the symbiotic-plant microbiome and its importance in plant

The Symbiotic-Plant Microbiome and Its Importance in Plant Health

Prof. Sharon L. DotyUniversity of Washington, Seattle

The Plant Microbiome: Microbial communities within a plant

Benefits from endophytes

Stress tolerance

Drought

Temperature

Salt

Reduced

phytotoxicity of

pollutants

Organic pollutants

Inorganic pollutants

Growth Promotion

Hormones

Nutrients (N, P, Fe)

Pathogen resistance

Anti-microbial

compounds

Endophytes are microorganisms within plants

Sharon Doty, Andrew Sher, Will Chrisler, Galya Orr, unpublished

Y-axis

Z-axis

X-axis Z-axis

Roots are fixed with Methanol-Triton X

Dtained with SybrGold under vacuum

Intact roots are screened by microscopy

Roots were embedded in OCT and cryosectioned

Endophytes can be added to plantsEnter through cracks at lateral root junctions or

through leaves and can colonize throughout the plant

GFP and RFP tagged endophytes on a poplar root (Photo

credit: Andrea Firrincieli) Doty, et al. 2017

Root colonization after foliar spray

inoculation (Photo credit: Raymond Yap)

Hypothesis: Plants use symbiosis with endophytes to thrive in hostile conditions

Nutrient stress Flooding

Drought & HeatToxicity

Amazon River

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

Macronutrients N and P

Some endophytes can

solubilize phosphate

Some endophytes can fix

atmospheric dinitrogen gas

into usable forms

N2 + 8H+ + 8e- + 16ATP

2NH3 + H2 + 16ADP + 16Pi

Nitrogenase

Endophytic N-fixation in wild poplar

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

Direct assay: 15N incorporation

from 15N2

Indirect assay: Acetylene

reduction assay

Evidence of N2 fixation in some poplar using the 15N2 incorporation assay Andrew Sher

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

Hypothesis: The variability in activity is due to the variability of

the endophytic population in every region of the plant

0

20

40

60

80

100

120

Undosed Dosed

mmol ethylene per gram


Evidence of N2 fixation in some poplar using the acetylene reduction assay

The microbiota of wild poplaris highly variable & unevenly dispersed


NifH sequences included

Burkholderia, Sphingomonas,

Azospirillum Bradyrhizobium, Rhodospirillum, Methanococcus,

and more

Quantify mRNA copies in intact bacterial cells

“fliFISH” Fluctuation localization imaging-based FISH

Average nifH transcripts in:

Solo cells (0.6)

Aggregated cells (19)

Doty, et al. unpublished data

Endophytes in culture (NL-CCM)

260 280 300 320 340 360 380

240

260

280

300

320

340

Implications for this research:Helps Explain the Biology of pioneer plant species

NOTE: Poplar and willow are

not nodulating species

Endophytes can be cultured from poplar & willow and added to other plants

• Rhizobium tropici• Burkholderia sp.• Herbaspirillum sp.• Pseudomonas sp.• Rahnella sp.• Sphingomonas sp.• Acinetobacter sp.• Enterobacter sp.

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

Hybrid poplar inoculated with 6 strains from wild poplar received 65% of the foliar N from biological

nitrogen fixation (15N dilution assay)

Inoculated with wild

poplar endophytes

Un-inoculated controls

Knoth, et al (2014) New Phytologist 201:599-609

Jenny Knoth

Increased leaf greenness in inoculated poplar

****

*

Significant differences from the control: •, alpha = 0.1; *, alpha = 0.05; **, alpha = 0.01; n = 7

J. Knoth, et al (2014) New Phytologist 201:599-609

Jenny Knoth

Can N-fixing endophytes of poplar and willow promote growth of other non-

legume plants?

Increased growth and yields of inoculated pepper plants grown in low-N conditions

Khan, Z, Guelich, G., Phan, H., Redman, R., and Doty, S. L. 2012. ISRN Agronomy

Zareen Khan

Controls without added

endophytes

With endophyte added

Seeds were inoculated with endophytes or mock-inoculated, and

grown in nutrient-poor soil watered with N-free Hoagland’s solution

Increased tomato plant growth and fruit yields

Uninoculated controls


Treatment Chocolate Cherry variety

Glacier variety

Inoculated 106 95

Control 63 43

Dr. Zareen Khan

Cherry Tomatoes grown under severe nutrient deficiency after 3 months

Seeds were inoculated with endophytes or mock-inoculated, grown in nutrient-poor soil.

Seedlings were grown with no nutrients for 2 months and then given 1/10X Hoagland’s

solution for one month

Apples• Variety “Honey Crisp” trees (2yrs

old) inoculated with consortium of Salicaceae endophytes; grown with 1/4x Hoagland’s fertilizer solution

• Enhanced Ps light reactions

• Increased root mass

• Delayed leaf senescence

Tony Rho, Victor van Epps, Soo-Hyung Kim, and Sharon L. Doty, unpublished

Endophytes increased fruit weight and sucrose content of apple fruits (Honey Crisp)

)

Tony Rho, Victor van Epps, Soo-Hyung Kim, and Sharon L. Doty, unpublished

Turfgrass

37% increase in total N content and

42% increase in mass of Kentucky Bluegrass

inoculated with Burkholderia strain WPB

Gang Xin, et al. 2009 Biology and Fertility of Soils 45:669-674

Inoculated with WPB Mock-Inoculated

Endophytes improved turfgrass health and growth in low nutrient conditions

Kentucky bluegrass +/- PTD1

Wisconsin grass line +/- WP19


Zareen Khan

Colonization and growth enhancement of rice

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

Weig

ht

(g)

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

NIFNS

NIF n=24

NS n=12

0

1

2

3

4

Nu

mb

er o

f Ti

llers

(Significance * * at α = <0.01)

* *

# of Tillers

Increased growth of Douglas-fir in nutrient-poor soil in response to endophyte consortium (8 strains) from poplar & willow

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

Broad Host Range

No added

microbes

With added

endophytes

No added

microbes

With added

endophytesNo added

microbes

With added

endophytes



Stress tolerance

Drought

Temperature

Salt

Reduced

phytotoxicity of

pollutants

Organic pollutants


Growth Promotion

Hormones


Pathogen resistance

Anti-microbial

compounds

Many of the endophytes

produce the auxin, IAA

Addition of the endophytes from wild poplar and willow

increases the rooting of a variety of plant species

Without added microbes With the added microbes

Doty, SL, et al., unpublished Khan, Z, et al. 2012. ISRN Agronomy

Khan, Z.,

Ramos, D. Ettl,

G., Kim, S.H.,

and Doty, S.L.

2015 Forests

6:3582-3593

With added

endophytes



Stress tolerance

Drought

Temperature

Salt

Reduced

phytotoxicity of

pollutants

Organic pollutants


Growth Promotion

Hormones


Pathogen resistance

Anti-microbial

compounds

Hybrid poplar inoculated with endophytes from

wild poplar and willow have increased growth and

drought tolerance

Poplar inoculated with endophytes

one month without watering


0

5

10

15

20

25

avg control avg innoc

mas

s (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

Dr. Zareen Khan

Mock Inoculated

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

Aghai, M. M., Khan, Z., Joseph, M. R., Stoda, A. M., Sher, A. W., Ettl, G. J., and Doty, S. L. Front. Microbiol. 10:1353

Endophyte consortium from Salicaceae increased water use

efficiency (WUE) in rice

Tony Rho

Rho, et al. 2018 Frontiers in Plant Science 9:188

Non-stress Drought Stress

• Reduced transpiration

• Increased growth

• Overall increased WUE of

productivity

Stress tolerance

Drought

Temperature

Salt

Reduced

phytotoxicity of

pollutants

Organic pollutants


Growth Promotion

Hormones


Pathogen resistance

Anti-microbial

compounds



In vitro Bio-Control of Agricultural Pathogens by Salicaceae Endophytes

Shyam

Kandel

• Dual plate inhibition assays

• Burkholderia sp. strains had

especially strong activity

Pathogen

Kandel, S.L., Firrincieli, A., Joubert, P.M., Okubara, P.A., Leston, N., McGeorge, K., Mugnozza, G.S.,

Harfouche, A., Kim, S.H., and Doty, S.L. 2017. Frontiers in Microbiology Vol 8, #386

Bio-Control of Several Agriculturally-

Important PathogensRhizoctonia solani AG‐8

Pierre

JoubertGaeumannomyces graminisvar. tritici (GGT)

Fusarium culmorumPythium ultimum

Burkholderia strain WPB has strong anti-pathogen activities

Rhizoctonia

solani AG‐8

Pierre

Joubert

Fusarium

culmorum

Gaeumannomyces

graminis

Anti-fungal glycolipopeptide

occidiofungin (ofc) gene cluster in

the Burkholderia genomes

ofc mutant had reduced anti-fungal

activity (Joubert, unpublished)

Primary screen for isolation of new antifungal endophyte strains

Genus strainRhizoctoniasolani

Gaemannomycesgraminis

Pythiumultimum

Fusariumculmorum

Verticiliumdahliae

Rahnella AFE1 weak strong no weak no

Rahnella AFE3 no strong no no no

Bacillus AFE4A strong strong no strong weak

Pseudomonas AFE5 strong weak no strong weak

Pseudomonas AFE8 strong weak no strong no

Rahnella AFE9 weak strong weak no no

Aureobasidium AFE11 weak strong no no no

Pantoea AFE14 no strong no no no

Pseudomonas AFE16 weak strong no no no

Bacillus AFE21B strong strong no strong no

Rhanella AFE22 no strong weak No no

Phythium ultimum and Verticilium dahlia were the least susceptible pathogens

Most strains had a broad plant

host range, able to colonize

agriculturally-important crop

plants: Kale, Rice, Soy and

Wheat.

Species and In silico identification of secondary metabolites with antifungal activity

• Illumina MiSeq whole genome sequencing was used to classify our strains down to species level and identify biosynthetic gene clusters involved in the synthesis of secondary metabolites with know antifungal activity

According to the average nucleotide

identity, the strains AFE4A and AFE21B

(the strongest antifungal strains) clustered

together with other Bacillus belonging to

the species Bacillus amyloliquefaciens

Macrolactin, 100% completeness [1]

Bacillaene , 100% completeness [2]

Fengicyn 100% completeness [4]Bacillomycin, 100% completeness [3]

Difficidin 100% completeness [5]

Bacilysin, 100% completeness [6]

Bacillibactin, 100% completeness [8]Amylocyclicin 100%[7]

1. Macrolactin, inhibitor of prokaryotic cell

division

2. Bacillaene, inhibitor of prokaryotic protein

synthesis

3. Bacillomycin, antifungal that form a pore in the

cell membrane

4. Fengicyn, inhibits filamentous fungi unknown

mechanism of action

5. Difficidin, inhibitor of prokaryotic protein

synthesis

6. Bacilysin, inhibitor of cell wall biosynthesis

7. Amylocyclicin, inhibitor of bacteria unknown

mechanism of action

In silico identification of secondary metabolites with antifungal activity in Bacillus amyloliquefacies AFE4 and AFE21

• Bacillus amyloliquefacies AFE4 and AFE21 share 8 biosynthetic gene clusters involved in the synthesis of circular and linear small peptides with antifungal antimicrobial activity against crop pathogens:

Importance of the plant microbiome

Stress tolerance

Drought

Temperature

Salt

Elevated CO2

Reduced

phytotoxicity of

pollutants

Organic pollutants


Growth Promotion

Hormones


Pathogen resistance

Anti-microbial

compounds

Increased nutrient acquisition and growth

Increased drought tolerance and WUE

Biocontrol activity against pathogens

Reduction of carcinogenic pollution

Elizabeth Pennisi 2015 Sciencemag.org 348:6237

Joe Funk, editor Seed Today 3rd Quarter 2015

Samples of press in response to our research

UW licensed strains to IntrinsyxBio that is now using the strains

in collaboration with other companies

Salicaceae endophytes were licensed to Intrinxyx Bio

3rd party validated greenhouse and field trials consistently showed increased growth of a

variety of crop plants

Dr. John L. Freeman III

Chief Science Officer

Greenhouse studies:

Statistically significant increases in growth, final size and harvest yield

(ie. height, mass, stem diameter, chlorophyll) under nitrogen and

phosphorous deficient soil conditions in over a dozen types of crops

such as: Corn, tomato, canola, lettuce, soybean, broccoli, wheat using

both seed coats and in-furrow applications

Salicaceae endophytes increased growth of a variety of crop plants in low N/low P soils

Increased # soy pods from control (2)

to endophyte-inoculated (4-5); p<0.1

Increased Broccoli plant weight from control

(100g) to endophyte-inoculated (up to 140; p<0.05)

Increased lettuce leaf # from

control (4) to endophyte-

inoculated (6-9)

Contro

l

Contro

lC

ontro

l

UNPUBLISHED DATA

Field tests in California:

11-57% increase in strawberry fruit yield

10-20% increase in fresh market tomato fruit yields

7.26 bushel/acre increase in corn grain harvest yield under 70% less N

fertilizer than normal

+5.1 tons per acre increase in processing tomato yield under 70% less N

>10g increase per fruit in fresh market tomato weight under 70% less N

Strawberry field tests in California:

0

1

2

3

4

5

6

7

Control WW5 WW6 WW7 PTD1

Aver

age

Nu

mber

of

Mar

ket

able

Fru

it p

er P

lant

Albion Strawberry - Liquid Root Spray

Eagle Farms Strawberry Trial

**

**

UNPUBLISHED DATA: The average yield of harvestable fruit yield was measured for ten randomly selected plants

(n=10). P-values were calculated using a two-sample t-test in comparison to the control (*: p<0.1, **: p<0.05).

The data in figure 7 showed all liquid root spray inoculum treatments increased the average number of marketable fruit per

plant over the control; WW5 increased by 11%, WW6 by 23%, WW7 increased fruit by 57%, and PTD1 by 32%.

Field tests in Hollister, California:

Apricot hybrids on salt-impacted soils had improved

health when inoculated with the Salicaceae endophytes

• Increased leaf area

• Increased branch number

• Increased SPAD (chlorophyll)

• During 2017-2018 extreme heat and drought conditions

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

Biomass Production

Implications for this Research:

Climate Change Mitigation


• 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:

NSF Energy for Sustainability

program

McIntire-Stennis

program

Byron and Alice Lockwood Foundation

Professorship

AFRI bioenergy program and NIFA

climate change mitigation program

Doty Lab Website:

http://sites.uw.edu/sldoty

Students and staff involved in the presented research

Thanks to Intrinsyx for

moving our research to

the field

Andrew Sher Pierre Joubert

Zareen Khan

Shyam

Kandel

Jenny Knoth Tony Rho

Matthew Aghai Andrea Firrincieli

Also the undergraduate

researchers especially

Grant Guelich, Beverly

Hung, Daniela Ramos,

Jack Emery, Nick

Wegley, and Shruti

Parikh

Special thanks to Co-PI’s

Assoc. Prof.

Soo-Hyung Kim

and Greg EttlThanks to Illumina for

filming us! Adventures in

Genomics video, “Power of

Plant Partnerships”

the symbiotic-plant microbiome and its importance in plant

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