Endophytes Of Grasses Research Articles

Anti-herbivore factors of grass endophytes: new prospects from metabolomics

A defensive role for several of the major alkaloids of Neotyphodium spp. endophytes in forage grasses has been confirmed in genetic experiments. However, many aspects of the activity of endophyte-infected grasses towards herbivores remain unaccounted for, suggesting there are further anti-herbivore factors yet to be uncovered. The development of metabolomics has provided new tools for the discovery of novel metabolites without preconceptions of the likely chemical structural class. Prospects for the application of new methods from metabolomics to further elucidate the defensive chemistry of associations of Neotyphodium spp. endophytes with their grass hosts are reviewed. Keywords: Neotyphodium, Epichloë, defensive chemistry, invertebrate, vertebrate, gene deletion

Targeted introduction of point mutations in Epichloe festucae

AgResearch is a world leader in the commercial utilisation of endophytes in forage grasses. These fungal endophytes have a symbiotic relationship with their host grass and and have traditionally been an agricultural double edged sword. On one hand, they increase plant yield, provide resistance to abiotic stress, and produce insecticidal secondary metabolites. However, they also produce toxic mammalian alkaloids that seriously affect the health of the grazing livestock. AgResearch has identified and commercialised endophyte strains (AR1, AR37 and MaxQ/ MaxP) that lack the capacity to produce two of the main classes of toxic mammalian alkaloids. In addition to the successful commercial strains, AgResearch has characterised a number of strains with potentially beneficial traits that were not placed into a commercial development pipeline because they would lead to negative effects on livestock (e.g. lolitremB producers). As part of our strategy for developing the next generation of endophytes, we are looking for strains with novel activities against some of the insect pests that are not deterred by the current commercial strains. It is likely that some of these strains will be lolitrem B or ergovaline producers and therefore some means of eliminating these pathways is desirable.

Biosynthesis of insecticidal loline alkaloids by epichloe endophytes of grasses

Epichloë endophytes are protective mutualists of cool-season grasses.

Fungal grass endophytes, grass cultivars, nitrogen deposition and the association with colonising insects

Fungal endophytes associated with pasture grasses can have community-wide effects on insect consumers. Here we asked the question to what degree endophyte infection, simulated nitrogen deposition and grass cultivar influence the abundance of colonising herbivores and their natural enemies. In a fully randomised field experiment, consisting of four Lolium perenne monocultures of known endophyte infection status and a nitrogen addition treatment, we determined the abundance of colonising aphids, their parasitoids and predators, and other grass herbivores. The three colonising cereal aphid species did not respond to endophyte infection, possibly because peramine concentrations were relatively low (3.9 μg/g). There was a significant interaction between nitrogen addition and plant cultivar on the abundance of Sitobion avenae, suggesting a cultivar-specific response to nitrogen addition. Aphid predators were affected by an interaction between endophyte and plant cultivar, but abundance of aphid parasitoids and other grass herbivores was not affected by any treatment. The fungus Claviceps purpurea naturally infected our experimental plants and infection rates differed among cultivars and were more likely to occur on endophyte-infected plants, in particular on wild-type Samson. We conclude that strong effects of endophytes on insect abundance may not occur in systems built upon L. perenne because overall peramine levels rarely reach threshold levels for insect toxicity. Keywords: fungal endosymbionts, multitrophic interactions, field experiment, insect food webs, alkaloids

Occurence and genetic structure of Epichloe festucae endophytes in wild red fescue populations and their seed progenies

In the systemic grass endophyte associations only one fungal genotype is transmitted vertically to seed progeny, promoting stable interactions between the fungal genotype and the host lineage.

Occurrence and Genetic Structure of the Systemic Grass Endophyte Epichloë festucae in Fine Fescue Populations

Epichloë species are systemic fungal endophytes that usually specialize in a certain group of related grass species. We examined the infection frequency of Epichloë festucae in populations of two fine fescue species (Festuca rubra and F. ovina) in natural and seminatural habitats at 86 study sites (total=2514 plants) across Finland and northern Norway. Infection incidence varied significantly among grass species and populations. A substantial number of the F. rubra and F. ovina populations (53 out of 77 and 25 out of 30, respectively) were either endophyte-free or had very low (<20%) infection frequencies. The highest infection frequencies were found in subarctic areas. Moreover, infection incidence differed between habitats. In the area with the highest infection frequencies, we used microsatellite markers to study genetic diversity and the rates of gene flow of E. festucae among 12 F. rubra populations. Twenty out of the 25 fungal genotypes detected with four microsatellite markers were carrying multiple alleles in at least one locus, indicating multiple infections or vegetative hybridization of the fungus. One dominant genotype occurred in all 12 populations, representing 63.5% of all isolates. We found a moderate level of average genotypic variation and a low level of genetic differentiation (Fst=0.0814). There was no correlation between infection frequency and genotypic diversity. Although the existence of a dominant genotype and the detected linkage disequilibrium suggest that the fungus is mainly asexual and vertically transmitted, the multiallelic loci and variation of genetic diversity among populations indicate occasional contagious spread and sexual or parasexual recombination of the fungus in some populations. Furthermore, the genotypes carrying multiallelic loci suggest the possibility of multiple infections or hybridization of the endophyte.

Occurrence of endophytes in grasses native to northern China

AbstractA survey of grass species associated with Neotyphodium and Epichloë fungal endophytes was made in the permanent grasslands of northern China. A total of 41 grass species, 56 sites and 172 local populations were examined to reveal endophytic infection of native grasses. Twenty‐five of the forty‐one species of grasses (proportionately 0·61) were infected by endophytes: twenty‐two species had a mean infection rate of 0·01–0·50 and only three species had a mean infection &gt;0·50. Of the 172 local populations examined, however, proportionately 0·72 were not infected, 0·181 had an infection rate of 0·01–0·50 and 0·099 had an infection rate of 0·51–1·00. Two genera of grasses, Cleistogenes and Koeleria, as well as twenty plant species, were previously unknown endophytic hosts in China. High infection rates (0·86–1·00) of plants of Achnatherum sibiricum were detected at all eleven sites studied. Other grasses in which high infection rates were found were Agropyron mongolicum, Roegneria turczaninovii, Agropyron elongate, Poa palustris and Poaangustifolia. It was concluded that endophytes are found widely in natural grass populations and that endophyte–grass interactions may have effects on ecosystems and livestock in the grasslands of northern China.

Costs of fungal endophyte infection in Lolium perenne genotypes from Eurasia and North Africa under extreme resource limitation

The effects of asexual, leaf-inhabiting endophytes on the growth of Lolium perenne are notoriously inconsistent and appear to be contingent on environmental conditions and host genetics. Both positive and negative growth effects of endophytes have been documented in some forage crop cultivars. Because they utilize host photosynthates, it is hypothesized that endophytes would impart a growth cost to infected plants under extreme resource limitation. The potential costs of infection by the fungal endophyte Neotyphodium lolii to its host grass, L. perenne, were investigated in accessions from Italy, Turkey, Morocco and Tunisia, the native part of the host's range. Four to five infected (+E) genotypes of L. perenne per accession were cloned into ramets, and the endophyte exterminated from half of the ramets (−E). Six +E and six −E ramets per genotype were grown for 5 months in a nutrient-poor substrate (vermiculite) within temperature- and light-controlled growth chambers. Accession and genotype within accession affected dry mass. Endophyte infection significantly reduced root:shoot ratio and the proportion of the shoot that was alive and photosynthetic. Relative interaction intensity indexes were negative for most genotypes, indicating host–endophyte associations were mostly parasitic. However, there were significant infection by genotype interactions; thus, endophyte-mediated effects depend on particular host genotype–endophyte combinations. This research is one of the few that have documented costs to endophyte infection in a grass–endophyte symbiosis. This cost, manifested as reduced root:shoot ratio and photosynthetic shoot fraction, was evident for infected L. perenne growing under conditions of extremely poor substrate nutrient availability.

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.

Model systems in ecology: dissecting the endophyte–grass literature

Model systems can facilitate and focus research efforts but ill-chosen or inapt ones can distract or impede scientific progress. In this Opinion article, we pose the question: how can the literature provide appropriate general conclusions if the model systems upon which the literature is based are unrepresentative of the relevant biological diversity? A good example of this problem is the endophyte-grass symbiosis, which is considered to be a classic example of mutualistic interactions. Meta-analysis of the primary literature demonstrates that the conceptual framework for endophyte-grass interactions has largely been based on endophyte-plant-herbivore studies of two agricultural grass species, tall fescue and perennial ryegrass. Consistent with conventional wisdom, the meta-analysis indicates that endophytes slightly increase grass resistance to herbivores. By contrast, endophytes appear not to affect plant performance or competitive ability. The positive effects of endophytes appear to be dependent on genetic variation in the host and endophyte, and on nutrient availability in soils. Thus, the agronomic grass model systems fail to capture the breadth of variability inherent in wild grass-endophyte populations and communities.

Rapid detection of fungal endophytes in grasses for large‐scale studies

Summary Standard visual screening methods for determining the qualitative and quantitative presence of fungal endophytes are too time‐consuming for large‐scale ecological studies. We investigated whether commercially available immunoblot kits, using monoclonal antibody techniques and designed for rapid‐screening of the presence of Neotyphodium endophytes in fresh samples of the pasture grasses Festuca arundinacea and Lolium perenne, could be used for Neotyphodium detection using other grasses and preserved samples. We also determined whether immunoblot kits could provide quantitative information about the amount of Neotyphodium in the grass. The kits accurately detected endophyte presence in F. rubra, F. ovina, F. pratensis and F. altaica, in both preserved samples (dried and fixed), including 12‐year‐old stored, dried samples of F. rubra. Endophytes were detected in 7‐day‐old seedlings of Lolium perenne, 3 days (30%) earlier than previously recognized. The intensity of the coloured tissue prints on scanned immunoblot cards was significantly positively correlated with hyphal density, demonstrating a previously unrecognized accurate quantitative application. These findings greatly reduce logistical barriers to large‐scale field research into the broader ecological significance of Neotyphodium in temperate and arctic grasses in non‐agricultural ecosystems (particularly in remote areas) and suggest potential for estimating historical infection rates using stored and herbarium specimens.

A new species of Epichloë symbiotic with Chinese grasses

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of β-tubulin gene (tub2) introns and translation elongation factor 1-α gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.

A new species of Epichloe symbiotic with Chinese grasses

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.

Susceptibility of endophyte-infected grasses to winter pathogens (snow molds)

Neotyphodium endophytes are suggested to be mutualistic symbionts of grasses and regarded as potential biological plant protection agents. We examined the effects of the Neotyphodium endophyte of meadow ryegrass on snow molds in vitro with dual cultures of endophyte and Typhula ishikariensis , and on grass–snow mold interactions in vivo in a greenhouse and in a field experiment. In dual cultures, the endophytes formed an inhibition zone and retarded the growth of T. ishikariensis. In the field experiment, however, the endophyte-infected (E+) meadow ryegrasses were more susceptible to T. ishikariensis than the endophyte-free (E–) grasses. Endophyte infection increased the winter damage of grasses both in greenhouse and in field conditions. After winter, the growth of E+ grasses exceeded the growth of E– plants in the field experiment, indicating the marked tolerance of E+ grasses against winter damage. We detected differences in growth and pathogenesis between the different T. ishikariensis strains and found meadow ryegrass cultivar–endophyte status interaction in the growth of meadow ryegrasses, which highlight the effects of the genetic background of the participants on endophyte–grass–snow mold interactions.

Recent Molecular and Genomic Studies on Stress Tolerance of Forage and Turf Grasses

Improvement in stress tolerance of forage and turf grasses is a major breeding goal. Most forage and some turf grasses are grown on marginal lands under stressful environments with minimal inputs. In contrast, current high‐input turf grass production systems such as golf courses and lawns are expensive and often environmentally unfriendly. Cultivars with improved stress tolerance are necessary for the development of sustainable and environmentally friendly production systems. Until recently, decades of breeding and selection have resulted in limited improvements of stress tolerance of forage and turf grass species. Recent developments in molecular and genomic sciences suggest new methods to improve stress tolerance in many plants, but compared to major crop plants (e.g., rice [Oryza sativa L.], wheat [Triticum spp.], and maize [Zea mays L.]), the development of molecular and genomic resources for forage and turf grasses has been limited. In this review, we present an overview of recent molecular and genomic studies aimed at improving stress tolerance of forage and turf grasses, including endophyte grass interactions. Important molecular and genomic resources are now available for some forage and turf grasses, including ryegrasses (Lolium spp.) and fescues (Festuca spp.). Noteworthy progress is being made in improvements of both biotic and abiotic stress tolerances of these grasses, but the challenge is to simplify and streamline the molecular tools and new discoveries for cost‐effective and efficient application in forage and turf grass breeding. Stress tolerances of many forage and turf grasses are influenced by their mutualistic association with Neotyphodium spp. endophytes, and this area of research is discussed.

GENETIC DIVERSITY OF NEOTYPHODIUM ENDOPHYTES ISOLATED FROM &amp;lt;EM&amp;gt;ACHNATHERUM SIBIRICUM&amp;lt;/EM&amp;gt; POPULATIONS IN MID_ AND EASTERN INNER MONGOLIA STEPPE, CHINA

Background and Aims The fungal endophytes in grasses grow intercellularly and systemically in aboveground plant tissues. Vertically transmitted asexual endophytes (Neotyphodium) forming asymptomatic infections of cool-season grasses have been repeatedly derived from sexual species (Epichlo) that abort host inflorescences. The phylogenetic distribution of seed-transmitted endophytes is strongly suggestive of cocladogenesis with their hosts. Endophytes are considered plant mutualists: they receive nutrition and protection from the host plant while the host plant may benefit from enhanced competitive abilities and increased resistance to herbivores, pathogens, and various abiotic stresses. Most studies of grass-endophyte associations have been undertaken with two economically important plant species, Lolium perenne and Festuca arundinacea. The interactions between endophytes and other grasses in natural plant communities, however, are seldom concerned. Much less attention has been paid to native grasses in Inner Mongolia Steppe, which lies in the north of China and is an important part of Steppe in Eurasia, and only limited published symbiosis studies are available in this natural ecosystem. Thereby, a more comprehensive view of the ecology, diversity of endophytes and their interactions with host plants becomes essential. Methods Our research of grass (Achnatherum sibiricum) associated with Neotyphodium endophyte (a new grass-endophyte association) was carried out in the Inner Mongolia Steppe, China. Twenty-seven endophyte strains were isolated from four A. sibiricum populations including Hulingol (H), Xi Ujimqin Qi (X), Inner Mongolia Grassland Ecosystem Research Station of the Chinese Academy of Sciences (IMGERS-CAS) (I), and the Leymus chinensis plot of the IMGERS-CAS (L), respectively. Colony morphology, growth rate on potato dextrose agar (PDA) and conidial measurements of isolates were used to characterize diversity of endophytes hosted by A. sibiricum. The genetic polymorphisms of Neotyphodium species from the four A. sibiricum populations were also analyzed using random amplified polymorphic DNA (RAPD) markers. Key Results Four morphological groups of Neotyphodium species and five individual morphological types were described based on the colony morphology, growth rate, and conidial shape and size. A total of 463 bands were obtained using 20 RAPD primers, of which 461 bands were polymorphic. A total of 93 unique loci were obtained, accounting for 20.1% of the total amplified loci. Nei's diversity index (h) and Shannon's diversity index (I) were 0.238 and 0.387, respectively. In the four populations (H , X , I, and L), the value of h was 0.122 0, 0.180 4, 0.146 3 and 0.210 7, respectively. The four populations may be ranked in a descending order as L X I H, and the I of the four populations had a similar trend. The fixation index (F ST ), the coefficient of gene differentiation (G ST ), and the genetic differentiation (Hsp-Hpop)/Hsp (estimated by Shannon's diversity index) were 0.205 1, 0.300 4, and 0.355 0, respectively. The genetic variation of Neotyphodium species mainly existed within populations. Genetic similarities within each population were found to be in the range of 0.647 9-0.943 8 in similarity, and the average genetic distance among the four populations was 0.285 8, varying from 0.057 8 to 0.433 9. Clustering analysis based on Nei's unbiased genetic distance and UPGMA method showed that there were seven distinct RAPD genetic groups. Conclusions The high incidence of endophytic infection in A. sibiricum was indicative that the presence of the endophyte gave the grass a competitive advantage over non-infected grasses. The study also indicated that there were higher genetic diversity based on morphological and RAPD results, and that there were relatively high genetic variations among the four populations and higher genetic differentiation of Neotyphodium sp. within a geographical region. Most of the genetic groups were related to geographical origins of the strains. The dendrogram of genetic distance matched rather well with the morphological data, as reflected by the four morphotypes.

Transfer of endophyte‐origin defensive alkaloids from a grass to a hemiparasitic plant

AbstractPlants growing in natural environments experience myriad interactions with a diverse assemblage of pathogens, parasites and mutualists. Many of these interactions involve symbiotic bacteria and fungi, but they also include macroparasitic plants. In this study, we investigated the interactions among a host grass (Lolium pratense, ex., Festuca pratensis), its symbiotic endophytic fungus (Neotyphodium uncinatum), a root hemiparasitic plant (Rhinanthus serotinus) of the host grass and a generalist herbivore (aphid Aulacorthum solani) of the hemiparasite. We demonstrate that the hemiparasitic plant acquires defending mycotoxins produced by the endophytic fungus living within their shared host grass. The uptake of defensive mycotoxins from the endophyte‐infected host grass enhances the resistance of the hemiparasitic plant to the generalist aphid herbivore. Endophyte infection increases the performance of the hemiparasitic plant, but reduces the growth of the host grass. In other words, the mutualistic endophytic fungus becomes parasitic in the presence of the hemiparasitic plant. Our results suggest that the outcomes of grass–endophyte interactions are conditional on the complexity of community‐level interactions; thus, the outcome of multispecies interactions may not be predictable from pair‐wise combinations of species.

Reduced herbivore resistance from a novel grass–endophyte association

Summary Endophytic fungi in agricultural grasses confer agronomic advantages but can be detrimental to the health and production of grazing mammals. Attempts have been made to resolve this dilemma by creating novel associations of high‐yielding grass cultivars and ‘safe endophytes’ that do not naturally co‐occur. Recently, researchers created a novel association by combining a Moroccan strain of Neotyphodium coenophialum, called AR542, with the tall fescue Festuca arundinacea cultivar Georgia 5. This novel association is marketed commercially and has no doubt become naturalized; it is therefore important to assess its impact on other organisms. The bird‐cherry‐oat aphid Rhopalosiphum padi uses tall fescue as an alternative host. We assessed the performance of R. padi feeding on Georgia 5 that had been artificially infected with AR542, naturally infected by the common strain (CS) of N. coenophialum or was endophyte‐free (EF). We cross‐factored these three levels of endophyte infection with four levels of nitrogen fertilizer, and used two different methods of assessing aphid performance, clip‐cages and enclosed populations. Endophyte‐infected plants produced approximately 20% more dry mass than EF plants. In both the clip‐cage and enclosed‐population experiments, aphid populations grew fastest on the EF plants, slower on AR542‐infected plants and slowest (or not at all) on the CS‐infected plants. Synthesis and applications. Our results suggest that AR542 infection does not provide the same degree of protection from aphids as the commonly occurring CS infection. If similar results are found for other invertebrate herbivores, then the use of this novel endophyte association may be less useful than previously thought. The widespread conversion of tall fescue pastures, currently infected with the CS endophyte, to pastures infected with AR542 could result in a general increase in the prevalence of cereal aphids and, potentially, the diseases that they spread. However, such conversion might be beneficial to native invertebrate herbivores.

Whole genome amplification from filamentous fungi using Phi29-mediated multiple displacement amplification

The availability of genomic DNA of sufficient quality and quantity is fundamental to molecular genetic analysis. Many filamentous fungi are slow growing or even unculturable and current DNA isolation methods are often unsatisfactory. We have used multiple displacement amplification (MDA) to amplify whole genomes for two fungal species, Penicillium paxilli and the slow growing endophyte of grasses Epichloë festucae. Up to 10 μg of high molecular weight DNA was routinely amplified from less than 10 ng of template DNA obtained using glass bead-mediated disruption of fungal spores or alkaline lysis of mycelium. PCR was possible from MDA-generated DNA and amplicons up to 10 kb were successfully amplified. RFLP analysis was successful, with bands of up to 5 kb routinely detected. Hybridization of MDA-amplified DNA to a cosmid library illustrated that the MDA product amplified from E. festucae is representative of the genome. MDA is a reliable method that could be applied to applications ranging from high-throughput screening of deletion mutants to genomic library construction.

Origin, divergence, and phylogeny of epichloë endophytes of native Argentine grasses

The epichloë endophytes are systemic, constitutive, and often vertically transmitted fungal symbionts of grass species in subfamily Poöideae. Prior studies indicate that several asexual epichloë endophytes ( Neotyphodium species) have evolved directly from sexual ( Epichloë) species, whereas others evolved by hybridization between two or more endophyte species. In this paper, we investigate the phylogenies of 27 Neotyphodium spp. isolates from 10 native grass species (in 4 tribes) in 22 populations throughout Argentina. Relationships among these fungi and a worldwide collection of epichloë endophytes were estimated by phylogenetic analysis of sequences from variable portions (mainly introns) of genes for β-tubulin ( tub2) and translation elongation factor 1-α ( tef1). Most of the Argentine endophyte isolates were interspecific hybrids of Epichloë festucae and E. typhina. Only one isolate was a hybrid of a different ancestry, and three isolates were apparently non-hybrid endophytes. These results indicate that interspecific hybridization, which promotes genetic variation, was common during the evolution of the endophytes of Argentine grasses.