Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The microbiome and metagenome data produced in this project are now publicly accessible. They benefit academics, industry professionals, agricultural scientists, plant biologists, geneticists, and farmers. Additionally, the methods, tools, and resources created during this project can be used to analyze the microbiota of other plant parasitic nematodes or pests. This project will specifically aid nematologists and, more broadly, plant biologists. Furthermore, the project supported one PhD-level and one MS-level graduate student from underrepresented groups interested in STEM programs with a focus on plant molecular biology. Throughout the project, the students received training in advanced genomic technologies. Changes/Problems:As expected and proposed, we made significant progress in all four objectives (1 - 4). The work delayed under Objective 2 was accomplished last year (2023-2024), and we are grateful to USDA-NIFA for graciously providing us with a year of no-cost extension. What opportunities for training and professional development has the project provided?In the last year, this project supported and trained two graduate (one PhD-level and one MS-level) students and a project assistant in the advanced STEM areas, i.e., genomics and bioinformatics. In addition, we trained two undergraduate students through departmental funding. The students and professional staff received hands-on experience in next-generation sequencing technologies, followed by bioinformatics analyses (QIIME 2) while analyzing the data generated in this project. The students and professionals trained in this project wish to pursue their careers in STEM fields, thus contributing to the next generation's workforce development. How have the results been disseminated to communities of interest?The data and the research results generated under this project are presented at annual meetings and conferences, disseminated to collaborating institutes, and moreover plant research communities are interested in utilizing these resources. Also, we deposited our data generated at NCBI and published our data and analysis via reviewed peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the first three years (2020-2023), we made significant progress on all four objectives listed in the timeline. However, we encountered challenges with Objective 2, which became our primary focus last year (2023-2024). Despite these challenges, our team's dedication and resilience shone through as we worked tirelessly to accomplish the proposed work. We are grateful to USDA-NIFA for accepting our one-year no-cost extension request. Our efforts included a detailed study of the Reniform Nematode (RN)-specific microbiome. This involved culturing microscopically incised RN on a media, isolating microbial DNA, and amplifying it with specific PCR primers. The major classes of bacteria identified belonged to α-Proteobacteria, β-Proteobacteria, Verrucomicrobiae, and Sphingobacteriia. Further, we designed primers specific to RN and major microbial classes identified in RN microbiota. We surpassed our initial expectations for Objectives 1 and 2, identifying two intra-individual ITS1 rDNA sequence variants in Alabama's female and male reniform nematode populations. This significant achievement was published in a peer-reviewed journal, further validating our progress.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Nyaku, S. T., Karapareddy, S., Cebert, E., Lawrence, K., Eleblu, J. S., Sharma, G. C., & Sripathi, V. R. (2023). Two Intra-Individual ITS1 rDNA Sequence Variants Identified in the Female and Male Rotylenchulus reniformis Populations of Alabama. Plants, 13(1), 5.
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Progress 06/01/20 to 05/31/24
Outputs
Target Audience:The research results generated here will help better understand microbial communities associated with the Reniform Nematode (RN), soils, and plant roots (e.g., cotton, soybean, etc.) and their interactions. We also trained next-generation STEM scientists and strengthened our Molecular Biology Program at Alabama A&M University. The project's focus on the emerging science of genomics and bioinformatics is a testament to its commitment to staying at the forefront of research and development. This project's resources (microbiome and metagenome data) are publicly available and helpful for academia, industry, agricultural scientists, plant biologists, geneticists, and farmers. Also, data generated by this project helps develop resources that benefit limited-resource farmers in the southern US to cater to our nation's ability to achieve food security. The methods, tools, and resources created during this project can be used to analyze the microbiota of other plant parasitic nematodes or pests, expanding the project's impact. This project has specifically supported three MS-level and one PhD-level graduate students, a project assistant, six undergraduate students, four research interns, and four K-12 interns from underrepresented groups, providing them with valuable training in advanced genomic technologies and inspiring hope for a more diverse and inclusive future in STEM programs with a focus on plant molecular biology. The project benefits nematologists and soil microbiologists in particular and plant biologists in general, significantly improving the capacity of this 1890 institution. Changes/Problems:All four objectives progressed as expected, except for collecting pure cultures from the microbiota identified in the reniform nematode, a component of Objective 2. This was due to the unexpected departure of one of the graduate students recruited to undertake this experiment. However, with the support of NIFA, we were able to request and receive an extra year, which was instrumental in helping us accomplish our research goals. The lessons learned were: 1. It takes more time to recruit a competitive graduate student. 2. It is challenging to retain graduate students because of added financial and societal circumstances. 3. Finding novel ways to conduct research even during these challenging times of the COVID-19 pandemic. What opportunities for training and professional development has the project provided?More than what we proposed, this project supported and trained three thesis students (S. Etukuri, A. Craft, and A. Bandarupalli), one doctoral student (S. Karapareddy), a non-thesis (S. Tamatamu), six undergraduates (J. Arnold, C. McCoy, C. Holland, A. Leslie, J. Callaway, and M. Joyner), a project admin assistant (S. Steele), and a bioinformatician (Z. Gossett) in the advanced STEM areas, i.e., Genomics and Bioinformatics. We also trained five research interns (S. Siddi, P. Jampala, R. Chevendra, B. Puligujju, and B. Chalasani) and four K-12 interns (K. Harris, B. McCraw, P. Aliche, M. Harris-Battle) in these advanced areas. The students and interns listed above received hands-on experience in next-generation sequencing technologies, followed by bioinformatics analyses (microbiome and metagenome sequencing and analysis) while analyzing the data generated in this project. The students and professionals trained in this project currently wish to pursue their careers in STEM, thus contributing to the next generation of workforce development. Further, we conducted four QIIME2 Analysis Bioinformatics hands-on sessions during fall semesters and received a tremendous response from the College. As a result, we trained and helped 6-8 students analyze their research projects at AAMU. How have the results been disseminated to communities of interest?Results from this work provide a substantially more comprehensive understanding of the changes in microbial composition within the nematode and in the rhizosphere soils in infested and non-infested soils of Alabama. The microbial and metagenomic resources developed from this project were available to the user community in many ways. For example, 1) we initially presented posters at the Plant and Animal Genome (PAG) conference and the Beltwide Cotton Conference organized by the National Cotton Council of America, each attended by plant breeders from both public and private entities; 2) the outreach is not be limited to a mere presentation at the professional meetings. For example, each summer, groups of high school students (5 -10) from different states in the United States come to AAMU through the North Alabama Center for Education Excellence (NACEE). These students will visit our laboratories and be provided information on the projects outlined above; 3) the results were shared with our close collaborators in the southeastern United States, both in public and private plant breeding programs. We currently have formal collaborations with USDA ARS scientists at College Station (TX), Stoneville, and Starkville (MS), and at our sister land grant universities in Alabama (members in Alabama Land Grant Alliance or ALGA include Alabama A&M University, Auburn, and Tuskegee Universities); 4) in addition, both the undergraduate and graduate students meet, greet, and introduce our research to potential students on STEM Day, an annual event at AAMU; 5) a website has been developed as an outcome of this project, which will soon be available and maintained through AAMU's Center for Molecular Biology. Short video content will also be produced, posted on our website, and linked to other sites visited by potential students. Additionally, as students complete their research work, it will be expeditiously published in peer-reviewed journals; 6) the data and other information resulting from the specific objectives are shared through the NCBI website and published in regional and national journals. This project is innovative and informative because we used an integrated approach in combining soil microbial data with reniform nematode microbial data to better understand the variation in microbial composition associated with four different levels of nematode infestation (Heavy, Moderate, Low, and No Infestations). Microbial (16S rRNA) Sequencing, followed by microbiome analyses, was conducted in rhizospheric soil and nematode samples to infer the reciprocal relationships among plant roots, nematodes, and soil microorganisms. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Plant roots, nematodes, and soil microorganisms interact in the rhizosphere by exchanging or communicating through biomolecules or chemicals or signals. Some rhizospheric microbes process such compounds via biogeochemical cycles to improve soil fertility, promote plant growth and development, and impart plant stress tolerance. This study aimed to strengthen the capacity of microbiome and metagenome research by understanding reciprocal interaction among plant roots, nematodes, and soil microorganisms coupled with student training under four overarching objectives. During the project period (2020-2024), we progressed well on all four objectives (1 - 4), as listed in the timeline. Under Objective 1, to study and analyze the changes in microbial composition in the rhizospheric soils, we included four different locations in North Alabama. Based on the level of Reniform Nematode (RN) Infestation, these locations were classified as Group A-RN Not Detected (ND), Group B-RN Low Infestation (LI), Group C-RN Medium Infestation (MI), and Group D-RN High Infestation (HI). In total, we have collected and processed over 200 soil microbiome and metagenome samples. Also, studying the RN-specific microbiome was confirmed by culturing the microscopically incised RN on media, isolating the microbial DNA, and amplifying them with specific PCR primers similar to that outlined in Objective 2. Under Objective 3, we conducted bioinformatic analyses to understand reciprocal interrelationships between the soil and RN-associated microbes. Finally, under Objective 4, we trained four graduate and four undergraduate students to achieve the proposed objectives. First, we recruited one MS-level student and two sophomores to advance our research objective 1, and they graduated in 2022. Then, we recruited one PhD-level, two MS-level graduate students, and two sophomores and juniors to undertake Objectives 2 and 3. The PhD student continued with our research objectives, along with two sophomores, while two MS-level grads and two junior undergrads advanced their research and graduated in the Spring of 2024. Our research employed next-generation sequencing, a cutting-edge technique that has the potential to significantly advance our understanding of the rhizosphere's complex and reciprocal interactions among microbes, crops, and nematodes. This technique is one of the most widely used and cost-effective ways of determining the composition and diversity of microbiomes in such complex environmental samples. In this study, we specifically employed amplicon sequencing of 16S ribosomal RNA (16s rRNA) for bacteria and Internal Transcribed Spacer (ITS2) region for fungi to profile the soil microbiome in the rhizosphere of major crops grown in North Alabama. We isolated microbial DNA (ZymoBIOMICS) and sequenced 48 soil samples, including three replicates from 16 locations in North Alabama. Microbial diversity was assessed by sequencing (Illumina/NextSeq) paired-end libraries of amplified 16S rRNA and ITS2 regions and later analyzed with Phyloseq/Qiime2/Mothur. Our comparative analyses identified over 3,000 Operational Taxonomic Units (OTUs) from 16s rRNA and ITS2 datasets. We also identified the eight most abundant rhizospheric bacterial phyla, including Actinobacteria, Proteobacteria, Acidobacteria, Firmicutes, Planctomycetes, Chloroflexi, Bacteroidetes, and Unclassified bacteria. Moreover, we identified five abundant fungal phyla belonging to Ascomycota, Basidiomycota, Glomeromycota, Mucoromycota, and Unclassified fungi. These findings could revolutionize our approach to soil microbiology and nematology, aiding in developing strategies to improve soil fertility, promote plant growth and development, and impart plant stress tolerance. The publication related to this work, which provides a comprehensive overview of our research findings and their implications, has been submitted and is currently under review. More than we proposed in our research objectives, we also identified two intra-individual ITS1 rDNA sequence variants in Alabama's female and male reniform nematode populations. We trained more students than we proposed in the timeline to achieve objectives 1, 2, and 3. During the project, we recruited three MS-level and two PhD-level students. We graduated all three MS-level students, while a PhD-level student is in the advanced completion stage, and one left the project abruptly. Also, six undergraduate students worked towards achieving the project goals; four graduated, and the remaining two will graduate this Fall (2024).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Sripathi, V. R., Anche, V. C., Gossett, Z. B., & Walker, L. T. (2021). Recent applications of RNA sequencing in food and agriculture. Applications of RNA-Seq in Biology and Medicine, 97.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Etukuri, S. P., Anche, V. C., Ayubov, M. S., Walker, L. T., & Sripathi, V. R. (2022). Transcriptome Analysis Using RNA Sequencing for Finding Genes Related to Fiber in Cotton: A Review. Cotton.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Nyaku, S. T., Karapareddy, S., Cebert, E., Lawrence, K., Eleblu, J. S., Sharma, G. C., & Sripathi, V. R. (2023). Two Intra-Individual ITS1 rDNA Sequence Variants Identified in the Female and Male Rotylenchulus reniformis Populations of Alabama. Plants, 13(1), 5.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2024
Citation:
1. Karapareddy, S., & & Sripathi, V. R. (2024). Transcriptome and microbiome analyses in two contrasting genotypes of cotton in response to salt-stress. The Plant and Animal Genome XXXI Conference (PAG 31), San Diego, CA., January 12 17, 2024.
2. Karapareddy, S., & & Sripathi, V. R. (2024). Rhizosphere Microbiome Profiling of North Alabama Soils for Understanding Reciprocal Relationships. Seventeenth Annual STEM Day, Alabama A&M University, Huntsville, AL., March 28th, 2024.
3. Karapareddy, S., & & Sripathi, V. R. (2024). Identification and Functional Characterization of Fiber-related Genes in Four Gossypium Species. Twenty first Biennial Research Symposium of the Association of 1890 Research Directors, Inc. (ARD), Nashville, TN, April 6-9, 2024.
4. Karapareddy, S., & & Sripathi, V. R. (2024). Rhizosphere Microbiome Profiling of Soils Infested with Reniform Nematodes in North Alabama. Crops Conference, HudsonAlpha Institute of Biotechnology, Huntsville, AL, June 3-6, 2024.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Karapareddy et al., 2024. Rhizosphere Microbiome Profiling of Reniform Nematode-Infested and Uninfested Soils of North Alabama. Frontiers in Plant Science (Submitted).
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:This project's resources (microbiome and metagenome data) will be publicly available and valuable for academics, industry, agricultural scientists, plant biologists, geneticists, and farmers. In addition, the protocols, tools, and resources developed in this project can be applied to other parasites or pests on crops. The project will benefit cotton researchers in particular and plant biologists in general. Moreover, this project supports one underrepresented undergraduate and one graduate student interested in STEM programs emphasizing plant molecular biology. During the project duration, students will be trained with advanced genomic technologies. Changes/Problems:As expected and proposed, we made significant progress in all four objectives (1 - 4). Some of the work under Objective 2 was delayed, and we requested an extra year from NIFA, which helped us accomplish or progress toward our goals. The lessons learned were: 1. It takes more time to recruit a competitive graduate student. 2. It is challenging to retain graduate students because of added financial and societal circumstances. 3. Finding novel ways to conduct research even during these challenging times of the COVID-19 pandemic. What opportunities for training and professional development has the project provided?In the last year, this project supported and trained two graduate (one PhD-level and one MS-level) students, two undergraduate students, a bioinformatician, and a project assistant in the advanced STEM areas, i.e., genomics and bioinformatics. The students and professional staff received hands-on experience in next-generation sequencing technologies, followed by bioinformatics analyses (QIIME 2 and MicrobiomeAnalyst) while analyzing the data generated in this project. The students and professionals trained in this project wish to pursue their careers in STEM fields, thus contributing to the next generation's workforce development. We conducted four QIIME2 Analysis Bioinformatics hands-on sessions this semester and received a tremendous response from the College. As a result, we trained and helped 5-7 students analyze their research projects at AAMU. How have the results been disseminated to communities of interest?The data and the research results generated under this project will be disseminated to collaborating and plant research communities interested in utilizing the resources developed here. Also, we are publishing our data and analysis via reviewed publications or NCBI. What do you plan to do during the next reporting period to accomplish the goals?More than we proposed, the phylogenetic analysis of female and male nematodes to understand the variation in ITS1 regions, associated plant-parasitic behavior, and evolutionary relationships of these nematodes was investigated during the current reporting period. The publication related to this work has been submitted and is currently under review. Further, by the end of this project duration (May 2024), we plan to screen plant-parasitic genes specific to male and female nematodes.
Impacts
What was accomplished under these goals?
In the last year (2022-2023), we progressed well on all four objectives (1 - 4), as listed in the timeline. Under Objective 1, to study and analyze the changes in microbial composition within the nematode and the rhizosphere, we included four different locations, with soils representing four infestation levels (high, moderate, low, and no-infestation). As a result, we have collected and processed over 200 soil microbiome and metagenome samples. However, studying the Reniform Nematode (RN)-specific microbiome was confirmed by culturing the microscopically incised RN on media, isolating the microbial DNA, and amplifying them with specific PCR primers similar to that outlined in Objective 2. Under Objective 3, we conducted bioinformaticanalyses to understand reciprocal interrelationships between the soil and Reniform nematode-associated microbes. Finally, under Objective 4, we trained four students to achieve the proposed objectives. First, we recruited one MS-level graduate student to advance the research and to work in close association with an undergraduate, and they graduated in 2022. Then, we recruited one PhD-level and one MS-level graduate student to undertake Objectives 2 and 3, advance the research, and closely associate with two undergraduates who will graduate in Fall 2023.More than we proposed, we identified two intra-individual ITS1 rDNA sequence variants in Alabama's female and male reniform nematode populations. The publication related to this work has been submitted and is currently under review.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Nyaku, S. T., Karapareddy, S., Cebert, E., Lawrence, K., Eleblu, J., Sharma, G. C., & Sripathi, V. R. (2023). Two intra-individual ITS1 rDNA sequence variants identified in the female and male Rotylenchulus reniformis populations of Alabama. Plants (Under Review).
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:This project's resources (microbiome and metagenome data) will be publicly available and valuable for academics, industry, agricultural scientists, plant biologists, geneticists, and farmers. In addition, the protocols, tools, and resources developed in this project can be applied to other parasites or pests on crops. The project will benefit cotton researchers in specific and plant biologists in general. Moreover, this project supports one underrepresented undergraduate and one graduate student interested in STEM programs emphasizing plant molecular biology. During the project duration, students will be trained with advanced genomic technologies. Changes/Problems:We made significant progress in three objectives (1, 3 & 4) as expected and proposed. But we only made a little progress towards objective 2. The reasons are 1) one of the graduate students recruited to undertake this experiment left the project abruptly and 2) due to the delay in hiring graduate students and professional staff (Research Assistant/ Bioinformatician/ Post-Doc) during these challenging times of the COVID-19 pandemic. The lessons learned were: It takes more time to recruit a competitive graduate student. It is challenging to retain graduate students because of added financial and societal circumstances. Finding novel ways to conduct research even during these challenging times of the COVID-19 pandemic. What opportunities for training and professional development has the project provided?In the last year, this project supported and trained two graduate (one PhD-level and one MS-level) students, two undergraduate students, a bioinformatician, and a project assistant in the advanced STEM areas, i.e., genomics and bioinformatics. The students and professional staff received hands-on experience in next-generation sequencing technologies, followed by bioinformatics analyses (QIIME 2 and microbiomeSeq) while analyzing the data generated in this project. The students and professionals trained in this project wish to pursue their careers in STEM fields, thus contributing to the next generation's workforce development. How have the results been disseminated to communities of interest?The data and the research results generated under this project will be disseminated to collaborating and plant research communities interested in utilizing the resources developed here. What do you plan to do during the next reporting period to accomplish the goals?Even though our goal was to study Objective 2, i.e., screening the Reniform nematode-specific microbiome during its infective stage in the previous reporting year, it is still under development due to its submicroscopic complexity and the delay in hiring graduate students and professional staff (Research Assistant/ Bioinformatician/ Post-Doc) during COVID-19. Therefore, the expression profiles of female and male nematodes to understand the plant-parasitic genes and any horizontal gene transfer events from bacterial species will be sequenced and compared in the next reporting period of the project.
Impacts
What was accomplished under these goals?
In the last year (2021-2022), we progressed well on three objectives (1, 3, and 4), as listed in the timeline. Under Objective 1, to study and analyze the changes in microbial composition within the nematode and the rhizosphere, we included four different locations, with soils representing four infestation levels (high, moderate, low, and no-infestation). As a result, we have collected and processed over 200 soil microbiome and metagenome samples. Under Objective 3, we conducted bioinformatic analyses to understand reciprocal interrelationships between the soil and Reniform nematode-associated microbes. However, studying the Reniform nematode-specific microbiome during its infective stage outlined in Objective 2 is still under development due to its submicroscopic complexity and the delay in hiring graduate students and professional staff (Research Assistant/Bioinformatician/Post-Doc) during COVID-19. Finally, under Objective 4, we trained four students to achieve the proposed objectives. First, we recruited one MS-level graduate student to advance the research and to work in close association with an undergraduate, and they graduated in 2022. Then, we recruited one PhD-level and one MS-level graduate students to undertake Objectives 2 and 3, advance the research, and closely associate with two undergraduates who will graduate in 2023.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
Etukuri, S. P., Anche, V. C., Ayubov, M. S., Walker, L. T., & Sripathi, V. R. (2022). Transcriptome Analysis Using RNA Sequencing for Finding Genes Related to Fiber in Cotton: A Review. DOI: 10.5772/intechopen.104572.
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