No description available.

Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three putative knockouts were identified. Southern blot analysis of digested genomic DNA indicated that homologous gene replacement at dmaW2 locus took place while dmaW1 was still present. Chromosome separation followed by Southern-blot hybridization showed that the dmaW genes in N. coenophialum are located on different chromosomes. The aim of this study was to obtain a nontoxic endophyte free of marker genes that could be used to inoculate popular tall fescue cultivars. Therefore the Cre/loxP system developed in this study allows reusing the marker gene for sequential transformations. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum, or Epichloë festucae isolates, containing a floxed hygromycin phosphotransferase (hph) gene (loxP::hph::loxP), were transfected with a Crerecombinase expression plasmid and then cultured without selection. The marker was excised in 0.5-2% of the colonies, leaving a single loxP sequence. This strategy will help to reduce the concerns related to field release or commercialization of economically important grasses associated with manipulated fungal strains. It is expected that the technology will likely be adapted and applied in other fungal species. Manipulation of the ergot alkaloid (EA) gene cluster from C. purpurea and C. fusiformis by introducing and expressing its genes in different fungal-grass symbionts was also investigated. Heterologous expression of the ergot alkaloid cluster could result either in the synthesis of compounds similar to the ones produced by the host or in synthesis of novel compounds with new modes of action. Even though the results indicated that several EA genes were expressed in the new symbiota, none of the ergot alkaloids intermediates were detected.

Demands are placed on cattle producers to provide a steady supply of beef at a competitive price. Producers must maximize beef output while minimizing input expenses without compromising product quality. The use of growth implants has become a common practice among cattle producers. The objective of this study was to evaluate the effects of two implant strategies on the average daily gain of suckling calves rotationally grazed on Kentucky – 31 endophyte-infected tall fescue and Kentucky – 31 non-endophyte-infected tall fescue. Eighteen cows with spring calves (N = 18) were used in this study. Calves were grouped by birth date into four blocks. Within each block, calves were stratified by sex and 45d of age body weight into three implant treatment groups for a total of six calves per treatment (n = 6, control 90.3 ± 9.7 kg, zeranol 102.9 ± 10.9 kg, and progesterone (100 mg) and estradiol benzoate (10 mg) 92.4 ± 10.3 kg). Calves were weighed and re-implanted at 129 d of age (84 d after initial implant). Zeranol treated calves were re-implanted using the same implant as the initial implant. Progesterone and estradiol benzoate treated calves were re-implanted after reaching a minimum body weight of 181 kg with either 200 mg progesterone and 20 mg estradiol benzoate or 200 mg testosterone propionate and 20 mg estradiol benzoate depending on sex. Data were analyzed using the REPEATED function in the MIXED procedure of SAS. No interactions were found among sex and treatments for 84d weight gains and 140d weights. There were also no main effects found for 84d weight gains and 140d weight gains. Forage analysis suggested that low crude protein and energy content may have contributed to the low ADG. Low endophyte concentrations may also have played a role.

Bacteria that provide benefit to plants are considered to be plant growth-promoting bacteria (PGPB) and can facilitate plant growth by a number of different mechanisms. Plant growth-promoting bacteria that are able to utilize the plant compound 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen, as a consequence of possessing the enzyme ACC deaminase, can protect host plants from a number of environmental stresses. In addition to ACC deaminase, PGPB may utilize other mechanisms to facilitate plant growth including IAA synthesis, siderophore production, phosphate solubilization activity, ammonia production, and antibiotic production. Plant growth-promoting bacterial endophytes employ similar plant growth promotion mechanisms to those used by rhizospheric PGPB. In fact, bacterial endophytes are PGPB that go one step further and colonize the inside of the plant tissues and provide more efficient and prompted protection to their hosts compared to those that bind exclusively to the plant’s rhizosphere. Therefore, it is likely that endophytic plant growth-promoting bacteria will be superior to similar non-endophytic bacterial strains in promoting plant growth under a wide range of environmental conditions. In the work reported here, new bacterial endophytes were isolated and characterized. Among twenty-five ACC deaminase positive strains, two best strains were selected and ACC deaminase deficient mutants were constructed. The ability of two newly isolated 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant growth-promoting bacterial endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6 and their ACC deaminase deficient mutants was shown to 1) delay the senescence of mini carnation cut flowers and 2) to facilitate tomato plant growth under salinity stress. In the mini carnation flower senescence evaluation, the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, our results demonstrate that this enzyme is directly responsible for a significant delay in flower senescence. Despite containing ACC deaminase activity, the rhizosphere-binding PGPB Pseudomonas putida UW4 was not taken up by the cut flowers and therefore had no effect on prolonging flower shelf life. In evaluating the effect of bacterial endophytes under salt stress, tomato plants treated with either of the wild-type strains of the two selected bacterial endophytes demonstrated early flowering and fruiting and had significantly greater numbers of flowers, buds, and fruits than either the corresponding ACC deaminase mutant strain-treated plants or the control plants. Although both bacterial endophytes P. fluorescens YsS6 and P. migulae 8R6 showed significant plant growth-promotion capabilities, P. migulae 8R6 demonstrated better plant growth facilitation under salt stress than did P. fluorescens YsS6. P. migulae 8R6 treated tomato plants demonstrated the least sodium uptake, the highest chlorophyll content, and highest fresh and dry biomass. The results of the work presented here suggest that ACC deaminase containing selected bacterial endophytes could be employed as environmentally friendly adjuncts to agricultural and horticultural practice.

The environmental impact of bitumen mining in the Athabasca region of Canada is of growing concern. Among these concerns is the need and difficulty to remediate and reclaim affected land, including tailing sands (TS), a byproduct of the hot water extraction used to separate bitumen from solid materials. Current reclamation methods consist of multiple steps and take several decades to be effective. The primary reason for the difficulty in reclaiming disturbed land is the harsh environment found within the TS combined with the scale of the problem. TS are extremely nutrient poor, having below-detectable levels of NPK and extremely low C and S. In addition to this TS have pHs outside of environmental normals, and are hydrophobic due to residual hydrocarbons. Previously, an endophytic fungus, Trichoderma harzianum strain TSTh20-1, was isolated from pioneer plants growing naturally on TS sites, and was found to promote plant growth on TS. In my study TSTh20-1 was also found to increase the rate of drought recovery, and to enhance seed germination rates on a variety of soils. Suitable application methods were explored for this endophyte, including seed coatings, granules, as well as direct application to plant/soil. Regardless of method, TSTh20-1 was found to successfully colonize the plants. Twenty-four species of grasses, forbs, and legumes were tested for their ability to grow on TS. The four most successful species (Trifolium repens, Bouteloua gracilis, Medicago sativa, and Elymus trachycaulus) were put into a seed mixture for use in experiments. In mesocosm-scale experiments, plant health and soil parameters were measured after 2 months of growth. Hydrocarbon analysis of the first mesocosm showed a 2.7-fold increase in total hydrocarbons when TSTh20-1 and plants were present, suggesting degradation of large hydrocarbons beyond the scope of the analysis. A repeat experiment using a different source of tailings did not yield this same result. This is most likely due using a source of tailings that had substantially different chemical characteristics. TSTh20-1 was also analyzed for its ability to produce plant hormones or siderophores, to increase peroxidase enzyme activity, to protect plants from reactive oxygen species, and to solubilize phosphate precipitates from soil. All of these are known mechanisms microbes use to promote plant growth.

No description available.

Epichloë species and their anamorphs, Neotyphodium species, are fungal endophytes that inhabit cool-season grasses and often produce bioprotective alkaloids. These alkaloids include lolines, which are insecticidal and insect feeding deterrents. Lolines are exo-1-aminopyrrolizidines with an oxygen bridge between carbons 2 and 7, and are usually methylated and formylated or acetylated on the 1-amine. In previously published studies lolines were shown to be derived from the amino acids L-proline and L-homoserine. In addition the gene cluster involved in loline-alkaloid biosynthesis has also been characterized. In this dissertation a survey of plant-endophyte symbioses revealed a phenotype with only N-acetylnorloline. This phenotype provided insights into loline alkaloid production. This dissertation focuses on determining the steps to loline biosynthesis after the amino acid precursors. The study involves feeding isotopically labeled potential precursors to loline-alkaloid-producing cultures of Neotyphodium uncinatum, as well as RNA interference (RNAi) of N. uncinatum genes for steps in the pathway. Synthesized deuterated compounds were fed to loline-alkaloid-producing cultures of N. uncinatum to test their possible roles as precursors or intermediates in the loline-alkaloid pathway. N-Formylloline was extracted from the cultures and assayed by GCMS for incorporation of the deuterium label. The results indicated that N-(3-amino, 3-carboxy)propylproline and exo-1-aminopyrrolizidine are intermediates in the loline-alkaloid biosynthetic pathway. Plasmids were also designed for expression of double-stranded RNA homologous to loline-alkaloid biosynthesis genes, and introduced by transformation into N. uncinatum. This RNAi strategy resulted in fungal transformants altered in loline-alkaloid profiles. The RNAi results indicated that N-acetyl-1-aminopyrrolizidine is the intermediate before oxygen bridge formation. Based on the results of this study and the likely roles of the loline-alkaloid biosynthesis genes inferred from signature sequences of their predicted protein products, I propose a pathway of bond formation steps in loline-alkaloid biosynthesis.

Tall fescue is the most common cool-season grass in the eastern USA, with broad economic and ecological importance to the region. Tall fescue is known to associate with a fungal endophyte, Neotyphodium coenophialum, whose presence can decrease biotic and abiotic stress experienced by the plant. This thesis evaluates the response of tall fescue and the fungal endophyte symbiosis to predicted climate change. I participated in two multi-factor climate change projects where I investigated the response of tall fescue tissue chemistry and growth to various climate change factors. Endophyte-infected (E+) tall fescue had decreased alkaloid production under elevated CO2 but increased alkaloid production under elevated temperatures. Significant differences between E+ and E- (endophyte-free) tall fescue tissue chemistry were also found, suggesting the endophyte interacts with the plant response to abiotic stress. Although several studies have reported benefits of endophyte infection for tall fescue growing under drought stress, my research found no differences between E+ and E- total growth and surprisingly showed increased mortality of E+ individuals under elevated temperature. Taken together, my research indicates that this grass-fungal relationship will respond to climate change, and may produce dramatic and unforeseen results that question the widely believed mutualistic nature of the symbiosis.

Ten wether lambs were used to determine the effects of ergovaline consumption from endophyte infected tall fescue, on nutrient utilization and metabolism. Lambs were fed a diet of 23% endophyte free tall fescue seed (E-) and 77% concentrate from d -14 to -1 (adaptation phase). On d 0, five lambs were switched to an endophyte infected seed diet (E+) where they remained through d 14. Nutrient digestibilities tended to increase from adaptation through the acute (d 1 to 4) to subacute (d 10 to 14) phases when E- was fed. E+ digestibilities were highest (P < 0.05) in the acute phase. Lambs fed E+ had higher rectal temperatures in the acute (P < 0.01) and subacute phases (P < 0.05). Fecal recovery of ergovaline increased as day of collection increased in the acute and subacute phases. Lysergic acid fecal recovery increased with day of collection in the acute phase, but no effect was found in the subacute phase. Serum enzyme analyses did not indicate tissue damage from alkaloid consumption. These results demonstrate lambs try to adapt to endophyte infected fescue seed consumption through increased nutrient digestibilities and increased ergovaline and lysergic acid excretion.

Pinus radiata is the most planted tree in the southern hemisphere. The planted trees are especially susceptible to pathogens, but even the native population, nowadays limited tomerely five locations, are threatened by diseases caused by arthropods, fungi and dehydration. Endophytes are bacteria or fungi that reside inside healthy plant tissue, and often have a beneficial effect on their hosts. Endophytes can help plants adapt to abiotic stress such as drought and protect them against pathogens and insect pests. Given the roles that endophytes play in host stress responses, it is possible that without studying endophytes we may not fully understand a plant’s response to increased temperatures and climate-induced disease.Using Illumina-sequencing of the 16S rRNA-gene the bacterial endophyte community in 15 trees from three of the remaining native populations were studied. By investigating trees from several sites geographical community differences were discovered. The three overall most dominating bacterial taxa can all be connected with genera known to contain members withanti-fungal properties.