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
A. C. van der Giessen and A. P. M. den Nijs
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
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
Blacklight sporulation (+ or -)
Number of plates
Mean number of spores/plate x 106
Malt extract agar
V-8 juice agar
z Combined data of several experiment with five different
isolates; cultured in incubator at 22°C for 10 days.
- 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.