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Effectiveness of a detached leaf assay

for determination of the reaction of pea
plants to powdery mildew
a

T.D. Warkentin , K.Y. Rashid & R.C. Zimmer

Research Centre, Agriculture and Agri-Food Canada , Unit 100-101,

Route 100, Morden, MB, R6M 1Y5
Published online: 29 Dec 2009.

To cite this article: T.D. Warkentin , K.Y. Rashid & R.C. Zimmer (1995) Effectiveness of a detached leaf
assay for determination of the reaction of pea plants to powdery mildew, Canadian Journal of Plant
Pathology, 17:1, 87-89, DOI: 10.1080/07060669509500724
To link to this article: http://dx.doi.org/10.1080/07060669509500724

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Note
CANADIAN JOURNAL OF PLANT PATHOLOGY 17:87-89. 1995

Effectiveness of a detached leaf assay for determination of the reaction

of pea plants to powdery mildew
T.D. Warkentin, K.Y. Rashid, and R.C. Zimmer
Research Centre, Agriculture and Agri-Food Canada, Unit 100-101, Route 100, Morden. MB R6M IY5
Accepted for publication 1995 01 04
An assay was developed to rapidly determine the reaction of field pea (Piswn sativum) plants to powdery mildew {Erysiphe polygoni). Detached leaflets or stipules were inoculated with conidia, incubated for three days, then assessed for powdery mildew
reaction. Assay results were highly correlated with the disease reaction of whole plants. Using this assay it was determined that
resistance to powdery mildew in the cultivar AC Tamor is conditioned by a single recessive gene.

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Warkentin, T.D., K.Y. Rashid, and R.C. Zimmer. 1995. Effectiveness of a detached leaf assay for determination of the reaction of pea
germplasm to powdery mildew. Can. J. Plant Pathol. 17:87-89.
Une technique a t dveloppe pour dterminer rapidement la raction au blanc (Erysiphe polygoni) des pois {Piswn sativum)
cultivs aux champs. Des folioles ou des stipules dtaches ont t inocules avec des conidies, incubes pour trois jours et
ensuite values pour leur raction au blanc. Les rsultats obtenus avec cette technique sont en trs bonne corrlation avec l'intensit de la maladie sur les plants. Cette technique a t utilise pour dmontrer qu'un gne rcessif, de la varit AC Tamor, est
responsable de la rsistance au blanc.
Powdery mildew of pea {Pisum sativum L.), caused
by the fungus Erysiphe polygoni D.C., is as widely
distributed as the crop (Reiling 1984) and can reduce
yields by 25-30% (Kumar & Singh 1981). The area in
field pea production in western Canada in 1993 was
467 000 ha (Statistics Canada 1993). The majority of
this area was seeded to cultivars that are susceptible
to powdery mildew. Currently, the only resistant cultivars registered in Canada are AC Tamor, Tara, and
Highlight. The most desirable means of disease control is the use of powdery mildew resistant cultivars.
Currently, selection for resistance relies on the occurrence of natural epidemics in the field. These do not
occur in every season. Therefore, better methods to
screen for resistance are required.
Harland (1948) described a single recessive gene
<?/-, which provided resistance to powdery mildew in
pea. Heringa et al. (1969) confirmed that a single
gene, er{, could bring about complete resistance in the
field and glasshouse in The Netherlands. They also
suggested that a second gene, erv from a Peruvian
source provided leaf resistance. Kumar and Singh
(1981) reported that two r e c e s s i v e genes were
required to produce field resistance in F2 populations
from crosses between susceptible cultivars and a
resistant source, S-143.
Recently, Cohen (1993) described a leaf disk assay
for the detection of resistance to powdery mildew in
melons. This assay allowed for the mass selection of
resistant plants from large segregating populations. It
avoided field-related problems, including lack of uniformity of infection and environmental limitations to
disease development.

The objectives of this study were to develop a

detached leaf assay for determination of the reaction of
pea plants to powdery mildew; to determine the con-elation between detached leaf assay and whole plant reactions; and to use this assay to investigate the resistance
to powdery mildew in the field pea cultivar AC Tamor.
The field pea cultivar AC Tamor, developed at the
Agriculture Canada Research Centre in Morden, MB,
is resistant to powdery mildew (Ali-Khan 1993). AC
Tamor was used as the female parent in crosses to the
following susceptible cultivars: Montana, Danto,
Triumph, Ricardo, Express, Miko, and Stehgolt.
Crosses were made in 1991 or 1992 at the Morden
Research Centre and the resulting progeny were used
in subsequent tests. The susceptible cultivar Trump
was used in preliminary studies of methods for
screening for resistance. An isolate of E. polygoni collected in the field at Morden, MB in 1992 was maintained in the greenhouse on plants of Trump. This isolate was used in all indoor inoculation tests.
Preliminary studies with the cultivars Trump,
Triumph, Ricardo, Express, and Stehgolt revealed that
the reaction to powdery mildew was identical on stipules and leaflets of pea. Stipules were used in all subsequent experiments because the cultivars Montana,
Danto, and Miko are semi-leafless, and therefore lack
leaflets. Plants were tested for their reaction to E.
polygoni at the four- to eight-node stage. Two stipules
from the second or third node below the apex of the
plant were excised with a scalpel then placed on a
sheet of cotton in petri dishes containing 15 mL of 5%
sucrose solution. The stipules were oriented with the
adaxial side up. The source of inoculum was young
87

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88 CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 17. 1995

Figure 1. A) Powdery mildew on detached stipules of the susceptible field pea cultivar Express 3 days after
inoculation (x 30). B) Lack of powdery mildew development on AC Tamor 3 days after inoculation (x 30).
C) Mycelial development on the susceptible field pea cultivar Trump 3 days after inoculation (x 200).
D) Germinated conidium with germ tube on AC Tamor 3 days after inoculation (x 400).

leaflets that were 80-100% covered with powdery

mildew. One infected leaflet was used per stipule,
Conidia were dusted onto stipules using a small
brush. Petri dishes were then placed in a growth
chamber at 22C with a 16 h photoperiod (high-intensity fluorescent light, 40 umol nr 2 s 1 ). The development of powdery mildew hyphae on the stipules was
assessed by observation under a dissecting microscope 3 days after inoculation. Stipules with abundant
mycelial growth were scored as susceptible; those
with no mycelial growth were scored resistant. AC

Tamor and the susceptible parents were used as

checks in all tests.
The response of whole plants was compared to that of
detached stipules. Inoculum from infected plants of
Trump was dusted onto plants by gently brushing infected with noninfected foliage. The response was measured
in a greenhouse at 20-25C and 16 h photoperiod, or in
a growth cabinet with day/night temperature of 20/15C,
16 h photoperiod (high-intensity fluorescent light, 380
jomol nr2 s 1 ). Disease reaction of individual plants was
visually assessed 1 to 2 weeks after inoculation.

WARKENTIN ET AL.: FIELD PEA/POWDERY MILDEW

89

Table 1. Reaction ot parents and segregating F2 populations ot held pea to powdery mildew infection
in a detached leaf assay and on whole plants
Detached leaf assay

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Parents
AC Tamor
VIontana
Danto
Triumph
Ricardo
Express
Miko
Stehgolt
AC Tamor
AC Tamor
AC Tamor
AC Tamor
AC Tamor
AC Tamor
AC Tamor

X
X
X
X
X
X
X

Montana
Danto
Triumph
Ricardo
Express
Miko
Stehgolt

Observed
ratio
R:S*

Expected
ratio
Rs

Chisquare
probability

% of time leaf
assay matched
whole plant assay

42:0
0:6
0:6
0:6
0:6
0:6
0:6
0:6
20:79
22:78
32:68
19:81
26:74
24:76
29:71

10
0 1
0 1
0 1
01
0 1
0 1
0 1
13
13
13
13
13
13
13

1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.25-0.50
0.75-0.90
0.10-0.25
0.10-0.25
0.75-0.90
0.75-0.90
0.25-0.50

100
100
100
100
100
100
100
100
96
94
94
100
99
98
98

R = resistant, S = susceptible.

The development of powdery mildew mycelia was

observed on the surface of detached stipules of all susceptible field pea cultivars within 3 days after inoculation (Fig. 1, A,C). Symptoms did not develop on stipules of AC Tamor (Fig. 1, B,D). Stipules of all cultivars remained green and vigorous for at least 7 days
under incubation conditions.
The resistance reaction of AC Tamor occurred at a
point after the germination of conidia, i.e. conidia
were able to germinate, but the growth of hyphae was
arrested soon after (Fig. 1. D). In contrast, extensive
mycelial growth occurred on stipules of susceptible
parents (Fig. 1, C).
Segregating F2 populations from crosses with the
cultivars Montana and Danto were evaluated in the
greenhouse for both detached leaf and whole plant reactions. All other F2 populations were planted in the field
in 1993 and detached leaf reaction data were collected.
However, cool and wet conditions prevented the development of an adequate powdery mildew epidemic,
making it impossible to assess whole plant reaction.
Therefore, F2 plants were harvested individually and F3
progeny were evaluated in a growth cabinet.
Assay results showed that the cultivar AC Tamor
contains a single recessive gene for resistance to powdery mildew (Table I). All seven populations segregated in a ratio of 1 resistant: 3 susceptible plants.
Results obtained in the detached leaf assay closely
matched those obtained for whole plants (Table 1). For
parents, assay and whole plant reactions always
matched. The frequency of mis-matches ranged from 0
to 6% in segregating populations. Some detached
leaves or whole plants may have received insufficient
inoculum despite efforts to inoculate uniformly.
Another possible cause of mismatches may have been

seed mixtures during the harvesting of individual F2

plants in the field.
Powdery mildew epidemics do not occur every year
in western Canada. Therefore, selection in segregating
populations in the field is not always possible. The
detached leaf assay described here is rapid and convenient. It allows the assessment of powdery mildew
reaction at the seedling stage instead of waiting until
the adult plant stage in the field. Reliable results were
obtained using stipules collected in both the greenhouse and field. This assay will be useful in breeding
powdery mildew resistant field pea cultivars and in
studies of the genetics of this disease. It should be
noted that only one gene for resistance was used in
this study. It is possible that this technique may not be
effective for other genes for resistance.
The technical expertise of E. Nichol-Seymour, A. Sloan, and I.
Wolfe is gratefully acknowledged.
Ali-Khan, S.T. 1993. AC Tamor field pea. Can. J. Planl Sci.
73:203-204.
Cohen, R. 1993. A leaf disk assay for detection of resistance of
melons to Sphaerotheca fitliginea Race 1. Plant Dis.
77:513-517.
Harland, S.C. 1948. Inheritance of immunity to mildew in
Peruvian forms of Pisttm suiivum. Heredity 2:263-269,
Heringa, R.J., A. Van Norel, and M.F. Tazelaar. 1969
Resistance to powdery mildew (Erysiphe polygon! D.C.) in peas
(Pisum sativum L.). Euphytica 18:163-169.
Kumar, H., and R.B. Singh. 1981. Genetic analysis of adult planl
resistance to powdery mildew in pea (Pisum sativum L.).
Euphytica 30:147-151.
Reiling, T.P. 1984. Powdery mildew. Pages 21-22 in D.J.
Hagedorn, d., Compendium of Pea Diseases, The American
Phytopathological Society, St. Paul
Statistics Canada. 1993. September estimate of production of
principal field crops, 1993,