Some endophytes in mutualistic associations with Festuca, Lolium and other grass species produce insecticidal loline alkaloids (1-aminopyrrolizidines; LA). These loline alkaloids have a saturated pyrrolizidine ring system (two-rings sharing a carbon and nitrogen atom), a 1-amine substituted with methyl, acetyl, or formyl groups, and an oxygen bridge between C-2 and C-7. The development of a reliable system of production of LA in cultures of the Lolium pratense (meadow fescue) endophyte, Neotyphodium uncinatum, facilitated work on the LA biosynthetic pathway. N. uncinatum produced norloline, loline, methylloline, N-acetylnorloline (NANL), N-formylloline (NFL), and N-acetylloline as detected in culture filtrates. The total production of the two most abundant alkaloids, NANL and NFL, approached 1000 g ml-1 of fungal filtrate. 1H and 13C chemical shifts were previously reported for this group of alkaloids. Extraction and synthesis of sufficient quantities of the alkaloids allowed determination of previously unknown 15N chemical shifts of some LA. Knowledge of 13C and 15N chemical shifts allowed identification of precursors by feeding stable-isotope-labeled compounds. Initially, due to structural similarity to other plant pyrrolizidines, this study examined putrescine and spermidine as possible precursors to LA. Feeding of 14C putrescine to the fungal cultures failed to demonstrate any enrichment in the LA, but enriched spermidine. In contrast, cultures fed with positionally labeled 2H, 13C and 15N amino acids namely, L-ornithine, L-proline, L-aspartate, L-homoserine, and L-methionine demonstrated specific isotopic enrichment in NFL. Determination of the enrichment from the labeled amino acids utilized 13C and 15 N NMR (nuclear magnetic resonance) and gas chromatography-mass spectroscopy (GC-MS). This study allowed the biosynthetic origins of all carbons and nitrogens of NFL to be determined. NFL incorporated L-proline into the B-ring and L-homoserine into the A-ring and 1-amine. The results strongly indicated that polyamines are not precursors of LA and implicated a novel biochemical pathway for the synthesis of LA.

Two experiments were conducted at the University of Kentucky, Eden Shale Farm, Owenton, KY to evaluate the use of MTB-100TM (Alltech, Inc., Nicholasville, KY) to alleviate the symptoms of fescue toxicity when lactating Angus x Beefmaster cows and their calves grazed endophyte-infected KY-31 tall fescue. Experiment 1 provided a carbohydrate based toxin adsorbent, MTB-100TM, ad libitum in a commercial mineral supplement to project a daily consumption rate of 0, 20 or 40 g of MTB-100TM per cow. Cows were weighed, assigned a body condition score (BCS) and hair coat score (HC), rectal temperatures were recorded and fecal grab samples were taken for ergovaline (EV) and lysergic acid (LA) analysis every 35 days for three grazing seasons (May to September). Calves were also weighed and assigned a HC score. Although MTB-100TM did not improve cow or calf performance, cows older than 4 years and those with greater Beefmaster breeding exhibited a higher tolerance to fescue toxicity than 2 and 3-yr-olds and cows with greater Angus breeding. Experiment 2 was conducted to evaluate the response of lactating beef cows and their calves to strategic supplementation with MTB-100TM. MTB-100TM was mixed with a complete mineral so daily intake was projected to be 0 or 20 g/cow. The experimental period extended from May 5 to October 2 and was divided into 3 strategic periods: P1 = May 5 to July 5; P2 = July 5 to August 31; P3 = August 31 to October 2. Treatments were either 0 or 20 g•cow-1•d-1 MTB-100TM within a period (Treatment 1 = 0, 0, 0; Treatment 2 = 20, 0, 20; Treatment 3 = 0, 20, 0; Treatment 4 = 20, 20, 0; and Treatment 5 = 20, 20, 20). Cow and calf performance was measured the same as Exp. 1, but every 21 days. Supplementation early in the grazing season tended to improve cow weight gain and body condition; however, there was no effect on calf performance. Fecal output of EV and LA did not increase in either experiment with supplementation. In conclusion, strategically invoked MTB-100TM consumption can increase performance of cows grazing endophyte-infected tall fescue forage.

The decomposition of plant biomass is associated with changes of the associated fungal community which is crucial in the process of degradation due to their production of extracellular enzymes. Moreover, fungal communities as well as enzymatic activity are specific for soil microhabitats. This thesis focused on the importance of fungi associated with the roots of Norway spruce (Picea abies) trees. Forest with dominating Norway spruce, located in the School Forest Enterprise Křtiny area in Moravia, was selected for the experiment. The aim of this thesis was to compare the community composition of fungi in the roots, rhizosphere and bulk soil and to describe the gradual development of this community after the death of the roots. The next aim was to isolate, describe and characterize those fungi that decomposed roots 5 and 9 months after tree harvest. Identification of fungal isolates was based on DNA sequencing. Degradation capabilities were determined as the activity of enzymes involved in the degradation of lignocellulose. Composition of fungal communities was described by sequencing of environmental DNA on the Illumina platform. Before the flow of assimilates from the live trees stopped, communities of ectomycorrhizal fungi dominated with 60 % of all sequences. Increased abundance of saprotrophic...

Epicoccum nigrum é um fungo ubíquo conhecido por sua capacidade de produzir vários metabólitos secundários bioativos e pelo seu uso potencial como agente de biocontrole contra vários fitopatógenos. Entre os compostos produzidos por E. nigrum, epicolactona é um policetídeo com uma estrutura bastante complexa. O objetivo desta tese foi identificar e caracterizar genes relacionados à biossíntese da epicolactona em E. nigrum. Três mutantes defectivos para a produção de epicolactona anteriormente gerados por mutagênese aleatória foram analisados. Entretanto, os resultados mostraram que o T-DNA provavelmente estava inserido em regiões regulatórias. Usando ferramentas de bioinformática, seis genes de PKSs foram selecionados para deleção. A deleção do gene PKSi12 mostrou que os seus produtos estão relacionados à atividade antagonista. A análise química permitiu a identificação putativa dos preditos precursores da epicolactona, os quais tiveram sua produção afetada no mutante ΔPKSi12. Uma possível via de biossíntese de epicoccona B e epicoccina por E. nigrum foi proposta. / Epicoccum nigrum is a ubiquitous fungus mainly known for its ability to produce many bioactive secondary metabolites and for its potential use as a biocontrol agent against many phytopathogens. Among the compounds produced by E. nigrum, epicolactone is a polyketide with a very complex structure. The aim of this thesis was to identify and characterize genes related to epicolactone biosynthesis in E. nigrum. Three defective epicolactone mutants previously generated by random mutagenesis were analyzed. However, the results showed that the T-DNA was probably inserted in regulatory regions. Using a genome mining approach, six PKS genes were selected for deletion. The deletion of PKSi12 gene showed that its products are related to E. nigrum antagonistic activity against fungal phytopathogens. The chemical analysis allowed a putative identification of the previously proposed epicolactone precursors, which production was affected in the ΔPKSi12 mutant. A proposed biosynthesis of epicoccone B and epicoccine by E. nigrum was suggested.

No description available.

Some aspects of the presence of systemic endophytic fungi in agriculturally important New Zealand grasses were studied in relation to plant breeding. Seedling resistance to adult Argentine stem weevil feeding in perennial ryegrass, Italian ryegrass and tall fescue was found to be related to the presence of their respective Acremonium endophytes in the seed rather than to plant genetic resistance. In addition a study of perennial ryegrass revealed that this resistance was independent of endophyte viability. The seedling resistance conferred by the endophyte of Italian ryegrass was found to be beneficial for field establishment. This endophyte differs from that in perennial ryegrass and tall fescue in that it does not confer resistance to Argentine stem weevil on mature plants, but only on seedlings. The extent of plant genetic seedling tolerance to adult Argentine stem weevil feeding was limited to broad inter-specific differences, with tall fescue more tolerant than perennial ryegrass and both of these more tolerant than Italian ryegrass. This ranking corresponds with previous observations on feeding preference on mature plants. A study of factors affecting the concentration of endophyte mycelia in infected seed of perennial ryegrass revealed that plant genetic factors had little effect. The major factors studied were: 1) the endophyte concentration in the maternal parent plant directly influenced the endophyte concentration in the seed. 2) nitrogen fertilizer applications to a seed crop reduced the concentration of mycelia in the seed, with earlier applications having a greater effect. 3) application of the fungicide propiconazole (Tilt) to a seed crop reduced the endophyte concentration in the seed. 4) the endophyte concentration in the seed was found to directly influence the endophyte concentration in seedlings, six month old plants and that of seed harvested from a first year seed crop. As there have been no previous reports of tetraploid perennial ryegrass cultivars with endophyte an experiment was conducted to determine if these could be developed by the standard procedure of colchicine treatment. The results revealed that endophyte was retained following colchicine treatment.

No description available.

Green roof design has proceeded without integration of arbuscular mycorrhizal fungi (AMF). A literature survey was therefore conducted in order to determine which aspects of green roof functioning might be enhanced by AMF, and subsequently levels of AMF and endophyte colonization were determined for seven plant species used in green roofs. Plants were sampled from an experimental green roof and from the field. A commercial growing medium was also tested for AMF inoculum potential. Colonization was poor in both field and rooftop samples of the commercially popular succulent, Sedum acre, but significantly higher in Solidago bicolor, a proposed green roof species. The commercial growing medium was found to contain extremely low levels of viable AMF propagules. Although the apparent lack of mycorrhizal dependency of S. acre helps to explain its popularity as a green roof plant, its overuse precludes the important ecosystem services potentially provided by AMF symbioses.

Aircraft strikes are a significant safety hazard on airports worldwide. Wildlife management at airfields is the most effective tactic to reduce airstrike risk – to modify the habitat to be undesirable to animals. Tall fescue grasses containing a fungal symbiont may serve that purpose. They produce alkaloids that convey resistance to some grass-feeding invertebrates, which might in turn reduce incidence of insectivorous birds. A commercial endophytic grass (Avanex™) consisting of ‘Jackal’ tall fescue infected with a unique endophyte (AR 601) is purported to contain especially high levels of alkaloids and to reduce bird populations if planted at airports. I evaluated it against the common KY31 tall fescue with its wild-type endophyte for invertebrate and vertebrate deterrence. Invertebrate abundance, survival, growth, and development were generally similar on Jackal E+ or KY31 E+. Spanish goats and wild birds showed no avoidance of Jackal E+, nor did Jackal E+ contain significantly higher levels of alkaloids than did KY31 E+. The Avanex™ tall fescue was not any better than KY31 in deterring herbivores but the concept is sound. However, better understanding of the relationship between grass, endophyte, alkaloid, and herbivore is needed to inform how such grasses might be used to reduce bird strike hazard.

A wild-type endophyte (Neotyphodium coenophialum [(Morgan-Jones & Gams) Glen Bacon & Hanlin]) that infects tall fescue [Lolium arundinaceum (Schreb.) Darbysh. = Schedonorous arundinaceum (Schreb.) Dumort.] imparts tolerances to moisture, heat, and grazing stresses, but also produces ergot alkaloids that adversely affect performance and physiology of cattle. Novel endophytes, developed by AgResearch Ltd. NZ, can sustain fescue persistence and productivity, but do not produce toxic ergot alkaloids. University of Kentucky Plant Breeder, T. D. Phillips Ph.D, developed a tall fescue experimental population (KYFA9301) for the upper transition zone. A 2-yr grazing experiment was conducted with steers to evaluate steer performance and physiology, and forage quality and productivity of KYFA9301 infected with AR584 novel endophyte (AR584) compared with KY31 wild-type endophyte (KY31), endophyte-free KYFA9301 (EF9301) and AR542-‘Jesup’ (MaxQ). Fescue-endophyte combinations were assigned to 1.0-ha pastures in a randomized complete block design with three replications. Pastures were grazed with variable stocking (four testers) from 6 May to 23 July 2008 (76 d), and 2 April to 25 June 2009 (84 d). Shrunk bodyweights were taken at initiation and termination of grazing each year. Average daily gains among MaxQ, AR584, and EF9301 were similar and were greater (P < 0.10) than KY31. Rectal and skin temperatures were collected three times each year at approximately days 28, 56, and study completion, along with blood collection for serum prolactin assay. Rectal and skin temperatures among AR584, MaxQ, and EF9301 were similar and were lower (P < 0.10) than KY31. Serum prolactin concentrations were similar among the three nontoxic varieties and higher (P < 0.10) than KY31. Forage collections were taken at 2 week intervals throughout the study each year and nutritive quality analysis were conducted through wet chemistry to determine forage acid detergent fiber (ADF), neutral detergent fiber (NDF), crude protein (CP), and In-Vitro Dry Matter Digestibility (IVDMD). Nutritive analyses indicated no differences between fescue-endophyte combinations with the exception of EF9301 having higher ADF concentrations (P = 0.031) than KY31 during the dry year of 2008. Results indicated steer performance and physiological responses for KYFA9301, with and without AR584 were enhanced compared to KY31 and similar to those for MaxQ.