ELONGATION OF SECONDARY HYPI-IAE AND

TAAASLOGATIOA OF3 53 AND H FRGM HOST T0

PAAASITE DURI we PRIMARY INFECTION 0F WHEAT
BY ERYSILPHE GRAMINIS F. swam 7

Dissertation for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
RICHARD E. STUCKEY
1973

  
   

ml.

Micl‘rig: n 3113 :6
Univ srrsity

-~ Wall:

This is to certify that the

thesis entitled
ELONGATION OF SECONDARY HYPHAE AND TRANSLOCATION

F 355 AND 3H FROM HOST T0 PARASITE DURING PRIMARY

INFECTION 0F WHEAT BY ERYSIPHE GRAM‘INIS F. SP. TRITICI

presented by

RICHARD E. STUCKEY

has been accepted towards fulfillment
of the requirements for

PH.D. degree in PLANT PATHOLOGY

 

 

:27 ///é.zé flag
Major professor ()Q’J/

 

Date August 17, 1973

 

0-7639

 

ABSTRACT

ELONGATION OF SECOggARY HYEHAE AND
TRANSLOCATION OF S and H FROM
HOST TO PARASITE DURING PRIMARY

INFECTION OF WHEAT BY ERYSIPHE
GRAMINIS F. SP. TRITICI

BY

Richard E. Stuckey

Development of secondary hyphae >10u long is evidence

of successful infection of wheat by Erysiphe graminis f. sp.

 

tritici. The lengths of secondary hyphae at 20 to 30 hours
after inoculation varied with the parasite/host genotype.
There were differences in the kinetics of secondary hyphal
elongation on plants of compatible and incompatible genotypes.
Differences between incompatible genotypes suggest that the
single gene differences for incompatibility are operating

via different mechanisms rather than via altered specificity
of the same mechanism.

Rates of 358 transfer from wheat to E. graminis f. sp.

 

tritici differed with the parasite/host genotype, the stage
of parasite development and the environmental conditions.

Inoculated primary leaves cut at the base and given

358042- for five hour periods in the light had more than

twice the radioactivity of inoculated primary leaves given

3580 2-

4 in the dark. After considering the amount of

Richard E. Stuckey

label translocated to the epidermis as a result of the
environmental conditions used, greatest rates of 358 transfer
were found to occur at 18 to 20 hours rather than at 26 hours
after inoculation. The amount of 35$ uptake and translocation
to the epidermis was greater in noninoculated plants than in
inoculated plants from 6 to 24 hours after inoculation.

Reduction of 35

S activity in inoculated plants was independent
of spore concentration, parasite/host genotype and species of
Erysiphe used for inoculation.

_Little is known about the form in which label is

transferred from wheat to g, graminis f. sp. tritici. The data

 

suggest that transfer of both methionine and sulfate can occur.
The fungus was able to incorporate 8042- into a TCA-insoluble
form. Inoculated plants given 3H-methionine transferred 3H to
the parasite.. Five times the number of molecules appear to be
transferred when inoculated plants were given 3H-methionine

than when given 358042.. When inoculated plants were given

both 3H-methionine and 358042-, the presence of 3H—methionine

inhibited 358 transfer to the parasite by ca. 60 percent.
Whether this inhibition occurred at the plant cell membrane,
the fungal membrane, or both is not known.

The percent of label incorporated into the TCA-
insoluble fraction in the parasite and in the epidermis of
the host was much less than the percent of label incorporated
into the TCAéinsoluble fraction in the leaf tissue when cut
seedlings were given 358042-. A higher percentage of

incorporation into the TCA-insoluble fraction occurred when

Richard E. Stuckey

leaves were given 3H-methionine or 358042- at one to six

hours after inoculation than when leaves were given 3H-
methionine or 358042- at 21 to 26 hours after inoculation.
Environmental conditions prior to labeling and age of leaf
affected the increase in the TCA—insoluble fraction at one
to six hours after inoculation. Leaves given 3H-methionine
at 21 to 26 hours after inoculation had a higher percentage
of radioactivity in the TCA-insoluble fraction than did
leaves given 358042- for the same time period.

Thin layer chromatography of extracts from the epider-
mis and leaves showed a higher percentage of radioactivity
corresponding to methionine when plantsvxnxagiven 3H-methionine
than when plants were given 358042-.‘ Cut seedlings given
3H-methionine had nearly 38 percent of the radioactivity in
the epidermis corresponding to a value of methionine on TLC
plates. Only four percent of the radioactivity in the
epidermis corresponded to a value of methionine when cut
seedlings were given 35 2-

4 0
Rates of 358 transfer from host to parasite were

SO

greatly reduced when plants were labeled at 4 C instead of
22 C. An active transport system from host to parasite is

suggested.

ELONGATION OF SECONDARY HYPHAE AND
TRANSLOCATION OF 353 AND 3H FROM
HOST TO PARASITE DURING PRIMARY
INFECTION OF WHEAT BY ERYSIPHE

GRAMINIS F. SP. TRITICI

BY

Richard E. Stuckey

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1973

.1: AU. "'7? ..
." “Jr-:9 fly

l"..‘:F,

DEDICATED
to
My Wife, Judy
and

Son, Jeffrey James

ii

ACKNOWLEDGMENTS

Sincere appreciation is due to Dr. Albert H. Ellingboe,
my major professor, for his interest, guidance and encourage-
ment during this investigation and in the preparation of this
manuscript.

Special thanks are also due to my graduate committee,
Dr. R. P. Scheffer, Dr. W. G. Fields, and Dr. J. A. Boezi
for serving on my committee and their assistance in the
preparation of this manuscript.

I am indebted to Mr. Joseph L. Clayton for his tech—
nical assistance and to Dr. M. Jost for making his equipment
available to me.

I am deeply grateful to my wife, Judy, for her
understanding and assistance during the course of this
investigation.

Sincere appreciation is due to the many graduate
students and faculty members for meaningful discussions of my
research and to Dr. W. B. Drew for allowing me the opportunity
to study at Michigan State University.

Financial assistance for this investigation was
Obtained from the National Institute of Health for which I

am indebted.

iii

TABLE

DEDICATION . . . . .
ACKNOWLEDGMENTS . . .
LIST OF TABLES . . . .
LIST OF FIGURES . . .
INTRODUCTION . . . .
LITERATURE REVIEW . . .

MATERIALS AND METHODS .

OF

CONTENTS

Maintaining Powdery Mildew

Methods of Inoculation
Controlled Inoculatio
Inoculation by Dustin

Environmental Condition

Counting Efficiency of

CHAPTER

n
9

3'

for

Experiments
H-methionine and SO

I. GENETIC CONTROL OF MORPHOLOGICAL DEVELOPMENT OF

E. GRAMINIS ON WHEAT.

Introduction . .

Materials and Metho
Results . .
Discussion . .
Summary . .

35

ds

II. S UPTAKE BY WHEAT AND TRANSFER TO E. GRAMINIS
F. SP. TRITICI AS AFFECTED BY ENVIRONMENTAL

CONDITIONS . . . .

Introduction . .‘
Materials and Metho
Results. . . .
Discussion . .
Summary . . .

d

iv

Page
ii
iii
vi

viii

17

17
18
18
19
19
20

21

21
22
24
31
33

34

34
36
39
49
52

 

.III. UPTAKE OF RADIOACTIVE ISOTOPES BY WHEAT
LEAVES, THE DISTRIBUTION AND FORM OF
RADIOACTIVITY IN WHEAT LEAVES, AND TRANSFER
OF RADIOACTIVITY FROM HOST TO PARASITE . .

Effect Of Inoculation of 35$ Uptake in the
Leaf and Translocation to the Epidermis.
Uptake of H-methionine by Wheat Leaves
and Transfer to E. graminis f. sp.
Tritici . . .— . . . . . . . .
Solubility in TCA of Radioactivity in
Leaves, Epidermis and Parasite . . .
Thin Layer Chromatography of Radioactivit
in Leaves, Epidermis and3garasite. . .
Effect of Temperature on S Uptake in the
Leaf, Translocation to the Epidermis
and Transfer to the Parasite . . . .

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

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

LITERATURE CITED . - - ~ - ~ - - - - -

Page

54

54

67
71
81

91

96
108

110

Table

LIST OF TABLES

Page
Effect of genotype of host and pathogen on
development of secondary~hyphae . . . . . 28
Removal of 3580 2- solution from vials by
inoculated and noninoculated seedlings at
various times after inoculation . . . . . 60
Effect of iggculation density of E. graminis
tritici on S radioactivity in inoculated
wheat leaves . . . . . . . . . . . 65

ngparison of uptake and translocation of
S radioactivity by wheat leaves inoculated
with E. graminis tritici and E. ggaminis

poae . . . . . . . . . . . . . . 66

Effect of different concentrations of 3H-
methionine given to inoculated BEE wheat

leaves on the amount of transfer to the

fungus at 25 hours after inoculation . . . 68

 

 

Effect of different labeling times of 3H-
methionine (100 uc/ml) given to inoculated

BEE wheat leaves on the amount of radio-

activity (CPM) in the epidermis and in the

fungus . .. . . . . . . . . . 68

Comparison of radioactivity (CPM) in the
parasite, epidermis and leaf tissue of
gnoculated BEE wheat seedlings given
H-methionine (100 uc/ml) at 25 hours
after inoculation. . . . . . . . . . 69

Transfer of 35$ and 3H from inoculated pmx
wheat leaves to E. graminis tritici at
25 hours after inoculation . . . . . 70

Distribution of 358 by TCA fractionation
of inoculated pmx wheat leaves 25 hours
after inoculation. . . . . . . . . . 74

 

vi

Table Page

10. The percent of radioactivity in the TCA—
insoluble fraction in conidig gegminated
on agar in the presence of SO and in
EE§_wheat leaves and in the fungus at 25
hours after inoculation . . . . . 76

11. The percent of radioactivity in the TCA-
insoluble fraction in inoculated and
ggninggulateg pg§_wheat leaves labeled with
SO , or H-methionine at various times
afteg inoculation. . . . . . . . . . 76

12. The effect of host age on the percent of
radioactivity in the TCA-insoluble
fraction in iggculaged pg§_wheat seedlings
labeled with 804 . . . . . . . . . 78
13. The effect of light and dark preceding
labeling of ngginogglated wheat seedlings
labeled with 80 on the percent of

radioactivity in ghe TCA-insoluble fraction . 79
14. The effect of temperature on 358 activity

in mx inoculated leaves and transfer to E.

graminis tritici . . . . . . . . . . 93

 

vii

 

11,11"

Figure

LIST OF FIGURES

Confirmation of synchronous development
of Erysiphe graminis f. sp. tritici
on wheat leaves . . . . . . . . .

 

Effect of five different parasite/host
genotypes of the development of
secondary hyphae of E. graminis f.
sp. tritici . . T . . . . . . .

 

Effect of light and darkness on uptagg and
translocation to the epidermis of S in
cut wheat seedlings inoculated with
Erysiphe graminis f. sp. tritici under
environmental conditions used for
synchronous parasite development in
primary infection. . . . . . . . .

Effect of light on 358 transfer from
Chancellor wheat to E. graminis f. sp.
tritici from 20 to 26 hours after
inoculation . . . . . . . . . .

 

 

Effect of light on the percentage of
secondary hyphae (>10 u long) formed
by E. graminis f. sp. tritici. . . . .
Rates of 358 transfer from five near-
isogenic wheat lines to Erysiphe graminis
f. sp. tritici. . . . . . . . . .

Uptake of 358 and translocation to the
epidermis of noninoculated wheat leaves
and wheat leaves inoculated 6 to 26
hours previously with E. graminis f.
sp. tritici. . . .'—. . . . . . .
Uptake of 358 and translocation to the epi—
dermis in cut noninoculated wheat seedlings
and wheat seedlings inoculated 6 to 26 hours
previously with E. graminis f. sp.
triticif . . . . . . . . . . .

 

 

 

 

viii

Page

26

30

41

44

46

48

57

59

INTRODUCTION

Erysiphe graminis f. sp. tritici, the cause of
powdery mildew of wheat, is an obligate parasite found in
most of the wheat growing areas of the world. This disease
causes a significant reduction in grain yield in some areas
of the world (47, 93).

The best and least expensive method of controlling
powdery mildew has been the development and use of resistant
cultivars. As a result of the breeding programs, there are
many host genotypes with different degrees of resistance to
powdery mildew.

Much attention has been given to studies on the
comparative physiology and biochemistry of diseased and
healthy tissues in the search for understanding of disease
resistance (16, 72, 82, 91). Most of these workers used
tissues several days after inoculation. There are conflicting
data and interpretations. The probable reasons for conflicting
results are differences in procedures for production of
inoculum and plants, differences in times of observation, and
differences in environmental conditions.

A well defined system for studying the early infection

process of E, graminis f. sp. tritici on wheat has been

 

extablished (53, 58, 62). Several distinct morphological

stages in fungal development were described. The stages
required different environmental conditions for optimum
development. The fungus population was synchronized in
morphological development by manipulating the environmental
conditions, and a maximum number of successful infections
was achieved.

The objectives of this research were as follows:
(1) to collect quantitative data on the kinetics of
elongation of secondary hyphae as a function of time, and
(2) to determine if the secondary hyphae which do begin to
elongate with incompatible genotypes develop similarly to
the development of secondary hyphae with compatible geno—

35S

types; (3) to examine the uptake and translocation of
inthe host under the environmental conditions used for
synchronous parasite development, (4) to determine the rate
of transfer from host to parasite by considering the amount
of label theoretically available in the epidermis to the
parasite, and correlate rates of transfer with the morpho-
logical stage of development of the parasite; (5) to

35$ uptake by wheat

examine the effect of inoculation on
leaves, and (6) to determine the form in which labeled
materials may be transferred from host to parasite.

Objectives one through four are the objectives of two papers

(chapters 1 and 2) which have been submitted for publication.

L I TE RATURE REVI EW

The genetics, physiological and biochemical aspects
of disease development have been studied extensively, often
with contradictory conclusions. The powdery mildews are
not an exception. In this review, I will attempt to summarize
some of the important genetic, physiological and biochemical
developments that have been reported and that are related to
the results presented in this thesis.

It is clear from many investigations (32, 33, 57, 65)
that the interactions between host and parasite are controlled
by complementary genes possessed by both host and parasite.

Flor (30) found that the ability of Melampsora lini to grow

 

and produce disease symptoms on flax lines containing genes
for resistance was determined by specific corresponding
genes in the pathogen. The existence of one gene in the
pathogen for each gene in the host led to the development of
the gene-for—gene hypothesis (31, 67). The gene-for-gene
concept is simply that for each E gene in the plant there is
a corresponding E gene in the pathogen, and the interaction
of these two genes determines disease development (29, 30, 67).
With most diseases studied to date, resistance (E) and
avirulence (E) are dominant and susceptibility (E) and
virulence (p) are recessive (29, 51, 66).

The terms "compatibility" and "incompatibility" have
been used to describe the interactions between host and

parasite and infection types with particular parasite/host

3

genotypes (25). An incompatible reaction, a low infection
type, (resistance when viewed as a characteristic of the
host, avirulence when viewed as a characteristic of the
parasite) is established when at least one E gene in the
pathogen and one corresponding E_gene in the host are
present (E§[E§). “This terminology takes into consideration
the genes in both the host and pathogen that affect disease
development. I

Person 3E_§E. (67) has suggested that the comple~
mentary relationships between host and pathogen have evolved
from specific selection pressures which initially favored
host plants which could escape disease and, secondly,
selection of pathogen strains which could develop in the
presence of the E gene. Studies of pathogenic variability
have shown that many pathogens are capable of rapid genetic
adaption to newly introduced host lines possessing E genes
(68, 83, 84). The discovery of the gene-for-gene relation-
ship controlling parasite/host compatibility has recently
been extended to insect-plant relationships also (40).

Development of the powdery mildew disease is greatly
affected by environmental conditions, i.e. light intensity,
photoperiod, relative humidity, temperature, and so forth
(12, 13, 34, 73, 97). Germination and subsequent development
of conidia occur over a wide range of temperatures and
relative humidities. Several reviews concerning the effects
of various environmental factors on disease development

(53, 58, 73, 97) have been published, each stressing the

variability of the data and the necessity of considering
the role of the environment in altering disease development.
Several different optimal conditions for mildew develop-
ment have been reported (12, 37, 62, 73).

The development of E. ggaminis spores on the plant

 

surface can be divided into several morphologically
distinguishable stages: (1) germination, (2) production

of appressorial initials, (3) formation of mature
appressoria, (4) penetration of the host, (5) formation of
haustoria, (6) develOpment of secondary hyphae, (7) initia-
tion of secondary, tertiary, etc., infections, and (8)
sporulation (12, 37, 55, 97). Each stage in primary
infection has certain temperature, relative humidity, and
light condition requirements for synchronous development of
the parasite (54, 62). When optimum environmental conditions
for each stage are used, over 75 percent of the parasite
population prOceeds through the various developmental stages
with a high degree of synchrony (53, 54, 55, 56). The pro-
duction of elongating secondary hyphae (ESH) by the parasite
suggests that the fungus is securing nutrients and other
essential materials from the host to permit continued fungal
growth (55, 56). Conidia on non-host plants will germinate,
form appressoria, and attempt to penetrate epidermal cells,
but do not form either haustoria or ESH (53, 95). The number
of ESH formed on the host surface corresponds to the number
of haustoria in the host epidermal cells (55). The per-

centage of applied conidia that develOp ESH and, therefore,

form a functional relationship with the host is defined as
the infection efficiency (25, 26).

The fungal haustorium is defined as "a specialized
organ whichflis formed inside a living host cell as a branch
of an extracellular (or intercellular) hypha or thallus,
which terminates in that host cell, and which probably has
a role in the interchange of substances between host and
fungus" (9). Bushnell and co-workers (11) have shown that
fungus development on the leaf surface ceases after haustorial
destruction. Nutrients supplied exogenously have supported
fungal development for a brief time in the absence of a
haustorium. This may indicate an ability of the mycelium
to absorb a limited amount of nutrients.

The E.M. has increased our knowledge of structural.
changes resulting from infection. Some workers believe that .
infection pegs of fungi penetrate the walls of host cells
with the help of enzymatic digestion (22). Regardless of
the degree of dissolution of cuticle and wall, some
mechanical force probably is required for penetration by

the infection peg of E. graminis (85). Small portions of

 

the cuticle have been observed to be pushed inward in the
region immediately beside the infection peg (85). Akai
and co-workers (1) observed a crack in the host wall in the

E.M. section intersecting the infeCtion peg of E, graminis

 

Edwards and Allen (22) conclude that penetration consists
of (l) the enzymatic digestion of the cuticle and cellulose

portion of the epidermal wall by enzymes apparently secreted

by the develOping mildew infection peg, and (2) a mechanical
pushing of the infection peg through a layer of material,
called a papilla, which has been deposited by the host on

the underside of the epidermal wall. The papilla is believed
to be formed after the appressorium attaches to the outer
surface of the hoSt epidermal cell wall, but prior to the
formation of the infection peg (22).

The host may deposit materials, besides papillae,
that form a sheath around the neck and body of the haustorium.
However, papillae and sheaths are so commonly found that
they can be regarded as a normal response to haustorial fungi
and to other plant pathogens if the host cell remains alive
for a short time after penetration (9). Thus these reactions
are probably nonspecific responses to wounding on the part
of the host.

The development of a functional haustorium is
required for continued disease development (9, 25, 55). The
extrahaustorial membrane (the boundary between the host
cytOplasm and the haustorial apparatus), closely resembles
the plasmatmembrane of the host from which it is thought to
be derived (9). In powdery mildew fungi, small irregular
invaginations extend from the extrahaustorial membrane into
the extrahaustorial matrix (24). These invaginations may
be involved in movement of materials across the membrane
from the host cytoplasm to the extrahaustorial matrix, or
in the opposite directions (8, 9). The volume of the

extrahaustorial matrix is composed of (1) an aqueous

solution which contains small amounts of uniformly suspended
particulates and (2) components that do not intermix freely
with the aqueous solution. Hirata and Kojima (42) found a
sac-like membrane (analogous to extrahaustorial membrane)
that began to separate from the haustorial walls as the
haustorium matured. iThe separation occurred earlier if the
mycelium attachment to the haustorium had developed poorly.
From these observations he concluded that the haustorium is
more functional before, than after, the extrahaustorial
membrane separates from the haustorial wall, and that the
separation serves to protect the surviving host cell or
haustorium when one or the other degenerates.

Transfer of materials from host to parasite (44, 58,
71, 80, 87) and from parasite to host (24, 46) has been
shown. Theactual form in which the radioactive labeled
materials are moving across the membranes in the powdery
mildew host—parasite system is not known. If the exchange
of nutrients from host to parasite, and vice versa, are
through the extrahaustorial matrix, both the host and
fungal membrane may act semi-independent of one another. For
active transport of an ion across a membrane three distinct
steps occur (49, 64), namely, (1) formation of the ion—
carrier complex in the membrane surface, (2) translocation of
the complex to the opposite surface and (3) release of the
ion into solution. The translocation of the complex across

the membrane is described as a transport over an activation

energy barrier and is often the rate-determining step in
ion transport (49).

The use of radioactive tracers in studying host-
parasite relationships has made possible the detection of

very small changes during early disease development. Mount

and Ellingboe (59) studied the transfer of 32PO43- and

35 2- . . . . . .
SO4 from wheat to E. graminis f. sp. tr1t1c1 during

32 35

primary infection. The amount of P and S transferred to

the fungus was correlated with the morphological development

of haustoria in the host cells. Slesinski and Ellingboe (80)

358 transfer rates between compatible

35

showed a difference in
and incompatible parasite/host genotypes. Transfer of S
from host to parasite was greatest with the compatible geno-
type. The incompatible genotypes varied in their rates of
transfer. Upon closer examination of three compatible
genotypes (El/BEE, El/Efll' and pl/ggl) involving one pair

of corresponding genes, Slesinski and Ellingboe.(80) found a

35

reduced rate of S transfer with the compatible genotype

El/Bfll than with the other two compatible genotypes El/pgl
and El/Efll° I

Reisener EEIEI- (71) have presented evidence that
certain amino acids and carbohydrates are transferred from
wheat tissue to rust spores. They conclude that glutamic
acid, tyrosine, and hexose are taken up from the host by the
fungus without alteration and that a part of fungal alanine

' 1 4

also comes directly from the host. Furthermore, C in the

fungus was found first in free amino acids and later in

10

protein. This was interpreted to mean that the amino acids
were transported from host to parasite but that the majority
of protein in the fungus was synthesized by the fungus.
Thewfollowing is a brief review of the pathway of
solutes in a plant from a cut stem to the haustorium, and
of other factors that affect solute translocation. Transport
of solutes in the xylem and in leaves has received consider-
able attention (48). Sulfur transport in the xylem is mainly
as the inorganic ion, although methionine transport has been
reported (48). The classical concept that, in xylem vessels,
solutes move with the same velocity as water (mass flow) no
longer holds for Ca-2.+ and probably not for other divalant
and trivalent cations (48). Translocation between the xylem

2- 3-
4 and P04 occur

and phloem of various ions K+, Na+, SO
possibly by xylem rays (98), but little is known about the
mechanism. In the leaves, ions apparently are transported
along with water in the leaf free space which appears con-
fined to the cell walls (15). While being conveyed in the
free space in the direction of the transpiring water, ions
can be transported across the plasma membrane into the

inner compartments of the leaf cells by means of carrier
mechanisms (81). A certain fraction of the ions may not be
exported from the free Space but follow the lateral pathway
of water to the main regions of water loss, i.e. the stomata
and other epidermal cells (15). Some evidence exists that

transport of ions through the mesophyll of leaves may

utilize both the free space and the symplasm as pathways.

11

36Cl- was located in cell walls and the cytoplasm of the

mesophyll cells but not in the vacuoles (99).

Environmental and internal factors undoubtedly affect
the complexdphysiological phenomena of ion transport in
leaves. Many of these factors have not been clearly identi-
fied, but light has been demonstrated to affect movement of
ions in leaves (48). Rains (70) reported that light
increased the rate of K+ uptake in corn leaves possibly
through energy brought about by synthesis of ATP in cyclic
photOphosphorylation. Light may cause stomatal opening
through the regulation of K+ influx into the guard cells
(45). Light nOt only has a controlling influence on ion
transport in leaves by providing the energy for active trans-
port across cellular membranes, but also affects ionic
movements through activation of the phytochrome system (48).

Active transport involves an accumulation of
materials against a concentration gradient. Cells accumulat-
ing solutes should not be viewed only as a system of
selective ion pumps because cells operate via solute
absorption and secretion in regulating the internal activity
of their water. The cell may more accurately be regarded
as a complex type of "water pump" manipulating the
collective prOperties of its water (60).

Plant and fungal cells have been shown to take up
sulfate by an active process (5, 28, 39, 75). Hart and
Filner (39) reported that sulfate uptake in tobacco cells

is inhibited by L-methionine, L—cyst(e)ine, and

12

L-homocyst(e)ine. Bradfield 23.31; (5) reported that

inorganic sulfate entered the mycelia of Aspergillus and

 

Penicillium spp. by a permease transport system that is
affected byatemperature, energy, pH, ionic strength and
substrate concentration. The transport of sulfate was
unidirectional into the cell, resulting in an accumulation
against a concentration gradient. As in tobacco cells,

sulfate accumulation in cells of Aspergillus and Penicillium

 

 

is depressed in the presence of methionine or cysteine
(5, 75). Interpretations given were that either organic
sulfur partially or completely suppresses the sulfate
transport mechanism, or the organic sulfur is oxidised
intracellularly to sulfate causing a dilution of specific
activity, or a combination of these mechanisms operates
(75). Segel and Johnson (75) favored a combination of
several mechanisms because methionine or cysteine suppressed
the intracellular concentration of sulfate by 65 to 80

35

percent and S-methionine fed to mycelium had been con-

verted to free ionic sulfate. Puccinia graminis tritici

 

grew in a culture medium with cysteine as the sulfur source;
it also grew with other sulfur-containing amino acids, but
did not grow with inorganic forms of sulfur (43). Excessive
leakage of sulfur amino acids from the mycelium into the
medium, rather than insufficient sulfate uptake or inability
to convert inorganic sulfur to sulfur amino acids, was
thought to account for lack of growth with inorganic sulfur

as the sole source of sulfur.

l3

Uptake oqu—methionine by mycelium of Penicillium

 

chrysogenum was mediated by two distinctly different,
independently regulated, stereospecific membrane transport
systems (3), One of the systems is relatively specific
for L-methionine and develops under sulfur-starvation con-
ditions. The other is a general (nonspecific) amino acid
permease.v

The effects of both powdery mildew and rust diseases
on host plants appears to be diverse and numerous by several
days after inOculation. Durbin (19) stated that substances
tend to stay at infection sites instead of being recirculated
to growing parts of the plant. Patterns of long distance
translocation apparently are altered so that movement of
organic substances toward the infection site is favored.
Thatcher (89) provided evidence that the permeability of
host cells was increased by rust infection thereby facili-
tating the movement of solutes to cells that contain
haustoria. Edwards and Allen (21) have shown that the

infection site is the major site of 14C accumulation in

plants given 14C02. Transfer of carbon from host to para-

site tissue was observed 15 minutes after plants were

allowed to take up 14C02. The accumulation of 14C in the

parasite occurred after the photosynthetic ability of the

infected host tissue was reduced (2, 22, 74). Therefore,

14C must be translocated from healthy to diseased areas of

14

the plant. Edwards (20) stated that C given to primary

leaves accumulated at the infection site when the fugus

14

was sporulating. Healthy leaves had 14

14

C distributed uni—

formly in the leaf. C in secondary leaves did not move to

14

the infection sites on primary leaves; only the C in pri-

mary leaves moved to the infected sites on primary leaves.

358 was found to accumulate at infection sites of two to four

day old colonies but not ten day old colonies if 35

35

S was given

after colonies were established (96). If

before inoculation, accumulation of 358 was not detected at

S was given

infection sites by three days after inoculation, but was
detected by ten days (96).

Increased respiration was observed in areas of
barley leaves adjacent to the cells invaded by the fungus,
but these respiratory increases were notdetected until six
days after inoculation (2, 10). Respiratory indreases begin
with the appearance of visible infection flecks (76).
Transpiration rate and average stomatal aperature of primary
bean leaves were significantly reduced during the fleck
stage of rust development (18). Duniway and Durbin (18)
reported that from four to six days after inoculation,
diseased leaves had less transpiration than healthy leaves.
Transpiration at night was slightly less with diseased
leaves than healthy leaves until five and one half days
after inoculation. Greatly increased transpiration of
diseased tissue was observed after five and one half days.
The increased transpiration of diseased tissue was con-

sidered to be due primarily to excessive cuticular

15

transpiration caused by the disruption of the cuticle and
epidermis by the fungus (18).

When uredospores of Puccinia ggaminis labeled with

 

14C were inoculated onto wheat leaves, radioactivity was

transferred throughout the leaf by 24 hours after inoculation
(23). At the time of fleck reaction, about six days after

35

inoculation, most of the S was in the inoculated primary

leaves (46). However, some 358 was uniformly distributed
in tissues of the second leaves and the roots.

Increases in protein synthesis, especially of
particular enzymes, have been reported to occur anywhere
from two to ten days after inoculation of resistant tissues
,(72' 74, 82, 91, 94, 100). The amount of RNA increased

parallel to the increase in respiration following infection

of susceptible wheat leaves by E. graminis tritici (41).

 

Changes in the synthesis of soluble proteins were
reported as early as six to 18 hours after inoculation of

flax with Melampsora lini (92). The rates at which various-

 

sized proteins were synthesized varied for each of six
different parasite/host genotypes. In general, the net rate
of protein synthesis increased in the four incompatible
reactions but did not increase in the two compatible inter-
actions. Increases in enzyme activities have been reported

as early as six hours after inoculation (38, 69). Preliminary
studies (92) indicate the rate of RNA synthesis likewise is

increased within six hours in an incompatible interaction.

16

The latter was interpreted to suggest changes were occurring
at the transcriptional level.

Some of the metabolic alterations observed in diseased
tissues arer,similar to those occurring in detached leaves
(27). Thus, many of the parasitically induced metabolic
alterations of hosts are highly unspecific (27). One classic
example of how easy it is to confuse cause and effect
relationships is presented. Increase in peroxidase activity
had been attributed to activity of the S E locus to give

resistance in wheat to Puccinia graminis. Daly gE_§l. (l7)

 

compared two wheat lines with different genes, §£.§ and §£_EE,
and detected no significant changes in phenolic components
with resistant and susceptible plants to race 56 of E.

graminis tritici. The increase in total peroxidase activity

 

due to the same isozyme in resistant reactions was similart'
with E£_EE and §£.§° Because the genetic and physiological
bases for resistance controlled by E5 E; and §£.E are
distinct, it is concluded that increased activity of the same
isozyme in both instances is a result of a nonspecific event

analogous to wounding (l7).

MATERIALS AND METHODS

Maintaining Powdery Mildew

The strain MS-l of Erysiphe graminis D.C. f. sp.

 

tritici Em. Marchal was maintained on susceptible wheat,
(Triticum aestivum L) cv. ‘Little Club‘ for the production

of inoculum for all experiments. Thirty to 40 wheat seeds

of Little Club were planted in four inch pots daily. Wheat
plants were inoculated daily when they were seven days old.
Conidia produced seven days after inoculation were dusted
onto the leaves of Little Club wheat. Inoculated plants were
maintained in a controlled environmental chamber providing
adequate air circulation under the following environmental
conditions:

1. Light period was 15 hr/day. Total light intensity
was 700 to 800 foot candles (650 to 750 foot candles from
white VHO flourescent tubes and 50 foot candles from 25 watt
incandescent bulbs).

2. Temperature was 18 f l C during the light period
and 17 i l C during darkness.

3. Relative humidity was 65 : five percent during
the light period and 95 : five percent during darkness.

Plants were watered daily.

17

18

Mycelial growth was macroscopically evident by
three to four days after inoculation. Conidia produced on
the sixth day after inoculation were used for experimental

purposes in all experiments.

Methods of Inoculation

Controlled Inoculation

Single five-to-six-day-old plants in two inch pots
were inoculated by the rolling method (61) in all experi-
ments for study of morphological development of the fungus.
during primary infection. Conidia from six-day-old
inoculated plants were dusted onto clean glass slides and
transferred to the abaxial leaf surface of single wheat
plants with a cotton swab. Conidia on the glass slide were
examined directly with a compound microscope at 125x
magnification. If more than ten percent of the conidia
were collapsed, the experiment was terminated and fresh
conidia collected at a later time. This method of inocu-
lation provided a uniform distribution of single conidia
ranging from 100-200/cm length of leaf. .Only single, well
separated parasite units were examined at each observation
to eliminate any effect caused by spore crowding or
clumping (53). Infection kinetics are similar on either
side of the leaf by microscopic observations and removal

of host epidermal strips are easier on the abaxial side (58).

19

Inoculation by DuSting

Plants were inoculated by gently dusting conidia
produced six days after inoculation onto the leaves of five
to six-day-Qld wheat seedlings. Inoculation by dusting
resulted in 1200-1500 conidia applied/cm length of leaf.
The development of the fungus during primary infection was
similar following use of either method, but the efficiency

of infection was reduced by dusting.

Environmental Conditions for Experiments

Sherer-Gillett (Model CEL 512-37 and Model CEL 25-7)
growth chambers were used for all experiments. The conditions
used for high efficiency of infection and synchronous growth
of each developmental stage of infection are as follows:

1. From 0 to one hour after inoculation, plants
were kept in darkness at 18 i l C and at approximately 100
percent relative humidity (RH). .

2. From one to six hour, inoculated plants were kept

5 '1 (0.25 x

in a light intensity of 1.1 by 10 ergs cm-Zsec
105 ergs cm-zsec-1 from white VHO-fluorescent tubes and 0.9 x
105 ergs cmflzsec"1 from 25 watt incandescent bulbs), 22 : 1C,
and RH 65.: five percent.

3. From six to twenty hour, plants were kept at the
temperature and RH as stated in (2) above, but in darkness.

4. From 20 to 30 hour, conditions were the same as

in (2) above.

20

Changes in temperature and relative humidity during
experiments were monitored with wet and dry bulb thermometers
and with a recording hygrothermograph calibrated with a sling
psychrometer. Light intensity was measured at the distance
of the plant from the lights with a YSI Kettering Model 65
Radiometer.

Counting Efficiency of 3H-methionine
35 2-
and SO

 

 

NJ:-

Counting efficiency for H-methionine was found to

be from 14 to 24 percent depending on preparation of the
35 2-

SO averaged 77

cocktail. Counting efficiency for 4

percent.
Other experimental conditions are given with each

chapter of results.

CHAPTER I

GENETIC CONTROL OF MORPHOLOGICAL
DEVELOPMENT OF E. GRAMINIS
ON WHEAT

Introduction

 

A high percentage of spores of Erysiphe graminis

 

f. sp. tritici on a wheat leaf will germinate, produce
appressoria, haustoria, and elongating secondary hyphae if
given the appropriate environmental conditions and if the
parasite/host genotype specifies compatibility (54, 78).
With incompatible genotypes the same percentage of spores
germinate and produce appressoria but few of them produce
secondary hyphae, but only parasite units that have
haustoria appear to be able to produce secondary hyphae
longer than 10 u (55, 78). Secondary hyphae less than ca.
10 u have not been observed to produce secondary appressoria
and haustoria. Therefore, those elongating hyphae ceasing
growth before 10 u in length are considered to be none
functional.

Several of the incompatible parasite/host genotypes
affect the percentage of parasite units that produce
elongating secondary hyphae (78). A few elongating secondary
hyphae are formed with all genotypes, but the percentage that
develop is dependent on the particular parasite/host geno-

type (78).
21

22

The objectives of the research reported herein were
(1) to collect quantitative data on the kinetics of
elongation of secondary hyphae as a function of time after
inoculation and (2) to determine if the secondary hyphae
which do begin to elongate with incompatible genotypes
develop similarly to the development of secondary hyphae

with compatible genotypes.

Materials and Methods

 

Erysiphe graminis f. sp. tritici, genotype E; E;

 

Egg Eg_(51, 79) (MS-l) was maintained on wheat cultivar
Little Club. Environmental conditions for maintenance of
cultures, procedures for the production of inocula, and
procedures to obtain high infection efficiency and synchro-
nous development of the parasite were described previously
(54, 55, 56). Controlled inoculations on the abaxial leaf
surface were made by the rolling method (61).

The five homozygous lines of wheat used were
[designated as follows (6, 7):
pml pm2 pm3 pm4 Pml pm2 pm3 pm4

= (pmx or = Rflli
pml pm2 pm3 pg4 Chancellor): Pml pm2 pm3 pg4

 

 

 

 

 

 

pml Pm2 pm3 pm4 pml pm2 Pm3a pm4
= Pm2; = Pm3a;
pml Pm2 pm3 pm4 pml pm2 Pm3a pm4

 

 

pml pm2 pm3 2E4

 

= Pm4.
pml pm2 pm3 Pm4

.-

 

Each of the four loci (Pml, Pm2, Pm3a, and Pm4) are

 

 

known to condition different reactions to powdery mildew.

23

The host genotype is abbreviated in this study as Egl rather
than as a diploid Bfll.gfllv since the wheat lines used are
homozygous for these genes. The compatible parasite/host
genotype, Efi/EEEI was the standard to which all other geno-
types were compared. The incompatible genotypes were El/Efll'
EE/sz, P3a/Pm3a and Eg/EEE. Parasite/host combinations are
referred to by the corresponding genotypes which specify
compatibility or incompatibility of the relationship. The
El/Efll genotype specifies incompatibility. The Egl_gene in
the host recognizes only the El_gene in the pathogen, even
though the pathogen possesses many other E genes. A corres-
ponding gene pair which conditions incompatibility is
expressed in the presence of any number of other corresponding
gene pairs specifying compatibility (80). Only the part of
the parasite/host genotypes that specifies incompatibility
is given.

The lengths of secondary hyphae were determined at
various times after inoculation by direct microscopic
measurements of all single, isolated parasite units on leaf
sections (l-cm long) from 1-2 cm from the tip portion of
the leaf.

The data are presented as percent of the total
number of conidia applied to the leaf having secondary
hyphae according to length and time of observation. New
leaf sections were used for each observation. From 100—150

parasite units were counted on a single leaf for each

24

observation. Results are the averages of at least five

experiments repeated on different days.

Results
Meaningful interpretation of results in relation
to work already reported (55, 58, 62, 79) requires a common
departure point. The common base for the research reported
herein was the ability to reproduce the essential stages

of synchronous morphological development of E. graminis f.

 

sp. tritici on wheat during primary infection (55, 58, 62).
Nearly identical results were obtained for spore germination,
appressorial maturation, and elongation of secondary hyphae
as was reported by Slesinski (77) (Figure l). Thirty-five
percent of the spores germinated by one hour after inocula-
tion. By four hours after inoculation, 93 percent of the
spores had germinated. Mature appressoria were observed on
35 percent of the spores by six hours after inoculation and
92 percent by eight hours. Initiation of secondary hyphae
was observed between 16 and 18 hours after inoculation.
Secondary hyphae greater than 10u in length were observed
on 17 percent of the spores by 20 hours after inoculation.
The percentage of secondary hyphae lOu long increased rapidly
Ibetween 22 and 24 hours and reached 75 percent by 30 hours
after inoculation with a compatible parasite/host genotype.
At 20 hours after inoculation with the genotype
Efi/Efliv 68 percent of the parasite units had either no

secondary hyphae or secondary hyphae Su or less in length,

[III III

 

25

.sost menu me

OOCHDDQO muasmou Eonm A V
.Aeec mecemoem A rrrrrrrrrrrrrrr c

.mcoH SoaA mmnmwn mnmccoomm ADV

can .mwuommoummm OHDUNE Amy
.coflumcHEHom A¢v

.mm>moa Moons
co HOHUHHu .mm .m mflcHEmum onmflmmum mo
ucmEmoao>m© msocounocwm mo coaumEHHMCOUII.H onsmflm

26

.H muamwm

 

15 20.53302. 55 us:
8 cu «a as o. m o
Razz 4...; I II...
ol\\\ro

 

 

1 ‘IVI‘I;II| ii'vtlz.v :r a 91.33‘; tl‘

 

JOVINSDHJJ

27

16 percent were 6-10u, 16 percent were ll-lSu, one percent
were 16-20u, and no parasite units had secondary hyphae
greater than 20u long (Table 1, Figure 2). By 24 hours
after inoculation, more parasite units have secondary hyphae
ll-lSu long (38 percent) than in any of the other classes
of lengths. Thirty-three percent were 6-10u long. By 30
hours after inoculation 37 percent secondary hyphae were
greater than 20u long. The distribution of lengths of
secondary hyphae as a function of time after inoculation as
presented in Figure 2A shows that secondary hyphae elongation
occurred from 20 hours through 30 hours after inoculation.

The El/EEE genotype was shown to affect infection
efficiency (78, 79), and in this experiment 15-20 percent
of the _secOndary hyphae were longer than. 10 u.
(Table 1, Figure 2B). Of those secondary hyphae that did
get longer than lOu, only a few elongated to more than lSu.
There was no significant change in the distribution of
secondary hyphae from 24 to 30 hours after inoculation.

In none of the previous experiments (78) has it
been possible to demonstrate an effect of the EE/Egg_genotype
during primary infection. Most of the parasite units (77
percent) eventually produced secondary hyphae longer than
lOu, and most secondary hyphae became much longer than lOu
(Table 1, Figure 2C).

The Egg/E333 genotype reduced secondary hyphae that

got longer than lOu to ca. 30 percent (Table 1, Figure 2D).

2E3

TABLE l--Effect of genotype of host and pathogen on
development of secondary hyphae.

 

Genotypes Hour after % of parasite units with secondary Functional secondary

 

 

(parasite/ Inoculation hyphae of a given length hyphae (%)
host) O-Su 6-10u ll-lSu 16-20u >20u (>10u in length)
Ei/EEE 20 68 16 16 1 0 17
22 56 26 17 2 0 19
24 17 33 38 12 0 50
26 14 20 35 21 10 66
30 16 12 25 ll 37 73
El/Efll 20 83 10 6 O 0 6
22 74 19 7 l 0 8
24 66 18 14 1 O 15
26 56 25 14 4 1 19
3O 63 23 10 2 2 14
EE/EEE I 20 67 21 10 l 0 ll
22 54 28 16 3 0 19
24 18 28 41 ll 2 54
26 14 14 30 27 14 71
3O 12 11 14 10 53 77
233/3333 20 77 14 8 1 O 9
22 71 19 8 l O 9
24 50 31 16 2 O 18
26 45 24 16 10 4 3O
3O 47 23 17 6 6 29
Efi/Emfl 20 85 ll 4 O O 4
22 79 14 6 1 O 7
24 77 17 4 l O 5
26 76 17 5 2 O 7

3O 75 18 5 1 O 6

 

29

Figure 2.--Effect of five different parasite/host genotypes
of the develOpment of secondary hyphae of E.
graminis f. sp. tritici. (A) genotype E§7RE£7
(B) genotype Pl/Pml; (C) genotype P2/Pm2;

(D) genotype P3a7LfiBa; and (E) genotype —P4/Pm4.
Those secondary _hyphae grouped in class 25 refer
to all secondary hyphae greater than 20u long.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

31

The secondary hyphae on some of the parasite units became
more than 20 u long.

The Efl/Efli genotype reduced secondary hyphae that were
longer than 10 u to ca. 5 percent (Table 1, Figure ZE). Few
secondary hyphae were more than 15 u long and none were
longer than 20 u. The maximum percentage of secondary
hyphae longer than 10 u was reached by 22 hours after

inoculation.

Discussion

 

The data suggest that it is possible to observe,
with time, the distribution of a parasite population having
no, or predominantly very short, secondary hyphae to a dis-
tribution of a parasite population having predominantly
secondary hyphae greater than 10 u. The data with the
compatible genotype Ei/Efli (Figure 2A), or with the incom-
patible genotype EZ/EEZ.WhiCh has not been shown to affect
primary infection (Figure 2C), suggest that, once the
formation of secondary hyphae is initiated, secondary hyphae
elongate at the rate of about 5 u every two hours. This is
a much slower rate of elongation than observed with E.
graminis f. sp. hordei (44). The observation that some
secondary hyphae do not elongate more than 20 u is con-
sidered due to the initiation of secondary appressoria.
Formation of secondary appressoria are believed to begin
about 26lunnxsafter inoculation with the environmental

conditions used.

32

Three genotypes have been shown to affect the percent-
age of secondary hyphae that attain a length >10 u (78)(Figure
23, 2D, 2E). 32/221 not only causes a reduction in the
percentage of parasite units that produce haustoria and
elongating secondary hyphae, but also causes a collapse of
most parasite units that produce haustoria and a discoloration
of the host cell adjacent to the haustorium (77). The
collapse of the parasite units that have produced haustoria
was observed at about 21 to 22 hours after inoculation (77).
The data presented here suggest that there is no detectable
increase in the lengths of secondary hyphae subsequent to 22
hours after inoculation (Table 1, Figure 2D).

Bl/Efll was also observed to affect the percentage of
parasite units that produce haustoria and, therefore, capable
of producing secondary hyphae longer than 10 u (77). More
than half of the parasite units that produce haustoria
collapse about 26 to 28 hours after inoculation. The data
presented herein suggest that cessation of elongation of
secondary hyphae longer than 10 u occurred by 26 hours after
inoculation, or earlier.

BEE/£232 was also shown to affect the percentage of
parasite units that produce haustoria and elongating secondary
hyphae (77, 78). The data presented herein suggest that ca.
30 percent of the parasite units that do produce haustoria
also produce secondary hyphae that are longer than 20 u.

The relative proportions of secondary hyphae in the 11 to 15,

33

16 to 20, and >20 u length ranges is approximately the same
as with the compatible genotype EE/EEE through 26 hours
after inoculation. The parasite units which are successful,
in producing haustoria have secondary hyphae which appear
to elongate to the same extent as with a compatible genotype
the first 26 hours after inoculation.

We interpret results presented herein to provide
additional supporting evidence that the different genotypes
affect different stages in the ontogeny of interactions

between host and parasite (25).

Summary 7
The lengths of secondary hyphae of E. graminis f. sp.

tritici at 20 to 30 hours after inoculation varies with the
genotype of host and parasite. With a compatible parasite/
host genotype (EE/EE§)' or the incompatible genotype (EE/EEQ)
which does not affect primary infection, secondary hyphae
elongate at about two to three u/hours. The secondary hyphae
that do become longer than 10 u with the incompatible geno-
type, EEE/EEEE, appear to continue to elongate the first 26
hours at the same rate as do hyphae with the compatible
genotype. The secondary hyphae which begin to elongate on
the incompatible genotypes El/Egl_and 25/323! cease
elongation at about 26 and 22 hours after inoculation,

respectively.

CHAPTER II

358 UPTAKE BY WHEAT AND TRANSFER TO E.

GRAMINIS F. SP. TRITICI AS
AFFECTED BY ENVIRONMENTAL
CONDITIONS

Introduction

 

Compatible and different incompatible parasite/host
genotypes have shown altered rates of nutrient transfer from
host to parasite during primary infection (80). Much emphasis
has been placed on develOping environmental conditions that
favor a high degree of synchrony of the parasite during the
distinct morphological stages of parasite development that
occur during primary infection (55, 58, 62). A synchronous
parasite population allows the association of a particular
morphological stage of the parasite with the time of
greatest rates of nutrient transfer from the host to the
parasite. Unfortunately, little attention has been given to
the effect of the fungus on the host and to the effect on
the host of the environmental conditions used for synchronous
parasite development. Do the environmental conditions used
affect the amount of label (358) in the host that would be
available to the parasite? If so, would the apparent time

of greatest rates of 35

S transfer from host to parasite be
altered? The remainder of this chapter gives insight into
these questions and has been submitted for publication.

34

35

The environmental conditions necessary for synchronous
development of Erysiphe graminis f. sp. tritici on wheat
(54,62) during primary infection have been established. Sixty
percent of the spores germinate on seedlings kept in dark-

ness at 100 percent RH and 17 C. Forty percent of the spores

5 erg/cmZ/s of

applied to a host leaf germinate with 1.1 x 10
incandescent and fluorescent radiation, 65 percent RH, and
22 C. More than 90 percent of the spores germinate by using
the first treatment followed by the second treatment from
0-1 hours and 1-6 hours after inoculation, respectively (25,
54, 61). Appressorial formation and maturation are favored
by fluorescent and incandescent radiation totaling 1.1 x 105
erg/cmZ/s with 65 percent RH at 22 C. Penetration takes
place in darkness. The next morphologically visible stage
on the surface of the leaf is the formation and elongation
of secondary hyphae, which is also favored by light at

5

1.1 x 10 erg/cmZ/s. For the parasite population to develop

synchronously for each of the morphologically distinct
stages, the following environmental conditions following
inoculation are employed: 0-1 h, 17C, 100 percent RH,

5

darkness; 1-6 h, 22C, 65 percent RH, 1.1 x 10 erg/cmZ/s

radiation; 6-20 h, 22C, 65 percent RH, darkness; and

5 erg/cmZ/s radiation

20-30 h, 22C, 65 percent RH, 1.1 x 10
(54, 87).
These environmental conditions are known to induce

synchronous development of the parasite population on the

host but little is known about their effect on the host.

36

Use of environmental conditions that give parasite synchrony
make it possible to test the effects of parasite/host geno-
types on transfer of labelled materials to the parasite.
Previous workers (80) have reported that rates of 358
transfer from host to parasite are affected by the genotype
of both parasite and host. The rates of transfer were
assumed to be dependent primarily upon the parasite/host
genotype and the stage of morphological development of the
parasite. The rate of transfer from host to parasite was
determined more accurately by calculating rates of transfer/
10,000 spores (44, 87).

The Objective of this study was to: (1) determine

358 in the host under the

the uptake and translocation of
environmental conditions used for synchronous parasite
development; (2) place the rates of transfer from host to
parasite considering the amount of label in the host epi-
dermis that would, theoretically, be available to the

358 transfer with the

parasite; and (3) correlate rates of
morphological stage of development of the parasite. An
abstract describing part of this paper has been published

(87).

- Materials and Methods

 

Culture MS-l of Erysiphe ggaminis f. sp. tritici

 

was cultured on wheat (Triticum aestivum) cv. 'Little Club'

 

which has no known genes for resistance. The environmental

conditions under which these stock cultures were maintained

37

have been described (54, 62). Wheat plants were uniformly
inoculated with mildew by dusting five to six dayaold plants
with conidia from stock cultures. The five near-isogenic

wheat lines were designated as pmx (Chancellor), Pml, Pm2,

 

EEEE, and Egg (6, 7). The cultivar Chancellor is not known
to possess any BE genes and was the recurrent parent in the
establishment of each of the other host lines. Each of the
Eg_genes is known to be unique and independent for reaction
to E. graminis f. sp. tritici. Culture MS-l possesses the
£1.23,322.31 genes conditioning incompatibility with each
corresponding gene Egg. Egg. E333. or Egg, respectively,

in the host (51, 65).

The uptake and translocation of 35

S by wheat
seedlings were determined (87) at various times after
inoculation. Five day-old wheat seedlings were cut at the
crown with a razor blade (with a film of water on the cutting
edge) and placed into small vials (25 x 5mm) that contained
23S$04

buffer (pH 6.9). After five hours, each leaf was removed

0.1 ml of a 100 uc/ml solution of H in 0.1 M NarK-PO

4
from the vial, placed on a paper towel, the tip 1 cm was
discarded, and a section (1 to 2 cm long) taken below the
tip was coated with a thin film of 1.9 percent parlodion
(Mallinckrodt Chemical Works) in 60:40 (v/v) ether: alcohol
solution. The parlodion film (containing the eCOparasitic
portion of the fungus) was dried, carefully removed, and
placed in a scintillation vial for determination of radio-

activity by liquid scintillation counting. To determine

38

leaf activity, l-cm long leaf sections were taken from the
area from which the parlodion was removed and four sections
each from different leaves were placed in scintillation
vials and dried. Fifteen ml scintillation fluid ([5 g 2,
5-diphenyloxazole and 0.1 g l, 4-bis-2-(5-phenyloxazolyl)
benzene in 1 liter toluene]) were added to each vial. Radio-
activity was determined in a Beckman LS-133 Liquid Scintilla-
tion spectrometer. Epidermal sections were taken in the
same manner. After removal of the parasite from the leaf
surface, the epidermis was removed from the leaf with for-
ceps and a razor blade. All chlorOphyllous tissue was
removed from the epidermal strips and the percent from each
l-cm length of epidermis recovered estimated. The total
epidermal strips recovered from four 1-cm sections was
generally the equivalent of 2.0-3.0 intact sections. These
results were adjusted to 4.0 intaCt sections to compare the
radioactivity in the epidermis with the radioactivity of an
equivalent area of the leaf. The epidermal strips were
counted in the same manner described for the leaf sections.
The results for leaf section activity and epidermal activity
are the average of ten replications taken on five or more
different days.

To determine the rates of transfer from host to
parasite, four parlodion strips were placed in a scintillation
vial, dissolved with 0.2 ml 60:40 (v/v) ether : alcohol

solution at room temperature overnight, 15 ml scintillation

39

fluid was added and the radioactivity counted with a Beckman
LS-133 Liquid Scintillation spectrometer. The rate of

35

transfer of S from host to parasite without regard to

level of label in the host was plotted as reported earlier
(80, 87). These same results were also plotted as radio-
activity per 10,000 Spores and per 25,000 CPM in the
epidermis (87). Results are the averages of six repli-

cations each taken on a different day.

Results

The 35

S activity in the leaf sections and in the
epidermis after five hour uptake periods during primary
infection is presented in Figure 3. Each point represents
the radioactivity at the end of a five hour uptake period.
For example, the point six hours after inoculation in leaf
sections represents the activity in plants that have been
allowed to take up 355 from three to eight hours after
inoculation, thus having three hours (3-6 hours) in the
light and two hours (6-8 hours) in the dark. The points

at 12, l4, 16, 18, and 20 hours after inoculation represent

35

uptake of S in darkness. Data representing the radio-

activity in the leaf at 22, 24, and 26 hours after

35

inoculation have taken up S for three hours in dark and

two hours in light, one hour in dark and four hours in light,

and all five hours in the light, respectively. Results

35

show that plants allowed to take up S in the light have

more than two times more activity than plants taking up

358 in the dark.

40

Figure 3.--Effect of light and darkness on up§§ke and
translocation to the epidermis of S in cut
wheat seedlings inoculated with Erysiphe

raminis f. sp. tritici under environmental
conditions used for synchronous parasite
development in primary infection.

 

10"

(”5) CPM x

20

_g—l
MO

LEAF SECTION

EPIDERMIS

Cd

41

 

 

 

mwhuouco

 

l

8
TIME AFTER

 

 

/°\./°/

l l _‘1
I2 16 20
INOCULATION (h)

H_.|_
24 26

Figure 3.

 

 

42

Data for radioactivity in the epidermis show the
same relationship. Plants taking up 358 in the light have
three to four times more label in the epidermis than do

358 in the dark.

those plants taking up
About 20 percent of the label taken up by the leaf
in the light appears in the epidermal strip (Figure 3).

35

Leaves that took up 8 in the dark have a smaller.percentage

of the label in the epidermal strip.

The light has a striking effect on transfer of 35$
from host to parasite (Figure 4). When lights remained off
from 20 to 26 hours after inoculation, transfer remained

low (ca 500 CPM). When lights were on, about 2800 CPM

were transferred from Chancellor wheat to E. graminis f. sp.

 

tritici by 26 hours after inoculation. The difference in
transfer may be attributed in part to lower uptake and

35$ in the host in the dark. There also

translocation of
appears to be an effect of the light on morphological develop-
ment of the parasite (Figure 5). The fungus in the dark
(Figure 5) failed to produce secondary hyphae at the usual
rate (88).

The rates of 35

S transfer from the host to parasite
of five different parasite/host genotypes at various times
after inoculation are shown in Figure 6A. Transfer rates
for the three incompatible parasite/host genotypes (El/Egl.
222/2332! Bfl/Bfli) appear to level off after 18 to 22 hours.

The rates for the compatible parasite/host genotype (EE/pmx),

Figure 4.--Effect of light on 3

43

5S transfer from Chancellor

wheat to E. graminis f. sp. tritici from 20 to
26 hours after inoculation:

 

 

 

 

 

OBOE?

each

inoculated plants in light,

inoculated plants kept in

dark,

non-inoculated plants in light,
and

non-inoculated plants kept in
the dark.

44

3
D\
0%) 2
X
2.
Q.
U D
In” *
02_' 1’
I ;
. I
/O .-
g /° \0—0 .'
aafl’T”o I
I O /. I
.§O—_a—_———:'::————. I
o S ; .-. , J

o 16 18 ‘ 22 24 26
TIME AFTER INOCULATION (h)

Figure 4.

45

Figure 5.--Effect of light on the percentage of secondary
hyphae (>10 u long) formed by E. graminis f.
sp. tritici on Chancellor wheat 20 to 26 hours
after inoculation.

 

46

 

IOO
n—o LIGHT
o—o DARK o
2 ~ % ; V
A a 3
I 60 / I .1.
m i
>- I
I I
E:- - o |
g 40 I
Z ;
O ‘ .
U 0
Lu ? I
m / g
as 20 c e.
! T
0L. » ~ ~ ' 3
O 20 22 24 26

TIME AFTER INOCULATION (h)

Figure 5.

47

.umon on» no mmwuum HMEHOCHQO
on» 8e sec ooo.m~\oouoEm ooo.oe\zoo Ame

.mouomm ooo.oa\zmu Adv

.wofluwnp
.mm .m mflcflfimmm mnmwwmum on mOCHH ummsz
DecomOmfluumoc O>Hm Bonn Mommcmuu mmm mo mODMMTT.m ousmflm

 

 

.o Tasman

 

 

48

 

      

 

3 20.3302. «we? 22: 3 20.3302. 5:... as:
on Va «m cm 2 0— F: o— 0 on Va «N ow 2 o— 3 o— o
1 a q u . u u «q u u o m u u - - JLquu - O
v m w)
0 n
/... 6
.a
flu
.. m A d
W
X
N— .7. NW.
m u.
.O__OucO;Uun.ln .0
L0. w I\ VEmdllld .O—W
M" 00.:mdlll4 nu
:. N.bm0lllo c.
l\\\l ”H ppcmOIllo mw
18W .on
I

 

 

 

 

 

 

 

49

and the incompatible genotype BE/EEZ.WhiCh does not affect
primary infection, continue to increase with time after
inoculation.

The same transfer data were plotted as radio-
activity per spore considering the amount of label in the
epidermal strip (Figure 6B). Genotype differences are still
evident but the shapes of the curves are changed. Transfer
rates for all genotypes are maximum by 18 to 20 hours after
inoculation. The rates of transfer appear to decrease or to
level off after 20 hours with the EE/EEE_and EE/EEE geno-

35

types. Transfer of S definitely decreases after 20 hours

with the genotypes El/Pml, P3a/Pm3a, and Eg/Pm4.

 

Discussion

 

The effect of several different incompatible parasite/

host genotypes on rates of 35

S from the host to the parasite
were described in a previous report (80). There were also
attempts to correlate transfer rates to the morophological
stage of parasite development with compatible genotypes.

Work described here verifies the differences and correlations
observed earlier. In addition, we have shown that environ-
mental conditions, which are varied to obtain high efficiency
of infection and synchronized development of the parasite,

will greatly affect the amount of 35

S taken up by the plant.
The amount of label available in the host will affect the
observed rate of transfer to the parasite. Light appears to
be a major factor in uptake by the plant, and in transfer

from plant to fungus.

50

The amount of 35

S collected in the intact leaf and,
to a greater extent, in the epidermis is much higher in
light than in darkness (Figure 3). This is presumed to be
the result of greater transpiration rates by plants in the
light. The amount of residual solution left in vials showed
that plants labelled in the light had a greater loss than
those kept in the dark (unpublished data). Apparently,
there is no selective absorption because the amount of 358
remaining in vials was equal on a volume basis for plants

that had taken up 35

S in the light or in the dark. Approxi-
mately 20 percent of the label taken up by the plant under
light conditions is in the epidermal strips, whereas only

ca 10 percent is found in the epidermal strips of plants
labelled in the dark. The light, therefore, affects the

amount of 35

S taken up by the plant and also the amount
translocated to the epidermal strips.

Low rates of transfer from plant to fungus occurred
in the dark at 20 t026hours after inoculation (Figure 4) .
However, the fungus did not develOp secondary hyphae as
rapidly in darkness as in light during this period (Figure
5). Slower morphological development may have accounted for
part of the lower transfer activity. However, failure of
transfer activity to increase at 24 and 26 hours with
increasing percentages of elongating secondary hyphae in
the dark lead the authors to believe the absence of light

to be the major reason for failure to get increased

transfer rates.

51

Data on the effect of genotypes on transfer from host
to fungus confirm earlier results (80), but are believed to
be more precise because the data are calculated on a per
spore basis (Figure 6A). The two parasite/host genotypes
that do not affect primary infection, i.e., EE/EEE. EE/EEE'
give rates of transfer that continue to rise 26 hours after
inoculation. The three parasite/host genotypes that do
affect primary infection, i.e., El/Efll! BEE/£222! and Efi/EESL
give rates of transfer that level off at various hours after
inoculation.

The rates of transfer from host to parasite appear
quite different when the results are plotted as radio-
activity per spore per unit radioactivity in the epidermal
strips. At 18 to 20 hours after inoculation the rates of
transfer appeared to be low by radioactivity per spore
(Figure 6A) but adjustment for activity in epidermal strips
shows that this is the time of most rapid transfer in
primary infection.

The data (Figure 6B) indicate that the most rapid
rates of transfer occur earlier than was previously thought
‘ (59, 80). At 18. toZO hours after inoculation, the period of
Ithe most rapid rate of transfer, the haustorial bodies
are forming appendages and secondary hyphal growth is
initiated (80). It would be interesting to know whether
or not the rates increase again after secondary penetration,
haustorial formation, and further fungal growth. This

question is difficult to answer because we have no

52

method of maintaining synchrony of fungal development during
secondary infection. We are attempting to develop such
methods.

These data support previous conclusions that transfer
will vary with the morphological stage of fungal development,
and with genotypes of both host and fungus (59, 80). Also,
it is evident that the rate of transfer is affected by the
amount of radioactive label in the host epidermis. This
amount will vary with the environmental conditions during

primary infection.

Summary

Light is necessary at specific times during primary

infection for synchronous development of Erysiphe graminis

 

f. sp. tritici on wheat. The effects of the environmental

conditions on the uptake and translocation to the epidermis

of 358 and subsequent transfer to the parasite in the

inoculated host were examined. Inoculated wheat plants were

35

cut and fed S for various five hour periods beginning one

to 21 hours after inoculation. Radioactivity was determined
in l—cm leaf sections, epidermal strips, and the fungus on
the leaf surface. Leaf sections and epidermal strips

incubated in light yielded 20 and 4.5 x 104

CPM, respectively,
which is more than twice the CPM obtained for plants incubated
in darkness. Radioactivity in the fungus on the leaf sur-
face ranged from background to 2100 CPM. The rates of 358

transfer from host to parasite were affected by the amount:

53

of tracer absorbed by the plant, the stage of development
of the parasite during primary infection, and by parasite/
host genes for compatibility. There were differences in

rates of 35

S transfer between four different parasite/host
genotypes for incompatibility (El/Egl. EE/EEE, BEE/£222!
and Efi/Bfli) after considering the amount of label in the
host epidermis. Rates of 358 transfer from host to I
parasite during primary infection are greater at 18 to 20
hours after inoculation than at later times. Haustoria are

not fully developed at these times.

CHAPTER III

UPTAKE OF RADIOACTIVE ISOTOPES BY WHEAT
LEAVES, THE DISTRIBUTION AND FORM OF
RADIOACTIVITY IN WHEAT LEAVES, AND
TRANSFER OF RADIOACTIVITY FROM
HOST TO PARASITE

Previous research has raised questions about the
relationship between morphological development of the para-
site and the kinetics of 358 and 32P transfer from host to
parasite. Of particular interest has been to determine
(1) whether or not there is an interaction between host and
parasite prior to penetration of the host cell by the para-
site, (2) the chemical form of the radioactive label in the
host and the form in which label is transferred from host
to parasite, and (3) whether transfer is an active or a
passive process.

Effect of Inoculation on 358 Uptake in the
Leaf and Translocation to the Epidermis

 

 

Plants of the five near-isogenic host lines (pmx,

Pml, Pm2, Pm3a, and Pm4) were uniformly inoculated with

 

 

conidia of culture MS-l (genotype PlP2P3aP4). The uptake
35

 

and translocation of S into the epidermis of the leaf, and
into the remainder of the leaf, was determined during five
hour labeling periods at various times after inoculation. The

ectoparasitic portion of the fungus was removed in parlodion

54

55

from inoculated plants prior to removal of epidermal strips

from the leaves.

35

The S activity in the leaf sections and in the

epidermal strips of noninoculated and inoculated wheat,
genotype EE/Pml, is presented in Figure 7A.

358 in the inoculated than noninocu-

There was less
lated leaves with genotype El/Pml except for the 26 hour

observation (Figure 7A). The epidermal strips of inoculated
35

.. a“... _..._.._. - -

leaves had less S than epidermal strips of noninoculated I

leaves at all time periods tested. The same general trend

358 activity in the leaves and epidermal strips L“

of less
of inoculated than in noninoculated plants was observed with

each of the other parasite/host genotypes (EZ/szr P3a/Pm3a,

 

P4/Pm4 and Eg/pmx)(Figure 7 B-E). No differences between ‘

parasite/host genotypes were detected in the level of 35S

activity in the epidermal strips of inoculated or non-
inoculated leaves. The data in Figure 8 are the averages
of data shown in Figure 7 A-E. Except for the 26 hour

observation, inoculated leaf tissue and epidermal strips

35

had less S activity than noninoculated leaf tissue and

epidermal strips, respectively.

35 2-

The relative amount of SO4 removed from solution

by inoculated and noninoculated leaves from 16 to 26 hours
after inoculation was determined by measuring the decrease in

35 2

the height of SO4 - solution in the vials from the

beginning to the end of the labeling period. At most times

56

Figure 7.--Uptake of 35$ and translocation to the epidermis
of noninoculated wheat leaves and wheat leaves
inoculated 6 to 26 hours prgviously with E.
graminis f. sp. tritici. S activity in four
leaf sections (eacE 1 cm long) of:

1 noninoculated leaves,
8:—-o inoculated leaves,
noninoculated epidermal strips, and
E; E3 inoculated epidermal strips. Parasite/
host genotypes were: (A) Bl/Efll’ (B) EE/Em2;
(C) Eég/Pm3a; (D) Efi/Eflfi; and (E) Px/pg§.~ ' ‘
Each point is the average of two rEElications.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

58

.mud n ousmfiw ca mumc mcammuo>m Eoum one Dump one
.5 musmwm mom .Howuflhu .Qm .m mwcfiemum mm sues
man50w>mum meson mm on m CODMHDOOCH mmcwacmom
ummnz can mmnwacoom ummn3 Coumasoocflcoc udo cw _

mwfiumpwmm on» on cofiumooamcmuu can mmm mo mxmumsll.m Dusmflm

 

v-Ol x was 558

60

after inoculation the noninoculated leaves absorb more

35

$042— solution than did inoculated leaves (Table 2). There

was a high positive correlation (r = 0.88) between loss of

35SO42_ in the ambient solution in the vials and the radio-

activity translocated to leaves (Figure 9).

TABLE 2.--Removal of 35SO 2- solution from vials by inoculated

and noninoculated seedlings at various times after

 

 

 

inoculation.

Time after Loss Of35SOAZI=Solution (mm)
Inoculation (HR) Inoculated ‘ Noninoculated
16 5.88a 6.71
18 4.98 5.46
20 5.15 I 4.94
22 ' 6.55 7.13
24 8.91 8.42
26 8.15 7.02

 

8Values are averages for four seedlings per
replication, 26 replications.

The cut seedlings took up much more 358042- solution

in the light than in the dark (Figure 10). Greater uptake of

358042- solutions by plants in the light correlated well with

increased 358 activity found in such plants.
The effect of inoculation density (2,500 vs 7,500

35

spores/cm length of leaf on four leaves) on S radioactivity

in inoculated wheat leaves is shown in Table 3. Leaves

61

a
ea smm>aumm m

mm

.mHmH> Bonn COHDDHOD

om mo mmoa way can mO>NOH umoc3

com3umn mwsmcowumamn Onall.m Ousmflm

62

(mm) NOlln'IOS .J'osse :IO

 

10-4

CPM X

Figure 9.

63

Figure 10.--The amount of 3580 2- solution (mm) taken up

by four inoculated wheat seedlings from vials
(0.5 mm diameter) during the dark period

(16, 18, 20 hours) and during the light period
(24, 26 hours).

~‘Mrfl' I.

b

'vrw'Mr-“wvmmrm

   

Loss OF 355042' SOLUTION (mm)

'0
11rd-._,

r"*””“r”'“

 

64

 

LIGHT

 

   
     
    

V v.
- 3034

v
9.:
9

9
9
9

'V'V
.9 9
f.

99

.9

.V

9
.9
9

9
9

V.‘ v
. 9

9
9

9
9
9

9
9
9

’0

9’.
o
o
9

9:.
9
A9.

’0

'9
.9
9

:0
o

o
9

9..

9

9.

6
o

‘9
9 9
’0

0
0’9

.2

.0}.
9.0
o

o

A

         
     

9

9
9.9

3-
39

’3

 
    

.9

    
     
  

9 0’3 3
, o
, '9’...

9'9'
9
9:0
26
1.1.1.14... - ”A

DARK

Figure 10.

65

inoculated with spores at both inoculation densities had

35

lower S activity than did the control in the epidermal

strips at 6, 18 and 26 hours after inoculation. Furthermore,
leaves inoculated with 2,500 spores had as great a reduction

35

of 8 activity in the epidermis as did leaves with 7,500

spores. Total activity in leaf tissue in most cases appeared

also to be reduced equally well by both inoculation densities.

TABLE 3.--Effect of in culation density of E. graminis
tritici on S radioactivity in inoculated
wheat leaves.

 

 

 

 

No. of Spores Rep I Rep II
°n F°“r 6 hra 18 hr. 6 hr. 26 hr.
Leaves
1. 0
a. epidermis 83.0b 34.2 67.6
b. leaf 138.1 103.4 373.7 320.3
325.7 267.4
2. 2,500
a. epidermis 54.9 8.2 55.2
b. leaf 158.0 64.0 307.5 239.8
248.5 287.7
3. 7,500
a. epidermis 63.1 7.4 51.3
b. leaf 116.0 30.6 340.0 184.5

238.7 286.2

 

ahour after inoculation when 5 hour uptake time was
discontiBued. _3
radioactivity in CPM x 10 .

66

Wheat leaves were inoculated with E. graminis f. sp.
tritici or with nonpathogenic E. graminis f. sp. poae, and

were c ompared for the effects on uptake and translocation of

35$ to the epidermis (Table 4). There was no evident

35

difference between the fungi in their effects on S uptake

and translocation to the epidermis.

TABLE 4.--Comparison of uptake and translocation of 35$

radioactivity by wheat leaves inoculated with
E. graminis tritici and E. graminis poae.

 

 

Rep. I Rep. II

 

Fungus Tissue

 

6 hra 11 hr 26 hr 6 hr 26 hr

 

b

E. graminis epidermis 23.4 8.9 36.5 -- 54.2
oae
leaf 79.3 37.2 240.1 271.4 211.8
359.3 284.8
E. graminis epidermis 25.6 10.3 32.6 -- 53.3
trit1c1

leaf 108.7 67.7 97.8 324.2 212.2
243.6 286.9

 

ahr after inoculation at which 5 hr uptake time was
discontinued

bradioactivity in CPM x 10-3

67

Uptake of 3H-methionine by Wheat Leaves
and Transfer to E. Graminis
f. sp. Tritici

35

 

The form in which S is present in leaves and

35$ is transfered to the

parasite, and the form in which
parasite, is not known. The possibility that the kinetics

of uptake, distribution, and transfer of a radioactive label
given to the plant in an amino acid might be different was
examined.

Inoculated pg§_plants were allowed to take up 3H-
methionine (in 0.1M phosphate buffer, pH 6.9, specific activity
1.66 C/mM). Four activity levels (50, 100, 200 and 1000 uc/ml)
were used for a five hour labeling period of 20 to 25 hours
after inoculation. Inoculated wheat seedlings were also
allowed to take up 3H-methionine (100 uc/ml) for three, five
or seven hour periods corresponding to 22 to 25, 20 to 25, and
20 to 27 hours after inoculation, respectively.

Increasing concentrations of 3H-methionine given to
inoculated wheat leaves did not give a corresponding increase
in radioactive transfer to the fungus (Table 5). Greatest
rates of transfer were achieved using 1000 uc/ml 3H-methionine.
With the high specific activity and because of the expense

3

of H-methionine, 100 uc/ml was selected to be used for

other experiments.

The number of hours wheat leaves were allowed to take

up 3H-methionine did not greatly affect the amount of 3H

transferred to the parasite (Table 6). However, the amount

3

of H in the epidermal strips appears to increase with

68

TABLE 5.-—Effect of different concentrations of 3H—methionine
given to inoculated pmx wheat leaves on the amount
of transfer to the fungus at 25 hours after

 

 

inoculation.
Specific Activity Radioactigity No. Reps
(uc/ml) (CPM)
0 70b 5
50 80 7
100 179 29
200 150 5
1000 294 3

 

aCPM in the ectoparasitic portion of the fungus on
1 cm length of leaf on four leaves.

bCPM background.

TABLE 6.--Effect of Different Labeling Times of 3H-methionine
(100 uc/ml) Given to Inoculated BEE Wheat Leaves
on the Amount of Radioactivity (CPM) in the
Epidermis and in the Fungus.

 

 

 

 

Labeling Period Radioactivity (CPM)
Hr. after No. of Trags— Reps Epiger- Reps
Inoculation Hours fer mis
25-50 0 70° 2 7o 2
22-25 3 202 2 714 2
20-25 5 212 6 1049 5
20-27 7 228 2 1648 l

 

aCPM in the ectoparasitic portion of the fungus on 1
cm length of leaf on four leaves.

bCPM in the epidermis of 1 cm length of leaf on
four leaves. .

cCPM background.

69

increasing incubation times. Five hour uptake periods were
selected for use in later experiments.

The distribution of radioactivity between the para-
site and the host when 3H-methionine (100 uc/ml) was given to
inoculated plants from 20 to 25 hours after inoculation is

shown in Table 7. Although the CPM of plants labeled with

3H-methionine were much lower than when plants were labeled

with 358042., the distribution of radioactivity between

fungus, host epidermal strips, and host leaf tissues was
very similar. The radioactivity on a cOncentration per

volume basis was much less in the remaining solution after

3

plants were labeled with H-methionine. Apparently, selective

absorption of 3H-methionine occurred as contrasted to non-

selective absorption when plants were labeled with 355042-.

TABLE 7.--Comparison of radioactivity (CPM) in the parasite,
epidermis and le f tissue of inoculated EE§_wheat
seedlings given H-methionine (100 uc/ml) at 25
hours after inoculation.

 

 

Source Radioactivity (CPM) Reps
Background 70 8
Fungusa 185 35
Epidermisb 1,260 16
Leafc 6,580 6

 

aCPM in the ectoparasitic portion of the fungus on 1
cm length of leaf on four leaves.

bCPM in the epidermal strips of 1 cm length of leaf
on four leaves.

cCPM in the leaf tissue of 1 cm length of leaf on four
leaves.

70

When plants were given 50 uc/ml each of 3580 2— and

4
35$ and 3H radioactivity were transferred

35

3H-methionine, both

to the parasite. The actual number of S and 3H counts in

the parasite was computed (Table 8). All of the 285 CPM

above background (305-20) in the 14C channel was 35

35

S and this

was 75.8 percent of the total S counts in the parasite. In

the 38 channel, 91 (24.2 percent of 35

35

S activity) of the 180

CPM above background were S radioactivity. The remaining

3

89 CPM above background represent the H counts of radio-

activity in the parasite. There were a total of 376 CPM above

35

background of S and 89 CPM above background of 3H transferred

to the parasite (Table 8).

TABLE 8.--Transfer of 35$ and 3H grom inoculated pmx wheat
leaves to E. graminis tritici at 25 hours after

 

 

 

 

 

inoculatioH.

Labeled Source Radioactivity (CPM)
Spgglflg Ert1v1ty Reps 3H Chan— l4C Chan- i4of CPM in

uc m nel nel C Channel
background 20 20
358042“ 4 411 1248 75.8% I 2.1
3H-methionine 3 180 21
355042- + 3H-methio-

nine (50 uc/ml each) 9 200 305 60.1
corrected for background
3550 2' 91 285 75.8

4
H-methionine 89 0

 

71
Solubility in TCA of Radioactivity in
Leaves, Epidermis and Parasite
Efforts to show differences in proteins synthesized
in inoculated and noninoculated plants during the first 30
hours after inoculation have given variable results. There-
fore, after determining the distribution of 35S and 3H (given

to the plant as 358042- and 3

H-methionine) in leaf parts,
the proportion of the radioactive label that was TCA-
insoluble was examined. The proportion of the radioactivity
that is TCA-insoluble might give some basis for evaluating
the effect of inoculation, environmental conditions, age of
host leaves, etc., on the metabolism of the host and para-
site, and possibly the form of the radioactivity available
for transfer from host to parasite.

Conidia of E. graminis f. sp. Egigigi,WhiCh were
germinated on two percent water agar in the presence of 355042-.
and 35$ or 3H labeled leaf sections, epidermal strips, and

ectoparasite units on the leaf surface were assayed for per-

centage of label in the TCA-insoluble fraction. A flow

diagram of the extraction procedures is given in Figure 11
(4). The percent of TCA-insoluble radioactivity in samples
of two leaf sections (l-cm length), 2.0 epidermal equivalents
(see chapter 2) or 12 parlodion strips containing the ecto-
parasitic portion of the fungus was determined. These
materials were each ground with a small amount of acid-washed
sea sand in 0.8 ml Tris HCl buffer, pH 7.5, containing 0.1M

Tris HCl, 0.25 M sucrose, 10-4M sodium sulfate and 0.01M

72

Source (leaves, epidermal strips, or fungus)

0.8 ml Tris-HCl buffer
acid washed sea sand

Maceration (mortar and pestle)
Rinse (0.8ml Tris-HCl buffer)

Centrifuge (1,000 x g, 20 minutes)

Pe!let Supernatant (1.0 ml)

1' 1.0 ml 20% TCA
2.0 .1

‘ 0 C, 20 minutes

Filtrate (fiber-glass filters)

2.0 ml 5% TCA rinse,
5 times

 

Filter (TCA insoluble) 12.0 (TCA soluble)

dry 1.0 ml aliquot

15 m1 scintill-
ation fluid dry

Radioactivity (liquid 15 ml scin—
scintillation counter) tillation
fluid

Radioactivity
(liquid scin-
tillation
counter)

Figure lla-Flow diagram of procedures used to determine
the percent of the radioactivity that was
TCA-insoluble.

73

ascorbic acid, with a cold mortar and pestle. After macer-
ation of tissues, the mortar was rinsed with 0.8 m1 Tris
buffer, both 0.8 m1 aliquots combined and centrifuged at
1,000 x g for 20 minutes in a Sorvall SS-l centrifuge. One
ml of the supernatant was collected and an equal volume of
20 percent TCA added to make a 10 percent TCA solution. The
2.0 ml solution was allowed to stand for 20 minutes at 0 C,
then filtered with Millipore fiber-glass filters, and washed
five times with two ml of 5 percent TCA. The filter was
removed, dried, and put in a scintillation vial (TCA-
insoluble fraction). A 1.0 m1 aliquot was taken from the
filtrate, placed on a filter paper in a scintillation vial,
and allowed to dry completely. Fifteen ml of scintillation
fluid (see Chapter 2) was then added to each vial and
radioactivity determined in a Beckman LS-133 scintillation
spectrometer.

In a separate experiment the distributiOn of 35$
radioactivity of leaf tissue among the pellet, and TCA-
soluble and TCA-insoluble supernatant fractions was examined
(Table 9). Subsequent to maceration and rinsing of the leaf
tissue with a total of 1.6 ml Tris buffer, one 0.8 ml aliquot
was assayed directly for radioactivity. The other aliquot
was fractionated (Figure 11) and the pellet and TCA-soluble
and TCA-insoluble fractions assayed for radioactivity. The
amount of radioactivity associated with each fraction

(Figure 11) is shown in Table 9. The total radioactivity

74

in the centrifuged sample closely approximates that of the
noncentrifuged sample. Since a sizeable portion of the
.radioactivity was associated with the pellet, the pellet was
repeatedly washed with 5 percent TCA. Thorough rinsing of
the pellet with 5 percent TCA washed 90 percent of the
activity out of the pellet. Therefore, approximately the
same percentage of radioactivity of the pellet was TCA-
insoluble as in the supernatant after centrifigation. In

subsequent experiments, only the supernatant was examined.

TABLE 9.--Distribution of 358 by TCA Fractionation of
Inoculated pmx Wheat Leaves 25 Hours after
Inoculation.

 

 

 

Distributiona Rep gadi°aCtiVitY (%::)II

Noncentrifuged Sample 246,420 $175,836
Centrifuged sample

Pellet 29,925 36,613

Supernatant

TCA-insoluble 14,330 14,745

TCA-soluble 196,042 116,285

. Total 240,297 167,643

 

aSee flow diagram Figure 11.

The ability of the parasite to incorporate 358 into

TCA-insoluble material was examined. If the parasite cannot

utilize 358042-, then transfer from host to parasite must be

75

other than 358042- alone. To test the ability of the para-

35

site to convert 8042- into a TCA-insoluble form, conidia

were dusted onto two percent water agar plates and kept
under environmental conditions for parasite development (see
chapter 1). 35SO42- was placed onto the agar surface at
eight hours after dusting, and the plates were placed in the
dark for two hours followed by two hours in the light. Spores
were then scraped with a razor blade into cold mortars and
extracted as described above. Germinated spores bathed in
0.4 ml 100 uc/ml 358042- for four hours had approximately
one percent of the radioactivity in the TCA-insoluble
fraction (Table 10). The control, which consisted of germi-
nated spores flooded with 0.4 ml 35SO42-(100 uc/ml) and
harvested immediately, had 0.16 percent in the TCA-insoluble
fraction. Spores on the host surface from 20 to 25 hours
after inoculation had 1.55 percent radioactivity in the TCA-
insoluble fraction. Radioactivity in the epidermal strips
and leaf tissue was 0.64 and 6.7 percent, respectively, in
the TCA-insoluble fraction.

The percentages of label in the TCA-insoluble fraction
in leaf tissue of inoculated and noninoculated leaves labeled
with 355042- and 3H-methionine are shown in Table 11. Con-
siderable variability was observed in leaves labeled from one
to six hours after inoculation. There does not appear to be
a difference between inoculated and noninoculated leaves
35 2—

804 . Leaves

labeled with 3H-methionine from 21 to 26 hours after

labeled with either 3H-methionine and

76

TABLE 10.-- The percent of radioactivity in the TCA-
insoluble fraction in conid§§ germinated
on agar in the presence of SO and in BEE
wheat leaves and in the fungus 3t 25 hours
after inoculation.

 

 

TCA-insoluble (%) Reps
Germinated Spores 0 hr 0.16 2
Germinated Spores 4 hr 0.98 5
Spores on Wheat 5 hr 1.55 6
Epidermal Strips 0.64 10
Leaf 6.7 13

 

TABLE ll.--The percent of radioactivity in the TCA-
insoluble fraction in inoculated an§_noni§ocu1ated
BEE wheat leaves labeled with '80 or H-
methionine at various times after Inoculation.

 

TCA-insoluble Fraction (%)a

 

 

 

Hr. after 3Enocé_1eave§ A - 35_Nog:noc.3leaves

Inoculation SO4 H-Met ' SO4 H-Met
1-6 23.2: 10.8 27.1: 6.3 18.4: 10.5 ‘26.3: 10.3
21-26 5.5: 1.8 10.8: 3.0 5.0: 1.6 16.7: 5.7

 

aAll values are the average of 8 reps. The standard
deviations are indicated.

inoculation JTad a greater percentage of the label as TCA-

insoluble thanclnileaves labeled with 358042- for the same

time period. Table 11 shows that the percentage of label in

the TCA-insoluble fraction was less when plants were labeled

from 21 to 26 hours rather than one to six hours after

77

inoculation. Both labeling periods, one to six and 21 to
26, are times when plants are in the light (lights are used
at those times for synchronous parasite development). Perhaps
either the age of the plant or the difference in light and
dark periods prior to the labeling period could account for
less incorporation of radioactivity into the TCA-insoluble
fraction in the leaf tissue at 21 to 26 hours after
inoculation.

The effect of host age on the percentage of radio-
activity in the TCA-insoluble fraction is presented in
Table 12. The percentage of label in the TCA-insoluble
fraction is less in six to seven day old plants than five
to six day old plants at both one to six and 21 to 26 hours
after inoculation. The age of the leaf definitely influences
the percentage of label in the TCA-insoluble fraction in
the leaf. However, five to six day old plants at 21 to 26
hours after inoculation are nearly the same age as six to
seven day old plants at one to six hours after inoculation.
Therefore, the values 6.8 and 12.5 (Table 12) should be
nearly equal if plant age is the only determining factor in
the percent of the radioactivity that was in the TCA—
insoluble fraction.

Prior to inoculation, all wheat leaves have at least
an eight hour light period. Since lower activity in the TCA-
insoluble fraction also occurs in noninoculated leaves

(Table 11) noninoculated leaves were used to determine the

 

78

TABLE 12.--The effect of host age on the percent of radio-
activity in the TCA-insoluble fraction in 35
inoculated pmx wheat seedlings labeled with SO

 

 

 

4
. . a
Hr after TCA-insoluble Fraction (%)
Inoculation 5 - 6 Day Plants 6 - 7 Day Plants
1 — 6 29.8 : 14.2 12.5 : 2.5
21 — 26 6.8 : 0.4 2.6 : 0.2

 

aEach value is the average of 2 reps. The standard
deviations are indicated.

effect of the lights between hours six to 20. Except for
the lights, standard environmental conditions were used.
(Inoculated leaves would have introduced another variable,
namely, effect of synchronous vs nonsynchronous parasite

development.) Plants labeled from 21 to 26 hours with 3580

4
that were kept in the light prior to labeling had a higher
percentage of label in the TCA-Insoluble fraction (10.4%)
than those plants kept in the dark (2.5%) preceding labeling
(Table 13). Therefore, both the increased age of the leaf
and darkness prior to the 21 to 26 hour labeling period
contribute to decreased percentages of label in the TCA-
insoluble fraction in leaves labeled at 21 to 26 hours after

inoculation. The data from Tables 11, 12, and 13 are

summarized in Figure 12.

2-

79

TABLE 13.--The effect of light and dark preceeding labeling 2_
of noninoculated wheat seedlings labeled with so
on the percent of radioactivity in the TCA-insolubIe

 

 

 

 

fraction.
Hr after. TCA—insoluble Fraction (%)a
Inoculation
l - 6 17.0 : 5.7
Plants in dark Plants in light
6 ~ 20 hr 6 - 20 hr
21 - 26 2.5 : 0.4 10.4 : 2.5

 

aEach value is the average of 2 reps. The standard
deviations are indicated.

80

Figure 12.--The percent of radioactivity in the TCA-insoluble
fraction in inoculated ( D ) and noninoculated

( I )

pmx wheat leaves given label from 1 to 6

or 21 to 26 hours after inoculation:

(A)
(B)
(C)

(D)

leaves given 3H-methionine,
355042—’
comparison between 5 to 6 day °§§ plants

vs 6 to 7 day old plants given SO4 and

comparison between plants given 3580 2-
kept in light from 6 to 20 hours after
inoculation and plants kept in darkness
from 6 to 20 hours after inoculation.

leaves given

 

 

81

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TIME AFTER INOCULATION (hr)

Figure 12.

82

Thin Layer Chromatography_of Radioactivity
in Leaves, Epidermis and Parasite

Radioactivity is transferred from host to parasite
but the form in which transfer occurs is not known. An
attempt was made to determine the form in which the 3H and

35S was present in the host and parasite. If a plant is

allowed to take up 355042., what proportion of the radio-
activity is in methionine in the plant and parasite? If a
plant is allowed to take up 3H-methionine what proportion
is still in methionine in host and parasite? Answers to
these questions should help to determine the form in which
the label is available to be transferred from host to
parasite and the extent of turnover of the radioactivity in
host and parasite.

Thin layer chromatography (TLC) was used to separate
methionine from SO42— and other components. Prior to
spotting of materials, the thin layer plate was air dried and
the glass chamber saturated with the solvent components for
at least 30 minutes. Moistened filter papers lined the glass
chamber to insure saturation. Leaf sections of 358042-
or 3H-methionine-labeled plants, epidermal strips, and
dissolved parlodion strips containing the parasite were ground
in water:propanol (90:10 v/v) and spotted with 10 ul pipettes
two cm from the base of the plate, and dried with a fan. The
plate was then inserted into the glass moisture chamber at

a slight angle, the lid greased tightly with vacuum grease

and the plate allowed to develop. The solvent front moved

83

ten to twelve cm in a developing time of 3.5 hours. The
plate was then removed from the chamber, allowed to dry,
sprayed with ninhydrin reagent, and heated to expose amino
acid bands. The developed areas (1 cm x 10 cm) of the radio-
active TLC plates were divided in ten 1 cm sections. Ten
sections were obtained from each sample chromatographed on
TLC plates. Each section was scraped into a liquid
scintillation counting vial, and 15 ml of scintillation fluid
was added. Each vial was counted for at least ten minutes
in a Beckman liquid scintillation counter, model LS-l33,
equipped with an external standard to determine counting
efficiency.

Silica gel GL plates and a solvent of ethanol-2-
propanol - 1N HCl (2:2:1 v/v/v) were found to be the best

35SO42- and sulfur-containing

amino acids (Figure 13A, B). All of the 35SO42- remained

of about .58,

thin layer system to separate

near the origin while methionine had an RF

cystine and its residues had RF's of .46, .35, and .31, and

cysteine had an RF of .33. Inoculated wheat leaves fed a
0.1M phosphate buffer from 20 to 25 hours after inoculation

were ground in 0.1 ml water:propanol (90:10) and 0.1 ml

100 uc/ml 3SSO 2-. The radioactivity on the thin layer

4
plate was very similar to 358042- spotted in that no

activity was determined above a RF of .30 (Figure 13C).

35 2-

Figure 13 C clearly shows that SO4 is not carried along

with other compounds to a higher R

F
. 35 2-
found above RF .30 is not SO4 .

and that any activity

84

 

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86

Nearly four percent of the radioactivity of inoculated wheat
35 2-

labeled with 804 was found in an area corresponding to the
RF of methionine (Figure 14A, Section 6). Greater radioactivity
occurred regularly at an BF of .70 to .80 when the experiment
was repeated. That spot was not identified. The epidermis

of inoculated leaves labeled with 35S0 2- had a slightly

4
higher percentage (4.5 percent) of its activity at an RF
corresponding to methionine (Figure 14 B). The radioactivity

from leaf tissues with an R from .70 to .80 was not found in

F
the epidermis (Figure 14 B). The fungus on the leaf surface
of plants given 35SO42- from 20 to 25 hours after inoculation

had as much as 25 percent of its activity at an RF corres—
ponding to methionine (Figure 14 C).

A much lower total activity was observed on the
chromatogram with inoculated wheat leaves labeled with 100
uc/ml 3H-methionine from 20 to 25 hours after inoculation.
The percentage corresponding to methionine, however, was much
greater than with 358042- labeled leaves (Figure 15 A). Up
to 25 percent of the activity could be found in the area
corresponding to methionine. The epidermis of leaves
labeled with 3H-methionine contained up to 38 percent of the
total activity in the approximate area corresponding to the
RF of methionine (Figure 15 B). The results for methionine
in the epidermis were somewhat variable from day to day.

Inoculated wheat leaves labeled with either 3H-
methionine or 355042_ showed similar amino acid development

with the use of ninhydrin spray reagent. Four purple (two

heavy) bands developed with R ranging from .43 to .64.

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91

A yellow spot also developed to a RF of .33 corresponding to
cysteine or one of the cystine residues. In these experi-
ments it was impossible to accurately distinguish between
inoculated and noninoculated leaves in both amino acid con-
tent and the distributions of radioactivity on the thin
layer plates.

Effect of Temperature on 358 Uptake in the

Leaf, Translocation to the Epidermis
and Transfer to the Parasite

Three criteria for active mediated transport are:
(l) tranSport against a chemical or electrochemical gradient,
(2) transport dependent upon a source of metabolic energy,
and (3) transport that is unidirectional or vectorial in its
normal energy requiring function (50). In the powdery
mildew disease complex, criteria (1) and (3) would be
difficult to establish under normal conditions of primary
infection. Criterion (2), however, could provide suggestive
evidence for either active or passive transfer.

Plant and fungal metabolism is known to be extremely
low at four C. A clearer idea of the role of active trans-

35

port should be evident by comparing the amount of S in the

epidermis of labeled plants incubated at four C and 22 C and
in the fungus at both four C and 22 C. Rates of 35S transfer
from host to parasite have been shown to be dependent upon
the stage of morphological development of the parasite.

During the five hour host labeling period the fungus develops

considerably. Comparing rates of transfer when inoculated

92

plants were given 35SO42- at four C and at 22 C presented

a variable in fungal growth since growth of E. graminis is
very limited at four C. To allow for this variable, inoculated
35

pmx wheat leaves were given 100 uc/ml SO42” either in a

growth chamber in the dark at 22 C from 15 to 20 hours after
inoculation or in a well ventilated regrigerator in the dark,
four C, from 18 to 23 hours after inoculation. The average

stage of development of the parasite should be about equiva-

lent for these two periods. Determination of 35

S activity in
leaf sections, epidermal strips and in the parasite was as
reported in materials and methods, chapter 2.

Less radioactivity was found in inoculated pmx leaves
labeled at four C, as compared to the radioactivity in plants
labeled at 22 C (Table 14). Inoculated leaves labeled at four
C had 12,000 fewer 358 CPM than those leaves labeled at 22 C.
Radioactivity in epidermal strips of inoculated leaves labeled
at four C had less than half the activity of those labeled at

22 c. 35

S activity in the parasite was reduced to 20 percent by
labeling plants at four C compared to plants labeled at 22 C.
To determine if the parasite accumulates, retains or
loses 355 after the five hour labeling period of 19 to 24
hours, inoculated plants were transferred to vials with
distilled water. Radioactivity in the leaf tissue, epidermal
strips and in the parasite were determined at zero, two, four

and nine hour intervals thereafter. The radioactivity of

leaf tissue and epidermal strips remained the same when

35SO 2-

4 from 19 to 24 hours

inoculated plants labeled with

 

93

TABLE l4.--The effect of temperature on 355 activity in
pmx inoculated leaves and transfer to g.
graminis tritici.

 

 

 

 

35 . . .
S Radioacthlty (CPM)
source Reps Dark, 4 0 Dark, 22 0
Leaf tissue 7 34,000 46,000
Epidermis 2 5,000 10,500
Transfer to
Fungus 4 90 435

 

at 22 C were transferred to vials containing distilled water
for an additional zero, two, four, or nine hours (Figure ]6).

However, the 35

S activity in the parasite more than doubled
with an additional nine hour association with the host. The
radioactivity in the parasite as a result of prolonged
feeding on the labeled host failed to approach the activity
in the epidermis.

Parlodion strips taken from inoculated wheat seedlings
labeled with 358042-, and which contained the ectoparasitic
portion of the fungus, were placed in distilled water or a

5.0 mM CaSO4 solution to determine how much 35

S radioactivity
could be washed out or exchanged. The parlodion strips were
placed in the exchange solutions for 30 minutes and rinsed
twice. The 358 that does enter the parasite appears to be
bound and not readily free for exchange. Solutions of 5.0
mM CaSO4 and distilled water were able to leach out 6.3 and

three percent of the fungal radioactivity, respectively.

94

Figure 16.—-Accumulation of 358 in E. graminis tritici at
various times after removing wheat seedlings
from radioactive solution. Inoculated plants
were labeled from 19 to 24 hours after inocu-
1ation, then removed from radioactive solution

(time 0), and placed in distilled H20 for two,
four, or nine hours.

 

(A) radioactivity in fungus (10 reps),
(B) radioactivity in epidermis (5 reps), and

(C) radioactivity in leaf tissue (2 reps).

130
' 150

 

4

TIME (hr)

95

Figure 16.

”I.

DISCUSSION

The environmental conditions necessary for high
infection efficiency and synchronous parasite development
in the different stages of primary infection of wheat and
barley by E. graminis have been studied (55, 58, 62). This
defined system has permitted studies of the initial host-
parasite interactions during primary infection and could be
used in determining the effects of environment or other
factors upon the establishment of a successful infection (78).
The production of elongating hyphae >10 u by the parasite
population was used as a criterion of a successful infection.

Initial studies were concerned with the kinetics of
secondary hyphal elongation with time with compatible and
incompatible parasite/host genotypes. Discussion of the
results of this study are presented in chapter 1.

Another criterion for measuring successful infection
has utilized rates of 355 transfer from host to parasite (44,
58, 80, 87). Rates of 358 transfer from host to parasite
were shown to be reduced to varying degrees when comparing
incompatible with compatible parasite/host genotypes (80, 87).
More quantitative data on rates of transfer were desired.
Rates of 358 transfer were placed on a per spore basis, and

a per unit of label in the epidermis which was considered

96

97

available to the parasite. To achieve this perspective of
the data, spores inoculated onto the leaf were counted and

35

results of uptake and translocation of S in the host

with the environmental conditions used for synchronous
parasite development were examined. Rates of 358 transfer
were then correlated with the morphological stage of
development of the parasite. Discussion of the results of
this study are presented in chapter 2.

The effect of inoculation on host metabolism has
received considerable attention. Unfortunately many of
these studies have been directed at late stages of disease
development, long after compatibility or incompatibility
have been expressed. Little has been known about the
effect of inoculation on the host before or at the time
incompatibility is determined. Would inoculation on the host

affect 35$ uptake by the host, and if so, how would this

effect be expressed and would there be differences in 358
uptake between compatible and incompatible parasite/host
genotypes?

The data presented in this thesis show that one
effect of inoculation of E. graminis f. sp. tritici on wheat

35

is to reduce uptake of S by leaves during primary infection.

The reduction in uptake of 35

S by inoculated leaves appears
to be independent of the compatibility or incompatibility
of the parasite/host genotype and is expressed as early as

six hours after inoculation (Figure 7). Furthermore, this

98

effect appears to be nonspecific as spores of other form
species of E. graminis elicit the same response (Table 4,
personal communication with T. J. Martin). The reduction in

35

the uptake of S by inoculated leaves was not dependent upon

spore density at two spore concentrations tested. The
effect of inoculation to reduce 35$ uptake by the leaf is
not considered to 1x2 a physical effect, i.e. plugging of
stomates, since T. J. Martin (personal communication) found
that viable Erysiphe spores were necessary for reduction of
uptake. Spores killed by UV-irradiation or chalk dust did
not cause reduced uptake by inoculated leaves. These
results suggest the need for further investigations of host-
parasite interactions prior to penetration. Clearly there is
transfer of information from the parasite to the host prior
to penetration of the host by the parasite.

35

Rates of uptake of S by cut seedlings are primarily

thought to be a function of host transpiration rates. Good

35 2-
804

in vials and radioactivity taken up by leaves. A difference

correlations were observed between loss of solution
in radioactivity in leaves due to treatments such as light,
inoculation, etc., was accompanied by a correlation with the
amount of 358042- solution taken up from the vial. Duniway
and Durbin (18) found that transpiration rates and stomatal
aperatures of primary bean leaves infected with rust were
reduced four to six days after inoculation when compared

with healthy primary bean leaves. Majerink (52) also

reported that low water output with a high water deficiency

99

occurs in the whole plant during the pathogenesis of mildew.
During fungal development, total and stomatal transpiration
were lowered but cuticular transpiration increased with time
in fungus—infected tissues (52). The results here are con-
sistent with reports of Majerink (52) and Duniway and Durbin
(18) but suggest these effects are evident before fungal
penetration. It appears quite likely that spores inoculated
onto the leaf may result in the partial closing of the
stomates, perhaps as a result of production of some diffusate,
thus reducing transpiration and uptake by leaves. The
results presented herein imply that an effect of inoculation
is to put stress on the host regardless of the compatibility
or incompatibility of the parasite/host genotype.

The form in which labeled materials are transferred
from wheat to E. graminis f. sp. tritici has been unknown.
Experiments designed to answer this question were to: examine
the kinetics of 3H-methionine uptake by the host and 3H
transfer to the parasite, label the host simultaneously with
358042_ and 3H-methionine, and determine TCA solubility and
separation by thin layer chromatography of labeled compounds
in both the host and parasite. The data presented herein
suggest that both sulfate and methionine may be transported
across membranes.

Rates of transfer of 3H, when given to the plants as

3H—methionine, were lower than transfer of 35

plants as 358042_. One reason for these results is that the

S given to the

100

counting efficiency for 3H is low and that the specific

3 3SS(l.6 C/mM versus 43 C/mM).

activity is lower for H than
Computation of the actual number of molecules that are
transferred shows at least five times more methionine than
sulfate molecules being transferred assuming that all of

3H is transferred as methionine and all the 358 is

the
transferred as sulfate. The actual number of methionine
molecules transferred may be less than the calculated number
because the 3H label was on the methyl group (which is

easily removed). However, the data from thin layer chroma-
tography suggest that a large percentage of the 3H given to

the plants as 3H-methionine is still in methionine in the
epidermis.

Fungal mycelia have been reported (3, 5) to have the
ability to take up sulfate and methionine. Depression of
sulfate uptake in the presence of methionine has been
reported in both plants and fungi (5, 28, 39, 75). When plants

were given solution containing both 3580 2- and 3H-methionine,

4
35 3

transfer of both S and H to the parasite was observed

with a compatible parasite/host genotype (Table 8). With a
compatible parasite/host genotype there is an apparent
inhibition (2 60 percent) of 358 transfer from wheat to the
parasite in the presence of 3H—methionine. Considering
specific activity, nearly 20 methionine molecules were trans—
ferred for every sulfate molecule transferred, again assuming

35

all the 3H is in methionine and all the S in sulfate.

101

Whether the inhibition of sulfate uptake by methionine is
occurring at the plant cell membrane, fungal membrane, or
both in the results presented herein is unknown.

The TCA-insoluble fraction is assumed to be largely
protein, but the fraction was not characterized and there—
fore is referred to as label in the TCA-insoluble fraction.

E. graminis f. sp. tritici can take up and incorporate

358042- into a TCA-insoluble form (Table 10). The difference

35

in percentage of TCA-insoluble S between the parasite on

the surface of the host and the parasite on an agar surface
may be due to the mode of incubation or developmental stage

of the parasite. A slightly higher percentage (1.6 versus

0.7) of the total 358 taken up was converted to a TCA-

insoluble form in the parasite on the surface of the host

than in the epidermis, respectively. The low percentage of

35S that is TCA—insoluble suggests that very little of the

358 is in protein in either the host epidermis or in the

parasite on the surface of the host. The difference of 1.6

versus 0.7 suggests that there may be more protein synthesis
in the parasite on the surface of the host than in the host

epidermis. However, the haustorial bodies of the fungus in

the inoculated epidermis of the host may account for some

of the radioactivity in the TCA-insoluble fraction. The low

percentage of 358 in a TCA—insoluble form precludes any easy

35

tests on whether any S is transferred from host to parasite

35

in the form of protein. It seems more likely that S given

102

as 358042” is either transferred as 358042” or a sulfur—

containing amino acid.
Since methionine is more readily incorporated into a
TCA-insoluble form than is sulfate (90), a greater percentage

of radioactivity in the TCA-insoluble fraction may be expected

35 2-
SO4 .
Several interpretations of the results obtained are possible.

in plants given 3H—methionine than in plants given

At a time of presumably rapid protein synthesis (one to six
hours after inoculation of plants six days old) little if
any difference occurs in the percentage of label in the
TCA—insoluble fraction in leaf tissue between plants given

3H-methionine or 355042- . One interpretation is that there

are different pool sizes from which 3H and 358 may be drawn
for protein synthesis. For example, rapid efflux of 3H-
methionine from a large methionine pool would show similar
amounts of label in the TCA-insoluble fraction as would slow

efflux of 358042- from a small sulfate pool.

Plants labeled with either 3H-methionine or 358042-
had less radioactivity incorporated into the TCA-insoluble
fraction at 21 to 26 hours than at one to six hours after
inoculation. However, inoculated plants given 3H-methionine
from 21 to 26 hours after inoculation have twice the percentage
of radioactivity in the TCA-insoluble fraction as plants given
358042-. This may suggest a change in relative methionine
and sulfate pool sizes or that inoculation of plants given
35

8042- caused a greater decrease in protein synthesis in

plants from 21 to 26 than from one to six hours after

103

inoculation than in inoculated plants given 3H—methionine for
the same time period. The pool sizes of methionine and sul-
fate in the host were not examined. Measurement of pool
sizes may have given an explanation for these results. A
lower rate of protein synthesis from 21 to 26 hours as
compared to one to six hours after inoculation due to
inoculation of plants given either 3H-methionine or 358042-
may also be a result of increased translocation of nutrients
to the infection site. If this were true, then epidermal
strips of inoculated leaves should possess greater activity
than epidermal strips of noninoculated leaves. However,

data of 358 activity in epidermal strips of inoculated leaves

358 2— 35

given 04 show less 8 activity than epidermal strips

of noninoculated leaves given 358042-. This increase of
translocation of nutrients to the infection site is not a
satisfactory interpretation of these results.
Incompatibility of several parasite/host genotypes
(El/Em}, BEE/3933' Eg/Emg) is expressed prior to 26 hours
after inoculation. While the reduction of 358 uptake by
inoculation does not appear to depend on the compatibility
or incompatibility of the parasite/host genotype it would
be interesting to know in further experiments whether
incompatible genotypes given 35SO42- or 3H—methionine would
differ from compatible genotypes in percentage of label

in the TCA-insoluble fraction at 21 to 26 hours after

inoculation. Based on the data and interpretations

104

presented herein, one may expect a difference at 21 to 26
hours after inoculation.

Large differences in percentages of 3SS and 3H—
methionine in the TCA-insoluble fraction were shown to occur
with time after inoculation (Figure 12, Table 11). These
differences are attributed to both (1) the different age
of leaves (Figure 12, Table 12), i.e. those plants labeled
from 21 to 26 hours after inoculation have begun initiation
of a second leaf and are nearly one day older than plants
labeled from one to six hours after inoculation, and (2) to
the environmental conditions preceding the incubation period
(Figure 12, Table 13), i.e. plants labeled from one to six
hours had received at least eight hours of light preceding
label uptake while plants labeled from 21 to 26 hours received
14 hours (from 6 to 20 hours) darkness preceding label uptake.
Patterson and Smillie (63) have reported that the total RNA
was less in six-day-old than in five-day-old wheat seedlings.
The amount of radioactive leucine incorporated into fraction
I protein was less in seven-day-old than in six-day-old
wheat seedlings. Rates of uptake by rice, carrot and potato
tissues, and release of CO2 in the light, were also affected
by the prior conditions used during growth (day length and
night temperature), by the age of the leaves, and by the
developmental stages of the plants (86). Crafts (14) states
that there was a preferential delivery of nutrients to young
leaves. Older leaves were bypassed. The results presented

herein are consistent with these reports that age of the

105

plants and the environmental conditions prior to labeling
has a major effect on the results obtained. These variables

should be considered in all future work.

About four percent of the 358 extracted from wheat

35

leaves, which had been given 8042-, moved to a position

on TLC plates where methionine was expected. Therefore, it

35

appears that about four percent of the S is in methionine.

Similar results were obtained in extracts of epidermal

355 was in

strips. A slightly higher percentage of the
methionine in the parasite on the host leaf.
About 25 percent of 3H activity extracted from wheat
leaves which had been given 3H—methionine moved to a position
on TLC plates expected for methionine. About 38 percent of
the 3H activity in the epidermal cell was probably methionine.

35

Since the kinetics of transfer of S and 3H (given to the

plant as 358042- and 3H-methionine, respectively) are similar,
the results are consistent with the hypothesis that both
SO42- and methionine are transferred from host to parasite.

The presence of radioactivity in both leaf sections
and epidermal strips in plants given 35SO42— at four C
suggest some diffusive flow from cut stems into leaf sections
and epidermal strips. The data (Table 14) indicate that

diffusive flow of 35

S is a much greater factor in leaf
sections than in epidermal strips. Diffusion and uptake in
the leaves are probably by xylem vessels. The fact that

radioactivity in plants labeled at four C does occur in the

epidermis but at a reduced rate suggests that translocation

106

of 35S may occur both by cell free space and through the

symplasm (15). However, Giaquinta and Geiger (36) have
reported that inhibition of translocation in beans by cold
temperature is a result of physical blockage of sieve plates
(through displacement of cytoplasmic material lining the
sieve tube wall) rather than from direct inhibition of a
metabolic process. I can not distinguish between these two
possible explanations of the results presented herein.

On 20 percent as much transfer of radioactivity to
the parasite was observed from plants labeled at four C
compared to plants labeled at 22 C. If uptake by the fungus
were simply free diffusion, 50 percent of the activity would
have been expected since epidermal strips of plants labeled
at four C had ca. 50 percent of the radioactivity of epidermal
strips of plants labeled at 22 C (Table 14). The fact that
a small amount of radioactivity does occur in the fungus
when plants are labeled at four C suggests either some passive
transfer or that a limited amount of metabolism occurs at
four C in E. graminis.

Gerson and Poole (35) propose that in certain
instances anion absorption by plant cells may occur by
transport on a single carrier accompanied by parallel free
diffusion. The interpretation given based on the above
data, literature, and the fact that very little 358 was
removed from parasites during exchange experiments is that
transfer from wheat to E. graminis is largely by an active,

energy requiring transport mechanism.

107

The results presented in this thesis suggest that

inoculation of E. graminis tritici on wheat causes an

 

alteration of host metabolism which is dependent upon many
variables: age of leaf, time after inoculation, environ-
mental conditions employed, and in some instances the
elements assayed. Incompatible parasite/host genotypes have
been shown to uniquely affect the growth of the fungus on

358 is transferred

the leaf surface and the rate at which
from the host to the fungus. The expression of incompatible
parasite/host genotypes on host and parasite metabolism
awaits future investigation. The more variables that can be
identified from procedures used in studying host-parasite

relationships, the better our understanding and interpre-

tation of the results obtained will be.

SUMMARY

The data on the kinetics of secondary hyphae elonga-
tion with time after inoculation are interpreted to give
further evidence of separate distinct interactions, rather
than alteration of single interaction rates, of the four
different incompatible parasite/host genotypes tested. The
four different incompatible parasite/host genotypes differ
from each other in the kinetics of secondary hyphal elongation,
time of host—parasite interaction, transfer of 358 from host
to parasite, host reaction and final infection type.

The environmental conditions used for synchronous
parasite development of E. graminis f. sp. tritici on wheat
greatly affect the amount of label taken up by the host,
translocated to the epidermis, and transferred to the
parasite. Greatest 35S transfer activity is correlated with
an earlier stage of the morphological development of the
parasite than was previously thought.

A nonspecific interaction occurs before the time of
penetration by E. graminis f. sp. tritici on wheat which
results in decreased transpiration (as measured by less

358 uptake) by inoculated tissues.

Both 3H and 358 are transferred to the parasite

3 35 2-
804

S transfer.

H—methionione and
35

from inoculated plants given both
simultaneously. Methionine partially inhibits

108

109

E. graminis f. sp. tritici is able to incorporate

SO42— into a TCA-insoluble form. Leaves given 3H-methionine

or 35SO42- from one to six hours after inoculation have a

higher percentage of label in the TCA—insoluble fraction than
corresponding leaves given 3H-methionine or 358042- from
21 to 26 hours after inoculation. This is apparently due in
part to both the difference in environmental conditions
preceding labeling and to the difference in age of the leaf.
Leaves given 3H-methionine from 21 to 26 hours after inocu-
lation have a higher percentage of label in the TCA-insoluble
fraction than leaves given 35SO42- for the same time period.
More radioactivity on thin layer chromatograms

corresponds to the R value of methionine in the leaf and

F
epidermis when plants are given 3H-methionine than when plants
are given 358042-.
Less 358 transfer from host to parasite occurs when

plants are given 358042- at four C than at 22 C. Less 358

is transferred from host to parasite at four C than at 22 C
than would be expected if transfer occurred solely by simple
diffusion. A largely active transport system from host to

parasite is suggested.

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110

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