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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
sensitivity of isolates as EC 50 mg / L
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
sensitivity of isolates as EC 50 mg / L
>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
sensitivity of isolates as EC 50 mg / L
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
sensitivity of isolates as EC 50 mg / L
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
ProthioconazoleCyproconazoleEpoxiconazole
Med
ian
EC50
(mg/
L-1 )
Year
England
1990 1992 1994 1996 1998 2000 2002 2004 2006 20080.01
0.1
1
ProthioconazoleCyproconazoleEpoxiconazole
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
cyp51 genotype groups
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
cyp51 genotype groups
I II III IV V VI
log
EC
50 C
CZ
0.001
0.01
0.1
1
10
cyp51 genotype groups
I II III IV V VIlo
g E
C50
EP
Z0.001
0.01
0.1
1
10
cyp51 genotype groups
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
cyp51 genotype groups
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
cyp51 genotype groups
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
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