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Article

Antifungal Activity and Mechanism of Xenocoumacin 1, a Natural Product from Xenorhabdus nematophila against Sclerotinia sclerotiorum

by
Shujing Zhang
1,2,†,
Yunfei Han
1,3,†,
Lanying Wang
2,
Jinhua Han
1,3,
Zhiqiang Yan
1,3,
Yong Wang
1,3 and
Yonghong Wang
1,3,*
1
Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, College of Plant Protection, Northwest A&F University, 22 Xinong Road, Yangling 712100, China
2
Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, School of Tropical Agriculture and Forestry (School of Agricultural and Rural Affairs, School of Rural Revitalization), Hainan University, 58 People’s Avenue, Haikou 570228, China
3
Shaanxi Research Center of Biopesticide Engineering & Technology, College of Plant Protection, Northwest A&F University, 22 Xinong Road, Yangling 712100, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2024, 10(3), 175; https://doi.org/10.3390/jof10030175
Submission received: 9 January 2024 / Revised: 25 January 2024 / Accepted: 27 January 2024 / Published: 26 February 2024
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Abstract

:
Sclerotinia sclerotiorum (Lib.) de Bary, a polyphagous necrotrophic fungal pathogen, has brought about significant losses in agriculture and floriculture. Until now, the most common method for controlling S. sclerotiorum has been the application of fungicides. Xenocoumacin 1 (Xcn1) is a potential biopesticide having versatile antimicrobial activities, generated by Xenorhabdus nematophila. This study was intended to isolate Xcn1 from X. nematophila YL001 and clarify its efficacies for S. sclerotiorum control. Xcn1 demonstrated a wider antifungal spectrum against 10 plant-pathogenic fungi. It also exhibited a strong inhibitory effect on the mycelial growth of S. sclerotiorum with an EC50 value of 3.00 μg/mL. Pot experiments indicated that Xcn1 effectively inhibited disease extension on oilseed rape and broad bean plants caused by S. sclerotiorum. Morphological and ultrastructural observations revealed that the hyphae of S. sclerotiorum became twisted, shriveled, and deformed at the growing points after treatment with Xcn1 at 3.00 μg/mL and that the subcellular fractions also became abnormal concurrently, especially the mitochondrial structure. Moreover, Xcn1 also increased cell membrane permeability and decreased the content of exopolysaccharide as well as suppressing the activities of polygalacturonase and cellulase of S. sclerotiorum, but exerted no effects on oxalic acid production. This study demonstrated that Xcn1 has great potential to be developed as a new biopesticide for the control of S. sclerotiorum.

1. Introduction

Sclerotinia sclerotiorum (Lib.) de Bary is a polyphagous and necrotrophic fungal pathogen which infects more than 400 plant species worldwide, such as oilseed rape [1], lentils [2], sunflower [3], soybean [4], chickpeas [5], lentils [6], and lettuce [7], resulting in significant losses in agriculture and floriculture. S. sclerotiorum infects different plant tissues and produces sclerotia, which are the source of infection in the disease cycle. Moreover, sclerotia are structures formed from converged mycelia and can survive in the soil for up to 8 years [8,9]. Oxalic acid (OA), which inhibits plant defense, is a key pathogenicity factor of S. sclerotiorum [10]. Effectively eradicating S. sclerotiorum requires preventing the production of oxalic acid and the formation of sclerotia. Owing to the broad host range and the persistent sclerotia bodies of S. sclerotiorum, as well as its ability to infect several rotational crops, traditional strategies including crop rotation and breeding of resistant crops are not effective in controlling S. sclerotiorum [11]. Currently, the most common method of S. sclerotiorum disease management is the application of chemical fungicides [12]. However, their extensive use makes sclerotia generate drug resistance to many fungicides, such as mancozeb, propineb, and carbendazim [13,14]. In addition, the adverse side effects of these chemical fungicides have also resulted in serious threats to human health and environmental security [15,16,17].
Xenorhabdus nematophila, which is a form of entomopathogenic bacteria, lives in symbiosis with Steinernema nematodes [18]. Previous studies have shown that cell-free broth derived from X. nematophila has antifungal activity, but it is unknown which compounds have antifungal effects [19]. Xenocoumacin 1 (Xcn1), a type of xenocoumacin, is the main antimicrobial compound produced by X. nematophila [20]. A previous study has shown that Xcn1 exhibits a strong antimicrobial activity against five species of Phytophthora, with EC50 values ranging from 0.25 to 4.17 µg/mL [21]. The EC50 values of Xcn1 against mycelial growth and zoospore germination of Phytophthora capsici were 2.44 and 0.81 µg/mL, respectively [22]. The transcriptional responses of Bacillus subtilis to Xcn1 predicted that its functions were similar to the mechanisms of protein synthesis inhibitors [23]. However, information regarding Xcn1 relating to its efficacy against S. sclerotiorum is limited.
It is well known that living microbes and their metabolites are important resources for the development of new biopesticides. Thus, identifying new compounds from microbes and evaluating their antimicrobial activity and action mechanism are important. Our previous studies revealed that the culture supernatant of X. nematophila YL001 exhibited potent antimicrobial activities against many plant pathogens [24,25,26]. However, to date, few bioactive compounds have been obtained from X. nematophila YL001. We have obtained Xcn1 from cell-free broth derived from X. nematophila YL001. Therefore, the objectives of the present study were to (i) evaluate inhibitory effects of Xcn1 against plant pathogens in vivo and in vitro; (ii) verify the effects of Xcn1 on the hyphal morphology and ultrastructure of S. sclerotiorum; (iii) test the effects of Xcn1 on the cell membrane permeability and the exopolysaccharide (EPS) and oxalic acid (OA) production of S. sclerotiorum; (iv) determine the effects of Xcn1 on the activities of polygalacturonase (PG) and cellulose (Cx) of S. sclerotiorum. The above results provided basis information for the potential of Xcn1 as a new candidate for biopesticides, as well as its action mechanism against S. sclerotiorum.

2. Results

2.1. Structure Elucidation and Antifungal Activity Assay of Xcn1

Xcn1 was isolated and purified from cell-free fermentation broth derived from X. nematophila YL001 using X-5 macroreticular resin, cation-exchange resin 110, and CM-Sephadex-C-25 weak acid cation exchange resin consecutively. Its structure was identified correctly via 1H NMR, 13C NMR, and HR-MS spectra (Figures S1–S3) [20,21]. Its purity was determined to be 96.7% via the HPLC method (Figure S4).
A previous study described the satisfactory antimicrobial activity of Xcn1 against a species of Phytophthora [21]. On the basis of this research, we further determined Xcn1’s antifungal activity against ten plant-pathogenic fungi. Of the fungal pathogens tested, Xcn1 showed high inhibitory effects on S. sclerotiorum, R. solani, B. cinerea, and E. turcicum at 10 µg/mL, with inhibition rates of 89.30%, 88.5%, 86.7% and 83.2%, respectively (Table 1). S. sclerotiorum was the most sensitive to Xcn1 with an EC50 value of 2.86 µg/mL (Table 2), indicating the strong antifungal activity of Xcn1 against S. sclerotiorum.

2.2. Effects of Xcn1 on the Disease Development In Vivo

The disease control efficacies of Xcn1 against S. sclerotiorum in two host plants (broad bean and oilseed rape) are shown in Figure 1 and Figure 2, respectively. As shown in Figure 1 and Figure S5, Xcn1 effectively protected the broad bean leaves from S. sclerotiorum infection, with preventive efficacies of 62.27% at 100 µg/mL and 78.99% at 200 µg/mL (Figure 1A,C and Figure S5). Moreover, Xcn1 also exhibited high curative activity against S. sclerotiorum on the infected diseased leaves, with control efficacies of 66.51% at 100 µg/mL and 88.90% at 200 µg/mL, which is comparable to that of carbendazim (MBC) treatment at 100 µg/mL (92.01%) (Figure 1B,D and Figure S5).
The control efficacy of Xcn1 against S. sclerotiorum in the oilseed rape plants is shown in Figure 2 and Figure S6. Xcn1 possessed a favorable preventive efficacy, with a control efficacy of 58.40% at 100 µg/mL and an efficacy of 86.30% at 200 µg/mL, comparable to that of carbendazim (MBC) treatment at 100 µg/mL (91.30%) (Figure 2A,C and Figure S6). Xcn1 also exhibited an excellent curative activity, with a control efficacy of 45.79% at 100 µg/mL, comparable to that of the referenced carbendazim (MBC) at 100 µg/mL (48.92%) (Figure 2B,D and Figure S6). Taken together, the disease control assay indicated that Xcn1 exhibited a potent control efficacy against S. sclerotiorum in vivo.

2.3. Effects of Xcn1 on the Hyphal Morphology and Ultrastructure of S. sclerotiorum

Previous research has reported that Xcn1 brings about the plasmolysis and cytoplasmic condensation of P. capsici [22]. To better understand the mode of antifungal action of Xcn1, we observed the mycelial morphology and ultrastructure of S. sclerotiorum treated with Xcn1 via SEM and TEM, respectively. The SEM results showed that the control sample exhibited a fine morphology of smooth, uniform, and cylindrical hyphae with plump growing points (Figure 3A,B). The Xcn1-treated sample, however, showed an altered morphology characterized by twisted, deformed, and rupturing hyphae with a sparse distribution (Figure 3C–F).
The effects of Xcn1 on the hyphal ultrastructure of S. sclerotiorum were observed via TEM. The control sample displayed a normal hyphal ultrastructure of intact and smooth cell membranes, as well as clearly visible protoplasm and mitochondria (Figure 4A,B). In the sample treated with Xcn1, the hyphae’s cell membranes became rough and invaginated. Mitochondrial abnormalities, including hazy outlines and vacuolar degeneration, were also observed. Moreover, the cytoplasm became disordered and vacuolate (Figure 4C–F). These microscopic observations indicated that Xcn1 disrupted the hyphal morphology and ultrastructure of S. sclerotiorum to exert its inhibitory effect on mycelium growth.

2.4. Effects of Xcn1 on the Cell Membrane Permeability and Exopolysaccharide Secretion of S. sclerotiorum

As shown in Figure 5A, relative conductivity increased over time whether the strains were treated with Xcn1 or not. The relative conductivity of the Xcn1-treated group (3.00 μg/mL), however, was always significantly higher than that of the control group (Figure 5A). These results indicated that Xcn1 increased the cell membrane permeability of S. sclerotiorum.
The effects of Xcn1 on the EPS secretion of S. sclerotiorum were determined to be due to the phenol-sulfuric acid method and were reported as glucose equivalents. As illustrated in Figure 5B, there were no differences in the content of EPSs between the Xcn1-treated group and the control group at the 3th day after inoculation. However, the EPS contents of the Xcn1-treated group were lower than those of the control group at the 6th, 9th and 12th days, respectively (Figure 5B). Visually, the supernatant culture of S. sclerotiorum after treatment with Xcn1 for 9 days was more limpid than that of the control group (Figure S7).

2.5. Effects of Xcn1 on the Oxalic Acid Biosynthesis of S. sclerotiorum

Bromophenol blue is an acid–base indicator, which exhibits a yellow color at low pH values below 3.0 and a blue color at pH values above 4.6 [27]. S. sclerotiorum produced OA to acidize the medium (pH < 3.0) during its growth [28]. Thus, we evaluated the effect of Xcn1 on this acidification process on the PDA medium supplemented with bromophenol blue. As shown in Figure 5C, although the growth of S. sclerotiorum was obviously suppressed by Xcn1 compared with that of the control, the acidification process still occurred on the Xcn1-treated plates, albeit with a time delay. On the basis of this result, we further tested the effects of Xcn1 on the OA production of S. sclerotiorum. As shown in Figure 5D, there were no significant differences in the content of OA between the Xcn1-treated sample and the control sample on a timeline extending from 3 to 12 days. All these results reveal an interesting idea, namely that Xcn1 has no effects on the biosynthesis of OA in S. sclerotiorum.

2.6. Effects of Xcn1 on the Activities of Polygalacturonase and Cellulase in S. sclerotiorum

The PG and Cx activities of S. sclerotiorum treated with Xcn1 were measured with the results shown in Figure 5. The PG activities of the Xcn1-treated group were comparable to those of the control group at the 3rd and 6th days after inoculation. However, when the inoculation time extended to the 9th and 12th days, the PG activities of the Xcn1-treated group were much lower than those of the control group (Figure 5E). Moreover, the Cx activities of the Xcn1-treated group were significantly lower than those of the control group at the 6th, 9th and 12th days after inoculation, respectively (Figure 5F). All these results indicated that Xcn1 suppresses the activities of PG and Cx in S. sclerotiorum when the interaction time is over 6 days.

3. Discussion

3.1. Xcn1 Has the Potential to Be Developed as a Pesticide for the Control of S. sclerotiorum

Xenocoumacin 1 (Xcn1) has great potential to be used as a new biopesticide in agricultural production. A previous study has reported that Xcn1 displayed good antimicrobial activity against Phytophthora species, especially against P. capsici, with an EC50 value of 2.44 μg/mL [21]. In this study, Xcn1 was isolated and purified from X. nematophila YL001. To obtain more information about its pesticidal properties, we tested its antifungal activity in vitro and in vivo. Xcn1 exhibited broad-spectrum antimicrobial activity against 10 common phytopathogens in vitro (Table 1) and was especially effective against S. sclerotiorum, with an EC50 value of 3.0 µg/mL. Moreover, Xcn1 was demonstrated to possess a comparable in vivo control efficacy against S. sclerotiorum in two selected host plants at 200 µg/mL to MBC at 100 µg/mL (Figure 1 and Figure 2). These results indicate that Xcn1 has the potential to be developed as a microbiological pesticide for the control of P. capsici, S. sclerotiorum, and other pathogens.

3.2. Xcn1 Exerted Its Antifungal Activity against S. sclerotiorum through Disturbing Mycelial Growth and Infection

Xcn1 exerted its antifungal activity against S. sclerotiorum by disturbing the multiple physiological and biochemical processes. SEM and TEM observations revealed prominent morphological and ultrastructural disruptions of S. sclerotiorum hyphae treated with Xcn1 compared to the untreated controls (Figure 3 and Figure 4). Xcn1 increased the cell membrane permeability of S. sclerotiorum (Figure 5A), as well as reducing the secretion of EPSs (Figure 5B and Figure S7), an important virulence factor of many kinds of microorganism [29,30,31]. Plant pathogens produced such cell wall-degrading enzymes as cellulase, hemicellulase, and pectinase to break down the first barrier of the host plant cells during their invasion. Pectinase and cellulase have been demonstrated to play key roles during the infection of S. sclerotiorum [32,33,34]. In this study, we also found that Xcn1 suppresses the activities of PG and Cx in S. sclerotiorum effectively (Figure 5). However, more work is needed to further clarify its mechanism of action as a potent antifungal agent.

3.3. Xcn1 May Inhibit Protein Translation

Amicoumacin A (AMI), a close analog of Xcn1, has been shown to bind to the E-site of the ribosome [35]. A published study has indicated Xcn1 could be an inhibitor of ribosomal protein synthesis; however, there is no direct experimental evidence to support this viewpoint [36]. Xcn1 has a longer carbon chain than AMI and contains guanidine groups that AMI does not have. Therefore, it cannot be confirmed that the mechanisms of action of AMI and Xcn1 are the same. Blasticidin S has a long chain structure containing guanidine groups similar to Xcn1 and inhibits the hydrolysis of tRNA-carrying peptides, resulting in the inability to release synthesized peptides and cause translation termination [37,38]. These studies suggest that the long carbon chain and guanidine structure of Xcn1 may play an important role in inhibiting protein translation processes.

3.4. Combined Utilization of Xcn1 and Oxalate Synthesis Inhibitors Will Be a High Effective Strategy in S. sclerotiorum Control

Combined utilization of Xcn1 and oxalate synthesis inhibitors may achieve the effective control of S. sclerotiorum. The present knowledge points out that oxalic acid (OA) is a key factor in successful infection by S. sclerotiorum [39]. OA is an elicitor of programmed plant cell death in the process of S. sclerotiorum infection, and its deficiency reduces the pathogenicity of S. sclerotiorum [40]. However, Xcn1 has been demonstrated to have no effects on the production of OA in S. sclerotiorum (Figure 5C,D). Many fungicides, such as pyraziflumid [41], pyrisoxazole [1], cinnamic acid [42], volatile organic compounds from Bacillus spp. [11], and lansiumamide B [43], have been reported to reduce the content of OA to weaken the pathogenicity of S. sclerotiorum. Combination of these compounds with Xcn1 may be a highly effective control strategy using multiple modes of action.

4. Material and Methods

4.1. Strains and Growth Conditions

X. nematophila YL001 was obtained from its host nematodes, Steinernema sp. YL001, which were isolated from the soil of Yangling, China. Its morphological and molecular characteristics were identified [26,44]. X. nematophila YL001 was shaken in Luria–Bertani medium (LB: 1.0% Bacto tryptone, 0.5% yeast extract and 1% NaCl in water; pH 7.2) at 150 rpm at 28 °C and transferred to NBTA medium for incubation at 28 °C in darkness [26].
Ten plant-pathogenic fungi for testing (Sclerotinia sclerotiorum, Rhizoctonia solani, Exserohilum turcicum, Fusarium graminearum, Verticillium dahliae, Botrytis cinerea, Alternaria alternata, Colletotrichum gloeosporioides, Gaeumannomyces graminis and Fusarium oxysporum) were provided by the Shaanxi Research Center of Biopesticide Engineering & Technology. Their detailed information is presented in Table S1.

4.2. Microbial Fermentation

X. nematophila YL001 was inoculated in a 500 mL flask containing 200 mL fresh Luria–Bertani medium and cultured for 12 h at 28 °C while being shaken at 180 rpm. Then five liters of culture broth (OD600 = 0.8) was transferred as a seed into a 70-L fermenter (Eastbio, Zhenjiang, China) containing 35 L of TSB medium [25]. The system was cultured under identical conditions (28 °C, pH 7.2, agitation of 160 rpm/min, and 0.25 v/v/min of aeration) for 48 h. After that, X. nematophila YL001 cell-free culture broth was obtained via centrifugation at 10,000× g for 20 min. Isolation and characterization of Xcn1 are shown in the Supplementary Material [20,45].

4.3. Assessment of Antifungal Activity of Xcn1 In Vitro

The antifungal activity of Xcn1 against the mycelial growth of phytopathogenic fungi was performed as described, with a slight modification [26]. Briefly, Xcn1 was dissolved in distilled water to prepare a stock solution of 1000 μg/mL, and carbendazim (MBC) was dissolved in 0.1 M HCl with a concentration of 500 μg/mL. The stock solution of Xcn1 (150 μL) was sufficiently mixed with 15 mL of molten PDA medium at a low temperature (<40 °C), and then the mixture was poured into Petri dishes (60-mm diameter) to form plates of a 2–3 mm thickness (5 mL for each disc). The final concentration of Xcn1 in each plate was 10 μg/mL. A 5-mm dish of actively growing (2-day-old) culture of fungus was transferred from the edges of 2-day-old colonies to the center of the prepared plates (one for each plate). PDA plates supplemented with MBC (0.5 μg/mL) served as the positive controls. Mean growth values were measured and subsequently converted into an inhibition rate of mycelial growth in relation to the control treatment according to the following formula:
Inhibition rate (%) = [(Dc − Dt)/(Dc − 0.5)] × 100
where Dc and Dt represent the mycelial growth diameters (cm) of the control and the treatment group, respectively; 0.5 represents the diameter of mycelial plugs (cm).
The effective concentration for 50% inhibition (EC50) was derived from the data analysis of the concentration inhibition rate. Serial concentrations of Xcn1 solutions were separately mixed with the molten PDA medium to the final concentrations of 0.1, 0.5, 1, 2, 4, 6, 8 and 10 μg/mL, and were poured into Petri dishes (60 mm diameter) to form plates. After inoculation of a 5-mm fungal mycelia disc into the center of the solidified medium, the dishes were incubated in the dark at 25 °C for 2 days. The inhibition rate was calculated via the above method when the control fungal mycelium reached the edge of the dishes. All of experiments were independently performed three times under the same conditions.

4.4. Assessment of Antifungal Activity of Xcn1 In Vivo

The efficacy of Xcn1 on broad bean and rape seedlings infected with S. sclerotiorum was evaluated according to a previous study with some modifications [46]. Seedlings of rape and broad bean plants were grown in a growth chamber (25 ± 1 °C, 75 ± 10% RH, and a 12:12 LD photoperiod) for 25 days. Stocks of Xcn1 (100 and 200 µg/mL) were prepared in sterile water composed of 0.5% Tween 20 (v/v). All experiments were arranged in a plant growth chamber with three plants per treatment and repeated three times.
For the protective activity assay, rape seedlings and broad bean leaves were sprayed with water, MBC (100 µg/mL), and Xcn1 (100 and 200 µg/mL) separately using a manually operated mini-knapsack sprayer (5 mL for each plant) [42]. The sprayer was held 20 cm away from the plant. After incubation for 12 h, each leaf was wounded by a sterilize needle at the center and inoculated with a 5-mm mycelia agar disc. Finally, the plants were placed in a plant growth chamber. Images were captured after incubation at 25 °C for two days. The lesion area was quantified using Image J 1.38× (https://imagej.nih.gov/ij/ accessed on 18 March 2019), and the control efficacy was calculated according to the following formula:
Control efficacy (%) = [(Ac − At)/Ac − 0.25] × 100
where Ac and At represent the disease areas of the control group and the treatment group, respectively; 0.25 represents the area of the mycelial agar disc (cm2).
For the curative activity assay of agentia to the broad bean and rape seedlings a 5-mm mycelia agar disc was placed in the center of the leaves. The plants were incubated for 12 h (~3 mm lesion around the discs), and then they were sprayed with the drugs as described above. The following procedures were same as those used for the protective activity assay.

4.5. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) Observations

The variations of hyphal morphology and ultrastructure of S. sclerotiorum treated with Xcn1 was investigated according to the method used by Soner Soylu [47]. Mycelial agar discs that had been growing for two days were placed in the middle of PDA plates with 3.0 μg/mL (EC50 value) of Xcn1 (one disc for each plate) and incubated at 25 °C for 48 h in darkness. PDA plates without Xcn1 were used as controls.
A 2.5% glutaraldehyde fixation of mycelial discs (2 mm × 4 mm × 4 mm) in 0.1 M phosphate buffer (pH = 7.2) was conducted overnight for scanning electron microscopy (SEM) observations. Next, with an identical buffer for each disc, they were washed three times for 20 min to remove the glutaraldehyde. The fixed specimens were dehydrated in a gradually increasing concentration of ethanol series (twice at 30%, 50%, 70%, 80%, and 90%, respectively, and three times at 100%) for 20 min for each succession. After dehydration, the discs were dipped into isoamyl acetate for replacement three times for 20 min each. Finally, each specimen was dried using supercritical carbon dioxide. With double-sided tape, the dried samples were fastened to a sample stage and given a gold coating using an E1010 sputter-coating machine (Hitachi, Tokyo, Japan) for 90 s at 9 mA. The images were recorded using a JSM-6360LV SEM (JEOL, Tokyo, Japan).
For transmission electron microscopy (TEM) observations, the pre-fixed mycelial discs were prepared as described in the SEM method. The discs were fixed with 1% osmic acid for 2 h after washing. Then, the specimens were washed again, immediately followed by dehydration as previously described. Samples were dipped into ethanol twice (30 min each), after which the specimens were passed through a solution of epoxy resin/epoxy propane (1:1, v/v) for 1 h and embedded in epoxy medium at 55 °C for 48 h. Subsequently, blocks were cut into slices using a diamond knife with an Ultramicrotome blade (Leica-ULTRACUT, Wetzlar, Germany) into ultrathin sections of approximately 70 nm. The ultrathin sections were contrasted with 2% uranyl acetate and 2% lead citrate for 30 min prior to examination on a JEM-1230 TEM (Hitachi, Tokyo, Japan).

4.6. Determination of Oxalic Acid (OA) Production of S. sclerotiorum

The medium acidification assay was verified on bromophenol blue agar plates with some modifications [11]. Molten PDA medium (30 mL) with 50 µg/mL of bromophenol blue was sufficiently mixed with Xcn1 at a low temperature (<40 °C) to the final concentration of 3.0 µg/mL, and the mixture was poured into Petri dishes (90-mm diameter) to form plates of a thickness of 2–3 mm (10 mL for each dishes). Mycelial plugs (5 mm in diameter) were transferred from the edges of 2-day-old colonies to the center of the plates (one plug for each plate). The plates without Xcn1 served as the controls. The Petri dishes were incubated at 25 °C in darkness, and the pH change was observed at 24 h intervals as the color shifted from blue to yellow.
Oxalic acid released in PDB medium by S. sclerotiorum isolates was quantified using the protocol of a previous study, with minor modifications [48]. Briefly, ten agar plugs of S. sclerotiorum were grown in a 250 mL Erlenmeyer flask containing 100 mL fresh PDB medium. The flasks were incubated on a rotary shaker at 25 °C with 150 rpm. After 24 h, Xcn1 was added to the flasks, with the final concentration being 3.0 μg/mL. Flasks without Xcn1 were used as the controls. The flasks were then statically incubated over different time periods (3, 6, 9, and 12 d). After that, the filtrates were centrifuged at 5000× g for 20 min, and the supernatants were used for oxalic acid determination. The weight of the mycelia of each sample was determined after lyophilization. The oxalic acid concentration was calibrated using the dry mycelium weight of each sample.

4.7. Measurement of the Cell Membrane Permeability of S. sclerotiorum

For the measurement of cell membrane permeability, S. sclerotiorum colonies were processed according to the method described in [42]. Ten agar plugs of S. sclerotiorum isolates were grown in a 250 mL Erlenmeyer flask containing 100 mL PDB. After 24 h, Xcn1 was added to the flasks, with the final concentration being 3.0 μg/mL. Flasks without Xcn1 were used as the controls. The flasks were then incubated at 25 °C and shaken at 150 rpm. After the flasks were shaken for 72 h, the mycelia were collected and washed three times with sterilized water. Then 1 g of fresh mycelia per sample was suspended in 50 mL of distilled water. The conductivity of the system was measured at 0, 1, 2, 4, 6, 8 and 10 h intervals via a conductivity meter (CON510 Eutech/Oakton, Singapore). After that, the samples were boiled for 10 min to measure final conductivity. Each treatment had three replicates, and the experiment was performed twice. The relative conductivity of mycelia was calculated as follows:
Relative conductivity = Cd/Cf × 100%
Cd represents the conductivity at a certain time interval, Cf represents the final conductivity.

4.8. Determination of the Exopolysaccharides of S. sclerotiorum

The effects of Xcn1 on exopolysaccharides of S. sclerotiorum were determined via the phenol-sulfuric acid method [48,49]. To establish the standard curve of EPSs, 2 mL of different glucose solutions (0, 20, 40, 60, 80, 100, 120, 160 and 200 µg/mL) were added to the different tubes. Then 1 mL of 5% phenol solution was added to each of the above tubes, and the solutions in each tube were mixed gently. Finally, 5 mL of H2SO4 was added to each tube slowly. The solutions in each tube were mixed gently for 10 s, and then incubated at 25 °C for 30 min. A standard curve was generated by plotting the absorbance at 490 nm against the glucose concentration.
For determination of EPS, the sample supernatants were prepared as described in Section 4.7. EPS was precipitated from 10 mL of each supernatant with 30 mL of absolute ethanol and dried at 50 °C. The sediment of EPS was dissolved in 10 mL of distilled water and quantified by the method of the standard curve. Sterile-distilled water was used as a control. Each treatment was set three replications, and the test was repeated three times.

4.9. Measurement of Activities of Polygalacturonase (PG) and Cellulase (Cx) of S. sclerotiorum

The activities of PG and Cx of S. sclerotiorum were measured according to the method of a previous study, with minor modifications [39]. Ten mycelial discs (5 mm diameter) were placed in a 250 mL Erlenmeyer flask containing 100 mL of Czapek’s liquid medium (NH4NO3 0.2%, KH2PO4 0.1%, MgSO4 0.01%, yeast extract 0.05%, NaOH 0.1%, vitamin B1, 3 g/L; pH 5.0) with 1% citrus pectin (Sigma Chemical, grade I) as the carbon source [50]. After 24 h, Xcn1 was added to the flasks, with the final concentration being 3.0 μg/mL. Flasks without Xcn1 were used as the controls. Flasks were incubated on a rotary shaker at 25 °C and shaken at 150 rpm. Culture filtrates were collected at the fixed time periods (3, 6, 9, and 12 d). The filtrates were centrifuged at 8000× g for 20 min, and the supernatants were stored at −80 °C as the crude enzyme solutions for use.

4.10. Data Analysis

Multiple comparisons between treatments were evaluated according to the least significant differences (l.s.d) at p < 0.05. The EC50 values were calculated statistically by probit analysis using the probit package of SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). The lesion areas were quantified from the captured images with an Image J 1.38× software (https://imagej.nih.gov/ij/, accessed on 18 March 2019).

5. Conclusions

Xcn1 exhibited a wider antifungal spectrum against 10 plant-pathogenic fungi and was most effective against S. sclerotiorum with excellent antifungal activities in vitro and in vivo. Xcn1 disturbed the mycelial growth and infection of S. sclerotiorum and has great potential to be developed as a new biopesticide for disease control.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof10030175/s1, Figure S1: 1H NMR spectrum of Xcn1. Figure S2: 13C NMR spectrum of Xcn1. Figure S3: HR-MS spectrum of Xcn1. Figure S4: HPLC analysis of Xcn1. Figure S5: In vivo antifungal activity of Xcn1 and carbendazim against S. sclerotiorum on broad bean. Figure S6: In vivo antifungal activity of Xcn1 and carbendazim against S. sclerotiorum on rape seedlings. Figure S7: The appearance of the S. sclerotiorum culture treated with Xcn1 (3.0 μg/mL) or not (CK) for 7 days. Table S1: The plant pathogenic fungi used in this study.

Author Contributions

Conceptualization, S.Z. and Y.W. (Yonghong Wang); Data curation, S.Z. and Y.H.; Formal analysis, S.Z., Y.H. and J.H.; Funding acquisition, Y.W. (Yonghong Wang); Investigation, Y.H., J.H. and Z.Y.; Methodology, L.W.; Project administration, Y.W. (Yonghong Wang); Resources, Y.W. (Yong Wang); Software, L.W. and Y.W. (Yong Wang); Supervision, Y.W. (Yonghong Wang); Validation, Y.H. and J.H.; Visualization, Z.Y.; Writing—original draft, S.Z.; Writing—review and editing, Y.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Hainan Province, China (No. 320QN189), National Natural Science Foundation of China (No. 31171910, 32072474), the Key Research and Development Program of Shaanxi Province of China (No. 2019NY-189), and Natural Science Foundation of Shaanxi Province (No. 2014JZ004).

Institutional Review Board Statement

This article does not contain any studies with human participants performed by any of the authors.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Acknowledgments

We thank the Life Science Research Core Services (Northwest A&F University, Yangling, China) for assisting in the using of Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM).

Conflicts of Interest

All authors declare that there are no conflicts of interest in this work.

References

  1. Duan, Y.; Li, T.; Xiao, X.; Wu, J.; Li, S.; Wang, J.; Zhou, M. Pharmacological characteristics of the novel fungicide pyrisoxazole against Sclerotinia sclerotiorum. Pestic. Biochem. Physiol. 2018, 149, 61–66. [Google Scholar] [CrossRef] [PubMed]
  2. Vagelas, I. Diagnosis and management of Sclerotinia stem rot (white mould) of lentils in Greece. Arch. Phytopathol. Plant Prot. 2014, 47, 1209–1217. [Google Scholar] [CrossRef]
  3. Huang, H.; Dueck, J. Wilt of sunflower from infection by mycelial-germinating sclerotia of Sclerotinia sclerotiorum. Can. J. Plant Pathol. 1980, 2, 47–52. [Google Scholar] [CrossRef]
  4. Wegulo, S.N.; Yang, X.B.; Martinson, C.A. Soybean Cultivar Responses to Sclerotinia sclerotiorum in Field and Controlled Environment Studies. Plant Dis. 1998, 82, 1264–1270. [Google Scholar] [CrossRef] [PubMed]
  5. Sarma, B.; Basha, S.A.; Singh, D.; Singh, U. Use of non-conventional chemicals as an alternative approach to protect chickpea (Cicer arietinum) from Sclerotinia stem rot. Crop Prot. 2007, 26, 1042–1048. [Google Scholar] [CrossRef]
  6. Ahmed, A.; Akhond, M. First report of Sclerotinia rot caused by Sclerotinia sclerotiorum on Lens culinaris in Bangladesh. New Dis. Rep. 2015, 31, 23. [Google Scholar] [CrossRef]
  7. Jones, E.E.; Mead, A.; Whipps, J.M. Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: Control of Sclerotinia disease in glasshouse lettuce. Plant Pathol. 2004, 53, 611–620. [Google Scholar] [CrossRef]
  8. Purdy, L.H. Sclerotinia sclerotiorum: History, diseases and symptomatology, host range, geographic distribution, and impact. Phytopathology 1979, 69, 875–880. [Google Scholar] [CrossRef]
  9. Amselem, J.; Cuomo, C.A.; van Kan, J.A.L.; Viaud, M.; Benito, E.P.; Couloux, A.; Coutinho, P.M.; de Vries, R.P.; Dyer, P.S.; Fillinger, S.; et al. Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet. 2011, 7, e1002230. [Google Scholar] [CrossRef]
  10. Williams, B.; Kabbage, M.; Kim, H.J.; Britt, R.; Dickman, M.B. Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathog. 2011, 7, e1002107. [Google Scholar] [CrossRef]
  11. Massawe, V.C.; Hanif, A.; Farzand, A.; Mburu, D.K.; Ochola, S.O.; Wu, L.; Tahir, H.A.S.; Gu, Q.; Wu, H.; Gao, X. Volatile Compounds of Endophytic Bacillus spp. have Biocontrol Activity Against Sclerotinia sclerotiorum. Phytopathology 2018, 108, 1373–1385. [Google Scholar] [CrossRef]
  12. Budge, S.P.; Whipps, J.M.; Chitrampalam, P.; Wu, B.M.; Koike, S.T.; Subbarao, K.V.; Turini, T.A.; Matheron, M.E.; Pryor, B.M.; Figuli, P.J.; et al. Potential for Integrated Control of Sclerotinia sclerotiorum in Glasshouse Lettuce Using Coniothyrium minitans and Reduced Fungicide Application. Phytopathology 2001, 91, 221–227. [Google Scholar] [CrossRef]
  13. Singh, M.; Sharma, O.P.; Bhagat, S.; Pandey, N. Effect of systemic fungicides, culture media, temperature and pH on growth of Sclerotinia sclerotiorum (Lib.) de Bary causing White mold of Chick pea. Ann. Plant Prot. Sci. 2013, 21, 136–139. [Google Scholar]
  14. Wang, Y.; Hou, Y.-P.; Chen, C.-J.; Zhou, M.-G. Detection of resistance in Sclerotinia sclerotiorum to carbendazim and dimethachlon in Jiangsu Province of China. Australas. Plant Pathol. 2014, 43, 307–312. [Google Scholar] [CrossRef]
  15. Chen, X.; Pizzatti, C.; Bonaldi, M.; Saracchi, M.; Erlacher, A.; Kunova, A.; Berg, G.; Cortesi, P. Biological Control of Lettuce Drop and Host Plant Colonization by Rhizospheric and Endophytic Streptomycetes. Front. Microbiol. 2016, 7, 714. [Google Scholar] [CrossRef] [PubMed]
  16. Koehler, H.R.; Triebskorn, R. Wildlife ecotoxicology of pesticides: Can we track effects to the population level and beyond? Science 2013, 341, 759–765. [Google Scholar] [CrossRef]
  17. Lamberth, C.; Jeanmart, S.; Luksch, T.; Plant, A. Current challenges and trends in the discovery of agrochemicals. Science 2013, 341, 742–746. [Google Scholar] [CrossRef] [PubMed]
  18. Herbert, E.E.; Goodrich-Blair, H. Friend and foe: The two faces of Xenorhabdus nematophila. Nat. Rev. Microbiol. 2007, 5, 634–646. [Google Scholar] [CrossRef] [PubMed]
  19. Chen, G.; Dunphy, G.; Webster, J. Antifungal activity of Two Xenorhabdus species and Photorhabdus luminescens, bacteria associated with the nematodes Steinernema species and Heterorhabditis megidis. Biol. Control 1994, 4, 157–162. [Google Scholar] [CrossRef]
  20. McInerney, B.V.; Taylor, W.C.; Lacey, M.J.; Akhurst, R.J.; Gregson, R.P. Biologically active metabolites from Xenorhabdus spp., Part 2. Benzopyran-1-one derivatives with gastroprotective activity. J. Nat. Prod. 1991, 54, 785–795. [Google Scholar] [CrossRef]
  21. Yang, X.; Qiu, D.; Yang, H.; Liu, Z.; Zeng, H.; Yuan, J. Antifungal activity of xenocoumacin 1 from Xenorhabdus nematophilus var. pekingensis against Phytophthora infestans. World J. Microbiol. Biotechnol. 2011, 27, 523–528. [Google Scholar] [CrossRef]
  22. Zhou, T.; Yang, X.; Qiu, D.; Zeng, H. Inhibitory effects of xenocoumacin 1 on the different stages of Phytophthora capsici and its control effect on Phytophthora blight of pepper. BioControl 2017, 62, 151–160. [Google Scholar] [CrossRef]
  23. Zhou, T.; Zeng, H.; Qiu, D.; Yang, X.; Wang, B.; Chen, M.; Guo, L.; Wang, S. Global transcriptional responses of Bacillus subtilis to xenocoumacin 1. J. Appl. Microbiol. 2011, 111, 652–662. [Google Scholar] [CrossRef]
  24. Guo, S.; Zhang, S.; Fang, X.; Liu, Q.; Gao, J.; Bilal, M.; Wang, Y.; Zhang, X. Regulation of antimicrobial activity and xenocoumacins biosynthesis by pH in Xenorhabdus nematophila. Microb. Cell Factories 2017, 16, 203. [Google Scholar] [CrossRef]
  25. Zhang, S.; Fang, X.; Tang, Q.; Ge, J.; Wang, Y.; Zhang, X. CpxR negatively regulates the production of xenocoumacin 1, a dihydroisocoumarin derivative produced by Xenorhabdus nematophila. MicrobiologyOpen 2018, 8, e00674. [Google Scholar] [CrossRef]
  26. Zhang, S.; Liu, Q.; Han, Y.; Han, J.; Yan, Z.; Wang, Y.; Zhang, X. Nematophin, an antimicrobial dipeptide compound from Xenorhabdus nematophila YL001 as a potent biopesticide for Rhizoctonia solani control. Front. Microbiol. 2019, 10, 1765. [Google Scholar] [CrossRef]
  27. Flores, R. A rapid and reproducible assay for quantitative estimation of proteins using bromophenol blue. Anal. Biochem. 1978, 88, 605–611. [Google Scholar] [CrossRef] [PubMed]
  28. Xu, L.; Xiang, M.; White, D.; Chen, W. pH dependency of sclerotial development and pathogenicity revealed by using genetically defined oxalate-minus mutants of Sclerotinia sclerotiorum. Environ. Microbiol. 2015, 17, 2896–2909. [Google Scholar] [CrossRef] [PubMed]
  29. Kamoun, S.; I Kado, C. A plant-inducible gene of Xanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts. J. Bacteriol. 1990, 172, 5165–5172. [Google Scholar] [CrossRef] [PubMed]
  30. Kim, S.W.; Hwang, H.J.; Xu, C.P.; Choi, J.W.; Yun, J.W. Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii. Lett. Appl. Microbiol. 2003, 36, 321–326. [Google Scholar] [CrossRef]
  31. Wang, Y.; Duan, Y.; Zhou, M. Molecular and biochemical characterization of boscalid resistance in laboratory mutants of Sclerotinia sclerotiorum. Plant Pathol. 2015, 64, 101–108. [Google Scholar] [CrossRef]
  32. Baker, C.J.; Whalen, C.H.; Korman, R.Z.; Bateman, D.F. α-l-Arabinofuranosidase from Sclerotinia sclerotiorum: Purification, Characterization, and Effects on Plant Cell Walls and Tissue. Phytopathology 1979, 69, 789–793. [Google Scholar] [CrossRef]
  33. Bauer, W.D.; Bateman, D.F.; Whalen, C.H. Purification of an Endo-β-1,4 Galactanase Produced by Sclerotinia sclerotiorum: Effects on Isolated Plant Cell Walls and Potato Tissue. Phytopathology 1977, 77, 862–868. [Google Scholar] [CrossRef]
  34. Favaron, F.; Alghisi, P.; Marciano, P.; Magro, P. Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sclerotiorum in soybean hypocotyls as elicitors of glyceollin. Physiol. Mol. Plant Pathol. 1988, 33, 385–395. [Google Scholar] [CrossRef]
  35. Polikanov, Y.S.; Osterman, I.A.; Szal, T.; Tashlitsky, V.N.; Serebryakova, M.V.; Kusochek, P.; Bulkley, D.; Malanicheva, I.A.; Efimenko, T.A.; Efremenkova, O.V.; et al. Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome. Mol. Cell 2014, 56, 531–540. [Google Scholar] [CrossRef]
  36. Zumbrunn, C.; Krüsi, D.; Stamm, C.; Caspers, P.; Ritz, D.; Rueedi, G. Synthesis and structure-activity relationship of Xenocoumacin 1 and analogues as inhibitors of ribosomal protein synthesis. ChemMedChem 2021, 16, 891–897. [Google Scholar] [CrossRef] [PubMed]
  37. Svidritskiy, E.; Ling, C.; Ermolenko, D.N.; Korostelev, A.A. Blasticidin S inhibits translation by trapping deformed tRNA on the ribosome. Proc. Natl. Acad. Sci. USA 2013, 110, 12283–12288. [Google Scholar] [CrossRef]
  38. Svidritskiy, E.; Korostelev, A.A. Mechanism of inhibition of translation termination by Blasticidin S. J. Mol. Biol. 2018, 430, 591–593. [Google Scholar] [CrossRef]
  39. Marciano, P.; Di Lenna, P.; Magro, P. Oxalic acid, cell wall-degrading enzymes and pH in pathogenesis and their significance in the virulence of two Sclerotinia sclerotiorum isolates on sunflower. Physiol. Plant Pathol. 1983, 22, 339–345. [Google Scholar] [CrossRef]
  40. Heller, A.; Witt-Geiges, T. Oxalic Acid Has an Additional, Detoxifying Function in Sclerotinia sclerotiorum Pathogenesis. PLoS ONE 2013, 8, e72292. [Google Scholar] [CrossRef]
  41. Hou, Y.-P.; Mao, X.-W.; Lin, S.-P.; Song, X.-S.; Duan, Y.-B.; Wang, J.-X.; Zhou, M.-G. Activity of a novel succinate dehydrogenase inhibitor fungicide pyraziflumid against Sclerotinia sclerotiorum. Pestic. Biochem. Physiol. 2018, 145, 22–28. [Google Scholar] [CrossRef]
  42. Wang, Y.; Sun, Y.; Wang, J.; Zhou, M.; Wang, M.; Feng, J. Antifungal activity and action mechanism of the natural product cinnamic acid against Sclerotinia sclerotiorum. Plant Dis. 2019, 103, 944–950. [Google Scholar] [CrossRef]
  43. Yan, H.; Xiong, Z.; Xie, N.; Liu, S.; Zhang, L.; Xu, F.; Guo, W.; Feng, J. Bioassay-guided isolation of antifungal amides against Sclerotinia sclerotiorum from the seeds of Clausena lansium. Ind. Crop Prod. 2018, 121, 352–359. [Google Scholar] [CrossRef]
  44. Wang, Y.; Zhang, X. Isolation and identification of symbiotic bacteria associated with entomopathogenic nematodes. J. Northwest AF Univ. (Nat. Sci. Ed.) 2006, 34, 174–180. [Google Scholar]
  45. Huang, W.R.; Zhu, C.X.; Yang, X.F.; Yang, H.W.; Xu, H.Z.; Xie, Y.Y.; Jian, H. Isolation and structural identification of main component CB6-1 produced by Xenorhabdus nematophilus var. pekingensis. Chin. J. Antibiot. 2005, 30, 513–515. [Google Scholar]
  46. Yang, D.; Wang, B.; Wang, J.; Chen, Y.; Zhou, M. Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape. Biol. Control 2009, 51, 61–65. [Google Scholar] [CrossRef]
  47. Soylu, E.M.; Kurt, S.; Soylu, S. In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea. Int. J. Food Microbiol. 2010, 143, 183–189. [Google Scholar] [CrossRef]
  48. Duan, Y.; Ge, C.; Liu, S.; Chen, C.; Zhou, M. Effect of phenylpyrrole fungicide fludioxonil on morphological and physiological characteristics of Sclerotinia sclerotiorum. Pestic. Biochem. Physiol. 2013, 106, 61–67. [Google Scholar] [CrossRef]
  49. Rao, P.; Pattabiraman, T.N. Reevaluation of the phenol-sulfuric acid reaction for the estimation of hexoses and pentoses. Anal. Biochem. 1989, 181, 18–22. [Google Scholar] [CrossRef]
  50. Riou, C.; Freyssinet, G.; Fevre, M. Production of Cell Wall-Degrading Enzymes by the Phytopathogenic Fungus Sclerotinia sclerotiorum. Appl. Environ. Microbiol. 1991, 57, 1478–1484. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. In vivo antifungal activity of Xcn1 and carbendazim against S. sclerotiorum in broad bean. (A,C) Protective activity of Xcn1. (B,D) Curative activity of Xcn1. Preventive/curative efficacy: a sample solution was sprayed on broad bean leaves 12 h before/after inoculation with S. sclerotiorum. Xcn1-100, 100 µg/mL of Xcn1; Xcn1-200, 200 µg/mL of Xcn1; MBC: 100 µg/mL of carbendazim. The different lowercase letters in the bar chart indicate significant differences according to Fisher’s prtected (l.s.d) test (p = 0.05).
Figure 1. In vivo antifungal activity of Xcn1 and carbendazim against S. sclerotiorum in broad bean. (A,C) Protective activity of Xcn1. (B,D) Curative activity of Xcn1. Preventive/curative efficacy: a sample solution was sprayed on broad bean leaves 12 h before/after inoculation with S. sclerotiorum. Xcn1-100, 100 µg/mL of Xcn1; Xcn1-200, 200 µg/mL of Xcn1; MBC: 100 µg/mL of carbendazim. The different lowercase letters in the bar chart indicate significant differences according to Fisher’s prtected (l.s.d) test (p = 0.05).
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Figure 2. In vivo antifungal activity of Xcn1 and carbendazim against S. sclerotiorum in rape seedlings. (A,C) Protective activity of Xcn1. (B,D) Curative activity of Xcn1. Preventive/curative efficacy: a sample solution was sprayed on broad bean leaves 12 h before/after inoculation with S. sclerotiorum. Xcn1-100, 100 µg/mL of Xcn1; Xcn1-200, 200 µg/mL of Xcn1; MBC: 100 µg/mL of carbendazim. The different lowercase letters in the bar chart indicate significant differences according to Fisher’s prtected (l.s.d) test (p = 0.05).
Figure 2. In vivo antifungal activity of Xcn1 and carbendazim against S. sclerotiorum in rape seedlings. (A,C) Protective activity of Xcn1. (B,D) Curative activity of Xcn1. Preventive/curative efficacy: a sample solution was sprayed on broad bean leaves 12 h before/after inoculation with S. sclerotiorum. Xcn1-100, 100 µg/mL of Xcn1; Xcn1-200, 200 µg/mL of Xcn1; MBC: 100 µg/mL of carbendazim. The different lowercase letters in the bar chart indicate significant differences according to Fisher’s prtected (l.s.d) test (p = 0.05).
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Figure 3. Effects of Xcn1 on the hyphal morphology of S. sclerotiorum. (A,B) Healthy hyphae in control plates. (C–F) Hyphae treated with Xcn1 (3.0 µg/mL). Scale bar: 100 µm for (A,C,D); 10 µm for B; 50 µm for (E); 20 µm for (F).
Figure 3. Effects of Xcn1 on the hyphal morphology of S. sclerotiorum. (A,B) Healthy hyphae in control plates. (C–F) Hyphae treated with Xcn1 (3.0 µg/mL). Scale bar: 100 µm for (A,C,D); 10 µm for B; 50 µm for (E); 20 µm for (F).
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Figure 4. Effects of Xcn1 on the hyphal cell ultrastructure of S. sclerotiorum. (A,B) Healthy hyphae in control plates. (C–F) Hyphae treated with Xcn1 (3.0 µg/mL). Xcn1: plates treated with Xcn1 at 3.0 µg/mL; 1, the invaginated cell membrane; 2 and 4, vacuolation degradation; 3, mitochondrial abnormalities; 5, cytoplasmic disorders; M: mitochondria, ER: endoplasmic reticulum, N: nucleus, V: vacuole, CY: cytoplasm, and CM: cell membrane. Scale bar: 2 µm for (A); 500 nm for (B,C,E,F); 1 µm for (D).
Figure 4. Effects of Xcn1 on the hyphal cell ultrastructure of S. sclerotiorum. (A,B) Healthy hyphae in control plates. (C–F) Hyphae treated with Xcn1 (3.0 µg/mL). Xcn1: plates treated with Xcn1 at 3.0 µg/mL; 1, the invaginated cell membrane; 2 and 4, vacuolation degradation; 3, mitochondrial abnormalities; 5, cytoplasmic disorders; M: mitochondria, ER: endoplasmic reticulum, N: nucleus, V: vacuole, CY: cytoplasm, and CM: cell membrane. Scale bar: 2 µm for (A); 500 nm for (B,C,E,F); 1 µm for (D).
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Figure 5. Pharmacological characteristics of Xcn1 on the S. sclerotiorum. (A) The relative conductivity variations of the mycelia of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) for 72 h. (B) The exopolysaccharide (EPS) secretion contents of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). (C) Medium acidification assay of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (1, 2, and 3 d). (D) The OA contents of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). (E) Polygalacturonase (PG) activities of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). (F) Cellulase (Cx) activities of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). Statistical differences were detected between Xcn1 treatment and the CK with independent sample t tests (asterisk [*] indicates p < 0.05).
Figure 5. Pharmacological characteristics of Xcn1 on the S. sclerotiorum. (A) The relative conductivity variations of the mycelia of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) for 72 h. (B) The exopolysaccharide (EPS) secretion contents of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). (C) Medium acidification assay of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (1, 2, and 3 d). (D) The OA contents of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). (E) Polygalacturonase (PG) activities of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). (F) Cellulase (Cx) activities of S. sclerotiorum treated with Xcn1 (3.0 μg/mL) or not (CK) at different time intervals (3, 6, 9, and 12 d). Statistical differences were detected between Xcn1 treatment and the CK with independent sample t tests (asterisk [*] indicates p < 0.05).
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Table 1. The inhibition rates of Xcn1 against 10 plant-pathogenic fungi.
Table 1. The inhibition rates of Xcn1 against 10 plant-pathogenic fungi.
Tested FungiInhibition Rate (%)
Xcn1 aMBC b
Sclerotinia sclerotiorum89.30 ± 2.3095.17 ± 4.05
Rhizoctonia solani88.50 ± 3.1067.70 ± 3.15
Botrytis cinerea86.70 ± 3.2013.88 ± 2.28
Exserohilum turcicum83.20 ± 2.20-
Alternaria alternata75.00 ± 1.80-
Fusarium graminearum50.60 ± 1.2989.78 ± 3.08
Alternaria solani67.50 ± 2.1065.58 ± 3.52
Colletotrichum gloeosporioides47.01 ± 3.1056.80 ± 2.24
Gaeumannomyces graminis38.11 ± 3.4044.05 ± 2.12
Fusarium oxysporum11.80 ± 2.2034.54 ± 3.87
a The concentration of Xcn1 was set at 10 µg/mL. b The concentration of carbendazim (MBC) was set at 0.5 µg/mL. Data are represented as the mean of three replications ± standard deviation.
Table 2. Antifungal activity of Xcn1 against S. sclerotiorum.
Table 2. Antifungal activity of Xcn1 against S. sclerotiorum.
StrainRegression CurveEC50 a (CI95 b) (μg/mL)EC95 c (CI95 b) (μg/mL)Chi d
S. sclerotiorumy = 2.01x − 0.92 2.86 (2.38–3.32)18.71 (14.49–26.65)2.21
a Effective dose for 50% inhibition compared with the control. b 95% confidence intervals. c Effective dose for 95% inhibition compared with the control. d Chi-square value, significant at p < 0.05.
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Zhang, S.; Han, Y.; Wang, L.; Han, J.; Yan, Z.; Wang, Y.; Wang, Y. Antifungal Activity and Mechanism of Xenocoumacin 1, a Natural Product from Xenorhabdus nematophila against Sclerotinia sclerotiorum. J. Fungi 2024, 10, 175. https://doi.org/10.3390/jof10030175

AMA Style

Zhang S, Han Y, Wang L, Han J, Yan Z, Wang Y, Wang Y. Antifungal Activity and Mechanism of Xenocoumacin 1, a Natural Product from Xenorhabdus nematophila against Sclerotinia sclerotiorum. Journal of Fungi. 2024; 10(3):175. https://doi.org/10.3390/jof10030175

Chicago/Turabian Style

Zhang, Shujing, Yunfei Han, Lanying Wang, Jinhua Han, Zhiqiang Yan, Yong Wang, and Yonghong Wang. 2024. "Antifungal Activity and Mechanism of Xenocoumacin 1, a Natural Product from Xenorhabdus nematophila against Sclerotinia sclerotiorum" Journal of Fungi 10, no. 3: 175. https://doi.org/10.3390/jof10030175

APA Style

Zhang, S., Han, Y., Wang, L., Han, J., Yan, Z., Wang, Y., & Wang, Y. (2024). Antifungal Activity and Mechanism of Xenocoumacin 1, a Natural Product from Xenorhabdus nematophila against Sclerotinia sclerotiorum. Journal of Fungi, 10(3), 175. https://doi.org/10.3390/jof10030175

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