https://orcid.org/0009-0003-2506-4318
Figures
Abstract
Fusarium oxysporum (F. oxysporum) is one of the main pathogenic fungus causing maize ear rot. In this study, the aims were to screen highly effective pesticides for F. oxysporum, reduce peasants’ misunderstandings about pesticide application, improve disease control levels, and enhance economic efficiency. The toxicity of seven fungicides (carbendazim, pyraclostrobin, epoxiconazole, tricyclazole, azoxystrobin, difenoconazole, quintozene) on F. oxysporum were determined by the mycelium growth rate and the spore germination method, and single and compound fungicides with effective inhibitory effects on mycelial growth were screened. The RT-qPCR method was used to detect the expression levels of chitin synthetase V (ChsV), folate uptake block T (FUBT), superoxide dismutase (SOD), and peroxidase dismutase (POD) genes in pathogenic bacteria treated with the selected agents and combination of fungicides. The results showed that all seven fungicides had inhibitory effects on mycelial growth hyphae and spore germination of F. oxysporum. Epoxiconazole had the strongest inhibitory effect on mycelium growth and spore germination of F. oxysporum, with effective concentrations (EC50) of 0.047 and 0.088 μg/mL, respectively. The combination of pyraclostrobin and difenoconazole (P&D, combined at a mass ratio of 7:3) had the best inhibitory effect, with an EC50 of 0.094 μg/mL and an SR of 2.650. Epoxiconazole and the combination P&D could inhibit mycelial growth and spore germination by down-regulating ChsV, FUBT, and POD, causing oxidative stress in F. oxysporum, and reducing the occurrence of maize ear rot.
Citation: Xu D, Wang K, Li T, Wang J, Wang S, Kong F, et al. (2025) In vitro activity of seven antifungal agents against Fusarium oxysporum and expression of related regulatory genes. PLoS One 20(4): e0322206. https://doi.org/10.1371/journal.pone.0322206
Editor: Ravinder Kumar,, ICAR - Indian Agricultural Research Institute, INDIA
Received: June 28, 2024; Accepted: March 18, 2025; Published: April 29, 2025
Copyright: © 2025 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The datasets presented in this study are available from the Figshare database (10.6084/m9.figshare.27951405).
Funding: This study was supported by the Key Research and Development Program of Anhui, China (202204c06020060). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Maize ear rot, a prevalent and highly damaging fungal disease worldwide, significantly impacts maize yield and quality, and it brings great safety hazards to food and feed [1,2]. Changes such as the replacement of maize varieties, increased planting density, and alterations in cultivation practices have created favorable conditions for the occurrence and prevalence of maize ear rot [3,4]. This disease is caused by various fungal infections, with Fusarium graminearum (F. graminearum), Fusarium oxysporum (F. oxysporum), and Fusarium verticillioides (F. verticillioides) being the major pathogens [5,6]. Moreover, all three mentioned Fusarium fungi can produce fungal toxins that are associated with various diseases in both humans and animals [3,7]. Fusarium spp. can infect maize ears and grains, producing fungal toxins, a process regulated by related genes. The folate uptake block T gene (FUBT) can regulate the production of fusaric acid (FA) by F. oxysporum [8]. The chitin synthase gene (Chitin synthetase V, ChsV) can protect Fusarium from plant antimicrobial substances, and the absence of the ChsV gene leads to a loss of its pathogenicity [9]. Fusarium spp. itself possesses numerous protective enzyme genes, such as the superoxide dismutase (SOD) gene and the peroxidase dismutase (POD) gene [10]. SOD is a crucial enzyme in the antioxidant system, playing a central role in the elimination of reactive oxygen species [11], which helps defend Fusarium spp. from oxygen toxicity and oxidative damage. Under stress conditions, POD can efficiently eliminate H2O2, and its activity can reflect the metabolic status of the organism and its adaptability to the environment [12]. FA is a non-specific toxin produced by F. oxysporum, which can reduce host resistance by altering the permeability of host plant cell membranes, decreasing mitochondrial reactive oxygen species, inhibiting ATP synthesis, and suppressing plant root growth [13,14]. At present, research on FUBT and ChsV has made some progress in the fusarium wilt of watermelon, melon, and cotton caused by F. oxysporum, but there are few reports on maize ear rot [15,16].
As people’s awareness of health increases, there is a growing concern about fungal toxin contamination in maize. Addressing and preventing maize ear rot disease and reducing fungal toxin contamination has become a current research focus. Currently, there are few varieties resistant to maize ear rot, and biological control methods are not yet mature [17,18]; chemical control is the most widely applied measure [19]. Fungicides can effectively control the occurrence and spread of maize ear rot in the short term, significantly reducing the content of toxins in the kernels [18]. However, there are a wide variety of fungicides on the market, each with different chemical structures and mechanisms of action. Improper use can easily lead to phytotoxicity issues. Therefore, conducting toxicity tests for different fungicides on maize ear rot is the primary task to ensure the selection of appropriate agents. In order to reduce the dosage and frequency of fungicide use, delay the development of resistance, and minimize phytotoxicity, it is essential to develop rational combinations of fungicides. This study focused on the toxicity and synergistic effects of seven commonly used fungicides for maize ear rot on F. oxysporum. The best single and compound fungicides were screened, and their effects on the expression levels of ChsV, FUBT, SOD, and POD genes in F. oxysporum are further analyzed, providing reference points for the effective control of maize ear rot.
2. Materials and methods
2.1 Materials
Fusarium oxysporum B (F. oxysporum B) was provided by the Plant Protection Laboratory of the Tobacco Research Institute, Anhui Academy of Agricultural Sciences. The potato dextrose agar medium (PDA), potato glucose broth medium (PDB), and water agar medium (WA) were prepared following the method outlined by Fang Zhongda [20].
Fungicides: Carbendazim (95%, Shandong Huayang pesticide chemical industry group Co., Ltd.), Pyraclostrobin (98%, Anhui Kelihua Chemical Co., Ltd.), Epoxiconazole (98%, Ningxia Gree Fine Chemical Co., Ltd.), Tricyclazole (95%, Shandong Shangnong Agricultural Technology Co., Ltd.), Azoxystrobin (96%, Shandong Union Pesticide Industry Co., Ltd.), Difenoconazole (97%, Jiangsu Heben Biochemical Co., Ltd.), Quintozene (40%, Shanxi Nongfengbao Pesticide Co., Ltd.).
2.2 Toxicity determination of fungicides against F. oxysporum B
2.2.1 Action of a single fungicide on the mycelium of F. oxysporum B.
The impact of different fungicides on the mycelial growth of F. oxysporum B was determined by the growth rate method [21]. Specifically, according to the method described by Kowalska Krochmal et al. [22], the minimum inhibitory concentration test was conducted to prepare PDA plates containing different doses (as shown in Table 1) of fungicides. A mycelial disc with a diameter of 6 mm was inoculated at the center of each plate with F. oxysporum B. Then the plates were cultivated in the dark at 25°C with three replicates for each treatment, and PDA plates without any fungicide served as the control. After 3 days, the colony diameter was measured, and the mycelial growth inhibition rate (MGIR) was calculated according to formula (1).
Where: MGIR, mycelial growth inhibition rate; φc, colony diameter in control group; φt, colony diameter in the treatment group; 6, colony diameter of the initial mycelial disc.
The MGIR were converted into probability values of inhibition rate; the logarithm of the fungicide mass concentration was used as the horizontal axis, and the probability values were used as the vertical axis to fit the regression equation. Then the correlation coefficient (R2) and effective concentration (EC50) were calculated, and the toxicity levels of 7 fungicides against the mycelium of F. oxysporum B were compared.
2.2.2 Action of a single fungicide on the spore of F. oxysporum B.
The impact of different fungicides at a single dose on the germination of spores of F. oxysporum B was determined by the spread plate method [23]. Specifically, F. oxysporum B was cultured on PDA plates for 3 days. The mycelium was rinsed with sterile water, filtered through double layers of sterile gauze, and the filtrate was centrifuged at 4,000 rpm for 10 min. Then the spore was resuspended in sterile deionized water to prepare a spore suspension with a concentration of 106 spores/mL. 100 μL of spore suspension was spread on WA plates containing different doses (as shown in Table 2) of fungicides and incubated at 25°C in the dark. With three replicates for each treatment, and WA plates without any fungicide served as the control. When the spore germination rate on the control WA plate reached 90% or more, the number of germinated spores was recorded for different mass concentrations of the fungicide treatment. The spore germination rate (SGR) and spore germination inhibition rate (SGIR) were calculated according to formulas (2) and (3), respectively.
Where: SGR, spore germination rate; nt, the number of germinated spores; n0, the total number of spores; SGIR, spore germination inhibition rate; SGIRc, SGR of the control group; SGIRt, SGR of the treatment group.
The SGIR were converted into probability values of inhibition rate; the logarithm of the fungicide mass concentration was used as the horizontal axis, and the probability values were used as the vertical axis to fit the regression equation. Then the R2 and EC50 were calculated, and the toxicity levels of 7 fungicides against the spore of F. oxysporum B were compared.
2.2.3 Action of compound fungicides on the mycelium of F. oxysporum B.
Based on the measurement results of 2.2.1 and 2.2.2, compound the fungicide. The effects of compound fungicides were determined by the growth rate method [21]. Specifically, dilute each fungicide separately to 100 μg/mL, and then prepare different proportions of mixed solutions according to Table 3, and prepare PDA plates containing 10% compound fungicide solution. The procedures outlined in Section 2.2.1 were repeated, F. oxysporum B mycelial disc (whose diameter was 6 mm) was inoculated, and the MGIR for the composite fungicide was calculated. Then we fitted a regression equation, and the R2 and EC50 were calculated. Analyzing the synergistic enhancement effect of the combination agent based on the method proposed by Wadley [24,25], using the synergistic ratio (SR) for the analysis of combined enhancement effects (Formulas 4 and 5), SR<0.5 indicates antagonistic effects in the compound formulation of the two fungicides; 0.5≤SR≤1.5 indicates additive effects in the compound formulation of the two fungicides; SR>1.5 indicates synergistic effects in the compound formulation of the two fungicides.
Where: SR, synergistic ratio; A (or B): one type of fungicide; a (or b): the mass (or volume) ratio of fungicide A (or B).
2.3 mRNA expression analysis
A single fungicide and a compound agent were selected that have inhibitory or synergistic effects on F. oxysporum B. Prepare PDA plates containing a fungicide or compound agent and cultivate F. oxysporum B; and the normal PDA plate was used as the control group. After 3 days, mycelium was collected. The mRNA levels of ChsV (ChsV-F: 5’-TCTTTTCCCCATCAAGTGTCT-3’; ChsV-R: 5’-GTGATGTTGGTGTTTCCGGTTGT-3’), FUBT (FUBT-F: 5’-GGAGCCTGAAGACAGATTGC-3’; FUBT-R:5’-CCGATAATAGGGACGATCCA-3’), SOD (SOD-F: 5’-GGTCCTCACTTCAACCCTCA-3’; SOD-R: 5’-AGTCGGTGACAGAGCCCTTA-3’), POD (POD-F: 5’-CGAGGGATGGATCAAGGATA-3’; POD-R: 5’-GTAGCATCCTGCTGGTCGAT-3’) [16,26] in the mycelium of F. oxysporum B were measured. The TRIzol (TIANGEN Biotech (Beijing) Co., Ltd.) method is utilized to extract mRNA from the mycelium. Then a 1μg mRNA of satisfactory quality is selected for reverse transcription to obtain cDNA (ReverTra Ace qPCR RT Master Mix with gDNA Remover; TOYOBO Co., Ltd.), which is subsequently subjected to qPCR. The relative mRNA abundance of the target genes was normalized to Actin (Actin-F: 5’-CCGTGACATCAAGGAGAAGC-3’; Actin-R: 5’-GGAAAGTGGACAGGGAAGCA-3’) and was then calculated using the 2−ΔΔCt method.
3 Results
3.1 Inhibitory effect of a single fungicide on the mycelial growth of F. oxysporum B
The effects of a single fungicide on mycelial growth are shown in Table 4. It can be observed that the mycelial growth of F. oxysporum B is inhibited by all seven tested fungicides; the EC50 is 0.047~35.089 μg/mL. And the three fungicides with the strongest inhibitory effect on mycelial growth are epoxiconazole, difenoconazole, and carbendazim. Their EC50 are as follows: 0.047 μg/mL, 0.078 μg/mL, and 0.445 μg/mL. The inhibitory effects of azoxystrobin on mycelial growth are the weakest, with an EC50 of 35.089 μg/mL. These results indicate that epoxiconazole, difenoconazole, and carbendazim are effective in inhibiting the mycelial growth of F. oxysporum B.
3.2 Inhibitory effect of a single fungicide on the spore germination of F. oxysporum B
The effects of a single fungicide on spore germination are shown in Table 5. It can be observed that the mycelial growth of F. oxysporum B is inhibited by all seven tested fungicides; the EC50 is 0.088~42.720 μg/mL. And the two fungicides with the strongest inhibitory effect on mycelial growth are epoxiconazole and pyraclostrobin; their EC50 are as follows: 0.088 μg/mL, 0.249 μg/mL. The inhibitory effects of tricyclazole on mycelial growth are the weakest, with an EC50 of 42.720 μg/mL. These results indicate that epoxiconazole and pyraclostrobin are effective in inhibiting the spore germination of F. oxysporum B.
3.3 Toxicity determination of compound fungicides on F. oxysporum B
3.3.1 Inhibitory and synergistic effects of a mixture of epoxiconazole and carbendazim.
The range of EC50 values for a mixture of epoxiconazole and carbendazim with different ratios is 0.046 to 0.282 μg/mL, and the EC50 values of the mixture are all lower than the EC50 values of carbendazim (Table 6). The inhibitory effects of the mixtures are stronger than those of carbendazim. When the mass ratio of epoxiconazole and carbendazim is 8:2, the EC50 is 0.046 μg/mL, which is lower than the EC50 of epoxiconazole (0.047 μg/mL, 95% confidence interval: 0.037~0.059 μg/mL). At this mass ratio (8:2), the inhibitory effect is best. The SR values for a mixture of epoxiconazole and carbendazim at different mass ratios range from 0.413 to 1.236. When the mass ratio is 4:6 and 7:3, the SRs are 0.464 and 0.413 (SR<0.5), indicating an antagonistic effect. When the mass ratio is 1:9, 2:8, 3:7, 5:5, 6:4, 8:2, and 9:1, the SR are 0.855, 0.656, 0.921, 0.868, 1.159, 1.236, and 0.537, suggesting an additive effect (0.5≤SR≤1.5).
3.3.2 Inhibitory and synergistic effects of a mixture of carbendazim and quintozene.
The EC50 values for a mixture of carbendazim and quintozene with different ratios is 0.437 to 3.684 μg/mL (Table 7), and all of the EC50 values for the mixture are lower than those for quintozene alone. Specifically, when the mass ratio of carbendazim and quintozene is 8:2, the EC50 is 0.437 μg/mL, which is lower than the EC50 of carbendazim (0.445 μg/mL, 95% confidence interval: 0.424~0.464 μg/mL). For all other combinations, the EC50 values for the mixtures are higher than that of carbendazim. However, the SR values for the mixtures of carbendazim and quintozene at various mass ratios range from 0.643 to 1.330, indicating an additive effect (0.5≤SR≤1.5).
3.3.3 Inhibitory and synergistic effects of a mixture of pyraclostrobin and difenoconazole.
The EC50 values for a mixture of pyraclostrobin and difenoconazole at different mass ratios range from 0.044 to 0.176 μg/mL (Table 8). These values are significantly lower than the EC50 value for pyraclostrobin (4.533 μg/mL, 95% confidence interval: 2.037~9.385 μg/mL), indicating that the inhibitory effects of these combinations are significantly better than those of the single pyraclostrobin. When the mass ratio of pyraclostrobin and difenoconazole is 2:8, 3:7, 4:6, and 6:4, the EC50 values are 0.066 μg/mL, 0.044 μg/mL, 0.061 μg/mL, and 0.077 μg/mL. All these values are lower than the EC50 of a single propiconazole; this indicates that these four combinations have a better inhibitory effect on F. oxysporum B than a single pyraclostrobin. For combinations of mass ratios at 3:7, 4:6, 6:4, 7:3, 8:2, and 9:1, the SRs are 2.494, 2.120, 2.481, 2.650, 2.386, and 3.903, respectively, and these combinations indicate a clear synergistic effect (SR >1.5). Additionally, at mass ratios of 1:9, 2:8, and 5:5, the SR are 0.972, 1.474, and 0.873, respectively, suggesting an additive effect (0.5≤SR≤1.5). All SR values are greater than 0.5 in different mass ratios of pyraclostrobin and difenoconazole; these combinations indicate additive and significant synergistic effects, thus holding promise for practical application and further research.
3.3.4 Inhibitory and synergistic effects of a mixture of pyraclostrobin and carbendazim.
The EC50 values for a mixture of pyraclostrobin and carbendazim at different mass ratios range from 0.260 to 0.824 μg/mL (Table 9). And when the mass ratio is 1:9, 2:8, and 3:7, the EC50 values are 0.288 μg/mL, 0.260 μg/mL, and 0.379 μg/mL, all lower than the EC50 of single carbendazim (0.445 μg/mL, P ≤ 0.05). The mixture of pyraclostrobin and carbendazim, in the range of 1:9–3:7, has a better inhibitory effect on F. oxysporum B than a single carbendazim. When the mass ratio of pyraclostrobin to carbendazim is between 7:3 and 9:1, as the proportion of pyraclostrobin increases, the EC50 of the mixture gradually increases but remains significantly lower than the EC50 of the single pyraclostrobin (4.533 μg/mL, 95% confidence interval: 2.037~9.385 μg/mL). The SR gradually decreases as the mass ratio of pyraclostrobin to carbendazim changes from 2:8–5:5. And at the ratios of 4:6 and 5:5, the SR is 1.305 and 1.219, respectively, indicating an additive effect. However, in other mass ratios, SR is greater than 1.5, demonstrating a significant synergistic effect. All SR values are greater than 0.5 in different mass ratios of pyraclostrobin and carbendazim; these combinations indicate additive and significant synergistic effects. This combination formulation holds promise for practical application and further research.
3.4 Effect of fungicides on the expression of resistance genes in F. oxysporum B
Based on the inhibitory effects of single fungicides and compound fungicides on F. oxysporum B, the expression levels of ChsV, FUBT, SOD, and POD in the epoxiconazole group, pyraclostrobin and difenoconazole (7:3) group (P&D (7:3)), and control group (CK) were determined using RT-qPCR. The results are shown in Fig 1. Compared to the CK, both epoxiconazole and P&D (7:3) treatments significantly downregulated the relative expression levels of ChsV, FUBT, and POD in F. oxysporum B. Additionally, the relative expression level of SOD was significantly increased in both treatments.
4. Discussion
Maize ear rot occurs in the ears of corn, making it difficult to control. In maize-growing regions such as the United States, Canada, and South Africa, the primary causative agent of ear rot is often Fusarium [27–29]. In most regions of China, such as Jilin, Anhui, and Heilongjiang, the primary causative agent of maize ear rot is also Fusarium [30–33]. At present, there are many reports on the toxicity determination of pathogenic fungi such as F. graminearum and F. verticillioides in maize ear rot, while there are fewer reports on F. oxysporum [34,35]. This study found that epoxiconazole exhibited the most effective inhibition of the mycelial growth and spore germination of F. oxysporum, followed by difenoconazole and pyraclostrobin. While individual fungicides showed inhibitory effects on F. oxysporum, long-term use of a single fungicide can easily lead to resistance to the fungus. Therefore, it is recommended to use compound formulations to mitigate this issue. This experiment revealed that the combination of pyrazoxystrobin and difenoconazole exhibited significant inhibition and synergistic effects on the mycelial growth of F. oxysporum. When the mass ratio of pyrazoxystrobin to difenoconazole was 7:3 (P&D (7:3)), the EC50 was 0.094 μg/mL, and the SR was 2.650.
Contains various pathogenic genes in F. oxysporum chitin synthase enzymes (Fochs). Among them, Fochs V and Fochs II play crucial roles in the pathogenicity of the strain [36]. Their absence results in a decrease in the pathogenic ability of the strain, and the loss of Fochs V leads to the loss of pathogenic capability in the strain [37]. Fusaric acid is a non-specific toxin that causes plant wilting. Research has demonstrated the presence of fusaric acid in cotton, leading to the occurrence of wilt disease. There is also evidence indicating a positive correlation between the virulence of F. oxysporum and the production of fusaric acid [13,14]. Additionally, FUBT has been shown to promote the production of fusaric acid. Knocking out the FUBT gene results in a significant reduction in fusaric acid production by F. oxysporum [8]. Antifungal agents can inhibit plant pathogens and cause damage by inducing the excessive production of reactive oxygen species (ROS) in the pathogens [38,39]. POD and SOD are critical enzymes in the reactive oxygen species (ROS) system. They have the ability to reduce or impede the damage caused by reactive oxygen-free radicals to organisms [40]. The activity of POD and SOD serves as important physiological indicators, reflecting the induced resistance of cells to the antifungal agent as well as their response to environmental stress [41,42]. Compared to the CK group, after treatment with epoxiconazole and P&D (7:3), the expression levels of ChsV and FUBT in F. oxysporum showed a significant decrease, suggesting a reduction in the pathogenicity and virulence of the fungus, which suggests a decreased likelihood of maize ear rot occurrence. And the expression level of the SOD exhibited an increasing trend, suggesting that the two treatments induced oxidative stress in F. oxysporum. Conversely, the expression level of the POD showed a significant decrease, indicating a reduction in the POD activity in F. oxysporum, leading to a weakened ability to eliminate H2O2. Therefore, the oxidative stress response in F. oxysporum was enhanced after treatment with epoxiconazole or P&D (7:3), leading to a decrease in its resistance to fungicides.
5. Conclusion
This study demonstrates the effectiveness of specific fungicides and their combinations in controlling F. oxysporum, a major pathogen causing maize ear rot. Epoxiconazole emerged as the most effective single fungicide, exhibiting strong inhibitory effects on both mycelial growth and spore germination. And compound formulations, particularly the combination of pyraclostrobin and difenoconazole (7:3 mass ratio), showed significant synergistic effects, providing superior control compared to individual fungicides. These fungicides downregulated key genes (ChsV, FUBT, and POD) and induced oxidative stress in F. oxysporum, reducing its environmental adaptability, infectivity, and pathogenicity. By optimizing fungicide combinations, this approach enhances disease control while minimizing pesticide use, offering a sustainable strategy to mitigate maize ear rot and its economic impact.
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