• TABLE OF CONTENTS
HIDE
 Title Page
 Credits
 Table of Contents
 Introduction
 The disease
 Etiology
 The pathogene
 Two associated saprophytes
 Varietal susceptibility
 The physiology of helminthosporium...
 Summary
 Literature cited






Group Title: Bulletin - University of Florida. Agricultural Experiment Stations ; No. 267
Title: Studies on the ring spot disease of sugarcane
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00027204/00001
 Material Information
Title: Studies on the ring spot disease of sugarcane
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 76 p. : ill., charts ; 23 cm.
Language: English
Creator: Bourne, B. A ( Benjamin Arthur ), b. 1897
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1934
 Subjects
Subject: Sugarcane -- Diseases and pests -- Florida   ( lcsh )
Phytopathogenic fungi   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 73-76.
Statement of Responsibility: by B.A. Bourne.
General Note: Cover title.
General Note: Originally presented as: Thesis (Ph.D.)--Cornell University, 1933.
Funding: Bulletin (University of Florida. Agricultural Experiment Station)
 Record Information
Bibliographic ID: UF00027204
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000924128
oclc - 18207019
notis - AEN4734

Table of Contents
    Title Page
        Page 1
    Credits
        Page 2
    Table of Contents
        Page 3
    Introduction
        Page 3
    The disease
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
    Etiology
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
    The pathogene
        Page 24
        Page 25
        Page 26
        Page 27
    Two associated saprophytes
        Page 28
        Page 29
        Page 30
        Page 31
    Varietal susceptibility
        Page 32
        Page 33
    The physiology of helminthosporium ocellum and its associated saprophyte phyllosticta sorghina
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
    Summary
        Page 72
    Literature cited
        Page 73
        Page 74
        Page 75
        Page 76
Full Text


Bulletin 267


UNIVERSITY OF FLORIDA
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA
Wilmon Newell, Director










STUDIES ON THE RING SPOT

DISEASE OF SUGARCANE


By B. A. BOURNE









TECHNICAL BULLETIN








Bulletins will be sent free upon application to the
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA


May, 1934








EXECUTIVE STAFF

John J. Tigert, M.A., LL.D., President of the
University
Wilmon Newell, D.Sc., Director
H. Harold Hume, M.S., Asst. Dir., Research
Harold Mowry, B.S.A., Asst. Dir., Adm.
J. Francis Cooper, M.S.A., Editor
R. M. Fulghum, B.S.A., Assistant Editor
Ida Keeling Cresap, Librarian
Ruby Newhall, Administrative Manager
K. H. Graham, Business Manager
Rachel McQuarrie, Accountant


MAIN STATION, GAINESVILLE

AGRONOMY
W. E. Stokes, M.S., Agronomist**
W. A. Leukel, Ph.D., Agronomist
G. E. Ritchey, M.S.A., Associate*
Fred H. Hull, M.S., Associate
W. A. Carver, Ph.D., Associate
John P. Camp, M.S., Assistant

ANIMAL HUSBANDRY
A. L. Shealy, D.V.M., Animal Husbandman"*
R. B. Becker, Ph.D., Dairy Husbandman
W. M. Neal, Ph.D., Associate in Animal Nutri-
tion
D. A. Sanders, D.V.M., Veterinarian
M. W. Emmel, D.V.M., Assistant Veterinarian
W. W. Henley, B.S.A., Assistant Animal Hus-
bandman
P. T. Dix Arnold, B.S.A., Assistant in Dairy In-
vestigations

CHEMISTRY AND SOILS
R. W. Ruprecht, Ph.D., Chemist*'"
R. M. Barnette, Ph.D., Chemist
C. E. Bell, Ph.D., Associate
H. W. Winsor, B.S.A., Assistant
H. W. Jones, M.S., Assistant

ECONOMICS, AGRICULTURAL
C. V. Noble, Ph.D., Agricultural Economist"*
Bruce McKinley, A.B., B.S.A., Associate
Zach Savage, M.S.A., Assistant

ECONOMICS, HOME
Ouida Davis Abbott, Ph.D., Specialist"
L. W. G-ddum, Ph.D., Biochemist
C. F. Ahmann, Ph.D., Physiologist

ENTOMOLOGY
J. R. Watson, A.M., Entomologist'*
A. N. Tissot, Ph.D., Associate
H. E. Bratley, M.S.A., Assistant

HORTICULTURE
A. F. Camp, Ph.D., Horticulturist**
A. L. Stahl, Ph.D., Associate
G. H. Blackmon, M.S.A., Pecan Culturist
Roy J. Wilmot, M.S.A., Specialist, Fumigation
Research
R. D. Dickey, Assistant Horticulturist

PLANT PATHOLOGY
W. B. Tisdale, Ph.D., Plant Pathologist**
George F. Weber, Ph.D., Plant Pathologist
R. K. Voorhees, M.S., Assistant
Erdman West, M.S., Mycologist

*In cooperation with U.S.D.A.
**Head of Department.


BOARD OF CONTROL

Geo. H. Baldwin, Chairman, Jacksonville
A. H. Blanding, Bartow
A. H. Wagg, West Palm Beach
Oliver J. Semmes, Pensacola
Harry C. Duncan, Tavares
J. T. Diamond, Secretary, Tallahassee


BRANCH STATIONS

NORTH FLORIDA STATION, QUINCY
L. O. Gratz, Ph.D., Plant Pathologist in Charge
R. R. Kincaid, M.S., Asst. Plant Pathologist
J. D. Warner, M.S., Agronomist
R. M. Crown, B.S.A., Assistant Agronomist
Jesse Reeves, Farm Superintendent


CITRUS STATION, LAKE ALFRED
John H. Jefferies, Superintendent
Geo. D. Ruehle, Ph.D., Associate Plant Pathol-
ogist
W. A. Kuntz, A.M., Associate Plant Pathologist
B. R. Fudge, Ph.D., Associate Chemist
W. L. Thompson, B.S., Assistant Entomologist

EVERGLADES STATION, BELLE GLADE
A. Daane, Ph.D., Agronomist in Charge
R. N. Lobdell, M.S., Entomologist
F. D. Stevens B.S., Sugarcane Agronomist
G. R. Townsend, Ph.D., Asst. Plant Pathologist
B. A. Bourne, Ph.D., Sugarcane Physiologist
J. R. Neller. Ph.D., Biochemist
R. W. Kidder, B.S., Asst. Animal Husbandman
Ross E. Robertson, B.S., Assistant Chemist

SUB-TROPICAL STATION, HOMESTEAD
H. S. Wolfe, Ph.D., Horticulturist in Charge
W. M. Fifield, M.S., Assistant Horticulturist
Stacy O. Hawkins, M.A., Assistant Plant
Pathologist


WEST CENTRAL FLORIDA STATION,
BROOKSVILLE
W. F. Ward, Asst. Animal Husbandman in
Charge*


FIELD STATIONS

Leesburg
M. N. Walker, Ph.D., Plant Pathologist in
Charge
W. B. Shippy, Ph.D., Asso. Plant Pathologist
K. W. Loucks, M.S., Asst. Plant Pathologist
J. W. Wilson, Ph.D., Associate Entomologist
C. C. Goff, M.S., Assistant Entomologist
Plant City
A. N. Brooks, Ph.D., Plant Pathologist
R. E. Nolen, M.S.A., Asst. Plant Pathologist
Cocoa
A. S. Rhoads, Ph.D., Plant Pathologist
Hastings
A. H. Eddins, Ph.D., Plant Pathologist
Monticello
,Assistant Entomologist
Bradenton
David G. Kelbert, Asst. Plant Pathologist
Sanford
E. R. Purvis, Ph.D., Assistant Chemist, Celery
Investigations A L










STUDIES ON THE RING SPOT DISEASE

OF SUGARCANE'
By B. A. BOURNE

CONTENTS
Page
Introduction ............................................................ ........... 3
The D disease ............................................................ .... ....... 4
Etiology .............................................................. ................ 13
The Pathogene .................................... .................................... 24
Two Associated Saprophytes ......................................................... 28
V arietal Susceptibility ......................................... .................... .. 32
The Physiology of Helminthosporium Ocellum and Its Associated Saprophyte, Phyllos-
ticta Sorghina ................................................................... 34
Control .................................... .......................................... 66
Sum mary ........................................ ............................... 72
Literature Cited ...................................... ... ...... ................ 3


INTRODUCTION

A number of fungous diseases affecting the leaves of sugarcane
have been made the subject of intensive study during recent
years, more especially those caused by various species of the
genus Helminthosporium. Although existing records show that
the common "ring spot" disease was described about 40 years
ago, little detailed research has been carried on concerning this
malady. It has also been interesting to note that for a consid-
erable number of years after a causal organism had been assigned
to the disease, differences in opinion existed as to the identity
of the true parasite.
Subsequent to the researches of Butler(13)2 however, in 1918,
no further studies appear to have been made with this disease
other than an extension of our knowledge of its geographical
range by numerous plant pathologists and mycologists. These
latter, of course, were guided in their identification work by
previously published descriptions of the disease. The finding
of the fungus Leptosphaeria sacchari van Breda de Haan asso-

'Also presented to the Faculty of the Graduate School of Cornell Univer-
sity, June, 1933, as a major thesis in partial fulfillment of the requirements
for the degree of doctor of philosophy. (Abridged for publication.) Grate-
ful acknowledgement is made to Dr. L. M. Massey, Dr. C. E. F. Guterman,
Dr. H. E. Thomas and Professor H. H. Whetzel of the Department of Plant
Pathology and Dr. O. F. Curtis of the Department of Plant Physiology for
helpful suggestions and criticisms.
2Numbers in parenthesis (Italic) refer to "Literature Cited" in the back
of this bulletin.










STUDIES ON THE RING SPOT DISEASE

OF SUGARCANE'
By B. A. BOURNE

CONTENTS
Page
Introduction ............................................................ ........... 3
The D disease ............................................................ .... ....... 4
Etiology .............................................................. ................ 13
The Pathogene .................................... .................................... 24
Two Associated Saprophytes ......................................................... 28
V arietal Susceptibility ......................................... .................... .. 32
The Physiology of Helminthosporium Ocellum and Its Associated Saprophyte, Phyllos-
ticta Sorghina ................................................................... 34
Control .................................... .......................................... 66
Sum mary ........................................ ............................... 72
Literature Cited ...................................... ... ...... ................ 3


INTRODUCTION

A number of fungous diseases affecting the leaves of sugarcane
have been made the subject of intensive study during recent
years, more especially those caused by various species of the
genus Helminthosporium. Although existing records show that
the common "ring spot" disease was described about 40 years
ago, little detailed research has been carried on concerning this
malady. It has also been interesting to note that for a consid-
erable number of years after a causal organism had been assigned
to the disease, differences in opinion existed as to the identity
of the true parasite.
Subsequent to the researches of Butler(13)2 however, in 1918,
no further studies appear to have been made with this disease
other than an extension of our knowledge of its geographical
range by numerous plant pathologists and mycologists. These
latter, of course, were guided in their identification work by
previously published descriptions of the disease. The finding
of the fungus Leptosphaeria sacchari van Breda de Haan asso-

'Also presented to the Faculty of the Graduate School of Cornell Univer-
sity, June, 1933, as a major thesis in partial fulfillment of the requirements
for the degree of doctor of philosophy. (Abridged for publication.) Grate-
ful acknowledgement is made to Dr. L. M. Massey, Dr. C. E. F. Guterman,
Dr. H. E. Thomas and Professor H. H. Whetzel of the Department of Plant
Pathology and Dr. O. F. Curtis of the Department of Plant Physiology for
helpful suggestions and criticisms.
2Numbers in parenthesis (Italic) refer to "Literature Cited" in the back
of this bulletin.






Florida Agricultural Experiment Station


ciated with typical lesions was taken as final evidence that the
disease was present.
This paper is the outcome of investigations conducted to deter-
mine the true nature of the so-called "ring spot" disease of sugar-
cane and to make such physiological and basic studies as would
enable cane breeders to appreciate fully certain factors likely to
be significant in the process of developing new types resistant or
immune to the malady.
Most of the results reported were obtained during the period
1930-1933 in the Lake Okeechobee region of Florida," where one
of the largest sugarcane* developments in the State is in progress.
However, observations on the disease were made on a large num-
ber of cane varieties planted in scattered sections of the State so
as to gain some idea as to the influence of such environmental
factors as climate and soil. Further, some of the physiological
and mycological studies were made in the laboratories of Cornell
University, where adequate facilities were available.

THE DISEASE

NAMES, HISTORY AND GEOGRAPHICAL RANGE
The disease was first briefly described by Krtiger(37) in 1890
as the ring spot of the cane leaf. Later, 1892, van Breda de
Haan(56), described the malady in greater detail, retaining the
same name. Wakker and Went(58), Butler(13), Bancroft(7),
Johnston and Stevenson(35), Nowell(46), Cook(18, 19, 20), Lee
and Martin(41), as well as several others, have rigidly held to
the original name. In only one instance has the writer detected
a departure from this original designation and that is in a paper
by Horne(32) in 1905, well illustrating the typical lesions, but
making reference to it under the name of "eye spot" ("la enfer-
medad mancha de ojo"). Since the disease was first described
under the name of "ring spot" it would be most desirable to give
priority to this original name in all references to the malady in
order to avoid confusion.
3The use of laboratory and other facilities furnished cooperatively by
the Southern Sugar Company and the United States Sugar Corporation is
much appreciated.
*The use of the term "sugarcane" as one word instead of being hyphenated
(see original thesis, Cornell University Library), or as two words (as adopted
and published in the Proceedings of the 4th Congress of the International
Society of Sugar Cane Technologists, 1933: p. 150. Porto Rico), is one of
editorial policy for which the author is in no way responsible.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 5

By perusal of the literature, it is seen that since the malady
was first described from Java where it occurs especially in the
western portion rather than in the dry plains of East Java, it
has been recorded from practically every cane producing country.
The main references are to be found by Horne(32) 1905, But-
ler(12) 1906, Cobb(16,17) 1906 and 1909, Bancroft(7) 1910,
Averna-Sacca(6) 1916, Johnston and Stevenson(35) 1917,
Caum(14) 1921, Lee(38) 1924, Anderson et al.(3) 1926, Steven-
son(53) 1926, Abbott(l) 1929, Bell(9) 1929, Chardon, Toro et
al.(15) 1930 and Cook(21) 1931. According to these, the disease
has been found in Java, India, Singapore, Borneo, Hawaii, Philip-
pines, Australia, Fiji, South Africa, Argentina, Brazil, Peru,
Mauritius, Cuba, Santo Domingo, Porto Rico, British West Indies,
Panama (Canal Zone), Columbia (N.W. of Palmira) and Florida
and Alabama of the United States.

ECONOMIC IMPORTANCE
After reviewing the estimates of damage caused by the ring
spot disease as given by van Breda de Haan(56), Wakker and
Went(58), Horne(32) and Butler(13), it is evident that several
authors agree that while the disease as a whole has never been
considered as being of much economic importance, certain en-
vironmental conditions not thoroughly understood have been
associated with very serious local outbreaks. Bell(9) has also
noted that the disease materially affected some of the newer
Hawaiian seedlings and points out that like eye spot, it may
become of special importance to cane breeders.
The writer has seen very severe outbreaks of this disease on
susceptible varieties in Barbados, Porto Rico, Cuba and in several
places in Florida. The view taken by some, especially Lee and
Martin(41), Cook(20) and others that the disease occurs almost
entirely on the outer and older leaves and thus is not as impor-
tant as certain diseases due to Helminthosporium spp. which
attack the young leaves is not concurred in by the writer subse-
quent to the studies on the true nature of the etiology of the dis-
ease and a recognition of the fact that the parasite is able to
attack various parts of the host in addition to the leaves.
Because several commercial canes at present grown in Florida
are highly resistant to the malady, the loss in this State is not
considered to be of major importance for the most part. How-
ever, the damage to certain susceptible varieties is very severe,







Florida Agricultural Experiment Station


although difficult to evaluate properly. Again, it must be ad-
mitted that otherwise excellent new seedling varieties might have
to be discarded or severely limited to special locations in the
future on account of high susceptibility to this disease. There
can be no doubt that the disease would become of distinctly major
importance unless adequate precautions were taken to select only
highly resistant canes for use commercially.
SYMPTOMATOLOGY
MORPHOLOGIC SYMPTOMS AND EXTERNAL SIGNS
The Leaves:-Van Breda de Haan(56) and Wakker and
Went(58) all agree in their description of the morphological
symptoms of the disease. Butler(13) however, makes no men-
tion of dark green spots in the early stages of the malady, but
regards the first symptoms as being the appearance of small
discolored, generally purple spots visible on both surfaces of the
leaf. Cook(20) states that the first symptoms are exhibited by
the appearance of small yellow spots which increase in size and
show a black color in the center. The illustration published by
this latter author shows numerous almost entirely black spots
not at all typical of the ring spot as illustrated by several authors,
including Wakker and Went(58), Butler(13), Nowell(46) and
Bell(9).
In Florida, typical ring spots usually appear very frequently
on susceptible cane varieties in early December and January.
Using the color standards of Maerz and Paul(43), the young
spots first appear superficially as minute ivy green to bronze
brown, somewhat elongated flecks which enlarge to become lesions
varying in color from Burgundy and India red to Spanish raisin,
with a distinct yellow areola. Fig. 14 shows lesions occurring on
the varieties CP. 27-1395 and Otaheite, which bring out the varia-
tions which have been observed. Fig. 4 shows the typical appear-
ance of the early stage of infection on Otaheite leaves after 14
hours under artificially controlled conditions. While only the
straw-colored spots are typical of what have been described pre-
viously as ring spots, the darker lesions having similar shape
4Reproduced from a photograph in full color as filed with thesis.
5Cane varieties are designated differently in various countries. In
Florida, "CP" and "US" canes refer to those bred by the U. S. Dept. of
Agric. at their Canal Point Station, while the "F" series refer to those bred
for the Florida Agric. Exp. Sta. "POJ" and "Co" canes refer respectively
to those bred by the Java Exp. Sta. and the sugarcane Station at Coimbatore,
India.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 7


A B1

Fig. 1.-Typical ring spots on leaves of sugarcane. A, leaf of CP. 27-139;
B, two half leaf portions of Otaheite.

have also been found to be caused by the same pathogenic or-
ganism.
When about 2 mm. wide by 5 mm. long, the length being always
greatest in the direction of the leaf veins, an oval portion of the







Florida Agricultural Experiment Station


center often turns an antique bronze, outside of which is a Bur-
gundy or Spanish raisin colored narrow-oval border surrounded
by a yellow areola. By the time the spot enlarges to its full
size-2.5-4x10-12 mms. or sometimes larger-it usually loses its
oval form and becomes somewhat irregular in outline, the ends
becoming rather pointed and elongate, often giving the spots a
spindle-shaped appearance. At this stage; the center frequently
becomes peach blow to straw-colored, while the narrow margin
remains Spanish raisin in color. The yellowish areola often disap-
pears laterally as the spot reaches its full size, but is plainly visible
at both ends, in some cases extending 5 mms. or less as a pointed
yellow streak, mainly in the direction of the leaf tip.
It often happens that two or three spots will coalesce either
laterally or longitudinally, and the margins which come in contact
eventually disappear, leaving a large irregularly shaped blotch
with a Burgundy or Spanish raisin colored margin about 0.5 mms.
wide. The narrow margin is usually thicker at the ends than
laterally. Also, instead of forming fairly well defined lesions,
very irregular and much elongated streaks frequently occur,
especially toward the leaf tip, these streaks often having a typical
oxheart-colored appearance.
While the centers of the spots usually become straw-colored,
this phenomenon does not always take- place, as clearly illustrated
in Fig. 1, the spots remaining bronze brown, antique bronze or
Spanish raisin in color with perhaps only a mere fleck of a paler
portion at or near the center. Occasionally, spots enlarge con-
siderably while yet maintaining an ivy green color with a rather
sharp margin of bronze brown. Such "green" spots have been
mentioned by van Breda de Haan(56) in his original description
of the disease in Java.
The writer has also confirmed Butler's observations relative
to the withering of the leaf tissues in the neighborhood of the
spots in the later stages of the disease, so that in cases of heavy
infection the whole leaf withers prematurely.
Should the center of the spot become peach blow or straw
colored, minute black points are commonly visible on the upper
surface between the fine veins, both in the center and also bor-
dering on the Burgundy or Spanish raisin colored plesionecrotic
marginal area. These black points represent, as will be brought
out later, the fruiting bodies of one or both of two frequently
associated saprophytic fungi.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 9

While the mid-rib has never been found to have a typical spot
on it, infections starting on the border of this leaf portion often
encroach on it laterally.
Lesions may occur on any portion of the green leaf blade, both
on the central as well as outer whorl of leaves, but appear to be
more numerous as a rule from the central portion toward the
tip, especially among stools of canes along the edge of the field
or in locations favorable for the deposition of dew on the leaf
surface. Fig. 5 shows the appearance of typical lesions at the
base of the leaf blade.
The Leaf-Sheath:-Caum(14) in 1921, gave a description and
diagnosis of a leaf-sheath disease of sugarcane in Hawaii which
agrees well with certain stages of ring spot disease as it affects
the sheath in Florida. He also described the fungus associated
with the disease as one forming pycnidia on the spots. This
author states that the fungus attacks practically all the varieties
grown in Hawaii and is primarily a leaf-sheath parasite, attack-
ing the sheath at or near the point of attachment, killing the tis-
sues and causing the formation of characteristic spots.
In Florida, one of the same saprophytic organisms which has
been found associated with the ring spots of the leaf blade, form-
ing minute blackish points, also occurs on the leaf-sheath along
with the true parasite. The young spots appear as minute dis-
colored areas which soon turn a Burgundy to Spanish raisin color.
The lesions frequently become oval or irregular in outline and
the center may dry out, becoming peach blow or straw colored
as in the case of the leaf spots, having a sharp, narrow margin
of Burgundy or Spanish raisin colored plesionecrotic tissue.
Other lesions remain bronze brown or Spanish raisin in color and
show no drying out in the center. Fig. 5 shows some of the
lesions on sheaths of D.74 with and without dry straw-colored
centers. Occasionally the edge of the leaf-sheath is attacked
and the parasite kills it back throughout its length for a consid-
erable depth, leaving it very often straw-colored with a narrow
Burgundy or Spanish raisin colored margin next to the healthy
tissue.
The Stem:-Caum(14), as noted above, records the occurrence
of the sheath disease on the stem also, especially in the case of
particularly susceptible varieties. In fact, in some instances all
the rind on the internode was found destroyed by the organism
forming pycnidia which the author claims to be parasitic. The






Florida Agricultural Experiment Station


typical lesions on the stem, however, were neither described nor
figured.
The writer has found abundant lesions on the stems of many
susceptible varieties, these being especially large when stalks
are mature and ready for fabrication. Fig. 6 shows typical
lesions on three canes. Spots vary from minute, oval or roundish
dots to areas of considerable size. As in the leaf and sheath
spots, the outline is irregular, but there exists a tendency to
considerable elongation, sometimes extending to about 11 cms.
In most cases, the center of the spot is characteristically alumi-
num to straw colored. The edge of the lesion is sharply defined
against the normal tissue and may be Burgundy, Spanish raisin
or bronze brown in color. However, many lesions show no such
coloration in the plesionecrotic zone, but are aluminum colored
up to the very edge of the lesion. A hand lens reveals very
readily the protruding pycnidia of the associated saprophyte
scattered indiscriminately over the surface of the light-colored
areas, instead of being arranged in rows as in the case of the
leaf lesions.

HISTOLOGICAL SYMPTOMS AND INTERNAL SIGNS
The Leaf:-The histological symptoms and signs of the ring
spot disease have previously been described by van Breda de
Haan(56), Wakker and Went(58) and Butler(13), special em-
phasis in all cases being placed on signs of Leptosphaeria sac-
chari, which was considered the cause of the pathological con-
dition. In making a study of these symptoms, the writer used
material collected in the field as well as artificially induced spots
by infecting leaves, sheaths and stems with a pure culture of the
causal organism under controlled conditions. Material was cut
both free-hand in the living condition and by means of a micro-
tome, after fixation and imbedding in paraffin by the usual
methods. The staining schedule of Jackson(33), Delafield's
haematoxylin and safranin and Haidenhain's iron-alum haema-
toxylin proved the most valuable in histological studies.
The causal fungus was found to enter either through stomata,
or directly through the wall of the epidermis at any point. Should
the mycelium enter at the juncture between two cells, it passes
down between them and later goes directly through the walls of
the sub-epidermal tissue. It often happens, however, as shown
in Fig. 9, that the mycelium passes directly through the char-







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 11

acteristically thin epidermal wall of the colorless motor cells
which are adjacent to and partly surrounding the spongy paren-
chyma. These large cells, according to Artschwager(4) have
only peripheral protoplasm and a watery sap. The walls and
contents of the cells soon turn brown and as the fungus invades
the internal tissues, it is seen that the chlorophyll-bearing paren-
chyma surrounding the chlorophyll-bearing bundle sheath of the
vascular bundle is the first to lose its green color and turn brown.
Sections through the Burgundy-colored plesionecrotic margin of
the spot show the chloroplasts in the chlorophyll-bearing paren-
chyma to have completely lost their color and the cellular contents
to be much broken up and disintegrated into a Burgundy-colored
mass. Even after the chloroplasts in the chlorophyll-bearing
parenchyma have lost their normal color and turned brown, how-
ever, the much larger green plastids found in the almost spherical
cells of the chlorophyll-bearing bundle sheath are still pale green.
The large xylem vessels and phloem tissues now turn brown and
finally all the cells throughout the thickness of the leaf show their
contents to be disintegrated and brownish in color. With the
death of the cells, the walls collapse and the tissues shrivel up,
so that the diameter of the leaf in the center of the holonecrotic
zone may be less than one-half that of the healthy leaf.
In controlled inoculation experiments, using only a pure cul-
ture of the pathogene, it was found that after such a short period
as 24 hours under favorable conditions of temperature and mois-
ture, the mycelium had penetrated into practically every cell of
the leaf tissue except those of the vascular bundles. The typical
smoky or bronze brown mycelium could be easily traced in its
passage between and through the various cells. In its passage
through cells, it frequently had a very large diameter (see Fig. 9).
It was noted, however, that for the distance of one and some-
times two or more vascular bundles laterally, no mycelium could
be found, although the chlorophyll-bearing parenchyma was very
pale green and apparently dying from the effects of the fungus.
In field-collected material, however, the cells of the chlorophyll-
bearing parenchyma which were pale green in color, just out-
side of the Burgundy-colored plesionecrotic zone, were found to
be frequently invaded by a very fine, hyaline mycelium, distinct
from the rather large, smoky thallus of the true parasite. As
will be shown later, the fine hyaline mycelium is that of a fungus
which is not capable of penetrating healthy leaf tissue, but can







Florida Agricultural Experiment Station


only enter the zone of the lesion which has been killed or weak-
ened in advance by the true pathogene.
As in the case of a number of different leaf spots investigated
by Cunningham(22), no definite cicatrice is formed about the
edge of the lesion.
Transverse sections often show the pycnidia and perithecia of
certain saprophytic fungi imbedded in the leaf tissue, both in the
holonecrotic as well as plesionecrotic zones. Fig. 10 shows the
fruiting bodies of two distinct fungi most frequently encountered,
both forms opening to the upper surface. The perithecial form
is recognized as that of Leptosphaeria sacchari van Breda de
Haan and the pycnidial form that of Phyllosticta sorghina Sacc.
The perithecia of the former fungus were frequently observed
to open to both surfaces of the leaf rather than to the upper sur-
face only as recorded by Butler(13).
The Sheath:-Sections made of natural infections and stained
with Delafield's haematoxylin, show that the mycelium penetrates
directly through the outer epidermis, in many cases at the junc-
ture between cells. After entrance, the fungus behaves much
the same as in leaf infections. It passes between and through
the cells and in the plesionecrotic zone could be seen to cause the
Burgundy discoloration of the cell contents and walls. The
parenchyma surrounding the vascular bundles shows the contents
of the cells to be much disintegrated. Later, with the death of
the cells within the vascular bundles, all the cells throughout the
thickness of the sheath are seen to be much shrivelled and col-
lapsed. The thickness of the tissue in the holonecrotic region
becomes considerably less than that in the normal sheath.
With artificially controlled infections, it was found as in the
case of leaf infections that the fungus kills the cells for some
distance in advance of the thallus. This outer zone of damaged
cells was also found to be permeated with very fine, hyaline
hyphae in field-collected material. The cross-section of straw-
colored sheath spots frequently shows the fruiting bodies of
P. sorghina Sacc. although the perithecia of Leptosphaeria sac-
chari are occasionally met with.
The Stem:-Thin sections of field-collected material show
clearly that the fungus enters directly through the epidermal
wall. After entrance, it passes down between and through the
cells of the hypodermis, but could not be traced beyond the ligni-
fled cortical sheath. The cell walls and contents of the attacked







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 13

tissues turn bronze brown. At the outer edges of lesions, the
presence of a very fine hyaline mycelium was also observed as
in the case of leaf and leaf-sheath lesions. The presence of
pycnidia of P. sorghina Sacc. in stem lesions is also very common.
Signs of the causal organism cannot be detected with the naked
eye. With microscopic aid, however, especially after lesions on
leaves, leaf-sheaths and stems have been incubated in a damp
chamber for a day or two at temperatures between 22-24oC., the
typical smoky conidiophores and spores can be seen, usually
associated with the discolored plesionecrotic zone. The spores
are large, several-celled and slightly curved. Further, by dehy-
drating and clearing the tissues of lesions, typical much-branched
and closely septated, smoky or bronze brown mycelium charac-
teristic of the parasite in culture can be often seen when viewed
from the upper surface.

ETIOLOGY

REVIEW OF THE LITERATURE
Van Breda de Haan(56) in 1892 was the first investigator to
publish the results of study concerning the cause of the ring spot
disease. He records two fungi as being associated with the
spots-Leptosphaeria sacchari and a species of Dematium which
is regarded as being possibly the conidial form of Leptosphaeria.
Proof of a genetic connection between these two fungi is not fur-
nished. Forms of the conidial fungus were obtained in culture
and inoculation experiments were partly positive and partly
negative. Admission is made that perithecia were not always
found in spots and also that a perithecium-bearing culture was
not secured for inoculation trials. Although the title of van
Breda's paper ("Ring spot disease of cane leaves caused by
Leptosphaeria sacchari n.sp.") indicates that the cause of the
disease was established, definite proof of pathogenicity is not
furnished.
Wakker and Went(58) in their book published in 1898, also
claim that the ring spot disease is caused by Leptosphaeria sac-
chari van Breda. They mention having found a species of Acro-
thecium sometimes in the dead spots, undoubtedly the same fun-
gus as noted by van Breda de Haan under the name of Dematium
sp., but state that this conidial form is not in any way connected
with Leptosphaeria and produces an entirely different kind of






Florida Agricultural Experiment Station


Fig. 2.-Typical eye spot and ring spot lesions on sugarcane x sorghum
hybrids. A, F. 31-30 (POJ. 2725 x Kansas Orange sorghum); B, F. 31-8
(POJ. 2725 x Texas Seeded Ribbon sorghum).

mycelium. No conidial form of Leptosphaeria was found and,
although these authors claim to have grown this fungus in agar
cultures, no spores were ever observed for positive identification.
In summarizing the results of their observations and experiments,
these authors state that although infection experiments had not
been performed as yet, it is beyond doubt that the spores of Lep-
tosphaeria sacchari, when they come in touch with the young
leaves of sugarcane, cause, by penetration of the germ tubes, a
local necrosis of the leaves called ring spot disease.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 15

In 1905, Horne(32) described the ring spot disease from Cuba
under the name of "la enfermedad mancha de ojo," or eye spot
disease. He states that the malady is due to Leptosphaeria sac-
chari, but gives no experimental proof of this contention.
In 1906, Butler(12) recorded the ring spot disease from Bengal.
He described a conidial form which agrees with that of Dematium
by van Breda de Haan(56) and Acrotheciunm by Wakker and
Went(58), but made no attempt at classifying the same. State-
ment is made that this conidial fungus is capable of infecting
leaves by entering directly into the cells and that it is the more
important form directly concerned in the attack, the Lepto-
sphaeria stage being the "resting" form. No proof of the genetic
connection between the conidial and perithecial forms is given.
In 1909, Cobb(17) in Hawaii also described the ring spot dis-
ease as being due to Leptosphaeria sacchari, the conidial form
being of the same type as described by van Breda de Haan(56)
and Butler(12).
Later, however, in 1918, Butler(13), after reviewing the work
done by other investigators, rather leans toward the conclusion
that Leptosphaeria sacchari is the true cause of ring spot, the
conidial stage previously mentioned as being the chief agent in
the spread of the disease being considered to be merely of acci-
dental occurrence on the spots.
Subsequent mention of the ring spot disease in the literature
invariably refers to Leptosphaeria sacchari as being the cause,
a contention obviously having no sound experimental background
after weighing the evidence presented by various investigators
from the time of the original description of the malady in 1890.

MICROORGANISMS FOUND ASSOCIATED WITH THE DISEASE
EXPERIMENTAL PROCEDURE
Typical ring spots were collected in different localities and on
different varieties of sugarcane. Portions were imbedded in
paraffin for histological study, while other portions were used for
free-hand sectioning and the isolation of associated micro6rgan-
isms. Dried herbarium material was also preserved.
For isolation studies, "Difco" corn meal agar and glucose
potato agar were the standard media employed. The usual
method of superficial sterilization of the tissue, taken mainly
from the outer edge or plesionecrotic zone of diseased spots, by







Florida Agricultural Experiment Station


means of alcohol, 1-1,000 mercuric bichloride and thorough wash-
ing with sterile distilled water was employed previous to planting
such tissue in sterile perti dishes, freshly poured to the nutrient
agar. Only an instantaneous immersion of the tissue in the dis-
infectant was possible, since prolonged treatment invariably
resulted in sterile cultures. After isolation of pure cultures by
the tissue planting method, monosporous cultures were prepared
from these either by the method devised by Keitt(36) or
Mutch(45). The monosporous cultures were then employed for
inoculation and other studies. In order to secure single-spore
cultures of Leptosphaeria sacchari, typical lesions having the
fruiting bodies of this fungus in the holonecrotic zone were cut
out and stuck to the cover of the petri dish in an eccentric position
by means of a drop of melted nutrient agar. After pouring the
plate, the cover was rotated very slowly so that a complete revo-
lution was made in half an hour. By this means, ascospores
could be readily isolated and grown in pure culture.
The organisms isolated were grown mainly on corn meal agar
and glucose potato agar, although sterilized cane leaves with a
few drops of distilled water in the bottom of the tube proved an
unusually good medium for propagation and study.

RESULTS OF ISOLATION EXPERIMENTS
From 104 tissue plantings made from leaf spots on the vari-
eties S. C. 12-4, B.417, Otaheite, CP.27-139, Badila, D.74,
Ba.11569 and POJ.2725, Helminthosporium ocellum was isolated
19 times and it was secured in every batch of material plated.
The number of times isolations were successful in the case of
various other organisms was as follows: Phyllosticta sorghina 59,
Nigrospora sp. 44, Leptosphaeria sacchari 21, Spondylocladium sp.
7, Alternaria sp. 1, and a yellow motile bacterial organism 1.
Isolations from ring spots on the leaf sheath were made in the
case of the varieties POJ.2725, Ba.11569, CP.27-139 and D.74.
Twenty-three tissue plantings yielded Helminthosporium ocellum
11 times, its presence being noted in each batch, P. sorghina 14
times and Nigrospora sp. 5 times.
In the case of stem lesions, only the very susceptible varieties
B. 417 and D. 74 were used. From 28 tissue plantings, H. ocellum
was secured from each batch. P. sorghina was also isolated from
each lot of material studied and appeared to be almost constantly
associated with the true pathogene.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 17

An examination of the results of the isolation trials from a
great many typical lesions on leaf, leaf-sheath and stem, brought
out the fact that Helmiinthosporium ocellum Faris was isolated
from every batch of material used for study. Phyllosticta
sorghina Sacc., Spondylocladium sp., Alternaria sp., Nigrospora
sp. and Leptosphaeria sacchari van Breda de Haan were also
found associated with typical lesions, but P. sorghina and Nigro-
spora sp. were most frequently met with.
In certain instances, H. ocellum was known to be present on
the lesions by microscopic examination, yet the technique used
for isolation failed to recover the pathogene, undoubtedly due to
the temperature of incubation which favored the activities and
rapid growth of the associated organisms, especially P. sorghina.
Leptosphaeria sacchari, although frequently associated with
the disease on the leaves, was not always present and also was
not met with as frequently as P. sorghina. Further, it has not
been encountered in isolation trials from the stem, whereas P.
.-r,,' 1i;, was very often found on lesions occurring on all parts
of the host.

INOCULATION EXPERIMENTS WITH PURE CULTURES
ON SUGARCANE LEAVES
Pure cultures of sugarcane were prepared similar to the
method described by Faris(26). Instead of employing cylinders,
however, which have a rather small capacity for the development
of cane leaves, cuttings previously sterilized in either 1-1,000
mercuric bichloride or calcium hypochlorite solution as used by
Wilson(61) were transferred without rinsing to the culture jar
described herewith. This culture jar consisted of an ordinary
desiccator with bell jar fitted to the top instead of the usual cover.
The base was filled with soil soaked in Knop's solution and the
top of the jar fitted with glass tubing plugged with cotton at the
ends to provide for passing fresh filtered air through the culture
every two or three days to imitate nearly normal growing atmos-
pheric conditions. The whole culture jar was then sterilized
in the autoclave for two hours at 20 pounds steam pressure.
After removal of the jar from the sterilizer, the cork and tubing
joints were sealed with hot paraffin, while the ground glass joint
between the bell jar and desiccator was sealed with vaseline.
The transfer of cuttings was made under a hood and the cuttings
were not covered with sterile soil, this being found to be entirely







Florida Agricultural Experiment Station


unnecessary for good germination and growth. In passing air
through the culture jar, two wash bottles were employed filled
with 1-1,000 mercuric bichloride solution for the air intake, the
jar being connected with an ordinary water pump. In order to
make inoculations, the plant culture was removed to the hood, the
bell jar removed and the leaves either atomized with spores sus-
pended in sterile distilled water, or pieces of the agar or cane-
leaf culture were transferred aseptically to the leaf surfaces.
After inoculation, the leaves were finally atomized with sterile
distilled water before replacing the bell jar cover. Cultures were
grown in the greenhouse with partial shade or near a window of
the laboratory, where they could receive direct sunlight during
a part of the day.
The cultures of microorganisms used for inoculation were
those secured in the isolation trials and included Helmintho-
sporium ocellum, which had already been shown to be pathogenic
on cane leaves by Faris(26) and regarded by him as being the
cause of a distinct malady known as eye spot. Monosporous
cultures were prepared of Phyllosticta sorghina Sacc. by means
of the method described by Mutch(45), while the method of
Keitt(36) was found suitable for preparing similar cultures of
Spondylocladium sp., Helminthosporium ocellum and Lepto-
spaeria sacchari. Cultures of the last named fungus were also
prepared directly from the leaf by isolating discharged ascos-
pores in petri dishes as previously described. The species of
Nigrospora was not secured in monosporous culture, although
pure cultures were easily secured. Young, vigorous cultures
from one to two weeks old were always employed for inoculation.
In all, 145 inoculations of L. sacchari were made on the vari-
eties S. C. 12-4, D. 74, B. 417, CP. 27-139 and Otaheite; 21 of
Spondylocladium sp. on Otaheite and B. 417; 50 of Nigrospora
sp. on Otaheite and B. 417; 73 of P. sorghina on D. 74, B. 417,
Otaheite, S. C. 12-4 and CP. 27-139; 22 of the yellow Bacillus sp.
on B. 417 and Otaheite and 38 of Alternaria sp. on D. 74 and CP.
27-139. No positive infections were secured from any of the
above. Of 135 inoculations of H. ocellum made on D. 74, B. 417,
CP. 27-139, Otaheite and S. C. 12-4, however, all proved positive,
the incubation period ranging from 14 to 24 hours.
In addition to using portions of agar culture on the leaves, the
latter were sprayed with spores of P. sorghina and H. ocellum
in several trials. In all cases, H. ocellum gave countless numbers
of infections, while P. sorghina gave only negative results.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 19

In all inoculation experiments, two control plants were sprayed
with sterile distilled water and held under similar conditions to
those inoculated. In no instance did any of these control plants
become infected.
In numerous cases, H. ocellum was recovered in pure culture
from artificially reproduced lesions.

ON LEAF SHEATHS AND STEMS
Because of the difficulty of growing sugarcane in pure culture
to secure normally developed full-sized leaf sheaths and stems,
most of the experiments reported were made with apparently
healthy full-sized sheaths and stems of susceptible varieties col-
lected in the field. The sheaths and stems were rinsed in tap
and distilled water and placed in glass cylinders 25 cms. long x 50
mms. wide, fitted with rubber corks and previously sterilized
with 1-1,000 mercuric bichloride. About an inch of distilled
water was placed in the bottom of the cylinder to supply moisture
and prevent withering.
After placing selected material in the cylinders, it was incu-
bated for 4 days prior to inoculation in order to detect the devel-
opment of any incipient spots. Those developing lesions on
incubation were discarded. Two control sheaths and stems were
always held without inoculation throughout each trial.
Of 45 inoculations of P. sorghina on the leaf-sheaths of CP.
27-139, D. 74 and Otaheite and 33 on the stems of the former two
varieties, none proved positive. Similarly, 23 inoculations of
Nigrospora sp. on the leaf sheaths of CP. 27-139 and D. 74; 19 of
Alternaria sp. on leaf-sheaths of Otaheite and B. 417 and 15 of
the same fungus on the stems of D. 74 and CP. 27-139, all proved
negative. On the other hand, 38 inoculations of H. ocellum on
leaf sheaths of CP. 27-139 and D. 74 and 33 on stems of CP.
27-139, D. 74 and Ba. 11569, all proved positive. The incubation
period of H. ocellum on the sheaths varied from 18 to 36 hours
and on stems 49 to 96 hours.
All of the trials recorded above were made with field collected
material. However, two pure culture plants, such as were used
for leaf inoculations, were employed for similar trials on the
sheath. In all, 20 inoculations of the green sheaths were made
with H. ocellum. All proved positive in three days.
Artificially produced sheath and stem lesions by H. ocellum
were used for re-isolation of the pathogene. The fungus was







Florida Agricultural Experiment Station


recovered in numerous instances. The control sheaths and
stems also remained healthy throughout the period of the trials.
It became very clear as a result of numerous inoculation experi-
ments under controlled conditions that of all the organisms iso-
lated, only Helminthosporium ocellum was pathogenic. The type
of lesion formed by this organism on the leaves, however, was
typical of that of eye spot which had been well described and
figured by Faris(26). The problem presented, therefore, was
how to account for the characteristic ring spot lesions on leaves,
sheaths and stems. The results naturally suggested that one or
more of the accompanying saprophytic organisms might be re-
sponsible for the change of eye spot lesions to those of typical
ring spots. It had been established as a fundamental fact that
ring spot lesions always gave rise to more than one organism in
isolation trials, even when the plesionecrotic zone was super-
ficially sterilized and plated out. Among the organisms accom-
panying H. ocellum in ring spots, Phyllosticta sorghina, Nigro-
spora and Leptosphaeria sacchari were outstanding in the number
of times they were isolated.
Before making any inoculations with known mixtures, all the
isolated fungi were first studied physiologically in pairs in plate
cultures so as to gain information as to association effects.
The results of these trials, reported elsewhere in this paper,
indicated that P. sorghina and L. sacchari were both unaffected
in their associations with H. ocellum in culture, while Nigrospora
sp. and H. ocellum were mutually antagonistic. Thus inocula-
tions were first made with mixed cultures of H. ocellum and either
P. sorghina or L. sacchari.
INOCULATION EXPERIMENTS WITH CERTAIN MIXED CULTURES
These experiments were carried out both with field collected
leaves, sheaths and stems in sterilized glass cylinders as already
described, incubating the material for 4 days in each before
inoculation, and with leaves only, using pure culture methods for
growing the host. In all trials, inoculations were made in dupli-
cate series, one series with H. ocellum alone and the other with
the known mixture. Two controls without inoculation were kept
in each series.
Table I gives the results of these inoculation experiments using
known mixtures of isolated fungi.








Bulletin 267, Studies on Ring Spot Disease of Sugarcane 21


TABLE I.-SUMMARY OF
KNOWN MIXTURES OF
ALONE.


Mixture of
Organisms.
A-H. ocellum + P.
sorghina.





B-H. ocellium + L.
sacchari.


C-H. ocelluom + P.
sorghina.

D-H. ocellumn + L.
sacchari.

E--H. ocellum + P.
sorghina.


F-H. ocellanm
saccha ri.


Part
of
Host.
Leaf






Leaf


RESULTS OF INOCULATION EXPERIMENTS WITH
ISOLATED FUNGI COMPARED WITH H. OCELLUM

No. of
Variety Inocula- Results Compared with
Used. tions. H. ocellum Alone.


D. 74 35


D. 74


Sheath CP.
139.

Sheath CP.
139.

Stem CP.
139.

CP.
Stem 139.


22



28

30


17


29


Lesions enlarged very
rapidly, forming large,
irregular-shaped blotches
with ring spot appear-
ance after a week. The
H. ocellum lesions were
typical of eye spots and
were relatively small.
Practically no difference
could be noted in the
mixed cultures as com-
pared with H. ocellum
alone.
Results essentially the
same as with D. 74 leaves
in (A).
Results essentially the
same as with D. 74 leaves
in (B).
Lesions were significantly
larger and soon develop-
ed aluminum colored cen-
ters.
No significant difference
could be observed be-
tween the two series.


DISCUSSION OF RESULTS OF INOCULATION EXPERIMENTS ON
SUGARCANE

An examination of the results shows clearly that H. ocellum,
while it was the only organism isolated from ring spots capable
of producing infections on leaves, leaf-sheaths and stems of
susceptible varieties, yet by itself it failed to form lesions exhib-
iting the complete range of morphological symptoms character-
istic of the disease as it occurs in the field and as commonly
described in the literature. On leaves, the lesions formed by
H. ocellum were identical with those of eye spot.
Of the organisms which were mutually agreeable with H.
ocellum in culture, only Phyllosticta sorghina was found to be
effective in rapidly enlarging the eye spot lesions on various parts
of the host and changing their appearance to those of ring spots.
Fig. 3 shows the typical difference occurring in parallel inocu-
lations on leaves of Otaheite with H. ocellum alone (A) and mixed
with P. sorghina (B), even after the brief period of only five days.







Florida Agricultural Experiment Station


ISOLATION AND INOCULATION EXPERIMENTS WITH HOSTS
OTHER THAN SUGARCANE
Although repeated attempts at the isolation of H. ocellum from
typical ring-spot-like lesions on Holcus sorghum, Panicum dicho-
tomiflorum, Panicum maximum and Panicum sp. had failed, the
writer determined
to try inoculations
of this sugar-
cane pathogene
on Holcus sor-
ghum and also on
a number of inter-
generic hybrids
between Holcus
and Saccharum
which he had pro-
duced, since a
number of these
latter had shown
typical eye spot
and ring spot le-
sions as exhibited
in Fig. 2, and H.
ocellum had been
isolated several
times from them.
The importance
of these studies
is obvious when
considered in the
light of extended
host range and
the inheritance of
B resistance or sus-
ceptibility to the
Fig. 3.-A, leaf of Otaheite cane inoculated with disease from sor-
Helminthosporium ocelluh alone, after five days at ghum and sugar-
room temperature (28-30'C.). B, leaf of Otaheite
cane inoculated with H. occllin and Phyllosticta cane respectively
sorghina, after five days. Both A and B were inocu- in the interge-
lated at the same time and held under identical con-
ditions. neric hybrids







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 23

INOCULATIONS ON SORGHUM LEAVES
These experiments were carried out with healthy field-col-
lected leaves of the varieties Texas Seeded Ribbon and Kansas
Orange, using sterilized glass cylinders as already described and
incubating the leaves for 4 days prior to inoculation. Also, an
additional series of trials were made with young sorghum plants
grown under aseptic conditions from seed superficially sterilized
with calcium hypochlorite and reared in culture jars used for
sugarcane. Inoculations were made both by atomizing spores of
H. ocellum and using portions of agar cultures on the leaves.
Two control jars with young plants and cylinders with leaves
were used in each case for comparison, the same being sprayed
with sterile distilled water instead.
Results
Repeated trials with both varieties, on mature leaves and those
of young plants, failed to show any infections even after two
weeks.
INOCULATIONS ON LEAVES OF CERTAIN Saccharum Holcus
HYBRIDS WITH H. ocellum
These trials were conducted only with fully grown leaves of
plants, using sterile cylinders as in the case of sugarcane and
sorghum. The hybrids F. 31-8, F. 31-29 and F. 31-30 were used,
the first mentioned being the cross POJ. 2725 x Texas Seeded
Ribbon and the latter two POJ. 2725 x Kansa. Orange. That
these are genuine hybrids is shown by the marked sorghum
characters exhibited. Two controls were used in each case and
the experiments were repeated twice.
Results
In every instance numerous infections were secured after 24
hours. Reisolation of the pathogene was readily accomplished
in several instances. The control leaves showed no symptoms of
infection after 10 days.
DISCUSSION OF RESULTS
While the two varieties of Holcus sorghum, Texas Seeded Rib-
bon and Kansas Orange, have shown immunity to H. ocellum
under the conditions of the experiments, it is not known whether
other varieties would react similarly to this pathogene.
That several intergeneric hybrids of Saccharum x Holcus
should prove susceptible to the disease is of considerable interest,
especially since both male parents (sorghum) were immune.
However, only hybrids found susceptible under field conditions
were used in the inoculation trials in order to definitely establish







Florida Agricultural Experiment Station


the pathogenicity of the sugarcane fungus. Of equal significance
is the fact that certain promising hybrids from an agricultural
and commercial standpoint, e. g. F. 31-61 (POJ. 2725 x Kansas
Orange), have so far shown high resistance to the disease after
field exposure on Okeechobee muck soil for one year. Although
a very large number of hybrids have not been under experimental
observation, it has definitely been established, nevertheless, that
the above intergeneric hybridization work is of value from the
standpoint of breeding for resistance to the disease.

THE PATHOGENE
NAMES, HISTORY AND CLASSIFICATION
The pathogene belongs to the group Fungi Imperfecti, Order
Moniliales, Family Dematiaceae, Section Phragmosporae and
Genus Helminthosporium. Although the fungus has been grown
on a wide range of culture media and kept under observation for
more than two years, a perfect stage has never been observed.
According to Faris(26), who in 1928 gave the first description
of the species into which the fungus falls, the spore and conidio-
phore measurements agree fairly well with those given for Cer-
cospora sacchari by van Breda de Haan(56). He pointed out
that although Johnston and Stevenson(35) in 1917 made Helmin-
thosporium sacchari Butler a synonym of Cercospora sacchari
van Breda de Haan, the spore and conidiophore measurements of
the two fungi did not agree and further, no comparative study of
the two organisms was made to properly settle the question. The
same author also proposed the name Helminthosporium ocellum
n. sp. for the fungus and showed that it was distinct from H. sac-
chari Butler and H. stenospilum Drechsler.
The fungus which the writer has found as being the primary
cause of sugarcane eye spot as well as ring spot in Florida was
studied in monosporous culture for the purpose of identification
and comparison with the morphological description by Faris.
Conidiophore and spore measurements were made from typical
lesions artificially produced on cane leaves. The conidiophores
varied in length from 70.3 to 277.5 7t (average of 30-158.4 /) ;
the width varied from 3.7 to 9.25 y (average 6.3 y) and there were
from 3 to 8 septa (average 5.7). The spores varied in length
from 24.1 to 92.5 y (100 measurements average 58.7 /) ; width
varied from 11.1 to 16.7 y (average 14.0 y) and there were from
3 to 9 septa (average 6.7). The color of the spores and conidio-
phores and the marked bulbous base of the conidiophores, etc.,
were identical with that found by Faris.






Bulletin 267, Studies on Ring Spot Disease of Sugarcane 25

Even though distinct differences in average conidiophore and
spore measurements occur as well as color of spores in the three
well defined species of Helminthosporium on sugarcane, adequate
comparative information on the physiology of the various species
is lacking. For the purpose of this paper, only the physiology
of H. ocellum has been undertaken as a basis for further com-
parative data on other species and to definitely establish additional
characteristics of the pathogene as an aid in proper classification.

LIFE HISTORY
1. Inoculation:-The entire life cycle of H. ocellum is spent in
the tissues of the host which is attacked, as far as can be deter-
mined. The disease of the ring spot type has been found to
appear on the young leaves about two or three months after the
cane germinated, either as plant or ratoon crop. Such young
cane may occur in the usual scheme of planting and harvesting
at present, anywhere from December to June in Florida. As a
rule, however, serious outbreaks do not occur until the advent of
cool weather in October, November and December, accompanied
by very heavy dews, which frequently remain on the leaves until
noon in the Everglades region during this period. At this time,
the disease is especially abundant on the outer whorl of leaves,
the leaf sheaths and stems of susceptible varieties. Badly at-
tacked standing cane adjoining young plant or ratoon fields are
common sources of primary infection. However, the fact that
the fungus occurs on lesions on the stalk and also that seed pieces
having lesions when planted in isolation or even germinated
under a bell jar culture without sterilization will frequently show
leaf infections, is evidence that the seed piece is a common source
of inoculum and undoubtedly has been instrumental in carrying
the disease from one country to another in the past.
In the case of ratoon cane, the bed of trash in the field consist-
ing largely of dead leaves, leaf sheaths, tops left by the cutters
and even dead and dying stalks, is a very frequent source of
inoculum. Dead leaves, sheaths and portions of decaying stalks
having eye spot or ring spot lesions, on being placed in a damp
chamber and kept at temperatures between 22TC. and 25"C. will
often show conidiophores and conidia of the pathogene on the sur-
face after 24 to 48 hours.
It is evident that spores borne on the long conidiophores are
easily blown by the wind and carried by splashing rain to other
sugarcane leaves, sheaths and stems.







Florida Agricultural Experiment Station


Whether or not insects are instrumental in carrying spores
from one part of the host to another or from one plant to another,
has not been definitely determined. Dead leaves and even living
seed pieces without surface sterilization, when placed in a damp
chamber, soon show large numbers of mites associated with them.
Evidence that these animals travel in nature from decaying
material to the living parts of the host is available from numer-
ous observations of germinating seed pieces, when the green
leaves have frequently been observed to have mites on them.
Primary infections, once started on the living host, produce
abundant conidiophores and conidia for secondary infections
from the developing lesions. These spores can readily be blown
by the wind or washed by driving rain to other leaves, leaf sheaths
and stems, or may even be accidentally carried by insects crawling
over the
spore-bear-
ing lesions.
As a result
of numerous
inoculations,
it was found
that infec-
tion took
place just as
readily
whether the
inoculum
was placed
on the upper
or lower
surface of
leaves. One
of the most
essential
conditions
for infec-
tion, how-
ever, was
found to be
the presence
of free mois-
ture on the
Fig. 4.-Portion of Otaheite leaf 14 hours after inoculation
with spores of H. ocellum. surface.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 27

2. Incubation:-The period of incubation was found to vary
from about 14 hours to two days, depending upon temperature
and moisture conditions and the age of the leaf. On the whole,
young leaves were infected slightly sooner than physiologically
mature ones. Fig. 4 shows typical lesions on young leaves after
14 hours.
The germination of spores is shown in Fig. 7. Under suitable
temperature and moisture conditions, germ tubes are produced
in about two hours in distilled water, but in 1% sucrose solution,
they appear much sooner. Spores usually germinate from both
ends, but occasionally they put out a tube from one end only. The
anastomosing of germ tubes was commonly seen.
Penetration was studied in the case of several varieties, includ-
ing Otaheite, D. 74 and CP. 27-139. Spores from a young agar
culture were placed on unwounded leaves, leaf sheaths and stems,
incubated for various periods (at room temperature) ranging
from 14 hours to several days. Free-hand cross sections were
then made through the infection courts or thin sections were cut
parallel to the surface, mounted and examined direct with a com-
pound microscope. It was clearly seen in the case of leaf material
that the fungus can enter directly through the walls of the epi-
dermis as shown in Fig. 9 or it may enter through stomates.
Passage through the wall of a short epidermal cell is shown in
Fig. 8. After entrance, the mycelium passes inter and intra-
cellularly. Fig. 9 shows the thick mycelium passing directly
through certain cells. The intercellular mycelium is often much
smaller in diameter than that passing through cells. The mode
of entrance into leaf sheaths and stems was found to be essentially
the same as in the case of the leaf. In its passage through the
epidermis of the motor cell of the leaf shown in Fig. 9, it is seen
that the thickness of the mycelium is practically unaltered in the
cuticle layer. On reaching the primary cell wall beneath, how-
ever, the growing point becomes enlarged and a characteristic
peg-like strand passes through the wall as recorded in the case
of pathogenes which are regarded as effecting passage through
cell walls at least partly through mechanical pressure.
3. Infection:-The infection stage on the various parts of the
host lasts throughout the age of the part affected. In the case of
leaves and sheaths, spots of the eye spot or ring spot type may
exist for two months or longer, depending on the age of the part at
the time of infection and the rate of growth of the host as influ-
enced largely by the period of the year. With the shedding of






Florida Agricultural Experiment Station


leaves and sheaths, the fungus may live saprophytically on the
decaying tissues. Stem infections persist until the cane is har-
vested, which may occupy a period of more than a year from the
time of the initial infection.
Under favorable conditions, fruiting bodies may appear in a
few days after the lesions are formed and may continue to form
as long as the lesion exists and food is available.
The infection stage becomes evident with the formation of a
peculiar watery appearance of the infection courts. Sections
show that the cuticle as well as cell walls of sub-epidermal tissue
are colored a characteristic pink in the early stages of infection.
Later, these walls become Burgundy or bronze brown in color.
It is evident that the fungus damages the cells in advance
because it was impossible to find the mycelium of the parasite in
the pale green cells of the chlorophyll parenchyma adjoining the
Burgundy colored plesionecrotic zone with its dying and dead
cells containing mycelium.
The pink walls of the epidermis and other cells injured in ad-
vance by the parasite are undoubtedly weakened so that P. sor-
ghina, the frequently associated saprophyte, can readily pene-
trate and aid in the destruction. Mixed culture inoculations
show clearly that such weakened tissues in advance of the true
parasite have very fine hyphae passing into and through the cells.
In the mixture referred to, the other fungus was Phyllosticta
sorghina. Thus the histological phenomena appearing in the
case of natural lesions commonly called ring spots, can be repro-
duced at will by using mixed cultures containing H. ocellwm and
P. sorghina. Owing to the importance of the latter fungus in
changing typical eye spots during the infection stage to ring
spots, it was thought desirable to determine the classification and
identity of the fungus, its general physiological characters and
the host range.
Because of the frequent occurrence of L. sacchari in the late
infection stage and the opportunity afforded during the course
of the work for life history and certain physiological studies on
this fungus, interesting data on it are recorded in other sections.

TWO ASSOCIATED SAPROPHYTES
NAMES, HISTORY, LIFE HISTORY AND CLASSIFICATION
1. Phyllosticta sorghina:-This fungus saprophyte belongs to
the Group Fungi Imperfecti, Order Sphaeropsidales, Family
Sphaerioidaceae, Section Hyalosporae.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 29

Although the fungus has been grown on a wide range of cul-
ture media and kept under observation for two years, a perfect
stage has never been observed.
The first description of the species into which the fungus falls
was by Saccardo(49) in 1878, although the complete volume did
not appear until the following year. The first description was
from Sorghum vulgaris, but the same author(50) later mentioned
its occurrence on Sorgho halepensi. Allescher(2) also gave a
description of the organism, but showed the spore measurements
to be slightly different from those of Saccardo(49).
Not realizing that Saccardo(49) had already described the
same organism from Sorghum spp. several other workers have
described the fungus under different specific names as shown by
comparative cultural studies made by the writer. Spegazzini(52)
in 1896, published a description of Phyllosticta sacchari Speg.
(n. sp.) occurring on the leaves of sugarcane which falls readily
under the description of P. sorghina Sacc.
Young(62) in 1915 definitely records the presence of Phyllos-
ticta sacchari Speg. on Saccharum in Porto Rico and also de-
scribed Phyllosticta panici, sp. nov. from the leaves of Panicum
maximum, in the same island. Comparative cultural studies by
the writer have definitely shown that these organisms are iden-
tical with P. sorghina Sacc.
Caum(14) in 1921 described Phyllosticta hawaiiensis from the
sheath and stem of Saccharumn lici. r., i,, in Hawaii. As already
mentioned in this paper, the same organism occurs on leaf, leaf
sheath and stem of sugarcane in Florida associated with the
parasite Helminthosporium ocellum Faris and is properly to be
referred to Phyllosticta sorghina Sacc.
Ellis and Everhart(25), in recording the species of Phyllosticta
in North America up to August, 1900, do not mention any species
occurring on Saccharum, Zea, Sorghum or Panicum. In 1926,
however, Stevenson(53) records the occurrence of P. sorghina
Sacc. on Holcus ( if,, ,,.'. H. halepenis and H. sorghum in Russia,
India, Italy, Portugal and Argentina. He also mentions that the
fungus has been reported from Texas, but the exact species on
which it occurred, or whether it was considered parasitic or
saprophytic is not mentioned. However, this is the first record
which the writer has found of the occurrence of this organism
on the North American continent.
Judging from the small variations occurring in the enormous






Florida Agricultural Experiment Station


number of species of Phyllosticta which have been described, it
would appear to be impossible to place certain species definitely
on the basis of the morphological characters alone. The neces-


Fig. 5.-Ring spot and eye spot lesions on sheath and lower portions of leaf
blade of D. 74 sugarcane.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 31

sity, therefore, for detailed knowledge of the physiology of the
various species has encouraged the writer to investigate this
phase more fully with respect to P. sorghina so as to furnish a
proper basis for classification within the genus and also give
some information concerning its activities in changing eye spot
lesions to ring spots.
Figures 10, 11 and 12 show typical pycnidia of P. sorghina on
the upper surface of ring spot lesions of sugarcane in Florida.
Pycnidia are found to vary from 44 to 147 v in diameter and
spores 3.4-6.7 x 1.7-3.4 y.
As shown in Fig. 16, the spores on being placed in water or
weak nutrient solution, swell considerably and may reproduce by
budding in a yeast-like fashion. Germ tubes form from one or
both ends and anastomosing is frequent with the young mycelium.
On account of the swelling of spores in liquids and the formation
of secondary bud spores, considerable variation in size is likely to
occur and may account for the variations noted by Saccardo(49)
and Allescherf2) already mentioned in another section.
The same P. sorghina was isolated many times from apparent
ring spots occurring on Holcus sorghum, Panicumn dichotomi-
florium, Panicum max.imumr and Panicum sp. (B.P.I. No. 48154)
in Florida. Inoculation experiments, however, showed that this
organism is not parasitic on these hosts, but apparently occupies
a similar role to these diseases as it does to the ring spot of
sugarcane.
Comparative cultural and morphological characteristics on
different media show clearly that the fungus isolated from the
above-mentioned hosts is identical.
2. Leptosphaeria sacchari:-This fungus saprophyte has al-
ready been classified and described by van Breda de Haan(56)
in 1892 on the basis of the perfect stage among the Ascomycetes.
No proof of a conidial or imperfect stage of this fungus has been
furnished by any previous investigator. Wakker and Went(58)
mention that when grown in agar agar culture, no spores of any
kind could be observed.
Tissue plantings by the writer from lesions having Lepto-
sphaeria sacchari, and single-spore isolations from perithecia by
the method previously described, gave identical cultures. On
glucose potato agar at room temperature, growth is very slow,
the colonies never completely covering the surface and always
becoming much elevated. The consistency is leathery and the
edge of the colony a burnt umber in color, while the center is







Florida Agricultural Experiment Station


much darker. These colonies have in all cases produced on agar
media abundant pycnidia of a species of Phyllosticta (see Fig. 17)
having very similar shape, size and appearance of perithecia of
L. sacchari on the surface of ring spot lesions as shown in Fig. 13.
When freshly isolated ascospores were grown on sterile cane
leaves, the perfect Leptosphaeria stage was secured during the
first two months, but after this period, continued transfer to agar
or cane leaf cultures yielded only the Phyllosticta stage. Proof
that the two stages are connected genetically is furnished by the
fact that cultures secured from single ascospores of the Lepto-
sphaeria stage or pycnospores of the Phyllosticta stage yielded
identical cultures and also because single-spore ascospore cultures
yielded the perfect stage at first in cane leaves when first isolated
and later gave only the Phyllosticta stage.
The Phyllosticta stage agrees well with the description of P.
saccharicola by Hennings(31) on the leaves of sugarcane. That
some confusion exists with regard to this conidial stage is evident
from the observations of Johnston and Stevenson(35). These
authors describe what is apparently the same fungus under the
title of P. sacchari Speg. but correctly state that it is probably
not the same as the latter fungus, which, according to the descrip-
tion by Spegazzini(52) has spores less than half the length of
P. saccharicola and should properly be referred to P. sorghina
Sacc., in the opinion of the writer.
The germination of spores and anastomosing of germ tubes
(from the Phyllosticta stage) are shown in Fig. 15. Culturally,
this fungus is very distinct from P. sorghina and temperature
relations to growth in culture also reveal very significant differ-
ences as illustrated in Fig. 21.


VARIETAL SUSCEPTIBILITY

Because of the fact that the etiology of the ring spot disease
had not been properly understood hitherto, only varieties which
became accidentally infected with Leptosphaeria sacchari as a
saprophyte following the true causal organism, were considered
in susceptibility studies. It is obvious that accurate informa-
tion on varietal susceptibility can only be referred to the rec-
ords of susceptibility to Helminthosporium ocellum Faris which
has now been shown to be the cause of the primary stage of
ring spot, but which is known from the literature as the pri-
mary cause of eye spot. The formation of the ring spot stage






Bulletin 267, Studies on Ring Spot Disease of Sugarcane 33

is considered to
be dependent
mainly on Phyl-
losticta sorghina
Sacc., which may
be followed by
Leptosph a eri
saccharin and
other sapro-
phytes.
Faris(26) has
collected a large
amount of valu-
able data on vari-
etal susceptibility
in Cuba and other
tropical coun-
tries. The writer
has not had an
opportunity to
study compara-
tively all the va-
rieties investigat-
ed by Faris, but
nevertheless in-
formation col-
lected for a pe-
riod of nearly
three years, cov- '3 C
ering several sec-
tions of Florida. Fig. 6.-Ring spot and eye spot lesions on stems of
three varieties of cane-A, B. 417; B, Ba. 11569;
having different c, D. 74.
climatic and soil
conditions, may prove of value to cane breeders and commercial
cane growers in the sub-tropics. Table II gives the results of
combined observations from varietal plots established at Clewis-
ton, Lake Harbor, Belle Glade, Homestead and Lake Alfred.
Observations on plots established at Gainesville and Quincy
proved unsuitable for these comparative studies, owing partly
to the prevailing drought which occurred in these areas during
the growing seasons when observations were made.







Florida Agricultural Experiment Station


TABLE II.-COMPARATIVE SUSCEPTIBILITY OF VARIOUS SUGARCANE VARIETIES
TO RING SPOT (AND EYE SPOT) DUE TO H. ocellam FARIS.

Very Susceptible Varieties Susceptible Highly Resistant
Varieties* But Only Moderately Varieties
Damaged

Otaheite S. C. 12-4 Badila
Louisiana Purple POJ. 2714 POJ. 213
Louisiana Striped POJ. 2725 Co. 213
Cavengire Rayada CP. 27-139t Co. 214
Ba. 11569. POJ. 2883 Co. 281
B. 417. POJ. 36-M Co. 290
B. H. 10-12 POJ. 36 CP. 807
L. 511 CP. 27-108
D. 74 Uba
D. 95 Chunnee
POJ. 2878 U. S. 1694
POJ. 100 F. 29-416
F. 29-382 F. 29-7
H. 109
Crystalina

*Ba, B and BH canes refer to those bred in Barbados, while those with
the insignium D, H and S. C. refer to designations given canes in Demerara,
Hawaii and Saint Croix, respectively.
tThis variety, after extensive tests, proved to be relatively so very resistant
to the disease (as well as other diseases) on "sawgrass" and "willow" lands
in the Everglades that it was rapidly extended in 1933 and now occupies
about 1,000 acres of commercial plantings.
THE PHYSIOLOGY OF HELMINTHOSPORIUM OCELLUM
AND ITS ASSOCIATED SAPROPHYTE, PHYLLOSTICTA
SORGHINA
SOURCES OF ENERGY FOR GROWTH
Only single-spore strains of H. ocellum and P. sorghina were
employed in these studies.
In the case of the parasite, H. ocellum, information on the
physiology with regard to this phase of its nutrition is essential
to a proper understanding of its activities within the host tissues.
Further, as a basis for comparison with other species of the
same genus affecting sugarcane, the data would serve to distin-
guish these forms more definitely.
Because of the very intimate association of P. sorghina with
the causal organism, the physiology of this saprophyte seemed
particularly desirable from the standpoint of having a better
understanding of its activities in making use of substances within
the host tissues which have previously been damaged by H. ocel-
lum. A comparison could also be made of the biological activities
of the two organisms and perhaps some evidence secured of the
nature of mutual relationships existing between them. In addi-
tion, the data would furnish an adequate basis for comparing
species or strains of Phyllosticta morphologically identical, but
probably very distinct in their physiological characters.








Bulletin 267, Studies on Ring Spot Disease of Sugarcane 35

TABLE III.-GROWTH OF H. ocellUm AND P. sorghina (CANE LEAF STRAINS)
COMPARATIVELY ON A MINERAL NUTRIENT SOLUTION WITH DIFFERENT
SOURCES OF CARBON. (AFTER 10 DAYS AT 28-300C.).


Carbon Source

Monosaccharoses
Fructose
Xylose
d-Galactose
Glucose
d-Mannose
Arabinose
Rhamnose
Inosite
Inosite
Disaccharoses
Sucrose
Maltose
Lactose
Trisaccharoses
Raffinose
Raffinose
Polysaccharoses
Dextrine (white)
Starch
Dextrine (yel.)
Inulin
Gum arabic
Cellulose
Primary Alcohol
Ethyl alcohol
Tri-Acid Alcohol
Glycerine
Hex-Acid Alcohols
Mannite
Dulcite
Dulcite
Sorbite
Organic Acids
Citric acid
Tartaric acid
Oxalic acid
Acetic acid

Tannic acid
Formic acid

Salts of Organic Acids
Ammonium lactate
Sodium asparaginate
Amyl acetate
Proteins and their
derivatives, Oils, etc.
Pectin
Peptone
Gelatin
Olive oil
Casein
Blood fibrin


Concen-
tration


2%
2%
2%
2%
2%
2%
2%
0.45%
2%

2%
2%
2%

0.265%
2%

2%
2%
2%
2%
2%
2%

0.5% by vol.

2%

2%
0.254%
2%
2%

9.5%
0.5 and 0.01%
0.5 and 0.01%
0.5 and 0.01%
by vol.
0.5 and 0.01%c
0.5 and 0.01%
by vol.

2%
2%
2% by vol.

2%
2%
2%
2% by vol.
2%
2%


Average dry weight in mgs. of
mycelium from 50 cc. culture.
H. ocellum P. sorghina

152.0 136.0
136.0 167.0
111.0 329.5
109.5 215.5
101.5 140.5
26.5 63.0
10.0 32.5
5.0
141.0

115.0 189.0
100.0 255.0
69.5 153.5

51.5


161.0
113.5
72.0
71.5
30.5
Good growth

10.5

1.5

10.0
0.5


0.0
0.0
0.0
0.0

0.0
0.0

4.5
2.0
0.0


232.0
158.5
108.5
60.5
Good growth
Good growth


371.5

228.5
321.5
131.5
149.0
50.5
Good growth

69.0

197.0

147.5

11.0
6.0

2.0
0.0
0.0
0.0

0.0
0.0


28.0
7.0


264.0
211.0
152.0
255.0
Good growth
Good growth






Florida Agricultural Experiment Station


The fungi were grown on White's modified Richard's solu-
tion(60) with varying sources of carbon, mostly in a 2%, concen-
tration. Growth was measured where possible by dry weight
determinations made by filtering the cultures after growth for
10 days at room temperature (28-30C.) in diffuse light, into
previously prepared and weighed Gooch crucibles and then drying
in an oven at 105C. to constant weight. Notes were also made
of any special characteristics of the various cultures.
The comparative yields of dry mycelium after 10 days are
given in Table III. Special cultural and other characters are
also recorded.
NOTES ON CULTURES OF P. SORGHINA
Marked differences were observed on different media in the
case of P. sorghina. In the majority of cultures growth was
mostly superficial, but in the cultures of cellulose, adonite, man-
nite, dulcite, sorbite and pectin it was both superficial and sub-
terranean. Chromogenesis was evident in the case of the liquid
in many cultures, varying from pale yellow xylosee and lactose)
and flesh (glucose, maltose, sucrose, starch and inulin) to rattan
(ethyl alcohol) and pink 1T (glycerine). The peptone liquid was
dark brown and both the gelatin and casein cultures were nugget
bronze. In the cultures of rhamnose, dextrine (white), gum
arabic, cellulose, adonite, mannite, dulcite, sorbite, citric acid,
sodium asparaginate, olive oil and blood fibrin, after 10 days the
liquid had not altered in color.
On the whole, pycnidia were formed abundantly in all cultures
with the exception of rhamnose, mannite and dulcite which had
only a fair number, glycerine and sorbite only a few and adonite
and citric acid none at all.
The cellulose cultures formed pycnidia at first only where the
solid material protruded to the surface. After 50 days, the sur-
face also appeared somewhat translucent and gelatinous. The
glycerine cultures were also noted to form gelatinous masses in
the bottom of the flasks.
Large crystals containing magnesium ammonium phosphate
were suspended from underneath the fungus mats in the cultures
of peptone, gelatin and blood fibrin after about 12 days. Odor
of ammonia was also distinct and the solution strongly alkaline
to litmus in the gelatin cultures.
The growth in the casein cultures was characteristically mal-
low color, later a Java brown. They were also very malodorous,
but no H2S could be detected.






Bulletin 267, Studies on Ring Spot Disease of Sugarcane 37


I F




\ J -: .












Fig. 7.-Germination of spores of Helminthosporium ocellum in 1% sucrose
solution after two hours at 30C. (X 87.)
NOTES ON CULTURES OF H. OCELLUM
In the case of H. ocellum, none of the cultures developed any
chromogenesis as found in the case of P. sorghina, except in the
case of casein cultures, which, after about two months, assumed
the color of Arab rugby tan. The subterranean portion of the
growth at first was practically colorless, while the superficial
growth was ivy green in color. Spore formation was abundant
in all cultures which made growth.
The peptone cultures of H. ocellum showed large crystals con-
taining magnesium ammonium phosphate hanging from the sur-
face after only 10 days and the cultures were malodorous. No
crystals of this compound were noted in the cultures containing
either gelatin or casein, even after two months, but in the case
of blood fibrin they were very abundant after this extended
period. The gelatin cultures showed no odor of ammonia as did
those of P. sorghina, but the casein cultures were very malodor-
ous.
DISCUSSION OF RESULTS
Striking differences are seen to occur as regards the utilization
of various sources of carbon by the two organisms. For example,
among the monosaccharoses, fructose is preferred by H. ocellum,







Florida Agricultural Experiment Station


while d-galactose is preferred by P. sorghina. Among the di-
saccharoses, sucrose is preferred by H. ocellum, while maltose gave
the greatest growth for P. sorghina. Among polysaccharoses,
white dextrine is preferred by H. ocellum, while starch is pre-
ferred by P. sorghina.
Over a 10-day period, the order of utilization as measured by
dry weights of mats was as follows: H. ocellum-pectin, dextrine
(white), peptone, fructose, xylose, sucrose, starch, d-galactose,
glucose, gelatin, d-mannose, maltose, dextrine (yellow), inulin,
lactose, olive oil, raffinose, gum arabic, arabinose, ethyl alcohol,
rhamnose and mannite (equal), inosite, sodium lactate, sodium
asparaginate, glycerine and dulcite. For P. sorghina-raffinose,
d-galactose, starch, pectin, olive oil and maltose (equal), dextrine
(white), glucose, peptone, glycerine, sucrose, xylose, lactose,
gelatin, inulin, mannite, inosite, d-mannose, fructose, dextrine
(yellow), ethyl alcohol, arabinose, gum arabic, rhamnose, sodium
asparaginate, dulcite, amyl acetate, sorbite, adonite and citric acid.
Good growth was secured by both fungi on cellulose, casein and
blood fibrin, but on account of the nature of these substances dry
weight of the mycelium could not be secured.
The marked utilization of pectin, especially by H. ocellum, is in
keeping with pronounced intercellular growth by both the para-
site and saprophyte in the host as shown by histological studies.
It is significant that while the proteins and their higher deriva-
tives can be utilized readily as sources of carbon by both the para-
site and saprophyte as shown by good growth on gelatin, blood
fibrin, casein and peptone, certain organic acids and salts of
organic acids, e.g. sodium asparaginate, proved to be poor sources
of this element, especially in the case of H. ocellum, possibly due
in part to unfavorable pH of the medium. In fact, of all the
latter substances investigated, citric acid and amyl acetate were
utilized only by P. sorghina and the growth of this fungus on
sodium asparaginate was 14 times better than in the case of
H. ocellum.
The marked utilization of olive oil and glycerine by P. sorghina
as compared with H. ocellum indicates that certain fat com-
pounds and acid-alcohol derivatives in the host tissues are largely
destroyed by the saprophyte.
That ethyl alcohol, a product of the decomposition of glucose,
is utilized by both organisms, is of special interest. White(60)
also found that Fusarium lycopersici could make use of this pri-
mary alcohol in its metabolism and used this fact to explain the







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 39

continued increase in dry weight of the fungus, even after glucose
had disappeared from nutrient cultures containing no other
source of carbon.
The formation of ammonia gas by P. sorghina when gelatin
was used as a source of carbon, and magnesium ammonium phos-
phate in considerable quantities by H. ocellum and P. sorghina
when certain protein compounds and their higher derivatives are
decomposed in the presence of the mineral medium employed, is
worthy of emphasis and will be discussed in connection with the
work on toxic substances produced.
A comparative examination of the physiological activity of
H. ocellum and P. sorghina (see Table III) at room temperatures
(28-300C.) based on the average dry weight of fungus after
growth on identical media for the same length of time and under
similar environmental conditions, employing 25 different sources
of carbon, reveals the fact that in only one instance did H. ocellum
show superiority over P. sorghina and that is in the case of fruc-
tose. The total yield of dry mycelium on 25 media shows H.
ocellum 1,963.0 grams and P. sorghina 3,895.0 grams. Thus it
would appear that under the conditions of the above experiments,
the saprophyte is on the average twice as active physiologically
as the parasite in breaking down and utilizing organic carbon
compounds suitable for their life processes. Since the tempera-
ture factor was in favor of P. sorghina, however, it should be borne
in mind that the result would undoubtedly be very different if
the experiment was conducted at the optimum temperature for
the growth of the pathogene.

THE ENZYMES PRODUCED BY H. OCELLUM FARIS AND P.
SORGHINA SACC.
Since both the parasite and saprophyte were found to be able
to utilize effectively so many sources of carbon in their metab-
olism, it was therefore considered desirable to study certain of
the enzymes produced by them, not only as a confirmative mea-
sure, but also to gain some relative conception as to the activity
of the enzymes produced by the two fungi.
From the results already reported, indications exist that the
following important enzymes are produced, among others, by
both fungi: invertase, lactase, amylase, maltase, inulase, pecti-
nase, cellulase, lipase, erepsin, trypsin and pepsin. It was there-
fore decided to make confirmative tests for the majority of these
and study their activity.







Florida Agricultural Experiment Station


EXPERIMENTAL METHODS
The fungi were grown on White's(60) modified Richard's
solution, but containing 1% glucose instead of 5%. In place of
using plain distilled water, however, a sugarcane leaf decoction
was employed. (Twenty-five grams of fresh cane leaves cut into
small portions, boiled for 30 minutes in 6,500 cc. of distilled water
and then filtered.)
Pyrex Erlenmeyer flasks, each containing 200 cc. of the above
medium, were autoclaved for 20 minutes at 20 pounds pressure.
After incubation
for three days,
.-. they were inocu-
~l ated with a loop-
'* -. ful of spores from
a single-spore cul-
S. .ture of either H.
Socellun or P. sor-
Sghina, obtained
S, from cane leaves.
.. The growth of
both fungi was
S 'allowed to take
Al. place at room
Temperature (ap-
proximately
28'C.). Under
Stithese conditions
which greatly fa-
vored the rapid
growth of P. sor-
ghina as shown
later, in order to
secure approxi-
Smately the same
Fig. 8.-Surface view of D. 74 leaf section made amount of mycel-
parallel to the surface, showing penetration of H. ium from each
ocellum into short epidermal cell and also into a fungus, H. ocel-
stoma. (X 1108.)
lutun was allowed
to grow for 14 days and P. sorghina only 6 days. The mycelial
mats were then removed (each fungus being kept separate),
washed in running water for 15 minutes, squeezed out by hand
and treated with acetone and ether according to the method used







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 41

by Harter and Weimer(29) in their studies with Rhizopus tritici.
The dried mycelium was not stored, but was immediately pow-
dered in a clean mortar without the use of sand and a weighed
quantity used for the following experiments.
Experiment Series 1:-The experiments with the two fungi-
H. ocellum and P. sorghina-were conducted in the same manner.
Samples of powdered mycelium consisting of 0.25 gram were
placed in 125 cc. pyrex Erlenmeyer flasks which had previously
been carefully cleaned and dried. Twenty-five cc. of 1% solution
of sucrose, starch and maltose and of a 1% cellulose* suspension
as well as 25 cc. of 0.5% pectin, were placed in each of two flasks.
Likewise, 25 cc. of distilled water were added to the same amount
of mycelium in
each of four kz
flasks for the de-
termination of
the amount of au-
tolysis. Two con-
trol flasks con-
taining in each
case 25 cc. of each
of the above su- 0 "
crose, starch, mal-
tose and pectin
solutions and cel-
lulose suspension
were held as con-
trols. Five cc. of
toluol were added
to all the flasks as 1
an antiseptic and .
they were held in .
a constant tem-
perature incu-
bator at 40'C. for
24 hours. At the
end of this period,
the whole series
Fig. 9.-Cross-section of D. 74 leaf showing pene-
of flasks were tration of H. ocellum directly through wall of epi-
steamed for 15 dermis of motor cell. Also shows large mycelium of
minutes and their the pathogene within cell adjoining the chlorophyll-
minutes and their bearing parenchyma. (X 1108.)
*The weighed portion of genuine ashless "Whatman" filter paper No. 42
was thoroughly ground in a clean mortar with a small quantity of distilled
water before transferring to the flask.







Florida Agricultural Experiment Station


contents tested for reducing sugars by the general gravimetric
method employing Fehling's solution as recommended by the
Association of Official Agricultural Chemists(5).
For each fungus, the average amounts of reducing sugars pre-
sent in the two flasks minus that formed on the average by auto-
lysis and that in the original solutions are presented in Table IV.

TABLE IV.-THE AVERAGE QUANTITY OF REDUCING SUGARS IN MGS. PER 10
CC. AFTER 24 HOURS AT 400C. RESULTING FROM THE HYDROLYSIS OF CANE
SUGAR, STARCH, MALTOSE, INULIN, PECTIN AND CELLULOSE BY 0.25
GRAMS OF POWDERED HYPHAE OF H. OCELLUM AND P. SORGH1NA (SEPA-
RATELY) IN 25 CC. OF SOLUTION.
Percent Milligrams of reducing sugar
Chemical Substrate Concentra- per 10 cc.
tion H. ocellum P. sorghina
Sucrose 1.0 46.0 83.4
Starch 1.0 18.7 23.7
Maltose 1.0 7.6 16.5
Inulin 1.0 00.0 6.3
Pectin 0.5 00.0 6.3
Cellulose 1.0 00.0 00.0

Experiment Series 2:-The two fungi were grown on the same
medium and at the same temperature as employed for Series 1.
H. ocellum was grown for 27 days, while P. sorghina was grown
for only 14 days. In making the tests, the same amount of dry
mycelium of each fungus was employed as well as quantity of
substrate, preservative, etc., so that the results are comparable,
with the exception of any differences which might arise as a
result of the varying ages of the mycelial mats and the fact that
P. sorghina was grown nearer its optimum temperature than
was H. ocellum.
Erepsin:-Powdered mycelium-0.109 gms. + 25 cc. of 0.1%
peptone + 0.2 gm. thymol in each of two 125 cc. Erlenmeyer
flasks. Two controls were run, using autoclaved mycelium. All
were incubated at 40 C. for 24 hours and the solutions tested for
the Biuret reaction.
Results showed in the case of both fungi only a very faint
bluish color in the case of the cultures with normal mycelium,
while the autoclaved mycelium gave typical reddish-violet colo-
ration.
Trypsin: -Powdered mycelium-0.109 gm. + blood fibrin
(stained with 1% congo red and autoclaved for 15 minutes at 15
pounds pressure) 2 cc. of N/10 Na2CO:. + 1 mg. thymol in each







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 43

of two test tubes. Two controls were run, using autoclaved my-
celium. All were incubated at 40C. for 24 hours.
In the case of both fungi, results showed that the normal
mycelium cultures had a distinct objectionable odor, the fibrin
had lost color and swelled somewhat and the liquid was thick,
opaque and slightly pink. The control cultures had no odor and
showed no visible changes.
Pepsin:-Powdered mycelium -0.109 gm.- blood fibrin (pre-
pared as above) + 2 cc. of 0.2c HC1 + 1 mg. thymol in each of
two test tubes. Two duplicate control tubes prepared using auto-
claved mycelium. All cultures incubated at 40 C. for 24 hours.












Fig. 10.-Cross-section of leaf of CP. 27-139, showing fruit bodies of
both Leptosphaeria succhari (left) and Phyllosticta sorghina (right) in
the same section. (X 136.)

The H. ocellum cultures of normal mycelium became very malo-
dorous, with slight swelling and loss of color. The color of the
liquid was very slightly pink and it was thick and opaque.
No differences could be noted in the cultures of normal my-
celium of P. sorghina and the four control tubes also remained
unchanged. The P. sorghina cultures were incubated for 72
hours without any significant changes being observed.
Lipase:-Trials were conducted both with methyl acetate and
olive oil as substrates. In both cases, care was taken to mix the
powdered mycelium with the subtrate properly before adding
the acid as an activator, since the possibility existed that the
acid might destroy the enzyme as was found in the case of castor
bean lipase by Haley and Lyman(30).
A. Powdered mycelium -0.3 gm. + 10 cc. distilled water + 2 cc.
methyl acetate + 1 cc. of N'30 oxalic acid as an activator 5 cc.
toluol placed in each of two 125 cc. Erlenmeyer flasks. Two du-







Florida Agricultural Experiment Station


plicate cultures prepared with mycelium which had been boiled
for 15 minutes. All incubated at 400C. for 24 hours. After the
addition of 50 cc. of neutral 95% alcohol and 1 cc. of neutral 1%
phenolphthalein solution, each was titrated with N/10 KOH in
95% alcohol.
B. Powdered mycelium -0.3 gm. + olive oil -1.0 gm., these
thoroughly mixed and then 0.6 cc. of 1% acetic acid added. Toluol
1 cc. used as a preservative. Two test tube cultures prepared
as above and two control tubes prepared similarly, but mycelium
which had been boiled for 15 minutes was used instead. All were
incubated for 24 hours at 40C. After the incubation period, the
contents of each tube were carefully transferred to a 125 cc.
titration flask, using 50 cc. of neutral 95% alcohol, 1 cc. of neutral
1% phenolphthalein solution added and the mixture titrated with
N/10 KOH in 95% alcohol.
C. Four tubes prepared exactly as in B (two with normal
unheated mycelium and two controls with boiled mycelium) but
these incubated at 40C. for 43 hours instead.
The results of the trials under A, B and C for each fungus
separately are given in Table V.

TABLE V.-EXTENT OF HYDROLYSIS PRODUCED BY 0.3 GM. OF POWDERED
MYCELIUM OF H. OCELLUM AND P. SORGHINA (SEPARATELY) ON 2 CC. OF
METHYL ACETATE AND 1.0 GM. OF OLIVE OIL.
Normal or Incu- Average cc. of N/10
heated bation KOH used in titration
Exp. Substrate heated hours
mycelium at 40oC. H. ocellum P. sorghina

A. Methyl acetate Normal 24 3.8 9.8
Methyl acetate Heated 24 2.0 2.9
B. Olive oil Normal 24 6.9 4.5
Olive oil Heated 24 3.1 3.4
C. Olive oil Normal 43 8.0 6.0
Olive oil Heated 43 3.0 4.0


DISCUSSION OF RESULTS
Definite confirmative evidence of the presence of the enzymes
invertase, amylase, maltase, lipase, erepsin and trypsin has been
obtained for both H. ocellum and P. sorghina. Pepsin was also
definitely confirmed for H. ocellum, but could not be detected in
the case of P. sorghina. Inulase and pectinase were confirmed
for the latter fungus, but not for H. ocellum. Conclusive evidence






Bulletin 267, Studies on Ring Spot Disease of Sugarcane 45

of the presence of cellulase has not been obtained by the methods
used, although both fungi grew well on media with cellulose as
the sole source of carbon.
The activity of invertase and amylase is very pronounced for
both fungi, especially the former enzyme, and serves to empha-
size the part evidently played by it in causing inversion of sucrose
in various parts of the host.
The enzymes pepsin, trypsin and erepsin no doubt play a sig-
nificant role in the attack on cellular proteins and their deriva-
tives by the parasite. The saprophyte, by secreting the latter


- u '
-1 4.ab~P


0---





I ir~~---


Fig. 11.-Cross-section of leaf of CP. 27-139, showing pycnidium of P.
sorghina opening to the upper surface. (X 590.)

two enzymes, is also able to aid effectively in the destruction and
removal of complex organic substances, thus hastening the rapid
shrinkage and collapse of tissue in the holonecrotic zone of the
original eye spot lesions. The formation of ammonia and its
accumulation in the surrounding medium in the form of mag-
nesium ammonium phosphate is undoubtedly brought about by
the action of the above proteolytic enzymes.
Although P. sorghina made much greater growth on olive oil


ICs
-~ *~
(
V;- "i
i







Florida Agricultural Experiment Station


than H. ocellum under similar environmental conditions, the
lipase secreted by the latter is apparently more active on this
substrate than that of the former, although the reverse is seen
to hold true with the substrate methyl acetate. This latter be-
havior was rather to be expected, however, since H. ocellum failed
to grow at all on amyl acetate as a source of carbon, while P.
sorghina made some growth on this substrate.

THE RELATION OF ASSOCIATED ORGANISMS TO H. OCELLUM
AND TO ONE ANOTHER
During the isolation studies of ring spots on cane leaves,
sheaths and stems, a number of fungi other than H. ocellum
were very frequently found associated with such spots and later
work showed that these were nonpathogenic forms. Among
these were Phyllosticta sorghina, Leptosphaeria sacchari, Nigro-
spora sp., Spondylocladium (Acrothecium) sp. and Alternaria sp.
The latter fungus was encountered so seldom that it was omitted
from the association experiments performed.
The importance of studying the effects of associated organisms
to the growth and development of parasitic microorganisms has
recently been extensively reviewed by Fawcett(27). This author
cites several examples from his own work and from the literature
to show that when certain parasites are inoculated with their
associated saprophytes, definite and marked effects are produced.
In some combinations, lesions are formed more readily and de-
velop more rapidly, while in others, the parasite appears to be
inhibited in its action so that it accomplishes no appreciable
harm. The object of the following experiments was to determine
if possible whether any antagonistic or mutual relationships
existed between the above mentioned fungi. It was also hoped
that the information obtained would prove valuable in suggesting
combinations of fungi in known mixtures for pathogenic studies
and also help to explain the frequent occurrence of certain fungi
together in the same lesions.

EXPERIMENTAL METHODS
It was decided to study the effects at room temperature
(28-30C.) of association of pairs of fungi on potato glucose agar
in petri dishes. After pouring the plates inoculations were made
separately in each hemisphere of the dish with pure cultures of
the two fungi to be studied. Trials were made in triplicate and







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 47

the contour of each colony traced in ink as it developed. All pos-
sible combinations of the above fungi were tried in pairs. The
growth of L. sacchari proved to be so slow that this fungus was
started about a week in advance of inoculating the other fungus
opposite it. The results are given in Table VI.

TABLE VI.-RESULTS OF GROWING DIFFERENT PAIRS OF FUNGI IN POTATO
GLUCOSE AGAR AT 28-300C., WITH SPECIAL REFERENCE TO H. OCELLUM
AND ITS ASSOCIATED SAPROPHYTES.


Associated Fungi


H. ocellnm + L. sacchari



P. sorghina + L. sacchari

Spondylocladium +
L. sacchari

Nigrospora + L. sacchari



P. sorghina + Nigrospora


P. sorghina +


Spondylo-
cladium


P. sorghina + H. ocellum

P. sorghina + P. sorghina


Results


L. sacchari apparently not influenced by
presence of H. ocellunm, but the latter
definitely grew away from the former,
indicating that L. sacchari is antagonis-
tic to H. ocellum
Both fungi mutually agreeable

Both fungi mutually agreeable


Nigrospora not toxic to L. sacchari, but
the latter inhibited growth of former and
in most instances prevented close contact
of the two. L. sacchari is therefore defi-
nitely toxic to Nigrospora
Both fungi mutually agreeable

Both fungi mutually agreeable

Mutually agreeable. Both colonies finally
,rew in contact with each other
Mutually agreeable colonies


H. ocellum + Spondylocla- Mutually agreeable
dium
H. ocellum + Nigrospora Both fungi definitely toxic to each other


Spondylocladium + Nigro-
spora


Mutually agreeable


DISCUSSION OF RESULTS

The results indicate that P. sorghina, L. sacchari and Spondy-
locladium sp. may be mutually associated with H. ocellum in eye
spot or ring spot lesions. Because of the very rapid growth of
P. sorghina as compared with Spondylocladium and especially L.
sacchari, it is evident that the former is usually the first organism


Exp.
No.

A-1



A-2

A-3


A-4






Florida Agricultural Experiment Station


to follow the parasite, provided equal conditions for inoculation
are provided.
The experimental data clearly indicate also that under the
conditions investigated, Lepto-
sphaeria sacchari is not hindered
in its growth either by the patho-
Sgene H. ocellum or any of its
frequently associated saprophytic
fungi. In spite of its very
slow growth, if it becomes inocu-
lated into the eye spot lesions, it
Sis to be expected that it will even-
tually occupy a conspicuous place
Sin the dead tissue. This is in
Agreement with field observations
in the case of infected leaves,
for under conditions favoring the
dissemination of this saprophyte
it may be almost constantly asso-
ciated with the lesions on old dead
material, even though the other
saprophytes might have occupied
a significant role in the destruc-
tion of the diseased tissues be-
fore it.
The interesting mutual toxic
effect existing between H. ocellum
and Nigrospora sp. and the inhib-
Fig. 12.Linear arrangement of itory effect of L. sacchari when
Fig. 12.-Linear arrangement of
pycnidia of P. sorghina between associated either with H. ocellum
veins of leaf of B 417. (X 136.) or Nigrospora sp. in artificial cul-
ture, might not be so pronounced or might be absent entirely in
actual living tissues. Further, the results secured might be due
to changes in pH of the substrate rather than the production of
an actual toxin.
RELATION OF TEMPERATURE TO GROWTH
Halma and Fawcett(28) report experiments on the relation of
growth of H. sacchari to maintained temperatures. Lee(40) in
reviewing the above authors' work, refers to the fungus as the
one causing the eye spot in Hawaii, so that the possibility exists







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 49

that the fungus studied was identical with H. ocellum Faris,
although no spore or conidiophore measurements of the cultures
employed are available for comparison. Further, no statement
is made that the culture used was derived from a single spore,
therefore the results reported could possibly be based on a culture
secured by the ordinary tissue-planting method without any sub-
sequent purification.
As a result of their studies, Halma and Fawcett do not commit
themselves with regard to the optimum temperature any more
than to state that it was between 20 C. and 29 C. On the other
hand, Lee(40) in reviewing the above authors' work, states that
the fungus grows most rapidly at about 84F. (29C.). Cook(19),
however, states that field records in Porto Rico indicate that the
eye spot organism makes its best growth with an average out-
door temperature ranging from 73"F. to 770F. (22.8C. to 25'C.).
With regard to P. sorghina and Leptosphaeria sacchari, no
reference could be found giving studies on the temperature rela-
tions of these two fungi. It was therefore decided to study this
factor for both of these organisms also, since these data would
allow of a better conception of environmental conditions affecting
the growth of these saprophytes in the tissues of the host already
damaged by H. ocellum and the transformation of eye spot lesions
into typical ring spots.
EXPERIMENTAL METHODS
Single-spore strains of all three organisms were used in the
studies here reported. The medium consisted of hard potato-
glucose agar.* The temperatures desired were maintained in
special constant temperature chambers which did not show a
fluctuation of more than 0.5C. at any time during the work.
Enough agar medium was made up in one batch to run the com-
plete series in sextuplicate for each temperature. After prelim-
inary trials, the duration interval for the experiment was chosen
to be six days. With the lower temperatures, no difficulty was
experienced in keeping the humidity of the chambers such that
the medium did not dry out. In the case of temperatures above
29'C., however, battery jars with wet absorbent cotton in the
bottom and a cover over the top were found to be very satisfac-
tory in preventing the drying out of the medium.
*Two hundred gms. cubed potatoes boiled in 1000 cc. tap water. To
the strained potato broth was added 1% glucose and 2% shredded agar.
tThis work conducted with the aid of the facilities provided by the
Departments of Plant Pathology and Entomology, Cornell University.







50 Florida Agricultural Experiment Station

Spore suspensions were made from young vigorous cultures
one week old growing on the same kind of medium used for the
experiments. One small loop of the suspensions was used to
inoculate the center of each previously poured agar plate. The
poured plates were immediately placed in their respective tem-
perature chambers in the dark. After six days, the average
diameter of each colony was measured and the average from the
six cultures of each fungus in each chamber computed. The
agar medium as made up was found to have a pH of 6.47, this
figure being.determined electrometrically.
EXPERIMENTAL RESULTS
A summary of the results of the average diameters of the
colonies of the three fungi in the various temperature chambers
appears in Table VII. The results are also represented graph-
ically in Fig. 21 and photographically in Figs. 18, 19 and 20.
DISCUSSION OF RESULTS
The optimum temperature for growth of the three organisms
is seen to be as follows: H. ocellum 23.5C., P. sorghina 27.0C.
and L. sacchari 21.0C. It is significant that at the optimum
temperature, H. ocellum makes a greater growth than P. sor-
ghina at its optimum. Further, the maximum temperature for
H. ocellum lies at some point above 380C., while that for P. sor-
TABLE VII.-AVERAGE DIAMETER OF COLONIES OF H. OCELLUM, P. SORGHINA
AND LEPTOSPHAERIA SACCHARI AFTER SIX DAYS AT DIFFERENT TEMPERA-
TURES ON POTATO-GLUCOSE AGAR HAVING AN ACIDITY OF PH 6.47.

Temperature Average diameter of colony in ems.
"C.
H. ocellum P. sorghina L. sacchari
3.0 0.0 0.6 0.0
6.0 0.3 0.7 0.2
8.5 1.1
10.6 1.6
11.0 .. 2.6 0.6
14.5 2.1 4.1 0.7
17.7 4.2
18.0 5.5 0.9
21.0 6.9 6.7 1.1
23.5 9.0
24.0 ... 7.0 1.0
27.0 .. 7.8 0.9
28.0 6.1 .
29.0 ... 6.9 0.5
31.0 4.5
33.0 4.3
34.0 .. 0.6 0.0
35.0 1.2
38.0 0.8 0.0






Bulletin 267, Studies on Ring Spot Disease of Sugarcane 51

ghina lies between 34 and 38C. and for L. sacchari between 29
and 34C. The optimum temperature for growth of H. ocellum
in culture agrees well with observations in Florida and elsewhere
that eye spots and ring spots are especially abundant during the
cooler periods of the winter months when the average tempera-
tures range between 22C. and 25C. Higher temperatures be-
tween 300C. and 35"C. are seen to be very unfavorable to the
growth of the pathogene and practically inhibitory to L. sacchari,
an organism of very slow growth even under optimum conditions.
Photographs of H. ocellum in this series also bring out most
interestingly the characteristic appearance of the colonies at the


different temperatures. The typical
sectorial type of colony figured at
280C. (see Fig. 18) and mentioned
by Faris(26) as simulating dichoto-
mous branching and as being char-
acteristic of the eye spot fungus,
especially on 1% sucrose agar, is
here shown to be greatly influenced
by temperature relations and is ab-
sent at the optimum and toward both
extremes of minimum and maximum
temperatures. Further, the charac-
teristic smoky-colored mycelium is
absent at the extreme temperatures
of 6.0, 8.5, 10.6 and 38.0C. after
six days.
Notes on spore formation after
six days by the three organisms at
the various temperatures show that
H. ocellum failed to form conidia at
the lower temperatures up to and
including 10.6C. and at the higher
temperatures of 35 and 380C. At
all intervening temperatures spore
production was abundant, especially
at the optimum for growth.
In the case of P. sorghina, pyc-
nidia and spore formation occurred
only between 14.5C. and 29.0C.,


AI

r<


i




0i


Fig. 13.-Linear arrange-
ment of perithecia of Lep-
tosphaeria sacchari between
veins of leaf of CP. 27-139
in the holonecrotic zone of a
ring spot. (X 136.)






Florida Agricultural Experiment Station


while for L. sacchari, pycnidia and spores of the imperfect stage
occurred between 14.50C. and 27C.

RELATION OF HYDROGEN-ION CONCENTRATION TO GROWTH
Although the relation of acidity and alkalinity to growth has
been studied in detail for a large number of fungi, this informa-
tion is lacking in the case of H. ocellum and P. sorghina, the two
fungi of special importance in the case of ring spot.

EXPERIMENTAL METHODS
Standard "Difco" potato-glucose agar was employed, using
40 grams per liter as per directions. Enough medium was made
up at once to take care of four plate cultures for each hydrogen-
ion concentration in the case of each fungus. After sterilization,
the hydrogen-ion concentration of the normal medium was deter-
mined and the remainder adjusted with normal HC1 and NaOH
under aseptic conditions. The pH was determined electromet-
rically after dilution with neutral distilled water as mentioned
by Lisse et al.(42), using the quinhydrone electrode. For the
determination of alkalinities above pH 7.5, the "Hellige" colori-
metric outfit was employed.

TABLE VIII.-AVERAGE DIAMETER OF COLONIES OF H. OCELLUM AND P.
SORGHINA AFTER 10 AND Six DAYS RESPECTIVELY, GROWN AT DIFFERENT
HYDROGEN-ION CONCENTRATIONS ON POTATO GLUCOSE AGAR AT 280C.

pH of Average diameter of four plate colonies, cms.
Medium H. ocellum P. sorghina
3.1 1.7 1.7
3.2 2.5
3.4 2.5
3.7 2.6 3.9
4.1 3.0 4.8
4.5 3.2
4.7 .. 6.0
5.6* 3.3 7.3
5.8 3.4
6.0 ... 7.6
6.5 6.9
6.7 3.8
7.2 ... 3.7
7.4 1.5 3.4
8.0 1.3
8.2 0.7
8.4 ... 1.4
8.6 0.0
8.7 0.0 0.8
*Normal medium before the addition of either acid or alkali.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 53

After pouring the plates, they were incubated for 24 hours
before being inoculated with a loop of spores from an aqueous
suspension of spores of the particular organism in the center of
the plate. Plates were incubated at approximately 280C. for six
days in the case of P. sorghina and 10 days in the case of H. ocel-


8


7


6


5:
0
H
0
0 4



H)

..


o of ediu
pH of I-edium.


P. sorghina after 6 days, 28C.
- H. ocellum after 10 days, 280C.

Fig. 14.-Graphical representation of the relation of pH of medium to
growth of H. ocellum and P. sorghina after 10 and 6 days, respectively,
at approximately 28C.


+ fli+ ...... .......... .. ......
4 ''IIIH4+-+ . .
## 44-4 1


-W 4
V44 i IM I: ----------
Iff
Mm M
lff

++-+H++
#4 ........... M IUM -I M -
.. ..... 4++---F4 1
44+-+!,,
T 4L --4 4---
4+--
I Mr4+-::--


+
-U 4-:
r4f+-
:_-M 6W144-+- # -
........ ........
:--+4+4 44+----!++- V 4




A
T
H ..... R-H4-1+
HHH-1:
14 1 '64 ------ +

k--4 ---------- 4 ----



--- -------- ------ .......
-------- ---------
--- -------- ----------
tiff ------- ---
REEQ: ....


I


V jL






Florida Agricultural Experiment Station


lum. At the end of this period, the average diameter of the
colony in each plate was determined and the average for each
pH computed.
EXPERIMENTAL RESULTS
The experimental results are given in Table VIII and shown
graphically and photographically in Figs. 14, 22 and 23.

DISCUSSION OF RESULTS
The optimum growth for H. ocellum is seen to be at pH. 6.7,
while for P. sorghina it is at pH 6.0. It is significant that the range
for growth of the latter fungus extended further on the alkaline
side of the scale than in the case of H. ocellum, which made no
growth beyond pH 8.2 for the period investigated. Both fungi,
however, are seen to be able to tolerate rather acid media, even
as great as pH 3.1.
While no significant difference in color of colonies could be
observed in the case of H. ocellum, there was a definite correlation
between pH of medium and pigmentation (production of pink,
salmon and other shades) by P. sorghina. Orange peel colors
seemed to predominate between the range pH 3.1-4.1 and salmon,
cherry blossom and copper leaf between pH 4.7 and 7.4, while
no pigmentation occurred on the alkaline side at pH 8.4 and 8.7.
Since H. ocellum fails to produce in artificial media the pig-
ments necessary for inducing the characteristic Burgundy or
Spanish raisin shades frequently encountered in eye spot and
ring spot lesions, especially in the plesionecrotic zone, support
is given to the theory that these distinct colors are largely due to
the chemical reactions produced by the accompanying saprophytic
organism P. sorghina. It has already been noted that certain
cultures of H. ocellum gave rise to colored by-products having the
appearance of Arab rugby tan and it is indeed possible that
certain leaf proteins might react in a manner with this fungus
to give similar pigments.

RELATION OF LIGHT TO GROWTH
Since light has been noted to have a marked effect on the growth
and reproduction of a number of fungi and because ring spots on
the lower leaves develop well in the shade, information on this
factor seemed to be desirable. Priode(47) working with Hel-





Bulletin 267, Studies on Ring Spot Disease of Sugarcane 55


minthosporium sp., the cause of
the target blotch disease of su-
garcane leaves, claims that the
exclusion of light greatly retards
mycelium development, but the
production of conidia by the
fungus is greatly stimulated.
EXPERIMENTAL METHODS
Twenty-four petri dishes were
poured from a uniform lot of
fresh potato glucose agar having
a pH of 5.6. After the dishes
cooled, the center in each case
was inoculated with a loopful of
an aqueous suspension of spores,
12 dishes being inoculated with
H. ocellum and 12 with P. sor-
ghina. Immediately after inocu-
lation, six plates of each fungus
were placed in a large light-tight
metal can on a table in the lab-
oratory and the other 12 plates
spread out singly on the same
table which received at no time
anything but reflected sunlight
from the sky after passage
through ordinary window glass.
Direct rays from the sun were
not allowed to shine on the plates
or the box with the plates in the
dark because of the temperature
factor which would be introduced.


Fig. 15.-Germination of spores
of pycnidial stage (Phyllosticta
saccharicola Henn.) of Leptospha-
eria sacchari in distilled water after
23 hours at 280C. Note anastomos-
ing of germ tubes. (X 590.)


RESULTS
After nine days, the H. ocellum cultures in the dark showed
a mean average diameter of 2.8 cms., while those in the light
showed a mean average of 2.3 cms. Since variations in diam-
eters in each group occurred from 1.9 to 3.2 cms., it is evident
that light exerted no significant difference in vegetative growth.
Further, no difference could be observed in the production of
conidia by the two groups.






Florida Agricultural Experiment Station


After five days, the P. sorghina cultures showed a difference
in mean average diameter of colonies from the two series amount-
ing to only 0.1 cm., which
was more than the varia-
Stion occurring in colonies
Sin each group. However,
-l the cultures in the dark
Showed no zonated rings
of pigmented mycelium,
the colonies being a uni-
form copper-leaf color.
e Colonies in the light, on
sr the other hand, were all
bright salmon colored in
the center and possessed
two zonate rings of pink-
ish brown outside of the
center. Between the
rings as well as the outer
edge of each colony was
almost white.
Abundant pycnidia
Fig. 16.-Germination of spores of Phyl-
losticta sorghina in 1% sucrose solution af- and spores were found in
ter 20 hours. The spores are now much the light and dark, no dif-
swollen. Note budding in yeast-like fashion,
as well as germ tube formation. (X 450.) ference in these respects
being detectable.
Because of the difference occurring in pigmentation and zona-
tion by colonies of P. sorghina in the light and darkness, a further
experiment was made with the cultures after the five-day expo-
sure to the two conditions. Two plates from the dark were placed
in diffuse light on the laboratory table, while two plates from
the light were placed in the dark can. The remaining four plates
in each case were left in their original locations for controls.
After four days it was found that the two plates removed from
the light and placed in the dark, failed to develop any further
rings, while the controls in the light formed two more rings
during the period. In the case of the two plates removed from
the dark and placed in the light, rings were very evident and a
definite pink pigmentation had developed. The controls left in
the dark showed no zonate rings or pigmentation.






Bulletin 267, Studies on Ring Spot Disease of Sugarcane 57

DISCUSSION OF RESULTS
While no difference of significance in the amount of vegetative
growth or repro-
duction occurred
in light or dark-
ness, light was
associated with
the formation of
zonate rings and
the development
of a pink pigmen-
tation in the case
of P. sorghina
only, after a pe-
riod of five days.
Zonation of H.
ocellum cultures
after prolonged
periods of several
weeks in the
light has been Fig. 17.-Portion of culture of Leptosphaeria sac-
observed, but chari (on sterilized cane leaves) after 43 days, show-
ing the pycnidia of the conidial stage, Phyllosticta
whether such zo- saccharicola. (X 60.)
nation occurs in
cultures grown for such extended periods in the dark has not
been determined.
STUDIES ON NITROGEN FIXATION
As Waksman(59) has recently pointed out, careful investiga-
tions have established the fact that common soil fungi are unable
to fix atmospheric nitrogen. However, few parasitic fungi have
been studied with respect to this phenomenon. Since positive
fixation has been claimed for Phoma betae by Duggar and
Davis(23) and P. radicis by Ternetz(54) and because of the arti-
ficial nature of the generic segregation of Phoma and Phyllosticta,
it seemed desirable to make tests on the important saprophyte
P. sorghina as well as H. ocellunm.
EXPERIMENTAL METHODS
A modified Richard's solution* was employed, using 1% sucrose

*K2SO4-10 grams; KH2 P04-5 grams; MgSO4-2.5 grams; FeC1:-20
mgs; Sucrose-10 grams; distilled water-1000 cc. Only C.P. chemicals em-
ployed.







Florida Agricultural Experiment Station


U
8.5


3 C8.0


Fig. 18.-Relation of temperature (degrees C.) to growth of Helmintho-
sporium ocellum in culture.

(C.P.) as a source of carbon and omitting a source of nitrogen
other than that of the atmosphere.
Sixteen 500 cc. pyrex Erlenmeyer flasks were thoroughly
cleaned and rinsed in tap and distilled water. After thoroughly







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 59

mixing the nutrient solution, 250 cc. was placed in each flask
and steam sterilized for 10 minutes at 15 pounds. The flasks
were incubated for 48 hours to detect contamination and then
inoculated with a small loopful of an aqueous spore suspension
of the organism selected. Six flasks were inoculated with H.
ocellum, six with P. sorghina and four held as controls without
inoculation. All cultures were then placed in diffuse light of
the laboratory. The intention was to make total nitrogen deter-
minations of each flask after good growth had been made in the
inoculated flasks.
RESULTS

After four weeks at room temperature, a very faint weft of
mycelium was visible on the surface of each inoculated flask.
The controls were still sterile.

DISCUSSION OF RESULTS
It is evident that H. ocellun or P. sorghina cannot grow in a
nutrient solution free from nitrogenous substances. The con-
clusion is drawn that the fungi cannot fix atmospheric nitrogen
under the conditions of the experiment.

RETENTION OF VIRULENCE OF PARASITE IN CULTURE
The loss of virulence by both parasitic bacteria and fungi has
been noted by several workers in the past. Smith(51) noted that
parasitic bacteria frequently lost their virulence when grown for
a long time on culture media and even considered that recovery
of the host from disease might be due to this phenomenon. Edger-
ton et al.(24) working with a parasitic species of Pythium caus-
ing a root rot of sugarcane, showed that the virulence of the
fungus becomes much attenuated after a period of two years in
artificial culture. Burkholder(1O, 11), also found that when
the parasite Fusarium martii phaseoli is grown in culture (pure)
for some time its virulence is considerably reduced, although such
an attenuated culture could be restored to normal by growing it
for several months on its host, the bean plant.
In April, 1930, H. ocellum was isolated from a sugarcane leaf
and after single-spore purification, grown in artificial culture
until December, 1932, or more than 21/2 years. The same strain,
subcultured and used for inoculation purposes and reisolated from
diseased tissue several times, was used for parallel infection trials







Florida Agricultural Experiment Station


Fig. 19.-Relation of temperature (degrees C.)
to growth of Phyllosticta sorghina in culture.


for comparison with the culture which had lived saprophytically
for 21/ years. Repeated trials showed clearly that the culture
after such a long period of saprophytic existence could infect
leaves, leaf sheaths and stems just as readily as the strain which
had been allowed to live parasitically for various periods.







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 61

EXPERIMENTS ON THE PRODUCTION OF TOXIC SUBSTANCES BY
THE PARASITE H. OCELLUM
Lee(39) in an extensive paper on the toxic substance produced
by the eye spot fungus of sugarcane, H. sacchari Butler, claims
that the wilting of cut cane leaves immersed in filtrates of cul-
tures of the fungus is closely correlated with the presence of
nitrites in such filtrates. Further, dilute solutions of potassium
nitrite brought about the same reaction of susceptible cane leaves
as the filtrates of cultures of the eye spot fungus in media con-
taining the lower nitrogen compounds.
The writer is unable to agree with the conclusions drawn by
Lee on the basis of experiments performed. In the first place,
the actual concentration of nitrites in various cultures was not
determined in a single instance for comparison with the artificial
concentrations arbitrarily tried and found to be toxic. Further,
although no nitrites could be detected in the peptone medium on
which H. sacchari grew, the cane leaves wilted decidedly before
the controls, strongly indicating that some factor other than
nitrites might have been involved in the wilting phenomenon.
During the physiological studies on H. ocellum, the formation
of magnesium ammonium phosphate in considerable quantities
from certain cultures, e. g. peptone and blood fibrin, led the
writer to suspect that perhaps ammonia or ammonium compounds
might be involved in the toxic action of the fungus. In fact,
when grown for 25 days on White's(60) modified Richard's solu-
tion containing 2% peptone, only traces of nitrites could be de-
tected, whereas tests for nitrogen as "free ammonia" showed
1,000 parts per million in the filtrate. It was therefore decided
to test the toxicity of filtrates of cultures of H. ocellum to cane
leaves and to make quantitative determinations of both nitrites
and nitrogen as "free ammonia" in these cultures at the time
of test.
EXPERIMENTAL METHODS
A modified Richard's mineral solution was made up as follows:
KNO:; 24 grams; KH2PO4 4 grams; MgSO4 2 grams; FeCl: 96
mgs; distilled water, 4,800 cc. This was divided into four equal
parts and the following added to each part separately: A, gelatin
24 gms.; B, casein 20 grams; C, blood fibrin 20 grams; and D,
sucrose 24 grams. Each part was then divided into 200 cc. por-
tions in 500 cc. Erlenmeyer flasks, steam sterilized, incubated
for 4 days to detect contamination and half the number of flasks















/o





I I 0
C r














Fig. 20.-Relation of temperature (degrees C.) to growth
of Leptosphaeria sacchari in culture.
F| z

4 ;'
s







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 63

of each kind inoculated with a loop of spores of H. ocellunm. At
the end of varying periods, as shown in Table IX, the filtrate of
each part containing a specific source of carbon was tested for
toxicity to freshly cut outer leaves of Otaheite cane, after filtra-
tion through a steam sterilized Berkefeld filter. At the same
time, quantitative determinations were made for nitrites and
nitrogen as "free ammonia" according to the methods described
by Mason(44). In each series of toxicity tests, 12 wide mouth
bottles thoroughly cleaned were used. Three bottles were used
for the full concentration of fungus filtrate, three for 50% con-
centration of filtrate diluted with distilled water, three for unin-
oculated culture medium and three for distilled water, all being
set up at the same time for comparison.
RESULTS
In not a single instance did wilting of the leaves occur until
after the cultures became cloudy due to bacterial action, which
almost invariably occurred in the nutrient cultures 24 to 48 hours
after the experiment was set up. The writer has therefore been
unable to detect any significant toxic action of the filtrate of H.
ocellum on any of the substrates tested, either in the form of
wilting of the leaves due to collapse of the motor cells or by the
discoloration of tissues.
The chemical tests of the filtrates used are reported in Table IX.
TABLE IX.-QUANTITATIVE RESULTS OBTAINED FOR NITRITES AND NITRO-
GEN AS "FREE AMMONIA" IN FILTRATES OF CULTURES OF H. OCELLUM
GROWN ON MODIFIED RICHARD'S SOLUTION WITH VARIOUS SOURCES OF
CARBON.
Carbon Age of Nitrogen as Nitrogen as
Medium No. Source culture nitrite. "free ammonia".
(days) p.p.m. p.p.m.
A Gelatin 34 0.3 300.0
B Casein 35 0.0 300.0
C Blood fibrin 38 0.0 300.0
D Sucrose 40 Trace 25.0

DISCUSSION OF RESULTS
The results show that H. ocellum does not produce nitrites in
culture in sufficient quantity to even arouse suspicion that this
chemical is of importance as far as the toxic action of the fungus
is concerned. On the other hand, depending on the amount of
KH2PO4 and MgSO4 used in the cultures containing certain pro-
tein substances, nitrogen as "free ammonia" may amount to as
much as 1,000 parts per million. The possibility of the fungus






Florida Agricultural Experiment Station


liberating free ammonia in toxic quantities in the living tissues
is therefore considered to be of sufficient significance to warrant
further study.
It should be borne in mind, however, that the toxic action of
the fungus in the tissues can be explained from other angles.
The fact that the fungus requires nitrates or some source of
nitrogen other than from the atmosphere for its metabolism
means that the removal of this element from the invaded cells
upsets the physiological balance and undoubtedly hinders further
synthesis of complex nitrogenous compounds. The pronounced
action of invertase is especially significant and indicates that this
readily soluble enzyme may diffuse out in advance of the growing
mycelium and invert sucrose produced synthetically in the leaf
tissue.
GENERAL DISCUSSION OF CERTAIN PHYSIOLOGICAL PHASES OF
THE RING SPOT PROBLEM
It has been shown that true ring spot symptoms as previously
described by most authors are brought about by the action of
certain saprophytic fungi which follow the primary pathogene
Helminthosporium ocellum. Among these, Phyllosticta sorghina
has been shown to be especially active, even to the point of causing
lesions of the pathogene under certain conditions to develop more
rapidly than when absent.
The ability of P. sorghina to tolerate a greater intensity of
alkalinity in the medium would appear to be of significance in
relation to its favorable association with H. ocellum. Both fungi,
because of their pronounced ability to form ammonia from cer-
tain higher protein substances and their derivatives, tend to
create an alkaline environment especially unfavorable to the
growth of the pathogene. It is true that heavy dews and rain
undoubtedly leach out unfavorable by-products from the ple-
sionecrotic and holonecrotic portions of the lesions, but P. sor-
ghina nevertheless can continue to destroy more effectively these
tissues even after the action of the pathogene has been almost
inhibited by an increase in alkalinity of the substrate.
Since the optimum temperature for the growth of H. ocellum
has been found to be 23.50C. and that for P. sorghina 27.0C., it
is obvious that temperature conditions are more favorable for
the growth of the saprophyte during a considerable part of the
24 hour cycle in Florida, even during the cool months of the year.
The fact that H. ocellum passes directly through the wall of








Bulletin 267, Studies on Ring Spot Disease of Sugarcane 65


rfl

0
H

0


C,
C)
ii
Hr


0 10 2U ou qu
Temperature 0C.
P. sorghina after 6 days.
H. ocellium after 6 days.
-.-..- L. sacchari after 6 days.

Fig. 21.-Graphical representation of the relation of temperature to the
growth of H. ocell'm, P. sorghina and L. sacchari in culture. All meas-
urements made after six days.

the epidermis, especially in the case of the motor cells of the
leaf, and also travels through the cell walls of the sub-epidermal
tissues, suggests that the thickening of these walls might be a
primary factor in resistance or immunity of certain varieties to
the disease. The application of nitrogenous fertilizer has been
noted to have a marked correlation with increase in susceptibility


i FH
T 4+M
i4:r+ TFF 'FH
-H' FFF+!
TIR H+H~"" "m," ~ -
UUM MM41411






7


6 t

5' .





Ti -T

UN
i t W L LL/







Florida Agricultural Experiment Station


of the host. Since nitrogen as a factor in plant nutrition has
been shown by Thatcher(55), Russell(48) and others to produce
a marked growth of soft sappy tissue having thinner walls, it
becomes evident that resistance may be largely a mechanical
phenomenon, although changes in the composition of the cells as
regards food materials, hydrogen-ion concentration and osmotic
pressure undoubtedly play a role of special importance.

CONTROL
Since the eye spot and its later stage, the ring spot, are well
established in practically every sugarcane country of the world,
and the parasite is capable of living saprophytically for more
than 21/. years without loss of virulence, attention must be
directed to the protection of the existing host varieties from
infection, or to the breeding of new types resistant or immune
to the malady.
The same correlation between the virulence of the disease and
nitrogen nutrition as has been observed for eye spot in Hawaii
by Lee(39) has been observed in Florida. The disease has never
been observed to be severe on sandy or clayey soils rather deficient
in nitrogen. On the other hand, in sections of the Florida Ever-
glades where cane is grown on peat and muck lands having a
high total and at times a very available supply of nitrogen, the
disease often assumes a role of considerable importance. The
practical control measure mentioned by Lee(39), whereby nitro-
genous fertilizers are avoided during seasonal periods in which
climatic conditions favor fungus infection-during the cool fall
and winter months in Florida when temperatures approach the
optimum of 23.5C. for the pathogene-is of course only possible
in regions where the nitrogen supply can be controlled. In
sections of the Florida Everglades where the control of the nitro-
gen content of the soil is impossible, use of resistant or immune
varieties is the only practical solution to the problem.
THE BREEDING OF VARIETIES RESISTANT TO THE DISEASE
No information has been available in the literature to show
the most desirable mode of attack in the problem of breeding
for resistance to this disease. It is true that many varieties
of the true sugarcane group Saccharum officinarum have shown
very serious susceptibility to the malady, indicating that atten-
tion should be directed toward observation on and possible use






Bulletin 267, Studies on Ring Spot Disease of Sugarcane 67


Fig. 22.-Relation of pH of medium to growth of H. occllumn. Photo after
10 days at approximately 28C.

of certain varieties of other species of Saccharum, such as S.
spontaneum (wild cane) or S. barberi, in a program of breeding
for resistance or immunity. The fact that certain saccharatus
varieties of Holcus sorghum are apparently immune to the disease
under conditions in the Florida Everglades, also suggests the
desirability of investigating fully the possibilities of the inter-
generic hybridization of Saccharum and Holcus.
During the past seven years, the writer has conducted exten-
sive breeding experiments with sugarcane varieties in a section
of Florida where the ring spot is most prevalent and has there-
fore had an unusual opportunity to study the disease among
progeny from many different combinations. While relatively







Florida Agricultural Experiment Station


few intergeneric hybrids between sugarcane and sorghum have
been produced for study, the results are not without interest.
The more important combinations which have been studied,
together with the number of progeny which have been under
observation in each case and their genealogical history are pre-
sented below, together with a brief discussion of the nature of
the results secured. The parentage of the Javanese canes has
been secured from the published work of Jeswiet(34) and Ban-
nier(8) and that of the Indian varieties from Venkatraman and
Thomas(57).
A. Sugarcanes with the Blood of S. officinarum and S. barberi.
Crystalina ......... D. 74 1
La. Purple X 16 seedlings.
X POJ. 213 .........U. S. 1694 J
Chunnee
(S. barber)
The number of progeny studied is too few to form any idea as
to the direct value of this combination. Nevertheless, some of
the seedlings e. g. CP. 27-34, 35 and 38 have shown a satisfactory
resistance to ring spot and have been used for further crosses
with promising results, largely because of the desirable early
blood of D. 74.
B. Sugarcanes with the Blood of S. officinarum, S. barberi and S. spontaneum.
1. Bandjarmasin hitam ]
X POJ. 100
Loethers J
X POJ. 2364 1
La. Purple ) |
X Kassoer J x POJ. 2725 1 101
S. spontaneum. J X seed-
E.K E.K. 28 U. S. 1694 J lings
POJ. 100 J
Although only a few progeny have been studied, it has been
noted that moderately susceptible varieties are to be found in
this group, e. g. CP. 27-139, undoubtedly due to the marked sus-
ceptibility of POJ. 100 in the ancestral lineage. On the other
hand, an equal number of progeny appeared to be very resistant,
e. g. F. 29-7, so that the combination has proved a valuable one
for securing resistant types under a system of rigid selection.
2. POJ. 2364 POJ. 2725 1
X [ 5,873 seedlings.
X CP. 27-108 J
U. S. 1694 ....... CP. 726







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 69

Such a large number of progeny of this combination have been
studied that a definite opinion can be rendered as to its value.
Most of the seedlings showed marked resistance to the disease
and some of the most promising agricultural types are at present
included here, e. g. F. 30-16, 20, 26, 35, 89 and others.
3. POJ. 2725 1
D. 74 1 X 2,800 seedlings.
X CP. 27-35 J
U. S. 1694 J

The number of progeny studied has been adequate for judging
the merits of this combination. The majority showed marked
resistance to the disease and in addition this cross has given the
largest stalked and apparently the most desirable agricultural
types so far developed, e. g. F. 31-407, 425, 426, 566, 572, etc.,
which are now in the process of agronomic trials.
.4. POJ. 2725 ]
D. 74 1 X [ 3,496 seedlings.
X } CP. 27-34
U. S. 1694

The majority of the progeny showed marked resistance to the
disease, but otherwise this combination was definitely inferior
from an agricultural standpoint to No. 3, where the brother
variety CP. 27-35 was used.
5. La. Purple I
X f POJ. 213 1
Chunnee J
X [ Co. 281
Ashy Mauritius 1 C
X ) Co. 206 J
C] X 763 seedlings.
S. spontaneum J
POJ. 2364 1
x [ CP. 27-108
CP. 726 J

The majority of the progeny were highly resistant and some
practically immune to the disease. Further, this combination






Florida Agricultural Experiment Station


is one of the few involving the early maturing Co. 281 which has
given indications of a good proportion of satisfactory commercial
types for selection.

6. La. Purple
X POJ. 213
Chunnee
X Co. 281
Ashy Mauritius 1
X [ Co. 206 J X 797 seedlings.
S. spontaneuc J |
POJ. 213 U. S. 1964 J

This combination has given rise to progeny a large number of
which are very susceptible to the disease. Practically all of the
seedlings are thin stemmed, "grassy" and unattractive from the
standpoint of commercial utilization for sugar making.
Certain facts stand out clearly as a result of the breeding work
with sugarcane varieties so far conducted. The most desirable
combinations from the standpoint of proportion of resistant
progeny, combined with indications of satisfactory commercial
habits are: POJ. 2725 x CP. 27-35, POJ. 2725 x CP. 27-108 and
Co. 281 x. CP. 27-108. It is also significant that those combina-
tions involving a high proportion of Chunnee (S. barber) blood
have given rise to a greater degree of susceptibility among the
progeny, whereas those having a high proportion of S. sponta-
neum blood have given seedling populations markedly resistant
to the disease.
With reference to the Saccharum x Holcus hybrids, only about
30 of these have been under observation, which number is rather
limited for an adequate study. The hybrids consist of about an
equal number of progeny of crosses between POJ. 2725 sugar-
cane and the Holcus sorghum saccharatus varieties Texas Seeded
Ribbon and Kansas Orange. While certain types, e. g. F. 31-61,
are showing marked resistance to the malady, others, e. g. F. 31-8,
are proving to be rather susceptible, indicating that rigid selec-
tion will have to be made from progeny of such intergeneric com-
binations if their utilization is intended either for the Florida
Everglades or for locations having environmental conditions
favorable for the development of the disease.






Bulletin 267, Studies on Ring Spot Disease of S,,,i,, i' t,, 71


;


pH-U.4


n- *


Fig. 23.-Relation of pH of medium to growth of P. sorghina. Photo after
six days at approximately 28C.






Florida Agricultural Experiment Station


SUMMARY
1. The names, history and geographical range of the ring spot
disease are reviewed. The economic importance from the stand-
point of commercial canes and the breeding of new varieties is
discussed.
2. The morphologic and histologic symptoms of the disease
on leaves, leaf sheaths and stems are given.
3. A summary of the organisms associated with the disease
in nature and their relative frequency of occurrence is recorded.
4. A review of the etiological history of the disease is given.
Proof is furnished that Helminthosporium ocellum Faris is the
primary cause of the disease, whose lesions at first are identical
with eye spot. Later, with the development of such saprophytes
as Phyllosticta sorghina Sacc. and Leptosphaeria sacchari van
Breda de Haan, the ring spot symptoms are brought out.
5. There is some evidence that a mixture of H. ocellum and
P. sorghina develop lesions more rapidly under certain conditions
than H. ocellum alone.
6. The names, history and classification of the parasite are
given and the life-history is discussed. The organism is seen
to gain entrance into the leaf directly through the epidermal
wall, especially in the case of the motor cells. Entrance also
occurs through stomates. The mode of entrance into sheath and
stem portions is accomplished in a manner similar to that of
the leaf.
7. The most important and frequently associated saprophyte,
P. sorghina, is discussed from the standpoint of names, history
and classification. Its occurrence on several other hosts, e. g.,
Panicum maximum, Panicum dichotomiflorum, Panicum sp. and
Holcus sorghum is recorded in association with similar ring spot
lesions due to other fungi.
8. The life-history of Leptosphaeria sacchari is reviewed and
proof given for the genetic connection between Phyllosticta sac-
charicola Henn. and the perfect stage.
9. The comparative susceptibility of a number of commercial
and other sugarcanes is recorded for Florida.
10. The physiology of both H. ocellum and P. sorghina as re-
gards the relation of temperature, pH of medium and light;
growth on different sources of carbon; enzymes produced; rela-
tion to associated organisms and to each other and nitrogen fix-
ation is given. The optimum temperature for the development







Bulletin 267, Studies on Ring Spot Disease of Sugarcane 73

of the parasite in culture has been shown to be 23.5'C. The
virulence of this organism is seen to be maintained unchanged
even after 21, years under saprophytic conditions.
11. In toxicity studies with filtrates of H. ocellum on several
media, no significant wilting of the leaves was secured. Among
the substances produced in suspicious quantities, magnesium
ammonium phosphate deserves special mention and this suggests
the production of ammonia in toxic quantities in the host tissue.
12. The host range has been extended from varieties of Sac-
charum officinairui to include hybrids having the blood of S.
officinarum, S. spontaneum and S. barber. Further, although
Holcus sorghum varieties Texas Seeded Ribbon and Kansas
Orange appear to be immune, a number of intergeneric hybrids
between them and sugarcane have proved to be susceptible to
the disease.
13. Breeding experiments for the production of resistant and
immune varieties, covering a period of seven years, are briefly
reviewed. The genealogical histories of some of the most favor-
able and unfavorable breeding combinations yet investigated are
recorded. Combinations having a high proportion of Chunnee
(Saccharum barber) blood have given rise to a greater degree
of susceptibility among the progeny than those having a high
proportion of Saccharum spontaneum blood, the seedling popula-
tions of which show a marked resistance to the disease.

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