Characterization of botrytis cinerea resistance to
CHARACTERIZATION OF BOTRYTIS CINEREA RESISTANCE TO FUNGICIDES IN CALIFORNIA STRAWBERRIES SCOTT COSSEBOOM
Outline ■ Introduction ■ Fungicide resistance screening
■ Fungicide use survey ■ Species Identification ■ Field Trial
Show Where where strawberries strawberries areare grown
1
When strawberries are Season in California grown
2
3
The biology Botrytis Maybe showoflife cycle
4
Cultural management
Fight The Chemical management
5
Fungicide resistance
6
Resistance management
7
Resistance
Recent research
8
Strawberry production
Three districts grow 95% of strawberry fruit in CA
Northern District
Central District
Southern District
20
Production (millions of trays)
California production regions
18 16
Northern district Central district Southern district
14 12
10 8 6 4 2
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month
Life cycle of Botrytis
Adapted from: Agrios 2005
Cultural management Show where strawberries are grown
Season in California
Maybe show life cycle
Chemical management ▪ Most effective method when weather is favorable for the pathogen ▪ Multiple applications are made per season ▪ Three types: Site-specific, multi-site, biological
Photo: G. Holmes
Fungicide resistance
Adapted from: Deisling, H. B. et al. 2008.
sensitive individual
fungicide application
resistant individual
regeneration
Resistance management
■ Rotate modes of action ■ Tank-mix ■ Use fungicides less
Previous research Eastern U.S. ▪ High levels of resistance to important fungicides for Botrytis gray mold control in strawberries ▪ Resistance changing over time ▪ Isolates resistant to multiple modes of action California ▪ Resistance reported to frequently used fungicides ▪ Resistance increasing in a population within a season
FUNGICIDE RESISTANCE SCREENING
• 47 fields
• 888 isolates collected • 2 sampling times
U.S. Department of Agriculture, National Agricultural Statistics Service
20
Production (millions of trays)
18 Northern district Central district Southern district
16 14 12
10 8 6 4 2 0
Jan Feb Mar Apr May Jun Early-season sampling
Jul Aug Sep Oct Nov Dec Late-season sampling
Table. Fungicides labeled for Botrytis gray mold of strawberry in California Example trade name
Active ingredient(s)
FRAC code(s)
Topsin
Thiophanate-methyl
1
Rovral
Iprodione
2
Fontelis
Penthiopyrad
7
Kenja 400
Isofetamid
7
Luna Sensation
Fluopyram
Trifloxystrobin
7
11
Luna Tranquility
Fluopyram
Pyrimethanil
7
9
Pristine
Boscalid
Pyraclostrobin
7
11
Merivon
Fluxapyroxad
Pyraclostrobin
7
11
Scala
Pyrimethanil
Switch
Cyprodinil
Elevate
Fenhexamid
17
Ph-D
Polyoxin-D
19
active ingredient was not tested in this study
9 Fludioxonil
9
12
1 No fungicide Cyprodinil Iprodione Fludioxonil Fenhexamid T-methyl Boscalid Fluopyram
Penthiopyrad Isofetamid Experimental 1
Experimental 2
2
3
4
5 6 Fernández-Ortuño, D. et al. 2014.
Boscalid
Fludioxonil
Fenhexamid Control
California conventional
Frequency of resistance (%)
100
Early-season (n=340) Late-season (n= 362)
90 80 70 60 50 40 30 20 10 0 T-methyl
Iprodione
Boscalid
Penthiopyrad
Isofetamid
1
2
7
7
7
Fluopyram
7
Active ingredient FRAC code
Cyprodinil
Pyraclostrobin*
Fludioxonil
Fenhexamid
9
11
12
17
Early-season (n=92)
California organic
Late-season (n=94)
Frequency of resistance (%)
100 90
80 70 60 50 40 30 20
10 0 T-methyl
Iprodione
Boscalid
Penthiopyrad
1
2
7
7
Isofetamid
Fluopyram
7 7 Active ingredient FRAC code
Cyprodinil
Pyraclostrobin*
Fludioxonil
Fenhexamid
9
11
12
17
7 Early-season Late-season
Average CCR
6 5 4
a b bc
3
c
2
1 0 Conventional
Organic
Production type
Early-season Late-season
Frequency of phenotype (%)
50 45 40 35 30 25 20 15
10 5 0 0
1
2
3
4
Chemical class resistances
5
6
7
Genetics behind resistance Chemical Class Hydroxyanilides Dicarboximides MBCs QoIs SDHIs
Gene ERG27 Bos1 Beta-tubulin Cytochrome b SDHb
Genotype observed F196C, F412I, F412S I356N, I365N, I365S E198A G143A H272R, H272Y, N230I, P225F
Genotypes matched phenotypes 94%
FUNGICIDE USE SURVEY
Active ingredient
Peroxyacetic acid Hydrogen dioxode Aureobasidium pullulans Thiophanate-methyl Bacillus amyloliquefaciens Streptomyces lydicus Thiram Extract of Reynoutria Sachalinensis Bacillus subtilis Trifloxystrobin BLAD Fluopyram Extract of Neem Oil Polyoxin D Fluxapyroxad Sodium Tetraborohydrate Decahydrate Penthiopyrad Pyrimethanil Fenhexamid Boscalid Fludioxonil Cyprodinil Pyraclostrobin Captan
▪ 15 applications per season ▪ 12 day interval
0
1
2
3
4
5
6
7
Avg. no. applications per season
8
SPECIES IDENTIFICATION
FIELD TRIAL
No fungicides applied
Fungicides applied weekly 1
1
2
3
4
2
5
7
6
3
8
10
9
4
11
12
Week
5
Sampling time (collection)
Resistance testing 1) non-amended control 2) Endura (boscalid) 3) Scholar (fludioxonil) 4) Elevate (fenhexamid)
100
Fenhexamid
Timing of isolate collection I
90
II
III
IV
V
Frequency of resistance (%)
80 70 60 50 40
30 20 10 0
Non-treated
Fenhexamid
Field treatment
Rotation
Tank-mix + rotation
100
Fludioxonil
90
Timing of isolate collection I II III IV V
Frequency of resistance (%)
80
70 60 50
40 30 20 10 0 Non-treated
Cyprodinil + fludioxonil
Rotation
Fungicide treatment
Tank-mix + rotation
100
Boscalid
Frequency of resistance (%)
90
Timing of isolate collection I II III IV V
80 70 60 50 40 30 20 10 0 Non-treated
Boscalid + pyraclostrobin
Rotation
Fungicide treatment
Tank-mix + rotation
Summary ■
79 isolates were B. cinerea and one isolate was B. mali.
■
Fungicide resistance frequency was high to FRAC 1, 11, 17
■
–
Low frequency of resistance to FRAC 12
–
Moderate frequency of resistance to FRAC 2, 7, 9
Multi-fungicide resistant isolates were present in all three growing districts. CCR3 was the most common. Few isolates were CCR0 or CCR7.
■
■
There was an average of 15 applications per season. –
Captan, a multisite fungicide, was applied the most frequently
–
Site-specific fungicides were applied less than twice per season
A within-season increase in CCR frequency was observed in conventional fields.
Conclusions ■
Botrytis cinerea is the primary causal agent of strawberry gray mold in California. Other species may exist at low frequencies.
■
The frequency of resistant isolates in a population is a.i. dependent
■
■
–
73% statewide frequency of resistance to fenhexamid
–
2% statewide frequency of resistance to fludioxonil
–
Fenhexamid was applied less (1.5 times per season) than fludioxonil (2.2)
The survey, the CCR shift, and the field trial all indicate that populations of Botrytis may respond rapidly to a single application of a site-specific fungicide –
The survey: Site-specific fungicides applied < 3 times per season
–
CCR shift: Within-season increase within conventional fields
–
Field trial: Rapid increase in fenhexamid resistance frequency following either one (rotation treatment) or three (fenhexamid treatment) exposures to fenhexamid
The field trial indicated that after rapid selection for resistance, the population may revert back to being sensitive if the selection pressure is removed (e.g., fenhexamid).
Outline ■ Introduction ■ Fungicide resistance screening
■ Fungicide use survey ■ Species Identification ■ Field Trial
Show Where where strawberries strawberries areare grown
1
When strawberries are Season in California grown
2
3
The biology Botrytis Maybe showoflife cycle
4
Cultural management
Fight The Chemical management
5
Fungicide resistance
6
Resistance management
7
Resistance
Recent research
8
Strawberry production
Three districts grow 95% of strawberry fruit in CA
Northern District
Central District
Southern District
20
Production (millions of trays)
California production regions
18 16
Northern district Central district Southern district
14 12
10 8 6 4 2
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month
Life cycle of Botrytis
Adapted from: Agrios 2005
Cultural management Show where strawberries are grown
Season in California
Maybe show life cycle
Chemical management ▪ Most effective method when weather is favorable for the pathogen ▪ Multiple applications are made per season ▪ Three types: Site-specific, multi-site, biological
Photo: G. Holmes
Fungicide resistance
Adapted from: Deisling, H. B. et al. 2008.
sensitive individual
fungicide application
resistant individual
regeneration
Resistance management
■ Rotate modes of action ■ Tank-mix ■ Use fungicides less
Previous research Eastern U.S. ▪ High levels of resistance to important fungicides for Botrytis gray mold control in strawberries ▪ Resistance changing over time ▪ Isolates resistant to multiple modes of action California ▪ Resistance reported to frequently used fungicides ▪ Resistance increasing in a population within a season
FUNGICIDE RESISTANCE SCREENING
• 47 fields
• 888 isolates collected • 2 sampling times
U.S. Department of Agriculture, National Agricultural Statistics Service
20
Production (millions of trays)
18 Northern district Central district Southern district
16 14 12
10 8 6 4 2 0
Jan Feb Mar Apr May Jun Early-season sampling
Jul Aug Sep Oct Nov Dec Late-season sampling
Table. Fungicides labeled for Botrytis gray mold of strawberry in California Example trade name
Active ingredient(s)
FRAC code(s)
Topsin
Thiophanate-methyl
1
Rovral
Iprodione
2
Fontelis
Penthiopyrad
7
Kenja 400
Isofetamid
7
Luna Sensation
Fluopyram
Trifloxystrobin
7
11
Luna Tranquility
Fluopyram
Pyrimethanil
7
9
Pristine
Boscalid
Pyraclostrobin
7
11
Merivon
Fluxapyroxad
Pyraclostrobin
7
11
Scala
Pyrimethanil
Switch
Cyprodinil
Elevate
Fenhexamid
17
Ph-D
Polyoxin-D
19
active ingredient was not tested in this study
9 Fludioxonil
9
12
1 No fungicide Cyprodinil Iprodione Fludioxonil Fenhexamid T-methyl Boscalid Fluopyram
Penthiopyrad Isofetamid Experimental 1
Experimental 2
2
3
4
5 6 Fernández-Ortuño, D. et al. 2014.
Boscalid
Fludioxonil
Fenhexamid Control
California conventional
Frequency of resistance (%)
100
Early-season (n=340) Late-season (n= 362)
90 80 70 60 50 40 30 20 10 0 T-methyl
Iprodione
Boscalid
Penthiopyrad
Isofetamid
1
2
7
7
7
Fluopyram
7
Active ingredient FRAC code
Cyprodinil
Pyraclostrobin*
Fludioxonil
Fenhexamid
9
11
12
17
Early-season (n=92)
California organic
Late-season (n=94)
Frequency of resistance (%)
100 90
80 70 60 50 40 30 20
10 0 T-methyl
Iprodione
Boscalid
Penthiopyrad
1
2
7
7
Isofetamid
Fluopyram
7 7 Active ingredient FRAC code
Cyprodinil
Pyraclostrobin*
Fludioxonil
Fenhexamid
9
11
12
17
7 Early-season Late-season
Average CCR
6 5 4
a b bc
3
c
2
1 0 Conventional
Organic
Production type
Early-season Late-season
Frequency of phenotype (%)
50 45 40 35 30 25 20 15
10 5 0 0
1
2
3
4
Chemical class resistances
5
6
7
Genetics behind resistance Chemical Class Hydroxyanilides Dicarboximides MBCs QoIs SDHIs
Gene ERG27 Bos1 Beta-tubulin Cytochrome b SDHb
Genotype observed F196C, F412I, F412S I356N, I365N, I365S E198A G143A H272R, H272Y, N230I, P225F
Genotypes matched phenotypes 94%
FUNGICIDE USE SURVEY
Active ingredient
Peroxyacetic acid Hydrogen dioxode Aureobasidium pullulans Thiophanate-methyl Bacillus amyloliquefaciens Streptomyces lydicus Thiram Extract of Reynoutria Sachalinensis Bacillus subtilis Trifloxystrobin BLAD Fluopyram Extract of Neem Oil Polyoxin D Fluxapyroxad Sodium Tetraborohydrate Decahydrate Penthiopyrad Pyrimethanil Fenhexamid Boscalid Fludioxonil Cyprodinil Pyraclostrobin Captan
▪ 15 applications per season ▪ 12 day interval
0
1
2
3
4
5
6
7
Avg. no. applications per season
8
SPECIES IDENTIFICATION
FIELD TRIAL
No fungicides applied
Fungicides applied weekly 1
1
2
3
4
2
5
7
6
3
8
10
9
4
11
12
Week
5
Sampling time (collection)
Resistance testing 1) non-amended control 2) Endura (boscalid) 3) Scholar (fludioxonil) 4) Elevate (fenhexamid)
100
Fenhexamid
Timing of isolate collection I
90
II
III
IV
V
Frequency of resistance (%)
80 70 60 50 40
30 20 10 0
Non-treated
Fenhexamid
Field treatment
Rotation
Tank-mix + rotation
100
Fludioxonil
90
Timing of isolate collection I II III IV V
Frequency of resistance (%)
80
70 60 50
40 30 20 10 0 Non-treated
Cyprodinil + fludioxonil
Rotation
Fungicide treatment
Tank-mix + rotation
100
Boscalid
Frequency of resistance (%)
90
Timing of isolate collection I II III IV V
80 70 60 50 40 30 20 10 0 Non-treated
Boscalid + pyraclostrobin
Rotation
Fungicide treatment
Tank-mix + rotation
Summary ■
79 isolates were B. cinerea and one isolate was B. mali.
■
Fungicide resistance frequency was high to FRAC 1, 11, 17
■
–
Low frequency of resistance to FRAC 12
–
Moderate frequency of resistance to FRAC 2, 7, 9
Multi-fungicide resistant isolates were present in all three growing districts. CCR3 was the most common. Few isolates were CCR0 or CCR7.
■
■
There was an average of 15 applications per season. –
Captan, a multisite fungicide, was applied the most frequently
–
Site-specific fungicides were applied less than twice per season
A within-season increase in CCR frequency was observed in conventional fields.
Conclusions ■
Botrytis cinerea is the primary causal agent of strawberry gray mold in California. Other species may exist at low frequencies.
■
The frequency of resistant isolates in a population is a.i. dependent
■
■
–
73% statewide frequency of resistance to fenhexamid
–
2% statewide frequency of resistance to fludioxonil
–
Fenhexamid was applied less (1.5 times per season) than fludioxonil (2.2)
The survey, the CCR shift, and the field trial all indicate that populations of Botrytis may respond rapidly to a single application of a site-specific fungicide –
The survey: Site-specific fungicides applied < 3 times per season
–
CCR shift: Within-season increase within conventional fields
–
Field trial: Rapid increase in fenhexamid resistance frequency following either one (rotation treatment) or three (fenhexamid treatment) exposures to fenhexamid
The field trial indicated that after rapid selection for resistance, the population may revert back to being sensitive if the selection pressure is removed (e.g., fenhexamid).
