temporal changes in soil microbiome in response to soil ...€¦ · soil microbial communities and...

Temporal Changes in Soil Microbiome in Response to Soil TreatmentsShashika S. Hewavitharana PhD

Assistant Professor

Horticulture and Crop Science Department

Cal Poly State University San Luis Obispo

Gulya et al. 2006

https://ucanr.edu/

Microbiome is ubiquitous

bacteria of the human gut microbiome

Credit: Wyss Institute at Harvard University

community of bacteria from human nose

Credit: Steve Gschmeissner/SPL

Temporal Changes in Skin Microbiome

Oh et al., 2016, Cell 165, 854–866

Soil Microbial Communities and Molecular Interactions

Jansson and Hofmockel, 2018Current Opinion in Microbiology 2018, 43,162–168

• Supporting interdisciplinary research to answer fundamental questions about microbiomes in diverse ecosystems.• Developing platform technologies that will generate insights and

help share knowledge of microbiomes in diverse ecosystems and enhance access to microbiome data.• Expanding the microbiome workforce through citizen science and

educational opportunities.

Source: https://obamawhitehouse.archives.gov/

Appl

es • ~68% national production in WA (NASS, 2019)

• ~90% of national organic crop in WA (NASS, 2016)

• Value ~$ 2.2 billion

Stra

wbe

rrie

s • ~90% of the national production in CA (NASS, 2019)

• ~98% of the crop value of organic strawberry in CA (NASS, 2016)

• Value $2.9 billion

Extent of the Industries

6

Apple replant disease

• Caused by a pathogen complex– Rhizoctonia solani AG-5, AG-6– Cylindrocarpon spp.– Pythium spp.– Phytophthora cactorum– Pratylenchus penetrans

http://www.ces.ncsu.edu

Disease symptoms

Manson, WAMoxee, WA

Courtesy Mark Mazzola

Soil-borne Pathogens of Strawberrya. Fusarium wilt - Fusarium oxysporum f. sp. fragariae

b. Macrophomina crown rot - Macrophomina phaseolina

c. Verticillium wilt - Verticillium dahliae

d. Phytophthora crown rot - Phytophthora spp.

a http://ucanr.edu b http://ucanr.edu c http://www.omafra.gov.on.ca https://pnwhandbooks.orgd

Need of alternatives for disease management

• Emergence of new soilborne diseases• Restrictions on soil fumigation• Demand for long-term disease control • Increasing extent of organic industry

Anaerobic Soil Disinfestation (ASD)

11

Anaerobic Soil Disinfestation (ASD)

12

Grass application Amending into soil Irrigation

Tarping Tarp Removal

C source Rate /acre Irrigation Cost ($) acre -1

Rice bran (RB) 2 t Field capacity 240

Brassica juncea seed meal (SM)

2 t Field capacity 3200

Grass clippings (GR)(Dactylis glomerata L.)

9 t Field capacity Mowing + spread

Composted steer manure (CM)

4.5 t Field capacity 153

Ethanol (ET) 80 gal ½ Field capacity 900

ASD Carbon SourcesTreatments after one

week incubation

Soil amendment rates 13

Fungal growth after ASDin treated orchard soil

Ethanol Control Pasteurized Grass clippings

Composted Steer manure

Rice bran

Brassica juncea seed

meal

Resident soil microbiome changes

15

0

0.5

1

1.5

2

C ET GR SM RB CM

Seed

ling

wei

ght/

g

C treatment

b b

aa

b b

R. solani AG-5

Fung

al C

omm

unity

Bact

eria

l Com

mun

ity

C source

Rice bran (RB)

Brassica junceaseed meal (SM)

Grass clippings (GR)

Composted steer manure (CM)

Ethanol (ET)

Hewavitharana and Mazzola, 2016

Experimental Set-up

(Swenson et al. 2015)

16

Day

0

Day

1

Day

2

Day

3

Day

7

Day

11

Day

15

Control

ASD Soil DNA

Illumina® sequencing

Polar and non-polar metabolite analysis

Volatile analysis

Swenson et al. 2015

5,634 OTUs

689 metabolitesHewavitharana et al. 2019

Changes in O2 and CO2 levels in the Headspace

17

0

5

10

15

20

25

30

35

0 50 100 150 200 250 300 350 400

Rela

tive

head

spac

e co

ncen

trat

ion

(%)

Hours after set-up

O2 ASD O2 Control CO2 ASD CO2 Control

Day1

5----

-<---

Day0

Day0---------->--------Day15

Day1

5----

--<---

-----D

ay0

Day0--------->---------Day15ASD

ASD

Cont

rol

Control

Bacteria

Similar

Dissimilar

Fungi

Day1

5----

--<---

-----D

ay0

Day1

5----

-<---

Day0

Cont

rol

ASD

Changes in Soil Microbiome

18Similar

Dissimilar

Changes in Relative Abundance of Bacterial Divisions

19

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

ASD.

Day0

.Rep

1.

ASD.

Day0

.Rep

2.

ASD.

Day0

.Rep

3.

ASD.

Day0

.Rep

4.

ASD.

Day0

1.Re

p1.

ASD.

Day0

1.Re

p2.

ASD.

Day0

1.Re

p3.

ASD.

Day0

1.Re

p4.

ASD.

Day0

2.Re

p1.

ASD.

Day0

2.Re

p2.

ASD.

Day0

2.Re

p3.

ASD.

Day0

2.Re

p4.

ASD.

Day0

3.Re

p1.

ASD.

Day0

3.Re

p2.

ASD.

Day0

3.Re

p3.

ASD.

Day0

3.Re

p4.

ASD.

Day0

7.Re

p1.

ASD.

Day0

7.Re

p2.

ASD.

Day0

7.Re

p3.

ASD.

Day0

7.Re

p4.

ASD.

Day1

1.Re

p1.

ASD.

Day1

1.Re

p2.

ASD.

Day1

1.Re

p3.

ASD.

Day1

1.Re

p4.

ASD.

Day1

5.Re

p1.

ASD.

Day1

5.Re

p2.

ASD.

Day1

5.Re

p3.

ASD.

Day1

5.Re

p4.

Perc

enta

ge o

f lib

rary

Incubation duration

VerrucomicrobiaThermotogaeThermodesulfobacteriaTenericutesSynergistetesSpirochaetesProteobacteriaPlanctomycetesNitrospiraeNitrospinaeIgnavibacteriaeGemmatimonadetesFusobacteriaFirmicutesFibrobacteresElusimicrobiaDeinococcus_thermusDeferribacteresCyanobacteriaChloroflexiChlorobiChlamydiaeCandidatus saccharibacteriaBacteroidetesArmatimonadetesAquificaeActinobacteriaAcidobacteria

Changes in Relative Abundance of Fungal Divisions

20

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

ASD.

Day0

.Rep

1

ASD.

Day0

.Rep

2

ASD.

Day0

.Rep

3

ASD.

Day0

.Rep

4

ASD.

Day0

1.Re

p1

ASD.

Day0

1.Re

p2

ASD.

Day0

1.Re

p3

ASD.

Day0

1.Re

p4

ASD.

Day0

2.Re

p1

ASD.

Day0

2.Re

p2

ASD.

Day0

2.Re

p3

ASD.

Day0

2.Re

p4

ASD.

Day0

3.Re

p1

ASD.

Day0

3.Re

p2

ASD.

Day0

3.Re

p3

ASD.

Day0

3.Re

p4

ASD.

Day0

7.Re

p1

ASD.

Day0

7.Re

p2

ASD.

Day0

7.Re

p3

ASD.

Day0

7.Re

p4

ASD.

Day1

1.Re

p1

ASD.

Day1

1.Re

p2

ASD.

Day1

1.Re

p3

ASD.

Day1

1.Re

p4

ASD.

Day1

5.Re

p1

ASD.

Day1

5.Re

p2

ASD.

Day1

5.Re

p3

ASD.

Day1

5.Re

p4

Perc

enta

ge o

f lib

rary

Incubation duration

ZygomycotaNeocallimastigomycotaMonoblepharidomycotaGlomeromycotaEntomophthoromycotaCryptomycotaChytridiomycotaBasidiomycotaAscomycota

Changes in Relative Abundance of Classes of Division Ascomycota

21

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

ASD.

Day0

.Rep

1

ASD.

Day0

.Rep

2

ASD.

Day0

.Rep

3

ASD.

Day0

.Rep

4

ASD.

Day0

1.Re

p1

ASD.

Day0

1.Re

p2

ASD.

Day0

1.Re

p3

ASD.

Day0

1.Re

p4

ASD.

Day0

2.Re

p1

ASD.

Day0

2.Re

p2

ASD.

Day0

2.Re

p3

ASD.

Day0

2.Re

p4

ASD.

Day0

3.Re

p1

ASD.

Day0

3.Re

p2

ASD.

Day0

3.Re

p3

ASD.

Day0

3.Re

p4

ASD.

Day0

7.Re

p1

ASD.

Day0

7.Re

p2

ASD.

Day0

7.Re

p3

ASD.

Day0

7.Re

p4

ASD.

Day1

1.Re

p1

ASD.

Day1

1.Re

p2

ASD.

Day1

1.Re

p3

ASD.

Day1

1.Re

p4

ASD.

Day1

5.Re

p1

ASD.

Day1

5.Re

p2

ASD.

Day1

5.Re

p3

ASD.

Day1

5.Re

p4

Perc

enta

ge o

f lib

rary

Incubation duration

xylonomycetes

sordariomycetes

schizosaccharomycetes

saccharomycetes

pezizomycotina

pezizomycetes

orbiliomycetes

leotiomycetes

lecanoromycetes

eurotiomycetes

dothideomycetes

ascomycota

arthoniomycetes

archaeorhizomycetes

Temporal Changes in Soil Microbiome and Metabolome in Response to ASD

Microbiome shifts Metabolome shifts

Bacterial Community Fungal Community

Hewavitharana et al. 2019

Co-abundance Correlation Microbial Network-ASD treatment


Co-abundance Correlation Metabolite Network-ASD treatment


Volatile acids, aldehydes, alcohols, and hydrocarbons produced during simulated anaerobic

soil disinfestation (ASD)


Key Functional Attributes of ASD


Host challenged by the pathogen complex of replant disease

Enhanced host growth and long-term disease suppression by managing rhizosphere microbiome and metabolome

Anaerobic soil disinfestation

Induced proliferation of beneficial microorganis

ms

Anaerobiosis Competition

Toxic volatiles and

metabolites

How does ASD Work?

Mazzola and Hewavitharana, 2019

Temporal Changes in Soil Microbiome in Response to Soil Fumigation in Strawberry

Production Systems

Objectives:

• Study temporal changes in microbiome� Post-treatment� At planting� One month after planting� At peak fruit production� End of the season

• Identify biomarker organisms that could be targeted for enrichment

https://ucanr.edu/

Post-treatment Soil SamplingFumigant/Treatment Active IngredientsAlly 33 Allyl Isothiocyanate (64%), Chloropicrin (31%)Dominus Allyl Isothiocyanate (96%)K-Pam HL Potassium N-Methyldithiocarbamate (54%)Tri-Clor Chloropicrin (99%)No-treatment Control NA

Transplanting in July 2019

Effect of Soil Treatments on V. dahliae Soil Population

0

1

2

3

4

5

6

7

8

9

Vert

icill

ium

dah

liae

CFU

g-1

soil

Soil treatment*time point

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Vert

icill

ium

dah

liae

CFU

g-1

soil

Soil treatment*(Time point 1-Time point 2)

Effect of Soil Treatments on Plant Mortality

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Ally Dominus KPAM Triclor Control

Perc

ent p

lant

mor

talit

y

Soil treatment

11/7/2019

11/23/2019

12/12/2019

1/5/2019

Marketable Yield Differences among Treatments

0.000

0.005

0.010

0.015

0.020

0.025

Ally33 Dominus K-PAM Tri-Clor Control

Yiel

d pl

ant-1

kg-1

Soil treatment

30-Oct

15-Nov

Concluding Remarks

• Biology mediated soilborne disease management is complex• Efficacy of ASD is dependent on various factors that need to be

tailored based on the pathosystem• ASD mediated disease suppression is connected to interaction

between soil microbiome and metabolome• Potential for identifying beneficial microorganisms after soil

fumigation that can be enriched with additional practices

• Dr. Mark Mazzola, Dr. David Rudell, Dr. Jim Mattheis, Dr. Loren Honaas, Dr. Rachel Leisso- USDA-ARS Wenatchee• Dr. Gerald Holmes, Dr. S. Mansouripour, Kyle Blauer, Drew Summerfield,

Farm crew- Cal Poly Strawberry Center• Cal Poly Students: Olivia Mann, Maria Debaros, Abby Maxwell, Maria

Sanchez, Angela Cruz, Lauren Tallichet• Funding agencies and industry partners:

Acknowledgement

Thank you

37

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