The overall goal of this project is to develop tangible options to mitigate and adapt to climate change impacts on agriculture and forestry more quickly than could be reached by relying solely on crop improvement approaches via breeding or transgenic.

1. The Use of Diazotrophic Endophytes as a
Means for Climate Change Mitigation and
Adaptation In Agroecosystems
Project Director: Sharon L. Doty, Professor
Co-PD: Soo-Hyung Kim, Assoc. Professor
University of Washington, Seattle
NIFAAgroclimatology Project Directors Meeting
December 16-18, 2016

2. The Plant Microbiome:
Microbial communities within a plant
Benefits from endophytes
Stress tolerance
Drought
Temperature
Salt
Elevated CO2
Reduced
phytotoxicity of
pollutants
Organic pollutants
Inorganic pollutants
Growth Promotion
Hormones
Nutrients (N, P, Fe)
Pathogen resistance
Anti-microbial
compounds

3. Hypothesis: Pioneer tree species use symbiosis
with N-fixing (diazotrophic) endophytes to thrive
in low-nutrient conditions
Rivers in the Pacific Northwest (USA) are
fed by snow-melt from the mountains.
Total N is only 0.05 to 0.3 mg/L

4. N2 fixation in wild poplar was demonstrated
using the direct 15N2 incorporation assay and
the indirect acetylene reduction assay
Doty, S.L., Sher, A.W., Fleck, N.D., Khorasani, M., Bumgarner, R., Ko, A., Khan, Z.,
Kim, S.H., and DeLuca, T. H. 2016 PLOS ONE 11(5):e0155979
• NifH sequences included Burkholderia,
Sphingomonas, Azospirillum, Bradyrhizobium,
Rhodospirillum, Methanococcus, and more

5. Poplar & willow endophytes isolated from
surface-sterilized branches
• Rhizobium tropici
• Burkholderia sp.
• Herbaspirillum sp.
• Pseudomonas graminis
• Rhanella sp.
• Sphingomonas sp.
• Acinetobacter sp.
• Enterobacter sp.
• Rhodotorula graminis
Doty, S. L., et al. (2005) Symbiosis 39: 27-35
Doty, S. L., et al. (2009) Symbiosis 47: 23-33
Xin, G., et al. (2009) Biology and Fertility of Soils 45:669-674
Firrincieli, et al. (2015) Frontiers in Microbiology 6: 978

6. 15N dilution assay indicated that hybrid poplar
inoculated with strains from wild poplar received 65%
of the foliar N from biological nitrogen fixation
Inoculated
with wild
poplar
endophytes
Un‐inoculated
controls
Knoth, et al (2014) New Phytologist 201:599-609
USDA AFRI grant # 2010-05080

7. Implications for this research:
Helps Explain the Biology of Populus
NOTE: Poplar and willow are
not nodulating species

8. Can we use the N-fixing endophytes of wild
poplar and willow to promote growth of non-
legume crop plants and forest trees?

9. Increased growth and yields of inoculated
plants grown in low-nutrient soil
Khan, Z, Guelich, G., Phan, H., Redman, R., and Doty, S. L. 2012. ISRN Agronomy
Dr. Zareen Khan
Controls without added
endophytes
With endophyte added

10. Colonization and growth enhancement of rice
with diazotrophic endophytes
S. Kandel, N. Herschberger, S.H. Kim, and S.L. Doty (2015) Crop Science 55:1765-1772
Shyam
Kandel
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Total Root Shoot
Weight(g)
Inoculated (NIF) vs Non-Inoculated (NS) Dry
Weight
NIF
NS
NIF n=24
NS n=12

11. Increased growth of Douglas-fir in
nutrient-poor soil
No added
microbes
With added
endophytes
Zareen Khan
Khan, Z., Ramos, D. Ettl, G., Kim, S.H., and Doty, S.L. 2015 Forests 6:3582-3593
N=12

12. The Plant Microbiome:
Microbial communities within a plant
Benefits from endophytes
Stress tolerance
Drought
Temperature
Salt
Reduced
phytotoxicity of
pollutants
Growth Promotion
Hormones
Nutrients (N, P, Fe)
Pathogen resistance
Antimicrobial compounds
Solubilize
phosphate Produce siderophores

13. Poplar inoculated with endophytes from wild poplar have
increased growth and survive drought stress longer
Poplar inoculated with endophytes
one month without watering
Uninoculated controls
0
5
10
15
20
25
avg control avg innoc
mass (g)
Total mass (dry)
* p << 0.01
Khan, Z., Rho, H., Firrincieli, A., Hung, S.H., Luna, V.,
Masciarelli, O., Kim, S.H., and Doty, S.L. 2016.
Current Plant Biology 6:38-47
USDA AFRI grant # 2010-05080

14. Several possible mechanisms for the
endophyte-conferred drought tolerance
• Phytohormone production
– IAA, ABA,
• Increased nutrients
• Reduction of ROS
• Genetic evidence for:
– Osmolyte trehalose
– VOCs Acetoin and 2,3-
butanediol
Inoculated Control
Khan, Z., Rho, H., Firrincieli, A., Hung, S.H., Luna, V.,
Masciarelli, O., Kim, S.H., and Doty, S.L. 2016. Current
Plant Biology 6:38-47.

15. Endophyte consortium increased
water use efficiency (WUE) in rice
and in maize
Tony Rho
Hyungmin Rho, Sharon Doty, and Soo-Hyung Kim (unpublished)
Unpublished data was removed for posting

16. Reduced mortality of
conifers under
drought conditions
• Western redcedar (Thuja plicata)
• After two seasons of simulating
seasonal moisture fluctuations
• No mortality in wet treatment
conditions
• 50% mortality reduction in normal
conditions
• 90% mortality reduction in drought
conditions
Matthew Aghai
Matthew Aghai, Sharon Doty, and Gregory Ettl (unpublished)
Unpublished data was
removed for posting

17. Reduced mortality of conifers
under drought conditions
• Douglas-fir (Pseudotsuga
menzeisii)
• 10-40% mortality reduction
across all moisture conditions
• Endophyte treatments were
significant (P<0.05)
• Black pineleaf scale outbreak
– inoculated seedlings still had
improved survival
Matthew Aghai
Matthew Aghai, Sharon Doty, and Gregory Ettl (unpublished)
Unpublished data was
removed for posting

18. Improved salt tolerance using an
estuary plant endophyte Andrew Sher
Andrew Sher, Sharon Doty, and Alisa Huffaker (unpublished)
Arabidopsis Maize
Unpublished data was
removed for posting

19. The Plant Microbiome:
Microbial communities within a plant
Benefits from endophytes
Stress tolerance
Drought
Temperature
Salt
* Elevated CO2
Reduced phytotoxicity of
pollutants
Organic pollutants (ie. TCE, PAHs)
Inorganic pollutants (ie. As)
Growth Promotion
Hormones
Nutrients (N, P, Fe)
Pathogen resistance
Antimicrobial compounds

20. ELE (800 ppm)ELE (800 ppm)AMB (400 ppm)AMB (400 ppm)
Douglas Research Conservatory, Seattle, WA
Co-Director: Assoc. Prof. Soo-Hyung Kim

21. Alleviation of photosynthetic down-
regulation in rice under elevated CO2
Hyungmin Rho, Sharon Doty, and Soo-Hyung Kim (unpublished)
Unpublished data was
removed for posting

22. Implications for this research:
Endophytes For Sustainable Agriculture, Forestry, and
Biomass Production

23. Elizabeth Pennisi 2015 Sciencemag.org 348:6237
Joe Funk, editor Seed Today 3rd Quarter 2015
Samples of press in response to our research

24. Outreach
Created a Plant Microbiology Lab class–
students characterized new isolates
Licensed strains to Intrinsyx
Technologies; sublicensed to
Verdesian Life Sciences,
Performance Ag, Indigo,
BioConsortia, ICL, etc.
- Testing on strawberries,
broccoli, maize, tomatoes, grapes,
jalapenos, lettuce, beets, bell
pepper, stone fruit trees, etc.

25. Implications for this Research:
Climate Change Mitigation
Uninoculated controls
• Plants can increase C
sequestration only if
nutrients and water are
not limiting
• Using more chemical
fertilizers is inconsistent
with mitigation
Endophytes can promote plant growth naturally
through improved nutrient acquisition,
phytohormone production, & stress tolerance

26. Funding and technology provided by:
NIFA climate change
mitigation program
Grant # 2012-68002-19824
Byron and Alice Lockwood
Foundation Professorship
Conifer studies additionally
funded by:
Corkery Endowed Chair
(University of Washington)
McIntire Stennis Cooperative
Forestry Research Funding

27. http://depts.washington.edu/envaplab
Dr. Zareen Khan
(Research Scientist)
Students and research staff involved
in the presented research
Undergraduate researchers especially Grant
Guelich, Jack Emery, Beverly Hung, Daniela
Ramos, Nick Wegley, and Pierre Joubert
Special thanks to Dr. John Freeman
(Intrinsyx) and Dr. Mike Blaylock
(Edenspace) for their partnerships in
moving the research from lab to field
Previous grad students and research staff:
Jenny Knoth, Mahsa Khorasani, Neil Fleck,
and Andrea Firrincieli
Shyam Kandel
(grad student)
Special thanks to Assoc. Prof. Greg Ettl for
collaboration on the conifer research
Andrew Sher
(grad student)
Matthew Agai
(grad student)
Hyungmin Rho
(grad student)