Gummy Stem Blight – General Information
Gummy stem blight or fruit and stem rot (Didymella bryoniae, Phoma cucurbitacearum)
Didymella bryoniae (Auersw.) Rehm (syn. Mycosphaerella citrullina (C. O. Sm.) Gross.). Imperfect conidial state has been classified as Phoma cucurbitacearum (Fr.:Fr.) Sacc. (syn. Ascochyta, Phyllosticta or Diplodinia).
Hyphae brown, white, green or gray, forming concentric circles with alternating day/night light conditions; black pycnidia.
Septate hyphae, conidia 3X longer than wide, clear, usually 1 celled.
M. Havey, Plant Pathology Department, University of Wisconsin, 1630 Linden Dr., Madison, WI 53706.
Mutates quickly. Take care to constantly select for isolates which form large numbers of pycnidia which exude grey to light pink spore masses.
Most isolates will be pathogenic. New isolations from plant tissue may include, or may be mostly, Phoma exigua. See CMI descriptions to identify the differences between P. exidua and P. cucurbitacearum.
Storage and Retrieval:
Cultures can be stored 2-3 months at 14-15°C. Stock cultures of conidia in sterile distilled water stored at 4-5°C remain viable for one year max. Make new stocks every year. I lost some isolates because they did not last past one and a half years. It would be a very good idea to freeze-dry a number of samples of each isolate that we have. I planned to do that, but never did. Currently, we have each isolate stored in water and in soil. One water tube and one soil tube of each isolate is stored at room temperature and one water tube and one soil tube of each isolate is stored at 5 C. To retrieve, remove a single colony from the agar or a loop full or conidia from the water and streak on PDA or MEA. Isolate kept under oil loses pathogenicity.
Select one virulent isolate of Didymella bryoniae to use for screening. Inoculate petri plates containing 10 ml of cucumber malt extract agar (CMEA), using mycelial plug inoculation. Each liter of CMEA is made by mixing 250 ml of cucumber purée and 750 ml of distilled water with 30 g of malt extract agar (Difco). The purée, made from whole, freshly-harvested cucumber fruits, must be tindalized and then stored at 0 C (32 F) until needed. Incubate inoculated plates for 10 to 14 days at 24 ± 2 C (75 ± 4 F) under alternating periods of 12 h florescent light (40 to 90 µmol.m-2.sec-1 PPFD) and 12 h darkness until spore-producing pycnidia form. I tried UV light, but it did not significantly increase production. Grow on malt extract agar or V8 agar at 20-24°C. Expose plates for 12 hours a day to fluorescent or UV light. Be sure isolates remain virulent. Most conidia producing isolates will be virulent.
Flood plates with 5 to 10 ml of acidified, sterile-distilled water and scrape the surface of the agar with a rubber spatula or finger. The water should be acidified to a pH of 3.5 to 4.5 using lactic acid and should also contain 20 drops/liter Tween-80, a surfactant. The low pH and the surfactant increase spore discharge from pycnidia and greatly reduced spore agglutination. Filter suspension through 4 layers of cheesecloth to remove mycelia, pycnidia, and dislodged agar. Standardize suspension to a concentration of approximately 1 ¥ 106 spores/ml using a hemacytometer. The final pH of the inoculum is unadjusted and may range from 5.4 to 6.8. The inoculum can be held at 5 C for approximately 15 h until use. Add several ml of deionized water to 10-12 day old cultures. Remove spores by rubbing with a rubber policeman or forefinger to bring spores into suspension. Rinse plates with additional deionized water, add to contents of flask. Induce pycnidia formation on Didymella by cutting across mycelial mat with probe in crisscross pattern (like tic-tac-toe game). Pycnidia will form at wounds. Slight amount of acetic acid (very dilute) will break open the pycnidia to release spores.
Count spores with a hemacytometer. Adjust spore concentration to 1X105 or 1X106 (best) spores/ml with deionized water. Add one drop Tween 80/100 ml suspension to ensure good contact with the plant.
Inoculum Distribution and Delivery:
Plants are ready to be inoculated when they reach the 3 to 4 true leaf stage. For 1 or 2 days prior to inoculation, plants should be watered excessively and shaded from direct sunlight, to promote guttation on the day of inoculation. Immediately prior to inoculation, Tween-80 (10 drops/liter), casein hydrolysate (0.5 g/liter, Sigma Chem.), and sucrose (1 g/liter, Sigma Chem.) are added to the inoculum. The spore suspension should be kept well mixed during inoculation. Plants are inoculated by spraying the inoculum onto all upper leaf surfaces at dawn. It is important to inoculate actively guttating plants and to avoid inoculum run-off. The inoculum should be delivered as a fine mist using spray pressures from 200 to 275 kP (30 to 40 psi). Inoculated plants are immediately placed into a chamber without light and maintained at 22 to 26 C (72 to 78 F) and 95 to 100% RH for 48 h. Plants must be reaclimated to the above greenhouse conditions over a period of 24 h.
Plants are ready to be inoculated when they reach the vine tip-over stage (6 to 8 true leaves). On the day prior to inoculation, plants should receive approximately 0.5 in of irrigation to promote guttation on the day of inoculation. Immediately prior to inoculation, Tween-80 (10 drops/liter), casein hydrolysate (0.5 g/liter, Sigma Chem.), and sucrose (1 g/liter, Sigma Chem.) are added to the inoculum. The spore suspension should be kept well mixed during inoculation. Plants are inoculated by spraying the inoculum onto all upper leaf surfaces at dawn. It is important to inoculate actively guttating plants and to avoid inoculum run-off. The inoculum should be delivered as a fine mist using spray pressures from 200 to 275 kP (30 to 40 psi).
Method 1) Spray the spore suspension over the plants with a pressure sprayer.
Cucumis sativus L., cucumber.
Source of Resistance:
PI200818, Homegreen#2, Slice, M 17
Differentials – Controls:
Straight 8, Supergreen (susceptible)
Growth of Host:
Method 2) Grow by direct seeding in bands in flats (4″ pots work better) until the plants have the third true leaf about 5 cm (2 in) in diameter.
Inoculate the plants when third true leaf is about 5 cm in diameter.
Incubate plants in the dark for 24-48 hours at 24-26°C and 100% relative humidity. The humidity is a real important factor in the infection of this pathogen. Subsequently place plants in greenhouse at 24-26°C for 4-5 days.
First symptoms are apparent 4-5 days after inoculation. After 7-14 days rate plants individually according to the following scale: 0 = no visible symptoms; 1-3 = slight infection, light brown lesions along the margins of the first true leaf, 4-6 = moderate infection, mostly brown lesions on the leaves, 7-8 = heavy infection, usually severe damage of the growing points; plant can recover from axillary buds, 9 = plant dead. Allow disease to develop for 7 to 14 days. Increased humidity (80 to 95% RH) is beneficial during this period. Rate leaf necrosis when resistant controls begin to show symptoms and susceptible controls have advanced symptoms. Necrosis is rated as an average of the 2 oldest leaves from 0 (no necrosis) to 9 (complete necrosis). Each unit of the scale is approximately equal to an 11% increase in necrotic leaf area.
Allow disease to develop for 10 to 15 days. Rate leaf necrosis when resistant controls begin to show symptoms and susceptible controls have advanced symptoms. Leaf necrosis is rated as an average of the oldest leaves from 0 (no necrosis) to 9 (complete necrosis). Each unit of the scale is approximately equal to an 11% increase in necrotic leaf area. Stem necrosis may also be rated on the lower 30 cm (12 in) of main stems from 0 (no necrosis) to 9 (complete necrosis). Each unit of the scale is approximately equal to an 11% increase in necrotic stem area. Optimal stem symptoms often require more time to develop (3 to 4 weeks) than do leaf symptoms. However, stem symptom development is usually sufficient at the time of leaf rating
Plants with response 0-3 can be transplanted to sterilized soil.
Procedures Developed by:
Todd C. Wehner
Dept. Hort. Sci., Box 7609
North Carolina State Univ.
Raleigh, NC 27695-7609
The following method gives satisfactory results:
Euphytica 27:861-864. 1978.
For gummy stem blight [Didymella bryoniae (Auersw.) Rehm or Mycosphaerella citrullina (C.O. Sm.) Gross]
The fungus is grown on malt extract agar (oxoid or Difco) in Petri plates at a temperature of 20°C. In order to get a good spore production the plates are exposed at the same time to near-UV light during 12 hours a day. Care must be taken to work with virulent isolates.
Plants are grown (in pots) in a greenhouse till they have a first true leaf of about 5 cm diameter. At this stage the plants are covered with transparent plastic at about 10.00 h. Inoculation (spraying) takes place 24 hours later with a spore suspension of 107 spores per ml. Subsequently the plants are covered again with transparent plastic and on top of that with a layer of black plastic. During incubation the temperature is kept at about 26°C and the relative humidity as near to 100% as possible. Two days after inoculation the black plastic is removed at 08.00 h.
Symptoms, which are a browning and dying of parts of the true leaves and, sometimes, of the growing point (Fig. 1) appear within a week after incubation. When plants are tested in the winter period additional light is given daily from 0700 to 1900 hours. HPI/T-400 Watt (Philips) lamps, are situated 1.0 m above the plants at a density of one per m2.