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Spinach and lettuce downy mildew epidemiology
Steve KlostermanUSDA ARS, Salinas, CA
Krishna Subbarao,University of California, Davis
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Lettuce downy mildew – Bremia lactucae
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Spore trap system to collect airborne spores
of Bremia lactucae(design and control box from Dr. Walt Mahaffee, USDA, Corvallis)
Coupled with B. lactucae-specific DNA assay
(Kunjeti et al. 2016. 106: 1426-1437)
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Downy mildew threat predictions may be useful
for more cost-effective fungicide applications
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Experiment to evaluate for reductions in fungicide applications
based on Bremia lactucae detection
Three 80” beds
Four replicated plots x 30 feet
Spore traps, at north and south ends of field
Spore trap sampling every 72 and 96 hour
1. Untreated
2. Calendar spray
3. Spore trap advised
(Three trials in 2016)
Bed 1 Bed 2 Bed 3
Rep
D
Rep
C
Rep
B
Rep
A
spore trap 1
spore trap 2
N
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Lettuce downy mildew fungicide sprays -- Calendar vs
spore trap advised sprays, (2016, Trial 3)
1. Untreated 2. Calendar 3. Spore trap
spray 1 … 22-Aug …
Manzate 2.1 lb/acre
spray 2 … 2-Sep …
Manzate 2.1 lb
spray 3 … 9-Sep …
Reason 8.2 fl oz
spray 4 … 19-Sep 19-Sep
Revus 8 fl oz Revus 8 fl oz
Conclusion:
Average savings of 1.7 fungicide
applications for all three trials, but
did lose half of the disease control.
Symptomatic plants (Sep 28)
Untreated
Calendar
spray
Advised
spray
Plants + 211/648 45/648 104/648
Percent of
total 33% 7% 16%
Counted plant positive even if a single lesion.
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Downy mildew on spinach
Objective: detection in leaves prior to symptom development
Symptoms: Chlorotic Signs: typically grey downy
spots on top of leaves. masses of spores on the
underside of leaf
50 μm
From Klosterman, 2016. Progressive Crop Consultant, 1:12-15.
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Klosterman,
Subbarao,
Zischke,
unpublished.
7-10 days
before
symptoms
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Objective:
- Examine spinach leaves to detect and determine downy
mildew infection prior to symptom appearance.
What is the value to the spinach industry?
Organic – determine if plants infected,
harvest before loss.
Conventional – assess whether plants infected
to time spray application for disease
control.
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Spinach plot to detect downy mildew infection
prior to symptom appearance
USDA ARS, SalinasC. Subbarao, K. Subbarao, A. Anchieta, S. Kunjeti
December 21, 2015
1
12
24
13
25
3637
48
Four weeks after planting – prior to leaf symptom development.
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1 2 3 4 5 6 8 9 10 11 12131516 17181920 21 222324 + -controls
2526 272829 3031323334 3536 37383940 414243444546 4748 + -
December 28 sampling date (five weeks after planting)
Plot examined January 4, pathogen and symptoms first observed January 8, 2016
PCR amplification of spinach downy mildew DNA from
infected leaves prior to appearance of plant symptoms
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January 15, 2016
Spinach plot to detect downy mildew infection
prior to symptom development
USDA ARS, Salinas
Seven weeks after planting - much of field showing symptoms.
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Spinach plot to detect downy mildew infection
prior to symptom appearance
USDA ARS, Salinas
November, 2016
1
12
24
13
25
3637
48
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1 0 10 40 70
2 0 10 35 65
3 0 15 40 50
4 0 15 35 45
5 0 5 25 40
6 0 10 20 40
7 0 15 30 30
8 0 10 25 30
9 0 15 35 30
10 0 5 20 35
11 0 10 35 30
12 0 10 35 40
0 10.8 31.3 42.1Average
Date
10/5 10/12 10/19 10/26
Downy mildew disease incidence over time, October 2016
Sample
10/5 DNA gelControls
+ -
10/19 DNA gel
Controls
+ -
Visual, number
of plants
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- Two field plot experiments indicate:
Detected the pathogen in leaves 7 days
prior to symptom appearance.
- Future work (2017)
One more experiment, and then make the assay
available to a private company.
Summary: DNA assay for detection of spinach
downy mildew pathogen in leaves
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Why the concern?
- Increased survival time of P. effusa (on seed, soil?, etc.)
May explain sudden appearance of new pathotypes
in different areas.
- Presence of oospores indicates sexual recombination, which provides
mechanism for rapid generation of new traits in populations, such as
new pathotypes.
Oospore transmission on spinach seed
14
12
10
8
6
4
2
01800 1850 1900 1950 2000 2050
Year
Ra
ce
Discovery of downy mildew races
Correll et al. 2011. European Journal
of Plant Pathology. 129:193–205.
CA spinach 30-40%
organic
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50 µm
In total, we have detected one or more oospores in 28
out of 133 spinach seed lots (21%).
Seed lot # 49
September, 2014
But are the oospores viable?
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A B
Etxeberria et al. 2011. Determination of viability of Phytophthora capsici oospores with the tetrazolium bromide
staining test versus a plasmolysis method. Rev. Iberoam Micol. 28:43-49.
NaCl
Test for oospore viability
Untreated
Oospore
Plasmolysis
Add NaCl
H2O rushes out
= cell membrane shrinks
Cell in a solution
H2O H2O
H2O
H2O
Na
Na
Na
Cl
Cl
Cl
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Plasmolysis time course of P. effusa oospores from spinach seed Kunjeti et al. 2016. Plant Disease 100:59-65.
Okay, but do the spinach downy mildew oospores germinate and infect spinach?
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Jakob Eriksson. 1918. Arkiv For Botanik. v.15
Jakob Eriksson - characterization of spinach downy mildew, 1918
(Eriksson prize – award to only 11 plant pathologists since 1923)
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Keimung der Oosporend = “Germination of the Oospores”
Jakob Eriksson. 1918.
Arkiv For Botanik. v.15
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“F. Surgical rule against disease.
Whoever wants to assure a healthy spinach in advance, he
only exempts such seeds of which he knows with certainty that
it has been harvested from healthy spinach plants.”
Jakob Eriksson. 1918. Arkiv For Botanik.
Google translated from German to English
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Conclusions:
1) Detected oospores in modern spinach seed lots (28/133
lots, or 21% of them).
*Could explain rapid appearance of disease in remote areas, and new
pathotypes in areas where previously not observed.
*Evidence in literature that oospores germinate (Eriksson. 1918)
14
12
10
8
6
4
2
01800 1850 1900 1950 2000 2050
Year
Ra
ce
Discovery of downy mildew races
Why so many new
races, so quickly?
What is the driving
factor?
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Conclusions:
2) Detected downy mildew DNA in spinach leaves in two
field plots, one week before symptom appearance.
*Application may be valuable to help the grower determine to:
a) harvest before loss, or
b) apply spray for disease control
3) Spore traps/DNA assays can be useful on smaller spatial
scale (ranches) for downy mildew detection and a reduction
in fungicide applications
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Acknowledgments Recent/current downy mildew team
Nikhilesh Dhar, UC Davis (Salinas, CA)
Sridhara Kunjeti, UC Davis (Salinas)
Amy Anchieta, USDA-ARS, Salinas
Lorena Ochoa, USDA-ARS, Salinas
Daniel Machado, USDA-ARS, Salinas
Sergio Jimenez, Hartnell College intern, Salinas
Ruben Pena, Hartnell College intern, USDA-ARS, Salinas
Chaitra Subbarao, USDA-ARS (currently USC)
Roberto Ornelas, CSUMB intern, Monterey, CA
Recent/current collaborators on downy mildew projects:
Allen Van Deynze, UC Davis, Davis, CA
Charlie Brummer, UC Davis
Neil McRoberts (and former student Robin Choudhury), UC Davis
Juliana Osorio Marin, UC Davis
Steve Koike, UCCE, Salinas
Frank Martin, USDA ARS, Salinas
Marco Thines, Biodiversity and Climate Research Center, Frankfurt, Germany
Hermann Voglmayr, Univ. of Vienna, Austria
Walt Mahaffee, USDA ARS, Corvallis, OR
Beiquan Mou, USDA ARS, Salinas
Richard Michelmore, UC Davis, Davis
Dale Krolikowski, Germains Seed Technology, Gilroy, CA
Alan Fox (Fox Weather) Fortuna, CA
Funding
California Leafy Greens Research Program
California Department of Food and Agriculture