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In a second experiment, three 30-year-old Scots pines located in the Orldans forest were inoculated with O. brunneo-
ciliatum to study the kinetics of the trees' phenolic responses. Each tree received 33 inoculations distributed in 5 rings, each
separated by 35 cm. After 3, 7, 14, 30 and 60 days, the reaction zones around respectively 15, 7, 5, 3 and 3 inoculation points
Inoculations were always performed with 5 mm diameter discs of culture placed in cambium-deep, equally sized
holes
per tree
made
were
with
measured
a cork borer,
and sampled.
and the
At
bark
days
plugs
30
were
and 60,
put
samples
back into
of
the
unwounded
holes, hnmediately
phloem were
after
also
being
collected
collected
from
from
each
the
tree.
trees, the phloem samples were frozen in dry-ice. In the laboratory, all samples were freeze-dried.
second experiment, zone
Phenols Extraction and Analysis
3
which were pooled all together (day 3) or by groups of 1 to 3 samples (day 7) to have sufficiently a high
quantity of material for further chemical analyses. Extractions were performed at 4°C on ground samples. In order to
remove resinous compounds (Alcubilla 1970), phloem powder was first washed with pentane before extraction of phenolics
using 80% methanol. Pentane treatment was checked to have no significant effect on the compounds analyzed in our study.
Gallic acid was used as internal standards for HPLC analyses.
Analyses were performed with reversed HPLC (Waters 600E, Photodiode array detector 991), in a 250 mm column
and 4 mm internal diameter. Stationary phase was a C-18 grafted silica (Merck, Lichrospher RRP18) with 0.005 mm porosity.
Mobile phase was a mixture of a 1% acetic acid in ultra pure water (solvent A) with a solution of pure methanol, acetonitrile,
acetic acid (49.5:49.5:1) (solvent B). The gradient is represented in Fig. 1. Readings were made at 310 nm. Results were
expressed in internal standard equivalents per gram of freeze-dried powder.
"" 100
o,_
.¢
= _?IP.,l
0.6 ....................... _ .....................................................
c_
ca ca gll
ca ' 50
0 4 ..... "_ = ........................ ° _I ................. ; ..... "'- s ................................
_ "_
• _I ca . o _
c_ = ...... -._
:,.7,, ; 6 =
t •_ $" .."
_' "
= __ li !_ -r,
.... :_ .......... i................. ! .................. i_ ................ _"' = ........ i........ u
: 2 : "_ : "=, !
02-, . _:.
,v,,,,,. " '" I .... I " " " " I " '-" ' r • _"i'" I " " " " I " " "'"" I ...... ; ' i'''' " " " " " " _
0 20 40 60 80 100 (ran)
time (ram)
Figure 1._Chromatogram of a reaction zone in Scots pine phloem and HPLC gradient. Solvent A = acetic acid, water
(1/99); Solvent B = methyl alcohol, acetonitril, acetic acid (49.5/49.5/1).
179