Vernicia fordii leaf extract inhibited anthracnose growth by downregulating reactive oxygen species (ROS) levels in vitro and in vivo

Preparation of extracts from V. fordii flowers, leaves, and husks

The samples of paulownia flowers, leaves, and fruit shells were ground with liquid nitrogen (30 Hz, 1.5 min) using an MM 400 grinder to powder form. After adding 200 mL of 80% acidic ethanol (pH adjusted to 3.0 with HCl) to each powder sample, further soaking was allowed for 24 h. After 1 h of sonication (SB-120DTN; 120W, Dongkang, Tianjin, China), the extract was filtered (DP-01; 20W, Scientz, Ningbo, Zhejiang, China). The extraction was then evaporated using a rotary evaporator, and the remaining paste was dissolved in three mL 0.1% DMSO before filtering through a 0.45 µm organic filter membrane (Liu et al., 2009).

Component identification (UPLC-MS/MS)

All chromatographic separations were carried out utilizing an ultra-performance liquid chromatography (UPLC) system coupled with an Agilent SB-C18 column (1.8 µm, 2.1 mm ×100 mm) (USA). The mobile phase consisted of solvent A (ultrapure water with 0.1% formic acid, v/v) and solvent B (acetonitrile), subjected to an elution gradient initiated at 5% solvent B and gradually increasing to 95% over 9 min, followed by a 1-min hold before decreasing to 5% and maintaining for 3 min. The flow rate was set at 0.35 mL/min, column temperature at 40 °C, and the injection volume was 4 µL.

The MS settings consisted of: an ESI source operating at a temperature of 550 °C, a mass spectrometer voltage of 5,500 V, and a gas curtain set at 206.8 kPa. CAD with high parameter settings was used in the triple quadrupole (QQQ) instrument, with each ion pair scanned and detected based on optimized declustering voltage and collision energy.

Quantification of major alkaloids in leaves of V. fordii

Concentrate a sufficient volume of VFLE into methanol to achieve a detection solution with a concentration of 10 µg/mL, utilizing an HPLC method concerninga standard curve consisting of trigonelline, stachydrine, O-phosphocholine, and betaine. The alkaloid content of V. fordii leaves was monitored using this procedure.

HPLC parameters included: Shimadzu Corporation’s Shim-pack XR-ODS III column (75 mm × 2.0 mm, 1.6 µm), a column temperature maintained at 35 °C, and a flow rate of 0.2 mL/min. The mobile phase consisted of solvent A (a 0.1% aqueous solution of formic acid) and solvent B (acetonitrile). An initial 5% B ratio was used for 6 min, rising linearly to 15%, followed by a 3-min equilibrium. Next, the B ratio dropped to 5% and remained steady for another 5 min.

Inhibition of extraction on pathogenicity of C. fructicola

The pathogenicity of the test strains was determined by conducting a detached leaf inoculation assay on C. oleifolia leaves, as described by reference Qian et al. (2009). Fresh and uniform-sized young leaves of C. oleifolia were collected and washed with distilled water. The leaves were then placed in Petri dishes lined with moist filter paper, with a piece of degreased cotton moistened with distilled water covering the leaf stalk. In the treatment group, a sterilized needle was heated using an alcohol lamp and cooled. Small holes were punctured on both sides of the C. oleifolia leaves, and in these holes, fungal mycelial plugs (6 mm in diameter) obtained from culture using 600 µg/mL extracts of V. fordii flowers, leaves, and fruit husks were inoculated. The control group consisted of six mm fungal mycelial plugs cultured on a standard PDA medium. The sealed Petri dishes were incubated at a temperature of 28 °C (GNP-9050; Sanfa, Shanghai, China). After 4 days of dark incubation at 28 °C, the leaves were removed, and the diameters of the fungal mycelial growth were measured using a vernier caliper with a cross measurement. Each treatment had three replicates. The average value was used to calculate the growth inhibition rate of the detached leaf lesions:

Inhibition rate of lesion growth $=\frac{\text{Lesion area of control group}-\text{Lesion area of treatment group}}{\text{Lesion area of control group}}\times 100$%.

Study of the anti-potency of VFLE against C. fructicola

VFLE at various concentrations was treated in the culture medium where the C. fructicola fungus was then inoculated. The impact of VFLE on the parasitic mycelium of C. fructicola was evaluated by quantifying colony growth. The study used a PDB medium prepared using 3.5 g of potato dextrose agar + 100 mL of distilled water, sterilized at 121 °C for 20 min. Once cooled, VFLE at final concentrations of 0, 100, 200, 400, 600, 800, and 1000 µg/mL, respectively, was added. Fungal cubes (6 mm) of C. fructicola were placed and the medium was then incubated at 28 °C. Each test involved three repeats.

SEM observation

The mycelia of C. fructicola were cultivated in the presence of 600 µg/mL VFLE and subsequently subjected to sequential washing with phosphate buffer salt solution (PBS) solution on two occasions. Subsequently, the pellet was resuspended in PBS and subjected to centrifugation (5000 rpm, 4 °C, 5 min) to separate the supernatant. The recovered mycelial biomass was then fixed in a 3% glutaraldehyde solution at 4 °C for 24 h, after which it underwent thorough rinsing with sterile water for 20 min. Subsequently, the samples were subjected to graded ethanol dehydration, with sequential immersion in 30%, 50%, 70%, and 95% ethanol solutions for 20 min each, culminating in a final dehydration step in absolute ethanol for 45 min. The treated mycelia were subsequently freeze-dried, gold-coated, and examined under a scanning electron microscope (SEM-6380LV; Yishu, Shanghai, China) (Cai et al., 2014). All experiments had three replicates.

Evaluation of cell wall integrity and cell membrane permeability

One hundred milliliters of PDB were supplemented with 100, 200, 400, and 600 µg/mL of VFLE, respectively. Subsequently, a six mm mycelium mass of C. fructicola was added to the medium and cultivated for 3 d at 28 °C in an oscillating incubator with 160 rpm. Supernatant and mycelium were isolated to assess extracellular alkaline phosphatase (AKP) activity and cell wall integrity, respectively. Three replicates per treatment were performed.

To examine cell membrane permeability, 0.10 g of mycelium was weighed and steeped in one mL PBS (37 °C, 5 min) and then rinsed thrice. Electrical conductivity was determined using a conductivity meter (DDSJ-319L; INES, Shanghai, China). Three replicates were used for each treatment.

Assessment of oxidative stress

An amount of 0.10 g of mycelium was weighed and assayed for intracellular ROS levels using the non-specific ROS probe DCF-DA (Gemes et al., 2016). Absorbance was measured at 450 nm (ELISA reader, F-4500; Hitachi) to determine ROS concentration. MDA content was detected following the procedures outlined in the MDA assay kit purchased from Jiangsu Enzyme Free Company (China), with three replicates per treatment.

Cellophane penetration assay

Mycelia measuring 2 mm ×2 mm were excised from VFLE-cultured colonies of C. fructicola and transferred to cellophane-coated PDA plates. Plates were maintained for 48 h at 28 °C after which cellophane was removed. Three replicates per treatment were included.

Gene expression analysis

The PDB medium was supplemented with VFLE at final concentrations of 0, 100, 200, 400, and 600 µg/mL, followed by the addition of six mm mycelial discs. Cultures were incubated at 28 °C and 160 rpm for 48 h before samples were subjected to RNA extraction. RNA was designated as CK A, 0.5A, 1A, 2A, 3A, and preserved at −80 °C. Custom RT-qPCR primers (Table S1) were designed for the detection of HAC1, MKK1, RAB7, VAM7, and VPS39 gene expressions. qRT-PCR was conducted on the CFX96™ real-time PCR system (Bio-Rad, Hercules, CA, USA) with SYBR^® Premix Ex Taq™ II (AG) using three replicates per treatment.

Statistical analysis

Each experiment was repeated thrice. Mass spectrometry data were processed utilizing the software SCIEX Analyst version 1.6.3. Experimental data, including AKP enzyme activity, extracellular conductivity, malondialdehyde content, reactive oxygen species (ROS) activity, anthracnose lesion size in detached leaves of C. oleifera, and the size of C. fructicola colonies on alkaloid standard culture plates, were processed and statistically analyzed via Excel 2016, IBM SPSS Statistics for one-way ANOVA, Image J, and Origin software. The expression levels of the selected genes were calculated using the 2^−ΔΔCt method.

Characterization of principal components in V. Fordii leaves

UPLC-MS/MS analysis identified 571 metabolites, with the percentage contribution of bioactive components such as phenolic acids, flavonoids, lignans, coumarins, tannins, alkaloids, and terpenoids exceeding 50%. Alkaloids and flavonoids have shown anti-bacterial and anti-inflammatory properties. The pH of the V. fordii leaves oil, tested in water and ethanol, was found to be above 8.0, indicating mainly alkaline properties (Table 1).

Characteristics of key alkaloids in V. fordii leaves

Morphosate targeted metabolomics analysis established the substantial presence of several active alkaloid metabolites such as trigonelline, stachydrine, betaine, and O-phosphocholine (Table 2).

To unearth the predominant alkaloids in the antifungal efficacy of VFLE, a comprehensive HPLC analysis was performed. As shown in Fig. 1, the VFLE showed four peaks corresponding to standard compounds: trigonelline, stachydrine, betaine, and O-phosphocholine, respectively.

Peak area-based methods were used for quantitative analysis of trigonelline, stachydrine, betaine, and O-phosphocholine, revealing that VFLE exhibited the highest stachydrine content at 13.455 mg/L (Table 3).

V. fordii extracts inhibited mycelial growth

Observations in Figs. 2A and 2D illustrate that lesions on C. oleifera leaves treated with pathogenic fungi were reduced upon exposure to extracts of V. fordii flower, leaf, and husk, respectively, indicating a reduction in virulence of C. fructicola, thereby constraining lesion expansion. Importantly, VFLE decreased fungi infectivity to a significant extent, showing only 18.75% lesion area compared to the control group (Table 4). These findings suggest VFLE as an effective mitigant of C. oleifera anthracnose, laying the groundwork for future development.

The results of antifungal activity tests on C. fructicola by primary alkaloids from V. fordii are shown in Figs. 2B, 2E, and Table 5. Notably, all four alkaloid criteria significantly inhibited C. fructicola, with stachydrine and trigonelline showing superior suppression compared to betaine and O-phosphocholine, resulting in significantly reduced colony diameters compared to the control group. The efficacy experiment revealed reduced virulence of the fungus post-cultivation with alkaloid standards, yielding smaller lesions compared to untreated control (Fig. 2C). Specifically, stachydrine and betaine showed significantly higher inhibitory effects on lesion production than trigonelline and O-Phosphocholine, resulting in lesion areas only one-fourth the size of the control group (Fig. 2F). Thus, it appears that the primary bioactive alkaloids responsible for limiting C. fructicola growth in VFLE are trigonelline and betaine.

VFLE inhibition concentration

VFLE’s effect on C. fructicola growth, at various concentrations, is demonstrated in Fig. 3 and Table 6. C. fructicola mycelia ceased growth after 2 d steeped in a PDB liquid medium with VFLE concentration reaching 600 µg/mL (Fig. 3E). On further incubation for two more days on PDA plates with VFLE above 600 µg/mL, C. fructicola growth completely ceased at a concentration of 800 µg/mL (Fig. 3I). This illustrates VFLE’s potent inhibition of C. fructicola growth and its positive antifungal effects.

VFLE compromised the cell wall and membrane of C. fructicola

To examine the anti-fungal effects of VFLE on C. fructicola, cell membrane integrity and wall integrity were examined. Initially, C. fructicola mycelium subjected to various treatments was visualized by scanning electron microscopy. The surface morphologic alterations induced by VFLE are depicted in Fig. 4. The untreated control group presented a smooth, complete, and intact structure, showing even thickness and robust growth (Fig. 4A). Conversely, with 600 µg/mL VFLE, there were rough, irregular, and prominent wrinkles (Fig. 4B). Next, wall damage was estimated through AKP activity. The experiment showed dramatic augmentation in the VFLE-treated group, reaching 0.111 ± 0.005 U/mL, significantly higher than the untreated control group (0.07 ± 0.09 U/mL, p < 0.05) (Fig. 4C). This significant increase signifies compromised cell wall integrity, causing AKP release from the periplasmic space into the extracellular space. In addition, the cell membrane’s response was evaluated by investigating relative electrical conductivity shifts. As shown in Fig. 4D, the treated group showed an appreciable increase in relative electrical conductivity compared to the untreated control group, and the conductivity escalated progressively with VFLE addition. Noteworthy, a sharp increase within the 200–400 µg/mL VFLE dose range was seen, subsequently reaching a plateau with escalating concentration, indicating saturated electrolyte leakage and slow conductivity increment.

VFLE induced an increase in oxidative stress in fungal mycelia

Considerable increases in malondialdehyde (MDA) were observed in the VFLE-treated mycelium, as depicted in Fig. 5. Notably, the MDA levels escalated with elevated VFLE concentration, particularly at 200–400 µg/mL inducing substantial apoptosis of C. fructicola mycelium. Furthermore, the introduction of 100 µg/mL VFLE to the medium resulted in a marked rise in the mycelium’s reactive oxygen species (ROS) content, suggesting the accumulation of ROS post-VFLE treatment in C. fructicola mycelium.

VFLE influenced the colonization capacity of C. fructicola

Cellophane mimicry of C. oleifera served as a substrate on which cultures with varying levels of VFLE were inoculated. Observations demonstrated that post-cellophane removal, non-VFLE, and 100 and 200 µg/mL VFLE-treated fungi consistently formed colonies. Conversely, 400 and 600 µg/mL VFLE-treated strains failed to proliferate (Fig. 6), suggesting VFLE-restricted C. fructicola penetration.

VFLE affected the expression of key pathogenic genes

The addition of varying concentrations of VFLE significantly altered the expression levels of five major pathogenic genes in C. fructicola (Fig. 7). These genes showed their peak expression at an extract concentration of 100 µg/mL, then gradually declined with increased extract concentration. This observation indicates that VFLE, at a concentration of 100 µg/mL, amplifies the pathogenicity of C. fructicola in C. oleifera. However, as VFLE concentration increases, C. fructicola growth in C. oleifera is inhibited, resulting in reduced pathogenicity. The most profound effect was observed for RAB7, followed by HAC1 and VPS39.