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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 Introduction
 Culture of dothidella ulei
 Pathogeniticity of two races of...
 Discussion
 Summary
 Literature Cited
 Biographical sketch
 Copyright














Title: Culture and pathogenicity of Dothidella ulei
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    Introduction
        Page 1
        Page 2
    Culture of dothidella ulei
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
    Pathogeniticity of two races of dothidella ulei to hevea sp
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
    Discussion
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
    Summary
        Page 32
        Page 33
        Page 34
    Literature Cited
        Page 35
    Biographical sketch
        Page 36
        Page 37
    Copyright
        Copyright
Full Text












CULTURE AND PATHOGENICITY OF

DOTHIDELLA ULEI












By

KENNETH R. LANGDON


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA
April, 1963













ACKNOWLEDGMENTS


The author is grateful to the Firestone Plantations

Co., Mr. B. H. Larabee, President, for a Grant-in-aid

which made this work possible. He wishes to thank

Mr. M. J. Vonesh, Firestone Plantations Co., Retalhuleu,

Guatamala; Dr. K. G. McIndoe, Firestone Plantations Co.,

Leesburg, Florida; Mr. W. E. Klippert, Vice President and

General Manager, Rubber Plantations, Goodyear Tire and

Rubber Co., Akron, Ohio; and Mr. H. Echeverri, Speedway

Estate, Goodyear Tire and Rubber Co., San Jose, Costa

Rica, for their respective parts in providing plants and

diseased material to be used in this research.

The author also wishes to express his appreciation

to Dr. D. A. Roberts, Committee Chairman; Dr. Phares Decker,

Head, Plant Pathology Department; Dr. G. F. Weber,

Dr. V. G. Perry, and Dr. J. H. Davis, Committee Members,

for their help and guidance during the course of this

research and study and for their help in the preparation

of the manuscript.













TABLE OF CONTENTS


Page
ACKNOWLEDGMENTS . . . . . . . . . ii

LIST OF TABLES. . . . . . . . . v

INTRODUCTION. . . . . . . . . . 1

CULTURE OF DOTHIDELLA ULEI. . . . . .. 5

Isolation and Growth in Pure Culture . . .. 3

Establishment and maintenance of the fungus
on trees in the greenhouse . . . .. 3

Isolation on agar . . . . . . 4

Observations of conidial formation and
germination. . . . . . . . 5

Cultural strains. . . . . . . 6

Nutrition. . . . . . . . . . 7

Procedure . . . . . . . . 7

Comparison of measurement systems and colony
types. . . . . . . . . 9

Carbon source . . . . . . . . 10

Hevea leaf extract. . . . . . . 11

Growth substances and amino acids . . . 13

Prepared media. ... . . . . 14

Other nitrogen sources. . . . . . 14

Recommended media . . .. . . . .. 16


iii










Page
PATHOGENICITY OF TWO RACES OF DOTHIDELLA ULEI TO
HEVEA SP. . . . . . . . .. . 18

Procedure . . . . . . . .... 18

Results . . . .. . . . . . . 20

Inoculation from culture . . . . . 20

Reactions of rubbertree clones to two races of
Dothidella ulei . . . . . . . 22

DISCUSSION . . . . . . . . .. . 25

SUMMARY. . . .. . . . . . . . 32

LITERATURE CITED . . . . . . . . .. 35

VITA . . . . . .. . . . 36













LIST OF TABLES

Table Page

1. Weights and Diameters of Colonies of
Dothidella ulei . . . . . . . 12

2. Peptone-Sucrose Agar for Growth of Dothidella
ulei in Culture . . . . . . 17

3. Ammonium Nitrate Agar for Growth of Dothidella
ulei in Culture . . . . . . 17

4. Classes of Resistance of Hevea brasiliensis to
Dothidella ulei used by the Firestone
Plantations Co. . . . . . . . 21

5o Reactions of Various Rubbertree Clones to Two
Races of Dothidella ulei. . . . . 24













INTRODUCTION


The devastating South American leaf disease of para

rubbertrees, Hevea brasiliensis (Muell.) Arg., caused by

Dothidella ulei P. Henn., is presently restricted to

tropical America where it is the main limiting factor for

rubber production (5,6), The disease has caused great

concern among rubber growers because it limits production

throughout its present range and would drastically curtail

rubber production in any area to which it might spread.

Media used previously (1,2,6) to culture D. ulei

failed to support satisfactory growth. Langford (6)

obtained growth to 5 mm diameter. The best colony

available from Blazquez's work (1,2) measured 20 mm and

weighed 3.6 mg dry weight. Any colony weighing less than

8 mg dry weight after three month's growth would not be

considered satisfactory. An improved medium would be very

useful for studies of the physiology of the fungus and to

provide a source of inoculum for pathogenicity tests.

Langford (7,8) reported the occurrence of a new

race of D. ulei which attacks progeny of clone F 4542

(Hevea benthamiana), previously resistant to all known

races of D. ulei. Pathogenicity of races grown in pure











culture has not been tested under controlled conditions,

and only rarely have reports of their natural occurrence

been made.

Objectives of this research were 1) to determine

some of the nutritional requirements of D. ulei in order

to develop satisfactory media for its culture, and 2) to

determine disease reactions of various greenhouse-grown

rubbertree clones inoculated under controlled conditions

with spores of two races of D. ulei grown in culture as

pure lines.













CULTURE OF DOTHIDELLA ULEI


Isolation and Growth in Pure Culture


Establishment and maintenance of the fungus on

trees in the greenhouse.--Leaves of Hevea brasiliensis

infected with a wild race of Dothidella ulei were obtained

from M. J. Vonesh, Firestone Plantations Co., Retalhuleu,

Guatamala. Conidia from these leaves were picked up with

a wet camel's-hair brush and deposited on the abaxial

surfaces of young leaves of trees in the greenhouse (2).

The entire flush of growth was covered overnight with a

moistened plastic bag to maintain high humidity. The

trees used were seedlings from Tjir 1 X Tjir 16, an

eastern cross susceptible to all known races of D. ulei.

Several sporulating lesions developed from inoculations

made from one shipment of leaves. Additional inoculations

to maintain the fungus were made with spores from these

lesions.

Leaves of clone IAN 717 with many sporulating

lesions of a new race of the fungus described by Langford

(7,8) were received from H. Echeverri, Speedway Estate,

Goodyear Rubber Plantations Co., Apartado 2050, San Jose,

Costa Rica. Conidia from these leaves were used to











inoculate successfully other trees in the greenhouse.

Both strains were maintained in vivo by serial inoculations,

No mixing of races occurred during the two-year course of

these investigations.

Isolation on agar.--D. ulei was isolated from

diseased Hevea leaves by two methods after it had been

established on trees in the greenhouse and was sporulating

well. Spores in one series of trials were seeded on water

agar plus 100 mg/1 quebrachitol (2) and germinated at 210C.

The germinated spores with small pieces of agar were then

transferred to tubes of leaf extract agar (2.5 g malt

extract, extract from 20 g young Hevea leaves boiled for

20 min in 200 ml of water, and 20 g agar, all in distilled

water to make 1 liter) or to tubes of Difco lima bean agar

with 200 mg/1 quebrachitol added (2). Pieces of young leaf

tissue approximately 5 mm square containing diseased areas

were, in a second series of isolations, surface sterilized

in 1:1000 HgC12 for about one minute, rinsed in sterilized

distilled water, and placed on Hevea leaf extract agar

slants. The Costa Rican (new) race was isolated only from

spores, whereas the Guatamalan (wild type) race was

isolated by both methods. Four cultures of the Costa

Rican race and three cultures of the Guatamalan race

developed from spores, and two cultures of the Guatamalan

race developed from leaf lesions. No cultural differences









5

were detected immediately among the nine original isolates,

but, as will be pointed out later, two distinct morpho-

logical strains were subsequently observed in each isolate.

Observations of conidial formation and germination.--

Conidia, as observed on hundreds of conidiophores in

undisturbed cultures, were produced singly and terminally,

but often appeared to be attached laterally. Apparently

each conidium was formed terminally, but was pushed to a

lateral position as the conidiophore continued to grow

longitudinally from the edge of the point of attachment

of the conidium. Ultimately, another conidium was formed

terminally and the cycle was repeated. Sometimes five or

six conidia could be seen along a single conidiophore at

one time.

Germ tubes from conidia germinating on the surface

of any agar medium tested grew aerially with only the

spores contacting the medium. The observation (2) that

germ tubes cease to develop if they contact the agar was

confirmed, Germ tubes which remained aerial soon branched

near the base, and these branches then grew on and into the

agar to form colonies. Transferring single germinated spores

before branching of the germ tubes usually resulted in

contact of the aerial germ tubes with the agar surface and

ultimate failure of the fungus to.grow. It was essential,

therefore, to allow sufficient time (3 days or more) for the











germ tubes to branch well before spores were transferred.

The only successful method tested, which was also used by

Blazquez (1,2), for transferring surface-germinated spores

earlier was to transfer clumps of spores. It was later

found that germ tubes from spores germinated below the

surface rather than on the surface of agar grew directly

into the medium with no apparent difficulty. There they

established colonies with a much higher degree of success

than did those from surface-germinated spores.

Cultural strains.--Two different morphological

strains or growth types, one a non-sporulating but compara-

tively rapidly growing mycelial type and the other a slower

growing but freely sporulating stromatic type, were observed

in cultures of both isolates after they had been maintained

for several months on Hevea leaf extract agar. The two

types were selected from the mixed cultures and maintained

separately. All attempts at inoculation with mycelia

failed to give infection, and it soon became obvious that

the non-sporulating mycelial type would be of little value

generally and of no value in pathogenicity tests.

The first two nutritional experiments were conducted,

as were those reported by Blazquez (1,2), with a mixture of

mycelial and stromatic growth forms derived from multispore

isolations or transfers. This mixture produced highly

variable results. The stromatic growth form of each race










was selected as a source of spores to inoculate trees and

was also selected for uniformity of growth among different

transfers on any given medium. The mycelial type of each

race was subsequently discarded, and the sporulating

stromatic type was maintained for further tests.

The stromatic forms of both races were used in

several experiments, and, since no significant differences

occurred between the growth rates of the two races on any

of 47 different media formulations, the Costa Rican race

was selected arbitrarily for use in subsequent tests.


Nutrition

Procedure.--Blazquez (2) found that extract from

50 g of Hevea leaves per liter of agar medium as used by

Langford (6) inhibited growth of D. ulei. The best

culture medium listed by Blazquez (2) contained 2.5 g

malt extract and boiled aqueous extract from 20 g young

Hevea leaves per liter. This was the first successful

medium used in these experiments. Blazquez1 later stated

that the optimum concentration of leaf extract was 2-5 g/1.

Most subsequent formulations, therefore, were prepared with

5-4 g/1 leaf extract, which proved much more favorable to
growth of the fungus than did the higher concentration.


C. H. Blazquez. 1962. Personal communication.











The basal semisynthetic medium used by Blazquez (2) was

less favorable for growth than was the leaf extract medium,

hence it was not used in these tests.

Uniform conditions were maintained for each test.

Cultures were grown in a temperature controlled room at

210C in the dark except for occasional use of room lights.

Two formulations previously tested as solid media were

also tested as liquid media. All other media contained 2

per cent agar. Cultures of the fungus were selected for

uniform growth characteristics on any given medium, and

only rarely did a variant appear. All transfers for a

given test series were made from a single culture tube.

A piece of stroma approximately 0.5 mm in diameter was

transferred to each slant. Most transfers were made from

3-month-old cultures. Almost all transfers from 3-month-

old or younger cultures grew, but transfers from older

cultures often failed to grow. Diameter measurements were

made two and three months after transfers had been made.

Dry weights of colonies were determined in later

experiments after diameter measurements had been made.

Enough water to cover the colonies to be weighed was placed

in the tubes. The agar was then melted and the colonies

were carefully picked from the melted agar with a transfer

needle, rinsed in hot water to remove as much agar as

possible, placed on filter paper, and dried overnight at











1000C. Dry colonies were weighed individually on an

analytical balance accurate to 0.1 mg.

Comparison of measurement systems and colony types.--

It became evident during the course of investigation that

diameter as a measure of growth was not fully satisfactory.

Diameter could be measured accurately only to 0.5 mm and

there was no satisfactory way to allow for differences in

thickness or density of the stroma. Dry weight would

account for all differences in growth, and colonies could

be weighed accurately to 0.1 mg. Accuracy was increased

with the measurement error being reduced from about 3 per

cent for the greatest diameter to about 0.2 per cent for

the heaviest colony weight.

Two mycelial colonies from earlier work (2) grown

on 2.5 g/1 leaf extract agar had been preserved in 10 per

cent formalin. These were measured and weighed for

comparison with the stromatic colonies used in these

experiments. The preserved colonies measured 19 and 20 mm

in diameter and weighed 3.5 and 3.6 mg, respectively. The

large diameters were not supported by equivalent weights.

These measurements may be compared with average measurements

of the stromatic type in this study (Table 1): 4.6 mm and

1.6 mg on 5 g/1 leaf extract agar with 3 g/1 sucrose,

8.1 mm and 9.0 mg on 1 g/1 NHRNO3 agar with 4 g/1 leaf











extract and 3 g/1 sucrose, 6.9 mm and 12.8 mg on 10 g/1

peptone agar with 4 g/1 leaf extract and 3 g/1 sucrose,

7.1 mm and 22.6 mg on 10 g/1 peptone agar with no leaf

extract and 10 g/1 sucrose, 11.2 mm and 55.5 mg on PDA

plus 10 g/1 peptone, and 17.0 mm and 17.7 mg on 7.5 g/1

phytone agar with 5 g/1 dextrose and 0.5 g/1 yeast extract.

The heaviest average colony weight herein reported was 15.4

times the weight of the larger preserved colony, despite

the fact that the preserved colony was almost twice (1.8

times) as wide as the former.

Any direct comparison of diameters reported by

Blazquez (2) with those given herein would be invalid.

Allowance must be made for the fact that the growth form

of D. ulei used in the tests herein reported was different

from that of the strain used in tests reported earlier (2).

The stromatic growth form of the fungus produces colonies

of smaller diameter and of greater weight in proportion to

the diameter than does the mycelial type. Also, the more

favorable the medium to total growth, usually the heavier

are the colonies proportional to their diameters.

Carbon source.--The amount or kind of carbon source

used in 3 g/1 leaf extract agar in early experiments,

whether malt extract, maltose, dextrose, or sucrose, at

1-10 g/1 caused no important differences in the growth rate

of Do ulei. Three g/1 sucrose appeared satisfactory, and











was used in most subsequent tests. Results of later experi-

ments, however, showed that the amount of the carbon source

could be limiting when other limiting factors were corrected.

For example, sucrose promoted significantly better growth

at 10 g/1 than at 3 or 5 g/1 (Table 1) when used with

10 g/1 peptone and 4 g/1 leaf extract. Sucrose, dextrose,

and malt extract promoted approximately the same amount

of growth when 4 g/1 leaf extract was present in 0.5-1.0

g/1 NH4NO3 agar. Growth was nil with either sucrose or

dextrose without leaf extract. Good growth was obtained

when malt extract was the sole source of carbon (and

other factors) in the ammonium nitrate medium.

Hevea leaf extract.--Extract from young Hevea

leaves tested at 2, 3, 4, 5, 6, 8, 10, 13, 16, and 19 g/1

with 3 g/1 sucrose proved most favorable at 4-6 g/1 (3.4-

4.6 mm at 76 days). No measurable growth of the fungus

was obtained with 15 g/1 or more leaf extract. Leaf

extract was of significant benefit when neither peptone

nor malt extract was used, but with a medium containing

10 g/1 peptone and 10 g/1 sucrose (the best medium tested)

leaf extract was of questionable benefit (Table 1). These

results indicate that some substance common to peptone,

malt extract, and leaf extract is indispensable to good

growth of D. ulei.










TABLE 1
WEIGHTS AND DIAMETERS OF COLONIES OF DOTHIDELLA ULEI GROWN ON DIFFERENT MEDIA

Colony
Expt. age Medium formulation Av. dia. Av. wt.
no. (days) ingredients, amount per liter ,(mm) (mg)
1 92 10 g peptone, 10 g sucrose 7.1 22.6
10 g peptone, 10 g sucrose, 4 g leaf extr. 8,7 21.5
10 g peptone, 5 g sucrose, 4 g leaf extr. 7.4 14.6
10 g peptone, 5 g sucrose, 4 g leaf extr. 7.8 10.3
2 100 10 g peptone, 3 g sucrose, 4 g leaf extr. 6.9 12.8
8 g peptone, 5 g sucrose, 4 g leaf extr. 7.4 10.9
1.0 g NH NO 5 g sucrose, 4 g leaf extr. 8.1 9.0
0.5 g NH NO 3 g sucrose, 4 g leaf extro 8.2 8.6
3 g sucrose, 4 g leaf extr. 4.4 lo4
5 100 39 g Difco PDA, 10 g peptone 11.2 5505
39 g Difco PDA, 0.8 g NH4NO5 10.2 27.4
39 g Difco PDA 9,8 1501
10 g peptone, 10 g sucrose 9.0 52.6
0.8 g NH NO 10 g sucrose, 4 g leaf extro 7.6 13.6
0.8 g NHLNO 5 g sucrose, 4 g leaf extro 8.0 13.0
0,8 g NH NO 5 g malt extr., 4 g leaf extro 9.4 10.3
0.8 g NH NO' 5 g malt extr. 9.4 10.5
0.8 g NH NO, 5 g sucrose Insufficient growth
4 to measure
4 47 7.5 g phytone, 5 g dextrose, 005 g yeast extr. 17.0 17.7
5 90+ Blazquez's (2) leaf extract agarb 20.0 5.6


aAll media contained 20 g/1 agar. Those which contained


NH4NO5 also contained,


per liter, 0.3 g KH2PO4, 0.5 g K2HPO4, 003 g MgSO4o7H20, and 0.005 g FeS04o73
preserved colonies on agar containing 2.5 g/1 leaf extract and probably
2.5 g/1 malt extract.


120.











Growth substances and amino acidso--Quebrachitol,

i-inositol, pyridoxine, p-aminobenzoic acid, nicotinic acid,

and riboflavin used in concentrations centered around those

used by Blazquez (1,2) had no noticeable effect on the

growth rate of D. ulei.

Glutamic acid, glutamine, leucine, and glycine,

used singly at 21 mg/1 (2) did not support good growth of

D. ulei. Glycine promoted best growth of any of these

compounds (average colony diameter 4.8 mm in 62 days),

leucine medium (4o1 mm), and glutamic acid and glutamine

poorest growth (3.9 mm), though none were significantly

better than the leaf extract agar check, in which the

average colony diameter was 4.2 mm. Combinations of two

or three amino acids, each at a concentration of 21 mg/1,

promoted improved growth over that by any single amino

acid or by any medium containing no other source of

nitrogen. Glutamine plus leucine, and glutamine plus

glycine promoted good growth (5.6 and 5.5 mm, respectively),

leucine plus glycine better (6.3 mm), and all three

together promoted best growth (6.6 mm). The total amount

of nitrogen present was larger in media containing the

combinations than in those with the compounds added singly.

This fact would probably account for the significantly

improved growth with all three compounds over that with

any one alone.










Prepared media.--Growth was poor on Difco nutrient

agar and lima bean agar, but was good on PDA (9.8 mm and

15.1 mg in 100 days). The addition of 10 g/1 peptone
improved the growth (11.2 mm and 55.5 mg) as did 0.8 g

NH NO3 (10.2 mm and 27.4 mg), but in all cases the fungus

produced only superficial stromas on PDA and did not grow

into the agar as it did with other media, Some stromas

on PDA plus peptone were 5 mm high and very dense, which

would account for the exceptionally heavy weights.

Other nitrogen sources.--Beef extract at 0.5-3.0 g/1
at intervals of 0o5 g and casein hydrolysate at 2, 5, and

10 g/1 were of little or no benefit in any formulation.

They were actually inhibitory when used in improved media

containing peptone or inorganic nitrogen.

Casein at 1, 2, 4, 6, 8, and 10 g/1 was of some

benefit, but less so than was peptone. Colonies on agar
containing 10 g/1 casein (the best concentration tested),

3 g/1 leaf extract, and 3 g/1 sucrose averaged 6.8 mm at
106 days compared with 8.3 mm with 10 g/1 peptone and 3.8 mm

on leaf extract agar only. Peptone promoted improved growth

of D. ulei at all concentrations tested, but was best at

8-10 g/1. A medium containing, per liter, 5 g dextrose,

0.5 g yeast extract and 7.5 g phytone (a papaic digest of
soya meal) promoted exceptionally good diameter growth

(17.0 mm) and good weight (17.7 mg in 47 days). The










colonies consisted of flat felt-like mats rather than dense

raised stromas as on PDA. Additional tests with phytone

are now underway.
It was deemed desirable to test the effect of

inorganic nitrogen in the medium on the growth of D. ulei,
since all products tested which improved the growth rate
contained nitrogen. Blazquez (2) obtained some growth on

Czapek's sucrose nitrate agar, but the amount of growth

was not considered significant. Ammonium nitrate at 0.5-

1.0 g/1 supplemented with 0.3 g KH2PO4, 0.3 g K2HP04,

0.3 g MgSO047H20, 0.005 g FeSO4-7H20, 4 g sucrose, and 4 g
leaf extract per liter proved highly favorable for growth

of D. ulei. Colonies on agar containing 0.5 g/l NH4NO3

averaged 8,2 mm in diameter and 8.6 mg weight at 97 days,

and those with 1 g/1 NH4NO3 averaged 8.1 mm and 9.0 mg.

These may be compared with colonies 4.5 mm and 1.4 mg on
leaf extract agar, 7.4 mm and 10.9 mg with 8 g/1 peptone,
and 6.9 mm and 12.8 mg with 10 g/1 peptone.
Colonies on 0.8 g/1 NH4NO3 agar containing 5 g/1

sucrose and 4 g/1 leaf extract measured 8.0 mm and weighed

13.0 mg average after 100 days growth compared with 3.5 mm
and 1o9 mg for those on the same medium except that it
contained 1.34 g/1 (NH4)2S04 instead of NH4NO3. Colonies

on the same medium except with 1.7 g/1 NaN03 (all 3 media

contained 0.02 M nitrogen) grew to 8.2 mm, but were










extremely thin with a very sparce, loose type of growth

weighing only 2.6 mg average. Ammonium nitrate was the

only inorganic nitrogen source tried which proved satis-

factory for growth of D. ulei.

Recommended media.--Two media containing different

nitrogen sources based on the above information are hereby

recommended for the culture of D. ulei. One contains

10 g/1 peptone as the nitrogen source, the other 1 g/1

NH4NO3 (Tables 2 and 3). Extract from 3 or 4 g young

Hevea leaves may be added per liter to either medium if

desired. The peptone-sucrose agar is simple to prepare and

would be best to use for general culture. The ammonium

nitrate agar is well suited for physiological studies of

the fungus Sucrose or other carbon source may be substi-

tuted for the malt extract in the ammonium nitrate agar

provided that leaf extract or some other source of the

growth factor present in peptone, leaf extract, and malt

extract is added, or that some test substance is added to

see if it would promote growth. Growth and sporulation

are good on either of the recommended media.

Two additional media, which the author has not as

yet had time to test sufficiently, may support even more

growth than either of the above media. One is PDA plus

10 g/1 peptone. The other contains, per liter, 5 g

dextrose (for which another carbon source could likely be










substituted), 0.5 g yeast extract, and 7o5 g phytone. Re-

sults of preliminary tests with these media appear promising.

TABLE 2
PEPTONE-SUCROSE AGAR FOR GROWTH OF DOTHIDELLA ULEI IN CULTURE


Ingredient Amount

Peptone 10 g
Sucrose 10 g
Agar 20 g
Distilled water to make 1000 ml



TABLE 3
AMMONIUM NITRATE AGAR FOR GROWTH OF DOTHIDELLA ULEI
IN CULTURE

Ingredient Amount

NH4NO3 1.0 g
KH2PO4 0.5 g
K2HPO4 0.5 g
MgS04 7H20 0.3 g
FeSO4*7H20 0.005 g
Malt extract 10 g
Agar 20 g
Distilled water to make 1000 ml












PATHOGENICITY OF TWO RACES OF DOTHIDELLA ULEI TO HEVEA SP.


Procedure

Rubbertrees of clones IAN 710, IAN 713, IAN 873,

FX 25, FX 232, all without F 4542 (Hevea benthamiana)

parentage; IAN 717, FX 637, FX 664, FX 2831, FX 3810,

FX 5925, all progenies of F 4542; P 122, a Madre de Dios

selection; and numerous seedlings from Tjir 1 X Tjir 16,

an eastern cross susceptible to all known races, were

planted in ground beds in the greenhouse. Clones FX 232,

FX 2851, P 122, and the Tjir 1 X Tjir 16 seedlings have

been used previously (1,2,5). All other clones were

furnished by M. J. Vonesh. The soil mix used was half

peat and half sandy soil. Shading was applied to the

glass to reduce sunlight intensity to slightly over 1000

ft-c. This prevented excessive heating in the summer,

but permitted good growth of the trees. The trees were

cut back periodically to keep them at workable heights.

An intermittent mist system with 28 Spray Systems Co.

TeeJet % TTX 1 nozzles operating 2 sec during each minute

was installed to increase the relative humidity to promote

disease development.











Inoculations were made by picking up conidia from

sporulating leaf lesions with a wet camel's-hair brush and

by brushing them on the abaxial surfaces of young leaves (2).

Inoculations at early stage "B" (4) resulted in best lesion

development and sporulation. Stage "B" is defined as the

period in the early leaf development from the time the

leaflets turn down until they lose their reddish color and

become a light yellowish green. Early stage "B" is

approximately the first one-third of this period (leaflets

less than 50 mm long). Generally only a portion of each

leaflet was inoculated in order to reduce leaf drop.

After inoculation the entire flush of growth was covered

with a plastic bag moistened inside to maintain a saturated

atmosphere. The bag was usually removed 16-24 hours after

inoculation.

Simultaneous inoculations with each race on separate

leaves of the same flush of growth were made to test the

reactions of the different clones to each race of the

fungus. One or more leaves in a flush were inoculated

with one race and tagged, then other leaves were inoculated

with the other race, tagged, and the entire flush was

bagged overnight. The disease severity was noted 2-3 weeks

later.

Trees in the greenhouse were rated for resistance

according to systems developed by Langford (6) and by the










2
Firestone Plantations Company. Trees were rated by the

first method for resistance on a scale from one (immune) to

ten (very highly susceptible) and for sporulation by the

fungus from 0 (no spores) to ++++ (heavy sporulation). The

Firestone system was devised for field screening for

resistance in Guatamala. It consisted of four classes

(Table 4): R (resistant), HR (high resistance), MR (medium

resistance), and S (susceptible). The Langford system

would have to be modified for use in greenhouse work under

controlled conditions. For instance, the criterion for

class ten in terms of field testing (6) is death of the

tree. No trees in the greenhouse were killed by the fungus,

even though trees normally rated class ten under field

conditions were present and infected. The Firestone system

was used without modification, and it or some similar

system is recommended for use in greenhouse testing of

trees for resistance.


Results

Inoculation from culture.--Trees were inoculated with

conidia obtained from serial subcultures about six months

after the original isolation. There was no apparent loss of

pathogenicity of the fungus Inoculations with the fungus


2. G. McIndoe. 1962. Personal communication.











TABLE 4

CLASSES OF RESISTANCE OF HEVEA BRASILIENSIS TO DOTHIDELLA
ULEI USED BY THE FIRESTONE PLANTATIONS COMPANYa


Desig-
nation Class Description

R Resistant Clones exhibiting no disease
lesions or a virtual absence
thereof

HR High resistance Clones exhibiting only a few
nonsporulating lesions

MR Medium resistance Clones showing more lesions
than those of the HR class,
often on the borderline of
susceptibility

S Susceptible Clones exhibiting many lesions
on stems, peticles, and leaves


provided by K. G.


McIndoe.










after a year in culture, however, were not nearly so

successful. Only a few infections occurred after each

inoculation. Sporulating lesions developed only rarely,

but inoculation with spores from these lesions resulted in

formation of a moderate number of sporulating lesions.

Many sporulating lesions developed after the third serial

inoculation, indicating a reselection for normal patho-

genicity. These results indicate that continued subculture

of D. ulei favors selection and propagation of nonpathogenic

(avirulent) strains of the fungus. Satisfactory inoculum

can be obtained from subcultures generally for only 6-12

months after isolation of the fungus from diseased leaves.

Reactions of rubbertree clones to two races of
Dothidella ulei:--Most of the Tjir 1 X Tjir 16 seedlings

on which both races of the fungus were maintained were

completely susceptible to both races. Clones IAN 710, IAN

715, IAN 873, and FX 232, all without F 4542 parentage,
were rated HR (3-0 to 4-0) to both races (Table 5). Clone

P 122, a Madre de Dios selection, was rated R (2-0) to

both races and was, therefore, the most resistant clone

tested. The F 4542 progenies, clones IAN 717, FX 657, FX
664, FX 2831, FX 3810, and FX 3925, were rated from HR (4-0)

to R (2-0) for the Guatamalan race and from S (6+++) to

MR (4+) for the Costa Rican race. All clones with F 4542

parentage were more susceptible to the Costa Rican race








23

than to the Guatamalan race, but those without F 4542

parentage were equally resistant or susceptible to both

races (Table 5).

These findings under controlled conditions

substantiate those made in the field by Langford (7,8).

Different races of the fungus exist and at least one race

can overcome the H. benthamiana (F 4542) type of resistance.









TABLE 5


REACTIONS OF VARIOUS RUBBERTREE CLONES TO TWO RACES OF DOTHIDELLA ULEI


Clone
IAN 710
IAN 715
IAN 717
IAN 873
FX 25
FX 252
FX 657
FX 664
FX 2831
FX 3810
FX 3925
P 122

Seedlings


Parentage
PB 86 X F 409
PB 86 X F 409
PB 86 X F 4542
PB 86 X FA 1717
F 351 X AV 49
F 551 X PB 186
F 4542 X Tjir 1
F 4542 X Tjir 1
F 4542 X Tjir 1
F 4542 X AV 365
F 4542 X AV 563
Ho brasiliensis
selection
Tjir 1 X Tjir 16


Resistance ratings from 1 (immune) to 10 (very highly susceptible, plant
killed by repeated defoliation) and sporulation ratings from 0 (no spores) to
++++ (very heavy sporulation)o
R resistant, HR high resistance, MR medium resistance, S =
susceptible,


Y


Resistance rating using
Langford's systema
Costa Rican Guatamalan
3-0 5-0 to 4-0
3-0 3-0
7+++ 4+
3-0 to 4-0 3-0 to 4-0
3-0 3-0
3-0 5-0
4+ 2-0
4+ 2-0
6+ 2-0
5++ 2-0 to 3-0
6+++ 4-0
2-0 2-0

6+ to 10++++ 8++ to 10+++


Resistance rating using
Firestone's system
Costa Rican Guatamalan
HR HR
HR HR
S MR
HR HR
HR HR
HR HR
MR R
MR R
MR R
MR R
S HR
R R


---


.













DISCUSSION


Dothidella ulei is at best a slow-growing fungus.

The stromatic type used in these experiments reached a

maximum diameter of only 17 mm on one medium and a maximum

dry weight of 56 mg on another medium after growth for more

than 90 days on the best media tested. The best available

measurements for the mycelial type were from a colony

grown by Blazquez on leaf extract agar and preserved with

formalin. It measured 20 mm and weighed 3.6 mg.

Possibly some improvement in growth rate can still

be achieved by additional changes in the recommended media.

It is possible that certain synthetic or naturally occur-

ring growth substances might stimulate growth of D. ulei

in culture.

It was necessary in these experiments to have all

variables controlled within reasonable limits in order to

detect relatively small differences in growth rates of

D. ulei resulting from the use of different media. A

major source of variability in culture studies results

from the fact that masses of spores always contain some

which give rise to nonsporulating, mycelial colonies and

others which develop into sporulating, stromatic colonies.











Thus, when masses of spores are used to infest culture

media, the two strains which develop are in competition.

The mycelial strain would predominate in some cultures

whereas in others the stromatic strain would predominate.

Diameter measurements in nutritional experiments where

mass spore transfers were used varied as much as 300 per

cent (2) within a single treatment (5-16 mm in 26 days).

Diameter measurements within a single treatment usually

varied only 10-35 per cent in these tests where pure lines

of the stromatic type were used. Moreover, variation in

dry weights within a treatment were usually only 20-30 per

cent. Dry weight measurements were more reliable than

colony diameter measurements as criteria for fungal growth

because stromatic colonies and probably mycelial colonies

also varied between treatments with respect to height and

density as well as diameter, and these variations were not

necessarily in direct proportion to diameter.

The reason for formation of aerial germ tubes when

conidia of Do ulei are germinated on the surface of agar

and the reason 'for their cessation of growth upon contact

with the agar remain unknown It is possible that aerial

germ tubes may have much higher osmotic concentrations

than the medium, such that they would take up water and

rupture on contact with the agar. The continued develop-

ment of germ tubes from subsurface germinated spores might










be accounted for by the fact that, before germination,

the spores reach an osmotic equilibrium with the medium.

D. ulei is capable of using inorganic nitrogen for

growth, but in tests so far it has produced the most dry

weight when grown on agar containing peptone. The nitrogen

source is one of the main factors affecting growth of D.

ulei. A second important but unknown factor, or growth

substance, is available from peptone or malt extract or

Hevea leaf extract. The several growth substances tested,

however, had no noticeable effect on growth. The medium

used at the time these growth substances were tested was

lacking in a proper nitrogen source; hence, nitrogen was

limiting. Tests of these substances in ammonium nitrate

agar might yield different results.

The development of a satisfactory semisynthetic

growth medium (ammonium nitrate agar) has opened the way

for more accurate physiological studies of D. ulei. The

ammonium nitrate medium with sucrose in place of the malt

extract would be ideal as a base *to which to add test

ingredients to determine if they can substitute for the

growth factor or factors in peptone, malt extract, and

Hevea leaf extract.

Experiments of this nature are needed to identify

the beneficial substance or substances common to these

three sources.










The value of the leaf extract is still in some doubt,

but the extract appears to be of little or no benefit when

used with peptone or malt extract. It is required, however,

for good fungus growth in the absence of peptone and malt

extract.

The recommended media (Tables 1 and 2) can also be

used as liquid media. Growth rate and form are similar in

liquid to those on solid media except that multiple

colonies tend to form. Growth in liquid shake culture is

about twice as rapid as in still, but in shake culture

part of the fungus adheres to the walls of the container

and is not continually bathed in the medium. Accurate,

valid quantitative measurements from liquid culture are

difficult because of complicating factors, such as the

tendency of the fungus to produce a variable number of

colonies, poor growth in still culture, and the tendency

of a variable portion of the fungus to adhere to the glass

and to be poorly nourished in shake culture. These

difficulties must be overcome if reliable measurements are

to be made of the fungus grown in liquid culture,

The loss of pathogenicity after repeated serial

transfers in culture likely resulted from inadvertent

selection of strains better adapted to growth in culture

than to parasitic growth in vivo. Reselection for wild-

type pathogenicity from 6-12-month-old cultures was











accomplished on the trees. It is recommended, therefore,

that isolations of the fungus from the trees be made

approximately every six months in order to maintain

virulent cultures of D. ulei.

The new race of D. ulei from Costa Rica is pathogenic

to some extent on all F 4542 progenies, in which only one

source of resistance was incorporated. Similar material

incorporating additional sources of resistance to that

from F 4542 might well be resistant to all known races.

Clone P 122, although its growth characteristics are poor,

possesses a very high degree of resistance (R, 2-0) to

both races tested, and would likely be a very good source

of resistance for use in breeding programs. Other clones

of the Madre de Dios selections have more desirable growth

characteristics than P 122. Many of these clones might

be equally as resistant as P 122, and might be profitably

included in an expanded breeding program incorporating

multiple sources of resistance to D. ulei.

Results of tests reported here demonstrate the

possibility of identifying races of Do ulei under partially

controlled conditions. Actually, it may be possible to

control conditions sufficiently in nursery plantings in

the tropics to permit conducting a testing program similar

to the program herein described for greenhouse testing.

This would entail isolation of test plantings to minimize










natural infections. Also, young trees not under test could

be sprayed with fungicides to reduce the amount of natural

infection. Emerging shoots of test trees might be pro-

tected from infection prior to and until after their

inoculation with pure lines from culture by a mechanical

barrier such as a plastic cover. Such a system, should it

prove practical, would make possible rapid testing of

clones to as many races of the fungus as are available.

Also, race identification would be simplified, since

pathogenicity of each race or collection on hand could be

tested on many genetically different clones in the same

environment.

A number of differential clones must be selected

before any extensive race identification program can be

initiated. One or more of the F 4542 progeny would be

needed as a differential for identification of the race

designated herein as the one from Costa Rica. Clone P 122

or others resistant to both races tested here could be

included to detect hitherto unknown races. A third race

of Do ulei is known to infect a number of Ford clones,

including F 409 and F 1619, in the vicinity of Belterra,

Brazil (7). At least one of these clones should be

included among the differentials. Early selection of

differential clones, a major problem in itself, and the

establishment of a race identification program are strongly








51
recommended to protect the already considerable investment

in the program of breeding Hevea resistant to D. ulei. It

is further recommended that a program be initiated to test

promising clones for their reactions to as many different

races as can be obtained.













SUMMARY


Results of nutritional experiments with various

amounts of four carbon sources, three amino acids and

glutamine, six growth substances, five peptide sources,

and three sources of inorganic nitrogen, used singly and

in various combinations, revealed that the requirements

of Dothidella ulei are relatively simple. Earlier diffi-

culties in culturing the fungus resulted partly from lack

of a proper nitrogen source in the medium and partly from

the use of media too rich in certain organic compounds
for this slow-growing fungus (maximum stromatic colony

diameter on one medium 17 mm, dry weight on another medium

56 mg in 3 months). Do ulei grew well and sporulated
freely on media containing per liter: 1) 5-10 g sucrose
and 8-10 g peptone; 2) 5-10 g sucrose, 5-4 g Hevea leaf

extract, and 1 g NH4NO3 with other mineral salts (0.3 g

each of KH2PO4, K2HP04, and MgSO407H20; and 0.005 g

FeSO4.7H20); 3) 5-10 g malt extract and 1 g NH4N03 with

other mineral salts as described above; 4) PDA plus 10 g/1

peptone; 5) 5 g dextrose, 0.5 g yeast extract, and 7.5 g

phytone. The only complex organic compounds required were

supplied by peptone or malt extract or Hevea leaf extract.








33

Rate of growth of D. ulei in culture was not affected

by nicotinic acid, riboflavin, pyridoxine, E-aminobenzoic

acid, quebrachitol, or i-inositolo In media provided with

peptone, the fungus grew equally well with glucose, maltose,

sucrose, or malt extract as the major carbon source. Three

amino acids and glutamine, in quantities sufficient to

provide nitrogen equivalent to 1 g/1 NH4NO3, promoted good

growth when used with leaf extract and malt extract. The

fungus did not grow well in media containing lesser amounts

of the amino acids and glutamine. Casein was of slight

benefit, whereas casein hydrolysate and beef extract were

inhibitory. Ammonium nitrate was the only inorganic

nitrogen source tested which promoted satisfactory growth.

Two growth types of the fungus were readily separated

in culture: 1) a nonsporulating, moderately rapidly growing

mycelial type, and 2) a slower growing, freely sporulating

stromatic type. Selective transfers were made to propagate

the stromatic type to maintain sporulation so that trees

could be inoculated with spores from pure cultures of a

particular strain.

More than 35 seedlings of Tjir 1 X Tjir 16 and 44

trees representing 12 rubbertree clones were tested for

their reactions to two races of Do ulei in over 100 separate

multiple inoculations on both clones and seedlings and in

many single inoculations on the seedlings. Each race was










inoculated on separate leaves of the same flush of growth

for simultaneous testing with more than one race. All

seedling trees were susceptible to both races. The six

clones, IAN 717, FX 664, 637, 3810, 3925, and 2831, which

were progeny of F 4542 were resistant to the wild type

from Guatamala but susceptible to the new race from Costa

Rica. Six clones, P 122, IAN 710, 713, 873, and FX 25 and

232, all without F 4542 parentage, were resistant to both

races. Clone P 122, a Madre de Dios selection, was the

most resistant one tested.












LITERATURE CITED


lo Blazquez, C. H. 1959. Host-parasite relations of the
fungus Dothidella ulei P. Henn. on the Hevea rubber
tree. Ph.D. Dissertation, Univ. of Florida,
Gainesville. 97 po
2, Blazquez, C. H., and J. H. Oweno 1957. Physiological
studies of Dothidella ulei. Phytopathology 47:727-
732.

3. Blazquez, C. H., and J. H. Owen. 1963. Histological
studies of Dothidella ulei on susceptible and
resistant Hevea clones, Phytopathology 53:58-65.

4. Dijkman, M. Jo 1951. Hevea, thirty years of research
in the Far East. Univ. of Miami Press, Coral Gables,
Florida. 329 p.

5. Hilton, R. N. 1955. South American leaf blight.
J. Rubber Research Inst. Malaya 14 (293):287-337.

6. Langford, M. H. 1945. South American leaf blight of
Hevea rubbertrees. U. S. Dept, Agr. Tech. Bull.
882, 51 p.
70 Langford, M. H. 1960. A new strain of leaf blight on
rubbertrees in Costa Rica. (Mimeo.) Report to AID,
Washington, D. C.

8. Langford, M. H. 1961. A new strain of leaf blight on
rubbertrees in Costa Rica (second report). (Mimeo.)
Report to AID, Washington, D. C.












VITA


Kenneth R. Langdon was born August 20, 1928, at

Cache, Oklahoma. He graduated from Cache High School in

May, 1946. He worked as a farmer and also as a radio

repairman from then until October, 1951. He was inducted

into the army in October, 1951, and served in the Korean

campaign until his discharge in October, 1953. He entered

Cameron State Agricultural College, Lawton, Oklahoma in

September, 1954. There he received the Associate of Arts

degree in May, 1956. He entered Oklahoma State University,

Stillwater, Oklahoma in September, 1956, and received the

Bachelor of Science degree with a major in Botany, in May,

1958. He began graduate work immediately at the same

university and received the Master of Science degree with

a major in Plant Pathology, in May, 1960. He entered the

University of Florida in September, 1960, on a research

fellowship provided by Firestone Plantations Co., and

pursued his work in Plant Pathology toward the degree of

Doctor of Philosophy to be granted in April., 1963.

Mr. Langdon is married to the former Gertrude

Wenzel Hooker, and has three children. He is a member of

Phi Kappa Phi, Sigma Xi, the American Phytopathological

Society, and the Society of Nematologists,

36











This dissertation was prepared under the direction

of the chairman of the candidate's supervisory committee

and has been approved by all members of that committee. It

was submitted to the Dean of the College of Agriculture and

to the Graduate Council, and was approved as partial

fulfillment of the requirements for the degree of Doctor

of Philosophy.


April 20, 1963


Dean, College of Agriculture



Dean, Graduate School

Supervisory Committee:


Chairman
Oe *< r\pr










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