Spinach and lettuce downy mildew epidemiology

Steve KlostermanUSDA ARS, Salinas, CA

Krishna Subbarao,University of California, Davis

Lettuce downy mildew – Bremia lactucae

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)

Downy mildew threat predictions may be useful

for more cost-effective fungicide applications

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

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.

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.

Klosterman,

Subbarao,

Zischke,

unpublished.

7-10 days

before

symptoms

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.

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.

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

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.

Spinach plot to detect downy mildew infection

prior to symptom appearance

USDA ARS, Salinas

November, 2016

1

12

24

13

25

3637

48

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

- 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

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

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?

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

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?

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)

Keimung der Oosporend = “Germination of the Oospores”

Jakob Eriksson. 1918.

Arkiv For Botanik. v.15

“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

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?

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

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