Source: SOUTH DAKOTA STATE UNIVERSITY submitted to
PLANT GROWTH PROMOTION AND PROTECTION THROUGH BENEFICIAL PLANT MICROBE INTERACTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1013140
Grant No.
(N/A)
Project No.
SD00H642-18
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2017
Project End Date
Mar 22, 2021
Grant Year
(N/A)
Project Director
Bucking, HE.
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Biology & Microbiology
Non Technical Summary
Agricultural crop productivity is threatened by numerous stresses, including poor soil fertility and nutrient deficiency, drought, salinity, pathogens, and herbivores. The production of sufficient food resources for an increasing world population will only be possible when the agricultural productivity on declining arable land resources can be significantly increased. Currently, agricultural production systems depend on a continuous supply with nitrogen (N) and phosphate (P) fertilizers, because these nutrients most often limit plant growth. The extensive use of fertilizers in the past has left behind substantial environmental degradation, including the reduction in water quality and eutrophication of marine ecosystems, the development of photochemical smog and increasing concentrations of the greenhouse gas nitrous oxide. The use of N fertilizers, for example, is responsible for more than 30% of the non-renewable energy consumption, and for 70% of the greenhouse gas emissions in U.S. corn production. The high energy demand for the production process has led to rapidly escalating prices for N fertilizers in recent years, but the production of P fertilizer will become even more precarious in the long term, because P rock is a non-renewable resource and the current known reserves are expected to be depleted in 50-100 years. Therefore, the development of alternative strategies to improve the nutrient supply, and the abiotic and biotic stress resistance of important crop species should represent an urgent research priority. We will focus in this project on two overarching goals: to investigate the effect of (1) plant growth promoting endophytes and of (2) tripartite interactions (mutualistic interactions between plants, fungi and bacteria).Endophytes are microorganisms that reside inside their host plant without causing any disease symptoms. Many endophytes have plant growth promoting capabilities and our specific objectives include:Isolate and identify bacterial and fungal endophytes from crops that play an important role in South Dakota (corn, soybean, wheat) and that are grown under different stress environments (e.g. low soil fertility, salinity);Screen these endophytes for their plant growth promoting capabilities in vitro;Develop pipelines for the selection of suitable microbial candidates for different capabilities (microbial fertilizers, microbial pesticides);Test the capability of these endophytes to improve plant growth and yield in different stress environments; andIdentify the mechanisms that contribute to these plant growth benefits.The second overarching goal is to investigate tripartite interactions that play a key role for the nutrient uptake and yield efficiency of legumes, such as soybean. In tripartite interactions, the plant is colonized with two important groups of beneficial microorganisms: (1) nitrogen fixing Rhizobia bacteria that are able to provide the host with nitrogen, and (2) arbuscular mycorrhizal (AM) fungi that contribute to nutrient acquisition and abiotic and biotic stress resistance of their host. In general, plants gain more from tripartite interactions than from the inoculation with either symbiont, and the colonization with both symbionts leads to a synergistic effects. However, antagonistic responses can also be observed, but the mechanisms that control the benefit of these interactions for the host are largely unknown. This goal has the following specific objectives:Determine the carbon costs of N fixing and AM interactions per nutrient gain (particularly in terms of N);Examine whether the host plant changes its carbon allocation strategy to both root symbionts depending on its nutrient demand;Test whether and how the co-colonization of the root system with both symbionts affects the symbiotic benefit that each symbiotic partner is able to provide; andExamine whether the competition with N-fixing symbionts in tripartite interactions affects fungal nutrient allocation strategy in common mycorrhizal networks.The overall aim of the project is to contribute to a better understanding of how beneficial plant microbe interactions can be used to improve yield and stress resistance of the plant under low input conditions. We expect that the program will lead to the isolation of endophytic bacteria or fungi with commercial potential, and will improve our knowledge about how tripartite interactions can be used to improve the yield of legumes. The project will also provide training opportunities for undergraduate and graduate students in a biotechnological field in which trained professionals are in high demand.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1024099110340%
2032499110360%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
4099 - Microorganisms, general/other; 2499 - Plant research, general;

Field Of Science
1103 - Other microbiology;
Goals / Objectives
Goal 1. Plant growth promotion through bacterial or fungal endophytesEndophytes are defined as organisms that live inside plant hosts for at least part of their lives, without causing apparent disease symptoms in the host as a result of this colonization. Fungal and bacterial endophytes are nearly ubiquitous across all groups of vascular plants but there is a large biological diversity among endophytes, and it is not rare for some plant species to host hundreds of different endophytic species. Our understanding of the role of these organisms in ecosystems and agroecosystems is incomplete, since, regarding agroecosystems, research on endophytes has largely been limited to the development of biocontrol techniques.Nitrogen fixing bacterial endophytes. Atmospheric N is fixed by several bacterial phylogenetic groups, among them, members of the Rhizobiales have been largely studied and employed for improving legume production. However, other unrelated genera also have the ability to fix N. Azotobacter, Azospirillum, Klebsiella, Rhodobacter and others are some examples of non-rhizobia N-fixing bacteria. These microorganisms are found in a wide variety of habitats including free-living in soil and water, in actinorrhizal associations with woody plants, and as endophytes of many plants. In addition to their capability to fix gaseous nitrogen, many bacterial endophytes also have other plant growth promoting capabilities. For example, bacterial endophytes or bacteria in the root rhizosphere can play an important role in the P nutrition of the host plant. A high percentage of P in soils is trapped as inorganic P (iron, aluminum and calcium phosphates) or as organic P (phytates, organic molecules). Soil/root inoculations with P solubilizing bacteria can increase the solubilization of fixed soil P, and make it plant available. P solubilizing bacteria mobilize soil P through the production of organic acids, phosphatases, phytases, proton efflux, and extracellular oxidation of glucose. Bacterial endophytes can also affect plant growth by their capability to produce plant hormones, e.g. indole acetic acid, or improve the resistance of plants against fungal pathogens by the production of antimicrobial substances. There is evidence in the literature that demonstrates that endophytes can have a positive effect on basically all stresses that a plant can be exposed to (e.g. drought, salinity, N, P or potassium deficiency, fungal and bacterial plant pathogens, and insect herbivores).This goal of the project is to address the following specific objectives:Isolate and identify bacterial and fungal endophytes from crops that play an important role in South Dakota (corn, soybean, wheat) and that are grown under different stress environments (e.g. low soil fertility, salinity);Screen these endophytes for their plant growth promoting capabilities in vitro;Test the capability of these endophytes to improve plant growth and yield in different stress environments; andIdentify the mechanisms that contribute to these plant growth benefits.2. Plant growth promotion through tripartite interactionsLegumes, such as soybean, cowpea and Medicago, are among the most important crop species worldwide, and are grown on approximately 180 million ha, or 15% of the arable land on Earth. Legumes account for 27% of the world´s primary crop production, and 33% of the dietary N needs of humans. Despite their significance, improvements in legume crop yields have not kept pace with those of cereals. While the yield for wheat continuously increased, yield gains in the most significant legumes were much lower. An increase in the nutrient efficiency of legumes therefore represents an urgent research priority to ensure cost-effective and environmentally sustainable crop production in the future.Approximately 88% of legumes form tripartite interactions, and are simultaneously colonized with N-fixing bacteria and AM fungi. It is well known that these interactions can substantially contribute to the nutrient efficiency of legumes, and that tripartite interactions increase the fitness of both the host and the different root symbionts. It is also established that a synergy of benefits can occur, and that plants typically gain more from tripartite interactions, than from single inoculations with either symbiont. The N-fixing capability of rhizobia bacteria is often limited by the P availability and it has been shown that AM fungi by their positive effect on P uptake, can stimulate root nodulation, and N fixing activity. Similarly, nod signals produced by rhizobia bacteria have been shown to enhance AM colonization and both symbiotic interactions share parts of a common signal transduction pathway. However, negative effects have also been observed, and the prior inoculation by either rhizobia or AM fungi can limit the subsequent colonization by the other symbiont. It has been suggested that plants control the extent of root colonization by both symbionts by an auto-regulatory mechanism, possibly to limit the high carbon cost associated with these interactions. Our current knowledge about the resource exchange dynamics among the different partners in tripartite interactions are poorly understood. We seek to enhance our understanding of the ecological, physiological and molecular factors that determine the benefits (or lack thereof) of a simultaneous colonization by different species of competing N-fixing bacteria and fungal symbionts. This information will be key in the development of strategies and future attempts to simultaneously maximize both root symbionts for increased yield of legumes under low or reduced fertilizer input conditions.Our specific objectives include:Determine the carbon costs of N fixing and AM interactions per nutrient gain (particularly in terms of N);Examine whether the host plant changes its carbon allocation strategy to both root symbionts depending on its nutrient demand;Test whether and how the co-colonization of the root system with both symbionts affects the symbiotic benefit that each symbiotic partner is able to provide; andExamine whether the competition with N-fixing symbionts in tripartite interactions, affects the fungal nutrient allocation strategy in common mycorrhizal networks?By the year 2040 the demands of a growing human population are projected to require 40 and 20 million metric tons of additional N and P fertilizers, to meet global food production needs on declining arable land resources. This trend can be reversed but it will require the development of alternative strategies to industrial fertilizers or pesticides. There is growing evidence that the unique capabilities of microorganisms can be harnessed to increase nutrient availability, to enhance crop growth, and to protect against pathogens and pests. Endophytic bacteria and AM fungi both have the capability to increase the nutrient efficiency of their associated hosts and have the potential to serve as effective biofertilizers or biopesticides in environmentally sustainable agriculture. However, in order to use these strategies to their full potential, a better understanding of these plant growth promoting interactions is required.
Project Methods
Goal 1. Plant growth promoting endophytesObjective 1: Isolate and identify bacterial and fungal endophytes from crops that play an important role in South Dakota (corn, soybean, wheat) and that are grown under different stress environments (e.g. low soil fertility, salinity). Plant samples of different crops will be taken at three different time points during the growing season: in spring shortly after plant establishment, during growth, and shortly before harvest. We will emphasize crops of importance in SD, and crop plants that are grown in low fertile soils or other stress environments. Root, shoot and soil samples will be collected and prepared for metagenome analysis, endophyte isolation, or 15N natural abundance analysis. This technique is based on the relatively low natural abundance of 15N in atmospheric N2 compared to the higher natural abundance of 15N in the plant available N sources in the soil. Formass spectrometric analysis, plant samples will be homogenized and measured for their 15N content. We will use culture based techniques to isolate bacterial and fungal endophytes from the collected plants. The bacterial endophytes will be isolated by washing surface sterilized and cut plant samples with a saline solution and plating the wash solution onto plates filled with nutrient agar or N free Bridges and N free WAT4C media and incubatedfor several days. Fungal isolates will be isolated by plating cut plant material onto PDA agar. After species isolation and DNA extraction, the endophyte species will be identified by sequencing 16S or 18S amplicons and comparisonto the NCBI database.Objective 2: Screen these endophytes for their plant growth promoting capabilities in vitro. All endophytes will be tested for their plant growth promoting capabilities with established in vitro screening techniques (P solubilization, biosynthesis of plant growth hormones, suppression of fungal pathogens, 1-aminocyclopropane-1-carboxylate (ACC) deaminase production).Objective 3: Test the capability of these endophytes to improve plant growth and yield in different stress environments.To test the effect of the isolated endophyte strains on N nutrition and biomass production, we will conduct greenhouse studies with seedlings, or with adult plants. Short-term experiments with seedlings will be conducted in growth chambers that are constructed from Petri dishes or in commercially available magent boxes using these systemsthe root and shoot system of the plant can be continuously observed.These experiments will allow us to study and observe the effects of endophytes on plant growth parameters (root and shoot biomass e.g. by phytohormone production) or fungal pathogen suppression in planta. In addition, we will conduct pot experiments, in which the plants will be grown with or without endophyte inoculation.Objective 4: Identify the mechanisms that contribute to these plant growth benefits. We will sequence the whole genome of the most promising candidates (see objective 2 and objective 4). The whole genome will allow us to predict gene function, to identify target genes of interest (e.g. nif genes, ACC demaninase genes), and to study the expression of these genes in greater detail and under different conditions (e.g. under different in vitro conditions, or after inoculation in different plants).Goal 2.Tripartite interactionsObjective 1: Determine the carbon costs of N fixing and AM interactions per nutrient gain (particularly in terms of N). The C costs per N transferred will be determined in specialized growth pots with one plant (PC) and one fungal compartment (FC) filled with Turface. The FC will be separated from the PC with a double nylon mesh that allows the extraradical mycelium (ERM) of the fungus to grow into the FC, but not the roots. The plants will be inoculated with the AM fungus R. irregularis (AM) or either of two strains of Sinorhizobium meliloti, one with the capability (Fix+) and one without the capability (Fix-) to fix N, or with both symbionts simultaneously (AM and Fix+ or Fix-). The comparison with non-inoculated controls will allow us to measure the growth and nutritional benefits for the host by each root symbiont individually or after dual inoculation. The N gain of the plant through BNF or through the AM fungus will be determined after venting the PC with 15N2:O2 (Fix+, Fix-) or after addition of 4 mM 15N-NH4Cl to the FC (AM), respectively, and the carbon investment of the host will be measured after labeling of the shoot with 13CO2.Objective 2. Examine whether the host plant changes its carbon allocation strategy to both root symbionts depending on its nutrient demand. The carbon investment of the host into each root symbiont will be examined under various nutrient supply conditions after labeling of Medicago plants with 13CO2 in split root chambers. This will allow us to examine shifts in the C allocation to each partner in fully co-colonized root systems. One root half will be inoculated with AM or Fix+ or Fix-,whilethe other root half will be non-inoculated, or one half will be inoculated with AM and the other root half with Fix+ or Fix-. The plants will first be grown under low P or N supply conditions, but one week before the plants will be labeled with 13CO2 we will change the nutrient availability by a supply of P or of N or different combinations of both ranging from low, via moderate to high P or N demand conditions.Objective 3: Test whether and how the co-colonization of the root system with both symbionts affects the symbiotic benefit that each symbiotic partner is able to provide. The co-colonization of the root with two symbionts can have an effect on the nutritional benefits that each partner is able to provide. We will test these interactions in a split root system and will examine how the co-colonization of the root system with N fixing bacteria affects the transport of N or of P via the ERM or the fixation of N by N-fixing bacteria. These split root systems with one mycorrhizal root compartment (MC), one rhizobial root compartment (RC) and an additional FC, will allow us to control the nutrient supply to each individual partner in tripartite interactions individually and will provide us with an excellent opportunity to study how nutrient and carbon transport is controlled in tripartite interactions.Objective 4: Examine whether the competition with N-fixing symbionts in tripartite interactions, affects the fungal nutrient allocation strategy in common mycorrhizal networks. In contrast to the N-fixing symbionts, AM fungi do have access to additional carbon sources, and form AM interactions with multiple host plants simultaneously. CMNs can connect plants of the same or of different plant species and of different developmental stages, and plants interact and 'communicate' via these CMNs and exchange infochemicals with other plants. This experiment will study how the competition with N fixing bacteria in tripartite interactions changes the nutrient allocation strategy of an AM fungus. For these experiments, multi-compartment systems will be used in which two independently colonized plants share one CMN. One PC will contain a plant that is co-colonized with AM and Fix+. The Medicago plant in the other PC will only be colonized by the CMN or the CMN and Fix-, or the Medicago plant will be replaced by wheat or maize plants, both non-nodulated crops. Both PCs share a common FC to which then 33P or 15N will be added. This will allow us to measure how the presence of a tripartite interaction influences the allocation of resources within a CMN.

Progress 10/01/17 to 03/22/21

Outputs
Target Audience:PI transferred to University of Missouri and did not complete final report after several communication attempts. Filing this report to close out project. Changes/Problems:PI transferred to University of Missouri and did not complete final report after several communication attempts. Filing this report to close out project. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? PI transferred to University of Missouri and did not complete final report after several communication attempts. Filing this report to close out project.

Publications


    Progress 10/01/19 to 09/30/20

    Outputs
    Target Audience: Nothing Reported Changes/Problems:PI transferred to University of Missouri and did not complete reports after several communication attempts. Filing this report to close out project. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? PI transferred to University of Missouri and did not complete reports after several communication attempts. Filing this report to close out project.

    Publications


      Progress 10/01/18 to 09/30/19

      Outputs
      Target Audience:Target audiences for this project include (1) farmers and stakeholder groups, (2) scientific community, and (3) industrial partners. 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, February 04-05, 2019 at Iowa State University Oral presentation at the Ag Innovation Group Meeting, March 07, 2019, in Minot, North Dakota Oral presentation at the SD Soybean Research and Promotion Council Meeting, April 08, 2019, in Brookings, South Dakota Oral presentation about the importance of mycorrhizal fungi in crop and grazing systems. Soil Health Workshop, September 12, 2019, in Dickinson, North Dakota In addition, we 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, I presented the importance of agricultural research at South Dakota State University 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 for soil health. Scientific community: We provided updates about our research program to other scientists by publishing manuscripts in peer reviewed journals (Raths et al., 2019a, b; Peta et al., 2019, Ma et al., 2019; Cope et al., 2019, Chen et al., 2019), and by publishing review chapters (Kafle et al., 2019) (see information below). In addition, lab members shared their results at two international conferences (ICOM 10 in Merida, Mexico; Rhizosphere 5 in Saskatoon, Canada). The following poster or oral presentations were provided at these conferences: Poster presentation: Soupir A, Peta V, Bücking H. 2019. Finding the needle in a haystack - the development of microbial fertilizers or pesticides for environmentally sustainable agriculture. Rhizosphere 5, Saskatoon, Canada, July 11, 2019. Oral presentation: Peta V, Soupir A, Bücking H. 2019. The plant microbiome of Brassica carinata and its potential to increase plant growth and yield. Rhizosphere 5, Saskatoon, Canada, July 11, 2019. Oral presentation: 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. Oral presentation: 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. Oral presentation: 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. Oral presentation: 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. Industrial partners: We continue to collaborate with two industrial partners, Indigo and Novozymes, with the goal to identify microorganisms with commercial potential that could serve as microbial fertilizer or pesticide. We provide regularly updates about our research progress to our industrial partners via video conferences. In addition, I traveled with two Ph.D. graduate students (Alex Soupir and Vincent Peta) to Indigo in Boston and provided an oral presentation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for two undergraduate students (Corbin Ketelsen, Dylan Blomme), two M.S. students (Rachel Raths, and Jeffery Bartel), three Ph.D. students (Jaya Yakha, Alex Soupir, and Vincent Peta), and one postdoctoral associate (Kevin Cope). The students were trained in experimental design, data and statistical analysis, and the writing of scientific reports or publications in peer reviewed journals. The students also presented their work orally or as posters at international conferences, and developed sections for book chapters. The postdoctoral associate is involved in the ongoing experiments, has submitted a postdoctoral fellowship application under my guidance, and two publications (one publication was selected for the cover of Plant Cell). The postdoctoral scientist was trained for his first faculty interview at Utah State University. Unfortunately, he was not selected for the position, but he is currently working on multiple publications that should further improve his competitiveness for faculty positions. How have the results been disseminated to communities of interest?Journal and review articles were published in peer-reviewed journals, and presentations were made to different audiences. What do you plan to do during the next reporting period to accomplish the goals?Plant growth promotion through bacterial or fungal endophytes We will continue our efforts to identify endophytes and other microorganisms with plant growth promotion potential from different crop species in SD, and will continue our collaboration with different industrial partners to evaluate the commercial potential of these bacterial or fungal isolates. In addition, we started another collaboration project with the industrial partner Indigo with the goal to test the effect of our isolates, and of isolates from our industrial partner on arbuscular mycorrhizal communities in the soil. Objective 1: Isolate and identify bacterial and fungal endophytes from crops that play an important role in South Dakota (corn, soybean, wheat) and that are grown under different stress environments (e.g. low soil fertility, salinity). We are not planning to isolate new bacteria until the screening of the bacterial strains in our culture collection is completed. However, if collaboration partners provide us with plants that show unique traits in stress environments, we will isolate additional bacteria for our culture collection. Objective 2: Screen these endophytes for their plant growth promoting capabilities in vitro. We will continue to screen bacteria in our culture collection for their plant growth promoting capabilities. These screening tests typically include testing bacteria for their ability to synthesize IAA, fix gaseous nitrogen, solubilize recalcitrant phosphate, and inhibit the growth of fungal pathogens. Objective 3: Test the capability of these endophytes to improve plant growth and yield in different stress environments. We were able to isolate several promising phosphate solubilizers from corn plants, and subsequently found that these strains were able to promote the growth of soybean plants when phosphate was supplied as plant unavailable calcium phosphate. We are planning to test these microorganisms on a broader range of crops, including wheat and corn, also grown under phosphate deficient conditions. We will first conduct greenhouse experiments with these promising candidates, and compare the biomass characteristics of the plants with our positive control strain (Pseudomonas aeruginosa) and with negative controls (bacteria from the same genera, but with no phosphate solubilization activity on PVK medium). Objective 4: Identify the mechanisms that contribute to these plant growth benefits. The phosphate solubilizing strains showed differences in their release of organic acids, and the genome sequencing of Tr3R3 and M2R1 revealed interesting candidates that could be involved in the phosphate solubilization activity of both strains (e.g. alkaline phosphatase, and a glycerophosphodiester phosphodiesterase). We are planning to conduct experiments to study the regulation of these candidate genes in greater detail. For example, we are interested to learning about the phosphate supply conditions under which these genes are up-regulated or down-regulated, and whether the gene expression activity is correlated to the phosphate solubilization activity under these conditions. Plant growth promotion through tripartite interactions We will focus on tripartite interactions and will continue our experiments to evaluate carbon to nutrient exchange processes in these interactions. Tripartite interactions play a crucial role for the productivity of different legume crops and a better understanding of the resource exchange processes in these interactions, can provide us with important insights in how the nutritional benefits of these beneficial root symbioses can be maximized. Objective 1: Determine the carbon costs of N fixing and AM interactions per nutrient gain (particularly in terms of N). We hoped to answer this question with experiments in which 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 over longer time periods to N15. Objective 2: Examine whether the host plant changes its carbon allocation strategy to both root symbionts depending on its nutrient demand. We are interested in exploring the conditions and timing under which the host plant changes its carbon allocation strategy. For example, our previous results have 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: Test whether and how the co-colonization of the root system with both symbionts affects the symbiotic benefit that each symbiotic partner is able to provide. 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 4: Examine whether the competition with N-fixing symbionts in tripartite interactions, affects the fungal nutrient allocation strategy in common mycorrhizal networks. We conducted an experiment to answer the question 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? Plant growth promotion through bacterial or fungal endophytes Objective 1: Isolate and identify bacterial and fungal endophytes from crops that play an important role in South Dakota (corn, soybean, wheat) and that are grown under different stress environments (e.g. low soil fertility, salinity). (75% Accomplished) We isolated 70 bacteria from corn plants: 20 from the root rhizosphere, 22 from the bulk soil, and 28 were endophytic bacteria. We examined the strains for their plant growth promoting capabilities, and particularly for their ability to solubilize phosphate. Prairie cordgrass is a perennial grass species native to SD, and it is adapted to high salinity. We isolated 60 endophytes from different tissues of prairie cordgrass plants growing at a site with a very high salt concentration to identify bacterial endophytes that are adapted to high salt stress. Objective 2: Screen these endophytes for their plant growth promoting capabilities in vitro. (30% Accomplished) The isolated microorganisms or endophytes were screened for different plant growth promoting capabilities, including their ability to: 1) solubilize recalcitrant phosphate, 2) fix gaseous nitrogen, 3) produce plant growth hormones such as indole acetic acid, 4) grow under high salinity, and 5) suppress different fungal pathogens. The 70 corn isolates were first tested on Pikovskaya (PVK) agar for their ability to solubilize phosphate, and 15 isolates tested positive. We sequenced the 16S rRNA of these bacteria, together with 6 isolates that were negative on PVK agar. The following genera were identified from the sequenced isolates: Pantoea, Paenibacillus, Bacillus, Ochrobactrum, Kosakonia, Curtobacterium, Enterobacter, and Klebsiella. Based on PVK clearing zones and genetic variability, 7 isolates were further tested for phosphate solubilization compared to Pseudomonas aeruginasa ATCC 27853 (positive control with known phosphate solubilization activity). Using CaPO4 as phosphate source, one Kosakonia (Tc3So2), Enterobacter (Tr3R3), and Raoultella (M2R1) strain were able to solubilize a statistically (P≤0.05) greater amount than all the other isolates, including the positive control. These strains also tested positive for nitrogen fixation and indole-3-acetic acid (IAA) biosynthesis. From prairie cordgrass, 13 endophytes were able to maintain growth when exposed to high salt concentrations, or were able to grow better under salt than under control conditions. These strains belong to the genera Bacillus, Pantoea, Agrobacterium, Pseudomonas, and Brevibacillus. Objective 3: Test the capability of these endophytes to improve plant growth and yield in different stress environments. (50% Accomplished) We studied the effects of different phosphate solubilizing bacteria on the growth of soybean plants under low P levels (phosphate supplied as calcium phosphate that is not plant available). Three isolates had a higher ability to solubilize phosphate than the positive control strain Pseudomonas aeruginosa. Tr3R3, M2R1, and Tc3So2 increased soybean root biomass and changed the root architecture of soybean plants. In one experiment Tr3R3 also led to a statistically significant increase in shoot biomass. We tested the effect of salt tolerant endophytes on the growth of wheat and prairie cordgrass under high salinity. Contrary to our expectations, none of the endophytes provided growth benefits for wheat under high soil salinity, and instead led to growth depression. A repetition of this experiment is currently ongoing. A trial with prairie cordgrass failed due to a low seed germination rate. Objective 4: Identify the mechanisms that contribute to these plant growth benefits. (30% Accomplished) To identify potential pathways by which phosphate solubilizing bacteria promote plant growth, we assessed the release of organic acids from these bacteria. Compared to the positive control (Pseudomonas aeruginosa), the three phosphate solubilizers (M2R1, Tr3R3, and Tc3So2) showed different organic acid profiles. The control strain released high levels of malic acid, but low levels of succinate acid, while the three corn isolates showed the opposite behavior. Whole genome sequencing of two of the endophytes revealed genes encoding an acid and an alkaline phosphatase, and a glycerophosphodiester phosphodiesterase that can all contribute to phosphate solubilization. Plant growth promotion through tripartite interactions Objective 1: Determine the carbon costs of N fixing and AM interactions per nutrient gain (particularly in terms of N). (60% Accomplished) Multi-compartment systems were used to evaluate how carbon allocation to different root symbionts changes when the fungal partner is able to provide nitrogen to its host plant in direct competition with N fixing root nodules. We found that the fungal partner becomes a stronger competitor for host plant carbon when the fungus had access to N. When the fungus is competing with rhizobia bacteria, the host plant transfers more carbon to the fungal partner when the fungus has access to an exogenous nitrogen source. We conducted a similar experiment in which we compared N transport from the arbuscular mycorrhizal fungus to its host plant, when the fungus is in competition with rhizobia that can fix gaseous nitrogen (fix +) versus rhizobia that form root nodules but don't fix nitrogen (fix -). When the fungus competes with fix + rhizobia, N is transferred by the fungus to the root, but is not transferred across the mycorrhizal interface to the host. The host plant allocates more carbon to root halves that are colonized with fix + rhizobia than to fix - root halves, and a N supply to the fungal symbiont increases the carbon allocation to the arbuscular mycorrhizal colonized root halves. Objective 2: Examine whether the host plant changes its carbon allocation strategy to both root symbionts depending on its nutrient demand. (75% Accomplished) No further experiments were conducted in the reporting period. Objective 3: Test whether and how the co-colonization of the root system with both symbionts affects the symbiotic benefit that each symbiotic partner is able to provide. (10% Accomplished) We used multi-compartment systems to compare the following systems: 1) both root halves non-inoculated, 2) one root half non-inoculated and one root half inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis, 3) one root half non-inoculated and one root half inoculated with rhizobia, and 4) one root half inoculated with the arbuscular mycorrhizal fungus and one root half inoculated with rhizobia bacteria. We provided the fungus with access to 15NH4Cl to test whether the transport of N from the fungus to the host is affected by the competition with rhizobia bacteria. As reported above, arbuscular mycorrhizal fungi transfer more N to plant hosts that are colonized with fix - strains, than to host plants that are colonized with fix + strains, and are better supplied with N. This indicates that under these conditions the fungus transfers other nutrients across the mycorrhizal interface to successfully compete with rhizobia bacteria for host plant carbon. We also found that the N fixing activity by fix + rhizobia is increased in systems that are also colonized with an arbuscular mycorrhizal fungus. Objective 4: Examine whether the competition with N-fixing symbionts in tripartite interactions, affects the fungal nutrient allocation strategy in common mycorrhizal networks. (0% 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 observed cross contaminations in our experiments. We are currently trying to design experiments through which this question can be answered.

      Publications

      • 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: Submitted Year Published: 2020 Citation: Peta V, Raths R, B�cking H. 2020. Novoherbaspirillum sperare gen. nov. sp. nov., a novel species of the Oxalobacteraceae. International Journal of Systematic and Evolutionary Microbiology, submitted.
      • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Raths R, Peta V, B�cking H. 2020. Massilia arenosa sp. nov., a novel addition to the Massilia genus, isolated from the soil of a cultivated maize field. International Journal of Systematic and Evolutionary Microbiology, under revision.
      • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Peta V, Raths R, B�cking H. 2020. Draft genome sequence of OM1, a potential novel bacterial species isolated from farm soil. AMC Microbiological Resource Announcement, under revision.
      • 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.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: Peta V, Raths R, B�cking H. 2019. Draft genome sequence of Massilia hortus sp. nov., a novel bacterial species of the Oxalobacteraceae family isolated from garden soil. ASM Microbiological Resource Announcement 8 (32): e00377-19; DOI; 10.1128/MRA.00377-19.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: Raths R, Peta V, B�cking H. 2019. Draft genome sequence of Duganella sp. strain DNO4, isolated from cultivated soil. ASM Microbiological Resource Announcement 8 (32): e00848-19; DOI: 10.1128/MRA.00848-19.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: Raths R, Peta V, B�cking H. 2019. Draft genome sequence of Massilia sp. Strain MC02, isolated from a sandy loam maize soil. ASM Microbiological Resource Announcement 8 (32): e00410-19; DOI: 10.1128/MRA.00410-19.
      • 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, November 01.
      • 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, 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, North Dakota, 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 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 Mycorrhiza, Merida, Mexico, July 02.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: B�cking H. 2019. Fair trade in beneficial plant microbe interactions. Indigo, Boston, May 14.
      • 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, 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, North Dakota, March 07.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Soupir A, Peta V, B�cking H. 2019. Finding the needle in a haystack  the development of microbial fertilizers or pesticides for environmentally sustainable agriculture. Rhizosphere 5, Saskatoon, Canada, July 11.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Raths R, Soupir A, Peta V, B�cking H. 2019. Phosphate solubilizing bacteria  a novel strategy to improve the phosphate nutrition of crops. 2019 Soil Health Conference, Iowa State University, February 04-05.


      Progress 10/01/17 to 09/30/18

      Outputs
      Target Audience:Target audiences for this project include (1) farmers and stakeholder groups, (2) scientific community, and (3) industrial partners. 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 presentations and leading discussion groups and workshops at the following meetings: Annual Meeting of the Midwest Cover Crop Council: March 13-14, 2018. Fargo, ND Meetings with Ukrainian producer groups: March 01, 2018. Kiev, Ukraine. Meeting with Ukrainian producer groups: May 31, 2018. Kansas City, Kansas. Soil health workshop at the Dickinson Research Extension Center (invited by Douglas Landblom, Research Center Beef Cattle Specialist): September 12, Dickinson, North Dakota. Ag Horizon Conference in Pierre, South Dakota (2018). Primary focus in these presentations was on the application potential of microbial fertilizers, and education of producers about the significance of arbuscular mycorrhizal communities for soil health. Scientific community: We provided updates about our research program to other scientists by publishing manuscripts in peer reviewed journals (Kafle et al., 2018; Neupane et al., 2018), and by publishing two review chapters (Kafle et al., 2018, Becquer et al., 2018) (see information below). Industrial partners: We continue to collaborate with the two industrial partners, Indigo and Novozymes, with the goal to identify microorganisms with commercial potential that could serve as microbial fertilizer or pesticide. Both industrial partners visited the SDSU campus in 2018, and we provided them with an update about the progress on our collaborative projects, discussed other scientific projects with interest for them, and invited other scientists from the SDSU campus to these discussions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for two undergraduate students (Jackson Pond, Marie Zander), four Ph.D. students (Arjun Kafle, Jaya Yakha, Alex Soupir, and Vincent Peta), and two postdoctoral associates (Kevin Garcia, Kevin Cope). The students were trained in experimental design, data and statistical analysis, and the writing of scientific reports or publications in form of posters at conferences, publications in peer reviewed journals, and to develop summaries for book chapter publications. One postdoctoral associate who was trained on this project 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 second postdoctoral associate has recently joined South Dakota State University, and is involved in the ongoing experiments, has submitted a postdoctoral fellowship application under my guidance, and is currently preparing the first publications (one review article has recently been submitted to Agronomy). For more information about other training opportunities that the project provided, see more information under target audiences. How have the results been disseminated to communities of interest?Journal and review articles were published in peer-reviewed journals, and presentations were made to different audiences. What do you plan to do during the next reporting period to accomplish the goals?We will continue our efforts to identify endophytes and other microorganisms with plant growth promotion potential from different crop species in SD, and will continue our collaboration with different industrial partners to evaluate the commercial potential of these bacterial or fungal isolates. In addition, we will focus on tripartite interactions and will continue our experiments to evaluate carbon to nutrient exchange processes in these interactions. Tripartite interactions play a crucial role for the productivity of different legume crops and a better understanding of the resource exchange processes in these interactions, can provide important insights how nutritional benefits of these beneficial root symbioses can be maximized. We are planning to publish several papers, and to contribute to conferences and other meetings, when invited.

      Impacts
      What was accomplished under these goals? Plant growth promotion through bacterial or fungal endophytes Objective 1: Isolate and identify bacterial and fungal endophytes from crops that play an important role in South Dakota (corn, soybean, wheat) and that are grown under different stress environments (e.g. low soil fertility, salinity). (50% Accomplished) In the last growing season, we isolated endophytes from multiple plant species, including wheat, corn, soybeans, and prairie cordgrass. Prairie cordgrass is a perennial grass species, native to SD, and it is highly adapted to high salinity. For example, we isolated endophytes from different tissues of cordgrass that is growing at a site with a very high salt concentration, to identify bacterial endophytes that are particularly adapted to high salt stress. In addition, we identified endophytes from corn, and are particularly interested in their capability to solubilize phosphate. Objective 2: Screen these endophytes for their plant growth promoting capabilities in vitro. (30% Accomplished) The endophytes were screened for different plant growth promoting capabilities, including their ability (1) to solubilize recalcitrant phosphate, (2) to fix gaseous nitrogen, (3) to produce plant growth hormones such as indole acetic acid, (4) to grow under high salinity, and (5) to suppress different fungal pathogens. We also screened the ability of the bacterial isolates from prairie cordgrass to grow in liquid medium at a range of different salt concentrations, and found several endophytes that grow better at high salt concentrations than without salt. These endophytes will enter a planned growth response experiment with two wheat cultivars under salt stress. In addition, isolates from corn were screened for their ability to solubilize phosphate, and from these tests different isolates were identified that showed the same or higher potential to solubilize phosphate than a reference strain with known P solubilizing activity. Objective 3: Test the capability of these endophytes to improve plant growth and yield in different stress environments. (10% Accomplished) We conducted an experiment with soybean plants that were grown under low P conditions (phosphate was supplied as calcium phosphate that is not plant available), and found that one bacterial isolate increased plant growth and contributed to changes in the root architecture of soybean plants. These experiments will be repeated with different plant species to test the range of host plants that are positively affected. Other experiments to test the effect of endophytes on the salt resistance of prairie cordgrass and wheat are currently in planning. Objective 4: Identify the mechanisms that contribute to these plant growth benefits. (0% Accomplished) Nothing to report Plant growth promotion through tripartite interactions Objective 1: Determine the carbon costs of N fixing and AM interactions per nutrient gain (particularly in terms of N). (40 % Accomplished) Custom-made multi-compartment systems were used to evaluate how the carbon allocation to different root symbionts changes when the fungal partner is able to provide nitrogen to its host plant, and is in direct competition with N fixing root nodules. We studied C allocation in four different systems with soybeans: (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). The study also compared systems in which the fungus had access or had no access to an exogenous supply of N. We found that the fungal partner becomes a stronger competitor for host plant carbon when the fungus had access to N. These results are published in Plant, Cell and Environment (Kafle et al., 2018). Additional experiments are planned to further investigate this question. Objective 2: Examine whether the host plant changes its carbon allocation strategy to both root symbionts depending on its nutrient demand.(75 % Accomplished) We conducted experiments in custom-made multi-compartment systems using a split root design. The root system of the Medicago plants was divided into two equal parts, and each root halve was placed into an individual soil compartment. One of the root halves was inoculated with rhizobia bacteria, while the other root half was inoculated with the arbuscular mycorrhizal 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 (LPLN, HPLN), the plants allocated significantly more carbon to the rhizobial root halves, while when the plants had access to N, more carbon was allocated to the root halves that were colonized with the arbuscular mycorrhizal fungus. The expression pattern of SUT and SWEET transporters was consistent with these changes in carbon allocation, and demonstrates that several of these transporters are involved regulating the carbon allocation to different root symbionts. The results were published in Plant, Cell, and Environment (Kafle et al., 2018). Objective 3: Test whether and how the co-colonization of the root system with both symbionts affects the symbiotic benefit that each symbiotic partner is able to provide. (10 % Accomplished) Experiments to answer this question are currently ongoing. We used custom-made multi-compartment systems and compared the following systems: 1) both root halves non-inoculated, 2) one root half non-inoculated and one root half inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis, 3) one root half non-inoculated and one root half inoculated with rhizobia, and 4) one root half inoculated with the arbuscular mycorrhizal fungus and one root half inoculated with rhizobia bacteria. We labeled the plants with 15N2 gas to test whether the ability to fix gaseous N and to transfer this N to the host plant is affected by the competition with an AM fungus, or we provided the fungus with access to 15NH4Cl to test whether the transport of N from the fungus to the host is affected by the competition with rhizobia bacteria. All systems were labeled with 13CO2 to follow the carbon allocation to different root halves in these systems. The plants are harvested and we are currently analyzing the data of these experiments. If successful, the results of the experiment will allow us to determine whether root symbionts change their nutrient allocation strategy when they compete with other root symbionts for the carbon supply from the host. Objective 4: Examine whether the competition with N-fixing symbionts in tripartite interactions, affects the fungal nutrient allocation strategy in common mycorrhizal networks. (0 % Accomplished) Nothing to report.

      Publications

      • Type: Book Chapters Status: Published Year Published: 2018 Citation: Becquer A, Guerrero-Gal�n C, Eibensteiner JL, Houdinet G, B�cking H, Zimmermann SD, Garcia K. 2018. The ectomycorrhizal contribution to tree nutrition. In: Molecular physiology and biotechnology of trees. Franscisco C�novas (ed.) Advances in Botanical Research, Vol. 89
      • 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. 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.
      • 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. May 31, 2018. Kansas City, KS
      • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Eibensteiner J, Garcia K, B�cking H. 2018. Do arbuscular mycorrhizal interactions and bacterial endophytes have an effect on wheat root disease? Annual Meeting of the North Central Branch of the American Society for Microbiology, September 28-29. Mankato, MN.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: B�cking H. 2018. Fair trade in beneficial plant microbe interactions. University of Wisconsin, December 11. Madison, WI.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: B�cking H. 2018. Beneficial plant microbe interactions and their potential as microbial fertilizers and pesticides in environmentally sustainable agriculture. Ag Horizons Conference. November 28. Pierre, SD.
      • 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: Kafle A, Garcia K, Wang X, Pfeffer PE, Strahan GD, B�cking 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: Journal Articles Status: Published Year Published: 2018 Citation: Neupane A, Feng C, Feng J, Kafle A, B�cking H, Lee Marzano S-Y. 2018. Metatranscriptomic analysis and in silico approach identified mycoviruses in the arbuscular mycorrhizal fungus Rhizophagus spp. Viruses 10(12): 707; doi: 10.3390/v101020707