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| UNITED STATES

DEPARTMENT or AGRICULTURE

DEPARTMENT CIRCULAR 376

Washington, D. C. January, 1 926

A METHOD FOR THE CONTROL OF CROWN
GALL IN THE APPLE NURSERY

M. B. WAITE

Senior Pathologist in Charge; and

E. A. SIEGLER

Assistant Pathologist, Office of Fruit-Disease Investigations, Bureau of Plant Industry

CONTENTS
Page Page
TT 5 = lec. Lele a 1 TOT SCR SEO S292 ee oe) TI cl. ts het 5
Experiments and results__________-- 2 | Recommendations for control________ 6
INTRODUCTION

The problem of crown gall in the root-grafted apple nursery is a
very serious one. The nurseryman who has to discard only 5 to 10 per
cent of his trees on careful inspection at digging time is usually
considered fortunate. Losses of 25 to 50 per cent are not uncommon,
and certain lots of trees in extreme cases have even shown a loss
of 95 per cent. ‘These serious losses to nurserymen and also the
general interest of orchardists in obtaining disease-free stock for
ylanting have made the writers feel the importance of this phase
of the crown-gall problem. Present-day nursery stock, especially
when grown by the root-grafting method, appears to contain a
higher percentage of infection than formerly, and there also appears
to be more trouble in recently planted apple orchards than in those
planted a generation ago. Whether this supposition is true or not
may be open to question. It may be that nursery and orchard trees
are examined more critically as increased attention is given to
orchard diseases.

Before the bacterial cause of this disease was known George G.
Hedgcock, in his extensive studies of crown gall, tried many experi-
ments in the propagation of root-grafted apple trees and made many
observations which resulted in progress toward the control of this
disease. The discovery of the bacterial nature of this previously

7TH646—26

2 Department Circular 376, U. S. Dept. of Agriculture

obscure disease by Erwin F. Smith and his associates in 1907 marked
a real epoch in the history of crown gall and appeared to open possi-
bilities of finding satisfactory and practical remedies for the trouble.
Nevertheless, in so far as the control of crown gall in the apple
nursery is concerned, no very extensive practical applications of
these important discoveries have as yet materialized. |

This circular is published for the purpose of making immediately
available to nurserymen and others interested in the propagation
and growing of root-grafted apple trees in nurseries a new method
of greatly reducing the attacks of crown gall. This preliminary

report, to be followed later by a technical paper containing full .

details, also discusses briefly certain problems concerning the diag-
nosis of crown gall. It is hoped that the results of these experiments

: will tend to answer some
of the questions concerning
the identification of this
disease.

EXPERIMENTS AND
RESULTS

As early as 1909 the sen-
lor writer used a formal-
dehyde solution as a dip
for apple stocks and scions
before grafting, as a means
of controlling crown gall.
Observations always indi-
cated the practicability of
this method, and accord-
ingly nurserymen have been
advised in correspondence
as to this treatment for at
least a decade. The need
of more exact knowledge
. ck was so apparent that in
Fic. 1—Typical trees classified as clean 1921 experiments were ini-
tiated in an attempt to ob-
tain more definite information on this phase as well as on related
phases of the subject. This investigation, covering a period of five
years, appears to confirm conclusively the observations of the writers
extending over a much longer period. In these experiments this
formaldehyde-solution treatment with modifications has consistently
proved of value in controlling the crown-gall disease on apple grafts.
For the past three years one of the organic-mercury compounds has
also been tested as a dip and has proved distinctly more efficacious
than the formaldehyde-solution treatment. Mercury compounds are
among the most powerful of germicides; but the morganic com-
pounds, such as corrosive sublimate, are often very injurious to
plant life, even in extremely dilute solutions. A number of new
organic-mercury compounds have recently been put on the market
and have proved of value in the control of other plant diseases.
This particular one was tried in the hope that it would be effective
in controlling crown gall and would be noninjurious to the apple

;

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<iié s ay
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Crown-Gall in the Apple Nursery 3

grafts. This proved to be the case. In a number of experiments
performed in 1925 complete ¢onfirmation of results of experiments
initiated in 1921 with the formaldehyde solution and in 1923 with the
organic-mercury preparation has been obtained. In practically all
of these experiments the grafts were wrapped either with raffia or
with a cheap grade of muslin in such a manner as to cover completely
the graft unions. The trees were dug as 1-year grafts. Neither the
stand nor the amount of growth was reduced by the use of the or-
ganic-mercury compound as compared with untreated grafts grown
as checks. The trees were classified into three grades: (1) Galled
trees, with further subdivision into those with large and those with
small galls; (2) doubtful trees, that is, trees exhibiting symptoms so
slight as to be open
to question or differ-
ence of opinion; and
(3) clean trees, that
is, those trees which
showed no symptoms
of gall or hairy-root.
It should be stated
that practically all
of these so-called
doubtful trees would
have been classed as
clean trees in an or-
dinary _inspection.
The accompanying
illustrations were
made from _ photo-
graphs of trees
grown in the experi-
ments of 1925. Fig-
ure 1 illustrates ex-
amples of clean
trees. Figure 2 il-
lustrates the type of
tree which has been gre. 2.—Type of tree classified as possessing a small gall
classified as possess-

ing a small gall, whereas Figure 3 illustrates two types of trees classi-
fied as having large galls, which have invariably encircled over 40
per cent of the circumference of the tree. Figure 4 represents one of
the types of trees classified as doubtful.

TABLE 1.—Comparison of results obtained with all. grafts treated with the
organic-mercury compound and untreated grafts grown as checks

Treated grafts . Untreated grafts

| Percentage of galls Percentage of galls
Number Number

Large Total Large Total

eRe si a ei es Se oe 2.0 Giele 2 GLO eer een ee Tae 28. 7 | 32. 6

4 Department Circular 376, U. S. Dept. of Agriculture

Fic. 3.—Typical trees classified as possessing large galls, one (left) showing the
woolly-knot type and the other a simple form of crown gall

Fig. 4.—One of the type of trees classified as
doubtful

In the experiments of 1925
the organic-mercury treat-
ment was given to 2,619
grafts, representing a num-
ber of different varieties,
with the result that the total
number of trees obtained at

digging time with both large

and small galls amounted to
6.1+per cent, as compared
with 32.6 per cent on 2,619
untreated grafts of the same
varieties which were used as
checks. The proportion of
the total number of large
galls in the treated grafts of
all varieties was 2 per cent
and in the untreated or
check grafts 28.7 per cent, as
shown in Table 1.

Figure 5 represents a typi-
cal lot of trees resulting
from the use of the organic-
mercury compound. ‘This
lot was selected because it
illustrates about the average
control obtained in the ex-
periments in 1925, whereas
Figure 6 shows the corre-

-

-

Crown-Gall in the Apple Nursery 5

sponding lot of trees resulting from untreated grafts which were
used as checks. The variety used in each case was Summer
Rambo. In both illustrations the galled trees are on the left, the
doubtful trees in the middle, and the clean trees on the right.
Figure 7 shows a lot of Ben Davis trees selected as a sample of
some of the best results obtained from grafts treated with the
mercury compound, and Figure 8 shows the corresponding lot of
Ben Davis trees resulting from untreated grafts used as checks.
Reading from left to right, in both illustrations, it will be seen
that there were no galled trees grown from the treated grafts, as
compared with 48 galled
trees in the check lot.
This, of course, represents
an example of absolute
control that has not been
met with very often in the
experiments.

DISCUSSION

Recently Riker and
Keitt + have advanced the
working hypothesis that
“the malformations dealt
with in the rejected nur-
sery trees were not induced
by the crown-gall organ-
ism.” This hypothesis
was formulated because of
their negative results in
attempts to isolate the or-
ganism from 175 trees
which had been rejected
on account of malforma-
tions supposedly due to
crown gall. Since appar-
ently similar malforma- frig. 5.—one of the lots of trees which, after

} 2 classifying, represented approximately the
tions WEKE not only con average of results obtained with the organic
trolled SRS VAS but also ee aan’ etek cede y Pe

ambo. Reading from left to right: Galle
were largely prevented trees (5), doubtful (11), clean (77)

from forming by the ac-

tion of germicides in the writers’ experiments, their results are not
in accord with the hypothesis of Riker and Keitt. It is difficult to
assume that these malformations are due to any agency other than
a pathogenic organism, since they are so definitely prevented by
germicices.

Referring to Riker and Keitt’s statement that malformations
occur on grafts treated as aseptically as possible and “that gall-
like formations may develop without the intervention of Bacterium
zumefaciens,” attention is called to the fact that Hedgcock? per-

1 Riker, A. J., and Keitt, G. W. Crowngall in relation to nursery stock. Jn Science,
vol. 62, p. 184-185. 1925.

2 Hedgecock, G. G. Field studies of the crown-gall and hairy-root of the apple tree.
U. S. Dept. Agr., Bur. Plant Indus. Bul. 186, 108 pp., illus. 1910.

6 Department Circular 376, U. S. Dept. of Agriculture

formed almost identical experiments over 15 years ago and arrived
at the following significant conclusions: ““The two regions where
the formation of callus in root grafts was most abundant * * *
coincide exactly with the position of more than 90 per cent of the
galls, * ™ * indicating a close relation between the formation
of callus and the development of the disease.” All of the writers’
observations and experiments furnish additional evidence to sup-
port these conclusions of Hedgecock.

It should also be noted that the failure to find a parasitic organism
or agency in diseased tissue at any given time does not necessarily
justify the conclusion that the organism or agency is not present or
that it has not been present
at some previous. time.
Until conclusive evidence
is brought forth proving
that malformations “sup-
posedly crown gall” are
not due to the strongly
pathogenic organism Bac-
tertum tumefaciens Sm.
and Tn., the present rigid
inspection and rejection of
crown-gall trees should be
maintained. It is consid-
ered that this particular
question is of vital impor-
tance to the orchardist.

RECOMMENDATIONS FOR
CONTROL

Seedling apple trees of
l-year’s growth ordinarily
show a certain amount of
hairy root and crown gall.
These seedling trees with

si 4 s
Fic. 6.—Lot of untreated Summer Rambo trees either type of the disease
grown as checks for those shown in Figure 5. when lined out for budding

Speer (Gy, enn ae Galledwerees 29)7) or whem sed wor erafting
continue to carry the dis-
ease, in the great majority of cases at least. The experience of the
writers is that the apple-seedling growers inspect the seedling stocks
rather critically. Only an occasional gall and a few hairy-root trees
may escape their scrutiny. However, when these diseased stocks are
planted out or cut up into pieces for piece-root grafting, the nursery-
man is simply propagating the disease. The best practice, therefore,
is to inspect critically the seedling stocks before using them for
grafting and to burn all the obviously infected ones. It has been
generally accepted by both pathologists and nurserymen, at least since
the work of Hedgcock, that the selection of roots free from crown-
gall and hairy-root infection is an important factor in the control of
this disease.

Crown-Gall in the Apple Nursery 7

Since this disease is
caused by a germ easily
killed by germicides when
it can be reached and since
this germ may be present

over the surface of the
) roots, the problem of kill-

i) ing the organisms present
on the surface and of dis-
infecting the material re-
solves itself into the ques-
tion of finding a suitable
germicide or fungicide and
a suitable dosage which
will kill the bacteria and
not materially injure the
roots of the trees. In this
respect the organic-mer-
cury compound, used as a
dip in these experiments,
represents a distinct im-
provement over the for-

Fic. 7.—Lot of Ben Davis trees selected as show-

maldehyde solution. ing some of ee lees pete neh a control-
ing crown ga y the use of the organic-
Well-made grafts prop- mereury compound. Reading from Jett to right:
: 7 alled trees (none in this case), doubtful (1),

erly fitted, especially those cette:

which have no overhang
at the lower end, were shown by Hedgcock to be less susceptible
to crown gall. ‘They have, in fact, less exposed callus. The wrap-
ping, of course, should be thoroughly done, whatever method is
selected. Since the material
has to be handled so much
and since the freshly made
grafts will stand this disin-
fectant, part of the writers’
experiments also included
dipping the grafts the same
day they were made, in
order to disinfect and satu-
rate the wrappers with the
germicide and to give a
second coating to the grafts
themselves.

Since there is still a pos-
sibility after the grafts have
been stored and the young
tender callus developed that
this may carry incipient in-
fections and as it is desir-
able to give another dose of
the germicide just before

Fic. 8.—Trees from untreated Ben Davis grafts planting, a third dipping

grown as checks for the lot illustrated in Was tried and found to be
Figure 7. Reading from left to right: Galled

trees (48), doubtful (4), clean (61) noninjurious to the graft

8 Department Circular 376, U. S. Dept. of Agriculture

and helpful in the control. Melhus and Maney ® also report the use
of Bordeaux mixture as a dip for apple grafts.

There is a possibility that the grafting knives or other tools and.
the benches and tables may become infected. It is not probable that:
all obscure cases will be eliminated in the inspection, and dormant
infection would, of course, escape notice. Furthermore, the knives:
may cut through bacterial infection beneath the thin outer bark
which was not reached by the disinfectant. Although there is no
definite proof that stock can be contaminated with crown gall by
means of grafting knives, there is proof that the pear-blight or-
ganism can be carried on grafting or pruning knives. In view of
the fact that it is rather easy occasionally to disinfect the tools
and grafting benches, it is thought good practice to utilize this
method of control, even though its value is not fully proved.

The following treatment of apple grafts for the control of crown
gall is recommended as a result of a large number of experiments.
over a number of years:

(1) Select seedlings free from hairy-root and crown-gall infection.

(2) After washing, dip for 10 minutes uncut seedlings and scions in a
solution consisting of 1 part of hydroxymercurichlorophenol * to 400 parts of
water (approximately at the rate of 1 ounce to 8 gallons). In this as well
as in subsequent dips do not rinse with water, and keep the solution in either’
a wooden or a nonmetallic container.

(3) Dip the grafts, which should be well fitted and carefully wrapped, in
this same freshly made solution for about 5 seconds.

(4) Store grafts under cool conditions and dip grafts for about 5 seconds.
in a freshly made solution of the mercury compound immediately before
planting.

(5) Keep the bench on which the grafting is done, as well as all grafting
tools, disinfected by frequent washings with a germicide.

shee Seg nD et

3 Melhus, I. E., and Maney, T. J. A study of the control of crown-gall on apple grafts
in the nursery. Iowa Agr. Exp. Sta. Research Bul. 69, pp. 159-172. 1921.
4 Obtainable on the market in powdered form under the trade name “ Semesan.”’:

—————

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OF THIS PUBLICATION MAY BE PROCURED FROM
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GOVERNMENT PRINTING OFFICE
WASHINGTON, D. C.
AT
5 CENTS PER COPY

V

4

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