Improved seed germination and stand establishment in sweet corn carrying the sh2 gene

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Material Information

Title:
Improved seed germination and stand establishment in sweet corn carrying the sh2 gene
Physical Description:
xi, 137 leaves : ill., photos ; 29 cm.
Language:
English
Creator:
Parera, Carlos Alberto, 1956- ( Dissertant )
Cantliffe, Daniel J. ( Thesis advisor )
Stofella, Peter J. ( Reviewer )
Hildebrand, Peter E. ( Reviewer )
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:
Copyright Date:
1990

Subjects

Subjects / Keywords:
Horticultural Science thesis M.S   ( lcsh )
Dissertations, Academic -- Horticultural Science -- UF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
Consumers demand sweet corn with a significantly high sugar content. Sweet corn hybrids carrying shrunken-2 (sh2) gene, also called supersweets, have high levels of sugar in the endosperm and high sugar retention after harvest. However, supersweet corns have poor germination and seedling vigor leading to reduced stand and yields because of the high susceptibility to seed and soil borne diseases, and poor seed vigor. The factors which affect the germination in sh2 corns and seed treatment to enhance germination and stand establishment were examined. The fungi isolated by seed incubation test included the genus Fusarium spp., rhizopus sp., Penicillium spp., Aspergillus sp, and Pythium spp.. Sodium hypochlorite was found to be an effective seed disinfected treatment.Laboratory germination tests had a high negative correlation with seed imbibition and electrolyte leakage. Basically three seed treatments, 1) biological seed treatment with Tichoderma harzianum, 2) fungicide combination (imazalil, apron, thiram, and captan), and 3) Solid Matrix Priming, were used to enhance seed emergence and stand establishment. The Trichoderma treatment was not effective however; the fungicide seed treatment enhanced stand establishment in all sh2 cultivars tested. The Solid Matrix Priming treatment, combined with a sodium hypochlorite, improved the seed emergence to the level of fungicide treatment. These results suggest that Solid Matrix Priming combined with sodium hypochlorite might be an alternative seed treatment to fungicide to improve stand establishment in sh2 sweet corns.
Thesis:
Thesis (M.S.)--University of Florida, 1990.
Bibliography:
Includes bibliographical references (leaves 121-136).
General Note:
Typescript.
General Note:
Vita.
Funding:
Electronic resources created as part of a prototype UF Institutional Repository and Faculty Papers project by the University of Florida.
Statement of Responsibility:
by Carlos Alberto Parera.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved, Board of Trustees of the University of Florida
Resource Identifier:
aleph - 001576405
oclc - 22914731
notis - AHK0258
System ID:
UF00054866:00001

Table of Contents
    Title Page
        Page I
    Dedication
        Page II
    Acknowledgement
        Page III
    Table of Contents
        Page IV
        Page V
    List of Tables
        Page VI
        Page VII
    List of Figures
        Page VIII
        Page IX
    Abstract
        Page X
        Page XI
    Introduction
        Page 1
        Page 2
        Page 3
    Review of literature
        Page 4
        The shrunken - 2 (sh2) endosperm mutant of corn endosperm mutants of corn
            Page 4
            Page 5
            Page 6
        Seed borne diseases and poor seedling emergence
            Page 7
            Page 8
            Page 9
            Page 10
            Page 11
            Page 12
            Page 13
            Page 14
            Page 15
            Page 16
        Seed imbibition, seed leakage and germination
            Page 17
            Page 18
            Page 19
            Page 20
            Page 21
            Page 22
            Page 23
        Seed germination and vigor analysis
            Page 24
            Page 25
        Presowing seed treatment: seed priming
            Page 26
            Page 27
            Page 28
            Page 29
            Page 30
            Page 31
    Improved stand establishment of shrunken-2 sweet corn by seed treatment
        Page 32
        Page 33
        Materials and methods
            Page 34
            Page 35
            Page 36
            Page 37
        Results and discussion
            Page 38
            Page 39
            Page 40
            Page 41
            Page 42
            Page 43
            Page 44
            Page 45
            Page 46
            Page 47
            Page 48
            Page 49
            Page 50
        Summary
            Page 51
            Page 52
    Imbibition, electrolyte leakage germination, and seed disinfection in sweet corn hybrids carrying sh2 mutant endosperm
        Page 53
        Page 54
        Page 55
        Materials and methods
            Page 56
            Page 57
            Page 58
            Page 59
        Results and discussion
            Page 60
            Page 61
            Page 62
            Page 63
            Page 64
            Page 65
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            Page 73
            Page 74
            Page 75
            Page 76
            Page 77
        Summary
            Page 78
            Page 79
    Improved stand establishment of sh2 sweet corn by solid matrix priming and seed disinfection treatments
        Page 80
        Page 81
        Page 82
        Materials and methods
            Page 83
            Page 84
            Page 85
            Page 86
        Results and discussion
            Page 87
            Page 88
            Page 89
            Page 90
            Page 91
            Page 92
            Page 93
            Page 94
            Page 95
            Page 96
            Page 97
        Summary
            Page 98
            Page 99
    Appendix
        Page 100
        Page 101
        Page 102
        Page 103
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        Page 118
        Page 119
        Page 120
    Literature cited
        Page 121
        Page 122
        Page 123
        Page 124
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        Page 135
        Page 136
    Biographical Sketch
        Page 137
    Signature page
        Page 138
        Page 139
Full Text












IMPROVED SEED GERMINATION AND STAND ESTABLISHMENT
IN SWEET CORN CARRYING THE sh2 GENE

















By

CARLOS ALBERTO PARERA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


1990




































TO MY FAMILY















ACKNOWLEDGEMENTS


I would like to express my acknowledgements to Dr.

Daniel J. Cantliffe, chairman of my supervisory committee,

for his understanding, guidance and support. Appreciation is

also extended to the other members of the supervisory

committee, Dr. Peter Hildebrand and Dr. Peter J. Stoffella,

for their assistance.

I would also like to thank faculty, staff, and students

in the Vegetable Crops department who helped throughout my

graduate studies. Special thanks to go Marie Bieniek, Dr.

Raymond Chee, and Daniel Leskovar for their support and

interesting discussions.

My gratitude is extended to the Instituto Nacional de

Tecnologia Agropecuaria (INTA) for the economic support

during my graduate program.

Finally I want to thank my wife, Carol Troilo, and our

children, Carlos and Victoria, for their encouragement,

help, and love.


iii
















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS ..................................... iii

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

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

ABSTRACT ............................................. x

CHAPTERS

1 INTRODUCTION ........................ ............ 1


2 REVIEW OF LITERATURE ............................ 4

The shrunken-2 (sh2) Endosperm Mutant of Corn.... 4
Seed Borne Diseases and Poor Seedling Emergence.. 7
Seed Imbibition, Seed Leakage and Germination.... 17
Seed Germination and Vigor Analysis.............. 24
Presowing Seed Treatment: Seed Priming............ 26



3 IMPROVED STAND ESTABLISHMENT OF shrunken-2
SWEET CORN BY SEED TREATMENT..................... 32

Materials and Methods............................ 34
Results and Discussion........................... 38
Summary........... ................................ 51



4 IMBIBITION, ELECTROLYTE LEAKAGE GERMINATION, AND
SEED DISINFECTION IN SWEET CORN HYBRIDS CARRYING
sh2 MUTANT ENDOSPERM.............................. 53

Materials and Methods............................. 56
Results and Discussion............................ 60
Summary......................................... .. 78









5 IMPROVED STAND ESTABLISHMENT OF sh2 SWEET CORN
BY SOLID MATRIX PRIMING AND SEED
DISINFECTION TREATMENTS........................... 80


Materials and Methods............................. 83
Results and Discussion........................... 87
Summary ................................................ 98

APPENDIX............................................... 100

LITERATURE CITED...................................... 121

BIOGRAPHICAL SKETCH.................................... 137















LIST OF TABLES


Table Page

3-1. Emergence Rate Index and emergence percentage
for September, October, and November 1988
combined field trials............................ 39

3-2. Seedling height (17 DAP) and dry weight
(19 DAP) for combined September and October
1988 field trials................................ 39

3-3. Effect of seed treatments on ERI and emergence
percentage in 'How Sweet It Is' and 'Crisp
N'Sweet 711' planted in September, October,
and November, 1988.............................. 40

3-4. Effect of sowing date on ERI and emergence
percentage in cv 'How Sweet It Is' and
'Crisp N'Sweet 711' planted in September,
October, and November, 1988.................... 41

3-5. Emergence Rate Index, emergence percentage,
seedling height (17 DAP), and dry weight
(19 DAP) in March and April 1989 field
plantings of two sweet corn cultivars............ 43

3-6. Yield of two sweet corn cultivars in four
field trials planted in March and April 1989.... 43

3-7. Effect of seed treatment on ERI, emergence
percentage, seedling height (17 DAP), and dry
weight (19 DAP) in four field trials planted
in March and April 1989......................... 44

3-8. Effect of sowing date on ERI, emergence
percentage, seedling height (17 DAP), and dry
weight (19 DAP) in four field trials planted
in March and April 1989........................ 46

3-9. Effect of sowing date on yield characteristics
in 'How Sweet It Is' and 'Crisp N'Sweet 711'
planted in March and April in 1989............... 47









3-10. Effect of seed treatment on yield
characteristics in 'How Sweet It Is' and
'Crisp N'Sweet 711' planted in March and
April 1989................... ................. 49

4-1. Total soluble sugar in seeds and seed leachate
characteristics (50 seeds/50 ml water) after 6
hours at 25 oC and germination percentage in a
rolled towel test of 'How Sweet It Is' and
'Crisp N'Sweet 711'............................. 64

4-2. Correlation coefficients among germination
percentage, imbibition electric conductivity,
potassium and total sugar of the seed leachate
in 'Crisp N'Sweet 711' and 'How Sweet It Is'.... 65

4-3. Effect of cultivar and temperature on
imbibition rate (50 seeds/50 ml water),
electric conductivity,potassium and total
sugar in the leachate after 6 hours of
imbibition......... ................ ............. 65

4-4. The effect of seed disinfection treatments on
germination (rolled towel test at 15 OC for 7
days) in sweet corn 'Crisp N' Sweet 711' and
'How Sweet It Is'............... ........ ........ 75

4-5. The effect of seed disinfection treatments on
seedling dry weight in sweet corn 'Crisp N'
Sweet 711' and 'How Sweet It Is'................ 77

5-1. Effect of seed treatments and cultivars
on emergence in a cold test...................... 92

5-2. Effect of seed treatments and cultivar on
Emergence Rate Index and emergence percentage,
calculated 7 days after planting, in a field
experiment planted in October 26, 1989 at
Gainesville, Fl................................. 94

5-3. Effect of seed treatment and cultivar on
plant height, 17 days after planting in a
field experiment planted in October 26, 1989
at Gainesville, Fl............................... 95

5-4. Effect of seed treatment and cultivar on
seedling fresh and dry weight, 19 days after
planting, in a field experiment planted in
October 26, 1989 at Gainesville, Fl............. 97


vii















LIST OF FIGURES


Figure Page

3-1. Maximum and minimum soil temperature (5 cm
deep) at the Horticultural Unit, Gainesville,
Florida, 1988................... ............... 48

3-2. Maximum and minimum soil temperature (5 cm
deep) at the Horticultural Unit, Gainesville,
Florida, 1989................................... 48

4-1. Scanning electron micrographs of 'Crisp N'
Sweet 711' (top) and 'How Sweet It Is'
(bottom) seeds. Note separation between
pericarp and aleurone layer in 'How Sweet
It Is' .. ....................................... 61

4-2. Seed surface of 'Crisp N' Sweet 711' (top)
and 'How Sweet It Is' (bottom)................. 62

4-3. Tetrazolium test in 'Crisp N' Sweet 711' (top)
and 'How Sweet It Is' (bottom). The red color
of the embryo was more uniform and intense in
'Crisp N' Sweet 711' depicting greater seed
vigor............................................ 67

4-4. Imbibition rate (50 seeds/50 ml water) in
'Crisp N' Sweet 711' (top) and 'How Sweet
It Is' (bottom) at 5 OC and 25 oC.
Significant at 5 % (*) or 1 % (**) level ....... 68

4-5. Electric conductivity of the leachate
(50 seeds/ 50 ml water) in 'Crisp N'Sweet 711'
(top) and 'How Sweet It Is (bottom) at 5 C
and 25 oC. Nonsignificant (ns) or significant
(**) at 1 % level.............................. 69

4-6. Imbibition rate (50 seeds/50 ml water) at
5 OC (top) and 25 oC (bottom) in 'Crisp N'
Sweet 711' and 'How Sweet It Is' (bottom).
Nonsignificant (ns), significant at the
5 % (*) or 1 % (**) level...................... 70


viii









4-7. Electric conductivity of the leachate
(50 seeds/50 ml water) at 5 oC (top) and 25 C
(bottom) in Crisp N'Sweet 711' (top) and 'How
Sweet It Is (bottom). Significant (**) at
1 % level ..................................... 71

4-8. Seeds of 'Crisp N' Sweet 711' treated with
sodium hypochlorite (top) and without
treatment (bottom), 10 days after
incubation.............. ....................... 73

4-9. Seeds of 'How Sweet It Is' treated with sodium
hypochlorite (top) and without treatment
(bottom), 10 days after incubation............... 74

5-1. Imbibition (top) and leakage conductivity
(bottom) in four sh2 sweet corn, after 4
hours soaking in distilled water. Means in
each data followed by the same letter are not
significantly different at the 1 % level
by LSD test..................................... 89

5-2. Imbibition (top) and leakage conductivity
(bottom) in primed and nonprimed seeds, after
4 hours soaking in distilled water.
zSignificantly different at 5 % level.......... 90

5-3. Electron micrographs of primed (a) 'Crisp N'
Sweet 711', (b) 'How Sweet It Is', and
nonprimed seeds (c) 'Crisp N' Sweet 711',
(d) 'How Sweet It Is'.......................... 91















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science


IMPROVED SEED GERMINATION AND STAND ESTABLISHMENT
IN SWEET CORN CARRYING THE sh2 GENE

by

CARLOS ALBERTO PARERA

May 1990
Chairman: Daniel J. Cantliffe
Major Department: Horticultural Science


Consumers demand sweet corn with a significantly high

sugar content. Sweet corn hybrids carrying shrunken-2 (sh2)

gene, also called supersweets, have high levels of sugar in

the endosperm and high sugar retention after harvest.

However, supersweet corns have poor germination and seedling

vigor leading to reduced stand and yields because of the

high susceptibility to seed and soil borne diseases, and

poor seed vigor. The factors which affect the germination

in sh2 corns and seed treatment to enhance germination and

stand establishment were examined.

The fungi isolated by seed incubation test included the

genus Fusarium spp., Rhizopus sp., Penicillium spp.,

Asperqillus sp, and Pythium spp.. Sodium hypochlorite was

found to be an effective seed disinfected treatment.









Laboratory germination tests had a high negative correlation

with seed imbibition, and electrolyte leakage.

Basically three seed treatments, 1) biological seed

treatment with Trichoderma harzianum, 2) fungicide

combination (imazalil, apron, thiram, and captain and 3)

Solid Matrix Priming, were used to enhance seed emergence

and stand establishment. The Trichoderma treatment was not

effective however; the fungicide seed treatment enhanced

stand establishment in all sh2 cultivars tested. The Solid

Matrix Priming treatment, combined with a sodium

hypochlorite, improved the seed emergence to the level of

fungicide treatment. These results suggest that Solid

Matrix Priming combined with sodium hypochlorite might be an

alternative seed treatment to fungicide to improve stand

establishment in sh2 sweet corns.















CHAPTER I
INTRODUCTION


In 1986 the total cultivated sweet corn (Zea mays L.)

area in the USA was 90,000 ha and the total value of

production was $206 million. Florida is the nation largest

producer of fresh market sweet corn, the total cultivated

area in 1988 was 27,000 ha, and the total production value

was $72 million (USDA, 1989). The Everglades and Zellwood

muck soils are important areas for sweet corn production in

Florida, especially in the fall and spring seasons. Dade

county is other important area for winter production.

Consumers demand sweet corn with a significant sugar

content to give the corn a taste of sweetness when eaten.

Sweet corn hybrids carrying shrunken-2 (sh2) gene, also

called supersweets, have higher sugar contents in the

endosperm and the sugar retention after harvest is longer

when compared to sugary su corn. Kernel sucrose contents in

shrunken-2 hybrids is among the highest when compared with

other maize mutants genotypes (Garwood et al., 1976).

However, hybrids with this gene have poor germination and

seedling vigor, which has severely reduced stands and yields

(Hannah and Cantliffe, 1977; Andrew, 1982; Tracy and Juvick,

1988). Germination and seedling vigor are especially reduced









2

in cold and wet soil. Soil borne diseases also contribute

to poor stands.

Different alternatives have been proposed to improve

germination and stand establishment of supersweet corn.

Seed treatment with fungicides have reduced soil and seed

borne diseases and improved stands (Berger and Wolf, 1974;

Cantliffe et al., 1975; Pieczarka and Wolf, 1978). Another

option is the use presowing seed treatments (hardening,

moisturizing, priming or osmoconditioning) to increase the

rate, uniformity and/or level of germination (Bodsworth and

Bewley, 1981; Bennet and Waters, 1987a; Sabota, 1987; Harman

et al., 1989). However, all of these methods have had

limited success. Compensated rate seeding also has been

proposed as a method to increase plant uniformity (Guzman et

al., 1983), although this method increases the production

cost, it does not achieve the yield maximization. A method

to consistently enhance germination and stand establishment

of supersweet corn under varying environmental conditions

are urgently needed to improve sweet corn production in

Florida.

The objective of the present study was to improve

germination, stand establishment, and yield in sweet corns

carrying the sh2 gene by fungicide seed treatment or

biological control of soil and seed borne diseases, solid

matrix priming (SMP) to increase germination rate, and a

combination of both techniques. Physiological parameters of










3

sh2 seed germination were evaluated and related to seed

vigor.















CHAPTER II
LITERATURE REVIEW


The Shrunken-2 (sh2) Endosperm Mutant of Corn

Endosperm mutants of corn

Corn (Zea mays L.) has a fruit caryopsiss) composed of

a thin pericarp (seed coat) enclosing the endosperm and

embryo (Wolf et al., 1952). Endosperm is classified as a

nonembryonic storage tissue. The size, chemical

composition, and other characteristics of the endosperm

differ among species and cultivars. The endosperm of seed

corn represents approximately 80% of the total seed dry

weight (Wolf et al., 1952a). A thin outer layer of cells

aleuronee layer) and a large inner part which contains

starch and proteins composes the endosperm (Wolf et al.,

1952b). The only living tissue at maturity in maize

endosperm is the aleurone layer. Food mobilizing enzymes

are produced and secreted by the aleurone layer to hydrolyze

the reserves stored during germination (Bewley and Black,

1985).

There are endosperm maize mutants that present

variations and alterations in the starch-synthetic pathways,

which modifies the carbohydrate composition of the endosperm

(Bewley and Black, 1985). Endosperm maize mutants sugary















CHAPTER II
LITERATURE REVIEW


The Shrunken-2 (sh2) Endosperm Mutant of Corn

Endosperm mutants of corn

Corn (Zea mays L.) has a fruit caryopsiss) composed of

a thin pericarp (seed coat) enclosing the endosperm and

embryo (Wolf et al., 1952). Endosperm is classified as a

nonembryonic storage tissue. The size, chemical

composition, and other characteristics of the endosperm

differ among species and cultivars. The endosperm of seed

corn represents approximately 80% of the total seed dry

weight (Wolf et al., 1952a). A thin outer layer of cells

aleuronee layer) and a large inner part which contains

starch and proteins composes the endosperm (Wolf et al.,

1952b). The only living tissue at maturity in maize

endosperm is the aleurone layer. Food mobilizing enzymes

are produced and secreted by the aleurone layer to hydrolyze

the reserves stored during germination (Bewley and Black,

1985).

There are endosperm maize mutants that present

variations and alterations in the starch-synthetic pathways,

which modifies the carbohydrate composition of the endosperm

(Bewley and Black, 1985). Endosperm maize mutants sugary









5

and sugary-2 (su, su2) form kernels with high levels of

reducing sugars and water soluble polysaccharide (Creech,

1956). Waxy (wx) gen, described by Collins (1909),

generates endosperm that is nearly all amylopectin (Bates et

al. 1943). The opaque-2 (o2) gene increases the lysine and

tryptophan in endosperm (Mertz et al., 1964).

Shrunken-2 endosperm mutant

Shrunken (sh) gene was characterized by Hutchison

(1921). The original seeds were collected from the Niobara

Indian Reservation in Nebraska. The physical

characteristics of the kernels (shrunken) give the name to

the gene which was described as a single factor pair. Mains

(1949) reported that shrunken-2 (sh2) gene was linked with

the al factor for aleurone color in chromosome 3 and blocked

the conversion of sucrose to water soluble polysaccharides,

therefore if was different from the gene described by

Hutchinson (1921). Shrunken kernels have a high

concentration of sugars in the endosperm, primarily sucrose

(Laughnan, 1953). Shrunken-2 genotypes have 7 to 10 times

more sucrose than normal genotypes (Creech, 1956). The

shrunken-2 gene affects carbohydrate synthesis in the

endosperm, increasing the levels of sucrose and decreasing

water soluble polysaccharides and starch at maturity

(Creech, 1956; Wann et al., 1971). The sucrose content in

several hybrids carrying different endosperm mutants was in

sh2 36.5%, 24.8% for ae du wx, 18.5% for ae wx and 14.4%











for su (Garwood et al., 1976). Sweet corn ('Florida Sweet'

and 'F-449') carrying the sh2 gene have more sugar than

'Iobelle' (su) and brittle-A (bt) hybrid at late

developmental stages (Hannah and Cantliffe, 1977). Wann et

al. (1971) determined that after 7 days of storage at 18 C

hybrids with (sh2) endosperm had more sugar than standard

(sul) sweet corn. Kerr (1988) reported that after harvest,

shrunken-2 sweet corn had two times more sugar than standard

sugary types.

Seeds of maize mutant shrunken-2 are less uniform, and

smaller than standard endosperm seeds. The kernel are

normally wrinkled, flat, and easily damaged. The seeds of

sh2 sweet corn were significantly lighter than seeds with su

endosperm (Styer and Cantliffe, 1983b). The ratio of

endosperm to embryo dry weight was two times greater in su

than sh2 hybrids (Styer and Cantliffe, 1981). Shrunken-2

seeds are lighter compared with normal genotypes since the

lower starch content in the endosperm, and the embryos also

weigh less than su or normal counterparts (Styer and

Cantliffe, 1984). Andrew (1982) reported that germination

in sh2 hybrids was superior in round compared with smaller

flat graded kernels.

Early acceptance by growers of sweet corn cultivars

containing the sh2 gene has been attributed to the poor

vigor of the seeds and susceptibility to soil-borne

diseases. Germination tests and vigor determinations in









7

seeds with shrunken-2, sugary, brittle, and normal endosperm

established that sh2 performed the poorest in field and

laboratory tests (Styer et al., 1980). Normal genotypes

have more vigor in intact kernels than sh2, however when

embryos were grown in agar, no differences were detected,

suggesting that the lower vigor might be related to small

endosperm (Wann, 1980). Final field stands of two hybrids

with sh2 endosperm was 34 and 60 % when compared with 77 %

in normal endosperm and 83 % in a bt hybrid (Hannah and

Cantliffe, 1977). Considerable variation in final stand,

between 50 and 95 %, was observed among 17 supersweet

cultivars and advanced breeding lines, in field trials (Howe

and Waters, 1988). Hancik et al. (1989), reported a high

variation (18.8 % and 93.8 %) in final stand from a

supersweet corn cultivar trial grown in Zellwood, Fl.

Seed Borne Diseases and Poor Seedling Emergence

Seed borne pathogens can produce serious crop losses in

vegetables, legumes, industrial crops, fiber crops, etc. In

the Graminae and Leguminosae families, the seeds are

important source of disease transmission.

In corn, many fungi are transmitted by seeds (Fusarium

spp., Pythium spp., Penicillium spp. and Diplodia maydis

(Berk.) Sacc. Neergard (1987) compiled a complete list of

seed borne pathogens in corn. The major seed-borne

pathogens in maize are Fusarium moniliforme, Fusarium

araminearum, Diplodia spp. and Drechlera mavdis.









8

The genus Fusarium is widely distributed all over the

world. It can survive in diverse types of soils, climates

and substrates. Fusarium moniliforme is located in humid

and subhumid temperate zones. The Gramineae family is

highly susceptible to this fungus, exhibiting diseases such

as seedling blight, foot rot, and kernel rot (Booth, 1971).

Seedling disease resulting from Fusarium moniliforme

infected seed corn includes seedling blight, damping-off,

leaf spot, and kernel rot (Shurtleff, 1977).

Fusarium moniliforme was the most prevalent specie

isolated from corn seed samples from the United States

(Manns and Adams, 1921; Wicklow, 1987), from Queensland,

Australia (Blaney et al., 1986), and from Kenya (Khare,

1985). In Mississippi Acrenomium spp. (Ochor et al., 1987)

and in Kenya Aspergillus niqer, and Penicillium spp. were

the predominant fungi isolated in addition to Fusarium

moniliforme (Khare, 1985).

Berger and Wolf (1974) reported that poor stands in

shrunken-2 (sh2) sweet corn hybrids was associated with seed

rot and damping-off. Isolation from damped-off sweet corn

seedlings in Florida commonly yielded Fusarium spp,

Penicillium spp., Rhizoctonia solani, and Pvthium spp.

(Pieczarka and Wolf, 1978). Sweet corn seeds infected with

Fusarium moniliforme have extremely poor germination under

stress conditions (Styer and Cantliffe, 1984). Using

electron microscopy Styer and Cantliffe (1981) located









9

fungus between the pericarp and aleurone in shrunken-2 (sh2)

sweet corn hybrids. Severe infections may affect both the

endosperm and embryo. Fusarium moniliforme was isolated

from embryo, endosperm and pericarp in maize (Li and Wu,

1986). The sources of inoculum for seedling infection in

sweet corn were the seeds and soil (Anderegg and Guthrie,

1981).

There are no external symptoms of internal infection in

seeds contaminated with Fusarium moniliforme (Cristensen

and Wilcoxon, 1966). Four species of Fusarium: F.

oxysporum, F. moniliforme, F. semitectum, and F. solani,

were isolated from 30 sorghum seed samples. Fusarium

solani, F. moniliforme, and F. semisectum were detected and

distributed in all seed tissues, and F. oxysporum was

isolated from the pericarp (Gopinath and Shetty, 1985).

Fusarium moniliforme was detected in endosperm and embryo of

sorghum (Mathur et al., 1975). Surface contamination of

Fusarium moniliforme in corn kernels was reported by El-

Meleigi et al., (1981).

Control of seed borne diseases

The first antecedent of seed treatment was in the 17

th. century, when salt-brine was used to control bunt of

wheat (Tilletia care) (Neergard, 1977). After that,

several physical and chemical treatments were utilized to

control seed-borne fungi. An adequate seed treatment must

eliminate the pathogens present in the seed disinfestationn)









10
and protect the seed and seedling against soil borne

pathogens during germination (protection). Recently,

biological control offers a new perspective on control of

seed borne diseases.

Chemical seed treatments: Chemical seed treatment is

an inexpensive method of plant disease control. Copper

compounds replaced salt-brine in the 19th century (Neergard,

1977). The 20th century began a modern era of chemical seed

treatment, with the utilization of organic mercurial

compounds (Neergard, 1977). Several chemical treatments

have been utilized, and now dithiocarbamatos (thiram),

heterocyclic nitrogen compounds captain) systemic

fungicides benomyll), or combinations of these have been

used as seed treatments.

Captan and thiram do not penetrate to the embryo. They

control fungi located in the seed coat (Ellis et al., 1977).

Carrot germination was improved when seed was treated with

captain (Perry and Hegarty, 1971). Seed treatment with

thiram increased field emergence compared with untreated

seeds in dry beans (Ellis et al., 1976), and peas (Short et

al., 1977). Under stressful environmental conditions,

supersweet corn seeds treated with a combination of captain,

thiram, imazalil, and metalaxil or captain, thiram, imazalil,

and captafol improved emergence compared to nontreated seeds

(Cantliffe and Bieniek, 1988). Application of captain (200

g/100 kg seed) in maize seeds was effective for Fusarium









11
graminearum control (Draganic et al., 1984). Seed corn

treatments with benlate and captain were the most effective

to control seed borne diseases in Kenya (Khare, 1985). Seed

treated with benomyl, captain or carbendazim M had improved

germination percentage compared with untreated maize seeds

(Moreno-Martinez et al., 1985)

Systemic fungicides can move throughout the seed and

seedling via translocation. They can control fungi in the

seed coat or in the internal parts of the seed. Benomyl

seed treatments controlled Phomopsis spp. in soybean (Shortt

and Sinclair, 1980). Singh et al. (1971) reported effective

control by benomyl of Fusarium moniliforme and

Cephalosporium acrenomium in corn. Benomyl plus captafol

seed treatment controlled seed rot and damping-off in sweet

corn (Berger and Wolf, 1974). Cantliffe et al. (1975)

reported that combinations of difolatan + dexon and benlate

+ dexon were the effective seed treatments for sh2 sweet

corn. Seeds of sweet corn 'Florida Staysweet' treated with

benlate + difolatan and banrot + difolatan had the higher

stands and yields than untreated control (Pieczarka and

Wolf, 1978).

Sodium hypochlorite has been used as a seed

disinfestant. Sweet peppers seeds soaked for 30 minutes and

germinated in potato-dextrose agar were free of

contaminants, whereas the non-treated seeds developed

colonies of Alternaria sp. (McCollum and Linn, 1955). Sweet









12

corn seeds surface contaminated with Fusarium moniliforme

were successfully disinfected with sodium hypochlorite

(Schoen and Kulik, 1977). Kernels of sweet corn soaked in a

mixture of sodium hypochlorite and ethanol eliminate surface

contamination of Fusarium moniliforme (El-Meleigi et al.,

1981).

Physical seed treatments: Hot water treatment has been

reported as an effective method in controlling some seed-

borne diseases. Hot water seed treatments are used in wheat

and barley to control Ustilago nuda and Ustilago tritici

(Neergaard, 1977). Heterosporium eschscholtziae was

eliminated from california poppy seeds treated with hot

water (Davis, 1952).

Anaerobic water treatment, dry heat treatment, solar

heat treatment and aerated-steam treatment, are also

examples of physical seed treatments, but the results are

not totally satisfactory (Neergaard, 1977).

Biological control of seed borne diseases

In modern agriculture, the extensive use of pesticides,

often excessive, has resulted in a variety of harmful and

undesirable effects on the environment, including man and

wildlife. Deterioration of the environment and the human

risk produced by chemical products, including agricultural

pesticides (insecticides, fungicides, herbicides, etc.) is a

major world concern. Biological control is an alternative

to chemical control of pests and diseases.









14
covered with spores of Penicillium oxalicum were protected

against infection of Fusarium spp., Pythium spp.,

Aphanomyces spp., and Rhizoctonia spp. (Windels, 1981).

Seed treatments in garbanzo beans with Penicillium oxalicum

was an efficient antagonist of damping-off (Cook and Baker,

1983). An isolate of Pseudomonas fluorescens applied to sh2

sweet corn seed controlled Pythium ultimum damping-off when

the seeds were sown in naturally infested soil (Callam et

al. 1989).

The genus Trichoderma belongs to sub-division

Deuteromycotina, family Gloisporae. According to Rifai

(1969), this genus includes nine species. All the species

are distributed worldwide and found in all types of soils

(Cook and Baker, 1983). The first report about the

antifungal properties was generated by Weidling and Emerson

(1936), who worked with Trichoderma viridae to control

damping-off in citrus seedlings. The mechanisms of

Trichoderma spp. antagonism on soil borne diseases are not

clear (Martin et al., 1985). The production of antibiotics

by Trichoderma was reported by Weidling and Emerson (1936).

Chet et al. (1979) reported the production of the cell wall

degrading enzyme B-(l,3)-glucanase and chitinasein when

Trichoderma harzianum controlled Sclerotium rolfsii and

Rhizoctonia solani. Mycoparasitic activity and not

antibiotic activity was detected toward Rhizoctonia solani

and Phytium spp. by Hadar et al. (1979), and Harman et al.










13

Biological control is defined as a method of pest and

disease control that relies on natural enemies to reduce

pest and disease to tolerable levels. The biological

control is the reduction of the amount of inoculum or

disease-producing activity of a pathogen accomplished by or

through one or more organisms other than man (Cook and

Baker, 1983).

Seed treatment with antagonistic agents is an

attractive method for introducing biological control into

the soil-plant environment (Chao et al., 1986). Biological

seed treatments protect seed and roots instead of protection

by chemicals (Chang and Kommendal, 1968). Several organisms

have been reported as potential biological control agents

for seeds. The most effective fungus used as biological

control treatments of seeds have been species of Chaetomium,

Penicillium, and Trichoderma (Cook and Baker, 1983).

Kernels of corn coated with Chaetomium globosum, sowed

in a greenhouse in soil infected with seedling blight

(Fusarium roseum 'Graminearum'), had higher emergence than

the nontreated seeds (Chang and Kommendahl, 1968). During

three years of field experiments, corn seed treated with

Chaetomium qlobosum exhibited improved emergence and stands

compared to nontreated seeds (Kommendahl and Mew, 1975).

Penicillium oxalicum has been reported as an effective

seed treatment against root fungus in peas (Windels and

Kommendahl, 1978; Kommendahl and Windels, 1978). Pea seeds










15

(1980). Lifshitz et al. (1986) suggested that Trichoderma

spp. produced toxic metabolites, which controlled damping-

off in peas.

Mycoparasitism is defined as a complex biological

system that involves chemotropic growth of Trichoderma,

identification of the host fungus, excretion of

extracellular enzymes and finally lysis of the host (Chet,

1987). Wells et al. (1972) isolated Trichoderma harzianum

from sclerotia of Sclerotium rolfsii. When applied to the

soil the isolated pathogen controlled Sclerotium rolfsii in

tomato and peanut plants. Trichoderma harzianum applied to

greenhouse soil infested with Rhizoctonia solani and

Sclerotium rolfsii suppressed damping-off in beans,

eggplants and peanuts (Chet et al., 1979).

Harman et al. (1980) using a Methocel slurry treatment

of pea and radish seed with Trichoderma hamatum which

controlled Pythium spp. and Rhizoctonia solani. Dent corn

and soybeans seeds treated with Trichoderma harzianum

yielded seed of the same quality as that produced using

chemical treatments (Kommendahl et al., 1981). Trichoderma

koningii and harzianum isolated from Arkport soil protected

seeds and root of pea, snap bean, and cucumber against

phytium infection (Hadar et al., 1984).

The addition of specific elements to the seed jointly

with Trichoderma spp. increased the biocontrol efficacy of

the fungi. Cell walls of Rhizoctonia solani or chitin added










16

to the seed increased the activity of Trichoderma hamatum to

control Phytium spp. and Rhizoctonia solani in pea and

radish (Harman et al., 1981). Pea seeds treated with

Metalaxil prior to infection with Trichoderma harzianum

increased the percentage of conidia in the rhizosphere

(Papavizas, 1981). The biocontrol by Trichoderma koningii

and harzianum of phytium seed rot in peas was enhanced by

the addition of organic acids, polysaccharides, and

polyhydroxy alcohols to seed treatment (Nelson et al.,

1988). Soil treatment with methyl bromide (200 kg/ha)

combined with seed treatment of Trichoderma harzianum

reduced the incidence of Rhizoctonia solani in beans (Chet,

1987)

A new biotype of Trichoderma harzianum induced by

ultraviolet irradiation showed tolerance to fungicide and

more effective biocontrol than wild strains (Papavizas et

al., 1982). Recently Harman et al. (1988) reported that

protoplast fusion of two strains of Trichoderma harzianum

lead to more effective progenies for biocontrol on seeds.

The combination of the new Trichoderma strains and the use

of Solid Matrix Priming in cotton, cucumber, pea, snap bean,

sweet corn and wheat seed increased plant stand in New York

soils infected with Fusarium graminearum and Phytium ultimum

(Harman et al., 1988).










17

Seed Imbibition, Seed Leakage and Germination

Seed imbibition

Imbibition or seed rehydration is the first step in the

germination process. Water is an indispensable solvent for

inorganic and organic compounds and vital in all biological

processes. Water potential is an expression of the energy

status of water (Bewley and Black, 1978). The movement of

the water is from higher to lower osmotic potential regions.

Air dried seed has an extremely low water potential -50 to

-100 Mpa (Hegarty, 1978). Seed water potential can be

divided in three components; osmotic potential due to the

solutes dissolved in the cells, matrix potential due to the

capacity of the protein bodies and cell wall to secure

water, and turgor potential due to the pressure of the water

to the cell wall when it enters into the cell (Bewley and

Black, 1985), (Woodstock, 1988). The osmotic and matrix

potential have negative and turgor potential positive

values. The water potential of pure water is zero.

Seed imbibition under optimal germination conditions

has been characterized by Bewley and Black (1985) in three

phases: Phase I, or imbibition phase, is a consequence of

matrix forces of the seed cell walls and the penetration of

water into crevices and interstices of the seeds (Hadas,

1982). The water uptake in phase I is passive and not

influenced by the viability or dormancy stage of the seed.

Phase II, or lag phase, is identified by an active










18

metabolism of the seed. It is a critical phase of

imbibition processes, any modification in this phase can

affect the germination of the seed (Hadas, 1982).

Phase III, or growth phase, is reached only by viable

seeds. It is associated with protrusion of the radicle or

visible germination. Water uptake increases rapidly because

of the formation of low molecular weight metabolites inside

the cells (Bewley and Black, 1985).

Water absorbed during imbibition differs in the various

seed parts. Stiles (1948) reported that after 96 hours of

imbibition in corn cv 'Sure Cropper', the percentage of

water absorbed per gram of dry weight was 154 %. In the

embryo the water percentage increased 1113 %, in the

scutellum 237 %, in the pericarp 131 %, and 67 % in the

endosperm. In dent corn when the total seed water content

reached 75 %, the embryo increased to 261 % while the rest

of the seed only reached 50 % (Blacklow, 1972).

The seed coat protects the inner parts of the seed

against physical damage. It is also a barrier to fungus

invasion and insect attack. The seed coat plays a vital

role in the regulation of imbibition. Passive, imbibitional

processes, and hygroscopic properties of the cell are

responsible for absorption of water by the seed coat

(Stiles, 1948). The anatomy of the seed coat varies among

species and cultivars. The differences in the cuticle and

protective layers results in different levels of










19

impermeability to gases and water (Bewley and Black, 1978).

The seed coat of lima bean protects against low temperature

and moisture stress (Pollock and Toole, 1966). Rapid water

uptake, in dry pea seeds imbibed without the seed coat,

produced dead cells on the surface of the cotyledons (Powell

and Matthews, 1978). Physical was more important than

chemical damage when rapid imbibition occurred. Oliveira et

al. (1984) reported that damaged seed coats in soybeans were

negatively correlated with field emergence and concluded

that there was an association between a split seed coat,

rate of imbibition, and dead area in the cotyledons. White

cultivars of dwarf bean imbibed water more rapidly than

brown cultivars, and there was a direct correlation with

germination percentage and growth of the seedling (Powell et

al., 1986)

Hardseeds generally have seed coat covers with waxy

substances and elongated pores (Yaklich et al., 1986;

Woodstock, 1988). By removing the seed coat, hardseeds

become more permeable (Arachevaleta-Medina and Snyder,

1981). Seed coat permeability normally was associated with

the thickness of the coat. In soybean, permeability of the

seed coat can be related to the seed coat/embryo ratio

(weight basis). Soybean genotypes with a ratio lower than

0.1, at maturity, were more permeable (Yaklich et al.,

1986). Davis and Porter (1936) noted that absorption of

water and germination rate in corn were faster when the seed










20

were placed embryo side down on wet blotters.

The size and structure of the seed also affects the

rate of imbibition. In corn seed, flat small kernels imbibe

water faster than round kernels (Shieh and McDonald, 1980).

Patil and Andrews (1983) determined in cotton seeds that the

differential rates of imbibition were a consequence of seed

size, chemical composition, and seed coat permeability.

Temperature influences the rate of water imbibition.

Under low temperature the viscosity of water increased, and

slow water uptake resulted (Murphy and Nolan, 1982;

Vertucci, 1989). The imbibition in corn seed was influenced

by temperature since changes occurred in the fluidity of the

water (Blacklow, 1972).

Cell membranes are constructed via protein and

phospholipid molecules organized in a bilayer structure

(Simon, 1978). This organization is an equilibrium between

water and the component molecules. At lower water content

(< 20%) the lamellar structures change (Luzzati and Husson,

1962), and become organized in a hexagonal phase (Bewley and

Black, 1985). When tissues are rehydrated membrane bilayer

structure is reconstituted. According to Woodstock and Tao

(1981), if the water absorption is reduced during the first

step of imbibition in soybean, the tissues develop in an

organized manner and giving extra time for membrane

rearrangement. Complete disorganization of cell membrane in

dry embryos of pea during rapid water absorption have been










21

reported by Powell and Matthews (1978). Thomson and Platt-

Aloia (1982) determined in cowpea seeds that plasmalemma is

permeable in both directions during the early stages of

imbibition.

Numerous factors such as seed viability and vigor,

membrane permeability, chemical composition of the seed,

seed size, presence and/or condition of the seed coat, and

physical constants can affect the imbibition process in

seeds. The influence of each of these factors in the seed

imbibition processes can not be analyze separately

(Woodstock, 1988).

Seed leakage

Concomitant with imbibition, there may be leakage of

solutes from seeds. Solutes such as sugars, organic acids,

amino acids, ions were detected in seed leachate. Potassium

generally is an important constituent of the seed leachate.

The most frequent cation released from cotyledons of legumes

seeds was potassium (Powell and Matthews, 1977). In pea

seeds potassium accounted for 25 to 50 % of the total

leachate (Mullet and Considine, 1980). Seed leakage was a

characteristic of each seed (Simon and Srimathi Mathavan,

1986; Matthews and Bradnock, 1968). Styer and Cantliffe

(1983b) reported decreases in leakage from dried corn seeds

at different stages of development. Leakage from a sh2 corn

seeds was greater than from su genotypes (Wann, 1986; Styer

and Cantliffe, 1983b).










22

Leakage can promote growth of pathogens in and around

seeds. Seed coat cracks in beans increased damping off, as

the exudates provided essential nutritive substances for

fungi development (Schroth and Cook, 1963). Axes of lima

beans, injured by low temperature imbibition, resulted in

lost organic constituents, which increased soil

microorganism growth (Pollock and Toole, 1966). Extracts

from black bean seed coats contained phenolic compounds,

which increased the development of Rhizoctonia solani

(Prasad and Weigle, 1976). Carbohydrates and cyclitol

leached by soybeans seeds may have encouraged the

colonization of Rhizobium species in the soil surrounding

the seeds (Nordin, 1984).

The seed coat has been well defined as a barrier

against imbibition, and the same role is associated with

seed leakage. Seeds with intact seed coats do not leak in

the same proportion as those with cracked seed coats. If a

seed coat suffers mechanical injury during harvest,

processing, or storage, subsequent leakage can increase

dramatically upon imbibition (Simon, 1978). Protein in the

leachate was associated with broken.pericarp and high

membrane permeability in sh2, ae, du, and wx endosperm

genotypes of corn (Wann, 1986). Leachate from pea seeds

without seed a coat has more solutes than leachate from

seeds with an intact seed coat (Larson, 1968). Cell rupture

and membrane alteration can affect the rate of solutes in










23

the leachate (Senaratna and Mc Kersie, 1983). The rate of

electrolyte was related to membrane leakiness (Woodstock,

1988). The first electrolytes which leak from seeds are the

result of passive diffusion, whereas the presence of

intracellular macromolecules in the leachate are the

consequence of the loss of membrane permeability (Duke et

al. 1983). Dry seeds lose their membrane integrity during

desiccation. When dry seeds were imbibed, solutes leak

prior to membrane reorganization (Simon and Raja Harun,

1972). The rate of potassium leakage in sunflower seeds has

was rapid in the first hour of imbibition, and reached a

maximum in three or four hours. In small seeds, (celery,

carrot, fennel) the maximum percentage of potassium leakage

was achieved after 15 minutes of imbibition (Simon and

Mathavan, 1986).

A high level of seed exudation was associated with low

germination in 11 cultivars of wrinkled-seeded peas and 16

cultivars of french beans (Matthews and Bradnock, 1968).

The rapid liberation of electrolytes of bean seed was

correlated with the susceptibility to soaking injury (Mullet

and Cosidine, 1979). The amount of seed leakage was greater

in sh2 seeds grown in the field than in the greenhouse

(Styer and Cantliffe, 1983). Tatum (1954) reported a

negative correlation between amount of corn seed exudate and

germination. An increased length of imbibition, in sweet

corn, corresponded to an overall increased leakage










24

conductivity; however this varied according to endosperm

type and inbred background (Schmidt and Tracy, 1989).

The measure of seed leakage conductivity is an

alternative method to determine seed quality. Presley

(1958) used seed leakage characteristics to determine cotton

seed viability. The higher the electric conductivity, the

lower the vigor. The leakage conductivity of low vigor axes

was six times greater than in high vigor axes of soybean

seeds (Woodstock and Tao, 1981). The measurement of seed

leakage conductivity in soybeans was a more effective

prediction of field emergence than laboratory germination

tests (Oliveira et al., 1984). Tao (1980) reported a high

correlation between seed leakage conductivity and field

emergence in corn. Waters and Blanchette (1983) reported a

high correlation between seed leakage conductivity and field

emergence in sweet corn hybrids.

Seed Germination and Vigor Analysis

Seed vigor has been defined by the International Seed

Testing Association (ISTA) as the sum of those properties

which determine the potential level of activity and

performance of the seed or seed lot during germination and

seedling emergence (The seed vigor test committee, AOSA,

1983).

Several methods of evaluating seed vigor are available.

The cold test is a widely used method of determining seed

vigor and predict field emergence in corn. The test is










25

based on the capacity of the seeds to germinate under cold-

wet soil conditions. Physiological conditions of the seed,

seed treatments, mechanical injury, and seed heredity are

the primary factors that affect seed germination. The cold

test reflects the combination of these effects on the seeds

(The seed vigor test committee, AOSA, 1983).

The seedling growth rate test has been recommended by

ISTA to evaluate seed vigor in corn and soybean. The test

measures seedling growth using a standard rolled towel

germination test (The seed vigor test committee, AOSA,

1983). Numerous authors have reported seed vigor to be

correlated with seedling development. Low-vigor soybean

seeds generated the smallest plants in the first stages of

development (Edje and Burris, 1971). Derwyn et al. (1967),

working in Phalaris sp., reported a high correlation between

a seed vigor and a seedling growth rate test.

The tetrazolium test has been used to determine seed

vigor and viability. It is a fast method and is applicable

to many different seed species. The test was originated in

Germany and now is widely used as a quick determination of

seed viability (The Tetrazolium Testing Committee, AOSA,

1970). The test is based on the characteristics of the

dehydrogenase enzymes to liberate hydrogen ions, which

change the color of the tetrazolium salt (2, 3, 5-triphenyl

tetrazolium chloride) to red. The dead cells remain

colorless. Moore and Goodsell (1965) reported high










26

correlation between the cold test and the tetrazolium test

in corn.

The conductivity test is based in the measure of the

electrical conductivity of the seed leakage. Membrane

damage affects numerous biochemical processes in the living

cells. Vigorous seeds generally have a greater capacity to

repair membrane damage during imbibition (Simon and Raja,

1972; Short and Lacy, 1976). Numerous authors reported a

high correlation between leakage conductivity and field

emergence (Tracy and Juvik, 1988; Oliveira et al., 1984;

Waters and Blanchette, 1983; Matthews and Bradnock, 1967).

Presowing Seed Treatments: Seed Priming

The time period between planting and emergence of

seedlings is critical for stand establishment and eventual

yield in many crops. Physical stresses, such as extreme

temperature, excess or deficit of water, salinity or soil

crusting and biological stresses, including pathogens and

insects, can all adversely affect germination and seedling

growth (Bradford, 1986). The uniformity and percentage of

emergence of direct seeded crops can have a major impact on

final yield and quality (Wurr and Fellow, 1983).

The 'hydration' presowing seed treatments (hardening,

moisturizing, priming) to increase the rate, uniformity

and/or level of seed germination have received considerable

attention in the past 15 years

An osmotic seed treatment, also referred to as










27

'priming' (Heydecker and Coolbear, 1977) or

'osmoconditioning' (Khan et al., 1978), has been the most

successful presowing hydration method. It consists of

imbibing seeds in an osmotic solution that allows seeds to

imbibe water and go through the first steps of germination

but which does not permit radicle protrusion through the

seed coat (Cantliffe, 1981).

Salt solutions had previously been used for this

purpose (Ells, 1963), but in recent years the use of

polyethylene glycol (PEG) 6000 first (Michel and Kaufman,

1973) and more recently PEG 8000 (Michel, 1983) replaced the

use of salt for priming seeds.

Priming treatments have been reported by numerous

authors as a successful presowing seed treatment. Heydecker

et al. (1973) reported promising results on onion (Allium

cepa L.). Improved germination rate in lettuce (Lactuca

sativa L.) at high temperature after priming was reported by

Guedes and Cantliffe (1980). Szafirowska et al.(1981)

improved carrot (Daucus carota L.) emergence time, stand

size, uniformity of the stand in the field, and yield in

cold soil by osmoconditioning. Osmotic priming of seeds

with solutes of PEG can lead to rapid and synchronous

germination at cool temperature (Bodworts and Bewley, 1981).

Khan and Taylor (1986) improved emergence rate and final

stand in beet (Beta vulgaris L) when seeds were amended with

PEG 8000. Priming may reduce poor stand establishment of










28
Brassica cultivars in cold and wet soils (Rao et al., 1987).

The mechanisms involved in osmoconditioning treatment

are not entirely understood (Khan et al., 1980; Bradford,

1986). Some of the physiological and biochemical changes in

primed seeds were investigated by Khan et al.(1978). They

reported that RNA and protein metabolism were enhanced by

osmoconditioning, and also suggested that seed storage

materials such as carbohydrates, fat and proteins were

mobilized. The increment of RNA, or the improved ability of

the treated seed to synthesize RNA during subsequent

germination may be a function of activation and\or synthesis

of enzymes of RNA metabolism (Khan et al, 1981). Coolbear

and Grierson (1979) working in tomato (Lvcopersicon

esculentun Mill.) reported extensive accumulation of nucleic

acids, following an osmotic presowing treatment. Rye

embryos, imbibed for 3 to 6 hours and dehydrated to the

original percentage of water, exhibited a high rate of

protein synthesis. When the embryos were rehydrated there

was also an enhancement of RNA synthesis (Sen and Osborne,

1974). Corn embryos, primed with K2HPO4 for three days at

20 oC, had more absolute amount of phospholipids and sterols

than non-primed embryos (Basra et al., 1988). One of the

phospholipids detected, diphosphatidylglycerol, may indicate

enhanced organization of the mitochondrial membrane ATP

accumulation.

There have been few attempts to use osmoconditioning










29

treatments in corn. Corn seeds primed with PEG solution

(-10 bars at 10 oC for 6 days) obtained early germination in

laboratory under cool conditions (10 oC), however when the

seeds were dried some of the beneficial characteristics were

lost (Bodsworth and Bewley, 1979). Seeds of corn, pea, and

soybean were imbibed and dehydrated for different time

periods. The loss of desiccation tolerance, in the three

species, was coincident with a loss of oligosaccharides,

which can prevent sucrose crystallization. Also there was

an increase in monosaccharides, which may cause protein and

DNA damage through the Maillard reaction (Koster and

Leopold, 1988). Osmotic seed priming, with potassium salts

or polyethylene glycol, accelerated the germination of corn

cv Partap under chilling conditions (10 OC) (Basra et al,

1988).

Bennet and Waters (1987 b), compared the effect of

priming, soaking and moisturizing seed treatments on stand

establishment of a normal (su) 'Jubilee' and two supersweet

(sh2) 'Sweetie' and 'Sugar Loaf' genotypes of sweet corn.

The primed treatment (33 % PEG solution, 7 days at 20 oC)

affected germination negatively while soaking and

moisturizing treatments enhanced emergence. They reported

similar results using the same treatments on three different

seed vigor classes of sweet corn (Bennet and Waters, 1987a).

Solid Matrix Priming: The application of priming

methods in large seed species is reduced because of aeration










30

problems, large volume of solution per seed and the large

amount of seeds required for commercial use.

Solid Matrix Priming (SMP) consists of seeds mixed with

an organic or inorganic carrier and water. The moisture

content of the mixture brought to a level just below that

required for radicle protrusion (Harman and Taylor, 1988).

The first attempt at using solid media in seed priming was

reported by Peterson (1976), who mixed onion seeds with PEG

6000 solution and vermiculite in a polyethylene bag.

Problems encountered included the separation of the media

from'the seeds and aeration, but the technique was adaptable

to a large quantity of seeds. Successful germination of

sweet corn and watermelon seed in cool soil was reported by

Sabota (1987), using seeds which were presoaked in an

aqueous preparation of Terra-Sorb GB for 24 hours. Sweet

corn seeds hydrated in moist vermiculite for 24 hours or

soaked in water for 16 hours before sowing demonstrated

early emergence and high seedling vigor compared to primed

and control seeds (Bennet and Waters, 1987 b). Seeds of

supersweet 'Florida Staysweet' and standard sweet corn

'Silver Queen' presoaked in an aqueous solution of Terra-

Sorb GB for 24 hours had high plant uniformity and stand

establishment (Sabota et al., 1987).

Tomato, onion and carrot seeds primed via solid matrix

priming, with Agro-Lig, had higher seedling emergence, lower

time to 50 % of emergence, and higher plant dry weight










31

compared with traditional priming methods (Taylor et al.,

1988).

The rate and uniformity of stand establishment in sweet

corn 'Crisp N' Sweet' were not improved by Solid Matrix

Priming treatments technique developed by J. Easting

(Kamterter, Lincoln, NB) (Cantliffe and Bieniek, 1988).

Solid Matrix Priming (SMP) provides ideal conditions to

deliver other products to the seeds. Harman and Taylor

(1988) combined SMP technique with biological control

agents. The association between SMP and Trichoderma was the

most effective treatment in tomato and cucumber seeds sowed

in Phytium infested soil. The SMP combined with biocontrol

agents can replace traditional chemical seed treatment

(Harman et al. 1989).

In summary, seed germination and stand establishment

are influenced by many factors such as soil and seed borne

diseases, seed imbibition, and electrolytes in the seed

leachate. Seeds of supersweet corn cultivars are severely

affected by these factors, reaching poor germination and

stand establishment under field conditions. Thus, the

primary goal of this study was to find an effective and

consistent seed treatment in sh2 sweet corn to achieve an

appropriate germination level and stand establishment under

varying environmental conditions.















CHAPTER III
IMPROVED STAND ESTABLISHMENT OF shrunken-2 SWEET CORN
BY SEED TREATMENTS


Sweet corns containing the shrunken-2 (sh2) gene have

high market and postharvest eating quality due to their high

sugar contents at edible maturity and their low sugar

conversion to starch rates (Garwood et al., 1976). However,

the use of supersweet corn has been limited in the past

because of poor stand establishment, aggravated under stress

conditions (Hannah and Cantliffe, 1977; Styer et al., 1980).

Poor germination in sh2 hybrids is attributed to low seed

vigor and susceptibility to seed and soil borne diseases

(Styer et al., 1984; Wann, 1980; Berger and Wolf, 1974; Wann

et al., 1971).

Seed-rot and post emergence damping off cause severe

stand losses in supersweet corn (Berger and Wolf, 1974).

Isolation of pathogens from damped-off sweet corn seedlings

in Florida commonly yielded Penicillium spp., Fusarium spp.,

Rhizoctonia solani, and Pythium spp. (Pieczarka and Wolf,

1978). Seeds of sh2 sweet corn infected with Fusarium

moniliforme had low germination and emergence in cold soil

(Styer and Cantliffe, 1984).

Different chemical treatments have been proposed to

reduce fungi incidence in sweet corn seed. Effective

32










33
disease control was reported by Berger and Wolf (1974) using

benomyl and captafol in a slurry seed treatment. The

fungicide combinations difolatan + dexon and benlate + dexon

were the most beneficial seed treatment in 'Florida Sweet'

corn to control seed and soil borne diseases (Cantliffe et

al., 1975).

Seed treatments with antagonistic agents are an

attractive method to introduce biological disease control

into the soil-plant environment (Chao et al., 1980).

Species of Trichoderma fungi have been reported as active

biostppressive agents. Trichoderma harzianum controlled

damping off in bean, peanut and eggplant (Chet et al.,

1979). Trichoderma harzianum and Trichoderma koninqii

reduced damping-off incidence in peas induced by Pythium

spp. (Lifshitz et al., 1986). Pea and radish seed,

inoculated with Trichoderma hamatum, did not become infected

with seed rot symptoms in soils infected with Pythium and

Sclerotium rolfsii (Harman et al., 1981). The combination

of Trichoderma strains and Solid Matrix Priming in tomato

seeds reduced damping off occurrence (Harman and Taylor,

1988). Seeds of cotton, cucumber, pea, snap bean, sweet

corn, and wheat treated with two strains of Trichoderma

harzianum increased the stands compared to control (Harman

et al., 1989).

Solid Matrix Priming (SMP) is a relatively new

procedure, where in seeds are mixed with an organic or










34

inorganic carrier instead of osmotic solutions in order to

improve germination rate and stand establishment (Kubik et

al., 1989). The treatment has the same advantages as

traditional priming, but the technique is easier and more

applicable to large seeds (Harman and Taylor, 1988). Seeds

of carrot, cucumber, lettuce, onion and tomato primed by

this method had superior seedling emergence characteristics

than nonprimed seeds (Taylor et al., 1988). However, seeds

of corn 'Crisp N'Sweet' primed via SMP (J. Eastin, Kamterter

Inc., Lincoln, NB) were not improved in germination and

stand characteristics as compared to nontreated seeds in

field trials (Cantliffe and Bieniek, 1988).

The objective of this work was to evaluate the effect

of Solid Matrix Priming (SMP), Trichoderma harzianum,

fungicides, and a combination of SMP and Trichoderma seed

treatments on field stand establishment of sh2 sweet corns.

Materials and Methods

The study was conducted during 1988 and 1989, at the

IFAS Horticultural Unit in Gainesville, Florida on an

Arredondo fine sand soil (loamy, silaceous, hyperthermic

Grossarenic Palenundult). The field had sweet corn grown in

it the previous two seasons, and residues were incorporated

into the soil to ensure a potentially high level of pathogen

development.

Seed treatments

Seeds of two sweet corn hybrids (Zea mays L.) 'Crisp N'











Seed treatments

Seeds of two sweet corn hybrids (Zea mays L.) 'Crisp N'

Sweet 711' and 'How Sweet It Is' were treated with captain:

(N-[(trichlorometyl)thio]-4-cyclohexene-l,2-dicarboximide,

and the combination of captain + carboxin: Carboxin(5,6

dihydro-2-methyl-l,4 oxathiin-3-carboxinilide) + metalaxil:

N-(2,6-dimethylphenyl)-N-(methoxyacetyl) alanine methyl

ester + Imazilil: (l-(2-(2,4-dichlorophenyl)-2-(2-

propenyloxy)ethyl)-lh Imidazole at commercial rate. The

biological treatment consisted of two seed inoculation

methods for adding Trichoderma harzianum (Strain Y). In

inoculation method 1 (TI) conidiospores of Trichoderma were

suspended in a 10 % (w/v) aqueous suspension of Pelgel

(Nitragin Co, Milwaukee, WI). The final solution

concentration was 107 108 conidia/ml and 1 ml of solution

was used to treat 6 g of seed. In method two (T2),

introduced in the 1989 trials, a wettable powder formulation

of conidiospores was added to Pelgel at a 5 % (w/w) rate.

In 1988, the SMP treatment consisted of a mixture of

seeds and Leonardite shale (2:3 w/w ratio) (Agro-Lig,

American Colloid Co., Agronomic Div., Arlinton Heights, IL)

with water (60 % moisture content), incubated 4 days at 20

C. The Agro-Lig moisture content in 1989 was reduced to 40

% and seeds were incubated in the mixture for one day. The

last seed treatment was a combination of SMP and

Trichoderma, where seeds previously inoculated with spores









36
as T1 in 1988 and T2 in 1989 were used for the SMP method

1988 and 1989 respectively (Dr. G. E. Harman personal

communication).

Field studies

The sowing dates in fall 1988 were: September 20,

October 19 and November 17. The seed treatments were:

captain (CA), fungicide combination of captain, carboxin,

metalaxil, and imazalil (CC), solid matrix priming (SMP),

Trichoderma (Strain Y) (T), a combination between SMP and

Trichoderma (SMP+T) and a control (nontreated) (C). In

spring 1989, the sowing dates were March 17, March 31, April

14 and April 28. The seeds were treated with: fungicide

combination (CC), Trichoderma inoculation method 1 (Tl),

Trichoderma inoculation method 2 (T2), combination between

Solid Matrix Priming and Trichoderma (SMP+T), and a control

(C).

The plot length was 7.6 m on beds separated by 1.22 m,

with each bed 0.70 m wide and 0.20 m in height. Two seeds

were seeded 4 cm deep, every 30 cm in each plot (50

seeds/plot). Overhead sprinkler irrigation was applied as

needed. Fertilization, growing practices, and pest control

were done according to Florida Agricultural Extension

Service recommendations (Showalter, 1986). Weed control was

mechanical and manual to avoid any effect of herbicides on

emergence and stand uniformity.

Data collection










37

Emergence Rate Index (ERI) was calculated according to

Shmueli and Goldberg (1971), (ERI= E Xn (c-n), where Xn=

number of seedlings/m of row, counted on day n; c= number of

days from planting until emergence ended; n= day on which

counts are made, expressed as the number of days after

planting). Seedling height, measured from the soil level to

the top of the seedling, was recorded 17 days after planting

for 10 plants per plot. Dry weight was recorded 19 days

after sowing by cutting 10 plants per plot at the soil level

and oven drying at 75 oC for 72 hours. The ears were

harvested manually from 6 m in each plot on June 6, 16, 20,

and July 1 and classified according to USDA (1954) quality

standards, then counted and weighed. Maximum and minimum

temperatures were recorded every day at three different soil

levels (15 cm deep, 5 cm deep, and soil surface) during the

course of the experiment. Due to early frost, in the fall

of 1988, only ERI and emergence percentage data were

recorded for the three sowing, and plant height and dry

weight were recorded for the September and October planting

dates.

Statistical analyses

All trials were conducted in a randomized complete

block design, with each treatment replicated four times.

The emergence percentage were analyzed as a square root arc

sine transformation. A Statistical Analysis System (SAS)

(1987) software program was used to analyze the data. Main










38

Results and Discussion

Fall 1988: The main effects of cultivar, treatment and

sowing date were significantly different for ERI, emergence

percentage and plant height; only cultivar was significantly

different for seedling dry weight (Appendix, Table 1, 3, 5,

6). Since the interactions cv x treatment, and cv x sowing

date were significant for most of the data, main effects

were partitioned and analyzed for each cultivar.

'Crisp N' Sweet 711' had greater ERI, emergence

percentage, seedling height and dry weight than 'How sweet

It Is' in the 1988 trials (Table 3-1 and 3-2). Seed

treatments had no significant effect on emergence rate and

percentage in 'Crisp N' Sweet 711' (Table 3-3). However, in

'How Sweet It Is' fungicide seed treatments captainn or

fungicide combination) significantly increased ERI and

emergence percentage. The Trichoderma seed inoculation,

SMP, and SMP+T treatments did not improve rate of emergence

and stand establishment over the control.

Emergence rate was more rapid in September and October

than November for both cultivars (Table 3-4). Total

emergence was unaffected by planting date in 'Crisp N' Sweet

711'; however, the emergence was higher in the September

planting of 'How Sweet It Is' than the October or November

plantings (Table 3-4).

Spring 1989: The main effects of cultivar, treatment,

and sowing date were significantly different for ERI,
















Table 3-1. Emergence Rate Index and emergence percentage for
September, October, and November 1988 combined field
trials.


Cultivar ERI Emergence (X)

Crisp N' Sweet 711 164.7 81

How Sweet It Is 90.3 47

Significance ** **

Significant at the 1% Level (**).
Data pooled over seed treatments and planting dates.


Table 3-2. Seedling height (17 DAP) and dry weight (19 DAP)
for combined September and October 1988 field trials.


Seedling Height Dry Weight
Cultivar (cm) (mg)

Crisp N' Sweet 711 11.8 197

How Sweet It Is 8.2 66

Significance ** **

Significant at the 1X level (**).
Data pooled over seed treatments and planting dates.












40




Table 3-3. Effect of seed treatments on ERI and emergence
percentage in 'How Sweet It Is' and 'Crisp N'Sweet 711'
planted in September, October, and November, 1988.


Cultivars

How Sweet It Is Crisp N'Sweet 711

Seed Treatment ERI Emerg.(%) ERI Emerg.(%)

CC 127.4 67 166.7 84

CA 119.1 57 165.8 85

T+SMP 80.9 44 165.8 81

T 73.7 39 167.0 87

SMP 74.4 39 165.8 81

C 66.4 44 152.3 73

Orthogonal Contrasts

C vs T, T + SMP ns ns ns ns

C vs CC, CA ** ** ns ns

C vs SMP ns ns ns ns

CC, CA vs T, T+SMP ** ** ns ns

T + SMP vs T ns ns ns ns

Nonsignificant (ns) or significant (**) at the 1% Level.
Data pooled over planting dates.
CC: Fungicide combination
CA: Captan
T+SMP: Trichoderma + SMP
T: Trichoderma
SMP: Solid Matrix Priming
C: Nontreated

















Table 3-4. Effect of sowing date on ERI and emergence
percentage in 'How Sweet It Is' and 'Crisp N'Sweet 711'
planted in September, October, and November, 1988.


Cultivars

How Sweet It Is Crisp N' Sweet 711

Planting Date ERI Emerg. (%) ERI Emerg. (X)

September 20 134.3 62 189.7 83

October 19 96.9 38 227.2 81

November 17 39.8 41 77.1 81

Orthogonal contrasts

Sept. vs Oct., Nov. ** ** ** ns

Sept. vs Oct. ** ** ** ns

Oct.vs Nov. ** ns ** ns

Nonsignificant (ns) or significant (**) at 1% level.
Data pooled over seed treatments.










42
Spring 1989: The main effects of cultivar, treatment,

and sowing date were significantly different for ERI,

emergence percentage, plant height, dry weight, yield,

yield/plant and number of ears/plant (Appendix, Table 7, 9,

11, 13, 15, 17, 19). Since the interaction cv x treatment

was significant for each variable measured main effects were

partitioned and analyzed for each cultivar.

Similar to the 1988 experiments 'Crisp N' Sweet 711',

had significantly greater emergence and seedling performance

than 'How Sweet It Is' (Table 3-5). The total marketable

yield was two times greater in 'Crisp N' Sweet 711' than

'How sweet It Is' (Table 3-6).

Final stand of both cultivars, emergence rate and

seedling vigor in 'How Sweet It Is' were significantly

improved when seeds were treated with a combination of

fungicides (CC) (Table 3-7). The two inoculation methods

for seed treatment of Trichoderma (Tl and T2) and the

combination of the biosupressive agent with Solid Matrix

Priming (SMP+T) had no effects on plant stands. However,

seedling height (17 DAP) in both cultivars and dry weight

(19 DAP) in 'Crisp N' Sweet 711' were significantly improved

compared to the control when Trichoderma + SMP were

utilized.

The emergence rate, final stand, seedling vigor and

marketable yield were also significantly affected in 1989 by

the sowing date. In 'Crisp N' Sweet 711' early sowing
















Table 3-5. Emergence Rate Index, emergence percentage,
seedling height (17 DAP), and dry weight (19 DAP) in
March and April 1989 field planting of two sweet corn
cultivars.


ERI Emergen. S.H. D.W.
Cultivar (X) (cm) (mg)

Crisp N'Sweet 711 176.9 90 21.3 332

How Sweet It Is 70.5 41 15.7 122

Significance ** ** ** **

Nonsignificant (ns) or significant (**) at the 1% level.
Data pooled over seed treatments and planting dates.


Table 3-6. Yield of two sweet corn cultivars from field
trials planted in March and April 1989.



Marketable yield
Weight Yield/plant ears/plant
Cultivar (t.ha ) (g.plant ) N

Crisp N'Sweet 711 53.1 308 0.796

How Sweet It Is 23.8 377 1.053

Significance ** ns **

Nonsignificant (ns) or significant (**) at the 1% level.
Data pooled over seed treatments.

















Table 3-7. Effect of seed treatment on ERI, emergence
percentage, seedling height (17 DAP), and dry weight
(19 DAP) in four field trials planted in March and
April 1989.


Cultivar

How Sweet It Is Crisp N' Sweet 711

ERI Emerg. S.Height D.Weight ERI Emerg. S.Height D.Weight
Seed Treatment (%) (cm) (mg) (%) (cm) (mg)

CC 147.8 82 18.5 196 183.6 94 21.7 316

T1 47.9 27 16.5 103 179.8 89 22.8 349

T2 52.4 33 16.2 109 172.8 90 20.7 256


SMP+T 47.1 29 14.0 87 171.9 90 21.2 255

C 57.9 36 12.7 98 176.6 89 19.9 253

Orthogonal contrasts

CvsT1,T2,SMP+T ns ** ns ns ns **

C vs CC ** ** ** ** ns ** **

CCvsT1,T2,SMP+T ** ** ** ** ns ** ns ns

SMP+T vs T1,T2 ns ns ** ns ns ns ns ns


Nonsignificant (ns) or significant at 5X (*) or 1% (**) Level.
Data pooled over planting dates.
CC: Fungicide combination
T1: Trichoderma inoculation method 1
T2: Trichoderma inoculation method 2
T+SMP: Trichoderma + SMP
C: Nontreated









45
(March) had more rapid emergence and final stand than the

April sowing (Table 3-8). The marketable yield in both

cultivars, regardless of seed treatment, was also

significantly higher in the early sowing (Table 3-9).

Temperature is an important factor in corn germination

and seedling development (Alessi and Power, 1971). In 1988,

the mean maximum and minimum soil temperature at 5 cm deep 1

week after sowing were in September 39 oC and 24 C

respectively, whereas in October temperatures were 25 OC and

17 OC, and in November 26 C and 17 oC (Figure 3-1). The low

temperature was coincident with a reduction of ERI and final

stand in 'How Sweet It Is'. In March 17 (1989), the mean

maximum and minimum soil temperature 1 week after sowing

were 32 oC and 19 oC respectively, and 39 oC and 16 oC in

March 31. In April 14, the mean maximum and minimum soil

temperature were 36 C and 19 C respectively, and 37 OC and

19 C in April 28 (Figure 3-2). The lower seedling dry

weight, regardless of cultivar, reached in March 31 sowing

could be explained by a low temperatures one week after

sowing.

Differences were also evident in the total marketable

yield among the treatments (Table 3-10). This was probably

related to variability in final stands as affected by

treatment. With 'How Sweet It Is' the fungicide combination

(CC) treatment significantly increased the total marketable

yield compared to control, biological or SMP treatments.

















Table 3-8. Effect of sowing date on ERI, emergence
percentage, seedling height (17 DAP), and dry weight
(19 DAP) in four field trials planted in March and
April 1989.


Cultivar

How Sweet It Is Crisp N' Sweet 711

ERI Emerg. S.Height D.Weight ERI Emerg. S.Height D.Weight
Seed Treatment (%) (cm) (mg) (%) (cm) (mg)

CC 147.8 82 18.5 196 183.6 94 21.7 316

T1 47.9 27 16.5 103 179.8 89 22.8 349

T2 52.4 33 16.2 109 172.8 90 20.7 256


SMP+T 47.1 29 14.0 87 171.9 90 21.2 255

C 57.9 36 12.7 98 176.6 89 19.9 253

Orthogonal contrasts
CvsT1,T2,SMP+T ns ** ns ns ns* **

C vs CC ** ** ** ** ns ** **

CCvsT1,T2,SMP+T ** ** ** ** ns ** ns ns

SMP+T vs T1,T2 ns ns ** ns ns ns ns ns


Nonsignificant (ns) or significant at 5% (*) or 1% (**) Level.
Data pooled over planting dates.
CC: Fungicide combination
T1: Trichoderma inoculation method 1
T2: Trichoderma inoculation method 2
T+SMP: Trichoderma + SMP
C: Nontreated
















Table 3-9. Effect of sowing date on yield in 'How Sweet It
Is' and 'Crisp N'Sweet 711' sweet corns planted in
March and April in 1989.


Marketable yield

How Sweet It Is Crisp N' Sweet 711

Weight Yield/plant Ears/plant Weight Yield/plant Ears/plant

Planting Date (t.ha-1) (g.plant"1) N (t.ha-1) (g.plant-1) No

March 17 36.0 476 0.670 75.7 417 0.676

March 31 24.3 424 1.332 48.5 269 0.810

April 14 17.1 429 1.480 50.3 312 0.924

April 31 17.7 186 0.731 37.7 232 0.775

Orthogonal contrasts

March vs April ** ** ns ** ** **

March31 vs Other ns ns ns ** ** ns

Nonsignificant (ns) or significant (**) at the 1% level.
Data pooled over seed treatments.












































Figure 3-1. Maximum and minimum soil temperature (5 cm deep)
at the Horticultural Unit, Gainesville, Florida, 1988.





Temperature oC

Max. Mi i i i n.
40e Horticultural Unit, Gainesville, Florida, 1989.


30-





10


3/11 4/1 6/1 6/1 6/21
Date






Figure 3-2. Maximum and minimum soil temperature (5 cm deep)
at the Horticultural Unit, Gainesville, Florida, 1989.
















Table 3-10. Effects of seed treatment on yield in 'How Sweet
It Is' and 'Crisp N'Sweet 711' planted in March and
April 1989.


Marketable yield

How Sweet It Is Crisp N' Sweet 711

Weight Yield/plant Ears/plant Weight Yield/plant Ears/plant

Planting Date (t.ha 1) (g.plant"1) N (t.ha-1) (g.plant-1) N0

March 17 36.0 476 0.670 75.7 417 0.676

March 31 24.3 424 1.332 48.5 269 0.810

April 14 17.1 429 1.480 50.3 312 0.924

April 31 17.7 186 0.731 37.7 232 0.775

Orthogonal contrasts

March vs April ** ** ns ** ** **

March31 vs Other ns ns ns ** ** ns

Nonsignificant (ns) or significant (**) at the 1X level.
Data pooled over seed treatments.









50
The biological and SMP treatments did not increase yield

compared to the control. The results reported confirm the

efficacy of fungicide seed treatments in sh2 sweet corn,

previously reported by numerous authors (Cantliffe and

Bieniek, 1988); (Cantliffe et al., 1975); (Piezcarka and

Wolf, 1978) (Berger and Wolf, 1974).

There were large differences in performance between the

two cultivars. Hancik et al. (1988) reported that 'How

Sweet It Is' had the poorest plant stand (18 %) in a field

trial in Florida. The fungi detected in seeds in both

cultivars were Fusarium spp., Rhvzopus sp., Penicillium

spp., Aspergillus sp., and Pythium spp. where the fungi

infection was more severe in 'How Sweet It Is' (Chapter IV).

The high response of this cultivar to fungicide treatments

in both trials note that seed-borne disease is an important

factor affecting seed emergence in sh2 sweet corns.

The biological seed treatments have been reported as

less effective than traditional fungicide treatments

(Kommendal et al., 1981). In the past five years, seed

inoculation with Trichoderma has been reported as an

effective seed protection method (Harman et al., 1989),

(Chet, 1987). Despite these results, several details about

microorganism survival under different soil conditions such

as texture, ph, temperature, and biotic status, and the

influence of pathogenic infection level of the seed require

additional investigations. The living condition of










51

Trichoderma after seed inoculation, in 1988 and 1989 seed

treatments, could affect the fungi activity (John Barnes,

Kodak Co. personal communication)

Solid matrix priming was not a successful seed

treatment in either season or cultivar. The emergence rate

and percentage and the marketable yield was lower than the

control in spring 1989 trials. Cantliffe and Bieniek (1988)

also reported, in a field trials, low effect of SMP on

emergence percentage in sh2 sweet corn compared to control.

However, since promissory results have been reported in

others vegetable seeds through this technique (Taylor et al,

1988), (Kubick et al., 1988), further investigation are

necessary in order to adjust the method to sweet corn seeds.

Summary

Seeds of two sweet corn hybrids carrying sh2 mutant

endosperm (Crisp N' Sweet 711 and How Sweet It Is) were

inoculated with Trichoderma harzianum by two different

methods; treated with Captan or a combination of Captan +

Carboxin + Metalaxil + Imazilil; primed via Solid Matrix

Priming; or treated by SMP and Trichoderma. The seed

treatments were evaluated during the fall 1988 (three

sowing) and spring 1989 (four sowing) in Gainesville,

Florida.

'Crisp N' Sweet 711' germinated better than 'How Sweet

It Is' in all seed treatments and sowing dates. Fungicide

seed treatments were the most effective method to improve










52

emergence rate, emergence percentage, seedling performance

and total marketable yield. Solid Matrix Priming,

Trichoderma, and the combination of both treatments

generally did not improve seed performance. The response of

different species and cultivars to SMP, and Trichoderma

survival after inoculation is in need of further studies.
















CHAPTER IV
IMBIBITION, ELECTROLYTE LEAKAGE, GERMINATION AND SEED
DISINFECTION IN SWEET CORN HYBRIDS
CARRYING sh2 MUTANT ENDOSPERM


Water and imbibition rate play decisive roles in seed

germination (Vertucci, 1989). Seed imbibition mechanisms

are influenced by environmental factors (initial moisture of

the seed and temperature), and by the genetic

characteristics of the seeds (seed coat permeability,

chemical composition of the tissues) (Vertucci, 1989;

Woodstock, 1988). The environmental factors can be

controlled, however the seed characteristics determine the

sensibility to stress imbibition. Seed genotype affected

seed imbibition in sweet corn; water uptake was higher in

seeds of sh2 than su genotype (Styer and Cantliffe, 1983).

Seed coats can regulate water absorption in seeds. Pea

seeds with intact seed coats had low rates of imbibition

when compared to decoated seed (Powell and Matthews, 1978).

The seed coat protects the lima bean seed against chilling

injury during imbibition (Pollock and Toole, 1966).

Throughout the imbibition process, seeds loose a wide

variety of sugars, nutrients, ions, proteins, and organic

acids (Nordin, 1984; Powell and Matthews, 1977). The

solutes leaked out of the seeds when the cellular membrane










54

was reconstituted during imbibition (Simon, 1978). Cell

rupture and the level of membrane disorganization affected

the rate of electrolyte leakage (Senaratna and McKersie,

1983; Woodstock, 1988). Temperature also had an important

influence on leakage. Legume seeds, imbibed at 5 OC and 40

oC, leaked more potassium and other electrolytes than seeds

imbibed at 25 oC (Mullet and Considine, 1979). The seed

coat also plays an important role in controlling seed

leakage. Sweet corn seeds with broken pericarp had higher

conductivity values than seed with intact pericarp (Wann,

1986). Presley (1958) reported a high correlation between

electrolyte leakage conductivity and seed vigor in cotton

seeds. Matthews and Bradnock (1968) used the conductivity

of the leachate as a vigor test, to estimate field emergence

in garden peas. Oliveira et al. (1984), Tao (1980), and

Waters and Blanchette (1983) reported that the conductivity

test was an appropriate method to predict field emergence

respectively in corn, sweet corn and soybeans seeds.

Seed borne diseases severely affect emergence and stand

establishment in sh2 sweet corns (Cantliffe and Bieniek,

1988; Cantliffe et al., 1985; Hannah and Cantliffe, 1978).

Fungi isolated from seeds and seedlings of sh2 'Florida

Sweet' corn included Fusarium spp., Rhizopus sp., and

Penicillium spp., Rhizoctonia solani, and Pvthium sp.

(Berger and Wolf, 1974). Similarly, Pieczarka and Wolf

(1978) reported that the same pathogens caused damping off










55
in 'Florida Staysweet' seedlings. Fusarium moniliforme S.

was the most prevalent fungus specie isolated from corn seed

samples from the United States (Manns and Adams, 1921).

Kernels of sh2 sweet corn, infected with Fusarium

moniliforme, had extremely poor seed and seedling vigor

under stress conditions (Styer and Cantliffe, 1984). In

dent corn cv. 'Reid Yellow Dent', Fusarium moniliforme

development was faster on kernels with broken pericarp

(Alberts, 1927). Fusarium moniliforme was reported to

penetrate sh2 kernels via small cracks in the pericarp

and/or by appressoria, then localized between the pericarp

and aleurone layer, and eventually moved into the endosperm

and embryo (Styer and Cantliffe, 1984). The cracks and

natural openings in corn kernels were the primary areas of

infection for Fusarium moniliforme (El-Meleigi et al.,

1981). Hyphae of Aspergillus flavus var columnaris were

observed to penetrate cracks localized in the surface of

damaged kernels (Mycock et al., 1988).

Sodium hypochlorite has been used as an effective

disinfestant method in seed. Pepper seeds treated with a 2%

solution of sodium hypochlorite were free of contaminants

after proper incubation (McCollum and Linn, 1955). Seeds of

pepper 'Early Calwonder' soaked in 1 % sodium hypochlorite

had higher germination rate and seedling dry weights than

nontreated seeds (Fieldhouse and Sasser, 1975). Kernels

treated with sodium hypochlorite apparently had all Fusarium










56
moniliforme infection eradicated (Schoen and Kulik, 1977).

The combination of captain, sodium hypochlorite (1 %), and

hot water (65 75 OC) seed treatments did not eradicate

Fusarium moniliforme from corn seeds, however when seeds

were soaked in 1:1 mixture of sodium hypochlorite (1 %) and

ethanol at 65 C for 45 seconds, effective disinfection was

obtained (El-Meleige et al.,1981). Similarly, 'Iochief' and

'Earlivee' sweet corn seedlings were less infected with

Fusarium moniliforme after seed treatment with sodium

hypochlorite than untreated seeds (Anderegg and Guthrie,

1981).

The objectives of the present investigations with sh2

sweet corn were to analyze the relationship among seed

imbibition, electric conductivity, total sugar, potassium

content in seed leachate and germination of two sh2 sweet

corn hybrids; to determine the effect of temperature on

imbibition and seed leakage conductivity; to characterize

pathogen infection on and in seeds; and to determine the

effective control of seed borne pathogens with different

non-contaminant seed disinfection methods.

Materials and Methods
Plant material:

Seeds of two sh2 sweet corn (Zea mays L.) cultivars,

Crisp N'Sweet 711 and How Sweet It Is, were used in these

studies. The seeds were supplied by Crookhan Seed Company










57
(Caldwell, Id) without chemical treatment, and were stored

after arrival at 10 oC and 45 % RH.

Imbibition and leachate analysis

Fifty seeds were imbibed in 50 ml of distilled water

for 6 hours at 25 oC, to correlate germination, seed

imbibition, electrolyte conductivity, potassium

concentration, and total sugar in the leachate. Also, 50

seeds were imbibed in distilled water, at 5 OC and 25 oC, to

determine the effect of temperature on imbibition rate,

characteristic of the leachate and electrolyte conductivity.

Imbibition was measured as the increase in fresh

weight, after seed surface blotting, and was measured every

hour then expressed as a percentage of fresh weight.

Conductivity was measured at room temperature (25 +/- 1 oC),

with a conductivity meter (Lecto Mho Meter, Lab Line

Instruments Inc., Ill). The data were expressed as umhos/g

of seed. Potassium concentration was determined by a Perkin

Elmer/232 flame spectrophotometer (Chapman and Prat, 1961)

and expressed as ppm/g of seed. Total sugar was analyzed by

a colorimetric phenol method (Dubois et al., 1956), where 1

ml of seed leachate was diluted in 250 ml of distilled

water. One milliliter of phenol (80 %) was added to 2 ml of

diluted sample, glucose standard, and distilled water

(blank). The test tubes were mixed and 5 ml of sulfuric

acid was added. The tubes were mixed again and placed in a

water bath (25 oC) for 15 minutes. Absorbance was read at










58

490 nm in a Beckman DU-20 spectrophotometer, and expressed

as g sugar/100 ml.

Soluble sugar analysis in dry seeds

Total soluble sugars were determined by mixing in a

mason jar 5 g of finely ground dry seeds with 85 ml of

ethanol (95 %) and homogenizing 1 minute with a blender at

high speed (Styer, 1982). The samples were sealed, and

placed in boiling water for 20 minutes to avoid enzyme

activity, then stored at -20 oC overnight to precipitate

insoluble materials. Each sample was filtered through

Whatman 1 qualitative filter paper in a Buchner funnel. The

filtrate was used to assay soluble sugars by the

colorimetric phenol method described above.

The germination and vigor tests

A rolled towel germination test was used according to

Association of Official Seed Analyst procedures (1983).

Fifty seeds were placed on a three moist non-toxic

germination papers (Anchor paper Co. St. Paul, MN). The

papers were rolled, placed in plastic containers (21.5 x

32.5 x 5.5 cm), and incubated in a dark germinator at 25 +/-

1 OC for 7 days.

Seed vigor was tested by a tetrazolium test

(Association of Official Seed Analyst, 1970). The seeds

were moistened at 30 oC for 18 hours, bisected

longitudinally through the embryo with a sharp single edge

razor blade, and soaked in a 0.25 % solution of 2,3,5-










59
triphenyl tetrazolium chloride for 6 hours. The seed halves

were rinsed with tap water and refrigerated at 10 oC for 24

hour before examination under a light microscopy.

Seed disinfection treatments

Two hundred seeds of each cultivar were enclosed in

cheesecloth bags and soaked for 15, 30, and 60 minutes in a

1 % and 10 % solution (v/v) of Clorox with Tween-20 (0.05 %

and 0.5 % available chlorine respectively), hot water (45

oC) or tap water (25 +/-1 OC) as a control. After each

treatment, the seeds were rinsed three times with tap water,

and air-dried (25 +/-1 OC, 45 % RH) for 1 hour prior to the

germination or incubation experiments.

Seed incubation test

This experiment was designed to determine the seed

pathogenic infection before and after seed treatment. Four

seeds of each cultivar and treatments were plated in an

acidified potato-dextrose agar (APDA). The plates were

incubated at 25 OC for 10 days under continuous fluorescent

light (5,000 lux).

Scanning electron microscopy

Seeds of each cultivar were cut in half with a sharp

single edge razor blade and dehydrated in an ethanol series

(70 % to 100 %). The seeds were rinsed with absolute

ethanol and dried in a critical-point dryer. The samples

were mounted on aluminum stubs by double-stick tape,

sputter-coated with gold palladium, and stored in a










60

desiccator. Observations and photographs were made on a

Hitachi S-420 scanning electron microscope at 20 KV

accelerated voltage.

Statistical analysis

All the experiments were conducted as a randomized

complete block design with each treatment replicated four

times. Statistical Analysis System (SAS) (1987) software

package was used for data analysis. Percentage data were

analyzed as square root arc sine transformation.

Results and Discussion

Seed micrographs revealed more separation between

pericarp and aleurone layer in 'How Sweet It Is' than 'Crisp

N' Sweet' (Figure 4-1). The air spaces between the seed

coat and aleurone layer may increase cracking in the

pericarp during seed harvest and transport of sh2 seeds

(Styer and Cantliffe, 1983). The seed coat of 'How Sweet It

Is' appeared to have more cracks and crevices than 'Crisp N'

Sweet 711' (Figure 4-2)

Seed leakage and imbibition may be controlled by seed

coat characteristics (Woodstock, 1988; Simon, 1978; Powell

and Matthews, 1978). Previous work reported that seed coat

characteristics, also influence fungi infection during

kernel development and storage (Mycock et al., 1988; Styer

and Cantliffe, 1984; Alberts, 1923).

In the present study, the cultivar How Sweet It Is had

significantly high imbibition rate, potassium, total sugar



























dl


Figure 4-1. Scanning electron micrographs of 'Crisp N' Sweet
711' (top) and 'How Sweet It Is' (bottom) seeds. Note
separation between pericarp and aleurone layer in 'How
Sweet It Is'.




























































Figure 4-2. Seed surface of 'Crisp N' Sweet 711' (top)
and 'How Sweet It Is' (bottom).










63

concentration in the leachate, and electrolyte conductivity

after 6 hour soaking in distilled water than Crisp N' Sweet

711 (Table 4-1). Similarly, the fungi infection and

development were more severe in seeds of 'How Sweet It Is'.

The air spaces between seed coat and aleurone layer, and the

pericarp cracks and crevices could promote more rapid water

imbibition and would give ideal conditions for fungi

penetration and infection. Also, the greater seed leakage

may provide an appropriate nutritive substrate for fungi

development. The total soluble sugar percentage in seeds

was also significantly higher in 'How Sweet It Is' (Table 4-

1). The high levels of sugar in the seed may result in an

increase in imbibition rate because of an increase in the

osmotic water potential of the seeds.

Tracy and Juvick (1988); Waters and Blanchette (1983);

Matthews and Bradnock (1968) reported that, in many species,

a negative correlation existed between seed electric

conductivity of the leachate and laboratory germination test

or field emergence. In the present study, negative

correlations occurred among germination percentage and

imbibition, electric conductivity, potassium concentration,

and total sugar of the seed leachate (Table 4-2). 'How

Sweet It Is' had more rapid imbibition, greater electrolyte

conductivity, potassium and total sugar concentration in the

leachate than 'Crisp N'Sweet 711', and the lower

germination. In contrast, there was a high positive
















Table 4-1. Total soluble sugar in seeds and seed leachate
characteristics (50 seeds/50 ml water) after 6 hours at
25 oC and germination percentage in a rolled towel test
of 'How Sweet It Is' and 'Crisp N'Sweet 711'.


Cultivar

How Sweet It Is Crisp N'Sweet 711 Signif.

Total sugar 0.184 0.077 **
seed (g/100 mL)

Imbibition 95 66 **
(% FW)

Conductivity 98.1 34.6 **
(umhos g. seed)

Potassium 42 13 **
-1
(ppm g. seed)

Total sugar 0.318 0.069 **
Leachate (g/ml)

Germination 76 97 *
(X)

Significant at 5% (*) or 1% (**) Level.
















Table 4-2. Correlation coefficients (r) among germination
percentage, imbibition electric conductivity, potassium
and total sugar of the seed leachate in 'Crisp N'Sweet
711' and 'How Sweet It Is'.


Seed Leachate

Total sugar Potassium Conductivity Imbibition
-1 -1
(g/ml) (ppm.g seed ) (u hos.g seed ) (% FW)


Germination -0.76z -0.75 -0.76 -0.92
(X)

Total sugar 0.97 0.99 0.98
(g/ 100 mt)

Potassium 0.99 0.99
(ppm.g seed )

Conductivity 0.99
-1
(umhos.g seed )
z ALL values are significant at the 1% level.


Table 4-3. Effect of cultivar and temperature on imbibition
rate (50 seeds/50 ml water), electric conductivity,
potassium and total sugar in the leachate after 6 hours
of imbibition.


Imbibition Conductivity Potassium Total sugar

(X FW) (umhos.g seed -1) (ppm.g seed 1) (g/100 ml)


5 oC 25 oC Sign. 5 oC 25 OC Sign. 5 C 25 oC Sign. 5 C 25 oC Sign.


How Sweet It Is 79 95 ** 93 98 ns 38 42 ns 0.175 0.318 **


Crisp N' Sweet 711 57 66 ** 31 34 13 13 ns 0.027 0.069 **


Significance ** ** ** ** ** ** ** **


Nonsignificant (ns), significant at the 5% (*) or 1% (**) level.








66

correlation among imbibition, electric conductivity,

potassium concentration and total sugar. The tetrazolium

test (Figure 4-3) also related possible differences of seed

vigor between the two cultivars. The red color of the

embryo was more uniform an intense in 'Crisp N'Sweet 711'

than 'How Sweet It Is'. The negative correlation measured

in the present work between a laboratory germination test

and electric conductivity, confirmed the confidence of the

method as an effective indicator of seed germination in

sweet corn.

Imbibition damage was more severe in a rapidly imbibing

cultivar of dwarf bean seeds (Powell, 1986). Rapid

imbibition may induce disruption of cell membranes (Powell,

1978; Wann, 1986). The rate of imbibition was higher in

'How Sweet It Is' and high levels of sugar and potassium

were found in the seed leachate as compared with 'Crisp

N'Sweet 711'. The alteration in cell membrane structure

caused by a rapid water uptake in seeds of 'How Sweet It Is'

could lead to the high concentration of electrolytes in seed

leachate.

Slow rate of hydration, in soybean seeds, prevented the

lost of germination by imbibition damage and reduced

electrolyte leakage (Tilden and West, 1985). Imbibition at

low temperature (5 C) (Table 4-3) (Figure 4-4, 4-5, 4-6 and

4-7) significantly reduced the imbibition rate and total










67























--<



: .. .

























Figure 4-3. Tetrazolium test in 'Crisp N' Sweet 711' (top)
and 'How Sweet It Is' (bottom). The red color of the
embryo was more uniform and intense in 'Crisp N' Sweet
711' depicting greater seed vigor.











68










Imbibition (% FW)
70

60 -

50

40

30:

20

10- -B-5 oC --25 oC

0 -----------'---------------
1 2 3 4 5 6
Time (hour)










Imbibition (% FW)
100 *-


80 -


60 **


40


20
S5 oC 25 oC


1 2 3 4 5 6
Time (hour)


Figure 4-4. Imbibition rate (50 seeds/50 ml water) in 'Crisp
N' Sweet 711' (top) and 'How Sweet It Is' (bottom) at 5
C and 25 oC. Significant at 5 % (*) or 1 % (**) level.



















umhos/g seed


1 2 3 4 5 6
Time (hours)


3 4
Time (hours)


Figure 4-5. Electric conductivity of the leachate (50 seeds/
50 ml water) in 'Crisp N'Sweet 711' (top) and 'How
Sweet It Is (bottom) at 5 C and 25 C. Nonsignificant
(ns) or significant at 1 % (**) level.





















100


80 -


60 -


40 -

ns
20
-- Crilp N'Sweet 711 How Swel It Is


1 2 3 4 5 6
Time (hour)








Imbibition (% FW)
100


80 -


60 -


40 -


20 -
Crisp N'Sweet 711 X How Sweet 11 Is


1 2 3 4 5 6
Time (hour)


Figure 4-6. Imbibition rate (50 seeds/50 ml water) at 5 C
(top) and 25 C (bottom) in 'Crisp N' Sweet 711' and
'How Sweet It Is'. Nonsignificant (ns) or significant
at 5 % (*) or 1 % (**) level.









































1 2 3 4 5 6
Time (hours)










umhos/g seed
120


100 -
ns
ns
80
ns

60 -


40 -


20 -
205 oC 5 25 oC


1 2 3 4 5 6
Time (hours)

Figure 4-7. Electric conductivity of the leachate (50 seeds/
50 ml water) at 5 oC (top) and 25 oC (bottom) in 'Crisp
N'Sweet 711' and 'How Sweet It Is. Significant (**) at
1 % level.









72

sugar in the leachate in both cultivars, and electric

conductivity of the leachate in 'Crisp N' Sweet 711'.

Fungi detected by the seed incubation test included

Fusarium spp., Rhizopus sp., Penicillium spp., Asperqillus

sp, and Pythium spp. Seeds treated with sodium hypochlorite

(Clorox 1 % and 10 %) had low or no fungi infection after

incubation in both cultivars (Figure 4-8 and 4-9). When the

seeds were disinfected with sodium hypochlorite and hot

water, the main effects of cultivar, treatment and time were

significantly different for germination percentage. Since

the cultivar x treatment interaction was significant, main

effects were partitioned and analyzed for each cultivar.

Germination percentage was significantly higher in 'Crisp N'

Sweet 711' than 'How Sweet It Is' in all seed treatments

(Table 4-4). However, seed treatments did not improve

germination percentage over the control in 'Crisp N' Sweet

711'. The non-significant response was representative of

achieving maximal germination under the conditions of the

experiment. In 'How Sweet It Is', germination percentage

was significantly improved in seeds treated with Clorox 1 %.

However, both Clorox 10 % and hot water, significantly

reduced germination compared to the control. Germination

had a linear decrease in response to time. The results

suggest that the imbibition rate of the seeds could affect

treatment effectiveness. Higher concentrations of sodium

hypochlorite (Clorox 10 % treatment) or time, might have




























































Figure 4-8. Seeds of 'Crisp N' Sweet 711' treated with
sodium hypochlorite (top) and without treatment
(bottom), 10 days after incubation.


























































Figure 4-9. Seeds of 'How Sweet It Is' treated with sodium
hypochlorite (top) and without treatment (bottom), 10
days after incubation.

















Table 4-4. The effect of seed disinfection treatments on
germination (rolled towel test at 15 oC for 7 days) in
sweet corn 'Crisp N' Sweet 711' and 'How Sweet It Is'.


Seed Treatment

Clorox 1 %

CLorox 10 %

Hot Water

Water


Cultivar

How Sweet It Is Crisp N' Sweet 711

Germination (X) Significance


66 98

80 96

Orthogonal Contrast


Clorox 1 X vs other ** ns

CLorox 1 X vs CLorox 10 % ** ns

Hot water vs Water ** ns

Time Linear **

Time quadratic ns

Nonsignificant (ns) or significant (**) at the 1% Level.


**

**

**

**









76
toxic effects on the embryo of 'How Sweet It Is', which was

shown to have higher imbibition rate. Heat from the hot

water treatment increased imbibition damage (Appendix,

Tables 32, 33, 34, 35)

The main effects of cultivar and treatment were

significantly different for seedling dry weight. Time was

not significantly different, and no cv x time and cv x

treatment interactions occurred (Appendix, Table 36). 'Crisp

N' Sweet 711' had significantly higher seedling growth in

all seed treatments than 'Crisp N'Sweet 711' (Table 4-5).

The 1 % Clorox seed treatment led to an increase in seedling

dry weight regardless of cultivar. There were no

differences in seedling dry weights among the other three

treatments.

The results in this study confirmed that the

differences between imbibition rate and seed leachate

characteristics in both cultivars cannot be attributed to

only one factor. The genetic seed characteristics (total

soluble sugar in the seeds) and the physical seed structure

(cracks, separation between seed coat and aleurone)

influenced the imbibition rate and the concentration of

electrolytes in the leachate. The high concentration of

electrolytes in the leachate can increase infection and

development of fungi during germination. The temperature

where the seeds were soaked modified seed imbibition and

conductivity of the leachate. Fusarium spp., Rhizopus sp.,















Table 4-5. The effect of seed disinfection treatments on
seedling dry weight in sweet corn 'Crisp N' Sweet 711'
and 'How Sweet It Is'.


Seed Treatment

Clorox 1 %

CLorox 10 %

Hot Water

Water


Cultivar

How Sweet It Is Crisp N' Sweet 711

Dry Weight (mg/seedling)

21 31-

16 27

15 26

15 27


Significance
**

**

**

**


Orthogonal Contrast

CLorox 1 % vs other **

CLorox 1 X vs Clorox 10 % **

Hot water vs Water ns

Time linear ns

Time quadratic ns

Nonsignificant (ns) or significant (**) at the 1% Level.


I









78

Penicillium spp., Asperaillus sp, and Pythium spp. can

infect sh2 sweet corn. Sodium hypochlorite (0.05 % chlorine

available) was an effective surface seed disinfectant for

sh2 sweet corn.

Further work should be done, to clarify the influence

of seed leachate on fungi susceptibility and development

both on and in the seed, and to determine a method to reduce

imbibition and seed leakage in sh2 sweet corns to improve

germination.

Summary

Cracks in the seed coat were more frequent in 'How

Sweet It Is' than 'Crisp N' Sweet', which also had high

levels of soluble sugar in the seeds. The low seed water

potential and the physical damage of the seed coat in 'How

Sweet It Is' apparently led to an increase in the imbibition

rate and increased imbibitional damage, denoted by the high

potassium and total sugar concentration in the leachate,

electrolyte conductivity, and low seed germination and

vigor. Imbibition, total sugar in the leachate, and

conductivity of the leachate from the seed increased as

temperature increased from 5 C to 25 oC.

Fungi isolated from seeds in both cultivars were

Fusarium spp., Rhizopus sp., Penicillium spp., Asperqillus

sp, and Pvthium spp. 'How Sweet It Is' had more fungal

infection than 'Crisp N'Sweet 711'. The differences

measured in seed infection between the two cultivars could









79
be associated also with the characteristics of seed pericarp

and electrolyte loss in the leachate. Sodium hypochlorite

(0.05 % available chlorine) was an effective seed

disinfected treatment in 'How Sweet It Is' and 'Crisp N'

Sweet 711', since germination percentage was increased in

the first, and seedling dry weight improved in both

cultivars in a rolled towel test.















CHAPTER V
IMPROVED STAND ESTABLISHMENT OF sh2 SWEET CORN
BY SOLID MATRIX PRIMING AND SEED
DISINFECTION TREATMENTS


Sweet corn hybrids carrying shrunken-2 mutant

endosperm, also called supersweet, have excellent eating and

postharvest storage quality. Until 1985, the acceptance by

growers was low because of poor stand establishment in the

field. The poor seed emergence has been attributed to low

seed vigor, high seed-borne disease infection, and high

susceptibility to soil-borne pathogens (Cantliffe and

Bieniek, 1988; Guzman et al., 1983; Hannah and Cantliffe,

1977; Cantliffe et al., 1975) .

Seeds of supersweet corn are less uniform and smaller

than normal or sugary (su) seeds. The kernel is wrinkled

and easy to damage during harvest and shipping. The embryo

size of sh2 was smaller than (su) and normal endosperm

(Styer and Cantliffe, 1984). Shrunken-2 sweet corn had

lower final germination percentage and seedling vigor in

laboratory and field trials as compared with (su), (bt), and

normal genotypes (Styer et al., 1980). The lower seed vigor

was initially thought to be related to small endosperm

(Wann, 1980).

Stand losses in a supersweet corn 'Florida Sweet' was









81

attributed to high seed and soil borne infection (Berger and

Wolf, 1974). Kernels of the sh2 sweet corn were heavily

infected by Fusarium moniliforme early in their development.

The fungi was located in pericarp crevices and eventually

moved into the endosperm (Styer and Cantliffe, 1984). Fungi

isolated from supersweet seeds were Rhizopus sp., Fusarium

spp., Penicillium spp., and Phvtium spp. (Berger and Wolf,

1974; Pieczarka and Wolf, 1978). Fungicide seed treatments

have been reported to improve stand establishment and

uniformity in supersweet corn seeds, (Berger and Wolf, 1974;

Cantliffe et al., 1975; Pieczarka and Wolf, 1978; Cantliffe

and Bieniek, 1988). Sodium hypochlorite has been used as a

seed disinfestant in species such as pepper (McCollum and

Linn, 1955; Fieldhouse and Sasser, 1975), and corn (El-

Meleige et al., 1981). Sweet corn seeds treated with a

Clorox solution (0.5 % chlorine for 5 min) had less Fusarium

moniliforme seed infection than non treated seeds (Anderegg

and Guthrie, 1981). Fusarium moniliforme was apparently

eradicated when corn seeds were disinfected with sodium

hypochlorite (1 % chlorine for 1 min) (Schoen and Kulik,

1977).

Seed priming is a treatment which enhances

germination readiness and consists of imbibing seeds in an

osmotic solution that allows seeds to imbibe water and go

through the initial germination stages, however does not

permit radicle protrusion through the seed coat (Cantliffe,









82
1981). The purpose of seed priming is to increase

germination rate, improve stand establishment, and increase

yield (Khan et al., 1981). Heydecker et al., 1973, reported

favorable emergence rate in primed onion seeds. Priming

overcame thermodormancy problems in lettuce germinated at

high temperature (Guedes and Cantliffe, 1980). In cold and

wet soil, the emergence time, stand uniformity, and yield

were higher in primed carrot seed than nontreated

(Szafirowska et al., 1981). Seeds of beet amended with PEG

8000 had a high emergence rate and final stand in cold wet

soils (Khan and Taylor, 1986).

Priming corn seed, however lead to variable results.

The emergence rate of corn germinated at cool temperatures,

was improved by seed priming in a polyethylene glycol

solution (Bodsworth and Bewley, 1981). Osmotic seed

treatment in corn cv Partap accelerated germination at 10 C

in a laboratory test (Basra et al., 1988). Seed of (su) and

(sh2) sweet corn genotypes primed with PEG 8000 for 1 week

at 20 oC had lower field emergence than a control (Bennett

and Waters, 1987a, 1987b).

Solid matrix priming (SPM) is another seed priming

method to improve rate, uniformity and/or level of seed

emergence under stress conditions. Seeds are moistened for

a given time at constant temperature seeds in an organic or

inorganic carrier to which water has been added (Harman and

Taylor, 1988). The Solid Matrix Priming method utilizes the









83

osmotic and physical characteristics of the solid carrier to

restrict water absorption (Kubik et al, 1988). The Solid

Matrix Priming method improved emergence of tomato and

pepper seed sown in soil under cool or warm temperatures in

a growth chamber (Kubik et al., 1988). Tomato, carrot and

onion seeds primed via SMP had superior or equal

characteristics of seedling emergence compared with normal

solution priming (Taylor et al., 1988). The SMP method did

not improve rate and stand uniformity in sh2 sweet corn sown

in the field (Cantliffe and Bieniek, 1988). Seedling

emergence was improved by SMP in 'Jubilee' sweet corn, but

was lower in SMP 'Florida Staysweet' corn than in untreated

seeds (Harman et al., 1989).

The objective of this study was to develop a SMP

treatment which would consistently improve emergence rate

and total emergence of various sh2 sweet corn cultivars

planted under stressful environmental conditions. In order

to be effective with sh2 sweet corn the SMP treatment has to

control seed borne pathogens after drying back the seeds

prior to planting, and the treatment can not be deleterious

to seed quality after storage.

Materials and Methods

Seeds of four sh2 sweet corns (Zea mavs L.) 'How Sweet

It Is', 'Crisp N'Sweet 711' (Crookhan Seed Co. Caldwell,

Id), 'Sweet Belle', and 'XPH 2644' (Asgrow Seed Co) were

included in this study.










Seed treatments

The seed treatments consisted of fungicide

combinations, surface disinfection by sodium hypochlorite

(NaOCl), SMP, and SMP with sodium hypochlorite. After

treatment the seeds were dried at room temperature (25 1

OC, 45 % RH) to their initial moisture content (6 %). The

seeds were stored before or after treatment at 10 OC and

45 % RH.

Chemical fungicide seed treatment: The seeds (200 g)

were soaked for two minutes in 1 1 solution of imazalil: (1-

(2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl)-1H

Imidazole) (0.653 ml/kg seed), captain: N-

[(trichloromethyl)thio]-4-cyclohexene-l,2-dicarboximide

(1.958 ml/kg seed), apron: N-(2,6-dimethylphenyl)-N-

(methoxyacetyl)alanine methyl ester (0.488 ml/kg seed), and

thiram: Tetramethylthiuram disulfide (3.264 ml/kg seed).

After treating, the seeds were dried as previously

discussed.

Sodium hypochlorite seed disinfection: In a

cheesecloth bag 200 g of seed were enclosed and soaked for

15 minutes in a 1 1 of 1 % solution (v/v) of Clorox (0.05 %

available chlorine). After the treatment, the seeds were

rinsed three times with tap water and dried.

Solid Matrix Priming: The seeds (3 g) were mixed with

6 g of calcined clay (Emathlite, Mid-Florida Mining Co.

Lowell, Fl.), and 2.5 ml of distilled water ('How Sweet It









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Is', 'Sweet Belle' and 'XPH 2644') or 2 ml ('Crisp N'Sweet

711) in a closed container (Nalgene, Filtunit Typ. Ta CA).

The containers were rotated continuously at 0.22 rpm

(Rotator Lab-Line Instruments. Melrose Park, Ill.) and

incubated at 5 oC for 6 hours, then transferred to 25 C for

24 hours. After 30 hours, 2 ml ('How Sweet It Is', 'Sweet

Belle', and 'XPH 2644') or 1.5 ml ('Crisp N'Sweet 711') of

Clorox (0.05% available chlorine) or distilled water was

added and incubated 15 hours. After priming, the seeds were

separated from the clay with a mesh sieve and dried as

previously described.

Seed imbibition and leakage conductivity studies

The objective in this experiment was to determine

differences in seed imbibition and leakage conductivity

between primed and non-primed seeds. Ten seeds were soaked

in 25 ml of distilled water at 25 oC. After 4 hours

soaking, the leachate was filtered and electrical

conductivity measured at room temperature (25 +/-1 OC) using

a conductivity meter (Lecto Mho-meter, Lab-Line Instruments

Inc., Melrose Park, Ill.) and expressed as umhos/g of seed.

Imbibition was determined gravimetrically measuring the

increase in fresh weight after surface blotting water from

the seeds.

Scanning electron microscopy

Scanning electron micrographs, using a Hitachi S-450

electron microscopy with 20 KV accelerated voltage, were









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viewed to determine differences between primed and non-

primed seeds. Seeds of 'How Sweet It Is' and 'Crisp N'

Sweet 711' were cut in halves and dried in a critical point

drier. The samples were mounted on aluminum stubs by double-

stick tape and sputter-coated with gold palladium.

Cold germination test

To determine the effect of treatments on seed emergence

under stress conditions, a cold germination test was

performed according to AOSA procedures (1983). Twenty seeds

were sown in a plastic box (18.7 x 12.5 x 9 cm). Which was

filled with 2.5 cm of Arredondo fine sand soil (loamy,

silaceous, hyperthermic Grossarenic Palenundult) from a

field which had corn grown on it for two seasons. The soil

was compacted and another 2.5 cm of soil was placed on top

of the seeds. The medium was adjusted to 70 % of its water

holding capacity. The containers were sealed and incubated

at 10 oC for 7 days, then transferred to 25 oC for 4 days.

Total percent of emergence was calculated. Seedlings with

leaves 2 mm in length above the soil were considered

germinated.

Field studies

Field plots were established on October 26, 1989 at the

IFAS Horticultural Unit in Gainesville, Florida on an

Arredondo fine sand soil (loamy, silaceous, hyperthermic

Grossarenic Palenundult). Corn had been grown on the plots

continuously for 18 months prior to planting to promote the









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development of soil borne pathogens. The plots were 7.6 m

long on beds 1.22 m apart, with each bed 0.70 m wide and

0.20 m in height. Two seeds were seeded 4 cm deep, every 30

cm in each plot (50 seeds/plot). Overhead sprinkler

irrigation was applied as needed. Fertilization, cultural

practices and pest control were according to Florida

Agricultural Extension Service recommendations (Showalter,

1986). Emergence Rate Index (Shmueli and Goldberg, 1971)

and percent emergence were calculated. Plant height, from

the soil to the top of the plant, was measured 17 days after

planting. Fresh and dry weights were determined 19 days

after planting, the seedlings were cut at the soil level and

dried at 75 OC for 72 hours. Minimum and maximum soil

temperature at 15 cm, 5 cm deep, and soil surface were

recorded.

Statistical analyses

The laboratory tests and field experiment were

conducted as a randomized complete block design, with four

replications. Percentage data was converted and analyzed as

square root arc sine transformation. Statistical Analysis

System (SAS) (1987) software program was used for data

analysis. Main effects of treatments were partitioned in a

single degree of freedom in an orthogonal contrast.

Results and Discussion

After 4 hours, imbibition was significantly higher in

'How Sweet It Is' and 'XPH 2644' than 'Crisp N' Sweet' and









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'Sweet Belle' (Figure 5-1). Seed leakage was significantly

higher in 'How Sweet It Is' compared with the other

cultivars (Figure 5-1). Regardless of cultivar, SMP

significantly reduced seed imbibition and leakage (Figure 5-

2). The reorganization of membranes during priming and the

increase of metabolic products that change the permeability

properties of the membranes may have affected the imbibition

rate in primed seeds and reduced damage during imbibition.

Tilden and West (1985) interpreted seed priming as a

metabolic repair of membranes in soybean seeds. Basra et

al. (1988) reported an increase of phospholipids and

esteroles in imbibed corn seeds. Reduced loss of organic

constituents in the leachate might further reduce substrate

availability for pathogen development.

Variations in seed anatomy were not detected between

primed and non-primed seeds in 'How Sweet It Is' and 'Crisp

N' Sweet 711' (Figure 5-3). Scanning electron micrographs

revealed differences between cultivars, where a higher

separation between pericarp and aleurone layer was observed

in 'How Sweet It Is'.

Under cold stress conditions the cultivar Crisp N'

Sweet 711 had the highest emergence percentage and 'Sweet

Belle' the lowest (Table 5-1). Seed treated with a

fungicide combination had a significantly higher emergence

percentage than the control in the cold test. The SMP +

sodium hypochlorite treatment significantly improved