In planta studies of the corn pathogen Pantoea stewartii subsp. stewartii and applications of a corn-based industrial byproduct

• Main Author: Bartholomew, Holly Packard
• Other Authors: Biological Sciences
• Format: Others
• Published: Virginia Tech 2020
• Subjects: Pantoea stewartii; Stewart's wilt; phytopathogen; transcription regulation; corn; ethanol production; Bacillus species; aquaculture
• Online Access: http://hdl.handle.net/10919/99356

Corn is a valuable agricultural commodity in the United States and in the world. The causal agent of Stewart's wilt disease in corn, Pantoea stewartii subsp. stewartii, is a bacterial phytopathogen that is vectored into the plant by the corn flea beetle, Chaetocnema pulicaria. After entering the apoplast of the leaf, the bacteria cause water soaking symptoms before traveling to the plant xylem to form a dense biofilm, thereby blocking water transport and inducing necrosis and wilt. This results in reduced crop yield and may even lead to death of the corn plant. To better understand the in planta requirements of this pathogen, a whole transcriptome study was performed via RNA-Seq to determine genes differentially expressed in the bacteria while inside the corn. It was found that nutrient transporters and stress response genes were upregulated specifically when the bacteria are in their host plant, suggesting a response to nutrient availability and host defense in the xylem. Further elucidation of the genes required for the P. stewartii in planta lifestyle was performed via a reverse genetics approach where in-frame gene deletions and the corresponding complementation strains were constructed for genes that had shown a fitness defect in corn based on a previously published Tn-Seq study: genes encoding seven transcription factors, nsrR, iscR, lrp, nac, DSJ_00125, DSJ_03645, and DSJ_18135, as well as a hypothetical protein DSJ_21690. Investigation of the physiological role of these genes was performed using in planta virulence and competition assays for all strains. An in planta qRT-PCR analysis of bacterial gene transcription was also completed for the strains with deletions in nsrR and iscR. In vitro assays were performed on all strains to determine their capsule production and motility phenotypes. Taken together, it was seen that iscR is important for colonization capabilities in planta, both NsrR and IscR act as regulators, and lrp is important for full disease capabilities, perhaps due to reduced capsule and motility phenotypes. These findings lay the groundwork for finding potential disease intervention strategies not only against P. stewartii, but also other xylem-dwelling bacterial phytopathogens. In addition to exploring ways to enhance crop yield, an additional research area was on repurposing a byproduct of corn ethanol production, syrup. It was hypothesized that this corn-based syrup could be utilized as a carbon source to grown bacteria. In turn, the resulting bacterial biomass could then be added as a fish feed supplement in aquaculture. Syrup was tested as a growth medium for individual soil bacterial isolates as well as a full mixed bacterial community consortium to determine which bacteria could grow most efficiently, both in rate and yield. It was found that the highest growth rate and yield was from Bacillus species, some of which may have probiotic benefits to fish. Ultimately, the collective outcomes from these projects in basic research about a bacterial corn pathogen and applied research about beneficial microbes grown on a corn-based substrate are expected to improve scientific endeavors as well as agricultural practices. === Doctor of Philosophy === Corn is a top agricultural commodity in the United States, as a food for human consumption, a primary nutrient source used in animal feed, and a substrate consumed during biofuel production. These various corn-based industries are impacted by bacteria in multiple ways; in some cases, bacteria may cause disease that reduces crop yield, but other bacteria serve beneficial roles that enhance health. This dissertation research describes studies about the bacterium that causes Stewart's wilt disease in corn, Panteoa stewartii subsp. stewartii. In an initial experiment, the genes that P. stewartii expresses at the highest levels when it grows inside the corn plant were identified. These genes were deduced to be important for the ability of the bacterium to live successfully in this environment. This work was followed up with a more specific approach that examined the role of certain genes that were predicted to be master regulators of the expression of other genes in the ability of the P. stewartii to colonize the plant and/or cause disease. By identifying key bacterial genes, disease intervention strategies to combat Stewart's wilt and other similar bacterial plant pathogen diseases might become possible. Protecting corn yields is important for ethanol production. The final study of this dissertation examined the ability of bacteria to grow on a byproduct of ethanol production called syrup. The goal was to then use the biomass of these beneficial microbes as a food source for animals being produced in aquaculture facilities. Among the species tested, the highest growth rate and yield was from Bacillus subtilis, a safe-to-eat bacterium that has known beneficial health properties when consumed by fish. Overall, the research studies that were completed for this dissertation have the potential to improve agricultural practices by decreasing corn disease leading to increased corn yield and developing new downstream corn-based animal feed products.