III

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IIIIIIIII

INHERITANCE 0F RESISTANCE TO COVERED SMUT
IN SEVERAL HYBRIDS OF BARLEY

Thesis Ior ”19 Degree of pI'I. D.
MICHIGAN STATE UNIVERSITY

Charles Cottingham
1958

IHESIS

This is to certify that the
thesis entitled
DIHERI'BLNCE CF RESIS'mNCE '10 COVERED SMUT
IN SEVERAL HIBRIDS 0F BARLE!

presented by

CHARLES COTmIIGHAM

has been accepted towards fulfillment
of the requirements for

P1111). degree in Plant PaflIOJ-Og

 

Major pr essor

Date «Inna 20. 1958

0-169

 

 

   
     

  

L! B R A R Y
Michigan State
University

 

INHERITANCE OF RESISTANCE TO COVERED SMUT

IN SEVERAL HYBRIDS OF BARLEY

by

CHARLES COTTINGHAM

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1958

Approved by .

 

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ABSTRACT Charles Cottingham

1

The probable origin of barley has been suggested as
Ethiopia and Asia. Barley is attacked by many fungi which’are
instrumental in decreasing yields. Covered smut.caused by
Ustilago hordei (Pers.) Lagerh. is one of these fungi.

This investigation was concerned with the inheritance of
resistance to race 6 of Q. hordei, and the inheritance of morpho-
logical characters, in four non-commercial varieties of barley:
Pannier, Odessa, Chevron and Jet. Pannier and Odessa have 6-row
heads, covered caryopsis and blue aleurones. Chevron has a 6-row
head, covered caryopsis and a white aleurone. Jet has a 2-row
head, naked caryopsis, and a black aleurone.

Inoculations of seeds were made by the vacuum, and the
hand-dehulled methods. Theseimethods placed the covered smut
spores within close proximity of the elongating coleoptiles which
was necessary to initiate infection.

Inheritance of resistance to Q. hordei race 6 in the crosses
between Pannier x Odessa, Pannier x Chevron and their reciprocals,
and Pannier x Jet was explained on the basis of two factor pairs.
One factor pair was found to govern the characters, covered versus

naked caryopses, and black versus blue aleurone in the cross,

Pannier x Jet.

INHERITANCE 0F RESISTANCE TO COVERED SHUT

IN SEVERAL HYBRIDS OF BARLEY

by

CHARLES COTTINGHAM

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1958

J f)-

N

h\

ACKNOWLEDGMENTS

This study was carried out under the guidance of Dr. Richard

L. Kiesling and I wish to express my sincere gratitude for his aid.
The author is particularly indebted to Dr. John E. Grafius for his
help in analyzing the data. Appreciation is extended to Dr. W. D.
Baten for help with the statistical analysis. I express a special
note of gratitude to my wife, Laura, and my sister, Ethel, for

their assistance and encouragement throughout the entire program.

To all others who contributed to this study but go unmentioned, I

am deeply grateful.

TABLE OF CONTENTS

PAGE

INTRODUCT I ON 0 O O O O O O O O O O O O I O O O O O O O O O C 1

 

REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . 2
A. History of Covered Smut Organism Ustilagp hordei

(PerSO) Lagerh. O O O O O O O 0 O O O O O O O O O 2

B. Environmental Relationships . . . . . . . . . . . . . 3

C0 InOCUIation MCthOdS o o o o o o o o o o o o o o o o o 5

D. ReSiStant varieties O O O O O O O O O O O O O O O O O 7

E. Inheritance of Morphological Characters . . . . . . . 8

Colored versus Naked Caryopses . . . . . . . . . . . 8

BlaCk versus Blue Aleurone o o c o o o o o o o o o 8

F. Inheritance of Resistance to Race 6 g. hordei . . . . 8

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . 11

A. Crosses Inv01ved o o o o o o o o o o o o o o o o o o o 11
B. Field Trials or F3 Plants 0 o o c o o o o o o o o o o 12

C. InOCUIation plethOdS o o o e o o o o e o o o e o o o o 12
D. Greenhouse Test of the F4 Plant . . . . . . . . . . 13
E. Classification of Diseased Plants . . . . . . . . . . 13

EHERIMEINTAIJ RESULTS 0 O O O O O O. O O O O O O O O O O O O O 15

A. Reactions of Parental Varieties, Odessa, Jet, Chevron,
andPannier....................15
B. Inheritance of Resistance to Race 6 of Q. hordei . . . 15

Pannier x Odessa Crosses . . . . . . . . . . . . . . 15
Pannier x Chevron o o o o o o o o o o o o o o o o o 17
Pannier x Jet . . . . . . . . . . . . . . . . . . . 19

C. Inheritance of Agronomic Characters in F3 Families . . 19

Black versus Blue Aleurone . . . . . . . . . . . . . 19
Covered versus Naked Caryopses . . . . . . . . . . . 19

DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O O O C 24
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . 30

LITERATIJRE CITFJD O O O O O O O I O O O O O O O O O O O O O 31

LIST OF TABLES

TABLE PAGE

1. SynonyIIBOfgohordei ooooooooooooooo 4

2. Reaction of Parental Varieties to Ustilago hordei
(Pers.) Lagerho Race 6 o o o o o o o o o o o o 10

3. Reaction of Parental Varieties Used as Checks in the
F4 Progeny Tests to Race 6 of Ustilago hordei . 16

4. Chi-square Test of F2 Progeny of the Cross Pannier
XOdessa....................16

5. Chi-square Test of F Families of the Cross Pannier
x Odessa InoculaEed with Race 6 of g. hordei . . 18

6. Chi-square Test of F4 Families of the Cross Pannier
x Chevron Inoculated with Race 6 of Q. hordei . . 18

7. Chi-square Test of F4 Families of the Cross Pannier
x Jet Inoculated with Race 6 of U. hordei . . . 20

8. The F3 Segregation of Black and Blue Color of Kernels
in Barley Hybrids of the Cross Pannier x Jet . . 20

9. Agronomic Characteristics and Segregation of F

Families in the Cross Pannier x Jet . . . g

. . . 21

10. F3 Segretation of Covered Versus Naked Caryopses
in Hybrids of the Cross Pannier x Jet . . . . . 23

11. Proposed Genotypes for Pannier, Jet, Chevron, and
Odessa 00.0000000000000000026

12. The Reaction of F4 Progeny from F3 Genotypes of the
Cross Pannier x Jet to Race 6 of U. hordei . . . 28

INTRODUCTION

Barley was cultivated as early as the Stone Age by the
lake dwellers of Switzerland. The probable origin of barley has
been suggested as EthiOpia and Asia. The cultivated species of

barley may have originated from Hordeum agricorithon, a wild 6-row

 

barley with a brittle rachis found in eastern Tibet or Hordeum
spontaneum, Koch a 2—row type.

Barley is grown throughout the world, and thrives best in
the temperate regions where cool climates prevail. Barley ranks
fourth in economic importance among the cereal crops, and is ex-
ceeded in value only by wheat, oats and corn. The principal barley-
producing states are Minnesota, North Dakota, California, South
Dakota, Nebraska, Wisconsin and Michigan. More than 70 million
bushels of barley are used annually for malting purposes. Barley
is also used as food for humans, and feed for animals.

Barley is attacked by many microorganisms. Among the im-
portant fungi attacking barley are the smuts, loose, semi-loose,
and covered smut. Covered smut of barley, caused by Ustilago
hordei (Pers.) Lagerh. attacks the barley plant and replaces the
head and kernels with smut spores. The purpose of this thesis is
to study the inheritance of resistance to race 6 y. hordei in four
varieties of barley, as well as the inheritance of specific morpho-

logical characters in one of the crosses (Pannier X Jet).

REVIEW OF LITERATURE

A- History of Covered Smut Organism Ustilago
hordei (Pers.l Lagerh.

A history of Q. hordei was prepared by Kellerman and
Swingle (21). They reported that Pliny and Theophrastus probably
knew of oat smut; but in their writing, all smuts were confused
with the stinking smut of wheat. Tragus illustrated the organism
in 1552, and placed it in the genus, Ustilago.

Lobeluir, in 1591, was one of the first investigators to
separate the smuts of barley from those of oats. He referred to

the forms on barley as Ustilago polystichi, and g. hordei distychi.

 

There was no means of knowing whether he included one or both of
the barley smuts under these names. Bauchin, in 1596, also separ-
ated barley from oat smut, calling the former Ustilago hordeace.
Persoon, in 1801, was the first to give a valid description of the
barley covered smut fungus. His description of the organism was
"Uredo (Ustilago) segetum 2 Uredo hordei, Pseudoperidia subelliptica
reggloso sulvere latente." This reference to the powder as latente
would indicate that he had the barley covered smut fungus.

After Persoon, many investigators copied his varietal names
without change. Tulasne, in 1847, called the smut of barley
Ustilago 22523 g Vulgaris g Hordeacea. Jensen, in 1888, was the
first to clearly separate covered smut from the other smuts on
barley under the names Ustilago segetum var. hordei Essie Jensen,

2

and Ustilago segetum var. tecta. He later called it Ustilago
tecta hordei Jensen. This classification differentiated between

Ustilago hordei and Ustilago nuda, but not Ustilago nigra.

 

 

Kellerman and Swingle reported the synonyms of Ustilago
hordei as in Table 1 (21). However, in 1889, the binomial was

changed to its present name, Ustilago hordei (Pers.) Lagerh. (12).
B. Environmental Relationships

Infection by covered smut cannot begin until two prelimin-
ary processes are completed (20). First, the coleoptile must be
exposed. This process required from 24-72 hours. Secondly, the
smut spore must germinate and produce sporidia and infection tubes.
This process was completed within 48 hours. Infection of the cole-
optile was no longer possible after the coleoptile had ruptured and
the first green leaf had pushed one centimeter beyond it (20).

Planting the inoculated kernels deeper in the soil has
been shown to produce more smut infected plants than shallow plant-
ints (20, 40). This was probably due to the longer exposure to the
pathogen by the susceptible tissues (20, 40). When seeds were
planted deeply in wet soil the balance of host and parasite was
disturbed. The fungus became destructively parasitic, killing some
of the already weak seedlings at an early age. This prevented a
portion of the infected plants from showing smutted heads in the

crop (11, 20). A four year experiment produced 2.? to 115 times

TABLE 1

SYNONYMS OF g. HORDEI

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Year Synonym

1552 Ustilago fragus - De Stisp

1591 Ustilago polystichi - Lobelius

1591 Ustilago hordei distichi - Lobelius

1596 Ustilago hordeacea - C. Bauhin

1767 Chaos Ustilago - Linne

1791 Reticularia segetum - Bulliard

1801 23232 (Ustilago) segetum a Uredo Hordei
- Persoon

1809 Caeoma segetum - Link

1813 Ustilago segetum - (Bulliard), Dittmar

1815 Uredo carbo 2 Hordei - De Candolle

1833 Erysibe uera 3 Hordei - Wallroth

1837 EEEEE carba Hordei - Philippar

1847 Ustilago carbo 2 Vulgaris g Hordeaceae -
Tulasne

1856 Ustilago segetum b Hordei - Rabenhorst

1888 Ustilago segetum var. £3332 - Jensen

1888 Ustilago segetum zgr. hordei tecta - Jensen

1888 Ustilago hordei - Rabenhorst

1889 Ustilagg hordei var. tests - Jensen

1890 Ustilago tecta hordei - Jensen

1890 Ustilago Jensenii

 

 

as much covered smut in Tennessee Winter barley sown at three inches
intiepth as in the same variety sown at a half inch (20).

Hyphae from spores on the outer surfaces of the caryopses
of covered barley varieties were delayed in penetrating the host
(36). Although the spores are normally present in that location,
penetration was prevented by adverse conditions occurring at emer-
gence time in very shallow planted seed (36). A higher percentage
of infection was obtained in experiments with hand-dehulled barley
when the temperature and moisture of the soil were under control
during the period of infection. The amount of infection was in-

fluenced by the low soil temperature at the time of planting (10).
C. Inoculation Methods

Methods of increasing the percentage of infection by H.
hordei in field trials were needed to study the inheritance of
resistance of barley varieties (42). Attempts to obtain infection
by applying spores to the kernels at planting time met with little
or no success (42). Some varieties showed slight infection while
others did not (18, 33).

Barley kernels were then dehulled by hand, and spores were
aF’Pllied directly to the kernels. This method produced approxi-
‘31:er 25 percent more diseased heads than the checks which were
dusted with the smut spores without dehulling (1, 18, 42). High
infection rates were obtained by the hand-dehulled method in the

8“Sceptible variety Chevron (22).

Barley kernels dehulled with concentrated sulphuric acid
and inoculated with the covered smut fungus produced approximately
the same percentage of infected plants as with hand-dehulled,
inoculated kernels. Complete dehulling was not found necessary
for good infection. A thin hull over the entire kernel produced
optimum results (4, 5, 6). However, the use of concentrated sul-
phuric acid led to complications which made exact analysis of the
reactiontfi’the hybrids impossible (19). Increased severity of
infection by.y. hordei aided by the removal of the hull resulted
in extreme distortions of seedlings and consequent failure to
emerge (19). Barley seed dehulled with a smut dockage machine
produced no greater infection than unhulled seed (45).

Techniques for inoculation without dehulling have been
reported (8, 28, 34, 37, 38). The spore suspension (vacuum)
method of inoculation produced 75 percent infection in field
plantings of spring and winter barleys (28, 34, 38). Eighty to
100 percent infection was obtained by employing a Waring blender
equipped with dull non-cutting blades (8). The seeds were placed
ill‘the blender which contained a spore suspension and churned for
15 to 30 seconds. The time of churning depended upon hull tough-
ness (8).

The most effective covered smut inoculum consisted of
sP‘iil‘es and extensive mycelial ramifications on the pericarps of
the caryopses beneath the seed hull (37). The occurrence of this

s“bluill inoculum in nature explainedwhy surface disinfectants of

seed 1

trol

Leior:
tere I
to c0‘

cover.

covert

Iutt.
{teen
able

mas
Wrce
Itdia
Iign
Virie
IIOgE
tier”

Bath!

seed barley have been relatively ineffective in covered smut con-

tI‘Ol (37) o

D. Resistant Varieties

O. A. C. Number 21, Atlas, Velvet, Sacramento, Glabron,
Leiorrhynchum, Wisconsin Barbless Number 38, Shaw, Sol and Success

were six-row, covered barley varieties which showed some resistance

to covered smut (1). Spartan, Golden Pheasant, and Huron two-row

covered varieties, and the hull-less varieties Himalaya, New Era,
Russian, Mongolian, and Burbank were moderately resistant to
covered smut (l).

The susceptible variety Odessa (C. I. 934) showed fewer

smutted spikes when inoculated kernels were field planted than

greenhouse planted inoculated kernels. However, under more favor-

able environmental conditions the smut percentage in greatly in-
creased. The variety Chevron (C. I. 1111) produced intermediate
Percentages of covered smut (22). Jet (C. I. 967) was an inter-
Iediate resistant variety with resistance governed by both morpho-
1"Sical and physiological characters (22). The highly resistant
Vamfiiety Pannier (C. I. 1330) produced no smutted plants in its

Progeny (22). These differences may be based upon gene combina-

tions which govern degrees of resistance or susceptibility to the

pathogen (22) .

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E. Inheritance of Morphological Characters

Covered versus Naked Caryopses
A study of the cross between Canadian ThrOpe x Guy Mayle

produced a 3:1 ratio in the F generation with covered caryopses

2
dominant. Canadian ThrOpe was a 2-row, covered variety and Guy
Maylewas a 6-row, naked variety. It was concluded that a single
factor difference existed between covered and naked caryopses (25).

Similar results have been found by other investigators (3, 7, 9,

Bladk versus Blue Aleurone

 

Black versus blue aleurone was governed by a single factor
pair in a number of crosses reported. Black was dominant (3, 7,

9, 15, 16, 27, 29, 31, 44).
F. Inheritance of Resistance to Race 6 2. hordei

Numerous morphological differences among barley varieties
led to a study of the relationships between these morphological
characters and resistance or susceptibility to covered smut (14).
Little or no correlation was found between factors for smut sus-
ceptibility and those for height of plant in a cross between the
barley varieties Glabron x Trebi (14). Little or no correlation
between lemma color and other morphological characters and the fac-
tor for resistance was found (14).

No infection was found in the F generation of a cross be-

2

tween Cape x Kwan (28). Infection ranged from O to 27.5 percentage

classe

tien.

tance
Brachy
Iajor
cross,
crosse:

reacti<

classes in the F3 families. Sixty-one families showed no infec-

tion. It was concluded that Kwan contained more than two dominant
genes for resistance to covered smut (28). Inheritance of resis-
tance to race 6 covered smut in the cross between Chevron x
Brachytic and Colsesstrachytic was found to be governed by one

major factor pair. In the F1 generation of the Chevron x Brachytic

cross, resistance was dominant. A 3:1 ratio was obtained in both

crosses by segregation of the F based upon F progeny tests. Smut

2 3

reaction was found to be independent of five plant characters (32).

Iariet]

Odessa

Jet

Caei'rc

Pannit

3I’eri

Ila:

10

TABLE 2

REACTION OF PARENTAL VARIETIES TO USTILAGO
HORDEI (PERS.) LAGERH. RACE 6 (22)

 

 

 

Smut
C. I. Smut Inf.
Variety No. Reaction Temp. Plants-
Percent
Odessa 934 Susceptible 24 C 94
Jet 967 Resistant-
Intermediatea 24 c 20
Chevron llll Intermediateb 24 C 53
1330 Resistant 24 C 0

Pannier

 

aPericarp removed over embryo in this variety.

bFlag leaf only showed smut sori.

COVE!

“I“!

this

MATERIALS AND METHODS
A. Crosses Involved

The covered smut, resistant variety Pannier (C. I. 1330),
was crossed with a susceptible variety, Odessa (C. I. 934), a sus-
ceptible variety, Chevron (C. I. 1111), and a resistant variety,

Jet (C. I. 967). (Table 2) After pollination the heads were
covered with aluminum foil to prevent chance pollination. F plants

1

were grown in pots in the greenhouse to provide F2

Because of the differences in the morphological character-

seed.

istics of Jet and Pannier, seeds from individual F plants were

2

grown in separate rows in the field. Seed from individual F2 plants
of the other crosses was also grown in separate rows in the field,

but no studies were made of morphological characters.

A portion of the F seed of Pannier x Odessa and its recip—

2
chal was inoculated with race 6 Q. hordei and grown to maturity

in the greenhouse in 1956. A random sample of these F plants

2
furnished seed for the F3 progeny which were field grown to observe
morphological characteristics. Two tests were conducted with the

remaining portion of the F2 seed to determine its reaction to cov-

ered smut. The F2 plants of the Pannier x Jet and Pannier x Chevron

crosses were not tested for smut reaction.

11

12

B. Field Trials of F3 Plants

In the spring of 1957 seeds from single F2 plants were
planted in the field in separate rows. These rows were designated
as F6 families. The F3 families were harvested and packaged as
individual plants within a family. Each envelope contained the
seed from a single plant of a single F3 family. Readings of plant

type were made for the plant characteristics of each F3 family.
C. Inoculation Methods

Inoculations were made by the vacuum method in the F2

the Pannier x Odessa cross. The barley kernels were placed in a

of

spore suspension in vials and inoculated with g. hordei. Following
inoculation, the seeds were spread out on blotting paper and allowed
to dry before planting in 6-inch pots in the greenhouse. Readings
were made on the number of infected plants.

The hand-dehulled method was used for the other tests,
since this method was found to give the highest percentage of in-
fected plants (22). The pericarps were removed from the embryos
in Jet, which removed the morphological resistance factor. Spores
of race 6 of Q. hordei were applied to moistened kernels of barley
to obtain the maximum spore load. The emerging coleoptiles were
dusted with spores twice before they elongated the length of the
caryOpses. This elongation required from 48 to 72 hours. Dehulled,
inoculated kernels were placed in petri plates which contained

moistened filter paper inside both the cover and bottom of the plates.

a...

13

Water was added periodically to maintain the required humidity
for maximum spore germination as well as maximum seed germination.
The plates were wrapped in wet paper towels and waxed paper and
incubated at 24° C.

Seedlings were removed from the petri plates on the third
or fourth day and transplanted into rows in the soil of a green-
house bench. The seedlings were planted with the coleoptiles
pointed upward. Care was taken not to plant the seedlings more
than one inch deep. However, some of the plants had distorted
leaves and grew in an abnormal manner under the soil surface. Such
plants were uncovered and exposed, but many failed to recover. Sim-
ilar results by other workers have been reported (10, 19, 32). The
seedlings were grown at temperatures above 70° F. to aid maximum
disease development. Fluorescent lights were provided to illuminate

the plants twelve hours per day.

D. Greenhouse Test of the F4 Plants

In the fall of 1957, the F4 seeds were hand-dehulled and

inoculated with race 6 of Q. hordei. The F4 seeds were planted in

a series of five pots, and each pot contained five seeds from a

single F3 plant. Therefore, each series contained a total of 25

seeds from five F plants and represented a single F

4

E. Classification of Diseased Plants

The number of smutted plants was recorded after maturity.

Plants with a stunted appearance were examined for smut by splitting

14

the sheaths open and exposing the heads. If any diseased head or
tiller appeared, the plant was classified as smutted. Plants on

which disease readings could not be taken were omitted from the

counts.

TOT.

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at

I
8

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EXPERIMENTAL RESULTS

A. Reactions of Parental Varieties, Odessa,
Jet, Chevron and Pannier
The reactions of the parental varieties Odessa, Jet, Chev-
ron and Pannier to race 6 of g, hordei in the tests of the F4 progeny
are shown in Table 3. Odessa was highly susceptible to covered smut.
Jet, showed a resistant-intermediate reaction, while Chevron was

susceptible in its reaction to covered smut. Pannier produced no

infected plants and was classified as resistant to covered smut.
B. Inheritance of Resistance to Race 6 of g. hordei

Pannier x Odessa Crosses

Inoculation of the F2 seedlings of the cross Pannier x
Odessa and its reciprocal produced 5.2 percent infected plants as
compared to 57 percent infected plants in the susceptible Odessa
Check. This ratio of 456 non—infected to 25 infected plants in the
F2 gives a mathematical ratio of 18.2 to 1 which lies within the
Probability of a 15:1 ratio. Therefore, the results of the F2
test indicate that two factors govern the resistance to race 6 of
2. hordei in the cross Pannier x Odessa.

A chi-square test (Table 4) was applied to the data to de-
termine the closeness of fit for a theoretical ratio of 15:1. The

Chi-square was .921 and the probability was .35 . This indicated

15

 

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NEH-null)... 4/

16

TABLE 3

REACTION OF PARENTAL VARIETIES USED AS CHECKS IN THE
F PROGENY TESTS T0 RACE 6 OF USTILAGO HORDEI

 

 

 

 

4
Smut
Variety C' 1‘ smut. Temp. Inf.
No. Reaction
Plants
Percent
Odessa 934 Susceptible 24 C 80
Jet 967 Resistant-
Intermediatea 24 C O
Chevron 1111 Susceptible 24 C 33
Pannier 1330 Resistant 24 C 0

 

aPericarp removed over embryo of this variety.

TABLE 4

CHI—SQUARE TEST OF F2 PROGENY OF THE CROSS PANNIER x ODESSA

 

 

cross g. hordei Char- Obs. Theoretical Calc. No. x2 p
Race acters Freq. Ratio Freq. Value

 

Pannier Res. 456 450.9
x

Odessa 6 Susc. 25 15:1 30.1 .921 .35

 

 

 

E “ Ill—As

 

17

a good fit for a 15:1 ratio if resistance in the cross Pannier x
Odessa is governed by two factor pairs in the F2 progeny.

Inoculation of the F’ progeny of 17 F3 families of the

4
cross Pannier x Odessa demonstrated that 5 of the F3 families
came from homozygous resistant F2 plants. The 12 remaining F3
families came from 12 F2 plants that were either heterozygous re-

sistant or homozygous susceptible. This group of 12 plants cannot
be separated into its components with certainty. This gave a
mathematical ratio of 5 homozygous resistant to 12 either hetero-
zygous resistant or homozygous susceptible families. These data
indicated that two factor pairs were Operating for resistance to

race 6 of U. hordei in the F of the cross Pannier x Odessa.

4

The chi-square test for closeness of fit for a 7:9 ratio
was 1.378 (Table 5) with a probability of .25. This indicated a

good fit for a two factor ratio, and agreed with the F tests.

2

gPannier x Chevron

Fifteen of the 31 F3 families inoculated with race 6 of

g. hordei proved to be homozygous resistant and came from homozygous

resistant F plants (Table 6). The remaining 16 F families were

2 3
derived from either heterozygous resistant or homozygous susceptible
F2 plants. The latter group of 16 F:3 families cannot be further
classified with accuracy. This gave a ratio of 15 homozygous re-
sistant to 16 heterozygous resistant or homozygous susceptible F3
families which fits a 7:9 ratio with a chi-square of .256 and a

probability of .63 that resistance in the cross Pannier x Chevron

was governed by two factor pairs.

 

“

18

TABLE 5

CHI-SQUARE TEST OF F FAMILIES OF THE CROSS PANNIER x ODESSA
INOCULATED WITH RACE 6 OF E. HORDEI

 

 

cross g; hordei Char- Obs. Theoretical Calc. No. x2 p

 

 

Race acters Freq. Ratio Freq. Value
Pannier Res. 5 7.4
x
Odessa 6 Susc. 12 7:9 9.6 1.378 .25
TABLE 6

CHI-SQUARE TEST or F4 FAMILIES OF THE CROSS PANNIER x CHEVRON
INOCULATED WITH RACE 6 or g. HORDEI

 

Cross 2. hordei Char- Obs. Theoretical Calc. No. x2 p
Race acters Freq. Ratio Freq. Value

 

Pannier Res. 15 13.6
x
Chevron 6 Susc. 16 7:9 17.4 .256 .63-

 

l9

Pannier x Jet

The F; progeny of individual F3 families of the cross

Pannier x Jet were inoculated with race 6 of g. hordei. Nine of

these F3 families proved to be totally resistant which indicated

that they came from homozygous resistant F2 plants (Table 7). The

remaining 13 F3

sistance or homozygous for susceptibility based upon the reaction

families proved to be either heterozygous for re-

of the inoculated F4 plants. The latter two groups were classified
together. This gave a ratio of 9 resistant to 13 susceptible
families which fits a possible 7:9 ratio with a chi-square of .066

and a probability of .79 that a two factor inheritance was involved

(Table 7).

C. Inheritance of Agronomic Characters in F3 Families

Black versus Blue Aleurone

 

The results of the chi-square test are shown in Table 8.
Five F3 families were homozygous black and two F3 families were
homozygous blue. The remaining 15 F3 families were either segre-
gating for black or blue color and could not be further classified
with accuracy (Table 9). The chi-square (Table 8) was .052 with

a probability of .11 that black versus blue aleurone is governed

by a single factor pair, and the black character is dominant.

Covered versus Naked CaryOpses

In the cross Pannier x Jet, 6 F3 families were homozygous

covered and four F3 families were homozygous naked. The remaining

20

TABLE 7

CHI-SQUARE TEST OF F4 FAMILIES on THE CROSS PANNIER x JET
INOCULATED WITH RACE 6 or g. HORDEI

cross 2. hordei Char- Obs. Theoretical Calc. No. x2 p

 

 

 

 

 

Race acters Freq. Ratio Freq. Value
Pannier Res. 9 9.6
x
Jet 6 Susc. 13 7:9 12.4 .066 .79
TABLE 8
THE F3 SEGREGATION 0F BLACK AND BLUE COLOR 0F KERNELS
IN HARLEY HYBRIDS OF THE CROSS PANNIER x JET
Cross Char- Obs. Theoretical Calc. No. x2 p
acters Freq. Ratio Freq. Value
Pannier Black 5 5.25
x

JCt Blue 2 331 1.75 .082 all

 

TABLE 9

21

AGRONOMIC CHARACTERISTICS AND SEGREGATION OF F3 FAMILIES
IN THE CROSS PANNIER x JET

 

 

Kernel Color

Kernel Characteristics

 

Blue Black Covered Naked
1 20 23
2 17 19 5
3 9 10
4 33 33
5 5 15 ll
6 3 7 7 3
7 20 20
8 3 6 2 7
9 l5 l3 2
10 10 10 2
ll 7 15 2O 2
12 16 O 13 3
13 8 21 23 6
14 4 ll 15
15 7 19 17 9
l6 6 19 2O
17 4 13
18 9
19' 2 10 12
20 1 10
21 3 2
22 23 23

 

 

22

F3 families were either segregating for the covered or the naked
characteristic (Table 9). A chi-square test was applied to the
homozygous characters to determine the closeness of fit for a
3:1 ratio where a single factor pair is involved (Table 10).

The chi-square was 1.20 with a probability of .28 that
covered versus naked caryopses in the cross Pannier x Jet was
governed by a single factor pair, and the covered character was

dominant.

 

23

 

 

 

TABLE 10
F3 SEGREGATION OF COVERED VERSUS NAKED CARYOPSES
IN HYBRIDS OF THE CROSS PANNIER x JET
cross Char- Obs.. Theoretical Calc. No. x2 p
acters Freq. Ratio Freq. Value

Pannier Covered 6 7.5

x
Jet Naked 4 3:1 2.5 1.20 .28.

 

 

DISCUSSION

The study of inheritance of covered smut resistance in
barley was complicated by several factors. The coleoptile must
be penetrated by the fungus in its early growth to produce infec-
tion. Therefore, a method of placing the spores of the fungus in
close proximity of the emerging coleoptile must be employed.

Either the vacuum or the hand-dehulled method was employed for
this purpose. However, the hand-dehulled method presented further
complications because the fungus became increasingly destructive
when applied directly to the young coleoptiles. This resulted in
severe distortions of the leaves, failure of many plants to emerge
properly, and death of many plants before and immediately after
emergence. Other investigators have found similar results (10,
19, 32).

A second complication was the lack of an inoculation method
which would consistently give 100 percent infection with susceptible
individuals. Consequently, only 57 percent infection was obtained
by the vacuum method in the susceptible Odessa checks with F2 popu-
lation studies, while 80 percent infection was obtained in the sus-

ceptible Odessa checks in the F population studies using the hand-

4

dehulled method.
Inoculation of F2 seedlings of the cross Pannier x Odessa

produced only 5.2 percent infection from a population of 481 plants.

24

25

The low percentage of covered smut infection in this cross was
partially explained by the inefficiency of the vacuum method of
inoculation, and the death of inoculated susceptible plants. Foot

rot and seedling blight caused by Helminthosporium sativum and

 

FUsarium sp. reduced the number of surviving individuals in certain
rows.
1 E

The barley variety Pannier had an immune reaction to race 1'"
6 of E. hordei. No attempted breakdown of its resistance to race
6 has been successful. In the three other varieties, Odessa,
Chevron, and Jet increased infection was obtained by increasing
the inoculum load and raising the temperature from 16' C. to 28' C.
However, infection was obtained in all crosses made between Odessa,
Chevron or Jet and Pannier. A 15:1 ratio for resistance to race 6
of Q; hordei was obtained in all of these crosses with Pannier.
Consequently, it was concluded that an allelic series (P1 P1) ex-
isted in Odessa, Chevron, Jet and Pannier which provided weak pro-
tection of race 6 of g. hordei. In addition Pannier had a gene
(P2 Pé)which provided immunity to race 6 of H. hordei. The weakest
character was found in Odessa (P1'"' P1""), the next weakest in
Chevron (P1"' 131'"), and the strongest in Jet (P1" P1"). (See Table
IL) When severe inoculation techniques were employed the resistance
of all three varieties to race 6 of Q. hordei was broken (22).

The proposed genotypes of Pannier, Jet, Chevron and Odessa
for reaction to race 6 of E. hordei are presented in Table 11. In

the F2 test of the cross Odessa x Pannier, the data was obtained

PROPOSED GENOTYPES FOR PANNIER,

TABLE 11

JET, CHEVRON, AND ODESSA

 

 

 

Variety Genotypes

Pannier P1 P1 P2 P2
" 0'

Jet P1 P1 p2 p2
O" I"

Chevron P1 P1 p2 p2
N" "H

Odessa P1 P1 p2 p2

 

26

 

IRE ntm¢za : FA

'7’:

-. 55'

‘F

27

by the direct reaction of the F embryo. However, in the F

2 3

family ratings, F4 plants were read for smut reaction. The

genotypes of the F families of the cross Pannier x Jet which

3

would give resistant and/or susceptible plants in the F4 are

shown in Table 12. Any combination of homozygous (P1 P1), or

(P2 P2) produced all resistant plants. All other combinations
produced some susceptible plants for a 7-resistant to 9-susceptible
ratio in the F4 generation of all crosses.

Since Pannier was totally resistant, the homozygous con-
dition of P1 P1 P2 P2 prevailed. In all cases of crosses between
Jet, Chevron or Odessa x Pannier, infection was obtained in the
offspring. Jet was resistant in its reaction to g. hordei as a
check in this experiment. However, its resistance has been broken
under severe inoculation techniques (22). Consequently, a weaker
allele was pr0posed without the benefit of the stronger Pannier
gene (P1" P1" p2 p2). The allele for Chevron was P1"' P1"' p2 p2 .
The weakest allele (Plvm 1’1"" p2 p2) was found in Odessa which was
highly susceptible to race 6 of 2. hordei.

The study of morphological characteristics in the cross
Pannier x Jet was conducted because Jet was different in more than
one morphological characteristic. One factor pair was found to
govern covered versus naked caryopses, and black versus blue

aleurone. The factors for covered and black aleurone were dominant

over those for naked and blue aleurone.

 

28

TABLE 12

THE REACTION OF F4 PROGENY FROM F3 GENOTYPES

OF THE CROSS PANNIER x JET
'ro RACE 6 or g. HORDEI

 

 

Genotypes’ Reaction to Race 6
of Q. hordei

 

P1 P1 P2 P2 1 All resistant plants
P1 P1 P2 p2 2 All " "
I. 9'
P1 P1 p2 p2 1 A11
H H '0
P1 P1 P2 P2 2 All
H .
P1 P1 P2 p2 4 Some susceptible plants
P1 P1" p2 p2 2 Some " "
H H ’
P1 P1 P2 P2 1 All resistant plants
I! M -
P1 P1 P2 p2 2 Some susceptible plants
P1" P1" p2 p2 , 1 All susceptible plants

 

P1 P1 , good resistance

P2 P2 , good resistance

P1" P1", fair resistance

p2 p2 , poor resistance

29

The chi-square for black versus blue aleurone was .052.
This was within the probability range for closeness of fit for a
3.1 ratio, if these two characters are governed by a single factor

pair, and black is dominant.

SUMMARY AND CONCLUSIONS

1. Ustilago hordei (Pers.) Lagerh. was the causal organism

 

of covered smut disease presented in this study. Pannier, Chevron,
Odessa and Jet were the parental varieties of barley used. Pannier
was resistant, and Chevron had a susceptible reaction. Odessa was
highly susceptible, and Jet was resistant in its reaction to covered
smut. The added morphological resistance factor of Jet was elimin-
ated in this experiment by removing the pericarps from the caryopses.

2. The F2 and FA families in all crosses produced ratios
which indicated that resistance was governed by two factor pairs.

3. The chi-square tests indicated that one factor pair
controlled the morphological characters for covered versus naked
caryopsis and black versus blue aleurone.

4. A reduction in stand counts was apparently caused by

destruction of some susceptible plants by Q. hordei, as well as

other soil borne organisms.

1.’

8.

10.
11.

12.

13.

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.-L..

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34

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