sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ ORMAN FAKÜLTESİ DERGİSİ
dissecting the trunk into discs. Samples of discolored or decayed wood were taken
from each second disc from eight cankered trunks. One trunk was analyzed in
detail and samples were taken from each disc. At least three samples of healthy
wood (showing no discoloration) from each trunk were taken as a control. When
taking samples, attention was given to the margin of decayed wood, the various
colors of decayed wood, the different phases of decay, and, in some cases, to the
dried zone of wood observed around the margin of the decayed wood. The samples
were taken with a boring machine with a wood borer of 12 mm diameter. Between
each boring the borer was sterilized by flaming with 96% (v/v) ethanol and was
then cooled down by dipping it in cool distilled water for few seconds. First, a 1–3
mm deep hole was made to eliminate possible contaminants and after flame
sterilization of the borer the sample for fungal isolation was taken. The boring
chips were collected in sterile Petri dishes. Up to five samples were taken per disc.
From each sample four isolations on 2% (w/v) malt extract agar (MEA) were made
with two repetitions. The sample was represented by a boring chip 2–3 mm × 2–3
mm in size. Altogether, 2276 isolations were made. Pure cultures were made of
every fungus growing from the sample. Cultures were incubated at 24°C for 4
weeks.
2.5. Density of perithecia and ascospore discharge
Three different densities of perithecia were distinguished when the area of the
Eutypella canker was measured. This enabled an assessment of ascospore
production of the canker in a more accurate way. Three different densities of
perithecia were then sampled. Each sample was checked for the maturity of its
perithecia before it was used in the test. The maturity test was performed on the
bark area just next to the area of perithecia that was taken into the test of ascospore
discharge. Mature perithecia are full of dark brown ascospores (Davidson and
Lorenz, 1938), young perithecia are white inside, and old perithecia are empty
when moistened. After the maturity test, the samples were cut to approximately 1
cm 2 . The exact area of the sample was determined after the test using analySIS
software. Because the samples had been air-dried they had to be immersed in water
for at least 30 min (Johnson and Kuntz, 1979). We immersed the samples in
distilled water for 1 h, and then the bark samples with perithecia were put on moist
filter paper for 3 hours, which is necessary for ascospore discharge to begin
(Lachance, 1971; Johnson and Kuntz, 1979). The sample with underlaying moist
filter paper was fixed to a rubber stopper with thin needle. Ascospores were
discharged into test tubes with a 2 cm diameter to which 2 ml 0.01% (v/v)
detergent Tween ® 80 solution had been added. Ascospores were allowed to
discharge for 3 hours at room temperature about 22°C. Ascospores were counted
using a Bürker-Türk counting chamber. The calculation of the number of
ascospores discharged per cm –2 h –1 was corrected using the exact area of the
sample. The samples were also histologically examined and the number and
maturity of the perithecia were determined.
153