Loline alkaloids are made by the endophytic fungus Neotyphodium sp. as part of a symbiotic relationship to keep insect herbivores from consuming their tall fescue host. Previous research has indicated that some epiphytic bacterial colonizers of tall fescue can use these alkaloids as a sole carbon source. Such bacteria grow to higher population sizes on loline containing grasses than other bacterial strains. It is thought that a multi-trophic symbiosis is occurring on the grass phyllosphere between the loline catabolizing bacteria, the host, and the endophytic fungus. This project will test the potential positive impact of loline catabolizing bacterial epiphytes on growth of the host. The bacteria will be screened for production of the plant growth regulators, auxin and cytokinin, through growth assays, microscopy, and biochemistry. Additionally, the protective impacts of loline catabolizing bacteria on the host grasses will be investigated by screening them for the antimicrobial production. The ability of the bacteria to protect their hosts via niche exclusion will also be examined. A combination of culturing and biochemistry techniques will be conducted for these tests. Finally, the potential to take advantage of the affinity of loline catabolizing strains to Neotyphodium sp. harboring grasses for the bioremediation of the major pollutant Atrazine will be tested.
The data collected from this work will have broad implications on the current scientific knowledge concerning microbe:microbe interactions. Potentially, this work may result in an efficient way to remove atrazine from the environment, which would generate safer ground water for future generations.
Introduction Previous studies revealed a unique relationship between Burkholderia ambifaria and leaves of endophyte infected Festuca arundinaceae (Tall fescue). The bacteria exhibited significantly higher population sizes when grown on grasses harboring fungal endophytes produce N-Formylloline (NFL). NFL is a loline alkaloid, which is produced as part of a symbiotic relationship between the fungus and its grass host to ward off insect herbivores. Interestingly, B. ambifaria and a few other bacterial species have been found to catabolize NFL, using it as a nutrient source. Alternatively, when inoculated on leaves of grasses infected with mutant fungi unable to produce alkaloids, the bacterial populations were significantly smaller. Therefore, it is thought that B. ambifaria and other loline catabolizing bacteria may have special properties that are being selected for by the fungus in order to help support the plant host. An additional hypothesis was that such bacteria may be useful in bioremediation of the soil pollutant atrazine since the production of lolines would support their growth and eliminate the need to add additional food sources. To test these hypotheses several studies were conducted during the 2012-2013 academic year. In fact, undergraduate students under my tutelage conducted all of the experiments. Thus completing one of the primary goals of this award. As the experimental work was performed, I was able to teach the students many skills that are vital to research science. Two of my students hope to continue their studies in graduate programs. Given their positive feedback, I’m confident that my students will go on to productive academic careers. An additional goal of this award was to create an online research archive. This project is underway, as I’m currently working with a student to create a website. This project has allowed me to teach my student proper scientific presentation skills, which are being applied to the web design. Conclusions Results from in vitro experiments to test the impact of the bacterial strains on growth of plant pathogenic bacteria and fungi were not fruitful. These preliminary tests showed no change in growth rate of the pathogenic strains with the application of secondary metabolites from B. ambifaria and other loline catabolizing strains. During the spring 2014 semester, I will repeat these experiments with whole bacterial cells instead of secondary metabolites. Unfortunately, our experiments with B. ambifaria in the Tall Fescue rhizosphere had several uncontrollable set backs and need to be repeated. However, competition assays with B. ambifaria were successfully conducted on bean plants. Plants inoculated with the pathogenic bacterium P. agglomerans showed lower levels of plant height than plants co-inoculated with B. ambifaria (Fig 1). Similarly, the addition of B. ambifaria allowed the plants to attain higher dry weights than plants inoculated with the pathogen alone (Fig 2). This suggests a protective benefit of B. ambifaria in the bean rhizosphere. A similar trend is expected to occur in the rhizosphere of tall fescue. In another successful experiement, a loline using strain, P. aureofaciens as well as a mutant strain unable to use the alkaloids as a nutrient source were inoculated into the rhizosphere (area around the roots) of Tall Fescue (Festuca arundinaceae). After a week of growth the loline catabolizing strain, P. aureofaciens was able to maintain high population sizes (Fig. 3) while a loline catabolism mutant, Burkholderia ambifaria LCMS1 suffered a drop in population. After 48 hours of growth in the rhizosphere, both strains of bacteria increased their populations, likely as a result of many nutrients deposited into the rhizosphere from the plant roots. Roots are notoriously leaky when it comes to sugars and flavonoids. So it is thought that between 48 and 720 hours the bacteria used up the easy to consume sugars and flavonoids, leaving only loline alkaloids which likely escape into the rhizosphere as well. Given that only P. aureofaciens can consume lolines, it is probable that this strain is able to maintain its population size by consuming this additional resource. The beauty of these findings is that they support the idea that loline consuming bacterial strains will be able to outcompete other bacteria in the rhizosphere. This would be particularly important in a bioremediation scheme, which can fail when unintended bacteria take over an area that scientists want the bioremediator strain to inhabit. The benefit of using grass that harbor loline producing grasses is that we can likely expect our bioremediator to outcompete other bacteria, thus allowing for a self sustaining bioremediation model. The fungus lives symbiotically in the plant and automatically produces the loline alkaloids. The loline alkaloids leach out of the roots into the rhizosphere, providing a nutrient source for only a few bacteria. Loline consuming bacteria genetically modified to chemically convert the wide scale pollutant atrazine into a none-toxic form will thrive in the tall fescue rhizosphere and continue to grow and thus remove the pollutants over time.
