250
Differences
BetweeJl Douglas-fir
Genoty pes ill
Relation
to Browsing Preference by Black-tailed Deer M. A. RADWAN Forestry Sci(,IICt'S Lahol'UlOry, Pacific Nortll1l'('s/ Forq'/ alld Rallge Erp<,rilll(,l1/ S/a/ioll, U.S.D.A. FUN's/ Senice, OIYll1pia, Washillg/oll, U.S.A.
Reccivcd December 21, 1971
RADWAN, M. A. 1971. Differences between Douglas-fir genotypes i n relation to browsing prefer­
ence by black-tailed deer. Can. J. Forest Res. 2, 250-255.
Tissue digestibility, essential oils, and levels of scyeral chemical constituents were compared ill
foliage of Douglas-fir. Genotypes with different susceptibilities to deer browsing were used, and
foliage was collected during the Jormant season. In general, clones resistant to deer browsing had
lower dry-matter and cellulose digestibilities, essential oils with greater inhibitory action on rumen
microbial activity, higher content of fats, total phenols, fiavanols, and lcueoanthocyanins, and lower
levels of chlorogenic acid. Results suggest that these resistance characteristics, especially the chlo­
rogenic acid content, might be used instead of conventional bioassays with captive deer, to screen
Douglas-fir breeding stock for resistance to deer browsing.
RADWAN. M. A. 1972. Differences between Douglas-fir genotypes in relation to browsing prefer­
ence by black-tailed deer. Can. J. Forest Res. 2, 250-255.
La digestibilite des tissus, les huiles essentielles et les taux de differents constituants chimiques
du feuillage de Sapins de Douglas ont ete compares. Des genotypes possedant differentes suscepti­
oilites au broutement des cerfs ont ete utilises et Ie feuillage a etc recolte durant la saison morte.
])'une fa on gencrale, les clones resistant au broutement des cerfs montrent une digestibilite moin­
dre en ce qui concerne la matiere seche et la cellulose, ils contiennent des huiles essentielles it forte
action inhibitrice sur l'activite microbienne du rumen, un taux plus eleve de graisses, de phenols
(tota]), de flavanols et de leucoanthocyanines, et des taux plus faibles d'acide chlorogenique. Ces
resuitats conduisent it proposer que ces caracteristiques de resistance, et spccialement Ie taux d'acide
chlorogenique, soient utilisees it la place des essais conventionnels chez lcs cerfs en captivite, en vue
de la selection genetique des stocks de Sapins de Douglas resistant au broutement des cerfs.
[Traduit par Ie j ournai]
Introduction Feeding by black-tailed deer (Odocoileus
hemionus colulllbianus Richardson) can se­
riollsly damage Douglas-fir (Pset/{lotsuga Ii/en­
::iesii (Mirb.) Franco) regeneration in the
Pacific Northwest (4, 8). Much work has been
done on measures to alleviate deer browsing,
and reasonable success has been achieved
with some control methods (25). However,
'
recent 0 bjections to use of synthetic chemicals
and the high cost of mechanical protection
dictate that new approaches to the problem
be considered. Tree breeding for resistance
to deer browsing is one approach now under
consideration.
Recent tests with captive black-tailed deer
by Dimock (11) showed that different Doug­
las-fir genotypes varied widely in susceptibility
to browsing. The minor morphological dif­
ferences between these genotypes suggest
that the key to racial variation in traits affectConodian Journal of Forest Research. 2, 250 (1972)
ing deer preference may reasonably be sought
through comparative studies of the trees'
chemical characteristics. In addition to provid­
ing knowledge basic to a better understanding
of plant-animal relationships, information
obtained could contribute significantly to
screening tests necessary in breeding for less
damage-prone Douglas-fir.
This investigation, therefore, deals with
foliage from different Douglas-fir. clones
previously rated for susceptibility to deer
browsing. Comparative tests included both
chemical analyses and in vitro fermentations
by deer rumen.
Materials and Methods
Plal/l Material
Foliage was obtained from 10-year-old, IS-foot-tall
Douglas-fir trees grown at the Olympic National Forest's
Dennie
Ahl Seed
Orchard
in
western
Washington.
Three grafted clones (SD-13, SD-19, SD-22) were used
in all work except in the separation of phenolic com-
RADWAN: DOUGLAS-I'm GE:-;OTYPES
pounds where a fourth clone (SD-IO) was added. De­
creasing order of foliage susceptibility to deer browsing
as determined with captive deer in 2 consecutive years
was: S D-19, SD-IO, SD-22, and SD-13 (II). Two com­
posite samples of about (JOO g each were collected in
mid-morning from each clone during the dormant seasons
(February-March) of 1968 and 1969. Each sample was
taken from 10 to 15 trees selected at random each year
and consisted of 3-in. tips of secondary laterals at a
height of about 5 ft up the trecs. Reproductive buds
were removed if prescnt, and samples were individually
sealed in glass containers and brought to the laboratory
in a portable cooler.
Sample Preparation for Analysis
Fresh foliage was chopped into small pieces. Sub­
samples were taken for determination of moisture, total
phenols, flavanols, and leucoanthocyanins, also for
chromatographic separation of phenolic compounds
and distillation of essential oils. Remaining tissue was
dried to constant weight at 65°C, ground to 40 mesh in
a Wiley mill, and stored in closed containers at -15°C
until used.
Digestibility Determillatiuns
Digestibilities of dry matter and cellulose were estimat­
ed by the ill vitro rumen fermentation technique. Deter­
minations were made on Douglas-fir oven-dried tissue
and on cellulose. Solka-F1ocl was used as the cellulose
source in investigating effects of Douglas-fir essential
oils.
(a) Rumen Source
Rumen fluid was obtained from the captive black­
tailed deer herd used in evaluating the clones' relative
susceptibility to browsing. Rumen fluid was obtained
by squeezing rumen contents, from a freshly sacrificed
deer, through four layers of cheesecloth. The fluid was
immediately gassed with COl while maintained at 39°C:
and after allowing the microorganisms to settle in about
1 h, the fluid in the bottom layer was withdrawn for use
as inoculum.
(b) Essential Oils
Foliage was chopped into small pieces, mixed with
minimum amount of distilled water, and steam-distilled
at atmospheric pressure for about 4 h. Distillates were
saturated with NaCI and the oils extracted with ether.
Extracts were dried over anhydrous Na2S04, and ether
was removed by distillation. Essential oils obtained were
stored at - 15°C until used.
(e) Ferfllentaiio/l COllditiollS
The ill vitro technique used was essentially that de­
scribed by Kutches et al. (19). Each fermentation was
conducted in triplicate on substrates of 0.7 g tissue or
0.2 g Solka-Floc in 100-ml lipless beakers fitted with
gas release valves. the buffer -nutrient solution for each
vessel was 35 ml of C02-saturated McDougall's solution
(22) containing 22 mg urea. Seven milliliters of rumen
fluid served as inoculum, and essential oils used in the
Solka-Floc runs ranged from 0 to O. 10 ml. After addition
I Mention of chemical companies and their products
does not represent endorsement by the Forest Service
or by the Department of Agriculture.
251
of the inoculum, the vessels were swept with C02, stop­
pered, and incubated at 39°c' All samples were fermented'
for 48 h when microbial activity was stopped by adding
3 ml 2 N H2S04 to each vessel. Appropriate unfermented
controls were used to correct results.
(tf) Dry Mattcr ami Ce{{lIlose Digestibilities
Following fermentation, samples were filtered in
sintered glass crucibles and dried to constant weight at
65°C for determination of dry matter. Cellulose in the
residual material was then estimated by the method of
Crampton and Maynard (9). Digestibility percentages
were calculated from losses of dry matter and cellulose
as compared to the unfermented controls.
Chemica! Analysis
Moisture was determined in a forced-air oven at 65°C,
and ground tissue was ashed in platinum, crucibles at
500-550°C, Sugars, extracted with 80o/t, ethanol, and
those resulting from extraction of total available carbo­
hydrates with 0.2 N H2S04 (27) were determined as
glucose by the cerie sulfate method (14). Analyses of
other tissue components were made as follows: crude
fat from loss in tissue weight after extraction with ether
in Soxhlet (15); acid-detergent fiber and acid-detergent
lignin according to Van Soest (32); cellulose by the method
of Crampton and Maynard (9); nitrogen by the micro­
Kjeldahl technique (15); calcium by a titrimetric method
(1 5); magnesium by the magnesium ammonium phos­
phate method (7); phosphorus according to the colori­
metric method of fiske and Subbarow (12); and total
phenols, flavanols, and leucoanthocyanins, obtained by
extraction i n Soxhlet with 80% methanol, by the tech­
niques of Swain and Hillis (31).
All determinations were made in duplicate on each
of two replicate samples.
Chromatography of Phenolic COll1pOlllld
The tissue was defatted with petroleum ether (30-60 0c)
prior to extraction in Soxhlet with 80% methanol. Ex­
tracts were then evaporated to dryness in vaCI/O, and
residues were taken up in 80% methanol for chromato­
graphy.
Separations were made on Whatman No. 1 paper.
Two-dimensional chromatograms were developed with
II-butanol : acetic acid: water,
RAW, 4: I :5, upper
phase (A), or BAW, 4 : I : 2.:2 (B) in the first direction
and with 270 aqueous acetic acid, HOAc, (C) in the
second direction. Separations were also accomplished
by one-dimensional chromatography on paper freshly
washed with 5 c methanol using the solvent methyl­
isobutylketone : formic acid : water, KFW, 14:3:2 (D)
(18). Phenolics \\ere located on the chromatograms by
examination under ultraviolet light (250 and 350 nm)
before and after fuming with NflJ and by colors devel­
oped with several chromogenic sprays (I, 3, 5, 6, 13, 20,
26) .
Positive identification' of chlorogenic acid was made
from Rr measurements; colors with specific spray rea­
gents; and by direct comparison with authentic material
by co-chromatography in fouf solvents, and spectral
analysis. Additional proof was obtained by hydrolysis
of the compound with alkali (2 N NaOH for 2 h under
N2 at room temperatllfc) and with acid (7. N HCI for I h
at 100°C). This was followed by chromatogmphic separa­
tion and identification of the prodUcts.
252
C.\:\.\Dl.\
.1On :\ ..\L UF FOI EST RESEARCH. VOL. 2. 1<),1
rollowing identification, chlo ogenic acid was elutcd
from the chromatograms and quantifieJ spectl'Ophoto­
metrically at 3280111 using apprnpriak blanks. A\'erage
concentrations in the tissues w re caJculakd b a sed lIpon
two replicates with three chromatogr<1Il1s per replicate.
Statistical Allalysis
Data wcrc subjected to analysis of va riance , and flleanS
were separated (lccording to Tuk e y' s tcst as required (28).
Results
In Vitro Digestibilities of Foliage
Avcragc dry-matter and cellulose digcstibil­
ities of all clones were 15.3(;; and 13. 2S·;,
respectively (Table I). Dry-matter values
are in agrcement with those obtained by others
(10) and indicate that Douglas-fir, at least
during the dormant season, has a relatively
low digestibility.
Foliage of SD-19, the clone most susceptible
to deer browsing, showed the highest digest­
ibilities. However, there were no significant
differences between the other two clones.
EfJeet of Essential Oils
011
Cellulose Digest-
ibility ill Vitro
Without Douglas-fir oils, microorganisms
of deer rumen digested 62. 5(; of the cellulose
TABLE 1. 111 vitro digestibilitics * of Douglas-fir ge­
notypes by rumen of black-tailed deer (%)
Douglas-fir c10nest
Dry matter
Cellulose
SD-13
SD-19
SD-22
14.4 a
18.6 b
12.9 a
11.4 a
15.8 b
12.4 a
'Determined by the itt vilro fermentation technique. Values
afC means of three determinations, and means within columns
followed by the same letter designation do not differ significant!,·
at tit(' 5l)b Jevel, using Tukey's te:-it.
tRelative susceptibility of foliage to deer browsing: SD-19 >
SD-22 > SD-B (I I).
TABLE 2.
substrate (Table 2). Digestion was alTected
when the oils were rrescnt, and efTects varied
with the level and sourcc of oil. 111 general,
digestibility was slimulated by low oil levels
and inhibited by high concentrations. Effects
of SD-19 oil, however, were chiefly stimulatory
but those of oils frolll the clones less suscept­
ible to deer browsing, SD-13 and S D-22,
were mostly inhibitoI y, Similar difTerential
effects by essential oils on deer-rumcn activity
havc rL'cently been dcmonstrated with oils
isolated from ncedles of old and ncw growth
of Douglas-fir (2'1).
Selected Chemical Constituents ill Foliage
Concentrations of the chemical constituents
of Douglas-fir foliage, summarized in Table
3, are exprcssed on a fresh-weight basis to
allow comparison of levcls as the animal
encounters them in feeding.
Variations among Douglas-fir clones were
significant in only four of the chemical con­
stituents. Foliage of S D-19, the clone most
susceptible to deer browsing, had lower fat,
total phenols, flavanols, and leucoantho­
cyanins than foliage of the other two clones.
Because of the difTerences obtained with
the phenols, it was decided to study this
group of compounds further and to include
one more clone, SD-IO. The paper chromato­
graphic method was chosen for this study.
Examination of chromatograms of the
methanol extracts of the different clones
showcd that Douglas-fir contained several
important phenolic and non-phenolic com­
pounds. Preliminary survey and provisional
identification showed that the main com­
pounds present in all extracts included poly-
Effect of increasing amounts of essential oils isolated from Douglas-fir genotypcs on digestibility of cellulose
substrate by rumen of black-tailed deer ill l'itro ('10)*
Clonal source of essential oilsl
Essential oils
added to
substratet
(ml)
Digestibility
Relative
digestibility
Digestibility
Relative
digestibility
Digestibility
Relative
digestibility
0.00
0.02
0.05
0.10
62.5 a
72.6 b
59.0 a
l.4 c
100.0
116.2
94.6
2.2
62.5 a
74.9 b
72.0 b
59.1 a
100.0
119.8
115.2
94.6
62.5 a
70.6 b
45.9 c
0.2 d
100.0
113.0
73.4
0.3
SD-l3
SD-22
SD-19
·Dig('stibiIity determined by the in l';lro frrmr-ntation t('("hnique. Values are mean s of three determinations and tn{'al1s
within columns
.
by the same lelter designation do not differ significantly at the $';0 leYel, ,,,ing Tukey's
. test.
tOils Were stearn-distilled from foliage. and Solka-Floc ""rvcd as substrate. :Relative susceptibility of foliage to deer browsing: SD·19 > SD-22 > SD-13 (II). followed
RADW,\N: DOUGLAS·FIR GE:-IOTVPES '
TABLE 3.
Concentration of selected chemical constitucnt
Douglas-fir genotypes (%)*
253
"
in the fresh folillge of
Douglas·fir clonest
Chemical constituent
SO-13
Moisture
Ash
Total sugars
Total available carbohydrates
Crude fat
Acid-detergent fiber
Acid-detergent lignin
Cellulose
Total nitrogen
Calcium
Magnesium
Phosphorus
Total phenols
Flavanols
Leucoanthocyanins
53.14 a
1.34 a
5.06 a
7 .26 a
4.10 a
1 3 .88 a
7.80 a
6.79 a
0,67 a
0.34 a
0.07 a
0.06 a
2.11 ab
1.66 ab
0.36 a
SD-22
54.74 a
1.38 a
4.74 a
7,05 a
3.68 b
13.90 a
7.64 a
6.56 a
0,60 a
0.35 a
0.07 a
0.06 a
1 .7 9 b
1.30 b
0.28 b
53.24 a
1.41 a
4.62 a
6.90 a
4.00 ab
14.16 a
8.12 a
6.50 a
0.60 a
0.37 a
0.08 a
0.06 a
2.34 a
1.80 a
0.36 a
-------- ------
'Values are averages of two composite samples from 10-15 trees each. Means within each chemical
constituent followed by the same letter designution(sl do not differ significantly at the 5';;;' le\·cl.
using Tukey's test.
tRelative susceptibility of foliage to deer browsing: SD-19 > SD·22 > SD-13 (11).
meric polyphenols, catechins, ftavonoids, dep­
sides of phenolic acids, glycosides, esters,
caffeic acid, quinic acid, and shikimic acid.
However, it appeared that clonal variations
occurred only i n two of these compounds;
one remains unknown and will be reported
after identification, and the other was ident­
ified as 3-0-caffeoylquinic acid (chlorogenic
acid),
Identification and Quantification of Chlol'ogcnic
Acid (eGA)
The suspect compound and authentic CGA
were
identical
in
physical
and
chemical
characteristics. Thus, fluorescence under ul­
traviolet light,
and reactions
(C) for quinic acid, and 0.79 (A) and 0.25,
0.61 (C) for catfeic acid.
Average concentration of CGA (ppm)
in the fresh foliage varied significantly (p
0.05) among the four clones as follows:
SD-19, 390; SD-lO, 170; SD-22, 113; and
SD-13, 75. These data indicate that Douglas.
fir foliage contained Jow to moderate con­
centrations of eGA compared to other
plant species (29), More important, results
clearly show the association of eGA with
susceptibility to deer browsing. Thus, accord­
ing to both parameters, the studied clones
rank as follows: SD-19 > SD-IO > SD·22 >
=
SD-13.
with several
spray reagents were typical of CGA (16,
26, 30). Co-chromatography with authentic
CGA gave one spot in each of 3 solvents and
a pair of sp'ots for the cis and trans forms,
characteristic of cinnamic aeid derivatives,
in 2% HOAe (33). Average Rf values were:
0.60 (A); 0.64 (B); 0.54, 0.72 (C); and
0,49 (D). Absorption spectra were similar
to those in the literature (2, 16), Amax ( 70S
328 nm with shoulders at 300,
methanol)
233, and 216 n m; l'max (KBr)
1260, 1630,
and 1720 cm-I. Further, both acid and
alkaline hydrolyses yielded quinic and caffeic
acids. Rf values were: 0.26 (A) and 0.89
=
=
Discussion and Conclusions
Results show that digestion of Douglas.flr
foliage by deer rumen in vitro Was minimal
during the dormant season; digestibilities
were well below those for pelle ted alfalfa
(10). Although this may be due to the essential
oils, which at high concentrations inhibited
rumen microbial activity, effects of other
substances present in the tissue cannot be
ruled out. Some phenolic compounds, for
example, have been reported as inhibitory
(21).
Comparison of the different Douglas-fir
genotypes indicated variations in tissue digest­
254
CANADIAN JOllRNAL OF FOREST RES ;:\RCH. VOL. 2,
ibili ty in effects of essential oils on cellulose
fermentation, and in chcmical composition,
,
Thus, clones with relatively hi h resistance
to deer browsing had lower dry-matter and
ccllulose digestibilities; essential oils with
greater inhibitory action on rumen microbial
function: higher
content of fals, toti\l phenols,
y
.
ftavanols, and Jeucoanthocyiln1l1S; and lower
levels of chlorogenic acid than the genotypes
preferred by deer. Obviously, it is impossible
to state conclusively the factor or combina,
tion of factors which were actually responsible
for the preferential differences or the mech.
anlsm(s) by which any of these factors were
operating. It is possible that essential oils
and perhaps some phenols affected deer
selectivity through their anti-rumen microbial
action (23). It is also possible that flavanols
and leucoanthocyanins, which are astringent
(17), influenced browsing through their effect
on taste. However, the role of chlorogenic
acid remains obscure, perhaps because its
presence has not previously been reported
in Douglas-fir and its physiological function
in animals has not been investigated.
Because of the scarcity of Douglas-fir clones
ranked for browsing susceptibility, only four
clones were studied. We have recently ex­
panded our work with additional clones
and progeny of the same genotypes as material
became available. Preliminary data from this
work support findings reported here and
show that the resistance characteristics stu­
died so far are transmittable through breeding.
•
Clearly, therefore, the results Ilppear to have
utility in breeding Douglas-fir for resistance
to deer browsing. Instead of the conventional
testing with captive Mer, one or more of the
d er-browsi/1g resistance characteristics found
in the present study might be u;,cd to select
the desired trees. For this purpose, selection
based upon the chlorogenic acid content seems
most promising.
Thanks are due to Dr. J. Corse, Western Regional
Research Laboratory, U,S. Department of Agriculturl),
Berkeley, California, for the authentic sample of chloro.
genic acid and to E. Dimock, Forestry Sciences L>lbora­
tory, and V. Allen, Olympic National Forest, U.S.
Forest Service, Olympia, Washington, for providing the
Douglas-fir clonal material.
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Reproduced by the FOREST SERVICE
U. S. Department of Agriculture For Official Use from Canadian Journal of Forest Research
Vol. 23 No. 3 19?2
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