Progress 03/15/17 to 03/14/22
Outputs
Target Audience:Target audience outlined in this project include research community, students of agriculture and biology and farmers. This project provided experiential learning opportunities to students at Alcorn State University (ASU) and Kansas State University (KSU). Over the entire project, eight female students and five male students were mentored, of which 11 were undergraduates and four graduate students. Six students were African American students. Two master's theses one each from ASU and KSU were developed. The conservation agriculture plots were part of Alcorn's classroom instruction in seven courses in Agronomy at ASU in 2017, 2018 and 2019, and at least 150 students have visited the plots. Further, the plots have served the extension and outreach of ASU by serving as demonstration plots to 11 visiting international scientists and farmers (Cochran Fellowship program of USDA Foreign Agricultural Service), 20 farmers from MS, two visiting international students, and USDA NRCS staff. Changes/Problems:The major problem encountered in the project was 1) Objective 1 could not be initiated as planned due to the early germination of one of the varieties in 2017 prior to awarding of the grant. So, the samples had to be collected in the following year, and modifications were made to the objectives as outlined previously. These modifications resulted in more data acquisition, overall resulting in a suite of microbiome, soil, biomass, and biofuel data. 2) the unexpected and multiyear COVID-19 pandemic restrictions in 2020 and 2021 presented substantial challenges to effectively continue our research efforts especially regarding objective 2. Travel and research restrictions hampered our ability to acquire data although the samples have been available. The metaproteome work was to be carried out at the Pacific Northwest National Laboratory (PNNL) Environmental Molecular Sciences Laboratory (EMSL) to acquire the compatible proteome data using their mass-spectrometry facility. Uncertainty of lab access and PNNL other priorities made these analyses challenging to arrange. However, for all other objectives (except Objective 2), we were able to obtain not just one year's data as originally proposed but two year's data which has given valuable insights into switchgrass conservation agriculture. 3) although not a problem, due to the COVID-19 restrictions and uncertainties, theses defense and manuscript submissions were also subjected to delays and the published manuscript information will be submitted past the submission of final report as and when they become available. For example, one manuscript will be submitted this month end. What opportunities for training and professional development has the project provided?Training activities: Students at ASU were trained in sample collection, sample processing, data collection and data entry; field experimental designs; collecting plant phenology data using the AccuPar and Ceptometer for recording percent canopy cover and leaf area index; soil compaction data; recording biomass production under two densities and processing samples for compositional estimation; soil sample grinding and sieving based on standard protocols; biomass pretreatment, sugar estimations using HPLC and cellulosic ethanol fermentation using shake flasks and 7L bench-top fermenters using processing controls. At KSU, students were trained in DNA extraction, PCR, DNA visualization, DNA quantitation, high throughput library preparation and trouble troubleshooting, use of common statistical inference tools for community analyses on statistical software platform R, script writing for statistical analyses in R, MiSeq pipeline analyses and multivariate tools to analyze complex multidimensional community data. In both institutions, students gained training in making oral and poster presentations, and masters students grained experience in manuscript writing. Professional development: The PI attended four workshops to enhance skillsets in pertinent statistical analyses like Multivariate Data Analysis Using PC?ORD, Statistical Analysis using R, Virtual IGS Microbiome Analysis Workshop and Experiment and Treatment Design Matter Workshop. How have the results been disseminated to communities of interest?The scientific data generated from this study has been shared with scientific community via conference presentations, master's thesis oral defense and approved thesis submission, and via manuscripts. Different manuscripts are at different stages of development and likely published in fall 2022 and spring 2023. The conservation agriculture plots have been used as demonstration plots for undergraduate and graduate courses, visiting international scientists and students, local farmers and legislators, and continue to serve as demonstration plots for future extension and outreach activities. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: The original proposed goal was to collect soil and leaf samples until from week 1, 2, 4 and week 8 since leaf emergence in spring of 2017 resulting in total of 256 samples (4cultivars*2densities*4blocks*4timepoints*2parts). Root and soil samples were collected from 32 experimental units on week 1, 2, 4, 8, 16 and 38 since leaf emergence in March 2018 resulting in 384 samples (4cultivars*2densities*4blocks*6timepoints*2parts). We extended the collection efforts to include week 16 (July, peak biomass) and week 38 right before biomass harvest to capture the dynamics of microbiomes through peak biomass and senescent biomass. The collection of root microbiomes was not outlined originally. We opted to include rhizobiome assembly (soil and root microbiome) in 2018 and conduct phytobiome (soil, root and leaf) assembly in 2019. These modifications were incorporated to ensure understanding of the origin and assembly of switchgrass microbiomes. In 2019, 576 samples were collected (4cultivars*2densities*4blocks*6timepoints*3parts). In 2018, fungal and bacterial microbiomes of roots and soils were determined, whereas in 2019, fungal and bacterial microbiomes of soils, roots and leaves were determined from six time points. Due to voluminous data and computational complexity, data were analyzed separately for the two years. The 2018 data indicated that the early temporal dynamics of bacterial and fungal communities dominate the cultivar and density effects of switchgrass under conservation agriculture. Indicator taxon analysis of early season compositional differences among the cultivars identified putative pathogens and potential beneficial members of the microbiome, suggesting that cultivars may differ in their potential disease susceptibility or ability to attract beneficial commensals or mutualists during the early growing season. The 2018 microbiome data is part of masters thesis defended and published in Fall 2021 by student Anna Kazarina (Kansas State University). It is also part of a manuscript entitled "Seasonal dynamics of Panicum virgatum associated bacterial and fungal communities trump variety and planting density effects" ready for submission. Due to COVID-19 imposed restrictions on faculty and student activity, the 2019 microbiome dataset which is voluminous is undergoing statistical analyses and expected to be a separate manuscript. Objective 2: We used our 2019 community data to identify the leaf-associated communities that differed most. Based on these data we chose two of the four cultivars included in our experiments (Alamo and BoMaster) and the two planting densities with four biological replicates each for a total of 16 experimental units for metagenome and -proteome sampling. COVID-19 restrictions in 2020 and 2021 limited our ability to generate metagenome and metaproteome sampling at PNNL from the Alamo and BoMaster samples which were selected based on the most diverse leaf-associated communities. Samples have been archived for future analyses. Objective 3: Major activities: Biomass samples from 32 units were harvested at the end of the season in 2018 and 2019. Samples were saccharified by acid-pretreatment. Saccharified biomass samples were then subjected to cellulosic ethanol production. Biomass yield was recorded. Data collected: Both 2018 and 2019 biomass yield was recorded per unit area. Biomass samples from 2018 were processed and analyzed for cellulose, hemicellulose and lignin. Saccharified samples were evaluated for glucose release and subsequent ethanol production by HPLC. Summary statistics: Biomass yield did not vary significantly between the years but was impacted by variety and density. When analyzed separately by years, significant impact of both variety and density was seen with significant interaction only for 2018. As predicted, in both years high density planting resulted in significantly higher yield than low-density planting. In 2019, varieties varied as Bomastera>Alamob>Colonyc>Kanlowc whereas in 2018 varieties varied as Alamoa>Bomastera>Colonyb>Kanlowb Overall, BoMaster and Alamo were top yield varieties in terms of biomass with 51-52 metric tons per hectare yield in 2018 for high density planting and 40-42 metric tons/hectare for low density planting. Cellulose, hemicellulose and lignin did not vary significantly over varieties or densities. About 39-41% W/W cellulose was seen in switchgrass biomass. Biomass total C and N also did not vary significantly. Glucose and xylose release upon saccharification were not impacted by variety or density but were impacted by their interaction. This was mainly driven by the difference in sugar yields between Alamo-HDP and Kanlow HDP with the latter resulting in highest glucose. The ethanol yield was significantly impacted by variety and variety*density interaction. The highest ethanol yield was from Kanlow-HDP and least was from Alamo-HDP. Alamo-LDP was comparable in ethanol yield with Kanlow-HDP and Kanlow-LDP. Literature review suggests that Kanlow is the best variety for ethanol production and confirmed in our study as well. Key outcomes: Biomass yield from conservation agriculture is 2-10 times higher than that reported for similar lowland varieties in other parts of the U.S. Our ethanol yields were low but is attributed to shake flask conditions. Scale-up in fermenters will result in higher ethanol yields. Considering biomass yield and ethanol yield, our ethanol production ranged from 3,969L/ha in BoMaster-HDP to 2,878L/ha in Kanlow-LDP. This is comparable to ethanol yield reported from other studies (3,880 and 3,517 L/ha for Kanlow and BomAster; Scagline-Mellor et al 2018) with our system having the advantage of high biomass yield without inputs of fertilizer, chemicals or intensive management practices. Objective 4: The original proposed goal was to collect soil pH, Ca, Mg, K, P, OM, CEC and nitrate-N from 128 soil samples in 2017 covering week 1, 2, 4 and week 8 since leaf emergence (4cultivars*2densities*4blocks*4timepoints). However, due to the modifications made to objective 1 and to provide soil data corresponding to the time points in objective 1, 384 soil samples were collected (4cultivars*2densities*4blocks*6timepoints per year*2years). Instead of the proposed eight soil factors, we measured 12 soil factors namely pH, OM, CEC, S, P, Ca, K, Mg, Cl, NO3-N, NH4-N, total N, total C, along with four extracellular enzymes (EEA) and PLFA for measuring overall microbial activity. EEA was measured at the Crop Production Systems Research Unit at USDA ARS, Stoneville, MS. The soil dataset was analyzed overall and separately by years. For example in 2018, any cultivar and/or planting density effects occurred, they did so exclusively early in the growing season (weeks 1-8). The greatest number of abiotic attributes varied among the management choices in the first sampling (week 1) when soil pH, K(ppm), Mg(ppm), total K%, Ca%, H% and K:Mg varied among the switchgrass cultivars. Later in the growing season, after week 8, there was no evidence for any switchgrass cultivar effects on soil chemistry. Planting densities differed in neither soil PLFA-inferred microbial biomasses nor EEAs. In contrast, PLFAs varied among the switchgrass cultivar, but did so only in the first half of the growing season (weeks 2, 4 and 16). Data from objectives 3 and 4 were outlined as part of the master's thesis by Joseph bridges to be defended in fall 2022 at ASU. This will also be published separately as a journal article.
Publications
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Progress 03/15/20 to 03/14/21
Outputs
Target Audience:Target audience outlined in this reporting period include research community, and students.This project provided experiential learning opportunities to students at Alcorn State University (ASU) and Kansas State University (KSU):Two Undergraduates at ASU assisted in sample processing and data processing until March 2020. Similarly, KSU engaged one undergraduate in this project.Graduate student at ASU worked on soil data analysis and is developing his thesis.Graduate student at KSU extracted DNA from all leaf, root and soil samples from 38 weeks of sampling for a total of 192 samples for each of leaf, root, and soil compartments. The graduate student mentored the undergraduate student and oversaw her activities in the laboratory for during the spring 2020 semester. Note that COVID-19 pandemic resulted in lab closures and limited research activity. All samples have now been sequenced and the developed pipelines have completed all analyses of samples from 2018. These data are now being written into a manuscript that will become a substantial component in the graduate student's thesis. The 2019 samples are currently being analyzed. Even though these data matrices are large, we are currently developing means to analyze Amplicon Sequence Variants (ASVs). We also acquired samples for metagenome and proteome samples that remain in archive. Our National Laboratory (PNNL) partners are still under limited operations and the future of these analyses remains under negotiation. Overall, in 2020, this project saw the participation/training of 3 undergraduate students and 2 graduate students. Changes/Problems:The pandemic has presented a substantial challenge to effectively continue our research efforts. All graduate student supervision has moved to virtual resulting in delay in communications. Travel and research restrictions have hampered our ability to acquire data although the samples have been available. Similarly, our training activities have been substantially impacted. Undergraduate engagement has become more challenging and our new CURE activity has been delayed. We hope that we can resume these activities in the fall 2021 or spring 2022 at the very latest. For our metaproteome work, we continue to expect some delays. CoPI Jumpponen has requested user agreements with the Pacific Northwest National Laboratory (PNNL) Environmental Molecular Sciences Laboratory (EMSL) to acquire the compatible proteome data using their mass-spectrometry facility. Uncertainty of lab access and PNNL other priorities have made these analyses challenging to arrange. In the meantime however we will analyze all of our datasets for microbiomes, soil properties, microbial biomass and ethanol production as we have 2 years data. What opportunities for training and professional development has the project provided?Training Activities: Alcorn State: Two undergraduate students were trained in sample collection, sample processing, data collection and data entry until March 2020, prior to COVID lockdown.Kansas State: One undergraduate student was trained in sample processing, nucleic acid extraction, and molecular methods including PCR and high throughput library preparation. Based on the current research activities, we also designed a Course-based Undergraduate Research Experience (CURE) activity to be delivered in General Microbiology (BIOL455) and Biology of Fungi (Biol604) for a total of more than 400 students annually. We had agreed on a pilot CURE during summer of 2020, when enrollment is ~ 20% of that in the spring and fall semesters. The pandemic restrictions led to delay of this activity and we are planning to revisit the pilot run during summer 2021 in BIOL455 or fall 2021 in BIOL604. How have the results been disseminated to communities of interest?Due to COVID we were unable to conduct any field related tours or outreach activities. Abstracts were submitted to the American Microbiological Society (ASM), but the meeting was cancelled. ASU students presented at the University research symposium held in Feb 2020 prior to COVID lockdown. What do you plan to do during the next reporting period to accomplish the goals?We continue to seek user agreement with the Pacific Northwest National Laboratory (PNNL) Environmental Molecular Sciences Laboratory (EMSL) to acquire the proteome data using their mass-spectrometry facility.PIhas initiated data analyses in consultation with statistician. To conduct multivariate data analyses, we will need the remainder of the PLFA and NLFA data which we hope to receive by early May 2021. We will analyze the overall soil data, microbial EEA and PLFA data (objective 3 & 4) for 2 years. Graduate student from ASU would have defended his thesis.
Impacts
What was accomplished under these goals?
Objectives 1:We have extracted DNA from 384 leaf, root and soil samples and those have now been sequenced. First data have been analyzed and indicate that communities in the leaves, roots and soils diverge in composition over time, i.e they overlap more early in the growing season than they do later. Although the microbiomes in the cultivars differ, they do so only in the early growing season. The current analyses are consistent with the preliminary analyses and indicate that the planting densities have a lesser effect. In sum, these data improve our understanding of microbiome seasonal dynamics as well as the effects of management decisions in conservation agriculture. Further analyses are underway to determine whether our data support the soil origin of plant microbiomes hypothesis. Objective 2:We used our community data to identify the leaf-associated communities that differed most and chose two four cultivars with most distinct microbiomes (Alamo and BoMaster) with four biological replicates each for a total of 16 experimental units for metagenome and -proteome sampling. The COVID-19 restrictions have limited our ability to generate metagenome and metaproteome data. We continue to archive our samples and look forward to pandemic situation resolving. Objective 3: We ascertained the impact of varieties and densities on biomass sugar and ethanol yield from 2019 switchgrass samples and compared the same with that from 2018. Graduate student Bridges will submit master's thesis and defend in spring 2021. Due to COVID delays and backlog, we are still awaiting biomass compositional data from external service provider and we expect data in April 2021. Objective 4: Week 38 samples from 2019 were subjected to Extracellular Enzyme activities (EEA) for cellobiohydrolase, phosphatae, beta glucosidase and beta N glucosaminidase. PLFA and NLFA data from 192 soil samples from 2019 were sent to the external service provider and due to COVID delays, short staffing due to social distancing, only 25% of the sample data were received and we are awaiting data from the remainder samples. We expect data by May 2021.
Publications
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Kazarina A*, Mandyam K, Jumpponen A, Panicker G (2020) Panicum virgatum tissue associated microbiomes diverge from soil inoculum in the course of a growing season. American Society of Microbiology, June 18-22, Chicago (P; IN) COVID cancellation
2. Bridges J*, Okoro C**, Mandyam K, Nanjundaswamy A, Panicker G (2020) Cellulosic ethanol production from switchgrass cultivated for soil conservation is impacted by variety and planting density. 6th Centers for Research Excellence, Alcorn State University, Feb 6, 2020 (P; L)
3. Bridges J*, Omole T**, Kazarina A*, Mandyam K, Nanjundaswamy A, Panicker G (2020) Soil organic matter is impacted by host genotypes and planting densities under switchgrass Cultivated for Soil Conservation. 6th Centers for Research Excellence, Alcorn State University, Feb 6, 2020 (P; L)
4. Dea, H., Kazarina, A., Hansen, P., Ismert, K., Platt, T., Sikes, B., Jumpponen, A. 2020. Does prairie restoration also restore prairie bacterial communities? American Society of Microbiology Microbe Annual Meeting, Chicago IL. June 18-22, 2020. Poster presentation and an Abstract. COVID-19 CANCELLATION.
5. Jumpponen, A., Loecke, T., Hui, N., Seta?la?, H. 2020. Plant-associated microbiomes in human altered systems. American Society of Microbiology Microbe Annual Meeting, Chicago IL. June 18-22, 2020. Invited symposium presentation and an Abstract. COVID-19 CANCELLATION.
6. Okoro C*, Mandyam K, Bridges J*, Kazarina A*, Nanjundaswamy A, Panicker G (2020) Understanding the impact of switchgrass Soil Conservation on soil microbial biomass. 6th Centers for Research Excellence, Alcorn State University, Feb 6, 2020 (P; L)
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Progress 03/15/19 to 03/14/20
Outputs
Target Audience:Target audience outlined in this reporting period include research community, and students. This project provided experiential learning opportunities to students at Alcorn State University (ASU) and Kansas State University (KSU): Two Undergraduates at ASU assisted in sample processing over the 38 week period. Graduate student at ASU collected samples over the 38 week period and processed all the 192 soil samples for analysis. He also processed biomass samples from 2018 for compositional analysis and cellulosic ethanol production. He harvested biomass in December 2019 and is presently processing those for ethanol production Graduate student at KSU extracted DNA from all leaf, root and soil samples from 38 weeks of sampling for a total of 192 samples for each of leaf, root, and soil compartments. The graduate student mentored two undergraduate students and oversaw their activities in the laboratory for a period of two semesters. All samples have now been either sequenced of submitted to sequencing. With the faculty scientist, the graduate student is currently building computer pipelines and scripts to expediently analyze sequence data once all sequencing is complete. We also acquired samples for metagenome and proteome samples. We are presently negotiating with the Pacific Northwest National Laboratory the arrangement to complete metagenome and -proteome analyses. Overall in 2019, this project saw the participation/training of 6 undergraduate students and 2 graduate students. Changes/Problems:For our metaproteome work, we expect some delays. CoPI Jumpponen currently is negotiating user agreement with the Pacific Northwest National Laboratory (PNNL) Environmental Molecular Sciences Laboratory (EMSL) to acquire the compatible proteome data using their mass-spectrometry facility. In the meantime however we will analyze all of our datasets for microbiomes, soil properties, microbial biomass and ethanol production as we have 2 years data. What opportunities for training and professional development has the project provided?Training Activities: Alcorn State: Two undergraduate students were trained in sample collection, sample processing, data collection and data entry. Two undergraduate students were trained in measuring plant data in the field. Graduate student was trained in soil sample processing based on standard protocols, biomass pretreatment and cellulosic ethanol fermentation. He was trained in operating 7L bench-top fermenters using processing controls. Kansas State: Two undergraduate students were trained in sample processing, nucleic acid extraction, and molecular methods including PCR and high throughput library preparation. This training continues with data analyses. How have the results been disseminated to communities of interest?A tour of the plot and concepts of soil erosion prevention, C4 grass physiology and anatomy and use of field instrumentation like soil compaction meter, moisture meter, AccuPar and Ceptometer was provided to visiting students from Puerto Rico in summer 2019. Our data has been presented in national meetings (Mycological Society of America and American Society of Agronomy). What do you plan to do during the next reporting period to accomplish the goals? Jumpponen has secured metagenome sequencing at the University of Kansas core facility to generate the metagenome data that are helpful to assemble metaproteomes. He currently is negotiating user agreement with the Pacific Northwest National Laboratory (PNNL) Environmental Molecular Sciences Laboratory (EMSL) to acquire the compatible proteome data using their mass-spectrometry facility. CoPI Nanjundaswamy and graduate student will ascertain the impact of varieties and densities on biomass quality and ethanol yield from biomass samples from 2019 and compare the same with that from 2018. Overall impact of conservation agriculture practices on biomass yield and ethanol yield will be analyzed. Graduate student is expected to submit master's thesis and defend in summer 2020. Mandyam will process soil samples from 2019 and submit them for PLFA and NLFA. Data from 2018 and 2019 will be analyzed to draw overall inferences regarding impact of conservation practices and switchgrass varieties and densities on soil parameters and soil microbial biomass. Correlation regression analyses will be conducted to draw big picture inferences.
Impacts
What was accomplished under these goals?
Objective1: We have extracted DNA from 192 leaf, root and soil samples and those have now been either sequenced or submitted to sequencing. Our preliminary data analyses show that total and core communities occupying the leaves, roots and soils diverge in composition over time, i.e they overlap more early in the growing season than they do later. Moreover, some cultivars differ in community compositions but the planting densities appear to have a lesser effect. These data improve our understanding of microbiome seasonal dynamics as well as the effects of management decisions in conservation agriculture. Objective 2: We used our community data to identify the leaf-associated communities that differed most. Based on these data we chose two of the four cultivars included in our experiments (Alamo and BoMaster) and the two planting densities with four biological replicates each for a total of 16 experimental units for metagenome and -proteome sampling. We have secured metagenome sequencing at the University of Kansas core facility to generate the metagenome data that are helpful to assemble metaproteomes. We are currently negotiating user agreement with the Pacific Northwest National Laboratory (PNNL) Environmental Molecular Sciences Laboratory (EMSL) to acquire the compatible proteome data using their mass-spectrometry facility. Objective 3: Major activities: Biomass samples from 32 units were harvested at the end of the season in 2018 and 2019. Samples were saccharified by acid-pretreatment. Saccharified biomass samples were then subjected to cellulosic ethanol production. Biomass yield was recorded. Data collected: Both 2018 and 2019 biomass yield was recorded per unit area. Biomass samples from 2018 were processed and analyzed for cellulose, hemicellulose and lignin. Saccharified samples were evaluated for glucose release and subsequent ethanol production by HPLC. Summary statistics: Biomass yield did not vary significantly between the years but was impacted by variety and density. When analyzed separately by years, significant impact of both variety and density was seen with significant interaction only for 2018. As predicted, in both years high density planting resulted in significantly higher yield than low-density planting. In 2019, varieties varied as Bomastera>Alamob>Colonyc>Kanlowc whereas in 2018 varieties varied as Alamoa>Bomastera>Colonyb>Kanlowb Overall, BoMaster and Alamo were top yield varieties in terms of biomass with 51-52 metric tons per hectare yield in 2018 for high density planting and 40-42 metric tons/hectare for low density planting. Cellulose, hemicellulose and lignin did not vary significantly over varieties or densities. About 39-41% W/W cellulose was seen in switchgrass biomass. Biomass total C and N also did not vary significantly. Glucose and xylose release upon saccharification were not impacted by variety or density but were impacted by their interaction. This was mainly driven by the difference in sugar yields between Alamo-HDP and Kanlow HDP with the latter resulting in highest glucose. The ethanol yield was significantly impacted by variety and variety*density interaction. The highest ethanol yield was from Kanlow-HDP and least was from Alamo-HDP. Alamo-LDP was comparable in ethanol yield with Kanlow-HDP and Kanlow-LDP. Literature review suggests that Kanlow is the best variety for ethanol production and confirmed in our study as well. Key outcomes: Biomass yield from conservation agriculture is 2-10 times higher than that reported for similar lowland varieties in other parts of the U.S. Our ethanol yields were low but is attributed to shake flask conditions. Scale-up in fermenters will result in higher ethanol yields. Considering biomass yield and ethanol yield, our ethanol production ranged from 3,969L/ha in BoMaster-HDP to 2,878L/ha in Kanlow-LDP. This is comparable to ethanol yield reported from other studies (3,880 and 3,517 L/ha for Kanlow and BomAster; Scagline-Mellor et al 2018) with our system having the advantage of high biomass yield without inputs of fertilizer, chemicals or intensive management practices. Objective 4: Major activities: 192 soil samples from year 2019 were collected to coincide with weeks 1, 2, 4, 8, 16 and 38 since time of grass emergence in spring. Data collected: Soil samples were processed and analyzed for 12 most important factors: pH, OM, CEC, S, P, Ca, K, Mg, Cl, NO3-N, NH4-N, total N, total C. Fresh soil samples were subjected to Extracellular Enzyme activities (EEA) for cellobiohydrolase, phosphatae, beta glucosidase and beta N glucosaminidase. Soil samples were freeze-dried and processed for PLFA and NLFA analyses. Summary Statistics: A total of 384 soil samples from years 2018 and 2019 were analyzed. The tested edaphic factors varied significantly over time, and only soil organic matter was impacted by varieties and densities. EEA data from 384 samples from both years varied significantly over time but were unaffected by varieties or densities. PLFA and NLFA data from 192 soil samples from 2018 indicated that both arbuscular fungi biomass and total microbial biomass vary over time with peak biomass coinciding with peak plant growth in summer (week 16); overall, the fungi:bacterial biomass was <1 indicating low organic inputs (confirmed by soil data) and a soil system dominated by bacteria; bacterial community was balanced and indicated greater diversity; by summer the soil system had less stress with more stable microbial communities. Statistical analyses is ongoing to understand the impact of varieties and densities on microbial PLFA and NLFA. Key outcomes: Soil organic matter is impacted by varieties and densities as predicted.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Mandyam K, Jumpponen A, Kazarina A*, Bridges J*, Nanjundaswamy A, Panicker G (2019) Switchgrass root mycobiome dynamics and responses to management in a conservation ecosystem. ASA community-Soil health; ASA Section- Land management and Conservation; American Society of Agronomy, Crop Science Society of America, Soil Science Society of America (ASA, CSSA and SSSA) 2019 Annual Meeting, Nov 10-13, 2019, San Antonio, TX (Oral; IN)
2. Bridges J*, Kazarina A*, Omole T**, Mandyam K, Nanjundaswamy A, Panicker G (2019) Exploring cellulosic ethanol production feasibility from switchgrass cultivated for soil conservation in Mississippi. ASA community- Bioenergy Systems, ASA Section- Agronomic Productions Systems. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America (ASA, CSSA and SSSA) 2019 Annual Meeting, Nov 10-13, 2019, San Antonio, TX (Oral; IN)
3. Omole T**, Bridges J*, Kazarina A*, Mandyam K, Nanjundaswamy A, Panicker G (2019) Seasonal variation of arbuscular mycorrhizal fungal abundance in switchgrass in response to management in a conservation agroecosystem. ASA community- Bioenergy Systems, ASA Section- Agronomic Productions Systems. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America (ASA, CSSA and SSSA) 2019 Annual Meeting, Nov 10-13, 2019, San Antonio, TX (Poster; IN)
4. Mandyam K, Tyler H, Bridges J*, Kazarina A*, Nanjundaswamy A, Panicker G (2019) Seasonal variation of microbial enzyme activities in response to switchgrass cultivation in a conservation agroecosystem. ASA section- Land Management and Conservation American Society of Agronomy, Crop Science Society of America, Soil Science Society of America (ASA, CSSA and SSSA) 2019 Annual Meeting, Nov 10-13, 2019, San Antonio, TX (Poster; IN)
5. Kazarina A*, Mandyam K, Jumpponen A, Panicker A (2019) Seasonal dynamics of mycobiomes associated with the grass Panicum virgatum growing under conservation agriculture conditions. Mycological Society of America, St. Paul, MN, Aug 11-14, 2019 (Oral; IN)
6. Berry J**^, Panicker G, Mandyam K, Cary T (2019) Assessment of the relationship of leaf area index (LAI), percent canopy cover (PCC) and dry biomass of switchgrass (Panicum virgatum) for erosiron prediction models. 83rd Annual Meeting of Mississippi Academy of Sciences, Hattiesburg, MS, Feb 21-22, 2019 (Oral; L)
7. Berry J**, Panicker G, Mandyam K, Cary T (2019) Assessment of the relationship of leaf area index (LAI), percent canopy cover (PCC) and dry biomass of switchgrass (Panicum virgatum) for erosiron prediction models. Section- Renewable energy, natural resources and environment. Association of 1890 Research Directors (ARD) 19th Biennial Research Symposium, Mar 30-Apr 3, 2019, Jacksonville, FL (Oral; N)
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Progress 03/15/18 to 03/14/19
Outputs
Target Audience:Target audience outlined in this reporting period include research community, and students. Efforts: Formal classroom instruction: CoPI Panicker taught undergraduate course PS 459 Soil Fertility and graduate course PS599 Advanced Soil Fertility. In both courses, students were introduced to conservation agriculture and its benefits to soils and visited the experimental plots. This project provided experiential learning opportunities to the following students at Alcorn State University (ASU) and Kansas State University (KSU): Ms. Anna Kazarina, Undergraduate, ASU The student collected and processed soil, root and leaf samples as outlined in Objective 1 on week 1, week 2, week 4 and week 8 of switchgrass germination. She also visited Dr. Heather Tyler's lab in USDA-ARS, Stoneville, MS to process samples for soil extracellular enzyme assays (EEA). Upon graduation from ASU in spring 2018, she was recruited to the Master's program in Biology in co-PI Dr. Jumpponen's lab at KSU in summer 2018 to continue to work on this project. Mr. Jy' Quavis Berry and Ms. Kelsey Henry, Undergraduates, ASU Students were involved in recording plant phenology data as part of long-term data collection and participated in biomass harvest in December 2018. Ms. Jessica Ransom and Ms. Temitope Omole, Undergraduate, ASU The students assisted in sample collection and processing of soil samples for EEA analysis for Objective 1. Mr. Michael Felton, Graduate, ASU He assisted in sample collections in spring and summer 2018 over the eight-week period as outlined in Objective 1. The student also participated in EEA at Dr. Tyler's lab in ARS, Stoneville, MS. The student is pursuing a doctoral program in soil science at Alabama A & M since fall 2018. Mr. Joseph Bridges, Graduate, ASU He collected samples for week 16 and week 38 and processed all the 192 soil samples for analysis. He is also processing biomass samples for compositional analysis and cellulosic ethanol production for Objective 3. Ms. Anna Kazarina, Graduate, KSU The student started her M.Sc. program in June 2018 and continued working on the samples obtained during her undergraduate at ASU. During the reporting period she 1) extracted nucleic acids from obtained samples; 2) built MiSeq libraries for bacterial and fungal communities from these samples; 3) acquired skills to analyze MiSeq generated sequence data; and 4) began training data manipulation and analyses to address the questions outlined in Objective 1. Ms. Ayanna Castro-Ross, Undergraduate, KSU The student was recruited to assist in MiSeq library generation. She has acquired skills in DNA extraction, PCR, DNA visualization, DNA quantitation, and trouble shooting. She will assume more active role once training has been completed. Overall in 2018, this project saw the participation/training of three male (minority) students and five female students of which three were minority students. Changes/Problems:The major problem encountered for Objective 1 was that the standard protocol for obtaining leaf discs for phyllosphere microbiome investigations could not be applied uniformly for all the time points. Younger leaves from week 1 and 2 were easily processed whereas for subsequent time points it was difficult to apply the protocol consistently. To avoid method biases, we decided to defer leaf sampling to year 2019 and continue to process the roots and soils for 2018. CoPI Jumpponen and student Kazarina evaluated a modified protocol for grass leaf processing and expect that leaves will be processed effectively. Originally, Objective 2 regarding phyllosphere metaproteomes was to be conducted in year 2 after accomplishing Objective 1. By introducing a modified protocol, we will be able to repeat accomplish both Objective 1 and 2 in year 2019. This modification does not alter the outcome of the project. We will address all the objectives as outlined and the soil and root time series microbiome data from 2018 will be a stand-alone contribution and lay the foundation for the data from 2019. Originally, the plan was to sample from week 1 to week 8 (April) since leaf emergence. We included two additional time points, week 16 (July, peak biomass) and week 38 right before biomass harvest to capture the dynamics of microbiomes through peak biomass and senescent biomass. The collection of root microbiomes was not outlined originally. We have included root and soil microbiome (rhizobiome) assembly for 2018 and phytobiome assembly for 2019. What opportunities for training and professional development has the project provided?Training Activities: Ms. Anna Kazarina was supported by the AFRI grant until spring 2018 at ASU as an undergraduate researcher. As a part of mentoring, the student was required to enroll in the undergraduate courses PS 490 Research Methods and PS 494 Biology of Fungi at ASU offered by the PI to have a broad understanding of the research methodology and fungal biology. Ms. Anna Kazarina started her M.Sc. training at KSU in fall semester 2018. She has taken graduate level classes in community ecology and statistics as well as gained expertise in MiSeq pipeline analyses and multivariate tools to analyze complex multidimensional community data. Students - Bridges, Omole and Ransom were trained in sample collection, sample processing, data collection and data entry. Bridges was trained in soil sample grinding and sieving based on standard protocols, biomass pretreatment and cellulosic ethanol fermentation. He was trained in operating 7L bench-top fermenters using processing controls. As a part of mentoring, Bridges took a course on Advanced Research techniques (PS 590). Students Berry and Henry were trained in collecting soil compaction data and using the AccuPar and Ceptometer for recording percent canopy cover and leaf area index. Ayanna Castro-Ross was trained in DNA extraction, PCR, DNA visualization, DNA quantitation, and trouble shooting. She will assume more active role once training has been completed. How have the results been disseminated to communities of interest?CoPI Panicker provided a tour of the plot and introduced concepts of soil erosion prevention, C4 grass physiology and anatomy and use of field instrumentation like soil compaction meter, moisture meter, AccuPar and Ceptometer to 41 high school students from neighboring counties on 6/14/2018. This outreach activity was conducted in partnership with a Community Collegeto introduce agriculture and STEM research as academic and career choices to high school students. What do you plan to do during the next reporting period to accomplish the goals? Soil, leaf and root samples will be collected again in 2019 soon after leaf emergence in spring. We will ensure that leaves are collected and processed for both microbiome work and proteome work simultaneously. We envision use of commercially available kits that permit simultaneous analyses of genomes, transcriptomes and proteomes. We will build the phyllosphere microbiome and ascertain the possible contributions of the root and/or soil microbiome in shaping the phyllosphere microbiome. As outlined in the proposal the goal is to identify the time point when the phyllosphere microbiome diverges from soil and/or root microbiome. Using the microbiome data we will determine when the phyllosphere microbiomes will stabilize and further analyze those samples for proteomes to determine if the communities also differ in functional attributes. CoPI Jumpponen and student Kazarina will analyze data for the soil and root bacterial and fungal microbiomes from year 2018. In addition, Jumpponen and Kazarina will continue the fruitful collaboration and process samples from 2019 efforts to accumulate the complementary microbiome and proteome data. Mandyam and Panicker will provide soil data for the respective time points to help build the secondary data matrix. CoPI Nanjundaswamy and Bridges will ascertain the impact of conservation agriculture practices on biomass quality and ethanol yield. In 2019, they will collect leaf samples at the required time points to ascertain the leaf sugars and conduct compositional analyses. Mandyam and students Bridges, Ransom and Omole will be involved in collecting soil, root and leaf samples, measuring plant phenology data and conduct EEA studies at Dr. Tyler's lab at ARS Stoneville, MS. We anticipate collecting 192 soil samples for respective time points as in year 2018.
Impacts
What was accomplished under these goals?
Objective 1 was initiated as outlined in the proposal. The original goal was to collectsoil and leaf samples until week 8 in April. Root, leaf and soil samples were collected from 32 experimental units on week 1, 2, 4, 8, 16 and 38 since leaf emergence in March 2018. We extended the collection efforts to include week 16 (July, peak biomass) and week 38 right before biomass harvest to capture the dynamics of microbiomes through peak biomass and senescent biomass. The collection of root microbiomes was not outlined originally. We opted to include rhizobiome assembly (soil and root microbiome) in 2018 and conduct phytobiome (soil, root and leaf) assembly in 2019. These modifications will ensure understanding of the origin and assembly of switchgrass microbiomes. Soil samples were additionally processed for soil testing of the 12 most important factors: pH, OM, CEC, P, Ca, K, Mg, Cl, NO3-N, NH4-N, total N, organic C, S and Extracellular Enzyme activities (EEA). DNA from the acquired samples has been extracted and submitted to be MiSeq sequenced. The libraries generated from samples have been preliminarily analyzed at the KSU Genomic core facility and their quality was determined adequate for sequencing. As a result, the four MiSeq libraries (bacteria and fungi from both roots and soils) are in queue for sequencing once machine time is available. Change of Knowledge: We acquired rhizosphere soil, root and leaf tissue samples from the experiment to address the proposal's objectives. However, during processing, we learned that the outlined plan for leaf processing was unsuitable for our samples collected after week 2 - we opted to return to the experiment in 2019 to repeat sampling with a modified and improved sampling protocol. Change of Action: Graduate student Kazarina evaluated methods for processing leaves for microbial DNA extraction. We found that storing leaf disks on site in DNA extraction buffer was superior for nucleic acid extraction, while preserving a larger aliquot for additional analyses. Objective 3: Biomass was harvested on 12/11/2018 from 32 experimental units and yield recorded. Last year (2017), we found that i) overall high density planting (HDP) resulted in significantly higher yield than low density planting (LDP), ii) varieties were significantly different with Alamo resulting in highest and Kanlow the lowest yield with Colony and BoMaster with intermediate yield and iii) there was no significant interaction between density and variety. We anticipate similar results for year 2018. Biomass will be subjected to compositional analysis and cellulosic ethanol production. Objectives 2 and 4 will be conducted in 2019.
Publications
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Mandyam K, Jumpponen A, Panicker G, Nanjundaswamy A (2018) Pre- and postharvest switchgrass root microbiomes and the effect of planting densities. 21st Penn State Plant Biology Symposium: Wild and Tamed Phytobiomes, Penn State University, State College, PA, June 19-22, 2018
Mandyam K, Jumpponen A, Panicker G, Nanjundaswamy A (2018) Pre- and post harvest switchgrass root microbiomes and the effect of planting densities. 48th Earth Day, The Charles Capps Center, Delta Research & Extension Center, USDA, Stoneville, MS, April 19, 2018 (Invited)
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Progress 03/15/17 to 03/14/18
Outputs
Target Audience:Target audience outlined in the project initiation include research community, farmers and students. Efforts: In year 1 (March 2017 to March 2018): Co-PI Dr. Girish Panicker taught 13 credits of Agronomy courses in spring and fall 2017; In spring 2017, he taught PS122 Crop production (Undergraduate; 3 credit hours; 36 students) and PS122L Crop Production Lab (Undergraduate; 1 credit; 37 students) and PS548 Advanced Soil Management (Graduate; 3 credits; 3 students). In fall 2017, he taught PS459 Soil Fertility (Graduate; 3 credit hours; 4 students) and PS599 Advanced Soil Fertility (Graduate, 3 credits; 6 students). All these courses covered concepts of sustainable agriculture and a total of 50 students visited the switchgrass plots to understand concepts of conservation agriculture, cover crop and soil conservation. Co-PI (Nanjundaswamy) offered two courses in spring 2017: PS448 Soil Management (Undergraduate: 3 credit hours; 17 students) and PS 320 Biomass and Bioenergy (undergraduate; 3 credit hours; 4 students). In the former course, the co-PI introduced concepts of sustainability using the switchgrass plots as a model, and in the latter course the switchgrass varieties served to introduce concepts in biomass production for bioenergy. Overall, in the first year of the study, the switchgrass plots were part of Alcorn's classroom instruction for a total of 71 students participating in seven courses in Agronomy. Majority of the students in these five courses were minority, African-American students. Our research plots also served as conservation agriculture demonstration plots to i) visiting Jordanian farmers as a part of Cochran Fellowship program of USDA Foreign Agricultural Service. At least seven Jordanian farmers visited ASU from July 8-22, 2017. The Fellowship named after U.S. Senator Thad Cochran from MS was initiated in 1984, has provided training to more than 17,500 fellows from 125 countries since its initiation, with 600 fellows annually visiting US Universities, government agencies and companies for 2-3 week to gain short-term training in agriculture-related technical knowledge or skills; ii) over 20 farmers from MS visited the plots on June 16, 2017 as a part of field day conducted by the Alliance for Sustainable Agricultural Production located in Holmes County, MS. ASU is one of the partners and sponsor of Alliance field days. Several participating farmers were minority, small- or resource-limited farmers; iii) MS legislators and NRCS staff visited the plots May 18-19, 2017; and iv) Indian High Commission in Atlanta, GA accompanied a team of four farmers from the agricultural co-op from the state of Maharashtra, India and visited the switchgrass plots on June 28-29, 2017. The farmers were specifically interested in the feasibility of cultivating switchgrass for prevention of soil erosion in Maharashtra. This project provided experiential learning opportunities to the following students at Alcorn State University (ASU) and Kansas State University (KSU): 1. Ms. Rebecca Becker, Undergraduate, ASU The student worked with co-PI Dr. Panicker and was trained in collecting soil and plant samples; processing samples for nutrient analysis; collecting plant parameters like percent canopy cover and leaf area index; data were collected before grass harvest in November 2016 and during grass emergence in March 2017. Student made presentations at the 2017 Annual Mississippi Academy of Sciences and also at the 2017 1890 Association of Research Directors (ARD) Research Symposium. She is now employed as Agronomist with the Office of Agriculture Commissioner, MS, and is likely to pursue Masters in Agronomy starting fall 2018. 2. Ms. Anna Kazarina, Undergraduate, ASU The student worked with PI Dr. Keerthi Mandyam since spring 2017 and was trained in collecting plant and soil samples from the switchgrass research plots, and processing samples for microbiome investigations. In summer 2017, the student along with the PI travelled to KSU and were hosted for two weeks by co-PI Dr. Ari Jumpponen. First, the student was introduced to Long Term Ecological Research (LTER) by undertaking a field visit to Konza Prairie, a native tallgrass prairie and one of the 24 LTERs in North America. The goal was to understand switchgrass as a component of native grasslands and its cultivation for conservation agriculture. The student isolated root colonizing fungi from native switchgrass to compare them with those isolated from switchgrass cultivated for conservation in Mississippi. The student isolated more than 55 cultures on seven different selective media. She is presently screening these fungi for a specific group of root colonizing endophytes, namely dark septate endophytes by testing Koch's postulates. Since the student will undertake sample collection and processing for microbiome analysis as outlined in Objective 1 in spring 2018, the student was provided hands-on training in DNA extraction, DNA quantification, and PCR amplification of DNA. We are currently recruiting Ms. Kazarina into MSc program in Dr. Jumpponen's laboratory at KSU. The envisioned plan is to engage Ms. Kazarina within the current program and assume primary responsibilities for characterizing switchgrass microbiomes as outline in objective 1. 3. Mr. Jy' Quavis Berry, Undergraduate, ASU The student has been a part of this project since summer 2017 and has been collecting data since. He has gained experience in using the Accupar to collect Leaf area index, percent canopy cover and in 2018, will be trained in using the chlorophyll meter. Additionally, he collected biomass data after harvest in November 2017. His data indicated the strong positive correlation of leaf area index and percent canopy cover with biomass. 4. Mr. Michael Felton, Graduate, ASU The student has volunteered to assist in sample collections in spring 2018 over the eight-week period as outlined in Objective 1. The student has assisted in sample collection from switchgrass plots in the past and is well versed with the design and sample collection procedure. 5. Mr. Christopher Reazin, Undergraduate, KSU The student was an integral resource providing hands-on expertise on building the first switchgrass MiSeq libraries for microbiome characterization. He worked closely with Dr. Mandyam and Ms. Kazarina during their visits to KSU. 6. Mr. Kyle Ismert, Undergraduate, KSU The student was introduced to pure-culturing and manipulation of fungi in pure-culture. Within the reporting period, the student participated in developing and designing tools and experimental approaches to evaluate fungal tolerance in vitro. 7. Ms. Samantha Fox, Graduate, KSU The graduate student shared responsibilities for assisting ASU visitors during their collaborative efforts at KSU. Mr. Joseph Bridges, Graduate, ASU The student was recruited for the master's program in Agronomy, ASU in fall 2017 to work towards this project. He will coordinate data collection with the other students and the main objective of his thesis will be to understand changes of edaphic factors during the 7th and 8th year of switchgrass conservation agriculture. He will be advised by the PI and co-PI (Panicker). Of all the student participants, three are minority (male) students and three female students. Changes/Problems:Objective 1: To identify core phyllosphere microbiomes and their potential origin from soil microbiomes from four switchgrass varieties cultivated under conservation agriculture This objective was to be accomplished by sampling for switchgrass soil and leaf samples during weeks 1, 2, 4 and 8 since grass emergence in spring 2017 (year 1). Each week a total of 32 soil and 32 leaf samples were to be collected. Usually, switchgrass emergence occurs during the first week of March. However, in 2017, due to unusually warmer conditions, one of the varieties of switchgrass (Bomaster) emerged unexpectedly early in the second week of February. Samples could not be collected as outlined in the project since we received funding only during first week of March 2017. Collecting samples during second week of March 2017 would have compromised the experimental design including the exclusion of one of the varieties completely. Also, Objective 2 is dependent on the results from Objective 1. Since we could not capture all of the samples from the time of emergence in year 1, we decided to defer the collection of samples for objective 1 to spring 2018 (year 2). This does not alter the objectives or experimental design or the goals of the project, but merely extends the end-date of the project probably by six months to a year. We are carefully monitoring our plots to ensure timely collection of potentially early emerging samples in spring 2018. What opportunities for training and professional development has the project provided?Training Activities: Ms. Anna Kazarina is supported by the AFRI grant. The PI and Co-PI (Jumpponen) trained undergraduate student from ASU Ms. Kazarina in gaining hands-on experience in DNA extraction and quantification; generation of PCR libraries, and provided insights into switchgrass ecology in native tallgrass prairie (Konza LTER) during her visit to Konza and KSU in summer 2017. The student was introduced to basic grassland ecology, species composition of a tallgrass prairies, the importance of switchgrass in a native tallgrass prairie, and as a native grass its use in conservation agriculture. Since our experimental plot in ASU is based on the randomized complete block design (RCBD), the student was familiarized with Experimental Designs by the PI and co-PI (Panicker) at ASU. As a part of mentoring, the student was also required to enroll in the Undergraduate course PS 495 Experimental Design at ASU offered by the PI to have a broad understanding of the designs used in an agriculture setting. Ms. Rebecca Becker and Mr. J Berry were trained in collecting leaf area index, percent canopy cover, soil compaction, biomass production under two densities and processing samples for compositional estimation. Both undergraduate students were mentored by co-PI (Panicker). Ms. Becker graduated and is employed as Agronomist with Office of Agriculture Commissioner, MS. Mr. Kyle Ismert (KSU) was trained in pure-culture manipulation and experimental design. In addition, the student enrolled into Jumpponen's Biology of Fungi to prepare and continue training as a graduate student in 2018-2019 academic year. Ms. Samantha Fox (KSU) has acquired skills and continues training towards fluency in the use of common statistical inference tools for community analyses on statistical software platform R. She has been integral ion assisting in experimental designs and continues to serve as a resource to assist script writing for statistical analyses in R. Mr. Berry will continue to be associated with project and will collect plant phenology and soil data. How have the results been disseminated to communities of interest?As a part of outreach activities, information regarding switchgrass cultivation for soil conservation was disseminated through the Alliance of Sustainable Farms (Holmes County, MS) field days which are sponsored annually by consortium of several institutions in MS, including ASU. Farmers from all over MS participate in these field days and over 20 farmers were part of the Alliance field day held in ASU in 2017. To reach a wider audience, co-PI (Panicker) partners annually with NRCS staff and USDA Foreign Agricultural Service (FAS). For example, through the 2017 Cochran Fellowship a program with USDA (FAS), a team of 7 farmers from Jordan were given short-training course in conservation agriculture at ASU. Ari Jumpponen presented to the microbiome communities at Pacific Northwest National Lab (PNNL) and University of Colorado in Jan2018 and March 2017 regardingFungal rhizobiomes of common grasses: core species, functions, and potential drivers respectively. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting year (March 2018 to March 2019), we will accomplish the following: 1. Collect soil and leaf samples for microbiome investigations as outlined in objective 1. A total of 128 soil and leaf samples respectively will be collected by graduate and undergraduate students at ASU and the PI. Leaf samples will be processed at ASU and all samples will be shipped to KSU, where processed for MiSeq community analyses by KSU graduate and undergraduate students (below). 2. At least two undergraduates and one graduate student have been identified to assist with the project. Mr. J Berry (undergrad) will continue to assist co-PI (Panicker). Undergrad Ms. Kazarina will continue to participate in the project until spring 2018 in the PI's lab. A new graduate student Mr. Bridges was hired and will participate in the project. 3. Jumpponen is currently recruiting two MSc students (Ms. Anna Kazarina (ASU) and Mr. Kyle Ismert (KSU)) to work on the project. Both are already familiar with the project and have some limited experience on building metabarcode sequencing libraries for community analyses. Additional undergraduate students will be considered once demand may emerge. 4. Soil samples will be collected for testing soil parameters of importance under conservation agriculture by ASU students and by co-PI Dr. Girish Panicker. Also, measurements of relevant plant characteristics like percent canopy cover, leaf area index and chlorophyll content will be continued. 5. Graduate/undergraduate students at KSU along with co-PI Dr. Ari Jumpponen will undertake the generation of bacterial and fungal libraries for sequencing. A graduate student will be hired in summer 2018 to work towards Objectives 1 and 2 of this project. 6. Core Bacterial and fungal phyllosphere communities will be identified and their potential origin from soil microbiomes will be determined. 7. Dr. Heather Tyler, Research Microbiologist, USDA ARS Stoneville, MS, has agreed to mentor ASU graduate and/or undergraduate students on the project with estimating soil microbial N and C, and extracellular enzyme assays on soil samples collected for objective 1. This was not a part of the original objective but will provide additional soil data that can complement soil health assessment presently outlined in the objective. The PI and Dr. Tyler will coordinate the student activities.
Impacts
What was accomplished under these goals?
Objective 1 was to be initiated in year 2017-2018 by collecting soil and phyllosphere samples from four grass varieties cultivated under two densities. However, this task could not be initiated as planned due to the early germination of one of the varieties in 2017 prior to awarding of the grant. Instead, we conducted an investigation to compare the bacterial and fungal communities of switchgrass roots collected before harvest in Nov 2015 and from March 2016 after grass emergence. The PI conducted this experiment in summer and fall 2017 at the the co-PI's (Jumpponen) lab at KSU. A total of 64 root samples were ground in liquid nitrogen and total DNA extracted from known volume of root samples. Two DNA extraction kits from Qiagen were used to compare extraction efficiency, and Qiagen PowerSoil DNA kit was selected because it provided higher DNA yield. The bacterial and fungal libraries that were generated were sequenced at The KSU Integrative Genomics Facility. The rhizobiomes were not influenced by switchgrass varieties but differed before and after harvest. Additionally, bacterial rhizobiomes were also differed among the planting densities before harvest but not after. In contrast, fungal rhizobiomes were less sensitive: they were influenced neither by variety nor planting density either pre- or post-harvest. Among the major bacterial phyla, Actinobacteria, Chloroflexi and Aramatimonadetes were more abundant before harvest, whereas Firmicutes, Planctomycetes, Bacteroidetes were more abundant after harvest. Proteobacteria, Nitrospirae and Acidobacteria remained unchanged pre and postharvest. Among fungal phyla, Glomeromycota and Basidiomycota were more abundant before harvest, whereas Ascomycota and Chytridiomycota were more abundant after harvest. Basal taxa formerly assigned to Zygomycota did not differ among samples collected before and after harvest. Analyses of a large number of indicator species and common (core) communities of both bacteria and fungi are ongoing. Change in knowledge: The information generated comparing fungal and bacterial microbiome of switchgrass roots under conservation agriculture before and after harvest is significant enough to be a part of a publication. Change of action: the protocol for generating fungal and bacterial libraries was selected based on the methods standardized and published previously by the co-PI (Jumpponen).
Publications
- Type:
Other
Status:
Accepted
Year Published:
2017
Citation:
R. L. Becker *, G. K. S. Panicker, W. L. Mims, M. T. Martinez and Timothy Carry (2017) Plant Density Effect on Biomass Development and Rate of Residue Decomposition of Four Varieties of Switchgrass (Panicum virgatum L.) on Memphis Silt Loam. Mississippi Academy of Sciences, Hattiesburg, MS. (Student won second place); 1890 Association of Research Directors (ARD), Atlanta, GA
Ari Jumpponen (2018) Fungal rhizobiomes of common grasses: core species, functions, and potential drivers. Pacific Northwest National Laboratory, Richland WA. January, 2018.
Ari Jumpponen (2018) Fungal rhizobiomes of common grasses: core species, functions, and potential drivers. University of Missouri, Columbia MO. March, 2017.
Kazarina A**, Mandyam K, Nanjundaswamy A and Panicker G (2018) Macromorphological and Micromorphological Diversity of Root-Associated Fungal Endophytes of Giant Miscanthus and Switchgrass. 82nd Annual Meeting Mississippi Academy of Science, Hattiesburg, MS, February 22-23, 2018 Poster
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