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1. CONFIDENTIAL
A. Evenhuis, G.B.M. van den Bosch & H.T.A.M. Schepers
Testing the efficacy of different fungicides to
control different strains of potato late blight
Praktijkonderzoek Plant & Omgeving (Applied Plant Research), part of Wageningen UR
Business Unit AGV PPO no.
3250281500
August 2013

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© 2013 Wageningen, Foundation Stichting Dienst Landbouwkundig Onderzoek (DLO) research institute
Praktijkonderzoek Plant & Omgeving. All rights reserved. No part of this publication may be reproduced, stored in a
retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or
otherwise, without the prior written permission of the DLO, Praktijkonderzoek Plant & Omgeving (Applied Plant
Research), Business Unit AGV
The Foundation DLO is not responsible for any damage caused by using the content of this report.
Project no PPO: 3250281500
Applied Plant Research (Praktijkonderzoek Plant & Omgeving),
part of Wageningen UR
AGV Research Unit
Address : Edelhertweg 1, Lelystad, The Netherlands
: PO Box 430, 8200 AK Lelystad, The Netherlands
Tel. : +31 320 - 291 111
Fax : +31 320 - 230 479
E-mail : info.ppo@wur.nl
Internet : www.ppo.wur.nl

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Table of contents
page
1 INTRODUCTION ............................................................................................................................ 5
2 MATERIALS & METHODS............................................................................................................... 6
2.1 Potato plants and leaf discs.................................................................................................... 6
2.2 Spraying ............................................................................................................................... 6
2.3 Treatments ........................................................................................................................... 7
2.4 Inoculation P. infestans........................................................................................................... 7
2.5 Disease observations P. infestans.......................................................................................... 7
2.6 Statistical analyses ................................................................................................................ 7
3 RESULTS ..................................................................................................................................... 8
3.1 Necrotic foliage..................................................................................................................... 8
3.2 Sporulation.......................................................................................................................... 11
4 DISCUSSION AND CONCLUSIONS................................................................................................ 13
APPENDIX 1 DISEASE OBSERVATIONS (RAW DATA EXP. 1)................................................................... 14
APPENDIX 2. PD-RECOGNITION........................................................................................................... 16

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1 Introduction
Late blight, caused by Phytophthora infestans, is the most important disease in potato production because
of its capacity to destroy foliage very rapidly and its ability to infect tubers. To avoid infection of the foliage,
fungicides are used frequently. In the Netherlands, in some years up to 15 sprays are necessary to prevent
the crop from infection by the pathogen.
New P. infestans isolates are found regularly. These isolates have new properties, such as new virulences,
overcoming cultivar resistance. Also new isolates can be more aggressive than isolates of the old P.
infestans population. Even the susceptibility to fungicides might have changed.
In the two experiments described in this report the efficacy of mancozeb, maneb, fluazinam and metalaxyl
against four P. infestans isolates representing different clonal lineages was tested in laboratory experiments
by request of United Phosphorus Ltd.

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2 Materials & methods
2.1 Potato plants and leaf discs
The cultivated potato plants (cv. Bintje) were grown in pots. The pots with a content of 5 litre were filled with
soil and the potato tubers were placed at a depth of 10 cm on 10 June 2013. From emergence until the
experiment, the plants were placed in the greenhouse. Leaves were picked on 30 July and leaf discs with a
diameter of 1.0 cm were punched from these leaves. Five potato leaf discs were placed in a Petri dish with
Water Agar (Figure 1). In total 384 Petri dishes were used in the two experiments.
Figure 1. Petri dish with leaf discs ready to be sprayed.
2.2 Spraying
The leaf discs were sprayed with the different fungicides in a spraying cabin developed by Applied Plant
Research (PPO) on 30 July. The fungicides were sprayed using a spray boom with three spray nozzles,
placed 50 cm apart, which is moving approximately 40 cm over the top of the potato plants. Spray volume
was 250 l/ha.

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2.3 Treatments
In Table 1 the fungicides used and dose rates are presented. Each product was tested in two dose rates:
the full dose (100%) and a 10% dose rate. The untreated control was sprayed with tap water. The
experiment was carried out twice on 30 July 2013. For each experiment the spray solutions were prepared
separately.
Table 1. The fungicides used in the experiments and the applied dose rates (2013)
Code Fungicide Dose rate Dose rate l or kg per ha
A Water (untreated) - -
B Mancozeb 500 g/l SC 100% 3.0
C Mancozeb 500 g/l SC 10% 0.3
D Mancozeb 75% WG 100% 2.0
E Mancozeb 75% WG 10% 0.2
F Maneb 75% WG 100% 2.0
G Maneb 75% WG 10% 0.2
H Metalaxyl 25% WG 100% 0.8
J Metalaxyl 25% WG 10% 0.08
K Fluazinam 500 g/l SC (Shirlan) 100% 0.4
L Fluazinam 500 g/l SC (Shirlan) 10% 0.04
2.4 Inoculation P. infestans
The four P. infestans isolates used were VK98014 (old population) and isolates of the Blue 13 (NL11147),
Green 33 (NL10328), and Pink 6 (NL11179) genotype. The Blue 13 isolate is resistant to metalaxyl. The
Green 33 isolate is controlled less effectively by fluazinam in field trials. The isolates were cultivated on
agar plates and potato slices. An inoculum suspension was made by rinsing a one week old culture of P.
infestans with tap water. The inoculum density was set at approximately 10,000 sporangia per ml.
Inoculation of the leaf discs was carried out by spraying the leaf discs over head with approximately 0.1 ml
of inoculum on 1 August 2013, two days after spraying. Each experiment was inoculated separately. After
inoculation Petri dishes containing leaf discs were incubated in a climate chamber for one week.
2.5 Disease observations P. infestans
Late blight disease severity was assessed one week after inoculation on 8 August 2013. Percentage
necrotic foliage per leaf disc was estimated. Sporulation of P. infestans was assessed in three classes.
0= no sporulation; 1= sparsely sporulating; 2= profusely sporulating.
Disease severity of treatments K and L inoculated with Green33 were also observed on 12 August, 11 days
after inoculation.
2.6 Statistical analyses
The experiment was carried out with four replications and two consecutive experiments. Each replication
consisted of one Petri dish containing five leaf discs. Analysis of variance on the late blight assessments
was made based on the percentage necrotic foliage and sporulation using GENSTAT 15th Edition.
Transformation of data was carried out when necessary. In order to improve the discrimination between the
treatments, the untreated control was not included in the final analysis.

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3 Results
3.1 Necrotic foliage
The late blight disease severity expressed as the percentage necrotic foliage per leaf disc is presented in
Table 2 (experiment 1), Table 3 (experiment 2) and table 4 and Figure 2 (experiment 1+2). The infection
level of untreated discs was in both experiments and for all isolates > 90%.
The efficacy to control potato late blight depended on the fungicide used and was also influenced by the P.
infestans. Almost all fungicide treatments reduced the percentage necrotic foliage compared to the
untreated control, regardless the used P. infestans isolate. Only in experiment 1, Metalaxyl 10% did not
show a significant difference in the percentage of necrotic foliage compared to the untreated control after
inoculation with Blue 13.
In both experiments it was observed that isolate Blue 13 resulted in the highest percentage of necrotic
foliage when all treatments were analyzed together.
When the leaf discs were inoculated with isolate Blue 13, the highest percentage infected foliage was
observed at Metalaxyl in both dose rates. All other treatment resulted in a significantly lower level of
necrotic foliage compared to Metalaxyl.
When the leaf discs were inoculated with Green 33, the highest percentage of necrotic foliage was
observed at 10% Mancozeb SC and 10% maneb WG, only in experiment 1 the differences with other
treatments were significant. Between all other treatment no significant differences were observed. Both
dose rates of Metalaxyl and Shirlan completely controlled late blight in both experiments. However when
disease assessment was made 11 days after inoculation in 9% late blight severity was found on Fluazinam
sprayed leaf disc at a full dose rate.
When the leaf discs were inoculated with Pink 6, the highest percentage of necrotic foliage was observed at
10% maneb WG in both experiments. Both dose rates of Metalaxyl and Shirlan completely controlled late
blight in both experiments.
When the leaf discs were inoculated with VK98014, all treatments controlled blight equally well in
experiment 1. In experiment 2 there were indications that the lower dose rates of Mancozeb and maneb
resulted in a higher level of necrotic foliage compared to the other treatments.
Table 2. Experiment 1: Percentage necrotic foliage per treatment 7 days after inoculation with 4 different
P. infestans isolates.
1) Values followed by the same letter are not significantly different (p=0.05).
Blue 13 Green 33 Pink 6 VK98014 Total
A Water (untreatedl) - 100.0 h 95.0 h 96.0 h 95.5 h 96.6 f
B Mancozeb 500 g/l SC 100% 25.0 bcdef 5.0 a 23.2 bcde 5.0 a 14.6 de
C Mancozeb 500 g/l SC 10% 5.0 a 28.0 cdef 9.0 ab 3.5 a 11.4 cd
D Mancozeb 75% WG 100% 14.5 abcd 0.0 a 5.0 ab 0.0 a 4.9 abc
E Mancozeb 75% WG 10% 6.0 a 10.0 ab 15.0 abcd 4.5 a 8.9 bcd
F Maneb 75% WG 100% 0.0 a 0.0 a 4.8 a 4.5 a 2.3 ab
G Maneb 75% WG 10% 10.0 ab 40.0 fg 29.0 def 11.5 ab 22.6 e
H Metalaxyl 25% WG 100% 48.5 g 0.0 a 0.0 a 0.0 a 12.1 cd
J Metalaxyl 25% WG 10% 87.0 h 0.0 a 0.0 a 0.0 a 21.8 e
K Fluazinam 500 g/l SC (Shirlan) 100% 1.5 a 0.0 a 0.0 a 0.0 a 0.4 a
L Fluazinam 500 g/l SC (Shirlan) 10% 5.0 a 0.0 a 0.0 a 0.0 a 1.3 ab
total 27.3 c 15.9 ab 18.3 b 11.2 a

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Table 3. Experiment 2: Percentage necrotic foliage per treatment 7 days after inoculation with 4 different
P. infestans isolates.
1) Values followed by the same letter are not significantly different (p=0.05).
Table 4. Experiment 1+2: Percentage necrotic foliage per treatment 7 days after inoculation with 4
different 4 different P. infestans isolates.
1) Values followed by the same letter are not significantly different (p=0.05).
Blue 13 Green 33 Pink 6 VK98014 Total
A Water (untreatedl) - 99.5 h 98.5 h 95.0 h 86.8 gh 94.9 e
B Mancozeb 500 g/l SC 100% 19.3 bcde 5.0 ab 11.5 abcd 0.0 a 8.9 ab
C Mancozeb 500 g/l SC 10% 15.0 abcde 0.0 ab 9.0 abcd 31.3 ef 13.8 bc
D Mancozeb 75% WG 100% 20.0 bcdef 5.0 ab 5.0 ab 5.0 ab 8.8 ab
E Mancozeb 75% WG 10% 6.3 abc 10.0 abcd 0.0 a 8.5 abcd 6.2 ab
F Maneb 75% WG 100% 0.0 a 0.0 a 0.0 a 5.0 ab 1.3 a
G Maneb 75% WG 10% 8.8 abcd 14.0 abcd 36.0 f 22.0 cdef 20.2 c
H Metalaxyl 25% WG 100% 19.3 bcde 0.0 a 0.0 a 0.0 a 4.8 a
J Metalaxyl 25% WG 10% 71.3 g 0.0 a 0.0 a 0.0 a 17.8 c
K Fluazinam 500 g/l SC (Shirlan) 100% 5.0 ab 0.0 a 0.0 a 0.0 a 1.3 a
L Fluazinam 500 g/l SC (Shirlan) 10% 10.0 abcd 0.0 a 0.0 a 0.0 a 2.5 a
total 28.9 b 12.1 a 15.0 a 15.1 a
Blue 13 Green 33 Pink 6 VK98014 Total
A Water (untreatedl) - 99.8 k 96.8 k 95.5 k 91.1 k 95.8 f
B Mancozeb 500 g/l SC 100% 22.1 efg 5.0 abc 17.4 def 2.5 ab 11.8 c
C Mancozeb 500 g/l SC 10% 10.0 abcd 14.0 bcde 9.0 abcd 17.4 def 12.6 c
D Mancozeb 75% WG 100% 17.3 def 2.5 ab 5.0 abc 2.5 ab 6.8 bc
E Mancozeb 75% WG 10% 6.1 abcd 10.0 abcd 7.5 abcd 6.5 abcd 7.5 bc
F Maneb 75% WG 100% 0.0 a 0.0 a 2.4 ab 4.8 ab 1.8 ab
G Maneb 75% WG 10% 9.4 abcd 27.0 fgh 32.5 gh 16.8 def 21.4 de
H Metalaxyl 25% WG 100% 33.9 h 0.0 a 0.0 a 0.0 a 8.5 c
J Metalaxyl 25% WG 10% 79.1 j 0.0 a 0.0 a 0.0 a 19.8 d
K Fluazinam 500 g/l SC (Shirlan) 100% 3.3 ab 0.0 a 0.0 a 0.0 a 0.8 a
L Fluazinam 500 g/l SC (Shirlan) 10% 7.5 abcd 0.0 a 0.0 a 0.0 a 1.9 ab
total 28.1 c 14.0 ab 16.7 b 13.2 a

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Figure 2. Experiment 1+2: Percentage necrotic foliage per treatment 7 days after inoculation with 4
different P. infestans isolates.

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3.2 Sporulation
The sporulation is presented in Table 5 and Figure 3. The sporulation index of the untreated control was
significantly higher compared to the fungicide treatments.
All fungicide treatments reduced the sporulation index compared to the untreated control, regardless the
used P. infestans isolate.
In both experiments it was observed that isolate Blue 13 resulted in the highest sporulation index when all
treatments were analyzed together.
The influence of the treatments on the sporulation index was similar compared to the influence of the
treatments on the percentage necrotic foliage.
Table 5. Experiment 1: Sporulation intensity per treatment 7 days after inoculation with 4 different P.
infestans isolates.
1) Values followed by the same letter are not significantly different (p=0.05).
Blue 13 Green 33 Pink 6 VK98014 Total
A Water (untreatedl) - 2.00 m 1.95 m 1.92 m 1.87 m 1.93 g
B Mancozeb 500 g/l SC 100% 0.42 efgh 0.07 abc 0.27 cdef 0.05 abc 0.20 cd
C Mancozeb 500 g/l SC 10% 0.20 abcde 0.25 bcdef 0.20 abcde 0.42 efgh 0.26 d
D Mancozeb 75% WG 100% 0.32 defg 0.02 ab 0.10 abcd 0.05 abc 0.12 abc
E Mancozeb 75% WG 10% 0.15 abcd 0.15 abcd 0.15 abcd 0.15 abcd 0.15 bcd
F Maneb 75% WG 100% 0.00 a 0.00 a 0.05 abc 0.10 abcd 0.03 ab
G Maneb 75% WG 10% 0.20 abcde 0.52 ghi 0.65 hi 0.45 fgh 0.45 e
H Metalaxyl 25% WG 100% 0.90 jk 0.00 a 0.00 a 0.00 a 0.22 cd
J Metalaxyl 25% WG 10% 1.57 l 0.00 a 0.00 a 0.00 a 0.39 e
K Fluazinam 500 g/l SC (Shirlan) 100% 0.05 abc 0.00 a 0.00 a 0.00 a 0.01 a
L Fluazinam 500 g/l SC (Shirlan) 10% 0.15 abcd 0.00 a 0.00 a 0.00 a 0.03 ab
total 0.58 b 0.27 a 0.34 a 0.29 a

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Figure 3. Experiment 1: Sporulation intensity per treatment 7 days after inoculation with 4 different P.
infestans isolates.

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4 Discussion and conclusions
The infection level and the sporulation index of untreated discs was in both experiments and for all isolates
> 90% and 1.92 respectively, indicating that the artificial inoculation was successful for all four P. infestans
isolates.
All the fungicide treatments were able to reduce the percentage necrotic foliage and sporulation intensity
compared to the untreated control, regardless the used P. infestans isolate.
Conclusions P. infestans:
Isolates
• Overal the level of necrotic foliage and the sporulation index was higher in the treated leaf discs
inoculated with the Blue 13 isolate compared to the other isolates. These findings confirm that Blue
13 isolates are often more agressive than isolates from other clonal lineages;
• The poor control of the Blue 13 isolate by metalaxyl confirms that this isolate is rated as a
metalaxyl-resistant isolate;
• In both experiments the Green 33 isolate was controlled effectively by fluazinam. In some field
experiments with a polycyclic epidemic and frequent treatments of fluazinam, fluazinam showed a
lower efficacy to control Green 33. But in laboratory tests both in vitro and on leaf discs this
correlation was not clear. The mechanism that causes these differences is still unknown.
Treatments and dose rates
• There are indications that the percentage necrotic foliage and the sporulation index at Mancozeb
WG is lower than at Mancozeb SC but the differences are not always significant.
• There are indications that the percentage necrotic foliage and the sporulation index at 100% Maneb
WG is lower than at Mancozeb WG and SC but the differences are not always significant.
• The dose rate of Mancozeb SC, Mancozeb WG and Shirlan did not significantly influence both the
percentage of necrotic foliage and the sporulation index.
• The lower dose rates of Maneb WG and metalaxyl resulted in both a significantly higher percentage
of necrotic foliage and a higher sporulation index.

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Appendix 1 Disease observations (raw data Exp. 1)
Late blight severity averaged over 5 potato leaf disc per replicate within a treatment, and experiment
Treatment Exp. 1 Exp. 1 Exp. 1 Exp. 1 Exp. 2 Exp. 2 Exp. 2 Exp. 2
Blue
NL11147
Green
NL10328
Pink
NL11179
VK98014 Blue
NL11147
Green
NL10328
Pink
NL11179
VK98014
A
1 100 100 100 96 98 96 100 100
2 100 100 100 90 100 98 80 57
3 100 100 84 96 100 100 100 100
4 100 80 100 100 100 100 100 90
B
1 20 20 12 0 0 0 0 0
2 40 0 16 20 21 0 26 0
3 20 0 26 0 56 20 20 0
4 20 0 39 0 0 0 0 0
C
1 20 0 0 0 20 0 0 40
2 0 54 16 10 40 0 16 40
3 0 20 20 0 0 0 0 41
4 0 38 0 4 0 0 20 4
D
1 0 0 20 0 20 20 20 20
2 20 0 0 0 0 0 0 0
3 20 0 0 0 60 0 0 0
4 18 0 0 0 0 0 0 0
E
1 0 0 0 0 20 0 0 0
2 20 40 20 0 0 0 0 19
3 4 0 0 18 0 20 0 0
4 0 0 40 0 5 20 0 15
F
1 0 0 19 0 0 0 0 0
2 0 0 0 0 0 0 0 0
3 0 0 0 18 0 0 0 20
4 0 0 0 0 0 0 0 0
G
1 0 60 40 26 0 26 39 36
2 20 60 0 20 15 0 25 0
3 20 20 40 0 0 0 20 22
4 0 20 36 0 20 30 60 30

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Treatment Exp. 1 Exp. 1 Exp. 1 Exp. 1 Exp. 2 Exp. 2 Exp. 2 Exp. 2
Blue
NL11147
Green
NL10328
Pink
NL11179
VK98014 Blue
NL11147
Green
NL10328
Pink
NL11179
VK98014
H
1 23 0 0 0 10 0 0 0
2 42 0 0 0 29 0 0 0
3 53 0 0 0 9 0 0 0
4 76 0 0 0 29 0 0 0
J
1 92 0 0 0 60 0 0 0
2 80 0 0 0 80 0 0 0
3 98 0 0 0 75 0 0 0
4 78 0 0 0 70 0 0 0
K
1 0 0 0 0 0 0 0 0
2 0 0 0 0 20 0 0 0
3 6 0 0 0 0 0 0 0
4 0 0 0 0 0 0 0 0
L
1 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0
3 20 0 0 0 40 0 0 0
4 0 0 0 0 0 0 0 0

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Appendix 2. PD-recognition