Host-Parasite Relations of the
Fungus Dothidella ulei P. Henn
on the Hevea Rubber Tree
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
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 author Is Indebted to the Firestone Tire and Rubber
Company for a Grant-in-Aid Fellowship which made this research
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.
. .. ..
. . ..
. . . .
. ... 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
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
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
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)
. . 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
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
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
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-
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
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
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
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
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,
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
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
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
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
Figure 1. Stages of develop-
ment. (1) Stage Stage (2 Stage I1, (3)
Stage IIi, (4) Stage IV, (5) Stage V,
(6) Stage VI.
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
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
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
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
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
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
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
7. Moderately susceptible: Defoliation ranging up to 50 per cent.
8. Susceptible: Severe to complete defollation--more than 50 per
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
-- = 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).
--- =Moderate sporulation: Very noticeable sporulation on the
most diseased flush of leaves,
usually some on several other
= 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
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.
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.
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
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
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
Although many cytological studies have been conducted
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
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
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
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.
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-
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
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
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
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
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
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
Figure 10. Collapsed cells in
the center of a resistant leaflet
second type was typical of resistant and almost Immune leaflets
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-
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
DEGREES OF RESISTANCE OF HEVEA CLONES TO D. ULEI
AND AMOUNT OF FUNGUS SPORULATION
According to Langford (15)
Tjir I x Tjir 16
10. Very highly
3. Highly resistant
2. Very highly
_ _____I___ __I_ _______I_ __ _______ ___1_ ____
Figure 11. Cleared leaf section of a young
susceptible leaflet soon after penetration, showing
primary hyphae branching profusely and growing
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,
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.
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
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
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
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
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
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.
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
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-
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
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
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
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-
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
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,
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-
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
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
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
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
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
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
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
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
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
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.
U- n a
0n L r
- o -
O S *-
0 U -
' 0 "
'o 'E *
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
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
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
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.
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.
Resistant F 232
Highly reslstant P-122
Highly resistant P-122
Diseased highly resistant
P-122, 6-month-old leaves
Hevea benthamlana Muell. Arg.
F 4542 2-month-old
F 4542 6-month-old
3 3 3 10
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.
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
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.
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.
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 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
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,
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.
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
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),
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
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.
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.
- C 0 Lt\
0 0 0 0
ro\ a a o
0 0 0 CD
4' *- LIN
U C I
0 -- -
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
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.
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
o C C C
E a .
2 e ) 0
0 3 o
11 4 20 7 5 15 18 14
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.
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-
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
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
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.
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
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
extensive experimentation to formulate a hypothesis dealing with
the nature of the host-parasite relationships between Dothidella
ulel and the Hevea rubber tree.