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Effectiveness of a detached leaf assayfor determination of the reaction of peaplants to powdery mildewT.D. Warkentin a , K.Y. Rashid a & R.C. Zimmer aa Research Centre, Agriculture and Agri-Food Canada , Unit 100-101,Route 100, Morden, MB, R6M 1Y5Published online: 29 Dec 2009.

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

To link to this article: http://dx.doi.org/10.1080/07060669509500724

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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.

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 été développée pour déterminer rapidement la réaction au blanc (Erysiphe polygoni) des pois {Piswn sativum) cultivés aux champs. Des folioles ou des stipules détachées ont été inoculées avec des conidies, incubées pour trois jours et ensuite évaluées pour leur réaction au blanc. Les résultats obtenus avec cette technique sont en très bonne corrélation avec l'inten­sité de la maladie sur les plants. Cette technique a été utilisée pour démontrer qu'un gène récessif, de la variété AC Tamor, est responsable de la résistance 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 recess ive 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

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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 Tamor and the susceptible parents were used as mildew. One infected leaflet was used per stipule, checks in all tests. Conidia were dusted onto stipules using a small The response of whole plants was compared to that of brush. Petri dishes were then placed in a growth detached stipules. Inoculum from infected plants of chamber at 22°C with a 16 h photoperiod (high-inten- Trump was dusted onto plants by gently brushing infect-sity fluorescent light, 40 umol nr2 s1). The develop- ed with noninfected foliage. The response was measured ment of powdery mildew hyphae on the stipules was in a greenhouse at 20-25°C and 16 h photoperiod, or in assessed by observation under a dissecting micro- a growth cabinet with day/night temperature of 20/15°C, scope 3 days after inoculation. Stipules with abundant 16 h photoperiod (high-intensity fluorescent light, 380 mycelial growth were scored as susceptible; those jomol nr2 s1). Disease reaction of individual plants was with no mycelial growth were scored resistant. AC visually assessed 1 to 2 weeks after inoculation.

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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

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* 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

Detached leaf assay Expected

ratio R 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1

s 0 1 1 1 1 1 1 1 3 3 3 3 3 3 3

Chi-square

probability 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

% of time leaf assay matched

whole plant assay 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

WARKENTIN ET AL.: FIELD PEA/POWDERY MILDEW 89

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, éd., Compendium of Pea Diseases, The American Phytopathological Society, St. Paul

Statistics Canada. 1993. September estimate of production of principal field crops, 1993,

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