Distribution and variation of extractable total phenols and tannins in

1030
Distribution and variation of extractable total phenols and tannins in the logs
of four conifers after 1 year on the ground
R ICK G. K ELSEY'
Department of Entomology, Oregon State University, Corvallis, OR 97331, U.S.A.
AND
MARK E. HARMON
Department of Forest Science, Oregon State University, Corvallis, OR 97331, U.S.A.
Received November 17, 1988
Accepted April 21, 1989
KELSEY, R. G., and HARMON, M. E. 1989. Distribution and variation of extractable total phenols and tannins in the
logs of four conifers after 1 year on the ground. Can. J. For. Res. 19: 1030-1036.
Concentrations of extractable total phenols and tannins have been analyzed in the outer bark, inner bark, sapwood,
and heart wood from logs of four conifer species, after 1 year on the ground. To estimate decay rates, initial tissue
densities were compared with those after 1 and 2 years of decomposition. The four species studied, Pacific silver fir
(Abies amabilis Dougl. ex Forbs), western hemlock ( Tsuga heterophylla (Raf.) Sarg.), Douglas-fir (Pseudotsuga menziesii
(Mirb.) Franco), and western red cedar ( Thuja plicata D. Don) represent a gradient of decay resistance. Within a species,
outer bark contained the greatest quantities of extractable total phenols followed by inner bark, heart wood, and sapwood. Outer barks also had the highest extractable tannin concentrations. Woody tissues contained very low concentrations of tannin compared with the barks. Total phenol concentrations were highest in the heart wood of red cedar,
the most resistant, and lowest in the heart wood of silver fir, the least resistant. There was no obvious relationship
between tannins, in any tissues, and the resistance gradient. Density measurements indicated minimal decay in all tissues
after 1 year. At 2 years, the inner barks of Douglas-fir, western hemlock, and silver fir had decreased significantly.
In general, tissues with the highest extractable phenols appear to be decomposing most slow ly.
KELSEY, R. G., et HARN1ON, M. E. 1989. Distribution and variation of extractable total phenols and tannins in the
logs of four conifers after 1 year on the ground. Can. J. For. Res. 19 : 1030-1036.
Les concentrations en phenols totaux et tanins extractibles ont ete analysees dans l'ecorce externe et interne, l'aubier
et le bois parfait de billes de quatre essences de conifere apres 1 an au sol. Afin d'evaluer les deares de pourriture,
les densitès de tissus initiaux ont ete comparèes a celles apres 1 et 2 annees de decomposition. Les quatre essences
6tudiees, le Sapin gracieux ( A bies amabilis Dougl. ex Forbs), la Pruche de l'Ouest ( Tsuga heterophylla (Raf.) Sara.),
le Sapin de Douglas (Pseudotsuga men:iesii (Mirb.) Franco) et le Thuya geant ( Thuja plicata D. Don) representent
un gradient de resistance a la pourriture. A l'interieur d'une essence, l'ècorce externe contenait les plus grandes quantitës
de phenols totaux extractibles suivie par l'ècorce interne, le bois parfait et l'aubier. Les ecorces externes avaient egalement
les plus fortes concentrations en tanins extractibles. Les tissus li g neux contenaient de três faibles concentrations en
tanins en comparaison des ecorces. Les concentrations en phenols totaux etaient les plus elevees dans le bois parfait
du Thuya g6ant, le plus resistant, et les plus faibles dans le bois parfait du Sapin gracieux, le moins resistant. Dans
aucun des tissus ne fut observèe une relation 6vidente entre les tanins et le gradient de resistance a la pourriture. Les
mesures de densitê ont indiquè une pourriture minime dans tous les tissus aprês 1 an. Apre . s 2 ans, les ecorces internes
du Sapin de Douglas, de la Pruche de l'Ouest et du Sapin gracieux avaient diminu6 de facon significative. En gèneral,
les tissus avec la plus forte teneur en phenols extractibles semblent se &composer le plus lentement.
[Traduit par la revue]
Introduction
Over the past half century, the importance of plant
secondary chemicals as mediators of ecological interactions
between plants, and between plants and animals, has become
increasin g ly apparent (Rice 1984; Rosenthal and Jansen
1979; Harborne 1982). In conifers, terpenes and phenols
protect livin g trees and impart decay resistance after death
(Scheffer and Cowling 1966). Durability of dead trees
depends upon their chemical composition at death and any
subsequent changes until metabolic activities cease. The
complete breakdown of a dead tree into its elemental components involves complex long-term interactions between
decomposers, environment, and substrate composition. The
extent to which allelochemics influence initial colonization,
community structure, and subsequent succession of the
( Author to whom all correspondence should be addressed.
Present address: USDA Forest Service, Forestry Sciences
Laboratory, 3200 Jefferson Way, Corvallis, OR 97331, U.S.A.
Printed in Canada Imprirne au Canada
decomposer organisms, however, is largely unkno\s 11.
Moreover, the fate of these compounds during decomposition has not been thorou g hly investi gated. The rates of
nutrient cyclin g and long-term forest productivity are dependent upon decomposition processes. Understandin g these
processes will allow more precise and efficient long-term
management of forest ecosystems.
Various heart wood extractives in trees are fungitoxic and
impart natural resistance to decay (Rudman 1962, 1963,
1965). Scheffer and Cowlin g (1966) point out that most toxic
heart wood components are phenolics. In this paper, we
report the relative concentrations of extractable total phenols
and tannins in the tissues from 1-year-old logs of four
conifer species and relate them to chan ges in tissue density
after 1 and 2 years of decay. These species represent a gradient of decay resistance from weak to very strong. This is
part of a long-term study in which lo g decomposition is
being investigated (Carpenter et al. 1988).
KELSEY AND HARMON
Study area
This study was conducted at the H.J. Andrews Experimental
Forest on the west slope of the Cascade Range in the Willamette
National Forest. The climate is maritime, with relatively mild, wet
winters and dry, cool summers. Mean annual precipitation is
2300 mm with 753/4 as rain between October and March. Mean
annual temperature is 8.5°C. Soils are deep, well-drained typic
dystrochrepts with slope gradients ranging from 20 to 60°7o. The
undisturbed vegetation is classified into two major zones, the
western hemlock (Tsuga heterophylla (Raf.) Sarg.) zone,
300-1050 m elevation, and the Pacific silver fir (Abies amabilis
Dougl. ex Forbs) zone, 1050-1550 m elevation. Douglas-fir
(Pseudotsuga menziesii (Mirb.) Franco) and western red cedar
(Thuja plicata D. Don) are also major components in both zones
(Dyrness et al. 1976).
Methods
Log sites
Six sites were located between 500 and 1000 m elevation in oldgrowth Douglas-fir and western hemlock forest, approximately
450 years of age. They represented intermediate conditions along
temperature and moisture gradients. Live, healthy trees of Douglasfir, western hemlock, western red cedar, and Pacific silver fir were
felled at two clear-cuts and two partial cuts near the sites, in
September-October 1985. Logs were cut 5.5 m in length and ranged
from 45 to 60 cm in diameter. To standardize initial conditions,
only logs without heart rot and with >95° bark cover were used.
Twenty logs of each species were placed side by side on the forest
floor at a spacing of 3 m along an access trail at each site. An additional 10 equal-sized logs of Douglas-fir and western hemlock were
placed at four of the six sites and individually enclosed in 0.5-mm
polyester mesh A-frame tents to exclude wood-boring insects (Leach
et al. 1934; Schmitz and Rudinsky 1968), particularly the ambrosia
beetles (Coleoptera: Scolytidae).
Sample collection and preparation
After placement in 1985, cross sections were removed from the
end of each log to determine the initial volume, density, moisture
content, and nutrient composition of the outer hark, inner bark,
sapwood, and heart wood.
One log of each species, at each site, was sampled for chemical
analyses in September 1986, 1 year after placement. Three sample
points were located on each lo g , one at the center and 0.5 m in
from both ends. At each sample point, outer bark, inner bark,
sapwood, and heart wood tissues were collected. Small squares of
outer and inner bark were removed with a chisel. Sapwood and
heart wood were sampled with an increment borer and electric drill.
Tissues were sealed in plastic bags and stored on dry ice for
transporting to the laboratory. Outer and inner barks were processed as described later. For each log, all pieces of a given tissue
were pooled into one sample.
To evaluate chemical chan g es in logs during the 1st year, one
tree of each species was cut at two of the sites (except Pacific silver
fir which was not present at one) in early March 1987. The felled
trees were cut into 4-m logs starting 1 m aboveground. An 8- to
10-cm cross section was removed from the first and fourth logs,
at approximately 1 and 17 m tree height. The discs were immediately transported to the laboratory in plastic bags where they were
stored in a cold room before processing. Pieces of outer and inner
bark were chiseled from the top, bottom, and two sides of the logs.
The disc was split along perpendicular lines connecting the chisel
points to yield four thin pieces of sapwood and heart wood. Each
tissue was pooled into a single sample, giving two samples per tree.
A 4°C cold room was used to separate, clean, and grind the
samples. To prevent contamination of tree tissues, sawed edges
were trimmed with a band saw. Bark samples were clipped into
small pieces and coarse ground in a Wiley mill without a screen.
All tree tissues and the inner bark and sapwood from logs had to
1031
be frozen with dry ice for grinding to 20 mesh with a Wiley mill.
Samples were sealed in two plastic bags and stored frozen. Moisture
contents of all tissues were measured by drying duplicate samples
at 105°C overnight.
Total phenol analyses
Extractable total phenols were quantified using fresh tissues with
dry weight equivalents of 40 to 320 mg. Samples were extracted
for 16 h at room temperature using 10 mL of aqueous acetone
(80°7o). Outer-bark extracts from Douglas-fir and western hemlock
were diluted 1:10 before analyses. Aliquots (100-200 kiL) were
diluted to 2 mL with water and then assayed with the FolinCiocalteu phenol reagent as described by Julkunen-Tiitto (1985).
Standard curves were prepared with phenol.
Tannin analyses
To quantify extractable tannin concentrations in the tissues, fresh
weight equivalents of 100 mg dry wei g ht were extracted with 0.75
or 1.0 mL aqueous acetone (80N) for 1 h. An aliquot (20 to
130 AL) was applied to wells in agarose plates containing 0.1°70 protein (Hagerman 1987). The diffusion rin g s were measured after
a minimum of 96 h at 30°C. Standard curves were prepared for
each species with the purified tannins (described later), and commercial tannic acid was purified as described by Ha german and
Klucher (1986).
Preparation of tannin standards
Bulk samples of fresh bark were collected in mid-April 1987 from
trees felled in March. Bark was removed from a 1-m section at
approximately 20-25 m tree hei g ht, sealed in plastic ba g s, and
returned to the laboratory where it was cleaned (at 4°C) and stored
frozen. Douglas-fir bark was removed from a tree immediately after
felling in the McDonald Forest near Corvallis. Frozen subsamples
of each bark were ground to 20 mesh as described earlier.
Three hundred grams fresh weight of ground bark were extracted
by soaking in 1 L of aqueous acetone (80'/7o) for 4 h. The tissue
was extracted twice again with the same volume of solvent, first
for 2 h, and then for 1 h. Each solution was filtered through glass
wool. A bilayer was formed by adding excess NaCI to the combined
extracts and shaking vi g orously. The acetone was recovered and
evaporated at 40°C under vacuum. This left a reddish brown water
solution and sticky residue. Filterin g with glass wool removed the
residue, which was rinsed several times with water and discarded.
The combined water solution and rinses were washed with
3 x 300 mL of diethyl ether. The water was evaporated in a 40`C
water bath under vacuum. The reddish brown residue was dissolved
in methanol (MeOH), transferred to a clean flask, and the solvent
evaporated as above. Five to 10 g of dry tannin residue were loaded
on a Sephadex LH-20 (50 g dry wei g ht) column equilibrated with
1:1 MeOH:H 2 0. Impurities were eluted with MeOH:H 2 0 (1:1)
collected in 500-mL fractions until two consecutive fractions were
of similar color, in all cases the fifth and sixth fractions. The tannin
was washed from the column with acetone:water (8:2). After
evaporatin g the acetone, residual water was removed by freezedrying to yield the final product.
Dihydroquercetin analyses
Dihydroquercetin (taxifolin), one of the major phenolic constituents in Dou g las-fir tissues (Hancock 1957; Gardner and Barton
1960), was measured by high-pressure liquid chromatography
(HPLC). Fresh weight equivalents of 100 mg dry tissues were
extracted with aqueous acetone (80 o)for approximately 16 h. Ten
millilitres of solvent was applied to outer bark and heart wood,
whereas only 2 mL was used for inner hark and sapwood. The
extracts were filtered through a 0.45-pm nylon 66 membrane before
analyses. Epicatechin was added to the solvent for an internal
standard. Although epicatechin has been reported to occur
naturally in Douglas-fir tissues (Hergert 1960; Holmes and Kurth
1961), our preliminary analyses indicated it was not present in sufficient concentrations to interfere. The HPLC column was an
1032
CAN. J. FOR. RES. VOL. 19. 1989
TABLE 1.
Extractable total phenol concentrations One equiv./g dry wt.) in the tissues from trees and 1-year-old logs of tour
conifer species
Douglas-fir
Logs
Trees
OB
IB
73.0
141.4
48.3
37.5
Pacific silver fir
Western hemlock
SW HW OB/IB SW HW OB
6.7ab
6.0
18.5c
26.0
45.6
52.7
5.0b
3.0
8.6
8.9
Western red cedar
IB SW HW OB 113 SW HW
171.3
192.8
15.8
37.7
7.9u
4.0
14.8c
18.7
27.7
25.7
4.7
14.9
7.6a
3.9
34.7
29.8
NOTE: OB, IB, HW, SW, OB/IB = outer bark, inner bark, heart wood, sapwood, and outer and inner bark combined, respectisely. Underscored means
of log tissues within a species are not significantly different (p < 0.05). Means of the same log tissue in different species followed by the same letter are not
significantl y different (p < 0.05). Tree data were not analyzed statistically. Log means base been adjusted for missing values and unequal sample sizes.
TABLE
2. Extractable tannin concentrations (mgig dry wt.) in the tissues from trees and 1-year-old logs of four conifer species
Douglas-fir
OB
Logs
Trees
65.9
109.7
Pacific silver fir
Western hemlock
IB SW HW OB/IB SW HW 013
77.8
44.3
12.0
2.4
5.5a
3.3
43.6b
30.7
4.5c
4.5
4.5a
1.9
151.9
168.7
IB
19.3d
25.6
Western red cedar
SW HW 013
5.3c
4.0
3.4a
2.8
49.1 b
53.0
113
13.2d
26.5
SW HW
3.1c
1.1
5.9a
4.6
N OTE: OB, IB. HW, SW, OB IB = outer hark, inner hark, heart wood, sapwood, and outer and inner bark combined, respecti‘ely. Underscored 111Carl,
of log tissues within a species are not significantly different (p < 0.05). Means of the same log tissue in different species followed by the same letter arc not
significantly different (p < 0.05). Tree data were not analyzed statisticall y . Log means have been adjusted for missing values and unequal sample sizes.
Alltech
Lichrosorb RP-18, 25 cm x 4.6 mm, maintained at
35 c C. The solvent gradient was the same as that used by Vande
Casteele et al. (1982) with an 18-min hold at 80 crlo solvent B in A.
The floss rate was 1 mLimin, with the UV detector set at 280 nm.
Density measurements
At each of the six sites in September 1986 and 1987, one log
of each species was sampled to determine changes in tissue density.
These were the same logs sampled for chemical analyses. Five cross
sections were removed along the length of each log sampled, and
the density of the outer bark, inner bark, sapwood, and heart wood
was measured. Outer-bark volume was measured using water
displacement after soaking for 24 h. Inner-back volume was determined by measurin g the dimensions with calipers. Wood volume
was measured on a subset of blocks cut to a standard size
(100 cm') on a table saw.
Statistical analyses
This experiment was designed as a split-split plot in time to
accommodate additional sampling in future years. Each of the six
sites represents a block, and the four log species is the main plot
effect. Tissues (outer bark, inner bark, sapwood, and heart wood)
are the subplot effects. For these 1st-year samples, time was not
a factor in the analyses.
All chemical data were analyzed by the General Linear Models
Procedure using the SAS Institute Inc. (1987) statistical program.
Significant differences between means were detected by Fischer's
(protected) least significant difference, p = 0.05. Residual plots
indicated heteroscedacity, making it necessary to transform the data
using square roots for tannins and natural logs for total phenols
and dihydroquercetin. Reported log means have been adjusted to
accommodate the missing values and unequal sample sizes.
Changes in tissue density data were assessed by comparing the
initial density and that at 1 and 2 years of decay using a one-tailed
t-test. Values for each log were avera g ed before the test was made.
Because the live trees were not true time-zero samples and
because of their limited sample numbers, these data have been
included for comparison, but not analyzed statistically.
There were no significant differences between enclosed and open
logs for any of the components measured. Consequently, they were
treated as a single population in all subsequent statistical analyses.
Results
Total phenols
Concentrations of extractable total phenols in the tissues
of Dou g las-fir, western hemlock, and Pacific sill er fir all
exhibited the same patterns (Table 1). Outer bark had the
highest quantities followed by inner bark, heart wood, and
sapwood. In nearly every instance, the differences between
tissues were si g nificant. In western red cedar, the greatest
quantity of total phenols occurred in the heart \\ ood , but
was not si g nificantly greater than that in the outer bark. The
lowest quantities occurred in the red cedar inner hark.
Comparing each tissue across species, the outer bark of
western hemlock contained 2.3 times more total phenols
than Dou g las-fir with the second hi g hest quantity. Western
red cedar outer bark had the lowest quantity, with only 16().-o
of the amount in hemlock (Table 1). Red cedar also had the
lowest inner-hark phenols of all species, with only 10(ro of
the concentration present in the top-rankin g Douglas-fir.
Outer- and inner-bark phenol concentrations were significantly different between each species. Western red cedar
heart wood contained 2 to 4 times more phenols than any
of the other taxa; the lowest quantities were found in silver
fir. There was no difference between Dou g las-fir and western
hemlock heart woods. Of all four tissues, sapwoods were
the least variable between species in their levels of extractable
total phenols.
In g eneral, phenol concentrations in the lire trees were
similar to those in the 1-year-old logs, with the exception
of Dou g las-fir outer bark, and the inner hark and sapwood
of hemlock and red cedar. Some differences can be expected
because the two sets of trees were cut in different seasons
and the sample size was smaller for the live trees. It appears,
however, that lo g total phenols have changed little in 1 year.
Tannins
ins
Within each species, the highest extractable tannin concentrations were found in the outer bark. Inner-bark quantities were significantly lower than in the outer bark for
western red cedar and western hemlock, whereas in Douglas-
CAN. J. FOR. RES. VOL. 19, 1989
1034
TABLE 4.
Percent change in tissue density from 1- and 2-year-old logs of four conifer species
Douglas-fir
Year 1
Year 2
OB
IB
+ 1.6
+ 1.6
-0.7
-21.2
Pacific silver fir
Western hemlock
SW HW OB/IB SW HW
OB
1B
+0.2
-5.3
+ 1.1
-0.6
+0.7
-26.5
+3.9
+1.4
+0.7
-12.9
-2.6
-2.6
-0.5
-1.1
Western red cedar
SW HW OB
-3.4
0.0
-3.5
-1.5
+5.2
-0.9
IB
-0.5
+3.4
SW HW
-2.6
-1.3
-3.6
-0.3
NOTE: OB, 1B, HW, SW, OB/IB = outer bark, inner hark, heart wood, sapwood, and outer and inner bark combined, respectively. Underscored means indicate siunificant
differences from initial tissue density based on a one-tailed r-test (p < 0.05).
plicatic acid, are the most abundant extractives in red cedar
heart wood, together with at least 10 other lignans (MacLean
and Gardner 1956b; Swan et al. 1969; Barton and
MacDonald 1971 and references cited within; MacDonald
and Barton 1973). This water-soluble phenolic fraction, with
the thujaplicins carefully removed, was fun gistatic (Roff and
Atkinson 1954). It is likely that all these individual lignans
exhibit the same broad biolo gical activities reported for other
lignans (MacRae and Towers 1984), including toxicity to
fungi and inhibition of specific enzymes. Another important g roup of red cedar extractives is the tropolones, and
ti thujaplicin (0-1.2%, in about equal proportions). Both
are fungitoxic, and nearly as active as pentachlorophenol
(Rennferfelt 1948; Roff and Whittaker 1959; Barton and
MacDonald 1971; Trust and Coombs 1973). These compounds chelate Cu and Fe (Cook et al. 1951; MacLean and
Gardner 1956a), making them potent inhibitors of enzymes
containin g these metals (Goldstein et al. 1964; Kahn and
Andrawis 1985).
Conidendrin, matairesinol, and hydroxymatairesinol are
western hemlock heart wood li g nans that coat tracheal walls
and sometimes fill tracheid lumens (Krahmer et al. 1970).
The last two compounds inhibited Forties annosus (Fr.)
Krast growth in vitro, and hydroxymatairesinol is partially
responsible for reaction zone resistance of Norway spruce
(Picea abies (L.) Karst.) to F. annosus (Shain and Hillis
1971). One percent matairesinol in wood substrate inhibited
growth of the wood decay fun g i Lentinus lepideus Fr.
(Rudman 1965). This compound also inhibits the activity
of cyclic AMP phosphodiesterase (Nikaido et al. 1981).
Douglas-fir heart wood phenolics are primarily flavonoids
(Hergert 1960), with dihydroquercetin (0-1.5%) the major
constituent (Hancock 1957; Gardner and Barton 1960).
Moderate decay resistance of this tissue has been attributed
to dihydroquercetin (Kennedy 1956). Rudman (1963)
reported low fun g al toxicity for numerous flavonoids and
considered them unimportant in decay resistance. He su g
-gestd(Ruman1962)hDogls-firetancud
from low water absorptive capacity, and not fungitoxicity
of dihydroquercetin. Nevertheless, tissue concentrations
(Table 3) are inversely correlated with reported tissue decay
rates (Maser and Trappe 1984). Dihydroquercetin also is not
a strong antibacterial a g ent (Mori et al. 1987).
Chemical components in silver fir heart wood have not
been as thoroughly investigated as the other species. Barton
and Gardner (1962) reported matairesinol, hydroxymatairesinol, conidendrin, and other unidentified components
by paper chromatography. Our phenol analyses (Table 1)
indicate their total concentrations in silver fir are below the
quantities in western hemlock. This may explain, in part,
the weaker decay resistance of silver fir.
It is not uncommon to find low concentrations of heart
wood extractives in the sapwood (Hancock 1957; Gardner
and Barton 1960; Barton and Gardner 1966; Swan et al.
1969; Barton 1970), but this tissue is so susceptible to decay
that differences in resistance amon g species have been
considered negligible (Scheffer and Cowling 1966). Our total
phenol and tannin data show only small differences between
taxa, suggesting sapwoods' decay rates will be more uniform
than heart woods'. When treated with various organisms,
in vitro, the decay rates of Douglas-fir, western hemlock,
and red cedar sapwood were quite similar (Ellyn and Highley
1976).
Inner and outer conifer barks are chemically more similar
to each other than to sapwood or heart wood, yet are still
quite distinct (Fen gel and Wegener 1984). No consistent
pattern for total phenols was observed amon g species for
the inner bark relative to heart wood and sapwood. However, tannin concentrations were consistently hi g her in the
inner bark. Other differences from sapwood and heart wood
include lower cellulose and higher li g nin concentrations in
the inner bark of all species, with the exception of red cedar
inner bark, which had the lowest lignin content (M.E.
Harmon, unpublished data). Tissue density changes indicate
inner bark may be less resistant to decay than sapwood; this
may result not because it lacks defensive chemicals, but
because of hi gher concentrations of carbohydrates, nitrogen,
and minerals that are readily available to the decomposers
(Maser and Trappe 1984; Harmon et al. 1986). Moreover,
a greater portion of inner bark may be inoculated with decay
organisms by bark beetles than sapwood by ambrosia beetles
(Carpenter et al. 1988). It is interestin g that western red cedar
inner bark decomposes slowly. Zhon g and Schowalter (1989)
found this species the least attacked by bark and ambrosia
beetles. This is not likely a phenol or tannin effect, because
red cedar had the lowest concentrations of both. It also had
the lowest lignin and highest cellulose content of all inner
barks (M.E. Harmon, unpublished data).
Outer bark contained the highest concentrations of total
phenols (except red cedar) and tannins (except Douglas-fir)
within each taxa. It was very distinct from the heart wood
and sapwood. High concentrations of these compounds in
the outer bark is consistent with their function in chemical
defenses. After 2 years, density changes in the outer barks
of Dou glas-fir and western hemlock were insi g nificant compared with their inner barks.
Total quantities of defensive chemicals such as phenols
will not necessarily show a stron g correlation with decay
resistance in all instances. For whole dead trees, there are
many mediating and confounding factors, including
(i) different rates of tissue colonization by insects and
inoculation by microorganisms (initial insect attack and
subsequent microbial establishment is confined to the inner
bark and sapwood); (ii) qualitative and quantitative variation in the chemical components between tissues; (iii) differential toxicities of tissue chemicals for various micro-
KEE SLY AND HARNION
1033
200
TABLE 3. Dihydroquercetin concentrations (mg/g
dry wt.) in the tissues of Douglas-fir trees and
1-year-old logs
Logs
Trees
OB
IB
SW
HW
17.7
0.4
1.1
52.6
0.4
1.9
8.4
17.3
NorF: OB, HW, SW, IB = outer hark, heart wood, sapwood,
and inner hark, respecti\ ely. Means of log tissues ,A ere all
significantly different (p < 0.05). Tree data were not analyzed
statistically. Log means have been adjusted for unequal sample
sizes.
fir there were no differences between the two (Table 2). In
all species but Douglas-fir, there were no differences in the
heart wood and sapwood tannin levels. Sapwood of
Dou g las-fir had more tannins than the heart wood. For all
species, both bark tissues contained much greater quantities
of tannins than the sapwood or heart wood.
Comparing tissues across species, the outer bark of
hemlock had more than twice the tannins of any other
species. Inner hark of Dou g las-fir had 4 and 6 times more
tannins than the inner barks of hemlock and red cedar,
respectively. Dou glas-fir was the only species with a sapwood
tannin content si g nificantly different from the others. There
were no differences between species in heart wood tannins.
It was apparent from the color and the appearance of the
diffusion rin g s that the bark tannin components were not
the same as those in the sapwood and heart wood. Bark
extracts formed distinct rin g s that were colored: shades of
yellow or whitish yellow for Douglas-fir and silver fir, and
salmon pink for hemlock and red cedar. Rin gs from sapwood
and heart wood were white to nearly clear, and the edge of
these rin g s were often fuzzy and indistinct. Tannic acid rings
were opaque white with sharply defined edges.
Except for Douglas-fir, the tannin concentrations of trees
and 1-year-old lo g s were similar. Differences for Dou g lasfir tissues were most likely the result of limited sample size
for the trees. As for the phenols, it appears that the tannin
concentrations probably did not change much in the log
tissues during the 1st year on the ground.
As demonstrated by Wisdom et al. (1987), the source of
a tannin standard utilized in preparing calibration curves
can have a significant effect on the results of the analyses.
Tannin standards we isolated produced distinct curves with
the exception of Dou g las-fir and western red cedar (Fi g . 1).
They were all appreciably different from purified commercial tannic acid, a commonly utilized standard (Cates and
McElroy 1987; Hagerman 1987).
Dihydroquercetin
Concentrations of dihydroquercetin in Dou g las-fir were
hi g hest in the outer bark, followed hy the heart wood,
sapwood, and inner hark (Table 3). Outer bark had 44 times
more dihydroquercetin than the inner bark. Concentrations
in tree tissues decreased in the same order, but quantities
in the outer bark and heart wood were twice the amounts
in logs. Similar concentrations have been reported for the
woody tissues (Hancock 1957; Gardner and Barton 1960).
Tissue-density changes
Despite 2 years of decay, lo g s lost <1.5 07o of their initial
mass. After 1 year, lo g tissue density was not significantly
different from initial values (Table 4). After 2 years, the den-
Tannic acid
Pacific silver fir
Douglas fir
lE
150
• Western redceder
0
0
o Western hemlock
E
E
7-2) 100
ct
50
0
100
200
300
400
Micrograms of tannin per well
F IG. 1. Tannin standard curves measured by radial diffusion
(Hagerman 1987).
sity of Douglas-fir, silver fir, and western hemlock inner
bark decreased si g nificantly (21.2, 12.9, and 26.5 0 'o, respectively). Sapwood density of silver fir and Douglas-fir
decreased 2.6 and 5.3 0ro, respectively, whereas that of red
cedar and hemlock remained unchan g ed. Lack of decay in
red cedar inner bark may be a reflection of a limited attack
by insects.
Discussion
The four conifer species studied represent a decay-resistant
gradient, with silver fir the least and red cedar the most resistant. Western hemlock and Douglas-fir are intermediate.
Our analyses show that the concentration of extractable total
phenols in heart wood corresponds to species decay
resistance ratings (Scheffer and Cowlin g 1966). Red cedar
heart wood contained four times more total phenols than
silver fir. However, our tannin analyses (Ha g erman 1987)
showed no si g nificant differences bet \\ een species.
Therefore, the mode of action of phenol-mediated resistance
is not merely the formation of tannin precipitated protein
complexes. Polyphenols in cedar heart wood resemble
tannins in appearance, but do not have protein precipitating
(tanning) properties (Barton and MacDonald 1971). Heart
wood phenols may function as inhibitors of extracellular
enzymes produced by microorganisms (Loomis and Battaile
1966; MacRae and Towers 1984) and (or) re g ulators of
fun gal growth (Rennerfelt 1948; Shain and Hillis 1971; Trust
and Coombs 1973; Haars et al. 1981).
Concentrations of biolo gically active compounds in heart
wood could be a key factor controlling lo g decay rates,
because this tissue is usually most abundant in coarse woody
debris. Western red cedar contains hi g h levels of heart wood
extractives. Water-soluble phenolics, primarily the lionan
1036
CAN. J. FOR. RES. VOL. 19, 1989
and ATKINSON, J.M. 1954. Toxicity tests of a watersoluble phenolic fraction (thujaplicin-free) of western red cedar.
Can. J. Bot. 32: 308-309.
ROFF, J.W., and WHITTAKER, E.I. 1959. Toxicity tests of a new
tropolone, /.3-thujaplicinol (7-hydroxy-4-isopropyltropolone)
occurring in western red cedar. Can. J. Bot. 37: 1132-1134.
ROSENTHAL, G.A., and JANZEN, D.H. (Editors). 1979. Herbivores: their interaction with secondary plant metabolites.
Academic Press, New York.
RUDMAN, P. 1962. The causes of natural durability in timber. IX.
The antifungal activity of heartwood extractives in a wood
substrate. Holzforschung, 16: 74-77.
1963. The causes of natural durability in timber. XI. Some
tests on the fungi toxicity of wood extractives and related compounds. Holzforschung, 17: 54-57.
1965. The causes of natural durability in timber. XVIII.
Further notes on the fungi toxicity of k■ood extractives.
Holzforschung, 19: 57-58.
SAS INSTITUTE INc. 1987. SAS/STAT guide for personal computers, version 6. SAS Institute Inc., Cary, NC.
SCHEFFER, T.C., and COWLING, E.B. 1966. Natural resistance of
wood to microbial deterioration. Annu. Rev. Phytopathol. 4:
147-170.
ROFF, J.W.,
R.F., and RUDINSKY, J.A. 1968. Effect of competition
on survival in western Oregon of the Douglas-fir beetle. Oreg.
State Univ. For. Res. Lab. Res. Pap. No. 8.
SHAIN, L., and HILLIs, W.E. 1971. Phenolic extractives in
Norway spruce and their effects on Fames annosus.
Phytopathology, 61: 841-845.
SWAN, E.P., JIANG, K.S., and GARDNER, J.A.F. 1969. The
lignans of Thrift] plicata and the sapwood-heartwood transformation. Phytochemistry, 8: 345-351.
TRUST, T..1., and COOMBS, R.W. 1973. Antibacterial activity of
13-thujaplicin. Can. J. Microbiol. 19: 1341-1346.
VANDI. CASTEELE, K., GEIGER, H., and VAN SUMERE, C.F. 1982.
Separation of flavonoids by reversed-phase high-performance
liquid chromatography. J. Chromatogr. 240: 81-94.
WISDOM, C.S., GONZALEZ-COLOMA, A., and RLNDEL, P.W.
1987. Ecological tannin assays: evaluation of proanthocyanidins,
protein binding assays and protein precipitating potential.
Oecologia (Berlin), 72: 395-401.
ZHONG, H., and SCHOVs ALTER, T.D. 1989. Conifer bole utilization by wood-boring beetles in western Oregon. Can. J. For.
Res. 19: 943-947.
SCHNIITZ,
KELSEY AND HARMON
organisms; (iv) varying concentrations of substrates essential
for the growth of microorganisms; (v) ecological interactions within microbial communities; and ( vi) the physical
environment, i.e., water, CO 2 , 02 , and temperature, which
all interact to determine decay rates (Harmon et al. 1986).
In general, tissues with the highest extractable total
phenols within a species, outer bark and heart wood, decay
most slowly, as indicated by density changes. Total phenol
concentrations in the heart wood also correspond to the
reported decay resistance rating between species. Although
highest in the outer bark, extractable tannin concentrations
do not seem to correspond with tissue decay rates within,
or between, species. Inner bark has greater quantities of
phenols and tannins than sapwood, but is less resistant, most
likely due to other factors, such as rate of insect and
microbial colonization and substrate components favoring
the growth of decay organisms. As demonstrated in this
study, decay resistance of dead tree tissue correlates with
phenol concentrations, providing strong evidence that these
compounds are mediators in the decomposition of coarse
woody debris.
Acknowledgments
The authors thank the National Science Foundation for
financial support from grants BSR-8516590 and
BSR-8514325 and also the USDA, Forest Service, Pacific
Northwest Research Station, Portland. Oregon. They thank
Dr. Joe Karchesy for advice on tannin isolation, Liz Gerson
for her assistance in the laboratory, and Tom Sabin for his
help with the statistical analyses.
BARTON, G.M.
1970. New polyoxyphenols from western hemlock
sapwood. Wood and Fiber, 2: 144-150.
BARTON, G.M., and GARDNER, J.A.F. 1962. The occurrence of
matairesinol in mountain hemlock ( Tsuga mertensiana), western
hemlock ( Tsuga heterophylla), and balsam (Abies amabilis).
J. Org . Chem. 27: 322-323.
1966. Brown stain formation and the phenolic extractives
of western hemlock ( Tsuga heterophylla (Rai.) Sarg.). Can. For.
Branch Dep. Publ. No. 1147.
BARTON, G.M., and MAcDoNAI I), B.F. 1971. The chemistry and
utilization of western red cedar. Can. For. Serv. Publ. No. 1023.
CARPENTER, S.E., HARMON, M.E., INGHAM, E.R., KELSEY,
R.G., LATTIN, J.D., and SCHOW ALTER, T.D. 1988. Early pat-
terns of heterotroph activit y in conifer logs. Proc. R. Soc. Edinb.
Sect. B (Biol. Sci.), 94: 33-43.
CATES, R.G., and McEt ROY, D.C. 1987. Secondary chemistry
among sympatric plant species and its relationship to insect
herbivory. USDA For. Serv. Gen. Tech. Rep. INT-222.
pp. 18-29.
COOK, J.W., G IBB, A.R., RAPHAEL, R.A., and SOMERVILLE,
A.R. 1951. Tropolones. 1. The preparation and general
characteristics of tropolone. J. Chem. Soc. pp. 503-511.
DYRNESS, C.T., FRANKLIN, J.F., and MoiR, W.H. 1976.
A preliminary classification of forest communities in the central portion of the western Cascades in Oregon. U.S./IBP Conif.
For. Biome Bull. No. 4.
ESLYN, W.E., and HIGHLEY, T.L. 1976. Decay resistance and
susceptibility of sapwood of fifteen tree species. Phytopatholoey,
66: 1010-1017.
FENGEL, D., and WEGENER, G. 1984. Wood: chemistry,
ultrastructure, reactions. Walter de Gruyter, Berlin.
GARDNER, J.A.F.. and BARTON, G.M. 1960. The distribution of
dih y droquercetin in Douglas-fir and western larch. For. Prod.
J. 10: 171-173.
1035
E., and MCKEREGHAN, M.R. 1964. The
inhibition of dopamine-(-hydroxylase by tropolone and other
chelating agents. Biochem. Pharrnacol. 13: 1103-1106.
HAARS, A., CHET, 1., and HUTTERMANN, A. 1981. Effect of
phenolic compounds and tannin on growth and laccase activity
of Fomes annosus. Eur. J. For. Pathol. 11: 67-76.
HAGERMAN, A.E. 1987. Radial diffusion method for determining
tannin in plant extracts. J. Chem. Ecol. 13: 437-449.
HAGERMAN, A.E., and KLUCHER, K.M. 1986. Tannin-protein
interactions. In Plant flavonoids in biology and medicine:
biochemical, pharmacological, and structure-activity relationships. Edited by V. Cody, E. Middleton, Jr., and J.B. Harborne.
Alan R. Liss, Inc., New York.
HANCOCK, W.V. 1957. The distribution of dihydroquercetin and
a leucoanthocyanidin in a Douglas-fir tree. For. Prod. J. 7:
335-338.
HARBORNE, J.B. 1982. Introduction to ecological biochemistry.
2nd ed. Academic Press, London.
HARMON, M.E., FRANKLIN, J.F., SWANSON, F.J., SOI.LINS, P..
GREGORY, S.V., L ATTIN, J.D., ANDERSON, N.H., Cl !NE, S . P.,
GOLDSTEIN, M., LAUBER,
ADMEN, N.G., SEDELL, J.R., LIENKAEMPER, G.W., CROMACK,
K., J R., and CuI\IlNS, K.W. 1986. Ecology of coarse woody
debris in temperate ecosystems. Adv. Ecol. Res. 15: 133-302.
H.L. 1960. Chemical composition of tannins and
polyphenols from conifer wood and bark. For. Prod. J. 10:
610-617.
HOLMES, G.W., and KURTH, E.F. 1961. The chemical composition of the newly formed inner bark of Douglas-fir. Tappi, 44:
893-898.
JULK1 NEN-TIITTO, R. 1985. Phenolic constituents in the leaN es of
northern willows: methods for the analysis of certain phenolics.
J. Agric. Food Chem. 33: 213-217.
KAHN, V., and ANDRAWIS, A. 1985. Inhibition of mushroom
tyrosinase by tropolone. Phytochemistry, 24: 905-908.
KENNEDY, R.W. 1956. Fungicidal toxicity of certain extraneous
components of Douglas-fir heartwood. For. Prod. .1.6: 80-84.
KRAHNIER, R.L., HEMINGWA), R.W., and Hii I is, W.E. 1970.
The cellular distribution of lignans in Tsuga heterophylla wood.
Wood Sci. Technol. 4: 122-139.
LEACH, .1.G., ORR, L.W., and CHRISTENSEN, C. 1934. The interrelationships of bark beetles and blue-staining fungi in felled
Norway pine timber. J. Aerie. Res. 49: 315-341.
L OOMIS, W.D.. and BATTAILE, J. 1966. Plant phenolic compounds and the isolation of plant enzymes. Phytochemistry, 5:
423-438.
NIACDON.ALD, B.F., and BARTON, G.M. 1973. Lignans of western
red cedar ( Thuja plicata Donn.) XI. 0-Apopli,:atitoxin. Can. J.
Chem. 51: 482-485.
NlAcLEAN, H., and GARDNER, J.A.F. 1956a. Analytical method
for thujaplicins. Anal. Chem. 28: 509-512.
1956b. Distribution of fungicidal extractives (thujaplicin
and water-soluble phenols) in western red cedar heartwood. For.
Prod. J. 6: 510-516.
MACRAE, W.D., and TOWERS, G.H.N. 1984. Biological activities
of lignans. Phytochemistry, 23: 1207-1220.
MASER, C., and TRAPPE, J.M. 1984. The seen and unseen world
of the fallen tree. USDA For. Serv. Gen. Tech. Rep. PNW-164.
NloRi„A., N ISHINO, C., ENOKI, N., and TAw A t A, S. 1987.
Antibacterial activity and mode of action of plant flavonoids
against Proteus vulgaris and Staphylococcus aureus.
Phytochemistry, 26: 2231-2234.
NIKAIDO, T., O HNIOTO, T., KINOSHITA, T., SANRAwA, U.,
N ISHIBE, S., and N ISADA, S. 1981. Inhibition of cyclic .AMP
phosphodiesterase by lignans. Chem. Pharm. Bull. (Tokyo), 29:
3586-3592.
RENNERFEI.T, E. 1948. Investigations of thujaplicin, a fungicidal
substance in the heartwood of Thuja plicata. D. Don. Physiol.
Plant. 1: 245-254.
R ICE, E.L. 1984. Allelopathy. Academic Press, New York.
HERGERT,