Endophyte-free Research Articles

Fungal endophytes Fusarium solani SGGF14 and Alternaria tenuissima SGGF21 enhance the glycyrrhizin production by modulating its key biosynthetic genes in licorice (Glycyrrhiza glabra L.).

To identify promising fungal endophytes that are able to produce glycyrrhizin and enhance it in licorice and the mechanisms involved. Fifteen fungal endophytes were isolated from Glycyrrhiza glabra L. rhizomes among which SGGF14 and SGGF21 isolates were found to produce glycyrrhizin by 4.29 and 2.58µg g-1 dry weight in the first generation of their culture. These isolates were identified as Fusarium solani and Alternaria tenuissima, respectively, based on morphological characteristics and sequence analysis of internal transcribed spacer, TEF1, ATPase, and CAL regions. Subsequently, G. glabra plants were inoculated with these fungal isolates to examine their effect on glycyrrhizin production, plant growth parameters and the expression of key genes involved in glycyrrhizin pathway: SQS1, SQS2, bAS, CAS, LUS, CYP88D6, and CYP72A154. Endophytes were able to enhance glycyrrhizin content by 133%-171% in the plants. Natural control (NC) plants, harboring all natural endophytes, had better growth compared to SGGF14- and SGGF21-inoculated and endophyte-free (EF) plants. Expression of SQS1, SQS2, CYP88D6, and CYP72A154 was upregulated by inoculation with endophytes. LUS and CAS were downregulated after endophyte inoculation. Expression of bAS was higher in SGGF21-inoculated plants when compared with NC, EF, and SGGF14-inoculated plants. Two selected fungal endophytes of G. glabra can produce glycyrrhizin and enhance glycyrrhizin content in planta by modulating the expression of key genes in glycyrrhizin biosynthetic pathway.

Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Effect of Epichloë gansuensis Endophyte on Seed-Borne Microbes and Seed Metabolites in Achnatherum inebrians.

The seed-borne microbiota and seed metabolites of the grass Achnatherum inebrians, either host to Epichloë gansuensis (endophyte infected [EI]) or endophyte free (EF), were investigated. This study determined the microbial communities both within the seed (endophytic) and on the seed surface (epiphytic) and of the protective glumes by using Illumina sequencing technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. In addition, metabolites of seeds and glumes were detected using liquid chromatography-mass spectrometry (LC-MS). Unlike with the seeds of EF plants, the presence of E. gansuensis resulted in significant changes in the content of 108 seed and 31 glume metabolites. A total of 319 significant correlations occurred between seed-borne microbiota and seed metabolites; these correlations comprised 163 (147 bacterial and 16 fungal) positive correlations and 156 (136 bacterial and 20 fungal) negative correlations. Meanwhile, there were 42 significant correlations between glume microbiota and metabolites; these correlations comprised 28 positive (10 bacterial and 18 fungal) and 14 negative (9 bacterial and 5 fungal) correlations. The presence of E. gansuensis endophyte altered the communities and diversities of seed-borne microbes and altered the composition and content of seed metabolites, and there were many close and complex relationships between microbes and metabolites. IMPORTANCE The present study was to investigate seed-borne microbiota and seed metabolites in Achnatherum inebrians using high-throughput sequencing and LC-MS technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. Compared with endophyte-free plants, the content of 108 seed and 31 glume metabolites of endophyte-infected plants was significantly changed. There were 319 significant correlations between seed-borne microbiota and seed metabolites and 42 significant correlations between glume microbiota and metabolites.

Evaluation of Implants, Clover, and Fescue Variety on Stocker Steers – Year 3

Sixty-four growing steers were used in a split-plot experiment, where the whole plot was pasture, and the split-plot was the implant level. Whole plot treatment was a 4 × 2 factorial with four levels of fescue (High Endophyte, Low Endophyte, Novel, or Endophyte Free) and two levels of legume (Legumes or No Legumes). The split-plot included four implant levels (No Implant, Synovex One Grass, Revalor-G, or Ralgro). Data collected were weights and ultrasound carcass characteristics when steers were coming off grass. Steers on High Endophyte had a lower average daily gain (ADG) and final weight than steers on novel endophyte, with those grazing pastures that had low endophyte and endophyte-free were intermediate. There was no difference in cattle gains or carcass measurements based on the addition of legumes or the type of implant the steers received. The drought restricted the days on grass and potentially reduced the positive effects of implants.

Modification of Cuticular Wax Composition and Biosynthesis by Epichloë gansuensis in Achnatherum inebrians at Different Growing Periods

Cuticular wax plays a critical role as a plant protectant against various environmental stresses. We predicted that the presence of the mutualistic fungal endophyte Epichloë gansuensis in Achnatherum inebrians would change both the composition of leaf cuticular wax as plants aged during the growing season and the gene expression levels associated with the wax biosynthesis pathway. Endophyte-infected (EI) and endophyte-free (EF) A. inebrians plants were established for a four-month pot experiment. In agreement with our prediction, the presence of E. gansuensis can change the composition of leaf cuticular wax at different growing periods, particularly the proportion of esters, fatty acids and hydrocarbons. The proportion of fatty acids in EI plants was lower than that in EF plants. The proportion of hydrocarbons increased and esters decreased as plants grew. Furthermore, we found 11 DEGs coding for proteins involved in cuticular wax biosynthesis, including FabF, FAB2, ECR, FAR, CER1, ABCB1 and SEC61A. The present study highlights the significant contribution of E. gansuensis to leaf cuticular wax composition and biosynthesis in A. inebrians plants.

Cuticular Wax Modification by Epichloë Endophyte in Achnatherum inebrians under Different Soil Moisture Availability.

The cuticular wax serves as the outermost hydrophobic barrier of plants against nonstomatal water loss and various environmental stresses. An objective of this study was to investigate the contribution of the mutualistic fungal endophyte Epichloë gansuensis to leaf cuticular wax of Achnatherum inebrians under different soil moisture availability. Through a pot experiment and gas chromatography−mass spectrometry (GC−MS) analysis, our results indicated that the hydrocarbons were the dominant components of leaf cuticular wax, and the proportion of alcohols, aldehydes, amines, and ethers varied with the presence or absence of E. gansuensis and different soil moisture availability. Amines and ethers are unique in endophyte-free (EF) A. inebrians plants and endophyte-infected (EI) A. inebrians plants, respectively. By transcriptome analysis, we found a total of 13 differentially expressed genes (DEGs) related to cuticular biosynthesis, including FabG, desB, SSI2, fadD, BiP, KCS, KAR, FAR, and ABCB1. A model is proposed which provides insights for understanding cuticular wax biosynthesis in the association of A. inebrians plants with E. gansuensis. These results may help guide the functional analyses of candidate genes important for improving the protective layer of cuticular wax of endophyte-symbiotic plants.

Epichloë Endophyte Infection Changes the Root Endosphere Microbial Community Composition of Leymus Chinensis Under Both Potted and Field Growth Conditions.

Epichloë endophytes can not only affect the growth and resistance of the host plant but also change the biotic and abiotic properties of the soil where the host is situated. Here, we used endophyte-infected (EI) and endophyte-free (EF) Leymus chinensis as plant materials, to study the microbial diversity and composition in the host root endosphere and rhizosphere soil under both pot and field conditions. The results showed that endophyte infection did not affect the diversity of either bacteria or fungi in the root zone. There were significant differences in both bacterial and fungal communities between the root endosphere and the rhizosphere, and between the field and the pot, while endophytes only affected root endosphere microbial communities. The bacterial families affected by endophyte infection changed from 29.07% under field conditions to 40% under pot conditions. In contrast, the fungal families affected by endophyte infection were maintained at nearly 50% under both field and pot conditions. That is to say, bacterial communities in the root endosphere were more strongly affected by environmental conditions, and in comparison, the fungal communities were more strongly affected by species specificity. Endophytes significantly affected the fungal community composition of the host root endosphere in both potted and field plants, only the effect was more obvious in potted plants. Endophyte infection increased the abundance of three fungal families (Thelebolaceae, Herpotrichiellaceae and Trimorphomycetaceae) under both field and potted conditions. In potted plants, endophytes also altered the dominant fungi from pathogenic Pleosporales to saprophytic Chaetomiaceae. Endophyte infection increased the relative abundance of arbuscular mycorrhizal fungi and saprophytic fungi, especially under potted conditions.Overall, endophytes significantly affected the fungal community composition of the host root endosphere in both potted and field plants. Endophytes had a greater impact on root endosphere microorganisms than the rhizosphere, a greater impact on fungal communities than bacteria, and a greater impact on root endosphere microorganisms under potted conditions than at field sites.

Drought Responsive Putative Marker-Trait Association in Tall Fescue as Influenced by the Presence of a Novel Endophyte.

Tall fescue (Festuca arundinacea Schreb.) is one of the most important cool-season perennial obligatory outcrossing forage grasses in the United States. The production and persistence of tall fescue is significantly affected by drought in the south-central United States. Shoot-specific endophyte (Epichloë coenophiala)-infected tall fescue showed superior performance under both biotic and abiotic stress conditions. We performed a genome-wide association analysis using clonal pairs of novel endophyte AR584-positive (EP) and endophyte-free (EF) tall fescue populations consisting of 205 genotypes to identify marker-trait associations (MTAs) that contribute to drought tolerance. The experiment was performed through November 2014 to June 2018 in the field, and phenotypic data were taken on plant height, plant spread, plant vigor, and dry biomass weight under natural summer conditions of sporadic drought. Genotyping-by-sequencing of the population generated 3,597 high quality single nucleotide polymorphisms (SNPs) for further analysis. We identified 26 putative drought responsive MTAs (17 specific to EP, eight specific to EF, and one in both EP and EF populations) and nine of them (i.e., V.ep_10, S.ef_12, V.ep_27, HSV.ef_31, S.ep_30, SV.ef_32, V.ep_68, V.ef_56, and H.ef_57) were identified within 0.5 Mb region in the tall fescue genome (44.5–44.7, 75.3–75.8, 77.5–77.9 and 143.7–144.2 Mb). Using 26 MTAs, 11 tall fescue genotypes were selected for subsequent study to develop EP and EF drought tolerant tall fescue populations. Ten orthologous genes (six for EP and four for EF population) were identified in Brachypodium genome as potential candidates for drought tolerance in tall fescue, which were also earlier reported for their involvement in abiotic stress tolerance. The MTAs and candidate genes identified in this study will be useful for marker-assisted selection in improving drought tolerance of tall fescue as well opening avenue for further drought study in tall fescue.

Transcriptome Analysis Revealed Plant Hormone Biosynthesis and Response Pathway Modification by Epichloëgansuensis in Achnatheruminebrians under Different Soil Moisture Availability.

The present study was designed to explore the effects of the endophyte Epichloë gansuensis on gene expression related to plant hormone biosynthesis and response pathways and the content of salicylic acid (SA) and jasmonic acid (JA) hormones of Achnatherum inebrians, under different moisture conditions. Through a pot experiment and transcriptome analysis, we found a total of 51 differentially expressed genes (DEGs) related to hormone biosynthesis and response pathways, including 12 auxin related genes, 8 cytokinin (CTK) related genes, 3 gibberellin (GA) related genes, 7 abscisic acid (ABA) related genes, 7 ethylene (ET) related genes, 12 JA related genes and 4 SA related genes. Furthermore, key genes of JA and SA biosynthesis and response pathways, such as LOX2S, AOS, OPR, ACX, JMT, JAZ, PAL, NPR1, TGA and PR-1, showed different degrees of upregulation or downregulation. Under 60% soil moisture content, the JA content of endophyte-free (EF) A. inebrians was significantly (p < 0.05) higher than that of endophyte-infected (EI) A. inebrians. Under 30% and 60% soil moisture content, the SA content of EF A. inebrians was significantly (p < 0.05) higher than that of EI A. inebrians. SA content of EI A. inebrians under 30% and 60% soil moisture content was significantly (p < 0.05) higher than that under 15% soil moisture content. With both EI and EF plants, the SA and JA levels, respectively, are very similar at 15% soil moisture content. This study has revealed that E. gansuensis differentially activated plant hormone synthesis and signal transduction pathways of A. inebrians plants under different soil moisture availability.

46 Effects of Implants, Clover, and Fescue Variety on Stocker Steers

Abstract Fescue thrives due to a symbiotic relationship with a fungus called endophyte. Endophyte releases ergot toxins leading to metabolic issues, such as vasoconstriction, rough hair coats and poor cattle gains. Developments have occurred to remove or limit the toxins. The objective of this project was to identify management practices while grazing fescue to improve production and toxicity issues. Sixty-four growing steers (283 ± 14 kg BW) were used in a split-plot experiment, where the whole plot was pasture and the split plot was implants. Whole plot treatment was a 4 x 2 factorial with four levels of fescue (High Endophyte, Low Endophyte, Novel, Endophyte Free) and two levels of legume (Legumes or No Legumes). Split plot included four implant levels (No Implant, Synovex One Grass, Revalor-G, Ralgro). Data collected were weights, hair coat scores, hair length, rectal temperature (every 28d) and ultrasound carcass characteristics last day on pasture. Data were analyzed using the Glimmix Procedure of SAS (v. 9.4, Cary, NC). Steers on high endophyte had the lowest ADG, longest hair, and highest temperature as compared to steers on all other fescue types (P &amp;lt; 0.05). The gain differentiation was observed beginning at D56 through the end of the study (P &amp;lt; 0.05). Overall ADG was not impacted by addition of legume nor implant type (P &amp;gt; 0.10). Steers that were not implanted had a longer hair length throughout many measurement dates (P &amp;lt; 0.05). Steers grazing pastures with legume tended (P &amp;lt; 0.10) to have a higher ultrasound measured marbling score and less muscle depth. This study found that the best management strategy for fescue toxicity is to use non-endophyte or non-toxic varieties of fescue pasture. Contrary to previous research, the addition of implants and legumes for this project showed no improvement in cattle gains.

Removal of Soil Microbes Alters Interspecific Competitiveness of Epichloë Endophyte-Infected over Endophyte-Free Leymus chinensis.

Epichloë endophytes may not only affect the growth and resistances of host grasses, but may also affect soil environment including soil microbes. Can Epichloë endophyte-mediated modification of soil microbes affect the competitive ability of host grasses? In this study, we tested whether Epichloë endophytes and soil microbes alter intraspecific competition between Epichloë endophyte-colonized (EI) and endophyte-free (EF) Leymus chinensis and interspecific competition between L. chinensis and Stipa krylovii. The results demonstrated that Epichloë endophyte colonization significantly enhanced the intraspecific competitive ability of L. chinensis and that this beneficial effect was not affected by soil microbes. Under interspecific competition, however, significant interactions between Epichloë endophytes and soil microbes were observed. The effect of Epichloë endophytes on interspecific competitiveness of the host changed from positive to neutral with soil microbe removal. Here higher mycorrhizal colonization rates probably contributed to interspecific competitive advantages of EI over EF L. chinensis. Our result suggests that Epichloë endophytes can influence the competitive ability of the host through plant soil feedbacks from the currently competing plant species.

Nitrogen fertilization for hay and seed from endophyte‐free and endophyte‐infected tall fescue

AbstractTall fescue [Schedonorus arundinaceus (Schreb.) Dumort.] is an important forage, but a fungal endophyte often found in the aboveground biomass can negatively affect animal production. Nitrogen also affects production, but data are limited regarding N fertilization effects on hay and seed yields from endophyte‐free (EF) compared with endophyte‐infected (EI) tall fescue grown on claypan soil in the eastern Great Plains. This study's objective was to determine the effects of N fertilizer timing and rate on EF and EI tall fescue yield of R4 (anthesis) hay, seed, and hay residual after seed harvest (stubble hay, SH). Endophyte infection did not affect R4 hay, seed, or SH yields and did not interact with N timing, N rate, or year for any measured parameters. Across the 3‐yr study, N fertilizer applied in late fall resulted in 18% greater seed production than late winter, with split late fall/late winter applications being intermediate. The effect of N timing on R4 and SH varied by year. Annual fescue seed yield response to N rate varied, but required 112 kg N ha−1 or more to achieve near 300 kg ha−1 in better‐yielding years. Hay measured at R4 or SH responded to N up to at least 168 kg ha−1. Hay N concentration was diluted with initial increments of N fertilizer but increased as yield response diminished with greater N rates. Simple return functions suggest that the N rate to achieve maximum economic return is reduced when N fertilizer costs are high and when seed/hay selling prices are low.

Effects of endophyte-infected tall fescue seed and protein supplementation on stocker steers: I. Growth performance and hemodynamic responses

Fescue toxicosis is a multifaceted syndrome common in cattle grazing endophyte-infected tall fescue and is detrimental to growth and performance. Recent research has shown that supplementing protein has the potential to enhance growth performance in weaned steers. Therefore, the objective of this study was to evaluate the effect of supplemental CP on physiological parameters in stocker steers experiencing fescue toxicosis. Thirty-six weaned Angus steers (6 mo of age) stratified by weight (196.1 ± 3.6 kg) were assigned to a 2 × 2 factorial arrangement for 56 d: endophyte-free (EF) seed and 14% CP (EF-14; n = 9), EF seed and 18% CP (EF-18; n = 9), endophyte-infected (EI) seed and 14% CP (EI-14; n = 9), and EI seed and 18% CP (EI-18; n = 9). Steer growth and hemodynamic responses were collected weekly during ergot alkaloid exposure. On day 14 of the trial, iButton temperature data loggers were subcutaneously inserted in the lateral neck region to record hourly body temperature for 42 d. Data were analyzed using PROC MIXED of SAS with repeated measures. No differences were observed in DMI, BW, ADG, F:G, or BCS during the treatment period (P > 0.05). Hair shedding scores, rectal temperatures, surface temperatures, and respiration rates were greater in EI steers compared to EF steers regardless of supplemental CP (P < 0.05). However, subcutaneous body temperature was greater in EI-14 steers (37.94 °C) compared to other steer groups (37.60, 37.68, 37.72 ± 0.04 °C for EF-14, EF-18, and EI-18, respectively; P < 0.05). Prolactin concentrations tended to be greater in EF steers when compared to EI steers (P = 0.07). Heart rate and hematocrit were reduced for EI-18 steers compared to other steer groups (P < 0.05). Caudal artery diameter was reduced in EI-18 steers compared to EI-14 steers (2.60 vs. 2.75 ± 0.05 mm, respectively; P < 0.05) and caudal vein diameter was reduced in EI-18 steers (3.20 mm) compared to all other steer groups (3.36, 3.39, 3.50 mm for EF-14, EF-18, and EI-14, respectively; P < 0.05). However, there was no difference observed in systolic or diastolic blood pressure during the treatment period (P > 0.05). Based on the data, exposure to low to moderate levels of ergot alkaloids during the stocker phase had a negative impact on hemodynamic responses and supplemental CP had minimal impact to alleviate symptoms. Therefore, feeding additional protein above established requirements is not expected to help alleviate fescue toxicosis.

158 Long term impact of ergot alkaloids on fetal heifer development and subsequent growth and reproductive performance

Abstract Previous studies from our laboratory have demonstrated physiological differences in mid-gestation cows grazing either endophyte-infected (EI) or endophyte-free (EF) tall fescue. The objective of this study was to investigate the effects of in utero exposure (TRT) of either EI or EF on heifer growth, reproductive tract development and onset of puberty. Pregnant Angus and SimAngus cows were blocked by age, BW, and breed; and then randomly assigned to graze either EF (n = 27) or EI (n = 27) pastures. At parturition, heifer birthweight was recorded and placental tissues were collected to assess placental characteristics. Heifer BW, height, frame score were recorded at weaning. Assessment of puberty via measurement of BW, BCS, reproductive tract scores (RTS) and pelvic area (PA) started at 10 mo of age. Data were analyzed using a MIXED procedure of SAS and examined for effects of TRT, sire, and age. Statistical significance was determined at P &lt; 0.05 and a tendency at 0.05); however, there was a sire by treatment interaction for ADG during onset of puberty (P &lt; 0.05). While TRT did not affect PA or RTS (P &gt; 0.05) at 10 mo of age, sire significantly influenced RTS (P &lt; 0.05) thus impacting of onset of puberty. While consumption of EI fescue impacts cow performance, calf birthweight, and ratio of cotyledon to placenta weight, it has minimal effect on postnatal growth and development through 10 mo of age. Further studies are needed to determine the impact on heifer reproductive performance.

Effects of endophyte-infected tall fescue seed and protein supplementation on stocker steers: II. Adaptive and innate immune function.

Fescue toxicosis is a multifaceted syndrome common in cattle grazing endophyte-infected tall fescue that affects performance; however, little information is available pertaining to its effects on immunity. Recently, it has been shown that supplemental CP can improve performance in weaned steers postvaccination. Thus, the objective of this study was to evaluate the effect of supplemental CP on innate and adaptive immune responses in stocker steers chronically exposed to ergovaline. Angus steers (n = 12 pens; 3 steers/pen) were stratified by weight and assigned to a 2 × 2 factorial arrangement to examine crude protein levels of supplement (14% or 18%) and ergovaline exposure (0 or 185 μg ergovaline/kg BW/d via ground endophyte-free (EF) or endophyte-infected (EI) tall fescue seed, respectively) on immune response. Consumption of low to moderate concentration of ergovaline from EI tall fescue seed was sufficient to induce mild symptoms associated with fescue toxicosis. Blood samples were collected at day 0, 42, and 56 to evaluate infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea virus (BVDV) type 1b titers following vaccine challenge. Additionally, serum cytokine concentrations were evaluated using Quantibody Bovine Cytokine Arrays on day 0, 28, and 42. Data were analyzed using PROC MIXED of SAS with repeated measures. Regardless of treatment, no differences were observed in IBR and BVDV-1b seroconversion following vaccine challenge (P > 0.05). Regardless of crude protein concentration, EI steers had greater concentrations of proinflammatory cytokines (TNF-α, IFN-γ, IL-1α), chemokines (CCL2, CCL4, MIG), anti-inflammatory cytokines (IL-2, -13, -15, -21), and various growth factors (FGF-1, IGF-1, VEGF-A) when compared to EF steers (P < 0.05). Furthermore, VEGF-A and IGF-1 concentrations were greater in EI-14 steers on day 28 compared to EI-18, EF-14, and EF-18 steers (P < 0.05), however, this difference was not observed on day 0 or 42 (P > 0.05). Based on these data, steers exposed to ergovaline have an increase in pro- and anti-inflammatory cytokines and supplemental CP had minimal impact to mitigate this response. However, in the current study, exposure to ergovaline had little to no effect on adaptive immunity and response to vaccination. Together, chronic exposure to ergovaline results in a hyperactive innate immune response, which may lead to an immuno-compromised animal.

A simultaneous effect of selfing and Epichloë endophyte on forage, seed yield and turf characteristics of perennial ryegrass (Lolium perenne L.)

AbstractRyegrass (Lolium perenne L.) is a putative turf and forage plant characterized as mainly outcrossing in which the extent of self‐fertilization and its consequences has not been investigated. In this study, selfed and half‐sib family seeds were obtained from ten clonally propagated ryegrass plants among which two were endophyte‐free (EF) and eight were endophyte‐infected (EI). Then, 20 seed populations plus two commercial varieties of Speedy and Vigor were sown and evaluated in a biennial experiment for their turf and forage characteristics. Selfing caused significant inbreeding depression for plant fresh and dry weight and leaf length and width in the first year for both EI and EF genotypes. In the second year, only leaf length remained depressed among these characters. Also, for the most growth, turf and reproductive traits, EI populations showed less depression. The presence of endophyte in self‐pollinated populations made an increasing effect for the number of seed per spike and days to heading, although seed weight was reduced. Some of the populations derived from self‐pollination and infected with fungal endophyte including S9 and S10 were even superior to the two commercial varieties in terms of both seed production and turf characteristics. However, for seed‐related characteristics including number of spikes per plant, seed weight and seed yield, EF populations including Speedy and Vigor were more reproductive than EI populations. It is concluded that selfing may increase the discrimination between EI and EF ryegrass plants because endophyte infection may mitigate the negative consequences of inbreeding depression in prone populations.

Endophyte Infection and Methyl Jasmonate Treatment Increased the Resistance of Achnatherum sibiricum to Insect Herbivores Independently.

Alkaloids are usually thought to be responsible for protecting endophyte-infected (EI) grasses from their herbivores. For EI grasses that produce few alkaloids, can endophyte infection enhance their resistance to herbivores? Related studies are limited. In the Inner Mongolian steppe, Achnatherum sibiricum is highly infected by Epichloë endophytes, but produces few alkaloids. Locusts are the common insect herbivores of grasses. In this study, A. sibiricum was used as plant material. Methyl jasmonate (MJ, when applied exogenously, can induce responses similar to herbivore damage) treatment was performed. The effects of endophyte infection and MJ treatment on the resistance of A. sibiricum to Locusta migratoria were studied. We found that locusts preferred EF (endophyte-free) plants to EI plants in both choice and no-choice feeding experiments. Endophyte infection enhanced the resistance of A. sibiricum to locusts. Endophyte infection decreased soluble sugar concentrations, while it increased the total phenolic content and phenylalanine ammonia lyase (PAL) activity, which may contribute to the resistance of A. sibiricum to locusts. There was an interaction effect between MJ treatment and endophyte infection on the growth of the host. MJ treatment was a negative regulator of the plant growth-promoting effects of endophyte infection. There was no interaction effect between MJ treatment and endophyte infection on the defense characteristics of the host. In groups not exposed to locusts, MJ treatment and endophyte infection had a similar effect in decreasing the soluble sugar content, while increasing the total phenolic content and the PAL activity. In groups exposed to locusts, the effect of MJ treatment on the above characteristics disappeared, while the effect of endophyte infection became more obvious. All of these results suggest that even for endophytes producing few alkaloids, they could still increase the resistance of native grasses to insect herbivores. Furthermore, endophyte infection might mediate the defense responses of the host, independent of jasmonic acid (JA) pathways.

Effects of Epichloë sinica on Roegneria kamoji seedling physiology under PEG-6000 simulated drought stress

Some Epichloe species are beneficial to their plant hosts by improving fitness and resistance to stress. The natural, seedborne, asymptomatic symbiotic endophyte Epichloe sinica is specific for Roegneria spp., which are perennial grasses found in grasslands or meadows; many species of Roegneria are excellent forage plants. This study was designed to reveal the biological function of E. sinica via seedling assays with drought simulation using polyethylene glycol (PEG)-6000. Endophyte infected (EI) and endophyte free (EF) R. kamoji seeds (obtained by heat treatment) were germinated and grown on 0, 10, 15 and 20% PEG-6000 for 10 days; seedling and root system morphologies were investigated, and reactive oxygen species (ROS), which act as signaling molecules in plant responses to abiotic stress, were detected. E. sinica enhanced both the germination potential and rate of R. kamoji seeds treated with high concentrations of PEG-6000. After PEG treatment, shoot and root morphologies differed significantly between EF and EI seedlings. Additionally, lower amounts of ROS concentration were detected in the leaves of EI seedlings treated with high concentrations of PEG-6000. In conclusion, E. sinica promoted PEG-6000 simulated drought stress resistance in R. kamoji seedlings.

Arbuscular mycorrhizal fungus inoculation reduces the drought-resistance advantage of endophyte-infected versus endophyte-free Leymus chinensis.

Grasses can be infected simultaneously by endophytic fungi and arbuscular mycorrhizal (AM) fungi. In this study, we tested the hypothesis that endophyte-associated drought resistance of a native grass was affected by an AM fungus. In a greenhouse experiment, we compared the performance of endophyte-infected (EI) and endophyte-free (EF) Leymus chinensis, a dominant species native to the Inner Mongolia steppe, under altered water and AM fungus availability. The results showed that endophyte infection significantly increased drought resistance of the host grass, but the beneficial effects were reduced by AM fungus inoculation. In the mycorrhizal-non-inoculated (MF) treatment, EI plants accumulated significantly more biomass, had greater proline and total phenolic concentration, and lower malondialdehyde concentration than EF plants. In the mycorrhizal-inoculation (MI) treatment, however, no significant difference occurred in either growth or physiological characters measured between EI and EF plants. AM fungus inoculation enhanced drought resistance of EF plants but had no significant effect on drought resistance of EI plants, thus AM fungus inoculation reduced the difference between EI and EF plants. Our findings highlight the importance of interactions among multiple microorganisms for plant performance under drought stress.

Effect of Endophyte Infection and Clipping Treatment on Resistance and Tolerance of Achnatherum sibiricum.

It is well-documented that endophytes can enhance the resistance of agronomical grasses, such as tall fescue and perennial ryegrass to herbivory. For native grasses, however, the related reports are limited, and the conclusions are variable. Achnatherum sibiricum is a grass native to the Inner Mongolian steppe. This grass is highly infected by endophytes but does not produce detectable endophyte-related alkaloids known under normal conditions. In this study, the contributions of endophytes to the resistance of A. sibiricum to Locusta migratoria were studied. We found that locusts preferred EF (endophyte-free) plants to EI (endophyte-infected) plants, and the weight of locusts fed on EI plants was significantly lower than those fed on EF plants. Hence, endophyte infection significantly enhanced the resistance of the host to L. migratoria. Endophyte infection significantly decreased the concentration of soluble sugar and amino acids while significantly increased the concentration of total phenolic content, and these metabolites may contribute to herbivore resistance of the host. The clipping treatment further strengthened the locust resistance advantage of EI over EF plants. After clipping, the weight of the locusts fed on EI plants significantly decreased compared with those fed on unclipped plants, whereas the weight of the locusts fed on EF plants increased significantly. The results suggested that endophyte infection could increase herbivore resistance while decreasing the tolerance of the host grass by mechanisms apart from endophyte-conferred alkaloid defense.