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STUDIES UPON THE BLACKLEG DISEASE OF THE
POTATO, WITH SPECIAL REFERENCE TO THE RELA-
TIONSHIP OF THE CAUSAL ORGANISMS

By W. J. Morse,
Plant Pathologist, Maine Agricultural Experiment Station
HISTORICAL REVIEW

The fact that the potato (Solanum tuberosum) is subject to maladies
like that under consideration in this paper was noted at a comparatively
early date in the literature of bacterial diseases of plants. De Jubain-
ville and Vesque (8) mentioned in 1878 a ‘“cellular-rot”’ of potatoes,
radishes, carrots, and beets as occurring in the soil and in cellars; but
bacteria are not mentioned as the cause. Their paper and the one
following are reviewed by Smith (32).

‘In 1879 Reinke and Berthold (30) pointed out quite clearly that a
-wetrot of potatoes could occur without the presence of any fungi.
According to Smith, they apparently did not have any active parasite
and did not work with pure cultures. However, they were able to
inoculate and cause a decay of healthy tubers by means of the watery
fluid taken from diseased potatoes, provided especially favorable condi-
tions were supplied in the line of moisture. They were also able to
demonstrate the constant association of bacteria with the wetrotting
of the tubers.

Prillieux and Delacroix (28) described a disease of the potato stem
from France in 1890 and gave the name “Bacillus caulivorus”’ to the
organism which they considered to be the cause. Nothing is said in
this paper as to the isolation of the organism and the growth of it in

pure cultures, although later Prilliewx (27, V. 1, Pp. 16) credits it with
producing a green coloration of certain culture media. Prunet (29), who
reviewed the literature on this subject in 1902, called attention to the
fact that Laurent (21) had stated in 1899 that B. caulivorus was prob-
ably nothing other than B. fluorescens liquefaciens Fliigge, a common
soil saprophyte. Pethybridge and Murphy (26), who have more recently
written on the subject, state that—

Later on Delacroix, in dealing with B. caulivorus, speaks of it as most probably
identical with Bacillus fluorescens liquefaciens Fliigge, a common saprophytic form,
which, he suggests, may perhaps under certain special conditions become parasitic.

It is evident that Prillieux and Delacroix (28) did not secure the
organism responsible for the disease. However, there is every reason
to believe that they were concerned with a malady similar to that under

1 Reference is made by number to “‘ Literature cited,” pp. 124-1 26.

Journal of Agricultural Research, Vol. VIII, No. 3
Washington, D.C. Jan. 15, 1917
2V Key No. Me.—9

(79)

80 Journal of Agricultural Research Vol. VII, No. 3

consideration, for it was characterized by a decay of the stem beginning
at the base and extending upwards.

It is to Smith (31) that we owe our first tnorough study and careful
description of a bacterial disease of the potato stem and tuber. How-
ever, the disease, caused by Bacillus solanacearum Smith, belongs to a
distinctly different type than blackleg.

As far as the writer has been able to discover, Frank, in Germany,
(13, 14) appears to be the first writer definitely to connect the blackleg
disease or ‘“‘Schwarzbeinigkeit”’ of the potato stem and the accompanying
wetrot of the tuber with a bacterial parasite. It is evident that the dis-
ease had been known and recognized there for some time, but it had
been associated with various fungi. Frank’s description of Schwarz-
beinigkeit is definite and clear, and it agrees very closely with certain,
but not all, of the characters of the blackleg as observed by the writer
in Maine, Vermont, New York, and in some of the Middle West and
Rocky Mountain States.

The organism, to which the specific name ‘Micrococcus phytophthorus”
was applied in the second paper, was found to be constantly associated
with the disease and capable of producing it when inoculated into
healthy stems. It is interesting to note that all other organisms described
as causing a similar type of disease are bacilli and are of greater length
than the diameter, 0.54, which Frank gave for the cocci he described.
In this connection, however, it may be mentioned also that Frank
stated that for four years material studied by him from different regions
invariably showed organisms of the type he described.

In 1901 Delacroix (9, 11) described what appears to be still another
type of bacterial disease of the potato stem for which was suggested the
name “‘brunissure,’’ or browning. He gave the name “Bacillus solan-
icola”’ to the causal organism. As in the case of blackleg, the first visible
signs of disease are a yellowing of the foliage and cessation of growth, fol-
lowed by the gradual death and drying up of the plant. The stems die
from the base upward, but are described as browned rather than black-
ened; and cross sections show transparent brown patches, which may
extend some distance up the stem. A prominent characteristic is the
development of a gummy material and tyloses in the wood vessels of the
stems of attacked plants.

It is evident that this disease, like that caused by Bacillus solanace-
arum, is of a type entirely distinct from blackleg and therefore need not
be considered further, especially since cultures of the organism are no
longer available for comparison. It may be mentioned, however, that
Delacroix considers it to be a soil organism, and the chief source of
infection is from the soil by means of wounds. Tubers are also attacked,

the disease entering from the stem end and causing a browning of the

tissues.

Jan. rs, 1917 Blackleg Disease of Potato 81

In 1902 Van Hall (15) in Holland described a bacterial disease of the
potato which was similar to that described in Germany by Frank. He
named the causal organism “‘Bacilus atrosepticus.” ‘This will be con-
sidered more in detail in the discussion of the organisms studied by the
writer.

From 1902 to 1906 Appel published a series of several papers (1-7)
upon his studies of the Schwarzbeinigket as he had observed it in Germany.
His most complete account of the disease and the description of the
organism was published in 1903 (4). He expressed a view at that time
that possibly more than one organism might be associated with the dis-
ease and suggested that the term ‘Schwarzbeinigke:t’”’ should not be
considered as applying to a specific disease but as descriptive of a patho-
logical symptom. He also mentioned that not all organisms studied by
him which were able to cause decay of the tuber were capable of pro-
ducing the stem disease as well. However, he pointed out that the
organism causing Schwarzbetmgkeit, which he studied, was probably
identical with the one described by Frank. This appears to be the most —
generally accepted view, although Appel found it to be a bacillus. He
published a fairly complete account of it and named it “Bacilus phy-
tophthorus.”” Smith (33) has more recently repeated and extended
Appel’s studies, working apparently with his original culture, and his
description of B. phytophthorus is the most complete one that we have.

In a general account of potato diseases and the disease resistance of
potatoes, Jones (19), in 1905, describes blackleg and summarizes his
observations upon its occurrence in Europe. He mentions it as being
found in Germany, Holland, Belgium, France, and England.

In 1906 Delacroix (12) reported on the presence of the Schwarzbeinig-
keit in France and compared the appearance of plants affected with the
latter and those showing “‘brumssure.’’ He states that blackleg is
primarily a disease of the late spring or early summer, while the malady
caused by Bacillus solamicola, as a rule, appears in midsummer or early
autumn. However, he says, in effect, that the exterior characters are
such that one can hardly separate the two diseases by them.

In the same yeag Johnson (18) stated that he had clear evidence of
the existence of Bacillus phytophthorus as a general cause of ‘‘yellow-
blight,’ blackleg, and potato tuber-rot in Ireland, but Pethybridge and
Murphy (26) are quite inclined to doubt his evidence.

Jones (20) records for the first time the occurrence of blackleg in the
United States, also in 1906. The disease agreed in every respect with
that which he had observed in Europe. This was in Vermont on a farm
where he had been studying potato diseases for nearly 20 years and had
not previously observed it. The seed tubers used for planting came
from Maine.

In 1906 Harrison (17) described a potato disease of the same type
which he stated to be of wide distribution in Canada and of much eco-

82 Journal of Agriculiural Research "Vol. VIII, No.3

nomic importance there. He regarded the causal organism as a new and
distinct species to which he gave the name “Bacillus solanisaprus.”
His study was the most detailed and complete of any which had
appeared up to this time upon an organism associated with blackleg.

The writer came to Maine in 1906, too late in the season to find the
disease that year, but in'1907 it was found and its presence recorded (22).
Preliminary accounts regarding the nature of the disease, economic
importance, distribution, manner of dissemination, and means of control
were published in 1909 (23) and 1911 (24).

In 1911 Pethybridge and Murphy (26) published a review of the litera-
ture on the subject and described a similar type of disease in Ireland.
These workers also considered the organism responsible for the type of
malady there to possess sufficient distinctive characters to be considered
a separate species, although they felt that it was closely related to
- Bacillus phytophthorus. . They named it “‘Bacillus melanogenes.”’

CHARACTER AND APPEARANCE OF THE DISEASE

Blackleg is, as has already been indicated in the preceding section, a
bacterial disease of the stem and tuber of the potato. The various com-
mon names which have been applied to it have been given largely as a
result of the quite characteristic signs of the disease which are exhibited
by the attacked stems. In Germany, where the troyble was first described,
it has been known for years as Schwarzbeinigkeit, although the term
“‘ Stengelfdule’”” has also been applied to it. It would seem from the
literature that the former name or a somewhat free translation of it is
the one which has been in common use in Holland, Belgium, France,
and England. Jones (19, 20) described it under the name “blackleg,” the
name with which he had become familiar. in England and Germany.
Smith (33) prefers “basal stemrot.” Pethybridge and Murphy (26,
p. 9-10) state that—

Since, however, the term “‘Blackleg”’ is one which is already in common use in
this country for a disease prevalent among cattle, it seems strongly advisable, in

order to avoid confusion, not to use the term ‘“‘blackleg”’ for the present disease; and
we have, therefore, decided to distinguish it by the name of ‘‘ Black Stalk-rot.”’

Were the writer describing this type of disease for the first time, he
would endeavor to select a somewhat more appropriate name than black-
leg and would prefer black stalkrot or black stemrot to this. However,
he has found the objections to the former term to be of more theoretical
than real importance.

Blackleg, as the name indicates, is characterized in its typical form by
a pronounced blackening of the base of the stem of the affected plant.
However, such plants frequently show other visible signs of disease
before this character is apparent above ground. ‘They first appear more
or less unthrifty and usually undersized. The branches and leaves,

Jan, 15, 1917 Blackleg Disease of Potato | 83
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instead of spreading out normally, tend to grow upward, forming a more
or less compact top, frequently with the young leaves folded and curled
up along the midrib. Later they become lighter green and even yellow,
and the whole plant gradually dies. If the disease progresses rapidly,
quite a different picture is presented, and the plant may fall over sud-
denly and wilt with very little previous signs of disease. At first sight
the general aspect of the affected plants does not differ from that pro-
duced from several other causes which injure or kill the parts at or below
the surface of the ground, such as the attacks of fungi or insects, or even
mechanical injury at or near the base of the stem. Occasionally, and
more {requently some seasons than others, when the plants are attacked
before setting tubers and when the progress of the disease is slow, numer-
ous aerial tubers are formed on the stalk at the surface of the ground or
in the axils of the leaves above. As is well known, this condition may
arise from certain other injuries of the stem as well.

The diagnosis of suspected cases is easily confirmed by pulling up the
diseased plants. Stems attacked by blackleg show an inky-black dis-
coloration extending from the base of the stem, where it is attached to
the seed piece, up to the surface of the soil, except in the early stages,
and very frequently 1, 2, or even 3 inches above the ground. Some
seasons, under favorable weather conditions, the disease may with con-
siderable frequency follow up the stem for several inches or even out on
the larger branches, destroying the entire stem with great rapidity.
This is more likely to occur when the plants are growing in a naturally
moist soil and during periods of moist, cloudy weather.

If two or more stalks arise from one seed piece and one develops the
disease, the remainder will invariably, sooner or later, succumb to it also.
Very frequently during the last of July, or during August in Maine, when a
diseased stalk is observed, a careful search will reveal close beside it the
dried remains of another which died some time earlier. It is these stalks
that are not visibly affected till later in the season which usually show
the rapid and complete invasion of the above-ground parts of the stem
by the bacteria. However, there is no evidence to indicate that the
disease is ever communicated from one stalk to another by traveling
down one and up the other. Ina large number of inoculations of living
potato plants in flowerpots in the greenhouse and in boxes of soil in the
field, including all regions of the plant from the petioles of the leaves to
parts of the stem below the surface of the soil, the writer has seen little
evidence, except in the case of certain inoculations on below-ground parts
made by Mr. G. B. Ramsey, the writer’s associate, in the summer of
1916, of progress of the disease downward on the stem. It is always
upward.

With one exception, the writer has not observed blackleg occurring in
patches or localized areas. ‘The attacked plants are scattered promiscu-
ously over the field. The pieces of seed tubers from which such diseased

84 Journal of Agricultural Research Vol. VIII, No. 3
plants have grown are invariably found to have been attacked by a soft
wetrot, or may have been destroyed entirely before the examination is

‘made. At this time and for some weeks thereafter the seed pieces from
which surrounding, healthy plants have sprung are entirely sound. If
young tubers have formed before the plants are destroyed, the disease
frequently passes along the stolons upon which they are produced and
infects these also, producing a rapid softrot. This is by no means the
invariable rule, as might be inferred from the statements of some writers.
However, as will be shown later, it is apparently such cases of infection
of the growing tubers which are responsible for the propagation and
spread of the disease, either directly or indirectly.

The exception noted above, suggestive of the spread of the disease in
the field, occurred in Dover, Me., in 1908, on the same field from which
pure cultures of the causal organism were first obtained. There was
very little blackleg in the entire field of 20 acres, except on one spot of a

‘few square rods where all of the plants were diseased. It was first noted
by the owner near the center of the affected area, from which it gradu-
ally spread outward. The season had been excessively wet, and this
area coincided with a low, undrained pocket or depression in the field,
where water would stand for a few hours after‘each heavy rainfall.

Sometimes the attacked plants attain a height of only 2 or 3 inches
above the surface of the soil, and the fact that many hills are entirely
missing on fields which show a high percentage of diseased’ stems indi-
cates that in such cases either the seed pieces decay before sprouting or
the sprouts are killed before they reach the surface. Asa rule, however,
the plants first begin to show signs of disease when they are 6 or 8 inches
high and are growing rapidly. In northern Maine this is about the first
of July, although the disease frequently makes its first appearance some
time in June. Again, the plants may attain nearly full size before many
of them on a field appear to be attacked.

The progress of the disease is markedly influenced by weather condi-
tions. Very moist, cloudy weather during the first few weeks of growth
may tend to favor rapid progress, resulting in the early death of the young
plants, so that only the dead stalks remain scattered among the healthy
plants within a month or six weeks or even a less time after its first
appearance. A period of dry weather coming on after the disease is well
established below the ground may check its progress but cause the death
of the plant at an equally early period on account of its inability to with-
stand the lessened water supply. Again, conditions between these ex-
tremes may prolong the attack well into August. More blackleg is
observed in wet than in dry seasons.

Fields which showed quite a percentage of blackleg early in July may
give a decidedly different impression to the observer by the middle or
last of August. At that time the diseased plants may have entirely dis-
appeared; and on account of the scattered occurrence of the former, the

Jan. 15, 1017 | Blackleg Disease of Potato 85

healthy plants, which have reached practically their full development,
obscure the vacant areas, giving the appearance of almost a perfect stand
over the entire field.

Soil conditions also are factors which influence outbreaks of blackleg.
All other things being equal, the disease is more likely to occur in wet
than in dry soil, and is more prevalent when the early part of the growing
season is characterized by abundant rainfall.

Different varieties of potatoes show a marked difference in their sus-
ceptibility to the disease. This is well shown in the case of the Irish
Cobbler and the Green Mountain, which represent by far the greater
proportion of the potatoes grown in Maine at the present time. The
former is an early variety, largely grown for seed purposes for southern
planting, and is highly susceptible. The latter is a late variety which is
primarily raisedfor table use and is seldom severely affected with blackleg.
In fact, as long as Maine potato growers planted this variety almost
exclusively, blackleg was of minor consequence. Harrison (17) has shown
by means of inoculation tests that these differences in ability to resist
the attacks of blackleg are exhibited by quite a number of varieties.

It should be understood that the above characterization of the dis-
ease is made without reference to the published descriptions of this and
similar types of potato-stem disease as they occur elsewhere and is based
entirely on personal observations which have been made largely in the
State of Maine.

There is a somewhat similar form of potato-stem disease which the
writer has seen with some frequency in certain Western States, but which
is not common in Maine. It differs from the ordinary type of blackleg
in that while the stem is blackened and discolored above ground there is
little or no external evidence of disease below ground. In all the cases
observed, however, a closer examination has shown that the pith has
been destroyed between the base of the stem and the externally diseased
area above ground.

A few typical cases of this trouble, which some have been inclined to
consider as not identical with blackleg, were seen in Presque Isle, Me.,
in the summer of 1916. In some instances the disease had spread through
the stolons to the young tubers. From one of these tubers Mr. G. B.
Ramsey isolated a bacterium which produced typical blackleg when
inoculated into young, growing stems.

GEOGRAPHICAL DISTRIBUTION OF THE DISEASE

The fact that blackleg or a very similar appearing disease of the potato
stem and tuber has been observed in Germany, France, Belgium, Holland,
England, Ireland, Canada, and the United States has been pointed out
in the previous section.

From the accounts of Frank (13-14) and Appel (1-7) it is evident
that the disease is common, widespread, and often destructive in Ger-

86 Journal of Agricultural Research Vol. VII, No. 5

many: Delacroix (12) stated in 1906 that he had not seen it in France
until within two years and that it was not widespread at that time.
At the same time he pointed out the marked similarity of blackleg to
the brunissure of the stem previously described by him. The possi-
bility is thus suggested that the malady first described in Germany may
be of wider distribution in France than has been reported and that the
two have been confused. In the same paper Delacroix says that black-
leg exists also in Denmark and Russia.

In writing of the occurrence of blackleg in England, Jones (19, p. 17)
stated in 1905 ‘that— }
it is said to be common, though apparently less troublesome than Appel reports

it from Germany. Reference to the agricultural papers of England during recent
years indicates that the disease is of quite widespread distribution in that country.

Harrison (17), in Canada, reports what is probably a much wider terri-
torial distribution of the type of disease he described than was the case
with the previous writers. Hestates that he had found it throughout the
Province of Ontario. Its presence had been reported from Nova Scotia,
New Brunswick, and Quebec, and one case had been reported from the
Northwest Territory.

In the United States the writer has been collecting information regard-
ing its occurrence during the past nine years. It has been reported in
most and doubtless occurs in all of the Atlantic seaboard States from
Maine to Texas. It probably occurs also on the Pacific coast, and has
been recorded from Ohio, Wisconsin, Minnesota, Colorado, Utah, Mon-
tana, and Idaho in the interior. In other words, blackleg has been
definitely reported from nearly all the great potato-growing centers or
States in this country.

There is reason to believe that Maine was one of the first places in
which the disease was introduced into the United States, and in connec-
tion with the question of geographical distribution it is of interest to
speculate on how this came about. It seems reasonable to believe that
it was introduced into Maine from Canada and into the Dominion from
England. As will be pointed out in a succeeding section, blackleg is
carried by seed tubers. Importation of seed stock into Canada from
England would naturally be much more common than into the United
States; and the wide distribution of the disease in Canada, reported by
Harrison, would indicate that it existed there for some time previous
to its entry into Maine. Maine’s greatest potato-producing section,
- where the disease was first discovered, is immediately adjoining the
Province of New Brunswick. Many of the potato growers in this region
formerly resided on the other side of the boundary line, and with the
constant intercourse and traffic between the adjoining sections of the
two countries it is readily conceivable that blackleg was imported in
this way.

Jan. rs, 1917 Blackleg Disease of Potato 87

In all probability the disease has been introduced in the same way
into other States along the northern border. It was carried to the
Southern States by northern seed. There is one authentic instance of
the introduction of blackleg into this country directly from England.t
At Kingston, R. I., in the summer of 1907 five hills of potatoes showing
blackleg were found. These potatoes were grown from seed tubers
which were obtained from England in the spring of the same year. This
was the first and only case of blackleg that had been reported from
Rhode Island up to that time.

One interesting and apparent case of importation of the disease from
Europe and its transference in this country is as follows: In the summer
of 1912 it appeared commonly on a field in Parkman, Me., in a part of
the State where the disease was practically unknown, and on a farm
where the owner had never observed it before. The field was planted
with a variety known as Delmany Challenge, which came from Twin
Falls, Idaho. Inquiry showed that this variety had been grown on a
farm near Twin Falls for three years, having been shipped there from
Carbondale, Colo., where it had been planted for two years. The Car-
bondale grower imported the original seed tubers from Scotland. The
writer has since visited the farms mentioned in Carbondale and Twin
Falls and found blackleg present in the potato fields on each.

ECONOMIC ASPECTS OF THE DISEASE

Wherever the type of disease under consideration occurs, those who
have described it emphasize its economic importance. Apparently a
loss of from 5 to 10 per cent of the plants on affected fields is not an
uncommon occurrence in Germany, and much greater losses are reported.
Frank (13) reports cases where at least 75 per cent of the plants on a
given field were killed from this cause alone. Pethybridge (25) reports
the result of experimental trials in which apparently sound tubers taken
from an infected crop of the previous season produced 94 per cent of
diseased plants.

During the past nine years the writer has had opportunity to make
some rather extensive observations on this subject. tn Aroostook
County, Me., in a fairly continuous area, there are about 50,000 acres of
potatoes grown annually. In this region there are few potato fields of
less than 10 acres, and usually the acreage of individual farmers runs
from 20 to 50, although fields of 60 to 80 are not uncommon and there
are those which run to 100 acres or more.

Considerable time was spent in this and other potato-growing sections
of the State each year, and during the first five years large areas of
potatoes were inspected annually. In many fields only scattered plants
have been observed and not infrequently these amount to 1 or 2 per cent,

1 Reported by Prof. G. E. Adams, of Rhode Island State College, in correspondence with the writer.

88 Journal of Agricultural Research Vol. VIII, No. 3

5 per cent of diseased plants being considered by. the growers as repre-
senting a severe attack. However, losses amounting to 10 or 15 per cent
or more are by no means unknown; and the writer saw one case in IgII
where 50 per cent of the plants had been killed by blackleg or the pota-
toes had failed to germinate. It should be mentioned that in this
instance the seed tubers apparently had been stored under very adverse
conditions and were not in a fit condition to plant. The writer has never
seen anything like what Pethybridge has described (25), where appar-
ently sound seed tubers, even if they came from a diseased crop, pro-
duced a large percentage of diseased plants. As will be shown in a later
section, it has been found that the selection of sound tubers from a
diseased crop is an important factor in reducing the amount of blackleg
in the following crop.

Much less loss has been observed on high, well-drained soils that were
well adapted to growing potatoes than on low, wet, undrained soils.
Likewise greater damage has resulted from blackleg in seasons of excessive
rainfall than in dry seasons. Much greater losses in Maine apparently
have been experienced since the advent of the southern seed-growing
industry. As has already been mentioned, certain of the early varieties
desired for seed purposes by the growers in the South are more susceptible
to the disease. Blackleg also appears to be more destructive in the South
than in Maine.

The above discussion has been limited largely to the losses resulting
from the failure of the seed tubers to germinate; or if they do germinate,
to produce plants which will live to mature a crop. The losses which
result from tuber decay either in the field or in storage should also be
considered.

Nearly all writers who have discussed blackleg have laid special stress
upon the amount of loss which it occasions through destruction of the
tubers. Harrison (17) estimates a total loss from rot of from 10 to 75
per cent of the crop in the Province of Ontario. Taking the lower
figure and allowing 40 cents per bushel, he states that this would
amount to $720,000 in that Province alone.

All of the blackleg-producing organisms described, including those
isolated and studied in Maine, are capable of causing a rapid and com-
plete decay of the tubers, especially when they are immature, or im-
mediately following harvesting. There is no doubt that they can and
do cause some decay in storage and may be responsible for a considerable
amount if storage conditions are unfavorable. The writer is fully aware
that the same disease may produce radically different results in different
countries under different climatic conditions. However, if he were to
base his opinion upon his observations of conditions in the northeastern
part of the United States and adjacent portions of Canada during the
last 15 or 20 years, he would say that the losses from tuber decay in the
field and in storage from this source have been largely overestimated.

Jan. 15, ror7 Blackleg Disease of Potato ; 89

It is believed that much of the rot of potato tubers, which in some in-
stances has been attributed to the same organism which produces the black-
leg of the stem, is due to entirely different causes. In the Northeastern
States epidemics of potato softrot in the field and in storage are quite
common, but these invariably follow and are associated with outbreaks
of lateblight on the foliage caused by Phytophthora infestans De Bary.
The decay caused by this fungus, as is well known, is of a dry nature.
However, if a tuber lying in a moderately damp soil becomes infected
with the fungus, various soil bacteria at once enter into the lesions thus
made and a rapid, soft, foul-smelling rot results. Where such decay
starts in the field, it is very likely to follow and be very destructive in
storage, particularly if the conditions of storage are damp. ;

Repeated experiments by Jones and his associates in Vermont, Stewart
in New York, and Woods and the writer in Maine have demonstrated
conclusively that epidemics of such bacterial softrot can be prevented
entirely if the parts above ground are kept free from Phytophthora in-
festans, by proper spraying with Bordeaux mixture, until they are ripe
or are killed by the frost. Moreover, epidemics of softrot of potato
tubers following lateblight were common in New England long before
blackleg made its appearance and now occur in sections which are en-
tirely free from blackleg. As has been stated previously, the latter
disease has been under observation by the writer to a greater or less
extent in Maine each summer since 1907. ‘Two severe epidemics of
tuber decay, and other less destructive ones, have been experienced dur-
ing the same period, but this was entirely controlled where thorough
spraying with Bordeaux mixture was practiced. On the other hand, no
severe outbreaks of tuber softrot have been observed which were not
preceded by lateblight on the foliage, regardless of the amount of black-
leg which occurred on the plants in the fields.

It must be conceded, therefore, that ordinarily there is no serious loss
from tuber decay from the blackleg organism in Maine. However, it
must do some damage of this nature, for there is ample evidence that the
infected seed tubers are responsible for the propagation of the disease
from year to year.- Also it is a natural supposition that conditions
which enable the various soil organisms to complete the destruction of
the tubers, begun by the lateblight fungus, would also favor decay by the
blackleg bacteria. If decay is once started in storage or the tubers are
kept under too warm, moist conditions and the latter organisms are
present, they certainly would be an important factor in causing the rot.
However, the writer has made many attempts to isolate organisms from
softrot of potatoes following attacks of lateblight on the foliage; but in no
case has he been able to secure a bacterial organism capable of causing
the decay independently and alone.

90 Journal of Agricultural Research Vol. VIII, No. 3

SOURCES OF INFECTION AND MEANS OF DISTRIBUTION

Various other agencies have been suggested as being responsible for
the spread of the disease; but a long series of observations have convinced
the writer that in Maine the ultimate source of the trouble is, either
lirectly or indirectly, infected seed tubers. A large amount of evidence
bearing on this point has been collected during the last nine years, both
by observation in Maine and by correspondence with others where
Maine potatoes are used for seed. As has already been mentioned, only
‘me case has been observed where the disease appeared to spread in the
field; and this is easily explained by peculiar local conditions. Since
the organisms, as will be shown later, are killed fairly readily by drying,
they are probably incapable of existing for any length of time in a living
state on the dry surfaces of potatoes. It is the writer’s opinion that they
are carried over the winter in decaying, bruised, cracked, or otherwise
imperfect seed tubers.

In Maine, potatoes are not stored in covered pits in the ground, as is
the case in Ireland and in certain other parts of Europe. They are always
kept in dark, cool, well-ventilated cellars or specially constructed potato
houses, usually the latter. In these houses considerable attention is
paid to ventilation. If possible, no excessive moisture is allowed to
develop, and the temperatures are held as low throughout the winter as is
compatible with safety to the potatoes. In laboratory experiments the
organisms in beef-broth cultures were found alive after a period of 10
months, or until nearly all of the moisture had evaporated from the cul-
ture tubes. Temperatures a few degrees above freezing, or in the ice
box, have been found to be most favorable for keeping this and similar
organisms alive for long periods of time in cultures. Under these condi-
tions the organisms retain their vitality, but multiply at a compara-
tively slow rate. Therefore it seems probable, if the bacteria are able to
enter the interior tissues of the tubers either by natural infection in the
field shortly before harvesting, or through wounds or cracks made in har-
vesting, or even through lesions produced by other parasitic organisms,
that, so long as they are supplied with a small amount of moisture, they
will remain alive. ‘The low temperatures of storage prevent their rapid
multiplication and the resultant decay of the tubers. It is undoubtedly
those only slightly affected potatoes which are responsibie for the propa-
gation of the disease.

Unfortunately, where blackleg is observed for the first time, it is not
always possible to trace the seed tubers to determine whether the disease
occurred the season before on the field where they were produced. In
all cases where this was possible the answer has been found to be in the
affirmative. ‘The only criterion by which to judge matters of this kind
is whether or not the characteristic blackening and death of the growing
stems occurred. As has already been pointed out, the fact that a large

Jan. 15, 1917 Blackleg Disease of Potato ‘OI

amount of the previous crop had been lost by decay, whether of the
nature of a softrot or not, is not necessarily an indication of the presence
of the blackleg disease.

Out of a large number a single case will be cited here as evidence that
the disease is first introduced by means of infected seed tubers. In 1907
a 4-acre field on the University farm at Orono, Me., where blackleg had
not previously appeared, was planted with seed tubers from several
different sources. Along one side three barrels of potatoes, each from a
different source, were planted. Quite a percentage of diseased plants
were found where one of these barrel lots was used; but a careful search
several times during the summer failed to reveal any such on the remainder
of the field.

Observations in Maine indicate that under the climatic conditions
which exist there infected seed potatoes are the sole source of infection
and distribution and that the disease does not live over the winter in the
soil. Planting the same field with potatoes two years in succession is
. quite frequently practiced. Where the disease occurred the first year
and sound seed tubers for the second crop were carefully selected and
then disinfected with formaldehyde as described in the following section,
blackleg was either entirely eliminated or was much reduced, depending
upon the thoroughness of the treatment.

On fields which are planted with potatoes the second time in succession
there is usually quite a percentage of volunteer plants which spring
from tubers which remained in the soil over the winter. These plants
are frequently easily recognized by their irregular occurrence on the
sides of the rows or between the hills. The writer has never seen such
plants affected by blackleg. This observation was quite unexpected,
for it seems not unreasonable to suppose that, if the tubers were suffi-
ciently protected from frost to be able to germinate, the bacteria causing
the disease might live over in the soil in such infected tubers as well as

in storage
CONTROL MEASURES

Laboratory studies showed that the organisms associated with the
disease did not form spores, were not resistant to drying, and were
readily killed by germicides. This, together with the fact that the dis-
ease did not spread from hill to hill in the field and that under Maine
climatic conditions decayed, diseased, or otherwise imperfect seed tubers
appeared to be the ultimate source of infection, suggested the probability
that blackleg might be easily and cheaply controlled or even eliminated
from a given farm, field, or locality. The most feasible measures which
presented themselves were careful sorting before and at seed cutting, and
rigid rejection of all diseased or imperfect seed tubers, especially those
which showed any blackened or decayed areas, supplemented as an
added precaution by disinfection with formaldehyde or mercuric chlorid.

g2 Journal of Agricultural Research Vol. VIII, No. 3

After certain preliminary experiments and with the realization that no
control measures would be of much practical use unless they were effec-
tive in the hands of the potato growers themselves, it was decided to
ask the cooperation of those interested in the work in order to test these
control measures on a large scale under actual field conditions. In
IQII, Cooperative experiments were conducted by eight practical potato
growers in three separate towns. On the eight different farms approxi-
mately 300 acres of potatoes were under experiment. Collectively these
gentlemen selected and disinfected seed tubers sufficient to plant 142
acres. Forimaldehyde solution was used for 88 acres and formaldehyde
gas for 54 acres. Detailed statements of the methods used and of the
results secured from these experiments have been previously published
(24).

The experimental fields were carefully watched by the owners for the
appearance of diseased plants. The writer visited some of the farms
several times and during the first and third weeks in July madea thorough
examination of each experimental field, counting each time the number
of diseased plants upon representative areas.

The question might naturally be raised that only two careful counts
of diseased plants on each part of the experimental fields during the
season would not accurately show the actual amount of blackleg on
them, for, as has already been pointed out, the time of the greatest evi-
dence of the disease on a given field may be materially influenced by
seasonal climatic conditions. That there may be something to this ob-
jection is freely granted; but the writer maintains that it in no way
influences the relative number of diseased plants on the different plots
at the time the counts were made, and this seems to be a fair way of
judging the efficiency of the different methods of treatment employed.
Moreover, if it were to appear at all on the plots planted at the same
time with selected and treated tubers, there seems to be no valid reason
why blackleg should not show up equally early in the season as on the
check plots planted with unselected and untreated tubers from the same
lots or bins. As a matter of fact, the times at which these counts were
made were selected because they coincided with the period at which
blackleg was most in evidence in surrounding fields that season. Also,
as has already been stated, other visits made to the fields at various
times during the season and the observations made by the owners of
the fields themselves furnish plenty of data to confirm the conclusions
derived from the counts of diseased plants.

Taken as a whole, the results of the cooperative experiments were
sufficiently clear-cut and conclusive to indicate that the preventive meas-
ures outlined are exceedingly efficient if properly carried out. In fact,
the uniformity of the results was surprising, since so many individuals,
including the men who were employed to cut the seed, were respon-
sible for them. In every case where both carefully selected and treated

Jan. 15, 1977 Blackleg Disease of Potato 93
Sn tn RELA re eB nak 0 DA NC en Se
seed tubers were used the disease was absolutely eliminated. In two
instances this occurred on fields planted the second year in succession,
in which considerable blackleg occurred the year before, and also
appeared upon the check plots used in the experiment. Also in every
case where either disinfection or selection was practiced alone and proper
check plots were planted for comparison, the amount of blackleg was
materially reduced, except on one field where small inferior tubers were
purposely sorted out and planted after first being disinfected with
formaldehyde solution.

An analysis of the data furnished by the experiments did not lead to
any very definite conclusions as to the relative value of selection of sound,
perfect seed potatoes for planting as compared with disinfection with
formaldehyde alone. There is no doubt that both are necessary. The
writer believes that careful selection of seed tubers and rejection for
planting all that are in any way cracked, bruised, discolored, or decayed
is absolutely essential, and no amount of disinfection with the present
known methods can be relied upon to take the place of it. On the other
hand, the formaldehyde treatment appears to be equally essential and
must be practiced to supplement selection of seed. No one familiar with
bacterial softrots of vegetables would assume or suggest that formalde-
hyde solution or gas could be relied upon to disinfect entirely or even
approximately a tuber the interior of which is partially decayed. The
writer has never maintained that this could be done, and it would be
unnecessary to refer to it or emphasize the point were it not for the fact
that in some instances it has been assumed that he did recommend
simply seed disinfection with formaldehyde for the control of the disease.

Unfortunately, the way the experiments worked out there was no
opportunity to compare on the same field the relative efficiency of formal-
dehyde gas and solution. In every case where formaldehyde gas was
used the results were less efficient than those where formaldehyde solu-
tion was used. For this and other reasons the gas treatment is not
recommended for general use.

Numerous other cases have come to the writer’s attention where careful
selection of sound, healthy seed tubers, followed by disinfection with
formaldehyde has either eliminated or largely reduced the amount of
blackleg in the resulting crop, but only one of these will be mentioned.

In ro1r the Maine Agricultural Experiment Station purchased two
lots of Irish Cobbler potatoes for planting at Highmoor Farm in the
central part of the State. These were disinfected with formaldehyde,
but no special care was used in selection before planting. In both cases
quite a percentage of the resulting plants were attacked by blackleg..
The following season the farm superintendent personally saw that the
writer’s recommendations relative to seed selection and disinfection
were rigidly carried out. A clean crop resulted, and since then no
blackleg has been observed on this farm.

94 Journal of Agricultural Research Vol. VIII, No. 3

In 1913 some of the crop raised from these selected and disinfected seed
tubers were sent to Director T. C. Johnson, of the Virginia Truck Experi-
ment Station, for planting at Norfolk. Out of over 3,000 plants pro-
duced from this seed only two doubtiul cases of blackleg were reported.
At the same time two lots of seed tubers purchased in the open market
gave 6.5 and 7.9 per cent, respectively, of diseased plants, and another
lot sent in for testing gave 16.8 per cent.

Undoubtedly much can be gained by uprooting and destroying all
blackleg plants in the field as soon as seen, taking care to remove also all
tubers which have formed; but the writer has no experimental data
bearing on this point. It should be recommended not as a substitute for
seed selection and disinfection, but rather to supplement them, for the
latter are, of course, equally important in the control of several other
diseases which are carried by the tubers.

COMPARATIVE STUDIES OF THE CAUSAL ORGANISMS

Of the various organisms which have been isolated and described in
different parts of Europe and in Canada as being the cause of Schwarz-
beinigkert, or blackleg of the stem of the potato, and the attendant decay
of the tubers, the following are available for study: Bacillus atrosepticus
Van Hall, B. phytophthorus Appel, B. solantsaprus Harrison, and B.
melanogenes Pethybridge and Murphy. As far as could be learned from
reading the published articles and descriptions, the different investi-
gators who named the species in question made no comparative, cultural
studies in the laboratory, under identical conditions, of the previously
described species and the organisms which they themselves had isolated.
They based their conclusions with regard to the nonidentity of the species
upon the manner in which they found their own organisms to differ from
the descriptions previously published.

Therefore, when the writer began his studies of this disease in Maine
in 1907, several interesting questions presented themseives. From both
a scientific and a practical point of view the most important question was,
Is the disease in the United States, especially in Maine, caused by one or
more of the named species of blackleg-producing bacteria or by one
differing from each of them in certain well-defined characters? In other
words, if the previously described organisms were collected together and
subjected to the various differential tests usually employed in describing
a species of bacteria under exactly identical laboratory conditions, would
these differences still hold or would they all prove to be identical with
each other and with those found associated with the disease in Maine?
The exerience of the writer (16) in a similar study of the very closely
related group of bacteria causing a softrot of various vegetables sug-
gested that the last question possibly might be answered in the affirma-
tive. Accordingly, after considerable preliminary study had been made
of some 18 different strains of organisms isolated in different parts of

Jan, 15, 1917 Blackleg Disease of Potato 95
Maine, in order to show that these were probably identical with each
other and very closely related to B. solanisaprus three of these represent-
ing isolations made from diseased plants from widely different parts of
the State were selected for detailed study and an attempt was made to
obtain all of the available named species for comparison. The names
of the organisms thus secured and the sources from which they came
are given in a later section.

While the work was in progress, Smith (33) published his report on his
studies of B. phytophthorus, mentioned below. It should be noted here
that while he did not at this time publish in detail on his findings with
regard to B. solanisaprus he states that: “Bacillus solanisaprus Harrison
is a very closely related, but not identical organism, causing a similar
disease in potatoes.” In a letter to the writer, about two weeks before
this paper was read, dated December 18, 1909, he stated that he had for
three years been making a comparative study of B. phytophthorus, Be
solanisaprus, and B. solanacearum. It is evident then that the sentence
quoted above was based upon a series of long and careful comparative
studies. ‘Therefore the writer is warranted in the beginning in assuming
that, while very closely related, there are in the opinion of Smith at
least two separate types or species to be considered.

For the purpose of comparison it seems necessary to state as briefly
as possible, without sacrificing accuracy of statement, the descriptions of
the four previously named species which have been included in the
present comparative study with the organisms isolated in Maine by the
writer. For convenience, since Smith’s description of B. phytophthorus
(33) is more recent than Appel’s, and since Harrison’s (17) is the first
extended and most complete description of the type of the orgarism
that the writer has found in Maine, these will first be given in detail,
followed by a brief statement of the characters by which the descriptions
of B. atrosepticus and B. melanogenes would appear to differentiate the
last two named from each of the first and from each other.

DESCRIPTION OF ORGANISMS PREVIOUSLY DESCRIBED
SMITH’S DESCRIPTION OF BACILLUS PHYTOPHTHORUS APPEL *

The organism is a non-sporiferous rod, variable in length, usually occurring singly of
in pairs, but also forming chains of several individuals; taken from young agar cultures
the diameter is about 0.6 to 0.8u, the length 1.5 to 2.54 ; actively motile by means of
peritrichiate flagella; stains readily with ordinary stains, but not by Gram’s method;
rots potatoes (stems and tubers), cucumbers, tomatoes, etc.; aerobe and facultative
anaerobe; organism grayish white on agar and slightly bluish opalescent by trans-
mitted light; surface colonies, on thinly sown --15 agar, mm. or less in diameter in 48
hours at 20° to 23° C., 2 to 3 mm. broad in 4 days; round, smooth, wet-shining, inter-
nally reticulated at first, amorphous under 16 mm. and 12 ocular, or with small flocks
in the older portion; the buried colonies appear brownish under the microscope, also

1 Quotation 748 from a paper read at the meeting of the American Phytopathological Society, Boston,
Dec. 30 and 31, 1909. (33-)

96 Journal of Agricultural Research Vol. VIII, No. 3

granular in the center; margin of buried colonies sharply defined; liquefaction of +10
gelatin moderate to rapid; circular white colonies with regular margins on gelatin
plates, visible in 18 hours at 30° C., in 26 hours at 21° to 23° C.; on thin-sown gelatin
plates colonies grow rapidly and are frequently 2 centimeters in diameter at the end
of fourth day at 22° C.; alkaline reaction in gelatin cultures to which litmus has
been added; on sterilized potato slow white to yellowish white growth; characteristic
rapid white growth and black stain on raw potato (when streaked from agar); grows vig-
orously and with great rapidity on all neutral and feebly alkaline media; clouds to cc.
of +15 bouillon in 6 hours at 30° C. and in 24 hours at 13° to 14° C., when inoculated
with one 3-mm. loop from a bouillon culture 4 days old at 24° C.; especially good
growth on neutralized potato-juice gelatin in which stab-cultures rapidly develop a
funnel-shaped liquefaction, but less rapid in my hands than in +10 peptonized beef-
gelatin; gradual clouding of salted peptonized beef-bouillon, and production of
chains therein and pellicle on undisturbed old cultures; no indol reaction; tolerates
in beef-bouillon a considerable amount of sodium chloride (5 per cent) and of sodium
hydrate (+50); very active growth in potato-juice with formation of thick pellicle
and heavy precipitate; rapid clouding of closed end of fermentation-tubes containing
potato-juice, but no production of gas; no growth in Cohn’s solution; slight greenish
tinge in Fermi’s solution on long standing; moderate production of hydrogen sul-
phide ; distinct and persistent nitrite reaction in nitrate bouillon but no gas; grows in
peptonized beef-bouillon from —50 to +16 and beyond, also in potato broth acidulated
to +-46 with citric acid, but no growth when acidulated to +45 with oxalic acid; slow
(acid) coagulation of milk with precipitation of the casein; slight reddening and final
reduction of litmus in milk; slight production of gas in shake-cultures in some beef-
agars; grows in bouillon over chloroform; in streak-cultures it reddens litmus agar
decidedly in 48 hours at 20° C. in presence of either dextrose, saccharose, lactose,
gelactose or maltose; it blues plain litmus agar decidedly in 48 hours and does not
promptly redden the same with addition of dextrine or glycerine; no reddening of
litmus in gelatin-cultures; the acid persists on boiling; produces small quantities of
gas from innosit (muscle sugar), lactose and mannit; optimum temperature 28° to 30°
C.; little growth below 4° to 5° C.; minimum temperature for growth in +15 beef
bouillon 1° C. or under; maximum temperature for growth in +15 beef-bouillon about
36° C.; thermal death-point in +15 beef-bouillon 47° C.; 90 per cent destroyed by
freezing in bouillon. Appel reports loss of virulence in some of his cultures, but I
have not observed any during a period of three years. * * * The following are
recommended as quick tests for differential purposes: Very thin sowings on gelatin
plates; streaks from agar to sterile raw potato; behavior in blue litmus milk; behavior
in nitrate bouillon and in Cohn’s solution. The right organism should produce big,
round, white colonies promptly on thin sown gelatin plates, and should rot potato

tubers promptly.
BACILLUS SOLANISAPRUS HARRISON

The following is a brief of Harrison’s original description of this
organism (17). No effort has been made to follow the original form, but
care has been taken to preserve accuracy of statement:

Bacillus, with slightly rounded ends, variable in size, according to the media and
temperature in which it was grown. From freshly infected potato stems and tubers
it varies in size from 1.5 to 4u long and about 0.6 too.gu wide. From beef-peptone-
agar cultures 24 hours old at 20° C. the bacteria were short and stout, rather variable
in length, ends rather square but rounded, vacuolated with average dimensions 1.2
by 0.64. From beef-peptone gelatin +5, 24 hours old at 20° C. the bacilli were
short and stout, ends slightly rounded, some vacuolated, dimensions 1 to 2.5 by 0.6
too.8u. Involution forms, long swollen bent rods in 24 hours at 37° C.; endospores

Jan. 35, 1017 Blackleg Disease of Potato 97

not seen; flagella, 5 to 15 or more, peritrichously disposed; stained by Van Ermen-
gem’s method; organism stained readily with the usual anilin colors; Gram’s method
was negative, but positive with amyl alcohol; growth in agar abundant, filiform,
spreading, elevation raised, luster glistening, opalescent; good persistent growth on
potatoes, slightly raised and spreading, dull waxy and pale cream in color which
subsequently changed to dirty white; growth in Loeffler’s blood serum good and
slightly spreading, raised a little with the edges some higher than the center, waxy
and pale yellow in color; uniform growth in gelatin stab cultures, filiform, liquefac-
tion begins in 35 days and is not complete in 44 days; strong growth in nutrient
broth +15 at 25° C., slower at 20° C.; ring in surface growth; clouding strong and
persistent, fluid turbid, fine sediment; prompt coagulation of milk in 48 hours,
extrusion of whey in 3 days, afew minute bubbles of gas visible; litmus milk acid,
with partial slow reduction; growth in gelatin colonies slow, round to elliptical
or egg-shaped in form; elevation, surface colonies flat; edge first entire, later lobate
or erose; color faint brown by reflected, and bluish white by transmitted light; agar
colonies round to lenticular, surface moist, shiny; elevation flat; edge entire; internal
structure finely granular; growth in Fermi’s solution similar to that in Uschinsky’s
medium, but slightly less in amount; copious growth in Uschinsky’s solution, ring,
sediment, liquid appeared bluish; the best media for long continued growth was found
to be beef bouillon and Uschinsky’s; in fermentation tubes gas was produced only in
bouillon containing mannit and lactose; growth in the closed arm with production of
acid in the presence of each of the following: Dextrose, saccharose, lactose, maltose,
glycerin, mannit, and levulose; at the end of 1o days the closed arm with mannit,
glucose, and lactose was clear, the other tubes remained clouded in the closed arm,
but with less turbidity than was observed on the second day of growth; nitrates
in nitrate broth were reduced, nitrites present; indol production moderate to feeble;
growth in bouillon +16, Fuller’s scale, with hydrochloric acid and not at +18; growth
in bouillon —16 with NaOH but not at —18; vitality on culture media, long; thermal
death point 54° C., the optimum temperature 25° to 28°, slight growth at 37°, none
at 42°; maximum temperature for growth 37.5°; minimum temperature for growth
about 0°; in poured plates sensitive to sunlight, 90 per cent being killed in 30 min-
utes and roo per cent being killed in 50 minutes; pathogenicity proven in the follow-
ing vegetables: Potato, tomato, Jerusalem artichoke, cucumber, red carrot, white
carrot, radish, red beet (sl.), sugar beet (sl.), parsnip, cauliflower, cabbage, celery,
mangel-wurzel, Swede turnip, and white turnip; also in living plants of potato,
tomato, common red pepper, and slightly in cucumber and physalis.

BACILLUS ATROSEPTICUS VAN HALL

The description of this species was published in Dutch (15) as a part of
a dissertation for the doctor’s degree. The following is based on a trans-
lation of a part of this article, made for the writer by Dr. R. de Zeeuw.

Bacillus occurring in 2-day-old bouillon cultures at 27° C., almost exclusively as
single rods, sometimes in pairs; size variable, 0.8 to 1.64 long by 0.2 to 0.4u wide,
stained with gentian violet; many small zoogloea; very motile at 27° C. in young cul-
tures containing 0.025 per cent of potassium phosphate and 0.25 per cent asparagin;
flagella stained from such cultures by Loefiler’s method, length 10 to 154; Gram’s
stain decolorized. Growth weak on malt agar and malt gelatin; gelatin liquified, but
rapidity is variable, does not take place on unneutralized meat gelatin and is some-
times slight on one weakly alkaline. Strong coagulation of casein in milk. No
diastasic action on starch. Gas production weak or absent except where mannit is
used as a source of carbon. A medium consisting of “‘duinwater’’ (water out of the
dunes, filtered through sand and gravel) plus 0.025 per cent of dibasic potassium phos-

98 Journal of Agricultural Research Vol. VII, No. 3

phate (K,HPO,), 1 per cent peptone, and 3 per cent, respectively, of different carbo-
hydrates gave the following results in three successive trials: Saccharose, 0.1, 0, 0.2;
glucose, 0, 0, 0.1; lactose, 0, o, 0; mannit, 0.4, 0.6, 0.3; glycerin, 0, o, o; galactose not
changed; nitrates reduced to nitrites; reduction of methylene blue weak; sodium
selenite reduced very rapidly; indol production not observed. Growth in bouillon
acidulated till the reaction is 0.5 per cent normal with citric and malic acids does not
stop growth, but 1 per cent entirely prevents it. Thermal death point for a 24-hour-
old culture between 51° and 52°C. Optimum temperature not accurately determined,
but strong growth takes place at 27° C. Readily killed by drying. Pathogenicity
to potato stems and tubers somewhat variable, but apparently imperfectly tested.

BACILLUS MELANOGENES PETHYBRIDGE AND MURPHY

Pethybridge and Murphy described their organism in considerable
detail (26), but it is sufficient for our present purpose to simply state
wherein they found it to disagree with published descriptions of pre-
viously described organisms. They stated that it was larger in size than
B. atrosepticus (0.7 to 0.9 by 1.3 to 1.84) and instead of occurring chiefly
singly was found more frequently in pairs, also that its action upon milk
appeared to be different. As contrasted with Harrison’s description of
B. solanisaprus, it possessed less flagella, formed gas in glucose and cane
sugar, did not form a distinct ring on the surface of potato juice, and did
not produce a raised, creamy white growth on cooked potato. They stated
that their organism showed marked resemblance to B. phytophthorus and
that they were strongly tempted to regard it as only a variety of the latter.
However, it did not produce a pellicle on Appel’s sterile potato juice, and in
nitrate broth it produced a small quantity of gas. In milk it caused the
separation of the curd as a not very compact mass and produced a dis-
tinct acidity in a comparatively short time. According to Appel’s
description, B. phytophthorus produces a strong pellicle on sterile potato
juice, apparently produces no gas in nitrate broth, and causes milk to
change only on long standing, forming then a compact cylinder of pre-
cipitated curd, and giving a reaction which is amphoteric to litmus.

SOURCES OF THE CULTURES USED

Blackleg was first observed by the writer in Maine at Sherman, on
July 30, 1907. It was seen later at Orono and Dover; but in all cases the
disease was in its last stages, and attempts to isolate the causal organ-
ism that season resulted in failure.

In 1908 diseased plants were obtained from Dover, Piscataquis County,
on July 24, which showed the affected tissues filled with actively motile
bacteria. Several subcultures were made from plates poured from these
stems and all proved to be nonpathogenic.

At the same time the diseased portions of the stalks were mashed up
with distilled water in a mortar. Healthy potato tubers, bearing shoots

1 The author also presents considerable data relative to experiments conducted to determine the avail-

able sources of nitrogen and carbon, using a considerable number of different substances, apparently added
to the culture medium singly or in combination.

Jan. 15, 1917 Blackleg Disease of Potato 99

2 or 3 inches long, were thoroughly moistened with this watery extract
of the diseased tissues and at once planted in boxes of soil which had
previously been thoroughly wet down. A part of the remaining watery
extract was then poured over each box. ‘These artificially infected tubers
put up shoots very rapidly and on August 15, 22 days after the planting,
one stalk began to show the characteristic signs of the disease. On the
18th two others were also plainly affected. Examination showed only
motile, rod-shaped organisms in the diseased tissues. Poured plates were
made from these stems and 9 subcultures were obtained, all of which
proved to be pathogenic to potato tubers and stems, producing typical
blackleg when inoculated into the latter. Some preliminary work,
especially in the line of fermentation studies, was done with all of these,
but one strain, designated as “IIIA,” was selected for detailed study.

On August 12, 1908, a potato stem about 18 inches long was received
from the University Farm, Orono, Penobscot County, which showed a
soft, colorless decay which apparently was progressing very rapidly. The
general appearance was decidedly different from the picture presented
in typical cases of blackleg. The stem was affected nearly its entire
length and there were no signs of blackening. ‘The disease was confined
almost entirely to the parenchyma cells of the pith within the vascular
ring. Apparently the water conducting system had not been materially
affected, as there was no yellowing or other abnormalities of the foliage and
very little signs of wilting, although one or two other stalks in the same
hill had begun to fall over.

The affected portion had been reduced toa pulpy, watery mass; appar-
ently the middle lamella had been destroyed between the cells which
latter had collapsed into irregular, shapeless masses. The liquid between
the cellsewas filled with large numbers of motile bacteria. From plates
made from this stem 5 subcultures were obtained, all of which proved to
be pathogenic to potato tubers and stems. However, in all inoculation
tests extending over a period of eight years, typical cases of blackleg were
produced, differing materially from the appearance of the original stem.
One strain, marked ‘‘SE,’’. was studied as a representative of this type.

In August, 1910, another series of four cultures, of which “IIP’’ was
selected as a representative, were isolated from a typical blackleg stem
received from Presque Isle, Aroostook County.

Thus, it will be seen that the organisms which were used for the detailed
studies were obtained from somewhat widely separated localities, repre-
senting two typical cases of blackleg and a third which looked like a radi-
cally different type of stem disease, characterized by a more rapid and
much more extensive decay of the stem without the development of any
blackening or discoloration.. As will be seen later, these proved to be,
except for slight variations in size, all of the same type as B. solanisaprus
which was not unexpected in view of the fact that Harrison has found this
organism of wide distribution in Canada and there is considerable reason

100 Journal of Agricultural Research Vol. VIII, No. 3

to believe that the disease was introduced into Maine from Canada.
It should be noted, however, that Smith (33) reports having isolated
B. phytophthorus, which he considers to be closely related to but not
identical with B. solanisaprus, from potatoes grown in Maine.

At the beginning of these comparative studies in 1908 an attempt was
made to collect cultures of all the available organisms which had been
described as causing a similar type of disease upon potato stems and
tubers.

So far as could be learned, B. atrosepticus Van Hall was the earliest
described species available. A culture so named was finally obtained
from Kral’s Bacteriologisches Laboratorium, Prague, Austria.

Difficulty was experienced in obtaining cultures of B. phytophihorus,
The cultures were requested of Dr. E. F. Smith, of the United States
Department of Agriculture, and of Dr. O. Appel, of the Biologische-
Anstalt, Dahlem. Dr. Smith, while expressing a willingness to pass
along the culture, stated that he preferred not to part with it till he
had published on the subject. Two cultures bearing this name were
received from Dr. Appel. These were apparently identical. Neither of
them proved to be pathogenic, either to potato tubers or stems, on
repeated inoculations. Apparently Dr. Appel had lost his original culture,
from which, presumably, Dr. Smith’s strain was obtained, and writing
under date of February 14, 1910, he stated that it had been necessary
to make a fresh isolation of the organism before he could send it. The
writer again wrote to Berlin in the summer of 1911, and a second culture
was sent him. This reached the writer in February, 1912, by hand of
Dr. W. A. Orton, of the United States Department of Agriculture, who
kindly consented to bring it from Germany. Accompanying the culture
was the following statement: :

B. phytophthorus is isolated from potato tubers by Dr. Schuster, 1911, at Dahlem,
Berlin, whose paper on rot of potato tubers is going to be published.

This culture was also found to be nonpathogenic to potato stems and
tubers, and, as will be shown later, showed certain well-marked differences
in its behavior on culture media when grown side by side with the culture
received earlier from Dr. Appel under the same name. It should be
remarked here that Dr. Appel recorded the fact that with him some
cultures of B. phytophthorus showed a less virulence after growing for
some time on artificial media. On the other hand, Dr. Smith (33, p. 749)
states: ‘‘I have not observed any during a period of three years.”” None
of the other organisms studied by the writer have shown any loss of
virulence on long-continued cultures, stock cultures being carried either
in potato broth or beef-extract bouillon.

My request to Dr. Harrison for a culture of B. solanisaprus was referred
to Prof. S. F. Edwards, then of the Ontario Agricultural College, who
furnished me with a virulent culture in March, 1909.

Jan. 15, 1917 Blackleg Disease of Potato IOI

B. melanogenes was received from Dr. Pethybridge in 1911. This has
also shown a good degree of virulence and like B. solanisaprus has re-
peatedly produced on inoculation from pure cultures a rapid and com-
plete softrot of potato tubers and a typical decay of the stem resembling
in every particular the blackleg as it occurs in the field.

METHODS USED

In the present investigation the writer has endeavored to follow as
closely as possible the procedures outlined by the Committee on Standard
Methods of Water Analyses ‘ and those recommended by Smith (32, v. 1).

For what seemed good and sufficient reasons, certain deviations were
made from the procedures recommended above; but wherever this has
been done, mention has been made of the fact and each named organism
or cultural strain has been treated exactly like all others. The most
important change has been the substitution of Liebig’s extract of meat
for infusion of lean beef as a basis for ordinary bouillon, gelatin, agar,
and bouillon containing carbohydrates for fermentation purposes.

‘The reasons for this are that the meat extract is a standard product,
compounded on a large scale by a reliable concern, and can be purchased
the world over, while, on the other hand, no two lots of lean beef obtained
from the market have exactly the same composition with regard to
amount of fat or fiber present, or decomposition products developed
from the different periods of storage before use. The chief objection to
the use of lean beef as a basis of fermentation broths is that it is first
necessary to remove.the muscle sugar from the meat infusion by inocula-
tion with Bacillus coli. Consequently the test for fermentation which
follows is made, not in a culture medium most favorable to the growth
of the organism to be tested, but in one containing the various metabolic
by-products of another. Each lot of meat-extract bouillon before being
used for fermentation purposes was first tested for fermentable carbo-
hydrates by filling a fermentation tube with it and inoculating it with
B. coli. Inno case was any gas produced. Moreover, in many tests of
bouillon made from Liebig’s extract during the last 15 years, in only one
instance did any gas develop and then only a small bubble in the end of a
fermentation tube, the closed arm of which contained approximately 25
c.c. of medium.

However, a careful comparison was made as to the rate of develop-
ment and vigor of growth of the various organisms in bouillon made
from an infusion of Jean beef, and in that made from Liebig’s extract.
In ordinary bouillon no difference was observed between the two in this
respect. In fermentation broth made from the meat extract a better

1 Report of Committee on Standard Methods of Water Analysis to the laboratory section of the Ameri-

can Public Health Association, presented at the Havana meeting, Jan. 9,1905. 141p. Chicago, Ill., 1905.
Reprinted from Jour. Infect. Diseases, Suppl. 1, 1905.

102 Journal of Agricultural Research Vol. VIII, No. 3

growth and more constant results were obtained than with that made
from lean beef and freed from fermentable carbohydrates by use of B. colt.

All chemicals employed in making media or testing reactions were
either chemically pure or were of the highest purity obtainable. In
addition to Liebig’s extract, Witte’s peptone, “Gold label” gelatin or
Nelson’s photographic gelatin No. 1 were used, the latter entirely during
the last of the work. For bouillon 5 gm. of Liebig’s meat extract and
10 gm. of Witte’s peptone were used for each liter of distilled water. To
this were added 15 gm. of agar shreds or flour, or 100 gm. of gelatin if a
solid medium was desired. No sodium chlorid other than that contained
in the meat extract was added. All media unless otherwise specified
were made neutral to phenolphthalein. Distilled water was used for all
culture media and Griibler’s dry stains formed the basis for all staining
liquids.

Tubes of ordinary bouillon and agar were sterilized by heating once
in an autoclave for 15 minutes at from 3 to 6 or 7 pounds’ pressure.
All vegetable media or media made from vegetables or containing sugars
were sterilized by flowing steam, for tubes, 15 to 20 minutes at from
99° to 100° C. on each of three consecutive days. In the earlier part of the
work gelatin was also sterilized by the discontinuous steaming method.
Later it was found that it was less likely to become liquid at ordinary
room temperatures if the tubes were sterilized by one exposure in the
autoclave to steam under 5 to 7 pounds’ pressure for 15 minutes.

At the beginning of the comparative work with the assembled cultures
each was transferred to a fresh potato-broth culture every 24 hours
for several successive days and then gelatin plates were poured from
the last potato-broth culture. From these plates transfers were made
from single colonies to tubes of sterile bouillon to furnish fresh
subcultures with which to work, after proving that the subcultures
thus obtained were pathogenic. This was to insure that the cultures
were in as near a uniform condition of vigor as possible before beginning
to work. Attempts were made to revive in this way the pathogenicity
of the two strains which were received as B. phytophthorus, but without
success. A separate series of stock cultures of each strain which had not
been put through this revivifying process were kept in reserve.

All transfers to media to test cultural features were made from young
broth cultures about 48 hours old, when the cloudiness had reached its
maximum density. For stab cultures to agar and gelatin a straight
platinum needle of approximately 0.5 mm. in diameter was uged. All
transfers to liquid media were made with a 2-mm. loop of the same
sized wire. Unless otherwise specified the cultures were incubated at
20° C. In making records of the detailed features of the morphology,
cultural, physical, and biochemical features, etc., of the various organ-
isms the descriptive chart or card adopted by the Society of American
Bacteriologists in December, 1907, was used as a model and guide.

Jan. 15, 1917 Blackleg Disease of Potato 103

DETAILS OF COMPARATIVE STUDIES

MORPHOLOGY

B. atrosepticus, B. solanisaprus, B. melanogenes, and the three organ-
isms studied which were isolated from Maine potato stems were found
to agree in all essential morphological characters except for certain vari-
ations in size. They are motile, rod-shaped organisms with peritrichiate
flagella, approximately three times as long as broad, occurring singly
and in pairs, and in unstained preparations from young cultures often
showing chains of several individuals. Frequently, however, rather
thick rods with very little difference in the two dimensions were observed,
more especially in preparations made from the decayed tissues of inocu-
lated tubers. The organism obtained from Dr. Appel as B. phytoph-
thorus, while agreeing in other morphological characters, was distinctly
smaller in size and the ratio between length and width was decidedly
less. No evidence of spore formation was obtained. Involution forms
were not observed, although Harrison (17) reports them for B. solani-
saprus when grown at 37° C. In this connection it may be mentioned,
as is stated later, that at this temperature the writer has been unable to
secure visible growth of any of the pathogenic strains either on agar
slants or in tubes of beef broth.

For making measurements all of the different strains studied, except
that obtained from Dr. Schuster as B. phytophthorus (which was a non-
pathogenic organism plainly of an entirely different type), were stained
with aqueous gentian violet, anilin water gentian violet, aqueous methy-
lene blue, alkaline methylene blue, aqueous fuchsin, and carbol fuchsin.
These preparations were made from agar slants 36 to 48 hours old.
Flagella were stained by a modification of the Pitfield method.

The results of these measurements are given in tabular form below.
Table I gives for each organism the extremes in length and breadth and
the average size obtained as shown by all the stains collectively. The
second presents the same data for each organism for each separate stain
used.

TABLE I.—Extreme and average measurements (in microns) of each organism as obtained
from using six different stains

Organism, Width. Length. Average size.
BS GtOSEPLICUS. halve docsatneNe See ovate oils erates 0.4too8}] 1.3t02.5.| o5by 18
TB SOLGRASE PTUSE icp Aa IORI haere ey nets Bnet Dy eeeatOl ea 7a lm. OuCOnay 2 Bist oiaacy:
ESENMNEVANOGENES Ses re cin os o/h ste nal ais ates eis otenshsta ie SANtol) 23) vr. 2) toe.) 5 Dekel
SIATATCAUES SF iS yanebcidatetar orencres syn chen euaiavers te erty axel aia eae rans 2G COMnas eters tise eu 2
Eerste ya ceitercis ratera abt easte srecbio ais ante el g arepeenegs 74 to? 2S) | T etoeae .6by 2
5 (NG) By Saal ctl ah eee eARS TT ard tev RRO PARCOMe OF |) tin aecOl25/0 .6by 1.9
B. phytophthorus from Appel............... Ss tony, 15 ton 6 ag by S90

1 The writer wishes to acknowledge the aid rendered by Messrs. M. Shapovalov and A. Strauss in assist-
ingin making many of these preparations, particularly the laborious task performed by the latter in making
the large number of measurements of stained organisms, which is the basis of the data here presented,

104 Journal of Agricultural Research

Vol. VIII, No. 3

TABLE II.—Measuremenis (in microns) of each organism arranged according to method
of staining

AQUEOUS GENTIAN VIOLET

Organism. Width. Length. Average size.
IBN OULOSEPTICUS: ich lk aeh utente Cah Pe sae ioe: O15 tOvO.'7) |) TO tOl2 oe oNOubiye2
EP SOLANISOPTUS.. new te Aes Pe OS ree ee = ANEO) tye, 7 || ch Te LOUENG oly longs:
ES SMMCLANO GENES 85 she SET ARE CATR RON eee a ES AMLOl eal maeian cones Sw cuby. Iso
1D Dao eR eOm aS tea raatte te Sivek Yee RRC Aer SS iLO 71 TRORtO ss: .6by 2.1
Se erisiat leivt ess ance c an eceened ed ea OBO - 5 Ov. S ||) TO rere .6by 2
TEP ee duke We ae a CoO ER ri emer DP ANLOM a7 Nt S Ona G 6 by 1.9
B. phytophthorus from: Appel). 22. os... ey OG (6 .6 tor.4 SSR DAVIE REG
ANILIN WATER GENTIAN ‘VIOLET
B AGUOSCPHCUS Rien te aE aT Seog ese @: 4 tore: 7#)| "55 3702 0. 5 by 1.6
By. SOMME PRUs aces eRe OE eleva 298d 4) TON 25 |p tO tOuE/6 .5 by 1.2
EX ONELEN GGON ES he ee PII EE is ey SEIS h e6. con ues AMATO af |) aig Gyn KOS at 2S DY. a7
je ee eee Sa eee a Oe A RR SH PLON Onli 5 tones .6by 2
OE toaie sion sds, OM Da eles 5 ccytals wGitos 18) \TyOitoven's .6by 2
UD es SPR eae te as RAN AD a SEO! eg) ede AtOLOTO 6by r.9
B. phytophthorus from Appel............... TALON) AG 25 to,6 5 by
AQUEOUS METHYLENE BLUE
TS OUGO SEPRIEUS cele Seyi p ya kic epee eee ee 0: 4°t0'0.'8 13 402.3) Ors by 16
BASOLANESOPTUS Aiea OO MAS oboe eink 14 40°05) Te tong 25 Dy mo4,
Ee mnelamg gemiese e.) cit sep motel tic davis ke syayer 2 AS LOWE 7 | adeegntOye ~§ DY 2.7
HOD) es SR NES Hs ys Ra eG LORE UN GP 1H) S083 6 by 2.3
SES UALS cp rn eR aeoen anes & EASIER EEE oe 25 tor So a Goes -7 by 2:1
UO) Se PP RR ace UCI cyte To OE RR ee ee 5410) Ol Wet OMtOl276 6 by 2
B. phytophthorus from Appel............... utOl ee ALO 5 by .9
ALKALINE METHYLENE BLUE
TB GU OSC DUSCUS ete cept ae By yh lays epee ea OyANLOLON 7 sel AgtOl2 435 Ons yatta
ID SOMIMESAPTUS a ee rea cee ete Tee S14 CON Zee cOretes G54 ON Tig S
Bi QNClanoGenesmicp nee: een Re nic. Ls et we 4to .8] 1.3to2.4 -5 by 1.9
ST TEAo.2x2 baal oe eee eects. ct 2d. OR spa 5 tor I, 7 :tO;2: .&8by 2.2
Nhe le, Hy stat ae eater a tbs ts (Py ae 5604.85) 1 tg tore. 5 .6by2
Ee oe icles baal Ge eke a nN Seating ACOM ION Wihela G tOR2 Eo 2OMDva2
B. phytophthorus from Appel............... ANcOMns 7 LOL Ted -§ bY .9
AQUEOUS FUCHSIN
Bi GiOSe PUES Aa pir IRE Noel i's Oi hha ae 0:4: 10'0N7 | vig 8O 223M) Lous by, te
B SOLUNG A HIS sonsD EMA mig es hse so oaths SAMO} 7All eke iLO -5 by 1.4
IB mealanagenes i Aaeeree ene. vce Seat TAL LO 77a | edna tore 5 by 1.6
TTA G oc stile Satae ct a eames tates ai) 0. Dea 2/5. £0) 8. jt OnE ays 7 by 2
CS ear i URS Su NAC a ah ge A tO 7 a. tO 2.2 6by 1.9
LBD ai Me Sn a ale 2 V2 Ne ee 4 tO(2 7a) eg toes s 5 by 1.8
B. phytophthorus from Appel............... na ton6 5 ntOums Sbys 29

Jan, 15, tor7 Blackleg Disease of Potato 105

TaslEe II.—Measurements (in microns) of each organism arranged according to method
of staining—Continued

CARBOL FUCHSIN

Organism. Width. Length. Average size.
BEN GITOSCDULCUS aan cL etary. [alec Meet ee siene 2 eta ske O74) too; 7 | °r. 4 toes x 0.5 by 1.7
IB USOLENTSAD TUS acikiens tas AARC A RT OR oer .4to .6 TO tO#E.@ 7h DVL. 3
BEVINCLANOGENES a Mater, st Gates lod RARE. hs fies ais AGO L 2) tOLe Selon, 155
ATT Aer costs) Sekcrar east ako) catenin a sh amenysrala Septet 5 WS) ney || TON, ZED, eS
BES. UN SAE IU Nh RRP ae dein Lager Shs WEES AG, 27ht Tr 3htotes 7 5 by 't:6
f UIC) POR Mote te ie Se Beste ane AL Wem rN ta TCU ARLOna 7a er ke ae CONQEET SO ae
B. phytophthorus from Appel............... ATtOn)6 .6to 1.2 = 5 DY £O

If the strain known as B. phytophthorus from Appel be omitted, it
will be seen that, while there are minor variations with different stains,
the three organisms from Maine run a little larger than those previously
named and described. Of these, IIIA is slightly larger than the other
two. Of the remaining three, while B. solanisaprus is plainly somewhat
smaller, on the average this difference is very slight. Taken as a whole
it seems difficult to separate the six different strains on the basis of
the morphological characters. It is true that the measurements given
differ somewhat from those previously recorded for the described organ-
isms. The writer is not in position to say which are the more accurate,
but this does not appear to him to be the important point under con-
sideration. What is given here is the relative measurements from
stained preparations of the different organisms, made under as nearly
uniform conditions as possible, and measured by the same individual,
using the same apparatus for all.

CULTURAL FEATURES

AGAR STROKES.—On agar slants the growth characters of B. atrosep-
ticus, B. solanisaprus, B. melanogenes, 111A, SE, and IIP were identical
in every respect except as noted below. The growth was moderate,
filiform, with a slight tendency to produce pseudopod-like branches on
the surface, if the latter was slightly moist, or such branches might grow
up from the bottom of the slant from the condensation water. Eleva-
tion flat to slightly raised, luster glistening, topography smooth. Slightly
but distinctly bluish opalescent to transmitted light (a smear on the
surface of slants with a loop would produce a decidedly opalescent
growth). Color pearly white, odor absent, consistency butyrous, slightly
viscid in the case of B. solanisaprus and B. melanogenes. No discolora-
tion of the medium was observed in any of the cultures of the organisms
named above.

B. phytophthorus from Appel differed from the above in that the growth,
though moderate, was spreading and very finely plumose, giving a char-.
acteristic appearance under a low-powered hand lens. B. phytophthorus

106 Journal of Agricultural Research Vol. VIII, No. 3

from Schuster also showed characters on agar slants which differentiated
it from all of the other cultures, including the one just mentioned. The
growth while filiform at first was more abundant, and later it spread out
in a broad band one-third to one-half the width of the slant. It was
thicker, somewhat convex and yellowish, and not opalescent. It very
soon imparted a very distinct yellowish-brown color to the substratum.

AGAR STABS.—The first six organisms mentioned in the above section
produced the same appearance on agar stabs. The growth was fairly
uniform but slightly best at the top, although perhaps not more than
would result from the heavier inoculation of the upper layers of the
medium. ‘The entire surface was covered in from a few days to about
a week, depending upon the amount of free moisture present. The
line of puncture was filiform to very slightly papillate.

The two organisms carried under the name “B. phytophthorus”’ grew
decidedly best at the top, and the one from Schuster caused a distinct
browning of the medium, beginning at the surface and diffusing down-
ward.

COOKED POTATO CYLINDERS.—The character of the growth on the
cooked potato has been a prominent one among those given by different
authors as differentiating from the others in the group the organisms
which they have described, consequently considerable -attention was
given to this topic.

For the work potatoes were chosen which were either freshly dug or
had been in storage only a few weeks. In all cases the tests were made
on slanted cylinders of sterilized potato in test tubes containing a small
amount of distilled water. The tubes were inoculated as soon as sterility
was proven, by making a single stroke with a straight needle along the
center of the slant.

Inoculations made to tubes of this kind gave practically identical
growth characters for B. atrosepticus, B. solansaprus, B. melanogenes,
IIIA, SE, and IIP, which are as follews:

At 20° C. little or no growth apparent in 24 hours; needle growth
plainly evident in 48 hours, and if the slant is rather moist, the growth
may have spread out by this time to cover a considerable portion of the
lower part; in three days, growth moderate to abundant, filiform to
spreading, depending upon the amount of water in the substratum; one
week, growth abundant and spreading, usually covering the entire surface
of the slant. Elevation first convex, then slightly raised to flat; luster
glistening; topography smooth to slightly rugose; color yellowish white,
resembling pus in appearance, later dirty white; odor not apparent at
first, but later slight odor of decayed potatoes; consistency butyrous,
with a slight tendency to viscidity in the case of B. solanisaprus and B.
melanogenes. Medium at first slightly grayed, and at the end of a week
plainly grayed.

Jan. 15, 1917 Blackleg Disease of Potato OF,

The organism received from Appel as B. phytophthorus produced no
apparent growth upon potato cylinders, but the liquid below was clouded,
and the substratum grayed or slightly browned.

The organism received from Schuster as B. phytophthorus upon cooked
potato agreed with the detailed description of the group given above,
except that in the earlier stages the growth was more abundant, and at
all times it had a distinct yellow color, especially on the more moist por-
tions of the slant. This agreed quite closely with the “honey-yellow”’
color which Appel originally described for B. phytophthorus. ‘The medium
was considerably grayed and had a brownish tinge.

GELATIN STAaBs.—The behavior of the various named species with
reference to liquefaction in gelatin stab cultures had been used also as a
differential character. While all are reported as liquefiers, Harrison (17),
who did a large amount of work with B. solanisaprus, reports this organ-
ism as very slow in this respect, liquefaction not beginning until 35 days,
and not complete in 44 days. ‘The others are reported to liquefy gelatin
much more rapidly, although van Hall (15) stated that with B. atrosep-
ticus liquefaction was variable. _

Therefore in the present studies particular attention was paid to the
behavior of the various organisms in gelatin stabs. With regard to B.
atrosepticus, B. solanisaprus, B. melanogenes, IIIA, SE, and IIP, the
behavior was somewhat erratic on this medium, especially when the
“gold label’? gelatin was used; sometimes one would liquefy the medium
more slowly, and sometimes another. For example, in one case with
B. solanisaprus liquefaction began on the third day, and was not com-
plete in 60 days. In another series the same organism, incubated at the
same temperature, 20° C., showed the beginning of liquefaction in less
than 24 hours, which was complete in 7 days, or the shortest time ob-
served for any of the series. After Nelson’s photographic gelatin, No. 1,
was adopted, the results were much more uniform. So close were these
at times that in a series of duplicate or triplicate cultures of the 6 organ-
isms named it would be impossible to pick out those which were of the
same name, except for the labels on the tubes.

Therefore the final conclusion was that B. atrosepticus, B. solanisaprus,
B. melanogenes, and the three organisms from Maine present no well-
defined differential characters upon gelatin stab which would separate
them from each other. The observed characters of the six on gelatin
may be briefly summarized as follows: A needle growth was apparent
in 12 hours at 20° to 22°C. Liquefaction as a rule begins in from 18 to
24 hours, and may not begin until the third day. It is usually complete
in about 10 days, but may be delayed for some time longer. Usually
liquefaction is infundibuliform to slightly napiform and later may assume
a saccate appearance. Occasionally liquefaction starts off rapidly at the
surface, giving a distinct crateriform appearance, which is quite likely to

108 Journal of Agricultural Research Vol. VII, No. 3

change to stratiform, and progresses very slowly from that time on.
These variations were common to all of the pathogens and were not
confined to any one of the series. No change of color or fluorescence was
observed. ‘The liquefied medium is quite turbid at first, and a copious
flocculent or granular, whitish deposit settles out into the bottom of the
funnel.

Of the two cultures received as B. phytophthorus, that from Appel did
not liquefy gelatin and that from Schuster produced first a crateriform
and then a stratiform liquefaction of only the upper portion of the
medium, which was browned.

NUTRIENT BROTH.—In neutral nutrient broth (10 c. c. inoculated with
a 2-mm. loop of young broth culture) the clouding in the case of B.
atrosepticus, B. solanisaprus, B. melanogenes, IIIA, SE, and IIP is very
rapid. It isapparent in 13 to 16 hours at 20° C., varying somewhat with
different strains, very evident in 18 hours, and is quite marked in from
24 to 48 hours. Later moderate to strong, persistent, the fluid being
frequently turbid in old cultures. Usually a very slight ring may be
observed in young cultures, and occasionally a very slight, granular
pellicle is formed during the first few days of growth which readily breaks
up and falls to the bottom if the tube is handled or even slightly jarred.
The sediment is compact, scant, granular, and dirty white. No odor
was observed and no discoloration of the medium. As a rule,a very
slight viscidity in both sediment and liquid was noted in the case of B.
solanisaprus and B. melanogenes. Otherwise the six above-named
organisms were exactly identical in respect to observed characters upon
bouillon.

The organism received from Appel as B. phytophthorus differed from
the above in that with it the sediment is very adhesive. While it was not
strictly viscid it resisted pulling apart with the needle and then appeared
very stringy. There was no evidence of viscidity of the medium itself
in cultures of this organism. The organism received from Schuster as
B. phytophthorus differed from all the others in that in old cultures the
medium was distinctly browned. While the sediment was dirty white
it was rather more abundant, and appeared more stringy and viscid than
was the case of the other culture carried under this name.

GROWTH IN POTATO BROTH.—The potato broth used was made as
follows: Sound, fresh, recently harvested potato tubers were used and
after peeling and washing, 500 gm. were grated directly into 1000 c. ¢.
of distilled water. This potato pulp and water was heated for one hour
at a temperature of 55° C. and filtered first through cotton and then
through filter paper toremove thestarch and pulp. The filtrate was then
placed in the steamer for 40 minutes at 99° to 100° C., and again filtered
through paper. ‘Titration showed the natural acidity to be equivalent
to +10 Fuller’s scale and this was not changed. ‘The medium was then
tubed and sterilized by fractional steaming—15 minutes at 99° to 100° C.

Jan. 15, 1017 Blackleg Disease of Potato 109

on each of three consecutive days. It gave a faint blue color on the addi-
tion of iodin solution, showing the presence of a small amount of starch.

In ordinary culture tubes. the appearance produced by B. atrosepticus,
B. solanisaprus, B. melanogenes, ILA, SE, and IIP was absolutely iden-
tical, except that the last named produced somewhat more turbidity with
less cloudiness. The characters shown were essentially those recorded
for beef bouillon except that the growth was somewhat more vigorous
and clouding appeared more rapidly, with a greater tendency to produce
a ring or pellicle on the surface. This latter was of a granular nature
and was less easily broken up than was the case with the pellicle formed
on beef bouillon.

In fermentation tubes filled with potato broth there appeared a
thorough, uniform clouding of both bulb and closed arm in from 18 to
24 hours, but no gas of any kind was formed.

With the cultures received as B. phytophthorus from Appel and as B.
phytophthorus from Schuster the growth in ordinary culture tubes was
the same as in beef bouillon except that with them it was also some-
what more vigorous. In fermentation tubes containing the potato
broth B. phytophthorus from Appel showed a marked contrast in the
appearance of the same organism in fermentation tubes of ordinary
beef bouillon containing carbohydrates. With the latter it was never
observed to produce any visible clouding of the closed arm of the tube,
while in the tubes of potato broth there was a faint clouding of the closed
arm with a very perceptible growth on the third day. In the case of
B. phytophthorus from Schuster the characters were not essentially dif-
ferent from those observed with‘ordinary bouillon plus carbohydrates.
There was a rapid and heavy clouding ot the bulb, followed by a slow
clouding of the closed arm.

After the cultures had grown for 10 days, the broth was tested with
iodin solution, but in no case did there seem to be any diminution of the
amount of starch present.

MiLkx.—Very little difference could be observed in the action of B.
atrosepticus, B. solanisaprus, B. melanogenes, IIIA, SE, and IIP upon
fresh skimmed milk sterilized by fractional steaming, except that SE
produced a slow but evident digestion of the curd. With this excep-
tion noted the record for the group when grown at a temperature of
20° C. is: Coagulation somewhat delayed, not appearing for about 7
days; extrusion of whey does not take place until the end of 10 to 14
days, and only a slight amount is formed. Coagulum solid and appar-
ently not digested, but readily breaks apart when probed with the needle,
not slimy nor viscid, and when shaken forms nearly a solid mass with
the whey. It will be noted that both Harrison (17) and Pethybridge
and Murphy (26) report coagulation in 48 hours, but this is at 25° C.
The latter gentlemen, however, recorded coagulation not appearing for
five days at laboratory temperatures.

110 Journal of Agricultural Research Vol. VIII, No. 3

The two organisms carried under the name of B. phytophthorus were
not tested upon milk and litmus milk.

LITMUS MILK.—On litmus milk the six organisms mentioned above
produced at 20° C. a rather slow, moderate acid development at first,
but later the cultures became strongly acid. At this temperature reduc-
tion or bleaching begins to appear at the end of one month’s time. If
the tubes were then heated sufficiently to kill the organisms, the red
color promptly returned.

GELATIN COLONIES.—Colonies upon gelatin plate cultures made from
meat-extract broth plus ro per cent by weight of Nelson’s photographic
gelatin No. 1 and made neutral to phenolphthalein, incubated at 20° C.
were identical in the case of B. atrosepticus, B. solanisaprus, B. melano-
genes, and the three Maine organisms. ‘These characters were: Growth
rapid, buried colonies in 24 hours, round, margins entire. Under a
16-mm. objective and 12 eye-piece, the colonies at this time appeared
nearly white and finely granular, usually with a faint indication ot
liquefaction at the margin. In 48 hours, in every case, a well-defined
crateriform liquefaction had appeared and heavily oo plates would
be largely liquefied by this time at 20° C.

AGAR COLONIES.—The organisms mentioned in the preceding para-
graph likewise gave characters identical with each other upon neutral
meat extract agar. In 24 hours, viewed under a 16-mm. objective and
6 ocular, the colonies were brownish-yellowish in color, finely granu-
lar, margins entire, lens shaped to slightly ovoid or spherical. Viewed
under a hand lens the colonies were slightly yellowish in color. The
surface colonies were pearly white, bluish opalescent to transmitted
light, flat, circular, and occasionally amoeboid in shape if the plate were
thinly seeded and the surface moist.

Some preliminary work was done, but no detailed studies were made
with regard to the colonies produced upon gelatin and agar with the
two organisms received and studied as B. phytophthorus.

FERMI’S SOLUTION.—In liquid cultures of Fermi’s solution all the
organisms showed a slight clouding in 24 hours. In 48 hours this was
very well defined, but moderate, in the case of B. atrosepticus, B. solani-
saprus, B. melanogenes, IITA, SE, and IIP, with a tendency to form a
pellicle, which in the case of SE was frequently quite well defined. B.
phytophthorus from Appel and B. phytophthorus from Schuster at the
same: time had produced only a faint trace of cloudiness in the medium
which later disappeared. Later observations showed considerable in-
crease of cloudiness, almost to opaqueness in the case of the first six
organisms named. ;

FERMI’S SOLUTION PLUS AGAR.—No attempt was made to grow the
various organisms on Fermi’s solution in silicate jelly, but one trial was
made in which one and one-half per cent of agar was added and the
sterilized tubes of media, slanted as in ordinary beef-peptone agar. On

Jan. 35, 1917 Blackleg Disease of Potato 111

these agar slants in 24 hours after being inoculated with a single stroke
of a straight needle there was no visible growth. In 48 hours and later
it was well defined and of uniform appearance in the case of B. atro-
septicus, B. solanisaprus, B. melanogenes, IIIA, SE, and IIP. ‘The
character of the growth may be described as moderate, filiform, flat to
slightly raised, smooth, glistening, plainly opalsecent, later opaque,
color whitish or pearly, later creamy, odor absent, consistency buty-
rous—slightly viscid in the case of B. solanisaprus and B. melanogenes.

The two strains carried as B. phyiophihorus produced only a very faint,
semitransparent, filiform growth on this medium. No discoloration of
the medium was observed in either case.

CoHN’S SOLUTION.—No growth was obtained with any of the organisms
when inoculated into Cohn’s solution.

USCHINSKY’S SOLUTION.—The organisms received from Dr. Schuster
as B. phytophthorus gave little evidence of growth in this medium, while
all of the others produced a copious growth and behaved alike in it.
Visible clouding with the latter was less during the first 24 hours than
in broth tubes inoculated at the same time, but at the end of three days
the medium was quite heavily clouded and milky in appearance.

SODIUM CHLORID IN BOUILLON.—One, two, three, four, and five gm. of
sodium chlorid to each 100 c. c. of culture media were added to neutral
beef infusion bouillon and narrow tubes each containing 5 c. c. of this
salted medium were inoculated with a 2-mm. loop in the usual way.

At the end of 18 hours there appeared to be no inhibition of growth of
any of the cultures in the tubes containing 1 and 2 per cent of sodium
chlorid. In 3 per cent, all were slightly but plainly less clouded than the
check tubes inoculated at the same time, and no growth had appeared
in the 4and 5 percent. At the end of 48 hours all the tubes containing
3 per cent of sodium chlorid were apparently as well clouded as the
checks. All showed growth in the presence of 4 per cent, but with some
inhibition. In 5 per cent no growth was apparent in any of the tubes
except those of B. phytophthorus from Appel, which showed about half-
normal clouding for bouillon cultures of the same age. In three days B.
atrosepticus, B. solanisaprus, B. melanogenes, IIIA, SE, and IIP in 5 per
cent of sodium chlorid showed a faint but well-defined clouding for the
first time and appeared quite uniform in appearance. B. phytophthorus

rom Appel was quite heavily clouded, while no growth could be detected
in the culture of B. phytophihorus from Schuster.

GROWTH IN BOUILLON OVER CHLOROFORM.—This test was made with
tubes containing ro c. c. of sterile bouillon into each of which were intro-
duced 5 c. c. of chloroform and the whole thoroughly agitated at inter-
vals for two days. After allowing the tubes to incubate for several days
to prove sterility they were inoculated with a 2-mm. loop of broth cul-
ture in the usual way, care being taken this time to agitate the medium

112 Journal of Agricultural Research Vol. VU, No. 3

as little as possible and to introduce the inoculating loop only into the
upper layers of the culture liquid.

In 18 hours there was no growth in any except B. phytophthorus from
Schuster which showed a little clouding in the upper layers. Daily
observations were made following this for several days. 7

In 36 to 48 hours as compared with normal broth cultures inoculated
at the same time those of B. atrosepticus, B. solanisaprus, B. melanogenes,
IITA, SE, and IIP were much restrained, but there was an evident slight
clouding all through, slightly stronger at the top. The same condition
prevailed as long as the cultures were under observation, except that
after three or four days the three orgasiisms from Maine showed a more
vigorous growth than the other three and produced a distinct, moderate
clouding all through the bouillon. The appearance of the first three
named was identical in all respects.

The growth in the cultures known as B. phytophthcrus from Appel.and
from Schuster after about 48 hours was unrestrained and appeared
equally as abundant as that in the check tubes.

BEST MEDIA FOR LONG-CONTINUED GROWTH.—Of the various media
used, neutral beef bouillon, made either from meat infusion or from
Liebig’s extract, proved to be the best for long-continued growth.

PHYSICAL AND BIOCHEMICAL FEATURES

FERMENTATION OF CARBOHYDRATES.—The ability of the different
organisms in this group to ferment various carbohydrates has furnished
perhaps the most important differential characters upon which the pre-
viously named species have been erected. Therefore in the present
studies more attention has been given to this subject than to any other.
The results obtained are not based upon single trials, but upon repeated
tests of each organism. All the fermentation tests were made in uni-
form fermentation tubes, having a small neck, a large bulb, and a
capacity of about 25 c. c. in the closed arm. ‘These were made to order
and very closely conform to the’ illustration given by Smith (32, v. 1,
p- 53). Both meat-infusion bouillon, previously freed from muscle sugar
by inoculation with B. coli, and meat-extract bouillon made from Lie-
big’s extract and tested for the absence of fermentable carbohydrates,
as previously described, were used. The meat-extract bouillon was used
for a large proportion of the work, for it was found to be more satisfac-
tory. For the fermentation work 1 per cent of the carbohydrate used
was added to the culture medium and the tubes containing the media
were sterilized by fractional steaming. The following substances were
tested for fermentation in this way: Dextrose, saccharose, lactose, mal-
tose, glycerin, mannit, and dextrin. In addition some of the organisms
were tested in the neutral-red lactose broth which is commonly used in
water work as one of the presumptive tests for B. coli. The culture
medium in each case was made neutral to phenolphthalein.

Jan. 15, r9r7 Blackleg Disease of Potato LYS

To save unnecessary repetition it may be stated in the beginning that
the two organisms received and carried under the name of B. phytoph-
thorus produced no gas or acid in the presence of any of these substances
and imparted a slight alkalinity to the culture medium. The one
received from Dr. Appel in no case produced any visible clouding of the
closed arm of the fermentation tube. The one received from Dr. Schuster
produced a delayed clouding of the closed arm in all cases except with
glycerin, but even with this the clouding was later nearly as complete
as in the bulb.

The results obtained with the remaining organisms—namely, B. atro-
septicus, B. solantsaprus, B. melanogenes, IIIA, SH, 1{1P—were alike and
constant with regard to gas formation, growth in the closed arm, and
production of acid. On repeated trials they all invariably produced gas
in the presence of dextrose, saccharose, lactose, maltose, and mannit.
They produced no gas from glycerin and dextrin. With dextrose this
amount of gas was small, ranging from 3 to 5 per cent. With saccharose
the percentage of gas was higher, usually being from 7 to 9 per cent.
Except with glycerin, there was always a prompt clouding of the closed
arm, though this was usually less than in the bulb. This clouding in
the closed arm was persistent, although a slight ciearing at the top of the
tube was observed in old cultures containing maltose. Acid was pro-
duced in the presence of all the carbohydrates used.

Very little of the gas which was formed was absorbed by a 2 per cent
solution of sodium hydrate. The remainder of the gas was explosive.
When expressed in the terms of H:CO,, as accurately as could be deter-
mined, this varied from 1:0to5 or 6:1. Noattempt was made to deter-
mine further the nature of this gas. Gas in all cases usually appeared
on the second or third day and reached its maximum before the tenth,
usually on the sixth or seventh.

It will be noted ‘that this constant appearance of gas in dextrose,
saccharose, and maltose with B. solanisaprus was contrary to Harrison’s
original description (17). Likewise the production of gas with B. airo-
septicus from lactose and dextrose differs from the results recorded by
Van Hall. In gas production B. melanogenes agreed in every respect to
Pethybridge and Murphy’s description (26).

In neutral-red lactose fermentation broth the amount of gas obtained
in each case with the 6 different organisms was the same as with the
ordinary lactose broth. In 48 hours the entire contents of the tube
appeared distinctly more red than the check. On the third day the
closed arm took on a yellowish olive tinge, which later changed to a
canary-yellow, stronger even than that produced by B. coli, run at the
same time for comparison. This color persisted for one month, or as
long as the cultures were under observation. During the same time the
bulb showed a more pronounced red than the check tube.

114 Journal of Agricultural Research —— Val. VII, No.3

AMMONIA PRODUCTION.—Cultures in Dunham’s peptone solution were
tested at the end of one, two, three, and four days as follows: In clean
test tubes of the same internal diameter there were placed toc.c. of
ammonia-free water. Into each was placed one 2-mm. loopful of the
culture to be tested, taken from the top of the tube, and then six drops
of Nessler’s solution was added to each tube of water and culture dilu-
tion. The color of the tubes was observed by looking vertically through
them upon a white background. No color developed in any of the tubes
except in those containing some of the culture of B. phytophthorus
from Schuster. The latter gave a distinct yellow color.

Pethybridge and Murphy (26) have recorded the appearance of small
amounts of gas in cultures of B. melanogenes in tubes of a 2 per cent
potassium-nitrate broth having a plug of vaseline on the top. For the
purpose of testing all of the organisms in this respect, fermentation tubes
were filled with the nitrate broth described in the next section, sterilized
by fractional steaming, and then inoculated. Growth appeared in these
rather slowly but at the end of 48 hours all were uniformly though some-
what faintly clouded with the exception of the two strains carried under
the name of B. phytophthorus. These showed only growth in the bulb.
Later they showed some clouding of the closed arm of the tube, but this
was exceedingly faint in the case of B. phytophthorus from Appel. No gas
whatever appeared in the closed arm of the tubes with any of the organ-
isms studied. Tubes of this same broth, tested at the end of five days,
with Nessler’s reagent by dropping five or six drops of the reagent directly
into the tubes gave no qualitative test for ammonia, except in the case of
B. phytophthorus from Schuster. Cultures of the latter when treated with
the reagent produced a very distinct, yellow reaction. That the Nessler’s
solution used was of good quality was indicated by the fact that it would
produce the characteristic reaction in the presence of very minute quanti-
ties of ammonia, artificially introduced into distilled water.

In connection with these tests it should be noted that Pethybridge and
Murphy used a 2 per cent potassium-nitrate broth, while the writer used
that specified by the Committee on Standard Methods of Water Analysis,? .
which carries only 2 gm. of potassium nitrate per liter.

Where Nessler’s solution was added directly to young cultures in
Dunham’s peptone solution, in no case was there any more color observed
to result than where the uninoculated check tube of the same medium
was so treated, except in the case of B. phytophthorus from Schuster.
Cultures of this organism gave a yellow color reaction.

REDUCTION OF NITRATES.—Cultures in nitrate broth, consisting of
2 gm. of chemically pure potassium nitrate, 1 gm. of Witte’s peptone and
1 liter of water, were tested by the starch-iodin method after five days. All

1 Report of committee on standard methods of water analysis to the laboratory section of the American
Public Health Association, presented at the Havana meeting, January 9, 1905. 141 p. Chicago, IIl., 1905.
Reprinted from Jour. Infect. Diseases, suppl. 1, 1905.

Jan, 15, 1917 Blackleg Disease of Potato I15

Dee a eee eee cece ee ——e—eeo

except B. phytophthorus from Appel when so tested at onceshoweda deep-
blue color reaction, indicating the presence of nitrites. No such color
appeared in the uninoculated check tubes of the same medium tested
at the same time.

From what is said in the preceding section it might be inferred that
B. phytophthorus from Schuster is apparently able to reduce nitrates to
nitrites and then toammonia. However, it should be remembered that
the nitrate broth used contained a small quantity of peptone, and it has
been shown that the organism is able to produce ammonia from peptone.
Doubtless this was the source of the ammonia reaction in the nitrate
broth.

INpoL PRoDUCTION.—Cultures of B. atrosepticus, B. solanisaprus,
B. melanogenes, the three Maine organisms and of the one received from
Schuster as B. phytophthorus gave a positive reaction to the indol test.
That received from Appel as B. phytophthorus and the check tubes always.
gave a negative reaction. The pink color, while well defined, and some-
times in old cultures being quite marked, was very much less than that
produced by B. coli, which was tested at the same time for comparison.
In the case of the positive-reacting organisms the color was very slight or
absent in cultures four to seven days old and was most marked in cul-
tures two months old or more. Therefore, indol production where it
occurred would be classed as being feeble to moderate.

TOLERATION OF acips.—In the present studies only hydrochloric
acid has been tested. Normal hydrochloric acid was added to meat-
extract broth which in the beginning was neutral to phenolphthalein,
in sufficient quantities to make it +10, +20, +30, +40, and +50
Fuller’s scale.

In from 12 to 18 hours all of the organisms showed equally good growth
in the check tubes of neutral broth. At +10 the amount of clouding
was nearly equal to that produced in the checks. At this time there was
a marked falling off in the clouding at +20 as compared with the check
tubes, and no growth had appeared in the presence of the larger amounts
of acid.

At 48 hours and later there was no change in the relative amounts of
cloudiness exhibited by the different organisms, except that cultures of
B. phytophthorus from Appel were slightly less cloudy and B. phytoph-
thorus from Schuster were slightly more cloudy at + 20 than those of B.
atrosepticus, B. solanisaprus, B. melanogenes, IIIA, SE, and IIP. The
latter were all alike in their behavior toward hydrochloric acid. None
of the various organisms produced any growth at +30 or beyond.

TOLERATION OF SODIUM HYDRATES.—Cultures of all of the organisms
were inade in meat-extract bouillon, neutral to phenolphthalein, in com-
parison with the same medium made —10, —20, —30, — 40, and —50
Fuller’s scale with normal sodium-hydrate solution.

116 Journal of Agricultural R esearch Vol. VIII, No. 3

At the end of 12 to 18 hours the cultures in the —10 bouillon were as
heavily clouded as the neutral; those at —20 were well clouded but
plainly less than at —10. At this time B. atrosepticus, B. solantsaprus,
B. melanogenes, IIIA, SE, and IIP were practically identical in the
amount of cloudiness they produced on the different media, but showed
no growth beyond —20. B. phytophthorus from Appel and B. phytoph-
thorus from Schuster showed a decreasing amount of cloudiness up to
and including —4o0, but they showed no growth in the tubes of media
with a reaction of — 50.

In 48 hours the last two named had produced a faint but evident
growth in the medium having a reaction of —30. At the end of two
weeks all showed growth at — 40 and all but B. atrosepticus and SE at
— 50.

OPTIMUM REACTION FOR GROWTH IN BOUILLON.—This is approxi-
_mately the same for all of the organisms studied. It lies very close to
the neutral point of phenolphthalein, but is apparently on the alkaline
side. Observations to determine this must be made with these organ-
isms during the first 12 to 18 hours of growth. Broth cultures with a
reaction of +10 with hydrochloric acid and —10 with sodium hydrate,
Fuller’s scale, were practically equally well clouded in 24 hours, but the
acid cultures in the early stages of growth were observed to cloud slightly
more slowly. Sodium hydrate restrains the growth much less than does
hydrochloric acid, but in both aikaline and acid media of stronger reaction
than that first mentioned there was a very decided falling off in the cloud-
ing during the first 24 hours as compared with that obtained with media
of neutral reaction.

VITALITY ON CULTURE MEDIA.—Cultures of B. solanisaprus and the
three organisms from Maine in neutral beef-extract bouillon, stored at a
temperature of from 15° to 18° C., were found to be alive at the end of 8
to 10 months, provided the moisture had not entirely evaporated. ‘Tests
were made with the other organisms, but with somewhat younger cul-
tures and in each case they were found to be alive in the bouillon. Cul-
tures in liquefied gelatin were also found to possess long vitality, but upon
agar slants, milk, and the various carbohydrate broths, the organisms
were killed out much more readily. Those in milk were frequently found
to be entirely dead at the end of three months.

TEMPERATURE RELATIONS.—The optimum temperature for growth in
the six pathogens studied is not far from 25° C., although no attempt
was made to determine this within a variation of 5° C. above and below.
Numerous tests were made of all the strains at the same time in seven
different incubator chambers, running from 5° to 35° C. Considerable
differences were noted in the rates of growth at lower temperatures, even
in different tests of the same strain, but as the optimum was approached _
a striking uniformity was obtained. ‘This is well illustrated by Table III,
which gives the results secured from a single series. In the temperature

Nt al
”

Jan. 15, 1927 Blackleg Disease of Potato 117

columns of this table are given the number of hours which elapsed from
the time a 1o-c. c. broth culture was inoculated with a 2-mm. loop of a
24-hour-old broth culture, before the first visible traces of clouding of
the medium could be observed, observations being made hourly. In all
cases the tubes of media were placed in the incubator at the desired tem-
peratures for a few hours before inoculation, and care was taken not to
remove them therefrom any longer time than was necessary to make
the inoculations and observations.

TaBLE III.—Effect of temperature on rate of growth of the organisms

First evidence of clouding of broth cultures at—

Organism.
BC Orem ees tt zor Can osc Cowles ee BomaCe

Hours. | Hours. | Hours. | Hours. | Hours.| Hours. Hours.
BBE GHOSE PUEUS 0 c'cc-ajaia's a 160s 84 27 22 13 ie 12 | No growth.
Bi Solantsaprus tees ees ees 96 27 22 13 vig 12 Do.
IB Mclanogenes-shiaaeiefen tie. 52 oy 22 13 II 12 Do.
DUG) is eat pat As edhe MO tN LE 84 28 2I I4 1 12 Do.
SANG: Wa Nau es Ae eS 96 24 21 16 II 12 Do.
Nr ry ethene sieeve acne Sy aire 192 59 27 16 13 16 Do.
B. phytophthorusfrom Appel.| 192 64 30 18 13 12) | 16!
B. phytophthorus from Schus-| 192 60 22 13 8 in | ae

tens

It will be seen that while the optimum for the two nonpathogenic
strains carried under the name of B. phytophthorus is about the same
temperature as for the six pathogens, the former grew at 35° C., while
the latter did not.

Both agar slants and beef-broth cultures were used to determine the
maximum temperature for growth in the case of B. atrosepticus, B.
solanisaprus, B. melanogenes, and the three pathogenic strains isolated in
Maine. On beef-extract broth neutral to phenolphthalein this was not
far from 33° C.; if anything, slightly below this. None of the six pro-
duced visible clouding of broth at 34° C. and above. On agar slants the
growth was scanty or absent at 32° and 32.5°, B. atrosepticus, SE, and
IIP giving the most frequent failures to produce growth on agar at these
temperatures. All of the pathogenic strains would show very evident
growth in broth at 32° in 18 hours, and also clouding in the same period
of time, with less regularity and with some failures to produce clouding
at all at 32.5°. Undoubtedly the somewhat lower maximum tempera-
ture for growth on agar was due to the rapid drying out of the surface of
the medium. ‘The results given above were checked a number of times,
since they were unexpected on account of Harrison having reported
growth with B. solanisaprus at 37°.

The thermal death point for B. atrosepticus, B. solanisaprus, B. melano-
genes and the three pathogenic strains isolated from Maine was found |
to be approximately 46.5° C. Retests at this temperature did not

TIS 5 Journal of Agricultural Research Vol. VII, No. 3

always prevent later clouding of the inoculated and heated tubes of broth.
In no case, except with a single tube of B. melanogenes, did growth occur
after heating at 47° and above, but clouding always appeared after an
exposure to 46° C. and below. Transfers from well-clouded broth
cultures 36 to 48 hours old appeared more resistant than those a few
days older. The strains carried under the name of B. phytophthorus
showed a much higher thermal death point. For the culture received
from Dr. Appel it was between 51° and 52°, while that from Dr. Schuster
was killed at 54° but not at 53°.

In making the thermal-death-point determinations thin-walled test
tubes of 16 to 17 mm. internal diameter and approximately 16 cm. long
were employed. Each tube contained 10 c. c. of meat-extract broth,
+15 Fuller’s scale.

Immediately after inoculation they were transferred to a specially
constructed water bath, provided with a stirring apparatus and an accu-
rate, certified thermometer, and immersed nearly their entire length in
the heated, constantly moving water. The period of immersion was 10
minutes and the bath during this time was maintained within one-tenth
of 1 degree C. of the required temperature.

EFFECTS OF DRYING.—The effect of drying was tested as follows: A
2-mm. loop of a 24-hour broth culture was removed to and spread upon
small, sterilized, cover glasses in sterile petri dishes. These were allowed
to dry at the same temperature as incubation for varying periods after
the last trace of moisture had disappeared from the cover glasses. Then
the latter were picked up with flamed forceps and dropped into tubes of
sterile bouillon. Only B. solanisaprus, IITA, and SE were tested in this
way. ‘The results were somewhat variable. Usually one or two minutes’
drying were sufficient to produce sterility, although in one or two cases
growth appeared after 10 and 15 minutes’ drying. However, in each
case like this the growth was much retarded.

EFFECT OF SUNLIGHT.—The effect of sunlight was tested only in the
case of the three organisms mentioned in the preceding paragraph. The
usual method was followed: The freshly inoculated’ and half-covered
petri-dish cultures were exposed on blocks of ice to bright sunlight at
midday in agreenhouse. There was no diminution of resulting colonies
on the exposed side in 1o-and 20-minute exposures, slight diminution at
30 minutes, while at 60 minutes all the organisms on the exposed side were
killed, and no colonies developed. This test was made in November
and consequently the conditions were not favorable. Doubtless the
same exposure outdoors in midsummer would have shown sunlight to
be more effective in killing the organisms.

EFFECT OF GERMICIDES.—In testing the effect of germicides, only
mercuric chlorid and formaldehyde were employed, and only B. solan-
isaprus, IIIA, and SE were tested. The results were the same with

Jan. 15, 1977 Blackleg Disease of Potato 119g

all three organisms. In the case of the mercuric-chlorid test one 2-mm.
loop of a deeply clouded, 24-hour broth culture was transferred to
check tubes of sterile distilled water and to others containing varying
dilutions of the poison, the weakest being 1 to 50,000. After one hour
transfers were made from these.to fresh broth tubes. In no case was
growth obtained except from the inoculated check tubes of pure water.
Where mercuric chlorid was added directly to broth tubes it was found
that it required a concentration of 1 to 10,000 to produce the same results.

With formaldehyde no attempt was made to determine how weak a
solution would be effective. It was simply tested and found that one
part in 250 in distilled water or beef broth was sufficient to kill all or-
ganisms in an hour’s exposure. This was somewhat weaker than is
usually used for disinfecting potato tubers and the time one-half as long.

PATHOGENICITY

As has already been stated, all of the strains studied were tested at
various times to determine their ability to produce the characteristic
blackleg disease of the stem and softrot of the tuber. From the beginning
to the close of the work B. solanisaprus, B. melanogenes, and the three
strains from Maine were found to be pathogenic to potato stems, leaf

. stalks, and tubers.

When first received in March, rg10, the culture of B.atrosepticus either
failed to attack or caused a slow decay of pieces of potato tubers pro-
duced the season before, which had been cut under aseptic conditions and
placed in tubes of sterile water. However, after several repeated trans-
fers from one 24-hour-old potato-broth culture to another it was found to
produce a fairly rapid decay when inoculated into potato tubers, especially
those which were immature and recently dug. This strain has continued
to rot tubers, provided they were not too mature and care was taken not
to allow the inoculated portion to dry out too rapidly..

It has been the custom whenever the pathogens were tested on growing
stems of potato plants to inoculate a plant each time with each of the
nonpathogenic strains. B. atrosepticus was repeatedly used in this way,
but the results were negative previous to June 15, 1916. A plant inoc-
ulated on this date by puncturing the young, growing stem near the
base with the needle of a hypodermic syringe and inserting a small
amount of a 24-hour-old culture in the tissues of the pith, was observed
five days later to be showing slight blackening around the point of inoc-
ulation. Closer examination showed that the pith had been softened and
decayed for a short distance above the puncture. This diseased condi-
tion did not progress much farther, however. About a month later
several other young stems were inoculated with transfers of the stock
strain of B. atrosepticus and this time the positive results were more
marked. ‘The inoculated stems were blackened on the surface for a dis-

120 Journal of Agricultural Research Vol. VIII, No. 3

tance of 3 to 4 inches and finally fully decayed and cut off, resulting in
the death of that part of the plants above the points of inoculation.+

As has been clearly indicated in the previous pages, inoculations of
potato stems and tubers with the cultures received and carried under
the name of B. phytophthorus produced no disease whatever. The punc-
tures into which the cultures were inserted dried out rapidly and were in
every way similar to those into which sterile broth or distilled water had
been inserted as checks.

Some work was done to test the pathogenicity of the strains used, upon
plants closely related to and unrelated to the potato. It will be unnec-
essary to go into the details of this since the present studies are pri-
marily concerned with a comparison of the various morphological, cul-
tural, physical, and biochemical features of the different organisms.

GROUP NUMBERS

The numerical system employed in the descriptive chart or card
adopted by the Society of American Bacteriologists furnishes a means
whereby a considerable number of contrasted, salient characters may be
brought together in a small space to facilitate comparisons. ‘This
decimal system of group numbers is useful as a quick method of showing
close relationships and important differences within the genus, but is not
a sufficient characterization for any organism. However, it does record
in a very compact manner many of the important differential characters.

GROUP NUMBERS BASED ON THE PRESENT STUDIES

Ba trOge PLCS INfADL ERA i osha tyticj ick bodys sere ea Sint ane SP geerE 221, 1113522
Bola nisa Pras uILAnt ASO Ie 2 She cy ase sity seb estate oh ete) secur: B21. ThE zin22
B. melanogenes Pethybridge and Murphy................. 221. 1113522
STATEAT S Pence tS Betas bial si BAS ed cnsye Varct Seta] ele Baa nietenere eva erate 227. Rinse?
SH EUG ae nse OSE BING ROMS. PUREE SUE Ma Oa eae 221. 113522
TD 4S eee ratty cenae tr algol Web ceue <a sila altos eieuane tle” Brcbetaren se 221. 1113522
B. phytophthorus ( as received from Appel)................ 222. 3333033
B. phytophthorus (as received from Schuster). ............ 221. 3333533

Some question might be raised with regard to whether the figure in
the fifth place to the right of the decimal point in the first six group num-
bers should be a5 orao. Here the only yellow color that developed was
on potato, and this was by no means strong. It is possible also that the
next figure to the right in the same group numbers should be 3, for the
diastatic action on potato starch is doubtful.

1 The writer has no explanation to offer for this unexpected manifestation of pathogenicity on the part
of this culture of B. atrosepticus. "The system used in keeping and transferring the stock cultures is such
that any possibility of getting the different strains mixed is practically eliminated. Thefact that the stock
cultures have always been carried in beef broth with occasional transfers to potato broth may have some-
thing to do with it. Others who have kept their stock cultures of these and the closely related softrot
bacteria of the B. carotovorus type on agar have reported loss in pathogenicity. Including those mentioned
in this paper the writer has carried over 20 different strains of blackleg organisms for from 5 to 9 years, but
with no permanent evidence of loss of pathogenicity with any.

Jan. 15, 1917 Blackleg Disease of Potato Lat

In the case of the organism received from Schuster and carried under
the name of B. phytophthorus, the yellow growth was apparent on both
agar and potato. Upon the latter the growth was of a very distinct
lemon yellow, considerably deeper than that produced by the other

organisms.
NOMENCLATURE AND RELATIONSHIP

An analysis of the data obtained as the result of these comparative
studies seems to point to but one conclusion. When subjected to the
various differential tests at the same time and under the same condi-
tions, the cultures received under the names ‘‘ Bacillus atrosepticus Van
Hall,” “Bacillus solanisaprus Harrison,’’ and ‘“‘ Bacillus melanogenes Pethy-
bridge and Murphy” and the three strains of organisms isolated from
potato plants affected with the blackleg disease from widely separated
parts of Maine appear to be identical.

It is the writer’s opinion that the pathogenic organisms studied should
be classed as one species, or at the most strains of the same species. The
only constant differences noted were slight variations in size, shown more
particularly by B. solanisaprus and IIIA, and the production of a slight
viscidity on different kinds of media shown by B. solanisaprus and B.
melanogenes, but not by any of the others.

It is evident that the two organisms which the writer received and
studied under the name of Bacillus phytophthorus Appel were not alike,
nor were they the same as the one originally described by Dr. Appel.

There is nothing in the data here presented which bears on the rela-
tionship between the organism originally described by Dr. Appel as B.
phytophthorus and the other strains of blackleg bacteria. As has already
been stated, Dr. Smith states that it and B. solanisaprus are not iden-
tical, but are closely related. While the writer regrets that he was
unable to get an authentic, virulent culture of this organism for compari-
son in time to make use of it he has no reason for questioning this state-
ment, coming from so good an authority.

While the present studies are primarily concerned with relationship,
the results obtained make it impossible to entirely ignore the question
of nomenclature. If it is granted that B. phytophthorus differs from B.
solanisaprus and consequently from the others under consideration, which
of the three other names should be retained? On grounds of priority,
B. melanogenes is excluded and the choice is between B. atrosepticus
and B. solanisaprus. Also on the grounds of priority alone it would
seem that the accepted name should be B. atrosepticus, but for certain
reasons the writer was at first inclined to favor B. solanisaprus. These
reasons are as follows: According to present standards B. atrosepticus was
not very fully described in the beginning. Moreover, the culture used by
the writer was obtained from Kral’s laboratory with no statement regard-
ing its origin or authenticity. Also its pathogenicity was erratic or weak,

122 Journal of Agricultural Research Vol. VIII, No. 3

and for a long time it entirely failed to produce typical blackleg of the
stem upon inoculation. This was in accord with the impression one
would gain as to Van Hall’s results relative to pathogenicity on reading
certain reviews of his paper (15). When Dr. De Zeeuw’s translation of
Van Hall’s paper became available, the results of his inoculation experi-
ments appeared in quite a different light. These were quite limited, on
account of a lack of a sufficient amount of proper material. As is the
case with any of the pathogenic strains, the inoculation experiments with
mature stems and tubers gave erratic results or failures. He was able
in some instances to produce typical blackening and decay when young
parts of stems were inoculated.

B. solanisaprus, on the other hand, was originally described in much
detail, and authentic, virulent cultures are to be obtained by anyone who
may wish to study it. That used by the writer is as pathogenic to-day
to potato stems and tubers as when described by Harrison 10 years ago.

In recommending the dropping of the name “B. melanogenes,” the
writer does not wish to be understood as casting any reflections upon the
work of Pethybridge and Murphy, or upon their conclusions, based upon
the results obtained by them when compared with published descrip-
tions. The data obtained in the present studies have checked in every
essetitial detail with their description of this organism except that the
writer secured a positive test for indol and was unable to get the forma-
tion of gas in nitrate broth. However, attention has already been called
to the fact that Pethybridge and Murphy used a broth containing a much
greater percentage of potassium nitrate than was used by the writer.
It should be noted that the agreement between B. solanisaprus and B.
melanogenes resulted from the writer’s obtaining certain results, especially
with the fermentation of the carbohydrates and the liquefaction of gela-
tin, which were different from those reported by Harrison. Also Harri-
son reports the thermal death point for B. solanisaprus to be 54° C.,
while Pethybridge and Murphy say that for B. melanogenes it lies between
45° and 50°. The writer’s results showing that the thermal death point
is approximately 46.5° are then in accord with the latter statement.

REVISED DESCRIPTION OF BACILLUS ATROSEPTICUS VAN HALL, GROUP NUM-
BER 2211113522"

I, MORPHOLOGY

Vegetative cells. Medium used was an agar slope at 20° C. 1 to 2 daysold. Form,
short rods, long rods, short chains, long chains. Limits of size in microns (stained
preparations) 0.4 to0.8 by rto2or more. Size of majority 0.5 to 0.6 by 1.5 to 2.
Ends rounded.

Endospores, none.

Flagella, few, not over 6 or 8. Attachment, peritrichiate. Stained by modified
Pitfield method.

i See statement following the list of group numbers on p. 120.

Jan. 15, 1917 Blackleg Disease of Poiato E23

Capsules, none.
Pseudozoogloea, present, slight. Involution forms, not observed. (Reported
by Harrison for B. solanisaprus, at higher temperatures. )

Staining reactions. Stains well in aqueous gentian violet, anilin water gentian
violet, aqueous methylene blue, alkaline methylene blue, aqueous fuchsin,
carbol fuchsin. Gram, negative.

.

II. CULTURAL FEATURES

Agar stroke. Growth, moderate. Form of growth, filiform. Elevation, flat to
slightly raised. Luster, glistening. Topography, smooth. Optical characters,
slightly but distinctly bluish opalescent. Chromogenesis, pearly white. Odor,
absent. Consistency, butyrous—some strains slightly viscid, others not.

Potato. Growth, moderate to abundant. Form of growth, filiform to spreading.
Elevation, first convex, then slightly raised to flat. Lwuster, glistening. Topog-
raphy, smooth to slightiy rugose. Chromogenesis, yellowish white, later dirty
white. Slight odor of decayed potatoes in old cultures. Consistency, butyrous—
some strains slightly viscid, others not. Medium, slightly grayed at first, later
plainly grayed.

Agar stab. Growth, slightly best at top, abundant, widespreading. Line of punc-
ture, filiform to slightly papillate.

Gelatin stab. Growth, best at top. Line of puncture, filiform. Liquefaction,
infundibuliform to slightly napiform, later may be saccate, occasionally crateri-
form to stratiform. Begins in 1 to 3 days at 20° C. Complete in 7 to 10 days
with some cultures, in others not complete in 60 days.

Nutrient broth. Surface growth, usually slight ring and occasionally slight granular
pellicle in young cultures. Clouding, moderate to strong, persistent. Medium,
not discolored. Odor, absent. Sediment, compact, scant, granular; and dirty
white—some strains slightly viscid, others not..-

Milk. Coagulation, usually not until the seventh day at 20° C. Coagulum, not
digested or at the most very slowly peptonized. Medium, not discolored.

Litmus milk. Acid, litmus reduced.

Gelatin colonies. Growth rapid at 20° C. Form, round. Edge, entire. Liquefac-
tion, saucer.

Agar colonies. Growth, rapid at 20° C. Form, round, occasionally irregular, buried
colonies, lens-shaped to slightly ovoid or spherical. Surface, smooth. Eleva-
tion, flat to slightly raised. Edge, buried colonies, entire, surface colonies,
entire to undulate. Internal structure, finely granular. Color buried colonies,
brownish yellow under a 16 mm. objective and 6 ocular, slightly yellowish under
hand lens, surface colonies, pearly white, bluish opalescent to transmitted light.

Fermi’s solution. Growth, first moderate, later abundant.

Cohn’s solution. Growth, absent.

Uschinsky’s solution. Growth, copious.

Sodium chloride in bouillon. Growth inhibited, 3 per cent, slightly, 4 per cent, some,
and 5 per cent, considerably.

Growth in bouillon over chloroform. Growth, much restrained at first, later moderate.

Best medium for long-continued growth. Neutral beef bouillon.

Fe lI. PHYSICAL AND BIOCHEMICAL FEATURES

Fermentation tubes. Gas produced from dextrose, saccharose, lactose, maltose, and
mannit, but not in glycerin and dextrin. Growth in the closed arm with dex-
trose, saccharose lactose, maltose, mannit, and dextria, but either absent or
slight with glycerin. Acid produced from dextrose, saccharose, lactose, mal-
tose, mannit, and glycerin.

124

Journal of Agricultural Research Vol. VIII, No. 3

Ammonia production, absent.
Nitrates in nitrate broth reduced to nitrites.
Indol production, moderate in old cultures, absent or feeble in young cultures.

Toleration of acids.

Grows in broth at +20 Fuller’s scale and not at +30 with hydro-

chloric acid.
Toleration of sodium hydrate, great.
Optimum reaction for growth in bouillon in terms of Fuller’s scale, o or slightly

alkaline.
Vitality on culture media.

Relatively brief on agar and media containing carbo-

hydrates; long on bouillon.

Temperature relations.

Thermal death point, approximately 46.5° C. Optimum

temperature for growth, about 25° C. Maximum temperature for growth, about
33° C. Minimum temperature for growth, below 5° C.
Effects of drying. Readily killed.

Exposure to sunlight.

Exposure on ice at midday in November, slight diminution

at 30 minutes, roo per cent killed at 60 minutes.

IV. PATHOGENICITY

Virulent strains produce rapid decay accompanied by a pronounced blackening of
the surface tissues when inoculated into young, growing potato stems; also causes
a rapid softrot of potato tubers at moderate temperatures and may produce decay
when inoculated into a considerable number of fleshy vegetables.

(1) APPEL,

1902.
(2)

1902.
(3)

1902.
(4)

1903.
(5)

1904.
(6)

1906.
(7)

1906.

LITERATURE CITED
Otto.
Der Erreger der ‘‘Schwarzbeinigkeit’’ bei den Kartoffeln.
Deut. Bot. Gesell., Bd. 20, Heft 2, p. 128-129.

In Ber.

Untersuchungen tiber das Einmiethen der Kartoffeln. In Arb. Biol.
Abt. K. Gsndhtsamt., Bd. 2, Heft 3, p. 373-436, 8 fig., pl. 9.

Zur Kenntniss der Bakterienfaiule der Kartoffeln. Jn Ber. Deut. Bot.
Gesell. Bd. 20; Heit 1, p. 32-35.

Untersuchungen itber die Schwarzbeinigkeit und die durch Bakterien
hervorgerufene Knollenfaule der Kartofieln. Jn Arb. Biol. Abt. K.
Gsndhtsamt., Bd. 3, Heft 4, p. 365-432, 15 fig., pl. 8 (col.).

Die Schwarzbeinigkeit und die mit ihr zusammenhangeade Knollen-
faule der Kartoffeln. K. Gsndhtsamt. Biol. Abt., Flugbl. 28, 4 p.,

illus.

Biologie des Einmietens und Einkellerns von Kartoffeln, Rtitben und

Gemiissen. Jn Lafar, Franz. Handbuch der technischen Mykologie.
Aufl. 2, Bd. 2, Lig. 12, p. 337-361, fig. 22-30. Jena. Literatur, p.
360-361.

Neuere Untersuchungen iiber Kartoffel- und Tomatenerkrankungen.
In Jahresber. Angew. Bot., Jahrg. 3, 1904/05, p. 122-136, 2 fig.

(8) ARBOIS DE JUBAINVILLE, M. A. D’., and VESQUE, Julien.

1878.

Les Maladies des Plantes Cultivées, des Arbres Fruitiers et Forestiers,
Produite par le Sol, 1’Atmosphére, les Parasites Vegetaux . . . 328p.,
illus., 7 pl. Paris.

Jan, 15, 1917 Blackleg Disease of Potato 125

(9) DELAcRorIx, Georges.
rgo1. Contributions a 1’étude d’une maladie nouvelle de la pomme de terre
produite par le Bacillus solanincola nov. sp. In Compt. Rend. Acad.
Sci. [Paris], t. 133, NO. 24, p. 1030-1032.

(ee hing
rgor. Rapport sur une maladie bactérienne nouvelle de la pomme de terre.
In Min. Agr. [Paris] Bul., ann. 20, no. 5, p. 1013-1033.
(11) ;
rgor. Sur une maladie bactérienne de la pomme de terre. In Compt. Rend.
Acad. Sci. [Paris], t. 133, no. 9, p. 417-419.
(12)

1906. Sur une maladie de la pomme de terre produite par Bacillus phytoph-
thorus (Frank) O. Appel. In Compt. Rend. Acad. Set Paris]:
143, No. 9, p. 383-384.
(13) FRANK, A. B.
1297. Kampfbuch gegen die Schadlinge unserer Feldfriichte. 308 p., 46 fig.,
2zocol. pl. Berlin.

(14)
1899. Die Bakterienkrankheiten der Kartofieln. In Centbl. Bakt. [etc.],
Abt. 2, Bd. 5, No. 3, p. 98-102, 2 fig., No. 4, p. 134-139.
(zs) Hay, C. J. J. van. 5
1902. Bijdragen tot de Kennis der bakterieele Plantenziekten ... 1098 p.
Amsterdam. Inaug. Diss.
(16) Harpinc, H. A., and Morse, W. J.
1909. The bacterial soft rots of certain vegetables. I: Part I. The mutual
relationships of the causal organisms. Jn N. Y. State Agr. Exp. Sta.
Tech. Bul. 11, p. 251-290. Also in Vt. Agr. Exp. Sta. Bul. 147 (Dec.
1909), P- 243-282. IgI0.
(17) Harrison, F. C.
1906. A bacterial rot of the potato, caused by Bacillus solanisaprus. Jn Centbl.
Bakt. [etc.], Abt. 2, Bd. 17, No. 1/2, p. 34-39; No. 3/4, p. 120-128;
No. 5/7, p. 166-174; No. 11/13, p. 384-395, 8 pl. ;
(18) Jounson, Thomas.
1907. Some injurious fungi found in Ireland. In Econ. Proc. Roy. Dublin
Soc., Vv. I, pt. 9, p- 345-379, 5 fig., pl. 32-35.
(19) JonEs, L. R.
1905. Disease resistance of potatoes. U.S. Dept. Agr. Bur. Plant Indus. Bul.

87, 39 p-

(20)
1907. The blackleg disease of the potato. In Vt. Agr. Exp. Sta. 19th Ann.
Rpt. 1905/06, p. 257-265.
(21) LAURENT, Emile.
1899. Recherches expérimentales sur les maladies des plantes. Jn Ann. Inst.
Pasteur, ann. 13, no. 1, p. 1-48.
(22) Mors#, W. J.
1907. Potato diseases in 1907. Maine Agr. Exp. Sta. Bul. 149, p. 287-339,
fig. 45-50.
(23) ‘
1909. Blackleg. A bacterial disease of the stem of the Irish potato. Maine
Agr. Exp. Sta. Bul. 174, p. 309-328.

(24)
r911. Control of the blackleg or black-stem disease of the potato. Maine Agr.
Exp. Sta. Bul. 194, p. 201-228, fig. 93.

126 Journal of Agricultural Research Vol. VII, No. 3

(25) PETHYBRIDGE, G. H.
rgi1. Investigations on potato diseases. In Dept. Agr. and Tech. Instr.
Ireland Jour., v. 11, no. 3, p. 417-449, 14 fig.
and Murpuy, P. A.
torr. A bacterial disease of the potato plant in Ireland. Jn Proc. Roy. Irish
Acad., v. 29, sect. B, no..1, p. 1-37, pl. 1-3. Bibliography, p. 35-36.
(27) PRILLIEUX, EH. EH.
1895-97. Maladies des Plantes Agricoles ... 2v. Paris.
and DELACROIX, Georges.
1890. La gangréne de la tige de la pomme de terre, maladie bacillaire. In
Compt. Rend. Acad. Sci. [Paris], t. 111, no. 3, p. 208-210.
(29) PRUNET, A.
1902. Jes maladies bactériennes de la pomme de terre. In Rev. Vit., ann. 9,
t. 17, NO. 433, P. 379-385.
(30) REINKE, Johannes, and BERTHOLD, G. D. W.
1879. Zersetzung der Kartoffel durch Pilze. 100 p.,g pl. Berlin. (Unters.
Bot. Lab. Univ. Gottingen, Heft 1.)
(31) SmirH, Erwin F.
1896. A bacterial disease of the tomato, eggplant, and Irish potato (Bacillus
solanacearum n.sp.). U.S. Dept. Agr. Div. Veg. Physiol. and Path.
Bul. 12, 26 p., 2 pl.

(26)

(28)

(32)
1905-11. Bacteria in Relation to Plant Diseases. v.1, 1905; Vv. 2, 1911. Wash-
ington, D.C. (Carnegie Inst. Washintgon Pub. 27, pt. 1-2.)

(33)
1910. Bacillus phytophthorus Appel. Jn Science, v. 31, no. 802, p. 748-749.

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