ENDOPHYTIC FUNGI FOR BIOLOGICAL CONTROL, PLANT PERFORMANCE ENHANCEMENT, AND SOURCES OF NEW COMPOUNDS FOR AGRICULTURAL AND HUMAN HEALTH A

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0213280
• Project Start Date: Jan 15, 2008
• Project End Date: Jan 14, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Plant Biology

Non Technical Summary
All plants maintain associations with fungal endophytes and epibionts. These associations between fungi and plants are generally a cryptic phenomenon in nature. Fungal endophytes may inhabit tissues of roots, stems, branches, twigs, bark, leaves, petioles, flowers, fruit, and seeds, including xylem of all available plant organs. These fungi have been found to impact on the ecology of plants, frequently enhancing capacity of host plants to survive and resist environmental and biological stresses through mechanisms that are only partially understood. In this project we will explore endophtes and their secondary metabolite products in a broad range of plants that have not yet been explored for these symbionts; and we will evaluate the roles of reactive oxygen species and secondary metabolites in the hypha-plant cell interactions in the clavicipitacious endophyte-grass symbiosis. Endophytes frequently appear to function as defensive mutualists to their grass hosts helping plants to survive drought, insect attack or pathogen infections. Much of the work of this proposal is targeted at identifying the defensive secondary metabolites and evaluating their utility as biorationals for agricultural applications in insect or microbial pathogen control and for medicinal applications. We will also seek to develop a more complete understanding of the ecology and physiological interactions of endosymbiotic fungi and their host plants. This research will directly impact New Jersey stakeholders in that our model system for examining the endophyte-plant interaction are the grass endophytes that are present in many turfgrasses. One of the key ecological outcomes of endophytes in turfgrasses is enhanced disease resistance. We will seek to understand the mechanism of disease resistance enhancement. Because the turfgrass industry is important in New Jersey and the United States this research has the potential to develop information that may be useful in producing more disease resistant turf cultivars and have a positive impact in the industry.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	0613	1020	25%
206	0730	1102	25%
215	0620	1070	25%
314	0680	1102	25%

Knowledge Area
206 - Basic Plant Biology; 204 - Plant Product Quality and Utility (Preharvest); 314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals; 215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
0730 - Mountain grasslands, meadow, and alpine; 0680 - Other products of the forest; 0620 - Broadleaf forests of the North; 0613 - Mixed conifer-broadleaf forests;

Field Of Science
1070 - Ecology; 1102 - Mycology; 1020 - Physiology;

Keywords

fungal endophytes

plant stress resistance

new pesticides

new fungicides

new medicines

ecology

Goals / Objectives
In this project we will explore endophtes and their secondary metabolite products in a broad range of plants that have not yet been explored for these symbionts; and we will evaluate the roles of reactive oxygen species and secondary metabolites in the hypha-plant cell interactions in the clavicipitacious endophyte-grass symbiosis. Endophytes frequently appear to function as defensive mutualists to their grass hosts. Much of the work of this proposal is targeted at identifying the defensive secondary metabolites and evaluating their utility as biorationals for agricultural applications in insect or microbial pathogen control and for medicinal applications. We will also seek to develop a more complete understanding of the ecology and physiological interactions of endosymbiotic fungi and their host plants. Specific objectives of this research are as follows: 1) Conduct studies to examine the effects of endophyte-produced reactive oxygen species (ROS) and secondary metabolites and test the hypothesis that these endophyte products play key roles in nutrient acquisition by endophytic mycelium from host cells and secondarily enhance performance of plants in enhancing disease resistance and stress tolerance in general; 2) Identify plants that naturally host epibionts and endophytes. Research on endophytes and epibionts in the fungal family Clavicipitaceae suggests that both epiphytes and endophytes are potentially valuable as producers of secondary metabolites. We intend to begin collection and screening of epibionts in the next period of this project; 3) Continue development of the strain collection library for natural product screening and agricultural applications; 4) Culture fungi on a range of media (including those containing known elicitors and screen extracts for bioactivity in program screens (Rutgers University and partner countries) and in collaboration with industrial partners; 5) Pursue scientific outputs using sampling and data-mining strategies employing the NAPIS database, including descriptions of new species, testing of the hypothesis that bioactive endophytes are encountered more frequently in plants in favorable habitats, and testing of the hypothesis that epiphytes of specific plant tissues rich in plant nutrients (e.g., meristems) are also rich as sources of bioactive metabolites. The nutritional abundance hypothesis predicts that endophytes or epibionts in or on plant tissues where they are exposed to abundant nutrients expend more energy in production of secondary metabolites.

Project Methods
Surveys of plants for endophytes and epibionts in several natural vegetation regions will be conducted through use of the grow out technique to assess for the presence of the symbiotic fungi. Comparative studies will be conducted and should permit us to increase support for either the secondary metabolite fungal disease defense mechanism or the ROS fungal defense mechanism. Many of the secondary metabolites produced by endophytes are alkaloids. The ergot alkaloids have been shown using animal cell systems to affect electric potentials of cells by inhibiting the proton pumping system in cell membranes. We intend to evaluate whether alkaloids are affecting plant cell membrane electric potentials using the fluorescent dyes as a probes to detect membrane potential variations. These studies will permit us to test the hypothesis that endophyte-produced secondary metabolites function, at least in part, as modifiers of host cell physiology to permit acquisition of nutrients by endophytes from the closely associated host cells through a membrane depolarization mechanism resulting in increased leakage of nutrients from plant cells.

Progress 01/15/08 to 01/14/13

Outputs
OUTPUTS: In this project we worked to resolve questions regarding systematics and distributions of endophytic fungi and to work out aspects of the biology of the organisms. For the Epichloe/Neotyphodium endophytes we demonstrated that the fungi produce cryptic conidial states on plants that may serve to vector the endophytes between plants. We also described a new species of endophyte Epichloe poae. A significant body of knowledge was developed on the endophytes and pathogens belonging to ascomycete family Gnomoniaceae. PARTICIPANTS: Dr. Mariusz Tadych (Research Associate), Dr. Monica Torres (Instructor) and Dr. Marshall Bergen (Program Associate) worked on aspects of this project. Several undergraduate (Robert Mattera and Emily Moscowitz) and graduate students (Chris Zsambell, Richard Chen and Mohini Somu) contributed to aspects of this project. TARGET AUDIENCES: Scientists in agriculture and plant science are the primary targets for this research. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
For this project findings included: 1. Many clavicipitaceous endophytes possess cryptic conidial stages employes to vector endophytes between hosts; 2. Many species of endophytic fungi impact the ecologies of host plants world-wide. 3. Endophytic bacteria have the potential to impact the nutritional status of host plants. These major findings have an impact on how we perceive the importance and potential applications of endophytic microbes of plants. As a result it is clear that endophytic microbes may be employed to modify plant performance through enhancing oxidative stress tolerance of hosts, and potentially providing nitrogenous nutrients in the case of diazotrophic bacterial endophytes.

Publications

• Tadych, M., Ambrose, K.V., Bergen, M.S., Belanger, F.C, White, J.F., Jr. 2012. Taxonomic placement of Epichloe poae sp. nov. and horizontal dissemination to seedlings via conidia. Fungal Diversity 1-15.
• White, J.F., Jr., Crawford, H., Torres, M.S., Mattera, R., Bergen, M., Irisarry, I. 2012. A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria. Symbiosis Volume 57, Issue 3 (2012), Page 161-171; Open Access Electronic: DOI 10.1007/s13199-012-0189-8.
• Tadych, M. Bergen, M.S., Johnson-Cicalese, J., Polashock, J.J., Vorsa, N., White, J.F., Jr. 2012. Endophytic and pathogenic fungi of developing cranberry ovaries from flower to mature fruit: diversity and succession. Fungal Diversity 1-16.
• White, J.F., Jr., Bacon, C.W. 2012. The secret world of endophytes in perspective. Fungal Ecology 5: 287-288.
• Walker, D.M., Castlebury, L., Rossman, A.Y., White, J.F, Jr. 2012. New molecular markers for fungal phylogenetics: Two genes for species-level systematics in the Sordariomycetes (Ascomycota). Molecular Phylogenetics and Evolution.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: In this period we conducted work on endophytes of grasses and other plants. In terms of fungal endophytes we demonstrated that the Epichloe/Neotyphodium endophyte in the grass Poa ampla may spread contagiously from plant to plant. This work resulted in one publication and a proceedings paper. We also conducted work on bacterial endophytes of plants. We are particularly interested in bacterial endophytes that are degraded by plants for nutrients. In this vein we have shown that many bacterial endophytes in grasses and common forest vines are degraded by plants using reactive oxygen. Our hypothesis is that the bacteria are a source of nitrogen/nutrients that offsets soil deficiencies in nitrates. PARTICIPANTS: Participating in this work were Drs. Marshall Bergen, Maruisz Tadych, Monica Torres, and graduate student Ivelisse Irizarry. In addition undergraduates Robert Mattera and Emily Moskowitz participated in the work. TARGET AUDIENCES: This work is targeted at other scientists working on plant protection and applications of endophytes to enhance plant performance. PROJECT MODIFICATIONS: In this period we have increased our focus on bacterial endophytes and their significance in plants.

Impacts
Our research demonstrating potential for horizontal dissemination by clavicipitalean endophytes leads to a better understanding about how endophytes spread in grasses. It also emphasizes that such endophytes possess unseen ecologies that enable them to be successful in nature and that could limit our capacities to contain them in specific hosts. Our work on bacterial endophytes in plants may find applications in development of technologies to cultivate crops with reduced nitrogen inputs. We are currently conducting experiments to measure the nutritional contribution of bacterial endophytes to plant needs.

Publications

• White, J.F., Jr., Crawford, H., Torres, M.S., Mattera, R., Bergen, M., Irisarry, I. 2012. A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria. Symbiosis Volume 57, Issue 3. 2012, Page 161-171, Open Access Electronic DOI 10.1007/s13199-012-0189-8
• Tadych, M. Bergen, M.S., Johnson-Cicalese, J., Polashock, J.J., Vorsa, N., White, J.F., Jr. 2012. Endophytic and pathogenic fungi of developing cranberry ovaries from flower to mature fruit: diversity and succession. Fungal Diversity 1-16
• White, J.F., Jr., Bacon, C.W. 2012. The secret world of endophytes in perspective. Fungal Ecology 5: 287-288.
• Tadych, M., Ambrose, K.V., Bergen, M.S., Belanger, F.C, White, J.F., Jr. 2012. Taxonomic placement of Epichloe poae sp. nov. and horizontal dissemination to seedlings via conidia. Fungal Diversity 1-15

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We have identified a microbial endophyte system in desert plants of the families Agavaceae and Cactaceae involving bacteria. It is our hypothesis that these symbioses function to provide nitrogen to plants when soil is dry and moisture and nutrients cannot be absorbed from the soil. We have observed that endosymbiotic bacteria are held within the cytoplasm of plant root cells. The accumulation of reactive oxygen species in the vesicles results in oxidation/digestion of the bacteria. We have termed this process 'Oxidative Nitrogen Scavenging' because nitrogen fixed by the bacteria are believed to be the primary product of these endosymbioses. This symbiosis may involve nutritional (nitrogen) support but also may result in enhanced stress tolerance for the desert plants. Oxidative nitrogen scavenging from bacterial endosymbionts may be an important mechanism for plants that grow and thrive in highly arid and infertil soils. We have also found a similar symbiosis in turf grasses, where bacteria are carried on the surfaces of seeds and colonize seedlings epiphytically after germination. Preliminary N15 gas studies with agave, yucca and fescue grass seedlings support the nitrogen assimilation function for these symbioses. PARTICIPANTS: Participants include: Monica Torres (Instructor) Rutgers University, Ive Irizarry (Graduate Student) Rutgers University, Marshall Bergen (Program Associate) Rutgers University. TARGET AUDIENCES: The target audiences include agriculturists and companies interested in growing plants in dry and infertile lands. Ecologists interested in how plants grow in deserts also would be interested in this research. PROJECT MODIFICATIONS: With this work we have expanded the project to include bacterial endophytes as well as fungal endophytes.

Impacts
The endosymbiotic microbes may provide an example of habitat adaptive symbiosis. Here the plants may not be able to survive in the low nutrient deserts without its nitrogen-assimilating symbiosis. In the continuation of this project we will be gathering data to identify all microbes involved, document their distributions in plants, and further support the 'oxidative nitrogen scavenging' hypothesis. This work is important because it may lead to a better understanding of how plants grow and flourish in low nutrient environments. It may lead to new technologies for cultivating crops in infertile and dry soils.

Publications

• Torres, M.S., White, J.F., Hinton, D.M., Bacon, C.W. 2011. Endophyte-mediated adjustments in host morphology and physiology and effects on host fitness traits in grasses. Fungal Ecology. DOI:10.1016/j.funeco.2011.05.006.
• Alvarez-Loayza, P., J. F. White Jr., M. S. Torres,N. Gil, J-C. Svenning, H. Balslev, T. Kristiansen. 2011. Light converts endosybiotic fungus to pathogen, influencing seedling survival and recruitment of host. PLoS ONE 6(1):e16386.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We have identified an endophyte system in Agave involving bacteria and fungi that may be important in facilitating growth of Agave in deserts. This symbiosis may involve nutritional support and enhanced stress tolerance for the desert agaves. This may be an example of habitat adaptive symbiosis. This project is just beginning. We are also testing the hypothesis that disease suppression effects as well as abiotic stress resistance in some grass-endophyte combinations is primarily the result of production of enhanced resistance to oxidants that are generated during disease and abiotic stress situations. We hypothesize that production of auxins by endophytes is responsible for triggering plants to produce antioxidants. This higher antioxidant level reduces the oxidative bursts that are associated with some diseases, drought tolerance, and heavy metal toxicity. In the case of the dollar spot pathogen in endophyte-free grasses, the pathogen produces oxidative hydrogen peroxide that normally triggers a hypersensitivity response in the grass. This hypersensitivity reaction is the result of an oxidative burst triggered by the H2O2 produced by the pathogen. The pathogen grows in the tissues killed by the oxidative burst and forms necrotic tissue in the turf. In endophyte-infected fine fescue grass the enhanced levels of antioxidants counteract the hydrogen peroxide produced by the pathogen and prevent the oxidative burst hypersensitivity reaction and necrotic tissues from developing. Thus the endophyte appears to truncate the process of infection through oxidative burst suppression. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The agave symbiosis may provide an example of habitat adaptive symbiosis. Here the agave may not be able to survive in the low nutrient deserts without its nitrogen-assimilating symbiosis. We have also supported this concept with the work we conducted on the palm endophyte that converts to a pathogen in full sunlight due to production of ROS by the endophyte. We have proposed that most of the benefits that stem from endophyte infection in turfgrasses may be the result of enhanced antioxidant production by endophytes or other mechanisms for suppression of cell death. In the continuation of this project we will be gathering data to further support this hypothesis. We have also demonstrated that endophytes in cranberry may play some role in stimulating plants to resist disease through stimulation of antioxidants and resistance to ROS secreted by the rot fungi.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Progress to date on this project has resulted in development of the hypothesis that disease suppression effects as well as abiotic stress resistance in some grass-endophyte combinations is primarily the result of production of enhanced resistance to oxidants that are generated during disease and abiotic stress situations. Enhanced antioxidant production by plants appears to be a reaction to production of reactive oxygen species (superoxide) by endophytes. Superoxides produced by mycelium in the plant meristems may cause the plant to adjust levels of antioxidants upward. This higher antioxidant level reduces the oxidative bursts that are associated with some diseases, drought tolerance, and heavy metal toxicity. In the case of the dollar spot pathogen in endophyte-free grasses, the pathogen produces oxidative hydrogen peroxide that normally triggers a hypersensitivity response in the grass. This hypersensitivity reaction is the result of an oxidative burst triggered by the H2O2 produced by the pathogen. The pathogen grows in the tissues killed by the oxidative burst and forms necrotic tissue in the turf. In endophyte-infected fine fescue grass the enhanced levels of antioxidants counteract the hydrogen peroxide produced by the pathogen and prevent the oxidative burst hypersensitivity reaction and necrotic tissues from developing. Thus the endophyte appears to truncate the process of infection through oxidative burst suppression. We are continuing to gather data to support or refute this hypothesis. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We have proposed that most of the benefits that stem from endophyte infection in turfgrasses may be the result of enhanced antioxidant production by endophytes or other mechanisms for suppression of cell death. In the continuation of this project we will be gathering data to further support this hypothesis. Another way that endophytes may be enhancing stress tolerance is through suppression of programmed cell death (PCD) or apoptosis through stimulation of repressor proteins for programmed cell death. The cell death suppressor Bax inhibitor-1 (BI-1) is an endoplasmic reticulum (ER) membrane protein that exists in a wide range of organisms (Watanabe and Lam, 2007). BI-1 is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced cell death. The ER is associated with generation of reactive oxygen species (ROS). BI-1 can protect cells by inhibiting ER stress proteins and preventing the accumulation of ROS. By stimulation of BI-1 and antioxidants the endophytes could effectively reduce cell death and enhance the host plants resistance to plant pathogens and other types of oxidative stress, including drought and heavy metal stress.

Publications

• Rodriguez RJ, White JF Jr, Arnold AE, Redman RS.. 2009. Fungal endophytes: diversity and functional roles. New Phytol. 2009;182(2):314-30.
• Tadych, M., M. S. Torres and J. F. White. 2009. Diversity and Ecological Roles of Clavicipitaceous Endophytes of Grasses. In Defensive Mutualism in Microbial Symbiosis J. F. White, Jr. and M. S. Torres, eds., CRC Press, Boca Raton, Florida, pp. 247-256.
• White, J.F. Jr. and M.S. Torres 2009. Is plant endophyte-mediated defensive mutualism the result of oxidative stress protection Physiologia Plantarum In press.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: We screened numerous Central Asian and South American plants for presence of endophytes; and we screened numerous of these endophytes for bioactive compounds. We conducted a survey of the Clavicipitaceae for production of ergot alkaloids and lolines. We attended and presented data in several conferences, including a plenary presentation and a symposium at the 6th Congress of the Latin American Mycological Association in Mar del Plata, Argentina. PARTICIPANTS: Participating in this research were Dr. Monica Torres, a female post-doctoral student; and Patricia Alvarez, a Peruvian graduate student in J. White's lab. TARGET AUDIENCES: The target audiences are ecologists, plant biologists, plant pathologists, agronomists, and scientists in drug discovery. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
During this period work was accomplished on understanding how some endophytes are triggered by environmental factors to become pathogens in plants. We found that one endophyte, Diplodia mutila, of the palm Iriartea deltoidea may be both pathogen and mutualist, depending on light exposure. In low light Diplodia mutila is a mutualist, protecting the host from insect predators; while in high light conditions the fungus reverts to a pathogen stage and may be mortal to seedlings of its host. This is the first report of an endophyte being transformed to a pathogen by exposure to light. Work was also accomplished on understanding how endophytism affects the production of secondary metabolite production by endophytic fungi. We developed the 'Nutritional Abundance Hypothesis' to explain enhanced production of secondary metabolites by endophytic fungi. In this hypothesis the abundance of plant nutrients in the endophytic niche permits endophytes 'the luxury' to produce secondary metabolites for defensive purposes.

Publications

• Torres, M.S. and J.F. White Jr. 2008. Diversity in the family Clavicipitaceae: insect pathogens to plant endophytes. Encyclopedia of Microbiology, 3rd edition. In press
• Torres, M.S., A. Singh, N.Vorsa and J.F. White. 2008. Phylogeny and ergot alkaloid production in the Clavicipitaceae Family. Symbiosis 46: 2-9
• Alvarez, Patricia, White Jr, James, Gil, Nataly, Svenning, Jens-Christian, Balslev, Henrik, and Kristiansen, Thea. Light converts endosymbiotic fungus to pathogen, influencing seedling survival and host tree recruitment. Available from Nature Precedings (2008)