GENETIC CONTROL OF PRIMARY
INTERACTIONS DURING INFECTION
OF WHEAT BY ERYSIPHE GRAMINIS

F. SP. TRITICI

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
RONALD STEPHEN SLESINSKI

1969

interns.

LIBRARY E

Michigan State
University

 
  

firm-raw"!-

  

This is to certify that the
thesis entitled

Genetic Control of Primary Interactions
During Infection of Wheat by
Erysiphe graminis f. sp. tritici

presented by

Ronald Stephen Sle s inski

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Botanx and Plant
Pathology

fl‘M/flzzlé" A. H. Ellingboe

/ Major professor

 

Date—August 1: 1969

 

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ABSTRACT

GENETIC CONTROL OF PRIMARY INTERACTIONS
DURING INFECTION OF WHEAT BY
ERYSIPHE GRAMINIS F. SP. TRITICI

By

Ronald Stephen Sle sinski

The objective of this research was to study powdery mildew
development during the initial stages of the host-parasite interaction.
Two aspects of fungus development were examined: 1) the effects
of the environment upon the initiation of secondary infections by the
parasite population, and 2) the effect of specific genes in the plant
and pathogen upon host-parasite compatibility.

Formation of secondary appressoria (SA) and the initiation of

secondary infections by Erysiphe flaminis f. sp. tritici was found

 

to be influenced by environmental conditions. High light (2600 ft-c),
temperature (27C), or photoperiod (240 ft-c during 20-30 hr after
inoculation followed by O ft-C during 30-40 hr) each affected the
percentage of parasite units which produced SA. Exposure of
inoculated plants to a combination of 27C and 2600 ft-c beginning at
26 hr; or 27C beginning at 26 hr and O ft-C beginning at 30 hr; or

2600 ft-c beginning at 26 hr and O ft-c beginning at 30 hr, allowed
more SA to form than with either the control (22C, 240 ft-c, 20-40 hr

photoperiod) or with each condition singly. The optimum environmental

Ronald Stephen Sle sinski

conditions for SA production appeared to be a combination of 27C
and 2600 ft-c starting at 26 hr after inoculation with 22C and 0 ft-c
starting at 30 hr.

The genetic control of the host-parasite relationship was
studied with thirteen near-isogenic wheat lines each of which con-
tained a single dominant fin gene conditioning reaction to mildew
development. These lines represented genes at a minimum of four
different loci as well as genes at the same locus but derived from
different sources. Chancellor and Little Club, which contain no
known P_m genes for incompatibility to mildew development, were
used as the standards to which mildew development on the various
lines was compared. Production of elongating secondary hyphae
(ESH) by the parasite pOpulation was used to quantitate the percentage
of applied conidia which established a successful parasitic relationship.

Mildew development was similar on all host lines during the
stages of germination and formation of appressoria. Differential
compatibility was first observed at or near the time of penetration
of the host cell and was reflected in the percentage of ESH produced
by the parasite population. With the fl/Enll and EMT—”.11 parasite/host
genotypes, incompatibility was expressed at or near the time of
penetration. The lowest percentages of ESH were produced with these
combinations. The E61112} genotypes (except P3c/Pm3c) inhibited

only 60-70% of the parasite units from forming ESH. The remaining

Ronald Stephen Slesinski

30-40% of the parasite population developed as in compatible combin-
ations. The 33/231} genotype did not affect mildew development
during primary infection. Incompatibility with this genotype was
expressed as a chlorosis and necrosis of host cells 3-4 days after
inoculation. Development of ESH with the four possible parasite/host
genotypes involving the gnu—1 locus (fl/3111' P_l/p£i__l, 11/221, Pl/EZLI)
was altered only with the incompatible (fl/P_m_l_) combination.

S transfer was used as a criterion of the movement of materials
from the wheat leaf to the external mycelium of I}. graminis f. sp.
_tr_it_i_c_i_. Transfer of 355 was studied with four different corresponding

gene pairs (_I_3_l_/Pml, E/PmZ, P3a/Pm3a, and Bil/Pmll) conditioning

 

incompatibility to mildew development with the compatible combination
MS-l/Chancellor as a basis for comparisons. The amount of 358
transfer with the four incompatible genotypes was found to reflect

the inhibition of fungus developMent previously measured by the
reduction in the percentage of ESH. In comparison with the MS-l/
Chancellor combination, 355 transfer was slightly greater with

fl/PmZ, intermediate with P3a/Pm3a, and lowest with _I:_’1_/Pml

 

and 33/13311'

With the four genotypes involving the £21; locus, the least
quantity of 358 was transferred with Pl/P_m_l, an intermediate amount
with fl/Rnll, and the greatest amounts were transferred with the

21/me and Kl/Eml genotypes. Although morphological development

Ronald Stephen Sle sinski

of the fungus was similar in each of these latter three compatible
combinations, 358 transfer with Ll/Pml was lower than the other

two (p_l/pml, fl/pml) compatible genotypes.

GENETIC CONTROL OF PRIMARY INTERACTIONS
DURING INFECTION OF WHEAT BY
ERYSIPHE GRAMINIS F. SP. TRITICI

BY

Ronald Stephen Slesinski

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1969

(150103“);
m 9 0
p’7 7

DEDICATED
TO

MY FAMILY

ii

ACKNOWLEDGEMENTS

I wish to thank the numerous faculty and graduate students that
I have known at Michigan State University that have helped to make
graduate education a lasting and meaningful experience.

I especially thank my friend and major professor Dr. A. H.
Ellingboe for his understanding and guidance during my graduate work.

Iwish to thank Dr. N. E. Good for his genuine interest in my
research and other problems.

I wish to thank Dr. R. P. Scheffer for his critical reviews of
my Thesis and other publications.

I thank Dr. C. .T. Pollard, Dr. N. E. Good, and Dr. W. G.
Fields for the use of their equipment and their interest in my research.

Thanks to Mr. J. L. Clayton for his technical assistance and
many lessons in applied Plant Pathology.

I would finally like to express my deep appreciation to my wife
and to my parents for their understanding and support.

Financial assistance for this research was obtained from the
National Institures of Health, Predoctoral Fellowship No. l-F-l-GM-

36, 191-01 and Grant AIO6420.

iii

T ABLE OF CONTENTS

Page

IN TR ODUC TION

LITERATUREREVIEW ................. 3
MATERIALS AND METHODS . 19

Production of inoculum. . . . . . . . . . . . . . . . 19
Methods of inoculation . . . . . . . . . . . . . . . . 21
Lines of wheat studied . . . . . . . . . . . . . . . . 21
Gene symbols and designation of parasite/host genotypes. , 22
Environmental conditions. , . , . . . . . . . . . . . 24
Examination of fungal structures. . . . . . . . . . . . 25

S labelling of wheat leaves , , , , , , , , . . . , , 25
Detection of 355 transferred to E. graminis using

parlodion. . . . . . . . . . . . . . . . . . . . . 27
Sample size and replication of experiments . . . . . . . 28

RESULTS 0 O O O O O O O 0 O O O O O O O O O O O O O O 29

Fungus Development During Primary Infection , , , . , . 29
Replication of Previous Work, , , , . , . , . . , 29
Effect of the Environment on the Formation of

Secondary Appressoria , . . . , . . . , . . . 29
Effect of temperature on SA formation . . . . 32
Effect of high light on SA formation. . . . . . 35
Effect of light period on SA formation. . . . . 35
Effect of combined high light and high

temperature on SA formation . . . . . . . 42

Genetic Control of Mildew Development. . . . . . . . . 42
Genotype of Mildew Cultures MS-l and MS-76 . . . . 49
Effect of Different Parasite/Host Genotypes on

Mildew Development During Primary Infection . . 51
Time of fungus inhibition . . . . . . . . . . 51
Compatible parasite/host combinations , . . . 51

iv

Table of Contents (cont'd.)

Incompatible parasite/host genotypes
involvingP_ml..............
Incompatible parasite/host genotypes
involvinngZ.............
Incompatible parasite/host genotypes
involvinng3.............
Incompatible parasite/host genotypes
involvinng4.............
Gene-for-Gene Specificity Expressed During
FormationofESH..............
Transfer of 35$ from Wheat Leaves to E graminis
f.s§.tritici ................
55 transfer with incompatible parasite/host
combinations............ ..
Transfer of 35$ with the four genotypes
involvingam_l..............

DISCUSSION 0 O O O O O O O O O O O O I O C O O O I I 0
SUMMARY. 0 O O O O O O O O O O O O O O O O O O O O O

LITERATURECITED..............o...

Page

56
61
61
66
71
74
74
81
90
99

101

LIST OF TABLES

Table Page

1. Near-isogenic lines with single Pm genes for
incompatibility to mildew development. . . . . . . . . 20

2. Infection types produced 7 days after inoculation of
near-isogenic wheat lines with cultures MS-l and
MS-76 of Erysiphe graminis f. sp. tritici . . . . . . . 50

 

vi

Figure

l.

10.

LIST OF FIGURES

Development of Erysiphe graminis during the stages
Of primary infeCtlon o o o o o o o o o o o o o o o o

 

The four possible parasite/host genotypes involving a
single locus governing compatibility in the plant and
the pathogen O O O O O O O O O I O O O O C O O O O 0

Development of Erysiphe graminis f. sp. tritici during
primaryinfection..................

 

Effect of various temperatures, beginning 26 hr after
inoculation, upon the formation of secondary appressoria .

Formation of secondary appressoria (SA) following 27C
temperature treatments starting at various hours
afterinoculation... . .. . . . .. .. ..

Effect of high light intensity (2600 ft-C) beginning 20,
26, or 30 hr after inoculation upon the formation of
secondary appressoria (SA) . . . . . . . . . . . . .

Effect of duration of light period (240 ft-c) upon the
formation of secondary appressoria (SA). . . . . . . .

Effect of combined increased light (2600 ft-c) and
temperature (27C) treatments, beginning 26 hr after
inoculation, upon formation of secondary appressoria (SA)

Effect of combined treatments of increased light
intensity (2600 ft-c) and duration of light period upon
formation of secondary appressoria (SA). . . . . . . .

Effect of combination of increased light (2600 ft-c),

temperature (27C), and duration of light period upon
the efficiency of formation of secondary appressoria (SA).

vii

Page

14

30

33

36

38

40

43

45

47

Figure Page

11. Similar development of appressoria during primary
infection with either compatible or incompatible
paraSlte/hOSt combinations. 0 o o o o o o o o o o o o o 52

12. Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici (MS-l) on cultivars 54
Chancellor and Little Club ' ° ' ° ° ' ° ° ° ° ° ° ' °

 

13. Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. triticiwith the following
compatible parasite/host combinations: MS-l/Chancellor,
p_(Unknown)/l_3_rrl(Unknown), and 2(Mich. Amber)/_Pfl(Mich.
Amber).......................57

 

l4. Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici (MS-l) with the following
incompatible parasite/host genotypes: Til/Ern—MChancellor)
(compatible control), P_l/E§1_1(Axminster), P_l/_P_m_l(AsII),
fl/gm_1(Noz-ka), and fl/P_m_l(C.I. 13836). . . . . . . . . 59

 

15. Formation of elongating secondary hyphae (ESH) by
Erysiihe graminis f. sp. tritici (MS-l) with the following
incompatible parasite/host genotypes: P_2_/p_m_2(Chancellor)
(compatible control), EE/Pm2(Ulka), and
fl/Pm2(C.I.12632) . ... ... .. .. . .. . .. 62

 

l6. Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici (MS-l) with the following
incompatible parasite/host genotypes:
P33.P3bP3c/pm3(Chancellor) (compatible control),
P3a/Pm3a, P3b/Pm3b, and P3c/Pm3c . . . . . . . . . 64

 

 

 

 

1?. Formation of elongating secondary hyphae (ESH) with the
following incompatible parasite/host combinations:
MS-l/Chancellor (compatible control), fig(MS-l)/Pm3a,
andP__3a(MS-1*)/pm3a. . . .. . . . . . . . —. . . . . 67

18. Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici (MS-1) with the following
incompatible parasite/host genotypes: fi/En_‘uChancellor)
(compatible control), Pi/PmMKhapli), and _P_4/Pm4(Yuma) . 69

 

viii

Figure Page

19. Similarity of production of elongating secondary hyphae
(ESH) with the following compatible parasite/host
genotypes involving Pml. . . . . . . . . . . . . . . . 72

20. Transfer of 35$ from wheat to the external mycelium of
Erysiphe graminis f. sp. tritici with the following
parasite/host genotypes: fl/flMChancellor) and
fl/Efl(Axminster)..................75

 

21. Transfer of 35$ from wheat to the external mycelium of
Erysiphe graminis f. sp. tritici with the following
parasite/host genotypes: fl/mfi(Chancellor) and
EE/PmflUlka)....................77

 

22. Transfer of 358 from wheat to the external mycelium of
Erysiphe graminis f. sp. tritici with the following
parasite/host genotypes: P3a P3bP3c/pm3(Chancellor)
andP3a/Pm3a....................79

 

 

 

23. Transfer of 35$ from wheat to the external mycelium of
Erysiphe gaminis f. sp. tritici with the following
parasite/host genotypes: fl/M(Chancellor) and
fl/Pm4(Khapli)...................82

 

24. Transfer of 35$ from wheat to the external mycelium of
Erysiphe graminis f. sp. tritici with the four possible
genotypes involving the Pml locus. . . . . . . . . . . o 84

 

25. Effect of light upon the transfer of 358 from wheat to

ETYSlphe graminis f. Sp. tritICi o o o o o o o o o o o o 87

 

ix

INTRODUCTION

The powdery mildew disease of wheat results from the
infection of a host plant (Triticum sp.) by the obligately parasitic

fungus Erysiphe graminis f. sp. tritici. The environment plays

 

an essential role in determining the progress of disease development
and it affects the host, the pathogen, and the combination of the
two organisms, the 'aegricorpus' (34). The effects of the
environment upon disease development have been studied for the
last 70 years or more and the results are numerous and often
conflicting. Methods of controlling disease development have also
received extensive study. Cultivars 'resistant' to mildew
development are available and represent the primary and least
expensive control measure. Studies of the inheritance and
expression of disease 'resistance', pathogenic variability, and
the physiology of 'resistance' are voluminous. Many of these data
are confusing because researchers have not always recognized
the necessity of defined environmental conditions, inocula, and
host plants.

A defined system for examining the early infection processes

of E. graminis on wheat and barley has been reported (39, 47, 48).

The development of the fungus was shown to consist of a number of
developmental stages which can be distinguished by changes in
morphology and differential sensitivity to various environmental
conditions. With the appropriate environmental conditions, the
synchrony of morphogenesis can be increased and the percentage
of successful infections can be maximized. The effect of additional
variables in the system can be measured by the alteration of the
infection process.

The objectives of this study were the following: 1) to determine
the environmental conditions necessary for increasing the synchrony
of formation of secondary appressoria by the parasite, 2) to study
the interaction of specific genes in the host and parasite which
alter development of the relationship, and 3) to characterize
the transfer of materials from the host to the fungus in various
compatible and incompatible parasite/host combinations utilizing

35504- as a radioactive tracer.

LITERATURE REVIEW

Powdery mildew of cereals is an important but frequently
underestimated agricultural problem in the United States. The
disease is conservatively estimated to produce a 1-2% reduction in
yield of wheat (32, 33). This reduction is very significant on a
nation-wide basis if we consider the total monetary value of the wheat
crop (l. 8 billion dollars in 1966) (65). Studies of the disease have
been aimed at understanding both the biology of the relationship and
in developing effective control measures. The physiological, genetic,
and biochemical aspects of disease development have been studied
extensively. I will attempt to summarize some of the important
developments in this review.

Powdery mildew disease development is affected by environmental
conditions and has been correlated with differences in climate. The
disease is more prevalent in humid regions such as the Great Lakes
states and the U. S. Pacific Northwest (68). Light intensity, light
period, relative humidity, temperature, and the genetic constitution
of the host and pathogen are some of the variables known to affect
disease develOpment (ll, 12, 58, 72). Germination and subsequent

development of conidia occur over a wide range of temperatures and

relative humidities. Several different optimal conditions for mildew
development have been reported (11, 25, 48, 58). There are several
excellent reviews concerning the effects of various environmental
factors on disease development (39, 47, 58, 72) and I will not attempt
to duplicate these efforts. One of the points made in each review,
however, is the variability of the present data and the necessity of
considering the role of the environment in altering disease development.

Growth of E. graminis on the plant surface can be divided into
a number of developmental stages: 1) germination, 2) production of
'club-shaped' appressorial initials, 3) formation of 'mature' appressoria,
4) penetration of the cuticle and epidermal cells, 5) formation of
haustoria, 6) development of elongating secondary hyphae (ESH),
7) initiation of additional infections, and 8) sporulation. This
sequential development, depicted graphically on Figure 1, has been
described by previous workers (11, 25, 37, 72).

Each stage of the infection process differs in the requirement
for particular temperature, RH, and light conditions (37, 48).
When the Optimum conditions for each stage are used, a high percentage
_ of the parasite population undergoes these various developmental
stages with an increased synchrony at each stage (37, 47, 48). The
term 'infection efficiency' will be used to mean the percentage of the
applied conidia which develop on the plant surface and form a

functional (1?) relationship with the host.

 

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Increased synchrony and efficiency of infection can be obtained by
using the optimum conditions for fungus development during primary
infection (37, 47, 48). The production of elongating secondary
hyphae (ESH) by the parasite apparently indicates that the fungus is
obtaining nutrients from the host which permit the continued growth
of the parasite (38, 40). Conidia on non-host plants will germinate,
form appressoria, and attempt to penetrate the epidermal cells,

but do not form either haustoria or ESH (39, 70). Masri (38) observed
that the number of ESH formed on 'resistant' lines of barley corre-
sponded to the number of mature (lobed) haustoria in the host
epidermal cells. The ability of the fungus to continue development
after the formation of an appressorium appears to depend on the
formation of a functional haustorium within the host.

Haustoria are thought to be specialized fungal structures for the
uptake and transport of nutrients from the plant to the external
mycelium (10, 11, 72). Fungus development on the leaf surface
ceases after the destruction of the haustorium (10). Exogenously
supplied nutrients, however, supported fungal development for a brief
period in the absence of a haustorium. This may indicate an ability
of the mycelium to directly absorb a limited amount of nutrients.

Electron microscopic studies have shown that the haustorial wall
of various pathogens is separated from the host cytoplasm by a sac-

like membrane (4, 16, 42, 51). The cytoplasm of the host and parasite

appear never to come into direct contact and materials transported
from the host to the developing fungus must cross both the sac-like
haustorial sheath and the haustorial membrane. Very little is known
concerning the transfer of materials from the host cell to the
haustorium. Recently developed techniques have made possible:

1) the isolation of intact haustoria of E. graminis f. sp. hordei from
barley (l4), and 2) the removal of the host cytoplasm from an
infected cell and replacement with nutrient solutions (10). These
techniques may prove useful for studies of the movement of metabo-
lites into the haustorium.

The effects of various experimental treatments upon mildew
development have been studied by observing final infection types.
Quantitating the effects of various treatments during primary
infection has the advantage of precisely measuring the alteration of
parasite development shortly after the treatment. The effect of
ultraviolet (UV) light upon fungus development has been studied by
measuring the percentage of the parasite population inhibited
during primary infection. Inhibition of mildew development by UV
light depended upon both UV intensity and the time after inoculation
that the treatment was given (44, 45). The fungus was more sensi-
tive to inhibition by UV during 6-12 hr after inoculation than during
the 14-20 hr period. The difference was correlated with the

appearance of a nucleus in the haustorium where it presumably was

more protected from the UV radiation. Similar studies of the effects

of various other environmental treatments were possible by observing
the development of ESH as a measure of the effects of the treatments

on parasite development (46, 48).

The effects of the powdery mildew disease on the host plant
appear to be diverse and numerous. Changes in respiratory rates,
alteration of the enzyme levels in the plant tissues, and the appearance
of new isozymes have been reported (2, 29, 59). The activity of many
enzymes normally present in the healthy plant were altered as a
result of infection by powdery mildew (36). One criticism of many
of these studies is that the analyses in most cases were made as late
as 7-12 days after inoculation. The data may simply reflect death or
physical disruption of the host cells. This criticism appears to be
valid since similar changes can be observed in non-infected senescing
wheat leaves (18). More data are needed concerning the physiological
changes occurring during early infection, when compatibility or
incompatibility is determined.

The use of radioactive tracers in studying host-parasite relation-
ships has made possible the detection of very small changes during

early disease development. Mount (46) used 32PO4 and 35

804
to study the transfer of materials from wheat to E. graminis f. sp.

tritici during primary infection. The amount of 32P and 358

transfered to the fungus was correlated with the morphological

10

development of haustoria in the host cells.

Radioautography of bean leaves inoculated with either Uromyces

 

Lhaseoli, Erysiphe polygoni, or Puccinia helianthi demonstrated

 

 

35

that 358 derived from H2 S preferentially accumulated around the

sites of infection (71). Shaw (60) observed similar accumulations of
32

P—phosphate and 14C-sugars around the infection sites of Puccinia

graminis and Erysiphe graminis on wheat and barley. These

 

accumulations appeared to be in the mesophyll cells and not in the

14

fungus mycelium. Less COZ fixation was observed around the
parasitized areas of the leaf. Other workers (23, 28, 30, 66) have
shown that radioactive labels, applied to an uninoculated portion of
the leaf, preferentially accumulate in infected areas even before
macroscopic fungus development is evident. It appears that one of
the immediate effects of disease development is the alteration of
metabolite distribution within the plant.

The nature of the systems which determine 'resistance' or
'susceptibility' to the powdery mildew disease is a matter of con-
jecture. Biochemical studies are difficult since they involve the
metabolic systems of two inseparable organisms which are both
changing continuously. Many changes in infected plants can be
observed by utilizing various chemical or biochemical assays, but

the cause and effect relationship of these alterations as determinants

of disease development is unclear. There are a few diseases for

11

which causal factors are known. The 'blight' of oats is incited by

Helminthosporium victoriae which produces a host-specific toxin

 

necessary for disease production (52). The toxin affects only
varieties of oats possessing the 113 gene which is thought to code for
a receptor site for the toxin molecu1e(57, 69). 'Resistance' to the
'Onion smudge' disease has been determined to be due to the presence
of toxic compounds in the outer scales of colored onion varieties

which inhibit the development of Colletotricum circinans, the

 

incitant of the disease (67). 'Resistance' to powdery mildew has
been studied physiologically and biochemically, but no clear cut
determinants to 'resistance' of the plant or 'pathogenicity' of the
fungus have been found.

Aqueous extracts of E. graminis conidia have been observed to
prOduce disease-like symptoms (9). However, no specificity of these
extracts was demonstrated and other substances, such as yeast ex-
tract, were shown to produce similar results. Many toxic compounds
have been isolated from 'resistant' plants (64), but again they lack
sufficient specificity and have not been shown to segregate with
the specific genes conditioning incompatibility.

The terms 'resistance' and 'susceptibility' are frequently used
loosely, in an agronomic sense, and can be very misleading. A
plant pathologist might view the lack of disease development after

inoculation as 'resistance' of the plant, while a mycologist might

12

view the same phenomenon as non-pathogenicity or avirulence of the
fungus. The production of a disease depends on the genotype of _139__t_l_1_
the host and pathogen; a very specific and defined terminology must
be developed to emphasize this fact. The following terminology has
been suggested by various people (l3, 19, 34). 'Resistance' and
'susceptibility' are used to describe the reaction of a particular
cultivar to infection by a particular strain of the pathogen. The
terms virulence and pathogenicity are both used to describe the
ability of an organism to incite disease; however, pathogenicity is
a general attribute of all plant pathogens, while virulence (E) and
avirulence (E) defines the compatibility of a particular strain of the
pathogen in relation to a particular host genotype. With most diseases
studied to date, resistance (3) and avirulence (E) are dominant and
virulence (p) and susceptibility (_r) are recessive (19, 34).

The combination of host and parasite (the 'aegricorpus' or
'sick body') (34) is the observable manifestation of disease and is
described by an infection type scale ranging from low to high mildew
development. 'Resistance' and 'susceptibility', therefore, refer
solely to alternate genes for reaction in the plant, while compatibility
and incompatibility describe the resulting infection type with par-
ticular parasite/host combinations. The terms 'compatible' and
'incompatible' have been suggested to describe various parasite/host

combinations (1?). This terminology takes into consideration the

13

genes present in M the host and pathogen that are involved in
affecting the progress of disease development.

Flor (20) observed that the ability of Melampsora lini to grow
and produce disease symptoms on flax lines containing genes for
incompatibility 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 hypo—
thesis (21, 49). The concept has been found to apply to many other
host-parasite systems (43, 50, 54). The gene-for-gene concept
simply states that each B gene for incompatibility in the plant inter-
acts with a specific corresponding E gene in the pathogen to determine
mildew development or parasite/host compatibility (19, 20, 49).
The interaction of pairs of corresponding genes as specific
pathogen/host genotypes leads to a single phenotypic expression by
which the presence or absence of the corresponding gene in either
organism may be recognized (19). The interaction of a single
corresponding pair of genes in the plant and in the fungus is
diagrammed in Figure 2. Incompatibility, or a low infection type,
is specified only when the corresponding parasite/host genotype
contains at least one E gene in the fungus and the corresponding B
gene in the plant (Ii/fl). Compatibility or high infection type and
unrestricted disease development is specified with the remaining

P_l/r_l, Ll/El, and 11/2 parasite/host genotypes (34, 56).

l4

 

 

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socom HImEInH .ucma 65 cm meow HIM 65 o» mcwpcommOMHoo
cowofmm 65 5 mOHOZN Oumcuofim of and HIAH pom MM pom.
Ema of 5 mofiozm 3.953? of mud flm pom mm. .oomofiuma
65 was 2.39 Os”. 5 >fifinfldm800 mcmcnocrom msOoH Cams;

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15

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w4n_h<mzoo

:5 Q
‘7 395.500

 

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2.9.19
392.38

 

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16

Person (49) has suggested that these complementary relationships
have evolved from specific selection pressures which initially favored
host plants which could escape disease and secondly, specific
pathogen strains which could develop in the presence of the B gene.
Studies of pathogenic variability have shown that many pathogens are
capable of rapid genetic adaptation to newly introduced host lines
possessing E genes for incompatibility (52, 62, 63). The discovery
of the gene-for-gene relationship controlling parasite/host com-
patibility has applicability to defined studies of disease development.
Rowell e_t_a_l (56) have proposed a biological test termed the 'quadratic
check' to study the physiological and biochemical effects of disease.
The combination of various host genotypes and strains of the pathogen
(similar to Figure 2) could be used to give a 4-way (quadratic) check
in which the molecular phenomena should parallel the biological
phenomena. This test should be valuable for studying many diseases
including the powdery mildew disease of cereals which is governed
by a gene-for-gene relationship (43, 54).

The study of the bases of parasite/host incompatibility has
involved the determination of the types of possible incompatible
responses. There is evidence that the different incompatible
”responses which can be observed are determined by the specific
interaction of particular flg/w genotypes. Many workers have

observed phenotypically different types of responses to infection by

17

E. graminis. Parasite development was affected at various stages
of disease development (12, 24, 35, 61). A similar wide range of
incompatible phenotypes was observed with various rust diseases.
Cells of 'resistant' safflower collapsed when invaded by a haustorium

(73). No difference in the development of Puccinia sorghi on near--

 

isogenic corn lines was observed with compatible parasite/host
combinations. With incompatible genotypes, reduced numbers of
haustoria, encapsulation of haustoria in the cells, and fungal lysis
were observed (27). The rapidity of cell collapse has been suggested
as the basis for resistance to disease development (70). However,
cell collapse appears to be only one of the many possible responses
which can be observed. The necrotic response of many varieties is
absent at elevated temperatures but the compatibility of the
parasite/host relationship is not increased (1, 22).

Parasite/host incompatibility has been studied on a quantitative
basis during primary infection of wheat and barley by E. graminis.
The production of secondary hyphal initials was not affected by
major genes which alter mildew development on barley (40), but
formation of ESH was. affected by these same genes (38). The per-
centage of the parasite population which established a functional
relationship with the host was estimated by the number of parasitic
units which produced ESH. The genes in barley affecting mildew

development were observed to inhibit the development of only a

l8

proportion of the parasite population during primary infection. The
inhibition of final development of mildew (infection type) was similar
to the inhibition of the formation of ESH during primary infection
(38, 40). The utilization of this defined host-parasite-environment
system has allowed the study of fungal morphogenesis and incom-

patibility responses previously described only qualitatively.

MATERIALS AND METHODS

Production gg inoculum--Two strains of Erysiphe graminis f. sp.

 

Mwere used. Michigan strain-l (MS—l) and Michigan strain-76
(MS-76) were collected in Michigan and maintained on Triticum
aestivum L. 'Little Club' wheat in different growth chambers. Each
of these strains was twice purified by isolating and increasing single
pustules formed on lightly inoculated Little Club wheat. The strains
were checked periodically for uniformity by scoring infection type on
a set of inoculated differential host lines (Table 1).
Little Club wheat was grown in 4-in. pots in the greenhouse
and inoculated with E. graminis f. sp. _t_1;1t_1c_1 at 6-7 days after
planting. Stock cultures of the mildew fungus were maintained by
inoculating each day by dusting with 7-day-old conidia. The stock
cultures were maintained under the following environmental conditions:
1. Light--700 to 800 ft-C (650 to 750 ft-c from white VHO-
fluorescent tubes and 50 ft-c from 25 watt incandescent
bulbs). Light period, 15 hr/day.
2. Temperature--18I1C during the light period and l7JElC
during darkness.

3. Relative humidity--80I5% during the light period and 95I5%

during darknes s .

l9

20

Table l. Near-isogenic lines with single Pm genes for incompatibility
to mildew development.

 

 

 

Designation of Former Gene C. I. (I)

Near-isogenic line Gene Involved Symbol Number
. 8(2) .

Axminster X Cc Pml(Axminster) Mlt 14114
AsII x Cc8 Pml(AsII) M1t 14116
Norka x CC8 Pm1(Norka) -—- 14117
C.I. 13836 x CC8 Pml(C.I. 13836) Mlt 14115
Ulka X Cc8 Pm2(Ulka) Mlu 14118
C.I. 12632 x Cc8 Pm2(C.I. 12632) Mlx 14119
Asosan X Cc8 Pm3a Mla 14120
Chul x CC8 Pm3b MIC 14121
Sonora x Cc8 Pm3c M1s 14122
Khapli x CC8 Pm4(Khapli) 14123
Yuma x Cc8 Pm4(Yuma) --- 14124
Unknown X Cc8 Pm(Unknown) --- -----
Mich. Amber x CC8 Pm(Mich. Amber) 14033
Chancellor(Cc) pmx --- 12333

 

 

 

 

(l) Cereal Investigations accession number.

(2) Cc refers to the 8 backcrosses to the cultivar Chancellor

21

4. Continuous air circulation.
Conidia produced on the 6th day after inoculation were used in all
experiments.

Methods 93 inoculation-~The 'rolling method' of inoculation (48)

 

was used in all experiments studying the development of mildew
during primary infection. Conidia were dusted onto a clean glass
slide and transferred to a single wheat plant with a cotton swab. Only
the lower (abaxial) leaf surface of each plant was inoculated. The
progress of the infection process is similar on either side of the leaf
(39), but microscopic observations and the removal of the host
epidermis are much easier on the lower side of the leaf. A uniform
distribution of single conidia ranging from lOO-300/cm length of leaf
was obtained by this method. Only single separated parasitic units
were counted at each observation to eliminate the possibility of
inhibition due to crowding (39).

With experiments studying the transfer of 35S—radioisotope from
the host to the parasite, plants were inoculated by lightly dusting
conidia onto the plants. This method was used because inoculation of
many plants by the 'rolling method' required too much time. The
development of the fungus during primary infection was the same with
both methods, but the efficiency of infection was reduced.

Lines giwheat studied-—The cultivars Chancellor and Little Club,

 

which contain no known major genes affecting mildew development,

22

were used as the standards to which mildew development on other
lines was compared. The thirteen backcross-derived lines of wheat
(Table l) which contained E__m genes affecting mildew development
were obtained from L. W. Briggle. The data concerning the source
of these genes, the results of various genetic tests, and their
identification have been published (5, 6, 7, 8, 41, 55). Chancellor,
a soft red winter wheat, provided a common genetic background for
each of these lines.

The plants for experimental studies were grown in 2-in. pots in
the greenhouse. Shortly after emergence (about 3 days after planting),
seedlings were transferred to the following standardized conditions:

1. Light--2600 ft-c (2400 ft-c from white VHO-fluorescent tubes

and 200 ft-c from incandescent bulbs) over a 15 hour day.

2. Temperature--20I1C during the light period and 1811C during

darkness.

3. Relative humidity, approximately 65%.

The plants grown under these conditions were inoculated approximately
6 days after planting. Plants grown solely at the greenhouse (especially
during the hot summer months) were less uniform and possessed tall

spindly leaves unsuitable for inoculation and microscopic observation.

 

 

Gene symbols and designation _o_fparasite/host genotypes--
The Pm symbols used to designate the E genes conditioning reaction

in the host lines (Table 1) follow Briggle's suggested terminology (7, 8).

23

The genes determined to be at distinct loci have been designated

Pml, PmZ, etc. Genes at the same locus thought to be either allelic

 

or very closely linked are followed by the letters a, b, c, . . .. when
positive evidence for their distinctness is available (eg. Pm3a, Pm3b,
E_m_3c). The remaining E11 genes at particular loci, derived from
different sources, but for which positive evidence of distinctness is

not available, are followed by the source of the gene (eg. Pml(Axminster),

I : r 4»‘ A. m ‘41 ‘_.
a
. . .'~.l

fln_I_(AsII), etc. ). The alternate alleles for each _F’il gene will be
referred to by their respective recessive designations (m, m, etc. ).
The genotypes of the host will be written as Emit rather than writing
the complete genotype of the diploid as mm since we are
dealing with reasonably homozygous host lines containing dominant
genes.
The symbols for genes in the pathogen conditioning pathogenicity
follow the terminology suggested by Loegering (34). The E(.P_m_1_{_)
or HM) genes for 'avirulence' and 'virulence' in the pathogen,
corresponding to the various host loci, will be shortened in this
manuscript to Eior a. The symbols for 'avirulence' and 'virulence'
will be followed by the corresponding host locus and allele designation
where applicable (eg. E(Erlil) = P_l, E(P_m_33) = E, etc. ).
Designation of particular parasite/host combinations studied
will utilize the preceeding gene symbolism to refer to the specific

genes conditioning the interaction (eg. fl/Pml = incompatible, or

24

Ll/Pml =‘ compatible). This abbreviated parasite/host symbolism is
possible since each host line contains only a single Pm gene for
incompatibility. The remaining host loci, with a Pml line for example,

are all for compatibility (ie. Pml pm2 pm3ajm3b pm3c ...... ).

 

Environmental conditions--The control of the environmental

 

conditions during experiments was obtained using Sherer-Gillett ' r:

.15”: . '

(Model CEL 512-37 and Model CEL 25-7) growth chambers. The

I"?— -. .

conditions necessary for a high efficiency of infection and synchronous
growth of the parasite population have been published and are used in
all of the following experiments (37, 47, 48). Briefly, they are the
following:
1. From 0 to 1 hr, inoculated plants were maintained in darkness
at 1811C andat approximately 100% relative humidity (RH).
2. From 1 to 6 hr, inoculated plants were maintained in 260 ft-c
of light (200 ft-c from white VHO-fluorescent tubes and
60 ft-c from incandescent bulbs), 22T1C, and RH of 65T5%.
3. From 6 to 20 hr, temperature and RH were the same as
2, above, but with darkness.
4. From 20 to 30 hr, conditions were the same as in 2.
In experiments to determine the environmental conditions necessary
for increasing the synchrony of formation of secondary appressoria,
the environmental conditions in the 20-30 hr period were varied.

Utilizing these environmental conditions, 80-90% of the applied conidia

25

proceed through the various stages of development (ie. germination,
formation of appressorial initials, formation of mature appressoria,
and production of secondary hyphal initials and elongating secondary
hyphaeL

Changes in temperature and RH during experiments were

monitored with a hygrothermograph calibrated with a sling psychro-

Tmr
J

meter and wet and dry bulb thermometers. Light intensity was

:9 “nun”

_..1

measured at the distance of the plants from the lights with a Weston
Model 756 light meter.

Examination o_f_ fungal structures--Observations of the percentage

 

of the parasite population in each stage of development were performed
by direct microscopic counts using the light microscope (160X). At
various times after inoculation, the development of the parasitic units
on 1 to 2 cm leaf sections (excluding the 1 cm tip section) was examined,
the percentage of parasitic units in each stage of development recorded,
and the leaf discarded. Approximately 100-125 parasitic units were
counted at each observation.

355 labellingo_fwheat leaves--Wheat leaves were allowed to take

 

 

up an 355 solution for 5 hr periods starting at different times after
inoculation with E. graminis f. sp. t_ri_£}_c_i. The 5 hr uptake period
was used to expose the developing fungus to approximately equal
amounts of 35$ in the wheat leaf. The amount of transfer to the fungus

with the 5 hr 'pulse', therefore, should reflect the development of the

26

host-parasite relationship and not the exposure to varying amounts
of 355.. A 5 hr uptake period provides more reproducible data than

shorter labelling times. The initial rapid rate of appearance of

 

radioactivity in the leaf was decreased by 4 hr and the leaves are
usually uniformly labelled by 5 hr (47).

Inoculated wheat leaves were cut at the base using a razor blade Fifi
dipped in water (the water on the cutting edge is necessary to prevent
the formation of air bubbles in the xylem tubes of the cut leaf).

The leaves were placed in small vials (25 mm x 5 mm I. D.) con-

. taining 0.1 ml of a 100 pc/ml solution of sulfur-35 (H23SSO4) in
0.1M phosphate buffer (pH 6. 9-7. 0). Frequently, 2 or 3 cut leaves
were placed in a single vial. Leaves were allowed to take up the
radioactive solution for 5 hr periods during the infection process
prior to the times that the fungus was removed. Inoculated plants
and cut leaves were maintained in growth chambers during the
experimental period under the optimum conditions for primary
infection described earlier. The darkness period, however, was
extended for two hours (6-22 hr) since light during the labeling
period resulted in a high percentage of shrivelled leaves after
application of parlodion. The 6-22 hr darkness period alters the
percentage of ESH production only slightly (47). The shrivelling of
leaves at the 24th and 26th hr time of fungus removal was greatly

reduced by placing the cut leaves at 100% RH and darkness for

27

10 minutes at the end of the labeling period prior to parlodion
application.

Radioactivity was determined using a Packard Model 3003
Tri-Carb Liquid Scintillation Spectrometer. Scintillation fluid was

a mixture of 5 g/liter PPO (2, 5 diphenyloxazole) and 0.1 g/liter

....

POPOP (l, 4-bis-(2-(5-phenyloxazolyl) )-benzene) in toluene.

“9‘1"? 1

Detection 52 35$ transferred t_OE. graminis using parlodion--

 

 

In...” .‘
I

The method of using parlodion for removal of ectoparasitic fungi was
described by Delp (15) and modified by Mount and Ellingboe (40).

A l. 9% solution of parlodion (celloidin) was prepared by dissolving
pieces of purified parlodion in a 60:40 v/v mixture of ethyl ether and
95% ethyl alcohol. The inoculated, radioactively labelled wheat
leaves were cut, placed lower surface up in plastic petri plates, and
the solution applied in a thin layer on the leaf surface with a Pasteur
pipette. Care was taken to avoid applying parlodion to either the cut
end of the leaf or to the leaf tip. The parlodion dried in minutes and
contained the parasitic units embedded in a thin film. The parlodion
strips were removed from the plant with forceps. Four strips were
placed in each scintillation vial, dissolved in 0. 2 m1 of 60:40
etherzalcohol, and the vials filled with 15 ml of scintillation fluid.
The samples were allowed to stand at least 24 hr at room temper-

ature before counts were taken to permit the parlodion to dissolve.

28

Sample size and replication 9_f_ experiments--The experiments

 

 

characterizing the development of E. graminis f. sp. tritici on the
host lines with different Efl genes were repeated a minimum of four
times on different weeks. The studies to determine the conditions
necessary for increasing the synchrony of formation of secondary
appressoria were repeated 4 to 6 times on different days. Data

presented in the Figures represent the mean of these observations.

t“~‘-r-r:—::=r.—-.tr—‘-
..£ ml

The number of parasite units counted at each observation varied from
80-125 depending upon the density of inoculation on each plant. The
experiments were repeated on different days using different plants to
eliminate small variations in either the plants or the inoculum.

The experiments involving the transfer of sulfur-35 from the
host to the parasite were performed on a minimum of 5 different days.
The average of at least 15 separate vials (4 parlodion strips/vial or
60 different plants) was plotted. Occasional vials containing parlodion
strips which had been contaminated during removal of the parasite
from the plant were discarded. Contamination occurs when the
parlodion solution flows over the cut end of the leaf and is not
observed during removal of the parlodion strip from the plant.
Distinguishing these contaminated vials was not a problem since they
contained greater than 20 times the mean counts per minute (CPM)

for that particular hour.

RESULTS

Fungus Development During Primary Infection

Replication 9_f_Previous Work I”

 

 

The development of E. graminis f. sp. tritici during germination, ,
formation Of appressorial initials, formation of mature appressoria,
and the production of elongating secondary hyphae, previously
studied by other workers (39, 47, 48), was re-examined. The optimum
environmental conditions for these stages of development have been
reported and the development of the parasite population presented in
Figure 3 is in close agreement to the earlier data. With the optimum
environmental conditions utilized, approximately 80-85% of the
applied conidia produced elongating secondary hyphae (ESH) by 26 hr
after inoculation. The attainment of this stage of development by the
fungus reflects production of mature, lobed haustoria in the host cells
and was used in later experiments as the criterion for the formation

of a functional parasitic relationship (38, 39, 40).

Effect o_f the Environment 313 the Formation 9i Secondary Appressoria

 

 

Environmental conditions are known to be important for the
development of ESH (47), but little was known concerning the environ-
mental conditions necessary for secondary infection. I was interested

29

 

30

.Ammmv Omani»: >Hmpcooom mcflmwcofio mo COSMEHOH 33
was £331.75 i393 >HNUCOOOm mo con—macho“ An:
:mTHOmmonmmm 6.35.68 mo COSOEHOH ADV .3363“ HmCOmmOHAEm

mo COSMEHOH Am: .coEmEEuom 33 .203035 scanning

 

mownsp 33.73 A? .w mmcwccmhw Osmwmtmum mo “Cogmogocwofl .m Ohdmwh

31

20.533002.
88888 a. 6.

amp“? «.1
Eugenem

 

 

 

 

 

39V1N3083d

32

to learn whether a high percentage of the parasite units with ESH

were capable of initiating secondary infections. Secondary appressoria
(SA) form by the differentiation of the elongating secondary hyphae

and represent the infection of additional host cells by each parasitic
unit. The effects of the environment upon the development of
secondary appressoria were examined.

Production of secondary appressoria with the optimum environ-

f-L‘n- .' . "'13 I I p -
."
.I i

mental conditions determined for formation of ESH at 22C is shown
in Figure 4. With these conditions (240 ft-c, 22C, 65% RH),
approximately 40% of the applied conidia formed secondary appres-
soria and initiated secondary infections by 38 hr after inoculation.

Effect _oi temperature o_n§£ formation--Temperatures of 17C

 

and 27C starting at 26 hr after inoculation were compared for their
effect on SA formation (Figure 4). The 26th hr was chosen for
changing the temperature from 22C since by this time 80-85% of
the parasite population had formed ESH. More SA formed at 27C
than at 22C and less SA formed at 17C than at 22C or 27C. Other
temperatures above 27C and below 17C decreased the percentage of
SA formed and were not studied further. The slight increase of SA
formation at 27C was the first indication that the efficiency of
secondary infections could be increased by environmental treatments.
The time that the exposure to 27C was started also affected the

efficiency of SA production. A greater percentage of the population

33

Figure 4. Effect of various temperatures, beginning 26 hr after
inoculation, upon the formation of secondary appressoria
(SA). 22C standard temperature (control) (O—O),

17C (Q—Q), and 27C (A—A).

 

PERCENT SA

34

 

HR AFTER INOCULATION

262830323430

35

formed SA when 27C was started at 26 hr, than at either 20 or 30 hr
(Figure 5). The 26th hr chosen for changing the environmental
conditions in the initial experiments appeared to be a good choice.

Effect o_f _h_igli_ Lighggn SAformationnHigh light (2600 ft-c) is
known to have an inhibitory effect upon the production of ESH (47),
but the effect on secondary infections were unknown. The percentage
of SA production increased following exposure of inoculated plants to
high light during the formation of SA (Figure 6). The increase was
greatest when 2600 ft-c was started at 26 hr, less when started at
30 hr, and no effect was observed when started at 20 hr in comparison
to the standard ESH conditions (22C, 240 ft-c, 20-40 hr). It appeared
thata change in either the temperature or light conditions at 26 hr
could be used to increase the production of SA. The lack of a
stimulation of SA formation with 2600 ft-c starting at 30 hr was
taken as possible indication of a light sensitive portion of the second-
ary infection process.

Effects 91th 1923.19.91 _o_r_1 .S_A_ formation--The possibility of an
increased sensitivity to light during the formation of SA was studied
by varying the light period during the formation of secondary
appressoria. A greater percentage of SA formed with 0 ft-c starting
at 30 hr than with O ft-c starting at 26 hr or with continuous light
(240 ft-c) (Figure 7). The formation of SA appeared to be affected by

both light and temperature at different times after inoculation.

36

Figure 5. Formation of secondary appressoria (SA) following 27C
temperature treatments starting at various hours after

inoculation. Control (22C) (D—D), 20 hr (.——.),

26 hr (O—O). and 30 hr (gm-.0).

PERCENT SA

80

7O

60

50

4O

3O

20

37

 

 

 

26 28 3O 32 34 36 38

HR AFTER INOCULATION

 

38

Figure 6. Effect of high light intensity (2600 ft-c) beginning 20, 26,
or 30 hr after inoculation upon the formation of secondary
appressoria (SA). Control (240 ft-c) (.—.),

2600 ft-c 20 hr (or—O). 2600 ft-c 26 hr (A——A),

and 2600 ft-c 30 hr (D—-—D).

PERCENT SA

39

 

20 20 30 32 34 36 30
HR AFTER INOCULATION

  

40

Figure 7. Effect of duration of light period (240 ft-c) upon the

formation of secondary appressoria (SA). 20-40 hr

 

(.——.), 20-30 hr (1:) CI), and 20-26 hr (O—O).

 

PERCENT SA

 

.I

 

7o -
60 -
so /D/D
D O
40 /%g%gé.
o /

30
/ /'
20-

.6. /

 

 

 

01+114Lll
26 28 30 323436 38

HR AFTER INOCULATION

42

Effect 3{ combined high light and high temperature 0 S

formation--Both high light (2600 ft-c) and high temperature (27C)

 

beginning at 26 hr increased the production of SA. Combinations of
high light and high temperatures were tested to determine if the
effects were additive. Combined high light and high temperature at
26 hr resulted in a greater percentage of SA formation than was
obtained with either treatment alone (Figure 8). Similarly, high light
at 26 hr with darkness starting at 30 hr was also somewhat additive
and resulted in more SA formed in less time than with either
condition alone (Figure 9). The combination of all three treatments;
2600 ft-c and 27C starting at 26 hr with O ft-c and 22C starting at

30 hr gave the highest efficiency and synchrony of SA production to
date (Figure 10). This combination of environmental conditions
increased both the efficiency and synchrony of SA production above
previous values and should be suitable for additional studies of

secondary infections.
Genetic Control of Mildew Development

The development of the powdery mildew disease is known to be
controlled by specific genes in the host and pathogen. But only a few
studies have attempted to determine when the various gene combina-
tions interact to alter development of the pathogen (3, 27, 38, 61).

Various experiments were performed to characterize the alteration

43

Figure 8. Effect of combined increased light (2600 ft-c) and
temperature (27C) treatments, beginning 26 hr after
inoculation, upon formation of secondary appressoria (SA).

Control (22C, 240 ft-c) (A—A), 2600 ft-c (o—o),

27C (Q—Q), and 27C with 2600 ft-c (kn-A).

 

44

.....

PERCENT SA

 

26 28 30 32 34 36 38
HR AFTER INOCULATION

 

45

 

Figure 9. Effect of combined treatments of increased light intensity
(2600 ft-c) and duration of light period upon formation
of secondary appressoria (SA). Control (240 ft-c 20-40 hr)
(.—.), 240 ft-c 20-30 hr and darkness 30-40 hr

(O—O), 2600 ft-c 26-40 hr (.---.), and 2600 ft-c

26-30 hr and darkness 30-40 hr (A—A).

 

PERCENT S A

 

46

26 28 30 32 34 36 38
HR AFTER INOCULATION

47

Figure 10. Effect of combination of increased light (2600 ft-c),
temperature (27C), and duration of light period upon
the efficiency of formation of secondary appressoria (SA).
Control (22C, 240 ft-c 20-40 hr) (.—-—.), and 2600 ft-c

and 27C 26-30 hr with darkness and 22C 30-40 hr

(O—O) .

 

PERCENT SA

48

 

26 28 30 32 34 36 38
HR AFTER INOCULATION

49

of parasite development during primary infection with various
incompatible parasite/host gene combinations. The initial studies
involved the characterization of the genotype of the pathogen strains

to be used in later experiments.

Genotype ngildew Cultures MS-l and MS-76

Culture MS-l was a clonally propagated isolate of £3. graminis
f. sp. £13.12 used in the previously described experiments. Culture
MS-76 was a second isolate selected because of its ability to develop
on the host lines with a m gene.

The various host lines were inoculated with cultures MS-l and
MS-76 and mildew development was recorded 7 days later. The
infection types produced 7 days after inoculation are presented in
Table 2. An infection type lower than that observed on Chancellor
was used as evidence that the gene in the fungus corresponding to the
1m gene in the host was for incompatibility (2). Based upon these
data the following statements could be made: 1) Isolate MS-l had the
corresponding 2(Unknown) and p(Mich. Amber) genes for virulence
since this isolate developed as well on host lines with a £r_r_1(Unknown)
or _P_rr_1(Mich. Amber) gene as on Chancellor. 2) Isolate MS-76 had
the corresponding El and 2(Mich. Amber) genes because it developed
as well on host lines with a _P_rrll or _PgfiMich. Amber) gene as on
Chancellor. The corresponding gene to £2.32 was difficult to

characterize since inhibition of mildew development with Pm3c has

50

Table 2. Infection types produced 7 days after inoculation of near-
isogenic wheat lines with cultures MS-l and MS-76 of
Erysilhe Eaminis f. sp. tritici.

 

 

Infection type”)

 

Near-isogenic line MS-l MS-76
Axminster x Cc8 (Pml) , 0,1 4
AsII x Cc8(Pm1) 0,1 4
Norka X Cc8 (Pml) 0,1 4
c.1. 13836 x Cc8 (Pml) 0,1 4
Ulka x Cc8 (PmZ) 2 2
C.I. 12632 x Cc8 (sz) 2 2
Asosan X Cc8 (Pm3a) 3 3
Chul x Cc8 (Pm3b) 3 3
Sonora x Cc8 (Pm3c) 4- 4-
Khapli x Cc8 (Pm4) 0,1 0,1
Yuma x Cc8 (Pm4) 0,1 0,1
Unknown x Cc8 ('2) 3, 4 2
Mich. Amber x Cc8 (7) 4 4
Chancellor (Cc) (pmx) 4 4

 

(”Infection type: O--no observable mildew development, l--chlorotic
flecking, no pustules, 2--chlorosis, necrotic reaction, 3--significant
reduction in mildew development, 4--abundant mildew development
('compatible').

51

been reported to be distinguishable only at the third or later leaf

stage (5, 7, 55). Both strains MS-l and MS-76 were assigned the
BE genotype, since in our study there was both a slight but noticeable
decrease of mildew development on seedling leaves and a significant
reduction of mildew on older leaves. The remaining wheat lines were
characterized by decreased mildew development. The corresponding
genotypes of the two mildew cultures, based on mildew development
on the host lines with various 23 genes, appeared to be the following:

MS-l _P_l_ BE P3a P3b P3c B4 2(Unknown) 2(Mich. Amber)

 

MS-76 Ll I12 P3a P3b P3c fl I_3(Unknown) 2(Mich. Amber)

 

Effect of Different Parasite/Host Genotypes on Mildew Development

During Primary Infection

 

Time o_f_ fungus inhibition--The development of appressoria

 

with various incompatible combinations (g/M) was essentially
identical to development in the absence of specific 31?} genes
(Figure ll). Neither the synchrony of development nor morphogenesis
of the parasite was affected prior to the time when penetration of the
epidermal cell is thought to occur (39, 45). Later stages of parasite
development were altered in a specific manner depending upon the
particular gene combinations which interacted.

Compatible parasite/host combinations--The formation of ESH
appeared to be essentially identical on the cultivars Little Club and

Chancellor (Figure 12). The optimum environmental conditions

Figure 11.

52

Similar development of appressoria during primary
infection with either compatible or incompatible parasite/
host combinations. MS-l/Chancellor (compatible control)
(An-A), fl/ErgyAxminster) (Q—Q). gg/glngmka)
(O—O). flit/M (A-—-A), and E/P—mflKhapli)
(on-o).

 

53

 
  

IOO >-

P

w

 

 

m

   
 

_IILIIL F+
m m m n w

4 E0 mmmmaad Pzwommm

 

HR AFTER INOCULATION

 

54

.AOIIOV 8:0 3:4 nan 3'9 8:835

 

mumzugo co Sumzv MUST: .mm .m mMEEMHm ofimwmrm

>3 Ammm: owe—93 naumpcoomm mcfimwdofio mo cofimfiuoh

:2 $st

55

Azo_b<4:ooz_ cut: 5.: NSF

1km QT mm mm s to

 

 

 

H83 lNBOUBd

56

determined for parasite development with the MS-l/Little Club
compatible combination (37, 47, 48) appeared to be satisfactory when
studying other parasite/host combinations which lacked pairs of
corresponding genes affecting compatibility.

The production of ESH was essentially identical with the
B(Unknown)/P_m(Unknown) and p(Mich. Amber)/_Pf_r2(Mich. Amber)
genotypes as observed with Chancellor and Little Club (Figures 12, 13).
Development of the parasite was similar in combinations with
Pi/BBCFi—x genotypes, or with compatible combinations (ESE/EEK or
25/311) in the absence of specific 5 genes altering compatibility.

A much wider variation in effects upon the formation of ESH was
observed on the remaining ten wheat lines.

Incompatible parasite/host genotypes involving Pml-—The
development of secondary hyphae was strongly inhibited with the
fl/Efll genotypes (Figure 14). The few secondary hyphae which
formed elongated briefly and attained a length of only 15-20 microns.
Approximately 24-28 hr after inoculation, elongation ceased and many
of the parasitic units collapsed. The host cells surrounding the
penetration sites frequently appeared brownish and discolored.

A few parasitic units formed what appeared to be small distorted
haustorial bodies. Mature haustoria with elongated appendages were

notfound.

 

57

.alg Anna—Ea. ..fin2vfl:§8< £35m can
.3. :9 Ansonxnovflgsonxnam .20383 AOIIQ

HOZmocm303nmé/H umcofimfiagoo «mos\ofimm.umm 3939800

 

mate/020m on» 5:? 35.73 .mm .H 358th 0:33me

>3 Ammmv vocal»: thumpcouom mdflmwdofio mo cofimfinoh

.2 8:3

58

On

AZO_F(JDOOZ. cut‘ 8.: N1;
u .N g \. .‘

 

n

8828»?

H83 ““0834

59

Figure 14. Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. £r_i_ti__c_l (MS-1) with the following
incompatible parasite/host genotypes: P_l/pgi_l(Chancellor)
(compatible control) (O—O), P_l/P_m_l_(Axminster) (.—.),
fl/_P_m_l(AsII) (l—fl), P_1/P_m_1_(Norka) (A---A), and

fl/Pml(C.I. 13836) (ID—El).

 

......

60

    
 

w
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U)
u 0
g...
2
u 0
0
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u 0
I.
to o
/

     

20 22 24 26 as
Tm: (HR AFTER INOCULATION)

61

Incompatible parasite/host genotypes involving PmZ--The
production of ESH with the £_m_2 genotypes (Figure 15) was essentially
identical to that observed with Chancellor or with lines with a fig}
gene but for which the parasite had the appropriate corresponding p_
gene for compatibility (Figure 13). Continued development of the ESH
at later hours appeared to be similar to the compatible genotypes
studied earlier. Haustoria observed in epidermal strips at 20, 26,
and 30 hr after inoculation corresponded in number and development
to those observed with Chancellor or Little Club. Approximately
3-4 days after inoculation, chlorotic flecking became macroscopically
evident on the inoculated leaves. After 2 more days, necrosis of the
leaf was more obvious and only a very limited amount of sporulation
by the fungus occurred.

Incompatible parasite/host genotypes involving Pm3--Of the
three distinct £91 genes available at the §_m_3 locus, only two were
found to operate during the stages of primary infection at the first
leaf stage of plant development. The third, m, had been reported
to be effective only in plants at the third or later leaf stage (5, 7, 55)
and no effect upon primary infection was observed. The development
of ESH with the P_3/_E_IT_1_3 genotypes is shown in Figure 16. The

P3a/Pm3a and P3b/Pm3b genotypes reduced the percentage of ESH

 

 

which formed. The parasite units which did produce ESH continued

development in subsequent hours and limited sporulation was usually

62

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, .ACIIICV Awfiovmfim\m .AOIQ 293:8 6353803

..A.~o:oucmnva8m\m ”moguocom “mos\ofimmumm ozfldmgoocm

 

9:30on 05 5:3 Suwgv SETS .mm .u 358de osmwmwum

>3 Ammm: mung: >Hmpcoomm mcfidmcoao mo coBmEuoh

.3 Barn

63

 

TIME (0“ AFTER INOCULATION)

64

.aILS IIIUMEEflM van .5119 £85an
.CIIQ mama .AOIIQ 28:8 0352503

Anozoodmsuvmfla\umnfinmm .mmnm "moguocom umo£\ofimmn.mm

 

 

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.2 ondmwh

65

 
  

 

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66

observed 7 days later. However, both infection type and continued
development were variable and a strong effect of the environment was
evident. With the standardized environmental conditions employed

in this study, the P3a/Pm3a and P3b/Pm3b genotypes appeared to

 

lower the efficiency of infection but not to completely inhibit the
development of the fungus.

The genetic uniformity of the mildew culture was tested in two
experiments. Results obtained with a re-purified isolate of culture
MS-l were essentially identical to those presented in Figure 16.
Also, when conidia from mildew which developed on the 2313 lines
were increased on Little Club, ESH production and infection type on
the £213 host lines were the same as observed using the standard
stock culture of MS-l (Figure 17). It appears that the partial
inhibition of development of the fungus with the P3a/Pm3a and

P3b/Pm3b genotypes is characteristic of the interaction and is not

 

due to mixed inocula.

Incompatible parasite/.1323 genotypes involving I_3_m_4_--The
_P_4/P_m:1_ genotypes were characterized by rapid and complete inhi-
bition of development of the parasite. The production of ESH on the
lines studied is presented in Figure 18. Only a very low percentage
of parasitic units produced ESH and those ceased elongating after
attaining a length of 15-20 microns. Approximately 20-24 hr after

inoculation, most of the parasitic units had collapsed and the host

Figure 17.

67

Formation of elongating secondary hyphae (ESH) with
the following incompatible parasite/host combinations:
MS-l/Chancellor (compatible control) (‘——‘),
ggms-n/m (0—0, and _ggng-Iawgngg
(.—--.). MS-l* was obtained from the mildew (MS-l)

which sporulated on the Pm3a wheat line.

 

 

68

 

b k b .
m w w m
Imw hzmomwd

   

22 24 26 28
INOCULATION

20
HR AFTER

 

69

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can .AOIIIQ Emnsmmmflmxm .5119 203:8
63339803 Auozoocmsuvvg\flm ”moguocvw «m0£\ofim.mh.mm
0352885 2:30:03 2: at? 2.36 a .mn q a

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«20333002. Kuhn: 5.: ml:

 

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H83 1N3083d

71

cells around the sites of penetration were discolored. Examination
of epidermal strips revealed that some parasitic units had produced
what appeared to be rudimentary distorted haustorial bodies. Mature
(lobed) haustoria were not observed. Darker staining areas were
frequently observed around the penetration sites during examination
of epidermal strips and may indicate a hypersensitive collapse of
host cells. Based upon the very low number of ESH observed, it
appeared that this gene combination interacted during or soon after

penetration of the host cell.

Gene-for-Gene Specificity Emessed During Formation of ESH
Cultures MS-l and MS-76 which possess the corresponding El
and L1 genes, respectively, were used to study mildew development
on wheat lines containing either _P_ml or am} Development of ESH
with the El/EELI genotype, a compatible combination, was similar to
that observed with compatible combinations in the absence of specific
El genes (21./221.1 or P_1/p_r2_l_) (Figure 19). The formation of ESH was
inhibited only with the 11/2321 incompatible combination. The
development of ESH with the four genotypes involving _E_rr_1_1_(.1_3_1/£’£1_,
fl/EELI. gym. Ll/ml) corresponded to the predictions of the gene-
for-gene hypothesis developed using infection type data (20, 21). In
addition, the development of ESH during primary infection corre-
sponded to the 'quadratic check' proposed by Rowell ital (56). The

'quadratic check' has been suggested to correlate the biochemical

72

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H83 1N3983d

74

and physiological data concerning host-parasite interactions with the

gene-for-gene specificity controlling the relationship.

Transfer of 355 from Wheat Leaves to E. graminis f. sp. tritici

355 transfer with incompatible parasite/host combinations--The
movement of 35$ from radioactively labelled wheat leaves to the
external mycelium of the fungus was examined with various incom-
patible (EL/PE) parasite/host combinations. Inoculated wheat leaves
were allowed to take up an 355 solution for five hour periods during
primary infection. The fungal mycelium on the surface of the leaf
was removed after the 5 hr period with a parlodion solution and the
radioactivity contained in the parlodion strip was determined. With
the fl/BrgyAxminster) genotype, which affects parasite development
at or near the time of penetration, only a small amount of 355 was
transferred as compared to the compatible El/pfllehancellor)
combination (Figure 20). With the £_2_/_P;m_2(Ulka) genotype, which
did not inhibit the formation of ESH, the amount of 355 transfer was
somewhat greater, but essentially very similar, to transfer with the
compatible fl/fl(Chancellor) combination (Figure 21). The amount

of 355 transfer with the P3a/Pm3a combination, in which the devel-

 

opment of only a fraction of the parasite population was inhibited,

was intermediate to that observed with the P3a/pm3a(Chancellor)

 

compatible combination (Figure 22). With the Ei/Pm4(Khapli)

combination, in which the formation of ESH was greatly inhibited,

75

Figure 20. Transfer of 355 from wheat to the external mycelium of
Erysiphe ggminis f. sp. tritici with the following
parasite/host genotypes: fl/pmMChancellor) (H)

and fl/PmHAxminste r) (.—.) .

 

3O

8

N
O

(353) COUNTS/MIN xno“
a

O

76

/

7 fi

68 |0l2l4 . = 0222426
HR AFTER INOCULATION

t

Figure 21.

77

Transfer of 355 from wheat to the external mycelium
of Erysiphe graminis f. sp. tritici with the following
parasite/host genotypes: El/BmZKJhancellor) (0—0)

and _l_?_2/Pm2(Ulka) (.——.).

.....

B

(”5) COUNTS/MIN x no"
a

,/
n...-
o 3,...

6 8 l0l2l4|6l020222428
HR AFTER INOCULATION

79

Figure 22. Transfer of 358 from wheat to the external mycelium

of Erysiphe graminis f. sp. tritici with the following

 

parasite/host genotypes: P3a P3bP3c/pm3(Chancellor)

(0—0) and Egg/Pm3a (.——.).

(3‘s) COUNTS/MIN x I04

8

3

5

80

 

6 0 DB H|6l8202224fl
HR AFTER INOCULATION

81

only a small amount of 355 transfer was detected (Figure 23). The
amount of 355 transfer was essentially identical to the transfer with
EBA/_P_nll (Figure 20). The transfer of 355 with the various genetically
controlled combinations appeared to be a very good indicator of the
inhibition of morphological development of the parasite on the surface

of the host.

 

Transfer 933 w the four genotypes involving P_m_l--The
movement of 358 from the wheat plant to the external mycelium was
studied with compatible and incompatible genotypes involving the BE
locus. Three of these combinations (El/REHChancellor),
LUflMChancellor), and pl/EflUXxminsterH resulted in a compatible
relationship indistinguishable by either infection type or by alteration
of the development of ESH (Table 2 and Figure 19). The remaining
incompatible El/EgiyAxminster) genotype inhibited parasite
development and allowed only a low number of ESH to form (Figure 14).
The transfer of 355, studied earlier with the P_l/_P_LI_1_1(Axminster) and
fl/pgil(Chancellor) combinations (Figure 20), was found to reflect the
inhibition of development of the parasite during primary infection.
Culture MS-76 was used to study the remaining two compatible
pl/_P_nll_(Axminster) and p_l_/p_ni(Chancellor) genotypes.

The transfer of 35$ with all four genotypes is shown in Figure 24.
The least quantity of 355 was transferred with the incompatible P_1/_li_n_i_l

genotype, an intermediate amount was transferred with the pl/Pml

82

Figure 23. Transfer of 35$ from wheat to the external mycelium

of Erysiphe graminis f. sp. tritici with the following

 

parasite/host genotypes: Eg/EmflChancellor) (0—0)

and fi/PmflKhapli) (.-—-.).

(3‘s) COUNTS/MIN x IO“:

~\
0

‘w
.

-
O

5

O

83

C
4.”. ‘

h'

ODIMIJ

HR AFTER

I NOOULATION

84

I5

Figure 24. Transfer of 35$ from wheat to the external mycelium
of Erysiphe graminis f. sp. tritici with the four possible
genotypes involving the Pml locus. P_l/Em1(Chancellor)

(A——A), g/mmhanceuor) (ID—El).

Ll/PmHAxminster) (.—.), and Til/Pml(Axminster)

(O—O).

N
on

8

85

 

6 8
HR AFTER

IOIZHB

  
  

 

 
  

z 20 22 24 26
INOCULATION

  

86

genotype, and the greatest and similar amounts were transferred with
the film and film genotypes. The transfer of 35$ in the com-
patible EMF—ml genotype was lower than the other two compatible
combinations. The reduced transfer of 355 with ELF—ml is not
explainable by variations in either environmental conditions, plants,
or the inoculation densities since the plants for both combinations
were grown together, inoculated at the same time, and maintained
in the same growth chamber each time the experiment was performed.
Furthermore, the difference in transfer is not due to inhibition of
parasite development, since the development of ESH by the fungus was
essentially identical with all the compatible genotypes.

The shape of the transfer curves in these compatible com-
binations appeared to reflect a two component process involved in
the movement of 355 from the plant to the fungus. There may be
two explanations for this result: 1) the 6-22 hr after inoculation are
in darkness and turning the lights on at the 22 hr may have an effect
on transfer, or 2) the host-parasite relationship is in a state of rapid
development during this period of the infection process and the steps
in the curve may reflect either the morphogenesis of fungal haustoria
or a lag period preceeding the initiation of secondary hyphae. The
first alternative was tested by leaving the lights off for the 22-26 hr
period to see if a step was still obtained. The results in Figure 25

demonstrate that a step still exists in continuous darkness, but the

Figure 25.

87

Effect of light upon the transfer of 35$ from wheat to

Erysiphe graminis f. sp. tritici. Darkness 6-22 hr

with 240 ft-c 22-30 hr (.—.), and darkness 6-26 hr

with 240 ft-c 26-30 hr (o—o).

 

 

(35$) COUNTS/MIN x IO'2

N
U!

8

G

5

88

, d——-

6802

“6320222428

HR AFTER INOCULATION

89

step was delayed two hours. This suggests that the step is a
reflection of parasite morphogenesis and treatments which alter

the synchrony of morphogenesis also affect the transfer data.

DISCUSSION

The primary stages of infection of wheat and barley by Erysiphe
graminis have been studied by employing the optimum environmental
conditions necessary for a high infection efficiency and synchrony
of parasite development (37, 47, 48). This defined system has allowed
studies of the initial host-parasite interactions during primary in-
fection and could be used in determining the effects of environmental
or other treatments upon the establishment of a successfdl infection.
The production of elongating secondary hyphae by the parasite pop-
ulation was used as the criterion of a functioning infection and was
believed to result from a dynamic interaction between the host and
parasite which allowed continued fungal development. This latter
assumption was tested in later experiments by measuring the transfer
of 355 from host to parasite.

Initial studies were concerned with the development of ESH
which eventually form SA and initiate secondary infection sites on
the plant. The production of SA was found to be affected by both
light and temperature during the 20-30 hr period after inoculation.

An increase in the production of SA following environmental changes

indicated that the formation of SA had particular environmental

90

91

requirements for maximum efficiency. High light or high temperature
beginning at the 26 hr was found to increase SA production. Light
period also appeared to have a definite effect. Increased percentages
of SA were formed if the lights were turned off at 30 hr after inocu-
lation. This inhibitory effect of light upon SA is similar to the
inhibition of the formation of primary appressoria which require an
initial 4 hr period of light (240 ft-c) followed by darkness for maximum
efficiency (48).

Combinations of various environmental conditions were employed
to further study the effects of light, darkness, and temperature upon
SA formation. Combination of high light (2600 ft-c) and high
temperature (27C) beginning at 26 hr after inoculation or high light
at 26 hr followed by darkness at 30 hr resulted in a greater percentage
of SA formed than with any of the conditions tested alone. Combination
of all three conditions (2600 ft-c and 27C beginning at 26 hr with
0 ft-c and 22C at 30 hr) was also somewhat additive and resulted in
the highest percentage of SA formation attained.) ‘This additive effect
of combining single environmental conditions each known to increase
infection efficiency is similar to that observed in studies of germina-
tion of E. graminis conidia (48). In those studies the optimum
conditions for germination required a combination of certain light,
humidity, and temperature conditions during 0-6 hr after inoculation.

The possibility exists that the combination of environmental conditions

92

utilized for increased SA production were additively affecting
physiologically distinct portions of the parasite population. This
possibility must be studied further since physiological non-uniformity
has been demonstrated during germination with an otherwise
genetically uniform clonal population of E. graminis conidia (48).
The inhibition of E. graminis development during the stages
of primary infection was examined with various near-isogenic wheat
lines containing single 293 genes altering compatibility to mildew
development. The phenotypic expression of various parasite/host
combinations indicated that the gene-for-gene specificity governing
the relationship was also reflected in the mode of interaction of the
particular genotypes studied during primary infection. The effects
of this interaction with each parasite/host genotype studied could
be observed at a specific stage of pathogenesis. The earliest effects
were observed during or soon after the penetration of the host epi-
dermal cell and are reflected in the low number of parasitic units
which produced ESH in combinations of either 31/2311 or P_4/§£4.
The morphological similarity of the responses conditioned by these
two incompatible combinations may indicate that a similar basis for
the reaction is involved. The fl/Erfli genotype was thought to
interact at slightly earlier stages of the infection process since a
lower percentage of ESH formed with 25./£215. than with P_l/§£l.

The development of the parasite with the EE/sz genotype was

93

especially interesting because compatibility was not altered during
primary infection. The effects of the incompatible interaction were
first observed 72-96 hr after inoculation, long after a functional
host-parasite relationship had formed. These results are somewhat
similar to delayed hypersensitive responses observed by other workers
(12, 27, 35, 38), but unique in that no inhibition of parasite
development could be observed during primary infection, even by

using a very sensitive system of measurement. The Pk/Mlk

 

genotype with barley mildew specifies a delayed hypersensitive
reaction 3-4 days after inoculation with E. graminis f. sp. hordei,

but it also inhibits the development of 60-70% of the parasite
population during primary infection (38). The P3c/Pm3c combinations
are somewhat similar to fl/m genotypes, but the expression of

the P3c/Pm3c interaction is a function of host maturity. Pm3c

 

appears to inhibit mildew development of pathogen strains with the
corresponding £23 gene only at the third or later leaf stage of plant
development. The determination of the bases for these delayed
responses should be possible. The use of a defined host-parasite-
environmental system together with biochemical and genetic studies
may provide some insight concerning the systems operating to alter
parasite/host compatibility.

The results with the P3a/Pm3a and P3b/Pm3b genotypes are

 

somewhat confusing since only a proportion of the parasite population

94

is inhibited during primary infection. Continued development of the
30% of the parasite units which do form ESH appears to be strongly
affected by later environmental conditions. Additional studies of these
genotypes at later developmental stages are necessary to clarify

this preliminary observation.

The inhibition of fungal development with the various incom-
patible gene combinations was tested further by observing the transfer
of 355 from the plant to the fungus. These methods were developed
by Mount (46) with the MS-l/Little Club compatible combination. The
amount of 355 transfer to the fungus was found to parallel the develop-
ment of the fungal haustoria in the host cells. The transfer of 358
possible with incompatible combinations, therefore, should reflect
the time that fungal development and the host-parasite relationship
was altered.

The amount of 355 transfer was found to correspond to the
morphological development of the fungus with the various incompatible
genotypes studied. 358 transfer was greatest with genotypes which
appeared fully compatible, as evaluated by the production of ESH
during primary infection. The reduction of transfer of 358 to the

fungus with the P_l/Pml, 133/sz, P3a/Pm3a, and Ei/Pm‘l incom-

 

 

patible combinations was similar to the inhibition of the percentage

of ESH with each combination. The correspondence of the amount of

355 transfer and the relative production of ESH during primary

95

infection suggests that the development of ESH is closely related to
the transfer of materials from the plant to the fungus. This move-
ment of materials and nutrients is presumed to allow continued
fungal development after the production of appressoria.

The correspondence between fungal development and 358
transfer appeared useful to further characterize the four £1111 geno:
types studied during primary infection. The characterization of these
genotypes would complete the 'quadratic check' on three levels:

1) by final mildew development (infection type), 2) by parasite
development during primary infection, and 3) by the amount of 358
transfer with each gene combination. The data on 355 transfer
demonstrated, however, that not all of the three compatible
combinations were the same, and that the pl/Efn—l genotype was
distinct from gum and Ll/pnll. This difference is considered to be
extremely interesting. since it indicated a unique interaction with the
Elm genotype. The difference appeared to be real, since the
Ll/P_m_l_ and Ll/Eml combinations were investigated on the same days,
with simultaneous inoculations, and identical environmental conditions
in the same growth chamber. Furthermore, the results were not
subjectively biased since the experiment was initially expected to
demonstrate the similarity of the compatible combinations. The
significance of demonstrating the Ll/_P_m_l genotype to be unique lies in

the applicability to model systems attempting to describe the basis of

96

host-parasite compatibility. The systems specified by the an; gene
involved in altering compatibility are apparently overcome or by-passed
by strains of the fungus with the correspondingfl gene. The _p_l/_Pln_l
combination is apparently fully compatible on a morphological basis

35S transfer. This

but can be shown to be different with respect to
difference offers many suggestions for future studies to determine the
basis for the altered transfer which does not appear to be due to
inhibition of parasite development. Additional parasite/host genotypes
and radioactive tracers must be investigated to corroborate these
observations. Further experiments will eliminate the possibility that
the inhibition of 355 transfer is due to modifier genes with only a

slight effect on fungus development.

The nature of the systems operating to alter compatibility
remains a matter of conjecture. Several people have speculated that
the induction of specific enzyme systems is involved in the incompatible
response (26, 31). The inducer is thought to be produced by pathogenic
strains possessing a §_x gene which activates the correspondingfii
system in the host. Induction does not occur in the absence of an};
gene and the inducer is either altered or absent in the pathogenic
strain containing the BE gene. This possibility is interesting but
unfortunately it has been recently applied to the production of

'phytoalexins' for which genetic specificity has not been demon-

strated (26). Furthermore, 9e possibility overlooked by this type of

97

model is that the E locus in the pathogen is involved in altering
sensitivity of the fungus to a substance produced by theE locus in the
plant. Sensitivity may be controlled at the membrane level as has been
proposed for the mechanism of toxic action of E victoriae toxin on
oat tissue (57), or in the breakdown of an inhibitory substance in the
plant by the developing pathogen. The ability to produce the inhibitor
would be controlled by the E genes in the plant and sensitivity to the
toxic effects would be determined by the 2 gene in the fungus.

The specificity in the operation of the gene-for-gene interactions
and the large number of possible E and E loci involved demand a
corresponding specificity in the operation of the incompatibility
mechanisms. The presence or absence of a toxic substance would
neither explain the specificity observed nor the various effects upon
parasite development with the various possible parasite/host
genotypes. Ellingboe (17) has suggested that if the incompatibility
system is operating via the DNA-RNA-protein pathway, then alleles
at a particular E locus should operate similarly since the structure
or function of the same protein would be involved. This suggestion is
consistent with the incompatible responses observed in this study for

the P3a/Pm3a and P3b/Pm3b genotypes known to represent distinct

 

 

alleles at the Pm3 locus in the plant. Whether this result is generally
true for other allelic genes at other Pm loci must be studied further.

The genetic determination of compatibility can be used as a

98

tool for estimating both the number and nature of the systems
involved in the establishment of a host-parasite relationship. The
diversity of the incompatible responses observed in this study suggests
that the systems controlling parasite/host compatibility may be based
on a number of possible mechanisms. The range of different in-
compatible responses and phenotypes observed should serve as a
guide for selection of particular genotypes for biochemical studies.
The eventual biochemical characterization of the particular systems
involved in the incompatible response, however, must be shown to
segregate with the genes governing compatibility and to operate at
the time of the earliest observed effects of the incompatible inter-

actions.

SUMMARY

The infection process of Erysiphe graminis on wheat consists
of a number of morphological stages of development (39, 47, 48).
Experiments demonstrated that the formation of secondary appressoria .
(SA) could be altered by various environmental conditions. The
efficiency and synchrony of SA production were increased by
environmental alterations. Sixty-five percent of the applied conidia
produced SA following exposure of inoculated plants to high light
intensity and high temperature (2600 ft-c and 27C, respectively) at
26 hr after inoculation followed by 22C and darkness at 30 hr after
inoculation.

Mildew development was studied during primary infection with
various compatible and incompatible parasite/host combinations.
The development of E. graminis f. sp. mon near-isogenic
wheat lines containing single genes affecting mildew development
was altered in a specific manner with each gene combination. The
development of the strain of the pathogen possessing the comple- .
mentary L1 gene(s) on host plants with the P_rr_g gene(s) (Ll/293$)
was indistinguishable from compatible gene combinations involving

pml (fl/pml or El/pml). With the four possible parasite/host

99

100

genotypes involving the E_m_llocus (El/gall, fl/ELI' Ll/_E_m_l, p_l_/En_l_),
alteration of both infection type and the development of elongating
secondary hyphae (ESH) was observed only with the incompatible
P_l/P_ml_ genotype.

355 transfer from the wheat leaf to the developing fungus was
less with incompatible genotypes than with compatible genotypes.
The amount of 355 transfer was greatest with parasite/host
combinations which did not alter the processes of primary infection
and was least with combinations which greatly inhibited fungus
development. Measurement of 355 transfer with the four ELIE
genotypes (El/m, 21./m, Ll/Efllg EL/Em—l) revealed that the
compatible al/le genotype could be distinguished from the com-
patible _Ifl/p_m_l and Ll/m genotypes. The amount of transfer was

lowest with P_l/Pml, intermediate with pl/Pml, and greatest with

I_D_1_/Em1 and Ll/pml.

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