Sample Collection and Storage Sample quality is paramount to the success of laboratory testing, and results are only as reliable as the sample itself. Improperly collected, stored, or shipped samples may result in a false negative. False negative means the tree has oak wilt, but the presence of the fungus was not confirmed by laboratory testing. Following the procedures outlined in this section will help to ensure sample quality and increase the likelihood of an accurate diagnosis. 1. Select branches that are partially wilted, with symptomatic leaves progressing from the tip of branches inward to the trunk. Be sure that branches are not totally wilted, dry, or dead. 2. Collect symptomatic leaves from selected branches. Be sure to package leaf samples separately from branch samples. In white and red oaks, look for leaves that are partially wilted and have brown discoloration that moves inward from the leaf margins and progresses from tip to the base of the leaf (Figure 1a,b). In live oak, leaves develop yellow veins that eventually turn brown, producing a symptom called veinal necrosis (Figure 1c).

Figure 1. Leaf symptoms of oak wilt. a) White oak showing marginal browning. b) Red oak showing marginal browning. c) Live oak showing veinal necrosis.

3. Look for discoloration in the sapwood of partially wilted branches. Discoloration may be evident in a cross sectional view of infected branches (Figure 2). Figure 2. Cross sectional view: black dots of discoloration are evident in the sapwood.

In some cases, particularly with red oak, discoloration is not evident in a cross sectional view of the branch and is detectable only after the bark is removed and the sapwood is longitudinally sectioned (Figure 3). In the field, remove the bark from symptomatic branches and check carefully for streaks or flecks of discoloration in the underlying sapwood. If sapwood discoloration is not present in one branch, check other partially wilted branches. Be careful not to remove all of the bark on the branch samples you submit for testing. Figure 3.Figure 3. Longitudinal view: streak of discoloration evident only after removal of the bark.

4. Select samples from up to three symptomatic branches per single tree. It is critical that the sapwood is moist to the touch, and the inner bark is still alive and green. In the field, check for this by removing the outer bark on a small section of the branch sample (Figure 4). Avoid collecting samples from the extreme tips of branches: this tissue wilts first and is often too dry for successful isolation. Figure 4. Remove the bark on a small section of the branch: check that the inner bark is alive and green, and the sapwood is moist to the touch.

Branch samples should be at least 1-inch in diameter, cut into 6- to 8-inch lengths, and placed in large resealable plastic bags (Figure 5). Smaller diameter branch samples are acceptable only if the sapwood is very moist, and samples have been kept cool. Samples from each suspect tree should be labeled separately and placed in individual resealable plastic bags. Figure 5. Cut branch samples into 6- to 8-inch lengths and store in a resealable plastic bag.

5. Bole samples may need to be taken in some cases when symptomatic branches are too high to reach with available equipment. Bole sampling wounds the main trunk of the tree and should be used only when tree health has already been compromised by excessive wilting or heavy defoliation. Bole samples are not the preferred sample type for laboratory testing because the wood is harder and more difficult to chisel out subsamples from, and contamination by saprophytic fungi is more common.

If bole samples are taken, a "window" must be cut through the outer and inner bark, exposing a clean sapwood surface (Figure 6). Strips of discolored sapwood tissue can then be cut from the outer growth rings with an axe or chisel. If discoloration is not present in the exposed sapwood, check other location(s) on the bole. Avoid including bark in the sample bag because the oak wilt fungus competes poorly with fungal contaminants found on the outer and inner bark. Figure 6. A "window" cut through the outer and inner bark, exposing the sapwood.

6. Keep samples cool during sampling, shipping, and storage, but do not freeze. Sample temperatures should never exceed 85-90º F. Along with resealable plastic bags, be sure to take a cooler and ice packs (avoid ice cubes and dry ice) with you to the field (Figure 7). Never leave samples unrefrigerated or exposed to direct sunlight. Figure 7. Mandatory field supplies for storing samples.

7. Provide background information about the tree(s) such as symptoms observed and when they first occurred; age of the tree; proximity to other trees and buildings; any damage caused by storms or other events. Use data collection forms supplied by your local diagnostic laboratory whenever possible. 8. Ship samples by overnight mail or deliver in person to the laboratory. Samples should remain in resealable plastic bags and be shipped in a disposable ice chest with ice packs. Mail samples early in the week to ensure arrival at the laboratory in ample time to culture the sample before the weekend, and to avoid sample delivery on the weekend. 9. Stop collecting samples after annual leaf fall begins in the fall. In Minnesota, the annual cut-off date for oak wilt testing is typically mid- September. In the spring, sampling can begin after the leaves have flushed. If sampling is done during peak infection periods (late April, May, or June in Minnesota), wound dressing should be applied to all pruning cuts.