Location: Characterization and Interventions for Foodborne Pathogens
2021 Annual Report
Objectives
1: Increase efficiency of AM fungus inoculum production and utilization to enhance vegetable production and reduce inputs.
1A. On-farm production and utilization of AM fungus inoculum to enhance vegetable crop yields and reduce inputs.
o Increase propagule density of inoculum produced when the indigenous community is used as starter inoculum. (Douds)
o Demonstrate use of AM fungus inoculum produced on-farm for eggplant production.
1B. Examine the use of black plastic mulch upon indigenous AM fungus activity and evaluate relative to opportunities to utilize AM fungus inoculum. (Douds)
2: Determine the effects of arbuscular mycorrhizal fungus species on improving crop physiological responses that enhance nutrient uptake, increase production of polyphenols, and enhance plant resistance to insect pests and diseases.
2A. Quantify the impact of AM fungus colonization upon production of polyphenols of known health benefit in different crops.
2B. Enhance the utilization of the AM symbiosis through studies of basic physiology and nutrient uptake.
o Measure the flow of C, N, and P between plants of varying photosynthetic rates via common, interconnected AM fungus networks and correlate to expression of plant P and NH4 transporter genes.
o Determine the impact of newly-identified root exudate signal molecules (abietic acid and dehydroabietic acid) on carbon uptake and lipid synthesis by germinating spores of AM fungi via NMR spectroscopy.
o Determine the impact of semi-purified root exudate signals in combination with low oxygen concentrations upon AM fungus germination and growth.
3: Develop management practices that minimize the potential negative effects of off-site transport of pathogenic bacteria in integrated crop/livestock production systems.
3A. Inactivate pathogenic bacteria, originating from animal manure, moving in surface water as a result of heavy rainfalls, before they can enter farm irrigation ponds by using erosion control socks amended with fast pyrolysis biochar.
Approach
Arbuscular mycorrhizal [AM] fungi are obligate symbiotic soil fungi that form a mutualistic symbiosis with the majority of crop plants. Better utilization of this symbiosis will enable farmers to increase or maintain yields while reducing synthetic chemical inputs. Earlier, we developed a method for the on-farm production of inoculum of AM fungi using bahiagrass as the host plant. This method will be refined for the production of AM fungi indigenous to the farm and its usefulness demonstrated for the production of eggplant. The obligate symbiotic nature of these fungi require that a host plant be present during the production of inoculum for use by farmers. Large scale production of pure inoculum of these fungi (i.e. growth of the fungi by themselves, in the absence of a plant) is possible if the physiological limitations that require colonization of a host plant could be overcome. We will attempt to overcome these constraints by growing the fungi in the presence of a combination of environmental factors (root exudate signals, high CO2, and low oxygen) more representative of the environment in which they naturally grow rather than typical laboratory conditions. Other studies will examine the impact of AM fungus colonization upon mineral nutrient uptake and the production of human health-promoting compounds in plants. Farms with integrated livestock and crop production are faced with regulatory pressures designed to minimize the risk of contamination of produce with bacteria originating in animal manure. We will explore the idea that erosion control socks containing biochar, previously shown to reduce the population of pathogenic bacteria in soil by 100 fold, could be used to intercept rainwater runoff from pastures and inactivate bacteria before they contaminate the water in an irrigation pond.
Progress Report
This is the final report for project, “Production, utilization, and importance of arbuscular mycorrhizal fungi in sustainable vegetable production” completed the OSQR Peer Review Process and was approved on October 14, 2016. In FY2017, progress was made on all objectives, all of which fall under National Program 212 Research Components: 1. Management and stewardship of soil resources and 3. Reducing environmental risk of agricultural operations. Problem Statement 1B: Agricultural management for resilient soil, Problem Statement 1C: Enhancing Soil Biodiversity and Functions, Problem Statement 3A: Reducing pathogens and antibiotic resistance. Arbuscular mycorrhizal (AM) fungi are soil fungi which form a symbiosis with the majority of crop plants, assisting roots in nutrient uptake, drought tolerance, and disease resistance. Better utilization of this symbiosis in agriculture through the contributions of this project should result in decreased synthetic chemical input and increased profits and sustainability. A number of field experiments were conducted to demonstrate the ability of AM fungus inoculum produced on-farm to enhance yield of crops in real-world situations. Experiments with eggplant concluded in autumn 2016 and showed increases in yield of AM fungus inoculated vs. non-inoculated plants of 6 to 18% (or 1 to 1.5 fruit per plant). Also completed was one replicate of a large scale experiment to determine which farm habitat is best for collection of field soil as starter inoculum for the on-farm production of indigenous isolates of AM fungi. Results indicated that a field previously used to produce a row crop subsequently planted to an overwintering small grain produced the most potent inoculum. The replicate of this experiment is underway. Biochemical studies were expanded to investigate levels of different polyphenols of (human) nutritional quality in plants (e.g., leeks, pepper, corn, tomato, lettuce) as a result of AM symbiosis. Mechanistic studies with collaborators at South Dakota State University examined how mineral nutrient flow to a given plant is governed by the relative photosynthetic strength of that plant compared to the others.
In FY2018, progress was made on Objective 1A, Increase propagule density of inoculum produced when the indigenous Arbuscular mycorrhizal (AM) fungus community is used as starter inoculum. An experiment was conducted in 2017 utilizing soil collected from habitats at the Rodale Institute farm. Soil from the pasture produced higher levels of AM fungi than it did the previous year (300 propagules cm-3 vs. 140 in 2016). Soil from fields used to produce small grain the prior year also produced satisfactory levels of inoculum. Progress was also made on Objective 2B, Measure the flow of Carbon (C), Nitrogen (N), and Phosphorus (P) among component organisms in the tripartite symbiosis of legumes. Legumes live in symbiosis with both AM fungi (supplying P) and Rhizobia (supplying N), with the plant supplying carbon in both instances. Physiological experiments conducted with a legume grown in compartmented chambers, separating the symbioses, showed that when N or P were externally applied chemically, the plant supplied carbon (sugar) preferentially to the organism supplying the nutrient that was in the most limited supply. This result helps us further understand the working of the symbiosis in order to put it to use for the sustainability of agriculture.
In FY2019 and FY2020 a new SY has been recently hired, therefore progress will be reported in the FY2020 annual report. Related to Objective 1 [Increase efficiency of arbuscular mycorrhizal (AM) fungus inoculum production and utilization to enhance vegetable production and reduce inputs] quantitative PCR (qPCR) assays were developed for the identification and quantification of 11 different species of AM fungi. Previously, it has been shown that mixed-species arbuscular mycorrhizal fungi (AMF) inoculum results in beneficial yield increases of multiple crops, but the measurement of impact of individual species was not possible. Identification and quantification of AM fungi from soil samples previously required isolation of spores from the soil, morphological identifications, and time-consuming plant-based colonization assays. Measurement of the colonization efficiency of individual species of AM fungi from a mixed inoculum was not possible. To develop the qPCR assays, amplicon sequencing of the internal transcribed spacer (ITS) and glomalin related protein gene was completed from >40 accessions of AM fungi in the international culture collection of (vesicular) arbuscular mycorrhizal (INVAM) repository and several cultures from the Eastern Regional Research Center (ERRC) greenhouse. This sequence data was utilized to identify species-specific targets within the genomes of the 11 different species represented. Specificity testing confirmed the assays do not cross react with DNA from non-target AM fungi. qPCR enables the rapid detection and quantification of AM fungi directly from soil DNA extracts, and enables species-specific quantification of AM fungi colonization of plant roots. Samples can be extracted and analyzed within a single day, and future work will involve multiplexing the assays for increased throughput. The availability of qPCR will enable more precise field and laboratory studies of the relationship between individual species of AM fungi and crops, and guide the selection of AM species for development as biofertilizers to improve nutrient acquisition and water uptake and reduce susceptibility to pathogens and disease. Related to Objective 1.B (Examine the use of black plastic mulch upon indigenous AM fungus activity and evaluate relative to opportunities to utilize AM fungus inoculum), experiments were delayed due to critical vacancies, but an opportunity to pursue this objective with at an alternate site has arisen. A Pennsylvania Specialty Crop Block Grant was awarded to the Rodale Institute to study the impact of black plastic mulch in Rodale Vegetable Systems Trial. ARS is a collaborator on this 3-year grant. The crops to be tested include sweet corn and butternut squash; the impact of AMF on yield and nutritional content of the produce will be evaluated. On-farm propagation of AMF inoculum has begun during 2020 for use in the 2021 growing season. Related to Objective 2.B.3 (Enhance the utilization of the AM symbiosis through studies of basic physiology and nutrient uptake, evaluation of the combination of low O2 tensions and root exudate signals on growth and carbon utilization by germinating spores of AM fungi), preparations for these experiments have been made. Past vacancies have caused delays in initiating these experiments, but it is anticipated that much of the original proposed work can be completed before the end of the project cycle. Root exudates have been prepared for use and stored, AM fungi have been propagated for these experiments, and equipment (new CO2 incubator, hypoxia chambers) are on site.
In FY2021, we initiated highly controlled, species-specific inoculation studies utilizing arbuscular mycorrhizal fungi on various vegetable crops (including sweet corn, winter squash, beans, and potatoes) to identify individual species or combinations of species of fungi that promote favorable increases (or detrimental decreases) in yield and nutrient content of vegetables. These trials are being conducted in collaboration with a non-profit agricultural research institute; some field trials will incorporate additional variables such as tillage and organic or conventional management practices. The overarching goals of these trials are to identify new formulations of mycorrhizal inocula that exceed the previously demonstrated 15% yield increase from mixed-species mycorrhizal inoculation and enhance crop nutrient quality. A modified version of this Subobjective 1B (Examine the use of black plastic mulch upon indigenous AM fungus activity and evaluate relative to opportunities to utilize AM fungus inoculum) is being addressed in collaboration with the Rodale Institute. This objective was planned to be addressed during the 12-36 months period but was not started then. An opportunity to revisit this subobjective arose in connection with a Specialty Crop Block Grant to Rodale to fund field studies involving sweet corn and butternut squash grown on black plastic mulch beds and reduced tillage and under both organic and conventional practices. ARS collaborates on the project for conducting the mycorrhizal analysis of both the native community and the outcomes of the seedling mycorrhizal analysis. Field studies commenced in May 2021 and continue through September 2022. Yield and nutritional analysis of the crops inoculated with mycorrhizal fungi and grown under the different tillage and fertility practices will help inform the optimization and development of mycorrhizal inoculants for vegetable crop production.
Accomplishments
