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Article

Effects on Powdery Mildew and the Mutualistic Fungal Endophyte Epichloë gansuensis When Host Achnatherum inebrians Plants Are Sprayed with Different Fungicides

by
Yue Zhu
1,
Keke Cao
1,
Kelin Wu
1,
Michael J. Christensen
2,†,
Jianxin Cao
3,
Yanzhong Li
1,
Xingxu Zhang
1,* and
Zhibiao Nan
1
1
State Key Laboratory of Herbage Improvement and Grassland Agro-Ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China
2
Grasslands Research Centre, Private Bag 11-008, Palmerston North 4442, New Zealand
3
College of Agriculture and Ecological Engineering, Hexi University, Zhangye 734000, China
*
Author to whom correspondence should be addressed.
†
The author is retired.
Agriculture 2025, 15(14), 1565; https://doi.org/10.3390/agriculture15141565
Submission received: 29 June 2025 / Revised: 16 July 2025 / Accepted: 17 July 2025 / Published: 21 July 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Versions Notes

Abstract

A study was conducted to examine the effects of the spray application of nine antifungal products, including microbial-derived fungicides, plant-derived fungicides, and chemical fungicides, on the grass Achnatherum inebrians that was either host to Epichloë gansuensis (E+) or E. gansuensis-free (E−) and that was exposed to Blumeria graminis, the fungal pathogen causing powdery mildew. The Epichloë endophyte is a seed-borne mutualistic biotrophic fungus whose growth is fully synchronized with the host grass. Bl. graminis is a biotrophic pathogen that continually infects leaves and stems via conidia, the formation of appressoria, leading to the presence of haustoria in epidermal cells. Prior to fungicide application, the presence of endophytes significantly increased the resistance of A. inebrians to powdery mildew and was able to increase the chlorophyll content. However, the positive effects of the Epichloë endophyte on the plant were suppressed with the use of some fungicides and the increase in the number of sprays, but the reciprocal relationship between the Epichloë endophyte and the plant was not significantly disrupted.

1. Introduction

Achnatherum inebrians (drunken horse grass) is a perennial grass that is mainly distributed in northern and northwestern alpine and subalpine grasslands in China [1,2,3,4]. Nearly 100% of A. inebrians plants growing in these grasslands are host to an Epichloë endophyte [4]. The two endophytes identified at the present time in A. inebrians plants are Epichloë gansuensis [5] and E. inebrians [6]. Symbiosis between vertically dispersing asexual endophytic fungi, such as Epichloë endophytes, and grasses is common and generally considered to be a mutualistic association [7]. Epichloë species are transmitted within seeds of host grasses and, following seed germination, colonize developing leaves and new tillers that develop from the basal apical meristems [8,9]. Among the dividing cells of the shoot, apex hyphae grow at their tips and branches, but when in the basal leaf expansion zone, hyphae attached to the adjacent plant cells elongate by intercalary extension at the same rate as the cells are elongating [9]. When no longer among dividing or elongating grass cells, hyphae cease branching and elongating and form no new compartments or nuclei but remain metabolically active for the life of the leaf in which they are located. This retention of metabolic activity, the ongoing supply of nutrients, and the removal of waste products result in hyphae becoming wider and increasingly complex as they age [10]. Host grasses supply the endophyte with essential nutrients, offer a stable internal environment for colonization, and ensure its vertical transmission through host seeds [9]. In return, the presence of the Epichloë endophyte provides competitive advantages to grasses, including the production of protective alkaloids [11]. The presence of an Epichloë endophyte can improve the tolerance of A. inebrians to drought [12,13,14], salt [15], heavy metal stress [16], pests [17], and pathogens [18,19,20,21].
Powdery mildew, a disease caused by a large pathogenic ascomycete group, is distributed throughout the world and causes economically important losses in the production of numerous plant crops [22]. Powdery mildew often occurs on A. inebrians plants from June to August in Lanzhou, China. The powdery mildew that occurs on A. inebrians, like other powdery mildew diseases, is characterized by symptoms of a white powdery growth on the upper and lower surfaces of the leaves and on the stems [23]. The disease is caused by Blumeria graminis, a specialized biotrophic fungus that causes disease when spores attach to the surface of the host and germinate [24]. Bl. graminis can cause powdery mildew in 634 Poaceae species, resulting in severe economic losses in many crops [25,26,27]. This pathogen competes with plants for nutrients, increases plant respiration, enhances transpiration, and significantly reduces photosynthesis and carbohydrate accumulation, resulting in reduced yields [19,21,28].
Powdery mildew is a widespread leaf disease, and in practice, chemical control—particularly the use of fungicides from multiple chemical classes—remains the most effective tool for its management [22,26,29]. The application of different types of fungicides to control the powdery mildew disease of grasses that are host to a mutualistic Epichloë endophyte carries the risk that, in addition to preventing the growth of Bl. graminis, the beneficial functions of the Epichloë species could be adversely affected. There is potential for this to happen because systemic fungicides belonging to the methyl-benzimidazole carbamates (MBCs), including benomyl and thiophanate methyl, are used to treat seeds carrying an Epichloë endophyte (E+ seeds) to obtain endophyte-free (E−) seedlings that can be grown to obtain E− seeds [20,30]. Such seeds are valuable to set up E− plants in trials to assess and compare the function of E+ and E− plants arising from the same population [18,19]. Other types of fungicides may also inhibit the growth and function of Epichloë endophytes in germinating seeds, which cannot be dismissed even when applied as a spray to mature plants. We investigated how the application of different types of fungicides would affect the nature of the two very different types of associations that the two biotrophic fungi have with A. inebrians (Table 1 and Table S1).
In this study, we sprayed nine representative fungicides (Table 1) on powdery mildew-infested E+ and non-infested E− A. inebrians plants, measured the percentage of diseased leaves, and calculated the disease index to assess the effects of the different treatments on the disease. To assess if the fungicides had any adverse effects on the Epichloë endophyte, in addition to effects on the severity of the powdery mildew disease, we measured the chlorophyll content of leaves, the plant height, the tiller numbers, and the biomass of E+ A. inebrians plants compared with E− plants, as a reduction in these parameters would also likely indicate a loss in plant growth enhancement of the Epichloë endophyte [13,18,19,20,21]. We carried out the assessments for the effects of the fungicides on Bl. graminis and on the growth of the E+ and E− A. inebrians plants before and a week after the three weekly spray applications. We also examined the stalks near the base of the inflorescences of A. inebrians a week after the third spray to determine if hyphae were present. The goal of this research was to investigate how the application of these different types of fungicides to E+ and E− A. inebrians plants would affect the levels of powdery mildew disease, spread by ongoing leaf infection from conidia, and if the beneficial effects of the mutualistic seed-transmitted endophytic fungus would be affected by these fungicide applications. Although the grass–endophyte symbiosis may initially appear peripheral to the study of economically important plant diseases, it offers a valuable model for understanding how symbiotic microbes can enhance host resistance to pathogens—insights that are crucial for developing sustainable disease management strategies in agriculture. Grasslands are ecologically and economically important, particularly as rangelands for grazing. The symbiosis between A. inebrians and E. gansuensis provides a unique opportunity to investigate plant resistance mechanisms against Bl. graminis, the causative agent of powdery mildew, which causes substantial economic losses in many crops [31,32]. While A. inebrians itself is not a major crop species, the resistance traits observed in this system may inform the development of novel and sustainable approaches to enhance powdery mildew resistance in economically valuable plants.

2. Materials and Methods

2.1. Plant Material and Experimental Design

The E+ and E− A. inebrians seeds were collected from individual A. inebrians plants in an experimental field of the College of Pastoral Agriculture Science and Technology, Yuzhong campus of Lanzhou University (104°390′ E, 35°890′ N, altitude 1653 m), in 2021. Seeds from individual plants were stained with aniline blue and examined microscopically to determine the presence or absence of an Epichloë endophyte, and this enabled the establishment of two populations of A. inebrians seeds that differed in that one population had 100% infection (E+) and the other had 0% (E−) endophyte infection, and these were stored at 4 °C until the trial. A pot trial was conducted from 20 May to 15 August 2023 in the greenhouse of the College of Pastoral Agriculture Science and Technology, Yuzhong campus of Lanzhou University. On 20 May, three healthy, full-looking seeds were sown into each of the pots (100 pots E+ and 100 pots E−, diameter: 24 cm; height: 15 cm), which were filled with vermiculite (75 g) that had been sterilized in an oven at 150 °C for 3 h. The pots were randomly placed in the constant-temperature greenhouse (temperature 26 ± 2 °C; humidity 42% ± 2%) and watered sufficiently until the surface of the vermiculite was moist. After the emergence of the second fully expanded leaf, Hoagland’s solution was applied every 7 days.
On 11 July, a small amount of powdery mildew disease was observed on the leaves of A. inebrians plants of this trial. On the same day, the infection status of each plant was determined by the microscopic (Olympus, Japan) examination of aniline-blue-stained leaf sheath slices. One hundred plants (50 with E+ and 50 with E−) of uniform growth were selected for the following experiment, and these were all trimmed to 15 cm above the vermiculite surface. The experiment consisted of ten treatments comprising nine fungicides and a water control (Table 1), with 10 × 2 (E+ and E−) treatments and five replicates per treatment. On 25 July, prior to the application of fungicides, the percentage of diseased leaves, disease index, and chlorophyll content of A. inebrians plants (5 with E+ and 5 with E−) was determined.
On the afternoon of July 25, the first application of fungicides was made, with two further applications after 7 and 14 days. The fungicides were applied by spraying using a 500 mL atomizer to the extent that the fungicides gathered into drops and flowed down at the leaf tips; tap water was sprayed on the control using the same criteria. The plants were watered with tap water as needed throughout the test period. Before the second and third fungicide applications, the percentage of diseased leaves, disease index, and chlorophyll content of the A. inebrians plants were again determined. The plant growth and biomass of A. inebrians plants of this trial were measured on 15 August, a week after the third application of fungicides, as described in Section 2.4.

2.2. Disease Investigation

Five pots of A. inebrians under each treatment were selected, and these were used for the three weekly assessments of the severity of powdery mildew infection. Three leaves in each pot that were attached to the upper, middle, and lower parts of the plant tillers were selected in each pot, and the number of lesions on individual leaves and the percentage rating of leaf area affected by the disease were counted. The pathogen was identified using the same method as Xia et al. [18] and confirmed as Bl. graminis. The disease index (DI) and control efficacy were also determined. The disease severity was categorized into nine different levels based on the percentage of leaf area on individual leaves affected by the disease. DI severity values of 1, 5, 10, 20, 30, 40, 60, 80, and 100% were calculated using the following formula:
D I % = Σ ( x × t ) s × Σ t × 100
where x is the disease severity degree (1–9), t is the total number of leaves of each degree of disease severity, and s is the highest degree of disease severity observed.
The control efficacies to the powdery mildew index were calculated as
C t r e a t m e n t % = C = D I c h e c k − D I t r e a t m e n t D I c h e c k × 100
where DIcheck is the disease index of the control plots without fungicide treatment and DItreatment is used as the control treatment to calculate the control efficacy of different fungicides on E+ and E− plants.

2.3. Determination of Chlorophyll Content

The chlorophyll content of the same five plants under each treatment was measured using a chlorophyll meter (SPAD-502Plus, Konica Minolta Sensing, INC, Osaka, Japan) from 9:00 to 11:00 a.m. on July 25, August 1, 8, and 15. Three leaves were selected from the upper, middle, and lower parts of tillers, and each leaf was measured three times at the tip, middle, and base of the leaf blade. The average of the values of the 3 points of each leaf was used in the subsequent data analysis, and the relative value of chlorophyll content was called the SPAD value.

2.4. Growth and Biomass of Plants and Epichloë Endophyte Retention Rate Detection

When the percentage of diseased leaves, disease assessment, and chlorophyll content measurements were completed, all plants under all treatments were carefully removed from the vermiculite on the 15th August, and the leaves and roots were washed with water. Plant height, number of tillers, and fresh weight values of each plant under each treatment were collected by blotting out the water from the plants with filter paper. Before measuring the dry weight, six tillers per plant were examined for the presence of hyphae using a scalpel to make an oblique cut in the stalk near the base of the A. inebrians inflorescence. A small amount of the pith was removed and stained with 0.8% lactic aniline blue staining solution for a few moments. The presence of hyphae characteristic of an Epichloë endophyte and the appearance of the cytoplasm were assessed by examining under a light microscope at 40× [33]. All samples were dried at 80 °C to a constant weight, and then the plants under each treatment were weighed to determine the dry weight values of each plant.

2.5. Statistical Analyses

A three-way analysis of variance (ANOVA) was used to analyze the percentage of diseased leaf, disease index, control efficacy, chlorophyll content, and plant biomass of A. inebrians under conditions of different fungicides, different number of sprays, and presence or absence of E. gansuensis (E+ or E−) using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Simple main effect analyses were conducted for statistically significant two-way interactions. All values are expressed as mean ± standard error (SE).

3. Results

3.1. The Percentage of Diseased Leaves

The interaction of endophyte, fungicide, and times of spraying with the percentage of diseased leaves of A. inebrians was highly significant (p < 0.001). In addition, the interaction of endophyte × fungicide, endophyte × times of spraying, and fungicide × times of spraying were significant (p < 0.05) with respect to the percentage of diseased leaves of A. inebrians (Table 2). Before fungicide spraying, the presence of Epichloë endophyte significantly reduced the percentage of diseased leaves of A. inebrians (Figure 1a). After the first fungicide spray, the presence of Epichloë endophyte under 1% Osthole, 16% Polyoxin B, 25% Myclobutanil, and 25% Azoxystrobin (B, D, E and G) treatments significantly reduced the percentage of diseased leaves of A. inebrians (Figure 1b). The presence of Epichloë endophyte under treatments 15% Triadimefon, 0.5% Physcion, 16% Polyoxin B, 25% Myclobutanil, 0.5% Matrine, 1 × 1011 cfu·g−1 Bacillus subtilis, 5% Carvacrol, and Untreated (A, C, D, E, F, H, I and J) after the second fungicide spray significantly reduced the percentage of diseased leaves of A. inebrians (Figure 1c). The presence of the Epichloë endophyte significantly reduced the percentage of diseased leaves of A. inebrians under treatments 15% Triadimefon, 1% Osthole, 16% Polyoxin B, 25% Myclobutanil, 0.5% Matrine, 25% Azoxystrobin, 1 × 1011 cfu·g−1 Bacillus subtilis, 5% Carvacrol, and Untreated (A, B, D, E, F, G, H, I, and J) after the third fungicide spray (Figure 1d). E− plants under treatments 15% Triadimefon and 1% Osthole (A and B) significantly reduced the percentage of diseased leaves of A. inebrians as the number of fungicide sprays increased (Figure 1a–c). In addition, the percentage of diseased leaves of E− A. inebrians under 5% Carvacrol (I) treatments was higher after the third fungicide application than after the second (Figure 1b,c).

3.2. Disease Index of A. inebrians Under Different Fungicides

The interaction of sprays of endophyte, fungicide, and times of spraying on the disease index of A. inebrians was highly significant (p < 0.001). In addition, the interaction of endophyte × fungicide, endophyte × times of spraying, and fungicide × times of spraying were significant (p < 0.05) with respect to the disease index of A. inebrians (Table 2). Before fungicide spraying, the presence of Epichloë endophyte significantly (p < 0.05) reduced the disease index of A. inebrians plants (Figure 2a). After three times of fungicide spraying, nine fungicide treatments A~I significantly (p < 0.05) reduced the disease index of A. inebrians plants (Figure 2b–d). After the third fungicide spray, the presence of Epichloë endophyte under the I treatment significantly (p < 0.05) reduced the disease index of A. inebrians plants (Figure 2d).

3.3. Effect of Different Fungicides on the Control of Powdery Mildew

The interaction of endophyte, fungicide, and times of spraying with the control efficacy of powdery mildew on A. inebrians was highly significant (p < 0.001). In addition, the interactions of endophyte × fungicide, endophyte × times of spraying, and fungicide × times of spraying were significant (p < 0.05) with respect to the efficacy of control of powdery mildew on A. inebrians (Table 2). There was no significant change in control efficacy with increasing number of fungicide sprays under the remaining fungicide treatments, except for the 16% Polyoxin B (D) treatment, which showed a significant increase followed by a decrease in the control efficacy of powdery mildew with an increasing number of fungicide sprays (Figure 3a–c). Moreover, the presence of the Epichloë endophyte did not play a significant role in the efficacy of control of powdery mildew on A. inebrians plants under each fungicide treatment (Figure 3).

3.4. Chlorophyll Content

The interaction of endophyte, fungicide, and times of spraying with the chlorophyll content of A. inebrians was significant (p < 0.05). In addition, the interactions of endophyte × fungicide, endophyte × times of spraying, and fungicide × times of spraying were highly significant (p < 0.001) with respect to the chlorophyll content of A. inebrians (Table 2). The presence of endophyte significantly (p < 0.05) increased the chlorophyll content of A. inebrians before fungicide spraying (Figure 4a). The presence of the Epichloë endophyte significantly (p < 0.05) increased the chlorophyll content of A. inebrians under treatment with 0.5% Physcion (C) after the first fungicide spray (Figure 4b). After the second spraying, the presence of presence of Epichloë endophyte had no significant (p > 0.05) effect on the chlorophyll content of A. inebrians under the treatment of A~I, nine fungicides, and CK (Figure 4c). The presence of the Epichloë endophyte significantly (p < 0.05) increased the chlorophyll content of A. inebrians under treatments 1% Osthole and 16% Polyoxin B (B and D) after the third fungicide spray (Figure 4d).

3.5. Plant Growth and Biomass

The fungicide and the Epichloë endophyte each had significant (p < 0.05) effects on plant height, tillering, dry weight, and fresh weight of A. inebrians. In addition, there were significant interactions between fungicides and Epichloë regarding the dry weight and fresh weight (Table 3). However, there was no significant interaction between fungicides and Epichloë with respect to the tiller number and plant height. The presence of Epichloë endophyte significantly (p < 0.05) increased the plant height of A. inebrians under different fungicide treatments (Figure 5a). Also, the treatments 15% Triadimefon, 1% Osthole, 0.5% Physcion, 25% Azoxystrobin, and 1 × 1011 cfu g−1 Bacillus subtilis (A, B, C, G, and H) significantly (p < 0.05) increased plant height compared to the control (J) (Figure 5a). The presence of Epichloë endophyte significantly increased the number of tillers of A. inebrians under treatments 1% Osthole, 0.5% Physcion, 16% Polyoxin B, 25% Myclobutanil 25% Azoxystrobinm, and 5% Carvacrol (B, C, D, E, G and I) (Figure 5b). In addition, the 0.5% Physcion and 1 × 1011 cfu·g−1 Bacillus subtilis (C and H) treatments significantly (p < 0.05) increased the number of tillers in the plants as compared to the control (J) (Figure 5b). The presence of the Epichloë endophyte significantly (p < 0.05) increased the dry weight of A. inebrians under the 15% Triadimefon, 1% Osthole, 16% Polyoxin B, 25% Myclobutanil, 0.5% Matrine, 25% Azoxystrobin, 1 × 1011 cfu·g−1 Bacillus subtilis, 5% Carvacrol, and Untreated (A, B, D, E, F, G, H, I and J) treatments (Figure 5c). The presence of the Epichloë endophyte significantly (p < 0.05) increased the fresh weight of A. inebrians under the 15% Triadimefon, 1% Osthole, 16% Polyoxin B, 25% Myclobutanil, 0.5% Matrine, 5% Carvacrol, and Untreated (A, B, D, E, F, I and J) treatments (Figure 5d).

3.6. Epichloë Endophyte Retention

Hyphae, densely stained by aniline blue, characteristic of normally functioning Epichloë endophytes, were present in all the examined stalks near the base of the inflorescences of E+ A. inebrians after three weeks of continuous spraying with nine fungicides (Figure 6a). No hyphae characteristic of Epichloë endophytes were observed in E− A. inebrians plants (Figure 6b). Although hyphae of E. gansuensis were clearly observed in E+ plants after fungicide treatments, future studies should consider quantifying hyphal density using image analysis or molecular techniques such as qPCR to provide more detailed insights into endophyte colonization dynamics under fungicide exposure.

4. Discussion

In this study, nine products with activity against fungi were applied to E+ and E− A. inebrians for three successive weeks during the incidence season of powdery mildew disease to explore the effects on Bl. graminis and E. gansuensis, the interaction effects of the two fungi, and on the growth of the A. inebrians plants. The results indicate that the presence of the Epichloë endophyte significantly decreased the percentage of diseased leaves and the disease index of plants (Figure 1a and Figure 2a), even in the absence of any fungicide, and improved the plant height and chlorophyll content of plants (Figure 4a and Figure 5a) under pathogen stress. The control of powdery mildew by Ba. Subtilis (H) was significantly greater than that by 0.5% Matrine (F), 25% Azoxystrobin (G), and 5% Carvacrol (I) (Figure 3).

4.1. How Fungicides Affect Powdery Mildew and Thus Promote E+/E− Plant Growth

Ba. subtilis induces the excitation of ISR (induced systemic resistance) by colonizing the plant or the rhizosphere. In most cases, the Bacillus that induces ISR also triggers plant growth promotion. The elicitation of ISR by Bacillus that enhances plant defenses and promotes plant growth results from the activation of phytohormones and the regulation of defense genes [34,35,36]. The effect of Ba. subtilis on powdery mildew control improved as the number of spray applications increased, reaching a maximum effect of 74.16% on E+ plants (E− plants 70.47%) after the third spraying–after the first spray, it was 67.35% E+ plants, 66.76% E− plants).
The mechanisms by which microorganisms such as Ba. subtilis cause the death of plant-pathogenic fungi include blocking and disrupting the synthesis of fungal cell walls or forming pores in lipid membranes, and some responses of fungal mitochondria and intracellular nucleic acids can also lead to death [37,38,39,40]. Bacillus species synthesize many potent amphiphilic and surfactant lipopeptides based on secondary metabolites, including bacillomycins, iturins, and mycosubtilin, with specific activity against plant pathogens [41]. The iturins cause cell leakage by inserting their hydrophobic tails into the cytoplasmic membrane and forming pores [42]. Romero et al. [43] demonstrated that the iturin and fungomycin families of lipopeptides in Ba. subtilis play a key role in the antagonism to Podosphaera fusca and reduce the germination of conidia. Xie et al. [44] also revealed that Ba. subtilis significantly reduced the powdery mildew of wheat at a concentration of 4 × 105 cfu·g−1 and inhibited the germination of conidia and the normal development of appressoria.
The plant-derived fungicides used in this study are also effective in the control of powdery mildew. The results of the experiment revealed that Physcion was effective in the control of powdery mildew (Figure 3). In addition, compared to the control, Physcion treatment significantly increased the chlorophyll content, plant height, and tiller number of A. inebrians with Epichloë endophyte (Figure 4b–d; Figure 5a,b). Physcion suppresses Blumeria graminis f. sp. hordei (DC.) Speer by inhibiting conidial germination and appressorium formation while also inducing host resistance through the upregulation of defense-related genes such as leaf-specific thionin [45,46,47]. Thionins are defense proteins with antimicrobial ability and are believed to fight plant pathogens, including bacteria and fungi, by disrupting biological membranes [48]. Resistance in susceptible plants can be induced by prior infection with pathogenic organisms or by treatment with various abiotic agents [49,50]. Physcion induced localized resistance rather than systemic resistance against powdery mildew [45] based on the fact that it failed to inhibit the germination of Bl. graminis conidia in vitro but significantly inhibited the germination in vivo. Physcion has been developed as a commercial herbal fungicide for the treatment of powdery mildew, downy mildew, and grey mold on plants in China [45].
Osthole inhibited the spread of powdery mildew by reducing the uptake of reducing sugars by the hyphae to suppress the formation of fungal cell wall chitin and the hyphal growth [51,52], thus promoting the enhancement of the biomass of A. inebrians. It has been reported that Osthole has significant inhibitory effects against Sphaerotheca fuliginea and Fusarium graminearum [51,52,53,54]. The fungicidal active ingredient of Matrine has a broad spectrum of fungicidal activity and is effective against a variety of diseases such as downy mildew, powdery mildew, and grey mold. Matrine inhibits spore germination and mycelial growth [55,56], alters the permeability of fungal cell membranes, inhibits the fungal glycolysis pathway, enhances aerobic respiration, and significantly increases the content of reactive oxygen species (ROS) and the activity of the protective enzymes (SOD, CAT, POD) [56]. In this experiment, Matrine and Carvacrol were generally effective in controlling the disease (Figure 3). It is interesting to note that the combination of Osthole and Matrine is effective in controlling Sorghum purple spot [57] and inhibiting mycelial biosynthesis [51,55]. In the current study, all products were used singularly; however, combining fungicides has been shown to improve control of plant diseases, but mixing plant-derived fungicides is not common [58].
Although more environmentally friendly alternatives than chemical fungicides have been found, in practice, chemical control is the most effective method of disease control, and chemical fungicides are known as an effective and reliable agricultural management measure for controlling diseases [59,60]. This study demonstrates the effectiveness of Triadimefon and Myclobutanil (DMI-fungicides) in controlling the persistent spread of powdery mildew. The principle of action of triazole fungicides is complex but functions mainly by inhibiting the catalytic action of cytochrome P450, which impairs the demethylation of ergosterol in the fungal cell membrane, thereby inhibiting or interfering with the development of appressoria and haustoria and interfering with mycelial growth and spore formation [61,62].
DMIs are widely used as fungicides in agriculture, mainly for the control of powdery mildew in cereals, grapes and other economically important crops [29], but triazole fungicides pose a great threat to the environment. Triadimefon, when applied to plants, can leach into the soil with rainwater, leading to environmental pollution and posing a hidden threat to the ecological balance. Powdery mildew resistance to QoI fungicides has been reported in plants from different regions [63,64], and resistant pathogen isolates have shown greater adaptability than sensitive strains, with long-lasting stability of resistance to QoI [65,66].

4.2. How Fungicides Affect the Epichloë Endophyte in Host Plants

The presence of Epichloë endophyte significantly decreased the disease index and increased the chlorophyll content of A. inebrians before fungicide spraying. However, the endophyte-fungus-mediated reduction in disease index after the third fungicide application was observed only in the Carvacrol treatment (Figure 2b). In addition, after the first fungicide spray, an increase in chlorophyll content of A. inebrians by Epichloë endophytes was observed only in the Physcion treatment (Figure 4b). Epichloë-endophyte-mediated increases in fresh weight were observed in the untreated samples but not with the Physcion, Azoxystrobin, and Bacillus subtilis fungicides (Figure 5b). However, the chlorophyll content of A. inebrians under the Osthole and Polyoxin B treatments was also increased by Epichloë endophyte after the third fungicide spray (Figure 4d). These diverse findings suggest that some fungicide treatments may lessen the positive effects of endophyte and that this inhibition is influenced by the number of fungicide applications.
Meanwhile, the chlorophyll content of E− A. inebrians decreased significantly as the number of fungicide applications increased, especially when treated with Polyoxin B and Bacillus subtilis, and the chlorophyll content of E− plants was significantly higher after the second fungicide spray than the third (Figure 4c,d). However, E+ plants did not show significant changes in chlorophyll content under the same treatments, suggesting that the presence of the Epichloë endophyte helped to maintain chlorophyll content under fungicide treatments. At the same time, it also suggests that although the endophyte was inhibited by fungicides, the reciprocal relationship between it and the plant was not disrupted. This was confirmed by the fact that the Epichloë endophyte still significantly increased plant height, tillers, dry weight, and fresh weight under most fungicide treatments (Figure 5) and that the Epichloë endophyte was retained in all of the reproductive tillers of E+ plants that were examined.

5. Conclusions

We singularly sprayed nine products with activity against powdery mildew on A. inebrians plants, with and without an Epichloë endophyte, and found that the presence of the Epichloë endophyte increased plant resistance to powdery mildew, and the control of powdery mildew differed with different fungicides and the number of fungicide sprays applied. Further studies are needed to assess any potential adverse effects of fungicide treatments on beneficial endophytes such as E. gansuensis, as well as on host plant growth and physiological functions. In particular, the examination of tillers formed in the weeks after the cessation of fungicide applications is needed to ensure that hyphae present in the shoot apex of tillers can still colonize the axillary buds from which new tillers can arise. Similarly, the examination of seeds formed in the weeks after the conclusion of the fungicide applications to ensure that they are Epichloë-endophyte-infected and if seeds formed on plants sprayed with each individual fungicide will give the same rate of seedling infection as with seeds from the no-fungicide control plants. Further, the study of the gene expression of hyphae in mature leaves prior to and following exposure to fungicides could reveal if selective treatments from this study have any adverse effects on physiological processes.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture15141565/s1. Table S1. Active ingredients, modes of action, and characteristics of fungicides used in this study.

Author Contributions

Conceptualization, Y.Z. and X.Z.; Methodology, Y.Z.; Software, Y.Z. and X.Z.; Validation, Y.Z.; Formal analysis, Y.Z.; Investigation, Y.Z., K.C., J.C., Y.L. and K.W.; Resources, X.Z.; Data curation, Y.Z. and X.Z.; Writing-original draft preparation, Y.Z.; Writing-review and editing, Y.Z., M.J.C. and X.Z.; Visualization, Y.Z. and M.J.C.; Supervision, X.Z. and Z.N.; Project administration, X.Z.; Funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Nature Science Foundation of China (32061123004) and the Fundamental Research Funds for the Central Universities (lzujbky-2022-ey21), Lanzhou University.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.

Acknowledgments

We wish to thank the editor and anonymous reviewers for their valuable comments. We thank Daniel A. Bastías for his help with data statistics. We also thank Cory Matthew, a native English speaker, for his language assistance.

Conflicts of Interest

The authors declare no competing interests.

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Agriculture 15 01565 g001
Figure 1. The percentage of diseased leaves of A. inebrians with (E+) and without (E−) Epichloë endophyte under before fungicide spraying (a), 7d after the first fungicide spray (b), 7d after the second fungicide spray (c), and 7d after the third fungicide spray (d). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
Figure 1. The percentage of diseased leaves of A. inebrians with (E+) and without (E−) Epichloë endophyte under before fungicide spraying (a), 7d after the first fungicide spray (b), 7d after the second fungicide spray (c), and 7d after the third fungicide spray (d). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
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Figure 2. Disease index of A. inebrians with (E+) and without (E−) the Epichloë endophyte before fungicide spraying (a), 7d after the first fungicide spray (b), 7d after the second fungicide spray (c), and 7d after the third fungicide spray (d). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
Figure 2. Disease index of A. inebrians with (E+) and without (E−) the Epichloë endophyte before fungicide spraying (a), 7d after the first fungicide spray (b), 7d after the second fungicide spray (c), and 7d after the third fungicide spray (d). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
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Figure 3. Control of powdery mildew by A. inebrians with (E+) and without (E−) Epichloë endophyte under 7d after the first fungicide spray (a), 7d after the second fungicide spray (b), and 7d after the third fungicide spray (c). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
Figure 3. Control of powdery mildew by A. inebrians with (E+) and without (E−) Epichloë endophyte under 7d after the first fungicide spray (a), 7d after the second fungicide spray (b), and 7d after the third fungicide spray (c). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
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Figure 4. Chlorophyll content of A. inebrians with (E+) and without (E−) the Epichloë endophyte before fungicide spraying (a), 7d after the first fungicide spray (b), 7d after the second fungicide spray (c), and 7d after the third fungicide spray (d). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
Figure 4. Chlorophyll content of A. inebrians with (E+) and without (E−) the Epichloë endophyte before fungicide spraying (a), 7d after the first fungicide spray (b), 7d after the second fungicide spray (c), and 7d after the third fungicide spray (d). Note: A~I: nine different fungicide treatments: J, CK (Table 1). Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05.
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Figure 5. Effect of different fungicides on the height (a), tiller number (b), dry weight, and fresh weight values (c,d) of E+ (E. gansuensis) and E− (E. gansuensis-free) A. inebrians. Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05. The asterisk (*) indicates a significant difference (p < 0.05) between the endophyte-infected and noninfected.
Figure 5. Effect of different fungicides on the height (a), tiller number (b), dry weight, and fresh weight values (c,d) of E+ (E. gansuensis) and E− (E. gansuensis-free) A. inebrians. Values are mean ± standard error (SE), with bars indicating SE. Columns with non-matching letters indicate a significant difference at p ≤ 0.05. The asterisk (*) indicates a significant difference (p < 0.05) between the endophyte-infected and noninfected.
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Figure 6. Under a light microscope at 40× power, the stalks near the base of the inflorescences of E+ (E. gansuensis, (a)) and E− (E. gansuensis-free, (b)) A. inebrians stained with aniline blue.
Figure 6. Under a light microscope at 40× power, the stalks near the base of the inflorescences of E+ (E. gansuensis, (a)) and E− (E. gansuensis-free, (b)) A. inebrians stained with aniline blue.
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Table 1. List of treatments used in fungicide assay.
Table 1. List of treatments used in fungicide assay.
TableFungicideDose (g/L)Active Ingredient (s)FormulationFRAC Code
A15% Triadimefon1.666TriadimefonWettable powder3
B1% Osthole1.666OstholeWater emulsionsBM01
C0.5% Physcion3.334PhyscionHydrotropeBM01
D16% Polyoxin B1.428PolyoxinSuspensions19
E25% Myclobutanil1.500MyclobutanilHydrotrope3
F0.5% Matrine1.000MatrineWater emulsionsBM01
G25% Azoxystrobin0.666AzoxystrobinSoluble agents11
H1 × 1011 cfu·g−1 Bacillus subtilis1.428Bacillus subtilisWettable powder44/BM02
I5% Carvacrol1.334CarvacrolWettable powderBM01
CKUntreated (CK)
Note: Fungicide Resistance Action Committee (FRAC).
Table 2. Three-way ANOVA for the effects of Epichloë endophyte, fungicides (F), and times (T) on the percentage of diseased leaves, disease index, control efficacy, and chlorophyll content of A. inebrians.
Table 2. Three-way ANOVA for the effects of Epichloë endophyte, fungicides (F), and times (T) on the percentage of diseased leaves, disease index, control efficacy, and chlorophyll content of A. inebrians.
TreatmentsThe Percentage of Diseased LeavesDisease IndexControl EfficacyChlorophyll Content
FpFpFpFp
E265.921<0.001207.677<0.00178.922<0.001385.107<0.001
F105.934<0.001104.803<0.00120.961<0.001145.270<0.001
T796.205<0.001805.787<0.00133.320<0.00120.757<0.001
E × F4.098<0.0014.536<0.00112.998<0.00139.222<0.001
E × T2.7130.0454.6560.0037.7140.0018.215<0.001
F × T18.575<0.00116.421<0.0012.905<0.0016.211<0.001
E × F × T3.401<0.0012.896<0.0013.229<0.0012.2470.001
Table 3. Two-way ANOVA for the effects of fungicides (F) and Epichloë endophyte (E) on plant heights, tiller number, and dry and fresh weight values of A. inebrians.
Table 3. Two-way ANOVA for the effects of fungicides (F) and Epichloë endophyte (E) on plant heights, tiller number, and dry and fresh weight values of A. inebrians.
TreatmentsdfPlant HeightsTillers Per PlantDry Weight Per PlantFresh Weight Per Plant
FpFpFpFp
F825.621<0.0013.1620.006217.700<0.0015.042<0.001
E1153.270<0.00121.246<0.0013005.696<0.001120.710<0.001
F × E81.4640.1950.8450.58090.832<0.0012.3390.032
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Zhu, Y.; Cao, K.; Wu, K.; Christensen, M.J.; Cao, J.; Li, Y.; Zhang, X.; Nan, Z. Effects on Powdery Mildew and the Mutualistic Fungal Endophyte Epichloë gansuensis When Host Achnatherum inebrians Plants Are Sprayed with Different Fungicides. Agriculture 2025, 15, 1565. https://doi.org/10.3390/agriculture15141565

AMA Style

Zhu Y, Cao K, Wu K, Christensen MJ, Cao J, Li Y, Zhang X, Nan Z. Effects on Powdery Mildew and the Mutualistic Fungal Endophyte Epichloë gansuensis When Host Achnatherum inebrians Plants Are Sprayed with Different Fungicides. Agriculture. 2025; 15(14):1565. https://doi.org/10.3390/agriculture15141565

Chicago/Turabian Style

Zhu, Yue, Keke Cao, Kelin Wu, Michael J. Christensen, Jianxin Cao, Yanzhong Li, Xingxu Zhang, and Zhibiao Nan. 2025. "Effects on Powdery Mildew and the Mutualistic Fungal Endophyte Epichloë gansuensis When Host Achnatherum inebrians Plants Are Sprayed with Different Fungicides" Agriculture 15, no. 14: 1565. https://doi.org/10.3390/agriculture15141565

APA Style

Zhu, Y., Cao, K., Wu, K., Christensen, M. J., Cao, J., Li, Y., Zhang, X., & Nan, Z. (2025). Effects on Powdery Mildew and the Mutualistic Fungal Endophyte Epichloë gansuensis When Host Achnatherum inebrians Plants Are Sprayed with Different Fungicides. Agriculture, 15(14), 1565. https://doi.org/10.3390/agriculture15141565

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