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LIBRARY
Michigan State 3
University ‘

This is to certify that the

thesis entitled

F. SP. CEPAE IN CULTIVATED ONIONS

presented by

Jeffery w. Bacher

of the requirements for

2,?Zé/7M

INHERITANCE 0F RESISTANCE T0 FUSARIUM OXYSPORUM

has been accepted towards fulfillment

Masters degree in Horticulture

fioywtggmfi

Major professor

 

MSUisan Affirm/"inn ‘ ' "1

n, ', Institution

 

 

 

INHERITANCE OF RESISTANCE TO FUSARIUM OXYSPORUM

 

F. SP. QEBAE IN CULTIVATED ONIONS

by

Jeffery W. Bacher

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1989

 

ABSTRACT

INHERITANCE OF RESISTANCE TO S
SP. QEEAE IN CULTIVATED ONIONS

By

Jeffery W. Bacher

The inheritance of resistance to Fusarium szsgggum f.
sp. ggpag was studied to increase the effectiveness and
speed of introduction of Fusarium resistant cultivars. The
effect of inbreeding on the level of resistance was also
investigated.

Two genes, £29; (A) and Eggz (E), are proposed to
govern resistance to E. Q. gggag. Results indicated that
both A and 5 genes were partially dominant (AA > A; and BB >
Eh) and that the interaction between loci was additive (8835
> AABh or AaBB > Ath). Resistance was not epistatic;
plants with genotypes AAbb, Aabb, aaBB and aaBb were
susceptible. Three genotypes resulted in a resistant
phenotype; AAEfl, AABh, and AaBB. Genotype AgBh was
moderately resistant.

Three cycles of screening for resistance and selfing of
line 6701 resulted in an 89% reduction in the level of
resistance to E. Q. ggpag. The original level of resistance
was not restored by sib mating of S, (selfed once)
generation plants. Lethal genes did not appear to be the

major cause of this decline.

ii

    

“-' ..' . : -- 1915-253 .W quark-9L

ACKNOWLEDGEMENTS

I wish to express my sincere thanks and appreciation to
my guidance committee; Dr. M. L. Lacy, Dr. Jim Hancock and
Dr. Lowell Ewart. I would also like to thank Uma Gupta for
taking the time to discuss my research and his helpful
suggestions.

Mostly I wish to express my love and appreciation to my
family... Susan, Aaron and Nora... they are my source of
love and strength.

To Susan... for your patience and love... I dedicate my

work and my life to you.

 

TABLE OF CONTENTS

Page
LIST OF TABLES ............... .. ..... ..... ....... ....... vi
LIST OF FIGURES ................ ........ ............ .... viii
INTRODUCTION ................. .......................... 1
LITERATURE REVIEW ......... . ................. . ....... ... 3
' Pathogen: WW f. sp. seesaw. 3
I Reduction in yield ..................... . ........ 4
I Disease Management with Resistance ........... 4
I Inheritance of Resistance to Formae Speciales
of Eusarium gxxspgrum ........................... 5
I Permanence of Genetically Inherited Resistance
to Fusaria ...................................... 7
I Mechanisms of Resistance ......... . ............. . 8
I Sources of Resistance ........................... 14
MATERIALS AND METHODS ........................ .......... 16
I Screening for Resistance to E. g. gggae ......... 16
I Screening Procedure for E. Q. cgpae Resistance
in Onion ....................................... 17
I Pathogenicity Tests .......................... ... 18
I Flower and Seed Production ...................... 19
I Culture .................................... 19
I Pollination .......................... ...... 19
I Pollination Technique and Timing ........... 21
I Cleaning and Storage of Seed ............... 22
I Selection of Resistant and Susceptible Parents.. 22
I Nomenclature ................................... 23
I F1, F2, Backcross and Selfs Used in Inheritance
Study ........................................... 23
I Effect of Inbreeding on Resistance .............. 24
I Statistical Analysis ............................ 24
RESULTS AND DISCUSSION ................................. 25

I Pathogenicity of Six Single Spore Isolates of
E. Q. cepae ...................................... 25
I Screening Results for Resistance to E. Q. cepae.. 26

iv

 

 

    

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Progeny Tests of Parental Lines 6701-1CRES),

1849(SUS) ........................................ 28
Use of Genotype Frequencies to Calculate Ratio of
Resistant to Susceptible Offspring ............... 28

Gene Action of Proposed Resistance Genes Egg; (A),
EQQZ (B) and Lethal Gene 1 ......................
Chi-square Tests for F1, F2 and Backcross

Families ......................................... 38
Effect of Inbreeding on Resistance to E. Q. gepae 41

SUMMARY AND RECOMMENDATIONS ............................. 44
I Summary of Inheritance of Resistance to
E. g. gegag ...................................... 44
I Recommendations for Breeding Onions for Increased
Resistance to E. a. usage ........................ 45
LITERATURE CITED ........................................ 48

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LIST OF TABLES

Page

Pathogenicity of Single Spore Isolates of Engaginm
oxygggggm f. sp. gggag on 5 Onion Lines..... ....... 26

Analysis of Variance of F1, F2 and BC Families..... 26

Summary Statistics of Screening Data for Resistance
to Fusarium oxysgorum f. Sp. cepae..... ............ 27

Expected Genotype Frequencies of Parents, F1, F2
and Backcross Families for Cross 1849 x 6701—1.
Model I. A and B Genes are Both Partially
Dominant. Resistant Genotypes are Aflflfl, AABh and
Aaflfi .. ..

Expected Genotype Frequencies of Parents, F1, F2

and Backcross Families for Cross 1849 x 6701-1.

Model II. A Gene is Partially Dominant and B

Gene is Dominant. Resistant Genotypes are AAEE

and AAEQ .............. . . . ....... . ....... ..... 32

Expected Genotype Frequencies of Parents, F1, F2

and Backcross Families for Cross 1849 x 6701-1.

Model III. Both A and B Genes are Partially

Dominant. Resistant Parent 6701—1 is Heterozygous

for Lethal Gene 1 ............................... ... 33

Expected Genotype Frequencies of Parents, F1, F2

and Backcross Families for Cross 1849 x 6701—1.

Model IV. Both A and E Genes are Partially

Dominant. Both Parents are Heterozygous for lethal
gene 1 ............................................ 34

Chi—Square Tests of Genetic Models for Resistance
tommmwwf.sp.§_em ................. 38

Chi-Square Test for F1, F2, Backcross Resistant

and Susceptible Progenies Resulting from Cross

1849 x 6701—1. Model I. Both A and B Genes

Partially Dominant ............................... .. 39

vi

 

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12.

Expected Frequency of Resistant Phenotypes for
Genetic Models of Complete Dominance and/or
Recessiveness .................................... .. 40

Analysis of Variance of SD, SI, S2 and S 3
Generations ........................................ 42

The Effect of Selfing on the Level of Resistance
on Resistant Line 6701 ............................. 42

 

LIST OF FIGURES

Figure Page

1.

Summary of Genetic Studies on Inheritance of
Resistance to Formae Speciales of
mum ..... 6

Cultural Procedure to Decrease Generation
Time in Onions Grown From Seed... ....... ........... 20

The Effect of Genotype on Resistance to
W f sp sagas. Model I Both A and
E Genes are Partially Dominant ..................... 36

The Effect of Genotype on Resistance to Egggzium

oxygpgzum f. sp. geese. Model II. A Gene is
Partially Dominant and E is Dominant ............ ... 36

viii

 

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INTRODUCTION

Thirty to thirty—five percent of world crop production
is lost annually to diseases, pests, and weeds (22,44).
About 10 to 12 percent of this loss is attributable to
plant diseases (22,44,66). The USDA (86) estimated the

average annual loss from all diseases of onions grown in

the United States at 20 percent. Fusarium basal rot caused
by Eusarium oxysgorum f. sp. ggpag (E. Q. ggpag) is
responsible for 4 percent of this loss. The United States
is the fourth largest onion producer in the world, with a
crop valued at $428 million (87). A 4 percent yield

reduction from E. g. gepae would represent a loss of $17
million annually. The State of Michigan ranks eighth in
the country in production of onions, with a crop valued at
$16.4 million (87). If 4 percent of the crop were lost to
E. g. ggggg that would be a loss of $0.66 million annually.

The need to control losses due to disease is obvious.
Millions of dollars are spent annually on chemical
applications to reduce or prevent these losses. This adds
to the cost of production and is ultimately passed on to
the consumer in terms of increased cost and pollution to

our environment.

 

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One of the most appealing solutions to losses from
disease is the use of disease resistant cultivars. The
advantages of resistance as a control strategy are primarily
its effectiveness and relatively low cost. Resistant
cultivars do not require the application of chemicals, or
at least reduced levels of them, and are therefore
environmentally sound. Often there may be no effective and
economical chemical control available to growers, which has
been the case with E. g. genie.

The incorporation of disease resistance into onion
breeding lines is one of the major objectives of the onion
breeding program at Michigan State University. Eggaxium
gxyspgxgm f. sp. gggag, a fungal disease which causes the
onion bulb to rot in the field or in storage, is a
potentially serious disease. Because of the length of time
necessary to develop new cultivars, foresight must be used
in development of resistant cultivars before wide-spread
epidemics and serious losses occur. Knowledge of the
inheritance of disease resistance to E* 9* QgQae would
greatly increase effectiveness and speed of introduction of
Fusarium resistant cultivars. Therefore, a study of the

genetics of resistance to E* 9* 9:21: was undertaken.

    

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LITERATURE REVIEW

PATHOGEN: EUSABLHH QXXSEQBUM F. SP. QEBAE (E. 2. 2325s)
Formae speciales of Fusarium Qxxgpgzum occur worldwide
and are the most commonly found Fusaria in soils,
accounting for 40 to 70 percent of the total Fusarium
species present (32). Most of these species are beneficial
saprophytes which decompose dead organic matter (82). E.
g. cease is a facultative saprophyte; it can parasitize a
host plant or remain dormant in decaying plant material
until conditions are favorable for germination (62).

Dissemination can occur by wind, water, farm equipment or by

use of infected onion sets (4,62). Katan (51) also,
reported that E. 2. cepag can be carried on onion seed.

The pathogen invades onion bulbs through the root tips
or injuries caused by other organisms (2). The
deterioration begins at the base of the bulb with a mealy
decay of all tissues at the basal plate and progresses
upward into the bulb center (26,55). This area may become
covered with white to purple cottony mycelium (55). If the
bulb loses water rapidly, it will become tough, leathery

and mummified. Bulbs may be completely rotted by harvest

 

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4
or slowly decay during transit and storage (26). Foliar
symptoms of E. g. gepag begin with a chlorosis and dieback
of leaf tips (18,45). Part or all of a leaf may become

chlorotic and die from the tip to the base.

REDUCTION IN YIELD

Research on yields of onion cultivars on Midwestern
organic soils artificially infested with E. g. ggggg
showed a 53 to 68 percent reduction in yield of the two
cultivars most commonly grown in Michigan; Spartan Banner
and Krummery Special (54). Both numbers of plants and
weight of bulbs per hectare were significantly reduced in
infested soils. There was a 60 percent mean reduction in
yield in the E. 2. ggggg infested trials compared to the
non-infested trials.

E. Q. 9:232 resistance trials conducted in Michigan on
naturally infested soil reported losses due to Fusarium
basal rot in field plus normal storage conditions of 30
percent in Spartan Banner 80, 41 percent in Krummery Special
and 37 to 54 percent in Sweet Sandwich (97). Yield
reductions at harvest ranged from 0 to 23 percent (6.8%
mean reduction) and from 3 to 67 percent loss in storage
(16.2% mean loss). The combined average loss for both

field and storage was 23 percent.

DISEASE MANAGEMENT WITH RESISTANCE
The traditional method of control of E. 2. cepae has

been through disease resistance (54). The advantages of

 

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resistance as a control strategy are: (1) that resistance
is inherited and therefore is present in the plant, (2) it
is not detrimental to the environment, (3) it is highly
selective, (4) non-phytotoxic, (5) can be highly effective
in disease control, and (6) requires low maintenance costs
once cultivars are developed. (31). On the other hand,

breeding for disease resistance can take many years and is

often only partially effective. In some cases resistance
genes are not available in germplasm which will cross
normally with Allium ce a. Because of the existence of

different races of a pathogen a cultivar may be resistant
in one growing area and not in others. A mutation to
virulence in the pathogen can also result in a loss of
resistance in a cultivar that was previously resistant to

the disease.
INHERITANCE OF RESISTANCE TO FORMAE SPECIALES OF EyfiABlflm
QXEEQBLM

The inheritance of resistance in plants to many
Eugglium Qxygpgrum species has been studied. This knowledge
of the nature of inheritance suggests a pattern with which
to compare the onion host-pathogen system. A partial
summary of genetic studies on inheritance of resistance to
Eugapium gxyspgrnm is given (Figure 1).

Resistance to plant diseases is generally inherited
dominantly and virulence of the pathogen inherited
recessively (27,79,90). Sidhu (79) reviewed 1042 papers on

the inheritance of disease resistance and found 927 (89%)

    

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Figure 1. Summary of Genetic Studies on Inheritance of Resistance
to Formae Speciales of Engaginm gxxfingznm.

 

 

 

Common Author Monogenic Monogenic Partial Polygenic
Name Dominant Recessive Dominant
Alfalfa Hijano(36) x x
Beanz Ribeiro(72) race 1 race 2
Bravo(10) x x
Cabbage Walker(92) x
Celery Orton(67) x
Cotton Netzer(64) x
Kappleman x
(50)
Cowpea Rigert(73) race 1
race 2
race 3
Cucumber Netzer(63) x
Pea Haglund(33) x
Chickpea Sindhu(80) x
Musk- ZinkC99) x
melon
Radishz Peterson(70) x
Sweet Collins(20) x
Potato
Tomato Circu11i(19) race 1
race 2
Tulipz Eijk(9l) x
Water— Netzer(65) x
melon
TOTAL 14 1 3 5

 

 

 

 

 

 

 

 

2

Host of root rot Fusaria (all others are hosts of vascular
wilt Fusaria).

  

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reported resistance due to dominant genes and 108 to
recessive genes and that inheritance was usually monogenic.
Sidhu (79) speculated that many reported cases of multigenic
resistance might have been due to the use of pathogen
populations composed of many races in the inheritance
studies.

Most of the crops summarized in Figure l are hosts for
vascular wilt Fusaria. Inheritance of resistance to these
wilt Fusaria is almost exclusively monogenic dominant. Of
the crops listed which are hosts for root rot Fusaria (bean,
radish, tulip) inheritance was found to be polygenic. Bravo
(10) found that resistance in beans (Ehasgglus,xulgari§) was
largely but not fully dominant over susceptibility.
Estimates of the number of genes controlling resistance
ranged from 3 to 7. In a study on resistance in radish to
W W f. We F1 progenies of
susceptible x resistant plants expressed a level of
resistance intermediate between the parents (70). The F2
progenies and backcrosses showed resistance of about 15%

suggesting that resistance was polygenic in nature.

PERMANENCE OF GENETICALLY INHERITED RESISTANCE TO FUSARIA

Van der Plank (88) has classified all plant disease

into two epidemiological categories depending on whether
their epidemics are mathematically analogous to simple
interest or compound interest in money. Eggazium is

considered to be a typical example of simple interest

diseases and is therefore predicted to spread slowly,

 

  

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8
perhaps taking decades for a new race to become widespread.
E. 2. QQEEE is a soil-born disease with no known sexual
cycle (60). Without a sexual cycle, recombination of genes

to produce more virulent races is greatly reduced and

mutation, a much slower process, provides the organisms
main method of variation (60). In such cases monogenic
resistance is often long lasting (74). Vertical
resistance, usually involving resistance mechanisms whose

inheritance is governed by single genes, is considered to be

generally effective against formae speciales of Eggggium

gxyspggum (74,89,90). Robinson (74) states that Eusazium
ggyspgggm can be completely controlled by vertical
resistance provided that: the host is not an annual, that
at least one ’strong’ gene is known, and that rotation is

practiced.
Grill (23) reports that monogenic resistance to race 1
was completely effective in preventing losses from

Fusarium wilt for 11 years (from 1949 to 1960) in Florida‘s

tomato crop. Race 2 was discovered in 1960 in Florida, but
by the time it had become a major state wide problem
varieties with monogenic resistance to race 1 and 2 were
developed.

Monogenic dominant “Type A” resistance has successfully

controlled cabbage yellows, caused by Eggarium Qxyspgxgm fiE

SE conglutinans, for more than 50 years (9).

 

MECHANISMS OF RESISTANCE

Onion lines varied in their response to infection by
E. g. cease by age (40). In general older plants showed
the greatest resistance to infection, followed by young
seedlings, then bulbs. It has been suggested that if
different types of resistance operate during different
stages of development then resistance may be polygenic in
nature (40). Soki (81) hypothesized that testing for
resistance at the bulb stage would be the most accurate
measure of resistance.

Onion cultivars susceptible and resistant to E. g.
gggag were found to be anatomically similar and equally
susceptible to E. g. cepae in their root and stem plate
tissue (1). This agrees with Beckman (7,8) who reported
that wilt—producing Fusaria grow equally well in resistant
and susceptible tissue. Resistance appears to depend
primarily on a physical localization of the parasite (7).
It was suggested that systemic distribution of the pathogen
in a susceptible type interaction is dependent upon fungal
products which rapidly degrade vascular gels and which
inhibit respiratory or growth metabolism of host cells.

Vascular occlusion is common and has been considered a
major factor in disease development and resistance to many
wilt diseases caused by E. oxvsporum (7,8). The blocked
vessels reduce or eliminate the spread of the pathogen
through the vascular system, thereby localizing the fungus.

Beckman (7) reported that tolerant varieties exhibited more

 

10
and earlier vascular occlusion than susceptible varieties
when infected with vascular wilt Fusaria. Tylose formation,
occlusion of xylem vessels and hypertrophy were reported
by Shalaby (75) in E. g. gepae infected onion tissues. Abawi
(1) found that xylem vessels were clogged by tylose-like
structures in both tolerant and susceptible varieties.

Pennypacker (69) reported that vascular wilt Fusaria
and the root rot Fusaria use different mechanisms to attack
their hosts. Root rot Fusaria are generally confined to
the cortical areas of their host, only penetrating the
vascular system late in the disease process. The two groups
of Fusaria induce different anatomical responses in their
hosts; gums, gels, and tyloses follow invasion by the
vascular wilt Fusaria, whereas hypertrophied and
hyperplastic tissue formation is generally found in
response to infection by the cortical rot Fusaria.

Plant pathogens produce an array of enzymes capable of
attacking plant cell components. It has been demonstrated
that pectic enzymes can macerate and kill plant tissues in
a manner similar to that occurring in soft-rot diseases and
that fragments released from the cell wall can elicit plant
defense reactions (21).

Collmer (21) reviewed the role of pectic enzymes in
plant pathogenesis and outlined the following steps which
occur during the interaction of a pectolytic pathogen and a

potential host:

...(1)the entering pathogen possesses structural genes

11
encoding pectic enzymes, (2)these genes are expressed
in a characteristic manner in the infected tissue,
(3)the enzymes are exported from the pathogen
cytoplasm to the host tissue, (4) in some tissues the
enzymes encounter inhibitors or protected substrates,
(5) in other tissues the enzymes are active and cleave
structural polymers in the primary cell wall and middle
lamella, facilitating pathogen penetration and

colonization.”

E. g. gegae produces two types of pectic enzymes,
exo—polygalacturonase (exo-PG) and endo pectin trans
eliminase (endo-PTE) on a mineral medium supplemented with
onion cell walls (41). These enzymes are also synthesized
in vivo and have been extracted from infected onion tissue
in which the onset of bulb rot was correlated with the
presence of endo-PTE.

Endo-PTE is the main enzyme responsible for host tissue
maceration and cell death (42). However, Endo—PTE showed
little activity during the early stage of onion stem plate
infection, even though in culture the stem plate cell walls
induced extensive production of pectic enzymes. Low pH of
stem plate tissue could suppress production or activity.

Similar patterns of fungal distribution and enzyme
production have been found during the early stages of
infection of both susceptible and tolerant plants (41),
The fungus rapidly invaded stem plate tissue then spread

more slowly to the outer bulb scales and eventually to base

 

12

of the inner leaf sheaths. Different genotypes vary
markedly in their ability to contain the pathogen to the
stem plate, thereby delaying bulb decay. Susceptible
plants restricted the pathogen to the stem plate from 2 to
3 months, whereas tolerant plants contained it for up to 9
months. This suggests that additional factors may operate
in bulb scale and leaf sheath tissue of tolerant plants to
interfere with pectic enzyme activity and thereby delay
spread of pathogen.

It has been shown that cell walls from different parts

of onion bulbs and from bulbs of genotypes differing in
resistance to E. g. gepag differed in the extent to which
they induced the fungus to produce endo-PTE (42). These
patterns of enzyme induction were correlated with

resistance and susceptibility and the different patterns
of host tissue colonization by the fungus.

Pectic enzymes secreted by plant pathogens in vivo are
influenced by host sugar content (29,43). Sugar contents of
equivalent parts of two onion genotypes susceptible and
resistant to E. g. gepae were similar, but different parts
showed marked differences. Sugars are present in bulb
scales and leaf sheaths in sufficient concentrations to
repress pectic enzyme synthesis by the fungus; whereas
lower sugar concentrations, equivalent to those in mature
bulbs and stem plate tissue, are too low to suppress
endo-PTE synthesis. Different parts of the onion bulb

differ in their susceptibility to bulb rot. Bulb scales and

    

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leaf sheaths are resistant (high sugar levels), stem plates

susceptible (low sugar levels). It was suggested that
differences in sugar content could help to explain
differences in susceptibility between different parts of
the bulb.

H012 and Knox-Davis (42) hypothesized that the slow
spread of the pathogen from the stem plate to the outer
bulb sheaths was due in part to the low pectic enzyme
inducing properties of the cell walls of these tissues.
This delay in endo-PTE synthesis would minimize cell damage
and enable host defense mechanisms to localize the fungus
to the stem plate tissue. Suggested defense mechanisms of
tolerant genotypes which could result in further
localization of the pathogen to the stem plate tissue are:
(1) phytoalexin production(52,53); antifungal substances are
produced in inoculated onion bulb scales and leaf sheath
tissue(1), (2) formation of structural barriers; tyloses
and gum—like material are formed in uninvaded xylem of the
leaf base adjacent to infected stem plate tissue (8,75),
(3) enzyme inhibitors or protected substrates(21), (4)
hypersensitive reaction or necrotic responses(27,75).

Changes in cell wall structure and resistance of plant
tissue occur with age (98). Zink (98) reported a marked
decrease in the calcium content of onion bulbs with age. In
field studies on onion bulb rot it was found that tolerant
genotypes lost their resistance in calcium deficient soils.

Histological studies revealed that young bulb scale and

 

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leaf sheath tissues from bulbs grown in these soils were
readily invaded by the pathogen. In onion plants, calcium
mobilization and the type of pectic substances formed in
bulb scale and leaf sheath tissue could influence pectic
enzyme induction by the host cell walls and therefore also

the resistance and susceptibility of the tissue.

SOURCES OF RESISTANCE

There are many sources of partial resistance or
tolerance to Fusarium basal rot that have been reported in
the literature (1,40,81,96). However, since multiple races
of the pathogen F. g. gegge may exist, cultivars screened in

one growing area may not prove to be resistant in other

areas. Also cultivars need to be adapted to local
environmental conditions such as daylength, temperature,
length of growing season, etc., and may not grow well in

areas that differ greatly from those in which they were
developed.

Lacy and Zandstra (54,97) evaluated onion cultivars in
Michigan growing areas for yield response on soils naturally
and artificially infested with E. g. gepag. Two of the
cultivars most commonly grown in Michigan, Spartan Banner 80
(which showed a 53% reduction in yield in artificially
infested fields) and Krummery Special (which showed a 68%
reduction) were among the higher yielding cultivars tested.
In naturally infested fields Spartan Banner 80 had a 30%
reduction in yield, Krummery Special a 41% reduction and

Sweet Sandwich 25%.

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Another source of resistance to disease exists in
related Allium species. Jones (47) found that Allium
{isgulgsum carried a high degree of resistance not only to
Fusarium basal rot, but also to pink root, downy mildew,
smut, yellow dwarf virus and thrips. Abawi and Lorbeer (1)
compared commercial onion cultivars with other Allium
species and interspecfic hybrids and found that the
cultivars possessing the highest resistance were Beltsville
Bunching (AE fistulgsgm x AE ggpa) and Japanese Bunching
(AE fingngyflD which had 85 and 98 percent survival rates
respectively.

Among the inbred lines developed by the USDA at
University of Wisconsin, line 6701 has shown the highest
level of resistance to E. 2. 932;; with 97% of seedlings
screened surviving (68). Line 6701 was included in this

inheritance study as a resistant parent.

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MATERIALS AND METHODS

SCREENING FOR RESISTANCE TO EHSARLUM QXXSRQRQM F. SP. CEEAE

Greenhouse screening for resistance to E. Q. gggag was
selected over field testing because of the need for
increased control over environmental conditions, inocula—
tion levels, race(s) of pathogen used, and the flexibility
of conducting screening trials when desired. A high
positive correlation between greenhouse and field testing
for resistance to E. 9. gggag (71) indicates that results
obtained under greenhouse conditions will be applicable to
field situations.

Disease development increases with increasing inoculum
concentrations of E. g. gepae (3,58); therefore an inoculum
concentration must be chosen which will cause serious
disease in susceptible but not resistant lines. Inoculum
concentrations of 20,000 to 40,000 microconidia per gram of
sand, typically used for E. Q. ggpae trials by others (68),
were found to be too extreme for this study.

Inoculum levels of 10,000 microconidia per gram of sand
were used in this study. This level was selected based on
inoculum concentration experiments (68) conducted on inbred

lines 1849 and 6701 (the same lines used in this study) at

16

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University of Wisconsin and on preliminary screening trials
which showed that seedlings from susceptible line 1849 were
all killed at concentrations as low as 10,000 microconidia
per gram of sand.
SCREENING PROCEDURE FOR EHSARIHM QXXSERQQM F. SP. CEEAE
RESISTANCE IN ONION.

The screening procedure was based primarily on the

method developed by Mary Palmer and Paul Williams of the
Department of Botany and Plant Pathology, University of
Wisconsin (68). Single spore isolate F593—1, obtained from
a E. g. gggge culture designated as PHW 593-19, was used in
this study. The culture was originally isolated from a
field in Wisconsin and was obtained from Dr. M. L. Lacy,
Department of Botany and Plant Pathology, Michigan State
University.
E. g. QQEQQ cultures are maintained on sterilized muck
soil at 4°C. Infested soil was transferred to potato
dextrose agar plates and grown at 24°C. A 7 mm diameter
piece of colonized agar was aseptically added to 50 ml of
potato dextrose broth in a 125 ml flask then incubated on a
shaker at 24 to 26°C for 3 to 6 days. The mycelia and
broth were blended at low speed for 2 minutes, centrifuged
for 10 minutes at 3400 rpm and the pellet was resuspended in
distilled water. The microconidia were then counted with a
hemacytometer to determine concentration.

An inoculation concentration of 10,000 microconidia

per gram of sand was used. Twenty kilograms of white

    

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silica sand was weighed out, placed in a 50 x 29 x 11 cm
stainless steel pan and sterilized in an autoclave. The
appropriate amount of spore suspension was added to
distilled water to give 2000 ml total volume and provide
10,000 spores per gram of sand. The inoculum was mixed
thoroughly with the sand. Grooves 15 mm apart and about 5-7
mm deep were made across the pan. One hundred seeds per
row were planted and covered. The pan was covered with
aluminum foil and placed in a controlled temperature water

bath set at 24°C. under high intensity discharge lights for

a 15-16 hour photoperiod. The sand temperature was
maintained at 20-22°C. The foil was removed after the
seedlings had emerged. When cotyledons were about 1 cm

tall, the sand temperature was increased to 26-28°C (the
optimum temperature range for the growth of E. g. cepae in
culture is 24 to 27°C (3,26,93)). Plants were watered as

needed and 15:16:17 fertilizer was applied weekly after two

weeks at a rate of 200 ppm Nitrogen. The number of
seedlings were counted 10 days after sowing and again 21
and 28 days later. On day 28 the seedlings were evaluated

and counted as either healthy or diseased.

PATHOGENICITY TESTS

Pathogenicity tests were conducted with six isolates of
E. g. ggpgg. The isolates used were obtained from Dr. M.
L. Lacy, Department of Botany and Plant Pathology, Michigan
State University. Three isolates originally obtained from

Wisconsin were designated as F593, F156-A, and F156-B; two

 

 

19
were isolated from New York fields, FllO-A and F110-B; one
was isolated from an infested onion field in Michigan,
FOC—l—l. An inoculation concentration of 20,000
microconidia per gram of sand was used. The isolates used
were single spored to assure genetic homogeneity, otherwise
the screening procedure described previously was followed.
Sixty seedlings(each) from three resistant and two suscep-
tible inbred lines selected from preliminary E. Q. ggnae

screening trials were used.

FLOWER AND SEED PRODUCTION
Culture

The onion plants were raised in growth chambers and
grown under environmental conditions designed to decrease
generation time by bypassing the bulbing stage (Figure 2).
This method was based on preliminary research and work done
by Shishido (76-78), Brewster (11-15) and others
(5,6,34,35,37,38,49,57,83,84,95). Fungicide (benomyl) was
applied as a spray and as a drench to reduce E. g. gggag in

the non-inoculated control plants.

Pollination

The pollination procedure used was similar to that
reported by Jones and Emsweller (47,48). To optimize seed
set two aspects of flower development were considered: the
flowering period and stigma receptivity.

Flowering period: the inflorescence is a roughly

spherical umbel bearing 50 to more than 1,000 flowers on

 

I‘. ' "..’:

20

Figure 2. Cultural Procedure to Decrease Generation Time
in Onions Grown From Seed.

 

1. Growth Environment (growth chambers)
A. Temperature 17°C (63’F)
B. Light 200 micromoles m""’s’1
C. Photoperiod 24 hrs cool-white fluorescent
D. Nutrition 100 ppm 15:16:17
E. Time Period 90 days
F. Container 18 cell flats
G. Media Artificial peat—based media
II. Flower Initiation (vernalization)
A. Temperature 9°C (49°F)
E. Light 200 micromoles m‘z's‘1
C. Photoperiod 24 hrs cool-white fluorescent
D. Nutrition No fertilizer
E. Time Period 30-40 days
III. Flower Bud Development (move to greenhouse)
A. Temperature 17°C (63°F) optimal
B. Light Natural daylight
C. Photoperiod 14+ hrs natural daylight
D. Nutrition 125 ppm 15:16:17
E. Time to Bloom 50—60 days
F. Container 6” clay pots
G. Media Artificial peat-based media

 

 

 

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21
an aggregate of cymes consisting of from 5 to 10 perfect
flowers (47). The flowering of a single umbel may extend
over 3 to 4 weeks (25,47). Flowering follows a general

pattern where at first the number of flowers opening per

day is small, but this rapidly increases, giving a peak
which lasts for a average of 4 to 10 days (25). During
this peak 50 or more flowers may open in a single day

depending upon the temperature and the hours of light that
day and the previous day (59).
Stigma Receptivity: Currah and Ockendon (25) found that

the onset of stigma receptivity was closely connected with

the production of stigmatic exudate. Pollen tubes grew in
the stigma before presence of exudate, but few adhered to
the stigmatic surface. They suggested the major role of

the stigmatic exudate was pollen capture and that it did

not have an essential function in pollen germination or
tube growth. Moll (61) found that the stigma was most
receptive on the 3rd, 4th, or 5th day after opening. The

duration of stigma receptivity is strongly influenced by

temperature (16,17).

Pollination Technique and timing

The umbels of the plants to be crossed were bagged as
soon as the first flower opened. Plants destined to be
pollen parents were allowed to flower. Open flowers on
plants used as seed parents were removed until 20 or more
flowers opened on a single day; thereafter, the anthers

were removed from any open flower. Emasculation was

     
 

  

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continued each morning and afternoon for a period of 3 to
5 days. At this point all the remaining unopened flowers
were removed and a flower head of the pollen parent was
tied with the emasculated head, and both are enclosed under
the same bag. One-half to one teaspoon of fly pupae were
added and within a few days adults emerged and pollinated
the female flowers. The heads were bagged separately when
pollination was completed, usually 5 to 7 days after flies
had hatched. Plants to be self pollinated were bagged
individually as soon as the first flower opened and flies

were added as pollinators.

Cleaning and Storage of Seed

Once the seed was thoroughly dried it was separated

from the receptacle through gentle grinding on a ribbed
rubber pad. Separation of light seed and chaff was
accomplished with forced airscreen cleaning. This not only

removed fragments of receptacle, but also had the effect of
removing the poorest quality seed.

Storage of onion seeds in normal temperature and

humidity results in fairly rapid deterioration in
viability, preceded by a rise in the incidence of
cytological damage (24). To prevent deterioration the onion

seed was stored at 2°C and 40% relative humidity

SELECTION OF RESISTANT AND SUSCEPTIBLE PARENTS
Based on preliminary screening trials resistant line

6701 and susceptible line 1849 were chosen as parents for

 

23
this inheritance study. Flowering plants from each of the
lines were self pollinated (selfed) and progeny tested to
determine level of resistance to E. g. 2223;. The resistant
parent chosen (6701—1) was a single plant selection showing
the highest level of resistance of those tested. Inbred
lines 6701 and 1849 originated from the University of
Wisconsin’s breeding program. Line 6701 had been screened
several generations for resistance to E. Q. 9321; and line
1849 has been used as a susceptible check in screening

trials.

NOMENCLATURE

Selfed progeny are designated by line number followed
by a hyphenated number which represents the nth plant to be
selfed (i.e. 6701-1). This nomenclature is extended for 2nd
and 3rd generations of selfing (i.e. 6701—1—4-7). Resistant
parent 6701-1 is also designated as 6701-1(RES) or RES.

Susceptible line 1849 is designated as 1849(SUS) or SUS.

F1, F2, BACKCROSSES, AND SELFS USED IN INHERITANCE STUDY

To study the inheritance of resistance to E. 2. gegég
crosses between resistant line 6701—1 and susceptible line
1849 were made using pollination techniques previously
described. F2’s, backcrosses to the resistant and
susceptible parents and selfing of parents were also

performed.

 

24

EFFECT OF INBREEDING 0N RESISTANCE

Alligm ggpge is naturally a cross pollinated species.
Inbreeding or selfing of onions results in a decrease of
vigor and changes in other traits (47,48). To determine if
inbreeding had an effect on the level of resistance, plants
from line 6701 were first screened then selfed. This cycle
was repeated for three generations, in each case those
p1ant(s) showing the highest level of resistance were
selected as parents for the next generation. Progeny from

each generation were screened for resistance to E. g. 9323:.

STATISTICAL ANALYSIS

Chi Square analysis was used to test the hypothesized
segregation ratios of F1, F2 and backcross families for
goodness of fit to the observed data. Yates correction for
continuity was used to calculate chi-square values (adjusted
X2 = Z (lobserved - expected] - 0.5)2 + expected).

Heterogeneity tests were also performed on all families
to determine population heterozygosity (heterogeneity X2 =
total X2 - pooled X2 ).

A completely randomized experimental design was used in
screening trials with a varying number of repetitions
depending upon availability of seed. Analysis of variance
was performed to test null hypotheses of no differences in
the level of resistance to E. g. cepae between F1, F2 and
backcross families. Because data were in the form of
percents, arc-sin transformation was performed to assure

homogeneity of variance.

 

 

RESULTS AND DISCUSSION

PATHOGENICITY OF SINGLE SPORE ISOLATES OF Eflfibfilflm QXXEEQBEM
F. SP. CEEAE

The purpose of the pathogenicity test was to determine:
(1) which E. g. ggpgg isolates were the most virulent, (2)
in which isolate there was the greatest range between the
most resistant and the most susceptible lines and (3) which
isolate gave results approaching the ideal situation
(resistant line having 100% survival rate and susceptible
line 0% survival rate).

Isolate 593 was chosen for further screening because,
while not being the most virulent strain tested, it showed
the greatest range between the most resistant (67% in 6701)
and most susceptible (0% in 1849) lines (Table 1). Also
lines 6701 and 1849 had been selected for resistance and
susceptibility to E. g. Egggg using isolate 593.

SCREENING RESULTS FOR RESISTANCE TO Eflfiflglflfl QXESEQREM F.
SP.

Parent lines, F1. F2 and backcross families were rated
for resistance based on the percent survival of those
seedlings emerging (Table 3).

The analysis of variance test found a significant
difference between the level of resistance to E. Q. gepgg in

the parents , F1, F2 and BC families (Table 2).

25

 

 

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Table 1.

  

Pathogenicity of Six Single

26

Spore Isolates of

12mm mm :2. :2. sense on 5 onion lines.

r

Percent Surviving'

 

 

 

 

 

 

 

 

 

 

Pedigree %Germ 593 156-A 156-B llO-A llO-B FOC-l—l
6701 95 67 32 19 14 19 37
6693 70 55 50 52 43 38 69
2399 60 28 17 8 8 3 83
1849 80 0 0 0 0 0 8
611-1 70 0 0 0 0 0 12

 

‘ % of emerging seedlings that survived 21 days.

 

 

 

Table 2. Analysis of Variance of F1, F2 and BC Families.
DF Sum of Error F-value Prob.
Squares Mean Square
Between 36 35469.70 985.27 10.77** 0.000
Within 179 16382.29 91.52
Total 215 51851.98

 

** Highly significant (a=0.5)

 

 

 

 

27

 

 

 

 

Table 3. Summary Statistics of Screening Data for Resistance to
MR1 r‘ Wf- Sp. asses.
CROSS # PLANTS %SURVIVE MEAN MEAN STD VARIANCE
TESTEDz CONTROLY %R D21“ %R D28 ERR” %R D28”
1849(SUS) 126 94 0.00 0.00e 3.91 0.00
SUS(selfed) 223 83 0 48 0.00e 3.62 0.00
6701 136 97 74 70 52.97a 3.91 23 94
RES-1(6701-1) 76 80 82 89 -— -- ~—
Fl 53 96 37.02 10.03de 5.52 173.95
F2 331 96 35.79 15.40b 4.28 35.34
leF1 175 98 20.11 9.15de 4.28 162.78
BC-RES 255 96 65.59 37.46a 4.78 43.32
BC—SUS 202 100 8 57 1.22de 4.28 41.03
F1 (recip) 30 100 37.00 -- -- -—
F2 (recip) 309 94 19.96 8.74bc 4.28 38.67
F1xF1(recip) 134 —— 13.26 2.72cde 5.52 50.86
BC—RES (recip) 114 98 68.59 37.17a 5.52 99.59
BC—SUSl (recip) 82 98 2.51 0.00e 4.78 0 00
RES—1xRES-lu 72 100 30.44 6.25 5.40 225.00
RES(se1f 1x)u 1088 91 39 52 17.77 2.01 131.29
RES(se1f 2x)u 932 80 27.54 7.08 1.94 108.72
RES(self 3x)u 395 89 24 35 6.05 2.48 115.53

 

C(IX(N

Number of seedlings emerged.
% of emerging seedlings surviving 28 days in the control.
% resistance on day 21.
Standard error for transformed data from day 28.
Variance of arc—sin transformed data from day 28.
% resistance corrected for lowered survival rate due to

lethal genes by following formula:

(% resistance) x (1 + (1 ~ frequency of survival in control)).

- BC-SUS is backcross susceptible,
Recip is reciprocal cross 6701-1x1849.
- Any two means with the same letter (abcde) are not

resistant.

significantly different by LSD means separation test (u=0.05).

BC-RES is backcross

 

28
The resistance values for cross 1849 x 6701~1 and
reciprocal cross 6701-1 x 1849 were not significantly
different as determined by LSD (a=0.5) test (Table 3).
Therefore, it was concluded that resistance to E. 2. 222;;
was not maternally inherited in the lines used in this

study.

PROGENY TESTS OF PARENTAL LINES 6701-1(RES) and 1849(SUS)

Segregation of progeny for resistance to E. 9. 92253
indicated that the parental line 6701-1 was heterozygous at
resistant loci and that line 1849 was homozygous
susceptible. Progeny tested from selfed 6701-1 plants
segregated for resistance and ranged in values from 0% to
42% surviving with a mean of 6%. The mean value for the
resistant parent was probably low because of inbreeding
depression and will be discussed later. Line 1849 appeared
to be homozygous with all progeny testing susceptible.
USE OF GENOTYPE FREQUENCIES TO CALCULATE RATIO OF RESISTANT
TO SUSCEPTIBLE OFFSPRING

Because of the heterozygosity of the parental line
6701-1 the ratio of resistant to susceptible offspring for
individual F1, F2 and backcross families was expected to
vary depending upon genotypes of the parents involved.
Therefore, the approach taken was to start with hypothetical
parental genotypes then calculate expected genotype
frequencies for the different families. These were compared
to the observed frequency of plants in each class (i.e.

number of plants surviving on day 10, 21 and 28). Mean

 

   

      

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29

percent resistance data for a F1, F2 or backcross families
were compared to expected frequencies to test different
hypotheses of gene action using chi-square analysis.
Parental genotypes AABh for 6701—1 and aahh for 1849 gave
the best fit to the observed data.

GENE ACTION OF PROPOSED RESISTANCE GENES E991 (A), E992 (E)
AND LETHAL l GENE

Two genes; Focl (A) and Foc2 (E), are proposed to

 

 

govern disease resistance to Ensaxium gxxspgzum f. sp.

ggggg. Several models are proposed for their gene action:
ugdgl I. (see Table 4 for expected gene frequencies).
Both A and B genes are partially dominant (AA >> A; and
EE >> Eh) and the interaction between loci is additive
(AABB > AAEh or 3333 > Ath). Resistance is not
epistatic; both resistant alleles A and B must be
present for the plant to remain healthy in screening
trials past 28 days. Plants with genotypes AAEB, AAEh
and Aéflfi are considered resistant. Plants heterozygous
at both loci (AaEE) are intermediate in resistance and
remain healthy for 21 days, then become diseased and
may die. Genotype aahh is completely susceptible with
all plants screened dying around day 10.
Mgdel 11. (Table 5). A gene is partially dominant and
E gene is dominant. The interaction between loci is
additive. This gene action results in only two

resistant genotypes, AAEB and AAEE.

 

 

30

Mgle 111. (Table 6). Both A gene and E genes are
partially dominant. The interaction between loci is
additive. In addition, a lethal recessive gene 1
results in death of seedlings after emergence. Gene 1

occurs only in the resistant parent.

Mgfigl 11. (Table 7). Both A and E genes are partially
dominant. The interaction between loci is additive. In
addition, recessive lethal gene 1 occurs in both
resistant and susceptible parents in heterozygous state

(Ll).

 

31

Table 4. Expected Genotype Frequencies of Parents, F1, F2

and Backcross Families for Cross 1849(Pl) x 6701-
1(P2). Model L. A and E Genes are Both Partially
Dominant. Resistant Genotypes (3) are AAEE, AAEE,
and AaEE.

 

TYPE P1 FREQ P2 FREQ CROSS FREQ FREQUENCY

OBSz EXP”

 

 

Fl aabb 1.00 AABb 1.0 AaBb 0.500 0.094 QEQQQ
Aabb 0.500
F2 AaBb 0.50 AAEE QEQQL 0.151 QElifi
Aabb 0.50 AABb 11.9.6.3.
AAbb 0.156
BABE
AaBb 0.125
Aabb 0.313
aaBB 0.031
aaBb 0.063
aabb 0.156

 

BC—R AaBb 0.50 AABb 1.0 5555 9.953 0.396 9.325
11.21%:

Aabb 0.50 m};

Aabb 0.188

 

 

BC-S AaBb 0.50 aabb 1.0 AaBb 0.125 0.015 QEQQQ
Aabb 0.50 Aabb 0.375
aaBb 0.125
aabb 0.375
RES" AABb 1.00 AAEE QEZEQ 0.820 9,259
MB}; (1.5.00. (D21) "

AAbb 0.250

 

'ix

Observed (OBS) is the observed frequency of resistant
seedlings after 28 days of screening.

Expected (EXP) frequency of resistance = freq of
resistant genotypes (underlined) X (1 — frequency

of lethals(ll).

Resistant parent line 6701-1.

Day 21 data used because day 28 data not available.
FREQ = frequency, BC-S AND BC-R = backcross susceptible
and resistant.

Resistant genotypes and their frequencies are underlined.
Parental genotypes: 1849 = aabb, 6701-1 = AABb.

 

32

Table 5. Expected Genotype Frequencies of Parents, F1, F2

and Backcross Families for Cross 1849(P1) x 6701-1
(P2). Mgdgl 11. A Gene is Partially Dominant and
E Gene is Dominant. Resistant Genotypes (2) are

BABE and am.

 

TYPE Pl FREQ P2 FREQ CROSS FREQ FREQUENCY

OBS' EXPy

 

 

 

 

 

F1 aabb 1.00 AABb 1.0 AaBb 0.500 0.094 QEQEQ
Aabb 0.500
F2 AaBb 0.50 AAEE 0.931 0.151 QEflflfi
Aabb 0.50 53512 0105.3
AAbb 0.156
AaBB 0.063
AaBb 0.125
Aabb 0.313
aaBB 0.031
aaBb 0.063
aabb 0.156
BC—R AaBb 0.50 AABb 1.0 AAEE 0,Q§§ 0.396 9,313
Aabb 0.50 AAEE QEQEQ
AAbb 0.188
AaBB 0.063
AaBb 0.250
Aabb 0.188
BC—S AaBb 0.50 aabb 1.0 AaBb 0.125 0.015 QEQQQ
Aabb 0.50 Aabb 0.375
aaBb 0.125
aabb 0.375
RES" AABb 1.00 5852 0,250 0.820 Q,7§Q
AABb 11.5.0.0 (D21)w

AAbb 0.250

 

Observed (OBS) is the observed frequency of resistant
seedlings after 28 days of screening.

Expected (EXP) frequency of resistance = freq of
resistant genotypes (underlined) X (l — frequency

of lethals(ll).

Resistant parent line 6701-l.

Day 21 data used because day 28 data not available.
FREQ = frequency, BC—S AND BC—R = backcross susceptible
and resistant.

Resistant genotypes and their frequencies are underlined.
Parental genotypes: 1849 = aabb, 6701—l = AABb.

 

33

Table 6. Expected Genotype Frequencies of Parents, F1, F2 and

Backcross Families for Cross 1849(P1) x 6701-1(
Model

P2).

Both A and E Genes are Partially Dominant

and Resistant Parent 6701—1 is Heterozygous for
Lethal Gene 1.

 

 

TYPE Pl FREQ P2 FREQ CROSS FREQ FREQENCY
OBS ‘ EXPY
F1 aabbLL 1.00 AABbLl 1.0 AaBbLL 0.330 0.094 9‘999

AaBbLl 0.170
AabbLL 0.330
AabbLl 0.170

 

 

 

F2 AaBbLL 0.33 AAEE;; 9,993 0.151 9,143
AaBbLl 0.17 AAEE;; £1953
AabbLL 0.33 AAbb-- 0.156
AabbLl 0.17 AaEB—— 9,063
AaB —— 0.125
Aabb-- 0.313
aaBB—- 0.031
aaBb-— 0.063
aabb—- 0.156
(———-11 0.083)

BC—R AaBbLL 0.33 AABbLl 1.0 AAEE;; 9,993 0.396 9,354
AaBbLl 0 17 AAEE;; 9‘219
AabbLL 0 33 AAbb-- 0.188
AabbLl 0 17 Aa —— 0,969
AaBb—— 0.250
Aabb-— 0.188
(----11 0.055)

BC-S AaBbLL 0.33 aabbLL 1.0 AaBbL- 0.125 0.015 9‘999
AaBbLl 0.17 AabbL- 0.375
AabbLL 0.33 aaBbL- 0.125
AabbLl 0.17 aabbL— 0.375

 

2 Observed (OBS) is the observed frequency of resistant
seedlings after 28 days of screening.

y Expected (EXP) frequency of resistance = freq of
resistant genotypes (underlined) X (l — frequency
of lethals(ll).

- Resistant genotypes and their frequencies are underlin

I Parental genotypes: 1849 = aabb, 6701-l = AABb.

ed.

 

 

34

Table 7. Expected Genotype Frequencies of Parents, F1,
Backcross Families for Cross 1849(P1) x 6701-1(P2).
flgggl 11. Both A and E Genes are Partially Dominant
and Both Resistant Parent (6701-1) and Susceptible
Parent (1849) are Heterozygous for Lethal Gene.

F2 and

 

 

 

 

 

TYPE P1 FREQ P2 FREQ CROSS FREQ FREQENCY

OBS ' EXPy

Fl aabbLl 1.00 AABbLl 1.0 AaBbL- 0.500 0.094 9*999
AabbL- 0.500
(—---11 0.25)

F2 AaBbL- 0.50 AAEE;; 9,039 0.151 9,137
AabbL- 0.50 AAEE;; 9‘99}
AAbb-- 0.156
Aafitlm
AaBb—- 0.125
Aabb-— 0.313
aaBB-- 0.031
aaBb-- 0.063
aabb—— 0.156
(----11 0.125)

BC-R AaBbL- 0.50 AABbLl 1.0 AAEE;; 9‘993 0.396 9*329
AabbL- 0.50 AAEE;; 9‘299
AAbb—— 0.188
Aaflflzzflalléfi
AaBb-- 0.250
Aabb-- 0.188
(——--11 0.125)

BC-S AaBbL- 0.50 aabbLl 1.0 AaBbL— 0.125 0.015 9*999
AabbL- 0.50 AabbL- 0.375
aaBbL- 0.125
aabbL— 0.375
(*'--11 0.125)

 

Observed (OBS) is the observed frequency of resistant
seedlings after 28 days of screening.

Expected (EXP) frequency of resistance = freq of
resistant genotypes (underlined) X (1 ~ frequency

of lethals(ll).

Parental genotypes: 1849 = aabb, 6701-l = AABb.

 

 

35

By matching the expected genotype frequencies for the
various families with the observed frequencies of resistance
on days 10, 21 and 28, the length of time a plant with a
specific genotype survived during screening was estimated
(Figure 3 for models I, III and IV and Figure 4 for model
11). This time probably varied with differences in
environmental conditions and also with the genetic
background of the plant.

The existence of a post—emergence lethal gene was
indicated by the lower than expected survival rates in the
uninoculated control plants. It is hypothesized that this
post—emergence recessive lethal gene resulted in the death
of seedlings after emergence when in the E; condition. The
survival rates of uninoculated selfed progeny from line
6701-1 (80%) and 1849 (83%) were close to that expected from
plants contained a heterozygous lethal gene (Ll) segregating
3:1 {75%) for normal and lethal phenotypes. This lethality
was observed in uninoculated selfed plants but, not in the
crosses (F1, F2 and BC families) where the average survival
rate in the control was 97.7%. Therefore, it is likely that
the two parents contained different lethal genes which
remained concealed in a heterozygous state in the crosses
and only segregated homozygous recessive in progeny of

selfed plants.

 

 

36

Figure 3. The Effect of Genotype on Resistance to
m f. sp- asms. Model I. Both A and B
Genes are Partially Dominant.

 

H 1 E D S 'v' I
0 10 21 28

AABb
AaBB
AaBb
AAbb
aaBB
Aabb
aaBb

aabb

 

 

 

 

 

healthy

diseased

No line = death

Number of days seedlings survive in E. o. cepge

inoculated sand from emergence to death.

 

 

 

NIII

 

 

 

37

Figure 4. The Effect of Genotype on Resistance to

Qxygpgxum f. sp. sagas. Model II. A Gene is
Partially Dominant and E is Dominant.

 

 

Ngmbgg 9f Days Sggvivingz

0 10 21 28
AABB

AABb
AaBB
AaBb
AAbb
aaBB
aaBb
Aabb

aabb

 

 

 

 

NIII

 

healthy

diseased

No line = death

Number of days seedlings survive in E. o. cepgg
inoculated sand from emergence to death.

 

 

 

CHI-SQUARE TESTS FOR F1,

The

38

F2 AND BACKCROSS FAMILIES

chi—square values for

F1, F2, BC

and SUS selfed

families for all models (Table 8) along with a more complete

table for

generated

Model I

the best

(both A and B gene

The chi—square

This

supports

(Table

fit to

values in

the

partially dominant and
all cases

hypothesis

9) are

the observed

of two

given.

The model

data was

were not

genes

that

model I

no lethal genes).

significant.

governing

resistance with gene action being partially dominant at both

 

 

 

 

 

 

 

 

 

loci.
Table 8. Chi-Square Values of Genetic Models for
Resistance to ' W f. 3p. mas.

Family Model I Model II Model 111 Model IV
F1 0.38 0.38 0.38 0.38
F2 0.03 12.20* 0.12 0.52
BC-R 0.40 7.65* 1.79 5.08*
BC-S 0.03 0.03 0.03 0.03
SUS 0.00 0.00 0.00 0.00

I Model I both A and E genes incompletely dominant.

I Model II A gene incompletely dominant and E gene
dominant.

I Model III
resistant line.
I Model IV
both parents.
* X2

 

same as model I. with lethal gene in
same as model I. with lethal genes in

> 3.841 is significant at the 5% level (df=l).

 

    

  
  

  

um _ ' .tfr‘r?“-'-‘§
on (I offlofi A
an

' labs-u: 2»: 23:5 Nev-gearin- on: as an '- MM!

 
    
   
   

not: em 91!? mar-'5?"
"z.

uran: .--._ ;.- ....._ --:.:...=n..q on” n has 1 _

_ _ ,._ .‘ .___ ._.-; 1. ... - .. m'gsuplrifla. _
Hague ntdi‘

".'.-I!

39

 

 

 

 

 

 

 

Table 9. Chi-Square Test For F1, F2, Backcross Resistant and
Susceptible Progenies Resulting from Cross 1849 x
6701-1'. Model I. Both A and E Genes Partially
Dominant.
PARENTS CROSSES PROB OBSERVED SUM DF X2 PROB
RES. SUS RES
SUS SUS-20x 0.000 39 0 39 1 0.00 1.00
PARENT SUS—21x 32 0 32 1 0.00 1.00
(selfed) SUS—24x 29 0 29 1 0.00 1.00
SUS-31x 51 0 51 1 0.00 1.00
SUS—42x 33 0 33 l 0.00 1.00
SUS-46x 21 0 21 l 0.00 1.00
SUS—48x 18 0 18 1 0.00 1.00
TOTAL 223 0 223
EXPECTED 223 0 223 l 9‘99 1‘99
RES 0.750
PARENT TOTAL 17 59 76
(6701-1) EXPECTED 19 57 76 1 9,19 9,29
F1 RES-1-55xSUS-27 0 000 16 3 19 1 0.33 0.60
RES—l-SSxSUS-27 15 0 15 1 0.00 1.00
RES-1-55xSUS-27 12 2 14 1 0.16 0.70
TOTAL 43 5 48
EXPECTED 48 0 48 1 9,39 9‘99
F2 F1-4x 0 156 62 4 66 1 3.87 0.05
Fl-8x 48 15 63 1 2.63 0.10
Fl-9x 60 8 68 l 0.50 0.50
F1—10x 57 15 72 l 1.13 0.30
F1—14x 54 8 62 1 0.17 0.70
TOTAL 281 50 331
EXPECTED 279 52 331 1 9,03 9E99
BC—RES Fl-lxRES-1—54 0.375 43 28 71 1 0.05 0.85
F1—7xRES*1—36 46 27 73 1 0.01 0.95
F1-29xRES—1-27 36 37 73 1 4.87 0.03
F1—30xRES—1-26 29 9 38 1 2.53 0.10
TOTAL 154 101 255
EXPECTED 159 96 255 1 9‘59 9,39
BC—SUS F1-22xSUS—42 0.000 40 0 40 l 0.00 1.00
F1-33xSUS-46 67 0 67 1 0.00 1.00
Fl—34xSUS—43 21 0 21 1 0.00 1.00
F1—37xSUS~48 25 0 25 l 0.00 1.00
F1—4leUS—10 46 3 49 1 0.13 0.75
TOTAL 199 3 202
EXPECTED 202 0 202 1 9,93 9‘99

 

Proposed genotype

aabb x AABb.

> 3.841 is significant at the

The chi—square

values in the

5% level (df=l).
F2 and BC-RES families

 

 

 

40

increased as the number of parents containing lethal genes
increased from 0 to 2 (in models I, III, and IV) resulting
in a progressively poorer fit (Table 8). This indicates
that the lethal genes were not expressed and, therefore, did
not effect the survival rates. This supports the earlier
statement that the lethal genes in the two parents were
different genes.

Other models of inheritance were tested; single gene
dominant, single gene recessive, two genes completely
dominant and three genes completely dominant resulted in a
poor fit between observed and expected values (Table 10).
Table 10. Expected Frequency of Resistant Phenotypes for

Genetic Models of Complete Dominace and/or
Recessiveness.

 

 

r e c o R 'stance
OBS 1 DOM
FAMILY FREQz l DOM 1 REC 2 DOM l REC 3 DOM
RES 0.830 0.750 100.0 0.750 0.750 0.750
Fl 0.094 0.500* 0.000 0.500* 0.000 0.500*
F2 0.151 0.375* 0.250* 0.282* 0.094* 0.220*
BC-R 0.396 0.625* 0.500* 0.626* 0.313* 0.625*
BC-S 0.015 0.250* 0.000 0.125* 0.000 0.025*

 

 

 

 

Observed frequency of resistant plants after 28 days of

screening for resistance to E. g. gegag.

I Parental genotypes for models: monogenic dominace (l DOM)
= Aa x aa, monogenic recessive (l REC) = aa x AA, 2 genes
dominant = AABb x aabb, 1 gene dominant + 1 gene
recessive = aaBb x AAbb, and 3 genes dominant = AABBCC x
aabbbcc.

* An asterisk indicates that the expected frequency of

resistant phenotypes predicted by a given model was

significantly different than the observed frequency of
resistance (by chi—square analysis).

 

      

inn. maintain“ .
.. . .‘.-w

bib .9soioueui .hnn hes:o:§ao sen 610w undid

 
 
   

II I.”_
Tai(.th :fi” 2:181:31 'tiflT .uuiss I‘VIV1BI I13 9

      

"-- ir'IQ'JI if“. I‘d, .‘..
.uonop dussoii§§}‘
.‘. l '-.. ..'; H :21.)

a : ...“:imob

‘z- '..ifl'.'l)b

41

Heterogeneity tests supported the hypothesis that
parental line 6701—l was heterozygous for resistance genes
and line 1849 homozygous susceptible. Heterogeneity chi-
square values were significant for F2 and backcross
resistant families.

EFFECT OF INBREEDING ON RESISTANCE TO EUSARIUM OXYSPORQM F.
SP. QEEAE

A cycle of screening and selfing of line 6701 was
repeated for three generations. Those plants showing the
highest resistance were selected as parents for the next
cycle.

The level of resistance to E. 9. 932g; in selfed
progeny decreased dramatically over the three generations
from that of the original parent line. Analysis of variance
test (Table 11) showed a significant difference between the
percent resistance in the progeny of the selfed families.
The greatest reduction occurred in the lst(SL) and 2nd(&.)
generations of selfing (Table 12) The difference between
the 2nd (8:) and 3rd (83) generations was not significantly
different at the a=0.5 level as determined by LSD test.
This loss of resistance, in part, was probably due to
expression of recessive lethal genes in the homozygous
state.

The trend of decreasing resistance in selfed progeny
is still apparent after separating the effect of any post-
emergence lethal gene (Table 12). The values given have
been corrected to reflect a “true” (effect of lethal genes

removed) level of resistance. Selfing of line 6701 for

 

42

 

 

 

 

 

 

 

Table 11. Analysis of Variance of So, 8,, SE, and S;
Generations.
DF Sum of Error F-value Prob
Squares Mean Square

Between 4 13273.16 3318.29 25.10** 000

Within 84 11103.58 132.19

Total 88 24376.74 H
** 7

Highly significant (a=0.01).

 

 

Table 12. The Effect of Selfing on the Level of Resistance
on Resistant Line 6701.
SO Si slxsl S2 53

No. plants selfed —— 29 5 32 19
No. seeds screened 136 1088 72 932 395
No. plants selected —— 1 -- 6 ~-
for next generation

% survivingx 97 91 100 80 89
in control

% resistance D 21“ 74.7 39.5 30.4 27.5 24.4
% resistance D 28“ 53.0a 17.8b 6.30 7.1c b.1c

 

 

 

 

 

 

 

 

z % of emerging seedlings that survive for 28 days in non—
inoculated conditions.

x (% resistance) x (1 + (1

control)).

' a.b.

and c are significantly different (LSD d=0.5)

frequency of survival in

 

 

 

 

43
three generations resulted in a 89% decrease in the number
of plants resistant to E. 2. ggggg. Since the reduction in
survival rate due to lethal genes was removed this decrease
in resistance was attributed solely to the effect of
inbreeding.

3; plants were sib mated to see if this would restore
some resistance that might have been lost due to other
effects of inbreeding depression besides recessive lethals.
The mean % resistance value was not significantly different
between S. x S. progeny and S2 progeny (Table 12). This
lack of effect from crossing sister plants to restore vigor
and/or resistance was also observed in the F2 generations
(Table 3) where F1 sib families were mated and compared to
F1 selfed families. This effect would be expected if a
high degree of homozygosity between sister plants existed.

The possibility of a lethal gene linked to one or
more of the resistance genes was considered, but this would
have resulted in a much lower level of apparent resistances
in BC-RES families. Also an increase in the level of
lethals in the control in BC-RES families was not observed

(96 to 98% of the BC—RES plants survived in the control),

 

 

 

 

SUMMARY AND RECOMMENDATIONS
SUMMARY OF INHERITANCE OF RESISTANCE TO EESARIHM_QKX§EQEEM
F.SP.QEEAE

Two genes, Egg; (A) and E292 (E), are proposed to
govern resistance to E. Q. ggnag. The A and E genes are
partially dominant resulting in the homozygous genotypes AA
and EE being more resistant than the heterozygous genotypes
A; and EE, respectively. There appears to be an additive
effect between loci with the order of resistance as follows:
M >Mor8fl>ba§b> AAbboraafli>Aabboraa§b>
aabb.

Resistance is not epistatic; at least one resistant
allele (A and E) at both loci must be present for a
resistant phenotype to occur. Plants with genotypes AAbb
and aaBB are susceptible.

Three genotypes result in a resistant phenotype: AAEE,
AAEE, and AQEE (plants survived free of symptoms of E. g.
ggggg for 28 day in screening trials). Plants heterozygous
at both loci (AgEE) appear to have some resistance,
surviving around 21 days in screening trials, thereafter,
developing symptoms of E. Q. genae.

The percent of plants resistant from cross 1849 x

6701—1 and reciprocal cross 6701—l x 1849 was not

44

 

45

significantly different. Therefore, it was concluded that
resistance to E. 2. ggpgg was not maternally inherited.

The existence of recessive lethal genes in both of the
parents was indicated by the lower than expected survival
rates in the uninoculated control plants. Survival rates in
the uninoculated selfed progeny from line 6701-1 and 1849
were close to a 3:1 (normal:lethal) segregation ratio, or
75% survival rate, expected from segregating plants
containing a heterozygous recessive lethal gene (L1). This
lethality occurred in selfed plants but, not in crosses
where the survival rate in the control was approximately
98%. It was concluded that the two parents contained
different lethal genes and, therefore, they did not effect
the survival rates of the F1, F2 and BC families screened

for resistance to E. Q. ggEag.

RECOMMENDATIONS FOR BREEDING ONIONS FOR INCREASED RESISTANCE
TOWF. SP. QEEAE

Assuming the proposed genetic model for resistance to
E. g. ggggg is true, it would be beneficial to increase the
screening time from 21 days to 28 (or more) to eliminate the
genotypes heterozygous for resistant genes. Genotypes AAEE,
AAEE, AgEE, AaEE, AAEE and aaEE survive for 21 days in
screening trials (see Figure 3.) while only genotypes AAEE,
AAEE and AQEE survive for 28 days. Therefore, increasing
the screening time would eliminate those plants with
genotypes AgEE and AAEE, decreasing the frequency of

recessive susceptible genes in the population. Considering

 

 

 

46
the partial dominant gene action proposed it is likely that

genotypes AABb and AaBB might also be reduced or eliminated

 

 

if screening time were extended.

Another effective way to increase selection pressure
for resistance genes is to increase inoculation
concentration (3,58,68,70). A combination of increased
screening time and inoculum concentrations is perhaps an
even better solution. Optimal screening time and inoculum
concentrations for selection of resistance genes could be
determined experimentally.

The effect of inbreeding on level of resistance should
be considered when selecting for resistance to E. g. aspag
in onions. Development of inbred lines requires a certain

amount of selfing to increase homozygosity and homogeneity

in the population. The effect of inbreeding can confound
attempts to select for increased resistance. Some
inbreeding depression, however, can be tolerated and

evidence from the selfing study on line 6701 indicates that
the decline in resistance will taper off after the 2nd
generation of selfing with selection. Massing of selected
plants and cross pollination will usually restore vigor
(45,46); whether the level of resistance will also be
restored will require further study. The use of large
populations (1000—2000 seeds) for screening should allow
heavy selection while retaining enough surviving plants to

recombine to restore heterozygosity and vigor.

 

 

 

 

47

The pattern of resistance observed in this study and
the proposed genetic model suggest that a hybrid with a
relatively high level of resistance to E. Q. gggag can be
produced by crossing a resistant parent with either
another resistant or intermediate level resistant parent.
Crossing a resistant parent with a susceptible parent should
result in a hybrid with little or no resistance since the

trait is not completely dominant.

 

10.

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