SOME ASPECTS OF PATHOGENIC AND
MORPHOLOGICAL VARIABILITY EN

CERCOSPORA APH

Thesis for ”to Degree of M. 5.
MICHIGAN STATE UNIVERSITY

Roger M. Knutson
19 61

 

LIBRARY

Michigan State
University

 

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ABSTRACT

SUCH ASPECTS OF PA'IHOCENIC
ANDHMORPHOLOGICAL VARIABILITT’
IN CBRCOSPORA APII

By Roger M. Knutson

The fungal genus Chrcospora_includes a large number of species

 

differentiated primarily on the basis of their morphology on host plants.
The present work is an investigation of variability in pathogenic cap-
acity or host range, combined with an evaluation of the constancy of
taxonomic characters when the fungi are cultured on artificial media.

Isolates of different Cercospora Species were obtained from several

 

widely separated geographic areas, cultured on steamed carrot leaf decoc-
tion agar which induced Sporulation of most Species, and test inoculated

to a number of taxonomically unrelated hosts. Most inoculations were made
on excised leaflets floated on tap water in covered petri plates to avoid

cross contamination by the different species of Cercospora used.

 

 

Experimental results indicate that Cercospgra agii and several
other Cercospgra species are capable of infecting a number of taxonomically
different hosts in the greenhouse. FUrthermore, pathogenicity on plants
other than the original host can be increased by repeated inoculation to
the new host.

length and width measurements of conidia of various species of
Cercospora were compared. Conidial characteristics were variable in
Culture; however, measurements from a number of Species showed similarities

in length distribution. On the basis of width measurements it was

Roger M. Knutson
possible to separate several pathogenically and morphologically distinct
lines from.an original 9. 22;; culture.

The presence of numerous nuclei in the conidia and the possibility
of nuclear transfer from one species to another indicates that hetero-

karyosis may be the mechanism of variability in the gercospora Species

 

investigated.

SOLE ASPECTS OF PATHOGENIC
AND MORPIDLOGICAL VARIABILITY

IN canoosm APII
By

Roger M. Knutson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTEROP SCIENCE

‘Department of Botany and Plant Pathology

1961

ACKNOWLEDGMENTS

The author wishes to express his appreciation to Dr. H. H.
Murakishi for originally suggesting the problem, and for con-
‘tinuing material and intellectual aid. Among the many graduate
students and faculty who offered suggestion and criticism,

Dr; R. L. Kiesling and Dr. E. H. Barnes should be singled out
as major contributors. Thanks are also due to my wife for
direct help as proofreader and critic and indirect aid as a

brewer and server of coffee.

Introduction. . . . . .

TABLE OF CONTENTS

Literature review . . . . . .

Materials and methods . . . .

Sources of host plants and fungal cultures .

Culture media and inoculation procedures . .

Conidial measurements . . .

ExpermentsandreSUItSoooooococo...

Effects of passage through different hosts
on the cross infectivity of g. apii . . . .

Comparative Spore morphology of different
species of gercospora on a single medium .

 

Effects of passage through different hosts

on spore morphology of 9,

84.211

Germination and penetration experiments .. .

Do Cercospora species show heterkaryotic potential?.

Discussion and conclusions

Bibliography

iii

0 O O O O O O O

Page

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15
17

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29
32
36
44

47

LIST OF TABLES
Page

Table 1- Results of inoculation of intact plants with
seven isolates of Cercospora from different hosts . . . l9

 

Table 2- Effectiveness of the atomizer inoculation
technique on excised leaflets . . . . . . . . . . . . 22

Table 3- Comparison of per cent infection of susceptible
lines of celery with Q. apii on intact plants
and CXCised leaflets o o o o e o o o o o o o o o o o o 22

Table 4- Results of serial inoculations of excised celery
leaflets with Cercospora isolates from five
differenthOStplantSooeooooooooooooozz

 

Table 5- Results of inoculations of excised leaves of
Malva rotundifolia, Althea rosea and Beta vulgaris
with g. apii cultures reisolated from various
hOSt plants 0 o o o o o o o o o o o o o o o o o o o O 24

 

Table 6- Results of inoculation of intact plants with
isolates obtained from infections on excised leaflets . 24

Table 7- Results of inoculations of épium graveolens and
Melilotus officinalis with six single spore isolates
From Cllltures 0f mixed 9. aEii and E. daViSii o o o o o 43

 

 

iv

Fig.

Fig.

Fig.
Pig.
Pig.
Fig.
Fig.

Fig.

3?

9...

LIST OF FIGURES

Diagram showing the lesion counts from successive
transfers of two forms isolated from celery
following inoculation with C. malvarum

obtained from Malva rotundifoliaf-t-tfz . . . . .

 

Diagram showing lesion counts from successive
transfers of E, davisii obtained from Melilotus
OffiCinalis . o o a o o o o o o o o o o o o o o 0

length distributions of conidia . . . . . . . . .

glength distributions of conidia . . . . . . . . .

camera lucida drawings of conidial tips and bases

Width distributions of conidia of Q. apii
from various hOStS o o o o o o o o o o o o o o o

Cbmparison of conidial width distributions for
C. apii from original culture and summation curve
for g. apii isolated from four other hosts . . .

Camera lucida drawings of representative conidia

Camera lucida drawings of representative conidia

10- Multinucleate conidium . . . . . . . . . . . . .

ll- Multinucleate conidium . . . . . . . . . . . . .

12- Multinucleate conidiophore . . . . . . . . . . .

l3-

Conidium germinating by the formation of conidiophores

Page

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30

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34
35
38
39
40

41

INTRODUCTION

Early blight, incited by Cercospora apii’Fres. is an economically
important disease wherever celery is grown. The disease and its causal
organism have been intensively studied by a number of workers since the
first report of their occurrence in the United States by Galloway (22)
and since the earliest cultural work by Duggar (15). The history of the
disease and its epidemiology have been reviewed by Klotz (44) and others.
Studies of the organism and its relationship to celery and celeriac, the
only reported hosts, have aided our understanding of the disease, and have
made possible a measure of control through the use of fungicides and the
development of tolerant varieties (54, 86).

Early workers were aware that the fungus might parasitize other hosts,
but aside from reports of unsuccessful inoculations on other members of
the family umbelliferae (44), there are no indications that wide host

range studies were undertaken. Later workers, attempting cross infection

with other species of the genus Cercospora, occasionally included celery

 

in their host ranges, but no further investigations of.§.Iggii itself as
a possible pathogen of other plants were made.

.Early inoculation work was hampered by difficulties in inducing
sporulation of the fungus in pure culture. Until the discovery by
Kilpatrick and Johnson in 1956 (43) that a steamed carrot leaf decoction

agar induced sporulation in a number of Cercospora isolates, inoculum was

 

obtained directly from infected plant parts or from growth of the fungus
on autoclaved plant parts. There are scattered reports of sporulation of
some isolates on potato-dextrose agar (PDA), but variations were observed
between isolates of the same species at different times, and the conidia

were seldom abundant enough for inoculation.

l

In addition to these technical difficulties encountered in the study

of Cercospora Species, the morphological similarity of isolates from

 

different hosts often made species identifications impossible unless

they were made directly from.known hosts. Chupp's monograph (8) provides
us with a listing and catalogue of almost 2000 named and described species,
but the overlapping of morphological characters and general impossibility
of identification without the host plant reduce its value as a tool.

Many of the fungi falling into the Class Fungi Imperfecti demon-
strate a great variability under differing conditions, and it is not to
be supposed that the genus Cercospora is unusual in this respect.
Cultural variations in the growth and appearance of g. 32.3; were observed
in the earliest pure cultures of the organism. Other fungi, more easily
manipulated in culture, have been studied in an attempt to elucidate the
causes for such variability. What was originally referred to as salts-
tion or sectoring in cultures was explained to a degree by the postula-
tion of the dual phenomenon by Hansen (26, 28), and by later explanations
involving heterokaryosis and the non-sexual recombination of genetic
factors. The effect of such phenomena on the parasitic potential of
fungal organisms has not been thoroughly explained, but should be con-
sidered in any explanation of the parasitic abilities of fungi demon-
strating cultural variability.

The morphological similarity of many of the named species in the
genus Cercospora, combined with reports in the literature of their wide
capacity to parasitise unrelated hosts:has led to the consideration that
Cercospora.apii might also be able to act as a pathogen on plants other
than celery. The present work involves the examination of 2, £233 and

several similar species with regard to host range, adaptibility of the

organism to various hosts, and the possible reasons for the observed
variability of the fungus. On the basis of this work, no definition of

a complete host range is possible, nor are there bases for an explanation
of the genetic relationships of the organisms studied. Further work
along the same lines, however, might well make possible the characteriz-
ation of species on physiological and pathological bases, as well as the

morphological criteria used at present.

LITERATURE REVIEW

Early work with 9, 222} and other species described the establish-
ment of infection, symptoms on the host and growth of the fungus under
various culture conditions (44). For the present work it should be suf-
ficient to mention that the conidia of the fungus are transported to the
leaves of a Susceptible host by wind, splashing water, workers clothing,
and insects (23), and that these conidia germinate at relative humidities
near 100% and send slender germ tubes across the leaf surface. A small
percentage of these germ tubes enter the leaf through the stomates or
guttation pores. Mycelium develops intercellularly and eventually pro-
_duces a necrotic Spot of a size and color varying with the host plant.
Within this necrotic area a mycelial stroma fills the substomatal spaces
and pushes out clumps of conidiophores through the stomates, rupturing
the guard cells. Conidia are produced on these conidiophores at relative
humidities from 85-100%, the mature conidia are detached, and the infec-
tion precess may be repeated on the same or on another leaf. These steps

are essentially the same for all Cercospoga species treated in this paper,

 

although little information, other than taxonomic description,is avail-
able for the large number of species commonly found on weed hosts and
plants of little economic importance.

Many attempts have been made to induce sporulation of _C_. 32}; and
other species on agar media. From the earliest efforts by Duggar (15, 16)
until the 1950's sporulation was sporadic and conflicting results were
often obtained with isolates from the same host plant. Duggar produced
conidia of g, apii on sterile bean stems or celery petioles (15), but was

unsuccessful with E. beticola. In studies of the leaf blotch disease of

potato Caused by E, concors, Jones and Pomeroy (41) obtained slow growth
of the fungus on artificial and natural agar media, but reported no con-
idia produced in their cultures. .§° brunkii was observed to Sporulate
sparingly on corn meal agar (23), and Hopkins (30) produced abundant
conidia by the partial drying of cultures of _C_3_. medicaginis. Coons and
Larmer (10) sporulated Q. beticola on a Specially prepared beet leaf agar.
Nagel (58) reported abundant sporulation of some isolates of a number of
Species on PDA, while other isolates produced no conidia on any of 30
agar media used. Nagel considered transfers every three days as the only
means of maintaining sporulating cultures in the laboratory. Various
Specialized media and techniques were found to induce sporulation in some
cases (12,18,77), while trials of over 30 media were unsuccessful for
other workers (55). Lewis obtained abundant sporulation of Q. §£_i_i_ on a
number of different sorts of autoclaved plant parts, and Kilpatrick and

Johnson (43) reported that a number of different Cercospora Species spor-

 

ulated well on steamed carrot leaf decoction agar. In 1958, Jenes (40).
following a method outlined by Calpouzos (4), obtained a sporulating
culture of.9. kikuchii by selectively picking out only those sectors of
his cultures that were sporulating abundantly. Seven repetitions of this
selection gave a culture that sporulated in 62 of 70 single spore sub-
cultures. The combination of carrot leaf agar and selective sub-cultur-
ing has worked well in the present studies and seems to be a method ap-

plicahuzto a large number of Cercospora Species.

 

Since a number of the Cercosporae with acicular hyaline conidia are
similar in the morphology of conidia and conidiophores, the thought that
isolates parasitizing different plants might be the same species occurred

repeatedly to investigators. Many attempts to establish host ranges

as a means of obtaining such information have been made. As early as
1892, Atkinson (1) observed that variation in host leaves seemed to exert
an influence on the form of the fungus, and while he did not draw the
conclusion that the same fungus might be infecting different hosts and
appearing there as morphologically different fungi, his observation
would support such a conslusion.

Most information concerning Cercospora host ranges has been accum-

 

ulated for 2. beticola. Schmidt (71.72.73) obtained infection and lesion
development with this fungus on Urtica dioica, Atropa_belladonna, 3:22:
aeolum £3122, Atriplex spp. and a number of Chenopodium species. He

also noted that passage of the fungus through E. dioica did not reduce

its pathogenicity to beet. In an extensive series of greenhouse inocula-
tion experiments, Vestal (87) infected 26 species from 12 widely separated
families with conidial suspensions of g. beticola. Infection was very
light on some hosts, and Vestal admitted the possibility that the fungus
did not parasitize actively growing tissue, but existed as a saprophyte

on the dead or dying leaves of some hosts. In addition to the weed hosts,
commercial plants infected were figium'ggaveolens, ngmea batatas, Lactuca
sativa, §212Lm3§ and Solanum tuberosum. There is also a single report of

a Cercospora isolated from clover being pathogenic on beet (67). More

 

recently, Prandsen (19) used extensive cross inoculations as a taxonomic

tool, and concluded that Q. anthelminthica and E. chengpodicola were syn-

 

onymous with g.‘beticola. He found that‘g. beticola could infect Urtica
urens, Plantago major and all Chenopodium.species tested. [He rejected
as inconclusive the work of Jehnson and Valleau (37) in which they pro-

posed that 45 common species of Cercospora be reduced to synonymy on the

 

basis of similar cultural morphology and host range.

In contrast to the apparent wide host range of 9. beticola, Jones (38)
and Baxter (2),in work on sweet clover and alfalfa reSpectively, report
negative results in attempting to infect leguminous hosts of different
genera with isolatesof E. davisii and _C_:_. medicaginis. Nagel (57) has
presented evidence, however, that a Ceregspora from Melilotus infected
Medicggo and Trifolium. Such divergence of results suggests that isola-
tion from a particular host does not guarantee that the fungus will be
physiologically identical in all respects, and that strains or isolates

of Cerc05pora may differ in their pathogenic ability (19). Other

 

Cercospora isolates from leguminous hosts have been reported to have host

 

ranges including several genera (47, 51). Kilpatrick and Jehnson (42)

have reported that the typical purple staining of legume seeds can be

induced artificially with a large number of Cercospo£§_isolates from

 

different hosts. A later report (39) indicates that similar coloration
can be obtained by the field inoculation of the pods of legumes with
various isolates of cercospora.

Probably the most striking report of host ranges crossing genus and
family lines is that of Emmons g£.§g,(l7) of the isOlation of a cerCOspora-
like fungus from human facial lesions. The fungus sporulated on corn
meal agar, and produced sporulating lesions on lettuce tomato and potato
plants in moist chambers. Reisolations were made of the same fungus from
the lettuce leaves. Chupp (9), however, states that this is not a
gerCOSpora. Chupp has accumulated a number of such host range reports,
and mentions them in his monograph (8); however, most of them are given

little consideration in his differentiation of species.

The taxonomy of the genus Cercospora has received attention from

 

several workers. Solheim (79, 80) was among the first to attempt the

classification of all species known at that time, by separating them into
sections, and carefully describing conidial and conidiophore characteris-
Otics. welles (89) felt that the number of species was excessive, and
proposed that differences in host caused variation in what was essentially
the same fungus. He found that various species on the same host were
identical in morphology, and distinguishablevonly on different hosts

(90, 91). Welles (89) considered only behavior on artificial media and
host range as valid taxonomic characters. Although there is much positive
evidence for the variability of Cercospora morphology in different envi-
ronments,there continues to be disagreement as to the specificity of a
given species for a certain host plant. New species are often described
on the basis of host plant (29,34,60). Statements similar to the followb
ing are found in the literature (63, 92): "As far as the writer has been
able to determine no Cercospora has ever been described on this host.

The pathogen is therefore described as a new species." and "No record
was found in the literature of any species of Cercospora on this host.

Furthermore, no Species of Cercospora reported on other members of the

 

Oleaceae could be matched with this one on Forestiera. It is therefore

 

considered worthy of designation as a new species." This approach can
only lead to a proliferation of described Species without adding to the
understanding of the genus as a whole, and without finding valid criteria
for separation of the species. Frandsen's (19) position considering
natural choice of hosts at the family level as a valuable taxonomic tool
is probably the most useful at present. Chupp's monograph (8) serves as
a basis for further work, but represents more of a catalogue of the des-

cribed species than an attempt to determine genetic relationships.

Since the goal of taxonomy is the discovery and elucidation of gen-
etic relationships, the nuclear condition of the fungus and the genetic
composition of the numerous nuclei in any fungal culture is of vital
importance to taxonomy. If all nuclei are identical, the problem is
simplified; however, a number of recent studies indicate that much of the
variability exhibited by fungi is best eXplained by the postulation of
heterokaryosis, the manifestation of genetically different nuclei within
a single thallus of the fungus. In such cases the use of single spore
techniques to obtain specific lines may be open to question, since a
single conidium may contain nuclei which are not identical genetically.

Hansen's work (26) on what he referred to as the dual phenomenon
gave the earliest indication that the variation observed in many of the
imperfect fungi could best be explained on the basis of genetically
variable nuclei in the conidia. Using Botrytis cinerea (28) and later a
number of other imperfects (26), he demonstrated that many conidia con-
tained a number of nuclei and that these nuclei could migrate from one
thallus to another through anastamoses. His work was based on the obser-
vation of three basic culture types: a dark sporulating culture (conidial
or C type), a white non-sporulating culture (mycelial or M type), and an
apparent mixture of these types (MC). Single spores of the C type pro-
duced only sporulating cultures, and transfers of the M type gave rise to
only mycelial cultures. Those cultures with characteristics of both the
above types (MC cultures) gave M, C, and MC types in varying proportions
when single spores were plated out. Similar cultural types and segrega-
tions of the same sort were observed by others (46, 93).

Jinks (35, 36) proposed that heterokaryosis was a widespread mecha-

nism for variation in naturally occurring fungi. Three major lines of

10

evidence for this wide occurrence were proposed. First, nearly all
imperfect fungi examined have multinucleate cells during active growth;
second, genetically unlike nuclei can occur in single hyphae, as demon-
strated by the dual phenomenon; and third, two biochemically deficient
homokaryons can form a heterokaryon which grows on media which supports
growth of neither homokaryon. Jinks and his co-workers (68) established
that heterokaryons occur in nature and that these heterokaryons may have
advantages over homokaryons. There would then be selection in the direc-
tion of maintaining this condition, that is, selection for multinucleate
spores, hyphal anastamoses and other variations tending to retain this
‘condition. Further work with heterokaryons of Penicillium demonstrated
that nuclear division rates vary with cytoplasm physiology and that this
physiology varies with the medium (68). Such differential rates would
give the different numbers of each type of nucleus in the heterokaryon.

A step toward greater understanding of variation in imperfect fungi
was taken by Pontecorvo (64, 65) in his examination of non-sexual recom-
bination or parasexuality. The recombination mechanism he proposes in-
volves fusion of vegetative haploid nuclei, multiplication of the result-
ing diploid nucleus and finally vegetative haploidisation. Recombination
occurs as a result of mitotic cross over or reassortment of chromosomes.
The first and last steps in this process have not been directly observed,
but are postulated from genetic analysis.

Among plant pathogenic fungi demonstrating heterokaryosis, several
species of the genus Helminthosporium have received the most intensive
study (7,24,33). The vegetative cells and conidia of H. sativum and
g. gramineum have been shown to be multinucleate. Numbers of nuclei from

the mycelium have been observed to enter the developing conidia (33), and

11

anastamoses have been demonstrated between hypae and germ tubes (24).
Further observations of germinating conidia have established the migration
of a number of nuclei from the conidium into the growing germ tube. These
would provide a mechanism for establishing and maintaining heterokaryosis
in the fungus.

Mycelial cells and conidia of other fungi have been observed to be
multinucleate (13, 61), and the migration of nuclei from cell to cell is
apparently possible (14). In some cases it has been proposed that dis-
sociations of such nuclei are the source of variation observed in imper-
fect fungi (49). Ryker (70) has stated that the cells of the conidia of

several common Cercospora Species are uninucleate and that cultural var-

 

iation in these fungi must be the result of mutation. However, migration

of nuclei in the mycelium and the fact that conidia of ggrcospora contain

 

a number of nuclei would provide a mechanism for the maintainance of
heterokaryosis once it is established.

The effects of heterokaryosis on the host range of fungi without a
true sexual stage is not clearly defined, although Buxton (3) has invest-
igated it. It is known that Helminthosporium Species with heterokaryotic
potential may have a wide capacity to parasitize (82), and that cultural
variants of a number of related fungi demonstrate differences in patho-
genicity (6, 53). Lerner and Coons (46) working with Q. beticola observed

that the behavior of the fungus in culture was similar to Helminthosporium

 

with regard to sectoring and morphological variation. Variants with
widely divergent conidial Sizes were isolated, some of which were non-
pathogenic or only weakly pathogenic on sugar beet. Where infection was
possible, passage through the sugar beet returned the aberrant fungus to

more normal cultural characteristics. Frandsen (20) also found variants

12

of Q, beticola, and noted that they were generally less pathogenic to
sugar beet than the normal isolates.

Variation such as noted above has seldom been taken into account in
taxonomic treatments of imperfect fungi, although it has been pointed out
that a Species description must take into account the whole range of var-
iation for a given fungus (5). In a study of the genus Glomerella, exam-
ination of life histories and forms of the fungus isolated from 36 dif-
feremgihosts resulted in referral of 34 of these to g, cingulata. Shear (75)
noted in this work that most morphological and physiological characters
were extremely variable, and that there was little restriction of a given
form to a particular host. While similar reduction of the number of

species in Cercospora would be preferable, basing species solely on par-

 

asitic ability would be as much in error as basing them solely on the
morphology of the fungus on its various hosts. An understanding of the
nuclear conditions and compatibilities of the various isolates might
simplify the problem of classification.

Since reports of wide and very restricted host ranges have been made
for the same Species of Cercospora in some instances, a sort of Variation,
not readily detected in all cases by morphological change, is a possibility.
In at least a few cases (72, 87), the plants reported as hosts for C.
beticola are either weeds commonly found in areas where sugar beets are
grown, or plants related to some such weed plant. Rotations of various
types have been shown to have no effect on the incidence of Q. beticola
infection (45); however, cross infections with local weeds allow the
possibility that Q. beticola is capable of transfer of pathogenicity from
an economically important crop to a local weed and back to the crop plant.

Such easily altered pathogenic capacity, if generally true of Cercosporae,

13

would have great im
portance both econo '
mically and in the
understanding

of genetic relationships within the genus

MATERIALS AND METHODS

Sources of host plants and fungal cultures.

The celery plants used for inoculation and other experiments were
all of the suSceptible variety Utah 103. Seed for other potential hosts
was obtained from commercial sources in the case of pepper (Capsicum annuum),
watermelon (Citrullus vulgaris), sugar beet (B333 vulgaris), Soybean (S213,
E§§)' and tobacco (Nicotiana tabacum). For weed hosts and other local
plants, collections of seed were made from the same plants that provided
the original Cercospora isolates. Collections from hollyhock (Althea
£2353), sweet clover (Melilotus officinalis), and mallow (Malva rotundifolia)
here made locally. The same host varieties or seed sources were used for
all succeeding experiments. Host plants were grown in the greenhouse at
approximately 22°C under the best cultural conditions available.

Isolates of the fungus were obtained from a number of locations.
The watermelon isolate (g. citrullina) was obtained from Florida, the
pepper isolate (g. capsici) from Missippi, and the tobacco isolate
(E. nicotianae) from Nerth Carolina. The several 9. apii isolates were
provided by Dr. H. H. Murakishi.from cultures of known history. Cultures
from other hosts were isolated from infected plant material collected
locally in most cases, and all were checked for pathogenicity on their
original hosts. Cultures obtained from other sources had often ceased
to sporulate if they had been maintained in culture for any length of
time. Such cultures would produce a very few conidia, but heavily spor-
ulating lines could not be separated by any method attempted. Only heavily

sporulating cultures were used in experiments. All cultures used were
from original single spore isolations, with selection of heavily sporu-

lating colonies in the first few transfers. Cultures continued sporulation

l4

15

at a high level even with fairly long periods between transfers, provid-
ing only sporulating material was transferred. Such isolates could be
revived and would sporulate heavily even after six to nine months in the

culture tube, when they were completely dried out, as Takimotos (84) work

 

suggests. Identifications of Cercospora species were made on the basis of
Chupp's (8) descriptions.

Culture media and inoculation procedures.

 

After a few unsuccessful attempts at maintaining cultures in a Spor-
ulating condition on PDA, Kilpatrick and Johnson's steamed carrot leaf
decoction agar (43) was used as the medium for the remainder of the work.
Fresh carrot leaves were frozen in 300 gram quantities per package, the
amount Specified for one liter of media, and the same source of leaves
used for all media prepared. Since the medium is sterilized by steaming
(43), contamination by spore forming bacteria became a serious problem.
Steaming the medium for one hour on each of three consecutive days reduced
the contamination to approximately 20%.of the slants prepared, however the
waste of time and materials led to a search for some other means of con-
trolling the contamination. Chloromycetin (Chloramphenicol: Parke-Davis
and Co.)fwas incorporated into the medium at lOOppm to control the un-
wanted bacterial growth. Comparative tests of sporulation, rate of growth,
and culture appearance demonstrated no effect of the Chloromycetin on the

development of any Cercospora isolate. This level of chloromycetin was

 

therefore maintained in all media used for the remainder of the work
described.

The chamber used for inoculation and incubation of whole plants was
a polyethylene film enclosed area of about 12x6x5 feet, with temperature

and humidity controls. Constant 100%»relative humidity was maintained

16

by an electrically controlled humidifier. Temperature was maintained by
_a one horsepower refrigeration unit with cooling coils at the top of the
chamber. Miole plants were inoculated and maintained at 26° C and 100%
relative humidity in this chamber. Following inoculation and incubation
for 72 hours plants were placed in greenhouses at approxomately 22° C
or 26° C until disease development was observed.

In experiments utilizing detached leaves or leaflets, inoculations
were made in petri dishes maintained at 26° C, with humidity provided by
10 ml of tap water in the bottom of the dish. Humidity was thus assumed
to be at or near 100% for the total incubation period of about two weeks.

Spores for inoculation were harvested from nine or ten day old
cultures grown in 250 ml flasks or in agar slants depending on the quan-
tity of inoculum required. Sterile distilled water was added to the cul-
tures and the culture vessel agitated for a few moments to dislodge the
conidia. Spore concentration was checked microscopically for initial
experiments until it was determined that concentration could be estimated
visually. Approximately 10,000 conidia per ml was used thereafter for all
inoculations. Spore suspensions were applied to the plants or excised
leaflets with an atomizer with pressure supplied by a compressed nitrogen
tank or by a hand bulb. Following application of the conidia, leaves were

allowed to dry in the normal lower humidity of the greenhouse until most
0f the water applied had evaporated, and then placed in the chamber at
1007. relative humidity. Spores were thus placed in direct contact with
the leaf, and the possibility of run-off reduced. Flats or pots of plants
inoculated with different isolates were always separated by at least two
feet in the chamber to reduce the chances of cross contamination to a

minimum. Flats and pots of identical uninoculated plants were similarly

17

separated and remained free of infection. The amount of contamination
was assumed to be neglegible under the conditions described.
Conidial measurements.

Measurements of conidia were made with a callibrated ocular micro-
meter disc from mounts in lactophenol on semipermanent slides made by
ringing the coverglass with clear nail polish. Comparative measurements
of width involving relatively short distances were made by visually
estimating the nearest tenth of a division on the ocular micrometer.

Other measurements were made to the nearest whole division.

EXPERIMENTS AND RESULTS
Initial experiments involved the inoculation of host plants grown
in flats or large pots. Difficulties were encountered in timing the
plants so that all different species in a Single flat were of equivalent
Size at the time of inoculation, but all plants had at least four or five
healthy leaves. Six or seven plants of each host were planted in rows
in the flats and four or five plants per pot where these were used.
Plants were inoculated, placed in the moist chamber for 72 hours, and
then removed to a greenhouse bench at approximately 22° C. After 14
days at this temperature, the plants were carefully inspected and leaves
showing necrotic spots or lesions were collected and placed on moist
filter paper in petri plates. An infection was assumed to have been
established in the leaf if Cercospora conidia were found on the lesions
‘within 48 hours (Table l). lesions observed on celery leaves were
:identical regardless of which Cercospora species served as inoculum.
After the first inoculation experiments had been completed, plants
<3f Urtica dioica were observed growing as weeds in flats of celery Seed-
lings inoculated with g. 222. As the disease developed on the celery
I31ants, circular lesions were noted on U. dioica leaves. A Cercospora
Similar in culture to 9. 3.21.; was isolated from these leaves. Celery
I’lants inoculated with this fungus developed typical symptoms of
Eagrcospora infection.
Since infection on host plants inoculated in the first experiment
“His often slight or on older leaves, the results presented are Open to
Some question. Chance infections were also possible, since the house
used for incubation was cooled by outside air. Conditions were not

OD timum for disease development during the total period of infection and

18

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20
incubation. Fer these reasons the detached leaf technique (32,76,88,95,
96) was used.

Detached leaves of the seven hosts used in the first experiment
were floated on distilled water, tap water, one per cent and five per
cent sucrose solutions. Results indicated that leaves of celery, sweet
clover and tobacco could survive for periods of three weeks or more on
tap water and for lesser periods on distilled water and sucrose solutions.
leaves of M rotundifolia, Althea m, and §_e__t_a_ vulgaris remained
healthy longer on sucrose solutions; however, young leaves of these
Species were green and apparently healthy for two weeks on tap water.
Conidia of E. 3231 germinated and formed small floating colonies on the
sucrose solutions but not on distilled or tap water. Tap water was
therefore used in all succeeding eXperiments.

The effectiveness of the atomizer inoculation method for detached-
leaves was determined by spraying two ml of a spore suspension of known
concentration on glass slides in petri plates and calculating the conidia
in the inoculum deposited per unit area (Table 2).

Since only one surface of the detached leaflets could be sprayed
with the spore inoculum, eXperiments were conducted to determine which
leaf surface of celery leaves was most susceptible to infection with the
inoculation method used. Wherever infection was established, the lower
surface was most susceptible. consequently, the lower surface of leaf-
lets was inoculated in all following experiments.

As a further check on the validity of excised leaf inoculations,
Comparisons were made between inoculations of intact celery plants of
Various susceptible lines (56), and excised leaflets from plants of the

Same lines. A representative sample of 20 leaflets was taken from each

21

line and inoculated with the same 9. 32;; culture used in the intact
plant test. Susceptible lines show approximately equal infection whether
inoculated as intact plants or excised leaflets (Table 3).
Effects of_passage through different hosts on the cross infectivity of
C. apii.

Inoculation of detached leaflets by this method should then provide

a more efficient means for further determinations of the infective ability

 

of Cercosppra isolates, while reducing the possibility of chance infection
and the bias introduced by growth of the fungi as saprophytes on dead or
dying leaves of host plants. With this method it has been possible to
inoculate, reisolate and reinoculate leaves from the same plant in series
to determine whether apparent pathogenicity would be increased by pas -
sage through a series of genetically identical host leaves.

Table 4 gives the results of experiments with such reisolation and
reinoculation procedures. Leaflets atomized with sterile distilled
water remained free of infection in every case, and provided a check on
the deterioration of uninoculated leaves.

Cultures were obtained from the inoculations reported in Table l,
and further inoculations of excised leaves were made with these. The
plants named in the left column of Table 5 are those from which the
culture used was isolated after their inoculation with E. apii. Cultures
originally isolated from celery are shown in Table 5 to be capable, after
a single passage through a different host, of infecting both that host
and celery.

The isolates of different cultural appearance were isolated from the

original celery plants inoculated with the isolate from E, rotundifolia

 

(C. malvarum) shown in Table 1. One culture was dark, nearly black, and

22

Table 2- Effectiveness of the atomizer inoculation technique on excised
leaflets. Results based on three replications.

 

 

 

Spore Spores Spores deposited. Spores
concentration deposited on on five average deposited on
of inoculum glass slides celery leaflets leaflets

No./ml No./cm3* No. %.of total
avg. range
16,000 73 68-82 2700 8.4
10,600 47 44-56 1700 8.0
.3,800 ll 7-12 400 5.3
3,400 15 11-20 540 8.0

Table 3- Comparison of per cent infection of susceptible lines of celery
with C.a a2 ii on intact plants and excised leaflets.

 

 

Per cent offexcised Per cent ofkintact
Celery line leaflets infected plants infected (56)
P 50 62
C 79 86
E 80 95
H 100 100

Table 4- Results of serial inoculations of excised celery leaflets with
Cercos ora isolates from five different host plants. Results
are rom two replications. Symbols as in Table l.

 

 

 

 

 

 

 

Original host and Excised celery leaflets

Cercospora species First Second Third
inoculation inoculation inoculation

Malva rotundifolia + + *+
C. —m§Ivarum

Althea rosea + +1 ++
g. althaeina 2

Nicotiana tabacum + 0 0
Q. nicotianae

Melilotus officinalis * ++ ++
Q. £1______avisii

Beta vulgaris + *+ tt
C. bet1cola

épim mgraveolens ++ ++ ++
g; apii

l-Only one lesion on all leaflets inoculated.
Z-The culture ceased to sporulate.

23

the other light gray. Both sporulated well on steamed carrot leaf decoc-
tion agar. A series of inoculations to both E. rotundifolia and celery
leaves was made with these isolates. The results are indicated diagram-
matichy'in Fig. l. The number of disease lesions produced is noted for
each inoculation in this case, since the comparative numbers produced by
the same inoculum may represent a significant difference between the
isolates. 'The figures noted are total numbers of lesions detected on

20 excised leaves or leaflets for each inoculation. A.similar series of
inoculations with an isolate from sweet clover produced results of the
same sort, as indicated in Pig. 2.

In both preceeding experiments a certain relative degree of path-
ogenicity on the two different hosts seems to be retained through sev-
eral reisolations and inoculations.

Several cultures were selected from the foregoing experiments and
inoculated to intact plants to eliminate the possibility that the infec-
tions observed on detached leaves could have been the result of gross
physiological changes in the leaves after detachment from the host plants.
Difficulty in timing the host plants in each flat so that all would be
as nearly comparable in size as possible allowed testing of only four
cultures in this way: the original Q, 32;; culture and three reisolations
from excised leaves (Table 6).

Comparative spore morphology of different species of Cercospora on a
single medium.

Since the experiments reported above demonstrated that cross infec-
tion was possible with Cercospora species from several different hosts,
an attempt was made to determine whether or not morphological features

used to distinguish the fungi on their respective hosts retain their

24

Table 5- Results of inoculations of excised leaves of Malva rotundifolia,
Althea rosea and Beta vulgaris with g. apii cultures reisolated
from various host plants. Symbols as in Table 1.

Source of isolate

 

 

 

 

 

 

 

 

 

and CetCospora species Host leaves inoculated
Malva Althea Beta Apium
rotundifolia rosea vulgaris ggaveolens
Malva rotundifolia + 0 0 ++
9. apii
Althea rosea I I 0 II
C. apil
CifEhllus vulgaris O O 0 ++
'9. apii '
Beta vulgaris 0 0 + ++
Co 51211 ‘
5213a graveolens 0 0 0 ++

 

g. apii

 

Table 6- Results of inoculation of intact plants with isolates obtained
from infections on excised leaflets. Symbols as in Table l.

 

 

 

Host plants
Culture -
number 1 épium Malva Althea.uelilotus Nicotiana Beta
graveolens rotundifolia rosea foicinali§_tabacum vulgaris
1. "' 0 0 0 0 "
2. ++ + + 0 0 0
3. ++ 0 0 + 0 +
4. ++ 0 0 + 0 0

 

l-Culture number 1 was an Althea isolate (C. althaeina) selected
from the second inoculation to celery shown in Table 4. Culture
number 2 was a.Malva isolate (Q. malvarum) from the second step
of the experiment reported in Fig. 1. Culture number 3 was a
Melilotus officinali§_isolate (Q. davisii) twice inoculated.to
excised celery leaflets. Culture number 4 was the original
9. apii isolate.

25

 

Celery 52 Celery 43 Celery 130
Malva O Malva 6 Malva 2
dark
Culture
Cercospora scelery
malvarum '
11;;%\
culture
Celery 5 Celery l7 Celery 13
:Malva 27 Malva 50

Fig. l.- Diagram showing the lesion counts from successive transfers of
two forms isolated from celery following inoculation with
E, malvarum obtained from Malva rotundifolia.

Cercospora

daViSH —9Celery 4:0316ry 69
Celery 60

SWQCt clover 9 Y
Sweet clover 2

 

Pig. 2.-Diagram showing lesion counts from successive transfers of
g. davisii obtained from Melilotus officinal_i_s_.

 

26

reported uniformity when the isolates are grown on identical media under
similar conditions of environment. Most characters used to distinguish
the separate species on their host plant are not readily observed when
the fungus is grown in pure culture. However, Chupp (8) believes, after
an almost lifelong study of the genus, that the conidia are the most
reliable single source of characteristics, and conidia can be obtained
from sporulating cultures. 0f the conidial characteristics used in tax-
onomic studies, only length, width, and number of septations are readily
measured objectively. While these are known to be variable with envi-
ronment(3l,91,94), they might be uniform enough to separate species in
identical environments.

Single spore cultures of a number of species, each isolated from a
different host, were grown under identical conditions of environment on
slants of carrot leaf decoction agar plus chloromycetin. Slides were
prepared of a fragment from the edge of the developing colony at five and
ten days. Tb avoid the effects of any disturbance of the culture from
the five day harvest, the conidia harvested at ten days were taken from
a replicate set of cultures. Measurements from ten day cultures were
much more uniform and only these are reported in the following figures.
Distribution of length of conidia for six cultures used in previous
experiments is plotted in Figs. 3 and 4. Although average lengths and
range of lengths varied for the individual cultures, when distributions
of length for 100 conidia are plotted, the modes of the curves obtained
fall within ten microns of each other for five of the six cultures.
Repetition of the measurements resulted in similar distributions, although
' averages varied as did range of lengths. The cultures could not be dis-

tinguished on the basis of conidial length.

 

 

27

 

 

 

24 -
Q. apii
20 ~
16 ~
No. of
conidia
12 -
8 r-
4 _
L J 1 l
50 100 150 200 250
length in microns
28r-
Beta vulgaris
isolate
24L £0 beti601a
20-
16-
No. of
conidia
er
8 _
4 -
J If I J”\ 1
50 100 150 200 250

. Fig. 3.- Length distributions of conidia.

16

12

28

24

16.

12

 

Althea rosea
solate

 

 

E, althaeina
l I A I
50 100 150 200

Length in microns

Melilotus officinalis
isolate

g. davisii

1
50 100 150 200

20

16
No. of
conidia

12

I

 

16
No. of
conidia

12

 

28

Malva rotundifolia
isolate

g. malvarum

I l l I 1A1

50 100 150 200 250 300
‘length in microns

Citrullus vulgaris
0 ate

9. citrullina

I I L I I /q\\/\LL_
50 100 150 200 250 300 350
Length in microns

Fig. 4.-Length distributions of conidia.

29

Observations on the character of the apices and bases of conidia
were also made in an attempt to distinguish the cultures. Chupp (8)
gives some weight to these particular characters. The camera lucida
drawings of Fig. 5, however, indicate that basal and apical characteris-
tics may be too similar to allow'separation of the isolates when they
are grown on the same medium. Furthermore, the conidial characteristics
seemed to vary more within each isolate than between isolates.

Measurements of conidial width revealed some interesting similarities
and differences in the cultures studied. While width demonstrated a
degree of constancy not seen for length, the cultures could not be dis-
tinguished on this basis.
Effects of passage through different hosts on spore morphology of C. apii;

Using the character of conidial width as a criterion, the variation
in the original culture of g. 322:1; as inoculated to and isolated from
four different hosts was investigated. These four isolates were grown
from single spores, conidia harvested at ten days, and.measurements of
width taken of 50 conidia chosen at random. The width distributions for
these four cultures and the parent 9°.EEii culture are shown in Fig. 6.
A comparison of the two graphs shows that the somewhat irregular distri-
bution for the original 9. 32311. culture coincides roughly with that of
the four reisolations, although no single reisolation covers the wide
range of widths seen in the original culture. Since width distributions
for these curves (Pig. 6) do not approach normality in all cases, com-
parison of mean widths is probably somewhat misleading. However, mean
values for three of the four isolates in the lower graph show significant

variation at the five per cent level from the mean of width distribution

for the parent 9. apii culture. The six possible comparisons between

 

525-1 A1 tttttt _ SS _ 3 M alva ot di
e Isolate
eina g. malvarull

MW “MM MGM

 

 

Men loituwac:c cinal Citrullus W
isolate
be ”9125.11 c mmmmmmm

 

 

31

 

 

 

 

101- 5
Original culture of
9.. 22.11
8 .—
6 e
No. of
conidia
4 I-
2 I-
I 1 I 1 L L L 14 I L I l i
ll 13 16 l 21 2 27 2 32 35 38 40 43 45 48 51 53 56
Width in microns
10
C. apii reisolated from;
16 _ ----- Beta vulgaris
; Malva rotundifolia
14.. ;‘ --—--—A1thea rosea
I ""“""“'
,’ I‘ —-——Citrullus vulgaris
12'-
10-
No. of
conidia
8 h-
6 .-
4 _
2 .-
/
/
/
V l

 

 

 

ll l3 16 19 21 24 27 29 32 35 38 40 43 45 48 51 53 56
Width in microns

Big. 6.-Wfidth distributions of conidia of 9. apii from various hosts.

32

means for the isolates in the lower graph all show significant differences
at the same level, and may be assumed to represent different populations.
The reisolation from Citru11us vulgaris (watermelon) and the parent cul-
ture have essentially the same mean, but differ significantly in vari-
ance about that mean, and may also represent truly different populations.

If what is demonstrated by these graphs is the actual separation
from the original culture of isolates with differing conidial morphology,
then the original culture would have been a mixture of these types, even
though it was derived from a single conidium. The width distribution
curve of the original culture might approximate the cumulative ranges in
width of the four isolates, if these are the only lines.separable from
the parent 9. £2253. culture, and if they were originally present in about
equal quantities. Since the summation curve represents the width distri-
bution of 200 conidia, it is compared with a curve for 200 conidia from
the original culture (Fig. 7). Major peaks in both curves coincide,
lending credence to the idea that the original single spore culture of
' E, apitias actually a mixture of morphological and pathogenic types of
the fungus. Camera lucida drawings of representative conidia from the
four reisolation cultures and the original culture further demonstrate
the differences observed in spore morphology among these isolations from
what had originally been a single spore culture of 9, apii (Figs. 8 and19).
Germination and_penetration experiments.

Thomas (85),working with E. carotae, has indicated that only about
one per cent of the conidia applied to a leaf actually penetrate the host

tissue. From the results obtained with the Cercospora isolates in this

 

work, the penetration percentage seems no greater. Spore germination and

penetration of host leaves was observed by utilizing a collodion film

33

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a” 00 'Illl

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

(I
L
fl
Original culture
of 9: agii
C. apii isolated C. gii isolated
from Althea rosea from Beta vulgaris

Pig. 8.-Camera lucida drawings of representative conidia. 500x

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

36

impression technique (50, 62). leaves were inoculated with conidial
suspensions and collodion impressions taken at intervals from four to
48 hours following inoculation. Penetration through stomates was ob-
served very rarely with any of the host-fungus combinations and no con-
clusions are possible. Germination was uniformly near 100 per cent re-
gardless of the combination of host leaf and Cercospora culture.
Parallel germination experiments were conducted with the expressed and

filtered sap of Malva rotundifolia, Althea rosea, Melilotg§_officinalis,

 

Beta vulgaris and Apium graveolens. No inhibition of germ tube devel-

 

opment in any isolate could be demonstrated with the sap of any of these
host plants, although a low air supply to the spore could prevent ger-
mination. This was demonstrated when a cover glass was placed over the
drop of sap in which conidia were germinating. The overall per cent of
germination was cut to 50 or 60 per cent, with no germination near the
center of the cover glass, and nearly 100 per cent germination near the
edge. Without cover glasses, numerous repetitions of the experiment
gave germination of 95 per cent plus in every case.

Do Cercospora species show heterokaryotic potential?

Since spore selection on the basis of germinability or penetration
of the leaves could not be demonstrated, the possibility of selection at
the nuclear level was considered. The presence of genetically different
nuclei in the mycelium of a single isolate would provide a mechanism for
the differences in morphology and pathogenicity observed. Such mechanisms
would require that several nuclei be present in the mycelial cells, con-
idia and conidiophores of the fungus, and that some means be present for

the transfer of these nuclei between the mycelium of different isolates.

Wbrk with Helminthospgrium (7,24,33,74) has shown that conidia of this

37

fungus contain several nuclei per cell, and in a few instances anasta-
-moses between germ tubes have been observed. These have been interpreted
as a means for the transfer of whole nuclei from one thallus to another.
If such nuclei are retained and can reproduce in the new thallus, the
chances of genetic heterogeneity reflected in the variation of morph-
ological and physiological characters would be greatly increased.

Some preliminary experiments were conducted in an attempt to deter-
mine whether or not a.mechanism for heterokaryosis and/or nuclear trans-
fer might be present in the Cercosporae. Fuelgen stains, as normally
used to detect nucleic acids, were negative. Hematoxylin stains (25)
were then attempted with greater success. Structures having the staining
characteristics of nuclei were observed within the conidia, conidiophores
and germ tubes. Figs. 10, 11, and 12 show the structures observed. Germ
tube anastamoses could not be demonstrated.

In the course of the staining procedures unusual germination patterns
were observed in some conidia. Solheim (79) reported that conidia
occasionally produced conidiophores directly and might hear secondary
conidia. Fig. 13 pictures a conidium giving rise to conidiophores and
producing both secondary and tertiary conidia. It is conceivable that
such a process could result in segregation of nuclei if the original
conidium were heterokaryotic.

Since nuclear transfer could not be demonstrated microscopically,
an eXperiment was designed to show whether a character such as patho-
genicity might possibly be exchanged between two different isolates.

The Cercospora isolates, one originally obtained as a single spore
isolate from celery and another obtained in the same way from a naturally

infected sweet clover plant, were selected in culture over several

38

 

 

50 microns

Fig. 10- Multinucleate conidium.

39

50‘microns

Fig. 11- Multinucleate conidium.

40

 

 

50 microns

Fig. 12- Multinucleate conidiophore.

41

, A» Primary conidium
B- Secondary conidium

C- Tertiary conidium
x. Conidiophores

Qua-u...)

A
—

 

Fig. 13- Conidium germinating by the formation of conidiophores.

42

generations-for uniformity of gross cultural characteristics and spor-
ulation. Both isolates were pathogenic on their original hosts at the
beginning of the experiment. Conidia were harvested from both isolates
in sterile distilled water and concentrations adjusted to the same level.
Equal amounts of suspensions were mixed under sterile conditions and
placed on agar medium in four 250 ml flasks. After 14 days of growth,
conidia were harvested from these cultures, mixed and placed in four more
250 ml culture flasks. Ten days later 25 single spores were picked at
random from the four flasks and placed on slants of the same medium. As
the cultures developed, six which demonstrated the greatest diversity of
observable cultural characteristics were selected. These cultures were
increased and inoculated to excised celery and sweet clover leaflets.

The inoculation was repeated twice, with ten leaflets in each of two
petri plates for each inoculation.(Table 7). The results presented in
Table 7 are in general agreement with the hypothesis that nuclear ex-
change has taken place, and that this exchange has had an effect on the
pathogenic capabilities of the isolates tested. More extensive work is

needed to establish the true picture.

43

Table 7- Results of inoculations of Apium graveolens and Melilotus
officinalis with six single spore isolates from cultures of
mixed 2. apii and E, davisii. Symbols as in Table l.

 

 

 

 

 

CulturesI Excised Leaflets inoculated
Apium graveolens Melilotus officinalis
Rep. 1 Rep. 2 Rep. 1 Rep. 2
A ++ ++ + +
B + + + +
C +4. +4- 0 0
D ++ ++ 0 0
F + O O 0
G ++ ++ o 0
Original from
521““ ++ ++ 0 0
Original from
Me 15.10.1315 0 0 +4 4+

4*

l-Letters A through G designate the single spore isolates from
mixed cultures

DISCUSSION AND CONCLUSIONS

The fungal genus Cercospora affords the research worker the oppor-
tunity to observe and examine virtually thousands of isolates described
as species, each capable of establishing a parasitic relationship with
one or more hosts. While demonstrating morphological differences on
their respective hosts and presumably physiological differences controlling
their parasitic capabilities, their similarity has led to the suggestion
by Jehnson and Valleau (37) that 45 common species should be reduced to
synonymy, and by Horsfall (31) that another six be treated similarly.
conflicting results and observations regarding morphology, host range
and pure culture characteristics indicates a need for some consistent
explanation.

The results of inoculation eXperiments on both intact plants and

excised leaves indicate that a Cercospgra isolated from celery is cap-

 

able of establishing a parasitic relationship with a number of genetically
different hosts, and that isolates from some of these hosts have a similar
capacity. Repeated inoculations to a host other than the original host

of the fungus increase apparent pathogenicity on the new host, and it

has been demonstrated that a particular isolate may be separated into

two lines, each having a different but relatively constant capacity to
parasitize at least two genetically different hosts.

The above results might be interpreted in several ways. lhe
Cercospora isolates utilized may be composed of several genetic types
separable on the basis_of differences in pathogenicity, which in nature
would likely retain the capacity to parasitize a number of widely di-
vergent host plants. Alternatively the original isolates may each be

a single genetic entity with a wider host range than had been supposed.

44

45

Considering uniformity of conidial size as an index of genetic uni-

formity, measurements indicate that a number of Cercospora Species show

 

basic similarities in distribution of conidial length when sporulated in
pure culture on the same medium. Measurements of a more constant char-
acter, conidial width, support the first hypothesis presented above,
that the original cultures were mixtures of genetic types. These are
separable to some extent on the basis of infectivity on the different
hosts tested.

Since the above results could be interpreted as a manifestation of
heterokaryosis, mechanisms for the initiation and maintainance of this
condition are important in an explanation of the phenomena observed.
Multinucleate cells in conidiophores, conidia and germ tubes were observed,
but no mechanism for transfer of nuclei could be demonstrated microscopic-
ally. Mixing of cultures and reisolations of single conidia to determine
whether pathogenic capacity'might be transferred gave some positive
evidence. Considering the limited number of cultures used, the phenom-
enon, if present, may be very common. If single conidial isolates from-
a culture obtained by mixing two pathogenically different isolates
demonstrate characteristics of both original cultures, some mechanism
for the transfer of genetic characters is present. Whether this mech-
anism involves the hyphal or germ tube anastamoses ordinarily presumed
to be present for the establishment of heterokaryoSis cannot be stated
without cytological evidence. The possibility of segregation from one
of the isolates giving rise to the several types observed cannot be
eliminated on the basis of the evidence presented here. Nbr can mutation

of one or the other culture be ignored as a possibility.

46

The results are consistent with the hypothesis that collections of

Cercospo£a_described as species may actually be heterokaryotic mixtures

 

of morphological and physiological types, capable of segregating and
recombining, and as a result demonstrating a great variation in their
ability to establish parasitic relationships with a number of host genera
and families. The accumulation of further evidence of the same sort

with a larger number of CErcospora isolates could possibly lead to a new

 

concept of the species in this complex and fascinating group of fungi.
From the practical point of view, the work has significance in that
he causal agent of an important disease has been shown to have a wider
host range than had been previously SUpposed. Control of the disease,
whether involving the most efficient use of chemical materials or the
proper approach to breeding for resistance, may profitably be evaluated

in this light.

7.

9.

10.

11.

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