Author: Kristen Gates              Major: Biology

Kristen Gates heating bacterial samples to determine the effect of heat-killing bacteria on antimicrobial activity.

My name is Kristen Gates and I am a biology major and African and African American studies minor in the Fulbright College of Arts and Sciences. In the spring semester of 2021, I conducted research in Dr. Clemencia Rojas’ lab in the department of entomology and plant pathology. The focus of my project is isolating and identifying antimicrobials secreted by symbiotic bacteria in order to control Bacterial Panicle Blight (BPB) in rice. Over the course of the semester, our lab made progress in refining the isolation of antimicrobial compounds by using minimal media to grow beneficial plant bacteria. I plan on continuing to refine the isolation process in order to obtain a quality sample to submit for mass spectrometry.

Bacterial Panicle Blight is a disease in rice caused by the bacterium Burkholderia glumae. BPB causes grain yield and economic losses that are likely to be exasperated due to global warming. Antimicrobials secreted by the beneficial plant bacterium Pseudomonas protegens PBL3 can be lyophilized or freeze-dried for storage. The antimicrobial compounds present in the freeze-dried powder can be reconstituted and applied as a biopesticide to control BPB in rice. We are currently exploring the lyophilization of P. protegens PBL3 in minimal media.

The opportunity to work in the lab of Dr. Clemencia Rojas was brought to my attention by Honors College Dean Lynda Coon. In the summer between my freshman and sophomore year, I worked in Dr. Rojas’ lab as a research assistant. I performed antimicrobial growth inhibition assays to determine the extent of the antimicrobial activity exhibited by P. protegens PBL3. Dr. Clemencia Rojas introduced the prospect of working on a cross-disciplinary research project that required biological and chemical separations. Prior to joining the lab, I had been introduced to separation methods such as SDS-PAGE and affinity chromatography in the biochemistry laboratory of Dr. T. K. S. Kumar. I was interested in the antimicrobial isolation project proposed by Dr. Rojas because it combined my interests in pharmaceutical science and sustainability.

This past semester, most of my time in the lab was spent plating serial dilutions for growth inhibition assays using several strains of Pseudomonas bacteria that exhibit antimicrobial activity. Comparing the results for the different strains of Pseudomonas, I could determine which bacterial strains exhibit the most antimicrobial activity. The bacterial strains that exhibited the most antimicrobial activity were grown in minimal media and lyophilized. We switched to minimal media because the samples that we previously submitted to mass spectrometry contained compounds that were too complex to produce a meaningful result. We also opted to switch the media that we had been using to plate the serial dilutions to a media selective for B. glumae. There were many unsuccessful attempts at creating a media selective for B. glumae and I had to keep reminding myself that it is okay to make mistakes— historically, sometimes what was originally viewed as a mistake turned out to be a great scientific discovery. I will not claim that the attempts at creating a selective media led to a great discovery, but I still believe that it is possible for great discoveries to be made under the guise of a blunder.

Laura Ortega, a graduate student in the lab, determined that growing P. protegens PBL3 in a larger volume in an initial step would result in a lyophilized sample with greater antimicrobial activity. Laura lyophilized samples and familiarized me with new protocols. The initial experimentation with solid phase extraction in the Rojas lab was performed by Casey Patrick. Casey worked on the initial phases of the antimicrobial isolation project and showed me how to perform solid phase extraction using a vacuum manifold. Dr. Clemencia Rojas encouraged me to apply for the Honors College Research Grant, looked over my drafts numerous times, and made invaluable suggestions. Dr. Rojas gave me and every member of the lab helpful feedback at our weekly lab meetings. The weekly lab meetings provided us an opportunity to present our findings from the past week and our research plan for the upcoming weeks. Dr. Rojas would sometimes ask me to perform experiments that would lead me to revise the protocols. These revisions saved me from repeating the same mistakes and allowed me to make progress in isolating the antimicrobial compounds.

I submitted a presentation to the Arkansas Crop Protection Association (ACPA) Annual Research Conference that took place in early December 2020. The ACPA Research Conference gives undergraduate, masters, and doctoral students in Arkansas a chance to present their research. The event was held virtually this year due to the pandemic. My presentation placed in the undergraduate category. I am looking forward to Plant Health 2021 this summer. Plant Health is the annual meeting for the American Phytopathological Society where plant scientists from across the country present their recent findings. My future research consists of refining the isolation process and submitting a sample for mass spectrometry. Identifying the antimicrobial compounds present in the sample will aid in the development of an effective biopesticide. If time allows, I hope to test the efficacy of a potential biopesticide in preventing the development of BPB in rice.