• TABLE OF CONTENTS
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 Title Page
 Table of Contents
 Introduction
 Distribution and present taxonomic...
 Materials and methods
 Morphology
 Cross-inoculation studies
 Taxonomy and synonymy
 Discussion
 Summary
 Literature cited






Group Title: Bulletin - University of Florida Agricultural Experiment Station -
Title: Life history and taxonomy of the fungus Physalospora rhodina
CITATION PAGE IMAGE ZOOMABLE PAGE TEXT
Full Citation
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Permanent Link: http://ufdc.ufl.edu/UF00026714/00001
 Material Information
Title: Life history and taxonomy of the fungus Physalospora rhodina
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 91 p. : ill. ; 22 cm.
Language: English
Creator: Voorhees, R. K ( Richard Kenneth ), 1907-
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1942
 Subjects
Subject: Physalospora rhodina   ( lcsh )
Plants -- Variation   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references.
Statement of Responsibility: by R.K. Voorhees.
General Note: Cover title.
General Note: Originally presented as: Thesis (Ph.D.)--University of Minnesota, 1941.
Funding: This collection includes items related to Florida’s environments, ecosystems, and species. It includes the subcollections of Florida Cooperative Fish and Wildlife Research Unit project documents, the Florida Sea Grant technical series, the Florida Geological Survey series, the Howard T. Odum Center for Wetland technical reports, and other entities devoted to the study and preservation of Florida's natural resources.
 Record Information
Bibliographic ID: UF00026714
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000925172
oclc - 07515297
notis - AEN5816
lccn - 44044259

Table of Contents
    Title Page
        Page 1
        Page 2
    Table of Contents
        Page 3
        Page 4
    Introduction
        Page 5
    Distribution and present taxonomic status of the fungus
        Page 6
        Page 7
        Page 8
        Page 9
    Materials and methods
        Page 10
        Source of races
            Page 10
            Page 11
        Procedures
            Page 12
        Cultural aversion in physalospora rhodina
            Page 15
            Occurrence of the phenomenon
                Page 15
                Page 16
            Stability of aversion reactions
                Page 17
                Page 18
                Page 19
                Page 20
            Races obtained from individual specimens
                Page 21
                Page 22
            The pycnidium as the source of races
                Page 23
            Perithecia and asci as sources of races
                Page 24
                Page 25
                Page 26
                Page 27
                Page 28
            Relation of aversion and sex in physalopora rhodina
                Page 29
                Page 30
                Page 31
                Page 32
        Cultural characters of races
            Page 33
            Page 34
            Page 35
            Page 36
            Page 37
            Page 38
        Effect of temperature on rate of growth in culture
            Page 39
            Growth of races in monoconidial origin at 7 temperatures
                Page 39
                Page 40
                Page 41
            Growht of races of monoconidial origin at 3 temperatures
                Page 42
                Page 43
                Page 44
            Growth of races of monoascospore origin at one temperature
                Page 45
                Page 46
        Effect of temperature on rate of decay in host material
            Page 47
            Page 48
    Morphology
        Page 49
        Conidial and ascigerous stages in the life history
            Page 49
            Mycelium
                Page 50
                Page 51
                Page 52
            Pycnidia and conidia
                Page 53
                Page 54
                Page 55
                Page 56
                Page 57
                Page 58
                Page 59
            Perithecia, asci, and ascospores
                Page 60
                Page 61
                Page 62
        Spore measurements
            Page 63
            Effect of substrata on size of conidia
                Page 63
                Page 64
                Page 65
                Page 66
                Page 67
            Effect of natural host source on size of conidia
                Page 68
                Page 69
    Cross-inoculation studies
        Page 70
        Page 71
        Page 72
        Page 73
    Taxonomy and synonymy
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
    Discussion
        Page 82
        Page 83
        Page 84
        Page 85
    Summary
        Page 86
        Page 87
    Literature cited
        Page 88
        Page 89
        Page 90
        Page 91
Full Text


Bulletin 371


UNIVERSITY OF FLORIDA.
AGRICULTURAL EXPERIMENT STATION, -h *
WILMON NEWELL, Director
GAINESVILLE, FLORIDA









Life History and Taxonomy

of the

Fungus Physalospora Rhodina

By R. K. VOORHEES










TECHNICAL BULLETIN







Single copies free to Florida residents upon request to
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA


May, 1942




EXECU STAFF
John J. Tigert, M. A., LL. D., President
of the University3
Wilmon Newell, D.Sc., Directors
Harold Mowry, M. S. A., Asst. Dir.,
Research
W. M. Fifield, M. S., Asst. Dir., Admin.4
J. Francis Cooper, M. S. A., Editors
Clyde Beale, A.B.J., Assistant Editors
Jefferson Thomas, Assistant Editor'
Ida Keeling Cresap, Librarian
Ruby Newhall, Administrative Managers
K. H. Graham, Business Managers
Rachel McQuarrie, Accountants

MAIN STATION, GAINESVILLE
AGRONOMY
W. E. Stokes, M.S., Agronomist'
W. A. Leukel, Ph.D., Agronomists
Fred H. Hull, Ph.D., Agronomist
G. E. Ritchey, M.S., Associate2
W. A. Carver, Ph. D., Associate
Roy E. Blaser, M.S., Associate
G. B. Killinger, Ph.D., Associate
John P. Camp, M.S., Assistant
Fred A. Clark, B.S.A., Assistant
ANIMAL INDUSTRY
A. L. Shealy, D.V.M., An. Industrialist'1,
R. B. Becker, Ph.D., Dairy Husbandmans
E. L. Fouts, Ph.D., Dairy Technologists
D. A. Sanders, D.V.M., Veterinarian
M. W. Emmel, D.V.M., Veterinarians
L. E. Swanson, D.V.M., Parasitologist
N. R. Mehrhof, M.Agr., Poultry Husb.'
T. R. Freeman, Ph.D., Asso. in Dairy Mfg.
R. S. Glascock, Ph.D., Asso. An. Husb.
D. J. Smith, B.S.A., Asst An. Husb.'
P. T. Dix Arnold, M.S.A., Asst. Dairy
Husbandman'
L. L. Rusoff, Ph.D., Asst. in An. Nutr.s
L. E. Mull, M.S,, Asst. in Dairy Tech.
O. K. Moore, M.S., Asst. Poultry Husb.
ECONOMICS, AGRICULTURE
C. V. Noble, Ph.D., Agr. Economists.3
Zach Savage, M.S.A., Associate
A. H. Spurlock, M.S.A., Associate
Max E. Brunk, M.S., Assistant
ECONOMICS, HOME
Ouida D. Abbott, Ph.D., Home Econ.1
Ruth O. Townsend, R.N., Assistant
R. B. French, Ph.D., Asso. Chemist
ENTOMOLOGY
J. R. Watson, A.M., Entomologist'
A. N. Tissot, Ph.D., Associate
H. E. Bratley, M.S.A., Assistant
HORTICULTURE
G. H. Blackmon, M.S.A., Horticulturist'
A. L. Stahl, Ph.D., Associate
F. S. Jamison, Ph.D., Truck Hort.
R. J. Wilmot, M.S.A., Asst. Hort.
R. D. Dickey, M.S.A., Asst. Horticulturist
J. Carlton Cain, B.S.A., Asst. Hort.4
Victor F. Nettles, M.S.A.. Asst. Hort.'
Byron E. Janes, Ph.D., Asst. Hort.
F. S. Lagasse, Ph.D., Asso. Horticulturists
H. M. Sell, Ph.D., Asso. Horticulturist3
PLANT PATHOLOGY
W. B. Tisdale, Ph.D., Plant Pathologist1',
George F. Weber, Ph.D., Plant Path.3
L. 0. Gratz, Ph.D., Plant Pathologist
Erdman West, M.S., Mycologist
Lillian E. Arnold, M.S., Asst. Botanist
SOILS
R. V. Allison, Ph.D., Chemistl.3
Gaylord M. Volk, M.S., Chemist
F. B. Smith, Ph.D., Microbiologists
C. E. Bell, Ph.D., Associate Chemist
H. W. Wingor, B.S.A., Assistant Chemist
J. Russell Henderson, M.S.A., Associates
L. H. Rogers, Ph.D., Asso. Biochemist4
Richard A. Carrigan, B.S., Asso. Chemist
Geo. D. Thornton, M.S.. Asst. Chemist
Thos. Whitehead. Jr., M.S.A., Asst.
R. E. Caldwell, M.S.A., Soil Surveyor
Olaf C. Olson, B.S., Soil Surveyor


BOARD OF CONTROL
H. P. Adair, Chairman, Jacksonville
R. H. Gore, Fort Lauderdale
N. B. Jordan, Quincy
T. T. Scott, Live Oak
Thos. W. Bryant, Lakeland
J. T. Diamond, Secretary, Tallahassee
BRANCH STATIONS
NORTH FLORIDA STATION, QUINCY
J. D. Warner, M.S. Agron. in Charge
R. R. Kinkaid, Ph.D., Asso. Plant Path.
R. W. Wallace, B.S., Asso. Agron.
J. H. Wallance, M.A., Asso. Agron.
Elliott Whitehurst, B.S.A., Asst. An.
Husb.3
W. C. McCormick, B.S.A., Asst. An.
Husb.
Jesse Reeves, Asst. Agron., Tobacco
W. H. Chapman, M.S., Asst. Agron.4
CITRUS STATION, LAKE ALFRED
A. F. Camp, Ph.D.. Horticulturist in Chg.
V. C. Jamison, Ph.D., Soils Chemist
B. R. Fudge, Ph.D., Associate Chemist
W. L. Thompson, B.S., Associate Ento.
F. F. Cowart, Ph.D., Asso. Horticulturist
W. W. Lawless, B.S.. Asst. Horticulturist4
R. K. Voorhees, Ph.D., Asso. Plant Path.
H. O. Sterling, B.S., Asst. Hort.
T. W. Young, Ph.D., Asso. Hort., Coastal
EVERGLADES STA.. BELLE GLADE
J. R. Neller, Ph.D., Biochemist in Chg.
J. W. Wilson, Sc.D., Entomologist
F. D. Stevens, B.S., Sugarcane Agron.
Thomas Bregger, Ph.D., Sugarcane
Physiologist
G. R. Townsend, Ph.D., Plant Pathologist
R. W. Kidder, M.S., Asst. An. Husb.
W. T. Forsee, Ph.D., Asso. Chemist
B. S. Clayton, B.S.C.E., Drainage Eng.3
F. S. Andrews, Ph. D., Asso Truck Hort.4
Roy A. Bair, Ph.D., Asst. Agron.
SUB-TROPICAL STA., HOMESTEAD
Geo. D. Ruehle, Ph.D., Plant Pathologist
in Charge
S. J. Lynch., B.S.A., Asst. Horticulturist
E. M. Andersen, Ph.D., Asst. Hort.
W. CENT. FLA. STA., BROOKSVILLE
W. F. Ward, M.S., Asst. An. Husband-
man in Charges
RANGE CATTLE STA., ONA
W. G. Kirk, Ph. D., An. Husb. in Charge
E. M. Hodges, Ph.D., Asso. Agron.
Gilbert A. Tucker, B.S.A., Asst. An.
Husb.4
Floyd Eubanks, B.S.A., Asst. An. Husb.
FIELD STATIONS
Leesburg
M. N. Walker, Ph.D., Plant Pathologist
in Charge
K. W. Loucks, M.S.. Asst. Plant Path.
E. E. Hartwig, Ph.D., Asst. Agron. &
Path.
Plant City
A. N. Brooks, Ph.D., Plant Pathologist
Hastings
A. H. Eddins, Ph.D., Plant Pathologist
E. N. McCubbin, Ph.D., Asso. Truck
Horticulturist
Monticello
S. O. Hill, B.S., Entomologist2 4
A. M. Phillips, B.S., Asst. Entomologist'
Bradenton
Jos. R. Beckenbach, Ph.D., Truck Horti-
culturist in Charge
E. G. Kelsheimer, Ph.D., Entomologist
F. T. McLean, Ph.D., Horticulturist
David G. Kelbert, Asst. Plant Pathologist
Sanford
R. W. Ruprecht, Ph.D., Chemist in
Charge, Celery Investigations
W. B. Shipoy. Ph.D.. Asso. Plant Path
Jack Russell, M.S., Asst. Ento.
Lakeland
E. S. Ellison, Meteorologists
'Head of Department
2In cooperation with U. S.
'Cooperative, other divisions. U. of I.
'On leave for military service.


Gift, T-of "ifi










CONTENTS
INTRODUCTION ----- -- ---.....- ---------------- 5

DISTRIBUTION AND PRESENT TAXONOMIC STATUS
OF THE FUNGUS --......--------... ---- ------------------.. ---------- 6.. 6

MATERIALS AND METHODS ----------......---- ..--- 10
Source of Races .. ..--..---------------- -- 10
Procedures ---- ----.----------------- 12

PHYSIOLOGY ----.---------- ----------------------- 13

Terminology and Possible Origin of Races _------- -- 13

Cultural Aversion in Physalospora rhodina -- ---. --- 15
Occurrence of the phenomenon -----..~....----- 15
Stability of aversion reactions -------------- 17
Races obtained from individual specimens --------------- 21
The pycnidium as the source of races ....-..- .......... -------23
Perithecia and asci as sources of races.-...... -...-- 24
Relation of aversion and sex in Physalospora rhodina -...------ 29

Cultural Characters of Races ........... ...........-------------------- 33 33

Effect of Temperature on Rate of Growth in Culture -...----.- 39
Growth of races of monoconidial origin at 7 temperatures..-- 39
Growth of races of monoconidial origin at 3 temperatures.- 42
Growth of races of monoascospore origin at one temperature 45

Effect of Temperature on Rate of Decay in Host Material ...-----. 47

MORPHOLOGY ----. ----~..--------............- ---- 49
Conidial and Ascigerous Stages in the Life History---------- 49
Mycelium ---....__---_....__--- .... -. ------..-- 50
Pycnidia and conidia ..____---...-- ------- ----- 53
Perithecia, asci, and ascospores --..----. ..---... ---------.---...- 60

Spore Measurements _.---.----_--------------------- 63
Effect of substrata on size of conidia .---------- ....---- 63
Effect of natural host source on size of conidia. ----------.. -- 68

CROSS-INOCULATION STUDIES ----..--...------------------ -... 70

TAXONOMY AND SYNONYMY ......----.----------------- 74

DISCUSSION ..----------------------.. ----------------.... ---------. 82

SUMMARY --------------..-.----------------- 86


LITERATURE CITED


.... --- 88


















































Fig. 1.-Map of the world showing the geographical distribution ofPhysalospora rhodina which cover
an area extending roughly from 300 North to 30* South of the Equator.








LIFE HISTORY AND TAXONOMY OF
THE FUNGUS PHYSALOSPORA
RHODINA'
By R. K. VOORHEES

INTRODUCTION
The complexity and variability of the genus Diplodia and
related genera offer one of the chief taxonomic problems con-
fronting mycologists and plant pathologists working with this
group of fungi. However, continued collection and study of
different forms of this group on different tropical and sub-
tropical hosts has convinced the writer that, primarily, differ-
ent forms or physiologic races of only a few specific organisms
are being dealt with. The large number of destructive diseases
caused and the wide distribution both geographically and in the
host relationships of the many forms considered has led to a
multiplicity of names, many of which might well be included
under one binominal. The attainment of this end, however, is
made somewhat difficult by the large number of taxonomic en-
tities comprised in this group and their variability in certain
morphological and physiological characters under certain con-
ditions, as well as lack of information on their sexual stages.
Many investigators have merely reported or described an
individual collection or the disease caused by a particular form,
while the attention of many other investigators has been drawn
to the above problems in the consideration of many different
or similar forms of one specific organism. Among the latter
are Petch (54, 55, 56)2, Bancroft (3, 4), Taubenhaus (77),
Nowell (51), and Stevens (67,68), who studied either the taxon-
omic or pathogenic phase or both, and the variation in cer-
tain forms and its bearing on an adequate system of classifi-
Acknowledgments.-Thanks are due to the Florida Agricultural
Experiment Station for permission to use the project "A Comparative
Study of Forms of Diplodia Resembling Diplodia frumenti" as thesis material
to the Graduate Faculty of the University of Minnesota; members of the
Departments of Plant Pathology of the Florida Agricultural Experiment
Station and the University of Minnesota for many valuable suggestions
and criticisms during the progress of this work and the preparation of
this manuscript; and the many correspondents furnishing materials for
comparison in this investigation.
'From a thesis submitted in partial fulfillment of requirements for
the Doctor of Philosophy degree in the Graduate School of the University
of Minnesota. The experiments were conducted at the Florida Agri-
cultural Experiment Station.
'Italic figures in paranthesis refer to "Literature Cited" in the back
of this bulletin.







Florida Agricultural Experiment Station


cation. Eddins (29), and Eddins and Voorhees (30), also studied
the taxonomic and pathogenic phases of certain forms, results
of which led to the present investigation.
The present studies were undertaken primarily to demon-
strate that collections of Diplodia on different tropical and sub-
tropical hosts made by the writer and other workers should be
included under Physalospora rhodina (B. and C.) Cke. It was
also demonstrated that these Diplodia forms are physiologic
races, which are variable or stable in certain physiological and
morphological characters under different environmental condi-
tions. More specifically, this investigation has entailed studies
on the possible number and distribution of these races, on their
possible origin, on their stability or variability on artificial and
host media, on their physiology and morphology, and their abil-
ity to infect different hosts by artificial inoculation.

DISTRIBUTION AND PRESENT TAXONOMIC STATUS OF
THE FUNGUS
The various conidial forms which agree with those of
Physalospora rhodina and which are considered as different
collections of it are apparently widely distributed both geograph-
ically and in their host relationships. Considerable of the con-
troversy on the taxonomic position of the fungus has been due
to this fact. The fungus is generally distributed throughout
the tropical world, having been reported from North, South,
and Central America, the East and West Indies, tropical Africa,
Ceylon, Malaya, Philippines, Samoa, and certain sections of the
Orient. As shown in Figure 1 the geographical distribution
covers an area extending roughly from 300 North to 30 South
of the Equator. In tracing the distribution and taxonomic status
Sof the fungus it is apparent that it has been described or re-
ported under many different names and on various host species.
Certain workers have reduced many of these names to syno-
nyms, which are also included under P. rhodina in the present
investigation.
In 1892 Patouillard (52) described Botryodiplodia theo-
bromae on fruits of Theobroma cacao from Ecuador. Prilleux
and Delacroix (61) figured and described a fungus on the roots
of cacao from Central America in 1894 which was said to be
particularly injurious to the plant and which they named Macro-
phoma vestita. Then followed Henning's description of Diplodia
cacaoicola on branches of Theobroma cacao from the Cam-






Life History and Taxonomy of the Fungus Physalospora Rhodina 7

eroons; it was under this name that Howard (45).first investi-
gated the parasitism of the fungus in connection with the die-
back disease of cacao in the West Indies. In 1906 Butler (13)
also reported this species from India as it occurred on sugar-
cane, but concluded that the fungus must be referred to as
Botryodiplodia because of the stromatic condition of the pyc-
nidia. In the same year Appel and Laubert (1) described a
fungus associated with diseased stems of Theobroma cacao and
Carica papaya in Samoa, and named it Lasiodiplodia nigra.
In the meantime Petch (53), working in Ceylon, described
a fungus on Hevea brasiliensis and Castilloa elasticae which
he named Botryodiplodia elasticae, and another form on pods
of Theobroma cacao which he named Chaetodiplodia grisea.
After a further study of these forms he (54) showed that B.
elasticae was identical with Diplodia cacaoicola; and was also
convinced that his Chaetodiplodia grisea was merely a hirsute
form of Botryodiplodia theobromae Pat. (56). During this same
period Charles (16) recorded a Lasiodiplodia as being parasitic
on Theobroma cacao and Mangifera indica in San Domingo, and
although not named at that time it was suggested that it might
be the same as L. tubericola E. & E. (17). In 1909 Griffon and
Maublanc (37), working in the French Congo, showed by ex-
amination of material of the above species that they were iden-
tical, and included them under their combination L. theobromae.
Petch (56) also mentioned a Chaetodiplodia recorded by Van
Hall and Drost on cacao pods in Surinam, which he includes
with all of the above species in his synonomy of Botryodiplodia
theobromae. Although not included in this synonomy Petch (56)
concludes that Diplodia rapax described by Massee (47) as
causing a disease of Para rubber in the Malay States is also
the same as Botryodiplodia theobromae.
On the basis of certain variable characters of these forms
Petch (56) suggested the abolition of the genera Lasiodiplodia
and Chaetodiplodia, the former to be included under Botryo-
diplodia and the latter under Diplodia. According to Petch (56),
Van Hall and Drost arrived at similar conclusions when they
suggested the abolition of the genus Lasiodiplodia on the ground
that the characters separating it from Diplodia are not con-
stant. Thus, they retained the old name Diplodia cacaoicola P.
Henn. instead of Botryodiplodia theobromae. At about the same
time Bancroft (3) considered several of the above species as
synonyms of Lasiodiplodia theobromae (Pat.) Griff. and Maubl.






Florida Agricultural Experiment Station


However, in the following year, Bancroft (4) reports a more
detailed study of this group, which he considered- to be the
Diplodia condition and the parasitic form of a perithecial fun-
gus described by him as Thyridaria tarda. No further record
of this fungus was made until 1929, when Tunstall (79) asso-
ciated it with the dieback disease of tea in India. However, the
connection which was not fully demonstrated in either case,
lacks confirmation, and the name has not been generally adopted.
The genus Lasiodiplodia as described by Ellis and Everhart
in 1896, in a paper by Clendenin (17), has been considered in
the review of certain species up to this time, and especially
in the controversy over L. theobromae and Botryodiplodia theo-
bromae. However, the type species Lasiodiplodia tubericola as
described by Ellis and Everhart (17), on sweet potato from
Java in 1894, was barely considered by the workers mentioned
above. Also, the fungus Diplodia natalensis described by Evans
(34), on Citrus fruits from South Africa in 1910, was not con-
sidered in the synonymy of this group until a later date. Evans
compared this species with other described Diplodia species on
Citrus and other hosts, but considered it distinct from all of
them except possibly D. ccacoicola, which it closely resembled.
Another species described earlier but not considered in the syn-
onymy of this group until later is Diplodia gossypina, described
by Cooke (24) in 1879, on cotton from India.
Taubenhaus (77) was one of the first to investigate this
group of fungi in this country. In comparing the growth of
Lasiodiplodia theobromae, L. tubericola, Diplodia gossypina
Cke., and D. natalensis Evans on sweet potatoes in a moist
chamber, he concludes that these species are congeneric. He also
concludes that the four genera Diplodia, Chaetodiplodia, Lasio-
diplodia, and Botryodiplodia "are not tenable", and all should
be combined with the genus Diplodia. Accordingly his combina-
tion of the sweet potato fungus is Diplodia tubericola (E. & E.)
Taub. Sometime later Petrak (57) examined and described a
similar fungus on potato tubers from the herbarium of J. A.
Stevenson, Washington, D. C. He concludes that the previous
work of Taubenhaus with this fungus in culture has no weight
and was quite misplaced. Thus, he referred the fungus to
Botryodiplodia and named it B. tubericola (E. & E.) Petr.
About the.same time Nowell (51) reported many of these forms
as they occurred on various crop plants in the Lesser Antilles
and suggested that the genus Lasiodiplodia and the fungus L.






Life History and Taxonomy of the Fungus Physalospora Rhodina 9

theobromae are not based on constant characters. Thus, he in-
cludes the forms considered in the synonymy by Petch (56)
and Bancroft (3) as synonyms of his combination Diplodia
theobromae (Pat.) Now. He also considers that the Diplodia
fungus on cotton bolls and D. natalensis on Citrus are prob-
ably identical with D. theobromae.
Most of the critical work with this group of fungi during
the next decade was done in this country. Beginning in 1925,
Stevens (67) reported the results of his investigations with
certain tropical forms of this group, the pycnidial stages of
which are quite similar morphologically. He described Physalos-
pora gossypina from cotton and other hosts in the Southern
United States, and considered it to be the perithecial stage of
Diplodia gossypina Cke. In the same paper he suggests that
Botryodiplodia gossypii, as described by Ellis and Bartholomew
(31) from cotton stems, is identical with Diplodia gossypina.
The following year Stevens (68) described Physalospora fusca
with brown ascospores along with a hyaline-spored Physalos-
pora on Citrus and other hosts, the conidial stages of which are
practically indistinguishable. He considered the latter Physalos-
pora to be P. rhodina (B. & C.) Cke., and included D. natalensis,
D. gossypina, and his previous combination P. gossypina as
synonyms of it. Stevens also suggested that Diplodia cacaoicola,
so generally parasitic on Hevea and Cacao, was probably iden-
tical with P. rhodina.
More recently Eddins (29) showed that D. tubericola, D.
natalensis, and D. gossypina caused a rot of ears of corn similar
to that caused by D. frumenti E. &. E. (32), and caused a sim-
ilar rot of other crop plants. In a similar manner, Eddins and
Voorhees (30) obtained positive infection on 31 species of
woody and herbaceous plants representing 24 families with cul-
tures of D. tubericola, Physalospora zeicola and P. rhodina. They
showed that D. frumenti on corn was morphologically similar
to certain Diplodia species on other crop plants, and that it
was the conidial stage of Physalospora zeicola E. &. E. (33).
Other workers (27, 36, 49, 77) also have demonstrated that
many of the Diplodia forms, occurring in the southern United
States, can pass readily from one host to another.
In the above review of what the writer considers different
forms of Physalospora rhodina the fungus has been traced from
one of its earliest described forms, Botryodiplodia theobromae
Pat., to the present investigation. Although certain other species






'Florida Agricultural Experiment Station


considered in the taxonomy and synonymy of this fungus were
described earlier than B. theobromac Pat., the controversy on
the taxonomic position of the fungus was first centered, and
still is to a great extent, around this species. Other species not
given particular attention in the above review, but still con-
sidered to be identical with or closely related to P. rhodina, are
referred to under the taxonomy and synonymy section. The
above review indicates the need of further research on the life
history and taxonomy of the organism. For instance, knowl-
edge on the physiology is especially lacking, and a correlation
between the variability of certain physiological and morpho-
logical characters is certainly in order. These are given atten-
tion in the following researches.

MATERIALS AND METHODS
SOURCE OF RACES
In the course of this investigation several hundred mono-
conidial isolations of Physalospora rhodina were made from
various host collections obtained from the southern United
States and other parts of the tropical world. A few cultures
which were single-spored before being used in this study were
also obtained from various correspondents. In most cases five
or more monoconidial isolations were made from each collec-
tion and kept in culture for some time. In practically every case
the isolates from a single collection proved to be culturally alike,
and all but one were discarded. Of the many isolations made by
the writer and received from other investigators, only 58 were
actually used for comparison in this study. As shown in Table
1, these isolations were made from different plant parts of 25
different hosts. Collection numbers 6, 8, and 9 on Zea mays
might well be Diplodia frumenti, the ascigerous stage of which
is somewhat distinct from Physalospora rhodina. However, the
pycnidial forms are not distinct and may occur on the same host
species. Thus the isolations made from these three collections
are included under P. rhodina in this study. Many of the isolates
were alike in certain cultural characters but were all distinct
physiologically by the aversion reaction between them. Accord-
ingly, they are all considered as different physiologic races of
P. rhodina and are designated as such by the collection numbers
shown in Table 1. In addition, many other monoconidia, as well
as monoascospore isolations, were made in the latter part of
this study and are referred to in the physiology section.






'Florida Agricultural Experiment Station


considered in the taxonomy and synonymy of this fungus were
described earlier than B. theobromac Pat., the controversy on
the taxonomic position of the fungus was first centered, and
still is to a great extent, around this species. Other species not
given particular attention in the above review, but still con-
sidered to be identical with or closely related to P. rhodina, are
referred to under the taxonomy and synonymy section. The
above review indicates the need of further research on the life
history and taxonomy of the organism. For instance, knowl-
edge on the physiology is especially lacking, and a correlation
between the variability of certain physiological and morpho-
logical characters is certainly in order. These are given atten-
tion in the following researches.

MATERIALS AND METHODS
SOURCE OF RACES
In the course of this investigation several hundred mono-
conidial isolations of Physalospora rhodina were made from
various host collections obtained from the southern United
States and other parts of the tropical world. A few cultures
which were single-spored before being used in this study were
also obtained from various correspondents. In most cases five
or more monoconidial isolations were made from each collec-
tion and kept in culture for some time. In practically every case
the isolates from a single collection proved to be culturally alike,
and all but one were discarded. Of the many isolations made by
the writer and received from other investigators, only 58 were
actually used for comparison in this study. As shown in Table
1, these isolations were made from different plant parts of 25
different hosts. Collection numbers 6, 8, and 9 on Zea mays
might well be Diplodia frumenti, the ascigerous stage of which
is somewhat distinct from Physalospora rhodina. However, the
pycnidial forms are not distinct and may occur on the same host
species. Thus the isolations made from these three collections
are included under P. rhodina in this study. Many of the isolates
were alike in certain cultural characters but were all distinct
physiologically by the aversion reaction between them. Accord-
ingly, they are all considered as different physiologic races of
P. rhodina and are designated as such by the collection numbers
shown in Table 1. In addition, many other monoconidia, as well
as monoascospore isolations, were made in the latter part of
this study and are referred to in the physiology section.








Life History and Taxonomy of the Fungus Physalospora Rhodina 11

TABLE 1.-SoURCES OF PHYSIOLOGIC RACES OF Physalospora rhodina USED IN THE
PRESENT INVESTIGATION.


Plant Part
Host from which Locality
Isolated


Col-
lection
No.
1
3
6
8
9
10
11
12
14
16
18
20
22
23
25
27
28
29
30
31
33
35
36
37
38
39
41
44
45
50
51
52
55
56
57
58
59
60
69
72
87
89
91
95
101
103
104
105
107
108
109
111
112
114
115
116
120
122


Pinus taeda
Theobroma cacao
Hevea brasiliensis


Arachis hypogaea Stem Gainesville, Fla.
Arachis hypogaea Stem Gainesville, Fla.
Zea mays Stalk Gainesville, Fla.
Zea mays Ear Gainesville, Fla.
Zea mays Ear Gainesville, Fla.
Gossypium hirsutum Boll Gainesville, Fla.
Gossypium hirsutum Boll Madison, Fla.
Gossypium barbadense Boll Gainesville, Fla.
Pyrus communis Branch Gainesville, Fla.
Pyrus communis Branch Gainesville, Fla.
Pyrus communis Branch Jacksonville, Fla.
Ipomoea batatas Tuber Pensacola, Fla.
Ipomoea batatas Tuber Gainesville, Fla.
Citrus aurantifolia Branch Ft. Lauderdale, Fla.
Citrus aurantifolia Branch Homestead, Fla.
Citrus sinensis Fruit Lake Alfred, Fla.
Citrus sinensis Fruit Lake Alfred, Fla.
Citrus sinensis Fruit Lake Alfred, Fla.
Fortunella margarita Branch Gainesville, Fla.
Citrus aurantifolia Branch Homestead, Fla.
Citrus aurantifolia Branch Lake Alfred, Fla.
Citrus grandis Branch Bradenton, Fla.
Citrus aurantium Branch Gainesville. Fla.
Citrus grandis Branch DeSoto City, Fla.
Citrus aurantifolia Branch Lake Placid, Fla.
Citrus aurantifolia Branch DeSoto City, Fla.
Citrus aurantifolia Branch Winter Haven, Fla.
Citrus aurantifolia Branch Sneads Island, Fla.
Citrus aurantifolia Branch Homestead, Fla.
Aleurites fordii Nut Milton, Fla.
Aleurites fordii Branch Gainesville, Fla.
Aleurites fordii Branch Monticello, Fla.
Aleurites montana Branch Gainesville, Fla.
Persea americana Branch Gainesville, Fla.
Persea americana Fruit Cocoa, Fla.
Ficus carica Branch Gainesville, Fla.
Hicoria pecan Branch Gainesville, Fla.
Hicoria pecan Branch Belle Glade, Fla.
Albizzia julibrissin Branch Reddick, Fla.
Pittosporium undulatum Branch Gainesville, Fla.
Phoenix canariensis Frond Gainesville, Fla.
Stained pine lumber Bogalusa, La.
Theobroma cacao Pod Dominican Rep.
Arachis hypogaea Stem Blountstown, Fla.
Cocos nucifera Root Key West, Fla.
Cocos nucifera Trunk Gainesville, Fla.
Citrus sp. Branch Nicosia, Cyprus
Cocos nucifera Trunk Key West, Fla.
Citrus aurantium Root Trinidad
Cocos nucifera Nut Pod Kuala Lumpur, F.M
S Australia
Theobroma cacao Pod Trinidad
Theobroma cacao Pod Philippine Is.
Cocos nucifera Nut Pod Key West, Fla.
Natal, S. Africa


.S. Alston
Young
Voorhees
Voorhees
Voorhees
Doidge


S Australia Young
Branch Terr. New Guinea Voorhees
Branch Terr. New Guinea Voorhees


1937

1937
1938
1938
1938
1938
1939
1939


Note: Leaders indicate host or plant part unknown.


I


By whom Year of
Iso- Isola-
lated tion
Voorhees 1933
Voorhees 1933
Voorhees 1936
Voorhees 1936
Voorhees 1936
Voorhees 1933
Voorhees 1935
Voorhees 1936
Voorhees 1935
Voorhees 1935
Voorhees 1936
Voorhees 1933
Voorhees 1937
Voorhees 1934
Voorhees 1935
Voorhees 1937
Voorhees 1937
Voorhees 1937
Voorhees 1935
Voorhees 1935
Voorhees 1936
Voorhees 1936
Voorhees 1936
Tisdale 1936
Tisdale 1936
Tisdale 1936
Voorhees 1936
Tisdale 1936
Tisdale 1936
Voorhees 1934
Voorhees 1935
Voorhees 1935
Voorhees 1936
Voorhees 1935
Voorhees 1936
Voorhees 1935
Voorhees 1935
Voorhees 1934
Voorhees 1935
Voorhees 1934
Voorhees 1936
Verrall 1937
Voorhees 1937
Voorhees 1937
Voorhees 1937
Voorhees 1937
Voorhees 1938
Voorhees 1938
Baker 1937






Florida Agricultural Experiment Station


PROCEDURES
A random sample of pycnidia from each collection was
crushed in a drop of water on a sterile glass slide. From this
mount the spores were taken up with a piece of sterile cotton
twisted around the end of a sterile dissecting needle, which
was streaked several times across the surface of agar in a petri
dish. The spores were allowed to germinate until the germ
tubes were approximately 100 to 200 [ long, which assured
the isolation of viable spores. After the petri dish cover was
removed the dish was placed upon the stage of a microscope
for examination. In examining a given spore streak the dish
was moved across the microscope stage until a germinating
conidium was found isolated from all other conidia. Then with
a sharp flat-pointed dissecting needle a small bit of agar in-
cluding the single conidium was picked out and transferred to
another petri dish containing potato-dextrose agar. After this
transfer the germinating conidium was allowed to grow for
approximately one day for further observation before being
transferred to an agar slant.
In the process of isolating single ascospores at random from
a given specimen the above procedure was followed. In a like
manner random samples of conidia and ascospores were iso-
lated from single pycnidia and perithecia. However, in attempt-
ing to isolate all eight ascospores from a given ascus it was
difficult to tease the spores apart without injuring some of
them. For this reason the ascospores were allowed to germinate
until the germ tubes were approximately 200 A in length. Then
the tip of each germ tube was severed with the dissecting needle
mentioned above, and a small bit of agar including the hyphal
tip was picked out and transferred to another petri dish and
subsequently to an agar slant. Even by this method consider-
able difficulty was encountered in attempting to isolate hyphal
tips of all eight ascospores of a given ascus, and this was suc-
cessful in only one case. However, the hyphal tips of sets of
four and five ascospores were isolated from several different
asci. For convenience the hyphal tips isolated from germinating
ascospores are referred to as monoascospore isolates. However,
if the characteristic aversion reaction developed between the
resulting colonies of any two isolates they are referred to as
different physiologic races.
Except where special media were used the principal sub-
stratum used in the cultural work was 2 percent potato-dextrose






Life History and Taxonomy of the Fungus Physalospora Rhodina 15

to produce the sexual stage of this fungus in the present study,
it does not preclude the possibility that these races or lines cross
or hybridize in nature and give rise to new physiological races.
The fact that physiologic forms or races of various fungi arise
through hybridization has been well established by many work-
ers, including Blakeslee (6), Hanna (39), Rodenhiser (62,63),
Christensen (18, 19, 20), Craigie (26), Dodge (28), Stakman,
et. al. (74, 75), and others. The probability that this does occur
in P. rhodina is suggested by the multiplicity of physiologically
differentiated races in this species. However, hybridization in
this species and other Ascomycetes would not be quite analogous
to that occurring in the Ustilaginaceae and certain other fungi,
in the sense that it is not a prerequisite to normal infection.
In the early part of this investigation many monoconidial
isolates from a number of host collections of P. rhodina were
paired in various combinations in an attempt to produce the
ascigerous stage. Although this stage was not produced at that
time, the stability of these monosporous mycelia indicated that
the segregation of the factor or factors for certain characters
had occurred previously in the ascigerous stage. Thus, even be-
fore viable material of the sexual stage was available for study
it was apparent that at least some of the physiologic races had
originated in this stage in nature. When the ascigerous stage
was finally investigated it was obvious that the characteristic
aversion reaction between certain monoascosporic mycelia in
culture was not distinct from that occurring between the mono-
conidial mycelia. This substantiated the contention that many
of the physiologic races of P. rhodina originate in its sexual
stage.
CULTURAL AVERSIONS IN PHYSALOSPORO RHODINA
OCCURRENCE OF THE PHENOMENON
In the course of combining several of the monoconidial iso-
lates of Physalospora rhodina shown in Table 1, as well as sev-
eral monoascospore isolates under different environmental con-
ditions, in an attempt to produce the ascigerous stage arti-
ficially, a typical aversion or inhibition between many of the
isolates was observed. Although the ascigerous stage was not
observed in any of the combinations on any substratum under
any condition, the aversion phenomenon was outstanding and
consistent. In plates containing two or more individual mono-
conidial isolates and certain monascospore isolates there was
usually an antagonistic reaction between the mycelium at the






Life History and Taxonomy of the Fungus Physalospora Rhodina 15

to produce the sexual stage of this fungus in the present study,
it does not preclude the possibility that these races or lines cross
or hybridize in nature and give rise to new physiological races.
The fact that physiologic forms or races of various fungi arise
through hybridization has been well established by many work-
ers, including Blakeslee (6), Hanna (39), Rodenhiser (62,63),
Christensen (18, 19, 20), Craigie (26), Dodge (28), Stakman,
et. al. (74, 75), and others. The probability that this does occur
in P. rhodina is suggested by the multiplicity of physiologically
differentiated races in this species. However, hybridization in
this species and other Ascomycetes would not be quite analogous
to that occurring in the Ustilaginaceae and certain other fungi,
in the sense that it is not a prerequisite to normal infection.
In the early part of this investigation many monoconidial
isolates from a number of host collections of P. rhodina were
paired in various combinations in an attempt to produce the
ascigerous stage. Although this stage was not produced at that
time, the stability of these monosporous mycelia indicated that
the segregation of the factor or factors for certain characters
had occurred previously in the ascigerous stage. Thus, even be-
fore viable material of the sexual stage was available for study
it was apparent that at least some of the physiologic races had
originated in this stage in nature. When the ascigerous stage
was finally investigated it was obvious that the characteristic
aversion reaction between certain monoascosporic mycelia in
culture was not distinct from that occurring between the mono-
conidial mycelia. This substantiated the contention that many
of the physiologic races of P. rhodina originate in its sexual
stage.
CULTURAL AVERSIONS IN PHYSALOSPORO RHODINA
OCCURRENCE OF THE PHENOMENON
In the course of combining several of the monoconidial iso-
lates of Physalospora rhodina shown in Table 1, as well as sev-
eral monoascospore isolates under different environmental con-
ditions, in an attempt to produce the ascigerous stage arti-
ficially, a typical aversion or inhibition between many of the
isolates was observed. Although the ascigerous stage was not
observed in any of the combinations on any substratum under
any condition, the aversion phenomenon was outstanding and
consistent. In plates containing two or more individual mono-
conidial isolates and certain monascospore isolates there was
usually an antagonistic reaction between the mycelium at the






Florida Agricultural Experiment Station


line of contact. This interaction varied from a complete inter-
mingling, without any apparent antagonism, to complete inhibi-
tion of growth at the point of meeting. This antagonistic reac-
tion usually resulted in a heavy line of demarcation or a dark-
ening of the substratum between the averting colonies. The
.aversion was observed to occur only between mycelium of dif-
ferent isolates and never between two colonies of the same
monospore mycelium. This phenomenon was considered from
several viewpoints and, aside from its fundamental aspects, a
further study seemed justified from the standpoint of a possible
physiological differentiation between the various isolates of P.
rhodina. Thus, studies were made on the stability of the aver-
sion reaction of various isolates following successive mycelial
transfers and following re-isolation from host material; and
on the number of different races from individual specimens,
pycnidia, perithecia, and asci.
Results of these studies at once suggested that the many
asexual forms of P. rhodina existing in nature were a com-
plex group of physiologic races. Also, regardless of the similar-
ity that existed between some of the races in their morphology
and physiology, the aversion reaction between them indicated
certain genetic and physiochemical differences. A study of the
literature reveals several cases of the aversion phenomenon,
some of which have been ascribed to the metabolism of one my-
celium causing the liberation of certain chemical substances
which are unfavorable to the development of the other mycelium
concerned. In other cases it has been reported that the aversion
between individual mycelia is not a haphazard occurrence but
that it depends upon inherited characters.
Cayley (14), in 1923, first directed particular attention to
this phenomenon in her studies with Diaporthe perniciosa. Per-
haps the first critical analysis of the cause of mutual. aversion
was made by Vandendries (80), in 1932. He demonstrated that
in Pleurotus columbinus aversion is perfectly correlated with
genetic constitution. At about the same time Cayley (15), in
further studies with Diaporthe perniciosa, considered the aver-
sion to be the outward and visible sign of a physiological hetero-
thallism, as distinguished from sex heterothallism, and con-
sidered this to be a heritable character. In the same paper she
cites as additional cases those described by Vandendries in
Coprinus micaceus, C. radians, and Paneolus complanatus; by
Brunswick in Coprinus fimetarius and in C. friesii; by Nakata







Life History and Taxonomy of the FungUs Physalospora Rhodina 17

in Selerotium rolfsii; and by Mounce in Fomes pinicola. More
recently cases of intraspecific aversion have been reported by
Vandendries and Brodie (81) in Lenzites betulina and by Brodie
(9) in Corticum calceum. Results obtained by Hoppe (44) on
the intraspecific and interspecific aversion in Diplodia are quite
analogous to the situation in Physalospora rhodina.

STABILITY OF AVERSION REACTIONS
Several different tests were made, using various monocon-
idial isolates, in studying the stability of the aversion reaction
of physiologic races of this fungus. Plating mycelial inoculum
of any individual race at opposite sides in a petri dish always
resulted in a free intermingling or overlapping of the hyphae
at the point of meeting, although the same individual may be
capable of showing aversion to other mycelia from different
sources. On plating mycelial inoculum from two averting races
at opposite sides in a petri dish, the characteristic aversion al-
ways occurred. Thus, mycelial inoculum of an individual race
was plated against itself as well as against that of other races
in all possible combinations.. Stability of reaction was main-
tained in each race, (1) following mycelial propagation on dif-
ferent media, and (2) following re-isolations from inoculated
host material.
Stability Following Mycelial Propagation.-In the first series,
mycelial inoculum from each of 30 of the monoconidial isolates
was plated in groups of seven in each culture plate (Fig. 2).
They were plated at random but it was made certain that each
isolate was paired with itself and in combination with every
other isolate in at least one instance. This method was employed
through each of three successive mycelial transfers on potato-
dextrose agar and through one mycelial transfer on each of
malt-extract and Czapek's media, making a total of five succes-
sive mycelial transfers. In the second series 35 of the monoco-
nidial isolates shown in Table 1 were employed in a similar man-
ner, but only through each of two successive mycelial transfers
on potato-dextrose agar. Stability of the aversion reaction be-
tween isolates was maintained on all three media through each
successive transfer in the first series and on potato-dextrose
agar through each of two successive transfers in the second
series. One series each of 36 and 42 of the isolates shown in
Table 1 were employed through one mycelial transfer on potato-
dextrose agar.






Florida Agricultural Experiment Station


Fig. 2.-Plate cultures showing the aversion reaction between various monoconid-
ial isolates of Physalospora rhodina from various host collections. According to
their reaction, all of the isolates represent different physiologic races.

All of the 58 isolates shown in Table 1 were employed at
least once in this study and several were employed in each of
the above series. In several cases the aversion reaction between
two different isolates was questionable because their mycelia
had not made contact when the results were recorded. When






Life History and Taxonomy of the Fungtgs Physalospora Rhodina 19

such questionable combinations were repeated and carefully
checked a certain degree of aversion usually was observed. The
aversion reaction was particularly questionable between isolates
3 and 12, which were isolated at about the same time from
hosts in two fields in the same locality. They suggested that any
given race of P. rhodina may at least be distributed in the same
vicinity. This point was further substantiated in a test where
a number of oranges were picked from several trees in a grove
and held in storage for the development of stem-end rot. Isola-
tions of P. rhodina made from many of the decayed fruit and
paired on potato-dextrose agar showed that the same race was
represented on trees throughout the grove. Also, we might ex-
pect that if a large number of isolates were made from several
different hosts in separated localities one or more physiologic
races of this fungus might be found to have a fairly wide dis-
tribution. Hoppe (44) demonstrated this point in Diplodia zeae
when he isolated 21 strains out of 25 cultures from one field,
and found 24 strains out of 25 cultures of the same fungus
from widely separated points throughout the corn belt. This
point is also clearly demonstrated in the results obtained with
several monoascospore isolates of Physalospora rhodina.
Stability Following Re-isolation from Inoculated Host Material.
-Cultures of all the monoconidial isolates from the third my-
celial transfer on potato-dextrose agar in the first series were
used for inoculations in studying the possible changes in re-
action following re-isolation from host material. As a conven-
ient and desirable host for this test, freshly picked Valencia
oranges were used. To avoid any possible natural infection en-
tering these oranges through the cut stems, they were debut-
toned shortly after being picked and the surface was sterilized.
The inoculations were made by removing a small round plug
of the rind from the stylar ends of the fruits with a sterilized
cork borer, then placing in these small cavities 5 mm. disks of
mycelial inocula of each race growing on potato-dextrose agar.
The plugs were then replaced and the wounds covered with a
small piece of nurseryman's tape. This was done in triplicate
for each of the 30 races used.
The re-isolations of each strain were first plated together
against the original stock cultures which proved that no change
in the individual races had occurred. Then one of the three re-
isolations of each race was again combined with all of the
races in groups of seven as in the previous test. Here again no






Florida Agricultural Experiment Station


aversion reaction occurred, indicating that the stability of the
races through the pathogenic phase of their life cycles was still
maintained. This was also demonstrated by Hoppe (44) in
Diplodia zeae after re-isolations from inoculated corn plants.
Results of this and previous tests show that these races are
different in their physiochemical reactions, and that such re-
action of any individual race may be different on different
media but that their original character is not greatly altered.
The stability of reaction would indicate that it is a heritable
character and that fixed genetic factors determine the physio-
chemical behavior which is maintained in these asexually prop-
agated lines. There is apparently no definite relationship be-


aj i


Fig. 3.-Plate cultures showing 15 monoconidial isolates of Physalospora rhodina.
isolated from 15 different pycnidia on a specimen of Citrus aurantifolia, and paired
in all possible combinations on potato-dextrose agar. According to their reaction,
one race occurred 13 times and two others occurred once each.






Life History and Taxonomy of the Fungus Physalospora Rhodina 21

tween the capacity of races for inhibiting the development of
others and their degree of pathogenicity.
RACES OBTAINED FROM INDIVIDUAL SPECIMENS
Some information on the number of physiologic races of
P. rhodina occurring on individual specimens was obtained
from tests in which monoconidial and monoascospore isolates
selected at random were paired on potato-dextrose agar in all
possible combinations. One monoconidial isolate was made from
each of 15 different pycnidia from each of six varied host speci-
mens. Each set of 15 monoconidial isolates was combined in
groups of seven on potato-dextrose agar, as in the previous
tests. Results from the combinations in one set showed that
one race occurred 13 times and two others occurred once each,
making a total of three different physiologic races in this set
(Fig. 3). On the other hand, there was apparently only one
race involved in each of the other five sets. The dominance of
one race over another in such cases may depend somewhat on
the aversion reaction or antagonism between these races in the
host plant, but not necessarily. This point was demonstrated
by Hoppe (44) when he artificially inoculated ears of corn with
a mixture of three strains of Diplodia zeae and almost consis-
tently re-isolated only one of the three strains. The antagonis-
tic action of one fungus or a race of a fungus toward another
may be due to a variety of causes and may result in numerous
modifications of the organisms involved, but no detailed dis-
cussion of this will be presented at this time.
Fifteen monoascopore isolates selected at random from each
of several citrus limbs were employed through each of two suc-
cessive mycelial transfers on potato-dextrose agar. Stability of
the aversion reaction between the averting colonies was main-
tained through each mycelial transfer. When two transfers of any
one monoascosporic mycelium were plated together there was al-
ways the free intermingling of the hyphae at the point of meet-
ing, in exactly the same manner as with the monoconidial iso-
lates. However, unlike any of the monoconidial isolates in the
above series, several of the monoascospore isolates were com-
patible and did not show the characteristic aversion reaction
between them. This indicated that these particular isolates
were of the same physiologic race. In one set of 15 random
monoascospore isolates numbers 3, 5, and 15, 9 and 10 showed






22 Florida Agricultural Experiment Station

no aversion reaction when combined on potato-dextrose agar
as shown in Table 2.

TABLE 2.-RESULTS OF PAIRING IN ALL POSSIBLE COMBINATIONS 15 MONOASCOSPOROUS
MYCELIA ISOLATED AT RANDOM FROM EACH OF TWO DIFFERENT CITRUS SPECI-
MENS (A AND B).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 0 + + + + + + + + + + + + + +
2 + 0 + + + + + + + + + + + + +
3 + + 0 + 0 + + + + + + + + 0
4+ ++ 0 + ++ +++ + ++++
5 + + 0 + 0 + + + + + + + + + 0
6 + + + + + 0 + ++ +++++ +
7 ++++++ 0 +++++++ +
8 +++++++ 0 + +++++ +
9 ++++++ + + 0 0 +++ ++
11 + + + + + + + + ++ 0 + + + +
12 + + + + + + + + + + + 0 + + +
13 + + + + + + + + + + + + 0 + +
14 + + + + + + + + + + + + + 0 +
15 + + 0 + 0 + + + + + + + + + 0
+ = Aversion 0 = No aversion

Since the monoascospore isolates used in this test were
selected at random, it was not known how many were from the
same ascus or perithecium in either set. However, certain iso-
lates of each set showed no aversion reaction when combined
on potato-dextrose agar, which would indicate that such iso-
lates were probably of the same physiologic race and that these
races probably originated in the sexual stage of this fungus.
This was the first definite evidence that the physiological dif-
ferences among the races might be controlled by genetic fac-
tors. Hoppe (44) considered aversion in Diplodia zeae to be
an interaction between two physiologically different strains,
and since the evidence indicated genetic differences among the
strains there was probably an undescribed sexual stage of this
fungus. The writer arrived at similar conclusions while work-
ing with D. zeae and D. macrospora during 1931-35, and was
also led to believe that D. macrospora might be a mutation or
hybrid of Diplodia zeae.
The above tests indicate that the multiplicity of physiologic
races of Physalospora rhodina on individual specimens bearing
the sexual stage is perhaps due mainly to the segregation of
the factor or factors for aversion. However, this does not pre-
clude the possibility that the occurrence of two or more races







Life History and Taxonomy of the Fungus Physalospora Rhodina 23

on the same specimen might be due to multiple infection. This
is apparently the case where the sexual stage is not involved,
since there seems to be very little if any delayed segregation
of factors for aversion in the asexually propagated lines. This
was also suggested by Cayley (14), and she (15) later sug-
gested that this was not improbable but that the presence of
averting strains in the same host must be mainly due to the
segregation of the factor or factors for aversion carried by
the mycelia.
Porter (60) showed that antagonism between fungi becomes
more marked as the phylogenetic relationships are widened.
Thus, it would seem reasonable to assume that the occurrence
of two or more races of a fungus together in nature would be
far more common than the occurrence of races of two or more
different fungi. The dominance of one fungus over another
in mixed cultures will also depend somewhat on the inhibition
or antagonism between these fungi. This was demonstrated by
Savastano and Fawcett (64) when they inoculated lemons and
oranges with mixtures of several different fungi, including
Diplodia natalensis, and found a complete dominance of the
external color and odor characteristic of D. natalensis and re-
isolated this fungus alone in 38 out of 40 cases.
THE PYCNIDIUM AS THE SOURCE OF RACES
In a manner similar to the previous test, 15 monoconidial
isolates were made from each of several pycnidia from differ-
ent host specimens. When these isolates from a given pycnidium
were combined on potato-dextrose agar the typical interming-
ling of their hyphae at the line of contact always resulted. This
indicates a uniformity in reaction for all spores from a given
pycnidium of Physalospora rhodina, which might be expected
at least in the asexual reproductive bodies of some fungi where
the genetic factors of an individual asexual line or race appear
to be fixed or maintained through asexual reproduction. How-
ever, in Sphaeropsis malorum, a fungus systematically close to
the pycnidial stage of Physalospora rhodina, Mohendra and
Mitra (50) demonstrated that two mycelial types may arise
from the spores of a single pycnidium or even a single spore.
They suggest that in such cases there must have been previous
segregation in the type of bud variation. Hansen (40) pointed
out the cohesive nature of the spore mass in a pycnidium and
that the spores of many members of the Sphaeropsidales ana-






24 Florida Agricultural Experiment Station

stomose readily, greatly minimizing the possibility of distribu-
tion of individual spores.
In determining the nuclear condition of the conidia of P.
rhodina, the young hyaline spores were observed to be multi-
nucleate but the nuclear condition of the hyphae of germinating
mature conidia was clearly demonstrated, as shown in Figure
6. For the most part the staining was done 12 hours after
germination and most of the septa were either obscure or had
not been formed during this period. In a few cases where the
hyphae had become septate during this period the cells were
multinucleate (Fig. 6). In all probability the conidia were at
least originally uninucleate, but regardless of their nuclear con-
dition the stability of the aversion reaction of all spores of a
given pycnidium would lead one to believe that they are all gen-
etically identical, or at least as far as the factors for aversion
carried by their mycelia are concerned. Granting the possibility
of the existence of nuclei of different genetic constitution, a
random assortment of nuclei by virtue of the frequently oc-
curring phenomenon of anastomosis would not preclude the pos-
sibility of conidia of different nuclear make-up. According to
Hansen and Smith (41) the frequently-occurring multinuclear
condition in hyphal cells and spores of certain fungi where
anastomosis or fusions have occurred, has suggested the possi-
bility of an irregular distribution of genetically dissimilar nu-
clei in cell division or conidial formation. Moreover, this phen-
omenon has been the basis of considerable speculation in rela-
tion to the origin of variation in fungi. Although anastomosing
of the hyphae of P. rhodina has been observed in the course of
this investigation, the stability of reaction of all the spores of
a given pycnidium is maintained for the most part through sub-
sequent conidia or mycelial generations. This is in agreement
with the results obtained by Hoppe (44) in Diplodia zeae, and
by Cayley (14,15) in Diaporthe perniciosa.
PERITHECIA AND ASCI AS SOURCES OF RACES
To obtain further information on the stability of the aver-
sion reaction between monoascosporic mycelia of Physalospora
rhodina, several series of monoascospore isolations were made
from different perithecia and asci on Citrus limbs. The first
series included 15 monoascospore isolates from the same speci-
men; 1-7 each from a different perithecium, 8-11 selected at
random from a single perithecium, and 12-15 from a single







Life History and Taxonomy of the Fungus Physalospora Rhodina 25

ascus. When these isolates were plated in all possible combina-
tions on potato-dextrose agar the characteristic aversion re-
action occurred between all of them, except between isolates
12 and 15 and 13 and 14, as shown in Table 3. Thus, accord-
ing to the aversion reaction of the four isolates from this single


TABLE 3.-RESULTS OF PAIRING IN ALL POSSIBLE COMBINATIONS 15 MONOASCOSPOROUS
MYCELIA ISOLATED FROM A CITRUS SPECIMEN*.


1 2 3 4 5 6 7
1 0 +++ ++ +
2 + 0 + + + + +
3 ++ 0 + + + +
4 + + + 0 + + +
5 + ++ 0 + +
6 + + +++ 0 +
7 + + + + + + 0
8 + +++++ +
9 +++++++
10 + + + + + + +
11 + + + + + + +
12 + + + + + + +
13 + + + + + + +
14 + + + + + + +
15 + + + + + + +


8 9 10 11 12 13 14 15
++++ + +++
++++ + + + +
+ + + + + + + +
+ + + + + + + +
+ +++ + + + +
++++ ++++
+ + ++ + + +
+ + + + + + + +
0 +++ ++ +
+ 0 ++ ++++
++ 0 + + + + +
+ + + 0 + + + +
++++ 0 ++ 0
+ + + + + 0 0 +
+ + + + + 0 0 +
+ + + + 0 ++ 0


*Each of isolates 1-7 from a different perithecium, 8-11 from a single
perithecium, 12-15 from a single ascus.
+ = aversion 0 = no aversion


ascus, two races were involved (Fig. 4-A). There was no aver-
sion between two colonies of the same monoascospore mycelium
and the free intermingling of the hyphae at the line of contact
always occurred as in certain monoconidial mycelia. The sta-
bility of the aversion reaction between the isolates of this series
as well as some of the following series was maintained through
each of three successive transfers on potato-dextrose agar. This
indicates that the factor or factors for the aversion of the my-
celium from any ascospore are fixed and maintained through
subsequent conidial and mycelial generations.
The second series included nine monoascospore isolates se-
lected at random from a single perithecium from the same
specimen used in the first series. The aversion reaction oc-
curred between all of the monoascospore mycelia of this series,
except between that of isolates 1 and 8 and 4 and 7, as shown
in Table 4. Thus, according to the aversion reaction between






Florida Agricultural Experiment Station


is, .F.


I,'


Fig. 4.-Plate cultures showing the aversion reaction between monoascospore
isolates of Physalospora rhodina from a Citrus specimen. (A) Isolates 1-7 from a
different perithecium, 8-11 from a single perithecium, and 12-15 from a single ascus.
Isolates 12 and 15; 13 and 14 are of the same race (See Table 3). (B) Nine mono-
ascospore isolates from a single perithecium. Isolates 1 and 8; 4 and 7 are of the
same race (See Table 4).






Life History and Taxonomy of the Fungus Physalospora Rhodina 27
TABLE 4.-RESULTS OF PAIRING IN ALL POSSIBLE COMBINATIONS 9 MONOASCOSPOROUS
MYCELIA ISOLATED AT RANDOM FROM A SINGLE PERITHECIUM.
_1 2 3 4 5 6 7 8 9
1 0 + + + + + + 0 +
2 + 0 + + + + + + +
3 + + 0 + + + + + +
4 + + + 0 + + 0 + +
5 + + + + 0 + + + +
6 + + + + + 0 + + +
7 + + + 0 + + 0 + +
8 0 + + + + + + 0 +
9 + + + + + + + + 0
+ = aversion 0 = no aversion
the isolates in this series, five races occurred once each and
two races occurred twice each (Fig. 4-B). A similar type of
intra-perithecial aversion was demonstrated in Diaporthe per-
niciosa by Cayley (15), which she considered was different
from the inter-racial aversion between biologic races, although
the two forms of aversion were macroscopically indistinguish-
able. The type of aversion reaction between monoascosporic
mycelia of Physalospora rhodina is indistinguishable from that
occurring between certain monoconidial mycelia. Moreover, the
evidence obtained thus far does not indicate any difference be-
tween the type of aversion occurring within perithecia or asci
and that occurring between asexual races in nature.
To ascertain further whether there was any difference in
the type of aversion between certain monoascosporic mycelia
and that occurring between certain monoconidial mycelia, num-
erous attempts were made to isolate and study the behavior of
all eight monoascosporic mycelia from several different asci.
Several sets of four and five spores were readily isolated from
various asci but considerable difficulty was encountered in
attempting to isolate all eight spores of a given ascus, and this
was successful in only one case. The third series of mycelia
combinations in this section included 20 of these monoascospore
isolates, four from each of five different asci from the same
perithecium. Each set of four monoascosporic mycelia repre-
senting a given ascus was numbered consecutively, as 1-4, 5-8,
etc., and was paired in all possible combinations on potato-
dextrose agar. Results of these pairings showed that two kinds
of mycelia were involved in one set of four isolates and that
three kinds of mycelia were involved in each of the other four
sets of isolates, as shown in Table 5. Also, all of the mono-
ascosporic mycelia of any given set showed aversion to all of
the mycelia of all of the other sets (Fig. 5).





Florida Agricultural Experiment Station


A




























Fig. 5.-Plate cultures showing the aversion reaction between monoascospore
isolates of Physalospora rhodina from a Citrus specimen. (A) Four isolates from each
of five different asci from a single perithecium. All combinations are not shown, but
each pair of isolates 1 and 3, 5 and 8, 9 and 12, 10 and 11, 15 and 16, 17 and 18
are of the same race (See Table 5). (B) Eight isolates from a single ascus paired in
all possible combinations, four of which are of one race and four of another (See
Table 6).







Life History and Taxonomy of the Fungus Physalospora Rhodina 29

These results suggest that regardless of whether this aver-
sion reaction is a sign of sex or physiological heterothallism,
the factor or factors for it are carried by the ascospores in
different ratios in the ascus and that segregation for same
occurs in either the first or second nuclear division. The re-
sults also indicate that the eight monoascosporic mycelia of
one ascus are not compatible with any of the eight monoasco-
sporic mycelia of another ascus, regardless of whether the two
asci involved are from the same or different perithecia.

TABLE 5.-RESULTS OF PAIRING IN ALL POSSIBLE COMBINATIONS 20 MONOASCOSPOR-
OUS MYCELIA, FOUR FROM EACI OF FIVE DIFFERENT ASCI FROM A SINGLE PERI-
TIIECIUM FROM A CITRUS SPECIMEN.


1 2 3 4 5 6 7 81 9
S+ 0+ + ++ + +
1+0++ ++++ +
0 +0+ ++++ +
+++o ++++ +
I++++ 0 + + 0 +
++++++0+++
++++++0++
++++ ++ 0 +
++++ ++++ 0
++++ ++++ +
++++ ++++ +
++++++++ 0
++++ ++++ +
++++ ++++ +
+++ ++++ +
++++ ++++ +
+++ ++++ +
+++ ++++ +
++++++++ +
+ = aversion


10 11 12 13 14 15 16 117 18 19 20
+ + ++++ ++++
+++ ++++ ++++
+++ ++++ ++++
++ + + + .+ + + + +
+++ ++++ ++++
+++ ++++ ++++
+ + ++++ ++++
+++ ++++ ++++
++ 0 ++++ ++++
0 0+ ++++ ++++
S0 + +++ ++++
++ 0 ++++ ++++
+++ 0 ++ ++++
++++ ++ ++++
+++++ o0 ++++
+++++ 0o ++++
+++ + + + 0 0 + +
++ + ++ ++ 0 0 ++
+++ ++ + ++ 0 +
+++++ +++++ 0
0 = no aversion


RELATION OF AVERSION AND SEX IN PHYSALOSPORA RHODINA
The manifestation of the aversion phenomenon between cer-
tain monoascospore and monoconidial isolates in this study has
led the writer to believe that it is probably a sign of a physio-
logical difference between physiologic races of P. rhodina, and
that it is inherited in a definite manner. Along this line it was
hoped that some definite scheme could be arrived at as to the
inheritance of aversion and the correlation, if any, between
sex and the capacity for showing aversion. Such a scheme in
this investigation has been hampered by the lack of cytological
evidence and by the fact that the sexual stage could not be pro-
duced artificially either in culture or on host material. Al-


1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20






Florida Agricultural Experiment Station


though not available in the present study, Eddins and Voor-
hees (30) showed that certain monoascospore cultures of the
genetically related fungus P. zeicola produced perithecia on
inoculated cornstalks, which would suggest homothallism. On
the other hand, the physiologically differentiated types of
ascosporic isolations of P. rhodina would suggest some type of
heterothallism, but valid proof that such physiological differ-
ences depend on differences in genetic constitution can be ob-
tained only by crossing tests.
Except in one case considerable difficulty was encountered
in attempting to isolate the eight spores of an ascus, or even
the hyphal tips of the germinating spores. On account of the
biserrate arrangement of the ascospores in the asci of P. rhodina
it was practically impossible to isolate the eight spores in any
definite order, and the eight spores of the ascus concerned
were isolated at random. After isolation it was found that four
of the isolates were of one race and four of another. Thus, the
four isolates of one race were given the odd numbers and the
four isolates of the other race were given the even numbers.
This is shown in Table 6, and was determined by pairing the
monoascosporic mycelia in all possible combinations on potato-
dextrose agar, as illustrated in Figure 5.

TABLE 6.-RESULTS OF PAIRING IN ALL POSSIBLE COMBINATIONS 8 MONOASCOSPOROUS
MYCELIA ISOLATED AT RANDOMg FROM A SINGLE Ascus.

I1 2 3 4 5 6 7 8
1 0 + 0 + 0 + 0 +
2 + 0 + 0 + 0 + 0
3 0 + 0 + 0 + 0 +
4 + 0 + 0 + 0 + 0
5 0 + 0 + 0 + 0 +
6 + 0 + 0 + 0 + 0
7 0 + 0 + 0 + 0 +
8 + 0 +. 0 + 0 + 0
+ = aversion 0 = no aversion

The results of this test show that the aversion reaction of
the eight monoascosporic mycelia of this particular ascus oc-
curred in a 1:1 ratio. Regardless of whether this aversion re-
action is a manifestation of sex or physiological heterothallism,
evidence reported herein indicates that the segregation for same
occurs in either the first or second meiotic divisions, which is
in agreement with the inheritance for sex reported in many
species of Ascomycetes. Cayley (15) observed in Diaporthe







Life History and Taxonomy of the Fungus Physalospora Rhodina 31


2oA


Fig. 6.-(A-E) Nuclear condition of ascospores and hyphae of Physalospora
rhodina. (F-J) Nuclear condition of conidial hyphae of P. rhodina; (I) multinu-
cleate condition of hyphal cells. All approximately 12 hours after germination.






Florida Agricultural Experiment Station


perniciosa that the monoascospore cultures remained stable but
that certain monoascus cultures showed sectoring. Thus, she
concludes that the spores of an ascus of this species are not all
genetically the same. She also found that the monoascospore
isolates from the same perithecium may segregate in a very
definite manner in the second and subsequent ascospore genera-
tions. Since sex can be distributed among the nuclei of the ascus
in several different ways, as reported in many species of As-
comycetes, it seems justifiable to assume that other segregating
characters or factors can be distributed in a like manner.
The essential cytological criteria for sex, namely, nuclear
fusion and the subsequent meiotic divisions with or without a
reduction in the number of chromosomes, have long been known
and described by various workers in many species of As-
comycetes. Although cytological studies of ascospore formation
in Physalospora rhodina have not been undertaken, we may
assume, as is conceded to be the case in most Ascomycetes pos-
sessing 8-spored asci, that each ascospore initially contains a
single haploid nucleus. Ascospores of P. rhodina were stained
approximately 18 hours after germination at room tempera-
ture and were observed to be multinucleate at this stage, as
shown in Figure 6. Thus, the haploid nucleus in each ascospore
divides several times before or shortly after spore germination,
which is also probably the case in the conidia. The septa in the
ascosporic hyphae were either obscure or had not been formed
during this period but the hyphal cells are probably multi-
nucleate by virtue of the distribution of the nuclei and by
analogy of the multinucleate condition of the hyphal cells of
germinating conidia. Although the ascosporic hyphae are multi-
nucleate, all of the nuclei have their origin in the mother nuc-
leus of the cell from which the hypha was produced. There-
fore, they should be genetically alike.
Evidence reported herein on the stability of the monoco-
nidial and monoascospore lines of P. rhodina indicates that all
of the nuclei involved in any given line are genetically identical.
This is corroborated by the fact that there was very little if
any evidence of sectoring in any of the monosporic mycelia,
whether due to heterocaryosis or any other condition. Although
unobserved in the present study, this does not preclude the pos-
sibility that such variations do occur in this fungus. The ques-
tion of heritable variations that may arise in the vegetative
stage of this fungus remains to be further investigated.






Life History and Taxonomy of the Fungus Physalospora Rhodina 33

CULTURAL CHARACTERS OF RACES
Other workers (67, 68, 72) have indicated that there are
distinct cultural races or strains of Physalospora rhodina, but
no extensive study has ever been made of the cultural behavior
of a large number of races of this fungus. The same races
used in the rate of growth studies at room temperature and
shown in Table 9 were used in the present test. These races
from various sources were grown on potato-dextrose agar and
malt-extract agar. Although some of these races were quite
similar in this rate of growth (Table 8) and their cultural char-
acteristics (Table 7), they were all distinctly different in their
aversion reaction. Thus, the cultural characteristics of all these
races were considered in the present study.
The mycelium of all of the races is usually light colored in
young cultures but becomes darker with age. There is consid-
erable contrast in the shades of color of both the mycelium and
substratum between some of the races, but very little contrast
is evident between many of them. Varying intensities of gray3,
green, and black were observed, as shown in Table 7. The type
of growth in some races was somewhat oppressed, but in the
majority the mycelium was a loose, cottony, aerial growth. In
general, pycnidia formation was more abundant on malt-extract
agar than on potato-dextrose agar, but very few of the races
produced pycnidia on either medium until five or six days after
inoculation. As indicated in Table 7, certain races produced
small single or aggregated pycnidia, which were produced
either slightly below the surface of the medium, on the surface,
or on the aerial mycelium. Also, certain races produced stro-
matic masses on the surface of the medium and pycnidia were
formed in the surface layers of these masses. Further treat-
ment on the pycnidia production of these races will be found
later in this paper.
Some of the races of this fungus may vary considerably in
color and type of mycelial growth when grown at different tem-
peratures as well as on different media. As indicated in Tables 8
and 9, the majority of the races studied showed chromogenesis
when grown at 360 C. or above, and the mycelium as well as
the substratum may appear pink or red at these higher tempera-
tures. As indicated in Table 8, certain races showed no growth
at 40 C., but the inoculum itself was able to produce chromo-
"Colors given are those described in Ridgway's Color Standard,
Washington, D. C., 1912.








TABLE 7.-CULTURAL CHARACTERISTICS OF 33 RACES OF Physalospora rhodina OF MONOCONIDIAL ORIGIN GROWN ON POTATO-DEXTROSE AGAR
AT ROOM TEMPERATURE AFTER 10 DAYS.

Race Type of
Number Growth Color of Mycelium Type of Pycnidia

8 Cottony Light olive-gray to dark olive-gray In small stroma, scattered over surface of medium.
9 Cottony Olivaceous black (3) In large stroma, scattered over surface of medium and around "
edge of dish.
11 Woolly Olivaceous black (3) to dark ivy- None produced.
green
12 Cottony Olivaceous black (1) to dark ivy- In large stroma, scattered over surface of medium.
green
14 Cottony Olive-gray to dusky olive-green None produced.
16 Cottony Olivaceous black (2) to dusky Some large stroma, scattered around edge of dish, mostly
olivee-green single or grouped, without stroma, scattered over surface.
18 Cottony Olivaceous black (3) to dark ivy- In large stroma, scattered around edge of dish.
to woolly green
22 Cottony Ivy-green to dark olive-gray In small stroma, scattered around edge of dish.
30 Cottony Dusky olive-green None produced.
31 Cottony Olivaceous black (3) 7.,l:: separate, without stroma, superficial to aerial, scat-
tered around edge of dish.
33 Cottony Light olive-gray to dark olive-green None produced.
35 Cottony Dusky olive-green to olive-gray In small stroma, scattered around edge of dish.
36 Cottony Olivaceous black (3) In large stroma, scattered over surface of medium.
to woolly
37 Cottony Dusky olive-green to olive-gray In small stroma, scattered over surface of medium.
39 Cottony Olivaceous black (2) In large stroma, scattered over surface of medium and
around edge of dish.
44 Cottony Dark olive-gray Some in small stroma, mostly separate or aggregated, with-
out stroma, immersed to superficial, well distributed over
medium.







52 Cottony Olivaceous black (3)


Cottony
Cottony

Cottony
Cottony

Cottony
Appressed
to velvet
Cottony

Appressed
to woolly
Cottony


103 Appressed
to cottony
104 Cottony

105 Cottony
107 Cottony
108 Woolly
to cottony
111 Cottony

112 Cottony


Olive-gray
Dark olive-gray

Olivaceous black (3)
Olivaceous black (3)

Olive-gray to dark olive-gray
Light olive-gray

Olivaceous black (3) to dusty olive-
gray
Dirty white

Olivaceous black (3)

Olivaceous black (2) to ivy-green


Deep olive-gray

Olivaceous black (1)
Olivaceous black (3)
Olivaceous black (3)

Olivaceous black (3)
green
Olivaceous black (1)


to ivy-green




to dark ivy-


Separate or aggregated, without stroma, immersed to super-
ficial, distributed over surface and aggregated around edge
of dish.
In large stroma, scattered over surface of medium.
Separate, without stroma, superficial to aerial, scattered over
surface of medium.
In long stromatic necks, scattered over surface of medium.
In large stroma, scattered over surface of medium, and around
edge of dish.
None produced.
None produced.

None produced.

None produced.

In large stroma, scattered over surface of medium and around
eedge of dish.
Some small stroma, mostly single or grouped, immersed to
superficial, scattered over medium.
Single to aggregated, without stroma, well distributed over
surface of medium.
In large stroma, scattered around edge of dish.
In large stroma, around edge of dish.
Very small, separate to aggregated, without stroma, immersed
to superficial, abundant over surface of medium.
None produced.

Some large stroma, mostly separate or grouped, superficial to
aerial, scattered over surface of medium and around edge
of dish.










TABLE 8.-EFFECT OF TEMPERATURE ON GROWTH OF 12 RACES OF Physalospora rhodina oF MONOCONIDIAL ORIGIN ON POTATO-DEXTROSE AGAR.
(Results given in millimeters as average diameter of colonies 60 hours after inoculation)


Race Average Colony Diameter
No.
10, C. 150 C. 200 C. 25 C. 30 C. 35 C. 40 C.

3 12.0 1.26 46.3 1.00 69.6 + 1.16 82.3 1.36 85.0 .10 80.3 1.83 13.7 .65*
9 Trace 25.0 .10 60.3 1.07 78.3 1.31 83.6 1.49 75.6 1.43 0*
12 3.0 .09 41.0 .62 58.3 .83 80.3 1.16 81.0 .08 63.0 1.24 0*
18 10.7 .18 31.0 1.09 48.3 .77 66.3 1.28 74.0 .08 55.0 1.27 0
22 Trace 36.3 .71 53.0 .62 78.3 1.13 83.0 1.09 64.3 1.32 0*
36 0 30.0 .55 62.3 1.22 82.3 1.16 85.0 .08 76.3 1.55 Trace*
52 4.3 .98 25.0 .62 53.6 1.09 72.3 1.28 74.3 -t 1.28 67.0 .08 12.0 .63*
57 7.0 + 1.38 20.3 .85 54.3 .98 71.7 .76 69.3 .96 48.3 .78 4.0 .59*
58 3.7 .44 29.0 .10 48.3 .78 79.0 .60 79.7 .51 63.0 1.09 0
59 6.3 .77 29.7 .78 55.0 .10 81.0 1.09 85.0 .10 74.3 .10 0*
103 0 36.0 .10 66.3 1.26 78.3 1.16 82.3 .98 59.7 1.39 Trace*
111 7.7 .07 34.3 .68 58.3 1.47 78.3 1.65 79.0 .10 55.0 .84 0

*Races showing chromogenesis at 400 C. after 60 hours.








Life History and Taxonomy of the Fungus Physalospora Rhodina 37

TABLE Q.-AVERAGE DAILY GROWTH OF RACES OF Physalospora rhodina OF MONO-
CONIDLAL ORIGIN ON Two DIFFERENT MEDIA AT DIFFERENT TEMPERATURES.


(Results given in millimeters as average diameter
after inoculation)


of colonies 60 hours


First Test Second Test

Room temperature
30 C. 36' C. (av. 29 C.)
Race Av. daily growth on Av. daily growth cn Av. daily growth on
No. Potato- Malt- Potato- Malt- Potato- Malt-
dextrose extract dextrose extract dextrose extract
agar agar agar agar agar agar


8
9
11
12
14
16
18
22
25
30
31
33
35
36
37
39
44
52.
55
56
57
58
59
60
91
95
101
103
104
105
107
108
111
112
114
Average


33.2
30.8
34.4
27.6

35.6
35.6
34.0
30.0
25.2

26.4
34.8
31.6
29.6
35.2
34.8
26.4
27.6
27.6
33.6
36.0
34.8
29.2
29.6
30.8
29.6


36.0
36.0
35.6
34.4
36.0
32.1+.38


28.4
26.4
28.0
26.0

31.6
28.4
32.4
27.2
26.0

24.0
30.0
28.0
27.2
30.8
27.6
26.0
28.4
22.8
30.0
36.0
30.8
25.6
29.2
26.8
27.2


32.8
34.8
31.2
28.0
36.0
29.0+-.41


32.4*
16.0*
27.6*
25.6

29.6
26.4*
9.6*
13.6
22.8

24.4
31.6*
25.6
24.8*
29.2*
17.2*
14.0*
10.8*
18.8*
27.2*
35.6*
6.8*
26.4*
13.6*
27.2
27.6*


30.0
9.2*
21.6
30.4
35.2*
23.0 .99


24.8*
14.8*
25.6*
24.0*

28.8
24.4*
9.2*
12.8
19.2

24.8
28.0*
21.2
21.2*
28.8*
12.8*
7.6*
5.2*
18.8*
20.8*
31.2*
3.2*
28.0*
9.2*
25.6
25.2*


29.2
4.8*
19.2
27.6
22.4*
20.0.98


34.8
35.6
29.2
34.8
31.6
34.0
30.4
33.2

36.0
34.0
31.6
34.8
35.6
35.6
33.2
36.0
32.0
29.2
26.8
31.6
33.2
36.0
24.8
32.0
36.0
33.2
36.0'
36.0
36.0
35.6
33.6
24.4
33.2

33.0+.40


30.8
30.4
26.0
30.0
27.2
30.4
25.2
25.2

31.6
29.6
27.2
28.8
28.8
28.8
27.2
30.8
26.0
25.2
24.0
25.2
27.2
33.2
17.2
26.8
33.6
24.0
31.6
S27.6
34.0
28.8
27.2
26.8
14.4

27.6-.47


*Races showing chromogenesis at 36 C. after 60 hours.






Florida Agricultural Experiment Station


genesis in the substratum at this temperature. In these tests
none of the races showed this character at 350 C. or below,
even when held at these lower temperatures for a considerable
length of time. Repeated tests with these races on the same
medium under similar conditions indicate that pigment pro-
duction is definitely characteristic of certain forms regardless
of their source. Stock cultures of several of the races on potato-
dextrose agar stored in a refrigerator at approximately 170
C. also showed chromogenesis. However, when transfers were
made to potato-dextrose agar in petri dishes from the stock
cultures showing this character and held at 350 C. or below,
this character did not appear. Although this phenomenon is
apparently not correlated with type or rate of growth, the mani-
festation of same is correlated with temperature relations.
The ability of certain fungi to produce pigments in the my-
celium or the substratum has been shown by various workers.
Stevens and Wilcox (72) and Stevens (67) showed that many
strains of P. rhodina turned the medium a bright pink or red
when grown on potato-dextrose agar at 370 C. Christensen (21)
showed that certain varieties of corn attacked by Ustilago zeae
react by producing anthocyan, so that their tissues appear to
be colored red. Christensen and Graham (22) showed that many
intergrading shades of color were produced in the substratum
by different races of Helminthosporium gramineum. The iden-
tity of the red pigment produced by certain races of Physa-
lospora rhodina was not determined, but it is probably an
anthocyan. It was also observed in cultures of several races in
association with bacterial and fungous contaminations. This
association seemed to stimulate or induce pigment production
by certain forms but not by others.
The dual heterocaryotic condition of many fungi in nature
is referred to by Hansen (40) as a "dual phenomenon", in
which single-spore series of many fungi give rise to three cul-
ture types, namely, M (mycelial), C (conidial), and Mc, an
intermediate type. Similar types are represented in the races
of P. rhodina, but there was no indication that any of the mono-
conidial isolates were dual in nature. The mycelium of a few
races when first isolated was dark as in the other races, but
through the process of transferring the mycelium for several
generations it gradually changed from cottony and dark to
appressed and white, as indicated by races 60 and 95 in Table
7. This change was gradual and not sudden as would be ex-






Life History and Taxonomy of the Fungus Physalospora Rhodina 39

pected in the segregation of characters in a heterozygous line
or a line dual in nature. The loss of capacity for pycnidia
formation was also evident in these races. However, certain
other races showing no change in mycelial color have never
sporulated from the time of isolation. This gradual change in
the color of the mycelium and the loss of capacity for pycnidia
formation in certain races is probably due to a gradual loss of
the factors controlling these characters that appear to be fixed
in the other lines. A similar condition was observed in cultures
of Sphaeropsis malorum by Mohendra and Mitra (50), in
which spores from a single pycnidium or even a single spore
might produce two kinds of daughter colonies, black and white.
They suggest that "this is a gradual replacement of a vigor-
ously spring black type of mycelium by a poorly spring white
type because of certain physiological differences in the course
of a number of generations." The mycelial colonies of certain
forms of Physalospora rhodina were often slightly irregular in
form or rather evenly lobed, especially at the higher tempera-
tures, as shown in Figures 8 and 9. Such irregularities in va-
rious colonies frequently resemble sectors or mutations, but
they do not behave as such.

EFFECT OF TEMPERATURE ON RATE OF GROWTH IN CULTURE
GROWTH OF RACES OF MONOCONIDIAL ORIGIN AT 7 TEMPERATURES
The effect of temperature on rate of mycelial growth of 12
races of Physalospora rhodina of monconidial origin on potato-
dextrose agar was determined. Petri dishes of uniform size
were selected and sterilized, and 15 cc. of sterile potato-dextrose
agar was poured into each dish. Three dishes were inoculated
in the center with 5 mm. disks of mycelial inocula of each form
and placed at the following temperatures: 100, 15, 200, 250,
300, 350, and 400 C. Temperature of the chambers varied ap-
proximately 10 C. Measurements from which averages were
obtained were made 60 hours after inoculation. From the
average diameter of the three colonies of each race at each
temperature, the diameter of the inocula (5 mm.) was sub-
tracted and the remainder was taken as the value for the in-
crement for this period.
Races used in this test were isolated from several different
hosts from several localities, as shown in Table 1. Results ob-
tained from this test are summarized in Table 8. It is to be
noted that some of the races varied considerably in rate of






Life History and Taxonomy of the Fungus Physalospora Rhodina 39

pected in the segregation of characters in a heterozygous line
or a line dual in nature. The loss of capacity for pycnidia
formation was also evident in these races. However, certain
other races showing no change in mycelial color have never
sporulated from the time of isolation. This gradual change in
the color of the mycelium and the loss of capacity for pycnidia
formation in certain races is probably due to a gradual loss of
the factors controlling these characters that appear to be fixed
in the other lines. A similar condition was observed in cultures
of Sphaeropsis malorum by Mohendra and Mitra (50), in
which spores from a single pycnidium or even a single spore
might produce two kinds of daughter colonies, black and white.
They suggest that "this is a gradual replacement of a vigor-
ously spring black type of mycelium by a poorly spring white
type because of certain physiological differences in the course
of a number of generations." The mycelial colonies of certain
forms of Physalospora rhodina were often slightly irregular in
form or rather evenly lobed, especially at the higher tempera-
tures, as shown in Figures 8 and 9. Such irregularities in va-
rious colonies frequently resemble sectors or mutations, but
they do not behave as such.

EFFECT OF TEMPERATURE ON RATE OF GROWTH IN CULTURE
GROWTH OF RACES OF MONOCONIDIAL ORIGIN AT 7 TEMPERATURES
The effect of temperature on rate of mycelial growth of 12
races of Physalospora rhodina of monconidial origin on potato-
dextrose agar was determined. Petri dishes of uniform size
were selected and sterilized, and 15 cc. of sterile potato-dextrose
agar was poured into each dish. Three dishes were inoculated
in the center with 5 mm. disks of mycelial inocula of each form
and placed at the following temperatures: 100, 15, 200, 250,
300, 350, and 400 C. Temperature of the chambers varied ap-
proximately 10 C. Measurements from which averages were
obtained were made 60 hours after inoculation. From the
average diameter of the three colonies of each race at each
temperature, the diameter of the inocula (5 mm.) was sub-
tracted and the remainder was taken as the value for the in-
crement for this period.
Races used in this test were isolated from several different
hosts from several localities, as shown in Table 1. Results ob-
tained from this test are summarized in Table 8. It is to be
noted that some of the races varied considerably in rate of






Florida Agricultural Experiment Station


growth at the different temperatures (Fig. 7) and that the
temperature range of certain races was wider than that of
others. All of the races grew well between 150 C. and 350 C.,
but they showed little or no growth at 100 and 400 C.
The optimum temperature, when considered as that tem-
perature at which there is best growth during a given time,
was found to be close to 300 C. for all races. However, in con-
sidering growth rates for consecutive observation periods, the
optimum would probably lie between 250 and 300 C. The maxi-
mum temperature of some races appeared to be at 400 C. or
above for the first day after inoculation, but appeared to be
displaced to below 40 C. for the second and third days after
inoculation. However, the maximum temperature of races 3,
52, and 57 was apparently slightly above 400 C., as there was
no apparent shifting or displacement, but a slight increase in
mycelial growth after each consecutive period. Since all of the
races grew well at 35 C. it is apparent that the maximum
temperature for all of the races was close to 400 C. The sig-
nificance of the shifting or displacement of the maximum tem-
perature of certain races is questionable. It would seem reason-
able to assume that the slight growth produced at 400 C. by
certain races for the first 24-hour period only, was merely
that growth made up to the time the cultures became adjusted
to this temperature. As shown in Table 8, the minimum tem-
perature for certain races was below 100 C., while for others
it was above 100 C. There was no shifting or displacement of
the minimum temperature of any of the races. Also, there was
either no growth at 100 C., or a definite increase of growth at
this temperature after each consecutive observation period.
These results agree in general with previous reports on the
growth-temperature relations of different cultures of the asex-
ual stage of this fungus. Fawcett (35), working with a culture
of Diplodia natalensis from Citrus, found that the maximum
temperature changed from about 460 C. for the first 24-hour
period to about 350 C. for the third 24-hour period, and that
the optimum temperature was displaced from about 310 C.
for the first day to about 270 C. for the third day. Stevens and
Wilcox (72) showed that forms of D. natalensis isolated from
Citrus would grow somewhat at 370 C., and that certain strains
when grown on potato-dextrose agar showed chromogenesis at
this temperature. During the same year Stevens (67) showed
that certain forms of D. (t..,in;,, isolated from cotton would






Life History and Taxonomy of the Fungus Physalospora Rhodina 41


-v

-I


Fig. 7.-Effect of temperatures indicated on the rate of growth of three mono-
conidial races of Physalospora rhodina after 60 hours on potato-dextrose agar.






Florida Agricultural Experiment Station


not grow at this high temperature, and only slowly at 310 to 320
C. Later, he (68) showed that these two species were synony-
mous and that they were the asexual forms of Physalospora
rhodina; also, that both the low and high temperature forms
occurred on various hosts.
Eddins (29), in working with the related form Diplodia
frumenti from corn, showed that the optimum temperature oc-
curred between 250 and 320 C., and that growth was slight at
150 and 390 C., at the end of a 48-hour period. Stevens (70),
in his studies on the temperature relations of certain fungi,
found that out of 10 different fungi a form of D. natalensis
was one of the two fungi that showed growth at 350 C. at the
end of a 24-hour period, but showed no growth at 100 C. for
the same period. According to Young (85), the minimum, opti-
mum, and maximum temperatures for growth of a strain of
D. natalensis were 12.0', 31.50, and 41.00 C., respectively. More
recently Verrall (82) showed that isolates of D. natalensis
tested from cotton, pear, and tung from Louisiana and Mis-
sissippi grew rapidly at 350 to 370 C., as did those from lum-
ber. These temperature relations are quite similar to those
observed for certain races of Physalospora rhodina in the pres-
ent study, as shown in Table 8. Since this species is widely
distributed throughout the tropics it is not surprising that it
has a high maximum temperature.

GROWTH OF RACES OF MONOCONIDIAL ORIGIN AT 3 TEMEPRATURES
Rate of growth for a number of the races of Physalospgra.
rhodina shown in Table 1 was obtained by measuring the in-
crease in diameter of colonies on potato-dextrose agar and malt-
extract agar. Petri dishes of uniform size were sterilized and
15 cc. of sterile media was poured into each dish. These dishes
were inoculated in the center with 5 mm. disks of mycelia in-
ocula of each race and incubated at the temperatures indicated
below. In the first test the cultures were held in constant tem-
perature chambers at temperatures of 30 and 360 C. In the
second test the cultures were held in a large transfer room in
the laboratory, the average temperature of which was 29 C:,
with a variation of more than +1 C. The average colony di-
ameter of each race at each temperature was measured at the
end of 60 hours after inoculation. The average increase per
day was determined from these data and is presented in Table 9.
Marked differences in the rate and character of growth




Life History and Taxonomy of the Fungus Physalospora Rhodina 43


0-


A


D


Fig. 8.-Twenty-four monoconidial races of Physalospora rhodina from various
host collections. Grown for 60 hours on potato-dextrose agar at 300 C.





44 Florida Agricultural Experiment Station


Fig. 9.-Twenty-four monoconidial races of Physalospora rhodina from various
host collections. Grown for 60 hours on potato-dextrose agar at 360 C.






Life History and Taxonomy of the Fungus Physalospora Rhodina 45

existed among the races at the various temperatures (Figs.
8, 9). As shown in Table 9, rates of growth of these races were
influenced by the medium upon which they were grown. In
general, all races grew more rapidly on potato-dextrose agar
than on malt-extract agar, but only rarely did the difference
in daily growth of any race on the two media exceed 10.0 mm.
The greatest difference in growth rates between races of
this fungus occurred at 360 C., while the least difference on
either medium occurred at room temperature. As indicated in
Table 9, certain races showed chromogenesis at 360 C. at the
end of 60 hours, but this character is apparently not correlated
with the growth rates. Taking all forms into consideration at
each temperature, the average daily growth on potato-dextrose
agar at 300 C., 360 C., and room temperature was 31.1 .38
mm., 23.0 .99 mm., and 33.0 .40 mm., respectively, and
on malt extract agar at these temperatures it was 29.0 .41
mm., 20.0 .98 mm., and 27.6 .47 mm., respectively. It is
interesting to note how close the average daily growth of most
races on both media in the first test at 300 C. approaches that
of the races in the second test at 290 C. In general, the higher
temperature forms indicated in these studies at 360 C. are the
same races which produced the highest percentage of decay in
oranges at 340 C. (Table 11), but this correlation is not con-
sistent. While the results of these tests point definitely to phys-
iological differences among the various races, it is doubtful
whether separation of the races could be made on the basis of
temperature relations.
Cultures used in the above test held for 10 days after in-
oculation showed considerable difference in the mycelial char-
acters of some races. The mycelium of some of the lower tem-
perature forms held at 360 C. had still not covered the entire
surface of the media at the end of this time.

GROWTH OF RACES OF MONOASCOSPORE ORIGIN AT ONE TEMPERATURE
A test was conducted to determine whether the type and
rate of growth of various monoascospore isolates varied as it
did with various monoconidial isolates. The 15 isolates referred
to in the first series of the monoascospore aversion studies
were used in the present test. The rate of growth was deter-
mined by measuring the diameter of colonies of each isolate in
triplicate, after being held for 60 hours at 360 C. The average
diameter of the three colonies of each isolate was recorded and






Florida Agricultural Experiment Station


is presented in Table 10. The average increase in colony di-
ameter per day was not determined as in the previous test.

TABLE 10.-RATE OF GROWTH OF RACES OF Physalospora rhodina OF MONASCOSPORE
ORIGIN AFTER 60 HOURS ON POTATO-DEXTROSE AGAR AT 360 C.

Isolate Number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Aver. Colony
Diam. in mm. 30* 77 73* 73 29* 83* 67 41 27* 64 50 24* 67 70 68*

Isolates 1-7 isolated at random from a Citrus specimen, 8-11 isolated
at random from a single perithecium, and 12-15 isolated at random from
a single ascus.
*Races showing chromogenesis at 36 C.


Fig 10.-Nine of the 15 monoascospore races of Physalospora rhodina from a
Citrus specimen: 5-7 from different random perithecia; 8, 10, 11 at random from
one perithecium; 12, 13, 14 at random from one ascus. Grown for 60 hours on
potato-dextrose agar at 36 C. (See Table 10).






Life History and Taxonomy of the Fungus Physalospora Rhodina 47

There was considerable difference in type and rate of growth
of some of these isolates, as shown in Figure 10. This varia-
tion in type and rate of growth was in general the same as that
manifested in various monoconidial isolates at 360 C. shown in
Figure 9. This difference occurred among certain monoasco-
spore isolates, namely, those isolated at random 1-7, among
those isolated from the same perithecium 8-11, and even among
those from the same ascus 12-15. As pointed out earlier in this
paper, isolates 12 and 15 are of one race and 13 and 14 of an-
other. As shown in Table 10, the difference in rate of growth
between isolates 12 and 15 is probably significant, that between
isolates 13 and 14 is not. This indicates that a difference in rate
of growth may exist between different monoascospore isolates
of the same physiologic race of P. rhodina. Thus, in considering
the fact that different monoascospore and monoconidial isolates
of the same race, as well as monoascospore and monoconodial iso-
lates of different races, may have different temperature rela-
tions, it is not surprising that both low and high temperature
forms have been reported for this species.
According to results presented in Table 10, some of the
monoconidial isolates showed chromogenesis at 360 C. in the
same manner as some of the monoconidial isolates in the prev-
ious test. It is interesting to note that this phenomenon devel-
oped in isolates 12 and 15, which are of the same race, but did
not develop in isolates 13 and 14 which are of another race
but from the same ascus. This indicates that the factor or fac-
tors responsible for this character are inherited and perhaps
segregated in a definite manner. This character occasionally
develops in stock cultures held at room temperature or at lower
temperatures, but for the most part develops only at 360 C.
and above, and then only in certain cultures. From results ob-
tained there is no evidence that chromogenesis is correlated
with rate of growth in any of the races. It was not determined
whether this character is correlated in any way with sex or
aversion but this point should be investigated.
EFFECT OF TEMPERATURE ON RATE OF DECAY IN
HOST MATERIAL
The relation of temperature to the rate of decay by several
races of Physalospora rhodina was determined. Freshly picked
Valencia oranges of uniform size, which were debuttoned and
surface sterilized shortly after picking, were used for inocula-
tion in this test. The inoculations were made by removing a






Florida Agricultural Experiment Station


small round plug of the rind from the blossom end of each fruit
with a sterilized cork borer. A 5 mm. disk of mycelial inoculum
from a culture on potato-dextrose agar was placed in this small
cavity, the rind plug was replaced, and the wound was covered
with a small piece of nurseryman's tape. This was done in trip-
licate for each of the 29 races used at each temperature. As a
control, three oranges were similarly inoculated with sterile
potato-dextrose agar and placed at each of the three tempera-
tures. Since the optimum temperature for mycelial growth of
most of the races in culture was found to be close to 300 C.,
this temperature was used in this test. Also, a temperature
approaching the optimum on either side was used.
Results of this test are summarized in Table 11. Rate of
decay of each race is measured in terms of percentage of the

TABLE 11.-AVERAGE ;PERCENTAGE OF THE CIRCUMFERENCE OF VALENCIA ORANGES
DECAYED FOUR DAYS AFTER INOCULATION WITH SEVERAL RACES OF Physalospora
rhodina AT DIFFERENT TEMPERATURES.

Race Percentage of Circumference Decayed when Inoculated at
No. 26 C. 300 C. 340 C.

Check 0.0 0.0 0.0
9 77.7 87.0* 33.7
11 79.0 100.0* 87.7
12 71.0 92.0* 75.0
14 71.0 96.0* 68.7
18 58.0 83.7* 75.0
22 79.0 86.0* 30.7
25 67.0 96.0 22.0
30 79.0 100.0* 91.7
31 71.0 92.0* 87.0
35 79.0 96.0* 87.7
36 92.0 92.0* 75.0
37 92.0 100.0 66.7
39 87.7 100.0" 66.7
44 75.0 87.7* 35.0
52 92.0 100.0* 92.0
55 75.0 92.0* 100.0
56 78.7 100.0* 92.0
57 83.0 100.0* 96.0
58 87.7 100.0* 60.0
59 77.7 100.0* 79.0
60 69.3 70.0 20.0
91 92.0 100.0* 100.0
95 87.0 100.0* 33.7
101 62.7 100.0* 63.0
103 71.0 96.0* 66.7
107 87.7 100.0* 43.7
108 75.0 87.7 46.0
111 88.7 100.0* 100.0
112 82.7 100.0* 100.0
Average 78.9- 1.70 94.9-.98 68.8 -3.07
*Races producing pycnidia on oranges 20 days after inoculation.






Life History and Taxonomy of the Fungus Physalospora Rhodina 49

circumference of the fruit rotted at the temperatures indicated.
Measurements from which the averages were obtained were
made four days after inoculation. Inoculated fruits were quite
uniform in size so that any difference in percentage decay be-
tween races was not due to variation in size. It is to be noted
that most of the races produced the highest percentage of de-
cay at 300 C., and that percentage decay was approximately
the same at 260 and 340 C. for some races but varied consid-
erably in others. No decay was observed in any of the check
fruit at any of the temperatures used. Taking all of the races
into consideration, the average percentage of decay was 78.9
1.70, 94.9 .98, and 68.8 3.07 percent, at 260, 300, and
340 C., respectively. In a similar manner, Savastano and Faw-
cett (64) inoculated oranges and lemons with various fungi at
different temperatures, and showed that a form of Diplodia
natalensis produced decay more rapidly at temperatures of 300
to 330 C. than any of the other fungi used.
Certain physiological differences between some of the races
as well as differences in pathogenicity are indicated. After
the results were recorded at the end of four days the inoculated
fruit in the 300 chamber was removed and held at room tem-
perature for 16 days. At the end of this time observations were
made on the condition of the fruit, and pycnidia production
of the various races was noted. The races producing pycnidia
at the end of this time are indicated in Table 11, and are in
general the same races that produced pycnidia in wheat cul-
tures and on potato-dextrose agar, as shown in Table 7.

MORPHOLOGY
CONIDIAL AND ASCIGEROUS STAGES IN THE LIFE HISTORY
A review of the literature reveals that many collections of
the pycnidial stage of Physalospora rhodina, on the same or
different hosts, have been described as distinct species of Dip-
lodia and related genera. Much of the controversy on the taxon-
omic position of various collections of this fungus undoubtedly
has been due to its wide distribution and host range, and con-
versely to the variation which exists in certain characters. After
correlating the variation in some morphological and physio-
logical characters, it is not surprising that such controversy
exists and is perhaps justifiable in some cases. However, it is
the opinion of the writer and certain other workers that many






Life History and Taxonomy of the Fungus Physalospora Rhodina 49

circumference of the fruit rotted at the temperatures indicated.
Measurements from which the averages were obtained were
made four days after inoculation. Inoculated fruits were quite
uniform in size so that any difference in percentage decay be-
tween races was not due to variation in size. It is to be noted
that most of the races produced the highest percentage of de-
cay at 300 C., and that percentage decay was approximately
the same at 260 and 340 C. for some races but varied consid-
erably in others. No decay was observed in any of the check
fruit at any of the temperatures used. Taking all of the races
into consideration, the average percentage of decay was 78.9
1.70, 94.9 .98, and 68.8 3.07 percent, at 260, 300, and
340 C., respectively. In a similar manner, Savastano and Faw-
cett (64) inoculated oranges and lemons with various fungi at
different temperatures, and showed that a form of Diplodia
natalensis produced decay more rapidly at temperatures of 300
to 330 C. than any of the other fungi used.
Certain physiological differences between some of the races
as well as differences in pathogenicity are indicated. After
the results were recorded at the end of four days the inoculated
fruit in the 300 chamber was removed and held at room tem-
perature for 16 days. At the end of this time observations were
made on the condition of the fruit, and pycnidia production
of the various races was noted. The races producing pycnidia
at the end of this time are indicated in Table 11, and are in
general the same races that produced pycnidia in wheat cul-
tures and on potato-dextrose agar, as shown in Table 7.

MORPHOLOGY
CONIDIAL AND ASCIGEROUS STAGES IN THE LIFE HISTORY
A review of the literature reveals that many collections of
the pycnidial stage of Physalospora rhodina, on the same or
different hosts, have been described as distinct species of Dip-
lodia and related genera. Much of the controversy on the taxon-
omic position of various collections of this fungus undoubtedly
has been due to its wide distribution and host range, and con-
versely to the variation which exists in certain characters. After
correlating the variation in some morphological and physio-
logical characters, it is not surprising that such controversy
exists and is perhaps justifiable in some cases. However, it is
the opinion of the writer and certain other workers that many






Florida Agricultural Experiment Station


of the collections resembling the sexual stage of P. rhodina are
merely strains or physiologic races of it.

MYCELIUM
Morphological characters of the mycelium of this fungus
have been described and illustrated on different substrata by
various workers. Variations existing in many of the described
characters can be attributed to environment and strain or race
differences. As pointed out earlier in this paper, there is con-
siderable variation in color, type and rate of growth of the
mycelium of several races on different substrata. In culture the
young mycelium is hyaline and granular, becoming septate and


Fig. 11.-Pycnidial characters of four races of Physalospora rhodina after 30
days on wheat cultures at room temperature. Only a few pycnidia formed by race
37; small or aggregated pycnidia formed by race 44; pycnidia formed in surface
layers of stromatic masses in races 12 and 39. Slightly reduced.






Life History and Taxonomy of the Fungus Physalospora Rhodina 51


W55;;2


Fig. 12.-Pycinidial characters of four races of Physalospora rhodina, after 15
days on potato-dextrose agar at room temperature. (52) Small, single or aggre-
gated, embedded to aerial pycnidia, without stroma; (104) Small, single or aggre-
gated pycnidia with spore tendrils, formed on surface of medium; (36) Pycnidia
formed in surface layers of stromatic masses. Note water drops at ostioles of
pycnidia (36); and white (immature) and black (mature) spore tendrils (9). All X 7-






52 Florida Agricultural Experiment Station


Fig. 13.-Pycnidial characters of six races of Physalospora rhodina when grown
in cultures on Malvaviscus stems. All X 7.


18.'






Life History and Taxonomy of the Fungus Physalospora Rhodina 53

branched 12 to 48 hours after spore germination with multi-
nucleate cells, producing a loose flocculent, white, aerial growth.
As it grows older it becomes dark, more septate, branched and
heavily granular, often filled with refractive oil globules. Dark-
colored, thick-walled intercalary cells frequently occur either
singly or in chains of the main hyphae. These intercalary cells
or chlamydospores are essentially nothing but swollen cells of
these septate hyphae which have become dark-colored and thick-
walled.
In various host tissues the mycelium is hyaline at first but
soon becomes dark, helping to produce a characteristic dark
coloration in the host tissues. The mycelium may be both inter-
and intra-cellular, as described by Evans (34) and Yu (86) in
Citrus fruit, by Higgins (43) in plum wood, and by Wardlaw
(84) in banana fruit. These workers have also illustrated my-
celial branches in the cells of certain tissues and described them
as haustoria or branches of similar function. Intercalary cells
or chlamydospores are also common in the mycelium in various
host tissues.

PYCNIDIA AND CONIDIA
There is some variation in the morphology of the pycnidia
of this fungus on different substrata, as indicated by the va-
rious races used in this study and as reported by other work-
ers. The pycnidia of certain races of this fungus produced on
various culture media may be small, single or grouped, and
formed either below the surface, on the surface, or on the aerial
mycelium. In other races the pycnidia may be clustered in the
surface layers of stromatic masses formed over the surface of
the substratum. As illustrated in Figures 11, 12, and 13, these
stromatic masses are typical of many races of this fungus and
are formed readily on different substrata under humid condi-
tions in the laboratory. Under such conditions the pycnidia pro-
duced on nutrient media or host material are frequently covered
with stiff, branched hyphae as shown in Figure 13.
The pycnidial characters of many forms of P. rhodina have
been described on different host collections by various workers.
As mentioned previously, many of these forms are described as
distinct species of Diplodia and related genera but many of
them are considered to be synonymous. In general, the pycnidia
as they occur on various hosts are simple or compound, varying
in size and complexity with the bark of the host, as shown in






Florida Agricultural Experiment Station


Fig. 14.-Showing cut perithecia and pycnidia of Physolospora rhodina on vari-
ous hosts. (A) Perithecia of P. zeicola on Zea mays; (B) Perithecia of P. rhodina
on Gossypium barbadense; (C) Perithecia on Citrus grandis; (D) Pycnidia on Arachis
hypogaea; (E) Pycnidia on Citrus aurantifolia; (F) Pycnidia on Padus virginiana;
(G) Pycnidia on Pyrus communis; (H) Pycnidia on Thea sinensis; (I) Pycnidia on
Zea mays. All approximately X 7.







Life History and Taxonomy of the Fungus Physalospora Rhodina 55


I


Fig. 15--Pycnidial characters of Physalospora rhodina on various hosts: (A) on
pod of Cocos nucifera, (B) on stalks of Zea mays, (C) on branch of Hicoria pecan,
(D) on branch of Pyrus communis, (E) on branch of Ficus carica, (F) on branch of
Citrus aurantifolia, (G) on fruit of Citrullus vidgaris, (H) on fruit of Persea ameri-
cana. All X 80.


rAft







TABLE 12-A SUMMARY OF CERTAIN MORPHOLOGICAL CHARACTERS OF VARIOUS PYCNIDIAL FORMS WHIIC AGREE W1TH THOSE OF Physalo-
spora rhodina.


Binomial Reported
Under

Diplodia
gossypina Cke. (24)
Botryodiplodia
theobromae Pat.. (52)
Botyrodiplodia
theobromae Pat. (56)

Botyrodiplodia
theobromae Pat.(79)

Lasiodiplodia
theobromae (Pat.)
Griff. & Maubl. (37)
Diplodia
theobromae (Pat.)
Now. (51)
Macrophoma
vestita Prill. & Del. (61)
Diplodia
cacaoicola P. Henn. (42)
Diplodia
ccacoicola P. Henn. (45)


Spore Di- Pycnidia
Substratum mensions Diam. in
in Microns Microns


Gossypium
sp.
Theobroma
cacao
Grevillea
sp.

Thea
sinensis

Theobrama
cacao

Theobroma
cacao

Theobroma
cacao
Theobroma
cacao


Other Characters


22 x 12 Pycnidia gregarious, erumpent. Spores elliptical, olivaceous-brown.

25-35 x 200 Pycnidia gregarious, more or less villous, in a villous stroma. Spores ellip-
12-15 tical, 1-septate, brown.
24-30 x 250-400 Pycnidia immersed, scattered or clustered, or in erumpent glabrous or vil-
12-15 lous stroma, globose. Spores oval, black to black-brown; paraphyses
numerous.
29.5 x Pycnidia subglobose to globose, partly embedded in bark or in stroma on
14.1 surface, frequently covered with hairs. Spores oval to ovoid, with stri-
ations, liberated in tendrils; paraphyses present.
20-30 x Pycnidia immersed at first, then erumpent. Spores brown, 1-septate, not
11-15 constricted; paraphyses numerous.

25-30 x 200-400 Pycnidia immersed in cortex, single or grouped, sometimes in a stroma,
12-15 pilose in moist air. Spores ovate or ellipsoid, often with longitudinal
striations; paraphyses present.
30 x 15 300 Pycnidia simple and isolated. Spores hyaline, nonseptate.

22-28 x Pycnidia scattered, immersed in cortex. Spores ellipsoid-oblong, 1-septate,
12-14 constricted.


Saccharin 20 x 10
officinarumn
& Theobroma
cacao


Pycnidia breaking through rind in vertical series, frequently single, fre-
quently in a stroma or in a web of hyphae. Spores 1 septate, brown.










Lasiodiplodia
t ubericola
Ell. & Ev. (17)

Botryodiplodia
tubericola
(E & E) Petr. (57)

Botryodiplodia
gossypii E. & B. (32)

Lasiodiplodia
nigra Appel. and
Laub. (1)
Botryodiplodia
elasticae Petch (54)

Chaetodiplodia
grisea Petch (54)

Diplodia
rapax Massee (47)
Diplodia
natalensis Evans (34)

Physalospora
rhodina (B. & C.) Cke.
(Diplodia natalensis
Evans) (68)
Lasiodiplodia
triflorae Higgins (43)


Impomoca 18-22 x
batatas 11-14


Ipomoea
batatas


Gossypiumn
sp.
Theobroma
& Carica

Hevea and
Castilloae
Theobroma
cacao
Hevea
sp.
Citrus
sp.

Various
Hosts


Prunus
sp.


23-32 x
10-14


15-22 x
12

28-32 x
18-21


25-30 x
14-15
24-28 x
13-14

32-35 x
15-16
24 x 15



20-33 x
10-18


22-25 x
13-16


250-350 Pycnidia globose, covered with branched hyphae, stromatically connected
in a small hemispherical erumpent tubercle. Spores elliptical, 1-septate,
not constricted; paraphyses present.

250-300 Pycnidia projecting as flask-shaped pustules from loose or compact, often
coalescing, stromata. Spores oblong, ellipsoid, egg-shaped, scarcely or very
slightly constricted; paraphyses present.

1-2 mm. Pycnidia in a semi-erumpent stroma, arranged in single or double rows in
in longitudinal cracks. Spores oblong-elliptical, brown and 1-septate.


Pycnidia in black pilose stromata.


250-400 Pycnidia in glabrous stromata, or single or in groups, embedded in the
bark. Spores among paraphyses.
Pycnidia scattered and clothed with long hairs. Spores among long para-
physes.

160-180 Pycnidia aggregated globose, glabrous. Spores elliptical, 1-septate.


150-180 Pycnidia scattered, subglobose, papillate. Spores elliptical, 1-septate, not
constricted, with longitudinal str:ations.


1-7 mm. Pycnidia simple or compound, varying in size and complexity with host.
Spores 1-septate, usually with longitudinal striations.


Pycnidia embedded in cortex, scattered or aggregated, glabrous or covered
with hairs. Spores oblong, intermixed with paraphyses.






Florida Agricultural Experiment Station


Figures 14 and 15. Some of the more variable characters as
manifested by a number of forms of this fungus and recorded
by various investigators are summarized in Table 12. The py-
cnidia of the Diplodia forms studied in this investigation may
be subglobose to globose, scattered to gregarious, single or
grouped in a stromata, immersed to erumpent or almost super-
ficial, hairy or glabrous, and papillate. The dimensions of py-
cnidia and the presence or absence of stromata are also variable
characters in the forms of this fungus, as shown by the arrange-
ment of the pycnidia on several hosts in Figures 14 and 15.
The pycnidia when cut across, especially before the spores are
fully mature, have the snow-white appearance characteristic
of the perithecia, as shown in Figure 14. According to Stevens
(67) this is a characteristic of the genera Botryosphaeria and
Physalospora which makes these fungi so easy to distinguish
and collect. When the conidia are mature, however, they are
brown in color and the contents of the pycnidium then appear
dark.
From results obtained with the various races of P. rhodina
in the present study it is apparent that the pycnidial characters
are more variable than the spore characters. There are signifi-
cant differences between the spore dimensions of a few races
on the same and different substrata (Tables 13 and 14). How-
ever, the differences for the most part are characteristic for a
race except in certain races showing differences due to environ-
mental conditions. Some forms are slow in attaining the final
coloration or septation of their spores, and the consideration
of immature spores of such forms has also led to confusion in
the taxonomic position of this fungus. Under certain environ-
mental conditions the conidia of a number of races may be
liberated from the pycnidium before they are mature. This has
been considered a generic distinction by some workers. Fre-
quently the spores are found collected or heaped around the
ostiole of the pycnidium, and in some cases they are liberated
through the ostiole in the form of tendrils, the color of which
may be white or black depending upon the maturity of the
spores (Fig. 12). In general the young conidia are nonseptate,
hyaline, and granular, becoming brown and 1-septate at ma-
turity; and may or may not be interspersed with paraphyses.
The conidia representing several host collections examined in
this investigation measured 17-34 x 10-18 t. In the process of
germination, after 2-6 hours, the spores swell at one or both






Life History and Taxonomy of the Fungus Physalospora Rhodina 59


Fig. 16.-Conidia characters of six races of Physalospora rhodina produced in
culture on Malvaviscus stems (3, 10, 91. 112), and two races produced in culture on
potato-dextrose agar (9, 44). All approximately X 450.






Florida Agricultural Experiment Station


ends and the wall is slit in a ragged manner as the germ tube
emerges. A slight swelling of the germ tube is usually visible
where it leaves the spore. The young hyaline spores are multi-
nucleate, and although the nuclear condition of the dark mature
spores is obscure, the hyphae therefrom are multinucleate, as
shown in Figure 6.
A study of Table 12 will show that differences exist in the
size and shape of the conidia of the forms of P. rhodina re-
ported by various workers. However, a study of Figure 16 will
show only slight differences in conidial characters of the races
used in the present study, and that the conidia of these races
are morphologically similar for the most part. Although there
are significant mean differences in the spore dimensions of a
few races of this fungus, the spores themselves provide one of
the most nearly constant and dependable characters that we
have. Longitudinal striations on the exospore of the mature,
brown, 1-septate conidia are a typical and fairly constant spore
character, but are not apparent in the conidia of all collections
of this species. The presence or absence of paraphyses has also
been used in the separation of forms of P. rhodina. However,
since they vary in number and visibility with the degree of
maturity of the fruiting body, and since they are frequently
difficult to distinguish, even when present, the use of these
structures as diagnostic characters is unreliable.
In view of the plasticity of some morphological characters
of various conidial collections considered in the present study,
many of these collections could be placed in either Diplodia,
Botryodiplodia, Chaetodiplodia, or Lasiodiplodia, as has been
done. Although the last two genera are now considered invalid
by most workers, they have played a part in the controversy
which exists in the literature. Since each worker has classified
the fungus according to the form he happened to have, the
differences of opinion cannot necessarily be attributed to any
fault on the part of the describer. However, it does demon-
strate that there is a tendency to separate species and genera
without a sufficient conception of what the variation within a
species or genera might be under different conditions.
PERITHECIA, ASCI, AND ASCOSPORES
The perithecial stage of Physalospora rhodina is apparently
not as common in nature as is the pycnidial stage. The writer
has frequently found the former stage on dead Citrus wood on






Life History and Taxonomy of the Fungus Physalospora Rhodina 61

the ground, but collections on other hosts have been limited.
Although this stage occurs occasionally in completing the life
cycle and perhaps in giving rise to new physiologic races, it
apparently plays a minor role in the perpetuation and distri-
bution of the fungus. Aside from the few collections of this
stage made by Stevens (68) in Florida and Cuba, very few
other collections have been reported. As pointed out by Stevens
(68), collections of this fungus may be confused with certain
collections of Botryosphaeria, although rather prominent stro-
mata are usually present in the latter genus. Likewise, the peri-
thecial stage of Physalospora obtusa (69) is quite similar and
perhaps genetically related to P. rhodina, but they can usually
be differentiated by their conidial stages.
Physalospora rhodina was first collected by M. A. Curtis
on rose in North Carolina and was briefly described by Cooke
(25) in 1889. In 1925 Petrak and Sydow (58) examined the
type material of this species at Kew and found that the fungus
on the Carolina specimens was very scant, thus their descrip-
tion is based on examination of a Pennsylvania collection. They
concluded that this fungus was a form of Melanops quercuum
(Schw.) Weese. The following year, Stevens (68) concluded
that a species of Physalospora collected by him on different
hosts from Cuba and Florida was identical with Physalospora
rhodina, and included several other described forms as syn-
onyms of it.
The perithecia of P. rhodina are single or gregarious to
closely crowded, scattered or in semi-parallel rows. When cut
across, they have the same white appearance of the cut py-
cnidia containing immature conidia (Fig. 14), which, accord-
ing to Stevens (68), is characteristic of the genus Physalospora.
They are externally carbonous, globose, papillate, with a black
pseudoparenchymatous wall which is continuous with an inner
structure of hyaline pseudoparenchyma enclosing the hymen-
ium and from which the latter appears to arise. Fifty peri-
thecia from a Citrus specimen measured 225-300 p in diameter;
as compared with 200-350 recorded by Petrak and Sydow
(58) and 250-300 p recorded by Stevens (68).
The asci are clavate-cylindrical, sessile, typically eight-
spored and seldom four-spored, with spores crowded-biserrate.
One hundred asci from a Citrus specimen measured 75-110
x 18-25 u; as compared with 65-85 x 20-25 p recorded by Petrak
and Sydow (58) and 90-120 p long recorded by Stevens (68).






62 Florida Agricultural Experiment Station

The young ascus has its granular contents enclosed in a sac
that lies in the space enclosed by the outer wall of this struc-
ture. After the spores are formed and mature, the wall of the
'sac remains to enclose the spores while the outer wall dis-
appears or becomes obscure, as illustrated in the related form
P. zeicola by Eddins and Voorhees (30), and observed in P.
rhodina by the writer.
The ascospores are hyaline, one-celled, elliptical to ellipsoid,
often tapering to narrow, rounded ends; and frequently pre-
senting one flattened and one curved side, as illustrated by
Stevens (68). According to Petrak and Sydow (58), the asco-
spores are 21-30 x 9-12.5 [; and 24-42 x 7-17 /, mostly 30-35
x 11-14 A as recorded by Stevens (68). In the present study
200 ascospores from a Citrus specimen measured 27-38 x 9-17,
averaging 30.5 x 13.7 it. Ascospores of P. rhodina measured
from other collections also have similar dimensions.
The Diplodia stage of the ascigerous fungus found on corn-
stalks in Florida, and considered as Physalospora zeicola by
Eddins and Voorhees (30), is not distinguishable from the va-
rious Diplodia forms of P. rhodina. However, the perithecia
of P. zeicola on cornstalks are significantly smaller and in a
somewhat different arrangement from those of P. rhodina on
various dicotyledonous hosts, as shown in Figure 14-A, B, and
C. Also, the asci and ascospores of the former are significantly
smaller than those of the latter. Although the two species are
quite similar in certain other morphological characters and
may be genetically related, they should be considered distinct
for the present.
In a similar manner, Stevens (68) showed that the Diplodia
stage of Physalospora fusca is practically indistinguishable
from that of P. rhodina. When young the perithecia and asco-
spores of P. fusca are hardly distinguishable from those of
P. rhodina; but when the ascospores of the former are mature
they are brown in color, the center of the perithecium appears
dark through the paraphyses, and the surrounding tissue of
the perithecium still remains white.
Although the perithecial stage of P. rhodina, P. fusca, and
P. zeicola are quite similar in many respects, they can be dif-
ferentiated in certain morphological characters and should be
considered distinct for the present at least. In all probability
there are other undescribed perithecial stages of the Diplodia
forms under consideration, but until such stages are described






Life History and Taxonomy of the Fungus Physalospora Rhodina 63

the writer is inclined to include these Diplodia forms under
P. rhodina.
SPORE MEASUREMENTS
As stated previously, the variation in certain morphological
characters of different conidial collections of P. rhodina has
led to considerable controversy over their taxonomic position.
The wide differences in some pycnidial characters have perhaps
been largely responsible for such controversy, but the differ-
ences in spore characters have also played a part. The varia-
tion in spore dimensions of many collections of this fungus as
reported by various workers has been wide enough to merit
specific distinction, as shown in Table 12. This was perhaps
justifiable in many cases, especially without a sufficient con-
ception of what the variation within this species might be under
different conditions.
The differences in spore dimensions of many collections may
appear to be significant offhand, but when analyzed statisti-
cally they are not significant. Thus, the significance of the
mean differences in the spore dimensions of many of the col-
lections under consideration was determined, as well as the
effect of a nutrient medium, host medium, and the natural host
source on spore size of the races of this fungus.

EFFECT OF SUBSTRATA ON SIZE OF CONIDIA
In all cases measurements were made with a Leitz micro-
scope, using the 4 mm. objective and a 15X eyepiece, with the
tube length adjusted so that one space on an ocular micrometer
was equal to one micron. On the basis of a preliminary test the
measurements of 200 or even 100 spores were found to be
enough to give a representative sample of a given population
or race. Thus, a random sample of pycnidia of each race from
each substratum was crushed separately on glass slides and
made into semi-permanent mounts.
In this series of spore measurements the dimensions of 200
conidia were determined from each of eight races from three
different substrata. These races were considered as a repre-
sentative sample in demonstrating the variations in spore di-
mensions of races of P. rhodina on the different substrata. The
potato-dextrose agar and Malvaviscus stems represented a nutri-
ent and a woody or host medium, respectively, which were
inoculated with mycelial inoculum of each race. The third sub-
stratum was the host material from which each race was origin-






Life History and Taxonomy of the Fungus Physalospora Rhodina 63

the writer is inclined to include these Diplodia forms under
P. rhodina.
SPORE MEASUREMENTS
As stated previously, the variation in certain morphological
characters of different conidial collections of P. rhodina has
led to considerable controversy over their taxonomic position.
The wide differences in some pycnidial characters have perhaps
been largely responsible for such controversy, but the differ-
ences in spore characters have also played a part. The varia-
tion in spore dimensions of many collections of this fungus as
reported by various workers has been wide enough to merit
specific distinction, as shown in Table 12. This was perhaps
justifiable in many cases, especially without a sufficient con-
ception of what the variation within this species might be under
different conditions.
The differences in spore dimensions of many collections may
appear to be significant offhand, but when analyzed statisti-
cally they are not significant. Thus, the significance of the
mean differences in the spore dimensions of many of the col-
lections under consideration was determined, as well as the
effect of a nutrient medium, host medium, and the natural host
source on spore size of the races of this fungus.

EFFECT OF SUBSTRATA ON SIZE OF CONIDIA
In all cases measurements were made with a Leitz micro-
scope, using the 4 mm. objective and a 15X eyepiece, with the
tube length adjusted so that one space on an ocular micrometer
was equal to one micron. On the basis of a preliminary test the
measurements of 200 or even 100 spores were found to be
enough to give a representative sample of a given population
or race. Thus, a random sample of pycnidia of each race from
each substratum was crushed separately on glass slides and
made into semi-permanent mounts.
In this series of spore measurements the dimensions of 200
conidia were determined from each of eight races from three
different substrata. These races were considered as a repre-
sentative sample in demonstrating the variations in spore di-
mensions of races of P. rhodina on the different substrata. The
potato-dextrose agar and Malvaviscus stems represented a nutri-
ent and a woody or host medium, respectively, which were
inoculated with mycelial inoculum of each race. The third sub-
stratum was the host material from which each race was origin-







Florida Agricultural Experiment Station


ally isolated. Thus, the spore measurements made from the
original host specimen consisted of a random sample of the
spores of each race as it occurred on this particular host in
nature.
The spore measurements of each race were arranged in a
frequency table from which the means and the standard errors
of the means were calculated. The means of the spore measure-
ments of the eight races from each of the three substrata are
compared in Table 13. From these comparisons the mean dif-
ferences and the standard errors of these differences were cal-
culated and are also presented in Table 13. The mean differ-
ences between races divided by the standard errors of these
differences gave a "t" value which was used to enter Fisher's
"t" table to determine significant differences. Accordingly, the
races with a significant mean difference in Table 13 are indi-

TABLE 13.-SUMIARY OF DIFFERENCES IN THE MEAN DIMENSIONS OF 200 CONIDIA
OF EIGHT RACES OF Physalospora rhodina ON THREE DIFFERENT SUBSTRATA.


Sub- Races
stratum Compared

9 & 12
9 & 18
9 & 22
9 & 39
9 & 52
9 & 91
9 & 104
12 & ,18
12 & 22
12 & 39
12 & 52
12 & 91
12 & 104
18 & 22
S 18 & 39
18 & 52
18 & 91
3 18 & 104
22 & 39
22 & 52
22 & 91
22 & 104
39 & 52
39 & 91
39 & 104
52 & 91
52 & 104
91 & 104
*Significant.


Mean Difference
(in microns)


Length
1.74 2.55
2.99 2.66
1.01 2.36
2.47 2.29
2.36 2.73
1.78 2.38
2.55 2.30
4.73 2.73
.73 2.44
.73 2.37
.62 2.80
.04 2.46
.81 2.39
4.00 2.56
5.46 2.50'
5.35 2.90
4.79 2.75
5.54 2.50
1.46 2.17
1.35 2.62
.77 2.26
1.54 2.18
.11 2.56
.69 2.19
.0'8 2.10
.58 2.63
.19 2.57
.77 2.20


Width
2.87 1.49
.34 1.37
1.84 1.65
1.80 1.52
2.42 1.53
2.89 1.51
3.58 1.43


2.53
1.03
1.07
.45
.02
.71
1.50
1.46
2.08
2.55
3.24
.04
.58
1.05
1.74
.62
1.09
.78
.47
1.16
.69


Length I Width
6.82 1.926
1.124 .248
.428 1.115
1.079 1.184
.864 1.582
.748 1.913
1.109 2.503*


1.732
.299
.308
.221
.016
.339
1.563
2.184*
1.394
1.734
2.216*
.673
.515
.341
.706
.043
.315
.038
.221
.074
.350


2.239*
.710
.823
.338
.015
.597
1,128
1.259
1.748
2.217*
3.115*
.027
.387
.714
1.252
.456
.826
.639
.348
.920
.566







Life History and Taxonomy of the Fungus Physalospora Rhodina 65


Sub- Races
stratum Compared

9 & 12
9 & 18
9 & 22
9 & 39
9 & 52
9 & 91
9 & 104
12 & 18
12 & 22
12 & 39
b 12 & 52
12 & 91
S 12 & 104
b 18 & 22
18 & 39
18 & 52
18 & 91
18 & 104
2 22 & 39
22 & 52
22 & 91
22 & 104
39 & 52
39 & 91
S39 & 104


Mean Difference
(in microns)


Length I Width I Length Width


.14 : 2.52 1.07 1.56
3.61 2.71 1.27 1.47
2.94 2.36 .98 1.54
.99 2.64 .37 1.61
2.01 2.45 .49 1.54
1.11 2.91 .36 1.57
4.84 2.51 1.55 1.57
3.75 2.66 2.34 1.29
2.80 2.31 .09 1.37
.85 2.60 .70 1.46
1.87 2.40 .58 1.37
.97 2.87 .71 1.41
4.70 2.47 .48 1.41
6.55 2.51 2.25 1.26
4.60 2.78 1.64 1.35
5.62 2.60 1.76 1.26
4.72 3.04 1.63 1.31
8.45 2.66 2.82 1.31
1.95 2.42 .61 1.46
.93 2.23 .49 1.34
1.83 2.73 .62 1.39
1.90 2.30 .57 1.39
1.02 2.53 .12 1.43
.12 2.43 .01 1.47
3.85 2.59 1.18 1.47


52 & 91 .90 2.81 .13
52 & 104 2.83 2.40 1.06
91 & 104 3.73 2.87 1.19
9 & 12 .95 2.52 .59
9 & 18 4.36 2.54 1.01
9 & 22 1.25 2.31 .75
9 & 39 .88 2.62 .01
9 & 52 3.50 2.32 .96
9 & 90 1.03 2.47 .09
9 & 104 4.59 2.36 .90
12 & 18 5.31 2.54 1.60
12 & 22 .30 2.31 .16
12 & 39 .07 2.62 .60
12 & 52 2.55 2.32 .37
12 & 91 .08 2.47 .50
12 & 104 3.64 2.36 .31
18 & 22 5.61 2.32 1.76
18 & 39 5.24 2.64 1.00
18 & 52 7.86 2.37 1.97
18 & 91 5.39 2.48 1.10
18 & 104 8.95 2.37 1.91
22 & 39 .37 2.41 .76 _
22 & 52 2.25 2.08 .21
22 & 91 .22 2.24 .66
22 & 104 3.34 2.12 .15
39 & 52 2.62 2.42 .97
39 & 91 .15 2.57 .10
39 & 104 3.71 2.46 .91
52 91 2.47 2.26 .87 -
52 & 104 1.09 2.14 .06


1.39
1.39


.056 .686
1.332 .864
1.246 .636
.375 .230
.820 .318
.381 .229
1.928 .987
1.410 1.814
1.212 .066
.327 .749
.779 .423
.338 .504
1.903 .340
2.610* 1.790
1.655 1.215
2.162* 1.397
1.553 1.244
3.177* 2.153*
.806 .349
.417 .366
.670 .446
.826 .410
.403 .084
.494 .007
1.486 .803
.320 .094
1.179 .763
1.300 .832
.377 .468
1.717 .856
.541 .577
.336 .007
1.509 .821
.417 .071
1.945 .709
2.091* 1.290'
.130 i .119
.027 .414
1.099 .301
.032 .379
1.542 .233
2.418* 1.375
1.984* .725
3.316* 1.713
2.173* .887
3.776* 1.528
.154 .514
1.082 .165
.098 .485
1.575 .110
1.083 .708
.058 .069
1.508 .623
1.093 .702
.509 .048
1.548 .609


I 91 & 104 3.56 2.30 .81 1.33
*Significant.


,,t"






Florida Agricultural Experiment Station


cated by the "t" values with an asterisk. These calculations were
also made for each of the eight races compared on two different
substrata, as shown in Table 14.
As pointed out earlier in this paper, more than one physio-
logic race of P. rhodina may be represented on a given collec-
tion or host specimen, and especially when the ascigerous stage
is present. Although this stage was not observed on any of the
host collections used in the present study (Table 1), the conidial
stage may represent more than one race on such collections, as
shown in Figure 3. With this in mind, it might appear some-
what misleading to compare dimensions of a random sample of
spores from a host collection with the artificially produced
spores of a monoconidial isolate from the same collection. How-
ever, in the present study this was done purposely to determine
the magnitude and significance of the difference obtained in
such comparisons.
It is to be noted (Table 13) that mean differences in spore
lengths and widths between a few races are significant, but
mean differences of the majority of the races compared on
either of the three substrata are not significant. The mean
differences in both dimensions of races 18 and 104 are signfii-
cant on all three substrata, except for the widths of the spores
from Malvaviscus stems. The mean differences in lengths of the
spores of races 18 and 22, and 18 and 52, from potato-dextrose
agar and Malvaviscus stems, were significant. In a similar in-
stance mean differences in lengths of the spores of races 18
and 39, from potato-dextrose agar and Malvaviscus stems, were
significant. Aside from the significant mean differences be-
tween races 18 and 104, there were significant differences in
the spore widths of only three other comparisons of the races
shown in Table 13. The lack of significance or the similarity
in one or both dimensions of certain races when compared
on different substrata may be correlated with the apparent
identity of these races in other morphological characters, but
this correlation is by no means consistent. Regardless of the
identity of certain races in their morphological characters they
can usually be separated as physiologic races by the aversion
reaction between them in culture.
To determine the effect of different substrata on the spore
dimensions of certain races, each of the eight races represented
in Table 13 were compared separately on the three substrata.
From the results of the comparison presented in Table 14,







Life History and Taxonomy of the Fungus Physalospora Rhodina 67

it is interesting to note than none of the eight races differed
significantly in either spore dimension when compared on any
two of the three substrata. The greatest differences in both
dimensions were obtained between the races compared from
the host material and potato-dextrose agar. This might be ex-
pected in view of the fact that the random sample of spores
from any host specimen could represent more than one race,
while only one race was represented by the spore sample from
either of the artificially inoculated substrata. In general, the
least differences in the lengths and widths were obtained be-

TABLE 14.-SUMMARY OF DIFFERENCES IN THE MEAN DIMENSIONS OF 200 CONIDIA
OF EIGHT RACES OF Physalospora rhodina WHEN COMPARED ON Two DIFFERENT
SUBSTRATA.

Mean Difference "
Substrata Race (Iin microns)
Length Width I Length I Width


9 .29 2.52 .53 1.70 .105 .312
12 1.31 2.55 1.27 1.34 .514 .948
18 .33 2.84 1.08 1.07 .116 1.009
g 22 2.22 2.19 .33 1.48 1.014 .223
S 39 1.19 2.42 .90 1.42 .492 .634
o 52 .06 2.66 1.40 1.36 .023 1.029
9 91 .38 2.79 2.00 1.37 .136 1.460
0 104 2.58 2.30 1.50 1.28 1.122 1.087
0


E 9 .48 2.50 1.09 1.46 .192 .747
12 .31 2.62 1.19 1.29 .118 .922
02 18 .80 2.71 .26 1.06 .295 .245
S22 .72 2.15 .00 1.50 .335 .000
m 39 1.11 2.42 .72 1.45 .459 .497
o 52 1.62 2.56 .37 1.23 .633 .301
w 91 .27 2.34 1.71 1.32 .115 1.295
104 2.52 2.15 1.59 1.23 1.172 1.293


.19 2.55
1.00 2.50
.56 2.71
1.50 2.10
.08 2.72
1.68 2.22
.11 2.89
.06 2.32


.56 1.48
.08 1.36
.82 1.17
.33 1.36
.18 1.54
1.03 1.25
.29 1.38
.09 1.39






Florida Agricultural Experiment Station


tween the races compared from potato-dextrose agar and Mal-
vaviscus stems. Some variation or even significant differences
in the spore dimensions of the same race might be expected
when compared from such widely different substrata, but per-
haps this possibility is minimized when such substrata are in-
oculated with the same monoconidial isolate. From the statistics
of the spore measurements of the eight races considered, it is
evident that only a few were significantly different when com-
pared from the same substratum and that none of the races
were significantly different when compared from two different
substrata.

EFFECT OF NATURAL HOST SOURCE ON SIZE OF CONIDIA
In addition to the many conidial collections of P. rhodina
made and compared by the writer, specimens of species of
Diplodia and related genera received from correspondents in
in different parts of the world on various tropical hosts were
also compared. The fungus present on many of these host
collections is considered as some form of P. rhodina, as based on
certain morphological and physiological characters. As shown
earlier in this paper (Table 1), monoconidial isolations were
made from many of these collections, and are considered to
be different physiologic races of P. rhodina. The writer also
received samples of several collections from various workers,
including some type material, which are not included in Table 1.
A representative sample of these latter collections will be con-
sidered at this time.
To demonstrate further certain differences which exist be-
tween the conidial dimensions of different host collections of
P. rhodina, conidia measurements were made from several of
the collections mentioned above. In addition to the measure-
ments made by the writer from these collections which agree
with P. rhodina in spore character, the measurements re-
corded by Stevens (68) from four different collections of
P. rhodina, and by Eddins and Voorhees (30) from one col-
lection of P. zeicola are compared in Table 15. The conidia
measurements made by the other workers are included primar-
ily to show that the mean differences between the spore popula-
tions measured by them are also insignificant. The data re-
corded in Table 15 were taken from the following collections:







Life History and Taxonomy of the Fungzts Physalospora Rhodina 69

1-4. Physalospora rhodina (B. & C.) Cke., on (1) Melia sp., (2) Citrus sp.,
(3) Gossypium sp., and (4) Persea sp., as recorded by Stevens (68).
5. Physalospora zeicola Ell. & Ev., on Zea mays as recorded by Eddins
and Voorhees (30).
6. Diplodia frumenti Ell. & Ev., on Zea mays, Louisiana, 1886. No. 493.
Ellis and Everhart (32) (Type material).
7. Botryodiplodia theobromae Pat., on Theobroma cacao, Philippine
Islands, July, 1914. No. 3778, Sydow.
8. Diplodia theobromae (Pat.) Now., on Theobroma cacao, Brazil, Feb-
ruary, 1937. Herb. da Seccao de Fitopatologia, Sao Paulo, Brazil,
No. 2741, Bitancourt.
9. Botryodiplodia theobromae Pat., on Hevea brasiliensis, Philippine
Islands, June, 1921, No. 40015, Sydow.
10. Botryodiplodia theobromae Pat., on Hevea brasiliensis, Kamerun, May,
1938, Sydow.
11. Botryodiplodia theobromae Pat., on Thea sinensis, Assam., January,
1917, Herb. Crypt. Ind. Orient., New Delhi, India, Tunstall.
12. Lasiodiplodia triflorae Higgins, on Prunus sp., Georgia, March, 1915,
No. 468, Higgins (43) (Type material).
13. Diplodia theobromae (Pat.) How., on Ficus retusa nitida, Brazil, Jan-
uary, 1936. Herb. da Seccao de Fitopatologia, Sao Paulo, Brazil,
No. 2160, Goncalves.

The spore measurements recorded from the different host
collections in this study were made in a manner similar to the
previous series, except that 100 spores were considered enough
to give a representative sample of each collection. In the re-
sults of this series of conidia measurements the mean dimen-
sions recorded by the writer agree with those of the other
workers. From the comparison of the mean dimensions of the
spores from these collections recorded in Table 15, it is in-
teresting to note that the only significant mean differences
were between the spore lengths of collections 4 and 12, and
11 and 12. Collections number 5 and 6 are the pycinidial stages
of P. zeicola and are not distinct from those of P. rhodina, but
the perithecial stages are distinct. Thus, it is of interest to
note that the mean dimensions of these two collections are not
significantly different from the collections of P. rhodina. From
these data it is obvious that the spore dimensions of most of
these conidial collections identified under different names are
not significantly different from most of the conidial collections
of P. rhodina. Conversely, a detailed examination of the col-
lections identified under different names showed that the struc-
ture of the pycnidia and the size and shape of the conidia are
not generally distinct from the collections of P. rhodina.







Florida Agricultural Experiment Station


CROSS-INOCULATION STUDIES

Various Diplodia forms of Physalospora rhodina have been
reported on numerous unrelated hosts throughout the tropics
and southern United States. The prevalent opinion among those
who have studied these Diplodia forms in the field is that they
can pass readily from one host to, another and that they are
wound parasites or secondary organisms. Many of the Diplodia

TABLE 15.--SUMMARY OF DIFFERENCES IN THE MEAN DIMENSIONS OF 100 CONIDIA
FROM DIFFERENT HOST COLLECTIONS WHICH AGREE WITHIN THOSE OF Physalospora
rhodina.


Collections
Compared

1& 2
1& 4
1& 5
1& 7
1 & 11
1 & 12
2& 4
2& 6
2& 12
4& 5
4& 6
4& 9
4& 12
5& 6
5& 9
5& 12
6& 8
6& 12
6& 13
7& 8
7 & 11
7& 12
8& 9
8& 12
9& 10
9& 11
9& 13
10 & 11
10 & 12
11 & 12
11 & 13
12 & 13
*Significant.


Mean Difference
(in microns)


1.2;
1.9
.1(
1.41
2.21
.7:
.7;
.8!
.51
1.8'
1.6
.2
1.2:
.21
1.6:
.6!
1.71
.3!
1.61
.61
.81
.7:
.4.
1.3!
.0!
.5:
.3:
.6;
.8!
1.5:
.2;
1.2\


Width
5 1.85
7 1.61
0 1.52
6 1.42
6 1.54
5 1.55
2 1.85
9 1.92
0 1.80
7 1.53
1 1.69
4 1.48
2 1.56
6 1.61
3 1.39
5 1.47
8 1.43
9 1.64
8 1.59
8 1.32
3 1.35
1 1.36
1 1.37
9 1.46
9 1.44
3 1.40
1 1.36
2 1.50
9 1.52
1 1.48
2 -- 1.43
3 : 1.44


Length
.800
1.377
.796
.162
1.350
.674
.528
.464
1.053
.580
1.200
.982
2.042*
.674
.371
1.470
1.011
.664
1.053
.966
1.144
.813
.768
1.753
.122
.951
.821
.763
1.209
2.021*
.019
1.793


Width
.676
1.224
.066
1.028
1.468
.484
.390
4.64
.278
1.222
.953
.162
.782
.161
1.173
.442
1.245
.238
1.057
.515
.593
.522
.299
.952
.063
.379
.228
.413
.586
1.020
.154
.896


Length
1.63 3.04
3.25 2.36
1.87 2.35
.39 2.41
3.16 2.34
1.57 2.33
1.62 3.07
1.50 3.23
3.20 3.04
1.38 2.38
3.12 2.60
2.21 2.25
4.82 2.36
1.74 2.58
.83 2.24
3.44 2.34
2.84 2.81
1.70 2.56
2.98 2.83
2.58 2.67
2.77 2.42
1.96 2.41
1.93 2.50
4.54 2.59
.28 2.29
2.12 2.23
2.07 2.52
1.84 2.41
2.89 2.39
4.73 2.34
.05 2.62
4.68 2.61


I






Life History and Taxonomy of the Fungus Physalospora Rhodina 71

forms described as distinct species are considered to be similar
or identical by various workers. Taubenhaus (77) reported
that D. natalensis and D. gossypina which, according to Stevens
(68), are synonyms of Physalospora rhodina, caused a typical
Java black-rot of sweet potatoes. He later (78) showed that a
strain of Diplodia causing blossom-end rot of watermelons was
identical with D. tubericola. Also, Meir (49) found that D.
tubericola from sweet potato would cause the stem-end rot of
watermelons. Fawcett and Burger (36) discovered that Diplodia
isolated from gumming peach trees caused a gumming of orange
trees and Diplodia isolated from orange trees caused a gum-
ming of peach trees. Crawford (27) showed that a strain of
D. gossypina isolated from cotton bolls produced a typical rot
of sweet potato caused by D. tubericola.
Cross-inoculation studies made by Eddins (29) showed that
D. tubericola, D. natalensis, and D. gossypina caused a rot of
ears of corn similar to that caused by D. frumenti, and each
caused a similar rot of oranges, grapefruit, sweet potatoes,
cotton bolls, and watermelons. In a similar manner, Eddins
and Voorhees (30) obtained positive infection on 31 species of
woody and herbaceous plants representing 24 families with
cultures of D. tubericola, Physalospora zeicola, and P. rhodina.
More recently, Verrall4 has shown that Diplodia natalensis caus-
ing stain in lumber and logs is, from a practical point of view,
the same fungus isolated from cotton, tung, pear, and Citrus.
Various workers outside of the United States also have
obtained similar results with these or related forms, which the
writer considers as synonyms of P. rhodina. Howard (45)
showed by inoculation that Diplodia cacaoicola, the cause of the
common dieback disease of cacao in the West Indies, was iden-
tical with a Diplodia form from sugarcane. He also considered
that Botryodiplodia theobromae reported on cacao pods in
Ecuador was identical with Diplodia cacaoicola in the West
Indies. Nowell (51) is of the opinion that D. natalensis, causing
dieback and stem-end rot of Citrus and boll-rot of cotton in the
Lesser Antilles, is identical with D. theobromae, the cause of
a disease of cacao. Petch (55) reported that Botryodiplodia
theobromae is a very common tropical fungus causing damage
as a wound parasite or secondary to other fungi on Hevea,
cacao, dadap, papaw, sugarcane, and coconut. Baker (2) con-
'Unpublished work by Dr. A. F. Verrall of the Bureau of Plant In-
dustry, Division of Forest Pathology, New Orleans, Louisiana.






Florida Agricultural Experiment Station


siders B. theobromae as a wound parasite causing wastage of
grapefruit in storage in Trinidad, and that it is probably iden-
tical with D. natalensis.
In a manner similar to the methods described by Eddins
and Voorhees (30), several cross-inoculation tests were made
in the present study. These inoculations were made on eight
different hosts or host parts, with mycelial inoculum of most
of the races of monoconidial origin represented in Table 1.
Of the eight different hosts or host parts used in these tests,
four were inoculated in the field (Table 16-A, C, F, H) while
the others were inoculated under laboratory conditions. Prac-
tically all of the hosts were inoculated with each race in trip-
licate, along with checks which were treated similarly to those
that were inoculated, except that inoculum of the different
races was not introduced. All inoculation wounds were first
covered with a piece of cellophane, then wrapped with nursery-
man's tape to avoid natural infection.
From the results of these tests (Table 16) it is to be noted
that practically all of these races, isolated from different
hosts or different collections of the same host, showed positive
infection when introduced artificially into different hosts. In
a few cases all three of the inoculations made with certain
races were not positive, but in most cases all of the inocula-
tions showed some degree of infection. There was no indication
of any natural infection occurring in any of the check inocu-
lations. It should also be stated that there was some variation
in the time required for many of the races to cause infection
on one or more of the hosts inoculated. A longer time was also
required for the fungus to fruit on the woody hosts than on
the herbaceous ones.
There was a slight difference in type of infection produced
by some of the races on certain hosts, but in general this was
comparable on any given host. As characteristic of this fungus,
there was a darkening of the host tissues and the production
of numerous pycnidia on the surface of the host in most cases.
Frequently the infection in certain woody hosts inoculated in
the field did not spread very far, the inoculation wounds be-
coming calloused and preventing further spread of the fungus;
while the picked fruits or plant parts were usually completely
decayed a certain length of time after inoculation. Since the
general symptoms of the decay produced by this fungus on
practically all of these hosts have been described and illustrated







Life History and Taxonomy of the Fungus Physalospora Rhodina 73

TABLE 16.-RESULTS OF CROSS-INOCULATIONS WITH 40 RACES OF Physalospora
rhodina.

Results on Different Hosts
A B C D E F G H

Source "
Race of
No. Races

.Na 2
E; C~ t~


Arachis hypogaea
Zea mays
Zea mays
Gossypium hirsutum
Gossypium hirsutum
Gossypium barbadense
Pyrus communis
Pyrus communis
Pyrus communis
Ipomoea batatas
Ipomoea batatas
Citrus aurantifolia
Citrus aurantifolia
Citrus sinensis
Citrus sinensis
Citrus sinensis
Fortunella margarita
Citrus aurantifolia
Citrus aurantifolia
Citrus grandis
Citrus aurantium
Citrus grandis
Citrus aurantifolia
Citrus aurantifolia
Citrus aurantifolia
Aleurites fordii
Aleurites fordii
Aleurites montana
Persea americana
Persea americana
Ficus carica
Hicoria pecan
Hicoria pecan
Theobroma cacao
Arachis hypogaea
Cocos nucifera
Citrus aurantium
Cocos nucifera
Theobroma cacao
Theobroma cacao


+ + + + + +
+ + + + + + +
+ + + + + + + +
+ + + + + 0 +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + 0 +
+ + + + + +
+ + + + + + + +
+ + + + + +
+ + + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + + 0 +
+ + + + + + + +
+ + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + 0 +
+ + + + +
+ + + + + + + +
+ + + + + + 0 +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + +
+ + + + +
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +


+ Indicates positive infection; 0 indicates no infection; indicates no tests were
made.






Florida Agricultural Experiment Station


by various workers, they will not be considered at this time.
The main feature to be considered from the results of this
study is the fact that practically all of the races were capable
of causing some degree of infection when introduced arti-
ficially into several different hosts. Also, that in spite of the
slight differences in pathogenicity of some of the races, it is
very doubtful whether they could be separated on this basis.
In other words, the various races studied in this investigation
show no definite host specialization.

TAXONOMY AND SYNONYMY
A review of the literature shows that there are many
species of Diplodia and related genera which might well be
included under one specific organism. The controversy existing
over the taxonomic position of many of these forms has been
due, in part at least, to certain fallacies in the present classifi-
cation of the Sphaerioidaceae-Phaeodidymae, which appears
quite definite but in practice proves quite artificial. The fact
that these forms are widely distributed both geographically
and in their host relationships has also added to the confusion.
There has also been the general tendency of separating certain
forms without a sufficient conception of the variations which
might exist under different conditions. No attempt has been
made in the present study to rework the above classification
nor to monograph the genus Diplodia or any related genus. On
the contrary, the aim has been primarily to demonstrate that
certain described forms of Diplodia and related genera might
well belong to one specific organism such as Physalospora
rhodina.
As pointed out earlier in this paper, various workers have
reduced certain Diplodia forms to synonyms, many of which
are also to be included in the synonymy of P. rhodina. Since
type material of many of these forms included as synonyms
by other workers was not available for comparison in this
study, they will be included in the synonymy of P. rhodina as
based on descriptions and conclusions of other workers. Thus,
only those forms bearing on the present taxonomic position of
P. rhodina will be considered in detail.
Many of the earlier-described species were first included in
a synonymy of Botryodiplodia theobromae by Petch (56). In re-
gard to the variability existing in these forms he concludes






Life History and Taxonomy of the Fungus Physalospora Rhodina 75

that the present system of classification of the genus Diplodia
is based to a certain extent upon characters which are not con-
stant. Thus, the presence or absence of a stroma in separating
Diplodia and Botryodiplodia is not a valid distinction in most
cases. Regarding the distinction between Chaetodiplodia and
Lasiodiplodia, Petch (56) states "A Chaetodiplodia is simply
a pilose Diplodia, while Lasiodiplodia is a pilose Botryodiplodia.
But if Diplodia cacaoicola, i.e., Botryodiplodia theobromae in
its simple form, is grown on a cacao pod in a moist chamber,
it becomes a Chaetodiplodia, while the Botryodiplodia form
grown under equivalent conditions becomes Lasiodiplodia."
Thus, he suggests the abolition of Lasiodiplodia and Chaeto-
diplodia, the former to be included under Botryodiplodia and
the latter under Diplodia.
Many of these same forms were examined by Bancroft (3)
and he came to the conclusion that the variability of the char-
acters upon which divisions between the genera in question
are based has caused considerable confusion in the nomen-
clature, besides serious multiplication of names for the same
fungus. Accordingly, he includes several of the forms consid-
ered by Petch (56) as synonyms of Lasiodiplodia theobromae.
In the following year, Bancroft (4) reports a more detailed
study of this group, and especially on what he considered the
Diplodia condition of a perithecial fungus, which he named
Thyridaria tarda. From this study he concludes that three dif-
ferent spore forms are included in the life history of the fungus;
First, a Diplodia sp., which appears to be the parasitic form;
second, a Cytospora sp., which develops on the host sometime
after it has died; and third, the perithecial form Thyridaria
tarda.
No further record of this fungus was made until 1929, when
Tunstall (79) working on the dieback of tea, found a few
specimens of a perithecial fungus which he identified as T.
tarda. However, he failed to produce this stage in culture, and
was able to obtain only immature pycnidia of the Diplodia form
from ascospore cultures. Thus, the supporting evidence ob-
tained by Tunstall cannot be considered entirely satisfactory,
for an element of doubt still remains until mature pycnidia
are produced in pure culture. In the writer's correspondence
with Tunstall regarding more recent information on this fungus,
he states "For a number of years, I continued to call the tea
Diplodia, Thyridaria tarda, but for some years now I have re-






Florida Agricultural Experiment Station


verted to Diplodia sp., as my faith in T. tarda is much weaker."
Thus, the connection which was not fully demonstrated, lacks
confirmation, and the name has not been generally adopted.
Tunstall is also of the opinion that there is no difference be-
tween Botryodiplodia, Lasiodiplodia, and other genera, as forms
may be found on the same bit of dead wood.
In his studies on the validity of Diplodia and certain related
genera, Taubenhaus (77) included several forms under his com-
bination D. tubericola (E. & E.) Taub. In comparing the growth
of Lasiodiplodia theobromae, L. tubericola, Diplodia gossypina,
and D. natalensis on sweet potatoes in a moist chamber, he
concludes that these species are congeneric. On the same basis
he concludes that the genera Diplodiella, Chaetodiplodia, Lasio-
diplodia, and Botryodiplodia, "are not tenable", and should be
included under the genus Diplodia. Taubenhaus suggests the
abolition of the genus Lasiodiplodia, for instance, because the
Diplodia, species under consideration produced hirsute pycnidia,
and the pycnidia of D. gossypina contained paraphyses on sweet
potato in a moist chamber.
In his examination of a similar fungus on sweet potato,
Petrak (57) concludes that the previous work of Taubenhaus
was quite misplaced, and that the sweet potato fungus is clear-
ly a Botryodiplodia, which he named B. tubericola (E. & E.)
Petr. In general he relates that the systematic value of fungus
genera should never be figured from the abnormal forms on
artificial media; and whether or not a stroma is present, wheth-
er it is resolved from the same hyphae or consists only of par-
enchyma, has no generic value. He claims that Diplodia spores
become dark and septate very early, while those of Botryo-
diplodia remain hyaline very long and are seldom septate be-
fore becoming dark colored.
Nowell (51) has also studied this group of fungi and sug-
gests that the genus Lasiodiplodia and the fungus L. theo-
bromae are not based on constant characters. Accordingly, he
includes the forms considered in the synonymy by Petch (56)
and Bancroft (3) as synonyms of his combination Diplodia
theobromae Now. He also considers that the Diplodia fungus
on cotton bolls and D. natalensis on Citrus are probably not
different from D. theobromae.
The fungus Lasiodiplodia triflorae, described by Higgins
(43) in 1916, has not been previously considered in the dis-
cussion of this group of fungi. An examination of the type






Life History and Taxonomy of the Fungus Physalospora Rhodina 77

material shows that it is similar or identical with many of the
forms examined in this investigation. The spore dimensions
from this material were 19-26 x 10-15 /, averaging 22.25 x
13.04, as compared with 22-25 x 13-16 reported in the original
description. Although the mean of the spore lengths was sig-
nificantly smaller than the spore lengths of two other forms
compared in Table 15, the lengths and widths are within the
dimensional limits of the various forms included under P.
rhodina. Higgins (43) considered this fungus to be closely re-
lated to L. theobromae and D. natalensis, but favored their dis-
tinction because of slight morphological differences between
them. However, the fungus is not morphologically distinct from
many of the pycnidial forms of Physalospora rhodina, and is
therefore included in the synonymy of it.
In general, results of the present investigation are in agree-
ment with those obtained by Stevens (67, 68) and Stevens and
Wilcox (72) on certain tropical forms of Diplodia which are
all quite similar morphologically. Stevens (67) first described
Physalospora gossypina from cotton and other hosts in the
southern United States as the perithecial stage of Diplodia
gossypina, and concluded that Botryodiplodia gossypii was
identical with it. The following year Stevens (68) described
Physalospora fusca and P. rhodina from Citrus and other hosts,
the Diplodia stages of which are quite similar morphologically,
and the perithecial stages of which are quite similar except
for the brown-colored ascospores of the former. At the same
time he included Diplodia natalensis, D. gossypina and his
previous combination P. gossypina as synonyms of P. rhodina.
More recently in this country, Eddins (29) showed that D.
tubericola, D. natalensis, D. gossypina, and D. frumenti are
all quite similar morphologically and can pass readily from one
host to another. In a similar manner, Eddins and Voorhees
(30) showed that D. tubericola, Physalospora zeicola and P.
rhodina can pass readily from one host to another. They also
showed that P. zeicola was the ascigerous stage of Diplodia
frumenti on corn and other hosts. Although the Diplodia.stages
of P. rhodina and P. zeicola are not generally distinct, the
perithecial stages differ in certain morphological characters
and must be considered as distinct species for the present.
Granting that many of the pycnidial forms considered in the
present study might well be the Diplodia stages of these and
undescribed perithecial forms, the writer is inclined to include






Florida Agricultural Experiment Station


them under P. rhodina until more information is obtained on
their perithecial stages.
At this time it is evident that there exists in the literature
a multiplicity of names for various Diplodia forms which are
morphologically similar or identical with the Diplodia stage of
Physalospora rhodina. This has been due to the wide distribu-
tion of these forms, both geographically and in their host re-
lationships; to the variation existing under different condi-
tions; and to the present system of classification of this group
of fungi. Various workers have shown that these forms can
pass readily from one host to another, and consider many of
them to be identical. Many of the forms considered in the
present investigation are identical in certain physiological and
morphological characters, but the reaction between them in
culture indicates that they are different physiologic races.
Thus, the writer firmly believes that many of these forms are
synonyms of P. rhodina, as based on examination and study of
type or authentic material, and various collections and cultures.
This is also based to some extent on the descriptions of many
of these forms, certain morphological characters of which are
summarized in Table 12.
The present synonymy of P. rhodina includes the synonyms
of Botryodiplodia theobromae recorded by Petch (56), of Lasio-
diplodia theobromae by Bancroft (3), of Diplodia tubericola
by Taubenhaus (77), and of Diplodia theobromae by NowelI
(51). The fungus Lasiodiplodia triflorae Higgins (43), and
Botryodiplodia tubericola (E. & E.) Petr. (57) are also in-
cluded. The present synonymy of P. rhodina is an extension of
that initiated by Stevens (68) in 1926, but it is by no. means
complete.
PHYSALOSPORA RHODINA (Berk. and Curt.) Cooke,
Grevillea 17:92. 1889.
Type specimen collected by M. A. Curtis on Rosa rubiginosa in North
Carolina. In Herb. Cooke, Kew. Authentic material collected by N. E.
Stevens on Citrus and other hosts in Florida and Cuba. In Herb. U. S.
Dept. Agr., Washington.
Pycnidia simple or compound, single or grouped, stroma present or
absent, immersed to erumpent or almost superficial, hairy or glabrous,
varying in size and complexity with substratum, frequently papillate
with prominent ostiole, 1/2-6 mm. in diameter; conidia borne on short
conidiophores, nonseptate, hyaline and granular when young, becoming
brown and 1-septate at maturity, and usually with longitudinal stri-
ations, 17-34 x 10-18 p; paraphyses present or absent.
Perithecia single or gregarious to closely crowded, scattered or in
semi-parallel rows, immersed to erumpent, papillate, opening by a black
rounded ostiole, externally carbonous, globose, 225-300 A in diameter; asci







Life History and Taxonomy of the Fungus Physalospora Rhodina 79

clavate-cylindrical, stalked, sessile, double-walled, the inner wall re-
maining to enclose the mature spores, typically eight-spored with spores
crowded-biseriate, 75-110 x 18-25 u; spores hyaline to dilute olivaceous,
one-celled, elliptical to ellipsoid, often tapering to narrow, rounded ends,
27-38 x 9-17 p.
Synonyms based on examination of type or authentic materials:
Diplodia gossypina Cke. Grevillea 7:94-96. 1879. U. S. Dept. Agr. Herb.,
and Roy. Bot. Gard., Kew.
Botryodiplodia theobromae Pat. Bull. Soc. Myc. France 8:113-140. 1892.
Botryodiplodia gossypii Ellis and Bartholomew. Jour. Myc. 8:173-178.
1902. Fungi Columbiani No. 1510.
Chaetoplodia grisea Petch. Ann. Roy. Bot. Gard. Peradeniya 3:(1), 6.
1906. Roy. Bot. Gard. Herb., Kew.
Diplodia natalensis Evans. Trans. Dept. Agr. Sci. Bul. No. 4. 1910.
Lasiodiplodia triflorae Higgins. Ga. Agr. Exp. Sta. Bul. 118. 1916. Ga.
Agr. Exp. Sta. Herb. No. 468.
Physalospora gossypina N. E. Stevens, Mycologia 17:191-201. 1925. U. S.
Dept. of Agr. Herb.
Synonyms based on descriptions and various host collections:
Diplodia cacaoicola P. Henn. Bot. Jahrb. Engler. 22:80. 1897.
Lasiodiplodia tubericola Ell. and Ev. Bot. Gaz. 21:92. 1896.
Lasiodiplodia nigra Appel. and Laub. Arb. aus der Kaiserl. Biol. Inst.
fur Land und Fortswirts. 3:147. 1906.
Botrydoplodia elasticae Petch. Ann. Roy. Bot. Gard. Peradeniya. 3:(1),
7. 1906.
Lasiodiplodia theobromae (Pat.) Griff. and Maubl. Bul. Soc. Myc.
France 25:51-58. 1909.
Diplodia rapax Massee. Kew. Bul. No. 1, p. 3. 1910.
Diplodia tubericola (Ell. and Ev.) Taub. Amer. Jour. Bot. 2:324-331.
1915.
Diplodia theobromae (Pat.) Now. Diseases of Crop Plants in the Les-
ser Antilles. London. 1923.
Botrydoplodia tubericola (E. & E.) Petr. Ann. Myc. 21:182-335. 1923.

This synonymy is certainly not at variance with the views
of many other workers. After a study of the literature and
correspondence with various investigators it is the unanimous
opinion of those who have studied the tropical and sub-tropical
species of "Diplodia" that many of them are similar or identical.
Accepting the abolition of the genera Lasiodiplodia and Chaeto-
diplodia and including these forms under Diplodia and Botryo-
diplodia, the greatest difference in opinion on the above synony-
my would probably be on the combination of the group con-
sisting of Botryodiplodia theobromae, Diplodia theobromae, and
D. cacaoicola, with the group consisting of D. gossypina, D.
tubericola, and D. natalensis. However, it is the unanimous
opinion that there is no distinction between many of these
forms. This is suggested in part by Nowell (51), Stevens (67,
68, 71), Shear (65), and many others.






80 Florida Agricultural Experiment Station

In addition to the forms included in the above synonymy
of Physalospora rhodina, an examination of the material of
many other described forms might reveal a similarity or iden-
tity. For example, the host species associated with and the de-
scription of such forms as Diplodia hesperidica Speg., D. man-
giferae Koord., D. caricae Sacc., Chaetodiplodia diversispora
Em., D. cococarpa Sacc., and Botryodiplodia sorghii P. Henn.,
as recorded by Saccardo in his Sylloge Fungorum, would sug-
gest some relation. Such forms as D. citrina Died., D. indica
Died., Botryodiplodia manihotis Syd., B. persicae Died., and B.
saccharine Died., as recorded by Sydow et. al. (76) would also
suggest a similarity. This synonymy might also include Chaeto-
diplodia arachidis Maubl. (48), Diplodia epicocus Cke. and
Sphaeropsis palmar'um Cke. (23), and Botryodiplodia palm-
arum (Cke.) Petr. & Syd. (59).
In regard to the fungus Physalospora fusca -described by
Stevens (68) in 1926 on Citrus and other hosts, it is probably
genetically related to P. rhodina but must be considered distinct
for the present on the basis of certain morphological char-
acters. As pointed out by Stevens, it is very close to P. rhodina,
except for the brown-colored ascospores, and their' Diplodia
stages are hardly distinguishable. Although the writer has not
collected this fungus in nature nor worked with it in culture,
he has had opportunity to examine certain collections of it,
including the type material. Through the courtesy of E. W.
Mason, of the Imperial Mycological Institute, the writer ex-
amined material of this fungus (I. M. I. No. 2108), collected
by C. G. Hansford on Tephrosia vogelii in Uganda. Ascospores
from this collection measured 32-42 x 13-18 1, averaging 37.2
x 15.2 p., as compared with the dimension 29-37 x 11-16 .,
mostly (more than half) 31-34 x 13-14 p recorded by Stevens
(68). Although the spore dimensions of the African collection
are slightly larger than those recorded by Stevens, it is ap-
parently the same fungus. The occurrence of this fungus in
South Africa was suggested by Stevens (68) in 1926, as was
the suggestion that it might be the perithecial stage of D. na-
talensis. N. E. Stevens has since made a new combination of
this fungus to avoid adding another title to botanical litera-
ture, and suggested to the writer that it be included in this
paper. Thus, the writer is glad to include this new combina-
tion by Stevens at this time:







Life History and Taxonomy of the Fungus Physalospora Rhodina 81

"E. W. Mason of the Imperial Mycological Institute called my at-
tention to the apparent identity of the fungus described by Berkeley in
1869 as Sphaeria abdita and the one to which I gave the name Physalospora
fusca in 1926. Mason's suggestion was, of course, based on examination
of the type in the Kew Herbarium, which was loaned to us by the di-
rector for examination.
The type is represented in the Mycological Collections of the Bureau
of Plant Industry by two slides, No. 3190 and No. 3191, made by C. L.
Shear a number of years ago and labelled
Sphaeria abdita B. & C.
Wright Cuba "No. 432"
From type herb. Berk.
These slides contained numerous brown ascospores, some in asci
and a few outside. The latter show the two-septate condition common in
old ascospores of this group. There are also a few pycnospores, typically
septate and striate. Ascospores in this material measure 24-28 x 10-13 p.
These measurements are somewhat larger than those given by Berkeley
".0001-.0006 inches long, .0005-0003 wide" (approximately 25-15 x 12-8 0).
Even according to my measurements, spores of the type are some-
what smaller than those in the specimens on which the description of
P. fusca was based, but if at that time I had known of S. abdita and had
the type material before me, I would certainly have considered the
two as belonging to the same species. The logical procedure now would
seem to be to propose the new combination and slightly revise the de-
scription.
Physalospora abdita (B. & C.) comb. nov.
Pycnidia simple or compound; pycnidial stromata black, variable in
size, often 1-2 mm. in diameter; pycnospores at first one-celled and
hyaline, becoming dark brown and septate, usually with irregular long-
itudinal striations, 20-28 x 11-16 j, mostly (more than half) 23-25 x
12-13 A.
Perithecia scattered to gregarious, black externally, 225-250 I in
diameter; asci typically eight-spored, clavate, 120-150 A long; ascospores
one-celled; 24-37 x 10-16 A, mostly (more than half) 26-37 x 11-16 p,
brown when mature.
Synonyms:
Sphaeria abdita B. & C. in Berk. Jour. Linn. Soc. Bot. X, p. 388. 1869.
Anthostomella abdita (B. & C.) Sacc. Sylloge I, p. 290. 1882.
Physalospora fusca N. E. Stevens in Mycologia 18:210. 1926.

Type specimen in Herb. Kew, labelled
"432. Sphaeria abdita B. & C. Cuba. C. Wright. On Congo Bean."
Literature Citations
Berkeley, M. J. On a collection of Fungi from Cuba. Part II, pp.
341-392. 1869.
Saccardo Syl. Fungi. Vol. I, p. 290. 1882.
Stevens, Neil E. Two species of Physalospora on citrus and other
hosts. Mycologia 18:206-218. 1926."






Florida Agricultural Experiment Station


Although certain Diplodia forms considered in the present
study and by other workers have their ascigerous stage in the
genus Physalospora, it is entirely possible that certain forms
may have their ascigerous stage in other genera. For example,
Saccardo in his Sylloge Fungorum gives Diplodia as the pycnid-
ial stage of species of Cucurbitaria, Massaria, Otthia, Melan-
omma, Pleospora, Thyridaria, and Gibberidia, but the genetic
relations have not been confirmed in most cases. In the case
of Thyridaria Bancroft (4) considered T. tarda as the peri-
thecial stage of the Diplodia condition studied by him, and was
confirmed to a certain extent by Tunstall (79). However, the
connection was not fully demonstrated in either case and the
name has not been generally adopted. Shear (65) demonstrated
that certain Diplodia forms occur in the life history of cer-
tain species of Tryblidiella, and ascospore culture from an In-
dian collection produced mycelium and pycnidia very similar
in appearance to that of species of Physalospora. However, the
Diplodia forms produced in ascospore cultures from other col-
lections were quite distinct from those of species of Physa-
lospora, which was confirmed by the writer (83) in 1939. At
any rate it is obvious that further collection and study of peri-
thecial forms having a Diplodia stage in their life history
should not be long delayed.

DISCUSSION
From the literature it is quite apparent that there is con-
siderable confusion in the taxonomic position of the forms of
Diplodia and related genera found on many tropical and sub-
tropical hosts. Moreover, it is apparent that this confusion has
been due to certain fallacies that exist in the present system
of classification of this group of fungi. The fact that many
of these forms are variable in certain characters under certain
conditions and that they are widely distributed both geograph-
ically and in their host relationships was not recognized by
many of the early investigators. Nevertheless the difference of
opinion cannot necessarily be attributed to any fault of the
describer, since many of these early investigators were field
mycologists without the time or facilities for supplementary
experiments.
More recently, work on the life history of many of these
Diplodia forms has shown that they are widely distributed and






Life History and Taxonomy of the Fungus Physalospora Rhodina 83

that they can pass readily from one host to another. It has
been shown also that many of these forms are variable in cer-
tain morphological and physiological characters under certain
conditions, but in general they are similar in appearance. How-
ever, they are all physiologically distinct by the aversion re-
action between them in culture, and are thus properly desig-
nated as physiologic races of Physalospora rhodina.
The evidence in the present investigation, while as yet in-
complete, indicates that at least some of the physiologic races
have their origin in the sexual stage, and that they are dis-
tinct by the aversion reaction between them. The stability of
the aversion reaction between races is maintained through suc-
cessive conodial and mycelial transfers on nutrient media, and
through inoculated host material. This indicates that the factor
or factors for the aversion of the monoconidial or monoasco-
spore mycelia are fixed and maintained through subsequent.
conidial and' mycelial generations. No difference was observed
in the manifestation of the aversion phenomenon of the mono-
conidial and monoascospore mycelia of P. rhodina in this in-
vestigation. Thus, the two types of aversion reported by Cay-
ley (15) in Diaporthe perniciosa, namely, inter-racial aversion
between biologic races and intra-perithecial aversion between
mycelia from the same fruiting body, are not recognized by
the writer in Physalospora rhodina.
In the literature dealing with intraspecific aversion the as-
pect of the problem studied most extensively concerns the re-
lationship between the phenomenon and sex. Several workers
have investigated the inheritance of the phenomenon and its
sexual relationships in the Ascomycetes and Hymenomycetes.
However, only a few cases need be cited to illustrate that the
aversion phenomenon in most fungi is conceded to be due to
genetic constitution rather than to a chemical interaction, which
is also assumed to be the case in P. rhodina. Cayley (15), in
her studies with Diaporthe perniciosa, concludes that "inter-
racial aversion is a form of sterility between biologic races,
and that intra-perithecial aversion is a peculiar form of physio-
logic self-sterility other than sex." According to Cayley (15),
Nakata concludes that the aversion between geographical
strains of Sclerotium rolfsii is not due to a substance formed
by the action of the fungus on the medium, but by a substance
which is produced by the mycelia irrespective of the medium.
Although the aversion reaction between strains of Diplodica






Florida Agricultural Experiment Station


zeae was not definitely correlated with sex of this species,
Hoppe (44) concludes that the differences among the strains
is genetic.
The aversion phenomenon in the Hymenomycetes has been
ascribed to the liberation of certain chemical substances, but
more generally to inherited characters. The first critical
analysis of the cause of neutral aversion in the Hymenomy-
cetes was made in 1932 by Vandendries (80), who demon-
strated that aversion is perfectly correlated with genetic con-
stitution in Pleurotis columbinus. In working with monospor-
ous cultures of Lenzites betulina, Vandendries and Brodie (81)
showed that aversion is entirely dependent upon the genetic
constitution of the mycelia, and concluded that it is difficult
to explain the results of their experiments by the assumption
that the repulsive effect is of a chemical nature.
Although it was not definitely determined whether the
aversion phenomenon in P. rhodina was correlated with sex,
the results indicate that the factor or factors for this char-
acter are carried by the ascospores in different ratios in the
ascus. Various workers have demonstrated that sex can be
distributed among the nuclei of the ascus in several different
ways in certain Ascomycetes, and that other segregating char-
acters or factors can be distributed in a like manner. Regard-
less of whether there is any correlation between the segrega-
tion for sex and aversion in P. rhodina, the aversion phenom-
enon is considered to be a manifestation of an inherited phys-
iological difference between physiologic races of this species.
Although not quite analogous to the haploid lines in cer-
tain fungi, the physiologic races of P. rhodina are actually
haplonts. Thus, it might be somewhat misleading to assume
that all of the different haplonts of P. rhodina are physiologic
races. On the other hand, it might be just as misleading to
assume that this species comprises so many different sexes.
Since certain points in question must necessarily await further
investigation, a simple and workable hypothesis at this stage
is the conception of two sexes in P. rhodina and that segrega-
tion for sex and aversion may or may not be independent.
Aside from the aversion reaction between the various mono-
conidial and monoascospore isolates referred to as physiologic
races in the present investigation, there may be only slight
physiological differences between them. In certain races, how-
ever, there is a distinct difference in their cultural characters,







Life History and Taxonomy of the Fungus Physalospora Rhodina 85

and their ability to show chromogenesis at 36 C. and above.
There is also considerable difference in the pycnidial char-
acters of certain races. Although the majority of the races
studied are not sufficiently specialized to be consistently dif-
ferentiated on this basis, many of them could be grouped ac-
cording to certain characters. From the rate-of-decay tests at
different temperatures the most apparent difference occurred
at the higher temperature (340 C.), which indicates that cer-
tain races are high temperature forms. For the most part,
however, they exhibit the same general cultural characters and
temperature relations as shown in other conidial collections
of P. rhodina by previous investigators.
The variation or difference in the morphological characters
of certain races studied is just as pronounced as in their phys-
iological characters. Although relatively few of them can be
differentiated as individuals on this basis, it is evident that
they can be grouped to a certain extent according to the struc-
ture and aggregation of their pycnidia and especially on arti-
ficial media. The pycnidial characters of many of the conidial
collections vary according to the conditions under which they
are grown. This has been overlooked by many investigators
in describing them as distinct species. Although the conidia
provide one of the most constant morphological characters in
that they are all similar in appearance, they may differ sig-
nificantly in size. Many investigators are of the opinion that
many of the conidial collections merit specific distinction as
based on this character, but such views are inevitably based
on the study of relatively few collections. It seems probable
that discrepancies between quantitative data secured by com-
petent workers often may be charged directly to the basing of
studies on only one or a few individuals from species in which
variation may exist.
The fact that the many races or conidial collections of
Physalospora rhodina are so widely distributed in their host
relationships and that they may vary in certain morphological
characters has led to a multiplicity of names in the literature.
This seems inevitable in view of the large number of races
existing:in nature, and that they can pass readily from one host
to another by cross-inoculation, without showing any definite
host specialization. Admitting that many of the described
species are very similar in certain characters, many investi-
gators, including Grove (38), would differentiate them by their






Florida AgricultUral Experiment Station


host plant. In opposition to this view, Stevens (71) has very
adequately expressed the writer's opinion in stating, "Admit-
tedly each fungus is a special case and each may behave dif-
ferently from any other, but such observations as these here
reviewed seem to me to suggest that it is more probable that
such fungi growing on dead or weakened plant parts, and which
grow readily on a wide variety of culture media, have a wide
host range, than that a distinct fungus species grows on each
host species." The observations reviewed by Stevens (71) were
made during the course of his work in this group of fungi
(67, 68, 69) and of work by Stevens and Wilcox (72), which
have been confirmed in the present investigation.
Although many of the conidial collections studied in the
present investigation and included in the synonymy of Physa-
lospora rhodina are quite similar in appearance, they may not
be distinct from forms having their sexual stage in other species
of Physalospora. There are apparently at least three distinct
species of Physalospora, namely, P. fusca, P. rhodina and P.
zeicola, having very similar Diplodia stages. To avoid adding
another title to botanical literature the writer has gladly in-
cluded a new combination of P. fusca, compiled by N. E. Stev-
ens, in the present paper.
In lieu of the plasticity of the present system of classifica-
cation, many of the forms investigated have been placed in
either Diplodia, Botryodiplodia, Chaetodiplodia or Lasiodiplodia.
This situation has been corrected to a great extent by various
investigators in the abolition of the latter two genera and the
reduction of many of the described forms to synonymy. In an
attempt to correct the situation further, the writer has re-
duced more of these forms to synonyms of Physalospora
rhodina. However, in view of the chaotic situation which still
exists in this group of fungi, more detailed studies should not
be long delayed.
SUMMARY
The many Diplodia forms to be included under Physalospora
rhodina are widely distributed throughout the tropics, grow-
ing as wound parasites or secondary organisms on numerous
tropical and sub-tropical hosts..
The taxonomy of these Diplodia forms has long been in
question, but many of them have been reduced to synonyms
by previous investigators.






Life History and Taxonomy of the Fungus Physalospora Rhodina 87

Several hundred monosporous isolates of P. rhodina were
made from material obtained from the southern United States
and other parts of the tropical world. Approximately 100 of
these isolates were studied in considerable detail.
The results indicate that P. rhodina comprises an indefinite
number of races which may be very similar in appearance but
may differ from one another in one or more characters.
No definite evidence was obtained on the possibility of new
races arising from sectors or mutations, but it was quite evi-
dent that many races arise in the sexual stage in nature. Prac-
tically all of the races are differentiated by the aversion re-
action between them in culture. The stability of this reaction
was maintained through one or more mycelial transfers in
culture and inoculated host material.
It was not determined whether the aversion phenomenon
in P. rhodina was correlated with sex, but the results indicate
that the factor or factors for sex are carried by the ascospores
in different ratios in the ascus. The results also suggest that
this phenomenon is a manifestation of an inherited physio-
logical difference between physiologic races.
In addition to the aversion reaction many of the mono-
sporous isolates or races are distinguished in culture by one
or more of the following characteristics: Nature and amount
of mycelial growth, color of mycelium, rate of growth, pro-
duction of chromogenesis at 360 C. and above, and type of
pycnidia produced.
The general morphological characters of the conidial and
perithecial collections of P. rhodina are described. The conidial
dimensions may be an additional aid in distinguishing races of
P. rhodina. There were statistically significant differences in
the conidial dimensions of a few of the races compared on dif-
ferent substrata, including the natural host source. The sub-
stratum did not have a significant effect on conidia size of a
given race, but it did on the difference in conidia size between
certain races.
Results of cross-inoculation studies with different isolates
or races of P. rhodina are in agreement with those obtained
by previous investigators with different Diplodia forms of this
fungus. Certain races showed slight differences in pathogen-
icity but no definite host specialization.
The difference in the morphology of many of the conidial
collections of P. rhodina studies is not considered wide enough






Florida Agricultural Experiment Station


to merit specific distinction. Thus, the collections studied in this
investigation and certain forms described as distinct species
by other investigators are included in the synonymy of P.
rhodina.
There are at least three distinct species of Physalospora
that are not distinguished by their Diplodia stages. Thus, it is
entirely possible that many of the Diplodia forms included un-
der P. rhodina have their sexual stages in undescribed species
of Physalospora and other genera.


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