NE1602: Explorations in the Turfgrass Phytobiome: Understanding Microbial Associations and Developing Tools for Management
(Multistate Research Project)
NE1602: Explorations in the Turfgrass Phytobiome: Understanding Microbial Associations and Developing Tools for Management
Duration: 10/01/2016 to 09/30/2021
Statement of Issues and Justification
Turfgrasses encompass many facets of everyday life through use in home lawns, commercial landscapes and parks, roadsides, athletic fields, golf courses and sod farms. While these aspects provide social benefit as a surface for exercise and recreation, the turfgrass sector also has a valuable economic impact through employment opportunities in the green industry, commerce, economic development, tax revenue for communities, and increased home values. In a 2002 study conducted by scientists at the University of Florida, the industry generated over 820,000 jobs in the U.S., with an estimated economic footprint of nearly $58 billion. A more recent survey estimates the economic impact of the golf industry alone at nearly $70 billion per year, so the overall influence of the industry has clearly increased in the past decade. In addition to the social and economic impacts, the extensive network of above- and below-ground turfgrass tissues have many environmental benefits including, dust and dirt removal from air, reduced soil erosion, surface water filtration, carbon sequestration, oxygen production, and heat, glare, and noise reduction. Furthermore, turfgrass areas are home to a variety of microorganisms and insects while golf courses, landscapes, and parks provide habitats to support numerous wildlife species.
The breadth of environmental benefits are best realized in well-managed, healthy turfgrass stands. Like all plants, turfgrasses are subject to abiotic and biotic stressors that require regular cultural, and sometimes chemical, management actions to ensure turf performance. Some examples of cultural management practices include irrigation, fertilization, and mowing while chemical management includes application of herbicides, insecticides, and fungicides. Integrated pest management (IPM) includes a combination of cultural and chemical management practices that limits destructive insects, pathogens and weed species while also limiting the non-target economic and environmental impacts of such practices. Turfgrass scientists are constantly researching new management tactics to produce healthy turfgrass stands with fewer inputs and environmental consequences by developing improved grass varieties, fertilization schedules, and new pesticides with fewer non-target effects. At the same time, an emerging trend among some state and local governments is legislation restricting the use of pesticides, water and some fertilizers inputs on turfgrass areas. These new laws eliminate important tools for maintaining healthy turfgrass stands, and require practitioners to rely on less well known strategies for nutrient and pest management. This legislation has resulted in an increased interest among turfgrass managers for research to better understand effective organic and pesticide-free management approaches. A related area of growing research interest involves understanding and developing applications in the turfgrass phytobiome. Defined as the microbial community that resides both on and within a turfgrass plant, the phytobiome can encompass many different species including archaea, bacteria, fungi, oomycetes, nematodes, and viruses. Previous research shows individuals and groups within the microbial community have strong interactions with plants and can significantly impact plant health through cell-to-cell signaling, plant defense, and physiological development. By furthering our knowledge of these complex interactions, we hope to foster beneficial plant-microbe relationships and reduce our reliance on fresh water, synthetic fertilizers and pesticide inputs necessary to maintain acceptable turfgrass stands.
With the advent and improvement in next-generation sequencing technologies, the ability to explore entire microbial communities has advanced significantly in recent years. Phytobiome research is growing with new projects being initiated in many plant science disciplines. As with any burgeoning research area, difficulties exist in developing technologies and understanding the wealth of data that comes with microbial community analysis. As we venture into this new research area, members of the turfgrass scientific community must collaborate to develop standardized research methods for phytobiome study. One certainty is that the phytobiome composition is impacted by many factors including, but not limited to, environment, plant host, and management inputs. However, our knowledge of how each of these inputs directly impacts different species is limited and indicates the need to initiate research in this area. Members of our proposed research group encompass multiple disciplines within turfgrass science and offer the necessary expertise to answer critical questions and advance this area of turfgrass management. Research generated within the turfgrass phytobiome will ultimately improve our knowledge of plant-microbe interactions, translating into improved turfgrass varieties and effective biocontrol strategies for limiting pathogen development. Providing a group forum to develop and share research ideas will broaden our ability to understand the complex relationships among microbial communities within turfgrass systems. Collaborative efforts will ultimately improve our ability to extend research results to turf practitioners in the field and develop applications to harness the full power of the turfgrass phytobiome.
Need for project as indicated by stakeholders:
In June 2015, a symposium on turfgrass phytobiomes was sponsored by The Grass Roots Initiative for Turfgrass Science and held at the National Arboretum in Washington, D.C. This meeting was held in conjunction with ‘Phytobiomes 2015: Designing a New Paradigm for Crop Improvement’, which was sponsored by USDA-NIFA, NSF, APS, US-DOE, Bayer Crop Science, the Samuel Roberts Nobel Foundation, and the U.S. Forest Service. Priority topics discussed in the turfgrass symposium included: (1) developing standardized research methods for applications in the turfgrass phytobiome, (2) providing an open source database for housing and analyzing metagenomic data generated from turfgrass phytobiome research, and (3) developing outreach methods to educate turf practitioners on future phytobiome applications. Moreover, from ongoing discussions with industry professionals, we know that turfgrass practitioners are increasingly aware of the environmental impacts of turfgrass management and are eager to utilize new methods to jointly promote the health of the turfgrass plant and the surrounding ecosystem. Numerous ongoing conversations with lawn care operators, golf course superintendents, and sports field managers has shown great interest in learning how the microbial community can provide practical benefits for turfgrass management, and potentially reduce the need for fresh water, fertilizers and pesticide inputs. These industry practitioners are committed to promoting environmental stewardship through developing sustainable management practices, which will be further improved with applications in the turfgrass phytobiome.
Importance of work and consequences if not done:
Research in phytobiomes is growing rapidly across the field of plant science on the whole. However, previous work has shown that there is a urgent need to formulate standardized research methods and share common resources. If the proposed research project on turfgrass phytobiome development is not conducted, consequences could include: (1) further reliance on natural resources and pesticide inputs to effectively manage turfgrass pests, (2) increased potential for turf loss due to environmental stressors, weed and disease development particularly where legislation restricts management inputs (3) continued potential for non-target effects associated with traditional management strategies to control turfgrass pests, and (4) loss of function and revenue among all sectors of the turfgrass industry through widespread turf failure due to restricted use of management inputs. From a scientific standpoint, the proposed standardization and sharing of common resources will allow for more rapid improvements to be translated into the field. Without a common, standardized and shared platform, research efforts may be duplicated, opportunities to share information will not exist, research efforts will proceed at a slower rate, and our ability to translate data into stakeholder deliverables will be hindered.
Scientists currently involved in developing the proposed research projects bring together diverse set of skills and expertise. Members of the group offer expertise in bacteriology, bioinformatics, breeding, agronomy, mycology, plant pathology, and plant physiology. In addition to the scientific expertise offered by group members, many offer valuable experience in outreach through successful careers in extension. The technical ability offered by all individuals in the research group will lead to a successful research project and offer results for developing future applications within the turfgrass phytobiome.
Advantages of a multi-state effort:
Many turfgrass species are utilized throughout the various geographic regions in the U.S. Diverse environments and climates require use of these differing adapted species, and also subject them to a wide range of abiotic and biotic stressors including soil type, temperature extremes, bacteria, fungi, and insect pests. Developing projects across geographically diverse regions will enhance our understanding of the microbial communities that inhabit turfgrasses, as previous research has illustrated the significant impact environment can have on these communities. While diverse scientists encompass our group, this research project will facilitate exchange of knowledge and research techniques. Moreover, the results generated from this work will provide the framework for future research developing applications in the turfgrass phytobiome. Ultimately, applications developed within the turfgrass phytobiome will be a fundamental component of integrated pest management and plant health programs. Using this approach, we can come together to manage turfgrass using sustainable practices that reduce our reliance on fresh water resources, synthetic fertilizers, and pesticide inputs while still producing functional and aesthetically pleasing golf courses, landscapes, and home lawns. Project members will communicate research results to industry practitioners through regional and national seminars, symposia, peer-reviewed publications, research field days, and web-based platforms.
Advances in understanding the microbiome of the human gut has had far reaching impacts in gastrointestinal health and transformed the field of personalized medicine. We anticipate that impacts from our study of the turfgrass phytobiome will be similarly transformative for the turfgrass industry, ultimately enabling a more predictive and systems-based approach in the management of turfgrass. Our research approach to studying the turfgrass phytobiome involves a series of interdependent projects across multiple environments and will provide a basis for turfgrass research in this growing area. The microbial composition of the turfgrass phytobiome will be classified at a higher level, allowing researchers to pinpoint candidates for plant health promotion and subsequent future research. Moreover, the effects of cultural and chemical inputs on the turfgrass phytobiome will be elucidated, and stewardship of this microbial resource will be communicated to turfgrass managers. The project will bring together researchers from diverse backgrounds and result in improved exchange of information between turfgrass agronomists, plant pathologists, computer scientists, bioinformaticians, and breeders. In our attempts to improve turfgrass management through applications in the phytobiome, it is imperative that outreach projects are effectively constructed to educate practitioners. New management concepts can be met with apprehension and confusion, but the strong extension experience offered by project participants will ensure dissemination of research results in a manner that practitioners can use, be it on golf courses, home lawns, or athletic fields. Social media and web-based tools are popular within all sectors of the turfgrass industry as a means to communicate ideas, local trends, and even management tools. With this in mind, a website will be developed in an effort to provide the most up to date information on the proposed project, along with research descriptions and a forum for questions related to our research. In addition to web-based tools, collaborations and independent work amongst the group will be broadcast through peer-reviewed publications, seminars at national meetings, research field days, and trade journal publications. Opportunities to share findings with the general public through mass media will also be sought. Continued updates will help to spread new research concepts, promote our research goals, and ultimately provide new tools for practitioners that are effective and environmentally friendly. Applications developed through our collaborative effort will result in improved sustainable management practices for major biotic and abiotic stressors of turfgrasses managed in our respective regions and throughout the U.S. Turfgrass growers will be surveyed throughout our region in year 1 and 4 of this multistate project in an effort to evaluate project impacts.
Related, Current and Previous Work
The concept of research in the phytobiome is a relatively new topic, but previous research has advanced our knowledge of microbial communities in turfgrass systems. Early perceptions assumed less numerous and diverse microbial populations associated with sand-based root zones commonly used in golf course green construction due to low clay and organic matter contents (Alexander, 2007; Nunan et al., 2003; Ranjard et al., 2000). However, Bigelow et al. (2002) showed that microbial populations increased rapidly in newly established golf course greens with high sand content root zones. Bartlett et al. (2007) illustrated that significant differences are observed in microbial communities on golf courses with respect to soil physical structure; sand content had the largest impact on microbial communities but management practice intensity (i.e., golf course green vs. fairway) was also a determination in microbial community structure. Similarly, Elliot et al. (2007) illustrated rich taxonomic diversity of culturable rhizosphere bacteria associated with USGA putting greens in Alabama, Florida, North Carolina, and South Carolina. While these and additional work have provided insight into the rich microbial diversity associated with turfgrass systems, even in golf course putting greens, a common determinant of results involved culturable identification techniques. Limitations exist in relying solely on phenotypic variations for identifying plant and soil microbiota as only a small percentage (~1%) are believed to be culturable using specialized media (Bakken, 1997). Moreover, previous research comparing traditional culturing techniques with culture-independent techniques for community analysis can yield significantly different results (Yang et al., 2001). Therefore, new studies would benefit by the ability to better understand all microbes associated with a given plant or soil sample and not solely relying on culturing ability for downstream processes.
Advances in next generation sequencing have significantly improved our ability to study individual microorganisms and entire microbial communities. Although, previous research has shown that analyses of microbial communities using next generation sequencing technologies have many limitations with respect to microenvironment, plant host, sampling methods and experimental reagents (Bent and Forney, 2008; Prakash and Taylor, 2012; Yang et al., 2001). Peiffer et al. (2013) showed that recovered maize rhizosphere microbial diversity was significantly impacted by 16S primer selection as the relative abundance of phyla recovered varied according to primers associated with different variable regions of the 16S subunit. Therefore, previous and ongoing research of microbiomes reflects a growing concern for the development of standardized research practices. Standardized research methods will allow for a better comparison of data across different environments and subsequently allow for more robust recommendations in the future.
Develop standardized research methods for studying microbial community dynamics within turfgrass phytobiomes.
Characterizing geographic and temporal norms of turfgrass phytobiomes.
Assess the impact of turfgrass management systems on phytobiome preservation and development.
Identifying constituents of turfgrass phytobiomes which confer improved abiotic and biotic stress tolerance.
Methods1. Developing standardized research methods for studying microbial community dynamics within the turfgrass phytobiome. Ongoing research of microbiomes reflects a growing concern for the development of standardized research practices. Previous research has shown that analyses of microbial communities using next generation sequencing technologies are significantly impacted by microenvironment, plant host, sampling methods and experimental reagents. Data generated from different research studies may not be comparable if substantitively different methods are applied, as methodological differences is a potential source of variation. Variation based on methodology reduces our ability to generate experimentally valid, systems-based conclusions about the turfgrass microbiome. Furthermore, the unique characteristics of the turfgrass ecosystem may present unique experimental challenges, particularly in cases where the environment is intensively managed and chemical residues are abundant. Our collaborative research group will partner to provide a more thorough understanding of experimental methodology and its impact on the turfgrass phytobiome, and develop a set of core recommendations for microbiome research in this area. 1.1 Plant and soil sampling, environmental DNA/RNA extraction, and sample processing Identify and develop standardized methods for plant and soil sampling for microbial community analysis, environmental DNA processing, and sample processing for next generation sequencing platforms. Due the strong environmental impact on microbial communities, special care is needed when sampling both plant and soil for microbial communities. Laboratory technical controls are vital, due to the sensitivity of next generation sequencing technology. There is increasing data that shows even experimental reagents such as DNA extraction kits can contribute contaminant sequences to microbiome analyses. Environmental conditions can have significant impacts on the ability to extract nucleic acids suitable for next generation sequencing, especially where chemical inputs are abundant and soil organic matter is reduced. 1.2 Data analysis and metadata storage As turfgrass scientists develop research to further understand turfgrass phytobiomes, there is a growing need for a common database to store and share sequence data. A significant challenge in the application of next generation sequencing tools for studies of the microbiome is the enormous amount of data produced. Millions of sequencing reads are generated from each project. Infrastructure is needed for data storage, and specialized training and expertise in bioinformatics is required to effectively manage and analyze these data. Custom computer tools and scripts are often needed for analyses and general processing. We will partner to develop a cloud-based computer platform for maintenance and storage of turfgrass data generated on the metagenomic and genomic scale. An open-source database will allow for more efficient analysis within turfgrass microbiomes and facilitate application development within the phytobiome. A primary limiting factor for turfgrass microbial community analyses is the paucity of available genome-scale resources for common pathogens, endophytes and free-living inhabitants of the turfgrass ecosystem. True metagenomic analyses, where the gene expression of all resident microorganisms is evaluated, are stymied by our inability to provide sequence identification from reference databases, as no such reference data yet exists. Individual research groups are beginning to generate genome-scale resources for some of the key turfgrass pathogens, however these resources represent only a fraction of the organisms shown to inhabit these environments. Research from the Crouch lab shows that soil within a single golf course putting green can host 103,000 unique bacteria and 46,600 unique fungi in a single growing season (unpublished data), yet the vast majority of these organisms cannot even be identified to the genus or species level using standard ribosomal DNA signatures. To understand how these organisms function at the systems level to impact turfgrass health, simple identification is an essential first step. As a group, we will partner to further develop genome resources for turfgrass-inhabiting microorganisms, and make these resources readily available through a common platform. 2. Characterizing geographic and temporal norms of turfgrass phytobiomes. Initial research projects exploring phytobiomes in turfgrass systems are expanding our knowledge of microbes that develop with plants and how management practices are impacting community development. Further research will improve our knowledge of these plant microbe interactions and result in downstream applications to reduce the need for fresh irrigation water, synthetic fertilizers, and pesticides necessary to maintain turfgrass. The diversity among constituents of turfgrass phytobiomes will be characterized in mature and immature cool-season turfgrass stands in the Northeast, Mid-Atlantic, and North Central United States (CT, MA, MD, MO, WI). Emphasis will be placed on identifying operational taxonomic units (OTUs) conserved across all regions which may serve as keystone representatives of the turfgrass phytobiome. Temporal changes in microbiome diversity will be assessed coincident with peak spring and fall root growth and summer root decline of perennial cool-season turfgrasses in all regions (CT, MA, MD, MO, WI). Selection of turfgrass sites will be standardized among collaborators, and specific details about each site will be recorded. Methodologies for sample collection and metagenomic analysis developed by MD-Beltsville in Objective 1.1 will be employed by collaborators. MD-Beltsville will examine the diversity of epiphytic and endophytic microbes in/on seed of various turfgrass species and cultivars. Plants will be grown under sterile conditions to compare the contribution of seed-borne organisms versus environmental cohorts in shaping the turfgrass phytobiome. Data will be shared among all collaborators in an open-source database (objective 1.2). 3. Assess the impact of turfgrass management systems on phytobiome preservation and development. Cultural management practices are integral to reducing disease and maintaining turf function on golf courses, athletic fields, and home lawns. Initial research has shown how fertilization practices can impact soil microbial communities in annual bluegrass with specific links to reductions in anthracnose disease. Moreover, research has alluded to chemical management impacts on culturable microbes in the creeping bentgrass phyllosphere. Future projects will evaluate additional cultural and chemical management practices, including organic methods for their impact on the turfgrass phytobiome. The intense management and perennial growth nature of turfgrass can also provide a model system for understanding the impact of management on the phytobiome. Moreover, identification of beneficial relationships associated with changes in management will be employed in future applications. CT, MA, MD, and NC will study the impact of turfgrass management on diversity and composition of the phytobiome. Collaborators will focus investigations on various turfgrass uses from intensively managed golf course turf to no-input residential lawn turf. CT will compare the influence of management systems including calendar-based fertilizer and pesticide lawn care, integrated pest management based, organic based, and no-input residential lawn maintenance programs on the phytobiome composition. All management systems were established at the same site in 2014; which provides a unique opportunity to study phytobiome evolution under various management inputs in the same environment. Metagenomic and bioinformatic analyses of CT samples, conducted by MD-Beltsville, will seek to identify unique OTUs among management systems; with an emphasis on those which proliferate in high performing turf stands with minimal inputs. MD, NC, and WI will evaluate the effect of various commonly used fungicide active ingredients on the phyllosphere and soil microbiome of high maintenance golf course turf. Studies will seek to address the short- and longer-term impact of fungicide use on various partitions of the turfgrass phytobiome. MA will continue to assess organic and conventionally managed golf courses for correlations between the soil microbiome and resident pathogenic and non-pathogenic nematode populations. MD will assess the role of the turfgrass phytobiome on the utility of effluent water sources as an irrigation source. The ability of various organisms to degrade, sequester, or transform common anthropogenic contaminants (e.g., personal care products, pharmaceuticals, etc.) of treated waste water may be important to expanding the use of this underutilized resource. Results will be shared and discussed among all collaborators to identify common OTUs that are promoted or suppressed by various management systems or practices. 4. Identifying constituents of the turfgrass phytobiome which confer improved abiotic and biotic stress tolerance. Candidate OTUs associated with improved turfgrass performance from phytobiome characterization studies will be identified. CT, MA, MD, MO, MD-Beltsville, NC, and WI will use bioinformational approaches developed by MD-Beltsville to screen candidates for putative plant health functions. Microbiome constituents associated with phytohormone production or regulation, siderophore production and nutrient availability, enhanced water use, or allopathic compounds will be targeted. Microbes involved in disease suppression through induced systemic resistance, antimicrobial compound production or other mechanisms will also be identified. These attributes could contribute to greater sustainability of turfgrass systems through reductions in water, nutrient, and pesticide use. In vivo and/or in situ studies will be developed to characterize plant health effects of candidate OTUs as well as mechanisms for potential application development.
Measurement of Progress and Results
- Standardized sampling procedures
- Standardized environmental DNA/RNA analysis protocols
- Open source turfgrass phytobiome database
- An index of key conserved turfgrass phytobiome constituents
- Scientific and trade journal articles
- Presentations on research results at national and regional seminars, symposia, and university field days
Outcomes or Projected Impacts
- Comprehensive knowledge of the diversity of turfgrass phytobiomes as influenced by geography, time, and management practices
- Identification of microbial candidates for use in applications to enhance sustainability of turfgrass management
Milestones(1):Develop standardized sampling and processing procedures Develop DNA analysis protocols for meta analysis
(2):Continue to develop and implement sampling, processing, and analysis protocols for phytobiome research in turfgrass systems Initiate multistate turfgrass phytobiome sampling survey
(3):Accumulate and assess turfgrass phytobiome survey results Examine the impact of current cultural and chemical management practices on phytobiome development
(4):Determine methods for impacting phytobiome development to benefit turfgrass health
(5):Continue to develop improved methods for implementing phytobiome research in turfgrass systems
Projected ParticipationView Appendix E: Participation
1. Prepare and disseminate pre- and post- regional project surveys to practitioners from all sectors of the turfgrass industry in Mid-Atlantic, Northeastern, and Mid-Western states.
2. Conduct regional and national workshops at turfgrass conferences each year to educate stakeholder groups on the latest project results.
3. Use social media and additional web-based platforms, including web-casts, to educate new clientele on phytobiome research.
4. Development and distribution of publications (fact sheets, refereed journal articles, trade articles, etc.) through a dedicated regional project website.
5. Employ surveys to document changing trends in management across the turfgrass industry and assess the impact of phytobiome research applications
The organization of the regional research project will be established on the premise of shared governance where all members of the Technical Committee are active participants. Each participating institution or agency will be designate a voting member of the Technical Committee, with approval of the institution’s or agency’s director. Other individuals and interested parties from each participating institution or agency are encouraged to participate as non-voting members of the committee. Each year, members will elect a Secretary. The Secretary, whose duties begin the following year, becomes Chair-elect in year two, followed by Chair the third year.
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