Development of a Seedling Test for Resistance to Gummy Stem Blight in Cucumber

Cucurbit Genetics Cooperative Report 15:23-27 (article 9) 1992

Zoila G. Abad and Todd C. Wehner
Department of Horticultural Science, Box 7609, North Carolina State University, Raleigh, NC 27695-7609

Gummy stem blight, caused by Didymella bryoniae and possibly by Phoma exigua, is the second most important pathogen of field grown cucumber (Cucumis sativus L.) in North Carolina (2). The disease is caused by didymella bryoniae (teleomorph stage of Phoma cucurbitacearum, usually reported as Ascochyta cucumis). Gummy stem blight causes conspicuous ‘V’ shaped yellow-brown lesions on leaves, and pale-fawn lesions on stems which are present mainly at the nodes. On fruits the disease causes a soft rot with black discoloration of the skin (exocarp). In the lesions of different organs, pycnidia of the anamorph stage and perithecia of the teleomorph can be observed easily as abundant black dots. The disease causes much defoliation of the cucumber crop in the late stages of production. Gummy stem blight is a serious disease of greenhouse cucumbers in The Netherlands, where it causes fruit rot (5).

The disease organism is apparently stimulated by volatile compounds contained in plants of species belonging to Cucumis and Cucurbita (1). Infection caused by D. bryoniae appears to be dependent on relative humidity, with more infection occurring at 95% than at 50% relative humidity. Greatest infection is produced by free-standing water on leaves. In the case of older leaves, wounding of the leaves is essential for infection (6). A high incidence of main stem lesions and fruit rot is also apparent in plants that are grown with a long period of high humidity and free water on the fruits, for example when changing greenhouse conditions to normal day conditions in late morning (5).

There was no correlation between gummy stem blight on seedlings, and fruit rot caused by gummy stem blight in 2- to 3-month-old plants (4). A seedling test can be used to show differences between susceptible and resistant cultigens (cultivars, breeding lines, plant introduction accessions populations and families) of cucumbers, using a virulent isolate of Didymella bryoniae inoculated at the first true leaf stage of plants kept at 100% relative humidity for 2 days (3).

There is little evidence that field tests for gummy stem blight are correlated with seedling tests. Two tests involving 954 and 1208 cultigens, inoculating at the 2nd and 3rd true leaf stage and repeated 9 days later, rating hypocotyl and stem cankers along with foliar lesions, showed no significant correlations with field ratings for the same cultigens grown in Florida and Wisconsin (7). However, several resistant cultigens were identified from the tests. The ability to distinguish large differences in resistance to gummy stem blight was demonstrated in another seedling test using seedlings at the 2nd and 3rd true leaf stage incubated for 2 days at 100% relative humidity, and rated for leaf lesions 4 days after inoculation (7).

A reliable seedling test should reveal significant differences among cultigens and a significant correlation with field test results. The objective of this study was to continue the development of a reliable seedling test for resistance to gummy stem blight in cucumber.

Methods. A field test, run in 1983 to determine resistance in cucumber to gummy stem blight, was used for comparison with the seedling tests. Three seedling tests were run in the greenhouse in 1988 and 1989 to evaluate several methods of inoculation, incubation and plant injury, and to choose the most pathogenic isolate. PI 200818, ‘Slice’, ‘Poinsett 76’; M 12 and M 17 were used as resistant checks, while ‘Marketmore 76’, Wis.SMR 18, ‘Addis’ and ‘Colet’ were susceptible checks. All but ‘Colet’ and PI 200818 are monoecious inbreds.

The field test was planted at the Horticultural Crops Research Station in Clinton, N.C. Plots were 1.5 m long with 1.5 m alleys at each end, and were arranged in rows 1.6 m apart. Plots were planted on 25 May with 15 seeds each, and thinned on13 June to 10 plants. The field was surrounded by susceptible Wis.SMR 18 border rows. Plots were inoculated on 26 June with one selected isolate of Didymella bryoniae. Also, there was natural disease in the field for gummy stem blight, and to a lesser extent for anthracnose (Colletotrichum orbiculare (Pass.) Ellis & Halst. the weather conditions of the summer season with high humidity helped to spread the inoculum.

Plots were rated on 12 and 19 July for foliar lesions using a 0 to 9 scale () = no disease, 1 to 2 = a trace of infection, 3 to 4 = few small lesions, 5 to 6 = 20 to 50% of leaves covered with small lesions, 7 to 8 = plant wilted and covered with 50% or more with lesions, 9 = plant dead). Ratings were averaged over the 10 plants in a plot.

Inoculum. In a preliminary test of 12 Didymella bryoniae isolates, we chose those that were virulent and that represented diverse sources of the pathogen. three isolates of Phoma cucurbitacearum collected in 1988 (two from cucumber fields in North Carolina and one from California) were used in greenhouse tests. the isolates were increased on malt extract agar medium at 20 ˚ C grown near a window for a 16 hr day (3). The fungus was incubated for 7 days until abundant pyenidia formed (an important characteristic for virulent isolates). Inoculum was prepared in 100 ml of distilled water by flooding plates with 15 ml of sterile distilled water, scraping the surface of the agar with a rubber spatula, and collecting the spores from one plate per isolate. Inoculum concentration was standardized using a hemacytometer to 106 spore/ml in 100 cc/ml. One drop of Tween 80 was added to the suspension before inoculation. The spore suspension was applied with an artist’s airbrush until a fine film covered the inoculated leaves.

Of the many tests run, three will be described here. The first involved seedlings of 6 cultigens using 4 isolates (2 from North Carolina, 1 from Wisconsin and 1 from Arizona), 3 humidity chamber durations (1, 2 or 3 days) and 3 preinoculation treatments (Carborundum, florist’s frog, or none). Preinoculation damage of the leaf produced mechanical damage in addition to the disease symptoms. the experiment was a split-plot with 2 replications of 4 plants each. Whole plots were chamber durations, subplots were leaf damage treatments, and sub-subplots were cultigens. Plants were inoculated with a hand-pumped mist sprayer until droplets formed on the leaf. The Carborundum treatment involved dusting the 1st true leaf with the finely-powdered compound before spraying, the rubbing lightly to damage the leaf epidermis. Damage by the florist’s frog (9 needles mounted in a 20 mm diameter base) was produced by pressing lightly on the leaf surface before spraying, leaving a grid of small holes.

For all tests, seedlings were thinned to 1 plant per pot 7 days after seeding, inoculated at first true leaf stage 7 days after thinning, and placed in a dark humidity chamber at 100% relative humidity and 18 to 21 ˚ C air temperature. Plants were removed after 1 day of dry-out (chamber doors open, humidifiers off) and rated for leaf damage 7 to 10 days after inoculation (21 to 24 days after seeding).

The second test involved seedlings of 5 cultigens, 3 isolates, r humidity chamber durations (0, 1 or 2 days) and 2 inoculum concentrations (106 or 107 spores*ml-1). The experiment was a split-plot with 2 replications of 1 plant each. Whole plots were chamber durations, subplots were isolate-concentration combinations, and sub-subplots were cultigens.

The third test involved seedlings of 5 cultigens, 3 isolates, and 2 inoculum concentrations (105 or 106 spores*ml-1 ). The experiment was a split-plot with 2 replications of 1 plant each. Whole plots were isolate-concentration combinations, and subplots were cultigens. Seedlings were incubated 1 day in the humidity chamber.

Results. The coeefficient of variability was 33% for the gummy stem blight rating in the 1983 field test, which is not unusually large for disease ratings. Occasional anthracnose lesions on the plants in that study made it more difficult to rate some plants for gummy stem blight. There may also have been an interaction between the two diseases. However, it is difficult to keep anthracnose out of field tests, since gummy stem blight and anthracnose occur naturally at about the same time in cucumber production areas of North Carolina. That is one of the reasons for our interest in developing a seedling test.

The first test showed no significant differences among treatments (Table 1). The only obvious trend was that longer durations in the humidity chamber produced more leaf lesions. The test was included to show that many of the seedling tests we conducted did not provide useful results., It was not possible to get correlation analysis between field and seedling tests since the seedling test did not differentiate the cultigens.

The second test showed that 1 day in the chamber provided greater differences among cultigens than 2 days, isolate DB-H-23 was more virulent than isolates DB-H-21 and DB-H-22, and cultigens PI 200818 and M 17 were more resistant than ‘Colet’ (Table 2). ‘Slice’ and M12 were variable in their reaction. A high concentration was used in the test (107 spores*ml-1 ), but proved too much for the seedlings, since all plants had ratings of 7 to 9 (data not shown).

The third test had much variability (CV = 66%) so there were few significant differences among treatments (Table 3). The low inoculum concentration (105 spores*ml-1 ) provided few differences among cultigens, whereas the high concentration caused reactions similar to the ranking in the second test. Also, isolate DB-H-23 was not as virulent as in the second test.

Seedling tests for gummy stem blight resistance work well for us only half the time, so they are not as reliable as we would like. However, they are useful for plant breeders wishing to test large numbers of plants off-season, or where limited greenhouse space is available. Based on the tests conducted, we recommend that seedlings be inoculated by spraying undamaged plants in the 1st or 2nd true leaf stage with 106 spores *ml-1 of an isolate that sporulates well, followed by 1 day of incubation in 100% relative humidity. Seedlings can be rated 7 to 10 days after inoculation if held at 25 to 30 ˚ C after incubation. Progeny of selections that are resistant in seedling tests should be tested in the field to verify resistance.

Table 1. Disease rating of seedlings of 6 cucumber cultigens inoculated using 1 isolate of Didymella bryoniae 3 incubation times (1, 2 or 3 days in the humidity chamber) and 3 damage treatments (Carborundum powder prior to inoculum spray, florists frog to puncture the leaf prior to inoculum spray, or none) compared with field rating^z.

	1 day in chamber			2 days in chamber			3 days in chamber			1983 Field
Cultigen	Carb.	Frog	None	Carb.	Frog	None	Carb.	Frog	None
Slice	1	1	2	3	5	5	5	4	5	2.9
Poinsett 76	1	1	0	4	4	6	5	3	5	4.0
M 17	0	1	1	4	4	5	6	4	5	4.1
Marketmore 76	1	2	2	4	4	7	5	4	5	5.3
Wis. SMR 18	1	1	0	6	1	7	8	4	6	5.6
Addis	1	1	1	4	6	5	5	4	6	6.2
LSD (5%) for row and column comparisons						NS				0.4
CV (%)						43				16

^z Seedling data are means of 2 replications of 4 plants each (field ratings are means of 6 replications).
^y Foliage damage rated 0 to 9 (0 = no disease, 1 to 2 = a trace of infection, 3 to 4 = few small lesions, 5 to 6 = 20 to 50% of leaves covered with small lesions, 7 to 8 = plant wilted and covered with 50% or more with lesions, 9 = plant dead) 7 days after inoculation at the first true leaf stage.

Table 2. Disease resistance of seedlings of 5 cucumber cultigens sprayed with 106 spores*ml-1 using 3 isolates of Didymella bryoniae and 3 incubation times (), 1 or 2 days in the humidity chamber).^z

	1 day in chamber			2 days in chamber			0 days in
Cultigen	DB-H-21	DB-H-22	DB-H-23	DB-H-21	DB-H-22	DB-H-23	Chamber	Water
PI 200818	2	2	6	5	6	9	0	0
M17	4	4	8	7	4	9	0	0
Slice	4	9	9	6	7	9	0	0
M12	4	6	6	6	8	9	0	0
Colet	6	7	9	6	9	9	0	0
LSD (5%) for row and column comparisons					3
CV (%)					40

^z Data are means of 2 replications of 1 plant each. A high concentration (107 spores*ml-1 ) was used which provided less consistent results (data not shown).
^y Foliage damage rated 0 to 9 () = no disease, 1 to 2 = a trace of infection, 3 to 4 – few small lesions, 5 to 6 = 20 to 50% of leaves covered with small lesions, 7 to 8 = plant wilted and covered with 50% or more with lesions, 9 = plant killed) 7 days after inoculation at the first true leaf stage.

Table 3. Disease resistance of seedlings of 5 cucumber cultigens sprayed with 106 spores *ml-1 using 3 isolates of Didymella bryoniae and 3 incubation times (), 1 or 2 days in the humidity chamber).^z

	DB-H-21		DB-H-22		DB-H-23		Water control
Cultigen	105	106	105	106	105	106
M 17	6	4		2	4	0	0
PI 200818	3	6		7	0	4	0
Slice	3	6		9	2	4	0
M 12	6	8		2	3	6	1
Colet	6	9		9	2	8	0
LSD (5%) for row and column comparisons				5
CV (%)				66

^z Data are means of 2 replications of ? plants each.
^y Foliage damage rated 0 to 9 ()0 = no disease, 1 to 2 = a trace of infection, 3 to 4 = few small lesions, 5 to 6 = 20 to 50% of leaves covered with small lesions, 7 to 8 = plant wilted and covered with 50% or more with lesions, 9 = plant killed) 10 days after inoculation at the first true leaf stage.

Literature Cited

1. Pharis, V.L., T.R. Kemp and D.E. Knavel. 1982. Host plant-emitted volatiles as a factor in susceptibility in vitro of Cucumis and Cucurbita spp. to the fungus Mycosphaerella melonis. Scientia Hortic 17:311-317.
2. St. Amand, P.C. and T.C. Wehner. 1991. Crop loss to 14 diseases in cucumber in North Carolina for 1983 to 1988. Cucurbit Genet. Coop.Rpt. 14:15-17.
3. Van Der Meer, Q.P., J.L. Van Bennekom and A.C. Van Der Giessen. 1978. Gummy stem blight resistance of cucumbers (Cucumis sativus L.). Euphytica 27:861-864.
4. Van Steekelenburg, N.A.M. 1981. Comparison of inoculation methods with Didymella bryoniae on Cucumis sativus. Euphytica 30: 515-520.
5. Van Steekelenburg, N.A.M. 1985. Influence of time of transition from night to day temperature regimes on incidence of Didymella bryoniae and influence of the disease on growth and yield of glasshouse cucumbers. Neth. J. Pl. Path. 91:225-233.
6. Van Steekelenburg, N.A.M. 1985. Influence of humidity on incidence of Didymella bryoniae on cucumber leaves and growing tips under controlled environmental conditions. Neth. J. Pl. Path. 91: 277-283.
7. Wyszogrodzka, A.J., P.H. Williams and C.E. Peterson. 1986. Search for resistance to gummy stem blight (Didymella bryoniae) in cucumber (Cucumis sativus L.). Euphytica 35: 603-613.