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4

dissertation entitled

INTEGRATED CROP MANAGEMENT AND INDUCED
DISEASE RESISTANCE IN ONIONS

presented by
Jorge E. Arboleya

has been accepted towards fulfillment
of the requirements for the

Ph D degree in Horticulture

gQCM/é/ / {fit/”79.2

Dr. Ber rd Zandstra
Major Professor’s Signature

Afif‘cni-AQ :2. g 1, (“C 3

Date

MSU is an Affinnative Action/Equal Opportunity Institution

 

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6/01 c:/ClRC/DateDuep$5p.15

INTEGRATED CROP MANAGEMENT AND INDUCED DISEASE RESISTANCE IN
ONIONS

By

Jorge E. Arboleya

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of
DOCTOR OF PHILOSOPHY
Department of Horticulture

2003

ABSTRACT

INTEGRATED CROP MANAGEMENT AND INDUCED
RESISTANCE IN ONIONS

By
Jorge E. Arboleya

Growing conditions, plant vigor, and crop management factors may increase the
susceptibility of onion to pathogens. In Michigan onions are normally direct seeded on
flat beds on muck soils. Most onion fields are subject to flooding after heavy rains, which
results in reduced stands and/or increased foliar and bulb diseases. Growers apply
fungicides to reduce foliar diseases and maintain disease-free bulb through harvest and
storage. Induced resistance is an alternative approach to disease control, and is based on
the activation of the plant defense system.

Necrosis was induced on onion leaves in the greenhouse with cucumber
pathogens Colletotrichum orbiculare or Didymella bryoniae. D. bryoniae caused
localized necrosis and induced resistance to Alternaria porri more effectively than C.
orbiculare. Purple blotch lesions were significantly smaller on D. btyonz'ae induced
plants compared to untreated control plants. Under field conditions methyl jasmonate
(MeJ A) was the best inducer of resistance against A. porri in onion in both years. DL-B-
amino-n-butyric acid (BABA) had inconsistent results between years, and acibenzolar-S-
methyl treated plants had more lesions than the control. Marketable yield was reduced
with Me] A application in 2002. Therefore, onion plants could experience a resistance
cost. Me] A did not have any negative effect on onion storage in both years of this study.

From 140 rotted onion bulb samples, 15 were identified as Burkholderia cepacia,

2 Were identified as B. gladioli pv. alliicola and 1 was identified as Pantoea ananatis,

using a DNA extraction method with slight modification and specific oligonucleotides for
bacteria identification. Plants treated with MeJ A plus minimum rate of fungicides had the
least number of bulbs (1) infected with B. cepacia compared to plants treated only with
fungicides (6), suggesting that Me] A could help to reduce rot of bulbs caused by B.
cepacia.

A study comparing onions grown on flat and raised beds at the Muck Soil
Research Station indicated that flat beds contributed to lower plant stands than raised
beds, 34% and 22% reduction in plant stand in 2001 and 2002, respectively, 40 days after
planting (DAP). Raised beds had less purple blotch disease incidence, 55% and 54% of
plants with the disease 121 DAP, compared to 60% and 68% for flat beds in 2001 and
2002. Purple blotch severity was 4 for flat beds (scale O=no disease; 5 > 70% of the third
green leaf from the base affected with the disease) compared to 3 for raised beds, 100
DAP in 2001. Onion yield was higher on raised beds in 2001 than on flat beds.

Foliar desiccation of onion tops with herbicides indicated that diquat, paraquat,
and carfentrazone desiccated onion leaves in the field very well but caused internal decay
of bulb quality in storage. Endothall was partially effective for onion desiccation and did
not reduce marketable bulbs in storage. Bromoxynil desiccated onion foliage 7 0-97%,
depending on the year, without inducing rot or reducing the percentage of marketable
bulbs in storage. Without good desiccation, onion growers must depend on good soil
management practices and drainage, use of reasonable population densities, adequate
weed, fertilizer and irrigation management practices to facilitate onion maturation, cure,

and storage.

ACKNOWLEDGMENTS

I am grateful to Dr. Bernard Zandstra, my major professor, for inviting me to
come to Michigan State University for my Ph.D. program and for his guidance and
support during my studies at MSU. I thank Dr. Raymond Hammerschmidt for his advice
in the induced resistance part of my dissertation. I thank Dr. Irvin Widders for inviting
me to come to MSU and for his guidance during this study. I thank Dr. Darryl Warncke
and Dr. Randolph Beaudry for their words of encouragement and advice. A special thank
to Dr. Andrea da Rocha for her help in Dr. Hammerschmidt‘ lab. Thank Dr. William
Kirk, Dr. Donald Penner, and Dr. George Garrity for their advice and suggestions. Thank
Dr. James Lorbeer from Cornell University, and Dr. Ronald Walcott and Anita Castro
from University of Georgia, for allowing me to learn techniques related with onion
bacterial diseases. I would like to thank the support staff of Dr. James Kelly and Dr.
Hammerschmidt for their help during my laboratory and greenhouse work, specially Dr.
Marcio Ender. Special thanks to Dr. Joe Masabni, Mike Particka, Robert Uhlig, Robert
Scheffer, and Ronald Gnagey for their friendship and help in my field work at the Muck
Soil Research Station. Thanks to HORT and CSS graduate students for their friendship
during my stay at MSU. Thanks to Latin American and Brazilian community for their
friendship and encouragement. Thank to the Uruguayans, especially Dr. Felix Marti and
his wife Elida F ranceschi, and Dr. Ruben Hernandez for their support, advice and
encouragement. Thanks to my family that always encouraged me in my degree program.

Thanks to the National Research Institute of Agriculture (INIA) and my major

professor for fimding my program.

iv

TABLE OF CONTENTS

LIST OF TABLES .............................................................................. VIII

LIST OF FIGURES ............................................................................. XHI

CHAPTER I

GENERAL INTRODUCTION
Integrated crop management ............................................................. 2
Importance of onion in Michigan ........................................................ 2
Characteristics of an onion bulb ......................................................... 3
Harvesting, curing and onion storage ................................................... 4
Rest and dormancy ........................................................................ 5
Onion production constrains ............................................................. 6
Purple blotch disease ...................................................................... 8
Induced resistance .......................................................................... 9
Types of induced resistance ............................................................ 10
Chemical activation of resistance ....................................................... 12
Bibliography ............................................................................... 1 5

CHAPTER II

BIOLOGICAL INDUCTION OF RESISTANCE IN ONION AGAH\I ST Altenaria porri

ABSTRACT ............................................................................... 21

INTRODUCTION ........................................................................ 22

MATERIALS AND METHODS ........................................................ 26

RESULTS ................................................................................... 30

DISCUSSION .............................................................................. 33

BIBLIOGRAPHY ......................................................................... 39
CHAPTER III

EFFECT OF CHEMICAL RESISTANCE ACTIVATORS ON PURPLE BLOTCH
DISEASE OF ONION

ABSTRACT ............................................................................... 52

INTRODUCTION ........................................................................ 54

MATERIALS AND METHODS ........................................................ 59

RESULTS ................................................................................... 64

DISCUSSION .............................................................................. 68

BIBLIOGRAPHY ......................................................................... 75
CHAPTER IV

EFFECT OF FOLIAR-APPLIED DISEASE RESISTANCE ACTIVATORS ON
PURPLE BLOTCH OF ONIONS AND BACTERIAL ROT IN STORAGE

ABSTRACT ................................................................................ 92

INTRODUCTION ......................................................................... 93

MATERIALS AND METHODS ......................................................... 96

RESULTS ................................................................................. l 00

DISCUSSION ............................................................................. 1 02

BIBLIOGRAPHY ........................................................................ 104
CHAPTER V

CULTURAL PRACTICES AFFECT PLANT STAND, YIELD AND PURPLE
BLOTCH INFECTION OF ONIONS

ABSTRACT .............................................................................. 114
INTRODUCTION ....................................................................... 116
MATERIALS AND METHODS ...................................................... 1 20
RESULTS ................................................................................. 1 26
DISCUSSION ............................................................................. 1 32
BIBLIOGRAPHY ........................................................................ 139

vi

CHAPTER VI

FOLIAR DESICCATION OF ONIONS WITH HERBICIEDS

ABSTRACT .............................................................................. 1 7 1
INTRODUCTION ....................................................................... 1 72
MATERIALS AND METHODS ...................................................... 1 77
RESULTS AND DISCUSSION ....................................................... l 81
BIBLIOGRAPHY ................................................................................ 1 86
CONCLUSIONS .................................................................................. l 95
APPENDIX ......................................................................................... 1 97

vii

LIST OF TABLES

Table 2.1. Number of necrotic lesions and lesion length measured at four, six and nine
days after inoculation (DAI) with D. bryom‘ae on onion cultivar T-439, 2002 ............. 44

Table 2.2. Number of necrotic lesions and lesion length measured at two and three days
after inoculation (DAI) with D. bryoniae on onion cultivars Hoopla, T-439 and
Altisirno .............................................................................................. 44

Table 3.1. Effect of chemical resistance activators on the number of plants infected with
purple blotch, number of lesions per plant, and lesion length of purple blotch of three
onion cultivars, 13 days after inoculation, Muck Soil Research Station, Laingsburg MI,
2001 ................................................................................................... 80

Table 3.2. Effect of chemical resistance activators on the number of plants infected with
purple blotch, number of lesions per plant, and lesion length of purple blotch of three
onion cultivars, 25 days after inoculation, Muck Soil Research Station, Laingsburg MI,
2001 ................................................................................................... 81

Table 3.3. Effect of chemical resistance activators on total and marketable yield of three
onion cultivars, Muck Soil Research Station, Laingsburg MI, 2001 ......................... 82

Table 3.4. Effect of chemical resistance activators on the percentage of large and medium
bulbs of three onion cultivars, Muck Soil Research Station, Laingsburg MI, 2001........83

Table 3.5. Effect of chemical resistance activators on the percentage of marketable bulbs
of three onion cultivars, 138 and 173 days after harvest (DAH), Muck Soil Research
Station, Laingsburg M1, 2001-2002 ............................................................... 84

Table 3.6. Interaction of chemical resistance activators and cultivars on the percentage of
marketable bulbs, 173 days after harvest (DAH), Muck Soil Research Station, Laingsburg
M1, 2001-2002 ...................................................................................... 85

Table 3.7. Effect of chemical resistance activators on the number of plants infected with
purple blotch, number of lesions per plant, and lesion length of purple blotch of three
onion cultivars, 16 days after inoculation, Muck Soil Research Station, Laingsburg MI,
2002 ................................................................................................... 86

Table 3.8. Effect of chemical resistance activators on the number of plants infected with
purple blotch, number of lesions per plant, and lesion length of purple blotch of three
onion cultivars, 22 days after inoculation, Muck Soil Research Station, Laingsburg MI,
2002 ................................................................................................... 87

Table 3.9. Effect of chemical resistance activators on total and marketable yield of three
onion cultivars, Muck Soil Research Station, Laingsburg MI, 2002 ......................... 88

viii

Table 3.10. Effect of chemical resistance activators on the percentage of large and
medium bulbs of three onion cultivars, Muck Soil Research Station, Laingsburg MI,
2002 ................................................................................................ 89

Table 3.11. Effect of chemical resistance activators on the percentage of marketable bulbs
of three onion cultivars, 133 and 161 days after harvest (DAH), Muck Soil Research
Station, Laingsburg M1, 2002-2003 ........................................................... 90

Table 4.1 Sequences for the oligonucleotides primers used to detect bacteria associated
with bulb rots, 2002 ............................................................................. 106

Table 4.2. Identification and distribution of bacteria from samples of onion that showed
symptoms of decay, 2002 ....................................................................... 106

Table 4.3. Number of samples that tested positive for B. cepacia according to the
chemical resistance activators applied in the field, 2002 .................................... 107

Table 5.1. Rainfall and irrigation from April to September, Muck Soil Research Station,
Laingsburg, MI, 2001 ............................................................................. 142

Table 5.2. Rainfall and irrigation from April to September, Muck Soil Research Station,
Laingsburg, MI, 2002 ............................................................................. 142

Table 5. 3. Historical average weather records for Laingsburg, MI, with a minimum
period of records of 30 years .................................................................... 143

Table 5.4. Disease severity rating used to evaluate purple blotch foliar disease caused by
Altemaria porri .................................................................................... 144

Table 5.5. Number of live plants in 3 m of bed on flat or raised beds, with or without
irrigation, Muck Soil Research Station, Laingsburg, MI, 2001 ........................... 145

Table 5.6. Effect of irrigation, bed type, and cultivar on the percentage of plants infected
with purple blotch 106 and 120 days after planting, Muck Soil Research Station,
Laingsburg, MI, 2001 ............................................................................ 146

Table 5.7. Effect of irrigation, bed type, and cultivar on Alternaria porri severity 106 and
120 days after planting, Muck Soil Research Station, Laingsburg, MI, 2001 ............ 147

Table 5.8. Effect of irrigation, bed type, and cultivar on total, marketable, large bulb and
medium bulb yield, Muck Soil Research Station, Laingsburg, MI, 2001 .................. 148

Table 5.9. Effect of irrigation, bed type, and cultivar on marketable bulbs 142 and 177
days after harvest (DAH), Muck Soil Research Station, Laingsburg, MI, 2001-2002...149

Table 5.10. Number of live plants in 3 m of flat and raised beds, with or without
irrigation, Muck Soil Research Station, Laingsburg, MI, 2002 .......................... 150

Table 5.11. Effect of irrigation, bed type, and cultivar on the percentage of plants infected
with purple blotch 100 and 120 days after planting, Muck Soil Research Station,
Laingsburg, MI, 2002 ........................................................................... 151

Table 5.12. Effect of irrigation, bed type, and cultivar on Alternaria porri severity 100
and 120 days after planting, Muck Soil Research Station, Laingsburg, MI, 2002 ...... 152

Table 5.13. Effect of in'igation, bed type, and cultivar on bulbing ratio (minimum
pseudostem diameter/maximum bulb diameter) 81, 85 and 89 days after planting (DAP),
Muck Soil Research Station, Laingsburg, MI, 2002 ......................................... 153

Table 5.14. Interaction between irrigation and bed type on bulbing ratio 81 and 85 DAP,
Muck Soil Research Station, Laingsburg, MI, 2002 ........................................ 154

Table 5.15. Effect of irrigation, bed type and cultivars on leaf, pseudostem and bulb dry
weight 64, 74, 100 and 125 days afler planting, Muck Soil Research Station, Laingsburg,
MI, 2002. ......................................................................................... 155

Table 5.16. Interaction between irrigation and bed type on bulb dry weight 125 day after
planting” Muck Soil Research Station, Laingsburg, MI, 2002 ............................. 156

Table 5.17. Interaction between bed type and cultivar on leaf dry weight 125 day after
planting” Muck Soil Research Station, Laingsburg, MI, 2002 ............................. 156

Table 5.18. Interaction between bed type and cultivar on pseudostem dry weight 125 day
after planting” Muck Soil Research Station, Laingsburg, MI, 2002 ....................... 157

Table 5.19. Interaction among irrigation, bed type and cultivar on bulb dry weight 125
day after planting” Muck Soil Research Station, Laingsburg, MI, 2002 .................. 157

Table 5.20. Effect of irrigation, bed type, and cultivar on total, marketable, large bulb and
medium bulb yield, Muck Soil Research Station, Laingsburg, MI, 2002 ................. 158

Table 5.21. Effect of irrigation, bed type, and cultivar on marketable bulbs 151 days after
harvest (DAH), Muck Soil Research Station, Laingsburg, M1, 2002-2003. ............. 159

Table 5.22. Effect of fungicide, row spacing, and cultivar on the number of plants
affected with purple blotch, severity of purple blotch, and yield of onion, Muck Soil
Research Station, Laingsburg, MI, 2001 ...................................................... 160

Table 5.23. Interaction between spacing and fungicide on disease severity 119 days after
planting, Muck Soil Research Station, Laingsburg, MI, 2001 .............................. 162

Table 5.24. Interaction between spacing and cultivars on disease severity 119 days after
planting, Muck Soil Research Station, Laingsburg, MI, 2001 ............................... 162

Table 5.25. Interaction between spacing and cultivars for medium sized bulbs yield,
Muck Soil Research Station, Laingsburg, MI, 2001 .......................................... 163

Table 5.26. Interaction between spacing and fungicide on small sized bulbs, Muck Soil
Research Station, Laingsburg, MI, 2001 ....................................................... 163

Table 5.27. Interaction between spacing, fungicide and cultivar on small sized bulbs 2,
Muck Soil Research Station, Laingsburg, MI, 2001 .......................................... 164

Table 5.28. Comparison of fungicide and no fungicide, row spacing and cultivars on the
number of plants affected with purple blotch, severity of purple blotch, total, marketable,
large, medium yield, Muck Soil Research Station, Laingsburg, MI, 2002. ............... 165

Table 5.29. Interaction between fungicide and cultivars on the number of plants infected
with purple blotch incidence 100 days after planting, Muck Soil Research Station,
Laingsburg, MI, 2002 ............................................................................. 167

Table 5.30. Interaction between ftmgicide and cultivars on purple blotch severity 120
days after planting, Muck Soil Research Station, Laingsburg, MI, 2002 ................... 167

Table 5.31. Interaction between fungicide and cultivars on large sized bulb yield, Muck
Soil Research Station, Laingsburg, MI, 2002 .................................................. 168

Table 6.1. Onion foliar desiccation 11 days after treatment (DAT), and percentage of
marketable bulbs 105 and 171 days after harvest (DAH), Muck Soil Research Station,
Laingsburg, M1, 1993-1994 ...................................................................... 1 89

Table 6.2. Onion foliar desiccation eigth DAT, and percentage of marketable bulbs 155
DAH, Muck Soil Research Station, Laingsburg, M1, 1994-1995 ........................... 190

Table 6.3. Onion foliar desiccation 11 DAT, and percentage of marketable bulbs at
174 DAH, Muck Soil Research Station, Laingsburg, M1, 1995-1996 ...................... 191

Table 6.4. Onion foliar desiccation 14 DAT, and percentage of marketable
bulbs 127 and 166 DAH, Muck Soil Research Station, Laingsburg, M1, 2001-2002. . ..192

Table 6.5. Onion foliar desiccation 15 DAT, percentage of marketable bulbs out of total
bulbs stored at 159 DAH, and apparently good bulbs with internal rot 193 DAH, Muck
Soil Research Station, Laingsburg, M1, 2002-2003 ........................................... 193

Table 7.1. Analysis of variance for plant stand 19 days after planting, Muck Soil
Research Station, Laingsburg, MI. 2001 ....................................................... 198

Table 7.2. Analysis of variance for plant stand 40 days after planting, Muck Soil
Research Station, Laingsburg, MI. 2001 ...................................................... 199

Table 7.3. Analysis of variance for plant stand 60 days after planting, Muck Soil
Research Station, Laingsburg, MI. 2001 ....................................................... 200

Table 7. 4. Analysis of variance for plant stand 79 days afier planting, Muck Soil
Research Station, Laingsburg, MI. 2001 ....................................................... 201

Table 7.5. Analysis of variance for plant stand 99 days after planting, Muck Soil
Research Station, Laingsburg, MI. 2001 ....................................................... 202

xii

CHAPTER I

GENERAL INTRODUCTION

 

INTRODUCTION

Integrated crop management

Integrated pest control is an ideal combination of biological, agrochemical,
chemical, physical and other methods of plant protection against the entire complex of
pests and diseases in a specific eco-geographic zone of a certain crop, whereby a number
of hannful species are brought down to economically insignificant levels with
maintenance of the activity of natural useful organisms (Fadeev et al., 1987).

Sustainable agriculture, integrated crop management, and organic farming are all
part of an alternative agriculture movement that promotes the use of biological
interactions and cultural practices in place of agricultural chemicals (Grubinger, 1999).
He stated that sustainable agriculture is judged not only by its effects on short term yield
and profit, but also on water quality, soil productivity, human health, and the local
viability of communities.

Integrated pest management (IPM) is a pest control strategy that promotes the use
of a variety of tactics, including pest resistant cultivars and biological, cultural, and
physical controls (Hoffrnann et al., 1996).

Integrated crop management (ICM) requires farmers to employ the best management
practices, which optimizes the use of inputs to enhance yields without threatening to
deplete or pollute resources (Grubinger, 1999).

Importance of onion in Michigan

The major onion production countries are China, India, US, Turkey and Iran,
respectively (F AOSTAT, 2003). U. S. farmers plant approximately 66,000 ha (145,000
acres). The US. onion industry accounts for 2.5 % of the world onion area and 7% of the
world onion production (NOA, 2003). Onion is produced commercially in 16 states
(USDA 2003). Michigan was a major producer in the past but now has about 1,600 ha
with a total production of 41,000 tons per year with a total value of 9 million dollars
(USDA 2003). Michigan ranked tenth in US. onion production based on planted onion
acreage (National Onion Association, 2003). The onion production in Michigan is
located mainly in the west central, southwest and south central parts of the state
(Kleweno and Matthews, 2002).

The most important vegetables consumed in U. S. are potato (21.3
kg/capita/year), lettuce (10.8 kg/capita/year), dry bulb onion (8.2 kg/capita/year) and
tomato (8.2 kg/capita/year) (ERS-USDA 2003; PMA, 2003). Onion consumption has
risen over 69 % in the last two decades, from 4.9 kg/person in 1981 to 8.2 kg/person in

2001 (NOA, 2003).

Characteristics of an onion bulb

The edible part of an onion is the bulb, which is composed of the swollen base of
the leaves. The growing point is in the center of the compressed true stem, which is at the
base of the bulb (Brewster, 1994). An onion plant continually produces new leaves fiom
the center of the stem, and new roots. Leaf bases of successively produced leaves are

larger in diameter and in length. Bulbing is induced by several factors, including plant

age, size, daylength and temperature (DeMason, 1990). Long day, storage onions such as
those grown in Michigan are induced to bulb with about 14 hours daylength.

When the bulb approaches maturity leaves soften at the neck and become less
turgid, which causes the leaves to fall over (Brewster, 1994).

Onion bulbs continue to gain size and weight as the photosynthates are moved
from the remaining green leaves to the bulb (Tiessen et al., 1981; Voss, 1979; and
Zandstra et al., 1996).

Harvesting, curing and onion storage

Once onion leaves have fallen over and turn yellow, and the necks have sealed,
bulbs should be harvested (Hoffrnann et al., 1996). Yields may increase 10% to 40%
from initial bending of leaves to totally dry foliage (Brewster, 1990; Wall and Corgan,
1994). At full matmity, all leaves are brown and desiccated, and the bulb becomes
dormant. In some years, onions do not reach maturity and leaves remain green beyond the
normal harvest period as a result of late planting, improper cultivar selection, incorrect
cultural practices, inadequate heat during the growing season, or a damp, cool fall
(Tiessen et al., 1981; Johnson, 1986; Pelter et al., 1992). Excessive green leaves make
harvesting difficult, and lack of maturity results in poor storage quality.

After harvest, onions are cured to prepare the bulbs for shipping or storage. Curing is the
process of natural or forced-air drying to dry out outer scales and shrink the neck.
(Hoffrnann et al., 1996). Onion must be cured to obtain maximum storage life and to

retain quality (Matson et al, 1985). During curing onions lose about ten percent of their

fresh weight.

Onions that are not well dried in the field are susceptible to sprouting and decay during
and afier storage; therefore, it is necessary to dry the bulbs and shrink the necks as
rapidly as possible to prevent pathogens fi'om entering before storing onions (Kaufman
and Lorbeer, 1967; Jones and Mann, 1968).

Rest and dormancy

The differences between rest and dormancy sometimes are confused. Meyer and
Anderson (1952) defined dormancy as "a state where seeds fail to germinate even if
placed under such conditions that all environmental factors are favorable for
germination”. Chong (1995) defined rest "like a process that prevents seeds in their
natural habitat from germinating in unseasonal times". Lang (1987) stated that "dormancy
is the temporary suspension of visible growth of any plant structure containing a
meristem". Because of the confusion between terms he differentiates between
ecodormancy, which describes the dormancy when one or more environmental factors are
not suitable for growth, and endodorrnancy or dormancy due to physiological factors
within the dormant structure.

Mehdinaqui (2002) stated that dormancy "can be considered as the ability to
retain viability while having minimal metabolic activity and no visible growth". After
bulb formation growth ceases and onion bulbs become dormant. Mitosis at the shoot apex
decreases after harvest and curing (Brewster, 1997). During onion bulb growth, inhibitors
that are responsible for maintaining subsequent dormancy are translocated from the
leaves to the bulb. If the harvest is too early, less of the sprouting inhibitor is translocated
to the growing point of the bulb. On the other hand, harvesting too late may allow the

destruction of the inhibitor. Early defoliation or leaf desiccation results in earlier

sprouting in storage since inhibitory substances are not fully translocated from the leaves
to the bulbs before defoliation (Stow, 1976).

Storage onions may be treated with maleic hydrazide, a sprout inhibitor, to extend
storage life of the bulbs. This compound should be applied when about 50 to 60 percent
of the tops have fallen over and onions still have at least five actively growing leaves to
trarrslocate the compound to the bulb (Walz and Burr, 1977; Tiessen et al., 1981; Voss,
1979; Hoffrnann et al., 1996). Maleic hydrazide does not improve the storability of
onions, it only reduces the incidence of sprouting at the end of the dormant period.
Without a sprout inhibitor, bulbs of some cultivars begin to sprout as soon as they are
removed from storage and exposed to ambient conditions.

Growers have used several methods to hasten onion maturity in the field,
including rolling the tops with a lightweight roller, undercutting the roots with a knife or
rod weeder (Longbrake et al., 1974; Voss, 1979; Tiessen et al., 1981), and chemical
desiccants (Harmer and Lucas, 1955).

Onion production constrains

In commercial onion production, several factors affect yield and quality.
Environmental and abiotic stresses can adversely affect plant grow and predispose plants
to pathogens and insects (Schwartz and Bartolo, 1995).

In Michigan, onions are normally direct seeded on flat beds on muck soil. Because of
significant subsidence of muck soils (Lucas, 1982), most Michigan onion fields now lie
below drainage ditches and consequently are flooded for periods of time after heavy
rains. Therefore, plant stand can be reduced after heavy rain during germination and early

development.

Purple blotch caused by Alternaria porri, downy mildew caused by Peronospora
destructor and Botrytis leaf blight caused by Botrytis squamosa are the most
economically important foliage diseases (Schwartz and Mohan, 1995). Plant diseases
tend to be more severe when a pathogen is highly virulent, the plant is susceptible, and
the environment is favorable (disease triangle) for pathogen infection over an extended
period (Hart and Jarosz, 2000). Each of these three components can vary considerably
and as one component changes it affects the degree of severity within an individual plant
or within a plant population (Agrios, 1997). Changing any side of the triangle, such as
adding an unfavorable environment or using a disease-resistant cultivar, can significantly
reduce disease development (Agrios, 1997; Hart and J arosz, 2000).

Botrytis neck rot caused by Botrytis allii, Fusarium basal rot (caused by F usarium
oxysporum f. sp. cepacea) and bacterial soft rot are the most important postharvest
diseases of onion. The bacteria causing bacterial rots often occur in conjunction with
other diseases such as Botrytis neck rot, mostly as secondary invading organisms
(Hoffmann et al, 1996). Bacterial infection and spread are favored by moisture and
wounds that may occur during mechanical cultivation and storms (Schwartz and Bartolo,
1995; Wright and Grant, 1998). Bacteria infected onion bulbs develop symptoms of sour
skin, slippery skin, soft rot, or center rot in storage. In the literature, these symptoms have
been associated with infection by Burkholderia cepacia, B. gladioli pv. alliicola, Erwim'a
carotovora subsp. carotovora, (Schwartz and Mohan,l995) and Pantoea ananatis
(Schwartz and Otto, 2000), respectively.

Bulbs affected by slippery skin, caused by B. gladioli pv. alliicola, do not show

any external decay in the early stages of the disease, only a slight softening of the neck

region. One or two inner fleshy scales appear soft and have a cooked or water soaked
appearance (Schwartz and Mohan, 1995). Bulbs affected by sour skin (caused by B.
cepacia) are characterized by a pale yellow to light brown decay and breakdown of one
or a few inner bulb scales. The adjacent bulbs scales and the center remain firm
(Schwartz and Bartolo, 1995). The decay symptoms caused by B. cepacia and B. gladioli
pv. alliicola frequently overlap and are difficult to categorize (Tesoreiro et al. 1982,
Hoffnrann et al. 1996). Center rot caused by P. ananatis is characterized by rot of the
neck tissue and between the scales (Schwartz and Otto, 2000). Soft rot caused by Erwim'a
carotovora subps. carotovora shows water-soaked and pale yellow to light brown scales
that become soft as the rot progresses (Schwartz and Otto, 2000).

Because it generally is easier and more advantageous to prevent pests than to
eliminate them, crop sanitation always should be practiced. Crop rotation, selection of
resistant cultivars, and use of appropriate cultural practices should be used to produce
good yields of good quality onions (Schwartz and Bartolo, 1995).

Purple blotch disease.

Purple blotch, caused by Alternaria porri, is a very important disease of onions
that affects foliage. It was suggested that it could cause yield reduction from 30 to 50%
(Nolla 1927), and even up to 100 % (Skiles 1953). Spores of A. porri collected in traps
increased with leaf wetness periods of twelve or more hours (Miller and Amador 1981,
Miller 1983, Amador and Miller 1985, Everts and Lacy 1996), and the levels of damage
caused by purple blotch were higher on older than in younger leaves (Miller 1983, Everts

and Lacy 1990).

 

The first symptoms appear as numerous small, white, circular or irregular spots or
flecks. Under favorable conditions they gradually enlarge and become dark purple or
brown. High relative humidity promotes the development of purple blotch, while
prolonged periods of low relative humidity after infection produce only white flecks
(Bock 1964). Under optimum humidity (90% or higher), lesions are produced between 17
and 25 °C, a few are produced at 35° C (Bock 1964), and below 13° C little bacterial
growth occurs (Schwartz and Mohan 1995).

Induced resistance

The defense of plants to pathogens comprises constitutive barriers (Thomma et
al., 1998; Metraux et al., 2002) present in plants prior to any contact with pathogens or
herbivores, and inducible defense (Thomma et al., 1998). Exposure to various
microorganisms or other forms of stress can lead to the activation of defense mechanisms
(Metraux et al., 2002). Induced resistance depends on the recognition of a pathogen or
stress by the plant. Disease will occur if the growth of the pathogen is faster than induced
response, if no elicitors are produced or if suppressors prevent the plant defense reactions
(Metraux et al., 2002).

Induced resistance is the phenomenon that a plant, once appropriately stimulated,
exhibits an enhanced resistance upon challenge inoculation with a pathogen. So the term

induced resistance emphasizes the fact a triggering factor (inducing agent) is needed to
achieve this enhanced defense capacity (Van Loon 1997).

Induced disease resistance is a tool for disease management, which can be

distinguished from conventional chemical disease control and biological procedures in

plant protection by the lack of toxicity of the inducing agents toward the pathogens.

Therefore, the protection of the plant is based in the activation of plant defense
mechanisms or on the enhancement of their activity rather than on the elimination of the
pathogens. Induced resistance is considered one of the biological plant protection
procedures, where the plant is the target of the procedure, not the pathogens (Steiner and
Schonbeck 1995).

Induced resistance, based on natural defense mechanisms of plants is a promising
alternative approach to control plant diseases. This approach is effective and economical
and would reduce the dependence on agrochemicals (Suo and Leung, 2002). Induced
resistance is generally characterized by a reduction in the size and or number of lesions
that develop after inoculation of the induced plant with virulent and sometimes,
hypersensitive response inducing avirulent forms of pathogens (Hammerschmidt, 1999).

Types of induced resistance

All plants posses active defense mechanisms against pathogen attack. These
mechanisms fail when the plant is infected by a virulent pathogen because the pathogen
avoids triggering or suppresses resistance reactions, or evades the effect of activated
defenses (van Loon et al., 1998).

Two types of resistance mechanisms have been described in plants, systemic
acquired resistance (SAR) and induced systemic resistance (ISR).

SAR is defined by Ryals et al. (1996) as "a distinct signal transduction pathway
that plays an important role in the ability of plants to defend themselves against
pathogens". The resistance expressed is generally effective against a broad range of
pathogens, is associated with the production of pathogenesis related (PR) proteins and is

increasing endogenous salicylic acid. PR proteins block the development of fungal,

10

oomycete and bacterial pathogens through hydrolytic action on pathogen cell walls or by
antimicrobial activity. In the case of viruses, SAR is explained by an effect on viral
replication or viral movement, resulting in decreased disease expression
(Hammerschmidt, 1999).

Lucas (1999) summarized the main biological features of SAR as a) induced by
agents or pathogens causing necrosis; b) delay of several days between induction and full
expression; c) protection of tissue not exposed to inducer inoculation; (I) expressed as
reduction in lesion number, size, spore production, or pathogen multiplication; e)
protection is long-lasting, often weeks or even months; 1) protection is non-specific
(against pathogens unrelated to inducing agent); g) the signal of SAR is translocated and
graft-transmissible; and h) protection not passed on to seed progeny.

The other type of resistance, induced systemic resistance (ISR), is mediated by
jasmonic acid and ethylene and does not involve expression of PR proteins
(Hammerschmidt, 1999). Certain strains of rhizosphere bacteria are referred to as plant
growth-promoting rhizobacteria (PGPR) because their application can stimulate growth
and improve plant stand under stressful conditions (van Loon et al., 1998; Pieterse et al.,
2001). Increased plant productivity results in part from the suppression of deleterious
microorganisms and soil born pathogens by PGPR. Fluorescent Pseudomonas spp. are
among the most effective bacteria in reducing soil-bome diseases in disease suppressive
soils (van Loon et al., 1998).

According to Kuc (2001), ISR is a phenomenon whereby resistance to infectious

disease is systemically induced by localized infection or treatment with microbial

11

components or products. He also reported that ISR is most effective against frmgi, less
effective against bacteria and least effective against systemic viruses.

Rhizobacteria-mediated ISR resembles pathogen-induced systemic acquired
resistant in that both types of induced resistance render uninfected plant parts more
resistant towards a broad spectrum of plant pathogens. Some rhizobacteria trigger the
salicylic acid (SA) dependent SAR pathway by producing SA at the root surface. In other
cases, rhizobacteria trigger a different signaling pathway that requires responsiveness to
the plant hormones jasmonic acid and ethylene (Pieterse et al., 2001).

Van Loon et al. (1998) summarized the main biological features of ISR as a) an
absence of toxic effects of the inducing agents on the challenging pathogen; b) the
suppression of the induced resistance by a previous application of specific inhibitors,
such as actinomycin D (AMD), which affect gene expression of the plant; 0) the necessity
of a time interval between application of the inducer and the onset of protection of the
plant; (1) the absence of a typical dose-response correlation known for toxic components;
e) nonspecifity of protection; f) local as well as systemic protection; and g) a dependence
on the plant genotype.

Chemical activation of resistance
Chemical activation of disease resistance in plants represents an additional option for
growers to protect their crops from losses due to plant diseases. Against some pathogens,
like bacteria and viruses, it may be the best option for chemical control where genetic
resistance is not available or not sufficient. The integration of this new technology of
activating broad-spectrum plant defenses with genetic resistance and fungicides seems to

offer a more sustainable plant health management system against dynamic fungal

12

pathogens with a history of adaptation to fungicides or to resistant cultivars (Oostendorp
et al., 2001). There are indications that such adaptation of pathogens to genetically
resistant cultivars can indeed be slowed down by plant activators such as acibenzolar-S-
methyl (ASM) (Romero et al., 1998).

Systemic acquired resistance (SAR) is a potentially desirable strategy in achieving
1PM since it involves enhancing natural defense mechanisms in crops. Certain biological,
physical or chemical elicitors can be used to activate and/or boost natural disease
resistance in non-infected plant tissue. For example, grey mold on strawberry fruit was
suppressed by application of plant activator acibenzolar-s-methyl (ASM) (Terry and
Joyce, 2000)

The accumulation of PR proteins in response to ASM may lead to increased
protection against infection by pathogens in rose (Suo and Leung, 2002). Cucumber
plants treated with ASM became resistant to scab caused by Cladosporium cucumerinum,
and chitinase was rapidly induced. (Narusaka et al., 1999).

To optimize resistance activation and yield benefits as well as to avoid negative
side effects on plant growth, the use of chemical resistance activators has to be adjusted
for each crop. The symptoms of powdery mildew on wheat were greatly reduced when a
mixture of the fungicides propiconazole and fenpropidin was compared to ASM alone.
However resistance activated by ASM gave longer lasting protection (Oostendorp et al.,
2001)

D, L-[i-amino-n-butyric acid (BABA) or its 3-(S)-enantiomer has been reported to
activate disease resistance (Oostendorp et al., 2001). On grapes the non-protein amino

acid BABA induced local and systemic resistance against downy mildew (Cohen et al.,

13

1999). Low concentrations of BABA (10 mM) were sufficient to protect lettuce against
downy mildew caused by Bremia lactucae, and the protection lasted for at least 15 days
and it was systemic (Pajot et al., 2001). Pea, cucumber, cotton, tobacco, pepper, tomato,
grape, melon, sunflower, kohlrabi, corn, pearl millet, cauliflower and Arabidopsis
thaliana have been reported to be induced by BABA (J akab et al., 2001).

Methyl jasmonate had a protective effect against Alternaria brassicicola in
Arabidopsis thaliana (Thomma et al., 1998; Ton et al., 2002). J asmonic acid is
responsible for the induction of many changes in plant resistance that occur following
herbivore attack (Thaler, 2002). In tomato for example, plants induced with jasmonic
acid had 40% less damage due to the herbivore compared with control plants (Thaler,
1999). Plant traits can act as defenses against herbivores both by reducing herbivore
performance directly and by increasing the effectiveness of the natural enemies of

herbivores.

14

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l8

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19

CHAPTER II

BIOLOGICAL INDUCTION OF RESISTANCE IN ONION AGAINST

Altemaria porri.

20

ABSTRACT

Induced disease resistance is an alternative approach to consider in addition to
disease control which has been studied nominally in onions. Necrosis was induced on
onion leaves in the greenhouse with the cucumber pathogens Colletotrichum orbiculare
or Didymella bryom’ae. The first non-senescent leaf of plants at the seventh leaf stage was
' inoculated at 10 sites with 10 pl of a conidial suspension of each fungus (1x106
spores/ml). D. bryom’ae caused localized necrosis and induced resistance to A. porri more
effectively than C. orbiculare. Purple blotch (Altemaria porri) lesions were significantly
smaller on D. biyoniae-induced plants, compared to untreated control plants. In our
research we found a significant reduction in the number of lesions of A. porri alter the
plants were induced with D. bryoniae. However, the length of lesions was less affected.
Therefore, we assume the mechanism of induced resistance in onion against A. porri was

to inhibit pathogen penetration rather than against pathogen spread.

21

INTRODUCTION

Purple blotch, caused by Altemaria porri, is a disease of onions that affects
foliage. Severe infection may cause partial or total yield loss (Nolla, 1927: Skiles, 1953).
Spores of A. porri collected in traps increased with leaf wetness periods of twelve or
more hours (Amador and Miller, 1981; Miller, 1983; Amador and Miller, 1985; Everts
and Lacy 1996) and the levels of damage caused by purple blotch were higher in older
than in younger leaves (Miller, 1983; Everts and Lacy, 1990).

Chemical application is the usual method to control purple blotch. Fungicides
such as chlorothalonil, copper oxychloride, metalaxyl plus mancozeb, mancozeb,
tebuconazole, azoxystrobin, iprodione give some control of purple blotch (Tahir et a1.
1991, Pena et al. 1992, Sugha and Tyagi 1994, Bird et al, 2002). During a severe disease
outbreak, mancozeb reduced disease intensity by 6% and increased yield by 11% (Borkar
and Patil, 1995). Although chemical control has been used against purple blotch, the
broad and often excessive use of pesticides could cause damage to the environment, and
may lead to pathogen resistance (Ozeretskovskaya, 1995; Hadisustrino et a1. 1996). The
development of alternative methods to control purple blotch, such as the use of climatic
data to begin fungicide spray, is important in order to prevent purple blotch outbreaks and
to reduce the number of sprays (V incelli and Lorbeer, 1987; Hadisustrino et al. 1996).
For example, “blight-alert” has been developed to more effectively time fungicide
applications on onion against Botrytis squamosa based on weather prediction (V incelli
and Lorbeer 1989). "Blitecast" has broad use in potatoes (Anderson et. al 2001;

Katsurayama et al., 1996; Pscheidt 1986), as well as "Tomcast" in tomatoes (Gastelum et.

22

al, 2002; Mills et al., 2002). Tomcast has been adapted to asparagus (Meyer et al. 2000)
and carrot (Bounds and Hausbeck, 2003; Dorrnan and Hausbeck, 2003).

According to Agrios (1988), resistance is the ability of an organism to exclude or
overcome, completely or in some degree, the effect of a pathogen or other damaging
factor. Biological induction of systemic acquired resistance usually follows plant cell
death elicited by an avirulent pathogen or from disease necrosis caused by a virulent
pathogen (Strobel et al., 1996). Host cell death, which is manifested as rapid, localized
necrosis of cells at the infection site and which occurs in resistant plants in response to
pathogenic viruses, bacteria, fungi or nematodes, is called the hypersensitive response
(HR). This response accompanies many incompatible interactions (the pathogen is
recognized by the host and fails to infect the tissue), limits the spread of the invading
microorganism (Alvarez, 2002), and is considered one of the important mechanisms
leading to resistance (Kombrink and Schmelzer 2001). Disease resistance is manifested
by limited symptoms, reflecting the inability of the pathogen to grow or multiply and
spread. Sometimes it takes the form of HR, in which the pathogen remains confined to
necrotic lesions at the site of infection (Van Loon 1997).

Induced disease resistance is another tool for disease management in plants,
differing from conventional fungicides by the lack of toxicity of the inducing agents
toward the pathogens. The protection of the plant is based on the activation of plant
defense mechanisms or on the enhancement of their activity rather than on the
elimination of the pathogens (Ozeretskovskaya, 1995; Steiner and Schonbeck, 1995). A
triggering factor (inducing agent) is needed to achieve this enhanced defense capacity

(Van Loon, 1997). Acquired resistance points to a change in the physiology of the plant

23

resulting from an added property (Ross 1961). Induced resistance can be localized or
systemic. It can be induced by pathogen infection, by avirulent pathogens, and by
chemicals (Metraux et al., 2002; Van Loon, 2000). Necrotizing pathogens, non—pathogens
or root colonizing bacteria are potential biological inducers (Oostendorp et al., 2001).
There are two types of induced resistance, systemic acquired resistance (SAR) and
induced systemic resistance (ISR). Ryals et al. (1996) defined SAR as a distinct signal
transduction pathway that plays an important role in the ability of plants to defend
themselves against pathogens. The expression of SAR depends on the accumulation of
salicylic acid (SA) and it is associated with the production of pathogenesis-related
proteins (PRs), (Kombrink and Schmelzer, 2001; Metraux et al., 2002).

Lucas (1999) summarized the main biological characteristics of SAR, which are:
a) induction by agents or pathogens causing necrosis; b) delay of several days between
induction and full expression; c) protection of tissue not exposed to inducer inoculation;
d) reduction in lesion number and size, spore production, pathogen multiplication; e)
protection is long-lasting, often weeks or even months; 1) protection is non-specific
(against pathogens unrelated to inducing agent); g) the signal of SAR is translocated and

graft-transmissible; and h) protection is not passed on seed progeny.

The second type of induced resistance, ISR, develops systemically as a response
to root colonization by plant growth promoting rhizobacteria (PGPR). It is mediated by
jasmonic acid and ethylene, which are essential steps in the signal-transduction pathway
leading to ISR, and does not involve the expression of PRs (Hammerschmidt, 1999; Kuc,

2001; Van loon, 1998).

24

Induced resistance can be expressed as a decrease of fungal penetration (J enns
and Kuc, 1977; Richmond et al., 1979; Cohen et al., 1981; Sahashi and Shishiyama,
1985; Stenzel et al., 1985; Dean and Kuc, 1986; Lucas, 1999), lesion expansion,
penetration (Jenns and Kuc, 1977; Lucas, 1999; Stenzel et al., 1985) or sporulation
(Stenzel et al., 1985). Velasquez (2002) found that cucumber plants treated with
acibenzolar s-methyl (ASM) and challenged with Didymella bryoniae reduced the
number of successful penetrations rather than the spread of the fungus within the tissue.
He also reported that the majority of the cultivars were more successful reducing the size
of the lesions against C. orbiculare, while the number of lesions was reduced when
plants were challenged with D. bryom'ae. Descalzo (1990) found that D. bryoniae was
more effective than C. lagenarium in inducing the resistance of cucumber against gummy
stem/leaf blight caused by D. bryoniae.

The mechanism of induced resistance, such as exhibited in tobacco (Cohen and
Kuc, 1981), and the time between induction and challenge is important. The longer this

period, the less effective the protection in the plant (Dalisay and Kuc, 1995).

Although induced resistance has been studied in many crops (Sticher et al., 1997),
there is little information about phytoalexins in onion (Dnritriev et al., 1989, 1990;
Tverskoy et al., 1991), but nothing on induced resistance in onion. The objective of this
study was to determine if prior infection with necrosis-inducing pathogens would induce

resistance in onion foliage against Altemaria porri.

25

MATERIALS AND METHODS

Greenhouse experiments were conducted in the Plant Science Greenhouse at
Michigan State University, East Lansing MI, during 2002. The experiments were initiated
on February 16, April 8, April 26 and May 13.
Plant material

Onion cultivars Hoopla (Seed Works, San Luis Obispo CA), Altisirno (Bejo
Seeds Inc. Geneva NY), and T-439 (Takii Seed Co., Salinas CA), were planted in trays
containing Baccto High professional planting mix (Michigan Peat Co., Houston, Texas)
in the greenhouse. Onion seedlings were transplanted singly to 4 x 4-cm pots when they
reached the one true leaf stage. Treatments were applied at the seven leaf stage. All the
experiments were designed as randomized complete blocks with six replications in
experiment one, five in experiment two, ten in experiment three, and five in experiment

four. One plant constituted one replication.

Inducing Inoculations

In the first experiment, cucumber pathogens Didymella bryom’ae and
Colletotrichum orbiculare, which are not pathogenic to onion, were used as biological
inducers of resistance in onion. A solution containing deionized water, 0.05 g/ 100 ml of
casamino acid, and lg/ 100 ml of sucrose was prepared for spore suspension of D.
bryoniae. Spores were harvested from a culture kept in quarter strength potato dextrose
agar media (PDA). Spore concentration was adjusted to 1 x 106 spores/ml. For C.
orbiculare, spores were harvested fiom a culture kept in V-8 media, and adjusted to the
concentration of 1 x 106 spores/ml. Polyoxyethylene sorbitan monolaurate (Tween-20), at

the rate of 1 drop/ml was added to both spore suspensions in order to assure better

26

adhesion to the plant and to prevent run-off. Onion seedlings of cultivars Hoopla and T-
439 were inoculated with 10 ul of spore suspension at 10 sites on the most recently
developed leaf showing no symptoms of senescence. Control plants were inoculated with
distilled water containing only polyoxyethylene sorbitan monolaurate (1 drop/ml). Plants
were kept in a plastic wet chamber using a cool mist humidifier (Holmes HM-1975), to
assure free moisture for the infection process.

In the second and fourth experiments, onion seedlings of cultivars Hoopla,
Altisirno, and T-439 were induced as described above. Onion seedlings of cultivar T-439
were used in the third experiment, and they were induced as previously described.

The number of necrotic lesions and lesion length caused by the biological
inducers D. biyoniae and C. orbiculare were measured at four and seven days after
inducing (DAI) in experiment one; at four and eight DAI in experiment two; at four, six,
and nine DAI in experiment three; and at two and three DAI in experiment four. Lesion
length was measured in all the experiments using a digital caliper (Mitutoyo model CD-

6" CS).

Challenge inoculations

Experiment One. Ten days after inoculation with the inducing agent, the leaf above the
treated leaf on each plant was detached and placed in a petri dish (a 100 mm x 15 mm)
with Whatrnan # 1 filter paper, wetted with 3 ml of deionized water. This leaf was
inoculated in ten sites with 10 ul of a conidia suspension of A. porri (8,500 spores/ml)

containing polyoxyethylene sorbitan monolaurate (1 drop/ml). Petri dishes were covered

27

with a plastic bag to maintain damp conditions, and kept at room temperature
(approximately 18 C) until disease assessment was performed five days after inoculation.
Experiment Two. At 11 days after application of the inducing treatments, the leaf above
the induced one on each plant was detached and challenged as described in experiment
one. At the time we detached the leaves for challenge inoculation, onion seedlings were
very healthy, therefore we decided to inoculate the plants in the greenhouse in addition to
inoculating the detached leaves. In the other experiments conducted in the greenhouse,
onion seedlings were affected by thrips or showed tip die-back and yellowing at the end
of challenge evaluations. This probably was due to the environmental conditions in the
greenhouse and in the humid chamber used for the inoculations. For these reasons it was
not possible to challenge another leaf of the same plants in other experiments.

The second leaf above the induced leaf was then inoculated, as described above,
with the same concentration of A. porri, and the onion plants were maintained as
described above. Disease assessment was performed at four days after challenge on
detached leaves in the laboratory, and seven days after challenge on the same plants in
the greenhouse.

Experiment Three. At nine days after application of the inducing treatments, onion
seedlings were challenged as described in experiment one. Disease assessment was
performed at nine, ten and twelve days after challenge.

Experiment Four. At eight days after application of the inducing treatments, the leaf
above the induced one was detached and challenged as described in experiment one.

Disease assessment was performed six days after challenge.

28

Statistical analysis

Analysis of variance was performed with MSTAT-C (MSTAT-C, , 1988). Mean
separation was performed with Fisher's LSD. Arcsin transformation was used to
homogenize the variance for the percentage of necrotic lesions (Snedecor and Cochram,

1967)

29

RESULTS

Experiment One.

The number of necrotic lesions caused by Didymella bryoniae was similar to that
caused by Colletotrichum orbiculare, and T-439 had higher number of lesions compared
to Hoopla at four DAI (Figure 2.1A). D. bryoniae had a higher number of necrotic lesions
at seven DAI on cultivar T-439, while C. orbiculare had similar number of necrotic
lesions on cultivar Hoopla. Although D. bryom’ae caused a greater number of necrotic
lesions than C. orbiculare, the lesion length was not significantly different. Nevertheless,
the necrotic lesion length was slightly larger on D. bryoniae treated plants compared to C.
orbiculare in cultivar T-439 (Figure 2.1 B).

Inoculation with D. bryoniae or C. orbiculare caused a reduction in the number of
lesions and lesion length of A. porri compared to the control in cultivar T-439 (Figure
2.2). There were no differences in the number of lesions or lesion length in cultivar

Hoopla among D. bryoniae, C. orbiculare or the control.

Experiment Two.

The number of necrotic lesions caused by D. biyom'ae was higher compared to the
number of necrotic lesions caused by C. orbiculare four DAI for cultivar Hoopla (Figure
2.3A). No differences were found between D. bryoniae and C. orbiculare for cultivars
Altisirno and T-439 four DAI (Figure 2.3A). D. bryoniae had significantly higher number
of necrotic lesions than C. orbiculare eight DAI for the three cultivars. The length of
lesions caused by D. bryoniae was larger on cultivar Hoopla and Altisimo compared to C.

orbiculare four DAI (Figure 2.3B). No differences were found for cultivar T-439. D.

30

bryom'ae also caused significantly larger necrotic lesions on onion leaves compared to C.
orbiculare on the three cultivars eight DAI (Figure 2.3B). D. bryoniae appeared to be a
more effective inducer of necrosis than C. orbiculare.

Plants previously induced with D. bryom‘ae had fewer lesions when challenged
with Altemaria porri, both in detached leaves at four days after challenge (DAC) (Figure
2.4A), and also in plants in the greenhouse at seven DAC (Figure 2.4B). T-439 had the
lowest number of lesions per detached leaf compared to cultivar Altisimo, while Hoopla
did not differ from them (Figure 2.4 A). Moreover, Altisimo was not different from the
control treatment. Plants previously induced with C. orbiculare were not different from
the control. Cultivars Hoopla and T-439 had a lower number of lesions of A. porri
compared to Altisimo in the greenhouse experiment (Figure 2.4 B).

The length of lesions caused by A. pom“ was smaller on plants previously induced
with D. bryoniae compared to those induced by C. orbiculare or the control plants, in the
detached leaf experiment for cultivar T-439 (Figure 2.4 A), but these differences were not
observed when leaves were challenged in the greenhouse experiment (Figure 2.4 B)

Experiment Three.

Necrotic lesions on plants induced with D. bryom'ae appeared at four DAI. The
number of inoculation sites with necrotic lesions and lesion length increased from four to
nine DAI (Table 2.1).

After challenge, the number of lesions caused by A. porri was smaller in the
induced plants compared with the non-induced at 9, 10 and 12 DAC, (Figure 2.5A). The

lesion length was smaller in induced plants at 10 DAC and a similar response was

31

observed at 12 DAC (Figure 2. B). This shows that T-439 onion plants were effectively
induced by inoculation with D. bryoniae.

Experiment Four.

Necrotic lesions appeared two days after induction treatment on all three onion
cultivars used in this experiment. The number of necrotic lesions and the lesion length
caused by D. biyom'ae were similar among the three cultivars at two and three DAI
(Table 2.2).

At challenge, the number of lesions caused by A. porri was smaller in the induced
plants compared to the non-induced plants in cultivar Hoopla and no differences were
found in cultivar T-439. Cultivar Altisimo tended to have fewer number of lesions on
induced plants but differences were not significant (Figure 2.6 A). This shows that onion
plants were effectively induced with D. bryoniae on cultivar Hoopla. However, lesion

length was. similar among treatments (Figure 2.6 B).

32

DISCUSSION

Localized treatment of plants with virulent or avirulent pathogens that cause
necrotic lesions can result in the local or systemic induction of disease resistance in the
treated plant to subsequent pathogen attack (Hammerschnridt, 1999). According to
Kombrink and Schmelzer (2001), necrosis accompanies many non-host interactions and
is considered to be an important mechanism of resistance. Both pathogens induced
localized necrosis, but D. bryoniae caused more necrosis than C. orbiculare in cultivar T-
439 at seven DAI in Experiment One, (Figure 2.1A). The number of necrotic lesions
caused by D. bryoniae was higher at four and eight DAI in experiment two for cultivar
Hoopla (Figure 2.3A). Cultivar T-439 had a slightly higher number of necrotic lesions
eight DAI although the differences were not significant. D. bryoniae had a trend to cause
slightly larger lesions than C. orbiculare in cultivars Hoopla and T-439 in Experiment
One (Figure 2.13). D. bryoniae caused significantly larger lesions in cultivar Hoopla in
Experiment Two, at four and at eight DAI, and larger lesions in cultivars T-439 and in
cultivar Altisimo eight DAI. (Figure 2.3B).

The level of protection afforded by induced resistance may depend on the
organism used for induction and even the extent of necrosis caused by the inducing
pathogen (Sticher et al., 1997). Inoculations with D. bryom’ae were more effective in
inducing of necrosis than C. orbiculare. Plants previously induced with D. bryoniae
showed fewer lesions when challenged with Altemaria porri on cultivar T-439, (Figure
2.2). Similar results were found on detached leaves in cultivars Hoopla and T-439 and on

plants inoculated in the greenhouse in Experiment Two for the three cultivars (Figure

33

2.4A and 2.4B). The lesion length was smaller on plants previously induced with D.
bryoniae on detached leaves for cultivar T-439 in Experiment Two (Figure 2.4A). It was
a clear trend to have a smaller number of lesions and smaller lesion length on plants
previously inoculated with D. bryoniae in this experiment. However, in the greenhouse
experiment the lesion length showed no differences among cultivars or inducing
treatments (Figure 2.4B). Cucumber plants prior inoculated with Colletotrichum
orbiculare became systemically protected against subsequent challenge by the same
pathogen, and showed a reduction in number of lesions and lesion length at challenge
(Kuc et al., 1975). Lesion length caused by A. porri was reduced where plants were
induced with D. btyom'ae in Experiment Three at 10 DAC.

Duration of induced resistance expression can also be a limitation for its practical
use in the field. Penetration of C. lagenarium was reduced on cucumber plants by
systemic resistance induced by inoculating the first leaf with the same fungus. This
reduction occurred when plants were challenged 7 to 14 days after induction. When
plants were challenged 21 to 28 days after induction that reduction did not occur (Dalisay
and Kuc, 1995). Therefore, plants were protected for only a certain time after being
induced. In the greenhouse in Experiment Three, after inducing with D. bryoniae,
resistance lasted for 12 days after T-439 was challenged with A. porri. (Figures 2.5A,
2.5B). The number of lesions (Figure 2.5A) caused by A. porri was less on the induced
plants compared to control plants 9, 10 and 12 DAC in this experiment. The length of
lesion was significantly smaller 10 DAC (Figure 2.5B) and tended to be smaller 12 DAC.
This means that once the mechanism of induced resistance was activated, the ability to

restrict initial infection was present up to 12 days in our experiment. In our results,

34

different cultivars had different levels of induction. For example, after challenge,
previously induced Hoopla and T-439, had a similar number of lesions caused by A.
porri, and no differences in the lesion length in Experiment One (Figure 2.2). After
challenge on detached leaves in Experiment Two, T-439 previously induced with D.
bryom'ae, had fewer lesions per leaf than Altisimo.

There were no significant differences in the number of lesions per leaf between
Altisimo and Hoopla, or between Hoopla and T-439 in Experiment Two. Altisimo did not
differ from the control treatment in the number of lesions per leaf. Nevertheless, there is a
clear trend that plants previously induced with D. bryom'ae had slightly fewer lesions
compared to control and plants induced with C. orbiculare. There were no differences in
lesion length among the three cultivars or between control and inducing treatments
(Figure 2.4A). After challenge on onion plants in the greenhouse in Experiment Two,
after induction with D. bryom'ae, Altisimo had the highest number of lesions of A. porri
compared to H00pla and T-439. There were no differences in lesion length among the
three cultivars (Figure 2.43).

Sticher et al., (1997) stated that the protection of plants against diseases is
dependent on not only the organism used for the inducing treatment, but also on the
extent of the necrosis. Cohen and Kuc (1981) reported that the mechanism of induced
resistance in tobacco exhibited specificity because plants previously induced with
Peronospora tabacina were protected against P. tabacina, whereas plants that were
induced with Phytophthora parasitica. var. nicotiana or Thielaviopsis basicola were not
protected. In our experiments D. bryoniae was more effective in inducing the resistance

than C. orbiculare. D. bryoniae caused a higher number of necrotic lesions in T-439 in

35

Experiment One compared to C. orbiculare. In Experiment Two, D. bryom‘ae also had a
significantly higher number of necrotic lesions and greater lesion length compared to C.
orbiculare. The greater the number of necrotic lesions on the induced leaf, the higher the
resistance of the cucumber plants after challenging with C. orbiculare (Hammerschmidt
and Yang-Cahman, 1995). This higher number of necrotic lesions observed on onion
seedlings after inducing with D. bryom'ae could explain why D. bryoniae was more
effective in inducing the resistance than C. orbiculare. In addition, the length of lesions
caused by D. bryom'ae was greater than those caused by C. orbiculare, such as in
Experiment two. This is in accordance with Descalzo et al. (1990), who reported that C.
lagenarium was less effective than D. bryoniae in inducing the resistance of cucumber
against gummy stem/leaf blight caused by D. bryoniae under laboratory conditions.

Pathogens like Altemaria brassicicola, Botrytis cinerea and Pythium sp. use a
common virulence strategy that involves rapidly killing plants cells to obtain nutrients,
referred to as necrotroph pathogens (Kunkel and Brooks, 2002). D. bryoniae uses
pectolytic enzimes for penetration (Chilosi and Magro, 1998) while C. orbiculare infects
their host through formation of appressoriurn (Kovats et al., 1991). These different
mechanisms for host invasion could explain the differences of induction of resistance of
onion seedling between D. bryoniae and C. orbiculare.

Cultivar Altisimo is related to Sweet Spanish onion type, which is commonly
more susceptible to diseases. Altisimo has light-green foliage, a characteristic related to
more disease susceptibility compared with cultivars of dark green color, which have

thicker cuticles, and this provides more resistance to A. porn“ (Bock, 1994).

36

In Experiment Four there were no differences among the three cultivars in the
lesion length caused by A. porri after inducing with D. biyoniae (Figure 2.6).

According to Dean and Kuc (1986) disease resistance may be expressed at many
levels such as reduced penetration and establishment, lesion expansion, and sporulation.
J enns and Kuc (1977) reported that cucumber inoculated with tobacco necrotic virus was
protected against C. lagenarium. They found a reduction in the lesion number and lesion
size, and suggested that the failure of pathogens to penetrate the tissue might explain the
reduction in lesion number and size. Richmond et al. (1979) also found a reduction in the
number of lesions on induced leaves of cucumber compared with the control. However,
infection hyphae that succeeded in penetrating appeared no different in induced and
control tissue.

Velasquez (2002) stated that cucumber cultivars induced with acibenzolar-s-
methyl (ASM) responded differently when they were inoculated with C. orbiculare or D.
bryoniae. When the leaves were infected with C. orbiculare all the cultivars had the same
number of lesions but they differed in their ability to restrict lesion development after
initial infection. When plants were challenged with D. bryoniae he found differences in
the number of lesions among cultivars but not in the length of lesions. He concluded that
the response of the plants against D. bryom'ae was more effective in stopping the initial
infection rather than the spread of the fungus within the tissue.

Stenzel et a1. (1985), studying induced resistance on the efficiency of powdery
mildew haustoria in wheat and barley, stated that the formation of primary haustoria was
reduced, the colonies on induced plants remained smaller, and reproduction was reduced

by 70% compared to non-induced plants. Sahashi and Shishiyarna (1985) found that on

37

barley plants previously induced against Erysiphe gramim's, the induction of resistance
was expressed as a decrease of frmgal penetration and restriction of hyphal growth. In our
research we found significant reduction in the number of lesions of A. porri after the
plants were induced with D. bryom‘ae. However, the length of lesions was less affected.
Therefore, we assume that the mechanism of induce resistance in onion against A. porri
was effective against the site of pathogen penetration rather than against pathogen spread.
This agrees with the results of Richmond et al (1979).

Our study demonstrates that it is possible to induce resistance in onions against
Altemaria porn“, and inoculation with D. bryoniae was more effective than C. orbiculare

to induce resistance in onion plants against A. porri.

38

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induced by rhizosphere bacteria. Annu. Rev. Phytopathol. 36:453-483.

Van Loon. LC. 2000. Systemic induced resistance. In: Slusarenko, A.; Fraser, R. S.
S.; and Van Loon, L.C (eds), Mechanisms of resistance to plant diseases, 521 -
574, Kluver, Netherlands.

Velasquez, L. A. 2002. The pathogenesis related protein, chitinase, and its role in the
systemic acquired resistance phenotype in cucumber plants (Cucumis sativus L.).
Dissertation.1365 89 THS Mich. State Univ. East Lansing Mich.

Vincelli, P. C. and J. W. Lorbeer. 1987. Sequential plant for timing initial fungicide
application to control Botrytis leaf blight of onion. Phytopathology,
77:1302-1303.

Vincelli, P. C. and J. W. Lorbeer. 1989. Bligth-Alert: A weather-based predictive

system for timing fimgicide applications on onion before infection periods of
Botrytis squamosa. Phytopathology 79:493-498.

43

Table 2.1. Number of necrotic lesions and lesion length measured at four, six and nine

days after inoculation (DAI) with D. bryoniae on onion cultivar T-439, 2002.

 

Number of necrotic lesions Lesion length (mm)
on induced leaf

 

4 DAI 2.4 3.6
6 DAI 4.5 5.9
9 DAI 6.7 6.0

 

Table 2.2. Number of necrotic lesions and lesion length measured at two and three days

after inoculation (DAI) with D. bryoniae on onion cultivars Hoopla, T-439 and Altisimo.

 

Number of necrotic lesions

 

 

 

 

on induced leaf Lesion length (mm)

2 DAI 3 DAI 2 DAI 3 DAI
Hoopla 8.0 9.2 4.3 6.3
T-439 7.6 8.4 4.1 5.2
Altisimo 5.8 9.2 2.6 4.9
LSD 5% NS NS NS NS

 

44

 

 

 

 

 

 

   

 

 

 

 

 

   

 

 

 

 

Lesion length (mm)

 

 

 

 

 

 

 

. (A)
7 - — :4 DAI
. - 7 DAI
:5 6 ‘
g. 5 - m
g , LSD 5 °/.
E 4 -
'8
o .
g 3 1
c...
o I
.—
‘E 2 ‘
Z .
1 d
0 ':_.,.=_.,_
COMIOI Control D. bryoniae D. bryoniae C. orbiculare C. orbiculare
T-439 Hoopla T-439 Hoopla T-439 H00pla
2.5 d (B) [:14 DAI
F" - 7 DAI
2.0 -
1'5 ‘ LSD 5%
1
1.0 -
0.5 -
d
0.0 r r r '
Control Control D. bryoniae D. bryoniae C, orbiculare C. orbiculare
r-439 Hoopla T-439 Hoopla r-439 Hoopla

Figure 2.1. Number of lesions per leaf (A), and lesion length (B) caused by Didymella
bryoniae or Colletotrichum orbiculare on onion leaves cvs. T-439 and Hoopla,

Experiment One, measured at four and seven days after inoculation.

45

Number of lesions per leaf

 

l7-
16—
15—
14—
13-1
12¢
11:
10—
9..
3..
7..
6-l
5H
4..
3...
2—1
1...

L

 

 

 

Figure 2.2 Number of lesions and lesion length caused by A. porri in detached onion

leaves on cvs. T-439 and Hoopla, five days afier challenge, inducing with Didymella

Control
T-439

 

Control
Hoopla

 

DI

LSD 5%

 

 

Hoopla

 

 

1:] Number of lesions
- Lesion length

 

 

 

D. bryoniae D. bryoniae C. orbiculare C. orbiculare
T439

T439 Hoopla

bryoniae or Colletotrichum orbiculare, Experiment One.

46

Lesion length (mm)

 

 

 

 

 

 

 

 

6
(A) [340!“
E 5.. LSDS% 8M
8' ‘1
8
.2 4.
E ‘
“5
g 3- Connol Corbiculare Dbryom'ae
E
Z 2_
T-439 l-kxpla AltisirmT-439 prla Altisirm T-439 Ibopla Altisirm
6..
. (B) 1:]4DAI
-8DAI
5-
E 41 LSDS°/o
go Cmtrol Cafiarlare Dbiyoniae
.1! 3-
a . |

 

 

 

 

r439 IboplaAlusinnT439 l-bquaAltrsirmT 39 l-bquaAltrsirm

Figure 2.3. Number of lesions per leaf (A), and lesion length (B) caused D. bryoniae
or C. orbiculare, on onion leaves cvs. T-439, Hoopla and Altisimo, Experiment

Two, measured at four and six days after inoculation.

47

Number of lesions per leaf

 

 

Number of lesions per leaf

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

16 16
. (A) :Number of lesions .
14 _, - Lesion length _ 14
12 _ l:| C.orbiculare l- 12
j LSD 5% E
10 - Control D. bryoniae 10 a
I 2
8 4 8 r:
.3
U)
6 - 6 '3
l
4 - 4
2 - d 2
0 . 0
T439 Hoopla Altisimo T-439 Hoopla Altisimo T-439 Hoopla Altisimo
" (B) .
[:1 Number of lesions
1ST - Lesron length _15
- Control D. bryoniae C.orbiculare E
V
10 - F10 fl,
LSD 5% E)
. s
m
a)
..J
5 - 5
0 - 0

 

       

 

 

 

 

 

1-439 Hoopla Altisimo T-439 Hoopla Altisimo T-439 Hoopla Altisimo

Figure 2.4. Number of A. porri lesions and lesion length on detached onion leaves at four
DAI (A), or in the leaf above the detached one in plants in the greenhouse seven DAI (B), on
cvs. T-439, Hoopla, and Altisimo, after induction with D. bryoniae or C. orbiculare,

Experiment Two. Experiment Two, measured at four and six days after inoculation.

48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

‘ (A) [:IControl
-Induoed
7- P=0.025
. P=0.003
5-
J
5..
.13
c... 4-
o
'- 4
.8
S 3:
Z .
2d
11
0 r
9 DAC 10 DAC 12 DAC
8
. (B) |:IControl
-Induced
7+
P=009
6" P=0.03

 

 

Lesion length (mm)
A

 

 

 

 

 

 

 

 

 

9 DAC 10 DAC 12 DAC

Figure 2.5 Number of A. porri lesions (A) and lesion length (B), on onion leaf cv.
T-439, Experiment Three, after induction with Didymella bryoniae, measured at

nine, ten and twelve days after challenge.

49

11

 

. (A)
10-

1:] Control
- lnducded

9-

 

 

8-l
7-r LSDSV.
6—l

5-l

 

4-l

Number of lesions per leaf

3-l

2-

 

 

 

 

 

 

 

 

Hoopla T-439 Altisimo
Cultivars

 

‘ (3) 1: Control
‘8 - - Induced

16-

 

14-

 

" LSD 5%
12 -l

 

10-

 

Lesion length (mm)

 

 

 

 

 

    

 

l
Hoopla T-439 Altisimo

Cultivars

Figure 2.6. Number of lesions (A), and lesion length (B), caused by Altemaria porri in
onion leaves on cvs. Hoopla, T439 and Altisimo during challenge at six days, after

induction with Didymella bryoniae, Experiment Four.

50

CHAPTER III

EFFECT OF CHEMICAL RESISTANCE ACTIVATORS ON PURPLE BLOTCH
DISEASE OF ONION

51

ABSTRACT

Purple blotch, caused by Altemaria porri, is a disease of onions that affects
foliage. Severe infection may cause partial or total yield loss. Fungicide application is the
usual method to control purple blotch. A new technology for plant disease control is
based on the activation of the plant defense system. Disease resistance can be activated in
plants by exposure to certain microorganisms or by treatments with chemicals that trigger
specific genes associated with Systemic Acquired Resistance (SAR). Chemicals:
acibenzolar-S-methyl (ASM) at 20 ppm, DL—B-amino-n-butyric acid (BABA) at 10 mM,
and methyl jasmonate (MeJ A) at 10 mM were sprayed 3 times. All three treatments
included crop oil concentrate (Herbimax 1% "v/v"), and were compared to fungicide
chlorothalonil (1.68 kg a.i./ha); crop oil concentrate 1%; and untreated control, in 2001.
For 2002, the ASM treatment was deleted and MeJA plus a minimum rate of fungicide
was included. An A. porri spore suspension (8,500 spores/mL) was sprayed on the plants
82 days after planting. The inoculation occurred 7 days after the third application of all
chemicals. At 25 days after inoculation (DAI), fungicide and methyl jasmonate treated
plants had 10 and 11 lesions/plant, while untreated plants had 20 lesions/plant. At this
time, plants sprayed with ASM had 24 lesions/plant, and plants treated with BABA had
15 lesions/plant, which was not significantly different from the untreated plants in 2001.
In 2002, at 22 DAI, fungicide, MeJ A alone and MeJA plus fungicide had fewer lesions
than the untreated control. Although plants treated with fungicide or methyl jasmonate
showed less disease symptoms, there was no difference in total and marketable yield in

2001. Total and marketable yield was reduced in 2002 with Me] A application. The

52

percentage of marketable bulbs in storage was not different among chemical resistance

activators in both years. Spartan Supreme stored better than Altisimo and T-439.

53

INTRODUCTION

Purple blotch, caused by Altemaria porri, is a disease of onions that affects
foliage. Severe infection may cause partial or total yield loss (Nolla, 1927; Skiles, 1953).
Chemical application is the usual method to control purple blotch. Fungicides such as
chlorothalonil, copper oxychloride, metalaxyl plus mancozeb, mancozeb, and
tebuconazole, have been suggested to control purple blotch (Tahir et al., 1991, Pena et al.,
1992; Sugha and Tyagi, 1994). During a severe disease outbreak, mancozeb reduced
disease intensity by 6% and increased yield by 11% (Borkar and Patil, 1995). Although
chemical control has been used against purple blotch disease, the broad and often over
use of pesticides could cause damage to the environment, and may lead to resistance in
pathogens (Ozeretskovskaya, 1995; Hadisustrino et al., 1996).

The defense of plants to pathogens comprises constitutive barriers (Thomma et
al., 1998; Metraux et al., 2002) present in plants prior to any contact with pathogens or
herbivores, and inducible defense (Thomma et al., 1998). Exposure to various
microorganisms or other forms of stress can lead to the activation of defense mechanisms
(Metraux et al., 2002). Induced resistance depends on the recognition of a pathogen or
stress of the plant. Disease will occur if the pathogen is faster than induced response, if
no elicitors are produced or if suppressors prevent the plant defense reactions (Metraux et
aL,2002)

A new technology for plant disease control is based on the activation of the

defense of the plant system with the aid of low molecular weight synthesis molecules

54

(Cohen et al., 1999). Induced resistance, based on natural defense mechanisms of plants
is a very promising alternative approach to control plant diseases. This approach is
effective and economical and would reduce the dependence on pesticides (Suo and
Leung, 2002). Induced resistance is generally characterized by a reduction in the size and
or number of lesions that develop after inoculation of the induced plant with virulent and
sometimes, hypersensitive response inducing avirulent form of pathogens. The resistance
expressed is generally effective against a broad range of pathogens, is associated with the
production of PR proteins in the case of systemic acquired resistance (SAR), and is
mediated via salicylic acid dependent process (Hammerschmidt, 1999).

Chemical activation of disease resistance in plants represents an additional option
for growers to protect their crops from losses due to plant diseases. Against some
pathogens, like bacteria and viruses, it may be the best option for chemical control where
genetic resistance is not available or not sufficient. Against dynamic fungal pathogens
with a history of adaptation to fungicides or to resistant cultivars, the integration of this
new technology of activating broad-spectrum plant defenses with genetic resistance and
fungicides seems to offer a more sustainable plant health management system
(Oostendorp et al., 2001). There are indications that such adaptation of pathogens to
genetically resistant cultivars can indeed be slowed down by plant activators such as
acibenzolar-S-methyl (ASM) (Romero et al., 1998).

Systemic acquired resistance (SAR) is a potentially desirable strategy in achieving
IPM since it involves enhancing natural defense mechanisms in crops. Certain biological,
physical or chemical elicitors can be used to activate and/or boost natural disease

resistance in non-infected plant tissue. For example, gray mould on strawberry fruit was

55

suppressed by application of plant activator ASM (Terry and Joyce, 2000). The
accumulation of PR proteins in response to ASM may lead to increase protection against
infection by pathogens in rose (Suo and Leung, 2002). Cucumber plants treated with
ASM became resistant to scab caused by Cladosporium cucumerinum, and chitinase was
rapidly induced (N arusaka et al., 1999).

To Optimize resistance activation and yield benefits as well as to avoid negative
side effects on plant growth, the use of chemical resistance activators has to be adjusted
for each crop. The reduction of symptoms of powdery mildew on wheat was stronger
reducedwhen the mixture of the fungicides propiconazole and fenpropidin was compared
to ASM alone. However resistance activation by ASM showed a superior long lasting
protection (Oostendorp et al., 2001).

D, L-B-amino-n-butyric acid (BABA) or its 3-(S)-enantiomer has been reported to
activate disease resistance (Oostendorp et al., 2001). For example induction of PR1 has
been found in tomato after applications of this compound (Cohen et al., 1994). It was
also reported that activation is dependent of SA accumulation and lesion formation
(Siegrest et al., 2000).

On grapes, the non-protein amino acid BABA induced local and systemic
resistance against downy mildew, while other five isomers did not protect grapes against
downy mildew fungus. Concentrations of 25 ug/mL (0.25 mM) of BABA were sufficient
to prevent tissue colonization by the fungus. No differences were found between cultivars
in their response to BABA. Activity of BABA lasts up to 14 days after treatment with the
chemical resistance activator on leaf discs. The persistence of BABA in intact growing

plants will probably decline with time due to dilution and (Cohen et al., 1999). Low

56

concentrations of BABA such as 10 mM were sufficient to protect lettuce against downy
mildew caused by Bremia lactucae, and the protection lasted for at least 15 days and was
systemic (Pajot et al., 2001). BABA concentrations higher than 20 mM provided
complete protection against downy mildew, caused by Phytophthora parasitica, but they
were phytotoxic (Silue et al. 2002).

Pea, cucumber, cotton, tobacco, pepper, tomato, grape, melon, sunflower,
kohlrabi, corn, pearl millet, cauliflower and Arabidopsis thaliana have been reported to
be induced by BABA (J akab et al., 2001). Cohen et al. (1999) demonstrated that
resistance in grapes induced by BABA depends on the amount that was translocated,
which varied from 3.5 to 30% from treated to non-treated leaves. They showed that the
variation was age-dependent and young treated leaves translocated BABA better than did
older leaves.

Disease resistance is regulated by multiple signal transduction pathways in
Arabidopsis. Salicylic acid, jasmonic acid and ethylene function as key signaling
molecules (Clarke et al., 2000). Methyl jasmonate had a protective effect against
Altemaria brassicicola in Arabidopsis thaliana (Thomma et al., 1998; Ton et al., 2002).
MeJ A induced a defense response that was activated in the plant and was not the result of
negative effect on the pathogen, (Thomma et al., 1998). Moreover, Vijayan et al. (1998)
reported that mutants of Arabidopsis that are not able to accumulate jasmonates have
been reported to be extremely susceptible to root rot caused by Pythium mastophorum
(Drench). Exogenous application of Me] A protected the plants and reduced the incidence
of the disease similar to that of the wild type plant. Young rice seedlings had induced

resistance against Pyricularia grisea (Woo et al., 2001).

57

Jasmonic acid is responsible for the induction of many changes in plant resistance
that occur following herbivore attack (Thaler, 2002). In tomato, for example, plants
induced with jasmonic acid had 40% less damage due to the herbivore compared with
control plants (Thaler, 1999). Plant traits can act as defenses against herbivores both by
reducing herbivore performance directly and by increasing the effectiveness of the
natural enemies of herbivores.

Disease resistance can be activated in plants by exposure to certain
microorganisms or by treatments with chemicals that trigger specific genes associated
with Systemic Acquired Resistance (SAR). The objective of this work was to determine
the effect of chemical resistance activators on purple blotch disease caused by Altemaria

porri, and on onion yield and quality.

58

MATERIALS AND METHODS

Location

The experiments were carried out at the Muck Soil Research Station, Laingsburg,
MI, during 2001 and 2002. The soil was Houghton Muck, Euic, mesic, typic Medisaprist
80% organic matter, pH 6.3.
Cultural practices.
The plots were maintained with fertilizer and pesticides throughout the growing season,

as recommended commercially (Bird et al., 2002; Zandstra et al., 1996).

2001 Experiment. 'Spartan Supreme" onion, Seed Works, San Luis Obispo, CA;
'Altisimo' onion, Bejo Seeds Inc., Geneva, NY; and 'T-439' onion, American Takii,
Salinas, CA, were planted on May 3, 2001. Seeds were planted on raised beds for all
plots at approximately 2 cm with a Gaspardo precision vacuum planter. The plot size was
1.62 m wide and 15 m long, with three rows, 41 cm apart per bed. The experiment was
designed as a split plot, with four replications. Each bed was the main plot, where
chemical resistance activators were applied. Every row per bed was a subplot,
corresponded to each cultivar. Six treatments were applied on the main plot in 2001: crop
oil concentrate solution 1% "v/v", fungicide chlorothalonil 1.68 kg.ha", Acibenzolar-S-
methyl (ASM) 20 ppm (Syngenta Crop Protection, Greensboro, NC); DL—B-amino-n-
butyric acid (BABA) at 10 mM, (Sigma Chemical Co. St Louis, MO); methyl jasmonate

at 10 mM (MeJ A) (Aldrich Chemical Company, Inc. Milwaukee, WI); and untreated

59

control. The three chemical resistance activators contained crop oil concentrate (COC)
1 % "v/v".

Chemical sprays were applied three times before inoculation with a C02 backpack
sprayer with 2 flat fan 11002 nozzles on the boom, which delivered 234 L.ha'1, at a
pressure of 234 KPa, and a speed of 5.7 km/h. The first spray occurred at the 3rd leaf
stage, 50 days after planting (DAP) (June 22); the second application at the 5th leaf stage,
60 DAP (July 2); and the third application at 6th -7th leaf stage at 75 DAP (July 17). One
week after the third application all plots were sprayed with Altemaria porri (8,500
spores/mL). Plants received irrigation (22 mm) the afternoon prior to inoculation, which
occurred at 7 pm (the temperature was 32 C, and relative humidity was 90%). All plots
were irrigated every other day the week following inoculation to assure good humidity
conditions for the infection process. Purple blotch symptoms appeared approximately 10
days after inoculation. Fungicide was sprayed on treatment two on July 20, 27 and
August 3, 10, 15 and 21, using chlorothalonil (1 .68 kg a.i. ha“), alternated with copper
hydroxide (0.84 kgha") and manzate 1.09 kg. ha".

Parameters measured

To determine disease incidence, 30 consecutive plants per plot were evaluated for the
presence of Altemaria porri, and the percentage of disease incidence was calculated.
Disease severity was evaluated 13 and 25 days after inoculation (DAI) in 2001. The
length of lesions of each spot in each leaf was measured in ten consecutive plants, using a

digital caliper (Mitutoyo model CD-6" CS, Mitutoyo, Aurora IL).

Harvest, curing and storage evaluations.

60

Twelve meters of row for each cultivar were harvested on October 4, 2001. The
onions were hand-pulled, and bulbs were topped in the field with a roll topper, and cured
for approximately two weeks in ambient air. Onions were then graded and weighed using
three categories, small (< 50 mm), medium (2 50mm 5 80mm), and large (2 80 mm).
The sum of these three categories corresponded to total yield, while the sum of medium
and large bulbs was the marketable yield. Two crates (approximately 80 kg) of bulbs
greater than 50 mm in diameter were placed in common storage. Storage temperature
declined as ambient temperature declined, and was maintained at 1-3 °C during all
storage period.

At 138 days after harvest (DAH), bulbs were graded as marketable, rotted or
sprouted for each plot. Fifty visually good bulbs were cut at 173 DAH and graded as
marketable or rotted due to internal decay. The percentage of marketable bulbs was
calculated.

Statistical analysis

Analysis of variance was performed with MSTATC. Mean separation was
performed with Fisher’s LSD. The percentage of marketable bulbs was transformed used
arcsine square rot of the percentage (Snedecor and Cochran, 1967) to obtain a normal

distribution of means, then was analyzed by analysis of variance.

2002 Experiment. The same cultivars used for 2001 experiment were planted on April
28, 2002, as described above. The experiment was designed as a split plot, with three
replications. Six beds of each cultivar were the main plots and on each bed the chemical

resistance activators, the subplots, were applied as described above. COC solution 1%

61

"v/v", fungicide chlorothalonil 1.68 kg.ha’l, DL-B-amino-n-butyric acid (BABA) at 10
mM, methyl jasmonate (MeJ A) at 10 mM, methyl jasmonate (MeJ A) at 10 mM plus
fungicide (minimum rate) chlorothalonil 0.84 kg a.i. ha'l, and untreated control were
applied to each cultivar. The three chemical resistance activators included COC 1% 'v/v".
Treatments were applied three times before inoculation as described above. The first
spray was applied at the 3rd leaf stage, 52 DAP (June 19); the second application at the
5th leaf stage, 66 DAP (July 1); and the third application at 6th -7th leaf stage, 73 DAP
(July 8). On the treatment combining the minimum rate of firngicide and MeJ A,
chlorothalonil was applied at least two days after the Me] A spray to avoid any possible
negative interaction between the two chemicals. After the third application, all plots
were sprayed with Altemaria porri (8,500 spores/mL), 80 DAP. Plants received irrigation
(10.5 mm) in the afternoon previous to inoculation, which occurred at 7 pm.
Temperature was 30 C, and relative humidity was 90%. All plots were irrigated every
other day the week following inoculation to assure good humidity condition for the
infection process. Purple blotch symptoms appeared approximately 8 days after
inoculation. Fungicides were sprayed on onion plants for fungicide treatment and for
MeJA plus fungicide at minimum rate, on August 5, 12, 19 and 26, with chlorothalonil at
2.53 or 1.26 kg a.i.ha'l, respectively; alternated with maneb (2.68 or 1,34 kg. ha'l), or

iprodione (0.56 kg. ha'1 or 0.28 kg. ha").
Parameters mea_sr_1red.

Disease incidence and disease severity was evaluated as described above, 15 and 22 DAI.

Harvest, curing and storggg evaluations.

62

Nine meters of row of each cultivar were harvested on September 19, 2002. The
onions were hand-pulled, topped, cured, graded and stored as described above. At 161
and 189 DAI-I all bulbs were evaluated for internal decay. The percentage of marketable
bulbs was calculated for each date. The data was transformed using arcsine square root of

the percentage (Snedecor and Cochran, 1967), then was analyzed by analysis of variance.

63

RESULTS

2001 Experiment. The incidence of purple blotch was significantly lower on fungicide
treated plants compared to other treatments 13 days after inoculation (DAI) (Table 3.1).
At 25 DAI plants treated with fungicide had the lowest disease incidence (Table 3.2).
There was not a significant interaction among cultivars and chemical resistance
activators. Although Me] A was not significantly different from the untreated control, it
had less disease incidence compared to other treatments at 25 DAI. MeJ A had less
disease incidence compared to COC at 25 DAI. MeJ A tended to have less disease
incidence than ASM and BABA at 25 DAI. Disease incidence was not different among
cultivars in 2001, and Altisimo had higher disease incidence compared to Spartan
Supreme or T-439 25 DAI.

The number of lesions of purple blotch per plant was significantly lower on
fungicide treatment at 13 DAI and 25 DAI and the differences were significant at P =
0.08 at 25 DAP (Table 3.1 and 3.2).

ASM did not differ from the untreated control or COC 13 DAI, and it had more
lesions (24) than the untreated control (20) at 25 DAI. Plants treated with BABA did not
differ from untreated control in the number of lesions per plant both 13 and 25 DAI.
MeJA did not differ from untreated control 13 DAI and 25 DAI. However, it tended to

reduce the number of lesions per plant (11) compared to untreated control plants (20),

and MeJ A was significantly different fi'om COC 25 DAI. Plants treated with BABA and
MeJA did not differ in the number of lesions per plant 13 and 25 DAI.

There were no differences in the number of lesions of purple blotch among the
cultivars at 13 DAI (Table 3.1). Altisimo had significantly higher number of lesions than
Spartan Supreme and T-439 25 DAI (Table 3.2). There was not a significant interaction
between cultivars and chemical resistance activators.

No differences were observed among cultivars or chemical resistance activators at
13 and 25 DAI (Table 3.1, 3.2) in length of lesions.

Total and marketable yield (Table 3.3) was not significantly different among
cultivars or chemical resistance activators in 2001. There was no significant interaction
between cultivars and chemical resistance activators. The percentage of large and
medium bulbs was not significantly different among chemical resistance activators (Table
3.4). Altisimo had higher percentage of large bulbs compared to Spartan Supreme and T-
439.

The percentage of marketable bulbs at 138 and 173 DAH was not significantly
different among chemical resistance activators. BABA and Mel A treatments did not
result in negative effect on storage quality of onion bulbs in 2001 (Table 3.5). Spartan
Supreme stored better at 138 DAH (98% marketable bulbs) and similar to cultivar
Altisimo (93%). T—439 was not significantly different compared to cultivar Altisimo
(87%). At 173 DAH, Spartan Supreme stored significantly better than cultivars Altisimo
and T-439. There was a significant interaction between cultivars and chemical resistance

activators at 173 DAH (Table 3.6).

65

Plants treated with ASM had the highest percentage of marketable bulbs in
cultivar Altisimo (94%) compared to Spartan Supreme (87%) and T-439 (82%), but we
do not have a clear explanation for this. MeJA treated plants had no differences in the
percentage of marketable bulbs 90%, 92% and 91% for S. Supreme, Altisimo and T-439
respectively. Altisimo had the lowest percentage of marketable bulbs with BABA (80%),
while Spartan Supreme and T-439 had 90% and 87% of marketable bulbs respectively.

2002 Experiment. The incidence of purple blotch was significantly lower on
fungicide, BABA, Me] A, and Me] A plus firngicide treated plants compared to untreated
control or only surfactant at 16 DAI (Table 3.7). At 22 DAI, fungicide, MeJ A alone and
MeJ A plus chlorothalonil at minimum rate also had less disease incidence compared to
untreated control or only surfactant (Table 3.8). BABA had significantly more disease
incidence than fungicide, MeJ A, and Me] A plus firngicide 22 DAI.

The number of lesions of purple blotch per plant was significantly lower on
fungicide (10), MeJA alone (11) or on Me] A plus chlorotalonil at minimum rate (7)
compared to untreated control (29) or crop oil concentrate (29) 16 DAI (Table 3.7). The
number of lesions on BABA treated-plants (16) was not significantly different from
chlorothalonil and Me] A 16 DAI. The number of lesions of purple blotch was
significantly lower on chlorothalonil (22), on MeJA (18) and on MeJ A plus
chlorothalonil treated plants (14) compared to untreated control (63) or crop oil
concentrate (54) 22 DAI (Table3.8). The number of lesions was not significantly different
among cultivars 16 and 22 DAI.

The lesion length was smaller on firngicide treated plants compared to the

untreated control at 16 DAI (Table 3.7). Me] A and MeJ A plus chlorothalonil did not

66

differ significantly fi'om chlorothalonil-treated plants in the length of lesion 16 DAI.

Me] A and MeJ A plus chlorothalonil were not superior to untreated control in the length
of lesion, but they were better than BABA-treated plants. The lesion length was similar
among chemical resistance activators, although it was significantly higher on firngicide
treated plants 22 DAI. There were no differences in the length of lesions among cultivars
at 16 and 22 DAI. There was no interaction between chemical resistance activators and
cultivars on disease incidence, number of lesions or lesion length of purple blotch.

Total and marketable yield was significantly reduced by Me] A and MeJ A plus
chlorothalonil compared to the untreated control, crop oil concentrate, and chlorothalonil
treated plants (Table 3.9). Altisimo had significantly higher total and marketable yield
compared to Spartan Supreme and T-439. There was no significant interaction between
chemical resistance activators and cultivar on total and marketable yield.

The percentage of large bulbs was significantly reduced by MeJ A and MeJ A plus
fungicide compared to other treatments. (Table 3.10)

The percentage of marketable bulbs 133 and 161 DAH was not significantly
different among chemical resistance activators (Table 3.11). Cultivar Altisimo had the
lowest percentage of marketable bulbs among the three cultivars while Spartan Supreme
stored the best also in 2002. There was not significant interaction between chemical
resistance activators and cultivars on the percentage of marketable bulbs in storage 133

and 161 DAH.

67

DISCUSSION

Inbar et al. (1998) stated that elicitors of plant defensive systems (such as ASM)
could induce biochemical changes that enable the plant to reduce the incidence of
diseases. In our research MeJ A treated plants had a slight decrease in the incidence of
Altemaria porri 25 DAI in 2001 (Table 3.1). In 2002 MeJA alone and MeJ A plus
fungicide at minimum rate significantly decreased the incidence of the disease 16 and 22
DAI (Table 3.7, 3.8). This agrees with the finding of Inbar et a1. (1998).

Induced resistance is expressed as reduction in lesion number, size, spore
production or pathogen multiplication (Lucas, 1999). Cucumber plants inoculated with
Colletotrichum orbiculare became systemically protected against subsequent challenge
by the same pathogen, and showed a reduction in number of lesions and lesion length
after challenge (Kuc et al., 1975). The number of lesions caused by Altemaria porri was
reduced by chlorothalonil application 13 DAI in 2001 but was not significantly reduced
at this time by the chemical resistance activators. At 25 DAI, chlorothalonil and MeJ A
tended to reduce the number of purple blotch lesions (Table 3.2). Altisimo had higher
number of lesions of purple blotch at this time. Spartan Supreme, with deeper dark green
foliage was found to be less susceptible to diseases compared to cultivar Altisimo, while

cultivar T-439 was in-between.

68

ASM treated plants tended to have a slightly higher munber of lesions of purple
blotch than untreated controls 25 DAI in 2001 (Table 3.2). BABA treated plants were not
significantly different compared to MeJ A in number of lesions in 2001.

The number of lesions of purple blotch was significantly reduced compared to
untreated control by chlorothalonil, Me] A and Me] A plus chlorothalonil at the minimum
rate 16 and 22 DAI in 2002 Experiment (Table 3.7 and 3.8). BABA had slightly higher
number of lesions of purple blotch compared to MeJ A, and significantly higher than
chlorothalonil and Mel A plus chlorothalonil at minimum rate, 16 DAI in 2002 (Table
3.7). Tobacco plants induced with salicylic acid reduced the number of lesions of tobacco
mosaic virus as well as lesion size (Raskin, 1992). On cucumber, plants prior inoculated
with Colletotrichum orbiculare became systemically protected against subsequent
challenge by the same pathogen, and showed a reduction in number of lesions and lesion
length at challenge (Kuc et al., 1975). The reduction in the number of lesions of purple
blotch found in our experiments with MeJ A application agrees with the finding of these
authors.

The lesion length was not different among treatments in 2001 (Table 3.1, 3.2) but
it was smaller (5 mm) on chlorothalonil treated plants compared to the untreated control
(7 mm) 16 DAI in 2002 (Table 3.7). However, 22 DAI chlorothalonil had longer lesion
length compared to the untreated control (Table 3.8). There were no significant
differences among other treatments 22 DAI. Therefore, there is no clear explanation for
the difference with chlorothalonil treatment. These results do not agree with Kuc et al.

(1975) but are in accordance with Lucas (1999).

69

Cucumber cultivars induced with ASM responded differently when they were
inoculated with C. orbiculare or D. bryoniae. When the leaves were infected with C.
orbiculare all the cultivars had the same number of lesions but they differed in their
ability to restrict lesion development after initial infection (Velasquez, 2002). When
plants were challenged with D. bryoniae there were differences in the number of lesions
among cultivars but not in the length of lesions. Velazquez concluded that the response of
the plants against D. bryoniae was more effective in stopping the initial infection rather
than the spread of the firngus within the tissue. The different strategy of infection of each
fungus might be involved in the differential resistance against D. bryoniae in cucumbers
(Velasquez, 2002). C. orbiculare form appressorium (swollen tip of a hypha or germ tube
that facilitate attachment and penetration to the host tissue; Agrios, 1997) to infect plant
material (Kovats et al, 1991) and D. bryoniae uses pectolytic enzymes to enter the host
(Chilosi and Magro, 1998). Cucumber plants induced with C. lagenarium seem to stop
pathogen spread through reduction of appresoria and increasing ligrlification of cells
(Kovats et al., 1991). Velasquez (2002) stated that overexpressing the SA dependent
pathway by ASM application could have detrimental effects on the jasmonic/ethylene
pathway that confers resistance against insects and pathogens. He hypothesized that the
jasmonic acid dependent pathway could be a better fit to expression of resistance against
D. bryoniae because of its mode of infection. In his experiments he had reduction in the
number of lesions of D. bryoniae and reduction in the lesion size with C. lagenarium. In
our experiments with Me] A application we observed reduction in the number of lesions.

This supports Velasquez’ finding.

70

Foliage wetting and coverage, solubilization of the leaf epicuticular wax, and
increasing herbicide penetration are properties of adjuvant. Therefore, the addition of
adjuvant such as surfactants or crop oil concentrates to spray solutions, increase the foliar
absorption and activity of the chemical. The COC Herbimax we used had the ability to
solubilize and alter the epicuticular wax of quackgrass (Elitrigia repens) leaf cuticle
(Wanamarta, 1987). In our study we found plants treated with COC had a higher number
of lesions caused by Altemaria porri l3 and 25 DAI in 2001 (Table 3.1 and 3.2),
suggesting that it might favor the pathogen penetration into the leaf tissue in this
treatment. Moreover, COC did not affect the inducing of resistance by the chemical used
in these experiments.

Even though benzothiadiazole is reported as a good chemical activator in different
crops such as tobacco, wheat, Arabidopsis (Lawton et al., 1996) the application of ASM
did not work effectively in onion against Altemaria porri. Systemic acquired resistance
(SAR) pathway has been reported not to work against Altemaria species (Thomma et al.,
1998). However Thomma et al. (1998) showed that resistance to Altemaria brassicicola
may be boosted by exogenous application of MeJ A. He stated that it might be feasible to
develop jasmonic acid-mimicking compounds that protect crops against economically
important pathogens. The activity of such compounds may be complementary to that of
salicylic acid-mimicking chemicals such as INA and benzothiadiazoles.

Jasmonic acid and methyl jasmonate caused an increase in the expression of
defense response genes on cell suspension cultures (Gundlach et al., 1992). In

Arabidopsis thaliana, Systemic Acquired Resistance (SAR) and Induced Systemic

71

Resistance (ISR) are effective against pathogens such as Pseudomonas syringae pv.
tomato (Clarke et al., 2000; Van Wees at al., 2000).

The salicylic acid dependent defense responses seem to be effective against
biotrophic pathogens (that need living cells to get their food) such as Peronospora.
parasitica. Resistance to this type of pathogen is associated with a hypersensitive
response leading to nutrient depravation of the pathogens that thrive on them. In contrast,
J A and ethylene- dependent defense responses seem to be more effective against
necrotrophic pathogens such as B. cinerea. J A—deficient and J A insensitive mutants of
Arabidopsis display higher susceptibility to the species of necrotrophic soil borne
pathogen Pythium (Thomma et al., 2001).

Altemaria brassicicola, Botrytis cinerea and Pythium Sp. use a common virulence
strategy that involves rapidly killing plant cells to obtain nutrients, and are referred to as
necrotrophic. Necrotrophic pathogens are normally controlled by J A pathway (Kunkel et
al, 2002). Traw et al. (2003) reported that herbivore damage and necrotrophic pathogen
infection cause rapid increases in jasmonic acid, whereas biotrophic pathogen infection
causes rapid increases in salicylic acid. In our experiments we found inducing of
resistance against Altemaria porri using Me] A but not ASM. Therefore, we could
assume that SAR is not involved in induced resistance in onions against Altemaria porn",
and could be mediated throughout jasmonate pathway.

In order for induced resistance to be used as a pest management technique, the
effects on plant yield must be considered (Thaler, 2001). Ideal inducer treatments should
not cause any detrimental effects such as reduction in growth or yield by using energy for

defense instead of growth or production (Descalzo et al., 1990). Plant defense should

72

provide benefits for the defended plant. However, investment in defense can lead to
unavailable resources for grth or other important processes (Heil, 2001) The steps of
the signaling pathway that lead to induced resistance require gene expression and
consequently consume energy (Buell, 1999). Smedegaard-Petersen and Stolen (1981)
reported that a successfirl resistance response by barley against powdery mildew requires
energy, and finally leads to a reduction in grain yield.

Although disease incidence and the number of lesions of purple blotch were
reduced by fungicide or MeJ A treatments, total and marketable yield was not
significantly different in 2001 (Table 3.3). The lack of differences between treatments
could be due to the considerable capacity of onion plants to recover from loss of leaf area
without decrease in yield, as long as growing conditions allow the plant to recover (Wien
et al, 2002). In 2002 total and marketable yield was significantly reduced in MeJA and
Me] A plus firngicide treated-plants compared to other treatments (Table 3.9). It might be
possible that onion plants experienced a resistance cost with Me] A application. Induced
expression of chemical defenses is physiologically costly to total seed production in
Arabidopsis thaliana. Total seed mass was reduced by 15 % (Cipollini, 2002). Tomato
plants sprayed with jasmonic acid had 25-30% fewer flower buds than control plants
(Thaler, 1999).

We assumed that a higher incidence and number of lesions of disease might allow
bacteria to enter the bulb and produce more rot in storage. However, the percentage of
marketable bulbs 138 and 173 days after harvest was not different among chemical
resistance activators in 2001 (Table 3.5). The same trend was observed in 2002 (Table

3.11). Therefore, under the conditions of our experiments the higher number of lesions of

73

purple blotch did not allow bacterial diseases to develop in storage. This trend could
reverse and more disease could be found in storage after more humid seasons.

Our results revealed that MeJ A was the best chemical inducer of resistance
against Altemaria porri in onion in both years of research. BABA had inconsistent
results between years. Marketable yield was reduced with Me] A application in 2002.
Onion plants may have experienced a resistance cost, which was reported in other species
including barley, tomato, and Arabidopsis (Smedegaard-Peterson and Stolen, 1981;
Thaler 1999; Cipollini, 2002). Therefore, lower rates of MeJ A in combination with
flmgicides could help to establish the best rate without affecting the yield. The higher
number of lesions observed on untreated control treatments did not cause a higher
incidence of decay in storage. However, seasons with more humid conditions from
maturity to harvest could favor the incidence of bacteria and later development of decay

in storage.

74

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Table 3.1. Effect of chemical resistance activators on the number of plants infected

with purple blotch, number of lesions per plant, and lesion length of purple blotch of

three onion cultivars, 13 days after inoculation, Muck Soil Research Station,

 

 

 

Laingsburg MI, 2001.
Treatments Infected plants Number of Lesion length
with purple lesions (mm)
blotch per plant
(%)

Chemical resist. giv. (T) ** ** NS
Crop oil concentrate (1%) 54 25 15
Fungicide 23 7 1 1
ASM (20 ppm) 59 18 13
BABA (10 mM) 57 16 12
MeJA (10 mM) 57 20 12
Untreated control 59 18 14

LSD 5% 11 7 NS

Cultivars (C) NS NS NS
Spartan Supreme 51 17 13
Altisimo 48 17 13
T-439 53 18 12

LSD (5%) NS NS NS

T x C NS NS NS

CV (%) 18 43 26

 

NS: Nonsignificant; *"' : Significant at P < 0.01.

80

Table 3.2. Effect of chemical resistance activators on the number of plants infected with
purple blotch, number of lesions per plant, and lesion length of purple blotch of three

onion cultivars, 25 days after inoculation, Muck Soil Research Station, Laingsburg MI,

 

 

2001.
Treatments Infected plants Number of Lesion length
with purple lesions (mm)
blotch per plant
(%)

Chemical resist. activ. (T) *"‘ *** NS
Crop oil concentrate (1%) 65 31 19
Fungicide 29 10 16
ASM (20 ppm) 50 24 18
BABA (10 mM) 45 15 24
MeJA (10 mM) 38 11 18
Untreated control 47 20 21

LSD 5% 16 9 NS

Cultivars (C) ** ** NS
Spartan Supreme 42 15 20
Altisimo 56 27 20
T-439 40 13 1 8

LSD (5%) 7 8 NS

T x C NS NS NS

cv (%) 28 61 24

 

'” : Nonsignificant; " : significant at P < 0.01, “*z significant at P < 0.10 .

81

Table 3.3. Effect of chemical resistance activators on total and marketable yield

of three onion cultivars, Muck Soil Research Station, Laingsburg MI, 2001.

 

 

 

Treatments Total yield (t/ha) Marketable yield
(W3)
(W3)
Ch_errgcpl resist. pctiv. (T) NS NS
Crop oil concentrate (1%) 43 42
Fungicide 44 43
ASM (20 ppm) 46 45
BABA (10 mM) 43 42
MeJ A (10 mM) 43 42
Untreated control 41 40
LSD 5% NS NS
Cultivars (C) NS NS
Spartan Supreme 43 42
Altisimo 46 45
T-439 41 4O
LSD (5%) NS NS
T x C NS NS
cv (%) 31 31

 

T : Nonsignificant.

82

Table 3.4. Effect of chemical resistance activators on the percentage of large and medium

bulbs of three onion cultivars, Muck Soil Research Station, Laingsburg MI, 2001.

 

 

 

Treatments Large bulbsz Medium bulbsy
(%) (%)
Chemical resigpgtig, (T) NS NS
Crop oil concentrate (1%) 46 52
Fungicide 50 48
ASM (20 ppm) 48 50
BABA (10 mM) 50 45
MeJA (10 mM) 44 49
Untreated control 52 46
LSD 5% NS NS
9% (C) "‘ *
Spartan Supreme 49 47
Altisimo 61 37
T-439 35 62
LSD (5%) 5 5
T x C NS NS
cv (%) 1 8 18

 

x: large bulbs > 80 mm; ’: medium bulbs 250 mm 5 80 rmn; "5 : Nonsignificant, * : significant at P < 0.05.

83

Table 3.5. Effect of chemical resistance activators on the percentage of marketable
bulbs of three onion cultivars, 138 and 173 days after harvest (DAH), Muck Soil

Research Station, Laingsburg M1, 2001-2002.

 

 

 

Treatments Marketable bulbsz Marketable bulbsy
138 DAI-I 173 DAH
(%) (%)
Chemicgl resist. activ. (T) NS NS
Crop oil concentrate (1%) 93 87
Fungicide 89 88
ASM (20 ppm) 96 88
BABA (10 mM) 91 86
Me] A (10 mM) 93 91
Untreated control 92 89
LSD 5% NS NS
Cultivars (C) * *
Spartan Supreme 98 91
Altisimo 93 i 86
T-439 87 87
LSD (5%) 7 4
TxC NS *
cv (%) 10 7

 

x: All bulbs were graded at this time; y: Fifty visually good bulbs were cut and evaluated for internal decay;

"5 : Nonsignificant; "' Significant a t P < 0.05.

84

Table 3.6. Interaction of chemical resistance activators and cultivars on the percentage
of marketable bulbs, 173 days after harvest (DAH), Muck Soil Research Station,

Laingsburg MI, 2001-2002.

 

 

Treatments Spartan Supreme Altisimo T-439
Marketable bulbs (%)

Crop oil concentrate 1% 92 82 88
Chlorothalonil 91 85 88
ASM (20 ppm) 87 94* 82
BABA (10 mM) 90 80 87
MeJA (10 mM) 90 92 91
Untreated control 96 82 89
LSD (5%) 9

 

* : Significant at P < 0.05

85

Table 3.7. Effect of chemical resistance activators on the number of plants infected
with purple blotch, number of lesions per plant, and lesion length of purple blotch

'of three onion cultivars, 16 days after inoculation, Muck Soil Research Station,

 

 

Laingsburg MI, 2002.
Treatments Infected plants Number of Lesion length
with purple lesions (mm)
blotch per plant
(%)

Chemical resist. activ. (T) ** ** **
Crop oil concentrate (1%) 45 29 7.7
Fungicide 20 10 5.5
BABA (10 mM) 31 16 8.0
MeJA (10mM) 22 11 6.0
MeJA (10mM) + fungicide 13 7 6.2
Untreated control 49 29 7.3

LSD 5% 9 5 1.5

Cultivars (C) NS NS NS
Spartan Supreme 29 15 7.5
Altisimo 3 1 l 8 6. 1
T -439 29 1 7 6.7

LSD (5%) NS NS NS

T x C NS NS NS

CV (%) 31 32 26

 

is : Nonsignificant, " : significant at P < 0.01.

86

Table 3.8. Effect of chemical resistance activators on the number of plants infected

with purple blotch, number of lesions per plant, and lesion length of purple blotch

of three onion cultivars, 22 days after inoculation, Muck Soil Research Station,

 

 

 

Laingsburg MI, 2002.
Treatments Infected plants Number of Lesion length
with purple lesions (mm)
blotch per plant
(%)

Waist. gptiv. (T) ** ** *
Crop oil concentrate (1%) 62 54 6.4
Fungicide 34 22 7.8
BABA (10 mM) 48 43 6.5
MeJA (10mM) 36 18 5.6
MeJA (10mM) + fungicide 26 14 5.5
Untreated control 61 63 6.2

LSD 5% 9 15 1.3

Cultivars (C) NS NS NS
Spartan Supreme 45 39 6.3
Altisimo 45 37 6.4
T439 44 32 6.2

T x C NS NS NS

LSD (5%) NS NS NS

cv (%) 20 42 21

 

"3 : Nonsignificant, "' Significant a t P < 0.05, ** : significant at P < 0.01.

87

Table 3.9. Effect of chemical resistance activators on total and marketable yield

of three onion cultivars, Muck Soil Research Station, Laingsburg MI, 2002.

 

 

 

Treatments Total yield Marketable yield
(t/ha) (t/ha)
Chemicai resist. activ. (T) ** **
Crop oil concentrated (1%) 45 38
Fungicide 43 38
BABA (10 mM) 43 36
Me] A (10 mM) 36 29
MeJA (10mM) + fungicide 34 28
Untreated control 44 36
LSD 5% 5 5
Cultivars (C) * *
Spartan Supreme 37 30
Altisimo 50 45
T-439 34 29
LSD (5%) 8 6
T x C NS NS
cv (%) 13 14

 

‘1‘? Nonsignificant, * Significant a t P < 0.05, .. : significant at P < 0.01.

88

Table 3.10. Effect of chemical resistance activators on the percentage of large and

medium bulbs of three onion cultivars, Muck Soil Research Station, Laingsburg MI,

 

 

2002.
Treatments Large bulbsz Medium bulbsy
(%) (%)
Chemical resist. actv. (T) ** NS
Crop oil concentrate (1%) 18" 75
Fungicide 20 75
BABA (10 mM) 20 71
MeJ A (10 mM) 12 76
MeJA (10mM) + fungicide 12 75
Untreated control 20 73
LSD 5% 5 NS
Cultivars (C) NS NS
Spartan Supreme 12 75
Altisimo 20 76
T-439 19 72
T x C NS NS
LSD (5%) NS NS
CV (%) 28 8

 

2: Large bulbs > 80 m; y: Medium bulbs 250 mm 5 80 mm; "2 Values in this column are arcsin \%

"s : Nonsignificant, *"' : significant at P < 0.01.

89

Table 3.11. Effect of chemical resistance activators on the percentage of marketable
bulbs of three onion cultivars, 133 and 161 days after harvest (DAH), Muck Soil

Research Station, Laingsburg M1, 2002-2003.

 

 

 

Treatments Marketable bulbsz Marketable bulbsy
133 DAH" 161 DAH
(%) (%)
Chemical resist. pctiv. (T) NS NS
Crop oil concentrate (1%) 84 78
Fungicide 85 80
BABA (10 mM) 83 74
MeJ A (10 mM) 81 75
MeJA (10mM) + fungicide 86 78
Untreated control 82 74
LSD 5% NS NS
Cultivars (C) * **
Spartan Supreme 88 84
Altisimo 74 67
T-439 89 79
LSD (5%) 3 4
T x C NS NS
CV (%) 6 7

 

1' y: All bulbs were graded at this time; ": DAH: days after harvest; ”5 : Nonsignificant. * Significant at P <

0.05, "”" : significant at P < 0.01.

90

CHAPTER IV

EFFECT OF F OLIAR-APPLIED DISEASE RESISTANCE ACTIVATORS ON

PURPLE BLOTCH OF ONIONS AND BACTERIAL ROT IN STORAGE

91

ABSTRACT

Bacterial and firngal diseases cause economic loss in onion. Excess moisture and
physical damage to bulbs may favor bacterial infection and bulb rot. Foliar infection by
Altemaria porri may create wounds through which bacterial pathogens can enter and
cause bulb rot in storage. The objective of this work was to identify the species of
bacteria in bulbs from a field where resistance activators were tested against A. porri.
Plants sowed in 2002 on a Houghton Muck Soil were sprayed with crop oil concentrate
1% “v/v”; Methyl J asmonate (Mel A) 10 mM; B-amino-butyric acid (BABA) 10 mM; and
MeJA 10 mM plus chlorothalonil (0.84 kg a.i. ha’l) to control Altemaria porri. MeJ A and
chlorothalonil reduced A. porri infection but had no effect on bulb rot. Total DNA was
extracted from decayed bulbs. From 140 rotted onion bulb samples we detected 15 as
Burkholderia cepacia, 2 as B. gladioli pv. alliicola, and l as Pantoea ananatis using the
polymerase chain reaction , respectively. Erwinia carotovora ssp. carotovora was not

detected.

92

INTRODUCTION

Onion yield and quality can be affected by fungal and bacterial diseases that
consequently cause economic loss. The bacteria that cause bulb rots often occur in
conjunction with other diseases such as Botrytis neck rot, mostly as secondary invading
organisms (Hoffrnann et al, 1996). Bacteria infection and spread are favored by excess
moisture and wounds that may occur during mechanical cultivation and storms (Schwartz
and Bartolo, 1995; Wright and Grant, 1998). Wounding at topping is common in onions
when the necks of these onion bulbs are not completely dry (Wright et al, 1993).
Bacterial infected onion bulbs develop symptoms of sour skin, slippery skin, soft rot, or
center rot in storage. In the literature, these symptoms have been associated with
infection by Burkholderia cepacia, B. gladioli pv. alliicola, Erwinia carotovora subsp.
carotovora, (Schwartz and Krishna Mohan 1995) and Pantoea ananatis, (Schwartz and
Otto, 2000) respectively. Bulbs affected by slippery skin, do not display external decay
in the early stages of the disease, except for an occasional softening of the neck region.
One or two inner fleshy scales appear soft with a cooked or water soaked appearance
(Schwartz and Mohan, 1995). Bulbs affected by sour skin are characterized by a pale
yellow to light brown decay and breakdown of one or a few inner bulb scales. The
adjacent bulb scales and the center remain firm (Schwartz and Bartolo, 1995). The decay
symptoms caused by B. cepacia and B. gladioli pv. alliicola frequently overlap and are
difficult to categorize (Tesoreiro et al. 1982, Hoffmann et al. 1996). Center rot symptoms

include rot of the neck tissue and between the scales (Schwartz and Otto, 2000). Soft rot

93

caused by Erwinia carotovora ssp. carotovora shows water-soaked and pale yellow to
light brown scales that become soft as the rot progresses (Schwartz and Otto, 2000).

The general methods used to isolate phytopathogenic bacteria are of limited use
when applied to rotten onion bulbs. Onion bulbs are exposed to a broad spectrum of
bacteria present in the field (including the bacteria from the soil, irrigation water,
equipment, etc.), and then are stored for about 4-5 months. Hence, it is particularly
difficult to isolate the pathogens in the presence of numerous opportunistic saprophytes.
Other limitations of conventional plate assay to identify bacteria associated with bulb rot
diseases include a small number of samples; it is time consuming, and expertise and
experience is needed to recognize the target bacteria.

Purple blotch, caused by Altemaria porri, is a disease of onions that affects both
foliage and bolting stems. Onion yield reductions due to purple blotch can range from 30
to 50% (Nolla 1927), and up to 100 % (Skiles 1953). The damage produced by purple
blotch, increases with leaf and plant age (Everts and Lacy, 1996); therefore, we postulate
that wounds caused by A. porri could be infection courts for phytopathogenic bacteria
(I-Ioffmann et al., 1996).

One alternative strategy for managing onion disease is induced resistance.
Localized treatment of plants with biotic chemicals can result in local or systemic
induction of resistance to pathogen attack in the plants (Kuc, 1982). Induced resistance
to pathogens can be classified as systemic acquired resistance (SAR) and induced
systemic resistance (ISR), (van Loon, 1998). Both are characterized by being effective

against a broad range of pathogens (Hammerschmidt, 1999).

94

The integration of induced resistance with genetic resistance and chemical control
offers a more sustainable plant disease management strategy against fungal pathogens
with a history of adaptation to fungicides (Oostendorp et al., 2001). On the other hand,
chemical activation of disease resistance in plants represents an additional option to
protect the crop against bacterial and viral pathogens, for which genetic resistance is not
available (Oostendorp et al., 2001). This is also important since no effective chemical
control measures are available for bacterial soft rot of onions (Wright et al. 1993,
Hoffmann et al., 1996).

We hypothesize that inducing resistance to A. porri may reduce the incidence of
purple blotch and subsequently, the damage associated with bacterial infection of onion
bulbs. The goal of this research was to determine the effects of resistance activators on

purple blotch of onion and bacterial rot of stored onions.

95

MATERIALS AND METHODS

Field experiment

The experiment was carried out at Muck Soil Research Station, Laingsburg, MI.
On April 28, 2002 onion cultivars 'Spartan Supreme", Seed Works, San Luis Obispo, CA;
'Altisimo' onion, Bejo Seeds Inc., Geneva, NY; and 'T-439', (American Takii, Salinas,
CA), were planted on three rows of raised, 41 cm apart. Seeds were planted
approximately 2 cm apart in each row. The experiment was designed as a split-plot, with
three replications. Six beds of each cultivar were the main plots and on each bed the
chemical resistance activators were assigned randomly. Crop oil concentrate (COC)
solution 1% "v/v", fungicide chlorothalonil 1.68 kg.ha'l, DL-B-amino-n-butyric acid
(BABA) at 10 mM, methyl jasmonate (MeJ A) at 10 mM, methyl jasmonate (MeJ A) at 10
mM plus fungicide (minimum rate) chlorothalonil 0.84 kg a.i. ha'l, and untreated control
were applied to each cultivar. The three chemical resistance activators included CDC 1%
'v/v". Chemical sprays were applied three times before inoculation with a C02 backpack
sprayer with two flat fan 11002 nozzles on the boom, which delivered 234 L.ha’1, at a
pressure of 234 KPa, and a speed of 5.7 km/h. Treatments were applied three times
before inoculation as described above. The first spray was applied at the 3-leaf stage, 52
days after planting (DAP); the second application was at the 5-1eaf stage, 66 DAP; and
the third application was at 6 -7-leaf stage, 73 DAP. On the treatment combining the
minimum rate of fungicide and Me] A, chlorothalonil was applied two days after the
Me] A spray to avoid possible negative interactions between the two chemicals. After the
third application (90 DAP), all plots were sprayed with A. porri (8,500 spores/mL).

Plants were irrigated (10.5 mm) in the afternoon before inoculation ((1900h).

96

Temperature was 30 C, and relative humidity was 90%. All plots were irrigated every 48
h during the week following inoculation to promote purple blotch development. Purple
blotch symptoms appeared approximately 8 days after inoculation.

Beginning on August 5, chemicals were applied at the recommended commercial
rates (Bird et al., 2002). Chlorothalonil (2,53 kg. ha") was alternated with maneb (2.68
kg. ha") or iprodione (0.56 kg. ha" ) on August 5, 12, 19, and 26. Chlorothalonil (1.26
kg. ha") was alternated with maneb (1.34 kg. ha’l) or iprodione (0.28 kg. ha") on August
5, 12, 19, and 26 for MeJA plus fungicide at minimum rate treatment.

Field assessment of pplgple blotch.

Lesion number and lesion length were evaluated on ten plants/plot at 22 days after
inoculation (DAI). Lesion length was measured with a digital caliper Mitutoyo model
CD-6" CS, Mitutoyo, Aurora Ill). At harvest bulbs were graded and rotten bulbs were
kept in a cold room until the bacteria present could be identified by the polymerase chain
reaction (PCR).

DNA extraction a_nd (PCR) detection of bioterig in rotten onion mg;

Onion bulbs with symptoms of decay were kept at 5 C for approximately 30 days
after harvest. Extraction of bacterial DNA fi'om 140 onion bulbs was performed using the
method described by Walcott et al. (2002) with slight modification. Onion bulbs were
peeled with a sterile knife and a sample of approximately 10 g of the decayed fleshy
tissue and surrounding area was collected and put in a bag (model 400, bags 6041,
Steward Limited, London, United Kingdom). Twenty milliliters of sterile high pressure
liquid chromatography (HPLC)-grade water (J. T. Baker, Phillipsburg, NJ) was added to

the tissue and the tissue was crushed for approximately five minutes in a Stomacher lab-

97

blender 400 (Tekrnar Co., Cincinnati, Ohio), and then filtered through two layers of
cheese cloth and one layer of #1 filter paper (Whatrnan UK). The filtrate was centrifuged
for 5 min. at 5000 rpm (Centrifuge RC5C, Sorvall Instruments, Newtown, CT), and the
supernatant was removed. The pellet was resuspended in 20 ml of sterile water added and
this procedure was repeated. After the second rinse, the pellet was resuspended in 200
uL of sterile HPLC-grade water. The suspended bactererial cells were incubated at 100 C

for 15 minutes on a standard heat block (Dry bath Incubator, Fisher Scientific, Pittsburg,

PA) to lyse bacterial cells and release DNA.

Two microliters of DNA from boiled cell suspensions were amplified in 25 uL of PCR
master mix containing PCR beads, puRe Taq Ready-To-Go, (Amersham® Biosciences,
Piscataway, NJ), 22 uL of sterile water, 0.5 uL of primer one, and 0.5 uL of primer two.
DNA amplification was carried out in a programmable thermal cycle (Gene Amp® PCR
System 2700, Applied Biosystem, Foster CA)

The PCR conditions included denaturation at 95°C for 5 min, 30 cycles of
denaturation at 95°C for 30 s, annealing of primers at 55°C for 30 s, and elongation at
72°C for 5 min. for P. ananatis. Annealing temperature of 57°C for B. cepacia and B.
gladioli pv. alliicola °C (Bauemfeind et al. 1998), and 56°C for Erwinia carotovora

subsp. carotovora (Parda, 2001). The sequences for the oligonucleotide primers used to

detect the bacteria associated with onion bulb rots are as shown in Table 4.1.

98

Results of the PCR were determined by electrophoresis (80 V for 1,5 h in 1x Tris acetate
EDTA buffer) of 10 11L of the PCR product in a 1% agarose gel, followed by staining

with ethidium bromide and visualization with ultraviolet illumination.

99

RESULTS

The number of lesions of purple blotch per leaf was smaller with chlorothalonil
treatment, methyl jasmonate (10 mM), and combinations of methyl jasmonate (10 mM)
and chlorothalonil at the minimum rate (Figure 4.1A) that the crop oil concentrate or non-
treated control. This trend was similar for cultivars Spartan Supreme, Altisimo and T-
439. On cultivar T-439, the chlorothalonil treatment resulted in longer lesion as compared
to the other five treatments, and there were no differences in length of lesions in Spartan
Supreme and Altisimo among chemical resistance activators. (Figure 4.1B).

The percentage of rotten bulbs did not differ significantly among cultivars or
chemical resistance activators. However, on cultivar T-439 methyl jasmonate and methyl
jasmonate plus fungicide tended to have a slightly lower percentage of rotten bulbs. The
same trend was observed on cultivar Altisimo when treated with methyl jasmonate plus
fungicide (Figure 4.2).

A pale yellow to light brown decay and breakdown of one or a few inner bulb
scales, were observed on bulbs possibly affected by sour skin, while adjacent bulb scales
and the center remain firm at the beginning but then the rot progressed to adjacent scales
(Figure 4.3A). Rot at the neck tissue and among the scales was observed on bulbs
possibly affected slippery skin (Figure 4.3B). Rot of the neck tissue and between the
scales was observed on bulbs possibly affected by center rot (Figure 4.3C).

Sour skin caused by Burkholderia cepacia was identified in 15 samples (11%),
Burkholderia gladioli pv. alliicola was identified in one sample (0.7%) and Pantoea

ananatis was identified in 2 samples (1.4%), while Erwinia carotovora subs. carotovora

100

was not identified (Table 4.2). Of the samples that tested positive for Burkholderia
cepacia, 6 came from the fungicide treatment (40%), 4 from COC treatment (27%), 2
from BABA treatment (13%) and 2 from untreated control treatment (13%). Only 1 of
the B. cepacia infected onion bulbs came fiom plots treated with methyl jasmonate (7%),
and no bacteria were identified in plots treated with methyl jasmonate plus fungicide at

the minimum rate (Table 4.3).

101

DISCUSSION

Methyl jasmonate and Me] A plus fungicides reduced the number of lesions of
purple blotch (Figure 4.1), but there was no difference in the lesion length. This suggests
that the induction of resistance was more effective in preventing fungal invasion than
colonization. Although the percentage of rotten bulbs did not differ among chemical
treatments, cultivar T-439 tended to have a slightly lower percentage of rot in Me] A and
Me] A plus firngicides at minimum rate treatment than other treatments. A similar trend
was found when MeJ A plus fungicide was applied on cultivar Altisimo, suggesting that
this treatment could have a beneficial effect against postharvest bulb decay (Figure 4.2).

Symptoms of sour skin caused by B. cepacia (Figure 4.3A) were similar to those
described by Schwartz and Krishna Mohan (1995). Bulbs affected by slippery skin
caused by B. gladioli pv. alliicola, do not display obvious external bulb symptoms
(Schwartz and Mohan, 1995). Nevertheless, Tesoriero et. al (1982) stated that artificial
inoculation of onions with B. gladioli pv. aliicola, and B. cepacia, produced
indistinguishable soft rot symptoms. Our results are in agreement with the symptoms
described by Schwartz and Krishna Mohan (1995) (Figure 4.3 B).

Center rot caused by P. ananatis is characterized by rotting of the neck tissue and
between the scales (Schwartz and Ottto, 2001), and this was observed in our experiment
(Figure 4.3 C).

Although fungicides were able to reduce A porri incidence, they did not reduce
the incidence of bacterial soft rots in onion bulbs (Tables 4.2 and 4.3). From the samples

identified as being infected with B. cepacia, 40% and 7% came from plants treated with

102

fungicide and MeJ A in the field, respectively. This suggests that MeJ A could help to
reduce sour skin on onion bulbs (Table 4.3). There were other bacterial rots that were not
identified throughout the course of this experiment.

Although MeJ A and fungicides reduced A. porri infection, they had no effect on
onion bulb decay. However, plants treated with MeJ A showed reduced number of bulbs

with sour skin when compared with plants treated with fimgicide.

103

BIBLIOGRAPHY

Bauemfeind, A., I. Schneider, R. Jungwirth, and C. Roller. 1998. Discrimination of
Burkholderia species detectable in cytis fibrosis patients by PCR. J. of clinic
microbiol. 36:2748-2751.

Chun, W. and J. B. Jones. 2001. II Gram-negative bacteria, Burkholderia. In: Shaad, N
W., Jones J ., B., and Chun, W. (eds). Laboratory guide for identification of plant
pathogenic bacteria. APS Press p. 139-150.

Everts, KL. and M. L. Lacy. 1996. Factors influencing infection of Onions leaves by
Altemaria porri, and subsequent lesion expansion. Plant Dis. 80, 276-280.

Hammerschmidt, R. 1999. Induced disease resistance: how do induced plants stop
pathogens?. Physiol.Mol Plant Pathol. 55, 77-94

Hoffinann, M. P., C. H. Petzoldt, and A. C. Frodshrnan. 1996. Integrated pest
management for onions. Cornell Coop. Ext. Service. IPM program
publication 119. Geneva, YN.

Kuc, J. 1982. Induced immunity to plant disease. BioScience 32, 854-860.

Nolla, J. A. B. 1927. A new Altemaria disease of onions (Allium cepa L.).
Phytopathology 17, 1 15-135.

Oostendorp, M.; Kunz, W.; Dietrich, B.; and Staub, Theodor. 2001. Induced disease
resistance in plants by chemicals. European Journal of Plant Pathology 107, 19-
28.

Parda K. 2001. Development and detection of bacterial soft rot of Hosta spp. Tratt. by
Erwinia carotovora subs carotovora. Masther thesis, Athens University of

Georgia Athens, GA.

Schwartz, H. F. and M. E. Bartolo. 1995. Colorado onion production and integrated pest
management. Cooperative Extension Resource Center. Colorado State University.
Bulletin 547 A.

Schwartz, H. F., and K. Mohan, S. 1995. Compendium of onion and garlic diseases.
APS p. 32-33.

Schwartz, H. F. and K. Otto. 2000. First report of leaf blight and bulb decay of onion by
Pantoea ananatis in Colorado. Plant Disease 84 (7) 808.

Skiles, R. L. 1953. Purple and brown blotch of onions. Phytopathology 43: 409-412.

104

Tesoriero, L. A., P. C. Fahy, and L. V. Gunn. 1982. First record of bacterial rot of onion
in Australia caused by Pseudomonas gladioli pv. alliicola and association with

internal browning caused by Pseudomonas aeruginosa. Australasian Plant
Pathology Vol. 11(4):56-57.

Van Loon, L.C., P. A. H. M. Bakker, C. M. and Pieterse, C. M. 1998. Systemic resistance
induced by rhizosphere bacteria. Annu. Rev. Phytopathol. 36:453-483.

Walcott, R. R., R. D. Gitaitis, A. C. Castro, F. H. Sanders Jr., and J. C. Diaz-Perez.
2002. Natural infestation of onions seed by Pantoea ananatis, causal agent of
center rot. Plant Disease Vol 86 (2) 2106-111.

Wright, P. J ., R. G. Clark, and C. N. Hale. 1993. A storage soft rot of New Zealand
onions caused by Pseudomonas gladioli pv. alliicola. Zealand Journal of Crop
and Horticulture Science 21:225-227.

Wright, P. J. and D. G. Grant. 1998. Evaluation of Allium germplasm for susceptibility

to foliage bacterial soft rot caused by Pseudomonas marginalis and Pseudomonas
viridiflava. New Zealand Journal of Crop and Horticulture Science 26: 17-21.

105

Table 4.1 Sequences for the oligonucleotides primers used to detect bacteria associated

 

 

 

 

 

 

with bulb rots, 2002.

Bacteria Name of the Sequence of primers
oligonucleotide
primers

Pantoea ananatis PanIT S1 (5 ’-GTC TGA TAG AAA GAT AAA GAG-3’)
ECS (5’-CGG TGG ATG CCC TGG CA-3’)

B. gladioli pv.alliicola CMG-23-1 (5’-ATA GCT GGT TCT CTC CGAA-3’)
G-23-2 (5’-CCT ACC ATG CAY ATA AAT-3’)

B. cepacia CMG-23-1 (5’-ATA GCT GGT TCT CTC CGAA-3’)
CM-23-2 (5’-CT C TCC TAC CAT GCG YGC-3’)

Erwinia carotovora ECCZW (5’-CAT CAC TCA CAC CAC ATT ACT -3 ’)

subs. carotovora ECC 1K (5 ’-TGT TGA TGC GAT GAG TGA CA-3 ’)

 

 

 

 

Table 4.2. Identification and distribution of bacteria fiom samples of onion that showed

symptoms of decay, 2002.

 

 

 

 

 

 

Bacterial rot Number of samples from 140 rotted
bulbs

B. cepacia 15

B. gladioli pv. alliicola 2

P. ananatis 1

E. carotovora ssp. carotovora 0

 

 

 

106

Table 4.3. Number of samples that tested positive for B. cepacia according to the

chemical resistance activators applied in the field, 2002

 

 

 

 

 

 

 

 

Treatments Number of samples that tested positive
for B. cepacia

Crop oil concentrate 1% 4

Fungicide 6

BABA (10 mM) 2

MeJ A (10 mM) 1

MeJA (10 mM) + fungicide minimum 0

rate

Untreated 2

 

 

107

 

Number of lemons pa pl-Il

 

I I I I. .. ____-___

10.1

 

[£30m length ( mm)

 

 

 

Purple blotc]

onion culliv;

ML 2002_

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. (A) 11;; 2:01 fpr chemical resistance activators 5mm "‘
e- “h": gnarl-r ,
7. 7 3mm“
. / 7
/ /
i p /
, / 7
l / / /
“6 / / L: /
i E: / / : /
. - a a a
E\_; / E /
:\ / : /
EN / é =§/
8. Supreme Altisimo T439
13 1 (B) P < 0.01 for chemical resistance activators 5mm 1%
12 _' NS for cultivars 3:23:33:
11-' grandam
10:
9-:
g: e
r E 7
‘5 3e =. 3 Z 5 *1?
a 2 / ES /
3': :1 / / Z§ /
2- -—+ / V :\ /
13 E / ? E§ é
o ; —\ / \ A —
S. Supreme Altisimo T-439

Orltivan

Figure 4.1. Effect of chemical resistance activators (A) on the number of lesions of
purple blotch per plant, and (B) on the length of lesions of purple blotch of three
onion cultivars, 22 days after inoculation, Muck Soil Research Station, Laingsburg,

MI, 2002.

108

 

NS for chemical resistance activators and cultivars

.—|
O\
l

-Cropoil oonwntratel%
ZZFungicide
-BaBa10mM
CjMeJalOmM
mMeJa 10mM+Fungicide
[ZZUntreatedoontrol

l—a
.Ds
I

1

t—d

N
I

V

 

p...
O
l

 

Percentage of rotted bulbs
00
l

 

 

\ \\\\\\\\\\\\\\\\\\\\\\\\l

 

 

 

 

 

 

 

 

 

 

 

 

 

S. Supreme Altisimo T-439
Cultivars

Figure 4.2. Effect of chemical resistance activators on the percentage of rotted bulbs of

three onion cultivars, Muck Soil Research Station, Laingsburg, MI, 2002.

109

 

(C) Center rot caused by P. ananatis

Figure 4.3. Observed symptoms of bulb decay in onions, after 30 days of cold room
storage (A) sour skin caused by Burkholderia. cepacia, (B) slippery skin caused by

Burkholderia gladioli pv. alliicola, and (C) center rot caused by Pantoea. ananatis.

110

M123456789101112M

 

Figure 4.4. Agarose gel showing PCR products, where: M= marker, DNA ladder; l=
positive control for B. gladioli pv. alliicola (388 bp); 2 = positive control for E.
carotovora sp. carotovora (330 bp); 3 = positive control for B. cepacia (375 bp);

5, 6, 7, 8, 9 = negative controls (water); 10 = sample that tested positive for B. gladioli
pv. alliicola (3 88 bp); 11 =sample that tested positive for P. ananatis (368 bp); 12 =

sample that tested positive for B. cepacia (3 75 bp).

111

M123456M M123456M M123456M

   

A

(A) M= marker, DNA ladder;1= positive control for P. ananatis (368 bp); 2= negative
control (water); 3= reaction with primers for B. gladioli pv alliicola; 4= reaction
with primers for B. cepacia; 5= reaction with primers for E. carotovora sp.
carotovora; 6= reaction with primers for P. ananatis (sample that tested positive).

(B) M= marker, DNA ladder;l= positive control for B. cepacia (375 bp); 2= negative
control (water); 3= reaction with primers for B. gladioli pv alliicola; 4= reaction
with primers for P. ananatis; 5= reaction with primers for E. carotovora sp.
carotovora; 6= reaction with primers for B. cepacia (sample that tested positive).

(C) M= marker, DNA ladder;1= positive control for B. gladioli pv. alliicola (3 88 bp); 2=
negative control (water); 3= reaction with primers for P. ananatis; 4= reaction
with primers for B. cepacia; 5= reaction with primers for E. carotovora sp.
carotovora; 6= reaction with primers for B. gladioli pv. alliicola (sample that

tested positive).

Figure 4.5. Agarose gels showing PCR products, where specific primers were used for

detection of P. ananatis, B. cepacia, and B. gladioli pv. alliicola.

112

CHAPTER V

CULTURAL PRACTICES AFFECT PLANT STAND, YIELD AND PURPLE

BLOTCH INFECTION OF ONIONS

113

ABSTRACT

Growing conditions, plant vigor, and crop management factors may increase the
susceptibility of onions to pathogens. Raised beds improve drainage and aeration in the
root zone, and may improve plant stand. In Michigan onions normally are direct-seeded
on flat beds on muck soils. Most onion fields are subject to flooding after heavy rains,
which results in reduced stands and/or increased foliar and bulb diseases. In one study we
compared flat and raised beds, with or without irrigation for onion production and
quality. In another study we compared two to three rows per bed, and two onion cultivars
with and without fungicide application. For the first study three onion cultivars, Hoopla,
Spartan Supreme and T-439 were direct seeded on May 3, 2001, and Spartan Supreme,
Hoopla and Norstar were seeded on April 28 2002, for the first study. Spartan Supreme
and Altisimo were seeded on the same dates for the second study. The experiments were
located at the Muck Soil Research Station, Laingsburg, MI. Each bed had 3 rows
separated 41 cm apart. Seed was planted approximately 2 cm apart in rows. Irrigation (an
average of 13mm per week) was applied beginning July 5 in 2001 and June 24, 2002.
Plant stand, Altemaria porri incidence and severity, yield, and bulb storability were
evaluated in the first study. Purple blotch incidence and severity, yield and quality were
evaluated in the second study. Cumulative rainfall of 140 and 128 mm during May and
June 2001, respectively, caused very wet soil conditions during germination and first
stage of development. Our preliminary study in 2000 showed that plant stand was
reduced and yield was lower on flat than in raised beds. There was a 34% and 22%
reduction in plant stand in the flat beds relative to raised beds in 2001 and 2002,

respectively 40 days after planting (DAP). Raised beds had less purple blotch disease

114

incidence. There was 55% and 54% disease incidence on raised beds 121 DAP, compared
to 60% and 68% for flat beds in 2001 and 2002 respectively. Disease severity rating was
4 for flat beds (scale 0: no disease; 5: 2 70% of the area of the third green leaf from the
base affected with the disease) compared to 3 for raised beds at 100 DAP in 2001. Raised
bed had greater yield in 2001. The percentage of marketable bulbs stored after harvest
was not significantly affected by irrigation or bed types. Spartan Supreme stored better
than the other cultivars in 2001. Purple blotch incidence and severity was reduced in

2001 in two rows spacing compared to three rows spacing. Total, marketable and large
sized bulb yield was significantly higher in three-row spacing compared to two row

spacing in 2001 but not in 2002.

115

INTRODUCTION

Dry bulb onion is the third largest consumption vegetable crop in the USA with
per capita consumption of 8.2 kg/year (Economic Research Center -U.S. Department of
Agriculture 2003 (ERS-USDA); Fresh Product Consumption, 2003). Onions are
produced commercially in 16 states (U .S. Department of Agriculture, 2003). Michigan
has been a major onion producer in the past with over 5,000 ha but now has about 1,600
ha with a total production of 45,000 t per year (Kleweno and Matthews, 2002) with a
total value of 9 million dollars (U .S. Department of Agriculture A, 2003).

There are several reasons to consider alternative disease control methods in onion
including development of pesticide resistance in onion pests, loss of registered pesticides,
public pressure against the use of pesticides and need to reduce costs to meet increasing
competition fiom other regions (Hoffrnann et al, 1996).

Purple blotch, caused by Altemaria porri, is a disease of onions that affects
foliage. Severe infection may cause partial or total yield loss (Nolla, 1927; Skiles, 1953).
Although chemical control has been used against purple blotch, the broad and often
excessive use of pesticides could cause damage to the environment, and may lead to
pathogen resistance (Ozeretskovskaya, 1995; Hadisustrino et al. 1996). Changes in
cultural practices may reduce purple blotch incidence and improve quality and yield.

Subsidence of muck soil or the lowering of surface elevation is influenced by
several factors such as biological oxidation, height of water table, compaction, wind and

water erosion, shrinkage and dehydration, and cropping systems. After an organic soil is

116

developed for agricultural production, oxidation increases due to more favorable
conditions for aerobic nricroflora. Wind erosion has been one of the predominant causes
of subsidence; a soil can lose over 3 cm of soil in a severe storm (Lucas 1982).

In Michigan, onions are normally direct-seeded on flat beds on muck soil.
Because of significant subsidence, most Michigan onion fields now lie below drainage
ditches and consequently are flooded for periods of time after heavy rains. This could
result in reduced stand, more foliar disease, reduced yields, and poor storage quality.

Environmental conditions and cultural management practices may increase the
susceptibility of onions to pathogens. Plant diseases tend to be more severe when a
pathogen is highly virulent, the plant is susceptible, and the environment is favorable for
pathogen infection over an extended period. Changing any side of the pyramid, such as
adding an unfavorable environment or using a disease resistant variety, can significantly
reduce disease development (Hart and J arosz, 2000).

Damping-off disease of seedlings occurs worldwide in valleys and forest soils, in
tropical and temperate climates. Losses vary considerably with soil moisture and
temperature. The invaded areas become soaked and discolored, and they soon collapse.
The basal part of the seedling stem becomes softer and much thinner than the uninvaded
parts above it; consequently the seedling falls over on the soil (Agrios, 1997). Pythium
spp. of onion is most severe under conditions of high soil moisture and soil temperatures
below 18 °C. Cultural practices such as land leveling, installing tile drains or planting on
raised beds, minimize periods of excessive soil moisture (Schwartz and Mohan, 1995).
Good air circulation around plants, avoiding excessive application of nitrate forms of

nitrogen, and crop rotation are helpful in reducing the amount of infection (Agrios, 1997 ).

117

Lettuce growing on ridges of 10.2 cm high and 17.8 cm wide had the lowest
degree of severity of bottom rot, caused by Rhizoctonia solani Kuenh, compared to
nonridged lettuce in both years of research. There were also a higher percentage of
harvested plants in the trials on lettuce growing on ridges (Pieczarka and Lorbeer, 1974).
They attributed this result to the better aeration and drainage of the ridged lettuce.
Moreover, they concluded that fungicides were most effective on ridge lettuce because of
the better spray coverage of the basal portions of the plant in addition to the maintenance
of drier conditions under the plants that suppress disease development (Pieczarka and
Lorbeer, 1974).

Fritz and Honma (1987), found that Chinese cabbage (Brassica campestris group
pekinensis) growing on raised beds had less incidence of soft rot caused by Erwinia
carotovora ssp. carotovora compared to flat beds. The raised beds may have allowed for
better ventilation in the foliage and decreased humidity. Raised beds also may have
decreased the soil moisture level and the amount of free moisture present in the foliage.
Soil moisture had been excessive (field capacity) for periods of two to three days during
the growing season on Chinese cabbage growing on flat beds (Fritz and Honma, 1987).

The raised bed system reduced the incidence of initial plant mortality of raspberry
affected by Phytophthora root rot. Raised beds, and raised beds plus metalaxyl increased
the yield compared to flat beds. Metalaxyl applied to flat beds did not increase the yield
(Maloney et al., 1993). They indicated that raised beds might aid control of fungal
disease of the fruit and foliage of raspberry by improving air circulation and drying

within the canopy (Maloney et al., 1993). Wilcox et al., (1999) reported that raised beds

118

reduced Phytophthora root rot of red raspberry to less than 10% compared to 50% on flat
beds. Moreover, red raspberry yield increased on raised beds compared to flat beds.

Foliar and fruit bacterial diseases of tomato caused by Pseudomonas syringae pv.
tomato and Xanthomonas campestris pv. vesicatoria, were reduced on plants growing in
single and equidistant row arrangements compared to double rows and other
arrangements (Warner et al., 2000).

The incidence and severity of Ascochyta blight of peas caused by Ascochyta pisi
were positively correlated with the number of plants and number of rows per plot
(Dhiman et al., 1987). Finger millet (Elusine coracana) leaf blast caused by Pyricularia
grisea, and leaf spot caused by Cylindrosporium sp. tended to be more severe with an
increase in plant population (Adipala et al, 1994).

Purple blotch disease management recommendations include crop rotation, onion
debris and cull sanitation, planting clean seed, application of firngicides, and moderate
plant densities (Schwartz and Bartolo, 1995).

Raised beds may improve drainage and aeration of onion, which should result in
improved plant stand, less disease, greater yields, and less rot in storage. Wider plant
spacing will improve air circulation between plants and reduce the incidence of diseases.
The objective of this research was to compare the effects of flat and raised beds, with or
without irrigation, on disease incidence, yield, and quality of onion; and to compare the

effects of two and three rows per bed on disease incidence, yield and quality of onion.

119

MATERIALS AND METHODS

Location

The experiments were conducted at the Muck Soil Research Station, Laingsburg,
Michigan, on a Houghton Muck soil, Euic mesic typic Medisaprist, 80% organic matter,
and pH 6.6.
The effect of bed type and irrigation on onion quality and yield.
The plot size was 1.6 m wide, and 15 m long with 3 rows, 41 cm apart per bed. Seeds
were planted at a spacing of approximately 2 cm. The plots were maintained with
pesticides throughout the growing season, as recommended commercially (Bird et al.,
2002; Zandstra et al., 1996).
2001 Experiment
Treatments and experimentgl desigp

The experiment was designed as a split-split plot, with irrigation and no irrigation
as the main plot. Each main plot was split into three raised (23 cm height) and three flat
beds of 1.6 m wide and 15 m long, which were assigned randomly. Each flat and raised
bed had three rows, and each row was assigned randomly to a cultivar. 'Hoopla' (raw
seed treated with Pro-Gro and Trigard), Seed Works, San Luis Obispo, CA.; 'Spartan
Supreme' (raw seed treated with thiram), Seed Works, San Luis Obispo, Cal; and 'T-439'
(Pro-Gro film coated), Takii Seeds Inc., Geneva N.Y.; were planted on May 2, with a
Gaspardo precision vacuum planter. Lorsban 15 G at the rate of 3.7 ounces (86 g) per

1000 feet (305 m) of rows was applied at planting. The field was irrigated with 7.6 cm

120

irrigation pipelines and Rain Bird 30H sprinklers (13/64 x 1/8 20 degree), which
discharged 6.4 mm water per hour. Irrigation schedule is shown in Table 5.1.
Parameters measured

The number of plants in three rows per plot in three linear meters was counted on May
22, June 12, July 2 and 19, and August 8 (19, 40, 60, 79 and 99 days after planting).
Disease incidence and disease severity

To determine the disease incidence of purple blotch, caused by Altemaria porri,
50 plants were evaluated in each plot 106 and 120 days after planting (DAP). A plant was
considered infected if it had one lesion. Disease severity was determined visually with a
disease rating from zero to five on ten plants per plot 106 and 120 DAP (Table 5.4). The
third green leaf from the outside base of the plant was evaluated each time. At 120 DAP
it was a different leaf compared with the leaf evaluated at 106 DAP, because the leaf next
to the base was senesced at that time.

Hive—st-

Nine meters of three rows on raised beds and 12 m of three rows on flat beds were
harvested on October 9, 2001. Because there were fewer onions on the flat beds, we
harvested a larger area to have enough bulbs for storage evaluations. The onions were
hand-pulled, and bulbs were topped in the field with a roll topper, and cured for ten days
in ambient air. Onions were then graded and weighed using three categories: small (< 50
mm), medium (2 50mm 5 80mm), and large (2 80 mm). The sum of these three
categories corresponded to total yield, while the sum of medium and large bulbs was the

marketable yield. Two crates (approximately 40 kg) of bulbs greater than 50 mm in

121

diameter were placed in common storage. Storage temperature declined as ambient
temperature declined, and was maintained at 1-3 C.

At 142 days after harvest (DAH) bulbs were graded as marketable, rotted, and
sprouted. Fifty visually good bulbs were cut at 173 DAH and evaluated for internal
decay. The percentage of marketable bulbs was calculated.

Statistical analysis

Analysis of variance was performed with MSTATC (MSTATC, 1988). Mean
separation was performed with Fisher’s LSD. The percentage of marketable bulbs was
transformed with arcsine square root of the percentage (Snedecor and Cochran, 1967) to
obtain a normal distribution of means.

2002 Experiment

Treatments and experimental desigp

The experimental design was the same in 2002. Spartan Supreme and Hoopla
were used again. Norstar onion seed pro gro coated (Takii Seeds Inc, Geneva NY.) was
used as substitution for T-439, which was not available that year. Onion seed was planted
on April 28, as described above. Irrigation schedule is shown in Table 5.2. Historical
average weather records for Laingsburg, MI., with a minimum period of records of 30
years is shown in Table 3 (The weather channel, 2003).

Parameters measured

The number of plants in three rows per plot in three linear meters of each row was

counted on June 6, June 27, July 18, and August 6 (39, 60 81 and 100 DAP,

respectively).

122

Disease incidence and disease severity were evaluated August 6 and 26 (100 and
120 DAP) as described above.

The minimtun diameter of the pseudostem and the maximum diameter of the bulb
was measured on July 18, 22, and 26 (81, 85 and 89 DAP) in ten plants in each plot with
a digital caliper Mitutoyo model CD-6" CS, and the neck/bulb diameter ratio or bulbing
ratio was calculated (Brewster, 1994).

wei t

Five plants per plot were sampled 64, 74, 100 and 125 days after planting to
determine dry weight. At 64 and 74 DAP plants were separated into leaf and stem (we
discarded roots because we were interested in the above-ground part of the plant). At 100
and 125 DAP plants were sectioned into bulb, pseudostem and foliage (we discarded
roots). We dried the samples in a forced air at 65 C for two to five days until constant
weight.

E31198;-

Six meters of three rows per plot were harvested on September 23, 2002. The
onions were hand-pulled, topped and graded as described above. Onions were cured for
24 days in ambient air and two crates (approximately 80 kg) of bulbs greater than 50 mm
in diameter were placed in storage as described above.

At 151 days after harvest (DAH) all bulbs were graded as described above. The
percentage of marketable bulbs was calculated, and the data was transformed as
described above.

The effect of fungicide and row spacing on onion quality and yield

Treatments apd experimental desigp

123

The experiment was designed as a split block, with two by two factorial in the
main plot. Fungicide used for commercial onion production (Bird et al, 2002), or no
fungicide was one main treatment, and the other main treatment was cultivar. 'Spartan
Supreme' Seed Works, San Luis Obispo, CA (raw seed treated with thiram), and
“Altisimo” onion, (Gro Coat, treated with Pro-Gro and Trigard; Bejo Seeds Geneva NY).
Lorsban 15 G at the rate of 86 g per 305 m of row was applied at planting. Half of each
block, (1.6 m wide and 15 m long), was sprayed with fungicides commonly used to
control onion diseases (Bird et al., 2002), and the other half was not sprayed. Each main
plot was split into two or three row treatments per raised bed and row treatments were
assigned randomly.

Onion cultivars were planted 41 cm apart in three row treatments and 81 cm apart
in the two row treatments in 2001. For 2002 the same distance was used for the three row
treatment, but for the two rows treatment onions were planted 61 cm apart to avoid the
damp conditions close to the edge of the raised bed experienced in 2001. Seeds were
planted at a spacing of approximately 2 cm in the three row treatments and at 1.3 cm in
the two row treatments in order to maintain the same plant population per unit area. The
plots were maintained with pesticides throughout the growing season, as recommended
commercially (Bird et al., 2002; Zandstra et al., 1996).

Parameters measured

Purple blotch incidence and severity were evaluated 107 and 119 DAP in 2001,

and 100 and 120 DAP in 2002, as described for bed type experiment.

Harvest

124

Six meters of two or three rows per plot were harvested on October 3, 2001, and
on September 23, 2002. The onions were hand-pulled, topped and graded as described
above.

Statistical analysis

Analysis of variance was performed as described above. Large and small bulb

yields were transformed by square root of x+1 due to small counts in these categories in

2002, and then analysis of variance was performed.

125

RESULTS

The effect of bed type and irrigation on onion quality and yield.
2001 Experiment.

Raised beds had a significantly higher number of plants compared to flat beds 19,
40, 60, 79 and 99 days after planting (DAP). There were no significant differences
between irrigation and no irrigation (Table 5.5). Hoopla had significantly lower number
of plants compared to Spartan Supreme and T-439. There was a significant interaction
between irrigation and bed type (Table 7.1, 7.2, 7.3, 7.4, 7.5 in the appendix). Flat beds
plus irrigation had a significantly lower number of plants compared to flat beds without
irrigation, or to raised beds with or without irrigation (F ig.5. 1).

The percentage of plants with purple blotch was not significantly different
between irrigation and no irrigation or between bed types 106 DAP (Table 5.6). Flat beds
had a significantly higher percentage of plants infected with purple blotch compared to
raised beds 120 DAP. Hoopla and Spartan Supreme had a higher percentage of plants
with purple blotch compared to T-439 106 DAP. The percentage of plants with purple
blotch was not significantly different among cultivars 120 DAP (Table 5.6).

Purple blotch disease severity was not different between bed types or among
cultivars 106 and 120 DAP. Irrigated plants had slightly higher disease severity than non-
irrigated plants 120 DAP (Table 5.7). There was no significant interaction between
irrigation, bed type and cultivars on purple blotch incidence or severity.

Total and marketable yield was not significantly different between irrigation

treatments (Table 5.8). Irrigated plants tended to produce larger bulbs compared to non-

126

irrigated plants, but the difference was not significant. Raised beds had significantly
higher total and marketable yield compared to flat beds. Moreover, plants growing on
raised beds had significantly higher large and medium bulb yield compared to flat beds.

Spartan Supreme had the highest total yield but was not significantly different
from T-439. Spartan Supreme and T-439 had higher marketable yield than Hoopla (Table
5.8). Spartan Supreme and Hoopla had more large bulbs than T-439.

The percentage of marketable bulbs 142 days after harvest (DAH) was not
significantly different between irrigation or between bed type (Table 9). Spartan Supreme
had the highest percentage of marketable bulbs (96%) compared to T439 (90%) and
Hoopla (72%). The same trend was observed 177 DAH (Table 5.9).

2002 Experiment.

Raised beds had a significantly higher number of plants compared to flat beds 39,
60, 81, and 100 DAP (Table 5.10). Irrigation treatments did not affect the plant stand and
there was no interaction between irrigation and bed type. Spartan Supreme had higher
number of plants compared to Hoopla and Norstar. There was no significant interaction
among irrigation, bed type and cultivars.

The percent of plants infected with purple blotch was not significantly different
between irrigation treatments 100 and 120 DAP (Table 5.11). Raised bed had
significantly lower number of plants with purple blotch compared to flat beds both at 100
and 120 DAP. There were no significant differences among cultivars 100 DAP. Hoopla
had the least percent of plants with purple blotch 120 DAP.

Purple blotch severity was not significantly different between irrigation

treatments 100 DAP (Table 5.12). Non irrigated plants had less purple blotch severity

127

 

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120 DAP. Raised beds had significantly lower disease severity compared to flat beds 100
DAP. Purple blotch severity was not significantly different between bed types 120 DAP.
Spartan Supreme had lower purple blotch severity compared to Hoopla and Norstar 100
DAP. At 120 DAP Hoopla had lower purple blotch severity than Spartan Supreme or
Norstar.

The bulbing ratio was not different between irrigation or bed type treatments 81 ,
85 and 89 DAP. Norstar reached bulbing stage earlier than Spartan Supreme and Hoopla,
as indicated by a lower bulbing ratio (Table 5.13). There was a significant interaction
between irrigation and bed type 81 and 85 DAP. Plants growing on flat beds without
irrigation reached bulbing later (0.55) compared to plants growing on raised beds without
irrigation (0.47) 81 DAP. Similar trend was found 85 DAP (Table 5.14).

Leaf, pseudostem and bulb dry weight were not significantly different between
irrigation or bed type treatments 64, 74, 100 and 125 DAP (Table 5.15). Norstar had less
leaf dry weight compared to Spartan Supreme and Hoopla 100 and 125 DAP. Pseudostem
was also smaller in Norstar compared to other two cultivars 100 and 125 DAP. Bulb dry
weight was smaller on Norstar than Spartan Supreme at 125 DAP (Table 5.15).

There was a significant interaction between bed type and irrigation on bulb dry
weight 125 DAP (Table 5.16). Plants growing on flat beds without irrigation had the
lowest dry bulb weight, and there were no significant differences between raised or flat
beds with irrigation. There was a significant interaction between cultivar and bed type on
leaf and pseudostem dry weight 125 DAP. H00pla had higher leaf dry weight on raised
beds compared to flat beds, while leaf dry weight for Spartan Supreme or Norstar were

not significantly different between raised or flat beds (Table 5.17). The same trend was

128

found for pseudostem dry weight (Table 5.18). There was a significant interaction among
irrigation, bed type and cultivar 125 DAP (Table 5.19).

Total and marketable yield was not significantly different between irrigation and
no irrigation. Total yield was not significantly different between raised and flat beds
(Table 5.20). Large and medium bulb sizes were not significantly affected by irrigation or
bed type treatments (Table 5.20). Norstar had significantly lower total and marketable
yield compared to Spartan Supreme and Hoopla. Marketable yield was higher in Spartan
Supreme than Hoopla (Table 5.20). Hoopla had significantly higher large sized bulb than
Spartan Supreme, and Norstar had the lowest large sized bulb of the three cultivars.
Spartan Supreme had more medium sized bulbs of the three cultivars.

The percentage of marketable bulbs 151days after harvest was not significantly
different between irrigation and bed type or among cultivars (Table 5.21).

The effect of fungicide and row spacing on onion quality and yield.
2001 Experiment

The number of plants infected with purple blotch was not different between plants
treated or not with fungicides 107 DAP. There were no differences in the number of
plants affected with purple blotch between Altisimo and Spartan Supreme (Table 5.22).
Plants growing in the three row spacing had significantly higher number of plants
infected with purple blotch (28%) compared with those plants growing in two row beds
(23%) 107 DAP. Plants growing in the three row spacing had higher incidence of purple
blotch (64%) compared to plants growing in the two row spacing (55%), 119 DAP (Table

5.22).

129

Plants not treated with fungicides had higher incidence of purple blotch (70%)
compared to fungicide treated plants (50%) 119 DAP. Spartan Supreme had slightly but
not significantly higher incidence of purple blotch (61%) compared to Altisimo (59%) at
this time.

Disease severity was not different for fungicide and cultivar treatments, but it was
greater on plants growing in three rows (3.1) compared to those growing in two rows
(2.3) 107 DAP. At 119 DAP, disease severity was higher in three rows compared to two
rows, and there were no differences between flmgicide or no fungicide treated plants at
this time. Spartan Supreme had higher disease severity (1.9) compared to Altisimo (1.6)
119 DAP.

Total, marketable, large, medium and small sized bulb yields were not
significantly different for fungicide treatments. Altisimo had higher total, marketable and
large sized bulbs compared to Spartan Supreme. Two row spacing produced lower total,
marketable, large and medium sized bulbs compared to three rows spacing. There were
no significant interactions between fungicides and cultivars on all parameters measured.
There were significant interactions spacing x firngicide (Table 5.23), spacing x cultivar
(Table 5.24) for disease severity 119 DAP; spacing x cultivars for medium sized bulbs
(Table 5.25); spacing x fungicide (Table 5.26) and spacing x fungicide x cultivars for
small sized bulbs (Table 5.27).

2002 Experiment
There were no significant differences between firngicide, cultivar and spacing

treatments on the incidence of purple blotch 100 DAP (Table 5.24). Plants not treated

130

with fungicides had higher incidence of purple blotch (94%) compared to fungicide
treated plants (69%) 120 DAP.

Purple blotch severity was only higher on plants not treated with frmgicides 120
DAP (Table 5.28).

There were no significant differences in total and marketable yields between
fungicide, cultivar and spacing treatments. Plants not treated with fungicide had lower
yield of large sized bulbs (5 t/ha) compared to fungicide treated plants (11 t/ha). Altisimo
had higher yield of large sized bulbs (13 t/ha) compared to Spartan Supreme (3 t/ha).
There was a significant fungicide x cultivar interaction for purple blotch incidence 100
DAP (Table 5.29), firngicide x cultivar for purple blotch severity 120 DAP (Table 5.30),
and fungicide x cultivar for large sized bulbs (Table 5.31). There were no other

significant interactions.

131

DISCUSSION

Raised beds have been proposed for upland and lowland agricultural systems of
southeastern Indonesia for several crops such as soybean (Glycine max), sorghum
(Sorghum vulgare), maize (Zea mays), pigeonpea (Cajanus cajan), and cassava (Manihot
esculenta) to overcome the constraint of watterlogging in the wet season. Excess rainfall
is common in that area when the northwest monsoons are at their peak. Higher annual
production was obtained using this production system (Van Cooten and Borrell, 1999).

Onion fields are often flooded for several days after heavy rains especially during
spring in Michigan. Cumulative rainfall of 140 mm in May and 128 mm in June 2001
(Table 5.1) led to very wet soil condition during germination and early stages of
development. The average precipitation history in May and June for Laingsburg, MI,
(with a minimum of 30 years of records) are 78.5 mm and 85.1 m (Table 5.4). It rained
78% and 50% more in May and June 2001 compared with the historical average. Raised
beds improved plant stand during 2001, when heavy rain flooded flat beds during May
and June. Flat beds had 34% reduction in plant stands in 2001 at 40 DAP.

Cumulative rainfall of 98 mm was registered in May 2002, 25% more than
historical average. Raised beds improved the plant stand again in 2002. Flat beds had
22% reduction in plant stands in 2002 at 39 DAP. Damping off disease of seedlings
occurs worldwide and conditions of high soil moisture and cool temperatures are
favorable for the disease (Schwartz and Mohan, 1995). We observed symptoms of this

disease in our plots especially on flat beds.

132

Chinese cabbage growing on raised beds had less incidence of soft rot caused by
Erwinia carotovora ssp. carotovora compared to flat beds (Fritz and Honma, 1987). In
our study flat beds had more incidence of Altemaria porri than raised beds at 120 DAP in
2001, and 100 and 120 DAP in 2002. The raised beds may have allowed for better
ventilation and decreasing humidity. Maloney et al., (1993) stated that raised beds may
help to decrease stem and foliar diseases in raspberry by reducing humidity within the
canopy. Purple blotch severity was higher on flat beds 100 DAP in 2001, which support
the assumptions of Maloney et al. (1993).

Although total and marketable yields were not significantly different between
irrigation and no irrigation, both tended to increase in irrigated plots (Table 5.8, 5.20).

Raised beds had significantly higher total and marketable yield in 2001 compared
to flat beds. Although there were no significant differences in 2002, both total (32 t/ha)
and marketable yield (29 t/ha) tended to be reduced on flat beds compared with total (37
t/ha) and marketable yield (33 t/ha) on raised bed. Total yield increase was not
significant. Most muckland crops are affected adversely by heavy rainfalls, and onions
often show severe loss of roots, and can develop tip burn on the leaves (Lucas, 1982).
Higher rainfall experienced during May and June 2001 could affect root growth of plants
growing on flat beds. However, in 2002 there was only a short period of rainfall above
normal, and onion roots probably were not affected. Therefore, plants grew better in that
year than in 2001, and probably this was the reason why we did not find differences in
yield between raised and flat beds. Moreover plant dry weight was not different between

raised and flat beds 64, 74, 100 and 125 days after planting (Table 5.12).

133

Hoopla had less stand (Table 5.5) compared to Spartan Supreme and T-439 in
2001. Therefore, this cultivar yielded less than the others. Hoopla is related to Sweet
Spanish onion type, characterized by producing big bulbs. This cultivar had less yield of
medium bulb compared to Spartan Supreme and T-439 in 2001 (Table 5.8). In 2002
Hoopla had higher yield of large bulbs compared to Spartan Supreme and Norstar. The
higher yield of medium bulbs in Spartan Supreme in 2002 (Table 5.20) was related to the
higher stand of plantsof this cultivar compared to Hoopla. Norstar is a medium season
cultivar and produces mostly medium sized bulbs and it reached bulbing earlier than
other cultivars (Table 5.13). It also had less leaf dry weight 100 and 125 DAP (Table
5.15).

We assumed that on flat beds and wet soils we would find more rot in storage
compared to raised beds. The percentage of marketable bulbs 142 and 17 7 days after
harvest (DAH) were no different between irrigation and no irrigation or bed type
treatments (Table 5.9). Similar results were found in 2002 (Table 21). Compared with
historical rainfall records in Laingsburg (Table 5.4), rainfall was 44% less in August, and
65 % less in September in 2001, and 58 % less in August and 54 % less in September in
2002. Therefore, weather at harvest time helped to prevent later losses in storage. Humid
conditions at harvest could increase the percentage of umnarketable bulbs from plots
under irrigation on flat beds. Raised beds can be argued to have disadvantages such as
equipment needs, bed construction costs, increased irrigation needs (Maloney et al.,
1993). However, the advantages of raised bed on plant stand, disease incidence and
severity, and yield will compensate in getting better yield and quality onion bulbs

especially in wet seasons. In two of three years we had flooded conditions in muck soil in

134

Laingsburg during our research, therefore we consider raised bed as a good cultural
practices in an integrated onion management system.

Although disease incidence was not significantly different between fungicide and
no fungicide treated plants 107 DAP in 2001 and 100 DAP in 2002, in the fungicide,
cultivar and spacing experiment, plants without fungicides tended to have higher purple
blotch incidence than fimgicide treated plants (Table 5.22, 5.28). This could be worse in
damp and warm years. Plants growing in three row spacing had higher incidence of
purple blotch 119 DAP in 2001. This agrees with the findings of Dhiman et al. (1987),
Adipala et al. (1994), and Warner et a1. (2002).

Purple blotch severity was higher on plants growing in three row spacing 107 and
119 DAP in 2001, but there were no differences in 2002. Although there were no
significant differences in disease severity between fungicide and no fungicide treated
plants 100 DAP in 2002, plants not treated with fimgicides tended to have higher disease
severity (3.6) compared to fungicide treated plants (2.8).

Total, marketable, large, and medium sized bulbs were greater on plants growing
in 3 rows spacing compared to plants growing in two rows spacing in 2001, but there
were no differences in 2002 (Table 5.22, 5.28). Plant population at harvest was
significantly higher in three rows spacing (389,736 pl/ha) compared to two rows spacing
(238,313 pl/ha) in 2001. However, in 2002 there were no significant differences in plant
population between two rows spacing (403,794 pl/ha) and three rows spacing (391,599
pl/ha) (Table 5.22, 5.28). During 2001 rainfall was higher during May and June, as
explained previously. This higher rainfall during germination and early stage of

development might affect plant stand especially in the two row spacing where the two

135

lines were at the edge of the beds. Water accumulated between beds after heavy rains and
affected onion germination and early development close to the edge of the beds. In 2002
onion rows were planted closer to the center of the bed to avoid that problem in two row
spacing. Damping off could be favored in this situation with the damp condition observed
in 2001. In 2002 we planted the seeds in the two lines 61 cm apart instead of 81 cm used
in 2001, to prevent the damp condition at the edge of the beds. This could explain the
differences in plant stand, besides rainfall was not as high in 2002 as it was in 2001.

Altisimo had higher total and marketable yield than Spartan Supreme in 2001 and
tended to be higher than Spartan Supreme in 2002, which is expected because Altisimo is
a cultivar that produces larger bulbs than Spartan Supreme. Altisimo produced higher
yield of large sized bulbs compared to Spartan Supreme.

Flat beds with irrigation significantly reduced plant stand in 2001 compared to flat
beds without irrigation or raised beds with or without irrigation. However, in 2002 there
was no significant interaction. Rainfall was higher in Spring 2001 than in 2002,
producing watterlogging during germination and early stage of onion development. This
reduction in plant stand was reflected in total and marketable onion yield in 2001 where
flat beds had significantly lower yield than raised beds. Not only the yield was affected
but also the quality. Yield of large sized bulbs also was reduced in flat beds in 2001.

Norstar had a lower bulbing ratio than Spartan Supreme and Hoopla (Table 5.12),
which is expected because Norstar is an earlier cultivar compared to Spartan Supreme
and Hoopla. The significant interaction found in bulbing index between irrigation and
bed type (Table 5.14), revealed that onion plants growing on flats beds without irrigation

tended to reach bulbing later than in flat beds with irrigation or in raised beds with or

136

without irrigation. Taking into account that purple blotch incidence and severity was
favored on plants growing on flat beds, and considering that in flat beds bulbing was
reached later, yield and large sized bulb could be negatively affected on plants growing in
flat beds.

Leaf and pseudostem dry weight was smaller on flat beds as compared to raised
beds in 2002 for Hoopla (Table 5.17, 5.18). If we take into account that 2002 was a much
drier year than 2001, that effect of flat beds on plant growth could be worse in years with
more rainfall and damp conditions. Consequently, stand and plant growth both could be
negatively affected in wet seasons on onions growing on flat beds. Moreover, purple
blotch infection was favored on onion growing on flat beds in 2001 and 2002.

Therefore, a wet season not only could contribute to less stand on flat beds, but also to
increased disease infection that could affect plant grth by reducing leaf area. Bulbing
is related to plant growth and if plants are smaller at the time of bulbing yield and bulb
size would be reduced. In addition, during wet seasons more disease could contribute to
more rot in storage.

In the experiment comparing fungicide, spacing and cultivar, we found a
significant interaction between spacing and fungicide on purple blotch severity 119 DAP.
Purple blotch severity was higher on plants growing in three row beds compared to two
row beds without fungicide (Table 5.23). Besides, Altisimo had higher purple blotch
severity without fungicide than Spartan Supreme 119 DAP (Table 5.24). Therefore,
establishing good management practices that improve soil drainage and air circulation
could contribute to reduced disease incidence and severity similar to that which we found

in our experiments during 2001 and 2002.

137

Yield of medium sized bulbs was reduced in Altisimo and Spartan Supreme
growing in two row spacing in 2002, but this effect was greater in Altisimo than in
Spartan Supreme, which produces bigger bulbs than Spartan Supreme.

All these results suggest that cultural practices that improve plant stand, reduce
disease infection, favor plant growth and contribute to larger sized bulbs and therefore
higher total and marketable yields should be adopted in order to reduce pesticide
application, water and environmental pollution, and to make onion production more

profitable.

138

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Skiles, R. L. 1953. Purple and brown blotch of onions. Phytopathology 43: 409-412.

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Agri. 39:1035-1046.

Warner J., X. Hao, and T. Q. Zhang. 2002. Effects of row arrangement and plant density

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on yield and quality of early, small-vined processing tomatoes. Canadian J. Plant
Sc. 82:765-770.

Wilcox, W. F, M. P. Pritts, and M. J. Kelly. 1999. Integrated control of Phytophthora root
rot of red raspberry. Plant Dis.83:1149-1154.

Zandstra, B., E. Grafius, M. Lacy, and D. Wamcke. 1996. Commercial vegetable
recommendations. Onions. Mich. State Univ. Ext. Ext. Bull. E-1307 (revision).

141

Table 5.1. Rainfall and irrigation from April to September, Muck Soil

Research Station, Laingsburg, MI, 2001.

 

 

Month Rainfall Irrigation
(mm) (mm)

April 32

May 140

June 128

July 31 95

August 54 25

September 81 0

Total over the season 466 120

 

Table 5.2. Rainfall and irrigation from April to September, Muck Soil Research

Station, Laingsburg, MI, 2002.

 

 

Month Rainfall Irrigation
(mm) (mm)
April 68 0
May 98 0
June 78 25.6
July 70 73.6
August 40 35.2
September 45 9.6
Total over the season 399 144

 

142

Table 5.3. Historical average weather records for Laingsburg, MI, with a minimum

period of records of 30 years.

 

 

Month Avg. high Avg. low Mean temp. Avg.
temp. (C ) temp. (C ) (C ) Precipitation
(nun)
January - 2 -10 -6 42.4
February 0 -9 -4 35.6
March 6 -4 1 55.6
April 13 2 7 82.3
May 21 8 14 78.5
June 26 13 19 85.1
July 28 16 22 75.7
August 27 14 21 96.0
September 22 10 16 97.0
October 16 4 10 73.4
November 8 -l 3 66.3
December 1 -7 -3 49.8

 

Source: The weather channel 2003, <http://www.weather.com/weather/climatology/monthly/48848/>.

143

Table 5.4. Disease severity rating used to evaluate purple blotch foliar disease

caused by Altemaria porri.

 

Disease rate*

 

0= no disease

1= less than 5% of leaf area affected by the disease
2 = 2 5 to S 20 % of leaf area affected by the disease
3= 2 20 to S 50% of leaf area affected by the disease
4= 2 50 to S 70% of leaf area affected by the disease

5= > 70 % of leaf area affected by the disease

 

*Determined by visual observation

144

Table 5.5. Number of live plants in 3 m of bed on flat or raised beds, with or without

irrigation, Muck Soil Research Station, Laingsburg, MI, 2001.

 

 

 

Number of plants
Treatment 19 DAP2 40 DAP 60 DAP 79 DAP 99 DAP
Irrigation (1) NS NS NS NS NS
Irrigation 237 143 123 120 114
No irrigation 234 153 137 135 130
Bed type (B) an *1: u *1: an
Raised bed 275 178 149 144 137
Flat 196 118 111 110 108
Ix B atol- u u an an:
gm (C) u u n n u
Hoopla 194 117 103 100 95
S. Supreme 252 157 141 136 129
T-439 262 169 146 145 143
LSD (5%) 30 23 23 24 21
CV (%) 15 19 21 22 21
C XI NS NS NS NS NS
C x B NS NS NS NS NS
C XI X B * NS NS NS NS

 

2: DAP: days after planting, NS: Nonsignificant, ‘: significant at P s 0.05, “: significant at P s 0.01.

145

Table 5.6. Effect of irrigation, bed type, and cultivar on the percentage of plants
infected with purple blotch 106 and 120 days after planting, Muck Soil Research

Station, Laingsburg, MI, 2001.

 

 

Treatments Infected plants Infected plants
106 DAPz 120 DAP
(%) (%)

Irrigation (I) NS NS
Irrigation 26 59
No Irrigation 30 57

Bed type (B) NS "'
Raised bed 27 55
Flat 30 60

Ix B NS NS

mm. (C) *"‘ NS
Hoopla 29 60
Spartan Supreme 33 59

T-439 23 54

LSD (5%) 5 NS

CV (%) 22 13

C XI NS NS

C x B NS NS

C x I x B NS NS

 

2 DAP: Days after planting. NS: Nonsignificant, *: significant at P S. 0.05, “i significant at P S 0.01.

146

Table 5.7. Effect of irrigation, bed type, and cultivar on Altemaria porri severity

106 and 120 days after planting, Muck Soil Research Station, Laingsburg, MI, 2001.

 

 

Treatments Disease severity 7‘ Disease severity
106 DAPy 120 DAP
(leaf 3 from the base) (leaf 4 from the base)x

Irrigation (I) NS *
Irrigation 1.5 0.97
No Irrigation 1.7 0.75

Bed type (B) NS NS
Raised bed 1.5 0.9
Flat 1.7 0.8

I x B NS NS

Qu_lti_vil; (C) NS NS
Hoopla 1.6 0.9
Spartan Supreme 1.6 0.9
T-439 1.6 0.8

LSD (5%) NS NS

CV (%) 15 20

C x I NS NS

C x B NS NS

C x Ix B NS NS

 

z : Disease rating 0: no disease, 5: 2 70 % of foliage affected by the disease
’ DAP: Days after planting; NS: Nonsignificant,‘ "" significant at P 50.05.

x: By 120 DAP leaf 3 from base was senesced.

147

Table 5.8. Effect of irrigation, bed type, and cultivar on total, marketable, large

bulb and medium bulb yield, Muck Soil Research Station, Laingsburg, MI, 2001.

 

 

Treatments Total yield 2 Marketable y Large bulbs " Medium
(t/ha) yield (t/ha) bulbs V
(V113) (V113)
Irrigation (I) NS NS NS NS
Irrigation 42 41 25 16
No Irrigation 36 35 18 17
Bed 2126 (B) u u *4: u
Raised bed 45 44 24 20
Flat 34 33 19 14
I x B NS NS NS NS
m (C) at: a: all at:
Hoopla 33 32 22 10
S. Supreme 43 42 24 1 8
T-439 41 40 1 8 22
LSD (5%) 6.5 6.4 3.7 4.0
CV (%) 20 20 20 28
C x I NS NS NS NS
C x B NS NS NS NS
C x Ix B NS NS NS NS

 

2: Large, medium and small bulbs; y: Large plus medium bulbs; ": Large bulbs: > 80 mm in diameter;

v: Medium bulbs: 2 50 nrrn 580, NS: Nonsignificant,‘ * : significant at P S 0.05, "z significant at P S 0.01

148

Table 5.9. Effect of irrigation, bed type, and cultivar on marketable bulbs 142 and 177

days after harvest (DAH), Muck Soil Research Station, Laingsburg, M1, 2001-2002.

 

 

Treatments Marketable bulbs Marketable bulbs
142 DAHz 17 7 DAHy
(%) ““0
Irrigation (I) NS NS
Irrigation 85 75
No Irrigation 88 75
Bed type (B) NS NS
Raised bed 88 74
Flat 85 75
Ix B NS NS
9.11.111!!! (C) 4* an:
Hoopla 72 61
Spartan Supreme 96 88
T-439 90 76
LSD (5%) 3 6
cv (%) 5 10
C x I NS NS
C x B NS NS
C x Ix B NS NS

 

z All bulbs were evaluated at this time. y: Fifty visually good bulbs were cut and evaluated for internal

decay. NS: Nonsignificant, "z significant at P < 0.05..

149

Table 5.10. Number of live plants in 3 m of flat and raised beds, with or without

irrigation, Muck Soil Research Station, Laingsburg, MI, 2002

 

 

Treatment 39 DAP 60 DAP 81 DAP 100 DAP
Irrigation (I) NS NS NS NS
Irrigation 193 162 156 148
No irrigation 203 163 159 147
Bed type (B) 4* an: #11! *4:
Raised bed 223 185 177 168
Flat 173 140 138 126
I x B NS NS NS NS
_C_ultiV_ar (C) ** *4: an slur:
Hoopla 181 146 141 135
S. Supreme 253 210 207 190
Norstar 160 129 125 117
LSD (5%) 19 32 32 26
CV (%) 11 23 24 20
C X I NS NS NS NS
C X B NS NS NS NS
C x I x B NS NS NS NS

 

2: DAP: days after planting; NS: Nonsignificant, 31: significant at P S 0.01.

150

Table 5.11. Effect of irrigation, bed type, and cultivar on the percentage of plants

infected with purple blotch 100 and 120 days after planting, Muck Soil Research

Station, Laingsburg, MI, 2002.

 

 

Treatments Infected plants Infected plants
100 DAPz 120 DAP
(%) (%)
Irrigation (1) NS NS
Irrigation 27 65
No Irrigation 23 58
Ms (B) ** u
Raised bed 21 54
Flat 29 68
I x B NS NS
Cm (C) NS **
Hoopla 26 56
S. Supreme 22 63
Norstar 27 66
LSD (5%) NS 6
CV (%) 25 11
C x I NS NS
C x B NS NS
C x I x B NS NS

 

2: DAP: Days after planting. NS: Nonsignificant, ": significant at P .<_ 0.01.

151

Table 5.12. Effect of irrigation, bed type, and cultivar on Altemaria porri severity 100

and 120 days after planting, Muck Soil Research Station, Laingsburg, MI, 2002.

 

 

Treatments Disease severity Disease severity
100 DAPz 120 DAP
Qeaf 3 from the base) (leaf 4 from the base)x

Irrigation (I) NS **
Irrigation 3.4 3.0
No Irrigation 2.9 2.1

Bed type (B) ** NS
Raised bed 2.7 2.6
Flat 3.6 2.6

Ix B NS NS

gu_1ti_v_ar_(C) . ..
Hoopla 3.3 2.0
Spartan Supreme 2.7 2.8
Norstar 3.4 2.9

LSD (5%) 0.4 0.3

cv (%) 17 16

C XI NS NS

C x B NS NS

C x I x B NS NS

 

2: DAP: Days after planting, NS: Nonsignificant, *: Significant at P S 0.05; "z Significant at P s 0.01

x: By 120 DAP leaf 3 from base was senesced.

152

Table 5.13. Effect of irrigation, bed type, and cultivar on bulbing ratio (minimum

pseudostem diameter/maximum bulb diameter) 81, 85 and 89 days after planting (DAP),

Muck Soil Research Station, Laingsburg, MI, 2002.

 

 

 

Bulbing ratioz
Treatments 81 DAPy 85 DAP 89 DAP
Irrigation (I) NS NS NS
Irrigation 0.52 0.50 0.45
No Irrigation 0.51 0.49 0.47
M (B) * NS NS
Raised bed 0.49 0.48 0.45
Flat 0.54 0.50 0.46
I x B * * NS
Cplflyar (C) *4: an: an
Hoopla 0.52 0.49 0.47
S. Supreme 0.58 0.57 0.53
Norstar 0.45 0.41 0.37
LSD (0.05) 0.05 0.05 0.07
CV (%) 10 13 18
C XI NS NS NS
C x B NS NS NS
C x Ix B NS NS NS

 

2: bulbing ration: minimum pseudostem diameter/maximum bulb diameter": DAP: Days after planting.

NS: Nonsignificant, *: Significant at P s 0.05, “Significant at P S 0.01.

Table 5.14. Interaction between irrigation and bed type on bulbing ratio 81 and 85 DAP,

Muck Soil Research Station, Laingsburg, MI, 2002.

 

Raised bed Flat bed Raised bed Flat bed

 

Bulbing ratio2 81 DAPy Bulbing ratio 85 DAP

 

 

Irrigation 0.52 0.52 0.51 0.48
No Irrigation 0.47 0.55 0.45 0.53
LSD (5%) 0.05 0.06

 

z: bulbing ration: minimum pseudostem diameter/maximum bulb diameter.

y: DAP: Days after planting.

154

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Table 5.16. Interaction between irrigation and bed type on bulb dry weight 125 day

after planting. Muck Soil Research Station, Laingsburg, MI, 2002.

 

 

 

Bulb dry weight (g) z
Raised bed Flat bed
Irrigation 50.1 54.8
No Irrigation 55.2 45.5 *
LSD (5%) 9.4

 

I : dry weight of five plants, *: Significant at P < 0.05.

Table 5.17. Interaction between bed type and cultivar on leaf dry weight 125 day

after planting” Muck Soil Research Station, Laingsburg, MI, 2002.

 

 

 

Leaf dry weight (g) Z
Spartan Hoopla Norstar
Supreme
Raised bed 21.7 28.2 12.0
Flat bed 25.8 21.2 * 11.8
LSD (5%) 4.6 4.6 4.6

 

z: dry weight of five plants, ’2 Significant at P < 0.05.

156

Table 5.18. Interaction between bed type and cultivar on pseudostem dry weight

125 day after planting” Muck Soil Research Station, Laingsburg, MI, 2002.

 

 

 

Pseudostem dry weight (g) 2
Spartan Hoopla Norstar
Supreme
Raised bed 5.6 7.0 3.0
Flat bed 6.7 4.9* 2.9
LSD (5%) NS 1.7 NS

 

z : dry weight of five plants, NS: Nonsignificant; *: Significant at P s 0.05

Table 5.19. Interaction among irrigation, bed type and cultivar on bulb dry weight

125 day after planting,, Muck Soil Research Station, Laingsburg, MI, 2002.

 

 

 

Bulb dry weight (g) Z
Raised bed Flat bed
S. Hoopla Norstar S. Hoopla Norstar
Supreme Supreme
Irrigation 48 56 46 72 41 5 1
No Irrig. 63* 52 51 50* 47 39
LSD (%) 16

 

Z: dry weight of five plants, I": Significant at P .<_ 0.05

157

Table 5.20. Effect of irrigation, bed type, and Cultivar on total, marketable, large bulb

and medium bulb yield, Muck Soil Research Station, Laingsburg, MI, 2002.

 

 

Treatments Total yield 7‘ Marketable y Large bulbs x Medium
(t/ha) yield (t/ha) bulbs V
(t/ha) (t/ha)
Irrigation (I) NS NS NS NS
Irrigation 38 36 10.8 24.9
No Irrigation 30 27 5.9 21.0
Bed type (B) NS NS NS NS
Raised bed 37 33 8.5 24.5
Flat 32 29 8.2 21.3
Ix B NS NS NS NS
m (C) an: an: an slur:
Hoopla 37 35 15.8 19.2
S. Supreme 46 42 7.5 34.3
Norstar 20 17 1.8 15.2
LSD (5%) 6 6 2.1 4.7
CV (%) 20 20 30 24
C x I NS NS * NS
C x B NS NS NS NS
C x Ix B NS NS NS NS

 

‘: Large, medium and small bulbs, yz Large plus medium bulbs, ": Large bulbs: > 80 mm in diameter; ":

Medium bulbs: 2 50 mm 580; NS: Nonsignificant,’ *: significant at P $0.05, *"': significant at P $0.01.

158

Table 5.21. Effect of irrigation, bed type, and cultivar on marketable bulbs 151 days

after harvest (DAH), Muck Soil Research Station, Laingsburg, M1, 2002-2003.

 

 

Treatments Marketable bulbs
1 51 DAHz
(%)
Irrigation (1) NS
Irrigation 93
No Irrigation 94
Bed type (B) NS
Raised bed 93
Flat 94
Ix B NS
Cilitiw (C) **
Hoopla 89
S. Supreme 98
Norstar 92
LSD (5%) NS
cv (%) 4
C x I NS
CxB NS
C x I x B NS

 

Z: All bulbs were evaluated at this my“: Nonsignificant.

159

 

 

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161

Table 5.23. Interaction between spacing and fimgicide on disease severity 119 days

after planting, Muck Soil Research Station, Laingsburg, MI, 2001.

 

 

Treatments Two rows Three rows
Disease severityz Disease severity

F ungicide 1.23 1.58

No fungicide 1.70 2.4

LSD (5%) 0.18

 

2: Disease severity rating: Ozno disease, 5:> 70% of the third green leaf from the base affected with the

disease.

Table 5.24. Interaction between spacing and cultivars on disease severity 119 days

after planting, Muck Soil Research Station, Laingsburg, MI, 2001.

 

 

Treatments Spartan Supreme Altisimo
Disease severityz Disease severity

Fungicide 1.7 2.0

No fungicide 1.3 1.9

LSD (5%) 0.18

 

2: Disease severity rating: Ozno disease, 5:> 70% of the third green leaf from the base affected with the

disease.

162

Table 5.25. Interaction between spacing and cultivars for medium sized bulbs yield,

Muck Soil Research Station, Laingsburg, MI, 2001.

 

Treatments Two rows Three rows

Medium sized bulbsz (t/ha) Medium sized bulbs (t/ha)

 

Spartan Supreme 23.9 29.6
Altisimo 10 25.8
LSD (5%) 4.02

 

2: Medium bulbs: 2 50 mm 580 mm in diameter

Table 5.26. Interaction between spacing and fungicide on small sized bulbs, Muck Soil

Research Station, Laingsburg, MI, 2001.

 

Treatments Two rows Three rows

Small sized bulbsz (t/ha) Small sized bulbs (t/ha)

 

Fungicide 0.8 1.8
No fungicide 1.5 1.7
LSD (5%) 0.49

 

2: Small bulbs: < 50 mm in diameter.

163

Table 5.27. Interaction between spacing, fungicide and cultivar on small sized

bulbs ‘, Muck Soil Research Station, Laingsburg, MI, 2001.

 

 

Treatments Spartan Supreme Altisimo

Two rows Three rows Two rows Three rows
F ungicide 1.3 2.5 0.3 1.1
No fungicide 2.5 2.0 0.5 1.3
LSD (5%) 0.69

 

2: Small bulbs: < 50 mm in diameter.

164

 

 

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166

Table 5.29. Interaction between fungicide and cultivars on the number of
plants infected with purple blotch incidence 100 days after planting, Muck Soil

Research Station, Laingsburg, MI, 2002.

 

Treatments Spartan Supreme Altisimo

Infected plants with p. Infected plants with p.

 

blotch (%) blotch (%)
F ungicide 27 20
No fungicide 31 36
LSD (5%) 1 1

 

Table 5.30. Interaction between fungicide and cultivars on purple blotch severity

120 days after planting, Muck Soil Research Station, Laingsburg, MI, 2002.

 

Treatments Spartan Supreme Altisimo

Purple blotch severityI Purple blotch severity

 

Fungicide 3 .1 2.3
No fungicide 4.3 4.1
LSD (5%) 0.9

 

ZI Disease rating 0: no disease, 5: 2 7O % of foliage affected by the disease

167

Table 5.31. Interaction between fungicide and cultivars on large sized bulb yield,

Muck Soil Research Station, Laingsburg, MI, 2002.

 

Treatments Spartan Supreme Altisimo

Large bulb (t/ha)z Large bulb (t/ha)z

 

Fungicide 3.8 18.1
No fungicide 2.1 7.9
LSD (5%) 1.4

 

‘: Large bulbs: > 80 mm in diameter.

168

 

 

  
 
  
   

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onion cultivars, Muck Soil Research Station, Laingsburg, MI, 2001.

169

CHAPTER VI

FOLIAR DESICCATION OF ONION WITH HERBICIDES

170

ABSTRACT
Onion maturity is often delayed as a result of adverse weather and uncontrolled weeds. If
onion leaves are mowed before harvest, the bulbs are susceptible to invasion by bacterial
soft rot, which develops in storage. If chemicals are used to stop foliar growth and
desiccate onion leaves, to mimic natural senescence, the incidence of bacterial soft rot in
storage may be reduced. However, some herbicides applied as preharvest desiccants may
injure onion bulbs and allow bacterial soft rot to develop. The objective of this study was
to evaluate the effects of different compounds on desiccation of onion foliage and on
stored bulb quality. Experiments were conducted in 1993, 1994, 1995, 2001 and 2002 at
the Muck Soil Research Station, Laingsburg, MI. Chemical desiccants were applied when
onion leaves were 25-80% down and 50-70% green. Foliar desiccation was rated on a
scale 1-100, where 1 = no desiccation, 100 = complete desiccation of all leaves. After
harvest, bulbs were topped, cured for two weeks in ambient air and placed in storage.
After varying periods of time in storage, bulbs were evaluated for rot. Diquat and
paraquat had desiccated onion leaves in the field very well but they caused internal decay
of bulbs in storage. Carfentrazone was highly effective in desiccation of onion foliage but
reduced the percentage of marketable bulbs in storage to 66 % on average. Bromoxynil
desiccated onion foliage 70 to 97%, depending on the year, without inducing rot or

reducing the percentage of marketable bulbs in storage.

171

INTRODUCTION

Dry bulb onion is the third largest vegetable crop consumed in the USA with per
capita consumption of 8.2 kg/year (ERS-U.S. Department of Agriculture 2003, PMA
2003). Onions are produced commercially in 16 states (U .S. Department of Agriculture,
2003). Michigan has been a major onion producer in the past with over 5,000 ha but now
has about 1,600 ha with a total production of 45,000 t per year (Kleweno and Matthews,
2002) with a total value of 9 million dollars (U .S. Department of Agriculture A, 2003).

The principle edible part of an onion is a bulb, which is composed of the swollen
base of the leaves. The growing point is in the center of the compressed true stem, which
is at the base of the bulb (Brewster, 1994). An onion plant continually produces new
leaves from the center of the stem and new roots. Leaf bases of successively produced
leaves are larger in diameter and length. Bulbing is induced by several factors, including
plant age, size, daylength and temperature (DeMason, 1990). Long-day storage onions
such as those grown in Michigan are induced to bulb with about 14 hours daylength.

During the bulbing process, young leaves stop developing but continue to form
swollen bulb scales. During bulbing, the photosynthetic reserves that have been held in
the leaf tissues are translocated to the leaf bases to form the bulb. As the bulb matures,
the outermost leaf sheaths become dry and thin and form the protective scales of the bulb.
When the bulb approaches maturity leaves soften at the neck and become less turgid,
which causes the leaves to fall over (Brewster, 1994).

Onion bulbs continue to gain size and weight as the photosynthates are moved
from the remaining green leaves to the bulb (Tiessen et al., 1981; Voss, 1979; and

Zandstra et al., 1996). Yields may increase 10% to 40% from initial bending of leaves to

172

 

totally dry foliage (Brewster, 1990; Wall and Corgan, 1994). At full maturity, all leaves
are brown and desiccated, and the bulb becomes dormant.

The differences between rest and dormancy sometimes are confused. Meyer and
Anderson (1952) defined dormancy as "a state where seeds fail to germinate even if
placed under such conditions that all environmental factors are favorable for
germination”.

Lang (1987) stated that "dormancy is the temporary suspension of visible growth
of any plant structure containing a meristem". Because of the confusion between terms
he differentiates between ecodormancy, which describes the dormancy when one or more
environmental factors are not suitable for growth, and endodormancy or dormancy due to
physiological factors within the dormant structure. Mehdinaqui (2002) stated that
dormancy "can be considered as the ability to retain viability while having minimal
metabolic activity". Chong (1995) defined rest" like a process that prevents seeds in their
natural habitat from germinating in unseasonal times".

Onion bulbs are morphologically dormant from near harvest through a certain
period of storage, regardless of whether the apical meristem of bulbs is mitotically
inactive or not (Brewster, 1990; 1994). After a period of dormancy, the onions remain at
rest if still in storage. Moving resting onions to ambient conditions will cause them to
sprout. For a chemical desiccant to be effective, it should induce dormancy rather than
rest, without killing or injuring the bulb meristem. The bulb should then survive storage
and a period of shelf life afterwards.

After harvest, onions are cured to prepare the bulbs for shipping or storage.

Curing is the process of natural or forced-air drying to dry out outer scales and shrink the

173

neck. (Hoffmann et al., 1996). During curing onions lose about ten percent of their fresh
weight.

In some years onion do not reach maturity and leaves remain green beyond the
normal harvest period as a result of late planting, improper cultivar selection, incorrect
cultural practices, inadequate heat during the growing season, or a damp, cool fall
(Tiessen et al., 1981; Johnson, 1986; Pelter et al., 1992). Excessive green leaves make
harvesting difficult, and lack of maturity results in lack of dormancy and poor storage
quality.

During onion bulbing, growth inhibitors that are responsible for maintaining
subsequent dormancy are translocated from the leaves to the bulb. If the harvest is too
early, less of the sprouting inhibitor is translocated to the growing point of the bulb. On
the other hand, harvesting too late may allow the destruction of the inhibitor. Early
defoliation or leaf desiccation results in earlier sprouting in storage since inhibitory
substances are not fully translocated from the leaves to the bulbs before defoliation
(Stow, 1976).

Storage onions may be treated with a maleic hydrazide, a sprout inhibitor, to
extend storage life of the bulbs. This compound should be applied when about 50 to 60
percent of the tops have fallen over and onions still have at least five actively growing
leaves to translocate the compound to the bulb (Walz and Burr, 1977; Tiessen et al.,
1981; Voss, 1979; Hoffrnann et al., 1996). Maleic hydrazide does not improve the
storability of onions, it only reduces the incidence of sprouting at the end of the dormant

period; i.e., it extends the rest period. Without a sprout inhibitor, bulbs of some cultivars

174

begin to sprout as soon as they are removed from storage and exposed to ambient
conditions.

Onions that are not well dried in the field are susceptible to sprouting and decay
during and after storage. Before storing onions, it is therefore necessary to dry the bulbs
and shrink the necks as rapidly as possible to prevent pathogens from entering (Kaufman
and Lorbeer, 1967; Jones and Mann, 1968). Growers have used several methods to hasten
onion maturity in the field, including rolling the tops with a lightweight roller,
undercutting the roots with a knife or rod weeder (Longbrake et al., 1974; Voss, 1979;
Tiessen et al., 1981), and applying chemical desiccants (Harmer and Lucas, 1955).

An effective desiccant for onions must dry the leaves relatively rapidly to avoid
entrance of bacterial or fungal diseases; it should induce dormancy of the bulb without
killing the growing point; and it must dissipate rapidly so there are no excess chemical
residues in the onion bulbs, which will be used for human consumption. Several
chemicals have been used commercially over the past 60 years to desiccate onion leaves
before harvest. These include xanthogen disulfide (sulfasan) and diethyl dithiobis
(thionoformate), (Herbisan 5®) [F ike Chemicals], (Harmer and Lucas, 1955; Meister et
al., 1983). Onion growers also used dinoseb as a pre-harvest desiccant, but it was
removed from the market in 1986 by the US. Environmental Protection Agency
(Masiunas, 1991). No safe and effective onion desiccants have been identified since then.

Researchers have tried several chemicals for onion desiccation. Isenberg and
Abel-Rahman (1972) applied neo-decanoic acid (NDA) to desiccate onion foliage and
allow for an earlier machine harvest. They found that two weeks after NDA application

plants were ready to harvest while plants treated with water and adjuvant were still green.

175

Onion leaves treated with NDA contained 42% moisture compared with 85% in control
plants. The tops of onions treated with paraquat dried very rapidly but they had a higher
percentage (47%) of rot than the control (1%). Endothall caused rot in storage (29%)
when applied at 4.5 kg.ha'1, but onions treated with 1.1 kg.ha’1 had only 4% rot
(Richardson et al., 197 7). Similar results were reported by Zschau and Bottcher (1968)
using paraquat for desiccating onion foliage. Bubl et al. (1979) found that paraquat-
treated bulbs had a significantly higher incidence of neck rot and bacterial diseases than
controls.

In onion set production, paraquat desiccated 80% of onion shoots within 3 days,
while diquat desiccated 60% in 10 days. Both herbicides injured the growing point in the
bulbs, which resulted in bulb rot, so they were considered not acceptable for foliar
desiccation in onion set production (Masiunas, 1991).

The purpose of this research was to identify chemicals that would desiccate onion

leaves and induce dormancy, while having no adverse effects on the bulbs.

176

MATERIALS AND METHODS

All the experiments were conducted at the Michigan State University (MSU)
Muck Soil Research Station, Laingsburg, Michigan, on a Houghton Muck soil, Euic
mesic typic Medisaprist, 80% organic matter, and pH 6.6. The plot size was 1.6 m wide,
and 15 m long on raised beds with 3 rows, 41 cm apart per bed. Seeds were planted at a
spacing of approximately 2 cm. Chemical sprays were applied with a C02 backpack
sprayer with 4 flat fan 8002 nozzles on the boom, which delivered 187 L.ha'1, at a
pressure of 207 KPa, and a speed of 5.1 km.h".

The experiments were designed as a randomized complete block with three
replications. The plots were maintained with pesticides throughout the growing season, as
recommended commercially (Bird et al., 2002; Zandstra et al., 1996). Onion foliar
desiccation was rated on a scale of 1-100, where l = no desiccation, 100 = complete
desiccation of all leaves. Alter harvest bulbs were topped, cured for two weeks in
ambient air, and placed in common storage. Storage temperature declined as ambient
temperature declined, and was maintained at 1-3 C. The bulbs were taken out of storage
and evaluated after at least 15 weeks of storage.

1993 Experiment 'Sweet Sandwich' onion, Asgrow Seed Co., Kalamazoo,

Mich.; was planted on April 27 , 1993 with a Gaspardo precision vacuum planter. The
plots were maintained with pesticides throughout the growing season, as recommended
commercially. The desiccant treatments were applied on August 27, when onion leaves
were 50% down and most leaves were still green. The treatments were paraquat 0.53 kg

a.i.ha", diquat 0.28 kg.ha", bromoxynil 0.56 kg. ha“, endothall 1.12 kg.ha", copper

177

sulfate 4.5 kg.ha'1, urea ammonia nitrate (UAN 28%) 46.8 L.ha'1, sodium borate 11.2
kg.ha", ammonium nitrate 22.4 kg.ha", calcium chloride 33.6 kg.ha’1, pelargonic acid 7.6
L.ha’1, ammonium chloride 5.6 kgha", and untreated control. Onions were rated for
foliar desiccation 11 days afier treatment (DAT). Four crates of large, hard onions,
approximately 91 kg from each plot were harvested on September 14, 1993. The onions
were cured in ambient air for two weeks and placed in storage on September 28. Fifty
onions from each plot were cut and evaluated for rot on December 28, 1993, and another
50 onions were cut and evaluated on March 4, 1994, at 105 and 171 days alter harvest
(DAH).

1994 Experiment. 'Spartan Banner 80' onion, Asgrow Seed Co., Kalamazoo Mich., was
planted on May 3, 1994, as described above. Desiccant treatments were applied on
August 25, when onion leaves were 25% down and 80-90 % green. The treatments were
bromoxynil 1.12 kg a.i.ha'l, bromoxynil 1.12 kg.ha'l plus organosilicone surfactant
(OSS) 0.5% "v/v" (Silwet L-77, Loveland Industries, Greeley, CO); endothall 1.12 kg.ha’
1, endothall 1.12 kg.ha'1 plus OSS 0.5% "v/v"; copper sulfate 5.6 kg.ha'1, copper sulfate
11.2 kg.ha’1, pelargonic acid 18.7 L.ha", pelargonic acid 37.4 L.ha'l, calcium EDT A 9.35
L.ha'l, calcium EDTA 9.35 L.ha'l plus OSS 0.5% "v/v"; sodium molybdate 1.12 kg.ha‘1
plus copper sulfate 4.48 kg.ha’1, and an untreated control. Onions were rated 8 DAT. Two
crates of approximately 40 kg of onions from each plot were harvested on September 21.
The onions were cured in ambient air for two weeks and placed in storage on October 5.
Fifiy onions from each plot were cut and evaluated for rot on February 23, 1995 (155

DAH).

178

1995 Experiment. 'Comanche' onion, Rispens Seed Co., Beecher 111., was planted on
May 10, as described above. Desiccant treatments were applied on September 1, when
onion leaves were 80% down and 60-70% green. The treatments were bromoxynil 1.12
kg.ha", plus OSS 0.5% "v/v" (Silwet L-77, Loveland Industries, Greeley, CO); endothall
1.12 kg.ha", endothall 1.12 kg.ha'l plus OSS 0.5% "v/v"; copper sulfate 11.2 kg.ha'l plus
OSS 0.5% "v/v"; copper sulfate 11.2 kg.ha", plus fatty acid esters with emulsifiers 1%
"v/v" (Hasten®, Wilfarrn L.L.C., Nappanee, IN); pelargonic acid 37.4 L.ha'1, calcium
EDTA 18.7 L.ha'l plus fatty acid esters with emulsifiers 0.5% "v/v"; potassium carbonate
2.24 kg.ha'l plus ethyloleate 1% "v/v" (Ee-Muls-Oyle®, Victorian Chem. Co.,
Richmond, Vic., Aust.); potassium hydroxide 2.24 kg.ha'l plus ethyloleate 1% "v/v";
paraquat 0.56 kg.ha'1 plus nonionic surfactant (NIS) 0.5 % "v/v" (Activator 90, Loveland
Industries, Greeley, CO.), and an untreated control, Onions were rated 11 DAT. Two
crates of approximately 40 kg of onions from each plot were harvested on September 19.
The onions were cured in ambient air for two weeks and placed in storage on October 3.
Fifty onions fi'om each plot were cut and evaluated for rot on March 12, 1996 (174
DAH).

2001 Experiment. 'Hustler' onion, Harris Moran Seed Co., Modesto Calif, was planted
on May 3, 2001 as described above. Desiccant treatments were applied on September 4,
when onion leaves were 70—80% down with 60-70% green leaves. The treatments were
paraquat 0.56 kg.a.i.ha‘l plus OSS 0.5% "v/v" (Silwet L—77®, Loveland Industries,
Greeley, CO); endothall 1.12 kg.ha'l plus liquid ammonium sulfate (AMS) (38%) 2.5%
"v/v" (Bronc®, Wilbur-Ellis Co, Union Gap, WA); diquat 0.56 kg.ha'l plus OSS 0.5%

"v/v"; carfentrazone 0.22 kg.ha'l plus OSS 0.5% "v/v"; carfentrazone 0.22 kg.ha’l plus

179

pelargonic acid 5% "v/v"; glufosinate 0.49 kg.ha'l plus AMS (38%), 2.5 % "v/v" plus
OSS 0.5% "v/v"; and an untreated control. Onions were rated 14 DAT. Two crates of
approximately 40 kg of onions from each plot were harvested on October 1, 2001. The
onions were cured in ambient air for two weeks and placed in storage on October 15. All
bulbs in each plot were evaluated for rot on February 5, 2002 at 127 DAH. Fifty visually
good bulbs were cut and evaluated for rot on March 16, 2002 (166 DAH).

2002 Experiment. 'Cortland' onion, Seedway Inc., Elizabethtown, Pa., was planted on
April 30. Desiccant treatments were applied on August 28, when onion leaves were
70-80% down with about 60-70% green leaves. The treatments were paraquat 0.56 kg
a.i.ha'1 plus OSS 0.5% "v/v"; (Freeway®, Loveland Industries, Greeley, CO); diquat
0.56 kg.ha" plus OSS 0.5% "v/v"; endothall 1.12 kg.ha'1 plus OSS 0.5% "v/v";
carfentrazone 0.12 kg.ha'l plus OSS 0.5% "v/v"; carfentrazone 0.22 kg.ha'1 plus OSS
0.5% "v/v"; bromoxynil 0.56 kgha'l plus OSS 0.5% "v/v"; flumioxazin 0.11 kgha'1 plus
OSS 0.5% "v/v"; flumioxazin 0.22 kg.ha'l plus OSS 0.5% "v/v"; sulfentrazone 0.22
kg.ha'l plus OSS 0.5% "v/v"; piraflufen ethyl 0.0098 kg.ha'l plus LI-700® (Loveland
Industries, Greeley, CO) 0.25% "v/v"; and untreated control. Onions were rated 15 DAT.
Two crates of approximately 40 kg of onions from each plot were harvested on
September 12. The onions were cured in ambient air for 11 days and placed in storage on
September 23. All bulbs were graded on February 18, 2003 (159 DAH). Two hundred
visually good bulbs were selected at this time and evaluated for internal rot, 50 at 159

DAH and 50 at 193 DAH.

180

RESULTS AND DISCUSSION

Paraquat and diquat were the most effective compounds for desiccation of onion
foliage in 1993 with 90% and 93% desiccation, respectively, 11 DAT (Table 6.1).
Bromoxynil was the only other treatment with better desiccation than the untreated
control. However, paraquat and diquat caused significant bulb rot alter storage. None of
the other treatments caused more rot than the control.

In the 1994 trial, none of the treatments gave a sufficiently high level (>90%) of
desiccation. However, bromoxynil and endothall alone gave 67% desiccation, which was
significantly more than the control (Table 6.2). The addition of organosilicone surfactant
(OSS) to bromoxynil or endothall had no significant effect on desiccation. Pelargonic
acid at 37 .4 L. ha'1 and calcium EDTA plus OSS also gave significant improvement in
desiccation, 60% and 53%, respectively. All other treatments were no better than the
untreated control. None of the 1994 treatments reduced storage quality more than the
untreated control.

In the 1995 trial, paraquat gave 97% desiccation ll DAT. Other treatments with
significant desiccation over the untreated were bromoxynil plus OSS (77%), endothall
(63%), endothall plus OSS (67%), copper sulfate plus OSS (63%), copper sulfate plus
Hasten® (63%), and pelargonic acid (63%). While these treatments gave significant
desiccation, it was insufficient for good onion harvest conditions. Only paraquat reduced

marketable bulbs in storage (72% vs 91% for untreated) among these treatments

181

(Table 6.3).

In the 2001 trial, carfentrazone plus pelargonic acid gave significant desiccation
(87%), but it was not significantly different from paraquat plus OSS (73 %), endothall
plus AMS (70%) and carfentrazone plus OSS (77%) (Table 4). Glufosinate plus AMS
gave 67% desiccation. Diquat gave 60% desiccation and it was not significantly different
fiom the untreated control.

Carfentrazone plus OSS, carfentrazone plus pelargonic acid, diquat, and
glufosinate plus OSS plus AMS reduced marketable bulbs to 40%, 48%, 55%, and 59%,
respectively at 166 DAH. The paraquat treatment had 79% marketable bulbs 166 DAH
in 2001-2002, which was lower than the control (96%) but not statistically significant
(Table 6.4). It is interesting to note that paraquat and diquat were not as effective in
desiccation in 2001 as in previous years. Yet diquat caused significant storage rot, while
paraquat also caused a trend toward higher level of rot than the control. Evidently the
condition of the onion foliage at time of application had some effect on uptake of the
chemicals.

In the 2002 trial, paraquat, diquat, endothall, carfentrazone, and bromoxynil, all
gave significant onion leaf desiccation at 15 DAT. Paraquat, diquat and carfentrazone
treatments resulted in reduced percentage of marketable bulbs 159 DAH (Table 6.5).
Several researchers have reported that paraquat or diquat applied to onion foliage as
desiccants caused rot in storage (Bubl et al., 1979; Zschau and Bottcher, 1968;
Richardson et al., 1977). Our results support their conclusions that paraquat or diquat can
not be used as an onion desiccant if onions are to be stored. Bubl et al. (1979), and

Richardson et al. (1977) also reported that endothall was only partially effective as a

182

desiccant, but it was safe, in that it did not cause an increase in storage rot at the normal
use rate.

Bubl et al. (1979) postulated that desiccants may be acting on the leaf and neck
tissue in different ways, such as physical disruption of the cuticle, changes in stomatal
function, alteration in the synthesis, stability, and transport of phenolic compounds, and
physical toxicity to plant cells, and favor pathogen infection.

Carfentrazone was very effective in desiccating onion foliage but it caused
increased rot in storage. Scales of carfentrazone-treated bulbs had a white, transparent
appearance and some were rotten (Fig. 6. l D). The percentage of marketable bulbs was
also low, 40 to 48 % in 2001-2002 at 166 DAH (Table 6.4), and 68-74 % in 2002-2003 at
159 DAH. F lumioxazin, sulfentrazone and pyraflufen ethyl were not effective for leaf
desiccation at rates applied and did not increase rots in storage.

Glufosinate decreased the percentage of marketable onion bulbs in storage to 74%
at 127 DAH in 2001-2002. When visually good bulbs were cut, only 59% were
marketable at 166 DAH in 2001-2002. Flumioxazin, sulfentrazone and pyraflufen ethyl
did not affect onion bulb quality in storage in 2002-2003 (Table 6.5).

Bromoxynil at 0.56 kg/ha gave 87% onion foliage desiccation ll DAT in 1993. It
desiccated onion foliage from 67% to 70% in 1994, when applied alone or plus OSS. It
desiccated 77% of onion foliage in 1995 at 1.12 kg/ha. Applied at 0.56 kg/ha plus OSS in
the 2002 trial, it desiccated onion foliage 77% to 97% at nine and 15 DAT, respectively.
In all the experiments bromoxynil did not reduce the percentage of marketable bulbs in

storage compared to the untreated control.

183

 

In 1994, Calcium EDTA plus OSS gave better foliage desiccation than the
untreated control. In 1995, copper sulfate plus OSS or plus fatty acid esters with
emulsifiers gave slightly better desiccation than the control. All other inorganic salt
treatments (Calcium EDTA alone, sodium molybdate plus copper sulfate, and copper
sulfate) were ineffective in onion leaf desiccation. Pelargonic acid at 18.7 L.ha'l gave
73% foliage desiccation 11 DAT in 1993 (Table 6.1) and 43% eight DAT in 1994 at the
same rate (Table 6.2). At 37.4 L.ha'1 it desiccated 60% and 63% of onion foliage in 1994
and in 1995 respectively (Table 6.2 and Table 6.3). Pelargonic acid did not reduce the
marketable bulbs in storage, but foliage desiccation was insufficient.

An ideal chemical desiccant should dry remaining green foliage, turn the growing
point dormant, stop all foliar growth, and maintain the bulb in a dormant state after
removal from storage. Since xanthogen disulfide and DNBP were removed from the
market, no safe compounds have been available for safe preharvest desiccation in onion
production (Masiunas 1991). Based on the results of the present work, and taking into
account the results of other researchers, pre-harvest onion foliage desiccation may
adversely affect storage quality of onions. Of all the chemicals tested in our experiments,
only bromoxynil gave sufficient onion desiccation with no adverse effects on onion
quality in storage. Bromoxynil is currently recommended for postemergence weed
control in onion, and it may be difficult to register as a post harvest desiccant because of
the very short post harvest interval.

Without good desiccation onion growers must depend on good soil management

practices and drainage, the use of appropriate population densities, adequate weed,

184

fertilizer and irrigation management practices to facilitate onion maturation, cure, and

storage.

185

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188

Table 6.1. Onion foliar desiccation 11 days after treatment (DAT), and percentage of
marketable bulbs 105 and 171 days after harvest (DAH), Muck Soil Research Station,

Laingsburg, M1, 1993-1994.

 

 

Treatment Onion foliage Marketable Marketable
(kg.ha") desic. bulbsz bulbsy 171
11 DAT 105 DAH DAH
(%) ( %) (%)
Paraquat 0.53 90 71 67
Diquat 0.28 93 81 81
Bromoxynil 0.56 87 93 95
Endothall 1.12 83 97 95
Copper Sulfate 4.5 67 99 97
Urea ammonium nitrate 43 99 94

(28%) 46.8 L.ha"

Sodium borate 11.2 57 93 97
Ammonium nitrate 22.4 57 95 95
Calcium chloride 33.6 63 98 97
Pelargonic acid 10% "v/v" 73 93 92
Ammonium chloride 5.6 53 93 96
Untreated control 60 97 93
LSD(0.05) 27 7 7

cv (%) 23 4.5 4.7

 

2‘ y :Fifty bulbs per plot were cut in half and evaluated for internal rot.

189

Table 6.2. Onion foliar desiccation eigth DAT, and percentage of marketable bulbs 155

DAH, Muck Soil Research Station, Laingsburg, MI, 1994-1995.

 

 

Treatment Onion foliage Marketable bulbsZ
(kg/ha) desiccation 155 DAH
8 DAT (%)
(%)
Bromoxynil 1.12 67 88
Bromoxynil 1.12 + OSSy 0.5% "v/v" 7O 86
Endothall 1.12 67 82
Endothall 1.12 + OSS 0.5% "v/v" 6O 83
Copper sulfate 5 .6 33 87
Copper sulfate 11.2 37 88
Pelargonic acid 18.7 L .ha‘1 43 83
Pelargonic acid 37.4 L .ha" 60 86
Calcium EDTA 9.35 L.ha'1 27 90
Calcium EDTA 9.35 L.ha" + 053 53 83
0.5% "v/v"
Sodium Molybdate 1.12 + 43 90
Copper sulfate 4.48
Untreated control 30 88
LSD (0.05) 17 NS
CV (%) 20 6

 

2: Fifty bulbs per plot were cut in half and evaluated for internal decay.

’: OSS: organosilicone surfactant.

190

Table 6.3. Onion foliar desiccation ll DAT, and percentage of marketable bulbs at

174 DAH, Muck Soil Research Station, Laingsburg, MI, 1995-1996.

 

 

Treatment Onion foliage Marketable
(kg/ha) desic. bulbsZ
11 DAT 174 DAH
(%) (%)
Bromoxynil 1.12 + OSS y 0.5 % "v/v" 77 87
Endothall 1.12 63 89
Endothall 1.12 + OSS 0.5 % "v/v" 67 87
Copper Sulfate 11.2 + OSS 0.5 % "v/v" 63 89
Copper Sulfate 11.2 + F.A.E.E" 1 % "WV" 63 92
Pelargonic acid 4.48 63 91
Calcium EDTA 2.24 + F.A.E.E 0.5 % "v/v" 47 89
Potassium carbonate 2.24 + Eemulsoyle 1% "v/v" 47 90
Potassium hydroxide 2.24 + Eemulsoyle 1%" v/v" 37 9O
Paraquat 0.56 + NIS 0.5 % "v/v" 97 72
Untreated control 43 91
LSD (0.05) 12 8
CV (%) 12 4O

 

2: Fifty bulbs per plot were cut in half and evaluated for internal rot.

y: OSS: organosilicone surfactant.

": F .A.E.E: fatty acid esters with emulsifiers.

191

Table 6.4. Onion foliar desiccation 14 DAT, and percentage of marketable bulbs

127 and 166 DAH, Muck Soil Research Station, Laingsburg, M1, 2001-2002.

 

 

Treatment Onion Mark. bulbs Marketable

(kg/ha) foliage 7‘ bulbs y

 

desiccation 127 DAH 166 DAH

 

 

l4 DAT (%) (%)
(%)

Paraquat 0.56 + OSS" 0.5% "v/v" 73 90 79
Diquat 0.56 + OSS 0.5% "v/v" 6O 81 55
Endothall 1.12 + AMS (38%) 2.5% "v/v" 7O 97 94
+ OSS 0.5% "v/v"
Carfentrazone 0.22 + OSS 0.5% "WV" 77 57 40
Carfentrazone 0.22 + 87 68 48
Pelargonic acid 5% "v/v"
Glufosinate 0.49 +AMS (38%) 2.5% 67 74 59
"v/v" + OSS 0.5% "v/v"
Untreated control 43 98 96
LSD (0.05) 18 18 22
CV (%) 15 13 18
2 All bulbs were graded at this time.

’ Fifty visually good bulbs were selected, cut and evaluated for internal rot.

": OSS: organosilicone surfactant.

192

 

Table 6.5. Onion foliar desiccation 15 DAT, percentage of marketable bulbs out of total

bulbs stored at 159 DAH, and apparently good bulbs with internal rot 193 DAH, Muck

Soil Research Station, Laingsburg, M1, 2002-2003.

 

 

Treatment Onion foliage Marketable Apparently
(kg/ha) desiccation bulbs 2 good bulbs
15 DAT 159 DAH with internal
(%) (%) rot 193 DAH
Paraquat 0.56 + OSSx 0.5% "WV" 100 92 2.5
Diquat 0.56 + OSS 0.5% "v/v" 100 76 4.1
Endothall 1.12 + OSS 0.5% "v/v" 100 98 1.5
Carfentrazone 0.12 + OSS 0.5% "v/v" 100 74 3.5
Carfentrazone 0.22 + OSS 0.5% "v/v" 100 68 2.7
Bromoxyni10.56 + OSS 0.5% "v/v" 97 99 0.65
Flumioxazin 0.1 + OSS 0.5%"v/v" 73 99 0.0
Flumioxazin 0.2 + OSS 0.5% v/v" 7O 99 0.0
Sulfentrazone 0.2 + OSS 0.5% "v/v" 57 98 05
Pyraflufen ethyl 0.0098 + 53 99 0.3
Untreated control 67 99 1.2
LSD (0.05) 22 13 1.5
CV (%) 15 9 56

 

’ : All bulbs were graded at this time.

y : 200 visually good bulbs were selected, and 100 were cut to evaluate internal decay. : Values in this

column are ‘1%.

x 2 OSS: organosilicone surfactant.

193

 

Figure 6.1. Cross section of onion bulbs (A) untreated control; (B and C) paraquat and
diquat showing decay of inner scales and at the growing point; and (D)

carfentrazone, showing decay of outer scales.

194

CONCLUSIONS

Didymella bryoniae was more effective on plants than Colletotrichum orbiculare
to induce resistance in onion against Altemaria porri. There was a significant reduction
in the number of lesions of A. porri after the plants were induced with D. bryoniae.
However, the length of lesions was less affected. Therefore, we assumed that the
mechanism of induced resistance in onion against A. porri was effective against the site
of pathogen penetration rather than against pathogen spread.

Methyl jasmonate (Me! A) was the best chemical inducer of resistance against A.
porrz' in onion. BABA had inconsistent results between years. Marketable yield was
reduced with Me] A application in 2002. Therefore, onion plants could experience a
resistance cost. Lower rates of Me] A in combination with fungicides could help to
establish the best rate without adversely affecting yield. The higher number of lesions
observed on untreated control treatments did not cause higher incidence of decay in
storage. However, seasons with more humid conditions from maturity to harvest could
favor the incidence of bacteria and later development of decay in storage.

Although the percentage of rotted bulbs did not differ among chemical resistance
activators in 2002, cultivar T-439 tended to have a slightly lower percentage of rotten
bulbs in Me] A plus fungicide at minimum rate treatments compared to other treatments.
A similar trend was found when Me] A plus fungicide was applied on cultivar Altisimo,
suggesting that this inducing treatment could have a beneficial effect against post harvest
bulb decay. From 140 rotted onion bulb samples tested, 15 were identified as being
infected with B. cepacia, 2 were identified as B. gladioli pv. alliicola, and 1 was

identified as P. ananatis. From the samples that tested positive for B. cepacia, six of them

195

came from plants treated with fimgicides and only 1 from plants treated with Me] A plus
minimum rate of fungicides, indicating that this compound could help to reduce sour skin
(caused by B. cepacia) on onion bulbs.

Raised beds improved plant stands in both years of research, increased total and
marketable yield and reduced purple blotch disease. A two row spacing reduced purple
blotch incidence and severity compared to the three row spacing in 2001. Yield was not
affected when the plant stand was similar between two and three row spacing. The
importance of choosing a good cultivar to get good yield and quality was demonstrated
with Spartan Supreme that produced good yield and stored better than the other cultivars.

Diquat, paraquat, and carfentrazone desiccated onion leaves in the field very well
but they caused internal decay of bulbs in storage. Bromoxynil desiccated onion foliage
70-97%, depending on the year, without inducing rot or reducing the percentage of
marketable bulbs in storage. Bromoxynil is recommended for postemergence weed
control in onion, but it may be difficult to register as a post harvest desiccant because of
the very short post harvest interval. Without a good desiccation onion growers must
depend on good soil management practices and drainage, use of reasonable population
densities, adequate weed, fertilizer and irrigation management practices to facilitate onion

mattu'ity, curing, and storage.

196

APPENDIX

197

Table 7.1. Analysis of variance for plant stand 19 days afier planting, Muck Soil

Research Station, Laingsburg, MI. 2001

 

 

Source Degrees of Sum of Mean F value Probability
fi'eedom squares square

Replication 2 235.39 1 17.69 0.0349

Irrigation (I) 1 72.25 72.25 0.0214

Error 2 6,747.17 3,373.58

Bed type (B) 1 55,617.36 55,617.36 45.33 0.0000

1 x B 1 13,728.03 13,728.03 11.19 0.0032

Cultivar 2 32,300.06 16,150.03 13.16 0.0002

C x I 2 102.17 51.08 0.042

C x B 2 1,112.06 566.03 0.45

C x B x I 2 4,130.39 2,065.19 1.68 0.2111

Error 20 24,541.44 1,227 .07

Total 35 138,586.31

 

CV: 15%

 

 

Table 7.2. Analysis of variance for plant stand 40 days after planting, Muck Soil

Research Station, Laingsburg, MI. 2001

 

 

Source Degrees of Sum of Mean square F value Probability
freedom squares

Replication 2 729.17 364.58 0.087

Irrigation (I) 1 880.1 1 880.1 1 0.2099

Error 2 8,387.39 4,193.69 42.46 0.0000

Bed type (B) 1 32,280.11 32,280.11 14.78 0.0010

I x B 1 l 1,236.0 11,236.00 11.38 0.0005

Cultivar 2 17,299.5 8,649.75 0.69

C x I 2 1,051.06 525.53 0.55

C x B 2 833.39 416.69 4.12

C x B x I 2 6,265.17 3,132.58 0.0317

Error 20 15,2061 1 760.31

Total 35 94,1680

 

CV: 19%

199

Table 7.3. Analysis of variance for plant stand 60 days after planting, Muck Soil

Research Station, Laingsburg, MI. 2001

 

 

Source Degrees of Sum of Mean square F value Probability
fi'eedom squares

Replication 2 649.39 324.69 0.077

Irrigation (I) 1 1,778.03 1,778.03 0.422

Error 2 8,423.39 4,211.69

Bed type (B) 1 13,263.36 13,263.36 17.92 0.0004

I x B 1 8.070.03 8,070.03 10.90 0.0036

Cultivar 2 13,662.39 6,831.19 9.23 0.0014

C x I 2 102.39 51.19 0.069

C x B 2 247.72 123.86 0.17

C x B x I 2 3,805.06 1,902.53 2.57 0.1015

Error 20 14,802.56 740.13

Total 35 64,804.31

 

CV: 21%

200

Table 7. 4. Analysis of variance for plant stand 79 days after planting, Muck Soil

Research Station, Laingsburg, MI. 2001

 

 

Source Degrees of Sum of Mean square P value Probability
fieedom squares

Replication 2 533.72 266.87 0.066

Irrigation (I) 1 2,055.11 2,055.11 0.508

Error 2 8,092.39 4,046.19

Bed type (B) 1 10,885.44 10,885.44 14.25 0.0012

Ix B 1 ' 6,724.00 6,724.00 8.80 0.0076

Cultivar 2 13,286.89 6,643.44 8.69 0.0019

C x I 2 130.89 65.44 0.086

C x B 2 238.89 119.44 0.16

C x B x I 2 3,700.67 1,850.33 2.42 0.1143

Error 20 15,281.89 764.09

Total 35 60,929.89

 

CV : 22%

201

Table 7.5. Analysis of variance for plant stand 99 days afier planting, Muck Soil

Research Station, Laingsburg, MI. 2001

 

 

Source Degrees of Sum of Mean square F value Probability
fi'eedom squares

Replication 2 417.17 208.58 0.056

Irrigation (I) 1 2,352.25 2,352.25 0.63

Error 2 7,480.17 3,740.08

Bed type (B) 1 7,367.36 7,367.36 11.77 0.0026

Ix B 1 4,511.36 4,511.36 7.21 0.0143

Cultivar 2 14,446.17 7,223.08 1 1.54 0.0005

C x I 2 178.17 89.08 0.14

C x B 2 300.72 150.36 0.24

C x B x I 2 3,388.72 1,694.36 2.71 0.0912

Error 20 12,522.67 626.13

Total 35 52,964.75

 

CV: 21%

202

    

     
 
 

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