v ._ v_< 4.— 7 _

 

INVESTIGATIONS ON BACTERIAL

RINGROT OF POTATOES CAUSED

BY PHYTOMONAS SEPEDONICA

{SPIECKERMANN AND KOTTHOFF]
BERGEY ET AL

Thesis for the Degree of M. S.
MICHIGAN STATE COLLEGE

Joseph Anthony Rupert
I94!

INVESTIGATIONS ON BACTERIAL RINGROT OF POTATOES CAUSED BY

PHYTOMONAS SEPEDONICS (SPIECKERMANN AND KOTTHOFF) BERGEY ET AL

 

by

JOSEPH ANTHONY EETERT

A THESIS

Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfillment of the
requirements for the degree Of
Master Of Science
Department of Botany
June 1941

ACKNOWLE EMENT

The writer wishes to express his indebtedness
to Dr. J. H. Muncie for advice in carrying out
this study, to Mr. Ralph Lewis and Dr. E. H.
Newcomer for aid in the photographic work, and
to Drs. J. H. Muncie and E. A. Bessey for criticism

and correction of the manuscript.

NOTE

Due to the writer's eligibility under
the Selective Service Act Of 1940 these
studies were cut short. Consequently

the material herein prepared represents
incomplete data.

Contents
Introduction
History and Distribution
Economic Importance
Hosts
Symptoms
Foliar
Tuber
Diagnosis
Gram's Staining Method
Ultra-violet Light
Etiology
Proof Of Pathogenicity
Description Of the Organism
Cultural Characteristics
Pathological Histology
Overwintering
1940 Field Experiments
Disinfectants and Control
Penetration Of Dowicide "A"
Spread Of the Disease in the Field
Dissemination
Varietal Resistance
Control
Summary

Literature Cited

INTRODUCTION

The potato ranks sixth among the crops grown in
the United States as determined by acreage and value
of the crop produced (1). If it is considered on
the basis of a table fOOd product, it ranks second
only to wheat.

In Michigan the potato crop ranks fourth in total
value being exceeded only by corn, hay, and oats as
determined by the 1930-1939 average. During this ten-
year period the average annual yield for the state was
25,778,000 bushels with an average annual income Of
$14,789,000 (2). As a cash crop in the state, the
potato is second to none (5).

A serious new disease of the potato was reported
in this country in 1957 (4). Recently this malady,
bacterial ringrot Of potato, has spread into Michigan
and is causing grave concern among the growers. The
swiftness with which this disease has spread to all
potato growing states is unique in the annals Of

potato diseases.

Name of the Disease
Savile and Racicot (5) first suggested the name

"Bacterial Wilt and Hot". Bonde (6) proposed "Bacterial

Wilt and Soft Rot". This led to some confusion as

"bacterial wilt is the common name associated with

 

a disease of potatoes caused by Phytomonas solanacearum
while "soft rot" is associated with the organism Erwinia

carotovora. Hence the Committee to Coordinate Research

 

on New and Unusual Potato Diseases recommended that
the name "Bacterial Ringrot" or "Ringrot", a direct

translation of the German "ringfaule", be adopted (7).

History and Distribution

Spieckermann Of Germany was the first to report
bacterial ringrot Of potatoes in 1906 (8). In this
country E. F. Smith received some affected tubers from
Massachusetts in 1918 which he believed to be related
tO Spieckermann's ringrot disease (9). His bacterial
isolates yielded no definite results on inoculations
into the potato and tomato. His excellent photographs
Of these tubers show no close resemblance to bacterial
ringrot today. In conclusion he states, "When the
plates were made, I believed the disease due, probably,
tO bacteria, but now I have no definite opinion as to
its cause." The name Of "net necrosis" was given this
disease.

In 1951 Conners (10) reported the presence of ring-

rot in Canada where it has since become widely distributed.

The first report Of the disease in this country was made
by Bonde Of Maine in 1937 (4) although he states that he
had observed the disease as early as 1932. Burkholder (11)
in 1938 established the identity of the causal agent Of

potato ringrot as Phytomonas sepedonica (Spieckermann

 

and Kotthoff) Bergey et al.

In Michigan the disease was first reported in 1959
by Muncie (12). However, the first serious outbreak
occurred on the Knoblauch farm near Blissfield in the
summer of 1940. When this field of Chippewas was ex-
amined in July, approximately 40% Of the hills had failed
to emerge. At digging time this plot yielded so bushels
per acre; this same grower's adjacent field of Cobblers
produced 240 bushels per acre.

In September Of the same year a severe infection
was found on the Schoenfeld Brothers' farm near Imlay City.
A careful checkup by random sampling in the field revealed
that 64% of the hills were diseased. This 20 acre field
of Chippewas was grown on poorly-drained muck soil. The
disease here was much more severe than On the well-drained
sandy loam.Of the Knoblauch farm.

Both of these 1940 outbreaks were in fields grown
for table stock planted to Maine select Chippewa seed

stock. In 1941 in Oakland County another lot Of the same

kind Of seed stock showed ringrot.

Aside from these two cases no other destructive out-
breaks have been Observed. Several mild instances Of the
disease were found. In all cases these growers have
agreed to dispose Of their present seed stock and to
Obtain a fresh supply of reliable seed.

At present the disease has been found either in the
field, in storage, or in seed stock in eleven widely scat-
tered counties in the state as follows:(see map)

Lapeer
Lenawee
Monroe
Oakland
Tuscola
Oceana
Emmet
Menominee
Cheyboygan
Marquette
Economic Importance

Losses from ringrot may vary from a trace to almost
total loss. Growers of Maine (6) and Florida (13) have
suffered heavy losses. In Michigan losses have been small
to date but the importance of the disease cannot be Judged
alone from the losses that have been sustained. This dis-
ease constitutes a potential threat to the increasingly

effective potato certification program that has been built

up by decades Of untiring effort and to the to the produc-

MAY 1941

r

    

mm
M ..
I

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Lie—a;

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DISTRIBUTION OF BACTERIAL RINGROT IN MICHIGAN

 

 

tion of quality table stock potatoes. The certification
program has taken on an added significance since the ap-
pearance Of ringrot and is playing a decisive role in

the fight to control the disease.

Hosts
All commercial varieties Of the Irish potato,

Solanum tuberosum, are susceptible to ringrot infection

 

(14) and (15). In addition the tomato, Lycopersicum

esculentum, is susceptible upon artificial inoculation

 

and shows typical ringrot symptoms Of the foliage (5).
The following plants were tested in the green-
house for susceptibility to the ringrot organism:
1. Black nightshade (Solanum nigrum)
2. Jimson weed (Datura stramonium)
5. Wild tobacco (Nicotiana rustica)
4. Sticky tobacco (Nicotiana glutinosa)
5. Red pepper (Capsicum annuum)
6. Egg plant (Solanum melongena)
7. Pyrathorn solanum (Solanum pyracanthus)
8. Tomato (Lycopersicum esculentum)
With the exception Of tomato, no definite infection
developed (Fig. 1).
Symptoms
Foliage
The first symptom to appear, aside from the general

unthriftiness Of the plant, is a slight inward rolling

 

Fig. 1A. John Baer tomato plant showing wilting of
lower leaves 32 days after inoculation at
base of stem with inoculum from ringrot
infected potato. Healthy plant on the
right. - A

 

Fig. 18. Same plant as in Fig. 1A (left) 59 days
after inoculation.

Of the leaves somewhat resembling the leaf-roll virous
disease at first glance. The rolled leaves are soft
and pliable in contrast to the leathery and brittle
leaves typical Of leaf-roll. Partial wilting follows
during the hottest part of the day, the plant recover-
ing during the night. The leaves become chlorotic and
marginal necrosis develops followed by death Of the
shoot (Fig. 2). On splitting the diseased stem, no
characteristics Of diagnostic value can be seen; bacter-
ial ooze is absent and the tissues appear normal in
color in contrast to the typical brown vascular dis-
coloration Of Fusarium Wilt or the black discoloration
of blackleg. Plants infected with blackleg may be
further distinguished from ringrot infected plants by
their characteristic black basal stem rot. This is
entirely absent in ringrot infected plants.

It has been Observed that in some instances the
plant infected with ringrot does not wilt, although
upon microscopic examination Of a stem smear, a rich
abundance Of the causative bacteria is found. More-
over, not infrequently one shoot may become severely
diseased while the remainder Of the plant grows nor-
mally to maturity.

The field symptoms are variable and come late in

the season when late and early blights and insect in-

 

e If»:
“or";
w _ -1. “11*

Fig. 2A. A naturally infected Chippewa tuber
(Schoenfeld stock) planted in the
greenhouse showing wilting typical
of ringrot.

 

 

 

Fig. 23. Chippewa potato plant showing wilt symptoms
47 days after stem inoculation using a pure
culture of Phytomqggg secedonica.

Control plant on the left.

 

if.

,Q,.4
.

.4...
~”

,5..

Juries may be prevalent and when the vines begin tO show
signs of maturity. Fusarium wilt and blackleg also may
cause confusion Of diagnosis. Consequently this masking
Of the true symptoms makes field detection of ringrot ex-
tremely difficult
Tuber

On digging an infected plant, usually all stages of
decay are present in the tubers - from complete decom-
position tO apparent soundness (Figs.5,4,& 5). In the
early stages only the vascular tissue may be affected
by a soft, yellow creamy, or more Often, crumbly rot.
Upon clipping and squeezing the stolon end Of such a
tuber, the decaying but odorless vascular tissue crumbles,
showing the consistency of a cooked potato. At the same
time pressure separates the cortex from the vascular ring.

When secondary soft-rotting bacteria gain entrance,
the Whole tuber rapidly disintegrates into a foul-smelling,
slimy mass (Fig. 6). Numerous less destructive secondary
invaders such as the Fusaria and saprophytic bacteria,
enter the ringrotted tuber in the later stages, more or
less obliterating any typical ringrot symptoms (Fig.5 ).
At times the vascular decay spreads to the center Of the
tuber, leaving only the outer shell, a portion Of which
may be decayed. This is particularly true Of potatoes

in storage.

 

 

 

Fig. 5. Cut tubers showing light infection of the
vascular tissue. Such lightly infected

tubers easily escape detection by visual
examination. ‘

 

 

 

Fig. 4. Advanced decay following ringrot infection.

 

-v-..—.—.~.

 

 

V

Fig. 5. Internal tuber decay following: severe
ringrot infection and invasion by
secondary organisms.

 

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Fig. 6.

Outward appearance of a tuber Showing
advanced decay of the stem end due to
ringrot infection. Cracking of the
tuber permits the entrance of various
secondary soil organisms which hasten
decay.

In the spring the more severely infected tubers in
the bin fail to sprout, the eyes appearing blackened

and dried (Fig. 7)

Diagnosis

Gram's Method Of Staining

Since diagnosis by field inspection is difficult,
it is important that some reliable laboratory method
be available. The Gram stain answers this purpose.
The suspected tuber or stem tissue is smeared on a
microscopic slide, stained by Gram's method and ex-
amined under the Oil immersion Objective. If the smear
reveals an abundance of Gram positive rods appearing
singly, in pairs, and, less Often, in short chains,
0.3 - 0.5 x 0.6 - 1.0 microns in size, then the presence
of ringrot in the stock has been established. Gram pos-
itive bacteria larger than one micron are Of no signifi-
cance in the diagnosis as they are harmless soil bacteria.
This procedure, as Outlined by Racicot, Savile, and Con-
ners (16) has been found to be highly satisfactory.

Often badly decomposed samples are sent to the
laboratory by growers who request a diagnosis for ring-
rot infection. On staining a sample of this kind, such
a mixed bacterial flora is seen under the microscope

that accurate diagnosis is quite impossible

 

 

 

Fig. 7. Outward appearance of ringrot decay.
Infection begins at the stem end and
gradually progresses toward the eye
and of the tuber.

 

 

as is also the isolation of the pathogen. If care is
taken in collecting samples to select, insofar as is

possible, only slightly infected tubers, a clear-cut

picture of the pathogen is Obtained on staining.

The Ultra-Violet Light

The introduction by Iverson and Kelly (17) Of the
ultra-violet light as a method of identifying potato
tubers free from ringrot has facilitated tuber exam-
inations. The method is rapid and permits the inspec-.
tion Of the entire vascular ring rather than a small
portion as in the case Of the staining method. The
greenish fluorescence emitted by ringrotted tuber tis-
sue On exposure to a G.E. 8H4 ultra-violet lamp consti-
tutes the diagnostic basis for the detection Of the
disease. Examinations are made at storage temperature
(40°F. or lower) in a totally dark room.

In limited tests conducted by the writer, several
tubenswhich had passed the macroscopic examination as
"healthy" showed a slight silvery-green fluorescence
Of the vascular tissue under the ultra-violet light.
To these tubers the Gram stain test was applied and
gave positive results, indicating that ultra-violet
light is successful in detecting infections that are
easily overlooked by examination under ordinary light.

In other instances tubers showing a suspicious greenish

-10-

fluorescence under the ultra-violet lamp gave negative
results upon Gram staining.

Unfortunately, satisfactory results can be Ob-
tained only after considerable experience and even
then it is difficult to distinguish the greenish
fluorescence of ringrot from other similar fluores-
cences which are encountered (15). This method is
valuable in making rapid preliminary checks but it

cannot replace the staining method.

Etiology
Proof Of Pathogenicity

The writer's first isolation Of the causal organ-
ism Of ringrot was from tubers furnished by C. H.
Metzger of Colorado. Proof of pathogenicity was es-
tablished as demanded by Koch's postulates. Young
potato plants growing in the greenhouse were inocu-
lated by placing a drop of the bacterial suspension
on the base Of the stem and pricking it in with a
needle.

Six weeks later the first symptoms, that is,
slightly upward rolling Of the leaves and partial
wilting Of the shoot, appeared and the organism was
isolated both from the stem and from the daughter
tubers using the dilution plate method. These re-

isolates were used tO inoculate another set Of potato

-11-

plants and typical symptoms were again produced, this
time in seven weeks.
Description of the organism

Phytomonas sepedonigg, the causal agent of potato

 

 

ringrot, is a short, Gram positive,non-motile, non-
encapsulated, non-spore-forming rod, averaging
0.3 - 0.5 x 0.6 - 1.0 micron in size. It is aerobic,
occurs singly, in pairs, and in short chains, and grows
best at 21°C. On potato dextrose agar small pin—point
colonies appear in about 10 to 12 days, while on the
special medium devised for this organism by Burkholder
(11) colonies appear a light yellow; 1 to 2 mm. in
diameter, smooth, glistening, circular, and slightly
raised. Gelatin is not liquified and growth in Burk-
holder's broth is cloudy, producing a granular sedi-
ment, but not producing a pellicle. Growth on cooked
potato is slimy and brownish. In litmus milk there
is growth with slight reduction of litmus but no clear-
ing. Starch hydrolysis is slight. No gas is produced.
Life history of the causal organism.

The pathogen overwinters normally in diseased
tubers, in storage. In the spring the slightly in-
fected tubers easily escape detection and are planted,

giving rise to a diseased crop of daughter-tubers.

All available evidence appears to indicate that the

-12-

organism cannot overwinter in the soil in Maine (18),
North Dakota, Florida, and California (15). Eide (15)
has shown that the ringrot organism may overwinter on
the sacks. It is likewise probable that storage bins
and implements which come in contact with infected stock
may still be contaminated with the virulent pathogen
even after three to four months.
Cultural Characteristics

In pure culture the bacterium was found to grow
poorly on all ordinary media. Nutrient agar gave no
growth, beef-extract-peptone agar supported a sparse
growth, while potato-dextrose agar supported a fair
growth but the organism.was found to lose its viability
rapidly on this medium. -Growth was materially en-
hanced on addition of.2% asparagine. Burkholder's
(11) medium, developed specifically for this organism,
supported a luxuriant growth and viability was main-
tained by transferring the cultures every two weeks.
Virulence, however, was found to diminish to a greater
or lesser extent on all media, including Burkholder's.
Workers in Colorado, Oregon, and West Virginia agree
that in pure culture the organism loses some of its
disease-producing power (15). On the other hand, work-
ers in California and Beltsville (15) report a high

percentage of successful pure-culture inoculations.

-15-

Metzger, Click, and Kreutzer (20) report that a
mixed culture from partly decomposed tubers appears
to be more virulent than a pure culture. In the field
experiment conducted by the writer in the summer of
1940, but 17 infections out of 150 pure culture inocu-
lations were obtained. Dorrel, Marten, and Leach (19)
report the same experience in their 1940 field experi-
ments. They conclude that "the results raise some
question as to the methods by which seed stocks be-
come infected."

Two factors which may be in part responsible for
the small percentage of infection resulting from pure
culture inoculations are l, diminished virulence caused
by growth on artificial medium; and 2, a synergistic
relationship existing between Phytomonasvsepedonica
and the various secondary soft-rotting organisms.

Inoculation tests carried out in the greenhouse
seem to indicate that both factors may affect the viru-
lence of the pathogen. Eight young tomato plants and
an equal number of.potato plants were inoculated with
an actively-growing, week-old agar slant suspension
by pricking the base of the stem. Some time later
another set of these plants was inoculated as previously
except that a suspension of the soft, cheesy vascular
tissue from an infected tuber was used as the inoculum.

The results are reported as follows:

-14-

Table l. Inoculations of Potato and Tomato Using Pure
Culture and Inoculum from Infected Tuber

Number of Plants Infected

 

 

 

Host
Pure Culture Infected Tuber
Severe Medium None Severe Medium None
Tomato 0 O 8 5 3 0
Potato 2 5 l 6 2 O

 

It should be stated that in subsequent inoculations
of tomato plants using freshly isolated cultures infec-
tion resulted. However, in the above inoculations no
tomato infection was obtained. The first symptoms ap-
peared on the potato plants after 42 days when pure
cultures were used. On the other hand, when inoculum
from an infected tuber was used, all of the tomato
plants became infected, the first symptoms appearing
after only 25 days; the potato plants in this series
showed symptoms after 55 days.

Experiments conducted in Colorado (15) have shown
that when the soft-rotting organism, Erwinia carotovora
was used to inoculate a tuber already infected with
ringrot, a serious and rapid decay followed; whereas
when this soft-rotting organism was used to inoculate
a healthy tuber, no infection developed.

In order to observe this phenomenon and to test

the Gram negative contaminant frequently occurring in

-15-

the isolation plates, the following experiment was run:
Healthy and ringrotted tubers were disinfected,

sliced aseptically, and the slices placed in sterile

petri dishes. The treatments and results are given

in the table below:

Table 2. Effect of Several Organisms on Healthy and
Ringrot Tuber Tissue

 

Amount of Decangroduced after 5 Dayg

 

 

 

 

Healthy Ringrotted
Inoculum Tuber Slices Tuber Slices
Complete
1. Erwinia carotovora* Slight Disintegration
2. Phytomonas sepedonica* None Slight
Complete
5. Gram negative Contaminant* Slight Disintegration
4. Check (5 c.c. water sterile) None Slight

 

* 5 cc. of 5 day old broth culture

The results show a pronounced acceleration of decay
when the soft-rotting bacteria were added to ringrotted
tubers. The cause of this phenomenon is not clearly
understood. It may be that a combination of enzymes
is essential to produce the severe type of tuber decay.

The idea of synergistic associations among plant
disease pathogens is by no means a new one. Erwin F.

Smith (21) points out that Phypomonas campestris, the

 

causal agent of black-rot of crucifers, and Phytomonas

 

solanacearum, causal agent of brown rot of potatoes,

 

are often aided by soft white rots.

~15-

Fawcett (22) has appealed to plant pathologists
to investigate more thoroughly mixtures of organisms.
He states, "This necessary insistence on pure cultures
of single organisms has led, perhaps unconsciously, to
a feeling that to allow the use of a mixed culture in
plant pathological work is extremely unscientific if
it is not actually a 'deadly plant pathological sin'...
We are not, it would seem, getting the whole story by
working with pure cultures of single organisms. Nature
does not work with pure cultures alone but most fre-
quently with associations...Pure cultures we must, of
course, continue to use as a basis for the known mix-

tures and as controls on the activity of the mixtures."

Pathological Histology

Histological sections of infected stems were pre-
pared using chrom-acetic killing solution. Dehydration
and infiltration were carried out in the usual manner.
Sections 10 microns thick were cut on the microtome
and stained according to Jones' method GB).

The sections prepared from infected stems revealed
that the entire vascular bundle ring is seldom invaded
by the bacteria. In cross section there are frequently
‘one or more bundkxawhich appear to be free of the in-
vading bacteria. The number of bacteria present in

the vessels varies greatly. Some of the vessels con-

-17-

tain only a small number of organisms while others are
occupied by masses that completely occlude the lumen
(Figs. 8,9). Moreover, completely occluded vessels
exist side by side with vessels which to all appear-
ances are entirely free of bacteria. None of the slides
show any infection of the parenchymatous tissue adjacent
to the vessels of the stem.

Invasion of the tuber is primarily vascular. In
time the bacteria escape from the vessels into the ad-
joining intercellular spaces of the parenchyma, caus-
ing a separation and breakdown of this tissue (Fig. 10).
As this breakdown continues, the tissue of the vascular
ring assumes a characteristic yellow, cheesy aspect.

Often the infection does not extend for more than
1 or 2 centimeters in depth from the stem end of the
tuber. When such a tuber is planted, Sprouts origin-
ating from the non-infected end may develOp their own
root system and by rotting of the seed piece may become
independent of the mother tuber before the infection of
the seed piece advances to the point where the young
sprouts become infected. This apparently explains the
occurrence of healthy and diseased stems and tubers in

the same hill.

 

 

 

 

Fig. 8A. Cross section of a potato stem in the
vascular region showing vessels occupied
by Phytomongg sepedonic‘.

b.:~‘. g-. ,

 

 

 

Fig. 8B. A portion of the same section as in
Fig. 10A as viewed under oil immersion
showing various degrees of bacterial
occlusion of the vessels.

 

 

 

;

Fig. 'QAJ Longitudinal section thru an inoculated
. potato stem showing the dense masses of
Phytomonas sepedonica restricted to the

vessels. .

 

 

 

Fig. QB. A detail from Fig. J9A showing the
individual bacteria of the occluded
vessel.

 

 

.
. .f .\., ..q .
.. . .-.. :.,_.r»~¢v
. .
. . a... r
a.) Ix
. . .6. .2.
. . . 4
.
.1 .
....
.

33.,

5‘
‘. ~" 1.

 

 

 

 

 

Fig. 10A. Section of a tuber infected with ringrot
shewing masses of bacteria in the inter-
cellular spaces of the parenchyma.

 

Fig. 10B. A portion of the same section as in Fig. 10A
but under 011 immersion to show the individual
bacilli.

-18-

1940 Field Experiments

I. Disinfectants and Control

A field experiment was run for the purpose of de-
termining the comparative bactericidal value of several
disinfectants. Because no naturally infected stock was
available at the time, pure cultures of the causal or-
ganism were used to inoculate healthy tubers. The path-
ogenicity of these cultures had been established several
months previously by stem inoculations of potato plants
growing in the greenhouse.

To week-old sub-cultures on agar slants 5 c.c. of
sterile tap water were added to make a turbid bacterial
suspension. The tubers were inoculated by dipping the
flamed scalpel into this suspension and then cutting the
seed piece.

One lot of inoculated tubers was treated in various
disinfectants and planted the day after inoculation,
while a second lot was stored for four days before treat-
ing to determine the effect of suberization of the cut
surface on the incidence of the disease.

The plots were under observation for foliar symp-
toms throughout the summer. Stems of suspected plants

were cut from time to time, smears made on clean slides,

stained, and examined. Only two diseased hills were

detected in this manner. At harvest time the hills

-19-

were dug by hand and the tubers were examined by clip-
ping the stem end.

Because of the small number of infected hills,
the tubers from the individual plots were bagged and
stored for approximately four months to allow any
slightly infected tubers to develop a more advanced
decay.

At this second examination no increase in the
amount of infection could be observed after a careful
scrutiny in which all suspicious tubers were stained
and examined under the microscope.

The results are indicated in the following table:
Table 5. Seed Treatment Experiment with Lot of

Inoculated Chippewa Potatoes Treated

Immediately after Cutting and 4 Days
after Cutting.

 

Number of Hills Infected
Treated Treated
Disinfectant Immediately After 4 Days

 

 

Improved Semesan Bel*
Dowicide A. l-lOOO
Dowicide A. l-2000
Corrosive sublimate 1-500
Acid mercurysa

Check - inoculated

Check - not inoculated

 

()1

(J1
0010 00100000

Total no. inoc. checks
Total no. infected checks
4% successful inoculations

CDCDO OCDOOOOO

l-'
H

 

* 1 pound to 7% gallons water
** Hg 012 1-500 + 1% Hcl

All treatments applied for 15 minutes

-20-

Because of the small amount of successful inocula-
tion, the comparative value of the disinfectants tested
or the effect of suberization could not be established.
It is interesting to note that in a similar experiment
run concurrently in West Virginia by Dorrel, Marten,
and Leach (19), the same experience is reported. They
state that the amount of successful inoculation was
"extremely small."

However, the results raise some question concern-
ing the effect of artificial culture upon the virulence
of the organism. Likewise the question of virulence in
mixed versus pure culture is brought to the fore. These
questions are discussed further under Cultural Charac-

teristics.

Penetration of Dowicide A into Tuber Tissue

Because the infection of the ringrot-diseased tuber
often affects the vascular ring tissue of the entire tuber,
it was decided to determine the depth to which Dowicide
A will penetrate into the cut surface of halved tubers,
and to determine also whether the disinfectant is germi-
cidal at different depths of penetration.

Accordingly, healthy and diseased tubers were cut
in half and soaked in the disinfectant. Depth of pene-
tration was established by using a spot test technique

as outlined by G. R. Anderson of The Dow Chemical Com-

pany. The following formula was applied:

Reagents (A) Dissolve'0.05 grains of 2,6 Dibromo
quinone chloroimide in 10 ml. ethyl
alcohol. Prepare fresh daily.

(B) Buffer. Dissolve 15 grams anhydrous
sodium tetraborate in 900 m1. of dis-
tilled water; add 5.27 grams sodium
hydroxide in the form of a strong
solution, and make up to one liter.

Procedure Put a drop of solution "A" on the
cut surface of the potato. Then
put a drop of buffer on the same
spot. A blue stain shows the pres-
ence of Dowicide A.

 

Successive thin slices were cut from
the surface of the treated tubers and
the spot test applied until the freshly
exposed surface no longer gave a posi-
tive reaction.
In addition, diseased tissue from various depths
of the treated infected tubers was plated to determine
germicidal action of the disinfectant. The results

follow:

-22-

Table 4. Depth of Penetration of Dowicide A into
Tuber Tissue of Potato

 

Plate Readings
Dilution Time Penetration Depth Growth

 

 

 

 

 

(mine) (We) (m‘)
l-lOOO 15 5 Surface -
l-lOOO 50 7 Surface -
l-lOOO 45 7 2 +
l-lOOO 6O 10 5 +
1-2000 15 2 Surface +
l-ZOOO 5O 4 2 +
l-2000 45 5 2 +
l-2000 60 8 5 +
l-SOOO l5 1 Surface +
l-SOOO 50 2 l +
1-5000 45 2 1 +
1-SOOO 50 5 2 +
1-4000 15 Less than 1 Surface +
1-4000 50 Less than 1 Surface +
l-4000 45 1 Surface +
1-4000 60 3 2 +

 

The results indicate that the usefulness of this
disinfectant is limited to surface disinfection. The
lowest dilution, l-lOOO, was found to be effective at
the surface but the pathogen was not destroyed at a
depth of 7 mm. even though the spot test showed that
the disinfectant had penetrated to a depth of 10 mm.

It appears that the action of the disinfectant may have
been neutralized by the organic matter of the potato
tissue, or diluted by the water content of the tuber.

Penetration into the diseased vascular tissues

of infected tubers was not perceptibly greater than

-25-

in the case of healthy vascular tissue. In no case was

any penetration through the intact periderm observed.

II. Spread of the Disease in the Field

To gain some idea as to the amount of spread in
the field and the manner in which this spread takes
place, alternate hills of healthy and inoculated tubers,
50 of each, were planted in a row on well-drained sandy
loam. On each side of this row healthy check rows were
planted. No above-ground symptoms could be observed
during the growing season. At harvest time the hills
were dug by hand and the tubers were examined macro-
scopically by clipping the stem end. Out of the 50
inoculated seed pieces which were planted, only four
diseased hills were produced or 8%. As in the preced-
ing experiment the healthy and inoculated tubers were
bagged separately and stored for four months. Again
no increase in disease could be ascertained on this
second examination after a careful check, indicating
that the inoculations did not produce infection. N0
disease was found in any of the tubers originating from
healthy seed pieces.

It is concluded that, insofar as the experiment is

capable of revealing, no field spread took place.

-24-

Dissemination

Undoubtedly the chief means of dissemination of
ringrot from one state to another is through ship-
ment of infected seed stock. Both of the serious
outbreaks in Michigan in the summer of 1940 origin-
ated from Maine seed. Drastic steps are being taken
by the certification industry to insure the shipment
of ringrot-free seed to other states by eliminating
ringrot from their own seed plots.

Spread in the field occurs in alarming amounts
by means of the cutting knife. Experiments in Cali-
fornia have demonstrated that a knife contaminated by
cutting through a diseased tuber is capable of trans-
mitting ringrot to the 24th healthy tuber cut immed-
iately thereafter. When the picker type planter is
used, two to three times the original amount of ring-
rot may result (15).

Contaminated bins, containers, and.graders are
known to be responsible for spreading infection (14).

Bonde (23) and Metzger and Brinkley (24) have
advanced experimental evidence showing that some spread
occurs through the agency of soil water.

Contact of healthy and diseased tubers in the bin,
in sacks, or in crates likewise has been shown to re-

sult in substantial spread (15).

-25-

All attempts to transmit the disease by insects
have been unsuccessful. In North Dakota (25) care-
fully planned experiments to determine possible dissem-

ination by grasshoppers yielded negative results.

Varietal Resistance

In Wyoming (15) 25 commercial varieties were
tested for resistance to ringrot and all were found
to be susceptible. However, Russet Rural was found
to be significantly more resistant than the Katahdin
(or Chippewa. Irish Cobbler and Bliss Triumph were
among the most susceptible.

These workers tested 540 seedlings for resis-
tance and out of this number 28 seedlings showed no
symptoms of ringrot. These were saved for further
testing.

Bonde (20) tested 145 seedlingsbut found all of
them to be susceptible.

The development of resistant varieties is an im-
portant phase in the control program. Preliminary re-

ports of resistance are distinctly encouraging.

Control
Primary controls
I. Eradication of ringrot infected stock appears

to be the most promising method of complete control.

Since the main source of infection is the infected seed
tuber, and since there is little evidence of the organ-
ism overwintering in the soil, it is apparent that the
weakest link in the life cycle of this pathogen is the
overwintering phase, and that an attack on the disease
from this angle offers the most hopeful prospect of
control. Exclusive use of ringrot free seed stock is,
therefore, of supreme importance as is also the strict-
est observance of the zero toleration by all certifying
agencies.

The following method is proposed for selecting a
disease-free lot of seed:

1. Disinfect the whole tubers by dipping
in l-lOOO Hg012 for 10 minutes.

2. Clip the stolon end of each tuber and
examine macroscopically. Discard all
tubers exhibiting any vascular dis-
coloration and disinfect the cutting
knife in HgCl 1-500 for 5 seconds
after each cu .

5. Examine under the ultra-violet light
all tubers passing the macroscopic
inspection. This should be done at
temperatures of 40°F or lower and in
a totally dark room. Discard all
tubers showing a greenish fluores-
cence in the vascular region and also
all suspicious or doubtful tubers.

4. From.each of the tubers surviving the
first two tests cut a slice contain-
ing a portion of the vascular ring
and press it lightly on a numbered
slide. Place the tuber into a cor-
respondingly numbered paper sack.

Disinfect the knife with each new
cut and wipe the blade off with a
small wad of absorbent cotton, using
fresh cotton each time.

5. Fix smears by passing the slide through
the flame several times and then stain
by Gramls method as outlined by Racicot,
Savile, and Conners (16).

6. Examine slides under microscope using
the oil immersion objective. Dis-
card all tubers which yield slides
displaying the morphologically typical
Gram positive bacilli.

7. Tubers yielding smears which appear to
be negative may be considered "ringrot-
free". These should be planted in
tuber units in a field which is well
isolated from other potato fields and
which has not been planted to potatoes
the previous year. Inspect and rogue
the plot at frequent intervals during
the summer.

8. At the time of digging cut a stem at
the base from each hill and press the
cut surface on a numbered microscopic
slide. Dig each unit separately and '
tag to correspond to the slide number.

9. Fix, stain, and examine smears. Dis-
card all units from which any smear
reveals morphologically typical bac-
teria.

10. The remaining tubers may be used for
increase, the following season under
supervision of the certification ser-
vice.

On farms where ringrot is present, all equipment,

such as cutting knives, containers, planter, etc.,

should be disinfected before handling clean seed.

-28..

Two pounds of copper-sulphate to ten gallons of water
or one pint of 40% formaldehyde to fifteen gallons of
water may be used.

This is admittedly a laborious and time-consuming
process but pending the development of a more rapid
and less cumbersome method the production of ringrot-
free seed stock should be expedited with the existing
methods.

II. The breeding of resistant varieties has
indications of becoming an important factor in eventual
control as was pointed out under Varietal Resistance.
This is a long-time proposition and should be carried
on to supplement the eradication program and to hasten

the realization of the objectives of eradication.

Secondary Controls

Under this heading are controls which do not
eliminate ringrot from seed stock but which merely
prevent further spread. These methods should be of
interest only to growers of table stock who have pur-
chased the best seed possible but in which some ring-
rot is present. Seed treatment cannot be recommended
to certified seed growers to control this disease
unless it is to prevent accidental infection from

outside sources.

-29-

1. Treat cut seed for 10 minutes with
l-lOOO HgClg, Acid mercury (HgClg
1-500 + 1% Hcl), or formaldehyde,

1 pint to 50 gallons of water.

2. Plant whole tubers. This eliminates
the spread of the disease by the cut-
ting knife but obviously does not

eliminate the disease from the seed
stock.

SUL‘IMARY

Bacterial ringrot is a serious new disease of
potatoes. It was first reported in the United States
in 1957 by Bonde of Maine, and since then has been re-
ported from 37 other states. It has been known in
Germany since 1908.

In Michigan, where the disease has been reported
in 11 counties, losses have been small. However, the
disease constitutes a potential threat to the potato
industry and the potato certification program, hence
its importance cannot be overestimated.

All commercial varieties of Irish potato are
susceptible. In addition, the tomato develops typical
symptoms upon artificial inoculation.

Foliar symptoms are variable and come late in the
season when early and late blight and insect and drought
injury may be prevalent. Masking of ringrot by symptoms

of other diseases and injuries makes detection difficult.

-30-

Of more diagnostic value is the soft, yellow, crumbly
vascular ring tissue of the infected tuber.

The Gram stain serves as a reliable laboratory
method for identifying diseased tubers. The recent
introduction of the ultra-violet light test has facili-
tated detection of infected stock.

Phytomonas sepedonica, the causal agent, is a
short, non-motile, Gram positive rod, measuring
0.5 - 0.5 x 0.6 - 1.0 microns.

The bacteria overwinter primarily in diseased
tubers in storage. Available evidence indicates that
the organism does not overwinter in the soil except
on volunteer plants.

In field experiments conducted during the summer
of 1940, inoculation of healthy tubers with pure cul-
tures resulted in a surprisingly small percentage of
infection. Diminished virulence of the bacteria follow-
ing growth on artificial media and a possible synergis-
tic relationship between Phytomonas sepedonica and
secondary soft-rotting bacteria which, of course, were
absent from the pure culture, may in part account
for the small amount of successful infection.

The chief means of geographical dissemination is

through the shipment of infected seed stock from one

state to another. Spread in the field occurs mainly
by means of the cutting knife and the picker-type
planter. No insect vector has been found.
Eradication of infected stock appears to be the
most promising method of control. Minor controls
such as seed treatment and planting whole tubers
serve to prevent spread but obviously do not elim-

inate the disease from the stock.

l.

2.

3.

5.

6.

7.

10.

11.

Literature Cited

Stuart, h. The Potato. J.B. Lippincott Company,
New York. 1957.

U.S.D.A. and Michigan Department of Agriculture.
1940 Crop Report for Michigan.

Moore, H.C. Better Potatoes for Michigan. Mich.
Ext. Bul. 49. 1959.

Bonde, Reiner. A Bacterial Wilt and Soft Rot of
the Potato in Maine. Phytopath.
27:106-108. 1957.

Savile, D.B.O. and H.N. Racicot. Bacterial Wilt
and Rot of Potatoes. Sci. Agric.
17:518-522. 1957.

Bonde, Reiner. Bacterial hilt and Soft Rot of
the Potato in Maine. Maine Agric.
Exper. Sta. Bul. 596.

Leach,J.G., et al. Report of the Committee to
Coordinate Research on New and Unusual

Potato Diseases. Amer. Potato Jour.
17:81-88. 1940.

Spieckermann, A. Ueber eine noch nicht beschriebene
bacterielle Gefasser Krankung der Karto-
felpflanze. Centralb. f. Bakt. pp. 205-
208. June 1, 1910.

Smith, E.F. An Introduction to Bacterial Diseases
of Plants. pp. 219-222. W. B. Saunders
Company. Philadelphia, 1920.

Conners, I. L. Canad. Plant Disease Survey. Ann.
Report. 11:49. 19320

Burkholder, W.H. The Occurrence in the U.S. of
the Tuber Ringrot and Wilt of Potato.
Amer. Potato Jour. 15:245-245.

12. Muncie, J.H. Occurrence of Bacterial Ringrot on

Potato in Michigan. U.S.D.A. Bur.
Plant Indus. 24:6. 1940.

15.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Eddins, A.H.

Some Characteristics of Bacterial
Ringrot of Potatoes. Amer. Potato
Jour. 16:509-522. 1959.

Brentzel, W.E.Bacteria1 Ringrot, a new disease of

Dykstra,T.P.

Racicot,H.N.,

potatoes. Bimo. Bul. North Dakota
Agric. Exper. Sta. 2:12-14. 1940.

Results of Experiments in Control of

Bacterial Ringrot of Potatoes in 1940.
(A compilation). Amer. Potato Jour.
18:27-55. 1941.

D.B.O. Savile, and I.L. Conners.
Bacterial Wilt and Rot of Potatoes -
Some Suggestions for Its Detection,
Verification, and Control. Amer.
Potato Jour. 15:512-518. 1958.

Iverson,V.E. and H.C. Kelly. A New Method of

Bonde, R. and

Dorrel, w.w.,

Dykstra,T.P.

Smith,E.F.

FaVJcett, HOS.

Identifying Potato Tubers Free from
Bacterial Ringrot and Other Types
of Tuber Decay. Montana Agric.
Exper. Sta. Mimeo. Cir. 20.

O.L. Wyman. Potato Ringrot. Maine
Agric. Exper. Sta. Ext. Bul. 286.
1941.

E.A. Marten, and J.G. Leach. Bacter-
icidal Efficiency of Sodium Hypochlorite
and Other Chemicals against Phytomonas
sepedonica. Phytopath. 51:7.

 

 

Results of Experiments in control of
Bacterial Ringrot of Potatoes in 1959.
U.SoDvo Euro or Plant Indus. 19400

Bacteria in Relation to Plant Diseases.
Carnegie Institution of Washington.
Washington, D.C. 1:72-75.

The Important of Investigations on the
Effects of Known Mixtures of Micro-
organisms. Phytopath. 21:545-550.
1951.

25.

24.

25.

Bonde, R. Bacterial Wilt and Soft Rot of the
Potato . Amer. Potato Jour. 16:109-
114. 1959.

Metzger,C.H. and A.M. Brinkley. Some Evidence
on the Spread of Bacterial Wilt.
Amer. Potato Jour. 17:198-201. 1940.

Brentzel, W.E. and J.A. Munro. Bacterial Ringrot
of the Potato: Investigation on Pos-
sible Dissemination by Grasshoppers.
North Dakota Agric. Exper. Sta. Bul.
295. 1940.

Jones, F. R. Winter Injury in Alfalfa. Jour.
Agric. Res. 57:189-211. 1928.

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