Biological control of Pythium aphanidermatum

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Biological control of Pythium aphanidermatum

• Impacts of the seed colonizing microbial
  community on zoospore pre-infection events

Allison L. H. Jack Dr. Eric B. Nelsons research
group April 8, 2009
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Outline

• Background on zoospore pre-infection events
• Disease suppressive vermicompost and vermicompost
  extracts
• Investigations into the mechanism behind observed
  suppression
• Vermicompost use in horticulture

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Zoospore pre-infection events
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Pythium aphanidermatum
germinating sporangium
sporangium
direct
asexual
zoosporangium
zoospores
indirect
DISEASE
vegetative hyphae
Germinating oospore
oogonium
sexual
antheridium
oospore
oogonium
modified from Matthews 1931
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Microbial interactions in the spermosphere
Gradient of seed exudates
Pythium aphanidermatum zoospore responding to
seed exudates with chemotaxis
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Eukaryotic flagella
Play 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
Mitchell 2004
Rosenbaum Whitman 2002
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Zoospore signaling
Chemotractant
GPCR
Ga
PsCAM1 PsCMK3 PsCMK4
PIPK
Ga
Calmodulin dependent protein kinases
Phytophthora sojae
Hua et al. 2008
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P. aphanidermatum zoospores

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

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

Donaldson Deacon 1993
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Cucumis sativum cv. Marketmore 76

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

Liu et al. 2007
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Zoospore pre-infection events(chemotaxis)
?
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Disease suppressive vermicompost
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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

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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?
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• Height of water column determines matric
  potential in growing media

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Sand or Sand/compost mixture
Sterile glass fiber filter
Sand or Sand/compost mixture
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Non-inoculated
Inoculated
Sand
Sterile Batch 3
Batch 1 2006
Batch 2 2007
Batch 3 2008
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Health rating
a
a
a
a
a
a
a
b
c
d
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Total seedling stand
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Conclusions

• Suppression of disease caused by P.
  aphanidermatum is relatively consistent from
  batch to batch
• Suppression is dependent on a biological factor

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Compost extracts

• Traditional agricultural practice
• Extensively studied in Europe in the 1980s
  Weltzien 1989, Trankner 1992
• Recent literature exists Scheuerell Mahaffee
  2004, 2006
• Most published methods use 15 110 ratios of
  compost to water

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Compost extracts provide soluble nutrients,
especially when plug size limits compost
amendment in certified organic systems
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Vermicompost Extract 15
Water
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Chemical characteristics

• 1 week extracts, B. 2 week extracts
• DO dissolved oxygen in ppm
• EC electrical conductivity in mS cm-1

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Non-aerated vermicompost extract

• 160 ratio of vermicompost to water (by mass)
• Circulation for 5 min 2 x per day
• Strained through 4 layers of cheesecloth

sump
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Non-inoculated
Inoculated
Sand
Sterile VC Extract
VC Extract
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Health rating
a
a
a
b
c
c
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Seedling stand
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Future directions

• Lyophilize the extract
• Reconstitute
• Use as seed treatment
• Consider adding as a treatment for follow up
  experiments with seed colonizing microbial
  community

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How are zoospores prevented from infecting the
seeds?
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When do P. aphanidermatum zoospores reach the
seed surface?
Harvest
H
SAND
Inoculate
Transplant
H
SAND INOC
H
T
SAND INOC T8
H
T
SAND INOC T16
H
T
SAND INOC T24
2
3
1
4
5
6
7
Time (d)
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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

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When does the suppressive community develop on
the seed surface? (P. ultimum on wheat)
Pythium inoculation
Shoot height (mm)
0
30
60
90
120
150
Sand
Suppressive compost
Seed
Microbes
Chen Nelson 2008
7d
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Zoospore attraction assays with microbially
modified seed exudates (MMSE)
Hypothesis Seed colonizing microbes modify
exudates which alters zoospore behavior.
Seeds removed, exudate sterile filtered
Seeds rinsed in sterile water
24 hr incubation in water
Microbially modified seed exudate (MMSE)
24 hr germination in Sand Vermicompost
(40) Sterile water filter paper
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Zoospore attraction assay
Agar plugs on a microscope slide infused with
exudates
Zoospore solution
Slides are removed after 30 min, imaged and
encysted zoospores are counted
19x
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40 vv amendment of vermicompost
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Are lower numbers of encysted zoospores due to
the presence of a repellant, or the absence of an
attractant?
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Dose response curve
Predictions for vermicompost MMSE
Unmodified exudate
Repellant present
Attractant missing
Dilution of seed exudate
Regression p lt 0.001
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Chemotaxis The zoospore maze

• Imaging the zoospores as they respond to
  exudates in real time

Perfusion chamber
Entire chamber filled with 275uL zoospore
suspension
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Short videos taken after 5 minutes
Unmodified exudate
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Vermicompost MMSE
2
Water (no seed)
3
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Are additional stages of zoospore pre-infection
behavior affected by seed colonizing microbes?
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Zoospore pre-infection events(chemotaxis)
?
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Interaction with plant cellsRoot border cells
P. dissotocum on cotton
Hawes Pueppke 1986
Goldberg et al. 1988
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Time lapse of interaction with a single root
border cell
19x
T 0
19x
T 50 m
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Only certain cells attract zoospores
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Root border cell viabilityFluorescein diacetate
staining
7.6 x
7.6 x
Larkin 1976
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Cucumber border cells with zoospores
7.6 x
7.6 x
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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

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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?

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Horticultural applications
Cabbage transplants 19 DAP, Growers 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.
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Acknowledgements
Nelson Lab Mary Ann Karp Eric Carr Hillary
Davis Monica Minson Liang Chen Sarah Arnold Dave
Moody
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
My committee Eric Nelson (PPPMB) Anthony Hay
(MICRO) Anu Rangarajan (HORT) Kathie Hodge
(PPPMB) Scott Peters (EDUC)
The Worm Guy Tom Herlihy RT Solutions
Boo Boo Steffen Jack
Kent Loeffler photo credits