Biological control of Pythium aphanidermatum

Impacts of the seed colonizing microbial community on

zoospore pre-infection events

Allison L. H. Jack

Dr. Eric B. Nelson’s research group

April 8, 2009

Outline

• Background on zoospore pre-infection events

• Disease suppressive vermicompost and vermicompost extracts

• Investigations into the mechanism behind observed suppression

• Vermicompost use in horticulture

Zoospore pre-infection events

vegetative hyphae

sporangium

germinating sporangium

zoosporangium zoospores

antheridium

oogonium

oogonium oospore

Germinatingoospore

asexual

sexual

direct

indirect

DISEASE

[modified from Matthews 1931]

Pythiumaphanidermatum

Microbial interactions in the spermosphere

Gradient of seed exudates

Seed colonizingmicrobes

Pythium aphanidermatumzoospore responding to seed exudates with chemotaxis

[Rosenbaum & Whitman 2002] [Mitchell 2004]

Eukaryotic flagellaPlay a crucial role in sensing the extracellular environment and transmitting signals to the cell body

Oomycete zoospores have specific receptor ligand interactions as encystment cues

PIPK G

GPCR

G PsCAM1PsCMK3PsCMK4

Calmodulin dependent protein kinases

[Hua et al. 2008]

Chemotractant

Zoospore signaling

Phytophthora sojae

P. aphanidermatum zoospores

• Known chemotractants:– L-aspartate– L-glutamate– L-glutamine– L-alanine– D-mannose– Sucrose– Maltose– D-fucose

[Donaldson & Deacon 1993]

If the solution contains a high enough background concentration of an amino acid, then chemotaxis is abolished

[Liu et al. 2007]

Cucumis sativum cv. Marketmore 76

• Exudates contain– Carbohydrates– Organic acids– Amino acids– Many other compounds

Zoospore pre-infection events(chemotaxis)

?

Disease suppressive vermicompost

Brief history of disease suppression research

• Late 1800s: suppressive soils documented [Huber & Schneider 1982]

• 1930s – 1940s: Link made between composts and soil health [Howard 1942]

• 1959: Biological nature of suppression documented [Menzies 1959]

• 1970s - 1980s: Extensive work done on suppressive composts [Hoitink & Kuter 1986, Weltzein 1989]

Vermicompost• Separated dairy manure solids• Hot composted for 5 days under forced aeration• Fed in thin layers to continuous flow through

worm beds• Harvested out the bottom after 65 days• Highly controlled process leads to a material

with consistent properties

Can vermicomposted dairy manure consistently

suppress Pythium damping off?

Height of water column determines matric potential in growing media

Sterile glass fiber filter

Sand or Sand/compost mixtureCucumber seeds

Sand or Sand/compost mixture

Sand

Sterile Batch 3

Batch 12006

Batch 22007

Batch 32008

Non-inoculated Inoculated

Health rating

0

1

2

3

4

5

6

sand CDVC3(St) CDVC1 CDVC2 CDVC3

Av

era

ge

he

alth

ra

tin

g 5

= h

ealt

hy

(n

=3

0)

NININa a a a a a

a

b

c

d

Total seedling stand

0

5

10

15

20

25

30

35

sand CDVC3(St) CDVC1 CDVC2 CDVC3

To

tal s

ee

dlin

g s

tan

d (

ou

t o

f 3

0)

NININ

Conclusions

• Suppression of disease caused by P. aphanidermatum is relatively consistent from batch to batch

• Suppression is dependent on a biological factor

Compost extracts

• Traditional agricultural practice• Extensively studied in Europe in the

1980’s [Weltzien 1989, Trankner 1992]

• Recent literature exists [Scheuerell & Mahaffee 2004, 2006]

• Most published methods use 1:5 – 1:10 ratios of compost to water

Compost extracts provide soluble nutrients, especially when plug size limits compost amendment in certified organic systems

WaterVermicompostExtract 1:5

Chemical characteristics

A.1 week extracts, B. 2 week extracts

DO = dissolved oxygen in ppm

EC = electrical conductivity in mS cm-1

Non-aerated vermicompost extract

• 1:60 ratio of vermicompost to water (by mass)

• Circulation for 5 min 2 x per day

• Strained through 4 layers of cheeseclothsump

Sand

Sterile VC Extract

VC Extract

Non-inoculated Inoculated

Health rating

0

1

2

3

4

5

6

sterile extract sand extract

Ave

rag

e h

ea

lth

ra

tin

g (

5 =

he

alt

hy

) n

=1

08

NININa a a

b

cc

0

20

40

60

80

100

120

sterile extract sand extract

To

tal s

eed

ling

sta

nd

(o

ut

of

108

)

NININ

Seedling stand

Future directions

• Lyophilize the extract– Reconstitute – Use as seed treatment

• Consider adding as a treatment for follow up experiments with seed colonizing microbial community

How are zoospores prevented from infecting the seeds?

When do P. aphanidermatum zoospores reach the seed surface?

H

H

H

H

H

Inoculate

Harvest

Transplant

1 2 3 4 5 6 7Time (d)

SAND

SAND INOC

SAND INOC T8

SAND INOC T16

SAND INOC T24

T

T

T

8 hours after sowing in sand

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 2 3 4 5 6

Proportion of 10 seeds with Pythium present in specific sections

16 hours after sowing in sand

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 2 3 4 5 6 7

Proportion of 10 seeds with Pythium present in specific sections

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1P

rop

ort

ion

of

see

ds

wit

h P

yth

ium

pre

sen

t

24 hours after sowing in sand

Proportion of 10 seeds with Pythium present in specific sections

Conclusions and next steps

• Pythium is present on most seeds within 24 hours

• Surface sterilize to distinguish between presence and infection

• Add seeds sown in vermicompost as a comparison– will this change the timing of zoospores reaching the

seed surface?

• Confirm results with qPCR once time frame is worked out in detail

Shoot height (mm)

0 30 60 90 120 150

[Chen & Nelson 2008]

Pythiuminoculation

Sand Suppressive compost

a7d

a8h 7d

b8h 7d

b7d

Non-inoculated a7d

When does the suppressive community develop on the seed surface? (P. ultimum on wheat)

Seed Microbes

a7d

24 hr incubation in: water

Seeds rinsed in sterile water

Seeds removed, exudate sterile filtered

Microbially modified seed exudate (MMSE)

Zoospore attraction assays with microbially modified seed exudates (MMSE)

24 hr germination in:SandVermicompost (40%)Sterile water & filter paper

Hypothesis: Seed colonizing microbes modify exudates which alters zoospore behavior.

Zoospore attraction assay

Zoospore solution

Agar plugs on a microscope slide infused with exudates

Slides are removed after 30 min, imaged and encysted zoospores are counted

19x

0

200

400

600

800

1000

1200

1400

1600

1800

novermicompost

batch 1 batch 2 batch 3 water

To

tal #

en

cyst

ed z

oo

spo

res

in 4

fie

lds

of

view

(a

vera

ge

of

6 re

ps)

40% v:v amendment of vermicompost

Are lower numbers of encysted zoospores due to the presence of a repellant, or the absence of

an attractant?

Dose – response curve

Regression p < 0.001Dilution of seed exudate

Predictions for vermicompost MMSE:

Attractant missing

Repellant present

Unmodified exudate

Chemotaxis – The zoospore maze

Imaging the zoospores as they respond to exudates in real time

Perfusion chamber

Entire chamber filled with 275uL zoospore suspension

27

2

3

Unmodified exudate

VermicompostMMSE

Water (no seed)

Short videos taken after 5 minutes

Are additional stages of zoospore pre-infection behavior affected by

seed colonizing microbes?

Zoospore pre-infection events(chemotaxis)

?

Interaction with plant cells:Root border cells

[Hawes & Pueppke 1986]

[Goldberg et al. 1988]

P. dissotocum on cotton

Time lapse of interaction with a single root border cell

19x 19xT = 0 T = 50 m

Only certain cells attract zoospores

Root border cell viability:Fluorescein diacetate staining

[Larkin 1976]

7.6 x 7.6 x

Cucumber border cells with zoospores

7.6 x 7.6 x

Conclusions

• Zoospore attraction appears to be affected by seed colonizing microbes from vermicompost which may account for the observed suppression of disease

• Whether this is due to an attractant missing or the presence of a repellant remains to be determined

• Time frame of when zoospores reach the seed and the nature of their interactions with root border cells need to be refined

Burning questions

• Which microbial taxa / functional genes are present on the seed surface during the critical time frame when suppression is expressed?

• How exactly are these seed exudates being modified?

Cabbage transplants 19 DAP, Grower’s mix (A.) with bloodmeal (B.), 10% vermicompost (C.), 10% vermicompost & bloodmeal (D.), Cornell base mix (E.) with bloodmeal (F.), 10% vermicompost (G.), 10% vermicompost + bloodmeal (H.). Treatments D and H had the highest transplant biomass of all treatments tested.

Horticultural applications

AcknowledgementsNelson Lab:Mary Ann KarpEric CarrHillary DavisMonica MinsonLiang ChenSarah ArnoldDave Moody

My committee:Eric Nelson (PPPMB)Anthony Hay (MICRO)Anu Rangarajan (HORT)Kathie Hodge (PPPMB)Scott Peters (EDUC)

Financial support:Department of Plant Pathology and Plant Microbe Biology

USDA BARD

Knight Institute for Writing in the Disciplines

New York Farm Viability Institute

NYSTAR Center for Advanced Technology & USDA SBIR Phase I (with RT Solutions)

Organic Farming Research Foundation

Organic Crop Improvement Association

Andrew W. Mellon Fellowship

The “Worm Guy”Tom Herlihy – RT Solutions

“Boo Boo”Steffen Jack

Kent Loeffler – photo credits