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Article

Evaluation of the Antifungal Activity of Polysubstituted Cyclic 1,2-Diketones against Colletotrichum gloeosporioides

by
Qiuyue Wang
1,
Xiangtai Meng
2,
Meiling Sun
1,
Zhi Wang
1,
Jiao He
1,
Shenlin Huang
2 and
Lin Huang
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2
International Innovation Center for Forest Chemicals and Materials, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(6), 1172; https://doi.org/10.3390/f14061172
Submission received: 24 March 2023 / Revised: 25 April 2023 / Accepted: 11 May 2023 / Published: 6 June 2023
(This article belongs to the Section Forest Health)
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Abstract

:
(1) Anthracnose caused by Colletotrichum damages crops, ornamentals, and forest trees severely, resulting in enormous economic losses to agricultural and forestry systems. Overusing traditional chemical fungicides leads to fungicide resistance, environmental pollution, and potential risks to public health. Therefore, priorities should be given to developing efficient and environmentally friendly approaches to phytopathogens management, including anthracnose. (2) In this study, the antifungal activity of botanical derivative polysubstituted cyclic 1,2-diketones (FPL001) against C. gloeosporioides was examined. (3) FPL001 significantly inhibited the vegetative growth of C. gloeosporioides with an EC50 of 160.23 µg/mL. When the concentration of FPL001 reached 30 µg/mL, the conidial germination and appressorium formation of C. gloeosporioides were completely inhibited. FPL001 also significantly suppressed the invasive hyphae development and plant infection of C. gloeosporioides. FPL001 did not exhibit toxicity to model organisms such as alfalfa and silkworm larvae. (4) These results indicate that compound FPL001 is a potential and efficient agent for green control of C. gloeosporioides.

1. Introduction

Fungi are the most damaging agents in agricultural and forestry ecosystems. To reduce losses from plant diseases, fungicides have been widely applied in agriculture and forestry [1]. Although traditional chemical fungicides have been used to control a variety of plant diseases for over 100 years, the long-term use of traditional chemical fungicides is a threat to human health and brings negative impacts on non-target organisms [2,3]. For example, ziram has negative effects on neural and visual disturbances [4]. Mancozeb reduces the egg hatch of Amblyseius andersoni [5]. Azoxystrobin and copper sulfate are toxic to fish [6,7]. Therefore, it has become a research priority to develop alternative methods for phytopathogens management.
Biofungicides have been given increasing attention due to their being environmentally friendly, non-toxic and without residues. Many compounds of biofungicides can attack various targets, which makes it worth exploiting multi-functional fungicides [8]. Biofungicides, derived from plants, have fewer side effects compared to most chemical fungicides. Thus, the intelligent use of biofungicides produces no damage to ecosystem stability and biodiversity [9]. Furthermore, botanical fungicides lack cross-resistance due to their different mode of action than chemical fungicides, which can effectively overcome fungal resistance [10]. Thus, botanical fungicides are considered a sustainable alternative to traditional chemical fungicides.
3-Methyl cyclopenten-1,2-dione (MCP), a natural cyclic alkene, is widely distributed in different plants, including maple, tobacco, papaya, and roasted coffee [11,12,13]. It was reported that MCP is an essential intermediate in the production of fungicides and pharmaceuticals, and possesses important biological activity and medicinal value in its antibacterial, anti-inflammatory, and antitumor effects [14,15,16,17]. Compounds of polysubstituted cyclic 1,2-diketones are also commonly found in various natural products such as bruceantin, terpestacin, acenaphtenequinone, benzils, and bruceollines [18,19,20,21,22]. It is extensively used in chemical and biopharmaceutical fields, but its applications in plant protection deserves further studies.
Colletotrichum gloeosporiodes, a notable pathogen, causes anthracnose on crops and trees in more than 470 different host species [23], including primary host plants such as Capsicum spp., Pyrus spp., Camellia spp., and Citrus spp. [24,25,26,27]. In the process of C. gloeosporioides infection of host plants, conidia germination falls on host cells, generates appressoria to penetrate the plant cell, and then develops invasive hyphae (IH) to colonize. However, at present, reducing economic losses by C. gloeosporioides mainly relies on chemical fungicides such as benzoyl, maneb, and chlorothalonil [28]. Although these fungicides effectively suppress the development of symptoms in most hosts, long-term overuse of fungicides leads to potential threats of fungicide resistance, environmental pollution, and potential risks to public health. Thus, it is necessary to highlight and exploit advanced and non-pollutive biofungicides for anthracnose caused by C. gloeosporioides.
In this study, we screen a safer, more effective fungicide from the antifungal activity of the MCP derivative polysubstituted cyclic 1,2-diketones. One bio-compound, FPL001 (2-Hydroxy-5-methyl-5-(2-(propylthio)benzofuran-3-yl)cyclopent-2-en-1-one), drastically suppressed mycelial growth of C. gloeosporioides. Furthermore, it was also observed that FPL001 inhibits conidial germination, appressorium formation, and invasive hyphae development. In addition, the inoculation assay demonstrated that FPL001 prevents the development of lesions in different hosts (Liriodendron chinense × tulipifera, Populus × euramericana cv. ‘Nanlin895’, and Cunninghamia lanceolate). Therefore, it is suggested that FPL001 is a potential non-pollutive with low toxicity to humans and can serve as an advanced biofungicide.

2. Materials and Methods

2.1. Fungal Pathogen

Colletotrichum gloeosporioides strain SMCG1#C provided by the Forest Pathology Laboratory of Nanjing Forestry University (Nanjing, China) was adopted as the fungal pathogen. It was isolated from the diseased leaves of Chinese fir with anthracnose symptoms [29]. This fungus was grown at 25 °C on Potato Dextrose Agar (PDA) plates.

2.2. Preparation of Compounds of Polysubstituted Cyclic 1,2-Diketones

Polysubstituted cyclic 1,2-diketones (FPL001, FPL002, FPL003, FPL004, and FPL005) were synthesized following previously reported procedures (3-Methyl cyclopenten-1,2-dione MCP) from the Chemical Engineering Laboratory of Nanjing Forestry University (Nanjing, China) [30]. Compounds were purified by silica gel column chromatography. Polysubstituted cyclic 1,2-diketones were dissolved in methanol containing Tween-80 (0.1%). The primary antifungal activity of MCP derivative compounds was analyzed through the mycelia growth inhibition method [31]. Chemical structures of polysubstituted cyclic 1,2-diketones (FPL001, FPL002, FPL003, FPL004, and FPL005) are shown in Figure S1.

2.3. Mycelial Growth Assays

The inhibitory activity of FPL001 on the mycelial growth of C. gloeosporioides was determined as described previously [31]. Mycelial blocks (6 mm in diameter) were inoculated onto PDA medium plates containing 100, 150, 200, 250, and 300 µg/mL FPL001, respectively. Mycelial blocks inoculated onto PDA without FPL001 were the negative control. Additionally, all plates were kept at 25 °C in dark for 5 days. The concentration inhibiting mycelium growth by 50% (EC50) was calculated by fitting a regression equation of the probability value corresponding to the inhibition rate and logarithm of the concentration of 10. The experiment was carried out three times with five replicates.
The inhibition rate of FPL001 against C. gloeosporioides was calculated according to the following formula:
Inhibition rate (%) = ((R1 − R2)/R1) × 100
where R1 represents the colony diameter of control, and R2 the colony diameter of C. gloeosporioides treated with FPL001.

2.4. Conidial Germination and Appressorium Formation Assays

The effects of FPL001 on conidial germination and appressorium formation were exploited [32]. Mycelial blocks SMCG1#C were inoculated into liquid complete medium (CM) and shaken in dark at 200 r/min for 1 day at 25 °C. Conidia of C. gloeosporioides were collected through two layers of Miracloth (EMD Millipore, Bellerica, MA, USA). Conidial suspensions were adjusted to 1 × 105/mL. FPL001 was added into the conidial suspensions to obtain the mixture with the final FPL001 concentration of 10, 15, 20, 25, and 30 µg/mL, respectively. Ten microliters of conidial suspensions of each treatment were placed on the glass coverslip (Fisher Scientific, St. Louis, MO, USA). The conidial germination rate was calculated at 4 h after inoculation. Additionally, appressorium formation rate was tested at 12 h after inoculation. The control was conidial suspensions without compound FPL001. This experiment was conducted three times with at least 100 structures per replicate.
The conidial germination rate (GR) and appressorium formation rate (FR) were calculated by the following formulas:
GR (%) = G/S × 100
FR (%) = F/S × 100
GR represents the conidial germination rate while FR the appressorium formation rate. G means the number of germinated conidia, F the number of developed appressoria and S the number of all conidia observed.

2.5. Pathogenicity Tests In Vitro

The effect of compound FPL001 on pathogenicity of C. gloeosporioides was tested [33]. Conidial suspensions containing 250 µg/mL of FPL001 were prepared. Ten microliters, ten microliters, and five microliters of conidial suspensions were inoculated on the leaves of Liriodendron chinense × tulipifera, Populus × euramericana cv. ‘Nanlin895’, and Cunninghamia lanceolate, respectively. The leaves were kept in an incubator at 25 °C with high humidity, and lesion size was measured at 5 days post inoculation (dpi). The experiment was conducted three times, and each treatment had six replicates.

2.6. Vegetative Growth Assays

The effect of FPL001 on the vegetative growth of C. gloeosporioides was evaluated [33]. FPL001 was prepared as described above and added to liquid potato dextrose (PD) at a final concentration of 250 µg/mL. Nine mycelial blocks (2 mm in diameter) were inoculated with the PD and shaken at 150 r/min at 25 °C. The PD without FPL001 was the negative control. Mycelia of C. gloeosporioides were collected, freeze-dried, and weighed at 3 dpi. The diameter of mycelial balls of C. gloeosporioides was measured with a Zeiss stereo microscope (SteRo Discovery v20). The experiment was carried out three times with three replicates.

2.7. Invasive Hyphae Assays

The effect of FPL001 on invasive hyphae was determined [34]. Conidial suspensions containing 25 µg/mL of FPL001 were inoculated onto the onion epidermal layers and incubated in a chamber at 25 °C. The invasive hypha was observed at 24 h post inoculation. Conidial suspensions without FPL001 were used as a control. Thirty invasive structures were observed under a Zeiss Axio imager A2m microscope (Carl Zeiss, Jena, Germany). Invasive hypha was divided into four types (type I, no penetration; type II, IH with one branch; type III, IH with not less than two branches and limited expansion; and type IV, IH with massive branches and extensive hyphal growth). The experiment was conducted three times.

2.8. Safety Test of Compound FPL001 on Alfalfa and Silkworm

The effect of FPL001 on seed germination of alfalfa (Medicago sativa) was performed [35]. The alfalfa seeds were surface-disinfested by immersion in 70% ethanol for 10 min, washed with sterilized deionized water, and air-dried. Twenty-five alfalfa seeds were placed in Petri dishes (90 mm in diameter) with two layers of filter papers. Then, 5 mL of FPL001 solution at concentrations of 125, 250, 500, and 1000 µg/mL was added to Petri dishes, respectively. The control was cultured in the solution without FPL001. Subsequently, seeds were kept in an incubator at 25 °C under a 12 h cycle of light and darkness. After 48 h, seed germination rate, root length, and stem length were measured. This experiment was conducted three times, and each treatment had three replicates.
The effect of FPL001 on silkworm (Bombyx mori) larvae was performed analyzed following the method as described previously [36]. Cleaned mulberry leaves were immersed into dilutions with different concentrations of FPL001 of 250, 500, 1000, and 2000 µg/mL, respectively. After 30 s, these leaves were air-dried and employed to feed 20 size-matched second-instar silkworms. Leaves dipped into dilutions were used as the control. The treated mulberry leaves were fed to silkworm larvae for 96 h and the survival rate of silkworm larvae was calculated. The experiment was conducted three times, and each treatment had three replicates.

2.9. Statistical Analysis

All experiments in this study were independently repeated at least three times with similar results unless otherwise noted. Statistical significance was analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test by SPSS 23 software. Pairwise comparisons were performed with Student’s t-test using GraphPad Prism 8 software [37].

3. Results

3.1. Suppression of the Mycelial Growth by Antifungal Compounds of Polysubstituted Cyclic 1,2-Diketones

To screen the effective botanical antifungal compounds, similar mycelial blocks of C. gloeosporioides (SMCG1#C) inoculated onto the PDA plates containing the tested 250 µg/mL polysubstituted cyclic 1,2-diketones (FPL001, FPL002, FPL003, FPL004, and FPL005) grown at 25 °C. These results showed that the colony diameter of fungus grown on the PDA plates containing different compounds was smaller than the control (Table 1). Among these compounds, FPL001 showed the strongest inhibition of mycelial growth. Thus, FPL001 was selected for further study on its antifungal activity against C. gloeosporioides.
When the C. gloeosporioides was exposed to FPL001 of different concentrations of 100, 150, 200, 250, and 300 µg/mL, the growth of SMCG1#C was inhibited by 41.88, 47.64, 53.79, 59.46, and 61.58%, correspondingly (Figure 1a). The virulence regression equation of FPL001 for C. gloeosporioides is Y = 1.094 X + 2.588, and the EC50 is 160.23 µg/mL (Figure 1b). These results indicated that FPL001 significantly inhibited the mycelial growth of C. gloeosporioides.

3.2. Inhibitory Effects of FPL001 on Conidial Germination and Appressorium Formation of C. gloeosporioides

Conidial germination and appressoria formation are key infection structures in the early stage of C. gloeosporioides. Conidia of C. gloeosporioides were firstly treated with FPL001 of different concentrations (0, 10, 15, 20, 25, and 30 µg/mL) (Figure 2a,b). When the concentration of FPL001 reached 30 µg/mL, conidial germination was completely inhibited 4 h post inoculation (Figure 2a,b). Similarly, FPL001 had a significant negative effect on appressorium formation. Appressorium development was also completely inhibited 12 h after inoculation in FPL001 of a concentration of 30 µg/mL. (Figure 2a,c). These data indicated that FPL001 suppressed conidial germination and appressorium development, which can prevent infection of C. gloeosporioides.

3.3. Compound FPL001 Reduced Pathogenicity of C. gloeosporioides

The inhibitory effect of FPL001 on the development of anthracnose was investigated using detached leaves of Liriodendron chinense × tulipifera, Populus × euramericana cv. ‘Nanlin895’, and Cunninghamia lanceolate. Prepared conidial suspensions of C. gloeosporioides with FPL001 were inoculated on the leaves of different host plants. The pathogenicity assay showed a smaller lesion size caused by conidia of C. gloeosporioides when treated with FPL001 (250 µg/mL) compared with the control in three host plants (Figure 3). Inoculated ddH2O was the negative control (Figure 3). These results suggest that FPL001 reduced the anthracnose development on different host plants.

3.4. Effect of FPL001 on the Biomass of C. gloeosporioides

The effect of FPL001 was further evaluated on the biomass of C. gloeosporioides. Mycelial blocks of C. gloeosporioides were inoculated into liquid PD medium with or without compound FPL001 of 250 µg/mL. The average diameter of the mycelial balls of C. gloeosporioides treated with FPL001 was 3.6 mm, significantly smaller than that (6.9 mm in diameter) of the control (Figure 4a–d). Similarly, the average dry weight of the mycelial balls of C. gloeosporioides treated with FPL001 was 0.012 g, which was markedly lower than that of the control of 0.081 g (Figure 4a–c,e).

3.5. Effects of FPL001 on Invasive Hyphae of C. gloeosporioides

To test the FPL001-suppressed invasive hypha development, the conidial suspensions containing FPL001 were inoculated onto onion epidermal layers and incubated in a chamber at 25 °C. The results showed that most invasive hyphae belonged to type IV, which had numerous branches and extensive hyphal growth (Figure 5a,b). The ratio of type III and type IV invasive hyphae (25.56%, 42.22%) in FPL001 of 25 µg/mL was lower than the control not treated with FPL001 (18.89%, 68.89%) (Figure 5a,b). These data indicated that FPL001 inhibited the invasive hyphae development of C. gloeosporioides.

3.6. FPL001 Safe for Planting Alfalfa and Silkworm

The effects of FPL001 were tested on alfalfa seed germination, root growth, and stem growth at 125, 250, 500, and 1000 µg/mL. With increasing concentration, the seed germination of alfalfa was not significantly inhibited (Figure S2a,b). Consistently, FPL001 had no effects on root growth and stem length (Figure S2a,c,d). These results suggested that FPL001 was not phytotoxic to the seed germination and seedling growth of alfalfa.
The effects of FPL001 were tested on silkworm larvae at 250, 500, 1000, and 2000 µg/mL. The survival rate of silkworm larvae fed mulberry leaves treated with FPL001 of different concentrations was not significant after 96 h (Figure S3). These data showed that FPL001 was not remarkably toxic to silkworm larvae.

4. Discussion

Various chemical fungicides, though highly effective in preventing plant diseases, have also caused serious environmental pollution and posed a threat to the health of humans and animals. Thus, it is the requirement that biofungicides effectively prevent pathogen infection and reduce damage to the environment. In this study, a botanical derivative FPL001 was successfully screened, which suppressed lesion expansion by means of delaying the development of invasive structures.
Many botanical derivatives, such as camptothecin and taxanes, are relatively safe for the environment and human health [38]. It was reported that the plant extract 3-Methyl cyclopenten-1,2-dione (MCP) possessed antibacterial activity to suppress the growth of different bacterium, such as Mycobacterium tuberculosis and Staphylococcus aureus [15,17]. However, it remains unclear whether MCP and its derivatives can effectively prevent infection from fungi. Firstly, it was found that the MCP derivative FPL001 more strongly inhibited the mycelial growth of C. gloeosporioides on the PDA plates compared with other derivatives (FPL002, FPL003, FPL004, and FPL005). Thus, compound FPL001 was selected for further study. Inoculation assays demonstrated that compound FPL001 restricted disease lesions caused by C. gloeosporioides in different host plants, including Liriodendron chinense × tulipifera, Populus × euramericana cv. ‘Nanlin895’, and Cunninghamia lanceolate. These results showed that compound FPL001 can be applied in various plants. Fungicides inhibit diverse biological processes in fungi by destroying cell membranes, interfering in protein synthesis, and preventing signal transduction, respiration, mitosis, and nucleic acid synthesis [39]. In further study, the efficient MCP derivative FPL001 not only inhibited vegetative growth but also delayed the development of invasive structures, including appressoria and invasive hyphae. These data indicated that compound FPL001 inhibited the pathogenicity of C. gloeosporioides by interfering with the development of invasive structures of C. gloeosporioides, but the molecular mechanisms of inhibition of C. gloeosporioides by FPL001 call for future studies.
The long-term overuse of chemical fungicides has brought about serious environmental pollution and ecosystem crisis in recent years [40]. It was reported lately that many botanical derivative compounds have little impact on the environment and human health, making them potential candidates as novel antimicrobial agents [41]. He et al. found that the 5% microemulsion of Uvaria grandiflora extract can effectively control cucumber powdery mildew, and it also promoted the rapid growth of cucumbers [42]. These data demonstrated the advantages of botanical derivative compounds as biofungicides in plant disease prevention and control. To ensure advances in compound FPL001, safety experiments indicated that compound FPL001 did not suppress the seed germination and seedling growth of alfalfa at concentrations lower than 1000 µg/mL. Additionally, there were no significant negative effects on the survival rate of silkworm larvae when the compound FPL001 reached the concentration of 2000 µg/mL. Therefore, it is suggested that FPL001 is environmentally friendly, and can be used as an advanced alternative fungicide.
In general, botanical products derived from plants are multi-functional because their chemical complex structures contain diverse groups [43]. On the other hand, the vast plant resources provide abundant sources to exploit various effective and broad-spectrum novel fungicides [44]. These biofungicides are rich in biologically active secondary metabolites, such as phenols, tannins, terpenes, saponins, alkaloids, flavonoids, and other compounds [45]. The active substances from plants are usually obtained by optimizing the extraction process or by chemical methods. MCP is widespread and abundant in various plants. In a hexane extract from Citrus aurantiifolia, the contraction of MCP reached 8.27% [15]. In this study, the MCP derivative polysubstituted cyclic 1,2-diketones showed an effective inhibition of the mycelial growth, invasive structure development, and disease lesion extension of C. gloeosporioides. Benzofurans, a class of heterocyclic compounds, are considered significant antimicrobial agents [46]. The compound FPL001 containing a benzofuran ring may play vital pharmacological and biological role in inhibiting disease. Therefore, the antifungal activity of polysubstituted cyclic 1,2-diketones may be improved through the introduction of different groups or by actively targeting moieties in the future.
Botanicals are becoming one of the most essential fungicides in sustainable agriculture. Various botanical derivatives, such as limonoids and osthol, inhibited different severe diseases caused by fungi [47,48]. In this study, compound FPL001 inhibited the mycelial growth of C. gloeosporioides more effectively than plant-derivative fungicides, such as halopyrazole matrine derivatives [49]. Conidial germination, appressorium formation, and invasive hyphae development are critical phases in the early stages of infection of host plants by C. gloeosporioides to suppress fungal infection. The compound FPL001 effectively hinders the development of these pivotal invasive structures. In the safety analysis, it was found that compound FPL001 only inhibited the pathogenicity of C. gloeosporioides, but it had no effects on the seed germination and root and stem elongation of alfalfa at higher than normal concentrations of FPL001. In addition, there were no significant effects on the survival rate of silkworm larvae fed with mulberry leaves treated with FPL001 of 2000 µg/mL. Thus, compound FPL001 was non-pollutive to the environment. Based on these advances, it is asserted that FPL001 is a potential, exploitable, and important advanced botanical fungicide.

5. Conclusions

In this research, we screened out a newly superior botanical fungicide polysubstituted cyclic 1,2-diketones FPL001 derivative from 3-Methyl cyclopenten-1,2-dione (MCP). The precursors of compound FPL001 were widely distributed in natural materials, including most plants. It was found that compound FPL001 inhibited anthracnose disease caused by C. gloeosporioides in different hosts through suppressed fungal growth, conidial germination, appressorium formation, and the development of invasion hyphae. In further study, it was also demonstrated that FPL001 was not significantly toxic to the growth of alfalfa and the survival rate of silkworm larvae. These results indicated that FPL001 is thus a potential, exploitable, and advanced alternative botanical fungicide.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f14061172/s1, Figure S1: Chemical structure of polysubstituted cyclic 1,2-diketones; Figure S2: Safety test of FPL001 on Medicago sativa; Figure S3: Toxicity test of compound FPL001 at 0, 250, 500, 1000 and 2000 µg/mL to silkworm larvae.

Author Contributions

L.H., Q.W. and M.S. designed the research; Q.W. and Z.W. performed experiments; L.H., S.H. and X.M. contributed new reagents/analytical tools; Q.W., J.H. and X.M. analyzed data; and Q.W. and J.H. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Science Foundation of China (31870631, 32171724), the Qing Lan Project, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare that they have no conflict of interest.

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Figure 1. Inhibition of FPL001 on mycelial growth of C. gloeosporioides. (a) Colonies of C. gloeosporioides on PDA plates supplemented with different concentrations (0, 100, 150, 200, 250, and 300 µg/mL) of FPL001 and the fungus was photographed at 5 dpi. (b) The virulence regression equation was generated by GraphPad Prism 8 software.
Figure 1. Inhibition of FPL001 on mycelial growth of C. gloeosporioides. (a) Colonies of C. gloeosporioides on PDA plates supplemented with different concentrations (0, 100, 150, 200, 250, and 300 µg/mL) of FPL001 and the fungus was photographed at 5 dpi. (b) The virulence regression equation was generated by GraphPad Prism 8 software.
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Figure 2. Effects of FPL001 on conidial germination and appressorium formation in C. gloeosporioides. (a) Photos of conidial germination and appressorium formation were observed in FPL001 of different concentrations (0, 10, 15, 20, 25, and 30 µg/mL) at 4 h post inoculation (hpi) and 12 hpi, respectively. (b) Conidia germination rates of C. gloeosporioides were calculated when treated with FPL001 of different concentrations (n ≥ 100). (c) Appressorium formation rates of C. gloeosporioides were conducted in FPL001 of different concentrations(n ≥ 100). Bar = 10 µm. Letters from a to f indicate significant differences among different treatments (one-way ANOVA, Duncan’s multiple range test).
Figure 2. Effects of FPL001 on conidial germination and appressorium formation in C. gloeosporioides. (a) Photos of conidial germination and appressorium formation were observed in FPL001 of different concentrations (0, 10, 15, 20, 25, and 30 µg/mL) at 4 h post inoculation (hpi) and 12 hpi, respectively. (b) Conidia germination rates of C. gloeosporioides were calculated when treated with FPL001 of different concentrations (n ≥ 100). (c) Appressorium formation rates of C. gloeosporioides were conducted in FPL001 of different concentrations(n ≥ 100). Bar = 10 µm. Letters from a to f indicate significant differences among different treatments (one-way ANOVA, Duncan’s multiple range test).
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Figure 3. FPL001 inhibited infection of C. gloeosporioides (SMCG1#C). Stars indicate the inoculation sites. The black vertical line represents the scale. Bar = 1 cm. (a–c) Anthracnose lesions on leaves of the Liriodendron chinense × tulipifera (a), Populus × euramericana cv. ‘Nanlin895’ (b), and Cunninghamia lanceolata (c), at 5 dpi. (d–f) Lesion size was measured on leaves of the Liriodendron chinense × tulipifera (d), Populus × euramericana cv. ‘Nanlin895’ (e), and Cunninghamia lanceolata (f) at 5 dpi. Data are presented as means ± SD (n = 6). Letters show significant differences among different treatments (one-way ANOVA, Duncan’s multiple range test).
Figure 3. FPL001 inhibited infection of C. gloeosporioides (SMCG1#C). Stars indicate the inoculation sites. The black vertical line represents the scale. Bar = 1 cm. (a–c) Anthracnose lesions on leaves of the Liriodendron chinense × tulipifera (a), Populus × euramericana cv. ‘Nanlin895’ (b), and Cunninghamia lanceolata (c), at 5 dpi. (d–f) Lesion size was measured on leaves of the Liriodendron chinense × tulipifera (d), Populus × euramericana cv. ‘Nanlin895’ (e), and Cunninghamia lanceolata (f) at 5 dpi. Data are presented as means ± SD (n = 6). Letters show significant differences among different treatments (one-way ANOVA, Duncan’s multiple range test).
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Figure 4. FPL001 inhibited the biomass of C. gloeosporioides. (a–c) Mycelial balls of C. gloeosporioides inoculated into liquid potato dextrose (PD) medium with or without compound FPL001 of 250 µg/mL. (d,e). The diameter of mycelial balls (d) and dry weight (e) of C. gloeosporioides treated with compound FPL001 of 250 µg/mL, at 3 dpi. Bar = 2 mm. Error bars represent the means ± SD, and asterisks indicate significant differences (**, p < 0.01; two-sided t-test).
Figure 4. FPL001 inhibited the biomass of C. gloeosporioides. (a–c) Mycelial balls of C. gloeosporioides inoculated into liquid potato dextrose (PD) medium with or without compound FPL001 of 250 µg/mL. (d,e). The diameter of mycelial balls (d) and dry weight (e) of C. gloeosporioides treated with compound FPL001 of 250 µg/mL, at 3 dpi. Bar = 2 mm. Error bars represent the means ± SD, and asterisks indicate significant differences (**, p < 0.01; two-sided t-test).
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Figure 5. Effects of FPL001 on invasive hyphae development. (a) Morphology of type I, II, III, and IV invasive hyphae. A, C, and IH indicate appressorium, conidia, and invasive hyphae, respectively. Bar = 10 µm. (b) Distribution of different types of IH in SMCG1#C with or without FPL001 treatment. The colors, from light to dark, represent invasive hypha type I, II, III and IV in that order. Error bars represent the means ± SD (n = 30).
Figure 5. Effects of FPL001 on invasive hyphae development. (a) Morphology of type I, II, III, and IV invasive hyphae. A, C, and IH indicate appressorium, conidia, and invasive hyphae, respectively. Bar = 10 µm. (b) Distribution of different types of IH in SMCG1#C with or without FPL001 treatment. The colors, from light to dark, represent invasive hypha type I, II, III and IV in that order. Error bars represent the means ± SD (n = 30).
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Table
Table 1. Effects of different compounds of polysubstituted cyclic 1,2-diketones on colony diameters and inhibition rates of C. gloeosporioides.
Table 1. Effects of different compounds of polysubstituted cyclic 1,2-diketones on colony diameters and inhibition rates of C. gloeosporioides.
TreatmentColony Diameter (cm)Inhibition Rate (%)
FPL0012.16 ± 0.06 a58.85 ± 1.30 a
FPL0022.29 ± 0.07 ab56.44 ± 1.25 b
FPL0032.40 ± 0.05 b54.43 ± 1.73 b
FPL0042.81 ± 0.13 c46.62 ± 1.61 c
FPL0053.23 ± 0.15 d38.51 ± 2.15 d
Control5.26 ± 0.29 e0
The colony diameter and inhibition rate represent the means of five independent experiments ± standard deviation (SD). According to Duncan’s multiple range test, letters from a to d indicate significant differences in colony diameters and inhibition rates of C. gloeosporioides-inoculated PDA containing different compounds (one-way ANOVA, Duncan’s multiple range test).
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MDPI and ACS Style

Wang, Q.; Meng, X.; Sun, M.; Wang, Z.; He, J.; Huang, S.; Huang, L. Evaluation of the Antifungal Activity of Polysubstituted Cyclic 1,2-Diketones against Colletotrichum gloeosporioides. Forests 2023, 14, 1172. https://doi.org/10.3390/f14061172

AMA Style

Wang Q, Meng X, Sun M, Wang Z, He J, Huang S, Huang L. Evaluation of the Antifungal Activity of Polysubstituted Cyclic 1,2-Diketones against Colletotrichum gloeosporioides. Forests. 2023; 14(6):1172. https://doi.org/10.3390/f14061172

Chicago/Turabian Style

Wang, Qiuyue, Xiangtai Meng, Meiling Sun, Zhi Wang, Jiao He, Shenlin Huang, and Lin Huang. 2023. "Evaluation of the Antifungal Activity of Polysubstituted Cyclic 1,2-Diketones against Colletotrichum gloeosporioides" Forests 14, no. 6: 1172. https://doi.org/10.3390/f14061172

APA Style

Wang, Q., Meng, X., Sun, M., Wang, Z., He, J., Huang, S., & Huang, L. (2023). Evaluation of the Antifungal Activity of Polysubstituted Cyclic 1,2-Diketones against Colletotrichum gloeosporioides. Forests, 14(6), 1172. https://doi.org/10.3390/f14061172

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