fundamental aspects for the development of resistance to ......dec 19, 2007 · the biosynthesis of...

Fundamental Aspects for the Development of Resistance to Fungicides

Eric Tedford and Ulrich Gisi

Syngenta Crop ProtectionGreensboro, USA, and Stein, Switzerland

Soybean Rust Symposium, Louisville, December 2007

2

Many of the current ASR fungicides are either strobilurinsor DMI’s

Azoxystrobin S Tetraconazole D

Boscalid

S

D

D

S&D

Prop & Azoxy D&S

Chlorothalonil Cyproconazole D

Pyraclostrobin Azoxy & Cypro S&D

Propiconazole Bosc & Pyraclo A&S

Tebuconazole Flusilazole

Myclobutanil Flutriafol

Tri & Propi Metconazole D

S = strobilurin; D = DMI

3

mitochondrium

III AOX

Q-Pool

IV

Cytochrome bc1 is target site of QoIs

IIIQi

Qo

FeS

Cyt c

ATP-synthase

H+ H+H+ H+

ADP + P ATP

I

NADH NAD+

succinate fumarateTCA cycle

Cytochrome b gene is part of mitochondrial genome

Cytochrome bc1 is target site of QiIs

Succinatedehydrogenaseis target site of

SDIs½ 02+ 2 H+ H20

Cyt b

positive outer side

negative inner side

Schematic diagram of mitochondrial respiration and flow of electrons and protons

Strobilurins

4

Mode of action (single site inhibition):Inhibition of cytochrome bc1 (complex III) at Qo pocket in mitochondrial respiration (encoded by the cyt b gene)

Mechanisms of resistance (monogenic, separation s/r): 1. G143A mutation (complete resistance, loss of disease control if QoI solo) Blumeria (= Erysiphe) graminis, wheat and barley Mycosphaerella graminicola (= Septoria tritici), wheat Pyrenophora (= Drechslera, Helminthosporium) tritici-repentis (DTR), wheat Plasmopara viticola, grape Alternaria alternata, pistacio

2. F129L mutation (“partial“ resistance, reduced disease control if QoI solo) Pyrenophora (= Drechslera, Helminthosporium) teres, barley Alternaria solani, potato (USA)

3. NO mutations (no resistance) Puccinia, Uromyces, Phakopsora, Hemileia, different crops

Mode of action and mechanism of resistance for QoIs

5

Sterol biosynthesis and site of action of DMIs

squalene

ergosterol cholesterol stigmasterol

lanosterol cycloartenol

fungi animals oomycetes

plants

DMI activity

6

Mode of action (single site inhibition):Inhibition of cytochrome P 450-dependent lanosterol-C14α-demethylase in the biosynthesis of fungal sterols such as ergosterol (encoded by the cyp51or erg11 gene)

Mechanisms of resistance (polygenic, multi-allelic, sensitivity shift):1. Mutations in cyp51 gene: V136A, Y137F, A379G, I381V (and others and combinations thereof) and thus altered binding

2. Over-expression of cyp51 gene and thus, increased production of target enzyme

3. Up-regulation of transporter genes and thus, increased activity of specific membrane (ABC-) pumps exporting DMIs out of cells

Mode of action and mechanism of resistance for DMIs

All 3 mechanisms of resistance can co-exist and contributeto decreasing sensitivity

7

Fungicide resistance development: Selection models for QoI and DMI fungicides

monogenic, single allelic resistance at target site, disruptive selection, high risk

freq

uenc

y of

isol

ates

in %

0

10

20

30

40

50

60

70

80

0.001 0.01 0.1 1 10 100 1000 10000

year 1

year 2

year 4

year 5

separation

QoIs

sensitivity of isolates as EC 50 mg / L

Loss of Control

>500x

100 – 1000 fold

• Always starts with a gradual shift but rapid disruptiveselection only occurs with a monogenic mechanism of resistance

• Therefore, the risk is HIGH

8

Fungicide resistance development: Selection models for QoI and DMI fungicides

polygenic, multi allelic resistance at target site, continuous selection, moderate risk

monogenic, single allelic resistance at target site, disruptive selection, high risk

0

10

20

30

40

50

60

70

80

0.001 0.01 0.1 1 10

year 1

year 3year 5

year 7

year 9

shift

stabilization

DMIs <20x


freq

uenc

y of

isol

ates

in %

freq

uenc

y of

isol

ates

in %

0

10

20

30

40

50

60

70

80

0.001 0.01 0.1 1 10 100 1000 10000

year 1

year 2

year 4

year 5

separation

QoIs


>500x

Loss of Control

Reduced Control

• Fitness cost• Polygenic inheritance

(dilution)

9

QoI Structure of cytochrome b gene and position of introns (arrow) and G143A and F129L mutations*

129 143

F G G143 (1474 – 1734 bp)

F G G143 (1657 bp)

Uromyces appendiculatus

Phakopsora pachyrhizi Y132

Puccinia (9 spp.)F G G143 (1474 – 1734 bp)

F G G143 (1657 bp)Hemileia vastatrix

Y132

1

120 150140130

F A

L AMagnaporthe grisea

Mycosphaerella graminicola

Plasmopara viticola

120 150140130

F A

Blumeria graminisL A

aa position

F AAlternaria alternataPistachioAlternaria solaniPotato

L G G143 (2157 bp)A126 V146

* Grasso, Palermo, Sierotzki, Garibaldi & Gisi, Pest Manag. Sci 62: 465-472 (2006)

FF

AA

L

= wild type (phenylalanine)= mutation from glycine to alanine= mutatipion to lycine

Cyt b

10

Structure of cytochrome b gene fragment coding forsensitivity to QoI fungicides (with/without intron)

CYT b

phenotype

cyt b DNA

cytcytcyt b mRNA

Trp142 Gly 143 – Ala144

sensitive to QoIs

–Trp142 Gly 143 – Ala144

sensitive to QoIs

–Trp142 Gly 143 – Ala144

sensitive to QoIs

–

TGA G142 143 144

TTGA GG GCA

translation

T

exon intron exon

//

transcription, splicing

lethal

non-functional cytochrome b respiratory deficient

TGA G142 143 144

TTGA GC GCA

T

translation

codon 143 mutated

T

exon intron exon

//

transcription, but no splicingintron cannot be excised

codon 143 not mutated

Puccinia recondita Alternaria solani

Puccinia recondita Alternaria solaniPhakopsora pachyrhizi

Trp142 – Ala144– – Ala144– Gly 143Ala 143 – Ala144

sensitive to QoI’sresistant to QoI’s

–

TGA GGT

142 143

GCA

144

TGA G TTGA GGT GCA

TTGA GG

CT GCA

translation

codon 143 wt or mutated

Mycosphaerella graminicolaAlternaria alternata

transcription

Consequence – 143 res will never occur for ASR!

1 2 3

11

Summary: Molecular mechanisms of QoI resistance

Alternaria solani: Intron at 143, NO G143A mutation, but F129L was detected (moderate risk) (Pasche et al., 2002; Rosenzweig et al., 2006).

Alternaria alternata: NO Intron at 143, G143A possible (high risk). G143A was detected in resistant isolates (Ma & Michailides, 2004).

All rusts (Puccinia, Uromyces, Phakopsora, Hemilia) as well as Alternaria solani and Pyrenophora teres CANNOT acquire QoIresistance (based on G143A), because specific gene structure does not allow it (intron at position 143): Low resistance risk (Grasso et al., 2006). The F129L mutation was not found in rust species. In bioassay, NO QoI resistance found in rusts (more than 5000 P.recondita isolates tested – past 10 years).

For the first time, it can be predicted, based on molecular information, whether resistance may appear or not in a particular pathogen.

Now have a molecular tool

12

Published cases of decreased sensitivity to DMIs and molecular explanations in plant pathogens

Mutations in cyp51 geneUncinula necator, grape, Delye et al. 1997, Y136F

Erysiphe (Blumeria) graminis fsp. hordei, barley, Delye et al. 1998, Y136F

Erysiphe (Blumeria) graminis fsp. tritici, wheat, Wyand & Brown 2005, Y136F, K147Q

Mycosphaerella graminicola, wheat, Cools et al. 2007, Leroux et al., 2007, Chassot et al., 2007,

V136A, Y137F, A379G, I 381V (also detected but less important are L50S, V136C, S188N and several

deletions and exchanges at aa positions 459, 460, 461)

Over-expression of cyp51 gene (insert or repeats in promotor)Venturia inaequalis, apple, Schnabel & Jones 2001

Penicillium digitatum, citrus, Hamamoto et al. 2000

Mycosphaerella graminicola, wheat, Stergiopoulos et al. 2003, Chassot et al. 2007

Blumeriella jaapii, cherry, Ma et al. 2006

Up-regulation of ABC transporter genesBotrytis cinerea, grape, Hayashi et al. 2003

Mycosphaerella graminicola, wheat, Stergiopoulos et al. 2003; Cools et al. 2005; Chassot et al. 2007

Monilinia fructicola, peach, Schnabel et al. 2003

13

Fungicide resistance development: Shift in sensitivity to DMI fungicides

0

10

20

30

40

50

60

70

80

0.001 0.01 0.1 1 10

year 1

year 3year 5

year 7

year 9

shift ~ 20x

stabilization

DMIs


freq

uenc

y of

isol

ates

in %

summation, cumulative frequency of log-normal distribution

log-normal distribution

0%

20%

40%

60%

80%

100%

0.001 0.01 0.1 1

cum

ulat

ive

freq

uenc

y

0%

20%

40%

60%

80%

100%

0.001 0.01 0.1 1

cum

ulat

ive

freq

uenc

y


Year 1 Year 7

10 X

14

Shift in sensitivity to DMIs for Mycosphaerella graminicolaisolates collected in Europe between 1988 and 2006

0%

20%

40%

60%

80%

100%

0.001 0.01 0.1 1 10 100log EC50 cyproconazole (mg ai/l)

Cum

ulat

ive

freq

uenc

y

1988 - 1998 (n =15)

2003 - 2006 (n = 96)

0%

20%

40%

60%

80%

100%

0.001 0.01 0.1 1 10 100log EC50 epoxiconazole (mg ai/l)

Cum

ulat

ive

freq

uenc

y 1988 - 1998 (n =15)

2003 - 2006 (n = 96)

0%

20%

40%

60%

80%

100%

0.001 0.01 0.1 1 10 100log EC50 prothioconazole (mg ai/l)

Cum

ulat

ive

freq

uenc

y

1988 - 1998 (n =15)

2003 - 2006 (n = 96)

0%

20%

40%

60%

80%

100%

0.001 0.01 0.1 1 10 100log EC50 prochloraz (mg ai/l)

cum

ulat

ive

freq

uenc

y

1988 – 1998 (n = 9)2003 – 2006 (n = 17)

► sensitivity shift for ALL DMIs

Chassot, Hugelshofer, Sierotzki, and Gisi. In Press

15

Slow but continuous shift in sensitivity between 1990 and 2006. Stabilization since 2005? Sensitivities of recent populations are lower than in the 1990‘s for all DMIs in all countries.

Shift of sensitivity (mean EC 50 of population) in Mycosphaerellagraminicola to cyproconazole, epoxiconazole and prothioconazolein Europe between 1990 and 2006

Germany

Year1990 1992 1994 1996 1998 2000 2002 2004 2006 2008

Med

ian

EC50

(mg/

L-1 )

0.01

0.1

1

ProthioconazoleCyproconazoleEpoxiconazole

France

1990 1992 1994 1996 1998 2000 2002 2004 2006 20080.01

0.1

1


Med

ian

EC50

(mg/

L-1 )

Year

England

1990 1992 1994 1996 1998 2000 2002 2004 2006 20080.01

0.1

1


Year

Med

ian

EC50

(mg/

L-1 )

How to explain the shift? Genotypes

Sequenced cyp51 gene

Chassot, Hugelshofer, Sierotzki, and Gisi. In Press

16

Frequency of cyp51 genotypes in Mycosphaerella graminicola in Europe between 1988 and 2006 (n = 257)

► wild type isolates disappeared from recent populations► number of mutations increases over the years► increase of isolates carrying I 381V and A379G mutations

A379G + I381V I381V V136A complex Y137F wt

Relative genotype frequency

9

1

3

1

13

1

4

7

45

4

28

2

5

2

32

25

7 2

76

60

3 2 4 352

0%

20%

40%

60%

80%

100%

1988-1999(n=18)

2002 (n=23)

2003 (n=12)

2004 (n=22)

2005 (n=16)

2006 (n=166)

years

perc

enta

ge in

gro

ups

group VIgroup Vgroup IVgroup IIIgroup IIgroup I

1

17

cyp51 genotype groups

I II III IV V VI

log

EC

50 P

TZ

0.001

0.01

0.1

1

10


I II III IV V VI

log

EC

50 T

BZ

0.001

0.01

0.1

1

10

Sensitivity of the 6 cyp51 genotypes of M. graminicola to different DMIs CCZ: cyproconazole; EPZ: epoxiconazole; PPZ: propiconazole; TBZ: tebuconazole; PTZ: prothioconazole; CTL: chlorothalonil (n = 211, isolates from 1988 to 2006)

TBZ PTZ

a c d b e f a ab b c c c


I II III IV V VI

log

EC

50 C

CZ

0.001

0.01

0.1

1

10


I II III IV V VIlo

g E

C50

EP

Z0.001

0.01

0.1

1

10


I II III IV V VI

log

EC

50 P

PZ

0.001

0.01

0.1

1

10

CCZ EPZ PPZ

a b bc c cd cd a bc b cd de e a b c c c d

► range of variation (boxes): CTL << CCZ = PPZ < EPZ ~ PTZ << TBZ► differences between genotypes: small for CCZ, PPZ, EPZ, PTZ; big for TBZ► the combination of CCZ + PPZ + CTL is expected to impose low selection

Chlorothalonil


III IV V VI

log

EC

50 C

hlor

otha

loni

l

0.01

0.1

1

CTL

18

Sequence analysis of cyp51 promoter and sensitivity of genotypes to DMIs in Mycosphaerella graminicola

447 bp 394 bp 19 bp 775 bp

1 447 504 897 1003 1021 1132 1906

L50S S188N A379G

I381V

459-461

insertion of around 1000 bp

200 bp

5‘ upstream sequence

► insertion in promotor present only in genotype V isolates (in about half of the type V isolates and in very few isolates of type VI)► an insertion in the cyp51 gene results in an over-expression of the gene►as a consequence, genotype V isolateswith an insertion in the promoter are significantly less sensitive to DMIs


III IV V - V + VI

log

EC50

CC

Z

0.001

0.01

0.1

1

10

a b a

19

0.001

0.01

0.1

1

10

I (n=3) II (n=2) III (n=8) IV (n=7) V (n=7) VI (n=8)

log

EC50

CC

Z

Influence of promazine (40 mg/ L, inhibitor of ABC transporters) on the sensitivity of Mycosphaerella graminicola genotypes to DMIs

- transporter protein inhibitor

+ transporter protein inhibitor (40 ppm promazine)in vitro growth assay

cyp51 genotypes

I(n=3)

II(n=2)

III(n=8)

IV(n=7)

V(n=7)

VI(n=8)

inhibitors of ABC transporters slightly increase the sensitivity to DMIs, suggesting that ABC transporters may be involved in the decrease of sensitivity to DMIs

20

Summary

• Strobilurins have specific, monogenic resistance mechanisms for resistance. Therefore, they are High Risk if this occurs.

• Resistance due to G143A mutation will never occur of Phakopsora pachyrhizi because this would be a lethal mutation due to non splicing of introns.

• Resistance to DMI’s can occur via several mechanisms. However, this is a gradual shift which does not result in loss of control.

21

Summary

•A slow but continuous shift in sensitivity has been observed to all DMIs in many pathogen populations since the introduction of DMIs resulting in an erosion of DMI product performance

• In Mycosphaerella graminicola, the sensitivity shift to DMIs observed between 1988 and 2006 is due to a change in genotype distribution in field populations (mutations in cyp51 gene)

• Because the more recent genotypes (V and VI) of M. graminicola are somewhat less sensitive to triazoles than isolates from the 1990’s, fungicide rates must be kept high to ensure robust disease control

22

Summary

• The presence of a promotor in the cyp51 gene (over-expression of cyp51 gene) and the action of ABC transporters (up-regulation of ABC transportergenes) further decrease the sensitivity to DMIs

• Research results currently “In Press” support the notion that mixtures of DMI’s and/or DMI’s with other fungicides should provide a good resistance management strategy – I.E. Absolute; Alto and Quadris tank mix;

Stratego; Quadris Xtra

fundamental aspects for the development of resistance to ......dec 19, 2007 · the biosynthesis of...

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