UPTAKE OF 32P BY TRITICUM AESTWUM

32 AND GENETIC CONTROL OF
P TRANSFER TO ERYSIIPHE GRAMINIS
DURING PRIMARY INFECTION

Dissertation for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
TERRY JOE MARTIN
1974

 

 

 

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ABSTRACT
UPTAKE OF 32P BY TRITICUM AESTIVUM AND GENETIC

CONTROL OF 32P TRANSFER TO ERYSIPHE GRAMINIS
DURING PRIMARY INFECTION

By

Terry Joe Martin

Environmental conditions necessary for synchronous development

of Erysiphe graminis f. sp. tritici Em. Marchal during primary infec-

 

tion of wheat were shown to affect the amount of 32P taken up and
translocated to the epidermis of excised leaves placed in a 32P solu-
tion. Light increased transpiration, which in turn increased the
amount of 32F taken up by the leaves and the amount of 32P transferred
to the parasite.

Rates of 32P transfer from host to parasite were determined
for compatible and incompatible parasite-host interactions during
primary infection. Transfer rate reflected the relative compatibility
of the host-parasite interaction studied. Interactions with little

fungal development had lower rates of transfer, while fully compatible

interactions had high rates of transfer.

Terry Joe Martin

The four possible parasite/host genotypes (the quadratic check)
for each interacting gene pair were evaluated for their effects on
final infection type seven days after inoculation, on 32F transfer
from host to parasite, and on elongating secondary hyphae. With all
gene pairs the final infection type of the three compatible interac-
tions were similar. Only the incompatible interaction (Eg/Emg) re-
sulted in a low infection type. The compatible interactions involving
El_and Eml, Eg_and 3mg, E§b_and Em§b_had similar rates of_32P transfer,
but this was not true for the compatible interactions involving £&_and
Egg, The pfi/fimfi_genotype had a reduced rate of transfer when compared
to pfl/pmfi_and Efl/pmfl, The production of elongating secondary hyphae
was also delayed with pfl/Emfi, Two other isolates containing p§_re-
sulted in a reduction in the percent of parasites that produced elon-
gating secondary hyphae. Thus the mutations from Efl_to pfl_in the para-
site did not completely negate the change in the host from pmfi_to 393,
This indicates that specificity of gene-for-gene interactions is for
incompatibility, not compatibility. It also demonstrated how the
gene-for-gene hypothesis might play a role in general or horizontal
resistance.

Germinating conidia of g, graminis were shown to reduce tran-
spiration rates of the host before penetration occurred. This was a
result of stomate closure induced by the germinating conidia.

2

Terry Joe Martin

Extracts of germinating conidia did not reduce transpiration rates and
abscisic acid was not detected in the conidia. The reduction in tran-
spiration continued through primary infection and was shown to be re-
sponsible for less 32F uptake by inoculated plants than noninoculated
plants during primary infection.

There were no indications for the presence of metabolic sinks
induced by g, graminis, which concentrated 32P at infection sites in
the epidermis of inoculated plants during primary infection. Accumu-
lation of 32F in the epidermis was apparently due to increased cuti-
cular transpiration caused by disruption of the cuticle by the devel-

oping fungus.

UPTAKE OF 32R BY TRITICUM AESTIVUM AND GENETIC

2

CONTROL OF 3 P TRANSFER TO ERYSIPHE GRAMINIS

DURING PRIMARY INFECTION

By

Terry Joe Martin

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

1974

DEDICATED
to

Lorna, my wife

ii

ACKNOWLEDGMENTS

I am deeply in debt to Dr. A. H. Ellingboe for his guidance
and assistance during my stay at M.S.U. and in the preparation of
this thesis.

I am sincerely grateful to Dr. Gene Safir for his advice,
assistance, and loan of equipment which enabled a large portion of
this research to be done.

I am also grateful to the members of my guidance committee.

Thanks is also given to Joe Clayton for his active support
and advice given to my research program.

The help and advice received from my fellow graduate students
were extremely valuable throughout my research program, and I thank
them for it.

I also want to recognize the moral and financial support given
to me by my mother and father. If not for their love and support this
thesis would never have existed.

Financial assistance for this investigation was obtained from
the National Institute of Health and the National Science Foundation

for which I am grateful.

TABLE OF CONTENTS

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

LIST OF FIGURES .........................

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . .
LITERATURE REVIEw ........................
MATERIALS AND METHODS ......................
Production of Inoculum ....................
Methods of Inoculation ....................
Environmental Conditions for Experiments ...........
Designation of Genotypes ...................
Examination of Fungal Structures ...............

Detection of 32

P in Leaf Sections, Epidermis, and Parasite. .
uptake of 32F ........................
32F Transfer from Host to Parasite ..............
Quadratic Checks .......................
Effects of Inoculation on 32F Uptake by Detached Leaves . . .
Effects of Inoculation on Transpiration ...........

Relative Diffusion Resistance ................

iv

Page
vi

vii

l8
I8
20
20
21
22
23
24
25
26
27
28

29

TABLE OF CONTENTS (cont.)

Page
Extraction of Germinated Conidia. .............. 29
Effects of E, graminis on Uptake and Translocation of
32? During Primary Infection of Wheat ........... 3i
Transpiration Rates of Intact Inoculated and Noninoculated
Plants During Primary Infection .............. 3l
Replication and Statistics .................. 32
RESULTS .................. . .......... 33
Replication of Previous Work ................. 33
32F Uptake and Translocation to the Epidermis ........ 33
Transfer of 32F to the Parasite ............. . . 4l
Quadratic Checks ....................... 52

Effects of Inoculation on 32F Uptake by Detached Leaves . . . 75

Transpiration Rates of Detached Leaves ............ 78
Relative Diffusion Resistance ................ 78
Interaction Specificity .............. . . . . . 79
Extracts of Germinated Conidia ................ 80

Effects of E, graminis on Uptake and Translocation of
32F During Primary Infection ................ 80

Transpiration Rates of Inoculated and Noninoculated
Near-Isogenic Lines of Wheat During Primary Infection . . . 84

DISCUSSION. . . . . . . . . . . . . . . . ............ 96
SUMMARY ............................. l09
LITERATURE CITED ......................... 112

V

Table

LIST OF TABLES

Page
Infection type produced seven days after inoculation of
five near-isogenic wheat lines with six cultures of
g, graminis f. sp. tritici.. ............... l9

The amount of 32F taken up by one cm leaf sections as
determined by three methods of preparing the sample . . . 36

Transpiration rates of intact noninoculated and inoculated
near-isogenic wheat seedlings three and six hours after
inoculation with Erysiphe graminis f. sp. tritici (MS-l). 79

The average transpiration rate (mg cm'2 hr") of near-
isogenic wheat lines, the percent decrease, and the P
value for the difference between inoculated and non-
inoculated plants 20-30 hours after inoculation with

E, graminis f. sp. tritici ................ 95

vi

Figure

1.

LIST OF FIGURES

The four possible parasite/host genotypes involving a
single gene pair governing compatibility of host and
parasite. Rx_and rx_are alternate alleles in the
host. Ex_and px_are alternate alleles of the para-
site. Ex/Rx_specifies incompatibility while 25/35,

95/35, and px/rx_specify compatibility .........

The pattern of interaction with a multiple allelic series

in the host ........................

The two types of gene products following crossing over

between two closely linked genes or within one cistron .

Development of Erysiphe graminis f. Sp. tritici during

primary infection of wheat leaves ............

The effect of light on 32F uptake in inoculated wheat
leaves one through l0 hours after being placed in 32F

solution (32F CPM X lO‘5 cm‘] leaf section) .......

The effect of the environmental conditions used for
synchronous parasite development during primary
infection on 32F uptake during five hour uptake
periods by wheat leaves and translocation to the
epidermis in wheat leaves inoculated with Erysiphe

graminis f. sp. tritici .................

Rates of 32F transfer from five near-isogenic wheat
lines to Er Si he raminis f. sp. tritici culture

MS-l (Ex) ( P CPM X 10' /5,000 spores applied) .....

Rates of 32F transfer from near-isogenic wheat lines of
the Em§_allelic series to Erysiphe graminis f. sp.
tritici culture MS-l (Ex) (34F CPM X lO'3/5,OOO spores

applied) ........................

vii

 

Page

12

T4

34

37

39

42

45

LIST OF FIGURES (cont.)

Figure
9.

10.

11.

12.

13.

14.

15.

16.

Rates of 32? transfer from five near-isogenic wheat lines
to‘Erysiphe graminis f. sp. tritici culture MS-l (Ex)
(32p CPM x 10-3/5,ooo spores applied/4,500 32p CPM in
the epidermis) .....................

Effect of light given from 20 to 30 hours after inocula-
tion on 32F transfer from Chancellor wheat (pmx) allowed
to take up 32F for five hours to Erysiphe graminis f.
sp. tritici culture MS-l (E29 ..............

Rates of 32F transfer from four near-isogenic wheat lines
to Erysiphe graminis f. sp. tritici culture MS-l (Ex)
s32? CPM x 10-37s,ooo functional parasite units/4,500

2P CPM in the epidermis) ................

Rates of 32F transfer from wheat to Erysiphe graminis f.
sp. tritici with the four possible parasite/host geno-
types involving El_and Eml_(32P CPM X 10'3/5.OOO spores
applied) ........................

Rates of 32? transfer from wheat to Erysiphe graminis f.
sp. tritici with the four possible parasite/host geno-
types involving Eg_and Emg_(32P CPM X 10'3/5,OOO spores
applied) ........................

Rates of 32F transfer from wheat to Erysiphe graminis f.
sp. tritici with the four possible parasite/host geno-
types involving g§_and gm§g_(32p CPM x 10-3/5,OOO spores
applied) ........................

Rates of 32F transfer from wheat to_Erysiphe graminis f.
sp. tritici culture MS-l (£5) and MS-Z (95) with the
four possible parasite/host genotypes involving Eg_and
gmg_( 2? CPM x 10-3/s.ooo spores applied) ........

Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici cultures MS-l (35)
and MS-Z (£5) with the four possible parasite/host
genotypes involving Pg_and Emg_ .............

viii

Page

47

50

53

55

58

60

62

64

LIST OF FIGURES (cont.)

Figure

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Page

Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici culture MS-Z (93)
on four near-isogenic wheat lines three of which
contained a dominant Em_gene .............. 67

Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici cultures MS-l (£2)
and MS-Z (£2) with the four possible parasite/host
genotypes involving 3mg. ................. 69

Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici cultures MS-l (£3)
and KhXCc7 (95) with the four possible parasite/host
genotypes involving Efl_and Emg_ ....... . . . . . . 7l

 

Formation of elongating secondary hyphae (ESH) by
Erysiphe graminis f. sp. tritici cultures MS-l (£3)
and MS—3 (23) with the four possible parasite/host
genotypes involving Pfi_and Emg_ ............. 73

 

The uptake of 32F in one cm leaf sections of wheat
seedlings one to six hours after inoculation and the
percent germination of conidia of Erysiphe graminis
f. sp. tritici exposed to various doses of ultraviolet
irradiation ....................... 76

Uptake and translocation of 32F to the epidermis of wheat
given 32F for five hours during primary infection. . . . 8l

Average transpiration rates of Chancellor (pmx) wheat
seedlings at different time periods during primary
infection ........................ 85

Average transpiration rate of Eml_wheat seedlings at
different time periods during primary infection ..... 87

Average transpiration rate of ng_wheat seedlings at
different time periods during primary infection ..... 89

Average transpiration rate of Pm3b wheat seedlings at
different time periods during primary infection ..... 9l

ix

 

LIST OF FIGURES (cont.)

Figure Page

27. Average transpiration rate of fimfi_wheat seedlings at
different time periods during primary infection ..... 93

INTRODUCTION

Many attempts have been made to correlate disease development
and physiological or biochemical changes that occur in resistant or
susceptible host tissue. Unfortunately most of these correlations
have been made during late stages of disease deveIOpment. Compati-
bility or incompatibility of an interaction is usually established
during early stages of the infection process. The purpose of this
study was to examine the interactions between host and parasite during
the initial stages of the infection process.

Powdery mildew of wheat is caused by the obligate parasite

Erysiphe graminis f. sp. tritici Em. Marchal. The disease has re-

 

sulted in significant reductions in grain yields in some areas of the
world (24, 53). The most practical means of controlling this disease
is the development and use of resistant cultivars. Selection for
disease resistance has made available many host cultivars with varying
degrees of resistance to powdery mildew. The inheritance of resistance
to E, graminis has been studied (6) and near-isogenic wheat lines which

differ by only single genes for resistance have been developed (7). A

large number of isolates of the fungus that differ in respect to
virulence are also available.

A well defined system for studying the primary infection
process of E, graminis on wheat has been described (27, 32, 36).

The process was divided into distinct stages, based on morphological
development of the fungus. Each stage required specific environmental
conditions for the parasite population to attain a high degree of
synchrony. Synchrony of the parasite population allowed correlation
of physiological and biochemical changes to morphological development
of the parasite.

Slesinski (49) measured the rates of 355 transfer from wheat
leaves to the hyphae of E, graminis during primary infection. Stuckey
(50) demonstrated the effect of the environment on the rates of 353
transfer and also put the data on a quantitative basis by determining

the amount of 35

S transferred to each parasite unit. He also took
into consideration the amount of 35S theoretically available for
transfer. The rates of transfer were correlated with the morphological
development of the parasite and were shown to be dependent on para-
site/host genotype.

The objectives of my research were: I) to examine the uptake
and translocation of 32F by the host under the environmental condi-

tions used for synchronous parasite development; 2) to determine the

rates of 32P transfer from host to parasite by considering the amount

of label theoretically available in the epidermis for transfer to the
parasite, and to correlate these with the morphological stage of
development of the parasite with both compatible and incompatible
parasite/host genotypes; 3) to use the criteria of 32P transfer,
infection efficiency (production of elongating secondary hyphae), and
final infection type to determine if the three compatible parasite/host
genotypes (Eszmg, px/me, and EE/pmé) involving a single pair of cor-
responding genes in the parasite and host are identical; and 4) to

32

determine why less P is taken up by inoculated excised wheat leaves

than by noninoculated excised wheat leaves.

 

LITERATURE REVIEW

In the following review I will attempt to summarize the
important developments in the physiology and genetics of disease
development that bear on the results reported herein.

The primary infection process of E, graminis on wheat has
been divided into distinct morphological stages: l) 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, and 6) development of elongating
secondary hyphae. Each stage differed in its requirement for temper-
ature, relative humidity, and light (28, 36). Under optimal condi-
tions over 75% of the parasite population moved through each stage
with a high degree of synchrony (28. 32, 36). The production of
elongating secondary hyphae (ESH) has been used as the criterion for
the establishment of a functional relationship between host and para-
site (29). For each elongating secondary hyphae (ESH) that formed

on the host surface a haustorium was produced in the epidermal cell
(29). Conidia on non-host plants germinated, formed appressoria,

and attempted to penetrate epidermal cells, but did not form either

haustoria or ESH (27, 54). The presence of the haustorium and ESH
indicated the transfer of nutrients and other essential materials
from host to parasite, thus the establishment of functional rela-
tionships. The percentage of applied conidia that produced ESH was
defined as infection efficiency (l4, l5).

Flor (18) found that the ability of Melampsora lini to grow

 

and produce symptoms on flax lines containing certain genes was deter-
mined by specific corresonding genes in the pathogen. The existence
of one gene in the pathogen for each gene in the host led to the
development of the gene-for-gene hypothesis (I9, 38). The gene-for-
gene hypothesis states that for every (3) gene in the host that con-
ditions resistance there is a corresponding (B) gene in the parasite
that conditions avirulence. The (E) gene interacts with the (3) gene
in the host to determine incompatibility (low infection type). In-
compatibility results only when a (E) gene in the parasite interacts
with its specific (3) gene in the host (El/El). Compatibility is
specified with the other possible parasite/host genotypes 31/51,
91/31, and pl/rl, With two alleles at one locus in a host (R_and 3)
and two at a corresponding locus in a parasite (E_or 9), there are
four possible interactions (Figure I). This basic scheme was proposed
(42) as a biological test to study physiological and biochemical
effects of disease development. By the use of different host geno-

types in combination with various pathogen genotypes, a four way or

Fig. l.-—The four possible parasite/host genotypes involving
a single gene pair governing compatibility of host and parasite.
Rx and rx are alternate alleles in the host. Px and p__are alter-
nate alleles in the parasite. Px/Rx specifies incompatibility while
Egg/pg, IDS/fl: and Up; specify compatibility.

 

H. .m—
w..._m<~_<m

Figure l

"quadratic check" is developed with which the observed phenomena can
be associated. This test is useful in studying disease development,
especially with the powdery mildew disease which followed the gene-
for-gene relationship (30, 39). The gene-for-gene relationship has
been demonstrated between plants and parasitic fungi, bacteria (8, 37),
viruses (IO), nematodes (23), and insects (20).

The effects of genes that condition incompatibility between
parasite and host have been Studied for both wheat and barley mildews
(29, 47). None of the parasite/host genotypes for incompatibility
(BE/Em§_for wheat mildew or Ex/ME5_for barley mildew) affected the
germination of conidia or the formation of mature appressoria. Three
of the four gene pairs affecting wheat mildew and all four of the gene
pairs affecting barley mildew affected the percentage of parasite units
which produced ESH (29) (one gene pair, EE/Emg, apparently did not
affect primary infection of wheat). The fate of paraSite units that
did form ESH with incompatible genotypes was also determined by the
gene pairs (29).

Mount and Ellingboe (34) studied the transfer of 32P and 35S
from wheat to E, graminis and were able to detect very small changes
during primary infection. The amount of 32F and 3SS transferred to
the fungus was correlated with the deveIOpment of haustoria. Slesinski

and Ellingboe (49) demonstrated differences in 358 transfer rates be—

tween compatible and incompatible parasite/host genotypes.

358 transfer during primary infection

Environment was shown to affect
(50). The increased rate of 355 transfer following 20 hours after
inoculation reported earlier (34, 49) was largely attributed to in-

355 being available for transfer at these times

creased amounts of
rather than to an actual increase in 35S transfer rate (50). The
time of the highest rate of transfer was found to be earlier than
previously reported but the same differences in 3SS transfer rates
were found between compatible and incompatible parasite/host genotypes.
Using the criteria of final infection type, 355 transfer, and
percent ESH, the quadratic check was completed with the El/Eml_gene
pair (48). The three compatible interactions El/pml, 211291, and
pl/pml_were indistinguishable by the criteria of percent ESH and final
infection type, but a reduced rate of 355 transfer was observed with
pl/Eml (48, 49). The differences observed may have been due to other
gene differences between the two near-isogenic host lines or between
the non—isogenic strains of the parasite. The quadratic check was
completed for Eg/MEg using the same criteria (23). Identical final
infection types, 35S transfer rates, and percent ESH were reported for
all compatible combinations of this gene pair, i.e.,Eg/m£g, pg/Mgg,
and pg/mgg, These quadratic checks are considered very important be-
cause they bear on the question of where the specificity of the gene-

for-gene interactions occurs. Is it with the incompatible or com-

patible interactions?

10

The simplest hypothesis is that specific host—parasite inter—
actions occur to give incompatible relationships. If this hypothesis
is true, no differences between the two compatible genotypes E5]Eߤ_
and El/Emég would be expected. The third compatible genotype EE/Emé,
might be expected to be distinct from the other compatible genotypes
EE/pm§_and pg/pm§_because there are no reasons to believe that the
transition in evolution from El_to pl_in the parasite would completely
negate the transition in evolution from pml_to Eml_in the host. The
pl/Emfl_interaction has only to have a selective advantage over the in-
compatible interaction El/Emfl, not the other two genotypes for compat-
ible interactions El/pml_and pl/pml, Thus if pl/Eml_resulted in a less
compatible interaction than pl/pml_or El/pml_it would indicate that the
pl product is an altered product of the El.gene with an altered
specificity for the Eml product.

Differences have been observed between the three genotypes for
compatibility with one gene pair (48), but not with a second pair (21).
Completion of the quadratic check with many gene pairs and with the use
of many strains of host and parasite is needed to decide which is more
universal.

The multiple alleles in barley at the MEg_locus (3l) that
determine reaction to E, graminis also demOnstrates that the simplest
explanation is that specific recognition occurs for incompatibility.

The alleles in barley were shown to be distinct by reactions to

11

different strains of E, graminis f. sp. hogggi, The corresponding
genes in the parasite are not allelic. The pattern of interactions
between the two alternate alleles in the parasite with each of six
alleles in the host is shown in Figure 2 (16). Only when Egl_inter-
acted with Mggl_did an incompatible interaction result. Egl_gave a
compatible interaction with hosts with the other MEg_alleles. If
specific recognition for compatibility occurs this would mean that
eleven different specific interactions or parasite/host genotypes
can give the same compatible relationship while only one cannot.

If specific genetic recognition occurs between host and para-
site to give an incompatible interaction, then intracistron recombi-
nation between two host alleles should yield a product that is not
recognized by either of the corresponding parasite E_genes. Two
classes of compatible interactions should result from recombination
between the two alleles. The means to distinguish between recombi—
nation between tightly linked genes and intracistronic recombination
has been developed (Figure 3) (l6). If recombination occurs between
two tightly linked genes El_and 5E, only one class of recombinants,
[1.32, would give a compatible interaction with a pathogen that pos-
sesses the corresponding El_and EE_genes for Rl_and 3;, If the two
genes were alleles with different specificities, for example E§_:_and
:_E§, and recognition occurred for incompatible relationships, there

would be two classes of recombinants that would give compatible

12

Fig. 2.--The pattern of interaction with a multiple allelic
series in the host. (-) = incompatibility, (+) = compatibility.

l3

HOST ALLELES

 

14

Fig. 3.--The two types of gene products following crossing
over between two closely linked genes or within one cistron.

15

Figure 3

X3 1-
+ X0
X3 X0
+ +

16

interactions with pathogens possessing the corresponding E_genes for
the host parental alleles. Crosses involving the Eg_and Elg_alleles
at the E_locus in flax did yield two classes Of recombinants which

gave compatible interactions with two cultures of Melampsora lini

 

which recognize the Eg_and Elg_alleles (45). These results give
additional evidence that the specificity in recognition between host
and parasite is for an incompatible relationship.

Temperature sensitivity has played an important part in under—
standing some of the basic processes in life (I3, 22, SI). Tempera-
ture sensitive mutations are believed to be missense mutations that
lead to changes in primary structure of proteins that affect the sta-
bility of the tertiary structure of the protein. The tertiary struc-
ture is especially affected at high temperatures. Normal or near
normal enzymatic activity of the protein may occur at low temperatures
but be greatly reduced or absent at high temperatures.

Temperature sensitive genes affecting stem rust of wheat have
been reported (25). The interaction between the §§§_gene and its
corresponding E§_gene in Puccinia graminis f. sp. tritici was temper-
ature sensitive. Of the four possible parasite/host interactions,
E§/§£§2 EE/grg, E§/§E§: and pE/ggg, only E§1§3E_was temperature sensi-
tive. At the low temperature, the E§/§E§_interaction resulted in
incompatibility. At the high temperature compatibility ensued. The

simplest explanation of this is that the low temperature allows for

17

a specific interaction between host and parasite and that the high
temperature did not allow specific interaction for incompatibility.
It is 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 compatible or in-
compatible relationships between host and parasite. A number of
physiological changes are known to occur following inoculation of
wheat with E, graminis. Respiration (I, 40), photosynthesis (43),
and translocation patterns of organic molecules (44) have been re-
ported to change two to lOdays after inoculation. However, previous
studies (47) have shown that the establishment of compatibility or
incompatibility often occurs before 24 hours after inoculation. It
is important, therefore, to determine the very earliest interactions

between a host and parasite.

MATERIALS AND METHODS

Production of Inoculum

All strains of Erysiphe graminis f. sp. tritici were maintained

 

in different growth chambers on Triticum aestivum L. 'Little Club.‘

 

Each strain was purified by isolating and increasing single pustules
from Little Club wheat. Each strain was periodically checked for
purity by scoring infection types on a set of differential host lines
(Table 1).

Wheat seedlings grown in the greenhouse for six days were
dusted with conidia from plants inoculated seven days earlier. The

cultures were kept under the following conditions.

I. Light--7OO to 800 ft-c (650-750 ft-c from white VHO-fluorescent
tubes and 50 ft-c from 25 watt incandescent bulbs), 16 hour

photoperiod.

2. Temperature--l8 i l C during the day and 17 i l C during the

night.

18

19

TABLE l.--Infection type produced seven days after inoculation of five
near-isogenic wheat lines with six cultures of E, graminis
f. sp. tritici.

 

 

Infection typea

 

Near-isogenic host line

 

Culture (Chancellor)
m): m m M Egg
MS-l 4 0 2 3 0
MS-2 4 0 4 3 4
MS—3 4 4 2 3 4
MS-4 4 4 2 3 0
MS-5 4 O 2 4 0
~~I<hXCc7 4 O 2 3 4

 

aInfection 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.

3. Relative humidity--80 i 5% during the day and 95 t 5% during
the night.
4. Continuous air circulation.

Conidia produced on the sixth day after inoculation were used

in all experiments. The wheat lines used in experiments were planted

20

in three inch pots of soil and kept in the greenhouse for five days

before being inoculated.

Methods of Inoculation

The rolling technique of inoculation (35) was used for all
studies of morphOlogical development of the fungus during primary
infection. The conidia were evenly dusted onto clean glass slides.
The conidial chains and dead conidia were removed by lightly blowing
across the slide. The remaining conidia were then transferred from
the slide with a cotton swab to the abaxial side of the leaf. Devel-
opment of the fungus was similar on both sides of the leaf (27).

Plants used in studies of 32R uptake and transpiration were
lightly dusted with conidia since the numbers of inoculated plants

required for those experiments were quite high.

Environmental Conditions
for Experiments

All experiments were done in Sherer—Gillett (Model CEL 512-37
and Model CEL 25—7) growth chambers. The following conditions were
used to obtain high infection efficiency and synchronous growth of the

parasite population during primary infection (0-30 hours) (46).

21

Zero to one hour after inoculation, plants were kept in the

dark at l8 i l C in approximately 100% relative humidity (RH).

One to six hours after inoculation, plants were kept under

l.0 X 105 ergs cm-2 sec.1 radiation (0.6 X l05 ergs cm-2 sec

from white VHO-fluorescent tubes and 0.4 X lo5 ergs cm'2 sec

from 25 watt incandescent bulbs) at 22.: l C and 65 i 5% RH.

Six to 20 hours after inoculation, plants were kept at the

same temperature and RH as in above, but with no light.

Twenty to 30 hours after inoculation, conditions were the

same as 2 above.

The above conditions were used for all experiments unless

otherwise stated. Light intensity was measured at the leaf tips with

a YSI Kettering Model 65 Radiometer. Temperature and RH were moni-

tored during experiments with wet and dry bulb thermometers and a

recording hygrothermograph calibrated with a sling psychrometer.

Designation of Genotypes

Briggle's terminology (6, 7) was used to designate the E_genes

conditioning reaction to E, graminis f. sp. tritici. Genes at a dis-

tinct locus have been designated 391, Eng, etc. Genes at the same

22

locus but thought to be allelic are followed by the letters 2, E, 9,
etc. Alternate alleles for each dominant Em_gene will be referred to
by their respective recessive designations (pml, 2mg, etc.). Since

the host lines are all homozygous, the genotypes will be written as
haploid (39;) rather than in the diploid form (Em§_Em§). The E;genes
in the parasite that result in incompatibility with host lines with
dominate Em_genes will be designated as El, BE, etc. The number refers
to its corresponding Em_gene in the host. The alternate alleles for
each E_gene will be referred to by their respective recessive designa-
tions (pl, 9g, etc.).

Parasite/host combinations are referred to by the corresponding
genotypes which specify compatibility or incompatibility. For example,
Efl/Emg_specifies incompatibility. The Em§_gene in the host recognizes
only the Efl_gene in the pathogen, even though the pathogen may possess
many other E_genes. A corresponding gene pair which conditions incom-
patibility is expressed in the presence of many gene pairs specifying
compatibility (49). Only the genes of the parasite/host genotype that

specify incompatibility are given.

Examination of Fungal Structures

Determination of the percentage of the parasite population at

each stage of development was made from counts on leaves with a

23

compound light microscope (l60X). The number of parasite units at
each stage of development was determined from a two cm length section
of leaf at two hour intervals after inoculation. A new leaf section
was used for each observation and 80—125 parasite units were counted

each time.

Detection of 32F in Leaf Sections,
Epidermis, and Parasite

The amount of 32

P was determined in a one-cm-long-section of
leaf, at least one cm from the leaf tip. Three methods were compared.
l) Leaf sections were placed directly into l5 mt of scintillation fluid
[5 g of 2,5-diphenyloxazole and 0.l g of l,4-bis-2-(5-phenyloxazolyl)
benzene in 1 liter toluene] and radioactivity was determined in a
Beckman LS-l33 Liquid Scintillation Spectrometer. 2) Four leaf sec-
tions were macerated in 5 mi of 0.l M Na-K—PO4 buffer in a glass tissue
grinder. A 0.1 mt aliquot was dried in the bottom of a scintillation
vial before scintillation fluid was added. Radioactivity was deter-
mined as described above. 3) The same procedure for preparation of
plant material was used as in 2 above, but a 0.l mt aliquot was dried
on a piece of Whatman #1 filter paper which was submerged in scintil-

lation fluid. The data are expressed as CPM in one leaf section.

Unless otherwise stated this last method was used in all experiments.

24

32P translocated to the epidermis was determined

The amount of
by stripping approximately 50% of the epidermis from the underside of
one leaf section. All green tissue was removed before placing the
epidermis tissue in scintillation vials. Scintillation fluid was added
and the radioactivity was determined. The data are expressed as the
CPM in the abaxial epidermis of a one cm leaf section.

The following procedure was used to separate the ectoparasitic
portion of the parasite from the leaf. A l.9% solution of parlodion
in ether:ethanol (40:60) was applied to a one cm section of a leaf
(ll, 34). The solvent evaporated in minutes and the parlodion film
was easily removed with forceps. The ectoparasitic portion of the
parasite was embeded within the plastic strip. Four plastic strips
were dried and placed into one scintillation vial. The radioactivity
was determined as described above.

32

Uptake of P

Twenty hours after inoculation wheat seedlings were cut at the
crown with a razor blade (a film of water was present on the cutting
edge) and placed into small vials (5 X 25 mm) that contained 0.l m2 of

32PO

3 4 4
One set of plants was kept in the dark and another set was kept in the

in 0.1 M Na-K-PO buffer (pH 6.9).

a 50 uCi/mx solution of H

light. Temperature and relative humidity were constant. After one, two,

25

three, four, five, six, eight, and l0 hours in 32P solution the radio-
activity in one cm length leaf sections was measured.

The amount of 32F taken up by the leaf and translocated to the
epidermis was also determined for five hour uptake periods beginning
at various hours after inoculation. Different plants were used for
each five hour uptake period. Inoculated wheat seedlings were cut and

32

placed in the P solution. Leaf sections and epidermal strips were

taken from different plants every two hours beginning at six and ending

32P in a leaf sec-

at 30 hours after inoculation. Thus,the amount of
tion at l0 hours after inoculation was the result of uptake from five
to l0 hours after inoculation.

32? Transfer from Host to Parasite

Rates of 32P transfer from host to parasite were determined.

Four parlodion strips were taken from inoculated plants which had taken

up 32F for five hours preceding the application of the parlodion.

These were placed in a single vial and the amount of radioactivity
determined. The results were plotted as radioactivity per 5,000 spores
applied to the leaves. The number of spores applied was determined by

making direct counts of parlodion strips made from plants inoculated

at the same time but not labeled with 32F. The radioactivity in

32

parlodion strips made from noninoculated leaves given P during the

26

same period as inoculated leaves was subtracted from the radioactivity

32P transfer during primary

transferred to the parasites. The rates of
infection were determined for one compatible and six incompatible
parasite-host interactions. These data were then corrected for the
amount of 32F theoretically available to be transferred (CPM/4500 CPM
in the epidermis) and the amount transferred per functional parasite

unit (47).

Quadratic Checks

The quadratic check includes all four possible parasite/host
genotypes involving one corresponding gene pair (Figure l). The cri-
teria of infection type seven days after inoculation and 32P transfer
from host to parasite during primary infection were used to complete
the quadratic checks for each of the following gene pairs, El/Eml,
EE/PmZ, P3b/Pm3b, and 35/395, Culture MS-l (El, EE, Egg, E5) was
used in all cases as the pathogen with the dominant E gene. Cultures
MS-4 and MS-S were used as the pathogens with pl_and 3b, respectively.
Culture MS-Z contained both pg_and 94, Chancellor wheat, the recurring
parent of the near-isogenic lines, carries no known (Em) genes. Chan-
cellor was used in all cases as the host carrying the pm; genes. Four
isogenic lines each containing one dominant Em_gene were used as the

alternate host lines.

27

The criteria of final infection type and primary infection
efficiency were used to complete the quadratic checks for EE/EmE_and
Eg/Emg, Three cultures, MS-2, MS-3, and KhXCc7, were used as repre-
sentatives of parasite cultures with the pfl_gene. Culture MS-2 was
also used as a representative culture with pg, The infection effi-
ciency of culture MS-Z was also determined on the near-isogenic lines
containing Eml, Egg, or gmgg.

Effects of Inoculation on 32P

Uptake by Detached Leaves

Chancellor wheat seedlings were inoculated heavily by dusting

32

with conidia, cut, and placed in the P solution. Noninoculated

seedlings were treated in the same manner. After five hours the

32F taken up and translocated to the epidermis was deter-

amount of
mined for both inoculated and noninoculated seedlings.

The effect of heavily dusting leaves with Puccinia graminis
uredospores, Helminthosporium victoriae conidia, E, graminis f. sp.
Egggg1_conidia, chalk dust, and carborundum on 32P uptake was also
determined.

The effect of nonviable conidia in eliciting an effect on the

host was determined by inoculating seedlings with conidia which had

been irradiated with ultraviolet (UV) light. Conidia were dusted onto

28

1)

for 0, IO, 30, 60, 120, and 240 seconds. The percent germination of

glass slides and exposed to UV radiation (l.2 X 105 ergs cm'2 sec—

conidia at each exposure was determined by controlled inoculations (35)
of wheat seedlings. The remainder of the conidia on the slides were
dusted onto wheat seedlings to be used in uptake studies. The seedlings
were cut and placed in 32F solution one hour after inoculation. The
amount of 32F in a one cm length leaf section and the percent germina-

tion was determined at six hours after inoculation.

Effects of Inoculation
on Transpiration

Transpiration rates were determined by placing cut inoculated
or noninoculated seedlings in 5 x 25 mm test tubes which contained
0.1 m2 of Na-K-PO4 buffer (pH 6.9) one hour after inoculation. The
test tubes were sealed with petroleum jelly and weighed on a Mettler
balance. At six hours after inoculation the plants were weighed to
determine water loss. The surface area of the leaves was obtained by
tracing an outline of each leaf and measuring the area with a Paragon
Compensating Polar Planimeter (Keuffel and Esser Co.).

Transpiration rates of intact plants were also measured.

Chancellor (pm!) and four near-isogenic lines of Chancellor, each

containing one known dominant gene Pml, Pm2, Pm3b, or Ema, were grown

29

in (l0 X 75 mm) test tubes. Test tubes containing inoculated or non-
inoculated five-day-old plants were sealed with petroleum jelly, and
weighed on a Mettler balance one, three, and six hours after inocula-

tion. The surface area of each leaf was determined as described above.

Relative Diffusion Resistance

Relative diffusion resistance of stomata to water vapor flow
was estimated from measurements of permeability to air for noninocu—
lated and inoculated wheat leaves with a modified Alvin's viscous flow
parameter (2). Data were expressed as the cube root of the time re-
quired for a standard l0 mm Hg Change in pressure (4). Because of the
small size of the primary leaf of wheat, the third leaf of two-month-

old plants was used.

Extraction of Germinated Conidia

Conidia were heavily dusted onto petri dishes containing l0 mi
of distilled water. The conidia were removed by filtration after five
hours at 22 C in the light. The filtrate was atomized onto intact
wheat seedlings. Distilled water was atomized onto control plants.
The rates of transpiration were determined for five hours after the
filtrates were applied. Triton X-lOO (one drop/200 mt extract) was

added to both filtrate and controls for some experiments.

30

Aqueous extracts were made from conidia which germinated on
distilled water. The extracts were tested for the presence of abscisic
acid (ABA). Conidia were allowed to germinate for five hours on dis-
tilled water before the spore suspension was sonicated for IS minutes.
The suspension was centrifuged to remove spores. The pH of the super-
natant was adjusted to 2.5 with l N HCl. The acidic solution was ex-
tracted four times with equal volumes of ethyl acetate in a separatory
funnel. The ethyl acetate was dried over anhydrous Na2504 and filtered
into a boiling flask and evaporated. The residue was taken up in
two mt of ethyl acetate. The solution was spotted onto a silica gel GL
plate for thin layer chromatography (TLC). ABA was also spotted onto
the plate. Ethyl acetatezchloroformzacetic acid (60:40:5) was used as
the solvent. The areas corresponding to the fluorescent spots of ABA
when exposed to ultraviolet light were scraped from the plate and ex-
tracted with ethyl acetate. This extract was then methylated with
diazomethane (3). The methylated extract was placed on a TLC plate
with methylated ABA. Hexane:ethyl acetate (lzl) was used as the

solvent. Ultraviolet light was used to determine the methylated ABA

regions on the plates.

31

Effects of E,Egraminis on

Uptake and Translocation
of432P during Primary
Infection of Wheat

32

The amount of P taken up and translocated to the epidermis

of inoculated and noninoculated plants during five hour uptake periods

from one to 30 hours after inoculation was determined. (The 32F in

the leaf at 10 hours after inoculation was the result of exposing

plants to 32F from five to 10 hours after inoculation, 32F in the

leaves at 30 hours was the result of uptake from 25 to 30 hours after
inoculation, etc.)

Inoculated and noninoculated plants were cut and placed in

the 32F solution in 100% RH 25 hours after inoculation. One set of

plants were also pre-treated from 22 to 25 hours after inoculation

in 100% RH before cutting. After cutting, the plants were allowed

32

to take up P from 25 to 30 hours after inoculation in 100% RH.

Radioactivity in the epidermis was determined.

Transpiration Rates of Intact~
Inoculated and Noninoculated
Plants during Primary Infection

'Transpiration rates were determined by measuring the water

loss from intact plants grown in 10 X 75 mm test tubes. The tubes

32

were sealed with petroleum jelly and weighed on a Mettler balance
at the following times after inoculation: one, three, six, 10, 15,
20, 22, 24, 26, 28, 30, and 34 hours. This allowed the determina-
tion of rates of transpiratibn during various periods of primary
infection. Transpiration rates were determined for inoculated and

noninoculated Chancellor (2E5) wheat and four near-isogenic lines

 

of wheat each with one of the following genes Pml, Pm2, Pm3b, or

Emi-

Replication and Statistics.

Experiments involving 32F uptake, translocation to the
epidermis, and transfer to the parasite were repeated at least
six times on six different days with two replications daily. Ex-
periments involving fungal morphological development and transpir-
ation rates were repeated On three or four different days. The
data are presented as averages of all replications. Statistical
analyses were done using a two-way analysis of variance, using

an F-test at the 5% level of significance.

RESULTS

Egplication of Previous Work

The development of E, graminis f. sp. tritici during primary
infection in a highly synchronous manner has been described (29, 32,
36). It was considered essential that this work be repeated because
meaningful interpretation of results reported herein is based on the
reproducibility of the earlier work. The Optimum environmental con-
ditions for germination, formation of appressorial initials, forma-
tion of mature appressoria, production of secondary hyphal initials,
and the production of elongating secondary hyphae were used as pre-
viously reported (51). The development of the parasite population
is presented in Figure 4. It is in close agreement with previous

findings.

32? Uptake and Translocation
to the Epidermis

The most efficient method of determining the radioactivity
in the leaves was to macerate the tissue in buffer and then dry an
aliquot on filter paper (Table 2). It appeared that drying the

33

34

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A V .Aemv vxmcpmm_m seem A--------------V "manna; xgaucoumm mcwummcopm

mo cowumseow Amy use .mpmwu_:w _mcax; agencoomm mo cowmeLow Any .mpeommmgnam weaves mo

cowpmseom Auv .mpawpwcw meeommmeaam mo cowpmsgom Amy .cowpmcwsemm A<V .mm>mm_ pews: mo
:owpommcw xemewea mcwezu wowpwep .am .m mwcAEamm mzawwxmm mo “cesao—m>mouu.e .mwm

 

 

 

35

e musmflm

1.: 20.53302. 5:: 22:

3 .3 ea «a as o.

m
x

o 0 V «O

lNiDlid

36

TABLE 2.--The amount of 32F taken up by one cmleaf sections (1.0 X 0.5
cm) as determined by three methods of preparing the sample.

 

 

Radioactivity (CPM)

 

 

Time after
Ino%:l;tion Direct Leaf Aliquot Dried In Aliquot Dried 0n
Section Count Scintillation Vial Filter Paper
20 39.292 38.350 53.000
28 -- 131,200 167,600
30 -- 116.176 149.500

 

aliquot in the bottom of a scintillation vial was no better than placing
the whole leaf section in the vials. Samples were dried on filter paper
throughout the remaining experiments.

The effect of light on 32? uptake by inoculated leaves was
determined (Figure 5). There was a very definite effect of light on

.32 32

P uptake. After five hours in P the leaves kept in the dark were

only 50% as radioactive as plants kept in the light. The rate of 32F
uptake during this 10 hour period appeared to be reasonably constant.
This was true for plants in the light or the dark.

The effects of the environment during primary infection on

32P and translocation to the epidermis were determined

uptake of
(Figure 6). The environmental conditions necessary for synchronous

parasite development are indicated across the top of the figure.

37

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m mm . a m w umuep :vmn emuem meson o_
5 song» use mm>mmp pawn: uwum_:uocv cw wxmpaa mum no a; __ eo powwow mzhuu.m .mrm

38

m musmflm

2.: 32:

0|.llo «TOD

. . Em:

 

“'2
c

O.—

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md.

‘91 x we: a“

39

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mcmvmxem spwz umpmrzuo:w mm>mm~ poms: cw mvELmuram mg» op cowpouormcmcp ucm mm>am_ paws:

xa meowema mxmua: Lao; m>wm mcwczc mxmua: awn co cowuumwcw xemsvea mcwcsu pamEQo—m>mu
mpvmmemq mzocoesuczm Low umm: mcowpwucou FaucmEcoLF>cm mg» yo pumwmm mghuu.m .mwm

 

40

o ousmflm

at: 20.533002. amhm< 5):...
ON «a 2. 3 O— o — o

O
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In
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41

Both curves show a major effect of light on 32F uptake or translo-

32

cation. The radioactivity in the epidermis of plants taking up P

from 21 to 26 hours after inoculation was eight times greater than

32P from 15 to 20

in the epidermis of those plants that took up
hours. Radioactivity in the leaf sections shows a fourfold in-
crease at the two different uptake times. The 32P in the epi-
dermis would be theoretically available for transfer to the para-
site. So there were large differences in the amount available to
be transferred to the parasite, depending on the time after inocu-
lation when the 32F was taken up.

Transfer of 32P to the Parasite

The rates of 32? transfer from wheat leaves to E, graminis
was determined, using near-isogenic wheat lines (Figure 7). Host

lines Pml, Pm2, Pm3b, and Emfl_are incompatible with culture MS-l (P )

 

while pm5_is compatible with culture MS-l (E5). The radioactivity
in parlodion strips from noninoculated plants which had taken up 32P
during the same time was subtracted from the amount of 32F in parlodion
strips from inoculated plants. This control averaged 53 to 81 CPM.

32F transfer from all wheat lines to the fungus started at
10 hours after inoculation and increased very slowly until 20 hours

after inoculation. There were no differences between near-isogenic

42

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xpw>wpomowume may vcm mawcpm coquo—ng cw um>osme cmsp we; mzmcze mgp we cowpcoa

o_pwmmeaa0pum use .meso; m>wm cow cowu=Fom awn a cw umompa new cowpmfiauocw Levee
meson mzovcm> omen as“ we use mew: mucmFQ .ammv Pumz me=p_:o Powpwcu .am .m mwcwsmem
mgmwmxcm op mmcw— paws: owcmmomw-emmc m>ve Eoee emwmcmep mum mo mmgmmuu.N .mwm

43

h madman

«an: 20....<.=..UOZ_ umhm<

 

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44

lines during this period. With the compatible genotype (BEE), the
rates increased sharply at 22 and 24 hours, then leveled off or
decreased slightly from 26 to 30 hours. The rates of transfer from
Eml were significantly less compared to pm5_at 24 hours. Transfer
continued to decrease from 22 to 30 hours. The transfer kinetics
were the same from Emg_and REE: Rates of transfer from Em§E_were
not significantly different from pm§_until 30 hours after inocula-
tion. Transfer rates from Emfi_were similar to me_until 28 hours
after inoculation, when the rate dropped rapidly.

The kinetics of 32F transfer from pm§_and three alleles of
the Em; locus were determined (Figure 8). Em§g_and Em§9_had sim-
ilar rates of transfer, while Em§g_did not differ from me, Em§g_
and Em§E_were both significantly different from pm§_and Em§g_30
hours after inoculation.

The rates of 32P transfer shown in Figure 7 were adjusted for

32P in the epidermis at each hour after inoculation

32

the amount of

(Figure 9). The amount of P in the epidermis is theoretically the

amount available to be transferred from the epidermis to the parasite

32

(Figure 6). The data were plotted as P CPM/5000 spores/4500 CPM

available in the epidermis at the end of the five hour uptake period.

32

Rates of P transfer from the five near-isogenic lines from Six to

45

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-opooe ocp oco mopepm copopopeoo cp oo>osoc mo: woman» osp mo coppeoo opppmoeooopoo
on» .meoo; o>pp com coppopom mum o op oooopo oco coppopooocp Loppo meson maopeo> omen
osp po poo oeoz mpcopo .Axov lez oeoppzo.flmflmflum .om .4 mpcpEopm.o;mexum op mopeom

oppo—po me; ogp mo mocpp poms: upcomompucooc Eoem eopmcoep mum mo mopomnu.w .mpm

46

on

 

1.:
on

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WY.

 
  

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47

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zpp>ppooopooe ocp coo mopcpm copopopeoo cw oo>oeoe cogp mo; momcom ogp mo coppeoo
opppmocooopoo o3» .meoo; o>pm so; coppspom mum o cp oooo—o coo coppopzoocp eopwo

mono; moopeo> omen ocp pm poo moo: mpcopo .mmmv —umz oeoppzo popppep .om .m.mmmflamum
osmpmxem op mocp— poms: upcomompxpoo: o>pp zoom poomcoep mum po mopomuu.m .mpm

 

48

on

m musmflm

:5 2923302. «up: as:
on «a o. 1 o—

.oilnlo. MWHHMM
dulllld — n—

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c_OI x was an

49

20 hours were not significantly different from each other, thus,
these data were averaged together and represented by a single line.
With the adjustments made (Figure 9), the same differences between
host lines which were shown in Figure 7 are seen again, but the
maximum rate of transfer from EEE."°W appears to occur from 20 to
22 hours instead of 24 to 26 hours after inoculation. A steady
increase in transfer rate occurred from ten to 22 hours and then
a gradual decrease occurred from 22 to 30 hours after inocula-
tion.

The rates of 32P transfer from pm5_in the light and dark
from 20 to 30 hours after inoculation were determined (Figure 10).
The data were plotted as CPM/5000 spores applied. No correction

32F in the epidermis was made. Transfer rates

for the amount of
in the light were similar to those reported in Figure 7. Transfer
rates in the dark at 23 and 29 hours were not different from the:
rate of 32F transferred at 20 hours. This gives support to the
argument that the adjustment of transfer data to take into consider-
ation the amount of 32F activity in the epidermis is reasonable.

If we assume that only functional parasite units (47), i.e.,
parasite units with haustoria and ESH, are taking up 32F from the
leaf we can further adjust the data in Figure 9 to give the rates of

32P transfer/functional parasite unit/CPM available in the epidermis

50

 

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soap psmpp oco mono; om op xpm sop» some .mmmv pumz oesppao.HmHMHum .om .m mpcpsoem

mmmmmmnmm op meson o>pm Lop mum on oxop op oozoppo «mamv poms: eoppoocosu soap commcoep
mum co coppopooocp Lopmo mason om op om soew co>pm pgmpp mo poompmuu.op .mpm

51

op ousopm

at: ZO:<._DU..OZ_ umhm< mirp.
on 8 «a 2 o. o

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(Figure 11). Infection efficiencies on pmx, Pml, Pm3b, and 394 were
80, 17, 30, and 4%, respectively. The adjustments indicated the rates

of 32

P transfer/successful primary infection were higher with incom-
patible genotypes than rates with compatible genotypes. At 30

hours after inoculation the rates of 32P transfer/successful primary
infection with the incompatible interactions with Eml_and Em§E_were

the same as the rate for the compatible interaction with 2mg, Transfer
rates from Emfl were not the same as from pm5_at 30 hours, but the

rate was decreasing rapidly. The adjustment was not applied to

transfer rates from Emg_because it gave results similar to transfer

from REE:

Quadratic Checks

The rates of 32F transfer from wheat to E, graminis with the

four possible parasite/host genotypes involving El_and Eml_were deter-
mined (Figure 12). The incompatible interaction El/Eml, which gives

32P transfer as shown pre-

a low infection type, had a low rate of
viously (Figure 7). The three compatible interactions, which all
gave the same high infection type, had similar rates of 32F transfer.
The final infection type of the four possible interactions

involving EE_and Egg followed the usual pattern for the quadratic

check, all four genotypes gave the same rates of transfer during

53

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oocpeeopoo app>ppooopooe new mopcpm copopopcoo cp oo>oeoe mo: mousse ogp mo coppeoo

opppmoeooopoo och .meso; o>pm pom coppopom mum o op oooopo coo :oppopooocp Loppo

meso; moopeo> omen asp po poo moo: mpcopo .mmmv pumz ogoppzo popppep .om .m mpcpEoem
ocmpmxcm op mocpp poms: upcomompupooc Loop Eoew eowmcoep own po mopomu-.—_ .mpm

54

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popppep .am .p mpcpeaom mompmpao op peas: soap amomeaap mom po mmpam--.~p .opm

 

56

hr.
7:. . . glib

mp mwoopm

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57

primary infection (Figure 13). EE/Emg_has not been shown to affect pri-

32P transfer supported that

mary infection efficiency (34). The rates of
conclusion.

The quadratic check with E§E_and Em§E_also resulted in one geno-
type with low infection type and three genotypes with high infection types.
Rates of 32P transfer (Figure 14) were similar with the three compatible
genotypes while transfer was lower after 26 hours with the incompatible
genotype.

The final infection types of the four possible genotypes involving
Efi_and Egg also followed the expected pattern of the quadratic check,
namely, one genotype gave low infection type and three genotypes gave high
infection types. The rates of 32F transfer with the incompatible genotype
were similar to earlier results (Figure 7). The rates of transfer for the
pfl/Emfi_genotype were reduced when compared to Eg/pmg_and pfi/pm4_(Figure 15).
The quadratic check using the same cultures was then completed using the
criterion of production of elongating secondary hyphae (i.e., infection
efficiency). A slower rate of development of elongating secondary hyphae
(ESH) with pfl/Emg_than with the other two compatible genotypes was ob-
served (Figure 16). The same infection efficiency was obtained but it was
delayed approximately two hours compared to the other compatible genotypes.
Standard deviations bracket each point (Figure 16). The behavior of MS-2

cannot be due to nonspecific genes for slow growth because normal kinetics

of the formation of ESH were observed on the host line with 2mg,

 

58

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poo mom: mpcopo .NEo oco No mcp>po>cp mmozpocmm pmo;\mppmogoo mpopmmoo Loom mcp opp:
popppep .om .p mpcpEoLm mgawwxom op poms: soc: empmcoep mum po mmpom--.mp .mpm

59

 

 

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poo mom: mpcopm .omEm oco mo mcp>po>cp mmoapocmm pmos\mppmoooo mpopmmoo Loom mcp opp:
popppep .om .p mpcpsopm mgapmzpu op poms: so»: empmcoep mum mo mmpoaul.¢p .opo

 

61

on

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a op omoopo oco coppopooocp mepo mpoo; moopgo> moon mgp po poo mom: mpcopo umam new on

mcp>po>cp mmoapocmm pmo;\mppmoeoo mpnpmmoo Loop mop cpp: away mum: one ammv pzmz mooppoo
popppep .om .p mpcpEoom mgmpmamm op poms: soap emmmcoep mum mo mmpomux.mp .mpm

 

63

mp ouampm

1.: 2253302. our? 22:
on cu «m .a— I c—

 

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N

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64

Fig. l6.--Formation of elongating secondary hyphae
(ESH) by Erysiphe graminis f. sp. tritici cultures MS-l (E4)
and MS-2 (94) with the four possible parasite/host genotypes
involving E3 and EDS:

65

100

(MS-l) Elm. ..... 0

H n H’ m 0—0
(Ms-2) gym . ...... .
u u 24/ anA a—a

80

 

 

60

ESH

R 40

2O

 

 

Time after inoculation (hr)

Figure 16

66

To further substantiate that culture MS-2 did not have genes
that merely conditioned slow growth, the infection efficiency of MS-2

was determined on near-isogenic lines containing pmx, Pml, Pm2, and

 

Emgg, The production of ESH (Figure 17) was similar to that reported
for MS-l (E5) on the same host lines (47).

The kinetics for production of ESH with the four possible
interactions involving EmE_was determined (Figure 18). Culture MS-2
also contained pE_and was used in this quadratic check to further
demonstrate that MS-2 wasn't just a slow growing culture. All four
interactions resulted in similar kinetics for the formation of elon-
gating secondary hyphae. This was additional evidence that a slower
rate of development was observed only when pg_was interacting with
Egg,

The quadratic check was completed again for Efl_and Emfl_but
culture KhXCc7 was substituted for culture MS-2. KhXCc7 carries 94,
thus gave a high infection type seven days after inoculation on host
plants with 3E5: An infection efficiency of 20% was observed for the

7 was used as the source of pg_(Figure 19).

251391 genotype when KhXCc
Apparently these 20% of the parasite units that did produce successful
primary infections continued to develop and produced a high final
infection type. A third culture with E4, culture MS-3, gave an in-
fection efficiency of 33% and a high final infection type when inocu-

lated onto a host line with Emg (Figure 20).

67

Fig. l7.--Formation of elongating secondary hyphae (ESH)
by Erysiphe graminis f. sp. tritici culture MS-2 (p4) on four
near-isogenic wheat lines three of which contained a dominant
Em_gene.

 

% ESH

100

80

60

40

20

68

Chancellor lgmx) 0 °

 

 

Time after inoculation (hr)

Figure 17

69

Fig. 18.-~Formation of elongating secondary hyphae (ESH)
by Erysiphe gEaminis f. sp. tritici cultures MS-l (P_2_) and MS-2
(E) with the four possible parasite/host genotypes involving Em_2_.

% ESH

7O

 

 

22 24 26 28

Time after inoculation (hr)

Figure 18

71

Fig. l9.--Formation of elongating secondary hyphae (ESH)
by Erysiphe g:aminis f. sp. tritici cultures MS-l (E3) and KhXCc7
(95) with the four possible parasite/host genotypes involving E3_
and Pm4.

72

(MS-l) Elm . ...... g
H n film 0—0
(KhXCc-7) pit/m . ...... .
u n 95/2014 A_A

 

 

 

 

22 24 26 23
Time after inoculation (hr)

Figure 19

73

Fig. 20.--Formation of elongating secondary hyphae (ESH)
by Erysiphe graminis f. sp. tritici cultures MS-l (E3) and MS-3
(93) with the four possible parasite/host genotypes involving
Eg_and Egg,

74

   

'00 (MS-l)l33/P . ...... .
II II EEF” ¢y___o
(MS-3) 93/211]. A ..... an

n u a/m A_A

80

 

 

20

22 24 26 23

Time after inoculation (hr)

Figure 20

75

32

Effects of Inoculation on P

Uptake by Detached Leaves

The average radioactivity in noninoculated and inoculated leaf

sections six hours after inoculation with E, graminis was 2.4 X 105 CPM
and 1.8 X 105 CPM, respectively. Twenty-five percent less 32P was

taken up by inoculated plants than by noninoculated plants. This ef-
fect was observed prior to attempted penetration of the plant by the

fungus. Epidermal strips from noninoculated and inoculated leaf sec- ,

tions contained 6.5 x 103 CPM and 3.2 x 103

32

CPM, respectively. There
was 51% less P taken up into the epidermis of inoculated plants than
noninoculated plants.

Puccinia graminis uredospores, Helminthosporium victoriae

 

conidia, Chalk dust, or carborundum heavily dusted onto leaves did
not reduce 32F uptake. Uredospores of E, graminis and E, victoriae
conidia did not germinate under the conditions used. E, victoriae
conidia which were atomized onto the leaves in distilled water germi-

nated but did not reduce the 32

P uptake when compared to leaves
inoculated with distilled water only.

The effect of UV-irradiated spores on 32P uptake was deter-
mined (Figure 21). As the UV dose increased the percentage germina-
tion decreased. Uptake of 32P increased with the decrease in germi-

nation. The uptake of 32P by plants inoculated with conidia that

76

Fig. 21.--The uptake of 32F in one cm leaf sections of
wheat seedlings one to six hours after inoculation and the percent
germination of conidia of Erysiphe graminis f. sp. tritici exposed
to various doses of ultraviolet irradiation.

'32P CPM x10"

3.0

2.5

\
O 10 3O

77

32P uptake by plants

Inoculated

 

Noninoculated ------------

% Germination

 

UV Dose (5)

Figure 21

60

100
90

7O

60
50
40
3O

20
10

O
120 240

% Germination

78

received a 10 second exposure of UV radiation was significantly
different at the 5% level from both the noninoculated and the
inoculated with no UV exposure. The 30, 60, 120, 240 second, and
noninoculated treatments were not different from each other. All
were different at the 5% level from the plants inoculated with
conidia that had no UV treatment. Calculation of germination per-
centage was based on the production of germ tubes by six hours.
The germ tubes produced following 30 second and 60 second UV treat- L

ments were usually malformed.

Transpiration Rates of
Detached Leaves

The average transpiration rates of noninoculated and inocu-
lated seedlings one to six hours after inoculation were 1.25 and
0.93 mg cm"2 hr-l, respectively. This represented a 25% decrease
in the transpiration rate of the inoculated seedlings. The dif-

ference was significant at the 5% level.

Relative Diffusion Resistance

The time required for a standard 10 mm Hg change in pressure
was 48 seconds for noninoculated and 367 seconds for inoculated wheat

leaves at six hours after inoculation. The relative diffusion

79

resistances of stomata were estimated to be 3.6 for noninoculated and
7.1 for inoculated leaves. This amounted to approcimately a 100%

increase for inoculated plants.

Interaction Specificity

The relationship of compatibility between host and parasite
and the reduction in transpiration rate was determined by observing
the rates of transpiration of inoculated and noninoculated near-
isogenic wheat lines which contain different Em_genes (Table 3).
TABLE 3.—-Transpiration rates of intact noninoculated and inoculated

near-isogenic wheat seedlings three and six hours after
inoculation with Erysiphe graminis f. Sp. tritici (MS-1).

 

 

 

Transpiration rate

 

 

(mg cm'2 hr-l)
Noninoculated Inoculated % decrease
from noninoculated
2mg_ 3 hr 1.11 0.85 24
6 hr 1.13 0.76 33
Eml_ 3 hr 1.11 0.90 19
6 hr 1.18 0.73 38
Egg. 3 hr 0.98 0.72 27
6 hr 1.35 0.87 36
Em§_ 3 hr 1.00 0.87 13
6 hr 1.18 0.95 20
Egg. 3 hr 0.90 0.61 32
6 hr 1.26 0.86 32

 

 

80

Intact plants were used to show that no artifacts were being intro-
duced by cutting the leaves. All inoculated lines showed a lower
transpiration rate than noninoculated plants after three hours. The
decrease in transpiration at six hours was comparable to that ob-
served for cut leaves. In all cases, except with Egg, the effect
increased from the third to the sixth hour after inoculation. The
effect of inoculation was the same at three and six hours for Emfi,

The uptake of 32

P by wheat seedlings inoculated with E,
graminis f. sp. hordei was reduced by 25%. This fungus was not

pathogenic on wheat, but did germinate and produce appressoria.

Extracts of Germinated Conidia

Aqueous extracts of germinated conidia did not affect tran—
spiration rates of wheat. Efforts to isolate ABA from germinating

spares or spore filtrates were not successful.

Effects of E, gggmjgjg on
Uptake and Translocation of

32F During,Primary,Infection

32

The amount of P taken up by wheat leaves and translocated

to the epidermis of inoculated and noninoculated leaves during
primary infection was determined (Figure 22). The amount of 32P

taken up by inoculated leaf sections six through 20 hours was

 

81

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82

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33

significantly less than that taken up by noninoculated leaves. At

22, 24, and 26 hours no significant differences existed. At hours

28 and 30 the differences were statistically significant. The 32F
translocated to the epidermis of inoculated plants was significantly
less than in the epidermis of noninoculated plants at the 5% level six
through 20 hours after inoculation. There was no difference at 22

32

hours but at 24 through 30 hours there was more P in the epidermis

of inoculated plants than noninoculated plants. The epidermis of

32F from 25

inoculated and noninoculated plants plants which took up
to 30 hours in 100% RH in the light contained 1,484 CPM and 3,870 CPM,
respectively. Thus, when the transpiration stress was removed, the
inoculated epidermis contained 62% less 32P than did the noninoculated
epidermis. If the plants were pretreated for two hours in 100% RH
before being cut and given 32P from 25 to 30 hours after inoculation
the epidermis of the inoculated plants contained 780 CPM and the non-
inoculated epidermis contained 1,616 CPM, 51% less 32P in inoculated

epidermis. This is approximately the amount of 32P that is taken up

in the dark (Figure 22).

84

Transpiration Rates of Inoculated
and Noninoculated Near-isogenic
Lines of Wheat During
Primary Infection

The average rates of transpiration during primary infection of
inoculated and noninoculated near-isogenic wheat lines were measured
(Figures 23—27). All lines have curves shaped similar to those seen
in Figure 22. Transpiration rates of inoculated plants were lower
through 20 hours. However, after 20 hours, the difference between
inoculated and noninoculated plants was not similar for all lines.
Table 4 gives the average transpiration rates of inoculated and non-
inoculated wheat lines from 22 to 30 hours after inoculation. It
also gives the percent differences between inoculated and noninocu-
lated lines and the P value for those differences. A P value of < .25
was shown for the difference between inoculated and noninoculated
Chancellor, while Em), Emg, and Em§E_inoculated and noninoculated

plants had P values of < .05 for their differences. There was no

difference between inoculated and noninoculated Emfl,

‘3'v.~ ' 4...‘\;.

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86

 

 

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95

TABLE 4.--The average transpiration rate (mg cm"2 hr']) of near-
isogenic wheat lines, the percent decrease, and the P value
for the difference between inoculated and noninoculated
plants 20-30 hours after inoculation with E, graminis f.
sp. tritici.

 

 

Host Genotype

 

(Chancellor)
R_m_x_ m. m2 PI". 32 m2
Noninoculated 1.36 1.03 1.41 1.35 1.06
Inoculated 1.14 1.32 1.08 1.04 1.04
% decrease from
noninoculated 17 32 23 23 2

P value < .25 < .005 < .05 < .05 > .75

 

DISCUSSION

The original goal Of this research was to study the genetic
control of 32P transfer from host to parasite. It was hoped that this
would give us a more sensitive measure of compatibility or incompati-
bility between host and parasite. It would also lay the basis for a
more detailed biochemical study on how compatibility or incompatibility
affects the different metabolic pathways involving phosphorus in both
host and parasite during primary infection.

In preliminary experiments, the environmental conditions
necessary for synchronous development of E, graminis f. sp. tritici
were shown to be quite influential on the uptake of 32F during primary
infection (Figure 6). It appears that light is the major factor. The
rate of 32P uptake by plants in the dark is only about 50% of the rate
of uptake by plants in the light (Figure 5). Earlier work (32) indi-
cated that leaves given 32P for four hours were saturated and addi-
tional uptake time would not greatly increase the amount of 32F in the
leaf. It was argued that if the leaf was already saturated with 32F,

the environment should not affect the amount of 32F in the leaf. The

data presented here (Figure 5) conclusively Shows that light does

96

 

97

32

affect P uptake and saturation does not occur for at least ten hours.

Earlier experiments were done with 100 uCi 32P/mt solutions, but the
50 uCi/ml increase over what was used in this study should have been
negligible because the 32F was in a 0.1 M phosphate buffer in both
studies. The differences between earlier studies and the data pre-
sented herein may be due to procedures used in this study which mini-

mize the possibility of getting air into the vascular system when

leaves were cut at the base and placed in solutions containing 32P.

35 32

If S and P were both transferred from host to parasite by

simple diffusion one would expect similar rates of transfer of each

isotope. However there are some important differences between the

32 35

data reported in this thesis for transfer of P and the data on S

transfer for a compatible parasite/host interaction (50). The earliest

32P was detected in the parasite was ten hours after inoculation, which

corresponds to the time haustoria can first be seen in the host cells

35

(32). S was first detected in the parasite at 16 hours, which is the

time that.haustoria begin to develop appendages (Personal communication
from Mary Joy Haywood). This may indicate that 32F simply diffuses
into the parasite as soon as the parasite penetrates the host cell
wall, while 35S may require a more complex transport system. The
possibility exists that the transport systems for 32P are merely func-

35 32 35

S. P and S transfer were very

tioning earlier than those for

98

similar from 18 hours onward. However the amount of 32F transferred

35

was much higher than the amount of S transferred. This could be a

function of the ability of 32

355.

P to move through the leaf faster than

When corrections are applied for the amount of 32F available
to be transferred we find that the highest rate of transfer occurs
from 20 to 22 hours after inoculation. Similar results were obtained

35 32P and 35

for S transfer (50). The rates of 5 transfer dropped off
after 22 hours. The validity of correcting transfer rates for the
amount of 32P available is supported by the fact that, when the lights
were left off from 20 to 30 hours, the same amount of 32P was available
for transfer after 20 hours as before, the rates of transfer in the
dark did not increase after 20 hours after inoculation. The dark
period from 20 to 30 hours does slow down development of ESH but only
for two hours (50). Thus slower rates of ESH development would not
explain the decreased transfer in the dark.

32

The amount of P transferred from host to parasite was found

to correspond to morphological development of the fungus with the
various genotypes. The reduction of the percentage of ESH for the
incompatible interactions (47), E1/Em1, EE/sz, P3b/Pm3b, and Efi/Emg,
was similar to the reduction in 32F transfer, however, the decreases

32

in P transfer were later than those observed for 355 transfer.

99

Decreases in 32P transfer were not seen until.after morphological

355 transfer was affected concurrent with or

differences were noted.
prior to effects of the different incompatible genotypes on morpho-
logical development of the fungus (50).

Results from the comparison of 32F transfer from host lines
with the allelic series of genes at the Em§_locus failed to differen-

tiate between Pm3a and Pm3b. If these genes are truely allelic, then i

one would expect them to affect parasite development in the same way.

“LC . h' :1”:

Pm3c had similar kinetics of transfer as 9mg, This was expected since
Pm3c does not have an apparent effect in the primary leaf (5, 6, 41).
If only functional parasite units transfer 32P, then the amount”

of 32

P transferred by the functional units in incompatible interactions
is greater than the rates per parasite unit in compatible interactions.
With the exception of 394, the rates of 32P transfer in incompatible
interactions eventually decreased to the same level as in the compat-

ible interactions. Rates of 32

P transfer from plants with Emg_were
higher at 30 hours, but they were decreasing and it is predicted that
the rates may eventually reach the same as in the compatible inter-
actions. An alternate explanation is of course possible. The non-
functional parasite units could be taking up 32P until 30 hours after
inoculation, at which time the process is halted. It is also possible,

especially with Eg_and Egg, that after collapse of the parasite, which

lOO

occurs approximately 22 hours after inoculation, that there was dif-

32P into the collapsed parasite. By 30 hours after

fusive flow of
inoculation this "dead" parasite may be such that 32F did not diffuse
into it or the necrotic host cell which the parasite had attempted to
penetrate.

The four possible parasite/host genotypes (the quadratic check)

' .. In". ...(Idunllll.

for each pair of interacting genes were evaluated for their effect on

 

rates of 32P transfer from host to parasite during primary infection.
The incompatible parasite/host genotype El/Eml_gave low rates Of 32P
transfer. The three compatible genotypes Eljpml, pl/Eml, and pl/pml,
all gave similar, high rates of transfer. The incompatible genotype
EEE/Em§2_gave low rates of transfer while the three compatible geno-
types P3b/pm3b, pSD/Pm3b, and Egg/Emgg, gave similar, high rates of
32P transfer from host to parasite during primary infection. The three
different compatible genotypes for each gene pair could not be dis-
tinguished on the basis of rates of 32F transfer.

There were no differences in the rates of 32P transfer for the
four genotypes EE/Emg, EE/ m2, pE/Emg, and pE/pmg during primary in-
fection. This was consistent with earlier observations that EE/Emg_
does not affect primary infection (47).

The genotype EE/Em&_gave a low final infection type, low rates

32

of P transfer from host to parasite during primary infection, and a

101

low primary infection efficiency. In this respect it was similar to
the El/Eml and EEQ/Em§9_interactions. The culture MS-Z, which by
definition has pfi_because it gives a high infection type seven days
after inoculation on the host line with Egg, gave a primary infection
efficiency similar to that observed on the host line with Emg_but the
formation of elongating secondary hyphae was delayed (Figure 16). The
rates of transfer of 32P from host to parasite during primary infec-
tion were also lower with this genotype, i.e., pfl/Emfl, than with the
other two compatible genotypes, Eg/pm&_and 95/239, If culture MS-Z
just had a gene for slower growth it should have developed more slowly
on the host line with pmg_also, but it did not. Therefore, the slower
development of MS-2 on host lines with Emg_is very probably related to
the presence of Egg,

When other cultures containing pg_were substituted into the
quadratic check, one was found to reduce infection efficiency to 20%
and a second to 33% with the pg/Emg_genotype. The pfi/pmfi_genotype
gave normal infection efficiency in both cases. These results are
important because they bear on where the specificity of gene-for-gene
interactions occur. They indicate that a unique interaction occurs
with the pfi/Emg_genotype. The simplest explanation of where the
specificity of gene-for-gene interactions resides is in the incom-

patible interaction (EE/Emg). The data indicate that the transition

‘23:, .. I1 .
l

102

in evolution from Efi_to pg_in the parasite did not completely negate
the transition in evolution from pmg_to Emg_in the host. There are
no reasons we should always expect complete restoration of compati-
bility since the pfi/Emg_genotype only has to have a selective advan-
tage for the parasite over the incompatible genotype EE/Emfl, The 94,
product is probably an altered product of E5 with an altered speci-
ficity for the Em4_product.

The results from these efforts to complete the quadratic checks
for several gene pairs also demonstrate how the gene-for-gene hypoth-
esis may play a role in nonspecific or general resistance. The first
pfl_isolate used was found to give a slow rate of ESH development in
combination with Egg, A high infection efficiency was reached but it
was delayed two hours. With the second two isolates with 94, a reduced
infection efficiency was obtained on host lines with Egg, These two
characteristics, i.e., slow mildew development and the development of
fewer normal pustules, have always been associated with "general"
resistance. This means that "general" resistance may result from
the accumulation of pE/EE_interactions. Techniques with high resolu-
tion are needed to detect the differences between the compatible
genotypes.

All pathogens which carried some form of pE_have probably been

selected and eventually the parasites with EE_were lost from the

103

population. The low frequency, or loss, of EE_in a population would
explain the difficulty to demonstrate the specific interactions that
. are probably occurring with "general" resistance. Manchurian barley
which was previously thought to contain no (E) genes for resistance
to powdery mildew was shown to possess (E) genes when inoculated with.
isolates from the regions of origin of barley (17). This shows that
if the avirulent (E) pathogen is available, the (E) genes could be
detected and selected. Varieties of potatoes introduced from Europe
in 1833 and maintained in the mountains of Basutoland in Africa under
almost no selection pressure from late blight were found to be much
more susceptible to blight than the susceptible varieties still grown
in Europe (52). This could be explained by the accumulation of 257E
interactions between Phytophthora igfestans and the European varieties
which have been under selection through the years. The mountain-grown
varieties would not be expected to possess these particular (E) genes
since they were never under selection pressure. The (E) gene in the
European varieties could be identified if the avirulent (E) pathogens
were still available.

If this hypothesis is true, then it should be possible to
identify a few of the known (E) genes which are not completely negated
by its corresponding (2) gene, similar to what I have done here. These

interactions should also be shown to have an additive effect. Then

i-

104

with the use of avirulent cultures, which would be readily available,
it should be possible to produce new sources of "general” resistance
from old sources of specific resistance. This would involve the use
of the same principles presently used in breeding for specific resis-
tance and would eliminate the hit and miss techniques presently used
to transfer "general" resistance from known sources to new varieties.

32F taken up by inoculated plants

The reduction in amounts of
one to six hours after incoulation showed that a pathogen could inter-
act with its host before the pathogen penetrated. Since chalk dust,
carborundum, E, graminis uredospores, and E, victoriae conidia did
not reduce the amount of 32F taken up by the leaves, the reduction in
label uptake was probably not due to the physical presence of par—
ticles on the surface of the leaves. As UV radiation decreased the

32

viability of E, graminis conidia there was an increase in the P

2P increased to the level of noninoculated

uptake. The uptake of 3
plants with 12% germination. However the 12% which germinated with
the 60 second dose of UV radiation had malformed germ tubes. Mount
and Ellingboe (33) reported lower UV doses given to E, graminis
conidia resulted in the formation of no elongating secondary hyphae.
Thus, the germinated conidia at the 60 second dose would probably

not have penetrated the host. We can clearly see that germinating

conidia on the surface of the leaf were responsible for the reduced

105

amount of label taken up by the leaves one to six hours after inocula—
tion. The decreased uptake of 32P by inoculated plants can be ex-
plained by the decreased transpiration rates. The reduced transpira-
tion rates can be attributed to the closure of stonates as determined
from measurements with a viscous flow porometer.

The transpiration rates of inoculated and noninoculated near-
isogenic wheat lines revealed lower transpiration rates by three hours
after inoculation in incompatible as well as compatible parasite/host
combinations. Inoculation of wheat with conidia of E; graminis f. sp.

32P taken up by the leaves. This

Egrg§j_also reduced the amount of
form-species on wheat represented an incompatible parasite/host inter-
action. The spores did germinate and form appressoria on wheat. Thus
this effect is not specific for the compatible or incompatible para-
Site/host genotypes.

Decreased transpiration rates have been reported for barley
infected with E, graminis f. sp. Eggggj_one to seven days after inocu-
lation (26). Decreased transpiration rates of bean two days after
infection by Uromyces phaseoli have also been reported (12). Our data
Show a decrease in wheat transpiration as early as three hours after
inoculation and before fungus penetration of the host, which is much

earlier than previously reported (26). The stomates of wheat were

shown to close upon attack by the germ tubes of leaf rust uredospores

106

which penetrate the host through the stomates (9). The data reported
here indicate that the stomates are responding to some stimulis(i)
produced by the germinating conidia of E, graminis before there was
direct penetration of host cells. The possibility may exist that, in
the previous study (9), many of the stomates may have been closing in
the near vicinity of the germinating uredospores and not just those
stomates being attacked by the pathogen.

Stomate closure induced by E, graminis may have been initiated
by diffusable substances produced by the germinating conidia, but
abscisic acid was not demonstrated to be present in germinating con-
idia. None of the extracts made of germinating conidia were shown
to reduce transpiration.

Inoculated plants were shown to have reduced transpiration
rates throughout the primary infection process. Even though inocu-
lated leaves transpired less, and took up less 32P, the epidermis

of inoculated plants contained more 32

P than did the epidermis of
noninoculated plants by 26 hours after inoculation. This could be
the result of a nutrient sink effect created by the developing fungus.
A sink effect has been reported during later stages of mildew devel-
opment (44). When transpiration stress was removed from the leaves

by placing them in 100% RH while taking up 32F from 25 to 30 hours

after inoculation, the epidermis of inoculated leaves contained

107

32 32

less P. The increase in P activity in the epidermis was probably

due to increased cuticular transpiration around the areas of penetra-

32P at the

tion. This would have the effect of concentrating the
epidermis. Transpiration effects have been overlooked in most reports
of nutrient sinks created by developing parasites. Transpiration
changes during primary infection of wheat have explained all the dif-
ferences in 32P uptake between inoculated and noninoculated plants.
The incompatibility of an interaction seemed to have an effect
on transpiration rates, especially at times when incompatibility is
being expressed. Transpiration rates of inoculated and noninoculated
me_plants are not significantly different from 22 to 30 hours. This
is probably due to increased cuticular transpiration due to fungal
development. With El/Eml_and EEE/EmEE, transpiration differences be-
tween inoculated and noninoculated plants were highly significant from
22 to 30 hours. This could be due to inhibition of fungal develop-
ment. Transpiration rates of noninoculated and inoculated Emg_plants
with a culture of EE_were identical from 22 to 30 hours. This might
be explained by the nature of the Eg/Emfl‘incompatible interaction. At
approximately 22 hours many of the parasite units collapse concomitant
with the browning of the host cell in which it had penetrated. The

possibility exists that the dead necrotic cell allows more water loss

or causes more disruption of the cuticle than the development of a

108

compatible interaction. Thus the transpiration rates of inoculated
and noninoculated plants would be expected to be similar.

The ability of a pathogen and its host to interact before
penetration of the host by the pathogen has clearly been demonstrated.
These results emphasize the need for more investigations into the
very earliest detectable interactions between parasite and host to
determine those interactions that are critical to the establishment

of compatible or incompatible parasite/host relationships.

 

 

SUMMARY

The environmental conditions necessary for synchronous

development of the parasite during primary infection were shown to

affect the amount of 32P taken up by the leaves and translocated

32

to the epidermis. The lights increased the amount of P in the

leaf and the amount transferred to the parasite.
32P transfer rates reflected the relative compatibilities

of the interactions studied. Interactions with little fungal devel-

Opment had lower rates of transfer, while fully compatible interac-

32P transfer did not

tions had high rates of transfer. Rates of
show differences between compatible and incompatible interactions
as early as did 355 transfer rates (50) or production of elongating

32

secondary hyphae (47). Rates of P transfer were observed earlier

35S transfer and the amount of 32F transferred was much higher.

than
The four possible parasite/host genotypes (the quadratic

check) for each interacting gene pair were evaluated for their effect

on final infection type seven days after inoculation, on 32P transfer

from host to parasite, and on the formation of elongating secondary

hyphae (ESH). With all gene pairs the final infection type of the

109

110

three compatible interactions were similar. Only the incompatible
interaction (EE/me) resulted in a low infection type. The compatible
interactions involving E1_and E91, EE_and Egg, E§E_and Pm3b had sim-

32 32

ilar rates of P transfer were not sim-

P transfer. The rates of
ilar for the compatible interactions involving Efl_and Egg, The pE/Emg_
genotype had a reduced rate of transfer when compared to gg/pmfl_and.
EE/pmfl, The production of ESH was also delayed with pE/Emfl, Two
other isolates containing pfi_resulted in a reduction in the percent

of parasites that produced ESH. Thus the mutations from Eg_to 91_in
the parasite have not completely negated the change in the host from
pmg_to Egg, This indicates that specificity of gene-for-gene inter-
actions is for incompatibility, not compatibility. It also demon-
strates how the gene-for-gene hypothesis might play a role in general
or horizontal resistance.

Germinating conidia of E, graminis were shown to reduce host
transpiration rates before penetration occurred. The transpiration
reduction was a result of stomate closure induced by the germinating
conidia. Extracts of germinating conidia did not reduce transpiration
rates and abscisic acid was not detected in the conidia. The reduc-
tion in transpiration continued through primary infection and was

32

shown to be responsible for less P uptake by inoculated plants than

noninoculated plants during primary infection.

 

111

Metabolic sinks induced by E, graminis, which concentrated

32P at infection sites in the epidermis of inoculated plants during

32

primary infection, were not demonstrated. Any accumulation of P

in the epidermis was due to increased cuticular transpiration caused

by disruption of the cuticle by the developing fungus.

 

 

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