Early Blight is caused by Alternaria solani. Although late blight, caused by Phytophthora infestans, is the most important disease in potato production Early Blight is regularly found in potato fields in the second half of the season. Farmers spray potatoes 2 to 4 times during a season to control Early Blight. Specific fungicides most commonly used in the Netherlands are Signum, Amistar and recently also Narita and Carial Star.
Efficacy of fungicides to control Early Blight genotypes
1. Efficacy of fungicides to control Early
Blight genotypes
Field experiment AGV7205
A. Evenhuis & H.T.A.M. Schepers
This study was carried out by Wageningen University & Research and was commissioned and financed by UPL
Europe Ltd
Wageningen Plant Research is the collaboration of Wageningen University and the foundation Stichting
Wageningen Research.
Wageningen, November 2016
CONFIDENTIAL
Report 3750331500
3. Contents
Contents 3
1 Introduction 5
2 Methods and materials 6
2.1 Lay out 6
2.2 Treatments 6
2.3 Inoculation 6
2.4 Disease observations 7
2.5 Sampling and genotyping 7
2.6 Statistics 7
3 Results 8
3.1 Field assessments 8
3.3 Genotyping 10
4 Discussion and conclusions 11
4.1 Early Blight 11
4.2 Soil coverage 11
4.3 Genotyping 11
4.4 Conclusions 12
Experimental lay-out 13Annex 1
nVWA recognition 15Annex 2
Raw data disease severity (%) 16Annex 3
Raw data soil coverage (%) 17Annex 4
Raw data genotyping 18Annex 5
Weather data 19Annex 6
4.
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1 Introduction
Early Blight is caused by Alternaria solani. Although late blight, caused by Phytophthora infestans, is
the most important disease in potato production Early Blight is regularly found in potato fields in the
second half of the season. Farmers spray potatoes 2 to 4 times during a season to control Early Blight.
Specific fungicides most commonly used in the Netherlands are Signum, Amistar and recently also
Narita and Carial Star. Furthermore a number of fungicides used to control potato late blight show also
some efficacy to control Early Blight. Especially products based on mancozeb can be mentioned here.
Resistance of A. solani to azoxystrobin has been reported in the United States (Pasche and
Gudmestad, 2008). This resistance is associated with the F129L mutation. This mutation has also been
reported in Germany (Leiminger et al., 2014), and one isolate with the F129L mutation has been
found in the Netherlands (Evenhuis, et al., 2013). Until now no obvious lack of control of azoxystrobin
has been found in agricultural practise (Leiminger and Hausladen, 2013). However in a field
experiment conducted in Valthermond in the Netherlands in 2014 the efficacy of Amistar and Signum
to control Early Blight was less than expected. It is not known what caused this effect. Fungicide
resistance management of A. solani was not investigated so far in the Netherlands.
In this experiment the efficacy of fungicides to control a mixture of A. solani isolates in a field
experiment was tested by request of UPL.
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2 Methods and materials
2.1 Lay out
The experiment was set up in a randomised block design. Details are described in Appendix 1.
2.2 Treatments
Fungicide applications were carried out using a trial site sprayer with Airmix 110.04 nozzles. Nozzles
were hanging approximately 50 cm above the foliage. Sprayings were carried out based on 300 l/ha.
Potato plants were sprayed for the first time when they reached a height of 20-30 cm when the rapid
growth started. Specific sprays to control Early Blight started Mid-July. Haulm killing was carried out
on 30 September 2016, despite natural senescence of the crop. In Table 1 the fungicides used and
dose rates are presented.
Table 1 The fungicides used in the 2016 experiment and the applied dose rates.
Fungicide Active ingredient Dose rate
L or kg /ha
Narita difenoconazole 250 g/l 0.5
Signum pyraclostrobin 6.7% + boscalid 26.7% 0.2
Penncozeb 80 WP mancozeb 800 g /kg 2.0
Treatment A is the untreated control and H the reference treatment chosen. Spray strategies are
given in Table 2.
Table 2 Spray strategies and date of application.
Code Fungicide 12-7 19-7 26-7 2-8 9-8 16-8 23-8
A UTC - - - - - - -
B Signum 0.2 S - S - S - S
C Penncozeb 80 WP 2.0 P P P P P P P
D Penncozeb 80 WP 2.0 + Signum 0.2 P+S P P+S P P+S P P+S
E Narita 0.5 N - N - N - N
H Signum 0.2 or Narita 0.5 S - S - N - N
2.3 Inoculation
A selection of 2 A. solani isolates was made. AltNL03003 was isolated in 2003 and belongs to
genotype 1 and is wild type. AltNL15002 belongs to genotype 2 and possesses the F129L mutation.
Both isolates were grown on wheat kernels separately. A mixture of 95% wildtype and 5% F129L type
was inoculated. Infested kernels were broadcasted in the field on 15 July 2016.
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2.4 Disease observations
Disease observations were carried out from mid-July. Depending on the disease development disease
observations were carried out with an interval of 1 to 2 weeks. The percentage necrotic foliage due to
A. solani per plot was estimated.
2.5 Sampling and genotyping
To assess Alternaria genotype and the presence of the F129L mutation leaves with Alternaria lesions
were collected. Per plot 8 leaflets with Alternaria lesions were sampled. Samples were taken four times
during the season in the last week of August and the first three weeks of September. Only the last
sample was genotyped. The samples were air dried and stored in Petri dishes until further processing.
At the laboratory monospore cultures were made on agar. Mycelium was scraped from the agar plate
after incubation. DNA was extracted from the mycelium. The genotype involved was assessed by
carrying out two PCR described by Pasche et al., 2004 and Lieminger et al., 2015. The PCR product
was extracted from the gel and sequenced. The nucleotide order was assessed and the presence of
the F129L mutation was established according to the publication of Lieminger et al., 2015.
2.6 Statistics
Analysis of variance on the parameters was made using GENSTAT 18th
Edition. The experiment was
carried out with four replications in a randomised block design. Each replication consisted of a plot.
Transformation of data was carried out when necessary.
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3 Results
3.1 Field assessments
The results are given in Table 3 and Table 4. On 22 July 2016 no Early Blight was found. A week later
on 29 July Early Blight was present in all plots at a low disease severity rate of 0.001% (data not
shown). From 5 August onwards the Early Blight epidemic started (Figure 1). After 17 September it
was not possible to assess Early Blight because it was too much entangled with natural senescence to
distinguish.
Phytotoxicity of the fungicides used was not observed. In this experiment the fungicides used were
crop safe.
Disease severity of all spray strategies was significantly lower than the untreated control. Based on
AUDPC, the efficacy of treatments D, E and H were significantly better than treatments B and C.
Table 3. Effect of fungicide spray treatments on Early Blight disease severity (%). Log 10 back
transformed Early Blight disease severity ratings.
Code 5-8 12-8 19-8 26-8 2-9 9-9 16-9 AUDPC
A 0.001 a 0.29 c 0.7 c 4.2 c 8.7 d 19.7 c 18.2 c 330 c
B 0.001 a 0.00 a 0.0 a 0.2 b 1.2 c 3.0 b 14.1 c 86 b
C 0.001 a 0.07 bc 0.0 ab 0.1 b 0.9 c 1.9 b 10.3 bc 68 b
D 0.001 a 0.03 abc 0.0 ab 0.0 a 0.0 a 0.1 a 5.1 b 21 a
E 0.001 a 0.02 abc 0.0 bc 0.1 b 0.2 b 0.2 a 1.2 a 12 a
H 0.001 a 0.01 ab 0.0 ab 0.1 b 0.1 b 0.2 a 1.5 a 10 a
1
) Values in columns followed by the same letter are not significantly different (P=0.05).
Figure 1 Early Blight disease severity (%)
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On average soil coverage (%) of treatments B to H was significantly higher than the untreated control
(A). No further differences between average soil cover were found.
Table 4 Effect of spray treatments on soil coverage (%).
Code 2-9 9-9 16-9 2
23-9 2
Average
A 43 a1
21 a 3 a 1.0 a 17 a
B 73 b 53 b 17 b 2.1 ab 37 b
C 71 b 46 b 18 b 4.1 bc 36 b
D 74 b 54 b 21 b 5.7 c 39 b
E 61 b 43 ab 13 b 4.8 bc 31 b
H 70 b 53 b 15 b 6.2 c 37 b
1
) Values in columns followed by the same letter are not significantly different (P=0.05).
2
) Values on 16 & 23-9 are logit back transformed data.
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3.3 Genotyping
The results are presented in Table 5 and Figure 2. No significant shift of the Alternaria solani genotype
was found when Penncozeb was used compared to the untreated control. When Signum (B) or Signum
followed by Narita (H) was sprayed, significantly more GII F129L types were found compared to the
untreated control.
Table 5 Effect of spray treatments on frequency of Alternaria genotypes.
Code Strategy GI WT GI F129L GII WT GII F129L
A UTC 56 b 0 0 a 44 a
B Signum 7 a 0 3 b 90 b
C Penncozeb 52 b 0 0 a 48 a
H Signum / Narita 13 a 0 0 a 86 b
1
) Values in columns followed by the same letter are not significantly different (P=0.05).
Figure 2 Alternaria solani genotype distribution (%)
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4 Discussion and conclusions
A field experiment was carried out in cultivar Agria. The Early Blight epidemic developed late despite
artificial inoculation. The weather in June was conducive for potato late blight and not so much for
Early Blight. Potato late blight was controlled with cover sprays using Revus 0.6 l/ha, Infinito 1.6 l/ha
and Ranman Top 0.5 l/ha. These fungicides have no known efficacy to control Early Blight.
Besides Alternaria spp. also Sclerotinia was found in the experiment. Sclerotinia severity was not
assessed. Nevertheless some treatments probably controlled Sclerotinia which together with Alternaria
and senescence account for soil coverage.
4.1 Early Blight
Early Blight disease severity in the untreated control was significantly higher than all other treatments
indicating that the inoculation was successful. Furthermore all spray strategies effectively controlled
Early Blight, although the efficacy varied between strategies.
Disease severity of treatment B and C increased more towards the end of the season compared to
treatments D, E and H. The last spray application was 23 August and the increase was observed from
9 September onwards. Interestingly when Penncozeb was added to Signum (or v.v.) the efficacy to
control Early Blight increased compared to the products used solo. However the efficacy to control
Early Blight was still less than using Narita or the combination Signum followed by Narita.
4.2 Soil coverage
Average soil coverage of all spray strategies was comparable and significantly higher than the
untreated control. On individual assessment data some minor differences between treatments
occurred.
4.3 Genotyping
The field was inoculated with wheat kernels with 2 Alternaria genotypes. Genotype I (GI) is wild type
and Genotype II (GII) possesses the F129L mutation. Predominantly these two genotypes were found
in the field. Only 1 isolate found was Genotype II, wild type. No Genotype I isolates were found
possessing the F129L mutation. In the untreated control 57% of the isolates were Genotype I and wild
type. The experiment was inoculated with 95% Genotype I wildtype. This could suggest that the
F129L mutation was already present in the natural A. solani population at the location of the field
experiment. Alternatively GII might be more aggressive than GI and therefore was found back more
frequently.
Leaves were picked at four times during the season. Alternaria solani was isolated only from the
leaves picked at the last sampling date. Leaves of the other sampling dates were stored under dry
conditions at room temperature and could be used for additional assessments.
When Signum or Signum followed by Narita was sprayed, significantly more GII F129L A. solani types
were found than in the untreated control and when Penncozeb was sprayed. This suggests that
selection towards GII F129L occurred under influence of spray strategies B and H. It is known that the
F129L mutation causes a reduced sensitivity to QoI fungicides. One of the active ingredients of
Signum is pyraclostrobin, a QoI fungicide.
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4.4 Conclusions
Phytotoxicity of the fungicides applied was not observed in this experiment. The used products are
crop safe.
Alternaria disease severity based on AUDPC was significantly higher in the untreated than all other
treatments.
Based on AUDPC, the efficacy of treatments D, E and H were significantly better than treatments
B and C.
On average soil coverage (%) of treatments B to H was significantly higher than the untreated
control.
No significant shift of the Alternaria solani genotype was found when Penncozeb was used
compared to the untreated control.
When Signum or Signum followed by Narita was sprayed, significantly more GII F129L types were
found compared to the untreated control.
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Experimental lay-outAnnex 1
Site Lelystad, The Netherlands
Soil texture Sandy loam
Previous crop onion
Pre previous crop
Fertilization 500 kg KAS on 17 May and 300 kg KAS on 22 June
Variety: Agria
Planting date: 3 May 2016
Seed Rate: 2.500 kg/ha
Herbicide treatment: 24 May
Fungicide application: see paragraph 2 and normal practice
Alternaria treatments: see paragraph 2 and normal practice
Crop desiccation: Reglone (diquat dibromide 200g/l) 2.0 l/ha 30 September 2016.
Harvest: -
Tuber assessments: -
Gross plot dimensions: Four rows (0.75 m) of 11 m length
Net plot dimensions: Two rows (0.75) of 11 m length
Demo design: Four replications in a randomized block design
Lelystad
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Project:
Experiment: AGV7205 Alternaria genotypes
Location: Lelystad
Field: G86-4
8 16 24 32
C F G D
7 15 23 31
D A B G
6 14 22 30
H B E F
5 13 21 29
A H C B
4 12 20 28
E G D C
3 11 19 27
B C H E
2 10 18 26
G D F A
1 9 17 25
F E A H
1.5 < 3m > < 3m > < 3m > < 3m > < 3m > < 3m > 1.5
3750331500
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I II III IV
26. Corresponding address for this report:
P.O. Box 16
6700 AA Wageningen
The Netherlands
T +31 (0)317 48 07 00
www.wur.nl/plant-research
Confidential Wageningen Plant Research
Report 3750331500
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