28.11.2014, GHJ

Trends in resistance against phytopharmaceuticals in plant production
What are the consequences for food safety?
28.11.2014, GHJ Kema
Philae
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•
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Comet probe
10 years
4 billion miles
Landing on the banana genome
Fungicide sensitivity
Wheat
Banana
Humans
Mycosphaerella
graminicola
Mycosphaerella fijiensis Aspergillus fumigatus
Black Sigatoka – Costa Rica
Environment
Resistance
Fungicides
(Agronomic)
measures
Disease
Host
Pathogen
Environment
Fungicides
Strobilurins &
azoles)
Host resistance
Sex
Septoria tritici blotch
Wheat
M. graminicola
Environment
Fungicides
Strobilurins &
azoles)
Host resistance
Sex
Septoria tritici blotch
Banana
M. fijiensis
Agrios 2005
Sampling locations Costa Rica
San Pablo = 246
Zent = 247
Cartagena = 234
Wild type = 98
Total =825
Resistance to strobilurins is complete and abundant

Diversity
 Anonymous markers



(VNTRs)
Mating type loci
Strobilurin resistance
(mt-cytb)
Azole resistance
(cyp51)



Very diverse,
plantations ‘isolated’
mat1-1:mat1-2=1:1
~ 100% resistant to
strobilurins, except WT
Fungicide resistance: azoles vs. strobilurins
Cyproconazole
Azoxystrobin
PPM
PPM
0.00
0.00
1.00
0.10
3.16
0.31
5.62
0.56
10.00
1.00
31.60
3.16
56.20
5.62
100.00
10.00
Azole sensitivity assays in a global population
mg/L
0.00
0.004
0.016
0.04
0.16
0.64
2.56
10.24
Strain
strain
strain
strain
Measurement of the
optical density of
mycelium growth
Calculation of the EC50
values to compare
differences in
sensitivity to the
compounds
Populations:
Costa Rica,
Colombia, Ecuador,
Philippines,
Martinique,
Guadalupe, Ivory
Coast and
Mozambique
Four different isolates
per plate, 2 repeats
per plate.
Azole fungicide use:
propiconazole,
epoxiconazol and
difenoconazol
Cross
resistance
mg/L
0.00
•
Three azoles
0.004
• Difeconazole
0.016
• Propiconazole
• Epoxyconoazole
•
•
SDHIs
Strobilurins
0.04
0.16
0.64
2.56
10.24
OSa-13
T52-2
OSSR-35
RNVP-4
-2
Cameroon WT
Taiwan WT
Brazil
Brazil
Burundi WT
Philippines WT
Gabon WT
N. Cledonia WT
Thailand WT
Brazil
Ecuador WT
Brazil
Brazil
Vietnam WT
Indonesia WT
Brazil
Costa Rica
Brazil
Tanzania WT
Costa Rica
Ecuador
Costa Rica
Costa Rica
Costa Rica
Ecuador
Costa Rica
Costa Rica
Costa Rica
Costa Rica
Costa Rica
Ecuador
Costa Rica
Ecuador
Ecuador
Costa Rica
Ecuador
Ecuador
Costa Rica
Brazil
Costa Rica
Costa Rica
Costa Rica
Log EC50
Sensitivity to Propiconazole in different
isolates
0,5
0
-0,5
-1
-1,5
Isolates
Common mutations and insertions in the
cyp51 promoter region
Promotor
0kb
572 bp
Gene (protein)
Y136F
Mutaciones
Y136F
A313G
Y461D
Y463D
Y463H
Y463N
Y463D
A313G
Y461N
I18J
n = 110
Resistant
(Pro EC50 >
1 mg/L)
Y463D
Y136F
Element found
18 ~ 20 bp
2.2 kb
A313G
40 bp
23 bp
Wild type gene
Reduced
sensitivity (Pro
EC50 > 0.4
mg/L)
Sensitive
(Pro EC50 < 0.04
mg/L)
PCR screening of insertions in the CYP51
gene promoter
2
6
3
1
1, 2, 3 and 6
insertions/copie
s of the
repetitions have
been found
PCR screening of different populations
T52-2
T52-2
Philippines
R
WT
Ecuador
Colombia
R
R
WT
RT-PCR and qRT-PCR experiments
Resistant
Wild type
Promotor and Gene Exchange
Hygro-GFP
CYP51 gene
Hygro-GFP
CYP51 promotor
E22-Promotor
Ca_516 Promotor
E22_Gene
Ca_516_Gene
CYP51 promoter swap
mg/L
0.00
0.004
0.016
0.04
0.16
0.64
2.56
10.24
Resistant
Susceptible
Transformant
Etopic
Growth room experiments
Adaptation of the infection index (IS): Calculated by
Towsend and Heuberguer equation (Orjeda, 1998):
IS= [Σ nb / (N-1) T] 100
Where:
IS= Infection Index
n= Number of leaves in each grade.
b= Grade.
N= Number of grades used in the scale*.
T= Total number of leaf scores.
* In this case the scale used for calculating the infection index was
the symptom stage of black leaf streak disease (Fouré E. 1982).
For the calculation of the Infection Index, the symptom stages were
scored for the cigar leaf and the leaves 1-4 over time. Score 0 was
given when no symptoms were visible.
Only cigar leaf and
leaves 1-4 were
scored
Profile plot of the Infection Index
Environment
Medicines
(azoles)
Hospital
Environment
Aspergillosis
Man
A. fumigatus
•
Maart 2007 “A new A. fumigatus resistance mechanism conferring in vitro
cross-resistance to azole antifungals involves a combination of cyp51A
alterations.” Mellado et al. AAC 2207
14 A. fumigatus isolates with reduced susceptibility showed a TR in
promoter region and L98H substitution in the Cyp51A gene.
•
April 2007 “Multiple-Triazole-Resistant Aspergillosis”
Verweij et al. NEJM 2007
12 out of 13 isolates cultured from 9 patients with invasive aspergillosis showed a
TR in promoter region and L98H substitution in the Cyp51A gene.
•
Juli 2007 “Aspergillosis-and a misleading susceptibility result”
Van Leer-Buter et al, Lancet 2007
A patient was infected with two A. fumigatus strains, one suscptible and one
resistant. The susceptible isolate was found at diagnosis, the resistant one at
autopsy
29
V13-09
V22-47
V12-73
V25-18
V56-42
V02-15
3004
V25-71
V07-37
V28-29
V23-02
V11-72
V32-09
V04-47
V37-35
V07-22
V13-16
V30-62
V33-63
2518
V54-73
V01-56
V06-59
V13-34
V44-58
V13-02
V27-28
V30-22
V17-17
V44-35
V56-65
V38-26
V45-07
V03-24
V20-78
V30-28, V30-40
V43-81
V45-12
V23-66
V30-44
V16-31
V28-74
V27-61
V52-18
V28-78
V52-35
3648
2830
V50-05
9717
V30-17
Whole genome STR analyses
3 clusters, MTR-strains in same
cluster
0.1
30
Epidemiology
Percentage of patients with an
ITZ+ A. fumigatus isolate
Number of isolates
400
8,0
350
7,0
300
6,0
250
5,0
200
4,0
150
3,0
100
2,0
50
1,0
0
0,0
1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
-10
year
31
32
33
34
Environmental strains
Isolate
v61-50
v61-79
v62-05
v62-09
v62-10
v62-14
v62-48
v62-63
v62-79
v63-08
V17-24
V22-76
V37-56
V61-56
V61-57
V04-22
V22-74
V22-80
V30-09
V57-35
V57-53
V62-04
V62-06
V62-07
V62-12
V62-13
V62-23
V63-01
V63-03
V63-04
v63-05
V63-06
Perkje Medische bieb (12 T)
Perkje Medische bieb (26A)
Boom Medische bieb (29C)
Perkje SEH (33)
Bloembak hoofdingang (36B)
Nieuwe heg kindergeneeskunde (43)
Zaden nachtschone (47)
Intratuin potgrond universeel (A1, 51)
Kwekerij 20 (60)
Intratuin potgrond universeel (A3, 51)
Luchtmonster HIP
Luchtmonster HIP
Luchtmonster HIP
filtrix mmb
filtrix mmb
Luchtmonster HIP
Luchtmonster HIP
Luchtmonster HIP
Controle
watermonster HIP
watermonster HIP
omgeving 29b
omgeving 29d
omgeving 29g
omgeving 36f
omgeving 36g
omgeving 44
omgeving 64
omgeving 67
omgeving 68
Grond passiebloem Intratuin (69)
omgeving 75
Mutations cyp51A gene
MIC (μg/ml)
Naam
ITC
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
16
>16
>16
>16
0,25
0,25
0,25
0,125
0,25
0,25
0,25
1
0,5
0,5
1
0,25
0,25
0,25
0,25
0,5
0,25
VOR
4
8
8
4
4
4
4
>16
0,5
4
1
16
2
4
4
0,5
0,5
0,5
0,5
0,5
0,5
1
4
0,5
4
2
1
0,5
1
0,5
4
1
POSA AMT
0,5
0,5
0,5
1
0,5
0,5
1
0,5
0,25
1
0,25
1
0,5
0,5
0,5
1
0,25
0,5
1
1
0,5
0,5
0,5
0,5
0,5
1
0,5
0,5
0,5
0,5
0,063
1
0,031
1
0,031
1
0,031
0,5
0,031
1
0,063
0,5
0,125
0,5
0,25
1
0,125
1
0,25
1
0,25
1
0,063
1
0,031
0,5
0,063
0,5
0,063
1
0,25
0,5
0,031
1
TER
0,25
0,016
0,125
0,063
0,125
0,063
0,25
0,031
0,5
1
0,031
0,031
0,125
0,25
0,063
0,063
0,063
0,5
0,125
0,063
0,016
0,25
0,5
0,063
0,031
0,5
0,25
0,125
0,063
0,016
0,5
0,25
CASPO*
>32
0,5
0,25
1
0,5
1
1
0,5
0,5
0,5
0,25
0,25
0,25
1
0,5
0,5
0,5
0,5
0,5
0,5
0,5
0,5
0,5
1
1
1
0,5
0,5
0,5
0,5
0,5
0,5
Tandem repeat
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Codon
98
CAC
CAC
CAC
CAC
CTC
CAC
CAC
CTC
CAC
CAC
CAC
CAC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
CTC
Other
----Q141H
----S52T
----S297T + F495I
---
------------F46Y, M172V, G98G, L358L, E427K, C454C
--F46Y, C454C
---
Red: resistance mechanism identical with strains from patients
35
Hypothesis
agriculture
compost
(Indoor) environment
Repeated exposure to triazole
fungicides with activity
against A. fumigatus
Inhalation of
Aspergillus conidia
Invasive aspergillosis
+/- triazole pressure
hospital
36
Agricultural cause?
• Tandem repeats in
promotor
• Cyp51 mutations
•
•
L98H
New mutation in
2010
• Not induced in the lab
• Role for sex?
cyp51 analogies in M. graminicola, M. fijiensis
and A. fumigatus
0kb
M. fijiensis
Promotor
572 bp
Gene (protein)
Y136F
2.2 kb
Y463D
A313G
M. graminicola
L98H
Resistant
(Pro EC50 >
1 mg/L)
A. fumigatus
Mutations
Y136F
A313G
Y461D
Y463D
Y463H
Y463N
Wild type gene
Sensitive
(Pro EC50 < 0.04
mg/L)
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