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.

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CONFIDENTIAL
A. Evenhuis, M.G. Förch, G.B.M. van den Bosch, C. van Tongeren, W. van den Berg
& H.T.A.M. Schepers
Testing the efficacy of fungicides on spore
germination of Alternaria solani
Praktijkonderzoek Plant & Omgeving (Applied Plant Research), part of Wageningen UR
Business Unit AGV PPO no.
37503111700
October, 2015

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Project no PPO: 3750311700
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 Treatments ........................................................................................................................... 6
2.2 Alternaria solani..................................................................................................................... 6
2.3 Germination assessment of A. solani....................................................................................... 6
2.4 Statistical analyses ................................................................................................................ 6
3 RESULTS ..................................................................................................................................... 7
4 DISCUSSION AND CONCLUSIONS................................................................................................ 13
APPENDIX 1 DISEASE OBSERVATIONS (RAW DATA).............................................................................. 15
APPENDIX 2. PD-RECOGNITION........................................................................................................... 17

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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.
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 Gudmested,
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 2014 in the Netherlands the efficacy of Amistar and Signum to control early blight was less
than expected. It is not known what caused this effect. Fungicide resistance of A. solani was not
investigated so far in the Netherlands.
A. solani isolates were collected in fields and experiment in a period from 2000 to 2014. These isolates
were stored in liquid nitrogen at Plant Research International part of Wageningen UR. In a laboratory
experiment the efficacy of Amistar, Signum and Penncozeb to control 15 isolates of Alternaria solani was
tested, by request of UPL.

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2 Materials & methods
2.1 Treatments
In Table 1 the fungicides used and active ingredients are presented.
Table 1. The fungicides used in the experiment.
Code Fungicide Active ingredient
A Amistar azoxystrobin 250 g/l
B Signum boscalid 26.7% + pyraclostrobin 6.7%
C Penncozeb mancozeb 75%
To establish the EC50 value a dose rate series was made. The efficacy of fungicides was tested at 0.01,
0.1, 1, 10 and 100 ppm, except for Penncozeb, which was not tested at the lowest dose rate 0.01 ppm
but at 1000 ppm instead. The ppm values were adjusted to the dose rate of the active ingredients. In the
case of Signum the dose rate of pyraclostobin and boscalid was added up. The fungicides in the proper
dose rate were added to cooling Water Agar and poured into Petri dishes (Figure 1). To Amistar and Signum
100 mg / l SHAM dissolved in methanol was added, regardless of the dose rate tested. SHAM is a known
inhibitor of the alternative oxidase (AOX) pathway that has been suggested as a possible mode of QoI
resistance in vitro in other fungi, therefor it was amended to the medium.
2.2 Alternaria solani
A selection of 15 A. solani isolates was made. Isolates were collected between 2006 and 2014.
The inoculum density was set at approximately 10,000 sporangia per ml. The spore suspension was
sprayed on the agar plates containing the fungicides. The plates were incubated for 6 hours at room
temperature (20°C) under day light conditions. After warm incubation the plates were transferred to a cool
chamber at 4°C in the dark until germination assessment.
2.3 Germination assessment of A. solani
The germination rate of A. solani was established by counting the number of germinated and not
germinated spores under a light microscope. The spores were considered germinated when the length of
the spore tube was at least the same as the diameter of the spore. The percentage germination was
calculated by division of the number of germinated spores with the total number of spores counted times
100.
2.4 Statistical analyses
The experiments were carried out with two replications. Each replication consisted of one Petri dish
containing spores of a certain A. solani isolate. Fungicide sensitivity was measured as the concentration at
which spore germination was inhibited by 50% relative to the untreated control (EC50 value) and was
determined for each isolate. Analysis of variance on Log10 (EC50) was made using GENSTAT 17th
Edition.

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3 Results
This experiment was designed to establish the EC50 values of fungicides to control different A. solani
isolates. The results are presented in Table 2 and Figure 1, Figure 2 and Figure 3.
Table 2. EC50 values of 3 fungicides for germination rate of 15 A. solani isolates.
Isolate Amistar Penncozeb Signum
Log
EC50
EC50 Log
EC50
EC50 Log
EC50
EC50
AltNL14019 2.54 .........j1
345.92 0.62 ......g...
4.1 0.87 ......gh..
7.44
AltNL14013 1.99 ........ij
98.2 0.61 ......g...
4.1 0.76 ......g...
5.71
AltNL14014 1.58 .......hi.
38.2 0.57 ......g...
3.7 0.63 ......g...
4.26
AltNL14027 0.71 ......g...
5.2 0.55 ......g...
3.6 -0.23 ..cdef....
0.59
AltNL0634 0.47 .....fg...
2.9 0.55 ......g...
3.6 -0.34 .bcde.....
0.45
AltNL08012 -0.21 ...def....
0.61 0.53 .....fg...
3.3 -0.39 .bcd......
0.41
AltNL09014 -0.36 .bcde.....
0.44 0.50 .....fg...
3.2 -0.39 .bcd......
0.40
AltNL08001 -0.46 .bcd......
0.35 0.50 .....fg...
3.2 -0.47 .bcd......
0.34
AltNL09015 -0.47 .bcd......
0.34 0.49 .....fg...
3.1 -0.49 abcd......
0.33
AltNL14001 -0.62 abcd......
0.24 0.48 .....fg...
3.0 -0.51 abcd......
0.31
AltNL14015 -0.67 abcd......
0.21 0.45 .....fg...
2.8 -0.60 abcd......
0.25
AltNL09011 -0.69 abcd......
0.20 0.44 .....fg...
2.8 -0.68 abcd......
0.21
AltNL0629 -0.75 abcd......
0.18 0.43 .....fg...
2.7 -0.93 abcd......
0.12
AltNL0635 -0.93 abcd......
0.12 0.42 .....fg...
2.6 -1.06 ab........
0.09
AltNL11002 -0.98 abc.......
0.11 0.38 ....efg...
2.4 -1.23 a.........
0.06
1
) Values followed by the same letter are not significantly different (p=0.05).
2
) EC-50 value obtained by extrapolation of the fitted dose response curve.

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Figure 1. Dose response effect of Amistar to control 15 isolates of A. solani. The untreated control was
placed at 0.0001 ppm.

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Figure 2. Dose response effect of Signum to control 15 isolates of A. solani. The untreated control was
placed at 0.0001 ppm.

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Figure 3. Dose response effect of Penncozeb to control 15 isolates of A. solani. The untreated control was
placed at 0.001 ppm.
The EC50 values for Amistar and Signum for A. solani collected in different years is given in Figure 4. The
EC50 values for Penncozeb for A. solani collected in different years are given in Figure 5. There is a strong
correlation for EC-50 values for spore germination rate of A. solani isolates under influence of different
fungicides (Figure 6).

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Figure 4. The EC50 value of Amistar and Signum of 15 isolates of A. solani, in order of sampling year.
Figure 5. The EC50 value of Penncozeb of 15 isolates of A. solani, in order of sampling year.

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Figure 6. Correlation between EC-50 values for spore germination of Amistar, Penncozeb and Signum

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4 Discussion and conclusions
Potato is a major crop in Europe. One of the most devastating diseases is caused by P. infestans. In the
second part of the season early blight occurs. Usually this disease is controlled by spraying fungicides.
Strobilurines, such as azoxystrobin (in Amistar) and pyraclostrobin (active ingredient of Signum next to
boscalid) are commonly used. It is known that fungicide resistance to strobilurines can occur. In the USA a
lack of efficacy to control early blight by strobilurine based fungicides has been reported (Pasche et al.,
2004). In Europe a lack of efficacy is not reported, but the F129L mutation thought to be responsible for
fungicide resistance was found (Leiminger et al, 2014). In this study the EC50 values for Amistar, Penncozeb
and Signum of 15 A. solani isolates was determined. The aim of the experiment was to establish whether a
shift in sensitivity to fungicides within the Alternaria population had occurred.
Within the Alternaria isolates tested the sensitivity for Penncozeb among the Alternaria isolates did not
significantly differ. Although the EC50 values for Penncozeb in most cases were higher than for Amistar and
Signum, the EC50 values were very stable for this active ingredient. This indicates that there was no shift in
sensitivity to mancozeb within the Alternaria population tested. Be it that the number of isolates tested was
limited and not necessarily randomly chosen.
For both Amistar and Signum the range of EC50 values was much larger. Based on the logarithmic analysis
significant differences in EC50 values between isolates for both Amistar and Signum were found. In the case
of Amistar AltNL14019, AltNL14013, AltNL14014, AltNL14027 & AltNL0634 had a moderate to high EC50
value. Data from Leiminger et al. (2014) show that in the sensitive population the EC50 value for Amistar
ranged from 0.04 to 0.07 ppm. In the population containing the F129L mutation the range was 0.2 to 0.3
ppm. In our study the EC50 value ranged from 0.11 to 0.61 ppm for isolates not significantly different from
each other and belonging to the susceptible group. Leiminger et al. (2014) used technical azoxystrobin for
their study whereas we used the formulated product. This might explain the small differences between EC50
values found in both experiments. We found much higher EC50 values for azoxystrobin compared to
Leiminger et al. (2014). However since the highest dose rate tested was 100 ppm, the values for
AltNL14013 en AltNL14019 were obtained through extrapolation.
For Signum a moderate EC50 value was found for AltNL14019, AltNL14013 and AltNL14014. Data from
Leiminger et al. (2014) show that the sensitivity for pyraclostrobin ranged from 0.04 to 0.08 ppm. In our
study the EC50 value ranged from 0.06 to 0.59 ppm for isolates not significantly different from each other
and belonging to the susceptible group. Leiminger et al. used technical pyraclostrobin for their study
whereas we used the formulated product Signum, which also contains boscalid. In our experiment both
active ingredients were added to calculate the dose rate (ppm) of Signum. In the study of Leiminger et al.
(2014) 100 ppm only refers to pyraclostrobin, whereas in our study it refers to pyraclostrobin + boscalid.
Thus the actual amount of pyraclostrobin is only 20% of the Signum dose rate tested. This might explain the
differences between EC50 values found in both experiments.
Alt1409 was collected in a field experiment in Valthermond in 2014 which was sprayed with Amistar. The
AltNL14013 and Alt14014 isolates were collected in field experiments in Lelystad in 2014. AltNL14027
was collected in the centre of the province of Drenthe in 2014. AltNL06034 was collected in 2006 from an
unknown location. The isolates were not tested for the presence of the F129L mutation. However EC50
value for Amistar of isolate NL09014 which has the F129L mutation was not exceptionally high.
Nevertheless it is recommended to test the isolates for the presence of the F129L mutation, which will give
the possibility to investigate whether there is a correlation between the presence of the mutation and the
fungicide sensitivity.
The results suggest that some of the A solani isolates became less sensitive to Amistar and to a lesser
extent to Signum. From these data there seems to be some sort of correlation for sensitivity of A. solani
isolates for the different fungicides tested. This does not necessarily mean that this is caused by cross

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resistance. It might also be caused by fitness of the Alternaria isolate as such. You would expect an isolate
that more readily germinates to do so under any circumstance compared to a less fit isolate. The slope of
the curve is much steeper when Signum and Amistar are compared than when the comparison is made with
Penncozeb. Furthermore no significant difference in Penncozeb sensitivity were found between the
Alternaria isolates. Thus cross resistance, between Penncozeb on the on the hand and Amistar or Signum
on the other is not likely. For both Signum and Amistar the EC50-value differs significantly between isolates
tested. In this case isolates less sensitive to Signum seemed to be also less sensitive to Amistar, which
suggest the possibility of cross resistance or sensitivity.
From the results it can not be concluded that higher EC-50 values also leads to lack of control in the field.
However isolate Alt14019 showed a very high EC50 value for Amistar, which may well be an indication for a
lack of control in the field.
Conclusions
- The EC50 value for Penncozeb was stable for all Alternaria isolates tested, which indicated that
there was no shift in sensitivity of Alternaria solani to mancozeb.
- The EC50 values for Amistar varied among the Alternaria solani isolates tested. Some EC50 values of
the 2014 isolates were very high indicating a reduced sensitivity of A. solani to azoxystrobin, and a
shift in the population.
- The EC50 values for Signum varied among the Alternaria solani isolates tested. Some EC50 values of
the 2014 isolates were moderately high possibly indicating a reduced sensitivity of A. solani to
pyraclostrobin + boscalid.
Literature
Evenhuis, A., H. Hausladen, P. van Bekkum, B. Adolf, H. Schepers, M. Förch & J. Leiminger2013. F129L
mutation found in Dutch Alternaria solani population EuroBlight Workshop Limassol May 2013.
Leiminger, J., B. Adolf and H. Hausladen, 2014. Occurrence of the F129L mutation in Alternaria solani
populations in Germany in response to QoI application, and its effect on sensitivity. Plant Pathology
(2014) 63, 640–650.
Leiminger, J. and H. Hausladen Kartoffelbau 5: 18, 2013.
Pasche J.S. and N.C. Gudmestad, 2014. Prevalence, competitive fitness and impact of the F129L mutation
in Alternaria solani from the United States. Crop Protection 27: 427-435.

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Appendix 1 Disease observations (raw data)

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Appendix 2. PD-recognition