Progress 05/01/17 to 04/30/23
Outputs
Target Audience:Target audiences for this project includes the broader scientific community. Broader scientific community: We provided updates about the research projects to other scientists by: Providing oral and poster presentations at national and international conferences: Cope et al., Plant and Animal Genome Conference, San Diego, California, January 12-17, 2023; Sethumadhavan et al., Singh et al., Mutyala et al., American Society of Plant Biologists Midwest sectional society meeting, Ames, Iowa, April 22-23, 2023 (more information can be found under products). Publishing research manuscripts and reviews in peer reviewed journals: Das et al., 2023, DOI:10.5772/intechopen.111540; Salomon et al., 2022 DOI:10.1016/j.isci.2022.104636; Gu et al., 2023 DOI:10.1016/bs.mie.2022.08.044 (more information can be found under products). Changes/Problems:Delays were caused by the long delivery times of experimental supplies, and the problems with hiring additional students for the project. What opportunities for training and professional development has the project provided?The project provided training opportunities for two Ph.D. students (Athira Sethu Madhavan, SDSU; Prema Mutyala, Univ. of Missouri), one M.S. student (Aniket Singh, SDSU) and two postdoctoral associates (Drs. Debatosh Das and Dhruv Aditya Srivastava; Univ. of Missouri). The Ph.D. and M.S. students have co-authored three conference presentations and have two manuscripts under revision/preparation (Cope et al., in prep; SethuMadhavan et al., in prep). Undergraduate student Hazem Khalaf Muhammed participated as a USDA-REEU summer student and continued on this project as a research student. He has trained well in research methodologies, documentation and data analysis; and has co-authored a conference presentation. Postdoctoral associates Drs. Debatosh Das and Dhruv Aditya Srivastava have led many of the experiments, analyzed data and manuscript preparation (tripartite studies, labeling studies, N-fixing root symbioses, transcriptome analysis), and writing of scientific manuscripts (Cope et al. in prep.; Das et al., in prep.). How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations to different audiences, webinars, and contributions to scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Tripartite interactions play a critical role in the nutrient uptake and overall performance of the majority of legumes. We conducted a set of carefully designed experiments to better understand how resource exchange processes in tripartite interactions are regulated. A better understanding of these processes is critical to maximize nutritional and other benefits of these root symbioses in agricultural settings. Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? (100% Accomplished) Carbon to nutrient exchange dynamics in these tripartite interactions are only poorly understood. We used split-root systems with Medicago truncatula that were colonized with different symbiont combinations of the AM fungus Rhizophagus irregularis and a nitrogen-fixing or a non-nitrogen fixing strain of Ensifer meliloti (Fix+ or Fix-) to examine the carbon allocation of the host in response to the contribution of different root symbionts to nitrogen uptake. We found that under nitrogen limiting conditions, plants transfer more carbon to root symbionts that provide higher nitrogen benefits. AM fungi that directly compete with a nitrogen-fixing rhizobia strain, transfer significant amounts of nitrogen to the root, but transfer less nitrogen across the mycorrhizal interface to the shoot. Tripartite interaction experiments in soybean also indicated that a similar carbon allocation mechanism might operate in other plant species as well based on root and nodule biomass accumulation, but direct carbon allocation was not measured in these experiments. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? (95% Accomplished) A time course experiment to examine how fast a host plant shifts its C allocation after it has been supplied with nutrients been completed and changes in carbon allocation between the symbionts has been measured. Tissues collected in parallel were used to evaluated global gene expression patterns through RNASeq. We will correlate these shifts in C allocation to changes in gene expression to identify the regulatory pathways involved in the C allocation to different root symbionts. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? (90% Accomplished) To evaluate the spatial expression of transporters that are putatively involved in nutrient exchange, in particular the C allocation to different root symbionts, transcriptional and translational fusion constructs were generated. Quantitative microscopy methods were optimized to evaluate cellular abundance and compartmentalization of transporter-GFP fusions between the cytosol and the symbiotic (periarbuscular and symbiosome) membranes. Upon completion of imaging and analysis, we expect to determine potential transcriptional and translational regulation associated with changes in nutrient and carbon allocation between the symbionts. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? (90% Accomplished) In a split root system, the root halves were non-inoculated (C) or inoculated with Rhizophagus irregularis (AM fungus) or with an Ensifer meliloti (rhizobium) Fix+ or Fix- strain. An N source containing 15N accessible via the fungal compartment was used to distinguish benefits provided by each symbiotic partner. Fix+/C plants had significantly higher shoot dry weights than C/C plants, C/AM and Fix-/C plants. When the fungus had access to 15N, the shoot weight of these plants was comparable to the Fix+/C plants. These results indicate that the growth of the plants was especially limited by N deficiency and that both root symbionts promoted plant biomass by supplying N to their host plant -- Fix+ E. meliloti through biological N fixation, and R. irregularis through the transfer of 15N. Evaluation of global gene expression patterns performed in Objective 2 are being used to evaluate potential symbiotic dialogue between the partners. For example, as reported in Objective 5, we discovered that the autoregulation of nodulation pathway plays a role in restricting nodulation by the ineffective nitrogen fixing strain versus the effective nitrogen fixer strain. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? (100% Accomplished) In a split root experiment, we used soybean plants that were non-inoculated, or inoculated with USDA 126 or USDA 110, two different Bradyrhizobium japonicum strains with a low or a high biological N fixation efficiency, respectively (Physiological results described in previous report). We evaluated transcriptomes of these roots to identify candidate genes involved in the host sanction mechanism. Gene groups belonging to Nodulation, Nitrogen Fixation, Oxygen Transport, Photosynthesis, Detection of hypoxia, and Anaerobic respiration were upregulated in roots inoculated with high N fixation capacity strain USDA110. Gene groups belonging to H2O2 catabolic process, Cellular response to iron, Cell wall modification, Pectin catabolic process, Iron sequestration, Iron/manganese transport, and Cell-cell junction assembly were enriched in roots inoculated with poor fixing strain USDA126 suggesting potential nodule senescence. Further, the abundance of transcripts encoding Rhizobia-Induced CLAVATA-like (RIC) peptides were higher in USDA110 (effective nitrogen fixer) root halves. These are signals that systemically suppress nodulation via the autoregulation of nodulation pathway. Conversely, transcripts encoding TOOMUCHLOVE (TML), an inhibitor of nodulation that acts downstream in this pathway were more abundant in USDA126 (ineffective nitrogen fixer) root halves. We hypothesized that effective nitrogen fixers might activate the AON pathway to suppress nodulation by ineffective fixers. Indeed, in NOD1-3 mutants impaired in AON pathway, effective fixer USDA110 was not able to inhibit nodulation by the ineffective fixer USDA126. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? (100% Accomplished) We asked whether the nitrogen-fixing efficiency of rhizobia would affect how AMF allocate nitrogen (N) to hosts plants connected by a common mycelial network. We hypothesized that AMF would allocate more N to host plants colonized by Fix- than by Fix+ rhizobia. To test this, we provided AMF with the stable isotope 15N to trace how much N the fungus would allocate to interconnected plants colonized by Fix- or Fix+ rhizobia. We found that tripartite interactions with Fix+ rhizobia led to synergistic growth responses due to the host plant's increased access to fixed N. However, co-inoculation with Fix- rhizobia and AMF or sole inoculation with AMF resulted in elevated 15N enrichment in the shoot of the host plant. These results indicate that AMF do not exchange as much N with host plants colonized by Fix+ rhizobia because their N demand is mostly fulfilled by the bacteria. Instead, they allocate most of the N they assimilate from the soil to the host plant with a greater N demand due to its lack of access to fixed nitrogen.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
D. Das, S. Tripathi, P. Mutyala, D. Aditya Srivastava, and H. B�cking (2023) Development and Resource Exchange Processes in Root Symbioses of Legumes, Symbiosis in Nature. IntechOpen. doi: 10.5772/intechopen.111540.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Salomon, M.J., Watts-Williams, S.J., McLaughlin, M.K., B�cking, H., Singh, B.K., Hutter, I., Schneider, C., Martin, F.M., Vosatka, M., Guo, L., Ezawa, T., Saito, M., Declerck, S., Zhu, Y-G., Bowles, T., Abbott, L.K., Smith, F.A., Cavagnaro, T.R., van der Heijden, M.G.A. (2022)
Establishing a quality management framework for commercial inoculants containing arbuscular mycorrhizal fungi,
iScience, 25(7):104636,
doi:10.1016/j.isci.2022.104636.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Mutyala, P., Das, D., Kafle, A., Yakha, J., Subramanian, S., Buecking, H., (2023) Carbon Allocation to Mutualists in Tripartite Interactions with Medicago Truncatula. American Society of Plant Biologists Midwest Section Meeting. Ames, IA. April 22-23.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Sethu Madhavan, A., Montanez-Hernandez, L. E., Hazem, K.F., Subramanian, S. (2023) Understanding Rhizobial competition for nodulation using split root assays. American Society of Plant Biologists Midwest Section Meeting. Ames, IA. April 22-23.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Singh, A., Singh, G., Gaillard, P., Petla, B., Subramanian, S. (2023) Cell-level auxin and cytokinin responses during root lateral organ development in Soybean. American Society of Plant Biologists Midwest Section Meeting. Ames, IA. April 22-23.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Hazem, K.M., Sethu Madhavan, A., Montanez Hernandez, L.E., Subramanian, S. (2023) Determining host plant responses towards competition between Bradyrhizobium elkanii strains. American Society of Plant Biologists Midwest Section Meeting. Ames, IA. April 22-23.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Gu L, Loya JR, Subramanian S, Graham C, Zhou R. (2023) Acetylene reduction assay for nitrogenase activity in root nodules under salinity stress. Methods Enzymol. 683:253-264.
doi:10.1016/bs.mie.2022.08.044. PMID: 37087191.
|
Progress 05/01/21 to 04/30/22
Outputs
Target Audience:Target audiences for this project includes the broader scientific community. Broader scientific community: We provided updates about the research projects to other scientists by: Providing oral and poster presentations at international conferences: Cope et al., 25th North American Symbiotic Nitrogen Fixation Conference, Madison, Wisconsin, 5-9 June 2022. Publishing research manuscripts and reviews in peer reviewed journals: Cope et al., 2022 Mycorrhiza 32, 281-303. (more information can be found under products). Changes/Problems:Delays were caused by the long delivery times of experimental supplies, and the problems with hiring additional students for the project. What opportunities for training and professional development has the project provided?The project provided training opportunities for one Ph.D. student (Jaya Yakha; SDSU), and one postdoctoral associate (Dr. Debatosh Das; Univ. of Missouri). The Ph.D. student Jaya Yakha has successfully defended her PhD. Dissertation, graduated. She has co-authored one publication (Cope et al., 2022), has a manuscript under revision (Yakha et al., being revised.), and two manuscripts under preparation (Cope et al., in prep; Yakha et al., in prep). Postdoctoral associate Dr. Kevin Cope a prior participant in the project has obtained a post-doctoral position at Oak Ridge National Laboratory. Postdoctoral associate Dr. Debatosh Das is being trained in experimental systems (tripartite studies, labeling studies, N-fixing root symbioses, transcriptome analysis), and writing of scientific manuscripts (Cope et al. in prep.; Yakha et al., in prep.). How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations to different audiences, webinars, and contributions to scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?Several experiments are currently ongoing to answer the following questions: Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? Complete data analysis and publication of manuscripts. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? Completion of the time course experiment and associated physiological and transcriptome analysis. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? A suite of reporter constructs where a constitutive promoter or native promoter drives the expression of selected transporters fused to a fluorescent protein have been constructed. Confocal microscopy will be used to precisely localize and quantify transporter expression to obtain more information about the potential role that these transporters play in the C allocation to individual root symbionts. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? Evaluate change in transport of phosphorus and potassium, or to repeat the experiment with labelled phosphate; and evaluate potential benefits of co-inoculation of the root system with AM fungi and rhizobia on biological nitrogen fixation. We will evaluate the hypothesis that AM-fungi can enhance nitrogen fixation by meeting the high P demand of root nodules. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? Complete data analysis and publication of manuscripts. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? Complete data analysis and publication of manuscripts.
Impacts
What was accomplished under these goals?
Tripartite interactions play a critical role in the nutrient uptake and overall performance of the majority of legumes. We conducted a set of carefully designed experiments to better understand how resource exchange processes in tripartite interactions are regulated. A better understanding of these processes is critical to maximize nutritional and other benefits of these root symbioses in agricultural settings. Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? (90% Accomplished) Carbon to nutrient exchange dynamics in these tripartite interactions are only poorly understood. We used split-root systems with Medicago truncatula that were colonized with different symbiont combinations of the AM fungus Rhizophagus irregularis and a nitrogen-fixing or a non-nitrogen fixing strain of Ensifer meliloti (Fix+ or Fix-) to examine the carbon allocation of the host in response to the contribution of different root symbionts to nitrogen uptake. We found that under nitrogen limiting conditions, plants transfer more carbon to root symbionts that provide higher nitrogen benefits. AM fungi that directly compete with a nitrogen-fixing rhizobia strain, transfer significant amounts of nitrogen to the root, but transfer less nitrogen across the mycorrhizal interface to the shoot. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? (80% Accomplished) A time course experiment to examine how fast a host plant shifts its C allocation after it has been supplied with nutrients is under progress. We will correlate these shifts in C allocation to changes in gene expression to identify the regulatory pathways involved in the C allocation to different root symbionts. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? (70% Accomplished) In the experiment described under Objective 1, We also studied the expression of transporters that are putatively involved in the C allocation to different root symbionts and found that the expression levels of MtSUT4.1 and MtSWEET3.3 showed the strongest consistency to the observed changes in carbon allocation. On the other hand, the expression of STR/STR2 that is putatively involved in the lipid transport to the AM symbiont, was not tightly linked to the carbon investment of the host into the AM symbiosis. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? (60% Accomplished) In a split root system, the root halves were non-inoculated (C) or inoculated with Rhizophagus irregularis (AM fungus) or with an Ensifer meliloti (rhizobium) Fix+ or Fix- strain. An N source containing 15N accessible via the fungal compartment was used to distinguish benefits provided by each symbiotic partner. Fix+/C plants had significantly higher shoot dry weights than C/C plants, C/AM and Fix-/C plants. When the fungus had access to 15N, the shoot weight of these plants was comparable to the Fix+/C plants. These results indicate that the growth of the plants was especially limited by N deficiency and that both root symbionts promoted plant biomass by supplying N to their host plant -- Fix+ E. meliloti through biological N fixation, and R. irregularis through the transfer of 15N. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? (80% Accomplished) In a split root experiment, we used soybean plants that were non-inoculated, or inoculated with USDA 126 or USDA 110, two different Bradyrhizobium japonicum strains with a low or a high biological N fixation efficiency, respectively (Physiological results described in previous report). We evaluated transcriptomes of these roots to identify candidate genes involved in the host sanction mechanism. Gene groups belonging to Nodulation, Nitrogen Fixation, Oxygen Transport, Photosynthesis, Detection of hypoxia, and Anaerobic respiration were upregulated in roots inoculated with high N fixation capacity strain USDA110. Gene groups belonging to H2O2 catabolic process, Cellular response to iron, Cell wall modification, Pectin catabolic process, Iron sequestration, Iron/manganese transport, and Cell-cell junction assembly were enriched in roots inoculated with poor fixing strain USDA126 suggesting potential nodule senescence. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? (80% Accomplished) We asked whether the nitrogen-fixing efficiency of rhizobia would affect how AMF allocate nitrogen (N) to hosts plants connected by a common mycelial network. We hypothesized that AMF would allocate more N to host plants colonized by Fix- than by Fix+ rhizobia. To test this, we provided AMF with the stable isotope 15N to trace how much N the fungus would allocate to interconnected plants colonized by Fix- or Fix+ rhizobia. We found that tripartite interactions with Fix+ rhizobia led to synergistic growth responses due to the host plant's increased access to fixed N. However, co-inoculation with Fix- rhizobia and AMF or sole inoculation with AMF resulted in elevated 15N enrichment in the shoot of the host plant. These results indicate that AMF do not exchange as much N with host plants colonized by Fix+ rhizobia because their N demand is mostly fulfilled by the bacteria. Instead, they allocate most of the N they assimilate from the soil to the host plant with a greater N demand due to its lack of access to fixed nitrogen.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Cope, K.R., Yakha, J.K., Huang, W., Ketelsen, K., Subramanian, S., B�cking, H. 2022. Physiological response of soybean to inoculation with Bradyrhizobium diazoefficiens strains USDA110 and USDA126 in a split-root system. 25th North American Symbiotic Nitrogen Fixation Conference. Madison, WI. June 5-9.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Cope, K.R., Kafle, A., Yakha, J.K., Pfeffer, P.E., Strahan, G.D., Garcia, K., Subramanian, S., B�cking, H. 2022. Physiological and transcriptomic response of Medicago truncatula to colonization by high- or low-benefit arbuscular mycorrhizal fungi. Mycorrhiza. 32, 281303. https://doi.org/10.1007/s00572-022-01077-2.
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:Target audiences for this project include 1) farmers and stakeholder groups, and 2) the broader scientific community. Farmers and stakeholder groups: In previous years we shared the data of this project with farmers and other stakeholder groups by presentations and by coordinating discussion groups and workshops. Unfortunately, the Covid-19 pandemic made it impossible to educate farmers and stakeholders about the importance of beneficial plant microbe interactions for crop production. Travel was restricted during the pandemic, and the majority of producer conferences were cancelled or postponed. Broader scientific community: In previous years, we typically shared updates about the outcomes of our studies at scientific conferences in form of oral or poster presentations. Due to Covid-19, many scientific conferences were cancelled or postponed, or were converted to virtual conferences. We shared our results with the broader scientific community, via: Oral and poster presentations at international conferences: 1) Physiological and transcriptomic response to Medicago truncatula to colonization with high and low benefit arbuscular mycorrhizal fungi. 11th Symposium of the International Society of Root Research, Virtual Meeting, May 24-28, 2021, 2) Physiological and transcriptomic response of soybean to strains of Bradyrhizobium diazoefficiens with high and low nitrogen fixation efficiencies. PB21 Plant Biology, Virtual Meeting, July 19-23, 2021, 3) Arbuscular mycorrhizal fungi and rhizobia compete for host carbon resources during a tripartite interaction with Medicago truncatula. 11th Symposium of the International Society of Root Research, Virtual Meeting, May 24-28, 2021, 4) A high-impact plant science workshop enhances youth interest in pursuing a career in plant science. PB21 Plant Biology, Virtual Meeting, July 19-23, 2021. Publishing research manuscripts and reviews in peer reviewed journals: 1) Thilakarathna et al., 2021; 2) Salomon et al., submitted (Nature Plants), and 3) Salomon et al., (currently under revisions for Applied Journal of Ecology). Changes/Problems:Progress on the project was affected by the Covid-19 pandemic and by the move of the PI of the project (Heike Bucking) to the University of Missouri. The South Dakota State University campus closure middle of March 2020 caused many interruptions and delays of experiments. During the campus closure no new experiments could be started, and there were significant delays in the delivery of chemicals and reagents for the lab, and significant delays in the outsourced 15-N and 13-C analysis of samples. The move of the laboratory of Dr. Heike Bucking to the University of Missouri in April 2021 also caused significant interruptions and delays. The new laboratory at the University of Missouri is now established, and with the hiring of the new postdoctoral scientist for the project, we expect that we will soon continue the project as planned. What opportunities for training and professional development has the project provided?The project provided training opportunities for one Ph.D. student (Jaya Yakha), one postdoctoral associate (Dr. Kevin Cope), and two undergraduate students (Corbin Ketelsen, Hailey Axemaker). The Ph.D. student Jaya Yakha has made significant progress towards graduation, and has significantly improved her skills in experimental design, various molecular techniques (e.g. q-PCR, localization of target genes in transformed roots, bioinformatics), data and statistical analysis, and the dissemination of scientific reports in form of presentations at international conferences (10th International Conference on Mycorrhizae, ICOM, Merida, Mexico), and publications in peer reviewed journals (three manuscripts will soon be submitted with her as first-, or co-author (Cope et al. in prep.; Cope et al., in prep.; Yakha et al., in prep.). The postdoctoral associate Dr. Kevin Cope was trained in experimental systems (tripartite studies, labeling studies, N-fixing root symbioses, transcriptome analysis), writing of scientific manuscripts (Cope et al., 2019; Cope et al. in prep.; Cope et al., in prep.; Yakha et al., in prep.), has participated in a grant writing workshop, has improved his teaching and mentoring skills, and has recently accepted a position at the Pacific National Laboratory. The postdoctoral scientist Kevin Cope mentored the two undergraduate students Hailey Axemaker and Corbin Ketelsen (student recently graduated). Hailey Axemaker is working on the design of gene constructs for target gene localization studies, and Corbin Ketelsen conducted a tripartite experiment with two competing N-fixing root symbionts, and performed most of the experimental analysis, learned confocal laser scanning microscopy, and is currently involved in the transcriptome analysis of this project. He is co-author on one manuscript (Salomon et al., submitted), and will become co-author on one of the manuscripts that are currently in preparation (Cope et al., in prep.). The search for a replacement is currently ongoing. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations to different audiences, webinars and contributions to scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?Several experiments are currently ongoing to answer the following questions: Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? We conducted an experiment from which we hoped to gain a complete representation of the C fluxes from a photosynthetic legume plant to its root symbionts. We used our split-root experiment approach and supplied the fungal partner with 15N-ammonium, and the rhizobial partner with 15N-gas, and studied the C allocation to each root half, and measured the 13C respiration of the different root halves. Unfortunately, the labeling with the 15N-gas was not sufficient to induce a significant labeling of the host plants. We are planning to repeat the experiment with a longer labeling time. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? We are currently planning to conduct an experiment that will provide us with more information how the nutrient demand alters the C allocation to individual root symbionts. In this time course experiment, we will examine how fast a host plant shifts its C allocation after it has been supplied with nutrients. We will correlate these shifts in C allocation to changes in gene expression to identify the regulatory pathways involved in the C allocation to different root symbionts. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? Based on our previous transcriptome and gene expression studies, we identified several SUT and SWEET transporters that are differentially regulated in tripartite interactions or in root systems that are colonized with high or low benefit root symbionts. However, only a few of these transporters have so far been functionally characterized, and we are currently using transformed root systems to localize some of the target SUT and SWEET transporters in mycorrhizal and nodulated roots. A more precise localization of the transporter expression will provide us with more information about the potential role that these transporters play in the C allocation to individual root symbionts. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? We conducted a first set of experiments that demonstrated that fungal partners reduce their nitrogen transport in response to a co-inoculation of the root system with a N-fixing root symbiont, but there are indications of a higher P transport to the host. We are planning to study this change in transport also with potassium, or to repeat the experiment with labelled phosphate. Other previously conducted experiments have shown that root nodules often benefit from the co-inoculation of the root system with AM fungi, due to an improved nutrition of the host plant with phosphate (biological nitrogen fixation is coupled to a high P demand of root nodules. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? We conducted a split root experiment with two N-fixing strains that colonize soybean plants, USDA 110 and USDA 126, that don´t differ in their ability to nodulate the roots of soybean plants, but differ greatly in their ability for biological nitrogen fixation. In addition to the simultaneous inoculation of both root halves with the different symbionts, we also used growth chambers in which the inoculation of the second root half was delayed. We are planning now to conduct similar experiments in Medicago inoculated with Ensifer meliloti (here we will affect the ability of the bacteria to fix N by exposing the root systems to Ar:02) and Rhizophagus irregularis inoculated root halves (here we will affect the fungal ability to take up N by washing the N out of the fungal compartment). Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? We conducted most of the planned experiments but have not yet analyzed all data. When the data analysis is completed, a journal publication of these experiments is planned.
Impacts
What was accomplished under these goals?
Tripartite interactions play a critical role for the nutrient uptake and overall performance of the majority of legumes. We conducted a set of carefully designed experiments to better understand how resource exchange processes in tripartite interactions are regulated. A better understanding of these processes is critical to maximize nutritional benefits of these root symbioses in agricultural settings. Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? (80% Accomplished) No additional experiments were conducted during the reporting period. The results are currently prepared for publication. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? (80% Accomplished) No additional experiments were conducted during the reporting period. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? (70% Accomplished) To get more information about the regulatory mechanisms that control the carbon allocation strategy of plants to different root symbionts, we focused on SUTs (sucrose uptake transporters) or SWEETs (Sugars will eventually be exported transporters) that in previous experiments showed a differential expression in response to the colonization with different root symbionts. Based on these studies, we are functionally characterizing eight sucrose transporters to evaluate their role in both the rhizobia-legume and arbuscular mycorrhizal symbiosis. We used Golden Gate cloning to build eight binary vectors for each sucrose transporter containing three constructs: 1) the native promoter of a given sucrose transporter fused to the coding DNA sequence of glucuronidase, 2) a constitutive promoter driving the expression of the coding DNA sequence of a sucrose transporter fused to green fluorescent protein, and 3) a red fluorescent protein that serves as a transformation marker. The binary vectors have been transformed into Agrobacterium rhizogenes and we are currently conducting plant transformations to identify the tissues in which the promoter is active and where the protein product is localized in different root symbioses. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? (30% Accomplished) No additional experiments were conducted during the reporting period. Previous results are currently in preparation for publication. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? (70% Accomplished) We studied how the efficiency with which root symbionts provide N affects the carbon allocation by conducting experiments with two strains of Bradyrhizobium diazoefficiens (USDA110, an efficient N-fixing strain; and USDA126, an inefficient N-fixing strain). In the first experiment, we inoculated one half of a split root system with USDA110 and the other with USDA126 at the same time. In two different treatments, we staggered the inoculation of either strain by seven days. In five control treatments, we either inoculated both root halves with the same strain (either USDA110 or USDA126), one side with one strain (either USDA110 or USDA126), but no inoculation on the other root half, or no inoculation on either root half. We followed the N uptake and carbon allocation in the plants, used acetylene reduction assays to measure nitrogen fixation in root nodules, and studied plant gene expression. Plants inoculated with USDA110 had higher chlorophyll content, and consequently a higher shoot biomass and an increased uptake of 13-C than USDA126-inoculated plants. When roots were inoculated with USDA110 on one root half and USDA126 on the other, the plant allowed USDA110 to form more nodules that were larger and contained more bacteroids, indicating that the plant was actively rewarding USDA110 and sanctioning USDA126. To determine if autoregulation of nodulation played a role in the USDA110-induced inhibition of nodulation by USDA126, we performed a second split-root experiment with a similar set up as the first experiment but we varied the time points of the inoculation of the second root halve: one root half was inoculated with USDA110 or USDA126, and the other root half was inoculated with either USDA110 or USDA126 on the same day or one, two, or four days later. USDA110 inoculation on day 0 inhibited USDA126 nodulation on days 0, 1, 2, and 4, but USDA126 inoculation only inhibited USDA110 nodulation on day 4. We evaluated the 15-N and 13-C contents of the nodules from each treatment and found that USDA126 nodules possessed a much higher 15-N content but lower 13-C contents than USDA110 nodules. These data collectively suggest that the durability of autoregulation of nodulation depends on the N benefits of the rhizobia strain, nodulation of USDA110 inhibits USDA126 independent on the inoculation day, but USDA126 only inhibits USDA110, when the inoculation occurred much earlier than USDA110 inoculation. We found distinct transcriptional responses of soybean to nodulation by USDA110 and USDA126 compared to control roots. Nearly twice as many genes were up and down-regulated in USDA110 compared to USDA126 nodulated roots, but some genes were similarly regulated in both. We used gene ontology (GO) enrichment analysis to determine global patterns of the type of genes differentially regulated by each treatment. The differentially expressed genes that were shared included those involved in nodulation, nitrogen fixation, and flavonoid biosynthesis. However, GO terms upregulated in USDA110-colonized roots included detection of hypoxia, anaerobic respiration, and stromule development, while those enriched in USDA126-colonized roots included many GO terms related to transmembrane transport. Also, metal ion transporters were more strongly upregulated in USDA110-colonized roots. The manuscript of these results is currently in preparation (submission planned to PNAS). Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? (75% Accomplished) To answer this question, we conducted two experiments. First, we inoculated Medicago plants with R. irregularis and/or with E. meliloti in separate growth chambers. After establishment of the plants, different combinations of growth chambers were connected by a bridge that allowed the AM fungus to crossover and form a common mycorrhizal network. We compared the following plant pairings: 1) non-inoculated control plants, 2)AM colonized plants, or 3) R. irregulars and E. meliloti colonized plants in both chambers, or one AM colonized plant paired with a Fix+/AM or a Fix-/AM plant (4 and 5). After the plants were established, 15-N-labelled ammonium was added to the interconnecting bridge. The 15-N labeling results indicate that the AM fungus transfers less 15-N, but more P when the fungus is competing with N-fixing root nodules. This indicates a change in the nutrient allocation strategy, when the fungus is in direct competition with N-fixing root symbionts, and when the host plant is likely in less N demand.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cope K. 2021. A high-impact plant science workshop enhances youth interest in pursuing a career in plant science. PB21 Plant Biology. Virtual Meeting. July 19-23.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cope KR, Yakha JK, Ketelsen C, Huang W, Subramanian S, B�cking H. 2021. Physiological and transcriptomic response of soybean to strains of Bradyrhizobium diazoefficiens with high and low nitrogen fixation efficiencies. PB21 Plant Biology. Virtual Meeting. July 19-23.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Yakha J, Cope KR, Kafle A, Subramanian S, B�cking H. 2021. Arbuscular mycorrhizal fungi and rhizobia compete for host carbon resources during a tripartite interaction with Medicago truncatula. 11th Symposium of the International Society of Root Research. Virtual Meeting. May 24-28.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Thilakarathna MS, Cope KR. 2021. Split-root assays for studying legume-rhizobia symbioses, rhizodeposition, and belowground nitrogen transfer in legumes. Journal of Experimental. Botany. 72: 5285-5299.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Salomon MJ, Demarmels R, Watts-Williams SJ, McLaughlin MJ, Kafle A, B�cking H, Cavagnaro TR, van der Heijden MGA. 2020. Global analysis of microbial inoculants for sustainable plant production. J Applied Ecology. (submitted).
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Salomon MJ, Watts-Williams SJ, McLaughlin MJ, B�cking H, Singh BK, Hutter I, Schneider C, Marin F, Vosatka M, Guo LD, Ezawa T, Saito M, Declerck S, Zhu YG, Bowles T, Abbott LK, Cavagnaro TR, van der Heijden MGA. 2021. Establishing a quality framework for commercial inoculants containing arbuscular mycorrhizal fungi. Nature Plants (submitted).
|
Progress 05/01/19 to 04/30/20
Outputs
Target Audience:Target audiences for this project include 1) farmers and stakeholder groups, and 2) the broader scientific community. Farmers and stakeholder groups: We continued to educate farmers and stakeholders about the importance of beneficial plant microbe interactions for crop production by providing presentations and leading discussion groups and workshops. During the reporting period, we provided the following presentations and workshops: 1) Importance of mycorrhizal fungi in crop and grazing systems. Soil Health Workshop, Dickinson, North Dakota, September 12, 2019; 2) Arbuscular mycorrhizal fungi - implications for management and conservation planning, Webinar, May 07, 2019; 3) Beneficial plant microbe interactions as tool to increase soybean yields in stressful environments. SD Soybean Research and Promotion Council, Brookings, April 08, 2019. Broader scientific community: We provided updates about the research projects to other scientists by: Providing oral presentations at other institutions: 1) Fair trade in beneficial plant microbe interactions, North Dakota State University, November 01, 2019; 2) Fair trade in beneficial plant microbe interactions, New Mexico State University, October 25, 2019; Providing oral and poster presentations at international conferences: 1) Bidirectional nutrient fluxes in tripartite interactions of Medicago truncatula are controlled by nutrient demand. Rhizosphere 5, Saskatoon, Canada, July 11, 2019; 2) Establishment and functionality of arbuscular mycorrhizal fungi in the root rhizosphere. Rhizosphere 5, Saskatoon, Canada, July 11, 2019; 3) How do arbuscular mycorrhizal fungi and rhizobia compete for host carbon resources in tripartite interactions with Medicago truncatula. 10th International Conference on Mycorrhizae, Merida, Mexico, July 03, 2019; 4) Friend or foe - How does a host plant distinguish among high or low benefit arbuscular mycorrhizal fungi? 10th International Conference on Mycorrhizae, Merida, Mexico, July 02, 2019; Providing oral presentations at meetings with industrial partners: 1) Fair trade in beneficial plant microbe interactions. Indigo, Boston, May 14, 2019. Publishing research manuscripts and reviews in peer reviewed journals (Garcia et al., 2020; Becquer et al. 2019; Chen et al., 2019; Cope et al. 2019, Kafle et al. 2019a; Kafle et al., 2019b; Ma et al. 2019), and in a producer magazine (Berti and Bücking, 2020) (more information can be found under products). Changes/Problems:In the time period relevant for this report, the effects of the Covid-19 pandemic were limited. However, the SDSU campus closure middle of March 2020 caused some interruptions and delays of experiments at the end of the reporting period. However, Covid-19 led to significant delays in the next reporting period, due to the extended period of time, in which access to laboratory and greenhouses and the number of allowed students per laboratory was severely restricted. Additional delays were caused by the long delivery times of experimental supplies, and the problems with hiring additional students for the project. What opportunities for training and professional development has the project provided?The project provided training opportunities for one Ph.D. student (Jaya Yakha), one postdoctoral associate (Dr. Kevin Cope), and two undergraduate students (Corbin Ketelsen, Hailey Axemaker). The Ph.D. student Jaya Yakha has made significant progress towards graduation, and has significantly improved her skills in experimental design, various molecular techniques (e.g. q-PCR, localization of target genes in transformed roots, bioinformatics), data and statistical analysis, and the dissemination of scientific reports in form of presentations at international conferences (10th International Conference on Mycorrhizae, ICOM, Merida, Mexico), and publications in peer reviewed journals (three manuscripts will soon be submitted with her as first-, or co-author (Cope et al. in prep.; Cope et al., in prep.; Yakha et al., in prep.). The postdoctoral associate Dr. Kevin Cope was trained in experimental systems (tripartite studies, labeling studies, N-fixing root symbioses, transcriptome analysis), writing of scientific manuscripts (Cope et al., 2019; Cope et al. in prep.; Cope et al., in prep.; Yakha et al., in prep.), has participated in a grant writing workshop, has improved his teaching and mentoring skills, and is currently actively applying for senior scientist and tenure-track faculty positions (two interviews). The postdoctoral scientist is currently mentoring the two undergraduate students Hailey Axemaker and Corbin Ketelsen (student recently graduated). Hailey Axemaker is working on the design of gene constructs for target gene localization studies, and Corbin Ketelsen conducted a tripartite experiment with two competing N-fixing root symbionts, and performed most of the experimental analysis, learned confocal laser scanning microscopy, and is currently involved in the transcriptome analysis of this project. He is co-author on one manuscript (Salomon et al., submitted), and will become co-author on one of the manuscripts that are currently in preparation (Cope et al., in prep.). How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations to different audiences, webinars and contributions to scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?Several experiments are currently ongoing to answer the following questions: Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? We conducted an experiment from which we hoped to gain a complete representation of the C fluxes from a photosynthetic legume plant to its root symbionts. We used our split-root experiment approach and supplied the fungal partner with 15N-ammonium, and the rhizobial partner with 15N-gas, and studied the C allocation to each root half, and measured the 13C respiration of the different root halves. Unfortunately, the labeling with the 15N-gas was not sufficient to induce a significant labeling of the host plants. We are planning to repeat the experiment with a longer labeling time. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? We are currently planning to conduct an experiment that will provide us with more information how the nutrient demand alters the C allocation to individual root symbionts. In this time course experiment, we will examine how fast a host plant shifts its C allocation after it has been supplied with nutrients. We will correlate these shifts in C allocation to changes in gene expression to identify the regulatory pathways involved in the C allocation to different root symbionts. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? Based on our previous transcriptome and gene expression studies, we identified several SUT and SWEET transporters that are differentially regulated in tripartite interactions or in root systems that are colonized with high or low benefit root symbionts. However, only a few of these transporters have so far been functionally characterized, and we are currently using transformed root systems to localize some of the target SUT and SWEET transporters in mycorrhizal and nodulated roots. A more precise localization of the transporter expression will provide us with more information about the potential role that these transporters play in the C allocation to individual root symbionts. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? We conducted a first set of experiments that demonstrated that fungal partners reduce their nitrogen transport in response to a co-inoculation of the root system with a N-fixing root symbiont, but there are indications of a higher P transport to the host. We are planning to study this change in transport also with potassium, or to repeat the experiment with labelled phosphate. Other previously conducted experiments have shown that root nodules often benefit from the co-inoculation of the root system with AM fungi, due to an improved nutrition of the host plant with phosphate (biological nitrogen fixation is coupled to a high P demand of root nodules. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? We conducted a split root experiment with two N-fixing strains that colonize soybean plants, USDA 110 and USDA 126, that don´t differ in their ability to nodulate the roots of soybean plants, but differ greatly in their ability for biological nitrogen fixation. In addition to the simultaneous inoculation of both root halves with the different symbionts, we also used growth chambers in which the inoculation of the second root half was delayed. This will allow us to study autoregulation, a process that allows legumes to prevent an over-nodulation of the root system, and to limit the carbon costs of the symbiosis for the host. We are currently analyzing the transcriptome in the roots and shoots of these plants, to better understand how the plant regulates its carbon flux to these root symbionts. We are also planning further experiments with Medicago in which, for example, Ar:O2 gas mixtures will be applied to the rhizobial root halves to examine how rapidly a host plant will change its C allocation when the rhizobia bacteria are unable to provide N to their host. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? We conducted some of the planned experiments, but have not had an opportunity to analyze all the data. When the data analysis is completed, we will decide, whether additional experiments will be need to be performed.
Impacts
What was accomplished under these goals?
Tripartite interactions play a critical role for the nutrient uptake and overall performance of the majority of legumes. We conducted a set of carefully designed experiments to better understand how resource exchange processes in tripartite interactions are regulated. A better understanding of these processes is critical to maximize nutritional and other benefits of these root symbioses in agricultural settings. Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? (75% Accomplished) We conducted experiments to evaluate how the carbon (C) allocation to different root symbionts is affected when the fungal partner is able to provide nitrogen (N) to its host plant, while acting as a direct competitor of N fixing root nodules. We studied this question in custom-made, three soil compartment systems, using Medicago as the host plant. We compared the C allocation in six different split-root systems with: 1) two non-inoculated root halves (C/C), 2) one non-inoculated root half and one inoculated with Rhizophagus irregularis (C/AM), 3,4) one non-inoculated root half and one inoculated with Fix+ or Fix- Ensifer meliloti (Fix+/C, or Fix-/C), and 5,6) two inoculated root halves, one inoculated with Rhizophagus irregularis and one inoculated with Fix+ or Fix- Ensifer meliloti (Fix+/AM, or Fix-/AM). The Fix- mutant of Ensifer meliloti is impaired in biological N fixation. The plants were later labeled with 13C labeled CO2, and after plant harvest, we analyzed the plants for biomass characteristics, 15N and 13C labeling, and gene expression. We found that the 15N uptake of the fungus from the soil compartment was independent of whether the other root half was colonized with the Fix+ or Fix- strain of Ensifer meliloti. However, a significant drop in the 15N transport to the shoot in Fix+/AM systems indicates a reduced 15N transport across the mycorrhizal interface when the fungus is in direct competition with a N-fixing root symbiont. Consistently with this reduced AM transport of 15N to the host, only in Fix-/AM systems more 13C was allocated to the fungal partner when the fungus had access to N. In Fix-/AM systems, the expression of the sucrose uptake transporter MtSUT1-1 was reduced, but the expression of MtSUT4-1 was induced when the fungus had access to N, while the Sugars Will Eventually be Exported Transporter (SWEET) expression was unaffected by the N supply. The results were presented at the last International Conference on Mycorrhizae, and are currently prepared for publication. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? (80% Accomplished) No additional experiments were conducted during the reporting period. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? (50% Accomplished) To get more information about the regulatory mechanisms that control the carbon allocation strategy, we conducted an experiment with Medicago plants that were non-inoculated, or inoculated with the AM fungus Rhizophagus irregularis 09 (Ri09), or Glomus aggregatum 165 (Ga165). Ri09 transfers more P and N to its host plant, and has a higher impact on plant growth than Ga165. We studied the transcriptome of the plants, and analyzed the differentially expressed genes (DEGs), and found that genes involved in the common symbiosis pathway, and particularly transporter genes are differentially regulated in Ri09 or Ga165. DEGs that could be responsible for differences in carbon allocation to these different root symbionts, included for example SWEETs. We found that the expression of SWEET1.2 was upregulated by both fungi in the roots, but especially by Ri09. SWEET3.3 and SWEET12 were only upregulated in Ri09-colonized roots, while SWEET7 and SWEET13 were only upregulated in Ga165-colonized roots. The variation in the expression patterns of these five SWEET genes suggests that sugar transport is strongly regulated in response to fungal benefit. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? (30% Accomplished) The experiment described under Objective 1 provides first answers to this question. The observed drop in the fungal 15N translocation to the shoot, when the fungus competes with the Fix+ Ensifer meliloti strain, indicates that the 15N that was taken up by the fungus was likely compartmentalized in the intraradical mycelium of the fungus and was not transferred to the host across the mycorrhizal interface. This indicates that the fungus changes its nutrient transport strategy, and exchanges other nutrients against carbon when in competition with a N-fixing root symbiont. Interestingly, we found that the P content in the shoots of the Fix-/AM, and the Fix+/AM plants were higher than in C/AM plants, indicating that the fungus provided more P when in competition with Fix- and Fix+ Ensifer meliloti. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? (50% Accomplished) In the first experiment (described under objective 1), we compared the carbon allocation to Fix+ and Fix- root halves when paired with a non-inoculated root half (Fix+/C or Fix-/C) or with an AM inoculated root half (Fix+/AM or Fix-/AM). The results clearly demonstrated that the host plant significantly reduced the carbon allocation and sanctioned the inactive Fix- root symbionts, compared to the Fix+ colonized root nodules. In the second experiment, we used a similar split-root approach as described above, but used soybean plants that were non-inoculated, or inoculated with USDA 126 or USDA 110, two different Bradyrhizobium japonicum strains with a low or a high biological N fixation efficiency, respectively. In addition, we examined chamber systems in which the roots of one chamber were inoculated with the second strain (USDA 126 or USDA 110) with a time delay of 4 or 7 days after the first strain was allowed to colonize the other root half. As expected, we found that USDA 110 increased chlorophyll concentration and shoot biomass, as well as the size, N fixation, and number of bacteroids in root nodules. Similarly, the host sanctioned ineffective root symbionts, and preferentially allocated carbon to USDA 110 root halves. We are currently working on the transcriptome analysis that will help us identify candidate genes involved in this sanction mechanism. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? (50% Accomplished) To answer this question, we conducted two experiments. In both experiments, we inoculated two Medicago plants with arbuscular mycorrhizal fungi or with Ensifer meliloti in two growth chambers. After establishment of the plants, both growth chambers were connected by a bridge to allow the AM fungus to crossover and to colonize the nodulated plant, and then 15N-labelled ammonium was added to the interconnecting bridge compartment. Unfortunately, Experiment 1 was not successful, since Ensifer meliloti used the hyphal connections to crossover and nodulated the AM colonized Medicago plant. To avoid these problems, we varied the experimental approach in Experiment 2. Here, we repeated the original set up of Experiment 1, but also added chamber systems, in which the AM inoculated plant was combined with a Bradyrhizobium japonicum colonized soybean plant (B. japonicum does not colonize Medicago plants), or with a Medicago plant colonized with a Fix- mutant of Ensifer meliloti. The analysis of this experiment is not yet completed, and we will include the data in the next report.
Publications
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Fair trade in beneficial plant microbe interactions. Invited seminar at North Dakota State University. Fargo, ND. November 07.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Fair trade in beneficial plant microbe interactions. Invited seminar at New Mexico State University. Las Cruces, NM. October 25.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Importance of mycorrhizal fungi in crop and grazing systems. Soil Health Workshop. Dickinson, ND. September 12.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Cope KR, Yakha J, Kafle A, Garcia K, B�cking H. 2019. Bidirectional nutrient fluxes in tripartite interactions of Medicago truncatula are controlled by plant nutrient demand. Rhizosphere 5. Saskatoon, Canada. July 11.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H, Mensah J, Fellbaum CR. 2019. Establishment and functionality of arbuscular mycorrhizal communities in the root rhizosphere. Rhizosphere 5. Saskatoon, Canada. July 11.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Peta V, Soupir A, B�cking H. 2019. The plant microbiome of Brassica carinata and its potential to increase plant growth and yield. Rhizosphere 5. Saskatoon, Canada. July 11.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Yakha J, Cope K, B�cking H. 2019. How do arbuscular mycorrhizal fungi and rhizobia compete for host carbon resources in tripartite interactions with Medicago truncatula. 10th International Conference on Mycorrhiza. Merida, Mexico. July 03.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Friend or foe How does a host plant distinguish among high or low benefit AM fungi? 10th International Conference on Mycorrhiza. Merida, Mexico. July 02.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Fair trade in beneficial plant microbe interactions. Indigo. Boston, MA. May 14.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H, Lehman M. 2019. Arbuscular mycorrhizal fungi- implications for management and conservation planning. Sponsored by: USDA NRCS Conservation Webinars. Available at: http://www.conservationwebinars.net/webinars/arbuscular-mycorrhizal-fungi-2013-implications-for-management-and-conservation-planning. May 07.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Beneficial plant microbe interactions as tool to increase soybean yields in stressful environments. SD Soybean Research and Promotion Council. Brookings, SD. April 08.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Cope K, Garcia K, Kafle A, Pfeffer PE, Strahan GD, Subramanian S, B�cking H. 2020. Physiological and transcriptomic response of Medicago truncatula to high and low benefit mycorrhizal fungi. BioRxiv, doi: https://doi.org/10.1101/2020.12.11.421693
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Salomon MJ, Demarmels R, Watts-Williams SJ, McLaughlin MJ, Kafle A, B�cking H, Cavagnaro TR, van der Heijden MGA. 2020. Global analysis of microbial inoculants for sustainable plant production. J Applied Ecology. (submitted).
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Garcia K, B�cking H, Zimmermann SD. eds. 2020. Importance of root symbiosomes for plant nutrition: new insights, perspectives, and future challenges. Lausanne: Frontiers Media: doi: 10.3389/978-2-88963-814-7.
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Berti M, B�cking H. 2020. Alfalfa productivity and nutrient uptake is related to its interaction with the soil microbiome. Forage Focus Magazine. March 2020, page 18-19.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Becquer A, Guerrero-Gal�n C, Eibensteiner JL, Houdinet G, B�cking H, Zimmermann S, Garcia K. 2019. The ectomycorrhizal contribution to tree nutrition. In: Advances in Botanical Research: Molecular Physiology and Biotechnology of Trees: 89, 77-126; DOI: 10.1016/bs.abr.2018.11.003.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chen B, Wang Q, B�cking H, Sheng J, Luo J, Chai Z, Kafle A, Hou Y, Feng G. 2019. Genotypic differences in phosphorus acquisition efficiency and root performance of cotton (Gossypium hirsutum) under low-phosphorus stress. Crop and Pasture Science 70(4): 344-358; DOI: 10.1071.CP18324
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Kafle A, Cope KR, Raths R, Yakha JK, Subramanian S, B�cking H, Garcia K. 2019. Harnessing soil microbes to improve plant phosphate efficiency in cropping systems. Agronomy 9(3): 127: DOI: 10.3390.agronomy9030127.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Cope KR, Bascaules A, Irving TB, Venkatshwaran M. Maeda J. Garcia K, Rush T, Ma C, Labbe JJ, Jawdy S, Steigerwald E, Setzke K. Fung E, Schnell KG, Wang Y, Schlief N, B�cking H, Strauss SH, Maillet F, Jargeat P, B�card G, Puech-Pag�s V, An�, J-M. 2019. The ectomycorrhizal fungus Laccaria bicolor produces lipochitooligosaccharides and uses the common symbiosis pathway to colonize Populus roots. Plant Cell 31:2386-2410; DOI: 10.1105/tpc.18.00676
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Ma Q, B�cking H, Gonzalez Hernandez JL, Subramanian S. 2019. Single-cell RNA sequencing of plant associated bacterial communities. Frontiers in Microbiology; DOI: 10.3389/fmicb.2019.02452
|
Progress 05/01/18 to 04/30/19
Outputs
Target Audience:Target audiences for this project include 1) farmers and stakeholder groups, and 2) the broader scientific community. Farmers and stakeholder groups: We continued our efforts to educate farmers and stakeholders about the importance of beneficial plant microbe interactions for crop production by providing poster and oral presentations at the following events: Poster presentation at the Soil Health Conference, Iowa State University, February 04-05, 2019; Oral presentation at the Ag Innovation Group Meeting, Minot, North Dakota, March 07, 2019; Oral presentation at the SD Soybean Research and Promotion Council Meeting, Brookings, South Dakota, April 08, 2019; Oral presentation about the importance of mycorrhizal fungi in crop and grazing systems. Soil Health Workshop, Dickinson, North Dakota, September 12, 2019. In addition, we recently submitted a short article to a Forage Magazine (Berti and Bücking, 2020) and produced a webinar with the title: Arbuscular mycorrhizal fungi - implications for management and conversation planning (May 07, 2019). The webinar was coordinated by the USDA NRCS Conservation Webinars and has reached 670 participants (365 participants followed the live webinar, 305 viewers watched the archived webinar, and 213 participants used the webinar to receive continuing education credits). The archived webinar is still available online. Furthermore, the importance of agricultural research at South Dakota State University was presented to South Dakota´s Congressional Delegates (staff members of Senator Thune and Senator Rounds), and to congressional delegates and funding agencies at the Agricultural Research Congressional Exhibit 2019 in Washington. Primary focus of all these presentations was the application potential of microbial fertilizers, and the significance of arbuscular mycorrhizal communities and other beneficial soil microorganisms for soil health. Scientific community: We provided updates about the research projects to other scientists by publishing manuscripts in peer reviewed journals (e.g. Ma et al., 2019; Cope et al., 2019, Chen et al., 2019, Salomon et al., submitted), and review chapters (Kafle et al., 2019) (see information below, additional manuscripts are currently in preparation). In addition, we shared our results at two international conferences (ICOM 10 in Merida, Mexico; Rhizosphere 5 in Saskatoon, Canada). The following oral presentations were provided at these conferences: Bücking H, Mensah J, Fellbaum CR. 2019. Establishment of functionality of arbuscular mycorrhizal communities in the root rhizosphere. Rhizosphere 5, Saskatoon, Canada, July 11, 2019; Cope KR, Yakha J, Kafle A, Garcia K, Bücking H. 2019. Bidirectional nutrient fluxes in tripartite interactions of Medicago truncatula are controlled by plant nutrient demand. Rhizosphere 5, Saskatoon, Canada, July 11, 2019; Bücking H. 2019. Friend or foe - How does a host plant distinguish among high or low benefit AM fungi? 10th International Conference on Mycorrhiza, Merida, Mexico, July 02, 2019; Yakha J, Cope K, Bücking H. 2019. How do arbuscular mycorrhizal fungi and rhizobia compete for host carbon resources in tripartite interactions with Medicago truncatula. 10th International Conference on Mycorrhiza, Merida, Mexico, July 03, 2019. In addition, I was invited to present two seminars at New Mexico State University, and North Dakota State University: Bücking H. 2019. Fair trade in beneficial plant microbe interactions. Invited seminar at New Mexico State University, October 25, 2019. Bücking H. 2019. Fair trade in beneficial plant microbe interactions. Invited seminar at North Dakota State University, November 01, 2019. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training opportunities for two Ph.D. students (Arjun Kafle and Jaya Yakha), three undergraduate students (Corbin Ketelsen, Dylan Blomme, Hailey Axemaker) and one postdoctoral associate (Kevin Cope). Both Ph.D. students were trained in experimental design, data and statistical analysis, and the dissemination of scientific reports in form of posters at conferences, publications in peer reviewed journals, and reviews for book chapter publications. One Ph.D. student graduated and joined the laboratory of Kevin Garcia at North Carolina State University as a postdoctoral associate. Kevin Garcia is now assistant professor but previously worked on the project as postdoctoral associate. The other postdoctoral associate was trained in experimental systems (tripartite studies, labeling studies), writing of scientific manuscripts, and has now submitted his first applications for assistant professor positions. The three undergraduate students work together with the postdoctoral associate Kevin Cope on independent experiments within this subject area. Corbin Ketelsen is working on the soybean experiment with two different rhizobia strains (USDA 110 and USDA 126), Hailey Axemaker is conducting one of the transporter localization studies, and Dylan Blomme is working on a time course study with Medicago truncatula plants. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations and scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?Several experiments are currently ongoing to answer the following questions: Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? We hoped to answer this question with experiments in which the carbon allocation to the root symbiont is measured when 15N-ammonium chloride is supplied to the fungal partner, or 15N gas is released into the rhizobial chamber. Unfortunately, we were only able to measure the 15N that was transferred from the fungal partner to the host plant, but not the 15N that was transferred via the root nodules. This could be due to leakage problems, or to the relatively short time period in which the rhizobial root halves were exposed to 15N gas. We are planning to repeat the experiment and expose the rhizobia root halves to N15 over longer time periods. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? We are interested in exploring the conditions and timing under which the host plant changes its carbon allocation strategy. For example, our previous results demonstrated that more carbon is allocated from the host plant to the fungus when the fungus is able to provide nitrogen to the host plant. We are currently preparing a time course experiment in which carbon allocation to the fungal partner is examined at different time points after nitrogen has been supplied to the fungal compartment. We will combine this study with a transcriptome analysis to identify the regulatory control of this allocation shift. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? See planned and ongoing experiments above. We are currently using transformed root systems to localize some of the target SUT and SWEET transporters in mycorrhizal and nodulated roots. A more precise localization of transporter expression will provide us with more information about the potential role of these transporters in the C allocation to individual root symbionts. In addition, we are planning transcriptome studies of Medicago and soybean plants in tripartite symbiosis. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? To examine this question, we conducted an experiment in which we provided the fungal partner with access to N15, and added 15N labeled gas to the rhizobial root compartment. Unfortunately, we were only able to measure the N15 that was added to the fungal compartment, but not the 15N that was added to the rhizobial chamber. We are planning to repeat this experiment. However, independently other results have demonstrated that the co-colonization of the root system with arbuscular mycorrhizal fungi increases the nitrogen fixation rate of rhizobia root halves (measured by acetylene reduction assays). Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? The Medicago experiment with the Fix + and Fix - rhizobia strains, and the soybean experiment with USDA 110 and USDA 126 provided us with first evidence how the C allocation strategy is affected by the inability of one partner to provide nutritional benefits. Further experiments are planned in which Ar:O2 gas mixtures will be applied to the rhizobial root halves to examine how rapidly a host plant will change its C allocation when the rhizobia bacteria are unable to provide N to their host. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? We conducted an experiment to answer the question of whether the fungal symbiont will change its nutrient allocation strategy in common mycorrhizal networks, but had problems with cross-contamination of rhizobia bacteria between the two plant compartments (one was inoculated with rhizobia bacteria, and the other one was not inoculated, but later showed root nodulation). We are currently examining different techniques to prevent the observed root nodulation (one pathway could be the addition of an antibiotic to the non-inoculated growth chamber).
Impacts
What was accomplished under these goals?
Tripartite interactions play a critical role in nutrient uptake and overall performance of the majority of legumes. We conducted a set of carefully designed experiments to better understand how resource exchange processes in tripartite interactions are regulated. Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? (50% Accomplished) We conducted experiments to evaluate how carbon (C) allocation to different root symbionts is affected when the fungal partner is able to provide nitrogen (N) to its host plant, while acting as a direct competitor of N fixing root nodules. Interactions with arbuscular mycorrhizal (AM) fungi are generally seen as less costly for the host plant, and we hypothesized that the host plant would shift its carbon allocation to the fungal symbiont when this symbiont is able to provide N to its host. We studied this question in custom-made, three soil compartment systems, using a split root design and Medicago as the host plant. We compared the C allocation in two different split root systems with: 1) two non-inoculated root halves (Ø/Ø), 2) one non-inoculated root half and one inoculated with Rhizophagus irregularis (Ø/AM), 3) one non-inoculated root half and one inoculated with Ensifer meliloti Fix + (N fixing Ensifer meliloti strain)(R+/Ø), 4) one non-inoculated root half and one inoculated with Ensifer meliloti Fix - (non-N fixing Ensifer meliloti strain)(R-/Ø), 5) two inoculated root halves, one inoculated with Ensifer meliloti Fix + and one inoculated with Rhizophagus irregularis (R+/AM), and 6) two inoculated root halves, one inoculated with Ensifer meliloti Fix - and one inoculated with Rhizophagus irregularis (R+/AM). In addition, we compared systems in which the fungus either had or did not have access to an exogenous supply of 15N labeled ammonium chloride, or the nodulated root halves either had or did not have access to N15 gas. The plants were later labeled with 13C labeled CO2, and after plant harvest, we analyzed the plants for biomass characteristics, 15N and 13C labeling, and gene expression. Root halves inoculated with the N-fixing Ensifer meliloti strain (Fix +) acted as strong C sinks and showed significantly higher 13C labeling than the non-inoculated root halves in R+/Ø systems, or the mycorrhizal root halves in R+/AM systems. In contrast, nodulated root halves that were inoculated with the Fix - strain, were not strong C sinks, and the host plants allocated more C into the AM root half. The AM root half became a stronger competitor for host plant C when the fungus had access to N. In tripartite symbiosis, the AM fungus transfers more N15 to the plant when the plant is colonized with the Fix- strain than with the Fix+ strain. Interestingly, this difference is particularly pronounced in the shoot system, while the 15N labeling in the AM root halve of the Fix + systems is still high, indicating that not the 15N uptake from the soil by the AM fungus is affected, but its translocation across the mycorrhizal interface to the host. Unfortunately, only the N15 labeling to the fungal partner led to a significant labeling of the plant tissue, while the N15 labeling of the rhizobial root halves did not result in a significant labeling of the plant tissues. This could be due to the relatively shorter labeling time that was used in this experiment (1 h exposure to 15N gas). Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? (80% Accomplished) No additional experiment were conducted during the reporting period. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? (50% Accomplished) We previously reported on expression levels of three plant sucrose transporters from the SUT family (MtSUT1-1, MtSUT2 and MtSUT4- 1) and seven transporters of the SWEET family (MtSWEET1b, MtSWEET6, MtSWEET9, MtSWEET11, MtSWEET12, MtSWEET15c, and MtSWEET15d). We found that in tripartite interactions, expression levels of MtSUT2 and MtSUT4-1 were positively correlated with C allocation to different symbiotic partners. MtSWEET11 is specifically expressed in root nodules, while MtSWEET1b is upregulated in mycorrhizal root halves. Similar to the changes in the gene expression patterns that were observed for the SUTs, the expression of MtSWEET12, MtSWEET15c, and MtSWEET15d were in agreement with the observed changes in C allocation to AM or rhizobial roots. This suggests that these transporters play an important role in sucrose transport to symbiotic sink tissues. We currently use multiple strategies to better characterize the importance of these transporters: Currently, we use GFP labeling to assess where these transporters are localized in transformed Medicago roots. After expression in transformed roots, confocal laser scanning microscopy will be used to visualize transporter expression in roots after colonization. We compared SUT and SWEET transporter expression in arbuscular mycorrhizal roots that were colonized with a high benefit fungus (Rhizophagus irregularis) and a low benefit fungus (Glomus aggregatum). This transcriptome study was consistent with our previous results: MtSWEET1b, MtSWEET3c, and MtSWEET 12 were specifically up-regulated in roots colonized with the high benefit fungus, while MtSWEET7 and MtSWEET13 are up-regulated in roots colonized with the low benefit fungus. We are currently completing a soybean experiment using a split root design in which root halves were inoculated with two different rhizobia strains (USDA 110 and USDA 126). While USDA 110 is highly beneficial for the host plant, the nitrogen fixation rate of USDA 126 is very low, and the plants show growth depression due to N limitation. We measured 13C labeling of the root halves after 13CO2 exposure of the host plant, and found that the host plant significantly allocates more carbon into the root halves that are colonized with USDA 110. A transcriptome study is currently underway to provide more information about the regulation of SUT and SWEET genes that are involved in the tripartite symbiosis with two different rhizobia symbionts. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? (40% Accomplished) Consistent with our previous findings, our experiments demonstrated that dual-inoculation with AM fungi increases the nitrogen fixation of rhizobia bacteria. Nitrogen fixation of Ensifer meliloti Fix + nodules were two-fold higher in plants that were also colonized with the AM fungus Rhizophagus irregularis than in non-mycorrhizal plants. However, we also found that dual inoculation with Fix + rhizobia bacteria reduces fungal N transport to the host plant. This could indicate that in competition with N fixing rhizobia bacteria, the fungus increases its competitiveness for host plant carbon through the transport of phosphate. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? (0% Accomplished) The first experiments to answer this question are currently ongoing. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? (20% Accomplished) We conducted an experiment but unfortunately had cross contaminations from nodulated to non-nodulated plants in our growth chamber systems. A recent article described that rhizobia can effectively use fungal hyphae to move through the soil, and this might explain the cross contamination we observed. We are currently trying to design experiments through which this question can be answered.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Fair trade in beneficial plant microbe interactions. Invited seminar at North Dakota State University. Fargo, ND. Nov 1.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Fair trade in beneficial plant microbe interactions. Invited seminar at New Mexico State University. Las Cruces, NM. Oct 25.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Importance of mycorrhizal fungi in crop and grazing systems. Soil Health Workshop. Dickinson, ND. Sept 12.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Cope KR, Yakha J, Kafle A, Garcia K, B�cking H. 2019. Bidirectional nutrient fluxes in tripartite interactions of Medicago truncatula are controlled by plant nutrient demand. Rhizosphere 5, Saskatoon, Canada, July 11.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H, Mensah J, Fellbaum CR. 2019. Establishment of functionality of arbuscular mycorrhizal communities in the root rhizosphere. Rhizosphere 5, Saskatoon, Canada, July 11.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Peta V, Soupir A, B�cking H. 2019. The plant microbiome of Brassica carinata and its potential to increase plant growth and yield. Rhizosphere 5, Saskatoon, Canada, July 11.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Yakha J, Cope K, B�cking H. 2019. How do arbuscular mycorrhizal fungi and rhizobia compete for host carbon resources in tripartite interactions with Medicago truncatula. 10th International Conference on Mycorrhiza. Merida, Mexico. July 03.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Friend or foe How does a host plant distinguish among high or low benefit AM fungi? 10th International Conference on Mycor-rhiza. Merida, Mexico. July 02.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. Beneficial plant microbe interactions as tool to increase soybean yields in stressful environments. SD Soybean Research and Promotion Council. Brookings, SD. April 08.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
B�cking H. 2019. The importance and management of mycorrhizal fungi for soil and plant health. Ag Innovation Group Meeting. Minot, ND. March 07.
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Cope KR, Garcia K, Kafle A, Pfeffer PE, Strahan GD, Subramanian S, B�cking H. 2020. The colonization with high or low benefit arbuscular mycorrhizal fungi leads to distinct transcriptional responses in Medicago truncatula. New Phytologist (In preparation).
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Kafle A, Cope KR, Garcia K, B�cking H. 2020. Beneficial effects in tripartite interactions in Medicago truncatula depend on nutrient status and carbon transport. Mycorrhiza. (In preparation).
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Salomon MJ, Demarmels R, Watts-Williams SJ, McLaughlin MJ, Kafle A, B�cking H, Cavagnaro TR, van der Heijden MGA. 2020. Global analysis of microbial inoculants: 84% of the tested products contained no active propagules. Agronomy Sustainable Development. (In preparation)
- Type:
Other
Status:
Submitted
Year Published:
2020
Citation:
Berti M, B�cking H. 2020. Alfalfa productivity and nutrient uptake is related to its interaction with the soil microbiome. Forage Focus Magazine. (submitted).
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chen B, Wang Q, B�cking H, Sheng J, Luo J, Chai Z, Kafle A, Hou Y, Feng G. 2019. Genotypic differences in phosphorus acquisition efficiency and root performance of cotton (Gossypium hirsutum) under low-phosphorus stress. Crop and Pasture Science 70(4): 344-358; DOI: 10.1071.CP18324.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Cope KR, Bascaules A, Irving TB, Venkatshwaran M. Maeda J. Garcia K, Rush T, Ma C, Labbe JJ, Jawdy S, Steigerwald E, Setzke K. Fung E, Schnell KG, Wang Y, Schlief N, B�cking H, Strauss SH, Maillet F, Jargeat P, B�card G, Puech-Pag�s V, An�, J-M. 2019. The ectomycorrhizal fungus Laccaria bicolor produces lipochitooligosaccharides and uses the common symbiosis pathway to colonize Populus roots. Plant Cell 31:2386-2410; DOI: 10.1105/tpc.18.00676.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Kafle A, Cope KR, Raths R, Yakha JK, Subramanian S, B�cking H, Garcia K. 2019. Harnessing soil microbes to improve plant phosphate efficiency in cropping systems. Agronomy 9(3): 127: DOI: 10.3390.agronomy9030127
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Ma Q, B�cking H, Gonzalez Hernandez JL, Subramanian S. 2019. Single-cell RNA sequencing of plant associated bacterial communities. Frontiers in Microbiology; DOI: 10.3389/fmicb.2019.02452
|
Progress 05/01/17 to 04/30/18
Outputs
Target Audience:Target audiences for this project include (1) farmers and stakeholder groups, and (2) the broader scientific community. Farmers and stakeholder groups: We continue to educate farmers and stakeholders about the importance of beneficial plant microbe interactions for crop production by providing presentations and leading discussion groups and workshops. During the reporting period, we provided the following presentations and workshops: (1) Annual Meeting of the Midwest Cover Crop Council: March 13-14, 2018. Fargo, ND; (2) Meetings with Ukrainian producer groups: March 01, 2018. Kiev, Ukraine. During these meetings, we shared information about the role that beneficial plant microbe interactions can play for the productivity of legumes, and discussed the effect of management practices on communities of beneficial microorganisms in soils. Scientific community: We provided updates about the research projects to other scientists by: (1) providing oral and poster presentations at an international conference, (2) publishing manuscripts in peer reviewed journals (Kafle et al., 2018; Neupane et al., 2018), and (3) publishing two review chapters (Kafle et al., 2018, Becquer et al., 2018) (see more information under products). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training opportunities for one Ph.D. student (Arjun Kafle) and one postdoctoral associate (Kevin Garcia). The Ph.D. student was trained in experimental design, data and statistical analysis, and the dissemination of scientific reports in form of posters at conferences, publications in peer reviewed journals, and reviews for book chapter publications. The postdoctoral associate was trained in experimental systems (tripartite studies, labeling studies), writing of scientific manuscripts, and has now moved on to a faculty position at North Carolina State University. The Ph.D. student has graduated and joined now the lab of this former postdoctoral associate at North Carolina State University. One new Ph.D. student (Jaya Yakha) and one new postdoctoral associate (Kevin Cope) are currently being trained on the project. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations and scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?Several experiments are currently ongoing to answer the following questions: Objective 1. What are the carbon costs of each symbiosis type per nutrient gain (here in terms of N)? We are currently analyzing samples from an experiment from which we hope to gain a complete representation of the C fluxes from a photosynthetic legume plant to its root symbionts. In contrast to previous experiments, we will also receive data about the C respiration of the different root halves in split root experiments. If successful, this will allow us to determine more precisely how much C the host plant will invest into N that is delivered by different root symbionts. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? We are currently planning to conduct an experiment that will provide us with more information how nutrient demand alters the C allocation to individual root symbionts. In this time course experiment, we will examine how fast a host plant shifts its C allocation after it has been supplied with nutrients. We will correlate these shifts in C allocation to changes in gene expression to identify the regulatory pathways involved in the C allocation to different root symbionts. Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? See planned experiments above. In addition, we are currently using transformed root systems to localize some of the target SUT and SWEET transporters in mycorrhizal and nodulated roots. A more precise localization of transporter expression will provide us with more information about the potential role of these transporters in the C allocation to individual root symbionts. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? To answer this question we are currently analyzing samples from another split root experiment using our multi-compartment systems. In addition to our previous experimental design, in which the root halves were inoculated with different combinations of root symbionts, in this experiment we added a Fix- mutant of Ensifer meliloti. This mutant is able to colonize the root system, but is unable to fix gaseous nitrogen. We labeled the nodulated root halves with 15N2 to study how the N fixation and N transfer of root nodules is affected by the co-colonization with AM fungi. We also supplied the AM fungus with access to 15N-labelled ammonium chloride to study how the fungal transport of N to its host is affected by the co-colonization of the root system with N fixing root nodules. The sample analysis of this experiment is currently ongoing. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? The experiment that is described above will also provide us with first evidence how the C allocation strategy is affected by the inability of one partner to provide nutritional benefits. Further experiments are planned in which Ar:O2 gas mixtures will be applied to the rhizobial root halves to examine how rapidly a host plant will change its C allocation when the rhizobia bacteria are unable to provide N to their host. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? Depending on the progress that will be made in answering the questions described above, we will decide whether these experiments can be conducted in the next reporting period.
Impacts
What was accomplished under these goals?
We conducted experiments to evaluate how the carbon (C) allocation to different root symbionts is affected when the fungal partner is able to provide nitrogen (N) to its host plant, while acting as a direct competitor of N fixing root nodules. Interactions with arbuscular mycorrhizal (AM) fungi are generally seen as less costly for the host plant, and we hypothesized that the host plant would shift its carbon allocation to the fungal symbiont when this symbiont is able to provide N to its host. We studied this question in custom-made, three soil compartment systems, using a split root design and Medicago as the host plant. We compared the C allocation in four different split root systems with: (1) two non-inoculated root halves (Ø/Ø),(2) one non-inoculated root half and one inoculated with Rhizophagus irregularis (Ø/AM), (3) one non-inoculated root half and one inoculated with Ensifer meliloti (R/Ø), and (4) two inoculated root halves, one inoculated with Ensifer meliloti and one inoculated with Rhizophagus irregularis (R/AM). In addition, we compared systems in which the fungus either had or did not have access to an exogenous supply of 15N labeled ammonium chloride.The plants were later labeled with 13C labeled CO2, and after plant harvest, we analyzed the plants for biomass characteristics, 15N and 13C labeling, and gene expression. Nodulated root halves acted as strong C sinks and showed a significantly higher 13C labeling than the non-inoculated root halves in R/Ø systems, or the mycorrhizal root halves in R/AM systems. The C allocation to the nodulated root half, however, was significantly reduced when the fungus had access to an exogenously supplied N source, and did not differ from the C allocation into the AM root halves. This indicates that the fungal partner became a stronger competitor for host plant carbon when the fungus was able to provide N under N limiting conditions. The detailed results of these experiments are published in Plant, Cell and Environment (Kafle et al., 2018). Additional experiments are planned to further investigate this question, and to examine when this shift in carbon allocation occurs. Experiments with the goal to determine the total C costs for N benefit are currently ongoing. Objective 2. How does the nutrient demand of the host affect the carbon allocation strategy to both root symbionts? (80% Accomplished) We again used our custom-made, multi-compartment system to assess how the nutrient demand of the host affects C allocation to the root symbionts. We divided the root system of Medicago plants into two equal parts, and placed each root half into an individual soil compartment. One of the root halves was inoculated with the N fixing root symbiont Ensifer meliloti, while the other root half was inoculated with the AM fungus Rhizophagus irregularis. Ten weeks after transplanting, the nutrient demand conditions of the plants were varied by adding a modified Ingestad nutrient solution with combinations of low (L) or high (H) phosphate (P) or nitrogen (N) concentrations to both root compartments (LPLN, LPHN, HPLN, HPHN). Three weeks later, we labeled the plants with 13CO2, harvested the plants the next day, and determined biomass characteristics, N and P concentrations in the different plant tissues, root nodulation or mycorrhizal colonization, the gene expression of sucrose uptake transporters (SUT), and SWEETs (Sugars Will Eventually be Exported Transporters), and the 13C allocation to the different root halves. The results clearly demonstrated that a host plant changes its carbon allocation to different root symbionts depending on its nutrient demand. When the plants were under N demand, the plants allocated only between 19.7% (LPLN) to 23.3% (HPLN) of their fixed C to their fungal partners, but under high N supply conditions, the plants transferred between 29.9% (LPHN) to 35.4% (HPHN) to the AM root half. This indicates that the host plant changes its C allocation strategy to different root symbionts depending on its nutrient demand conditions. The expression patterns of SUT and SWEET transporters were consistent with these changes in C allocation, and demonstrated that several of these transporters play a role for the regulation of the C allocation to different root symbionts. The results were published in Plant, Cell, and Environment (Kafle et al., 2018). Objective 3. What are the important regulatory mechanisms that control the carbon allocation strategy? (20% Accomplished) We analyzed the expression levels of three plant sucrose transporters from the SUT family (MtSUT1-1, MtSUT2 and MtSUT4- 1) and seven transporters of the SWEET family (MtSWEET1b, MtSWEET6, MtSWEET9, MtSWEET11, MtSWEET12, MtSWEET15c, and MtSWEET15d). The expression levels of MtSUT2 and MtSUT4-1 were positively correlated to the C allocation to different symbiotic partners. These transporters are not symbiosis-specific transporters, and are expressed in non-inoculated roots, and in AM and nodulated roots. MtSUT1-1 encodes a H+ -sucrose symporter. The high transcript levels of MtSUT1-1 (particularly in AM roots), and its upregulation in AM roots when the fungus had access to an exogenous N source, supports a possible role of this transporter in phloem unloading towards AM-colonized sink roots. MtSUT4-1 could play a role in the release of stored C sources from the vacuole towards symbiotic root sinks, and the high correlation of its transcript levels with the observed carbon allocation pattern clearly suggests a role of this transporter in symbiotic carbon flux to both root symbionts. We also determined the expression of six SWEETs in the roots of Medicago truncatula after colonization with different root symbionts. In contrast to MtSWEET11, which was specifically expressed in root nodules, none of the other SWEETs showed a mycorrhiza-restricted induction. However, three of the SWEETs (MtSWEET1b, MtSWEET6, and MtSWEET15d) were upregulated in AM roots compared to control roots. Although MtSWEET1b and MtSWEET6 were also highly expressed in rhizobial roots, the downregulation of both transporters in tripartite interactions, in which the AM colonization was suppressed, suggest a potential role of both transporters for sugar transport in arbusculated cells. Similar to the changes in the gene expression patterns that were observed for the SUTs, the expression of MtSWEET12, MtSWEET15c, and MtSWEET15d were in agreement with the observed changes in C allocation to AM or rhizobial roots, and suggest that these transporters play an important role for the sucrose transport to symbiotic sink tissues. The fact that MtSWEET12, MtSWEET15c, and MtSWEET15d all showed similar changes in their expression patterns indicates some level of redundancy in the function of these transporters. Additional studies are currently underway to get more information about the localization and potential function of some of the SUT and SWEET transporters. Objective 4. How does the co-colonization of the root system with both symbionts affect the symbiotic benefit that each symbiotic partner is able to provide, and is there symbiotic dialogue between partners in tripartite interactions? (0% Accomplished) Nothing to report yet. First experiments to answer this question are ongoing. Objective 5. How will the inefficiency of one partner to provide N affect the carbon allocation strategy of the host, and is the host plant able to change its carbon allocation strategy depending on its demand? (0% Accomplished) Nothing to report yet. The first experiments to answer this question are currently ongoing. Objective 6. Does the competition with N-fixing symbionts in tripartite interactions affect the fungal nutrient allocation strategy in common mycorrhizal networks? (0% Accomplished) Nothing to report yet. Experiments are planned in the nearer future.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Kafle A, Garcia K, Wang X, Pfeffer PE, Strahan GD, Bucking H. 2018. Nutrient demand and fungal access to resources control the carbon allocation to the symbiotic partners in tripartite interactions of Medicago truncatula. Plant, Cell and Environment; doi: 10.1111/pce.13359.
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Kafle A, Garcia K, Peta V, Yakha J, Soupir A, B�cking H. 2018. Beneficial plant microbe interactions and their effect on nutrient uptake, yield, and stress resistance of soybeans. In: Soybean Biomass, Yield and productivity. Intechopen: doi: 10.5772/intechopen.81396
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Becquer A, Garcia K, Amenc L, Rivard C, Dor� J, Trives-Segura C, Szponarski W, Russet S, Baeza Y, Lassalle-Kaiser B, Gay G, Zimmermann SD, Plassard C. 2018. The Hebeloma cylindrosporum HcPT2 Pi transporter plays a key role in ectomycorrhizal symbiosis. New Phytologist 220: 1185-1199. DOI: 10.1111.nph.15281
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Guerrero-Gal�n C, Garcia K, Houdinet G, Zimmermann SD. 2018. HcTOK1 participates in the maintenance of K+ homeostasis in the ectomycorrhizal fungus Hebeloma cylindrosporum, which is essential for the symbiotic K+ nutrition of Pinus pinaster. Plant Signaling & Behavior, DOI: 10.1080/15592324.2018.1480845
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Guerrero-Galan C, Delteil A, Garcia K, Houdinet G, Sentenac H, Zimmermann SD. 2018. Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum. Environmental Microbiology 20: 1873-1887
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Kafle A, Wang X, Strahan G, Pfeffer P, B�cking H. 2017. Biological markets control resource exchange in tripartite interactions in legumes. International Conference on Mycorrhizae. July 30 to August 04. Prague, Czech Republic.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
B�cking H. 2017. Inter- and intraspecific diversity in the arbuscular mycorrhizal symbiosis and the consequences for the composition of arbuscular mycorrhizal communities. International Conference on Mycorrhizae. July 30 to August 04. Prague, Czech Republic.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
B�cking H. 2018. Beneficial plant microbe interactions a tool to increase agricultural production and improve environmental sustainability. Vitazyme Company Seminar. March 01 02, Kiev, Ukraine.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
B�cking H. 2018. Beneficial plant microbes and their effect on crop productivity. Midwest Cover Crops Council Annual Meeting, March 13-14. Fargo, ND.
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