sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ Faculty of Forestry Journal
Table 1: Origin of Sphaeropsis sapinea sensu lato and endophyte isolates used in the
experiments.
Isolate Isolate no. a Species Pine host Geographic origin
P1 411 D. pinea b Pinus resinosa Clearwater Co., MN
P2 04-126 D. pinea b Pinus banksiana Wood Co., WI
S1 124 D. scrobiculata b Pinus banksiana Jackson Co., WI
S2 462 D. scrobiculta b Pinus resinosa Clearwater Co., MN
E1 08-08 Not determined Pinus banksiana Jackson Co., WI
E2 08-09 Not determined Pinus resinosa Jackson Co., WI
E3 08-10
Trichoderma atroviride
Karst. c
E4 08-13 Rosellinia subiculata
(Schwein.) Sacc. c
74
Pinus resinosa Jackson Co., WI
Pinus banksiana Jackson Co., WI
a
Culture collection numbers of M. A. PALMER (3-digit number) or G. R. STANOSZ
(04-xxx).
b
The isolates were previously characterized according to the specific primers given by
SMITH and STANOSZ (2006).
c
The most homologous species given in the GeneBank after introducing the ITS
sequence.
Diplodia isolates was evaluated in vitro. Identifications of these four endophytes
were carried out by obtaining the ITS region sequence and later comparison with
the GeneBank. The fungal DNA was extracted directly from mycelia previously
grown in PDB (Potato Dextrose Broth, 39 g·l -1 , Difco Laboratories, Detroit, MI) by
using the slightly modified method of Gilbertson et al. (1991) outlined in Smith
and Stanosz (1995). The extracted DNA was amplified using ITS1 and ITS4
primers, and the entire region containing both internal transcribed spacers (ITS)
was sequenced by the University of Wisconsin Biotechnology Center on an
Applied Biosystems 3730XL DNA sequencer.
2.3. Interaction between D. pinea and D. scrobiculta experiment
The elongating, asymptomatic terminal shoot of each seedling was inoculated in
the greenhouse approximately 11 weeks after transplanting in the case of red pine,
and 6 weeks in jack pine. On each shoot, two wounds (in the opposite sides of the
shoot) were made by removing a needle fascicle per side (by a sterile scalpel cut
flush to the stem) approximately 2 cm below the shoot apex. Inoculations were
made by placing fungus-side-down a 4-mm-diameter plug, cut from the margin of
an actively growing culture on PDA (Potato Dextrose Agar, 39 g·l -1 , Difco
Laboratories, Detroit, MI), on each of the two wounds. In the controls, seedlings
were inoculated with sterile PDA in both wounds. Another five nonwounded
seedlings for each host were incorporated in the experiments as additional controls
but those were not included in the statistical analyses. Parafilm (American National
Can Co., Chicago, IL) was wrapped around the shoots for 7 days. Five seedlings
per treatment combination (isolate and fungal species; Table 1) were used in each
of two trials separated by two weeks. Thus, for each tree species were inoculated