Laboratory Isolation Procedures The following procedures are used routinely in university diagnostic clinics throughout the Midwest and Texas. They are considered to be the best techniques for isolating the oak wilt fungus from branch and stem samples.

1. When samples arrive at the laboratory, keep them in resealable plastic bags and refrigerate immediately. Retain samples in refrigerated storage until culture results are completed, in case there is a need to re-culture the sample because of contamination or other reasons.

2. Surface sterilize branch and stem samples by spraying them with 95% alcohol and then flaming (Figure 8). Figure 8.  Spraying with 95% alcohol and flaming sterilizes the surface. 3. Remove the bark on branch samples with a knife that has been sterilized by dipping it in alcohol and flaming.

4. Look carefully for discoloration in the sapwood that appears as streaks or flecks. (Figure 9). If sapwood discoloration is not present in one branch sample, check other branch samples. Be diligent in your search because the likelihood of isolating the oak wilt fungus from discolored sapwood tissue is excellent, nearly 90%. If discoloration is not present, however, the likelihood of isolating the fungus in culture is very low. Figure 9. Bark peeled back to reveal discoloration in the sapwood. 5. With a sterile wood gouge or knife, remove strips of discolored sapwood and cut into 1/4- inch pieces.

Note: If the bark and/or strips of sapwood are removed from branch samples outside the sterile environment of the isolation hood, surface disinfect the sapwood strips before cutting them into 1/4-inch pieces and placing them on growth media (Step 6). To disinfect sapwood strips, submerge them in a 10% household bleach solution (1:9 ratio of 5% household bleach to sterile, distilled water) for 1 minute, rinse in sterile distilled water, and blot dry on sterile paper towels.

6. With a sterile forceps, place four sapwood pieces in each of 6 petri plates containing acidified potato dextrose agar (APDA) (Figure 10). See recipe for APDA on page 10. Figure 10. Small pieces of sapwood, cut and transferred with a sterile utensil, are placed on APDA media in a petri dish. 7. Incubate petri plates at ambient room temperature and lighting. Petri plates are best stored in an incubation hood, but can be stored on the lab bench if sealed with parafilm. Be sure to store plates in a "clean" lab, away from plant samples and other contaminants.

8. The conidial state of the oak wilt fungus, Chalara, should be evident in 5-14 days. Mycelial growth of the oak wilt fungus on APDA media first appears nearly transparent, but soon becomes gray, and often has a brown to green cast (Figure 11). Cultures emit a characteristic fruity odor that is easily detected when the petri dish lid is gently opened. A good identifying characteristic of Chalara is the presence of endoconidia that are produced within elongated hyphal structures called phialides (Figure 12). Endoconidia are rectangular, greenish when mature, and may be borne in chains. Figure 11. The oak wilt fungus growing on APDA media. Note the brownish-gray color of the colonies.

9. Hold plates for 14 days before interpreting results as negative. Plates should be monitored and read every 2-3 days, and any necessary transfers made. Figure 12. Rectangular endoconidia (a) and elongated phialides (b).

Recipe for APDA: 1. Add 39 grams of Difco R Commercial PDA (potato dextrose agar) to 1 liter of distilled water. 2. Autoclave for 20 minutes. 3. Add 25% lactic acid at a rate of 5 drops per 100 ml of agar. Add the lactic acid after the agar has cooled, just before you pour the mixture into plates. 4. Store plates inverted, in the refrigerator, to keep water droplets from dripping down on the agar surface.

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