• TABLE OF CONTENTS
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 Title Page
 Staff and board members
 Introduction
 The fungus
 Relation of soil temperature to...
 Control experiments
 Summary
 Literature cited
 Table of Contents
 Historic note














Group Title: phytophthora disease of tobacco
Title: A phytophthora disease of tobacco
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00027111/00001
 Material Information
Title: A phytophthora disease of tobacco
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: p. 157-219 : ill., charts ; 23 cm.
Language: English
Creator: Tisdale, W. B ( William Burleigh ), 1890-
Kelley, J. G ( John Grady )
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1926
 Subjects
Subject: Tobacco -- Diseases and pests -- Florida   ( lcsh )
Tobacco -- Diseases and pests -- Control -- Florida   ( lcsh )
Phytophthora nicotianae   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 217-218.
Statement of Responsibility: by W.B. Tisdale and J.G. Kelley.
General Note: Cover title.
General Note: "Contribution no. 2 from the Tobacco Experiment Station"--T.p.
 Record Information
Bibliographic ID: UF00027111
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000923479
oclc - 18172242
notis - AEN4030

Table of Contents
    Title Page
        Page 157
    Staff and board members
        Page 158
    Introduction
        Page 159 (MULTIPLE)
        Page 160
        Page 161
        Page 162
        Page 163
        Page 164
        Page 165
        Page 166
        Page 167
        Page 168
    The fungus
        Page 169
        Page 170
        Page 171
        Page 172
        Page 173
        Page 174
        Page 175
        Page 176
        Page 177
        Page 178
        Page 179
        Page 180
        Page 181
        Page 182
        Page 183
        Page 184
        Page 185
        Page 186
        Page 187
        Page 188
        Page 189
        Page 190
        Page 191
        Page 192
        Page 193
        Page 194
        Page 195
        Page 196
        Page 197
        Page 198
    Relation of soil temperature to the development of black shank
        Page 199
        Page 200
    Control experiments
        Page 201
        Page 202
        Page 203
        Page 204
        Page 205
        Page 206
        Page 207
        Page 208
        Page 209
        Page 210
        Page 211
        Page 212
        Page 213
        Page 214
    Summary
        Page 215
        Page 216
    Literature cited
        Page 217
        Page 218
    Table of Contents
        Page 219
    Historic note
        Page 220
Full Text


Bulletin 179


UNIVERSITY OF FLORIDA
Agricultural Experiment Station







A PHYTOPHTHORA DISEASE

OF TOBACCO


By

W. B. TISDALE AND J. G. KELLEY


TECHNICAL BULLETIN

(Contribution No. 2 from the Tobacco Experiment Station)








Bulletins will be sent free upon application to the Experiment Station
GAINESVILLE, FLORIDA


May, 1926






BOARD OF CONTROL
P. K. YONGE, Chairman, Pensacola
E. L. WARTMANN, Citra
E. W. LANE, Jacksonville
A. H. BLENDING, Leesburg
W. B. DAVIS, Perry
J. T. DIAMOND, Secretary, Tallahassee
J. G. KELLUM, Auditor, Tallahassee


STATION STAFF
WILMON NEWELL, D. Sc., Director
JOHN M. SCOTT, B. S., Vice Director and Animal Industrialist
SAM T. FLEMING, A. B., Assistant to Director
J. R. WATSON, A. M. Entomologist
ARCHIE N. TISSOT, M. S., Assistant Entomologist
H. E. BRATLEY, M. S. A., Asst. in Entomology
R. W. RUP.ECHT, Ph. D., Chemist
R. M. BARNETTE, Ph. D., Assistant Chemist
C. E. BELL, M, S. Assistant Chemist
E. W. COWAN, A. M., Assistant Chemist
J. M. COLEMAN, B. S., Assistant Chemist
O. F. BURGER, D, Sc., Plant Pathologist
G. F. WEBER, Ph. D., Associate Plant Pathologist
.. L. SEAL, M. S., Assistant Plant Pathologist
ROBERT E. NOLEN, M. S. A., Lab. Asst. in Plant Pathology
K. W. LOUCKS, A. B., Lab. Asst. in Plant Pathology
ERDMAN WEST, B. S., Lab. Asst. in Plant Pathology
D. G. A. KELBERT, Field Asst. in Plant Pathology
W. E. STOKES, M. S., Grass and Forage Crops Specialist
W. A. LEUKEL, Ph. D., Assistant Grass and Forage Crops Specialist
A. F. CAMP, Ph. D., Plant Physiologist, Cotton Investigations
W. A. CARVER, Ph. D., Assistant Cotton Specialist
EDGAR F. GROSSMAN, M. A., Assistant Entomologist, Cotton Investigations
RAYMOND CROWN, Field Asst., Cotton Investigations
A. L. SHEALY, D. V. M., Veterinarian
D. A. SANDERS, D. V. M., Assistant Veterinarian
C. V. NOBLE, Ph. D., Agricultural Economist
BRUCE MCKINLEY, B. S. A., Assistant in Agricultural Economics
H. G. HAMILTON, M. S., Assistant Agricultural Economist
OUIDA DAVIS ABBOTT, Ph. D., Head, Home Economics Research
GEORGIA WESTOVER, Assistant in Home Economics
HAROLD MOWRY, Assistant Horticulturist
G. H. BLACKMON, B. S. A., Pecan Culturist
IDA KEELING CRESAP, Librarian
J. FRANCIS COOPER, B. S. A., Editor
RUBY NEWHALL, Secretary
HENRY ZEIGLER, Farm Foreman
W. B. TISDALE, Ph. D., Plant Pathologist, in charge Tobacco Experiment
Station (Quincy)
J. G. KELLEY, B. S. A., Lab. Asst. in Plant Pathology (Quincy)
JESSE REEVES, Foreman Tobacco Experiment Station (Quincy)
L. O. GRATZ, Ph. D., Assistant Plant Pathologist (Hastings)
A. S. RHOADS, Ph. D., Assistant Plant Pathologist (Cocoa)
A. N. BROOKS, Ph. D., Assistant Plant Pathologist (Plant City)
STACY O. HAWKINS, Field Asst. in Plant Pathology (Miami)
J. H. JEFFERIES, Superintendent Citrus Experiment Station (Lake Alfred)
W. A. KUNTZ, A. M., Assistant Plant Pathologist (Lake Alfred)
GEO. E. TEDDER, Foreman, Everglades Experiment Station (Belle Glade)


K. H. GRAHAM, Auditor
RACHEL MCQUARRIE, Assistant Auditor








A PHYTOPHTHORA DISEASE OF TOBACCO


By
W. B. TISDALE AND J. G. KELLEY

INTRODUCTION
Upon the inception of the tobacco investigations in the Flor-
ida-Georgia district in 1922 the most serious disease of tobacco
observed at that time was that known to the tobacco growers
as "black shank." Altho the disease had been prevalent in cer-
tain localities for several years, the fields in which it first ap-
peared were somewhat isolated from the main tobacco-growing
sections and only a small percentage of the total acreage of the
district had become infested prior to 1922. Since that year it
has spread over the district at an appalling rate and now only a
few of the old fields remain free from infestation. As a result
the total acreage planted to shade tobacco in 1925 was only
about one-third that of previous plantings. Indeed, the culture
of the commercial types of cigar wrapper tobacco in this dis-
trict in the future will be a hazardous undertaking unless they
are planted on new land each year.
A search thru the literature revealed that black shank had
hitherto not been reported in the United States. Preliminary
investigations by the senior writer showed that the disease was
caused by a species of Phytophthora somewhat similar to Phy-
tophthora nicotianae Breda de Haan. A report of these investi-
gations was published in 1922 (11)'. Since that report was pub-
lished the junior writer has assisted in working out the details
of pathogenicity, physiology and morphology of the fungus and
in the field experiments which are discussed in this paper.
Various methods of soil treatment have been tested for con-
trolling the disease but none of them have proved effective.
Progress has been made in the development of a resistant strain
of wrapper tobacco but at present it is not considered sufficient-
ly resistant for commercial growing on old infested soil.

THE DISEASE
DESCRIPTION
Black shank is primarily a disease of the roots and basal
portion of the stem; hence the common name, black shank, given

1Reference is made by number (italic) to "Literature Cited," page 217.






Florida Agricultural E.'i, 1 ;1,tl1 Station


to it by the growers. Signs of the disease vary somewhat with
the age of the plant at the time of attack and with existing weath-
er conditions. If the plants are attacked within 10 days or two
weeks after they are transplanted, the disease is evinced as a
damping off of the stem at the surface of the soil (Fig. 88). Plants


affected in this


1.


manner drop over































-'0
.-* '


Fig. 88.-Damping-off of tobacco seed-
ling caused by the black shank
Phytophthora in three days after
the plant was transplanted to in-
oculated soil. The plant on the
left is the same age and was
transplanted to sterilized soil at
the same time.


and, during humid weather,
the disease advances in both
directions on the stem and
quickly involves the entire
plant. With less moisture
the invaded parts may dry
out so rapidly until the soft
decay is not apparent.
If the attack is delayed
until the plants attain a
height of 12 inches or more,
first signs of infection are
evinced by sudden wilting
of the entire plant (Fig.
89), which is usually per-
manent. Examination of
the underground parts of
such wilted plants reveals
a dark brown lesion on the
lower part of the main root
or crown, having the con-
sistency of a dry rot (Fig.
90).
Subsequent invasion de-
velops very rapidly so that
the entire root system and
basal portion of the stem be-
comes involved within a few
days. It is not unusual for
the stem to finally turn
brown or black for a foot
or more above ground.
Wilting is followed by yel-
lowing and drying out of the
lower leaves progressively
upward, and finally they
shrivel up and turn brown.






Bulletin 179, A Phytophthora Disease of Tobacco


There is never any distortion of the leaves or one-sided develop-
ment of the plant, as is characteristic of plants affected with
Granville wilt (Bacterium solanacearum). Occasionally, how-
ever, affected plants retain a few small green bud leaves and
blossom prematurely, even tho the stem be dead for several
inches above ground. If such plants are pulled, all of the
roots remain in the ground.
During periods of rainy weather of several days' duration
the lower leaves
are attacked by
the fungus, caus- i 4
ing large brown
spots. Such
weather condi- L
tions often obtain .
just prior to the
harvest season.
Early leaf infec-
tion is usually
characterized by i
more or less cir- A t
cular spots o f
paler green color
than the healthy
parts. Under V X
continued humid
conditions t h e
spots enlarge
very rapidly and
develop the con-
sistency of a soft
decay.
In this stage Fig. 89.-Wilt of tobacco plants resulting from the
the disease on invasion of the roots by black shank Phytoph-
thora. At this stage the wilted condition is the
certain varieties only sign of the disease apparent above ground.
of tobacco has The condition of the root system of this plant
t h e appearance may be seen in figure 90.
of late blight of potato. If the affected leaves touch the ground,
or if placed under a moist chamber, the entire leaf will be in-
vaded in two or three days. On the other hand, the spots on
leaves which do not touch the ground usually do not exceed one
or two inches in diameter before they dry out and become brittle.






162 Florida Agricultural Experiment Station

As the invaded tissues dry out the spots become marked with
bands of different shades of brown, the lighter color being in

S91). Sparse
aerial hyphae and
conidia of the
fungus may be
observed occa-
Ssionally on the
A ;spots, especially
on the leaves
S/ which touch the
ground.
Large blotches
Soften develop on
'''' the first leaves
harvested from
4 infested fie ds
i after they have
.& been hung in the
curing barns.
Enlargement of
the blotches is
very rapid dur-
S ing the first two
i/ or three days but
a, ceases after the
leaves begin to
S. ... cure out. When
S two or three of
/ the blotches oc-
cur on a leaf it is
rendered worth-
less for cigar
Fig. 90.-Root system of one of the wilted plants
shown in figure 89, showing the extent of invasion wrappers. These
at the time the parts above ground wilt. The blotches do not
lower part of the main root or crown is the usual
point of attack on plants of this size. show the same
characteris-
tic markings as the ones which develop on the leaves in the
field, but exhibit a greenish-bruised appearance that persists
in the cured leaves. The tissues of the affected parts are brittle
and often break out during the curing process.


_'MW






Bulletin 179, A Phytophthora Disease of Tobacco


After the lower leaves have been harvested from plants which
previously escaped the disease,- infection frequently occurs in
the leaf scars (Fig. 92). From these centers of infection the
disease advances on the stem in both directions, invasion be-
ing faster in the pith than in the vascular tissue and cortex. It


is not unusual to
find the pith in-
vaded and dried
out for a distance
of one to three
feet in advance
of the invaded
cortex. As the
pith dries out it
splits into disc-
like plates (Fig.
93). .The rate
of invasion of
young stems ap-
pears to be about
equal in all tis-
sues and they
dry out and
shrink together.
A cross section
of a young stem
i m e d i a t e-
ly above the dead
portion shows
brown areas in
the cambium re-
gion. When the
stem is split
lengthwise t h is
discolora-
tion shows as


J^ ^ -"^,





..





., .
"" .. ~. '~- '- ".













Fig. 91.-Spots on leaf of Big Cuba tobacco which
developed as a result of natural infection in the
field. Such spots develop late in the season dur-
ing periods of rainy weather.


brown streaks which extend from one to several inches in ad-
vance of the upper limit of the dead cortex. Examination
of these brown streaks with a microscope reveals the pres-
ence of fungus hyphae which, upon planting segments of such
diseased stems on agar plates, were found to be in part that






Florida Agricultural Experiment Station


of the Phytophthora and also Fusarium. The Phytophthora
hyphae were found to invade all tissues of young stems, leaves
and roots, being both inter- and intra-cellular. The pith cells
become filled with the
hyphae which coil about
within the cell. There
4 1 is no such evident accu-
!I mulation in the paren-
'I chymal tissues but the
Sci '! hyphae penetrate in all
Directions without much
evidence of resistance
by the membranes. Very
little of the fungous
growth appears on the
surface of diseased por-
tions of the plant and this
only during rainy
weather of several days'
duration or when the
parts attacked are
shielded from the sun-
light. Under these con-
ditions few conidia have
been found on long slen-
der conidiophores which
S are not distinctly differ-
ent morphologically from
t h e infertile hyphae.
Fe w chlamydospores
have also been found
under field conditions on
the surface of and with-
in the cortex and pith of
Fig. 92.-Sections of tobacco stems show-
ing infection of leaf scars which oc- diseased portions of the
curred after the leaves were harvested.
This type of infection is also occasion- stem, but this is by no
ally produced by the Phytophthora means a common occur-
working back through the leaf petiole. rence. Numerous con-
idia and chlamydospores have been found on artificially inocu-
lated plants in the greenhouse when the moisture content of
the soil was kept high.






Bulletin 179, A Phytophthora Disease of Tobacco


HISTORY AND GEOGRAPHIC
DISTRIBUTION

No one knows where black
shank came from, but the
evidence indicates that it
was introduced, not endemic.
Tobacco has been grown in
the Florida-Georgia district
for more than 60 years and,
according to information ob-
tained from the growers,
black shank was unknown un-
til about 1915. In that year it
appeared on two farms in
the southern part of Decatur
County, Georgia. The own-
ers of these farms stated
that they had imported no
tobacco seed nor stable
manure for at least two
years prior to that time, so
there is no clue as to how
the fungus was introduced.
In 1922 the disease was
observed on several farms
in the northern and western
part of Gadsden County,
Florida, and on two farms
in Leon County. Since that
year it has spread very rap-
idly from one farm to an-
other in Gadsden County
and by 1925 very few old
farms have escaped without
some infection. In 1923 the
disease appeared in Pasco
County, Florida, which is
more than 200 miles distant
from any other tobacco-
growing district of the
state. It was reported in


i


I





i
I;












i
I


en..

Z.,









I.
:7.


\ *' ..... -;;; t

i:

Fig. 93.-Tobacco stems split longi-
tudinally to show the character-
istic manner in which the pith
segregates as it dries out follow-
ing invasion by the Phytophthora.
It is not unusual for infected stems
at harvest time to show such in-
vasion of the pith for one to three
feet in advance of any discolora-
tion on the surface of the stem.






Florida Agricultural Experiment Station


1924 from Summerdale, Alabama, by a grower from that sec-
tion. Also, in that year specimens of bright leaf tobacco af-
fected with black shank were sent to the Tobacco Experiment
Station at Quincy, Florida, from two localities in Georgia. The
disease was reported from Virginia in 1924 (12). What ap-
pears to be the same disease of tobacco has been prevalent in
Java since 1895 (1). Also, a similar disease was reported from
Porto Rico in 1925 by G. H. Chapman and Mel T. Cook (by let-
ter to the senior writer). However, at that time neither of the
investigators had obtained the organism in culture so it could
not be compared with the black shank Phytophthora occurring
in Florida.
ECONOMIC IMPORTANCE
Black shank has no peer in economic importance among the
several field diseases of tobacco occurring in the Florida-Georgia
district. Connecticut Round Tip, a type of wrapper tobacco re-
cently introduced from Connecticut, is completely susceptible to
the disease and when it is planted in fields which showed previ-
ous infestation, the crop is always a total failure. The final
outcome of the local type, Big Cuba, under such conditions is
but little better. Consequently, it soon became common practice
to abandon fields for tobacco culture after the first year that
signs of the disease appeared. However, heavy losses, indeed
total failures, are frequently incurred during the year the dis-
ease makes its first appearance in a field. This condition was
especially common in 1924 and in 1925.
Therefore, the sudden appearance of the disease together with
its rapid spread, once it has been introduced, have made the cul-
ture of shade tobacco very risky and unprofitable. As a result,
the total acreage of shade tobacco grown in 1925 was only about
one-third of previous plantings. During the last two years a
few of the growers have been experimenting with cloth shades
and moving to new land each year. So far it is not known
whether this method will be profitable, as there is a question
about the quality of leaf produced under cloth alone.
Many fields have come under observation where black shank
appeared early in the season in one or more localized areas and
by the end of harvest from five to ten acres had become involved.
Figure 94 shows a portion of a field in which black shank ap-
peared in several areas about April 15 and advanced uniformly






Bulletin 179, A Phytophthora Disease of Tobacco


across the field, leaving only the portion of three rows by May 20
On the other hand, a few other fields have been observed where
the disease made very little headway during the season, even
tho the type of soil and methods of cultivation were practically
the same as in the fields in which the initial infested areas en-
larged very rapidly. Furthermore, several fields varying in
size from five to thirty acres have shown uniform initial infes-
tation and where Connecticut Round Tip had been planted the
crop was a total failure. Such sudden appearance and uniform
rapid dissemination of a soil-inhabiting parasite without previ-
ous warning can only be considered a phenomenon.


AI


Fig. 94.-Portion of three rows of Connecticut Round Tip tobacco under
a six-acre shade which have not yet been attacked by the Phytophthora.
Centers of infection appeared early in the season some distance to the
left of these rows and enlarged very rapidly, making a clean sweep
across the field in all directions.
Black shank causes little or no direct loss to seedlings in the
plantbeds in this district, while in Java and Porto Rico the beds
are reported to suffer great damage, varying with seasonal con-


5 I
!Ih~iC Il I;Cb .*r_.






Florida Agricultural Experiment Station


editions. The absence of the disease from seedbeds in the Flor-
ida-Georgia district seems to be due to the relatively low tem-
perature prevailing at that time. Also, most beds are sterilized
with steam or "fired" before they are seeded. So far the disease
has been found in only two beds and it appeared in these just
at the conclusion of the transplanting period. One of these
beds was overflowed by drainage water from an infested field,
and it seems probable that the fungus was introduced into the
other in soil adhering to the feet of laborers and the crates used
for transporting the plants to the fields.
Tobacco is susceptible to attack by the Phytophthora at any
stage of development. However, since the attack is inhibited
by low soil temperature, the plants set early reach a more ad-


.5;.
~*. +-~
20' Vt ~X'
p Jh. L1c.
;1
I
I'. :~~.c


B


it

A
U.~


_ ,:- ",


i _
''-M4
jc-
y C n. ^**-*


. 1


Fig. 95.-Portion of a field of bright leaf or cigarette tobacco killed by
black shank. This type of tobacco is highly susceptible to the disease
and once a field becomes infested the subsequent rate of spread is about
as rapid as on the more fertile shaded soils.

vanced stage of growth before the disease develops than do
plants set later in the season. Consequently, the loss has been
lighter in certain fields set early because part of the crop could
be: harvested before the fungus had spread-to all parts of the
field. However, there is no advantage derived from early plant-
ing in fields known to be uniformly infested. During normal
seasons the transplanting period extends from about March 20
to April 15, and first signs of black shank appear about April
10. If susceptible plants are set in infested soil after April 15,
the chances are that they will be attacked before they recover
from the shock of transplanting.






Bulletin 179, A Ph.t,1ipliftbotit, Disease of Tobacco


So far black shank has not been a serious menace to bright
leaf tobacco in this district, altho all types tested are highly
susceptible to the disease (See Table I). Bright leaf tobacco
was introduced into this district in 1924, and a few growers
made the mistake of planting it near old infested shades. The
crops planted in such locations suffered heavily, the disease
making a clean sweep across the field (Fig. 95). Also some loss
occurred in fields set with plants from one of the infested beds
referred to in a preceding paragraph. Since bright leaf tobacco
produces the best quality of leaf on poor sand land under a two-
or three-year rotation, it seems possible that the loss from
black shank can be kept at a minimum for the-next few years
at least.
THE FUNGUS
HOSTS
So far a search for natural host plants other than tobacco
has been in vain. None of the many species of weeds growing
in and around infested fields have shown any signs of attack by
the Phytophthora. The roots of several species of cultivated
plants, including tomato (Lycopersicum esculentum Mill.), egg-
plant (Solanum melongena Linn.), potato (Solanum tuberosa
Linn.) and beans (Phaseolus vulgaris Linn., Hort. var. Ken-


Fig. 96.-A vigorous crop of tomatoes growing on soil after a crop of to-
bacco had failed due to infection with Phytophthora.






Florida Agricultural Experiment Station


tucky Wonder) growing in infested soil have been examined for
the presence of the Phytophthora. The vigor of Kentucky
Wonder beans and tomatoes growing in infested soil is shown
in figures 96 and 97.


Fig. 97.-An excellent crop of Kentucky Wonder beans growing on soil
thoroly infested with the black shank Phytophthora. A crop of tobacco
which had failed was turned under when the land was prepared for the
beans.






Bulletin 179, A Phytophthora Disease of Tobacco 171

Of the several species of plants repeatedly inoculated with the
Phytophthora by different methods, tomato, potato, eggplant and
castor-oil plant (Ricinus communis Linn.), were the only ones
that developed any disease symptoms. When mycelium of the
fungus was inserted into young stems of these plants the region


FP. I
Ni


LW


~~4','


Fig. 98.-Young leaf of castor-oil plant (Ricinus communis) showing in-
fection produced by inoculating with zoospores of the black shank
Phytophthora. Photographed three days after inoculation.

around the points of inoculation in potato, eggplant and castor-
oil plant turned brown and dried out but the disease remained
local. No infection developed in older stems inoculated in this
manner. Neither did infection develop when seedlings were
transplanted to infested soil. Young twigs of eggplant bearing
blossom buds showed signs of infection on the buds in three
days when inoculated with a water suspension of zoospores and






Florida Agricultural Experiment Station


kept under a bell jar. Young tomato seedlings developed slight
infection in three days but the leaves were never seriously in-
vaded. Potato seedlings inoculated in a similar manner showed
few diseased spots on the leaves in three days and the entire
plants were invaded in seven days. Young castor-oil plant
seedlings showed signs of infection in two days when in-
oculated with a water suspension of zoospores and kept under
a moist chamber. After three days the spots were from one
to several centimeters in diameter and bore conidiophores and
conidia (Fig. 98).

Breda de Haan reported (1) that Amaranthus sp. and And-
rong, weeds growing in tobacco fields in Java, were affected by
P. nicotianae. Palm stated (8) that young plants of Ricinus
communis, Commelina nudiflora and tomato (Lycopersicum
esculentum) were susceptible to attack by P. nicotianae, while
potatoes were not susceptible.

SUSCEPTIBILITY OF DIFFERENT VARIETIES OF TOBACCO

During the four seasons black shank has been under investi-
gation, different types and varieties of tobacco, as they could
be obtained, have been tested under field conditions for resis-
tance to the disease. The object in making these tests was to
ascertain whether a naturally resistant type existed and, if it
were not a wrapper type, a cross might be effected between it
and a desirable type with the hope of combining resistance with
desirable wrapper characters in a new type. The results of
these tests are brought together for reference in Table I.

It may be seen from Table I that all cultivated varieties of
Nicotiana tabacum tested are highly susceptible to black shank.
The final observations, as recorded, were made a few days
before the end of harvest and most of the plants noted as
living at that time died before the seed matured. Nicotiana
rustica, on the other hand, proved highly resistant to. the dis-
ease. In the one test of this species the plants remained free
from any disease symptoms until the full blossom stage when
the percentage indicated in Table I suddenly succumbed. Fig-
ure 99 shows its appearance in the trial plot just prior to the
time the plants wilted. Examination of the roots of the plants
attacked by black shank revealed that they were attacked by






Bulletin 179, A Phytophthora Disease of Tobacco


nematode also. It seems pobable, therefore, that the Phytoph-
thora was unable to make a successful attack until the knots
caused by the nematode began to decay. This same condition
has been observed with the partially resistant strains of Big
Cuba. So far all efforts to cross Nicotiana iustica with differ-
ent types of cigar wrapper tobacco have been unsuccessful.

TABLE I. COMPARATIVE SUSCEPTIBILITY TO BLACK SHANK OF DIFFERENT
TYPES AND VARIETIES OF TOBACCO.


Variety or Type


Big Cuba (E Type).................
Big Cuba (P Type)................
Big Cuba (H Type)-..................
Conn. Round Tip.-...................
Haliday Havana.........................
1207 (W is. binder)........................
W hite Burley..................................
Conn. Broad Leaf....................
Zimmer .................. .....
Maryland Mammouth........
Conn. Havana----.......... ..........
Cuban (Conn. Shade)---.................
Adcock (Bright leaf)..................
Harrison Pryor (Bright leaf)..
Warne (Bright leaf) ...................
Big Oronoco (Bright leaf)...........
White Stem Oronoco (Bright
leaf) ......----....................... ......
Porto Rican (Var. name
not given......
Nicotiana rustica........................


Number of Condition at End of Season
Plants Living Diseased
percent percent
439 17 90
968 0.6 99.4
315 7 96
1,220 00 100
402 00 100
500 00 100
308 00 100
303 2 99.5
102 00 100
102 6 98
72 00 100
5 acres 00 100
90 00 100
82 00 100
34 00 100
67 00 100
82 00 100
00
119 7 95
54 70 30


AGENCIES OF DISSEMINATION
Because of the sudden introduction of the fungus from one
locality to another and its rapid spread over fields, once it has
been introduced, considerable effort has been made to determine
the chief agencies of distribution so that some means might be
devised to check further spread until methods could be perfected
whereby a crop could be grown on infested soil. Field surveys
over a period of four years, with cumulative experience of to-
bacco growers, afford abundant circumstantial and direct evi-
dence that the organism is carried from field to field in a variety
of ways. These include the following:

1. By diseased plants from infected beds. This mode of dis-
semination was clearly demonstrated in 1922 and again in 1925,
altho this is not a common way.






Florida Agricultural Experiment Station


2. By wa-
ter. This
mode has
been illus-
trated in sev-
eral instances
where drain-
age water
from infested
fields and
roadways
flooded a
seedbedor
swept across
depressions in
fields. In such
instances the
disease first
appeared
along the wa-
ter courses
(Fig. 100).
There is also
c i r cum-
stantial e v i-
dence indicat-
ing that the
fungus was
introduced in-


S*..~.-. ...


.:"" _r' -. f -_ -.




. .- -,.^ ...K









Fig. 99.-Row of Nicotiana rustica growing on in-
fested soil in a trial plot. Its high resistance to
black shank is indicated by the uniform stand in
contrast with the total failure of the types of cigar
wrapper tobacco planted in adjacent rows.
.Is~-. .r X!- I

Fig 9-Rw f ictina usic gowngonin
fete oi n rilplt.Is ih eisacet


to fields in water from stagnant ponds used in setting the plants.
3. By farm tools, animals and man. Many growers offer
circumstantial evidence indicating that the fungus has been
carried from field to field in soil adhering to plows and feet of
farm animals and laborers. There is also considerable circum-
stantial evidence indicating that contractors or buyers have
carried it from one locality to another on their feet while on in-
spection tours. It is customary for them to visit fields and in-
spect the crop several times during the growing season, visiting
a number of fields during the same day.
4. By wind blowing dust from field to field. The uniform
initial infestation of several fields indicates that this may be






Bulletin 179, A Phytophthora Disease of Tobacco


.1


Fig. 100.-Portion of a tobacco field illustrating the importance of surface
drainage water in disseminating the Phytophtora. Note the complete
failure of the plants along the watercourse.

an important agency of distribution. During March, after the
land has been prepared for planting, great clouds of dust may
be seen drifting out of the fields.
5. By barnyard manure. Altho all attempts to locate the fun-
gus in manure have failed, it is possible that this is an agency of
disseminating it to different fields on a farm. D'Angremond
reported (3) that he had obtained infection of tobacco leaves
with P. nicotianae by placing a paste on them made from man-
ure obtained from the native villages. He stated further that
the disease was more severe on land fertilized with such manure.
PERSISTENCE IN THE SOIL
Having ascertained that the fungus is a soil-inhabiting
parasite, the possibilities of its eradication thru crop rotation
were given early consideration. Upon inquiry among the grow-
ers, it was learned that no one had returned to infested soil
with tobacco once it had been abandoned, the reason being that
the initial outlay for growing a crop of shade tobacco is too
great to risk a chance of failure.






Florida Agricultural Experiment Station


Sufficient evidence has been accumulated since 1922 to show
that once the Phytophthora has been introduced into the soil,
it is very aggressive and persistent. It was formerly common
practice to grow tobacco many years in succession in the same
field, several cases having been reported where 15 to 18 good
crops were obtained. Now in most sections of Gadsden County
the soil may become infested the first year and, if the field be
planted the second year, the disease may be universal and cause
almost if not a complete failure. Indeed, several fields have
been observed which were universally infested the second year
without having shown any signs of the disease the previous
year. Once established in the soil, evidence shows that the fun-
gus persists for several years, just how many has not been as-
certained.
Tests made in two fields which had been abandoned for to-
bacco culture showed that the soil was still infested after three
and five years respectively. One of these fields grew the last
crop of tobacco in 1921 when it showed uniform infestation.
The land remained idle in 1922 and was planted to corn the two
years following. In 1925 the field was set with a partially re-
sistant strain of Big Cuba tobacco. The loss from black shank
was relatively light but the diseased plants were sufficiently
distributed over the field to show that the soil was still pretty
thoroly infested. The other field was last planted to tobacco in
1917 and the crop was almost a complete failure that year. In
1923 one acre of this field was planted to Connecticut Round Tip
tobacco. Precautions were taken to prevent reinfestation from
infested tobacco fields on the farm. Also no manure was used
as a fertilizer. The plants were set on April 23 and they began
to show signs of the disease by May 16. From that time on
the disease developed very rapidly and less than half a crop
was obtained, even tho the plants were topped low and the
leaves harvested early.
Altho these tests do not prove how long the Phytophthora
persists in the soil, they show that the period is too long to elimi-
nate it thru any rotation system practical with shade tobacco.
The account of the disease of tobacco in Java caused by P. nico-
tianae indicates that the life of the organism in the soil is in-
definite. De Bruyn reported (5) that P. nicotianae was known
to persist in the soil for three years. D'Angremond also reported
(4) that there are indications that this organism persists in the






Bulletin 179, A Phytophthora Disease of Tobacco


soil during the 15 months the land is devoted to growing a crop
of rice in rotation with tobacco.

RESISTANCE TO DRYING IN THE SOIL
In an experimental determination of the-effects of drying upon
the virulence of the fungus both natural and artificially inocu-
lated soils were used. Soil was brought into the laboratory from
an infested field in March, 1923, and placed in boxes 18 inches
square by 8 inches deep. About the same time two boxes of
sterilized soil were inoculated with pure cultures of the fungus.
Both lots of soil were then set aside on a bench and allowed to
remain in an air-dry condition until September, 1924, when it
was again watered and given an application of commercial fer-
tilizer. Ten Connecticut Round Tip seedlings were set in one
box of each lot of soil and the boxes were placed on a greenhouse
bench where the temperature was favorable for the develop-
ment of black shank. Thirty days later five of the plants in
each box were dead and the others showed signs of the disease.
This test shows that the fungus is capable of retaining its viru-
lence in air-dry soil for at least 18 months, altho it has not been
determined in what stage it holds over in the dormant condition.
Since it has been ascertained that the fungus will grow read-
ily on sterilized manure and to some extent on sterilized Nor-
folk loam soil, it seems possible that it is capable of living a
saprophytic life for some time under field conditions. This
is strongly emphasized by Jensen's account of P. nicotianae (6)
in which he showed that a very small amount of diseased to-
bacco stems was necessary to infect manure artificially.
ISOLATION
Considerable difficulty was encountered at first in isolating
the fungus in pure culture from young diseased plants. One
cause of failure was due to the large number of saprophytes
which follow closely the invading parasite. Besides bacteria,
Fusaria were the forms most commonly present. These follow
the parasite so closely thru all tissues of young stems and leaves
until no method of surface disinfection tried gave aid in destroy-
ing the saprophytes without at the same time killing the para-
site. Furthermore, examination of old diseased stems suggests
that the Phytophthora disintegrates after the host cells become
exhausted and destroyed. Less difficulty was encountered in
making isolations from plants which were about mature before






Florida Agricultural Experiment Station


they were attacked. Sections of such stems two or three inches
long were taken from the upper limit of the invaded portion,
treated with mercuric bichloride solution for several minutes
and then split lengthwise with a flamed scalpel. Small pieces of
the diseased pith were then removed and planted on poured
plates of potato-dextrose agar and oat agar. When the frag-
ments of pith were taken from the juncture of healthy and dis-
eased parts pure cultures of the fungus were obtained in a
high percentage of attempts.

PROOF OF PATHOGENICITY
Preliminary experiments to prove the pathogenicity of the
fungus were made by inoculating marked areas on detached
young tobacco leaves with drops of a water suspension of zoo-
spores. (The suspension of zoospores for these and subsequent
experiments in which zoospores were used was obtained by
pouring sterile water into a tube culture of the Phytophthora
and allowing it to remain until a microscopic examination re-
vealed an abundance of zoospores in the water, usually 15 to 20
minutes.) The inoculated leaves were kept in a moist chamber
at room temperature, 20 degrees Centigrade or above. Infec-
tion was usually apparent in 36 hours when the leaves were in-
oculated, on either the upper or lower surface. After four to six
days a greater portion of the leaves were invaded and numerous
conidia were present.
In December, 1922, four vigorous Big Cuba tobacco plants
about 18 inches high were atomized with a water suspension of
zoospores. The leaves of two of the plants were punctured
with a flamed steel needle before they were inoculated. After
they were inoculated one plant with uninjured and'one with
injured leaves were placed under bell jars partially lined with
moist filter paper, while the other two were left uncovered in
the laboratory room. Two other plants, one with punctured
leaves and one unpunctured,. were atomized with sterile water
and placed under bell jars for checks. After three days the
leaves of inoculated plants under bell jars showed numerous
water-soaked spots varying in size from one to two cm. There
was no evidence that the fungus was dependent upon wounds
for gaining entrance into the leaf tissues. The inoculated plants





Bulletin 179, A Phytophthora Disease of Tobacco


left in the open showed no signs of infection at this time or
later.
After six days the spots on several leaves had coalesced and
involved the entire leaf, extending back to the stem. The mar-
gins of the spots at that time were pale green and the tissues
appeared as if scalded, while the centers were darker colored
and had the consistency of a soft decay. A few long, sparsely
branched hyphae were present on the surface of diseased parts
but very few conidia were observed. Two days after the bell
jars were removed the spots had dried out and turned brown.
The check plants remained healthy thruout the experiment.
Several other in-
oculations have been -
made in a similar
manner at different
seasons of the year, .
using plants of dif- "
ferent ages and
growing in the
greenhouse. In f e c-
tion invariably devel-
oped except when the .,"
temperature was too i '
low or when the
plants were exposed.
to the sunlight dur- r' -
ing the summer.
Figure 101 shows -' '.
the nature, of infec-. ..
tion on a young leaf
after three days. "
Invasion of the ,
leaves was immedi-
ately checked when
the bell jar was re-
moved and the
plants exposed to the Fig. 101.-Leaf of tobacco seedling showing
sunlight. The spots disease produced by inoculating with a
unligt. water suspension of zoospores of the black
then dried out and shank Phytophthora and then placing in
a moist chamber. Photographed on the.
developed the differ- third day after inoculation.






Florida Agricultural Experiment Station


ent shades of brown color characteristic of the leaf spots which
develop in the field.
Infection invariably resulted when a small amount of my-
celium of the Phytophthora in pure culture was inserted into
the stems of tobacco plants at any point. During the spring and
summer months seedlings inoculated in this manner would drop
over at the point of inoculation within two or three days. In-
vasion was equally rapid in stems of older plants when the in-
oculation was made near the bud, but was slower in the older
parts.
Further proof of the pathogenicity of the Phytophthora was
obtained by inoculating sterilized soil with pure cultures of the
fungus and then transplanting tobacco seedlings to the soil.
Under these conditions and with favorable temperature, seed-
lings of the age and size for transplanting invariably showed
signs of the disease on the basal portion of the stem in three to
five days (Fig. 88), typical of the disease developing in the field.
Other experiments were made to ascertain how soon seed-
lings would be attacked when the seed were planted in infested
soil. In March, 1923, Big Cuba seed were planted in boxes of
infested soil kept out of doors under cloth covers. Seed was
planted in sterilized soil at the same time for check. A good
stand of seedlings appeared in both sterilized and infested soil
and they grew normally to the size for transplanting by April
15 without showing any signs of black shank. After a few days
of warm weather the seedlings began to die in the infested soil
with typical black shank symptoms.
In May, 1925, seed were planted in artificially inoculated soil
and in sterilized uninoculated soil for check. A good stand of
seedlings appeared in both lots of soil and no signs of black
shank developed until June 18 when the leaves were about the
size of a silver dollar. From that date the plants died very
rapidly in the inoculated soil. The seedlings in uninoculated
soil remained healthy. This indicates that the young seedlings
are resistant to the disease or that sunlight inhibits infection
until the roots have penetrated the top layer of soil and the
leaves are big enough to shade the soil, which appears more
probable.
INOCULATION OF OTHER PLANTS
The collection of plants selected for inoculating with the black
shank Phytophthora consisted primarily of the species and






Bulletin 179, A Phytophthora Disease of Tobacco


varieties commonly planted under tobacco shades during the
fall and winter months and ornamental plants grown in yards
around farm houses. The plants used were inoculated by one
or all three of the following methods: (1) Inserting mycelium
of the fungus from pure culture into young stems, (2) growing
seedlings in inoculated soil, and (3) atomizing the entire plant
with a water suspension of zoospores and placing the inoculated
plants under a bell jar. Most of the inoculations were made
during the fall and winter months, but a few were repeated at
different seasons of the year.
Vigorous young seedlings of tomato (Lycopersicum esculentum
Mill.), potato (Solanum tuberosum Linn.), eggplant (Solanum
melongena Linn.), Petunia, var., pepper (Capsicum annuum
Linn. var. Ruby King and Chinese Giant), bean (Phaseolus vul-
garis Linn. var. Kentucky Wonder and Red Valentine), castor-
oil plant (Ricinus communis Linn.), Dahlia, var. and two culti-
vated varieties of Aster were inoculated by inserting mycelium
of the fungus into different parts of the stems. The plants
were growing in boxes of sterilized soil in the greenhouse. Of
this group potato, egg-plant and castor-oil plant developed dis-
ease symptoms when the inoculations were made near the buds.
Small areas around the points of inoculation turned brown
after five days and the parts above, except castor-oil plant,
finally died. Stems inoculated near the ground developed
brown streaks in the inner cortex and vascular tissue, extend-
ing a short distance in each direction from the point of
inoculation, but the p 1 ants continued to g r o w normally.
No growth of the fungus appeared on the surface of the
diseased parts. No signs of disease developed on any of the
other plants and the wounds made for inoculation soon calloused
over.
In the fall of 1924, 10 pieces each of cut and whole potato
tubers were planted in artificially inoculated soil. The same
number of each were planted in sterilized soil at the same time
for checks. All whole tubers produced vigorous sprouts which
remained healthy for the duration of the experiment-60 days.
The seed pieces and roots and young tubers of the plants were
also healthy at that time. On the other hand, only two of the
cut tubers sprouted and at the end of 60 days these sprouts
were much smaller and less vigorous than the ones developing





Florida Agricultural Experiment Station


from the whole tubers, altho they showed no signs of disease.
The seed pieces were decayed, as well as the ones which failed
to sprout. All tubers planted in sterilized soil, both whole and
cut, produced healthy and vigorous plants. The experiment
was repeated in the spring of 1925 and with similar results, ex-
cept that none of the cut tubers sprouted. Potatoes have been
planted on a number of infested fields but there has been no
evidence of a reduced stand resulting from the decay of seed
pieces. This is perhaps due to the fact that potatoes are planted
in this locality in January and February and the soil tempera-
ture at that season of the year is too low for the Phytophthora
to be vegetatively active.
In the spring of 1925, 10 vigorous seedlings four to six
inches high each of Petunia and Zinnia were set in artificially
inoculated soil. That this soil was thoroly infested was indi-
cated by the fact that Connecticut Round Tip tobacco plants set
in it a few weeks earlier developed 100 percent infection. After
22 days the plants of both kinds had made good growth and
showed no signs of disease, on either stem or roots.
In May, 1924, seed of the following species of plants were
planted in boxes of artificially inoculated soil in the greenhouse:
cucumber (Cucumis sativus Linn.), lettuce (Lactuca sativa Linn.
var. Iceberg), cantaloupe (Cucumis melo Linn.), squash (Cu-
curbita moschata Duchesne, var. Summer Crook-neck), and
beans (Phaseolus vulgaris Linn. var. Red Valentine). The soil
used in this experiment was thoroly infested with the Phytoph-
thora, as evidenced by 100 percent infection of tobacco plants
which were removed immediately preceding the date of plant-
ing the seed. Seed of each species was planted in sterilized
soil at the same time for checks. All seed germinated equally
well in both inoculated and sterilized soil and at the end of 22
days the plants were vigorous and showed no signs of disease
on stems or roots.
Seedlings of potato, tomato, eggplant and castor-oil plant from
four to six inches high were atomized with water suspensions of
zoospores and placed under bell jars in the laboratory. Signs
of infection were evident on all four species in three days, altho
it was not abundant on the potato and eggplant. After seven
days all leaves of the potato plants were completely invaded and
the disease had the appearance of late blight. Sparse mycelial
.growth was present on the diseased parts but no conidia were ob-





Bulletin 179, A Phytophthora Disease of Tobacco


served. Invasion of the tomato leaves never became so extensive
but sparse mycelial growth developed on the diseased spots.
Young twigs of eggplant bearing blossom buds also showed in-
fection in three days. The buds turned dark brown and shed in
five days and narrow brown streaks extended back on the young
parts of the stems. Small spots developed on the youngest
leaves. Copious mycelium of the Phytophthora developed on
the blossom buds but it was less abundant ion the leaf spots.
No spores were observed on any of the diseased parts.
Leaves of castor-oil plant showed infection in two days and
after three days many of the spots had coalesced and involved
a greater part of the leaf area.


MORPHOLOGY
Mycelium.-In sec-
tions of recently in-
vaded tissues the
vegetative stage of
the parasite is re-
vealed as a hyaline
non-septate mycel-
ium, composed of hy-
phae, varying some-
what in diameter in
the different tissues.
Individually the hy-
phae are not subject
to abrupt fluctua-
tions in respect to
this dimension. In
the pith cells the hy-
p h a e are usually
slender and sparsely
branched, while in
other tissues branch-
ing is slightly more
frequent and part
of the branches
make little linear
growth, remaining
as short spurs on the
axial filaments. The


SI

{ -J






b
t ^ *



r ^- *


Fig. 102.-Outline drawings of clusters of con-
idia produced on potato-dextrose agar in
van 'Tiegham cells. (a)-P. parasitica,
(b)-black shank Phytophthora, (c)-P.
nicotianae. Drawings made with the aid of
a camera lucida.


:3






Florida Agricultural Experiment Station


fungus is largely intracellular in the pith, the cells often becom-
ing crowded with hyphae. In. the cortex and parenchyma
tissues of the leaf the hyphae are less abundant and they pass
promiscuously from cell to cell without much evidence of the
membranes providing any obstacles. There is little evidence of
constriction of the hyphae where they pass thru the cell walls.
In culture the aerial hyphae are uniform in diameter and
sparsely branched, while the hyphae on the surface of the med-
ium are profusely branched and part of the branches make lit-
tle linear growth.
Asexual reproduction.-Morphologically distinct conidiophores
have never been observed in nature. A comparatively few
conidia have been observed in the field on the leaf spots and
on the base of diseased stems during rainy weather and
they were attached to long hyphal branches which could not be
distinguished by any morphological characters from the infer-
tile hyphae. The fertile hyphae usually arise singly from the
hyphae within the diseased tissues and break thru the epidermis
at almost any point. Conidiophores observed on culture media
were similar to those produced on diseased parts of the plant,


*1 I


Fig. 103.-Different sizes and shapes of
of the black shank Phytophthora ob
in culture. Drawn with the aid of a
era lucida.


except in van Tieg-
ham cell cultures a
few loose clusters of
c o n i d i a developed
(Fig. 102). Occa-
sionally in culture
conidia are inter-
/. calar with the long
diameter at right
angles to the parent
hypha (Fig. 103).
-/ Conidia vary in
shape from almost
spherical to elongate-
ovate but on the av-
erage are of the
ovate type and com-
paratively lar ge
conidia (Fig. 103). Figures
served 104 and 105 show
cam- the classes f o u n d


/ N
A


/I


--



'I,






Bulletin 179, A Phytophthora Disease of Tobacco 185

in 400 measurements from oat agar cultures. It will be
seen from these graphs that a majority of the conidia produced
on oat agar fall within the limits of 28.8 to 39.3 by 21.8 to 32.3
microns. Using Rosenbaum's method (9), the ratio of length to
width was found to vary from 1.11-2.00, 1.4 occurring most fre-
quently. Papillae are mostly apical, an occasional lateral one be-
ing found. Also, a few conidia have been found with two papil-
lae (Fig. 103). The papillae are prominent, the average of 25
measurements from tobacco leaves being 6.4 microns. Conidia
produced on tobacco leaves artificially inoculated and kept under
a moist chamber varied in size from 17.5 by 21 to 42 by 80.5
microns, averaging 32 by 51.6 microns.






S _






tinae, P. parasi, -te -s k
.r. *- / -i ....
I ..... -4 -'- \, \- I- > -
-\ -



tianae, P. parasitica, and the black shank Phytophthora.
At room temperature conidia from 30-day old agar cultures
begin to germinate almost immediately after they are mounted
in water, and they are followed by others during a period
of 30 minutes or more. A small percentage of the conidia
germinate from younger cultures. On the other hand, con-
idia were produced in van Tiegham cell cultures in three days
at room temperature and began to germinate within 48 hours
after they were formed. In the absence of sufficient moisture
for the zoospores to swim away they emerged in a group near the






Florida Agricultural Experiment Station


apical end of the conidium and germinated, giving rise to a tuft
of mycelium. Some of the zoospores failed to pass out of the
spore case and germinated in situ. In such cases the germ tubes
penetrated the conidial wall and gave rise to miniature conidia


1.5 1 .N I .. A N-.: ?' 2 -C .i 4Z
\ '1 O,,'IDIA IN 1ICKONS


Fig. 105.-Graph showing the comparative widths of conidia of P. nico-
tianae, P. parasitica, and the black shank Phytophthora.


186






Bulletin 179, A Phytophthora Disease of Tobacco


just outside of the wall. The process of germination of conidia
from the tube cultures was typical of that reported in litera-
ture for certain other species of Phytophthora, so it is consid-
ered superfluous to give a detailed account of it in this connec-
tion. After emergence the zoospores swim about very freely
for 15 to 20 minutes, when they become inactive and almost
spherical. About 20 to 30 minutes later they send out one,
occasionally two, germ tubes.
Under humid conditions chlamydospores are produced on the
surface of diseased parts of the host plant and occasionally
within the tissues. They occur in great abundance in old cul-
tures on certain media. They are usually spherical and the
walls of the majority of them are about the same thickness as
the walls of conidia. At first they are about the same color as
the conidia but later they become more brownish on certain
media. They are borne at the end of slender hyphae or inter-
calary. Occasionally they occur in chains consisting of from
two to four spores. They vary in diameter from 14 to 36.7
microns, the average of 200 measurements being 24.6 microns.
They have
never been
observed to
germinate
unless it be .\ .
that one \
spore gives
rise to an-
other in the
formation
of chains. '
In cultures :-
from one to
six months
old many //
e m p t y
spores
have been Fig. 106.-Outline drawings of different types of chlamy-
ave een dospores of the black shank Phytophthora observed in
observed culture. Drawn with the aid of a camer lucida.
without any indication of the disposition of the, contents.
No oospores or sex bodies have been observed in culture or
on diseased parts of the host plant.






Florida Agricultural Experiment Station


TAXONOMY
Isolations of the fungus under consideration have been made
from diseased tobacco plants from several localities in Florida
and in no case has a sexual stage been observed under any cul-
tural conditions employed. Neither has it been observed in dis-
eased tissues from any source. Therefore, the fungus can be
compared only in part with those known species and strains of
Phytophthora which produce conidia of similar size and shape.
These characters may be too limited for specific identification,
as Leonian reported (7) that the members of this genus ex-
hibit a remarkable uniformity of morphological features and
that these characters are extremely variable.
However, a comparative study of several reported species of
Phytophthora in connection with these investigations has re-
vealed differences which seemed sufficient to eliminate all mem-
bers from consideration except two, namely, P. nicotianae Breda
de Haan and P. parasitica Dastur. Cultures of P. nicotianae
were obtained from Westerdijk of Holland, D'Angremond of
Java and from Leonian of West Virginia. Leonian stated that
he obtained his culture from Sherbakoff of Tennessee, who ob-
tained it from Holland. The culture of P. parasitica was obtained
from Leonian. Neither of these species produced oospores in
culture during the course of investigations.
The cultures of P. nicotianae from the different sources ex-
hibited slight differences in cultural characters. These differ-
ences, however, did not appear to be greater than one might
reasonably assume as being due to various lengths of time they
had been carried in culture, with different cultural methods or
to differences in strains. However, all strains produced similar
disease symptoms when inoculated into healthy tobacco plants.
In comparingP. nicotianae and P. parasitica with the black
shank Phytophthora it was deemed advisable to take into con-
sideration their reaction to certain cultural media and pathogen-
icity on certain plants as well as their morphology. Only the
outstanding similarities and differences will be presented in
this paper.
According to the description and pictures given by Breda de
Haan (1) P. nicotianae causes a disease of tobacco in Java very
similar to black shank in Florida. Furthermore, inoculations
with the three strains of P. nicotianae resulted in the produc-
tion of a disease of tobacco in Florida which could not be dis-


I






Bulletin 179, A Phytophthora Disease of Tobacco


tinguished from that caused by the black shank Phytophthora
under the same conditions. On the other hand, P. parasitica
could not be made to infect tobacco, which confirms Dastur's
experience with this organism (2). Moreover, Dastur reported
that castor-oil plant could not be infected with P. nicotianae
and used these differences in host relationship as a basis for
separating the two organisms. This difference was not sub-
stantiated by Palm, who reported in 1923 (8) that he obtained
infection of castor-oil plant seedlings by inoculating them with
P. nicotianae. Furthermore, inoculations under Florida condi-
tions showed that castor-oil plant seedlings were equally sus-
ceptible to infection by zoospores of P. nicotianae, P. parasitica,
and the black shank Phytophthora and the disease caused by
each fungus was apparently the same. The disease was also
similar to Dastur's description and pictures of the disease of
castor-oil plant caused by P. parasitica.' Dastur reported fur-
ther that seedlings of potato, eggplant and tomato were in-
fected by P. parasitica, while Palm reported that young tomato
plants were readily infected with P. nicotianae but potatoes
were not infected. Under Florida conditions when seedlings
of potato, tomato and eggplant were atomized with water sus-
pensions of zoospores of the three organisms and placed under
bell jars, signs of infection were apparent in three days. The
diseased areas on eggplant were only a few millimeters in di-
ameter after five days and on older plants infection was limited
to the blossom buds and pedicels and the signs of disease pro-
duced by each of the three organisms were similar. The to-
mato seedlings inoculated with P. parasitica were completely
invaded in four days, while those inoculated with P. nicotianae
and the black shank Phytophthora had numerous small diseased
areas which developed very slowly. All organisms produced
copious mycelium on the diseased tissues but P. parasitica
alone produced conidia and chlamydospores. The potato
seedlings inoculated with P. nicotianae were completely invaded
in five days. Initial infection was less abundant on the plants
inoculated with P. parasitica and the black shank Phytophthora
but enlargement of the diseased spots appeared to be as rapid
as those caused by P. nicotianae and the plants were completely
invaded in seven days. The disease had the appearance of late
blight except that no conidia and very little mycelial growth
occurred on the diseased parts.






Florida Agricultural Experiment Station


The three organisms also exhibited certain common charac-
teristics on culture media and on sterilized fruits and vege-
tables. All three forms' grew rapidly on potatoes, sweet pota-
toes, carrots, apples and partially ripe tomatoes and produced
similar symptoms of decay, as described for the black shank
Phytophthora. Where aerial mycelium was produced there was
a slight difference in its appearance as between the three forms,
altho it appeared more a difference in amount than in charac-
ter (Fig. 107). These results differ from Dastur's account of
P. parasitica in that he stated (2) that tomato fruits could not
be infected even when wounded. None of the organisms pro-
duced spores on any of the fresh fruits or vegetables.
All three organisms grew rapidly on oat agar, producing
similar dense white aerial mycelium (Fig. 108) which covered
the surface of the medium in petri dishes in five days and ex-
tended to the cover. Growth was a little slower on Lima bean
agar and the aerial mycelium was less dense and grayish-white
in color. All three forms produced little submerged growth on












Fig. 107.-A comparison of growth characters on fresh tomatoes of the
black shank Phytophthora (left), P. nicotianae, (center), and P. para-
sitica (right). The tomatoes inoculated with the black shank Phytoph-
thora and P. nicotianae have collapsed and spread out; hence the larg-
er size.

this medium, and P. nicotianae produced fewer spores than
either of the other two. It also produced fewer spores on potato-
dextrose agar and developed less aerial mycelium (Fig. 109).
All three forms produced luxuriant growth on steamed potato
slabs and bean pod plugs, but sporulated only after two or three
months.
The conidia were found to differ slightly in size but the differ-
ences among the three forms were not greater than our meas-






Bulletin 179, A Phytophthora Disease of Tobacco


urements differed from those given by other investigators for
the known species. Figures 104 and 105 show the comparative
sizes found in 400 conidia of each form from 18-day old oat
agar cultures. Dastur (2) gives 25-50 by 20-40 microns as the
average size of conidia of P. parasitica and 20-60 microns as the
size of chlamydospores. He also stated that the conidiophores
are simple except in water cultures and certain media where they
may be irregularly branched. Breda de Haan (1) gives 25 by 36
microns as the average size for the conidia of P. nicotianae and
stated that the conidiophores may be more or less branched.


Fig. 108.-Comparative growth on oat agar of the black shank Phytoph-
thora (upper center), P. nicotianae (lower left), and P. parasitica (lower
right). Photographed four days after the plates were inoculated.

Under our conditions the following dimensions were obtained
for each of the organisms on oat agar: P. parasitica, conidia,
14.75 by 15.75 to 42 by 52.5, averaging 25.86 by 31.25 microns;






Florida Agricultural Experiment Station


chlamydospores, 10.5 to 45.5, averaging 26.88 microns; P. nico-
tianae, Holland strain conidia, 14 by 21 to 33.25 by 66, averag-
ing 25.30 by 36.67 microns; chlamydospores, 14 to 33.25, aver-
aging 22.96 microns; Java strain, conidia 10.5 by 14 to 42 by
61, averaging 28.98 by 38.2; the black shank Phytophthora, con-
idia, 15.75 by 21 to 38 by 56, averaging 26.18 by 34.44 microns,
chlamydospores 14 to 36.75, averaging 24.64 microns. The size


Fig. 109.-Comparative growth on potato-dextrose agar of the black shank
Phytophthora (upper center), P. nicotianae (lower left), and P. para-
sitica (lower right). Photographed four days after the plates were in-
oculated.
of conidia of the latter fungus on diseased parts of tobacco
plants under humid conditions varied from 17 by 21 to 42 by
80, averaging 32 by 51.6 microns.
Studies of van Tiegham cell cultures on Lima bean agar and
on potato-dextrose agar revealed that conidiophores of the three






Bulletin 179, A Phytophthora Disease of Tobacco


forms are somewhat similar and usually indistinguishable from
the aerial hyphae. Conidia were usually borne singly but oc-
casionally clusters of from 2 to 6 were observed (Fig. 102). In
one culture of P. parasitica on Lima bean agar a high percent-
age of the conidia were borne in dense clusters. The germina-
tion of the conidia of all forms so far as could be distinguished
was the same.
The surface hyphae on culture media of P. parasitica and the
black shank Phytophthora were less profusely branched and
were more uniform in diameter than P. nicotianae. The surface
hyphae of the latter organism also produced small tuberculate
patches.
From this comparison it may be seen that the Phytophthora
causing black shank of tobacco has several characters in com-
mon with both P. nicotianae and P. parasitica. The most strik-
ing difference observed between P. parasitica and the black
shank Phytophthora was the difference in pathogenicity on to-
bacco, while the most apparent differences between the latter
organism and P. nicotianae were slight differences in the mor-
phological and physiological characters. Therefore, if patho-
genicity should be given consideration in the determination of
species, it seems that the black shank Phytophthora is more
closely related to P. nicotianae than it is to P. parasitica.
Leonian concluded from his studies of the physiology of most
of the known species of the genus Phytophthora (7) that the tax-
onomic status of P. nicotianae was not definitely established and
that it closely resembled P. parasitica both in its morphological
as well as in most of its physiological characteristics. Moreover,
on a basis of his physiological studies of the genus he proposed
to replace P. parasitica and several other related strains and
species by P. omnivora DeBy. Also, Smith and Smith in discuss-
ing (10) the status of Pythiacystis, Pythium and Phytophthora
stated that there is much variation in size, form, type and oc-
currence of reproductive bodies and many intermediate forms
are continually being discovered. Therefore, in view of this
apparent unsettled status of the genus and species the writers
do not wish to assume the responsibility of definitely placing the
black shank Phytophthora as to species. However, it seems
that, for the sake of convenience and to avoid further complica-
tions for the person who might undertake to reclassify the mem-






Florida Agricultural Experiment Station


bers of the genus, the black shank organism might be considered
tentatively as a strain of P. nicotianae Breda de Haan.

PHYSIOLOGY

The Phytophthora has been grown on several of the ordinary
culture media used in plant pathology investigations and also
on certain fresh fruits and vegetables for the purpose of com-
paring its morphological and physiological characters with cer-
tain other members of the genus. During the course of study
the following observations were made:

Oat agar (100 gms. rolled oats, 20 gins. Bacto agar). Growth on this
medium was more abundant than on any other agar used, covering the
surface of a 90 mm. plate in five days with dense white aerial mycelium.
Slight submerged growth developed. Conidia began to appear in six days
and became abundant after two weeks. Chlamydospores were abundant
in old cultures.
Corn meal agar (100 gms. corn meal, 20 gms. Bacto agar). Surface
growth spreading and faster than on oat agar; no submerged growth.
Conidia and chlamydospores were abundant after 10 days.
Lima bean.agar (100 gins. cracked Lima beans, 20 gins. Bacto agar).
Aerial mycelium grayish-white, loose and fluffy, extending to cover of
plate in center of colony; no submerged growth. Conidia and chlamydo-
spores began to appear after 10 days to two weeks but were produced less
abundantly than on either oat agar or corn meal agar.
Potato-dextrose agar. Growth rather slow for the first few days produc-
ing loose, grayish-white mycelium. A thin, irregular film or mat developed
on the surface of the medium around the point of inoculation, and hyphae
penetrated several mm. below the surface of the medium of acid reaction.
After 10 days to two weeks the surface of the medium was covered with
a loose, grayish-white or transparent mycelium; aerial hyphae sparsely
branched, surface and submerged hyphae profusely branched, the branches
being short and irregular in diameter; conidia and chlamydosporcs be-
gan to appear in five days and became abundant after 10 days, altho
very few would germinate before the culture was 30 days old.
Beef-peptone agar. Very slight surface growth; no spores.
Cooked potato slabs (In Roux tubes with water and absorbent cotton
on the bottom). Growth rapid, covering the slabs in five to eight days
with a dense, white and tough mycelium. No conidia or chlamydospores
appeared until after two or three months and then they developed in great
numbers.
Cooked sweet potato slabs (In Roux tubes with water and absorbent cot-
ton on the bottom). Growth l-ss raid and less abundant than on potato
slabs; aerial mycelium grayish-white, loose and short; hyphae in old
cultures filled with oil globules. Few conidia ard chiamyoospores ap-
peared in four days but never became abundant.
Cooked bean pod plugs. Vigorous growth of grayish-white, loose and
tough mycelium. No conidia and only few chlamydospores developed.

Inoculations were also made into fresh fruits and roots of
several plants. Whole specimens of the different varieties were
first treated with 1-1,000 mercuric bichloride solution for 10
minutes and then thoroly rinsed in sterile water. The inocula-





Bulletin 179, A Phytophthora Disease of Tobacco


tions were made by cutting sloping slits with a flamed scalpel
and inserting mycelium from a vigorous culture of the organism.
After inoculation the specimens were placed in sterilized moist
chambers and incubated at 24 degrees Centigrade. All inocula-
tions were made in duplicate and repeated twice.
After eight days most of the upper surface of carrots was
covered with loose, grayish-white mycelium 2-5 mm. high, and
cream colored, watery, stromatic masses were present in the
wounds made for inoculation. The epidermis and cortex were a
light brown color, watery and soft while the deeper tissues were
firm. No spores were observed.
Partially ripe tomatoes were attacked very readily, slight
growth being evident after one day and the invaded areas were
slightly depressed. After 10 days the entire fruits were col-
lapsed and most of the juice was leaked out. Stromatic masses
1 to 2 mm. in diameter occurred in and around the wounds made
for inoculation. Dense white mycelium 2 to 3 mm. high oc-
curred on top of the fruits and small tufts extended farther
down the sides (Fig 107). No spores were observed.
Slight growth developed around the edges of inoculation
wounds on potato in three days and after 10 days the entire
tubers were wilted and leaking. Cream colored stromatic
masses 2 to 4 mm. in diameter developed in and around inocu-
lation wounds. Thin, loose, white aerial mycelium developed
near the points of inoculation and small tufts of mycelium pro-
truded from different points on the surface. The epidermis and
internal tissues were unchanged in color when first cut but
the internal parts turned pinkish-brown on being exposed to
the air for 30 minutes. The internal tissues were watery and
crumbly.
No surface growth developed on sweet potato. The entire po-
tato turned brown in 10 days, the internal parts exhibiting a
spongy and slightly watery condition. A slight rose geranium
odor was observed.
Turnips developed only slight discoloration around the points
of inoculation in 10 days and the internal tissues were unaf-
fected.
Winesap apples were completely invaded in 10 days. A
small amount of granular growth developed around the' points
of inoculation. The epidermis and internal parts were brown
and the internal parts were crumbly and moist, exhibiting al-
most the consistency of a baked apple.






Florida Agricultural Experiment Station


EFFECT OF TEMPERATURE UPON TIE DEVELOPMENT OF THE
FUNGUS IN CULTURE

The fact that black shank develops most rapidly during the
summer months indicates that the fungus must be able to thrive
at comparatively high soil temperatures. In an experimental
determination of the thermal limits within which the fungus
will grow, potato-dextrose agar was used. The most favorable
reaction of the medium for growth had not previously been de-
termined, so several different reactions (Fuller's scale) were
used in this experiment. Petri plates of each reaction were pre-
pared in triplicate and uniformly inoculated in the center and
exposed to temperatures ranging from 8 to 37 degrees Centi-
grade. The temperatures 8, 13 and 20 degrees were obtained
in a refrigerator where the daily fluctuation was plus or minus
one degree. The others 25, 30, 35 and 37 were under thermo-
static control and were practically constant. The plates exposed
at 37 degrees were placed in moist chambers to prevent the
medium from drying out. The diameter of the colonies was re-
corded daily for one week and the average diameter of the three,
plates for three-day and seven-day periods is shown in Table II.

TABLE II. EFFECT OF TEMPERATURE UPON THE-RATE OF GROWTH OF THE:
BLACK SHANK PHYTOPHTHORA ON POTATO-DEXTROSE AGAR.
Reaction of medium Diameter of colonies in millimeters
(Fuller's scale) at the temperatures indicated.
Degrees Centigrade
3 days 7 days .
| 8113120125130135137[ 8113120125130135137
- 10 ....................................... 00 0 6111113 0 019353633 0
- 5 ............................... ....... 0 0 1015 1413 0 0 6305042 33 0
0 ...... --..................:................ 0 3 151822 11 0 0 936555738 0
+ 5 ............. 0 4 22332929 0 3 94657 5530 0
+ 10 ...................................... 0 015172417 0 537365321 0

It may be seen from these results that the fungus grows over
a wide range of temperature and that the optimum, as was ex-
pected, is high. There is also evidence that the optimum tem-
perature varies somewhat with the reaction of the medium.
Furthermore there was a difference in amount of growth on
different reactions at a given temperature. This suggested the
advisability of studying the behavior of the fungus on a wider
range of reactions of the medium.






Bulletin 179, A Phytophthora Disease of Tobacco


EFFECT OF HYDROGEN-ION CONCENTRATION OF CULTURE
MEDIA UPON THE RATE OF GROWTH OF THE
BLACK SHANK PHYTOPHTHORA

In the series of experiments for the determination of the ef-
fects of hydrogen-ion concentration upon the rate of growth of
the fungus, potato-dextrose agar was used. Different lots of
the medium were adjusted to hydrogen-ion concentrations rang-
ing between pH 4.4 and pH 8.5. Petri plates were prepared in
triplicate for each reaction and after they were inoculated they
were exposed at a constant temperature of 24 degrees Centi-
grade for nine days. The average diameters of the three plates
of each reaction for the periods indicated are given in Table III.

TABLE III.-EFFECT OF HYDROGEN-ION CONCENTRATION OF POTATO-DEX-
TROSE AGAR UPON THE GROWTH OF THE BLACK SHANK PHY-
TOPHTHORA.
pH readings 4.41 5.1] 5.7 6.01 6.51 7.0 7.4 8.01 8.5
-in mm. | !
Diameter of colonies -
after three days ................... 13 18 20 21 18 18 17 15 12
-in mm.
Diameter of colonies
after nine days......... ....... 36 42 60 157 63 60 51 53 51

These results show that so far as increase in diameter of the
colony is concerned the hydrogen-ion reaction most favorable
for growth of the fungus lies between pH 5.7 and pH 7.0. How-
ever, if the quantity of vegetative growth should be considered,
the optimum reaction would perhaps fall between pH 4.4 and
pH 5.1. There was also a noticeable difference in the growth
characters on the different hydrogen-ion concentrations. The
most outstanding differences observed at the end of 12 days may
be summarized as follows:
pH 4.4. Aerial mycelium abundant, white, cottony, 2-3 mm. high in cen-
ter of colony, slightly tufted around margins; hyphae straight, sparsely
branched; thin film or membrane formed on surface of medium; hyphae
penetrate medium, forming a thin membrane on bottom of plate; hyphae
on surface of and within medium profusely branched, the branches being
short and irregular in diameter. No spores.
pH 5.1. Mycelium abundant, grayish-white, 5 nmm. high in center of col-
ony. Slight submerged growth. Few conidia and chlamydospores.
pH 5.7. Aerial mycelium loose, grayish-white, 3-5 mm. high in center of
colony, more or less tufted around margin; no submerged growth. Hyphae
on surface of medium profusely branched, branches irregular in diameter,
enlarged near ends. Few chlamydospores occurring in chains.
pH 6.0. Aerial mycelium similar to that of pH 5.7, except a little more
tufted and branches of surface hyphae more enlarged. Few conidia and
chlamydospores.






Florida Agricultural Experiment Station


pH 6.5. Aerial mycelium thin and tufted, 1-3 mm. high. Surface growth
thin, hyphae very irregular in diameter. No submerged growth. No con-
idia. Few chlamydospores.
pH 7.0. Aerial mycelium very sparse, fluffy and tufted. Surface growth
thin and spreading, hyphae more uniform in diameter than that on more
acid medium, filled with large oil droplets. No submerged growth. No
spores.
pH 7.5. Growth similar to that on pH 7.0.
pH 8.0. Aerial mycelium short, thin and fluffy. Surface growth very
thin and spreading; hyphae profusely branched, forming witches brooms.
No submerged growth. No spores.
pH 8.5. Aerial mycelium very thin, fine and uniformly distributed;
surface growth thin, yellowish-gray; hyphae profusely branched forming
witches brooms. No spores.

EFFECT OF FERMENTATION OF INFECTED LEAVES
UPON THE FUNGUS

Since the disease was found to develop on the leaves after
they were hung in the curing barns and since some of the af-
fected leaves are placed on the market, it was deemed advisable
to ascertain whether the organism could be distributed in a
viable condition to different parts of the country on these leaves.
In an experimental determination of the effects of the fermen-
tation process, to which the cured leaves are subjected in the
sweat room, upon the viability of the Phytophthora, heavily in-
fected leaves were selected in the curing barn, tied in hands,
labeled and placed in a bulk in the usual manner. One lot of
the leaves was removed from the bulk after the fermentation
process was about half completed and the other lot was carried
thru the entire process. The two lots of leaves were then kept
in the laboratory until March of the following year. Twenty
leaves of each lot were then cut into small pieces and each lot
was mixed with the top inch of sterilized soil contained in two
boxes 16 x 20 x 9 inches. Two other boxes of the same kind of
soil to which no leaves were added were used as checks. All boxes
were then placed out of doors and watered as needed. On April
23 five Connecticut Round Tip seedlings were set in each box
and the boxes were kept under favorable conditions for growth
of the plants until July 13. On that date all plants, inoculated
and checks, were in full bloom and showed no signs of infection,
on either the stems or roots.
Two attempts were made to isolate the fungus in culture from
some of the fermented diseasead leaves but without success. In-






Bulletin 179, A Phytophthora Disease of Tobacco


deed the fungus has never been isolated from diseased leaves
when they are brought directly from the field and kept in the
laboratory for several days.

RELATION OF SOIL TEMPERATURE TO THE
DEVELOPMENT OF BLACK SHANK

Volunteer tobacco plants have been observed to grow on in-
fested soil during the winter months until they were finally
killed by frost without showing any signs of black shank. The
period for transplanting tobacco in this section usually extends
from March 20 to April 15 but is occasionally one or two weeks
later, depending on weather conditions. No continuous record
of soil temperature has been made during the growing season
but readings made at different hours of the day just prior to
and at the time earliest infection was observed showed that the
mean daily temperature was about 20 degrees Centigrade. It
was also observed that plants set in infested soil before April
1, attained considerable growth before they were attacked by
the fungus, while the ones set after April 15 may show signs ff
the disease within a few days of the time of planting.
These field observations have been supplemented with experi-
ments conducted at different seasons of the year. No appara-
tus for controlling the soil and air temperature was available,
so advantage was taken of the natural low and high tempera-
tures prevailing during different months. By this method only
the approximate minimum temperature for the development
of the disease could be determined. The first experiment was
begun in November, 1922, and concluded in March, 1923. Five
Big Cuba tobacco seedlings were set in each of two boxes of
artificially inoculated soil and the boxes were kept in an un-
heated room of a brick building. Five plants were set in each
of two boxes of sterilized soil for checks. The plants in inocu-
lated soil grew as vigorously as the checks and showed no signs
of disease until March 12. The average mean daily tempera-
ture up to March 1 was slightly below 20 degrees Centigrade,
the temperature at night frequently dropping as low as 10 to
15 degrees. Between March 1 and 12 the mean daily tempera-
ture ranged between 20 and 24 degrees Centigrade. The plants
were removed from the boxes on March 30 and at that time






Florida Agricultural Experiment Station


eight of the 10 plants in inoculated soil showed infection on the
roots and basal portion of the stems, while all 10 of the plants
in sterilized soil were healthy.
Immediately after these plants were removed the boxes were
reset with five seedlings each and the-boxes were left in the same
room. At that time the mean daily temperature of the room
was about 24 degrees Centigrade. Signs of the disease became
apparent in five days and after 11 days all of the plants in in-
oculated soil were dead. Subsequently, many plants have been
set in inoculated soil during the spring and summer months and
invariably the disease developed in two to five days.
Tobacco plants have also been inoculated at different sea-
sons of the year by inserting mycelium of the fungus into the
stems. In December, 1922, four vigorous plants about 18 inches
high were inoculated in this manner about four inches below
the buds and two others were inoculated about four inches above
the surface of the soil. The stems were wrapped at the points
of inoculation with saturated absorbent cotton to prevent rapid
drying out of the injured tissues. The plants were kept in a
room where the temperature during the first six days after the
plants were inoculated fluctuated between 9 and 16 degrees
Centigrade. No signs of disease became apparent during the
six-day period. During the two days following the temperature
of the room reached a maximum of 22 degrees Centigrade, and
during that time the stems turned brown around the points of
inoculation. One week later the tops of the inoculated plants
were wilted and the ones inoculated near the bud were dead.
The experiment was repeated during the winter of 1923 and
so long as the temperature remained below 20 degrees Centi-
grade no signs of the disease developed. On the other hand,
plants inoculated during the spring and summer months when
the temperature was high (35 to 40 degrees Centigrade in the
greenhouse) invariably showed signs of infection in two to
three days. The results of these limited experiments combined
with cumulative field observations indicate that the Phytoph-
thora is active over a wide range of temperatures. Altho the
minimum could not be definitely determined by the improvised
methods, evidence indicates that it is below 20 degrees Centi-
grade. There is usually only about 10 days to two weeks at the






Bulletin 179, A Phytophthora Disease of Tobacco 201

beginning of the growing season when the temperature is too
low for infection to take place and the temperature during the
remainder of the season never gets too high.

CONTROL EXPERIMENTS

STEAM STERILIZATION OF INFESTED FIELDS

Upon arrival in the Florida-Georgia district in 1922 it was
learned that one of the larger companies was completing the
sterilization of a 13-acre field by the inverted pan method. For
this huge undertaking they used a portable boiler and a battery
of six pans 6 by 21 feet, using a steam pressure of about 90
pounds for 45 minutes. A good crop of tobacco was produced
on the field that year but some signs of the disease appeared on
part of the field before the end of harvest. Not being easily
discouraged, the company ordered the infested portion of the
field, about six acres, re-sterilized the following winter. A
ditch was dug, separating the two parts of the field and tobacco
was planted on the entire field in 1923. Black shank appeared
early in the season in the portion of the field not re-steamed and


.' J .
l*. .


6\X~j I
L vk
b : 4 ,

d~~"


Fig. 110.-View of a portion of a 13-acre shade which was steamed in 1922
by the inverted pan method, showing the reduction in stand of the sec-
ond crop planted after the treatment. Photographed July 19, 1923 just
after harvest.


* .,
i, .


* l''K"
cr;~
ur:..






Florida Agricultural Experiment Station


caused considerable loss. Figure 110 shows the appearance of
the field at the end of harvest. The re-steamed portion remained
free from the disease until about the middle of May when water
broke over the ditch and a greater portion of the field was over-
flowed with water from that portion steamed in 1922. Ten
days later a high percentage of the plants showed infection and,
altho the first leaves were harvested at that time, some loss was
incurred before harvest was completed. The company reported
that the cost of the treatment was a little less than $200 per
acre, excluding the cost and depreciation of the boiler. There-
fore the cost of the treatment is prohibitive so long as it is
effective for only one season. Since the land around the edges
of the fields must be infested, it seems that it will be a matter
of only a few weeks before the sterilized area will become re-
inoculated.
EFFECT OF ORGANIC CONTENT OF THE SOIL UPON THE
DEVELOPMENT OF BLACK SHANK

Tobacco is considered one of the rankest feeders among the
cultivated crops, altho the period for its development is relative-
ly short. In order to produce a leaf with desirable qualities for
wrappers the plant must be supplied with sufficient available
nutrients to permit continuous rapid growth from the time it
is transplanted until harvest. To be reasonably sure of having
this supply of nutrient materials available, growers have been
accustomed to apply annually about 12 tons of manure per acre
in combination with from one to one and a half tons of commer-
cial fertilizer, the bulk of which is cottonseed meal. Because of
this high organic content of the soil it seemed probable that the
environment might be more favorable for the saphrophytic life
of the Phytophthora, especially since it was ascertained that the
fungus would grow on sterilized manure. Experiments were
planned to test the hypothesis.
The trials were made in boxes in the greenhouse in the spring
of 1925. Sterilized Norfolk sandy loam was placed in five boxes
with surface area of two square feet. Stable manure and com-
mercial fertilizer were applied, as indicated in Table IV, and
thoroly worked into the soil. Fifteen days later the soil was
inoculated with pure cultures of the Phytophthora and the
boxes were placed on a greenhouse bench for 30 days, adding






Bulletin 179, A Phytophthora Disease of Tobacco


water as needed. Ten vigorous Connecticut Round Tip seed-
lings were then set in each box and notes were made on their
condition at regular intervals for a period of 36 days. The
plants were then removed and 10 days later the boxes were
each reset with 10 healthy seedlings. Final notes were made
on this series at the end of 36 days and the combined results
of the two series are given in Table IV.
TABLE IV. EFFECT OF ORGANIC CONTENT OF THE SOIL UPON THE DEVEL-
OPMENT OF BLACK SHANK IN TOBACCO SEEDLINGS.
Fertilizer Treatment Total Percentage of Plants Diseased
(rate per acre) Plants at Intervals Indicated
6 days | 14 days 36 days
No fertilizer ..........- ......... 20 15 45 70
1 ton comm. fert .............. 20 65 80 95
10 tons manure -................. 20 45 55 80
1 ton comm. fert. plus 10 I
tons manure ................. 20 65 90 95
It may be seen from the preceding table that there is no evi-
dence of gain by eliminating stable manure from the fertilizer
so long as the soil is made sufficiently fertile to permit normal
growth of the plants. Therefore, since it is conceded by the
growers that manure is essential for the production of the de-
sired quality of leaf, the results offered little encouragement
for reducing the loss by eliminating manure from the fertilizer.
This conclusion was confirmed in 1925 when opportunity per-
mitted the observation of black shank in a field of bright leaf
tobacco. This type of tobacco is planted on the light sandy
soils and usually only 800 to 1,200 pounds of commercial fer-
tilizer is applied as a source of plant food. Thus, this crop is
grown on soil relatively low in organic matter. The disease
seemed to spread as rapidly and destructively under these con-
ditions as on the highly fertile soils under the shades (Fig. 95).
It appears, therefore, that the fertility or organic content of the
soil is not an important factor in the rate of spread or destruc-
tiveness of the disease.

EFFECT OF CERTAIN CHEMICALS UPON THE DEVELOPMENT
OF BLACK SHANK.

Since the introduction of black shank into this section the
growers have used such materials as land plaster, lime and sul-
phur in combination with the usual fertilizers in an effort to






Florida Agricultural Experiment Station


reduce the toll demanded by the disease. Inasmuch as conflict-
ing reports concerning the results of such treatments had been
received, it was deemed worth while to obtain definite informa-
tion on the effects of certain available materials.
Preliminary experiments were conducted in boxes in the
greenhouse during the fall and spring months of 1924 and 1925.
Norfolk sandy loam soil was used for the experiments. It was
first fertilized in the usual manner for growing shade tobacco
and then treated with a 1-50 solution of formaldehyde. After
allowing it to air-dry for two weeks the soil was placed in seven
boxes 18 inches square by 6 inches deep. Six of the boxes were
thoroly inoculated with cultures of the black shank Phytoph-
thora and the other was left uninoculated as a check. The
boxes were then placed on a greenhouse bench for 27 days and
water was added as needed. The inoculated sulphur was added
to one box at the rate of 400 pounds per acre; to another
Semesan was added at the rate of 400 pounds per acre, and to
a third land plaster was added at the rate of 1,000 pounds per
acre. The materials were thoroly worked into the top two
inches of the soil. One week later 10 Connecticut Round Tip
seedlings were set in each of the seven boxes and 50 cc. of water
was applied around each plant in all boxes except those in two
boxes of the inoculated soil. The plants in one of these boxes
were watered with 50 cc. of a 0.25 percent Uspulun solution
and those in the other box with a 0.50 percent Uspulun solution.
After 36 days the plants were removed and 10 other seedlings set
in each box. None of the boxes of soil were treated for the second
test except the ones treated with Uspulun solution. These were
treated with the same quantity and concentration of this mate-
rial as in the first test. Final notes were made on the second
series at the end of 36 days and the combined results of the two
series are given in Table V.
These results show that all of the materials used had a de-
cided inhibiting effect upon the development of black shank.
However, the plants in boxes treated with Uspulun and Seme-
san started growth more slowly than the ones in other boxes
and were considerably smaller and less vigorous at the conclu-
sion of the experiment Altho the number of plants in these
tests was small, the results appeared sufficiently significant to
warrant a field test.






Bulletin 179, A Phytophthora Disease of Tobacco


TABLE V.-EFFECT OF CERTAIN CHEMICALS UPON THE DEVELOPMENT OF
BLACK SHANK IN .TOBACCO SEEDLINGS.
Soil Treatment Total Percent of Plants Diseased
(rate per acre) Plants at Intervals Indicated
S[ 3 days 14 days 36 days
Check (Uninoc. untreat.) .......... 20 0 0 0
Inoculated, untreat. ................... 20 60 90 95
Inoculated sulphur-400 lbs....... 20 0 0 5
Semesan, 400 lbs. ...................... 20 0 0 0
Land plaster, 1,000 ...........-.......... 20 0 0 15
Uspulun, 0.25%, 50 cc. per plant 20 0 0 10
Uspulun, 0.50%, 50 cc. per plant 20 0 0 0
In the field tests all materials except those used in solution
were applied in the row with the commercial fertilizer and
thoroly mixed with the soil about 10 days before the plants
were set. The solutions were applied around the plants, about
150 cc. per plant in lieu of water. For convenience in figuring
the amount of materials for the given length of row the amounts
per acre were varied somewhat from that used in the green-
house experiments, as indicated in Table VI. Connecticut
Round Tip seedlings were used in the experiment. All mate-
rials except land plaster were tested in 1925, and it was tested
in 1924. In 1924 the plants were set on April 19, while in 1925
they were set on March 30. The results are shown in Table VI.
TABLE VI.-EFFECT OF CERTAIN CHEMICALS UPON THE DEVELOPMENT OF
BLACK SHANK IN TOBACCO SEEDLINGS IN THE FIELD.
Soil Treatment Number of Percent of Plants Diseased at
(rate per acre) Plants the Intervals Indicated
S| 14 days 30 days 90 days
Check (untreated) ................... 95 19 29 100
Inoculated Sulphur 180 lbs.... 96 35 41 100
Inoculated Sulphur 360 Ibs.... 95 9 21 100
Semesan, 80 lbs ..................... 110 3.6 10 99
Semesan, 0.5% sol. ................. 97 11 20 100
Land plaster, 600 lbs. ............. 173 91 100 100
Land plaster, 1,200 lbs............. 180 68 83 100
It appears from the results in Table VI that Semesan and in-
oculated sulphur inhibited the development of black shank to
a slight extent early in the season but the effect was not last-
ing. Since the tobacco plant is susceptible at any stage of its
development to attack by the Phytophthora, it is apparent that
for any soil treatment to be effective it must last thruout the
growing period of the crop. The slow development of the dis-
ease in the plants set in 1925 is perhaps due to the fact that
they were set early and to cool dry weather which obtained be-
tween April 4 and May 12.






Florida Agricultural Experiment Station


DEVELOPMENT OF BLACK SHANK-RESISTANT STRAINS OF
TOBACCO BY SELECTION
Outline of Plan.-During the first year after the inception of
the work it became evident that this was a promising line of
attack with the variety locally known as Big Cuba. In a 30-
acre field of this variety (Fig. 111) the plants were affected in
varying degrees and individuals here and there appeared normal
and healthy at the end of harvest season. On the other hand,
such variations as between individual plants of the Connecticut
Round Tip variety have never been in evidence. As. already
explained, when this variety is planted on infested soil, black
shank makes a clean sweep of the entire field.
If infection of the soil was uniform, it was obvious that the
varying degrees of infection in the Big Cuba variety was due
to different degrees of resistance of the individual plants. If
this was the case, the practical solution of the problem was fur-
ther conditioned upon finding such fixed disease-resistant char-
acter in combination with the desired qualities which charac-
terize the best commercial type of wrapper tobacco, namely,
yield, color, texture, taste, burn and aroma.



r -
.t .^ ,









A"'A




Fig. 111.-View of a portion of a 30-acre field of Big Cuba tobacco on
Phytophthora-infested soil. Part of the original selections for resist-
ance to black shank were made in this field.
During the initial blossom stage, selections were made in two
different fields, both badly diseased with black shank. Tobacco






Bulletin 179, A Phytophthora Disease of Tobacco


is normally self-pollinated but under field conditions some cross-
ing is effected by wind, insects and humming birds. Therefore,
the inflorescence was bagged to insure self-pollination. Pro-
ceeding in this manner about 100 plants were selected from the
two fields. Several of the selected plants died before the seed
matured and others showed signs of the disease above ground.
At the time the seed were harvested the plants were pulled and
the roots were carefully examined, and it was found that all
plants showed infection varying from slight injury to a few
small roots to complete destruction of the root system. How-
ever, seed was saved from those plants which showed least in-
jury, 23 from the two fields. The seed from each plant was
kept separate and planted separately in 1923. Since 1922, se-
lections have been made in other diseased fields and treated in
a similar manner.
Trials in 1923 of These First-Generation Selections.-The
trials in 1923 included all of the individual selections made in
1922, together with certain other selections made in non-in-



--4, '- k
".'^ y





r ) f*ty.* *- .













Fig. 112.-Comparative resistance to black shank of first generation selec-
tions in the trial plot of 1923. The row on the left center is strain
A-22-22, 100 percent diseased. Row on right center is E-22-14, 94 per-
cent living and 24 percent slightly diseased on parts above ground.
Photographed July 17, 1923 just after harvest.
sl. I: s-: r ~SL
rlj~c,









Photographed July 17. 1923 just after harvest.






208 Florida Agricultural Experiment Station

fested fields. Some of the stock mother seed was used for com-
parison. The trials were made on soil which had been planted
to tobacco for the past three years and where black shank was
very prevalent the last year. The seed was planted in sterilized
soil where all developed strong, healthy plants, altho the per-
centage germination varied among the different strains. A uni-
formly strong, healthy lot of seedlings was then selected from
each strain and all transplanted on the same day-April 16-
into the field described above, in parallel rows. Black shank be-
gan to develop within a few days of the time of transplanting
and continued to increase thruout the season. From the out-
set, a marked difference was shown in the amount of disease in
certain of the selected strains as compared with the mother
strain checks and with other select strains, which condition ob-
tained thruout the season. Figure 112 shows the difference in
resistance between two of the select strains at the end of har-
vest season. The results of the trials are given in Table VII.

TABLE VII.-RESULTS OF TRIALS IN 1923 OF THE SEED OF INDIVIDUAL SE-
LECTIONS MADE IN 1922.

Number of Strain Number of Condition at End of Season-
Plants Percent
Living Diseased Healthy
Commercial Checks ..--........ 520 77 82 18
P-22-3 -........................ ......... ---112 95 19 81
E-22-14 ....---..-........ ..--.....--- 400 93 24 76
E-22-2 ........................ .....-.... ---431 95 26 74
E-22-35 .................................. 84 95 10 90
P-22-4 ......................... .......... ---43 100 12 88
H-22-16a ...---..................... 153 90 28 72
Se-22-12a .................. ....... 28 93 39 61
A-22-5a ................................... 64 98 12 88
A-22-22a ................................ 96 40 100 00
P-22-1 .................................... 456 70 94 6
E-22-6 ......-............................... 61 89 77 23
E-22-22 .................................... 62 81 61 39
E-22-24 ................................... 64 52 93 7
E-22-19 .................................... 62 51 86 14
E -22-15 .................................... 58 62 93 7
E-22-3 ...................................... 71 66 95 5
E-22-34 ............-....................... 278 66 85 15
E-22-36 .................................... 259 60 92 8
E-22-28 .................................... 376 56 91 9
E-22-16 .............-........-.......-..-.. 150 68 85 15
E-22-18 ................................... 113 80 85 15
E-22-10 ........--.......................... 287 .68 65 35
E-22-21 .................................... 457 77 58 42
E-22-27 .................................... 607 67 71 29
E -22-4 ...................................... 148 47 93 7
a-Selections made on non-infested soil.






Bulletin 179, A Phytophthora Disease of Tobacco 209

They represent the condition of the above-ground parts of plants
at the end of harvest and are expressed in percentages of the.
original stand, except that deduction was made for the occa-
sional loss from cutworms. There was no disease other than
black shank responsible for the death of any plants, so that the
results as tabulated represent the loss due to the attack of
the Phytophthora. The inflorescence of the most promising
plants of the strains showing highest percentage resistance,
E-22-2, E-22-14, E-22-35, A-22-5, Se-22-12, P-22-3 and P-22-4,
was bagged. Upon examination of the root system when the
seed were mature, only a few were found free from some injury.
The results given in Table VII show that certain select
strains were highly resistant to black shank, producing almost
a normal yield, while others were even more susceptible than
the commercial strain from which the selections were made. It
is also shown that none of the selections were completely resis-
tant, only in degree. Upon examination of the roots at the time
the seed was harvested a very few of the plants were found
free from some injury.
Trials of Selected Strains in 1924.-The procedure this year
was the same as in 1923, to make the seedbed on disinfected
soil, select uniformly strong seedlings for planting, and trans-
plant to Phytophthora-infested soil. Because of the poor condi-
tion of the shade under which the trials were made in 1923, it
was necessary to locate the trial plots on other fields in 1924.
Two plots were used this year, each consisting of one acre. The
soil of one plot-Durden farm-was Norfolk sandy loam, the
other-Shaw farm-of the Tifton series. The crops on both
fields were almost total failures in 1923, due to black shank.
All strains were planted on each plot so far as the number of
seedlings would permit and several rows of the commercial
type were planted on each plot for checks. The plants were set
on the Durden farm plot on April 16 and on the Shaw farm
plot on April 19. The plants started poorly on both plots, due
to hot dry weather during the first few days after they were
transplanted, and it was necessary to replant the missing hills.
The results of the trials were based on the original number of
plants set of each strain. It became evident a few weeks after
the plants were set that the Norfolk sandy soil was badly in-
fested with nematode which resulted in the failure of all strains.
Therefore, the results of this trial will not be shown. The re-







210 Florida Agricultural Experiment Station

suits obtained from the Shaw farm plot are summarized in
Table VIII.

TABLE VIII.-RESULTS OF TRIALS OF SELECTED STRAINS IN 1924-SHAW
FARM.

Number of Condition at End of Season-
Strain Number Plants Percent

Living Diseased Healthy
Commercial check ................ 586 7 96 4
P-23-4g .................................... 115 67 37 63
P-23-6 .... ---- -..............-..... 180 48 64 36
P-23-4 ...................................... 205 36 66 34
P-23-9 ...................................... 96 36 64 34
P-23-11 .................................... --179 50 57 43
P-23-10 ....................-- ..............--178 38 70 30
P-23-8 ........................---............. 154 33 74 26
P-23-1 ......................................---- 157 27 77 23
A-23-3 ..................................... 205 22 81 19
A-23-4 ................................... --177 19 88 12
A-23-16 .... -............................ 229 3 100 00
Ho-23-1 .................................... 151 29 77 23
Se-23-3 ..................................... -209 7 96 4
H-23-2 ....... .............................. 211 3 99 1
H-23-3 ..................................---- 215 8 92 8
E-23-27 ..-..............................-- 120 8 95 5
E-23-4 ...................- ...... .......----175 8 95 5
E-23-34 ................................... 186 15 89 11
E-23-14 .................................. 211 12 90 10
E-23-43 ........................---.....---- 178 5 97 3
E-23-16 ...................................----170 18 84 16
E-23-42 ......-.....---.... .............---- 166 2 100 00
E-23-21 ....- .............................. 167 5 97 3
E-23-15 ...................... ......... 178 14 92 8
E-23-2 .................................. --- 164 17 88 12
E-23-8 ................................... -- 165 15 90 10

It is apparent from these results that none of the selected
strains in the 1924 trials were as resistant to black shank as
their parent strains of the first generation (Table VII). Al-
tho several of them were superior to the commercial checks,
the outcome was less encouraging than might have been hoped
for. However, it is possible that the soil on which this test was
made was more uniformly infested early in the season than that
used in 1923. Furthermore, there was evidence of infection by
Thielavia basicola early in the season, which perhaps compli-
cated the conditions. Even so, there was a higher percentage
of the best strains, P-23 series, free from root injury at the
time the seed was harvested than of the previous year.
Therefore, this prepared the way for focusing attention in the
trials of 1925 upon the relative merits of these most promising







Bulletin 179, A Phytophthora Disease of Tobacco


TABLE IX.-RESULTS OF TRIALS OF SELECTED STRAINS IN 1925-S. & C.
FARM.


Strain Number Numbero
Plants

Commercial checks ............. 223
P-25-11a .................................. 112
P-24-1 ..................................... 108
P-24-3ga ............................. ----105
P-25-3a ...................... ....-.... 111
P-25-5a .................................... 105
P-25-12a .................................. 110
P-25-4a ...............................-.... 112
P-25-8a .................................... 106
P-25-10a .................................. 53
P-25-14a .................................. 106
P-24-3 .....................................-------------------- 113
P-24-6 ...................................... 111
P-24-8 ...................................... 108
P-24-41 ....-..............--......... --111
P-24-51 .................................... 111
P-24-61 .................................... 107
P-24-52 .............. ........--- ....-... 106
P-24-33 .................................... 114
P-24-32 .....-.............- ..... ..... 116
P-24-19 .................................... 120
P-24-44 .................................... 111
P-24-54 ....-......................-........ 112
D -24-1 ...................................... 114
E -24-1 ...................................... 109
E -24-2 ...................................... 112
E-24-6 .........................---- ...-- --. 116
E-24-7 ....--...............................--- 112
M-24-2 ..............-.................... -115
M-24-4 ....-.....................-- .....- ... 109
M-24-6 .-...................................-- 112
M-24-8 ...................................---- 108
M-24-9 ..........................-........ 110
A-24-22 ..............-... -----.... 113
A-24-24 .-......-..............--....-- 113
A-24-26 ...............-.....---...... -107
A-24-27 .-...........- ....-- ....----- 105
A-24-30 ...--...- ......------------ 110
A-24-35 ....--....-- --....---- ---- 109
A-24-36 ..........-- ..--...---------- 111
H-24-1 -...---..-----...-........ ---- 111
H-24-8 .---.....-.....------------. 110
L. C.-24-1 .-..-.....-..---..----. 110
L. C.-24-2 ........-..---...---------- 110
aGained one year by growing seed
ter of 1924-25.


Condition


Living
4
83
79
74
73
70
76
58
55
60
50
64
67
63
70
73
26
39
29
41
62
46
69
10
42
31
74
49
69
33
50
27
60
36
24
36
37
28
44
46
33
34
90
83


at End of Season-
Percent
Diseased Healthy
99 1
36 64
40 60
40 60
43 57
48 52
49 51
53 47
54 46
53 47
58 42
56 44
51 49
55 45
55 45
43 57
86 14
76 24
76 24
67 33
50 1 50
66 34
52 48
97 3
78 22
82 18
56 44
60 40
52 48
84 16
70 30
81 19
70 30
77 23
80 20
90 10
88 12
86 14
76 24
73 27
78 22
75 25
10 90
26 74


in the greenhouse during the win-


individuals of the P-23 series. All other strains, except a few
promising individuals of the E-23 and A-23 series, were discon-
tinued.
Trials of Selected Strains in 1925.-In addition to the most
promising individuals of the strains described above a few se-







212 Florida Agricultural Experiment Station

TABLE X.-RESULTS OF TRIALS OF SELECTED STRAINS IN 1925-SHAW
FARM.

Strain N r Number of Condition at End of Season-
tran umPer lants Percent

] Living Diseased Healthy
Commercial checks ............. 568 18 82 18
P-25-11a ................ .... ......... 109 86 23 77
P-25-5a ...................................... 185 83 25 75
P-25-12a ..........-.......-.............I 102 74 39 61
P-25-4a ...................................... 184 78 35 65
P-25-8a .................................... 135 75 38 62
P-25-10a .................................... 95 88 35 65
P-24-51 .................................... 99 80 22 78
P-24-52 ...................................... 95 71 43 1 57
P-24-41 ...................................... 98 77 28 72
P-24-43 ...................................... 101 67 50 50
P-24-54 ...................................... 95 65 57 43
P-24-1 .--..................................... 95 78 24 76
P-24-3 ..... -.............................. 101 61 50 50
P-24-6 ........... ......................-.... 102 68 50 50
P-24-3ga .................................. 100 75 40 60
P-24-20 .................................... 98 77 40 60
P-24-29 .................................... 95 81 31 69
P-24-30 .................................... 102 67 38 62
P-24-32 .................................... 94 77 24 76
P-24-36 .................................... 99 62 50 50
P-24-60 .................................... 92 54 67 33
P-24-61 .................................... 95 42 77 23
P-24-62 .................................... 94 46 65 35
P-24-45 .................................... 94 69 40 60
D-24-1 ...................................... 96 49 73 27
E-24-1 ...................................... 84 66 46 54
E-24-2 ...................................... 103 25 91 9
E-24-6 .................................. ---95 64 47 53
E-24-7 ...................................... 93 64 40 60
M-24-2 ...................................... 97 82 28 72
M-24-4 .:...-....................-........... 96 60 64 36
M-24-6 .............................-------------- ...... 103 76 33 67
M-24-8 ...................................... 102 59 54 46
M-24-9 ..................................... 93 85 22 78
A-24-22 .................................... 89 82 42 58
A-24-26 .......................-............ 96 45 75 25
A-24-27 .............................-...... 97 63 42 58
A-24-30 .................................... 96 50 66 34
A-24-36 .................................... 94 63 50 50
A-24-35 ............................-....... 96 55 68 32
H-24-5 ...................................... 94 41 74 26
H-24-8 ...................................... 100 31 81 19
L. C.-24-1 .............................. 99 79 40 60
L. C.-24-2 .............................. 99 90 30 70
aGained one year by growing seed in greenhouse during winter of
1924-25.
elections made in commercial fields in 1924 were included in the
trials of this year. The seedlings were grown as heretofore
explained.
Two one-acre plots were used this year, one on the Shaw farm
in the same field but not in the same place as used in 1924, the






Bulletin 179, A Phytophthora Disease of Tobacco 213

other on the S. & C. farm, also on Tifton soil. This change of
location was made in an effort to avoid complications with
nematode and Thielavia. Because of the early season both
plots were planted earlier than in previous years, the S. & C.
farm plot being set on March 25 and the Shaw farm plot on
March 30. The plants started well but suffered severely later on
from dry weather, as there was no rainfall between April 4 and
May 12. Black shank began to develop early in April, as usual,
but was checked to some extent during the last two weeks of the
dry period. Within a few days after the rain on May 12 the
disease began to develop very rapidly and continued to increase
during the remainder of the season. The results are brought
together for reference in Tables IX and X.
It may be seen from the above tables that there is consider-
able variation in resistance as between individual strains in the
same series as well as between the different series. Certain
strains in any series are almost as susceptible as the commer-
cial checks, while others show marked improvement over their
parent strains in the 1924 trials. Moreover, the figures as
tabulated do not show the difference in vigor and uniformity


Fig. 113.-Trial of selected strains, 1925. Shaw farm. Row on the left
is the commercial type of Big Cuba from which the original selections
were made in 1922; row on the right is P-24-51, a vigorous and high-
ly resistant strain.






214 Florida Agricultural Experiment Station

between the commercial check and the best selections. Altho
they showed no signs of wilting or stem injury above ground,
many plants living of the commercial check and certain selected
strains remained small and produced few or no marketable
leaves. An examination of the roots of such plants revealed
that they were severely injured. On the other hand, the best se-
lected strains produced plants of uniform vigorous growth and
many of the plants which showed some signs of disease on the
stems produced almost, if not entirely, a normal crop of good
leaves. This difference in vigor and uniformity of growth be-
tween the commercial check and two of the resistant strains is
illustrated in figures 113 and 114.



.';, 1. _

~--














,,-. .. ..- -"
Fig. 114.-Trials of selected strains, 1925, S. & C. farm. Row on the left
is the commercial type of Big Cuba from which the original selections
were made in 1922; row on the right is strain E-24-6, a vigorous and
fairly resistant type.
A comparison of the results from the two fields will show
that the P-25 series is somewhat superior to any other of the se-
ries, while the P-24 series is a close rival. A comparison of these
two series as they grew side by side in the two fields shows
even greater superiority of the P-25 series than is evinced by the
figures in the tables. The summaries in Tables XI and XII serve
to bring out some of these points in tabular form.






Bulletin 179, A Phytophthora Disease of Tobacco


TABLE XI.-SUMMARY OF SIGNIFICANT RESULTS FROM TABLE IX-S. & C.
FARM.

Strains Total Condition at end of season-
Plants Percent
S Living I Diseased I Healthy
Commercial check--.......... ..... 223 4 99 1
P-25 series, av. all................ 815 66 49 51
P-24 series, av. all................. 1,553 57 58 42
E-24 series, av. all........ ........... 449 49 68 32
M-24 series, av. all................. 554 48 71 29
A-24 series, av. all ............... 768 38 81 19
TABLE XII.-SUMMARY OF SIGNIFICANT RESULTS FROM TABLE X-SHAW
FARM.
Total Condition at end of season-
Strains Plants Percent

SLiving Diseased Healthy
Commercial checks---............... 568 18 82 18
P-25 series, av. all................ 810 81 32 68
P-24 series, av. all ............. 1749 68 44 56
E-24 series av. all ................. 375 55 56 44
M-24 series, av. all ............... 491 72 40 60
A-24 series, av. all ............... 667 60 57 43

For further selection looking forward to the improvement of
the present standards, seed was saved from the most promis-
ing plants of the three best strains of the P-25 series from both
fields and these will be planted separately in infested soil in
1926. Practically all of the other series will be discontinued.
Whether a strain sufficiently resistant for successful commer-
cial growing on infested soil can be perfected by this method
from the strains now on hand, only further trials will determine.

SUMMARY

1. A disease of tobacco commonly known as black shank has
been prevalent in certain localities of the Florida-Georgia dis-
trict for several years and has been quite general for the past
two years. This disease is primarily a dry rot of the roots and
basal portion of the stem, but the lower leaves are frequently
attacked during the rainy season both while in the field and
after they are hung in the curing barns.
2. Outside of the Florida-Georgia district black shank has
been observed on shade tobacco in Pasco County, Florida and on
bright leaf tobacco from near Moultrie, Georgia. It has been
reported from Virginia and Summerdale, Alabama. A similar,
if not the same, disease has been prevalent in Java since 1895.






Florida Agricultural Experiment Station


3. Of the several diseases of tobacco occurring in the
Florida-Georgia district which have been distinguished, black
shank is the most important. It has caused total failures in
many fields the first year it appeared.
4. Investigations have revealed that black shank is caused by
a species of Phytophthora similar in many characters to P. nico-
tianae. It also has certain characters similar to P. parasitica.
Morphologically it seems to be intermediate between these spe-
cies. All three forms are similar in pathogenicity, except P.
parasitica could not be made to infect tobacco. On a basis of
these relationships it is proposed to consider tentatively the
black shank Phytophthora as a biologic strain of P. nicotianae
Breda de Haan.
5. All commercial types of tobacco (Nicotiana tabacum)
tested are highly susceptible to the disease at all stages of
growth. Nicotiana rustic proved fairly resistant to the dip-
ease. All efforts to make crosses between this and types of
wrapper tobacco have failed.
6. Tobacco is the only species of plant found attacked by the
Phytophthora under natural conditions. Disease symptoms
were produced in young seedlings of potato, eggplant and cas-
tor-oil plant by inserting mycelium of the fungus into the stems.
Also leaves of these plants and tomato seedlings were infected
by atomizing them with water suspensions of zoospores. When
freshly cut potato tubers were planted in inoculated soil in the
greenhouse they decayed before sprouting. No signs of decay
have been observed on cut tubers planted in infested fields.
This is perhaps due to the low soil temperature prevailing at the
time they are planted.
7. The Phytophthora persists in the soil for several years.
It is also resistant to drying in the soil, having survived 18
months in air-dry soil.
8. The optimum temperature for growth of the fungus in
culture is between 25 and 30 degrees Centigrade. The mini-
mum is about 12 and the maximum about 35 degrees Centigrade.
9. The minimum temperature for the development of the
disease is below 20 degrees Centigrade, but the exact degree has
not been determined. Neither have the optimum and maximum
temperature been determined. Field observations and green-
house experiments have shown that the soil temperature is usu-


216






Bulletin 179, A Phytophthora Disease of Tobacco 217

ally too low for development of the disease in the field until
about April 10.
10. The fungus does not survive the fermentation process
which the cured leaves undergo in the sweat room. Consider-
able evidence is at hand indicating that the fungus loses its vi-
tality in diseased leaf tissues within a few days after they are
brought from the fields-the time required for the leaves to
dry out.
11. Steam sterilization of infested fields has proved imprac-
tical because of the expense and the great probability of rein-
festation within a short time.
12. The amount of organic matter in the soil appears to
have little or no effect upon the spread of the fungus thru the
soil or upon the development of the disease. However, there
is a possibility of introducing the fungus into new fields in man-
ure from infested farms. It has been reported that the disease
in Java was more serious on land fertilized with manure.
13. No benefit was derived from the use of inoculated sul-
phur, Semesan, Uspulun or land plaster under field conditions,
altho all of these materials inhibited development of the dis-
ease for 36 days in greenhouse experiments.
14. Progress has been made in the development of strains
of Big Cuba tobacco resistant to black shank but they are not
considered sufficiently resistant to release for commercial
growing.
LITERATURE CITED
1-BREDA DE HAAN, J. VAN.
De bibitziekte in Deli-tabak veroorzaakt door Phytophthora nicotianae.
Mededeelingen Uit 'S Lands Plantentuin 15:5-107. 1895. illust.
2-DASTUR, JEHANGIR FARDUNGI.
On Phytophthora parasitica nov. sp. A new disease of castor oil
plant. Mem. Dept. Agr. India. Bot. Ser., v. 5, no. 4: 177-231.
1913. illust.
3-D'ANGREMOND, A.
Die Bekampfung von Phytophthora nicotianae in den Vorstlanden.
Meded. Proefstation voor Vorstenlandsche Tabak XLIII. (Abs. in
Zentralb. fur Agrikulturchemie LI, 8: 203-205. 1922).
4- .................. ...... .........
Bestrijding van Phytophthora nicotianae in de Vorstenlanden. I.
Mededeeling no. 39:3-59. 1920-1921. Proefstation voor Vorsten-
landsche Tabak.
5-DE BRUYN, HELENA L. G.
The saprophytic life of Phytophthora in the soil. Mededeeling van
de Landbouwhoogeschool. Waneningen, XXIV, paper 4:1-38. 1922.






Florida Agricultural Experiment Station


6-JENSEN, Hj.
De Lanasziekte en Hare Bestrijding II. Proefstation voor Vorsten-
landsche Tabak. Mededeeling no. 29:5-118. 1917.
7-LEONIAN, LEON H.
Physiological studies of the genus Phytophthora. Amer. Jour. Bot.
v. XII, no. 7:444-498. 1925. illust.
8-PALM, B. T.
Verslag van het Deli Proefstation over 1 July, 1922-30 Juni, 1923.
Meded. Deli Proefstation te Medan Sumatra Ser. 2, XXIX: 41 pp.
1923. (Reviewed in The R. of Appl. Mycol. vol. 3, Pt. 2:107. 1924.)
9-ROSENBAUM, J.
Studies of the genus Phytophthora. Jour. Agr. Res. vol. VIII, no.
7:233-276. 1917. illust.
10-SMITH, RALPH E. AND SMITH, ELIZABETH H.
Further studies of Phythiaceous infection of deciduous fruit trees
in California. Phytopath. vol. 15, no. 7:389-404. 1925. illust.
11-TISDALE, W. B.
Tobacco diseases in Gadsden County in 1922, with suggestions for
their prevention and control. Univ. Fla. Agr. Exp. Sta. Bul. 166:
77-118. 1922. illust.
12-WINGARD, S. A. AND GODKIN, JAMES.
Tobacco diseases in Virginia and their control. Va. Agr. Exp. Sta.
Bul. 90:3-31. 1924. illust.






Bulletin 179, A Phytophthora Disease of Tobacco 219




TABLE OF CONTENTS
PAGE
INTRODUCTION ....................--..................----------- .................... 159
THE DISEASE .....--...........-----....-- ....---..----------------.... ... 15
Description ...-...---...- ....-...--- .....-- ----.......---------------...-.... --.. --...-- 159
History and Geographic Distribution ..................-- .............. --- -- 165
Economic Importance .......-.....-- -.... ...............-..... 166
THE FUNGUS ... ----.......-....... ...-......-- ................ 169
Hosts ....--.......-...-- ...--. ....... ----............--- ...............----- 169
Susceptibility of Different Varieties of Tobacco..-............................ 172
Agencies of Dissemination ........................................ 173
Persistence in the Soil ............................................................ 175
Resistance to Drying in the Soil .................. ....... .............. 177
Isolation -..............---- ...- -....--......-- ..-- ..---- 177
Proof of Pathogenicity ............------ -- -------------................... 178
Inoculation of Other Plants ............-----.................. --- 181
Morphology ..........-- ...... ..-- ..------..--------- 18:3
Mycelium ..------------........ ...... ................. 183
Asexual reproduction ..............................--. ----.-- 184
Taxonomy .------.......... -- ----------------..................... 188
Physiology ...........--------------------..... ... ..................-......---- -- 194
Effect of Temperature Upon the Development of the Fungus in
Culture .....................---.......---.-- ....--- -- ..... ................ 196
Effect of Hydrogen-Ion Concentration of Culture Media Upon the
Rate of Growth of the Black Shank Phytophthora-----.................. 197
Effect of Fermentation of Infected Leaves Upon the Fungus-....... 198
RELATION OF SOIL TEMPERATURE TO THE DEVELOPMENT OF BLACK
SHANK .----- ----------...................-.-------------- 199
CONTROL EXPERIMENTS ..................................-.............. --- 201
Steam Sterilization of Infested Fields ..........-.............................. ... 201
Effect of Organic Content of the Soil Upon the Development of
Black Shank .........................---....--------------------..... 202
Effect of Certain Chemicals Upon the Development of Black
Shank ......................--..........------.............................---- 203
Development of Black Shank-Resistant Strains of Tobacco by
Selection ......-........--- ----- ..... ..................... 206
Outline of Plan ....----.................................-.-- .-- ------.---- 206
Trials in 1923 of These First-Generation Selections.................. 207
Trials of Selected Strains in 1924 ..........-....-............... ........ 209
Trials of Selected Strains in 1925 ...............-- --.... .................. 211
SUMMARY ..--......--- ....... ..-------.-.--- -..--------........ 215
LITERATURE CITED ......... .......- .............. ...- ...... -----.................. 21









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