Cucurbit Genetics Cooperative Report 4:6-8 (article 3) 1981

In Vitro and Sporulation of Didymella bryoniae and a Glasshouse Method for Screening for Resistance of Cucumber

A. C. van der Giessen and A. P. M. den Nijs

Institute for Horticultural Plant Breeding, P. O. B. 16, The Netherlands

At the Institute for Horticultural Plant Breeding (IVT) a breeding project for resistance against Didymella bryoniae (Auersw.) Rehm, syn. Mycosphaerella citrullina (C.O.Sm.) Gross. (common names: gummy stem blight, fruit and stem rot) has been in progress since 1969 in cooperation with the Research Institute for Plant Protection (IPO). This program has resulted so far in several partially resistant lines (1). Although the testing method was briefly described in the above paper, requests for additional information have convinced us that a more detailed description of the procedure would be worthwhile.

Preparing the Inoculum. Isolations of the fungus are made by preference from top internodes of infected plants. Stem pieces are disinfected in mercuric chloride 0.1% or sodium hypochlorite 1% during 30-60 sec, rinsed in sterile water and cut in segments of about 0.5 cm length. These are transferred to 9 cm petri dishes with malt extract agar (Oxoid). when we suspect heavy bacterial contamination of the plant material, we use prune agar (Difco) with 50 ppm oxytetracycline. The cultures are incubated at 22°C under exposure to blacklight (Philips or Sylvania F20t12-BLB; 12 hrs light/12 hrs dark). After several days we transfer agar discs from the edge of the developing colonies to fresh plates or slants with malt extract agar. After incubation for 10-12 days the plates are fully covered with sporulating fungus.

Cultures should be stored at a temperature of 14-15°C. Under these conditions they last for 2-3 months. To prepare the inoculum we flood fresh plates with distilled water and rub with he forefinger to bring the spores into suspension. The number of spores per ml is counted with a hemacytometer and the suspension is diluted to 107 spores/ml. We add one drop of Tween 80/100 ml suspension to ensure good contact with the plant.

Effects of the Environment on Spore Production. To get an insight into the effect of the type of culture medium and alternating blacklight during the incubation on the spore production, five isolates were transferred to two different culture media: malt extract agar and V-8 juice agar. The incubation took place under alternating blacklight or in the dark. From the full-grown plates a spore-suspension was made with a known quantity of water. The mean number of spores per plate could be obtained from the spore countings. From the figures in Table 1, it is clear that growing D. bryoniae on malt extract agar under alternating blacklight is by far the best way of obtaining large amounts of inoculation material. We have obtained comparable results with other Didymella species (D. lycopersici and D. chrysanthemi).

We generally observe much more fluffy mycelium growing on the plates in the absence of blacklight exposure. This type of mycelium does not contain pycnidia, and it has, therefore, little value as inoculum. Some isolates tend to form this fluffy mycelium more than others, even under blacklight. Usually sectors with sporulating pycnidia occur, and we always take material from these sectors for maintaining the isolate.

Inoculation. Plants are transplanted into 12 cm pots for 4-5 days after sowing. They are put under a transparent plastic tent when the first true leaf has a diameter of about 5 cm. After 24 hrs the spore suspension is sprayed over the plants with a propane gas operated knapsack sprayer as a pressure of 4 atm (60 psi). The optimal temperature for incubation is 26°C at a relative humidity of over 95%. For 36 hrs after the inoculation, the plants are kept in the dark by covering the tent with a sheet of black plastic. The transparent plastic is removed 4-5 days after inoculation. The first symptoms are then apparent. After 1-2 weeks the plants are assessed individually according to the following scale

0 - no visible symptoms
1 - slight infection, light brown lesions usually along the edge of the first true leaf
2 - moderate infection, mostly brown lesions on the leaves
3 - heavy infection, usually severely damaging the growing point; plants can recover from axillary buds

We exclude the cotyledons from the assessment because of earlier unreliable results. Individual plants without (severe) symptoms are selfed and intercrossed and the resulting progenies again tested. This way progress has been made in increasing the resistance level of young potted plants (1). We must, however, take into account the possibility of plants escaping effective inoculation, and we, therefore, compare lines as well. We also note differences in the overall severity of symptoms between tests, so we compare the reactions of breeding lines with that of check cultivars.

Table 1. Effect of culture medium and blacklight on sporulation of isolates of didymella bryoniae.^z

Culture medium	Blacklight sporulation (+ or -)	Number of plates	Mean number of spores/plate x 106
Malt extract agar	+	62	3017.8
"	-	35	254.4
V-8 juice agar	+	35	6.6
"	-	33	8.9

^z Combined data of several experiment with five different isolates; cultured in incubator at 22°C for 10 days.

Literature Cited

1. Meer, Q. P. van der, J. L. van Bennekom and A. C. van der Giessen. 1978. Gummy stem blight resistance of Cucumbers (Cucumis sativus L.). Euphytica 27:861-864.