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Article

Impact of Grass Endophyte on Leaf Spot in Perennial Ryegrass Caused by Bipolaris sorokiniana and Subsequent Aphids’ Feeding Preference

by
Ziyuan Ma
1,2,3,
Jia He
4,
Youlei Shen
1,2,3,
Yingde Li
1,2,3,
Ping Wang
5,* and
Tingyu Duan
1,2,3,*
1
State Key Laboratory of Herbage Improvement and Grassland Agro-Ecosystems, Lanzhou University, Lanzhou 730020, China
2
Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, Lanzhou 730020, China
3
College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China
4
School of Forestry and Prataculture, Ningxia University, Yinchuan 750021, China
5
Dingxi Vocational and Technical College, Dingxi 730500, China
*
Authors to whom correspondence should be addressed.
Agriculture 2025, 15(2), 116; https://doi.org/10.3390/agriculture15020116
Submission received: 28 November 2024 / Revised: 30 December 2024 / Accepted: 5 January 2025 / Published: 7 January 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
Grass endophytes (Epichloë) are important symbiotic microorganisms of perennial ryegrass, playing a vital role in plant resistance against various stresses. This study investigated the effects of grass endophyte on leaf spot disease caused by fungal pathogen Bipolaris sorokiniana and subsequent feeding preferences of aphids (Rhopalosiphum maidis) on perennial ryegrass, with a particular focus on how grass endophyte influence the interactions between pathogens and aphids. The results indicated that grass endophytes significantly increased the net photosynthetic efficiency of perennial ryegrass. The interactions among grass endophytes, pathogen, and aphids affected the activities of superoxide dismutase (SOD), peroxidases (POD), and catalase (CAT). Grass endophytes enhanced SOD and CAT activities in pathogen-infected ryegrass. While pathogen infection and aphid infestation decreased jasmonic acid (JA) and salicylic acid (SA) concentrations, grass endophyte increased SA levels. Correlation analysis revealed a negative relationship between shoot dry weight and plant transpiration rate, SOD, and CAT activities. Aphid feeding choice showed that grass endophytes attracted more aphid feeding when co-infected with pathogens. This preference correlated positively with H2O2 and SA levels but negatively with NO and JA concentrations. Overall, grass endophytes enhance perennial ryegrass resistance to leaf spot pathogens and aphids, offering a novel pest and disease management strategy in agriculture.

1. Introduction

Perennial ryegrass (Lolium perenne L.), characterized by vigorous growth and extensive tillering, is a globally popular cool-season (C3) turfgrass and pasture species and is one of the most popular varieties for lawn applications worldwide [1,2,3]. It grows wildly throughout almost all of Europe, northern Africa, temperate Asia, North America and Australia [4]. It is widely used as green manure crop to stabilize soils and prevent erosion [4,5], and as an alternative renewable bioenergy source plant [6]. Perennial ryegrass is also valued for its ability to establish resilient turf for sports and golf courses, making it a popular choice for lawn construction in urban [7,8].
Fungal diseases are a major limiting factor in the production and utilization of perennial ryegrass [9]. Common diseases affecting ryegrass include biotrophic pathogens disease stem rust (Puccinia graminis subsp. graminicola), crown rust (Puccinia coronata f.sp. lolii) [10] and powdery mildew (Blumeria graminis DC.), and necrotrophic pathogens disease dollar spot (Sclerotinia homoeocarpa F.T. Bennett), vascular wilt disease (Fusarium oxysporum) [11] and leaf spot (Bipolaris sorokiniana). The fungi of B. sorokiniana is pathogenic to many plants, with symptoms ranging from necrotic lesions to wilting or death [12]. Diseases caused by B. sorokiniana have been reported to cause significant reductions in crop yields in countries such as India, Nepal, Canada, Scotland and Brazil [13].
Insect infestations is another significant limiting factor affect perennial ryegrass growth, particular those associated with drought and overgrazing have severe impact on persistence of perennial ryegrass [14]. For example, the African black beetle (Heteronychus arator) instar larvae feed on grass roots near the crown ryegrass seedling caused severe damage when population exceeding 20 per square meters [15,16]. Additionally, root aphid (Aploneura lentisci) present throughout the year in New Zealand and can significantly reduce ryegrass yields [17,18,19,20]. Argentine stem weevil (Aploneura lentisci) larval attack caused the dry matter of biennial ryegrass (Lolium multiflorum) reduced by more than 40% in Chile [21].
Grass endophytes (Epichloë) are a diverse group of fungi that spend all or most of their life cycle within host grasses, often causing no visible symptoms. A large number of grasses are infested by grass endophytes [22]. The infected can positively impact plant growth and reproduction while also can enhance resistance of abiotic and biotic and stressors [23]. Conclusive field evidence indicates that endophyte-infected tall fescue (Festuca arundinacea) exhibit increased tolerance to drought [24], and greenhouse studies have demonstrated enhanced resistance to harmful insects [25,26]. Throughout their long-term evolution, endophytic fungi have developed a reciprocal symbiotic relationship with their host plants, promoting nutrient absorb and improving resistance to biotic stress such as pests and diseases [27].
The infection of grass endophyte activates the activities of defense enzymes and the accumulation of reactive oxygen species (ROS), such as peroxidases (POD), superoxide dismutase (SOD) and catalase (CAT), and these defense enzymes have a positive impact on plant response to fungal pathogen infection [28]. Additionally, grass endophytes produce alkaloids with anthelmintic activity: ergot alkaloids, ryegrass alkaloids, povidone alkaloids and indole diterpenoid alkaloids, with ryegrass alkaloids and povidone alkaloids being the most toxic [29]. It has been found that the colonization of grass endophyte reduced the survival rate of pest insect [30] and reduced the severity of fungal disease of ryegrass. For example, grass endophyte Epichloë festucae var. lolii promotes the growth of perennial ryegrass by providing protection against disease during the pre-emergence and foliar stage [28].
Leaf spot of ryegrass caused by B. sorokiniana and aphids are specialized and have a worldwide distribution that can cause severe damage to ryegrass. To cope with the specialized damage and feeding pattern of the pathogens and aphids, plants utilize distinctive recognition, signaling and defense strategies [31,32,33]. When a plant is damaged by pathogen infection or aphid feeding, the plant activates its own resistance mechanisms which include alteration in ROS, nutrients, secondary metabolites and phytohormones. These changes work together to enhance the plant’s defense against pathogens and aphids. For example, Epichloë festucae var. lolii can reduce leaf spot severity caused by B. sorokiniana by altering the levels of POD, SOD, CAT, malondialdehyde (MDA) and hydrogen peroxide (H2O2) [34].
Plant diseases and pests often occur simultaneously, exacerbating the damage to host plants [35,36]. Previous studies mostly investigated the individual effects of grass endophytes on plant infections with pathogenic fungi or insect pests [10,14,27,28,29]. However, research examining the effects of grass endophytes on the co-infestation effects of diseases and insect pests in perennial ryegrass is limited. Additionally, research found that the prior infection by pathogens will attract [37,38] or repel [39,40] the feeding priority of pest insects, for example, the infection of Phoma medicaginis with arbuscular mycorrhizal fungi in alfalfa attracted the feeding of aphid (Acyrthosiphon pisum) [41]. Ellsbury’s research shows the infection by pathogen Phytophthora sp. inhibits the infestation of pea aphid Acyrthosiphon pisum on the blackhawk arrowleaf clover (Trifolium vesiculosum) [42].
This study was designed to investigate the effects of grass endophyte on the response of perennial ryegrass to the co-infestation of B. sorokiniana and subsequent aphids (Rhopalosiphum maidis, Fitch). At the same time, to determine the effect of plant pathogens infecting plants on the subsequent feeding preference of aphids on perennial ryegrass, we assessed the aphids’ preference for infected versus uninfected plants. We also explored whether the aphid feeding preference is influenced by pathogen and grass endophyte. We hypothesized that (1) grass endophyte could mitigate the combination effects of pathogen and aphid infestation of perennial ryegrass by altering certain plant defense enzyme activities and signaling substances of perennial ryegrass. (2) The co-infection of pathogen and colonization of grass endophyte together will alter subsequent aphid feeding preferences and can attract aphid aggregation.

2. Materials and Methods

2.1. Plants and Fungi Used in the Study

The cultivar of perennial ryegrass was Lolium perenne cv. Samson, Epichloë-free (Nil), or Epichloë-infected (AR37). The seeds were provided by Grasslanz Technology Ltd., Palmerston North, New Zealand. The pathogen Bipolaris sorokiniana was provided by the State Key Laboratory of Herbage Improvement and Grassland Agro-ecosystems, Lanzhou University. Rhopalosiphum maidis (Fitch) was provided by the Quanying Insect Biology Inc., Jiyuan, China.

2.2. Potting Medium

The growth medium for plant growth contained 25% soil and 75% sand. The soil was collected from a field on the Yuzhong Campus of Lanzhou University, Lanzhou, China, while the sand was purchased from a local market. Both components were passed through a 2-mm sieve and dried at room temperature. The sand–soil mixture was then sterilized in an autoclave at 121 °C for 1 h, repeated twice within 3 days, and subsequently dried in an oven at 110 °C for 36 h.

2.3. Experimental Design

A fully crossed, three-factor experiment was designed: grass endophyte (infected or not infected) × B. sorokiniana (inoculated or uninoculated) × R. maidis (Fitch) (with or without) = eight treatments, each treatment with four replicates. The pathogen B. sorokiniana was inoculated onto half of each grass endophyte treatment at 9 weeks after plant emergence. Perennial ryegrass seeds were surface-sterilised in 10% NaClO for 10 min, and washed three times with sterilized distilled water. We placed the sterilized seeds on sterilized moist filter paper, and incubated them in the dark at 25 °C for 48 h to germinate. Six germinated seeds of perennial ryegrass with grass endophyte infection (E+) or without grass endophyte infection (E−) were planted in each pot. The size of the pot was 19 cm in height and 15 cm in width, and contained 1.5 kg of the above-mentioned soil and sand mixed growth medium. One week after emergence, the plants were thinned to five seedlings with similar size for each pot.
After 9 weeks’ growth in the greenhouse, half of the plants was used to establish pathogen treatments. Ten mL of suspension of B. sorokiniana containing a spore 1 × 106 mL−1 was sprayed onto each of the perennial ryegrass (B+). Ten mL of sterilised water was sprayed onto the other half of the plants to design the control treatments (B−). The pots with plants were covered with a black plastic bag for the following 36 h to retain moisture for pathogen infection.
One week after establishing the pathogen inoculation (B+ and B−) treatments, 10 aphids per pot (two aphids per plant) were introduced onto the perennial ryegrass plants to establish the aphid (Rhopalosiphum maidis (Fitch)) R+ treatments (E−B−R+, E−B+R+, E+B−R+ and E+B+R+). The remaining four pots in each group were not infested with aphids and treated as R- treatments (E−B−R−, E−B+R−, E+B−R- and E+B+R−). Each pot was individually placed in a tubular plastic cage, with one cage per pot for a week.
A week before inoculation with the pathogen, one week after inoculation with the pathogen, and one week after aphid infestation, the net photosynthesis rate (Pn, μmol·m−2·s−1) and stomatal conductance (Gs, μmol·m−2·s−1) of perennial ryegrass were measured with the Li-6400 Portable Photosynthesizer (LI-COR Inc., Lincoln, NE, USA) under normal air conditions from 9:00 a.m. to 11:00 a.m. on sunny days. One plant was randomly selected from each pot and the leaves near the new tillers of the plant were chosen for measurement. For each pot, one leaf was counted at 15-s intervals, and a total of five leaves were recorded in each pot. Four pots were measured for each treatment, and the average was then calculated.
The experiment was conducted in the greenhouse of Yuzhong Campus, Lanzhou University. The photosynthetic photon flux density during the experiment was in the range of 480–850 mmol/ m2·s. The average temperatures were 20–28 °C. The plants were watered with tap water every other day until reaching a permanent weight of 10% dry weight of the soil. The plants were harvested 14 days after infestation by aphid R. maidis (Fitch), a full 12 weeks after emergence of perennial ryegrass (Figure 1).

2.4. Plant Harvest and Measurement

At harvest, shoots were cut from the plants, one sub-samples were used to determine plant enzymes activities, including peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO). Another subsample was used to analyze plant signaling compounds including hydrogen peroxide (H2O2), salicylic acid (SA), jasmonic acid (JA) and nitric oxide (NO), and the rest of the leaves were oven-dried to obtain dry weights, which were used to calculate the total aboveground portion of the total dry weight of each plant in each pot. Two subsamples of plant defense enzymes and signaling chemicals were considered for the ratio of fresh and dry weights to be counted within the total aboveground dry weight. Additionally, the roots from each pot were washed and collected to determine their fresh and dry weight in an oven at 80 °C for 48 h.
The SOD activity of plant shoot was determined by the nitrogen blue tetrazolium (NBT) photochemical reduction method [43], POD activity was measured by the guaiacol colorimetric method [44], CAT activity was determined by the potassium permanganate titration method and PPO activity was determined by the colorimetric method [45]. The concentration of leaf H2O2 was measured based on the titanium peroxide complex absorbance change at 412 nm (uv-visible photometer, UV-2102PC, Unico Instrument Co., Ltd., Shanghai, China) [46]. Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of nitric oxide (NO), salicylic acid (SA) and jasmonic acid (JA) [47].

2.5. Olfactometer Test

Aphids assessed the tropism behavior of ryegrass volatile organic compounds (VOCs) released by plants under six treatments (E−B−R−, E+B−R−, E−B+R−, E+B+R−, E−B−R+, and E+B−R+). The aphid selection apparatus is illustrated in Figure 2. Two pots of ryegrass plants from each treatment were placed in four separate odor-source glass hoods. Air was drawn from a pump, purified by passing through silica gel and activated charcoal filters and humidified with distilled water bottles before entering the glass hoods. The airflow from the glass enclosure is directed into the glass disk, which is regulated by an airflow meter in the system to maintain an airflow of 100 milliliters per minute. Ten aphids were placed in the center of the glass disk, and their movement was observed for three replications of 25 min per trial. Whenever an aphid crawled to more than 2/3 of any side during the observation period, the movement of that aphid in this odor direction was recorded as a choice for that side. If the 2/3 was not reached, it was recorded as “no choice”.

2.6. Statistical Analysis

All data are presented as means and standard errors of the mean of four replicates. Microsoft Excel 2016 and Prism 10.0 Software (GraphPad, San Diego, CA, USA) were used to organize and summarize the data. Statistical significance between treatment and control groups was analyzed by one-way analysis of variance (ANOVA) via SPSS 26.0 (SPSS Institute Inc., Chicago, IL, USA). Interaction effects were analyzed using univariate analysis of general linear models in SPSS 26.0 (SPSS Institute Inc., Chicago, IL, USA). Tukey’s HSD all-pairwise comparisons were employed to compare treatment means at p < 0.05. To determine whether grass endophytes, pathogens and pre-aphid damage influenced aphid attraction or repulsion, insect selection data were analyzed using a single t-test in SPSS 26.0. (SPSS Institute Inc., Chicago, IL, USA).

3. Results

3.1. Plant Growth

At harvest, pathogen B. sorokiniana’s infection caused typical leaf spot on ryegrass, the E+ plant was successfully infected by grass endophyte. The pathogen significantly affected the aboveground biomass, and significantly affected the belowground biomass. The inoculation of R. maidis had very significant impact on the aboveground growth of perennial ryegrass (Table 1). In contrast, the infection of B. sorokiniana (B+) significantly decreased perennial ryegrass aboveground biomass by 69.18%, while the colonization of grass endophyte (E+) increased the aboveground biomass by 20.02% (Figure 3). The underground biomass showed no response to the grass endophyte (E+) regardless of the inoculation with B. sorokiniana or R. maidis (R+) inoculation (Figure 3).

3.2. Plant Photosynthetic Parameters

The interactions between grass endophyte and B. sorokiniana significantly affected transpiration rate (Table 1). Perennial ryegrass with grass endophyte (E+) showed a significant increase in net photosynthetic rate, increasing by 112.01~164.07%. In particular, the combination of grass endophyte colonization and aphid infestation given the highest value of net photosynthetic rate by 164.07%. No significant differences in plant transpiration rates were found between control (E−B−R−) and other treatment. However, the transpiration rate in E+B−R+ treatment was 75.15% less than that of control. In the case of aphid infestation, perennial ryegrass with the grass endophyte (E+) and infected with B. sorokiniana (B+) increased transpiration rate by 69.38% (Figure 4).

3.3. Plant Defense Enzymes Activities

The three-factor interactions of grass endophyte, B. sorokiniana and aphids extremely significantly affected the SOD, POD and CAT contents of ryegrass (Table 1). Perennial ryegrass co-infected with the grass endophyte (E+) and B. sorokiniana (B+) significantly increased the activities of SOD and CAT, but significantly decreased POD activity. The values of SOD activities in E−B+R+, E+B+R− and E+B+R+ treatments were 60.78%, 137.75% and 34.13% significantly higher than those of the control (E−B−R−). The value of POD in E−B+R− was 64.86% significantly higher than that of control, whereas it was 85.22% lower than the control in E+B+R−. The colonization of grass endophyte (E+) increased the PPO content of perennial ryegrass by 5.02~41.39% (Figure 5).

3.4. Plant Hormone Signal Compounds

The three-factor interactions of grass endophyte, B. sorokiniana and aphids extremely significantly affected the NO, SA and JA contents of ryegrass (Table 1). Grass endophyte, B. sorokiniana and aphids significantly influence H2O2 concentrations of perennial ryegrass. The H2O2 concentration was 16.39% higher in aphid infested ryegrass (E−B−R+) compared to the control (E−B−R−). The pathogen B. sorokiniana alone or in combination with aphids significantly decreased H2O2 concentration by 27.56% and 19.14%, respectively. The presence of grass endophyte (E+) significantly decreased H2O2 concentrations by 12.83~21.52%. There were no significant differences of H2O2 concentrations in grass endophyte colonized ryegrass, regardless of pathogen infection or aphid infestation (Figure 6).
Grass endophyte (E+) significantly increased NO activity by 14.40%. The value of NO activities in the E−B−R+, E-B+R+, E+B+R− and E+B-R+ treatments were 22.42%, 16.62%, 28.92% and 5.74% lower than those of the control (E−B−R−) (p < 0.05) (Figure 6). The JA activities in E−B−R+, E+B−R− and E+B+R− treatments were 17.51%, 13.54% and 28.66% less than those of the control (E−B−R−). The interactions of grass endophyte, B. sorokiniana and aphid resulted in a significant 11.31% reduction in SA concentration (Figure 6).
The infestation of aphid significantly increased SA activity by 40.63% in E− plant, which was given the highest value of SA activity across the treatments. The change is exactly the opposite of the variation in salicylic acid concentrations. Grass endophyte colonization alone or co-infected with pathogen or aphids, significantly increased SA activity by 26.97%, 26.84% and 20.25%, respectively (Figure 6).

3.5. Correlation Analysis

The correlation analysis revealed that shoot dry weight exhibited a significant negative correlation with transpiration rate, SOD activity and CAT activity. Conversely, shoot dry weight showed a significant positive correlation with root dry weight and hydrogen peroxide content. Additionally, root dry weight was positively correlated with SA content. The net photosynthetic rate demonstrated a significant positive correlation with PPO content, while it exhibited a significant negative correlation with hydrogen peroxide levels. Furthermore, the transpiration rate was positively correlated with both SOD and CAT activities (Figure 7).

3.6. Aphid Selective Behavior

In the olfactory choice test, 74.64% of the tested aphids made a choice among the various treatments offered for selection in the aroma of the air stream. The colonization of grass endophyte with the infection of B. sorokiniana (E+B+) plants significantly attracted aphids compared to the E−B+ plants (23.33% vs. 76.67%). Similarly, aphids preferred E+ plants compared to E− plants (34.48% vs. 65.52%). However, aphids showed no preference to the plant colonized by grass endophyte in the presence of aphids. Instead, it favors ryegrass that has been pre-infested with aphids. In addition, aphid also showed no preference to the ryegrass without grass endophyte regardless pathogen infected or not (Figure 8).

3.7. Correlation Analysis of Aphids Selective Behavior

Aphids showed a significant preference for pre-infested ryegrass in the E−B−R− and E−B−R+ selection tests. A correlation analysis revealed that this preference was positively correlated with hydrogen peroxide and SA contents, while it exhibited a significant negative correlation with NO and JA levels (Figure 9).

4. Discussion

To understand how the grass endophyte affects perennial ryegrass tolerance to pathogen infection and subsequent infestation by insect pests, we evaluated physiological and biochemical indicators including biomass, plant defense enzymes activities, photosynthetic indicators and signal compounds. Using the pathogen B. sorokiniana, the aphid R. maidis and perennial ryegrass with and without grass endophyte Epichloë in a glasshouse experiment, we revealed that grass endophyte reduced the negative impacts of pathogen, aphids, or the combination of both on the activities of SOD, POD, CAT, PPO and concentrations of H2O2, NO, SA and JA in perennial ryegrass. Furthermore, grass endophytes co-infected with pathogens significantly attracted aphid feeding preferences, with ryegrass pre-infested with aphids being more attractive to subsequent aphid feeding. Additionally, the preference of aphid for pre-infested ryegrass showed a positive correlation with H2O2 and SA levels, while exhibiting a significant negative correlation with nitric oxide (NO) and JA concentrations. Our hypothesis that grass endophytes can alleviate the combined effects of pathogen and aphid infestations in perennial ryegrass by modifying plant defense enzyme activities and signaling substances, while grass endophyte co-infection with pathogens influences aphid feeding preferences, was supported.
The leaf spot pathogen B. sorokiniana significantly affected ryegrass growth, resulting in a significant reduction in aboveground biomass. This is consistent with Li et al. [48], who also reveal that B. sorokiniana significantly reduced the dry weight of ryegrass across treatments. Aphid infestation did not significantly affect ryegrass biomass, which was probably due to the short duration of aphids feeding and low numbers per plant.
The grass endophyte significantly increased the photosynthetic rate of ryegrass, and this increase was further enhanced by the infection of pathogen or the infestation of aphids. This indicates that the colonization of grass endophytes results in improved photosynthetic efficiency, thereby increasing resistance to diseases and pests. The results align with Li et al., who found that the mycorrhizal fungi, as well as the grass endophyte Epichloë, significantly increased the net photosynthetic rate of ryegrass by more than 172.5% at all soil moisture contents [49]. The endophyte Epichloë typhina colonization increases the photosynthetic efficiency of Dactylis glomerata by enhancing the rate of carbon assimilation and PSII photochemistry to meet the higher energy demand of D. glomerata [50]. In contrast, interactions of grass endophyte, pathogens, and insects resulted in a decrease in the enhancement of photosynthetic efficiency compared to the previous two groups. It is hypothesized that the role played by grass endophyte is diminished due to the dual attack by the pest and disease.
The infection of B. sorokiniana increased the SOD and CAT contents, which were further enhanced by the presence of the grass endophyte. This demonstrates that the grass endophyte help host plant resist pathogen infection by enhancing their SOD and CAT contents. Tian et al. similarly found that the SOD activity of ryegrass was increased by grass endophyte under fungal pathogen Alternaria alternata, Bipolaris sorokiniana, Curvularia lunata and Fusarium acuminatum. The pathogens C. lunata and B. sorokiniana also caused significantly higher POD defense enzyme activities in grass endophyte infected plants than in un-infected plants [51].
The defense enzyme activities of ryegrass were all increased when co-infested by the pathogen and aphids. Interestingly, when ryegrass with grass endophyte was co-infested with pathogen and aphids, the defense enzyme activity of ryegrass decreased compared to the previous increase, suggesting that the grass endophyte may have increased the resistance of ryegrass, making it unnecessary to further increase the defense enzyme activities to defend against the dual stress of pathogen and aphids. Correlation analysis revealed a significant negative relationship between shoot dry weight and transpiration rate, SOD and CAT activities, suggesting that grass endophytes alter the plant’s trade-off between growth and defense in response to pathogen and aphid attacks.
In addition, grass endophyte exhibits a greater affinity for their host than fungi from soil or other environments. They are more likely to colonize and expand within the plant body, thereby providing enhanced protection and benefits to the plant [52]. Plant–pathogen interactions are dynamic and evolving, with the success of infection determined by a combination of multiple signaling pathways [33], include aerobic metabolism accumulate that disturbs the redox state of proteins and other molecules that are essential for cell signaling [53,54]. Our results showed that hydrogen peroxide (H2O2) concentrations were significantly reduced in all ryegrasses colonized by grass endophyte. Nassimi and Taheri found that the endophyte Piriformospora indica reduced hydrogen peroxide content in rice thereby increasing resistance to the sheath blight pathogen Rhizoctonia solani AG1-IA [55]. Notably, co-infection with the grass endophyte and B. sorokiniana resulted in the most significant decrease in NO activity, by 28.92%. In all other treatments, nitric oxide content was significantly higher in E+ plants than in E− plants. Xie et al. found that the endophyte Phomopsis liquidambari can increase the interaction between peanut–Bradyrhizobium interactions by enhancing H2O2/NO-dependent signaling crosstalk, which facilitates the alleviation of crop barriers by increasing peanut nodulation and N2 fixation [56].
Plant hormone salicylic acid (SA) and Jasmonic acid (JA) play crucial roles in plant defense responses to pathogens and insect herbivores [57,58,59,60]. In this experiment, JA levels were significantly lower in perennial ryegrass colonized by grass endophyte when the pathogen was present, during interactions between the pathogen and aphids, and even in the absence of infestation. Conversely, a study by He et al. found that perennial bunchgrass containing the endophytic fungus Epichloe gansuensis antagonized the SA pathway by increasing JA levels during feeding by the aphid Rhopalosiphum padi [61]. By disabling the SA mechanism of herbivore-mediated inhibition of plant growth, the endophyte induced plant tolerance to the herbivore. It is hypothesized that the reason for the differences may be related to the species of grass endophyte and plants.
Biotrophic pathogens and sap-sucking insects are thought to be an effective mechanism to modulate SA content to alter plant resistance [57,62,63]. In this experiment, SA content in ryegrass was significantly elevated when the grass endophyte interacted with pathogen and aphids, as well as when there were no stressor interactions. Interestingly, when aphids infested ryegrass, SA content increased significantly, however, when the grass endophyte interacted with aphids, this elevation was attenuated, indicating that the endophytes moderated the effects of aphid infestation on SA levels in ryegrass. Kou et al. also found that, when Achnatherum inebrians containing the grass endophyte Epichloë gansuensis infected by the biotrophic pathogen Blumeria graminis, the grass endophyte increased SA content and gene expression levels in response to SA hormones to improve resistance to the pathogen [64]. Scott et al. found that aphids (Rhopalosiphum padi) triggered higher SA concentrations which suppressed JA concentrations in the model grass Brachypodium distachyon. Both grass endophyte colonization and aphid infestation increased the SA content of perennial ryegrass, but the interaction between endophytes and aphids slowed down the trend of SA content increase [65].
The results of the aphid selection test indicated that ryegrass containing grass endophyte significantly attracted aphids when inoculated with B. sorokiniana. Similarly, ryegrass with grass endophyte was preferred by aphids when inoculated with pathogenic fungi. Gange’s study found that Acer pseudoplatanus, which harbors the endophytic fungus Rhytisma acerinum, positively affected aphid populations, resulting in higher numbers of aphids on infected leaves in late summer [66]. Hammon and Faeth suggested that late-season herbivores may be adapted to the chemicals induced or produced by endophytes in the host plant, thereby performing better on infected leaves [67]. Our research also reveals that aphids showed a significant preference for pre-infested ryegrass, this preference was positively correlated with hydrogen peroxide and SA contents, while it exhibited a significant negative correlation with NO and JA levels. This suggests that the induced defense response of the host plant, triggered by prior aphid infestation, plays a crucial ecological role in shaping the subsequent pest feeding preferences.
Studies have shown that Botanophila flies are attracted to the sesquiterpene alcohol chokol K emanating from the stroma of Anthoxanthum odoratum colonized with Epichloë sylvatica [68]. Higher levels of 1-octen-3-ol were produced in leaf sheaths of colonized tall fescue (Festuca arundinacea Schreb.) colonized by endophytes [69]. From the plant’s perspective, one defense consequence of establishing symbiotic interactions with these microorganisms is that symbiotic plants are often more vulnerable to biotrophic pathogens and certain species of sap-sucking insects [70,71]. The results of the aphid selection test are also in line with these results. However, there are fewer studies related to the effect of endophytic fungi on insect feeding after infection by pathogens, further study is required to clarify the mechanism that grass endophyte impact the interactions of pathogens and pest insects. Future research should also focus on the interactions between plant-grass endophyte dynamics, particularly how symbiotic grass endophytes influence the resistance mechanisms of ryegrass against the interactions of pathogens and aphids. This will provide insights into optimizing plant defense strategies. Additionally, exploring the ecological implications of induced defense responses on pest feeding preferences will enhance our understanding of integrated pest management approaches in agricultural systems.

5. Conclusions

The present study highlighted the impact of grass endophyte (Epichloë) on defense enzymes, hormones and other indicators in ryegrass in response to leaf spot diseases, aphids and their combinations. Our results demonstrated that grass endophytes significantly enhance the resistance of perennial ryegrass against the leaf spot and influence aphid feeding preferences. Grass endophytes notably increased the net photosynthetic efficiency and the activities of plant defense enzymes (SOD and CAT). While pathogen and aphid infections reduced JA and SA levels, grass endophyte increased SA concentrations, indicating a shift in the plant’s defense strategy. Additionally, aphids showed a preference for ryegrass pre-infested with aphids, which correlated with hydrogen peroxide, NO, SA and JA concentrations, suggests that the induced defense response of the host plant, triggered by prior aphid infestation, plays a crucial ecological role in shaping the subsequent pest feeding preferences. Overall, grass endophytes not only mitigate the physiological impacts of pathogens and aphids but also alter pest feeding behavior, suggesting a potential strategy for pest and disease management in agricultural practices.

Author Contributions

Z.M. performed the experiment and data analyses. Z.M., J.H., Y.S., Y.L., P.W. and T.D. wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

Funding for this research was provided by the China Modern Agriculture Research System (CARS-22 Green Manure).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All applicable data are published and referenced in the paper.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Flow chart showing the experimental design of the study.
Figure 1. Flow chart showing the experimental design of the study.
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Figure 2. Diagram of aphid odor selection apparatus. Experimental setup for directing the headspace air from the plants into the olfactometer. A four-arm olfactometer was used for the olfactometer experiments with an aphid. The insect was placed inside the central area of olfactometer and air was pulled through the apparatus by a suction pump. Headspace air from the odor chamber of plants is pumped out, directed to the arms of the olfactometer. At the center of the arena of the olfactometer, air was sucked out, thus creating a constant flow from each arm towards the middle. The air flow in the system was controlled by flow meters adjusted to maintain 200 mL /min airflow through each sidearm of the olfactometer. The left side of the setup is the suction pump and flow meters, the right side is the olfactometer, and the middle is the plant odor chamber.
Figure 2. Diagram of aphid odor selection apparatus. Experimental setup for directing the headspace air from the plants into the olfactometer. A four-arm olfactometer was used for the olfactometer experiments with an aphid. The insect was placed inside the central area of olfactometer and air was pulled through the apparatus by a suction pump. Headspace air from the odor chamber of plants is pumped out, directed to the arms of the olfactometer. At the center of the arena of the olfactometer, air was sucked out, thus creating a constant flow from each arm towards the middle. The air flow in the system was controlled by flow meters adjusted to maintain 200 mL /min airflow through each sidearm of the olfactometer. The left side of the setup is the suction pump and flow meters, the right side is the olfactometer, and the middle is the plant odor chamber.
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Figure 3. Shoot and root dry weights (DW) of perennial ryegrass (L. perenne) without (E−) or with (E+) grass endophyte, uninoculated (B−), or inoculated (B+) with B. sorokiniana and uninfested (R−) or infested (R+) with aphids. Different lowercase letter above the bar indicates there is significant difference between the aphid treatments at the p < 0.05 level for shoot dry weight. The asterisk means there is significant difference between the pathogen treatment at the p < 0.05 level for shoot dry weight or root dry weight.
Figure 3. Shoot and root dry weights (DW) of perennial ryegrass (L. perenne) without (E−) or with (E+) grass endophyte, uninoculated (B−), or inoculated (B+) with B. sorokiniana and uninfested (R−) or infested (R+) with aphids. Different lowercase letter above the bar indicates there is significant difference between the aphid treatments at the p < 0.05 level for shoot dry weight. The asterisk means there is significant difference between the pathogen treatment at the p < 0.05 level for shoot dry weight or root dry weight.
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Figure 4. Net photosynthetic rate (a) and transpiration rate (b) in perennial ryegrass (L. perenne) without grass endophyte (E−) or with grass endophyte (E+), uninoculated with B. sorokiniana (B−) or inoculated with B. sorokiniana (B+), and uninfested with aphid (R−) or infested with aphid (R+). Different lowercase letter above the bar indicates there is a significant difference between the grass endophyte treatments at the p < 0.05 level for net photosynthetic rate (a); different lowercase letter above the group of the bar indicates there is a significant difference between the grass endophyte and pathogen treatments at the p < 0.05 level for transpiration rate (b). The asterisk means there is significant difference between the pathogen treatment at the p < 0.05 level for transpiration rate (b).
Figure 4. Net photosynthetic rate (a) and transpiration rate (b) in perennial ryegrass (L. perenne) without grass endophyte (E−) or with grass endophyte (E+), uninoculated with B. sorokiniana (B−) or inoculated with B. sorokiniana (B+), and uninfested with aphid (R−) or infested with aphid (R+). Different lowercase letter above the bar indicates there is a significant difference between the grass endophyte treatments at the p < 0.05 level for net photosynthetic rate (a); different lowercase letter above the group of the bar indicates there is a significant difference between the grass endophyte and pathogen treatments at the p < 0.05 level for transpiration rate (b). The asterisk means there is significant difference between the pathogen treatment at the p < 0.05 level for transpiration rate (b).
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Figure 5. Defense-related enzyme activities of superoxide dismutase (SOD) (a), peroxidase (POD) (b), catalase (CAT) (c) and polyphenol oxidase (POD) (d) in perennial ryegrass (L. perenne) without grass endophyte (E−) or with grass endophyte (E+), uninoculated with B. sorokiniana (B−) or inoculated with B. sorokiniana (B+), and uninfested with aphids (R−) or infested with aphids (R+). Different lowercase letter above the bar indicates there is significant difference between the treatments at the p < 0.05 level for superoxide dismutase (a), peroxidase (b), catalase (c); different lowercase letter above the bar indicates there is a significant difference between the grass endophyte treatments at the p < 0.05 level for polyphenol oxidase (d).
Figure 5. Defense-related enzyme activities of superoxide dismutase (SOD) (a), peroxidase (POD) (b), catalase (CAT) (c) and polyphenol oxidase (POD) (d) in perennial ryegrass (L. perenne) without grass endophyte (E−) or with grass endophyte (E+), uninoculated with B. sorokiniana (B−) or inoculated with B. sorokiniana (B+), and uninfested with aphids (R−) or infested with aphids (R+). Different lowercase letter above the bar indicates there is significant difference between the treatments at the p < 0.05 level for superoxide dismutase (a), peroxidase (b), catalase (c); different lowercase letter above the bar indicates there is a significant difference between the grass endophyte treatments at the p < 0.05 level for polyphenol oxidase (d).
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Figure 6. (a) Hydrogen peroxide (H2O2), (b) Nitric oxide (NO), (c) jasmonic acid (JA) and (d) salicylic acid (SA) concentrations of perennial ryegrass (L. perenne) uninoculated with grass endophyte (E−) or inoculated with grass endophyte (E+), uninoculated with B. sorokiniana (B−) or inoculated with B. sorokiniana (B+), and uninfested with aphids (R−) or infested with aphids (R+). Different lowercase letter above the bar indicates there is significant difference between the treatments at the p < 0.05 level.
Figure 6. (a) Hydrogen peroxide (H2O2), (b) Nitric oxide (NO), (c) jasmonic acid (JA) and (d) salicylic acid (SA) concentrations of perennial ryegrass (L. perenne) uninoculated with grass endophyte (E−) or inoculated with grass endophyte (E+), uninoculated with B. sorokiniana (B−) or inoculated with B. sorokiniana (B+), and uninfested with aphids (R−) or infested with aphids (R+). Different lowercase letter above the bar indicates there is significant difference between the treatments at the p < 0.05 level.
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Figure 7. Plot of correlation analysis of measurement indicators. Positive correlation between two factors in red and negative correlation between two factors in blue. Significant difference is marked with an asterisk (* p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 7. Plot of correlation analysis of measurement indicators. Positive correlation between two factors in red and negative correlation between two factors in blue. Significant difference is marked with an asterisk (* p < 0.05; ** p < 0.01; *** p < 0.001).
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Agriculture 15 00116 g008
Figure 8. (a) Aphid selective behavior toward E−B−R− vs. E+B−R−, E−B+R− vs. E+B+R−, E−B−R+ vs. E+B−R+ groups perennial ryegrass (L. perenne). (b) Aphid selective behavior toward E−B−R− vs. E−B+R−, E+B−R− vs. E+B+R−, E−B−R− vs. E−B−R+, E+B−R− vs. E+B−R+ groups perennial ryegrass. Columns T and NS show the total number of tested aphids and the number of aphids making no selection, respectively. E−, perennial ryegrass without grass endophyte; E+, perennial ryegrass with grass endophyte; B−, perennial ryegrass uninfected with pathogen (B. sorokiniana); B+, perennial ryegrass infected with pathogen (B. sorokiniana); R−, perennial ryegrass not infested by aphids (R. maidis); R+, perennial ryegrass infested by aphids (R. maidis). Significant difference is marked with an asterisk (* p < 0.05; ** p < 0.01).
Figure 8. (a) Aphid selective behavior toward E−B−R− vs. E+B−R−, E−B+R− vs. E+B+R−, E−B−R+ vs. E+B−R+ groups perennial ryegrass (L. perenne). (b) Aphid selective behavior toward E−B−R− vs. E−B+R−, E+B−R− vs. E+B+R−, E−B−R− vs. E−B−R+, E+B−R− vs. E+B−R+ groups perennial ryegrass. Columns T and NS show the total number of tested aphids and the number of aphids making no selection, respectively. E−, perennial ryegrass without grass endophyte; E+, perennial ryegrass with grass endophyte; B−, perennial ryegrass uninfected with pathogen (B. sorokiniana); B+, perennial ryegrass infected with pathogen (B. sorokiniana); R−, perennial ryegrass not infested by aphids (R. maidis); R+, perennial ryegrass infested by aphids (R. maidis). Significant difference is marked with an asterisk (* p < 0.05; ** p < 0.01).
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Figure 9. (a) Hydrogen peroxide (H2O2), (b) Nitric oxide (NO), (c) jasmonic acid (JA) and (d) salicylic acid (SA) correlation analysis of the selective behavior of aphids towards E−B−R− and E−B−R+. The two different colored dots represent the data values for perennial ryegrass per pot for these two treatments.
Figure 9. (a) Hydrogen peroxide (H2O2), (b) Nitric oxide (NO), (c) jasmonic acid (JA) and (d) salicylic acid (SA) correlation analysis of the selective behavior of aphids towards E−B−R− and E−B−R+. The two different colored dots represent the data values for perennial ryegrass per pot for these two treatments.
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Table 1. ANOVA result for effects of grass endophyte (E), B. sorokiniana (B), R. maidis (R) and their interactions on the listed variables.
Table 1. ANOVA result for effects of grass endophyte (E), B. sorokiniana (B), R. maidis (R) and their interactions on the listed variables.
Source of VariationEndophyteB. SorokinianaR. MaidisInteraction
(E)(B)(R)E × BE × RB × RE × B × R
Aboveground biomassns *<0.0010.006nsnsnsns
Underground biomassns0.023nsnsnsnsns
Net photosynthetic rate<0.001nsnsnsnsnsns
Transpiration ratens<0.001ns0.006nsnsns
SOD<0.001<0.001<0.001<0.001<0.0010.025<0.001
POD<0.001<0.001ns<0.001<0.001<0.001<0.001
CATns<0.0010.010ns0.008ns0.012
PPO0.021nsnsnsns0.008ns
H2O2<0.001<0.0010.009<0.001<0.0010.008ns
NO0.006ns<0.001<0.001<0.001<0.001<0.001
SA<0.001<0.0010.005<0.001<0.001<0.0010.001
JA<0.001ns0.025<0.001<0.001<0.0010.021
* ns means there is no significant at p < 0.05 levels.
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Ma, Z.; He, J.; Shen, Y.; Li, Y.; Wang, P.; Duan, T. Impact of Grass Endophyte on Leaf Spot in Perennial Ryegrass Caused by Bipolaris sorokiniana and Subsequent Aphids’ Feeding Preference. Agriculture 2025, 15, 116. https://doi.org/10.3390/agriculture15020116

AMA Style

Ma Z, He J, Shen Y, Li Y, Wang P, Duan T. Impact of Grass Endophyte on Leaf Spot in Perennial Ryegrass Caused by Bipolaris sorokiniana and Subsequent Aphids’ Feeding Preference. Agriculture. 2025; 15(2):116. https://doi.org/10.3390/agriculture15020116

Chicago/Turabian Style

Ma, Ziyuan, Jia He, Youlei Shen, Yingde Li, Ping Wang, and Tingyu Duan. 2025. "Impact of Grass Endophyte on Leaf Spot in Perennial Ryegrass Caused by Bipolaris sorokiniana and Subsequent Aphids’ Feeding Preference" Agriculture 15, no. 2: 116. https://doi.org/10.3390/agriculture15020116

APA Style

Ma, Z., He, J., Shen, Y., Li, Y., Wang, P., & Duan, T. (2025). Impact of Grass Endophyte on Leaf Spot in Perennial Ryegrass Caused by Bipolaris sorokiniana and Subsequent Aphids’ Feeding Preference. Agriculture, 15(2), 116. https://doi.org/10.3390/agriculture15020116

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