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Copy 1
The University of Chicago

BLISTER CANKER OF APPLE TREES
A PHYSIOLOGICAL AND
CHEMICAL STUDY

A DISSERTATION

SUBMITTED TO THE FACULTY
OF THE OGDEN GRADUATE SCHOOL OF SCIENCE
IN CANDIDACY FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

DEPARTMENT OF BOTANY

BY
DEAN HUMBOLDT ROSE

Private Edition, Distributed By
THE UNIVERSITY OF CHICAGO LIBRARIES
CHICAGO, ILLINOIS

Reprinted from
THE Botanica GazeETTE, Vol. LXVII, No. 2
February 1919

The University of Chicago

BLISTER CANKER OF APPLE TREES;
A PHYSIOLOGICAL AND
CHEMICAL STUDY

A DISSERTATION

SUBMITTED TO THE FACULTY
OF THE OGDEN GRADUATE SCHOOL OF SCIENCE
IN CANDIDACY FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

DEPARTMENT OF BOTANY

BY
DEAN HUMBOLDT ROSE

Private Edition, Distributed By
THE UNIVERSITY OF CHICAGO LIBRARIES
CHICAGO, ILLINOIS

Reprinted from
THE BoTaNIcAL GAZETTE, Vol. LXVII, No. 2
February 1919

Git

Aut
MAR 13 1919

VOLUME LXVII NUMBER 2

6
BOTANICAL C4AZE7 FE

FEBRUARY igro

BLISTER CANKER OF APPLE TREES; A PHYSIOLOGE-
CAL AND CHEMICAL STUDY

CONTRIBUTIONS FROM THE HULL BOTANICAL LABORATORY 246
DEAN H. ROSE
(WITH TEN FIGURES)
Introduction

It is now generally recognized that among the most important
problems of plant pathology are those connected with the physiol-
ogy of diseases whose etiology is already known. It is also recog-
nized that this must be the physiology of the host, of the parasite,
and of the two in relation to each other, and, further, that such a
comprehensive view of all the factors involved furnishes the only
rational approach to an understanding of the principles underlying
immunity and disease resistance.

In the present paper are given the results of a physiological
study of the destructive disease known as Illinois or blister canker,
the etiology of which, including the identity of the causal organism,
Nummularia discreta (Schw.) Tul., was worked out by HAssEt-
BRING (22) in 1902. The work reported here is a continuation of
an earlier investigation by the writer (30) on the oxidase activity
of healthy and diseased bark; in addition there is included an
account of the catalase activity and microchemical and macro-
chemical analyses of both kinds of tissues. Further work is planned
on the chemistry of the disease, on the rdle of other enzymes than.
oxidases, and on the physiology of the fungus itself in pure culture.

105

106 BOTANICAL GAZETTE [FEBRUARY

The work was done-in part at the Missouri State Fruit Experi-
ment Station and in part in the Botany Department of the Uni-
versity of Chicago.

Historical

The problem of oxidation by plant and animal tissues or tissue
extracts has been studied by many investigators since the time of
the pioneer work by SCHONBEIN, the discoverer of ozone. An
immense literature has accumulated, for reviews of which the reader
is referred to publications by CLARK (14), KASTLE (24), BATTELLI
and STERN (5), and ATKINS (3). In this paper only those articles
will be cited which bear directly on the problem in hand.

That pathological conditions in plants are often accompanied
by increased oxidase activity has been shown repeatedly in recent
years. Woops (35) found greater oxidizing power in the chlorotic
portions of tobacco leaves affected with mosaic than in the green
portions; this has been confirmed by ALLARD (1) and by FREI-
BERG (20). SORAUER (31, 32) and Dosy (17), working with leaf-
roll of potatoes, found oxidase activity greater in diseased tubers
than in healthy ones, although the former makes the point that |
this greater enzyme activity is to be considered a symptom of the
disease rather than the cause. BUNZELL (11), working with the
curly-dwarf disease of potatoes, showed by an extensive series of
tests that “‘affected plants have a greater oxidase activity than
healthy ones of the same age, both in the juice of their tubers and
in the juice of their foliage.” Similar results were obtained by
BUNZELL (10) in work with curly-top of sugar beets. All 4 of
these diseases are of the so-called physiological type, and the
question is still unsettled for the last 3 whether the increased
oxidase activity is the cause of the disease or merely the result of
disturbances due to the real but at present unknown cause.

In the case of diseases whose cause is known the oxidase situa-
tion seems to be about the same as for those already mentioned.
REED (29) found that the juice of apples affected with bitter rot
(Glomerella cingulata) has greater oxidase activity than that of
sound apples. In his previous work the writer (30) found that
diseased apple bark shows greater oxidase activity than healthy
bark, and is at the same time less acid. This seems to indicate

1919] ROSE—BLISTER CANKER 107

that the oxidizing power of a tissue bears some relation to its acidity,
a relation which was rendered more probable by the fact that,
according to titration and indicator tests, the acidity rises in the
Bunzell apparatus during the course of an experiment at the same
time that oxidation gradually decreases and finally ceases: The
suggestion was made, therefore, that “the gradual slowing down
of oxidation in the Bunzell apparatus is brought about in part by
the accumulation of oxidation products, probably acetic and oxalic
acids in the case of pyrogallol, and not by a using up of the oxidase
through chemical combination between oxidase and oxidizable
substance.” The validity of this theory in the light of later investi-
gation will be discussed in the experimental part of this paper.

Experimental
OXIDASE ACTIVITY

EXTRACTS OF FRESH BARK.—An account will first be given of
that part of the work done at the Missouri State Fruit Experiment
Station. Extracts of fresh Ben Davis bark were used, prepared as
follows: limbs were brought in from the orchard, the bark quickly
ground in a meat grinder, and water and toluol added in the propor-
tion of 4.25 cc. of toluol for each too cc. of water. The mixture
was then allowed to extract at 28-30° C. for 1 hour, with frequent
stirring, and filtered through filter paper. The proportions of
water and toluol used, assuming that the fresh bark contained
50 per cent water, were such as to make the extracts very nearly
equivalent to those prepared for the earlier work (30) with dried
bark. All data were corrected to the basis of dry weight deter-
mined by weighing and drying samples of the ground bark in
triplicate to constant weight in a bath at 95—-99° C.

Measurement of the amount of oxidation was made by means
of the simplified Bunzell apparatus, using 1 cc. of the extract pre-
pared as just described, and either 4 cc. of a 1 per cent solution of
pyrogallol, o.04 gm. of benzidine, or 2 drops (0.025 gm.) of
guaiacol; water was added to make the final volume 6cc. The
various combinations of bark, oxidase reagent, and water were
run in duplicate.

108 BOTANICAL GAZETTE [FEBRUARY

After the experiment had been set up in the incubator, 1 hour
was allowed for the apparatus and solutions to come to a constant
temperature. The manometers were then closed and the solutions
mixed. No shaking machine was used, but the apparatus holder
was tipped back and forth several times whenever a reading was
taken. Allowance for temperature variations was made by run-
ning with each experiment a blank containing only water and cor-
recting the others by it.

Table I gives the results of two representative experiments,
showing the amount of oxidation of the 3 different reagents by

TABLE I

OXIDATION OF PYROGALLOL, BENZIDINE, AND GUAIACOL BY EXTRACTS OF HEALTHY
AND DISEASED BARK; MANOMETER READINGS CORRECTED AGAINST APPARATUS
CONTAINING ONLY WATER; TEMPERATURE 28-31° C.

EXTRACT OF DRIED

: kK
EXTRACT OF FRESH BAR Sane

Day OF Pyrogallol Benzidine Guaiacol Pyrogallol
TEST ; Sa =
Healthy | Diseased | Healthy | Diseased | Healthy | Diseased | Healthy | Diseased
Sample 38a|Sample 38)/Sample 41a|Sample 416|Sample 38a|Sample 386
LUN 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Dreyer 0.71 1.62 Onl7 0.82 0.02 0.43 0.26 0.57
Bipiio 1.38 2.46 0.22 E20 O.II TO 0.53 0.94
Ais orsciys 1 a7O 2.50 0.41 1.76 0.21 1.38 0.77 I.20
IB fave a 1.83 2.50 0.50 2.07 0.27 Si 0.86 T3230
OE ey ouch lisdumets ek earel ks Ce eaMes ence ell eae atebrad[Raboice usa odteiccy feel cure ed eco 1.07 I.50
Thee Digke 2.66 0.60 Ae Shit 0.32 P68" | os eis. seeker
STAC 2.20 Deeps) 0.74 De3 73 On32 TOD. fl tke ei |eeteetetene
Ojeveus 01s Dos 2.86 O.77 2.67 0.35 T3098). y) cede alent renee
TO! Sire 2.44 2.096 0.88 2.03 Oey) De ites) TES I.94
Ratio..| 1.00 to 1.21 T./00 sto 3.32 T00) (tO) 1540 Too) tOpaEes

extracts of both healthy and diseased bark. ‘There are included
also data from the earlier paper showing the amount of oxidation
of pyrogallol by extract of dried bark. The results indicate that
for approximately equal amounts of dry matter the dried bark is
considerably less active than the fresh (fig. 1). The decrease is
probably due to the drying; this is shown more definitely by data
to be presented later. It is to be noted that the oxidase activity
of diseased bark is definitely greater than that of healthy bark,

1919] ROSE—BLISTER CANKER feye)

although the ratio between the two is greater where benzidine or
guaiacol was used as oxidase reagent than where pyrogallol was
used. The writer prefers to follow BuNzELL in using the term
oxidase activity or oxidizing power rather than “oxidase.’”” Where
the latter term occurs in this paper, it is used only for the sake of
brevity, with no intent to imply any fixed notion as to the nature of
the agent which brings about the oxidation.

Titration and indicator tests on extracts of fresh bark showed
the healthy bark to be more acid than the diseased, exactly as
had been shown previously in the work with dried bark. No data

2 3 4 5 6 7 8 9

Fic. 1.—Oxidation of pyrogallol, guaiacol, and benzidine by extract of fresh
bark, healthy and diseased, and extract of dried bark, healthy and diseased: A,
pyrogallol and fresh healthy bark; B, pyrogallol and fresh diseased bark; C, ben-
zidine and fresh healthy bark; D, benzidine and fresh diseased bark; E, guaiacol
and fresh healthy bark; F,, guaiacol and fresh diseased bark; G, pyrogallol and dried
healthy bark; H, pyrogallol and dried diseased bark; H =healthy, D=diseased.

are given, since the true condition, at least for dried bark, was
determined more accurately by means of a potentiometer.
EXTRACTS OF DRIED BARK.—For the work at the University
of Chicago bark was used which had been dried at 35—-40° C. for
2-3 hours, ground fine enough to go through a 4o-mesh sieve, and
stored air dry in zinc-capped Mason jars. A few of the experiments
were tun with oxidases precipitated from an extract of this bark
powder, but in most of them the powder itself was used, 0.10 gm.
in each apparatus. The reagents tested were pyrogallol and pyro-
catechin, 4cc. of a 1 per cent solution; benzidine 0.05 gm.;

IT1o BOTANICAL GAZETTE [FEBRUARY

guaiacol 2 drops (0.025 gm.). Tests for any given set of condi-
tions were always run in duplicate, sometimes in triplicate, or even
quadruplicate. All experiments were shaken for 3 hours at the
rate of 106 complete excursions per minute in a constant tempera-
ture chamber provided with a fan driven from the outside, and
then allowed to stand for ro-90 hours. Temperature variations
were rarely greater than 0.5° during the shaking period, but some-
times amounted to as much as 1.o° afterward, owing to less perfect
control when the machinery was not in motion. Corrections for
temperature variations were made as before by comparison with
a blank containing only water.

Potentiometer measurements were made with a hydrogen
electrode like that described by Bovie (8), streaming hydrogen,
3 resistance boxes as described by MIcHAELIS (25, p. 131), a
saturated calomel electrode, a normal element checked against
another which had been calibrated by the United States Bureau
of Standards, and a Leeds and Northrup dead-beat galvanometer.
Hydrogen of high purity from a tank of the compressed gas was
run through an electrically heated combustion tube containing
platinized asbestos and then through the hydrogen electrode tube.
The latter, together with the capillary from the calomel electrode,
projected through a rubber stopper into the vessel containing the
solution to be tested. Escape of hydrogen was provided for by a
third opening in the stopper. An error was undoubtedly intro-
duced here, due to displacement of CO, from the solution, in
cases where the hydrogen ion concentration was less than 1075
(MICHAELIS, pp. 142-144), but since the only solutions showing
this slight degree of acidity were mixtures of bark, water, and pyro-
gallol for determination of hydrogen ion concentration before any
oxidation had taken place, and since all others were found to be
more acid, the error is probably negligible. It could have been
avoided entirely by using a Hasselbalch shaking electrode had it
and the time for using it been available.

Among the first experiments run was one designed to test fully
the oxidase activity of healthy and diseased bark when pyrogallol
was used as the oxidizing substance. ‘The results, given in table II,
are the average of 5 closely agreeing determinations. ‘These results

T919] ROSE—BLISTER CANKER 1A

agree well with those obtained without a shaking machine in show-
ing considerably greater oxidation by diseased than by healthy
bark. The ratio between the two, 1.00:2.19, is larger than that
found previously (1.00:1.28), the difference probably being due
to differences in drying or possibly to the shaking itself.

TABLE II

OXIDATION OF PYROGALLOL BY HEALTHY AND DISEASED APPLE BARK;
SAMPLES 3 AND 4; TEMPERATURE 27 C+1.7°C.

MANOMETER READINGS, EX- MANOMETER READINGS, EX-
| PRESSED IN CM. OF MERCURY, PRESSED IN CM. OF MERCURY,
1p 2 : | CORRECTED AGAINST BLANK 1: z - CORRECTED AGAINST BLANK
FIM OF | CONTAINING ONLY WATER DIME OF CONTAINING ONLY WATER
READING | eae en =e READING
| Healthy Diseased 4 . Healthy Diseased
sas | a
March 19_ OC |
2A Se YM. ya | 0.0 0.0 March 19 0.48 Ta
BrOO wa nGke> 4 0.0 0.2 AN AIS PMs 0.53 ness
ESS TES eden ue trees 0.10 0.48 SOO me ec p ee 0.61 1.45
PAO Messe) oo | 0.10 0.65 CERES ere suet: 0.64 Tesh
SAG a ska aes ow23 0.80 eeiO ened ere 0.59 1.49
AT OO me iniae tee 0.31 0.92 SNS Ge fone ued
ACTS dys Voce st tat | ©.41 O05 March 20 i, £0 2.41
ASS Obey «cts | 0.45 ny

In table III are summarized the results of an experiment to
test the oxidizing power of both diseased and healthy bark on
pyrocatechin, guaiacol, and benzidine.

A comparison of the figures in table III with those in tables I
and II shows that diseased bark causes greater oxidation of pyro-
gallol, pyrocatechin, benzidine, and guaiacol than does healthy
bark, and that both tissues cause greater oxidation of the first
two reagents than of the last two. It is further shown by tables I
and III that the amount of oxidation increases slowly for several
days; in fact table III shows that it is practically doubled for all
the combinations, except those containing pyrocatechin, during
the 64-hour period following the 3 hours’ shaking. This fact of
an increase of oxidation on standing was observed to a greater or
less degree with most of the bark material used in this work, and
is in direct contradiction to BUNZELL’s explicit and repeated
statement that oxidation in his apparatus comes to a definite end
after 3 or 4 hours’ shaking. The only exceptions the writer has

TG BOTANICAL GAZETTE [FEBRUARY

noted were in those cases where the bark powder showed low
oxidase activity to begin with, possibly due to injury of the “ oxi-
dase”’ during drying.

TABLE III

OXIDATION OF PYROCATECHIN, GUAIACOL, AND BENZIDINE BY HEALTHY AND
DISEASED BARK; TEMPERATURE 29.4~-29.7° C.

HEALTHY DISEASED
TIME OF READING iW rifee ii se j a
| Benzidine | Guaiacol Py Aaa Benzidine | Guaiacol Py. pee
June 8, 1:30P.M.... 0.0 0.0 0.0 0.0 0.0 0.0
4:30 after |
shaking 3 hours. . 0.08 0.33 Tene 0.65 0.75 Baily]
June 9, 8:10AM... 0.25 On3y; T.45 0.80 I.00 4.35
2120 PYM» 0.38. 0.48 1.65 0.98 |e O77 4.55
SU LOM Oe sG PAW ay 0.40 Ons5 1.85 1 9) TZ 4.87
Pe eT OROvAL Min ar 0.65 0.65 Dye it) ii avis 1.47 Bate

That the rate and temperature of drying have an effect on the
oxidase activity as well as on the hydrogen ion concentration is
clearly shown in table IV.

TABLE IV

EFFECT OF RATE AND TEMPERATURE OF DRYING UPON OXIDASE ACTIVITY AND
HYDROGEN ION CONCENTRATION OF HEALTHY AND DISEASED APPLE BARK

OxIDATION e
INITIAL ADA OES DEGREE OF
SAMPLE | ’ AND DURATION
After shaking| After stand- | After stand- Py OF DRYING BESOXUBS ERSTE
3 hours ing ro hours | ing 15 hours
4 diseased . : 1.49 Bete 2.78 (oat * 40°, 2 hours ste
: D2 PAT OYm Wid bane epte Ae Bray Ame? ig
ee Ba eres S TOME al eee ee Hom» vier, A Much
3 healthy 0.59 0.80 gO Gy | hte 2 Very little
5 . 1.07 SAO ae lich ots, ete ee 204) || :4On. 22 Slight
Gans 0.35 OMB Ste tales seas chee OOM | 50m 12 Very little
I f 0.62 Gna: tallowrar nations ABO) WesiS ened. Much

* This figure is the negative logarithmic exponent of 10 where the whole expression 1o=5® is a

measure of the hydrogen ion concentration in the solution.
hydrogen ion concentration it expresses.

The larger it is, therefore, the smaller the
In this particular case it can be written 2.454 107° (6.00—

5.61=0.39. Antilog 0.39 =2.454). In the amplified form this becomes 0.000002454 (normal).

Samples 1, 2, 5, 5a, and 6 were all run in one experiment.
Oxidation data for samples 3 and 4 are taken from table II and

from another experiment not recorded in this paper.

Samples 5

and sa were parts of the same lot of ground bark but received

1gt9] ROSE—BLISTER CANKER 113

different treatments as shown. The results show that oxidase
activity is much reduced by drying at 35—40° for 4 hours (sample 1,
healthy; sample 2, diseased), or at 50° for 2 hours (sample 5a,
healthy).

HyDROGEN ION CONCENTRATION. Hydrogen ion determinations
on mixtures of bark and water and of bark, water, and pyrogallol,
used in the same proportions as in the oxidase apparatus, showed
that pyrogallol has no effect on the reaction. It was found pos-
sible to get constant initial readings on all mixtures~containing
healthy bark and pyrogallol in 30-45 minutes; the same period
sufficed for mixtures containing diseased bark and pyrogallol after
they had been shaken in the oxidase apparatus, but not for similar
mixtures freshly made up and not shaken. In these cases the
potential increased slowly for an hour or two from about Py=5.60
to P,=5.40, but never reached the figure given by healthy bark.

CULPEPPER, Foster, and CALDWELL (16), working with normal
and diseased Red Astrachan apples, state that when titrations were
made on fruit pulp suspended in water “the diffusion of acids out
of the tissues continues for many hours and at slower rates in
diseased than in normal fruits,’ but in the light of the following
results the writer is inclined to think this increase of acidity was
due to oxidation going on in the solutions, and not to diffusion of
acids out from the tissues.

TABLE V

CORRELATION BETWEEN OXIDASE ACTIVITY AND HYDROGEN ION CONCENTRATION
OF MIXTURES CONTAINING PYROGALLOL, WATER, AND EITHER HEALTHY
OR DISEASED BARK; TEMPERATURE 29-30.5° C.

|

HEALTHY DISEASED
STAGE OF EXPERIMENT == = ———
Oxidation be Oxidation | ' Py
| |
[sYtorde: Cle Nas eee aoe wee ©.00 Goi 0.00 | 5.61
After shaking 3 hours...... (OY o ordi | Pave RRM iA 2.28 {pete cpeM oy her cihs
After standing 15 hours.... I.10 4.82 2.59 4.89
x ef AOwe Rtn p acne BROOM a) ||! ao tere Fi VELCly (x Rupa ‘ave cect tee Sear
* ROLL iat.) 2700" \ "| 4.29 4.96 4.20

INCREASE IN HYDROGEN ION CONCENTRATION DURING OXIDA-
TION.—Experiments designed to test more fully the theory that -
oxidation causes an increase in acidity are summarized in table V.

114 BOTANICAL GAZETTE [FEBRUARY

It is clear from table V that oxidation in these mixtures is
accompanied by a marked increase in hydrogen ion concentration,
and the conclusion certainly seems justified that there is a causal
relation between the two. It is also seen that when oxidation
comes to an end, both mixtures have the same reaction, Py=4.29,
a condition suggesting that at this point the hydrogen ion is the
limiting factor.

BUNZELL (12) and REED (28) have studied the effect of hydrogen
ion concentration on oxidation, but apparently neither of them has
realized that it might increase during the oxidation process (30).
They apparently assume that the hydrogen ion concentration
established at the beginning of an experiment remains constant
until the end, whereas the results given show that in these cases
it increased as long as the oxidation continued.

In order to discover, if possible, what relation exists between
oxidation and hydrogen ion concentration in the oxidase apparatus,
further experiments were tried with mixtures of bark, dry pyro-
gallol, and, instead of water, 5 cc. of buffer solutions containing
various amounts of N/r1o sodium acetate and either N/1o or
N/roo acetic acid. The initial reactions of these mixtures (before
shaking) and of the buffers alone are given in table VI and shown

graphically in fig. 2.
TABLE VI

REACTION, iar OF BUFFER SOLUTIONS AND MIXTURES OF BUFFER SOLUTIONS,
BARK, AND PYROGALLOL

= —— = = =
|

Solution | I 2 3 fe | 5 Oya 7 8 9

Buffer alone....... 6102 |5.73°| 5-41 | 5-07 |4.80.) 4.53) | 4025 |e Oouaeun
Buffer and healthy | | |
bark and pyro- | | | |
Pallolicra eee 15-50 | 5.52 | 55-36.) 5.25.) 4985 | er58al aed, |Rakosi aoe
Buffer and diseased | | |
bark and _ pyro-|
palloliemer tee | Sov | So 7 | Balto. | Goo

5.00) | A201 A 20 Anos se73

Graphs B and C in fig. 2 show that while diseased bark absorbs
H* ions to about the same extent as the healthy, the latter absorbs
more OH™ ions; that is, its titration acidity is greater, which
is exactly the condition found by titration with N/2o0 sodium
hydroxide (30). The P, values at points where B and C cross A

ROSE—BLISTER CANKER Thats

. Lek ACETIC ACID TOV CCL SOD. ACELA
*10.24 5.12 2.56 1.28 0. 0.32 0-16 0.08 0.04

Fic. 2.—P# of mixtures of bark, pyrogallol, and various buffer solutions before
and after oxidation had ceased: A, P,, of buffer solutions; B, P,, of mixtures of
buffer solutions, pyrogallol, and diseased bark before oxidation; C, P,, of mixtures
of buffer solutions, pyrogallol, and healthy bark before oxidation; D, P,, of mixtures
of buffer solutions, pyrogallol, and diseased bark after oxidation; E, P,, of mixtures
of buffer solutions, pyrogallol, and healthy bark after oxidation. *Only acetic acid

used here.

1160 BOTANICAL GAZETTE [FEBRUARY

(healthy bark about 5.10, diseased about 5.65) agree well with
those determined without the buffer (P, healthy = 5.15, diseased =
5.61); the latter are taken, therefore, to represent practically the
actual acidity in each case. This is based on the assumption that
if the acidity of a buffer solution is the same as that of a mixture
of bark, pyrogallol, and water, no change in acidity will take place
when the buffer is used instead of water.

EFFECT OF BUFFER SOLUTIONS.—The oxidations brought about
by mixtures of bark, pyrogallol, and the various buffer solutions
are given in table VII, together with the initial P, of these mixtures
and their P,, after oxidation had practically ceased.

TABLE VII

OXIDATION BY MIXTURES OF BARK AND PYROGALLOL WITH VARIOUS BUFFER
SOLUTIONS; TEMPERATURE 29-30° C.

HEALTHY DISEASED
BUFFER = a wut = =i ees Se
SOLUTION | | |
| Oxidation Initial P,, Final P, | Oxidation Initial P,, Final P,,
Tee ners hy Sp Pi cake, Seer SIS OWL | ect cmeaege rs dare | 4.68 5-76 4.85
Diss Semens YA | 268 Se 4.58 se 5.70 4.85
Ly tea Peers cho cal SPP Or gene RE SARC ONAN be cots os este aes Sa es Fe HOn > ||, stetecuente eres
Barolo 205 isietaels 4.34 4.36 Ba att 4.75
Re ase ats de = hada ies We ep eee GR oN Se My Ween dts atts Re lesa Gutbencach Al SOO ||| hornets
ORRT RES eke 18 5(0)55 4.58 3.98 4.48 Aba || — 9 (016)
Fh Cee ase tee 1.80 4.2 3.65 shit) 4.39 4.25
oie oases cee os S 3.98 BESGy ulleeh eka es | 4LOS, We | aercn ert
2 ees CREA 0.53 3.61 235 1.82 BTS 3.68

Check: "22. 2:22 i595 ths 4.2 4.27 Reon il Ala)

|
|

The principal fact shown by the results in table VII is that the
P, (4.29) reached by mixtures of pyrogallol, water, and either
healthy or diseased bark when oxidation comes to an end is not
sufficient to inhibit oxidation when the mixture has that P, value
to begin with; in fact, a greater degree of acidity does not inhibit
entirely, since a healthy bark mixture with an initial Py of 3.61
gave an oxidation (a mercury rise) of 0.53 cm., and a diseased
bark mixture with an initial P, of 3.78 gave an oxidation of
1.82cm. The check, bark, pyrogallol, and water gave, in the
former case, 2.22 cm. mercury rise, and in the latter 4.27 cm.

It might seem from this that the acidity brought about in
mixtures of bark, pyrogallol, and water is not the factor which

r9t9] ROSE—BLISTER CANKER 117

brings oxidation to an end. It seems more reasonable to suppose,
however, that the time factor is of importance here; that is, that
an acidity of Py=4.29 is more effective when reached gradually
than when established as a starting point. Looking at the situa-
tion from another angle, we may say that inhibition is total if the
initial hydrogen ion concentration is high enough, but will be only
partial if the concentration is lower; but since partial inhibition
means some oxidation, which in itself increases acidity, the process
in time necessarily comes to an end. The hydrogen ion concen-
tration at that point will depend on what it was in the beginning,
but will never be equal to that which causes total inhibition.

That this theory fits the facts is shown by table VII. Oxidation
took place in all the mixtures, the amount depending on the initial
hydrogen ion concentration, except where diseased bark was used
with buffer no. 4. Acidity increased in all the mixtures but one,
diseased bark with buffer no. 6 (see tables VI and VII). The
increase in acidity is shown graphically in fig. 2. It is unexpectedly
small for diseased bark except where the 3 most alkaline buffers
were used, a condition which suggests the need of further experi-
ments.

In figs. 3 and 4 are shown graphically the oxidation data given in
table VII, representing the final amounts of oxidation for each set
of tests (healthy and diseased bark with the different buffer solu-
tions). In addition there are shown graphs for several earlier stages
in each experiment. These graphs show that below 1X 1074(P,=4)
for healthy bark, and 2.5xX10~5 (Px=4.39) for diseased bark,
oxidation drops rapidly as acidity increases. Above these points
the changes are not so marked. The hydrogen ion concentration
for total inhibition, estimated by extrapolation to the base line,
lies between 3.55 and 3.80X10 4 for healthy and between 3.55
and 4.27107‘ for diseased bark. All these figures closely approxi-
mate those found by BuUNZELL (12) for potato oxidase, 2.1—
2.8X10 4, and by REED (28) for apple oxidase, 5 .o-7.0X 1074.

The results given in table VII show that hydrogen ion con-
centration is not the only factor effective in controlling oxidation
in the apparatus, and consequently that the lower hydrogen ion
concentration of diseased bark cannot account entirely for its

118 BOTANICAL GAZETTE [FEBRUARY

greater oxidizing power. For example, when both kinds of bark
were brought to approximately the same hydrogen ion concentra-
tion by buffer no. 6, the final amount of oxidation (mercury rise)
for healthy bark was 1.95 and for diseased 4.48, the final Pz 3.98
and 4.60 respectively. The total oxidase activity of the diseased
plant is the joint oxidase activity of the host and parasite, while

iS
O
fs
i)
”
de
>
a
3
18)
<
ie)
=

3 4 H) 6

Frc. 3.—Oxidation of mixtures of healthy bark, pyrogallol, and various buffer
solutions: A, after 3 hours; B, after 22 hours (19 hours without shaking); C, after
29 hours; D, after 48 hours; A, bark, pyrogallol, and buffer solutions as indicated by
numbers; B, initial P,,; points of plotting marked by vertical broken lines.

the oxidase activity of the healthy plant is that of the host alone.
This may account in part for the difference both in rate of activity
and in the P, concentration at the time the action ceases.
NATURE OF EQUILIBRIUM REACHED.—BUNZELL (13), in experi-
ments with potato peel powder, has obtained what he considers
evidence that “the activity of the plant powder is not paralyzed
by the products formed in the course of the reaction.” He found

1919] ROSE—BLISTER CANKER IIg

Mercury rise in cm.

3 4 5 6

Fic. 4.—Oxidation by mixtures of diseased bark, pyrogallol, and various buffer

solutions: A, after 23 hours (shaken 2 hours of this time); B, after 45.5 hours; C, after
69.5 hours; A and B as in fig. 3.

I20 BOTANICAL GAZETTE [FEBRUARY

that by adding a second portion of the powder to the apparatus
in which oxidation by the first portion had ceased he could cause
a further increase in oxidation, the amount of increase varying
with the oxidase reagent used. ‘The writer has found a similar
increase in oxidation when more oxidase reagent is added, after
oxidation ceases. ‘The results of an experiment of this kind are
summarized in table VIII. Results are given beginning with the

TABLE VIII

SUMMARY OF RESULTS FROM AN EXPERIMENT TO TEST EFFECT OF ADDING
FRESH SUPPLY OF OXIDASE REAGENT.

Increase in oxidation (cm. of

Stage of experi- mercury rise)

ment

Experiment

8 (a) Io II

Effect of adding 7 and 4 drops 1 per cent
benzidine to apparatus to and 11 on
ninth day, 8 and 9 as checks........ oth to 11th | 0.00 | 0.02 | 0.49 | 0.19

Effect of adding 10 drops 1 per cent ben- |
zidine to apparatus 10 and 11 on |

eleventh day, 8 and 9 as checks...... Trthy tomAthy or t74|,Os235 |e terol sora
Total effect of 1 per cent benzidine, 8 as

Check hee. cs Ley ea tana hemes oth to: 2oths)) 0-47 eee eee 1.95 | 1.34
Effect of adding 0.06 gm. of pyrogallol

to 8 on twenty-sixth day........... A WO HUGE || HAGE Wb og adcilecowccloscsos
Stas check eeeae cee ated reeset oe OHO 1K) AHO Nl CoAU Nhs co osallocowon|lous sos
Effect of adding 0.06 gm. benzidine to 9

on fourteenth day, 8 as check ...... ARMY UOS Auese |) Coit) | Oa774 loscaculla2deox

Effect of adding 1o drops absolute
alcohol to 9 on twenty-first day, 8 as
GHECK eee eetnsrceiiets bastete dic siete ats PATE UO AKIN || Cone || ©) osececieacoox

ninth day of the experiment. Up to that time oxidation in all
4 of the tubes was practically the same, the average being 3.12
(cm. of mercury rise). Alcohol was used at the beginning of the
experiment to discover whether it has an inhibiting effect on oxi-
dation, and later, when solid benzidine was added, to bring the
benzidine into solution more rapidly. The results show that, in
the small quantities used, the alcohol had no inhibiting effect
(table VIII, ninth day, apparatus 10 and 11), and probably did
bring the benzidine into solution (twenty-first-forty-first day,
apparatus 9).

The most important fact shown by these results is that after
oxidation had practically ended, the addition of more oxidase

1919] ROSE—BLISTER CANKER 121

reagent was followed by a marked increase in oxidation. For
example, in table VIII it is seen that from the ninth to the twenty-
sixth day oxidation in apparatus 8, containing pyrogallol and bark
extract, showed an increase of only 0.47 cm., while tubes ro and 11,
also containing pyrogallol and bark extract to which benzidine
solution was added later, showed an increase of 1.95 and 1.34 cm.
respectively. Equally marked excess over the check was obtained
when solid pyrogallol or solid benzidine was added. One might
infer that the oxygen admitted, when the tubes were opened to
introduce reagents, increased oxidation, but this effect could hardly
account for the difference observed. BUNZELL states that exhaus-
tion of oxygen is not the limiting factor, and experiments by the
writer have shown that, when a fresh oxygen supply is allowed to
enter the apparatus, the subsequent increase in oxidation is small.
The fact that after oxidation ends it can be started afresh
by the addition of fresh plant material or of fresh oxidase reagent
suggests that the equilibrium reached is a false one, like the third
case described by HOBER (23, p. 671), in which a reaction product
of the catalytic reaction brings about equilibrium by an inactiva-
tion of the catalyzer. A test for this condition according to HOBER
is that reaction begins again when more catalyzer is added, as in
the case of the hydrolysis of amygdalin by emulsin. The similarity
between the two reactions, however, does not prove that the oxida-
tion catalyst is an enzyme, for it may be non-enzymic in nature
and still be inactivated by the products of the catalytic reaction.
An idea of the nature of the oxidase reaction was obtained by
testing some of the data by the formula for unimolecular reaction,

k = log. —*__ In these calculations the total, amount of oxida-
a—%

tion (mercury rise) at the end of the shaking period was assumed
for the value of a, and the amount of oxidation at the end of each
15-minute interval for the value of «. The figures which should
be used, of course, are the total amount of pyrogallol at the begin-
ning of the experiment and the amount oxidized at the end of each
15-minute interval, but such figures would be difficult to obtain.
The writer sees no reason why the values used for a and x do not
truly represent the course of the reduction.

I22 BOTANICAL GAZETTE [FEBRUARY

In most cases the values of k given in these tables are fairly
constant and may be considered a strong indication that the
oxidase reaction is unimolecular. In table XI,column 3, table XII,

TABLE IX

HEALTHY BARK AND PYROGALLOL

t (min.) ® eas saat a—« k = log. ae
Tatas 0.14 onze 0.00514
ZO a misters aie 0.19 0.67 0.00361
ASR. Gr afersiemeas 0.24 0.62 0.00315
OO! chante cdoeis 0.34 0.52 0.00365
HGR) in. ctener ata 0.44 0.42 %}O.OO415
QOn ioe tases 0.49 One 0.00407

TOS Gis tcaeweys woe 0.54 0.32 ©.00409
120.) Meee etc 0.63 0.23 0.00477
P35. eet, 0.68 0.18 0.00503
TCO se eaetcdene 0.74 0.12 0.00570
TO Star Ney aere 0.70 0.07 0.00660
foe Yee RTS Oc Ov8Os T Sletten ce. eee
Mie an 53} al iserccecers ote Sage listers gery 0.00433

* Brackets in this and following tables indicate those values of k
which were considered in calculating the mean.

TABLE X

DISEASED BARK AND PYROGALLOL

t (min.) ASS a—x k= log.
Tic tette welce Gotha tee 0.13 Tey 0.00286
BO ste ainbbisee 0.30 1.08 0.00355
ZR ore era 0.50 0.88 0.00434
COR Rrartens 0.65 0.73 0.00461
TTA a, See 0.72 0.66 0.00427
(0s) ae 0.85 0.53 0.00461
TOS is, sty aneaeie 0.99 0.39 0.00522
E20 Fyre: I.04 0.34 0.00507
T'S hy hats ras 0.23 0.00576
CEO met westctecci: 1.18 0.20 0.00559
1H Oya tes ae Te25 Ome 0.00621
TORS ert: ESS 8 Vl eedectcesiseeae Oeil eget etre

Mica’, ite a| estat tay tebecalll yas nate sven 0.00478

column 1, and table XIV, column 1, the values for k show a gradual
increase throughout the experiment, and can scarcely be taken
to indicate a unimolecular reaction. Table XII, column 1, how-
ever, is checked by tables IX and XII, column 3, the mean value

1919] ROSE—BLISTER CANKER 123

of k being nearly the same in all 3 cases, although it is doubtful
whether a mean for table XII, column 1, is really significant.

TABLE XI

VALUES OF k CALCULATED FROM DATA OBTAINED IN EXPERI-
MENTS WITH APPLE BARK, K2CO3, AND PYROGALLOL

k
- Healthy bark, | Diseased bark
. K.CO; and K.;CO; and ?| a 5
# (min) pyrogallol pyrogallol EN parte

LG aes 0.00747 0.00552 ©.O01T4 0.00525
BOLE nn 0.00776 0.00803 0.00380 0.00600
AG sree 0.00774 0.00773 ©.00431 0.00615
OOS ear es 0.00785 0.00786 0.00368 0.00604
FIG ie aoe ars 0.00808 0.00819 0.00472 0.00606
Osc asee 40.00767 0.00750 0.00492 | +40.00628
TOS Mie. 0.00765 0.00709 0.00514 0.00642
L20 ach 2 0.00786 0.00890 0.00470 0.00657
L3G rpmetaeie 0.00811 ©.00941 0.00633 0.00700
DELO) ett aE ee 0.00768 0.00947 0.00663 0.00678
EOS et he ih oie 0.00805 0.00936 0.00692 0.00862

ESC cone i en ess eh eat Ant TPN ee enue
Mean 0.00781 0.00834 0.00482 0.00635

TABLE XII

VALUES OF k CALCULATED FROM DATA OBTAINED IN EXPERI-
MENTS WITH APPLE BARK, PYROGALLOL, AND PYROCATECHIN

k
¢ (min.) F Healthy bark | Diseased bark
Healthy bark | Diseased bark
ud aorallol Soe tt and Bea and det ere
ii ahs een One Ser | Do cnaee enee 0.00458 0.00502 0.00483
BOR ve camer 0.00246 0.00528 0.00429 0.00494
OS ERE Tans a 0.00277 0.00515 0.00357 0.00510
Okie ciccont. ©.00322 0.00515 0.00346 0.00488
TENS tiers 0.00383 ©.00510 0.00416 0.00526
QOMrs Wetescts 0.00493 0.00530 0.00433 0.00536
LOST seit 0.00460 0.00507 0.00426 0.00553
2 OLyats ea syoe ©.00501 0.00575 0.00434 0.00584
TB eters, wees 0.00584 0.00604 0.00483 0.00013
THON ered 0.00886 0.00744 0.00548 0.00690
HALOS card Eger sl [enue ches aiceseoie| (bea ete Ea ai 0.00590 0.00866
DO Or pelos ar Pllc ceat a: aPepe PNASII CAS yas PRA | Ma Rees eee cE
Mean ©.00430 0.00527 0.00451 0.00521

Confirmation of the results with apple bark is found in
table XIII and table XIV, column 2, based on data obtained by

BOTANICAL GAZETTE [FEBRUARY

124

BUNZELL (9, 13) with tulip tree leaves and with potatoes, although
the mean value of k in all 3 cases is much larger than that found for
bark. Attention has already been called to the fact that the data
in table XIV, column 1 (also from BUNZELL’s work), fail to fit the
equation for a unimolecular reaction. The fact of a marked rise

TABLE XIII

VALUES OF k CALCULATED FROM DATA PUBLISHED BY
BUNZELL (9) FOR POTATO JUICE
AND PYROGALLOL

¢ (min) k* t (min) kt
DOr e-nie O oeee 0.0315 TOME ae enie 0.0246
2Ocr i Sean 0.0266 BO magne Roe 0.0277
BOM o sess 0.0240 AG RR i tetorae 0.0199
TO ane Meee 0.0216 Oui Liens 0.0168
ISON Secs Chen 0.0244 7S ete 0.0174
(Yo MaInIrS Araya ace 0.0277 On eh trsgsen os 0.0233
TOs elo ee cca 0.0255 LOGE sah cill ne ae nee
BOs ets eee 0.0283 Mean 0.0208
QO crycastt tole ais ae eee

Mean 0.0262

© 23, p. 20, table VII, columns 1 and 4.
} 23, p. 26, table II, columns 5 and 7.
TABLE XIV

VALUES OF k CALCULATED FROM DATA PUBLISHED BY BUNZELL (13)

=

k k
t (min.) ; t (min.)

Spinach leaves |Tulip tree leaves Spinach leaves |Tulip tree leaves

and para-cresol | and phlorhizin and para-cresol | and phlorhizin
TG awe = acess oe 0.00374 0.0124 QOparr rere OJOIOLS: ¥en sere
BO lara inis Foie 0.00054 O.O1Ig TOS sts as endeeie (Sttas-b 4 Seatelsy | eee
Ast erent ae 0.00640 0.0133 T2O's 5 citer saya lish ya's ae eevee 36 SP oem
GOW sks see 0.00940 0.0137 TSG bc tlihra eos rece iets eueye tue he (eee
Stas SOR SIS OLCO AMA re pee facie tek Mean? s\n eke res 0.0137

in the value of k toward the end of the experiments with bark may
mean that at that point the “oxidase” oxidizes not constant frac-
tions but constant weights of pyrogallol in a given time (PHILIP 27,
p. 295). The data at hand, however, are insufficient for a veri-
fication of this hypothesis.

1910] ROSE—BLISTER CANKER 125

A unimolecular reaction is one in which the concentration of
only one substance is changed. If oxidation of pyrogallol by plant
material in the oxidase apparatus be such a reaction, the substance
whose concentration is changed is pyrogallol. The ‘oxidase’ then
appears as the catalyst, its concentration remaining unchanged
during the course of the reaction. Even at that it is not neces-
sarily proved to be an enzyme, since the linear relationship between
time and amount of change is also shown in the oxidation of pyro-
gallol by potassium carbonate.

EFFECT OF ADDING PROTECTIVE COLLOIDS.—BAyLiss (6) and
PERRIN (26) have suggested that the oxidizing enzyme is an active
form of the colloidal hydroxide of manganese, iron, or copper,
kept in this active state by an emulsion colloid such as gum or
albumin, acting as a protective colloid. Tables XV and XVI
show the effects of additions of gelatine and gum arabic. Table XV
shows that o.2 per cent gelatine increases considerably the oxida-
tion by healthy bark and only slightly that by diseased bark.
Three other experiments with pyrogallol and 2 with pyrocatechin
with o.2 per cent gelatine added showed similar results. The use
of o.8 per cent gelatine with pyrogallol also showed a similar effect.
Both o.2 and 0.8 per cent gum arabic had little or no effect on
healthy bark and a slight accelerating effect on diseased bark.

TABLE XV

EFFECT OF 0.2 PER CENT GELATINE ON OXIDATION OF PYROGALLOL BY HEALTHY
AND DISEASED BARK; TEMPERATURE 22-24° C.

HEALTHY DISEASED

TIME OF READING

Without gelatine] With gelatine |Without gelatine) With gelatine
WiayetS. 7303 PIM. a... a2 0.0 0.0 0.0 0.0
10:03 P.M. after
shaking 3 hours. . 0.77 0.81 2.22 2.24
SeRETOM OS TOVACMY 1. fea. cro- 0.94 Te 32 2.40 2.54
PE ZOO NG 5 ARMac aiake fo sc Tey 2.26 2.88 3.00
MEO Ter OTC OAUM oie syorsicicnes 1.65 2.74 ety BaP

Since gelatine is amphoteric, one might infer that it or its
splitting products act as buffers, thus reducing the rate of increase
of the hydrogen ion concentration with progress of the oxidation

126 BOTANICAL GAZETTE [FEBRUARY

(fig. 5). Table XVII, however, shows that gelatine has little
effect on the hydrogen ion concentration of oxidizing mixtures of
either healthy or diseased bark.

PRECIPITATED OXIDASES.—Experiments were run using pre-
cipitated ‘‘oxidases,”’ prepared as follows: 2gm. of bark were
allowed to extract with ro cc. of water and 5 drops of toluol for
t hour; the extract was then squeezed through moist cheesecloth
on to coarse filter paper, the beaker washed with five 1 cc. portions
of water and the filter paper finally with two more; 50 cc. of 95 per

han

w= — Mercury risei

ti 8 20 44

Fic. 5.—Effect of o.8 per cent gum arabic and o.8 per cent gelatine on oxidation
of pyrogallol by healthy and diseased bark: A, healthy bark; B, healthy bark and
gum arabic; C, healthy bark and gelatine; D, diseased bark; E, diseased bark and
gum arabic; F, diseased bark and gelatine.

cent alcohol were then added to the filtrate (concentration of alcohol
about 70 per cent), the whole allowed to stand for ro minutes and
the flocculent precipitate collected on a hard filter by gentle suction
with a filter pump; 150 cc. more alcohol were then added to the
filtrate (concentration of alcohol now about 90 per cent) and the
whole allowed to stand for 1 hour, since precipitation was slow,
before collecting this second fraction on the filter with the first.
The precipitate from diseased bark was much browner than that
from healthy bark. Whether this bears any relation to its greater
oxidase activity is not known.

1919] ROSE—BLISTER CANKER 127

For tests in the oxidase apparatus the combined precipitates
were dissolved in 20 cc. of water, and 2 cc. of this solution contain-
ing the precipitate obtained from o.1 gm. of bark was put in each
apparatus together with the usual amounts of pyrogallol and water.

TABLE XVI

EFFECT OF 0.2 PER CENT GUM ARABIC, 0.8 PER CENT GUM ARABIC, AND 0.8 PER
CENT GELATINE ON OXIDATION OF PYROGALLOL BY HEALTHY AND
DISEASED BARK; TEMPERATURE 21-23° (G.

HEALTHY DISEASED
TIME OF READING Gelutine Gum arabic qutene Gum arabic
No o.8 per No o.8 per
addition cent 0.2 per | o.8 per addition cent 0.2 per | o.8 per
cent cent cent cent
Atberinning...-| 0,00 |; 6.00 | o.00 | ‘0.00 |. 0.00 | 0.00.| 6.00 0.00
After shaking 3
ROUES sere eee ©2709) |e" O278: 4s 0265 ||) O. 75 Wats || 2528 Atte) || fos
After 18.5 hours! 1.03 1 Syl I.00 On 2.30 2.69 ey Git 2.46
Miter Ae yHOUTS..4|4 0.50 ph! 2.20 |i... .. 1.54 2729 he sek Ola Rae 2.05
Average of..... Dre Wer 8 owertel| Werte, «35 aliccte aetees Ze Men eres | So cbisa rie cio hoes

In table XVIII are given results showing the oxidizing power of
these solutions, with and without gelatine (fig. 6).

The relation observed with bark powder still holds here, that
diseased material is more active than healthy. On the other hand,
gelatine increases oxidation by the precipitate from extract of

TABLE XVII

REACTION OF MIXTURES OF BARK AND PYROGALLOL WITH GELATINE (o. 2 PER CENT)
AND WITHOUT AT VARIOUS STAGES OF OXIDATION PROCESS

HEALTHY DISEASED

TIME OF READING
Without gelatine} With gelatine | Without gelatine] With gelatine

AVA Ta ee cele fa op Sees seen: oat tits 5.61 5.60
PayAtter ns hours... 5... 2. 4.82 4.84 4.89 4.86
¥ (After 64 hours........ 4.29 4.35 4.29 4.52

diseased bark, but is without marked effect on that from healthy
bark, the reverse of the condition found when bark powder was

used.
There were indications in the preliminary work that the alco-

holic precipitate from bark extract was easily separated into 2

128 BOTANICAL GAZETTE [FEBRUARY

fractions, hence it seemed worth while to collect these separately.
This was done for both healthy and diseased tissue and gave

TABLE XVIII

OXIDATION OF PYROGALLOL BY AQUEOUS SOLUTIONS OF PRECIPITATED OXIDASE
FROM HEALTHY AND DISEASED BARK, WITH AND WITHOUT
GELATINE; TEMPERATURE 29.3-30.3° C.

HEALTHY DISEASED
‘TIME OF READING |

Without gelatine} With gelatine | Without gelatine} With gelatine
UNS De Resta a ob ab oc 0.0 0.0 0.0 0.0
PDO OMA SVALM fir sy rede ed 0.31 0.33 0.68 0.72
SS) arr AC ACMaaruer |
shaking 3 hours... 0.35 | 0.46 TOL T.24
Orne? O} Ones OUACMIs tans eteto« 0.42 0.46 1.08 1.56

Fic. 6.—Oxidation of pyrogallol by precipitated oxidases from healthy and
diseased bark, with and without gelatine, shaken only during period from A to B:
A, precipitate from healthy bark without gelatine; B, precipitate from healthy bark
with gelatine; C, precipitate from diseased bark without gelatine; D, precipitate
from diseased bark with gelatine.

precipitates whose air dry weights, determined by the use of tared
filters, were as follows:

From extract of From extract of
healthy bark diseased bark
RraGhionyir... iy. eee ©.0099 gm. | 0.0532 gm.
Bractiong2) 4..c eee: 0.0080 0.0164
Total Naatelos tee 0.0179 0.0696

The greater amount of precipitate from diseased bark may or
may not be directly connected with its greater oxidase activity.

1919] ROSE—BLISTER CANKER 129

Further study is necessary to show the facts. A test of these pre-
cipitates with pyrocatechin showed that while the 2 fractions from
healthy bark are about equal in oxidizing power the first fraction
from diseased bark is r1 times as active as the second (fig. 7).

Fic. 7.—Oxidation of pyrocatechin by precipitated oxidases from healthy bark,
without gelatine: A, fraction 1; B, fraction 2; C, fractions 1 and 2 tested together;
D, sum of fractions 1 and 2 tested separately.

Other precipitates were prepared using 25 cc. of alcohol for the
first fraction and 100 cc. more for the second. The oxidase activity
of these, tested separately and combined, with and without gelatine,
is shown in table XIX.

TABLE XIX

OXIDASE ACTIVITY OF FIRST AND SECOND FRACTIONS FROM BARK EXTRACT
TESTED SEPARATELY AND COMBINED; TEMPERATURE 29.5-30.0° C.

WITHOUT GELATINE WITH GELATINE

BARK EXTRACT Sum of fractions

Sum of.fractions
I and 2 tested |

Sail Asean Fractions t and 2

combined

Fractions rand 2
combined

separately separately
Healthy, after 23 hours... . 0.65 0.84 0.76 | 0.84
Disedsedey 538i. Fur Sh 1.82 2.00 2.64 3.06

The mechanism by which gelatine increases the oxidase activity
is not clear. It is evidently not through buffer action, as shown by
its lack of effect on the hydrogen ion concentration (table XVII,
figs. 8,9, 10). Special tests showed that there was no hydrolysis
of the gelatine to amino acids, in either healthy or diseased bark,
which would increase its buffer effect. If gelatine is effective
through its action as a protective colloid, its effect in this direction
must be very complex, as shown by its difference in effect on bark
mixtures and precipitated oxidases.

BOTANICAL GAZETTE [FEBRUARY

H
io)
Oo

NCTC syISe
Vere Aa) (uaa

Time in hours

Fic. 8.—Oxidation of pyrocatechin by first and second fractions of healthy bark,
with gelatine (for explanation of lettering see legend for fig. 7).

w
u
Pd
oO.
=
ry)
=

123 16 40
Fic. 9.—Oxidation of pyrocatechin by first and second fractions from diseased
bark, without gelatine (for explanation of lettering see legend for fig. 7); points of
plotting marked by vertical broken lines.

G |
3 bre a
i: | aah ee D
a= -_

_

Be at re A
p=

qQ —_

=| | |
| |

| | |
‘ | B
Era me n NOU

[23 16 40

Fic. 1o.—Oxidation of pyrocatechin by first and second fractions from diseased
bark, with gelatine (for explanation of lettering see legend for fig. 7); points of plotting
as in fig. g.

1919} - ROSE—BLISTER CANKER Tease

The difference in the effect of gelatine and of gum arabic on
oxidation by healthy bark may depend on differences in the col-
loidal solutions they form. An artificial oxidase prepared by
Dony-HENAULT (18) from manganese formate, sodium bicarbonate,
and gum arabic could be destroyed by heat; but one prepared by
TRILLAT (33) from albumin and manganese could not be so
destroyed. Baytiss (6, p. 585) thinks the difference here “clearly
depends on the nature of the emulsion colloid in association with
the metal.’ On the other hand, what little increase in oxidation
gum arabic produces may be due to an oxidase naturally present in
it (BoURQUELOT 7), although an experiment designed to test this
question gave negative results. One per cent gum arabic plus
I per cent pyrogallol, and pyrogallol alone, were placed in separate
oxidase tubes and shaken twice during each 24 hours. At the end
of 3 days thé mercury rise was 0.32 cm. in the first case and
o.20cm. in the second, a difference almost within the limits of
error in reading the manometers.

The data given in table XIX show that when the precipitate ©
is collected in 2 fractions, these fractions have a greater oxidase
activity if combined than if used separately. This condition seems
to be about the same as that described by Bacu and Cuopat (4)
for Lactarius vellereus. ‘They found that by the fractional pre-
cipitation of an aqueous solution of the oxidase of this fungus, by
alcohol, 2 fractions could be obtained possessing markedly different
properties. The first of these was almost insoluble in 4o per cent
alcohol and had the properties of a weak oxidase; the second was
soluble in 40 per cent alcohol but insoluble in pure alcohol and had
no oxidizing powers. ‘This fraction, however, was found to impart
greater activity to hydrogen peroxide as an oxidizing agent; it
was also found to increase markedly the oxidizing powers of the
first fraction. The chief difference between this situation and
that found in the work with apple bark is that in the latter
case the first fraction has more than a weak oxidase activity,
while the second, possibly because of incomplete separation of
the fractions, is not entirely without it. No tests have been
made of the behavior of the second fraction toward hydrogen
peroxide.

132 BOTANICAL GAZETTE [FEBRUARY

OXIDASE ACTIVITY OF THE FUNGUS IN PURE CULTURE.—A fungus
powder was prepared according to the method employed by
ReeEp (29) from mats of Nummularia mycelium grown in the
potato extract medium described by DuccaR (19). A test with
3 Bunzell tubes using o.1 gm. of fungus powder, 4 cc. of 1 per cent
pyrogallol, and 1 cc. of water gave after 4 days an average mercury
rise of 2.35 cm. Quantitative tests on the medium in which the
fungus had grown showed “oxidase” present there also. From
these results it appears probable that the greater oxidase activity
of diseased bark is due to a summation of the oxidase activity of
normal bark and of the canker fungus itself. This may also account
for the difference in behavior of the oxidases of the two.

The general conclusion to be drawn from the preceding data is
that diseased bark has greater oxidase activity than healthy bark,
probably because of lower acidity and greater degree of dispersion
of the oxidizing agent, and because of an actually greater oxidase
content. The lower tannin content of diseased bark (see macro-
chemical work) may also be a contributing factor, since tannins
are known to cause inhibition of oxidase action. This factor is
probably eliminated when precipitated oxidases are used.

In reference to the Bunzell apparatus it may be said that while
it gives valuable comparative measurements of oxidase activity,
those using it must realize its limitations Conditions within it
are artificial; with reference to hydrogen ion concentration, and
probably other inhibiting factors, they are unstable and continually
moving toward an equilibrium which, so far as we know, does not
coincide with the equilibrium obtaining in the plant.

Catalase

Determinations of catalase activity (table XX) were made on
12 samples of bark, of which nos. 9 and ro form a set from one
tree and nos. 13 to 20 a set from another tree. Nos. 3 and 4 each
came from different trees and are the ones used for most of the
oxidase work reported in this paper. ‘They were about 1 year old
when tested for catalase. The other samples were freshly prepared
for this work in December 1917 and January 1918. The limbs
from which they came were carefully cleaned to remove lichens,

1919} ROSE—BLISTER CANKER 133

Pleurococcus, etc., since microchemical work had shown that such
growths have a high catalase activity. The bark was then shaved
off, ground in a meat chopper, and allowed to dry on filter paper
at room temperature. In the case of samples 9, 10, 14, 16, 18, and
20, calcium carbonate was added during the grinding process at
the rate of 0.5 gm. to each 10 gm. of unground bark, to prevent
destruction of catalase by the acids of the bark (2) or of the hydro-
gen peroxide used. The dried bark was finally ground to a powder
and only that part used which passed through an 80-mesh sieve.

TABLE XX

CATALASE ACTIVITY OF APPLE BARK

POSITIVE PRESSURE IN CM.
ee DESCRIPTION OF SAMPLE EE z= = a
After 5 min. | After ro min.
Rie. Se Healthy, from sound limb, no car-
PRA LG ea cote tee eae ee Ra Bova OSLO Mn a ROnTO
A Rare ate Diseased se ROLCARDONALE ». is yee Cee eee 0.55 | 0.95
Optilkien Healthy, from sound limb, plus car-
DOUALES Sees Mess Sc om. 2: ted eee 21.0 Ouest |eeorE 2G
TO), Se Miseaseds plus carbonate. ..f)..2.% oo. 5002. SAO ete Skee
Tie it OR Healthy, from sound limb, no car- |
DORACE Grane, tees ee 21.0 OnS 2 sali inOn70
117. ets 3 ee Healthy, from sound limb, plus car-
IDOI ATEN SNe ay old Gea at Ne ae a ia LY am 1.83 Reo
Tecra rape es Healthy (?) 5 cm. from canker, no |
CARDONALE Lorrie ih erie by 7 2210 OoGh |) TGS
HORM ed: Healthy (?) 5 cm. from canker, plus |
carbonate. hula ee se. | eaten ora | aoe ee On 73 I.O1
Ley Secon ts Diseased,'no carbonate... ..., ii.. 20.5 0.55 o.81
TUS ere the Diseased, lis carbonates: *.0) Mie ey es es Doh
HO PRs scoceerey. Dead no carbonate. 25 21 t BOR 5.00 he Sul
DOs, oars | Dead, plus carbonate.............]....,..... 7.01 T2077,

Tests were made at room temperature by means of the simpli-
fied Bunzell apparatus, using 0.03 or 0.10 gm. of bark powder,
tcc. of water, and 4 cc. of 25 per cent hydrogen peroxide. After
the experiment was set up the apparatus was allowed to stand for
half an hour, when the manometers were closed and the solutions
mixed. The apparatus was shaken for 10 seconds at the end of
each minute and readings taken after 5 and 10 minutes. All tests
were made in duplicate or quadruplicate, a water blank being
included for temperature corrections as in the oxidase work.

e

134 BOTANICAL GAZETTE [FEBRUARY

A test for catalase was run also on the fungus powder pre-
viously mentioned, using 0.03 gm. in each tube and calculating
the results to the basis of o.10gm. ‘The average mercury rise
(positive pressure) produced in 3 tubes was 1.65 cm. in 5 minutes
and 2.57 cm. in 10 minutes, or, calculated to the basis of 0.10 gm.,
5.49 cm. in 5 minutes, and 8.55 cm. in ro minutes. It is worthy
of note that a powder prepared from Nummularia mycelium grown
in Raulin’s solution, which is acid to litmus, showed no catalase
activity. Experiments with different amounts of material showed
that the positive pressure varies directly with the amount of
material used. It was deemed legitimate, therefore, to calculate
all results to the basis of 0.10 gm. of bark powder, and the figures
for final tabulation were so calculated.

The results for samples 14, 16, 18, all from the same tree, show
that diseased bark (sample 18) had more than twice the catalase
activity of seemingly healthy bark 5 cm. away from the canker
(sample 16), but only nine-tenths of that of bark from a sound
unaffected limb on the same tree (sample 14). Dead cankered
bark from this tree (sample 20) had 4 times the catalase activity
of healthy bark, 12 times that of seemingly healthy bark next the
canker, and nearly 5 times that of diseased bark. In the case of
samples 9 (healthy) and 10 (diseased), the results are reversed,
since the diseased had a catalase activity nearly 7 times. greater
than that of the healthy bark. The reason for the discrepancy
between these two sets is not clear. The high catalase activity
of sample no. ro can hardly have been due to the presence of
lichens, etc., or of an admixture of really dead bark, for precautions
were taken when the samples were removed to avoid these sources
of error. From the present data the only conclusion that can be
drawn is that diseased bark from different trees varies considerably
in its catalase activity, and that in general the more completely
the bark is destroyed by the fungus the greater is its catalase
activity. This condition is probably to be explained by the
presence in the diseased bark of considerable amounts of mycelium
which, as shown, produces a catalase of its own.

The seemingly healthy bark near the canker when compared
with sound and with diseased bark appears to form an exception

1919] ROSE—BLISTER CANKER I
IS

in the series. Its catalase activity is less than that of either of the
others and seems to be less affected by tissue acids when no car-
bonate is added. It is possible that near the canker the host’s
catalase is injured by materials from the fungus, even in advance
of actual invasion by the hyphae. The fungus catalase may not
appear here at all, but only later in the diseased bark, and in
increasing amounts as the amount of mycelium increases.

The oxidase activity of samples 13, 15, 17, 19, together with the
catalase activity of samples 14, 16, 18, 20, identical with them
except for the addition of carbonate, are. given in table XXI.

TABLE XXI

CATALASE’ ACTIVITY OF APPLE BARK

|
/MANOMETER READINGS EXPRESSED
| IN CM. OF MERCURY USING 0.1

DESCRIPTION OF SAMPLE EES (D8 EMSS LON SABIOS

Catalase | Oxidase

|
Healthy tans be 8 | 3.02 ame 00)
Healthy (?) 5 cm. from canker) I.O1 1.47
Wiseasedie.. = y= Kamm ee yee | 2.50 | 1.95

Dead ert See Sone |e eeeriot ca | 0.88

It will be seen that there is a gradual increase in oxidase activ-
ity from healthy to diseased bark, but a marked decrease in the
case of dead bark. The catalase is considerably lower in apparently
healthy bark near a canker than in the bark of an unaffected limb,
but very much higher in the bark killed by the fungus than in
bark from a healthy limb.

Microchemical analysis

_ Tests for oxidase, peroxidase, and catalase were made on fresh
bark, all others on bark preserved in 50 per cent alcohol. The
results are given in table XXII.

In making the tests for oxidase (direct action) and peroxidase
(indirect action), the brownish purple color due to oxidation of
benzidine was found most marked at first, in both healthy and
diseased bark, in a zone 2 or 3 cells wide just inside the cork and
in the pith rays. Later it came to about the same intensity over

136 BOTANICAL GAZETTE [FEBRUARY

the whole section. Catalase, judging by evolution of gas when
H.O, was added, was evenly distributed in all the tissues. Tests
with FeCl, on sections of bark successively farther and farther

TABLE XXII

RESULTS OF MICROCHEMICAL TESTS ON HEALTHY AND DISEASED BARK

REACTION
SUBSTANCE REAGENT ee
| Healthy Diseased
Gellullosesvsepe IKI and 75 per
cent H,SO, ++ Ste
BRECtine eee | Ruthenium red + +p
TOIT eps ey seeks cPanel | Phloroglucin and
We ode Kel + in bast + in bast
ANG wovoth ween OAR o8 | Ferric chloride ara | ar :
INUETates? eae ere Diphenylamine in
lUne75 pen centiHsSO)|feracgerestantier « MR hi in Pkt o
Bats ence a ee | Sudan III + Especially in | + Same as for
| parenchyma next _ healthy bark
| to cork
Calcium (crystals)...)| 50 per cent acetic) +-+ Crystals sol- +-+ Crystals sol-
acid uble : uble
50 per cent H.SO, | ++ CaSO, formed) +--+ CaSO, formed
Calcium oxalate (crys- +-+ Crystals not | +-+ Crystals not
Palsy ete oy cee eee Oxalic acid changed changed
Direct reducing sugars Fliickigers reagent + Soe
Starchiee caine | IKI SPAr se
Cyanogenic gluco- | Picric acid and + RAs Pan Sea cc
side, probably | © Na,CO,
amyedalin. ceo .ske _ Berlin blue reaction ates AE OEE Ree
Oxidase (direct action) 1 per cent benzidine| + +
| tml ="50) pers cent
alcohol
Peroxidase (indirect
ACtiONn)/ 22s sages. _ 1 per cent benzidine’ es +
| and H,O,
@atalase--2e7 4. ae. | IBE{O} + | +

distant from the badly browned region showed steadily increasing
amounts of tannin. Pectin seemed to be present in about UE
amounts in both healthy and diseased tissues.

Macrochemical analysis

Six samples were analyzed. The analytical methods used for
4 of them are based on those employed by Kocu for the quantitative
study of animal and plant tissues (21, pp. 199-207). The differ-
ence in material required minor variations from these methods,
but it is not thought necessary to describe them here. The other

\

1910] ROSE—BLISTER CANKER 137

2 were analyzed according to a method devised by KRAYBILL
(unpublished work) in a study of the chemical composition of tomato
plants. Material for 4 of the samples, healthy 1 and 2 and diseased
1 and 2, was taken from 8-10 cm. apple limbs cut in January at
the Missouri State Fruit Experiment Station, and shipped from
there by express. As soon as these samples arrived they were pre-
pared as follows: bark designated as “healthy”? was removed from
sound limbs with a box scraper and cut into pieces half an inch
square; about 150 gm. were then weighed quickly on a torsion bal-
ance to hundredths of a gram and put into enough redistilled alcohol
(95 per cent) to give an alcohol concentration of approximately
85 per cent. . The bottles containing the samples were then set
into a steam bath until the alcohol came to a boil, then on top for
t hour longer, to inactivate the enzymes.

Bark designated as ‘“‘diseased’’ was taken from 8-10 cm.
limbs showing well developed but not old cankers, usually about
45cm. long. A strip of moist browned bark 2-3 cm. wide around
the outside of the canker was removed with the box scraper,
cut up, weighed, and preserved as described. This material usually
contained small portions of the seemingly healthy bark outside
of the canker, but never any part of the black dead material that
often covers the central part of the cankered areas. Healthy
samples 3 and 4 were taken from a 7 cm. limb cut in April when
the bark peeled easily, to avoid removing small shavings of wood
along with the bark, as was inadvertently done in the case of healthy
samples 1 and 2 (see discussion of table XXV). Healthy samples 3
and 4 were not extracted with hot alcohol and ether as in the
method described by HARvEy (21); instead the alcohol for pre-
serving was filtered into a rooo cc. flask and made up to volume.
One-twentieth aliquots were then pipetted off into small beakers,
evaporated to a syrup, and used later for dry weight and other
estimations. The partly extracted bark was dried as described
for the other samples, weighed, ground, allowed to come to air dry
condition, and one-twentieth aliquots weighed out as_ before.
This method of handling the material is much shorter than the
Kocu method and is very satisfactory if one is not interested in the
distribution of substances in the various fractions.

138 BOTANICAL GAZETTE [FEBRUARY

DRY WEIGHT.—One-tenth or one-twentieth aliquots, in tared
crucibles or beakers, were brought to constant weight in a vacuum
desiccator after intermittent drying for various lengths of time at
about too” C.

NITROGEN.—Estimations were made by the Kjeldahl-Gunning
method, modified to include the nitrogen of nitrates. For healthy
samples 1 and 2 and diseased samples 1 and 2 estimations were
made separately on fractions 2 and 3; no nitrogen was found in
fraction 2. Estimations for healthy samples 3 and 4 were made
on one-twentieth of the alcohol extract combined with one-twentieth
of the partly extracted bark.

CARBOHYDRATES.—Healthy samples 1 and 2, diseased samples 1
and 2: in the case of fraction 2, direct reducing sugars, and reducing
sugars after mild hydrolysis, were estimated by the Bertrand
volumetric method and calculated as dextrose by use of the MuNSON
and WALKER tables (34). The more important details of manipula-
tion, including precipitation of non-sugars, are given by CUuL-
PEPPER, Foster, and CALDWELL (16). The polysaccharides in
fraction 3 were estimated as dextrose, but after 2.5 instead of
5 hours’ hydrolysis (16).

Healthy samples 3 and 4: one-twentieth of the air dry, partly
extracted bark was further extracted on a filter with about 200 cc.
of water at 40° C., the filtrate being collected in a beaker containing
one-twentieth of the alcohol extract. Estimation of sugars and
polysaccharides in the combined extracts were then made as
already described. ‘The results of the analysis are given in tables
XXIII and XXIV and summarized in table XXV.

The most important differences shown in the tables, as between
healthy samples 1 and 2 and diseased samples 1 and 2, are as fol-
lows: diseased tissue contains 3.23 per cent more dry matter than
healthy, although here much depends on the manner in which the
sample is taken; on the basis of dry weight, fraction 1 is larger
in the diseased by 4.56 per cent (nearly doubled), indicating a
synthesis of lipoids by the fungus; fraction 3, the alcohol-water-
insoluble residue, is larger by 1.83 per cent, while fraction 2, con-
taining the alcohol-water-soluble substances, is smaller by 6.27 per
cent. These results are strikingly similar to those found by

1919] ROSE—BLISTER CANKER 139

CULPEPPER, Foster, and CALDWELL (16), working with black rot
of apples, caused by Sphaeropsis malorum. The increase in total

TABLE XXIII

RESULTS OF ANALYSIS OF HEALTHY BARK

Matera ee cease | er eeaiare cer eet a a
Sample r Sample 2 Sample r Sample 2
Motalisolids e) v.es.c 5. 0.28 lei SB. 55 (Sito): al ogee meal Hoolsors soko
se cs Bava eee A | 2.56 2.59 4.97 5.04
“ - Lees ctoleseieoeete | 14.84 13.26 28.79 25.85
: Ron cic oe ee 34.14 35-45 66.21 69.08
Motalenitroeen nena )s oe 0.23 0.2 0.45 0.40
Direct reducing sugars. .... TGS 1.60 3.06 2503)
Reducing sugars after mild
ny.drolysist: sey... jen. 0.55 0.54 1.07 1.05
Reducing sugars after strong
hydrolysis F, and F,..... TO] 0.22 2.08 stat
Reducing sugars after strong
My GKOlySIS iH sense ee 7.40 7.50 14.35 14.74
Reducing sugars after strong,
nydrolysiswtotal.. 0... 8.47 Vaan 16.43 16.45
| Sample 3 Sample 4 Sample 3 Sample 4
Motalsolidsre. 2:05, 24 825.6 2 Aon 13 ORCL se Al Oe OR ALA peas tin sicko
Hotal mitrogens 4.2). 6. 4.2. OL 257 0.231 0.46 0.50
Direct reducing sugars. .... 0.915 ©.949 1.96 2.05
Reducing sugars after mild
lin iOlWSe oo gpodsenoese 0.634 0.662 es 72 1.45
Reducing sugars after strong
inydralysiss 12200.) see 7.524 7.189 16.20 16:55
TABLE XXIV
RESULTS OF ANALYSIS OF DISEASED BARK
Sample 1 Sample 2 Sample r Sample 2
Material Percentage Percentage Percentage Percentage
wet weight wet weight dry weight dry weight
‘noi eo) Io Se gis oe aaa 54.20 GRE I Es ereta asf See win eee ere
ips SO Rae ee aces AD 4.69 oui 8.64 10.47
ee SO” Renee are Tr .52 II.52 Zit Ot 20.89
Mg COS Ras her aac 38.07 37.94 70.16 68.80
Miotalenitrogents -..: 5.2... 0.45 0.45 0.83 0.81
Direct reducing sugars... .. 1.42 1.506 2.62 2.83
Reducing sugars after mild
nsgelmoliysisteeera ty. a a2 0.66 ©.59 Tee 1.07
Reducing sugars after strong |
hydrolysis F; and F2..... 0.13 0.38 On23 0.70
Reducing sugars after strong
ny cdroliySishH =... 0: 13... 8.80 8.84 16.21 | Toros
Reducing sugars after strong
hydrolysis, total........ 8.93 Q. 22 16.44 16.74

I40 BOTANICAL GAZETTE [FEBRUARY

nitrogen in diseased bark may be due to fixation by the fungus
or to a withdrawal of nitrogen from the surrounding tissue. Fur-
ther data are necessary before a conclusion can be reached. CuL-
PEPPER, Foster, and CALDWELL found protein-nitrogen content
of fraction 2 for diseased apples larger than for normal ones, but
the total nitrogen for the whole tissue smaller for the former than

for the latter.
TABLE XXV

SUMMARY
AVERAGE PERCENTAGE WET WEIGHT| AVERAGE PERCENTAGE DRY WEIGHT
MATERIAL |
Healthy 1 | Healthy 3 |Diseased 1 | Healthy 1 | Healthy 3 | Diseased 1
and2 | and4 and 2 and 2 and 4 and 2
PRotalisolids weep rise Sit iye) 46.19 SAEZ. ||. Akeartraecs |) «1 cee ae | eee
os Samy Birk ania yee DAG OVEN ley: atonal Sole OO). chicane 9.56
‘“ oe iais esceys eet TACOS, len meee Taree Dips Cove ilMele é a 6 oh 21.05
. oN CEES ee od Gc EV Morovia geears o.o%e Doh ype al Orie Oe leriacte 5.6 c 69.48
Motalinitroveny..- a: oe- +l ouee 0.24 ©.45 0.46 0.48 0.82
Direct reducing sugars....| 1.59 0.93 1.49 | 3.00 2.00 oe)
Direct reducing sugars after
mild hydrolysis........ ©.55 0.65 0.62 1.06 I.59 Teas
Direct reducing sugars after
strong hydrolysis....... 8.12 oes 9.08 16.44 16.38 16.59

Results with healthy samples 3 and 4 furnish little of additional
interest. They show, however, that as far as total nitrogen and
starch are concerned, the small amount of wood in the other 2
healthy samples had no effect on the results. The difference in
the case of dry weight and reducing sugars before and after hy-
drolysis is probably due to the fact that samples 3 and 4 were taken
from a limb cut early in the growing season, while samples 1 and 2
were taken from limbs cut in the dead of winter.

Estimation of tannin

The method used was that of LOwENTHAL, as modified by
Proctor (34, p. 150). Material for analysis was taken from
8-12 cm. Ben Davis limbs cut in November, December, and
January. The bark was cut off as already described, ground in a
meat grinder, and transferred to a glass moist chamber at once.
About ro gm. were then weighed out and set to boil in 4oo cc. of

1910]

ROSE—BLISTER CANKER

141

water as required by the LOwENTHAL method; at the same time

duplicate samples were taken for moisture determination.

What-

ever may have been the errors introduced by this method, the

agreement between duplicates

taken for moisture determination

was very close in most cases, as is shown in table XX VI.

TABLE XXVI

PERCENTAGE OF DRY MATTER IN DUPLICATE SAMPLES OF
VARIOUS LOTS OF BARK ANALYZED

Sample Duplicate r Duplicate 2 Average
Healthy t2)t6 62 ¢o5<% 47.89 47.65 AW TT
ss Dee an ot 45.96 45.76 45.86
; = BR Meets 46.95 46.86 46.91
Diseased Wess. sce 50.44 49.53 49.99
? [sh Stele a eg oe 49.98 49.41 49.69
os Once eee 49.86 49.73 49.80

ID eadtinn teres ts ere GOA ark eee tan) ee eet pei
ee oly Ae OTTO 64.85 64.74 64.79
EER Oe Ae de rae ae 76.34 75.83 76.10

The results of the analysis
shown in table XXVII.

of 9 different samples of bark are

TABLE XXVII

PERCENTAGE OF TANNIN IN HEALTHY AND DISEASED
APPLE BARK

Description of sample oa eS
Healthy
STE Etec yonttete sya pat susie op ic Weber ey st aise oes 5.16
2a O—TOWcM rom) Canker seer een ae 3.64
3. From’same tree as '6and9.......... 3.38
AV ETAL ON treba sstoc wey seeks 4.06
Diseased
AEA SRAM INS cis Raber eeoh pedal sete nett 2.40
ge MHEOM, SAMMe IMD EAS) 2) ce seeder au) sheeted 3.50
Ose Mee mea astord cmeieroes karen te viene retary: 2.93
AVETAG Em caine 2 asrocuns ane taeaaabbons 2.99
Dead (from surface of canker)
7 Eromesame Litm Dias) Dyes serene = 0.25
2) en EN Dee Seer cage 28s a ae re BRN EF cr ech Coe I.14
Cie Geer PERSO apo DHE mera S ceamind Lage
ACTA CE) Walh cratere hel shore earthen 0.07

The LOwENTHAL method probably determines merely the
easily water-soluble tannins, but fails to reach those tied up with

142 BOTANICAL GAZETTE [FEBRUARY

the suberin. If suberin is for any reason more abundant in the
diseased bark, an error would thus be introduced which might
invalidate any comparisons based on the results obtained. Sub-
ject to this possible correction the results shown in table XX VII
confirm those obtained in the microchemical analysis; that is,
they show a progressive decrease in tannin as the bark is more and
more affected by the disease. Healthy bark was found to contain
on the average 4.06 per cent of tannin, diseased 2.99, and dead
0.97. Ifsample 1 healthy, which gave a high figure, and sample 7
dead, which gave a low figure, be eliminated, the averages become
healthy 3.51, diseased 2.99, dead 1.33. The figures for samples
3, 6, and 9, all from the same tree, are healthy 3.38, diseased 2.93,
dead 1.51. There is undoubtedly a difference between bark from
a sound limb and seemingly healthy bark from a limb that is badly
cankered. The latter is usually slightly browned throughout
when first cut off and rapidly becomes reddish brown on exposure
to air. Really healthy bark under such conditions shows only a
slight browning.

Whatever the results with apple bark may mean, they are not
in agreement with the statement made by KERR (see Cook and
WILSON, 15, p. 26, footnote) that because of the greater stability
of tannin and the disappearance of other constituents “‘all decayed
wood and bark give higher tannin contents, no matter what causes
the decay.” If confirmed by further analyses they would indicate
a different relation between host and parasite with reference to
tannin in the case of blister canker than obtained in any of the
cases studied by Kerr. Leaching of tannin may account for
the low percentage found in dead apple bark, as suggested by the
chemist of the Chestnut Tree Blight Commission (15, p. 6) for old
cankers of chestnut blight, but can hardly be responsible for the
condition found in diseased bark.

Summary

1. Measurements with the simplified Bunzell apparatus show
that apple bark attacked by Nummularia discreta causes about
twice as much oxidation of pyrogallol, pyrocatechin, guaiacol, and
benzidine as does healthy bark.

1919] ROSE—BLISTER CANKER 143

2. The gradual slowing down of oxidation in the Bunzell
apparatus is shown to be due, in part at least, to increasing hydrogen
ion concentration, brought about by the oxidation process itself,
The equilibrium reached in the oxidase apparatus seems to be a
false one, which can be disturbed by the addition of either fresh
oxidase reagent or plant material. When tested by the formula
for a unimolecular reaction, the oxidase reaction gives values for
k, which indicate clearly a linear relationship between time and
amount of change and suggest that the oxidase is a catalytic agent.

3. The hydrogen ion concentration of diseased bark (Py = 5.61)
is definitely less than that of healthy bark (Pa=5.15). Work
with buffer solutions shows that this difference is not great enough
to account for all of the difference in the oxidase activity of the
two kinds of material. When mixtures of the two are brought to
the same hydrogen ion concentration by means of buffer solutions,
diseased bark still shows greater oxidase activity.

4. The temperature and duration of drying have an effect on
the acidity and the oxidase activity of both healthy and diseased
bark.

5. Eight-tenths per cent gelatine increases the oxidase activity
of both kinds of bark. ‘This may be due to the action of gelatine
as a protective colloid which prevents precipitation of the “‘ oxidase.”
It is not due to buffer action.

6. The concentration of hydrogen ion necessary or complete
inhibition of oxidase activity of healthy bark lies between 3.55 and
3.80 X10 *; for that of diseased bark between 3.55 and 4.27 X107*.

7. Oxidation in the apparatus comes to an end only after several
days instead of after a few hours, as stated by BUNZELL.

8. When the ‘‘oxidase”’ is precipitated in 2 fractions, the first
has greater oxidizing power than the second, and the 2 combined
have slightly greater oxidizing power than when tested separately.

g. Catalase determinations gave the following results: healthy
3.02 (cm. positive pressure), seemingly healthy 5 cm. from the
canker 1.01, diseased 2.74, dead 12.17; results from oxidase
determinations for the same stages were 1.16, 1.47, 1.95, 1.85
(cm. negative pressure). ‘These results show some discrepancies,
but justify the general statement that the more severely the bark

144 BOTANICAL GAZETTE [FEBRUARY

is attacked by the fungus the greater is its catalase activity, and
that catalase activity in part is in indirect ratio to oxidase activity.

to. Microchemical tests indicate, for diseased bark, a partial
disintegration of cellulose, a disappearance of cyanogenic glucoside,
and a lower content of starch, calcium oxalate, and tannins.

11. Macrochemical analyses show that diseased bark has a
higher percentage of dry matter, lipoids, alcohol-water-insoluble
residue, and total nitrogen, but a lower percentage of alcohol-water-
soluble material than healthy bark. The percentage of carbo-
hydrates in both tissues seems to be about thesame. Differences
in tannin content are definite but not large. Sound healthy bark
contains more than diseased bark and diseased bark more than
dead bark from the surface of the canker.

12. The greater oxidase activity of diseased bark is probably
due to the combined activity of the oxidases of fungus and host,
lower acidity, and possibly to a greater degree of dispersion of the
oxidizing agent. ‘The lower tannin content of diseased bark may
also be a contributing factor.

The writer wishes to acknowledge his indebtedness to
Dr. WILLIAM CROCKER, Dr. F. C. Kocu, and Dr. Sopuia H.
ECKERSON for valuable suggestions and criticism during the course
of the investigation. Thanks are due to Dr. PAut Evans of the
Missouri State Fruit Experiment Station for bark material used
in the experiments.

U.S. DEPARTMENT OF AGRICULTURE
WASHINGTON, D.C.

LITERATURE CITED

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1919] ROSE—BLISTER CANKER 145

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