Biology of Dothidella ulei; I. in vitro production of toxin, II. Differential clones of Hevea for identifying races of the fungus

BIOLOGY OF DOTHIDELLA ULEI
I. IN VITRO PRODUCTION OF TOXIN
II. DIFFERENTIAL CLONES OF HEVEA FOR
IDENTIFYING RACES OF THE FUNGUS

By

JOHN WESLEY MILLER

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
April, 1965

ACKN0W1EDGMENTS

I am deeply indebted to the Firestone Plantations Co., B. H.
Larabee, President, for providing the fellowship which made this re-
search possible. Acknowledgment is made to Dr. K. G. Mclndoe, Fire-
stone Plantations Co., for his cooperation and advice during this
study and for his helpful review of the manuscript.

I wish to express my sincere gratitude to Dr. D. A. Roberts,
Chairman of the Supervisory Committee, for encouragement and guidance
throughout the course of this research and in preparation of the
manuscript. Acknowledgment is also made to Dr. P„ Decker, Dr. V. G.
Perry, and Dr. D. S. Anthony for their critical review of the manu-
script. I am indebted to Dr. R. E. Stall who suggested an injection
technique for bioassay of the toxin produced by Dothidella ulei.

ii

FOREWORD

The research reported in this dissertation concerned two dis-
tinct facets of the biology of Dothidella ulei. Therefore, each
phase was written in the form of a separate manuscript in the style
used for publication in "Phytopathology," the journal of the Ameri-
can Phytopatho logical Society. The "Style Manual for Biological
Journals" was followed for use of abbreviations, numerals, etc.

iii

TABLE OF CONTENTS

ACKNOWLEDGMENTS

FOREWORD •

LIST OF TABLES ......

PART I. IN VITRO PRODUCTION OF TOXIN

Introduction ... ............

Materials and Methods

Results

Discussion .. ...........

PART II. DIFFERENTIAL CLONES OF HEVEA FOR
IDENTIFYING RACES OF THE FUNGUS

Introduction

Materials and Methods .. ......

Results ........................

Discussion ........

LITERATURE CITED ........

BIOGRAPHICAL SKETCH . .............

Page

ii

iii

v

1

2

5

6

9

10

11

15

18

20

iv

LIST OF TABLES

Table Page

1. Number of times that Hevea clones gave indicated re-

actions to 3 races of Dothidella ulei 12

2. Differential Hevea clones and their reactions3 to 4

races of Dothidella ulei 14

v

PART I

IN VITRO PRODUCTION OF TOXIN
Introduction

DeBary (1) was among the first to show the pathological effect
of a fungal culture filtrate against plant tissue. Many toxic sub-
stances produced by various micro-organisms have since been dis-
covered; these include the host-specific (16) pathotoxins (19) Vic-
toria (14) and the Periconia toxin (17), the vivotoxins (5, 19) Fusaric
acid (7) and the wildfire toxin (21), and the phytotoxins (19) Alter-
naric acid (15), Colletotin (8), Victoxinine (18), and Lycomarasmin (20).
The literature on this subject has been reviewed (4, 5, 16, 19).

Penetration of para rubbertree leaves, Hevea spp., by Dothi-
della ulei P. Henn. , causal agent of South American Leaf Blight, occurs
within 24 hr after inoculation (3). A depressed area in the leaf forms
approximately 4 days later, and is the first symptom of disease (3).

The thin, chloranemic spots enlarge to diameters of 5-8 mm; sporulation
of the fungus begins 8-14 days after inoculation. The lesions are later
delimited by a thin, black margin and the centers usually drop out,
producing a "shot-hole" effect (9). This suggests that a toxin is pro-
duced by the fungus, since the histogenic demarcation that causes the
"shot-hole" symptom is said to be a specific defense reaction against
toxins produced by the parasite or by the host as a result of infec-
tion (6). The purpose of the research reported here was to determine

1

2

whether D. ulei produces £ toxin capable of inciting symptoms in never
leaves similar to those diagnostic of the disease caused by the fungus
itself.

Materials and Methods

Dothidella ulei was grown in culture on agar slants and in a
liquid medium. The ingredients, per liter, in the agar medium were: 39
g of "Bifco" potato-dextrose agar, 5 g of peptone, 5 g of phytone (a
papaic digest of Soya bean meal), 5 g of malt extract, C.5 g of yeast ex-
tract and enough deionized water to make 1,0CQ ml (11). Samples of ap-
proximately 10 ml of the culture medium were placed in test tubes, auto-
claved for 30 min at 15 psi, and were allowed to harden as agar slants.
Isolations of the fungus were made from infected leaves by scraping off
conidia and inserting them into the agar medium. Blazquez (2) observed
that germ tubes from conidia germinating on the surface of an agar med-
ium grew aerially; growth of the fungus ceased if the aerial germ tube
touched the agar before branches near its base had grown enough to estab-
lish a colony. Langdon (10) found that germ tubes from conidia germina-
ted below the surface of agar grew directly into the surrounding medium
with no apparent difficulty and colonies were established with a much
higher degree of success than with surface-germinated spores. Conse-
quently, the method of sub-surface germination was used for isolating
the fungus from leaves.

Colonies that developed in pure cultures were allowed to grow for
2-3 months, and the elevated, stroma tic bodies were picked off the agar.
These were ground for 1 min in the micro -attachment of the Servail Omni-

3

mixer while being kept cold in an ice water bath. The resulting sus-
pension was centrifuged at about 3500 g, and was filtered by suction
through a Seitz filter pad with a pore diameter of 0.1 p. The result-
ing fungal-free filtrate was tested later for toxicity to Hevea leaves.

To determine whether D. ulei produces an extracellular toxin,
the fungus was grown in a liquid culture medium similar to the agar med-
ium, except that the commercial potato-dextrose agar was replaced by
20 g of dextrose and by the broth from 200 g of potatoes cooked for 40
min in 500 ml water. Samples of 250 and 500 ml were placed in 500- and
1,000-ml flasks, respectively, and were autoclaved for 30 min at 15 psi.
In some experiments, a bacterial contaminant that could withstand auto-
claving grew profusely in the rich medium. Sterilization then was ac-
complished by autoclaving for 30 min at 15 psi, 3-5 times at daily inter-
vals or by autoclaving once for 2 hr at 15 psi. These treatments caused
the medium to darken, but had no apparent effects on subsequent growth
of the fungus.

The liquid culture' medium in each flask was infested with sporu-
lating colonies of D. ulei and was kept at 20° C for 7 weeks to 6 months
without shaking. Following the growth period, the cultures were filtered
aseptically by suction through a Seitz filter pad with pore diameter of
0.1 p. This provided a fungal-free culture filtrate; uninfested medium
of the same age was filtered in the same manner and served as a control.
Concentration, when desired, was accomplished by either of 2 methods.

The filtrates were placed in dialysis tubing and hung either in an incu-
bator equipped with a fan and with temperature controlled at approxi-

4

mately 5° C or in front of a fan in a room with the temperature con-
trolled at 20° C. The latter method was the more efficient, as concen-
trations of more than 20-fold were achieved in 3-4 days and fungal con-
tamination on the outside of the dialysis tubing was avoided. Contami-
nation occasionally occurred on the outside of the bags in the incuba-
tor. This was controlled by wiping the bags with a cloth saturated with
107o commercial Clorox.

Several methods of bioassay for toxic activity in mycelial ex-
tracts and culture filtrates were tried. No symptoms developed in leaves
floated on water and on which drops of test solutions were placed, even
though some leaflet tissues under the drops were wounded with a pin to
provide entrance into the leaf. Test solutions also failed to induce
symptoms after vacuum infiltrations of leaf disks.

Symptoms of toxic activity were observed, however, when the cut
ends of petioles of detached leaves were submerged in test solutions. Tip
and marginal curl of the leaflets, followed by drying and blackening,
developed in leaves placed in fungal culture filtrates 'or mycelial ex-
tracts. Symptoms frequently developed in control leaves placed in
sterile culture medium, although they were usually milder and were al-
ways slower in appearing. It was later observed that mechanical injury
also caused leaflet curl, drying, and blackening.

Distinct symptoms developed in leaves of greenhouse-grown Hevea
seedlings after mycelial extracts or culture filtrates had been injec-
ted into their petioles or stems. Seedlings of clones PB 86, AV 1126,

AV 1581, and Tjirandji 1, all susceptible to all known races of the

5

fungus, were used. Injections with a syringe and needle (size 26 or 27)
were made either into the stem just below the developing leaf flush or
into the petioles of leaves in Stage II or early Stage III (3). Stage
II in the growth of Hevea leaves occurs when the leaflets are folded
dorsally and are 1-2 cm long. Stage III occurs when the leaflets are un-
folding and are 2-10 cm long. In early Stage III the leaflets are 2-4
cm long. No symptoms were observed to develop when treatments were begun
on leaves past this stage of development. Injections were made once or
twice daily for 7-10 days. A total of 0.5-0.75 ml of solution was injec-
ted into each treated petiole or stem. Results reported below were ob-
tained from experiments in which the injection method of testing had been
used.

Results

Chloranemic spots with thin, transparent centers developed in sus-
ceptible leaflets 2-9 days after the first injection of mycelial ex-
tract or culture filtrate. The tissue surrounding the lesions soon be-
came savoyed, and affected leaflets became distorted because their normal
expansion was retarded near the lesions. These symptoms induced by fun-
gal-free mycelial extracts or by sterile culture filtrates were striking-
ly similar to those induced by the fungus itself (3, 9). Symptoms de-
veloped in 2-6 days after the first injection when the stem below the
developing leaf flush or when leaflet petioles were injected with the
fungal-free mycelial extract, whereas symptoms appeared in 4-9 days af-
ter the first injection with sterile culture filtrate. The variation in
time of symptom appearance was apparently related to the source of toxic

6

material, and may be due to differences in concentration in the differ-
ent materials.

The culture medium or deionized water occasionally caused small
flecks to form in leaflets when their petioles had been treated. There
was, however, neither chloranemia nor any adverse effect on the expan-
sion of leaflets. Leaf growth ceased when water or the culture medium
or test materials were injected into petioles before leaves had reached
Stage II of their development. Growth cessation was occasionally ob-
served in Stage II leaves, but never in Stage III leaves. Affected leaves
became anthocyanescent, distorted, and desiccated before they died some
7-10 days after the first appearance of symptoms.

In 3 experiments, the culture filtrate (concentrated 10X) was
either boiled for 1 hr or dialyzed against deionized water for 96 hr be-
fore injection of susceptible Hevea seedlings. In 2 experiments with
boiled material and in one with dialyzed material, neither chloranemia
nor leaf distortion occurred in leaflets that developed on treated peti-
oles. Thus, the symp tom- inducing principle that occurs in fungal-free cul
ture filtrates appears to be heat labile and dialyzable.

Discussion

Wheeler and Luke (19) proposed the term "pathotoxin" for those
toxins that play an important causal role in plant diseases. Criteria
for determining whether or not a toxin belongs in this category are:

"(a) the toxin, applied at concentrations which could be reasonably ex-
pected in or around the diseased plant, produces in a susceptible host
all the symptoms characteristic of the disease; (b) the pathogen and

7

the toxin exhibit similar suscept specificity; (c) the ability of the
pathogen to produce the toxin varies directly with its ability to cause
disease; (d) a single toxin is involved."

In the work reported here, symptoms produced by less than 1 ml
of the crude fungal-free filtrate or the crude mycelial extract appeared
to be the same as those induced by D. ulei itself. Fungal-free materials
thus far tested, however, have not caused the symptom of "shot-hole."
Thin, ch lor anemic spots and leaflet distortion occurred, regardless of
whether plants were treated with the toxic materials or inoculated with
conidia of the fungus. Since these results satisfy the modified (19,
p. 233) first, and most important, criterion of a pathotoxin, the host-
specific toxin of Pringle and Scheffer (16), the toxin produced by D.
ulei is temporarily classified as such. It definitely is not a phyto-
toxin, because it produces almost all of the symptoms incited by the
fungus, whereas a phytotoxin produces a few or none of the symptoms in-
cited by the pathogen (19). Admittedly, future research may show that
the toxin from D. ulei does not fit all of the criteria of a pathotoxin
and it might have to be classed then as a vivotoxin (5, 19).

Best evidence for iri vitro toxin production by D„ ulei is the
fact that the fungal-free mycelial extracts or culture filtrates induce
symptoms indistinguishable from those incited by the fungus itself;
this evidence is strongly supported, however, by the fact that boiled
or dialyzed culture filtrates had no toxic effects on Hevea leaves.
Moreover, only Stage II and early Stage III leaves were susceptible to
the toxin, and leaflets in these stages of development were also most
susceptible to attack by the fungus.

8

The high potency of mycelial extracts from colonies grown on a
solid medium indicates that the toxin diffuses but slowly into agar.

The toxin evidently is highly soluble in a liquid culture medium, how-
ever, because extracts prepared from the fungus grown in liquid cul-
ture showed no toxic activity by the injection method of bioassay.

If the toxin from D. ulei does indeed prove to be a pathotoxin,
a valuable program of testing Hevea clones for resistance could be de-
veloped; it might be possible to make such tests with the toxic prin-
ciple in the absence of the fungus itself. Thus, Hevea clones would
not have to be sent to infested areas (Central and South America) for
resistance testing and then returned to areas free of Dothidella (Africa
and the Far East) for further development. The time needed to develop
resistant, high-yielding clones for commercial use would thereby be re-
duced. These benefits would represent great economic savings for the
commercial rubber industry. Most important, however, would be reduction
of the danger of the fungus being carried as a contaminant to the unin-
fested regions, where establishment of the fungus could result in severe
damage to the plantation rubber industry.

It should be pointed out, however, that many problems involved in
using the toxic material in resistance testing would have to be solved.
Among these are how to obtain sufficient material for injection of thou-
sands of seedlings, how to purify the toxin and under what conditions
the toxic material retains its potency. Obviously, it would take years
of research to solve these and, possibly, other problems inherent in

such a project

PART II

DIFFERENTIAL CLONES OF HEVEA FOR IDENTIFYING
RACES OF THE FUNGUS

Introduction

Pathological specialization in Dothidella ulei has been suspec-
ted since 1946. Langford (13) observed near Belterra, Brazil, that
Ford clones (F 409 and F 1619) of Hevea brasiliensis , that were previous-
ly rated as resistant to the disease caused by D. ulei, were attacked
by a local strain of the fungus. In 1960, Langford (13) noted that
progeny of the resistant clone F 4542 (H. benthamiana) were infected by
a strain of the fungus in Costa Rica. This observation was confirmed
experimentally by Langdon (12), who was the first to obtain direct evi-
dence that pathological races of D. ulei exist. Working with isolates
from Guatemala and Costa Rica and under greenhouse conditions, he made
pathogenicity studies on clones with and without F 4542 parentage. The
isolate from Costa Rica attacked and sporulated heavily on all F 4542
clones tested and was designated Race 2. The Guatemalan isolate at-
tacked no F 4542 clones, except IAN 717, and was designated Race 1.

Since it had been confirmed that at least 2 races of D. ulei ex-
ist, the need for establishing a set of differential host clones for
race identification became apparent (12). Also needed is further test-
ing of established races and testing of new isolates of the fungus
against different clones that represent various sources of resistance.

9

10

The objectives of the research reported here were to determine which
clones could be used to differentiate among known existing races and to
identify additional races.

Materials and Methods

Race 1 of Dothidella ulei from central Guatemala had been estab-
lished in the greenhouse by Langdon (12). Race 2 was not used. Two new
isolates were sent by Dr. K. G„ Mclndoe from Navajoa, Guatemala, a third
was obtained from Mr. H. Echeverri, Goodyear Speedway Estates, Costa
Rica, and a fourth was received from the Instituto Agronomico do Norte,
Belem, Para, Brazil, All were established and maintained on seedlings
of the family Tjir. 1 X Tjir, 16 growing in the greenhouse. They were
also kept in culture on agar slants containing, per liter, 39 g of
"Difco" potato-dextrose agar, 5 g of peptone, 5 g of phytone (a papaic
digest of soya bean meal), 5 g of malt extract, and 0.5 g of yeast ex-
tract (11). In order to maintain a virulent culture of the races of D.
ulei (10), isolations from infected leaves were made about every 6
months, because pathogenicity decreased after 6 months and cultures be-
came avirulent at the end of approximately 12 months.

Inoculations of Hevea plants were made by picking conidia from
sporulating lesions with a wet camelhair brush and placing the conidia
onto the abaxial surface of leaflets in Stage III (3) of their develop-
ment. The leaf flush was then covered with a moistened plastic bag,
which was left in place 16-24 hr. On seedlings used to maintain inocu-
lum, the entire leaf flush was inoculated with a single race or isolate.
No mixing of the races occurred and on only 2 occasions did seedlings

11

that had not been inoculated by hand become infected. On test clones
a separate pair of leaves was inoculated with each race or isolate to
be tested, each inoculated leaf was labelled, and the entire leaf flush
was bagged overnight. This permitted testing of the different isolates
under very similar conditions.

Disease severity ratings on test clones were made 2-4 weeks af-
ter inoculation, using the Firestone Plantations Co. system, modified
for greenhouse use (12). Ratings under this system were divided into 4
classes: resistant (R), flecks or very small non-sporulating lesions;

highly resistant (HR), small non-sporulating lesions; moderately resis-
tant (MR), larger lesions with light or no sporulation; susceptible (S),
large lesions with heavy sporulation. In this research conducted under
conditions where air temperatures were held at 70° F or higher and where
humidity was maintained at high levels by an intermittent mist system,
light sporulation was considered necessary for a rating of MR,

Results

Further testing with Race 1 and with new isolates of the fungus
was carried out, with the results summarized in Table 1. The 2 isolates
from Navajoa, Guatemala, and the isolate from Costa Rica proved similar
and were designated as Race 3, All data on Race 2 were obtained from
research done by Langdon (10). Clone IAN 717, a clone having F 4542
in its parentage, was rated as susceptible to Race 2 and Race 3, but
was highly resistant or moderately resistant to Race 1, All other
clones with F 4542 parentage, however, were rated as highly resistant to
Race 3, but were moderately resistant (with sporulation) or suscepti-

12

Table 1. Number of times that Hevea clones gave indicated reac

tions to

3 races of Dothidella

ulei

Race 1

Race 3

Race 4

Clone

Parentagea

R HR MR S

R HR MR S

RR HR MR S

IAN 710

PB 86 X F 409

29

30

1 1

IAN 713

PB 86 X F 409

26

26

1

IAN 717

PB 86 X F 4542

11

2 10

4

IAN 873

PB 86 X FA 1717

15 4 1

11 6 1

5 1

FX 25

F 351 X A V 49

20

17

3

FX 232

F 351 X PB 186

4

3

FX 637

F 4542 X Tjir o 1

1 4

4

1

FX 664

F 4542 X Tjir. 1

3 12

14

2

FX 2831

F 4542 X Tjir. 1

7

6

2

FX 3810

F 4542 X A V 363

5 6

2 7

FX 3925

F 4542 X AV 363

22

25

3

MDF 72

Seedling Selection

1

1

MDF 138

Seedling Selection

4

2

MDF 158

Seedling Selection

8

9

1

MDF 180

Seedling Selection

22

23

1 4

MDF 232

Seedling Selection

10

10

1

MDF 350

Seedling Selection

11

6

MDF 363

Seedling Selection

3

4

1

MDX 13

AV 308 X MDF

2

1 1

P 122

Forest Selection

4

9

1

aMDF (Madre de Dios Firestone) clones were selected in Guatemala
from seedlings arising from seedlings obtained from the Madre de Dios
region of Peru. P 122 was obtained as budwood from a native tree of the
Madre de Dios region of Peru0

13

ble to Race 2; this clearly distinguished Race 2 from Race 3.

The isolate from Brazil was designated as Race 4. Ratings of sus-
ceptible in IAN 710 and IAN 713, clones of F 40S parentage, indicated
that this was the same or a similar isolate to the one from Brazil pre-
viously noted by Langford (13). These clones were highly resistant to
Races 1, 2, and 3, and can, therefore, be used to distinguish between
any of these races and Race 4. Also, FX 25, MDF 180, MDX 13, and P 122
were rated as moderately resistant (with sporulation) to Race 4. These
clones are from a variety of parentages (Table 1) and demonstrate the
ability of Race 4 to overcome several sources of resistance. Clones de-
rived from F 4542, however, generally shewed a high degree of resistance
to this race.

Based on reactions of the different clones to various races of
the fungus already identified, a set of differentials was established
(Table 2). IAN 717, with F 4542 as one of its parents, was rated as sus-
ceptible to Races 2 and 3. This differentiates these races from Race 1,
to which IAN 717 was rated as highly resistant to moderately resistant.

FX 3925, another F 4542 selection, differentiates Race 3 from Race 2,
because this clone is resistant to the former race and susceptible to
Race 2. IAN 710 and IAN 713, both F 409 progeny, and MDF 180, a Madre
de Dios selection of Firestone, separates Race 4 from all other races,
since this race was able to sporulate on these clones, whereas Races 1
and 3 could not. The clones derived from F 409 are also highly resis-
tant to Race 2 (10), but this race has not been tested on MDF 180.

IAN 873, a clone selected from a cross of PB 86 X FA 1717, which

14

Table 2. Differential Hevea clones and their reactions3, to
4 races of Dothidella ulei

Clone Race 1 Race 2 Race 3 Race 4

IAN 717

HR

S

S

HR

FX 3925

HR

S

HR

HR

IAN 710 or 713

HR

HR

HR

S

MDF 180

HR

b

HR

MR

P 122

R

R

R

MR

% resistant; HR, highly resistant; MR, moderately resistant;
S, susceptible.

^Race 2 not tested against MDF 180.

15

had previously rated as highly resistant to Races 1, 2, and 3, sudden-
ly began to exhibit sporulation and to show ratings of moderately re-
sistant or susceptible to Races 1, 3, and 4. This change occurred in
November, 1964, and was observed on several individuals of this clone
in the greenhouse. The change was not due to changes in the races be-
cause all other clones tested since then showed their normal reactions
to Races 1 and 3. Apparently IAN 873 has resistance that is more sensi-
tive than that of related clones to subtle changes in the environment.

In any event, its reaction to Races 1 and 3 is unpredictable. It was not
exposed to Race 2 after this variability in its response to other races
was noted. Also, it had not been inoculated with Race 4 prior to the
time of its apparent change.

Discussion

Multiple race testing of Dothidella ulei was continued and ex-
panded (Table 1), with the identification of 2 additional races of the
fungus. A set of 5 differential clones for race identification was also
established (Table 2). Four races of the fungus have now been identi-
fied. Some of these known races are able to break down sources of resis-
tance derived from F 4542, F 409, and/or MDF clones, when these sources
are used singly.

With 4 ratings on each of the 5 differential clones, it is theo-
retically possible to identify 5^, or 625, races of D. ulei. Undoubted-
ly, there are other races, as yet undiscovered, that attack clones of dif
ferent genetic makeup than those presently used as differentials (Table
2), and new clones to detect these races will need to be added. Moreover

16

there are only 2 groups of distinctive ratings, resistant (R and HR)
and susceptible (MR and S). The resistant group shows no sporulation,
whereas the susceptible group shows light to heavy sporulation. This
allows for ready identification of only 5 , or 25 races of the fungus.

Low humidity conditions, which often occur in the field, inhibit sporu-
lation on clones that would be rated as MR with light sporulation.

This absence of sporulation would result in the erroneous rating, HR.
Under the conditions of high humidity in the greenhouse used for this
research, sporulation occurred on all susceptible (MR and S) clones,
but never occurred on resistant clones. Such consistencies make the rat-
ings reported here appear reliable.

In one instance. Race 4 failed to sporulate on one individual of
the clone MDF 180 (Table 1). The mist system was out of order at the
time of this inoculation, and the relative humidity in the greenhouse was
lower than usual. Later, when high humidity was maintained. Race 4
sporulated on the first and one other individual of clone MDF 180. These
results are interpreted to mean that this clone is actually susceptible
(MR) to Race 4. They also emphasize the necessity for maintaining high
humidity around test plants.

The results reported in this research strengthen Langdon's (12)
argument for the need of using a race identification program and multiple
race resistance testing in conjunction with multiple source breeding for
resistance. The use of only single sources of resistance limits the
areas of usefulness of a particular clone to locations where the domi-
nant races are those to which that clone is resistant. Such a practice

17

also increases the chances that races which can attack clones with this
type of resistance will build up and perhaps destroy the commercial
planting. The use of multiple sources of resistance would probably in-
crease the geographical range and life of a particular clone developed

in this manner

18

LITERATURE CITED

1. Bary, A. de. 1886. Ueber einige Sclerotinien und Sclerotien-

krankheiten. Eotan. Ztg. 44: 409--426.

2. Blazquez, C. H», and J. H. Owen. 1957. Physiological studies

of Dothidella ulei. Phytopathology 47: 727-732.

3. Blazquez, C. H., and J. H„ Owen. 1963. Histological studies of

Dothidella ulei on susceptible and resistant clones. Phyto-
pathology 53: 58-63.

4. Braun, A. C., and R. B. Pringle. 1958. Pathogen factors in the

physiology of disease. Toxins and other metabolites, p.

88-99. In C. S„ Holton, et al, (eds„). Plant pathology, prob-
lems and progress, 1908-1958. Univ. Wisconsin Press, Madison.

5. Dimond, A. E., and P. E. Waggoner. 1953. On the nature and role

of vivo toxins in plant disease. Phytopathology 43: 229-235.

6. Gaumann, E. 1946. Types of defensive reactions in plants. Phy-

topathology 36: 624-633.

7. Gaumann, E. 1957. Fusaric acid as a wilt toxin. Phytopathology

47: 342-357.

8. Goodman, R. N. 1959. Observations on the production, physiological

activity, and chemical nature of Colletotin, a toxin from Colle-
to trichum fuscum Laub. Phytopathol. Z. 37: 187-194.

9. Hilton, R. N. 1955. South American leaf blight. J. Rubber Re-

search Inst. Malaya 14: 287-354.

10. Langdon, K. R. 1963. Culture and pathogenicity of Dothidella
ulei. Ph.D. Thesis. Univ. of Florida, Gainesville. 35 p.

19

11.

Lang don.

K. R.

1963.

Personal

Communication .

12.

Lang don.

o

o

1965.

Relative

resistance or susceptibility of

several clones of Heyea brasiliensis and H. brasiliensis X H.
benthamiana to two races of Dothidella ulei. Plant Dis. Reptr.
49: 12-14.

13. Langford, M0 H. 1960. A new strain of leaf blight on rubber trees

in Costa Rica. (Mimeo.) Report to AID, Washington, D. C. 4 p.

14. Meehan, Frances L. , and H. C. Murphy. 1947. Differential phyto-

toxicity of metabolic by-products of Helminthosporium victoriae.
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BIOGRAPHICAL SKETCH

John Wesley Hiller was born February 23, 1937, at Dilley,

Texas. He graduated from Dilley High School in May, 1955. He at-
tended Texas University, Austin, Texas, from September, 1955, until
May, 1958, majoring in Botany. He entered the Agricultural and Me-
chanical College of Texas, College Station, Texas, in September,

1958 and received the Bachelor of Science degree with a major in Plant
and Soil Science in May, 1960. He began graduate work immediately
at the same institution and received the Master of Science degree
with a major in Plant Pathology. He entered the University of Flor-
ida in September, 1962, and was on a research assistantship until July,
1963, when he was awarded a research fellowship provided by Firestone
Plantation Co. He pursued his work in Plant Pathology toward the de-
gree of Doctor of Philosophy to be granted in April, 1965. He is a
member of the American Phytopathological Society and Alpha Zeta
Honorary Fraternity.

20

This dissertation was prepared under the direction of the
chairman of the candidate’s supervisory committee and has been ap-
proved by all members of that committee. It was submitted to the
Dean of the College of Agriculture and to the Graduate Council, and
was approved as partial fulfillment of the requirements for the de-
gree of Doctor of Philosophy.

April, 1965

; ,-v/ v , ^ L t

^t'»-'I)ean, College of Agriculture

Dean, Graduate School

Supervisory Committee:

Chairman