Molecular biology and evolution of the grass endophytes.

Acremonium coenophialum Morgan-Jones et W. Gams is a maternally transmitted fungal symbiont (endophyte) of the important forage grass Festuca arundinacea Schreb. (tall fescue), and provides biological protection and enhanced fitness to its host, but its anti-mammalian ergot alkaloids detract from the usefulness of tall fescue as forage for livestock. Molecular genetic techniques and materials are being developed in order to specifically eliminate the gene(s) encoding the first enzyme in ergot alkaloid biosynthesis. These techniques will also facilitate basic studies, such as host-fungus compatibility or biosynthesis of insecticidal alkaloids. Molecular phylogenetics indicate that endophytes related to A. coenophialum have evolved on multiple occasions from strains of Epichlo? typhina (Ascomycotina, Clavicipitaceae), for which the sexual cycle is known. These studies also reveal significant diversity among seedborne endophytes in individual grass species. Thus, the endophytes are an important source of biochemical potential and genetic diversity in grass-fungus symbiota.     

Structural analysis of a peptide synthetase gene required for ergopeptine production in the endophytic fungus Neotyphodium lolii.

Lysergyl peptide synthetase 1 catalyzes the assembly of toxic ergopeptines from activated D-lysergic acid and three amino acids. The gene encoding this enzyme in the endophytic fungus Neotyphodium lolii was analyzed and compared to a homologous gene from the ergot fungus Claviceps purpurea. Each gene contained two introns, which were found in the same relative position within two modules of the gene. The 5' ends of the two genes were unusually divergent. Signature sequences determining substrate specificity were similar in adenylation domains that recognized identical amino acids but differed within the adenylation domain for the amino acid that varies between the major ergopeptines of the two fungi. Homologues were detected in several related endophytic fungi; the tall fescue endophyte Neotyphodium coenophialum contained a divergent, second copy of the gene. Our results provide new information on the structure and distribution of this important peptide synthetase involved in ergot alkaloid biosynthesis.     

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichlo? isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichlo? species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.     

Molecular characterisation and interpretation of genetic diversity within globally distributed germplasm collections of tall fescue (Festuca arundinacea Schreb.) and meadow fescue (F. pratensis Huds.)

Allohexaploid tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum [Schreb.] Darbysh.) is an agriculturally important grass cultivated for pasture and turf world-wide. Genetic improvement of tall fescue could benefit from the use of non-domesticated germplasm to diversify breeding populations through the incorporation of novel and superior allele content. However, such potential germplasm must first be characterised, as three major morphotypes (Continental, Mediterranean and rhizomatous) with varying degrees of hybrid interfertility are commonly described within this species. As hexaploid tall fescue is also a member of a polyploid species complex that contains tetraploid, octoploid and decaploid taxa, it is also possible that germplasm collections may have inadvertently sampled some of these sub-species. In this study, 1,040 accessions from the publicly available United States Department of Agriculture tall fescue and meadow fescue germplasm collections were investigated. Sequence of the chloroplast genome-located matK gene and the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) permitted attribution of accessions to the three previously known morphotypes and also revealed the presence of tall fescue sub-species of varying ploidy levels, as well as other closely related species. The majority of accessions were, however, identified as Continental hexaploid tall fescue. Analysis using 34 simple sequence repeat markers was able to further investigate the level of genetic diversity within each hexaploid tall fescue morphotype group. At least two genetically distinct sub-groups of Continental hexaploid tall fescue were identified which are probably associated with palaeogeographic range expansion of this morphotype. This work has comprehensively characterised a large and complex germplasm collection and has identified genetically diverse accessions which may potentially contribute valuable alleles at agronomic loci for tall fescue cultivar improvement programs.     

Symbiont-mediated changes in Lolium arundinaceum inducible defenses: evidence from changes in gene expression and leaf composition

Plants have multiple strategies to deal with herbivory, ranging from chemical or physical defenses to tolerating damage and allocating resources for regrowth. Grasses usually tolerate herbivory, but for some cool-season grasses, their strategy may depend upon their interactions with intracellular symbionts. Neotyphodium endophytes are common symbionts in pooid grasses, and, for some host species, they provide chemical defenses against both vertebrate and invertebrate herbivores. Here, it was tested whether defenses provided by Neotyphodium coenophialum in Lolium arundinaceum (tall fescue) are inducible by both mechanical damage and herbivory from an invertebrate herbivore, Spodoptera frugiperda (fall armyworm), via a bioassay and by quantifying mRNA expression for lolC, a gene required for loline biosysnthesis. Both mechanical and herbivore damage had a negative effect on the reproduction of a subsequent herbivore, Rhopalosiphum padi (bird cherry-oat aphid), and herbivore damage caused an up-regulation of lolC. Uninfected grass hosts also had significantly higher foliar N% and lower C:N ratio compared with infected hosts, suggesting greater allocation to growth rather than defense. For L. arundinaceum, N. coenophialum appears to switch its host's defensive strategy from tolerance via compensation to resistance.     

The effect of feeding tall fescue seed infected by Acremonium coenophialum on pregnancy and parturition in female rats

1. Female Sprague-Dawley rats (Rattus norvegicus) were randomly assigned to various dietary treatments containing: (1) 100% Purina rodent chow, ad libitum; (2) same as 1, but restricted to daily intake of 7; (3) 50% rodent chow (w/w) and 50% endophyte-free tall fescue (Festuca arundinacea) seed; (4) same as 3, but restricted to intake of 5; (5) 50% rodent chow, 25% endophyte-free tall fescue seed and 25% endophyte-infected (Acremonium coenophialum) tall fescue seed; (6) 50% rodent chow, 12.5% endophyte-free and 37.5% endophyte-infected tall fescue seed; and (7) 50% rodent chow and 50% endophyte infected tall fescue seed. 2. Average daily feed intakes and average daily weight gains decreased with higher levels of endophyte infected seed. 3. Frequency of litter production was affected by all endophyte-infected containing diets. 4. Conception was reduced only in dietary treatment (7). 5. Litter weights, number of pups per litter and weight per pup were proportionally reduced as higher levels of infected seed were incorporated in the ingested diets.     

Variation in Nitrogen Utilization in Acremonium coenophialum Isolates

Acremonium coenophialum, a fungal endosymbiont in tall fescue, is responsible for the production of alkaloid toxins. Animals grazing endophyte-infected tall fescue often show toxicosis. In marginal environments, the endophyte is important for long-term survival of tall fescue. Few differences in endophyte isolates from individual tall fescue plants have been reported. To aid development of a toxicosis-free tall fescue, it is important to identify differences in endophyte isolates. This report describes variation in nitrogen utilization in a defined culture medium by endophyte isolates from Kentucky-31 tall fescue. Overall, the best nitrogen sources for dry weight production of mycelium were proline and potassium nitrate. Thirty-four isolates grown on agar-solidified defined media with single nitrogen sources showed variation in nitrogen utilization. Fifty percent of the isolates were unable to utilize two or more amino acids. Manipulation of endophyte variation could lead to development of a toxicosis-free tall fescue cultivar.     

Influence of phosphorus on the growth and ergot alkaloid content of Neotyphodium coenophialum-infected tall fescue (Festuca arundinacea Schreb.)

Tall fescue (Festuca arundinacea Schreb.) plants infected by the fungal endophyte Neotyphodium coenophialum (Morgan-Jones & Gams) (Glenn et al., 1996) often perform better than noninfected plants, especially in marginal resource environments. There is a lack of information about endophyte related effects on the rhizosphere of grasses. In a greenhouse experiment, four endophyte-infected (E+) tall fescue clones (DN2, DN4, DN7, DN11) and their endophyte-free (E–) forms were grown in limed (pH 6.3) Porter soil (low fertility, acidic, high aluminum and low phosphorus content, coarse-loamy mixed mesic Umbric Dystrochrept) at three soil P levels (17, 50, and 96 mg P kg^-1 soil) for five months. Excluding the genotype effect, endophyte infection significantly increased cumulative herbage DM yield by 8% at 17 mg P kg^-1 soil but reduced cumulative herbage DM yield by 12% at 96 mg P kg^-1 soil. With increased P availability in the soil, shoot and root DM, and root/shoot ratio in E+ plants were significantly less when compared to E– plants. Endophyte infection increased specific root length at 17 and 50 mg P kg^-1soil. At soil P level of 17 mg P kg^-1soil, E+ plants had significantly higher P concentrations both in roots and shoots. Similar relationships were found for Mg and Ca. E+ plants had significantly higher Zn, Fe, and Al concentration in roots, and lower Mn and Al concentration in shoots when compared to E– plants. Ergot alkaloid concentration and content in shoot of E+ plants increased with increasing P availability in the soil from 17 to 50 mg P kg^-1 but declined again at 96 mg P kg^-1 soil. Ergot alkaloid accumulation in roots increased linearly with P availability in the soil. Results suggest that endophyte infection affects uptake of phosphorus and other mineral nutrients and may benefit tall fescue grown on P-deficient soils. Phosphorus seems also to be involved in ergot alkaloid accumulation in endophyte-infected tall fescue.     

内生真菌对草坪植物病原真菌抑制作用的比较

分别从野生牧草羽茅(Achnatherum sibiricum (L.) Keng)、栽培种高羊茅(Festuca arundinacea Schreb.)(品种Millennium)、栽培种黑麦草(Lolium perenne L.) (品种Justus)中分离出内生真菌Neotyphodium sp.、N. coenophialum和N. lolli,通过体外培养法比较了这3种内生真菌对草坪植物病原真菌的抑制作用.结果表明,从羽茅中分离的内生真菌Neotyphodium sp.在两菌相交前对所有供试的病原真菌都有一定的抑制作用,其中对枝孢霉属(Cladosporium sp.)、弯孢霉属(Curvularia sp.)和拟茎点属(Phomopsis sp.)病原真菌的抑制效果尤为显著,对峙培养3d后的抑菌率分别达70.1%、52.3%和30.9%,营养竞争作用、重寄生作用是其主要的拮抗机制;从高羊茅中分离的内生真菌N. coenophialum对枝孢霉属病原真菌存在一定的抑制作用;而从黑麦草中分离的内生真菌N. lolli与病原真菌对峙培养时,病原真菌菌落慢慢侵占整个营养空间,内生真菌停止生长并逐渐褐变死亡.体外培养结果说明Neotyphodium sp.对供试病原真菌的拮抗效果优于N. coenophialum和N. lolli,由此推测Neotyphodium sp.与宿主植物羽茅的共生可能有利于宿主植物抵抗病原真菌的侵扰.     

高羊茅和多年生黑麦草内生真菌的分子检测

根据GenBank上报道的内生真菌Neotyphodium coenophialum和N.lolii的Nc25基因序列,设计了2对特异性引物FM1/R1和F3/R652,建立了一套适合于从高羊茅和多年生黑麦草种子中检测内生真菌N.coenophialum和N.lolii的常规PCR和巢式PCR方法。     

A complex ergovaline gene cluster in epichloe endophytes of grasses

Clavicipitaceous fungal endophytes of the genera Epichlo? and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.     

Endophyte isolate and host grass effects on Chaetocnema pulicaria (Coleoptera: Chrysomelidae) feeding

Endophytic fungi belonging to the genus Neotyphodium, confer resistance to infected host grasses against insect pests. The effect of host species, and endophtye species and strain, on feeding and survival of the corn flea beetle, Chaetocnema pulicaria Melsheimer (Coleoptera: Chrysomelidae) was investigated. The grass-endophyte associations included natural and artificially derived associations producing varying arrays of common endophyte-related alkaloids or alkaloid groups, peramine, lolitrem B, ergovaline, and the lolines. Preference and nonpreference tests showed that C. pulicaria feeding and survival were reduced by infection of tall fescue with the wild-type strain of N. coenophialum, the likely mechanism being antixenosis rather than antibiosis. In the preference tests, endophyte and host species effects were observed. Of the 10 different Neotyphodium strains tested in artificially derived tall fescue associations, eight strongly deterred feeding by C. pulicaria, whereas the remaining two strains had little or no effect on feeding. Infection of tall fescue with another fungal symbiont, p-endophyte, had no effect. Perennial ryegrass, Lolium perenne L., infected with six strains of endophyte, was moderately resistant to C. pulicaria compared with endophyte-free grass, but four additional strains were relatively inactive. Six Neotyphodium-meadow fescue, Festuca pratensis Huds., associations, including the wild-type N. uncinatum-meadow fescue combination, were resistant, whereas three associations were not effective. Loline alkaloids seemed to play a role in antixenosis to C. pulicaria. Effects not attributable to the lolines or any other of the alkaloids examined also were observed. This phenomenon also has been reported in tests with other insects, and indicates the presence of additional insect-active factors.     

Endophytic fungal beta-1,6-glucanase expression in the infected host grass

Mutualistic fungal endophytes infect many grass species and often confer benefits to the hosts such as reduced herbivory by insects and animals. The physiological interactions between the endophytes and their hosts have not been well characterized. Fungal-secreted proteins are likely to be important components of the interaction. In the interaction between Poa ampla and the endophyte Neotyphodium sp., a fungal beta-1,6-glucanase is secreted into the apoplast, and activity of the enzyme is detectable in endophyte-infected plants. Sequence analysis indicates the beta-1,6-glucanase is homologous to enzymes secreted by the mycoparasitic fungi Trichoderma harzianum and Trichoderma virens. DNA gel-blot analysis indicated the beta-1,6-glucanase was encoded by a single gene. As a secreted protein, the beta-1,6-glucanase may have a nutritional role for the fungus. In culture, beta-1,6-glucanase activity was induced in the presence of beta-1,6-glucans. From RNA gel blots, similar beta-1,6-glucanases were expressed in tall fescue (Festuca arundinacea Schreb.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman) infected with the endophyte species Neotyphodium coenophialum and Epichlo? festucae, respectively.     

Endophyte-mediated resistance to black cutworm as a function of plant cultivar and endophyte strain in tall fescue

To improve Neotyphodium endophyte-mediated resistance to black cutworm Agrotis ipsilon (Hufnagel) (BCW), a series of experiments was conducted by using several different cultivars of tall fescue, Schedonorus arundinaceus (Schreb.) Dumort. in combination with several different haplotypes of the endophyte Neotyphodium coenophialum (Morgan-Jones & Gams) (plant cultivar × endophyte haplotype = plant line), each producing unique alkaloid profiles. BCW settling response, survival at 5 and 10 d, and larval biomass varied significantly among plant lines. In general, greater variation BCW performance was observed within a single plant cultivar infected with different endophyte haplotypes than among different plant cultivars infected with the same endophyte haplotype, but comparisons among the former were far more numerous. Although five endophyte-mediated alkaloids representing three alkaloid classes were quantified in the plants, the pyrrolizidine alkaloid N-acetyl norloline was consistently the single best predictor of BCW performance. BCW settling response, 5-d survival, and 10-d survival decreased as levels of the alkaloid N-acetyl norloline increased. The same three response variables also decreased with increasing levels of peramine, but increased with increasing levels of ergovaline. Minor variation in endophyte infection levels occurring among infected plant lines had no significant influence on BCW performance. Results indicate a potentially important role for N-acetyl norloline and peramine in providing resistance to black cutworm whereas ergovaline appears to be much less important. Therefore, endophyte haplotypes expressing high levels of N-acetyl norloline and peramine may be of particular importance for developing 'friendly' endophyte-enhanced turf and pasture grasses that resist challenging lepidopteran pests, although remaining safe for wildlife and grazing mammals.     

Genetic diversity in a world germplasm collection of tall fescue

Festuca arundinacea Schreb., commonly known as tall fescue, is a major forage crop in temperate regions. Recently, a molecular analysis of different accessions of a world germplasm collection of tall fescue has demonstrated that it contains different species from the genus Festuca and allowed their rapid classification into the three major morphotypes (Continental, Mediterranean and Rhizomatous). In this study, we explored the genetic diversity of 161 accessions of Festuca species from 29 countries, including 28 accessions of INTA (Argentina), by analyzing 15 polymorphic SSR markers by capillary electrophoresis. These molecular markers allowed us to detect a total of 214 alleles. The number of alleles per locus varied between 5 and 24, and the values of polymorphic information content ranged from 0.627 to 0.840. In addition, the accessions analyzed by flow cytometry showed different ploidy levels (diploid, tetraploid, hexaploid and octaploid), placing in evidence that the world germplasm collection consisted of multiple species, as previously suggested. Interestingly, almost all accessions of INTA germplasm collection were true hexaploid tall fescue, belonging to two eco-geographic races (Continental and Mediterranean). Finally, the data presented revealed an ample genetic diversity of tall fescue showing the importance of preserving the INTA collection for future breeding programs.     

内生真菌感染对宿主植物高羊茅锌耐受性的影响

以感染内生真菌(Neotyphodium coenophialum)和未感染内生真菌的高羊茅(Festuca arundinacea Schreb.)为实验材料,在营养液中加入ZnSO4进行锌胁迫实验,分析内生真菌对宿主植物锌耐受性的影响。与未感染内生真菌的植株相比,内生真菌感染对高羊茅的总生物量没有显著增益作用,但增加了分蘖数和叶片延伸生长累积值。内生真菌感染降低了高羊茅中Zn2+的总含量,改变了锌在高羊茅中的分配,增加叶鞘中锌的含量,减少叶片中锌的含量。在高锌浓度下,内生真菌感染对净光合速率的变化没有影响,但是显著提高了其宿主的PSⅡ光化学效率(Fv/Fm)。总体来看,内生真菌感染改善宿主高羊茅的锌耐受性。     

The Effects of Endophytes on Seed Production and Seed Predation of Tall Fescue and Meadow Fescue

Fungal endophytes of grasses are often included in agricultural management and in ecological studies of natural grass populations. In European agriculture and ecological studies, however, grass endophytes are largely ignored. In this study, we determined endophyte infection frequencies of 13 European cultivars and 49 wild tall fescue (Schedonorus phoenix) populations in Northern Europe. We then examined seed production and seed predation of endophyte-infected (E+) and endophyte-free (E?) tall fescue (in wild grass populations and in a field experiment) and meadow fescue (Schedonorus pratensis; in a field experiment only). Endophytes were detected in only one of the 13 cultivars. In contrast, >90% of wild tall fescue plants harbored endophytes in 45 wild populations but were absent in three inland populations in Estonia. In three wild tall fescue study sites, 17%, 22%, and 56% of the seeds were preyed upon by the cocksfoot moth. Endophyte infection did not affect seed mass of tall fescue in the field experiment. However, seed predation was lower in E+ than E? grasses in the two tall fescue populations with higher predation rates. For meadow fescue, the mean number of seeds from E+ plants was higher than E? plants, but E? and E+ seeds had equal rates of predation by the moth. Our results suggest that the effects of grass endophytes on seed production and cocksfoot moth seed predation vary considerably among grass species, and the effects may depend on herbivore pressure and other environmental conditions.     

How does the Fungal Endophyte Neotyphodium coenophialum Affect Tall Fescue (Festuca arundinacea) Rhizodeposition and Soil Microorganisms?

The goal of our study was to investigate the impact of fungal endophytes in tall fescue (Festuca arundinacea) on rhizodeposition and in turn, the soil microbial community. Sand-based, aseptic microlysimeter units were constructed for the collection of rhizodeposit solutions for chemical analyses from the roots of endophyte-free (E−) and endophyte-infected (E+) tall fescue plants. E+ plants were infected with Neotyphodium coenophialum, the most common endophyte found in tall fescue. Rhizodeposit solutions collected over nine weeks from E+ grass contained more organic carbon and carbohydrates than E−. These solutions were allowed to percolate through columns of plant-free soils to assess the response of the soil microbial communities. Soils to which solutions from E+ grass were applied had significantly higher respiration rates than those receiving solutions from E− grass, suggesting that microbial activity was stimulated by changes in the rhizodeposits. Culture-based assays of the soil microbial community (plate counts and community-level physiological profiling) suggest that the basic structure of the microbial community was not affected by application of rhizodeposit solutions from E+ plants as compared to E−. Our results indicate that the presence of a fungal endophyte may enhance rhizodeposition by tall fescue and could consequently influence microbial mineralization processes in the soil. In grasslands where nutrients may be limiting, hosting a fungal endophyte has the potential to enhance plant nutrient supply indirectly via a stimulatory effect on the soil microbial biomass. Megan M. Van Hecke and Amy M. Treonis - Both authors contributed equally to this work.     

Expression of a novel chitinase by the fungal endophyte in Poa ampla

Many wild and cultivated cool-season grass species are naturally infected with fungal endophytes of the genera Neotyphodium and Epichlo?. These associations generally are considered mutualistic with the plants benefiting from reduced herbivory and the fungi benefiting from nutrients supplied by the plants. The fungi secrete proteins that might have a role in the interspecies symbiosis. In the interaction between Poa ampla Merr. and the endophyte Neotyphodium sp., a fungal chitinase was detected in the apoplastic protein fraction. The chitinase was also the major protein secreted in culture. Sequence analysis of the chitinase revealed it has a low level of amino acid sequence identity to other fungal chitinases and one of the conserved active site residues is altered. DNA gel-blot analysis indicated the chitinase was encoded by a single gene. Expression of similar chitinases also was detected in endophyte-infected tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman). This is the first report of an endophyte chitinase expressed in the infected host grass. As a secreted hydrolytic enzyme, the chitinase might have roles in the nutrition, growth or defense of the endophyte.