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SOLLETIN. OF LTE

USDEPARIMENT OFAGRICULTURE

No. 40

Contribution from the Bureau of Plant Industry, Wm. A. Taylor, Chief,
January 15, 1914.

THE MOSAIC DISEASE OF TOBACCO.?

By H. A. ALLARD,
Assistant Physiologist, Tobacco and Plant-Nuirition Investigations.

INTRODUCTION.

* Mosaic” is one of the most serious and widespread diseases known
to affect the tobacco plant. This disease also is known locally as
“calico,” “gray-top,” “ mottled-top,” “mottling,” and “foxy” to-
bacco. Especially in southern tobacco sections the term “ frenching ” ?
is used to designate abnormal, sickly plants characterized by the de-
velopment of stringy, very thick, and leathery leaves which may be
free from mottling. It has not been determined whether this is dis-
tinct from mosaic or only a somewhat different phase of this
disease. In the tobacco sections of Kentucky and Tennessee “ wal-
loon” is often used to denote mosaic or other abnormal appearances
more or less closely resembling symptoms of the mosaic disease. The
disease has also been termed “chlorosis.” Finally, mosaic plants
sometimes are spoken of as “brindle” or “mongrel.” These terms

1In a study of the mosaic disease of tobacco facts have been learned which throw con-
siderable light on its nature and origin. It has been shown that it is communicable to
plants of many genera of the solanaceous family and that aphides are sometimes active
disseminators of infection both in the greenhouse and in the field. The fact that par-
ticular insects are involved in the spread of the disease not only helps to explain the
contradictory results obtained by previous investigators, but also serves to indicate
means by which the disease can, to a greater or less extent, be controlled under practical
field conditions.

Since insects are involved in the spread of the mosaic disease both in seed beds and
in the field and the practical control of this disease must depend primarily upon an
intimate knowledge of the habits and distribution of species which may act as capable
carriers of infection, this phase of the investigation has been taken up in cooperation
with the Bureau of Entomology.—Wwm. A. TAyI.or, Chief of Bureau,

2The term “ frenching” or “ frenched” is applied to sickly, spindling corn plants
and peach trees-as well as to tobacco plants. Its origin dates back to the settlement of
Maryland when the English settlers applied the term ‘‘ Frenchmen” to weak, spindling
tobacco plants as a term of contempt. See Clayton, John. A Letter ... to the Royal
Society, May 12, 1688, giving an account of several observables in Virginia... p. 19.
(in Force, Peter. Tracts and Other Papers, Relating Principally to the Origin, Settle-
ment, and Progress of the Colonies in North America, v. 8, Washington, 1844.)

131287-—-Bull, 4014-1

2 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

are usually descriptive of the characteristic mottling or otherwise
sickly appearance of plants affected with the disease. It is true,
however, that they are in many instances somewhat loosely applied
to a variety of abnormal appearances and symptoms not at all asso-
ciated with the specific mosaic disease of tobacco.

Both in Europe and America the mosaic disease of tobacco has
been the subject of wide inquiry. Hitherto no investigator has
been able to offer very conclusive evidence which would consistently
explain the baffling nature and mysterious origin of the disease.
Each in his own way, however, has emphasized a favorite opinion,
so that the literature of the disease is especially conspicuous for its
widely contrasted theories.

The view seems to be generally accepted that the disease is a
physiological or functiona! disorder, although it has long been known
that it is more or less infectious. For this reason the writer’s experi-
ments were at first planned along physiological lines. Facts soon
came to hght, however, which led to the conclusion that the disease
must be parasitic in its origin rather than physiological or functionai.

The writer has found that particular aphides can induce mosaic
symptoms in plants infested by them, and much evidence has accumu-
lated to show that aphides are responsible for serious outbreaks of
the disease both in the greenhouse and in the field. Since it has been
found that insects may act as carriers of the disease, this phase of the
problem has been taken up in cooperation with the Bureau of Ento-
mology with the hope that further hght may be thrown on this point.

APPEARANCE OF AFFECTED PLANTS.

The development of the mosaic disease of tobacco is attended with
various physiological and morphological changes in the leaves,
branches, and sometimes the flowers of all affected plants. The char-
acter and intensity of these symptoms vary greatly, depending upon
the age, habits of growth, species of plants affected, and external con-
ditions. The following symptoms are more or less characteristic of
different phases of the disease at one time or another:

(1) Partial or complete chlorosis.

(2) Curling of the leaves.

(8) Dwarfing and distortion of the leaves.

(4) Blistered or “ savoyed” appearance of the leaves.

(5) Mottling of the leaves with different shades of green.

(6) Dwarfing of the entire plant.

(7) Dwarfing and distortion of the blossoms.

(8) Blotched or bleached corollas (in Nicotiana tabacum only).

(9) Mosaic sucker growths.

(10) Death of tissues (sometimes very marked in Nicotiana rustica).

The extent and character of the first observable symptoms of the
disease depend upon the age and vigor of the plants at the time of

THE MOSAIC DISEASE OF TOBACCO. | Oo

Inoculation with the virus. In order to study this phase of the ques-
tion thoroughly the writer has inoculated tobacco plants at all stages
of growth from the cotyledon stage until the plants have matured
seed.

INITIAL SYMPTOMS IN YOUNG PLANTS.

In very young plants at the two-leaved or three-leaved stage and
in much larger plants in the rosette stage the first visible symptom
of the mosaic disease usually appears as a slight downward curling
and distortion of the smallest, innermost leaves, which at the same
time become more or less chlorotic.

As these leaves increase in size, small, abnormally dark-green spots
and areas quite generally make their appearance. If the plants are
given plenty of room and an abundance of plant food to stimulate
active growth, these dark-green portions of the leaves develop rapidly
into large, irregular, crumpled swellings, or blisters, more or less
characteristic of the so-called “ savoyed appearance.” At this period
these leaves very frequently grow to a size disproportionate to the rest
of the plant, becoming long, narrow, sinuous, or otherwise enlarged,
distorted, and irregular. On the other hand, if the young plants
affected with the disease are badly crowded and starved in the seed
_ bed, growth proceeds very slowly, so that the leaves may show only
a slight mottling. Under such unfavorable conditions of growth tiny
plants showing on the leaves the dark-green spots characteristic of
the true mosaic disease may remain unchanged in size and appear-
ance for months.

Although young plants sometimes appear to outgrow the marked
distortion and deformity affecting the immature leaves at this age,
the mottled phase of the disease makes its appearance and persists
with more or less intensity until all leaf development has finally
ceased. (PI. I, fig. 1, B.)

DEVELOPMENT IN PLANTS APPROACHING MATURITY.

Should tobacco plants approaching maturity become infected with
the mosaic disease, a characteristic phase known as “ mottled-top ”
or “gray-top” develops. (PI. I, fig. 2.) Although the number of
leaves affected and the intensity of the markings is considerably in-
creased if the disease develops at a comparatively early period in the
life of the plant, attenuated mottling in one or two of the small top-
most leaves is the only evidence of the malady in plants which
develop the disease just prior to final maturity. In many experi-
mental tests made by the writer upon greenhouse plants, typical
phases of “ mottled-top ” have almost invariably appeared whenever
the mosaic virus has been introduced into healthy plants just as the
small, immature flower head was beginning to emerge and to elon-

4 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

gate above the uppermost leaves. At this time the cluster of flower
heads is very small, compact, and less than 1 inch across. After an
incubation period of 10 to 15 days, the first symptoms of the disease
become manifest as a very noticeable wilting of the upper leaves,
especially in a hot sun. These leaves, especially in transmitted sun-
light, show a very fine mottling, which very rapidly develops into
characteristic “ mottled-top.”

If the leaves are examined closely, it will be seen that this appear-
ance is due to the distribution of the dark-green shades along the
fine anastomosing veins, while the lighter shades occupy the small
inclosed areas. The earlier manifestations of “mottled-top” in
some respects simulate certain color changes which accompany the
normal ripening of fully matured leaves. The “ mottled-top ” phase
of the disease in nearly mature plants produces little or no distor-
tion in the leaves which exhibit this appearance. Practical growers
do not consider this phase particularly serious.

If plants become infected just prior to the appearance of the
blossoms, the leaves of the main stalk, all of which at this time
have practically completed growth, are little, if at all, changed in
normal appearance. The characteristic symptoms, however, such as
distortion, etc., are reproduced in the secondary growths (suckers)
in much the same manner that young plants are affected.

ROOTS OF DISEASED PLANTS.

The roots of mosaic plants superficially appear quite as normal
in many instances as those of healthy plants. It is probable, how-
ever, that the roots as well as the leaves may be more or less seriously
impaired in form and function, depending upon the age and condi-
tion of the plant when affected with the disease.

LEAVES OF DISEASED PLANTS.

The development of the mosaic disease in tobacco plants produces
a great variety of changes in the normal color and form of the
leaves. The more evident changes are confined to the actively grow-
ing portions, such as the immature leaves of young plants and the
vigorous secondary growth of more mature plants. A character-
istic feature of the true mosaic disease, which seems to distinguish
it from nearly all unhealthy appearances of purely physiological
origin, is the distinctive blotched and mottled appearance of the
leaves, sometimes accompanied by distortion, especially if the leaves
are immature. In very immature plants the leaves often show
marked distortion and irregularities of growth, from which they may
almost recover as they become larger and more mature.

Fantastic leaf forms frequently arise in mosaic plants. In some
instances the development of the lamina is almost entirely suppressed,

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-GATILLOW,, GS1TIVO-OS AHL SNIMOHS DNIMOHS ‘4V4a7] OIVSOW ‘G ‘3v37 AHLIVSH ‘YW ‘WwnOoVvd
‘ANOVAV_L VNVILOOIN SO AVE] dDOL—'? ‘SIF -V 1 VNVILOOIN JO SSAVA7 OIVSOIN GNV AHLIVSH—'] “SI

PLATE I.

Bul. 40, U. S. Dept. of Agriculture.

Bul. 40, U. S. Dept. of Agriculture. PLATE II.

Fic. 1.—MALFORMED Mosaic LEAVES OF NICOTIANA TABACUM (A, B, C, AND D) AND
A HEALTHY LEAF (E).

(Somewhat reduced.)

Fic. 2.—Mosaic LEAVES OF NICOTIANA TABACUM, SHOWING THE DEVELOPMENT OF
PROMINENT BLISTERLIKE OUTGROWTHS OF THE LEAF TISSUES.

These swellings appear on the upper face of the leaf and are usually very dark green in
color in comparison with the rest of the leaf. (Natural size.)

Bul. 40, U. S. Dept. of Agriculture. PLATE III.

FIG. 1.—COROLLAS OF AMERICAN VARIETIES OF NICOTIANA TABACUM, SHOWING VARIOUS
DEGREES OF MOTTLING PRODUCED BY THE Mosaic DISEASE.
The dark areas represent the normal pink color; the light or white areas indicate a partial or

complete absence of red. A, Normal pink blossom; B, mosaic blossom which is entirely white.
(About natural size.)

Fic. 2.—BubDs OF NICOTIANA TABACUM, SHOWING DISTORTED AND OTHER DEPAUPERATE
BUDS PRODUCED BY THE MoSAIc DISEASE (A) AND NORMAL, HEALTHY BuDs (B).

(About natural size.)

THE MOSAIC DISEASE OF TOBACCO. 5

so that the leaf is little more than a twisted midrib. In other in-
stances a long, sinuous, ribbonlike leaf is produced by the growth of
a very narrow and irregular leaf blade (PI. II, fig.1,C.) <A very
peculiar and irregular leaf shape sometimes arise when the lamina
fails to develop beyond certain abnormally thickened veins, which
become the margins of the leaf. (PI. II, fig. 1, D.)

A very distinct and irregular phase of mottling is usually produced
in immature leaves of young plants by the development of abnormally
dark-green swellings, or blisters. These surface prominences may
be so small as to appear like mere dots, or so large and confluent as
to involve the greater portion of the leaf, and they sometimes occur
in definite lines along the midrib or lateral veins of a leaf. Other
leaves may appear perfectly normal in color, except for a few dots
scattered irregularly here and there. (PI. II, fig. 2, A, B, and C.)

In later manifestations of the disease the immature top leaves and
suckers become beautifully mottled; hence, the term “ mottled-top.”
This phase of mottling is typically developed in upper leaves which
have almost completed their growth and is not generally associated
with the development of surface swellings, or blisters.

Individual plants inoculated at the same age and in identically the
same manner with the same virus frequently exhibit markedly dis-
similar phases of the disease. In one plant the disease is of a par-
ticularly malignant type, greatly dwarfing and distorting all the
leaves; in another plant the symptoms may be shown only as a slight
mottling of the leaves throughout the growth of the plant.

BLOSSOMS OF DISEASED PLANTS.

The presence of the mosaic disease in all varieties of Nicotiana
tabacum usually affects the intensity and distribution of the pink
coloration of the blossoms in much the same manner that it affects
the intensity and distribution of the green coloration of the leaves.
For this reason an examination of the blossoms affords one of the
surest indications of the presence of the disease in a tobacco plant.
So far as the writer is aware, this distinctive appearance has not
before been mentioned. As a rule, in blossoms of mosaic plants the
normal pink coloration is present only in lines, specks, or conspicuous
blotches. In most instances these markings are very irregular in dis-
tribution, sometimes involving a portion or all of the lobes of the
corolla. (PI. III, fig. 1.)

A rather striking and symmetrical color pattern is sometimes
afforded by a blossom in which the normal pink coloration occurs
only as a fine line in the sinus of each corolla lobe. Other affected
plants produce blossoms all of which are devoid of color, so that
they appear quite white or very pale. More rarely, the presence of

6 - BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

the disease in a plant produces in the blossoms a pink-purple color
several shades darker than that which occurs in normal blossoms of
a healthy plant.

The development of the mosaic disease in tobacco plants is not
invariably indicated in the blossoms by all the color changes pre-
viously described. With regard to the typically blotched appear-
ance of the blossoms, however, the writer has failed to find an in-
stance when this appearance was not associated with the disease in
a plant. Experiments with healthy tobacco plants grown in green-
houses at Arlington, Va., during the winter of 1912, indicate, how-
ever, that high temperatures together with high humidity may pro-
duce very pale or almost white blossoms, indistinguishable from the
white or pale blossoms sometimes produced by mosaic plants.

Now and then tobacco plants show the most pronounced symptoms
of the disease in the upper leaves and suckers and at the same time
produce blossoms entirely normal in color. At other times the
disease appears on a single sucker, all the blossoms of which are
distinctly mottled, although the blossoms of the main flower head
and other suckers are perfectly normal in color. In other plants the
blossoms of the main stalk are mottled, while the suckers at the same
time produce normal pink blossoms. Even in the same flower head
some blossoms may show the most pronounced phases of mottling,
while others retain the uniform pink coloration of a healthy blossom.
In local manifestations of the disease the immature leaves as well
as the blossoms of affected branches usually indicate the disease,
although this is not an invariable rule.

By carefully timed inoculation the writer has caused the disease
to develop in healthy plants subsequent to the apperance of the
first open blossoms. Blossoms appearing in advance of the earliest
symptoms of the disease displayed the uniform pink coloration of
the normal bloom, while later blossoms becoming affected by the
disease displayed the characteristic blotched colorations. The first
observable symptoms have sometimes made their appearance in
the blossoms alone, and not until suckers had developed was the
disease manifest elsewhere in the plant.

In particularly malignant phases of the mosaic disease the plants
often produce depauperate and misshapen blossoms. In such in-
stances the corollas remain small and imperfectly formed, so that
the distorted, immature stigma and stamens extrude long before the
blossom has approached normal maturity. Likewise, on such plants
the seed pods are abnormally small and shrunken and may contain
very few viable seeds. (PI. III, fig. 2; Pl. IV, fig. 1.)

The irregular distribution of color in the blossoms of plants
affected with the mosaic disease seem to be confined entirely to the
pink-flowered varieties of Vicotiana tabacum. None of the greenish,

THE MOSAIC DISEASE OF TOBACCO. q

purplish yellow, or white-flowered species of Nicotiana observed by
the writer have shown this phenomenon in diseased plants. A few
of the more common species observed in this connection may be
mentioned here:

Nicotiana silvestris (blossoms white).

N. rustica (blossoms greenish yellow).

N. longifiora (blossoms purplish).

N. alata (blossoms white).

_ N. langsdorfii (white and yellow flowered forms).
N. paniculata (blossoms greenish).

The red-flowered forms of Nicotiana forgetiana have not been
modified by the disease.

The various manifestations in the intensity and unequal distri-
bution of color in the blossoms of mosaic tobacco plants bear a strik-
ing resemblance to the mottled color of peaches produced by trees
affected with peach yellows. Speaking of the mottled color of
peaches produced by diseased trees, Smith? says:

The amount of color appears to depend somewhat upon variety. Sometimes
there is comparatively little crimson spotting, and again, it is a very marked
feature, the skin being almost purple and the flesh of the deepest crimson,
even in pure white varieties. In an experience covering four years and includ-
ing a great many thousand trees diseased by yellows, I have never known but
one in which there was entire absence of red spotting in the fruit. This tree
bore premature insipid peaches and the characteristic shoots.

Farther ‘on he says:

In many cases the red-spotted, prematurely ripened fruits are the first indi-
cations of disease, or at least the first symptoms striking enough to attract
general attention. They are very often borne exclusively upon one or two limbs
of otherwise healthy-looking trees. These limbs are not different in appear-
ance from the rest of the tree. They bear vigorous shoots and full-grown,
smooth, dark-green foliage; often, also, green, half-grown fruits, which after-
wards ripen in a normal manner. There is no indication of disease except in
the fruit, which, in color and size, contrasts strikingly with the fine green foliage
and the normal, immature fruit.

LOCAL AND GENERAL MANIFESTATIONS OF THE DISEASE.

It is a matter of common observation that obvious symptoms of
the mosaic disease do not necessarily appear in all parts of a plant
simultaneously. In an affected plant individual leaves, branches, or
blossoms of the same flower cluster may show localized symptoms of
the disease. A leaf may appear quite healthy and normal except for
a few small isolated blisters. Branches healthy in appearance some-
times grow close beside those showing the most pronounced symp-
toms. Likewise, in a flower head, blossoms normal in color are often
intermingled with those showing extreme phases of mottling.

1Smith, Erwin IF. Additional evidence of the communicability of peach yellows and
peach rosette. U. S. Department of Agriculture, Division of Vegetable Pathology, Bul-
letin 1, 1891, p. 12-18.

8 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE,

In the “ mottled-top ” or “ gray-top” phase of the disease the top-
most growing leaves and suckers first manifest the disease. Leaves
lower down on the main stalk usually do not show symptoms of the
disease, since these completed their growth in advance of its develop-
ment.

This phenomenon, coupled with the fact that the disease in young
plants affects successively younger and later leaves as they appear
on the elongating main stalk, has led to the erroneous belief that the
course of the disease is upward and never downward.

By inoculating large, nearly mature plants at various points the
writer has brought out some very interesting details concerning the
occurrence of local and general manifestations. If the plants are
inoculated while young or at a period much in advance of maturity,
the mosaic disease in tobacco plants very rarely manifests itself as a
local trouble, since practically all suckers, which appear at a consid-
erably later period, become affected. On the other hand, large, well-
nourished plants at maturity usually produce an abundance of long,
leafy, vigorous suckers. Properly topping the plants at this period
stimulates a yet more vigorous sucker development. By introduc-
ing the virus into the top of one of these, very pronounced local
symptoms of the disease sometimes precede the general phase which
invariably follows.

Several weeks prior to blooming, when the immature flower heads
were only one-half inch to 1 inch across and suckers were only feebly
developed, several series of plants were inoculated with mosaic virus.
One series was inoculated by puncturing the main stalk deeply with
a long needle just above ground. A second series was inoculated by
puncturing the main stalk deeply at several points, beginning at the
ground and extending along about two-thirds of its length. A third
group of plants was inoculated at the very apex of the immature
flower head of the main stalk. Other healthy plants were allowed
to mature fully in order to obtain long, vigorous suckers, which, in
many instances, arose near the base of the plants and nearly or quite
equaled the main stalk in length. One of these was then inoculated
with mosaic virus at or near the apex of the immature buds.

When the virus was introduced into the main stalk at its base or
at several points: along its length all immature parts of the plants,
including the top leaves and suckers, usually developed pronounced
symptoms of mosaic simultaneously. When the virus was introduced
at the very apex of the immature flower heads of the main stalk a
pronounced mottling of the corollas of the main flower head in
several instances was the only noticeable evidence of the disease,
slthough at this time long vigorous suckers were not infrequently
present. Later, however, these also showed the usual disease symp-
toms. In one plant the writer, by antiseptic methods, carefully cut

THE MOSAIC DISEASE OF TOBACCO. 9

away the main stalk, showing only the mottled corollas. Two large,
vigorous, apparently healthy lateral suckers several feet in length
were left undisturbed, and in three days these also had developed all
the symptoms typical of the disease.

Local manifestations of the disease were readily induced in a
number of mature plants by inoculating the apex of a tall, vigorous
sucker. The earliest symptoms were frequently confined to the
branch inoculated and usually first appeared in the small secondary
suckers nearest the point of inoculation. Plate IV, figure 2, illustrates
a typical instance of this sort. <A tall plant of the Sumatra variety
was allowed to blossom, and it soon developed suckers freely. As
shown in the illustration, some of these were nearly as long as the
main stalk. On February 9 one of these (at A) was inoculated
thoroughly in the immature flower head (when only one-fourth inch
across) and at the first bald sucker just below. At this time the
flower head B of the main stalk had been in bloom several weeks
and all the blossoms produced were of a uniform pink color. The
branch or sucker (at C) had not yet blossomed. On February 21
the first symptoms of the disease appeared in the inoculated branch
A, the blossoms of which were pale white or conspicuously mottled.
Likewise, the small axillary shoots (at a and 6) were just showing
mosaic symptoms. No other indications of the disease were notice-
able in the plant. Examined again on March 4 the blossoms of the
branch A were distinctly mottled, although those of the main flower
head B and the branch (’ were still normal in color. On March 5 a
large immature basal sucker was showing mosaic symptoms, and on
March 25 this basal sucker was producing leaves and blossoms mot-
tled to a very pronounced degree. The main stalk B and the branch
( did not at any time show symptoms of the disease, either in the
small axillary suckers or in the color of the blossoms.

Although local mosaic symptoms may sometimes develop as the
result of certain methods of inoculation, the disease invariably
becomes general, appearing in other immature growing parts of
affected plants. In all respects this progressive development of
mosaic is strikingly similar to the behavior of peach yellows, as
described by Smith.1 He says:

There is also a progressive development of the disease, more and more of the
tree becoming involved. In other words, it appears first to be a local disease ;
then, later on,a general one. Quite often, the first season, only one limb or a few
limbs are diseased, but additional limbs are invariably affected the second year.
_ I know of no exception to this. The first year of attack it is comparatively
infrequent to find a tree diseased in all its parts; the second year it is some-
what rare to find many sound branches; often there are none.

1 Smith, Erwin F. Peach yellows, a preliminary report. U. S. Department of Agri-
culture, Botanical Division, Section of Vegetable Pathology, Bulletin 9, p. 91,

13128°—Bull. 40—14-—2

10 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.
SUSCEPTIBILITY OF SOLANACEOUS PLANTS TO THE DISEASE.

Although Mayer? established many important facts concerning
the mosaic disease, he states that he did not succeed in communi-
cating the disease from tobacco to other solanaceous plants.
Westerdijk? has also reported that the mosaic disease of tobacco
is distinct from the mosaic disease of tomato. By actual experi-
ment Clinton ® first proved conclusively that the mosaic disease of
tobacco was communicable to healthy tomato plants, and vice versa.
The writer’s experiments indicate that the two diseases are identical
and likewise communicable to the same plants throughout the
solanaceous family. The writer has readily transferred the disease
from tobacco to a great variety of solanaceous plants. By inocula-
tion the disease has been obtained in the following genera:

In Nicotiana (all varieties of NV. tabacum tested and many distinct
species of Nicotiana).

In Lycopersicon (several of the more distinct varieties of tomato).

In Petunia (Petunia violacea).

In Physalis (two distinct garden species).

In Datura (D. stramonium and D. tatula).

In Hyoscyamus (H. niger).

In Solanum (8S. nigrum and S. carolinense).

In Capsicum (several of the more distinct varieties).

Both in the greenhouse and in the field natural agencies produce
the disease in practically all the plants mentioned.

A mosaic plant of the species Solanum carolinense brought to the
writer’s attention indicates that the mosaic disease of tobacco some-
times occurs in strictly wild plants.

In the genera Solanum and Atropa, although the methods used to
inoculate these plants were particularly severe, all efforts to inocu-
late the common potato (Solanum tuberosum), the eggplant (So-
lanum melongena), and belladonna (Atropa belladonna) have failed
to produce any visible evidence of the disease.

In the genus Datura some species appear to be considerably more
resistant than others to the mosaic disease of tobacco. The disease
may be communicated to D. stramonium L., D. tatula L., and D.
quercifolia H. B. and K. The most persistent and rigorous methods
of inoculation have failed to produce any symptoms of mosaic in
the species D. fastuosa L.

1 Mayer, Adolf. Ueber die Mosaikkrankheit des Tabaks. Die Landwirtschaftlichen
Versuchs-Stationen, Bd. 38, p. 450-467, pl. 3, 1886. Abstract in Journal of Mycology,
v. 7, no. 4, p. 382-385, 1894.

2 Westerdijk, Johanna. Die Mosaikkrankheit der Tomaten. Amsterdam, 1910, 19 p.,
3 pl. (Mededeelingen uit het Phytopathologisch Laboratorium “ Wille Commelin Schol-
ten,’’ Amsterdam, 1.)

*Clinton, G. P. Notes on fungous diseases, etc., for 1908. Connecticut Agricultural
Experiment Station, Biennial Report, 1907/1908, p. 857-858, pl. 66, fig. 6, 1909.

THE MOSAIC DISEASE OF TOBACCO. ab

Evidence at hand indicates that the species Datura metalloides L.
and D. leichhardti F. Muell. may also be as resistant to the disease
as the species D. fastwosa. During the season of 1912 the occurrence
of the mosaic disease was noted in a plat of Datura species growing
at Arlington, Va. The relative susceptibility of these species to in-
fection was well illustrated in this plat. Late in the season prac-
tically all the plants of the species D. stramonium and D. quercifolia
had developed well-marked symptoms. On the other hand, the spe-
cies D. fastwosa, D. metalloides, and D. leichhardtit showed no
observable symptoms of mosaic throughout the season. ;

All efforts to communicate the mosaic disease of tobacco to plants
of other families than the Solanacee have always given negative
results. The following plants have been inoculated by the most
rigorous methods without any visible symptoms of the disease:

Polygonaceze—Sheep sorrel (Rumex acetosella L.).
Leguminos#—

White clover (Trifolium repens L.).

Red clover (Trifolium pratense I.).

Peanut (Arachis hypogea Ll.)

Soy bean (Glycine hispida Moench.).
Cruciferse—

Caulifiower (Brassica oleracea U..).

Peppergrass (Lepidiuwm sp.).
Plantaginacee—Rib-grass (Plantago lanceolota L.).
Labiatee—Dead nettle (Lamium amplexicaule L.).
Oxalidacee—Wood sorrel (Oaalis stricta L.).
Composite—Garden lettuce (Lactuca sativa L.).
Phytolaccacee—Pokeweed (Phytolacca decandra lL.)

SPECIES AND SPECIES HYBRIDS OF NICOTIANA.

A considerable number of distinct species of Nicotiana have been
inoculated at all stages of growth with the virus of the mosaic disease.
Of the various species tested all, with the exception of two or three,
readily became affected with the disease. It has been produced in
the following species: Nicotiana silvestris, N. rustica, N. longiflora,
NV. alata, N. plumbaginifolia, N. forgetiana, N. paniculata, and WN.
langsdor fit.

On the other hand, after the most persistent and rigorous methods
of inoculation, the species Vicotiana glauca and XN. viscosum, so far
as observable symptoms are concerned, do not seem to be affected by
the disease. WV. langsdorfii, which appeared to be immune, finally
developed the disease in one or two plants after a long series of
inoculations with the virus. Plants of this species when inoculated
through the stalk and petioles seem particularly susceptible to a very
destructive and progressive rot, which begins at the point of inocu-
lation and finally kills the plant by slowly involving the surround-

12 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

ing tissues. JV. viscosum is sometimes killed in exactly the same man-
ner, yet no attendant mosaic symptoms have ever appeared in the
affected plants. Although frequent inoculations have been made
deep into the tissues of all parts of V. glauca and NV. tabacum, these
species have never shown any indications of rot at the punctured
points. Plants of the species V. glauca and N. viscosum have been
repeatedly cut back and mosaic virus poured upon all cut surfaces.
This severe treatment has invariably failed to produce any indica-
tions of the disease.

Other experiments were carefully made to determine whether the
sap of mosaic plants could be rendered innocuous by mixing it with
different proportions of the sap of immune species. At first a num-
ber of trials were carried out by mixing equal quantities of mosaic
virus and the sap of. Vicotiana viscosum. As controls, the sap of
healthy plants was substituted for the mosaic virus and diluted in
the same manner. The mixtures were shaken thoroughly and allowed
to stand several hours. Later trials were made in exactly the same
manner, using the sap of the immune species of V. glauca. In pass-
ing, it may be remarked that the diluted virus in no instance lost
its infectious properties, since every plant developed the disease in
15 to 18 days following inoculation. All controls remained healthy.

The virus was still further diluted with the sap of Nicotiana
glauca and tested as follows: Eighteen young plants of Connecticut
Broadleaf were arranged in two series of nine plants each. One
series was inoculated with a mixture of one-tenth mosaic virus and
nine-tenths healthy glauca sap. This was shaken and allowed to
stand three hours at a greenhouse temperature of 80° to 85° F. before
using. Nine control plants were inoculated in the same manner,
using healthy tobacco sap diluted with glauca sap in the same pro-
portions. Needle inoculations in the leaves were made on December
22. Examined on January 9, eight of the series inoculated with the
diluted virus had developed the mosaic disease. All the controls
were healthy. It is evident that the virus was not rendered in-
nocuous.

In further tests with immune species the writer has made many
successful graftings of susceptible varieties of Nzcotiana tabacum
and WV. rustica with the immune species V. glauca and NV. viscosum.
The scions in both instances by subsequent inoculations readily be-
came diseased, but the immune stocks, which were constantly putting
forth vigorous branches, have never shown indications of the dis-
ease, although these were growing permanently and intimately in
contact with the badly diseased scion. Many inoculation experi-
ments have shown conclusively that the virus of. the mosaic scions is
likewise ‘present in the leaves and shoots of the V. glauca stock,
although no visible mosaic symptoms are produced. Furthermore,

Bul. 40, U. S. Dept. of Agriculture. PLATE IV.

Fic. 1.—NORMAL AND MOSAIC BLOSSOMS OF NICOTIANA TABACUM.

Top row, normal blossoms. Bottom row, blossoms from mosaic plants showing an ex-
tremely depauperate growth of the corollas, so that the stamens and pistils extrude.
(Greatly reduced. )

Fig. 2.—SUMATRA TOBACCO PLANT WHICH WAS INOCULATED (AT A) WITH THE
Mosaic DISEASE.

The first symptoms of the mosaic disease appeared ataand b. The disease became gen-
eral at a later period and large basal suckers showed pronounced symptoms of it.

ral

Bul. 40, U. S. Dept. of Agriculture. PLATE V.

Fic. 1.—ScCION OF .NICOTIANA GLAUCA (A) WHICH HAS BEEN GRAFTED UPON A MOSAIC
PLANT OF NICOTIANA RUSTICA (B).

Although the stock is badly diseased the graft shows no visible symptoms. (Reduced.)

FiG. 2.—HEALTHY AND MOSAIC PLANTS OF NICOTIANA LONGIFLORA. A, HEALTHY PLANT;
B, MOSAIC PLANT PRODUCED BY ARTIFICIAL INOCULATION.

(Greatly reduced.)

THE MOSAIC DISEASE OF TOBACCO. 3

the mosaic disease can be produced in healthy scions by inoculating
the virus into the tissues of V. glauca. Plate V, figure 1, shows a
scion of V. glauca growing upon a badly diseased stock of V. rustica.

As to the virulent nature of the sap of the NVecotiana glauca stock
bearing the mosaic scion of V. tabacum, the following experiments
afford conclusive evidence. On February 28 three groups of 25 to 30
small plants of the species V. silvestris were inoculated as follows:
One group, as a control, was inoculated with the sap of a healthy
NV. glauca plant. A second group was inoculated with the sap of a
stock of V. glauca upon which a mosaic scion of NV. tabacum was
growing. A third group was inoculated with the juice of the mosaic
scion itself. On March 11 the control series was perfectly healthy,
but a considerable number of mosaic plants had appeared in the other
two groups. On March 21 all the plants in the control lot were
healthy, while in the remaining two groups practically every plant
had become infected with the mosaic disease. Throughout the ex-
periment these plants were carefully screened in a cage so as to ex-
clude all insects.

The above experiment was repeated as follows: Thirteen young
Sumatra plants were inoculated with sap from the same stock of Vico-
teana glauca, and 10 other plants were inoculated with the sap of a
healthy plant of V. glauca to serve as controls. On April 1, 10 plants
of the 13 had developed the disease. Every plant of the control series
remained healthy. These experiments were repeated several times
at later dates and further confirmed the results of earlier experiments
that the sap of the V. glauca scion carried the mosaic virus.

Hybrids of several distinct species of Nicotiana also have been ob-
tained and grown in order to determine their immunity to the mosaic
disease during the first generation. It was not possible to carry
the work beyond plants of the first generation, since in every instance
the plants were absolutely sterile. With a single exception these
hybrids have not shown the least degree of immunity. In truth,
owing to their exceptionally rapid and vigorous growth, some of
these hybrids have proved to be especially good material for the study
of the disease in all its phases. The following species hybrids have
been made and tested:

Nicotiana tabacum 2° X N. silvestris 6.
NV. longifiora 9 X N. alata ¢.

NV. tabacum @ X N. glauca ¢.

N. tabacum 2 X N. viscosum ¢.

Owing to its extremely rapid and uniform growth, its numerous
leaves, and the readiness with which it succumbs to the mosaic disease,
the writer has used the hybrid Nicotiana tabacum ? X N. silvestris @
very extensively in the experimental study of the disease. The hybrid
NV. tabacum 2 XN. glauca 8 also readily succumbs to the disease,

14 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

although it has been impossible to produce observable mosaic symp-
toms in the male parent.

After many trials the writer finally secured an abundance of
viable seed from a single capsule of Nicotiana tabacum fertilized
with pollen of V. viscosum. This hybrid, in size of plant, in leaf and
blossom characters, etc., is an intermediate form between the two
parents. The most vigorous and persistent methods of inoculating
the mosaic virus into plants of this hybrid have invariably failed to
produce observable symptoms of the disease.

These hybrids, like the male parent. V. viscosum, are attacked by a
very destructive rot at the points of injury if the mosaic virus is
punctured into the stems.

MOSAIC DISEASE IN DIFFERENT SPECIES OF NICOTIANA.

In the genus Nicotiana the degree of expression of particular
mosaic symptoms depends to a considerable extent upon the species
affected with the disease.

All pink-flowered varieties of Nicotiana usually indicate the pres-
ence of the disease by a more or less conspicuously blotched appear-
ance of the corollas in addition to various foliage symptoms. So far
as the writer’s observations extend, no other plants manifest the dis-
ease in this characteristic manner.

The mottled phase cf the mosaic disease is particularly pronounced
in plants of the species Vicotiana longifiora, N. silvestris, and N.
plumbaginifolia. (Pl. V, fig. 2, and Pl. VI, fig. 1.) This distinctive
appearance of the foliage in its extreme form occurs in J. longifiora.
Likewise, in affected plants of this species the tendency to produce
abnormally long, sinuous, ribbonlike leaves is especially marked.
In mosaic plants of the species V. longijiora and NV. silvestris, as a
result of abnormal division in the region of the apical cells, bifur-
cated leaves not infrequently arise. These forms have not been
observed in any varieties of V. tabacum affected with the disease.

Nicotiana rustica (from seed known as the D’Amersfort) by ex-
perimental inoculation readily becomes affected with the mosaic
disease. In plants of this species the development of the disease is
particularly malignant, oftentimes in young plants completely dwarf-
ing the plants and inhibiting practically all foliage growth and
flower formation. (Pl. VII.) The mottled phase is less evident in
mosaic plants of this species than in any other species observed.
The “savoyed” appearance of the leaves, however, is an especially
striking feature of mosaic in plants of this species. Directly or in-
directly, as a result of the disease many affected plants are fre-
quently attacked by a progressive decay of the tissues, which some-
times involves the entire plant, lnlling it to the ground.

THE MOSAIC DISEASE OF TOBACCO. 15

In affected plants of the species Nicotiana paniculata the corolla
tube of the small greenish flowers frequently becomes bent or
crumpled in the middle.

VARIETIES AND VARIETAL CROSSES OF NICOTIANA TABACUM.

In connection with various inoculation experiments with the mosaic
disease, practically all the distinct varieties of Nicotiana tabacum
and crosses among them have been used. None of them has shown
immunity to the disease. The rapidity of development of the disease,
the intensity of the mosaic markings of the leaves of affected plants,
etc., are more or less dependent upon the variety, its vigor, the size
and abundance of the leaves, and their shade of green. The most
malignant symptoms of the disease are frequently associated with
the more distinctive White Burley variety. In affected plants of this
variety badly depauperate and bleached corollas and an intensely
chlorotic appearance of all the leaves are of common occurrence.

THE MOSAIC OF TOBACCO PROBABLY DISTINCT FROM POKEWEED
MOSAIC.

It is a fact of considerable interest to note that pokeweed (Phyto-
lacca decandra) is attacked by a mosaic disease which produces symp-
toms in every way similar to those produced by the mosiac disease in
tobacco plants. Although the mosaic disease of pokeweed is highly
infectious to healthy pokeweed plants, all experiments to date con-
firm the statement that the virus of mosaic pokeweed plants does not
produce mosaic symptoms in tobacco plants. Likewise the virus
of mosaic tobacco plants has never produced the disease in healthy
pokeweed plants.

Healthy pokeweed plants have been repeatedly cut back and in-
oculated for long periods with the virus of mosaic tobacco plants.
The same procedure was followed with other pokeweed plants, using
the virus of mosaic pokeweed. Im the first experiment mosaic
symptoms never appeared. The virus of mosaic pokeweed plants,
however, produced typical pokeweed mosaic, usually within 15 to
20 days following inoculation. —

The following experiments show the results of inoculating different
lots of tobacco plants in pots on the same date, some with the virus
of mosaic pokeweed plants, others with the mosaic virus from tobacco:
On June 18, 15 healthy tobacco plants 6 to 8 inches high were inocu-
lated with tobacco mosaic virus. The first symptoms appeared in
practically every plant June 22-23, 9 to 10 days later. On June 13,
28 similar tobacco plants were inoculated with pokeweed mosaic
virus. Symptoms of disease had not appeared in these plants which
were left undisturbed until July 11. On that date, 14 of these plants

16 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

were arranged in 2 rows of 7 each and inoculated with the virus of
mosaic tobacco plants. The first mosaic symptoms were showing in
these plants on July 23. On July 28, the entire 14 plants showed pro-
nounced mosaic symptoms. With the exception of a single plant, the
remaining 14 plants which had been inoculated with pokeweed mosaic
on June 13 were still healthy. These plants came into bloom on
August 13 and still remained free from the disease, with the exception
of one or two plants.

On June 14, 14 similar plants were inoculated with tobacco-mosaic
virus. The first mosaic symptoms showed in nearly every plant June
23 and 24, 9 to 10 days later. On June 14, 20 similar plants were
inoculated with pokeweed mosaic virus. The disease did not develop
in these plants.

On August 13, when all the plants were just coming into blossom,
2 rows of 7 plants each were selected from the plants treated on June
13 and 14 with the virus of mosaic pokeweed. One row of 7 plants
was then inoculated with the mosaic virus of tobacco. The remaining
row of plants was inoculated with the mosaic virus of pokeweed. On
August 26, 13 days after inoculation, 6 of the 7 plants receiving the
virus of tobacco mosaic had developed the disease. The entire lot of
7 inoculated with the pokeweed mosaic remained free from the disease
as late as October 9, when seed pods were fully ripened.

In October, inoculation experiments were again made with tobacco,
using the virus of mosaic pokeweed and of mosaic tobacco plants.
On October 10, 20 small Connecticut Broadleaf plants in pots were
inoculated with the juice of mosaic pokeweed plants. Using the same
mortar for extracting the juice, 20 similar plants were inoculated
with the virus of mosaic tobacco. The first mosaic symptoms began
to appear in these plants on October 21, 11 days later. On October
27, mosaic symptoms had developed in 17 of the 20 plants treated with
the virus of mosaic tobacco. On the other hand, not a single case of
the disease appeared in the series of 20 plants treated with pokeweed
mosaic, although these plants were kept under observation until
February 1, 1913, at which time they had reached complete maturity.

INCUBATION PERIOD OF THE DISEASE.

Following inoculation a rather variable period precedes the appear-
ance of the first visible symptoms. The length of this period depends
to a considerable extent upon the kind of plant, the age of the plant
when infected, and such external factors as may retard or accelerate
growth.

So far as the writer has been able to learn, eight days, reported by
Woods,! is the shortest period previously observed between the time

1 Woods, A. F. Observations on the mosaic disease of tobacco. U. S. Department of
Agriculture, Bureau of Plant Industry, Bulletin 18, 1902, p. 17.

Bul. 40, U. S. Dept. of Agriculture. PLATE VI.

Fic. 1.—HEALTHY AND Mosaic PLANTS OF NICOTIANA SILVESTRIS. A, HEALTHY PLANT;
B, DISEASED PLANT PRODUCED BY ARTIFICIAL INOCULATION.

(Greatly reduced.)

WR ast Sabi es ss

Fic. 2.—HEALTHY AND Mosaic PLANTS OF NICOTIANA TABACUM. A, HEALTHY PLANT;
B, Mosaic PLANT, SHOWING SYMPTOMS OF THE DISEASE FOLLOWING INFESTATION
WITH APHIDES.

Hundreds of mosaic plants were obtained in this manner as a result of aphid infestation.
(Greatly reduced. )

Bul. 40, U. S. Dept. of Agriculture. PLATE VII

HEALTHY AND MoSAIc PLANTS OF NICOTIANA RUSTICA. A, HEALTHY PLANT; B, DISEASED
PLANT OF NICOTIANA RUSTICA PRODUCED BY ARTIFICIAL INOCULATION FROM A Dis-
EASED PLANT OF NICOTIANA TABACUM.

(Greatly reduced.)

THE MOSAIC DISEASE OF TOBACCO. 17

of inoculation and the appearance of the first mosaic symptoms. |
Mayer‘ produced the disease in 10 to 12 days. In the writer’s experi-
ments the shortest period elapsing between inoculation and the
expression of the first visible symptoms was six to seven days. In ex-
periments during the years 1912 and 1913 many hundreds of tobacco
plants were inoculated with the mosaic virus at all stages of growth,
from the appearance of the cotyledons to complete maturity. The
results of some of these inoculations are as follows:

Ten good-sized plants of several American varieties of tobacco inoculated on
November 18, 1912. First symptoms noted in 16 to 17 days.

Five plants of Nicotiana tabacum 2 X N. glauca ¢ inoculated on December
8, 1912. First symptoms noted in 14 to 15 days.

One hundred good-sized plants of NV. tabacum 2 X N. silvestris ¢ inoculated
on December 8, 1912. First symptoms appeared in 10 to 11 days.

Four young plants of N. tabacum Q@ X WN. glauca ¢ inoculated on December
12, 1912. First symptoms noted in 12 days.

Six rather large plants inoculated on December 16, 1912. First symptoms
appeared in 20 days.

Twelve plants of NV. tabacum 2 X N. silvestris 6 inoculated on December 16,
1912. First symptoms noted in 20 days.

Nine Broadleaf plants inoculated on December 21, 1912. First symptoms
appeared in about 15 days.

Nine young plants of Connecticut Broadleaf inoculated on December 26, 1912,
First symptoms appeared in 11 days.

Twelve full-sized, nearly mature tobacco plants inoculated on February 8,
1912. First symptoms noted in 12 to 15 days.

Sixty-six small tobacco plants inoculated on May 8, 1912. First symptoms
were showing in nearly every plant on May 14 to 15, i. e., 6 to 7 days following
inoculation.

Hight plants of Nicotiana paniculata inoculated May 10, 1912, showed first
observable symptoms 20 days later.

Twenty-nine young tobacco plants inoculated on June 14, 1912, showed first
observable symptoms in practically all within 9 to 10 days.

Seventy young, vigorous tobacco plants inoculated on March 28, 1912, showed
first observable symptoms in 6 to 7 days.

Thirty young plants inoculated on December 9, 1912, showed practically every
plant diseased January 10 to 11, about 50 days later. ‘These plants were grown
in a very cool greenhouse, so that the rate of growth of the plants was very slow.

Thirty young plants inoculated on January 4, 1913, showed first symptoms
18 to 20 days later.

Six or seven days probably is about the minimum period required
to show the first visible mosaic symptoms in tobacco at whatever
stage of growth the plants are inoculated. The disease manifests
visible symptoms usually after 12 to 15 days. Low temperature con-
ditions that generally retard growth appear indirectly to retard the
visible expression of symptoms, which are usually associated with

actively growing cells and tissues.
1Mayer, Adolf. Op. cit.
13128°—Bull. 40—14——3

18 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

Numerous experiments have shown that the disease develops very
readily in 10 or 12 days in large, vigorous plants inoculated just
pricr to blossoming or even after all blossoms have ceased to appear
in the flower head of the main stalk. The progress of the disease is
then coincident with the development of suckers at one point or an-
other on the plant.

DISTRIBUTIGN OF VIRUS IN DISEASED PLANTS.

As already pointed out, obvious symptoms of the mosaic disease
do not necessarily appear simultaneously in all the leaves and branches
of an affected plant. If nearly mature plants become affected with
the disease, the topmost leaves and suckers usually develop unmis-
iakable symptoms, while the lowermost mature leaves always remain
perfectly normal in appearance. In many Instances a single sucker
at the base of a plant becomes typically diseased, while at the same
time others growing beside it show no symptoms whatever. Although
it has been proved experimentally many times that the sap of mosaic
leaves contains an infectious virus, it has not been definitely shown
that this virus may reside in all parts of the affected plants.

Many inoculation experiments made by the writer have shown con-
clusively that the apparently healthy lower leaves, the roots, and even
the corollas of mosaic plants contain the virus of the disease.

On December 26, 1911, 27 young, healthy Connecticut Broadleaf
plants growing in 4-inch pects were arranged in 38 series of 9 each.
One series was inoculated with the sap of the apparently healthy
lower leaves of a plant indicating mosaic disease in only the small
leaves at the top. The second series was inoculated with the sap of the
topmost mosaic leaves of this same plant. The third series, which
served as a control, was incculated with the sap of a healthy plant.
The inoculations in each plant of the three series were made in iden-
tically the same manner by puncturing the leaf blade and midrib of
one or two leaves. When examined on January 6, 12 days after inoc-
ulation, all 9 plants of the series treated with the sap of the topmost
mosaic leaves had developed the disease. Of the 9 plants in the series
inoculated with the sap of the apparently healthy lower leaves of the
same plant 6 had developed the disease. The 9 controls were per-
fectly healthy. When examined about a week later each series re-
mained as before.

On January 5 this experiment was repeated, using 4 series of 8
or 9 plants each. All plants were inoculated alike at two points by
placing the virus upon the stub of a leaf which had been cut off and
by puncturing the leaf blade of a second leaf, as follows:

Nine plants were inoculated with the sap of the mosaic topmost leaves of
a diseased plant.

Eight plants were inoculated with the sap of the apparently healthy lower-
most leaves of a diseased plant.

THE MOSAIC DISEASE OF TOBACCO. 19

Eight plants were inoculated with the sap of the roots of a diseased plant.
Nine plants were inoculated with the sap of a healthy plant.

When examined 16 days later 8 out of 9 plants in the series inocu-
lated with the mosaic topmost leaves of the diseased plant were badly
diseased. Of the 8 plants inoculated with the sap of the appar-
ently healthy lowermost leaves of this plant, 5 had also developed the
disease. Of the 8 plants inoculated with the juice of the roots, 6
plants had become diseased. ‘The 9 controls treated with the juice
of a healthy plant remained healthy.

Inoculations made with the juice of corollas of mosaic plants were
carried out in the following manner: Three series of 9 plants each
were arranged. for the experiment. These plants were grown in
4-inch pots and were of the same age, size, and variety as those used
in the two preceding tests. The plants of each series were inoculated
alike on June 11 by puncturing two or three leaf blades.

Nine plants were inoculated with the sap of mosaic leaves of a diseased
plant.

Nine plants were inoculated with the sap of the corollas of this same plant.

Nine plants were inoculated with the sap of a healthy plant, as a control.-

In 11 days (June 22) all of the 9 plants inoculated with juice
expressed from corollas of the mosaic plant had become diseased;
likewise all of the 9 plants of the series treated with the mosaic
leaves of this same plant were diseased, but the 9 controls receiving
the healthy juice remained healthy.

Similar results were again obtained in a second experiment. On
March 28, 20 young Sumatra plants growing in small pots were ar-
ranged in two rows of 10 each. One series of 10 plants was inoculated
with the expressed juice of corollas of a mosaic plant; the second
group of 10, serving as controls, was inoculated in the same manner
with the expressed juice of corollas of a healthy plant. On April 15,
6 of the 10 plants receiving the sap from corollas of a mosaic plant
had developed the malady. The 10 controls remained free from dis-
ease.

Various similar experiments have always confirmed these results.
It is evident at once that an active infectious principle resides in all
parts of mosaic plants, although such plants frequently indicate only
local symptoms of the disease.

DISSEMINATION OF VIRUS FROM THE POINT OF INOCULATION.

Tf the mosaic virus is pricked into a leaf of a healthy tobacco plant,
an infectious principle disseminates itself from this point into all
parts of the plant. The following experiment gives some idea of the
rate of dissemination of the virus from the point of inoculation:

Seventy young, vigorous Connecticut Broadleaf plants growing in
pots were arranged in 7 rows of 10 plants each. On March 28, 1912,

20 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

two leaves of every plant were inoculated in exactly the same manner
by injecting the virus into each leaf at two points, one on each side
of the midrib. These punctures were made toward the apex of the
leaf, about two-thirds the distance from the base to the tip.

By grasping each leaf near the apex and snapping it off quickly at
the point where the petiole joins the stem, the two inoculated leaves
were carefully removed from a row of 10 plants on each successive
day beginning with March 29. Using this method of detaching the
leaves, accidental infection could not be conveyed by the hand to the
exposed surface of the severed petiole.

By removing these two inoculated leaves on successive days the fol-
lowing results were obtained:

Row 1. Two leaves removed March 29; all plants remained healthy.
Row 2. Two leaves removed March 380; all plants remained healthy.

Row 38. Two leaves removed March 31; all plants remained healthy.
Row 4. Two leaves removed April 1; two plants only developed the disease.

Row 5. Two leaves removed April 2; eight plants became diseased.
Row 6. Two leaves removed April 3; eight plants became diseased. The
symptoms began showing in some of these plants on April 3, the day the

_ leaves were removed.

Row 7. The inoculated leaves of row 7 were not removed, since eight of the
plants were already diseased on April 4. ,

The results in row 3 indicate that the virus had not passed into
the stem from the inoculated leaves which were removed turee days
after inoculation, since none of these plants developed the disease.
Allowing the inoculated leaves to remain on the plant four days from
inoculation in some instances gave the virus time to pass into the
stem. All removals later than this failed to prevent the development
of the disease in the plants, showing that the virus had already passed
from the leaves into the main stem.

Experiments have shown that the sap obtained from infected plants
between the time of inoculation and prior to the appearance of the
first observable symptoms will produce the disease in healthy plants.

During the writer’s experiments the disease developed throughout
a series of controls following inoculation with the sap of a sup-
posedly healthy plant. Since this plant itself very soon developed
symptoms, it is clear that the sap having been obtained during the
incubation period of the disease in this plant contained the mosaic
virus, the disease coming into expression later.

DURATION OF THE DISEASE IN AFFECTED PLANTS.

As the result of their observations of the mosaic disease, Beije-
rinck and Woods have mentioned that plants under certain con-
ditions sometimes appeared to recover from the disease. Lode-

1 Some practical growers have claimed that the disease can be checked if taken at its
first appearance by pulling affected plants until they are loosened from the soil. There
is little in this view, however, to recommend its general adoption.

THE MOSAIC DISEASE OF TOBACCO. 21

wijks? claims to have effected a complete cure by treatment with
blue light. The young, mosaic portions of the treated plants were
covered, while at the same time the lower apparently healthy leaves
were subjected to blue light. He states that the blue hght stimu-
lated the healthy leaves to produce an antivirus which destroys or
renders inert the virus of the disease. Lodewijks also stated that red
light decreased the disease and that suffused light checked it. If the
malady in question was true infectious mosaic disease, one is inclined
to believe that covering the young plants temporarily reduced the
color contrasts of the mottled areas. These changes may have led
Lodewijks to conclude that a partial or a complete cure had been
effected in his experiments.

In the writer’s experimental tests many thousands of affected
plants have been kept under observation throughout all phases of
the disease. In no instance has there been a case of actual recovery
from true mosaic disease. Independent of all external conditions,
the disease remains more or less in evidence so long as the plant
continues to grow. Likewise the writer has never known a plant
of Nicotiana tabacum to die prematurely as a direct result of the
disease. Conditions favorable or unfavorable to vigorous growth
affect mosaic and healthy tobacco plants in much the same manner.
In fact, the most pronounced mosaic symptoms develop only in
well-nourished, succulent, rapidly growing plants, which at maturity
also are capable of supporting a vigorous secondary (sucker) growth.

In a seed-bed experiment mosaic plants which had become affected
when very small, even though growing in a poor soil, badly crowded,
and insufficiently watered, at the end of five months were still main-
taining themselves and elongating slowly in spite of the injurious
effects of the disease. Several of the more vigorous plants had out-
stripped their fellows by a few inches and were preparing to put
forth some blossoms. Other tiny plants were completely hidden
beneath the thickly crowded leaves of the more vigorous individuals.
The leaves of these stunted and overcrowded plants affected with
the disease, although distinctly blotched with dark-green areas, were
little, if at all, “blistered” or “savoyed.” Under conditions form-
ing rapid and vigorous growth these dark-green areas usually pro-
duce pronounced surface swellings.

No curative treatment has been found for the mosaic disease of
tobacco. Gile,? working with pineapple chlorosis in Porto Rico,
showed that this condition was completely overcome so long as

1 Lodewijks, J. A., jr. Zur Mosaikkrankheit des Tabaks. Recueil des Travaux Néer-
landais, v. 7, p. 107-129. Abstract in Botanisches Centralblatt, Bd. 114, No. 20, p. 518,
1910.

*Gile, P. L. Relation of calcareous soils to pineapple chlorosis. Porto Rico Agricul-
tural Experiment Station, Bulletin 11, p. 33, 2 pl., 1911.

22 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

solutions of iron salts were applied to the leaves. Likewise, crystals
of ferrous sulphate applied to the roots were effective. In the
writer’s experiments with tobacco, mosaic leaves in some instances
have been painted with a 1 to 2 per cent solution of ferrous sulphate.
This solution in different strengths has been introduced into the leaf
tissues and other parts of mosaic plants. In no instance has this
treatment produced any noticeable effect in checking the subsequent
course of the disease. Keeping mosaic plants in darkness serves to
induce marked chlorosis, which merely diminishes to some extent the
color contrasts in mottled leaves.

Although it does not appear possible to free a plant from the
mosaic disease, the expression of obvious symptoms depends almost
entirely upon the treatment which affected plants receive. During the
progress of the malady in small, stunted plants the leaves, when
young, are sometimes so slightly changed in appearance that many
of these finally appear to outgrow all traces of the disease. In such
plants this feeble and transitory expression of the disease makes it
appear as if there were a gradual migration of obvious symptoms
toward the top of the plant.

On the other hand, a rapid and extensive development of symptoms
is coincident with rapid growth. Likewise, conditions which retard
growth must of necessity check the further appearance of mosaic
leaves and branches.

EXCISION EXPERIMENTS WITH DISEASED PLANTS.

Various excision experiments have shown that the complete re-
moval by antiseptic methods of all portions of a plant showing
visible mosaic symptoms does not rid the plant of the disease. Pro-
nounced symptoms later appear in other growing portions of plants
thus treated. The excision of mosaic branches sometimes seems
actually to hasten the appearance of pronounced symptoms of the
disease in branches previously normal inappearance. Plants showing
mosaic symptoms only in the topmost leaves or even in the blossoms
alone may be cut completely to the ground, yet mosaic symptoms.
often of the most malignant character, again appear in the shoots
which arise from these stubs.

SOME PROPERTIES OF THE VIRUS OF THE DISEASE.

The sap of mosaic plants is highly infectious to all healthy sus-
ceptible plants. An extremely minute quantity of the virus in-
jected into the tissues is capable of initiating the disease. Tests
have also shown that simply pouring the sap of mosaic plants liber-
ally upon the leaves of healthy plants is oftentimes suflicient to
communicate the disease.

THE MOSAIC DISEASE OF TOBACCO. 23

Mayer has shown that the mosaic virus is killed near a temperature
of 176° F. Iwanowski? first demonstrated that the sap of mosaic
plants was still infectious after passing through a Chamberland
filter. Beijerinck? and Koning? later obtained the same results.
Beijerinck* states that he found the unfiltered juice, volume for
volume, more effective than the filtered. The writer has filtered the
virus through a Berkfeld filter, but the perfectly clear filtrate remains
as highly infectious as the unfiltered sap. Just how long this filtered
sap will retain its virulence has not been determined. Tests by the
writer show that it was still infectious after being bottled for four
months. Beijerinck, also, found the filtered sap infectious after being
kept more than three months and mentions that weak solutions of
formalin did not kill the virus, while heating to the boiling point
did. Beijerinck found that dried mosaic leaves retained their infec-
tious qualities for two years. The writer has found that air-dried
mosaic material, ground and bottled, was still highly infectious at
the end of 18 months. In fact, inoculation with dried material mixed
and macerated with water appears to be the surest method of com-
municating the disease to healthy plants.

Experiments with fresh mosaic leaves placed in soil in large pots
kept out of doors during the winter of 1911-12 seem to indicate that
the virus in this condition retained its infectious properties to some
extent. It was thought that these leaves would decay, but the con-
tinuous, severe freezing weather evidently kept this process in check.
Portions of these buried leaves, practically dry and green in color,
were ground into a paste with water and pricked into young, healthy
plants. The results of inoculations were far less certain than with
fresh juice or bottled mosaic material. Healthy leaves left out of
doors under the same conditions did not produce the disease at any
time.

Numerous experiments have also shown that the mosaic virus pre-
served in ether, toluene, or glycerin retains its infectious properties
for a considerable length of time. The mosaic virus preserved as
follows was still infectious to healthy plants on March 5:

10 ec. e. virus-+1 ¢. e toluene prepared December 9.

1Twanowski, D. Ueber zwei Krankheiten der Tabakspflanze. Land- und Forst-
wirtschaft, 1892. (Russisch.) Abstract in Beihefte, Botanisches Centralblatt, Jahrg. a,
p. 266-268, 1893. (Original not seen.)

2 Beijerinck, M. W. Ueber ein Contagium vivum fluidum als Ursache der Fleckenkrank-
heit der Tabaksblatter. Verhandelingen, Koninklijke Akademie van Wetenschappen, Am-
sterdam, sect. 2, deel 6, no. 5, 22 p. 2 pl., 1898. Abstract in Centralblatt fiir Bakteri-
ologie [ete.], Abt. 2, Bd. 5, No. 1, p. 27-338, 1899.

3 Koning, C. J. Die Flecken- oder Mosaikkrankheit des hollidischen Tabaks. Zeitschrift
fiir Pflanzenkrankheiten, Bd. 9, Heft 2, p. 65-80, fig. 1-2, pl. 2, 1899.

4 Beijerinck, M. W. De l’existence d’un principe contagieux vivant fluide, agent de la
nielle des feuilles de tabac. Archives Néerlandaises des Sciences Exactes et Naturelles,
8. 2, t. 3, livr. 2, p. 164-186, pl. 5-6, 1899.

24 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

8 c. c. virus+1 ¢. c. ether prepared December 9 (ether again added Decem-

ber 26).

5 c. @. virus+20 ¢c. ¢. glycerin prepared December 9.
§ ¢. @ virus+5 ec. ec. toluene prepared January 4.
6 c. c. virus+6 ¢. ¢. glycerin prepared January 4.

The original bottled sap used in these preparations and allowed to
undergo free fermentation was also infectious on March 5. Earlier
experiments have shown that bottled mosaic sap alone, after under-
going fermentation, is able 4 or 5 months later to produce infection.
Tests with fermented bottled virus kept for 15 months did not pro-
duce infection.

It is evident that the active causative agents in the sap of mosaic
plants show a high degree of resistance to the ordinary destructive
agencies.

ROOT INOCULATION WITH THE DISEASE.

The writer’s experiments show conclusively that the mosaic dis-
ease may be communicated to healthy plants by direct inoculation
of the virus into the roots. Several methods of root inoculation have
been tested. In some instances young plants have been pulled up and
their roots dipped into the sap of mosaic plants. The treated plants
were then reset. In other instances, the plants were pulled up and
their roots dusted with dried finely ground mosaic material before
resetting. Other plants when very young were transplanted into
healthy soil, and when these plants had become well established the
soil was removed from some of the roots and mosaic sap poured upon
the broken surfaces from a pipette.

In whatever manner the roots were treated with the virus, the
disease usually deweloped in the plants after 10 or 12 days. The
roots of a plant pulled forcibly from the ground are always more or
less broken, so that the virus in most instances probably came
directly in contact with the broken ends. Root inoculation, how-
ever, does not seem to be as certain to produce the mosaic disease in
plants as inoculation through the leaf tissues.

SOIL INFECTION WITH THE DISEASE.

All experimental data at hand indicate that the occurrence of the
mosaic disease in the seed bed is not so closely associated with soil
infection as has been supposed.

The following method has been used to test the effects of germi-
nating and growing tobacco plants in badly infected scil material:
Square, tightly made frames of wood 10 or 12 inches deep and with-
out bottoms were set into a carefully prepared, light. friable soil to
a depth of 2 to 3 inches. In some of these frames green mosaic

THE MOSAIC DISEASE OF TOBACCO. 25

material, consisting of stem and leaves, was finely chopped into the
soil and covered with a shallow layer of fine soil. Others, serving
as controls, were given the same soil preparation without the addi-
tion of plant material. These frames were kept carefully screened
until the plants outgrew them. As soon as the young plants ap-
peared fumigations with nicotine were made from time fo time to
prevent infestation by aphides. The size of each frame, the amount
of green mosaic stems and leaves chopped into the soil, and the
occurrence of the mosaic disease in each are shown below. The
plants were kept under observation at all stages of development until
the close of each test.

Frame No. 1, control, 18 inches square; nothing put into the soil; seed sowed
August 9. When examined on October 6 the plants were growing vigorously
and all were healthy. On December 9 the plants, 36 in number, were 2 feet
tall, mostly in bloom, and all healthy but one, which had developed the mosaic
disease. ;

Frame No. 2, 18 inches square; 2,000 grams mosaic stems and leaves chopped
into the soil; seed sowed August 9. On October 6 the plants were larger, of a
darker green, and more thrifty than the control frame No. 1. They were 12
to 13 inches high and badly crowded, conditions probably due to the fertilizing
effect of the decomposed plant material originally added to the soil. When
examined on December 9 the 45 plants were 3 to 4 feet high and many were in
bloom; all, from the smallest to the largest, were free from disease.

Frame No. 3, control, 18 inches square; nothing added; seed sowed August 9.
On October 6 the plants, 30 to 35 in number, were 7 to 8 inches high, dark
green, and free from disease. When examined again on December 9 the plants
were 2 to 4 feet high, many were in bloom, and all were free from disease.

Frame No. 4, 18 inches square; 3,000 grams of green mosaic stalks and leaves
chopped into the soil; seed sowed August 20. On October 6 the plants, although
thickly crowded, were 4 to 5 inches high and very green and healthy. Not a
single case of mosaic disease was evident. On December 9 the 75 plants aver-
aged 2 to 23 feet high, growing under badly crowded conditions; many plants
were budding. No mosaic disease appeared.

Frame No. 5, 18 inches square; 2,500 grams of mosaic material added to the
soil; seed sowed August 20. On October 6 the plants were 3 to 4 inches high
and badly crowded; none diseased. On December 9, 30 plants were counted;
all were about 2 feet in height and preparing to blossom; none diseased.

Frame No. 6, control, 18 inches square; nothing added to the soil; seed sowed
August 20. On October 6 the plants, although crowded badly, were 8 to 4
inches high and free from disease. On December 9 the plants, 45 in number,
were about 2 feet high and just ready to bloom; none diseased.

Frame No. 7, 10 inches square; 1,230 grams of green mosaic material added
to the soil; seed sowed August 19.eOn October 6, 40 to 50 plants, averaging
4 to 5 inches high, were growing in this space; badly crowded, yet none dis-
eased. On December 9 there were 35 plants, ranging from 6 inches to 8 feet
8 inches high. The largest plants were in bloom; others were budded; no
disease developed.

Frame No. 8, control to frame No. 7, 10 inches square; nothing added; seed
sowed August 19. On October 6 the plants were about 3 inches high and all
healthy. On December 9 these plants were still free from disease, and their
average height was 18 inches, a few reaching the maximum height of 2 feet.

26 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

The plants in this frame were considerably smaller than those in frame No. 7,
which had received the stems, etc.

In addition to the above tests, which were made under greenhouse
conditions, the following were made in the same manner, out of doors,
in the experimental tobacco plat:

Frame No. 9, 2 feet square; 2,200 grams of mosaic material chopped into the
soil; seed sowed August 4. On October 9 the plants, between 100 and 200 in
number, were 10 to 11 inches high; all were healthy but one, which had become
diseased and was carefully removed. On October 19 the piants were 12 to 15
inches high. One more plant had become diseased. The rest were fine, healthy
plants.

Frame No. 10, control, 2 feet square; seed sowed August 4. On October 9
the plants, several hundred in number, were 10 to 11 inches high and all healthy.
On October 19, 2 plants in this control frame were diseased. It is interesting
to note here that the mosaic plants in both frames were infested with aphides.
Throughout the season these aphides were present on the tobacco plants grow-
ing immediately around frames Nos. 9 and 10. Practically ail the plants in
this field became diseased following infestation. Since the screens protecting
these frames were removed on September 27, it is not surprising that these
aphides should appear on the young plants.

Many other tests of growing plants in infected soils have been
made in addition to these mentioned. Although large quantities of
green material have in some instances been added to the soil, this
very quickly decomposes and incorporates itself with the soil mate-
rial. In no instance has an outbreak of mosaic disease appeared in
plants germinated and grown in such soils. Plants when young have
been pulled and transplanted from infected soils to healthy soils,
where they were allowed to blossom, in oxder to simulate practical
methods of handling plants from soils supposedly infected. Out of
20 or 30 plants thus handled not a single plant developed the disease.

ORIGIN OF THE DISEASE DEPENDENT UPON SPECIFIC INFECTION.

Although earlier investigators have concluded that a true infectious
mosaic condition can be induced by simply cutting back the plants,
the writer’s experiments do not support this conclusion.

Many tobacco plants have been used to determine whether the
operation of severely cutting them back can in itself produce an in-
fectious mosaic disease. Good-sized individual plants were grown in
a screened inclosure, fumigated frequently to prevent infestation by
aphides, and cut back at frequent intervals.

Tobacco plant “A,” in a screened cage, was cut back on January
31, February 24, March 8, and March 25. This plant continued
healthy.

In other experiments tobacco plants were allowed to germinate and
to grow thickly crowded in carefully screened cages, which were
fumigated from time to time. These plants were cut back at frequent

THE MOSAIC DISEASE OF TOBACCO. Qi

intervals from the time the plants were 2 or 3 inches high. The oper-
ation of cutting back was continued as long as the plants were
capable of making growth.

In cage No. 1, 35 plants were cut back on March 8, March 21, March
28, April 10, April 28, May 11, and May 28. These plants remained
free from disease.

In cage No. 2, 25 plants were cut back on March 21, March 28, April
10, April 23, May 11, May 28, and June 25. These plants were still
free from disease on July 5.

All experiments indicate that plants may be cut back indefinitely
if they are free from the mosaic disease at the time of cutting and the
infectious principle of the disease is not accidentally introduced dur-
ing the progress of the experiments. Certain precautions, however, ~
must be very carefully observed. The plants must at all times be kept
screened and fumigated to exclude aphides and other insects. All
instruments must also be very thoroughly sterilized before cutting
back each plant. A flame has afforded the quickest and surest means
of sterilizing these instruments. :

INSECTS AS CARRIERS OF THE DISEASE.

In the course of the writer’s greenhouse experiments with tobacco
it was observed that the occurrence of the mosaic disease was asso-
ciated with aphid infestation. When certain colonies of these aphides
were introduced into cages containing healthy plants it was found
that the disease usually developed in every infested plant. (Pl. VI,
fig. 2.) On the other hand, control tobacco plants never developed
the disease if they were grown in screened cages fumigated at short
intervals to prevent aphid infestation.

Whether other insects than aphides disseminate mosaic infection
to healthy plants yet remains to be determined. This phase of the
question has been taken up by the Bureau of Entomology.

During the season of 1912 the occurrence of the mosaic disease
in tobacco plants in the seed bed and in the field at Arlington,
Va., was unquestionably associated with infestation of the plants
by aphides of the species Macrosiphum tabaci Perg. <A small colony
of these insects early established itself at a single point in the
seed bed. Somewhat later the disease developed in plants at this
point. Shortly after plants from this bed were transplanted to
the field one or two sporadic cases of the mosaic disease came into
evidence. The lower leaves of these diseased plants were infested
with the aphides. These insects sooner or later began to appear
beneath the lowermost leaves of practically every healthy plant in
the field. At the same time mosaic infection spread rapidly through-
out the field until the greater portion of all the plants had developed

28 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

the disease. Although these aphides were very common on the under
side of the ground leaves during early summer, it was interesting
to note that very few could be found a few weeks later.

The occurrence of the mosaic disease in the tobacco seed beds and
later in the field at Arlington can not readily be explained on any
other grounds than that of transmission by insects. The seed beds
were prepared from a good soil, largely mixed with clean sand. This
prepared soil was uniform throughout and had never been used
before. Nor can the widespread occurrence of the disease through-
out the field during the season of 1912 be attributed to mosaic refuse
from any previous tobacco crop, since the only crop of tobacco pre-
viously produced by this field, at least in recent years, was grown in
1911, and during that season only a very small portion of the field
was devoted to tobacco.

WINTER SURVIVAL OF MOSAIC INFECTION.

Tt would seem to be pretty clearly established that specific infection
is wholly responsible for the occurrence of the mosaic disease of
tobacco. During an outbreak of the disease an infectious principle
in some manner must be carried from plant to plant. Since it has
been shown that aphides are sometimes responsible for the spread
of the disease, the question arises as to how this infectious principle
survives the winter. Whether it can be carried over the winter by
hibernating insects yet remains to be determined, but it is hoped that
through cooperation with the Bureau of Entomology definite evidence
may be obtained on the point.

There is strong evidence in support of the conclusion that the mosaic
disease in an affected perennial, such as Solanum carolinense, persists
throughout the life of the plant. In the roots of such plants the
mosaic disease of tobacco doubtless is sometimes carried over the
winter. Although the writer has examined many hundreds of plants
of S. carolinense in various localities, with the exception of the
mosaic plant of S. carolinense previously referred to, every plant has
appeared to be free from the disease. It must be kept in mind,
however, that it is frequently very hard to discern external symptoms
of the disease in plants of this species. No other susceptible species
of the Solanacez has shown such slight symptoms of the disease as
S. carolinense.

It is of some interest here to consider the mosaic disease which
affects the perennial pokeweed (Phytolacca decandra). The writer
has observed that the disease persists from year to year in affected
plants of this species. Although all aerial portions of these plants
perish during the winter, the infectious principle responsible for the
disease is preserved in the underground portions until the following

THE MOSAIC DISEASE OF TOBACCO. 29

spring, when the expression of the disease is coincident with the
growth of new shoots. On several occasions the writer has observed
on these plants the occurrence in large numbers of aphides, which
may serve as an important agency in the dissemination of this
disease.

OCCURRENCE OF THE DISEASE UNDER PRACTICAL FIELD CONDITIONS.

So far as is known, the mosaic disease of tobacco occurs to a greater
or less extent in all countries where tobacco is grown. Mosaic plants
may first appear in the seed bed, or at any stage of growth after the
plants have been transplanted to the field. The extent of injury
caused by the disease depends very largely upon the age of the plants
at the time the disease manifests itself.

Not infrequently plants which appear healthy when cut down at
maturity give rise to mosaic sucker growths from the stubs through-
out a field. This late development and widespread occurrence of the
disease might be explained as due to the fact that the instrument
used to cut the plants carried infection from mosaic to healthy plants
during the process of cutting the stalks. Priming the leaves also
would serve to cause a widespread development of the disease in
sucker growths appearing very late in the season after the period of
harvesting.

The greatest injury is caused when the disease develops in young
plants, since these are as a result usually much dwarfed in all their
parts. The development of the disease in plants that are nearly
mature may in itself result in little or no loss to the grower, on account
of normal leaf growth having been already attained. It is a common
impression among many growers, however, that mosaic plants are
much more susceptible to rust, so that in the end this secondary effect
may sometimes occasion great loss.

In the field, the relative distribution of mosaic and healthy plants
is sometimes very peculiar. It is claimed that only alternate plants
in the rows throughout a field sometimes develop symptoms. It is
very evident that field conditions can not account for this peculiar
occurrence of the disease. Such uniformity in the distribution of
‘mosaic plants can be explained in only two ways. Since two men
riding on a setting machine drop plants alternately in the row, it
may be conceived that one man’s hands have become badly infected
in some way, so that the alternate plants, being the ones which he
has dropped, later develop the disease; but it is more reasonable to
believe that all the plants in the lot dropped by the one man have
been pulled from an infected portion of the seed bed.

Although the opinion seems to be quite generally held-that the
refuse of mosaic plants plowed into the soil may be responsible for

30 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

the disease, there is little evidence to support this view. The fact
is well ectablichent however, that the occurrence of mosaic in both
the seed bed and the field may bear no relation whatever to a previous
outbreak. It has frequently been observed that an epidemic of the
disease may occur on new land as well as on land long devoted to
tobacco. Both in seed beds and in the field the sporadic occurrence
of symptoms is one of the most:striking facts connected with the
history of the disease. This feature of the disease, together with the
fact of its highly infectious nature, has always proved a most serious
obstacle confronting all theories which have attempted to account
for the mosaic disease on a purely physiological basis.

METHODS OF CONTROL.

While conducting various greenhouse experiments with tobacco
during the season of 1911-12, the writer found it almost impossible
to grow plants which did not sooner or later become mosaic. At this
time it was observed that practically all the plants were infested with
aphides and that these insects were responsible for the occurrence
of the disease. By careful experiment it was later shown that cer-
tain colonies of these insects were the active disseminators of the
disease.

In order to secure conditions in every way favorable to the develop-
ment of mosaic in all unprotected plants, fumigations were with-
held until the entire house was overrun with aphides. Large num-
bers of young tobacco plants were then grown in isolation cages
screened with a fine cloth to exclude these insects. At frequent
intervals the inclosed plants were also fumigated with nicotine paper.
By observing such precautions it proved to be an easy matter to
grow plants free from the mosaic disease for any length of time.

Since these preliminary experiments were made, the writer, by
keeping the greenhouses thoroughly fumigated against aphides, has
grown many thousands of healthy tobacco plants during a period of
six months without the occurrence of a single sporadic case of the
mosaic disease. Throughout this period mosaic plants were growing
on every side in connection with various inoculation studies, yet it
was found quite unnecessary to isolate or to screen healthy plants so
long as fumigation was continued.

Although all evidence at hand indicates that the mosaic disease
can be readily controlled under greenhouse conditions, practical
means of preventing its occurrence in the seed bed by similar methods
of procedure have not been attempted. This phase of the question is
now under investigation in cooperation with the Bureau of Ento-
mology.

In any consideration of the control of mosaic under practical grow-
ing conditions two phases of the question present themselves. One is

THE MOSAIC DISEASE OF TOBACCO. ol

concerned with the origin of the disease in the seed bed; the other with
the origin and spread of the disease in the field. Observations at Ar-
lington, Va., during the season of 1912, indicate that if suitable insect
carriers are active, mosaic plants in a field serve as a source of infec-
tion to all surrounding healthy plants. Should the origin of these
diseased plants trace back directly to the seed bed, it is plain that any
means of keeping mosaic infection from seed beds is at the same time
a most important means of controlling the introduction and possible
spread of the disease in the field. Should the disease originate
directly in the field, the matter of successful control would be very
difficult. 2

In various field experiments different investigators have shown
conclusively that laborers in the practical operations of worming,
suckering, and topping tobacco plants readily communicate infection
from diseased to healthy plants. For this reason it is evident that the
prompt removal of all mosaic plants is a most important means of
preventing the spread of the disease in the field by human agencies.
Since insect agencies may also act as carriers of infection in the field,
the early removal of mosaic plants serves as an important check to
the spread of the disease from this source. In whatever manner in-
fection may be carried to healthy plants, it is obvious that mosaic
plants must always be considered infection centers so long as they
are allowed to remain in a field.

NATURE OF THE DISEASE.

Various theories have been advanced to explain the primary origin
of the mosaic disease of tobacco. The view most generally accepted
defines the disease as a disturbance of the enzymatic equilibrium in-
duced by unfavorable conditions of growth. An enzymatic disease,
is physiological in its nature, has it origin within the protoplasmic
complex, and results in a serious and sometimes permanent impair-
ment of the assimilative functions.

Although it has been shown by previous workers that the oxidase
and peroxidase content of mosaic leaves is higher than in normal,
healthy plants, this fact alone does not warrant its being considered
the initial cause of the disease, for it might well be an effect rather
than a cause. If it be considered that oxidases play an essential
part in the metabolism of the plant, it would seem reasonable to sup-
pose that the living cells suffering from lack of nutrition as a result
of the disease in question would be stimulated to secrete an increased
quantity of these enzyms, for it is a well-recognized physiological
principle that cells suffering from lack of nourishment develop an in-
creased amount of enzyms. It has been shown in the Office of To-
bacco and Plant-Nutrition Investigation, for example, that during

32 BULLETIN 40, U. S. DEPARTMENT OF AGRICULTURE.

the process of curing, the diastatic activity of tobacco leaves is greatly
increased, since under these conditions the leaf cells are undergoing
gradual starvation.

As a matter of fact, Bunzel,t from a biochemical study of the
curly-top of sugar beets, a malady in many respects similar to the
mosaic disease of tobacco, has recently shown that an abnormally
high oxidase content is not necessarily a function of the disease, since
his investigations show that plants subjected to various abnormal
growing conditions, such as drought, overwatering, etc., develop a
higher oxidase content than normal plants growing under favorable
conditions. It is apparent, then, that a high oxidase content simply
indicates disturbed metabolism arising from any one of a variety of
unrelated causes.

Although purely physiological mosaic symptoms attended by
chlorosis and various morphological changes in the leaves have been
- induced in tobacco plants by submitting them to sudden temperature
changes, by severely cutting them back, etc., such disturbances are
never infectious and do not usually persist under favorable condi-
tions of growth. In the light of recent facts brought out in our ex-
periments it is hard to see how the mosaic disease of tobacco can be
logically placed in the category of purely physiological disturbances.
The theory of parasitism accounts for the primary origin of the dis-
ease more consistently than the enzymatic hypothesis.

SUMMARY.

The mosaic disease of tobacco is communicable to a great variety
of solanaceous plants. A number of species appear to be immune to
the disease. In the susceptible group some species show a far greater
degree of resistance to infection than otbers. The disease does not
seem to be communicable to plants of other families. Although quite
similar in many respects, the mosaic disease of pokeweed appears to
be distinct from the mosaic disease of tobacco. Both diseases are
readily communicable to plants of their kind.

The incubation period of the mosaic disease is variable, depending
npon conditions favorable or unfavorable to the growth of the plants.
The mosaic virus permeates all parts of the plant, including the roots
and corollas as well as the foliage. The virus does not infect the
embryos of seed produced by mosaic mother plants; therefore, such
seeds produce healthy plants.

By artificial inoculation the mosaic disease can be induced to mani-
fest itself, at first locally. In the course of time, however, the disease
always becomes generalized.

1Bunzel, H. H. Biochemical study of the curly-top of sugar beets. U. S. Depart-
ment of Agriculture, Bureau of Plant Industry, Bulletin 277, 28 p., 1913.

THE MOSAIC DISEASE OF TOBACCO, By)

The visible expression of the disease is only an incidental feature
of obscure pathogenic conditions within the plant and is considerably
modified by external conditions.

Scions of Nicotiana glauca grafted upon mosaic stocks of V. tabac-
um show that the sap of V. glauca carries the virus even though this
species does not manifest visible symptoms. Solanum carolinense fre-
quently shows very slight visible symptoms. The character and in-
tensity of the symptoms depend considerably upon the morphologi-
cal structure of the leaf. Symptoms are more marked in thin, tender-
leaved plants.

The mosaic disease, once initiated, becomes a progressive, incurable
malady. Although many individuals escape the disease from a single
inoculation, no plant of a susceptible species appears to be perma-
nently immune.

The sap of mosaic plants after passing through a filter still retains
its infectious properties. Mosaic material ground and dried retained
its virulence 14 years. The virus preserved by ether, toluene, and
glycerin was virulent four months later, as was also the original
juice, which had been allowed to undergo natural fermentation during
this time.

Certain species of aphides are active disseminators of the mosaic
disease. Not all sucking insects appear to act as carriers of infection.
In greenhouses, where the disease is frequently very troublesome, the
writer has kept it under complete control by frequent fumigations
with nicotine to exclude aphides.

Observations in the field have shown that aphides of certain species
may sometimes be responsible for the occurrence of the disease in
the seed bed and its subsequent spread in the field.

Methods of controlling the mosaic disease of tobacco by fumigation
have not been attempted for practical growing conditions. If the
disease originates in the field after the plants have been transplanted,
its practical control will be exceedingly difficult.

The simple processes of cutting back, frequent repotting, or other-
wise subjecting plants to unfavorable conditions of growth are not
sufficient to produce infectious mosaic symptoms. All facts indicate
that the development of the disease depends entirely upon specific
infection. The disease is not in any sense an air-borne contagion.

Although unbalanced enzymatic activities and physiological toxins
have been considered responsible for the origin of the disease, these
theories do not harmonize with all the facts at hand. Although or-
ganisms responsible for the mosaic disease have not yet been isolated,
parasitism offers the more plausible explanation of the ultimate cause
of the disease.

©

WASHINGTON : GOVERNMENT PRINTING OFFICH : 1913