NC State and USDA Cucumber Disease Handbook
Cotyledon Inoculation for Anthracnose
This protocol is written to conduct virulence tests using four differential cultivars, cucumber cultivars Marketer and Arkansas Little Leaf (H-19), and, watermelon cultivars Black Diamond and Charleston Gray.
Day 1: Pre-germinate Black Diamond seed.
Day 3: Pre-germinate Charleston Gray (also Edisto and Butternut squash).
Day 4: Pre-germinate Marketer and H19 seed.
Day 5: Plant pre-germinated seed in 10 cm square pots in a 1:1 peat/perlite soil-less mix (Sunshine Mix # 1). Do not fertilize the plants.
Day 12: Approximately four days after emergence, or when the cotyledons on all cultivars were fully expanded, inoculate.
Day 20: Rate test using 0-9 scale.
Get isolates started on green bean agar (GBA) or Emerson medium. Isolates are stored in vials at 4 C as pieces of desiccated #3 Whatman filter paper.
If you start your isolates from stored paper, it will take about 5 -7 days until the colonies are big enough to use.
Pre-germinate seed of all cucurbit cultivars to be used in the test.
Soak seed for 5 minutes in deionized water and then pour off the water for treated seed. For seed that has not received a seed treatment rinse three times in deionized water. Put seed in a petri dish on moist filter of tissue paper. Add some water so that there is a little amount of free water (1 – 2 ml). Try not to have too much excess free moisture, i.e. the seed is submerged. Incubate seed in the dark at room temp (23-25 C). If you want to speed things up, incubate seed in the dark at 28 C. Now if you incubate seed at this temperature, then you will pre-germinate Charleston Gray on day 2, and the cucumbers on day three.
On day four you should streak plates of Emerson medium with the isolates you are testing. I got better success when I inoculated plates with four plugs of the fungus.
With a loop, collect spores off of the original plates started and thoroughly streak the inoculum plates. If there are no spores on the original plates, start the inoculum plates with several (up to five) transfer plugs. Grow cultures for 7 to 10 days at room temperature (23 C).
Always make at least 2 inoculum plates per isolate. For some isolates (15096, 15097, 15015, 15016, and AK2 you may need up to four plates).
Approximately four days after emergence, or when the cotyledons on all cultivars were fully expanded, inoculate.
Spores are washed off the agar surface and suspended in cold deionized water (4 C). The suspension is adjusted to a spore concentration of 8 x 104 spores per ml using a hemacytometer. Cotyledons were inoculated using a paint sprayer (15 PSI) until the entire surface of the cotyledons are covered.
After inoculation, incubate plants in dew chamber (or modified dew) for 24 hours at 21 to 25 C and maintained at 100% relative humidity. A hygrothermograph (or data lager) should be placed in the dew chamber during each inoculation to monitor temperature and relative humidity. After inoculation, the plants should be returned to the greenhouse, where temperatures ranged between 22 to 37 C. Observed plants daily for disease development.
Disease severity ratings are given by assessing the area of the cotyledon showing symptoms (chlorosis and necrosis) of infection whereby 0 = no infection; 1 = 1 to 10% of cotyledon area with visible symptoms; 2 = 11 to 25%, 3 = 26 to 50%; 4 = 51 to 75%; 5 = 76 to 90%; 6 = > 90%; and 7 = dead cotyledons.
Disease severity was rated daily from the onset of visible symptoms (usually three days) until seven days after inoculation. Although disease severity ratings were taken up to seven days after inoculation, the disease severity ratings collected seven days after inoculation were used in the statistical analysis because there was no change of symptom development for virulent isolates after six days.
Purified cultures are stored by transferring isolates to GBA medium containing a sterile Whatman # 3 filter paper disc on the surface of the medium. When the fungus fully colonizes the filter paper, remove it (the filter paper) and air dry for 14 days under room temperature on a clean bench. The desiccated paper cultures are stored at 4 C.
Green bean (Phaseolus vulgaris L.) agar (GBA) is prepared by adding 20 g agar and two jars (226 g) of green bean baby food (Gerber Production Co., Fort Smith, AR), to one liter of double deionized water.
Emerson medium is prepared by adding 4 g yeast extract; 15 g soluble starch;1 g potassium phosphate (dibasic); 0.5 g magnesium sulfate; and 16 g agar to one liter of water.