Transcript Pythium aphanidermatum - New York Farm Viability Institute

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
germinating sporangium
Pythium
aphanidermatum
sporangium
direct
asexual
zoosporangium
zoospores
indirect
DISEASE
vegetative hyphae
Germinating
oospore
oogonium
sexual
antheridium
oogonium
[modified from Matthews 1931]
oospore
Microbial interactions in the spermosphere
Gradient of seed exudates
Pythium aphanidermatum
zoospore responding to
seed exudates with
chemotaxis
Seed colonizing
microbes
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
[Rosenbaum & Whitman 2002]
[Mitchell 2004]
Zoospore signaling
Chemotractant
GPCR
Ga
PIPK
[Hua et al. 2008]
Ga
PsCAM1
PsCMK3
PsCMK4
Calmodulin dependent
protein kinases
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
Cucumis sativum
cv. Marketmore 76
• Exudates contain
–
–
–
–
Carbohydrates
Organic acids
Amino acids
Many other compounds
[Liu et al. 2007]
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
Sand or Sand/compost mixture
Cucumber
seeds mixture
Sand
or Sand/compost
Sterile glass fiber filter
Non-inoculated
Sand
Sterile
Batch 3
Batch 1
2006
Batch 2
2007
Batch 3
2008
Inoculated
Average health rating 5 = healthy (n=30)
Health rating
6
a
a
a
a
a
5
a
NIN
IN
a
b
4
3
c
2
1
d
0
sand
CDVC3(St)
CDVC1
CDVC2
CDVC3
Total seedling stand
Total seedling stand (out of 30)
35
NIN
IN
30
25
20
15
10
5
0
sand
CDVC3(St)
CDVC1
CDVC2
CDVC3
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
Water
Vermicompost
Extract 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
sump
• 1:60 ratio of
vermicompost to
water (by mass)
• Circulation for 5 min 2
x per day
• Strained through 4
layers of cheesecloth
Non-inoculated
Sand
Sterile
VC
Extract
VC
Extract
Inoculated
Average health rating (5 = healthy) n=108
Health rating
6
a
a
NIN
IN
a
5
b
4
3
2
c
c
1
0
sterile extract
sand
extract
Seedling stand
Total seedling stand (out of 108)
120
NIN
IN
100
80
60
40
20
0
sterile extract
sand
extract
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?
Harvest
H
SAND
Inoculate
Transplant
H
SAND INOC
SAND INOC T8
SAND INOC T16
SAND INOC T24
Time (d)
H
T
H
T
H
T
1
2
3
4
5
6
7
Proportion of 10 seeds with Pythium present
in specific sections
8 hours
after
sowing in
sand
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
1
Proportion of 10 seeds with Pythium present
in specific sections
0.9
0.8
16 hours
after
sowing in
sand
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
Proportion of seeds with Pythium present
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Proportion of 10 seeds with Pythium present
in specific sections
24 hours
after
sowing in
sand
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
When does the suppressive
community develop on the
Pythium
seed surface? (P. inoculation
ultimum on wheat)
Shoot height (mm)
0
30
60
90
120
7d
8h
a
7d
a
b
7d
8h
7d
a
b
7d
7d
Sand
Suppressive compost
150
Non-inoculated
Seed
Microbes
a
[Chen & Nelson 2008]
Hypothesis: Seed colonizing microbes modify
exudates which alters zoospore behavior.
Zoospore attraction assays with microbially
modified seed exudates (MMSE)
Seeds rinsed
in sterile water
24 hr germination in:
Sand
Vermicompost (40%)
Sterile water & filter paper
Seeds removed,
exudate sterile
filtered
24 hr incubation in:
water
Microbially modified
seed exudate (MMSE)
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
Total # encysted zoospores in 4 fields of view
(average of 6 reps)
1800
1600
1400
1200
1000
800
600
400
200
0
no
vermicompost
batch 1
batch 2
batch 3
40% v:v amendment of vermicompost
water
Are lower numbers of encysted
zoospores due to the presence
of a repellant, or the absence of
an attractant?
Dose – response curve
Predictions forFitted
vermicompost
MMSE:
Line Plot
total count = - 12.38 + 186.7 treatment
- 736 treatment**2 + 1343 treatment**3
S
R-Sq
R-Sq(adj)
900
800
Unmodified exudate
87.8409
90.9%
89.8%
total count
700
600
500
400
300
200
Repellant present
100
0
Attractant missing
0.0
0.2
0.4
0.6
treatment
0.8
Dilution of seed exudate
1.0
Regression p < 0.001
Chemotaxis – The zoospore maze
Imaging the zoospores as they respond to
exudates in real time
Perfusion chamber
Entire chamber filled with
275uL zoospore suspension
Short videos taken after 5 minutes
Unmodified
exudate
27
Vermicompost
MMSE
2
Water (no seed)
3
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
P. dissotocum on cotton
[Hawes & Pueppke 1986]
[Goldberg et al. 1988]
Time lapse of interaction with a
single root border cell
19x
T=0
19x
T = 50 m
Only certain cells attract zoospores
Root border cell viability:
Fluorescein diacetate staining
7.6 x
7.6 x
[Larkin 1976]
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?
Horticultural applications
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.
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
My committee:
Eric Nelson (PPPMB)
Anthony Hay (MICRO)
Anu Rangarajan (HORT)
Kathie Hodge (PPPMB)
Scott Peters (EDUC)
New York Farm Viability Institute
The “Worm Guy”
Tom Herlihy – RT Solutions
“Boo Boo”
Steffen Jack
USDA BARD
Knight Institute for Writing in the Disciplines
NYSTAR Center for Advanced Technology &
USDA SBIR Phase I (with RT Solutions)
Organic Farming Research Foundation
Organic Crop Improvement Association
Andrew W. Mellon Fellowship
Kent Loeffler – photo credits