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LIBRA RY

'"“ " Michigan State
University

 

This is to certify that the
thesis entitled
Formation of Heartwood and Discolored

Sapwood in White Oak and White Spruce

presented by

John Frederick Wardell

has been accepted towards fulfillment
of the requirements for

Ph - D . degree in leant
Pathology

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Cik and whit

ABSTRACT

FORMATION OF HEARTWOOD AND DISCOLORED
SAPWOOD IN WHITE OAK AND WHITE SPRUCE

BY

John Frederick Wardell

Formation of heartwood and discolored sapwood was

studied in white oak (Quercus alba L.) and white spruce

 

(Eige§.glauca (Moench) Voss). An increment borer was
used to induce formation of discolored sapwood.

Qualitative differences in ether and butanol-
soluble compounds were detected between sapwood (SW),
heartwood (HW) and discolored sapwood (D8) of white
oak and white spruce with paper chromatography. Few
qualitative and quantitative differences in ether-
soluble compounds were detected between US and HW of
white oak with thin-laYer chromatography. .Many qualita-
tive and quantitative differences were detected between
these tissues and SW. D8 of white spruce was quantita-
tively different from SW and HW which were similar to
each other. A

The degree of similarity of chemical constituents
between SW, HW and DS was determined for ether and

butanol-soluble compounds from both tree species. Every

“mound detec‘
master in CC
:3: of 5min:

:ak and White 5

:ezical compo

ietected durin'
Eatassium and 3
in Mg and Mn ‘
:;;‘:.est in SW.
iiiferent from
Pctassium incrl
3i. Phosphoro
and Al increas‘
decreased in D:
A8“ C0:
5"“ and lowest
tissues Were n
water eXtract ‘

q

%.
Ash conte

John Frederick Wardell

compound detected in a tissue was considered as a separate
character in constructing a similarity index. Calcula-
tion of similarity indexes between the 3 tissues of white
oak and white spruce showed that they were different in
chemical composition from each other.

Changes in levels of inorganic elements were
detected during formation of DS and HW in both tree species.
Potassium and P decreased in D8 and HW of white spruce,
but Mg and Mn decreased only in D8. Calcium and Mn were
highest in HW. Few changes, however, were significantly
different from levels of the same element in SW.
Potassium increased in D8 of white oak, but decreased in
HW. Phosphorous, Mg and Mn decreased in HW while Cu, B
and Al increased in D8. Calcium sometimes increased or
decreased in D8 and decreased in HW.

Ash content was highest in HW, intermediate in
SW and lowest in DS of white spruce. Differences between
tissues were not always significant. The pH of the cold
water extract was similar from SW and HW and lowest for
DS. Ash content was highest in DS, intermediate from
SW and lowest in HW of white oak. The pH of the cold
water extract was highest from DS, intermediate in SW
and lowest from HW.

An electron microprobe x-ray analyzer - scanning
microscope showed that changes in levels of elements

occurred at different radial positions in SW and HW of

John Frederick Wardell

both tree species. Differences in levels of elements were
detected between ray and non-ray cells. The distribution
of elements in DS was different from that in SW.
Phosphorous, Ca and Mg were studied in both tree species
while Mn, K, Cl, S and 0 were also studied in white oak.

Chlorine, S, 0 (white oak) and Mg (white spruce)
were not affected by changes in radial positions in SW or
HW. Phosphorous decreased from outer to inner SW and Ca
was lowest in outer SW of both tree species. Magnesium
and Mn decreased between inner SW and outer HW. Potassium
increased from outer to inner SW, but decreased from
inner SW to outer HW. Calcium, Mg, K and Mn increased
from outer to inner HW of white oak. In white oak, more
P, Mg (DS, SW), Ca, 5 (DS, sw, HW), Mn, K and Cl (sw, HW)
were detected in ray cells than in vascular elements. In
white spruce, more P (SW), Ca (SW, HW) and Mg (US) were
detected in ray cells than in vascular elements.

The distribution of elements in ray and non-ray
cells at different radial positions in D8 of white spruce
injured in spring and white oak injured in winter was not
very different from that observed in SW. Phosphorous
decreased between middle and inner DS while Ca was highest
in inner D8 of white spruce. Calcium and Mn were not changed
at different radial positions in D8 of white oak. More
drastic changes were observed in D8 of white oak injured

in spring. In ray cells, more P was detected in middle

John Frederick Wardell

than in outer D8 which had more P than inner DS. No
difference in P was detected at different positions in
non-ray cells. Differences in K were not detected at
radial positions in D8.

In both tree species, DS was more resistant than
SW (both) and heartwood (spruce) to decay by Poria_monticola
and Polyporous versicolor 4 months after mechanical injury
when trees were wounded in late April. Ellagitannins
were not responsible for the greater durability of D8 of

white oak to P. monticola.

 

Physiological condition of white oak at the time
of mechanical injury affected development of DS. Increases
in K and ash content and development of decay-resistant
discolored sapwood were not observed until 7 months after
mechanical injury when trees were wounded in December,
but increases in K and ash content and development of decay-
resistant discolored sapwood were observed within 4 months
after mechanical injury when trees were wounded in late
APril.

DS and HW are distinct tissues in both tree
sPecies. Formation of DS and HW of white spruce is much

different from that in white oak.

FORMATION OF HEARTWOOD AND DISCOLORED

SAPWOOD IN WHITE OAK AND WHITE SPRUCE

BY

John Frederick Wardell

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1971

PLEASE NOTE:

Some Pages have indistinct
print. Filmed as received.

UNIVERSITY MICROFILMS

 

Dee
for his COt
this invest
wuscript
3r. Eldon .'
Pass-.ussen

n
help in t1:
Paul RasmL
assistance

Tc

patience ,

ACKN OWLE DGMENTS

Deep appreciation is expressed to Dr. John H. Hart
for his counsel and assistance throughout the period of
this investigation and during the preparation of the
nmnuscript. Thanks are also due to Dr. James W. Hanover,
Dr. Eldon A. Behr, Dr. Gary R. Hooper and Dr. H. Paul
Rasmussen for their critical evaluation of the thesis.

Thanks are due to Dr. James W. Hanover for his
help in the paper chromatography studies and to Dr. H.
Paul Rasmussen and Mr. Vivion E. Shull for their
assistance with the electron probe microanalyzer studies.

To my wife, for her encouragement, help and

patience, I am indebted.

ii

LIST OF TAE
LIST OF PIC

INTRODUCTIc
Liter at

PART 1 .

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . .

Literature Cited . . . . . . . . . . . .
PART I. Phenolic Compounds as Criteria for

PART II.

Evaluation of the Chemical Relationship
between Discolored Sapwood, Heartwood and
Sapwood . . . . . . . . . . . .

Materials and Methods . . . . . .
Results and Discussion. . . . . .
Summary. . . . . . . . . . .
Literature Cited. . . . . . . .
Appendix I. . . . . . . . . .

Appendix II
Effect of Polyporou§_versicolor and
Poria monticola on PhenoIic Compounds
from White Oak and White Spruce . .
Literature Cited . . . . . . .

 

Changes in Mineral Content during Forma-
tion of Discolored Sapwood and Heartwood
in White Oak and White Spruce. . . . .

Materials and Methods . . . . . .
Measurement of Ash Content. . . .
Measurement of Levels of Inorganic
Elements with Optical Emission and

'Flame Photometry . . . . . . .
Measurement of pH. . . . . . .
Electron Probe Microanalyzer

Studies . . . . . . . . . .

iii

Page

12
39
41

44

69
95

97

102
102
104
104

105

PART III.

Results and Discussion. . . . .
Levels of Inorganic Elements, Ash
Content and pH of Tissues from
White Spruce . . . . . . .
Levels of Inorganic Elements, Ash
Content and pH of Tissues from
White Oak . . . . . . . .
Electron Probe Microanalyzer
Studies - White Spruce . . . .
Electron Probe Microanalyzer
Studies - White Oak . . . . .

Summary. . . . . . . . . .
Literature Cited. . . . . . .

The Durability of Sapwood, Heartwood
and Discolored Sapwood of White Oak and
White Spruce to Polyporous versicolor
and Poria monticola . . . , , , ,

Materials and Methods . . . . .
Measurement of Durability of
Discolored Sapwood, Sapwood and
Heartwood . . . . . . . .
Extraction of Tissue of White Oak
for Ellagitannins. . . . . .

Results and Discussion. . . . .
Durability of Discolored Sapwood,
Sapwood and Heartwood to Polyporous
versicolor and Poria monticola .
DistributiOn of_E11agitannins in
Tissues of White Oak. . . . .

 

smary O O O O O O I O O 0
Literature Cited. . . . . . .
Appendix

Analysis of Lignans in White Spruce

with Gas Chromatography. . . .
Literature Cited . . . . ,. .

iv

Page
106

106

113
124
125
151
155

159
163

163
164
166

166
170
177
179

182
190

Table

10

11

LIST OF TABLES

Compounds in fresh tissue of white oak which
separate discolored sapwood (DS) from heart-
wood (HW) and sapwood (SW) . . . . . .

Compounds in dried tissue of white oak which
separate discolored sapwood (DS) from heart-
wood (HW) and sapwood (SW) . . . . . .

Compounds in fresh tissue of white spruce
which separate discolored sapwood (DS) from
heartwood (HW) and sapwood (SW). . . . .

Compounds in dried tissue of white spruce
which separate discolored sapwood (DS) from
heartwood (HW) and sapwood (SW). . . . .

Similarity between phenolic compounds from
fresh and dried tissue of white oak . . .

Similarity between phenolic compounds from
fresh and dried tissue of white spruce . .

Rf values and color reactions of the ether—
soluble compounds from woody tissue of
white oak . . . . . . . . . . . .

Rf values and color reactions of the butanol-
soluble compounds from woody tissue of
white oak . . . . . . . . . . . .

Rf values and color reactions of the ether-
soluble compounds from woody tissue of
white spruce . . . . . . . . . . .

Rf values and color reactions of the butanol-
soluble compounds from woody tissue of
white spruce . . . . . . . . . . .

Weight loss (% dry weight) of sapwood (SW),
heartwood (HW) and discolored sapwood (08)
after 6 weeks exposure to Poria monticola and
Polyporous versicolor . . . . . . . .

Page

14

15

16

17

34

35

46

48

51

52

74

Table

12

13

14

15

16

17

18

19

20

21

22

23

24

Page

Number of compounds detected in blocks of
control and fungus-decayed tissue of
white oak . . . . . . . . . . . . . 75

Number of compounds detected in blocks of
control and fungus-decayed tissue of
white spruce . . . . . . . . . . . . 76

Rf values and color reactions of the ether-
soluble compounds from woody tissue of
White oak O O O O O I O O I O O O O 77

Rf values and color reactions of the
butanol-soluble compounds from woody tissue
of white oak . . . . . . . . . . . . 78

Rf values and color reactions of the ether-
soluble compounds from woody tissue of
white spruce . . . . . . . . . . . . 80

Rf values and color reactions of the butanol-
soluble compounds from woody tissue of
white spruce . . . . . . . . . . . . 80

Distribution of compounds from the ether
fraction in woody tissue of white oak. . . . 90

Distribution of compounds from the butanol
fraction in woody tissue of white oak. . . . 91

Distribution of compounds from the ether
fraction in woody tissue of white spruce. . . 93

Distribution of compounds from the butanol
fraction in woody tissue of white spruce. . . 94

Information about trees used in research in
Part II. 0 O I O O O I O O O O O O 103

Amounts of various inorganic elements in
sapwood (SW) heartwood (HW) and discolored
sapwood (D8) of white spruce. . . . . . . 108

Amounts of Cu, B and Al in sapwood (SW),

heartwood (HW) and discolored sapwood (D8)
of white spruce . . . . . . . . . . . 109

vi

Table
25

26

27

28

29

30

31

32

33

34

35

36

Page

Ash content of sapwood (SW), heartwood (HW)
and discolored sapwood (D8) of white spruce . 110

pH of the cold water extract of sapwood
(SW), heartwood (HW) and discolored sapwood
(D8) of white spruce . . . . . . . . . 111

Amounts of various inorganic elements in
sapwood (SW) heartwood (HW) and discolored
sapwood (D8) of white oak. . . . . . . . 114

Amounts of Cu, B and A1 in sapwood (SW),
heartwood (HW) and discolored sapwood (D8)
of white oak . . . . . . . . . . . . 116

Ash content of sapwood (SW), heartwood (HW)
and discolored sapwood (D8) of white oak. . . 118

pH of the cold water extract of sapwood (SW),
heartwood (HW) and discolored sapwood (D8)
of white oak . . . . . . . . . . . . 119

Relationship between K, Ca, ash content and
pH of the cold water extract in sapwood (SW)
and discolored sapwood (D8) of white oak. . . 123

Phosphorous levels at different radial
positions in sapwood, heartwood and discolored
sapwood of white spruce injured in spring . . 127

Calcium levels at different radial positions
in sapwood, heartwood and discolored sapwood
of white spruce injured in spring . . . . . 128

Magnesium levels at different radial positions
in sapwood, heartwood and discolored sapwood
of white spruce injured in spring . . . . . 129

Phosphorous levels at different radial
positions in sapwood, heartwood and discolored
sapwood of white oak injured in winter . . . 131

Calcium levels at different radial positions
in sapwood, heartwood and discolored sapwood
of white oak injured in winter . . . . . . 132

Table

37

38

39

40

41

42

43

44

45

46

Page

Magnesium levels at different radial

positions in sapwood, heartwood and dis-

colored sapwood of white oak injured in

winter . . . . . . . . . . . . . . 133

Manganese levels at different radial

positions in sapwood, heartwood and dis-

colored sapwood of white oak injured in

winter . . . . . . . . . . . . . . 135

Phosphorous levels at different radial

positions in sapwood, heartwood and dis-

colored sapwood of white oak injured in

spring . . . . . . . . . . . . . . 136

Calcium levels at different radial

positions in sapwood, heartwood and

discolored sapwood of white oak injured

in spring . . . . . . . . . . . . . 138

Magnesium levels at different radial

positions in sapwood, heartwood and dis-

colored sapwood of white oak injured in

spring . . . . . . . . . . . . . . 139

Manganese levels of different radial

positions in sapwood, heartwood and dis-

colored sapwood of white oak injured in

spring . . . . . . . . . . . . . . 140

Potassium levels at different radial

positions in sapwood, heartwood and dis-

colored sapwood of white oak injured in

spring . . . . . . . . . . . . . . 142

Chlorine levels at different radial

positions in sapwood, heartwood and

discolored sapwood of white oak injured

in spring . . . . . . . . . . . . . 143

Sulfur levels at different radial

positions in sapwood, heartwood and dis—

colored sapwood of white oak injured

in spring . . . . . . . . . . . . . 144

Oxygen levels at different radial positions

in sapwood, heartwood and discolored sap-
wood of white oak injured in Spring . . . . 145

viii

Table

47

48

49

50

51

52

53

54

Weight loss (% of oven-dry weight) of sap—
wood (SW), heartwood (HW) and discolored

(D8) of white spruce after 6 weeks exposure

to Poria monticola and Polyporous versicolor .

 

Weight loss (% of oven-dry weight) of

sapwood (SW), heartwood (HW) and discolored
sapwood (D8) of white oak after 6 weeks
exposure to Poria monticola and Polyporous
versicolor. . . . . . . . . . . . .

 

 

Characteristics of the ellagitannins from
woody tissue of white oak. . . . . . . .

Distribution of ellagitannins in sapwood
(SW), heartwood (HW) and discolored sapwood
(D8) of white oak . . . . . . . . . .

Relationship between pH of the cold water
extract and decay resistance of discolored
sapwood of white oak . . . . . . . . .

Growth of Poria monticola in a buffered,
defined liquid medium . . . . . . . . .

 

Heptane and methanol solubilities of
white spruce wood (% oven-dry basis) . . . .

Relative concentration of hydroxymatairesinol,
liovil and conidendrin in the sapwood (SW),
heartwood (HW) and discolored sapwood (D8)

of white Spruce . . . . . . . . . . .

ix

Page

167

168

171

172

174

176

184

187

LIST OF FIGURES

Figure Page

1 Chromatographic evidence for quantitative
changes in phenolic compounds after
mechanical injury to the sapwood. Compounds
38 and 41 from the butanol fraction of dried
tissue of white oak. . . . . . . . . . l9

2 Chromatographic evidence for quantitative
changes in phenolic compounds after
mechanical injury to the sapwood. Compound
54 from the butanol fraction of dried
tissue of white oak. . . . . . . . . . 21

3 Chromatographic evidence for quantitative
changes in phenolic compounds after
mechanical injury to the sapwood. Compound
9 from the ether fraction of dried tissue
of white oak . . . . . . . . . . . . 23

4 Chromatographic evidence for quantitative
differences in phenolic compounds of the
ether fraction from discolored sapwood
(D), heartwood (H) and sapwood (S) of dried
tissue of white spruce. Greater color
intensity is shown by darkened spots. Twenty-
five (left) and 12 (right) microliters of
each extract were used. . . . . . . . . 25

5 Chromatographic evidence for similarities
and differences in phenolic compounds of
the ether fraction from discolored sapwood
(D), heartwood (H) and sapwood (S) of dried
tissue of white oak (injured in spring).
Arrows show possible differences between
tissues. Twenty-five (left) and 12 (right)
microliters of each extract were used. . . . 27

Figure

10

11

12

13

14

15

16

Chromatographic evidence for similarities
and differences in phenolic compounds in
the ether fraction from discolored sapwood
(D), heartwood (H) and sapwood (S) of dried
tissue of white oak (injured in winter).
Arrows show possible differences between
tissues. Twenty-five (left) and 12 (right)
microliters of each extract were used. .

Composite chromatogram of the ether-
soluble compounds from fresh woody tissue
of white oak . . . . . . . . . .

Composite chromatogram of the butanol-
soluble compounds from fresh woody tissue
of white oak . . . . . . . . . .

Composite chromatogram of the ether-
soluble compounds from dried woody tissue
of white oak . . . . . . . . . .

Composite chromatogram of the butanol-
soluble compounds from dried woody tissue
of white oak . . . . . . . . . .

Composite chromatogram of the ether-
soluble compounds from fresh woody tissue
of white spruce . . . . . . . . .

Composite chromatogram of the butanol-
soluble compounds from fresh woody tissue
of white spruce . . . . . . . . .

Composite chromatogram of the ether-
soluble compounds from dried woody tissue
of white spruce . . . . . . . . .

Composite chromatogram of the butanol-
soluble compounds from dried woody tissue
of white spruce . . . . . . . . .

Composite chromatogram of the ether-
soluble compounds of control and fungus-
decayed tissue of white oak . . . . .

Composite chromatogram of the butanol-

soluble compounds of control and fungus-
decayed tissue of white oak . . . . .

xi

Page

29

54

55

58

60

62

64

66

68

83

85

Figure

17

18

Page
Composite chromatogram of the ether-
soluble compounds of control and fungus-
decayed tissue of white spruce . . . . . . 87
Composite chromatogram of the butanol-
soluble compounds of control and fungus-
decayed tissue of white spruce . . . . . . 89

xii

INTRODUCTION

Heartwood is composed of dead cells that originate
from physiological processes. The factor(s) responsible
for initiating the series of events which leads to
heartwood formation is unknown. Several factors have
been suggested which initiate the transformation process,
but none have conclusive support.

Stewart (1966) stated that the heartwood was a
depository for excretions from living cells in the sap-
wood. The extraneous materials, toxic to these cells,
were translocated to the center of the tree, accumulated
to lethal concentrations and caused the death of living
cells. The continued translocation of excretions
resulted in outward movement of the heartwood boundary.

The isolation of fungi from.heartwood of
Nothofagus cunninghamii Oerst., myrtle beech, and
Sloanea woollsii F. Mue1., yellow carbeen, led Chattaway
(1952) to believe that the primary stimulus in heartwood
formation was pathological. Prior to cell death, a
period of increased cell metabolism resulted in utiliza-
tion of surplus starch and subsequently the formation
of tyloses and gum plugs. After cell death, the break-

down of cellular membranes allowed the extractives to

1

escape from the cells. Changes in the air-moisture
relationships within the cells solidified the extractives.

Loss of water and entry of air have been consid-
ered responsible for heartwood formation. Abnormal
withdrawals of moisture reserves resulted in the inflow
of atmospheric oxygen into the tissue. The death of
parenchyma cells and formation of tyloses resulted from
continued withdrawal of moisture and accumulation of
atmospheric oxygen in vessel elements. Characteristic
discolorations were caused by oxidative processes
(Zycha, 1948).

Heartwood formation occurs in Fagus sylvatica,
European beech, trees when their age is between 80 and
100 years. Investigators have suggested that, for this
and other species of trees, inner cells of sapwood died
without external stimuli when they reached a certain
age (Zycha, 1948).

Mechanical injury to sapwood may result in dis-
coloration (discolored sapwood). The damaged cells
darken prematurely and resemble heartwood in color.

The nature of the wounding stimulus appears immaterial
(Hart, 1965). Some authors considered microorganisms
were responsible for some discolorations in sapwood.
More than one-fourth of the isolations from discolored
sapwood of Liriodendron tulipifera yielded bacteria,

but very few yielded fungi (Roth, 1950). Microorganisms,

however, are not a prerequisite for discoloration but
greatly enhance the discoloration processes (Shigo, 1965;
1968).

Formation of discolored sapwood may result from
the action of certain enzymes produced by wounded
parenchymatous cells. The enzymes are translocated various
distances and produce physiological reactions which result
in local necrosis (Hart, 1965).

The purpose of this investigation was to compare
heartwood with discolored sapwood in forest tree species.
Phenolic compounds were used to evaluate the chemical
relationship between discolored sapwood and heartwood
(Part I). Changes in mineral content were studied during
formation of heartwood and discolored sapwood (Part II)
and the durability of heartwood and discolored sapwood

to wood-decay organisms was investigated (Part III).

LITERATURE CI TED

LITERATURE CITED

Chattaway, M. 1952. The sapwood-heartwood transformation.

Hart, J. 1965. Formation of discolored sapwood in three
species of hardwoods. Quart. Bull. Mich. Agric.
Exp. Sta. 48: 101-116.

Roth, E. 1950. Discoloration in living yellow poplar
trees. J. For. 48: 184-185.

Shigo, A. 1965. The patterns of decays and discolorations
in northern hardwoods. Phytopathology 55: 648-652.

and E. Sharon. 1968. Discoloration and decay
in hardwoods following inoculations with hymenomycetes.
Phytopathology 58: 1493-1498.

 

Stewart, C. 1966. Excretion and heartwood in living
trees. Science 153: 1068-1074.

Zycha, H. 1948. Uber die Kernbildung und verwandte
Vorgange im Holz der Rotbuche (The formation of
heartwood and allied processes in Fagus sylvatica).
Forstwiss. Cbl. 67: 80-109.

 

PART I

PART I

PHENOLIC COMPOUNDS AS CRITERIA FOR EVALUATION OF THE
CHEMICAL RELATIONSHIP BETWEEN DISCOLORED

SAPWOOD, HEARTWOOD AND SAPWOOD

Qualitative and quantitative chemical differences
have been reported between discolored sapwood, heartwood
and sapwood. Hillis and Inoue (1968) have shown that
the composition of phenolic compounds in Sirex noctilo-

affected wood of Pinus radiate is different from that

 

of sapwood and heartwood. Pinobanksin and pinocembrin
were detected in heartwood, but not in S. noctilo-affected
wood. Pinosylvin was detected in S. noctilo-affected
wood, but not in sapwood. Damaged sapwood (cause unknown)
contained small amounts of unidentified phenols not
detected in other tissues.

Fungal heartwood formed in Prunus domestica

 

var. Victoria after attack by Stereum purpureum contained

 

significant quantities of scopoletin, a coumarin absent
from heartwood (Hillis and Swain, 1959). A similar
distribution for the lignan, isoolivil, occured in

Prunus jamasakura attacked by Polyporous versicolor

 

 

(Hasegawa and Shirato, 1959).

The reaction zone of Fomes annosus-infected

 

Pinus taeda and Picea abies is characterized by the

 

 

accumulation of phenolic compounds present in the heartwood
of both species of trees (Shain and Hillis, 1971).

Damaged sapwood of P. radiata has less pinosylvin than

its monomethyl ether; the reverse is true for S.
noctilo-affected (Hillis and Inoue, 1968).

Cells surrounding kino veins of Eucalyptus spp.

 

contained polyphenols different from those in uninjured
wood (Hillis, 1958; Skene, 1965). In cankers or galls

induced by Cronartium fusiforme on P, taeda. 16 compounds

 

 

appeared and 13 compounds disappeared compared with

healthy tissue (Rowan, 1970). Lignans detected in sap-

wood of E. abies were fewer in number and quantity than

those detected in the reaction zone (Shain and Hillis, 1971).
In my study, the distribution of phenolic

compounds was used to investigate the chemical rela-

tionships between discolored sapwood and heartwood or

sapwood in white oak (Quercus alba L.) and white spruce

 

(Picea glauca (Moench) Voss).

MATERIALS AND METHODS
The white oak were located 4 miles northwest of
White Cloud, Michigan and were co-dominant in the forest
Stand. The white spruce were located at W. K. Kellogg
Forest, Augusta, Michigan and were intermediate in a

Closed, pure stand of the species.

Trees were mechanically damaged with an increment
borer. Twelve holes in 3 rows or 20 holes in 4 rows
were bored into each tree approximately 1.5 m above ground
level. Two white oak were injured in late December,

1969 and felled in early January, 1971. Two white oak

and 2 white spruce were wounded in late April, 1970.

The oak were felled in early November, 1970 and the spruce
in early January, 1971. White oak were 13-17 cm in
diameter (DBH) and contained 10-18 growth rings of sap-
wood and 25-30 growth rings of heartwood. White Spruce
were 13 cm in diameter (DBH) and contained 24-27 growth
rings.

Trees were felled and a bolt was removed with the
rows of borer holes located in the middle. To prevent
desiccation the ends of the bolt and borer holes were
covered with alumninum foil. The bolts were cut, in a
_longitudinal plane, into sections 1-2 cm in thickness
and stored at -5 C.

Bulked samples of sapwood, heartwood and discolored
sapwood were used from oak wounded in spring (S), in
winter (W) and spruce wounded in spring. Both fresh and
oven-dried material were used.

Fifty g of fresh material were extracted for 48
hours on a rotary shaker with 500 ml of’hot water. The
initial 500 m1 of water was removed and extraction was

continued another 48 hours with 500 ml of cold water.

10

Both extracts were combined and reduced to 250 ml in a
rotary evaporator at 45 C.

Fresh material was dried 72 hours on a laboratory
bench and ground to pass through a 2 mm mesh screen in a
Wiley mill. The Wiley mill did not overheat during the
grinding process. The material was dried to a constant
weight in a 40 C oven. The method of Hanover and Hoff
(1966), with modifications, was used to extract phenolic
compounds from the oven-dried material.

Five 9 of oven-dried material were extracted 5
minutes with 100 ml of hot water. The mixture was
homogenized 4 minutes and filtered. The residue was washed
with.50m1 of hot water and all filtrates were combined.

Extracts from both methods were washed with five
50 ml portions of ethyl ether followed with five 50 ml
portions of butanol. The ether fraction was evaporated
to near-dryness under forced air and dissolved in ethanol.
Fractions were reduced to near-dryness in a rotary
evaporator at 45 C and brought to 1 ml with ethanol or
butanol, respectively.

Two-dimensional paper chromatography was used to
separate the phenolic compounds. Fifty microliters
of each extract were spotted with a micropipette onto
Whatman 3 MM chromatography paper. Papers were irrigated
in the first direction with 6% acetic acid and in the

second direction with benzene, acetic acid, water (6:7:3)

11

or butanol, acetic acid, water (4:1:5) for the ether and
butanol fractions, respectively.

Dried chromatograms were examined under ultra-
violet light, exposed to ammonia fumes and reexamined in
the ultraviolet. Chromatograms were sprayed with a
solution of 0.4 g of diazotized sulfanilic acid in 100 m1
of water and oversprayed with 2N NaOH. Several authentic
compounds were chromatographed separately or in combina-
tion with the extracts. Compounds which appeared on the
chromatograms were numbered and Rf values were determined
and characterized by their responses to the treatments.

Thin-layer chromatography was also used to
examine phenolic compounds in the ether fraction from
oven-dried material. Twelve and 25 microliters of each
extract were applied with a micropipette onto Silica
gel SF 254 thin-layer plates (Analtech, Inc., Wilmington,
Del.) and plates were developed to a height of 19 cm
with benzene, methanol, acetic acid (45:8:4). Dried
plates were viewed under short and longwave ultraviolet
light.

The distribution of sugars in fresh tissue was
studied in both white oak injured in winter. The water
fraction, material left after washing the water extract
with ether and butanol, was reduced to near-dryness in
a rotary evaporator at 45 C and brought to 3 ml with

water.

12

Twenty microliters of an extract were applied with
a micropipette onto Whatman No. l chromatography paper
and Umepapers were irrigated in one direction with butanol,
acetic acid, water (6:1:2). Glucose and sucrose (5 mg/ml)
were used for standards. Dried chromatograms were
sprayed with para-anisidine (2.5 g in 10 ml conc HCl and
100 m1 glacial acetic acid) and heated 3-5 minutes at
105 C. Spots which appeared were outlined under ultra-

violet light.

RESULTS AND DISCUSSION

Fourteen compounds were detected in the ether
fraction and 46 compounds were found in the butanol
fraction of fresh tissue from white oak. Nineteen com-
pounds were detected in the ether fraction and 46
compounds were found in the butanol fraction of dried
tissue from white oak. Twenty-seven compounds were found
in the ether fraction and 20 compounds were detected in
the butanol fraction of fresh tissue of white spruce.
Sixteen compounds were detected in the ether fraction
and 11 compounds were found in the butanol fraction of
dried tissue from white spruce.

R value and color reactions of each compound

f
are presented in Tables 7-10 of Appendix I. Composite
chromatograms of the ether and butanol fractions from both

tree species are shown in Figures 7-14 of Appendix I.

13

Other investigators have shown that compounds in
discolored sapwood were qualitatively and quantitatively
different from compounds in heartwood and sapwood (Hillis
and Inoue, 1968; Shain, 1967; Shain and Hillis, 1971).
Such differences have been used to support claims that
the processes leading up to death of living cells affect
the composition of extractives (Hart, 1968; Hillis, 1968).
In both tree species, I found differences in phenolic
compounds between discolored sapwood and heartwood or
sapwood (Tables 1—4). In addition to qualitative differ-
ences, quantitative changes in phenolic compounds were
detected between the 3 tissues using paper chromatography
(Figures 1-3). Compounds which accumulated during
formation of discolored sapwood did not always accumulate
during heartwood formation (Figures 2,3).

Thin-layer chromatography was also used to study
the ether fraction from dried material of white oak and
white spruce. Discolored sapwood of white spruce was
quantitatively different from both sapwood and heartwood.
Color intensity was strongest for most compounds detected
in the discolored sapwood (Figure 4). Few quantitative
or qualitative differences were detected between heartwood
and discolored sapwood of white oak. Qualitative and
quantitative differences were observed between these

tissues and the sapwood (Figures 5, 6).

TABLE 1.

14

Compounds in fresh tissue of white oak which

separate discolored sapwood (DS) from heartwood (HW) and

sapwood (SW).

 

Compound Fraction SW

135 (w)1

E

U

U)

A
m
V

 

12
20
21
22

8

N

other
It

I!
II
M

butanol

N
n
H
M
It
M
II
H
II
II
n
It
It
N
u
fl
0!
II
n
H
II
OI
u
M
It
It
H
II
It
l!
n
n

>>>>>w>w>>>>>>>w>m>wm>>>>w>mm>>>> >>>m>
w>>vm>>>wmwwwmm>>>>>mwv>w>we>vmmw wwww»
>vvm>w>>>m>>>>>>v>w>>m>w>>>>>>mmw >>>>>

>>>v>>m>>>>>>>>>>>>>>>>>>>>>>>>wm >>>>m

 

1(W) - injured in winter; (5) - injured in spring

IA - not detected; P - detected

15

TABLE 2.--Compounds in dried tissue of white oak which
separate discolored sapwood (DS) from heartwood (HW) and
sapwood (SW).

 

ES(S)

E

8

3
H

Compound Fraction SW

 

N

other
ll

2
3
(4' n
7 ll
8 II

10 n
11 u
in u
15 n
16 n
18 u
19 fl

mpv>>>>wwp>>

butanol

1

2 fl
3 n
a II
7 ll
8 N

10 u
15 N
16 u
20 "
21 "
22 "
2“, It
26 I!
28 v
31 II
32 n
uz "
“a N
“5 N
[‘6 u
“8 II
49 I!
55 "
56 It
so u
61 "

>>>>>>>>>>m>w>>>>mv>m>>>>>> >>>>>>>>>>>>
wwwmwmmmmw>>>ww>>wmm>>wwvwm mv>wwm>wmm>m
>v>>>>>>>pw>>>>>>>>wvww>ppp vmpm>>vm>mv>

>m>>>>>>>wmw>v>mm>>>vw>>www

 

1(W) - injured in winter; (S) - injured in spring

IA - not detected; P - detected

16

TABLE 3.--Compounds in fresh tissue of white spruce which
separate discolored sapwood (DS) from heartwood (HW) and
sapwood (SW).

 

%
E
8

Compound Fraction

 

H

other
It

1
u
5 n
6 I:
8 ll
9 It

10 u
n n
12 n
13 u
15 u
16 II
17 It
19 ll
21 n
22 n
23 u
2“ u
25 n
26 u
27 u
28 ..

‘UWJ'O’U‘U‘U’U‘U‘UTW‘U'Ut’Ti‘U‘UTJV'U’U‘U

butanol
It

1

3

5 n
6 n
7 n
8 n
9 u

11 "
12 "
13 N
14 "
15 N
17 It
18 "
19 N
20 v
23 II

>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>
>>>>>>>>>>>>>>w>> vv>>>w>mw>>>>m>>>>>>>>

'U‘U’U'U’UW'O‘U‘U‘U’U‘U‘U‘O>*U'U

1A - not detected; P - detected

17

TABLE 4.--Compounds in dried tissue of white spruce which
separate discolored sapwood (DS) from heartwood (HW) and
sapwood (SW).

 

E
8

Compound Fraction

 

4 other
5 n
6 fl
8 u

12 n
16 n
17 n
18 II
19 ll
22 h
25 n

>>>>>>>~u>~o~$ g3

*0‘U*U*U*U‘11"U’U‘U"U'O

3 butanol
7 H
10 II

>>>>>>> >>>>>>m>>>>

H
a>
>>>>wmw
~u*o*u*oa>*u*o

 

1A - not detected; P - detected

18

FIGURE l.--Chromatographic evidence for quantitative changes
in phenolic compounds after mechanical injury to
the sapwood. Compounds 38 and 41 from the
butanol fraction of dried tissue of white oak.

 

 

 

l9

 

20

 

FIGURE 2.—-Chromatographic evidence for quantitative changes
in phenolic compounds after mechanical injury
to the sapwood. Compound 54 from the butanol
fraction of dried tissue of white oak.

yu-fl‘wA—w‘ ‘3

 

‘_ A;H....-#_.

 

1.1m; .

22

FIGURE 3.--Chromatographic evidence for quantitative changes
in phenolic compounds after mechanical injury to
the sapwood. Compound 9 from the ether fraction
of dried tissue of white oak.

 

-
fib‘u—‘A

UN.

24

FIGURE 4.--Chromatographic evidence for quantitative differ-
ences in phenolic compounds of the ether fraction
from discolored sapwood (D), heartwood (H) and
sapwood (S) of dried tissue of white spruce.
Greater color intensity is shown by darkened
spots. Twenty-five (left) and 12 (right) micro-
liters of each extract were used.

25

 

 

 

 

H 0. 90000.00 0
s O. gooooco 000
D O O ‘0.’.. OOO

 

 

 

N
m H O 0 3800.00 0 R
m
m s o. 388808
a m
m 00030 m...

 

 

$me 9% uEu< soz<zmz mflwzum

 

 

26

FIGURE 5.--Chromatographic evidence for similarities and
differences in phenolic compounds of the ether
fraction from discolored sapwood (D), heart-
wood (H) and sapwood (S) of dried tissue of
white oak (injured in spring). Arrows show
possible differences between tissues. Twenty-
five (left) and 12 (right) microliters of
each extract were used.

lColor abbreviations: B - blue; P - purple;
f - fluorescence; p - pale; - - trace.

27

 

 

‘1

WHITE OAK

 

 

 

 

-

Ho 9 0.0 0 8Q
SQQ BOco Q Q ..-u
.00 ©0000% 8.0

 

 

 

TON

 

.. 50 060.0 mains;

N

m1 .0 s 30 a . 98

m m

m poo 0300 0.0 gum
. N

Van”? . . . OV<I...

 

 

28

FIGURE 6.--Chromatographic evidence for similarities and

differences in phenolic compounds in the ether
fraction from discolored sapwood (D), heart-
wood (H) and sapwood (S) of dried tissue of
white oak (injured in winter). Arrows show
possible differences between tissues. Twenty-
five (left) and 12 (right) microliters of each
extract were used.

lColor abbreviations: L - light; Br — brown;

G - green; B - blue; P - purple; R - red; p - pale;

29

 

 

WHITE A W1

 

 

 

nos 800 @.® 00 gig

see 800- so 8
000 030038 0.6

 

m Hoe 39.000 Q 08
m4 .8 $30.0 2 So 0
m D©® m

 

 

N

CTI

 

E g. . ..Or&- >.r

 

 

 

30

Paper chromatography showed that both fractions
of phenols had compounds which separated discolored sap-
wood, heartwood and sapwood from each other in oak and
spruce. The butanol fraction from white oak was more
useful than the ether fraction for separating the 3
tissues. Both fractions from white spruce were equally
useful for this purpose. Compounds detected in dis-
colored sapwood of white oak which separated it from
sapwood or heartwood, were also detected in heartwood
and sapwood, respectively. Compounds which distinguished
discolored sapwood from other tissues in white spruce
were often detected only in this tissue. Compounds
detected in sapwood and heartwood of oak were not detected
in discolored sapwood and helped to separate these tissues
from discolored sapwood. Few compounds in sapwood and
heartwood of white spruce separated these tissues from
discolored sapwood. These observations indicate that
formation of discolored sapwood might be different in the
2 species of trees. Other differences were observed
between discolored sapwood of white oak and white spruce
(Part II, III).

Thin-layer chromatography indicated that differ-
ences in the ether fraction between sapwood, heartwood
and discolored sapwood were largely quantitative for
both species of trees. Thin-layer plates were developed
with a different solvent system and in only one direc-

tion. Direct comparisons between results obtained by

31

both techniques are not easily made. Whether a similar
situation also exists with the butanol fraction is unknown.
Previous investigators (Hillis and Inoue, 1968; Shain,
1967) have used quantitative differences between tissues

as evidence that discolored sapwood was different from
sapwood and heartwood. The quantitative differences
observed between the 3 tissues support the conclusion that
they are chemically distinct from each other.

No previous literature was found concerning the
relationship between discolored sapwood, heartwood and
sapwood in both tree species. Some recent work with
gigga’abig§_will be published soon (Shain and Hillis,
1971). This work describes qualitative and quantitative
differences between lignans in the reaction zone and
sapwood and quantitative differences between the reaction
zone and heartwood. The reaction zone of g. abies was
produced by fungus infection and mechanical injury to
the sapwood.

I, however, found qualitative differences between
discolored sapwood and both sapwood and heartwood of
white spruce. The number of lignans detected by Shain
and Hillis was much less than the number of phenolic
compounds I detected. The chance for variability in
chemical constituents between tissues would be much

greater with phenolic compounds. Different conclusions

32

reached by their and my studies might be due to this
reason. The reaction zone of g. abig§_was much different
from discolored sapwood of white spruce in other charact-
eristics (Part II, III).

Other evidence used to evaluate the chemical
relationships between discolored sapwood, heartwood and
sapwood was the degree of similarity of chemical
constituents in both fractions for the 3 tissues. Every
compound detected in a tissue was considered as a separate
character in constructing a similarity index. The results
were expressed as the per cent of compounds in common
between the tissues using the formula (Wilkinson, 1970):

Compounds in common for tissue A+B 1000

Total compounds in tissue A+B

With fresh tissue, the ether fraction of dis-
colored sapwood from oaks wounded in spring and winter
was more like sapwood than heartwood while the butanol
fraction was similar to both tissues. When discolored
sapwood was combined, both fractions were similar to
those from sapwood and heartwood. Differences detected
in phenolic compounds between discolored sapwood<mf
oaks wounded in winter and spring were nearly as great
as that between heartwood and sapwood. Formation of

discolored sapwood usually caused fewer or similar changes

33

in phenolic compounds as heartwood formation. Most
pronounced changes occurred in the butanol fraction
during formation of discolored sapwood and heartwood
(Table 5) .

With dried tissue, the ether fraction of discolored
sapwood from oaks was more like heartwood than sapwood
while the butanol fraction was similar to both tissues.
Differences detected in phenolic compounds between dis-
colored sapwood of oaks wounded in winter and spring were
much less than between sapwood and heartwood. Formation
of discolored sapwood caused fewer or similar changes
in phenolic compounds as heartwood formation. Most
pronounced changes usually occurred in the ether fraction
during formation of heartwood and discolored sapwood
(Table 5).

With fresh tissue, the ether fraction of discolored
sapwood from spruces was more like heartwood than sapwood
‘while the butanol fraction was similar to both tissues.
Formation of discolored sapwood caused much greater
changes in phenolic compounds than heartwood formation.
Most pronounced changes occurred in the ether fraction
during heartwood formation, but in the butanol fraction

during formation of discolored sapwood (Table 6).

cclo,

"God ‘

34

TABLE 5.--Similarity between phenolic compounds from fresh
and dried tissue of white oak.

 

 

 

 

Fresh Tissue1
Tissue DS (S) DS (W) DS W
1.!'

Percent of Compounds in Connnonz—-— -—- ==--
DS (W) 82 63
DS -- -- -4 --
W 73 49 76 63 78 62
SW 84 51 89 60 80 58 78 56

 

Dried Tissuel

 

Tissue D5 (5) D6 (W) D5 HW

 

 

Percent of Compounds in Counonz—w- - — - w-==

DS (w) 77 82

ns -- -- -- --

KW 77 75 81 66 81 75

SN 55 77 66 65 61 7a 52 67

 

105 (S) -’discolored sapwood: injured in spring; DS (W) -dis-
colored sapwood: injured in winter; SW - sapwood; DS - discolored sap-
wood; KW - heartwood.

2'Fih'st number - ether fraction; second nunber - butanol fraction

35

TABLE 6.--Similarity between phenolic compounds from fresh
and dried tissue of white spruce.

 

FreshTissue1

 

Tissue D5 HW

 

 

Percent of Compounds in Common2--A—‘ _ _
Eu 9+ 26
SW 32 27 63 86

 

Dried Tissuel

A_

 

 

Tissue DS HW

Percent of Compounds in Common2___ ~ ——:= =-
KW 55 #6
SW 67 71 71 6O

 

1DS -)discolored sapwood; Hw - heartwood; SW - sapwood

2First number — other fraction; second number - butanol fraction

36

With dried tissue from Spruce, both fractions were
more like sapwood than heartwood. Formation of discolored
sapwood caused similar changes in phenolic compounds in
the ether fraction and fewer changes in the butanol fraction
than heartwood formation. Most pronounced changes
occurred in the butanol fraction during heartwood formation.
Both fractions were similarly affected during formation
of discolored sapwood (Table 6).

Calculation of chemical similarity between tissues
was an attempt to give a quantitative value to relation-
ships between the tissues. In addition, the effect of
mechanical injury to sapwood could be compared with heart-
wood formation. Fewer chemical similarities between
heartwood or discolored sapwood compared with sapwood
would indicate greater changes in phenolic compounds
during formation of either tissue.

Formation of discolored sapwood in white oak was
generally accompanied by similar or fewer changes in
phenolic compounds than heartwood formation. Formation
of discolored sapwood in fresh tissue of white spruce was
accompanied by greater changes in phenolic compounds
than heartwood formation. Similar or fewer changes in
phenolic compounds occurred during formation of discolored
sapwood compared with heartwood using oven-dried

material.

37

Different conclusions about whether discolored
sapwood was more like heartwood or sapwood for both
species of trees were reached depending on whether fresh
or dried tissue was used. Because of the small sample
size used in this study, the effect of technique upon
results is not completely understood. Further work in
this area should be encouraged. Calculation of similar-
ity of phenolic compounds between the 3 tissues showed
them to be different in composition from each other.
This observation supports the conclusion that discolored
sapwood is chemically different from sapwood and heart-
wood.

Analysis of the distribution of sugars showed
that sapwood and discolored sapwood were very similar
to each other in white oak. Glucose, fructose, sucrose
and l or 2 additional sugars (Rg values less than 1)
were detected in both tissues. Glucose, fructose and
1 additional sugar (Rg value greater than 1) were detected
in heartwood.

The distribution of sugars in oak followed a
different pattern than that of phenolic compounds. Sap-
wood and discolored sapwood were very similar to each
other. The number of sugars detected in woody tissue
was much less than the number of phenolic compounds. The

chance for variability would be much greater with phenolic

38

compounds. The chance for variability would be much
greater with phenolic compounds and might better reflect
the actual relationship between the 3 tissues. Hillis
and Inoue (1968) detected differences in phenolic com-
pounds, but not sugars in different tissues of P. radiata.

Because of the objectives of this study, it did
not appear necessary to identify the phenolic compounds
detected in tissues from both tree species. However,
several authentic compounds were chromatographed
separately and in combination with extracts in an attempt
to tentatively identify a few of the phenolic compounds.
Caffeic acid co-chromatographed with compound 9 in the
ether fraction (oak) and compound 54 in the butanol
fraction (oak). In addition, the color reactions of
caffeic acid, 9 and 54 were identical. Compounds 9 and
54 were tentatively identified as caffeic acid. Paper
chromatography indicated that large increases in com-
pounds 9 and 54 occurred during formation of discolored
sapwood, but not heartwood. Compounds such as caffeic
acid have been suggested as being important in bio-
synthesis of lignin, flavanoids and related compounds
(Neish, 1964).

Two-dimensional chromatograms of the ether
fraction were developed with butanol, acetic acid, water

(4:1:5), in addition to benzene, acetic acid, water (6:7:3).

39

Compounds 5, 9, l7 and 18 in the ether fraction were the
same as compounds 40, S4, 59 and 60 in the butanol fraction
of oak, respectively. In addition, compounds 38 and 41,
detected in the butanol fraction, were also detected in

the ether fraction. Few compounds detected in the ether
fraction of white spruce were also detected in the

butanol fraction. Because of the large amounts of these
compounds which were detected in the tissue, their

identification seems desirable.

SUMMARY

The chemical relationship between discolored
sapwood, heartwood and sapwood was studied in white oak
and white spruce. Fresh and oven—dried material was
used. A different water extraction procedure was used
with fresh than with oven-dried material. Two-dimensional
paper chromatography was used to separate phenolic
compounds in the ether and butanol fractions of each
tissue. Thin-layer chromatography was also used to
separate phenolic compounds in the ether fraction of
oven-dried material.

Some compounds were detected in the ether or
butanol fractions of fresh material, but not detected in
these fractions from oven-dried material. The opposite
situation also occurred. Other investigators (Wilkinson,

1970) have observed the same phenomenon. Similar

4O

conclusions, however, were obtained whether fresh or
oven—dried material was used.

Phenolic compounds from both fractions were
detected in discolored sapwood, heartwood and sapwood
of both tree species which separated these tissues from
each other. In white oak, few qualitative and quantitative
differences in phenolic compounds of the ether fraction
were observed between discolored sapwood and heartwood
with thin-layer chromatography. Qualitative and quantita-
tive differences were detected between these tissues
and sapwood. In white spruce, discolored sapwood was
quantitatively different from sapwood and heartwood which
were similar to each other. Color intensity of compounds
was greatest from discolored sapwood.

A similarity index was constructed with phenolic
compounds from both fractions. Every compound detected
in a tissue was considered as a separate character.
Calculation of similarity indexes between the 3 tissues
of white oak and white spruce showed that they were
different in chemical composition from each other.

The results presented here support the conclusion
that discolored sapwood is chemically different from

heartwood and sapwood in white oak and white spruce.

LITERATURE CITED

41

LITERATURE CITED

Hanover, J. and R. Hoff. 1966. A comparison of phenolic
constituents of Pinus monticola resistant and
susceptible to Cronartium ribicola. Physiol.
Plant. 19: 554-562.

 

 

Hart, J. 1968. Morphological and chemical differences
between sapwood, discolored sapwood and heartwood
in black locust and osage orange. For. Sci.

14: 334-338.

Hasegawa, M. and T. Shirato. 1959. Abnormal constituents
of Prunus wood: Isoolivil from wood of Prunus
jamaskura. J. Jap. For. Soc.: 41: 1-3.

v—‘wjr—

 

Hillis, W. 1958. Formation of condensed tannins in
plants. Nature 182: 1371.

and T. Swain. 1959. Phenolic constituents of
Prunus domestica. III. Identification of the
major constituents in the tissue of Victoria
plum. J. Sci. Food Agric. 10: 533-537.

 

 

. 1968. Chemical aspects of heartwood formation
Wood Sci. Technol. 2: 241-259.

 

and T. Inoue. 1968. The formation of polyphenols
in Pinus radiata after Sirex attack. Phytochemistry

7: m.

Neish, A. 1964. Major pathways of biosynthesis of phenols.
In. J. Harborne, Biochemistry of Phenolic Compounds.
Academic Press, New York. p. 295-359.

 

 

Rowan, S. 1970. Fusiform rust gall and canker formation
and phenols of loblolly pine. PhytOpathology
60: 1221-1224.

Shain, L. 1967. Resistance of sapwood in stems of loblolly

pine to infection by Fomes annosus. Phytopathology
57: 1034-1045.

42

43

and W. Hillis. 1971. Phenolic extractives in
Norway spruce and their effects on Fomes annosus.
Phytopathology 61: In press.

 

 

Skene, D. 1965. The development of kino veins in
Eucalyptus obliqua. Aust. J. Bot. 13: 367-378.

 

Wilkinson, R. 1970. Chemical analysis of the species
relationships in northeast American spruces.
Michigan State University Ph. D. thesis. 111 p.

APPENDIX I

44

45

1Color abbreviations: B - blue; P - purple;
W - white; Gr - grey; G - green; Y - yellow; R - red;
O - orange; L - light; NV - not visible; Br - brown;‘
V - violet; Pk - pink; b — bright; d - deep.

46

 

 

 

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53

FIGURE 7.--Composite chromatogram of the ether-soluble
compounds from fresh woody tissue of white oak.

54

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55

FIGURE 8.~-Composite chromatogram of the butanol—soluble
compounds from fresh woody tissue of white oak.

S LVE FR NT

 

 

 

 

 

 

 

 

57

FIGURE 9.--Composite chromatOgram of the ether-soluble
compounds from dried woody tissue of white oak.

58

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59

FIGURE lO.--Composite chromatoqram of the butanol-soluble
compounds from dried woody tissue of white oak.

\62/

 

 

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61

FIGURE ll.--Composite chromatogram of the ether-soluble
compounds from fresh woody tissue of white
spruce.

62

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63

FIGURE 12.--Composite chromatogram of the butanol-soluble
compounds from fresh woody tissue of white
spruce.

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65

FIGURE 13.--Composite chromatogram of the ether—soluble
compounds from dried woody tissue of white
spruce.

 

 

 

 

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SOL

 

 

67

FIGURE 14.--Composite chromatogram of the butanol-soluble
compounds from dried woody tissue of white
spruce.

68

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APPENDIX II

69

APPENDIX II

EFFECT OF POEYPOROUS VERSICOLOR AND PORIA MONTICOLA

 

 

ON PHENOLIC COMPOUNDS FROM WHITE OAK AND WHITE SPRUCE

Cowling (1961) studied decay of sapwood blocks
of Liquidambar styraciflua by Polyporous versicolor

(white-rot fungus) and Poria monticola (brown-rot fungus).

 

In all stages of decay, P, versicolor depolymerized

 

lignin and carbohydrates only as rapidly as the depoly-
merized products were used by the fungus. In initial

stages of decay, P, monticola degraded lignin and

 

carbohydrates more rapidly than the degraded products
were used. After a 20-30 per cent weight loss, carbo-
hydrates were depolymerized less rapidly than the
depolymerized products were used by the fungus.

In my study, I investigated the effect of P.

versicolor and P, monticola on the number of compounds

 

 

detected in discolored sapwood, heartwood and sapwood

of white oak (Quercus alba L.) and white spruce (Picea

 

glauca (Moench) Voss).

Location of both species of trees, methods of

mechanical injury and initial preparation of wood samples

70

71

is described in Part I. White oak injured in winter and
white spruce injured in spring which were used for
chromatographic investigations (Part I) were used for
this study.

The resistance of discolored sapwood, sapwood and
heartwood to decay was measured using the agar—block
method (McNabb, 1958). Three blocks of discolored sap—
wood and 5 blocks of heartwood and sapwood were exposed

to each wood-decay organism. Polyporous versicolor L.

 

ex Fr. (USDA isolate Madison 697), a white-rot fungus,

and Poria monticola Murr. (USDA isolate Madison 698), a

 

brown-rot fungus, were the test organisms used.

Blocks were dried 14 days at 40 C and weighed to
determine the initial dry weight of the blocks. After
incubation with the fungus, blocks were removed from
the decay chambers and the fungus was carefully scraped
from the blocks. Blocks were immediately weighed to
determine their moist weight. The final dry weight was
determined after drying the blocks 14 days at 40 C.
Weight loss was expressed as percent of the initial dry
weight of the block. Blocks which were not exposed
to the decay fungi served as controls.

Blocks of each tissue, approximately 15 x 8 x 8 mm
in size, were cut with the longest dimension parallel to
the radial axis of the tree. The blocks were steamed

20 minutes at 99 C and placed aseptically into the decay

72

chambers 14 days after the chambers had been inoculated
with the test organism. Preparation of decay chambers

is described in Part III. The decay chambers were incubated
at 26 C for 6 weeks.

Two g (oven-dried weight) of each tissue from
control blocks and blocks decayed by each fungus were
extracted for phenolic compounds. Preparation of the
wood sample, extraction of oven-dried material and
two-dimensional paper chromatography of the ether and
butanol fractions is described in Part I.

Heartwood from both white oak was more resistant
to decay than sapwood, but only discolored sapwood from
1 white oak was more resistant to decay than sapwood and
heartwood. The discolored sapwood of white spruce was more
resistant to decay than sapwood and heartwood. Sapwood
and heartwood were not different from each other in their
resistance to either decay fungus (Table 11).

More compounds were detected in the ether and
butanol fractions from sapwood, heartwood and discolored

sapwood of white oak exposed to P, monticola than to

 

g. versicolor (Table 12). Except for discolored sapwood,

 

similar results were obtained with white spruce. Only
1 or 2 compounds were detected from blocks of discolored

sapwood exposed to both decay organisms (Table 13).

73

Cowling (1961) showed that P. versicolor rapidly

 

assimilated depolymerized products, but initially P.

monticola did not. Similar results observed with my

 

chemical constituents might be for the same reason.

Polyporous versicolor is able to rapidly use lignin,

 

but P;_monticola only affects lignin to a minor extent

 

(Cowling, 1965). Increases in the number of compounds

detected in P. montocola-decayed material might result

 

from the ability of the fungus to degrade, but not
assimilate that portion of the lignin molecule. The
phenomenon observed by Cowling (1961) for sapwood was
also observed for heartwood and probably discolored
sapwood of white oaks. Resistant and susceptible tissues
of this tree species were affected in a similar fashion
by both wood-decay organisms.

The Rf value and color reactions of each compound
are given in Tables 14-17. Composite chromatograms of
the ether and butanol fractions from both tree species
are shown in Figures 15-18 while the distribution of
compounds in control and fungus-decayed tissue of oak

and spruce is given in Tables 18-21.

74

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75

TABLE 12.-—Number of compounds detected in blocks of control
and fungus-decayed tissue of white oak.

 

 

 

 

Treatmentl
Fraction Tie sue2 Control £338 ecsyed ELI-decayed
other SW 7 4 17
EN 13 10 18
us 17 ‘ 12 17
‘umumml SW 15 6 12
HR 15 8 27
DS 20 12 27
1

n-decsyed: decayed by g. versicolor: wowed: decayed by g.
monticola

2
SW’- sapwood; Hw - heartwood; D6 - discolored sapwood

76

TABLE 13.--Number of compounds detected in blocks of control
and fungus-decayed tissue of white spruce.

 

 

 

 

‘I‘reaIu-ewt.1
Fraction Tis sue2 Control flydecayed gm-decayed
0th”? SW 3 5 9
aw 1. I. 7
D6 “ 1 1
butanol sw 5 5 8
EM 3 (I 6
DS 5 1 1
1

2v_.deoayed: decayed by g. versicolor; Pg-decayed: decayed by g.
M

2
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82

FIGURE 15.--Composite chromatogram of the ether-soluble
compounds of control and fungus-decayed tissue
of white oak.

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84

FIGURE l6.-—Composite chromatogram of the butanol-soluble
compounds of control and fungus-decayed tissue
of white oak.

 

 

 

 

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86

FIGURE 17.--Composite chromatogram of the ether-soluble
compounds of control and fungus-decayed tissue
of white spruce.

87

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FIGURE 18.--Composite chromatogram of the butanol-soluble
compounds of control and fungus-decayed tissue
of white spruce.

89

SOLVENT FRONT

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TABLE 18.-Distribution of compounds from the ether fraction in woody tissue of

white oak.

 

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H~~amw>wsanmssnssssssasan

?

 

PIIddhuflwd:1|-1mflzddhlfind

us .- discolored sapwood: SH - sapwood: m - heartwood

TABLE l9.-Distribution of compounds from the butanol fraction in woody tissue of
white oak.

 

annual

.Dunyuihw

lkwdnlmnNdnoLI

Duuwndlur

IbgngyvnainusMMflpr

 

 

91

m<m<<<<mm<mmm<m<m<<m<m4<<m
<4<«<<<¢<<<m<4m<<<<<<<<<<4

9.9.0.”:mm<mm<<m<<<<dm<mm<m<¢<

A¢‘QC“<‘k‘fi‘4‘444<4<*¢‘<4
<4<‘4‘4‘444flo<¢<4§44“‘49¢11

DI¢ILDI¢<¢<“4GI<<<‘4"44fl-<<¢44

<¢44<44<fln¢<h‘fln“d€¢flo‘fi4‘<<
4<<<<<m<<<<m<m<<<<m<¢d<<<<

immedd<<<<<m<<<m<mmmdqdmm<

"~"a“vsweaqmnsnsaasaaasaaa

 

TABLE 19.-Continued.

 

W by
Porto monticola

Doooyod by
Fonz-on: vmicolor

Control

 

ml

Compound

 

92

flofla‘<fluflnfl-D¢‘¢‘hfln‘mfloflnflo‘<finfln4fla

““mm<fln“fiflo‘fifim“‘m“m

m<<<mmmm<mmm<mmm<m<mm<m

<«<<4<mm¢<<m<d<«<<<m<<m

“<1‘14‘G‘4fla<‘flo‘¢‘flflo<<h

<¢<¢¢<mm<<<<<¢m<<<<mmmm

<<<<¢¢mm<<mmm<mm¢dmmm<m

<<Dn0nfln¢flnmfln§floflfln‘4fl“flnfln“flu

$4m4<<mm¢<m<m<m<<<mmm<m

assaassspsssassgsssssss

 

P-dotootodzt-notdotootod

2

DB - dhoolorod sapwood: SH- oopvood: EH- hoortaood

l

883% no: I 4 .8838 I mu

QXEEQ-Axiodxzao.I“RA.§R§ngn..3m_;ooat:ia..2fiA

 

93

 

 

 

 

 

a a a m m m 4 4 4 3
a a 4 4 a 4 m m m a
4 4 4 a m 4 4 4 4 m
a a 4 4 4 4 4 4 4 A.
m a 4 4 4 4 m m m o
4 a 4 4 4 4 4 4 4 n
a a 4 4 4 4 4 4 4 4
4 a 4 4 4 4 4 4 4 m
a a 4 m m 4 a 4 .A N
m a 4 m .4 4 4 m lmw A
3m 3m 8 :4 gm 8 A34 3m 8 930948
Snood anal 178m so Hooaohob g .
.3 8538 .3 .8538 A288

 

.oosnmm ouws3
mo osmmAu >8003 nA cOAuooum Hocuo mnu scum mccsomeoo mo :OAusnAuuonII.o~ mnm4a

94

uktxabcnfiE_I.4«ogfilfibv.um

 

 

 

 

 

m

Aaoafnli IeaanXEEin.I3m 386192.49flzoo954I.mn

A
m m A m m m m m m {A
4 4 4 4 4 4 4 m m ma
4 4 4 4 m 4 4 4 4 «A
m m 4 4 4 4 m m A AA
m m m 4 4 4 4 4 4 ed
A m 4 4 4 4 4 4 4 m
4 m 4 4 4 4 4 4 4 m
m m 4 4 4 4 4 4 4 u
4 m 4 m m 4 4 4 4 e
m m 4 4 4 4 4 4 4 n
4 4 4 m m 4 4 4 4 a
4 4 4 m m 4 4 4 4 m
4 4 4 4 4 4 m m A m
4 4 4 4 4 4 4 m an .A

:4 3.... 8 i am 8 3. 3.0. A8 2898
ud2:358_$tfim BJBQflEPpmmflmmfimfl
3 1588 P 4.5.84 A038

 

.oosumm ouwns
mo mommflu moooz c4 coauomum Hosanna mnu Scum monsomfioo mo coflusnwnumonI.AN mqmda

LITERATURE CITED

95

LITERATURE CITED

Cowling, E. 1961. Comparative biochemistry of sweetgum

.McNabb,

by white-rot and brown-rot fungi. USDA For. Serv.
Tech. Bull. 1258. 79 p.

. 1965. Microorganisms and microbial enzyme
systems as selective tools in wood anatomy. In:
W. Cote, Jr., Cellular Ultrastructure of Woody
Plants. Syracuse University Press, Syracuse,
New York. p. 341-368.

Jr., H. 1958. Procedures for laboratory studies

on wood decay resistance. Proc. Towa Acad. Sci.
65: 150-159.

96

PART I I

97

PART II

CHANGES IN MINERAL CONTENT DURING FORMATION OF
DISCOLORED SAPWOOD AND HEARTWOOD

IN WHITE OAK AND WHITE SPRUCE

Ash content changes during formation of heartwood
and discolored sapwood. During heartwood formation, ash

content decreased in Quercus alba, Maclura pomifera and

 

Robinia pseudoacacia while it increased during formation

 

of discolored sapwood. Ash content did not change

during heartwood formation in Juglans nigra, but it

 

increased during formation of discolored sapwood. Ash
content increased during formation of heartwood and

discolored sapwood in Acer saccharinum with the greatest

 

increase in the latter tissue. An increment borer was
used to induce formation of discolored sapwood (Hart,
1965; 1968).

Investigators have reported changes in inorganic
elements during formation of heartwood and discolored
sapwood. Formation of heartwood in Quercus EBEEE was
accompanied by decreases in calcium, magnesium, potassium,

sodium and manganese (Ellis, 1967). PhosPhorous decreased

98

99

during heartwood formation in 3. pseudoacacia and M.

 

pomifera (Hart, 1968). Potassium and.phosphorous
decreased and manganese increased during heartwood forma-

tion in Picea abies (Bergstrom, 1959; Shain, 1971). In

 

Pinus taeda, calcium, sodium, magnesium, and potassium

 

increased from the cambium to the pith (McMillin, 1970).
Levels of trace elements were not changed during heart-
wood formation (Hart, 1968; Wasny and Waény, 1964).

Changes in levels of inorganic elements and nitrogen
have been reported for different radial positions of the
sapwood. Phosphorous and potassium decreased and calcium
increased from the outer sapwood into the heartwood of
Pinus radiata, Pi22§_nig£a_var. pgiretiana and Pinus

sylvestris except for the growth rings immediately surround-

 

ing the heartwood where phosphorous and potassium increased
and calcium decreased (Wright and Will, 1958; Orman and
Will, 1960). Merrill and Cowling (1966) showed that
nitrogen decreased from the cambium to the heartwood
boundary of several tree species. They postulated that
decreases in nitrogen resulted from apposition of cellulose
and lignin in cells, elution of nitrogen from vascular
elements by the transpiration stream and recovery of
nitrogen from dying parenchyma cells near the heartwood

boundary (Cowling and Merrill, 1966).

100

Accumulation of calcium, magnesium, potassium,
sodium, iron, manganese and numerous trace elements was

observed in decayed wood of Abies grandis (Ellis, 1967).

 

Potassium, calcium and magnesium increased in decayed
wood and the reaction zone of P. abies and decayed wood

of P. sylvestris (Rennerfelt and Tamm, 1962; Shain, 1971).

 

The reaction zone of P. abies was produced in response

to infection by Fomes annosus, other wood-decay fungi

 

and mechanical injury to the sapwood (Shain, 1971).
Potassium increased during formation of discolored sap-

wood in g. alba, A, saccharinum, g. nigra and 3. pseudoacacia.

 

 

Phosphorous did not change during formation of discolored

sapwood in M, pomifera and 3. pseudoacacia (Hart, 1968).

 

Changes in mineral constituents can alter pH of
woody tissue (Good, Murray and Dale, 1955). The pH of
cold water extract was highest in discolored sapwood,
intermediate in sapwood and lowest from heartwood of
Q. ElEE and g. nigra (Hart, 1965). The pH was lowest

from sapwood of A. saccharinum and R. pseudoacacia.

 

The pH of the cold water extract of heartwood and dis-
colored sapwood was similar or that from discolored sapwood
was highest in these tree species (Hart, 1965; 1968). The
pH of the cold water extract was highest from the reaction
zone of E, abies and similar from sapwood and heartwood

(Shain, 1971).

101

I studied changes in mineral content during forma-
tion of heartwood and discolored sapwood in white oak
(Quercus alb§_L.) and white spruce (Picea.glauca (Moench)
Voss). Levels of inorganic elements, ash content and the
pH of the cold water extract were measured in sapwood,
heartwood and discolored sapwood of both tree species.

An electron microprobe x-ray analyzer - scanning micro-
scope was used to detect differences in levels of elements
at radial positions in sapwood, heartwood and discolored
sapwood. Differences in levels of elements in ray and
non-ray cells were studied at these radial positions in
both tree species. The effect of physiological condition
of white oak at the time of injury and its effect on
changes in mineral content during formation of discolored
sapwood was also studied.

One objective of this research was to gain informa—
tion about formation of heartwood and discolored sapwood
in white spruce. The second objective was to gain more
information about changes in elements during formation
of discolored sapwood and heartwood in white oak, particu-
larly as it related to differences in levels of elements
at radial positions in the tree and between ray and non-

ray cells at these positions.

 

102

MATERIALS AND METHODS

Location of trees, method of mechanical injury
and procedures for initial preparation of the wood samples
are described in Part I. Information about the trees used
in this research is presented in Table 22. Blocks of
tissue were dried for 72 hours on a laboratory bench,
ground in a Wiley mill and passed through a 2 mm mesh
screen.

Measurement of Ash Content

Ash content was measured in tissue of both tree
species (Table 22). The dry weight of the crucibles was
determined by heating them for 2 hours at 575 C. After
cooling the crucibles in a desiccator over indicating
Drierite for 15 minutes, they were weighed to the nearest
0.1 mg. A 2.2-2.4 9 sample of tissue was added to each
crucible and they were heated at 105 C for 24 hours. The
cruciblesywue cooled in a desiccator for 15 minutes and
weighed to the nearest 0.1 mg to determine the oven-
dry weight of each sample. The samples were ashed for
3 hours at 575 C, removed and cooled in a desiccator for
15 minutes and weighed to the nearest 0.1 mg. Ash content
was expressed as per cent of the oven-dry weight of the

tissue for each sample.

 

 

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«auuoaouonm oEoAm can cOAmmAEo AMOAumo I m «uaoucoo cum I 4

"momaAmcm

 

 

M
woosuuomn I 3x “tOOBQom I 3m ”mammAu mo nchu nusoumw
.20 :A Ao>oA poncho m>onm noon m.4 um HoumEoApA
a.m.4 MM MA = g m =
Osmed 0N NH thh IH .. 4 .-
o.u.m.4 4m MA = a 4 =
o.m.4 4M MA = g M :
Demefl @N NH .. .. N :
6.4.4 4~ AA osImAIm 07314 A 893m 323
o.m.4 mm SA MA = g MA =
o.m.4 4N MA MA onINAIAA = AA =

m. a.o.m.4 3 4A A .. .. S ..

l D.U.m.< mm mA 4A .. .. m ..
o.m.4 3 A 4A .. .. m ..
o.m.4 MM NA NA onIbAIM o>Im~I4 h 440 ouAnz
Q.m.4 NM 4A MA = = 4A =
Q.m.4 MN cA MA AnIAAIA a MA =
u.m.4 AM MA oA ohIm IF = o =

U mm a MH Ohlv l0 : m :
Osmsfl mN HH NH Cbl.—”HI? : fl ..
o.m.4 MM 5A MA = a M =

Q on m ”H = 8 N .-

.o.m.4 MN oA NA ohIMAIM moIANINA A xmo ouAnz
Mom: 3: 3m Ammo ooAAom coocsoz done

 

.AH unmm dA zoummmmu dA com: mmeA usonm coAumEHomcHII.NN mqmsa

‘i‘fi 1.! SI

 

 

104
Measurement of Levels of Inorganic Elements with
Optical Emission and Flame Photometry

Optical emission was used to measure levels of P,
Na, Mg, Mn, Ca, Fe, Cu, B, Zn and A1 in tissue from both
tree species (Table 22). Samples of each tissue (equiva-
lent to one-half g oven-dry weight) were ashed for 8 hours
at 500 C, cooled to room temperature and 5 ml of an
internal cobalt standard were added to each sample. Levels
of each element (ppm) were measured on an Applied Research
Laboratory spectrograph (Quantograph).

Levels of K were measured with flame photometry.
Samples of each tissue (equivalent to one-fourth g oven-
dry weight) were extracted for 2 hours with 50 m1 of
distilled water. The samples were shaken every 30 minutes
during the extraction period. The levels of K (ppm)
were determined with a Beckman flame photometer.

Measurement of pH

The pH of the cold water extract was measured
for each tissue. The 3 tissues were bulked separately:
white oak 1-4; 7-10; 11 and 12; 13 and 14 (discolored
sapwood not bulked); white spruce 1-4; A and B (Table 22).
Four 9 of each tissue were placed into 100 m1 of distilled
water and soaked for 12 hours. The pH of the water extract
was measured with a Beckman zeromatic II pH meter.
Distilled water without tissue (control) was treated in

an identical fashion.

 

105

Electron Probe Microanalyzer Studies

Levels of inorganic and organic elements were
studied with an electron microprobe x-ray analyzer -
scanning microscope (Applied Research Laboratory) at
different radial positions in sapwood, heartwood and
discolored sapwood and in ray and non-ray cells at these
positions in white oak injured in winter (5, 6) and spring
(9, 10) and 1 white spruce injured in spring (4) (Table 22).
The distribution of P and Ca was studied in white oak 5,
6, 9 and 10 and white spruce 4. The distribution of Mg
was studied in all of the above mentioned trees except
white oak 5. Manganese was studied in white oak 6, 9 and
10 and K, 0, C1 and S in white oak 9 and 10 (Anderson,
1967).

Locations in ray and non-ray cells were analyzed
in the outer sapwood (youngest one-third), middle sapwood
(middle one-third), inner sapwood (oldest one-third),
outer heartwood (2-3 growth rings next to sapwood), middle
heartwood (middle one-third), inner heartwood (oldest one-
third), outer discolored sapwood (youngest one-third),
middle discolored sapwood (middle one-third) and inner
discolored sapwood (inner one-third). A second group
of positions, similar in location to those mentioned,
was analyzed on the opposite side of the tree. In each

section of tissue, measurements were made at 3 locations

 

106

each in ray and non-ray cells. A single 100 second count
was made for each element at a location.

Sections of fresh tissue, 25-30 microns in thick-
ness, were cut using a sliding microtome and immediately
frozen on dry ice. Sections of tissue were freeze-dried
at -55 F and 50-75 microns of Hg for 48 hours. Sections
were mounted on polished carbon discs with Scotch double
stick tape. A 100 x 80 micron area was scanned at 10
second intervals for 100 seconds. Measurements for

background and tape were carried out in identical fashion.

 

Beam current was 15 kV and sample current 25
nano-amperes for measuring levels of P, Ca, Mg, Mn and
K. For 0, Cl and S, the sample current was raised to
50 nano-amperes. Lead sterate dodecinate (O), potassium
acid pthalate (Mg), ammonium dihydrogen phosphate (K,
Ca, P, Cl, S) and lithium fluoride (Mn) were the crystals

used.

RESULTS AND DISCUSSION

Levels of Inorganic Elements, Ash Content and pH
of Tissues from White Spruce

Formation of heartwood and discolored sapwood did
not change levels of most inorganic elements in sapwood
of white spruce. For both groups of trees, levels of
K and P were reduced in discolored sapwood and heart—

wood while levels of Mg and Mn were reduced only in

”I

 

107

discolored sapwood. Levels of Ca and Mn were highest in
heartwood. The increase and decrease in level of elements,
however, was usually not significantly different from
those of the same element in sapwood (Table 23, 24).

Levels of ash were highest in heartwood, inter-
mediate in sapwood and lowest in discolored sapwood of
white spruce (Table 25). These differences, however,
were significant for only one group of trees. The pH
of the cold water extract was similar for sapwood and
heartwood and lowest from discolored sapwood (Table 26).

Shain (1971) reported decreases of P and K and
increases of Ca and Mn during heartwood formation in
g. abies. In white spruce, similar changes in
inorganic elements were observed during heartwood
formation. The changes, however, were usually not
sfignificant. Because Shain (1971) did not statistically
analyze his data, I do not know whether his differences
are significant. The inorganic elements which change
and the direction of change are similar for both tree
species.

The pH of the cold water extract from sapwood
and heartwood was similar for both tree Species. That
from g. abies was between 5.6-6.8 and from white spruce
was 5.3 or 6.0. With respect to these criteria, formation
of heartwood of white Spruce was not different from P.

abies.

 

 

108

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umwon muouumA ucoummon mm ooBOAA0m mommuo>m .mosAm> mo umm Sumo Mom

 

 

 

m
ooh» m Eoum osmmAu nomo mo mcoAuMCAEAmuot M mo mcmoEA
4.M m m p mm m 4m 2 mmm n 4 m mm m omm mo
0.0 m h m mm m ooA m 4MMA n M m om m 5AM Sm
m.m m AA m 4m m 00A m MAMA 4 4A m omA m 4mm 3m “Mm“ N
o.N m m.o m MN o no m hAm m 0A m om Q omm mo
>.m m m.m 6 M4 m mm m MNOA m m m o Q hAm Sm
m.o o N.A m 4M m an m mmo m w m SM 8 5A4 3m WMHW 4
unmAmB who mo Ema
omwmm>umm momma
:N mm :2 ms mo oz m smm mammAB UoHdncA «0 .oz

 

A

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UGM Azmv UOO3UHMOS 4 Azmv U003nmmm CH mfifiwfiwflm UHGMOHOCH mAAOflHMNV MO mUCSOEIIIMN Mdmdfi

109

TABLE 24.--Amounts of Cu, B and A1 in sapwood (SW), heart-
wood (HW) and discolored sapwood (D8) of white spruce.

 

2

 

No. of Injured Tissue Cu B Al
Trees Harvested
ppm of dry weight
6 4-70 SW 1.9 a 2.9 a 8.5 a
8-70
or HW 1.8 a 3.3 a 6.5 a
1-71
DS 2.1 a 2.9 a 7.1 a

 

1means of 3 determinations of each tissue from a tree

2For each set of values, averages followed by
different letters differ significantly (0.05 level).

 

110

TABLE 25.--Ash content of sapwood (SW), heartwood (HW) and
discolored sapwood (D8) of white spruce.

 

No. of Trees Injured Harvested SW2 HW DS

 

percent of dry weight
4 4-70 8—70 .362 a .370 a .327 a

2 4-70 1-71 .313 a .356 b~ .248 c

 

l . . .
means of 4 determinations of each tissue from a tree

2for each set of values, averages followed by differ-
ent letters differ significantly (0.05 level).

 

111

TABLE 26.--pH of the cold water extract of sapwood (SW), heart-
wood (HW) and discolored sapwood (D8) of white spruce.

 

 

No. of Trees Injured Harvested SW2 HW DS
4 4-70 8-70 5.3 a 5.3 a 4.0 b
2 4-70 1-71 6.0 a 6.0 a 5.0 b

 

1means of 4 determinations of each tissue from a tree

2For each set of values, averaged followed by differ—
ent letters differ significantly (0.05 level).

 

112

Levels of Ca, K and Mg increased greatly and the
pH of the water extract was between 7.0—7.7 in the reaction
zone of P, abie§_(8hain, 1971). Formation of discolored
sapwood in white spruce was not accompanied by increases
of these elements or pH of the water extract. Forma-
tion of this tissue in white spruce was very different
from formation of the reaction zone in P. abies. Dis-
colored tissue from both tree species, however, was
more resistant to decay than sapwood or heartwood (Shain,
1971, Part III).

The reaction zone of P. abies was produced in
response to infection by F. annosus, other decay fungi
(including brown-rot fungi) and mechanical injury to
the sapwood. It was not resin-soaked (acetone-
solubles ca. 5% of oven-dry weight) (Shain, 1971).
Discolored sapwood of white spruce was also produced in
response to mechanical injury, but it was resin—soaked
(heptane-solubles ca. 30%) (Part III, Appendix).

Fungus invasion and mechanical injury can expose
the interior of trees to air. Low levels of resin
(acetone or heptane-solubles) might not be able to
completely plug the Openings and evaporation of water in
the transpiration stream through openings could deposit
minerals in the discolored tissue. This would result in
increases in levels of inorganic elements and a higher

pH. High levels of resin might be able to plug the

113

openings and prevent evaporation of water. Increases in
levels of inorganic elements and pH might be prevented.
Differences observed in levels of inorganic elements and
pH between discolored tissue in P. abies and white
spruce may result from different levels of resin in each
tissue.

Levels of Inorganic Elements, Ash Content and pH
of Tissues from White Oak

Formation of heartwood and discolored sapwood
changed levels of inorganic elements present in sapwood
of white oak. Levels of K increased during formation of
discolored sapwood and decreased during heartwood forma-
tion. Levels of P, Mg and Mn decreased during heart-
wood formation. When all trees were statistically
analyzed together, levels of Cu, B and Al increased
during formation of discolored sapwood.

Calcium levels were sometimes higher in dis-
<3olored sapwood and lower in heartwood compared with
levels in sapwood. Levels of P decreased and Mg and
Mn increased or decreased during formation of dis-
colored sapwood. These levels, however, were usually
not significantly different from those observed for the

same element in sapwood (Table 27, 28).

 

114

 

 

 

 

m h M m M M 0 MM n MMA a MMMA 4 MM A MAN 0 oovm mm
m o.A m A.M n MM n MMA a com o M n o~A n MMM 3m AMIA
m M.» 4 «.4A m MM m MMM m comm m 4m m OMM m MMM 3m MMINA A
m M.M m 4.NA 0 NM m 00A 4 MMMA m MA m 0AM m MOM mm
m o.A m M.M a MN m MMA m com o MA A cm a MM4 Sm AMIA
o o.A m M.MA 0 MM m MMA m MMMA m 0A m own m MMM 3w MMINA A
m M.4 m M.MA m «M m MAA n MM4A m mm m MAA n MMMA mm
m M.M 3 MM a MA n MM m omoA m m n o m MMMA 3m onlh
m M.M m o.MA m An m 4AA m MMMA m NA m 54A m MMMA 3m MMINA A
m M.M m M.MA m AM m MMA m MMMA m MM 8 ovm m MMMA mm
m o.o m M.M Q MN m MMA m MMMA m M4 n MM Q MMM 3m omlv
m M.M m o.AA m MM m com o MMAA m MM m omm m NMAA 3m MMIMA M
unonB who mo Ema
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'7

 

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Mam Azmv Moosuumon .AZMV ooo3mmm GA mucoEvo OACMMHOQA msoAHm> mo muc5054II.h~ mqmda

115

.AAm>mA mo.ov MAucmoAMAcmAm
noMMAo muouqu ucmAwMMAo MQ MoBOAAOM mommum>m .mosAm> mo uom Sumo Mom

 

 

 

N
monp m Eonm oommAu Loom mo mcoAuchEuouoo M mo mcmoEA
m 0.0 m m.A m Mb Q mm m OOMA m 00 M 5m Q 000m mm
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m 0.0 m 0.0 m om m 40 m MMMA Q AMA m mm 0 mmmm mm
m 0.0 m 0.0 Q 4N Q 0 Q 004 m mH Q 0 Q mmm Sm Ohlm
m 0.0 m 0.0 m mm m Ah 0 MAOA m mm m 00 m MBAA 3m Ohlv 4
pnon3 MAM Mo 8mm
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.mochucooII.km mqm4a

116

TABLE 28.--Amounts of Cu, B and A1 in sapwood (SW), heart-
wood (HW) and discolored sapwood (D5) of white oak.1

 

No. of Trees Injured Tissue Cu2 B Al

 

ppm of dry weight

12 12-69 SW 4.9 a 4.0 a 7.7 a
or

4-70 HW 2.9 b 4.3 a 5.3 a

DS 8.0 c 4.9 b 21.6 b

 

l O I 0
means of 3 determinations of each tissue from a tree

2For each set of values, averages followed by differ-
ent letters differ significantly (0.05 level).

117

Levels of ash were usually higher in discolored
sapwood, intermediate in sapwood and lowest from heatwood.
In 1 of 2 white oak wounded in winter and harvested 1
year later, high ash levels were detected in discolored
sapwood. Discolored sapwood from the other tree had ash
levels between those of sapwood and heartwood (Table 29).

The pH of the cold water extract was lowest in
heartwood, intermediate in sapwood and usually highest
in discolored sapwood. In 1 of 2 white oak wounded in
winter and harvested 1 year later, high pH values were
detected in discolored sapwood. Discolored sapwood
from the other tree had a pH value between those of sapwood
and heartwood (Table 30).

Physiological condition of white oak at the time
of mechanical injury affected development of discolored
sapwood. Increases in K levels and ash content were not
observed until 7 months after mechanical injury when
trees were wounded in December, but increases in K levels
and ash content were observed within 4 months after
mechanical injury when trees were wounded in late April
(Table 27, 29).

Ellis (1967) reported decreases in Ca, Mg, K,

Na and Mn during heartwood formation in g. £2233. Heart-
wood of Q, albg_also had lesser amounts of K than sapwood

(Hart, 1968). During heartwood formation, P levels

 

118

TABLE 29.--Ash content of sapwood (SW), heartond (HW) and
discolored sapwood (D8) of white oak.

 

2

 

 

 

No. of Trees Injured Harvested SW HW DS
percent of dry weight

3 12-69 4-70 .464 a .374 a .482

1 12-69 7-70 .405 a .269 b .623

1 12-69 1-71 .441 a .167 b .328

1 12-69 1-71 .498 a .257 b 1.154

4 4-70 8-70 .406 a .225 a 1.419

2 4-70 11-70 .456 a .439 a 1.131

I; .439 a .299 b3

 

1means of 4 determinations of each tissue from a tree

2For each set of values, averages followed by differ-
ent letters differ significantly (0.05 level).

3For each set of values, averages followed by differ-
ent letters differ significantly (0.05 level).

119

TABLE 30.--pH of the cold water extract of sapwood (SW),
heartwood (HW) and discolored sapwood (DS) of white oak.

4

 

No. of Trees Injured Harvested SW2 HW DS
4 12-69 4-70 5.6 a 4.2 b 5.6 a
1 12-69 1-71 6.0 a 4.1 b 4.5 b
1 12-69 1-71 6.0 a 4.1 b 6.8 c
4 4-70 8-70 5.3 a 4.2 b 6.5 c
2 4-70 11-70 5.7 a 4.4 b 6.2 c

 

 

 

 

1means of 4 determinations of each tissue from trees
bulked as indicated in Materials and Methods section

2For each set of values, averages followed by differ-
ent letters differ significantly (0.01 level).

120

decreased in 3. pseudoacacia and M. pomifera while Mg

 

only decreased in the latter species (Hart, 1968).
Heartwood formation in white oak is accompanied
by similar changes in inorganic elements as that observed
with other species of hardwoods. VLevels of P, K, Mg,
Mn and sometimes Ca were lower in heartwood than in
sapwood. Cowling and Merrill (1966) postulated that
trees recovered N from cells in sapwood before their
transformation into heartwood for use elsewhere in
the tree. A similar mechanism may operate for inorganic
elements, especially for those important in cellular
:metabolism. Additional evidence to support this sugges-
tion is presented later in Part II.
Formation of discolored sapwood is also accompanied
Iby changes in levels of inorganic elements present in
‘the sapwood. In several species of hardwoods, K levels
increased during formation of discolored sapwood. Higher
levels of Ca and Mg were detected in discolored sap-
‘dOOd than in sapwood of M. pomifera and 5. pseudoacacia
(Hart, 1968).
Differences and similarities were observed
loetween formation of discolored sapwood in white oak
43nd that observed with other hardwood species. During
jformation of discolored sapwood, large increases in K
Eind smaller increases in Cu, B and A1 were observed.

IAeNels of Ca were sometimes higher in discolored sapwood

121

than in sapwood. Small decreases of P and increases or
decreases of Mg and Mn were also detected in discolored
sapwood. Changes in levels of Mg, P and Mn were usually
not significantly different from levels detected in
sapwood.

Ellis (1967) reported that microorganisms such
as decay and stain fungi change the mineral composition
in discolored tissue of trees. The role microorganisms
played in the accumulation of inorganic elements in
discolored sapwood of white oak was not investigated.
Because they are very effective at accumulating trace
elements in discolored tissue, increases in Cu, B and
Al may be primarily due to the presence of micro-
organisms in discolored sapwood of white oak. A wide
variety of microorganisms are associated with processes
of discoloration and decay in hardwoods (Shigo and
Sharon, 1970).

There is another possible mechanism for accumula-
tion of inorganic elements in discolored sapwood of
white oak. Borer holes remained unplugged during the
investigation period. Evaporation of water from the
transpiration stream of the tree at the surface of the
borer hole could deposit minerals in discolored tissue
and large increases in levels of inorganic elements

could result.

 

122

Hart (1965) reported that ash content and pH of
the cold water extract was lowest in heartwood, inter-
mediate in sapwood and highest in discolored sapwood
of white oak. Similar results were obtained with my
white oak.

Increases in levels of inorganic elements were
accompanied by increases in ash content and pH of the cold
water extract while decrease in levels of inorganic
elements were accompanied by decreases in ash content
and pH. Similar relationships between mineral content
and pH have been reported (Good, Murray and Dale, 1955).
In 3. pseudoacacia, however, heartwood had lower levels
of ash than sapwood, but a higher pH (Hart, 1968). High
K levels appear to be most responsible for increases
in ash content and pH of discolored sapwood in white oak.
Only when high levels of K were detected in discolored
sapwood, were increases in ash content and pH observed
(Table 31).

Physiological condition of the host at the time
of mechanical injury affected development of discolored
sapwood in white oak. One question was whether mechanical
injury in winter actually delayed formation of discolored
sapwood or whether formation of discolored sapwood did
not occur because the tree was dormant and proceeded at
the same rate as formation of discolored tissue did in

trees wounded in spring after tree growth had begun.

123

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A
«.8 AAA.A oomA coma s.m MM4. oMOA 444A osIAA osI4 m
M.M mA4.A MMMA MMMA M.M oo4. sAoA MAAA osIM o~I4 4
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M.M 4MA.A MMMA oo4~ o.M mm4. oosm AMM ARIA moImA A
462 MAM. mva oOAA maz mo4. smoA ommA ohIh mmImA A
M.M ~44. AMAA smoA M.M 4M4. MmAA mmAA osI4 AMINA m
It moons
mm :m4 mo 4 ma sm4 no A omumm>umm omuaflcA mo .02
mo 3m

 

A.xmo muA£3 mo Amav ooo3mmm vaOAOOMAM mam ASMV Moozamm GA
uomuuxo umuo3 UAoo on» no mm can ucmucoo nmm .40 .M aom3umn mAnmcoAuMAomII.AM mqmda

124

Potassium accumulation was a characteristic of discolored
sapwood and appeared to accumulate in discolored tissue
when the tree was growing. Significant increases in K

in discolored sapwood had occurred in trees wounded in
winter within 2 months after growth of the trees had
started. No accumulation was observed during the 5
months after mechanical injury in winter when the trees
were dormant. This observation suggests that mechanical
injury in winter to white oak does not actually delay
formation of discolored sapwood, per se. Accumulation

of K with upward movement of water in the xylem also
suggests that evaporation of water from the transpiration
stream through the borer holes might at least be partially
responsible for the high mineral content of this tissue.

Electron Probe Microanalyzer Studies
White Spruce

In white spruce wounded in spring, P levels
decreased (0.01 level) from outer to inner sapwood. Levels
of P in inner sapwood were not different from those in
heartwood. No difference in P levels was detected at
different positions in heartwood. Levels of P in inner
discolored sapwood were less (0.01 level) than those in

outer and middle discolored sapwood.

125

Levels of P were higher (0.01 level) in ray than
non-ray cells in sapwood, but not in heartwood and dis-
colored sapwood. The difference between levels of P
in ray and non-ray cells decreased (0.01 level) from outer
to inner sapwood (Table 32).

Levels of Ca were not different from each other
in outer and inner sapwood, but were greater in middle
(0.05 level) than in outer sapwood. Calcium levels in
outer heartwood were higher (0.01 level) than in middle
and inner heartwood which were not different from each
other. Levels of calcium in inner discolored sapwood were
greater (0.05 level) than in outer and middle discolored
sapwood. At every position sampled, Ca levels were higher
in ray than non-ray cells (0.01 level) of sapwood and
heartwood, but not discolored sapwood (Table 33).

Levels of Mg were not different from each other
in sapwood and heartwood and Mg levels were not different
from each other in discolored sapwood. Higher Mg levels
(0.01 level) were detected in heartwood than in sapwood.
Ray cells in discolored sapwood had higher (0.05 level)
levels of Mg than non-ray cells (Table 34).

Electron Probe Microanalyzer Studies
White Oak

In white oak injured in winter, P levels decreased
(0.01 level) from outer to inner sapwood. No change in
levels of P was observed at different positions in heart-

wood. Levels of P decreased (0.05 level) from outer to

46.

ABBREVIATIONS

The following abbreviations are used in Tables 32-

R
NR

OSW

ISW

OHW

IHW
ODS
MDS
IDS
BKGD
5%
1%

ND

ray cells

non-ray cells

outer sapwood

middle sapwood

inner sapwood

outer heartwood

middle heartwood

inner heartwood

outer discolored sapwood

middle discolored sapwood

inner discolored sapwood

background

significant (0.05 level) above background
significant (0.01 level) above background

not determined

126

 

127

TABLE 32.--Phosphorous levels at different radial positions
in sapwood, heartwood and discolored sapwood of white spruce
injured in spring.

 

 

 

R NR

Tissue CPlOOS 10310 Count CPlOOS Loglo Count
03W 390 2.592 251 2.400
MSW 257 2.410 219 2.342
13»: 215 2.333 201 2.305
cm 212 2.328 219 2.342
mm 223 2.350 216 2.336
110: 205 2.313 196 2.293
0135 590 2.733 325 2.513
MDS “97 2.697 377 2.577
IDS 223 2.350 223 2.350
BKGD 163 2.213
5 3% (SW. HW) 185 2.267
1 % (SW. PM) 195 2.290
5 13 (D6) 215 2.334

133(06) 242 2L385

‘1‘)

 

 

128

TABLE 33.--Calcium levels at different radial positions in
sapwood, heartwood and discolored sapwood of white spruce
injured in spring.

 

R NR

 

4'42.

 

Tissue CPlOOS Log1° Count CPlOOS Loglo Count
0511 2016 3.383 2080 3.318
MBW’ 2711 3.“33 2395 3.397
ISW' 2541 3.905 222“ 3.3“?
DIN 3199 3.505 2909 3.463
NEW 2680 3.028 2&78 3.39“
IHW 2755 3.000 2692 3. #30
DDS 2056 3.313 1893 3.27?
M05 2104 3.323 1862 3.270
IDS 2‘88 3.387 2285 3.357
BKGD 1M6 3.160
5 % (SW. HW) 1596 3.203
1 % (sw. aw) 1661» 3.221
5 1» (DS) 1630 3.212
1 % (DS) 1710 3.239

 

 

129

TABLE 34.--Magnesium levels at different radial positions in
sapwood, heartwood and discolored sapwood of white spruce
injured in spring.

 

 

 

 

11 NR
Tissue 0121005 10810 Count 091003 Loglo Count

05w 609 2.781 566 2.753
new 620 2.793 633 2.802
ISW 605 2.782 571 2.757
cm 708 2.850 601 2.807
mm 639 2.806 619 2.792
IHW 720 2.860 663 2.822
CD6 724 2L860 5%3 2%???
nos 712 2.853 620 2.793
IDS 605 2.810 558 2.707
BKGD 575 2.760

5 75 (SW. 1M) 629 2.799

1 95 (av. m) 656 2.816

5 56 (D8) 659 2.816

1 $ (US) 690 2.839

 

 

130

inner discolored sapwood. At each position sampled

in sapwood and discolored sapwood, P levels were greater
in ray than non-ray cells (0.01 level). No differences
were detected between P levels in ray and non-ray

cells of heartwood (Table 35).

Levels of Ca were lower (0.05 level) in outer
than in middle and inner sapwood. Calcium levels
decreased between inner sapwood and outer heartwood
and were highest in inner heartwood (0.01 level).

At each position sampled in sapwood, heartwood (0.01
level) and discolored sapwood (0.05 level), Ca levels
were higher in ray than non-ray cells (Table 36).

Levels of Mg did not change from outer to
inner sapwood. Magnesium levels decreased from inner
sapwood to outer heartwood and increased from outer
to inner heartwood (0.01 level). Higher Mg levels
were detected (0.01 level) in ray than non-ray cells
of sapwood and discolored sapwood, but not heartwood.
Levels of Mg in ray cells were greater (0.01 level)
in outer than in middle and inner discolored sapwood
(Table 37).

Levels of Mn decreased from outer to inner
sapwood and inner sapwood to outer heartwood (0.01 level).
No differences in Mn levels were observed at different

positions in heartwood and in discolored sapwood. At

 

131

TABLE 35.--Ph08phorous levels at different radial positions
in sapwood, heartwood and discolored sapwood of white oak
injured in winter.

 

 

 

R NR
lflsswu CEHXES Inglotkmmm (30003 Iogn,Chnnt

CB" 529 2L72“ 369 2.593
MSW 359 2.555 270 2.432
ISW 312 2.995 ' 23b 2.370
can 206 2.314 206 2.314
m ND ND ND ND
1m 218 2.339 207 2.316
ODS 440 2L640 285 25456
HOS 345 2.538 232 2.366
ms 281 2.450 220 2.343
31:00 169 2.229

5 6 (SW. PM) 181 2.258

1 % (SW. HW) 186 ‘ 2.270

5 5 (DB) 181 2.258

1-¢;(DS) 194 2&288

 

 

132

TABLE 36.--Calcium levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in winter.

 

 

 

 

R NR
Tissue 091005 1.0310 Count @1006 1.6glo Count.
0817 2850 3.456 2560 3.409
rsw 3060 3.487 2710 3.434
13»: 3070 3.488 2650 3.424
cm 2880 3.460 2380 3.377

101w ND ND ND ND
IHW Iyfim 3mg“, 3000 BJWQ
ODS 3&10 3.533 2870 3.359
was 3040 3.484 2700 3.432
108 3110 3.493 2790 3.446
BKGD 1707 3.232

5 % (5W) 3”) 1799 ' 3.255

1§$(SW,EM9 1837 1L26h

5 9% (DS) 1906 3.280

1 '35 (DS) 1996 3.300

 

133

TABLE 37.--Magnesium levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in winter.

 

R 1m _

 

 

no suo 0191005 Loglo Count 021005 Log10 Count

and 584 2L767 469 2L662
MSW 530 2.725 492 2.692
ISW 543 2.735 448 2.652
cm 389 2.590 374 2.573
mu ND ND ND ND
IHW 593 2.735 . 518 2.715
ODS 612 2.787 460 2.663
1405 501 2.700 451 2.655
IDS 489 2.690 431 2.635
BKGD 375 2.575

51$(3W.EM), 396 ZJEB

1 % (8w, aw) 405 2.608

5 9 (D5) 400 2.602

115(DB) 411 Zdflh

 

 

134

every position sampled in sapwood and heartwood, Mn
levels were higher (0.01 level) in ray than non-ray
cells. Levels of Mn in ray and non-ray cells of
discolored sapwood were not different from each other
(Table 38).

In white oak injured in spring, P levels decreased
from outer to inner sapwood (0.01 level). The difference
between levels of P in ray and non-ray cells also
decreased (0.01 level). No change in levels of P
occurred in heartwood. In discolored sapwood, P levels
in ray cells were greater (0.01 level) in middle than
in outer and inner discolored sapwood. Ray cells in
outer discolored sapwood had higher P levels (0.05
level) than those in inner discolored sapwood. No differ-
ences in P levels were detected at different positions
in non-ray cells of discolored sapwood.

Every position sampled in sapwood and discolored
sapwood had higher levels of P in ray than non-ray
cells (0.01 level). No differences were observed in
P levels between ray and non-ray cells in heartwood.

More P was present in sapwood than in heartwood (0.01
level) and a greater proportion of P was detected in
ray cells of discolored sapwood than in sapwood (Table 39).

Levels of Ca were not different from each other

at positions sampled in sapwood and discolored sapwood.

Levels of Ca increased from outer to inner heartwood

135

TABLE 38.--Manganese levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in winter.

 

 

 

a an
Tia sus 071005 1.0310 Count 091005 Log10 Count
05w 626 2.797 524 2.722
mu 572 2.758 459 2.662
ISW 555 2.745 476 2.678
on: 436 2.640 382 2.582
mm ND ND ND ND 9
me 407 2.610 352 2.547
003 656 2.817 467 2.670
ms 482 2.683 391 2.593
1:05 524 2. 720 521 2. 717
13x00 311 2.493
5 5 (3". H”) 336 2.527
1 as (SW. 10:) 348 2.542
5 76 (DB) 387 2.588

1 71» (DS) 424 2.628

 

136

TABLE 39.--Phosphorous levels at different radial positions
in sapwood, heartwood and discolored sapwood of white oak
injured in spring.

 

 

 

a 1m
Tissue CPlOOS Loglo Count CPIOOS Loglo Count
05w 1090 3.038, 447 2.651
new 662 2.821 375 2.574
ISW 567 2.75“ 362 2.559
cm 272 2.435 260 2.415
mm 258 2.413 258 2.413
mu 274 2.438 280 2.448
005 881 2.945 370 2.569
1405 1930 3.287 356 2.552
lbs 584 2.767 303 2.482
BKGD 187 2.273
5 5 (5w. m) 203 2.309
1 5 (5w, m) 211 2.325
5 16 (05) 226 2.355

1 5 (05) 245 2.390

 

137

(0.01 level). Except for discolored sapwood and outer
and middle sapwood, higher Ca levels were observed in
ray than non-ray cells (0.01 level) (Table 40).

No differences in levels of Mg and Mn were
observed at positions sampled in sapwood and discolored
sapwood. Levels of both elements decreased from inner
sapwood to outer heartwood and increased from outer
to inner heartwood (0.01 level) (Tables 41, 42).

Higher Mg levels were detected (0.01 level) in
ray than non-ray cells of sapwood and discolored sapwood.
The difference between levels of Mg in ray and non-
ray cells was greater (0.01 level) in outer than in
middle and inner sapwood. No difference in levels of
Mg were observed between ray and non-ray cells of heart-
wood (Table 41).

Higher levels of Mn were detected (0.01 level)
in ray than non-ray cells of sapwood and heartwood. No
differences between levels of Mn in ray and non-ray
cells were detected in discolored sapwood (Table 42).

Levels of K increased from outer to inner sap-
wood, decreased from inner sapwood to outer heartwood
and increased from outer to inner heartwood (0.01 level).
Changes in K levels at different locations in discolored
sapwood were not significant. Higher K levels were
detected in ray than non-ray cells in sapwood (0.01

level) and heartwood (0.05 level), but not in discolored

J

138

TABLE 40.--Calcium levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in spring.

‘3—

 

 

 

a 1m
Tissue CPlOOS Loglo Count CPlOOS LOGIC Count
0541 2230 3.350 2220 3.348
MS" 2300 3.363 2300 3.363
154 2330 3.368 2180 3.340
om 2230 3. 349 2050 3. 313
MEN 2300 3.363 2110 3.325
1114 2610 3.418 2320 3.366
005 2020 3.307 1910 3.283
MDS 1960 EL293 1960 EL293
IDS 1970 EL296 1920 ELZBS
8x00 1514 3.180
£5$1Gflh HW) 1593 2L202
1 5 (511. m) 1626 3.211
5 5 (no) 1633 3.213
1 5 (DS) 1683 3.226

 

139

TABLE 4l.--Magnesium levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in spring.

 

 

 

R . 1m
fflumuo CEEmC Jugdo(kmnt (34905 IoQu,Comfl;

0511 929 2.968 770 2.887
194 839 2.924 807 2.907
15»: 851 2.930 774 2.889
on: 620 2.793 629 2.799
m 690 2.840 688 2.838
nu 837 2.923 811 2.909
005 1050 3.023 765 2.884
was 1020 3.012 772 2.888
IDS 1170 3.0T1 699 24895
BKGD 575 2.760

5 5 (5w, 34) 606 2.783

1 1: (541, m) 620 2.793

5 5 (no) 656 2.817

1 5 (05) 693 2.841

 

140

TABLE 42.--Manganese levels at different radial positions
in sapwood, heartwood and discolored sapwood of white oak

injured in spring.

 

 

 

R
133cm: CREME IngNOCMMnt «unnos lhgio(kmmt
OSW 439 2.643 400 2.602
m 431 2.635 417 2.621
1511 420 2. 624 390 2. 592
01M 390 2.592 357 2.553
W 39“ 2.596 375 2.575
1114 434 2.638 ' 379 2.579
005 360 2.557 378 2.578
1018 358 2.554 342 2.535
IDS 369 2.568 353 2.598
BKGD 309 2.490
5 % (SW. HW) 328 2.517
1 fl» (SW. HW) 338 2.529
5 76 (05) 337 2.528
1 1% (DS) 350 2.544

 

 

141

sapwood. More K was present in sapwood (0.01 level)
than in heartwood (Table 43).

Levels of Cl, 8 and 0 were not different from
each other at positions sampled in sapwood, heartwood
and discolored sapwood. Levels of Cl were greater
(0.01 level) in ray than non-ray cells in sapwood and
heartwood, but not in discolored sapwood. Levels of S
were higher (0.01 level) in ray than non-ray cells of
all tissues while levels of 0 were not different from
each other in ray and non-ray cells of any tissue
(Tables 44, 45, 46).

I observed different patterns of distribution
for elements at different radial positions in sapwood
of white oak and white spruce. Phosphorous levels
decreased from outer to inner sapwood in both tree species.
In 1 group of oaks, Mn followed a similar pattern. The
distribution of P and, in l instance, Mn was the same
as that reported previously for N (Merrill and Cowling,
1966). In white oak, Mg and Mn followed a different
distribution than that of P. Decreases in these elements
occurred at the heartwood boundary. Even in the 1 group
of oaks where Mn had a similar distribution as P, Mn
also decreased significantly at the heartwood boundary.
Potassium levels in oak increased from outer to inner

sapwood. At the heartwood boundary, levels of this

142

TABLE 43.--Potassium levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in spring.

 

 

 

R NR

Pumas onums Zhuio(kmnt CEKDB 10%“,Cmmm
05w 5210 3. 717 2692 3. 430
11811 5795 3.763 2945 ‘ 3.469
ISW 6715 3.827 318‘! 3.503
ON 2559 3.408 2089 3.320
MB“ 2742 3.438 2618 3.418
In»: 3027 3.481 2891 3.461
005 9886 3.995 13102 4.118
was 13105 4. 119 7960 3. 902
105 11803 4.073 10698 4.029
3x00 984 2.993
5 5 (SW. PM) 1079 3.033
1 5 (5w, 8») 1122 3.050
5 5 (00) 1331 3.124
1 5 (05) 1511 3.179

 

143

TABLE 44.--Chlorine levels at different radial positions
in sapwood, heartwood and discolored sapwood of white oak
injured in spring.

 

 

 

R NR

Tis suo 071005 10310 Count CPlOOS 116g10 Count
08»: 2648 3.423 2297 3.361
usw 2884 3.460 2512 3.400
Isw 3090 3.490 2377 3.376 .
onw 2793 3.446 2444 3.388
mm 2898 3.462 2637 3.421
IHW 2979 3.474 2577 3.411
ODS 3891 3.590 3945 3.596
1105 3420 3.534 4678 3.670
IDS 3112 3.493 3468 3.540
BKGD 1280 3.107
:551aflh HW) 1406 2L148
1.% URL HW) 1463 1L165
5 5 (05) 1556 3.192
1 5 (DS) 1691 3.228

 

144

TABLE 4S.--Sulfur levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in spring.

 

 

 

 

R NR
T15 on. 091005 Log10 Count- 071005 1.0310 Count
0511 1055 3.023 800 2.903
now 950 2.978 781 2.893
15w 1017 4 3.007 765 2.884
0841 950 2.978 794 I 2.900

‘ mm 939 2.973 824 2.916
111w 944 2.975 822 2.915
005 1084 3.035 918 2.963 '
mas ‘ 1130 3.053 948 2.977
105 1117 3.048 897 2.953

81:00 606 2.783
5 5 (SW. 1'14) 654 ' 2.816
1 5 (511, m) 676 2.830
5 5 (05) 659 2.819

15%(DS) 682 2L834

 

145

TABLE 46.--Oxygen levels at different radial positions in
sapwood, heartwood and discolored sapwood of white oak
injured in spring.

 

 

 

R 1111
Thump CEMKB 2kmd0(kmnt caums Inqu,0mnm
054: 12701 4.104 11608 4.071
1154 12602 4.101 11007 4. 044
1541 13205 4.122 12405 4.095
OHW 14409 4.161 12306 4.092
11114 13109 4.120 12309 4.093
m 14309 4.158 14509 4.164
005 16303 4.213 18504 4.268
1015 16600 4.220 19105 4.282
ms 17901 4.253 18705 4.273
5100 2224 3.347
5 5 (541, HW) 2427 3.385
1 5 (511. m) 2518 3.401
5 5 (DB) 2383 3. 377
1 5 (05) 2455 3.390

 

 

146

element greatly decreased. The distribution of K in
sapwood of white oak is opposite to that reported for
N (Merrill and Cowling, 1966).

Levels of some elements remained unchanged at
different radial positions in sapwood and heartwood.
These included Cl, S, O (oak) and Mg (spruce). The
distribution of Mg in white spruce was different from
that of P. taeda where levels of Mg increased from
cambium to pith (McMillin, 1970). More Mg, however,
was detected in heartwood than in sapwood of white
spruce. Calcium levels were lowest in outer sapwood

of white spruce and 1 group of white oaks. The distri-

 

bution of Ca resembled that reported for several Pinus
spp where Ca levels increased from outer sapwood to the
heartwood boundary (Wright and Will, 1958; Orman and
Will, 1960).

My data show that elements are recovered from
cells in sapwood before or during their transformation
into heartwood. Phosphorous, K, Mg and Mn levels
decreased across the sapwood or at the heartwood boundary.
The mechanism for recovery must act very quickly.
Potassium levels were highest in inner sapwood, but
greatly decreased in outer heartwood. Different tree
species are not always able to recover the same elements.
White spruce was unable to recover Mg, but recovery

occurred in white oak during heartwood formation. Recovery

occurred from ray and non-ray cells.

147

Chlorine, S, O and Ca were not recovered before
heartwOod formation in white oak. Oxygen forms part
of the woody matrix of the tree and most of this element
is probably not available for recovery. Any recovery
of 0 would probably not be detected because of the small
percentage of the total 0 present in the cell actually
involved.

Studies with Hordeum vulgare, barley, (Greenway

 

and Thomas, 1965) and Phaseolus vulgaris, red kidney

 

bean, (Biddulph et a1. 1958) showed Ca, C1 and S were
not readily retranslocated from the original place of

deposit in these plants. If a similar situation occurred

 

with white oak this might explain why Ca, Cl and S were
not recovered. Ziegler (1966) showed, however, that I
355 was recovered from ray cells of 1 year old Fagus

sylvatica near the pith of the tree. Whether recovery

 

would occur in trees at the age of my oaks is not known.
Juvenile and adult stages in a tree are different in
several characteristics from each other and both oak
and beech, according to Robbins (1957) have juvenile
and adult forms.

Merrill and Cowling (1966) postulated 2 mechanisms
for recovery of N from dying parenchyma cells. Nitrogen
was removed along ray parenchyma back towards the

cambium or recovered by elution with the transpiration

148

stream. Some evidence exists to support the first idea.

Ziegler (1966) showed that significantly higher amounts

35

of 804 were present in ray cells than in vascular

elements in 1 year old E. sylvatica until the pith was

 

reached. Activity which remained in ray cells after

358 did not account for the

removal of the soluble
different levels observed between ray cells and vascular
elements. He concluded that translocation of S occurred
in ray cells and that removal of S from ray cells at

the pith occurred through rays in the tree. A mechanism
for transport of elements along rays was not proposed.
Retranslocation of P in H. vulgare was from mature to
immature leaves, but controlled by the older tissue
(Greenway and Gunn, 1966). Whether similar methods for
regulation of retranslocation of elements exist in both
tree species, is completely speculative.

Recovery of elements from dying cells is not
restricted to cells in sapwood. Recovery of nitrogenous
and phosphorous compounds has been observed from
senescencing leaves of forest trees (Zimmerman, 1964).
In annual plants, N, K, P, S, Cl and sometimes Mg were
recovered from senescencing tissue (Biddulph, 1959).
Calcium and Mn were not recovered. Similarities and

differences exist between elements which are recovered

and not recovered by the 2 phenomena.

Juli.

 

149

Elements which are required for cellular metabolism

were detected in greater amounts in ray cells of sap-
1nood. More P, Mg, Mn, Ca, K, Cl and S were detected in
ray cells than in vascular elements. Oxygen was the
only element studied which did not have higher levels
in ray cells. This element contributes to the woody
matrix of the tree and any accumulation in ray cells
would probably not be detected.

More Mn, Ca, K, S and Cl were observed in ray
cells than in vascular elements in heartwood. Sulfur,
Cl and Ca were not recovered from cells before their
transformation into heartwood and high levels of these
elements in ray cells of heartwood was expected. High
Mn and K levels in ray cells of heartwood suggest that
recovery of these elements might not have been completed
before heartwood formation occurred.

Levels of Ca, Mg and Mn increased from outer
to inner heartwood. Merrill and Cowling (1966) observed
a similar phenomenon with N. They suggested that
diffusion of N from the pith was 1 possible explanation.
Cells in annual increments nearest the pith are usually
shorter in length and have thinner walls. In these
cells, the higher ratio of cytoplasm to cell wall sub-
stance would result in increases in levels of N. If

a similar situation occurred with Ca, Mg or Mn, either

of their suggestions might also explain my results.

'11."?

 

150

Relationships between distribution of elements
at radial positions in sapwood were changed after
mechanical injury. Phosphorous levels decreased between
middle and inner discolored sapwood of white spruce
instead of across the entire tissue as in sapwood. In
ray cells of white oak injured in spring, P levels
were highest in middle discolored sapwood and higher
in outer than in inner discolored sapwood. Levels of
P in non-ray cells were not changed at different radial
positions in discolored sapwood. Levels of P decreased
from outer to inner sapwood. Differences in K and Mn
levels were not observed at radial positions in dis-
colored sapwood while levels of K increased from outer
to inner sapwood and levels of Mn decreased from outer
to inner sapwood.

Differences between levels of elements in ray
and non-ray cells of sapwood were changed after mechanical
injury. In white oak, differences were not observed
between levels of K, Mn and C1 in ray and non-ray cells.
Differences were detected in sapwood and heartwood.

Selective accumulation of elements might occur
in ray cells of discolored sapwood. Greater prOportions
of P (oaks injured in spring) and Mg (oaks injured in
spring, spruces) in discolored sapwood were detected

in ray cells than in sapwood. The central role of both

 

151

elements in cellular metabolism is well documented
(Devlin, 1966). Though highly speculative, this might
suggest that increased synthesis of chemical constituents
occurred in these cells.

Physiological condition of white oak at the time
of mechanical injury might have influenced changes in
distribution of P and Mg in ray and non-ray cells of
discolored sapwood. Greater prOportions of both elements
in discolored sapwood were detected in ray cells than in

sapwood only when trees were injured in spring.

SUMMARY

I measured levels of inorganic elements, ash
content and pH of the cold water extract of discolored
sapwood, heartwood and sapwood of white oak and white
spruce. Levels of P, Na, Ca, Mg, Mn, Fe, Cu, B, Zn
and Al were measured with optical emission and K with
flame photometry.

Changes in levels of inorganic elements were
detected in both tree species during formation of heart-
wood and discolored sapwood. Potassium and P decreased
in heartwood and discolored sapwood of white spruce,
but Mg and Mn decreased only in discolored sapwood.

Calcium and Mn were highest in heartwood. Few changes,

 

152

however, were significantly different from levels of
the same element in sapwood. Potassium increased in
discolored sapwood of white oak, but decreased in
heartwood. Phosphorous, Mg and Mn decreased in heart-
wood while Cu, B and Al increased in discolored sap-
wood. Calcium sometimes increased and decreased in
discolored sapwood or decreased in heartwood.

Ash content was highest in heartwood, intermediate
in sapwood and lowest from discolored sapwood of white
spruce. Differences between tissues were not always
significant. The pH of the cold water extracts were
similar from sapwood and heartwood and lowest from dis-
colored sapwood. Ash content was highest in discolored
sapwood, intermediate in sapwood and lowest in heartwood
of white oak. The pH of the cold water extract was
highest in discolored sapwood, intermediate from sap-
wood and lowest in heartwood.

An electron microprobe x-ray analyzer - scanning
micros00pe was used to detect differences in levels of elements
at radial positions in sapwood, heartwood and discolored
sapwood. Differences in levels of elements in ray
and non-ray cells were studied at these radial positions
in both tree species. Phosphorous, Ca and Mg were
studied in both tree species while Mn, K, Cl, S and 0

were also studied in white oak.

153

Chlorine, S, 0 (white oak) and Mg (white spruce)
were not affected by changes in radial positions in
sapwood and heartwood. Phosphorous decreased from
outer to inner sapwood and Ca was lowest in outer sap-
wood of both tree species. Magnesium and Mn decreased
between inner sapwood and outer heartwood. Potassium
increased from outer to inner sapwood, but decreased
from inner sapwood to outer heartwood. Calcium, Mg,

Mn and K increased from outer to inner heartwood in
white oak. In white oak, more P, Mg (DS, SW), Ca, S
(DS, SW, HW), Mn, K and Cl (SW, HW) were detected in
ray cells than in vascular elements. In white spruce,
more P (SW), Ca (SW, HW) and Mg (DS) were detected in
ray cells than in vascular elements.

The distribution of elements in ray and non-
ray cells at different radial positions in discolored
sapwood of white spruce injured in spring and white
oak injured in winter was not greatly different from
that observed in sapwood. Phosphorous decreased between
middle and inner discolored sapwood while Ca was highest
in inner discolored sapwood of white spruce. Calcium
and Mn were not changed at different radial positions
in discolored sapwood of white oak. More drastic changes

were detected in discolored sapwood of white oak injured

 

154

in spring. In ray cells, more P was detected in middle
than in outer discolored sapwood which had more P than
inner discolored sapwood. No difference in P was

detected at different positions in non-ray cells. Differ-
ences in K were not observed at radial positions in
discolored sapwood.

Physiological condition of white oak at the time
of mechanical injury affected development of discolored
sapwood. Increases in K levels and ash content were
not observed until 7 months after mechanical injury
when trees were wounded in December, but increases in
K levels and ash content were observed within 4 months
after mechanical injury when trees were wounded in late
April.

With respect to the criteria investigated in this
study, formation of discolored sapwood and heartwood
are different from each other in white oak. Formation
of discolored sapwood was sometimes different from
heartwood formation in white spruce. Formation of dis-
colored sapwood in white oak is very different from

that observed for white spruce.

LITERATURE CITED

155

 

 

   

LITERATURE CI TED

Andersen, C. 1967. An introduction to the electron
probe microanalyzer and its application to
biochemistry. In: D. Glick, Methods of
Biochemical Analysis. XV. John Wiley and
Sons, Inc., New York. p. 148-270.

Bergstr6m, H. 1959. The ash content and phosphorous
content of coniferous woods. Svensk PappTidn.
62: 160-161.

Biddulph, 0., S. Biddulph, R. Cory and H. Koontz. 1958.
Circulation patterns for phosphorous, sulfur and
calcium in the bean plant. Plant Physiol. 33:
293-300.

. 1959. Translocation of inorganic solutes.
In: F. Steward, Plant Physiology, A Treatise.
II. Plants in Relation to Water and Solutes.
Academic Press, New York. p. 553-603.

 

Cowling, E. and W. Merrill. 1966. Nitrogen and its
role in wood deterioration. Canad. J. Bot.
44: 1539-1554.

Devlin, R. 1966. Plant Physiology. Reinhold Pub. Corp.,
New York. 564 p.

Ellis, E. 1967. Minerals, crystals, and heartwood. XIV.
IUFRO Congress 9 (Sec 41): 1-7.

Good, H., P. Murray and H. Dale. 1955. Studies on heart-
wood formation and staining in sugar maple Acer
saccharum Marsh. Canad. J. Bot. 33: 31-41.

 

Greenway, H. and D. Thomas. 1965. Plant response to
saline substrates. V. Chlorine regulation in the
individual organs of Hordeum vulgagg during treat-
ment with sodium chloride. Aust. J. Biol. Sci.
18: 505-524.

156

157

and A. Gunn. 1966. Phosphorous retranslocation
in Hordeum vulgare during early tillering.
Planta 71: 43-67.

Hart, J. 1965. Formation of discolored sapwood in three
species of hardwoods. Quart. Bull. Mich. Agric.

. 1968. Morphological and chemical differences
between sapwood, discolored sapwood and heartwood
in black locust and osage orange. For. Sci. 14:
334-338.

McMillin, C. 1970. Mineral content of loblolly pine
wood as related to specific gravity, growth rate
and distance from pith. Holzforschung 24:
152-157.

Merrill, W. and E. Cowling. 1966. Role of nitrogen in
wood deterioration: amounts and distribution of
nitrogen in tree stems. Canad. J. Bot. 44:
1555-1580.

Orman, H. and G. Will. 1960. The nutrient content of
Pinus radiata trees. N. Z. J. Sci. 3: 510-522.

 

Rennerfelt, E. and C. Tamm. 1962. The contents of
major plant nutrients in spruce and pine attacked
by Fomes annosus (Fr.) Cke. Phytopath. Z. 43:
371-382.

 

Robbins, W. 1957. Physiological aspects of aging in
plants. Amer. J. Bot. 44: 289-294.

Shain, L. 1971. The response of sapwood of Norway spruce
to infection by Fomes annosus. Phytopathology
61: 301-307.

Shigo, A. and E. Sharon. 1970. Mapping columns of dis-
colored and decayed tissues in sugar maple Acer
saccharum. Phytopathology 60: 232-237.

 

I I
Wazny, H. and J. Wazny. 1964. Uber das Auftreten von
Spurenelementen im Holz (The occurrence of trace
elements in wood). Holz Roh-u. Werkstoff 22:
299-304.

Wright, T. and G. Will. 1958. The nutrient content of
Scots and Corsecan pines growing on sand dunes.
Forestry 31: 13-25.

 

158

Ziegler, H. 1966. The use of isotOpes in the study of
translocation in rays. Isotopes and Radiation in
Soil-Plant Nutr. Stud. Symp. Ankara, Turkey, 1965.
Int. Atomic Energy Agency, Vienna, 1966. p. 361-368.

Zimerman, M. 1964. The relation of transport to growth
in dicotyledonous trees. In: M. Zimmerman,
The Formation of Wood in Forest Trees. Academic
Press, New York. p. 289-301.

PART III

159

141-'4-

 

 

«Ii

 

PART III

THE DURABILITY OF SAPWOOD, HEARTWOOD AND DISCOLORED
SAPWOOD OF WHITE OAK AND WHITE SPRUCE TO

POLYPOROUS VERSICOLOR AND PORIA MONTICOLA

 

 

In most trees, the trunk is composed of sapwood
and heartwood. In living trees, sapwood is usually
more resistant than heartwood to decay fungi. After
harvest, sapwood of many tree species is more susceptible
to decay fungi than heartwood. Mechanical injury or
attack by microorganisms may cause discoloration to sap-
wood (discolored sapwood). Discolored sapwood may
be more resistant to wood-decay organisms than sapwood.
Considerable variation in decay resistance

has been reported for heartwood of Quercus alba. The

 

variability (6-36% of oven-dry weight) was as great
whether trees were from similar or widely scattered
locations (Scheffer, Englerth and Duncan,l949). The
sapwood of Q. alba is susceptible to decay (Hart, 1964;
Zabel, 1948) while heartwood of Pigga spp is susceptible

to decay (Scheffer and Cowling, 1966).

160

 

 

 

161

Discolored sapwood may be more resistant to

decay fungi than sapwood. Discolored sapwood of Q. alba,

 

Robinia pseudoacacia and Maclura pomifera was more

 

resistant to decay by Polyporous versicolor than sap—

 

wood (Hart and Johnson, 1970). The reaction zone of

Fomes annosus-infected Pinus taeda and Picea abies,

 

incipiently decayed wood (both species) and heartwood
(g. taeda) were more resistant than sapwood (both
species) and heartwood (g. abies) to decay by E. annosus
(Shain, 1967; 1971). Discolored sapwood of Juglans

nigra and Acer saccharinum, however, was as susceptible

 

as sapwood to decay by P. versicolor (Hart, 1964).
Chemical constituents which are deposited during

formation of heartwood and discolored sapwood are thought

to be responsible for the increase in decay resistance

of both tissues. Removal of methanol-soluble compounds

from heartwood of several eucalypts increased the

susceptibility of this tissue to decay fungi (Rudman

and Da Costa, 1961). Compounds which were soluble in

ether and methanol appeared to be responsible for the

durability of Tectona grandis heartwood to decay organisms

 

(Rudman and Da Costa, 1959). The hot water extract from
heartwood of g. alba tested in_vitro was toxic to
linear growth of Lenzites trabea (Zabel, 1948). Extrac-

tion of resistant tissues of F. annosus-infected P.

162

taeda with acetone increased their susceptibility to
decay by F. annosus (Shain, 1967). Shain and Hillis
(1971) suggested that hydroxymatairesinol and the
alkaline conditions of the reaction zone of F. annosus-
infected P. abies contributed to the durability of

this tissue to g. annosus.

I studied the development of decay-resistant
discolored sapwood in white oak (Quercus a1 a L.) and
white spruce (Picea glauca (Moench) Voss). One
objective of this study was to determine whether
mechanical injury to sapwood of white spruce produced
discolored sapwood that was more resistant to decay than
sapwood and heartwood. Another objective was to deter-
mine the effect of physiological condition of white
oak at the time of mechanical injury on the development
of decay-resistant discolored sapwood. Recently,

Hart and Hillis (unpublished data) suggested that the
durability of heartwood of g. alba to P, monticola
appeared to result from the presence of several
ellagitannins in this tissue. The role of ellagitannins
in the durability of discolored sapwood to g. monticola

was also investigated.

163

MATERIALS AND METHODS
Location of trees, method of mechanical injury
and initial preparation of wdod samples are described
in Part I. White oaks and white spruces which were
used in Part II were also utilized in this investigation.
Information about these trees is presented in Table 22
in Part II.

Measurement of Durability of
Discolored Sapwood, Sapwood and Heartwood

The resistance of discolored sapwood, sapwood
and heartwood to decay was measured with the agar block
method (McNabb, 1958). Tissues of white oaks and
white spruces were exposed to Polyporous versicolor
L. ex Fr. (USDA isolate Madison 697), a white-rot

organism, and Poria monticola Murr. (USDA isolate

 

Madison 698), a brown-rot organism.

Blocks were dried for 14 days at 40 C and
weighed to determine the initial dry weight of the
blocks. After incubation with the fungus, blocks were
removed from the decay chambers and the fungus was
carefully scraped from the blocks. Blocks were immediately
weighed to determine their moist weight. The final dry
weight was determined after drying the blocks for 48

hours at 105 C. Weight loss was expressed as percent

164

of the initial Oven-dry weight of the block. Blocks
*which were not exposed to the decay fungi served as
controls.

Blocks of each tissue, approximately 15 x 8 x 8 mm
in size, were cut with the longest dimension parallel
to the radial axis of the tree. The blocks were auto-
claved for 5 minutes at 121 C and placed aseptically
into the decay chambers 14 days after the chambers had
been inoculated with the test organism. The decay
chambers were incubated at 26 C for 6 weeks.

Eight oz. French square bottles capped with
unlined aluminum lids were used for decay chambers.

Each bottle contained 32 ml of 3% (w/v) malt extract
and 1.5% (w/v) agar in distilled water. Bottles were
autoclaved at 121 C for 40 minutes, cooled to room
temperature and inoculated with a small piece of
mycelium from the outer margin of a 2 week old culture
of the test organism. A 4 mm glass rod was placed on
the agar to support the block above the medium.
Extraction of Tissue of White Oak for Ellagitannins

White oak ll, 12, 13 and 14 (Part II, Table 22)
were used to investigate the distribution of ellagitannins.
Sapwood, heartwood and discolored sapwood of white oak
injured in spring (11, 12) were bulked separately

while sapwood and heartwood of white oak injured in

165

winter (13, 14) were bulked separately. Discolored
sapwood of these trees was extracted individually because
previous work (Part II) had showed that these tissues
were different from each other. Oven-dried material
prepared as described in Part I was used.

Four g of each tissue were extracted for 48
hours on a "wrist-action" reciprocal shaker with 250 ml
of acetone, water (9:1 v/v). The extraction was
repeated 2 more times and the extracts were combined.
They were reduced to 200 ml in a rotary evaporator at
40 C. Extracts were washed with six 50 ml portions
each of hexane, chloroform and ethyl acetate and the
washings were discarded. Extracts were reduced to
near-dryness in a rotary evaporator at 40 C and brought
to 1 ml with acetone, water (9:1 v/v).

Two-dimensional paper chromatography was used
to study the ellagitannins. Fifty microliters of each
extract were applied with a micropipette onto Whatman
3 MM chromatography paper. Papers were irrigated in
the first direction with 6% acetic acid and in the
second direction with butanol, 27% acetic acid (1:1).
Chromatograms were viewed under shortwave ultraviolet
light and sprayed with 0.05% p - nitrobenzediazonium

tetrafluoborate (pNA) and oversprayed with 20% sodium

166

carbonate. Chromatograms were also sprayed with NSSC
(15 g NaZSO3: 3.5 Na2C03: 350 ml H20) and examined at
3 minutes and 12 hours after spraying. The Rf value

and color reaction of each ellagitannin was determined.

RESULTS AND DISCUSSION

Durability of Discolored Sapwood, Sapwood and Heartwood
to Polyporous versicolor and Poria monticola

 

Discolored sapwood from both tree species was
more resistant than sapwood (oak) or sapwood and heart-
wood (spruce) to decay by g. versicolor and g, monticola
(Tables 47, 48). My results with white oak are the
same as those reported previously with this species
(Hart and Johnson, 1970). Shain (1971) reported that
the reaction zone of g. abies produced in response to
fungus invasion and mechanical injury to sapwood was
more resistant to decay by E. annosus than sapwood and
heartwood. My results show that mechanical injury to
sapwood of white spruce also produced discolored sap-
wood more resistant than sapwood and heartwood to decay
by at least 2 decay fungi. The mechanism for resistance
in each tissue is very different (Appendix).

Physiological condition of white oak at the time
of mechanical injury influenced the develOpment of

decay-resistant discolored sapwood. When trees were

167

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169

wounded in December, discolored sapwood durable to both
decay fungi was not observed until 7 months after
mechanical injury while durable discolored sapwood was
observed within 4 months after mechanical injury
‘when trees were wounded in late April (Table 48). Other
aspects about this subject are presented in Part II.

The effect of season on formation of discolored

sapwood has been investigated with Pinus sylvestris

 

(Lyr, 1967) and Pinus resinosa (Jorgensen, 1961). In

 

P, sylvestris, formation of discolored sapwood (resin

 

flow and accumulation of pinosylvin) occurred between
late April and early December. In g. resinosa,
formation of discolored sapwood (accumulation of
pinosylvin) occurred in November and January, but not

in June and July. In both cases, the durability of
discolored sapwood was not evaluated. My results appear

to be similar to those observed with P. sylvestris.

 

Formation of discolored sapwood in white oak, however,

was much different from that observed in P. sylvestris

 

(Lyr, 1967).
Discolored sapwood from white oak was more

resistant to g, monticola than to P. versicolor while

 

 

the reverse was true for white spruce. Rudman (1963)

showed that most extractives toxic to wood-decay fungi

170

had a limited spectrum of activity. If extractives
were responsible for durability of discolored sapwood
in either or both tree speices, they might be less
effective against one of the wood-decay organisms.
Mechanisms other than extractives may contribute to the
durability of discolored sapwood. As with extractives,
they could be less effective against one of the wood-
decay fungi.

Distribution of Ellagitannins in Tissues
of White Oak

Four ellagitannins were detected in tissues of
white oak. The Rf value and color reaction of each
compound are given in Table 49 and the distribution of
these compounds in the 3 tissues of the tree is presented
in Table 50. Three of the 4 ellagitannins were detected
in sapwood and discolored sapwood (one exception) and
all 4 ellagitannins were detected in heartwood. The
color intensity of ellagitannins in heartwood extracts
was greater than observed for the other tissues. These
compounds are the same as those detected in heartwood
of Q. alba (Hart, personal communication) and reported
to contribute to the durability of this tissue to

P, monticola (Hart and Hillis, unpublished data).

 

171

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172

TABLE 50.--Distribution of ellagitannins in sapwood (SW),
heartwood (HW) and discolored sapwood (DS) of white oak.

 

Compound SW(S)1 HW(S) 05(3) sw<w> HW(W) 05(w1) 05(w2)

 

 

l P P P P P P A
2 P P P P P P A
3 P P P P P P A
TZ A P A A P A A

1(S) - injured in spring; (W) - injured in winter:

SW, HW and D8 of 2 trees wounded in spring bulked while SW
and HW of 2 trees wounded in winter bulked, but US of these
trees extracted separately. Previous decay studies showed
that SW from white oaks was susceptible, HW was resistant
and DS resistant (8, W2) or susceptible (W1) to decay by
Poria monticola and Polyporous versicolor.

2P - detected; A - not detected

 

 

 

173

My results show that the presence of ellagitannins
is not necessary for discolored sapwood to be durable

to g. monticola. Discolored sapwood of 1 white oak (W 2)

 

was very resistant to decay by B. monticola (weight

 

loss of 0.3%), but ellagitannins were not detected in
this tissue. This observation suggests that the 4
ellagitannins are not responsible for formation of dis-
colored sapwood that is resistant to decay by g.

monticola.

 

Discolored sapwood of white oak was more resistant

to decay by g. monticola when the pH of the cold water

 

extract from this tissue was above 6.0 (Table 51). A
liquid medium was used to measure the effect of pH upon
in zitgg growth of this fungus. The buffered (0.05 M)
medium contained (mg/1): glucose, 20,000; asparagine,
2000; KH2P04, 1000; MgSO4-7H20, 500; biotin, 0.005;
thiamine, 0.1. Two ml of a micronutrient solution which
contained (mg/l): Fe(NO3)3-9H20, 723.5; ZnSO4-7H20,
439.8; MnSO4-4H20, 203.0 were added and the medium was
brought to 1 liter with distilled water. Portions of
the medium were adjusted to the proper pHs with NaOH

or 6 N HCl and sterilized by millipore filtration.

Twenty-five ml of medium were added to 300 m1 erlenmyer

¥.___1__

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174

TABLE 51.--Relationship between pH of the cold water extrait
and decay resistance of discolored sapwood of white oak.

 

 

 

 

 

 

Characteristic Tree SW DS HW2
pH 11, 12 5.7 a 6.2 b 4.4 c
13 5.9 a 4.5 b 4.1 C
14 6.0 a 6.8 b 4.1 C
Weight loss (% of 11, 12 12.6 a 3.0 b 4.7 b
oven-dry weight)
13 24.8 21.8 a 7.5 b
3° m°nt1°°1a 14 28.7 0.3 b 10.5 c
Weight loss (% of 11, 12 42.6 29.6 b 6.8 C
oven-dry weight)
13 32.7 20.9 b 7.8 c
3' ver51°°l°r 14 36.0 15.5 b 11.9 b

 

1

13, 14 - injured in winter

white oaks 11, 12 - injured

in spring; white oaks

2SW - sapwood; HW - heartwood; DS - discolored sap-
wood; For each set of values, averages followed by differ-
ent letters differ significantly (0.05 level).

175

flasks and the flasks were inoculated with 1 ml of mycelial
suspension (aerial mycelium ground in 100 ml of sterile
distilled water). Flasks were incubated on laboratory
shelves up to 3 weeks at 26 C.

At 7 day intervals, growth of g. monticola was
measured by filtering cultures through tared Whatman No. 3
filter paper which was previously dried for 12 hours
at 40 C, oven-dried for 1.5 hours at 105 C, cooled in
a desiccator over indicating Drierite for 15 minutes
and weighed. The filtered medium was saved and the pH
measured with a Beckman zeromatic pH meter.

Poria monticola grew between pH 3.5-6.0. Best

 

growth was achieved when the fungus was able to reduce
the pH of the medium during the incubation period
(Table 52). These results suggest that high pH might
play some role in the development of decay-resistant
discolored sapwood of white oak to E, monticola. The
effect of high pH on fungus growth ig_vitro, however,
may be very different from that observed in gigg,
Additional data is needed before firm conclusions are
possible. High K levels appear to be primarily res-
ponsible for the high pH of the cold water extract from

discolored sapwood (Part II).

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177

Discolored sapwood was resistant to decay (0.05

level) by g, versicolor when the pH of the cold water

 

extract from this tissue was 4.5 (Table 51). This
observation suggests that the agent(s) responsible for
durability of discolored sapwood to E, monticola and

E. versicolor may be different. Different factor(s)

 

appear to be responsible for the durability of heartwood

of Q. alba to g. monticola and E. versicolor (Hart and

 

Hillis, unpublished data).

S UMMARY

The durability of discolored sapwood of white oak
and white spruce to Poria monticola and Polyporous versicolor
was measured. The agar-block method was used. Discolored
sapwood from both tree species was more resistant than
sapwood (oak) or sapwood and heartwood (spruce) to decay
by both decay fungi.

Physiological condition of white oak at the time
of mechanical injury influenced the development of decay-
resistant discolored sapwood. Durability of discolored
sapwood to both decay fungi was not observed until 7
months after mechanical injury when trees were wounded
in December, but was observed within 4 months after

mechanical injury when trees were wounded in late April.

178

The distribution of ellagitannins in tissues of
‘white oak was studied with two-dimensional paper
chromatography. Four compounds were detected in heart-
‘wood and 3 were detected in sapwood and discolored sap-
wood (one exception). Ellagitannins were not necessary

for discolored sapwood to be durable to g, monticola.

 

High pH levels of discolored sapwood of white
oak might contribute to the durability of this tissue

to P, monticola. Growth of the fungus in vitro did not

 

occur above pH 6.0. Resistance of discolored sapwood

to decay by P, monticola was observed when the pH of the

 

cold water extract from this tissue was above 6.0.

.I. *3.

LITERATURE CITED

179

*a-
. .

LITERATURE CITED

Hart, J. 1964. Production of fungistatic substances by
mechanically damaged sapwood. Phytopathology
54: 895 (Abstr.)

and K. Johnson. 1970. Production of decay-
resistant sapwood in response to injury. Wood
Sci. Technol. 4: 267-272.

 

JorgenSen, E. 1961. The formation of pinosylvin and
its monomethyl ether in sapwood of Pinus resinosa
Ait. Canad. J. Bot. 39: 1765-1772.

 

 

Lyr, H. 1967. Uber den jahreszeitlichen Verlauf der
Schutzkernbildung bei Pinus sylvestris nach
Verwundungen (On the seasonal course of wound
heartwood formation in Pinus s lvestris after
wounding). Arch. Forstw. I6: 91-57.

 

McNabb, H., Jr. 1958. Procedures for laboratory studies
on wood decay resistance. Proc. Iowa Acad. Sci.
65: 150-159.

Rudman, P. and E. Da Costa. 1959. Variation in extractive
content and decay resistance in heartwood of
Tectona grandis L. f. J. Inst. Wood Sci. 3: 33-42.

and . 1961. The causes of natural
durability in timber. IV. Variation in the role
of toxic extractives in the resistance of durable
eucalypts to decay. Holzforschung 15: 10-15.

 

 

. 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.

 

Shain, L. 1967. Resistance of sapwood in stems of
loblolly pine to infection by Fomes annosus.
Phytopathology 57: 1034—1045.

 

. 1971. The response of sapwood of Norway spruce
to infection by Fomes annosus Phytopathology 61:
301-307.

180

181

and W. Hillis. 1971. Phenolic extractives in
Norway spruce and their effects on Fomes annosus.
Phytopathology 61: In press.

 

Scheffer, T., G. Englerth and C. Duncan. 1949. Decay
resistance of seven native oaks. J. Agric. Res.

and E. Cowling. 1966. Natural resistance of
wood to microbial deterioration. Annual Review
of Phytopathology 4: 147-170.

 

Zabel, R. 1948. Variations in the decay resistance of
white oak. N. Y. State College Forestry Tech.
Pub. No. 6& 53 p.

APPENDIX

182

APPENDIX

ANALYSIS OF LIGNANS IN WHITE SPRUCE WITH

GAS CHROMATOGRAPHY

Samples of sapwood, heartwood and discolored
sapwood were cut from white spruces A and B (Part II,
Table 22). Each wood sample was dried on a laboratory
bench for 72 hours and ground in a Wiley mill to
pass through a 2 mm mesh screen. Preliminary experi-
ments indicated that the high resin content of dis-
colored sapwood interfered with the chromatographic
analysis of lignans. Hence each sample was first
extracted for 6 hours in a Soxhlet apparatus with n-
heptane before being extracted with methanol. The
percentage of each sample which was soluble in the 2
solvents was determined (Table 53).

Quantitative estimates of lignans present in
each tissue were made by GLC using a Varian 2100 gas
chromatograph with flame ionization detectors. A glass
column 2 meters long of 3 mm inside diameter packed
with 3.6% Apiezon L on 80-100 mesh DMCS Chromosorb W

was used. The carrier gas (nitrogen) flow rate was

183

184

TABLE 53.--Heptane and methanol solubilities of white spruce
wood (% oven-dry basis).

 

 

Tissuel Heptane Methanol
A-SW 2.0 3.6
A-HW 1.2 1.15
A-DS 29.3 6.2
B-SW 2.4 3.4
B-HW 1.7 1.42
B-DS 34.0 10.0

 

l
sapwood

SW - sapwood; HW - heartwood; DS - discolored

185

50 ml/min, while hydrogen and air flow rates were
35 ml/min and 350 ml/min. Detector and injection temper-
atures were 250 C and initial oven temperature (200 C)
was increased 1 C per min to a final temperature of 240 C.
Seventy microliters of TMS mixture-
hexamethyldisilazane:trimethylchlorosilane:pyridine
(2:1:10 v/v/v) - and 30 microliters of BSA (N,O-bis
(tri-methylsilyl) acetamide) were successively added to
a known amount (approximately 1 mg) of the vacuum dried
methanol extract. This mixture was briefly heated with
a match and after 15 min 5 microliters were injected
into the chromatograph. Known amounts of matairesinol,
liovil and conidendrin were silylated in a similar
fashion for calibration purposes. Previous work (Shain
and Hillis, 1971; Krahmer, Hemingway and Hillis, 1970)
had shown that calibration curves for these lignans
showed a linear response over the range of concentration
that lignans were detected in wood. Authentic
hydroxymatairesinol and pinoresinol were also used to
establish relative retention times (RRT). The 5 authentic
silylated lignans were used as markers both alone and
in combination with the various silylated methanol
extracts. RRT for the silylated lignans were:
hydroxymatairesinol 1.00; liovil 0.80; matairesinol 1.13;
conidendrin 1.28; pinoresinol 1.68. Authentic lignans

and extracts were also run under previously reported

186

chromatographic conditions (Shain and Hillis, 1971;
Krahmer, Hemingway and Hillis, 1970) for comparative
purposes. Two or more analyses were made of each
sample.

Compounds with the same retention time as
authentic silylated liovil, hydroxymatairesinol,
conidendrin and pinoresinol (trace amounts) were present.
Matairesinol was not present in detectable amounts.
Relative values for peak areas for liovil, hydroxymatairesinol
and conidendrin are given in Table 54.

There is no indication from this work that
mechanical injury to the sapwood of white spruce resulted
in an increase in hydroxymatairesinol or in any of the
other lignans studied. At a concentration of approximately
0.10%, hydroxymatairesinol appears to have little effect
on the decay resistance of the wood against Pgria

monticola and Polyporous versicolor. Shain and Hillis

 

(1971) did report that 0.1% of hydroxymatairesinol in

an agar medium reduced growth of EQEEE annosus by 25%.

The increased decay resistance shown for discolored
sapwood of white spruce must be due to some other factor
than lignan concentration as the decay susceptible sapwood
and heartwood contained the same lignans in approximately
the same concentration as did the injured tissue. The

response of white spruce sapwood to mechanicl injury

187

TABLE 54.--Relative concentration of hydroxymatairesinol,
liovil and conidendrin in the sapwood (SW), heartwood (HW)
and discolored sapwood (D8) of white spruce.

 

 

 

A B
SW HW DS2 SW HW DS2
Hydroxy- l
matairesinol 172 72 67 215 142 207
Liovil 39 34 22 54 47 45
Conidendrin 20 29 25 4 32 50

 

1A value of 170 is equal to 0.10% of the oven-dry
wood.

2Amount of liovil in DS was difficult to measure
accurately because of other compounds with similar retention
times.

188

(no significant changes in lignans present and acid pH)

is in marked contrast to the response of Picea abies

 

sapwood to invasion by E. annosus (large increases in
hydroxymatairesinol and alkaline pH) as reported by
Shain and Hillis (1971). They also reported that
hydroxymatairesinol and conidendrin were not present in
detectable quantities in the sapwood of g. abies, but
liovil was present in similar amounts to those reported
here for white spruce.

The values reported in this study for the heart-
wood of white spruce agree very closely with the values
reported for the heartwood of g. abies by Freudenberg
and Knof (1957). They are also quite similar to the
data reported by Goldschmid and Hergert (1961) for the
sapwood of Tsuga heterophylla. Hydroxymatairesinol
(0.255%) was found to be present in a ratio of about 5:1
to that of conidendrin (0.05%). Only a trace of
pinoresinol (0.009%) and no matairesinol were present.
Freudenberg and Niedercorn (1958 as reported by Goldschmid
and Hergert) stated that the cambial sap of g. abies
contained hydroxymatairesinol and pinoresinol.

Weinges (1960) reported both quantitative and
qualitative changes in wound resin lignan composition of
g. abies after injury while Shain and Hillis (1971)

reported only an accumulation of normal heartwood lignans

189

after attack by E. annosus. In this study, neither
quantitative or qualitative differences were observed
in response to injury. Hence this work is additional
evidence to support the hypothesis that the accumula-
tion of lignans in plant tissue is quite variable as
previously reported by Krahmer, Hemingway and Hillis

(1970).

LITERATURE CITED

190

LITERATURE CITED

Freudenberg, K. and L. Knof. 1957. Die Lignane des
Fichtenholzes (Sprucewood lignans). Chemische
Berichte 90: 2857-2869.

and F. Niedercorn. 1958. Anwendung radioaktiver
IsotOpe bei der Erforschung des Lignins. VIII.
Umwandlung des Phenylanins in Coniferin und
Fichten Lignin (Application of radioactive
isotopes in investigations of lignin. VIII.
Transformation of phenylalanine into coniferin

and spruce lignin). Chemische Berichte 91: 591-
597.

 

Goldschmid, O. and H. Hergert. 1961. Examination of
western hemlock for lignin procursors. Tappi
44: 858-870.

Krahmer, R., R. Hemingway and W. Hillis. 1970. The
cellular distribution of lignans in Tsuga
heterOphylla wood. Wood Sci. Technol. 4: 122-139.

 

Shain, L. and W. Hillis. 1971. Phenolic extractives in
Norway spruce and their effects on Fomes annosus.
Phytopathology 61: In press.

Weinges, K. 1960. Die Lignane des Uberwallungsharzes

der Fichte (Lignans of the resin of spruce).
Tetrahedron Lett. 20: 1-2. '

191

   

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