Source: MICHIGAN STATE UNIV submitted to NRP
SOLVING THE MYSTERY OF ALFALFA AUTOTOXICITY: CAUSES AND SOLUTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031466
Grant No.
2023-70005-41079
Cumulative Award Amt.
$946,349.00
Proposal No.
2023-05909
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
Aug 31, 2026
Grant Year
2023
Program Code
[AFRP]- Alfalfa and Forage Program
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
PLANT SOIL MICROBIAL
Non Technical Summary
Autotoxicity is a type of intraspecific allelopathy in which alfalfa releases compounds that aretoxic to new alfalfa seedlings, causing poor germination or seedling death. Autotoxicityprevents renovation of thinning alfalfa stands and delays replanting and is widely recognized as asignificant challenge to alfalfa establishment. However, it is a complex problem with many poorlyunderstood interacting factors and has resisted an easy solution. Our overall research goal for thisproposal is to improve understanding of what causes autotoxicity in alfalfa so we can betterunderstand how it can be mitigated. Our specific research goals are to use a previously developedsoil bioassay to direct identification of compounds associated with autotoxic or non-toxic soils,elucidate how the shoot and root microbiome influence production and dissipation of theseautotoxins, investigate the potential influence of nutrient stress on production of autotoxins in rootexudates and plant tissue, initiate breeding of less autotoxic and more tolerant alfalfa cultivars, andengage farmers in extension efforts to understand and reduce yield losses caused by autotoxicity.This work will take advantage of technological advances in study of metabolomics, genomics, andthe microbiome to take a fresh look at the causes of autotoxicity. Improved understanding of *why*alfalfa becomes autotoxic will ultimately drive development of better management tools to reduceautotoxicity, improved alfalfa establishment, and better alfalfa cultivars, and therefore meet theASAFS goal of increasing alfalfa forage yields and reducing the cost of production.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2051640106067%
2021640108133%
Knowledge Area
205 - Plant Management Systems; 202 - Plant Genetic Resources;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1060 - Biology (whole systems); 1081 - Breeding;
Goals / Objectives
Autotoxicity is a type of intraspecific allelopathy in which alfalfa releases compoundsthat damage new alfalfa seedlings, with impacts including establishment failure or low lifetimestand productivity. Our overall research goal is to improve understanding of what causesautotoxicity in alfalfa and how it can be mitigated. This work is 100% applied to ASAFS ProgramPriority #1, "Increasing alfalfa forage and seed yields and forage quality through improvedmanagement practices, plant breeding, and other strategies to reduce biotic and abioticstresses and costs of production." Our specific research goals are to identify compounds thatcause autotoxicity, elucidate how the root microbiome and soil microbiology influencedevelopment and dissipation of these autotoxins, investigate the influence of nutrient stress onproduction of autotoxins, initiate breeding of tolerant or less autotoxic alfalfa cultivars, andengage alfalfa producers in extension education about autotoxicity. Results will ultimately helpalfalfa producers improve effectiveness of establishment and provide improved germplasm forbreeding efforts.Obj. 1. Identify causative compounds responsible for alfalfa autotoxicity.Obj. 2. Evaluate impact of environmental (alfalfa microbiome and soil biology) andmanagement factors (soil fertility) on autotoxicity.Obj. 3. Evaluate and select alfalfa genotypes for reduced autotoxin production andimproved autotoxin tolerance.Obj. 4. Engage alfalfa producers in extension education about autotoxicity.
Project Methods
Obj. 1: Identify causative compounds responsible for alfalfa autotoxicity. Sub-obj. 1.1. Identify autotoxic compounds in field soil. We will build from previous knowledge of tissue extract autotoxicity as described in the background section to address identification of autotoxic compounds as they are found in field soil.. Using our autotoxicity soil bioassay, we will collect soil samples from three alfalfa fields identified with high autotoxicity and three alfalfa fields with low autotoxicity. Untargeted metabolomics will be performed at the West Coast Metabolomics Center at UC Davis to identify and provide relative concentrations of all measurable metabolites including polyphenol-flavonoids from ten soil samples from each of the six field sites Metabolites that are more prevalent in high than in low autotoxicity soils will be targeted for further study in Expt. 2.Sub-obj. 1.2. Quantify presence of targeted metabolites from sub-objective 1.1 in autotoxic alfalfa plant tissue and root exudates. The metabolic profiles of vegetative plant extracts and root exudates will be analyzed using a hybrid soil hydroponic system to validate that alfalfa is the source of autotoxicity in the field soils from Expt. 1 using a commercial alfalfa cultivar previously determined to produce high autotoxicity. Vegetative extracts will be created and extracts and exudates will be stored at -80?F until they can be analyzed. Targeted metabolite analysis will be run on the root exudates and vegetative extracts through the MSU metabolomics core facility. Target metabolites will be determined from the identified compounds of interest from untargeted metabolomic testing of field soils and used to narrow the field of metabolites of interest in above-ground and below-ground tissues and direct our efforts in Experiment 3.Sub-Obj. 1.3. Determine autotoxicity thresholds in field soil for identified compounds. Once a group of likely suspect compounds is confirmed by targeted metabolomics, we will use the autotoxicity soil bioassay to identify threshold concentrations for inhibition by isolated compounds and mixtures of compounds. This will be a series of experiments with treatments informed by results from Expt. 2. Different concentrations of autotoxic compounds, mixtures of autotoxic compounds, or deionized water will be applied to control soil in the autotoxicity bioassay. This step will help us understand how much autotoxin is required to reduce alfalfa seedling performance and inform specific experiment designs and synthesis for Objectives 2 and 3. Obj. 2: Evaluate impact of environmental (alfalfa microbiome and soil biology) and management factors (soil fertility) on autotoxicitySub-Obj. 2.1. Characterize microbiome communities in autotoxic and non-toxic field soils. We will characterize the microbiome composition of the six bulk field soils identified with high autotoxicity or low autotoxicity from the bioassay in objective 1 by 16S rRNA and ITS amplicon sequencing to define the bacterial and fungal soil communities associated with these soils. We will compare the microbial communities in the bulk soil of high and low autotoxicity soils to identify microbes consistently associated with soils with greater autotoxicity. To identify those microbes that may help degrade autotoxins, we will instead focus on those microbes that are consistently found in soils with lower autotoxicity.Sub-Obj. 2.2. Determine whether autotoxins exuded during primary nutrient stress reduce growth of replanted alfalfa. In the stressed phase 1, alfalfa will be grown in the greenhouse in rhizoboxes as described in Objective 1, Expt. 2. The rhizoboxes will be filled with topsoil modified to four different fertility treatments: phosphorus deficiency, excess phosphorus, potassium deficiency, or nutrient sufficiency. After 90 d, the rhizobox will be opened and the taproot and other visible roots will be removed, leaving as much soil behind as possible. In the unstressed phase 2, remaining soil from Expt. 2 will be kept in its respective pots and a second round of alfalfa will be planted into the same soils and grown for 90 d. The growth and development of this "replanted" alfalfa will serve as another measure of the autotoxicity caused by the original fertility treatments and validate the results of the bioassay and targeted metabolomics at a more ecologically relevant scale.Sub-obj. 2.3. Determine whether soil microbial communities associated with nutrient stressed plants contain microbes capable of degrading autotoxins. We will characterize the microbiome composition of the rhizobox soils from Expt 2.2 and determine the presence of autotoxin-degrading microbes identified in Expt. 1. We will also test the soil fertility of the six bulk field soil samples from Expt. 1. Obj. 3. Evaluate and select alfalfa genotypes for reduced autotoxin production and improved autotoxin tolerance. In Year 1 of the project, we will evaluate half-sibling families from genetically diverse breeding populations from the Cornell University alfalfa breeding program for both autotoxic potential and response to autotoxicity, paired in a limited number of combinations with other half-sib families in an incomplete diallel design. 20-30 plants from each half-sib family will be grown in the greenhouse to produce plant extracts and tissue for genotyping. Then, 20-30 seeds from each half-sib family will be exposed to plant extracts from a limited set of other families and evaluated for germination, seedling growth, and other measures of response to autotoxicity. These data will be used to identify four groups of half-sib families: (1) high autotoxic compound production (HIGH), (2) low autotoxic compound production (LOW), (3) susceptibility to autotoxic compounds (SUS), and (4) tolerance to autotoxic compounds (TOL). Half-sib families in each of these groups will be intermated to form the base populations for a divergent recurrent selection program.In Years 2 and 3, half-sib families from the HIGH and LOW groups will be grown for extraction purposes and half-sib families from the SUS and TOL groups will be exposed to the plant extracts from a limited subset of HIGH and LOW families in an incomplete factorial design. In Year 3 of the project, bulked cycles of selection will be evaluated for autotoxic production (HIGH and LOW) and susceptibility (SUS and TOL) using targeted metabolomics and the autotoxicity bioassay (see methods in Obj. 1). In each cycle of selection, the performance of half-sib families will be evaluated using a general-specific mixing ability model where general mixing ability (GMA) represents the autotoxicity outcome of a half-sib family across all tested partners, and specific mixing ability (SMA) represents the outcome of specific pairs of families. In addition to identifying families with high/low autotoxicity and susceptible/tolerant families, the relative magnitude of GMA and SMA will indicate the degree of specificity of autotoxicity interactions within tested populations. Starting in Year 2, marker-derived genomic relatedness matrices will be incorporated in the models to increase prediction accuracy for the performance of untested family pairs. Results of the Year 3 bioassay will be used to calculate response to selection (R) and narrow-sense heritability (h^2).Obj. 4. Engage alfalfa producers in extension education about autotoxicity. Activities include a central hub for project information (MSU Forage Connection at www.forage.msu.edu) and a range of extension outputs including bulletins, factsheets, webinars, videos, presentations at producer meetings, and podcasts.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Crop and vegetable growers, extension educators, ag industry professionals, consumers, undergraduate students, graduate students, post-docs Changes/Problems:Sub-objective 1.1 We improved the bioassay for detection of autotoxicity in field soil by adjusting the protocol from a 4-d, soil-on-agar bioassay, to a 14-d soil-only bioassay. The agar base created challenges managing soil moisture during the bioassay. Additionally, greater volume of soil and longer growth period increase the sensitivity. Sub-objective 1.2 The hybrid-soil hydroponic system described in the initial grant, in which plants were grown in soil for 60-d before being transferred to a hydroponic system for root exudate collection, has been simplified. Plants are now grown in a semi-hydroponic system containing sand in sterile jars for 30-d before tissue collection and filtering root exudates through the sand. This system allows for greater control of fertility and the microbiome and decreases plant physical stress by eliminating the transfer step. In communication with the MSU Metabolomics Core, we have altered our metabolite identification approach. Our initial plan to conduct untargeted metabolomics on soils and compare the metabolites in high and low autotoxicity soils to identify 5-10 metabolites for further study would be limited by the complex nature of soils. Instead we have selected 8 metabolites identified as potentially autotoxic in existing literature for targeted metabolomics in both alfalfa tissues and soils. Sub-objective 2.2 We changed from a field soil growth medium to a clay-sand substrate. The clay-sand substrate allows for easier manipulation of fertility and cleaner extraction of metabolites than the field soil we initially planned to use. Sub-objective 2.3 For sub-objective 2.3, determining whether the soil microbial community associated with nutrient stressed plants contain microbes capable of degrading autotoxins, we will no longer characterize the microbiome of the rhizoboxes because of the change to the clay-sand substrate. Instead, we will inoculate an alfalfa extract seedling bioassay with an alfalfa synthetic community to identify which microbes degrade autotoxins. Then, we will incubate bulk soils from sub-objective 1.1 with alfalfa extracts and monitor shifts in the microbiome. Lastly, will inoculate alfalfa plants in our semi-hydroponic system with the synthetic community (sub-objective 1.2) to determine how microbes influence the production of autotoxins. The multivariate analysis of our collected field soils is a new addition that will further inform the influence of environmental and management factors on autotoxicity and creates greater continuity between experiments. What opportunities for training and professional development has the project provided?The project has provided training in scientific method, data management, and presentation skills for 2 graduate students and 5 undergraduates. How have the results been disseminated to communities of interest?Results were disseminated via written articles, online articles, webinars, professional abstracts and posters, and in-service trainings to scientists, students, professionals, and farmers. What do you plan to do during the next reporting period to accomplish the goals?Obj. 1. In the next year, we will run the soil-bioassay with our collected soil samples. Three rounds of the semi-hydroponic system for alfalfa tissue and root exudate collection are planned for the next year. Each round will also include autotoxicity bioassays with the collected extracts and metabolomics analysis. Work on sub-objective 1.3, determining autotoxicity thresholds in field soil for identified compounds, will commence once sub-objectives 1.1 and 1.2 are complete. Obj. 2. 16s and ITS amplicon sequencing for collected soils will be completed at the beginning of next year. Two rounds of the nutrient stress rhizobox experiments are scheduled for the next year. We will also inoculate an alfalfa extract seedling bioassay with an alfalfa synthetic community to identify which microbes degrade autotoxins. We also aim to complete the multivariate analysis of our collected field soils to determine factors driving performance in the bioassay and detection of autotoxicity. Obj. 3. We will generate the base populations for the autotoxicity recurrent selection experiment and conduct the first round of selection. Obj 4. We will continue to present updates at extension programs.

Impacts
What was accomplished under these goals? Obj. 1. During year one, our progress towards identifying the causative compounds responsible for alfalfa autotoxicity included protocol development and refinement and sample collection. To address sub-objective 1.1, identifying autotoxic compounds in field soil, we collected soil from 17 alfalfa fields for use in our soil autotoxicity bioassay. Regarding sub-objective 1.2, quantifying the presence of targeted metabolites autotoxic alfalfa plant tissue and root exudates, we refined and trialed a root exudate collection system and a tissue extraction protocol. One preliminary trial on procedures was completed for the plant growth and root exudate collection system. We also conducted preliminary trials on procedures for petri dish seedling bioassays with alfalfa tissue extracts. The extract bioassays will indicate the autotoxicity of extracts collected from the previously mentioned plant growth systems. Obj. 2. Evaluating the impact of environmental (alfalfa microbiome and soil biology) and management factors (soil fertility) on autotoxicity. Regarding sub-objective 2.1, characterizing the microbiome communities in autotoxic and non-autotoxic field soils, we have collected all soils for 16s and ITS amplicon sequencing. 16s amplicon sequencing is complete for 25% of the samples. In our work towards sub-objective 2.2., determining whether autotoxins exuded during primary nutrient stress reduce growth of replanted alfalfa, we have trialed and selected a clay-sand substrate for our rhizobox greenhouse trials. A preliminary trial growing plants under K deficiency is underway. We collected additional environmental and management characteristics from the alfalfa fields used in the soil bioassay in sub-objective 1.1. Multivariate analysis of factors including bioassay results, stand age, stand density, soil texture, soil fertility, soil metabolite content, and soil microbial community composition will be used to determine factors driving performance in the bioassay and detection of autotoxicity. Obj. 3. In the first year of the study, we first sought to determine whether dried alfalfa biomass samples retained autotoxic attributes when stored over long periods of time. To do this, we created extracts using samples from the same alfalfa cultivar (Vernal) sampled between 2004 and 2021 from Cornell's alfalfa yield trial program and also grew Vernal plants in the greenhouse and sampled tissue from these plants. We screened these extracts using two alfalfa cultivars (AFX 469 and SW315LH). We found that older ground samples and greenhouse grown samples show as much or more autotoxicity than more recently sampled ones. This finding will enable us to take advantage of the large number of stored alfalfa samples from both our research program and potentially other public yield trials to screen a larger number of cultivars for autotoxicity. We conducted a second round of testing to evaluate autotoxic production and susceptibility in a larger number of cultivars and Cornell breeding populations. The goal of this experiment was to determine the base populations used for a subsequent recurrent selection experiment which will select for high and low autotoxic compound production and for susceptible and resistant populations. We tested 18 genotypes (cultivars and populations) and included each genotype as an extract and as seeds germinated in extracts. We found more variability among extracts (i.e., production of autotoxic compounds) than among seed (i.e., susceptibility to autotoxic compounds), indicating it may be easier to select for the former than the latter, and/or that we may need to identify additional germplasm to find populations with significant resistance to autotoxic compounds. We used the information from this experiment to identify germplasm that will contribute to the base populations for our recurrent selection experiment. We planted these populations in the greenhouse to be recombined in early 2025. Obj 4. In the first year of the study we have engaged alfalfa producers via seven project-authored extension presentations using webinars, in-person conferences and service trainings, online articles, and print articles in trade magazines. Information obtained from these has been further featured in print and online articles written by trade professionals. Estimated total audience of all outputs based on attendance, views, and magazine circulation figures is 100,000 producers.

Publications