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archives@tulane.edu / The influence of environment on microbial community structure is of increasing interest, especially in coastal habitats where climate change is rapidly altering the landscape. In this dissertation, I characterize the microbial communities associated with a key wetland species, the baldcypress tree (Taxodium distichum), and examine the relationship between environment, geographic distance, and microbial community composition. In a culture-based study of T. distichum leaf and root endophytes, I found that both salinity and flooding contributed to bacterial and fungal endophytic community composition. Additionally, I report that diversity and endophyte isolation frequency were higher in roots than in leaves, with leaf bacteria being almost negligible. Using 16S Illumina profiling, I found that geographic distance correlated with rhizosphere but not root endosphere bacterial communities and that mean water level, mean salinity, and the volume of woody debris were correlated with both endosphere and rhizosphere bacterial communities of T. distichum. Finally, using salt challenge assays, I isolated five strains of extreme halotolerant endophytes and eleven strains of moderately halotolerant endophytes— a necessary first step towards using endophytes for restoration, or towards understanding the functions of some of these organisms in situ. This dissertation demonstrates a connection between environmental variables, plant symbionts, and a key restoration species and may help in predicting future outcomes of sea level rise for endophytes communities in baldcypress and other wetland plants. / 1 / Elizabeth Kimbrough

archives@tulane.edu / 1 / Peter Tellez

This dissertation comprises a series of studies designed to explore the associations between plants and the endophytic fungi they harbor in their above-ground tissues. By viewing endophyte diversity in ecologically and economically important hosts through the lenses of phylogenetic biology, microbiology, and biotechnology, this body of work links plant ecology with newly discovered symbiotic units comprised of endophytic fungi and the bacteria that inhabit them.This work begins with a large-scale survey of endophytic fungi from native and non-native Cupressaceae in Arizona and North Carolina. After isolating over 400 strains of endophytes, I inferred the evolutionary relationships among these fungi using both Bayesian and parsimony analyses. In addition to showing that native and introduced plants contained different endophytes, I found that the endophytes themselves harbor additional microbial symbionts, recovering members of the beta- and gamma-proteobacterial orders Burkholderiales, Xanthomonadales, and Enterobacteriales and numerous novel, previously uncultured bacteria. This work finds that phylogenetically diverse bacterial endosymbionts occur within living hyphae of multiple major lineages of ascomycetous endophytes.A focus on 29 fungal/bacterial associations revealed that bacterial and fungal phylogenies are incongruent with each other and did not reflect the phylogenetic relationships of host plants. Instead, both endophyte and bacterial assemblages were strongly structured by geography, consistent with local horizontal transmission. Endophytes could be cured of their bacterial endosymbionts using antibiotics, providing a tractable experimental system for comparisons of growth and metabolite production under varying conditions. Studies of seven focal fungal/bacterial pairs showed that bacteria could significantly alter growth of fungi at different nutrient and temperature levels in vitro, and that different members of the same bacterial lineages interact with different fungi in different ways.Focusing on one isolate, I then describe for the first time the production of indole-3-acetic acid (IAA) by a non-pathogenic, foliar endophytic fungus (Pestalotiopsis neglecta), suggesting a potential benefit to the host plant harboring this fungus. I show that this fungus is inhabited by an endohyphal bacterium (Luteibacter sp.) and demonstrate that mycelium containing this bacterium produces significantly more IAA in vitro than the fungus alone. I predict that the general biochemical pathway used by the fungal-endohyphal complex is L-tryptophan-dependent and measure effects of IAA production in vivo, focusing on root and shoot growth in tomato seedlings.

Plants are inhabited by diverse species of bacteria and fungi, which affect plant health and fitness. Endophytes are bacteria or fungi that live within plant host tissues without causing symptoms of disease, and mediate important plant traits in agriculture, such as nutrient acquisition, disease resistance, and abiotic stress tolerance. However, we know little about the general ecology of endophytes, including which factors determine their compositions within plants. Seedborne transmission may represent an important source of bacterial and fungal endophytes, which can significantly impact the plant microbiome and plant traits. However, seeds are also a vehicle for transmission of plant pathogens. Seeds are commonly treated to control against seedborne pathogens, and increasingly bacteria and fungi are inoculated onto seeds to serve as biological control against pathogens. My dissertation explores the theoretical and applied ecology of seedborne endophytes of maize, including their interactions with pathogenic Fusarium fungi, and with seed treatments designed to control Fusarium. In Chapter II, I examine factors that affect the transmission of seedborne fungal endophytes and Fusarium into maize seedlings, including the influence of soil microbiota, and the impact of disinfection and biological control seed treatments. In Chapter III, I determine the long-term effects of seed disinfection and biological control inoculants on maize bacterial and fungal endophytes and Fusarium pathogenicity across three different farms. In Chapter IV, I recruit maize seed growers across the Pacific Northwest and U.S. to participate in a broadscale study of seedborne endophytes. Across submitted seed samples, I find commonly occurring seedborne endophyte taxa, and delineate how maize varieties interact with environmental factors to affect the composition and diversity of seed-associated endophytes in seeds. Throughout these chapters, I explore the potential applications of seedborne endophytes in agriculture, particularly as a source for biological control against Fusarium in maize, and speculate how seed treatments can have significant, lasting impacts on the plant microbiome.

Loline alkaloids (LA) are secondary metabolites produced by Epichloandamp;euml; (anamorph, Neotyphodium) grass endophytes. They are toxic and deterrent to a broad range of herbivorous insects but not to livestock. This protective bioactivity has spurred considerable research into the LA biosynthetic pathway. LOL, the gene cluster containing nine genes, is required for LA biosynthesis. The regulation of LOL genes during LA production in culture and in symbio is of interest. In this study, coordinate regulation between LOL gene expression and LA production level was investigated in both MM culture and symbiota. Results showed that expression of LOL genes in N. uncinatum MM culture were tightly correlated with each other (p andamp;lt; 0.0005), and all presented a significant temporal quadratic pattern during LA production. Gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached before the highest amounts of LA were detected, and gene expression level declined to a very low level after amounts of LA plateaued. Observations suggested that the hierarchical clusters based on the correlation coefficient could help to predict the roles of LOL genes in the LA pathway. In symbiota, coordinate coregulation of LOL gene expression with LA was found in E. festucae-meadow fescue inflorescences and stromata, whereby lower LOL gene expression corresponded with the lower LA level in stromata. In N. uncinatum (or N. siegelii)-meadow fescue vegetative tissues, dramatically higher LA levels were found in younger leaf tissue than in older leaf tissue, yet no evidence was found to relate this difference to LOL gene expression differences. Instead, substrate availability may regulate the LA level. In particular, asparagine was more than 10-fold higher in young leaf tissue than in old tissue, although proline was significantly lower in young tissue. Therefore, different regulatory mechanisms underlie LOL gene expression and LA production in different circumstances. The GUS activity of Pro-lolC2-GUS and Pro-lolA2-GUS in Neotyphodium species was almost undetectable in culture, though the activity could be detected in symbiota. The mRNA of GUS did not exhibit the same pattern as lolC2 or lolA2 in culture during LA production time course. A Pro-lolC2-cre transgene was expressed in complex medium, in which lolC2 mRNA was not detectable. These results suggest that proper regulation of LOL genes in culture or symbiota is dependent on the LOL cluster.

Endophytic fungi have been found in every terrestrial plant investigated thus far. They can receive nutritional benefits from their host tissues. Two major modes of nutrition for endophytic fungi include biotrophy and saprotrophy. In chapter 1, I set out to determine whether endophytic fungi isolated from Quercus gambelii in Utah, USA were capable of saprotrophy and, if some were, how much variation in saprotrophic ability exists among them. Because saprotrophy requires moisture and, in Utah, moisture is largely unavailable outside winter and early spring, in chapter 2 I set out to determine if any of our isolated endophytic fungi were capable of saprotrophy under cold conditions (psychrotolerance). If any were, we would then determine how much variation exists in psychrotolerance among them. Additionally, I tested the hypothesis that a tradeoff exists between saprotrophic ability under winter and non-winter temperatures. During these studies, I observed differences in growth patterns among our isolates. Some grew radially quite rapidly but produced little biomass while others produced a great deal of biomass but grew radially slowly. Thus, in chapter 3 I investigated the possibility of a tradeoff between saprotrophic ability in terms of radial growth rate and saprotrophic ability in terms of biomass accumulation among our isolates at winter and non-winter temperatures. In chapter 1 I found that a majority of our isolates were saprotrophic at non-winter temperatures, and that there was a great deal of variability among them in saprotrophic ability. Surprisingly, I found more variability in saprotrophic ability among isolates within a species than among species, which is contrary to the assumption of phylogenetic conservation of important traits. Nevertheless, our results suggest that saprotrophic endophytic fungi have priority over non-endophytic fungi in litter consumption and, therefore, a significant impact on the decomposer community. In chapter 2 I also found that a majority of our isolates were psychrotolerant and that there was a great deal of variability among them in psychrotolerance, more of which was found among isolates within a species than among species. Additionally, I found that there was no tradeoff between saprotrophic ability at winter and non-winter temperatures, but that instead there was a positive correlation. Our results suggest that many of these endophytic fungi are capable of saprotrophy during the cold months of winter and early spring and thus, although the large degree of variation suggests that there may be a lack of phylogenetic conservation of physchrotolerance within species, play significant roles in overwinter decomposition and nutrient cycling. Lastly, in chapter 3, I found that there was no tradeoff between saprotrophic ability in terms of radial growth rate and saprotrophic ability in terms of biomass accumulation at either winter or nonwinter temperatures. In fact, I found no correlation of any kind between the two saprotrophic strategies. These results suggest that, at least theoretically, an isolate could simultaneously possess both rapid radial growth and rapid biomass accumulation and thus be highly competitive, or both slow radial growth and slow biomass accumulation and thus be poorly competitive, although our results did not include a significant number of isolates at either end of this spectrum.

Fescue toxicosis is a condition that afflicts livestock grazing endophyte-infected tall fescue and is particularly detrimental to pregnant mares. A two year evaluation study of a novel endophtye-infected cultivar, AGRFA-144 (A-144), did not induce fescue toxicosis in late-term pregnant mares. All mares delivered viable foals except in E+ group which had two viable foals, one stillborn-dystocia and one compromised foal which was euthanized at 72 h pp. Serum P4 concentrations were similar among A-144, NE+, and E- mares (p>0.05). Foal BW and foal/placental weight ratios were similar for A-144, NE+, and E- (p>0.05). Foal serum P4 was similar on 1 d and 2 d in all groups, but was lower (p = 0.049) in the A-144 group than the E- foals on day 0. Neutrophil/lymphocyte ratios were similar (~5:1) in all foals on d 0 and 2. IgG values were similar (p>0.05) among A-144, NE+, and E-.

For Virginia, the primary forage base is endophyte-infected tall fescue (Schedonorus phoenix (Scop.) Holub). However, the decreased animal performance and disorders caused by the presence of the fungal endophyte Neotyphodium coenophialum reduces its suitability for many forage-livestock producers. The endophyte, which is found in the inter-cellular space of tall fescue tissues, forms a mutalistic relationship with the plant and helps the fescue tolerate drought, insect predation, and grazing pressure. The production of toxic ergot alkaloids by the endophyte is associated with decreased performance of animals that graze endophyte infected tall fescue. The objective of the current experiment was to determine how tall fescue type (endophyte free vs novel endophyte) and orchardgrass-legume (alfalfa vs clover) mixture affect grazing behavior of weaned steers. Forty-eight weaned steers (218 ± 18 kg) were blocked by frame score (medium and large), and randomly assigned within block to four treatments with three replications in a 2x2 factorial design. Each treatment consisted of 2 paddocks with the combination of tall fescue and legume-orchardgrass mixture as follow: alfalfa-orchardgrass mixture (A)/E-, clover-orchardgrass mixture (C)/E- , A/E++ and C/E++. Each group of 4 steers had 24 h access to both tall fescue stand and legume-orchardgrass mixture. Botanical composition of paddocks was determined on d 0, and forage mass, sward height and nutritive value were determined on d 0, 17 and 33. Behavior of steers was scanned every 5 min on d 24 and 25 from 0700 to 1900. The proportion of legumes was higher (P = 0.001) in A paddocks(37 %) compared to C paddocks (6%), while the percent orchardgrass was lower (P = 0.040) in A (33 %) than C (53 %). The proportion of broadleaf and grassy weeds was higher (P = 0.013) in E- paddocks (11 %) as compared with E++ paddocks (3 %). Sward height and herbage mass were similar between treatments during the experiment (P > 0.05). Nutritive value (CP, NDF and ADF) did not differ between fescue paddocks (P > 0.05). However, within mixed swards, CP was higher and NDF was lower in A compared with C (P < 0.05). Acid detergent fiber was (P < 0.05) lower in A than C only on d 0 and 17. Average daily gain did not differ between treatments (P > 0.05). Steers spent higher (P = 0.002) proportion of the daylight time grazing in treatments with C (56 %) as compared with A (50 %), and in treatments (P = 0.023) with E++ (55 %) compared with E- (52 %). Time spent ruminating, idling and lying, as well as steps taken per day, did not differ between treatments (P > 0.05). Regardless of differences in weeds between E- and E++, these results indicate that fescue type affected grazing behavior of calves. Difference between mixed swards in proportion of ground cover with legumes and orchardgrass, as differences in nutritive value between A and C may have affected grazing behavior of calves between mixed swards, instead of a legume species effect within mixtures. / Master of Science

Les champignons endophytes sont des microorganismes non-pathogènes impliqués dans des associations mutualistes avec les plantes. Les endophytes foliaires, en particulier, représentent un groupe très divers, mais leurs interactions avec la plante hôte et ses micro-organismes associés sont peu connues. Des travaux préliminaires initiés par notre équipe, explorant la diversité microbienne foliaire du conifère Cephalotaxus harringtonia, ont permis d’isoler la souche fongique Paraconiothyrium variabile (Ascomycota), un antagoniste du phytopathogène Fusarium oxysporum. Au cours de leur interaction, on détecte des quantités moindres de beauvéricine, une mycotoxine synthétisée par F. oxysporum. En parallèle, on observe une augmentation de la synthèse de deux oxylipines, l’acide 13-hydroperoxyoctadécadiénoïque (13-HPODE) et l’acide 13-oxo-octadécadiénoïque (13-oxo-ODE), dans la zone de confrontation. L'objectif de ce travail était de comprendre les mécanismes conduisant à la diminution de la beauvéricine au cours de l'interaction et d'explorer le rôle des oxylipines dans la régulation de cette dernière. Dans mon travail de thèse, je montre la présence de deux gènes lox chez P. variabile (pvlox1 et pvlox2) codant tous deux pour des manganèse lipoxygénases, potentiellement à l'origine des acides 13-HPODE et 13-oxo-ODE. Pvlox2 est spécifiquement induit pendant l'interaction, et ces résultats sont corroborés par une synthèse accrue de 13-HPODE chez P. variabile. Par ailleurs, l'interaction avec l’endophyte, ainsi que l'ajout de l’oxylipine 13-HPODE, régulent positivement la voie de biosynthèse de la beauvéricine, comme l’indiquent les teneurs plus élevées en mycotoxines observées chez F. oxysporum. Enfin, nous avons montré que la beauvéricine inhibait la croissance de l’endophyte, mais que ce dernier était capable de dégrader la mycotoxine, expliquant ainsi les faibles quantités de beauvericine observées initialement dans la zone de compétition. Ce travail contribue à la compréhension du rôle des oxylipines dans la communication inter-microbienne. / Endophytic fungi are non-pathogenic microorganisms involved in mutualistic associations with their host. Foliar endophytes, in particular, represent a very diverse group but little is known about their interactions with the host and its associated micro-organisms. In preliminary work, exploring the leaf microbial diversity of the conifer Cephalotaxus harringtonia, our team isolated the fungal strain Paraconiothyrium variabile (Ascomycota), an antagonist of the phytopathogen Fusarium oxysporum. During their interaction, decreased amounts of the F. oxysporum mycotoxin beauvericin, and higher amounts of the two oxylipins, 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-oxo-octadecadienoic acid (13-oxo-ODE), were observed in the confrontation zone. The objective of the present work was to understand the mechanisms leading to beauvericin decrease during the interaction and to explore the role of oxylipins in beauvericin regulation. In my thesis work I show the presence of two lox genes in P. variabile (pvlox1 and pvlox2) coding both for manganese lipoxygenases, potentially at the origin of 13-HPODE and 13-oxo-ODE. Pvlox2 is specifically induced during the interaction, which lead to an increased synthesis of 13-HPODE in P. variabile. The endophyte itself, as well as the oxylipin 13-HPODE, up-regulated the beauvericin biosynthesis gene beas, which was paralleled by higher mycotoxin content in the mycelium of F. oxysporum. Finally, we showed that beauvericin inhibited the endophyte’s growth, but the latter was capable to degrade the mycotoxin, which explains the lower amounts of beauvericin found in the competition zone. This work presents pioneer undertaking to elucidate the role of oxylipins in inter-microbial crosstalk.