff. & Maubl.-induced disease development and pericarp browning of harvested longan fruit in association with membrane lipids metabolism. Food Chem 244, 93-101. https://doi.org/10.1016/j.foodchem.2017.10.020.
  71. Zhang T, Li Y, Zheng Z, Ye R, Tang BZ (2019) In situ monitoring apoptosis process by a self-reporting photosensitizer. J Am Chem Soc 141, 5612-5616. https://doi.org/10.1021/jacs.9b00636.