sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ ORMAN FAKÜLTESİ DERGİSİ
five seedlings per each one of the following treatments: 1) P1+S1, 2) P1+S2, 3)
P2+S1, 4) P2+S2, 5) P1+P1, 6) P2+P2, 7) S1+S1, 8) S2+S2, and 9) Agar A+A.
Then, a total of 45 seedlings per trial and tree species were inoculated. All
treatments were assigned randomly. Four weeks after inoculation, the distances
below the inoculation site at which necrotic needles and cankers were present were
measured (symptom severity). Most of the seedlings were carried to the lab and, by
means of a molecular analysis with specific primers (Smith and Stanosz, 2006), the
presence of D. pinea or/and D. scrobiculata was verified in order to assure they
were the causal agents of the necroses.
2.4. Interaction between D. pinea and several endophytes experiment
This experiment was carried out on 1-year-old jack pine seedlings. Shoots were
inoculated as explained above, although D. pinea was inoculated 2 cm below the shoot
apex and the endophyte 5 cm below the shoot apex. The endophytes were inoculated
one week before the Diplodia inoculation. Two types of controls were used: seedlings
inoculated with sterile PDA instead of the endophyte into the lowest wound (D+PDA)
and seedlings nonwounded in the lower part (D). Other nonwounded seedlings were
incorporated in the experiments as additional controls but those were not included in
the statistical analyses. Five seedlings per treatment combination (D. pinea isolate and
endophyte species) were used in each of two trials separated by two weeks. Thus, a
total of 60 seedlings were double-inoculated per trial and considered in the analysis.
All treatments were assigned randomly.
Two and four weeks after inoculation, the distances below the site inoculated with
D. pinea isolates at which necrotic needles and cankers were present (symptom
severity) were measured. At the end of the experiment, 40% of the seedlings were cut
and carried to the lab where, after needles were removed, cross sections, 1 cm long,
centered at 0, 1.5, and 3 cm from the site inoculated with Diplodia, were aseptically cut
and surface disinfected as described above. Each of the three cross sections was
transferred to a PDA plate and incubated for 1 week at 25ºC and light. The presence of
Diplodia isolates and endophyte species in the plates was recorded.
2.5. Statistical analysis
Symptom severity was analyzed by two-factor analysis of variance with
interaction. Factors used as main effects were treatment and trial. Fisher´s least
significant difference (LSD) test was used for multiple comparison among
treatments by means of the General Linear Model Procedure of SAS (Statistical
Analysis Software v. 9.1.3, SAS Institute Inc., Cary, NC) when significant
differences were found in the ANOVA table. The ln (x+1) transformation of the
response variable was used to stabilize the residual variance, although backtransformed
values are presented in tables and figures for clarification.
Assumptions of normality and homoscedasticity were assured by Kolmogorov-
Smirnov and Levene´s tests respectively. In the endophyte experiment, since
symptom severity was recorded at two and four weeks after inoculation, a repeated
measurements analysis was also applied by means of Repeated Procedure of SAS,
to test the effect of time on the symptom severity.
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