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Article

Epichloë Endophyte Alters Bacterial Nitrogen-Cycling Gene Abundance in the Rhizosphere Soil of Perennial Ryegrass

by
Munire Maimaitiyiming
,
Yanxiang Huang
,
Letian Jia
,
Mofan Wu
and
Zhenjiang Chen
*
Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Aairs, State Key Laboratory of Grassland Agro-Ecosystems, Engineering Research Center of Grassland Industry, Ministry of Education, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China
*
Author to whom correspondence should be addressed.
Biology 2025, 14(7), 879; https://doi.org/10.3390/biology14070879
Submission received: 15 April 2025 / Revised: 15 June 2025 / Accepted: 17 June 2025 / Published: 18 July 2025
(This article belongs to the Collection Plant Growth-Promoting Bacteria: Mechanisms and Applications)
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Simple Summary

Perennial ryegrass (Lolium perenne) is a widespread forage and turf grass. Epichloë endophytes have been widely found in the above-ground tissues of grasses, and the effects of endophyte infection on ryegrass physiology and ecology have been relatively well studied. The response of the soil microbial community and nitrogen-cycling gene to this relationship has received much less attention. This study demonstrated the impacts of foliar fungal endophyte on the abundance and diversity of nitrogen-cycling genes involved in soil nitrification and denitrification. We found that the endophyte increased the concentrations of soil available nitrogen in ryegrass rhizosphere by promoting the abundance of the AOB-amoA gene involved in soil nitrification and decreasing the abundance of the nosZ gene involved in soil denitrification.

Abstract

Perennial ryegrass (Lolium perenne), an important forage and turfgrass species, can establish a mutualistic symbiosis with the fungal endophyte Epichloë festucae var. lolii. Although the physiological and ecological impacts of endophyte infection on ryegrass have been extensively investigated, the response of the soil microbial community and nitrogen-cycling gene to this relationship has received much less attention. The present study emphasized abundance and diversity variation in the AOB-amoA, nirK and nosZ functional genes in the rhizosphere soil of the endophyte–ryegrass symbiosis following litter addition. We sampled four times: at T0 (prior to first litter addition), T1 (post 120 d of 1st litter addition), T2 (post 120 d of 2nd litter addition) and T3 (post 120 d of 3rd litter addition) times. Real-time fluorescence quantitative PCR (qPCR) and PCR amplification and sequencing were used to characterize the abundance and diversity of the AOB-amoA, nirK and nosZ genes in rhizosphere soils of endophyte-infected (E+) plants and endophyte-free (E−) plants. A significant enhancement of total Phosphorus (P), Soil Organic Carbon (SOC), Ammonium ion (NH4+) and Nitrate ion (NO3−) contents in the rhizosphere soil was recorded in endophyte-infected plants at different sampling times compared to endophyte-free plants (p ≤ 0.05). The absolute abundance of the AOB-amoA gene at T0 and T1 times was higher, as was the absolute abundance of the nosZ gene at T0, T1 and T3 times in the E+ plant rhizophere soils relative to E− plant rhizosphere soils. A significant change in relative abundance of the AOB-amoA and nosZ genes in the host rhizophere soils of endophyte-infected plants at T1 and T3 times was observed. The experiment failed to show any significant alteration in abundance and diversity of the nirK gene, and diversity of the AOB-amoA and nosZ genes. Analysis of the abundance and diversity of the nirK gene indicated that changes in soil properties accounted for approximately 70.38% of the variation along the first axis and 16.69% along the second axis, and soil NH4+ (p = 0.002, 50.4%) and soil C/P ratio (p = 0.012, 15.8%) had a strong effect. The changes in community abundance and diversity of the AOB-amoA and nosZ genes were mainly related to soil pH, N/P ratio and NH4+ content. The results demonstrate that the existence of tripartite interactions among the foliar endophyte E. festucae var. Lolii, L. perenne and soil nitrogen-cycling gene has important implications for reducing soil losses on N.

1. Introduction

Perennial ryegrass (Lolium perenne L.), widely cultivated in the cool season for forage supply and turfgrass, plays a crucial role in agricultural ecosystems [1]. It is known for its high nutritional value, cold tolerance and rapid regrowth after grazing or mowing. The symbiotic relationship between perennial ryegrass and the endophytic fungus Epichloë festucae var. Lolii has garnered significant attention due to its potential benefits for plant health and productivity.
Litter production and decomposition is a key ecosystem process that greatly influences the formation of soil organic matter, the release of nutrients for plants and microorganisms, CO2 and N2O fluxes and nutrient cycling in agricultural and forest ecosystems [2,3]. Nitrogen (N), P and approximately 60% of other mineral elements in forest ecosystems are typically recycled through litter decomposition [4,5,6,7]. An essential characteristic of the nitrogen cycle is that plant-available nitrogen predominantly derives from the microbial-mediated mineralization of organic matter (decomposition process) or biological nitrogen fixation [8,9]. The soil N cycle is mainly driven by a series of microbes (e.g., diazotrophs, nitrifiers and denitrifiers) which are collectively described as N-cycling microbes [10,11]. These nitrogen-cycling microorganisms mediate plant growth through biogeochemical transformations of nitrogen into bioavailable forms [nitrate (NO3−) or ammonium (NH4+)] that can be directly absorbed and used by plants [12]. Previous studies have demonstrated that litter addition (Typha latifolia) and litter quality significantly affected the abundance and diversity of two nitrifying genes (AOA-amoA and AOB-amoA) and three denitrifying genes (nirS, nirK and nosZ) involved in the N cycle [13,14].
Epichloë endophytes have been investigated with divergent outcomes regarding their impacts on host plant litter decomposition [15,16,17]. As with the effects of Epichloë endophytes on host–environmental stress factor interactions, effects on litter decomposition have been demonstrated to be less homogeneous, and have been found to vary from positive to negative [15,18]. Some toxic substances (e.g., endophyte alkaloids) produced by endophyte symbionts which have shown deterrent effects on herbivores result in litter becoming a low-quality substrate for many detritivores [19], and may exert a direct inhibitory effect on host litter decomposition. The changes in litter quality caused by Epichloë endophytes affect the decomposer community, and then affect litter decomposition [16]. Moreover, endophyte infection can have strong effects on the components of detrital food webs, including nematodes, earthworms, mites, collembola and soil microflora, and ultimately affect litter decomposition [16]. Results from both field and laboratory studies suggest that endophytes, which although mainly distributed in the above-ground parts of grasses, also affect the below-ground parts through influencing endophyte-mediated litter decomposition and root metabolism [16,20,21], may ultimately affect carbon and nutrient cycling. Previous studies have shown that leaf litter from Epichloë endophyte-infected ryegrass alters the abundance and diversity of the AOB-amoA, nirK and nosZ genes associated with soil N cycling in non-rhizosphere soil [22]. However, little is known about the effects of the Epichloë endophyte on the AOB-amoA, nirK and nosZ genes in the host rhizosphere soil of L. perenne plants after litter incorporation.
Epichloë endophytes (fungal genus Epichloë; formerly genus Neotyphodium) are a group of clavicipitaceous fungi that form hereditary symbioses with cool-season grasses from the subfamily Pooideae in temperate regions, and these symbioses are transmitted through successive host generations via vertical (seed) transmission [23,24]. Epichloë endophyte infection may increase host plants’ fitness by conferring stress tolerance to drought and poor soil, and/or resistance to herbivory and fungal diseases [25]. Fungal endophyte symbioses have generally been characterized as mutualistic [26,27]. However, it has been well established that leaf Epichloë endophytes can alter host phenotypes, expand ecological niches and increase fitness in some grasses, especially agronomic cultivars [28]. Documented effects of endophytes on wild host grasses in non-agricultural settings are highly variable [29,30]. The outcome of endophyte symbiosis appears to depend on the particular combination of host plant genotype, endophyte strain or species and environmental factors [31,32].
Here, we investigated the impact of the fungal endophyte E. festucae var. Lolii on the soil nitrogen-cycling gene involved in nitrification and denitrification. In particular, we aimed to understand how the soil nitrogen-cycling genes are influenced by the presence of endophytes, and whether these effects vary with different durations of litter incorporation in the rhizosphere soil. It was hypothesized that the effects of endophyte infection on soil nitrogen-cycling genes (AOB-amoA, nirK and nosZ) in rhizosphere soil would vary across litter incorporation times. The qPCR analysis was employed to estimate the total abundance of the AOB-amoA, nirK and nosZ genes coupled with amplicon sequencing to characterize the community composition of functional genes found in the rhizosphere soil collected from experimental endophyte-infected and endophyte-free L. perenne stands.

2. Materials and Methods

2.1. Plant Material

The ryegrass cultivar used was a turf-type grass selection of L. perenne Lanhei No. 1, which had been cultivated by our team over the last 18 years. This cultivar showed high tolerance to diseases [33] and low nutrient requirements [34]. Moreover, the infection frequency by the endophyte E. festucae var. lolii in seeds was 96.5% [35].
Seeds with high endophyte infection rate and seeds with genetically comparable and low endophyte infection rates were obtained by screening from primary material during three years in the field [35]. Briefly, seeds with an average endophyte infection rate of 62.5% were planted in September in 2014, and 128 single plants were harvested at maturity the next July. The 300 randomly selected seeds from each plant, along with all the tillers, were stained using aniline blue to determine the endophyte infection rate in seeds and tillers, respectively. Seeds with low (≤2%) and high (≥95%) infection rates in the tillers were designated as endophyte-infected (E+) and endophyte-free (E−) materials [35]. E+ and E− seed experimental plots were established at Yuzhong Experimental Station of Lanzhou University (104°12′ E, 35°85′ N, altitude 1400 m), Gansu Province, China, in 2015. In 2016 and 2017, the same method was used to determine the endophyte infection rate of each plant in E+ and E− seed fields, respectively. The high infection frequencies (E+) by E. festucae var. lolii in the tillers and seeds of L.perenne were 96.5%, and low infection frequencies (E−) were 1.1% [35]. In 2017, E+ and E− seeds of L. perenne were collected from the E+ and E− fields and stored at 4 °C to break seed dormancy and maintain endophyte viability.

2.2. Experiment Design

The experiments were conducted at the Yuzhong Experimental Station of Lanzhou University in Gansu Province, China, from 10 September 2017 to 1 November 2018. The test area was in a temperate continental climate, where the average annual temperature was 7.5 °C, the average annual rainfall was 358 mm and the frost-free period was 120 days/year during the test period [22]. The test site was evenly divided into 12 equivalent plots (3 m × 4 m). Initial soil was collected from experiment plots before planting by a five-point sampling method.
E+ and E− plots were established by bunch planting on pre-divided plots on 10 September 2017, using a 50 cm row spacing and distance between plants within rows. The experiment was laid out in a completely random design, with six replicates. Sprinkle irrigation was used, and weeds were removed by hand to avoid the effects of other organisms on soil properties. The endophyte infection status of seedlings in all plots was determined using the aniline blue staining method at 45 days after emergence, ensuring that the endophyte infection rates of plants were near 100% in E+ plots, and a mean 0% in E− plots. After 60 days, all plots were mowed to a height of 5 cm above the surface, and the harvested material was weighed to ensure that the weight of the returned fresh shoot litter was consistent across each plot. Leaf litters from E+ and E− (200 kg, each) were cut into 1 cm and incorporated by hand into soils at the respective row spacing of E+ and E− plots with a depth of 0–10 cm, respectively. Litter was incorporated average once every 120 days, totaling three times.

2.3. Soil Sampling and Chemical Analysis

The E+ and E− leaf litter materials were added to soil just after sampling. A total of four samplings were conducted: T0 (prior to first litter addition), T1 (post 120 d of 1st litter addition), T2 (post 120 d of 2nd litter addition) and T3 (post 120 d of 3rd litter addition). Four replicates of the endophyte and litter addition times were sampled for rhizosphere soil (i.e., soil adhering to plant roots) by five-point sampling. In each replicate, roots in multiple clumps of perennial ryegrass were moved out by inserting a soil corer in the center of the plant. Rhizosphere soil was collected by brushing roots with a sterilized paintbrush after gently shaking the soil around the roots [36]. Collected soil was immediately sieved (2 mm mesh) to remove any remaining root material and other impurities. Samples were divided into two parts; one part for DNA analysis was placed in a foam box with dry ice, transported to the laboratory and stored at −80 °C. The second part was brought back from the field at ambient temperature and stored at −20 °C to analyze soil chemical indicators.
Soil moisture content was measured gravimetrically after oven-drying soil (20 g) at 105 °C for 24 h [37]. Soil pH was determined using a pH meter (Sartorious PE10, Gottingen, Germany) with a suspension of soil and deionized water (1:2.5 = soil:water). Soil organic C (SOC 0.5 g) was determined through the K2CrO7-H2SO4 oxidation–reduction titration method [22]. To analyze soil ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N), a fresh sample (5.0 g) was extracted using 20 mL of 2 M KCl, then shaken for 1 h at 200 rpm and filtered through Whatman No. 42 filter paper into a 50 cm plastic bottle [38]. The contents of NH4+-N and NO3−-N in the extracts were determined using a flow injection system (FIAstar 5000 Analyzer, Foss, Denmark). To analyze soil total N and total P, soil samples were digested with H2SO4 and a catalyst (CuSO4:K2SO4, 1:10 mixture) at 420 °C for 2 h on a digestion block (Hanon Instruments, Co., Ltd., Jinan, China), and a flow injection system (FIAstar 5000 Analyzer, Foss, Denmark) was used to determine the TN and TP contents. The ratios of C/N, C/P and N/P in the soil were calculated.

2.4. Soil DNA Extraction and Quantitative PCR

DNA was extracted from 500 mg freeze-dried (−80 °C) soil samples using the Fast-DNA® Spin Kit of Soil Genome (MP Bio-medicals, Santa Ana, CA, USA), following the manufacturer’s instructions. The concentration and purity of extracted DNA was quantified using a Micro Nanodrop ND-1000 UV-Vis Spectrophotometer (NanoDrop™ 1000, Wilmington, DE, USA). To assess the change in total abundance of the AOB-amoA, nirK and nosZ genes, we used polymerase chain reaction (PCR)-based techniques to amplify following the approaches described in Chen et al. [22]. Briefly, we PCR-amplified DNA from three different gene markers to assess gene copy numbers of the AOB-amoA, nirK and nosZ genes. To examine functional genes involved in nitrification, the AOB-amoA gene was amplified and sequenced using bamoA1F (5′-GGGGTTTCTACTGGTGGT-3′) and bamoA2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′) barcoded primers [39]. For functional genes involved in denitrification, the nirK and nosZ genes were amplified and sequenced using Cunir3F (5′-CGTCTAYCAYTCCGCVCC-3′) and Cunir3R (5′-GCCTCGATCAGRTTRTGG-3′) [40], nosZ-2F (5′-CGCRACGGCAASAAGGTSMSSGT-3′) and nosZ-2R (5′-CAKRTGCAKSGCRTGGCAGAA-3′) barcoded primers [41]. In all three cases, samples were amplified in quadruplicate. qPCR reactions from all samples were conducted on an CFX96 optical real-time detection system (CFX96TM Thermal Cycler, Foster City, CA, USA). High amplification efficiencies of 96.8% for the AOB-amoA gene, 95.2% for the nirK gene and 98.8% for the nosZ gene were obtained on account of standard curves, as calculated using the formula Eff = [10(−1/slope) − 1] × 100%, respectively.

2.5. PCR Amplification and Sequencing

To characterize the diversity and composition of the AOB-amoA, nirK and nosZ genes in the rhizosphere soil of E+ and E− plants at T1 and T3 times for litter incorporation, we used PCR amplification and sequencing approaches. PCR products from all samples were recovered by gel cutting with the AxyPrepDNA Gel Recovery Kit (Axygen, Union City, CA, USA) following 2% agarose gel electrophoresis assay. Recovered PCR products were quantified using a QuantiFluor™ -ST blue fluorescent quantification system (Promega, Madison, WI, USA) and pooled together in equimolar concentrations. Amplicons were purified and concentrated using the QIAquick Gel Extraction Kit (Qiagen Sciences, Germantown, MD, USA). Samples were sequenced on an Illumina MiSeq PE300 instrument using a NEBNext® Rapid DNA-Seq Kit (Illumina Inc., San Diego, CA, USA) at the Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), with separate runs for the AOB-amoA, nirK and nosZ genes amplicon pools.

2.6. Bioinformatics Analysis

Sequence analyses of the AOB-amoA, nirK and nosZ genes were performed on the Majorbio Cloud Platform (www.majorbio.com (accessed on 15 May 2024)) using the QIIME 2 pipeline (http://qiime.sourceforge.net/ (accessed on 15 May 2024)) [42]. Briefly, low-quality sequences were removed by fastp (length < 50 bp or with a quality value < 20 or having N bases) [43]. The extraction of non-repetitive sequences from optimized sequences (http://drive5.com/usearch/manual/dereplication.html (accessed on 23 April 2024)) and the removal of single sequences without duplicates (http://drive5.com/usearch/manual/singletons.html (accessed on 18 May 2024)) facilitated the reduction in redundant calculations in the analysis [44]. Filtered, high-quality PE reads were clustered for operational taxonomic unit (OTU) based on 97% similarity using the UPARSE v.11 pipeline (http://drive5.com/uparse/ (accessed on 20 May 2024)), and chimeras were removed in the clustering process to obtain representative sequences of OTU [41]. The OTU abundance tables of samples were generated by mapping all optimized sequences to OTU representative sequences and selecting sequences with a similarity of 97% or more to OTU representative sequences based on a pipeline of USEARCH v7.1 software. To obtain the classification information corresponding to each OTU of the AOB-amoA, nirK and nosZ genes in E+ and E− soils, RDP classifier and QIIME (Version 1.7.0) software with the functional gene database from GeneBank were used to perform a taxonomic analysis of the representative sequences of OTUs [45]. To compare the differences in AOB-amoA, nirK and nosZ genes in the rhizosphere soil of E+ and E+ plants in this study, OTUs at the phylum and genus levels were primarily utilized, as they are equivalent in taxonomic classification. The community diversity of the AOB-amoA, nirK and nosZ genes was presented using alpha (Shannon and chao1 indexes) diversity.

2.7. Statistical Analysis

The factor endophyte status (E) was utilized as a categorical variable with two levels: E+ (endophyte-infected plants) and E− (endophyte-free plants). Sampling times (T) were also categorical variables with four levels: T0, T1, T2, and T3. We used two-way ANOVA to analyze the effects of endophyte status (E) and sampling times (T) on soil pH, soil organic C (SOC), total nitrogen, total phosphorus, ammonium nitrogen (soil NH4+), nitrate nitrogen (soil NO3−), the C/N, C/P and N/P ratios and the absolute abundance and alpha diversity of the AOB-amoA, nirK and nosZ genes. The differences in rhizosphere soil between E+ and E− plants were assessed using Tukey’s b-test at p ≤ 0.05 when a significant effect was detected. All data analyses of variance (ANOVA) were performed in the software SPSS v. 20. The effects of endophyte status (E) on soil pH, SOC, total N, total P, NH4+, NO3− and the C/N, C/P and N/P ratios were characterized under the same treatments (T0, T1, T2 and T3) through the independent sample t-test. We counted the species abundance of the AOB-amoA, nirK and nosZ genes in the rhizosphere soil of E+ and E− plants at the generic taxonomic level, and visualized the community composition by histogram. The Kruskal–Wallis H test was used to assess the significance level of differences in species abundance at p ≤ 0.05. Principal component analysis (PCA) was used to analyze the diversity of the AOB-amoA, nirK and nosZ genes at genus level between the rhizosphere soil of E+ and E− plants in two periods of sampling time (T1 and T3) to explore the similarities or differences in community composition between different grouped samples. To assess the correlation between microbial taxonomy and environmental factors, Pearson product-moment and Spearman’s rank-based correlation coefficients of the AOB-amoA, nirK and nosZ genes with environmental factors in the rhizosphere soil of E+ and E− plants was calculated using the Mantel test. We utilized canonical correspondence analysis (CCA) to characterize significant correlations between the top ten genera in the relative abundance of the AOB-amoA, nirK and nosZ genes and environmental factors, employing the CCA/RDA functions from the vegan package in R, as well as the Pearson product-moment correlation coefficient.

3. Results

3.1. Endophyte Infection Altered Soil Chemical Properties

Both endophyte status and sampling times exhibited significant effects on soil organic carbon (SOC), soil total P, soil NH4+, soil NO3− and the C/N, C/P and N/P ratios, but only sampling times significantly affected soil pH and the concentrations of soil total N (Table 1 and Figure 1). Compared with the rhizosphere soil of E− plants, sampling time (T2) significantly increased soil total P, soil C/P and N/P ratios in the rhizosphere soil of E+ plants (Table 2 and Figure 1C,H,I), and sampling times (T1, T2 and T3) significantly increased the SOC and NH4+ concentrations in the rhizosphere soil of E+ plants (Table 2 and Figure 1D,E). The soil NO3− content was significantly higher in the rhizosphere soil of E+ plants compared to that of E− plants at both T2 and T3 (Table 2 and Figure 1F), and the C/N ratio of E+ rhizosphere soil was significantly higher than that of E− rhizosphere soil at T3 (Table 2 and Figure 1H). Endophyte infection did not change soil pH and soil total N in comparison with endophyte non-infection under sampling times (T1, T2 and T3) (Table 2 and Figure 1A,B).

3.2. Endophyte Infection Affects Relative Abundances of Nitrification and Denitrification Functional Genes

There was a significant variation in the absolute abundance of the AOB-amoA gene between the rhizosphere soil of endophyte-infected (E+) plants and endophyte-free (E−) plants at both T0 and T1 times (Table 3). Endophyte infection significantly increased its abundance (Figure 2A). The combination of endophyte status and sampling times also significantly altered the relative abundance of the AOB-amoA gene (Table 4 and Figure 2D). The absolute and relative abundances of the nirK gene in the rhizosphere soil was only significantly affected by sampling times, rather than endophyte status or the interaction between both sampling times and endophyte status (Table 3 and Table 5 and Figure 2B,E). There was a significant effect of endophyte, sampling times and their interaction on the absolute abundance of the nosZ gene, which was significantly reduced at T0, T1 and T3 by endophyte infection (Table 4 and Figure 2C). The relative abundance of the nosZ gene in the rhizosphere soil was not significantly affected by endophyte status, sampling times and the interaction between both (Table 6 and Figure 2F).

3.3. Endophyte Effect on Alpha and Beta Diversity of the AOB-amoA, nirK and nosZ Functional Genes

Significant effects on the alpha diversity of the AOB-amoA, nirK and nosZ functional genes within communities were not observed based on endophyte status, sampling times or their interaction, as indicated by the Shannon and Chao1 indexes. (Table 3 and Figure 3). Endophyte infection significantly altered the beta diversity of the AOB-amoA functional gene in the T1 period of sampling times (Figure 4A), but the beta diversity of the nirK and nosZ functional genes did not show any significant difference between the rhizosphere soil of E+ and E− plants at different sampling times (T1 and T3) (Figure 3B,C).

3.4. Relationships Between the AOB-amoA, nirK and nosZ Functional Genes with Soil Properties

Major gradients in the abundance and diversity of AOB-amoA gene differentiation were visualized by CCA, with the first two axes explaining 95.75% of the variance in the abundance and diversity of the AOB-amoA gene following environmental properties (Figure 5A). CCA confirmed the larger effect of soil pH (p = 0.008) and soil NH4+ (p = 0.03) on the abundance and diversity of the AOB-amoA gene compared to the other environmental parameters, and the contribution of the two amounted to 34.3% and 30.8%, respectively (Figure 5A). Analyzing the abundance and diversity of the nirK gene indicated that changes in soil properties accounted for approximately 70.38% of the variation along the first axis and 16.69% along the second axis, with soil NH4+ (p = 0.002, 50.4%) and soil C/P ratio (p = 0.012, 15.8%) having a strong effect (Figure 5B). Our CCA analysis of the nosZ functional gene abundance indicated that changes in soil properties, particularly with litter returning, explained approximately 85.51% of the variation along the first axis and 11.83% along the second axis (Figure 5C). There was a clear differentiation of the abundance and diversity of the nosZ gene between endophyte-infected (E+) and endophyte-free (E−) plants at different litter addition times (T1 and T3) along with the first principal component, indicated by an obvious clustering of the study plots (Figure 5C). For the nosZ gene, soil pH (p = 0.03, 20.6%) and soil N/P ratio (p = 0.002, 68.5%) had significant impacts on the abundance and diversity compared to the other environmental parameters (Figure 5C).

4. Discussion

Shifts in soil microbial communities caused by plant symbiotic microorganisms, especially foliar Epichloë endophytes, have a direct or indirect impact on ecosystem functioning and soil fertility and crop productivity, which makes it a necessity to study how soil microbiomes respond to symbiotic microorganisms. Numerous studies have been conducted to understand the effect of foliar fungal endophytes on soil microbial communities, as well as on their interactions [46,47,48]. These studies provide essential knowledge of how Epichloë endophytes affect soil bacterial and fungal communities [49,50,51,52]. The current study revealed the effects of the Epichloë endophyte on the abundance and diversity of soil nitrogen-cycling genes (e.g., the AOB-amoA, nirK and nosZ functional genes) in host rhizosphere soil. The results indicated that infection with the endophyte Epichloë festucae var. lolii significantly increased the absolute abundance of the AOB-amoA gene and significantly decreased the absolute abundance of the nosZ gene in the rhizosphere soil of the host plant L. perenne at various sampling times (T0, T1 and T3), but not significantly affected the absolute abundance of the nirK gene. Meanwhile, the relative abundance and beta diversity of the AOB-amoA gene and the relative abundance of the nosZ gene in the host rhizosphere soil were affected by endophyte infection. These results were similar to our previous experiment, in which the leaf litter perennial ryegrass containing the fungal endophyte significantly altered the abundance and diversity of the AOB-amoA, nirK and nosZ genes in non-rhizosphere soil [22]. Concurrently, the absolute abundance of the AOB-amoA gene in host rhizosphere soil at the T1 sampling time, and the absolute abundance of the nosZ gene at T3 and T1 sampling times, were higher than those at T0 time. Overall, the Epichloë endophyte had an effect on the absolute and relative abundances and beta diversity of the AOB-amoA gene and the relative abundance of the nosZ gene in the rhizosphere soil of L. perenne plants.
Several reported studies showed that the Epichloë endophyte in Lolium multiflorum caused changes in the soil bacterial community structure by modifying host rhizodeposition [46]. Infection by Epichloë endophytes can modify litter decomposition by altering both the quantity and quality of the litter, or by changing the decomposer communities [14,17]. Our previous study also demonstrated that litter containing the fungal endophyte E. festucae var. lolii significantly altered the abundance and diversity of the AOB-amoA functional genes in non-rhizosphere soil compared to endophyte-free litter [22]. We found significant differences in the absolute and relative abundances and beta diversity of the AOB-amoA gene at T1 time between the rhizosphere soil of endophyte-infected plants and endophyte-free plants. Consistent with the previous findings by Jin et al. [53], the absolute abundance of the AOB-amoA gene was increased in the rhizosphere soil of Achnatherum inebrians by endophyte infection. These findings suggested that which pathways are mediated by endophytes impacts the abundance and beta diversity of the AOB-amoA gene involved in soil nitrification, which in turn affects nitrogen transformation. Extensive studies have confirmed that soil nitrification is strongly correlated with environmental factors, including aeration conditions, texture, temperature, soil moisture, pH value and fertilization factors. Soil pH is the main factor affecting the AOA-amoA or AOB-amoA gene communities [54]. Our study supports the previous literature suggesting that the abundance and diversity of the AOB-amoA gene was significantly correlated with soil pH and NH4+.
Although the denitrifying genes (nirS, nirK and nosZ) are the most extensively studied as markers for the composition and population of soil microbial communities in forest, wetland, pasture and agricultural soil [55,56], they are rare in the rhizosphere soil of endophyte-infected plants, raising concerns regarding Epichloë endophytes in beneficial effects of denitrification in soil. Our previous findings demonstrated that litter containing the fungal endophyte significantly reduced the abundance and alpha diversity of the nirK and nosZ genes involved in soil denitrification [22]. Jin et al. [53] showed that the change in the abundance of denitrification genes nirK and nosZ by Epichloë endophyte was dependent on host ecotypes. In the current study, no significant alterations in the abundance (Figure 3B,E) and diversity (Figure 4B and Figure 5B) of the nirK gene were observed between the rhizosphere soil of endophyte-infected plants and that of endophyte-free plants across various sampling times. Because of its importance as the final step in denitrification, nosZ was chosen to determine the effect of the Epichloë endophyte. The abundance but not diversity significantly changed in rhizosphere soil of the host ryegrass under Epichloë endophyte infection, compared to the rhizosphere soil of endophyte-free plants under different sampling times (T0, T1 and T3). A possible explanation for this finding could be that the effect of the Epichloë endophyte on nirK nosZ genes might related to host types, experimental treatments and soil types (e.g., rhizosphere soils and bulk soils).
This study provides new insights into how plant–endophyte interactions regulate soil microbial functional groups to influence nitrogen cycling. Future research should integrate multi-omics approaches (e.g., metagenomics, metabolomics) to elucidate the underlying molecular mechanisms and validate these findings across diverse soil environments and host systems.

5. Conclusions

To our knowledge, the response of the nitrogen-cycling gene in host rhizosphere soil to a symbiotic relationship between Lolium perenne plants and the fungal endophyte Epichloë festucae var. lolii. has received little attention. This study demonstrated the impacts of foliar fungal endophyte on the abundance and diversity of nitrogen-cycling genes involved in soil nitrification and denitrification. We found that the endophyte increased the concentrations of soil available nitrogen in ryegrass rhizosphere by promoting the abundance of the AOB-amoA gene involved in soil nitrification and decreasing the abundance of the nosZ gene involved in soil denitrification. The changes in the abundance and diversity of the AOB-amoA gene were associated with soil pH and NH4+ concentration, and the abundance and diversity of the nosZ gene was associated with soil pH and soil N/P ratio. The findings from this study may contribute to a new understanding of how endophytes affect functional genes involved in the soil nitrogen cycle, as well as the associated mechanisms in the rhizosphere soil of host plants. Further studies are necessary to elucidate the correlation among nitrogen-cycling gene, soil N transformation, and Epichloë endophyte-mediated litter decomposition and root exudation.

Author Contributions

Conceptualization, M.M. and Z.C.; methodology, M.M., Z.C. and L.J.; data curation, M.M., Y.H. and M.W.; writing—original draft preparation, M.M. and Z.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Basic Research Program of China (2014CB138702), the National Science Foundation of China (32201445), the China Postdoctoral Science Foundation (2021M701525), Program for Changjiang Scholars and Innovative Research Team in University, China (IRT17R50), Gansu Provincial Youth Science and Technology Fund Program (22JR5RA532), Gansu Province Outstanding Doctoral Students Project (22JR5RA434) and 111 Project (B12002). The authors are thankful for support from USDA-NIFA Multistate Project W4147 and the New Jersey Agricultural Experiment Station.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The nucleotide sequences of the AOB-amoA, nirK and nosZ functional genes from this study have been deposited the NCBI sequence read archive (Bioproject: PRJNA861957, PRJNA861959 and PRJNA861960, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA861957 (accessed on 25 July 2022), PRJNA861959 and PRJNA861960, respectively).

Acknowledgments

We thank the Weigen Huang of Institute of Soil Science, Chinese Academy of Sciences, Nanjing, for supporting this study with structures and software usage. We also thank the editor and anonymous reviewers for their valuable comments. We would like to express our sincere gratitude to J.F.W and K.M for their valuable contributions to manuscript revision and English language polishing. Their expertise significantly improved the quality of this work.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Effects of Epichloë endophyte and sampling times (T0, T1, T2 and T3) on rhizosphere soil properties of Lolium perenne. (A): Soil pH, (B): soil total N, (C): soil total P, (D): SOC, (E): soil NH4+, (F): soil NO3−, (G): the C/N ratio, (H): the N/P ratio and (I): the N/P ratio. Values are mean ± standard error (SE). Different lowercase letters indicate significant differences at p ≤ 0.05, among different sampling times (T0, T1, T2 and T3). Also, *, ** and *** mean significant difference at p ≤ 0.05%, p ≤ 0.01% and p ≤ 0.001%, respectively (independent t-test), between endophyte-infected (E+) and endophyte-free (E−) plant rhizosphere soil under the same addition times. Same as below.
Figure 1. Effects of Epichloë endophyte and sampling times (T0, T1, T2 and T3) on rhizosphere soil properties of Lolium perenne. (A): Soil pH, (B): soil total N, (C): soil total P, (D): SOC, (E): soil NH4+, (F): soil NO3−, (G): the C/N ratio, (H): the N/P ratio and (I): the N/P ratio. Values are mean ± standard error (SE). Different lowercase letters indicate significant differences at p ≤ 0.05, among different sampling times (T0, T1, T2 and T3). Also, *, ** and *** mean significant difference at p ≤ 0.05%, p ≤ 0.01% and p ≤ 0.001%, respectively (independent t-test), between endophyte-infected (E+) and endophyte-free (E−) plant rhizosphere soil under the same addition times. Same as below.
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Figure 2. Effects of Epichloë endophyte on the absolute and relative abundances (at the genus level) of the AOB-amoA, nirK and nosZ genes in rhizosphere soil properties of Lolium perenne under sampling times (T1 and T3). (A,D): The AOB-amoA gene, (B,E): the nirK gene and (C,F): nosZ gene.Also, Different lowercase letters indicate significant differences at p ≤ 0.05, among different sampling times (T0, T1, T2 and T3). *, ** and *** mean significant difference at p ≤ 0.05%, p ≤ 0.01% and p ≤ 0.001%.
Figure 2. Effects of Epichloë endophyte on the absolute and relative abundances (at the genus level) of the AOB-amoA, nirK and nosZ genes in rhizosphere soil properties of Lolium perenne under sampling times (T1 and T3). (A,D): The AOB-amoA gene, (B,E): the nirK gene and (C,F): nosZ gene.Also, Different lowercase letters indicate significant differences at p ≤ 0.05, among different sampling times (T0, T1, T2 and T3). *, ** and *** mean significant difference at p ≤ 0.05%, p ≤ 0.01% and p ≤ 0.001%.
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Figure 3. Effects of two levels of sampling times (T1 and T3) on Shannon and Chao1 indexes (at the genus level) of the AOB-amoA, nirK and nosZ genes in the E+ and E− rhizosphere soils under litter addition (T1 and T3). (A,D): The AOB-amoA gene, (B,E): the nirK gene and (C,F): nosZ gene.
Figure 3. Effects of two levels of sampling times (T1 and T3) on Shannon and Chao1 indexes (at the genus level) of the AOB-amoA, nirK and nosZ genes in the E+ and E− rhizosphere soils under litter addition (T1 and T3). (A,D): The AOB-amoA gene, (B,E): the nirK gene and (C,F): nosZ gene.
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Figure 4. Principal component analysis (PCA) (at the genus level) of the AOB-amoA, nirK and nosZ genes in the E+ and E− rhizosphere soils under sampling times (T1 and T3). (A): The AOB-amoA gene, (B): the nirK gene and (C): nosZ gene.
Figure 4. Principal component analysis (PCA) (at the genus level) of the AOB-amoA, nirK and nosZ genes in the E+ and E− rhizosphere soils under sampling times (T1 and T3). (A): The AOB-amoA gene, (B): the nirK gene and (C): nosZ gene.
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Figure 5. Canonical correlation analysis (at the genus level) of relative abundance and diversity of the AOB-amoA, nirK and nosZ genes in the E+ and E− rhizosphere soils under sampling times. Environmental factors include soil pH, SOC (soil organic carbon), TN (soil total nitrogen), TP (soil total phosphorus), soil NH4+, soil NO3−, C_N (the SOC and TN ratio) and C_P (the SOC and TP ratio). (A): The AOB-amoA gene, (B): the nirK gene and (C): nosZ gene.
Figure 5. Canonical correlation analysis (at the genus level) of relative abundance and diversity of the AOB-amoA, nirK and nosZ genes in the E+ and E− rhizosphere soils under sampling times. Environmental factors include soil pH, SOC (soil organic carbon), TN (soil total nitrogen), TP (soil total phosphorus), soil NH4+, soil NO3−, C_N (the SOC and TN ratio) and C_P (the SOC and TP ratio). (A): The AOB-amoA gene, (B): the nirK gene and (C): nosZ gene.
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Table 1. Two-way analysis of variance (two-way ANOVA) for the effect of endophyte status (E) and sampling times (T) on soil pH, soil organic carbon (SOC), total nitrogen, total phosphorus, ammonium nitrogen (soil NH4+), nitrate nitrogen (soil NO3−) and the C/N, C/P and N/P ratios in rhizosphere soil of Lolium perenne.
Table 1. Two-way analysis of variance (two-way ANOVA) for the effect of endophyte status (E) and sampling times (T) on soil pH, soil organic carbon (SOC), total nitrogen, total phosphorus, ammonium nitrogen (soil NH4+), nitrate nitrogen (soil NO3−) and the C/N, C/P and N/P ratios in rhizosphere soil of Lolium perenne.
TreatmentdfSoil pHSOCSoil Total NSoil Total PSoil NH4+Soil NO3−C/N RatioC/P RatioN/P Ratio
FpFpFpFpFpFpFpFpFp
Endophyte (E)11.4670.238384.473<0.0000.4310.5188.130 0.009 17.843<0.00038.522 <0.0007.2650.01314.4710.0012.8530.048
Litter addition times (T)34.4830.0121536.522<0.0005.1950.00718.725<0.00078.311<0.000979.759<0.0005.5640.0055.125 0.0076.3370.003
E x T30.1700.91655.246 <0.0001.6920.1951.981 0.144 1.150 0.349 6.158 0.003 4.7850.0097.675 0.0012.3960.093
Table 2. The independent sample t-test for the effects of endophyte status (E) on soil pH, SOC, total N, total P, NH4+, NO3− and the C/N, C/P and N/P ratios in rhizosphere soil of Lolium perenne under different sampling times (T0, T1, T2 and T3).
Table 2. The independent sample t-test for the effects of endophyte status (E) on soil pH, SOC, total N, total P, NH4+, NO3− and the C/N, C/P and N/P ratios in rhizosphere soil of Lolium perenne under different sampling times (T0, T1, T2 and T3).
Times Soil pHSOCSoil Total NSoil Total PSoil NH4+Soil NO3−Soil C/N RatioSoil C/P RatioSoil N/P Ratio
T0df777766766
F0.7165.3831.2594.6450.0370.1100.0000.0010.000
p0.4530.5080.4260.4350.7010.7180.8760.7580.571
T1df555566666
F0.0290.0591.0360.1750.8858.3940.1190.1488.568
p0.438<0.0000.0560.1310.0010.2340.3660.0800.402
T2df666666666
F2.9340.9550.0210.1350.0431.9312.0760.5520.363
p0.743<0.0000.1710.0210.003<0.0000.2640.0070.006
T3df666666666
F0.4273.3888.2731.3765.9608.2941.6571.1482.596
p0.314<0.0000.3260.4390.0490.0230.0170.7330.536
Table 3. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on alpha diversity indexes (Shannon and Chao1) of the AOB-amoA, nirK and nosZ genes at the genus level.
Table 3. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on alpha diversity indexes (Shannon and Chao1) of the AOB-amoA, nirK and nosZ genes at the genus level.
TreatmentdfAOB-amoA GenenirK GenenosZ Gene
ShannonChao1ShannonChao1ShannonChao1
FpFpFpFpFpFp
Endophyte (E)10.0080.9301.1250.3201.1420.3162.1790.1780.5520.4790.5250.489
Litter addition times (T)10.0130.9111.1250.3200.0920.7690.0020.9690.1030.7560.0750.791
E x T10.9690.3543.1250.1150.0960.7640.0650.8050.3970.5460.0040.952
Table 4. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on the relative abundance of the AOB-amoA gene at the genus level.
Table 4. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on the relative abundance of the AOB-amoA gene at the genus level.
TreatmentdfNnorank_f__Environmental_SamplesNitrosospiraUnclassified_k__Norank_d__BacteriaNorank_p__Ammonia_Oxidising_Bacteria_EnsembleUnclassified_o__NitrosomonadalesOther
FpFpFpFpFpFp
Endophyte (E)110.253 0.0134.9790.05662.421<0.0000.0680.8017.2630.0270.685 0.432
Litter addition times (T)17.834 0.02311.030 0.01149.618<0.0003.7450.0895.1960.052 0.992 0.348
E x T12.990 0.1226.3830.03524.4130.0010.6980.4288.3230.0201.013 0.344
Table 5. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on the relative abundance of the nirK gene at the genus level.
Table 5. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on the relative abundance of the nirK gene at the genus level.
TreatmentdfUnclassifiedUnclassified_d__BacteriaMethylobacteriumGemmataGeodermatophilusPseudomonasSinorhizobiumRhodococcus_f__Nocardiaceae
FpFpFpFpFpFpFpFp
Endophyte (E)10.3470.5723.1980.1120.4050.5420.1270.7301.1120.3220.4570.5180.9560.3571.3620.277
Litter addition times (T)10.1170.7420.217 0.6548.5640.0190.1650.6955.8660.0420.1650.6950.1390.7191.1040.324
E x T10.0020.9631.2060.3040.8350.3880.0030.9550.0060.9420.7950.3990.0120.9150.8270.390
TreatmentdfBradyrhizobiumArthrobacterNitrosomonasConexibacterEnsiterMicrovirgaPseudarthrobacterOther
FpFpFpFpFpFpFpFp
Endophyte (E)11.0620.3330.6490.4441.0670.3320.7240.4201.6920.2290.2450.6340.8700.3780.1070.752
Litter addition times (T)10.7240.4201.7740.2201.1740.3100.1880.6761.3710.2750.5570.4771.4760.2590.0940.767
E x T11.0200.3421.0180.3431.3420.2800.8390.3860.0000.9870.1540.7050.8700.3780.2220.650
Table 6. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on the relative abundance of the nosZ gene at the genus level.
Table 6. Two-way ANOVA for the effect of endophyte status (E) and sampling times (T) on the relative abundance of the nosZ gene at the genus level.
TreatmentdfUnclassified_p__ProteobacteriaUnclassified_c__AlphaproteobacteriaUnclassified_k__Norank_d__BacteriaUnclassified_o__RhizobialesUnclassified_f__RhodobacteraceaeRalstoniaOther
FpFpFpFpFpFpFp
Endophyte (E)11.3080.2860.0090.9260.0000.9939.6430.0158.8600.0181.7900.2181.0010.346
Litter addition times (T)112.022 0.00813.7700.0060.0860.77643.165<0.00035.847 <0.0001.7900.2183.7710.088
E x T17.1260.0280.2790.6120.4820.5071.4550.2628.9760.0171.7900.2180.7560.410
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Maimaitiyiming, M.; Huang, Y.; Jia, L.; Wu, M.; Chen, Z. Epichloë Endophyte Alters Bacterial Nitrogen-Cycling Gene Abundance in the Rhizosphere Soil of Perennial Ryegrass. Biology 2025, 14, 879. https://doi.org/10.3390/biology14070879

AMA Style

Maimaitiyiming M, Huang Y, Jia L, Wu M, Chen Z. Epichloë Endophyte Alters Bacterial Nitrogen-Cycling Gene Abundance in the Rhizosphere Soil of Perennial Ryegrass. Biology. 2025; 14(7):879. https://doi.org/10.3390/biology14070879

Chicago/Turabian Style

Maimaitiyiming, Munire, Yanxiang Huang, Letian Jia, Mofan Wu, and Zhenjiang Chen. 2025. "Epichloë Endophyte Alters Bacterial Nitrogen-Cycling Gene Abundance in the Rhizosphere Soil of Perennial Ryegrass" Biology 14, no. 7: 879. https://doi.org/10.3390/biology14070879

APA Style

Maimaitiyiming, M., Huang, Y., Jia, L., Wu, M., & Chen, Z. (2025). Epichloë Endophyte Alters Bacterial Nitrogen-Cycling Gene Abundance in the Rhizosphere Soil of Perennial Ryegrass. Biology, 14(7), 879. https://doi.org/10.3390/biology14070879

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