fusarium & verticillium wilts in spinach: research update€¦ · 06/02/2013 · baby leaf...
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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
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Fusarium wilt of spinach Fusarium oxysporum f. sp. spinaciae
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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
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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
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spinach potato winter wheat spinach
Limestone applications
Column1
2009 2010 2011 2012
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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
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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
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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
spinach potato winter wheat spinach
2009 2010 2012 2011
Limestone applications
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4.0
5.0
6.0
7.0
8.0S
oil
pH
0
1
2
0/2
spinach potato winter wheat spinach
2009 2010 2012 2011
Limestone applications
Effect of limestone rate on soil pH: 2009-2012
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4.0
5.0
6.0
7.0
8.0S
oil
pH
0
1
2
0/2
spinach potato winter wheat spinach
2009 2010 2012 2011
Limestone applications
Effect of limestone rate on soil pH: 2009-2012
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4.0
5.0
6.0
7.0
8.0S
oil
pH
0
1
2
0/2
spinach potato winter wheat spinach
2009 2010 2012 2011
Limestone applications
Effect of limestone rate on soil pH: 2009-2012
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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
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0x4 1x4 2x4 0-0-0-2
0
20
40
60
80
100
0 1 2
Incid
ence o
f w
ilt (
%)
Limestone rate (t/A)
Susceptible
Moderate
Resistant
Male
9 July 2012 14 July 2009
Wilt incidence
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0
1
2
3
4
5
0 1 2
Severity
of w
ilt (
0 to 5
scale
)
Limestone rate (t/A)
Susceptible
Moderate
Resistant
Male
0x4 1x4 2x4 0-0-0-2
9 July 2012 31July 2012
Wilt severity
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0x4 1x4 2x4 0-0-0-2
0
50
100
150
200
250
0 1 2
Dried p
lant w
eig
ht
(g)
Limestone rate (t/A)
Susceptible
Moderate
Resistant
Male
22 July 2009 11 July 2012
Spinach biomass (dry plant weight)
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0x4 1x4 2x4 0-0-0-2
0
400
800
1200
1600
0 1 2
Seed
yie
ld (
lb/A
)
Limestone rate (t/A)
Susceptible
Moderate
Resistant
2009 2012
Seed yield
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0x4 1x4 2x4 0-0-0-2
0
5
10
15
20
25
0 1 2
Fusari
um
-infe
cte
d s
eed (
%)
Limestone rate (t/A)
Susceptible
Moderate
Resistant
2009 2012
Harvested seed (%) infected with Fusarium
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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 (
%)
Limestone rate (t/A)
Susceptible
Moderate
Resistant
Male
2009 2012
Harvested seed (%) infected with Verticillium dahliae
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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
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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
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S
M
R
Soil A
S
M
R
Soil B
S
M
R
Soil C
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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
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C. 2011-12 bioassay Field (soil) sample
Partially resistant line Moderately-susceptible line Susceptible line
Risk assessment: Spinach Fusarium wilt soil bioassay
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Bioassay validation
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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
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Evaluation of Proline for Management of Fusarium Wilt in Spinach Seed Crops
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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
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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
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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
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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%
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0
500
1000
1500
Susceptible Moderate Resistant
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%
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0
5
10
15
Susceptible Moderate Resistant
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
Susceptible Moderate Resistant
Incid
ence o
f seed w
ith F
usari
um
(%
)
Spinach inbred line
Proline
No Proline
Fusarium Verticillium
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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
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2013 Spinach Fusarium Wilt Proline Field Trial
Suggestions? Questions? [email protected] or 360-848-6140
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Management of Verticillium in Spinach
Lindsey du Toit & Mike Derie, Washington State University
Jim Correll, University of Arkansas
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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
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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?
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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)
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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)
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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
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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)
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1 June 2010 14 June 2010
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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
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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
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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
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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
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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
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Mount Vernon, WA: 2010, 2011 CFU of V. dahliae in soil at spinach planting
Significant fumigation effect, but no seed treatment effect
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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.
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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.
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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
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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
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Salinas, CA: 2011, 2012 CFU of V. dahliae in soil at spinach planting
No significant fumigation or seed treatment effects
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Salinas, CA: 2011, 2012 CFU of V. dahliae in soil at lettuce planting
No significant fumigation or seed treatment effects
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Salinas, CA: 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
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• 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
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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.
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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)
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0x4 1x4 2x4 0-0-0-2
Wilt incidence
0
20
40
60
80
100
0 1 2
Incid
ence o
f w
ilt (
%)
Limestone rate (t/A)
Susceptible
Moderate
Resistant
Male
4 June 2009 29 May 2012