Search

This chapter discusses the problems associated with endophyte-infected Festuca L. and Lolium L. pasture grass species; the desired beneficial characteristics of infected grasses; and the selection, methods of introduction of desirable endophytes into grasses, and characterization of the new associations. It also discusses the progress and problems associated with manipulation of nonpathogenic endophytes with reference to cultivar improvement. Desirable properties of endophyte-infected forage grasses are the same as for endophyte-free herbage. Livestock toxicosis may be alleviated by appropriate pasture and animal management practices that reduce the intake of toxic substances, by reduction or elimination of the toxic compounds in the herbage and total diet, and by reduced susceptibility of the grazing livestock. Genetic manipulation is especially designed for well-characterized gene functions synthesis of toxic alkaloids and the introduction of known foreign genes. The identification of toxic alkaloids and their biosynthetic pathways is information that has facilitated the development of lolitrem and ergot minus symbiota.

Sixty-three male sprague Dawley rats were randomly assigned to dietary treatments containing 1) N-formylloline alkaloid (NFL), 2) N-acetylloline alkaloid (NAL), 3) NFL + NAL, 4) NFL + a mixture of ergot alkaloids, 5) NAL + a mixture of ergot alkaloids, 6) NFL + NAL + a mixture of ergot alkaloids, 7) a mixture of ergot alkaloids, 8) endophyte-free tall fescue seed (EFTF), and 9) endophyte-infected tall fescue (EITF) seed (negative control). All diets were prepared by mixing the prepared treatments and Laboratory Chow (1:1 ratio) and were fed at a maximum of 15 g per rat per day. All rats were killed at termination (d 18). Rats fed the EITF consumed less (P.05) than those fed all other treatments. Feed intake for rats fed the NFL + ergot alkaloids was lower than for those fed NAL, NAL + ergot alkaloids, NFL + NAL + ergot alkaloids, ergot alkaloids, and EFTF treatments. Average daily weight gains (ADG) followed a trend similar to feed intake with some exceptions. Rats fed the NFL + NAL treatments had higher (P.05) ADG than those fed all other treatments except the NFL + NAL + ergot alkaloid treatment. Rats fed the EITF had lower (P.05) ADG than those fed all other treatments except those fed ergot alkaloids and NAL + ergot alkaloids. Epididymides, testes, hypothalamus, corpus striatum weights, prolactin and alkaline phosphatase content were not altered by dietary treatments. Results suggest that loline alkaloids may have a slight depressing effect on feed intake.

The p-endophytes, previously identified as Gliocladium-like and Phialophora-\ike fungi, constitute a group of closely related, seed transmissible grass symbionts which have been found in several Festuca species and Lolium perenne, most often in cosymbiosis with mutualistic Epichloe-type (Acremonium) endo? phytes (e-endophytes). To help assess the ecological importance of p-endophytes, their incidence and the incidence of e-endophytes was determined in seed ac- cessions of Festuca and Lolium grass species in the U. S. Department of Agriculture Plant Introduction col? lection. Ten seeds of each accession were tested by tissue print immunoblot assay for the presence of e- or p-endophytes in separate seeds, or together in in? dividual seeds. A total of 370 accessions from 36 Fes? tuca species and five Lolium species were tested. The e-endophytes were present in 25 of the Festuca species and all of the Lolium species, while the p-endophytes appeared in five and one species, respectively. The p-endophytes were present in 18 Festuca accessions and six Lolium accessions, while the e-endophytes ap? peared in 121 and 43 accessions, respectively. The p- and e-endophytes were detected together in individual seeds in a total of five species and 13 accessions from both genera. Infectivity studies indicated that when p-endophytes from different species were introduced into their own and reciprocal hosts only the endo? phytes introduced into their own hosts produced sig? nificant levels of sustainable infections. Three conclu

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...

The objective of this study was to assess the effects of feeding endophyte-infected (Acremonium coenophialum) tall fescue seed on the growth and reproductive performance of Japanese quail (Coturnixcoturnixjaponica). One hundred and twenty female and 60 male chicks (5 weeks old) were selected from a larger group of chicks and were randomly allocated to one of three dietary treatments (Diet 1, Diet 2 and Diet 3). Two females and one male were housed in each of 60 battery breeder cages. A quail breeder diet was formulated to contain 45% fescue seed plus corn, soybean meal, vitamins and minerals. The fescue seed used for Diet 1 was 100% endophyte infected, while that used for Diet 2 and Diet 3 was not infected. Birds on Diet 1 and Diet 2 were fed on an ad libitum basis, while the feed intake of birds on Diet 3 was restricted to that of birds on Diet 1. The daily photoperiod of 6 hours was increased to 16 hours by 60 days of age, at which time the hens began to lay eggs. Dietary treatments were started at 35 days of age. Eggs were collected daily, set weekly and incubated through hatching. The results indicated no differences (P>0.05) in average daily gain (ADG) or average daily intake (ADI) among the 3 treatments. There was also no difference (P>0.05) in egg production among the 3 treatments. However, there was a significant reduction (P

Current knowledge of grass- Acremonium associations results from the discovery that animals grazing on infected grasses show an array of debilitating symptoms which lead to severe economic losses. Acremonium endophytes occur worldwide, infecting many cultivated and wild pooid grasses. It is also apparent that parasitism of different grasses by these fungi is complex. The fungal symbiont may be completely or partially endophytic and expression of parasitism may range from agonistic (the pathogenic phase of Epichloe typhina (Pers.) Tul.) to mutualistic. Regardless of how symbiosis is expressed, the associations can often benefit host grasses, improving their growth and survival. In addition, much information has been gained on the biology, ecology and physiology of specific grass-endophyte associations, the effects of infected (E+) grasses on herbivores (vertebrates and invertebrates), the chemistry of alkaloids responsible for herbivore resistance, tolerance of E + grasses to drought, and the management of endophytes for the benefit of turf and pasture grasses. Present information strongly suggests that most grass-endophyte symbioses must be maintained for optimum survival of the host grasses. Future research efforts can be clearly defined, on a pratical basis, as a continuation of studies that may lead to alleviation of toxicity to grazing animals, and the development of E+ grasses suitable for turf and pasture use. These goals may be accomp ished by manipulation of either livestock, endophyte or grass via genetic engineering, conventional breeding practices, or natural selection. Other approaches include improved plant and animal management and the use of chemical antidotes to reduce animal toxicoses. Specific research goals include continuation of studies on the biology, ecology, and physiology of symbiosis in cultivated and wild species of grasses. These studies should aid in elucidating the role of the symbionts in the host-fungus interactions (nutrition and metabolism) and mutualistic versus agonistic expression, as well as the origin and coevolution of the symbionts, mechanisms of biosynthesis of alkaloids, the bases for improved plant growth and resistance to biotic and abiotic stresses, and development of techniques for genetic manipulation of the symbionts. Pathophysiology studies using pure alkaloids, small animal bioassays, in vitro organs, tissue culture, and indicators of biochemical activity (enzyme and hormones) will clarify the mechanisms of alkaloid toxicity, potentially leading to chemical antidotes and improved systems of animal field, great strides have been made in understanding the role of Acremonium endophytes in animal toxicoses and the stress survival of E+ plants.

The close associations between grasses and endophytes, whereby the fungi constitute maternally inherited components of the symbiotic entities (symbiota), make genetic engineering and other genetic manipulations of the endophytes an attractive approach to improvement of forage, pasture and turfgrass cultivars. A number of protective alkaloids are produced by the endophytes. Although insecticidal, nematocidal and antimicrobial metabolites produced by the mycosymbionts are desirable agronomic characteristics, anti-mammalian activities which are problematic for livestock farmers need to be mitigated. As a necessary prerequisite for endophyte genetic engineering, the authors have developed a transformation protocol for the tall fescue symbiont Acremonium coenophialum Morgan-Jones et Gams. The approach used was to introduce the antibiotic resistance gene, hph, which was obtained from the bacterium Escherichia coli and modified in vitro to allow expression in fungi. Successful incorporation of this ‘chimeric’ gene into the fungal genome was detected by resistance of the transformants to hygromycin B and by DNA hybridization analysis. The wide variation in the amounts and/or types of protective alkaloids produced by different isolates of Epichloe typhina (Fr.) Tulasne (Acremonium typhinum Morgan-Jones et Gams), and other Acremonium spp., raises the prospect of employing the sexual cycle of E. typhina as an alternative genetic system for development of new endophyte strains. However, there is a lack of phenotypically variable characters to follow in Mendelian crosses. Therefore, restriction fragment length polymorphisms should be identified and used for genetic mapping. Preliminary studies of this approach are presented. Genetic manipulations and other approaches to cultivar development based on the endophytes should be done with an understanding of their evolutionary biology and the basis for host preference. Previous studies have added much to the understanding of the alkaloid production potential of different isolates, and the degree of host specificity of both choke pathogens and non-pathogenic endophytes. This paper presents a phylogenetic analysis using DNA sequence information. The results support four conclusions: (1) the endophytes have evolved from E. typhina; (2) the endophytes do not have a strict coevolutionary relationship with their grass hosts; (3) loline production, characteristic of certain symbiota, is not a good indicator of phylogenetic affinities of the fungal symbionts; (4) there is a close relationship between isolates of A. coenophialum from tall fescue, and Acremonium lolii Latch, Samuels et Christensen from perennial ryegrass and a non-pathogenic A. typhinum from red fescue.

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.

Acremonium 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 beta-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.

This study was undertaken to further determine the sensitivity of the mouse to a diet containing endophyte-infected (Acremonium lolii) perennial ryegrass (Lolium perenne L.) seed. The degree to which the mouse is affected by the endophyte will determine its value as a bioassay to study ryegrass toxicity in livestock. The focus of this study was to determine the effects of the endophyte on the reproductive performance of mice (Mus musculus) using a continuous breeding protocol for three parturitions. Thirty-six CD-1 mice were randomly assigned to one of three dietary treatments and housed separately by sex for 42 days before pairing. The diets contained 50% Purina rodent chow (w/w) and: (1) 50% endophyte-infected perennial ryegrass seed; (2) 25% infected seed and 25% un-infected seed; or (3) 50% un-infected seed. The intake for Diet 2 and Diet 3 was limited to the average daily intake of Diet 1. The mice were allowed to cohabit through three parturitions. Litter parameters assessed included dam we...

Propagule population densities of the nine mycorrhizal species detected in field plots planted to tall fescue free of the Acremonium coenophialum endophyte were more than double those in plots planted to fescue heavily infected with Acremonium . Seedlings of Acremonium -free fescue detected more propagules of most of the eleven mycorrhizal species detected in a field soil than seedlings infected with Acremonium . Sporulation by three mycorrhizal isolates over 17 weeks was higher on fescue seedlings free of Acremonium than on seedlings infected with Acremonium . Since mycorrhizal fungi are believed to be confined to roots and Acremonium endophytes to shoots, the inhibitory effect of Acremonium on mycorrhizal fungi may be due to the translocation to roots of alkaloids produced by the Acremonium-Festuca symbiosis.

Epichloë typhina is a clavicipitaceous ascomycete which systemically infects grasses, causes choke disease of host inflorescences, and is related to a group of mutualistic grass endophytes. Three plasmids of 7.5, 2.1 and 2.0 kilobase pairs were found in mitochondrial DNA preparations of an E. typhina isolate from perennial ryegrass (Lolium perenne). Results of nuclease digestion indicated that the plasmids, designated Et7.5L, Et2.1L, and Et2.0L, were linear, double-stranded DNAs with protein linked to their 5'-ends (plDNA). The plasmids shared little or no homology with each other, and were not integrated into the mitochondrial or nuclear genomes. No homologous plasmids were detected in isolates of E. typhina from other grass hosts, anamorphic endophytes, or other Clavicipitaceae. However, other plasmids were present in Balansia obtecta and Claviceps purpurea. A partial sequence of one of the E. typhina plasmids, Et2.0L, indicated an open reading frame when UGA was assumed to encode tryptophan. The inferred amino acid sequence had 24% identity over 258 amino acids in two regions of the reverse transcriptase encoded by the circular Mauriceville and Varkud plasmids of Neurospora spp. The homologies included six segments conserved in RNA template-dependent DNA or RNA polymerases.

(1991). Expression of Antifungal Activity in Agar Culture by Isolates of Grass Endophytes. Mycologia: Vol. 83, No. 4, pp. 529-537.

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,Epichloe 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.

A 3-yr study was conducted to determine the performance of midlactation multiparous and primiparous Holstein cows (Bos taurus) fed 3 endophyte-free varieties of tall fescue (Festuca arundinacea Schreb) soilage. Cultivars were maintained in a vegetative stage by removal of excess growth. Forages were «Johnstone», «Kenhy», and «Kentucky-31» (Ky-31) tall fescues (...)

The presence of the fungal endophyte Acremonium lolii in perennial ryegrass (Lolium perenne) has been linked to resistance of infected plants to attack by the Argentine stem weevil (Listronotus honariensis). Production in the infected plants of the alkaloid peramine, a feeding deterrent to adult weevils, is thought to be largely responsible for the resistance’. Isolation and identification of peramine was first reported by Rowan and coworkers’,*, but the method used was too time-consuming and difficult for use in routine analysis. Tapper et a1.3 recently reported an improved method for extraction, identification and measurement of peramine. Their procedure involved a two-stage extraction using methanolchloroform (1: 1, v/v); then hexaneewater (l:l, v/v) followed by cleanup of the extract on small ion-exchange columns. Peramine was detected on silica gel layers using Ehrlich’s reagent or quantified by a high-performance liquid chromatography (HPLC) procedure. We report here the development of a rapid and sensitive reversed-phase (RP) thin-layer chromatography (TLC) procedure for detection and quantitation of peramine in crude extracts of endophyte-infected grasses.

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 F1 progeny, and the frequencies of seedling infection and stability in adult plants were assessed. In the E8 × E2466 cross, the F1 progeny exhibited a wide range of infection frequency and stability in each parental host. In crosses E8 × E469 and E8 × E2467, where the orchardgrass-derived parents infected 5 to 13% of inoculated perennial ryegrass seedlings, the distributions of infection frequencies for the F1 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 × E469 cross had a few F1 progeny that infected orchardgrass at high efficiency (up to 81%). A Spearman rank correlation applied to the E8 × 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 × E2466 F1 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.

While the discovery that fungal endophytes were present in some pooid grass species was made at the turn of the 20th century (White et al. 1993), knowledge of their widespread distribution (Leuchtmann 1992; Chap. 12, Vol. V, Part B) and the ecological and evolutionary consequences for host and fungi (Clay 1994; Leuchtmann 1994; Schardl et al. 1994; Tsai et al. 1994; Chap. 14, Vol. V, Part B), as well as their impact on livestock production (Prestidge 1993; Strickland et al. 1993), is of more recent origin. The association of grasses with endophytes of the genus Epichloe has been shown to affect the growth and/or reproduction not only of mammalian herbivores, but also of insect herbivores (Latch 1993; Popay and Rowan 1994) and, to lesser extents, nematodes, (Latch 1993), plants (Petroski et al. 1990), and fungi (Guo et al. 1992; Gwinn and Gavin 1992). In many cases, the toxicity has been associated with specific secondary metabolites produced by grass/endophyte associations (symbiota). Additionally, endophyte compatibility and toxin production is under the influence of both host and fungal genomes (Leuchtmann 1992; Christensen et al. 1993; Leuchtmann and Clay 1993; Agee and Hill 1994; Roylance et al. 1994; Welty et al. 1994; Christensen 1995) as well as the environment (Agee and Hill 1994; Arechavaleta et al. 1992; Bacon 1993). These constraints upon toxin biosynthesis result in a wide spectrum of biological activity for symbiota and offer the opportunity for manipulation of the endophytes for improvement of the grasses when used in forage, turf, and soil conservation (Schardl 1994; Siegel and Bush 1994).

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

The mutualistic associations of tall fescue (Festuca arundinacea) with seed-borne fungal symbionts (endophytes) are important for fitness of the grass host and its survival under biotic and abiotic stress. The tall fescue endophytes are asexual relatives of biological species (mating populations) of genus Epichloë (Clavicipitaceae), sexual fungi that cause grass choke disease. Isozyme studies have suggested considerable genetic diversity among endophytes of tall fescue. Phylogenetic relationships among seven isolates from tall fescue, three from meadow fescue (a probable ancestor of tall fescue), and nine Epichloë isolates from other host species were investigated by comparing sequences of noncoding segments of the beta-tubulin (tub2) and rRNA (rrn) genes. Whereas each Epichloë isolate and meadow fescue endophyte had only a single tub2 gene, most tall fescue endophytes had two or three distinct tub2 copies. Phylogenetic analysis of tub2 sequences indicated that the presence of multiple copies in the tall fescue endophytes was a consequence of hybridization with Epichloë species. At least three hybridization events account for the distribution and relationships of tub2 genes. These results suggest that interspecific hybridization is the major cause of genetic diversification of the tall fescue endophytes.

Acremonium spp. endophytes are mutualistic fungal symbionts of many C3 grasses. They are anamorphs of Epichloe typhina (Clavicipitaceae) that have become strictly seedborne, heritable components of symbiotic units (“symbiota”). In order to test the possibility that endophytes may contribute to the genetic diversity of symbiota, a survey was conducted of plants from nine populations of Festuca arizonica in the southern Rocky Mountains. Sequence analysis of rRNA gene segments distinguished three Acremonium endophyte types. Parsimony analysis indicated at least two distinct evolutionary origins of the Acremonium endophytes from E. typhina. Either or both of these evolutionary lineages may have involved cospeciation with the host.

This study was designed to assess effects of a diet containing endophyte (Acremonium coenophialum)-infected tall fescue seed on litter growth and development in mice. Twenty pairs of mature CD-1 mice were allocated to dietary treatments containing 50% (wt/wt) rodent chow plus either 50% endophyte-infected tall fescue seed or seed that was not infected. At parturition, six pups of approximately equal BW and sex ratio were selected from each litter and fostered to each of the dams for suckling, resulting in four suckling groups. Pups born to dams fed the infected diet weighed less at birth and experienced developmental delays in BW gain and in eye opening (16.7 vs. 13.7 d) regardless of the foster dam's diet. Pups born of dams fed the diet that was not infected attained the greatest growth rates and weaning weights; those suckling dams fed the diet that was not infected demonstrated the largest gains. Pups born of and suckled by dams fed the infected diet demonstrated greatly reduced growth rates and weaning weights compared with the other groups. Results suggest that the congenital effect on pups born of dams fed an infected diet was significant in lowering the growth rate of the pups during suckling. The dam's ability to provide nourishment to the pups also was lowered when fed this diet. When these two factors were combined, the differences were greater than for either factor separately.

Relationships between grasses and endophytic fungi have recently received considerable attention because of the impact of certain of these associations on agricultural production, and because of unique features that make these relationships excellent models of the ecology and evolution of mutualistic associations. Fungal species of interest include those belonging to the tribe Balansieae, family Clavicipitaceae (Ascomycetes), species of which exhibit a range of host interactions that span a continuum from pathogenic to nonpathogenic. The fungal genera involved include Balansia, Myriogenospora, Atkinsonella and, Epichloe. The asexual state of Epichloe and one of the two asexual states of Atkinsonella are classified in the form genus Acremonium section Albo-lanosa, which also includes a number of imperfect (nonsexual), seed-disseminated endophytes. Many Acremonium fungi produce no disease on their hosts and are referred to as “true endophytes” (Siegel et al., 1987b).

Epichloe 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, seed-disseminated endophytes. As an initial study of gene structure and evolution in Epichloe and related endophytes, the beta-tubulin gene, tub2, of the perennial ryegrass choke pathogen (EtPRG) was cloned and sequenced. The coding sequence and the predicted beta-tubulin amino acid sequence were highly homologous to the Neurospora crassa homologs, and to one of the two beta-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 beta-tubulin coding sequence.

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.

The family Clavicipitaceae (Ascomycota) is comprised of fungi with perithecia borne on stro- mata, unitunicate asci, and filamentous, multiseptate ascospores. All are biotrophic symbionts, either mu- tualistic with plant hosts or pathogenic to plants, in- vertebrate animals or other fungi. Genera of plant- associated Clavicipitaceae (tribes Balansieae and Clavicipieae) are distinguished, in part, by stromal and ascus morphology, ascospore germination pat- terns, whether sclerotia are formed, and host inter- actions. Their anamorphs include enteroblastic mi- croconidial states, classified in anamorphic genera Neotyphodium and Sphacelia (for teleomorphs Atkin- sonella, Claviceps, Echinodothis, and Epichloe), and ho- loblastic macroconidia, classified in the anamorphic genus Ephelis (teleomorphs Atkinsonella, Balansia, and Myriogenospora). Epichloe' species often are mu- tualistic with grass hosts, and are ancestral to asexual, seed transmitted endophytes symbiotic with many cool-season grasses. Partial 28S nuclear rDNA se- quences were determined from isolates of five species and two undescribed mating populations of Epichloe, one asexual Epichloe hybrid (Neotyphodium coeno- phialum), and representatives of six other genera in the family. Results from phylogenetic analysis of the sequences supported monophyly of plant-associated Clavicipitaceae, with insect-pathogenic Cordyceps spe- cies more deeply rooted. Four clades were distin

A 232-bp DNA sequence was obtained from random amplified polymorphic DNA of Peronospora tabacina, the blue mold pathogen of tobacco. This sequence had homology to P. tabacina DNA but not to DNA of other downy mildew or oomycetous fungi tested. The fragment was determined to be part of a repetitive DNA sequence that was ubiquitous in a worldwide collection of P. tabacina. Oligonucleotides were designed for amplification of this sequence by polymerase chain reaction. The fragment was reliably amplified with amounts of DNA (1-10 fg) that are less than that contained in a single P. tabacina sporangiospore. Use of this technique enabled the detection of P. tabacina DNA directly in local lesions, systemic vascular infections, and other infected parts of tobacco plants [...]

An experiment was conducted to determine the effect of feeding tall fescue seed infected by an endophytic fungus Acremonium coenophialum on the reproductive performance of male rats. Thirty 70-day-old rats were randomly allocated to four treatments: (I) fed 50% rat chow and 50% healthy fescue seed ad libitum (control; N=9; (II) same as I but restricted to the daily feed intake of III (N=7; (III) 50% fungal-infected fescue seed and 50% chow (N=7); and (IV) a mixture of 50% laboratory chow, 25% healthy fescue seed and 25% fungal-infected fescue seed (N=7). The rats were fed these diets for 42 days. During this time, body weights were taken weekly and feed intake was taken daily. At the end of the experiment, the rats were sacrificed and the testes and epididymides were excised and measured. Sperm parameters were assessed (concentration, percentage of motility and progressive motility) at the site of the cauda epididymis; the testes were homogenized and assessed for daily sperm production potential (DSP). Concentrations of spermatozoa (× 106) among the various treatments were 645.6, 486.5, 387.4 and 457.1; motility and progressive motility measurements ranged from 48 to 50% and 2.3 to 2.4 (0–4), respectively. DSP values (per gram) and testicular weight were reduced (P

Three trials involving 184 male Sprague Dawley rats were conducted to identify the biologically active fraction of endophyte-infected tall fescue seed. In trial 1, seed infected (greater than 95%) with the endophytic fungus Acremonium coenophialum and shown to be toxic to cattle, was serially extracted with hexane, ethylacetate, and methanol, respectively. A second sample of this seed was extracted with chloroform only. Controls included a solvent treatment (spraying the various solvents on endophyte-free seed and carrying the mixture to dryness), the seed post-extraction, endophyte-infected seed and endo-phyte-free seed. Laboratory chow was mixed with all diets to provide 50% of the mix. Trial 2 examined the same treatments as trial 1 except that the chloroform treatment was omitted. Dosages 3 times (equivalent to 22.5 g seed) those used in trial 1 were applied to endophyte-free seed (7.5 g) in an attempt to accentuate any differences. Trial 3 examined the methanol fraction extracted serially, a methanol fraction extracted batchwise, and a water fraction extracted batchwise. Controls included seed residues post-extraction, solvent residue from methanol sprayed on endophyte-free seed, and endophyte-infected seed. Lowered feed intake and weight gains, and depressed serum prolactin concentrations were used as indicators of toxicity. Feed intake was lower in all three trials for rats fed the methanol fraction. In all three trials mean serum prolactin concentrations in rats fed the methanol extracts were not different from those fed endophyte-infected fescue seed. Rats fed the water extract in trial 3 had lower feed intakes than rats fed endophyte-free seed. However, rats fed the seed, post-water extraction, had serum prolactin concentrations similar to that of the negative control. Results suggest that the toxic factor(s) may be concentrated in the methanol extract.

Excretion and distribution of single and multiple intraperitoneal doses of [ 35 S]captan and [ 14 C]folpet were similar in normal and 70% hepatectomized male rats. After receiving the single dose of captan, the rats eliminate approximately 76% of the radioactivity in the urine after 72 hr. The elimination in the feces for the same time period was 13%. Normal rats administered single or multiple doses of [ 14 C]folpet excreted nearly 100% of the total dose in the urine within the first 24 hr. Nuclei isolated from the liver of normal and 70% hepatectomized rats receiving multiple doses of [ 35 S]captan contained 0.008–0.009 μg 35 S/g of tissue. Appreciable amounts of the radioactivity from [ 35 S]captan were bound by isolated nuclei from the livers of normal and partially hepatectomized rats. After a 1-hr treatment with [ 36 S]captan, the nuclei were fractionated into nuclear sap protein, deoxyribonucleoprotein (including histones), acidic ribonucleoprotein, and “residual” protein fractions. These proteins in normal nuclei bound 10, 14, 39, and 16% of the total label, respectively, with essentially the same results obtained with nuclei from regenerating rat liver. When compared by polyacrylamide gel electrophoresis, acidic nuclear proteins from treated and nontreated normal nuclei were characterized by band diffusion and the presence or absence of Amido Schwartz-staining bands. None of the abovementioned effects on histones from treated nuclei were observed. Captan treatment of isolated nuclei also altered the extraction characteristics of the nuclear protein fractions, presumably because of extensive aggregation of thiol-containing nuclear proteins.

The tenn endophyte (Greek: endo == within + phyte = plant) has been defined as an organism contained or growing (entirely) within the substrate plant, whether parasitically or not ( 100, 106). Using this tenn in its broadest sense, the subject of this chapter could include all fungi that spend all or nearly all of their life cycles in the host grasses. We do not discuss such endophytic fungi as the smuts and vesicular-arbuscular mycorrhizas that infect grass. Instead, our interests lie primarily with a specific group of c1avicipitaceous fungi that belong or are related to fungi in the tribe Balansiae (5, 24). These fungi either are true endophytes that never produce external fructifications on the plant or else may produce external mycelium andlor spores that affect flower and seed production. We focus specifically on those grass-endophyte complexes that may also cause maladies of grazing animals. Using the above criteria, two recent events involving grass-endophyte associations have important implications for the livestock industry. In 1977 Bacon et al (9) reported the close association of an endophyte (Sphacelia typhina) in infected tall fescue (Festuca arundinacea) and the incidence of

The reactions of captan, folpet, and thiophosgene with calf thymus histones and total rat liver histones were investigated. The binding of radioactivity from [ 35 S]captan or [ 14 C]folpet and changes in polyacrylamide gel patterns of captan-treated calf thymus histones were pH dependent. At pH 7.5, gel patterns of captan-treated histones were not different from those of non-treated histones; whereas, at pH 9.0 the Amido Schwartz-staining bands of captan- and thiophosgene-treated histones in polyacrylamide gels were characterized by band diffusion, loss of staining definition, band migration, and aggregate formation. Nearly equal amounts of radio-activity from [ 14 C]folpet and [ 35 S]captan were bound to lysine-rich histones at pH 9.0, while at pH 7.5 there was little binding of radioactivity to the proteins. Approximately equal amounts of radioactivity were removed from histones in the presence of the low-molecular-weight thiol, dithiothreitol. Reconstitution experiments using captan-treated lysine-rich histones and non-treated calf thymus DNA yielded less stable complexes, as well as smaller nucleohistone-nucleohistone complexes. These data suggest that the intact molecule binds to deprotonated sites on the lysine-rich histones. This binding causes changes in the configuration of the protein molecule which alters the ability of nuclear histones to stabilize DNA structure.

The purpose of this study was to examine the effects of endophyte (Acremonium coenophialium) toxic tall fescue seed on serum prolactin, daily feed intake, and average daily weight gain in male rats. Thirty Sprague-Dawley male rats (218 g) were randomly assigned to treatments of (I) rat chow (50%) plus an endophyte-free tall fescue seed (50%), (II) same as I only restricted feed intake as III, (III) rat chow (50%) plus endophyte infected tall fescue seed (50%) and (IV) rat chow (50%) plus endophyte infected tall fescue seed (25%) plus endophyte free tall fescue seed (25%). Dry matter intake and average daily gain were lower (P less than .05) for rats fed treatment II and III than for those fed treatment I and IV, with treatment IV being lower (P less than .05) than treatment I. Body weights on days 4, 11 and 21 followed the same trend with the rats fed treatment I having higher weights at each weigh period than those on treatments II, III and IV; and those fed treatment IV having higher weights at each period than those on treatment II and III. Baseline concentrations of serum prolactin were similar among treatments. Serum prolactin concentrations were lower (P less than .05) in rats fed treatment III and IV than in those fed treatment II, and those fed treatment I were not different from all others on day 4. On day 11, rats fed treatment I, III and IV had lower (P less than .05) serum prolactin concentrations than those fed treatment II.(ABSTRACT TRUNCATED AT 250 WORDS)

The reaction characteristics of chlorothalonil with glyceraldehyde-3-phosphate dehydrogenase (GPDH), from yeast, (EC 1.2.1.12) were studied in vitro. Enzyme inhibition was related to the amount of [14C]chlorothalonil bound to the protein. Kinetics of enzyme inhibition was non-competitive for the substrate glyceraldehyde-3-phosphate (GAP) (Ki = 0.42 muM). Reversal of enzyme inhibition could not be demonstrated with the low molecular thiol dithiothreitol (DDT), although the thiol did protect the protein against the toxic action of the fungicide. Because 5,5' dithiobis-(2-nitrobenzoic) acid (DTNB) reduced the binding of 14C-labeled fungicide by approximately 90% it is postulated that chlorothalonil affects catalytic activity by reacting with the 4 sulfhydryl sites (cysteine-149) responsible for the binding of GAP. Certain reaction characteristics of the trichloromethyl fulfenyl fungicides with GPDH were found to be similar to those of chlorothalonil. However, chlorothalonil differed from those fungicides in that it did not react with non-thiol groups of either GPDH or alpha-chymotrypsin (alphaCT) and had a slower reaction rate with the GPDH. It is suggested that the differences in reaction rates of the fungicides are due to the molecular size and the chemical nature of the reactive toxiphores.

This study was designed to assess the effect of endophyte-infected ( Acremonium coenophialum ) tall fescue (KY-31) seed (80% infected) on reproductive performance in CD-1 mice by continuous breeding. Twenty-four pairs of 70-d-old CD-1 mice were randomly allocated to four diets: 1) mouse chow ad libitum; 2) 40% infected fescue seed and 60% chow (w/w); 3) reduced intake (100% chow) similar to the intake, adjusted daily, in Diet 2; and 4) 60% infected fescue seed and 40% chow. Males and females were randomly paired (six pairs/treatment) and placed on the above diets. The mice were fed the corresponding diets for 80 d, although the pairs were separated on Day 60 (prior to the birth of the 3rd litter) and the females were monitored for one additional gestation period (20 d). The pregnancy data (litters produced) among the four treatments were 100.0 (18), 77.8 (14), 100.0 (18) and 80.0% (12) respectively. Similarly, the average number of pups born per litter among the four treatments was 11.8, 9.3, 10.1, and 9.8. When the chow treatment (1 and 3) and the fescue treatments (2 and 4) were pooled and compared, the percent pregnancy was 100.0 (n = 36) and 78.8 (n = 26), and the pups born per litter (means ± SEM) were 11.0 ± 0.5 and 9.5 ± 0.6, respectively. Also the intervals between the three litters born during the 60-d cohabitation period were 21.6 ± 1.1 and 24.5 ± 0.9 d for the chow and fescue treatments, respectively. The results point out that 40 and 60% infected fescue seed in the diet of mice does influence (P

Ultrastructural studies were made of Acremonium fungal endophytes of tall fescue and perennial ryegrass and two isolates of Epichloe typhina. Hyphae of these fungi were observed in leaf sheaths of their natural host plant grown from infected seed and in artificially inoculated nonhost Festuca and Lolium species of grasses. Hyphae were also studied from fungal colonies grown on cellophane on potato dextrose agar. Crystalline inclusions were found in all endophyte–host combinations with the exceptions of E. typhina from perennial ryegrass in its own host and in a nonhost tall fescue, and Acremonium coenophialum in a nonhost perennial ryegrass. Tubular complexes were seen only in Acremonium lolii when growing in both host and in a non-host tall fescue. Three structures not seen before in endophytes (bundles of microtubules, 55 to 60 nm diameter "tubes," and bilayered cell walls) were found in E. typhina growing only in perennial ryegrass, its natural host. None of the distinguishing ultrastructural components described were found in hyphae grown in culture. Our observations suggest that endophytes express specific ultrastructural features only when they are grown in the grasses. However, it was not readily discernible what function the components serve and what role, if any, the natural versus the nonhost grass plays in the expression of endophyte ultrastructure.

The objective of this study was to assess the effects of endophyte-infected tall fescue (Gl-307) seed (80% infected, by Acremonium coenophialum) on the estrous cycle in CD-I mice. Twelve 60-day-old CD-I female mice were randomly allocated to one of two dietary treatments containing 50% laboratory chow and (1) 50% endophyte-free (noninfected) fescue seed; (2) 50% endophyte-infected fescue seed. All mice were fed ad libitum. On day 35 of the study all mice were allowed access to a diet containing 100% laboratory chow for 7 days. Vaginal smears of each female were prepared daily throughout the 42 day trial period. All vaginal smears were stained and evaluated to determine phase of the estrous cycle. The days (±SE) to the first abnormal occurrence in the estrous cycle for treatments one and two were 27.0±5.6 and 16.7±5.2, respectively. The percent abnormal cycles of the total cycles observed between treatments one and two were 20.5±6.1 and 39.3±12.5, respectively. Mice on treatment two exhibited a gre...

When the petioles of detached tobacco leaves (10–17 cm2) were incubated in aqueous solutions containing [14C]metalaxyl, uptake of the fungicide was dependent on the temperature and photoperiod. Detached leaves took up 78% more [14C]metalaxyl at 26°C than at 16°C. The rate of uptake in the light at 21°C was linear, but after an additional 20h in the dark, there was only twice as much fungicide in the leaves. Different sized leaves contained the same amount of fungicide per cm2 area. Uptake by detached leaves of the 14C-labelled anilide lactones ofurace and RE-26940 [2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)acet-2′,6′-xylidide] was similar to that of metalaxyl. At the concentration of metalaxyl (66 ng ml−1) that controlled blue mould (Peronospora tabacina) on detached tobacco leaves, the amount of fungicide in the leaves was found to be 7.25 ng. Autoradiography showed that the distribution of [14C]metalaxyl in detached leaves after incubation for 23h was uniform, although higher concentrations of the label were present in the smaller veins of the leaves.

Ethanol and ethanol-water extracts of endophyte-infected tall fescue seed were effective for extracting toxin(s) responsible for feed intake and average daily weight gain (ADG) depression in Sprague Dawley rats. Although the ethanol extract of endophyte-infected seed depressed serum prolactin (Prl) concentrations, the data were less reflective of overall toxicity than feed intake and weight gain. Inclusion of commercially available ergonovine maleate, ergocryptine and ergotamine tartrate had no effect on rat feed intake, ADG or serum Prl at the levels tested. Dietary addition of a recombination of hexane, ethanol and ethanol-water extracts caused a toxicity response (depressed feed intake, ADG, and serum Prl) equivalent to that of the whole seed. The toxic factor(s) were extracted primarily in more polar solvents such as ethanol and ethanol-water. Rat assays can be used successfully in lieu of cattle assays as a routine evaluation of toxic components in endophyte-infected tall fescue. However, species differences dictate that eventually all suspected toxins be evaluated in the bovine.

The reactions of chlorothalonil with calf thymus histones, DNA, and isolated rat liver nuclei were studied utilizing labeled fungicide, polyacrylamide gel electrophoresis, and nucleohistone thermal denaturation. The reaction was dependent on pH, type of histones, and the histone and fungicide concentrations. In addition, two types of fungicide-histone binding patterns were observed. These patterns were characterized by the amount of label bound to protein after dialysis or after dialysis and acid precipitation. There was little binding of chlorothalonil to DNA. Polyacrylamide gel patterns of treated lysine-rich calf thymus histones were characterized by a loss of up to 80% of the stained protein without a change in the migration of the bands. Gel patterns of treated calf thymus whole histones were characterized by small variations in band density and minor loss of stained protein. The results of thermal denaturation experiments using chlorothalonil-treated lysine-rich histones and nontreated calf thymus DNA were not different from those using nontreated histones. Almost all of the radioactivity from [ 14 C]chlorothalonil was bound by isolated rat liver nuclei. The binding patterns observed with rat liver nuclei were very similar to those using purified histones. Fractionation of proteins from treated nuclei revealed that the label was unequally distributed between the nuclear sap protein, ribonucleoprotein, and deoxyribonucleoprotein.

The presence of an endophytic fungus, Acremonium coenophialum , in tall fescue ( Festuca arundinacea ) deterred aphid feeding by Rhopalosiphum padi and Schizaphis graminum. Both species of aphid were unable to survive when confined to endophyte-infected tall fescue plants. Feeding deterrents and toxic factors to R. padi and Oncopeltus fasciatus , large milkweed bug, were primarily associated with a methanol extract obtained when endophyte-infected tall fescue seed was serially extracted with hexane, ethyl acetate, and methanol. The concentrations of pyrrolizidine alkaloids were determined to be 30 to 100 times greater in the methanol extract than in the hexane and ethyl acetate extracts.

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.

The effects of different incubation conditions on toxicity and uptake of imazalil by mycelium of a wild-type (003) and fungicide-resistant (R264) strain of Aspergillus nidulans were determined. In agar plate and liquid shake culture, imazalil was 200 and 40 times less toxic to the R264 strain, respectively, than to the wild-type strain. Inhibition of C-4 desmethyl sterol (ergosterol) biosynthesis occurred rapidly in mycelium of both strains at minimum growth inhibitory concentrations. Imazalil was neither detoxified nor converted into a toxic compound by mycelial suspension. Increased uptake of imazalil by the two strains occurred as concentrations of the fungicide were increased. However, the percentage uptake of imazalil by the wild-type strain was highest at the lowest concentration. These results suggest that binding in the wild-type strain involved a small number of high-affinity sites which became saturated as fungicide concentrations increased; and that at higher concentrations considerable nonspecific binding occurred in both strains. Uptake of imazalil during the initial 10 min of incubation was considerably lower in resistant than in the wild-type strain. However, upon prolonged incubation, both strains took up near equal amounts of fungicide. Uptake of fungicide by both strains was not inhibited by incubation at low temperature but was stimulated by respiratory inhibitors. These data support, in part, the hypothesis that resistance to imazalil, as reported previously for fenarimol (M. A. de Waard and J. G. M. van Nistelrooy, Pestic. Biochem. Physiol. 13, 255 (1980)), is based on reduced uptake of fungicide by mycelium of the resistant mutant.

Two bioassay methods are described which use detached tobacco leaves to measure the sensitivity of Peronospora tabacina to systemic fungicides. Tobacco leaves (13–15 cm2), treated with fungicides before or after detachment from the plant, were inoculated with sporangia in water drops and, after incubation in beakers and Petri plates, the disease severity and/or production of sporangia was determined 4–7 days after treatment with the fungicides. Of 15 systemic fungicides applied to detached leaves, eight N-phenylamides at 0.066−1.0 μg ml−1 controlled blue mould; metalaxyl was the most effective fungicide. Isolates of P. tabacina, collected in the field from tobacco plants grown in soil treated with metalaxyl, were not resistant to the fungicide applied to detached leaves prior to inoculation. The fungicide, applied to leaves before detachment, was used to measure the efficacy of five systemic N-phenylamide fungicides sprayed on the basal and unsprayed distal portions of the leaves. Blue mould was controlled on the basal portion of the leaf by all the fungicides at 0.66−1.0 μg ml−1, but it required the application of 3–30 times more chemical on the basal portion to achieve comparable blue mould control on the distal part of the leaf.