Kristen Gates applying Parafilm to KB plates after plating serial dilutions for a sample.

My name is Kristen Gates and I am a biology major in the Fulbright College of Arts and Sciences. In the fall semester of 2020, 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 the antimicrobial compounds by determining causes of variation among lyophilized bacterial secretions. 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.

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 of sub-fractions obtained from solid phase extraction. Solid phase extraction is a separation technique that is commonly used before submitting a sample for further chemical analysis. It takes approximately three hours to plate a single sample and I often processed two samples a week. Before some new adjustments were made to the protocol, the plated bacteria would overgrow and produce inconclusive results. I learned to accept that most weeks my results would be inconclusive for one reason or another. Either there was variation among the samples, unfavorable conditions for bacterial growth, or an error was made in the hundreds of pipette transfers. I quickly learned that attitude is everything. If I was going to manage to stay positive through the process, I would need to bring a good attitude. I would consider myself a pretty positive person but the repeated failures challenged me. Sometimes frustration seemed fitting but it was best kept to a minimum. With time, performing serial dilutions became a form of meditation for me— mix, transfer, mix, change tips, mix, transfer, mix… until all samples are plated. Instead of becoming frustrated, I began to enjoy this time.

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. 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.