Ethanol, n-butanol, glucose, and agar powder were procured from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Air-dried loquat leaves (purchased from Zhangshu medicinal materials market and authenticated as leaves of Eriobotrya japonica Lindl) were finely pulverized and extracted with 50% ethanol (100 g of dry powder per 1 L) at 60°C for 1 h. The extracts were then filtered and concentrated under reduced pressure using a rotary evaporator (Rotavapor R-3, Buchi, Flawil, Switzerland) at 45°C. After that, the extracts were suspended in water and sequentially extracted with ethyl acetate and n-butanol. The n-butanol extract was filtrated, combined, and evaporated to obtain dried LLE. The dried LLE was stored in a cryovial at −20°C until further analysis.

The phytopathogenic fungi P. digitatum (CGMCC 3.15410) and P. italicum (CGMCC 3.4040) were bought to the laboratory from the center of China General Microbiological Culture Collection (Beijing, China). G. citri-auranti was provided by the Department of Plant Protection in Jiangxi Agricultural University (Nanchang, China) and identified by Prof. Junxi Jiang. D. citri was isolated from decayed citrus fruits with a representative disease symptom of Phomopsis stem-end rot and identified by DNA sequencing (Qingke Biotech, Changsha, China). Each fungus was cultured on a potato dextrose agar (PDA) medium (leaching solution of potato 200 g, glucose 20 g, agar powder 20 g, and deionized water, 1 L) at 25 ± 1°C for a reactivation period of 7 days. The spore suspension of P. digitatum was prepared based on the protocol of previous studies (1, 14).

The effect of LLE on microscopic morphological alterations in P. digitatum spore was observed by SEM (15). P. digitatum spores, which included the control and LLE-treated samples, were washed with a PBS buffer three times and subsequently fixed with 2.5% (v/v) glutaraldehyde at 4°C. After fixation for 48 h, the samples were rinsed three times in distilled water for 20 min and then dehydrated with sequential graded cold ethanol (30, 50, 70, and 90%) for 20 min, and finally with absolute ethanol for 45 min. The specimens were then sputter-coated with gold and observed with a scanning electron microscope (FEI Quanta 250 FEG, Hillsboro, OR, United States).

The effects of LLE on the cell membrane permeability of P. digitatum were investigated by assaying extracellular conductivity, cell lysis rate, and leakages of protein and nucleic acids. Briefly, 200 μl of a P. digitatum suspension was evenly added in 100 ml of PDB and incubated in a shaker for 2 days. About 2 g of fresh mycelium was re-suspended in 50 ml PDB having different LLE concentrations of 0 (set as the control), MIC (0.625 mg/ml), and MFC (1.25 mg/ml). The samples were taken at various time intervals (0, 30, 60, 90, 120, and 240 min). Extracellular conductivity was detected as reported by Tao et al. (16) with a conductivity meter (model ST3100C, Ohaus Co., Parsipanny, NJ, United States). Cell lysis rate was evaluated according to the method of Chen et al. (1). Leakages of protein and nucleic acid from P. digitatum hyphae were determined based on the method described by Huang et al. (17) to measure the optical density (OD) of the supernatant at 260 and 280 nm. The experiments were conducted twice with three replicates for each treatment.

The loss in intracellular constituents into the supernatant was measured according to the method described previously (18), with minor modifications. Soluble protein, reducing sugar, total lipid, and ergosterol contents in both the supernatants and the hyphae of P. digitatum were measured using bovine serum albumin standards, glucose, and cholesterol, respectively. The experiments were conducted twice with three replicates for each treatment.

The effect of LLE on the plasma membrane integrity of P. digitatum was determined following the method described by Xin et al. (19). Briefly, P. digitatum hyphae were treated with LLE at 0 and 1.25 mg/ml (MFC) for 120 min. Then, the control and LLE-treated mycelia were dyed with propidium iodide (PI) at 25°C in the dark. After staining for 30 min, the control and LLE-treated mycelia were washed three times with PBS and observed with a Ni-U fluorescence microscope (Nikon Corporation, Tokyo, Japan).

The lipid peroxidation of P. digitatum cell was quantitatively determined in terms of malondialdehyde (MDA) content using the thiobarbituric acid method described by Pasquariello et al. (20), and MDA content was calculated using the equation below:

where Vt is the total volume of the extract (ml), and FW is the frozen weight of P. digitatum (g).

The experiments were conducted twice with three replicates for each treatment.

The 2-day-old mycelia from 100 ml PDB were collected and resuspended in 50 ml PDB with LLE at various concentrations (0.625, and 1.25 mg/ml). β-1,3-glucanase (β-Glu) activity, after exposure to LLE for 0, 30, 60, 90, 120, and 240 min, was measured according to the previously developed method (1). According to the instructions of the manufacturer, the extracellular AKP activity of P. digitatum hyphae after exposure to LLE treatments was determined using a commercial AKP kit (Jiancheng Bioengineering Research Institute Co., Nanjing, China) (21), and enzyme activities were expressed as U/mg prot. The experiments were conducted twice with three replicates for each treatment.

Comparisons of data from different groups were analyzed by one-way analysis of variance (ANOVA) at 5% significance level with the SPSS 22.0 software (SPSS Inc. Chicago, IL, United States). The levels of significance were assessed as significant (p < 0.05) and highly significant (p < 0.01) using Prime and Excel 2010 software.

The treatments of LLE completely inhibited the mycelial growth of P. digitatum, P. italicum, and D. citri at the concentration of 0.625 mg/ml when incubated for 2 days. LLE (1.25 mg/ml) completely inhibited mycelial growth on the sixth day (Table 1). The results revealed that the MIC and MFC values of LLE against P. digitatum, P. italicum, and D. citri were 0.625 and 1.25 mg/ml, respectively. Moreover, LLE at the concentration of 0.625 and 2.5 mg/ml completely inhibited G. citri-auranti growth on the second and sixth days of incubation, respectively (Table 1). Hence, the MIC and MFC values of LLE against G. citri-auranti were 0.625 and 2.5 mg/ml, respectively.

Different LLE concentrations showed potent inhibitory effects on the mycelial growth of P. digitatum on the PDA culture, compared with the control group (Figure 1). After 2 days, the diameter of the colony decreased significantly (p < 0.05) with increased LEE concentration, and the extension of the P. digitatum colony disappeared completely at LLE concentrations ≥0.625 mg/ml (Figure 1). Besides, the mycelial growth rate, after 48 h of culture with LLE concentrations of 0.625 and 1.25 mg/ml, decreased to 57.5 and 20.3%, respectively, relative to the mycelial growth in the control group (Figure 1). Furthermore, the mycelial growth of P. digitatum was negligible over the incubation period when treated with LEE concentrations of ≥1.25 mg/ml.

As shown in Table 2, a significant inhibitory effect of LLE on the spore germination of P. digitatum in the PDB medium was seen in a dose-dependent manner, where higher LLE concentration resulted in a lower spore germination rate. LLE at a concentration of 0.625 mg/ml could significantly (p < 0.05) suppress the spore germination of P. digitatum by 78.95 ± 0.41% compared with the control group (Table 2); whereas at an LLE concentration of 0.156 mg/ml, only 17.62 ± 0.45% of inhibitory percentage was recorded. As the LLE concentration increased up to 1.25 mg/ml, P. digitatum spore germinated rate was <5%. The linear regression of the inhibitory percentage of P. digitatum (Y) on the log-transformed LLE-treated concentrations (X) was determined as Y = 1.75X−1.491, R² = 0.987, with the half-maximal effective concentration (EC₅₀ refers to the LLE dose causing 50% inhibition of the spore germination) of LLE against P. digitatum being 0.317 mg/ml.

The scanning electron microscopy images showed that the microscopical morphology of the P. digitatum spores was severely affected by LLE treatment. The untreated spores had an engorged globular shape, with a smooth surface (Figure 2A). However, LLE treatment at the MFC (1.25 mg/ml) altered the microscopical morphology of the P. digitatum spores, including sunken surface, loss of linearity, and malformation (Figure 2B).

The effect of LLE treatment on the cell membrane permeability of P. digitatum is presented in Figure 3. The extracellular conductivity in the 1.25 mg/ml LLE-treated P. digitatum cell suspension increased from 259.1 ± 1.03 μs/cm to 722.2 ± 11 μs/cm after 120 min of incubation (Figure 3A, p < 0.05). The cell lysis rate in LLE-treated P. digitatum suspensions significantly increased (p < 0.05), whereas the control group remained stable. After 240 min of incubation, the cell lysis rate in the P. digitatum suspension treated with 0.625 and 1.25 mg/ml LLE was 50.54 ± 1.71 and 74.23 ± 2.54%, respectively, compared with 20.35 ± 0.92% in the control group (Figure 3B). As illustrated in Figures 3C,D, LLE treatment significantly induced the leakages of nucleic acid and protein in P. digitatum hypha (p < 0.05). After 240 min of incubation, both nucleic acid leakage and protein leakage in P. digitatum suspensions treated with 1.25 mg/ml LLE were 3.22 times and 4.01 times higher than that of the control. These results might indicate that cell membrane permeability was likely to be one of the vital antifungal mechanisms for LLE in P. digitatum growth.

The fluorescence microscopy images of P. digitatum hypha treated with MIC and MFC LLE are shown in Figure 4A, and are consistent with the cell membrane permeability results described above. No visible red fluorescence was observed in the control P. digitatum hypha after 30 min of incubation. In contrast, robust red fluorescence was observed in the MIC and MFC LLE-treated samples, respectively.

Changes in intracellular reducing sugar, protein, total lipid, and ergosterol contents of P. digitatum exposed to LLE treatment at 0 mg/ml, MIC, and MFC are presented in Figure 5. A significant difference in intracellular reducing sugar content was observed after 60 min of exposure and gave a declining trend with increasing LLE concentrations (Figure 5A). After 120 min of incubation, the reducing sugar content in P. digitatum hypha was 15.95 ± 1.05 and 13.45 ± 0.27 mg/g at LLE concentrations 0.625 and 1.25 mg/ml, respectively, which was significantly lower than that in the control samples (21.7 ± 0.72 mg/g, p < 0.05). As demonstrated in Figure 5B, the protein content of control P. digitatum hypha was found to maintain a stable level during an incubation period of 0–240 min. In contrast, in the LLE-treated groups, the protein content significantly decreased. For instance, at 240 min after the treatment, the intracellular protein content in P. digitatum hypha treated with the MIC and MFC LLE was 17.95 and 34.62% lower than that of the control, respectively. In addition, the total lipid content in LLE-treated P. digitatum hypha decreased with increasing exposure time. In contrast, the total lipid content in the MIC and MFC LLE-treated samples after 240 min of incubation was 91.65 ± 0.62 and 63.69 ± 2.27 mg/g, respectively, which was significantly lower than that in the control group (121.78 ± 3.64 mg/g, p < 0.05, Figure 5C). Similarly, a continuous decrease in the ergosterol content of P. digitatum hypha after LLE treatment was observed throughout the whole incubation period, whereas the ergosterol content in the control group remained stable (Figure 5D). After 120 min of incubation, the ergosterol content in P. digitatum hypha treated with the MIC and MFC LLEs were 0.79 ± 0.06 and 0.54 ± 0.08 mg/g, respectively, which was significantly lower (p < 0.05) than that observed in the control group (1.2 ± 0.08 mg/g).

The effect of loquat leaf extract LLE treatment on the malondialdehyde (MDA) content of P. digitatum hypha is shown in Figure 4B. After 60 min of LLE treatment, a striking difference between the control and LLE-treated samples was observed, with the intracellular MDA content in the MIC and MFC LLE-treated groups being 2.61 ± 0.13 and 2.88 mmol ± 0.06 mmol/g, respectively, which was significantly higher than that in the control group (2.34 ± 0.06 mmol/g, p < 0.05). As shown in Figure 4B, the β-Glu activity in the control P. digitatum hypha was found to maintain a stable level during an incubation period of 0–240 min. In contrast, it rapidly increased in the LLE-treated groups. For instance, at 120 min of incubation, the β-Glu activity in P. digitatum hypha treated with the MIC and MFC LLEs were 1.47 times and 1.92 times higher than that of the control, respectively (Figure 4C, p < 0.05). The AKP activity of the MIC and MFC LLE-treated P. digitatum hypha was.27 ± 0.019 and 0.31 ± 0.015 U/mg, respectively, at 90 min after treatment, which was higher (p < 0.05) than that in the control samples. As the treatment time increased to 240 min, the AKP activity of P. digitatum hypha treated with LLE at MIC and MFC were 1.39 times and 1.68 times, respectively, higher than that of the control group (Figure 4D, p < 0.05).

The effects of LLE treatment on the ATP, ADP, and AMP contents in P. digitatum hypha are presented in Figure 6. As shown in Figure 6A, the ATP content of P. digitatum hypha treated with LLE at MIC and MFC is 110.1 ± 2.25 and 77.9 ± 1.05 mg/kg, respectively, which is much lower than that in the control samples after 120 min of incubation (150.2 ± 3.03 mg/kg), indicating that the ATP supply of P. digitatum hypha is hindered by LLE treatment. Figure 6D shows the effect of ATP content release when the P. digitatum cell suspension is treated with LLE at MIC and MFC. The ATP content of the P. digitatum cell suspension treated with MFC LLE was dramatically increased after 120 min of exposure, which was significantly higher (p < 0.05) than that in MIC LLE (36.7%) or the control (98.6%). Similarly, both the ADP and AMP contents of P. digitatum hypha significantly decreased with the MIC and MFC LLE treatments (Figures 6B,C, p < 0.05). In comparison, the release of ADP and AMP of the P. digitatum cell suspension significantly increased with the MIC and MFC LLE treatment (Figures 6E,F, p < 0.05). Those results suggested that the intracellular energy source of P. digitatum was infiltrated into the cell suspension after LLE treatment. Thus, the extracellular ATP, ADP, and AMP levels in the P. digitatum cell suspension exposed to LLE treatment was much higher than those of the control.