Source: UNIVERSITY OF WASHINGTON submitted to NRP
THE USE OF FUNGAL AND DIAZOTROPHIC ENDOPHYTES AS A MEANS FOR CLIMATE CHANGE MITIGATION AND ADAPTATION IN AGROECOSYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0229904
Grant No.
2012-68002-19824
Cumulative Award Amt.
$750,000.00
Proposal No.
2012-00931
Multistate No.
(N/A)
Project Start Date
Aug 15, 2012
Project End Date
Aug 14, 2017
Grant Year
2012
Program Code
[A3142]- Integrated Approaches to Climate Adaptation and Mitigation in Agroecosystems, AFRI
Recipient Organization
UNIVERSITY OF WASHINGTON
4333 BROOKLYN AVE NE
SEATTLE,WA 98195
Performing Department
Bioresource Science and Engineering
Non Technical Summary
This project will enable us to develop strategic adaptation plans for crop improvement using endophytes in response to the anticipated climate change. By establishing effective symbioses with nitrogen-fixing endophytic microbes, crops and trees will be able to utilize elevated CO2 while requiring less chemical fertilizers. Furthermore, improved water use efficiency and stress-tolerance by fungal endophytes are vitally important characteristics needed as the global climate changes. By utilizing beneficial microbial endophytes, tangible options to adapt to and mitigate climate change impacts on agriculture are likely to materialize more quickly than relying solely on crop improvement approaches via breeding to develop new lines. The team we have assembled for this project is multi-disciplinary, and each is an expert in the Project Aim that he or she is leading. This grant will bring together for the first time, two lines of parallel research to determine if N-fixing growth-promoting bacterial endophytes can work in concert with stress tolerance-imparting fungal endophytes. With the training of graduate students and numerous undergraduate researchers, this project will fulfill the goals of the Program, providing tools for plant adaptation to climate change, reduction of greenhouse gas emissions, increased carbon sequestration, as well as profound educational opportunities and effective outreach.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2032410110367%
2032410303033%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2410 - Cross-commodity research--multiple crops;

Field Of Science
1103 - Other microbiology; 3030 - Information and communication;
Goals / Objectives
By utilizing beneficial microbial plant symbionts to increase water use efficiency, provide essential nutrients, and improve plant growth and stress tolerance, the overall goal of this project is to develop tangible options to mitigate climate change impacts on agriculture and forestry more quickly than could be reached by relying solely on crop improvement approaches via breeding or transgenics. Using corn, rice, and Douglas-fir as model systems, we will 1) develop the most effective nitrogen-fixing endophytes for improved growth with minimal need for chemical fertilizers; 2) screen fungal endophytes to impart stress tolerance and increased water use efficiency; 3) assess the significance of endophytic symbiosis in mitigating the impacts of climate change; and 4) develop outreach programs and educational opportunities to insure that the knowledge gained in this research is widely disseminated. Through this study, we will develop optimized inoculum and methods for improved plant growth, stress tolerance, biomass, and yield of grain crops and timber forests with limited inputs of nutrients and water. Elevated CO2 stimulates crop growth most when N and water are not limiting. Thus, plants with symbiotic N fixation are most likely to capitalize on the benefits of increasing atmospheric CO2. We will evaluate the physiological benefits of the endophytic symbionts identified from Aim 1 and 2 under current and elevated CO2 conditions. Utilizing process-based crop physiology models and life cycle assessments, we will then evaluate agro-ecological and economic benefits of the use of endophytic symbionts. Outreach and dissemination of the research findings will also be a priority, through our teaching both at the university level and to K-12 groups and teachers, training graduate students and postdoctoral fellows, speaking frequently at international conferences, participation in key roles in the International Symbiosis Society, and engaging the agricultural community directly.
Project Methods
AIM 1: N2-fixing endophytes for improved growth to minimize chemical fertilizer inputs: a) Selection of endophyte inoculum: Seeds of rice and corn varieties will be selected based on agronomic interest. We will identify the optimum mixture of endophytes to achieve the best increase in plant growth under nitrogen limitation. b) Assessment of colonization: We will visualize the location of colonization. We will also test if the endophytes are within the resulting grain. c) Quantification of nitrogen fixation: Seedlings will be inoculated with the best-performing inoculum and divided into two experiments, one to assess total nitrogen under normal greenhouse conditions and the other to directly quantify nitrogen fixation using stable isotope labeling within chambers. d) Protein content and quality: We will have the corn and rice grain tested for protein content and quality. AIM 2: Fungal endophytes for stress tolerance and increasing water use efficiency: a) Stress tolerance versus growth and yield in the presence and absence of stress: We will perform temperature, salt, and water stress tests. We will examine the physiology of stress tolerance at three levels of organization: whole-plant, organ, and tissue. b) Seed germination in the presence and absence of stress: We will determine if exposing seeds with endophytes allows for seed germination under heat and salt stress. c) Bio-stacking for agricultural sustainability: We will join two areas of research: diazotrophic endophytes for reduced chemical N needs and fungal endophytes for improved stress tolerance. These experiments will determine if multiple benefits can be conferred to plants co-colonized with multiple endophytes and if co-colonization has a metabolic cost on the plant. Plants will be propagated, stress imposed and endophytes assessed. AIM 3: Effectiveness of endophytic symbiosis in elevated CO2 as an adaptive solution to climate change: Using the inoculum developed, we will assess the effectiveness of these endophytic symbiotic relationships in elevated CO2 as an adaptive solution to climate change with co-benefits of mitigating the impacts of climate change on agriculture. AIM 4: Education and Outreach: a) Local Education and Outreach: Two Ph.D. graduate students will be responsible for running a new lab course that will provide hands-on training in making media, streak-purifying bacterial colonies, inoculating media, measuring optical density, inoculating seeds, monitoring plant growth, quantifying plant health using physiological assays, and verifying colonization using fluorescent microscopy. Local outreach will be achieved through workshops for the public, high schools, and industry. b) National education and outreach: We will hold workshops to educate high school science teachers to bring symbiosis science into their classrooms. The workshops will provide access to protocols, curricula, and basic information about plant-microbe symbioses. The research funded through this grant will provide the necessary results to bring the technology to a national level. c) International education and outreach: The results from this grant will be disseminated through global networking.

Progress 08/15/12 to 08/14/17

Outputs
Target Audience:Scientists at conferences, undergraduate and graduate students in classes, general public at outreach events and through press releases; high school students, agronomic companies, foresters Changes/Problems:We were awarded a no-cost extension for an additional year. This was needed since the original co-PI Dr. Rodriguez and research scientist Regina Redman had left the project to focus on their symbiosis-based company utilizing the fungal endophytes that impart salt, heat, and drought tolerance. We attempted to isolate bacterial and yeast endophytes that confer these traits, and although we were successful, the projects require additional time to reach a commercialization stage for these new isolates. What opportunities for training and professional development has the project provided?6. Training and Development Graduate student, Shyam Kandel, completed his Ph.D. in August 2016. Graduate student, Hyungmin Rho, will complete his Ph.D. in 2018 Graduate student, Amber Hageman, was trained in Kim's lab in winter 2016 Visiting graduate student, Andrea Firrincieli, was trained in Doty's lab January-June 2015 Previous undergraduate researchers, Andrew Sher, Neil Fleck, and Pierre Joubert, were hired as research scientists, receiving further scientific training as research professionals Research scientist, Dr. Zareen Khan, gained mentoring experience working with dozens of undergraduate researchers on the project A total of 58 undergraduate researchers were trained in the laboratories of Prof. Doty and Prof. Kim. Many of these students presented their research not only at lab meetings but also at the UW Undergraduate Research Symposium, gaining experience in public speaking and scientific outreach. Pierre Joubert and Shruti Parikh were awarded Mary Gates Research Scholarships. How have the results been disseminated to communities of interest?7. Outreach Through press releases, book chapters, a book, oral and poster presentations, outreach events, and the filming of our research for the "Frontiers in Genomics" series, we disseminated our research findings broadly to the public At the global level, Doty is working with others in her role as VP for Education in the International Symbiosis Society to provide materials for course development on plant-microbe interactions (2017) Ms. Parikh presented at the Everett Public Schools STEM Fair (June 2017) Our laboratory website (http://depts.washington.edu/envaplab/) describes the projects and provides links to our press releases. A twitter account was set up for dissemination of lab news : @DotyLab What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aim 1. N2-fixing endophytes for improved growth to minimize chemical fertilizer inputs The project successfully developed microbial strains that increase the growth and resilience of major crop plants and forest trees with reduced inputs of fertilizer We published on the increased growth of rice in nutrient-limited soil with the endophyte inoculum (Kandel, et al. 2015) The strains were characterized for multiple symbiotic traits. This led to the discovery that some of the strains also have strong anti-fungal properties. This result led to a collaboration with USDA scientist, Dr. Okubara, and a current UW CoMotion Innovation Grant for commercialization of the strains. Some of the strains displayed in vitro antifungal activity to a variety of agriculturally-important fungal pathogens including F. culmorum, G. graminis var. tritici, R. solani and P. ultimum. A consortium of Salicaceae endophyte strains increased the growth of Douglas-fir in nutrient-limited soil (Khan, et al 2015). Additional endophyte strains were isolated from wild conifers. This project led to a more extensive study by graduate student, Matthew Aghai, who will submit a manuscript in Nov. 2017 Our manuscript on the genomic analysis of the strongest plant growth-promoting endophyte, WP1, was published in Frontiers in Microbiology in 2015. This research involved visiting graduate student, Andrea Firrincieli, with whom we have continued to collaborate on the genomic analyses of more of the endophyte strains. As part of Aim 1, we studied the colonization of the endophytes in multiple plant species and varieties. A key finding was that non-responsive maize lines were not deficient in colonization. A manuscript on the colonization and distribution of endophytes was submitted (Kandel, Joubert, and Doty, in review) Molecular analysis of N-fixation by the endophyte strains is continuing through collaborations with the Pacific NW National Labs EMSL and the Dept. of Energy JGI. It was discovered that nitrogenase gene expression is elevated in microbial aggregates, suggesting that quorum sensing is involved. Random barcoded Tn mutagenesis of one of our diazotrophic strains was conducted with successful saturation of the genome. Experiments to determine the genes required for colonization and N-fixation are in progress as part of JGI CSP project #502944 Aim 2. Endophytes for stress tolerance (drought, salt, heat) and increasing water use efficiency A consortium of the Salicaceae endophyte strains significantly increased drought tolerance in both poplar (Khan, et al 2016) and the conifer species, Douglas-fir and western redcedar (manuscript in preparation). Production of phytohormones and identification of microbial genes putatively involved in conferring drought tolerance including acetoin, butane-2,3-diol, and trehalose biosynthesis, suggests their potential roles in the modulation of the plant host stress response. The same consortium of strains increased water use efficiency in rice. A manuscript on this topic was submitted Nov 2017 (Rho, et al) A salt-tolerant endophyte of a marine estuary plant was identified as Bacillus megaterium and shown to increase the salt tolerance of maize varieties. Metabolic profiling through LCMS of endophyte-colonized maize roots versus un-inoculated revealed a number of altered metabolites, including increases in an oxylipin associated with defense and stress responses,10-oxo-11-phytoenoic acid. This work was conducted by UCSD graduate student, Andrew Sher, a previous undergraduate researcher and research scientist of Doty's lab. Undergraduate researchers, Shruti Parikh, in Doty's lab isolated an additional 15 salt-tolerant strains from marine estuary plants. These strains are currently being screened on poplar and bush beans to see whether they confer salt tolerance Experiments to isolate heat tolerance-conferring endophytes have been challenging but did result in an inoculum that significantly improved the resilience of rice to repeated and prolonged heat stress. Aim 3. Effectiveness of endophytic symbiosis in elevated CO2 as an adaptive solution to climate change Rice grown in a low N condition showed increases in expression level of rbcS gene in inoculated samples grown under both ambient and elevated CO2 (66.2 and 7.4% increases in gene copy number compared to controls, respectively). This suggests that N-fixing endophytes contributed to increases in photosynthetic capacity of rice. Under a low N regime, inoculated rice under ambient/elevated CO2 showed 4.7/11.8% increases in total biomass in an autumn growing season. Under a high N regime, total biomass gains of inoculated rice were increased by 46.6/8.9% under ambient/elevated CO2. There appears to be interaction effects between N level and CO2, which is further to be statistically tested in the future. Under a low N regime, in an autumn growing season, inoculated maize showed a 14.2% increase and a 9.2% decrease in total biomass under ambient and elevated CO2 conditions, respectively. Under a high N regime, in a spring growing season, a 5.8% decrease and a 26.8% increase in total biomass were observed in inoculated maize plants. Although there was a seasonal variation in the comparison, it is likely that maize benefits more from the symbionts in low N under ambient CO2 and high N under elevated CO2 conditions. Aim 4. Broader Impacts The project provided research training for 6 graduate students, 2 of whom were directly funded by the grant, and 57 undergraduate research students of broad ethnic diversity We established a new Plant Microbiology Lab course that taught a total of 85 students over 4 years We licensed the plant growth promoting microbial strains to multiple agricultural companies with broad global markets. While the grant project focus was on maize, rice, and Douglas-fir, the endophyte strains performed well in their greenhouse trials on such diverse crops as strawberries, kale, lettuce, broccoli, grapes, and stone fruit trees. Over the five years, we presented the results at numerous conferences, many of which were international To date, we have 12 manuscripts published or in press, and have 2 more currently in review and 1 to be submitted later this month.

Publications


    Progress 08/15/16 to 08/14/17

    Outputs
    Target Audience:Scientists at conferences, undergraduate and graduate students in classes, general public at outreach events and through press releases; high school students, agronomic companies, foresters Changes/Problems:We were awarded a no-cost extension for this additional year. This was needed since the original co-PI Dr. Rodriguez and research scientist Regina Redman had left the project to focus on their symbiosis-based company utilizing the fungal endophytes that impart salt, heat, and drought tolerance. We attempted to isolate bacterial and yeast endophytes that confer these traits, and although we were successful, the projects require additional time to reach a commercialization stage for these new isolates. What opportunities for training and professional development has the project provided? Graduate student, Shyam Kandel, completed his Ph.D. in August 2016. Graduate student, Hyungmin Rho, will complete his Ph.D. in 2018 Graduate student, Amber Hageman, was trained in Kim's lab in winter 2016 Previous undergraduate researcher, Pierre Joubert, was hired as a research scientist, receiving further scientific training as a research professional Research scientist, Dr. Zareen Khan, gained mentoring experience working with dozens of undergraduate researchers on the project Undergraduate researchers were trained in the laboratories of Prof. Doty and Prof. Kim. Many of these students presented their research not only at lab meetings but also at the UW Undergraduate Research Symposium, gaining experience in public speaking and scientific outreach. Pierre Joubert and Shruti Parikh were awarded Mary Gates Research Scholarships. How have the results been disseminated to communities of interest? Through press releases, book chapters, a book, oral and poster presentations, outreach events, and the filming of our research for the "Frontiers in Genomics" series, we disseminated our research findings broadly to the public At the global level, Doty is working with others in her role as VP for Education in the International Symbiosis Society to provide materials for course development on plant-microbe interactions (2017) Ms. Parikh presented at the Everett Public Schools STEM Fair (June 2017) Our laboratory website (http://depts.washington.edu/envaplab/) describes the projects and provides links to our press releases. A twitter account was set up for dissemination of lab news : @DotyLab What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Aim 1. N2-fixing endophytes for improved growth to minimize chemical fertilizer inputs The project successfully developed microbial strains that increase the growth and resilience of major crop plants and forest trees with reduced inputs of fertilizer The strains were characterized for multiple symbiotic traits. This led to the discovery that some of the strains also have strong anti-fungal properties. This result led to a collaboration with USDA scientist, Dr. Okubara, and a current UW CoMotion Innovation Grant for commercialization of the strains. Some of the strains displayed in vitro antifungal activity to a variety of agriculturally-important fungal pathogens including F. culmorum, G. graminis var. tritici, R. solani and P. ultimum. A consortium of Salicaceae endophyte strains increased the growth of Douglas-fir in nutrient-limited soil (Khan, et al 2015). Additional endophyte strains were isolated from wild conifers. This project led to a more extensive study by graduate student, Matthew Aghai, who will submit a manuscript in Nov. 2017 As part of Aim 1, we studied the colonization of the endophytes in multiple plant species and varieties. A key finding was that non-responsive maize lines were not deficient in colonization. A manuscript on the colonization and distribution of endophytes was submitted (Kandel, Joubert, and Doty, in review) Molecular analysis of N-fixation by the endophyte strains is continuing through collaborations with the Pacific NW National Labs EMSL and the Dept. of Energy JGI. It was discovered that nitrogenase gene expression is elevated in microbial aggregates, suggesting that quorum sensing is involved. Random barcoded Tn mutagenesis of one of our diazotrophic strains was conducted with successful saturation of the genome. Experiments to determine the genes required for colonization and N-fixation are in progress as part of JGI CSP project #502944 Aim 2. Endophytes for stress tolerance (drought, salt, heat) and increasing water use efficiency A consortium of the Salicaceae endophyte strains significantly increased drought tolerance in both poplar (Khan, et al 2016) and the conifer species, Douglas-fir and western redcedar (manuscript in preparation). Production of phytohormones and identification of microbial genes putatively involved in conferring drought tolerance including acetoin, butane-2,3-diol, and trehalose biosynthesis, suggests their potential roles in the modulation of the plant host stress response. The same consortium of strains increased water use efficiency in rice. A manuscript on this topic was submitted Nov 2017 (Rho, et al) A salt-tolerant endophyte of a marine estuary plant was identified as Bacillus megaterium and shown to increase the salt tolerance of maize varieties. Metabolic profiling through LCMS of endophyte-colonized maize roots versus un-inoculated revealed a number of altered metabolites, including increases in an oxylipin associated with defense and stress responses,10-oxo-11-phytoenoic acid. This work was conducted by UCSD graduate student, Andrew Sher, a previous undergraduate researcher and research scientist of Doty's lab. Undergraduate researchers, Shruti Parikh, in Doty's lab isolated an additional 15 salt-tolerant strains from marine estuary plants. These strains are currently being screened on poplar and bush beans to see whether they confer salt tolerance Experiments to isolate heat tolerance-conferring endophytes have been challenging but did result in an inoculum that significantly improved the resilience of rice to repeated and prolonged heat stress. Aim 3. Effectiveness of endophytic symbiosis in elevated CO2 as an adaptive solution to climate change Rice grown in a low N condition showed increases in expression level of rbcS gene in inoculated samples grown under both ambient and elevated CO2 (66.2 and 7.4% increases in gene copy number compared to controls, respectively). This suggests that N-fixing endophytes contributed to increases in photosynthetic capacity of rice. Under a low N regime, inoculated rice under ambient/elevated CO2 showed 4.7/11.8% increases in total biomass in an autumn growing season. Under a high N regime, total biomass gains of inoculated rice were increased by 46.6/8.9% under ambient/elevated CO2. There appears to be interaction effects between N level and CO2, which is further to be statistically tested in the future. Under a low N regime, in an autumn growing season, inoculated maize showed a 14.2% increase and a 9.2% decrease in total biomass under ambient and elevated CO2 conditions, respectively. Under a high N regime, in a spring growing season, a 5.8% decrease and a 26.8% increase in total biomass were observed in inoculated maize plants. Although there was a seasonal variation in the comparison, it is likely that maize benefits more from the symbionts in low N under ambient CO2 and high N under elevated CO2 conditions. Aim 4. Broader Impacts The project provided research training for 6 graduate students, 2 of whom were directly funded by the grant, as well as a number of undergraduate research students in our laboratories and in the plant microbiology lab classes We licensed the plant growth promoting microbial strains to multiple agricultural companies with broad global markets. While the grant project focus was on maize, rice, and Douglas-fir, the endophyte strains performed well in their greenhouse trials on such diverse crops as strawberries, kale, lettuce, broccoli, grapes, and stone fruit trees. To date, we have 12 manuscripts published or in press, and have 2 more currently in review and 1 to be submitted later this month.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Khan, Z., Rho, H., Firrincieli, A., Hung, S.H., Luna, V., Masciarelli, O., Kim, S.-H., and Doty, S.L. 2016. Growth enhancement and drought tolerance of hybrid poplar upon inoculation with endophyte consortia. Current Plant Biology 6:38-47. [Although this paper was on poplar, it was the test of the strains later used in rice and conifers by Rho, et al. and Aghai, et al., respectively]
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Shyam L. Kandel, Andrea Firrincieli, Natalie Leston, Kendra McGeorge, Patricia A. Okubara, Giuseppe Scarascia Mugnozza, Antoine Harfouche, Soo-Hyung Kim, and Sharon L. Doty. 2017. Bio-control and plant growth promotion potential of Salicaceae endophytes. Frontiers in Microbiology volume 8, article #386.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Hyungmin Rho, Marian Hsieh, Shyam L. Kandel, Johanna Cantillo, Matthew Flora-Tostado, Sharon L. Doty, and Soo-Hyung Kim. 2017. Do endophytes promote growth of host plants under stress? A meta-analysis on plant stress mitigation by endophytes. Microbial Ecology doi/10.1007/s00248-017-1054-3
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: P. Okubara, N. Kumar, L. Hohenwarter, D. Graham, S. Kandel, S.L. Doty, Ute Micknass, Karl-Heinz Kogel, and Jafargholi Imani. 2017. Inhibition of fungal and bacterial plant pathogens by VEGLYS�, an Allium-based antimicrobial formulation. J Biol. & Nature 8(2)40-51.
    • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Shyam Kandel, Pierre Joubert, and Sharon L. Doty. Bacterial endophyte colonization and distribution. Submitted 3Nov2017
    • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Hyungmin Rho, Soo-Hyung Kim, and Sharon L. Doty. Salicaceae endophytes modulate stomatal behavior and increase water use efficiency in rice. Submitted 7Nov2017
    • Type: Book Chapters Status: Published Year Published: 2017 Citation: Sharon L. Doty. 2017. Chapter 1: Functional Importance of the Plant Endophytic Microbiome: Implications for Agriculture, Forestry and Bioenergy. In Functional Importance of the Plant Endophytic Microbiome: Implications for Agriculture, Forestry and Bioenergy. Sharon L. Doty, editor. Springer doi: 10.1007/978-3-319-65897-1
    • Type: Book Chapters Status: Published Year Published: 2017 Citation: Sharon L. Doty. 2017. Chapter 2: Endophytic Nitrogen Fixation: Controversy and a Path Forward. In Functional Importance of the Plant Endophytic Microbiome: Implications for Agriculture, Forestry and Bioenergy. Sharon L. Doty, editor. Springer doi: 10.1007/978-3-319-65897-1
    • Type: Journal Articles Status: Other Year Published: 2017 Citation: Matthew Aghai, Zareen Khan, Gregory Ettl, and Sharon L. Doty. Evaluating microbial endophyte consortia for enhancing Douglas-fir and western redcedar regeneration. Manuscript in preparation.
    • Type: Other Status: Published Year Published: 2016 Citation: Sharon L. Doty. 2016. Key roles of the poplar microbiome. Poplar and Willow News. Issue #6.
    • Type: Other Status: Published Year Published: 2016 Citation: Sharon L. Doty. 2016. Editorial: Plant-microbe symbiotic interactions. Plant Molecular Biology 90(6)1.
    • Type: Book Chapters Status: Published Year Published: 2017 Citation: Hyungmin Rho and Soo-Hyung Kim. 2017. Chapter 4: Endophyte Effects on Photosynthesis and Water Use of Plant Hosts: A Meta-Analysis. In Functional Importance of the Plant Endophytic Microbiome: Implications for Agriculture, Forestry and Bioenergy. Sharon L. Doty, editor. Springer doi: 10.1007/978-3-319-65897-1
    • Type: Books Status: Awaiting Publication Year Published: 2017 Citation: Pierre M. Joubert and Sharon L. Doty. Endophytic Yeasts: Biology, Ecology and Applications. In Endophytes of Forest Trees: Biology and Applications. 2nd edition. Pirttila and Frank, eds. Springer. In press.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: " Prof. Doty gave an invited presentation on other aspects of endophytes (phytoremediation) at the Plant and Animal Genomics (PAG) conference in San Diego (Jan 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Prof. Doty gave poster presentations on N-fixation at the PAG conference (Jan 2016), the JGI Annual Genomics of Energy and Environment conference (March 22-24, 2016) and at the Symbiosis Conference in Yosemite (May 6-8, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Graduate student Andrew W. Sher gave a poster presentation at the Symbiosis Conference in Yosemite entitled Uncovering the molecular mechanisms of salt stress tolerance imparted by endophytic symbiosis (May 6-8, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Undergraduate researcher, Pierre Joubert, gave an oral presentation at the UW Undergraduate Research Symposium (May 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Undergraduate researcher, Victor Van Epps and Nick Chandler, gave an oral presentation at the UW Undergraduate Research Symposium (May 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Prof. Doty, Dr. Khan, and undergraduate researcher Pierre Joubert gave poster presentations at the International Congress on Molecular Plant-Microbe Interactions in Portland (July 17-21, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Prof. Doty gave an invited presentation at the International Congress on N-Fixation in Non-Legumes in Budapest, Hungary (Aug 22-25, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Prof. Doty gave an invited presentation at the International Poplar Commission meeting in Berlin, Germany (Sept 13-16, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Prof. Doty gave an invited talk and undergraduate researcher Pierre Joubert gave a poster presentation at the NW Branch of the American Society for Microbiology (Nov 18-19, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Prof. Doty gave an invited talk at the Plant and Animal Genomics conference (Jan 14-18, 2017). Although the primary topic was phytoremediation, the importance of the phytobiome for increased growth and health under stress (nutrient, drought, salt) was also described.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Prof. Doty and undergraduate researcher, Pierre Joubert, gave poster presentations at the annual DOE Joint Genome Institute meeting (2017)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Prof. Doty gave a talk at the Yosemite Symbiosis Workshop (March 6-7, 2017) entitled Drought tolerance in poplar, rice, maize, and conifers with endophytes of wild Salicaceae.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Undergraduate researchers, Joubert, Parikh, Joseph, and Scharffenberger gave poster presentations at the UW Undergraduate Research Symposium (May 2017)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Prof. Doty gave a plenary presentation at the International Congress on N-fixation in Granada, Spain (Sept 3-7, 2017)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Rho gave an oral presentation on endophyte effects on growth promotion and fruit quality improvements in apple at the American Society for Horticultural Science Crop Science in Waikoloa, Hawaii (Sep. 19-22, 2017).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Prof. Doty gave a presentation at the International Phytotechnologies Conference in Quebec, Canada (Sept 25-29, 2017).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Rho gave an oral presentation on meta-analysis of endophyte effects on host plant stress responses and ecophysiology at the Crop Science Society of America in Tampa, Florida (Oct. 22-25, 2017).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Undergraduate researcher, Shruti Parikh, presented on the projects in the Freshman Interest Group class-- UW General Studies 199 (Nov 2016), the UW Summer Undergraduate Research Symposium (August 2017), the UW Program on the Environment-- Introduction to Capstone Research Session (October 2017), and in 2017, the UW- Program on the Environment Capstone Symposium and at the CoMotion Mary Gates Innovation Seminar/Workshops
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Research Scientist, Pierre Joubert, gave an invited seminar presentation entitled, "The plant microbiome: Implications of the ecosystem within" at Washington State University as part of their Plant Pathology Seminar Series (2017)


    Progress 08/15/15 to 08/14/16

    Outputs
    Target Audience:Target audiences include scientists at conferences, undergraduate and graduate students in classes, the general public at the Pacific Science Center, high school students, agronomic companies, and foresters. Changes/Problems:We were awarded a no-cost extension for an additional year. This was needed since the original co-PI Dr. Rodriguez and research scientist Regina Redman had left the project to focus on their symbiosis-based company utilizing the fungal endophytes that impart salt, heat, and drought tolerance. We are focusing instead on bacterial and yeast endophytes that confer these traits, but they first needed to be isolated and characterized. What opportunities for training and professional development has the project provided?We have three Ph.D. students: one in Doty's lab (Kandel), one in Kim's lab (Rho), and one in our collaborator Huffaker's lab (Sher) who were involved in the projects, gaining training and experience in plant microbiology, physiology, microscopy, and molecular biology. Kandel completed writing his dissertation and successfully defended it on August 5, 2016. Graduate student Lisa Hannon in Doty's lab began testing the impact of diazotrophic endophytes from Hawaiian coffee plants on maize under nutrient-limited conditions. Undergraduate researcher Joubert joined Doty's lab in October 2015 and was trained in doing genomic DNA preps, sequence analysis, GenBank submissions, subcloning, Tn mutagenesis, fluorescent microscopy, and plant assays. Undergraduate researcher Nick Wegley learned a variety of plant physiology skills. Benjamin Austin joined Doty's lab in summer 2016 for his capstone project, and is learning plant microbiology techniques. Graduate student Amber Hageman was trained in Kim's lab in winter 2016 and gained experience in plant physiology and molecular biology during her rotation. Undergraduate researcher Victor Van Epps, who joined Kim's lab in the previous reporting year, continued his training with Rho and has been conducting capstone project experiments from summer 2015 throughout summer 2016. Undergraduate researcher Norah Hummel performed her experiment in summer 2015 and finished her capstone project in autumn 2015. Undergraduate researchers Alexander Francisco and Nick Chandler joined Kim's lab in summer 2015 and learned various plant microbiology, physiology, and statistical analysis techniques. Undergraduate researcher Victor Jackson and Erich Hoffer joined Kim's lab in 2016, and are learning plant physiology and microbiology skills. How have the results been disseminated to communities of interest?The syllabus and protocols for the Plant Microbiology Lab course developed by Doty and Kim with NIFA funding was provided to our new collaborator, Professor Huffaker for development into a course at UCSD. Approval was obtained to develop the course as a summer option taught by Doty's research scientist (Khan) at University of Washington next year. For Aim4b, impacts at the national level, several agriculture companies are commercializing the endophyte strains from Doty's lab. At the global level, Doty is working with others in her role as VP for Education in the International Symbiosis Society to provide materials for course development on plant-microbe interactions. Since July 2015, Doty gave presentations at the Agricultural Bioscience International Conference in Melbourne, Australia, the International Conference on Nitrogen Fixation at the Asilomar conference center in Pacific Grove, CA, a Science Café in Olympia, Washington, the International Congress on Frontiers in Biology in Brasilia, Brazil, the Plant and Animal Genomics conference in San Diego, the Symbiosis Workshop in Yosemite, and the International Congress on Molecular Plant-Microbe Interactions in Portland. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. N2-fixing endophytes for improved growth to minimize chemical fertilizer inputs Quantify N-fixation in rice inoculated with wild-type diazotrophic strains compared to with the nitrogenase mutant strains Submit a manuscript on the construction and characterization of the nitrogenase mutants and their impact on plant growth Complete the characterization of multiple symbiotic traits including anti-fungal activity of the diazotrophic endophytes and re-submit the revised manuscript Complete the colonization assays comparing the patterns of individual and consortia of endophyte strains on maize and rice with on the native host, and submit a manuscript Complete the conifer N study and submit a manuscript Continue the conifer drought study Aim 2. Endophytes for stress tolerance (drought, salt, heat) and increasing water use efficiency Complete the manuscript on water use relations in maize and rice, and submit it for publication Complete the analysis of the impact of the endophytes on Douglas-fir under normal and drought stress conditions. Submit a manuscript. Quantify the impacts of the salt-tolerant endophyte strain, AWS1, on germination of maize and rice. Complete the initial characterization of AWS1, repeat the maize salt stress experiments, conduct the rice salt experiments with and without AWS1, and submit a manuscript on endophyte-conferred salt tolerance Isolate endophyte strains from plants in the semi-arid, high temperature environments of eastern Washington, and screen for those that confer heat tolerance to rice. Begin characterization of the new isolates. Submit the revised meta-analysis manuscript: endophyte effects on plant stress physiology. Aim 3. Effectiveness of endophytic symbiosis in elevated CO2 as an adaptive solution to climate change Conduct qPCR analysis on rbcL and rbcS. Complete the manuscript on photosynthetic improvement with endophytes in rice under elevated CO2. Conduct a new chamber experiment for plant N cost analysis with a nifH mutant line developed in Aim 1. Aim 4. Broader Impacts Teach the Plant Microbiology Seminar in winter 2017 and the Plant Microbiology Lab class in summer 2017 Continue the training of graduate students, Rho and Sher, and undergraduate researchers, Joubert and Austin Doty is an invited speaker at the international conference on N-Fixation in Non-Legumes to be held in Budapest, Hungary in August 2016 Doty will give an invited presentation at the American Society for Microbiology NW branch meeting in October 2016. Joubert will give a poster presentation Doty will be giving an invited seminar for the Microbiology Department at the University of Washington in November 2016 Rho will give an oral presentation on photosynthesis and water relations improvements by endophytes at the Crop Science Society of America Conference in Phoenix, Arizona in November.6-9 2016. As part of the executive team of the International Symbiosis Society, Doty will be preparing teaching materials for the website and help to organize the next meeting in 2018 in Oregon

    Impacts
    What was accomplished under these goals? 1. N2-fixing endophytes for improved growth to minimize chemical fertilizer inputs 1a) Selection of inoculum. We assessed the strains for phosphate solubilization, siderophore production, phytohormone production, and antifungal activity to select the most beneficial strains for agricultural uses. By understanding the relative importance of N-fixation to these other symbiotic traits, we will be able to use genomic screening to isolate superior strains for inoculations. Our most active diazotrophic endophyte, WP9, was subjected to Tn mutagenesis and screened for the inability to grow on N-free medium. This project (Joubert) resulted in one mutant, AO1. We cloned the nitrogenase nifHD region from diazotrophic strain WP40 and did in vitro Tn mutagenesis. We previously used this procedure with WP5 to create a nifH mutant. In collaboration with the JGI/EMSL to identify the most active diazotrophic strains from wild poplar, cuttings were exposed to 15N2 gas and fixed in PFA for analysis by nifH FISH and NanoSIMS. 1b) Assessment of colonization. Kandel used fluorescent microscopy to address if the reason why some lines of maize and rice did not respond to the endophytes was due to an inability to colonize. He discovered all lines were well colonized. He verified the plant growth promoting endophytic yeast strain, WP1, is capable of migrating through these monocots by an as yet unidentified mechanism. 1c) Quantification of N-fixation. Under axenic conditions, rice plants inoculated with the WP5 nifH mutant grew poorly in N-limited medium, suggesting N-fixation was necessary for improved growth of endophyte-inoculated rice. There was reduced growth of rice inoculated with the mutant compared to the mock-inoculated controls, suggesting there may be a cost of the symbiosis to the plant. The rice seeds were germinated in ­15N labeled water, then quantified for ­15N dilution at the end of the experiment. Mock and mutant inoculated plants contained 7 times more ­15N than the wild type inoculated group suggesting a significant dilution of ­15N due to N-fixation by the wild type endophyte. Kandel conducted similar experiments in greenhouse conditions; differences were not statistically significant. It is likely the mock or mutant inoculated plants are colonized by diazotrophic strains present in the greenhouse. We initiated plant assays with WP9 mutant, AO1. Rice was germinated in 15N-labeled ammonium sulfate as for the WP5 mutant study and then used in an axenic experiment (Joubert) and a greenhouse study (Khan). These experiments are in progress. We isolated new endophytes from conifers and characterized them for symbiotic traits. We chose a consortia of the best strains to study effects of inoculation with and without the presence of fertilizer on two conifer species: Douglas-fir and Western redcedar, in long-term greenhouse experiments (Khan and Agahi). There is a trend with the inoculated plants being taller with more root diameter growth in the presence of fertilizer. Physiological data including chlorophyll fluorescence (fv/fm) and chlorophyll content (SPAD) during the growing season (2015-16) highlight trends suggesting the inoculated seedlings are benefitting from endophyte inoculations. Since the impact is only seen when N is added, it may be these diazotrophic strains are not effectively fixing N in the conifers. We collected more conifer samples from harsher environments in August 2016 and have begun processing the tissues to select more active strains. 2. Endophytes for stress tolerance and increasing water use efficiency A consortium of ten strains of the diazotrophic endophytes increased drought survival of poplar plants (Khan, et al 2016). Production of phytohormones and identification of microbial genes putatively involved in conferring drought tolerance including acetoin, butane-2,3-diol, and trehalose biosynthesis, suggests their potential roles in the modulation of the plant host stress response. The same consortium was used with maize (var. 29B7) and rice (var. M206). Inoculated plants used less water than did the mock-inoculated control plants throughout the growing season and had higher water use efficiency especially under drought conditions (Rho and Wegley). Aghai with the help of Dr. Khan set up a drought study with Douglas-fir and Western redcedar seedlings using the same "drought mix" inoculum. The seedlings were either inoculated or mock-inoculated and treated to moisture regimes that stimulated moist, normal or dry precipitation conditions respectively. Analysis is underway. To isolate salt-tolerance-conferring endophytes, research technologist Sher and Doty collected estuary plant endophytes that could grow on 3% NaCl. Dominant strains were streak-purified and cryogenically stored. In collaboration with the Huffaker Lab where Sher is now a graduate student, the most robust strain, AWS1, was identified as Bacillus megaterium and shown to increase the salt tolerance of maize varieties. Inoculated seedlings demonstrated a more than three-fold increase in foliar fresh weight biomass and a more robust root system under salt stress conditions as compared to un-inoculated plants. Metabolic profiling through LCMS of endophyte-colonized maize roots versus un-inoculated revealed altered metabolites, including increases in an oxylipin associated with defense and stress responses,10-oxo-11-phytoenoic acid. In a study done by Khan, there was a higher germination of rice when inoculated with AWS1 under high salt conditions in a sterile environment. Khan is conducting greenhouse experiments of rice with and without AWS1 in salty soil. Rho reviewed the literature on endophytes conferring stress tolerance on host plants and meta-analyzed 82 articles. Overall effect sizes on gained biomass of the host plants under nitrogen/salt/drought stress conditions were significantly positive, meaning endophytes increase stress tolerance of plants as a general response. 3: Effectiveness of endophytic symbiosis in elevated CO2 as an adaptive solution to climate change Further analysis of the 2014 (rice grown in low N condition) samples showed increases in expression level of rbcS gene in inoculated samples grown under both ambient and elevated CO2 (66.2 and 7.4% increases in gene copy number compared to controls, respectively). This suggests N-fixing endophytes contributed to increases in photosynthetic capacity of rice. We are repeating this assay to confirm the findings, including quantification of rbcL gene. We performed data analysis collected from the previous two years' CO2 chamber experiments with different growing conditions; maize/rice grown in low/high N regimes under ambient/elevated CO2 conditions. Under a low N regime, inoculated rice under ambient/elevated CO2 showed 4.7/11.8% increases in total biomass in an autumn growing season. Under a high N regime, total biomass gains of inoculated rice were increased by 46.6/8.9% under ambient/elevated CO2. There appears to be interaction effects between N level and CO2, which will be statistically tested. Under a low N regime, in an autumn growing season, inoculated maize showed a 14.2% increase and a 9.2% decrease in total biomass under ambient and elevated CO2 conditions, respectively. Under a high N regime, in a spring growing season, a 5.8% decrease and a 26.8% increase in total biomass were observed in inoculated maize plants. Although there was a seasonal variation in the comparison, it is likely maize benefits more from the symbionts in low N under ambient CO2 and high N under elevated CO2 conditions. From a nifH mutant (WP5) CO2 chamber experiment, we did not find significant differences between rice/maize inoculated with the wild-type and the mutant strain. In both cases, the plants inoculated with the mutant had the similar biomass gain as with the plants inoculated with the wild-type. There could be a possibility of cross-contamination between samples in greenhouse conditions.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Khan, Z., Kandel, S.L., Ramos, D.N., Ettl, G.J., Kim, S.-H., and Doty, S.L. 2015. Increased biomass of nursery-grown Douglas-fir seedlings upon inoculation with diazotrophic endophytic consortia. Forests 6:3582-3593.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Andrea Firrincieli, Robert Otillar, Asaf Salamov, Jeremy Schmutz, Zareen Khan, Regina S. Redman, Neil D. Fleck, Erika Lindquist, Igor V. Grigoriev, and Sharon L. Doty. 2015. Genome Sequence of the Plant Growth Promoting Endophytic Yeast Rhodotorula graminis WP1. Frontiers in Microbiology Vol 6, article #978
    • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Biocontrol and plant growth-promoting potential of Salicaceae endophytes. Kandel, S.L., Firrincieli, A., Leston, N., McGeorge, K., Okubara, P., Mugnossa, G.S., Kim, S.-H., and Doty, S.L. (In revision)
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: As part of a related research project funded by USDA/AFRI with Populus, we published a manuscript: Doty, S.L., Sher, A.W., Fleck, N.D., Khorasani, M., Bumgarner, R.B., Khan, Z., Ko, A.W.K., Kim, S.-H., and DeLuca, T.H. 2016. Variable nitrogen fixation in wild Populus. PLOS ONE 11(5):e0155979.
    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: As part of a related research project funded by USDA/AFRI with Populus, we submitted a manuscript: Khan, Z., Rho, H., Luna, V., Hung, B., Kim, S.-H., and Doty, S.L. Growth enhancement and drought tolerance of hybrid poplar upon inoculation with endophyte consortia (accepted with minor revisions)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Professor Doty gave an invited presentation at the Agricultural Bioscience International Conference in Melbourne, Australia (Sept. 7-9, 2015)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Professor Doty gave an invited presentation at the International Congress on Nitrogen Fixation at the Asilomar conference center (Oct. 4-9, 2015).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Professor Doty gave an invited seminar at the International Congress on Frontiers in Biology in Brasilia, Brazil (Nov. 10-13, 2015).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Graduate student Hyungmin Rho gave an oral presentation on water use efficiency increases by endophyte inoculation at the Crop Science Society of America Conference in Minneapolis, Minnesota (Nov. 15-18, 2015).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Professor Doty gave an invited presentation on other aspects of endophytes (phytoremediation) at the Plant and Animal Genomics (PAG) conference in San Diego (Jan 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Professor Doty gave a poster presentation on N-fixation at the Plant and Animal Genomics (PAG) conference in San Diego (Jan 2016).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Professor Doty gave a poster presentation on N-fixation at the JGI Annual Genomics of Energy and Environment conference (March 22-24, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Professor Doty gave a poster presentation on N-fixation at the Symbiosis Conference in Yosemite (May 6-8, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Graduate student Andrew W. Sher gave a poster presentation at the Symbiosis Conference in Yosemite entitled Uncovering the molecular mechanisms of salt stress tolerance imparted by endophytic symbiosis (May 6-8, 2016)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Professor Doty, Dr. Khan, graduate student Lisa Hannon, and undergraduate researcher Pierre Joubert gave poster presentations at the International Congress on Molecular Plant-Microbe Interactions in Portland (July 17-21, 2016)


    Progress 08/15/14 to 08/14/15

    Outputs
    Target Audience:Target audiences include scientists at conferences, undergraduate and graduate students in classes, the general public at the Pacific Science Center, high school students, agronomic companies, and foresters. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students, H. Rho and Shyam Kandel gained teaching experience as TA's for the Plant Microbiology Lab Class (winter 2015). Neil Fleck gained teaching experience by helping lead some of the activities in Doty's Pacific NW Ecosystems class and Plant Microbiology Lab class (winter and spring 2015). Graduate student Andrea Firrincieli from the University of Tuscia, Italy, worked in Doty's lab for six months to gain experience in plant microbiology and genomic analysis. Graduate student, Shyam Kandel mentored under graduate students Jennifer Zech, Ellie Tagen and Nicholas Wegley about research techniques that are used to inoculate crop plants and assess the growth benefits conferred by endophytes. Graduate student H. Rho earned a PhD candidacy with his dissertation proposal developed from the project. Also, H. Rho trained four undergraduate students: Marian Hsieh, Victor van Epps, Norah Hummel, and Alexander Francisco. How have the results been disseminated to communities of interest?Workshops were given for high school girls in Bellevue through the AAUW Expanding Your Horizons program that encourages girls to enter the fields of science and engineering. Doty's lab participated in the UW partnership days with the Pacific Science Center in downtown Seattle, running a booth on plant microbiology to inform the general public at this very popular event. Prof. Kim was invited to give a presentation on our climate change and crop-endophyte interactions research at the US-Korea Conference (UKC2014) in San Francisco, CA in August 2014. H. Rho gave the presentation and introduced the research to a Korean engineer and scientist society on behalf of him. What do you plan to do during the next reporting period to accomplish the goals?For the Douglas-fir studies, trial A will be evaluated for continuation into a second year during the fall of 2015 and spring of 2016. Trial B should produce enough viable stock for an outplanting in fall of 2015 or for a simulated outplanting to continue plant monitoring through 2016. Aim 2: We will start to assess impacts of the stress tolerance conferring endophyte consortium on plant physiology under drought, heat, and salt stress conditions in greenhouse experiments. The same rice and corn varieties will be used. Also, their impacts under elevated [CO2] will be tested if it is available. Aim 3: We will continue to assess plants and diazotrophic endophyte interactions in elevated [CO2] focusing on photosynthetic and stomatal responses. At the same time, the data accumulated will be processed and analyzed for publication.

    Impacts
    What was accomplished under these goals? Additional endophytes were cultured and detailed characterization was begun. The students in the Plant Microbiology Lab class 2015 screened the new isolates on corn for symbiotic effects and tested the strains for auxin production, phosphate solubilization, siderophore production, and growth characteristics. The symbiotic traits of our endophyte strains were quantified in Doty's lab by undergraduate researchers. Endophytes of salt-tolerant plants were isolated. Genomic sequencing of dozens of the endophyte strains was done or is in progress at the Department of Energy Joint Genome Institute. A manuscript on the genomic characterization of the endophytic yeast was recently accepted with minor revisions by Frontiers in Microbiology. Colonization pattern and growth benefit conferred by endophytes in rice plants was published in Crop Science (Crop Science 2015 55:1765-1772). In addition, growth response and colonization pattern in maize by these endophytes were assayed, and a manuscript is under preparation. Moreover, some endophytes showed antifungal property by inhibiting the growth of Rhizoctonia solani AG-8 (soilborne pathogen of cereal crops) in in-vitro condition. They were characterized through 16S rDNA sequencing. Currently, plant assays to test the anti-Rhizoctonia solani AG-8 and other fungal pathogens of different crop plants are underway. Two greenhouse trials have been initiated to test the effect of inoculation on the response of Douglas-fir under nitrogen stress and drought stress. The first trial (A) utilizes precision grown container forestry stock (1+0) that has been either mock-inoculated with endophyte consortia (drought mix) and contraol moisture using fixed irrigation regimes. From the second trial (B) surfact sterilized seeds that have been either mock-inoculated or inoculated with endophyte consortia (conifer nif mix) and control nutrient input using control release fertilizers. Germination response, plant heights, root collar diameter, health and vigour are being monitored every week. In addition, photosynthetic measurements including Fv/Fm, SPAD are also being recorded every 2-3 weeks. A data analysis of Year 1 and 2 experiments was carried out, which showed the diazotrophs mitigated the down-regulation of photosynthesis under high [CO2] both in high and low nitrogen conditions. To examine whether this benefit is derived from N-fixation ability of the bacteria or from other mechanisms of having the symbiotic relationship (e.g. bacterial IAA-production or bacteria as biological sinks of photosynthates), a chamber experiment using a nifH mutant strain WP5 is currently being performed. Overall conclusions will be made by the end of the experiment and written in a manuscript. To confirm the stomatal reactions with inoculation of diazotrophic endophytes from Year 2 experiment, a follow-up glasshouse experiment was conducted and the results showed decrease in stomatal conductance of inoculated rice leaf samples during daytime. These results are promising as all three diazotrophic bacterial strains were effective in decreasing the conductance, which probably related to the phytohormone production of the endophytes. Using diazotrophic endophytes can be suggested as a means to save water in host crops. With the results from Year 2, a manuscript is in preparation.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kandel, S., Herschberger, N., Kim, S.-H., and Doty, S.L. 2015. Diazotrophic endophytes of poplar and willow for growth promotion of rice plants in nitrogen-limited conditions. Crop Science 55:1765-1772.
    • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: The manuscript, Genome Sequence of the Plant Growth Promoting Endophytic Yeast Rhodotorula graminis WP1 by Andrea Firrincieli, Robert Otillar, Asaf Salamov, Jeremy Schmutz, Zareen Khan, Regina S. Redman, Neil D. Fleck, Erika Lindquist, Igor V. Grigoriev, and Sharon L. Doty was accepted with minor revisions by Frontiers in Microbiology for a Special Issue on Emerging Tools for Emerging SymbiosisUsing Genomics Applications to Study Endophytes
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty gave an invited keynote presentation for the International Association for Plant Biotechnology in Melbourne, Australia, August 10-15, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty gave an invited presentation at the American Society for Microbiology NW Branch meeting Oct 17-18, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Professor Doty gave an invited presentation at the Plant and Animal Genome Conference in San Diego Jan. 10-14, 2015.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Professor Doty gave a presentation at the Yosemite Symbiosis Workshop May 1-3, 2015.
    • Type: Conference Papers and Presentations Status: Submitted Year Published: 2015 Citation: H. Rho submitted an abstract of a planned presentation to the ACS meetings on Nov. 15-18, 2015 in Minneapolis, MN.
    • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Submitted manuscript  Increased biomass of nursery-grown Douglas-fir seedlings upon inoculation with diazotrophic endophyte consortia. Zareen Khan, Shyam L. Kandel, Daniela Ramos, Gregory J. Ettl, Soo-Hyung Kim, and Sharon L. Doty.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Co-PI Kim gave an invited presentation on endophyte-mediated improvements in host crop physiology at the Japan-Korea Joint Symposium of Plant Pathology organized by Korean Society of Plant Pathology in Busan, Korea, 2225 October 2014.


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

    Outputs
    Target Audience: Target audiences included scientists at conferences, undergraduate students in classes, the general public at the Pacific Science Center, high school students, agronomic companies, and foresters. Changes/Problems: Co-PI Rusty Rodriguez and Research Scientist Regina Redman are now focusing on their company, Adaptive Symbiotic Technologies, but will continue to serve as collaborators and consultants rather than being directly involved in this project. What opportunities for training and professional development has the project provided? Undergraduate researchers, Caitlyn Little and Christina Pom gained experience in photosynthetic measurements. Ivy Salim learned molecular biology skills and assisted in characterizing endophytes isolated from conifers. Graduate student Shyam Kandel gained mentoring experience as he trained three undergraduate researchers: David Delin, Ivy Salim, and Alex Batson about microbiology techniques and fluorescent microscopy. Undergraduate researchers, Neil Fleck and Andrew Sher, were hired as Research Technologists upon their graduation in March 2014. Both gained mentoring experience by training the new undergraduate researchers. Mr. Fleck gained teaching experience by leading activities in Professor Doty’s plant microbiology lab classes. How have the results been disseminated to communities of interest? Professor Doty gave workshops for high school girls in 2 cities (Bellevue and Edmonds) through the AAUW Expanding Your Horizons program that encourages girls to enter the fields of science and engineering. Doty’s lab participated in the University of Washington partnership days with the Pacific Science Center in downtown Seattle, running a booth on plant microbiology to inform the general public at this very popular event. What do you plan to do during the next reporting period to accomplish the goals? Aim 1. We will optimize the inoculation procedure for the selected maize and rice lines using fluorescent microscopy and biomass gain as the indicators of successful colonization. We will then focus on quantification of N fixation using total N and 15N labeling studies. We will begin long-term studies to assess the impacts on grain yield and quality. With the nifH mutant of WP5, we will determine the relative importance of N fixation in the endophyte-induced enhancement of plant growth. For the conifer study, Dr. Khan has started a long term greenhouse experiment with the seeds and will be focusing on nitrogen fixation by using the 15N dilution assay. Dr. Khan will be studying the effect of WP1 inoculation and plant responses under drought stress. We will complete the characterization of the symbiotic traits of the endophyte strains and use the JGI provided full genomic sequences of several of the strains to identify the genes for these traits. Aim 2. We have found that bacterial and yeast endophytes can confer stress tolerance to plants. We will continue to isolate and characterize these endophytes and assess their impacts on plants under drought and salt stress conditions. Aim 3. We will continue to assess plants and diazotrophic endophyte interactions in elevated CO2 and plant stress tolerances with bacterial and yeast endophytes. Aim 4. We will again teach the Plant Microbiology Lab class in winter quarter 2015 and train more undergraduate researchers. We will do outreach activities at the Expanding Your Horizons workshops and Pacific Science Center. We have several manuscripts in preparation on the effects of the endophytes on corn, rice, and Douglas-fir. We will continue to present the research at conferences around the world.

    Impacts
    What was accomplished under these goals? Following screening of several different genotypes of rice and corn for responsiveness to consortia of diazotrophic endophytes, we determined that corn lines 23A71, 29B17, and XR1634 and rice lines M206 and XL203 had the greatest biomass gain from the endophytes. Using fluorescent microscopy we demonstrated that the endophytes effectively colonized these varieties. A manuscript on the rice results is currently in review, and a manuscript on the maize results is in preparation. Additional endophytes were cultured from poplar and detailed characterization was begun. The students in the Plant Microbiology Lab class screened the new isolates on corn for symbiotic effects and tested the strains for auxin production, phosphate solubilization, siderophore production, and growth characteristics. These symbiotic traits were later quantified in Doty’s lab by undergraduate researchers. In order to assess the relative contribution of N-fixation compared to other symbiotic traits, a nifH mutant strain WP5 was constructed. The replacement of the wild-type copy of the gene was verified by PCR. The mutant was tested for N-fixation activity by growth in N-free medium, 15N incorporation, and the acetylene reduction assay. All three assays confirmed that the mutant is unable to fix N. It is now being tested on several plant species in comparison to inoculation with the wild-type strain, and a manuscript is in preparation. The effects of endophyte inoculation on 3 Douglas-fir ecotypes were assessed. After 8 months of growth in N-limited soil, significant increases in root length and shoot length were observed in the seeds inoculated with a consortium of wild poplar and wild willow and newly isolated conifer endophyte mixes. Dr. Khan developed protocols for DNA isolation from inoculated Douglas-fir and protocols for visualization of endophytes from inoculated Douglas-fir using fluorescence microscopy. A drought tolerance experiment with Douglas-fir is underway with an endophyte strain that conferred drought tolerance to poplar. A field study was initiated at Pack Forest, WA. About 250 seedlings inoculated with 5 different treatment groups were planted in May 2104. Plant heights, overall health, root collar diameter (RCD) and photosynthetic parameters (greenness, Fv/Fm) are being monitored. Professor Kim’s lab led the efforts to test if the physiological benefits of crop-endophyte symbiosis are enhanced in elevated [CO2]. Inoculation of diazotrophic endophytes appeared to eliminate the down-regulation of photosynthesis under high [CO2] condition in rice. This result is exciting and leads us to look further into the underlying mechanisms of this beneficial phenomenon. In addition, stomatal conductance of the rice plants were significantly decreased by the inoculation of diazotrophic bacteria during daytime both in ambient and elevated CO2 conditions. This result suggests that the use of bacterial endophytes in the future climate can be a way to improve water use efficiency of host crops (rice) while producing more photosynthates combined with the enhanced photosynthesis.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Findings from Aim 3 were presented at the ASA-CSSA-SSSA meeting on November 3-6, 2013 in Tampa, FL.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: An invited presentation on our climate change and crop-endophyte interactions research was made at the US-Korea Conference (UKC2014) in San Francisco, CA in August 2014.
    • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Kandel, S., Herschberger, N., Kim, S.-H., and Doty, S.L. 2014. Diazotrophic endophytes of poplar and willow promote growth of rice plants in nitrogen-limited conditions. (in review)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Research Scientist Dr. Zareen Khan presented at the Washington State Society of American Foresters Meeting at Pack Forest, WA (May 8-9,2014).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Undergraduate researcher M. Hsieh presented a poster at the ASA-CSSA-SSSA annual meetings in Long Beach, CA on November 2-5, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Graduate student Shyam Kandel and research technologist Andrew Sher gave poster presentations at the American Society for Plant Biology (ASPB) conference.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Graduate student Shyam Kandel and research technologist Andrew Sher gave poster presentations at the Northwest Branch of the American Society for Microbiology (ASM).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Graduate students Shyam Kandel and Hyungmin Rho presented their research at the ASA CSSA SSSA annual meeting 2014 Nov. 2-5., Long Beach, CA.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty presented the research findings at the Program Directors meeting in Gainesville, Florida in January, at NASA-Ames in March, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty presented the research findings at the Pan-American Congress on Plants and Bioenergy in Guelph in June, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty presented the research findings at the International Congress for Molecular Plant-Microbe Interactions in Rhodes, Greece in July, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty presented the research findings at the ASPB conference in Portland in July, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty presented the research findings at the International Congress for Plant Biotechnology in Melbourne, Australia in August, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty presented the research findings at the Northwest Branch American Society for Microbiology (ASM) meeting in Seattle in October, 2014.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Professor Doty presented the research findings at the International Symposium on Nitrogen Fixation with Non-Legumes in Chengdu, China in November, 2014.


    Progress 08/15/12 to 08/14/13

    Outputs
    Target Audience: Target audiences include scientists at conferences, undergraduate students in classes, the general public at the Pacific Science Center, high school students, and agronomic companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project initiated a new plant microbiology lab course that had 22 students in this first offering in winter 2013. The students learned a variety of microbiology skills as well as how to quantify the effects of the endophytes on plant growth and physiology. The course received a very high score in the course evaluations at the end of the quarter. In addition, about 20 undergraduate students engaged in undergraduate research projects within the scope of the NIFA project in Doty and Kim’s laboratories. The students worked individually or in small groups on either N fixation in conifers, corn, or rice; on molecular characterization of the endophytes; on other endophyte characteristics including ammonia production, phytohormone production, or phosphate solubilization; response to fungal endophytes; and photosynthesis and water use efficiency measurements. The project involves four Ph.D. graduate students who are working on different projects within the NIFA project. Two are funded directly by the grant and two are funded by their own fellowships. The students are learning plant microbiology and plant physiology skills. By being part of a large grant project, they have opportunities to mentor undergraduate researchers as well. How have the results been disseminated to communities of interest? Doty presented the data at the International Poplar Commission meeting in Dehradun, India in November 2012. She ran workshops for high school girls in 2 cities (Bellevue and Edmonds) through the Expanding Your Horizons program that encourages girls to enter the fields of science and engineering. Doty’s lab participated in the UW partnership days with the Pacific Science Center in downtown Seattle April 2013, running a booth on plant microbiology to inform the general public at this very popular event. Doty gave an invited seminar at the UW Genomics symposium on agriculture. In Washington D.C., Doty met with a DOE program manager on the importance of endophytes for promoting plant growth. She also discussed the data with two company owners at the DOE’s Biomass 2013 conference. Research Scientist, Zareen Khan, presented results at a symbiosis conference in California in April 2013. Graduate students, Shyam Kandel and Tony Rho, presented at the UW Graduate Student Symposium in March 2013. Undergraduate researchers, Daniela Ramos, presented her results on improved conifer growth with endophytes, and Sang Cho on crop-endophyte interactions in elevated CO2 in rice at the UW Undergraduate Research Symposium. What do you plan to do during the next reporting period to accomplish the goals? In Year Two, we will be optimizing the inoculum and inoculating procedures for corn, rice, and Douglas-fir. We will conduct the stress tests comparing inoculated and uninoculated plants. We will assess the physiological parameters under ambient and elevated CO2. We expect to publish several papers and participate in multiple conferences to disseminate the knowledge gained from this project. We will continue teaching the new plant microbiology lab course and doing outreach to high school students and to the general public.

    Impacts
    What was accomplished under these goals? The goals for year one in the proposed time line were to test the endophytes on a variety of corn and rice and Douglas-fir; to begin screening for effects under elevated CO2; to train graduate students and undergraduates in plant microbiology. For Aim 1, we received 6 hybrid rice varieties from RiceTec, 4 hybrid corn from Blue River Hybrids, 8 hybrid corn varieties from Pioneer Hi-Bred, and Douglas-fir seeds from 3 different locations. We tested the bacterial and yeast endophytes on many of these varieties (some tests are still underway as some seed varieties arrived this summer). The results were good, showing enhanced growth of the inoculated plants compared to controls. The collection of endophytes is being characterized in terms of phytohormone production, nitrogen fixation, and genetic identification. A nitrogenase mutant is being constructed in one of the best-performing endophytes. Transposon insertions in the nitrogenase gene were mapped and the mutated gene introduced into the strain for homologous recombination. Many of the endophytes were labeled with fluorescent markers (GFP and RFP) and used to verify colonization of the corn, rice, and Douglas-fir. For Aim 2, the goal was to verify that the corn and rice could be colonized by the fungal endophytes, and this was accomplished. For Aim 3, responses to elevated CO2 were tested in inoculated and uninoculated corn and rice. A set of physiology experiments that investigated leaf CO2 assimilation and water relations in elevated CO2 were completed. For Aim 4 that focused on education and training, we describe in the sections below.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Doty, S. "Improving biomass production and phytoremediation using natural endosymbionts of poplar and willow." Presentation to the 24th Session of the International Poplar Commission in Dehradun, India (Oct 29-Nov 2, 2012).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Khan, Z. "Poplar and willow endophytes improve growth in crop plants and grasses" at the Symbiosis Meeting at SNRI, Yosemite National Park from May 25-26,2013.