CHARACTERIZATION OF BOTRYTIS CINEREA RESISTANCE TO FUNGICIDES IN CALIFORNIA

STRAWBERRIES

SCOTT COSSEBOOM

Outline

■ Introduction

■ Fungicide resistance screening

■ Fungicide use survey

■ Species Identification

■ Field Trial

Recent research

Maybe show life cycleShow where strawberries are

grownSeason in California

Fight The

Resistance

When strawberries are

grownThe biology of Botrytis Cultural management

Chemical management Fungicide resistance Resistance management

Where strawberries are

grown1 2 3 4

5 6 7 8

Northern District

Central District

Southern District

Three districts grow 95% of strawberry fruit in CA

Strawberry production

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2

4

6

8

10

12

14

16

18

20

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Pro

du

ctio

n (

mill

ion

s o

f tr

ays)

Month

Northern district Central districtSouthern district

California production regions

Adapted from: Agrios 2005

Life cycle of Botrytis

Maybe show life cycleShow where strawberries are

grownSeason in California

Cultural management

▪ Most effective method when weather is favorable for the pathogen

▪ Multiple applications are made per season

▪ Three types: Site-specific, multi-site, biological

Chemical management

Photo: G. Holmes

Fungicide resistance

Adapted from: Deisling, H. B. et al. 2008.

sensitive individual

resistant individual

fungicide application

regeneration

Resistance management

■ Rotate modes of action

■ Tank-mix

■ Use fungicides less

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

Previous research

FUNGICIDE RESISTANCE SCREENING

• 47 fields

• 888 isolates collected

• 2 sampling times

U.S. Department of Agriculture, National Agricultural Statistics Service

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2

4

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16

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Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Pro

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mill

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s o

f tr

ays)

Early-season sampling Late-season sampling

Northern district Central districtSouthern district

active ingredient was not tested in this study

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 9

Switch Cyprodinil Fludioxonil 9 12

Elevate Fenhexamid 17

Ph-D Polyoxin-D 19

No fungicide

Cyprodinil

1 2 3 4 5 6

Iprodione

Fenhexamid

Fludioxonil

T-methyl

Boscalid

Fluopyram

Penthiopyrad

Isofetamid

Experimental 1

Experimental 2

Fernández-Ortuño, D. et al. 2014.

Boscalid

Fenhexamid

Fludioxonil

Control

0

10

20

30

40

50

60

70

80

90

100

T-methyl Iprodione Boscalid Penthiopyrad Isofetamid Fluopyram Cyprodinil Pyraclostrobin* Fludioxonil Fenhexamid

Fre

qu

ency

of

resi

sta

nce

(%

)

Active ingredient

FRAC code

California conventional Early-season (n=340)

Late-season (n= 362)

1 2 7 7 7 7 9 11 12 17

0

10

20

30

40

50

60

70

80

90

100

T-methyl Iprodione Boscalid Penthiopyrad Isofetamid Fluopyram Cyprodinil Pyraclostrobin* Fludioxonil Fenhexamid

Fre

qu

ency

of

resi

sta

nce

(%

)

Active ingredient

FRAC code

California organic Early-season (n=92)

Late-season (n=94)

1 2 7 7 7 7 9 11 12 17

0

1

2

3

4

5

6

7

Conventional Organic

Ave

rag

e C

CR

Production type

Early-season

Late-season

b

a

bc

c

0

5

10

15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7

Fre

qu

en

cy o

f p

hen

oty

pe (

%)

Chemical class resistances

Early-season

Late-season

Chemical Class Gene Genotype observed

Hydroxyanilides ERG27 F196C, F412I, F412S

Dicarboximides Bos1 I356N, I365N, I365S

MBCs Beta-tubulin E198A

QoIs Cytochrome b G143A

SDHIs SDHb H272R, H272Y, N230I, P225F

Genetics behind resistance

Genotypes matched phenotypes 94%

FUNGICIDE USE SURVEY

0 1 2 3 4 5 6 7 8

Captan

Pyraclostrobin

Cyprodinil

Fludioxonil

Boscalid

Fenhexamid

Pyrimethanil

Penthiopyrad

Sodium Tetraborohydrate Decahydrate

Fluxapyroxad

Polyoxin D

Extract of Neem Oil

Fluopyram

BLAD

Trifloxystrobin

Bacillus subtilis

Extract of Reynoutria Sachalinensis

Thiram

Streptomyces lydicus

Bacillus amyloliquefaciens

Thiophanate-methyl

Aureobasidium pullulans

Hydrogen dioxode

Peroxyacetic acid

Avg. no. applications per season

Ac

tive

in

gre

die

nt

▪ 15 applications per season

▪ 12 day interval

SPECIES IDENTIFICATION

FIELD TRIAL

Resistance testing1) non-amended control2) Endura (boscalid)3) Scholar (fludioxonil)4) Elevate (fenhexamid)

Week1 2 3 4 5 6 7 8 9 10 11 12

Sampling time (collection)

1 2 3 4 5

Fungicides applied weekly No fungicides applied

0

10

20

30

40

50

60

70

80

90

100

Non-treated Fenhexamid Rotation Tank-mix + rotation

Fre

qu

ency

of

resi

stan

ce (

%)

Field treatment

Fenhexamid

I II III IV VTiming of isolate collection

0

10

20

30

40

50

60

70

80

90

100

Non-treated Cyprodinil + fludioxonil Rotation Tank-mix + rotation

Fre

qu

ency

of

resi

stan

ce (

%)

Fungicide treatment

Fludioxonil

I II III IV V

Timing of isolate collection

0

10

20

30

40

50

60

70

80

90

100

Non-treated Boscalid + pyraclostrobin Rotation Tank-mix + rotation

Fre

qu

ency

of

resi

stan

ce (

%)

Fungicide treatment

Boscalid

I II III IV V

Timing of isolate collection

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).