sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ ORMAN FAKÜLTESİ DERGİSİ
caused by D. pinea by means of co-inoculations on P. resinosa and P. banksiana
seedlings, with the goal of evaluating their potential role as biological control
agents of Diplodia pinea.
2. MATERIALS AND METHODS
2.1. Plant material
Dormant, 1-year-old jack pine and 2-year-old red pine nursery seedlings were
lifted at the beginning of winter and transplanted into Deepot cones (conical tubes,
6.4 cm wide x 25.4 cm deep; Stuewe & Sons Inc., Corvallis, OR) in a soil mix (1:1
vol/vol) of Plainfield sand (containing 89% sand and 7% silt) from a 24-year-old
red pine plantation in central Wisconsin and Fafard growing mix no. 2 (Conrad
Fafard Inc., Inkerman, New Brunswick, Canada). Red pine seedlings had a mean
stem height of 16.9 cm 0.3 standard error (SE) and jack pine had a mean stem
height of 26.8 cm 0.6 SE at the time transplanted. Seedlings were placed in a
greenhouse supplemented with artificial light (maximum recorded ambient
greenhouse photon flux density was 1,560 µmol·s -1 ·m -2 ; supplemental photon flux
density averaged 132 µmol·s -1 ·m -2 ) to provide a 16-h photoperiod. The seedlings
were watered to field capacity every 2 to 3 days. The climatic conditions during the
experiments in the greenhouse were: average temperature (T) 30.6º C 0.7 SE,
average relative humidity (RH) 30.0% 1.5 SE.
2.2. Fungal material
Diplodia mycelia inoculum was produced for two monoconidial isolates of each
species, D. pinea and D. scrobiculata, collected from various pine species and
locations in the north central United States (Table 1). Thirty four endophytes were
isolated from vigorous external twigs located at 1.5 m above ground in the canopy
of 20–30 year old dominant red and jack pine trees. For isolations, needle-free
shoots were surface sterilized by immersion for 30 s in 95% ethanol, and then two
immersions for 2 min each in 1.05% NaClO plus two drops Tween-80 per litre.
This procedure is similar to those routinely used for detection of endophytic fungi
(Bills, 1996). Then, for each shoot, four 5-mm long plant segments including bark
were cut and plated into a Petri dish containing PDA (Potato Dextrose Agar, 39 g·l -
1 , Difco Laboratories, Detroit, MI) medium and incubated at room temperature for
1 week before examination. Outgrowing colonies were transferred to PDA and
incubated at 24º C. Four endophytes (Table 1) among the total were selected to
perform the experiments based on a preliminary study (data not shown) where the
antagonism between all of them and the
73