Title: Host-parasite relations of the fungus Dothidella ulei P. Henn on the Hevea rubber tree /
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Title: Host-parasite relations of the fungus Dothidella ulei P. Henn on the Hevea rubber tree /
Physical Description: viii, 97 leaves : ill. ; 28 cm.
Language: English
Creator: Blazquez, Carlos H
Publication Date: 1959
Copyright Date: 1959
 Subjects
Subject: Dothidella   ( lcsh )
Hevea   ( lcsh )
Plant Pathology thesis Ph. D
Dissertations, Academic -- Plant Pathology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1959.
Bibliography: Includes bibliographical references (leaves 95-97).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
Statement of Responsibility: by Carlos H. Blazquez.
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Bibliographic ID: UF00097999
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: alephbibnum - 000445668
oclc - 37973923
notis - ACK6649

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Host-Parasite Relations of the

Fungus Dothidella ulei P. Henn

on the Hevea Rubber Tree










By
CARLOS H. BLAZQUEZ


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
June, 1959












ACKNOWLEDGMENTS


Acknowledgments are made to Dr. John H. Owen for his guidance

In the pursuit of this problem and to Dr. George F. Weber for his

assistance In the preparation of this manuscript and to other staff

members and graduate students of the Plant Pathology Department of

the College of Agriculture for their advice and timely suggestions.

Appreciation is hereby expressed to all who have furnished,

supplied growing stock of the host, rare chemicals, and specimens of

the fungus.

The author Is Indebted to the Firestone Tire and Rubber

Company for a Grant-in-Aid Fellowship which made this research

possible.

























II
V, a'













TABLE OF CONTENTS


ACKNOWLEDGMENTS . . . .

LIST OF TABLES . . . . .

LIST OF FIGURES . . . .

INTRODUCTION . . . . .

DISEASE DEVELOPMENT ON LEAVES OF
STAGES OF CONTROL . . . .

Leaf Stage Designation. .
Macroscopic Observations on


HISTOLOGICAL STUDIES . . .

Method of Inoculation . .
Histology . . . . .
Early Stages of Infection
Development of Infection.


Page

Ii


v


1


DIFFERENT




Living Leaves


. .. ..

. . ..
. . . .
. ... o . o.
. .. . ..


BIOCHEMICAL STUDIES . . . . . . . . .

Media Containing Quebrachitol . . . . . .
Media Containing Latex Serum . . . . .
Effects of Carbohydrates on the Growth of the Fungus
Leaf Extract from Hevea Spp.. . . . . . .
Media Containing Hevea benthamlana Leaf Extract .
Media Containing Rubber Latex . . . ...
Chromatographical Studies . . . . . . .
Inosltol and Quebrachitol Determination . . .

DISCUSSION . . . . . . . . . . .

SUMMARY . . . . . . . . . . . .

LITERATURE CITED . . . . . . . . .












LIST OF TABLES


Page


4


4


18


38


63


71

81


Table


1 Designation of Clonal Selections Showing
Parentage and Resistance Rating . . . . .

2 Amounts of Chemicals Used as Fertilizers in
Stock and Watering Solutions . . . . . . .

3 Classes of Resistance of the Hevea Rubber Tree
to the Fungus Dothidella Ulel P. Henn . . . .

4 Degrees of Resistance of Hevea Clones to 0. Ulel
and Amount of Fungus Sporulation . . . . . .

5 Effect of Vitamin and Amino Acid Combinations with
Sugars on the Growth of D. Ulel . . . . .

6 Effect of Ethyl Alcohol and Water Extracts Added
to Malt Agar Media . . . . . . . . .

7 Rf Values of Quebrachitol, Inosltols, and Compound 5 .

8 Rf Values of Fluorescent Compounds Found in
Extracts from Leaves of Hevea Clones . . . . .











LIST OF FIGURES


Figure Page

I Stages of development. (1) Stage I, (2) Stage
II, (3) Stage III, (4) Stage IV, (5) Stage V,
(6) Stage VI. . . . . . . . . . 6

2 Stages of leaf development 2 months and 3 weeks
after inoculation. (2) Stage II, (3) Stage III,
(4) Stage IV, (5) Stage V . . . . . . 9

3 Translucent lesion with the halo effect of the
lighter green raised areas. . . . . . ... 11

4 Stages of leaf development one week after
Inoculation showing the velvety appearance of
sporulating lesions. (3) Stage III, (4) Stage
IV, (5) Stage V, (6) Stage VI . . . . . 12

5 Stages of leaf development 2 months and 3 weeks
after Inoculation. (4) Stage IV, (5) Stage V,
(6) Stage VI. . . . . . . . . ... .15

6 Stage III leaflets of resistant clones 3 days
after inoculation showing translucent lesions.
(2) Clone IAN 45-873, (3) Clone FX 232, (4)
Clone FX 2831, (5) Clone P-122 showing pin-
point centers of the lesions. . . . . .. 21

7 Stage III leaves 1 week after inoculation
showing the extent of leaf malformation in
highly susceptible clone Tjir 1 x TJIr 16.
Resistant clone IAN 45-873. Almost Immune
clone P-122 . . . . . . . .. .. . .23

8 Top (A) and bottom (B) Stage III leaflets of
(2) Resistant clone IAN 45-873, (3) Resistant
clone FX 232, (4) Almost immune clone FX 2831,
(5) Almost Immune clone P-122 . . . . .. 25

9 Photomicrographs of cleared leaves showing (A)
Direct penetration of epidermis from young sus-
ceptible leaflet, (B) Penetration of epidermis
from a FX 2831 resistant leaflet, (C) Direct
penetration of dpidermis from a FX 232 resistant
leaflet, (D) Direct penetration of epidermis
from a P-122 highly resistant leaflet. . . ... 34









LIST OF FIGURES--Continued


Figure

10 Collapsed cells In the center of a resistant
leaflet lesion . . . . . . .

11 Cleared leaf section of a young susceptible
leaflet soon after penetration, showing
primary hyphae branching profusely and growing
intercellularly . . . . . . . .


12 Cross section of a young susceptible leaflet
section 108 hours after Infection. Conidial
production has begun, and both the palisade
and parenchyma layers have collapsed .. ..

13 Cross section through a spermogonium, showing
the spermatia and the distinct pore ostiolee)


Page


. . 41


14 Cross section of an old diseased susceptible
leaflet showing yellow material plugging the
xylem vessels . . . . . . . ... . 43

15 Cleared leaf section of a i-year-old diseased
leaflet showing dark-brown mycelium with
typical septations. . . . . . . . ... 44

16 Collapsed epidermal cells near the point of
infection of resistant FX 232 leaf tissue . . .. 46

17 Cross section of a P-122 highly resistant
leaflet showing (A) A latlclferous cell, (B)
A black exudate near an infection point and
the complete surrounding of the subepidermal
layer of cells. . . . . . . . ... ..49

18 Photomicrographs of resistant host reaction to
Invasion of D. ulel. (A) Collapsed mesophyll
and palisade layers showing necrotic and dis-
organized cells. (B) Cross section of a lesion
from a highly resistant leaflet showing sclerenchyma-
like cells. (C) Yellow material observed near
mycelium of invaded resistant cells. (0) Cells
with a granular yellow appearance in the center
of the lesion, and dark sclerenchyma-like cells
found In the perimeter of the lesion. . . . ... 50








LIST OF FIGURES--Continued


Figure Page

19 Six month old cultures of D. ulel comparing
growth in media from (left to right) contaminated
quebrachltol; pure quebrachltol (Firestone Co.);
quebrachltol (Calif.); d-lnositol; with I-lnosltol
as control. . . . . . . . . ... 56

20 Nine-month-old cultures of D. ulel comparing
growth In media prepared from (left to right)
riboflavin plus sugars lactose 10 g; galactose
10 g; mannose 5 g; maltose 10 g; I-Inositol plus
lactose 10 g; galactose 10 g; maltose 5 g; and
mannose 10 g . . . . . . . . . . 65

21 Nine-month-old cultures of D. ulel comparing
growth In media prepared from (left to right)
nicotinic acid plus the sugars lactose 10 g;
galactose 10 g; maltose 5 g; mannose 5 g;
i-inositol plus lactose 10 g; galactose 10 g;
maltose 5 g; and mannose 10 g. . . . . . ... 66

22 Six-month-old cultures of D. ulel comparing
growth in media prepared from Hevea leaf
extracts (left to right) ethyl alcohol from
young, diseased susceptible leaves; water
extract from young, diseased susceptible
leaves; water extract from FX 232 leaves;
water extract from young P-122 leaves; water
extract from 6-month-old leaves; water extract
from 2-month-old F 4542 leaves of an
H. benthamiana clone . . . . . . . . 6

23 Chromatographic pattern of compound 5, which
was present in extracts from healthy leaves
(15, 20, and 7) but not on those from diseased
leaves (12, 10, and 1). Known standards are
of quebrachltol (Q), d- and i-lnositol (d-1,
i-I). Paper, Whatman No. 1; solvent,
n-propanol-acetic acid-water, 6:2:2. . . . .. 80

24 Chromatographic pattern of yellow compound 1
present In extracts from young leaves (11, 4,
20, and 7), but not in those from old leaves
(5, 15, 18, and 14). Paper, Whatman No. 1;
solvent, n-propanol-acetlc acid-water, 6:2:2 .... 84








LIST OF FIGURES--Continued


Figure Page

25 Chromatographic pattern of fluorescent compounds
(2, 3, and 4) present in extracts from young
susceptible leaves (11, 4, and 20), but not in
those from young resistant leaves (7, 8, 6, 13,
and 14).- Paper, Whatman No. 1; solvent,
n-propanol-acetic acid-water, 6:2:2. . . . .. 85


vi i












INTRODUCTION


Dothldella ulel P. Henn. causes a destructive disease of the

foliage of the Para rubber tree, Hevea brasiliensis Muell. Arg., and

is known as the South American leaf blight disease. It has been the

primary cause of the failure or near ruin of many plantations in the

Gulnas and the Ford plantations at Fordlandia, Belterra, Belem, Brazil.

Klippert (12) stated that leaf blight was a major factor in preventing

the establishment of a plantation rubber Industry in the Americas.

Kuyper (14), Stahel (25), Weir (30), and Rands (21) have stated

that the only susceptible parts of the trees to infection by Dothidella

ulel were rapidly growing leaves, shoots, fruits, and flowers.

Stahel (25) conducted a comprehensive study of the disease,

and concluded that leaves up to 2 weeks old were highly susceptible,

and that with continued growth they gradually became resistant to fun-

gus attack.

Hilton (10) stated that the symptom expression always varied

with the age of the leaf attacked, and this was particularly marked In

Hevea, in which the leaves pass through a series of well defined stages.

Langford (15) stated that leaves are at their maximum suscep-

tibility from the time of emergence until they are 7 to 10 days old.

Inoculations on 2-week-old leaves of susceptible clones induced


This grant-in-aid fellowship was sponsored by the Firestone
Tire and Rubber Co., Akron, Ohio.









numerous non-sporulating lesions after 15 to 20 days.

A system for the classification of Hevea clones for resistance

to D. ulel was proposed by Langford (15), based on the tolerance of

the trees to the disease and the partial to complete inhibition of

fungus sporulation.

Blazquez and Owen (2) reported that fungus growth was favored

by small concentrations of i-inositol and quebrachitol (2-mono-methyl

ether of I-inositol), but that at high concentrations of both 1-inositol

and quebrachltol growth of the fungus was inhibited. They stated that

quebrachitol was present In the latex of species of HavAa in concen-

trations of 0.5 to 2.0 per cent, and that it may be present In smaller

amounts in the latex of young leaves. They also stated that this may

be the reason why young Hevea leaves are susceptible to infection to

this fungus whereas older leaves are not Infected or only slightly

Infected.

The purpose of this work was to make comparative studies of

the host-parasite relationship of this disease using Hevea clones of

different degrees of resistance. Histological studies were made to

determine, if possible, what host reactions took place within the tis-

sues of diseased susceptible clones as compared to diseased resistant

clones. Biochemical investigations including chromatographic, nutri-

tional, and microchemlcal experiments were also conducted in an attempt

to find out whether or not certain compounds which might be antago-

nistic to development of 0. ulei existed in greater concentrations in

clones of a higher degree of resistance than in the highly susceptible

clones.












DISEASE DEVELOPMENT ON LEAVES OF DIFFERENT STAGES OF GROWTH


Four clonal selections of Hevea brasiliensis Muell. Arg. which

possessed varying degrees of susceptibility and resistance were ob-

tained from the United States Department of Agriculture, Plant Intro-

duction Gardens, at Coconut Grove, Florida. A number of TjIr 1 x

TJIr 16 seedlings were budded with buds from the 4 clonal selections.

After the buds had taken, the trees were topped, packed and shipped

to Gainesville, where they were planted In beds Inside a greenhouse.

Attempts were made to control the rolsture and temperature within the

greenhouse as near as possible to the optimum for disease development.

Budwood from each clone was brought to Galnesville and budded to seed-

lings growing under greenhouse conditions to insure representative

samples of each clone (Table 1).

Good growth of the seedlings and budded clones was maintained

by adding iron, magnesium, manganese, zinc, nitrogen, and potassium

In dilute solutions once a week.

The solutions were prepared following the recommendations of

Professor Seton N. Edson, of the Soils Department, University of

Florida. The amounts of chemicals added as fertilizers are shown In

Table 2.

The fastest growing trees were topped to keep them at workable


Courtesy of Mr. Jeffrey B. Shrum, Horticulturist, United States
Department of Agriculture, Plant Introduction Gardens, Coconut Grove,
Florida.









TABLE 1

DESIGNATION OF CLONAL SELECTIONS SHOWING PARENTAGE
AND RESISTANCE RATING


Clone Parentage Rating

Liberian Tjir I x TJIr 16 10 ////
IAN 45-873 PB-86 x FA-1717 8 //
FX 232 F 351 x PB-86 5 0/
FX 2831 F 4542 x TJIr 1 3 0
P-122 H. brasillensis (Iberia, Peru) 2 0


TABLE 2

AMOUNTS OF CHEMICALS USEDAS FERTILIZERS
IN STOCK AND WATERING SOLUTIONS


Stock solution Net amount of
Elements g of element In mi Stock solution element in
I liter of water in 3 gal of water lbs/acre

ZnSO 7H20 24.0 60 20
NaNO3 98.1 30 10
KNO3 45.9 60 20
MnSO4 H20 32.4 75 25

MgSO4 7H20 160.5 300 100

Fe0 as 1.3 3 2
Sequestrin









heights, and to cause them to produce more foliage. Shading of the

trees was provided by a netting at a height of 10 feet. Netting was

also hung from the sides of the greenhouse, both to prevent gusts of

wind from scattering the conidia and to increase the humidity. Flushes

of growth were produced continuously by most trees. During the months

of May until early October, when the minimum temperatures did not fall

below 650 F. the greenhouse vents were left open.

During the colder months of the year the greenhouse vents were

closed, and steam heat maintained a temperature of about 700 F. The

dryness of the atmosphere under this type of heating was prevented by

blowing air with an electric fan placed behind a mist sprayer In the

upper section of the greenhouse.


Leaf Stage Designation

Six arbitrary designations were set up to facilitate leaf age

classification depending upon growth characteristics of the Hevea

leaves.


Stage I Leaves

In the earliest leaf differentiation a flush of leaves Is com-

posed of 3 sets of leaves and each leaf Is composed In turn of 3 leaf-

lets. The leaflets are folded dorsally and point upward. The petrioles

are folded upward close to the growing point (Figure 1).


Stage ii Leaves

Stage II is characterized by the centripetally bending leaves,

eventually the tips are pointed straight down, and remain folded. The







1


*2


Figure 1. Stages of develop-
ment. (1) Stage Stage (2 Stage I1, (3)
Stage IIi, (4) Stage IV, (5) Stage V,
(6) Stage VI.


i









leaflets are very firm, have a reddish color, and are less than 3/4

of an inch in length. Some of the younger leaves of the flush may

still be in Stage I. The petioles are separated and are pointing

upward, away from the growing point at an angle of 300. Dijkman (5)

called this period stage "A" (Figure 1).


Stage III Leaves

Leaves are in this stage when pointing straight down, larger

than 3/4 of an inch in length, firm, very shiny, rich in anthocyanin

pigment, of a maroon color, part way open, and with the 3 leaflets

hanging with their lower surfaces tightly pressed against the lower

surfaces of the other leaflets, the petioles being bent 600 away from

the growing point (Figure 1). (Dljkman's stage "8")


Stage IV Leaves

The leaves unfolded and hung vertically with their lower sur-

faces lightly pressed against the lower surfaces of the other leaflets.

The reddish color of the leaflets changed to opaque olive green, and

the petioles are separated to about an angle of 600 from the growing

point (Figure 1). (Dijkman's stage "C")


Stage V Leaves

The expanding light-green leaves lose their olive-green color,

and the petioles are separated from the growing point at an angle of

700 (Figure 1). (Dijkman's stage "D")


Stage VI Leaves

The fully extended light-green leaves change to dark green









with firm laminae, and petioles are separating from the growing point

at an angle of 700 or more (Figure 1).

An experiment was designed to determine the effect of leaf age

on the variation of symptoms and disease development on susceptible

clonal selections.

Leaves of susceptible selections were inoculated simultane-

ously at various stages of development.

At suitable intervals after inoculation, data were taken on

the appearance and development of Infection on the Individual leaves.


Macroscopic Observations on Living Leaves

Stage I. Heavily inoculated leaves produced a black exudate

at the points of infection, which remained on the leaflets until they

blackened, shriveled, and fell off. If only a few spores were placed

on the leaves, the black exudate remained, adhered to the leaves, dis-

torting their shape. Generally severely infected leaves showed ex-

treme twisting and curling before shriveling and dropping off.

Stage II. Inoculated leaves showed minute raised areas after

3 days. Within a week they began to curl, and the fungus sporulated

abundantly. The reddish tinge of the normal leaves changed to dark

olive green as spores were produced on the under surface of the un-

folding leaves. They blackened and shriveled, hung onto the petioles

for less than a day and fell off, leaving the petioles still attached

to the shoot or to the stem of a tree (Figure 2).

Stage 11l. inoculated leaves larger than 3/4 of an inch,

maroon in color and very shiny, showed 2 types of symptoms, a












4 3
I ,


Figure 2. Stages of leaf development 2 monthss and 3
weeks after inoculation. (2) Stage I1, (3) Stage III, (4)
Stage IV, (5) Stage V.










translucent condition previously described by other workers (14,15,

21,27,30) and a humid condition. In the translucent condition the

lesions were clearly delimited by a marginal brownish-black line,

which sometimes was very pronounced. The tissues within the border

were very shiny and translucent. The green color of the leaf was much

darker within the lesion, and In most cases was only near the marginal

black line, but in others It was scattered throughout the lesion. Some

small maroon areas were readily seen, since they were not masked by the

opaque normal green color. The lesions were limited by the secondary

veins. Generally, the lesions had numerous raised areas surrounding

them which were lighter green than the normal tissues; however, these

raised areas were not translucent. The translucent lesions were flat

at first, with no visible raised areas, but as the disease progressed

the raised areas became visible and more abundant around the limiting

black margin of the lesion. The color of the raised areas gradually

changed to a lighter green giving the translucent lesions a halo effect

(Figure 3). The centers of the translucent lesions gradually became

dark, and after 2 to 3 days changed to olive in color, and a velvety

appearance developed due to the presence of spores. The raised mar-

ginal areas changed from a light-green color to olive, and spore de-

velopment produced the velvety appearance as previously noted (Figure

4). Any leaf movement resulted In the release of a small cloud of

spores from the under surface of the leaf.

Humid Condition.--The second type of symptom was observed only

under 100 per cent relative humidity. It was similar to the raised

areas surrounding a translucent lesion, except that the only raised














































Figure 3. Translucent lesion with the
halo effect of the lighter green raised areas.









































Figure 4. Stages of leaf develop-
ment one week after Inoculation showing the
velvety appearance of sporulating lesions.
(3) Stage 1ii, (4) Stage IV, (5) Stage V,
(6) Stage VI.










areas were visible, without any translucent parts or centers of the

lesions. Sporulation occurred in the same manner as previously de- -

scribed for the raised areas of the translucent lesions.

Macroscopic symptoms on Inoculated leaves were of two types.

The first and most common symptom was the gradual changing of the cen-

ters of olive lesions to dark green and ultimately to charcoal black.

This black lesion was stromatic in nature and was surrounded by vari-

ous distinct, erumpent, black dot-like stromatic structures, or sper-

mogonla. There was a small yellowish circular area between the small

spermogonia and the large black stromatic mass In the center of the

lesion. The second type of symptom was a gradual change in color,

from the outer perimeters of the raised areas surrounding the black

limiting line of the translucent lesion toward the center of the le-

sion and including the raised areas. There was a definite dying of

this Inner marginal tissue, which became light yellow and then orange

colored. The olive velvety mass formed on the translucent lesion

gradually became black and stromatic, with few erumpent dot-like sper-

mogonia in the center of the lesion. In some lesions there was a black

necrotic line around the ring of dried tissue, while in others this

black line was absent. On both types of lesions, masses of spores were

produced on the top surfaces of the leaves from stromatic masses in

the centers of the lesions.

Stage IV. Inoculated leaves showed small raised areas within

10 days after Inoculation. These raised areas became rapidly dis-

colored, and after 5 days black stromatic structures were present In

their centers. On the lower surface of the leaf, masses of conldla









appeared directly opposite the stromatic structures on the upper sur-

face. The number of lesions developed after inoculation was much less

than the number appearing after Inoculations at earlier stages of leaf

development. The fungus sporulated moderately and formed few coalesced

stromatic structures (Figure 5).

Stage V. Inoculated leaves showed symptoms similar to those

observed In Stage IV.

Stage VI. Inoculated leaves produced very few lesions, and

although they were similar to those developing on Stages IV and V they

formed few erumpent black spermogonia and did not produce spores.

Inoculated leaves of greater maturity produced no symptoms

although the dark olive-green masses of spores remained readily visi-

ble on the leaf surfaces.

Three types of symptoms were previously observed on the upper

surface of leaves Inoculated at Stages iii and IV. The first type

showed lesions with a black stromatic, erumpent, doughnut-like struc-

ture which may or may not have been surrounded by a light-green halo-

like border. The doughnut-like stromatic structures were I to 3 mm

wide, with dead and dry tissues ranging from light green to white in

color in the center. The centers of the lesions fell out, and the

characteristic "shot hole described by Rands (21) was produced. in

the second type the lesions had marked black necrotic margins sur-

rounded by dead tissue and the black, erumpent, doughnut-shaped stro-

matic structure Inside of the ring of dead tissues. There was a

slightly green discolored area surrounding the entire lesion. in the

third type of symptoms, no stromatic doughnut-shaped structure was













6


5













Figure 5. Stages of leaf development 2
m-onths and 3 weeks after inoculation. (4) Stage
IV, (5) Stage V, (6) Stage VI.









formed within the lesion, but rather a few black spermogonia were

scattered throughout the lesion's dead and dry tissue inside a small

light-brown necrotic marginal line. On the lower surfaces of the

leaves, only flat stromatic tissue was observed directly underneath

the spermogonia and the stromatic structures.

Inoculated leaves at Stage VI developed stromatic masses after

10 or more days which grew very slowly on the upper surface of the

leaves. Within I month after Inoculation black dot-like structures

appeared on the upper surface of the leaves forming a ring, the center

of which dried and either remained on the leaflet or fell away

(Figure 5).

Leaf petioles, leaf veins, and growing shoots all gave the

same type of symptoms at the various ages of the tissues.

Petioles and main veins of Stage I leaves produced the same

type of black exudate as the laminae when heavily Inoculated. The

petioles blackened before being shed. If not heavily inoculated they

often swelled, giving the leaves and growing shoot a twisted appear-

ance. As the disease advanced these swellings cracked and

sporulated heavy before turning black, shriveling and falling away.

Stage II petioles gave the same type of symptoms as the

lightly inoculated Stage I petioles; however, as the age and size of

the leaves increased, the swellings appeared as long streaks of spor-

ulating cankers. These cankers acquired a woody appearance with age.

The petioles of the Stage IV leaves and the lower parts of the grow-

ing shoots showed symptoms after Inoculation.

Kuyper (14) reported that the swelling of the petioles was








due to the phloem tissue hypertrophy, particularly of the phloem

parenchyma cells. Weir (30) stated that cankered stems bearing peri-

thecia and terminating in well-developed young leaf clusters were fre-

quently observed. Cankers were found on older stems, but the peri-

thecial stroma had deteriorated and no ascospores were found.

Rands (21) was of the opinion In 1924 that within such a

variable species as Hevea braslliensis substantial differences in

susceptibility occurred, and he reported that in every plantation

attacked by the disease a few trees had remained healthy, made excel-

lent growth and produced large canopies of dark-green foliage. He

considered these trees to be resistant to the disease.

Langford (15) determined that resistance to the disease was

exhibited in either or both of 2 forms: (1) tolerance of the disease

by resistance to leaf damage and defoliation, and (2) partial or com-

plete inhibition of the fungus. He proposed a system of classifica-

tion of Hevea clones for resistance or susceptibility based on the

extent of damage to leaves and the amount of fungus sporulation

(Table 3). The classes he set up were for damage resulting to plants

that were subject to heavy inoculation, and growing under conditions

extremely favorable for disease development.

Heavily Inoculated leaves of the resistant clone IAN 45-873

while at Stage I produced the same black exudate as highly susceptible

leaves. Infected petioles also produced the black exudate. Lightly

inoculated leaves at this stage produced a black exudate, but were not

shed and became distorted and misshaped. The leaves were more dis-

torted in areas where the black exudate was produced. Sparse








TABLE 3

CLASSES OF RESISTANCE OF THE HEVEA RUBBER TREE
TO THE FUNGUS DOTHIDELLA ULEI P. HENN
Proposed by Langford


1. rmmune:2 No evidence of infection.

2. Almost immune: Yellow flecks; no apparent damage.

3. Very highly resistant: Small necrotic lesions causing very
slight damage.

4. Highly resistant: Necrotic lesions causing slight damage.

5. Resistant: Necrotic lesions causing distinct damage; leaves
reduced in size and sometimes deformed.

6. Moderately resistant: Necrotic lesions causing conspicuous
damage; leaves dwarfed and deformed
or ragged.

7. Moderately susceptible: Defoliation ranging up to 50 per cent.

8. Susceptible: Severe to complete defollation--more than 50 per
cent.

9. Highly susceptible: Plant stem killed back. (New shoots may
arise from secondary buds.)

10. Very highly susceptible: Plant killed by repeated defoliations.

The five symbols designating degree of sporulation are as follows:

O0 No sporulation.

= Very sparse sporulation: A maximum of two or three weekly
sporulating leaf or petrloles le-
sions on the most diseased flush
of leaves.

-- = Sparse sporulation: A few weakly sporulating lesions on the
most diseased flush of leaves.


1Classes set up for damage under severe Infection (15).

2The term Immune Is used throughout this investigation follow-
ing Langford (15).





19



TABLE 3--Continued


--- =Moderate sporulation: Very noticeable sporulation on the
most diseased flush of leaves,
usually some on several other
flushes.

= Heavy sporulatlon: Conspicuous sporulation on the most
diseased flush of leaves; usually
some on most other flushes.









sporulation was observed on the lower surface of the leaves. After

3 weeks the leaves turned yellow and were shed. Leaves of Stages II

and III gave similar symptoms when heavily inoculated on the lower

surface. Lesions produced at these 2 leaf stages were similar to

Inoculations carried out on highly susceptible Stage IV leaves. On

the upper surfaces of the leaves stromatic structures were observed

with light-green areas and a light-brown center around raised lesions.

Sporulation was observed on the lower surfaces of the leaflets oppo-

site these brown centers, and as the disease progressed these areas

became necrotic, ceased sporulating and the leaflets acquired a crin-

kled and stunted appearance. Some lesions, very minute in size with

light-brown necrotic areas, which later turned into streak-like le-

sions,were observed on petioles and midribs of the leaves (Figure 6).

Leaves inoculated at Stage IV developed 2 kinds of lesions on

the lower surface. Lesions of the first type were raised, light-green

areas and yellow centers. Other lesions observed had Irregular nec-

rotic areas with large centers of dead tissue. Some sporulation was

observed on this clone near young lesions on the laminae. Some le-

sions were found on the secondary veins and velnlets. No stromatic

structures were observed on the veins or veinlets of the lower sur-

face of the leaflets. On the upper surface of the leaflet some lesions

showed only light-green depressed areas, while others had necrotic bor-

ders and large centers of dead, dry tissue. Inoculated leaves at older

stages produced no visible symptoms and remained normal. Heavily in-

oculated leaves of Stage I of resistant clone FX 232 produced the black

exudate observed on other resistant and susceptible clones. Leaves


















3















Figure 6. Stage ill leaflets of resistant
clones 3 days after Inoculation showing translucent
lesions. (2) Clone IAN 45-873, (3) Clone FX 232,
(4) Clone FX 2831, (5) Clone P-122 showing pin-
point centers of the lesions.









with black exudate adhering to their surfaces grew abnormally and were

twisted and wrinkled. The more conspicuous symptoms of the diseases

were the dot-like lesions on the under surface of the leaflets, which

were visible 6 days after inoculations. These lesions had no definite

shape, but varied in size and in the degrees of necrotic tissue pres-

ent in their centers (Figure 7). On the lower surfaces of the leaves,

a shiny green color was observed in areas near old lesions, instead

of the normal opaque green color.

Leaves inoculated at older stages produced no visible symptoms

of the disease and developed to normal size.

Almost Immune clone FX 2831, Stage I leaves, when heavily In-

oculated produced black exudate both on the leaflets and the petrioles.)

These leaves soon lost color, blackened, shriveled and fell within a

week. Lightly inoculated leaves produced very little black exudate,

formed some raised lesions with light-green areas, and later some

necrotic centers. The lesions were Irregular in shape, both depressed

and raised on the upper surface of the leaves. As the leaves Increased

in size, the diseased leaves became wrinkled and stunted. Leaves in-

oculated at Stages III and IV produced similar symptoms. Depressed

lesions with light- to dark-brown necrotic areas and with or without

yellowish-green margins were formed on the lower surface of the leaf-

lets. These lesions had no definite shape, and some coalesced, with

a light-yellow center of dead tissue surrounded by a black-brown nec-

rotic border and a light-green halo. No stromatic or pseudostromatic

tissue was observed on the surface of the lesions. Some very small

pin-point lesions were seen near some secondary velnlets, or the





































Figure 7. Stage III leaves I week after in-
oculation showing the extent of leaf malformation in
highly susceptible clone Tjir 1 x TJIr 16. Resistant
clone IAN 45-873. Almost Immune clone P-122.









midribs of the leaflets. On the upper surface of the leaflets, small

raised, necrotic lesions with no definite shapes were observed.

Almost Immune clone P-122 leaves inoculated at Stage I pro-

duced black exudate similar to the one formed on other resistant and

susceptible clones. As the leaves began to unfold, they showed

slightly depressed areas on the lower surface of the leaflets, with

dot-like necrotic centers. Some leaves with black exudate were mis-

shapen, and were somewhat wrinkled and slightly smaller than healthy

ones. The lesions did not continue to enlarge with further leaf de-

velopment, and only the dead tissue in the centers of the lesions in-

creased. No sporulation or stromatic structures were observed on the

developed leaflets. The only visible symptoms observed were pin-point

lesions surrounded by shiny green areas, Instead of the normal opaque-

green color of the under surface of the leaflets (Figure 8).

All of the lesions were slightly to deeply depressed with

brownish-red areas. When magnified the brownish red-areas appeared

as a group of Individual cells, similar to small pebbles, surrounding

an orange center. Lesions on the upper surface of the leaflets were

slightly raised, giving the leaflet a wrinkled appearance. Under

magnification the wrinkled surfaces of the leaves were not conspicuous,

and the only visible symptoms were the translucent white lesions with

pin-point necrotic centers. No lesions were observed on the leaf

etrioles or midribs, and no sporulation or signs of the fungus were

observed on the lesions produced on this clone.



























5 ,4


Figure 8. Top (A) and bottom (B) Stage iII leaf-
lets of (2) Resistant clone IAN 45-873, (3) Resistant
clone FX 232, (4) Almost Immune clone FX 2831, (5) Almost
Immune clone P-122.












HISTOLOGICAL STUDIES


The results of fungus infections in terms of the efficiency

of fungus nutrition and the range of congeniality of host-parasite re-

lationship of Dothidella ulel P. Henn. and the Hevea rubber are very

similar to those of invasion by many of the so-called obligate

parasites.

The establishment of a parasite will result after a success-

ful penetration of a fungus germ tube when the conditions within the

host are favorable. In many cases it has been observed that a fungus

spore will germinate and invade the tissue even of a resistant host,

but that the establishment of the parasite then falls and the fungus

dies.

Martin (18) stated that the hyphae of yellow rust (Pucclnia

glumarumyichmJ) Eriks and Henn. will penetrate the stomata of resist-

ant wheats but that further progress of the fungus is prevented by

some unfavorable condition within the host. He further stated that on

barley the mildew of wheat (Erysiphe graminis D.C.) will form haus-

toria, but after a few days the lack of receptivity of the host brings

about degeneration of the fungus.' The conditions which affect further

growth of a fungus must be presumably of a chemical or physiological

nature, and the ability of a fungus to thrive after penetration will

be dependent on the presence of the required food material in the

plant tissue.

Although many cytological studies have been conducted

26









frequently on the host-parasite relationships of various pathogenic

fungi, especially in the order Uredinales (1,26,29), little attention

has been given hitherto to similar studies within the order Dothideales

group of which Dothidella ulel is typical.

In early studies of the South American leaf blight disease of

rubber caused by D. ulel, Griffon and Maublanc (8) suggested that

fungi of genera (Fuslcladium, Aposphaeria, and Dothidella) previously

found associated with the disease might actually be a single genus and

not 3.

Stahel (25) Investigated thoroughly the various phases of

fungus infection, and by tracing mycellal strands was able to conclu-

sively prove that the 2 other stages frequently found with the perl-

thecia of Dothidella were a part of its life cycle.

Stahel's work laid the foundations for host-parasite relation-

ship studies, especially with reference to details of cuticle penetra-

tion and the early phases of establishment of the parasite. However,

he did not consider the distinctive phenomena of parasitism and patho-

genesis In the resistant and Immune trees.

The objectives of the present study was to compare the host-

parasite relations between resistant and susceptible Hevea clones that

might account for their disease reactions, and to determine any

histological differences.


Method of Inoculation

Spores used for Inoculation were obtained from diseased leaves

'of a highly susceptible clone TJIr 1 x TJir 16 used in previous inves-

tigations (2). They were collected from sporulating leaf lesions with









a wet camel-hair brush and inoculation was made by brushing them on

the moistened surface of young healthy leaves. It was necessary to

cover the inoculated tree with a plastic bag unless the humidity in

the greenhouse was high.

Leaves of the susceptible clone TJir 1 x TJir 16, resistant

clones IAN 45-842, FX 232, and Immune clones P-122, FX 2831, were In-

oculated at leaf Stages I, II, III, IV, V, and VI, and the ecological

data recorded.

Inoculation experiments were carried out In 2 ways, In one a

leaf was heavily Inoculated with high concentrations of conidia (200

conidia per drop of water), and In the other a leaf was lightly Inoc-

ulated with a less concentrated conidlal suspension (40 conidia per

drop of water).

Prior to Inoculation each tree was thoroughly washed with a

fine spray of tap water from a mist sprayer and gently shaken to re-

move the larger drops of water.

The conldia were brushed on the upper leaflet surface In one

experiment, and on the lower leaf surface In a second experiment.

After Inoculation the entire tree was covered with a plastic bag for

2 or 3 days until the first macroscopic symptoms were visible. During

the summer when the greenhouse vents were open the plastic bag was

kept on until after conldia had been produced and collected.


Histology

Infected leaf material was collected every 3 hours the first

day after Inoculation and at 12-hour intervals thereafter and fixed

In chemical solutions. The date and hour of fixationwere recorded and








each collection, with the fixed material, was labeled accordingly.

in preliminary experiment, various fixatives were tried in

combinations with different methods of dehydrating and staining. Best

results were obtained with Newcomer's cytological fixative (20) with

the following formula (by volume): 6 parts isopropyl alcohol, 3 parts

propionic acid, I part ether (petroleum), I part acetone, and 1 part

dioxane. The tertiary butyl alcohol of Johansen's (il),was employed

for dehydration and embedding. Sections were cut with a rotary micro-

tone 8 u thick. Heldenhain's iron alum hematoxylin was a satisfactory

stain for early stages because it brought out the infection hyphae

clearly. A safranin-fast green method was best for differentiating

advanced stages of the fungus. Duplicate slides were stained only

with safranin in advanced stages of disease development.

Various methods of leaf clearing were tried in combination

with different staining methods. Best results were obtained with

young tissues by clearing in a saturated solution of chloral hydrate

preceded by fixation and killing in Newcomer's fixative. Staining

was accomplished by gently warming the tissue on a glass slide

flooded with a 1 per cent cotton blue In lactophenol for 2 minutes,

counterstained with a i per cent acid fuchsin rinsed in 50 per cent

alcohol, mounting in chloral hydrate and ringing with Clearcol mount-

ing medium.1

This method was well adapted for studying certain stages of


'Clearcol mounting medium. H. W. Clark, 33 So. High Street,
Melrose 76, Mass.









conidlal germination, direct cutlcular penetration, and the establish-

ment of the fungus beneath the cuticle, as it afforded a means of ob-

serving these phenomena In toto on relatively large areas of leaf

surface.

Pieces of leaves were collected 6, 12, 18, 24, 36, 48, 60, 72,

96, and 144 hours, respectively, after Inoculation, fixed in Newcomer's

solution (20), strained,and mounted as previously described.


Early Stages of Infection

The germination of conidia, formation of appressoria and pene-

tration of the cuticle, as observed in cleared leaves examined In toto,

appeared to be similar In all the susceptible, resistant and almost

immune clones. Following penetration, however, there were distinctive

differences in the extent and nature of mycelium development In the

susceptible, resistant, and almost immune clones.

At the end of 12 hours, the germination of most of the conidia

had begun. The conidia were 2 celled, with the distal cell expanding

and developing a slight protuberance. The conidial wall, however, re-

mained intact, forming a thin slightly stained membrane about the

protuberance.

After 24 hours, germination was well advanced, with rudimen-

tary germ tubes (usually from the distal cell), penetrating directly

through the cuticle and epidermal layer of cells. In a large number

of cases appressoria were observed closely adhering to the cuticle.

Occasionally a germ tube branched and formed 2 distinct

appressorla, each of which functioned in actual penetration of the









host. Penetration from the proximal cell of the conidlum was gener-

ally observed after penetration from the distal cell had occurred.

In some cases penetration was observed to take place without the for-

mation of a germ tube or appressorium.

In many cleared leaves it was possible to observe direct pen-

etration of a primary hypha regardless of location on the leaflet.

The primary hypha stained with cotton blue, revealing a cross-

wall between it and the germination tube.

The single celled, Irregularly shaped, primary hypha found In

most 24 hour sections, rapidly branched out In all directions.

After 48 hours cells near the penetration hypha, stained with

cotton blue, showed a granular condition of the protoplasm, and even-

tually collapsed. At the end of 48 hours the development of the fun-

gus in leaflets of susceptible clones was more pronounced than that

of resistant and almost Immune clone leaflets. There was a consider-

able amount of granular protoplasm near the penetration sites, in

leaflets of,reslstant clone FX 232. In almost immune leaflets of

clones FX 2831 and P-122, the granular condition of protoplasm sur-

rounding penetration sites was very pronounced, with a manifestation

of necrosis that will be explained later. The position of these epi-

dermal cells was very Irregular, generally occurring in the highly

susceptible clone leaflets 48 to 60 hours after inoculation. Epider-

mal cell collapse was frequently observed in resistant and almost

immune clone leaflets. In highly susceptible clones, the mycelium

developed throughout the leaflet regardless of type of host cell en-

countered. It was possible to observe the mycellum crossing over and









under, and surrounding conductive tissues.

Mycellal development was strictly intercellular, and in no

Instance was it observed to become Intracellular. In leaflets of all

clones tested the mycelium surrounded the subepidermal layer of cells,

regardless of stage of leaf development.

In resistant and susceptible leaflets mycellal development was

accompanied by the formation of a yellowish material, which Is be-

lieved to be a reaction of the host tissue. The yellowish material

was observed in advanced stages of infection of susceptible leaflets,

next to collapsed cells, and near the point of Infection. In resist-

ant and almost Immune leaflets the yellowish material was formed where-

ever mycelium was present.

In susceptible leaflets the mycellum radiated centripetally

from the Infection initial Into the parenchyma layer after 96 hours,

and formed a depressed area in the leaflet which was the first visible

symptom of infection.

In resistant and almost imnmne leaflets, the average diameter

of the lesions was much smaller, lacking any depressed area. However,

the number of killed cells was greater than in susceptible leaflets,

and the lesion could only be discerned as a pin-point.

Host Penetration.--The germinating conidia of Dothidella ulel

penetrated the cuticle of the Hevea leaflets directly. This process

was similar In all details for all clones. There was no cytologlcal

evidence to show that a cutlcular barrier of any kind hindered the

establishment of the fungus on any clones. Such penetration always

occurred at a juncture epidermal cells, or at the base of a trichome.









No peeling back or mechanical rupturing of the epidermal layer and

cuticle were observed (Figure 9).

Germination of Conidla.--Present observations of conldlal ger-

mination on the host tissue were in accord with reports by Kuyper (14),

Stahel (25), and Blazquez and Owen (2). A germ tube was first pro-

duced by the distal cell of the conldlum, growing to about 200 u in

length. The proximal cell would then produce a germination tube which

developed similarly.

On susceptible leaflets the germination tube would grow to

about 700 to 800 u in length, forming an arc and producing an

appressorium when touching the surface of the leaf cuticle. Generally

the germ tubes that were observed to penetrate susceptible leaflets

directly were not as long as those forming appressoria.

In resistant leaflets from FX 232 and IAN 45-873 clones, as

well as in leaflets of almost immune clones P-122 and FX 2831, the

germination tubes were very short, not over 200 u, and sometimes pen-

etrated directly through the cuticle. Most germ tubes formed

appressoria upon coming in contact with the leaflet surface Instead

of penetrating directly.

Formation of an Appressorlum.--As the advancing tip of the

germ tube touched the surface of the leaflet, It became closely ad-

hered, and developed Into a more or less clearly differentiated

appressorium. The appressoria in cross section appeared to be round

or oval in outline and densely filled with cytoplasm, staining heavily

with safranin, or hematoxylin. A larger number of appressoria were

formed on leaflets of resistant and almost Immune clones than on














IU


E
1C
41
' '
><| C


i.


Figure 9. Photomicrographs of cleared leaves
showing (A) Direct penetration of epidermis from young
susceptible leaflet, (B) Penetration of epidermis from a
FX 2831 resistant leaflet, (C) Direct penetration of epi-
dermis from a FX 232 resistant leaflet, (D) Direct pene-
tration of epidermis from a P-122 highly resistant leaflet.









leaflets of susceptible clones. In some Instances conldia did not

form a germ tube, but formed instead an appressorium Immediately after

germinating. The appressoria were apparently held fast to the leaflet

surface by a mucilagenous sheath. The fixed remains of this structure

were usually found In the cytological preparations.

Primary Hypha.--The term primary hypha is used to designate

the first hyphe that is visible within the epidermal layer of cells

after penetration.

In host tissue, after penetration, the fungus developed an Ir-

regular, shapeless somewhat branched primary hypha. In the majority

of cases the only difference between a germ tube and a primary hypha

was a cross wall, and the affinity of the primary hypha for stains.

In susceptible leaflets the primary hypha was observed to be

adjacent or surrounding 1 or 2 epidermal cells showing a granular

protoplasm condition without a nucleus.

In resistant and almost immune leaflets all cells surrounding

appressoria showed disorganized protoplasm, and no nuclei. Cells in

the centers of older Infections had a heavy yellow granular appearance,

and In leaf Stages I, II, III, and IV, the cells were beginning to

collapse (Figure 10). Primary hypha were not easily visible due to

this rapid cell reaction.


Development of Infection

Two types of infections were observed on both the upper and

lower epidermis, palisade and mesophyll layers of Hevea rubber leaf-

lets. The first type was typical of susceptible leaflets, and the





36






























Ji





Figure 10. Collapsed cells in
the center of a resistant leaflet
lesion.









second type was typical of resistant and almost Immune leaflets

(Table 4).

Infection of Susceptible Leaflets.--Followlng penetration of

the epidermis of susceptible leaflets and the formation of a primary

hypha, I or 2 subepidermal cells changed in appearance. The primary

hypha developed intercellularly, branched profusely, formed normal sep-

tate mycelium, which rapidly penetrated deep Into host tissue. Vas-

cular and sclerenchyma tissues did not hinder mycelial development as

the mycellum was often observed to grow adjacent to vessels and sieve

tubes (Figure 11).

Susceptible leaflets Inoculated at Stages I and II were rap-

Idly invaded by the mycellum after 96 hours, and after 120 hours

mycelial hyphae had broken through the epidermis and cuticle forming

conidiophores and conldla. Host cells near rapidly expanding hyphae

were as normal as those beyond the area of Infection. Leaflets Inocu-

lated at Stage I produced a black exudate near the point of Infection,

which adhered to the surface of the laminae and prevented normal leaf-

let development.

The condition of chloroplasts was used to determine the degree

of deterioration of subepldermal cells. A more delicate indicator of

health according to Rice (24) would be the amount and condition of

chlorophyll present. Leaves which showed yellow, translucent areas

around water-soaked Infection loci at the time of Infection, were some-

times found to have Intact chloroplasts In all but the centers of the

infection loci. Thus the chlorophyll disappeared before the chloro-

plasts lost definition. In all leaf stages chloroplast disintegration

















TABLE 4

DEGREES OF RESISTANCE OF HEVEA CLONES TO D. ULEI
AND AMOUNT OF FUNGUS SPORULATION
According to Langford (15)


Clone

Tjir I x Tjir 16


IAN 45-873

FX 232

FX 2831

P-122


Sporulation


////


Degree of
Resistance

10. Very highly
susceptible

8. Susceptible

5. Resistant

3. Highly resistant

2. Very highly
resistant
(Langford's immune)


_ _____I___ __I_ _______I_ __ _______ ___1_ ____


I











































Figure 11. Cleared leaf section of a young
susceptible leaflet soon after penetration, showing
primary hyphae branching profusely and growing
Intercellularly.









preceded the formation of a yellow granular condition of the proto-

plasm. There were normal gradations in degeneration, from completely

cleared, though well-oriented turgid cells in the centers of Infec-

tions, to cells with chloroplasts coalesced but with the outlines of

the individual plastids still visible, to cells with well defined,

healthy chloroplasts.

Fastest mycellal growth in the earliest stages of Infection

appeared to be near the lower epidermal cells. This was observed in

all the leaflets inoculated, regardless of whether they were from re-

sistant or susceptible clones.

A yellow granular condition was observed near the point of

infection, and throughout the parenchyma layer adjacent to the epi-

dermis. Cell collapse began at the point of infection, and continued

until both the mesophyll and the parenchyma layers were completely

collapsed at the initial point of infection. This collapse produced

abnormal tensions within the cell layers and formed an irregular de-

pressed area. Conidial production was observed soon after conidiophore

formation on the lower epidermis (Figure 12). After sporulatlon, the

leaflet tissue appeared disorganized, wrinkled, dry, and finally dead.

Leaflets inoculated in Stages III, IV, and V showed that the

tissues were readily invaded by the fungus in the early stages of in-

fection; however, with continued leaf growth the areas of infection

became somewhat delimited. The fungus was well developed throughout

the mesophyll and palisade layers of the leaf laminae, and conldfal

production began 108 hours after inoculation. In some Instances spor-

ulation was first observed in the outer perimeter, and later throughout











































Figure 12. Cross section of a
young susceptible leaflet section 108
hours after infection. Conidral produc-
tion has begun, and both the palisade
and parenchyma layers have collapsed.








the entire lesion on the lower surface of the leaflet. Some conldia

were produced on the upper surface during later stages of the disease.

With the decrease in sporulation some stromatic Initials were

observed at the point of infection, Increasing in size until they cov-

ered the surface of the lesion. Griffon and Maublanc (8) called these

cells chlamydospores. When the lesion was larger than 1/4 of an inch

the central cells were often killed, and In these Instances the stro-

matic masses formed on the perimeter of the lesion. A few small round

erumpent structures were observed on the upper epidermis directly

above the stromatic mass. These structures were considered to be

spermogonia by Stahel (25) and Langford (15). These spermogonia were

filled with spermatia, which oozed out through a distinct pore

(Figure 13) approximately 2 months after Inoculation.

In a tangential section of a leaflet 2 months after Inocula-

tion, stained with safranin It was possible to observe a deeply

stained tannin-like material. In a cross section the Intra- and

intercellular presence of the yellow material was observed In the

&ylem vessels (Figure 14).

In lesions 1 year old, the mycelium was dark brown, with def-

inite septations, and grew Intercellularly (Flgure 15). A large part

of the stromatic masses was growing on the surface of collapsed or

semi-collapsed tissue. Spermogonia appeared to be empty, and no sper-

matla were visible In any of the sections prepared.

The perlthecial stage was not observed.

Leaflets Inoculated at Stage VI showed similar fungus pene-

tration to earlier stages. However, fungal development was not as




























Figure 13. Cross section
through a spermogonium, showing the
spermatia and the distinct pore (ostlole).


,--, "
..,.





Figure 14. Cross section of an
old diseased susceptible leaflet showing
yellow material plugging the xylem vessels.




























rv
p
,f,


Figure 15. Cleared leaf
section of a 1-year-old diseased
leaflet showing dark-brown mycelium
with typical septations.









vigorous, there was less severe Impoverishment of underlying host tis-

sues, and a decreased anmunt of necrosis. Cross sections showed myce-

lial invasion of the mesophyll and palisade layers 2 weeks after inoc-

ulation. The striking difference observed was the lack of conldial

production and the formation of stromatic tissue. Impoverishment of

the palisade layer appeared In the middle of the lesion 12 to 15 days

after inoculation manifested chiefly by the disappearance of plastids

and by marked vacuolation.

Infection of Resistant Leaflets.--Host reaction to the fungus

Invasion of IAN 45-873 and FX 232 leaflets was very similar, and they

therefore were considered as one type of reaction and will be described

together.

The immediate host reaction to fungus penetration was the

distinctive difference between susceptible and resistant foliage.

Epidermal and parenchyma cells of susceptible leaflets appeared to be

normal near primary hyphal development, while in resistant leaflets

1 or 2 epidermal cells collapsed, and a few parenchyma cells adjacent

to primary hypha appeared disorganized and soon collapsed (Figure 16).

In resistant clones, 108 hours after Inoculation the fungus

had developed throughout the various leaflet tissues. Cells adjacent

to mycelium showed some impoverishment with cell disorganization more

pronounced in the area nearest the point of infection.

A very pronounced host reaction of the resistant and Immune

clones was the production of a yellow material found throughout areas

where fungus hyphae had Invaded the tissues. In most of the disor-

ganized cells there was a granular appearance, yellowish In color,





































Figure 16. Collapsed epi-
dermal cells near the point of In-
fection of resistant FX 232 leaf
tissue.









which changed to dark brown in older lesions.

The subepidermal parenchyma of leaflets of clone IAN 45-873

showed protoplasm disorganization 108 hours after infection. Mycelial

hypha were well distributed throughout the subepidermal parenchyma

cell layer 6 days after infection.

The fungus continued to spread throughout the tissue, and

after 7 days most of the parenchyma layer of the mesophyll and some

palisade cells nearest the point of infection had been Invaded.

Parenchyma cells closest to infection points showed disorganized

chloroplasts and the appearance of yellow granular material. The

entire mesophyll layer was invaded 10 days after Inoculation, and

after 12 days the palisade layer showed an Impoverished granular

condition.

Stromatic tissue appeared on the lower surface of the leaf-

lets near the apparently dead cells, and after 13 days, condiophores

and conidia were formed in abundance. They were also formed on the

upper surface of the leaflet, directly over the discolored epidermal

and palisade cells. Spermogonia were not observed In any of the

inoculated leaflets.

Diseased tissues of FX 232 and IAN 45-873 were similar except

for a decrease In sporulation, Intercellular Invasion, and necrotic

tissue In clone FX 232.

Almost Immune Clones.--Histological observations of leaflets

considered to be almost immune revealed that the fungus was capable

of penetrating, colonizing, and killing leaflet tissue. The type of

host reaction observed was similar to the resistant type reaction.









Observations of almost inmune (hereafter designated as highly resist-

ant) leaflets will be described, and essentially considered to be of

the resistant type of host reaction.

Host reactions and tissue invasion of clones FX 2831 and

P-122 were considered similar and are described together. In these

2 clones inoculations of leaflets at Stage I produced a black exudate

on or near the lesions within 36 hours (Figure 17 A). Epidermal cells

near cell junctures where Infection hyphae penetrated collapsed soon

after penetration occurred (Figure 16). Rapid proliferation of the

fungus mycellum throughout the subepidermal layer was observed on the

lower surface of a leaflet inoculated 3 days previously. The host

nuclei In the underlying mesophyll cells retained their normal shape

and were not as heavily stained as the cells of the subepidermal

layer (Figure 17 8).

In cleared leaf sections a yellow material was observed inter-

cellularly throughout the diseased tissue. Collapsed cells appeared

yellow to brown before staining, and stained a dark blue with cotton

blue, and deep red with safranin (Figure 18 C)

Leaflets Inoculated on the upper surface showed collapsed

palisade cells after 3 days, mesophyll Invasion occurred after 9 days,

and a total collapse after 2 weeks.

Highly resistant leaflets inoculated at Stage 11 had similar

reactions as Stage I leaflets, except that cell collapse was not as

rapid or as extensive. Fungus growth appeared to be restricted to

the subepldermal layer for a longer period of time.

The striking differences between highly resistant and





































B
Figure 17. Cross section of a P-122
highly resistant leaflet showing (A) A lati-
ciferous cell, (B) A black exudate near an
Infection point and the complete surrounding
of the subepidermal layer of cells.


Ic\
















"".



B
4.
o-ff






A














C D

Figure 18. Photomicrographs of resistant host reaction to In-
vasion of D. ulei. (A) Collapsed mesophyll and palisade layers showing
necrotic and disorganized cells. (B) Cross section of a lesion from a
highly resistant leaflet showing sclerenchyma-like cells. (C) Yellow
material observed near mycelium of invaded resistant cells. (D) Cells
with a granular yellow appearance in the center of the lesion, and dark
sclerenchyma-like cells found in the perimeter of the lesion.










susceptible clones were the lack of conidiophores and conidia produc-

tion, the absence of leaf distortion, and the failure of the fungus

to produce stromatic tissues in the highly resistant clones

(Figure 18 A).

Six months after inoculation, mesophyll tissue appeared to be

normal, and only cells near the point of Infection had a granular

yellow appearance. Cells near the center of the lesion were dark,

tightly packed and associated with sclerenchyma tissue (Figure 18 D).

Leaves inoculated at Stages III, IV, and V also show these

types of cells, which appeared in a circular pattern surrounding a

point of infection, or scattered throughout the leaflet (Figure 18 B).

Cells adjacent to the sclerenchyma-like tissue and in the subepider-

mal layers were impoverished. The yellow material observed in younger

lesions was not observed in older lesions.

Slight depressions were observed on the outer perimeter of

the lesion, but generally there was no alterations of cellular

structures observed.

On the upper surface of the leaflets, epidermal and palisade

layers were normal in shape and yellowish-brown. Cells adjacent to

the palisade layer contained impoverished chloroplasts. In some cases

large lesions were observed to have parenchyma-ilke palisade cells,

instead of normal palisade cells.

No stromatic structures or fungus fructifications were ob-

served in the lesions of almost Immune leaflets. No specific mechan-

Ical tissues were observed to be formed In resistant and highly re-

sistant leaflets.





52



in highly resistant leaflets Inoculated in Stages II to VI

no large development of a lesion was observed. Generally epidermis

penetration occurred, but no further fungus development was observed.

in Stage III leaves, a few cells appeared to be impoverished In a

small area surrounding the point of infection but did not stain with

safranin.












BIOCHEMICAL STUDIES


In early microchemical work Rawlins (22) stated that micro-

chemical methods could be applied to identify resulting compounds

from fungi grown in synthetic culture media, as well as in the cells

of the pathogen or host.

With the rapid advances being made in the field of chromatog-

raphy and by use of newer ion exchange resins for the purpose of con-

centrating the samples, small volumes of plant extracts may be used

to find significant differences between healthy, diseased, resistant,

and susceptible plants.

Kuc et al. (14) studied the production of funglstatic agents

by potatoes In response to Inoculation with Helminthosporlum carbonum.

They prepared ethyl alcohol extracts of healthy and diseased potato

peel and pulp tissues. Part of the extracts were used for chromatog-

raphlcal analysis and part were added to potato-dextrose agar media.

Fife (6) found through the use of paper chromatography strik-

ing differences in the relative concentrations of certain amino acids

in the juices expressed from healthy and diseased beet leaves, and in

the phloem exudates collected from healthy and diseased sugar beet

roots.

Zscheile and Murray (32), using paper chromatography, found

specific differences of the amino acids and amounts present in wheat

ovules in relation to genes for disease resistance.

Blazquez and Owen (2) grew the fungus D. ulel on media prepared

53








from water extracts of Hevea brasillensis Muell. Arg. leaves. They

reported that both 1-inositol and quebrachltol (mono-methyl ether of

I-Inositol) greatly favored fungous growth In small concentrations

(100 mg. per liter), but that at high concentrations (above 400 mg.

per liter) no fungous growth was obtained. They stated that there

may be a relationship between the toxic effects of high concentration

of both compounds and disease development. Quebrachitol is present

In the latex of species of Hevea in concentration of 0.5 to 2.0 per

cent, and this high concentration of quebrachitol might account for

the susceptibility of young Hevea leaves to D. ulel, whereas older

leaves are immune or only slightly susceptible.

Resing (23) found that quebrachitol was only found in the

water-soluble impurity fraction of latex lipids. Bolle-Jones (3) in

the determination of sugars present in the laminae of Hevea

braslliensis found glucose, fructose, sucrose and relative large

amounts of i and 1-inosltol, but not quebrachitol.


Media Containing Quebrachitol

In previous nutritional experiments where media were prepared

with quebrachitol (2-mono-methyl ether of 1-inositol) as a vitamin

source, fungal growth was obtained In concentrations of 25 to 50 mg

of quebrachitol per liter. Using chromatographic methods it was de-

termined that the quebrachitol used in early experiments was not pure

and that It contained small amounts of 3- and 1- forms of Inositol.

It was therefore necessary to determine which of these 3 compounds,

pure quebrachitol, i-lnositol, or d-inosltol, were utilized by the










fungus. An experiment wras set up in which contaminated quebrachitol

(with I- and d-inositol), pure quebrachitol, d-inositol,2 quc-
2
brachitol, and i-inositol were used as the only vitamin source in a

semi-synthetic medium and galactose was used as the carbohydrate

source. The semi-synthetic media was prepared as in previous experi-

ments (2), with the vitamin source being used at a concentration of

20 mg per liter, and the carbohydrate source at a concentration of

10 g per liter. The media was tubed, plugged and autoclaved.

On contaminated quebrachitol media black stromatic tissue de-

veloped on the surface of the slant with some olivaceous mycelium

growing along the periphery of the colony. Conidial formation was

moderate with fair spermogonial development. The average diameter of

the colonies was 7 mm, and the average height was 5 mm after 6 months

(Figure 13).

On pure quebrachitol medium the fungus produced white myce-

lium from the black stromatic tissue on the surface of the medium.

Black stromatic tissue, either superficial or partly submerged grew

on the medium. Conldial and spermogonlal production was moderate.

Colonies grow to 5 mm In diameter, and 6 rm In height within 6 months.

On the quebrachitol medium dark-brown to black stronatic myce-

lium grew on the surface of the medium, with whitish mycelium growing


Samples of contaminated and pure quebrachltol were obtained
from Dr. 0. D. Cole of the Firestone Tire and Rubber Company, Akron,
Ohio.

2Samples of d-inosltol and quebrachitol were obtained from Dr.
H. J. Teas of the University of Florida, Agricultural Experiment Sta-
tion, In Gainesville, Florida.






































Figure 19. Six month old cul-
tures of D. ulel comparing growth in
media from (left to right) contaminated
quebrachitol; pure quebrachttol (Fire-
stone Co.); quebrachitol (Calif.);
d-inositol; with 1-inosltol as control.









on the surface of the stromatic mass of hyphae. Conldial and spermo-

gonial formation was very abundant. Colonies grew to 3 mm in diameter,

and 3 mm in height after 6 months.

Dark-brown to black stromatic mycelium grew on the surface of

the d-inositol media, with white mycelium growing from the periphery

of colonies. Conidial and spermogonial formation was fair.

On the i-inosltol medium mycelium developed on the surface

and grew deep Into the medium. Conidial and spermogonial formation

was fair. The fungus colonies were compact and small. They grew to

3 mm in diameter and 2 mm In height within 6 months.


Media Containing Latex Serum

Resing (23) In experiments with phosphatides from fresh un-

ammoniated latex found that quebrachitol was not present In the

alcohol-soluble fraction and was only able to detect it in the water-

soluble fraction of the latex. Smith (27) reported that quebrachltol

was present only in the water-soluble part of latex.

An experiment was devised to determine the possible presence

of growth-increasing substances (such as quebrachltol) in the water-

soluble fraction of the latex (serum). Serum from latex (Firestone

S-4) was obtained by a process of super-filtration as follows: A 3-

foot length of 1 1/8 In. wide cellophane dializing tube was soaked in

water for 10 minutes. One end of the casing was tied, the tube filled

with latex and the 2 ends tied together. The loop was then hung over

a glass rod and placed inside a bouyoucous cylinder. The casing was

kept moist by placing a large beaker over the cylinder to maintain









high humidity. As the rate of filtration decreased, the casing was

kneaded gently to remove a pasty deposit from Its walls.

The 226.5 ml of serum obtained by super-filtration from 1.35

liters of latex was transferred to a flat glass dish inside a larger

beaker containing IN sulfuric acid. Both beakers were covered with

a glass jar to remove any remaining ammonia from the serum portion of

the latex. The serum fraction was removed from the flat dish with the

sulfuric acid when ammonia fumes could no longer be detected. It was

then divided Into 2 fractions, a 100 ml fraction and a 140 ml fraction.

The 100 ml fraction was used to prepare media with 5 different con-

centrations of serum and was sterilized by autoclaving at 15 lbs per

square Inch of pressure for 30 min. The 140 ml fraction was used to

prepare media using 6 concentrations of serum and was sterilized by

filtering through a sterile Seltz filter. In the first serum frac-

tion, the serum was mixed with water agar in proportions which reduced

the volume of serum from 50 ml to 1 ml per 100 ml of medium at inter-

vals of 25, 10, 5 and 1 ml. No additional nutrients were added to the

serum media. In the second fraction, the water agar in the desired

concentrations, and the necessary glassware to mix and measure the

media were washed with tap water, rinsed twice with distilled water

and autoclaved. The mixing of sterile solutions was done in a trans-

fer chamber under aseptic conditions, and the Erlenmeyer and graduated

cylinders used were flamed before and after pouring the serum. Solu-

tions were made up to volume, mixed, tubed, plugged and slanted.

Conldia seeded on media containing concentrations of 1, 5, and

10 ml of serum germinated readily, but on the 25, 40 and 50 ml









concentrations no germination was observed. In the 1 ml concentration

mycelium grew from the surface of the stromatic tissue on the test

medium. Olivaceous mycellum developed on the surface of the media and

on the surface of the stromatic tissue. Conidlal production was fair

and spermogonial production was moderate. Colonies greu to 1 mm in

diameter after 50 days, and to 5 mm after 300 days.

In the media prepared from autoclaved serum no fungus growth

was obtained In concentrations above 10 ml of serum per liter.

Media with 10 ml of serum per liter produced light-brown to

yellow pseudo-stromatic tissue in 50 days. Black stromatic tissue

grew on the surface of the medium with olive mycelium developing on

the stromatic tissue. Conldial production was fair. Spermogonial

production was moderate. Colonies grew to 5 mm after 300 days.

Media prepared with I and 5 ml of serum gave similar Fungus

growth up to the 100 ml per liter concentration. Conidia germinated

more readily and growth appeared to be more vigorous. Conidlal pro-

duction was fair and spermogonlal production was moderate. Colonies

grew to 5 mm after 300 days.


Effects of Carbohydrates on the Growth of the Fungus

Carbohydrates are of extreme importance In the nutrition of

fungi as they are the main sources of carbon. Almost half of the dry

weight of fungous cells consists of carbon. Protoplasm, enzymes, the

cell wall, and reserve nutrients stored within the cells are compounds

of carbon. In addition to being the main structural elements, carbon

compounds play an equally important functional role.








Various fungi possess different abilities to use carbohydrates

according to Wolf and Wolf (31). Lilly and Barnett (17) stated that

the most common source of carbon reported was glucose. lost species

generally gave better yields on hexoses than on pentoses, although

Hawkins (9) found that Glomerella cingulata utilized 2 pentoses,

arabinose and xylose.

Moore (19) reported that certain pathogens possessed wide

capabilities for utilizing carbohydrates, whether mono-, dl-, or

polysaccharides. She determined that Phymatotrlchum ormlvorum used

glucose, fructose, galactose, maltose, sucrose, lactose, and mannitol.

An experiment was designed to test the effect that various

sugars might have on the growth of D. ulei. The sugars were tested

in a basal semi-synthetic media which was prepared as follows: Carbon

source, 10 g per liter; asparagine, 2 g; KH2P04, 1 g; Mg" (MgSO *

7H20), 0.5 g; Fe" (Fe2SO XH20), 0.2 mg; Zn' (ZnSO4 7H20), 0.2

mg; Mn" (MS04 H20), 0.1 mg; biotin, 5 ug; thiamine, 100 ug; 20 g

of agar; and distilled water to make 1 liter. One-half liter of dis-

tilled water was first warmed and the various Ingredients added until

dissolved and made up to 1 liter. The carbohydrates tested and the

concentrations per liter of the stock solutions were as follows: d-

Mannltol, 75 and 150 g; d (/) Mannose, 75 and 150 g; d (/) Maltose,

75 and 150 g; d (/) Galactose, 75 and 150 g; 1-Sorbose, 75 and 150 g;

d (Q) Xylose, 75 and 150 g; d (/) Arabinose, 75 and 150 g; d (/)

Lactose, 75 and 150 g; d (/) Raffinose (Hydrate), 75 and 150 g.

Blazquez and Owen (2) studied the effects of vltamln-mlno acid

combinations on growth of D. ulei. They determined that I-inosltol









- glutamic acid; nicotinic acid glycine; riboflavin glycine; and

p-amino benzoic acid were the best combinations for fungous growth.

With this in mind the following combinations of vitamins and amino

acid, and concentrations per liters in 100 ml of stock solutions were

prepared: I-inositol, 1.2 g; p-amino-benzoic acid, 15 mg; nicotinic

acid, 600 mg; riboflavin, 15 mg; 1-glutamic acid, 441.4 mg (added to

the I-lnosltol stock solution); glycine, 222.5 mg (one lot added to

the nicotinic acid stock solution and another added to the riboflavin

stock solution). Vitamin-amino stock solutions were made up to 2.7

liter lots and 60 g of agar were added to each lot.

It was necessary to have 2 aliquots from each 2.7 liter lot

since 2 concentrations of sugars were to be tested. Therefore each

lot was separated Into 2 aliquots of 1350 ml. From each aliquot 135

ml were placed into ten 250 ml Erlenmeyer flasks. Fifteen ml from

each 75 and 150 g concentrations of sugar stock solution was added to

the 135 ml Erlenmeyer flasks of the vitamin-amino acid aliquots. Thus

the total volume in each flask was 150 ml, and the final concentrations

of sugars were 5 and 10 g per liter. The final concentrations per

liter of the vitamin-amino acid combinations were: i-inositol 400 mg;

p-amino-benzoic acid, 5 mg; nicotinic acid, 200 mg; riboflavin, 5 mg;

1-glutamic acid and glycine, 21 mg of nitrogen equivalents. One small

lot of 150 ml of each vitamin-amino acid combination without sugars

was used as controls. The prepared media were then tubed, plugged,

sterilized, slanted and stored at 40 C. When conidia became available,

10 tubes of each sugar-vitamin-amino acid combination were planted and

seeded with conidla. Germination readings were taken periodically and








compared with those of other media previously prepared. After conid-

ial germination a small section of medium with germinating conldia

was then transferred to slants of the same sugar medium. These slants

were then incubated at 200 220 C. In order to evaluate the growth

of conidial and spermogonial development after 80 and 280 days, an

arbitrary numerical rating was used, with 4 being maximum and 0 min-

imum development.

Response of the fungus to the various sources of carbon

tested in basal semi-synthetic media varied greatly. There was no

significant difference between the vltamin-amnlno acid combinations

riboflavin-glycine; nicotinic acid-glyclne; and p-amino benzoic acid.

Only the combination I-Inositol-glutamic acid gave excellent growth

with all the carbon sources tested (Table 5).

The pentoses, arabinose, xylose, and sorbose gave fair growth,

while the hexoses, maltose, galactose, mannose, and the sugar alcohol,

mannitol gave the best growth (Figure 20). The discharlde, lactose

and the trlsaccharide, raffinose gave intermediate results between the

pentoses and hexoses (Figure 21).


Leaf Extract from Hevea Spp.

Leaf extracts for susceptible, resistant, and highly resist-

and Hevea brasiliensis, and from H. benthamiana were prepared with

distilled water and also with 80 per cent ethyl alcohol in a soxhlet

apparatus. The extracts were prepared as follows: 2 g of leaf lam-

inae were cut in small squares, placed in a No. 1 Whatman extraction

thimble and the thimble inserted into the soxhlet extraction tube.

One hundred ml of water were poured into an extraction flask, glass





63



TABLE 5

EFFECT OF VITAMIN AND AMINO ACID COMBINATIONS WITH
SUGARS ON THE GROWTH OF D. ULEI

Vitamin-amino Spermogonial Conidlal Av Diameter
Acid Plus Sugar Formation Productiona of Colony (mm)
Medium AD Bc Ab Ab Bc
5 10 5 10 5 10

I-Inositol-I glutamic
acid plus 1
Mannitol 1 I 2 3 7 5
Mannose 3 2 3 0 12 13b
Maltose 1 0 2 1 10d 10
Galactose 3 4 2 1 10 10
Sorbose I 2 6
Xylose 3 0 2 3 6 5
Arabinose 0 0 I 1 6 3
Lactose 1 2 2 2 8e 10
Rafflnose 0 0 2 0 7 5
Control 2 2 3 3 8 8

P-amino benzoic acid
plus
Mannitol I I 0 1 1 1
Mannose 1 2 0 0 6 3
Maltose 3 3 3 0 5 5
Galactose 0 1 0 0 2 3
Sorbose -
Xylose 0 0 0.1
Arablnose 0 0 0 0 0.1 0.1
Lactose 3 2 0 3 3 3
Raffinose 3 3 0 3 5 3
Control 2 2 3 3 5 5

Nicotinic acid-
qlycine
plus
Mannltol 0 1 0 I 1 2
Mannose 2 2 0 1 3 3
Maltose 3 3 3 3 6 6.5
Galactose I 1 1 1 5 4
Sorbose -
Xylose 1 0 0.1
Arabinose I I -
Lactose 3 2 3 2 2 2
Raffinose 3 0 0.5
Control I 1 3 3 4 4








TABLE 5--Continued


Vitamin-amino Spernogon al Conldlal Av. Diameter
Acid Plus Sugar Formation Productiona of Colony (mm)
Medium A5 B 10 Ab5 c0 A 5 0
5 10i0 5i 5

Riboflavin
plus
Mannltol 2 2 2
Mannose 1 2 0 1 5 3
Maltose 2 1 3 3 5 5
Galactose 0 2 0 2 2 3
Sorbose 0 0 2
Xylose 1 0 -
Arabinose 2 0 2 1 2 2
Lactose 1 0 3 3 4 4
Raffinose 1 2 0 1 2 2
Control 1 1 1 1 3 3


aArbltrary numerical
and conidial formation: 4,


rating for percentage of spermogonial
above 75 per cent; 3, 50-75 per cent;


2.25-50 per cent; 1, below 25 per cent; 0, none.


bMedia prepared with 5 g of sugar.

CMedia prepared with 10 g of sugar.




















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beads were added, and the apparatus was assembled. The extraction

process was of 1 hour duration. Alcohol extracts were made in the

same manner. To the water extracts were added 5 g of agar and 0.5 g

of malt extract, the extract solution was then made up to 250 ml. In

the case of the alcohol extracts, it was necessary to reduce the vol-

ume to about 30 ml, water was then added to make 100 ml, the agar and

the malt extract added and the solution made up to 250 ml. The leaf-

extract media were tubed, plugged, autoclaved at 15 lbs of pressure

for 30 min. and then stored until conidia became available for

inoculation.

Susceptible leaf extract media.--Extracts were made and tested

from a Far Eastern clone, TJir 1 x Tjir 16, using 6 month old dry

leaves; 36-day-old diseased leaves which had been inoculated when they

were 6 days old.

Water extract from 36-day-old diseased leaves.--Black stro-

matic mycelium grew on the surface and deep In the medium. Olive and

white mycellum were formed on the surface of the stromatic mycellum.

Conidlal and spermogonlal production was good. Colonies grew to a

diameter of 8 mm In 6 months (Figure 22).

Alcohol extract from 36-day-old diseased leaves.--Black stro-

matic mycellum was produced on the surface of the medium, with pseudo-

stromatlc tissue growing deep in the medium. Long thin hypha were

produced from the black stromatic mycellum, with small patches of

cottony-white mycellum In some areas. Abundant conidlal and sper-

mogonlal productions were observed. Spermatia oozed out of the sper-

mogonla in small droplets. Within 6 months colonies grew to 10 mm in































Figure 22. Six-month-old cultures of
D. ulel comparing growth In media prepared from
Hevea leaf extracts (left to right) ethyl alcohol
from young, diseased susceptible leaves; water
extract from young, diseased susceptible leaves;
water extract from FX 232 leaves; water extract
from young P-122 leaves; water extract from 6-
month-old leaves; water extract from 2-month-old
F 4542 leaves of an H. benthamiana clone.









diameter, and 4 mm In height (Figure 22).

Water extract from dry leaves.--Light-brown to yellowish myce-

Ilum was produced on the surface as well as deep in the medium. In

some colonies dark-brown pseudostromatic tissue grew on the surface

of the medium. No conidial or spermogonial production was observed.

Colonies grew to a diameter of 0.01 mm in 80 days.

Dry leaves alcohol extract.--Light-brown to yellowish pseudo-

stromatic mycellum was formed. Many colonies did not grow. No conld-

lal and spermogonlal production was observed. The average diameter

of the colonies was 70 u after 80 days.

Resistant leaf extract media.--Leaf extracts from a resistant

clone FX 232 were made with distilled water and 80 per cent ethyl

alcohol.

On water extract, olive and black stromatic mycelium grew on

the surface, with dark pseudostromatic tissue forming deep In the

medium. Conidlal and spermogonial production was good. The average

diameter of the colonies was 7 mm after 80 days (Figure 22).

Highly resistant extract media.--Water and 80 per cent ethyl

alcohol extracts were prepared from 4-day-old leaves, healthy 6-month-

old leaves, and diseased 6-month-old leaves of P-122, a highly resist-

ant clonal selection of Hevea brasiliensis Muell. Arg.

Water extract from 4-day-old leaves.--Light-brown to black

pseudostromatic masses of cells grew on the surface and deep In the

medium. Colonies appeared very flat, without any dumped stromatic

masses. Conldlal and spermogonial production was fair. Colonies grew

to 13 mm In diameter, and 5 mm In height after 6 months.









No growth was observed on media prepared from alcohol extract

of 4-day-old leaves (Table 6).

Water extract from healthy 6-month-old leaves.--Olive mycelium

grew on the surface of the slant, with black stromatic mycelium In the

center of the colony. The stromatic mycellum grew deep into the me-

dium. Conidial production was good. Spermogonial production was good.

Colonies grew to 7 mm In diameter, and 6 mm in height after 6 months.

Alcohol extract from healthy 6-month-old leaves.--Light-brown

pseudostromatic mycelium grew on the surface and deep in the medium.

No conidlal or spermogonial production was observed. The one colony

grew to 1 mm in diameter after 6 months.

Water extract from diseased 6-month-old P-122 leaves.--Olive

and black mycelium were formed on the surface of the medium. Black

stromatic tissue grew only on the surface of the slant. Conidial and

spermogonial production was abundant, and droplets of spermatia oozed

out of the spermogonla. Colonies grew to 7 mm in diameter within 6

months.

Alcohol extract from diseased 6-month-old leaves.--Light-brown

to yellowish pseudostromatic tissue was developed on the surface of

the medium. Fungal growth in this medium was scant and was not vig-

orous. No conidial or spermogonial production was observed. Colonies

grew to 1 mm in diameter within 6 months (Table 6).


Media Containing Hevea benthamiana Leaf Extract

Water and 80 per cent ethyl alcohol leaf extracts were prepared

from 6-month- and 2-month-old F 4542 leaves of an H. benthamiana Muell.

Arg. clone.









TABLE 6

EFFECT OF ETHYL ALCOHOL AND WATER EXTRACTS
ADDED TO MALT AGAR MEDIA


Spermogonlal Conldlal Av. Diameter
Extract Type Frmationa Formationa of Colony (mm)
A" B A0 B Ab B


Hivca brasllensis Muell. Arg.
Diseased 36-day-old
susceptible leaves


Dry 6-rnnth-)ld
leaves

Resistant F 232
6-day-old leaves


Highly reslstant P-122
4-day-old leaves

Highly resistant P-122
6-nmnth-old leaves

Diseased highly resistant
P-122, 6-month-old leaves

Hevea benthamlana Muell. Arg.
F 4542 2-month-old
leaves

F 4542 6-month-old
leaves

Control


3 3 3 10


2


4


- 3


2



2


-13


aArbltrary numerical rating for percentage of spermogonlal
and conldial formation: 4, above 75 per cent; 3, 50-75 per cent;
2, 25-50 per cent; 1, below 25 per cent; 0, none.

bAlcohol extract.


CWater extract.









Water extract media from 2-month-old leaves.--Black stromatic

tissue was formed on the surface of the medium. The medium surround-

ing the stromatic tissue was reddish-black in contrast to the light-

red color of the remainder of the medium. Deep in the medium, oliva-

ceous mycelium formed large clumps of pseudostromatic tissue. Long

thin hyphae were observed on the surface of the stromatic tissue.

Conidlal formation was moderate. Spermogonial formation was abundant.

Colonies grew to 6 mm in diameter and 5 mm in height within 6 months

(Figure 22).

Alcohol extract from 2-month-old leaves.--Light-brown to yel-

low clumps of mycelium grew on the surface and deep in the medium.

No conldlal or spermogonlal production was observed. Colonies grew

to 1 mm In diameter within 6 months.

Water extract from 6-month-old leaves.--Conidial seeded on

this medium germinated readily but produced no growth (Table 6).

Alcohol extract from 6-month-old leaves.--Light-brown to yel-

low clumps of pseudostromatic mycelium were observed on the surface

of the medium. No conidlal or spermogonlal production was observed.

Colonies grew to 1 mm in diameter In 6 months.


Media Containing Rubber Latex

An experiment was conducted to determine the presence of a

possible fungus growth source In Hevea latex.

Fubber latex Firestone S-41 (with an approximate rubber


IFurnished through the courtesy of Dr. 0. D. Cole of the Fire-
stone Tire and Rubber Company, Akron, Ohio.









content of 61 per cent dry rubber, and 62.5 per cent solid content),

preserved in 0.7 per cent ammonia was placed in a beaker Inside a

glass plate with IN sulfuric acid to remove the ammonia preservative.

After 2 days, when the ammonia preservative odor was almost gone, the

latex was mixed with water agar in proportions which reduced the

weight of dry rubber and solid content from 56.25 to 0.625 g per liter.

No growth was observed in any of the concentrations tested.

The conidia germinated very slowly in the low concentrations while

at higher concentrations, from 15.626 g to 56.25 g, no germination

was observed. The conidia formed long germ tubes in the typical man-

ner. At 2 and 4 weeks after seeding the conidia no further growth

was observed in any of the concentrations.


Microchemical Studies

Microscopic difference observed between fungus Invaded sus-

ceptible and resistant leaves was the abundant production of a yellow

substance soon after fungus penetration of resistant leaf tissue and

in old lesions of susceptible tissue. There was also observed the

presence of sclerenchyma-type cells in lesions of old resistant leaf

tissue. The chemical nature of the yellow material and of the scle-

renchyma-type cells was studied with the aid of microchemical methods.

Microchemical tests for glucosides, hemicellulose and lignin

were made with fresh and cleared leaf tissue.


Glucosides

The presence of saponins and tannins was studied following

Johansen's (11) tests. Saponin-cleared sections of diseased resistant









leaflet tissue were placed on a slide with 2 drops of concentrated

sulphuric acid. The sections changed from yellow to red, and ulti-

mately violet. The sections were then placed for 24 hrs. In a barium

hydroxide solution, washed in a weak aqueous solution of calcium chlo-

ride, and changed to a 10 per cent aqueous potassium bichromate. The

yellow material in diseased young leaflet tissue gave no reaction and

showed no change of color. The sclerenchyma cells gave a negative

saponin test, as they remained brownish-red. Johansen (11) stated

that tannin-containing cells became brownish-red during the reaction.

Tannin.--Fresh and cleared sections of fungus-invaded sus-

cepLille and resistant leaflet tissue were placed on a slide with a

10 per cent ferric chloride solution to detect the presence of tannins

following Johansen (11). The yellow material present In young dis-

eased resistant leaves near Infection sites did not stain with ferric

chloride. The sclerenchyma-type cells present in old diseased resist-

ant leafletsturned blue-black with ferric chloride. No blue, blue-

green, or black color was observed in lesions of young and old dis-

eased susceptible leaflets. The yellow material observed near the

old lesions gave a negative reaction with ferric chloride.


Hemicelluloses

Hemicelluloses are considered to be those compounds that yield

pentosans, galactose or mannose upon hydrolysis. Johansen (11) stated

that there are 2 kinds of hemicelluloses: (A) Those entering into the

constitution of permanent cell walls, and (B) those occurring in

storage organs.









The only hemicelluloses studied were those entering into the

constitution of cell walls.

Xylan, Araban.--The phloroglucln-hydrochloric test was used

to determine the presence of arabans and xylans. None of the fresh

and cleared leaflet pieces gave the cherry-red color typical of xylose,

arabinose.

Methyl Pentoses.--Fresh and cleared leaf sections of diseased

susceptible and resistant leaflets were placed on a slide with 2 drops

of acetone, 1 drop of concentrated hydrochloric acid was added, and

the sections warmed gently for 15 minutes. Sections of susceptible

leaflets gave negative results, as no cherry-red color was observed

in the yellow material. Resistant leaflet sections gave a cherry-

red color first and changed to dark brown-red later.


Liqnin

Fresh and cleared sections of Invaded susceptible and resist-

ant leaflets were tested for the presence of lignin using an ammonlcal

silver nitrate solution. None of the sections gave the typical black

color of the reaction.

Microchemical test of the abundant yellow material found near

lesions of young diseased resistant leaflets gave negative results.

ScIerenchyma-type cells observed In pin-point lesions of old diseased

resistant leaflets gave a positive test for tannins, and methyl

pentoses.









Chromatographlcal Studies

Extracts were prepared from susceptible, resistant, healthy

and diseased leaves of H. brasillensis and healthy H. benthamlana

leaves and were assigned numbers arbitrarily.

Chromatograms were prepared with quebrachitol, d-inosltol,

and i-inositol as references, and the prepared extracts spotted on

No. I Whatman filter paper and developed In 4 solvent systems follow-

ing Lederer and Lederer (16).

Solvent systems.--The best preparation of spots was obtained

with the following solvent systems v/v: n-propanol-acetlc acid-water

(A), 7:1:2; n-propanol-acetic acid-water (B), 6:2:2; and phenol-water

(C), 8:2. Spots were the sharpest with a n-butanol-ethanol-water (D),

10:1:2 solvent.

Dipping agents.--Various dipping agents have been used to

detect the presence of sugars in chromatograms. Resing (23) used a

strong ammonlacal silver nitrate to detect both sugars and alcohols.

For sugars detection only he used aniline-trichloroacetate. Smith

(27) in 1954 used the method described by Trevelyan (28), which is

based on Feigl's test for reducing sugars. Bolle-Jones (3) used

aniline phthalate and naphthoresorsinol In a hydrochloric phosphoric

acid mixture as dipping agents.

The general treatment used for the detection of carbohydrates

on the chromatograms was a modification of Trevelyan's reagents (28).

The first reagent solution was prepared by diluting 0.1 ml of satu-

rated aqueous silver nitrate solution to 20 ml of acetone. The aqueous

silver nitrate was added dropwise with shaking, until the silver









nitrate which separates on addition of acetone had redissolved.

Spreading of the spots was limited due to the sparing solubility of

sugars In acetone (0.014 per cent at 230 C. for crystalline glucose).

The second reagent, used also as a dipping agent Instead of a spray,

was prepared by dissolving 20 g of sodium hydroxide in 1 liter of 80

per cent ethanol (v/v). Upon dipping in this reagent, brown silver

oxide was Immediately produced.

Development procedure.--One dimensional chromatograms were

prepared by placing a total of 5 ul (1 ul at a time, allowing the

drops to dry, and repeating 5 times) along a line 2.5 cm from one end

of a 20 cm long and 29 cm wide street of No. 1 Whatman filter paper.

Ascending chromatograms were run In 1 gallon wide-mouth Jars, covered

with flat square pieces of glass, and vaseline on the rim of the bot-

tles to form an air-tight seal.

The solvents A, B, C, and D were placed In a gallon Jar and

allowed to stand for 4 hours. Prepared chromatograms were rolled so

that they would stand as a cylinder, clipped together at the uppermost

edge with paper clips and placed in the Jars so that the bottom edges

of the cylinder would not touch. The chromatograms were allowed to

run at rocm temperature (240 C) until the solvent fron reached a 1 cm

distance from the top edge of the paper. The chromatograms were

allowed to dry between dippings.

The chromatograms were first dipped continuously in the

aqueous acetone solution for about 3 minutes. After allowing to dry,

they were dipped In the ethanolic sodium hydroxide solution until the

spots became dark brown, taken out and rinsed in tap water before the









background became light brown. Rinsing of the chromatograms In tap

water was followed by dipping In a 50 per cent diluted solution of

sodium thiosulfate. The sodium thiosulfate was washed off with run-

ning tap water and the chromatograms dried and stored in the dark.

Preparation of leaf extract.--The following procedure was

found satisfactory for the preparation of leaf extracts suitable for

both the chromatographic examination and quantitative estimation of

the sugars present. Two g of fresh laminae were soxhlet extracted

with 120 ml of 80 per cent ethyl alcohol for 2 hours. The extract

was evaporated under reduced pressure at 30-400 to approximately 10

ml. This volume was partially clarified by filtering through dia-

tomaceous earth (Cellte), and made up to a volume of 50 ml with water.

Twenty-five ml of this solution was concentrated under reduced pres-

sure to a volume of 1 ml, and the remaining 25 ml were transferred to

a screw-cap vial and stored at 0 C.


Inosltol and Quebrachitol Determination

In early tests, It was determined that the quebrachltol used

by Blazquez and Owen (2) In previous nutritional studies of Dothidella

ulel P. Henn was not a pure compound, but that It contained large

amounts of i-lnositol and an unknown compound. It was suspected that

because of the close Rf values of the unknown and i-inositol, the com-

pound might have been the d- or I-enantiomorph of I-inositol.

The d-enantlomorph of 1-inositol and a purified form of que-

brachitol were obtained from the California Corporation for Biochemical









Research in an attempt to Identify the impurity found In the que-

brachltol used previously. An additional purified form of que-

brachltol was obtained from the Firestone Tire and Rubber Company.2

Solutions were prepared in concentrations of 4 mg per ml of

the 3 quebrachltol compounds: impure quebrachitol, purified que-

brachitol (California Corp.), purified quebrachitol (Firestone Co.);

and of the d- and I- forms of inositol. One dimension chromatograms

were run in solvents A, B, C, and D. In all the solvents tested, it

appeared that the unknown impurity of the first quebrachitol used was

d-inosltol, as the Rf values of the impurity and d-inosltol were the

same.

Chromatographical determinations of leaf extracts.--Chromato-

grams prepared for the detection of possible differences between leaf

extracts were run twice, with drying In between following Crossan and

Lynch (4). Unidirectional chromatograms of leaf extracts from Hevea

clones with varying degrees of resistance showed no significant dif-

ference In d- and i-inositol. The extracts from healthy and diseased,

resistant and susceptible, showed the presence of both d- and I-

Inositol (Figure 23). The Rf values for d- and 1-inositol are shown

in Table 7. The presence of quebrachitol could not be determined as

other sugars spots overlapped the area where the quebrachitol reference

was found. The Rf values of quebrachitol are shown In Table 7.


ICalifornia Corporation for Biochemical Research, 3625 Medford
Street, Los Angeles 63, California. Courtesy of Dr. H. J. Teas.

2Courtesy of Dr. 0. D. Cole. Firestone Tire and Rubber Com-
pany, Akron, Ohio.









5


0
0


O


Qo0
O C9


0


d-i
&I-9D


,a


0
0)


15 20 7 Q d-i 12 10 I
Figure 23. Chromatographic pattern of compound 5, which was
present in extracts from healthy leaves (15, 20, and 7) but not on
those from diseased leaves (12, 10, and 1). Known standards are of
quebrachltol (Q), d- and i-inositol (d-i, 1-1). Paper, Whatman No.
1; solvent, n-propanol-acetlc acid-water, 6:2:2.


0.7


04


0.2
0.1



































I
0

m


2
0








o

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r z
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o,
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00
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r


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-4








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ro\ a a o


















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00 0

U C I
0000








ci -
O O
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In unsprayed chromatograms a yellow spot (compound 1) was

detected in extracts of young leaves regardless of their degree of

resistance (Table 8). Upon treatment with diazotized anisidine the

yellow spot gave a pink color, and under ultra-violet light, long

wave Black-ray 8-100, It acquired a light absorbing dark-brown color

(Figure 24).

Under ultra-violet light and ammonia fumes 3 blue fluorescent

spots were observed. They were numbered 2, 3, and 4 in order of in-

creasing Rf value. Table 8 shows the Rf value of the 3 fluorescent

compounds. Extracts of young susceptible leaves showed the presence

of the 3 compounds, while young resistant leaves did not. The 3 com-

pounds did not react with diazotized anisidine or with aqueous sliver

nitrate in acetone (Figure 25).

Chromatograms prepared from extracts of diseased and healthy

leaf extracts showed a spot at Rf, 0.6 to 0.7, present only in healthy

leaf extracts. The spot (compound 5) did not fluoresce under ultra-

violet light, and did not react with diazotized anisidine. Upon treat-

ment with the silver-acetone reagent, the spot reacted with the silver

to give a dark-brown spot. Rf values for compound 5 are given in

Table 7. Trevelyan (28) reported that reducing sugars will reduce

sliver to silver oxide to form a dark-brown spot. It is possible that

compound 5 may be a reducing sugar.



























U-

0




t>
_J



-I
w




0



ci




I/
-J









0


c_
z





0
.3


0
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0
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:3
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-J

.4-


u ---


0 ..
4


0I

CL



C-











U-
c
Q. I





u








. 4.
0
C








0.C
gr
oM

41

S0









N
ocx


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C


0s.


a-


m o O o
; rc oo c0
Ln 0 0 0



















o o 0 00
u' 0 0 0
Co C0 0 C
















LA 0 0 0
-0 u0 r-

o 0 0 0














0 L0 0 0
0














o C C C

E a .
o o



OL -

C13
2 e ) 0

4.---
0 3 o


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4-






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-g
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41
-0











-0





L4
















0.



0.6














11 4 20 7 5 15 18 14
a?--
04

0.

0.1





11 4 20 7 5 15 18 14

Figure 24. Chromatographic pattern of yellow compound I
present In extracts from young leaves (11, 4, 20, and 7), but not in
those from old leaves (5, 15, 18, and 14). Paper, Whatman No. 1;
solvent, n-propanol-acetic acid-water, 6:2:2.








4

0
ED


4@
3 3

0 2
02 00


II 4 20


7- 8- 6 13 14
7 8 6 13 14


Figure 25. Chromatographic pattern of fluorescent compounds
(2, 3, and 4) present in extracts from young susceptible leaves (11,
4, and 20), but not In those from young resistant leaves (7, 8, 6,
13, and 14). Paper, Whatman No. 1; solvent, n-propanol-acetlc acid-
water, 6:2:2.


0.6


0.4


0.3
0.2
0.1


0

00O
D












DISCUSSION


Inoculation experiments carried out with 4 resistant clones

of Hevea rubber did not agree with Langford's work (15). Under 100

per cent humidity It was possible to observe the rapid symptom ex-

pression of susceptible as well as resistant clones. In highly re-

sistant leaflets (Langford's Immune) small dot-like lesions were

very conspicuous 3 days after inoculation. The normal reddish-brown

color changed to yellow in areas bordering the lesions. Langford

(15) stated that lesions on resistant and highly resistant leaves

were smaller than lesionson susceptible leaves, appeared much later,

and caused less extensive damage. It is probable that he might

have observed the development of lesions at less frequent intervals

missing the similarity on young resistant leaves, in which case his

findings would agree with the results obtained herein.

Lesions on resistant leaflets were similar to the susceptible

ones the first 24 hours after inoculation, after which discoloration

of marginal areas and some distortion occurred. These observations

did not agree with Langford's findings (15).

Hypertrophy of the center of the lesions, and the initiation

of sporulation was observed in susceptible leaflets, while the

lesions on resistant leaflets showed neither.

Very young leaflets of resistant and highly resistant clones

developed normally when heavily inoculated but were shed after 3

weeks. They remained on the tree longer than the young leaflets of

86









susceptible clones. The resistant and highly resistant leaflets

were capable of inhibiting the growth of the fungus, and were able

to grow normally although heavily Infected. The resistance may be

due to a hypersensitivity of the cells to fungus invasion. Attacked

cells became devoid of chlorophyll and gradually collapsed. Cells

bordering the lesions became disorganized and appeared to be filled

with a yellow granular material.

Microchemical tests Indicated that the nature of the yellow

material was not related to lignin or tannin, as tests for both were

negative.

It is believed that extraction of the yellow material and

identification of its chemical composition might be accomplished by

extraction with selected solvents, and testing for methyl pentoses,

pentosans, and hemlcelluloses.

Resistant leaves might produce or contain a substance which

could be changed to this yellow material by fungus enzymes soon

after penetration. In susceptible leaves the formation of the yellow

material occurred later and possibly required a higher concentration

of fungus enzyme. In susceptible leaves this yellow accumulation

occurred only in lesions of old leaves.

Certain factors may be involved in the phenomena of fungal

growth inhibition in the resistant and highly resistant leaves.

Host reaction may be a possible explanation, whereby a substance

might undergo a structural change when attacked by enzymes produced

by the fungus. The changed substance may be toxic to the fungus or









to the host, preventing any further development of infection because

of supersensitivity of the host or as inhibiting growth of the

fungus. r I

It Is quite possible that the yellow material may not be

growth inhibiting, but rather a waste product which merely accumu-

lated in the tissues wherever fungus mycellum penetrated. Growth

inhibition in this case may be due to the absence of a substance re-

quired for growth of the fungus. It is also possible that growth

Inhibition In resistant leaves may be due to the presence of a sub-

stance in larger amounts than required; however on susceptible

leaves this substance may be present In smaller quantities which

would allow growth of the fungus.

Cross sections of highly resistant leaflets 6 months after

inoculation showed no collapsed cells or necrotic areas. The only

signs of fungus invasion were groups of sclerids distributed near

points of infection. The presence of these sclerids may be paral-

lel to the formation of brachysclerids as reported by Foster (7).

Tissue that collapsed after Infection might have left definite gaps

in the leaf structure. These gaps might have been filled by

parenchyma cellswhich divided to varying degrees and were transformed

into brachysclerids, thus "repairing" the broken leaf structure,

Brachysclerids are often found near wound tissue according to Foster

(7). The sclerlds found throughout the diseased resistant leaf tis-

sue gave a positive tannin test with ferric chloride.

Growth of the fungus was inhibited In media prepared from









alcohol extracts of diseased highly resistant leaves, but was

favored in media from the alcohol extract of diseased suscepti-

ble leaves. Media prepared from water extracts of highly resistant

and susceptible, young and old leaves, greatly favored growth of

the fungus. This suggested that a growth Inhibitory substance

could be extracted with ethyl alcohol but not with water. This sub-

stance may either be the product of fungus enzyme action on host cell

contents or a secretion of the host tissue as a protective effort

when Irritated by the toxin produced by the Invasion of the fungus.

It was believed that an alcohol soluble substance which inhibited

growth of the fungus had been broken down by fungus enzymes in

diseased susceptible leaves. Thus it would allow fungus growth In

media from alcohol extracts of diseased susceptible leaves.

Media prepared from water extracts would allow growth of the

fungus due to the absence of the Inhibitory substance.

Similarly it might be said that If the water soluble sub-

stance were required for growth of the fungus, then it might be

possible that media prepared from water extracts would have suffi-

cient amounts of the substance and would favor fungus growth. Al-

cohol extracts, on the other hand, would lack the water soluble

substance and would not favor fungus growth. Growth on alcohol ex-

tract media from diseased susceptible leaves may have been due to

fungus enzymatic breakdown of the water soluble compounds into an

alcohol soluble form that would favor fungal growth.

A yellow spot (compound 1) was found in chromatograms from









extracts of healthy young leaves regardless of their degree of re-

sistance. It Is believed that this compound 1 may be of a steroid

nature, and not unlike the yellow compound found by Kuc et al. (13),

and later identified as chlorogenic acid.

Some of the differences could be attributed to substance

only present during each stage of leaf development, but distinct

differences were detected between the resistant and susceptible

leaf extract under ultra-violet light. It Is believed that addi-

tional work should be carried out with the ultra-violet fluorescent

compounds 2, 3, and 4, as they may be found to be of some importance

in the study of parasitism.

It is quite possible that many of these differences between

the young and old leaflets, between resistant and susceptible clones,

and between health and disease, may or may not be involved in the

phenomena of resistance. They are important and need further con-

sideration since they may help clarify the complicated biochemical

host-parasite relationship involved in the host resistance to D.

ulei.

Two solvents were used for leaf extraction, as a conse-

quence it should be pointed out that results obtained should be

compared with results obtained from extracts by other organic

solvents.

It is felt that extracts from leaves should be prepared

with various solvents and added to natural media to test fungus

growth stimulation or inhibition. Thus it would be possible after





91



extensive experimentation to formulate a hypothesis dealing with

the nature of the host-parasite relationships between Dothidella

ulel and the Hevea rubber tree.




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