Malcolm R. Siegel

Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response.

The occurrence of the alkaloidsN-formyl andN-acetyl loline, peramine, lolitrem B, and ergovaline and the response of aphids to plants containing these compounds were determined in species and cultivars ofFestuca,Lolium, and other grass genera infected with fungal endophytes (Acremonium spp., andEpichloe typhina). Twenty-nine of 34 host-fungus associations produced one or more of the alkaloids, most frequently peramine or ergovaline. Three alkaloids (lolines, peramine, and ergovaline) were found in tall fescue and in perennial ryegrass infected withA. coenophialum, while peramine, lolitrem B, and ergovaline were present in perennial ryegrass and in tall fescue infected withA. lolii and inF. longifolia infected withE. typhina. WhileA. coenophialum andA. lolii produced similar patterns of alkaloids regardless of the species or cultivar of grass they infected, isolates ofE. typhina produced either no alkaloids or only one or two different alkaloids in the grasses tested. Aphid bioassays indicated thatRhopalosiphum padi andSchizaphis graminum did not survive on grasses containing loline alkaloids and thatS. graminum did not survive on peramine-containing grasses. Ergovaline-containing grasses did not affect either aphid.

Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism.

Fungal endophytes provide grasses with enhanced protection from herbivory, drought, and pathogens. The loline alkaloids (saturated 1-aminopyrrolizidines with an oxygen bridge) are fungal metabolites often present in grasses with fungal endophytes of the genera Epichloë or Neotyphodium. We conducted a Mendelian genetic analysis to test for activity of lolines produced in plants against aphids feeding on those plants. Though most loline-producing endophytes are asexual, we found that a recently described sexual endophyte, Epichloë festucae, had heritable variation for loline alkaloid expression (Lol+) or nonexpression (Lol-). By analyzing segregation of these phenotypes and of linked DNA polymorphisms in crosses, we identified a single genetic locus controlling loline alkaloid expression in those E. festucae parents. We then tested segregating Lol+ and Lol- full-sibling fungal progeny for their ability to protect host plants from two aphid species, and observed that alkaloid expression cosegregated with activity against these insects. The in planta loline alkaloid levels correlated with levels of anti-aphid activity. These results suggested a key role of the loline alkaloids in protection of host plants from certain aphids, and represent, to our knowledge, the first Mendelian analysis demonstrating how a fungal factor contributes protection to plant-fungus mutualism.

Sexual compatibility and taxonomy of a new species of Epichloe symbiotic with fine fescue grasses

Mating tests among stroma-forming and nonstroma-forming strains of Epichloe from several fine fescue species, including Festuca rubra subsp. rubra and F. rubra subsp. commutata, indicated a distinc...

Molecular phylogenetic relationships of nonpathogenic grass mycosymbionts and clavicipitaceous plant pathogens

Acremonium sect.Albo-lanosa (Fungi Imperfecti) includes beneficial, endophytic mycosymbionts of various grasses of the subfamilyPooideae, and also the anamorph of the grass choke pathogen,Epichloë typhina (Clavicipitaceae, Ascomycotina). These fungi are seed-disseminated, thus stably maintained for many host generations. To investigate the possibility of long-term coevolution, isolates ofE. typhina and anamorphs were obtained from eight grass species, sequences of their rRNA gene internal transcribed spacers were aligned with those from otherClavicipitaceae, and cladograms were generated by maximum parsimony. The results indicated that the nonpathogenic endophytes have not necessarily coevolved with their host species and that they arose fromE. typhina on multiple occasions.

Chemistry, occurrence and biological effects of saturated pyrrolizidine alkaloids associated with endophyte-grass interactions

Abstract Saturated amino pyrrolizidine alkaloids with an oxygen bridge between C-2 and C-7 have been isolated from grasses and the chemistry and biology of these substances are reviewed in this paper. These substances are commonly referred to as loline alkaloids as they are derivatives of loline and originally were isolated from Lolium spp. The saturated amino pyrrolizidine alkaloids are not hepatotoxic and must be distinguished from the 1,2-unsaturated pyrrolizidine alkaloids which are very significant animal and human toxins and carcinogens of plant origin. Chemical synthesis of loline has been achieved based on starting materials of a nitrone and methyl 4-hydroxycrotonate. N-formylloline is made by addition of ethyl formate to loline and removal of excess ethyl formate and ethanol formed. N-acetylloline is synthesized by addition of acetyl chloride to loline in chlorofirm. N-acetylloline is partitioned into a dilute acid, then from a dilute base with CHCl3, and the CHCl3 removed to yield N-acetylloline. N-methylloline, norloline, N-formynorloline, N-acetylloline, and N-propionylnorloline are also readily formed from loline. Little is known about the biosynthesis of the loline alkaloids but from biosynthesis of other pyrrolizidine alkaloids a proposed biosynthesis is ornithine → putrescine → spermidine → → dialdehyde → → norloline → → loline. Loline was first isolated from Lolium temulentum L. (Lolium cuneatum Nevski) and later several derivatives were identified in tall fescue (Festuca arundinacea Schreb.). Chromatographic methodologies have been used to separate the pyrrolizidine alkaloids but 50-m capillary columns (0.32 mm i.d.) or 10-m wide-bore columns (0.53 mm i.d.) provide the greatest resolution of the many loline derivatives. Loline alkaloids have been found primarily in plants known to be infected witn Acremonium coenophialum Morgan-Jones and Gams. Lolines have been found in Lolium perenne L., F. arundinacea, and Poa autumnalus Muhl. ex Ell. infected with A. coenophialum and in Festuca gigantea (L.) Vill. infected with an unidentified Acremonium spp. Loline alkaloids were present in infected tall fescue in greatest amounts in seed, followed in decreasing amounts in the rachis, stem, leaf sheath, and leaf blade. Site of synthesis, plant and/or endophyte, is not known nor is the translocation tissue known. During the main part of the growing season there is little change in the accumulation of the loline alkaloids in leaf blade. Alkaloid levels increased with plant age and increased in regrowth tissue. The concentration of endophyte mycelium in leaf sheaths is positively associated with loline alkaloid accumulation. N-formylloline is toxic to several different insects from ingestion, topical and injected applications. Larger animal bioassay have not been conducted with pure compounds but the observations suggest some pharmacological activity in these systems.

The β-tubulin gene of Epichloë typhina from perennial ryegrass (Lolium perenne)

SummaryEpichloë typhina is a biotrophic fungal pathogen which causes choke disease of pooid grasses. The anamorphic state, Acremonium typhinum, is placed in the section Albo-lanosa along with related, mutualistic, seeddisseminated endophytes. As an initial study of gene structure and evolution in Epichloë and related endophytes, the β-tubulin gene, tub2, of the perennial ryegrass choke pathogen (EtPRG) was cloned and sequenced. The coding sequence and the predicted β-tubulin amino acid sequence were highly homologous to the Neurospora crassa homologs, and to one of the two β-tubulin genes of Emericella nidulans. However, two introns characteristic of the N. crassa and Em. nidulans genes were absent in the E. typhina gene. Furthermore, one of the remaining introns possessed the uncommon 5′ splice junction, GC. In contrast to published observations concerning other Ascomycetes, a mutant of EtPRG, selected for resistance to methyl-2-benzimidazole carbamate (benomyl), possessed no alteration of its β-tubulin coding sequence.

Defensive chemicals in grass-fungal endophyte associations.

The ability of organisms to form long term intimate and diverse relationships with each other (symbiosis) is now recognized as a common ecological phenomenon. Symbiosis, as a general term, does not imply detriment or benefit, but rather that the outcome (net effect) of species interaction exists within a symbiotic continuum or “species interaction grid” that includes agonism (predation and disease) and mutualism (benefits for both species).1,2 The continuum also includes pleotropic symbiosis, where net effects of species interaction vary spatially or temporally in relative agonism or mutualism.3 Grasses systematically infected with specific clavicipitaceous fungi are examples of species interactions that span the symbiotic continuum, profoundly affecting the ecological fitness of the hosts.4–8

Mechanism of action and fate of the fungicide chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) in biological systems: I. Reactions with cells and subcellular components of Saccharomyces pastorianus

Abstract The mechanism and sequence of reaction of chlorothalonil in cells of Saccharomyces pastorianus was investigated by the use of either 14C-labeled fungicide or Na235SO4-labeled cells. The initial uptake of fungicide resulted in rapid formation of substituted chlorothalonil-reduced glutathione (GSH) derivatives. Chlorothalonil reacted with proteins during derivative formation but decreased cell viability did not occur until all the GSH was reacted and inhibition of specific NAD thiol-dependent glycolytic and respiratory enzymes occurred. It is postulated that enzyme activity and cell viability are controlled either directly or indirectly by the concentration of GSH. The loss of GSH through derivative formation with chlorothalonil and the lack of regeneration of the reduced thiol could be an important feature in the sequence of toxicity of the fungicide. The general mechanism of action of chlorothalonil resembles the trichloromethyl sulfenyl fungicides in that treated cells accumulate large concentrations of fungicide; reactions involve both low and high molecular-weight thiols, with the formation of glutathione-fungicide derivatives; and toxicity resides ultimately with the inhibition of thiol-dependent enzymes.

Transformation of Acremonium coenophialum, a protective fungal symbiont of the grass Festuca arundinacea

SummaryAcremonium coenophialum is a mutualistic mycosymbiont and natural agent of biological protection of the widely distributed grass Festuca arundinacea (tall fescue). An electroporative transformation system was developed for A. coenophialum. Segments of DNA 5′ to the β-tubulin gene (tub2) of the closely related ascomycete Epichloë typhina, fused to the Escherichia coli hph gene encoding hygromycin B phosphotransferase, conferred hygromycin resistance when introduced into A. coenophialum by electroporation. The incorporation of the Emericella nidulans trpC terminator greatly increased protoplast germination on selective medium and improved transformation efficiencies 30–200% depending on the plasmid construct. Plasmid pCSN43, which incorporates the trpC controlling elements for hph expression, was also used to transform A. coenophialum. Southern blot analysis of ten pCSN43 transformants indicated the possibility of random integration of this vector into the genome.

Genetics of Host Specificity in Epichloë typhina.

ABSTRACT Epichloë typhina perennially and systemically infects grass plants, causing choke disease in which maturation of host inflorescences is suppressed. In seedling-inoculation tests, isolate E8 from perennial ryegrass established and maintained infection in this host but not in orchardgrass. In contrast, isolates E469, E2466, and E2467 from orchardgrass varied in infection frequency and stability in orchardgrass, but all were unable to establish stable infections in perennial ryegrass. To investigate the genetics of host specificity, isolate E8 was crossed with each of the isolates from orchardgrass. Seedlings of parental host species were inoculated with F(1) progeny, and the frequencies of seedling infection and stability in adult plants were assessed. In the E8 x E2466 cross, the F(1) progeny exhibited a wide range of infection frequency and stability in each parental host. In crosses E8 x E469 and E8 x E2467, where the orchardgrass-derived parents infected 5 to 13% of inoculated perennial ryegrass seedlings, the distributions of infection frequencies for the F(1) progeny wereskewed toward levels comparable to that of the parent from perennial ryegrass. In all crosses, most progeny had low frequencies of infection in orchardgrass. However, transgression was evident in a cross of E8 with E469, an isolate that infected orchardgrass seedlings at a low frequency (2 to 3%). The E8 x E469 cross had a few F(1) progeny that infected orchardgrass at high efficiency (up to 81%). A Spearman rank correlation applied to the E8 x E2466 progeny indicated a significant negative correlation between infection frequencies in perennial ryegrass and orchardgrass. Also, there was a significant correlation of infection frequency and stability in perennial ryegrass but not in orchardgrass. To test whether only a few genes governed infection frequency in perennial ryegrass, an E8 x E2466 F(1) progeny (designated E386.04), which had intermediate compatibility with this host, was backcrossed to E8. The progeny of this backcross exhibited a distribution of infection frequencies in perennial ryegrass between that of E386.04 and the backcross parent, suggesting that multiple genes may determine compatibility at the seedling infection stage. The results of these experiments indicated multiple genetic determinants of compatibility or incompatibility with each host, with intermediate or high heritability.