CIP’s Protocol for standard evaluation of potato clones for resistance to Late Blight (LB) in
the field: experimental design, evaluation, data analysis and interpretation
Introduction
Late blight is a polycyclic disease, having several cycles of infection and inoculum production
during one growing season. Thus, the infection may be expected to increase proportionally to
both the initial amount of inoculum present and the amount of new inoculum produced during
the growing season. The amount of inoculum produced is dependent on the host, pathogen,
environment and management conditions. When dealing with polycyclic diseases there are
several ways to measure resistance, and CIP recommends the Area Under the Disease Progress
Curve (AUDPC) to measure resistance to late blight. The AUDPC is a resistance parameter
calculated from the percentages of leaf area affected, which are estimated at different times
during the epidemic. The advantage of using AUDPC is that it is simple to calculate, uses
multiple evaluations, and does not rely on transformations, which often are based on assumptions
about the distribution of the data points. The AUDPC also has some disadvantages that are
discussed below.
Experimental Design
The evaluation of advanced clones should be done in a replicated trial (4 repetitions / 5 tubers
each repetition). If disease severity is variable within the field and the pattern of variation can be
anticipated then a blocked design may be useful. Otherwise a completely randomized design is
probably preferable. If potato genotypes (clones or varieties) of known levels of resistance are
available, a small number (1-5) of these should be included in the trial. CIP has developed a set
of standard cultivars with known resistance levels that are available for distribution.
In addition to blocking, there are other measures one can take to reduce or control the effects of
variability in disease severity within the field. One is inoculation, which is especially useful in
areas where natural inoculum may not be very high. Protocols for field inoculation are available
in the literature (Forbes et al., 1993). Another technique frequently used is to plant one or more
genotypes of potato at regular intervals in the crop to produce sources of inoculum. These are
often referred to as spreader rows and generally the genotype(s) used is susceptible. There are,
however, problems with spreader rows. If the genotype is very susceptible, it will tend to bias
results by making its immediate neighbors look more susceptible due to the large amount of
inoculum the spreader sheds. To avoid this, spreaders can be planted between each clone so that
all clones receive an equal amount of inoculum, but this effectively doubles the size of the
experiment.
Late blight occurs very early in many tropical and sub-tropical locations. If infections occur
when plants are very small, differences between resistant and susceptible genotypes may be
difficult to appreciate. Under these conditions, it is advisable to protect plants with fungicides
until they are of sufficient size for evaluation. It is difficult to say exactly when plants are large
enough for evaluation but a “rule of thumb” might be about 30% of their expected maximum leaf
area.
Evaluation of Disease
To obtain the AUDPC, disease readings are taken on the basis of the percentage of leaf area
affected by late blight. It is important to start taking readings as soon symptoms appear;
therefore frequent scouting is required at the beginning of the assay. Since it is difficult to know
exactly when symptoms will start to appear, the first reading is planned 30 to 40 days after
planting, during which time the field should be observed carefully for initiation of disease. A rule
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of thumb is to make the first reading on all plots when the most susceptible one reaches 10% LAI
(Leaf Area Infection). The frequency of readings is not really crucial, but if disease is advancing
quickly in susceptible genotypes readings should be done frequently (5-7 days). If the disease is
advancing slowly, the interval between readings can be longer (10 – 14 days). The objective is
to have readings at low, medium and high levels of disease in all genotypes, including
susceptible ones.
One of the strengths of the AUDPC is that readings do not have to be done on a regular schedule,
nor do all units of the experiment have to be evaluated on the same day for a particular reading.
Readings should continue until the susceptible cultivars reach high levels of disease, near 100 %.
It is best to record readings independently (i.e., without knowing the value given at the previous
reading) at each date, such as by having someone else write in the field book or by using a
cassette recorder.
When evaluating late blight most researchers estimate the percentage of total leaf area that is
affected by the disease. This is done by simply comparing the green and non-green portions
(assuming late blight is the only or dominant foliage disease). Thus, one mentally estimates the
percent of infected foliage in the plot. This is standard procedure and generally works well,
especially when readings are integrated into a measure like the AUDPC. Nonetheless, the
estimation of percent infection is subject to several sources of error. First, one only really
evaluates the portion of the disease that has produced visible symptoms and that is still on the
plant. Infected leaflets eventually fall off the plant and the point in time at which they fall is
probably cultivar-dependent. Much of the diseased tissue is green and symptomless and therefore
not seen at any particular moment of evaluation. Some of this infected green tissue may be
sporulating but this is not always visible unless one gets very close to the leaflet. That degree of
scrutiny is generally not employed in cultivar evaluations. A second source of error is related to
how people evaluate proportional differences. Researchers have shown that people estimate
disease severity more precisely at low and high levels than at intermediate levels. This leads
researchers to propose that evaluators use logarithmically based scales, however subsequent
research demonstrated that it is probably better to simply estimate percent foliage affected
directly without the aid of a logarithmic scale (Forbes and Korva, 1994).
Data Analysis
When data have been collected sequentially through the season, the AUDPC is calculated in
order to compare cultivars or treatments. The AUDPC is most frequently calculated using the
midpoint rule method (Campbell and Madden, 1990). The formula is:
AUDPC = i=1n-1 [(ti+1 – ti)(yi + yi+1)/2],
Or alternately
n 1
 yi  yi  1 
AUDPC   
ti  1  ti 
2

i 1 
Where “t” is time in days of each reading, “y” is the percentage of affected foliage at each
reading and “n” is the number of readings. The first equation is easier to apply to the Excel
example below. Here is a graphical representation of these equations.
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The AUDPC is calculated for each experimental unit (i.e., each plot). Note, the variable “t” can
be Julian days, days since planting or days since emergence, since only the difference between t
values (i.e., number of days between readings) that is used in the equation. The AUDPC can be
calculated using virtually any statistical analysis program or spreadsheet. Here we give an
example using Excel.
Enter the evaluation of the five clones under the corresponding days written in the second line of
the table. Next, locate the cursor in cell I4, which corresponds, to the area of clone 1, and
calculate the area using the following formulae.
Area = ((C4+B4)/2)*($C$2-$B$2)+((D4+C4)/2)*($D$2-$C$2)+((E4+D4)/2)*
($E$2-$D$2)+((F4+E4)/2)*($F$2-$E$2)+((G4+F4)/2)*($G$2-$F$2)+
((H4+G4)/2)*($H$2-$G$2)
Press [Enter] and the area (581) will appear in the cell I4. Then copy this cell to the other cells
from I4 to I8. Table 1 shows the results.
Table 1: Percentage of foliar area affected by late blight and corresponding AUDPC calculated
by a spreadsheet EXCEL.
1
2
3
4
5
6
7
8
A
Accessions
CLONE1
CLONE2
CLONE3
CLONE4
CLONE5
B
C
D
F
G
H
I
55
E
Days
64
42
49
71
78
85
AUDP
C
1
0
3
5
5
5
5
15
10
20
10
10
30
25
35
15
20
40
50
50
15
30
60
65
70
20
40
90
85
85
30
50
90
100
85
581
933
2018
2070
2193
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Interpretation of Parameters Obtained
The AUDPC estimates the area under the actual infection curve. It is expressed in %-days (i.e.,
the accumulation of daily percent infection values) and interpreted directly without
transformation. The higher the AUDPC the more susceptible the clone or variety. It is often
helpful to plot the percentage of leaf area infected versus evaluation date to get a better idea how
resistant and susceptible clones or varieties perform in the evaluation experiment (see figure 1).
100
%leaf area affected
90
80
70
60
Clone A
50
Clone D
40
Clone F
30
20
10
0
35
42
49
55
64
71
78
85
90
Evaluation days
Figure 1: Disease severity curves of three potato clones.
Data Recording and Summary
A LATE BLIGHT EVALUATION DATASHEET (see attachment Q) is included to facilitate
data recording. Measurements of plant development (vigor) are generally recorded in addition to
disease severity. If the AUDPC is to be calculated, the date of each disease severity reading
must also be recorded. Data are collected on each experimental unit (each clone or variety
within each replication) and the AUDPC is calculated for each experimental unit. Simple
statistics (e.g., mean, standard deviation) or analyses (ANOVA, regression) can be calculated
using the AUDPC values.
Getting Around Common Problems Associated with the AUDPC.
As mentioned, the AUDPC is robust in the sense that it allows for different intervals between
readings. However, the AUDPC can be biased if readings begin too long after disease initiation
because part of the disease progress curve of susceptible materials will not be considered in the
AUDPC. For this reason, it is import to start readings when disease begins.
The AUDPC is also sensitive to lack of uniformity of disease in the field. Methods for
controlling this type of error are discussed above. The AUDPC can be biased if readings are
taken too long after susceptible cultivars reach high levels of disease. If this occurs, difference
between resistant and susceptible materials may be underestimated. For this reason it is
important to stop readings when susceptible materials are severely infected. Alternatively,
different AUDPC’s may be calculated, one including all materials and a reduced number of
readings, and another including more readings but excluding the most susceptible materials.
The AUDPC is generally not comparable across experiments. In an effort to standardize the
AUDPC, researchers often use the relative AUDPC. This is calculated by dividing the AUDPC
by the “maximum potential AUDPC”. The maximum potential AUDPC is simply the AUDPC a
variety or clone would have if it had 100% infection at all readings. Therefore, the maximum
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potential AUDPC is calculated by multiplying the total number of days between the first and last
readings by 100.
References
Campbell, C. L., L. V. Madden. (1990): Introduction to Plant Disease Epidemiology. John
Wiley & Sons, New York City.
Forbes, G. A., J. T. Korva (1994): The effect of using a Horsfall-Barratt scale on precision
and accuracy of visual estimation of potato late blight severity in the field. Pla. Path.
43, 675-682.
Forbes, G. A., O. Trillos, L. Turkensteen, O. Hidalgo (1993): Field inoculation of potatoes
with Phytophthora infestans and its effect on the efficiency of selection for
quantitative resistance in the plants. Fitopatología 28, 117-120.
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