Recipient Organization
UNIVERSITY OF MIAMI
1320 S DIXIE HWY STE 650
CORAL GABLES,FL 331462919
Performing Department
(N/A)
Non Technical Summary
Sweet potatoes (Ipomoea batatas) are an emerging crop that is important for U.S. Agriculture. In 2020, consumption in the United States of America rose by 15% compared to the previous year and has risen steadily since 2000. It is the sixth most important food crop globally with China producing 70% of the world's sweet potato. In 2021, one-third of the fresh vegetable volume China shipped to the United States was sweet potatoes. North Carolina, California, Mississippi, and Texas produce the most sweet potatoes in the United States and, as of 2021, generate a value of approximately $680 million. Sweet potatoes are storage roots with a lot of Vitamin A and fiber as well as hardy plants with the ability to grow in less than ideal soil conditions; however, they are still susceptible to low nutrient conditions. My research will document how microbial communities stabilize themselves during low nutrient conditions and use this information to improve sweet potato stress tolerance. The research and associated products can eventually improve storage root production to domestically address the increasing demand for sweet potatoes.I will use culture-based techniques, synthetic biology, and manipulative field and hydroponic experiments to address the hypothesis that Ipomoea batatas rhizosphere microbial function is distinct before and after storage root production and the microbial community is more important for plant health before storage root production. The data and results from experiments will be shared in peer-reviewed publications, scientific conferences, and across social media in easily accessible infographics. Ultimately, this project will lead to more information about how microbial communities maintain their structure and function under nutrient limitations and potentially create microbial amendments that can be used to support sweet potato production
Animal Health Component
15%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
(N/A)
Goals / Objectives
The purpose of this project is to improve the production of sweet potatoes (Ipomoea batatas) for U.S. agriculture by understanding their response to phosphorus limitations. In addition, this project will facilitate the independence of the project director and provide essential experience in project management, science communication, and team building. The major goals that support this purpose include:Using the current skills of the project director on issues relevant to U.S. agricultureTraining personnel in molecular biology, synthetic biology, and collaboration skillsDeveloping pathways for undergraduates to gain research experience.The objectives that will achieve these goals include:Determining needs and barriers for sweet potato production by rural and urban farmers.Developing experimental plans for the major research aims in conjunction with key personnel such as mentors and collaborators. The major research aims are (1) Identify the structure and function of Ipomoea batatas rhizosphere microbial community at the fibrous to storage root transition under normal and stress conditions (2) Isolate and improve plant-growth-promoting properties of microorganisms associated with sweet potatoes (3) Quantify horizontal gene transfer (HGT) of the gcd gene - a gene important for phosphorus solubilization - under normal and low-phosphorus conditions in sweet potato microbiome.Collecting and analyzing experimental data with the help of paid undergraduate researchers.Documenting the scientific research process and sharing it with a broad audience to encourage transparency and interest in research.Disseminating results through peer-reviewed publications, popular media, and scientific conferences.
Project Methods
The project will be conducted using a balanced 2x7 factorial design that manipulates phosphorus availability and measures changes at seven time points after planting. The experiment will include the collection of soil from long-term P fertilization field sites or agricultural soil will be amended with differing concentrations of phosphorus to represent low- and high-phosphorus conditions. Sampling of the rhizosphere will occur at the start of the experiment as well as 3, 7, 14, 30, 60, and 90 days after planting (7 time points). A subset of the rhizosphere sample will be used for amplicon sequencing, metatranscriptomics, and metabolomics.The structure and function of the Ipomoea batatas rhizosphere microbial community will be identified under normal and stress conditions. To accomplish this, DNA and RNA will be extracted using commercially available extraction kits. DNA will be amplified using 16S rRNA primers, and mRNA will be enriched and reverse transcribed to create complementary DNA (cDNA). The DNA and cDNA will be gel purified and then sequenced on an Illumina platform. For metabolomics, metabolites will be extracted from soil using 3:3:2 (v/v/v) acetonitrile:isopropanol:water, and individual compounds will be identified using a gas chromatography-mass spectrometry (GC-MS) platform.The data collected will be analyzed using standard pipelines. For metagenomics and metatranscriptomics, the microbial community composition between time points and nutrient levels will be visualized using Non-Metric Multidimensional Scaling (NMDS) and statistically compared using a multivariate analysis of variance (MANOVA). For metabolomics, profiles will be processed, normalized, and a two-way ANOVA coupled with Fisher's LSD method will be used to identify significant differences between metabolite concentrations in soil and time points.The results will be evaluated by comparing the microbial community structure and function under normal and stress conditions, and identifying specific microbial taxa and metabolites that contribute to plant growth and stress tolerance. These findings will have important implications for developing strategies to improve crop yield and resilience in a changing climate.The methods for Aim 2 involve isolating and improving plant-growth-promoting properties of microorganisms associated with sweet potatoes. The isolation will be done using culture-based techniques and the ability of the isolates to solubilize phosphorus will be tested using Pikovaskya's colorimetric assay. The top 5 solubilizers will be tested individually and collectively as low and mid complexity PGPP communities, along with the whole rhizosphere microbial community. Experimental evolution will be done by adding the communities to solutions that decrease in phosphorus concentrations, and the activity of the gcd gene will be measured using RT-PCR as a proxy for adaptation to low-phosphorus environments. Sweet potatoes will be grown in high- and low-phosphorus conditions, and plant growth metrics will be used to assess the effectiveness of each amendment.The analysis will involve a 2x3 factorial design with complexity and phosphorus levels as independent variables and plant growth metrics as the dependent variable, which will be analyzed using a two-way ANOVA. A Before-After Control-Impact (BACI) analysis will also be used to explore the impact of experimental evolution of the microbial communities in low-phosphorus conditions on plant health.The methods used in this Aim involve several unique aspects that are not typically used in microbiome studies. The use of cat-RNA to record gene transfer in situ is a novel approach that allows for the detection of HGT events in real-time. Additionally, the use of publicly available data sets to obtain gene sequences related to phosphorus solubilization allows for a broad range of data to be used in the study. The transfer of the cat-RNA plasmid into an isolate obtained from Aim 2 allows for the gene transfer to occur in a more controlled environment.The incubation of the cat-RNA microbial slurries in water-saturated soils with differing concentrations of phosphorus allows for the investigation of the effect of phosphorus concentration on HGT events. The extraction of DNA for amplicon sequencing and RNA for transcript sequencing allows for the investigation of both the abundance and relative activity of the gcd gene under normal and low-phosphorus conditions.The analysis of the results will be done through standard metagenomic pipelines, which typically involves the quality control, filtering, and assembly of sequencing data followed by annotation and taxonomic classification. The analysis will also involve the comparison of gene transfer events under normal and low-phosphorus conditions to investigate the effect of phosphorus concentration on HGT. The results will be evaluated and interpreted in the context of the other aims to gain a comprehensive understanding of the sweet potato microbiome and its role in phosphorus solubilization.Efforts that support a change in knowledge of target audience include the above described methods as well as soliciting input from multiple stakeholders about project priorities and including in data collection and project documentation and broad dissemination. The success of my research, collaboration, and mentorship goals will be evaluated through various measures.Research Evaluation:Quantitative measures of success will include publication in high-impact journals, citations, and the number of conference presentations. Qualitative measures of success will include feedback from peers, mentors, and collaborators on the novelty and impact of my research.Collaboration Evaluation:Evaluation of my collaborations will be based on the successful completion of joint projects with collaborators from K-12 educators and environmental-based non-profits. A key milestone will be the successful implementation of a student mentorship program that engages undergraduate students in my research projects. Quantitative measures of success will include the number of collaboration proposals submitted, the number of joint publications and presentations, and the success of any grants received as a result of collaborations. Qualitative measures of success will include feedback from collaborators and the impact of joint projects on advancing research and promoting public outreach efforts.Mentorship Evaluation:The success of my mentorship efforts will be evaluated based on the successful completion of the student mentorship program and the number of mentees that have gone on to pursue careers in science. A key milestone will be the successful completion of the undergraduate research program, which includes training in experimental design, data collection and analysis, and scientific communication. Quantitative measures of success will include the number of students who complete the program and the number of mentees who go on to pursue advanced degrees in science. Qualitative measures of success will include feedback from mentees on the usefulness of the program in preparing them for careers in science and feedback from mentors on the impact of the program on advancing scientific research.Overall, the success of my project will be evaluated through a combination of quantitative and qualitative measures that relate milestones and indicators of success to expected project outcomes and impacts. The project evaluation plan will be regularly reviewed and updated to ensure that the project remains on track towards achieving its goals