Progress 03/01/19 to 02/28/24
Outputs
Target Audience:The target audiences for our project included a diverse range of individuals, groups, and communities, carefully selected to ensure maximum impact over the entire duration of the project. Our primary focus was on reaching the scientific communities, particularly those specializing in plant breeding, soil science, agronomy, biogeochemistry, and climate change mitigation. By sharing our research findings with experts in these fields, we aimed to advance knowledge and foster collaboration to address critical issues related to environmental sustainability. We also engaged a global audience through participation in a BNI international consortium led by scientists from the Japan International Research Center for Agricultural Sciences (JIRCAS). The consortium included scientists from four the Consultative Group on International Agricultural Research (CGIAR) institutions: the International Center for Tropical Agriculture (CIAT), the International Maize and Wheat Improvement Center (CIMMYT), the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), and the International Livestock Research Institute (ILRI), along with scientists from organizations and institutions from several other countries. In addition to engaging with scientific experts, we placed a strong emphasis on education. We hired two PhD students, one majoring in Plant Breeding and the other in Agronomy. These graduate students were instrumental in advancing the project through their dedicated research and academic contributions. Their involvement not only furthered the project's objectives but also provided them with valuable experience and expertise in cutting-edge agricultural research. Several undergraduate students also participated in the project, assisting with various tasks and gaining hands-on experience that enriched their educational journey and prepared them for future careers in science and agriculture. Additionally, we revised the graduate-level course SCSC607 Crop Physiology course taught by Dr. Rajan (PI) to incorporate results from this project on sorghum BNI, enhancing the curriculum with the latest research findings. Our outreach efforts extended beyond the academic and scientific communities to include a broader audience. We made presentations to sorghum commodity groups, sharing insights and practical applications of our research findings. This engagement was crucial in translating our scientific results into actionable strategies for those directly involved in sorghum cultivation. We also reached out to farmers and other agricultural practitioners, providing them with information on innovative practices involving BNI sorghum and strategies derived from our research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided extensive training and professional development opportunities, particularly for two Ph.D. students, Bal Krishna Maharjan (Plant Breeding major) and Dinesh Phuyal (Agronomy major), and several undergraduate students. Both Ph.D. students benefitted from dedicated one-on-one mentorship, enhancing their technical and professional skills. They participated in numerous conferences, networking events, and professional activities, including an international symposium on BNI in Japan where they interacted with BNI consortium members. Within the department, they assumed leadership roles, leading research projects and organizing events. Undergraduate students involved in the project gained hands-on experience and foundational knowledge by working closely with the Ph.D. students and mentors. How have the results been disseminated to communities of interest?The results of our project have been widely disseminated to various communities of interest through multiple channels. We presented our findings at several scientific conferences, including the international symposium on BNI, where we interacted with a global network of scientists specializing in BNI research. This facilitated the exchange of ideas and advancements with leading experts in the field. The project has also produced two dissertations, one published paper, one paper currently under review, and four more papers under preparation. Additionally, we engaged with sorghum commodity organizations such as the National Sorghum Producers and Sorghum Checkoff. These organizations were particularly eager to learn about our project, recognizing the potential of BNI as a climate-smart trait. Our data played a crucial role in convincing these groups of the project's value and garnering their support. Through these efforts, we have not only advanced scientific understanding but also increased public awareness and interest in climate-smart agricultural practices. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Project Impact:This project provided the first proof of concept that biological nitrification inhibition (BNI) is a viable trait in sorghum for climate change mitigation. Our experiments demonstrated that BNI in sorghum can effectively suppress soil nitrification, reduce soil N2O emissions, and enhance nitrogen uptake efficiency. By quantifying and characterizing the secretion of BNI compounds in sorghum, we identified key mechanisms and genetic traits involved. We evaluated the release of BNI compounds and their impact on soil nitrification inhibition, finding significant reductions in nitrification rates. In addition, our research examined how soil microbial communities are altered by sorghum BNI compounds, contributing to an improved understanding of BNI's impact on soil nitrifiers.Breeding programs can now target BNI in sorghum as a climate-smart trait. Our findings have significant implications for developing solutions for climate change mitigation and reducing the environmental footprint of agricultural activities. Indirect benefits to society include reduced greenhouse gas emissions and improved water quality, as less nitrate is leached from BNI sorghum fields. This project has advanced scientific understanding of BNI mechanisms and has the potential to revolutionize agricultural practices, making them more environmentally friendly and sustainable. Objective 1:We investigated the mechanisms behind the secretion of sorgoleone, the primary BNI compound in sorghum. Previous studies identified numerous lipidic vesicles in sorghum root hairs, but their exact roles were unclear. Our experiments focused on determining the expression and localization of sorgoleone biosynthesis enzymes. We found that the expression of these enzymes is induced in a specific root zone, indicating that sorgoleone secretion is developmentally regulated. We observed that the accumulation of internal vesicles precedes the peak of sorgoleone biosynthesis and secretion, suggesting these vesicles play a role in precursor storage rather than secretion. Our data also revealed that enzymes SbDES2, SbDES3, and SbARS1 interact to form a multi-enzyme complex on the endoplasmic reticulum surface. Lipid analysis of ars1/2 mutants and null-segregant lines showed that the internal vesicles in root hairs are oil bodies containing neutral lipids such as triacylglycerols, which serve as substrates for sorgoleone synthesis. To further investigate the mechanism of secretion, the impact of various inhibitors on sorgoleone secretion was studied. Sorgoleone secretion was sensitive to ATPase inhibitors but not to protonophores, suggesting that primary active transporters are involved. Further genetic and phenotypic studies identified such a transporter expressed in the root hairs and is essential for sorgoleone secretion. Collectively, our findings suggest that sorgoleone is synthesized from the precursors such as triacylglycerol that are stored in young root hairs, and that transporter-mediated export, rather than vesicle-mediated transport, is required for sorgoleone to be secreted from the root hair. The transporter can serve as a new target for modulating sorgoleone secretion through biotechnology. In addition to investigating the mechanisms behind sorgoleone secretion, we studied the nature and quantitative inheritance of this trait in sorghum. To achieve this,seed parents and pollinator parents from the sorghum breeding program at Texas A&M were crossed in an incomplete factorial design, generating 158 hybrids. We quantified sorgoleone secretion from both hybrids and inbreds. A linear mixed model analysis calculated general and specific combining abilities for the genotypes, detecting significant genetic effects for male, female, and male x female interactions (p<0.001). The heritability was calculated to be H² = 0.87, with heritability due to female effects (hf²) at 0.35 and male effects (hm²) at 0.39. These results indicate that sorgoleone exudation is primarily driven by additive genetic effects, despite significant dominance effects. These findings highlight the genetic variability in sorgoleone root exudation among elite grain sorghum genotypes and enable the selection of genotypes with enhanced BNI. Objectives 2, 3, and 4:We conducted multiple field experiments to investigate these objectives. In Experiment 1, we investigated the effects of different nitrogen fertilizer types on the BNI activity of sorghum and its associated climate-smart benefits over three years (2021-23) at the Texas A&M research farm using a high BNI genotype. The study evaluated the impact of four nitrogen sources, Ammonium Sulfate, UAN, and SuperU (DCD-treated Urea with Urease inhibitor). A no-fertilizer control was included. We found that Ammonium Sulfate fertilizer significantly lowered soil pH (up to 23%) and triggered BNI activity. BNI activity resulted in suppressed soil nitrification and kept ammonium in the soil throughout the growing season. This also resulted in higher nitrogen uptake efficiency (more than 20% higher uptake compared to other fertilizer sources), and reduced N2O emissions (up to 86% lower than other fertilizer sources) by decreasing soil nitrifier abundance.Experiment 2 focused on the effects of BNI in sorghum across different soil types, conducted as a two-year greenhouse study (2021 and 2022). The study highlighted that BNI activity was more prominent and stable in acidic soils, which triggered higher BNI activity and resulted in greater nitrogen use efficiency and improved plant biomass. All high BNI lines reduced both ammonia-oxidizing archaea and bacteria abundance, suggesting the genotypic effect on BNI activity was significant regardless of soil type.In Experiment 3, we examined the BNI activity of different sorghum genotypes under field conditions. Over two years (2022 and 2023), four sorghum genotypes from the Texas A&M breeding program, with varying capacities to secrete sorgoleone, were tested alongside a wild sorghum genotype with the highest BNI that we estimated in our growth chamber studies. The study revealed that high BNI sorghum lines maintained higher ammonium levels in soil, emitted less N2O, had a lower abundance of soil nitrifiers, and exhibited higher nitrogen uptake efficiency. Further, we observed the BNI activity increased as the plant progressed to grow. These results demonstrated that BNI trait can be effectively expressed in alkaline soil conditions, expanding the scope for using BNI to reduce nitrogen pollution and improve sustainable agricultural practices.Experiment 4 focused on looking at the weed suppression potential. All sorghum lines inhibited grass germination at twice the rate observed in control plots without sorghum. Additionally, high-sorgoleone producing sorghum lines exhibited efficacy in suppressing broadleaf plant germination compared to low-sorgoleone producing lines. Overall, sorghum lines with high sorgoleone production demonstrated greater effectiveness in controlling weed emergence than those with lower sorgoleone production throughout the 12-week growing season. This suggests that the enhanced allelopathic properties associated with higher sorgoleone production play a crucial role in the competitive suppression of weeds. Collectively, these experiments provided comprehensive evidence that BNI is a viable trait in sorghum for climate change mitigation. The findings suggest that integrating BNI trait into sorghum breeding programs can lead to the development of new cultivars that can reduce greenhouse gas emissions, improve nitrogen use efficiency, and promote water quality. By targeting the BNI trait, we can enhance sorghum's role as a climate-smart crop, contributing to environmental sustainability, climate change mitigation, and agricultural productivity.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2024
Citation:
Maharjan, B. K. (2024). Study of Sorgoleone secretion mechanism and its inheritance in sorghum (Sorghum bicolor L. Moench) [Doctoral Dissertation, Texas A&M University].
- Type:
Theses/Dissertations
Status:
Under Review
Year Published:
2024
Citation:
Phuyal, D. (2024). Investigating the Biological Nitrification Inhibition (BNI) Activity in Selected Sorghum (Sorghum bicolor L. Moench) Genotypes [Doctoral Dissertation in Progress, Texas A&M University].
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Maharjan, B., Vitha, S., & Okumoto, S. (2023). Developmental regulation and physical interaction among enzymes involved in sorgoleone biosynthesis. The Plant Journal, 115(3), 820-832.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Rajan, N. (2024). Sorghum: A Smart Choice for Reducing the Nitrous Oxide Footprint of Agriculture. Sorghum Improvement Conference of North America, Oklahoma City, OK. (Invited)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Schill, M. L., Phuyal, D., Maharjan, B. K., Rajan, N., Subramanian, N. K., & Bagavathiannan, M. (2022) Biological nitrification inhibition potential improves nitrogen availability in johnsongrass (Sorghum halepense) rhizosphere [Abstract]. ASA, CSSA, SSSA International Annual Meeting, Baltimore, MD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Phuyal, D., Rajan, N., Rooney, W. L., Antony-Babu, S., Casey, K. D., Okumoto, S., & Subbarao, G. V. (2023) Impact of Nitrogen Fertilizer Sources on Biological Nitrification Inhibition Activity of Sorghum [Abstract]. ASA, CSSA, SSSA International Annual Meeting, St. Louis, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Phuyal, D., Rajan, N., Rooney, W. L., Antony-Babu, S., Casey, K. D., Okumoto, S., & Subbarao, G. V. (2023) Exploring the Impact of Sorghum Biological Nitrification Inhibition Activity: Implications for Climate Change and Agriculture [Abstract]. ASA, CSSA, SSSA International Annual Meeting, St. Louis, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Schill, M. L., Phuyal, D., Subramanian, N., Rajan, N., & Bagavathiannan, M. V. (2023) Sorghum Halepense Promotes Biological Nitrification Inhibition in Its Rhizosphere [Abstract]. ASA, CSSA, SSSA International Annual Meeting, St. Louis, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Schill, M. L., Rajan, N., Rooney, W. L., & Bagavathiannan, M. V. (2023) Studies on the Allelopathic Weed Suppression Potential of Known Sorghum Bicolor Lines Differing in Sorgoleone Production [Abstract]. ASA, CSSA, SSSA International Annual Meeting, St. Louis, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Rajan, N. (2023). Biological Nitrification Inhibition: A climate-smart strategy to develop low-nitrifying production systems and tackle the global nitrogen problem. Ecological Society of America Annual Meeting. Portland, OR. (Invited)
|
Progress 03/01/22 to 02/28/23
Outputs
Target Audience:Targeted audience of this project during the reporting period included scientists, graduate students, undergraduate students, extension personnel, policy makers, and general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph. D. students and one undergraduate student are currently working on the project. They have received training in various activities related to conducting BNI-related experiments How have the results been disseminated to communities of interest?Results are being disseminated through presentations in scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue the research activities as outlined in the proposal.
Impacts
What was accomplished under these goals?
Our experiments under Objective 1 in previous years have revealed the subcellular localization of all known sorgoleone biosynthetic enzymes, the main BNI compound in sorghum. Our results showed that all membrane-bound enzymes (DES2, DES3, and CYP71AM1) are localized to the endoplasmic reticulum (ER), whereas the two soluble enzymes (ARS1 and OMT3) are in the cytosol. This raised a further question: how is the intermediate shuttled between separate cellular locations? This led us to hypothesize that some of these enzymes interact with each other at the ER to channel the metabolite. Such a mechanism has been previously discovered for chalcone synthase, an ARS1 homolog, which interacts with multiple enzymes localized in the ER. To examine our hypothesis, we tested the interaction between all enzymes using a mating-based split ubiquitin system. The results identified DES2, DES3, and ARS1 as potential interactors. This finding was further confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. These results indicate that sorgoleone synthesis occurs through a protein complex existing on the ER membrane. A manuscript is under preparation for submission based on these results. In order to identify potential transporter genes, we reasoned that the genes involved in sorgoleone secretion could be differentially regulated in ars1/2 plants. In collaboration with Dr. Baerson (USDA-ARS), root hair transcriptomes of the ars1/2 RNAi lines and the corresponding null-segregants have been obtained. Data analysis was performed using Qiagen CLC genomics Workbench (CLCGxWB, ver. 11). The results showed that in addition to ARS1 and 2 (the target of RNAi), DES2, DES3, and OMT3 genes were also downregulated in ars1/2 RNAi lines, indicating that other sorgoleone-related genes are affected in ars1/2 RNAi lines. We also identified multiple transporter genes, many of which belong to the ABC family. Since our previous work proved that the secretion is sensitive to ATPase inhibitor, those are deemed the primary candidate and we are currently pursuing those. Multiple CRISPR/CAS9 constructs are being designed now to create loss-of-function mutant, in order to examine their functions in sorgoleone secretion. In 2022, we conducted field experiments to investigate Objectives 2 to 4. The field was planted with a high BNI sorghum genotype. Treatments consisted of four types of nitrogen (N) sources: Urea (46-0-0), Ammonium Sulfate (21-0-0), UAN (32-0-0), and SUPERU (46-0-0). The experiment was conducted in a randomized complete block design with three replications. N fertilizer (112 kg N ha?¹) was applied in two split doses. Automated soil chambers (LI-8100, LI-COR Biosciences, Nebraska, US) integrated with a trace gas analyzer (LI-7820, LI-COR Biosciences, Nebraska, US) were used to collect the gas samples. Soil samples collected at 6, 10, and 11 weeks after planting showed the highest amount of soil ammonium in plots treated with ammonium sulfate, followed by SUPERU, which were not significantly different. The ammonium levels in urea and UAN-treated plots were significantly lower. High ammonium indicates lower nitrification rates and BNI, which was also evident in the soil microbial data. The abundance of ammonia-oxidizing bacteria (AOB), in terms of the number of amoA gene copies per gram of soil, was lowest in the SUPERU and ammonium sulfate-treated plots. The AOB abundance in UAN plots was significantly higher than the rest of the fertilizers indicating lower levels of BNI. The cumulative N2O emissions in ammonium sulfate-treated plots were 4%, 83%, and 73% lower compared to SUPERU, UAN, and Urea-treated plots, respectively. Application of ammonium sulfate also resulted in significantly higher above-ground biomass compared to other fertilizers.Our results indicated that ammonium sulfate-treated plots had the highest BNI expression under field conditions. As the same high BNI genotype was planted, we can safely conclude that the application of ammonium sulfate promoted the highest BNI effect, as indicated by high soil ammonium, high above-ground biomass,low AOB abundance, and low N2O emissions. A second field experiment was conducted withfour sorghum genotypes from the Texas A & M breeding program with varying sorgoleone secretion capacity (two high BNI and two low BNI), along with a wild sorghum genotype exhibiting the highest BNI secretion capacity.The experiment followed a randomized complete block design with three replications. Nitrogen fertilizer (112 kg N ha-1) was applied in two split doses. Soil samples were collected for analysis of soil NH??, NO3-, and soil nitrifier abundance. N2O samples were collected using automated soil chambers integrated with a trace gas analyzer. Similar to the previous field experiment, the genotype with the highest BNI retained significantly higher soil ammonium at the end of the growing season. Soil N2O emission was also the lowest in high BNI plots. The highest sorghum BNI genotype also showed significantly higher nitrogen update efficiency compared to other genotypes.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
[36] Phuyal, D., Rajan, N., Rooney, W. L., Antony-Babu, S., Schill, M., Subramanian, N. K., Okumoto, S., & Subbarao, G. V. (2022) Effect of biological nitrification inhibition activity of sorghum genotypes on nitrification in a field study [Abstract]. ASA, CSSA, SSSA International Annual Meeting, Baltimore, MD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
[35] Phuyal, D., Rajan, N., Rooney, W. L., Antony-Babu, S., Casey, K. D., Zapata, D., Schill, M., Subramanian, N. K., Salehin, S. M. U., Maharjan, B. K., Okumoto, S., & Subbarao, G. V. (2022) Biological nitrification inhibition activity of sorghum genotypes with differential sorgoleone secretion capacity [Abstract]. ASA, CSSA, SSSA International Annual Meeting, Baltimore, MD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
[40] Maharjan, B. K., Okumoto, S., Rajan, N., & Baerson, S. (2022) Understanding the mechanism of sorgoleone biosynthesis and secretion [Abstract]. ASA, CSSA, SSSA International Annual Meeting, Baltimore, MD.
|
Progress 03/01/21 to 02/28/22
Outputs
Target Audience:Targeted audience of this project during the reporting period included scientists, graduate students, undergraduate students, extension personnel, policy makers, and general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph. D. students and one undergardte student are currently working in the project. They have received training in various activities related to conducting BNI-related experiments How have the results been disseminated to communities of interest?Results are being disseminated through presentations in scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue the research activities as outlined in the proposal. The project team is also planning to attend the BNI international symposium organized by JIRCAS in Tsukuba, Japan.
Impacts
What was accomplished under these goals?
Objective 1: In the previous year, we have demonstrated that sorgoleone secretion first starts to form on the root hairs that are 2-3 cm away from the root tip. When we visualize the internal vesicles with a lipid staining dye (Nile red), on the other hand, younger root hair between 0-3 cm contained numerous internal vesicles. In the 4-5 cm zone where the root hairs showed extensive sorgoleone secretion, the number of those internal vesicles decreased significantly. This past year, we added further evidence for the timing of sorgoleone synthesis by investigating the expression of sorgoleone biosynthesis enzymes peaks. The results showed that the expression peak occurs in 3-4 cm zone, whereas in 0-1 and 1-2 cm zones are much lower. Therefore, we concluded that the internal vesicles form prior to the peak of sorgoleone biosynthesis and secretion. Therefore, we hypothesized that sorgoleone is exported from the cell via the activity of plasma membrane transporter rather than exocytosis. Past studies in bacteria, yeasts, animals, and plants identified ATP Binding Cassette (ABC) transporters, and in particular subfamily G (ABCG) transporters as the major players in exporting a wide range of hydrophobic metabolites, including lipids and amphiphilic lipid derivative. In addition, MATE efflux family proteins and Major Facilitator Submfamily (MFS) proteins are both largely driven by the electrochemical gradient (in plants, mostly proton gradient) and are involved in xenobiotic extrusion in bacteria and animal cells. To understand the nature of the transporter, we treated sorghum roots with either protonophore (CCCP) or ATPase inhibitor (vanadate) at the range of concentrations found effective on plant root. The results clearly showed that the secretion process is sensitive to the ATPase inhibitor but not to the protonophore, indicating that ABC transporters are the likely candidates. We have reasoned that the genes involved in sorgoleone biosynthesis and secretion could be differentially regulated in ars1/2 plants, allowing us to further narrow down the candidate genes for sorgoleone transporters. In collaboration with Dr. Baerson (USDA-ARS), root hair transcriptome of the ars1/2 RNAi lines and the corresponding null-segregants have been obtained. The reads were mapped to the current sorghum genome using HISAT2, then the reads mapped to the gene models were counted using Featurecounts function in Subread package. Differentially expressed genes (DEGs) were analyzed using DESeq2 package. A total of 176 genes were differentially regulated in the RNAi line compared to the null-segregant in at least two out of three pairs. GO-term analysis revealed that the DEGs are enriched for lipid and fatty acid metabolism, organic acid metabolism, and oxidation reductions. In addition, we have identified 10 differentially expressed ABC transporters, among which six belong to ABCG family. Homologs of lipid transfer proteins (LTPs) that are involved in wax secretion have also been found. These genes are currently being tested for their ability to transport sorgoleone, the major BNI compound in sorghum. Objectives 2-4: In the past reporting period, we conducted a greenhouse experiment with treatments that included four sorgoleone producing lines (two high BNI and two low BNI) from the TAMU breeding program, one wild sorghum genotype with very high BNI, and bare soil. The soil used in the study was a clay loam soil collected from a row-crop field with pH 6.2. Ammonium sulfate fertilizer was applied 30 days after planting at 168 kg N ha-1. Rhizosphere soil samples were collected 55 days after planting to test the effect of BNI activity on potential nitrification, soil nitrifier population, and N2O emission. The results showed that the high BNI lines can significantly (p < 0.05) reduce soil nitrification. Additionally, the BNI activity of sorghum decreased the soil nitrifier population in rhizosphere soil. The N2O flux from the rhizosphere soil of high sorgoleone producing lines was significantly lower than other sorghum genotypes. The result shows potential of BNI in reducing nitrification and N2O emissions. Results from a field study at the Texas A & M research farm evaluated BNI activity of sorghum on N2O emission under different N fertilizer types. The field was planted with a high BNI sorghum genotype. Treatment consisted of four types of N sources: Urea (46-0-0), Ammonium Sulfate (21-0-0), UAN (32-0-0), and SUPERU (46-0-0). The experiment was conducted in a randomized complete block design with three replications. N fertilizer (112 kg N ha-1) was applied in two split doses. Gas samples were collected before and after fertilizer applications and analyzed using a gas chromatograph. The results showed the lowest N2O flux from plots that received ammonium sulfate. Soil pH was low (~6.2)in those plots which could have triggered the release of BNI compounds and suppressed N2O emission compared to other fertilizer types. Emission was the highest in plots that received urea. These results suggest that N fertilizer types have an effect on BNI activity and N2O emission under the field conditions.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Phuyal, D., Rajan, N., Schnell, R., Rooney, W., Maharjan, B., Casey K., Zapata D., Okumoto, S., Subramanian, N., Kim, J., Chu, K. H., Peterson, J.A., & Subbarao, G.V. (2021). Biological nitrification inhibition (BNI) activity of sorghum genotypes on a typical field soil. Abstracts, ASA-CSSA-SSSA International Annual Meetings, 7-10 November, Salt Lake City, UT.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Phuyal, D., Rajan, N., Schnell, R., Salehin, S. M., Casey K., Rooney, W., Maharjan, B., Okumoto, S., & Subbarao, G.V. (2021). Effect of biological nitrification inhibition of sorghum on nitrous oxide emission under different nitrogen fertilizer types. Abstracts, ASA-CSSA-SSSA International Annual Meetings, 7-10 November, Salt Lake City, UT.
|
Progress 03/01/20 to 02/28/21
Outputs
Target Audience:Targeted audience of this project during the reporting period included scientists, graduate students, undergraduate students, extension personnel, policy makers, and general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph. D. students and one undergardte student are currently working in the project. They have received training in various activities related to conducting BNI-related experiments How have the results been disseminated to communities of interest?Results are being disseminated through presentations in scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue the research activities as outlined in the proposal. The project team is also planning to attend the BNI international symposium organized by JIRCAS in Tsukuba, Japan. This meeting is orginally scheduled for November 2022, but may get delayed due to the Covid-19 situation.
Impacts
What was accomplished under these goals?
Objective 1:Quantify and characterize the BNI compound secretion in sorghum: We studied the interaction between the biosynthetic enzymes of sorgoleone.Sorgleone biosynthetic enzymes have been identified by previous studies, but their localization in the cell had been unknown. In the previous year, we determined their cellular localization and figured out that the enzyme that catalyzes the committing step, AlkylResorcinol Synthase (ARS), is localized to the cytosol but also associates with the ER. This year, we have used two independent methods to show that ARS interacts with the enzyme that mediates the previous step, Fatty Acid Desaturase (DES)3, which resides in the ER. The results indicate that they might be a part of a multi-enzyme complex, which makes the channeling of metabolite possible, and also explains why the end product of the pathway nearly exclusively derives from the product of DES3 and not other unsaturated fatty acids, even though ARS can utilize other unsaturated fatty acids as substrates. The manuscript is in preparation. Identification of sorgoleone precursor storage mechanism was investigated.Previous studies using electron micrographs showed multiple electron-dense lipid bodies in sorghum root hair and assumed that these vesicles contain sorgoleone. To understand the relationship between these vesicles and sorgoleone biosynthesis, we performed lipid staining on the root hair at different developmental stages. The results showed that the vesicles accumulate before the start of sorgoleone secretion, suggesting that these vesicles contain the precursor, rather than sorgoleone. Moreover, inars1/2RNAi plants, in which the committing step of sorgoleone biosynthesis is inhibited by RNAi, the number and sizes of the intracellular vesicles were greatly increased. Our hypothesis was further confirmed by the lipid analysis of root hair; inars1/2RNAi a large amount of sorgoleone precursor (16:3 FA) was stored as triacylglycerol. RNAseq ofars1/2RNAi revealed further genes involved in sorgoleone secretion.Currently, the secretion mechanism through which sorgoleone is secreted is unknown. To identify such a mechanism, RNA from wild-type vsars1/2RNAi root hair have been sequenced in collaboration with Dr. Baerson from USDA-ARS. The results showed that several homologs of genes that are involved in xenobiotics and lipid secretion in other organisms are downregulated in thears1/2RNAi which does not secrete sorgoleone. We are currently in the process of cloning these candidate genes to perform biochemical analysis. Objective 2: Evaluate the release of BNI compounds and nitrification inhibition in soils: To evaluate the effect of BNI compounds on nitrification inhibition in soils, we collected soil from a row crop field located in Williamson County, TX. The soil had a clay loam texture with pH of 6.2. The soil was used in a greenhouse experiment with five sorghum genotypes including three high BNI (one wild genotype and two from TAMUbreeding program) and two low BNI lines (TAMU breeding program). The soil was filled in a 3.3L pipe column to achieve a bulk density of 1.44 g cm-3. Ammonium sulfatewas added as a fertilizer source (168 kg N ha-1). 50 days after seed emergence, plants were destructively harvested. Aboveground biomass, root biomass, bulk soil, and rhizosphere soil samples were separated. Soils were analyzed for ammonium and nitrate content before conducting potential nitrification experiment. 10 g air-dried rhizosphere soil was used in the potential nitrification study. 100 ml of 27.1 ppm as NH4-N was added to 10 g soil in 250 ml Erlenmeyer flasks. The flasks were covered with silicone sponge caps and placed in a shaking incubator at 140 rpm with an incubation temperature set at 25 °C. After 1 hr., the flasks were taken out and slurry pH was recorded. 5 ml representative sample was taken out with a pipette and centrifuged. The supernatant was filtered and analyzed for ammonium and nitrate. A similar sample collection was done Day 5 and Day 10 of the experiment. The wild sorghum genotype with the highest BNI showed the greatest nitrification inhibitory effect throughout the experiment compared to all the other genotypes and bare soil (soil without root exudate). On day 1 (24 hours after the incubation), Only 6% ammonium dropped in the incubated flask with rhizosphere soil collected from the wild genotype. However, the percentage drop of ammonium was 40% and 53% in the high BNI line treatments and low BNI line treatments respectively. On day 10, the wild genotype of sorghum resulted in 78% higher ammonium than the second-best BNI genotype. The highest BNI sorghum line showed consistently higher ammonium content and lower nitrate conversion throughout the experiment (p < 0.05) showing its potential for reducing nitrification rate. Objective 3: Evaluate how soil microbial community changes due to secretion of sorghum BNI compounds:For the determination of AOB and AOA abundance, the respective ammonia monooxygenase (amoA) gene copy number was quantified. We used destructively collected rhizosphere soil 50 days after emergence from the objective 2 experiment for this. Soil was stored at -20 °C until the analysis was done. Among the five genotypes used in the experiment, the wild genotype of sorghum genotype with the highest BNI resulted in the lowest (p < 0.001) amoA gene copy. The low BNI secretion line 'Hegari' resulted in the highest number of amoA gene copies. The amoA gene copy in the control (bare soil) was significantly higher than all the sorghum genotypes (p < 0.001). Results from this experiment showed that BNI sorghum was able to suppress the population of nitrifying bacteria in soil. Objective 4:Investigate how BNI properties will affectN2O emissions in production fields. Afield experiment to explore the BNI activity of sorghum on nitrous oxide emission under different nitrogen fertilizer types fwas conducted in summer 2021. Sorghum genotype with the higets BNI was planted at a seeding rate: 16,000 ha-1. Four N fertilizer sources (Ammonium sulfate, Urea, Urea-Ammonium Nitrate, and SuperU) with control (no fertilizer application) were applied in a randomized complete block design with three replications. The N fertilizer was applied at the same rate 112 kg N ha-1 applied in (two split doses: 44 DAP and 70 DAP). Static chamberswere installed parallel to the sorghum rows for gas sample collection. Gas samples were collected from 9 am to 10:15 am (CST) from 40 DAP to 74 DAP.Samples wereanalyzed using a gas chromatograph.Additionally, The soil samples were collected before initiating the experiment, and after the terminations of gas sampling (48 DAP, 75 DAP) and 101 DAP. Soil samples were collected from each plot and analyzed for pH, ammonium, and nitrate.The study showed the N2O flux spiked after fertilizer application 44 DAP and 70 DAP (except control) and decreased over time. The N2O emission from ammonium-based fertilizer was lower compared to other fertilizer types. Urea application resulted in the greatest N2O gas emission compared to other N fertilizer types. Throughout the experiment, N2O emission from plots treated with ammonium sulfate was significantly less than other treatments (p < 0.05) except control (no fertilizer application).Further, the soil sampling showed a decrease in soil pH in the plot treated with ammonium sulfate compared to other treatments. Also, the same plots showed higher soil ammonium content and lower soil nitrate (p < 0.05) throughout the sampling. As previously studies found, ammonium in the rootzone and low pH stimulate BNI root exudate secretion in sorghum. This might have been the reason for low N2O emission in plots that received ammonium sulfate fertilizer. This experiment will be continued in 2022 to invetsigate the effect of BNI on nitrogen uptake NUE, N2O emission and weed suppression.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Phuyal, D., Rajan, N., Rooney, W., Kim, J., Chu, K.H., Subramanian N., Maharjan B., Okumoto, S., Schnell, R., Peterson, J.A., and Subbarao, G.V. (2020). Climate Smart Farming: A Preliminary investigation of Biological Nitrification Inhibition (BNI) in selected sorghum genotypes. Abstracts, ASA-CSSA-SSSA International Annual Meetings, November 9-13, Virtual. (Won 2nd place in graduate student poster competition organized by the "Global Climate Change Community)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Phuyal, D., Rajan, N., Rooney, W., Kim, J., Chu, K.H., Subramanian N., Maharjan B., Okumoto, S., Schnell, R., Peterson, J.A., & Subbarao, G.V. (2020). Effect of biological nitrification inhibition (BNI) of sorghum on Weswood silt loam soil. Abstracts, Texas Plant Protection Conference, Dec 8, Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Rajan, N. (2020). Greenhouse gas emissions from grain cropping systems. Soil Critique Meeting, July 22, Virtual.
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:Targeted audience of this project during the reporting period includedscientists, graduate students, undergraduate students, extension personnel, policy makers, and general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph. D. students and one undergardtestudent are currently working in the project. They have received training in various activities related to conducting BNI-related experiments. How have the results been disseminated to communities of interest?PI Dr. Rajan has made a presentation at the 2019 ASA-CSSA-SSSA Annual Meeting. This presentation was titled "Climate Smart Agriculture: The Role of Biological Nitrification Inhibition". What do you plan to do during the next reporting period to accomplish the goals?We will continue the research activities as outlined in the proposal. The project team is also planning to attend the BNI international symposium organized by JIRCAS in Tsukuba, Japan. This meeting is orginally scheduled for November 2020, but may get delayed due to the Covid-19 situation.
Impacts
What was accomplished under these goals?
To quantify the BNI activity, we screened sorghum genotypes from Dr. Bill Rooney's sorghum breeding program at Dr. Subbarao's lab at JIRCAS (Tsukuba, Japan) using the bioluminescent Nitrosomonas europea culture. The purpose of these experiments were; 1) To confirm the repression of AMO activity by the root secretion of selected cultivars, and 2) To evaluate this method as the potential choice for screening additional cultivars for future work. Seedlings were grown in a growth chamber and roots and shoots were excised from sorghum seedlings after 8 days. Root exudates were then collected using organic solvents and analyzed for sorgoleone, a major BNI compound (hydrphobic) in sorghum. Out of the 89 genotypes, we discovered 5 genotypes with high sorgoleone production (above 50 micrograms per plant) and approximately half of the genotypes had sorgoleone production significantly higher than the positive control genotype we used which had medium level sorgoleone production (23 micrograms per plant). The experiment was repeated twice with 24 selected genotypes from the first experiment. The repetition showed consistent high Sorgoleone production in 3 genotypes (high BNI) and 12 genotypes had consistent low Sorgoleone (low BNI) production. We have selected 2 high BNI and 2 low BNI lines from this experiment for the soils experiment proposed as part of objective 2. Our experiments associated with investigating the molecular nature of Sorgoleone secretion in sorghum has provided interesting results. We treated the roots with different concentration of vanadate and observed changes in the cytosolic structure of the root hairs due to the inhibitor activity. We conducted qPCR for different genes (ARS1, DES2, DES3, CYP71AM1, and OMT3) involved in the secretion of Sorgoleone in the root hairs. We ran qPCR in 2 low secreting lines and 2 high secreting lines where we observed differential expression of those genes in the low and high secreting lines. Currently, we are continuing our experiments associated with objectives 1 -3. Objective 4 will be initiated in 2021 growing season.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Rajan, N., W. L Rooney, S. Okumoto, R. Schnell, M. Bagavathiannan, J. Aitkenhead-Peterson, J. Jifon, K. Chu, K. D. Casey, and G. V Subbarao. 2019. Climate Smart Agriculture: The Role of Biological Nitrification Inhibition. Abstracts, ASA-CSSA-SSSA Annual Meeting, November 10-13, San Antonio, TX.
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