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FIELD AND CULTURAL STUDIES WITH

PHYTOMONAS BETICOLA

THESIS

Submitted to the Faculty of the Michigan
State College of Agriculture and Applied
Science in partial fulfillment of the

requirements for the degree of master of

Science.

by

Harry A. Elcock

1928

 

‘ H
E
SIS

i

ACKNOWLEDGMENTS.

The writer is indebted to Doctors G. H.
Coons and E. A. Bessey for many valuable
suggestions throughout the course of ex-
periments, and for thorough criticism

and correction of the manuscript.

$36054

TABLE OF CONTENTS.
Introduction . . . . . . . . . . . . . . . . .
History and Occurrence . . . . . . . . . . . .
Geographical distribution . . . . . . . .
Economic Importance . . . . . . . . . . . . .
Sugar and purity percentage tables . . .
Symptoms . . . . . . . . . . . . . . . . . . .
Comparison of Tuberculosis and Crown-gall
Environmental Conditions . . . . . . . . . . .

PathOgenicity . . . . . . . . . . . . . . . .
Strain numbers . . . . . . . . . . . . .
Host Consideration . . . . . . . . . . . . . .
Taxonomy . . . . . . . . . . . . . . . . . . .

Description of the Organism

-
o
o
a
o
o
o
o
o

Gas production . . . . . . . . . . . . .
Acid production . . . . . . . . . . . . .
Thermal death point . . . . . . . . . . .
Agglutination Tests . . . . . . . . . . . . .
Overwinteri g of the Organism . . . . . . . .
Existence of P. beticola in Soil . . . . . . .
Agglutination method . . . . . . . . . .
Patel's medium method . . . . . . . . . .
Influence of PH Concentration on P. beticola .
Influence of Freezing on P. beticola . . . . .
The Anatomy of the Overgrowth . . . . . . . .
Summary. . . . . . . . . . . . . . . . . . . .
Literature Cited . . . . . . . . . . . . . . .

Explanation of Figures . . . . . . . . . . . .

Figures 0 u o o u a o a u o n o o c I a o a o

10

12

13

16

17

19

19

23

24

25

28

34

38

41

45

48

53

56

58

60

FIELD AND CULTURAL STUDIES WITH
PHYTOMDNAS BETICOLA.

INTRODUCTION.

It is commonly recognized that the sugar beet industry, es-
pecially in many western states, is in precarious condition. The small
margin of profit on the manufactured sugar, the distance from large
markets, and the vicissitudes of water supply together with many other
conditions, such as diminishing labor supply, have served not only to
hamper development of the industry, but even to threaten its existence.
But still more important than these economic and environmental factors
are the disease factors which in recent years have caused the closing
of many factories and which, if not controlled, will drive the beet in-
dustry from large areas in the west.

The epidemic diseases, leaf spot and curly top, stand first in
importance in causing losses to the sugar beet industry, but second only
to them is the group of diseases known collectively as the root diseases
of the beet. During the last few years the epidemic diseases of the beet
in certain areas are limiting factors in crop production, but each year
the root diseases levy a definite toll upon the crOp, and many fields are
rendered unprofitable. These root diseases are numerous, and some have
been but little studied. Their effects may be to cause complete destruc—
tion of the roots by rotting, such as is found in the Phoma and Rhiz-
octonia root rot, or there may be produced malformations which upset the
normal metabolism, as occurs in tuberculosis of the beet.

The continued existence of the sugar beet industry is a vital
necessity to successful agriculture in many western states. It is, there-

fore, of utmost importance that intensive study he made of all the im-

portant sugar beet diseases in order that proper methods of control for
each of the disease factors may be discovered so that the losses, which
year after year are jeopardizing the industry, may be reduced to a minimum.
This paper presents the results of experiments carried on during
1926 and 1927 upon the root disease which has been called sugar beet tuber-
culosis. The investigations here presented deal with the nature and etio-
logy of the disease, the effect of the disease upon the metabolism of the
sugar beet, the anatomy of the gall which is characteristic of the disease,
and includes studies upon the reaction and affinities of the causal organ-
ism as well as observations and experiments to throw light upon the sapro—

phytic existence of the organism in the soil.

HISTORY AND OCCURRENCE.

The recognition of tuberculosis of sugar beets as a distinct
disease is comparatively a recent thing.

In the course of his classic work on crownegall of plants, Dr.
Erwin F. Smith and associates (14) discovered the tuberculosis of beets
and called attention to it as a distinct disease. Turking with material
obtained from Colorado, Dr. Smith isolated and described the pathogen, a
yellow bacillus, and he proved that this organism produced an entirely
different type of overgrowth from that which is always found in the crown-
gall disease. Because of the anatomical similarity of the overgrowths
found in the newly discovered beet disease to tubercles found in tuber-
culosis of animals, Dr. Smith called the sugar beet disease tuberculosis,
whereas the crown—gall caused by Phytomonas tumefaciens (Smith and Town-
send) S. A. B. he considered plant cancer.

However, in spite of Dr. Smith's work, the sugar beet tubercu-

losis has continued to be confused with the true crown—gall, which also

occurs on beets. Both diseases are indiscriminately termed crown-gall by
field workers and growers.

With a single exception of a publication by Townsend (16) enti—
tled "Field Studies of the Crown-gall of Sugar Beets" in which the effects
of the disease upon the sugar content and the purity as well as upon the
actual per cent of sugar in the gall are given, there seems to have been
no further contribution to the literature dealing with this disease. Town—
send in his account gives no indication of the nature of the disease on the
particular specimens analyzed, but many of the photographs given are very
typical of tuberculosis as it occurs in beets in Colorado.

Such other references to the disease have been found, Sackett
(12) and Peklo (9). The first author gives an abstract of Dr. Smith's
work, while the latter worker worked with both the crownegall and tuber-

culosis organism sent to him by Smith.

GEOGRAPHICAL DISTRIBUTION.

The disease is known only from the United States, and the re-
ports of its occurrences are limited. As has been said, the first mater—
ial was received by Dr. Smith from Colorado at the time he was working on
the crown-gall problem. The material used by Dr. Townsend came from
Girard, Kansas. The disease was next collected in September 1926 by G. H.
Coons and Dewey Stewart from a field at Lamar, Colorado, in which some hail
injury had occurred. They reported at least ten per cent of the beets
affected in certain portions of the field. Since that time various col—
lections have been made at Rocky Ford, Colorado, and in other fields in the
Arkansas Valley, so that it can safely be said that the distribution of the

disease is fairly general in this area. Mr. Asa C. Maxson of the Great

Western Sugar Company has found the disease at Lowell, wyOming.**

Because of the common confusion of the tuberculosis of sugar
beets with crownegall and perhaps with overgrowths of other causes, it is
impossible to approximate the field distribution of the disease. Dr. G. H.
Coons states that he has not seen any gall resembling the tuberculosis gall
in Michigan material, although true crown-gall occurs frequently.*' Obser-
vations made_by the writer in 1926 and 1927 of beets grown at Michigan State
College showed a few cases of crown—gall, but no tuberculosis. Inspection
of nine carloads of Michigan beets as well as inspection at the factory in
Lansing, Michigan, of a large pile of beets revealed only one galled beet
which proved to be crown gall. During the 1926 beet harvest at Lansing,
Michigan, Mr. Lill of the U. S. Dept. of Agriculture sent in two crown-gall
beets.

It seems then that the distribution of tuberculosis is limited
to the western beet area, and it seems safe to predict that additional ob-
servations will undoubtedly reveal that the disease is more extensively
distributed in the western territory than the few collections made so far

would indicate.

ECONOMIC IMPORTANCE .

The occurrence of galled beets has been noted for many years,
and these beets have always attracted attention at harvest time. Townsend
(16) states that the crown-gall of beets has increased rapidly in recent
years and that it is still on the increase. From the meager data at pre-
sent available as to the distribution of the tuberculosis of sugar best as
distinct from crown—gall, it is obvious that no determination of actual

loss caused by this disease can be made. The nearest approach that can

* Verbal communication.

** Letter.

be had to a determination of the potentialities for loss from the disease

organismimust‘be based upon the studies which have been made of the actual
conditions in certain fields in Colorado. Estimations of prevalence in
various fields at Rocky Ford and Lamar, Colorado, have shown a 3 to 4 per
cen t infection, while one field was known to have run as high as 10 per
cent. It is doubtful if the percentage of galled beets in the general
factory run approaches 1 per cent, but the high percentage found in certain
fields indicates that under certain conditions the disease may become of
increasing importance.

Losses from the tuberculosis of beets may be considered under
three heads:

(1) Rotting: Although tuberculosis does not itself cause rotting
of the affected sugar beet, the loss which occurs from secondary invaders,
chiefly saprophytic fungi, may be serious. The ruptured epidermis and the
more or less disintegrated nature of the gall tissue afford easy avenues
of entrance for fungi, and considerable decay of affected beets is to be
expected in storage piles from these secondary invaders.

(2) waste from topping: The tuberculosis of the beet as will
be obvious from the photographs causes loss because of the wasteful tOpping
which is given to diseased beets. Since the crowns contain such a high
percentage of salts which prevent the sugar from crystallizing in the mill,
it is essential that all of the crown be removed just below the scars of
the lowest whorl of leaves. The tubercles are often on the crown and ex-
tending down onto the main part of the root. (Fig. 1). This means a very
large portion of the beet is likely to be cut off by the tOpper, thus
losing considerable tonnage for the grower, especially if the field is
running 5 to 10 per cent diseased.

(3) The effect of quality in roots: The tuberculosis of the beet,

however, produces its losses very largely because of the profound upset of

 

 

 

the sugar beet metabolism which it causes. Townsend has already for at
least two types of sugar beet gall called attention to the injurious effects L/
that some overgrowths produce upon the quality of the roots. He shows that
the galls themselves are low in purity and are detrimental in the milling
process.

In order to obtain further information On this point, the writer
while stationed at the U. S. Dept. of Agriculture Field Station at Rocky
Ford, Colorado, made analyses of galled beets and compared these with the
analyses of the nearest nearby normal beets of approximately the same size.
The samples were taken from a field of commercial beets of a high sugar
strain which was being sampled at weekly intervals for the purpose of de— “/
termination of the course of sugar production during the season. Whenever
a naturally occurring gall was found, a nearby beet (within a radius of
4 feet) of approximately the same size was chosen for a comparative test.
The beets were analyzed by the usual methods of handling individual beets

and before wilting had taken place to any marked degree.

TABLE I.
Comparative analyses of normal beets and of beets affected with
the tuberculosis disease. Analyses made when beets were approzimately

two—thirds grown.

Condition of Beets Date Weight in Sugar in Coefficient Size of
Pounds Juice of Purity** Cells
1927
1. Galled Beet 9 — 15 2 1/4 11.2 55.5 Small
ormal " 2 1/4 12.8 77.7 n
2. called " 9 - 15 5 1/4 10.8 n
Normal " 5 5/4 12.0 n
5. Galled " 9 - 21 11.4 n

5
Normal " 4 1/2 15.0 n

Condition of Beets Date Weight in sugar in Coefficient size of

Pounds Juice of Purity** Galls
1927
4. Galled Beet 9 — 25 2 1/4 10.2 -68.0 Small
Normal 1' 2 1/2 15.5 78.4 n
5. Galled " 9 - 25 2 5/4 11.2 "
Normal " 3 11.1 "
5. Galled " 9 - 25 5 11.0 "
Normal " 4 12.6 "
7. Galled " 9 — 25 2 1/2 11.0 "
Normal " 5 1/4 11.2 n
8. Galled " 9 - l5 2 10.8 63.0 Medium
Normal " 2 1/4 14.8 78.9 n
9. Galled " 9 - l5 3 10.4 64.5 "
Normal " 2 1/2 15.0 78.5 n
10. Galled " 9 — 50 2 1/2 10.4 55.2 "
Normal " 5 11.5 78.5 n
11. Galled " 9 — 25 5 1/4 11.2 "
Normal " 5 1/4 11.8 n
12. Galled " 9 — 15 4 1/4 9.4 Large
Normal " 5 5/4 15.0 v
15. Called " 9 — 21 4 10.0 55.2 "
Normal "
14. Galled " 9 - 21 2 1/2 9.2 "
Normal " 2 1/2 12.4 n
150 Gall-ed H 9 " 30 2 1106 7200 9‘
Normal " 4 1/4 11.5 78.0 n
15. Called H 9 - 25 4 1/2 9.8 54.0 "
Normal " 5 5/4 12.5 79.5 n
17. Galled " 9 - 25 4 10.5 "
Normal " 4 1/2 15.5 n
18. Galled " 9 — 25 5 9.2 n
Normal " 4 1/2 10.8 n
19. Galled " 9 — 25 2 1/2 10.5 "
Normal " 2 1/2 15.2 n
20. Galled " 9 - 15 6 10.6 51.2 n
Normal " 4 1/2 15.0 81.9 "

 

* The normal beet used as a check was adjacent to the galled one, except
where a considerable difference in size made it desirable to choose some

other beet within a radius of approximately four feet to serve as a check.
** Computed from solids in beet 511109 :19. determined 'hv rQ-Prnni-nmof-a-n

The sugar content and coefficient of purity are the factors which
determine the value of beets for sugar-making purposes. A considerable
percentage of the total weight of the beets consists of soluble solids,
of which sugar forms the largest portion. The galled beets show a very
much lower coefficient of purity due to the larger amounts of impurities
in the form of soluble solids other than sugar. The greater the proportion
of other soluble solids present the lower the purity, hence the more diffi-
cult it is to extract the sugar. These diseased beets with their low
sugar and purity percentage give syrup from which recovery of crystaliz-
able sugar is poor, and the handling of such beets in the factory is un-
profitable.

An additional experiment to the above was performed to determine
the odds that the tuberculosis disease is significant in the reduction of ./
the sugar content.

A number of artificially inoculated beets from three different
varieties of plots were used. These beets were inoculated at intervals
so that on August 29, 1927, when the sugar determinations were made, there
were three stages of the galls present i.e. small, medium, and large. The
checks were, in the majority of cases, the two adjacent beets; however, if
this was not practical, the checks were taken within a four foot radius.
Attempts were made to choose the normal and diseased beets as nearly the
same size as possible, as shown in photographs. (Fig. 2).

In computing the data obtained in this experiment, the galled
beet value is the actual beet sugar percentage, whibe the check value is
the average of the two adjacent normal beets. The following tables give
the odds calculated by Student's Eethod as to the significance of the

differences, grouping the data according to gall size, small, medium, and

large.

TABLE II.

Sugar percentages of diseased sugar beets contrasted with per-

centages found in normal beets.

Sugar per cent in juice
Normal
Beets

1927.
Variety Galled
Beets
12.5
50165 11.3
166 11.5
11.0
4088—23 10.0
T 9.5
10.0
9.6
11.1
F. F. 9.8
24 10.2
8.8
10.0
9.5

14.60
12.53
12.05
12.69

10.84
10.00
10.55
10.83
11.80

12.82
12.64
11.51
12.25
11.90

Difference

-2.10
-l.23
- .52
-1.69

- .84
- .50
- .21
-1.23
- .70

-3.02
-2.44
-2.71
-2.53
-2.40

Tested at Rocky Ford, Colorado, Summer

Size of
Gall Odds

Small

77 to 1

9999 to 1

9999 to 1

The analyses given in the tables I and II show such consistency

in the sugar and purity percentages found in beets affected with tubercu-

losis when compared with normal beets, and these differences as seen in

the tables are so significant even in the case of beets affected with

small galls, that the writer believes that the early statement of Townsend

(16) is confirmed.

Thus with these three sources of loss the disease may become of

very considerable economic concern to both the beet grower and the manu-

facturer.

10

SYMPTOMS.

The overgrowths of this disease are generally confined to the
crown, but they are also at times observed down almost to the tip of the
root. (Fig. 5).

There are as Smith (14) has pointed out two distinct types of
overgrowths occurring on the sugar beet roots, and these have been desig—
nated as "tumors and tuberculosis" Townsend (16). It is almost impossible
to differentiate the two types of galls apart unless certain well marked
characteristics of the older galls, which make it possible to identify the
two types of overgrowth are observed. In the tuberculosis of beet the
young tubercle is a smooth, yellowish white proliferation, which shows no
sign of outer cell disintegration. At this point it is almost impossible
to determine the two above types of galls apart without taking into consi-
deration internal structure, which will be described later. As the galls
mature the older overgrowths are characterized by their roughness and badly
fissured surfaces. The diseased root generally shows a tendenCy to be
turnip shape, that is the beet does not develop with the normal long taper-
ing root, but becomes short and very broad at the crown. Also a.marked
profusion of leaves may accompany this type of growth. The leaves come out
in groups over the entire crown, and often extend down onto the root proper.
Along with the flattening out of the crown, there generally arises swell-
ings which extend as ridges from the middle of the root up to the crown.
These ridges generally terminate in a large rough overgrowmh. (Fig. 1). The
primary tubercular focus Which is the cause of this ridge deve10pment is
generally deep within the tissue of the root with secondary tubercles at the
apex of the ridges. These aspects are in marked contrast to the condition
in other beet root galls, which generally are lateral fleshy out growths

upon the affected roots. Over the surfaces of the old galls, secondary

11

tubercles can be noticed, and these nodular growths together with the deep
fissure formation give the proliferation a very rough appearance. Near the
end of the growing season the tubercles show a more or less marked decay of
the outer layers of cells. The large fissures and cracks are found to
penetrate as deep as one inch into the tissue of the root. These openings
offer excellent avenues for entrance of secondary fungous invaders, and
these under ordinary conditions lead to additional decay of the crown.

The internal appearance of this disease is very distinct from that
of crown-gall. In this disease localized brownish watery spots with a
general distribution can be noticed, hence the name tuberculosis. The galls
also show hyperplasia and hypertrophy tissue which extends out in all direct-
ions. Upon microscopic examination of the spots, large masses of bacteria L.
are found to be present. The spots vary from 2 mm. to 10 mm. in diameter,
and extend up and down the gall 1 mm. up to 5 to 4 cm. These bacterial
pockets have a viscid consistency, and under moist conditions are sometimes
so very mucilaginous that the bacterial slime when touched may be drawn out
in sticky threads. (Fig. 4). The diameter of the galls varies from about
1 cm. in young infections to as large as 1 dm. in old beets.

On Petioles: The only galls observed on leaf petioles were re-
sults of artifical inoculations. The proliferations are at first small,
and appear to come from under the epidermis at the point inoculated. As
the gall becomes older, the epidermis begins to crack and small fissures
are formed. The overgrowth then takes much the same rough appearance as
the galls on the roots. The galls when young are green, but as the epider-
mis is ruptured the outer cells disintegrate, becoming brown and subse-
quently dry.

Since crown-gall and tuberculosis of the sugar beet have so

generally been confused, the following table is given. (See also the

 

photograph plates Figs. 1 and 5.)

12

TABLE III.

Comparison of Tuberculosis and Crown—Gall of Sugar Beets.

Cause

Tuberculosis.
A bacterial disease caused
by Phytomonas beticola (Smith

Brown and Townsend) S.A.B.

Symptoms on

1. Rough overgrowth pro—
duced.

2. Rough appearance due to
ruptured epidermis, and large
fissures over surface of over
growth.

5. Ridges extending from
middle of root to crown termin—
ating in the galled out growths.

4. Turnip shaped beet roots
generally results from this dis—
ease, generally with a large
amount of newly formed leaves
coming out over entire crown..

5. Secondary fungous in-

vasion of the beet commonly found.

Crown-Gall.
A bacterial disease caused by

Phytomonas tumefaciens (Smith and

 

Townsend) S.A.B.
the Root.

1. Smooth or at most wrinkled
overgrowth produced.

2. Contour uneven in old galls,
but epidermis intact without fissure
formation.

3. No ridges present; gall gener—

ally separated from root by stipe.

4. The shape of root not changed
except at place of tumor formation; no

extra growth of leaves.

5. No secondary fungous growth
generally associated with this type of

overgrowth.

 

15

6. Small newly developed galls 6. Small yOung galls white,
yellowish white, even contour, epi— even contour, epidermis intact, no
dermis intact, no fissures. fissures.

Internal Symptoms of Boots.

1. Numerous yellowish watery 1. No bacterial pockets present;
spots present within galls. (Bac— beet tissue white.
terial pockets) Surrounding tissue 2. Strands of tissue extending
white. in all directions within gall; outer

2. Strands not very noticeable, cells not decayed.
outer cells of overgrowth decayed,

fissures extending into tissue.

Environmental conditions necessary for the Development of the
Disease:

From field observations made in 1926 and 1927 in Colorado, it
appears that the incidence of the tuberculosis disease providing wounds
which occurs in young growing beets is strongly influenced by temperature
and water factors. The observed correlations with the above factors has
received positive confirmation from greenhouse tests in which temperature
and humidity factors have been found to play a role in the success of in-
oculations. Injury to plants by hail usually results in abundant infect-
ions, as the hail is generally accompanied by heavy showers which give the
organism exceptionally favorable conditions for penetration and estab-
lishment within the host tissue.

Various fieldmen for the western sugar beet companies have ob-
served that after a hail storm certain fields were found to run very high
in tubercle formation. It was also noticed by Coons, in the field in

which this type of gall was first brought to his attention, that a great

14

amount of hail injury was evident. A letter from Asa C. Maxson at Long—
mont, Colorado, stated that "Several times during the past 18 years I have
found this type of growth very common in fields badly injured by hail."
Observations made in the field by the writer indicated that an injury to a
beet root was necessary for entry of the organism into the host as is shown

by the following table.

TABLE IV.
Results of Field Experiment on wounded and sound sugar and garden

beet roots exposed to P. Beticola and P. tumefaciens.

 

 

Method of Inoculation No. of plants showing symptoms.

5 days 10 days 15 days 20 days

12 plants - check 0 O O O
P. beticola - No. l O 10 12 24
25 plants - Injured

10 plants - check 0 0 O O
P. beticola - No. 3 O 5 14 25

25 plants - Injured

P. beticola - No. 3 O O O O
25 plants not injured

No inoculum O O O O
25 plants - not injured

Beet organism - No. 2

25 plants - Injured O O O O
25 G. Beets — Injured O 3 4 9
P. tumefaciens - No. 146 O O 0 15
25 plants - Injured

P. tumefaciens - No. 146 O O O O

25 plants - not injured

This experiment shows that infection in the field took place from

seven days up to twenty days after inoculations were made, and that the

15

‘ghytomonas beticola cultures No. l and No. 3 are very virulent for sugar
beets, but the No. 2 strain is only pathogenic on garden beets. Phytomonas
tumefaciens (Smith and Townsend) S. A. B., M. S. C. No. 146, was also viru-
lent to sugar beets and produced typical crown-gall.

Mechanical injuries, such as made by a cultivator just before
irrigation water is applied to a field of beets, generally results in 50
to 60 per cent of the injured beets becoming infected. Mr. A. W. Skuderna
of the American Beet Sugar Company has reported a field of beets which
following rolling Showed practically 100 per cent tuberculosis infection.*

Attempts at Rocky Ford, Colorado, to isolate the organism from
irrigation water was unsuccessful, but it is evident that irrigation water
can serve to carry organisms in the soil to the wounds arising from hail,
rolling, or other cultural practices, and thus materially influencing in-
fection. On the other hand, beets injured under dry soil conditions in
the field have shown practically no invasion by the organism. The growing
season for beets in the western states affords favorable soil conditions
for the organism. The variation in weather conditions, such as tempera-
ture outside of the amount of hail, wdll show very little effect on the
epidemic nature of the disease.

Under greenhouse conditions in Michigan, however, temperature
has a definite effect upon disease production. Under cool conditions when
the beet root is in a very slow growing condition, the disease may not be
produced, even with direct inoculation. There seems to be a distinct
ratio between the rate of root growth and disease incidence. Under the
above circumstances then, it seems to indicate that temperature, root in-
jury, and soil water are important factors influencing the severity of

outbreaks of the disease.

 

*Verbal communication.

16

PATEDGENICITY.

The causal organism was isolated by pouring plates in the usual
manner, using a small amount of the yellow portion taken from a firm gall.
In 48 hours, when the gall was of the tuberculosis type, yellow organisms
such as described by Dr. Smith appeared in great abundance on the plates.
Potato dextrose agar was found to be a good medium for isolation, and for
continued growth of the organism.

In making inoculations ten rapidly growing beets wdth roots that a ng

15
showed the anomalous secondary growth were used. The method of inocu- E‘b;
lation was the puncturing of the beet crown with a needle whose point had
been dipped in a suspension of the bacteria to be tested. Ten beets of the
same type as above were used as checks. They were injured by puncturing
the same as the others except the needles were not dipped into the sus-
pension.

After twenty-six days all ten beets showed the symptoms of the
disease, while no check beet deveIOped overgrowths. The galls of the dis-
eased beets were cut Open, and numerous bacterial pockets were observed in
each one. Platings from these pockets produced a great abundance of yellow
colonies. A second series of inoculations of the reisolated organism into
ten beets gave typical tubercles again. The check plants again showed
negative results. There can be no question of the etiological relation of
the yellow organism isolated to the tuberculosis of the beet.

With the pathogenicity of the yellow organisms established on
sugar beets, the question of cross inoculation arose. The first cross in-
oculation tried was on ordinary garden beets (Beta vulgaris L.) Inocu-
lations both from agar slants and broth cultures were made on ten roots;
infection on these showed a 100% positive. Symptoms on these beets (Fig. 6)

showed up in 19 days under greenhouse conditions. The galls showed unusual-

17

ly large fissures, which were formed from an excess amount of splitting,
and foreign soil invaders were plentiful. Crown-gall organism, P. tume-
faciens, was inoculated as a check; they only showed an 83 per cent in—

fection. The first gall was noticed 32 days after inoculation, with an

average of 34 days for all.

In the course of the work a number of organisms have been iso-
lated and used in infection experiments. These organisms have also been
used in serologiCal investigations, as will be discussed later; these iso-
lations have shown certain characteristics. For convenience these organ-
isms have been called "strains". The following is the list of isolations
used with a summary of their outstanding peculiarities.

Strain No. 1. Isolated from sugar beet received from Rocky Ford,
Colorado, October 16, 1926. Good grower. Strong virulence until Sept.
1927, when strain died out.

Strain No. 2. Isolated by Miss M. C. Carpenter of Michigan
State College from a sugar beet sent from Lamar, Colorado, Oct. 13, 1926.
Good grower. Strong virulence on garden beets, but not virulent to sugar
beets.

Strain No. 3. Isolated from soil obtained at Lamar, Colorado,
by use of agglutination method later described. Good grower. Strong
virulence to both sugar and garden beets.

Strain No. 4. Reisolated from garden beet, Rocky Ford, 0010-
rado, which had been inoculated wdth strain No. 2. Same type of virulence
as No. 2.

Strain No. 5. Reisolated from beet inoculated with Strain No. 1.
Good grower. Strong virulence for sugar and garden beets, also to Swiss

Chard. "R" form seems to be present.

18

Strain No. 6. Reisolated from sugar beet artificially inocu-
lated with No. 5. Reactions same as No. 5. "S" forms probably present.
Strain No. 7. Same source as No. l but did not die out.

P. tumefaciens, M. S. C. No. 146. A culture obtained from Dr.

 

E. F. Smith. Isolated from hop. Has been grown for some years at Michi-
gan State College, and was used in comparison with the tuberculosis patho—

gen.

HOST CONSIDERATION.

The cultivated sugar beet (Beta vulgaris L.), as represented by
two varieties grown at Rocky Ford, Colorado, namely Pioneer and Flat
Foliage, was found very susceptible to the tuberculosis disease discussed
in this paper. No varietal differences as to resistance or susceptibility
were found in sixteen varieties of commercial beets which were tested by
artificial inoculation. The disease has also been produced on garden
beets (B. vulgaris L.) and Swiss chard (B. vulgaris L.). When inocula-
tions were made into tomatoes (Lyc0persicon esculentum), no typical gall
formation resulted. However, a small, smooth, yellowish spot on the stem
at the point of injection (Fig. 9) was noticed 28 days after inoculation.
Pure cultures of the organism were obtained from this spot at that time.
What probably is the case here is that the tomato can harbor the organism
without reacting to gall formation. Tobacco (Nicotiana Sp.), raspberry
(Rubus sp.), apple (Malus sp.), and geranium (Geranium sp.) were all in-
oculated but showed no symptoms of gall formation. In contrast to these
results, all the above plants with the exception of tobacco develOped over-
growths when inoculated with the crown-gall organism in a series of tests
paralleling the above mentiOned inoculations. NO‘Wlld host has been found,

although various weeds, many of them in the same family as the heat, were

 

19

examined in fields where beets were developing the disease naturally. '\»
So far as information goes at present, the tuberculosis disease
is confined to the members of the genus Beta, a group of plants commonly

thought to be a closely related species, if not members of the same species.

TAXONOMY.
The organism was first described by Dr. Smith in 1910 under the
name Bacterium beticolum n. sp. Smith, Brown, and Townsend.* According to
the revision of Migula's and Smith's classifications adopted by the Commit-

tee of the Society of American Bacteriologists in 1920 (10), the termino-

 

logy would be Pseudomonas beticola. In a later report (11) of another
committee of the same society the genus name Phytomonas was made to include

organisms of this character, whence the name Phytomonas beticola which is

 

used in this paper.**

DESCRIPTION OF THE ORGANISM.

The following description of the organism is based upon strain
No. l, isolated by the writer from Colorado beets. In a large part the
tests outlined follow the work reported by Dr. Smith; however, where sig-
nificant variation from Dr. Smith's results have been found and addition-
al information has been obtained, the statements are starred.

Morphological Characters: The organism in the host is a small
rod with rounded ends occurring singly, but at times in pairs or clumps;

in different media, however, it varies greatly. The measurements of

 

' The word 'cola' being a Latin noun meaning inhabitant, cannot be changed
to 'colum' as used by Dr. Smith and associates in naming the organism.
They assumed erroneously that this came from an adjective-—colus, a, um.
The species name when a noun cannot be changed to correspond with the
gender of the genus, this only taking place when the species name is an
adjective. Verbal explanation by Dr. E. A. Bessey.

** The species name in Bergey's Yanual is written 'betacola' instead of
'beticola'.

20

single rods vary from 0.5 to 0.9«by 1.0 to 1.94(, *the average size
being 0.6 by 1.4a,.

The organism is motile by the means of polar flagella, which
vary in numbers from one up to four. *The flagella are very tenuous and
range from 2.2 to 3.1q.in length, the length being much larger on the
large cells. The best results in staining flagella were obtained by using
a mordant of 1 part 2% Osmic acid and 2 parts 10% tannic acid followed by
standard carbol-fuchsin stain. (Fig. 7). No endospores were observed in
any cultures examined, which included 24 hour old cultures to one six
months old.

Capsules were demonstrated by the method given in Giltner's
manual, when the organism was grown in Uschinsky's solution. However, the
organism when grown upon nutrient broth or agar and potato dextrose agar
shows no capsule formation.

Zoogloeae occur when Uschinsky's solution was offered, also
psuedozoogloeae was produced in 48 hour old potato dextrose agar slant
cultures. Microsc0pic examination of such material shows the cells em—
bedded in a gelatinous mass.

Behavior toward Stains:

*The organism is gram positive, and stains readily with all the
ordinary basic anilin stains such as gentian—violet, carbol—fuchsin,
methyl—green, safranin, and methylene-blue.

Cultural characteristics:

Potato dextrose and beef bouillon proved to be very favorable for

prolonged growth.

Gelatine Stabs:

There is a slight granular growth along the entire line of the

 

21

puncture. The best growth is at the top where it is villous or papillate,
the length of the villi or papillae decreasing downward. After 18 to 21
days at 20°C. it shows a fair degree of liquefaction, but this is not com-
plete until sometime between the 30th and 40th day.

Gelatine Plates: Colonies of the organism when planted on these
plates showed up within 36 hours, being whitish at first then becoming
yellow. On the surface of the media the colonies appear round, glistening
with a smooth flat surface, but in the substratum they appear elliptical
in shape. Slight liquefaction begins in five days at a temperature of 20°C.
In thickly sown plates, liquefaction proceeded rapidly, becoming completely
so in eighteen to twenty-five days. In thinly sown plates the colonies
average size was 1.2 cm., and small saucer-shape liquefactions were formed.

Agar plates: *On potato dextrose agar plates Pb. 7.2 kept at
26° C. colonies appeared in 24 to 30 hours. They are at first white,
round, smooth, and after 48 hours they start to turn yellow. The margin
is entire without any conspicuous zonation; the yellow color is diffused
throughout. The individual colony is somewhat sticky to the touch of a
needle and becomes viscid in old cultures; it is glistening, somewhat
raised or convex, and internally appears amorphous. Submerged colonies
are elliptical in shape and showed small growth.

*Potato sucrose agar: Same as Potato dextrose, except colonies
become a more deeply yellow.

Plain nutrient agar plate: Same as potato dextrose, only the
growth is not nearly so abundant.

Plain nutrient agar slant: A streak culture will show moderate
normal filiform growth for the first five days then a change to filiform—

fimbriate type takes place. (Fig. 8).

 

22

Loeffler's blood serum: Growth restricted, whitish yellow, dull,
filiform, convexed slightly; medium was not liquified.

Maltose Agar: *Very scanty filiform growth, but the organism
secretes a very bright yellowabrown pigment producing more color than with
other sugars.

Lima bean agar: *Growth very feeble to nil.

Prune juice agar: *Scanty growth.

Nutrient broth: In beef-peptone bouillon Ph 7.2, there is a
moderate clouding in 24 hours, and heavy in 48 hours. A thin pellicle some-
times forms over the surface which readily breaks up and falls in thin
flakes. *But the usual case is moderate clouding settling to the bottom
with a formation of a scant amount of yellow precipitate.

Uschinsky's solution: A moderate to copious growth is produced
in this medium. Growth is best at the surface in the form of clouding,
zoogloeae, and a slight pellicle. After a few days the entire liquid is
very viscid, and stains show large thin walled capsules present. Color
is first white becoming yellow in old cultures.

. Cohn's solution: *Growth very scanty.

Nitrate Reduction: Nitrates are promptly reduced. Tests were
made on cultures in peptonized broth with use of Sulphanitic acid (sol.I)
and a-naphthylamin (sol.II).

Indol Production: Indol is produced in small but definite
amounts in 1% peptone, 0.5% disodium phosphate, 0.1% magnesium sulphate
in distilled water. Also good amounts were formed in 2% peptone water.

B. coli was used as a check and showed much stronger reaction.
Coon's synthetic: *Growth first white, not as abundant as on

potato dextrose; later turning yellow.

23

Carrot plugs (Raw): *It grows readily with a yellow color
covering the entire slab, but not rotting the tissues. The non-rotting
action indicates the absences of a pectic enzyme.

Steamed potato cylinders: It grows at a moderate rate, flat,
spreading, smooth, dull yellow, and somewhat glistening. *The potato
changes very slightly to a dry grayish brown color in 5 days. Diastasic
action is very marked. Tests were made by use of this method for dias-
tasic action on starch. At four and six days, tests were made by pouring
Lugol's iodine solution over the plates. Light areas appeared around
the colonies to a very marked degree. P. tumefaciens M. S. C. No. 146
streaks used as a check showed less diastasic reaction.

Hydrogen sulphide: *Hydrogen sulphide is not produced. Tests
were made by hanging pieces of lead acetate paper in mouths of tubes of

bouillon, potato, and milk cultures.

GAS PRODUCTION.

A basal solution was made by adding two per cent of peptone to
water. Six solutions were made from this, each containing one per cent
respectively of one of the following carbon compounds: glycerin, sucrose,
mannite, dextrose, maltose, lactose. Five fermentation tubes were filled
with each of these solutions and sterilized by heating for twenty-five
minutes on three successive days. Three tubes of each set were inocu-
lated and two were left as a control. The inoculated tubes each received
a one-millimeter loop of culture material from a twenty-four hour old
bouillon culture. Thirty-six hours after inoculation, a cloudiness
appeared in the Open arm of each of the inoculated tubes. This turbidity
increased with age, extending up into the closed arm in the dextrose, su-

crose, and maltose cultures, but not in the tubes containing glycerin and

24

lactose. There was a well-defined line between the growth in the bulb and
the clear liquid in the closed arm in the latter two tubes. This points
toward the facultative nature of the organism, except where certain sugars
are offered. No gas was produced in the closed arm in any of the sub-

stances listed during a twenty-five day period.

ACID PRODUCTION.

*Various experiments indicate that this organism does not pro-
duce gas in any of the ordinary nutrient media. In the presence of certain
sugars, however, the acid reaction was very marked, as shown in the followe
ing tables.

TABLE V.

Behavior of P. beticola strains and P. tumefaciens on various
sugars with an indicator of Bromo—Cresol—Purple.

Sucrose Man- Dex— Lac- Gly— LeVu- Mal- Galuc- Plain v
and ggs nose trose tose cerin lose tose tose

AcidGasJA_GA GA GAGAGAGA GA G

B. beticola
No. 5 + - + - + - - — — - + — + - + - _ _

 

P. tumefaciens

 

No. 146 + — - — - - - - - - - - - - - - - -
P. betiola
No.3 s-—-------+-£-+--_

 

Check - - - — - - - — - _ _ - - - - - _ _

 

25

TABLE VI.
Behavior of P. beticola on various sugars when the Quinhydrone

Method was applied.

 

Type of Carbohydrate Original Ph at end Ph at end Ph at end
Plus Nutrient Broth Ph of 24 hrs. of 5 days of 10 days
Plain Broth 7.15 7.46 7.90 — 8.00 -
Dextrose 1% 6.91 6.76 5.54 5.32
Sucrose 6.98 6.75 5.41 5.07
Lactose 7.51 7.45 7.55 7.62
Mannose 7.24 6.98 6.61 5.98
Glycerine 7.02 7.12 7.24 7.50
Sucrose

Dextrose - Check 6.91 6.91
Plain Broth — Check 7.13 7.12

 

THERMAL DEATH POINT.

In preliminary tests it was found that the thermal death point V
lies between 49° and 50° C. The following tests were made to determine
the death point more exactly. Seven sets of fourteen tubes each of pep-
tonized beef broth were inoculated wdth P. beticola from six day old
slants of potato dextrose cultures. The tubes were placed in water at a
constant temperature of 49° c, 50° c, 51° c, 52° C, 55° c, 54° c, and 55°
C, respectively. The tubes were removed at the end of ten minutes and
were kept at 25° C for two weeks. At the end of six days growth had taken
place in all the tubes of 49° c and eleven tubes of 50° c. Growth had
also appeared in two of the fourteen tubes that had been kept at 51° C.

No growth appeared at the end of two weeks in tubes that had been kept for

26

ten minutes at 52°, 55°, 54°, and 55°C., except one tube at 55°C.

The experiments at 50°, 51°, and 52°C. were repeated several
times with culture isolation strains No. 7, No. 1, No. 5, and No. 5, and
in most all cases there would be one to three tubes showing growth at 51°C,
but in no other series was growth observed in 55° or 54°C. *The thermal
death point, therefore, is between 50°C and 5P’C. Smith in 1911 placed
the thermal death point of P. beticola at 51°C.

Resistance to drying: Small drops of four day old bouillon cul-
tures were placed on sterile cover glasses in a covered sterile petri dish
and left at room temperature which ranged from 18°C to 25°C. At intervals
of twenty—four hours one of the cover glasses was transferred to a tube of
sterile bouillon. These tubes were used for each test. Growth occurred
up to the eighteenth day, after which the tubes remained sterile.

This experiment was repeated a number of times with bouillon
cultures from four to ten days old. As a rule there was no growth after
the eighteenth day; in one case, however, growth occurred on the twenty-
first, and another case on the sixteenth day.

*Optimum temperature. The Optimum temperature for growth
appears to lie between 25"and 28°C., preferably 26°C. Growth at 26°C on
potato dextrose and peptonized beef bouillon was considerably better than
at 5200. Growth at 20°C. and 57°C. was about the same but was considerably
less than at 52°C. and much less than at 26°C.

Maximum temperature: Four day old bouillon cultures of No. 5,
No. 6, and No. 7 isolations of the organism were used to inoculate slants
of potato dextrose medium. Six tubes were used for each culture; three
tubes of each lot were placed in an incubator at 59°C; and three were kept
at 26°C. All cultures, both in the incubator and at 26°C. made good

growths; however, those exposed to the higher temperature were of the color

27

0f the medium, while those in the lower temperature had developed the
normal yellow color after thirty-six hours. On the tenth day two of
those at 59°C. were placed at 26°C., and in twenty—four hours they took
on the normal yellow color. At 42°C. no growth occurred. *The maximum’
temperature for the organism for growth can be placed between 59°C. and
42°C.

Effect of sunlight: Moderately thick sowings of 24 hour broth
cultures were made on potato dextrose poured plates. One-half of each
plate was covered with black paper, and the plates were set on a white
enamel surface so that reflection would have effect also.

These plates were exposed to October sun for 2 1/2, 5, 10, 15,
and 20 minutes, duplicates being used for each time period. They were
then incubated at 26°C. No difference in number of colonies could be
detected for any of the periods in the exposed as against the covered
half. The results were rather unexpected since bacterial pathogens are
often described as sensitive to sunlight, certainly within the range
employed, 5 to 20 minutes being sufficient to kill or very much retard
colony development in a large number of forms. The experiment was re-
peated in July at Rocky Ford, Colorado, with exposures the same as above,
except additional exposure of 50, 40, and 45 minutes were included. The
su was exceptionally bright, but there was hardly more than 55 per cent
reduction in the number of colonies on plates exposed and those not ex—
posed. The organism does not appear to be so very sensitive to sunlight,
unless exceptionally bright and over considerable length of time.

Loss of virulence: Park (6) stated that, "bacteria differ -—-
as to ease and rapidity with which they grow in any nutrient substance

and the amount of poison they produce. Both of these properties not only

28

vary greatly in different members of the same species, but each variety
of bacteria may to a large extent be increased or diminished in virulence."

In October 1926 three cultures were isolated from sugar beets.
All the cultures were grown continually on potato dextrose medium. It was
observed that a culture designated as No. 1 was the most vigorously grow-
ing strain in hand. The inoculation work in the greenhouse at Michigan
State College showed its pathogenicity was very marked. Also the first
field inoculations at Rocky Ford, Colorado, in July, 1927 showed the marked
virulence of the organism. Late in the middle of August after a month
without new transfers the strain seemed to lose its vigor on culture media,
and the beet injury was also far less marked when it was inoculated into
beet roots. Only 45 per cent of the plants inoculated produced galls,
while formerly it was not uncommon to get 100 per cent infection with
rapidly growing cultures. The original culture finally died out, but re-
isolations from beets with this organism proved to be a rapid grower, pro-
ducing the same color on potato dextrose. It was designated as No. 5
which has a high virulence. From these results it seems possible that
the rate of growth of the pathoqen may be one Of the controlling factors
in the disease production of the organism.

AGGLUTINATION TEST.

As a method 0f Identification of a specific phytopathogenic bacteria.

The writer's researches are concerned with the production of
specific agglutinins of P. beticola, for use in diagnosis and for studies
on the relationships to other plant pathogenes.

Production of Antiserum: The methods followed were in general
similar to those given by Zinsser (17). However, in order to get a high
titre, modifications had to be made. It was discovered, as the following

data will show, that a dead organism produced a very low titre. It was

29

necessary to use living cultures to get a sufficiently high titre.
The organisms used for antigens were the various strains, listed

on page 17, of P. beticola, P. tumefaciens, and a culture of E. carotovora.

 

A 48 hour culture from potato dextrose agar (Pb 7.2) was washed from the
slant with normal saline, and after filtering through sterile cotton and
being shaken with beads, it was adjusted to standard density No. 2 by use
of McFarland nephelometer.

In the first attempts the standardized suspensions of P. beticola

 

were heated to 56°C. for one hour before the intravenous injections. The
titres obtained with this material were not very high, namely 1 to 950 and
l to 1200. In another series of rabbits the antigen was injected without
the preliminary heating. After 5 injections of 1 cc. of the antigen given
intravenously at 2 to 4 day intervals, serum of satisfactory titre was
obtained, 1 to 2200 and up to l to 3600.

The following are the titres obtained by the latter method of

rabbit serum immunized against the indicated antigens.

 

 

 

 

 

 

Rabbit 407 Titre l — 1200 Anti — P. beticola No. 1
Rabbit 239 " l — 2200 Anti - P. beticola No. 5
Rabbit 101 " l - 2750 Anti — P. tumefaciens " 146
Rabbit 397 " 1 — 3050 Anti - P. tumefaciens " 146
Rabbit 294 " l - 3250 Anti P. beticola No. 5
Rabbit 295 " 1 — 5600 Anti - P. beticola No. 2
Rabbit 595 " l - 650 (intraperitoneal) Anti 3. Care—

tovora.
Having obtained the high titre serum , various plant and soil
bacteria were subjected to cross agglutination tests. In the tests the
macroscopic method of determination was used. Some difficulty was experi-

enced with P. tumefaciens in that it had a tendency to form small clumps,

 

30

which formed spontaneously in the .85 per cent salt solution; however, with
a 0.5 per cent salt solution this was obviated.
The results of the various tests are shown in the following
tables:
TABLE VII.
Test of Phytomonas beticola Strain No. 5 antiserum against

various antigens.

 

Animal Antigen Dilutions

 

1/10 1720 1/50 1/100 1/200 iflsoo Check

 

239 P. beticola No.5 + + + + ++ + + + + + + -
P. beticola No.4 + t — - - _ -
P. beticola No.2 — — _ - _ _ _

B. noctuarum - - - - - - _

E. atroseptica (l)

E. atroseptica (2)
P. vignae + + - — _ _ -
B. sphingidis - - — _ _ - -

. carotovora t - - _ _ _ _

til

*P. tumefaciens + + + + + + + + + + + + + +

 

*Saline solution .85% caused floculation.

+ 4 Strong flucculation.
Medium "
Doubtful "
N0 u

|a+ +

31

TABLE VIII.

Test of Phytomonas tumefaciens No.

146 antiserum against various

 

 

 

antigens.
Animal Antigen Dilutions ,
1 /10 1/20 1/50 1/100 1 /200 1/500 Check
397 P. beticola No. 5 + - — - _ _ _
P. beticola No. 4 - - — _ - _ -
P. beticola No. 2 .t - - — _ - -
B. noctuarum - - - — - _ _
E. atroseptica (l) - - - — - - _
E. atroseptica (2) - — - - _ _ _
P. vignae + ‘1 — - - - _
B. sphingidis - — - — _ - -
E. carotovora — — — _ _ _ -
P. tumefaciens + + r + + + + + + + + + _ _

(In .05 saline)

 

TABLE IX.

Test of Erwinia carotovora antiserum against various antigens.

 

 

 

Animal Antigen Dilutions
1710 1 /720 1 /5o 1 [100 1 /zoo 1/500 Check
396 P. beticola No. 5 .1 — — - - - -
P. vignae - - — - _ - -
P. tumefaciens. + + .1 - _ - _
E. carotovora + + + + + + + + + + + + -

 

52

TABLE X.

Test of Phytomonas beticola Strain No. 2 antiserum against

various antigens.

 

 

 

Animal Antigen Dilutions
1 /10 1 /2o 1 Z50 17100 1/200 17500 Check
406 P. beticola No. 5 — - — - - _
P. beticola No. 4 + + + 4 4 + + .1 - _
P. tumefaciens - - — _ _ _
P. beticola No. 2 + + + 4 + + + 4 + +

 

Test of Phytomonas beticola Strain No.

TABLE XI.

 

ious antigens.

5 antiserum against var-

 

 

 

Animal Antigen Dilutions
1/10 1/20 1/50 1/100 1/200 1/500 Check
294 P. beticola N0. 5 + + + + + + + + + + + 4 _
P. beticola No. 7 + + + + + + + + + + + + -
P. beticola No. 2 - — - _ - _ _
E. atroseptica (l) - - — — - _ -
a. atroseptica (2) — — — — - _ _
E. amylovora - — - — - _ _
E. carotovora + — - — - _ _
Ps. flourescens — - - - - _ _
P. vignae .i - — _ - _ _
P. hyacinthi - — — _ _ - -
P. tumefaciens No. 146 + - - _ - -

|+

 

 

35

The tabulation of the first antiserum experiment (Table VII)

indicated that the antiserum of P. beticola No. 5 shows no very strong

 

reaction with any antigen except the homologous antigen. However, a
yellow organism reisolated from a garden beet at Rocky Ford, which was
_observed to have gall formation, showed a slight reaction. It is sus—
pected that this particular organism is also a soil organism of that

vicinity. E. carotovora showed a very slight tendency toward a reaction,

 

but could only be detected with microscopic methods. P. vignae showed a
slight reaction with serum from rabbit 259, but from 294 no reaction was
noticed. This organism, much the same as P. tumefaciens, has a tendenCy
to flocculate in saline .85% solution, which may have helped to cause the
conflicting results in the two.

The antiserum of P. tumefaciens proved to be very specific for
only its own antigen, except in the case of a 1-10 dilution reaction with

P. vigpae and P. beticola No. 5. The first reaction was very marked and

 

a group relationship between these two organisms seemed close. The agglu-
tination with P. beticola No. 5 was very slight, and no strong group
agglutination is apparent.

The antiserum E. carotovora from rabbit 396, which was the
result of intraperitoneal injections, showed a very low titre. However,

it was specific for its own antigen except when P. tumefaciens was used.

 

Here a very noticeable reaction was found. Link (5) has also observed

the same phenomena. The antiserum of P. tumefaciens showed no affinity,
however, for E. carotovora. This seems to indicate that a specific pro—
tein is present in E. carotovora for the production of antibodies which

will react with P. tumefaciens, but the production is not vica versa.

 

The antiserum of P. beticola strain No. 2 was specific to its

own antigen except when the No. 4 strain which was reisolated from the red

34

garden beet at Rocky Ford was used. No agglutination whatsoever was

noticed with P. beticola No. 5 or P. tumefaciens. These two are evidently

 

a soil organism from the Rocky Ford district which shows pathogenicity for
beets with a very low resistance.
The antiserum of P. beticola N0. 5 from rabbit 294 showed a

slight tendency toward agglutination with l — lO dilution of P. tumefaciens

 

and E. carotovora. No other reactions could be noticed. The above 3;
beticola No. 5 antigen showed a tendency for roughness (R), while a Culture
designated as No. 6 was obtained which seems to show smooth (8) appearance.
These two showed no difference in the agglutination reaction of the above
antiserum.

The work has shown that when titres are obtained which run above
1 - 1200, they are sufficient for diagnostic purposes, as given under the

identification experiment. The antisera of P. beticola and P. tumefaciens

 

 

are specific respectively for their own antigens to a very marked degree.
It can safely be said that in relatively high dilutions (1 - 500) the

identification of specific plant pathogenes studied can be made.

OVERWIRTERING OF THE ORGANISM.

In common agricultural practice, beets are topped in the field,
and it is obvious with a disease of the type of tuberculosis that debris
containing the organism.is thus scattered in the field. To determine if
the organisms lived over in the tops, two beets showing gall formation
were placed on Nov. 5, 1926 in sandy loam at a depth of six inches. One
of these beets was examined march 5, 1927 and the other April 29, 1927;
both were somewhat disintegrated. (Fig. 10). Isolations made from the
interior of the decaying galls gave a great variety of organisms, but in

dilutions of l - 10,000 a very great preponderance of yellow organisms

55

were found, which by cultural tests and inoculations were shown to be

P. beticola. The experiment was run under Michigan conditions, and the
air temperature during this time reached as low as 10 F below zero. It
is evident from these isolations that bacteria in the galls can withstand
severe conditions, as the soil around the beets was frozen from the last
of November to March.

Three tubercles from a beet that had been kept in the ice box
from October 16, 1926 to January 15, 1927 were ground in a meat grinder.
This material was mixed thoroughly with sterilized potted soil from the
greenhouse, having a PH reaction of 6.80. Ten pots were used and the
following quantities of the ground material were distributed to the vari-
ous pots of the series: 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 50.0, 100.0,
and 200.0 grams. Young sugar and garden beets showing about 7 sets of
leaves and whose root bundles had started to differentiate into the
collateral type were transplanted to the pots. After the plants were
firmly established, the dirt was carefully removed from one side of the
root. The crown and root was injured by scratching with a sterile needle,
care being taken to injure the roots by a longitudinal scratch so as to
expose the lower portions of the beet to the material to be tested. Over
a period of twentyeseven days the pots were watered uniformly with sterile
water; after this time the plants received ordinary care. The results of

the tubercle formation are given in Tables 12 and 15.

36

TABLE XII.
Infections secured in a 40-day period with varying amounts of
ground tubercle material. Young sugar beets exposed to infection after

being wounded at crown and along the roots.

 

 

 

 

Pot Quantity Symptoms showed mp
28 days 29 days 30 days 31 days 32 days 40 days
1 0.5 gms. 0 o o o o o
2 1.0 " 0 0 0 0 0 0
8 2.0 " 0 0 0 0 0 0
4 5.0 " 0 0 0 + + O
5 10.0 " 0 0 0 0 + Q
6 20.0 " 0 0 + 0 + +
7 30.0 " ‘1 + O + O +
8 50.0 " O O O ' + + +
9 100.0 " O O + O + +
10 200.0 " 0 0 .1 + + +
0 E0 symptoms.
+ New symptoms each.
‘3 Doubtful.

37

TABLE XIII.
Infections secured in a 40—day period with varying amounts of
ground tubercle material. Young garden beets exposed the same as prece-

ding.table.

 

 

 

Sample Dilutions Symptoms showedpup.
18 days 19 days 20 days 21 days 22 days 23 days
1 0.25 gms. 0 0 0 0 0 0
2 0.5 " 0 0 0 0 0 0
3 1.0 " O 0 0 O O 0
4 2.0 " 0 0 + 0 0 0
5 5.0 " 0 0 + + 0 0
6 10.0 " + 0 0 0 + 0
7 20.0 " 0 + O 0 + +
8 30.0 " O 0 + + + 0
9 50.0 " 0 r e 0 + 0
10 100.0 " + + + ’ o o +

 

The persistence of the organism capable of producing an infection
when harbored in a gall over a long period of time is clearly shown by the
above tables. It is evident that overwintering is particularly likely to
take place when infected crowns are left in the field, especially if covered
by soil so as to prevent excessive desiccation.

In another experiment 48—hour old broth cultures of P. beticola
were poured onto two flats of sandy loam soil. These two flats were left
outside from November 29, 1926 until January 20, 1927, after which sugar
beet seeds were planted in one of them. When the seedlings were at the
second leaf stage, the hypocotyls of a portion of the plants were injured

with a sterile needle, others were left uninjured. In the other flat older

38

sugar beet plants which had attained a root size of one half inch in dia-
meter were transplanted, and a portion of these were stabbed with a sterile
needle, some being left uninjured. The two series were allowed to run
forty-two days after injuring the plants. In the first flat, with seedlings,
no symptoms of the disease were noticed. In the second flat, with large
roots, two beets showed remarkable tubercle formation. It is shown by this
experiment that some of the organisms had lived over in the soil.

The above series of experiments indicate that the bacteria caus-
ing tuberculosis are capable of living overwinter in ooth beet debris and
soil. This datum, coupled with the finding of the organism in the field
soil one year after affected beets were observed, indicates that the organ-

ism may be of a saprophytic nature as well as parasitic.

EXISTENCE OF P. BETICOLA I? THE SOIL AS FREE ,IVING FORMS.
In determining the existence of the tuberculosis organism in the
soil, two different methods have been employed: first, the selectiOn from

yellow organisms obtained by platings by the use of P. beticola antiserum;

 

second, a method as employed by Patel (7) with use of his Crystal Violet
Bile medium for selection of P. tumefaciens in the soil.

It has been shown by Goldsworthy (2) that it is possible to
select specific organisms from soil by the use of an antiserum. After
positive results were obtained in agglutination test from laboratory ex-
periments, it was thought possible that Goldworthy's method for determin-
ing organisms from the soil would be helpful as an aid in the study of
western soil where the disease is observed.

Greenhouse test: In order to find out if P. beticola could be

reisolated from soil and to find out how serviceable the agglutinating

39

serum would be in this connection, the following preliminary work was done.

An antiserum of P. beticola with titre l - 1200 was used.* A composite

 

broth mixture of P. beticola No. 1 and several other undetermined yellow
organisms which were isolated from soil at Rocky Ford, Colorado, were
poured on soil in flower pots in which young sugar beet plants were grow-
ing. The beets were then punctured with a sterile needle and kept in a
very moist condition with sterile water; they later developed galls. Four—
teen days after the suspension was poured on the soil, cultures were made
in serial dilutions as shown in Table 14. The numerous yellow organisms
which developed on the plates were isolated and grown on potato dextrose

agar, and then tested with the P. beticola antiserum in the usual manner.

 

The results are given in column 3 of the table.

TABLE XIV.
Results of the selected number of yellow organisms on the dilu—

tion plates which showed agglutination reaction with P. beticola antiserum.

 

 

No. of Colonies

 

Soil Dilutions Used Yellow Colonies Agglutinated at Per cent
Selected l - 1000 titre
1/1,ooo 14 , 4 28
1/1o,ooo 9 3 33 1/3
1/100,000 1 o o
1/1,ooo,ooo 2 o o
1/2,ooo,ooo 1 o o
1/5,ooo,ooo 3 1 33 1/5

 

*See page 29.

With the organisms showing an agglutination reaction with the
antiserum, inoculations were made into rapidly growing sugar beets. One
month after inoculation, the following results were obtained: Out Of the
eight organisms which were agglutinated, seven produced typical tubercles

on inoculated beets, thus proving that P. beticola by the above technique

 

can easily be reisolated from a composite of soil organisms.

With the above evidence, field samples of soil were collected
from several fields at Rocky Ford and Lamar, Colorado. Some samples were
from beet fields where the disease, if present, was certainly not serious.
Other samples were from the field at Lamar, Colorado, which the previous
year had shown 10 per cent diseased in certain portions, and still other
samples were obtained in which so far as could be ascertained from the
records of the American Beet Sugar Company, beets had never been grown.
Water from two irrigation canals was also tested.

The first selection was twenty samples taken at random July 2,
1927 from the Lamar field. Serial dilutions of l - 10,000 to l - 2,000,000
were made of each sample in duplicate on potato dextrose agar (PH 7.56).
From these plates, organisms which showed the most typical pigment forma—
tion and growmh characteristics were picked out and cultured. The organ-
isms were then tested by use of an agglutinating serwn. Six organians
from the forty plates showed a strong agglutination reaction with anti—
serum at a dilution 1 to 1,000. The pathogenicity of each of the six
organisms was then tested by inoculating into rapidly growing beets in a
field with apprOpriate checks left. Five of the organisms thus selected
produced galls on sugar beets, whereas all of the organisms proved patho-
genic to garden beets.‘ It is evident that P. beticola had persisted over
winter in the soil under the dry conditions prevalent in the Colorado

district.

41

Other soil samples taken from the various Rocky Ferd, Colorado,
fields have only yielded one other definitely identified culture 0f.§;
beticola, although numerous platings have been made. Both Catlin ditch
. and Rocky Ford ditch irrigation water was tested by the above method, but
in all cases the results proved negative. Soil that as far as is known
may be termed virgin showed no tubercle producing organisms present; howb
ever, the amount of material worked with was insufficient to prove that

the organism is not present in that type of soil.

TABLE XV.

Results of the Agglutination test with P. beticola antiserum

 

and organisms isolated from the twenty samples obtained at Lamar, Colo.

 

 

Plates Antiserum Dilutions
Dilutions N0. of N0. yellow No. yellows showing Agg. Remarks
Colonies Colonies 1:5 1:50 1:100 1:500 1:1000
Avergge Average
l/l,000 Too numerous Too numerous None sel-
- ected
1/1 , 000 n n n n n n
1/10,000 96 1 27 2 1 0 0 0
1/10,000 103 15 6 4 2 2 2 Pathogenic
1/50,000 47 22 2 1 1 1 1 Path. on
Garden Beet
1/50,000 30 11 1 1- 0 0 0
1/100,000 12 7 1 1 1 1 1 Pathogenic
1/100,000 17 3 1 ? ? ? ? "
1/1,000,000 4 1 o 0 0 0 0
1/1,000,000 8 0 0 0 0 o 0
1/2,000,000 9 2 1 1 1 1 ? Pathogenic

1/2,000,000 5 0 0 0 0 0 0

 

The Patel's Crystal Violet Bile medium was next tested as to its

behavior when P. beticola was grown on it. The medium was produced by

 

Patel especially for the differentiation of P. tumefaciens from other organ-
isms in the soil. However, the reaction of P. beticola, that of a large
halo, is a very marked difference from either P. tumefaciens or any other
organism tested with the single exception of Ps._pyocyanea which in a few
cases showed slight halo formation. With the above demarcations showing
up and a tendency for P. beticola and Ps. pyocyanea to show somewhat the
same reaction, a modification of Patel's medium was made. This gave.PL
beticola distinctive reaction from all other organisms tested, as shown
in Table 16.
The modification of Patel's agar is as follows:
*4 grams Sodium Taurocholate.
*20 grams Sucrose.
20 grams Agar.
*7 grams Peptone.
2 cc. Crystal violet 1/1,000 aq. solution.

1000 cc. of water.

 

*Amounts were changed to make possible selectiveness.

43

TABLE XVI.

Behavior of various soil organisms when grown on a modification

of Patel's Crystal Violet Bile Medium.

 

 

 

Organisms Growth 60 hours 72 hours 84 hours Description
Started Growth in diameter
P. tumefaciens 48 hrs. 2.3 mm. 3.2 mm. 7.1 mm. Colony glossy.
P. beticola No. 5 44 hrs. 2.4 mm. 3.5 mm. 7.0 mm. Halo produced
4.25 cm. across.
P. beticola No. 2 72 hrs. 2.1 mm. Takes up dye.
P. beticola No. 4 None No growth.
B. prodiglosus 48 hrs. 2.04 cm. 2.52 cm. Red color.
B. violaceus 60 hrs. 1.7 mm. 2.0 and Deep purple.
Ps. radiciccola 72 hrs. 1.2 mm. Light purple.
P. pyocyanea 24 hrs. 1.92 cm. 2.20 cm. 2.29 cm. Col. spreading.
B. flourescens 60 hrs. 3.4 mm. 3.6 mm. Light purple.
B. mesentericus None ‘
B. subtilis None
B. mycoides None
Asp. niger . None
Pen. italicum 60 hrs. 3.3 mm. 7.5 mm. Very fuZZy.
M. varians None

 

A test was performed on field materials sent to Michigan from

Rocky Ford, Colorado, with the use of the modified medium.

The technique

was followed as given by Patel (7) for isolation of P. tumefaciens from

the soil.

From the ten samples on hand only one P. beticola colony was

obtained, and this from a field that had been in one year clover following

a beet crop.

 

 

 

 

 

 

 

 

 

The above experiments along with the overwintering results gives

some evidence that P. beticola can persist for eighteen months in Colorado

 

soils under ordinary climatic conditions. This evidence coupled with the
deve10pment of the disease here and there in the Arkansas Valley leads the
writer to the contention that with this organism we have to do with a patho-
gen capable of existence for a considerable period in the soil. .
INFLUENCE OF PH CONSENTRATION 0F SOIL AND ARTIFICIAL
MEDIA ON P. BETICOLA.
The effect of the hydrogen —ion concentration upon the growth of

P. beticola is very marked. The optimum range is between 7.00 and 7.60;

 

the complete range, however, where growth has taken place is approximately
between 5,80 and 8.60 1' Below 5.80 very scanty growth resulted, while
above 8.60 1 growth was not found. A check on different soil samples at
Rocky Ford-and Lamar, Colorado, showed that the pH. range in that locality
was between 7.42 and 8.10. This finding was observed to be very close to
that which, as stated above, was found best in cultural media for growth
of the organism.

After the determination of the PH values of western soil, local
beet soil from Michigan was obtained and the PH of it was obtained. It
was thought that the PH of a soil might be one of the deciding factors in
keeping the-organism within a definite area. Samples of the local beet
soil showed a range of 5.83 to 6.03.

It can plainly be seen that the soil which is known to have the
organism present is of a much more alkaline reaction than soil where the
disease has not been observed. This may be one of the factors restricting

the organisms to the west.

 

45

THE INFLUENCE OF FREEZING 0N P. BETICOLA.
Temperature is considered one of the cardinal factors influencing
life activities of micro-organisms. The majority of bacteria are unable
to exercise normal metabolism at temperatures below 6°C. 0r above 45°C.
according to Hilliard (3). In order to determine the resisting power of

P. beticola, experiments with both freezing and thawing were conducted.

 

Prudden (8) in 1887 found pure cultures of B.,prodigiosus and B.gproteus

(common soil organisms) to be sterile after fifty-one days at temperatures

 

ranging between -100 to 190. B. typhosus survived for at least one hundred
and three days at a temperature between 14” and 30°F. Keith (1923) con-
trary to Prudden has emphasized the importance of solid freezing as com-
pared with cold in relation to the death rate of bacteria. B. coli frozen
solidly in water at -20°C. shows 99 per cent killed in five days, but when
not actually frozen, a large per cent remained alive for months. When

P. beticola was frozen in diluted soil solutions, the death rate increased

 

with the dilution. If suspended in aqueous mixture of 5 to 40 per cent
glycerine, a large percentage of the P. beticola remained alive for three
months at temperatures ranging from a few degrees above zero to 412°F.

below.

46

TABLE XVII.
Results showing resisting power of both Freezing and Thawing of

P. beticola.

 

Distilled water Soil Solution

 

Frozen Solid Alternate Fz. Frozen Solid Alternate Fz.

Phytomonas beticola

 

Count per cc Count per cc Count per cc Count per cc
Before Freeze Before Freeze Before Freeze Before Freeze
50,428 50,428 50,428 50,428
Frozen 1 hr. *Fz. 3 thnes . Frozen 1 hr. *Fz. 3 times
15,600 920 39,042 20,049
Frozen 12 hrs. Fz. 5 times Frozen 12 hrs. F2. 5 times
1,620 116 16,422 1,940
Frozen 24 hrs. Fz. 6 times Frozen 24 hrs. Fz. 6 times
160 124 10,327 832
Frozen 3 days Fz. 8 times Frozen 3 days F2. 8 times
156 8 949 ' 73
Frozen 5 days Frozen 5 days Fz. 10 times
93 926 4
Various degrees Various degrees

 

* 10 min. at —lo°c.

47

TABLE XVIII.

Results of Resisting power to Freezing and Thawing of Senatia

 

 

 

 

 

marcescens.
Distilled Water Soil Solution
Frozen Solid Alternate Fz. Frozen Solid Alternate Fz.
Senatia marcescens (B. prodigiosus)
Count per cc_ Count per cc Count per cc Count per cc
Before Freeze Before Freeze Before Freeze Before Freeze
39,516 39,516 39,516 39,156
Frozen 24 hrs. *Fz. once Frozen 24 hrs. *Fz. once
6,417 5,257 24,007 1,673
Frozen 30 hrs. Fz. 2 times Frozen 30 hrs. Fz. 2 times
7,679 76 15,090 1,579
Frozen 3 days Fz. 3 times Frozen 3 days Fz. 3 times
1,973 15 11,730 973
Frozen 5 days Fz. 4 times Frozen 5 days Fz. 4 times
986 17 4,872 74
Various degrees Various degrees

 

* 10 min. at -lo°o.

 

It is apparent by the above data that P. beticola can endure a

 

continued freeze, especially in a soil solution. This compared favorably

with the common soil organism Serratia marcescens. The organism when in

 

the soil below six inches in depth is not subject to a very great fluctu-
ation during the winter; hence it is possible for them to overwinter as a
soil organism.

The distilled water gave a much higher mortality than the soil
solution. This is probably due to the lowering of crystallization and
possible mechanical crushing of the bacteria during the freezing of the

medium.

48

When in decaying vegetable parts, the beets may only be slightly
covered with soil, but the solution within the disease tissue will be suffi-
ciently concentrated to offer good living conditions. Thus it is further

shown that P. beticola has the ability to withstand conditions necessary

 

for successful overwintering of a soil organism.

THE ANATOMY OF THE OVERGROWTH.

Smith (15) before the Societe de Pathologie Vegetale et d'Entomo-'
logie Agricola de France stated that he believed that there were structures
produced in plant diseases which are analogous to the tubercles found in
diseased animals. He further stated that the P. savastanoi (E.F.S.) S.A.B.,

causing olive knot and P..Beticola, causing tuberculosis of sugar beet

 

furnished proof of his statement. In addition to the two already mentioned,

there is also to be included 3. leguminosum (Frank) S. A. B. which causes

 

the tubercles on plants of the Leguminosae. According to Smith's conception
then, there are three distinctly different organisms causing plant tubercles.
All three produce symptoms in plants which, if Smith's idea is accepted,

are closely analogous in structure to those growths caused by organisms
pathogenic to animals.

Macroscopic examination of a cross-section of the tuberculoSis
type of overgrowth in sugar beets reveal small, yellowish, watery, spots
surrounded by much distorted tissue. Upon microscopic examination it is
seen that these spots are bacterial pockets. The crown—gall type of pro-
liferation presents no such aspect.

A study was made of the overgrowth to determine the correlation
of the cellular behavior with the localization of organisms, and to com—
pare the type of pathological growth of the beet with animal tubercles.

The material selected for this study was fixed, and embedded in paraffin,

sectioned 10 in thickness, and stained with Haidenhain's Iron Alum Haema-

49

toxylin.

The organism causing the neoplasm was found in a wide range of
tissues including phloem, xylem, cambium, and parenchyma. The overgroumhs
appear to be the result of a stimulant produced by material secreted by the
bacteria. It was evident that the cells of the plant are also mechanically
pushed apart by bacteria passing from one place in the tissue to another,
this being especially true in the cambium region of the host. The over—
growths show a tuberculoma-like type of neoplasm, in that the tissue in-
volved tends to hold the parasite within localized areas. (Fig. 11). The
organism causes no decay of the tissues with which they come in contact,
but new host cells or enlarged old cells restrict the bacteria to small
pockets. This condition is somewhat analogous to granulomatous tissue of
animals.

Microsc0pic study of cross—sections of diseased material at
various stages of development reveal the origin of the bacterial pockets.
The organisms in early stages of infection, or as they are squeezing through
the tissue, are intercellular. They multiply at certain advantageous
points. Such points are usually in the cortex, where the newly differen-
tiated parenchyma cells with their commonly occurring intercellular Spaces
allow the colony of organisms to increase in size. (Fig. 12). A similar
growth may also be found between the cells of the cambium. As the bacteria
increase in numbers at these points, the pressure from the growing mass
crushes the bordering host cells, whose structures may also have been weak—
ened by seepage of bacterial by—products, thus forming the disorganized
cavities. When the bacteria are found located in the vicinity of the meris-
tematic cells, hyperplasia is the most characteristic type of proliferation.

When the bacterial cavities are surrounded by parenchyma, hypertrOphy of

50

the cells is the most common type of response. However, the stimulating
substance from the parasite has an effect over a zone at least ten to fif-
teen cells in thickness, and when this substance acts upon newly differen-
tiated parenchyma cells, hyperplasia often takes place. With such increases
in cell divisions, poly-nucleated cells are often found, and the rate of
division is accelerated (Fig. 15). These fast dividing cells often form
strand-like structures in the overgrowth which may either be purely hyper-
plastic or hypertrophic, or at times both growth types intermingled.

In a large number of sections the pockets were found within the
cambium layer, and this is especially true in the case of petiole infect—
ion (Fig. 14). The cells of the cambium are in a state of rapid division,
but with the stimulation produced by the organism these cells seem to
divide even more rapidly; thus abundant hyperplastic growth is caused.

The young rapidly dividing cells are very easily crushed, and hence we

find a great number of both large and small bacterial pockets. The rapid
internal cell production along with the enlargement of old cells, which are
the results of the stimulating effect of the colonies of the organism,
cause a great internal pressure to take place. Since no new outer cells
are being formed, the tubercle splits, thus forming the deep fissures over
the surface of the overgrowth.

The tubercles, besides having large fissures present, are also
very rough and show extremely large amounts of disintegration. This was
observed to be caused when the organisms are located in the old parenchyma
tissue, or the stimulant reaches these cells. The effect is mostly
hypertrOphic in nature (Fig. 15). These cells take on various shapes—-
cylindrical, polyhedral, and spherical with large intercellular spaces.
There is no evidence of an epidermal develOpment in later stages of tuber-

cle growth. As the proliferation develOps, the epidermis is ruptured so

 

51

that the large hypertrOphic cells with enlarged intercellular spaces are
exposed, which allows soil organisms to enter. This causes the very rough
and decayed surfaces spoken of earlier as a distinguished characteristic
under the external symptoms.

The parenchyma cells next to the xylem are thick walled, and are
spoken of by Artschwager (1) as the "sugar sheath". Cells of the "sugar
sheath" attacked by P. beticola lose their turgidity, and suffer partial
or complete collapse, producing primary bacterial pockets. The bacteria
are able to break.down the "sugar sheath" cells very easily, whereas the
strength of the tracheae and the tracheids will withstand the pressure from
the bacterial growth. However, the organisms at times do penetrate into
these tubes after the pockets have been formed in the "sugar sheath", and
travel along in them only to develop in large masses again, breaking out
of the tubes and forming new pockets. This condition is also present in
the bundles of the petiole which are of the Open collateral type, with the
cambium taking the place of the "sugar sheath" as the point of bacterial
invasion (Fig. 16). Certain pockets over two inches in length have been
obserVed in the oeet root. These pockets extend in longitudinal direction,
and are more or less associated with the vascular system.

The above considerations undoubtedly play the decisive role in
determining the nature and localization of the galls upon the sugar beet.
In the root tubercles the situation is as follows: A mediam horizontal
section through a beet shows a number of annular zones which are more or
less equidistant, except near the periphery, where they are very close
together. (of. Artschwager (1)). With the cambium located so near the
periphery, and being the most susceptible tissue to the attack of the

organism, it is evident that infection may take place following even a very

shallOW‘wound.

52

In the course of the studies of beet tuberculosis, the question
arose as to why at certain stages of beet growth there are apparently
different degrees of susceptibility. A structural difference is readily
observed when the age of beets is taken into consideration, and there are
probably physiological differences as well. In the hypocotyls of the young
beet seedlings, there is no differentiated cambium present which has been
seen in the tissue especially subject to attack by P. beticola. The corti—
cal cells prevent the organism from becoming established. In certain
artificial inoculation experiments, it has been noticed that the resistance
in young seedlings has been broken down and an infection secured, only to
have the cortex cells of the hypocotyl sloughed off, thus eliminating the
infection.

In petiole infection the shallow wounds which are sufficient for
beet infection are of no consequence. It will be recalled that petiolar
galls have not been found in nature, but it was found necessary to puncture
one of the bundles before disease symptoms could be produced. The histo—
logical composition of the vascular tissue, according to Artschwager (l),
is that of a typical collateral bundle (Fig. 14b). The phloem forms a
narrow zone composed chiefly of sieve tubes with their companion cells and
some phloem parenchyma with only a thin layer of cambium. Although this
cambium layer is favorable tissue for pocket formation, it is surrounded
b y thick walled cortex cells which are unfavorable for bacterial extension
and development. It would seem that the time explanation of the absence
of petiolar tubercles in nature is to be found in the anatomical structure
of the petioles.

As is evident from the entire discussion given of the pathological
anatomy and as is shown by the photomicrographs, the tubercle formed in

this disease fits the criteria used by animal pathologists for their concept

 

55

of a true tuberculosis, and the writer believes Dr. Smith's characteri-

zation of the galls as a similar structure is fully warranted.

SUMMARY.

Sugar beet tuberculosis, a disease characterized by overgrowths
on the crown and root of beets, is one of the many root diseases of this
plant.

The literature of this disease is limited to the original con-
tribution of Smith, Brown, and Townsend, and a few brief notes.

The disease up to the present time has only been reported from
Colorado, Kansas, and flyoming, and seems to be limited in its distribution
to the west.

The disease may eventually become of considerable economic
importance, since diseased beets are subject to rot in storage piles and
are likely to be wastefully topped. The diseased beets show consistently
lower sugar percentage and purity than normal beets.

The disease is distinguishable from crown-gall in that the
tubercles are more rough; they extend generally from ridges perpendicular
to the root; cross-section shows yellow watery spots present; and frequently
the roots assume turnip shape.

In the areas where P. beticola is in the soil, hail or mechanical

 

injury of some sort has been found to produce heavy infection on rapidly
growing roots. Irrigation water has been found to carry the organism to
the wounds.

The pathogenicity of the organism has proven according to
Koch's rules of proof. The bacterial pathogen was found to occur in large

masses in the yellowish, watery spots in the tubercles.

54

Cross inoculations have only proved successful on garden beets
and Swiss Chard, With tomatoes harboring the parasite for one month without
the production of the typical symptoms. Inoculations on many other hosts
gave negative results.

The classification as given by Bergey's Manual is followed;

hence the name used in this paper Phytomonas beticola.

 

The description of the organism is given. In a large part the
test outlined follows the work reported by Dr. Smith; however, some signi-
ficant variations have been noted, along with additional information.

The production of specific agglutinins of P. beticola for use
in diagnostic purposes and relationships of the organism to other plant
pathogens are of practic;1 use in this work.

The existence of tuberculosis organism in the soil was determined

by two methods; first by use of P. beticola antiserum, second by use of

 

Patel's Crystal Violet Bile medium (modified).

The organism causing the tubercles was found to be located in a
wide range of tissue, namely phloem, xylem, cambium, and parenchyma, with
the bacteria having a special preference for the cambium.

The overgrowths appear to be the result of a stimulatiOn of the
cells by material produced by the bacteria.

The overgrowths showed a tuberculoma-like type of neOplasm, in
that the tissue involved tends to hold the bacteria within localized areas.

HypertrOphic and hyperplastic tissue was found to make up the
greater part of the galls, hypertrOphy being associated with parenchyma
cells, and hyperplasia with the cambium cells.

The apparent resistance of the young seedlings to the disease
seems attributable to the absence of the cambium layer at this stage. When

infection was produced by artificial inoculation, the diseased parts are

55

usually eliminated from the plant at the time of the rupture and sloughing

of the primary cortical layer.

56

LITERATURE CITED.

1. Artschwager, Ernst

3.

5.

7.

8.

9.

Anatomy of the vegetable organs of the sugar beet. Jour. of
Agr. Res.-§§. 2:143 - 176. 1926.

Goldworthy, M. C.
Studies on the spot disease of cauliflower; a use of serum
diagnosis. PhytOpath.,l§: 878 — 885. 1926.

Hilliard, C. M. and Davis,.Mildred A.
The germicidal action of freezing temperatures upon bacteria.

Jour. of Bact. 5: 423. 1918.

 

Inoculation of shade trees against a deadly bacterial disease.
Science 64. 1717: Supp. xiv. 1927.

Link, Geo. K. K. and Link, Adeline De S.
Further agglutination test with bacterial plant pathogens.

Bot. Gaz. 85:178-197. 1928.

 

Pathogenic microorganisms. Park and Williams. Eighth Ed:160.
1924.

Patel, K.
Longevity of P's tumefaciens Sm. and Town. in various soils.
PhytOpath. 18:129. 1928.

Prudden, T. E.
Bacteria in ice and their relation to disease. Med. Rec. Mar. 26
and Apr. 2. 1887.

Peklo.
"Ueber die Smith' schen Rubentumoren". Zeitschrift fur Zucher-

industrie in Bohmen. Jahrg. XXXIX, 5 Heft, pp. 204-219, Feb. 1915.

10.

ll.

12.

13.

14.

15.

16.

17.

57

Society of American Bacteriologists.
Committee on characterization and classification of bacterial
types. The families and genera of the bacteria. Final report.

Jour. Bact. 5:191-229. 1920.

 

Bergey's manual of determinative bacteriology. p. 182. Balt. 1925.
Sackett, W. G.
Tuberculosis of Sugar Beet. Marshall, C. E. Microbiology 2nd Ed.
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Sardina, J. Rodriguez.
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Crown—gall of Plants: Its cause and remedy. U. 8. Dept. of Agr.
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Le crown-gall. Extrait de la Revue de Pathologie vegetate et
d'Entomologie agricola, t. XI, fasc. 4. 1924.
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Field studies of the crown—gall of sugar beets. U. 8. Dept. Agr.
Bur. Plant Ind. Bul. 203:1-8. (illus.) 1915.
Zinsser, Hans, Hopkins, J. G., and Ottenberg, Ruben.

A laboratory course in serum study. Bact. 208:66-84. 1921.

Fig.

Fig.

Fig.

10.

11.

58

EXPLANATION OF FIGURES.

Sugar beet tuberculosis caused by Phytomonas beticola. Beet from

 

a hail—injured field Lamar, Colorado, natural infection. Photo
by Dewey Stewart.
Type of diseased and normal beets used in report on Table II.
DiseaSe produced by artificial inoculation.
(a) Medium size tubercle. (b) Large size tubercles.
Natural infection of sugar beets following injury from field
practices. Tubercles produced on main part of root.
(a) Cross-section of crown tuberhles showing central, brownish,
water—soaked, bacterial areas surrounded by white flesh.
(b) Longitudinal section of beet showing bacterial pockets.

(a) Sugar beet crown—gall produced by inoculation with Phytomonas
0

 

I,

tumefaciens. M. S. C. strain 146.

 

(b) Cross~section of the same gall.

Tuberculosis on garden beet caused by Phytomonas beticola. Beet

 

was artificially inoculated at Rocky Ford, Colorado.

Phytomonas beticola stained to show flagella.

 

Typical growth of Phytomonas beticola on nutrient agar slants.

 

Growth characterized by brush-like projections.

Tomatoes inoculated with (a) thtomonas beticola, and (b) Phytomonas

 

 

tumefaciens.

Disintegrated condition of sugar beet left in soil overwinter.
Cross-section of a sugar beet tubercle with typical bacterial
cavities marked. Result of a pure culture inoculation using

Phytomonas beticola No. 5.

Fig.

Fig.

Fig.

Fig.

’31
Ho
0"?

12.

15.

14.

15.

16.

59

Tubercles on sugar beet showing pockets filled with rod shape
bacteria. Ten micron sections of a twenty day gall X1000.
Tubercles in young cortical parenchyma of a sugar beet petiole.
The rapidly dividing polynucleated cell may be seen. From a
pure culture inoculation.

Section through a sugar beet petiole showing (a) the cambium of
the bundle destroyed by the formation of a bacterial pocket,
(b) a normal bundle of the same petiole.

Cross-section of a tubercle showing large hypertrophic parenchyma
cells exposed, and the large intercellular spaces. Epidermis
has been ruptured and has fallen away.

Longitudinal section showing bacterial pockets produced at

intervals along the tracheae. From pure culture inoculations.

FIGURE 1.

 

 

 

FIGURE 2.

 

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