Title: Soybean seed-coat permeability as related to seed deterioration, fungal association, and seed size /
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Title: Soybean seed-coat permeability as related to seed deterioration, fungal association, and seed size /
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Creator: Hill, Henry Jacob, 1952-
Copyright Date: 1984
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SOYBEAN SEED-COAT PERMEABILITY AS RELATED TO SEED
DETERIORATION, FUNGAL ASSOCIATION, AND SEED SIZE















BY

HENRY JACOB HILL JR.


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1984




































This dissertation is dedicated

to my wife,

Shirley Davenport Hill,

whose love and support enabled

me to complete these studies.















ACKNOWLEDGMENTS


I extend my sincere appreciation to my major professor, Dr.

Shirlie Hill West, for his guidance, assistance, and moral support

during the course of these studies. He freely contributed many hours

of his time for me. His efforts will not be forgotten.

Special recognition and appreciation are extended to members of

my supervisory committee for their time and effort in guiding me

through the course of these studies, and for critically reading and

offering many valuable suggestions for this dissertation: Drs. Kuell

Hinson, Norman C. Schenck, Daniel J. Cantliffe, James W. Kimbrough,

and Robert H. Biggs.

I would also like to extend my thanks to members, both past and

present, of the Agronomy Seed Laboratory for their support during the

course of these studies, and to Mrs. Patricia French for typing this

manuscript.

Lastly, I want to thank my teachers, both in and out of the

classroom, for trying to instill in me an open mind, an appreciation

for work, and a curiosity for the world around me.














iii















TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS................................................ iii

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

LIST OF FIGURES................................................. ix

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

CHAPTER I INTRODUCTION...................... ...... ........... 1

CHAPTER II LITERATURE REVIEW.................................. 3

Factors Affecting Soybean Seed Quality Before
Harvest.................................................. 4
Factors Affecting Soybean Seed Quality After
Harvest........................................... 13
The Impermeable Seed Coat.......................... 17
Benefits of the Impermeable Seed Coat.............. 24

CHAPTER III MATERIALS AND METHODS.............................. 28

Introduction...................................... 28
Response of Impermeable- and Normal-Seeded
Genotypes to Field and Storage Environments....... 29
Response of Impermeable- and Normal-Seeded
Genotypes to Field Weathering and Seedborne
Fungi............................................ 33
Statistical Analysis............................... 39

CHAPTER IV RESULTS AND DISCUSSION............................. 40

Response of Impermeable- and Normal-Seed
Genotypes to Adverse Field, and Favorable and
Adverse Storage Environments..................... 40
Response of Impermeable- and Normal-Seed
Genotypes to Field Weathering and Seedborne
Fungi............................................ 67
Seedborne Fungi and Germination of Seeds
Differing in Permeability........................ 90
Impermeable Seed Studies........................... 101

CHAPTER V SUMMARY AND CONCLUSIONS............................ 119













APPENDIX A


APPENDIX B


APPENDIX C


FUNGAL INCIDENCE WHEN 2,4-D WAS ADDED TO THE
IMBIBING SOLUTION..................................

INFLUENCE OF HARVESTING METHOD ON IMPERMEABLE
SEEDS WITHIN SEEDLOTS OF DIFFERENT SIZES...........

IMPERMEABLE SEED OF F4 IMPERMEABLE-SEED LINES
GROWN IN 1980......................................


LITERATURE CITED................................................ 140

BIOGRAPHICAL SKETCH............................................ 150


PAGE















LIST OF TABLES

PAGE

1 Viability of seeds harvested at maturity and at one
month after maaturity................................. 41

2 Rainfall occurring before and after harvest maturity
during the field experiment............................ 43

3 The influence of delayed harvest on seed permeability
of 8735 (visual observation)........................... 46

4 The influence of delayed harvest on seed permeability
of 8735 (weight measurements).......................... 48

5 Seed viability after accelerated aging for seeds
harvested at maturity and 1 month after maturity....... 49

6 Influence of favorable storage on viability of seeds
harvested at maturity and at 1 month after maturity.... 51

7 Response of seeds harvested at maturity and at 1 month
after maturity to accelerated aging and emergence tests
after 18 months of favorable storage................... 59

8 Moisture content (wet weight basis) of seeds from
different harvests after various weeks in adverse
storage ................................. ............... 61

9 The effect of storage environments on permeability of
8735 seeds after soaking for various time periods
(weight measurements).................................. 65

10 Viability of seeds harvested at maturity and at times
after maturity.......................................... 69

11 Rainfall occurring before and after harvest maturity
for the individual entries.............................. 70

12 The influence of line and delayed harvest on seed
permeability after soaking for various time periods...... 72

13 The effect of delayed harvest on permeability of 8731 and
8745 seeds after soaking for various time periods (weight
measurements)...................................... ...... 73

14 Correlations for impermeable seed percentages after
various soaking times with normal germination after
delayed harvests for seeds of 8731 and 8745............... 74











15 Correlations between the percentage of impermeable seeds
at harvest maturity, after various soaking periods, with
normal germination after 2 months of delayed harvest...... 76

16 Viability of seeds harvested at maturity and at times
after maturity in the blotter test........................ 77

17 Incidence of total fungi at maturity and at times after
maturity................................................... 79

18 Incidence of Aspergillus flavus, Aspergillus niger, and
miscellaneous Moniliales at maturity and at times after
maturity.................................................. 80

19 Correlation between incidence of fungal organisms and
normal germination of seed in the blotter test over
harvests................................................... 82

20 Incidence of observed fungal growth from the start of the
blotter test (days) for seeds harvested at maturity and at
times after maturity...................................... 83

21 Correlations between incidence of observed fungal growth
on seeds after start of the blotter test and germination
measurements....................................... ......84

22 Incidence of Phomopsis spp. and Fusarium spp. at harvest
maturity and at times after maturity..................... 86

23 Incidence of Alternaria spp. at harvest maturity and at
times after harvest maturity ............................ 88

24 Recovery of microorganisms from embryonic axes at harvest
maturity (HM) and 2 months later (DH).................... 89

25 Influence of 2 months of delayed harvest on permeability
of seeds as determined by different periods of soaking.... 91

26 Correlations between germination measurements and
incidence of microorganisms with seeds, differing in
permeability, of 8731 and 8745 over harvests............... 93

27 Incidence of Phomopsis spp., Fusarium spp., and bacteria
with seeds differing in permeability harvested at
maturity and at 2 months after maturity.................... 95

28 Incidence of observed fungal growth from the start of the
blotter test (days) for seeds differing in permeability
harvested at maturity and at 2 months after maturity...... 97

29 Normal germination for seeds from impermeable-seed lines,
which differed in permeability, and seeds from a normal-
seed cultivar harvested at maturity and at 2 months after
maturity................................................... 98


1











30 Total germination for seeds from impermeable-seed lines,
which differed in permeability, and seeds from a normal-
seed cultivar harvested at maturity and at 2 months after
maturity................................................. 100

31 Mean percentage distribution within three seed sizes and
total percentage of these seed sizes for the nine
impermeable-seed lines.................................... 102

32 Regression equations for percent impermeable seed (% IS)
on seed size after soaking for various periods of time.... 105

33 Slopes of regression (b) and Pearson's correlation coef-
ficients (r) for percent impermeable seed with seed size
after soaking for various periods of time for the
impermeable seed lines.................................... 108

34 Permeability of seeds from the nine impermeable-seed
lines after soaking for various periods of time........... 110

35 Dry weight of seeds, seed-coat weights, and seed-coat
percentage of total seed dry weights for impermeable
seeds and seeds of low impermeability in different lines... 115

36 The influence of threshing method on permeability of seeds
within impermeable-seed lines after soaking for various
periods of time............................................ 118

37 Incidence of fungi at harvest maturity when the imbibing
solution contained 0.1% 2,4-D............................. 126

38 Incidence of fungi at the first delayed harvest when the
imbibing solution contained 0.1% 2,4-D..................... 127

39 Incidence of fungi after 2 months of delayed harvest when
the imbibing solution contained 0.1% 2,4-D................. 128

40 Impermeable seed of lines after various periods of
exposure to moisture when seeds were threshed by different
methods.. ................................................. 137

41 Impermeable seed from different plant sections of lines
after various periods of exposure to moisture.............. 138


viii















LIST OF FIGURES


PAGE

1 Normal germination for seeds of entries after various
weeks of adverse storage................................. 55

2 Total germination for seeds of entries after various
weeks of adverse storage ................................ 57

3 Permeability of 8735 seeds after different storage
periods in different storage environments after various
periods of soaking (visual observations)................. 62

4 Permeability of seed in the three seed-size classes
averaged across the nine impermeable-seed lines after
soaking for various periods of time...................... 103

5 Permeability of seed within different seed-size classes
of nine impermeable-seed lines after 2 hrs of soaking,
1981 .................................... ................ 106

6 Permeability of seed within different seed-size classes
of nine impermeable-seed lines after 72 hrs of soaking,
1981 ..................................................... 107

7 Permeability of seed within different seed-size classes
of nine impermeable-seed lines after 2 hrs of soaking,
1981 .................................... ............... 111

8 Permeability of seed within different seed size classes
of nine impermeable-seed lines after 72 hrs of soaking,
1982 ................................... ................ 112

9 Permeability of 8732 seeds in different seed sizes and
after different harvesting methods...................... 131

10 Permeability of 8736 seeds for different seed sizes and
after different harvesting methods...................... 132

11 Permeability of 8740 seeds for different seed sizes
and harvesting methods................................. 134

12 Permeability of 8743 seeds for different seed sizes and
after different harvesting methods...................... 135















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


SOYBEAN SEED-COAT PERMEABILITY AS RELATED TO SEED
DETERIORATION, FUNGAL ASSOCIATION, AND SEED SIZE

BY

HENRY JACOB HILL JR.

April 1984

Chairman: S. H. West
Major Department: Agronomy

An impermeable seed coat prevents a seed from imbibing water.

The value of the impermeable seed coat in preventing seed

deterioration and fungal incidence was investigated. The importance

of seed size and harvest method on seed permeability was also studied.

When harvested 30 days after maturity, germination of seeds from

an impermeable-seed line (8735) was higher than seeds of a normal-seed

cultivar, if cultivar plants did not receive field-applied benomyl.

Germination of 8735 seeds was also higher than seeds of the cultivar

after 3 months of adverse storage (270C, 90% RH) irrespective of

field-applied benomyl. Permeability of impermeable seeds increased

under delayed harvest and storage conditions.

In another study, seeds from two impermeable seed lines,

harvested at maturity and 2 months later were compared with seeds from

a cultivar which had received biweekly benomyl treatments. Seeds from

both lines varied in degree of permeability. After 2 months of

delayed harvest, the least permeable seeds did not decrease in


I











viability or increase in incidence of Phomopsis spp. Fusarium spp., or

bacteria. Benomyl-treated cultivar seeds were higher in viability

than the most permeable seeds, lower in viability than the least

permeable seeds, and approximately equal to mean seed viability for

the impermeable lines.

Seeds from nine impermeable-seed lines differed in permeability.

Smaller seeds within these lines were less permeable. Seed size is

important in determining the permeability of a seed, within these

lines. Seeds of 15 impermeable-seed lines were hand-threshed and

combined threshed. Combine-threshed seeds were more permeable than

hand-threshed seeds. Thus, mechanical harvesting does scarify some

impermeable seeds.

Under stress impermeable seeds retained viability better than

permeable seeds. However, seeds from an impermeable-seed line differ

in permeability, smaller seeds being the least permeable. For seeds

to retain viability under environmental stresses, they must be very

impermeable because stress environments increase permeability of seeds

that are slightly permeable.















CHAPTER I
INTRODUCTION


High temperatures, high relative humidity, and seedborne fungi

are considered by many researchers to be the most important abiotic

and biotic factors associated with seed deterioration in soybeans

[Glycine max (L.) Merr] and other crops. When these factors act in

concert their effect can be particularly severe. Improper storage of

seeds or seeds remaining in the field due to a delay in harvesting are

the situations where seeds most frequently encounter these factors.

Seed deterioration leads to a loss of vigor and viability resulting in

delayed, spotty, or inadequate stands if seeds are planted. This will

cause yield reductions for the crop unless replanting with high-

quality seeds is done immediately. Thus, seed deterioration and its

ramifications can impose a major constraint to the production of

soybeans and other crops.

The impermeable seed coat in soybeans is an inherited

characteristic which does not allow water to re-enter the seed. Since

the impermeable seed coat presents a barrier to water, microorganisms

may also be deterred from entering. Thus, two of the three major

factors associated with seed deterioration, high humidity and fungi,

may be nullified. Seeds expressing this trait may be able to retain

viability better than seeds which have a permeable seed coat when

these factors are present in the seed's environment.











While in theory the impermeable seed coat may provide a solution

to the problem of retaining high quality planting seed; few studies

have been published on how well this trait retains seed viability or

prevents fungal incidence. Furthermore, knowledge is limited on how

well this character prevents deterioration as compared to other seed

protection measures, how much impermeability is needed to prevent

deterioration in a seed, and what other seed factors may be associated

with the impermeable seed coat.

The objectives of the following studies were: to determine if

seeds with the impermeable seed-coat trait can retain viability under

delayed harvest and improper storage better than seeds with a normal

seed coat; to determine if the impermeable seed coat trait can protect

seeds against fungal incidence, and if this protection is better than

the application of foliar-applied benomyl on normal seeds; to

ascertain if the impermeable seed coat is equally expressed in all

seeds and what the influence of seed size is on expression; to

determine if harvest method is important in retaining the expression

of this character in seed lots. These studies will aid in determining

the benefits and limitations of this genetic trait to improved seed

quality. These studies will also advance the understanding of this

trait so that it can be used more effectively.















CHAPTER II
LITERATURE REVIEW



Seed quality has different meanings to different individuals.

The Federal Seed Act states that quality refers to the purity and the

germination of a seed lot (McDonald, 1980). To a farmer, quality may

imply a seed lot's vigor. To a grain-elevator manager, quality may

imply a seed lot's appearance. For the purposes of this literature

review, quality refers to a seed lot's capacity to germinate and/or

emerge from the soil when planted.

In storage, soybean seeds can lose their quality faster than any

other major crop seed except for shelled peanuts (Delouche, 1982).

Loss of seed quality in storage is often referred to as deterioration.

Soybean seeds can also deteriorate while "stored" in the field.

Deterioration in the field is commonly referred to as "weathering"

(Andrews, 1982). Seed deterioration is physiological and biochemical

in nature (Abdul-Baki, 1980; Osborne, 1980; Delouche, 1982). Besides

the process of deterioration other factors have been associated with

poor quality soybean seed. Some of these factors are: disease

(Sinclair and Shurtleff, 1975), mechanical or harvest methods (Paulsen

et al., 1981; Green et al., 1966) and insects (Todd, 1982). These

factors may disrupt physiological processes during germination or

accelerate the physiological or biochemical events occurring with

deterioration before or after seed maturity.


1










The external environment may affect both the initial quality and

the rate of deterioration of a seed lot. The primary abiotic

environmental influences on soybean seed quality are temperature and

humidity (Delouche, 1982). An important biotic influence is

microorganisms, mainly fungi.


Factors Affecting Soybean Seed Quality Before Harvest


Temperature and Relative Humidity


The earliest stage at which an accurate assessment of seed

quality can be measured is at physiological maturity (Copeland, 1976).

In soybeans, this stage is attained when the seed's moisture content

ranges from 50 to 55% (Delouche, 1982; Tekrony et al., 1979). This is

an important bench mark since subsequent loss of quality cannot be

measured without a starting point. Even at this stage, however, seed

quality may already be affected adversely by the environmental factors

of temperature and humidity (Tedia, 1976; Tekrony et al., 1980).

Temperature and humidity influence seed quality in the following

two situations. (1) Prevalence of high temperatures during seed

maturation: Green et al. (1965) reported that seeds maturing under

hot dry conditions were lower in germination than seeds which matured

after these conditions had passed. High temperatures may cause poor

quality soybean seed by accelerating their maturation. Soybean-seed

maturation is a metabolically active process involving protein

modification, starch utilization, production of soluble sugars (Adams

et al., 1982) and other processes. Soybean seeds which were harvested

prematurely and "fast-dried" remained green, leaked high amounts of










solutes upon rehydration, and did not contain germination-specific

enzymes such as malate synthase and isocitric lyase. Seeds which were

harvested prematurely but "slow dried" did not show these

abnormalities (Adams et al., 1983).

Tekrony et al. (1980) reported a significant decline in seed

germination during the 10-day period between physiological maturity

and harvest maturity in a year in which maximum air temperatures

exceeding 300C for six of these 10 days. Mondragon and Potts (1974)

erected screens over plants after the seeds had reached physiological

maturity to decrease sunlight by 50%. This treatment reduced the

temperature around the plants by an average of 2.20C and raised the

relative humidity by 2%, as compared to the control plants. Four

weeks later, seed germination from the shaded plants was significantly

higher than for seeds from the control plants. The authors suggested

that it is the fluctuations in temperature and relative humidity and

not specific temperatures (as suggested by Tekrony et al., 1980) that

causes the loss in germination capacity of soybean seeds.

(2) Periods of high rainfall characterized by higher humidities

and lower temperatures than the first situation. Mondragon and Potts

(1974) reported that a daily water spraying on soybean plants after

the seeds had reached physiological maturity resulted in lower

germination than the seeds from the control plants, after 5 weeks.

Tedia (1976) also reported that an increased humidity around maturing

soybean plants decreased seed germination. Tekrony et al. (1980)

reported that the decline in soybean seed vigor, as measured by

accelerated aging, was closely related to air temperature, R2 = 0.90,










maximum relative humidity, R2 = 0.94, and precipitation per day, R2

= 0.75, during delayed harvest. Alexander et al. (1978) noted that

seeds from the soybean cultivar 'Forrest' had a significantly lower

germination at maturity than 'Bragg', 'Cobb', and 'Hampton 266A'.

They attributed this difference to 'Forrest' being exposed to higher

rainfall than the other cultivars rather than genetic differences.

The decline in seed quality, during periods of high rainfall, has

been associated with an increase in fungal-infected seeds (Tedia,

1976; Alexander et al., 1978; Kilpatrick, 1957). This is not to say

that rainfall and relative humidity only act on seed deterioration by

promoting fungal infection. Rather, rainfall may act in "concert"

with fungi to cause deterioration (although the direct influence of

rainfall on seed deterioration has not been delineated for soybeans).

The influence of temperature and relative humidity on the degree

of fungal infection of soybean seeds have been elucidated by Spilker

et al. (1981). In growth chambers, soybean plants were sprayed with a

spore suspension of Phomopsis sp. (hereafter referred to as Phomopsis)

at the R4 stage (full pod, Fehr and Caviness, 1977). At the R6 stage

(full seed), different combinations of temperature and relative

humidity were imposed on the plants. At the R8 stage (full maturity),

seeds were harvested and percent germination and percent Phomopsis-

infected seeds were determined. Plants exposed to high relative

humidity, 90 4%, and high temperature, 280C, had the highest

Phomopsis-infected seeds, 49%, and the lowest seed germination, 32%.

Plants exposed to low relative humidity, 43 4%, and low temperature,

21C, had the lowest Phomopsis-infected seeds, 4%, and the highest










seed germination, 96%. At low relative humidity, Phomopsis-infected

seeds and seed germination were not different at either high or low

temperatures. The authors concluded that humidity is the most

important factor in determining Phomopsis infection of seed.

Reports of field experiments have also related the influence of

rainfall on fungal infection of soybean seeds. Kilpatrick and Hartwig

(1955) reported that early plantings of 'Ogden' had higher total

fungal- and Phomopsis-infected seeds than later plantings because of

weather conditions prior to plant maturity. Kilpatrick (1957) also

reported that more fungal-infected flowers, pods and seeds, occurred in

a year with the highest rainfall. Alexander et al. (1978) reported

significant increases in fungal-infected seeds after harvest maturity

if the seeds were exposed to normal or above-normal rainfall.

Since soybean seed quality can be impaired by exposure to

environmental factors, the seeds should be harvested as soon as

possible. Harvesting soybean seeds should be done as soon as the

moisture content is 13-14% (Delouche, 1972), although it may not

always be possible. In the "post-maturation pre-harvest" stage of

soybean seed production, the influences of environmental factors on

seed deterioration can be particularly severe (Delouche, 1982). In

Puerto Rico, delaying harvest of 24 genotypes by 4 weeks significantly

reduced sand and field emergence of soybean seed by 12 and 37%,

respectively (Paschal and Ellis, 1978). In Kentucky germination was

not, but vigor was reduced by delayed-harvest (Tekrony, 1980).

Several other studies have reported on the decrease in seed quality

with delayed harvest in soybeans and associated poor seed quality with










an increase in fungal infection (Wilcox et al., 1974; Ellis and

Sinclair, 1976; Athow and Laviolette, 1973; Alexander et al., 1978).

Some researchers, however, still question whether increased fungal

association with soybean seeds is a consequence or cause of delayd-

harvest weathering (Delouche, 1982; R. W. Yaklick, personal

communication).


Microorganisms

The association of seedborne microorganisms with reduced

germination of soybean seeds, as well as other crop seed, has been

established in many publications and review articles (Neergard, 1977).

In soybeans, fungi are the most prevalent microorganisms associated

with poor quality seed (Sinclair and Dhringa, 1975). However, other

types of organisms can be associated as well.

Six bacteria species are known to be associated with or seedborne

in soybean seeds (Sinclair, 1976). Two of the six species, Bacillus

subtilis (Ehrenberg) Cohn and Pseudomonas glycinea Coerper are serious

seed pathogens (Schiller et al., 1977; Tenne et al., 1977). The

virulence of B. subtilis on soybean seeds increases as germination

temperature increases from 25 to 45C (Ellis et al., 1977; Tenne et

al., 1977). Although B. subtilis is frequently isolated from the

hypodermis of the soybean seed coat (Tenne et al., 1977), increases of

bacterial-infected seeds may not be related to increases of fungal-

infected seeds (Sinclair, 1978).

At least eight viruses are associated with or seedborne in

soybean seeds (Sinclair, 1976). The most prevalent virus in soybeans

may be soybean mosiac virus (SMV) (Goodman, 1975). SMV-infected seeds










have lower germination than non-infected seeds (Dunleavy et al.,

1970). SMV-infected plants have a higher incidence of Phomopsis-

infected seed than non-infected plants (Hepperly et al., 1979). Seed

coat mottling or the "bleeding hilum" of soybeans is associated with

SMV-infected seeds or plants (Kennedy and Cooper, 1967). Seed coat

mottling also causes soybean seeds to be discounted at the elevator

because of lower market grades (Athow, 1976).

Although 66 fungal species have been associated with soybean

seeds or are seedborne (Sinclair, 1976), not all of them have been

shown to be deleterious. Increased recovery of Aspergillus flavus Lk.

ex Fr. from soybean seed lots was not correlated with decreased

germination (Dhingra et al., 1973). Although Alternaria spp. is

frequently isolated from soybean seeds they have not been associated

with reduced germination (McGee et al., 1980; Tenne et al., 1974;

Athow and Laviolette, 1973). Recovery from field infection of less

common fungi such as species of Chaetomium, Penicillium, and

Cladosporium may not be associated with either reduced laboratory

germination or field emergence (McGee et al., 1980).

Cercospora kikuchii (Mat. and Tomoy.) Gardner, causal agent of

purple stain in soybeans, has been associated with reduced germination

in some studies (McGee et al., 1980; Wilcox et al., 1973; Murakishi,

1951), but not in others (Hepperly and Sinclair, 1981; Roy and Abney,

1976; Sherwin and Kreitlow, 1952). The reason for this discrepancy

may be due to the antagonism between C. kikuchii and Phomopsis (Roy

and Abney, 1977). Seeds infected with C. kikuchii are higher in

germination than those infected with Phomopsis (Roy and Abney, 1977;








10

Hepperly and Sinclair, 1981). C. kikuchii-infected seeds do not

increase past harvest maturity (Athow and Laviolette, 1973; Hepperly

and Sinclair, 1981).

Phomopsis is generally regarded as being one of the most

prevalent (Dunleavy, 1976; Sinclair, 1975) and most virulent of fungal

organisms associated with soybean seeds (McGee et al., 1980; Paschall

and Ellis, 1978). Phomopsis sp. and Phomopsis sojae Leh. are the

causal agents of pod and stem blight in soybeans but are really only a

part of a disease complex with soybean seed. Phomopsis along with

Diaporthe phaseolorum (Cke. and Ell.) Sacc. var. sojae (Lehman) Whem.

(perfect state of Phomopsis and also causal agent of pod and stem

blight), and Diaporthe phaseolorum (Cke. and Ell.) Sacc. var.

caulivora Athow and Caldwell (the causal agent of stem canker) have

all been associated with poor germination of soybean seeds. Among

these organisms, Phomopsis is reported to be the most prevalent (Kmetz

et al., 1978) and the most virulent (Kmetz et al., 1974).

Colletotrichum dematium (Pers. ex Fr.) Grove var. truncata

(Schw.) Arx, Syn.: Colletotrichum truncatum (Schw.) Andrus and W. D.

Moore, hereafter referred to as Colletotrichum, has been associated

with both decreased seed yield and seed germination (Sinclair and

Shurtleff, 1976; Nicholson and Sinclair, 1973). Fusarium spp. has

been associated with poor seed germination in the laboratory (McGee et

al., 1980; Hepperly and Sinclair, 1981), but not with poor field

emergence (McGee et al., 1980).

Certain fungi can affect the appearance of soybean seed thus

reducing their market grade. Cracked and shriveled seed, partially or


T











completely covered with white mycelium is associated with Phomopsis

infection. Seed lots heavily infected with Phomopsis have a higher

percentage of seed classified as damaged kernels according to the

official grain standards of the United States (Athow, 1976); and seeds

also have lower test weights and poorer-quality oil (Hepperly and

Sinclair, 1978). C. kikuchii, may also reduce the market grade of a

soybean seed lot by causing purple-stained seeds (Athow, 1976).

The use of fungicides and antibiotics may control the amount of

fungal and bacterial damage in soybean seeds. Treating soybean seeds

with captain, thiriam, and benomyl, as a seed dressing, can

significantly increase germination. All fungicides reduced the

recovery of fungi from seeds. However, thiriam was ineffective in

controlling Aspergillus spp. and benomyl was not effective against

Alternaria sp. (Ellis et al., 1975). Spraying the crop with benomyl

can increase the germination of seeds if harvest is at maturity (Ellis

et al., 1974) or if harvest is delayed (Ellis and Sinclair, 1976).

However, fungicides have little influence in improving germination if

the seed lot already possesses a high germination percentage

(Sinclair, 1976; Mengistu et al., 1975). Treating seeds with

streptomycin sulfate will increase germination at high temperatures if

the seed lot is heavily infected with B. subtilis (Ellis et al., 1977;

Schiller et al., 1977).

The nature of fungal infection has been documented, to some

extent, for soybean seeds. Fungal organisms reported to be

deleterious to seed germination may be closely associated with the

mother plant throughout the vegetative and reproductive stages of a











plant's life cycle. Phomopsis can be isolated from soybean plants at

the first trifolate leaf stage (Kmetz et al., 1978). This early

infection can be due to carry-over from the germinating seed (McGee et

al., 1980) or induced by seed emerging through over-wintered soybean

straw (Kmetz et al., 1979). The prevalence of Phomopsis isolated from

soybean stems can increase throughout the vegetative growth stages

(Kmetz et al., 1978), but the mycelia remains close to the site of

inoculation until senescence begins (Hill et al., 1980).

Colletotrichum can be isolated from soybean pods, seeds, the epidermis

of leaves, and the cortex of stems. Similar to Phomopsis, mycelial

growth of Colletotrichum increases just as the plant approaches

maturity (Tiffany, 1951). From the R3 to R7 growth stages (beginning

pod to beginning maturity), the percentage of pods infected with

Phomopsis increases with the lower pods having earlier and higher

infection than the upper pods (Kmetz et al., 1978; Hepperly and

Sinclair, 1980). Seed infection is also earlier and greater in the

lower pods (Kmetz et al., 1978).

The importance of seed infection by fungi from the mother plant

versus the importance of seed infection by fungi from external sources

has been studied by Athow and Laviolette (1973). They inoculated

soybean plants 5 weeks before plant maturity with Phomopsis. At the

same time, they covered the pods with pollination bags. When seeds of

these different treatments were harvested at plant maturity,

germination (approximately 80%), and seed infection by Phomopsis,

Cercospora sp., Alternaria sp., and miscellaneous fungi for seeds of

these different treatments were similar. If the plants were left in











the field for 6 weeks after maturity there were differences among the

treatments. Seeds from unprotected pods had a significantly lower

germination than seeds from protected pods, 22 versus 75%.

Inoculation with Phomopsis was not as important to germination as

protecting the pods. Seed infection by Phomopsis followed the same

trends as seed germination, but was inversely related, with the

highest Phomopsis-infected seed from inoculated plants and unprotected

pods, and the lowest Phomopsis-infected seeds from inoculated plants

and protected pods. Alternaria sp. and miscellaneous fungi, mainly

Fusarium sp., was more than 400 and 300% higher for seeds from the

unprotected pods. These results indicate that seed infection by fungi

occurs primarily from external sources, and that germination can be

maintained, during delayed harvest, by protecting the seed with a

barrier to prevent fungi and rainfall from entering.


Factors Affecting Soybean Seed Quality After Harvest


Environmental factors that decrease viability in the field are

also influential in storage. Although temperature and relative

humidity would naturally fluctuate in an uncontrolled on-farm storage

or elevator situation, all studies reviewed used constant or near

constant conditions to ascertain the influence of temperature and

relative humidity on seed quality.

The temperature of the storage environment plays an important

role in regulating the rate of deterioration in soybean seeds.

Soybean seeds stored at 9.4% moisture content can maintain their

viability for more than 10 years if storage temperature is











100C. However, increasing temperature to 20'C will reduce germination

of a seed lot in 5 years, and increasing temperature still further to

30C will reduce germination of a seed lot in 1 year (Toole and Toole,

1946). Exposure of soybean seeds to short durations of extremely high

temperature (such as placing seed bags in the sun, or a temperature

buildup in storage due to extremely hot weather) may also adversely

affect germination. Normal germination of soybean seeds at 12%

moisture content can be reduced from 70-30% in only 24 hours if the

seeds are exposed to 550C (West, 1982).

"Soybean seeds are hydroscopic" (Delouche, 1982 p. 58). They can

absorb moisture from the air surrounding them. Soybean seeds when

stored can reach an "equilibrium moisture content" with the

surrounding environment (Delouche, 1973). This moisture content is a

function of the vapor pressure or the relative humidity at a specific

temperature. For example, at 250C and 60% RH, the moisture content,

in equilibrium, of soybean seeds is 9.4%; at 250C and 90% RH the

moisture content, in equilibrium, is 18.8% (Christensen, 1973;

Delouche, 1982). As the moisture content of soybean seeds increase

their rate of deterioration also increases. Soybean seeds stored at

100C and 9.4% moisture content can retain their germination for 10

years. If the moisture content is increased to just 13.4%,

germination cannot be retained past 3 years (Toole and Toole, 1946).

Soybean seed deterioration is a rapid process if seeds are stored

at a high temperature with a high moisture content. At 300C, seeds

stored at 18.3, 16.5, and 14.7% decreased to 50% germination in 10,











14, and 20 weeks of storage (Dorworth and Christensen, 1968). Of the

two factors, temperature and relative humidity, relative humidity is

considered to be the most important factor in determining the

longevity of a soybean seed lot (Delouche et al., 1973). There are

several equations developed for predicting longevity of seed in storage

(Delouche and Baskin, 1973). Harrington's rule-of-thumb (Harrington,

1959) states that the storage life of seeds increases two-fold for

each 1% decrease in seed moisture and increases two-fold for each

5.50C reduction in temperature, within the range of 0 to 450C. This

equation has been found to be essentially correct for soybean seeds

within the moisture contents of 6 to 16% (Delouche, 1968).

Accelerated aging is a technique originally developed to

determine a seed's ability to withstand deteriorative conditions found

in storage (Delouche and Baskin, 1973). The technique consists of

exposing a group of seeds to high temperature (usually 400C) and 100%

relative humidity for a preset time period. A standard germination

test is performed after this treatment to determine how the seeds

responded to these conditions. This technique has been reported to be

a good predictor for storability of soybean seeds (Baskin and Vieira,

1980), as well as other crop seeds (Delouche and Baskin, 1973).

The accelerated-aging technique has also been used to determine

basic physiological or biochemical changes which occur in a seed

during adverse storage to cause the seed to "age." Among these

changes, a damaged membrane system in seeds is a major cause of

deterioration. The formation of free radicals due to perioxidation of

unsaturated fatty acids as reported in peas, Pisum sativum L.











(Pammeter et al., 1974) is a cause of membrane impairment. The damage

prevents proper reformation of the membrane system in cells once

imbibition starts. Because membranes cannot reform properly there is

a loss of compartmentalization of cellular components; thus, the cell

cannot function effectively. Increased leakage of electrolytes from

seeds is evidence of membrane damage (Parrish and Leopold, 1979).

However, increased leakage from the seed may also be due to the

inability to utilize molecules due to impairment of other systems as

well (Abdul-Baki and Anderson, 1970). Loss of nucleic acid and

protein synthesis has been reported in accelerated-aged soybeans

(Abdul-Baki and Chandra, 1977), as well as decreased mitochondrial

efficiency (Abu-Shakra and Ching, 1977). Several other physiological

and biochemical changes have been reported to be due to accelerated

aging and are thought to be indicative of storage deterioration but

they will not be discussed. In addition to the ability to affect

physiological damage temperature and relative humidity may also

indirectly affect seed quality by promoting the activity of storage

fungi. The most common of these storage fungi are species within the

genera Aspergillus and Penicillium. These storage fungi do not

require free water to be active but require relative humidities above

65%. Moderate temperatures are also important for the activity of

these fungi. The optimum temperature for their development is 30-33C

(Christensen, 1973). These fungi cause reduced germination in

soybeans (Kennedy, 1964) and loss of oil quality (Christensen, 1967;

Kennedy, 1964).











The action of storage fungi on seeds to cause loss of germination

has been elucidated. In peas, Aspergillus ruber has been shown to

produce a toxin which will accelerate degradative changes within the

seed (Harmon and Nash, 1972). Mitochondrial damage and breakdown of

the plasmalemma was more severe in seeds infected with A. ruber than

seeds which were not. This resulted in higher solute leakage and a

slower increase in respiration during imbibition of the infected seeds

(Harmon and Drury, 1973; Harmon and Gravett, 1972).

Storage of soybean seeds can also improve germination, provided

that the environment is favorable. Under low relative humidities,

field fungi dehydrate and become inactive. If the length of storage

time is long enough, field fungi lose their ability to rehydrate.

Since field fungi cannot rehydrate during imbibition the germination

rate increases (Neergard, 1977). Wallen and Seamen (1963) stored

soybeans under "cool dry conditions" and recorded the Phomopsis

infection and seed germination of this lot at yearly intervals. In

the most heavily-infected seed samples, the percentage of Phomopsis-

infected seed dropped from an initial 73 to 40% in the first year, and

dropped to 4% the next. Seed germination increased, over this time,

from 20 to 96%.


The Impermeable Seed Coat


The pericarp or testa of a seed may delay germination or impose

dormancy due to a number of different mechanisms. These mechanisms

include preventing gaseous exchange between the embryo and the

environment, modifying the quality of light reaching the embryo,











preventing an inhibitor from leaching out of the seed or embryo, or

the testa may contain an inhibitor (Brewley and Black, 1982). In

soybeans, the testa or seed coat may impose a dormancy or delay

germination by restricting water uptake by the embryo. This

mechanism is found within several other genera of Leguminosae besides

Glycine, and is common within Melilotus, Trifolium, and Coronilla

(Ballard, 1973). In addition to Leguminosae, impermeable seeds have

been reported in other plant families including Malvaceae (Lee, 1975),

Liliaceae (Villiers, 1973), and Solonaceae (Toole, 1939).

The restrictive or impermeable seed coat can cause a condition of

hardseededness which is defined by the Association of Official Seed

Analysts (AOSA) as "seeds which remain hard at the end of a prescribed

test period because they have not absorbed water due to an impermeable

seed coat" (1978, p. 29). Since the test period for soybeans in the

standard germination test is 8 days only those seed which remain

impermeable for this time period can officially be termed "hard"

(Potts, 1978).

Impermeable or hard seeds occur in soybean cultivars (Carlson,

1973; Wolf et al., 1981; Green and Pinnell, 1968). Woodworth (1933)

first studied the inheritance of the impermeable-seed trait using

seeds from the F1 and F2 progeny of the cross PI 65388 x Dunfield. He

expressed the amount of impermeable seeds for a plant by calculating

an "average-bean-hour." The average-bean-hour was calculated on seeds

of each plant by multiplying the number of seeds swollen with the time

in hours it took for each seed to swell, adding the products, and

dividing by 10. The highest possible value was 192 and the lowest











value was three. Dunfield fell into the lowest class with a mid-point

of 10 and PI 65388 had a mid-point of 170. Seeds of the F1 plants had

a mid-point value of 50 which indicated partial dominance for normal

seeds. The distribution of the "average-bean-hour" for seeds of the

F2 was not normal. Green and Pinnell (1968) crossed 'Chippewa' and

'Harosoy 63' with PI 261469, PI 200499, and PI 229336. In this case,

they were interested in lowering the hard seeds of the American

cultivars to those of the PI's. Seeds of the F1 either showed

dominance or recessiveness for high hard-seed percentage. They

concluded, however, that the impermeable-seed trait was highly

heritable.

Kilen and Hartwig (1978) crossed 'Tracy' with D67-5679.

Individual plants of the Fl, F2, and the two parents were hand

threshed to avoid scarification [unlike the study of Green and Pinnell

(1968) in which plants were machine threshed]. Based on the results

of the F2 population, Kilen and Hartwig suggested that the normal seed

type was at least partially dominant. They also suggested that three

major genes control the variation in this trait, although some minor

genes may be present. Work by Srinives (1980) confirmed the results

by Kilen and Hartwig.

The seed coat of soybeans has long been considered to be the site

of permeability (Carlson, 1973). The seed coat of soybeans, similar

to other legumes, is maternal tissue differentiated from the outer

integuments of the ovule (Esau, 1965). Carlson (1973) reviewed the

anatomy of the soybean seed coat and reported it to be composed of

several different cell types and distinct layers. The epidermis or











palisade layer is the outermost layer of the seed coat. It is

composed of macrosclereid or malpighiam cells with thick secondary

cell walls and a small cell lumen. A cuticle is deposited on the

exposed outer wall of this layer. In the hilum region of the seed

coat are two palisade layers, the outermost layer derived from the

funiculus. The subepidermal, hypodermal, or middle layer is commonly

referred to as the hour-glass layer because it is composed of

osteosclereid cells (which have an hour-glass shape). Large

intercellular spaces, characteristic of this layer, are thought to be

the initial site of colonization for seedborne fungi (Schneider et

al., 1974; Ilyas et al., 1975) and seedborne bacteria (Tenne et al.,

1977). The inner layer commonly referred to as the parenchyma layer

is the most complex layer of the seed coat. This layer has an

articulated parenchyma of thick cell walls above a narrow zone of

vascularization. Below this zone of vascularization is a zone of

thin-walled parenchyma cells which lies above an endothelium (Thorne,

1981). Because the endothelium is living at seed maturity, it has

been thought to be a remnant of the endosperm layer referred to as the

aleurone (Carlson, 1973; Brewley and Black, 1978). New evidence

indicates that this is not endospermic tissue because it lies outside

the embryo sac (Thorne, 1981).

Several studies have attempted to define the anatomical or

histochemical differences in the seed coat which cause permeability in

soybean seeds. Duangpatra (1976) studied the ontogeny of the

impermeable and permeable seed coats to determine if any comparative

anatomical differences exist and to determine the most critical stage











for development of impermeability. Using two impermeable-seed lines

and a near-isogenic permeable-seed cultivar, seed coat development,

moisture content, and percent impermeable seeds before and after

drying were monitored from physiological maturity to 15 days after

harvest maturity. Complete histological differentiation of the seed

coat occurred in approximately 30 days after flowering for all lines.

At this time, no anatomical differences were noticed among the seed-

coat types. There was also no difference in the form or structure of

the "light line" between the permeable or impermeable seed coat. When

the seed coat was mature, approximately 50 days after flowering,

impermeable seeds were observed in seeds that were artificially dried.

This was 2 weeks earlier than in seeds dried naturally. In both

impermeable-seed lines, impermeable seeds were not detected in fresh

seeds until the moisture content dropped to 14%. These observations

are similar to observations on seeds of Trifolium reopens, Trifolium

pratense, and Lupinis arboreus. These seeds became impermeable at 14%

moisture (Hyde, 1954). In cotton, impermeable seeds were first

detected when seeds reaches 11% moisture (Christenses et al., 1960).

Although no anatomical differences were noted between permeable

and impermeable seeds (Duangpatra, 1976), there are histochemical

differences. High quantities of "fat" at the base of the palisade

layer penetrating upwards between the macrosclereids and a high

lignification of mascosclereid cell walls have been observed in

impermeable seeds of soybean cultivars grown in Romania (Baciu-

Miclaus, 1970). Histological studies by Duangpatra (1976) on

impermeable seeds from PI 163453 did not confirm Baciu-Miclaus'











observations. She observed suberin deposits in the inner palisade

layer of the hilum region forming a "continuous barrier" across this

layer. She speculated that these suberin deposits may account for the

impermeable-seed condition within the hilum region and that an

unidentified fatty acid accounts for the impermeable-seed condition in

the rest of the seed coat (Duangpatra, 1976).

Based on work with Pisum, the neccesity of a dry down for the

impermeable seed to occur in soybeans (Duangpatra, 1976) indicates

that the chemical barrier to water may be due to the presence of

phenolics. In Pisum, oxygen is necessary for the enzyme, catechol

oxidase, to oxidize phenolic compounds which are thought to cause the

impermeable seed (Marbach and Mayer, 1974). While the seed coat is

still high in moisture, enough free oxygen may not be available for

oxidation to occur.

Although the occurrence of lignin was not noted to be different

in the permeable or impermeable seeds studied by Duangpatra (1976).

The presence of lignin may be important in determining permeability.

In lima beans, white seeds have a lower lignin content than dark seeds

(Kannenburg and Allard, 1964) and white seeds were more permeable than

dark seeds (Morris et al., 1968).

The hilum fissure may be closed or open in soybean seeds and a

closed condition is thought necessary for the seed to be impermeable

(Yaklick, 1983). If this is true, the soybean hilum may act as a

hydroscopic valve (open when the seed's moisture content was less than

the surrounding air and closed when the seed's moisture content is

higher than the surrounding air) as found in species of the genera

Trifolium and Lupine (Hyde, 1954).











The presence of pores or pits has been reported as occurring on

the seed-coat surface of several commercial soybean cultivars (Calero

et al., 1981; Wolf and Baker, 1980; Wolf et al., 1981). These pores

are indentations on the surface which extend downward into the

palisade layer. There may (Calero et al., 1981) or may not (Wolf et

al., 1981) be an opening at the bottom of a pore. These pores can

occur with high frequency on the seed coat surface, 21 per 0.075 mm2

and varietal differences in the frequency of pores have been observed

(Calero et al., 1981). These pores may allow for faster uptake of

water by soybean seeds and may provide an entryway for fungal

infection of the seed coat (Calero et al., 1981; Hill and West, 1982).

A cavity through the palisade layer may also be associated with this

surface pore (Wolf and Baker, 1981; Yaklick, 1983; Calero et al.,

1981).

Using scanning electron microscopy, Calero et al. (1981) made the

startling discovery that impermeable seeds do not possess these pores,

and these seeds often have material embedded on the surface, which

appears different than the normal cuticle. They went on to conclude

that the shape, size, functionality of the pore along with material

embedded on the surface may account for permeability.

Barton (1965) reviewing the environmental factors determining

permeability in Leguminosae concluded that humidity appeared to be the

most important, but other factors such as harvest time, seed size, and

nutrition may also be influential. Baciu-Miclaus (1970) indicated that

low relative humidity and high temperatures during seed maturation

contributed to an increase in impermeable seeds within soybean











cultivars. Srinives (1980) grew impermeable-seed lines in the field

at Isabella, Puerto Rico. He observed that impermeable-seed lines

grown in the winter usually had higher impermeable-seed percentages.

He believed that impermeable seed differences were due to less

rainfall, disease, and insects in the winter season. He also reported

a significant environmental effect for impermeable-seed percentages

when growing lines in the same season but in different years, even

when the seeds were from the same homozygous parent.

Variation in impermeable seeds within a plant does occur.

Growing two determinate genotypes and two indeterminate genotypes,

Srinives (1980) reported that pod position may or may not be

associated with permeability. In the determinate genotypes,

impermeable seed percentage was higher on the upper nodes than the

lower nodes for one genotype and not for the other. In the

indeterminate genotypes, impermeable seed appeared in greater

frequency in the middle of the plant than at the sides. Again, this

was noted for one genotype and not the other. Seed.position within

the pod was not associated with permeability. Whether a plant is

grown from an impermeable or permeable seed has no effect on the

frequency of impermeable seeds from that plant.


Benefits of the Impermeable Seed-Coat Trait


Since the prevention of moisture reabsorption defines the

impermeable seed, this trait should prevent deterioration caused by

reabsorption of moisture. Besides preventing reabsorption of moisture

the impermeable seed coat should also prevent seed damage by











microorganisms. If water cannot enter through the seed coat, perhaps

neither can microorganisms. Even if microorganisms could enter an

impermeable seed, their activity should be limited because of limited

availability of moisture.

Seeds from an impermeable-seed line have been demonstrated to

reabsorb less moisture from the environment, after harvest maturity

than seeds of a normal-seed cultivar. Harvesting seed twice daily on

alternate days for one month, seeds from the normal-seed cultivar were

above 13% moisture on 13 of 24 harvests. Seeds from the impermeable-

seed line were above 13% moisture on only 4 of the 24 harvests. The

highest moisture recorded for impermeable seeds was 16.5 compared to

23% for permeable seeds. Not only did the impermeable seed have less

moisture reabsorption but less fluctuation in moisture content as well

(Duangpatra, 1976).

Impermeable seeds may also resist infection by fungi. Seeds of

an impermeable-seed line along with seeds of 'Dare' and 'Mack' were

bioassayed during three stages of seed development and maturity.

There were no significant differences among these entries, in fungal-

infected seeds, during the first and second stages of seed development

(flower, pod, and seed development). Significant differences were

reported, however, among entries, after the third stage (10 weeks past

physiological maturity). Seeds of the impermeable-seed line had a

lower infection percentage of both Phomopsis and total fungi than

seeds of either normal-seed cultivars (Potts, 1978).

Because of reduced moisture reabsorption and reduced fungal

infection during delayed harvest, impermeable-seeds should retain











their vigor better than normal seeds. To test this Duangpatra (1976)

allowed an impermeable-seed line and a normal-seed isoline to

"weather" for 2 months beyond maturity. In each of 2 years, the

impermeable-seed line had significantly higher percentages of "total

viable seed" than the normal-seed isoline.

Because of reduced hydroscopic capacity impermeable seeds may

also give the grower more flexibility in harvesting. An impermeable-

seed line can be harvested on more days, within a given period of

time, than a normal-seed cultivar. The moisture content of seeds from

the impermeable-seed line was below 12% on 18 days, as compared to 7

days for seeds of the normal-seed cultivar (Duangpatra, 1976).

Impermeable seeds withstand the adverse effects of accelerating

aging better than normal seeds. An impermeable-seed line had a

significantly higher percentage of total viable seed (germinated plus

impermeable seed) compared to a normal-seed cultivar after exposure to

the accelerated aging test for 96 hours, and twice the total viable

seed after 120 hours (Duangpatra, 1976). Impermeable-seed lines

stored under warm, humid conditions (250C, 75% RH) lost their

germination potential slower than their normal-seed isoline. The

impermeable seed lines were considered more "vigorous" even after 10

weeks of this storage treatment (Maxey and Delouche, 1980).

In the most ambitious screening of soybean genotypes for improved

storability, Minor and Paschall (1982) exposed 235 genotypes, of

maturity groups VIII, IX, and X, from the USDA soybean germplasm

collection to adverse storage conditions. Seeds were pretreated with

Thiabendazole and potassium penicillum G suspended in dicloromethane











for control of Phomopsis and B. subtilis in storage. Seeds were

stored in a walk-in chamber maintained at 30C and 80% RH. At the

start of storage and every 2 weeks thereafter seeds were removed,

exposed to 24 hrs of accelerated aging and then germinated. Although

16 of the 235 genotypes exhibited above average storability, only one

genotype exhibited outstanding performance. This genotype was

'Barchett' and only Barchett had any appreciable amount of hard seeds

(approximately 50%). The half-life values (period immediately

preceding and following a 50% loss in initial germination) for all

entries was 6.3 weeks. Barchett's half-life was 15.5 weeks. The

authors concluded that "seed-coat impermeability appears to offer the

most promise for use in the improvement of soybean storability under

conditions of high temperature and relative humidity" (Minor and

Paschall, 1982, p. 138).















CHAPTER III
MATERIALS AND METHODS


Introduction


Impermeable-seed lines used in these studies were developed and

supplied by Dr. Kuell Hinson, USDA-ARS, Gainesville, Florida. Fifteen

F lines were grown from individual F3-plant selections made in 1979.

These 15 lines were first evaluated for the presence of impermeable

seeds in 1980. Impermeable-seed percentage associated with pod

position and harvest method was also evaluated for these lines at this

time (see Appendix C). The 1981 and 1982 field experiments utilized

these lines without further selection; plants within these lines were

in the F5 and F6 generations, respectively. Since selection within

lines ceased at the F4 generation of plants (Dr. K. Hinson, personal

communication), lines are referred to as advanced F4-lines throughout

the text. The lines originated from the cross of D 65-8232 x D 77-

12480. The genetic trait responsible for impermeable seed was carried

in D 65-8232. PI 163453 is the original source of the genetic trait

and is a selection of Glycine soja, a wild relative of Glycine max

(Dr. K. Hinson, personal communication).

All field experiments were carried out at the Agronomy Farm,

University of Florida, Gainesville, Florida. The soil type of the

experimental plots was an Arredondo fine sand (a loamy siliceous

hyperthermic Grossarenic Paleudult). Recommended cultural practices











were followed in each field experiment. Climatological data for 1981

and 1982 presented were recorded at the Agronomy Farm Weather Station.


Response of Impermeable- and Normal-Seeded Genotypes to Field
and Storage Environments


For these experiments the impermeable-seed line used was 8735.

This line was selected because preliminary data from 1980 indicated

that it had a relatively high percentage of impermeable seeds as

compared to the other lines available (see Appendix C). Since no

isogenic normal-seed line was available for comparison, a normal-seed

cultivar, Bossier, was used. Bossier was selected from the normal-

seed cultivars available because it had a reported seed-quality

problem where produced or stored under adverse environmental

conditions (Dr. S. H. West, personal communication). An additional

entry in these experiments was provided by spraying a duplicate plot

of Bossier with the fungicide benomyl (Benelate 50W, courtesy of E. I.

Dupont de Nemours and Co., Inc., Wilmington, Delaware). This

treatment was devised to determine how well seed of an impermeable-

seed line could maintain their germination as compared to a normal-

seed cultivar, which was protected (to some extent) against the

adverse influence of seedborne fungi, but not humidity and rainfall in

a field environment.


Field Experiment


Seeds for these three entries (8735, Bossier, and Bossier-

sprayed) were planted on 15 June 1981. Single-row plots were 6.1 m

long and 0.91 m wide. The experiment was planted in a randomized











complete-block design with three replications. Duplicate plots of

each entry were included in each replication so seed germination could

be evaluated at two separate harvests.

Starting at flowering, every 6.1 m plant row of the Bossier-

sprayed entry was sprayed with 0.625 g of benomyl (Methyl 1-

(butylcarbamoyl)-2-benzimidazolecarbamate) suspended in 500 mL of

distilled water using a 7.6 L stainless-steel hand-pump garden-type

sprayer (Sears and Roebuck Co. Model 7K). Both sides of the plant row

were sprayed with the nozzle directed at the leaves when foliage was

abundant. After defoliation the spray was directed at pods. One

application was sprayed every 3 weeks until maturity. After maturity,

treated rows were sprayed every 2 weeks. Adjacent plots were

protected against possible benomyl drift by placing large cardboard

sheets between rows (cardboard sheets were supplied courtesy of

Storter Printing Co., Gainesville, Florida).

Plants in one-half of the plots were harvested at maturity (95%

of the pods were brown or buff, Fehr and Caviness, 1977). Bossier was

harvested 120 days after planting, Bossier-sprayed was harvested 126

days after planting. Plants within the remaining plots were allowed

to "weather." The second harvest (hereafter referred to as the

delayed harvest) Bossier was harvested 35 days after maturity,

Bossier-sprayed was harvested 33 days after maturity and 8735 was

harvested 30 days after maturity. The differences in days to maturity

and length of time in the field after maturity caused entries to be

exposed to slightly different amounts of rainfall (see Results and

Discussion).












After harvest, all plants were dried in a forced-air dryer for 24

hrs at 350C. Plants were then placed inside a building (under a roof)

until threshed. Preliminary data from 1980 indicated that combine

harvesting reduced impermeable seeds of the impermeable-seed lines

(see Appendix C). Because of the possibility of scarification, all

plants were threshed by striking the plants against the inside of a

round cardboard drum. Intact pods remaining were opened by hand and

the seeds were hand cleaned. This method of threshing is referred to

as "hand-threshed" in the remainder of the text.

Seed quality of entries from both harvests were evaluated 1 month

after harvest by a standard-germination test and an accelerated-aging

test. Impermeable seed percentages were determined approximately 8

months after harvest.


Storage Experiments


Favorable storage


Seeds were stored in a low-temperature low-humidity environment

(15.50C and 30% RH) for 18 months following harvest. The influence of

this storage period on seed quality was evaluated by standard

germination, accelerated aging and emergence tests. Impermeable seeds

were determined at the end of this storage period.


Adverse storage


Three-thousand seeds from entries of both harvests were placed in

open paper bags and stored in an environment of 281C and 90.44.1%











RH. Moisture content of seeds was determined initially, and after 5

and 9 weeks of this storage. Standard germination tests were

performed at the beginning and every 2 weeks thereafter during this

experiment. Impermeable-seeds were determined at the end of the

experiment.

Standard-germination test. All germination tests conformed to

the guidelines of the Association of Official Seed Analysts (AOSA,

1978) except 200 seeds (four samples of 50 seeds) were used from each

plot instead of 400, and the test was concluded at the end of 5 days

instead of 8; consistent procedures were used in the individual

germination tests. Seeds were first surface sterilized by soaking

them in a 1% solution of sodium hypochloride (NaOC1, Fisher Scientific

Co., Fairlawn, New Jersey) for 5 min followed by a 5-min rinse in

deionized or sterilized water (Grybanskas, 1979). Seeds from the

impermeable-seed lines were scarified for the germination test by

inserting a needle through the seed coat on the side opposite the

hilum.

Accelerated-aging test. Four samples of 50 seeds from each plot

were surface sterilized (as described previously) then placed on a

wire-screen supported by plastic tubes in a 150 x 25 mm Petri plate

with 40 mL of deionized water. These plates were then placed in a

chamber at 400C and 100% RH for 60 hrs. At the end of this time,

plates were removed from the chamber, impermeable seeds were

scarified, and a standard germination test performed on the seeds as

described (except no additional surface sterilizations).











Impermeable-seed determinations. Four samples of 25 seeds from

each impermeable-seed plot were weighed and each sample placed in a

separate 150 x 25 mm Petri plate on top of two, 136.5-mm Anchor heavy-

weight paper discs (Anchor Paper, Co., St. Paul, Minnesota). Twelve

milliliters of deionized water were added and the plates were placed

in an incubator, without lights, at 250C. Plates were removed at 2,

4, 8, 24, and 72 hrs and seeds were inspected visually for uptake of

water. A seed was recorded to be impermeable if no swelling of the

seed, in part or whole, occurred. After visual determinations, the

seeds were "patted" dry to remove excess moisture, and the group of

seeds weighed to the nearest 0.01 g.

Emergence test. Seeds from plots of 8735 were scarified, as

described, and 100 seeds from all plots were planted in soil at a 5-cm

depth. A seedling was recorded as emerged when the cotyledons rose

above ground level. Emergence was recorded on all plots at 4, 5, and

8 days after planting.

Seed-moisture determinations. Ten grams of seeds from each plot

were placed in a 1000C oven for 48 hrs. Samples were reweighed and

percent moisture was calculated on a wet-weight basis.


Response of Impermeable- and Normal-Seed Genotypes to
Field Weathering and Seedborne Fungal Infestation


Two impermeable-seed lines were used to compare the germination

of a high impermeable-seed line to a low impermeable-seed line after

field weathering. Line 8731 represented the high impermeable-seed

line because it had a higher impermeable-seed percentage than 8735 in

the 1981 field replicated experiment; and 8745 represented the low










impermeable-seed line because of results from the 1981 field

experiment, and availability of seeds. 'Hardee' was selected to

represent the normal-seed genotype because it's maturity was assumed

to be more closely related to the maturities of the impermeable-seed

lines than Bossier. Hardee-sprayed was included for the same reason

as Bossier-sprayed (as described in the previous section). Seed of

these entries were planted on 4 June 1982. The second field

experiment consisted of four replications instead of three and three

harvests instead of two.

Benomyl was first applied to Hardee 30 days after planting to

reduce fungal organism which "carried-over" from the seed. Spraying

resumed on a 2-week schedule when pods were 1.9 cm long and continued

on a 2-week basis until the plants were harvested. Benomyl was

applied as in the previous experiment. Thus, Hardee-sprayed harvested

at maturity was sprayed five times, Hardee-sprayed harvested 1 month

after maturity was sprayed seven times, and Hardee-sprayed harvested 2

months after maturity was sprayed nine times. Line 8745 matured 136

days after planting, 8731 matured 146 days after planting, Hardee

matured 150 days after planting, and Hardee-sprayed matured 152 days

after planting.

Plants were harvested at maturity, 1 month (except for 8731 and

8745, which were harvested 2 and 6 weeks after maturity,

respectively), and 2 months after maturity. Plants were threshed as

described. Standard germination tests and impermeable-seed

determinations were performed as described.











Recovery of Fungi From Seeds


A "blotter test" technique (Neergaard, 1977; N. C. Schenck,

personal communication) was employed to determine the presence of

seedborne fungi among the different entries. One-hundred seeds of

each plot were first soaked in 1% sodium hypochloride for 5 min

followed by a 5-min rinse in sterilized water (all seeds from 8731 and

8745 were scarified before surface sterilization). Ten seeds were

then placed on two autoclaved 136.5-mm paper discs in decontaminated

150 x 25 mm Petri plates. Fifteen milliliters of sterilized water

with 400 ppm of streptomycin sulfate (Sigma Chemical Co., St. Louis,

Missouri), for control of bacteria, was added to each plate. These

plates were randomized and placed in a walk-in chamber maintained at

250C with a 12-hr daylight regime. Lights were 1 m above plates and

consisted of two fluorescent 40-watt lamps and a Westinghouse 40-watt

sunlamp (Westinghouse Co., Inc., Pittsburgh, Pennsylvania).

Observable mycelium on seeds was recorded at 2 and 4 days after the

bioassay began. At the end of 7 days, seed germination and recovery

of seedborne fungi was recorded for each seed.

An additional bioassay was performed for all plots. The above

procedures were used except a 0.1% solution of 2,4-D (2,4-

dichlorophenoxy) acetic acid (courtesy of Dr. G. H. Teem, University

of Florida, Gainesville, Florida) was employed as the imbibing

solution for the seeds. The separate bioassay was used to obtain a

greater recovery of seedborne fungi, since 2,4-D prevents soybean seed

from germinating.











Impermeable-Seed Separations


To determine seedborne fungi associated with permeable and

impermeable seeds, seeds of 8731 and 8745 were separated in the

following procedure: 1000 seeds of each plot from the first and third

harvest were placed between two sheets of germination paper in a 27.9

x 20.3-cm glass dish. One-hundred milliliters of deionized water were

added to this dish and the excess was drained. Seeds which were

slightly swollen at the end of 2 hrs of exposure to moisture were

classified as "very-permeable". Seeds which had no visible water

uptake at the end of 2 hrs were classified as "slightly-permeable".

Seeds which had no visible water uptake at the end of 24 hrs of

exposure were classified as "non-permeable". A standard germination

test and a fungal bioassay were performed on these seeds as described.


Recovery of Fungi From Embryonic Axes


Fifty embryonic axes (except the plumule) were exised, in the dry

state, from seeds of each entry from the first and third harvests.

Embryonic axes were then surface sterilized with a 2-min soak in 1%

sodium hypochloride and a 1-min rinse in sterilized water. Embryonic

axes were then plated on a 1%-solution of water agar. Fungal

organisms associated with these embryonic axes were recorded after 1

week of incubation at room temperature.











Impermeable-Seed Studies


Field Experiments


Fifteen impermeable-seed lines were grown in a randomized

complete-block design with three replications in 1981 and 1982. These

experiments were planted on 25 June 1981 and 4 June 1982, in 2.44-m

rows with 0.91 m spacing between rows. Plants were harvested at

maturity with one-half of the plants from each plot hand threshed as

described previously. The other half of the plants were threshed

using a combine under normal operating conditions. Seeds were stored

in a low-temperature, low-humidity environment until used.

The percentage of impermeable seeds within each line for each

year and harvest method was determined as follows: 50 seeds from each

plot were placed in a 150 x 25 mm Petri plate on two 136.5-mm

germination paper discs. Twenty-five milliliters of deionized water

were added. Seeds not visibly swollen were recorded after 2, 4, 8,

24, and 72 hrs of incubation and classified as "impermeable."


Seed Permeability Associated with Seed Size


In 1982, nine of the advanced-F4 lines were used to examine the

relationship between seed size and frequency of impermeable seed. All

seeds from each plot plus seed from Hardee and Bossier were separated

into seed size classes by the use of round-holed screens. Only three

of the possible six seed sizes occurred in sufficient numbers in all

plots to be used for analysis. Seeds passing through a 6.35-mm screen

yet retained on a 5.95-mm screen were designated size 6.35 mm; seeds











passing through a 5.95-mm screen yet retained on a 5.56-mm screen were

designated 5.95 mm; seeds passing through a 5.56-mm screen yet

retained on a 5.16-mm screen were designated 5.56 mm. Seeds were

screened gently to avoid scarification. Four samples of 25 seeds from

each size were placed in a 150 x 25 mm Petri dish on top of two 136.5

mm diameter heavy-weight germination paper discs. Twelve milliliters

of double-deionized water were added to the plate and the plates were

placed in an incubator, without lights, at 250C. Impermeable seeds

were recorded after 2, 4, 8, 24, and 72 hrs of incubation as described

previously. This experiment was repeated again in 1983 for lines

8744, 8738, and 8733 grown in 1982. Frequency distributions of the

different seed sizes within lines were obtained prior to the

impermeable-seed determinations by screening 600 seeds per plot.


Seed-Coat Weights


Six impermeable-seed lines were studied to determine if the

weight of the seed coat differed between known impermeable seeds and

seeds with high permeability. Four samples of 25 seeds (known to be

impermeable after a 72-hr exposure to moisture) from three lines

(8732, 8740, and 8745) were used to represent impermeable seeds. Four

samples of 25 seeds from lines with low impermeable-seed percentages

(after 72 hrs of exposure to moisture) were used to represent

permeable seeds. Seed from lots of 8737 (6% impermeable seeds), 8743

(3% impermeable seeds), and 8738 (0% impermeable seeds) were used.

Initial seed weights were taken after drying seed in oven at 45C for

24 hrs.










After weighing, seeds were cut and placed in 20 mL distilled

water for 30 min to soften the seed coat for easy removal. Seed coats

were removed and then redried for 24 hrs at 45 C. Seed coats were

then weighed and percentage of total seed weight calculated.


Impermeable Seed Longevity Study


One-hundred seeds of each major seed class (5.16, 5.56, 5.95 mm)

from hand-threshed and combine-threshed subplots were used from four

lines (8732, 8736, 8740, 8743). Fifty seeds were placed in each 150 x

25 mm Petri plate with two Anchor paper 136.5-mm germination pads

(Anchor Paper Co., St. Paul, Minnesota). Twenty-five milliliters of

deionized water were added and approximately 20 mL of water added per

week thereafter, for 4 weeks, to each plate. Impermeable seeds were

determined approximately every 2 days for 30 days. Because of the

high variability among replications, the data are not discussed and

data are presented in Appendix B.


Statistical Analysis


All percentage data were first transformed using the arcsine

square-root transformation as proposed by Barlett (1947) and Steele

and Torrie (1960). Only transformed percentage data were used for

analysis.

Data, when applicable, were analyzed using the analysis of

variance. Significant differences between means were determined by

the Duncan's Multiple Range Test, and the Waller-Duncan k-ratio t

test. When differences were compared between two means, Tukey's

honestly significant difference (HSD) procedure was used.















CHAPTER IV
RESULTS AND DISCUSSION


Response of Impermeable- and Normal-Seed Genotypes to Adverse
Field, and Favorable and Adverse Storage Environments


The purpose of these experiments was to ascertain the ability of

seed from a soybean line, 8735, which had the impermeable seed-coat

trait, to retain viability under stress and non-stress environments,

as compared with seeds of a normal-seed soybean cultivar. The stress

environments included a delayed harvest or "weathering" period of 1

month past harvest maturity, and a moderate-temperature, high-humidity

environment referred to as "adverse storage." The non-stress

environment was a low-temperature, low-humidity storage environment

referred to as "favorable storage." Line 8735 along with two

treatments of the cultivar, Bossier, unsprayed and sprayed with

benomyl (hereafter referred to as Bossier and Bossier-sprayed), were

used in these experiments and are referred to as "entries" during the

course of the discussion. A secondary purpose was to determine the

effect that different environments may have on seed permeability.


Results of the Field Experiment


When seeds were harvested at harvest maturity, the mean normal

and total germination for entries were 86 and 98%, respectively.

Normal and total germination for seeds of Bossier and Bossier-sprayed

were significantly higher than for seeds of 8735 (Table 1). This














Table 1. Viability of seeds harvested at maturity and at one month
after maturity.



Harvest Delayed
Entry maturity harvest

% germination

Normal

Bossier 87.4 a At 46.6 b B

Bossier-sprayed 89.9 a A 72.5 a B

8735 80.3 b A 66.6 a B

Total

Bossier 98.6 b A 64.8 c B

Bossier-sprayed 99.7 a A 90.3 b B

8735 97.2 b A 93.8 a B


tMeans within a column followed by the same lower case letter are
not significantly different according to the Waller-Duncan k-ratio
t test, a = 0.05, k = 100. Means within a row followed by the same
upper case letter are not significantly different according to the
Tukey's HSD methods.











difference between entries may be due more to the nature of the

impermeable seed coat rather than a lower viability. Seeds of 8735,

during germination, were observed to have a slower rate of water

uptake and radicle protrusion than seeds of Bossier. The restrictive

nature of this seed coat may have resulted in a seedling becoming

abnormal or even dead. There was not a significant difference in

germination between seeds of Bossier and Bossier-sprayed at this first

harvest. The application of benomyl appeared to have no effect. The

effectiveness of a fungicide in preventing seed deterioration is due

to a number of factors including the level of disease pressure

(Sinclair, 1976). The environment prior to the first harvest was not

suitable for a serious disease problem to occur, and hence no increase

in viability for seeds receiving foliar-applied benomyl.

Normal and total germination of seeds from the delayed harvest

were significantly lower than for seeds at harvest maturity (Table 1).

The mean normal and total germination of seeds after 1 month of

delayed harvest were 62 and 86%, respectively. The 1 month period of

delayed harvest decreased normal germination more than total

germination. The greater decrease in normal germination was due to a

greater increase in abnormal seedlings than dead seeds, and is

indicative of the seed deterioration process (Delouche and Baskins,

1973). Results from delayed harvest demonstrate that soybean seed

viability was reduced when seeds were allowed to "weather" under the

delayed-harvest environment. The delayed-harvest environment was

characterized by rainfall five times higher than for the same time

period prior to harvest maturity (Table 2).















Table 2. Rainfall occurring before and after harvest maturity during
the field experiments.



Rainfall from
Rainfall during harvest maturity Total
the 30 days before to delayed rainfall during
Entry harvest maturity harvest periods


Bossier 20.6 123.9 144.5

Bossier-sprayed 20.6 123.9 144.5

8735Bossier-sprayed 20.6 123.9 144.5
8735 19.3 107.4 126.7










Bossier seeds were significantly lower in germination, after the

1-month delayed-harvest period, than Bossier-sprayed or 8735 seeds.

Bossier seeds also had the greatest decline in germination from

harvest maturity to delayed harvest. Seeds of 8735 were not

significantly different from Bossier-sprayed in germination.

Evidently, seeds from the impermeable seed-coat line did not maintain

viability any better than seeds from a normal-seed cultivar receiving

benomyl application.

The results of benomyl application to retard seed deterioration

under delayed harvest conditions agrees with other reports (Ellis and

Sinclair, 1976). The importance of fungi in causing seed decay is

reaffirmed by the differences in germination between Bossier and

Bossier-sprayed. In order for seeds of 8735 to have retained their

viability they must have also been able to prevent fungal seed decay.

Impermeable seed-coat lines have a lower incidence of fungal-infected

seed than normal-seed cultivars under conditions of delayed harvest

(Potts, 1978).

The comparison between seeds having the impermeable trait and

normal seeds sprayed with fungicide was performed to determine the

better seed-quality entry. Although worth consideration, this

experiment may not permit fair comparisons between these entries

because line 8735 differs from Bossier in characteristics other than

the impermeable seed-coat trait. These characteristics may have at

least some bearing on the rate of deterioration. For example, because

of differences in maturity, 8735 was exposed to slightly lower

rainfall (17 mm) as compared to Bossier-sprayed (Table 2). This might











have caused a more deleterious environment for Bossier-sprayed than

8735 seeds.

The ability of the impermeable seed-coat trait to retain seed

viability under the adverse influence of a delayed-harvest environment

is not fully realized. Not only were there differences among seeds of

8735 in permeability, but there were differences among impermeable

seeds in the degree of permeability (Table 3). There was an increased

percentage of permeable seeds within 8735 as the seeds were exposed to

increased soaking periods. These differences in permeability of 8735

seeds may influence the rate of deterioration of individual seeds.

Seeds that remain impermeable for 72 hrs may be better able to retain

their viability than seeds which are impermeable for only 2 hrs of

soaking. The gradual decrease in the percentage of impermeable seeds

within a seed lot has been reported (Kilen and Hartwig, 1978). The

cause is not known, but there is a possibility that some seeds

gradually absorb moisture. Over an extended period of time this

moisture can build-up and the swelling of the cotyledons causes a

break in the seed coat. Once this break occurs, the flow of water

into the seed can cause visual swelling.

Another problem in evaluating the ability of the impermeable

seed-coat trait to retain seed viability is an increased permeability

of 8735 seeds due to the delayed-harvest environment. The percentage

of impermeable seeds, within 8735, decreased significantly during

delayed harvest (Table 3). The cause of this decrease is unknown.

Factors in the field environment such as insects, microorganisms, or

rainfall may be responsible. These factors may render impermeable














Table 3. The influence of delayed harvest on seed permeability of
8735 (visual observations).



Time of Time of soak (hrs)
harvest 2 4 8 24 72

X impermeable seed

Harvest
maturity 92.3 at 89.3 a 82.7 a 55.0 a 49.3 a

1 month
later 72.7 b 62.7 b 45.3 b 36.0 b 30.7 b


tMeans within a column followed by different
different at 0.01 probabiity level.


letters are significantly










seeds permeable by causing small breaks in the seed coat. If the

decline in impermeable seeds did not occur during the delayed harvest

period, then the germination of 8735 seeds may have been higher (Table

1). The increase in seed permeability is confirmed by an increase in

water uptake of 8735 seeds (as measured by weight). Weights for seeds

from the delayed harvest were significantly higher than at harvest

maturity after 8 and 24 hrs of soaking (Table 4). The more permeable

seeds were expected to absorb more moisture and thus have a greater

weight after soaking.

Seeds at harvest maturity had a significantly higher normal

germination, 38.7%, than the delay-harvested seeds, 20.9%, after

accelerated-aging. Among entries, normal germination was

significantly higher for seeds of 8735 than either Bossier or Bossier-

sprayed seeds (Table 5). The lack of significant entry x harvest

interaction in the analysis of variance indicated that 8735 seeds

withstood the stress of accelerated aging better than Bossier.

Although not significantly different than Bossier-sprayed in

germination, accelerated aging results may indicate that seeds of 8735

had better vigor. However, seeds of 8735 were not scarified before

this test began; so they probably absorbed less moisture during

accelerated aging than Bossier. Because of less moisture absorption,

seeds of 8735 avoided the stress of accelerated aging. Therefore, for

comparisons among entries in vigor, the accelerated aging test may not

provide a true estimation of the vigor potential of 8735 relative to

the vigor potential of Bossier.












48











O .0 60



0 )
m o






.H I ar) H
(V 0 C0


























a)
3 a)






Co
W Co
Q u

'- 3- o n 14 1
SCO
.)t 0 --t cO




z o

















-4a
00 p
i co





o a .1
a) n aI 0 U)



O C4O1 0 0
SI 0 0 Ic





























0> > o
a) -4 41 )c








co m coz t 2:





p x = -e 04-
io a) T-

o Cll





iild Ui








-: 44 0






-^ a















Table 5. Seed viability after accelerated aging for seeds harvested
at maturity and 1 month after maturity.


Across harvests


% germination

Normal


Bossier


Bossier-sprayed


8735


Bossier

Bossier-sprayed


8735


21.2 b

22.3 b

45.7 a

Total


Harvest
maturity

83.0 b A

84.3 b A

92.0 a A


Delayed
harvest

32.5 c B

45.2 b B

84.7 a B


tMeans within a column followed by the same lower case letter are
not significantly different according to the Waller-Duncan k-ratio
t test, a = 0.05, k = 100. Means within a row followed by the same
upper case letter are not significantly different according to the
Tukey's HSD methods.


Entry











Results of the Storage Experiments


Favorable storage


A standard germination test conducted after 5 months of storage

(Table 6) revealed that significant differences in seed viability

among entries within harvests, and between harvests continued to exist

as those reported from the first germination test (Table 1). The only

exception was that total germination of Bossier-sprayed seeds was not

significantly different than 8735.

To determine the influence of a prolonged favorable-storage

period on seed viability another standard-germination test was

conducted after 18 months of storage and results compared to those

obtained after 5 months. Comparisons between tests reveal that there

was a significant increase in seed viability for entries from the

delayed harvest during the 13-month favorable-storage period (Table

6). Normal and total germination for seeds from the delayed harvest

increased by 15.1 and 7.4%, respectively. A significant increase was

not detected for seeds at harvest maturity. Although viability was

still significantly higher for seeds at harvest maturity than for

delayed-harvested seeds, germination percentages among entries from

the delayed harvest increased such that significant differences were

not detected.

The reason for the increase in normal and total germination for

seeds from the delayed harvest, over this storage period, may be due

to a decrease in the virulence of seedborne fungi. Seedborne fungi

originating from field infection dehydrate and become inactive in an




















CO

n m









co o0










CN u~l





Coo r


Al
0





4J:
0






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o u















0 \C


I-I
o\ a\






-< 0
0" 0


S41



0
u w



c4.J









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a q
0 0
SL"




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0 r























01 0
M0 0
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W c 4-
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52

environment with a relative humidity of less than 95% (Christensen,

1973). If these fungi are exposed to long periods of dehydration they

lose their ability to rehydrate. Since fungi can no longer cause

damage during germination, seeds become viable (Neergaard, 1977).

This phenomenon is true for soybeans infected by Phomopsis sp. Wallen

and Seaman (1963) reported that the percentage of Phomopsis-infected

seeds decreased from 73 to 40% in the first year, and decreased to 4%

by the end of the second, when seeds were stored under cool, dry

conditions. Seed germination increased, over these 2 years, from 20

to 96%.

The theory that this long storage period adversely affected the

virulence of the seedborne fungi and increased germination rates of

seeds is given credibility by examining which entries from which

harvests increased the most in germination. Seeds from entries most

severely infected by seedborne fungi show the greatest increase in

germination after storage. Seeds of Bossier from the delayed harvest

increased significantly in normal germination from 54.8 to 78% (Table

6) during this year of storage. The corresponding increase for seeds

of Bossier sprayed from 78 to 85.3% (Table 6) was not significant.

Seeds of Bossier should have more seedborne fungi than seeds of

Bossier-sprayed, because application of benomyl decreases seedborne

fungi (Ellis et al., 1974; Ellis and Sinclair, 1976). Across entries,

seeds from the delayed harvest had a significant increase in

viability, while seeds from the first harvest (Table 6) did not. Once

again, seeds from the delayed harvest would be expected to have more

virulent fungi than seeds at maturity (Ellis and Sinclair, 1978;

Wilcox et al., 1974).











Unlike results from the germination test, seeds of Bossier and

Bossier-sprayed had significantly lower normal and total germination

after accelerated aging, and lower emergence than seeds of 8735 after

18 months of favorable storage. Also, germination, after accelerated

aging, and emergence for 8735 seeds did not differ significantly

between harvests, whereas, Bossier and Bossier-sprayed did.

Evidently, the germination test for seeds of entries from the second

harvest did not give an adequate indication of their respective

vigors, or these stress tests may have favored impermeable seeds.


Adverse Storage


Normal germination for seeds of the normal-seed entries, from

either harvest, increased during the first 2 weeks of adverse-storage

period whereas normal germination for seeds of 8735 did not. This is

reflected in a significantly higher normal germination for seeds of

Bossier and Bossier-sprayed than seeds of 8735, within their

respective harvests, after 2 weeks of adverse storage (Fig. 1). These

differences were not detected at 0 weeks. The cause of this increase

may be due to a "priming" or conditioning effect caused by the initial

exposure to high relative humidity. This effect has also been

observed after short durations of accelerated aging of cotton seed

(Bourland and Ibrahim, 1982) and soybean seed (S. H. West, personal

communication). Total germination of the normal-seeded entries also

increased, but not as much as normal germination (Fig. 2).

Seeds of Bossier and Bossier-sprayed from the delayed harvest

declined in normal and total germination faster than seeds from




































Fig. 1. Normal germination for seeds of entries after
various weeks of adverse storage. H-l, harvested
at harvest maturity; H-2, harvested 1 month later
(delayed harvest).
tMeans within a column followed by the same letter
are not significantly different according to the
Duncan-Waller k-ratio t test, a = 0.05, k = 100.


























S.d \\ \
I ,\ \ o
z 60 a

LUI
( 50- \ \ b\

\\ \ \
Treatment Symbol 4b
40- b
40 Bossier H-l A---A b
O Bossier H-2 --- \ \
Z Bossier-sp H-1 A----IA
Bossier-sp H-2 i-----.
0- 30 8735 H- \
8735 H-2 \ \ \

\\ \ \\ \
20
\ \ ,

10-\ \
0 \ \\
Sd --.
0 I 2 3 4 5 6 7 8 9 10 11

TIME IN ADVERSE STORAGE (WEEKS)




































Fig. 2. Total germination for seeds of entries after various
weeks of adverse storage. H-1, harvested at harvest
maturity, H-2, harvested 1 month later (delayed
harvest).
tMeans within a column followed by the same letter
are not significantly different according to the
Duncan-Waller k-ratio t test, a = 0.05, k = 100.




















N\ \


100-


90-


80-


70


60-


50-


40-


30-


20


I0-


0-


Symbol
A----A


A-- -A
.--


\ \ \
\ \ \ b

\ \

\ \ \
\ \


\\ \

\\ \\
\ \d \.
\\ \\

\d \\
S cd


- I I I I


2 3 4 5 6 7 8
TIME IN ADVERSE STORAGE
(weeks)


Treatment
Bossier H-I
Bossier H-2
Bossier-sp-H-I
Bossier-sp-H-2
8735 H-I
8735 H-2


9 10 II











harvest maturity. The faster decrease in seed germination of the

normal-seeded entries from the delayed harvest as compared to seed

from harvest maturity is consistent with expectations of performance

in the adverse environment. Results from accelerated aging and

emergence tests (Table 7) indicated seed of the normal-seed entries

from delayed harvest had a lower vigor than seeds from harvest

maturity. Since the vigor of a seed lot is an important consideration

in determining longevity of that seed lot in storage (Delouche et al.,

1973; Delouche and Baskin, 1973), it would be expected that a

comparatively "weaker" seed lot would deteriorate first under

conditions of adverse storage (Delouche et al., 1973).

Normal and total germination for seeds of Bossier and Bossier-

sprayed decreased rapidly after 5 weeks of adverse storage (Figs. 1

and 2). Within their respective harvests, seeds of Bossier-sprayed

maintained a germination longer than Bossier. This result indicated

that the application of benomyl in the field increased seed quality

that viability was retained better than the unsprayed seed, for a

limited time period.

Seeds of 8735 had a slower decrease in normal and total

germination than Bossier or Bossier-sprayed during the course of this

experiment. By the seventh week, seeds of 8735 from the delayed

harvest had a significantly higher normal and total germination than

Bossier Pnd Bossier-sprayed from the delayed harvest. By the eleventh

week, seeds of 8735, from the delayed harvest, had a significantly

higher normal and total germination than Bossier and Bossier-sprayed,

from harvest maturity. Seeds of 8735 from harvest maturity had a















Table 7. Response of seeds harvested at maturity and at
1 month after maturity to accelerated aging and
emergence tests after 18 months of favorable storage.



Harvest Delayed
Entry maturity harvest


Accelerated aging
% normal germination

Bossier 83.7 a A+ 47.5 b B

Bossier-sprayed 86.8 a A 63.9 b B

8735 80.7 a A 81.7 a A

% Emergence

Bossier 89.0 a A 65.7 b B

Bossier-sprayed 84.0 a A 64.0 b B

8735 85.0 a A 81.0 a A


tMeans within a column followed by the same lower case letter are
not significantly different according to the Waller-Duncan k-ratio
t test, a = 0.05, k = 100. Means within a row followed by the same
upper case letter are not significantly different according to the
Tukey's HSD methods.










significantly higher normal and total germination than Bossier and

Bossier-sprayed from harvest maturity by the ninth week of this

experiment. At the end of 11 weeks, the normal and total germination

of Bossier and Bossier-sprayed from either harvest had decreased to

near 0%. Seeds of 8735 from the first and second harvests had normal

germinations of 57 and 41%, and total germinations of 73 and 52%,

respectively. Clearly, seeds of 8735 retained viability better than

the normal-seed entries. One reason that viability of 8735 seeds was

preserved was by the seeds absorbing significantly less moisture than

the normal seed cultivars in this adverse environment (Table 8).

Reduced moisture in seeds meant less physiological damage (Delouche,

1982) and less activity by storage microorganisms (Dorworth and

Christensen, 1968; Christensen, 1973).

Initially, there were no significant differences in normal or

total germination for seeds of 8735 from the different harvests (Figs.

1 and 2). However, from the second week on, seeds at harvest maturity

had significantly higher normal and total germinations than seeds from

the delayed harvest. The reason for this may be due to seeds of 8735,

from harvest maturity having a significantly higher percentage of

impermeable seeds than seeds from the delayed harvest (Table 3). The

higher the percentage of impermeable seeds within a seed lot, the

higher the germination.

The influence of the favorable, and the favorable followed by the

adverse-storage environment on the percentage of impermeable seeds

within 8735 is presented in Fig. 3. Means presented are averaged

across harvests. The percentage of impermeable seeds was















Table 8. Moisture content (wet weight basis) of seeds from different
harvests after various weeks in adverse storage.



Time of Weeks in adverse storage
Entry harvest 0 5 9

% moisture

Bossier Harvest maturity 7.3 at 17.8 a 18.0 a

1 month later 7.5 a 17.6 a 18.1 a

Bossier- Harvest maturity 7.1 a 17.6 a 18.8 a
sprayed
1 month later 7.5 a 17.8 a 17.8 a

8735 Harvest maturity 7.3 a 12.0 b 13.6 b

1 month later 7.1 a 13.6 b 14.7 b


tMeans within a column followed by the same letter are not signifi-
cantly different according to the Duncan-Waller k-ratio t test,
a = 0.05, k = 100.
























80-
a


70-









50
a
%


40- -- ------- -- a
40- Ac c b
3...........0


30:,


0 10 20 30 40
TIME OF EXPOSURE TO
(hours)


MO 60
MOISTURE


Fig. 3. Permeability of 8735 seeds after different storage periods
in different storage environments after various periods
of soaking (visual observations). Imnermeable seeds
after 8 months of favorabel storage (*---), Impermeable
seeds after 20 months of favorable storage (m---n),
Impermeable seeds after 17 months of favorable storage
plus 3 months of adverse storage (A-,-A).


I











significantly reduced after a 12 month period of favorable storage for

seeds impermeable after 2, 4, and 8 hrs of soaking. The percentage of

impermeable seeds was reduced even more by a 3 month period of adverse

storage. In the latter situation, permeability of seeds increased

significantly after every soaking period. These results indicate that

the adverse storage period of 3 months increased permeability more

than the 12-month period of favorable storage.

The significant reduction in impermeable seeds after 2, 4, and 8

hrs of soaking for the 20-month favorable storage period as compared

to the 8-month favorable storage period may be due to the influence of

time. Over time, the impermeable barrier may deteriorate even under

these moderate conditions.

One reason for the decrease in impermeable seeds after adverse

storage may be the high relative humidity of 90%. There is a distinct

possibility that no seed is completely impermeable to moisture if the

seed is in contact with free water for a long enough period of time.

Both of these storage treatments affected impermeable seeds which

could be termed "slightly-permeable", i.e., seeds which remained

impermeable for 2, 4, or 8 hrs of soaking, more than seeds which could

be termed "non-permeable", i.e., seeds which remained impermeable 24

or 72 hrs. At the end of the adverse storage experiment seeds which

were impermeable for only a short soaking were nearly exhausted, only

the seeds which were impermeable to soaking for 72 hrs remained. The

effect of storage periods on permeability of soybean seeds has not

been well documented. Minor and Paschal (1982) noticed a gradual

decrease in impermeable seeds over a storage period of 16 weeks with











80% RH and 300C. However, they did not separate impermeable seeds

into different classes of permeability so there is no way of assessing

what classes of seed permeability were affected by their adverse

storage environment.

Significant differences in weights for seeds from the different

storage environments, over soaking periods confirm results attained by

visual inspection for permeability (Table 9). Significant differences

between storage environments were first detected after only 2 hrs of

soaking. These differences were between seeds from the favorable

storage environments and seeds from the adverse storage environment.

This would be expected from Fig. 3 which indicated the largest

differences in impermeable seeds were among these same comparisons.

After 4 hrs of soaking, seeds from each storage environment were

significantly different in their weights. Again, this would be

expected since Fig. 3 indicates that seeds from each storage

environment differed significantly in the percentage of impermeable

seeds after 2, 4, and 8 hrs of soaking.

The results of the field and storage experiments indicate that

seeds from an impermeable seed-coat line retain their viability better

than seeds from a normal-seed cultivar under adverse field and storage

conditions. Several papers have discussed environmental factors that

are associated with seed deterioration in the field and in storage

(Andrews, 1982; Christensen, 1973; Delouche, 1980, 1982). These

papers all emphasize that temperature, relative humidity, and

microorganisms are the most important factors. To prevent seed

deterioration, these conditions need to be avoided. The impermeable




















C3 CU 0)
S0 I I t < a



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- (3 \ N cU
(N C-0 r ff L3








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m 1-oi


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O cc cc t hi m



a U 0c



w a) '.0 -4
C *
0 -i 0I -M )C


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0) 1-t
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) -HO




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cc t> 0t
m o o r*l r* ro O











S4
400i4-1












0 0 cc ti -

m (im -0 tim
E1 Q). 1-I
4-1




0 O) 0

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o U U t
S0 C r
nU) 0 o r- 110

"o oCU R 1H
S4-1 Ic > j
-H c 0 J ~ i
6 a) a) 1 1 -1







0) WE4
Wi 0 E o)

O b U 4 0 -4

4J -H 4a ) -4


4-i 4-0 ca o ":

CU) En O 3 -






WuE eCU








cU 4 C CU CU cC S:
EicJ) t &] i











seed-coat trait, by definition, prevents moisture reabsorption. This

trait may also avoid the adverse effects of microorganisms either by

the prevention of infection (Potts, 1978) or by reducing their

activity due to a low internal relative humidity (Christensen, 1973).

Because these factors are avoided, seed deterioration is reduced. The

reason why seed viability was not preserved at a more desirable level

may be due to the presence of permeable seeds within 8735, and loss of

impermeable seeds under an adverse environment. The significantly

higher impermeable-seed percentage and normal germination for seeds of

8735 from the first harvest compared to seeds of the second harvest

indicates that after adverse storage only impermeable seeds will have

preserved their viability, not just any seed from an impermeable seed-

coat line.

Favorable storage (i.e., low relative humidities and low

temperatures), over the period studied, was not an adverse environment

so benefits of the impermeable seed-coat trait were not realized.

Because of this, farmers who have adequate climate control for their

seed storage may not choose to use seeds with this trait.

Seeds from an impermeable-seed line were not any better than

seeds from a crop continually sprayed with benomyl in preserving their

seed quality under delayed-harveest conditions. Because of the

production problem of scarification, farmers may want to avoid

planting seed with the impermeable seed-coat trait and use benomyl

instead, if a delay in harvest of soybeans is a common occurence.

However, the application of benomyl on a soybean crop (applied before

harvest maturity and during delayed harvest) may well increase











cost of seed production beyond economic feasibility. An estimate of

$23/ha per application (material plus application costs) for benomyl

(Dr. Cliff Hiebsch, personal communication) would make the cost of the

benomyl treatment in this experiment approximately $138/ha. If

benomyl and motorized-application equipment were available in tropical

countries, these costs would double to $276/ha (Dr. Cliff Hiebsch,

personal communication). In situations where a fungicide is not

available and there are also storage problems involved with the crop,

the impermeable seed-coat trait may offer an acceptable solution to

the problem of poor-quality planting seed caused by adverse

environmental influences.


Response of Impermeable- and Normal-seed Genotypes to Field
Weathering and Seedborne Fungal Infection


The primary purpose of this field experiment was to look

specifically at the quantity of seeds associated with fungi during

delayed harvest for both impermeable- and normal-seed genotypes

(hereafter referred to as entries). Since the previous field

experiment indicated that the impermeable seed decreased under

delayed-harvest conditions, a secondary purpose was to determine the

viability and fungal infection of seeds, within the impermeable-seed

lines which remained impermeable during delayed harvest. The

assumption tested was if a seed managed to remain impermeable then

seedborne fungi would not be recovered from it.










Germination and Incidence of Seedborne Fungi After Delayed Harvest of
Seeds from Impermeable- and Normal-Seeded Entries


When harvested at maturity, seeds of 8745 were significantly

lower in normal and total germination than seeds of the other entries

(Table 10). This may have been caused by the different environments

in which seeds of 8745 and the other entries developed. Rainfall for

the month prior to maturity of 8745 was 49% higher than for the month

prior to maturity of the other entries (Table 11). Since the

impermeable seed-coat trait is not expressed until near maturity

(Duangpatra, 1976), seeds of 8745 had no protection against this

environment. Rainfall during the month prior to maturity for the

other entries was not sufficient to affect seed viability. This is

why the application of benomyl had no effect on seed viability of

Hardee-sprayed as compared to the unsprayed Hardee (Table 10).

Comparisons among entries in seed viability from the second

harvest will not be made because harvesting occurred at different

periods after maturity. Results from the second harvest will be

presented to show the initial seed response to delayed harvest. From

the first to the second harvest, seed viability did not decline

significantly for seeds of Hardee, Hardee-sprayed, or 8731. This may

have been due to insufficient rainfall. Seeds of Hardee were exposed

to only 42 mm of rainfall (Table 11) as compared to seeds of Bossier

which were exposed to 127 mm (Table 2) the previous year (Table 1).

Results from the standard-germination test revealed that in 2

months of delayed harvest seed viability declined significantly for

all entries. Normal germination of Hardee, Hardee-sprayed and 8731















Table 10.


Viability of seeds harvested at maturity and at times
after maturity.


Delayed harvest
Entries Harvest maturity 1 2

% germination

Normal

Hardee 77.0 a AS 70.0 A 12.4 b B

Hardee-sprayed 79.4 a A 81.6 A 30.4 a B

8731 82.6 a A 82.1 A 26.2 a B

8745 55.7 b A 30.5 B 18.5 b C

Total

Hardee 97.9 a A 88.0 A 27.1 c B

Hardee-sprayed 96.5 a A 95.6 A 54.5 A B

8731 94.7 a A 91.6 A 43.2 abB

8745 83.0 b A 48.5 B 33.4 bcB


tDelayed harvest-i was 1 month after harvest maturity for Hardee and
Hardee-sprayed, and 2 and 6 weeks for 8731 and 8745, respectively.
Delayed harvest-2, 2 months after harvest maturity for all entries.
tMeans within a column followed by the same lower case letter are
not significantly different according to the Waller-Duncan k-ratio
t test, a = 0.05, k = 100. Means within a row followed by the same
upper case letter are not significantly different according to the
Tukey's HSD methods.





























0
CO 0




U 4-4
0 o
UC.,

1-1

co
C,4 -1
to


>N m
4-1 e
o-i co

5 -
41



CO

0)
cti

4

nj a3


-I






0 ca
(u


*d



C0 0
-l 4-


c* a)
prf lu











declined by 87, 62, and 56%, respectively. Total germination declined

by 72, 43, and 54%, respectively. Seeds of 8745 which were exposed to

35% more rainfall than the other entries declined in normal and total

germination by 75 and 60%, respectively. Similar to the results for

the previous field experiment (Table 1), normal more than total

germination was affected by delayed harvest.

Impermeable-seed percentages were significantly higher in seeds

of 8731 than for 8745 as revealed by visual observations in the

soaking test (Table 12), but differences were not large. The

percentage of impermeable seeds across lines decreased significantly

over the 2 month delayed-harvest period. Increased seed weights

during the soaking test confirm the increase in permeable seeds for

8731 and 8745 (Table 13).

Although seed viability was low in both impermeable-seed lines

after 2 months of delayed harvest, there was still a significant and

highly positive correlation between the percentage of impermeable

seeds and normal germination at each delayed-harvest period for 8731

and 8745 seeds (Table 14). The high correlation between the

percentage of impermeable seeds after 4 hrs of exposure to moisture

and normal germination (r = 0.92**), indicates that seeds which were

only slightly permeable retained their viability. The high initial

correlation followed by a gradual decline in correlation coefficients

(as the periods of soaking increases) indicates that seeds that are

very impermeable to moisture, i.e., impermeable after 24 or 72 hrs of

soaking are not all of the seeds that germinate normally.

When the percentage of impermeable seeds at harvest maturity is

correlated with normal germination of seeds after 2 months of delayed
















Table 12.


The influence of line and delayed harvest on seed
permeability after soaking for various time periods.


Time of soak (hrs)
Line 2 4 8 24 72

% impermeable seed

8731 62.7 at 57.6 a 52.0 a 36.7 a 31.5 a

8745 59.2 a 50.4 b 46.1 a 29.2 b 25.0 b



Time of
harvest

Harvest
maturity 83.0 a 79.0 a 71.4 a 41.2 a 32.4 a

2 months
later 39.0 b 29.0 b 26.7 b 24.7 b 24.1 b


tMeans within a column followed by the same letter are not signifi-
cantly different at the 0.05 probability level.
















Table 13.


The effect of delayed harvest on permeability of 8731 and
8745 seeds after soaking for various time periods (weight
measurements).


Time of Time of soak (hrs)
Harvest 2 4 8 24 72

wt/100 seeds (g)

Harvest
maturity 13.09 at 13.28 a 13.65 a 14.28 a 16.81 a

2 months
later 12.77 a 13.77 b 14.47 b 15.38 b 18.40 b


tMeans within a column followed by the same letter are not signifi-
cantly different at the 0.05 probability level.
















Table 14.


Correlations for impermeable seed percentages after
various soaking times with normal germination after
delayed harvests for seeds of 8731 and 8745.


Time of soak (hrs)
2 4 8 24 72

r

Normal
germination .84** .92** .89* .49 .29


*,-*Significant at the 0.05 and 0.01 probability levels, respectively.












harvest, a different association is significant (Table 15). The

significant, although weaker, correlation coefficients suggest that

only seeds which are very impermeable, i.e., impermeable after 24 or

72 hrs of exposure to moisture, may germinate normally after the

delayed-harvest stress.

The quantity of fungi associated with seeds from the different

entries at harvest maturity and after periods of delayed harvest was

determined using a standard blotter test technique. This test is

similar to a germination test except that conditions were modified so

fungi which inhabit the seeds will grow and sporulate. Sporulation is

a necessary requirement for identification. This test measures both

the growth of the organism and the seedling so that the association of

the organism with the germination process can be measured (Neergaard,

1977). A separate bioassay was performed with the addition of 2,4-D

in the imbibing solution. However, the seeds from the second delayed

harvest were so heavily infected with bacteria that fungal growth was

severely repressed. Results of this bioassay are presented in

Appendix A.

Significant differences in normal and total germination among

entries from harvest maturity were similar in the blotter test (Table

16) to those differences reported from the standard-germination test

(Table 10). One striking difference between germination percentages

reported was that germination was lower in the blotter test than in

the germination test for each entry x harvest combination. Evidently,

the more favorable conditions for growth and development of the fungi

in the blotter test, affected adversely the germination process of the

seeds.
















Table 15.


Correlations between the percentage of impermeable seeds
at harvest maturity, after various soaking periods, with
normal germination after 2 months of delayed harvest.


Time of soak (hrs)
2 4 8 24 72

r

Normal
germination .65 .52 .56 .72* .75*


*Significant at the 0.05 probability level.











Table 16.


Viability of seeds harvested at maturity and at times
after maturity in the blotter test.


Delayed harvestt
Entries Harvest maturity 1 2

% germination

Normal

Hardee 66.0 a At 51.0 A 8.0 c B

Hardee-sprayed 72.0 a A 56.0 A 24.5 abB

8731 75.0 a A 62.0 A 30.5 a B

8745 39.5 b A 21.5 B 13.5 bcB

Total

Hardee 90.5 a A 62.7 B 17.2 b C

Hardee-sprayed 89.5 a A 76.2 B 45.5 a C

8731 92.3 a A 84.2 A 35.5 a B

8745 62.5 b A 36.7 B 19.2 b C

tDelayed harvest-1, 1 month after harvest maturity for Hardee and
Hardee-sprayed, and 2 and 6 weeks for 8731 and 8745, respectively.
Delayed harvest-2, 2 months after harvest maturity for all entries.
SMeans within a column followed by the same lower case letter are
not significantly different according to the Waller-Duncan k-ratio
t test, a = 0.05, k = 100. Means within a row followed by the same
upper case letter are not significantly different according to the
Tukey's HSD method.












Fungi were recovered from seeds at harvest maturity and the

percentage of seeds which bore fungi increased as the time of delayed

harvest increased (Table 17). There was no apparent advantage for

seeds from the impermeable-seed lines in preventing fungi from

becoming associated with them. It is assumed that fungi recovered

from seeds were actually borne within the seeds. Surface

sterilization prior to incubation was used to prevent germination of

fungal spores on the seed-coat surface.

Fungi belonging to at least 13 different genera were recovered

from seeds. The largest number of individual genera belonged to the

form-order Moniliales (Alexopulus and Mims, 1979). Except for species

in the genera Fusarium, Alternaria, and Aspergillus the counts of

individual organisms of this form order were low. Because of low

counts of Cercospora kikuchii, Cladosporium oxysporium, Curvularia

lunata, Curvularia prasadii, Dreshslera indica, Epicoccum sp.,

Pithomyces chartarium, Trichocladium canadense, and Nigrospora

sphaerica were summed and presented as miscellaneous Moniliales in

Table 18. Several species of Fusarium were recovered from seeds and

among these F. semitectum was tentatively identified. Although great

variation existed in the appearance of pycnidium bearing conidia which

were single celled, hyaline, and biguttulate, no speciation was

attempted. Aspergillus niger and Aspergillus flavus were readily

recovered from the seed coats. Although Alternaria spp. (hereafter

referred to as Alternaria) was one of the most common organisms

recovered, speciation was difficult. Almost all Alternaria recovered

appeared to be of the same species. Judging by sporulation and growth












Table 17. Incidence of total fungi
maturity.


at maturity and at times after


Delayed harvest
Entries Harvest maturity 1 2

% seeds affected

Hardee 48.7 10.0 73.4 12.1 88.0 5.1

Hardee-sprayed 38.6 8.4 81.0 8.4 82.4 5.7

8731 26.5 8.6 30.2 4.9 90.5 4.8

8745 66.5 7.1 79.1 19.6 104.2 2.7


Standard error of the mean.
'Delayed harvest-1, 1 month after harvest maturity for Hardee and
Hardee-sprayed, 2 and 6 weeks for 8731 and 8745, respectively.
Delayed harvest-2, 2 months after harvest maturity for all entries.












Table 18. Incidence of Aspergillus flavus, Aspergillus niger, and
miscellaneous Moniliales at maturity and at times after
maturity.


Delayed harvest
Entries Harvest maturity 1 2

% seeds affected


Aspergillus flavus


Hardee


Hardee-sprayed


8731

8745


6.5 2.7

1.7 1.0

3.7 3.4

8.0 5.4


7.7 5.0

18.5 6.1

1.5 0.6

6.0 4.7


Aspergillus niger


Hardee


Hardee-sprayed


8731

8745


39.0 7.7

34.7 19.5

14.7 4.2

30.5 7.4


30.0 7.6

45.5 5.5

12.0 4.6

25.7 7.9


0.7 0.2

2.0 0.9

33.7 7.4

39.0 6.4


Misc. Moniliales


Hardee


Hardee-sprayed


8731

8745


Standard error of the mean.
tDelayed harvest-i occurred when all entries reached their respective
maturities. Delayed harvest-2 occurred 4 weeks later for Hardee and
Hardee-sprayed, and 2 and 6 weeks later for 8731 and 8745, respec-
tively.


0.0


0.2 0.1

3.0 1.3


2.7 0.5


0.0


4.2 1.4

1.0 0.3


2.5 1.0

2.2 0.8

1.2 0.7

1.5 0.5


0.2 0.1

2.0 1.2

0.2 0.1

1.0 0.7












habit, two species of Rhizopus were recovered in approximately equal

numbers but they were not identified.

The recovery of several groups of fungi from soybean seeds does

not appear to be as important in seed deterioration as is the genera

to which the fungi belong. Only Phomopsis spp. and Fusarium spp.

(hereafter referred to as Phomopsis and Fusarium) appeared to

significantly decrease germination (Table 19). The presence of

Rhizopus spp. and Alternaria spp. also reduced germination but the

correlation coefficients were low and non-significant. Aspergillus

niger, Aspergillus flavus, and miscellaneous Moniliales appeared

superficially to enhance germination. However, this is probably

misleading due to a greater recovery of A. niger, A. flavus, and

miscellaneous Moniliales in seeds from harvest maturity which had

higher germinations. Because of their relative non-importance to seed

viability, their presence on seeds are reported (Table 18) but not

discussed in detail. The decreased recovery of A.niger, A. flavus,

and miscellaneous Moniliales may have been influenced by the increased

presence of more aggressive fungi such as Phomopsis and Fusarium during

delayed harvest. These organisms may have been antagonistic to the

Moniliales.

Preliminary observations of fungal growth in the blotter test

were recorded to ascertain the severity of fungal incidence with

seeds. A rapid growth of fungi on seeds was thought to indicate a

more severe infection than the absence of fungal growth (Table 20).

Correlation coefficients between these early observations and seed

germination were significant and negative (Table 21). The fungal

















Correlation between incidence of fungal organisms and
normal germination of seed in the blotter test over
harvests.


Fungal organism



Aspergillus niger

Aspergillus flavus

Alternaria spp.

Fusarium spp.

Misc. Moniliales

Phomopsis spp.

Rhizopus spp.


Normal germination


r

.27

.21

-.19

-.52**

.25

-.84**

-.11


*, Significant at the 0.05 and 0.01 probability levels, respectively.


Table 19.











83






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4-i m$o- U c o
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41 4.. i
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Cn a) M CB o)
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0 C C C n U U C ,.C C
>n :0 w co = o
( 4.o ca t C 4E

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a) cU tw C) to


.)0 CC co m ct 3
4-4---1 a 1 e to


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c c C > 3 0 C Cl 0 a
S14 0 r m C Q11 ) 4
Cd U I '0c *





X ::1 4 1 0 41
a to M m a< (f a)o 1m










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w -4-ii m 444 J
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SO O a Ci
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'4 m( (c *4 C -* *f2
0 I > 0





l ) a)C

> C (m c 4J m
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a C Q >B -I Cr 0a c
P"C C4 r- C- rW
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w 1"0 "4-1 en m uo
1 S cw C- a) u o
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cl 00 4o 4- ++
















Table 21.


Correlations between incidence of observed fungal growth
on seeds after start of the blotter test and germination
measurements.


Germination measurements
Normal Total

r

Observed fungal
growth after 2 days -.84" -.83**


Observed fungal
growth after 4 days -.87** -.88**


"Significant at the 0.01 probability level.












growth observed on the seed-coat surface (at least after 2 days of the

blotter test) in almost all recorded instances was later identified as

either Phomopsis, Fusarium, or Alternaria. Therefore, the means

presented may also give a relative indication of the combined

incidence of Phomopsis, Fusarium, and Alternaria in seeds of these

entries and harvests. In general, the percentage of seeds affected by

early fungal growth increased as seeds were delay harvested and means

are highest for seeds affected most by Fusarium, Phomopsis, and

Alternaria.

When seeds were harvested at maturity, the percentage of 8745

seeds affected by Phomopsis was significantly higher than for seeds of

the other entries (Table 22). This may have been due to more humid

conditions under which seeds of 8745 developed (Table 11) as compared

to the other entries. The percentage of seeds with Phomopsis

increased significantly as harvest was delayed. The impermeable seed

lines did not exhibit a lower incidence than the normal-seed entry

Hardee at the end of delayed harvest. Phomopsis incidence was lower,

however, for seeds of Hardee-sprayed. Evidently, benomyl application

was successful in reducing the percentage of seeds affected by

Phomopsis. This may explain the higher germination of Hardee-sprayed

seeds (Tables 10 and 16).

The percentage of seeds affected by Fusarium was significantly

lower in the impermeable seed-coat lines than in the normal-seed

entries (Table 22). This may be due to the presence of the

impermeable seed coat but genetic differences between the entries may

be equally responsible, because only 39% (Table 12) of the seed from























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the impermeable seed lines were observed to be impermeable when

harvested 2 months after maturity (Table 20). Application of benomyl

appeared to have no effect on preventing Fusarium incidence and this

may be why seeds of Hardee-sprayed significantly decreased in both

normal and total germination from the first to the second delayed

harvest (Table 10).

Another effect of benomyl application was a significantly higher

percentage of Hardee-sprayed seeds affected by Alternaria than for the

other entries at the second delayed harvest (Table 23). This increase

in Alternaria from seed sprayed with benomyl has been reported

(Jeffers et al., 1982), although not under delayed harvest conditions.

The presence of Phomopsis may inhibit the expression of Alternaria

similar to Cercospora sp. antagonism (Hepperly and Sinclair, 1982).

Why Alternaria levels were not higher previously is unknown.

Fungal infection of embryonic axes although low, followed the

same trend as seed infection (Table 24). One note of interest,

however, is the lack of embryonic axes infected by Phomopsis and

Fusarium from seeds of the first harvest. Evidently, Phomopsis and

Fusarium require time to reach the embryonic axes. Also, the increase

in total fungal infection for embryonic axes after 2 months of delayed

harvest may indicate the progressively greater severity of seed

infection after harvest maturity.

In general, seeds from the impermeable seed-coat lines did not

retain viability nor prevent fungi from becoming associated with them.

This may be due to the low quantity of impermeable seed remaining

after the delay-harvest period rather than a failure of the











Table 23.


Incidence of Alternaria spp. at harvest maturity and at
times after harvest maturity.


Delayed harvest
Entries Harvest maturity 1 2

% seeds affected

Hardee 0.0 a AS 0.2 A 0.5 b A

Hardee-sprayed 0.8 abB 3.0 B 35.2 a A

8731 1.5 abA 0.2 A 0.5 b A

8745 2.5 a A 0.2 A 0.2 b A


+Delayed harvest-1, 1 month after harvest maturity for Hardee and
Hardee-sprayed, 2 and 6 weeks for 8731 and 8745, respectively.
Delayed harvest-2, 2 months after harvest maturity for all entries.
SMeans within a column followed by the same lower case letter are
not significantly different according to the Waller-Duncan k-ratio
t test, a = 0.05, k = 100. Means within a row followed by the same
upper case letter are not significantly different according to the
Tukey's HSD method.




















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