biological control of pythium aphanidermatum impacts of the seed colonizing microbial community on...
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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. Nelson’s 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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vegetative hyphae
sporangium
germinating sporangium
zoosporangium zoospores
antheridium
oogonium
oogonium oospore
Germinatingoospore
asexual
sexual
direct
indirect
DISEASE
[modified from Matthews 1931]
Pythiumaphanidermatum
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Microbial interactions in the spermosphere
Gradient of seed exudates
Seed colonizingmicrobes
Pythium aphanidermatumzoospore responding to seed exudates with chemotaxis
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[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
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PIPK G
GPCR
G PsCAM1PsCMK3PsCMK4
Calmodulin dependent protein kinases
[Hua et al. 2008]
Chemotractant
Zoospore signaling
Phytophthora sojae
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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
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[Liu et al. 2007]
Cucumis sativum cv. Marketmore 76
• Exudates contain– Carbohydrates– Organic acids– Amino acids– Many other compounds
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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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Sterile glass fiber filter
Sand or Sand/compost mixtureCucumber seeds
Sand or Sand/compost mixture
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Sand
Sterile Batch 3
Batch 12006
Batch 22007
Batch 32008
Non-inoculated Inoculated
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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
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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
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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
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
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Compost extracts provide soluble nutrients, especially when plug size limits compost amendment in certified organic systems
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WaterVermicompostExtract 1:5
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Chemical characteristics
A.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
• 1:60 ratio of vermicompost to water (by mass)
• Circulation for 5 min 2 x per day
• Strained through 4 layers of cheeseclothsump
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Sand
Sterile VC Extract
VC Extract
Non-inoculated Inoculated
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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
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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
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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?
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
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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
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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
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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
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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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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
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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.
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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
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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
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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
Regression p < 0.001Dilution of seed exudate
Predictions for vermicompost MMSE:
Attractant missing
Repellant present
Unmodified exudate
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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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27
2
3
Unmodified exudate
VermicompostMMSE
Water (no seed)
Short videos taken after 5 minutes
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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 cells:Root border cells
[Hawes & Pueppke 1986]
[Goldberg et al. 1988]
P. dissotocum on cotton
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Time lapse of interaction with a single root border cell
19x 19xT = 0 T = 50 m
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Only certain cells attract zoospores
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Root border cell viability:Fluorescein diacetate staining
[Larkin 1976]
7.6 x 7.6 x
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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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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
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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