the use of diazotrophic endophytes as a means for climate change mitigation and adaptation in...

The Use of Diazotrophic Endophytes as a Means for Climate Change Mitigation and

Adaptation In Agroecosystems

Project Director: Sharon L. Doty, ProfessorCo-PD: Soo-Hyung Kim, Assoc. Professor

University of Washington, Seattle

NIFA Agroclimatology Project Directors MeetingDecember 16-18, 2016

The Plant Microbiome: Microbial communities within a plant

Benefits from endophytes

Stress toleranceDrought

TemperatureSalt

Elevated CO2

Reduced phytotoxicity of

pollutantsOrganic pollutants

Inorganic pollutants

Growth PromotionHormones

Nutrients (N, P, Fe)

Pathogen resistanceAnti-microbial compounds

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

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

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: 27-35Doty, S. L., et al. (2009) Symbiosis 47: 23-33Xin, G., et al. (2009) Biology and Fertility of Soils 45:669-674Firrincieli, et al. (2015) Frontiers in Microbiology 6: 978

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

Implications for this research:Helps Explain the Biology of Populus

NOTE: Poplar and willow are not nodulating species

Can we use the N-fixing endophytes of wild poplar and willow to promote growth of non-

legume crop plants and forest trees?

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

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

ShyamKandel

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0.5

1

1.5

2

2.5

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3.5

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4.5

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Total Root Shoot

Wei

ght (

g)

Inoculated (NIF) vs Non-Inoculated (NS) Dry Weight

NIFNS

NIF n=24NS n=12

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




TemperatureSalt

Reduced phytotoxicity of

pollutants



Pathogen resistanceAntimicrobial compounds

Solubilize phosphate Produce siderophores

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

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.

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

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)


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)


Improved salt tolerance using an estuary plant endophyte Andrew Sher

Andrew Sher, Sharon Doty, and Alisa Huffaker (unpublished)

Arabidopsis Maize





TemperatureSalt

* Elevated CO2

Reduced phytotoxicity of pollutants

Organic pollutants (ie. TCE, PAHs)Inorganic pollutants (ie. As)



Pathogen resistanceAntimicrobial compounds

ELE (800 ppm)ELE (800 ppm)AMB (400 ppm)AMB (400 ppm)

Douglas Research Conservatory, Seattle, WA

Co-Director: Assoc. Prof. Soo-Hyung Kim

Alleviation of photosynthetic down-regulation in rice under elevated CO2

Hyungmin Rho, Sharon Doty, and Soo-Hyung Kim (unpublished)


Implications for this research:Endophytes For Sustainable Agriculture, Forestry, and

Biomass Production

Elizabeth Pennisi 2015 Sciencemag.org 348:6237

Joe Funk, editor Seed Today 3rd Quarter 2015

Samples of press in response to our research

Outreach

Created a Plant Microbiology Lab class–students characterized new isolates

Licensed strains to IntrinsyxTechnologies; 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.

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

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

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)

the use of diazotrophic endophytes as a means for climate change mitigation and adaptation in...

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