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CONFIDENTIAL
A. Evenhuis & H.T.A.M. Schepers
Testing the efficacy of fungicides to control
different strains of Phytophthora infestans
Praktijkonderzoek Plant & Omgeving (Applied Plant Research), part of Wageningen UR
Business Unit AGV PPO no.
3250294400
May 2014
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
2
© 2014 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: 3250294400
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
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
3
Table of contents
page
1 INTRODUCTION ............................................................................................................................ 5
2 MATERIALS & METHODS............................................................................................................... 6
2.1 Potato plants......................................................................................................................... 6
2.2 Spraying ............................................................................................................................... 6
2.3 Treatments ........................................................................................................................... 6
2.4 Inoculation P. infestans........................................................................................................... 6
2.5 Disease observations P. infestans.......................................................................................... 7
2.6 Statistical analyses ................................................................................................................ 7
3 RESULTS ..................................................................................................................................... 8
4 DISCUSSION AND CONCLUSIONS................................................................................................ 13
APPENDIX 1 DISEASE OBSERVATIONS (RAW DATA EXP. 1)................................................................... 14
APPENDIX 2. PD-RECOGNITION........................................................................................................... 18
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
4
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
5
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 virulence’s,
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 this report the efficacy of mancozeb, fluazinam and metalaxyl-m against four P. infestans isolates
representing different clonal lineages was tested in a greenhouse experiment by request of United
Phosphorus Ltd.
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
6
2 Materials & methods
2.1 Potato plants
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 6 March 2014. From emergence until the
experiment, the plants were placed in the greenhouse in Lelystad.
2.2 Spraying
The potato plants were sprayed with the different fungicides in a spraying cabin developed by Applied Plant
Research (PPO) on 9 April. 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.
2.3 Treatments
In Table 1 the fungicides used and dose rates are presented. The untreated control was sprayed with tap
water. The experiment was sprayed on 9 April 2014. For each experiment the spray solutions were
prepared separately.
Table 1. The fungicides used in the experiments and the applied dose rates (2014)
Code Fungicide Dose rate l or kg per ha
A water (untreated control) -
B mancozeb 75% WG 2.0
C mancozeb 64% + metalaxyl-m 8% 2.5
D mancozeb + Shirlan 2.0 + 0.4
E metalaxyl-m 25% WG 0.8
F fluazinam 500 g/l SC (Shirlan) 0.4
2.4 Inoculation P. infestans
The four P. infestans isolates used were of different clonal lineages, known as Blue 13 (NL07041), Green
33 (NL10328), Pink 6 (NL11179) and Orange (NL12079) genotype. The Blue 13 isolate is resistant to
metalaxyl-m. 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 potato plants was carried out by spraying the leaves over head with inoculum on 15
April 2014, six days after spraying the fungicides. After inoculation the potato plants were incubated in a
climate chamber for one week.
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
7
2.5 Disease observations
Late blight disease severity was assessed five times after inoculation. Percentage necrotic foliage was
estimated. From the individual disease ratings a Standard Area Under Disease Progress Curve (stAUDPC)
was calculated.
2.6 Statistical analyses
The experiment was carried out with four replications. Each replication consisted of one potato plant.
Analysis of variance on the late blight assessments was made based on the percentage necrotic foliage and
sporulation using GENSTAT 16th 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.
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
8
3 Results
The late blight disease severity expressed as the percentage necrotic foliage is presented in Table 2 and
Table 3. The efficacy to control potato late blight depended on the fungicide used and was also influenced
by the P. infestans isolate. Almost all fungicide treatments reduced the percentage necrotic foliage
compared to the untreated control, regardless of the used P. infestans isolate. Only applying metalaxyl-m
(E) did not show a significant difference in the percentage of necrotic foliage compared to the untreated
control after inoculation with Blue 13.
Table 2. Percentage necrotic foliage per treatment 7, 9 and 10 days past inoculation (d.p.i.) with 4 different
P. infestans isolates.
Genotype Code disease severity (%)
7 d.p.i 9 d.p.i 10 d.p.i
Blue 13 A 48.8 l 67.5 k 65.0 j
B 14.3 fgh 18.8 defg 23.8 h
C 11.0 defg 18.8 defg 20.0 fgh
D 21.3 ij 22.5 fgh 23.8 h
E 48.8 l 71.3 k 77.5 k
F 20.0 hij 23.8 gh 27.5 h
Green 33 A 15.0 ghi 28.8 hi 40.0 i
B 1.8 abc 4.3 ab 6.8 abcd
C 0.0 a 0.3 a 0.3 a
D 2.3 abc 8.3 abc 13.3 cdef
E 0.8 ab 4.0 ab 7.3 abcd
F 4.8 abcd 12.3 bcde 22.5 gh
Pink 6 A 26.3 j 37.5 ij 48.8 i
B 4.8 abcd 5.8 ab 9.8 bcd
C 0.0 a 2.3 a 2.5 ab
D 4.3 abc 13.3 bcdef 14.5 defg
E 8.0 cdef 18.0 defg 18.8 efgh
F 12.5 efg 20.0 efgh 27.5 h
Orange A 33.8 k 45.0 j 47.5 i
B 7.0 bcde 15.5 cdefg 18.8 efgh
C 0.0 a 0.3 a 0.8 a
D 1.3 ab 5.5 ab 5.5 abc
E 1.5 abc 6.5 abc 9.8 bcd
F 4.8 abcd 9.5 abcd 10.5 bcde
1) Values followed by the same letter are not significantly different (p=0.05).
In Table 3 disease severity 13 and 15 days past inoculation is given and the stAUDPC is presented. The
stAUDPC is a weighted average of the individual disease ratings.
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
9
Table 3. Percentage necrotic foliage per treatment 13 and 15 days past inoculation (d.p.i.) with 4 different
P. infestans isolates and stAUDPC.
Genotype Code disease severity (%) stAUDPC
13 d.p.i 15 d.p.i
Blue 13 A 71.3 j 78.75 ij 67.1 j
B 27.5 efgh 28.75 defg 23.4 efg
C 23.8 defg 26.25 cdefg 20.6 defg
D 30.0 fgh 32.5 efg 26.3 fg
E 85.0 k 91.25 j 76.8 k
F 35.0 gh 37.5 g 29.5 g
Green 33 A 53.8 i 81.25 ij 44.2 h
B 16.3 bcde 16.75 bcd 9.9 ab
C 2.5 a 2.5 a 1.2 a
D 19.5 cdef 21.25 cde 13.9 bcd
E 23.8 defg 26.25 cdefg 13.4 bcd
F 28.8 efgh 36.25 fg 22.0 defg
Pink 6 A 66.3 ij 71.25 i 52.1 hi
B 12.3 abcd 15.5 abcd 9.9 ab
C 8.5 abc 15.5 abcd 5.6 ab
D 18.0 cdef 21.25 cde 14.9 bcde
E 38.8 h 53.75 h 27.9 fg
F 26.3 efgh 36.25 fg 24.9 fg
Orange A 63.8 ij 78.75 ij 54.3 i
B 25.0 efg 25 cdefg 19.4 cdef
C 4.5 ab 4.5 ab 2.2 a
D 8.8 abc 7.25 ab 6.2 ab
E 23.8 defg 22.5 cdef 14.1 bcde
F 12.0 abcd 14 abc 10.5 abc
1) Values followed by the same letter are not significantly different (p=0.05).
Table 4 gives the efficacy of the treatments to control potato late blight average over all genotypes.
However it must be kept in mind that not all genotypes are equally well controlled by the same fungicide
(Tables 1 to 3).
Table 4. Percentage necrotic foliage, expressed as stAUDPC, per treatment after inoculation with 4
different P. infestans isolates, including the untreated control..
Genotype Code stAUDPC
All water (untreated control) 54.4 e
mancozeb 75% WG 15.6 b
mancozeb 64% + metalaxyl-m 8% 7.4 a
mancozeb + Shirlan 15.3 b
metalaxyl-m 25% WG 33.0 d
fluazinam 500 g/l SC (Shirlan) 21.7 c
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
10
Figure 1 represents the efficacy of different treatments to control P. infestans isolates belonging to
different clonal lineages. Figure 2 to 5 present the disease severity of P. infestans in time of the different
clonal lineages.
Figure 1. Percentage necrotic foliage per treatment expressed as stAUDPC after inoculation with 4
different P. infestans isolates, representing large clonal lineages in Europe.
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
11
Figure 2. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate,
representing clonal lineage Blue13
Figure 3. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate,
representing clonal lineage Green33
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
12
Figure 4. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate,
representing clonal lineage Pink6
Figure 5. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate,
representing clonal lineage Orange
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
13
4 Discussion and conclusions
The infection level of untreated plants was 70-85% at the end of the experiment 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 compared to the untreated control, regardless the used P. infestans isolate,
except for metalaxyl-m when potato plants were inoculated with Blue13.
Conclusions P. infestans:
Isolates
• Overal the level of necrotic foliage was higher in the treated potato plants 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.
• In this experiment on average disease severity was higher when potato plants were inoculated with
Pink 6 compared to Green 33 and Orange.
• The absence of control of the Blue 13 isolate by metalaxyl-m confirms that this isolate is rated as a
metalaxyl-resistant isolate. The efficacy of all other treatments to contol Blue13 was better than
metalaxyl-m.
• The efficacy of mancozeb (B) to control the Blue 13 isolate was significantly better than the
efficacy of fluazinam (F) and metalaxyl-m (E).
• The Green 33 isolate was significantly better controlled with mancozeb + metalaxyl-m (C) than with
mancozeb + fluazinam (D), metalaxyl-m (E) and fluazinam (F).
• The Green 33 isolate was less effectively controlled by fluazinam (F) compared to mancozeb (B)
and manoczeb + metalaxyl-m (C). No singificant differences were observed between the other
treatments.
• The Pink 6 isolate was singinficantly better controled by mancozeb (B), mancozeb + metalaxyl-m
(C) and manczeb + fluazinam (D) compared to application of fluazinam (F) or metalaxyl-m (E).
• The Orange isolate was significantly better controlled with mancozeb + metalaxyl-m (C) than
mancozeb (b) or metalaxyl-m.
• The efficacy of mancozeb + fluazinam (D) to control the orange isolate was significantly better than
mancozeb (B).
Treatments
• The efficacy of mancozeb + metalaxyl-m to control P. infestans was significantly better than all
other treatments.
• The efficacy of mancozeb (B) and mancozeb + fluazinam (D) was comparable and significantly
better than the efficacy of fluazinam and metalaxyl-m. This is mainly caused by the fact that Green
33 is less effectively controlled by fluazinam and Blue13 is not controlled by metalaxyl-m.
• The efficacy of fluazinam to control potato late blight was significantly better than metalaxyl-m.
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
14
Appendix 1 Disease observations (raw data Exp. 1)
Pot Treat-
ment
Isolate Rep foliar blight (%) stAUDPC
7 9 10 13 15 Average
1 A Blue 1 40 60 65 65 70 60.0 61.6
2 E Orange 1 2 3 10 20 20 11.0 12.1
3 E Pink 1 15 25 25 55 60 36.0 37.5
4 E Blue 1 55 90 95 95 95 86.0 89.1
5 C Blue 1 7 15 15 20 25 16.4 16.8
6 D Orange 1 3 15 15 25 20 15.6 17.3
7 D Green 1 3 10 25 35 30 20.6 23.2
8 C Green 1 0 1 1 10 10 4.4 4.8
9 E Green 1 0 7 15 30 35 17.4 18.8
10 A Orange 1 35 50 55 65 80 57.0 57.8
11 A Green 1 20 40 45 70 95 54.0 55.0
12 F Orange 1 1 7 10 15 15 9.6 10.5
13 C Pink 1 0 5 5 15 25 10.0 10.0
14 D Pink 1 7 15 20 25 25 18.4 19.6
15 D Blue 1 25 25 25 35 35 29.0 29.4
16 A Pink 1 25 40 50 60 65 48.0 50.0
17 B Green 1 0 0 2 5 7 2.8 2.9
18 C Orange 1 0 1 1 10 10 4.4 4.8
19 F Blue 1 20 25 30 30 35 28.0 28.4
20 F Pink 1 15 25 35 20 40 27.0 26.6
21 F Green 1 5 15 25 25 35 21.0 21.9
22 B Pink 1 15 15 25 25 30 22.0 22.5
23 B Orange 1 5 15 25 25 25 19.0 20.6
24 B Blue 1 30 30 30 35 45 34.0 33.4
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
15
Pot
number
Treatment Isolate Rep foliar blight (%) stAUDPC
5 7 9 13 15 Average
25 B Blue 2 10 15 25 35 30 23.0 25.0
26 C Orange 2 0 0 0 0 0 0.0 0.0
27 A Green 2 15 25 35 45 75 39.0 38.8
28 A Orange 2 30 40 45 65 85 53.0 53.4
29 C Green 2 0 0 0 0 0 0.0 0.0
30 E Blue 2 55 75 70 80 85 73.0 74.1
31 F Blue 2 15 20 25 35 30 25.0 26.6
32 F Pink 2 15 25 35 45 55 35.0 36.3
33 D Green 2 5 20 25 35 45 26.0 27.2
34 A Pink 2 25 35 50 75 80 53.0 55.6
35 A Blue 2 55 70 65 70 80 68.0 68.1
36 D Orange 2 0 0 0 0 2 0.4 0.3
37 F Green 2 10 20 30 40 55 31.0 31.9
38 F Orange 2 15 25 25 20 30 23.0 22.8
39 D Pink 2 0 3 3 7 15 5.6 5.4
40 B Green 2 2 7 10 35 30 16.8 18.8
41 B Orange 2 15 25 25 35 40 28.0 28.8
42 E Green 2 1 3 5 25 25 11.8 12.9
43 E Pink 2 7 15 15 25 35 19.4 19.6
44 B Pink 2 3 5 7 10 15 8.0 8.1
45 D Blue 2 25 25 30 35 40 31.0 31.3
46 E Orange 2 0 1 4 20 15 8.0 9.3
47 C Blue 2 15 20 20 25 30 22.0 22.2
48 C Pink 2 0 1 1 4 7 2.6 2.6
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
16
Pot
number
Treatment Isolate Rep foliar blight (%) stAUDPC
5 7 9 13 15 Average
49 A Orange 3 35 40 40 55 65 47.0 47.2
50 D Orange 3 0 0 0 0 0 0.0 0.0
51 F Pink 3 10 15 25 15 25 18.0 18.1
52 C Blue 3 15 25 25 35 35 27.0 28.1
53 B Green 3 5 10 15 25 30 17.0 17.8
54 A Blue 3 45 65 60 70 80 64.0 64.7
55 B Orange 3 5 15 15 25 20 16.0 17.5
56 F Blue 3 25 30 30 40 45 34.0 34.4
57 F Green 3 3 10 20 25 30 17.6 18.8
58 D Pink 3 7 15 15 20 20 15.4 16.2
59 C Green 3 0 0 0 0 0 0.0 0.0
60 B Pink 3 0 1 2 7 7 3.4 3.8
61 A Green 3 10 20 35 45 80 38.0 37.8
62 E Pink 3 0 7 15 35 55 22.4 22.9
63 D Blue 3 15 15 15 25 20 18.0 18.8
64 A Pink 3 25 35 45 55 60 44.0 45.6
65 C Orange 3 0 0 1 5 5 2.2 2.4
66 D Green 3 1 2 2 5 5 3.0 3.2
67 E Green 3 1 5 7 25 25 12.6 13.8
68 F Orange 3 3 5 5 10 7 6.0 6.6
69 C Pink 3 0 2 3 10 15 6.0 6.1
70 B Blue 3 7 10 15 15 15 12.4 13.1
71 E Blue 3 50 70 75 90 95 76.0 78.1
72 E Orange 3 1 7 10 25 30 14.6 15.5
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
17
Pot
number
Treatment Isolate Rep foliar blight (%) stAUDPC
5 7 9 13 15 Average
73 F Orange 4 0 1 2 3 4 2.0 2.1
74 E Blue 4 35 50 70 75 90 64.0 65.9
75 C Pink 4 0 1 1 5 15 4.4 3.9
76 F Pink 4 10 15 15 25 25 18.0 18.8
77 B Blue 4 10 20 25 25 25 21.0 22.2
78 C Blue 4 7 15 20 15 15 14.4 15.3
79 E Orange 4 3 15 15 30 25 17.6 19.4
80 F Green 4 1 4 15 25 25 14.0 15.6
81 B Green 4 0 0 0 0 0 0.0 0.0
82 A Green 4 15 30 45 55 75 44.0 45.3
83 A Blue 4 55 75 70 80 85 73.0 74.1
84 D Blue 4 20 25 25 25 35 26.0 25.6
85 E Green 4 1 1 2 15 20 7.8 8.0
86 F Blue 4 20 20 25 35 40 28.0 28.4
87 C Green 4 0 0 0 0 0 0.0 0.0
88 A Orange 4 35 50 50 70 85 58.0 58.8
89 B Pink 4 1 2 5 7 10 5.0 5.2
90 D Green 4 0 1 1 3 5 2.0 2.0
91 C Orange 4 0 0 1 3 3 1.4 1.6
92 B Orange 4 3 7 10 15 15 10.0 10.8
93 D Pink 4 3 20 20 20 25 17.6 18.5
94 E Pink 4 10 25 20 40 65 32.0 31.6
95 A Pink 4 30 40 50 75 80 55.0 57.2
96 D Orange 4 2 7 7 10 7 6.6 7.3
© Praktijkonderzoek Plant & Omgeving
(Applied Plant Research)
18
Appendix 2. PD-recognition

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Testing the efficacy of fungicides to control different strains of Phytophthora infestans

  • 1. CONFIDENTIAL A. Evenhuis & H.T.A.M. Schepers Testing the efficacy of fungicides to control different strains of Phytophthora infestans Praktijkonderzoek Plant & Omgeving (Applied Plant Research), part of Wageningen UR Business Unit AGV PPO no. 3250294400 May 2014
  • 2. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 2 © 2014 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: 3250294400 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
  • 3. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 3 Table of contents page 1 INTRODUCTION ............................................................................................................................ 5 2 MATERIALS & METHODS............................................................................................................... 6 2.1 Potato plants......................................................................................................................... 6 2.2 Spraying ............................................................................................................................... 6 2.3 Treatments ........................................................................................................................... 6 2.4 Inoculation P. infestans........................................................................................................... 6 2.5 Disease observations P. infestans.......................................................................................... 7 2.6 Statistical analyses ................................................................................................................ 7 3 RESULTS ..................................................................................................................................... 8 4 DISCUSSION AND CONCLUSIONS................................................................................................ 13 APPENDIX 1 DISEASE OBSERVATIONS (RAW DATA EXP. 1)................................................................... 14 APPENDIX 2. PD-RECOGNITION........................................................................................................... 18
  • 4. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 4
  • 5. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 5 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 virulence’s, 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 this report the efficacy of mancozeb, fluazinam and metalaxyl-m against four P. infestans isolates representing different clonal lineages was tested in a greenhouse experiment by request of United Phosphorus Ltd.
  • 6. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 6 2 Materials & methods 2.1 Potato plants 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 6 March 2014. From emergence until the experiment, the plants were placed in the greenhouse in Lelystad. 2.2 Spraying The potato plants were sprayed with the different fungicides in a spraying cabin developed by Applied Plant Research (PPO) on 9 April. 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. 2.3 Treatments In Table 1 the fungicides used and dose rates are presented. The untreated control was sprayed with tap water. The experiment was sprayed on 9 April 2014. For each experiment the spray solutions were prepared separately. Table 1. The fungicides used in the experiments and the applied dose rates (2014) Code Fungicide Dose rate l or kg per ha A water (untreated control) - B mancozeb 75% WG 2.0 C mancozeb 64% + metalaxyl-m 8% 2.5 D mancozeb + Shirlan 2.0 + 0.4 E metalaxyl-m 25% WG 0.8 F fluazinam 500 g/l SC (Shirlan) 0.4 2.4 Inoculation P. infestans The four P. infestans isolates used were of different clonal lineages, known as Blue 13 (NL07041), Green 33 (NL10328), Pink 6 (NL11179) and Orange (NL12079) genotype. The Blue 13 isolate is resistant to metalaxyl-m. 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 potato plants was carried out by spraying the leaves over head with inoculum on 15 April 2014, six days after spraying the fungicides. After inoculation the potato plants were incubated in a climate chamber for one week.
  • 7. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 7 2.5 Disease observations Late blight disease severity was assessed five times after inoculation. Percentage necrotic foliage was estimated. From the individual disease ratings a Standard Area Under Disease Progress Curve (stAUDPC) was calculated. 2.6 Statistical analyses The experiment was carried out with four replications. Each replication consisted of one potato plant. Analysis of variance on the late blight assessments was made based on the percentage necrotic foliage and sporulation using GENSTAT 16th 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.
  • 8. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 8 3 Results The late blight disease severity expressed as the percentage necrotic foliage is presented in Table 2 and Table 3. The efficacy to control potato late blight depended on the fungicide used and was also influenced by the P. infestans isolate. Almost all fungicide treatments reduced the percentage necrotic foliage compared to the untreated control, regardless of the used P. infestans isolate. Only applying metalaxyl-m (E) did not show a significant difference in the percentage of necrotic foliage compared to the untreated control after inoculation with Blue 13. Table 2. Percentage necrotic foliage per treatment 7, 9 and 10 days past inoculation (d.p.i.) with 4 different P. infestans isolates. Genotype Code disease severity (%) 7 d.p.i 9 d.p.i 10 d.p.i Blue 13 A 48.8 l 67.5 k 65.0 j B 14.3 fgh 18.8 defg 23.8 h C 11.0 defg 18.8 defg 20.0 fgh D 21.3 ij 22.5 fgh 23.8 h E 48.8 l 71.3 k 77.5 k F 20.0 hij 23.8 gh 27.5 h Green 33 A 15.0 ghi 28.8 hi 40.0 i B 1.8 abc 4.3 ab 6.8 abcd C 0.0 a 0.3 a 0.3 a D 2.3 abc 8.3 abc 13.3 cdef E 0.8 ab 4.0 ab 7.3 abcd F 4.8 abcd 12.3 bcde 22.5 gh Pink 6 A 26.3 j 37.5 ij 48.8 i B 4.8 abcd 5.8 ab 9.8 bcd C 0.0 a 2.3 a 2.5 ab D 4.3 abc 13.3 bcdef 14.5 defg E 8.0 cdef 18.0 defg 18.8 efgh F 12.5 efg 20.0 efgh 27.5 h Orange A 33.8 k 45.0 j 47.5 i B 7.0 bcde 15.5 cdefg 18.8 efgh C 0.0 a 0.3 a 0.8 a D 1.3 ab 5.5 ab 5.5 abc E 1.5 abc 6.5 abc 9.8 bcd F 4.8 abcd 9.5 abcd 10.5 bcde 1) Values followed by the same letter are not significantly different (p=0.05). In Table 3 disease severity 13 and 15 days past inoculation is given and the stAUDPC is presented. The stAUDPC is a weighted average of the individual disease ratings.
  • 9. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 9 Table 3. Percentage necrotic foliage per treatment 13 and 15 days past inoculation (d.p.i.) with 4 different P. infestans isolates and stAUDPC. Genotype Code disease severity (%) stAUDPC 13 d.p.i 15 d.p.i Blue 13 A 71.3 j 78.75 ij 67.1 j B 27.5 efgh 28.75 defg 23.4 efg C 23.8 defg 26.25 cdefg 20.6 defg D 30.0 fgh 32.5 efg 26.3 fg E 85.0 k 91.25 j 76.8 k F 35.0 gh 37.5 g 29.5 g Green 33 A 53.8 i 81.25 ij 44.2 h B 16.3 bcde 16.75 bcd 9.9 ab C 2.5 a 2.5 a 1.2 a D 19.5 cdef 21.25 cde 13.9 bcd E 23.8 defg 26.25 cdefg 13.4 bcd F 28.8 efgh 36.25 fg 22.0 defg Pink 6 A 66.3 ij 71.25 i 52.1 hi B 12.3 abcd 15.5 abcd 9.9 ab C 8.5 abc 15.5 abcd 5.6 ab D 18.0 cdef 21.25 cde 14.9 bcde E 38.8 h 53.75 h 27.9 fg F 26.3 efgh 36.25 fg 24.9 fg Orange A 63.8 ij 78.75 ij 54.3 i B 25.0 efg 25 cdefg 19.4 cdef C 4.5 ab 4.5 ab 2.2 a D 8.8 abc 7.25 ab 6.2 ab E 23.8 defg 22.5 cdef 14.1 bcde F 12.0 abcd 14 abc 10.5 abc 1) Values followed by the same letter are not significantly different (p=0.05). Table 4 gives the efficacy of the treatments to control potato late blight average over all genotypes. However it must be kept in mind that not all genotypes are equally well controlled by the same fungicide (Tables 1 to 3). Table 4. Percentage necrotic foliage, expressed as stAUDPC, per treatment after inoculation with 4 different P. infestans isolates, including the untreated control.. Genotype Code stAUDPC All water (untreated control) 54.4 e mancozeb 75% WG 15.6 b mancozeb 64% + metalaxyl-m 8% 7.4 a mancozeb + Shirlan 15.3 b metalaxyl-m 25% WG 33.0 d fluazinam 500 g/l SC (Shirlan) 21.7 c
  • 10. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 10 Figure 1 represents the efficacy of different treatments to control P. infestans isolates belonging to different clonal lineages. Figure 2 to 5 present the disease severity of P. infestans in time of the different clonal lineages. Figure 1. Percentage necrotic foliage per treatment expressed as stAUDPC after inoculation with 4 different P. infestans isolates, representing large clonal lineages in Europe.
  • 11. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 11 Figure 2. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate, representing clonal lineage Blue13 Figure 3. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate, representing clonal lineage Green33
  • 12. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 12 Figure 4. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate, representing clonal lineage Pink6 Figure 5. Percentage necrotic foliage per treatment after inoculation with a P. infestans isolate, representing clonal lineage Orange
  • 13. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 13 4 Discussion and conclusions The infection level of untreated plants was 70-85% at the end of the experiment 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 compared to the untreated control, regardless the used P. infestans isolate, except for metalaxyl-m when potato plants were inoculated with Blue13. Conclusions P. infestans: Isolates • Overal the level of necrotic foliage was higher in the treated potato plants 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. • In this experiment on average disease severity was higher when potato plants were inoculated with Pink 6 compared to Green 33 and Orange. • The absence of control of the Blue 13 isolate by metalaxyl-m confirms that this isolate is rated as a metalaxyl-resistant isolate. The efficacy of all other treatments to contol Blue13 was better than metalaxyl-m. • The efficacy of mancozeb (B) to control the Blue 13 isolate was significantly better than the efficacy of fluazinam (F) and metalaxyl-m (E). • The Green 33 isolate was significantly better controlled with mancozeb + metalaxyl-m (C) than with mancozeb + fluazinam (D), metalaxyl-m (E) and fluazinam (F). • The Green 33 isolate was less effectively controlled by fluazinam (F) compared to mancozeb (B) and manoczeb + metalaxyl-m (C). No singificant differences were observed between the other treatments. • The Pink 6 isolate was singinficantly better controled by mancozeb (B), mancozeb + metalaxyl-m (C) and manczeb + fluazinam (D) compared to application of fluazinam (F) or metalaxyl-m (E). • The Orange isolate was significantly better controlled with mancozeb + metalaxyl-m (C) than mancozeb (b) or metalaxyl-m. • The efficacy of mancozeb + fluazinam (D) to control the orange isolate was significantly better than mancozeb (B). Treatments • The efficacy of mancozeb + metalaxyl-m to control P. infestans was significantly better than all other treatments. • The efficacy of mancozeb (B) and mancozeb + fluazinam (D) was comparable and significantly better than the efficacy of fluazinam and metalaxyl-m. This is mainly caused by the fact that Green 33 is less effectively controlled by fluazinam and Blue13 is not controlled by metalaxyl-m. • The efficacy of fluazinam to control potato late blight was significantly better than metalaxyl-m.
  • 14. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 14 Appendix 1 Disease observations (raw data Exp. 1) Pot Treat- ment Isolate Rep foliar blight (%) stAUDPC 7 9 10 13 15 Average 1 A Blue 1 40 60 65 65 70 60.0 61.6 2 E Orange 1 2 3 10 20 20 11.0 12.1 3 E Pink 1 15 25 25 55 60 36.0 37.5 4 E Blue 1 55 90 95 95 95 86.0 89.1 5 C Blue 1 7 15 15 20 25 16.4 16.8 6 D Orange 1 3 15 15 25 20 15.6 17.3 7 D Green 1 3 10 25 35 30 20.6 23.2 8 C Green 1 0 1 1 10 10 4.4 4.8 9 E Green 1 0 7 15 30 35 17.4 18.8 10 A Orange 1 35 50 55 65 80 57.0 57.8 11 A Green 1 20 40 45 70 95 54.0 55.0 12 F Orange 1 1 7 10 15 15 9.6 10.5 13 C Pink 1 0 5 5 15 25 10.0 10.0 14 D Pink 1 7 15 20 25 25 18.4 19.6 15 D Blue 1 25 25 25 35 35 29.0 29.4 16 A Pink 1 25 40 50 60 65 48.0 50.0 17 B Green 1 0 0 2 5 7 2.8 2.9 18 C Orange 1 0 1 1 10 10 4.4 4.8 19 F Blue 1 20 25 30 30 35 28.0 28.4 20 F Pink 1 15 25 35 20 40 27.0 26.6 21 F Green 1 5 15 25 25 35 21.0 21.9 22 B Pink 1 15 15 25 25 30 22.0 22.5 23 B Orange 1 5 15 25 25 25 19.0 20.6 24 B Blue 1 30 30 30 35 45 34.0 33.4
  • 15. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 15 Pot number Treatment Isolate Rep foliar blight (%) stAUDPC 5 7 9 13 15 Average 25 B Blue 2 10 15 25 35 30 23.0 25.0 26 C Orange 2 0 0 0 0 0 0.0 0.0 27 A Green 2 15 25 35 45 75 39.0 38.8 28 A Orange 2 30 40 45 65 85 53.0 53.4 29 C Green 2 0 0 0 0 0 0.0 0.0 30 E Blue 2 55 75 70 80 85 73.0 74.1 31 F Blue 2 15 20 25 35 30 25.0 26.6 32 F Pink 2 15 25 35 45 55 35.0 36.3 33 D Green 2 5 20 25 35 45 26.0 27.2 34 A Pink 2 25 35 50 75 80 53.0 55.6 35 A Blue 2 55 70 65 70 80 68.0 68.1 36 D Orange 2 0 0 0 0 2 0.4 0.3 37 F Green 2 10 20 30 40 55 31.0 31.9 38 F Orange 2 15 25 25 20 30 23.0 22.8 39 D Pink 2 0 3 3 7 15 5.6 5.4 40 B Green 2 2 7 10 35 30 16.8 18.8 41 B Orange 2 15 25 25 35 40 28.0 28.8 42 E Green 2 1 3 5 25 25 11.8 12.9 43 E Pink 2 7 15 15 25 35 19.4 19.6 44 B Pink 2 3 5 7 10 15 8.0 8.1 45 D Blue 2 25 25 30 35 40 31.0 31.3 46 E Orange 2 0 1 4 20 15 8.0 9.3 47 C Blue 2 15 20 20 25 30 22.0 22.2 48 C Pink 2 0 1 1 4 7 2.6 2.6
  • 16. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 16 Pot number Treatment Isolate Rep foliar blight (%) stAUDPC 5 7 9 13 15 Average 49 A Orange 3 35 40 40 55 65 47.0 47.2 50 D Orange 3 0 0 0 0 0 0.0 0.0 51 F Pink 3 10 15 25 15 25 18.0 18.1 52 C Blue 3 15 25 25 35 35 27.0 28.1 53 B Green 3 5 10 15 25 30 17.0 17.8 54 A Blue 3 45 65 60 70 80 64.0 64.7 55 B Orange 3 5 15 15 25 20 16.0 17.5 56 F Blue 3 25 30 30 40 45 34.0 34.4 57 F Green 3 3 10 20 25 30 17.6 18.8 58 D Pink 3 7 15 15 20 20 15.4 16.2 59 C Green 3 0 0 0 0 0 0.0 0.0 60 B Pink 3 0 1 2 7 7 3.4 3.8 61 A Green 3 10 20 35 45 80 38.0 37.8 62 E Pink 3 0 7 15 35 55 22.4 22.9 63 D Blue 3 15 15 15 25 20 18.0 18.8 64 A Pink 3 25 35 45 55 60 44.0 45.6 65 C Orange 3 0 0 1 5 5 2.2 2.4 66 D Green 3 1 2 2 5 5 3.0 3.2 67 E Green 3 1 5 7 25 25 12.6 13.8 68 F Orange 3 3 5 5 10 7 6.0 6.6 69 C Pink 3 0 2 3 10 15 6.0 6.1 70 B Blue 3 7 10 15 15 15 12.4 13.1 71 E Blue 3 50 70 75 90 95 76.0 78.1 72 E Orange 3 1 7 10 25 30 14.6 15.5
  • 17. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 17 Pot number Treatment Isolate Rep foliar blight (%) stAUDPC 5 7 9 13 15 Average 73 F Orange 4 0 1 2 3 4 2.0 2.1 74 E Blue 4 35 50 70 75 90 64.0 65.9 75 C Pink 4 0 1 1 5 15 4.4 3.9 76 F Pink 4 10 15 15 25 25 18.0 18.8 77 B Blue 4 10 20 25 25 25 21.0 22.2 78 C Blue 4 7 15 20 15 15 14.4 15.3 79 E Orange 4 3 15 15 30 25 17.6 19.4 80 F Green 4 1 4 15 25 25 14.0 15.6 81 B Green 4 0 0 0 0 0 0.0 0.0 82 A Green 4 15 30 45 55 75 44.0 45.3 83 A Blue 4 55 75 70 80 85 73.0 74.1 84 D Blue 4 20 25 25 25 35 26.0 25.6 85 E Green 4 1 1 2 15 20 7.8 8.0 86 F Blue 4 20 20 25 35 40 28.0 28.4 87 C Green 4 0 0 0 0 0 0.0 0.0 88 A Orange 4 35 50 50 70 85 58.0 58.8 89 B Pink 4 1 2 5 7 10 5.0 5.2 90 D Green 4 0 1 1 3 5 2.0 2.0 91 C Orange 4 0 0 1 3 3 1.4 1.6 92 B Orange 4 3 7 10 15 15 10.0 10.8 93 D Pink 4 3 20 20 20 25 17.6 18.5 94 E Pink 4 10 25 20 40 65 32.0 31.6 95 A Pink 4 30 40 50 75 80 55.0 57.2 96 D Orange 4 2 7 7 10 7 6.6 7.3
  • 18. © Praktijkonderzoek Plant & Omgeving (Applied Plant Research) 18 Appendix 2. PD-recognition