2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Modern agricultural practices make the soil less hospitable for beneficial organisms such as arbuscular mycorrhizal [AM] fungi by application of synthetic chemicals and excessive tillage. More efficient utilization of AM fungi, beneficial soil fungi which aid roots in taking up nutrients from the soil and which have been shown to impart disease resistance to plants, would allow for reduced use of synthetic chemicals in food production. Organic farming also can benefit greatly from the use of AM fungi since these organisms reduce the need for pesticides because mycorrhizal plants have a higher level of pathogen resistance than nonmycorrhizal plants. These fungi cannot be grown axenically (with no other organisms present) as other fungi can. This prevents the production of sufficient quantities of these fungi for large scale use. Thus, the emphasis of this project is to determine the critical metabolic components, chemical and physical factors that will allow us to produce large quantities of AM fungi either axenically, monoxenically (in dual culture), or through "on farm" methods and to effectively utilize the resulting inoculum in agricultural applications. Unraveling the recognition/communication events, metabolic pathways, and regulation of carbon and nitrogen flows between fungus and host are necessary for eventual axenic culture of AM fungi and optimization of current inoculum production technologies. This new information will lead us to more efficient inoculum production with the eventual goal of substantial quantities of AM fungi for high volume field applications. This CRIS was responsive to National Program 202, Soil Quality and Management (70%): in the Problem Areas 4.1 Soil Ecology, Goal: 4.1.1 Understand the ecological characteristics and processes of soil/root biota; Problem Area: 4.2 The Rhizosphere and Spermosphere, Goal: 4.2.2 Evaluate the effects of management practices on the biology and ecology of the rhizospheres; Problem Area: 4.3 Interactions Between Soil Management and Soil Biota, Goals:4.3.1 Determine the effect of management practices on soil biota , 4.3.2 Determine the role of soil biota interactions with soil chemicals and physical processes, 4.3.3 Develop management practices that emphasize management of the soil biota, 4.3.4 Develop methods for upgrading the biological quality of soils through the application of mass-produced beneficial organisms; and Plant Biological and Molecular Processes 302 (30%): Problem Area: Crop Plant Associations with Beneficial Organisms, Goal: Develop new basic knowledge about mechanisms of organismal interactions that will allow enhanced symbiotic associations. 2.List by year the currently approved milestones (indicators of research progress) FY 2002 Objective 2D On-farm production of AM fungus inoculum Milestone (i) Production round one, assay and testing. FY 2003 Objective 1A - Elucidate the metabolic pathways of carbon and nitrogen that are essential for establishing the obligate relationship between host plant and fungus using NMR spectroscopy and mass spectrometry in conjunction with isotopically labeled substrates. Milestone (i) Complete regulation of carbon translocation studies. Objective 2A - Adapt current in vitro culture techniques to purify and identify chemical constituents of root exudates, root mucilage, root border cells, and root extracts which may lead to the axenic culture of an AM fungus. Milestone (i) Identification of hyphal stimulators and inhibitors. Milestone (ii) Develop a second bioassay to detect stimulators of penetration hyphae formation. Objective 2D On-farm production of AM fungus inoculum Milestone (ii) Production round two, assay and testing. Objective 2E - Use of AM fungi in vegetable crop production Milestone (i) Growing season one. FY 2004 Objective 1A - Elucidate the metabolic pathways of carbon and nitrogen that are essential for establishing the obligate relationship between host plant and fungus using NMR spectroscopy and mass spectrometry in conjunction with isotopically labeled substrates. Milestone (ii) Determine sites of DNA Synthesis. Milestone (iii) Complete studies of lipid export in response to metabolic factors. Objective 2D On-farm production of AM fungus inoculum Milestone (iii) Production round three, assay and testing. Objective 2E - Use of AM fungi in vegetable crop production Milestone (ii) Growing season two. FY 2005 Objective 1A - Elucidate the metabolic pathways of carbon and nitrogen that are essential for establishing the obligate relationship between host plant and fungus using NMR spectroscopy and mass spectrometry in conjunction with isotopically labeled substrates. Milestone (iv) Determine role(s) of CO2 dark fixation. Objective 2B - Adapt current techniques for the monoxenic production of Gigaspora species and to study the effect of physical factors which may increase extraradical hyphal growth. Milestone (i) system for study of Gigaspora. Objective 2E - Use of AM fungi in vegetable crop production Milestone (iii) Growing season three. FY 2006 Objective 1A - Elucidate the metabolic pathways of carbon and nitrogen that are essential for establishing the obligate relationship between host plant and fungus using NMR spectroscopy and mass spectrometry in conjunction with isotopically labeled substrates. Milestone (v) Complete nitrogen transport studies in mycorrhizae and germinating spores. Objective1B - Use the information gained from metabolic studies to simulate the conditions and substrates necessary for the completion of the life cycle of the fungus in the absence of host roots. Milestone (i) Develop axenic culture methodology. Objective 2C - Increase the sporulation of Gigaspora species in monoxenic culture using the above results and those from Objective 1. Milestone (i) Increase sporulation of G. gigantea in vitro. Objective 2E - Use of AM fungi in vegetable crop production Milestone (iv) Growing season four. 4a.List the single most significant research accomplishment during FY 2006. Nitrogen transport in the AM symbiosis Arbuscular mycorrhizal fungi colonize plant roots, establishing a symbiotic relationship which increases crop yield with lower input of fertilizer and pesticides. The problem is how to maximize the efficient use of the organisms to deliver nutrients such as nitrogen to the host plants. Collaborative experiments between scientists at ERRC, Wyndmoor, PA; New Mexico State University, and Michigan State University demonstrated how the supply of photosynthate from the plant controls the movement and transfer of nitrogen to the host. The existence of this pathway and control mechanism and the high flux of nitrogen through it indicate that the AM symbiosis can transfer large amounts of nitrogen from the soil to plant roots. This means that the symbiosis has a more significant role in the global nitrogen cycle than had been widely believed. These findings, reported in the journal Nature, give us insight into how AM fungi take up and transfer nitrogen to their host crop plants, information that will allow us to determine the most efficient methods for implementing low input nitrogen utilization. (NP 302 Action Plan Component II (Biological processes that improve crop productivity and quality), Problem Statement 2A (Understanding growth and development); Performance Measures in the ARS Strategic Plan, 1.2.6). 4b.List other significant research accomplishment(s), if any. Penetration hyphae bioassay A bioassay needed to study the 4th step of the fungal life cycle, growth of penetration hyphae after appressium formation, was developed. This technique required the production of large pieces of host root cell walls in order to produce large numbers of appressoria in vitro. With this technique, host compounds can now be tested for their ability to induce penetration hyphae. In addition, we have demonstrated for the first time that compounds isolated and separated from carrot root exudates induce two different but morphologically pronounced responses by AM fungi. This finding adds to our knowledge of the growth of these organisms in the “asymbiotic” state and may be useful for eventual axenic culture of AM fungi. ( 302 Action Plan Component II (Biological processes that improve crop productivity and quality), Problem Statement 2B (Understanding plant interactions with their environment), Performance Measures in the ARS Strategic Plan, 5.2.2 and 5.4.2). 4c.List significant activities that support special target populations. Presentations to farmers and organizations: none 4d.Progress report. 1935-12000-007-01T This report serves to document research progress under a NRI competitive grant #20002-35318-12713 initiated 10/02. Additional details of this research can be found in the report for the parent CRIS 1935-12000-007-00D Monoxenic and Axenic Cultivation of Arbuscular Mycorrhizal (AM) fungi. Mycorrhizae enhance the N nutrition of their hosts although little is known about the mechanisms of uptake of various forms of N by germinating spores of AM fungi. We used 15N labeled inorganic and organic sources of N, in conjunction with mass spectrometric analysis of amino acids to study preferences in N uptake and metabolism in spores of Glomus intraradices. Uptake of inorganic N exceeded that of organic N. Further, the fungus preferred NH4 to NO3 nitrogen. Arginine and asparagine were the two most heavily labeled amino acids isolated from fungal tissues. These findings have provided us with insight into the most effective means of delivering nitrogen to axenically growing spores. 302 Action Plan component III Mechanisms of Plant Interactions with other Organisms, Performance Measures in the ARS Strategic Plan, 5.2.2. 5.Describe the major accomplishments to date and their predicted or actual impact. Carbon Metabolism in the Arbuscular Mycorrhizal Symbiosis. Our isotopic labeling studies combined with NMR spectroscopy have given us insight into the metabolic profiles of the AM symbiosis. Looking at each step of the fungal life cycle we have defined how carbon flows between and are metabolized by the symbionts. Gene expression studies have confirmed the existence of numerous active enzymatic pathways defined by our isotopic experiments and have provided insights into the mechanisms of nutrient delivery to the host and fungus. Most of this work can be summarized by our conclusion that the fungal mycelium, although a contiguous tissue, has completely different carbon metabolism and gene expression within vs. external to the root. Therefore, to complete its life cycle in the absence of a host, the fungus must up regulate genes that we have observed it to express after it grows into the root during its symbiotic phase. That is, it will need to take up hexose and synthesize lipid as it does within the root in order to grow and proliferate. This finding is significant because it defines the problem researchers have been tackling for 50 years- how to grow AM fungi in the absence of plant roots? Researchers in many countries are using the methods we devised to study the physiology of AM fungi. These studies will contribute toward the eventual “axenic” culture of AM fungi. Axenic culture will allow for the mass production of AM fungi for inoculum to be used in agriculture, making the many benefits of the symbiosis readily available to farmers, decreasing economic and environmental costs of crop production. (NP 302 Action Plan Component II (Biological processes that improve crop productivity and quality), Problem Statement 2A (Understanding growth and development). Nitrogen uptake and metabolism in the AM symbiosis. Another major accomplishment is the discovery of how the fungus takes up nitrogen from the soil and delivers it to the host plant. We have found that the fungus outside of the root can take up many forms of N, convert them into the amino acid arginine, and transport that arginine to the root where the N is released via the urea cycle. We established that AM fungi can provide a significant amount of the nitrogen used by the host plant. This finding is significant because it means AM fungi deserve a significant role in the nitrogen cycle in our environment. In addition, scientists will find the information of the N metabolism of AM fungi useful in achieving axenic culture. (NP 302 Action Plan Component II (Biological processes that improve crop productivity and quality), Problem Statement 2A (Understanding growth and development). Stimulating the early phases of the AM fungus’ life cycle in the absence of a root. As part of the quest to determine how to grow an AM fungus without it colonizing the roots of plants, we have examined the interaction of roots and the fungus prior to colonization. We developed the first rapid and sensitive bioassay to detect chemical compounds released by host roots that cause the first noticeable change in germinated fungal spores, i.e. increased hyphal branching. Hyphal branching is a critical step in the life cycle of AM fungi because they cannot colonize a root to complete their life cycle without first finding a host root in the soil. This assay is used by scientists in France, Canada, and several research labs in the U.S. to study the first interaction between the host root and fungus. We have semi-purified these signal molecules out of root exudates. In addition, we developed a way to highly purify biologically active root cell walls to study appressoria (anchoring fungal structures) formation. Ours was the first report to show appressorium formation in the absence of an intact root. Therefore, we have completed the first three developmental steps of the life cycle of AM fungi without the presence of an intact root. Recently, we have developed another bioassay, utilizing purified and sterile cell walls and a method to concentrate them, which allows us to the determine the factors which complete the fourth step, production of penetration hyphae. The impact of reproducing the early phases of the life cycle of AM fungi in vitro without host roots is that it brings us that many steps closer to axenic culture and completion of the life cycle of these fungi in vitro. This has potential for inoculum production and economic and environmental benefits to US agriculture. ( 302 Action Plan Component II (Biological processes that improve crop productivity and quality), Problem Statement 2B (Understanding plant interactions with their environment) Production and utilization of inocula of AM fungi. Production of inoculum of AM fungi currently is done either in greenhouse pot culture with potted plants or in the laboratory in Petri dishes with plant roots present. Nevertheless, utilization of AM fungus inoculum in large scale agriculture is inhibited by the costs associated with production, processing, and shipping the large quantities of inoculum necessary. Therefore, we sought to develop technology for “on-farm” production of inoculum to minimize these costs and make the technology available to small farms. Grass seedlings, precolonized by AM fungi, are transplanted into raised beds of compost and vermiculite mixtures. The plants grow for one growing season, during which the fungus spreads and proliferates in the bed. The fungus then overwinters naturally, and the mixture with AM fungi is available for use the following growing season. Upwards of one hundred million propagules of AM fungi have been produced in 0.1 m3 of media at a cost of less than $50. We have obtained prices from commercial suppliers and the cost of an equivalent number of propagules ranges from a low of $8750 to a high of $43,697. The on-farm system is currently being used by a core group of six farms participating in a pilot project. Other collaborative work with The Rodale Institute has demonstrated the potential economic benefit of inoculating vegetable crops with AM fungi. Green peppers showed upwards of a 34% increase in weight marketable fruit when seedlings were precolonized with mycorrhizal fungi. The application of the inoculum produced via the “on-farm” method to seed potatoes increased the total weight yield of tubers by 45% in 2002 and 10-15% in 2003 over uninoculated controls in plots under both conventional fertilizer and organic fertility regimes. Together, these accomplishments have the predicted impact of contributing to the widespread conversion from conventional, chemical based agriculture to low-input sustainable agriculture and the economic and environmental benefits that would accompany that change. (202 Action Plan Component II: Understanding and managing soil biology and rhizosphere ecology, Focus Area 2: Utilizing AM fungi and other biological processes to enhance productivity, profitability, and sustainability). 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Information has been placed in the hands of users (farmers) through lectures to farmers organizations (see 4C), at field days at The Rodale Institute, and through several feature articles on The Rodale Institute’s website (newfarm.org). Technology for on-farm inoculum production has been transferred to six farmers who are participating in a three-year pilot project. The basic scientific information regarding the metabolic interactions and nutrient flow of AM fungi has been widely disseminated in the scientific literature, including an important article in Nature, and through collaboration with many other scientists here and abroad. Also we have developed a rapid and sensitive bioassay which will allow for the identification of specific signal compounds from the host root that induce the growth and hyphal branching of AM fungi. This assay is widely used by scientists in Canada, Japan, France, and the United States as indicated by presentations at the 4th International Conference on Mycorrhizae at Montreal, Canada in 2003. This increasing knowledge base will ultimately allow us to achieve our goals for axenic culture of AM fungi for large scale low input agriculture. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presentations to farmers and organizations: 1. Aug. 13, 2004: invited oral presentation at the meeting of the Northeast Organic Farming Assoc., Amherst, MA, entitled “An introduction to AM fungi, management and utilization.” 2. Feb. 5, 2005: invited oral presentation to the Pennsylvania Association for Sustainable Agriculture annual meeting, State College, PA, entitled “Management and utilization of mycorrhizal fungi.” 3. July 22, 2005: two invited lectures and demonstrations at The Rodale Institute Experimental Farm Field Day- “On-farm production and utilization of arbuscular mycorrhizal (AM) fungi.” Popular press: Articles written about our work: “Cultivating diversity underground for better yields above” by Laura Sayre. On the NewFarm.org website [http://www.newfarm.org/depts/NFfield_trials/0903/daviddouds.shtml] “Cultivating beneficial fungi to increase yields” by Laura Sayre. On the NewFarm.org website [http://www.newfarm.org/depts/NFfield_trials/0404/mf_update.shtml] Fact Sheet: AM fungi- “Improve your soil, increase your yields, and reduce your expenses with AM fungi” by David Douds and Christine Ziegler. On the NewFarm.org website [http://www.newfarm.org/depts/NFfield_trials/0604/factsheet.shtml]