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Reduced blood flow to peripheral and core body tissues in sheep and cattle induced by endophyte-infected tall fescue.

Four wethers and 14 steers (environmentally heat stressed, 32 degrees C, 60% relative humidity) were evaluated for changes in blood flow induced by endophyte-infected tall fescue. Concentration of the ergopeptide ergovaline was used as an indicator of diet toxicity due to the endophytic fungus Acremonium coenophialum. Blood flow to specific tissues was measured using radiolabeled microspheres. Wethers received one of two dietary treatments for 30 d before determination of tissue blood flow: 1) a low-endophyte diet (less than .05 ppm ergovaline) or 2) a high-endophyte (1.18 ppm ergovaline) diet. Blood flows to the adrenal glands and skin covering the inner hind leg were less (P less than .10) in wethers consuming the high-endophyte diet than in those consuming the low-endophyte diet. Tissue blood flows in steers were determined on two occasions: 1) after steers had received a low- (less than .01 ppm ergovaline) or high-endophyte (.52 ppm ergovaline) fescue diet for 14 d and 2) 8 d after steers had been switched to a common, fescue-free diet. Blood flows to skin covering the ribs, cerebellum of the brain, duodenum, and colon were less (P less than .10) in steers consuming the high-endophyte diet. However, 8 d after consuming fescue-free diets, steers that had previously consumed the high-endophyte diet had greater (P = .08) blood flow to the coronary bands of the front hooves than steers that had consumed the low-endophyte diet. Blood flows to all other tissues were similar between treatments. We inferred from these experiments that the toxin(s) associated with endophyte-infected tall fescue caused decreased blood flow to peripheral and core body tissues and that this effect was abated within 8 d of removing the toxin(s).

Adrenal Glands↗

Comparison of two ammoniation procedures to reduce the toxicity of endophyte-infected tall fescue seed fed to rats.

To determine the effect of extending the duration of ammonia (2% dry matter basis) treatment from 1 to 5 wk on the toxicity of endophyte-infected tall fescue seed, 60 male Harlan Sprague-Dawley rats were randomly assigned to the following six treatments during a 28-d trial: endophyte-free (E-), endophyte-infected (E+), 1 wk ammoniated endophyte-free (1AE-), 1 wk ammoniated endophyte-infected (1AE+), 5 wk ammoniated endophyte-free (5AE-), and 5 wk ammoniated endophyte-infected (5AE+) tall fescue seed. The concentration of total pyrrolizidine alkaloids (N-acetyl and N-formyl loline) of E+ fescue was reduced from 4203 micrograms/g to 3009 and 2533 micrograms/g by the 1AE+ and 5AE+ treatments, respectively. Ergovaline was lowered from 3.77 to 1.57 micrograms/g by 1AE+ and eliminated by 5AE+. Endophyte-infected treatment groups had depressed (P < 0.0001) daily feed intakes (DFI), daily weight gains (DWG), feed efficiencies (G/F), primary antibody responses, and T cell and B cell mitogenic responses than endophyte-free treatment groups. Ammoniation of endophyte-infected fescue seed improved DFI and DWG (P < 0.0001) and G/F (P < 0.05); however, there was no difference in performance criteria between the 1-wk and 5-wk ammoniation treatments. Endophyte-induced depressions in immune function were not alleviated by ammoniation.

Acremonium↗

Comparison of two ammoniation procedures to reduce the toxicity of endophyte-infected tall fescue seed fed to rats.

To determine the effect of extending the duration of ammonia (2% dry matter basis) treatment ti'om 1 to 5 wk on the toxicity of endophyte-infected tall fescue seed, 60 male Harlan Sprague-Dawley rats were randomly assigned to the following six treatments during a 28-d trial: endophyte-free (E-), endophyte-infected (E+), 1 wk ammoniated endophyte-fee (1AE-), 1 wk ammoniated endophyte-infected (1AE+), 5 wk ammoniated endophyte-free (5AE-), and 5 wk ammoniated endophyte-infected (5AE+) tall fescue seed. The concentration of total pyrrolizidine alkaloids (N-acetyl and N-formyl loline) or E+ rescue was reduced from 4203 12 g/g to 3009 and 2533 I-tg/g by the 1AE+ and 5AE+ treatments, respectively. Ergovaline was lowered from 3.77 to 1.57 12 g/g by 1AE+ and eliminated by 5AE+. Endophyte-infected treatment groups had depressed (P < 0.0001) dally feed intakes (DFI), daily weight gains (DWG), feed efficiencies (G/F), primary antibody responses, and T cell and B cell mitogenic responses than endophyte-free treatment groups. Ammoniation of endophyte-infected rescue seed improved DFI and DWG (P < 0.0001) and G/F (P < 0.05); however, there was no difference in performance criteria between the 1-wk and 5-wk ammoniation treatments. Endophyte-induced depressions in immune function were not alleviated by ammoniation.

Acremonium↗

Characterization of culturable endophytes and microbial communities in the rhizosphere and pitcher fluid of the carnivorous plant Nepenthes khasiana.

Endophytes colonize plant tissues through roots and shoots without causing harm and can move throughout the plant via its vascular system. However, little is known about culturable endophytes, particularly bacteria, in pitcher plants, and their possible entry through pitcher fluid remains unexplored. To address this gap, we isolated endophytes from the pitcher plant Nepenthes khasiana, and performed metagenomic analysis of its rhizospheric soil and pitcher fluid, from which bacteria and fungi were also isolated, to investigate the possible origin of these endophytes. We found that culturable endophytic bacteria were predominantly associated with roots and seeds, whereas endophytic fungi were more abundant across the N. khasiana pitcher. Although most endophytes were restricted to specific tissues, some exhibited a broader distribution across nearly the entire plant. Several OTUs from the rhizospheric soil matched endophytes at the genus level, including some that were also detected in the pitcher fluid. Specifically, three bacterial genera - Enterobacter, Staphylococcus and Bacillus - and one fungal genus, Cladosporium, detected in the pitcher fluid, matched the isolated endophytes. These findings suggest that endophytes in N. khasiana most likely originate from the rhizosphere, with possible migration into the pitcher fluid.

Rhizosphere↗

Fungal epiphytes and endophytes of coffee leaves (Coffea arabica).

Plants harbor diverse communities of fungi and other microorganisms. Fungi are known to occur both on plant surfaces (epiphytes) and inside plant tissues (endophytes), but the two communities have rarely been compared. We compared epiphytic and endophytic fungal communities associated with leaves of coffee (Coffea arabica) in Puerto Rico. We asked whether the dominant fungi are the same in both communities, whether endophyte and epiphyte communities are equally diverse, and whether epiphytes and endophytes exhibit similar patterns of spatial heterogeneity among sites. Leaves of naturalized coffee plants were collected from six sites in Puerto Rico. Epiphytic and endophytic fungi were isolated by placing leaf pieces on potato dextrose agar without and with surface sterilization, respectively. A total of 821 colonies were isolated and grouped into 131 morphospecies. The taxonomic affinities of the four most common nonsporulating fungi were determined by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region: two grouped with Xylaria and one each with Botryosphaeria and Guignardia. Of the most common genera, Pestalotia and Botryosphaeria were significantly more common as epiphytes; Colletotrichum, Xylaria, and Guignardia were significantly more common as endophytes. Suprisingly, more morphospecies occurred as endophytes than as epiphytes. Differences among sites in number of fungi per plant were significant. Thus epiphytic and endophytic communities differed greatly on a single leaf, despite living only millimeters apart, and both communities differed from site to site. Significant correlations between occurrence of fungal morphospecies suggested that fungi may have positive or negative effects on their neighbors. This is the first quantitative comparison of epiphytic and endophytic fungal floras in any plant, and the first to examine endophytic fungi or epiphytic fungi in leaves of coffee, one of the world's most valuable crops.

Classification↗

Relationships among non-Acremonium sp. fungal endophytes in five grass species.

Many cool-season grasses (subfamily Pooideae) possess maternally transmitted fungal symbionts which cause no known pathology and often enhance the ecological fitness and biochemical capabilities of the grass hosts. The most commonly described endophytes are the Acremonium section Albo-lanosa spp. (Acremonium endophytes), which are conidial anamorphs (strictly asexual forms) of Epichloë typhina. Other endophytes which have been noted are a Gliocladium-like fungus in perennial ryegrass (Lolium perenne L.) and a Phialophora-like fungus in tall fescue (Festuca arundinacea Schreb.). Here, we report the identification of additional non-Acremonium sp. endophytes (herein designated p-endophytes) in three more grass species: Festuca gigantea, Festuca arizonica, and Festuca pratensis. In each grass species, the p-endophyte was cosymbiotic with an Acremonium endophyte. Serological analysis and sequence determinations of variable portions of their rRNA genes indicated that the two previously identified non-Acremonium endophytes are closely related to each other and to the newly identified p-endophytes. Therefore, the p-endophytes represent a second group of widely distributed grass symbionts.

Base Sequence↗

Growth rate and physiology of steers grazing tall fescue inoculated with novel endophytes.

Cattle grazing tall fescue (Festuca arundinacea Schreb.) often develop fescue toxicosis. This condition is thought to be caused by ergot alkaloids produced by the endophyte Neotyphodium coenophialum. Endophytes from wild tall fescue plants, which do not produce ergot alkaloids, were transferred into the endophyte-free tall fescue germplasm, HiMag. The novel associations also lacked the ability to produce ergot alkaloids. Our objective was to determine whether cattle grazing these novel endophyte associations showed signs of fescue toxicosis. At the Fayetteville, Arkansas location, tester steers (n = 72) were assigned to one of four pasture treatments: endophyte-free HiMag tall fescue (HiMag-); 'Kentucky-31' tall fescue infected with its native, toxic endophyte (KY+); and two novel endophyte-infected tall fescue associations, HiMag4 and HiMag9. At the Mount Vernon, Missouri location, steers (n = 54) were used to test three of the four cultivars (HiMag9 was not tested). Ergot alkaloid concentrations in the forage of HiMag4 and HiMag9 were low or undetectable. Respiration rate, rectal temperature, ADG, and hair scores were measured during the grazing period. Blood was collected via jugular venipuncture and used for prolactin, aspartate aminotransferase, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), cholesterol, triglyceride, and creatinine analysis. Weight gains by steers grazing HiMag4 and HiMag9 did not differ from those of steers grazing HiMag-, but were greater than gains (P < 0.05) by steers on the KY+ treatment. Steers grazing KY+ had higher (P < 0.05) respiration rates, rectal temperatures, and hair scores than did steers grazing novel endophyte and HiMag- pastures. Prolactin, ALP, cholesterol, LDH, and triglycerides all were suppressed (P < 0.05) in steers grazing KY+ compared with steers grazing novel endophyte and HiMag- pastures. Steers grazing the novel endophyte tall fescues did not suffer from the decreased weight gains and toxicities associated with fescue toxicosis, resulting in enhanced animal production.

Alkaline Phosphatase↗

Ecology, metabolite production, and substrate utilization in endophytic fungi.

Endophytic fungi are a taxonomically and ecologically heterogenous group of organisms, mainly belonging to the Ascomycotina and Deuteromycotina. The isolation methods affect the species composition of the endophyte assemblage in a given host. The number of endophyte taxa isolated from a host species is usually large; however, only few, normally host specific species or strains are dominant. Endophyte assemblages are specific at the host species level, but species composition and frequencies are significantly affected by site-specific conditions. Moreover, the relative importance and number of endophytic species vary among individuals within sites. In some cases, each individual could be considered a separate ecosystem. In general, however, 40 individuals with 30 to 40 sampling units per organ and individual should be enough to detect 80% of taxa present in a given host at one site. Endophytes usually produce the enzymes necessary for the colonization of plant tissues. Substrate utilization studies and isozyme analysis have demonstrated that most endophytes are able to utilize most plant cell components. The production of growth promoting factors and of metabolites useful in the pharmaceutical and agricultural industry is widespread among endophytic fungi. The usefulness of endophytes in agricultural and pharmaceutical research is briefly discussed.

Ecology↗

Fungal endophytes in seeds and needles of Pinus monticola.

Using a sequence-based approach, we investigated the transmission of diverse fungal endophytes in seed and needles of Pinus monticola, western white pine. We isolated 2003 fungal endophytes from 750 surface-sterilized needles. In contrast, only 16 endophytic isolates were obtained from 800 surface-sterilized seeds. The ITS region was sequenced from a representative selection of these endophytes. Isolates were then assigned to the most closely related taxa in GenBank. Although 95% of the endophytes in needles from mature trees belonged to the Rhytismataceae, 82 unique ITS sequences were obtained from at least 21 genera and 10 different orders of fungi. Significantly, none of the endophytes in seed were rhytismataceous (chi(2) = 180; P < 0.001). Similarly, needles of greenhouse seedlings yielded only non-rhytismataceous isolates, whereas seedlings of the same age that had naturally regenerated near older white pines in roadless areas were colonized by rhytismataceous endophytes almost to the same extent as in mature trees. Only one of 17 rhytismataceous isolates were able to grow on a medium containing only 0.17% nitrogen, whereas 25 of 31 non-rhytismataceous endophytes grew. Rhytismataceous endophytes are dominant in needles of P. monticola, but they appear to be absent in seed, and unlikely colonists of nitrogen-limiting host tissues such as the apoplast.

Ascomycota↗

Production of swainsonine by fungal endophytes of locoweed.

Consumption of locoweeds, legumes endemic in arid western USA, has long been associated with locoism, a disease of ruminant animals. To explore the relationship between fungi associated with locoweed and locoweed toxicity, 11 locoweed populations from various sites in New Mexico were assessed for endophytic fungi. Endophytes were isolated from the leaves, stems, seeds, and flowers of eight populations of the toxic locoweeds Astragalus mollissimus, Oxytropis lambertii, and O. sericea. Fungal cultures grew very slowly and sporadically produced subcylindrical conidia with very dark transverse septa. All cultured endophytes produced the alkaloid swainsonine, which causes locoism. Endophyte-infected locoweed populations produced swainsonine, and the swainsonine level of endophyte strains in vitro was highly correlated with the swainsonine level of their host plant populations. The rDNA ITS from mycelia from four endophyte isolates and beta-tubulin encoding regions from mycelia of 18 fungal endophyte isolates were amplified using PCR and the nucleic acid sequences were analyzed. The nucleic acid sequences of the beta-tubulin encoding regions were essentially identical among all the endophytes regardless of plant genus and locations. Morphological evidence and sequence analysis of the ITS region suggest that the endophytes are most closely related to Embellisia. However, with the paucity of Embellisia species represented in sequence databases, precise taxonomic placement will await further study.

Animals↗

Grass-herbivore interactions altered by strains of a native endophyte.

Many plants support symbiotic microbes, such as endophytic fungi, that can alter interactions with herbivores. Most endophyte research has focused on agronomically important species, with less known about the ecological roles of native endophytes in native plants. In particular, whether genetic variation among endophyte symbionts affects herbivores of plant hosts remains unresolved for most native endophytes. Here, we investigate the importance of native isolates of the endophyte Epichloë elymi in affecting herbivory of the native grass host, Elymus hystrix. Experimental fungal isolate-plant genotype combinations and endophyte-free control plants were grown in a common garden and exposed to natural arthropod herbivory. Fungal isolates differed in their effects on two types of herbivory, chewing and scraping. Isolates exhibiting greater sexual reproduction were associated with greater herbivore damage than primarily asexual isolates. Endophyte infection also altered patterns of herbivory within plants, with stroma-bearing tillers experiencing up to 30% greater damage than nonstroma-bearing tillers. Results suggest that intraspecific genetic variation in endophytes, like plant genetic variation, can have important 'bottom-up' effects on herbivores in native systems.

Animals↗

Effect of microbial inoculants on the indigenous actinobacterial endophyte population in the roots of wheat as determined by terminal restriction fragment length polymorphism.

The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected.

Actinomycetales↗

Physiological responses in rats fed extracts of endophyte infected tall fescue seed.

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.

Acremonium↗

Endophyte-infected tall fescue diet alters gene expression in heifer luteal tissue as revealed by interspecies microarray analysis.

Cattle consuming endophyte-infected tall fescue grass have an associated reduction in circulating progesterone and reduced reproductive rates. In this study, commercially available rat microarrays were used to analyze the gene expression in luteal tissues from heifers fed endophyte-free fescue, endophyte-infected fescue, or endophyte-infected fescue supplemented with the dopamine (DA) antagonist, domperidone. The number of hybridized spots represented approximately 40% of the total 10,000 rat genes/ESTs evaluated. Each luteal sample was analyzed in triplicate, resulting in within treatment correlation coefficients of >/=0.98. Median values of mRNA abundance from luteal tissue taken from the endophyte-infected fed heifers revealed 598 genes and ESTs that were down regulated and 56 genes and ESTs that were upregulated compared with luteal mRNA values from the endophyte-free treatment. There were fewer comparative differences between median values from luteal mRNA from the endophyte-free versus feeding endophyte-infected plus domperidone treated heifers. Only 19 genes and ESTs were upregulated and two were down-regulated.

Animal Feed↗

Growth and ovarian function of weanling and yearling beef heifers grazing endophyte-infected tall fescue pastures.

Growth and ovarian function of crossbred beef heifers grazing low and high endophyte-infected tall fescue pastures were studied for 2 successive years. In April of each year, 20 weanling and 20 yearling heifers were included in the study. All heifers were weighed at 28-d intervals for 112 d. Blood samples were collected from each heifer on Day 0 and +7 of each of five 28-d periods and analyzed for progesterone concentration. Heifers with progesterone concentrations >/= 1.5 ng/ml on either or both Day 0 and +7 were classified as having normal cyclic ovaries. High endophyte-infected fescue pastures adversely altered the ovarian activity (P < 0.05) of weanling heifers in both years. In each trial, average weight gains were lower (P < 0.05) in yearling and weanling heifers grazing the high endophyte-infected pastures than in heifers grazing low endophyte-infected pastures. In 1992, heifers were synchronized with PGF(2alpha) administered on Days 101 and 112. Blood samples were collected on 0, 4, 8 and 12 d after the second PGF(2alpha) injection for progesterone analysis. Heifers grazing high and low endophyte-infected pastures were pastured separately with 4 bulls each and were given heatmount detectors. At 96 h, less estrus activity was observed (P < 0.10; power=0.63) in weanling heifers grazing the high vs. low endophyte pastures although pregnancy rates were similar for all groups. Progesterone concentrations suggested that weanling heifers on the high endophyte pastures had a higher incidence of luteal dysfunction after PGF(2alpha) synchronization. In summary, high endophyte-infected pastures decreased growth in both weanling and yearling heifers, ovarian activity and luteal function were adversely altered in weanling heifers with subsequent decreased estrus response to estrus synchronization.

Journal Article↗

Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants.

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.

Alkaloids↗

Model systems in ecology: dissecting the endophyte-grass literature.

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

Animals↗

Endophytic fungi associated with cacti in Arizona.

21 cactus species occurring in various localities within Arizona were screened for the presence of fungal endophytes. 900 endophyte isolates belonging to 22 fungal species were isolated. Cylindropuntia fulgida had the maximum endophyte species diversity, while C. ramosissima harboured the maximum number of endophyte isolates. Alternaria sp., Aureobasidium pullulans, and Phoma spp. were isolated from several cactus species. The diversity of the endophyte assemblages was low and no host specificity among endophytes was observed. However, the frequencies of colonization of the few endophyte species recovered were high and comparable to those reported for tropical plant hosts. Species of Colletotrichum, Phomopsis, and Phyllosticta, which are commonly isolated as endophytes from plants of more mesic habitats, were absent from these cacti.

Agriculture↗