Effects of the endophyte Epichloë coenophiala on the root microbial community and growth performance of tall fescue in different saline-alkali soils

Soil salinization is detrimental to plant growth and yield in agroecosystems worldwide. Epichloë endophytes, a class of clavicipitaceous fungi, enhance the resistance of host plants to saline-alkali stress. This study explored the effects of the systemic fungal endophyte Epichloë coenophiala on the root microbial community and growth performance of tall fescue (Lolium arundinaceum) growing under different saline-alkali stress conditions. Structural equation modeling (SEM) was conducted to analyze the direct and indirect effects (mediated by root microbial community diversity and soil properties) of the endophyte on the growth of tall fescue under saline-alkali stress. The endophyte-infected plants produced higher shoot and root biomass compared to endophyte-free plants under saline-alkali stress (200 and 400 mM). Endophyte infection increased the fungal community diversity and altered its composition in the roots, decreasing the relative abundance of Ascomycota and increasing that of Glomeromycota. Furthermore, endophyte infection decreased the bacterial community diversity and the relative abundance of dominant Proteobacteria. SEM showed that endophyte infection increased the shoot and root biomass under saline-alkali stress (200 and 400 mM) by increasing the arbuscular mycorrhizal fungal diversity in the roots, and soil total nitrogen and phosphorus concentrations. Therefore, it is important to examine aboveground microbes as factors influencing plant growth in saline-alkali stress by affecting belowground microbes and soil chemical properties.     

Effects of the fungal endophyte Phomopsis liquidambari on nitrogen uptake and metabolism in rice

Phomopsis liquidambari can establish a mutualistic symbiotic relationship with rice. It promotes the growth and yield of the host plant and reduces the amount of nitrogen (N) fertilizer required for plant growth. However, the mechanisms responsible for the effects of the fungal endophyte on N use in rice are largely unknown. We conducted a hydroponic experiment to investigate the effects of P. liquidambari on N uptake and N metabolism in rice plants. Rice plants were cultivated in the presence or absence of P. liquidambari under three N levels. Under the low-N treatment, fungal infection significantly increased the biomass, and the total N, soluble protein, free amino acid, free NH₄ ⁺, and chlorophyll contents of rice roots and shoots. The activities of nitrate reductase and glutamine synthetase were increased in infected rice plants. Some genes related to N uptake (OsAMT1;1, OsAMT1;3, OsAMT2;2, OsAMT3;2, OsAMT3;3, OsNRT2;1) and N metabolism (OsNR1, OsGS1, OsGS2, OsNADH-GOGAT) were also up-regulated in infected plants under the low-N treatment. However, these effects gradually weakened as the N level increased. The colonization rate of the endophyte substantially decreased with increasing N levels. Taken together, these results suggest that low-N fertilization induces a physiological state in rice that is favorable for the P. liquidambari symbiosis. The greater extent of P. liquidambari colonization under low-N conditions stimulated the expression of several genes involved in N uptake and N metabolism in rice, thereby enhancing N utilization. These results have implications for enhancing plant growth in low-input systems at nutrient-poor sites.     

Epichloë endophyte affects the ability of powdery mildew (Blumeria graminis) to colonise drunken horse grass (Achnatherum inebrians)

The effect of the systemic seed-borne endophyte Epichloë gansuensis on the colonization by Blumeria graminis, the cause of powdery mildew disease, and the growth of the host grass Achnatherum inebrians, was studied under four soil water conditions. Infection incidence, disease lesion parameters, disease index, biomass production and growth parameters of the grass with and without the fungal endophyte were measured and counted after a period of disease. There was a significantly (P < 0.05) higher disease incidence and disease index for endophyte-free (E−) compared to endophyte-infected (E+) plants under different drought stresses. The presence of the endophyte significantly positively affected all of the host grass growth factors. The results of the present study demonstrate that the presence of the Epichloë endophyte reduced the ability of B. graminis to colonise A. inebrians and also conferred enhanced host plant growth at all soil water conditions tested.     

Endophyte inoculation enhances Ulmus minor resistance to Dutch elm disease

Certain fungal endophytes improve plant resistance to biotic stresses in forest trees. In this study, three stem fungal endophytes belonging to classes Cystobasidiomycetes, Eurotiomycetes and Dothideomycetes were selected from 210 isolates for their potential as enhancers of Ulmus minor resistance to Ophiostoma novo-ulmi. We evaluated phenotypic traits of these endophytes that could be beneficial for inhibiting O. novo-ulmi in the host plant. Under in vitro conditions, the Dothideomycetous isolate YCB36 strongly inhibited O. novo-ulmi growth, released antipathogenic VOCs, chitinases and siderophores, and overlapped with the pathogen in nutrient utilization patterns. These functional traits could explain the 40% reduction in leaf wilting due to O. novo-ulmi in elm trees pre-inoculated with this endophyte. Ulmus minor trees inoculated with this endophyte showed increased leaf stomatal conductance and higher concentrations of flavonoids and total phenolic compounds in xylem tissues, suggesting induction of defence metabolism.     

Fungal Endophyte (Epichlo? festucae) Alters the Nutrient Content of Festuca rubra Regardless of Water Availability

Festuca rubra plants maintain associations with the vertically transmitted fungal endophyte Epichloë festucae. A high prevalence of infected host plants in semiarid grasslands suggests that this association could be mutualistic. We investigated if the Epichloë-endophyte affects the growth and nutrient content of F. rubra plants subjected to drought. Endophyte-infected (E+) and non-infected (E−) plants of two half-sib lines (PEN and RAB) were subjected to three water availability treatments. Shoot and root biomass, nutrient content, proline, phenolic compounds and fungal alkaloids were measured after the treatments. The effect of the endophyte on shoot and root biomass and dead leaves depended on the plant line. In the PEN line, E+ plants had a greater S:R ratio than E-, but the opposite occurred in RAB. In both plant lines and all water treatments, endophyte-infected plants had greater concentrations of N, P and Zn in shoots and Ca, Mg and Zn in roots than E- plants. On average, E+ plants contained in their shoots more P (62%), Zn (58%) and N (19%) than E- plants. While the proline in shoots increased in response to water stress, the endophyte did not affect this response. A multivariate analysis showed that endophyte status and plant line impose stronger differences in the performance of the plants than the water stress treatments. Furthermore, differences between PEN and RAB lines seemed to be greater in E- than in E+ plants, suggesting that E+ plants of both lines are more similar than those of their non-infected version. This is probably due to the endophyte producing a similar effect in both plant lines, such as the increase in N, P and Zn in shoots. The remarkable effect of the endophyte in the nutrient balance of the plants could help to explain the high prevalence of infected plants in natural grasslands.     

The role of in vitro cultivation on symbiotic trait and function variation in a single species of arbuscular mycorrhizal fungus

In vitro propagation of AM fungi using transformed root cultures (TRC) is commonly used to obtain pure AM fungal propagules for use in research and industry. Early observations indicate that such an artificial environment can alter traits and function of AM fungi over time. We hypothesized that increased in vitro cultivation may promote ruderal strategies in fungi by enhancing propagule production and reducing mutualistic quality. To examine the effect of in vitro cultivation on the trait and function of AM fungi, we inoculated plants with 11 Rhizoglomus irregulare isolates which fell along a cultivation gradient spanning 80 generations. We harvested plants at 10, 20 and 30 d post inoculation to observe differences in fungal and plant traits post infection. In vitro cultivation led to increased spore production but reduced plant shoot phosphorus. Our results indicate that in vitro propagation may indirectly select for traits that affect symbiotic quality.     

Genetic Diversity and Structure of Neotyphodium Species and Their Host Achnatherum sibiricum in a Natural Grass–Endophyte System

Achnatherum sibiricum (Poaceae) is a perennial bunchgrass native to the Inner Mongolia Steppe of China. This grass is commonly infected by epichloë endophytes with high-infection frequencies. Previously, we identified two predominant Neotyphodium spp., N. sibiricum and N. gansuense. In the present study, genetic diversity and structure were analyzed for the two predominant Neotyphodium spp. as well as the host grass. We obtained 103 fungal isolates from five populations; 33 were identified as N. sibiricum and 61 as N. gansuense. All populations hosted both endophytic species, but genetic variation was much higher for N. gansuense than for N. sibiricum. The majority of fungal isolates were haploid, and 13% of them were heterozygous at one SSR locus, suggesting hybrid origins of those isolates. Significant linkage disequilibrium of fungal SSR loci suggested that both fungal species primarily propagate by clonal growth through plant seeds, whereas variation in genetic diversity and the presence of hybrids in both endophytic species revealed that although clonal propagation was prevalent, occasional recombination might also occur. By comparing genetic differentiation among populations, we found around 4–7-fold greater differentiation of endophyte populations than host populations, implying more restricted gene flow of endophytes than hosts. We proposed that endophyte infection of A. sibiricum might confer the host some selective advantages under certain conditions, which could help to maintain high-endophyte-infection frequencies in host populations, even when their gene flows do not match each other. Furthermore, we suggested that the same genotype of endophyte as well as host should be confirmed if the objective of the study is to know the influence of endophyte or host genotype on their symbiotic relationship, instead of just considering whether the plant is infected by an endophyte or not, since endophytes from the same host species could exhibit high levels of genetic diversity, which is likely to influence the outcome of their symbiotic relationship.     

Effect of an Epichloë endophyte on adaptability to water stress in Festuca sinensis

As water stress, including drought and waterlogging, can severely affect plant growth, this study investigated the effects of an endophyte from the genus Epichloë on two different ecotypes of Festuca sinensis grass under five soil water conditions in a controlled greenhouse experiment. Changes in F. sinensis plants grown with (E+) and without the endophyte (E−) were evaluated as they were subjected to different water treatments (20%, 35%, 50%, 65% and 80% relative saturation moisture content, RSMC). Growth parameters such as plant height, number of tillers, blade width, stem diameter, root length, total biomass, root-shoot ratio and relative water content were determined. The results showed that drought and waterlogging significantly (P < 0.05) inhibited the growth of F. sinensis. The presence of the endophyte significantly (P < 0.05) increased plant growth and root-shoot ratio under drought and waterlogged conditions. In addition, the plant height, number of tillers, blade width, stem diameter and total biomass in seedlings of both ecotypes reached the maximum at 65% RSMC, which suggests the optimal water condition. These findings also show that moderate drought (35% and 50% RSMC) could promote root growth of grass seedlings. Therefore, endophytic infections can result in enhanced host plant resistance to drought and waterlogged conditions.     

Environmental factors and host genetic variation shape the fungal endophyte communities within needles of Scots pine (Pinus sylvestris)

To determine the role of environmental and host genetic factors in shaping fungal endophyte communities we used culturing and metabarcoding techniques to quantify fungal taxa within healthy Scots pine (Pinus sylvestris) needles in a 7-y old provenance-progeny trial replicated at three sites. Both methods revealed a community of ascomycete and basidiomycete taxa dominated by the needle pathogen Lophodermium seditiosum. Differences in fungal endophyte taxon composition and diversity indices were highly significant among trial sites. Within two sites, fungal endophyte communities varied significantly among provenances. Furthermore, the communities differed significantly among maternal families within provenances in 11/15 and 7/15 comparisons involving culture and metabarcoding data respectively. We conclude that both environmental and host genetic variation shape the fungal endophyte community of P. sylvestris needles.     

Metabolic profiles of Lolium perenne are differentially affected by nitrogen supply, carbohydrate content, and fungal endophyte infection

Lolium perenne cultivars differing in their capacity to accumulate water soluble carbohydrates (WSCs) were infected with three strains of fungal Neotyphodium lolii endophytes or left uninfected. The endophyte strains differed in their alkaloid profiles. Plants were grown at two different levels of nitrogen (N) supply in a controlled environment. Metabolic profiles of blades were analyzed using a variety of analytical methods. A total of 66 response variables were subjected to a principle components analysis and factor rotation. The first three rotated factors (46% of the total variance) were subsequently analyzed by analysis of variance. At high N supply nitrogenous compounds, organic acids and lipids were increased; WSCs, chlorogenic acid (CGA), and fibers were decreased. The high-sugar cultivar 'AberDove' had reduced levels of nitrate, most minor amino acids, sulfur, and fibers compared to the control cultivar 'Fennema', whereas WSCs, CGA, and methionine were increased. In plants infected with endophytes, nitrate, several amino acids, and, magnesium were decreased; WSCs, lipids, some organic acids, and CGA were increased. Regrowth of blades was stimulated at high N, and there was a significant endophyte x cultivar interaction on regrowth. Mannitol, a fungal specific sugar alcohol, was significantly correlated with fungal biomass. Our findings suggest that effects of endophytes on metabolic profiles of L. perenne can be considerable, depending on host plant characteristics and nutrient supply, and we propose that a shift in carbon/N ratios and in secondary metabolite production as seen in our study is likely to have impacts on herbivore responses.     

Competition between Lolium perenne and Digitaria sanguinalis: Ecological consequences for harbouring an endosymbiotic fungus

Abstract. This study examined the influence of the fungal endophyte Neotyphodium lolii on the competitive interactions between its perennial, cool season host, Lolium perenne (perennial ryegrass), and a warm season, annual grass, Digitaria sanguinalis (large crabgrass), in densely planted stands (>1000 plant.m^?2) in the glasshouse. Endophyte infection had little or no effect on L. perenne tiller production, above‐ or below‐ground biomass or root: shoot ratio in monoculture. However, endophyte infection significantly reduced L. perenne tiller production and above‐ground biomass in mixtures with D. sanguinalis. Conversely, D. sanguinalis had significantly higher above‐ground biomass and yielded more seed (g) when competing with endophyte infected L. perenne. An apparent trade‐off between allocation of resources to reproductive vs root tissues was observed in D. sanguinalis– root: shoot ratio was significantly lower when competing with endophyte infected L. perenne. Results indicate negative ecological consequences for harbouring the fungal endophyte when competing with the fast growing annual grass in newly established stands. These findings underscore the existence of a physiological cost of harbouring the fungal endophyte which is often overlooked.     

不同浓度和形态磷处理下内生真菌感染对高羊茅的影响

以感染内生真菌(endophyte-infected,EI)和不感染内生真菌(endophyte-free,EF)的高羊茅(Festuca arundinacea Schreb.)为材料,在温室沙培条件下研究内生真菌对高羊茅适应缺磷及利用不同形态磷肥的影响。结果表明,1)缺磷条件下,高羊茅EI和EF植株生长差异不显著;正常供磷条件下,高羊茅EI植株拥有更多分蘖数和绿叶数。说明正常供磷条件下内生真菌改善了宿主高羊茅的生长。2)与水溶性磷相比,高羊茅根有机酸和酸性磷酸酶(acid phosphatase,APase)活性在难溶性磷条件下显著增加,而根总酚含量无显著变化。在水溶性磷条件下,高羊茅EI植株根总酚含量显著高于EF植株,此时EI植株比EF植株拥有更多分蘖数和绿叶数,说明在水溶性磷条件下内生真菌对宿主地上部生长具有一定贡献。在难溶性磷条件下,虽然高羊茅EI植株根总酚含量仍然高于EF植株,但同时EI植株根有机酸含量显著低于EF植株,因此内生真菌感染只是增大了宿主植物的根冠比,而对分蘖数和绿叶数等无显著影响,说明内生真菌对宿主利用难溶性磷贡献不大。可见,内生真菌对宿主植物的生长在水溶性磷条件下更有利。     

Unraveling the role of fungal symbionts in plant abiotic stress tolerance

Fungal symbionts have been found to be associated with every plant studied in the natural ecosystem, where they colonize and reside entirely or partially in the internal tissues of their host plant. Fungal endophytes can express/form a range of different lifestyle/relationships with different host including symbiotic, mutualistic, commensalistic and parasitic in response to host genotype and environmental factors. In mutualistic association fungal endophyte can enhance growth, increase reproductive success and confer biotic and abiotic stress tolerance to its host plant. Since abiotic stress such as, drought, high soil salinity, heat, cold, oxidative stress and heavy metal toxicity is the common adverse environmental conditions that affect and limit crop productivity worldwide. It may be a promising alternative strategy to exploit fungal endophytes to overcome the limitations to crop production brought by abiotic stress. There is an increasing interest in developing the potential biotechnological applications of fungal endophytes for improving plant stress tolerance and sustainable production of food crops. Here we have described the fungal symbioses, fungal symbionts and their role in abiotic stress tolerance. A putative mechanism of stress tolerance by symbionts has also been covered.Key words: abiotic stress, endophytes, fungal symbiont, mycorrhizal fungus, Piriformospora indica, stress tolerance, symbiosis     

Family issues: fungal endophyte protects host grass from the closely related pathogen Claviceps purpurea

Certain cool season grasses establish systemic and asymptomatic symbioses with clavicipitaceous fungi of the genus Neotyphodium, which affect multiple biotic interactions within host neighborhood. The presumed symbiont-mediated plant resistance to pathogens is mostly based on studies performed under laboratory and greenhouse conditions. Here we investigated, in two outdoor experiments, the relation between two fungi of the same family with opposite effects on Lolium multiflorum plants: the mutualist endophyte Neotyphodium occultans, and the pathogen Claviceps purpurea. Natural infection and its consequences on symbiotic and non-symbiotic plants were studied under varying conditions of stress by herbicide. In both experiments, N. occultans reduced significantly the infection by C. purpurea at population levels (70 % less). The percentage of spikes infected by C. purpurea was almost three times lower in endophyte-symbiotic plants than in non-symbiotic ones. However, the protective effect was not maintained under stress condition. Our results show that constitutive symbionts such as the systemic fungal endophytes mediate the interaction between host grasses and pathogens, although the effect may depend on the level of stress in the environment.     

Host genotype overrides endophyte infection effects on growth,physiology, and nutrient content of a native grass,Achnatherum sibiricum

The effect of infection by the fungal endophyte Neotyphodium, host genotype, and their interaction on growth and physiology, as well as photosynthesis, was investigated in the native grass Achnatherum sibiricum. We artificially inoculated the endophyte into mature tillers of endophyte-free A. sibiricum. Plants were clipped to 5 cm height after recording growth traits, and analyzed for total nonstructural carbohydrates (TNC %), the percentage of nitrogen (N %), and carbon (C %) in leaves before and after clipping. In our study, the prominent host genotype–endophyte infection interactions detected in A. sibiricum indicates that, for many growth and storage traits, endophyte infection can impact a little change. However, there is no overriding consistently positive effect of the endophyte on growth or storage in A. sibiricum before or after clipping. Our study showed that the interaction between endophyte and host grasses was highly contingent on plant genotypes. We found host genotype overrode fungal endophyte infection in influencing tiller number and photosynthetic properties of A. sibiricum before clipping. After clipping, host genotype accounted for more of the variation in regrowth and above-ground biomass of A. sibiricum than endophyte infection. Our study showed that host genotype affected the total nonstructural carbohydrates of A. sibiricum before and after clipping, whereas endophyte infection increased the carbon content after clipping. Genotype by infection interactions for plant height, leaf mass, total nonstructural carbohydrates, and photosynthetic characteristics indicated genotype-specific effects of endophytes on A. sibiricum physiology and photosynthetic capacity. The host genotype–endophyte infection interactions detected in A. sibiricum suggest that host genotype overrides fungal endophyte infection on growth, physiology, and nutrient content of this native grass. In contrast, endophyte effects did not appear to positively affect growth, physiology, or photosynthetic capacity before or after clipping.     

Plant-endophyte-herbivore interactions: More than just alkaloids?

A recent paper by Rasmussen et al., (New Phytol 2007; 173:787–97) describes the interactions between Lolium perenne cultivars with contrasting carbohydrate content and the symbiotic fungal endophyte Neotyphodium lolii at different levels of nitrogen supply. In a subsequent study undertaken by Rasmussen et al., (Plant Physiol 2008; 146:1440–53) 66 metabolic variables were analysed in the same material, revealing widespread effects of endophyte infection, N supply and cultivar carbohydrate content on both primary and secondary metabolites. Here, we link insect numerical responses to these metabolic responses using multiple regression analysis.Key words: Neotyphodium lolii, Lolium perenne, high sugar grasses, metabolomics, insect herbivoresPasture grasses are often infected with symbiotic fungal endophytes and benefits for host plants arising out of these associations are generally ascribed to endophyte produced anti-herbivorous alkaloids. We tested the effects of (i) infection with three strains of endophytes differing in their alkaloid profiles, (ii) high vs. low nitrogen (N) supply, and (iii) ryegrass cultivars with high vs. control levels of water soluble carbohydrates (WSCs) on numerical insect responses (aphids, thrips, mites). A difference in WSC content between the cultivars had no significant effect on insect numbers, whereas high N compared to low N supply increased mites, thrips and alate Rhopalosiphum spp., but decreased apterous Rhopalosiphum spp. The effect of endophyte infection was strain dependant and differed for the different insects.A total of 66 metabolic variables of the same plants analysed prior to insect treatment were linked to insect responses using multiple regression analysis. One of the major conclusions to be drawn is that alkaloids are not always the most important factor influencing numerical insect responses which will also be determined by other metabolites, clearly indicating the importance of metabolomics type studies to point the way toward a mechanistic explanation of grass-endophyte-herbivore interactions.Grass species are often hosts of symbiotic clavicipitaceous endophytic fungi¹ residing in the apoplastic spaces of above ground plant parts and usually not causing any visible symptoms of infection.^2–4 These fungal symbionts confer protection from insect herbivory to their host plants through alkaloids,^5–8 some of which (ergovaline, lolitrem B) are also toxic to grazing mammals.^9,10 Natural endophyte strains lacking these mammalian toxins, but still retaining at least some of their insect deterring features, have been commercialized and are now widely used in ryegrass and tall fescue based pastures.^11,12     

Consequences of simultaneous interactions of fungal endophytes and arbuscular mycorrhizal fungi with a shared host grass

Plants simultaneously associate with multiple microbial symbionts throughout their lifetimes. To address the question of whether the effects of simultaneous symbionts are contingent on the specific identities, we conducted a greenhouse experiment manipulating the presence and identities of arbuscular mycorrhizal fungi (AMF) and fungal endophytes on the shared host grass Elymus hystrix. Each plant host was inoculated with one of two AMF species having varying effects on host growth, or a sterile soil control. Further, we used naturally occurring endophyte‐infected (E+) and uninfected (E–) individuals from two populations of the endophyte Epichloë elymi that varied in their interaction with E. hystrix. We then measured responses of plants, AMF, and fungal endophytes. Overall, we found that the combined effects of AMF and fungal endophytes on plant growth were additive, reflecting the mutualistic quality of each symbiont independently interacting with host plants. However, fungal endophyte infection differentially altered hyphal colonization of the two AMF species and the identity of the coinfecting AMF species affected fungal endophyte fitness traits. The results of this study demonstrate that the outcome of interspecific symbiotic interactions varies with partner identity such that the effects of simultaneous symbioses can not be generalized.     

High nitrogen supply and carbohydrate content reduce fungal endophyte and alkaloid concentration in Lolium perenne

The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. Here, we infected two perennial ryegrass (Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.