Comparative virulence of Pyrenophora graminea Ito et Kurib isolates and the inheritance of resistance
to P. graminea in barley
by Richard Lee Ruff
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant
Pathology
Montana State University
© Copyright by Richard Lee Ruff (1984)
Abstract:
Studies were continued to identify genes for resistance in barley to barley leaf stripe disease (causal
organism : Pyrenophora graminea Ito et Kurib.). Because of the variability of infection, inoculation
techniques were studied. Eight media were developed to compare substrate effects upon infection of a
barley cultivar's seedlings by a P. graminea isolate. Although infection mean differences of the cultures
were not significant, cultures grown on diffusate from germinated barley seed had slightly increased
infectivity.
Virulence of 24 P. graminea isolates was tested to determine the spectrum of resistance in three barley
cultivars. Artificial inoculation of seeds was by the layered mycelium method. Both of the highly
susceptible cultivars, 'Lami' and 'Summit', were found to have a low level of resistance to some of these
isolates. 'Betzes' seeds inoculated with 18 of the isolates resulted in plants without symptoms.
However, up to 16% symptomatic seedlings were found after inoculation with six other isolates.
Specific resistance in these cultivars to the isolates was inferred.
The Turkish cultivars, Tokak and Yesilkoy, were each reciprocally crossed to Betzes to study the
inheritance of resistance in barley to leaf stripe disease. Tokak and Betzes are highly resistant to
infection, and Yesilkoy has a low level of resistance. Seeds of the crosses were produced through the
F3 and BC F2 generations and inoculated by the layered mycelium method. Seedlings were grown for
six weeks and divided into a class with disease symptoms or a class without symptoms.
Seedlings' reactions from the F1 and F2 generations of the Betzes X Tokak crosses fit models
indicating that a single dominant gene was inherited from each parent. The lack of fit of the data in the
BC F2 families indicated a modification of the dominant genes models. Seedlings' reactions indicated
that a recessive gene was inherited from Yesilkoy. Qualitative gene action with multiple genes or
quantitative gene action was inferred from the results of seed inoculations. A method to calculate the
effects of penetrance and expressivity on expected gene ratios was discussed. COMPARATIVE VIRULENCE OF PYRENOPHORA GRAMINEA
ITO ET KURIB ISOLATES AND THE INHERITANCE OF
RESISTANCE TO JL4. GRAMINEA IN BARLEY.
by
Richard Lee Ruff
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Plant Pathology
MONTANA STATE UNIVERSITY
Bozeman, Montana
December 1984
APPROVAL
of a thesis submitted by
Richard Lee Ruff
This thesis has been read by each m e m b e r of the
thesis c o mm it tee and has been found to be satisfactory
regarding content, English usage, format, citations,
bibliographic style, and consistency, and is ready for
submission to the College of Graduate Studies.
Date
Chairperson, Graduate Committee
Approved for the Major Department
Date
Approved for the College of Graduate Studies
Date
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of
the requi re me nts for a m a s t e r ’s degree at Montana State
University,
I agree
that
the
Library
shall
make
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Brief
quotations
special
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permission,
thesis are allowable
provided
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acknowledgement of source is made.
Permission
for
extensive
quotation
from
or
reproducti on of this thesis may be granted by my major
professor,
or
in
his
absence,
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Director
of
Li brari es when, in the opinion of either, the proposed
use
of
copying
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or
mat erial
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the
for
scholarly
material
in
purposes.
this
thesis
Any
for
financial gain shall not be all owe d without my written'
permission.
.
V
ACKNOWLEDGMENTS
I express sincere appreciation and gratitude
following
to the
persons and agencies (not in any particular
order) :
Dr. A. L . Scharen, for his continual encouragement,
confidence,
and personal
concern
as well
as material
facilities;
Dr's.
E . H . Hockett,
J . H . Riesselman,
and
Sharp for their assistance and advice during my
E. L .
graduate
studies;
the
st a f f
De partm ent
abbetment,
my
of
and
Plant
fellow
graduate
Pathology
for
students
their
of
the
assistance,
and perseverance;
mother
and
father,
family,
and
friends
emotional, moral, and physical support;
the Ground of my being for life;
US-AID and the U SN for financial support.
for
vi
TABLE OF CONTENTS
Page
APPROVAL
................ ............... ......... .....
ii
STATEMENT OF PERMISSION TO USE ..................... .
ill
VITA
....... ^ ......... ....... . . .'............. .
ACKNOWLEDGMENTS ..... ........................ ...... .
TABLE OF CONTENTS ...... ..................... .
LIST OF TABLES ....... ............................. .
ABSTRACT ................................ ..............
iv
v
vi
viii
xi
CHAPTER
I; INTRODUCTION ..................
I
CHAPTER
2: MEDIA EFFECTS ON INFECTION STABILITY OF
PYRENOPHORA GRAMINEA ISOLATES WHEN
ARTIFICIALLY CULTURED ................
Introduction ........................
Materials and Methods ...............
Results and Discussion ........... ......
8
8
10
13
3: VIRULENCE OF PYRENOPHORA GRAMINEA
ISOLATES ON SPECIFIC BARLEY CULTIVARS.
I n t r o d u c t i o n .................
Materials and Methods ........
Results and Discuss ion .......
19
19
21
24
4: INHERITANCE OF RESISTANCE IN THREE
BARLEY CULTIVARS TO TWO PYRENOPHORA
GRAMINEA ISOLATES. ...................
Introduction............... ........ .
Materials and Methods .........
Results ...................... ...........
Betzes - Tokak Crosses ...............
Betzes - Yesilkoy Crosses ............
Discussion ................
31
3I
33
35
37
47
57
CHAPTER
CHAPTER
. vii
TABLE OF CONTEMTS-Continued
Page
CHAPTER 5:
SUMMARY ...... . ..........................
65
REFERENCES C I T E D .... . . . ...................... ......
68
APPENDIX ................... ...... .............. .
74
viii
LIST OF TABLES
Tables
1.
2.
3•
4.
5.
6.
7.
J
Page
Percent of barley seedlings infected as a
r e s u l t of i n o c u l a t i o n w i t h c u l t u r e s of
Pvr eno pho ra gra minea isolate R PB grow n on
different media ...... ....... .
Iif
Barley plants
infected
by P v r e n o p h o r a
gra mi ne a as a
percentage of
total plants
emerged (average of three replications) .....
25
Barley plants
infected
by P v r e n o o h o r a
graminea as a
percentage of
total plants
emerged (average of three replications)
26
Barley plants
infected
by P v r e n o o h o r a
■finuajm-i.ne a as a
percentage of
total plants
emerged (average of three replications) .....
27
Number of barley seedlings derived from check
cultivars,
par en ts, and p r o g e n y
of tw o
crosses with and without s ymp to ms after
inoculation with two isolates of Pvrenophora
graminea ................... ..................
38
N u m b e r of s p e c i f i c p a r e n t and F g b a r l e y
seedlings with and without s y mpt om s after
inoculation" with Pvrenoohora graminea isolate
MT 6 and C h i - s q u a r e (X ^ ) values when fit to a
15:1 r a t i o .........................
41
Number of specific parent and F 2 barley seed­
lings with and without symptoms after inocu­
lation with Pvrenonhora graminea isolate Kaya
and C h i - s q u a r e ( X ^ ) values when fit to a 15:1
ratio .........................................
43
ix
LIST OF TABLES-Continued
Tables
8.
9.
10.
11.
12.
13 .
14.
15.
16.
Page
N u m b e r of b a r l e y s e e d l i n g s f r o m b u l k e d
parents and backcrossed F ^ ) seeds with and
without symptoms after inoculation with two
Pvre noohora gra minea isolates ............
45
Number of backcrossed Fg families resistant
and segregating after inoculation with two
Pvrenophora graminea isolates ...............
46
Number of barley seedlings derived from check
cultivars,
par ents, and p r o g e n y of two
crosses with and without sym pt om s after
inoculation with two isolates of Pvrenophora
graminea .......... .................... ....... .
48
Num be r of specific parent and Fg barley
seedlings with and wit hou t s y mpt om s after
ino culation w it h two Pvrenophora gra mi ne a
isolates and Chi-square values when fit to a
13:3 ratio ...................................
52
N u m b e r of b a r l e y s e e d l i n g s fr o m c h e c k
cultivars, bulked parents, and backcrossed
F(.j) seeds with and with out sym pt om s after
ino culation with two Pvrenophora gra mi ne a
i s o l a t e s ............................
55
Number of backcrossed Fg families resistant
and segregating after inoculation with two
Pvrenophora graminea isolates ............
57
Potato dextrose agar plus diffusate of germ­
ina te d, b a r l e y s e e d ......................
75
Potato dextrose agar plus extract of ground,
germinated,
barley seed ... .. ........ .
76
T w o - f a c t o r i a l a n a l y s i s of v a r i a n c e of
cultured isolate experiments and comparison
of
culture means
... ...................
77
X
LIST OF TABLES-Continued
Tables
17 •
18.
19 .
20.
Page
Co mpa ris on of e m e r g e n c e p e r c e n t a g e s of
barley seedlings from the parent and check
c u l t i v a r s ..... .
t
.
78
A n a l y s i s of c o v a r i a n c e w i t h e m e r g e n c e
percentages as the covariable and infection
percentages as the dependent variable of
seedlings
from
the
parent
and
check
cultivars .......... .
78
Number of Fo f amilies resistant and segrega­
ting after inoculation with two Pvrenophora
graminea isolates (30-80 seedlings were read
in each family) ................ .......... . .
79
Calculations of genetic ratios with the
effects of penetrance and expressivity ....
81
xi
ABSTRACT
Studies were continued to identify genes for resis­
tance in barley to barley leaf stripe disease (causal
organism : Pvrenophora g r amine a Ito et Kurib.). Because
of the variability of infection, inoculation techniques
were studied.
Eight media were developed to compare
substrate effects upon infection of a barley cultivar's
seedlings by a P. gra min ea isolate.
Although infection
mean differences of the cultures were not significant,
cultures grown on diffusate from germinated barley seed
had slightly increased infectivity.
r
Virulence of 24 JLt gra minea isolates was tested to
determine the spectru m of resistance in three barley
cultivars.
Artificial ino culation of seeds was by the
layered mycelium method.
Both of the highly susceptible
cultivars, 'L a m i 1 and 'Summit', were found to have a low
level of resistance to some of these isolates. lBetzes'
seeds inoculated with 18 of the isolates resulted in
plants without symptoms.
However, up to 16/6 symptomatic
seedlings were found after ino culation with six other
isolates.
Specific resistance in these cultivars to the
isolates was inferred.
The Turkish cultivars, Tokak and Yesilkoy, were each
reciprocally crossed to Betzes to study the inheritance
of resistance in barley to leaf stripe disease.
Tokak
and B e t z e s are h i g h l y r e s i s t a n t to i n f e c t i o n , and
Yesilkoy has a low level of resistance.
Seeds of the
crosses were produced through the Fg and BC F g gener a­
tions and inoculated by the layered myc e l i u m method.
Seedlings were grown for six wee ks and divided into a
class with disease symptoms or a class without symptoms.
Seedlings' reactions from the F^ and Fg generations
of the Betzes X Tokak crosses fit models indicating that
a single dominant gene was inherited from each parent.
The la c k of fit of the data in the BC Fg f a m i l i e s
indicated a m o d i f i c a t i o n of the dominant genes models.
Seedlings' reactions indicated that a recessive gene was
inherited from Yesilkoy.
Qualitative gene action with
multiple genes or quantitative gene action was inferred
from the results of seed inoculations.
A meth od to
calculate the effects of penetrance and expressivity on
expected gene ratios was discussed.
I
CHAPTER I
INTRODUCTION
Barley
systemic
leaf
in
barley,
Pvrenonhora
organism.
(Rab.
g ra mi ne um
Rab.
potentially
Ito
ana mor ph
Schlect.)
ex
disease
Hordeum
graminea
The
ex
stripe
is
vulsare
et
stage
is
Schlect.
Barley
thoughout the world.
in
The
is
causal
graminea
Helminthosnorium
leaf
stripe
barley
is
a
growing areas
Early in this century,
was one of the most significant,
and
fungus,
the
Drechslera
synonym
disease
L.
K u r i b.
Shoem.,
threatening
seed-borne
leaf stripe
yield reducing diseases
-
of barley.
mercury
In the
seed
1930's,
the introduction of organic
treatments
importance of leaf stripe.
use
of
these
effects
in
seed
decreased
has
commercial
Since the 1 970's restricted
treatments,
nature,
the
led
due
to
to their long term
increased
disease
incidence.
The
Pathogen.
Ito
(1930)
graminea as the teleomorph
on
barley
in
Japan.
The
stage
described
Pyrenophora
of Drechslera
description
was
graminea
b a sed
on
2
cultural
fungus.
characters
and
on
the
Alexopoulos and Mims
the family Pleosporaceae,
pathogenicity
(1979)
of
the
placed the genus in
subclass Loculoascomycetidae.
Pseudothecia of jL_ graminea are rarely reported in the
literature.
Smedegaard-Petersen
(1973)
s cle r o t i u m - Iike bodies on barley straw
leaf
stripe infected fields.
described
collected from
The sclerotium-like
bodies
were superfici all y su bme rged and elongated with dark,
rigid setae on the surface.
seldom found,
Using
Asci and ascopsores
were
and no physical characteristics were given.
inoculum
from
ascospore
cultures,
typical
leaf
stripe symptoms apparently developed on plants grown from
inoculated seed (Smedegaard-Petersen,
Shoemaker
characteristics
(1962)
1973)*
described
of the anamorph,
the
conidial
D. graminea.
were light green to medium yellowish brown.
Cells within
the conidia were shorter in length than width.
measured 55-85 X 16-20 microns.
regularly
formed
(Shoemaker,
1 962).
Historically,
Bioolaris
were
in
on
the
the
both
form
form
Secondary
apical
and
genera
genus
Conidia
Conidia
conidiophores
bas al
cells
Drechslera
and
Helminthosporium.
Drechslera and Bioolaris form species were later removed
from the H e l m i n t h o s p o r i u m form genus.
These two form
3
genera were differentiated from each other essentially by
two characters,
conidium shape and germination
pattern.
Alcorn (1983) questioned the validity of separating the
two
form
genera
reported
wide
by
the
g erm in ati on
variation
germinated in each
in the
conidium.
of
number
conidia.
of
Al corn (1 983)
He
cells which
proposed
the
point of origin of the germ tube from the basal cell and
the germ tube's direction of growth in relation to the
long
axis
of
the
characteristics.
conidium
as
distinguishable
graminea produces germ tubes growing
laterally from the basal cell of the conidium.
The H o s t .
of
barley
grew
Agriculture,
Research suggests that the
in
wes te rn
Asia
(U . S.
progenitors
Depa rtmen t
of
1979).
The world center for diversity in
barley is Ethiopia.
Barley, H . v u l a a r e . belongs to the
grass
family,
barley
is
Gramineae,
diploid
with
tribe
a basic
Hordeae.
chr om osom e
Cultivated
number
of
seven.
The
barley kernel consists of the caryopsis,
pale a , and
pericarp,
rachilla.
integuments,
kernel germinates,
emer ge s through
The
caryopsis
endosperm,
and embryo..
the embryonic root,
the coleorhiza.
shoot, or e picotyI, emerges.
consists
or radicle,
Later,
lemma,
of
the
As the '
first
the primordial
If
Barley grows best in well-drained,
loam
s o ils
with
tolerate
flooded
ripening
season
a pH
of
soils.
is
at
least
6.0.
B a r l e y/ grows
long
and
loam and clay-
cool,
It
best
and
does
not
where
the
rainfall
is
moderate. . In the lower latitudes or the temperate zones,
barley
is
drought,
often
grown
during
the
cool
season.
Where
summer frost, or alkaline soils are encountered,
barley is one of m a n ’s most dependable
cereal
crops.
In
recent years an average of 9 1 m i lli on hectares per year
have
been
devoted
to
barley
production
(U . S . Department of Agriculture,
The
become
Ho st-P ath oge n
infected
development.
with
Relationship.
the leaf
stripe
Mathre, I 9 82).
Barley
during
Platenkamp
in the pericarp of ung erminated
She described infection of the coleorhiza as
the kernel germinates.
From the coleorhiza,
grew
meristem.
into
(1 9 7 6 a),
kernels
organism
The embryo is not infected.
( 1 9 7 6 ) found m y c e l i u m
kernels.
1 979;
worldwide
the
using
apical
naturally
infected
the mycelium
Teviotdale
seed,
found
and
Hall
mycelium
in the seedling apex after 21 days of germination at 6 C.
Platenkamp
stems of
at 20 C.
(1976) found
12 -day-old
hyphae
plants
though.out the leaves and
naturally
infected
and
grown
5
Once the mycelium invades the culm, rapid elongation
of
the
internodes
infection
tissues.
loci
breaks
are
the
h y pha l
established
s t ran ds ,
in the elevated
The stem apex is invaded just before
and
plant
the spike
e m er ge s from the boot (Skoropad and A r n y , I 956).
high, h u m i d i t y
at
the
ti m e
of
hea d i n g ,
produced on leaves of infected plants.
windblown
to
nearby
he a d s
where
With
conidia
are
These conidia are
infection
of
the
developing kernels occurs.
Low soil tem per at ur es (6-14 C) during ger min at io n
and
early
growth
development.
higher
of
Prasad,
disease
seedlings
Leonard,
incidence
enhance
disease
and Murphy (1976) found
with
an
int ermed iate
soil
moist ure (-7 . 1 bars) compared with a wet soil (- 1 . 0 b a r )
or
a dry
soil
(-12.9
bars).
Irrigation
near
heading
enhanced kernel infection in the field (Metz and Scharen,
1 979).
Disease symptoms may appear soon after emergence of
seedlings,
but they are often not evident until six weeks
post-planting.
One
or
more
long,
chlorotic
stripes
develop parallel to the leaf ribs, often exte nding from
the
base
stripes
necrotic.
to
turn
the
tip
brown
The leaf
of
or
the
grey
blade
may
leaf.
as
the
Later
the
tissue
yellow
becomes
split and have a frayed
6
appearance.
retarded,
Growth
of
infected
seedlings
is
often
and plant death may occur at an early stage.
In the field, heavy attacks arrested spike emergence,
and
barren spikes or imp ro pe rly developed kernels resulted
(Smedegaard-Petersen,
197.6).
Yield reductions of 0.6% to
1.0% for each percent of leaf stripe infected
plants are
reported (Richardson, Whittle., and Jacks, 1 976; T e k a u z ,
1983).
Early
attempt s
to
control
leaf
stripe
cultural practices and seed treatments.
seed soak was an early control method.
involved
The hot water
With the advent
'
of
organic
merc ury
seed
effectively controlled.
available
for
Riess el ma n
seed
(1982)
treatments,
the
disease
was
Organic mercury is not currently
treatments.
found
Johnston,
Metz,
and
pro mi sing results in tests of
leaf stripe control with systemic chemicals.
The use of
these chemicals in many areas of continued leaf stripe
disease occurrence, for example North Africa and the Near
East,
is not practical due to infrastructure
Control
of
leaf, s t r i p e
disease
by
problems.
increasing
host
resistance is a viable alternative to chemical control.
This
study
r:,
was
initiated
to
determine
available
'
genes in barley for resistance to leaf stripe disease.
preliminar y
objective
was
to
improve
the
A
artificial
7
inoculation
techniques
barley kernels.
chapter one.
variation
several
of
the
disease
organism
onto
This prelim ina ry study is discussed in
In chapter two, data are presented on the
in virulence
sources.
Any
of
grami ne a isolates
attempts
to
develop
from
resistant
barley cultivars will have to take this variation into
acco un t.
In
the
th i r d
experiments
are
discussed
Chapter
on the
for resistance to the disease.
of
this
thesis,
determination
of genes
Two reciprocal crosses
were made,
and progeny were tested for resistance in the
backcross
(BC)
inoculation
and
to
tests indicated
Fg
generations.
dominant
and
Results
recessive
of
gene
action; however, these effects were not confirmed in BC
F 2 and Fg generations of crossed material.
8
CHAPTER 2
MEDIA EFFECTS ON INFECTION STABILITY OF PYRENOPHORA
G RAMINEA ISOLATES WHEN ARTIFICIALLY CULTURED
Introduction^
One
problem
with
artificial
inoculation
grami nea is the variability in infection.
does
not
readily
sporulate
in
researchers report various methods
of
the
Oswald,
Singh,
fungus
in
culture
(Paxton,
1922;
P.
P^_ graminea
culture,
to induce
of
although
sporulation
Hou sto n
and
1 9 46 ; Teviotdale and H a l l , I 976b; Sengupta and
1979;
Even with
Tekauz
these
and
methods,
C h i ko,
1980;
conidia
are
Grbavac,
not
1981).
produced
enough abundance for use as an inoculum source.
in
In the
literature (Ar ny and S h a n d s , 1 9 4 2 ; Houston and Oswald,
1 948;
Nilsson,
Jorgensen,
or
1 975;
Metz,
1982; Konak,
without
predominant
1 97 8 ;' Smedegaard-Petersen and
1983; Tekauz,
c o n i d i o sp ores,
are
inoculu m
grown
either
substrate or on artificial media.
1983) mycelia,
reported
to
on a cereal
be
with
the
kernel
9
Shands
and
hyphal-tip
Dickson
cultures
(193*0
of
reported
graminea.
variati on
they
in
reported
differences in culture morphology on PDA (potato dextrose
agar) , in
pathogenicity
tests,
and
in
symptoms
when
single spore cultures were transferred by hyphal-tips.
Shands and Arny (1944) and Arny (1945) used hyphal-tip
cultures
of £*. grami nea
artificial
were
media
stable
for
that
over
and virulent
had
twelve
been
years.
on a large
mai nt aine d
These
number
on
cultures
of barley
varieties.
Nilsson
(1975)
compared
the
virulence
of
newly
isolated P. graminea cultures and old cultures grown on
PDA.
Virulence of the stored cultures was determined
after
two and nine months.
cultures
lost
little
Nilsson
virulence,
showed
that
others
completely
while
some
lost the ability to induce disease sym pt om s on plants.
Metz
and Scharen (1979) identified barley cultivars with
various reactions
cultivars
varied
replications.
to different JiLt. graminea isolates.
in
They
their
disease
proposed
reaction
genetic
The
among
factors,
environmental conditions, or the inoculation technique as
causes for the variations in reaction.
Konak (1983) reported a dra ma ti c loss in virulence
(from
9 5 % to
2 % seedling
infection)
of
specific
graminea isolates inoculated to a single barley cultivar.
J.G.N.
Davidson
pure
(personal
isolates
communication)
of ILl graminea
found
impossible.
culturing
Therefore,
fresh isolates were obtained from infected leaves for
each experiment.
However,
different isolations made from
a single leaf at the same time in the same way differed
in
virulence
between
(0% -'I 0 0 %
experiments
infection).
produces
Such
results
variability
that
are
not
comparable.
As a pr eli min ary
task to my inheritance
study,
I
at tempt ed to devise a method to decrease the infection
variability of the barley leaf stripe disease organism.
I concentrated on the growth substrate used to culture P.
graminea
ot h e r
prior
to inoculation.
researchers,
ei g h t
With
media
the
were
cooperation of
developed
for
comparing the effects of the substrate upon infection
a specific
barley
cu lti var.
Aft er
culturing
of
a P.
grami nea isolate on these media for eight months,
only
small differences were detected in infection percentages.
Materials and Methods
A single
taken
from
conidiospore
naturally
breeder's barley seed,
isolate
infected
leaf
RPB 43971
of
gra mi ne a was
tissue
gro wn from
(obtained from
Mr. V.
Stewart,
Northwest
Agricultural
K a l i s p e l l , Montana).
The isolate
Research
was
first
Center,
grown
on
Two weeks later, a single 6 mm plug
water agar medium.
of mycelia and medium from the perimeter was transferred
I. water agar; 2 . barley
to the eight media that follow:
leaf piece agar (Teviotdale and Hall,
dextrose
1976a); 3. potato
agar (PDA) amended with 2 grams spring wheat
bran per liter of water;
4. PDA amended with 20 grams
spring wheat bran per liter of w a t e r ; 5. PDA amended with
germinated
barley seed ^diffusate (Appendix Table
12);
6.
PDA amended with germinated barley seed extract (Appendix
13); 7. V 8 juice; a n d , 8 . barley leaf extract
Table
in Konak
[ I 9 83 ] but
with
30 g/1
fresh
barley
(as
leaves).
Individual cultures were transferred by a single, 6 mm,
mycelial
bore
to their
respective
from January to August 1983.
again
isolated
media.
in
August
and
media every two weeks
A single conid io spore was
transferred
to the
same
These newly isolated cultures were compared to
the above cultures in ah inoculation test.
Inoculum for
the inoculation
tests was
prepared
by
autoclaving 30 g 'Ingrid' b a r l e y , C. I. 10083, with 30 cc
distilled water in I p t :Mason jars for 30 minutes at 121
C (Arny and Shands, 1942; Metz and Scharen, 1979; Konak,
1983).
The jars were shaken w i t h i n two to three hours
after
mats,
rem ov in g
from
the
autoclave
to loosen the seed
and then they were allowed to cool overnight.
The
sterile barley kernels were inoculated with three 6 mm
bores
of
m yc eli um
and medi u m
per
jar.
At
this
time
a p p r o xi mat el y 4 cc
sterile, distilled water was added
to
the
the
jars,
and
substrate
was
mixed
well.
Nonin oc ula te d checks were included in .each experiment.
The autoclaved barley kernels in jars were treated the
same;
however,
graminea
the kernels were not inoculated with P.
my celi a.
The jars containing inoculated and
n o n i n o cu la ted kernels were
incubated
at 12-14 C (12 h
light/dark cycles) for five to seven days.
•Summit* seed used in the expe rimen ts was obtained
from Dr. E .A. Hockett,
US DA , Montana
State University.
Prior to inocul ati ng the 60 - seed lots with Pj. graminea
mycelia,
the seeds were surface disinfected for three to
five min ute s in I 0 % Clorox (0.5% sodium hypochlorite) +
2% ETOH.
Surface disinfected seed lots were allowed to
dry on paper towels over night in a Micro void IIC clean
air
chamber.
The seed lots were
then mixed
with
the
previously prepared barley kernel inoculum.
One pt Mason jars of P. gra minea mycelia on kernel
substrate
and a S u m m i t
seed lot were
then placed in a
refrigerator at 4 + 2 C. The barley seeds were allowed to
I3
germinate in this substrate for 12-14 days at which time
most seeds had noticeable shoots.
germi na te d
kernels,
planted in a 1: 1
mycelia,
The entire mixture of
and
substrate
was
then
sand:soil mixture in 12 cm plastic pots.
The pots were placed in a growth chamber at 16 C / 2 C
+
I C
(12
seedlings
h light/dark
was evaluated
cycle).
at 10-14
Emergence
of
the
days after planting.
Number of symptomatic plants was read at three, four, and
six weeks.
The disease
were
readings
recorded as percent
taken six weeks after planting
symptomatic
emerged plants in each replication.
were
calculated for each
experiments
reisolation.
conducted
Analyses of variance
exp er imen t
over
plants of total
time,
and for
the
four
including
the
Analyses were conducted on the Superbrain
II microcomputer
with
statistics
programs written by R.
E. Lund (1983).
Results and Discussion
Table I lists the infection means (two replications
each) of .Ejl g raminea grown on each medium for the four
experiments.
No
significant
differences
(LSD
0.05)
between means of the cultures grown on different media
were
noted in any of
the
planting
times.
In
both
29
August
plantings
no significant differences (LSD 0.05)
were calculated between any of the means of the cultures
and the no ni no cul a te d ,check.
These data indicate that
the substrates did not affect
the stability of virulence
of this P. era minea isolate when serially cultured over
eight months.
Table I;
Per cent of barley seedlings infected as a
result
of i n o c u l a t i o n w i t h
cultures
of
Pvrenonhora eraminea isolate RPB grown on
different media.
Media
3 Mar
Noninoculated
Water agar
B L P Ab
PDA + Ic
PDA + IOd
PDA + seed dif.e
PDA + seed ext.^
V 8 juice
B L Ex t . 8
Meansb
00.0
30.0
20.0
20 . 0
Planting; Date
5 Jun
29 Aug
00.0
38.0
44.0
29 Auga
00.0
2.0
I.2
00.0
00.0
I.0
2.0
2.0
35.0
32.0
28.0
3.9
3.9
38.0
51 .0
2.0
35.0
32.0
30.0
44.0
45.0
35.0
I .3
2.9
0 0 .0
I .5
2.8
I.0
24.0
25.0
0.93
3.6
I .5
a Trial using reisolated JLa. eraminea cultures.
b Barley Leaf Piece Agar (Teviotdale and Hall, 1976a).
0 PDA amended with 2 g/1 spring wheat bran.
d PDA amended with 20 g/1 spring wheat bran.
e PDA amended with germina ted seed diffusate (see
appendix Table 12).
” PDA amended with germina ted seed extract (see
appendix Table 13).
S Barley leaf extract medium (Konak, 1983? but with
30 g / 1 fresh barley leaves).
" Means of symptomatic seedlings per total emerged
seedlings for each planting date.
A
two-factorial
exper im en ts
analysis
of
variance
for
the
conducted over time was also calculated.
Percentage of emer ged plants that had s y m p t o m s was one
factor,
was
and the date that the experiments were
the
second factor.
co mpari son
Table I 4.
The results,
using a LSD of 0.05,
conducted
including a mean
are
given in Appendix
The LSD (0.05) between culture means over time
in this analysis indicated that the inoculum
grown on
PDA amended with germinated, barley seed diffusate had
the highest percentage of infected
eight month
plants.
However,
the
cultured test and the reisolation test (both
planted on 29 August) resulted in extremely low infection
levels on the susceptible cultivar,
infection
levels
tended
to
Summit.
increase
These low
the
significant
differences found between the cultures over
the timed
experiments as indicated by the relatively large
for
planting
Prasad,
differences
dates
in
Leonard,
in
soil
the
and
water
analysis
Murphy
of
(1976)
potential
F-value
v a ria nc e.
found
affected
that
percent
diseased plants grown from artificially inoculated seeds.
Highest
infections
i n t e rm ed iat e
Infections
water
were
in
plants
potential
grown
(average
of
in
soils
-7. 1
of
bars).
decreased greatly in plants grown in soils
with high water potentials.
In my experiments water was
16
added to large pans containing up to 21 pots,
water was absorbed by the soil.
and the
Without monitoring the
soil water potential, high potentials were quite possible
during emergence
of the inoculated
seeds.
This may
explain the overall low infection levels obtained in the
plants in the last two planting dates.
Tekauz
w i th in
(1983)
found
replications
inoculated
barley
of
seed.
varying
his
He
levels
tests
with
proposed
of
infection
artificially
that
the lack of
good contact betw een the wheat kernel inoculum and the
seed,
due
to
clumping
of
the
decreased infection levels.
environmental
substrate^
Also,
variations
and
contamination as causes for the
ma y
he suggested minor
possible
partial
variability in infection
between replicates.
In continuing experiments,
isolated
of
low
barley kernels.
level's
have
bacteria
from
the
I have
autoclaved
Although mycelia were present on all the
substrates at the time of planting in these experiments,
partial
c o n t am ina ti on
that
would
hinder
infection is
possible.
This comparison of culture media was begun with
three
sraminea isolates. However, one isolate proved
difficult to maintain on all the substrates.
A second
isolate gradually lost the ability.to grow on some of the
17
substrates
so
that
by
October
1983,
it
was
ma intai ned on only three of the substrates.
data from only one isolate were analyzed.
differences
between
the infection means
bei ng
Therefore,
No significant
of the
cultures
were detected for any of the isolates.
In the inocul ati on meth od used in this study,
inoculated seed was treated harshly.
surface disinfected,
graminea
inoculum
germination,
mixture.
then dried,
First,
substrate.
tansplanted
into
Any injury of these germinated
trans planting
resulted
in
the seed was
and then placed with P.
growing on a kernel
the seeds were
the
decreased
After
a planting
seeds during
emergence.
The
production of inoculum on a substrate that can be mixed
with
the. planting
surface
mixture
disinfected
seed
would
could
be
then
beneficial.
be planted
into the inoculum and planting mixture.
production
method
Wilcoxson,
1984).
The
purpose
has
of
been
this
proposed
study
directly
Such an inoculum
recently
was
The
(Miles
to develop
and
a more
practical, efficient, and reliable method of artificially
in ocu latin g the leaf stripe organism onto barley seeds.
Konak
(1983)
presented
results
indicating
that
JL_
graminea grew more profusely on a rich medium, his barley
leaf
extract
medium.
More
recently,
J.G.N.
Davidson
(personal,
communica tio n)
stated
that
virulence
was
independent of the isolation met ho d or the m e d i u m
(V 8
juice
the
and water
agar).
My attempt
spectrum of substrates used.
active
and
s y s t e m ! c a l Iy
germination,
diffusate
was
Since jL_ graminea becomes
invades
the
I amended a proven medium,
and
extract
of
to extend
germinated
plant
PDA,
during
with the
barley
seed.
Although mean differences of the cultures were not highly
significant,
from
an indication was obtained that diffusate
germinated
pathogenicity.
continued
P.
barley
did
increase
c u ltu re
These results may provide impetus for
research into
graminea.
seed
better inoculation
methods
with
CHAPTER 3
VIRULENCE OF PYRENOPHORA GRAMINEA ISOLATES
ON SPECIFIC BARLEY CUL TIVARS
Introduction
Christensen
and
Graham
(cit ed
in
Stakman
and
Christensen, I 960) in a 1934 bulletin were the first to
detail variation in virulence among single spore isolates
of Pvrenoohora gramine a S. Ito & K u r i bay.
Virulence of
24 JL2. graminea isolates inoculated on lP e a t l a n d 1, C. I.
2613,
barley varied from almost 0 % to 7 7 % infection in a
greenhouse experiment.
Arny
(1945)
among three
cultivars.
detailed
physiologic
spe cialization
graminea isolates tested on five barley
The
results
showed
that
one
isolate
was
pathogenically distinct from two other isolates.
Kline
(1971) reported resistance levels to barley
leaf stripe disease in 82 winter
wrote
"Isolates
barley cultivars.
Kline
did not differentially attack cultivars,
and there was no evidence of pathogenic specializ ation
among these isolates on these cultivars."
20
Several researchers have hypothesized physiologic
specia liz ati on in pathogenic isolates
These
researchers include Nilsson
& Scharen (1979) in Montana,
in
Canada;
and,
Knudsen
of £*_ gr ami ne a.
(1975) in Sweden;
Metz
O S A ; Tekauz and Chiko (1980)
(1980)
in Denmark.
Knudsen
(1 9 8 0 ) stated that resistance in barley to his population
of JEj. gra minea
factors
was mainly
which
physiological
would
det er mine d
support
by quantitative
the
hypothesis
specialization in the fungus.
Smedegaard-Petersen and Jorgensen (1982)
on 28
experiments
single
spore
inoculation
technique.
of
barley cultivars using 15 j\_ graminea.
isolates
of
conducted
collected
seeds
was
in
by
Denmark.
the
layered
Artificial
mycelium
They found considerable variation in virulence
of JLt gra minea
isolates on specific
barley
cultivars.
The isolates also showed variati on in virulence to all
cultivars.
They
proposed
that
graminea isolates into different
the
separation
of
P.
pathogenic races was
indicated by these data.
Konak (1983) reported on virulence tests utilizing
the barley cultivar ’Summit' with 27 La, graminea isolates
obtained
from
Inoculation
of
Montana,
USA,
seed
by
was
Turkey,
a mod ified
and
Eth iop ia.
barley
kernel
21
inoculum
method.
Variation
in
virulence
of
these
isolates was 0% to 9 5 *3 % infection.
Virulence
paper
studies on
describe
the
gra min ea reported in this
variati on
in
virulence
of
24
P.
graminea isolates from several sources bn three barley
cultivars that exhibit varying degrees of resistance.
Materials and Methods
Leaf samples from plants symptomatic of barley leaf
stripe disease were collected in Tunisia, Egypt, Syria,
Turkey,
and Montana,
mo isten ed
after
filter
surface
USA.
paper
Leaf pieces were
or water
sterilization
in
(approximat ely three minutes).
conidia production,
water
agar.
Then,
placed on
agar in Petri
10%
Clorox
dishes
solution
After conidiophore and
masses of spores were transferred to
after
16-24
hours
g e r m i nat io n
at
approximately 15 C, a single conidium was transferred to
barley
1 976a).
leaf
piece
agar
(BLPA)
(Teviptdale
and
Hall,
Cultures were allowed to grow on BL PA for 12-16
days at 15 C , after which a single, six mm bore from the
perimeter of g r ow ing mycelia was placed in autoclaved
barley kernels (30 g 'Ingrid1, C. I. 1 00 83,
cc distilled water).
barley + 3 6
Mycelia grew on the barley kernels
for 9-12 days at 15 C. Then, a single barley kernel with
22
\
!
Ayceli a was placed on PDA + 4 ( 4 g/1 wheat bran) medium
in each plastic Petri dish.
Cultures were agai n' allowed
to grow at approximately 15 C for 12-15 days.
Three
barley
experiments:
cuItivars
lBetzes',
from Dr. E .A . Hockett,
Bozeman,
Summit
Montana,
seed,
were
used
in
these
C. I. 6398, originally obtained
was increased at the Post Farm,
and
harvested
originally
from
in
North
September,
I 982 ;
Ame ric an
Plant
Breeders, was increased at Mesa, Arizona and harvested in
April,
1983;
a n d , 'Lami 1 seed,
obtained from
Ms. Sally
Metz, was increased at Mesa and harvested in April,
Forty-seed lots were
surface
sterilized for
1983.
4-5 minutes
in I 0% Clorox plus 2 % E T O H , after which the seeds were
dried in a Microvo id IIC clean air chamber for eight to
twelve
hours.
Inoculation
was
by
the
layered
mycelium
method
(S h a n d s , 1934; Houston and Oswald, 1948; Nilsson, 1 97 5).
Individual seed lots (40 seeds each) were placed on top
of a layer of actively gro wing mycelia on PDA + 4 medium.
Another layer of mycelia growing on the medium was placed
over the top of the seeds.
Parafilm
strips
and
Petri dishes were sealed with
placed
in
a growth
chamber
at
approximately 8 C (12 h light/dark cycle) for seven days.
At that time the seeds had germinated,
and radicles were
23
0.5
to
Petri
1.5 cm
long.
The
seeds
were
dishes and planted in a 1:1
removed
sand:soil
from
the
(pasteurized
Bozeman silt loam) mixture in aluminum trays (20 X 30 cm,
3 seed
lots
in
each
divided
tray).
returned to the same growth chamber.
required to ma i n t a i n a moist
weeks,
The
trays
were
Water was applied as
soil surface.
After four
plants were removed to a greenhouse maintained at
16-20 C.
Symptomatic plants were first evaluated at four
weeks post-planting and every five to six days thereafter
until eight weeks post-planting,
were
pulled at each evaluation.
plants of the total
Symptomatic
Percent
plants
sym pt o m a t i c
plants emer ged from each seed lot
were recorded for analysis.
Three
same
different
procedures
graminea isolates,
split
were
(see
transferred
Chapter
I).
conducted,
each
using the
with
eight
P.
a check JLt eraminea isolate (RPB), and
a noninoculated check.
serially
plot exp er imen ts
The check isolate,
for more
The
RPB,
had been
than eight months
noninoculated
check
was
on BLPA
surface
sterilized seed lots germin ate d in an agar sandwic h as
above,
but with no fungus.
The noninoculated
checks had
no s y m p t o m a t i c plants in any of the experiments.
isolate-cultivar
combination
replications in each experiment.
consisted
of
Each
three
Analysis of the data
was by a split plot design with the isolates as the main
plot
and
cultivars
as
the
sub plot.
Analyses
we r e
calculated with a Superbrain microcomputer using programs
written by R.E. Lund ( I 9 8 3 )•
Results and Discussion
In
this
graminea
paper,
isolates
Tunisia (TN-
the
from
isolates),
isolates),
and
vari ation
Montana
Egypt
Turkey
(TK-
(E-
in virulence
(numbered
isolates),
isolates)
is
of
P.
isolates) ,
Syria (Sreported.
Percent s y m p t o m a t i c plants of total emerged
plants is
given for each isolate X cultivar interaction (average of
three replications each) in Tables 2, 3, and 4. Percent
emergence
of
the
cultivars
differed in the experiments.
Over the three experiments, average eme rgence for Lami
was 82.956, for Summit,
Betzes
seed
experiments.
lot
had
82.1?,
poor
Analysis
of
emergence as the independent
precision,
however.
taken into account.
and for Betzes,
emergence
5 9.9%.
The
throughout
the
covariance
covariable
with
p e rce nt
did not increase
The severity of s y m p t o m s was not
Rather, any sy m p t o m a t i c plant was
evaluated as infected and pulled.
25
Table 2:
Barley plants infected by Pvrenophora graminea
as a p e r c e n t a g e of tot al p l a n t s e m e r g e d
(average of three replications).
Isolates
VLami t
Noninoculated C k .
E- 8
TN-I
24-216
33-267
TN- 9
TN-6
24-212/19
33-313
RPB
Cultivar means
0.0
3.0
6 .6
19.1
22.5
44.1
47.6
49.4
71.5
73-4
33.7
Aa
A
A•
A
AB
BC
C
CD
DE
E
Cultivars
I1Summit'
0.0
0.0
4.9
2.0
9.6
I .0
2-7
4.9
2.0
3.5
3 .1
A
A
A
A
A
A
A
A
A '
A
•Betzes’
0 .0
I .2
0.0
0.0
0.0
16.4
0 .0
0.0
6.7
4.5
2.9
A
A
A
A
A
A
A
A
A
A
Isolate
Means
0.0
I .4
3.8
7.0
10.7
20.5
16.8
18.1
26 .8
27 •I
a Different capital letters o n two isolate X cultivar
interactions indicate a significant difference
between means (LSD 0.05).
26
Table 3:
Isolates
Barley plants infected by Pvrenbphora araminea
as a p e r c e n t a g e of total p l a n t s e m e r g e d
(average of three replications).
'Lami
Noninoculated Ck . 0.0
TN-4
0 .0
E-9
0.0
S- 2
10.2
E- 17
37-3
E-I It
45.7
E-IO
49-7
E-Il
66.8
96.2
E- I9
RPB
100.0
Cultivar means
40.6
I
Aa
A
A
AB
BC
CD
CD
D
E
E
Cultivars
1Summit1
0.0
I .0
0.0
0.0
25.8
10.4
11.7
20.2
36.2
11 .0
11.6
A
A
A
A
AB
AB
AB
AB
B
AB
lBetzesl
0.0
I .2
0.0
0 .0
0.0
1.5
0. 0
0.0
I .3
3-7
0. 8
A
A
A
A
-vA
A
A
A
A
A
Isolate
Means
0.0
0 .7
0.0
3.4
21.1 .
19.2
20.5
2 9.0
44.6
38.2 .
a Different capital letters on two isolate X cultivar
interactions indicate a significant difference
between means (LSD 0.05).
27
Table H :
Barley plants infected by Pvrenophora graminea
as a p e r c e n t a g e of total p l a n t s e m e r g e d
(average of three replications).
Isolates
Noninoculated Ck
TK-I 8
E-29
TK- 8
TK-I H
TK-I 3
TK-I I
S-Y .
TK- 9
RPB/MT6
Cultivar means
Cultivars
•Summit'
lLa m i '
O .0
0.0
0.9
I .I
I -7
5 .7
8.2
31 .3
32.6
94.3
17-6
Aa
A
A
A
A
A
A
B
B
C
0.0
0.0
0.0
0.0
I .0
0.9
0.0
6.4
4.0
0.0
I .2
A
A
A
A
A
A
A
A
A
A
'Betzes1
0.0
0.0
0 .0
0.0
0 .0
0.0
0.0
0.0
0.0
0 .0
0 .0
A
A
A
A
A
A
A
A '
A
A
Isolate
Means
0.0
0.0
0.3
0.4
0.9
2.2
2.7
12.6
12.2
31.4
a Different capital letters on two isolate X cultivar
interactions indicate a significant difference
between means (LSD 0.0 5).
28
• Virulence
of these
isolates
on the
cultivar Lami
varied from 09» to 10 0% infected plants.
Lami is highly
susceptible
Scharen,
1979;
and Jorgensen,
1982).
Knudsen,
to jL_ eramine a
1980;
However,
(Metz
and
Smedegaard-Petersen
infections on Lami did vary between isolates
which indicated a level of resistance to some isolates.
Summit has been reported to be highly susceptible to
P. graminea (Metz,
here,
however,
1978; Konak,
indicate
Summ it to P. g r a m i n e a .
tests
was
harvested
from
a leaf
prior
heading,
and
stripe disease
levels
of
infected
undertaken in this nursery
plants
lacking
selected for bulked seed harvest.
low
in
The Sum mit seed lot used in these
Intense roguing was
the
presented
a low level of resistance
nursery.
to
1983). The data
infection
symptoms
were
Selection may explain
when
the
isolates
were
inoculated onto this Summit seed lot.
Betzes barley had been reported to have 0% infection
by leaf stripe disease (Kline,
1 97 9;
Tekauz
and
C h i k o ,I 980) ,
described symptomatic
experiments.
produced
plants.
no disease
Metz and Scharen,
until
Konak
( 1 9 83 )
plants in artificial inoculation
Virulence
in these ex per im ent s
infected
1972;
of the 2*. graminea isolates used
on Betzes
Eighteen
symptoms
of
resulted
the
when
in
0%
2 4 isolates
inoculated
on
to
16%
tested
Betzes.
29
Konak
(1983)
dominant
proposed
that
Betzes
gene for resistance.
has
at
least
one
Inoculation tests with
Betzes crosses will be reported later.
The
data
interactions
presented
occurred
here
between
and Z jl gr ami nea isolates
The
significant
analysis
isolate
of variance
indicate
that
specific
from
differential
barley
infected
cultivars
barley
plants.
X cultivar interaction in the
for each
experiment
(P-values
less
than 0.01 for each experiment) indicated differential,
cultivar-sp eci fic
virulence.
cultivar-specific,
exper im en t
These
isolate virulence were
reaction
in
apparent in
I (Table 2) where a difference in isolate
cultivar infection ranking occurred.
tested,
differences
X
With most isolates
cultivar ranking from susceptible to resistant
was
L am i , Summit,
and
Betzes,
Isolates 33-313 and TN-9 reversed
the
respectively.
Summit
and Betzes
ranking such that Betzes was less resistant than Summit.
No
significant
found.
differences
The LSD's
bet wee n
between S u m m i t
had t-values
and greater than 0.50,
Smedegaard-Petersen
and
means
were
and Betzes infection
means for isolates TN-9 and 33-313
0.10 and 0.20,
these
between
respectively.
Jorgensen
(1982)
also
described variation in virulence of different Zjl. eraminea
isolates
on
barley
cultivars.
Even
with
relatively
30
susceptible
in
cultivars,
virulences
of
these
workers
specific
susceptibility to immunity."
reported
isolates
variation
from
"full
Data of Smedegaard-Petersen
and Jorgensen (1982), along with those reported in this
paper
and
Scharen,
by other
researchers
1979; Knudsen,
specific resistance to
(A r n y , 1945;
Metz
and
1980), indicate the occurrence of
sraminea.
From these data it can be concluded that, sources of
resistance
in
barley
to
leaf
s t rip e
disease,
are
available. These sources must be tested for resistance to
P. sraminea isolates specific to the intended
growing
area if they are to be utilized effectively in cultivar
development programs.
31
CHAPTER 4
INHERITANCE OF RESISTANCE IN THREE BARLEY CUL TIV ARS
TO TWO PYRENOPHORA ORAMINEA ISOLATES
Introduction
Barley
controlled
leaf
by
organic
number of years.
in
use
because
fungicides.
s t ri pe
disease
mercury
was
seed
efficiently
treatme nts
for
a
Mercury seed treatments have decreased
of
Since
the
the
envir on me nta l
1970's
leaf
effects
stripe
of
the
disease
has
again increased in occurrence.
The disease has recently
been
in
positively
identified
New
recommendation for treatment of new
(Arnst, Sheridan,
and Grbavac,
Zea l a n d ,
and
a
seed lines was made
1 978).
In Montana,
USA, a
potential increase in infected seed lots from I% to 4 3%
with in
three
197 9)•
In Denmark, leaf stripe is endemic with levels of
20%
years
was
predicted
to 25% infection reported,
(Metz
S c h a r e n,
and rare infections of up
to 70% have been observed (Tekauz, I 9 83).
also
and
reported w i d e s p r e a d occurrence
Tekauz (1 983)
of the disease in
32
Canada
with
diseased
infections
ranging
from
a trace
to
3%
plants.
Current
industry
mo v e m e n t
is
Fungicides
toward
for
the
in
the
specific,
control
and
systemic
chemical
fungicides.
of leaf stripe disease
becoming available (Johnston,
Smedegaard-Petersen
agricultural
Metz, and Riesselman,
Jorgensen,
because of economic and environmental
important
components
19 8 4),
resistant
of the
1982;
1982).
However,
concerns
and with
the de velop men t of integrated crop man ag e m e n t
(Nis se n and Juhnke,
are
systems
cultivars
remain
production formulae.
Also,
many barley producing areas lack the needed distribution,
marketing,
and treatment facilities,
farmers' seed.
are
to chemically treat
Cultivars resistant to prevalent diseases
an important
means
of increased production in those
areas.
The primary emphasis in my research was to identify
genes in barley which would provide resistance to barley
leaf stripe disease.
Crosses betw een three Turkish and
American cultivars were made.
Seed was produced through
the Fg and backcross Fg generations.
give
details
in ocula tio n
results.
of
of
experiments
plants
from
In this chapter,
with
these
the
crosses
I
artificial
and
discuss
33
Materials and Methods
In the s u m m e r
' B e t z e s 1 ( C . I.
Betzes
and
Research
of I 982 reciprocal
6 3 9 8)
'Yesilkoy'
Center,
made
'Tokak'
were
made
at
the
Arthur
MT.
At
the
same
onto
1 . 86 5 5),
Cahit Konak (1983).
from four parent
Mesa,
50%
Post
time,
X
The F^1) seeds were
The. seeds from these
crosses were harvested in Sep te mber 1982.
seeds (approximately
and
F 1 plants of the Betzes
Tokak and Betzes X Yesilkoy crosses.
provided by Mr.
(C.
between
and
Bozeman,
backcrosse s were
crosses
Four to ten
of the seed from a crossed head)
plants of each cross were
AZ in the fall of 1982.
planted in
F (2) and b a c k e r o s s (BC)
F( 2 ) seeds were harvested in April 1983.
F(2) seeds were
planted in Boz em an in the spring of 1983, and F (g) seeds
were
harvested
in
Inconsistencies
and
generations
September.
in
the
designation
often occur in
attempted to standardize
papers.
of
crossed
Ahokas
seed
(1976)
designation of generations.
He
proposed that the generation number of seed be placed in
parentheses.
The plants grown from that seed have the
same generation number,
but the parentheses
Inoculation experiments were
The layered
Chapter 2.
my c e l i u m
method was
are removed.
begun in January
1984.
used as described
in
Seeds of bulked and specific-plant parents,
34
F 1 , F2 , and Fg generations from one cross with at least
one susceptible cultivar were inoculated with a single
spore isolate of 1% graminea at the same time.
Seeds of
parents,
BC
the
recurrent
parents
F 1,
and
BC
F 2 generations
backcrossed onto
the
from
F1 plants
two
with
at least one susceptible cultivar were also inoculated
with a single spore isolate of P. -graminea at the same
time.
was
A check P. gra minea isolate, RPB (see Chapter I),
inoculated
experiments.
on t o
che ck
Inoculated
cultivar
seeds
were
see d s
in
germ inate d
all
in the
layered mycelium for 10-12 days with Pj. graminea isolate
MT6 and for 6-7 days with isolate K a y a.
isolates
were
obtained
inoculated by Konak
from
infected
Both of these
'Summit*
leaves
(19 83) .
After germination in the layered mycelium,
seed lots
were planted in a 1:1 s a n d :soil mixture and placed in a
growth
F(i),
chamber.
F(2 ), and
The growth
F(g ^ seed
chamber
lots
was
used for parents,
set
at
a constant
temperature of 8 ± 2 C with a 12 h/12 h light/dark cycle.
Lights were fluorescent tubes (Sylvania cool white, 95 W )
with
an
intensity
of
3.0-3.6
growth chamber used for parents,
X
IO2* e r g s / c m 2 sec.
The
BC F^1) and BC F^2) seed
lots was set at a constant tem perat ure of 9 ± I C with a
12 h/ I 2 h l ight/dark cycle.
Lig hti ng was also provided
35
by fluorescent tubes (Sylvania cool white, I 95 W) with an
intensity
allowed
and
of
2.8-4.0
to grow
then
X 10 ^ ergs/cm^sec.
for three weeks
they
were
Plants
were
in the growth chambers,
transferred
to
a
greenhouse.
T e m p e ra tur es in the greenhouse were no lower than I 8 C.
However,
during
sometimes
provided
did
the
summer, months,
exceed
30
with.' halogen
C.
lamps
midday
temperatures
Additional
lighting
as
to
needed
was
provide
a
m i n i m u m of 12 h days.
Diseased
plants
and
total
emerged
plants
counted at 4, 5, arid 6 weeks post-planting.
s h ow in g
striping
typical
counted, as infected
indefinite
or
and
of
leaf
stripe
destroyed.
atypical
symptoms
Rare
were
were
Any plant
disease
leaves
was.
with
transferred
to
barley leaf piece agar (BLPA) or moi st ened filter paper
and observed for sporulation.
plants per total eme rg ed
seed lot.
Percentage of
diseased
plants was recorded for each
Lots varied in number of seed from 10 seeds or
less for the.Fi generation to 100 seeds in some
bulked
of the
parent, Fg and Fg generations.
Results
Emergen ce
lo t s
in
all
of the seedlings
trials
t
conducted.
varied amo ng the seed
This
variation
was
36
lessened
when
the
seed
lots
were"
germina ted
layered mycelium for a shorter time.
in
the
Inoculation with P.
'
M T 6 was for 10-12 days,
g r amine a isolate
time
was
reduced
Emerg en ce
to
6-7
days
with
the
whe rea s this
Kaya
isolate.
percentages were higher in the inoculations
using
this
shorter
Tokak
X Betzes
time
and
period;
For example,
reciprocal
crosses
emergence of barley seedlings from 3 5 7
inoculated with isolate
the
in
the
average
seed lots when
MT 6 was approximately 5 9 %.
With
the same crosses after inoculation with isolate Kaya, the
average
of barley seedlings was about 7 7 % ( 221
emergence
seed lots).
Methods were
seedling
sought
infection
percentages
variable.
emergence
generations of crossed seed were limited in number.
So,
Seeds
conducted on parent and check cultivar bulked
(Appendix
em ergen ce
a d de d
using
the
seeds
an
percentages
the precision of
from
tests were
as
to increase
of
seedlings,
occurred with
susceptible
Table
the
17).
Com par ing
a drop of 6 %
ino culation
cultivar,
Summit,
of
the
six-weeks
to 7 %
emergence
seeds
and
in
of
the
the
highly
resistant
cultivar, T o k a k , versus the noninoculated seeds of the
respective
cultivars.
An analysis
of
covariance
was
calculated on these results with the percent emergence of
37
seedlings as an independent covariable and the percent
infection
of
seedlings
(Appendix Table
well
as
similar
seedlings
was
18).
the
dependent
variable
In the analysis of this data,
data
not
as
sets,
the
percent
significant.
emer gence
Precision
was
as
of
not
increased in the analysis of covariance with the addition
of the
covariable.
Therefore,
percentages of emerged
seedlings were considered random in this study.
Betzes - Tokak Crosses
Betzes and Tokak were both resistant to infection by
P. eraminea
(4.9%
respectively,
and
in these
7.4%
infection
trials).
of
seedlings,
Konak (1983) proposed
that each of these cultivars had one dominant gene for
resistance.
This proposal was based on the inoculation
of 115 Fg seedlings wi th
era min ea isolate M T 6 .
seeds were inoculated with two
F(I)
eraminea isolates in
trials with seeds from check cultivars, bulk parents, and
specific
parents
(seeds from
the female and male
plants
used for the cross) (Table 5). No symptomatic plants were
found in the Betzes X Tokak nor reciprocal crosses when
seeds were inoculated with the isolate MT6.
were
Results
similar when the same crosses were inoculated with
the isolate Kaya, except for one symptomatic plant in the
Betzes X Tokak
trial.
The leaves from this single
38
i
,plant
produced
on BLPA.
Pyrenophora-tvoe
cdnidia after
isolation
Some of the seedlings from specific parents of
these crosses also had no, or a few ^ s y m p t o m a t i c plants
in these inoculation trials.
Table 5:
Num be r of barley seedlings derived from check
cultivars , parents, and progeny of two crosses
with and without s y m p t o m s after inoculation
with two isolates of Pvrenophora graminea.
Seed Source
Isolate
No
Symptoms
Symptoms
Total
Betzes X Tokak trial3
Summit - Gk
Lami - Ck
Betzes - bulked
Tokak - bulked
MT6
MT6
MT6
MT6
73
59
86
65
I5
31
I
I
90
87
66
Betzes - spec.b
Tokak - spec.
B X T, V
MT6
MT6
MT6
19
25
2
I
O
O
'20
25
2
Betzes - spec.
Tokak - spec.
B X T, F 1
MT6
MT 6
MT6
37
57
2
O
5
O
37
62
2
Betzes - spec.
Tokak - spec.
B X T, F 1
MT6
MT6
MT6
58
76
I4
O
9
O
58
85
I4
88
39
Table 5 (cent’d ) .
Seed Source
Isolate
No
Symptoms
Symptoms
Total
95
75
53
24
Tokak K Betzes trial
Summit - Ck
L ami - Ck
Tokak - bulked
Betzes - bulked
MT6
MT6
MT6
MT6
69
48
52
24
26
27
I
O
Tokak - spec.
Betzes - spec.
T X B, F 1
MT6
MT6
MT6
33
56
6
I
4
O
Tokak - spec.
Betzes - spec.
T X B, F 1
MT6
MT6
MT6
41
47
2
I •
3
O
42
50
2
Tokak - spec.
Betzes - spec.
T X B, F1
MT6
MT6
MT6
30
1I
8
3
O
O
33
11
8
.
34
60
6
tzes X Tokak trial
Summit — Ck
Betzes - bulked
Tokak - bulked
Kaya
Kaya
Kaya
4
59
53
46
4.
7
50
63
60
Betzes - spec.
Tokak - spec.
B X T, F 1
Kaya
Kaya
Kaya
27
32
10
5
I
O
32
33
10
Betzes - spec.
Tokak - spec.
B X T, F 1
Kaya
Kaya
Kaya
26
24
IO
5
10
0
31
34
10
Betzes - spec.
Tokak - spec.
B X T, F 1
Kaya
Kaya
Kaya
36
46
14
4
5
I
40
51
15
40
Table 5 (cent1d ).
Seed Source
Isolate
No
Symptoms
Symptoms
32
68
5
O ■
34
69
61
45
Total
Tokak X Betzes trial
Summit - Ck
Lami - Ck
Tokak - bulked
Betzes - bulked
Kaya
Kaya
Kaya
Kaya
2
I
56
45 ■
Tokak - spec.
Betzes - spec.
T X B, F 1
Kaya
Kaya
Kaya
40
42
I4
O
O
O
40
42
I4
Tokak - spec.
Betzes - spec.
T X B, F 1
Kaya
Kaya
Kaya
19
36
7
3
5
O
22
4-1
7
Tokak - spec.
Betzes - spec.
T' X B , F 1
Kaya
Kaya
Kaya
36
38
II
5
3
O
41
41
11
a Each trial was conducted at a different time.
b Parents marked ‘spec.1 give reactions of seedlings from
the specific female and male plants that were crossed
to produce the associated
progeny.
. c The F . seedling reactions are given f oll ow ing their
specific female and male parent plants.
Fg populations of the B e t z e s X Tokak cross and its
reciprocal were inoculated with
and
Kay a.
Each
Fg
graminea
population
was
isolates MT6
composed
of
F2
seedlings derived from five or less F^ plants which were
derived from
a single mother
seeds with isolate
plant.
Inoculations of F^g)
MT6 produced results which fit a 15:1
genetic ratio whic h was expected for the two dominant
genes
model.
individual
The
reactions
of
F2 seedlings
plants of the cross Betzes X Tokak and its
reciprocal were fit to the 15:1 ratio,
Chi-square
Table 6:
from
(X2) was
and a homogeneity
calculated for each
cross
(Table
6).
N u m b e r of s p e c i f i c p a r e n t and F 2 b a rle y
seedlings with and without sym p t o m s after
inoculation with Pvrenoohora graminea isolate
MT6 and C h i - s q u a r e (X2 ) values whe n fit to a
15:1 ratio.
Seed Source
No
Symptoms
Symptoms
Total
X2
df
P>a
Betzes X Tokak
Betzes - spec. b
Tokak - spec.
402-a
402-b
402-c
402-d
Subtotal
28
50
27
21
27
I8
93
O
2
Betzes - spec.
Tokak - spec.
40 5- a
405-b
40 5- c
405-d
Subtotal
Total
Homoge neity
2
O
I
7
28
52
BI
23
27
19
IOO
2.34
0.23
I .80
0.03
4.40 '
I
I
I
I
4
0.10
0.50
0.20
0.70
0.25
19
25
62
55
34
36
I 87
I
O
2
2
0
4
8
20
25
64
57
34
40
I95
1 .07
0.11
2.27
0.96
4.41
I
I
I
I
4
0.30
0.70
0.10
0.30
0.25
2 80
I5
295
8.81
-0.68
8.13.
8
I
7
0.25
0.25
0.25
H
42
Table 6 (cent 'd)
Seed Source
Symptoms
No
Symptoms
Total
X2
df
P>a
Tokak X Betzes
Tokak - spec.
Betzes - spec.
4 I6-a
4 16-b
416-c
4 I6 -d
416-e
Subtotal
37
69
67
62
67
51
316
2
O
2
3
2
2
4
13
57
37
71
70
64
69
55
329
I .43
O .46
I .07
I .53
0.10
4.59
I
I
I
I
I
5
0.20
0.50
0.30
0.10
0.70
0.25
Tokak - spec.
Betzes - spec.
417-a
417-b
417-c
417-d
Subtotal
41
24
69
47
66
65
247
I
O
3
3
2
5
13
42
24
72
50
68
70
260
0.52
0.01
1.21
0.09
I .83
I
I
I
I
4.
0.30
0.90
0.20
0.70
0.75
Total
563
26
589
9
I
8
0.50
0.05
0.90
55
Homoge neity
6.42
-3. IQ
3.03
a Probability level of a larger Chi-square value,
k Disease readings bf seedlings from the specific parents
of the F 2 populations are given proceeding the
data.
Seedlings from the bulk parents and check cultivars
disease readings are given in Table 5. respective to the
specific cross and isolate.
After inoculation with JPi. graminea isolate Kaya,
seedlings
derived
from
th e
Betzes
X
Tokak
Fg
and
reciprocally crossed F^ plants did not fit a one, two, or
three,
dominant gene model.
Fg see tilings derived from
six individual F^ plants from the Betzes X Tokak cross
43
fit a 15:1
plants
r a t i o , but
had quite
high
the
progeny from
Chi-square values
three other
(Table
seedlings derived from eleven F 1 plants from
7 ).
F2
the Tokak X
Betzes cross also were not hom og enou s for the expected
ratio.
F 2 see tilings from
six of these eleven F 1 plants
did fit the 15:1 ratio.
Table 7:
Number of specific parent. F 2 barley seedlings
with and without sy m p t o m s after inoculation
with Pvrenophora graminea isolate Kaya and Chisquare (X^)Tralues when fit to a 15:1 ratio.
Seed Source
No
Symptoms
Symptoms
Total
X^
df
p>a
0.02
1
0.75
I .90
0.74
I
I
0.10
2.71
73-48
1
0 . 1-0
Betzes X Tokak
Betzes - spec . * 5
Tokak - spec.
402-b
402-c
402-d
37
57
41
50
25
4
8
'
3
6
3
41
65
44
56
28
32
33
117
Betzes - spec.
Tokak - spec.
403-b
403-c
27
32
I I4
73
5
I
3
27
100
Betzes - spec.
Tokak - spec.
405-a
405-b
40 5-c
405-d
36
46
54
4
5
13
5
13
3
40
51
I
67 19.78
I
1 I5 0.71
89 10.61 . I
60
0.16
I
110
76
57
I
0.25
e
e
0.25
e
0.50
Table 7 (cent *d ) .
No
Symptoms
Seed Source
X2
df
2.53
I
0.25
88 21 . 3 8
95 0 . 76
58 11 96
I
I
I
e
8
0
44
17
90
97
84
5.60
I
I
I
0.75
0.25
e
0.82
1. 21
I
I
0.25
•0.25
Symptoms
Total
Tokak X Betzes .
P>a
,
I
22
Tokak - spec.
Betzes - spec.
41 4-a
414-b
41 4-c
I9
36
38
13
9
3
5
4l5-ad
41 5-b
41 5-c
72
87
48
16
. 8
Tokak - spec.
Betzes - spec.
416-b
4 16 -c
4 I6 -e
39
16
85
89
84
5
I
5
4l7-bd
417-c
86
8
94
85
3
88
41
38
15
I0
0
2
I
10
C
C
.
0.07
0.66
0.25
-
6
a Probability level of a larger Chi-square value,
b Disease readings of seedlings from the specific parents
of the F 2 populations are given proceeding the F- data.
Seedlings* reactions from the bulk parents ana check
c u l tivars are given in Table 5 respective to the
specific cross and isolate.
0 Chi-square values were not calculated for populations
consisting of less than 16 plants.
^ Specific parents of these populations were not tested.
e P-values less than 0.05 were not calculated (X ^ > 3 •8 *1) .
F i plants
from
backerosses
to
both
parents
were
inoculated with both M T 6 and Kaya isolates (Table 8 ). A
few
symptomatic
inoculations.
plants
were
With a dominant
fou nd
after
these
gene derived from each
45
parent,
a 1 (resistant):© (susceptible) ratio is expected
in the backerossed
Table 8:
populations.
Number of barley seedlings from bulked parents
and backcrossed F(i) seeds with and without
symptoms after inoculation with two Pvrenophora
graminea isolates.
Seed Source
Isolate
■ ' No
Symptoms
Betzes - bulked
Tokak - bulked
MT6
MT6
87
77
Betzes/Tokak
X Tokak,.F 13
Betzes/Tokak
X Betzes, F1
MT6
Symptoms
Total
I
I2
88
89
20 :
I
21
MT6
21
I
22
Betzes - bulked
Tokak - bulked
Kaya
Kaya
69
62
2
4
71
66
Betzes/Tokak
X Tokak, F 1
Be tzes/Tokak
X Betzes, F 1
Kaya
37
O
37
Kaya
22
2
24
a Data from
seedlings
backcross.
the backcrossed
generation include
derived from
three F^ plants for each
Segregation occurred
they were tested with
among
the BC Fg families when
graminea isolates M Tb and Kaya.
Families in the backcrossed Fg generation did not fit the
expected ratios resulting from
model
(Table
9).
The
BC
Fg
the two dom inant
f a mil ie s
either as resistant or as segregating.
were
genes
classified
The percentage
46
infection of seedlings from the recurrent
parent was the
upper
Fam ilies
limit
greater
were
these
of
the
infection
classified
resistant
percentages
than
as segregating.
backcrossed
Fg
class.
fam il ies
with
the
recurrent
The
distribution
was
definitely
parent
of
skewed
towards higher susceptibility than either parent which
indicates gene action other than the dominant genes model
Table 9:
Number of backcrossed Fg families resistant and
segregating
after ' i n o c u l a t i o n
with
two
Pvrenophora eraminea isolates.
Seed Source
------------ Families-----------Resistant
Segregating
Total
Betzes/Tokak X Tokak - MT6
Tokaka : Infection = 10.2?
420b
421b
422b
423b
Total
I
3
I
0
5
4
3
5
4
16
Betzes/Tokak X Betzes - MT6
Betzes : Infection = 3.1?
427°
428c
42 9b
430b
Total
2
I
2
3
8
4
5
4
3
16
5
6
6
4
21
6
■6
6
6
24
47
Table 9 (cent *d ) .
Seed Source
Resistant
Betzes/Tokak X Tokak - Kaya
Tokak: Infection = 6 •7%
420d
421d
422d
423d
Total
Segregating
> Total
I
I
I
0
3
4
5
5
5
I9
5
6
6
5■
22
Betzes/Tokak X Betzes - Kaya
Betzes: Infection = 4 •2%
0
427e
428e
0
2
429e
430®
3
Total
5
5
2
4
3
I4
5
2
6
6
I9
a The average infection percentage of the respective
recurrent parent in each backcross trial was used as
the upper limit of the resistant class,
b Seed
lots consisted of 20-35 seeds each.
0 Seed
lots consisted of 10-20 seeds each.
^ Seed
lots consisted of 60-80 seeds each.
e Seed
lots consisted of 35-80 seeds each.
Betzes - Yesilkov Crosses
Betzes is highly resistant to infecti on by P .
graminea,
and
resistance
respectively,
Yesilkoy
(6.4%
and
has
I 8.1%
in these trials).
a much
lower
infection
of
lev e l
of
seedlings,
^(i) seeds of the Betzes
X Yesilkoy and reciprocal crosses were tested for disease
reaction after inoculation with two
(Table 10).
No symptomatic
2^.
graminea isolates
plants were observed after
48
inoculation
with
isolate
MT6.
When
inoculated
with
isolate K a y a , a few of the F 1 plants from the Betzes X
Yesilkoy and reciprocal crosses exhibited typical disease
symptoms.
These symptomatic
or mois te ned filter paper,
plants were
placed on BL PA
and typical Pvrenophora sp.
conidia were produced.
Table 10:
Number of barley seedlings derived from check
cultivars, parents, and progeny of two crosses
with and without sy m p t o m s after inoculation
with two isolates of Pvrenophora eraminea.
Seed Source
Isolate
No
Symptoms
Symptoms
Total
Betzes X Yesilkoy trial3
Summit - Ck
Lami - Ck
Betzes - bulked
Yesilkoy - bulked
MT6
MT6
MT6
MT6
70
28
48
71
I4
58
O
17
84
86
48
88
Betzes - s p e c . ^
Yesilkoy - spec.
B X Y, F 10
MT6
MT6
MT6
54
66
12
I
30
0
55
96
12
Betzes - spec.
Yesilkoy - spec.
B X Y, F 1
MT6
MT6
MT6
79
26
10
0
3
0
79
29
10
Betzes - spec.
Yesilkoy - spec.
B X Y, F 1
MT6
MT6
MT6
79
22
I9
4
I
0
83
23
I9
49
Table 10 (cent *d ) .
Seed Source
Isolate
No
Symptoms
Symptoms
Total
Yesilkoy X Betzes trial
Summit - Ck
Lami - Ck
Yesilkoy - bulked
Betzes - bulked .
MT6
MT6
MT6
MT6
52
II
64
62
44
83
10
3
96
94
74
65
Yesilkoy - spec.
Betzes - spec.
Y X B, F 1
MT6
MT6
MT6
47
38
7
8
O
O
55
38
7
Yesilkoy - spec.
Betzes - spec.
Y X B, F 1
MT6
MT6
MT6
38
44
5
4
2
O
42
46
5
Betzes X Yesilkoy trial
Summit - Ck
Lami - Ck
Betzes - bulked
Yesilkoy - bulked
Kaya
Kaya
Kaya
Kaya
I5
3
63
64
52
65
7
12
67
68
70
76
Betzes - spec.
Yesilkoy - spec.
B X Y, F 1
Kaya
Kaya
Kaya
58
21
I2
7
II
O
65
32
I2
Betzes - spec.
Yesilkoy - spec.
B X Y, F 1
Kaya
Kaya
Kaya
60
6
15
6
3
2
66
9
17
Betzes - spec.
Yesilkoy - spec.
B X Y, F 1
Kaya
Kaya
Kaya
41
31
12
1I
11
O
52
42
I2
50
Table 10 (cent *d ).
Seed Source
Isolate
No
Symptoms
Symptoms
Total
Yesilkoy X Betzes trial
Summit - Ck
Lami - Ck
Yesilkoy - bulked
Betzes - bulked
Kaya
Kaya
Kaya
Kaya
contaminated
2
66
16
45
62
2
68
61
64
Yesilkoy - spec.
Betzes - spec.
Y X B, F 1
Kaya
Kaya
Kaya
I8
53
4
2
6
O
.20
59
4
Yesilkoy - spec.
Betzes - spec.
Y X B, F 1
Kaya
Kaya
Kaya
20
56
3
24
3
2
44
59
5
Yesilkoy - spec.
Betzes - spec.
Y X B j F1
Kaya
Kaya
Kaya
7
53
7
2
II
O
9
64
7
Yesilkoy - spec.
Betzes - spec.
Y X B, F 1
Kaya
Kaya
Kaya
42
32
5
16
5
O
58 '
37
5
® Each trial was conducted at a different time.
b Parents marked 'spec.' give results of seedlings from
the specific female and male plants that were crossed
to produce the following
seedlings.
c The F 1 seedling reactions are given f oll ow ing their
specific female and male parent plants.
Disease reactions of F2 seedlings from these crosses
were variable.
the
Yesilkoy
F2 seedling reactions of populations from
X Betzes
cross
did
fit
an expected
Chi-
square ratio of 13:3 after inocul ati on with isolate M T 6
(Table 11). This ratio indicated the expected
dominant
gene inheritance from Betzes and a recessive gene from
51
Betzes
or
Ye sil koy .
calculated,
A homogeneity
Chi-square
and the Fg populations of this
found homogeneous.
was
cross were
Fg seedling reactions of populations
from the reciprocal cross (Betzes X Yesilkoy) did not fit
any expected ratios when inoculated with isolate
MT6.
The populations of Fg seedlings from six of the eight F^
plants
did
fit
an
expected
Chi-square
ratio
of
13:3,
however.
Populations of the inoculated Betzes X Yesilkoy
seeds fit an expected
g^
13:3 ratio when tested by the Chi-
square method and inoculated with isolate Kaya (Appendix
Table
11).
indicated
the
Calculation
that
the
populations
populations
of
Fg
of
the
homo genei ty
Chi-square
populations were homogeneous,
were
grouped
seedlings
from
tog eth er.
the
reciprocal
(Yesilkoy X Betzes) did not fit any dominant
after inoculation with isolate Kaya.
The
and
Fg
cross
gene ratios
52
Table 11:
Seed Source
N u m b e r of s p e c i f i c par e n t and F 2 b a rle y
seedlings with and without sy m p t o m s after
inocul ati on with two Pvrenonhora sraminea
isolates and Chi -square (X2 ) values when fit
to a 13:3 ratio.
No
Symptoms
Symptoms
Total
X^
df
p>a
Yesilkoy X Betzes - MT6
Yesilkoy - spec.b 48
Betzes - spec.
47
40 8- a
76
408-b
82
408-c
35
Subtotal
I 93
8
4
11
I2
6
29
56
51
87
94
41
222
409-a°
409-b
409-c
409-d
Subtotal
62
68
33
30
I 93
16
11
6
7
40
68
79
39
37
233
0.16
1.21
0.29
0.01
I
I
I
I
0.50
0.25
0.50
0.90
3 86 .
69
455
6.47
-3.84
2.63
7
. I
6
0.25
0.05
0.75
17
44
100
84
5
8
I0
6
22
52
I I0
90
1.74
0.22
1.62
3.36
I
I
I
I
0.10
0.50
0.10
0.05
24
20
66
10
30
25
I
2
I
I
2
3
25
22
67
11
32
28
8.84
6
I
d
I .35
0.15
I
I
0.10
0.50
Total
Homogeneity
2.13
2.21
0.46
I 0.10
.I 0.10
I . 0.25
Betzes X Yesilkoy - MT6
396-ac
396-b
396-c
396-d
Betzes - spe c .
Yesilkoy - s pe c i
398-a
398-b
398-0
398-d
53
Table 11 (cent1d )„
Seed Source
No
Symptoms
Symptoms
Total
X2
df
0.03
1 .36
9-98 .
I
I
I
0.75
0.10
d
P>a
Yesilkoy X Betzes - Kaya
l»08-ac
408-b
40 8- c
64
57
51
14
18
25
78
75
76
Yesilkoy - spec.
Betzes - spec.
409-a
409-b
40 9-o
409-d
42
32
60
65
54
46
16
5
13
II
21
28
58
37
73
76
75
74
0.04
0.91
4.21
.17.70
I
I
I
I
O'.7 5
0.75
Q
d
Yesilkoy - spec.
Betzes - spec.
411-a
411-b
411-c
18
53
68
75
70
2
6
10
4
11
20
59
78
79
81
I .80
9.02
I .42
I
I
I
0.10
d
I .66
0.18
0.03
0.71
I
I
I
I
0.10
Betzes X Yesilkoy - Kaya
Betzes - spec.
Yesilkoy - spec.
3 97- a
397-b
397-c
397-d
Subtotal
41
31
58
17
20
I4
109
II
11 .
19
3
5
5
32
52
42
77
20
25
I9
T 41
0 .10
0.50
0.75
0.25
54
Table 11 (cont1d ) .
No
Symptoms
Seed Source
Symptoms
Total
Betzes X Yesilkoy - Kaya (cont1d)
398-bG
26
3
398-c
76
14
398-d
44
9
Subtotal
I 46
26
Total
58
255
X2
df
29
90
53
172
I .35
0.60
0.11
I
I
I
0.10
0.25
0.50
313
4.64
-n . m
4.63
7
I
6
0.50
0.90
0.50
Homogeneity
P>a
a Probability level of a greater Chi-square value,
k Disease readings of the specific parents of the Fg
populations are given preceeding the F2 data. Seedlings
from the bulk
parents and check cultivars disease
readings are given
in Table 10 respective to the
specific cross and isolate.
0 Specific parents of this F2 population were not tested.
^ P-values less than 0.05 were not calculated (X^>3•84).
e Chi-square values were not calculated for populations
consisting of less than 16 plants.
The populations of BC F^ plants from these crosses
did
segregate
as
expected
for
a one
dominant
(Betzes) and one recessive gene model
(Table
symptomatic
the
BC
Betzes/Yesilkoy
inoculated
Betzes,
with
all
F1
X Betzes
isolate
plants
expected
to
be
seedlings
from
plant
grown
seeds
M T6.
backcross
three resistant BC F 1 plants
was
With
from
resistant.
the
from
the
.In
to
BC
the
12).
One
backcross
observed
the
gene
when
backcross
F(i)
seeds
populations
Y e s i l k o y , a ratio
to one
to
are
of
of
susceptible BC F 1
55
plan t
is
expected.
Seedlings*
reactions
of
BC
F1
populations which had been backcrossed to Yesilkoy fit an
expected
3:1
ratio
(P-value
>0.50)
after
inoculations
with both the MT6 and Kaya isolates.
Table 12:
Number
of b a r l e y s e e d l i n g s
from
check
cultivars, bulked parents, and backcrossed
F^ 1J seeds with and without s y m p t o m s after
inocul ati on with two Pvrenophora graminea
isolates.
No
Symptoms
Symptoms
Total
. MT 6
MT 6
MT 6
MT 6
30
13
65
84
92
3
114
105
81
12
MT 6
29
I
30.
MT 6 .
28
9
37
18
Seed Source
Isolate
Summit - Ck
Lami - Ck
Betzes - bulked
Yesilkoy - bulked
Betzes/Yesilkoy
X Betzes, F1a
Betzes/Yesilkoy
X Yesilkoy, F 1
.
68
93
76
75
6 5.
63
Summit - Ck
Lami - Ck
Betzes - bulked
Yesilkoy - bulked
Kaya
Kaya
Kaya
Kaya
O
62
56
58
75
3
17
Betzes/Yesilkoy
X Betzes, F 1
Betzes/Yesilkoy
X Yesilkoy, F 1
Kaya
23
O
23
Kaya
31
9
40
a Data from the backcrossed F 1 generation include
seedlings derived from three or four F 1 plants for each
backcross.
56
With the one dominant gene and one recessive gene
m o d e l , BC F 2 fam il ie s
ratio
of
two
are expected
ho moz ygo us
segregating families.
to segregate
resistant
families
to
in a
two
The BC F2 families did not fit the
expected ratios.
The BC F2 families were separated into
either resistant
or seg regating
infection
parent
percentage
was
the
of
upper
seedlings
limit
of
the
the
resistant
recurrent
parent were classified as segregating.
ratio,
found
so these BC F 2 famil ies
(Table 13).
percentages
homog eneou s
were
average
recurrent
with
were
infection
from
The
Famil ies
F 2 famil ies
greater
classes.
class.
than the
The BC
for fitting a 1 : 1
grouped
together
57
Num be r of backcrossed F 2 fam ilies resistant
and segregating after inoculation with two
Pvrenophora graminea isolates.
Table 13:
Seed Source
Resistant
--Families----Total
Segregating
Betzes/Yesilkoy
X Yesilkoy - MT 6
Yesilkoy: Infection = 13.0$a
Four F 1 Plantsb
9
Betzes/Yesilkoy
X Betzes - MT 6
Betzes: Infection = 5.0%
Four F 1 Plants *3
20
. 11
8
II
19
Betzes/Yesilkoy
X Yesilkoy - Kaya
Yesilkoy: Infection = 11.6%
Four F 1 Plants 0
7
10
17
I4
24
Betzes/Yesilkoy
X Betzes - Kaya
Betzes: Infection =,5.7%
Four F 1 Plants 0
.
10
® T h e a v e r a g e i n f e c t i o n p e r c e n t a g e o f s e e d l i n g s from the
respective recurrent parent in each baekcross trial
was used as the upper limit of the resistant class,
k 30-40 seedlings were read from each BC
plant.
0 60-80 seedlings were read from each BC F 1 plant.
Discussion
Konak (1983) proposed that Betzes and Tokak each had
at least one dominant gene for resistance.
determine
locus.
whether
The
data
or not these
of
seedlings
He
could not
genes were at the same
from
the
F1
and
Fg
generations do not refute that Betzes and Tokak each have
58
one dominant
graminea
gene for resistance when inoculated with P.
isolate, MT6.
specific
parents
expected
r a t i o , no
proposed.
and
However,
the
because
F2 generation
conclusions
as
seedlings from
fit
to gene
the
same
action
are
The infection reactions of seedlings from the
BC F2 families indicate that the suggested dominant genes
are not at the same locus, and that genes other than two
dominant genes are involved in this cross.
Dominant
gene
action is also one possibility in the
Betzes-Tokak crosses when seed is artificially inoculated
with the fa. grami nea isolate Kaya.
F i plant was observed from
from
Only one symptomatic
the inoculated
seeds
derived
this cross and its reciprocal.
The
F2
populations
from
the Betzes
X Tokak
and
reciprocal crosses do not fit a dominant gene ratio when
artificially
inoculated
populations
which
exhibited
plants.
do
with
not
the isolate Kaya.
fit
a dominant
greater
than
expected
Although
only
a
few
gene
numbers of
BC F 1 plants
The
ratio
infected
from
Betzes/Tokak backcrosses were infected with isolate
the
Kaya,
the BC F2 fam ili es from these backcrosses consisted of
greater
than
expected
numbers
of
infected
seedlings.
These findings indicate that any dominant gene action is
modif ied by other factors which
segregate in advanced
59
The lack of higher numbers of Fg and BC F2
generations.
families from these crosses prevent the analyses required
to determine oligogenic or quantitative gene action.
Betzes was
in
this
also
study.
crossed
Dominant
reciprocally
gene
action
with
was
Yesilkoy
again
not
refuted in the F^ plants and F 2 populations from these
crosses ,
but
the
similar ity
progenies'
reactions
in
conclusions
as to gene
action.
of
the se
parents'
and
their
crosses
prevented
No infected F^ plants
were found after artificial inoculation with JPj. sraminea
isolate
MT6 .
A few infected F^ plants occurred after
inoculation with the isolate Kaya.
The F 2 populations from the Betzes X Yesilkoy and
reciprocal
crosses were variable
in disease
reaction.
When inoculated with the isolate MT 6 , Fg populations from
the
Yesilkoy
ratio.
X Betzes
cross
did
fit
an expected
The F 2 populations from the reciprocal
13:3
cross did
not fit an expected ratio when inoculated with the M T 6
isolate.
Conversely,
after inoculation with
the isolate
Kaya, the F 2 populations from the Betzes X Yesilkoy cross
fit
an
expected
seedlings
from
ratio
the
of
13:3-
reciprocal
Populations
cross
did
not
of
F2
fit
an
expected ratio after inoculation with the Kaya isolate.
BC F 1 and B C F 2 seedlings' reactions supported
the model
60
with
Betzes
providing
a
single,
Yesilkoy providing a single,
Genetic
(Allard,
effect
I960).
in
gene,
is
th e
frequency
individuals
Expressivity
carrying
is
the
of
a
thes e
concepts
have
been
a
gene
degree
m a n i f e s t a t i o n of the genetic character (Allard,
Although
and
recessive gene.
penetrance
recognizable
dominant
of
I960).
discussed
in
inheritance studies (Metcalfe and Helgason, 1962; Boyd,
1 9 6 6 ; Khan,
calculate
1 96 9 ; A Io n , et
a l .,
1974),
a method
such effects is not wid el y accepted.
to
Wells
(1 9 5 8 ) proposed one method of calculating such effects in
an
inheritance
study
Lagerh.-Hordeum
of
vulgare
the
L.
Ostilago
horde!
interaction.
Another
(Pers.)
method
has been used in relation to the inheritance of barley
leaf
s t ri pe
Hockett,
disease
personal
Parent
from
crosses.
(Konak,
1983;
E.A.
communication).
s e e d l i n g s ’ reactions
distinguished
th e s e
resistance
and
The se
could
not
F g seedlings'
results
may
be
easily
be
reactions
in
due
to
penetrance and expressivity of the resistance genes.
the
A
method of calculating penetrance and expressivity effects
is presented in Appendix Table
20.
Calculations with
these modified expected ratios confirmed the dominant and
recessive gene model proposed for the.Fg populations from
61
the Yesilkoy X Betzes cross as previously presented.
calculated
r a ti o
populations
also
from
the
fit
the
Betzes
results
X Yesilkoy
of
The
the
cro s s
Fg
when
inoculated with isolate MT 6 .
The calculated ratios using
this method did not increase
the fit of the results from
the
Betzes
X
Yesilkoy
and . reciprocal
crosses
after
inoculation with the Kaya isolate.
These
support
results
to
the
with 2a. gramine a
dominant
proposed by Konak (1983).
also
gene
hypothesis
A distinct
proposed for the cultivar Tokak.
study
with
the
cultivar
Yesilkoy
M T 6 lend
isolate
in
Betzes
dominant
gene is
Results from this
indicate
that
a.
recessive gene for resistance is present. The occurence
of greater than expected numbers of susceptible plants in
the BC
F
2
fam ili es and a limited
number of Fg famlies
(Appendix Table 19) indicates that a more comp lex gene
action is involved.
The Fg fam il ie s from these crosses
were not analyzed because of the limitation in the number
of famil ies tested.
This l i m i t a t i o n suggests a major
hindrance in the study of resistance in barley to leaf
stripe
disease.
Multiple
gene
and quantitative
action have been proposed in resistant
cultivars.
able to propose and determine such gene models,
gene
To be
advanced
62
generations of crossed seed with sufficient numbers of
lines must be analyzed.
A great amount of growth chamber space was required
to artificially, inoculate barley seeds with the fungus
and
to
grow
action,
the
seedlings.
To
advanced lines might
verify
better
grown to mat uri ty in the field.
have
sufficient
statistically
the
in
gene
be inoculated and
One would
replications
increase
multiple
such
precision
a
of
be able to
stu d y
to
infection
percentage means.
Also, one would be able to read the
plants
through
for
disease
their
complete life
cycle.
Multiple disease readings would facilitate the method of
determining
resistance
proposed
by
(1982; Rosenkranz and S c o t t , 1984).
Scott
and
Rosenkranz
Field inoculations
of a non-native pathogen (in Montana) present problems.
Either the isolati on of such a disease
planting of
the
nursery
nursery in another area may
or the
provide a
solution.
. The
finding
gene
actions
presented
Isenbeck found
by
det er mine d
Isenbeck
Fg lines
here
(cited
agree
in
Ar n y ,
with
a
I 945).
that were more susceptible
than
either parent in a cross between two resistant varieties.
Arny
(1945)
inheritance
also
found
dom inant
of resistance
to leaf
gene
action
stripe
in
the
disease.
He
63
also proposed incomplete dominance along with a number of
factors,
partial
resistance.
Suneson (1950)
incomplete
resistance
dominance,
dominance
and
and
a mod if ying
proposed
two
a gene
recessive
to leaf stripe disease.
gene
for
providing
genes
for
Nilsson ( 1 9 7 5 ) has
also proposed major gene control for resistance in barley
to leaf stripe disease.
Knudsen
disease
(1980). found
reaction
of
I4 5
continuous
barley
var iation
cultivars
in the
that
were
naturally inoculated with JLt eraminea (to a population of
the pathogen).
Tekauz (1983) has suggested that the high
resistance
to
leaf
monogenic.
Although near isogenics of Betzes ('Shabet1,
lErbet*)
stripe
disease
are resistant to the disease
in
Betzes
is
not
(Metz and Scharen,
1979), a cultivar derived from a cross using Betzes and a
more
susceptible
('Klages').
than single,
especially
isolate,
pa re n t
Results
dominant
after
( ' D o m e n 1) was
susceptible
presented here indicate
that more
gene action is involved in Betzes,
inoculation
with
the
eraminea
Kaya.
In conclusion single, dom inant genes in Betzes and
Tokak and a recessive gene in Yesilkoy for resistance
P. erami nea were proposed.
to
The gene action seems to be
more complex than these prelim ina ry models.
Genes for
64
resistance
to P. gra min ea are available in barley,
but
the statement of Shands and Arny (I 94 4) remains valid:
"Before
should
ass um in g
a varietal
artificially inoculate
is interested,
using local
response
an investigator
the varieties in which
cultures..."
he
65
CHAPTER 5
SUMMARY
This
barley
study
to
on
the
Pvrenon h o ra
inheritance
eraminea
of
Ito
resistance
et
Ku rib.
in
was
a
continuation of previous research conducted at Montana
State University (Metz and Scharen,
and
Riesselman,
studies
oh
Va riabi lit y
I 9 82;
Konak,
1979; Johnston,
I 983).
Results
inheritance
have
of
percentages
infection
been
inoculation methods has been reported.
Metz,
of
the
inconclusive.
with
different
Therefore, a more
efficient and stable inoculation method was sought.
Konak
profusely
(1 9 83) reported
on
a rich
that JLx. eraminea
medium.
I used
this
grew
m e diu m
more
and
several other media to test the stability of virulence qf
P. eraminea
isolates
when
the isolates were
continuously on the media over time.
tested were amended with wheat bran.
of jLa. era min ea is dormant w i t h i n
barley kernel,
seemed
cultured
Two of the media
Since the mycelium
the
pericarp
of
the
the en han cem ent of growth on wheat bran
plausible.
JLl
eraminea
sys te mica ll y
infects
66
barley
plants
during
emergence.
Therefore,
diffusate and an extract of germinated,
barley
standard
leaf
culturing media
piece
agar).
Other media
(W A , V 8 juice,
Although
a
barley seeds were
used as an am en d m e n t for two other media.
used were
both
no
and
significant
differences between means of the cultured isolate were
found,
barley
it was indicated that the diffusate of germinated
seed
was
beneficial
to
inf activity
by
the
P.
graminea isolate tested.
Variab ili ty in disease reacton of specific barley
cultivars has also been reported in this host-path ogen
interaction.
I collected
a number
of _P_i_ g r a m i n e a
isolates from MT, USA, North Africa, and the Near East.
Twenty-four
inoculated
conclusions
virulence
variabilit y
of
to
thes e
three
were
barley
reached
associated
of
isolates
the
disease
artificially
cultivars.
as
with
were
to
No
different
particular
reaction
in
specific
levels
r e gio ns .
the
Summit indicated that a low level of resistance
of
The
cultivar
to some
isolates of £*. graminea is present in this cultivar. ,
Differential interactions between specific barley
cultivars and P. gra min ea isolates were recorded.
differences
betw een
means
of
the
isolate
X
The
cultivar
interactions were significant in an analysis of variance.
67
These
results
support
hypotheses
of
specific
resistance
in barley to certain virulence groups of JEi. araminea.
The
primary
purpose
of my thesis research was to
identify genes for resistance in barley to infection by
P. s r a m i n e a .
The m e c h a n i s m s of resistance in barley to
this disease are not understood.
To study inheritance of
resistance three parents were crossed,
and plants were
tested from the parent to the Fg and BC Fg generations.
A
standardized
inoculation
method
and
controlled
environment were used in testing seed lots for either the
presence
or
from
F 1 and
the
presence
of
disease
F 2 generations
symptoms.
did
not
R e sul ts
refute
the
of dominant gene action in Betzes and Tokak and
recessive
increased
families,
absence
gene action in Yesilkoy.
susceptib ili ty
inheritance
of
However,
seedlings
of resistance
in
with
the
BC
the
F2
in Tokak and Betzes
was determined to be more complex than the dominant genes
model.
68
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H e l m i n t h o s p o r i u m stripe resistance in
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Mathre, D. E.
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Inheritance
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S. G.
1978.
Etiology and epi dem io lo gy of the
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semi-arid environment.
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Metz, S. G., and A. L. Scharen.
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Production of
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cornmeal, and potato-dextrose agar.
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B.
1975.
Resistance
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Barley Genetics III.
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Nissen, S. J., and,M . Elliott Juhnke.
1984.
Integrated
crop management for dryland small grain production
in Montana.
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Paxton, G. E.
1922.
Studies on Helminthosnorium species
f o u n d on c u l t i v a t e d
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Platenkamp, Reyna.
1976.
Investigation on the infection
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Kgl. Vet. -og Landbohojsk. Arsskr, 1 976:4964.
Prasad, M. N., K. J. Leonard, and C. F. Murphy.
1976.
Effects of temperature and soil water potential on
expression
of
barley
stripe
incited
by
Helminthosnorium gramineum.
Phytopathology 66:631634 .
Richardson, M. J., A. M. Whittle, and M . Jacks.
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in cereals.
Pathology 25:21-30.
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Plant
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Rosenkranz, E., and
the number of
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Phytopathology
G. E. Scott.
1984.
Determination of
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74:71-76.
Scott, Gene E., and Eugen Rosenkranz. 1982.
A new
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P. K., and S. Amu Singh.
1979.
Inducing
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Temper atu re studies on stripe
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1944.
Stripe reaction of
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1934.
Variati on in
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Drechslera
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Can.
J. Hot.
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W. R., and D. C. Arny.
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Histologic
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The perithecial and
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_____ .
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Pathogenesis and genetics of net-spot
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Resistance
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I 9 8 3 . Reaction of Canadian barley cultivars
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Factors
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_____ . 1 976b.
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APPENDIX
75
Table 14:
6
P o t a t o d e x t r o s e aga r
germinated, barley seed.
80-seed lots
39 g
1000 cc
plus
diffusate
Summit barley seed
BBL Potato dextrose
Distilled water
of
agar
1.
Surface disinfect the seed lots
then, 3-4 min in 10% Clorox.
I min in 50% E T O H ;
2.
Dry the seed lots 8-12 h on paper towels in a clean
air chamber.
3•
Place each 80-seed lot on filter paper in a Petri
dish; add 5 cc sterile distilled water;
cover with
filter paper and Petri lid.
4.
Let seeds germinate at room temperature for 3 days.
5.
Soak ge rmi nat ed seed 10 min in 25 cc
water; strain liquid throug h.t wo layers
cloth.
6.
Add germinated seed diffusate to flask measuring the
liquid as part of the one liter water.
7.
Autoclave m ed i u m 20 min at 121 C.
distilled
of cheese
76
Table 15:
6
Potato dextrose agar plus extract of ground,
germinated, barley seed.
80-seed lots
39 g
1000 co
Summit barley seed .
BBL Potato dextrose agar
Distilled water
1.
Surface disinfect the seed lots
then, 3-4 min in 10 % Clorox.
I min in
2.
Dry the seed lots 8-12 h on paper towels in a clean
air chamber.
3•
Place each 80-seed lot on filter paper in a Petri
dish; add 5 cc sterile, distilled water;
cover with
filter paper and Petri lid.
4.
Let seeds germinate at room temperature for 3 days.
5.
Add 25 cc distilled water to each seed lot and grind
with a mortar and pestle; blend mixture for 3 min at
high s p e e d in a b l e n d o r (Wa rin g, e .g .) ; s t r a i n
mixture through two layers of cheese cloth.
6.
Add ground extract to flask m e asu ri ng the liquid as
part of the one liter water.
7.
Autoclave medium at 121 C.
50%
ETOH;
77
Table 16:
Two-factorial
analysis
of
variance
of
cultured isolate experiments and comparison of
culture means.
Source
Planting Dates
Media
Dates X Media
Error
df
3
8
24
36
S. S.
M . S.
I .57
0.27
0.31
0.19
0.52
0.03
0.01
F-value
99.34
6.42
2.49
P-value
. 0.00
0.00
0.007
0.005
Means Comparison:
LSD at 0.05 (using error mean square)
Nonihoculated
PDA + 2 °
PDA + 20°
B L P Ad
PDA + seed ext.®
Water agar
B L .Ext.f
V 8 juice
PDA + seed dif.6
cd
O
O
O
0.13
0.17
0 .17
0.17
0 .18 ,
0.18
0.19
0.23
A
B
BC
BC
BC
BC
BC
BC
C
.
a In the ANOVA, 0.00? was entered as 1.0 X IO-^.
b PDA amended with 2 g/1 spring wheat bran.
0 PDA amended with 20 g/1 spring wheat bran.
d Barley leaf piece agar (Teviotdale and Ha l l , 1976a).
e PDA amen ded with ground, germinated, barley seed,
extract (Appendix Table 13)«
f Barley leaf extract m e d i u m (Konak, 1 9 8 3 , but with 30
g / 1 fresh barley, leaves) . .
6 PDA amen ded with germinated, barley seed, diffusate
(Appendix Table 12).
78
Table 17:
Comparison of emergence percentages of barley
seedlings from the parent and check cultivars.
Cultivars
Germ.
Summit
Lami
Betzes
Tokak
Yesilkoy
98.7
97.9
91 .9
90.0
6-week
Emerg.a
Noninoc.
Emerg.^
I no c.
Emerg.
97.3
94.0
95.0
80 . 2
85.4
91 . 2
88.5
88.9
81 .9
83.9
85.0
87.6
91 .9
—— ——
90.0
Infection0
64.9
88.8
4 .2
7.3
17.5
a The 6 -we ek eme rge nce is the percentage of emerged
plants
that had ge rmi nat ed in the g erm in ati on test.
b Nonin oc ula te d em erg enc e is the percentage of emerged
plants 6 weeks old after seed lots had been placed in
the layered
PDA + 4 medium without fungus.
0 Average infection percentages of emerged plants are
given from the same seed lots that were counted for the
inoculated emergence.
Table 18:
Analysis
of
covariance
with
emergence
percentages as the covariable and infection
percentages as the dependent variable of
see tilings from the parent and check cultivars.
Source
Emergence 3
Cultivars
Isolates *3
Cv *s X Isolates
Error
df
S. S.
M. S.
F-value
I
4
0.012
6 .789
0.012
0.711
. 107.900
2
8
0.066
29
0.456
0.182
1.6 97
0.033
0.023
0.016
P-value
0 .406
2.096
0.000
0.140
1.443
0.221
a An arc sin t r a n sf orm at ion of the percentage data was
made before calculating the analysis of covariance,
b I s o l a t e s used in the i n o c u l a t i o n of see d w e r e P .
eraminea isolates MT 6 , Kaya , and RPB.
A noninoculated
check was not included in the calculations.
79
Table 19 • Number of Fg families resistant and -segregating
after inoculation with two Pvrdnobhora araminea
isolates (30-80 seedlings were read in each
family).
Seed Source
------------ Families----- ----Resistant
Segregating
Total
Betzes X Tokak - MT 6
Betzesa : Infection = 0.0%
Tokakb : Infection = 3-7%
i»0 2 -a c
35
5
40
3
37
.40
24
16
40
16
24
40
14
20
26
20
40
40
Betzes X Yesilkoy - MT 6
Betzes: Infection = 3.0%d
Yesilkoy: Infection = 19.1%
3 9 8 -a
12
28
40
Yesilkoy X Betzes - MT 6
Yesilkoy: Infection = 14.2%
Betzes: Infection - 8.0%
408-a
16
24
. 40
Tokak X Betzes - MT 6
Tokakb : Infection = 3-3%
Betzes: Infection = 0.0%
*116-3
Betzes X Tokak - Kaya
Betzes: Infection = 9.8%
Tokakb : Infection = 12.3%
402-a
Betzesb : Infection = 10.0%
Tokak:
Infection = 9.8%
405-a
Tokak X Betzes - Kaya
Tokakb : Infection = 11.4%
Betzes: Infection = 5.9%
416-a
4 16 -b
80
Table I 9 (cent *d ).
Seed Source
-------- ---- Families-------- Resistant
Segregating Total
Betzes X Yesilkoy - Kaya
Betzese : Infection = 10.0%
Yesilkoye : Infection = 15.8%
398-a
13
398-b
17
24
20
37
37
Yesilkoy X Betzes - Kaya
Yesilkoye : Infection = 26.2%
Betzese : Infection = 3.1%
40 8 - a
I
38
39
Yesilkoy: Infection = 27.6%
Betzes : Infection = 13 •5%
409-a
18
20
38
a Disease readings of seedlings from the specific parents
are given preceeding the Fg data.
Seedlings' reactions
from the bulk parents and check cultivars are given in
Tables 5 and 10 respective to the specific cross and
isolate.
b The greater infection percentage of the specific
parents was the upper limit of the resistant class.
0 Data from inocul ati on of Fp seedlings was given in
Tables 6 , 7, and 11 respective to the specific cross
and isolate.
^ The infection percentage of seedlings from the
resistant parent, Betzes, was used as the upper limit
of the resistant class.
e Specific parents were not tested due to insufficient
seed, numbers.
Infection percentages of bulked seed
lots are given.
81
Table 20:
C a l c u l a t i o n s of g e n e t i c r a t i o s w i t h
effects of penetrance and expressivity.
the
Yesilkoy X Betzes - MT 6
Lami (check):
Infection = 88.3$
Yesilkoy:
Average of bulked and specific parent
infections = 13.0$
B e t z e s : Average of bulked and specific parent i n f e c ­
tions = 4.20$
In a 13:3 ra t i o the 13 r e s i s t a n t p l a n t s have a
dom inant gene in 12 of the resistant plants (from
B e t z e s ) and a d o u b l e r e c e s s i v e in one of the
resistant plants (from Yesilkoy).
Therefore, the percentage of resistant plants that are
infected and show symptoms can be calculated.
12(0.04201
±
is
I (0 .I? Q 7 )
= 0 .048?
Susceptible plants without symptoms (escapes) are also
found.
I . 0 0 0 - 0.883 = 0.117
When more susceptible plants are without symptoms than
resistant plants that are with symptoms, a subtrac­
tion is made.
0.117 - 0.0487 = 0.06823
This fraction of symptomless, susceptible plants is
then taken from the susceptible class and added to
the resistant class.
13:3 = 0.8125:0.1875
or, with penetrance and expressivity calculations:
1 3 : 3 becomes
0.81 25 + (0.1 875 X 0.06 83): 0.1 87 5 - (0.1 875 X 0.06 83)
= 0.8253:0.1747
82
Table 20 (cont1d .).
Betzes X Yesllkoy - MT 6
Lami (check):
Infection = 67.4%
B e tz e s :
A v e r a g e of b u l k e d and s p e c i f i c
infections = I..9 2 %
Y e s i l k o y : Average of bulked and specific
infections = 15.7%
Calculations
ratio.
follow
as
abovefor
1 2 ( 0.01021
+
^g
pare nt
parent
an expected.
13:3
1 ( 0.1571
= 0.0298
I - 0.674 = 0.326
- 0 . 0 2 9 8 = 0.296 ( the fraction of susceptible
plants that appear resistant)
0.326
13:3 = 0.8125:0.1875
or, with penetrance and expressivity calculations:
13:3
becomes
0.81 25 + (0.1 87 5 X 0.296 ): 0.1 87 5 - (0.1 87 5 X 0.2 96)
= 0 . 8 6 8 :0.132
MONTANA STATC __________ _ __
■■
Iv v Iu o y o
^
N3T8 Ruff, R. L.
R838
Comparative virulence of
cop.2 Pyrenophora graminea Ito ...
I S SUED T O
D A T E
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MAIN
N378
R838
cop. 2