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

Material Information

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

Subjects

Subjects / Keywords:
Conductivity ( jstor )
Corn ( jstor )
Endosperm ( jstor )
Fusarium ( jstor )
Germination ( jstor )
Imbibition ( jstor )
Seed treatment ( jstor )
Seedlings ( jstor )
Seeds ( jstor )
Trichoderma ( jstor )
Dissertations, Academic -- Horticultural Science -- UF ( lcsh )
Horticultural Science thesis M.S ( 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.

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University of Florida
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University of Florida
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001576405 ( ALEPH )
22914731 ( OCLC )
AHK0258 ( NOTIS )

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
iv




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
v




LIST OF TABLES
Table Pagre
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
vi




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 C 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 C 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. ...... o ........ ........ 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 C 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 0C. Nonsignificant (ns) or significant
(**) at 1% level ................................ 69
4-6. Imbibition rate (50 seeds/50 ml water) at
5 0C (top) and 25 0C (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 C (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.
Significantly 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
ix




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.
x




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 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.
xi




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
1




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 stablishment, 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 (caryopsis) 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 (aleurone 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
4




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 (g2) 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%




6
for su (Garwood et al., 1976). Sweet corn ('Florida Sweet' and 'F-449') carrying the sh2 gene have more sugar than 'lobelle' (sgu) 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 oC 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 graminearum, Diplodia spp. and Drechlera maydis.




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 niger, 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 Pythium 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 ElMeleigi 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 carie) (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 (disinfestation)




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 (captan), systemic fungicides (benomyl), 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 captan (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 captan, thiram, imazalil, and metalaxil or captan, thiram, imazalil, and captafol improved emergence compared to nontreated seeds (Cantliffe and Bieniek, 1988). Application of captan (200 g/100 kg seed) in maize seeds was effective for Fusarium




graminearum control (Draganic et al., 1984). Seed corn treatments with benlate and captan were the most effective to control seed borne diseases in Kenya (Khare, 1985). Seed treated with benomyl, captan 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 seedborne diseases. Hot water seed treatments are used in wheat and barley to control Ustilago nuda and Ustilaqo 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 globosum 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 dampingoff 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 Leakagre 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 PlattAloia (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, _u, 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 coldwet 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).
Presowinr 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 (Lycopersicon 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 0C for 6 days) obtained early germination in laboratory under cool conditions (10 0C), 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 0C) (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 (au) 'Jubilee' and two supersweet (sh2) 'Sweetie' and 'Sugar Loaf' genotypes of sweet corn. The primed treatment (33 % PEG solution, 7 days at 20 0C) 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 TerraSorb 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 koningii 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'




35
Seed treatments
Seeds of two sweet corn hybrids (Zea mays L.) 'Crisp N' Sweet 711' and 'How Sweet It Is' were treated with captan: (N-[(trichlorometyl)thio]-4-cyclohexene-l,2-dicarboximide, and the combination of captan + 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-(2propenyloxy)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 10 10 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 oC. 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 TI 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: captan (CA), fungicide combination of captan, 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 recomendations (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/rn 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 0C 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 (captan 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,




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




41
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. (%)
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. vsOct. ** ** ** 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 (TI 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




43
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 (%) (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
Weigh Yield/plat 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.




44
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
Ti 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




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 0C and 24 0C respectively, whereas in October temperatures were 25 0C and 17 OC, and in November 26 OC and 17 0C (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 0C and 19 0C respectively, and 39 0C and 16 0C in March 31. In April 14, the mean maximum and minimum soil temperature were 36 0C and 19 *C respectively, and 37 0C and 19 OC 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.




46
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.
Cutltivar
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




47
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.ptant1) No (t.ha-1) (g.ptant-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.




48
Temperature oC
50
FMin. Max.
30 20
10a
0 .. .. .. .. .. "..."... ........
9/21 10/1 1V 12/1
Date
Figure 3-1. Maximum and minimum soil temperature (5 cm deep)
at the Horticultural Unit, Gainesville, Florida, 1988.
Temperature oC
-Max. -Min.
40
20 10
3/11 4/1 5/1 6/1 6/21
Date
Figure 3-2. Maximum and minimum soil temperature (5 cm deep)
at the Horticultural Unit, Gainesville, Florida, 1989.




49
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) N0 (t.ha-1) (g.ptant-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 1% 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
53




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 0C, leaked more potassium and other electrolytes than seeds imbibed at 25 0C (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 Pythium 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 captan, 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 oC 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 C and 45 % RH. Imbibition and leachate analysis
Fifty seeds were imbibed in 50 ml of distilled water for 6 hours at 25 C, 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 C and 25 C, 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 C), 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 C) 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 0C 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 0C for 7 days.
Seed vigor was tested by a tetrazolium test
(Association of Official Seed Analyst, 1970). The seeds were moistened at 30 0C 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 0C 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 0C) as a control. After each treatment, the seeds were rinsed three times with tap water, and air-dried (25 +/-1 0C, 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 0C 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




61
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'.




62
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 41). 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




64
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 1 98.1 34.6 **
(umhos g. seed)
Potassium 42 13 **
(ppm g. seed-1
Total sugar 0.318 0.069 **
leachate (g/mlt)
Germination 76 97 *
(X)
Significant at 5% (*) or 1% (**) Level.




65
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
(g/mL) (ppm.g seed ) (unhos.g seed ) (% FW)
Germination -0.76z -0.75 -0.76 -0.92
(X)
Total sugar 0.97 0.99 0.98
(g/ 100 mL)
Potassium 1 0.99 0.99
(ppm.g seed-1
Conductivity 0.99
(umhos.g seed )
z AtLL 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
(% FW) (umhos.g seed -1) (ppm.g seed -1) (g/100 ml)
5 0C 25 oC Sign. 5 oC 25 oC Sign. 5 oC 25 oC Sign. 5 oC 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 0C) (Table 4-3) (Figure 4-4, 4-5, 4-6 and 4-7) significantly reduced the imbibition rate and total




67
-sf"
A-
t 7
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
4030:
20
0 -- -5oC -x- 25 oC
0
1 2 3 4 5 6
Time (hour)
Imbibition (% FW)
100 *.
80
60 -**
40
20
5 oC 25 oC
0
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
oC and 25 oC. Significant at 5 % (*) or 1% (**) level.




69
umhos/g seed
35
ns
ns] 30- ns
25 -2 4
20
15
10
0
5 5 6oC ----25oC
1 2 3 4 5 6
Time (hours)
umhos/g seed
120
ns
100
ns
ns
80
ns
60
40
0 t5 (oC 25 oC
0
1 2 3 4 5 6
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 oC and 25 oC. Nonsignificant
(ns) or significant at 1 % (**) level.




70
Imbibition (% FW)
100
80
60
40
ns
Crisp N'Sweet 711 How Swel It Is o II
2 3 4 5 6
Time (hour)
Imbibition (% FW)
100
80
6o
40
20
Crisp N'Sweet 711 How Sweet 11 Is
0
1 2 3 4 5 6
Time (hour) Figure 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'. Nonsignificant (ns) or significant
at 5 % (*) or 1% (**) level.




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umhos/g seed
35 ns
ns] 30 ns
25
20,
15
100
0
5 5 oC 25 oC
1 2 3 4 5 6
Time (hours)
u umhos/g seed
120
ns
100
ns
ns
80
ns
60
AD
40
20 5 oC 25 oC
0
1 2 3 4 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., Aspergillus 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




73
Crisp N Sweet Clorox 1% (60 main.)
Crisp N Sweet Control
Figure 4-8. Seeds of 'Crisp N' Sweet 711' treated with
sodium hypochlorite (top) and without treatment
(bottom), 10 days after incubation.




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How Sweet It Is 1
Figure 4-9. Seeds of 'How Sweet It Is' treated with sodium
hypochlorite (top) and without treatment (bottom), 10
days after incubation.




75
Table 4-4. The effect of seed disinfection treatments on
germination (rolled towel test at 15 C for 7 days) in sweet corn 'Crisp N' Sweet 711' and 'How Sweet It Is'.
CuLtivar
How Sweet It Is Crisp N' Sweet 711
Seed Treatment Germination (%) Significance
Clorox 1 % 86 98- **
Clorox 10 % 70 97 **
Hot Water 66 98 **
Water 80 96 **
Orthogonal Contrast
Clorox 1 % vs other ** ns
Clorox 1 % vs Clorox 10 % ** ns
Hot water vs Water ** ns
Time Linear **
Time quadratic ns
Nonsignificant (ns) or significant (**) at the 1% Level.




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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.,




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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'.
Cultivar
How Sweet It Is Crisp N' Sweet 711
Seed Treatment Dry Weight (mg/seedLing) Significance
CLorox 1 % 21 31 **
Ctorox 10 % 16 27 **
Hot Water 15 26 **
Water 15 27 **
Orthogonal Contrast
CLorox 1 % vs other **
Ctorox 1 % vs Ctorox 10 % **
Hot water vs Water ns
Time Linear ns
Time quadratic ns
Nonsignificant (ns) or significant (**) at the 1% LeveL.




78
Penicillium spp., Aspergillus 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 oC to 25 oC.
Fungi isolated from seeds in both cultivars were
Fusarium spp., Rhizopus sp., Penicillium spp., Aspergillus sp, and Pythium 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
80




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 Phytium 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 (ElMeleige 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 0C in a laboratory test (Basra et al., 1988). Seed of (au) 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.




84
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)-lH Imidazole) (0.653 ml/kg seed), captan: 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




85
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 0C for 6 hours, then transferred to 25 OC 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 leakaQe 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 0C. After 4 hours soaking, the leachate was filtered and electrical conductivity measured at room temperature (25 +/-l 0C) 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




86
viewed to determine differences between primed and nonprimed 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 doublestick 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 0C for 7 days, then transferred to 25 0C 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




87
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 0C 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




88
'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 52). 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




89
Imbibition (% FW)
s80
a
a
60- b b
40
20
0
How Sweet it Is XPH-2644 Sweet Belle Crisp N'Sweet 711 Cultivar
Conductivity (umhos/g seed)
140
a
120
100
b
80 2
so
60 C C
4020
0
How Sweet it is XPH-2644 Sweet Belle Crisp N'Sweet 711 Cultivar
Figure 5-1. Imbibition (top) and leakage conductivity
(bottom) in four sh2 sweet corn cultivars, 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.




Full Text