Fusarium & Verticillium Wilts in Spinach: Research Updatemtvernon.wsu.edu/path_team/du-Toit-2013-WE-ACSC-mtg-spinach-Fus... · Fusarium & Verticillium Wilts in Spinach: Research Update

Fusarium & Verticillium Wilts in Spinach: Research Update

Wilbur-Ellis/Alf Christianson Seed Co. Seed Growers’ Mtg 6 February 2013, Burlington, WA

Lindsey du Toit, Mike Derie, Emily Gatch, & Barbara Holmes WSU Mount Vernon NWREC

Fusarium wilt of spinach Fusarium oxysporum f. sp. spinaciae

1. Assess potential for enhancing suppression of Fusarium wilt using annual applications of limestone and nitrate-N fertilizers, and the effects of limestone on Verticillium dahliae.

2. Develop a soil bioassay for spinach seed growers to determine the relative risk of a field for Fusarium wilt, using spinach parent lines with different levels of resistance.

3. Determine the effects of micronutrients and pH on F. oxysporum f. sp. spinaciae and host-pathogen interactions.

Emily Gatch’s PhD Research Objectives

Rep 1

NH4 NO3

Rep 2

NO3 NH4

Rep 4

NH4 NO3

Rep 3

NO3 NH4

Rep 5

NO3 NH4

O tons limestone

1 ton limestone

2 tons limestone

2 tons

1 ton

0 tons

2 tons

0 tons

1 ton

1 ton

2 tons

0 tons

0 tons

1 ton

2 tons

S R M S R M R S M R S M R M S R M S M R S M R S S M R S M R

2009 Field Trial

spinach potato winter wheat spinach

Limestone applications

Column1

2009 2010 2011 2012

Rep 1

Rep 2

Rep 4

Rep 3

Rep 5

0 tons limestone

1 ton limestone

2 tons limestone

2 tons

1 ton

0 tons

2 tons

0 tons

1 ton

1 ton

2 tons

0 tons

0 tons

1 ton

2 tons

S M R S M R M S R M S R M R S M R S R M S R M S S R M S R M

2012 Field Trial

Proline

Proline

Proline

Proline

Proline

1 ton limestone/acre each year

2 tons limestone/acre each year

0 tons limestone/acre each year 0 tons limestone/acre each year

+ 2 tons/acre in spring 2012

4.0

5.0

6.0

7.0

8.0S

oil

pH

0

1

2

0/2

Effect of limestone rate on soil pH: 2009-2012


2009 2010 2012 2011


4.0

5.0

6.0

7.0

8.0S

oil

pH

0

1

2

0/2


2009 2010 2012 2011



4.0

5.0

6.0

7.0

8.0S

oil

pH

0

1

2

0/2


2009 2010 2012 2011



4.0

5.0

6.0

7.0

8.0S

oil

pH

0

1

2

0/2


2009 2010 2012 2011



0x4 1x4 2x4 0-0-0-2

0

20

40

60

80

100

0 1 2

Incid

en

ce

of

wilt

(%

)

Limestone rate (t/A)

Susceptible

Moderate

Resistant

Male

19 June 2012 24 June 2009

Wilt incidence

0x4 1x4 2x4 0-0-0-2

0

20

40

60

80

100

0 1 2

Incid

ence o

f w

ilt (

%)


Susceptible

Moderate

Resistant

Male

9 July 2012 14 July 2009

Wilt incidence

0

1

2

3

4

5

0 1 2

Severity

of w

ilt (

0 to 5

scale

)


Susceptible

Moderate

Resistant

Male

0x4 1x4 2x4 0-0-0-2

9 July 2012 31July 2012

Wilt severity

0x4 1x4 2x4 0-0-0-2

0

50

100

150

200

250

0 1 2

Dried p

lant w

eig

ht

(g)


Susceptible

Moderate

Resistant

Male

22 July 2009 11 July 2012

Spinach biomass (dry plant weight)

0x4 1x4 2x4 0-0-0-2

0

400

800

1200

1600

0 1 2

Seed

yie

ld (

lb/A

)


Susceptible

Moderate

Resistant

2009 2012

Seed yield

0x4 1x4 2x4 0-0-0-2

0

5

10

15

20

25

0 1 2

Fusari

um

-infe

cte

d s

eed (

%)


Susceptible

Moderate

Resistant

2009 2012

Harvested seed (%) infected with Fusarium

0x4 1x4 2x4 0-0-0-2

0

5

10

15

20

25

0 1 2

V.

da

hlia

e infe

cte

d s

ee

d (

%)


Susceptible

Moderate

Resistant

Male

2009 2012

Harvested seed (%) infected with Verticillium dahliae

2. Develop a soil bioassay for spinach seed growers to determine the relative risk of a field site for Fusarium wilt, using spinach lines with different levels of resistance.

- Wilt ratings

- Spinach biomass

- Quantify F. oxysporum and V. dahliae on selective media

- Soil nutrient analyses: macro & micronutrients, pH, OM, CEC

- Texture analysis

- # of years out of spinach seed crops

- Quantify F. oxysporum f. sp. spinaciae with a real-time PCR assay

Research Objectives

2009-10: 26 soils submitted 2010-11: 42 soils submitted 2011-12: 39 soils submitted 2012-13: 40 soils submitted

Control soils included each year: Low (no), medium, and high Fusarium wilt risk

S

M

R

Soil A

S

M

R

Soil B

S

M

R

Soil C

Risk assessment: Spinach Fusarium wilt soil bioassay

B. 2010-11 bioassay Field (soil) sample

A. 2009-10 bioassay

Partially resistant line Moderately-susceptible line Susceptible line

Field (soil) sample

Fusa

rium

wilt

in

de

x

C. 2011-12 bioassay Field (soil) sample

Partially resistant line Moderately-susceptible line Susceptible line

Risk assessment: Spinach Fusarium wilt soil bioassay

Bioassay validation

Acknowledgements Puget Sound Seed Growers’ Association Washington State Commission for Pesticide Registration

Alfred Christianson Family Endowment, Robert MacDonald Fund

USDA Western Region IPM, Western SARE

Participating growers and seed companies

Todd Gordon Dan Nelson, Russ Duckworth

Mike Derie, Barbara Holmes, Avi Alcala,Sarah Meagher,

Ron Dralle, Jonathan Roozen, Martha Sudermann,

John Kuhn, Anita da Costa, Shawn Watkinson, Tammy Ennen,

Nathan McCartney, Tyler VanderPol

Evaluation of Proline for Management of Fusarium Wilt in Spinach Seed Crops

Proline and Fusarium Wilts Proline 480 SC = prothioconazole (triazole, Group 3) Spinach seed crops in DK: empirical observations? Transplanted watermelon Fusarium wilt: drench, drip trials

Zhou & Everts. 2009. PDMR 3:V092; Zhou et al. 2010. PDMR 4:V065 Everts et al. 2011. Phytopathology 101:S50-S51 (Abstract)

0

20

40

60

80

100

120

Wilt incidence 17 Jul

Plot vigor 7 Aug

Yield (kg/plot)abc a

d ab

cd bc cd cd

e

bcd

b

e

bc

cde

a

ab

abcd

e

a

cd

abc

bcd

a ab • 3 drench

applications

• 2-week

intervals from

transplanting

Rep 1

Rep 2

Rep 4

Rep 3

Rep 5

0 tons limestone

1 ton limestone

2 tons limestone

2 tons

1 ton

0 tons

2 tons

0 tons

1 ton

1 ton

2 tons

0 tons

0 tons

1 ton

2 tons

S M R S M R M S R M S R M R S M R S R M S R M S S R M S R M

2012 Spinach Seed Crop Fusarium Wilt Field Trial

Proline

Proline

Proline

Proline

Proline

Banded Proline applications (CO2-backpack sprayer) 10 May = 2 days after planting spinach 31 May = 3 weeks after planting 21 June = 6 weeks after planting

0

1

2

3

4

5

9 July 31 July

Severity

of w

ilt (

0 to 5

scale

)

Wilt assessment date

Proline

No Proline

Effect of Proline on severity of wilt

* 6.3%

* Denotes significant difference between Proline vs. no Proline plots at P <0.05

0

1

2

3

4

5

Susceptible Moderate Resistant

Severity

of w

ilt (

0 to 5

scale

)

Spinach inbred line

Proline

No Proline* 5.1%

* 8.2%

0

500

1000

1500


Se

ed

yie

ld (

lb/a

cre

)

Spinach inbred line

Proline

No Proline

* Denotes significant difference between Proline vs. no Proline at P <0.05

Effect of Proline on seed yield

* 18.4%

* 11.4%

6.9%

0

5

10

15


Incid

en

ce

of

se

ed

with

Ve

rtic

illiu

m (

%)

Spinach inbred line

Proline

No Proline

Effect of Proline on incidence of harvested seed infected with Fusarium and Verticillium

0

2

4

6

8

10


Incid

ence o

f seed w

ith F

usari

um

(%

)

Spinach inbred line

Proline

No Proline

Fusarium Verticillium

2013 Spinach Seed Crop Fusarium Wilt Proline Field Trial

• Field: acid soil, spinach seed crop last 4-5 yrs, proximity to NWREC

• Treatments: • Proline vs. no Proline

• 3 female spinach inbred lines (S, M, R)

• 2 tons limestone/acre (all plots)

• Apron + Thiram + Topsin seed treatment

• Proline applications: • Method: In-furrow drench + banded soil applications

• Number: 3 = maximum (sugar beet)

• Interval: 14-21 days

2013 Spinach Fusarium Wilt Proline Field Trial

Suggestions? Questions? [email protected] or 360-848-6140

Management of Verticillium in Spinach

Lindsey du Toit & Mike Derie, Washington State University

Jim Correll, University of Arkansas

Verticillium associated with spinach seed

1. Prevalence of Verticillium in commercial spinach seed lots (90% of lots tested: US, EU, NZ, Chile)

2. Pathogenicity of seed isolates to spinach

3. Seed-to-seed transmission

Plant Disease 89:4-11 Plant Disease 89:1305-1312

PDMR 1:ST003; PDMR 3:ST020; PDMR 4:ST038

Spinach/lettuce connection?

Atallah et al. 2010. Fungal Genetics & Biology. Gene flow among V. dahliae isolates from CA lettuce, pepper, strawberry,

artichoke & weeds, spinach seed, & WI ornamentals (not tomato)

?

S. T. Koike

S. T. Koike

Relative significance of infested spinach seed?

Evaluation of spinach seed treatments

B. Efficacy of spinach seed treatments for managing seedborne Verticillium (du Toit et al., 2009. PDMR 3:ST020, du Toit et al., 2010. PDMR 4:ST038)

1. Reduce incidence of seed infected/infested

2. Reduce rate of seed transmission

3. Reduce rate of soil infestation from planting infested seed

C. Influence of soil fumigation

A. Hot water & chlorine (du Toit & Hernandez-Perez, 2005. Plant Disease 89:1305-1312)

GH Trials: % Seeds with Verticillium - Trial 1

0

10

20

30

40

50

60

70

% s

ee

d w

ith

Ve

rtic

illiu

m d

ah

liae

Co

ntr

ol

AC

X80

2

AC

X80

1

Co

ron

et 2

00 m

l

Vo

rtex

Co

ron

et 4

00 m

l

AC

X80

4

Exp

. I

Inco

tec

I

AC

X80

3

Inco

tec

II

Exp

. II

Cap

tan

400

C

Th

iram

42-

S

See

d S

up

po

rt I

See

d S

up

po

rt I

I

See

dg

ard

BA

S 5

95 X

GF

Mer

tect

340

F

Far

m.D

300+

Mer

tect

To

psi

n M

70W

P

Conventional Organic

a a

ab abc

bc

cd d de

ef ef ef

g fg

hi ij kl ij

kl l l

gh

(du Toit et al., 2009. Plant Dis. Mgmt. Reports 3:ST020)

0

20000

40000

60000

80000

100000

120000

140000

CF

Us

V. d

ahlia

e/1

00 s

eed

ling

s

Co

ntr

ol

AC

X80

2

AC

X80

1

Co

ron

et 2

00 m

l

Vo

rtex

Co

ron

et 4

00 m

l

AC

X80

4

Exp

. I

Inco

tec

I

AC

X80

3

Inco

tec

II

Exp

. II

Cap

tan

400

C

Th

iram

42-

S

See

d S

up

po

rt I

See

d S

up

po

rt II

See

dg

ard

BA

S 5

95 X

GF

Mer

tect

340

F

Far

m.D

300+

Mer

tect

To

psi

n M

70W

P

To

psi

n 4

.5F

L

Co

ntr

ol,

soil

Co

ntr

ol,

pas

tzd

so

il

Conventional Organic

Not evaluated

ab a

ab

bc

ab

cd

e

cd

ef fgh e gh

d

cd

h efg

(du Toit et al. 2010. PDMR 4:ST038)

GH Trials: Seed transmission assay - Trial 2

Influence of soil fumigation/pH on Verticillium seed transmission & soil infestation

2010 WA field trial of baby leaf spinach & head lettuce 1. Main plots:

MeBr:Chloropicrin (57:43% at 350 gpa) No soil fumigation

2. Split plots: Acid soil pH (~5.7-5.9) 5 tons limestone/ha (pH 6.6-6.8)

3. Spinach split-split plots: No spinach seed Baby leaf spinach: No seed treatment Baby leaf spinach: Apron + Thiram Baby leaf spinach: Apron + Thiram + Topsin 4.5 FL Baby leaf spinach: Farmore D300 + Mertect 340F

Followed by head lettuce in each plot (‘Salinas’ type)

1 June 2010 14 June 2010

2011 & 2012 Field trials Mount Vernon, WA (2011) & Salinas, CA (2011, 2012)

Baby leaf spinach & head lettuce, RCBD, 5 reps 1. Main plots:

Fumigated vs. non-fumigated 2. Spinach split plots:

No spinach Non-treated seed Apron + Thiram Farmore D300 + Mertect 340F Apron + Thiram + Topsin 4.5 FL (WA 2011) Seed Support (WA 2011) ProGro (WA & CA, 2011), ProGro + Apron (CA, 2012)

Followed by head lettuce in each plot

Salinas, CA: 2011 & 2012

Schafer Ag Services Holaday Seeds

Rachel Yim Bobby Eugenia

Ocean Mist Farms (2011)

Christensen & Giannini (2012)

New Fresh

Germain’s Technology Group

Mount Vernon, WA: 2010, 2011 Effect of seed treatment & soil fumigation on

seed transmission of V. dahliae

CFUs V. dahliae/100 spinach plants after harvest of leaves

Significant fumigation & seed treatment effects

0

2

4

6

8

10

12

14

Heig

ht

(mm

) of

spi

nach

pla

nts

on

16 J

une 2

011

Soil fumigation treatment

Non-treated seed

Apron+Thiram

Apron+Thiram+Topsin

FarmoreD300+Mertect

ProGro

Seed Support

ab c

d

bc abc a

Mount Vernon, WA: 2011 Effect of seed treatment on spinach plant height

No significant seed treatment or fumigation effects, & no symptoms of Verticillium wilt on lettuce.

Mount Vernon, WA: 2010, 2011 % of lettuce stems infected with V. dahliae at harvest

Mount Vernon, WA: 2010, 2011 CFU of V. dahliae in soil at spinach planting

Significant fumigation effect, but no seed treatment effect

Mount Vernon, WA: 2010, 2011 CFU of V. dahliae in soil at lettuce planting

Significant fumigation & seed treatment effects in 2010, significant seed treatment effects in 2011.

Mount Vernon, WA: 2010, 2011 CFU of V. dahliae in soil 2-5 weeks after harvest of

lettuce & incorporation of lettuce residues

No significant fumigation or seed treatment effects in 2010.

Salinas, CA: 2011, 2012 Effect of seed treatment & soil fumigation on

seed transmission of V. dahliae

CFUs V. dahliae/100 spinach plants after harvest of leaves

Significant seed treatment effects, no significant fumigation effects

No significant seed treatment or fumigation effects, & no symptoms of Verticillium wilt on lettuce plants.

Salinas, CA: 2011, 2012 % of lettuce stems infected with V. dahliae at harvest

Salinas, CA: 2011, 2012 CFU of V. dahliae in soil at spinach planting

No significant fumigation or seed treatment effects

Salinas, CA: 2011, 2012 CFU of V. dahliae in soil at lettuce planting


Salinas, CA: 2010, 2011 CFU of V. dahliae in soil 2-5 weeks after harvest of

lettuce & incorporation of lettuce residues


• Soil fumigation increased colonization of spinach plants by V. dahliae 7-fold in 2010 in WA & 3-fold in 2012 in CA (not in 2011 in WA or CA sites)

• Seed treatments reduced seed transmission effectively in all 4 trials

• Best seed treatments = not planting spinach seed

• Mertect 340F: federal registration spring 2014 • Rancona Spinach (ipconazole): registered Jan. 2013 • Topsin M 70WP: SLN spinach stock seed in WA

• Planting infested spinach seed did not result in Verticillium wilt in head lettuce in all 4 trials, & had no significant effect on soilborne V. dahliae detected 2-5 weeks after incorporating lettuce residues

Summary

Pathogenicity, virulence, & vegetative compatibility grouping of

Verticillium isolates from spinach seed

Iglesias-Garcia, A.M., Villarroel-Zeballos, M.I., Feng, C., du Toit, L.J., & Correll, J.C. 201_.

Plant Disease: submitted January 2013.

Characterization of Verticillium dahliae from spinach seed, and screening for resistance to

Verticillium wilt in spinach. Villarroel-Zeballos, M.I., Feng, C., Iglesias, A., du Toit, L.J., & Correll, J.C. 2012.

HortScience 47:1297–1303.

Acknowledgements American Seed Research Foundation of ASTA

Puget Sound Seed Growers’ Assoc. WA State Comm. for Pesticide Registration

CA Leafy Greens Research Board Alfred Christianson Endowment

Participating seed & chemical companies International Seed Health Initiative – Root, Bulb & Leafy Vegetable

International Technical Group

Mike Derie, Barbara Holmes, Emily Gatch, Ana Vida Alcala, Ron Dralle, others …

Angela Iglesias-Garcia (MS), Maria Villarroel-Zeballos (MS)

0x4 1x4 2x4 0-0-0-2

Wilt incidence

0

20

40

60

80

100

0 1 2

Incid

ence o

f w

ilt (

%)


Susceptible

Moderate

Resistant

Male

4 June 2009 29 May 2012

Documents

Fusarium & Verticillium Wilts in Spinach: Research Updatemtvernon.wsu.edu/path_team/du-Toit-2013-WE-ACSC-mtg-spinach-Fus... · Fusarium & Verticillium Wilts in Spinach: Research Update