GENETIC CONTROL OF THE

DEVELOPMENT or HAusIomA: (II I} 13:7}: 5 , :1;

ERYSIPHE GRAMINIS F;-
SP. TRITICI 0N WHEAT

' ' Dissertation III the Dég’Iee Df PTI. D. _‘:,I
MICHIGAN STATE UNIVERSITY ‘

MARI IOI HAIwooo '
» 1925 " ~

 

This is to certify that the

thesis entitled

GENETIC CONTROL OF THE DEVELOPMENT OF
HAUSTORIA 0F ERYSIPHE GRAMINIS F. SP.
TRITICI ON WHEAT

presented by
Mary Joy Haywood

has been accepted towards fulfillment
of the requirements for

Ph,D_ degree in Botany and Plant Pathology

Major professor

Date August 5, 1975

0-7 639

 

 

ABSTRACT
GENETIC CONTROL OF THE DEVELOPMENT
OF HAUSTORIA OF ERYSIPHE GRAMINIS F.
SP. TRITICI ON WHEAT
BY

Mary Joy Haywood

Genes conferring resistance in wheat to Erysiphe graminis f.

 

sp. 3% have been shown to affect the ontogeny of interactions
between host and parasite. Direct microscopic observations made
every 2 hr from 8 through 30 hr after inoculation, following fixation
and staining of materials, with aniline blue, showed that, with the
compatible TEE/pg genotype, 87% of the parasite units form pri—
mary haustoria by 30 hr after inoculation. With the incompatible

genotypes, Pla/Pmla, P2a/Pm2a, P3a/Pm3a, and P4a/Pm4a,

 

 

the percentages of the parasite units that formed primary haustoria
by 30 hr after inoculation were 15, 66, 18, and 3, respectively.

The incompatible interaction P2a/Pm2a apparently does have an

 

affect earlier than previously believed. The earlier work was
based on the infection efficiency and development of elongating

secondary hyphae (ESH). With the incompatible genotype, P3a/Pm3a,

 

by 30 hr after inoculation, only 15% of the parasite units had haus-
toria greater than 35“ in length. A comparison of these results
with the infection efficiency of 30% obtained by direct measure-

ments of the elongating secondary hyphae, the percent of the

Mary Joy Haywood
parasite units that produced ESH was smaller and had to include
haustoria less than 35“ in length, thus, smaller haustoria are sup-
porting the development of ESH in this genotype. The percent of
the parasite units that produced ESH with the incompatible inter-

actions, Pla/Pmla and P4a/Pm4a are in close agreement with

 

 

previous findings. The data also showed that haustorial develop-
ment in the apparently incompatible interactions is not a clear—cut
phenomenon. When some infected cells were penetrated, develop-
ment of the parasite unit ceased rapidly. In other infected cells,

the parasite unit appeared to develop normally for a few hours and
development stopped, leaving a rudimentary haustorium. In these
latter reactions, the host cells picked up the dye indicating meso-
phyll collapse.

Light is necessary at specific times during primary and secon-

dary interactions for synchronous development of Erysiphe graminis

 

f. sp. tritici on wheat. Synchronous development of _F__.‘_. graminis
during secondary infection was increased when light periods (1. 3

2 sec-1, incandescent and fluorescent) were altered

ergs cm"
between 26 and 58 hr after inoculation. With a light period from
20-36 hr and a dark period from 36-44 hr followed by another light
period from 44-58 hr after inoculation, approximately 87% of the

13L. graminis conidia applied to wheat that produced primary haus-

toria also produced secondary haustoria.

Mary Joy Haywood
Establishing the optimal environmental conditions necessary
for secondary haustorial development of Egg/ping provided a standard
for determining the rate of development of the incompatible inter-
actions from 38-58 hr after inoculation. By 58 hr after inoculation,
52% of the compatible B/EE interaction had formed secondary
haustoria up to 35;; in length. The percent of secondary haustoria

formed with the incompatible parasite/host genotypes, Pla/Pmla,

 

P2a/Pm2a, P3a/Pm3a, and P4a/Pm4a were 6, 28, 5, and 1, res-

 

 

pectively. Greater than 75% of the host cells in each of the incom-
patible interactions had picked up the dye, indicating mesophyll
collapse and necrogenic protoplasts. These results provide addi-
tional evidence that the different genotypes affect different stages
in the ontogeny of the host/parasite interactions and that some of
these interactions affect development earlier than previously

reported .

GENETIC CONTROL OF THE DEVELOPMENT
OF HAUSTORIA OF ERYSIPHE GRAMINIS F.
SP. TRITICI ON WHEAT

BY

Mary Joy Haywood

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

1975

Dedicated
to

My Mother and Father

ii

ACKNOWLEDGEMEN TS

I am deeply grateful to Dr. Albert H. Ellingboe, my major
professor, for his guidance, assistance, and encouragement during
this investigation and in the preparation of this thesis.

Iwish to thank Drs. W. G. Fields, W. B. Drew, L. L.
.Bieber, and D. J. deZeeuw for serving on my committee and their
assistance in the preparation of this manuscript.

Moral and financial support given to me by the members of
my religious family, the Pittsburgh Sisters of Mercy, is gratefully
recognized.

I would also like to thank Mr. Joseph L. Clayton for his
technical assistance and Dr. T. Joe Martin for his assistance and
advice throughout my stay at Michigan State University.

My gratitude also to Mr. Walter W. Burinski for his assis-
tance in obtaining materials via the Interlibrary loan and to the
many faculty members and fellow graduate students for use of
equipment and meaningful discussions during my research.

This research received partial support from the National

Science Foundation for which I am indebted.

iii

 

TABLE OF CONTENTS

Page
DEDICATION ........................................... ii
ACKNOWLEDGEMENTS ................................ iii
LIST OF TABLES ..................................... VI.
LIST OF FIGURES ............................ . ......... vii
INTRODUCTION ......................................... 1
MATERIALS AND METHODS ............................. 3
Inoculum Production .................................. 3
Method of Inoculation ................................. 5
Environmental Conditions for Experiments -------------- 5
Gene Symbols and Designation of Parasite/ Host
Genotypes ...................................... 6
LITERATURE REVIEW .................................. 8
CHAPTER
I.. GENETIC CONTROL OF PRIMARY HAUSTORIAL
DEVELOPMENT OF ERYSIPHE ON WHEAT ----------- 15
Introduction .................................... 15
Materials and Methods .......................... 16
Results ....................................... 22
Discussion .................................... 34
Summary ..................................... 39

iv

 

Page

II. THE INFLUENCE OF LIGHT PERIODS ON THE
PRODUCTION OF SECONDARY HAUSTORIA BY

ERYSIPHE GRAMINIS ON SUSCEPTIBLE WHEAT °°°°° 41
Introduction ................................... 4 1
Materials and Methods .......................... 42
Results ....................................... 44
Discussion ..................................... 50
Summary ...................................... 51

III. GENETIC CONTROL OF SECONDARY HAUS—
TORIAL DEVELOPMENT OF ERYSIPHE

GRAMINIS ON WHEAT .............................. 5 2
Introduction ................................... 5 2
Materials and Methods .......................... 5 3
Results ....................................... 5 3
Discussion .................................... 6 1
Summary ...................................... 6 3

LITERATIJRE CITED .................................. 65

 

 

LIST OF TAB LES

Table Page

1. Near-isogenic lines with single Pm genes and
infection types produced seven days after
inoculation With MS_1 ............................ 4

2. Effect of genotype of host and pathogen on
development of haustoria through primary
infection ....................................... 25

3. Standard environmental conditions used for the
first 26 hr after inoculation ---------------------- 43

4. Experimental environmental conditions used
beginning 26 hr after inoculation ------------------ 43

5. Percent of parasitic units forming primary
haustoria which continued to form secondary

haustoria ....................................... 49

6. Effect of parasite/host genotype on development
of primary and secondary haustoria --------------- 54

vi

Figure

1.

LIST OF FIGURES

The four possible parasite/host genotypes
involving a single gene pair governing
compatibility of host and parasite --------------- 13

Photomicrograph of a primary haustorium,
with a compatible genotype, Px/pmx, in
wheat epidermal cell at 26 hfifter
inoculation .................................... 19

 

Drawing of a primary haustorium with a com-
patible genotype, Px/pmx, at 26 hr after
inoculation depictfi infection structures
and extensive hyphal growth --------------------- 19

 

The ectoparasitic structures of Erysiphe
graminis on wheat observed with a Scanning
Electron Micros cope ........................... 21

Development of Erysiphe graminis f. sp. tritici
during primary infection of wheat leaves ---------- 24

 

Effect of five different parasite/host genotypes
on the development of primary haustoria of
Erysiphe graminis f. sp. tritici on wheat --------- 27

Photomicrograph of the penetration process of
the incompatible interactions, Pla/Pmla,
P2a/Pm2a, P3a/Pm3a, and P4a/Pm4a ----------- 32

 

Drawing of the penetration process of the incom—
patible interaction Pla/Pmla, P2a/Pm2a,
P3a/Pm3a’ and P4a/Pm4a ...................... 32

 

Drawing of the incompatible interaction, P4a/Pm4a
showing rudimentary haustoria and mesophyll
collapse ....................................... 38

 

 

vii

Page

Figure Page

10. Formation of secondary haustoria by
Erysiphe graminis f. sp. tritici cultures
MS-l by varying the light periods --------------- 46

 

11. Drawing of a primary haustorium and a secondary
haustorium with a compatible genotype, Ex/pmx,
at 54 hr after inoculation ---------------------- 48

12. Effect of the five different parasite/host geno-
types of the development of secondary haus—
toria of Erysiphe graminis f. sp. tritici --------- 57

 

viii

 

INTRODUCTION

Plants that live in groups, or communities, compete with
one another at all stages of development. As a result of this com-
petition, certain plants, the more vigorous and better adapted ones,
survive. Others, less vigorous, are eventually suppressed or
eliminated. The host-parasite interactions of Erysiphe graminis

f. sp. tritici on Triticum aestivum can be recognized as competi—

 

tion between two organisms. The obligately parasitic fungus,

Erysiphe graminis f. sp. tritici em. Marchal, causes powdery

 

mildew of cereals and is responsible for a significant reduction in
grain yield in certain areas of the world (21, 50).

The most practical means of controlling this disease is the
development and use of resistant cultivars. This has been accom-
plished by selection for mildew resistance, which has produced
many host cultivars with varying degrees of resistance to powdery
mildew. The inheritance of resistance to _E_. graminis has been
described (2, 3), and near—isogenic wheat lines which differ by
only single genes for resistance have been developed (4). Many
isolates of the fungus that differ with respect to virulence are also
obtainable, though highly isogenic strains are not yet available (4).

l

2

Primary infection of wheat by Erysiphe graminis f. sp.
m has been divided into distinct morphological stages (26, 34,
36). Given the appropriate environmental conditions, the parasite
on a susceptible host undergoes each stage of development in a
highly synchronous manner, i. e. , one—six hours, germination;
six-12 hours, appresorial initials and mature appresoria; 12-20
hours, formation of haustorial bodies and secondary hyphal initials;
20-26 hours, formation of elongating secondary hyphae and develop-
ment of haustorial appendages. Under the same environment,
incompatible interactions indicate that the incompatibility of some
interactions is determined as early as 20 hr after inoculation (47).
The demonstration that incompatible genotypes affect primary infec-
tion stresses the importance of directing any physiological or bio-
chemical studies toward the very earliest interactions between a
host and its parasite.

The objectives of this research were to study some of the
events in the transition from two independent organisms to two
organisms that have or have not established compatible relation-
ships during primary infection. This was done by: (1) determining
the development of the primary haustorium as a function of time to
determine the earliest time incompatibility is expressed; (2) estab—
lishing the environmental conditions necessary for synchronous
development of secondary haustoria with compatible interactions,
and (3) determining the formation of secondary haustoria as a func-

tion of time with the incompatible interactions.

MATERIALS AND METHODS

Inoculum Production

 

The culture, Michigan strain (MS-1) of Erysiphe graminis

f. sp. tritici was used in all experiments. The MS—l strain was

 

collected in Michigan and maintained on _Triticum aestivum L.
'Little Club' wheat in growth chambers. Little Club has no known
genes for resistance to powdery mildew. The strain was checked
periodically for purity by scoring infection types on a set of differ-
ential host lines (Table 1) (25, 46).
Wheat seedlings grown in the greenhouse for six days were
dusted with conidia from plants inoculated seven days earlier.
The stock culture was 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 incan-
descent bulbs), sixteen hour photoperiod/day.
2. Temperature -- 181’1C during the light period and
17th during the dark period.
3. Relative humidity —- 80 i 5% during the light period
and 95 ”I 5% during the dark period.

4. Continuous air circulation.

Table 1. Near-isogenic lines with single Pm genes and infection types produced
seven days after inoculation with MS-l

 

 

 

Near-isogenic Designation of Gene Former Gene (1) Infection (2)
line Involved Symbol C. I. Type
Axminister x Cc8 (3) Pmla Ml 14114 0
(Axminister) t
Ulka x Cc3 Pm2a M_1’ 14118 2
(Ulka) u
Asosan x Cc8 Pm3a Ml 14120 3
a
Khapli x Cc8 Pm4a —. 14123 0
(Khapli)
Chancellor (Cc) pm x . . . 12333 4

 

(1) Cereal Investigation accession number.
(2) Infection type:

0-no observable mildew development
1—chlorotic flecking, no pustules

2-chlorosis, necrotic reaction

3-significant reduction in mildew development
4—abundant mildew development

(3) C08 refers to the 8 backcrosses to the cultivar
Chancellor.

 

 

5

Conid ia produced on the sixth day after inoculation were
used in all experiments.

All the wheat lines used in experiments were planted in
three inch pots and maintained in the greenhouse for five days

prior to inoculation.

Method of Inoculation

 

The 'rolling method' of inoculation (35) was used for all
studies of morphological development of powdery mildew during
primary and secondary infections. Healthy conidia, dusted onto
clean slides, were transferred to the abaxial or adaxial side of
leaves (26). This method provided a uniform distribution of
single conidia, with approximately one hundred conidia per centi-

meter of length of leaf.

Environmental Conditions for Experiments

 

All experiments were carried out in Sherer-Gillett (Model
CEL 512-37 and Model CEL 25—7) growth chambers. The following
environmental conditions were used to obtain a high infection
efficiency and synchronous growth of the parasite population
during primary infection (0-30 hours) (12, 47):
1. Zero to one hour, inoculated plants were maintained
in darkness at 18 “f 1 C and approximately 100% rela-

tive humidity (RH).

 

6
2. One to six hours after inoculation, plants were kept
under 1.0 x 105 ergs cm-zsec"1 radiation (0.6 x
105 ergs cm'zsec'1 from white VHO fluorescent
tubes and 0.4 x 105 ergs cm'gsec’1 from 25 watt
incandescent bulbs) at 22 i 1 C and 65 l 5% RH.
3. Six to 20 hours after inoculation, plants were kept
at the same temperature and RH as in the above,
but with no light.
4. Twenty to 30 hours after inoculation, conditions
were the same as in 2.
The above environmental conditions were used for all experiments
unless otherwise stated.

Light intensity was measured with a YSI Kettering Model 65
Radiometer.

Temperature and relative humidity were monitored during
experiments with wet and dry bulb thermometers and a recording
hygrothermograph calibrated with a sling psychrometer.

The experimental environmental conditions established for

synchronous development of secondary infection (38-58 hours

after inoculation) are given in Chapter 2.

Gene Symbols and Designation of Parasite/Host Genotypes

 

Briggle's suggested terminology (2, 3) was used to desig-

nate the Pm genes used to designate genes conditioning reaction

tog graminis f. sp. _t_r_‘it_ic_i. The symbols P_ml, Eng, etc.

have been used to designate genes at distinct loci. Genes con-
sidered to be either closely linked or allelic are followed by the
letters 3, b, c, . . . Alternate alleles for each Pm gene will be
referred to by their respective recessive designations, pm_1,

p_m2_, . . . As the genotypes of the host lines are all homozygous
for the designated gene, meme, or pmxpmx, they will be abbre-
viated and written as if haploid, m, or p_rrg.

Parasite/host combinations are referred to by their respec-
tive genotypes which specify compatibility or incompatibility.
E/p_mx_ is intended to imply compatibility specified by each gene
pair and fli/RIn—la is intended to imply that &/E specifies
incompatibility but all other gene pairs in that host and parasite

specify compatibility.

 

LITERATURE REVIEW

Approaches to research on the problem of obligate parasi-
tism have been varied. These range from studies of gross che-
mical differences between host varieties resistant or susceptible
to a given strain of pathogen to studies of the host-parasite
interface by electron microscopy. Each approach has certain
advantages, but none seem to be completely free from either
conceptual or technical problems. It has become increasingly
obvious in the review of research literature that there is no
direct road to an understanding of infections caused by obligate
parasites. Extensive studies of the disease have been aimed at
understanding the genetics of disease development and eluci-
dating its physiological and biochemical aspects. I will attempt
to summarize some of the important developments that relate to
the results reported in this study.

The powdery mildew fungus, Erysiphe graminis f. sp.

 

tritici, is an obligate parasite. Erys iphe graminis is character-

 

 

ized morphologically by the ectoparasitic conidium, appresorium
with an infection peg, and extensive mycelial growth on the sur-

face of the host. In a susceptible host, the infection peg forms a
haustorium inside the penetrated cell. The infection spreads via

8

 

9
formation of secondary hyphae from the original germ tube and
formation of additional infection pegs and haustoria. Only the
host epidermal tissue is infected with haustoria. In spite of
this localization, the disease is conservatively estimated to pro-
duce a 1-2% reduction in yield per year (21, 22).

Powdery mildew disease development is reportedly affected
by environmental conditions. Germination and subsequent
development of conidia occur over a wide range of temperature
and relative humidity. Several different optimal conditions for
mildew development and the effects of various environmental fac-
tors on disease development have been reported (46) and need not
be reiterated.

The primary infection process ofE. graminis on the plant
surface is conveniently divided into distinct morphological
stages: 1) germination, 2) production of 'club-shaped' appre—
sorial initials, 3) formation of mature appresoria, 4) penetration
of the cuticle and epidermal cells, 5) formation of haustoria, 6)
development of elongating secondary hyphae (ESH), 7) initiation
of secondary infections, and 8) sporulation. Each stage of the
infection process differs in its requirements for temperature,
relative humidity, and light (24, 27, 28, 36, 43). Under optimal
conditions, over 80% of the parasite population move through the
stages of primary infection with a high degree of synchrony (27,

28, 34, 36). The production of elongating secondary hyphae

 

10
(ESH) has been used as the criterion for the establishment of a
functional relationship in primary infection between host and
parasite (27, 28). For each ESH that formed on the host sur-
face, a haustorium was produced in the epidermal cell (27, 28).
The production of ESH by the parasite indicates that a functional
relationship had been formed and that transfer of nutrients and
other essential materials from host to parasite occurs. The
percentage of conidia applied that produced ESH is defined as
infection efficiency (10, 11, 12).

The formation of a functional haustorium is the crucial step
in the development of a compatible host-parasite relationship,
but it is very important to know the earliest stages of interaction
between host and parasite and to determine if there is a sequence
of events which is critical to the establishment of a compatible or
incompatible relationship.

A number of physiological changes are known to occur
following inoculation of wheat withE. graminis. Respiration (1),
photosynthesis (44), and translocation patterns of organic mole—
cules (9, 40, 53) have been reported to change two to 10 days
after inoculation. Previous studies (47) have shown that the
establishment of compatibility or incompatibility often occurs
before 24 hr after inoculation. Conidia placed on non-host plants
germinated, formed appresoria, and attempted to penetrate

epidermal cells, but they did not form either haustoria or ESH

11
(26, 51). Corner (8) observed that the development of powdery
mildew is arrested before ESH or haustoria are formed in the
resistant hosts. These interactions between the incompatible
parasite and host may lead to the death of the infected cell and
adjacent cells and act as a barrier to additional infections by the
parasite units (6, 8).

It is clear from many investigations (15, 16, 33, 38) that
the interactions between host and parasite are controlled by com-
plementary genes possessed by both host and parasite. Flor
(13, 14) found that the ability of Melamspora lini to grow and pro-
duce disease symptoms on genetically different flax lines was
determined by specific corresponding genes in the pathogen. The
finding of one gene in the pathogen for each gene in the host led
to the development of the gene-for-gene hypothesis which states
that for each B gene in the host that conditions resistance there
is a corresponding P gene in the parasite that conditions aviru-
lence. The _P gene interacts with the B gene to determine incom-
patibility, i. e. , low infection type. Incompatibility results only
when a P gene in the parasite interacts with a specific B gene in
the host, e. g. , P_1/R_1. Compatibility is specified with the other
posSible parasite/host genotypes, fl/r_1, p_1/E, and p_1/r_1.
With two alleles at one locus in a parasite, P or p and two alleles
at one locus in the host, B or 3, there are four possible interactions

(Figure 1).

 

Figure 1.

12

The four possible parasite/host genotypes
involving a single gene pair governing com-
patibility of host and parasite. £131 and Bm_1
are alternate alleles in the host. £11 and p_1_
are alternate alleles in the pathogen. ELF—HE
specifies incompatibility (—) while El/grnl,

El/Pml, and pl/pml specify compatibility (+).

HOST

 

F

91

._
a.|

ELLISVHVd

14
The basic scheme (41) was proposed to be used as a biological
test to study physiological and biochemical effects of disease
development. This test is useful in studying disease develop-
ment, especially with powdery mildew which follows the gene-

for-gene relationship (32, 37, 39).

 

CHAPTER I

GENETIC CONTROL OF PRIMARY HAUSTORIAL

DEVELOPMENT OF ERYSIPHE GRAMINIS ON WHEAT

Introduction

A high percentage of spores of Erysiphe graminis (DC. )
Merat f. sp. iriticii em. Marchal placed on a leaf of wheat will
germinate, produce appresoria, haustoria, and elongating secon-
dary hyphae (ESH) if given the appropriate environmental condi-
tions, and if the parasite/host genotype specifies compatibility
(27, 28, 47).

Genes conferring resistance in wheat to E graminis are
expreSSed at different stages in the ontogeny of interactions
between host and parasite. The effects of these genes have been
determined by measuring the production of ESH during primary
infection.

The production of ESH greater than 10p has been used as
the criterion of development of a functional relationship between
wheat and E graminis. It has been shown that for each ESH
greater than 10p, a normal haustorium was always present (48).

15

 

16

However, no direct studies had been done on the development of
haustoria during primary infection in either the compatible or
incompatible interactions.

The objectives of this research were: 1) to determine the
rate of development of primary haustoria of E. graminis on 5
near—isogenic lines of wheat containing P_m genes, and 2) to
correlate these results with the development of elongating secon-

dary hyphae.

Materials and Methods

 

The development of haustorial bodies was measured by
direct microscopic observations. Measurements were made
every two hours from eight through 30 hr after inoculation.
Environmental conditions were used which permitted synchronous
development of the ectoparasitic portion of the parasite (27, 28,
29). Conidia were applied via 'rolling technique' (35). Every
two hours after inoculation, beginning at hour 8, strips of the
abaxial epidermis were taken and fixed in Carnoy's solution for
five minutes. Extractable chlorophyll was removed by two or
three rinses of 100% methyl alcohol. The epidermal strips were
placed in 0. 06% aniline blue, specific for fungal material and
protoplasts (7), and incubated at 35 C for one hour to intensify
the dye in the fungal tissue. The tissue was transferred to 85%

lactic acid for an additional hour, then allowed to destain in

 

17
fresh 85% lactic acid at room temperature for one or two days.
The dark blue-stained haustoria were easily observable with
bright field microscopy (see Figure 2 and interpretive drawing,
Figure 3).

The microscopic determinations and observations were
done with Bausch & Lomb and Zeiss phase contrast microscopes.
The photomicrographs of the complete parasite unit was taken
with a Zeiss Photomicroscope II.

Spatial relationships, higher resolution, and a greater
depth of focus, were accomplished by use of the scanning elec-
tron microscope (SEM), Model AMR 900 (Figure 4).

Since wheat leaves possess a cuticle layer which is almost
impermeable to most liquids, the following procedure gave good
results for SEM: 1 cm length inoculated leaf sections were fixed
in 3:3 Glutaraldehyde (25%)/Acrylic Aldehyde (100%) solution for
1 hr under vacuum, followed by a phosphate buffer rinse. The
tissue was then fixed in 2% 0504 for 1 hr and dehydrated via the
iso-amyl acetate technique (18, 30, 31). A Model Bomar SPC—
900/Ex critical point drying instrument was then used to remove
any excess water from the tiss1e. The tissue was then mounted,
coated and examined with the SEM.

The data are presented as percent of total number of
conidia applied to the leaf that gave produced haustoria of various

lengths at different times during primary infection. The

 

 

 

Figure 2. Photomicrograph of a primary haustorium,
with a compatible genotype, E/pg, in wheat
epidermal cell at 26 hr after inoculation.

(A) Conidium
(B) Appresorium

(C) Point of penetration
(D) Haustorial body with appendages

Figure 3. Drawing of a primary haustorium with a com-
patible genotype, B/EEE’ at 26 hr after ino-
culation depicting infection structures and
extensive hyphal growth. The haustorial body

has the finger-like projections.

19

 

 

Figure 2 and Figure 3

 

20

Figure 4. The ectoparasitic structures of Erys iphe
graminis on wheat observed with a Scanning

Electron Micros cope .

(A) Conidium
(B) Appresorium with characteristic

'beak-like' appearance

21

r,

 

DIP:

Figure 4

22
experiments were repeated on four different days. When near-
isogenic lines were compared, all lines were tested on the same

day.

Results

It is imperative that a common base be established to
allow for meaningful interpretation of results in relation to the
work done by previous experimenters (27, 28, 34, 42). Optimum
environmental conditions for germination, formation of appre—
sorial initials, formation of mature appresoria, and production
of secondary hyphae were used (25). Figure 5 shows that the
development of the parasite population was in close agreement
with previous findings (25).

The rate of haustorial development was determined for five
different parasite/host genotypes with standard environmental
conditions (Table 2). At eight hours (Figure 6-A) after inocula-
tion with the compatible genotype, Pi/fl, (Chancellor), all of
the parasite units applied had attempted penetration and/or
formed haustoria 5;; or less in length. By 16 hr, 21% had formed
haustoria 5p in length, 56% had haustoria 5-15n long, and 25%
had haustoria 15'25M long. Formation of appendages on the haus-
torial bodies was evident at 18 hr after inoculation in all the com-
patible and incompatible interactions. By 30 hr after inoculation

with the compatible genotype, the percentage of parasite units

23

Figure 5. Development of Erysiphe graminis f. sp.

tritici during primary infection of wheat

leaves

(A)
(B)
(C)
(D)

(E

v

( ___________

(

Germination

Formation of appresorial initials
Formation of mature appresoria
Formation of secondary hyphal
initials

Formation of elongating secondary
hyphae (ESH)> 10“ in length

_________ ) Martin (25)

) results obtained in
this study

24

an

m onswfim

A2,: ZO_._.<._DUOZ_

on #N «N ON

0—

«Eh—d.

52....

.v

 

 

— — _ _

 

 

 

ON

0?

on

on

00—

39V1N3383d

25

Table 2. Effect of genotype of host and pathogen on development of haustoria through
primary infection

 

‘76 of parasite units with haustoria
Genotypes Hours alter of a given length (11) ’ 019- Col-
(parasite/host) inoculation 0-5 5-15 15-25 25-35 35-45 45-55 torted lapsed Total

 

fllpmx
— 8 100 304
10 99 1 267
12 90 10 388
14 22 73 3 2 256
19 21 56 25 2 221
18 13 25 57 4 1 228
20 8 9 57 27 239
22 4 27 63 2 232
24 7 5 19 50 20 235
26 5 3 14 50 26 2 189
28 5 4 8 19 59 6 195
30 1 1 9 43 44 209
Pla/Pmla
_ 8 100 299
10 100 298
12 91 8 2 314
14 69 21 2 5 3 221
16 38 25 33 2 2 231
18 50 8 29 4 9 3 219
20 29 7 28 24 7 5 249
22 25 1o 24 11 2 24 5 236
24 46 11 14 8 4 19 2 250
26 34 3 6 11 4 42 252
28 41 4 6 12 9 1 33 217
30 33 3 9 19 9 7 26 265
PZa/sza
— — 9 100 322
10 98 1 313
12 92 9 1 411
14 53 35 7 1 4 230
19 35 19 47 2 260
18 18 9 63 9 2 1 234
26 17 6 34 44 222
22 23 4 16 51 5 241
24 16 4 14 32 31 2 2 239
26 24 2 3 41 33 2 239
28 21 2 5 9 42 18 2 1 221
30 19 3 7 35 31 4 198
PSa/Pmaa
— ' 9 100 277
10 96 1 277
12 91 9 317
14 23 69 4 3 2 223
16 47 23 30 291
19 26 15 50 7 1 219
20 36 5 34 22 3 221
22 27 4 30 22 13 4 284
24 23 1 12 37 9 19 1 269
29 23 3 10 19 6 32 244
28 34 2 4 15 6 2 34 2 200
30 35 2 6 12 11 7 29 251
P4a/Pm4a
— 9 100 346
16 98 2 224
12 9o 10 372
14 52 38 9 1 2 233
16 58 20 21 2 205
18 51 7 30 9 4 2 271
20 7o 4 9 5 9 5 230
22 44 6 20 1o 11 8 291
24 46 2 11 15 4 14 9 295
26 64 3 4 3 1 16 8 231
28 49 2 4 3 3 1 38 198
30 60 2 4 3 2 1 19 10 199

 

 

 

26

Figure 6. Effect of five different parasite/host genotypes
on the development of primary haustoria of

Erysiphe graminis f. sp. tritici on Wheat

 

(A) Genotype Px/pmx

(B) Genotype fia7P—m1a
(C) Genotype P2a/Pm2a
(D) Genotype P3a/Pm33
(E) Genotype P4a/Pm4a

 

 

 

 

27

 

 

 

 

Figure 6

k . u _ _
\ \
\I77 1 1 \ \r 7
“' // \
I. \r 77—.“7 . //’\’ a _ .—
N
\
c . i. ,0 _ _
94 AI: 34: 29‘: an ‘5: mm m

28

 

 

 

 

Figure 6 (cont'd.)

29

 

 

Figure 6 (COHt'd-)

 

,‘amaunm

X

 

 

30

with haustoria of a given lengthwas: 5% 0-511, 2% 5—2511, 8%
25-3511, 43%. 35-45“, and 44% 45-5511.

' There was not a significant number of collapsed ecto-
parasitic structures by 30 hr after inoculation and no evidence
of distorted haustoria within the host cells. The mesophyll cells
showed no sign of necrosis and none of the host cells had picked
up the dye. Progressive development of the parasite units is
obvious by the extension of the peaks of increased haustorial
development up to 30 hr after inoculation.

With the incompatible genotype, flat/w, (Figure 6-B),
the percentage of parasite units with haustoria of a given length
was: 33% 0—511, 3% 5—15u, 8% 15-2511, 19% 25-3511, 8% 35-4511,
and 7% 45-55“, by 30 hr after inoculation. An additional 26% of
the parasite units were distorted (Figure 7). Distortion was
evident in the observation that haustoria tended to "ball up"
(Figure 8). The highest proportion of distorted haustoria was
at 26 hr after inoculation. Cells with distorted haustoria picked
up the dye more quickly than the other cells. By 30 hr after
inoculation, approximately 95% of the infected cells were readily
infiltrated with the dye. Collapse of the ectoparasitic portion of
the parasite was greatest at approximately 20-22 hr after inocu-
lation (Figure 6—B).

With the incompatible genotype, P_2a/P_m_23, (Figure 6-C),

19% of the parasite units had formed haustoria 511 or less in

 

31

Figure 7. Photomicrograph of the penetration process of
the incompatible interaction, Pla/Pmla, on
wheat at 26 hr after inoculation. The incom-
patible interactions, P2a/Pm2a, P3a/Pm3a,
and P4a/Pm4a, show similar results.

(A) Conid ium
(B) Point of penetration of the host by
the parasite unit

Uptake of the aniline blue by infected
host cell depicting mesophyll collapse

(C

v

Figure 8. Drawing of the penetration process of the incom-
patible interaction Pla/Pmla, with similar re-
sults for P2a/Pm2a, P3a/Pm3a, and P4a/Pm4a

(A) Penetration and failure of the para—
site unit to develop haustoria

(B) Mesophyll collapse and uptake of
aniline blue

32

 

Figure 7 and Figure 8

 

 

 

33
length and 66% had haustorial bodies 35-55;; in length by 30 hr
after inoculation: only 4% of haustoria were distorted and rela—
tively few of the host cells had picked up the dye. Another inter-
esting point was observed. With the incompatible E/PLZa
genotype, during appresorial development, the length of the
appresorium is much longer when compared with the appresorial
development of the other compatible and incompatible genotypes.

The development of haustoria with the _P_&a;/_E:m_33 (Figure
6—D), genotype showed that at 30 hr after inoculation, 35% 0-5u,
2% 5-1511, 6% 15-2511, 12% 25-35u, 18% 35-5511 in length, and
2% were distorted. The highest percentage of haustoria was
reached 28 hr after inoculation and almost all infected host cells
had picked up the aniline blue (see Figure 7 and interpretive
drawing in Figure 8).

The P4a/Pm4a (Figure 6-E), showed that very few of the
parasite units had haustoria longer than 51.1 in length by 30 hr
after inoculation. Approximately 6% of the parasite units had
haustoria 3511 or longer at this time; 18% of the haustoria were
distorted, and 10% had collapse of the ectoparasitic portion of the
parasite unit. As early as 14 hr after inoculation, groups of
mesophyll cells had collapsed beneath or near the penetrated
cell, and usually the highest percentage of collapsed cells ad-

joining the infected cell was observed 28 hr after inoculation.

34
Discussion

In this study the time at which different genotypes affected
the ontogeny of the host—parasite interactions during primary in-
fection was observed. The data with the compatible genotype,
fix/pm_x, (Figure 6-A), suggest both development of the haus-
toria and development of the ectoparasitic portion of the parasite
are reasonably synchronized (26, 27, 28, 29).

With the compatible E/pfix genotype, penetration by the
parasite unit occurs at approximately 8—10 hr after inoculation.
By 18 hr after inoculation, appendages are present on the pri—
mary haustorial body and relatively few of the ectoparasitic
structures of the parasite have collapsed. The fact that the
haustoria are not distorted and no detectable aniline blue has
infiltrated the host cells indicates that the infected cell is still
viable. By 30 hr after inoculation, 86% of the conidia applied
have produced primary haustoria, and this figure corresponds
to the percent of ESH reported at that time (48).

The 313/31313 genotype was observed to affect infection
efficiency (5) and the development of haustoria as well as the up—
take of aniline blue by host cells during primary infection. The
kinetics of haustorial development for Eli/Em—m show that 33%
of the parasite units had infected the host cell and formed haus-
toria 51.1 or less in length by 30 hr after inoculation. Twenty-six

percent of the parasitic units were distorted and their host cells

 

v

35
stained with aniline blue. Nineteen percent of the parasitic units
formed normal haustoria, a figure comparable to formation of
ESH>10u long by 26 hr after inoculation (48). It appears, there-
fore, that only haustoria that develop normally support the pro-
duction of ESH.

Published studies have not demonstrated an effect of the
BEE/£3333 gene interaction during primary infection (19). Most
of the parasite units, 77%, eventually produce ESH>10u (48).
The kinetics of haustorial development in this research show that
by 30 hr after inoculation, 19% of the parasite units had formed
haustoria 5H or less in length, while only 66% had haustoria
35-55).: in length. Four percent of the parasite units had distorted
haustoria and host cells which picked up the dye. While the per-
cent of ESH does not show an effect of Bit/M in primary
infection, a study of haustorial development does indicate that
P_281/_lfla_ genotype significantly differs from the compatible
interaction during primary infection.

The interactions of 132/3232 were similar to that of
E/m and Lm/m (Figure 6-B, D, E). The kinetics of
haustorial development for Pig/Eng are not in complete agree-
ment with the percent of parasite units that have formed ESH
more than 1012 long. Thirty percent of the parasite units have
haustorial development supporting growth of ESH) 1011 by 26 hr

after inoculation. In order for the P3a/Pm3a data to be in

36
agreement with both parameters, it was necessary to include
haustoria 25—3511, as well as those 35;; or longer in length.
Apparently, smaller than normal haustoria are supporting the
development of ESH.

The data also showed that haustorial development in the
incompatible interactions is not a clear-cut phenomenon (Figure
6-B, C, D, E). In some cases, the host cells were penetrated
and subsequent development of the parasite unit was rapidly
halted. In other infected cells, the parasite unit appeared to
develop normally for a few hours, then development stopped,
leaving a rudimentary haustorium (Figure 9). The uptake of the
aniline blue by the infected host cell in these interactions led to
the assumption that a large percentage of the infected cells were
nonfunctional. Only 18% of the infections with the incompatible
P_3a/fln_3§ genotype were normal 30 hr after inoculation.

BEND—“1449:. showed a reduction in the percentage of the para-
site units that produced primary haustoria as well as a discolora-
tion of the host cells adjacent to the infected cell (46, 47). Sixty
percent of the parasite units penetrated the host cells and stopped
growing. Eighteen percent of the parasite units were distorted;
rudimentary haustoria and stained host cells were observed.

Ten percent of the total number of parasite units applied showed
collapse of the ectoparasitic portion, and 3% had haustoria 35-5511

in length.

 

 

37

Figure 9. Drawing of the incompatible interaction,
P4a/Pm4a, showing rudimentary haustoria
and mesophyll collapse.

(A) Rudimentary haustoria
(B) Mesophyll collapse

38

 

 

Figure 9

 

39
The results presented here provide additional evidence
that the different genotypes affect different stages in the ontogeny
of interactions between host and parasite and that some of the in-
compatible interactions affect parasite development earlier than

previously reported .

Summary

The stages of haustorial development of Erysiphe graminis
f. sp. m from 8 to 30 hours after inoculation varies with the
genotype of the host and parasite. With the compatible genotype,
E/m, haustorial development occurred as was expected com-
pared with the length of ESH formed (Figure 6-A). The incom—
patible interactions, Elam—1a, PZ_a/Pr_n§, P_3a/P_m_3§, and
E‘La/EEIIE- are not as distinctly different as expected. With
Pia/m, the majority of the parasite units that produced haus-
toria collapsed 26-28 hours after inoculation, but the infected
host cells did not appear altered. Efi/M apparently does
have an interaction earlier than previously observed based on the
infection efficiency and the development of ESH as modes of com-

parison. In earlier experiments (48), P3a/Pm3a had 30% ESH

 

longer than 10p. The present data indicate that with this inter-
action, the percent of parasite units which produced full-sized
haustoria is less than 30%. Therefore, haustoria that are not

growing at the normal rate can support the production of ESH.

 

40
Results on the relationship between development of haustoria and
ESH with P4a/Pm4a are in close agreement with previous findings

(19).

 

CHAPTER II

THE INFLUENCE OF LIGHT PERIODS ON THE
PRODUCTION OF SECONDARY HAUSTORIA BY

ERYSIPHE GRAMINIS ON SUSCEPTIBLE WHEAT

Introd uction

The ontogenesis of haustoria was established for the pri—
mary infection processes with both the compatible and incom-
patible parasite/host genotypes in Chapter 1. The study was pos-
sible because conditions for reasonably synchronized primary
infection were known. The assumption has been made that, since
secondary hyphae longer than 10-15u are formed only if a haus-
torium was formed, the formation of ESH was indicative of the
establishment of a compatible, functional relationship between
host and parasite. It was considered necessary, therefore, to
establish that the secondary hyphae formed were capable of initi-
ating secondary infection.

The purpose of this part of the investigation was (1) to
define the various compenent stages that make up the process
of secondary infection, (2) to attempt to synchronize the parasite

41

42
population in the various stages of secondary infection by altering
the environment, and (3) to determine useful criteria for the
establishment of a compatible host-parasite interaction from

38—58 hr after inoculation.

Materials and Methods

 

Culture MS—l of Erysiphe graminis f. sp. tritici was main-
tained on Triticum aestivum cv. 'Little Club'. The environmental
conditions under which these stock cultures are maintained have

been described by others (27, 28, 35, 36) and in Chapter 1, as

 

have been the conditions for reasonably synchronous primary
infection (34). Some observations had indicated that if standard
conditions were used for the first 26 hr after inoculation, the
highest percentage of secondary appresoria would form if given
27 C and high light intensity for 4 hr followed by 22 C and dark-
ness (46, 47). No determination was made of the percent of
secondary appresoria which produced haustoria (46, 47).
Standard environmental conditions used for the first 26 hr
are presented in Table 3. Environmental conditions used begin-

ning 26 hr after inoculation are given in Table 4.

43

Table 3. Environmental conditions necessary for synchronous
development of parasite unit through primary infection

 

 

Hour 0-1 1-6 6-20 20-26
Environ- dark light dark light
mental 100% RH 65% RH 65% RH 65% RH
Conditions 17 C 22 C 22 C 22 C

 

Table 4. Experimental alterations of the light periods to obtain
synchronous development of the parasite unit through
secondary infection

 

 

Experiment 1

 

Hour 26—30 30-38 38-58
Environ— light dark light
mental 65% RH 65% RH 65% RH
Conditions 22 C 22 C 22 C

Experiment 2

 

Hour 26-36 36-44 44-58
Environ- light dark light
mental 65% RH 65% RH 65% RH

Conditions 22 C 22 C 22 C

44
Table 4 (cont'd.)

Experiment 3

 

Hour 26-30 30—44 44-54 54—58
Environ- light dark light dark
mental 65% RH 65% RH 65% RH 65% RH
Conditions 22 C 22 C 22 C 22 C

Experiment 4

 

Hour 26-36 36-58

Environ- light dark 4
mental 65% RH 65% RH ’
Conditions 22 C 22 C

 

Each set of experiments involving alterations of light periods
was repeated four times on four different days with four different
sets of inoculated plants. Epidermal strips were removed and
stained using the same procedures as in Chapter 1. The data
are presented as averages of all replications, and statistical
analyses (52) were done using a two-way analysis of variance

(Figure 10).

Results
The formation of secondary haustoria with the compatible
genotype, B/pmx, is shown in Figure 11. By 30 hr after inocu—

lation, 87% of the parasite units applied formed functional

 

45

Figure 10. Formation of secondary haustoria by

Erysiphe graminis f. sp. tritici cultures

MS—l by varying the light periods.

That

Applied

% Conidia
Formed Secondary Haustoria

46

 

100

80

60

40

2O

Experiment 1
Experiment 2

Experiment 3

- Experiment 4

 

 

I'D—Cl

o——o

H

 

 

 

 

Time After

46 50

Inoculation

Figure 10

(hours)

54

58

47

Figure 11. Drawing of a primary haustorium and secon-
dary haustoria with a compatible genotype,
B/pmx, at 54 hr after inoculation.

(A) Primary haustorium
(B) Secondary haustoria

48

 

Figure 11

49
primary haustoria (20). Of this 87%, approximately all of the
parasite units developed secondary haustoria given the optimal
environmental conditions for secondary infection (Figure 10).
The results of Experiments 1, 2, 3, and 4 (Table 4) may
be summarized as follows:

Table 5. Statistical analyses of the percent of parasitic units
forming secondary haustoria

 

Percent of parasitic units forming
Experiment Figure primary haustoria which continued
to form secondary haustoria

 

1 10 69. 00
2 ' 10 80. 00
3 10 87. 00
4 10 90. 00

 

Even though relatively high percentages of the conid ia
applied formed secondary haustoria in all the experiments, the
synchrony as evidenced by the normal growth curve, failed to
approach that observed in Experiment 3. Since synchrony and
infection efficiency through secondary haustorial development
were equally important considerations, the environmental condi—
tions of Experiment 3 were used in subsequent studies of the

development of secondary haustoria.

 

50
Discussion

The primary stages of infection of wheat and barley by _E_.
graminis have been studied by employing the optimum environ—
mental conditions necessary for a high infection efficiency and
synchrony of the parasite units (17, 22, 23). If a high infection
efficiency and synchronized development of the parasite with
compatible genotypes had not been attained, the identification of
the effects of the genotypes for incompatibility on primary infec-
tion would have been essentially impossible.

The percent of elongating secondary hyphae is an accurate
indication of the degree of success in establishing infection (46).
The data in Chapter 1 demonstrates the extent to which develop-
ment of primary haustoria is an even finer indicator of the success
in establishing an aegricorpus. Both methods delineate a time,
unique to each incompatible combination of genotypes, at which
the incompatibility is first expressed.

In incompatible interactions, however, a fraction of the
parasite units proceed past this critical time. How this occurs
is yet unknown. The small fraction that does produce successful
primary infections then takes part in the secondary infection.

To study this process, a reasonably synchronous population of
parasite units was needed. To this end, environmental conditions
giving some synchrony of secondary infection in the compatible

interactions were empirically developed.

51
Summary

Light is necessary at specific times during primary and
secondary infection for synchronous development of _E_. graminis
on wheat. Synchronous development of E. graminis during secon—
dary infection was increased with changes in the light periods
(1. 3 ergs cm'zsec_1, incandescent and fluorescent) between 26
and 58 hr after inoculation (34). With the optimal environmental
conditions (26-30 hr, light; 30-44 hr dark; 44-54 hr light; and
54-58 hr dark, and 65% relative humidity, 22 C for the period
26-58 hr after inoculation) established for the compatible inter-
action, E/m, approximately all of the parasite units that form
primary haustoria from 20—26 hr after inoculation formed secon—
dary haustoria by 58 hr after inoculation. The synchrony of
haustorial development was affected if the light schemes were
changed. Increased synchrony in development of secondary
haustoria on susceptible wheat makes it possible to determine
when the different incompatible parasite/host genotypes affect

the ontogeny of the host—parasite interactions.

 

 

CHAPTER III

GENETIC CONTROL OF SECONDARY HAUSTORIAL

DEVELOPMENT OF ERYSIPHE GRAMINIS ON WHEAT

Introduction

A disease caused by a parasite will usually develop so as S
to reflect the degree to which the parasite maintains growth and
reproduction in its interaction with the host.

The kinetics of observable interactions between E
graminis f. sp. m and wheat with compatible E/pinfi and

incompatible Pla/Pmla, P2a/Pm2a, P3a/Pm3a, and P4a/Pm4a

 

 

parasite/host genotypes have been described for the process of
primary infection the first 26 hr after inoculation (48).

Chapter 2 has described environmental conditions which
give reasonably synchronous development of secondary haus-
toria of the parasite with compatible parasite/host genotypes
during the period from 26—58 hr after inoculation.

The objectives of this investigaion were (1) to determine
the rate of development of secondary haustoria of E graminis
on the 5 near-isogenic lines of wheat containing genes for

52

53
reaction to E. graminis, and (2) to determine if and when the
different incompatible parasite/host genotypes affect secondary
infection. The latter should give a more complete understanding

of when and how the parasite and host genes interact.

Materials and Methods

 

The development of the haustorial bodies was observed
microscopically using the aniline blue dye technique described
in Chapter 1. Every four hours after inoculation, beginning at
hour 38, strips of the abaxial epidermis were removed, stained, ?
and examined microscopically as described previously, and the
number of haustoria recorded (Table 6). The environmental
conditions which gave the most synchronous development of the
ectoparasitic portion of the parasite through primary infection
(17, 22, 48), and synchronous development of the compatible
interaction for secondary infeption were used (see Chapter 2).

The data (Table 6) are presented as percent of the total
number of parasite units with primary haustoria which formed
secondary haustoria of any length. The experiments were

repeated on four different days.

Results
Observations of haustorial development with the compatible
parasite/host genotype, Px/pmx, (Table 6, Figure 11), showed

that, by 38 hr after inoculation, 83% of the parasite units had

 

54

 

 

 

 

 

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55
formed primary haustoria only, 4% also had formed secondary
haustoria 0-5/4 in length, and 3% had formed secondary haus-
toria 5-1511 in length (Table 6, Figure 12-A). Five percent of
the total number of conidia applied had formed rudimentary haus-
toria (Figure 12-F). Six percent of the total number of conidia
applied germinated, penetrated the host cells, and stopped
growth. By 58 hr after inoculation, 14% of the parasite units
had secondary haustoria 0—511, 14% 5-15u, 8% 15-2511, 6%
25—3511, 6% 35—4511, and 4% 45-5511 in length. Thirteen per—
cent of the parasite units had penetrated the host cell but did
not form haustoria. With the compatible genotype some secondary
haustoria reached a length greater than 55/4. The mesophyll cells
showed no sign of necrosis except for the 13% of the parasite
units that had penetrated the host cell but did not form haustoria.
The cells with infection pegs but no haustoria took up stain. Pro-
gressive development of the parasite unit is depicted by the con—
tinued growth of secondary haustoria up to 58 hr after inoculation
(Table 6, Figure 12-A).

With the incompatible genotype, 313/m, no parasite
units that had formed primary haustoria had formed secondary
haustoria by 38 hr after inoculation. Thirty—three percent of
the parasite units had formed rudimentary primary haustoria
while 48% penetrated the host and growth ceased. By 58 hr after

inoculation, 1% were 0—5u in length, 2% 5-15u, while 4% had

Figure 12.

56

Effect of the five different parasite/host
genotypes of the development of secondary

haustoria of Erysiphe graminis f. sp.

 

tritici.

 

(A) Genotype Px/pmx

(B) Genotype P'l—anr—nla

(C) Genotype P2a/Pm2a

(D) Genotype P3a/Pm3a

(E) Genotype P4a/Pm4a

(F) Penetration with rudimentary haus-
toria and penetration with no haus- .
toria on the compatible and incom-
patible interactions

 

 

 

 

57

 

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Figure 12

 

 

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58

 

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Figure 12 (cont'd.)

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Figure 12 (cont'd.)

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60
formed rudimentary primary haustoria, and 81% had penetrated
and stopped development. Secondary haustoria remained rela-
tively constant in size, never exceeding a length greater than
2511. by 58 hr after inoculation (Figure 12-B). Eighty-one per-
cent of the infected cells depicted mesophyll collapse indicating
necrogenic protoplasts by 58 hr after inoculation (Figure 12-F).

With the incompatible genotype, _PE/Pinga, by 38 hr
after inoculation, 1% of the parasite units had formed haustoria
0'5H in length. Thirteen percent of the parasite units had
formed rudimentary haustoria and 28% had penetrated the host
cell. By 58 hr after inoculation, 7% had secondary haustoira
0-5u in length, 10% 5-15/1, 9% 15-25u, and 2% 25-35/4.
Secondary haustorial development never exceeded 35].; in length
(Figure 12-C). Five percent formed rudimentary haustoria and
28% of the parasite units showed mesophyll collapse and uptake
of dye by 58 hr after inoculation (Figure 12-F).

In the incompatible aegricorpus, E/Piiia, no measur-
able number of the parasite units that had formed primary haus-
toria formed secondary haustoria by 38 hr post inoculation.
However, some 37% of the parasite units that had formed pri—
mary haustoria formed secondary haustoria, and some 43% had
simply penetrated the host before growth ceased. By 58 hr after
inoculation, only 2% of the parasite units had formed secondary

haustoria 0-511, 2% 5-15u, and 1% 15-2511 in length (Figure 12-D).

 

61
Seven percent of the parasite units had formed rudimentary
haustoria and 76% penetrated the host cell and picked up the
aniline blue (Figure 12-F).

With the incompatible P4a/Pm4a genotype, very few of

 

the parasite units had secondary haustoria 0-511 in length as late
as 58 hr after inoculation (Figure 12-E). Three percent of the
parasite units had formed rudimentary primary haustoria, and
93% had simply penetrated the host cell. Mesophyll collapse
was evident not only in the infected cells but in the adjoining

cells (Figure 12—F).

Discussion

Microscopic observations of haustorial development in
the early stages of infection were expected to elucidate the
mechansim underlying resistance and susceptibility (10). In
this study, the time at which different genotypes affect the
ontogeny of the host/parasite interactions was established.

The data from the compatible genotype, E/pfl,
(Figure 12-A) suggest that haustorial development is still
relatively well synchronized and that the parasite units will
continue to undergo normal development and to reach sporula-
tion. By 58 hr after inoculation, 52% of the parasite units have
formed secondary haustoria. Apparently cells undergoing nor-

mal development do not absorb aniline blue as the ectoparasitic

 

 

62
structures appear normal presenting no sign of imminent col-
lapse.

The kinetics of secondary haustorial development for
P1a/Pm1a indicate that approximately 6% of the parasite units
may reach sporulation. In primary infection, 81% of the para-
site units penetrated but failed to develop haustoria. Similarly,
of those 19% producing primary haustoria, 81% failed to pro-
duce secondary haustoria.

While 28% of the parasite units in the LZa/M inter—
action had formed functional secondary haustoria by 58 hr after
inoculation, the compatible interaction, Px/pmx, at the same
hour had 52% of the parasite units with secondary haustoria.
The Eli/£1313, P_3a/_P_m3_a, and 14%/BEE showed only 6, 5,
and 1% respectively of the parasite units with secondary haus—
toria at this time.

The incompatibility conditioned by P2a/Pm2a seems to be

 

intermediate between the extreme incompatibility of 313/m,
PE/Piiia, and _P4_a/P_m4_a_ and the compatibility of Bx/fl.

The observations of Pfi/Pm_3a_ development were essen-
tially identical to those of Pia/w. The kinetics of haustorial
development for FEE/M demonstrate very slow formation of
secondary haustoria; 6% at 58 hr was shown previously for
development of secondary haustoria. Some 76% of the parasite

units are stopped at the stage of secondary penetration, and an

 

 

63
additional 7% are stopped at the formation of a rudimentary
haustorium.

A greater reduction in the percentage of the parasite units
that produce secondary haustoria and of the infected and adjacent
cells that stained with aniline blue was observed in Kai/£48..
Ninety-three percent of the applied conidia penetrated, but
failed to form haustoria. Approximately 1% of the total number
of conidia applied continued to develop. Pia/Pings; had the
lowest secondary infection efficiency of all the interactions

studied. 3

Summary

The rate of development of secondary haustoria of Erysiphe
graminis f. sp. m on 5 near—isogenic lines of wheat con-
taining single genes for reaction to E. graminis (MS-1) were
observed microscopically after staining with aniline blue. Haus-
torial measurements, taken from 38-58 hr after inoculation
under environmental conditions which ensured synchronous
development of the parasite, showed that 52% of the parasite
units on the compatible host, E/pfl, had formed secondary
haustoria up to 55/4 in length. The percent of secondary haus—
toria formed with the incompatible parasite/host genotypes
ale/m. 1322/3129. 332/2193. and Eli/1111.49 were 6,

28, 5, and 1% respectively. More than 75% of the parasite units

64
were associated with necrotic host tissue as implied by uptake of
aniline blue. These findings provide additional evidence that
different genotypes affect different stages in the ontogeny of the
host/parasite interactions as well as indicating that the incom—
patible interactions can be demonstrated much earlier than pre—

viously reported.

 

 

LITERATURE CITED

 

LITERATURE CITED

ALLEN, P. J. and R. D. GODDARD. 1938. A respira-
tory study of powdery mildew of wheat. Amer. J. of
Bot. 25:613—621.

BRIGGLE, L. W. 1966. Three loci in wheat involving
resistance to Erysiphe graminis f. sp. tritici. Crop.
Sci. 6:461-465.

BRIGGLE, L. W. 1966. Transfer of resistance to
Erysiphe graminis f. sp. tritici from Khapli emmer and
Yuma duram to hexaploid wheat. Crop. Sci. 6:459-461.

BRIGGLE, L. W. 1969. Near-isogenic lines of wheat
with genes for resistance to Erysiphe graminis f. sp.
tritici. Crop. Sci. 9:70-75.

BUSHNELL, W. R., J. DUREK, and J. B. ROWELL.
1967. Living haustoria and hyphae of Erysiphe graminis
f. sp. hordei with intact and partly dissected host cells
of Hordeum vulgare. Can. J. Bot. 45:1719-1732.

CHEREWICK, W. J. 1944. Studies on the biology of
Erysiphe graminis DC. Can. J. Research. 22:52-86.

CLARK, G. 1973. Staining procedures. The Williams
and Wilkins Co. Maryland. 418 pp.

CORNER, E. J. H. 1935. Observations on resistance to
powdery mildews. New Phytologist. 34:180—200.

DURBIN, R. D. 1967. Obligate parasites, effect on the
movement of solutes and water. In The Dynamic Role
of Molecular Constituents in the PTant-Parasite Inter-
action. ed. C. J. Mirocha, I. Uritani, pp. 80-99.
St. Paul: Am. Phytopathol. Soc. 372 pp.

65

 

 

10.

11.

12.

13.

14.

16.

17.

18.

19.

20.

21.

22.

23.

66

ELLINGBOE, A. H. 1968. Inoculum production and infec-
tion by foliar pathogens. Ann. Rev. Phytopath. 6:317-330.

ELLINGBOE, A. H. 1972. Genetics and physiology of
primary infection by Erysiphe graminis. Phytopath.
62:401-406.

ELLINGBOE, A. H. 1973. Genetics and Physiology of
Plant Parasitism. National Science Foundation Research
Proposal. 17 pp.

FLOR, H. H. 1946. Genetics of pathogenicity in
Melamspora lini. J. Agr. Res. 73:335-337.

FLOR, H. H. 1955. Host-parasite interactions in flax-
rust--—its genetics and other implications. Phytopath.
45:680-685.

FLOR, H. H. 1956. The complementary genetic systems
in flax rust. Advan. Genet. 8:29-54.

FLOR, H. H. 1971. Current status of the gene-for—gene
concept. Ann. Rev. Phytopath. 9:275-296.

GRAF-MARIN, A. 1934. Studies on the powdery mildew of
cereals. Cornell Univ. Agr. Exp. Sta. Mem. 157. 48 pp.

HAYAT, M. A. 1970. Principles and Techniques of
Electron Microscopy. Van Nostrand Reinhold Co.
New York, 412 pp.

HAYWOOD, M. J. 1974. Proceedings of the American
Phytopath. Soc. Vol. 1: p. 67.

HAYWOOD, M. J. 1974. Proceedings of the American
Phytopath. Soc. Vol. 1: p. 126.

LARGE, E. C. and D. A. DOLING. 1962. The measure-
ments of cereal mildew and its effect on yield. Plant
Pathology. 11:47-57.

LARGE, E. C. and D. A. DOLING. 1963. Effect of
mildew on yield of winter wheat. Plant Pathology.
12:128-130.

LACHLI, A. 1972. Translocation of inorganic solute.
Ann. Rev. Plant Physiol. 23:197—218.

 

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

67

LOEGERING, W. Q. 1966. The relationship between
host and pathogen in stem rust of wheat. (Proc. 2nd.
Int. Wheat Symp. Lund. 1963) Hereditas, Suppl.
Vol. 2:167-177.

MARTIN, T. J. 1974. Uptake of 3213 by Triticum
aestivum and genetic control of 32P transfer to
Erysiphe graminis during primary infection. Ph. D.
Thesis. Michigan State University, 116 pp.

MASRI, S. S. 1965. The development of appresoria,
haustoria, and secondary hyphae during primary infec-
tion of wheat and barley by Erysiphe graminis. Ph. D.
Thesis. Michigan State University. 89 pp.

MASRI, S. S. and A. H. ELLINGBOE. 1966. Primary
infection of wheat and barley by Erysiphe graminis.
Phytopath. 56:389-395.

MASRI, S. S. and A. H. ELLINGBOE. 1966. Germination
of conidia and formation of appresoria and secondary
hyphae in Erysiphe graminis f. sp. tritici. Phytopath.
56:304-308.

MCCOY, M. S. and A. H. ELLINGBOE. 1966. Major
genes for resistance and the formation of secondary
hyphae by Erysiphe graminis f. sp. hordei. Phytopath.
56:683-686.

MOLLENHAUER, H. H. 1959. Permanganate fixation of
plant cells. J. Biophys. Biochem. Cytol. 6:431.

MOLLENHAUER, H. H. 1964. Plastic embedding mix—
tures for use in electron microscopy. Stain. Technol.
39:111.

MOSEMAN, J. G. 1959. Host-pathogen interaction of the
genes for resistance on Hordeum vulgare and for patho—
genicity in Erysiphe graminis f. sp. hordei. Phytopath.
49:46 9-472.

MOSEMAN, J. G. 1966. Genetics of powdery mildews.
Ann. Rev. Phytopath. 4:269-290.

MOUNT, M. S. 1968. Environmental effects and transfer
events during primary infection of wheat by Erysiphe
graminis. Ph.D. Thesis, Michigan State University. 112 pp.

 

 

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

68

NAIR, K. R. S. and A. H. ELLINGBOE. 1962. A method
of controlled inoculations with conidiospores of Erysiphe
graminis var. tritici. Phytopath. 52:714.

NAIR, K. R. S. and A. H. ELLINGBOE. 1965. Germi-
nation of conidia of Erysiphe graminis f. sp. tritici.
Phytopath. 55:365-368.

PERSON, C. 1959. Gene-for-gene relationship in host:
parasite systems. Can. J. Bot. 37:1101—1130.

PERSON, C., D. J. SAMBORSKI, and R. ROHRINGER.
1962. The gene-for-gene concept. Nature. 194:
561-562.

POWERS, H. R. and W. J. DANDO. 1960. Genetic con-
trol of the host-parasite relationship in wheat powdery
mildew. Phytopath. 50:454-457.

PRATT, R. 1938. Respiration of wheat infected with pow-
dery mildew. Sci. 88:62-63.

ROWELL, J. R., W. Q. LOEGERING, and H. R. POWERS,
JR. 1963. Genetic model for physiologic studies of
mechanisms governing development of infection type in
wheat stem rust. Phytopath. 53:932—937.

SAKO, N. and M. A. STAHMANN. 1972. Multiple mole-
cular forms of enzymes in barley leaves infected with
Erysiphe graminis f. sp. hordei. Physiological Plant
Path. 2:217-222.

SCHNATHORST, W. C. 1965. Environmental relation-
ships in the powdery mildews. Ann. Rev. Phytopath.
3:343-366.

SEMPIO, C. 1950. Metabolic resistance to plant diseases.
Phytopath. 40:799-819.

SHAW, M., S. A. BROWN, and D. R. JONES. 1954.
Uptake of radioactive carbon and phosphorus by parasi-
tized leaves. Nature. 173:769-798.

SLESINSKI, R. S. 1969. Genetic control of primary inter—
actions during infection of wheat by Erysiphe graminis
f. sp. tritici. Ph.D. Thesis, Michigan State University.
107 pp.

 

 

47.

48.

49.

50.

51.

52.

53.

69

SLESINSKI, R. S. and A. H. ELLINGBOE. 1969. The
genetic control of primary infection of wheat by Erysiphe
graminis f. sp. tritici. Phytopath. 59:1833-1837.

STUCKEY, R. E. and A. H. ELLINGBOE. 1975. Effect
of environmental conditions on 35S uptake by Triticum
aestivum and transfer to Erysiphe graminis f. sp.
tritici during primary infection. Physiological Plant
Pathology. 5:19—26.

STUCKEY, R. E. 1973. Elongation of secondary hyphae
and translocation of 35S and 3H from host to parasite
during primary infection of wheat by Erysiphe graminis
f. sp. tritici. Ph. D. Thesis, Michigan State Univer-
sity. 119 pp.

WALKER, J. C. 1957. The powdery mildews. I_nPlant
Pathology. pp. 312-318. McGraw-Hill Book Co., New
York.

 

WHITE, N. H. and E. P. BAKER. 1954. Host-pathogen Ii
relations in powdery mildew of barley. I. Histology of
tissue reactions. Phytopath. 44:657—662.

WOOLF, C. M. 1968. Principles of Biometry. D.
Van Nostrand Co. , Inc., New Jersey. 35-45 pp.

YARWOOD, C. E. and LOIS JACOBSON. 1955. Accumu-
lation of chemicals in deceased areas of leaves.
Phytopath. 45:43-48.