Effect of Calcium and Boron Nutrition on Grey Mould of

Capsicum (Capsicum annuum L.) and Fruit Quality

By

Thong Duc Le

A thesis submitted for the degree of Doctor of Philosophy

The University of Adelaide

Faculty of Sciences

School of Agriculture, Food and Wine

Waite Campus

Adelaide, South Australia

2014

i
Table of Contents

Abstract ................................................................................................................. vii

Declaration .............................................................................................................. x

Acknowledgements ................................................................................................ xi

Abbreviation .......................................................................................................... xii

CHAPTER ONE

Introduction ............................................................................................................. 1

CHAPTER TWO

General literature review

2.1 Capsicum ........................................................................................................... 3

2.2 Postharvest changes in capsicum ...................................................................... 3

2.3 Grey mould ....................................................................................................... 5

2.3.1 Development of grey mould disease .......................................................... 7

2.3.2 Infection pathways ..................................................................................... 8

2.4 Disease control methods ................................................................................... 8

2.4.1 Physical or cultural methods ...................................................................... 9

2.4.2 Chemical methods .................................................................................... 10

2.4.3 Biological methods ................................................................................... 10

2.5 Calcium and boron application to control disease .......................................... 12

2.5.1 Preharvest application .............................................................................. 13

2.5.2 Postharvest application ............................................................................. 16

2.6 Literature summary and aims of study ............................................................ 17

i
CHAPTER THREE

Infection pathway of Botrytis cinerea in capsicum fruit ....................................... 19

CHAPTER FOUR

Effect of preharvest boron applications

4.1 Introduction ..................................................................................................... 20

4.2 Materials and methods .................................................................................... 21

4.2.1 Plants and growth conditions ................................................................... 21

4.2.2 Nutrient solutions for B application ......................................................... 22

4.2.2.1 Soil application ......................................................................................... 22

4.2.2.2 Foliar B application ................................................................................... 23

4.2.3 Nutrient analysis of leaves and fruit ......................................................... 23

4.2.4 Inoculation of fruit preharvest or postharvest .......................................... 24

4.2.4.1 Isolation, maintenance and culture of B. cinerea ...................................... 24

4.2.4.2 Pre-harvest inoculation ............................................................................. 24

4.2.4.3 Postharvest inoculation of fruit ................................................................. 25

4.2.5 Assessment of grey mould development and postharvest quality in fruit 25

4.2.5.1 Grey mould development on fruit ............................................................. 25

4.2.5.2 Postharvest quality of fruit ........................................................................ 26

4.2.6 Statistical analysis and photography ........................................................ 28

4.3 Results ............................................................................................................. 28

4.3.1 Effect of B applied to soil on nutrient status of plant tissues, postharvest

quality of fruit and grey mould development on capsicum ............................... 28

4.3.1.1 Boron concentration in leaf and fruit tissues............................................. 29

ii
4.3.1.2 Postharvest quality of fruit ........................................................................ 29

4.3.1.3 Grey mould development on fruit derived from inoculated young fruit ... 36

4.3.1.4 Grey mould development on fruit inoculated postharvest ........................ 39

4.3.2 Effect of B as a foliar application on nutrient status of plant tissues,

postharvest quality of fruit and grey mould development ................................. 39

4.3.2.1 Foliar application at lower B concentrations ............................................ 39

4.3.2.2 Foliar application at higher B concentrations ........................................... 50

4.3.3 Negative correlation between lesion area on fruit and boron concentration
on leaf and fruit tissues ...................................................................................... 60

4.4 Discussion ....................................................................................................... 60

4.5 Conclusion ...................................................................................................... 66

CHAPTER FIVE

Effect of preharvest calcium applications

5.1 Introduction ..................................................................................................... 67

5.2 Methods and materials .................................................................................... 69

5.2.1 Plants and growth conditions ................................................................... 69

5.2.2 Nutrient solution for Ca application ......................................................... 69

5.2.2.1 Soil application ......................................................................................... 69

5.2.2.2 Foliar application ...................................................................................... 70

5.2.3 Nutrient analysis ....................................................................................... 72

5.2.4 Inoculation of fruit preharvest or postharvest .......................................... 72

5.2.4.2 Preharvest inoculation of young developing fruit ..................................... 72

5.2.4.3 Postharvest inoculation of fruit ................................................................. 72

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 5.2.5 Assessment of grey mould development and postharvest quality in fruit 72

5.2.5.1 Grey mould development on fruit ............................................................. 72

5.2.5.2 Postharvest quality of fruit ........................................................................ 73

5.2.6 Statistical analysis and photography ........................................................ 73

5.3 Results ............................................................................................................. 74

5.3.1 Symptoms of Ca deficiency in plants treated with low Ca concentration 74

5.3.2 Effect of Ca applied to soil on nutrient status of plant tissues, postharvest

quality and grey mould development on capsicum fruit ................................... 74

5.3.2.1 Soil application of Ca in the first trial (1.5, 4.0 or 8.0 mM Ca) ................ 74

5.3.2.2 Soil application of Ca in the second trial (0.0, 1.5 or 4.0 mM Ca) ........... 84

5.3.3 Effect of Ca as a foliar application on nutrient status of plant tissues,

postharvest quality and grey mould development on capsicum fruit ................ 94

5.3.3.1 Foliar application of Ca in the first trial [0.5, 0.75 or 1% Ca(NO₃)₂] ...... 94

5.3.3.2 Foliar application of calcium in the second trial [0.0, 0.5 or 1% w/v
Ca(NO₃)₂] ........................................................................................................... 103

5.3.4 Correlation between lesion area on fruit and calcium concentration on leaf
and fruit tissues ................................................................................................... 108

5.4 Discussion ..................................................................................................... 114

5.5 Conclusion .................................................................................................... 117

CHAPTER SIX

Effect of postharvest calcium dips or vacuum infiltration

6.1 Introduction ................................................................................................... 119

6.2 Materials and methods .................................................................................. 120

6.2.1 Plant material and growth conditions ..................................................... 120

iv
 6.2.2 Isolation, maintenance and culture of B. cinerea ................................... 120

6.2.3 Effect of calcium chloride on growth of B. cinerea in vitro .................. 121

6.2.4 Postharvest calcium dips ........................................................................ 121

6.2.5 Postharvest calcium vacuum infiltration ................................................ 121

6.2.6 Effect of infiltration method on absorption of Ca into flesh of fruit ...... 122

6.2.7 Postharvest inoculation and assessment of postharvest quality of fruit . 123

6.2.8 Statistical analysis and photography ...................................................... 123

6.3 Results ........................................................................................................... 123

6.3.1 Effect of calcium chloride on fungal growth of B. cinerea .................... 123

6.3.2 Effect of postharvest calcium dips on Ca uptake and nutrient status of fruit
......................................................................................................................... 124

6.3.3 Effect of postharvest calcium dips on postharvest quality and grey mould
development .................................................................................................... 129

6.3.4 Effect of postharvest Ca vacuum infiltration on Ca uptake and nutrient

status of fruit .................................................................................................... 134

6.3.5 Effect of postharvest Ca vacuum infiltration on postharvest quality and

grey mould development ................................................................................. 134

6.4 Discussion ..................................................................................................... 142

6.5 Conclusion .................................................................................................... 145

CHAPTER SEVEN

General discussion

7.1 Outcomes of study ........................................................................................ 147

7.2 Further research ............................................................................................. 152

7.3 Conclusions ................................................................................................... 153

REFERENCES.................................................................................................... 155

v
Appendix A.1 ...................................................................................................... 171

Appendix A.2 ...................................................................................................... 172

Appendix A.3 ...................................................................................................... 173

Appendix A.4 ...................................................................................................... 174

Appendix A.5 ...................................................................................................... 175

Appendix A.6 ...................................................................................................... 176

Appendix A.7 ...................................................................................................... 177

Appendix A.8 ...................................................................................................... 178

Appendix A.9 ...................................................................................................... 179

Appendix A.10 .................................................................................................... 180

Appendix A.11 .................................................................................................... 181

Appendix A.12 .................................................................................................... 182

vi
Abstract

Capsicum (Capsicum annuum L.) is mostly cultivated in humid and warm

conditions, which increases disease development, particularly grey mould caused
by Botrytis cinerea. Infection of capsicum fruit by B. cinerea often occurs
preharvest but symptoms of grey mould are not usually visible until after harvest
making the pathogen difficult to control. Appropriate fertilisation that ensures
calcium (Ca) and boron (B) is sufficient in plant tissues, especially in fruit, has
been suggested as an alternative to fungicides for disease management. This
research studied the infection pathway of B. cinerea and the effect of Ca and B on
grey mould development and quality of fruit in two capsicum cultivars (cv. Aries
and cv. Papri Queen).

Botrytis cinerea infected capsicum preharvest and flowers often died when
inoculated at anthesis. The number of dead flowers increased when inoculum
concentration increased. The extent of grey mould development on fruit
inoculated preharvest was not affected by timing of inoculation [at anthesis, 3
days after anthesis (DAA) or 6 DAA], but was dependent on inoculum
concentration and cultivar. When capsicum fruit were inoculated after harvest,
grey mould developed most rapidly in red (R) fruit from cv. Aries and breaker red
(BR) fruit from cv. Papri Queen. An inoculation of 10⁶ conidia mL¯¹ caused more
disease on fruit than 10⁴ or 10⁵ conidia mL¯¹. Cv. Aries was more susceptible to
B. cinerea than cv. Papri Queen regardless of whether inoculation occurred before
or after harvest.

The effect of both soil and foliar application of boron (B), at different
concentrations, on grey mould development and fruit quality of capsicum was
examined. Preharvest B application, from transplanting to harvest when fruit were
mature and red, using 0.05 or 0.1 mM H₃BO₃ via soil amendment or 2.0 or 7.0
mM H₃BO₃ as a foliar spray increased B concentration in leaves and fruit of both
cultivars. However, soil application was more effective than foliar application in
increasing B concentration in plant tissues. Foliar application of B at low
concentrations (0.025 or 0.075 mM H₃BO₃) did not increase B concentration in
plant tissue. Increasing B concentration in leaf and fruit tissue reduced grey mould

vii
development on fruit inoculated with B. cinerea preharvest compared to the
control, but did not affect grey mould development on red fruit inoculated with B.
cinerea postharvest. Preharvest soil application of B increased shelf life of fruit,
but did not affect quality of fruit including water content, firmness, total soluble
solid content (TSSC) and titratable acidity (TA) at harvest or during storage.
Symptoms of B toxicity were observed on leaves from plants that received high B
concentration (0.1 mM H₃BO₃) in the soil, but no effect was observed on fruit.

Preharvest application of calcium (Ca) via soil amendment [1.5, 4.0 or 8.0 mM
Ca(NO₃)₂] or as a foliar spray [0.5 or 1.0 % w/v mM Ca(NO₃)₂] increased Ca
concentration in leaves, but did not increase Ca concentration in fruit, regardless
of cultivar. Soil Ca application appeared to increase Ca concentration in leaf
tissue more effectively than the Ca foliar spray. Ca concentration in leaf tissue
from cv. Aries was significantly higher than in leaf tissue from cv. Papri Queen
when plants received the same amount of Ca, regardless of application method.
Ca treatment did not affect quality of fruit at harvest or during storage. Preharvest
application of Ca reduced grey mould development on fruit that had been
inoculated with B. cinerea preharvest, but did not reduce grey mould in fruit
inoculated postharvest. Symptoms of Ca deficiency were observed on plants that
received no Ca or low Ca concentration [1.5 mM Ca(NO₃)₂] from transplant to
fruiting.

Dipping and vacuum infiltration with calcium chloride (CaCl₂.2H₂O) did not
increase Ca concentration in flesh after treatment, but vacuum infiltration did
increase Ca concentration in flesh after 10 days of cool storage (10°C). Ca
treatment after harvest did reduce grey mould development on fruit, but did not
affect the quality of fruit during storage. A directly inhibitory effect of Ca on
fungal growth was responsible for reducing grey mould development on fruit.

In conclusion, capsicum was most sensitive to infection by B. cinerea at anthesis

and high inoculum concentrations caused a greater disease incidence in capsicum
fruit, regardless of whether inoculation occurred preharvest or after harvest.
Reducing inoculum concentration, especially during flowering, is therefore
recommended to reduce losses in capsicum. Preharvest application of Ca or B

viii
may be used as an alternative method to reduce grey mould on capsicum fruit, but
they had no effect on fruit quality. Postharvest application of Ca could also be
recommended for cv. Aries fruit before or during storage for controlling grey
mould on fruit. Findings in this research may therefore provide basic knowledge
for management of B. cinerea in the capsicum industry.

ix
Declaration

I certify that this work contains no material which has been accepted for the award
of any other degree or diploma in my name, in any university or other tertiary
institution and, to the best of my knowledge and belief, contains no material
previously published or written by another person, except where due reference has
been made in the text. In addition, I certify that no part of this work will, in the
future, be used in a submission in my name, for any other degree or diploma in
any university or other tertiary institution without the prior approval of the
University of Adelaide and where applicable, any partner institution responsible
for the joint-award of this degree.

I give consent to this copy of my thesis when deposited in the University Library,
being made available for loan and photocopying, subject to the provisions of the
Copyright Act 1968.

The author acknowledges that copyright of published works contained within this
thesis resides with the copyright holder(s) of those works.

I also give permission for the digital version of my thesis to be made available on
the web, via the University’s digital research repository, the Library Search and
also through web search engines, unless permission has been granted by the
University to restrict access for a period of time.

Thong Duc Le
2014

x
Acknowledgements

Firstly, many thanks to my supervisors, Associate Professor Amanda J. Able,

Professor Eileen Scott and Associate Professor Glenn McDonald for their
guidance, support and understanding throughout my study. I would like to thank
the external advisor James Stangoulis for his useful comments and suggestions.

Special thanks to the Agricultural Science and Technology Project of Vietnam for
awarding a PhD scholarship. Thanks to the University of Adelaide for granting a
top-up scholarship.

Thanks to all members of the Able laboratory, the past and present members, who
provided their help, support in my experiments and the fun they shared at morning
teas. I would like to acknowledge the Waite Analytical Services for nutrient
analysis and allowing the use of grinding facilities.

Thanks to Seminis and Fair Bank Seeds for supplying capsicum seeds. I would
also like to thank Sue Pederick (South Australia Research and Development) for
sharing her techniques for long-term storage of B. cinerea and Dr Olena Kravchuk
(the University of Adelaide) for her advice about multifactorial analysis.

I would like to thank all Vietnamese friends at Waite campus, particularly Trung
Dzung Nguyen, for their help and support during my study.

Finally, many thanks to my beloved wife Pham Thanh Thuy and my lovely
daughter Le Thanh Lan for their love, encourage and never-ending support.

xi
Abbreviation

Abbreviation Full term

ANOVA Analysis of Variance
AOAC Association of Official Analytical Communities
ASTA American Spice Trade Association
B boron
BR breaker red
°Bx °Brix
°C Degrees Celsius
Ca calcium
CaCl₂ calcium chloride
Ca(NO₃)₂ calcium nitrate
CuSO₄ copper sulphate
cv. cultivar
DAA days after anthesis
DAH days after harvest
DPI days post-inoculation
DG deep green
DW dry weight
EDTA ethylenediaminetetraacetic acid
et al. and others
e.g. for example
FW fresh weight
FAO Food and Agricultural Organisation
Fe³⁺-EDTA Ethylenediaminetetraacetic acid iron (III)
Fig Figure
g gram
h hour
H₃BO₃ boric acid
ICP-OES Inductively Coupled Plasma Optical Emission
Spectrometer
kg kilogram

xii
kgf kilogram force
kGy kiloGray
KCl potassium chloride
KNO₃ potassium nitrate
KH₂PO₄ potassium dihydrogen orthophosphate
KOH potassium hydroxide
K₂SO₄ potassium sulphate
L litre
LSD Least Significant Difference
MgSO₄ magnesium sulphate
MnSO₄ manganese sulphate
mg milligram
min minute
mL millilitre
mm millimetre
mm² square millimetre
mt million tones
N Newton
NaH₂PO₄ sodium phosphate dibasic
NH₄NO₃ ammonium nitrate
(NH₄)₆Mo₇O₂₄ ammonium molybdate tetrahydrate
PDA Potato dextrose agar
PE pectinesterase
PG polygalacturonase
pH power of hydrogen (negative log of H⁺ concentration)
ppm parts per million
RH relative humidity
RO reserve osmosis
SE standard error
sec seconds
TSSC total soluble solid content
TA titratable acidity

xiii
UC University of California
UV ultraviolet
ZnSO₄ zinc sulphate
w/v weight by volume
µM micromoles per litre
µL microlitre
% percentage

xiv
 CHAPTER ONE

Introduction

Capsicum (Capsicum annuum L.), also called pepper, is a warm-season crop that
is a member of the Solanaceae family. Capsicum fruit is the major source of red
food colourant and pungency for spice production and can also have medicinal
uses [Food and Agricultural Organisation (FAO), 2013]. For the period from 2001
to 2011 capsicum production increased by 17% such that by 2011 there was over
29.61 million tones (Mt) and 1.84 million hectares of cultivation (FAO 2013).
However, capsicum fruit are susceptible to fruit rots, especially grey mould
caused by Botrytis cinerea Pers. [teleomorph: Botryotinia fuckeliana (de Bary)
Whetzel], which causes high postharvest losses (20 - 25%) (Fallik et al. 1996). B.
cinerea is therefore a significant concern for growers and marketing operations in
many countries.

Botrytis cinerea also causes grey mould disease in a wide range of economically
important plants, and it has traditionally been considered as a non-specialised
necrotrophic fungus that multiplies on debris of a broad range of plant species.
Following infection during flowering and/or the early stages of fruit development,
B. cinerea may remain latent until environmental conditions are suitable and
ripening causes physiochemical and biochemical changes in the fruit, leading to
the development of grey mould (Droby and Lichter 2004), making fruit
unmarketable (Utkhede and Mathur 2003). Fungicide application is the most
commonly-used method to control fungal pathogens preharvest and postharvest.
However, fungicides may be toxic to human health and damage the environment.
Alternative means of control, such as nutrient application to plants, could
minimise the use of fungicides. The use of nutrient application might be a
potential way to control diseases as well as improving plant growth and fruit
quality (Fallahi et al. 1997; Hansch and Mendel 2009).

Calcium (Ca) and Boron (B) are essential nutrients for plant growth. Adequate Ca
in plants has been demonstrated to increase cell wall strength and thickness.
Calcium may also increase plant defence responses to infection by fungi
(Benhamou 1996). The role of B in maintaining the structural integrity of plant

1
membranes is well known (Sams 1999; Hewett 2006). There are some previous
studies describing the positive effects of these two nutrients on disease
development in plants and in fruits after harvest, such as bean and tomato (Elad
and Volpin 1993), table grape (Amiri et al. 2009) and strawberry (Wójcik and
Lewandowski 2003; Singh et al. 2007). However, there is a lack of understanding
about the effect of Ca and B on grey mould of capsicum and quality of capsicum
fruit. Therefore, this research aimed to investigate the effect of Ca and B nutrition
on grey mould of capsicum and fruit quality.

2
 CHAPTER TWO

General literature review

2.1 Capsicum

Capsicums (Capsicum annuum L.) originated in South and Central America (Burt
2005). Capsicums are primarily classified as one of three cultivar types: capsicum,
paprika and chilli. Capsicum is a sweet non-pungent fruit often called bell pepper
and used for fresh consumption. Paprika is non-pungent and used to produce
spice, while chillies are smaller and hotter than non-pungent capsicum (Bosland et
al. 1996 in Klieber 2000) and are used in both spice and sauces (Rajput and
Paruleke 1998).

Capsicums are planted world-wide and the production has increased steadily with
international trade being valued at approximately US$3.1 billion (FAO 2013).
Capsicum normally grow in warm conditions with 16°C to 21°C on average being
best for fruit setting, high yields and good quality fruit (Burt 2005). Asia produced
two-thirds of world’s capsicum in 2011 (20.51 Mt) (FAO 2013). Capsicum are
grown throughout Australia in tropical and subtropical areas (primarily in
Queensland, South Australia and Victoria) with the total capsicum production in
2012 of 36,600 tonnes (Australian Bureau of Statistic, 2013). Capsicums can be
grown on many different kinds of soil with a wide range of pH (5 - 9) but its
optimum is well-drained soil at a pH of 5.5 to 6.5 (Burt 2005).

Capsicum is self-pollinating and fruit are normally mature (fully grown and light
green in colour) approximately 30 to 35 days after anthesis, with fruit taking a
further 20 to 25 days to turn red depending on season (Burt 2005; Rajput, 1998).
Harvesting at the light green stage affects fruit quality because fruit colour does
not change properly to red, while fruit harvested at the breaker stage (40% to 50%
red surface colouration) will normally ripen and develop colour sufficiently
(Pham 2007). Postharvest changes in fruit will be discussed in the next section.

2.2 Postharvest changes in capsicum

Based on their postharvest physiology and biochemical changes, fruit have been
divided into two groups: climacteric and non-climacteric fruit (Biale 1981). In

3
climacteric fruit, respiration peaks before ripening and this is associated with
significant changes in sugar content, colour and texture. Ethylene production also
increases sharply to reach a peak and remains at a relatively high rate throughout
the ripening period (Rhodes 1980). The autocatalytic activity of ethylene plays an
important role in the coordination and completion of the ripening process of
climacteric fruit (Giovannoni 2001). In contrast, there is no peak in respiration or
ethylene production in non-climacteric fruit and ripening processes are not
sensitive to ethylene. Capsicum is difficult to classify as either climacteric or non-
climacteric due to variation amongst cultivars and species. Saltveit (1997) did not
consider capsicums to be climacteric because there was a lack of the typical
increase in respiration and ethylene production during ripening. Limited ethylene
production has been reported in the paprika-type cultivar, cv. Papri Queen, and
the bell pepper, cv. Aries (Pham 2007), while fruit from the chilli cv. Changjiao
did not respond to ethylene or have increased respiration during ripening (Lu et al.
1990). Even though bell pepper from cv. Maor at the green and red stages had a
significant increase in ethylene, there was very low respiration during storage
(Lurie et al. 1986). Together, these results suggest that most capsicum is probably
non-climacteric.

Changes in colour, firmness and sugar content during ripening of capsicum fruit
on the plant have also been widely investigated. Chlorophyll commonly decreases
and is absent from tissues when the fruit turns fully red (Mendez and Mosquera
2002). Firmness has also been shown to decline during ripening due to the
breakdown of the fruit cell walls (Brady 1987). These changes are caused by the
increasing activity of hydrolase enzymes, including polygalacturonase (PG) and
pectinesterase (PE) (Sethu et al. 1996). The hydrolase enzymes, especially PG, are
reported to be absent or inactive when fruit are not ripe, but to have high activities
when fruit are ripe (Harpster et al. 1997). During ripening, the neutral sugars
decreased dramatically, from 25% initially (1st day of harvest) to 15% (21st day of
harvest) (Sethu et al. 1996). The change in total soluble solid content (TSSC) in
capsicum fruit also depends on ripening stage (Pham 2007). The TSSC of fruits
which ripened on the plant increased greatly and reached a peak when fruit turned

4
full red and was higher than for fruits harvested at the deep and light green stages
in Papri Queen, Aries and Caysan cultivars.

Postharvest loss in capsicum is related to shriveling (weight loss), nutrient

reduction and rot caused by diseases. Although weight loss occurs, the extent of
this is limited because peppers have a thick waxy layer on their epidermis
(Kissinger et al. 2005). Disease is therefore the main factor limiting the shelf life
of capsicum (Sharma et al. 2009). Most postharvest diseases are caused by fungi,
including white mould caused by Fusarium solani, black mould caused by
Alternaria alternata and grey mould caused by Botrytis cinerea (Barkai-Golan
2001). Several species of bacteria, such as Bacillus polymyxa and Erwinia
carotovora ssp. carotovora, also cause rotting, which may occur in fruit from any
region growing crops (Snowdon 1991). Under suitable conditions, fungi
germinate on the surface of the host to penetrate into the host tissues and to
develop there (Barkai-Golan 2001). Seedlings and ripening fruit were reported to
be susceptible to infection by Colletotrichum truncatum and infected leaves
served as a potential primary inoculum source for fruit infection (Ranathunge et
al. 2012). Seed, placenta and pericarp can also be infected by fungi causing a
latent infection where symptoms are not observed until postharvest (Elad and
Shtienberg 1995). B. cinerea is considered to be the most important disease in
many countries and efficacy of fungicides is being lost because of development of
resistance. Development of alternative means to control fungi may therefore be
essential to contribute to sustainable production.

2.3 Grey mould

Grey mould (also called Botrytis fruit rot or ash mould) caused by Botrytis
cinerea Pers. [teleomorph: Botryotinia fuckeliana (de Bary) Whetzel], causes
many different symptoms. B. cinerea causes soft rot associated with destruction
and water-leaking of parenchyma tissues and then a grey mass of conidiophores
and conidia appears rapidly. These are the most common symptoms on leaves and
fruits with soft tissues (Williamson et al. 2007). Grey mould also might occur
anywhere on the fruit, including the stem-end and blossom-end (Snowdon 1991)
(Fig 2.1).

5
A B

Fig. 2.1 Grey mould on capsicum fruit from cv. Aries (A) and cv. Papri Queen
(B)

In capsicum fruit, infected tissue is water-soaked and brown-grey, and grey-brown

conidia can develop accompanied by small black sclerotia (Snowdon 1991).
Botrytis cinerea infects tissues at many developmental stages, even seedlings, and
remains latent for long periods before causing rot quickly when environmental
conditions are optimal and the host physiology changes (Droby and Lichter 2004).
The life cycle for B. cinerea can be divided into two main modes depending on
the weather and time of year (Fig 2.2). In the winter, the fungus can persist in
dead organs or the soil as resting bodies (sclerotia and mycelia) or in infected
plant debris (Snowdon 1991). In early spring when the weather is warmer, the
mycelia and sclerotia become active and germinate to produce conidiophores on
the surfaces of the infected plant debris. The conidia are disseminated from
conidiophores by wind, rain-splash, irrigation (Maas 1998) and by human hands
to fruit (Plakidas 1964; Barkai-Golan 2001). In the summer, the fungus does not
enter a resting period, but produces conidiophores directly on infected tissue. In
the presence of moisture, conidia on infected organs produce fresh mycelia which
quickly spread in the tissue, causing cells to collapse and the tissue to be
destroyed. Conidia commonly infect susceptible tissue first by germination and
form appressoria which allow penetration via wounds. Mycelia then spread to
other tissues when resistance of the fruit to the pathogen decreases (discussed in
more detail in Section 2.3.2).

6
Fig. 2.2 Lifecycle of Botrytis cinerea [modified from (Winedoctor 2010)]

B. cinerea re-enters the reproductive stage by generating conidiophores again and

releases fresh conidia. This process is constantly repeated during appropriate
conditions in the summer and the fungus only enters the over-wintering cycle
when cooler conditions occur (Williamson et al. 2007).

2.3.1 Development of grey mould disease

Conidia of B. cinerea are released from conidiophores by air currents in humid

weather and usually develop on dead plant organs and spread quickly into
susceptible plant parts, especially wounded tissues. In presence of nutrients and
moisture on wound sites, conidia germinate and hyphae colonise plant tissue
causing cell death. Moderate temperature (15 to 25°C) with high humidity
conditions or a wet surface are optimal for grey mould to develop (Maas 1998).
However, a number of conditions have been shown to slow disease development,
including cold temperature or hot and dry weather (Snowdon 1991; Williamson et
al. 2007). Light also has effects on B. cinerea at different wavelengths. Elad and
Shtienberg (1995) indicated that sporulation in B. cinerea was reduced by
increasing the ratio of blue to ultraviolet (UV) light which passed through

7
polyethylene film. In addition, fungal development decreases at low oxygen and
high carbon dioxide concentration. Mycelial growth of B. cinerea and Fusarium
roseum was inhibited by over 50% when the oxygen level was 4%, and
germination of B. cinerea was inhibited by more than 90% in an atmosphere of
16% carbon dioxide compared to ambient conditions (Wells and Uota 1970).
Because the carbon dioxide level is high inside capsicum fruit (~ 30 mg kg¯¹ h¯¹)
(Villavicencio et al. 1999), B. cinerea may favour development in the outer
tissues of capsicum fruit.

2.3.2 Infection pathways

Experiments in previous research have confirmed that flowering is the most

important time for B. cinerea to infect plants and cause subsequent infection of
fruit such as strawberry (Jarvis and Borecka 1968) and grapes (Keller et al. 2003).
Conidia can germinate in drops of free water on the petal or any part of the flower,
and then penetrate through the senescing parts, into the edge of the receptacle
where they form mycelia that then become dormant as a latent infection (Barkai-
Golan 2001). During fruit ripening the cell wall becomes softer due to activity of
the enzymes, PG and PE (Brady 1987). Mycelium of B. cinerea then invades and
develops in the fruit, causing rot after harvest (Williamson et al. 2007). When
tomato fruit were infected by B. cinerea during flowering, grey mould only
developed at the stem-end when fruit were stored (Lavy-Meir et al. 1989). B.
cinerea also can infect fruit directly in the field via airborne conidia (Barkai-
Golan 2001), especially where fruit have been damaged during harvest, handling
and transport causing wounds and bruises. However, the infection pathway of B.
cinerea in capsicum is not well understood and it is not known if the flower stage
is the most susceptible to B. cinerea. As such, the activity of B. cinerea in
capsicum was investigated in this study.

2.4 Disease control methods

Because capsicum is mostly grown in glasshouses or warmer regions in the field,

both of which favour development of B. cinerea, grey mould needs to be
controlled. Which methods are used depends on cultural practice and the desired
quality of products. However, three major methods of disease control commonly

8
exist; physical or cultural, chemical and biological methods (Elad and Shtienberg
1995).

2.4.1 Physical or cultural methods

As noted above, grey mould is stimulated by moderate temperature and high

humidity. Thus in crop management, an open canopy is necessary to provide good
air movement and high light interception so that free water dries as soon as
possible. Avoiding rainfall during the blossom period by covering the crop with
plastic can reduce disease in strawberry by 90% compared with open field plants
(Williamson et al. 2007). In addition, removal of plant debris after each growing
season can reduce the amount of inoculum (Elad and Shtienberg 1995). Hence,
removal of inoculum and making conditions unsuitable for survival of the
pathogen are effective ways to minimise infection by B. cinerea.

Physical methods after harvest, such as heat treatment, ionising radiation and UV
illumination, are used to kill and inhibit fungi and bacteria on fruit. Heated fruit
were often harder than non-heated fruits during storage (Klein and Lurie 1991),
helping fruit to resist pathogen infection. In a study by Fallik et al. (1996), the
authors treated red sweet pepper by dipping these fruit in hot water at 50°C for 3
minutes. Decay development caused by B. cinerea was completely inhibited in
both naturally-infected fruit and artificially-inoculated fruit. However, heat
damage was shown in fruit when dipping at a temperature hotter than 50°C for
more than 3 min.

Ionising radiation may be harmful for living cells of pathogens and it may also
prevent decay by delaying ripening and senescence (Barkai-Golan 2001). Barkai-
Golan et al. (1971) found that radiation of 2 kGy from CO⁶⁰ (cobalt) extended the
shelf-life of strawberry naturally infected by B. cinerea from 3 to 10 days at 15°C.
UV at low doses has a germicidal activity that can reduce diseases in a wide range
of fruits and vegetables (Wilson et al. 1994). Additionally, UV treatment has been
found to delay the ripening process in some commodities, such as tomato and
peach (Liu et al. 1993), leading to an indirect reduction in their susceptibility to
infection. Inoculated berries treated with UV-C doses of 0.125-0.5 kJ m⁻² had a

9
significantly lower incidence of infection and a reduction of B. cinerea compared
with control berries (Nigro et al. 1998).

2.4.2 Chemical methods

Although chemical methods of disease control include fungicides (compounds

lethal to fungi) and fungistatins (compounds that inhibit fungal growth), fungicide
application is more common on seed or plant surfaces either to kill fungal spores
or prevent their germination (Persley 1993). Pre-harvest fungicide application is
an effective way to reduce infections initiated in the field (Barkai-Golan 2001)
and to prevent the formation of latent infections in the young fruit (Sommer et al.
1973). However, preharvest fungicides do not protect fruit if wounded during
postharvest handling, so fungicide application is also needed in the postharvest
period. Postharvest fungicide application should be done as soon as possible after
harvest to prevent mycelial growth in the host tissue (Barkai-Golan 2001).

However, there are rising concerns about increased chemical use in agricultural
systems because of the development of strains of fungi resistant to chemicals
(Sharma et al. 2009). Using chemicals might be potentially harmful for human
health and damage the environment. Therefore, alternative and more
environmentally friendly methods such as biological control are now necessary to
reduce fungicide use in agricultural systems.

2.4.3 Biological methods

Biological control (biocontrol) has become a focus for research to reduce the
presence of fungicide residues in fresh food crops and to slow the development of
pathogens resistant to major fungicides. Biological control has been used to
antagonise preharvest and postharvest pathogens (Barkai-Golan 2001) and occurs
via four main modes of action: the secretion of an antibiotic compound by the
antagonist; competition with pathogens for nutrients at wound sites on the plant
tissue; secretion of enzymes to injure the pathogen; and induction of host defence
mechanisms (Droby et al. 1992; Wilson et al. 1994). The effectiveness of a
biological agent depends on factors such as time of application, presence of
moisture in the wound, and the number of pathogen conidia or antagonist
concentration. Smilanick (1994) stated that biocontrol should be applied to the

10
wound site prior to the arrival of the pathogen. The antagonist yeast Candida
oleophila controlled B. cinerea more effectively when applied to a fresh wound
than a dry wound (Mercier and Wilson 1995), and the greatest antagonist activity
of Trichoderma was observed at the highest concentration of the antagonist and
the lowest inoculum levels of the pathogens (Mortuza and Ilag 1999). Similarly,
an increase in the concentration of yeast Saccharomyces cerevisiae from 10⁵ to
10⁷ cells mL⁻¹ significantly enhanced their antagonistic activity to B. cinerea in
table grapes (Nally et al. 2012). Schena (1999) indicated that application of the
yeast-like fungus, Aureobasidium pullulans, at 108 and 107 cells mL⁻¹ controlled
B. cinerea, Rhizopus stolonifer and Aspergillus niger on table grape, and B.
cinerea and R. stolonifer on cherry and tomato. When wound sites in apple fruit
were inoculated with Candida oleophila prior to inoculation with B. cinerea, the
percentage of fruit with grey mould after 14 days storage was significantly
reduced compared with the control (Mercier and Wilson 1995). The application of
the yeasts Rhodotorula rubra and Candida pelliculosa to wounded tomato before
inoculation with B. cinerea was reported to reduce the diameter of lesions by
more than 60% compared to the control after 1 week (Dal Bello et al. 2008).

In addition, host protection plays a significant role in reducing the incidence of

disease. Plant have the ability to detect invading pathogens and respond by
producing toxic chemicals or defence-related proteins against the pathogen
(Ferreira et al. 2007). Capsidiol, a phytoalexin which is a natural defence
mechanism to protect against disease infection (Egea et al., 1996). However, the
ability of the host to protect against infection differs between crops and cultivars.
All capsicum cultivars grown in Australia are susceptible to B. cinerea. Although
some level of control is available by keeping the humidity low in the glasshouse
(Conrad, L., Seminis Sales, Monsanto, Personal Communication, March 2010),
with no true resistance, other alternative methods must be sought.

An adequate supply of nutrients, especially micronutrients, is important for plant

growth, fruit quality and disease resistance. The application of nutrients to plants
to improve their resistance is therefore considered to be a biological method to
reduce pathogen diseases. Calcium induces the signaling pathways that lead to
plant defence responses such as the production of lignin-like polymers that cannot

11
be easily degraded therefore preventing the pathogen from spreading (Benhamou
1996). Calcium (Ca) and boron (B) have been demonstrated to improve fruit
quality and reduce disease incidence in crops, such as bean and tomato (Elad and
Volpin 1993), table grape (Amiri et al. 2009), and strawberry (Wójcik and
Lewandowski 2003; Singh et al. 2007). However, quality of sweet pepper cv.
Orlando at harvest, including firmness, TSSC or acidity was not affected by Ca or
potassium nutrient treatments (Rubio et al. 2010). There has been limited research
on the effect of Ca and B on grey mould development and fruit quality in
capsicum. The role of these elements will be discussed below.

2.5 Calcium and boron application to control disease

Both Ca and B are essential nutrients for plant growth, but they also have a role in
protecting plants from disease. Calcium is an essential component of the plant cell
wall and confers structural rigidity and firmness (Maas 1998; Easterwood 2002).
Moreover, adequate Ca may increase the resistance of the plant to diseases
because the Ca²+ forms cross-bridges between adjacent pectic acids or between
pectic acids and other polysaccharides which makes the cell wall more resistant to
the action of the pathogen’s pectolytic enzymes (Preston 1979; Conway et al.
1994a). Calcium is thought to have a key role in the induction of plant defence
responses (Benhamou 1996). The role of B is linked to cell wall synthesis by
cross-linking of cell wall polysaccharides and the structural integrity of
biomembranes as well (Marschner 1995; Hansch and Mendel 2009). Therefore,
fruit becomes less susceptible to attack by the pathogen. Furthermore, application
of Ca and/or B significantly deceased the activity of the fruit softening enzymes
PG and PC (Dong et al. 2009). As a result, the fruit may display better resistance
to grey mould development.

Calcium and B concentration in plant tissue may be less than optimal because of
some major factors. Firstly, both Ca and B are immobile in plants and poorly
translocated to actively growing tissues and fruits. Therefore, Ca and B content
was often higher in older tissues than growing tissues and higher in the leaves
than in fruit (Gupta 1979; Reuter and Robinson 1997). Moreover, Ca is taken up
less effectively than some other nutrients, such as potassium (K), sodium (Na) and

12
ammonium (NH4) (Kirby and Pilbeam 1984). Boron uptake was less by roots of
aged plants than young plants (Marschner 1995). These factors cause blossom-end
rot and blackheart in celery, tomato and pepper (Geraldson 1957). In addition,
environmental factors also affect the uptake of Ca and B. Increasing soil pH and
wet winters both cause B deficiency (Mengel and Kirkby 1987). Salinity and high
temperature are also reported to reduce Ca and B content in plant tissues (Mengel
and Kirkby 1987; Taylor and Locascio 2004).

The interaction of Ca and B may affect plant growth and disease resistance. A
high Ca concentration in soil caused a B deficiency in tomato (Geraldson 1957;
Dong et al. 2005), while B sprays increased the mobility of Ca and the Ca
concentration of apple fruit as well (Shear and Faust 1971). Boron sprays during
blossoming effectively reduced bitter pit in apple, which is caused by Ca
deficiency (Dunlap and Thompson, 1959 in Mengel and Kirkby, 1987). In
addition, B has a role in keeping Ca within the wall which is important for
maintenance of cell wall integrity. The host would therefore have reduced
susceptibility to the pathogen (Stangoulis and Graham 2007).

Botrytis cinerea often infects fruit in the preharvest period as a latent pathogen
(see section 2.3.2) and grey mould symptoms are evident only in the postharvest
period. In order to minimise pathogen development, it is necessary to ensure Ca
and B are present at a high enough level in both the preharvest and postharvest
period.

2.5.1 Preharvest application

Calcium and B deficiencies cause serious problems in the plant. Calcium

deficiency was related to poor germination of seed, reduction in tissue growth and
absence of fruit in tomato and paper (Taylor and Locascio 2004). Calcium
deficiency also led to reduced firmness of tissue due to dissolution of the cell wall
(Mengel and Kirkby 1987), increased blackheart in celery and blossom-end rot in
watermelon, pepper, eggplant and tomato (Taylor and Locascio 2004). Boron
deficient plants have abnormal development of fruit and restricted flower and fruit
development (Jones 1998). In some plants, inadequate B causes fruit to be very
small and of poor quality (Mengel and Kirkby 1987).

13
The literature suggests a wide range of optimal concentrations for Ca in plants due
to variation in requirement among plant species. Generally, Ca concentrations in
plants and in fruit were from 0.1 to 5% and 0.2 to 0.3% of the dry weight of the
tissue, respectively (Taylor and Locascio 2004). In bell pepper, the concentrations
of Ca in the youngest mature leaf for deficient, adequate and high range of Ca
content are 1.00 - 1.29%, 1.30 - 2.80% and >2.80%, respectively (Reuter and
Robinson 1997). In contrast, B has a very narrow range between deficiency (23 –
24 mg kg⁻¹), adequately (25 – 75 mg kg⁻¹) and toxicity (>75 mg kg⁻¹) (Reuter and
Robinson 1997).

Preharvest Ca and B applications have been demonstrated to reduce grey mould

development and/or improve fruit quality in some crops (Table 2.1). Soil
application of Ca has occurred at concentrations ranging from 1.0 to 8.0 mM and
for B was from 0.01 to 0.2 mM. However, B has been reported to be toxic in
capsicum at concentrations higher than 1 mg kg⁻¹ (Nabi et al. 2006). Some salts
containing Ca or B were used in preharvest applications. Calcium nitrate
[Ca(NO₃)₂] is often used in industry or in research as a source of Ca because it is
more soluble than calcium sulphate (CaSO₄) or calcium chloride (CaCl₂), and
boric acid (H₃BO₃) is most commonly used as a boron-containing compound for
soil application.

Foliar Ca and B applications have been used widely as an effective method to

increase Ca and B concentration in fruit due to immobility of these elements when
applied to soil. Calcium chloride or Ca(NO₃)₂ has most commonly been applied as
a Ca-containing compound and Ca concentrations of foliar spraying solutions
have a range from 0.8 to 2 % CaCl₂ or 1 - 2% Ca(NO₃)₂ (Table 2.1). The B-
containing compound most commonly used in foliar sprays is H₃BO₃ at
concentrations ranging from 0.025 to 0.1%. The first time of spraying is often
when the petals drop, with later applications at 7-day intervals in strawberry and
14-day intervals in pepper (El-Tohamy et al. 2006; Singh et al. 2007).

Preharvest Ca and B applications have been reported to increase marketable yield

of fruit, improve quality, appearance and reduce blossom-end rot in capsicum

14
Table 2.1 The effect of preharvest Ca and B application in some fruit crops

Application Crop Treatment Effect

References
method
Bean and 1-3 mM CaCl₂ or Ca(NO₃)₂ Decreased severity of grey mould
(Elad and Volpin 1993)
tomato Reduced severity of fruit ghost spots
Apple 27 mg B kg¯¹ soil (H₃BO₃) Increased fruit yield and higher fruit soluble solid content (Wojcik et al. 2008)
Soil
amendment Ca(NO₃)₂ at low, medium and Increased marketable yield and improved fruit appearance at 4 and
Sweet (Rubio et al. 2010)
high level (1.5, 4 and 8 mM) 8 mM
pepper
(four B at rate of 0, 1, 2, 4, and 8 mg B Maximum crop biomass at ~1 mg B kg¯¹ (Nabi et al. 2006)
cultivars) kg-1 soil (H₃BO₃) Toxic for plants at higher than 1 ppm
CaCl₂ at 0, 0.8, 1.2, 1.6 and 2% Significantly improved berry firmness, colour and appearance
w/v associated with increase of Ca concentration
‘Asgari’
Reduced Botrytis infection and berry drops (Amiri et al. 2009)
grape
Leaf injury (lesions) was sometime observed at high Ca level (2%
w/v) but not observed on cluster or fruit
Foliar
0.0, 0.025, 0.05, and 0.1 % Increased in fruit yield and improved fruit physical properties and
spray Anna apple (Khalifa et al. 2009)
H₃BO₃ decreased in severity of blossom-end rot
 CaCl2 at rate of 2 kg Ca Significantly reduced albinism and grey mould for Ca and Ca + B
ha¯¹spray¯¹ treatments
 150 g B (H₃BO₃) ha¯¹spray¯¹ Significantly decreased malformation in B-treated fruits
Strawberry
 Ca + B in combination at same (Singh et al. 2007)
levels Increased fruit firmness and reduction in disorder incidence
compared to B alone and control

CaCl₂ at 1 and 2 % w/v Improved yield (El-Tohamy et al. 2006)

Pepper Ca(NO₃)₂ 1 - 2% + 0.5% Tween Significantly increased Ca concentration in leaf, fruit and reduced
(Schon 1993)
20 blossom-end rot

15
(Schon 1993; Keinan et al. 2000; Rubio et al. 2010). However, the effect of Ca
fertilisation on grey mould of capsicum is not well understood. Boron fertilization
at 1 mg kg⁻¹ H₃BO₃ was the critical level for growth of capsicum (Nabi et al.
2006), but the effect of B on grey mould of capsicum and fruit quality remains
unclear. Foliar application of Ca at 1.0 or 2.0% CaCl₂ improved growth
parameters of capsicum plants (plant height, number of branches and fresh weight
of plants) and yield significantly compared to the control (El-Tohamy et al. 2006).
However, the effect of foliar application of Ca on quality of capsicum fruit has not
been reported. Therefore, this study focused on the effect of preharvest Ca and B
application on grey mould development and fruit quality in capsicum.

2.5.2 Postharvest application

Both Ca and B have limited mobility in the plant phloem and the lowest
concentration is found in the fruit (Mengel and Kirkby 1987). Postharvest
application has been an effective way to increase concentration of nutrients in
fruit. Postharvest Ca application is a way of applying Ca directly to harvested fruit
via the standard practice of dipping fruit in solution at ambient pressure (normal
dipping) or active infiltration (pressure infiltration or vacuum infiltration)
(Conway et al. 1994a). Calcium chloride has been commonly used as a dipping
solution to increase firmness for whole fruit and fresh-cut commodities (Martín-
Diana et al. 2007). However, B is not commonly applied postharvest due to
toxicity of inorganic boron-containing compounds to human health if adults
consume more than 13 mg B per day (Nielsen 1997).

Normal dipping of fruit in a CaCl₂ solution increased Ca concentration of the skin

and outer layers of the fruit, but an active infiltration procedure was more
effective in increasing Ca concentration in flesh of apples (Conway et al. 1994a).
Increasing Ca concentration in apples by vacuum infiltration reduced incidence of
bitter pit and the quality of fruit during storage was better than fruit that were
dipped (Scott and Wills 1977). Conway (1982) examined the effect of postharvest
Ca application on decay of ‘Delicious’ apples. Fruit were treated with a range of
CaCl₂ solutions (0, 2, 4, 6 and 8%) by dipping, vacuum infiltration or pressure
infiltration for 2 min. Pressure infiltration of 8% CaCl₂ resulted in the least decay

16
and the highest concentration of Ca. Vacuum infiltration also increased Ca
concentration in flesh, but did not reduce decay. However, tissue damage prevents
the use of active infiltration of Ca in some soft fruit, such as capsicum, so dips are
the preferred method to improve postharvest quality of soft fruit and control
decay. For example, dipping in 1% CaCl₂ for 1 min recommended for diced Roma
tomato fruit to increase Ca concentration in fruit, enhance firmness and decrease
soluble pectin (Magee et al. 2003).

Calcium treatment delayed decay caused by pathogens due to either Ca increasing

cell wall strength or directly inhibiting the pathogen growth itself (Barkai-Golan
2001). Grey mould development in the strawberry cultivar ‘Selva’ was reduced
significantly by normal dipping in calcium lactate solution (1500 mg kg⁻¹ Ca) or
CaCl₂ solution (4500 mg kg⁻¹ Ca) (Naradisorn 2008). Although the chloride anion
from calcium chloride could have a potential effect on fungal growth, the
inhibitory effect of Ca cation on conidial germination and germ-tube elongation of
B. cinerea has been confirmed in vitro (Wisniewski et al. 1995).

Postharvest Ca treatment has potential benefits in reducing grey mould

development and extending fruit shelf life in some fruits, as discussed above.
However, the effect of this element on the storage life of capsicum fruit and grey
mould development is not well understood. Thus, its effect on postharvest quality
and grey mould development in capsicum fruit was examined in this study.

2.6 Literature summary and aims of study

Capsicum plants are susceptible to B. cinerea which causes grey mould. Grey
mould is responsible for the main losses during the preharvest and postharvest
periods. The flowers and wound sites on fruit have been demonstrated to be the
most susceptible to infection by B. cinerea in crops, such as grape, strawberry and
tomato. However, how B. cinerea infects the capsicum plant is unclear. Therefore,
aim of this study was to conduct a comprehensive study of how B. cinerea infects
capsicum fruit in both preharvest and postharvest periods (Chapter 3, Le at all.
2013).

17
Fungicide application has been used to control pathogens and diseases in both the
preharvest and postharvest periods. Using fungicide may cause a risk to human
health and the environment. The method of Ca and B application has been shown
a potential to improve fruit quality and decrease grey mould development in a
number of fruit crops. However, an understanding of the effect of Ca and B on
grey mould of capsicum and fruit quality remains unclear. Therefore, this study
aimed to examine the effect of preharvest and postharvest application of Ca
(Chapter 5 and 6) and preharvest application of B (Chapter 4) on postharvest
quality and grey mould development on capsicum fruit.

18
 CHAPTER THREE

Infection pathway of Botrytis cinerea in capsicum fruit

This chapter was published as Le at al. (2013) Australasian Plant Pathology

42:449 - 459 (DOI 10.1007/s13313-013-0204-4). The published paper may be
downloaded at http://link.springer.com/article/10.1007%2Fs13313-013-0204-4.
The statement of authorship is provided in Appendix A.1.

19
 Austnlasi¿n Plant Pathol. (2013) 42:449459
DOI I 0. I 007/s133 13-013 42044

Infection pathway oT Botrytís cínereø in capsicum fruit

(Cøpsícum ünnuum L.)
Thong D. Le. Glenn McDonald . Eileen S. Scott.
A Amanda J. Able

Le, T.D., McDonald, G., Scott, E.S. & Able, A.J. (2013) Infection pathway of Botrytis cinera in
capsicum fruit (Capsicum annuum L.).
Australasian Plant Pathology, v. 42(4), pp. 449-459
Received: 30 October 2012 /Accepted: l9 Fcbruary 2013 /Published online: 22 March 2013
@ Australasian Plant Pathology Society Inc. 2013

Àbstract Botrytis cinerea, which causes grey mould, rn- Keywords Preharvest and postharvest inoculation .

fects fruit of a number of horticultural crops during their Flowering . Fruit ripening . Latent infection . Grey mould
development but then remains latent until the ripening pro-
cess when the disease manifests. However, how B. cínerea
grows in capsicum fruit after harvest has not been fully Introduction
characterised. The present research has examined the
growth of ^8. cinerea in ñuit of two cultivars of capsicum Botrytis cinerea Pers. Fr. [teleomorph Botryotinia
(cv. Aries and cv. Papri Queen) that were inoculated either fuckeliana (de Barry) Whetzell, which causes grey mould
before or after harvest. Three concentrations of conidial offruit in a number ofhorticultural crops, can infect during
suspensions (104, 105 and 106 conidia ml-r) were used to flowering and/or the early stages of fruit development as
inoculate flowers at three preharvest stages - anthesis, 3 days well as mature fruit often via wounds. Following infection
after anthesis (DAA) and 6 DAA, and fruit at three of flowers, B. cinereø may remain latent until reactivated
postharvest ripening stages deep green (DG), breaker red
- when ripening causes physiochemical and biochemical
(BR) and red (R). Inoculation with water served as a control. changes in the fruit, leading to fruit rot and the development
Rot development was then monitored dailyNOTE:
during of grey mould if environmental conditions are suitable
This publication
postharvest storage at
is included
l0 "C by measuring after
the length and
page 19 in the print copy
(Droby and Lichter 2004). The processes of infection by
width of lesions. Cv. Aries was more susceptible to B. B. cinerea and development of grey mould are well
cinerea of the thesis held in the University
than cv. Papri Queen regardless of whether inocu- of Adelaide
established Library.
for some fruit, such as red raspberry
lation occurred preharvest or postharvest. Flowers often (Dashwood and Fox 1988), strawberry (Jarvis and Borecka

It is also available online to authorised users at:

died when inoculated at anthesis. Regardless ofcultivar, as
inoculum concentration increased the number of flowers
1968), grape (Keller et al. 2003) and tomato (Lavy-Meir et
al. 1989). Inoculation ofcrops such as strawberry, raspberry
that died also increased. However, disease development on (Jarvis and Borecka 1968; Dashwood and Fox 1988) and
http://doi.org/10.1007/s13313-013-0204-4
fruit was not affected by inoculum concentration or the grape (Keller et al. 2003) at flowering leads to a greater
timing of inoculation before harvest. When fruit were inoc- incidence of grey mould in ripe fruit after harvest than at
ulated after harvest, grey mould developed most rapidly in other stages of development. When tomato fruit were
BR fruit of cv. Papri Queen and in R fnrit of cv. Aries. The infected by B. cinerea during flowering, grey mould only
understanding of infection of B. cinerea revealed by this developed at the stem-end when fruit were stored (Lavy-
research and its implications for disease management are Meir et al. 1989). However, no relationship between flower
discussed. infection by B. cínerea and postharvest decay was found in
nectarine and plum (Fourie and Holz 1994). Infection by B.
cinerea occ't¡s via the flower parts, after which the fungus
T. D. Le. G. McDonald. E. S. Scott. A. J. Able (X) remains latent in plant tissue. Disease then develops after
School ofAgriculture, Food and Wine, The University
harvest or through wounding of fruit.
of Adelaide, Waite Research Institute, PMB l,
Glen Osmond, SA 5064, Auslrâlia Infection by B. cinerea and the timing of grey mould
e-mail: [email protected] development are therefore likely to be related to a number of

Q Sp.inger
 CHAPTER FOUR

Effect of preharvest boron applications

4.1 Introduction

Capsicum is mostly cultivated in the glasshouse or in the field in warmer regions,

both of which favour development of disease on fruit, particularly grey mould
which is caused by Botrytis cinerea. Botrytis cinerea infects flowers and young
fruit but then remains latent until ripening, such that grey mould symptoms are
usually not visible until after harvest (Chapter 3), making the pathogen difficult to
control. In addition, because the cultivars of bell pepper most commonly grown in
Australia are susceptible to B. cinerea, control options may be necessary.
Fungicide application has been the most commonly used method to control this
pathogen both preharvest and postharvest. However, using fungicides raises a risk
to human health and may damage the environment or allow development of
strains of fungi resistant to chemicals (Elad and Shtienberg 1995; Sharma et al.
2009). Therefore, alternative means to control B. cinerea are necessary to reduce
the use of chemicals in agricultural practice. Nutrient deficiency, especially
micronutrients have been demonstrated to reduce resistance of plants or elevate
pathogen development (Marschner 1995). Ensuring adequate nutrition may
therefore be an important way to support resistance of plants to B. cinerea. Boron
(B) is a key element that contributes to resistance of plants (Conway et al. 1994a;
Marschner 1995).

An adequate B level in the plant has been demonstrated to reduce disease

incidence in fruit, due to the involvement of B in the structural integrity of
biomembranes and in cell wall synthesis by cross-linking of cell wall
polysaccharides (Marschner 1995; Hansch and Mendel 2009). When B deficiency
occurs in plants, cell wall and membrane structure may be compromised and the
cell wall becomes more susceptible to attack by the pathogen (Cakmak and
Römheld 1997). Although B accumulates in growing tissues of plants, B
concentration is often higher in the leaves than in fruit (Gupta 1979; Reuter and
Robinson 1997) because B is phloem immobile (Blevins and Lukaszewski 1998).
Furthermore, B has a very narrow range between deficiency and toxicity. Mengel

20
and Kirkby (1987) indicated that soils with less than 1 ppm water soluble B may
not supply enough B for plant growth, while values above 5 ppm may be toxic.
Therefore, providing B at an optimal level is important. Postharvest B application
is not commonly used due to toxicity of inorganic boron-containing compounds to
human health if adults consume more than 13 mg B per day (Nielsen 1997).
Preharvest B application to the soil or as a foliar spray has been commonly used
to achieve optimal yield, greater postharvest quality of fruit and less disease
severity in a number of crops including apples (Peryea and Drake 1991; Omaima
and Karima 2007; Wojcik et al. 2008; Khalifa et al. 2009), peaches (Sotiropoulos
et al. 2010; Thomidis and Exadaktylou 2010; Kavvadias et al. 2012), strawberries
(Naradisorn et al. 2006; Singh et al. 2007) and tomatoes (Huang and Snapp 2004).
Boron fertilisation by the addition of 1 ppm boric acid to soil was critical for
capsicum to grow healthy (Nabi et al. 2006), but the role of this element in grey
mould development and fruit quality remains unclear. The effect of preharvest
application method by soil fertigation or foliar spray on postharvest quality of
fruit and disease in capsicum fruit is also unknown. Boron concentration in
‘Conference’ pear fruit was significantly increased by foliar B spray compared to
that using a soil B application (Wojcik and Wojcik 2003). Foliar B spray may also
be a more effective method to increase B concentration in capsicum fruit tissue,
particularly in cases when soil application causes phytotoxicity. Therefore, the
aim of this research was to examine the effect of preharvest soil or foliar B
application on grey mould development and postharvest quality of capsicum fruit.

4.2 Materials and methods

4.2.1 Plants and growth conditions

Capsicum cv. Aries and cv. Papri Queen were used in this study as per Chapter 3.
Seeds supplied by Monsanto® and Fairbanks Seeds (Australia), respectively were
sown in 6-pot trays (12 cm length x 8 cm width) in UC potting mix (University of
California at Davis) (Baker 1957) to germinate before transplanting to the larger
individual pots (15 cm diameter) containing Mount Compass sand. The sand was
passed through a 2-mm stainless steel sieve to remove organic matter and other
debris and washed with reverse osmosis (RO) water and then air-dried in the
glasshouse. Sieved Mount Compass sand (2.5 kg) was weighed into each
21
individual pot, into which young seedlings at the four leaf stage (6 to 7 weeks)
were planted. Soil B applications were initiated immediately after transplanting
seedlings. Foliar B application was initiated when plants had started flowering.

Capsicum plants were grown in a greenhouse as per Chapter 3. Pest and insects
control were implemented when necessary (usually at ~ 2 months before flower
buds and during fruit development). Spider mites mostly appeared on capsicum
plants of the experiments and two insecticides were used for control them
including Omite® at 2 g L⁻¹ - group 12C insecticide, Chemtura Australia Pty Ltd
or Secure® at 0.3 ml L⁻¹ - group 13A insecticide, Crop Care Australasia Pty Ltd.

4.2.2 Nutrient solutions for B application

4.2.2.1 Soil application

The basal nutrient solution for all treatments was modified from Hoagland’s
solution (Hoagland and Arnon 1938) as described by Rubio et al. (2010) in the
following form: 6.0 mM KNO₃, 4.0 mM Ca(NO₃)₂, 1 mM MgSO₄.7H₂O, 1 mM
KH₂PO₄ and 0.05 mM KCl. The micronutrients without B were applied similarly
in all the treatments, in the following form: 0.002 mM MnSO4.H₂O, 0.002 mM
ZnSO4.7H₂O, 0.0005 mM CuSO4.5H₂O, 0.0005 mM (NH4)6 Mo7O24.4H₂O and
0.02 mM Fe³⁺-EDTA. Boric acid (H₃BO₃) was used as the source of B to obtain
three B concentrations of 0.00, 0.05 and 0.1 mM H₃BO₃ in the Hoagland’s
solution. The pH was kept within the range of 5.5 to 6.0 by adding 1 µM or 0.5
µM KOH. The Hoagland’s solution at three B concentrations with micronutrients
(200 mL) was applied manually to each plant by pouring into the soil every 2 days
after transplanting, while reverse osmosis (RO) water was used to maintain soil
moisture, particularly on hot days during summer. RO water was used as a control
(0.0 mM H₃BO₃). Nutrient analysis showed that B concentration in control plants
was at adequate level (Table 4.1), which suggested contamination of the treatment
with B. The RO water was tested and found to have a B concentration of 0.08
ppm, but this was only known after finishing the experiments.

Twenty plants for each cultivar per treatment were divided into five replicate
groups, with four plants per treatment in each group arranged randomly in the
22
greenhouse. The experiment was conducted twice using the same plants: plants
were kept after the first experiment for a repeat experiment.

4.2.2.2 Foliar B application

The Hoagland’s solution plus the micronutrient without B, as described in Section

4.2.2.1, was applied to all treatments after transplanting. Plants treated with 0.1
mM H₃BO₃ in the soil showed symptoms of toxicity on their leaves (Fig 4.1),
therefore lower B concentrations were chosen for the first foliar application trial
to avoid toxicity. Three B concentrations; 0, 0.025 and 0.075 mM H₃BO₃, were
initially trialled. Capsicum plants were sprayed at weekly intervals by using a
hand-held sprayer pump at an average rate of 200 mL m⁻² (equivalent to 2000 L
per ha). Treatment with RO water was the control (0 mM H₃BO₃). Higher B
concentrations were selected for the second foliar application trial after analysing
the nutrient status of plant tissue at the end of the first trial. The contamination of
the RO water with B resulted in high B concentration in control plants and B
concentration was not significantly different among the three B treatments.
Therefore, plants were maintained after harvest for a second trial. In the second
trial, 0.0, 2.0 or 7.0 mM H₃BO₃ was sprayed on to plants as described earlier.
Twenty plants for each cultivar per treatment were divided into three treatment
groups for three B concentration applications. Each group including 60 plants was
arranged randomly in the greenhouse.

4.2.3 Nutrient analysis of leaves and fruit

Nutrient concentration of leaves and fruit were measured using Inductively

Coupled Plasma Optical Emission Spectrometer (ICP-OES) at Waite Analytical
Services (WAS), Waite Campus, University of Adelaide (Wheal et al. 2011).
Youngest mature leaves (~ 20 g) and three replicate fruit at the red stage were
randomly collected at harvest from a total of 20 replicated plants per treatment for
each cultivar for nutrient analysis. Samples were placed in a paper bag and dried
at 80°C ± 3°C in an oven until the moisture of samples was constant after
weighing twice. The dry samples were ground into fine grade powder by an
electric mill (Kika-werke GMBH, Germany) before providing them to WAS.

23
Each sample was analysed in triplicate and nutrient concentrations in plant tissues
were expressed relative to dry weight (DW).

4.2.4 Inoculation of fruit preharvest or postharvest

4.2.4.1 Isolation, maintenance and culture of B. cinerea

Isolation, maintenance and culture of B. cinerea were carried out as described in

Chapter 3.

4.2.4.2 Pre-harvest inoculation

Previous research showed that flowers of capsicum died at a greater rate when
they were inoculated with 10⁶ conidia mL¯¹, particularly in cv. Aries, which
appeared more susceptible than cv. Papri Queen (Table 1, Chapter 3). Moreover,
there was no significant difference in the development of grey mould on fruit
derived from flowers inoculated at 0, 3 or 6 days after anthesis (DAA) (Fig 1 and
2, Chapter 3). Therefore, in order to minimise the death of flowers following
inoculation, this research used flowers at 3 DAA.

Fruit on multiple flowering nodes on each plant were randomly selected and
tagged before direct application of 100 µL of conidial suspension onto the stem-
end of small fruit by using a pipette. Development of grey mould on fruit cv.
Papri Queen was much less than on fruit from cv. Aries (Fig 1 and 2, Chapter 3).
In order to ensure grey mould development on fruit from cv. Papri Queen after
harvest, a suspension of 10⁶ conidia mL¯¹ was used for inoculation of fruit from
cv. Papri Queen, while a suspension of 10⁴ conidia mL¯¹ was used for cv. Aries.
Conidial suspension was prepared as per Chapter 3. There were 15 or more
replicate young fruit on 15 or more plants per treatment to ensure that at least 10
red fruit were obtained for each treatment in each experiment to assess grey mould
development on fruit after harvest.

24
4.2.4.3 Postharvest inoculation of fruit

Symptomless, uniform red fruit (100% red with low intensity) were harvested
from treated plants for postharvest inoculation. Postharvest inoculation was
conducted as described by Fallik et al. (1996) and as per Section ‘Inoculation by
B. cinerea’ in Chapter 3. Briefly, fruit were surface-sterilised by dipping in 2%
sodium hypochlorite for 1 min and then air-dried and rinsed briefly with sterile
water. The surface-sterilised fruit were wounded on opposite sides to a depth of
1.5 mm by puncturing them with the point of a sterile 1.5 mm diameter nail. Each
wound site was inoculated with 40 µL of suspension containing 10⁵ conidia mL¯¹.
Ten fruit per treatment in each experiment were used in this study.

4.2.5 Assessment of grey mould development and postharvest quality in fruit

4.2.5.1 Grey mould development on fruit

Symptomless red fruit derived from young fruit inoculated preharvest were
harvested to assess development of grey mould during storage using the protocols
described in Section ‘Assessment of grey mould development’ in Chapter 3. The
fruit were monitored daily and the number of fruit exhibiting rot was recorded.
Rot development was quantified by measuring lesion area on individual fruit by
measuring the length and width of decayed areas using digital callipers (digiMax,
Switzerland) and multiplying them.

The correlation between lesion area on fruit and B concentration in leaves and
fruit of capsicum cv. Aries and cv. Papri Queen was determined based on 12
individual measurements of grey mould development on fruit and B concentration
in leaves and fruit from experiments involving soil or foliar B application. Lesion
area on fruit derived from young fruit inoculated preharvest was measured at 28
days after harvest (DAH), while lesion area on fruit inoculated postharvest was
measured at 10 DAH. Boron concentration in leaves and fruit was analysed at
harvest as described as in Section 4.2.2.3.

25
4.2.5.2 Postharvest quality of fruit

Healthy fruit were harvested at full red (100% red with low intensity) from 20
treated plants for assessment of postharvest quality during storage at 10°C at five
time points: 0, 5, 10, 15 and 23 DAH. Firstly, 10 fruit per treatment were
randomly collected to measure length and width by using a digital calliper and
then weighed (using a balance; Adventurer™, OHAUS, USA). Three fruit for each
time-point per treatment were then selected randomly and washed three times in
RO water to remove soil, dust particles and pesticide residues prior to placing in
clear plastic containers (30 cm length x 20 cm width x 10 cm height) and stored at
10°C.

Shelf life was assessed by using a 9-point general appearance (GA) scale (9 = the
best condition and 1 = the worst condition) as described by Able et al. (2002).
When fruit reached a GA of 5.5, they were considered to have reached the end of
their storage life. General Linear Regressions were fitted to data using GenStat
(14th Edition, VSN International Limited, UK) and shelf life was predicted as the
time to reach a GA score of 5.5 using a third degree polynomial.

Fruit firmness was determined by measuring penetration force in kilogram-force

(kgf) by a Fruit Pressure Tester (FT 110, Italy) equipped with a 4.5-mm diameter
plunger tip. A puncture test was performed on four sides of each fruit for cv. Aries
and two sides of each fruit for cv. Papri Queen by holding the fruit against a hard
surface before forcing the plunger tip into the fruit at a uniform speed so that the
depth of penetration was consistently to the line inscribed on the tip. Firmness (N
cm⁻²) was calculated by using following formula:

( )

Where: 1 kgf cm⁻² = 9.807 N cm⁻²

r² is radius of plunger tip

After measuring the firmness, the stem-end and seeds were removed from
individual fruit and the remains of whole fruit was cut into small pieces (2 cm
length x 5 mm width) for assessment of total soluble solids content (TSSC),

26
titratable acidity (TA) and extractable colour. Small pieces (20 g) were randomly
collected for TSSC and TA and 10 g for extractable colour.

TSSC was determined by using a hand-held refractometer (Stanley Limited,

Switzerland). Small pieces of fruit were ground using a porcelain mortar and
pestle prior to wrapping with cheesecloth and squeezing by hand. Juice samples
were then filtered through mira-cloth to make sure a clear juice was available for
measurement. A drop of juice sample was placed on the glass surface of the
refractometer using a micropipette. Two measurements per fruit were taken and
the TSSC was read and presented as ºBrix.

TA was determined by taking 6 g of the juice sample, prepared as described

above, diluting to 50 mL with nanopure water and using 0.1 N NaOH to titrate to
pH 8.1. Each fruit was titrated in triplicate. Results were expressed as percentage
of citric acid in the juice which is the predominant organic acid in capsicum
(Rubio et al. 2010) using the formula:
( ) ( )

Where acid milliequivalent factor for citric acid = 0.064 (Garner et al. 2005)

Extractable colour was measured based on the standard method of the American
Spice Trade Association (ASTA) and presented as ASTA units [Association of
Official Analytical Communities (AOAC 1997)] and American Spice Trade
Association (ASTA 1985)]. Ten g of tissue pieces from each fruit, prepared as for
TSSC measurement, were placed in a paper bag and dried at 40°C in an oven until
the moisture of samples was constant after weighing twice. The dry weight of
samples was used to calculate water content in fruit (for only the experiment of
foliar application with high B concentration) as per the following formula:

( ) ( )
( )
( )

The dry samples were then ground into fine grade powder as described above. The
ground powder was kept in sealed plastic bags and stored in the dark at 4°C using
a water absorbent (silica gel) for no longer than 2 weeks.

27
Seventy mg of the ground sample was placed in a 100 mL volumetric flask with
acetone. The flask was tightly stoppered, shaken and placed in the dark at room
temperature (22°C) for 16 h. The supernatant (containing the colour extract) was
transferred to 1 mL cuvettes and the absorbance at 460 nm (A₄₆₀) measured using
a spectrophotometer (SP8001, Metertech). The final ASTA value for each
measurement was calculated using the following formula:

( )

4.2.6 Statistical analysis and photography

Experiments were conducted twice and designed with no blocking using the
factors of cultivar and B concentration. Data from repeated experiments when B
was applied to the soil were statistically similar and therefore combined for
analysis. Data were subjected to repeated measurements analysis of variance
(ANOVA) using GenStat (14th Edition, VSN International Limited, UK) and
means of the treatments compared using the Least Significant Difference (LSD) at
a significance level of P ≤ 0.05. Means and standard errors were determined using
Microsoft Excel. Photography was by a digital camera (Canon D500, Japan).

4.3 Results

4.3.1 Effect of B applied to soil on nutrient status of plant tissues, postharvest

quality of fruit and grey mould development on capsicum

Toxicity symptoms were first observed on the leaves of plants from cv. Aries,
which had received a soil application of 0.1 mM H₃BO₃ in the first experiment
when plants had flower buds (Fig 4.1A). Symptoms (where older leaf margins
turned to a faded brown) on leaves of the same plants became greater in the repeat
experiment (Fig 4.1B). Toxicity symptoms did not show on leaves of plants
treated with 0.05 mM H₃BO₃ in either experiment. Toxicity symptoms were not
observed on leaves from cv. Papri Queen in any experiments. There were also no
toxicity symptoms on flowers or fruit of any plants, regardless of whether B was
applied to the soil or via foliar application.

28
4.3.1.1 Boron concentration in leaf and fruit tissues

Boron concentration in leaf and fruit tissues significantly increased with

increasing B concentration in the nutrient solution applied into the soil (Table
4.1). B concentration in leaves and fruit from plants treated with 0.1 mM H₃BO₃
was significantly higher (P<0.001) than B concentration in leaves and fruit from
plants treated with 0 or 0.05 mM H₃BO₃ for both cultivars. The B concentration
in leaves and fruit from plants treated with 0.05 mM H₃BO₃ was significantly
more than that in control plants. B concentration in leaf tissues of control plants (0
mM H₃BO₃) unexpectedly had B present at levels considered to be adequate for
growth (23 - 24 mg kg¯¹) (Reuter and Robinson 1997). Boron concentration in
leaf tissue from both cultivars treated with 0.05 or 0.1 mM H₃BO₃ was above
those usually regarded as toxic for capsicum (40 - 100 mg kg¯¹) (Reuter and
Robinson 1997). There were no significant differences in other nutrients,
regardless of cultivar or B concentration (Appendix A.2). However, copper
concentration in leaves from any of the three concentrations, regardless of
cultivar, appeared deficient compared to that considered to be adequate (6 - 25 mg
kg¯¹) (Reuter and Robinson 1997).

4.3.1.2 Postharvest quality of fruit

Length, width and weight were not significantly different among fruit from plants
treated with the three B concentrations, regardless of cultivar (Fig 4.2A, B).

The shelf life of fruit from cv. Aries plants treated with 0.1 mM H₃BO₃ in the soil
was significantly longer (P = 0.024) than that from plants treated with less boron
(Fig 4.3A), while shelf life of fruit grown in soil treated with 0.0 or 0.05 mM
H₃BO₃ was not significantly different from each other. For cv. Papri Queen, there
was no significant difference in the shelf life of fruit from plants treated with three
B concentrations (Fig 4.3B). The shelf life of fruit from cv. Papri Queen was
significantly shorter than for fruit from cv. Aries.

29
 A B

Fig. 4.1 Toxicity symptoms on the leaves of plants of cv. Aries growing in soil to
which 0.1 mM H₃BO₃ had been applied. (A) Plants at 60 days after transplanting
at flower bud stage in the first experiment; (B) Plants at 180 days after
transplanting at the harvest stage in the repeat experiment

30
Table 4.1 Effect of soil boron application on B concentrations in leaf and fruit
tissues of cv. Aries and cv. Papri Queen. Other nutrients are in a table in Appendix
A.2. The nutrient concentrations were determined using ICP-OES analysis.
Youngest mature leaves and red fruit for nutrient analysis were collected at harvest
from plants that received 0, 0.05 or 0.1 mM H₃BO₃ in Hoagland’s solution (200 mL
per plant every 2 days). Data are presented as means ± SE from n = 6 from two
experiments. For each cultivar, means with the same letters in each column were not
significantly different as determined using the LSD (P<0.05)
B concentration
B treatment
Cultivar (mg kg¯¹ DW)
(mM H₃BO₃)
Leaf Fruit
a
Control 55.73 ± 8.50 10.84 ± 0.21a
0.05 169.12 ± 21.65b 14.28 ± 0.27b
Aries
0.1 287.50 ± 18.87c 17.85 ± 0.37c
LSD (P value) 55.32 (<0.001) 2.16 (<0.001)
Control 50.59 ± 4.66a 11.74 ± 0.16a
0.05 193.19 ± 32.91b 14.28 ± 0.22b
Papri Queen
0.1 305 ± 40.31c 18.35 ± 0.43c
LSD (P value) 96.50 (<0.001) 2.16 (<0.001)

31
 A
180 180
0 mM H₃BO₃ 0.1 mM H₃BO₃
160 160
140 0.05 mM H₃BO₃ 140
120 120

mm per fruit

g per fruit
100 100
80 80
60 60
40 40
20 20
0 0
length width weight

180 B 180
160 160
140 140
120 120
mm per fruit

g per fruit
100 100
80 80
60 60
40 40
20 20
0 0
length width weight
Size of fruit

Fig. 4.2 Length, width and weight of fruit from cv. Aries (A) and cv. Papri Queen
(B) when plants received a soil application of 0, 0.05 or 0.1 mM H₃BO₃ in
Hoagland’s solution (200 mL per plant every 2 days) from transplant to harvest of
red fruit. Data are means ± SE from n = 20 across two experiments

32
 A
30

25

Predicted shelf life (days)

20

15

10

0
0 0.05 0.1
30
B
25
Predicted shelf life (days)

20

15

10

0
0 0.05 0.1
Soil boron application (mM H₃BO₃)

Fig. 4.3 Effect of soil B application on shelf life of fruit from cv. Aries (A) and cv.
Papri Queen (B) during storage at 10°C and relative humidity of >90%. Fruit were
harvested from plants that received a soil application of 0, 0.05 or 0.1 mM H₃BO₃ in
Hoagland’s solution (200 mL per plant every two days) from transplant to harvest of
red fruit. Shelf life of fruit was predicted at a general appearance (GA) of 5.5 by
using a third degree polynomial in GenStat. Data are means ± SE from n = 6 across
two experiments. LSDs (P ≤ 0.05) for among individual boron concentrations are
shown

33
Extractable colour of fruit from both cultivars increased significantly during storage,
regardless of soil treatment. Extractable colour of fruit was not significantly different
among the three B concentration treatments (Fig 4.4A, B). Fruit from cv. Papri
Queen had a significantly greater extractable colour than fruit from cv. Aries.

Firmness of fruit from cv. Aries significantly decreased (P<0.001) from 10 DAH to
23 DAH during storage compared with that of fruit at harvest in all three B
treatments (Fig 4.4C), while firmness of fruit from cv. Papri Queen decreased
significantly (P = 0.002) at 5 DAH but then significantly increased from 10 DAH to
23 DAH (Fig 4.4D).

TSSC in fruit from cv. Aries increased significantly (P<0.001) at 10 DAH compared
to that at harvest. Fruit from the control treatment showed a further significant
increase in fruit at 23 DAH, while there was no futher change in TSSC in fruit from
plants treated with 0.05 or 0.1 mM H₃BO₃ (Fig 4.4E). For cv. Papri Queen, initial
TSSC in fruit at harvest was not significantly different among B treatments, but
significantly different at 10 DAH when TSSC in fruit from control plants was
significantly lower (P = 0.009) than that from plants treated with two B
concentrations (Fig 4.4F). However, at 23 DAH TSSC in fruit from control plants
was significantly higher than that from plants treated with 0.1 mM H₃BO₃, but the
same with that from plants treated with 0.05 mM H₃BO₃. TSSC in fruit from cv.
Papri Queen was significantly higher than in fruit from cv. Aries.

TA in fruit during storage was not significantly different among B concentration

treatments for both cultivars (Fig 4.4G, H). For cv. Aries, TA in fruit from plants
treated with 0 or 0.05 mM mM H₃BO₃ increased significantly (P<0.001) from 5
DAH to 23 DAH, while TA in fruit from plants treated with 0.1 mM H₃BO₃
increased significantly from 5 DAH to 15 DAH but then there was no change at 23
DAH. TA in fruit from cv. Papri Queen decreased significantly (P<0.001) at 5 DAH
but then significantly increased from 10 DAH to 23 DAH.

34
 350 350
A B
Extractable colour (ASTA) 300 300
250 250
200 200
150 150
0 mM H₃BO₃
100 100
0.05 mM H₃BO₃
50
50
0.1 mM H₃BO₃ 0
0
0 5 10 15 20 25
0 5 10 15 20 25
120 120 D
C
100 100
Firmness (N cm⁻²)

80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
16
14
E 16 F
14
12 12
TSSC (°Bx)

10 10
8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 G 0.7 H
0.6 0.6
Citric acid (%)

0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 4.4 Effect of soil B application on postharvest quality of fruit during storage at
10°C and relative humidity of >90%: extractable colour (A and B); firmness (C and
D); TSS (E and F) and TA (G and H) for cv. Aries and cv. Papri Queen,
respectively. Fruit were harvested from plants that received a soil application of 0,
0.05 or 0.1 mM H₃BO₃ in Hoagland’s solution (200 mL per plant every 2 days) from
transplanting to harvest of red fruit. Data are means ± SE from n = 6 across two
experiments. LSDs (P ≤ 0.05) are shown for among individual B concentration for
each time-point during storage

35
4.3.1.3 Grey mould development on fruit derived from inoculated young fruit

Grey mould was not observed on fruit at harvest in either cv. Aries or cv. Papri
Queen. Grey mould development on fruit was mostly observed 14 days after harvest
(DAH) and then increased significantly (P<0.001) during storage, regardless of
cultivar or B treatment (Fig 4.5).

For cv. Aries, lesion area on fruit from plants treated with 0.1 mM H₃BO₃ was
significantly smaller (P = 0.009) than that from plants treated with 0.0 mM H₃BO₃
from 18 DAH to 28 DAH, whereas lesion area on fruit from plants treated with 0.05
mM H₃BO₃ was similar to that from plants treated with 0.1 mM H₃BO₃, but was
significantly smaller than that from control plants at 18 DAH (Fig 4.5A). The
number of fruit exhibiting rot was not significantly different among B treatments
(Fig 4.5B). For cv. Papri Queen, lesion area on fruit from plants treated with 0.1 mM
H₃BO₃ was significantly smaller (P = 0.039) than that from plants treated with 0.0
mM H₃BO₃ at 28 DAH, but was the same with that from plants treated with 0.05
mM H₃BO₃ (Fig 4.5C). The percentage of fruit exhibiting rot from plants that did not
receive B was significantly larger (P = 0.039) than those that received B (Fig 4.5D).
Lesion area on fruit from cv. Papri Queen, regardless of B treatment, was
significantly smaller (P<0.001) than that from cv. Aries.

Time to reach 50% of maximum lesion area on fruit from plants treated with 0.1 mM
H₃BO₃ was significantly longer (P = 0.001 for cv. Aries and P = 0.006 for cv. Papri
Queen) than that from control plants for both cultivars (Fig 4.6A and 4.6B). Time to
reach 50% maximum lesion area on fruit from plants treated with 0.05 mM H₃BO₃
was not significantly different from that from control plants or plants treated with 0.1
mM H₃BO₃ for cv. Aries, but was significantly longer than that from control plants
for cv. Papri Queen.

36
 B
A
0 mM H₃BO₃ 100
9000
90
8000 0.05 mM H₃BO₃
80

Fruit exhibiting rot (%)

7000 0.1 mM H₃BO₃
Lesion area (mm²)

70
6000
60
5000
50
4000
40
3000
30
2000
20
1000 10
0 0
14 16 18 20 22 24 26 28 0 0.05 0.1

D
C 100
9000 90
8000 80
Fruit exhibiting rot (%)

7000 70
Lesion area (mm²)

6000 60
5000 50
4000 40
3000 30
2000 20
1000 10
0 0
14 16 18 20 22 24 26 28 0 0.05 0.1
Soil boron application
Time during storage (days)
(mM H₃BO₃)

Fig. 4.5 Effect of soil B application on grey mould development and the number of
fruit of capsicum cv. Aries (A and B) and cv. Papri Queen (C and D) exhibiting rot
during storage at 10°C and relative humidity of >90%. Fruit were derived from
young fruit inoculated with 100 µL suspension of 10⁴ conidia mL¯¹ for cv. Aries and
10⁶ conidia mL¯¹ for cv. Papri Queen at 3 DAA when plants received a soil
application of 0, 0.05 or 0.1 mM H₃BO₃ in Hoagland’s solution (200 mL per plant
every 2 days) from transplant to harvest red fruit. Data are means ± SE from n = 20
across two experiments. LSDs (P ≤ 0.05) are shown for among individual boron
concentrations for each time-point during storage

37
 35
A

Time (days) to reach 50% of maximum

30

25

lesion area
20

15

10

0
0 0.05 0.1
35
Time (days) to reach 50% of maximum

30 B

25
lesion area

20

15

10

0
0 0.05 0.1
Boron concentration (mM H₃BO₃)

Fig. 4.6 Time after harvest for lesions to reach 50% of maximum size on fruit of
capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at 10°C and relative
humidity of >90% for 28 days when plants received a soil application of 0, 0.05 or
0.1 mM H₃BO₃ in Hoagland’s solution (200 mL per plant every 2 days) from
transplant to harvest red fruit. Fruit were derived from young fruit inoculated with
100 µL suspension of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv.
Papri Queen at 3 DAA. Data are means ± SE from n = 20 across two experiments.
LSDs (P ≤ 0.05) for among individual B concentrations at 28 days after harvest are
shown

38
4.3.1.4 Grey mould development on fruit inoculated postharvest

Symptoms of grey mould on fruit which were inoculated postharvest were observed
4 days post-inoculation (DPI) for both cultivars, with 100% of fruit exhibiting rot
after 10 DPI. Lesion area on fruit was significantly different among B treatments for
both cultivars (Fig 4.7A and 4.7B). Lesion area on fruit from plants which were
treated with either B concentration was significantly smaller than that from control
plants from 8 to 10 DPI for cv. Aries (P = 0.013) and at 10 DPI for cv. Papri Queen
(P<0.001). However, lesion area was similar on fruit from plants of cv. Aries which
were treated with either B concentration. For cv. Papri Queen, lesion area on fruit
from plants treated with 0.1 mM H₃BO₃ was significantly smaller than that from
plants treated with 0.05 mM H₃BO₃.

4.3.2 Effect of B as a foliar application on nutrient status of plant tissues,

postharvest quality of fruit and grey mould development

4.3.2.1 Foliar application at lower B concentrations

Symptoms of B toxicity or deficiency were not observed on leaves or fruit from

plants treated with the three B concentrations of 0, 0.025 or 0.075 mM H₃BO₃ as a
foliar spray from flowering to harvest of red fruit.

B concentration was not significantly different in leaves or fruit from the control or
plants sprayed with B and all values in the leaves were above the critical level for
deficiency (25 -75 mg kg¯¹ ) (Reuter and Robinson 1997) (Table 4.2). There were no
significant differences in other nutrients in leaf and fruit tissues, regardless of
cultivar or B concentration (Appendix A.3).

Length, width and weight were not significantly different among fruit from plants
treated with the three B concentrations, regardless of cultivar (Fig 4.8A, B).

39
 4000 0 mM H₃BO₃
A
3500 0.05 mM H₃BO₃

3000 0.1 mM H₃BO₃

Lesion area (mm²) 2500

2000

1500

1000

500

0
4 6 8 10

4000 B
3500

3000
Lesion area (mm²)

2500

2000

1500

1000

500

0
4 6 8 10
Time post-inoculation (days)

Fig. 4.7 Effect of soil B application on grey mould development on postharvest-

inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at
10°C and relative humidity of >90% when plants received a soil application of 0,
0.05 or 0.1 mM H₃BO₃ in Hoagland’s solution (200 mL per plant every 2 days) from
transplant to harvest red fruit. Fruit were wounded both sides and inoculated
postharvest with 40 µL suspension of 10⁵ conidia mL¯¹. Data are means ± SE from n
= 20 across two experiments. LSDs (P ≤ 0.05) are shown for among individual B
concentrations for each time-point post-inoculation

40
Table 4.2 Effect of foliar B application on nutrient status in capsicum plant tissues in
the first trial (low B concentrations). Other nutrients are in a table in Appendix A.3.
The nutrient concentrations were determined using ICP-OES analysis. Youngest
mature leaves and red fruit for nutrient analysis were collected at harvest from plants
that received a foliar application: 0.0, 0.025 or 0.075 mM H₃BO₃ from flowering to
harvest of red fruit. Plants were watered with Hoagland’s solution without B (200
mL per plant for every 2 days). Data are presented as means ± SE from n = 3. There
were no significant difference between B treatments within tissue types (P values are
shown)

B concentration
B treatment
Cultivar (mg kg¯¹ DW)
(mM H₃BO₃)
Leaf Fruit
Control 87.54 ± 0.60 12.67 ± 0.99
0.025 72.34 ± 0.98 10.77 ± 0.4
Aries
0.075 76.23 ± 1.4 10.16 ± 0.32
P value 0.06 0.074
Control 66.79 ± 0.60 11.40 ± 0.47
0.025 67.92 ± 1.41 11.57 ± 0.36
Papri Queen
0.075 77.53 ± 0.49 11.51 ± 0.33
P value 0.07 0.95

41
 180 0 mM H₃BO₃ 0.075 mM H₃BO₃ 180
A
160 0.025 mM H₃BO₃ 160
140 140

mm per fruit 120 120

g per fruit
100 100
80 80
60 60
40 40
20 20
0 0
length width weight

180 B 180
160 160
140 140
120 120
mm per fruit

g per fruit
100 100
80 80
60 60
40 40
20 20
0 0
length width weight
Size of fruit

Fig. 4.8 Length, width and weight of fruit from cv. Aries (A) and cv. Papri Queen
(B) when plants received a foliar application of 0, 0.025 or 0.075 mM H₃BO₃ in the
first trial (lower B concentrations). Plants were sprayed from flowering to harvest of
red fruit at weekly intervals by hand-held sprayer pump at an average rate of 200 mL
m⁻² (equivalent to 2000 L ha⁻¹). Data are means ± SE from n = 10

42
The shelf life of fruit from plants that had B applied to them as a foliar spray
appeared to be longer than those from control plants, but the difference was not
significant, regardless of cultivar (Fig 4.9A, B). The shelf life was similar for fruit of
cv. Aries and fruit of cv. Papri Queen, regardless of B concentration.

Application of B as a foliar spray did not affect extractable colour of fruit during
storage regardless of cultivar (Fig 4.10A, B). Extractable colour increased steadily
during storage and was significantly higher at 10 DAH than at harvest, regardless of
B treatment or cultivar.

Firmness of fruit during storage was not significantly different among three B
treatments, regardless of cultivar (Fig 4.10C, D). Firmness of fruit from cv. Aries
decreased significantly (P<0.001) from 10 DAH to 23 DAH, while firmness of fruit
from cv. Papri Queen increased significantly (P<0.001) from 10 DAH to 23 DAH.

TSSC in fruit of both cultivars generally increased steadily from 10 DAH to 23 DAH
(Fig 4.10E, F). For cv. Aries at 5 DAH, TSSC in fruit from plants treated with 0.075
mM H₃BO₃ as a foliar spray was significantly lower (P = 0.038) than that for fruit
from plants treated with 0 mM or 0.025 mM H₃BO₃ (Fig 4.10E). At 15 DAH TSSC
in fruit from plants treated with 0.025 mM H₃BO₃ was significantly higher (P =
0.038) than that of fruit from control plants, but the same as that for fruit from plants
treated with 0.075 mM H₃BO₃. For cv. Papri Queen, foliar B application had no
effect on TSSC in fruit during storage (Fig 4.10F).

43
 25
A

20

Predicted shelf life (days)

15

10

0
0 0.025 0.075

25
B
Predicted shelf life (days)

20

15

10

0
0 0.025 0.075
Foliar B application (mM H₃BO₃)

Fig. 4.9 Effect of foliar B application on shelf life of fruit of capsicum cv. Aries (A)
and cv. Papri Queen (B) during storage at 10°C and relative humidity of >90% in the
first trial (lower B concentrations). Fruit were harvested from plants that received a
foliar application of 0, 0.025 or 0.075 mM H₃BO₃. Plants were sprayed from
flowering to harvest of red fruit at weekly intervals by hand-held sprayer pump at an
average rate of 200 mL m⁻² (equivalent to 2000 L ha⁻¹). Shelf life of fruit was
predicted at a general appearance (GA) of 5.5 by using a third degree polynomial in
GenStat. Data are means ± SE from n = 10. LSDs (P ≤ 0.05) for among individual
boron concentrations are shown

44
TA in fruit during storage was not significantly different among B concentration
treatments, regardless of cultivar (Fig 4.10G, H). For cv. Aries, TA in fruit increased
significantly (P<0.001) from 5 DAH to 10 DAH but then did not change from 15
DAH to 23 DAH, while TA in fruit from cv. Papri Queen increased significantly
(P<0.001) from 10 DAH to 23 DAH.

Lesion area on fruit derived from young fruit that had been inoculated preharvest
was not significantly different for cv. Aries, regardless of B concentration used (Fig
4.11A). However, for cv. Papri Queen, lesion area on fruit from plants sprayed with
either 0.075 or 0.025 mM H₃BO₃ was significantly smaller (P<0.001) from 20 DAH
to 28 DAH compared to those from control plants (Fig 4.11C). The percentage of
fruit exhibiting rot from plants that were sprayed with 0.075 mM H₃BO₃ appeared to
be lower than those that were not sprayed or sprayed with lower B concentration,
regardless of cultivar (Fig 4.11B, D).

Time to reach 50% of maximum lesion area on fruit derived from young fruit that
had been inoculated preharvest was not significantly different among three B
treatments for cv. Aries (Fig 4.12A). In contrast, for cv. Papri Queen time to reach
50% of maximum lesion area on fruit from control plants was significantly shorter
(P<0.001) than that on fruit from plants sprayed with 0.025 or 0.075 mM H₃BO₃ (Fig
4.12B). However, time to reach 50% of maximum lesion area was similar on fruit
between plants sprayed with either B concentration.

Lesion area on fruit inoculated postharvest was not significantly different, regardless
of B concentration or cultivar (Fig 4.13A, B).

45
 350 350
A B
Extractable colour (ASTA) 300 300
250 250
200 200
150 0 mM H₃BO₃ 150
100 0.025 mM H₃BO₃ 100
50 0.075 mM H₃BO₃ 50
0 0
0 5 10 15 20 25 0 5 10 15 20 25
120 120
C D
Firmness (N cm⁻²)

100 100
80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
16 16 F
14 E 14
12 12
10 10
TSSC (°Bx)

8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 0.7
0.6 G 0.6 H
0.5 0.5
Citric acid (%)

0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 4.10 Effect of foliar B application on postharvest quality of fruit during storage
at 10°C and relative humidity of >90%: extractable colour (A and B); firmness (C
and D); TSS (E and F) and TA (G and H) for cv. Aries and cv. Papri Queen,
respectively in the first trial (lower B concentrations). Fruit were harvested from
plants that received a foliar application of 0, 0.025 or 0.075 mM H₃BO₃ from
flowering to harvest of red fruit at weekly intervals by hand-held sprayer pump at an
average rate of 200 mL m⁻² (equivalent to 2000 L ha⁻¹). Data are means ± SE from n
= 3. LSDs (P ≤ 0.05) are shown for among individual B concentration for each time-
point during storage

46
 9000 A 0 mM H₃BO₃ 100 B
8000 90
0.025 mM H₃BO₃
7000 80

Fruit exhibiting rot (%)

Lesion area (mm²)

0.075 mM H₃BO₃ 70
6000
5000 60
50
4000
40
3000 30
2000 20
1000 10
0 0
14 16 18 20 22 24 26 28 0 0.025 0.075

9000 C 100 D
8000 90
80

Fruit exbiting rot (%)

7000
Lesion area (mm²)

6000 70
60
5000
50
4000
40
3000 30
2000 20
1000 10
0 0
14 16 18 20 22 24 26 28 0 0.025 0.075
Foliar boron application
Time during storage (days)
(mM H₃BO₃)
Fig. 4.11 Effect of foliar B application on grey mould development and percentage
of fruit exhibiting rot of capsicum cv. Aries (A and B), cv. Papri Queen (C and D)
during storage at 10°C and relative humidity of >90% in the first trial (lower B
concentrations). Fruit were derived from young fruit inoculated with 100 µL
suspension of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri
Queen at 3 DAA when plants received a foliar application of 0, 0.025 or 0.075 mM
H₃BO₃. Plants were sprayed with foliar B from flowering to harvest of red fruit at
weekly intervals by hand-held sprayer pump at an average rate of 200 mL m⁻²
(equivalent to 2000 L ha⁻¹). Data are means ± SE from n = 10. LSDs (P ≤ 0.05) are
shown for among individual boron concentration for each time-point during storage

47
 35
A

Time (days) to reach 50% of maximum

30

25

lesion area
20

15

10

0
0 0.025 0.075
35
Time (days) to reach 50% of maximum

30 B

25
lesion area

20

15

10

0
0 0.025 0.075
Foliar boron application (mM H₃BO₃)

Fig. 4.12 Time after harvest for lesions to reach 50% of maximum size on fruit of
capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at 10°C and relative
humidity of >90% for 28 days in the first trial (lower B concentration) when plants
received a foliar application of 0, 0.025 or 0.075 mM H₃BO₃. Plants were sprayed
with foliar B from flowering to harvest at weekly intervals by hand-held sprayer
pump at an average rate of 200 mL m⁻² (equivalent to 2000 L ha⁻¹). Fruit were
derived from young fruit inoculated with 100 µL suspension of 10⁴ conidia mL¯¹ for
cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3 DAA. Data are means ± SE
from n = 10. LSDs (P ≤ 0.05) for among individual B concentrations at 28 days after
harvest are shown

48
 6000 0 mM H₃BO₃
A
0.025 mM H₃BO₃
5000
0.075 mM H₃BO₃

Lesion area (mm²)

4000

3000

2000

1000

0
4 6 8 10 12

6000
B
5000
Lesion area (mm²)

4000

3000

2000

1000

0
4 6 8 10 12
Time post-inoculation (days)

Fig. 4.13 Effect of foliar B application on grey mould development on postharvest-

inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at
10°C and relative humidity of >90% in the first trial (lower B concentrations) when
plants received a foliar application of 0, 0.025 or 0.075 mM H₃BO₃. Plants were
sprayed with foliar B from flowering to harvest of red fruit at weekly intervals by
hand-held sprayer pump at an average rate of 200 mL m⁻² (equivalent to 2000 L
ha⁻¹). Fruit were wounded both sides and inoculated postharvest with 40 µL
suspension of 10⁵ conidia mL¯¹. Data are means ± SE from n = 10. LSDs (P ≤ 0.05)
are shown for among individual B concentrations for each time-point post-
inoculation

49
4.3.2.2 Foliar application at higher B concentrations

Symptoms of B toxicity or deficiency were not observed on leaves and fruit from
plants treated with three higher B concentrations of 0, 2.0 or 7.0 mM H₃BO₃ from
flowering to harvest of red fruit.

B concentration in leaf tissues from control plants indicated the plants were deficient
and spraying with 2.0 or 7.0 mM H₃BO₃ significantly increased the boron
concentration in leaves of both cultivars. Boron concentration in leaves from plants
sprayed with 7.0 mM H₃BO₃ was significantly greater (P<0.001) than that of leaves
from plants sprayed with 2.0 mM H₃BO₃ (Table 4.3). For cv. Aries, B concentration
in fruit from plants treated with 7.0 mM H₃BO₃ was significantly higher (P = 0.048)
than that from control plants, but was not significantly different between plants
sprayed with 2.0 mM H₃BO₃ and control plants. For cv. Papri Queen, spraying plants
with 2.0 or 7.0 mM H₃BO₃ did not affect B concentration in the fruit. There were no
significant differences in other nutrients in the leaves and the fruit, regardless of
cultivar or B concentration (Appendix A.4).

Length, width and weight were not significantly different among fruit from plants
treated with the three B concentrations, regardless of cultivar (Fig 4.14A, B).

The shelf life of fruit was not significantly different among the three B applications,
regardless of cultivar (Fig 4.15A, B).

Extractable colour in fruit increased steadily during storage for all three B
applications, regardless of cultivar. Extractable colour in fruit from cv. Papri Queen
was significantly greater than that for fruit from cv. Aries from 15 to 23 DAH.
Application at 2.0 or 7.0 mM H₃BO₃ as a foliar spray had no effect on extractable
colour of fruit during storage, regardless of cultivar (Fig 4.16A, B).

50
Table 4.3 Effect of foliar B application on nutrient status in capsicum plant tissues in
the second trial (higher B concentrations). Other nutrients are in a table in Appendix
A.4. The nutrient concentrations were determined using ICP-OES analysis.
Youngest mature leaves and red fruit for nutrient analysis were collected at harvest
from plants that received a foliar application: 0.0, 2.0 or 7.0 mM H₃BO₃ from
flowering to harvest of red fruit. Plants were watered with Hoagland’s solution
without B (200 mL per plant for every 2 days). Data are presented as means ± SE
from n = 3. For each cultivar, means with the same letters in each column were not
significantly different as determined using the LSD (P<0.05)

B concentration
B treatment
Cultivar (mg kg¯¹ DW)
(mM H₃BO₃)
Leaf Fruit
Control *14.98 ± 0.01a 4.99 ± 0.54a
2.0 32.86 ± 0.41b 5.61 ± 0.24ab
Aries
7.0 54.98 ± 0.43c 6.77 ± 0.34b
LSD (P value) 1.55 (<0.001) 1.37 (0.048)
Control *18.38 ± 0.28a 4.05 ± 0.3a
2.0 25.50 ± 0.02b 4.35 ±0.27a
Papri Queen
7.0 32.83 ± 0.19c 4.33 ± 0.26a
LSD (P value) 0.88 (<0.001) 0.95 (0.70)

*deficient as stated by Reuter and Robinson (1997)

51
 180 A 180
0 mM H₃BO₃ 7 mM H₃BO₃
160 160
140 2 mM H₃BO₃ 140
120 120
mm per fruit

g per fruit
100 100
80 80
60 60
40 40
20 20
0 0
length width weight

180.00 B 180.00
160.00 160.00
140.00 140.00
120.00 120.00
mm per fruit

g per fruit
100.00 100.00
80.00 80.00
60.00 60.00
40.00 40.00
20.00 20.00
0.00 0.00
length width weight
Size of fruit

Fig. 4.14 Length, width and weight of fruit from cv. Aries (A) and cv. Papri Queen
(B) when plants received a foliar application of 0, 2 or 7 mM H₃BO₃ in the second
trial (higher B concentrations). Plants were sprayed from flowering to harvest of red
fruit at weekly intervals by hand-held sprayer pump at an average rate of 200 mL
m⁻² (equivalent to 2000 L ha⁻¹). Data are means ± SE from n = 10

52
 25 A

20

Predicted shelf life (days)

15

10

0
0 2 7
25
B

20
Predicted shelf life (days)

15

10

0
0 2 7
Foliar boron application (mM H₃BO₃)

Fig. 4.15 Effect of foliar B application on shelf life of fruit of capsicum cv. Aries (A)
and cv. Papri Queen (B) during storage at 10°C and relative humidity of >90% in the
second trial (higher B concentrations). Fruit were harvested from plants that received
a foliar application of 0, 2 or 7 mM H₃BO₃. Plants were sprayed from flowering to
harvest of red fruit at weekly intervals by hand-held sprayer pump at an average rate
of 200 mL m⁻² (equivalent to 2000 L ha⁻¹). Shelf life of fruit was predicted at a
general appearance (GA) of 5.5 by using a third degree polynomial in GenStat. Data
are means ± SE from n = 10. LSDs (P ≤ 0.05) for among individual boron
concentrations are shown

53
Firmness of fruit during storage was not affected by application at 2.0 or 7.0 mM
H₃BO₃ as a foliar spray, regardless of cultivars (Fig 4.16C, D). Firmness of fruit
from cv. Aries decreased significantly (P =0.001) at 10 DAH but then did not change
at 15 DAH and significantly increased at 23 DAH. In contrast, firmness of fruit from
cv. Papri Queen increased significantly (P<0.001) from 10 DAH to 15 DAH but then
remained constant to 23 DAH.

Water content in fruit at 23 DAH was significantly lower (P<0.001) than that in fruit
at harvest from 10 DAH to 23 DAH, regardless of cultivar or B treatment (Fig4.16E,
F). Water content in fruit (53.89%) from cv. Aries was significantly greater (P =
0.047) than that in fruit (47.24%) from cv. Papri Queen, regardless of B
concentration (Fig 4.16E, F). Foliar B application did not affect water content in fruit
from both cultivars.

For fruit from cv. Aries at 0 DAH, TSSC in fruit from control plants was
significantly greater (P = 0.006) than that for fruit from plants sprayed with 2.0 mM
H₃BO₃, but the same with that for fruit from plants sprayed with 7.0 mM H₃BO₃ (Fig
4.16G). At 23 DAH, TSSC in fruit from control plants was significantly (P = 0.006)
lower than that from plants sprayed with either of the two B concentrations. For fruit
from cv. Papri Queen, TSSC in fruit was not affected by application 2.0 or 7.0 mM
H₃BO₃ as a foliar spray (Fig 4.16H).

Titratable acidity (TA) in fruit during storage, regardless of cultivar, was not
significantly different among B concentrations (Fig 4.16K, I). For cv. Aries, TA in
fruit decreased significantly (P<0.001) at 5 DAH then increased at 10 DAH,
followed by a steady increase from 15 DAH to 23 DAH. For cv. Papri Queen, TA in
fruit increased significantly (P<0.001) from 10 DAH to 23 DAH. Amount of acid in
fruit from cv. Papri Queen was generally higher than in fruit from cv. Aries.

54
 350 350 B
A
Extractable colour (ASTA)
300 300
250 250
200 200
150 0 mM H₃BO₃
150
100 2 mM H₃BO₃
100
50 7 mM H₃BO₃ 50
0 0
0 5 10 15 20 25 0 5 10 15 20 25
100 100 D
C
Firmness (N cm⁻²)

80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
60 60
Water content (%)

40 E 40
F
20 20

0 0
0 5 10 15 20 25 0 5 10 15 20 25
14 14 H
12 G 12
10 10
TSSC (°Bx)

8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 0.7 I
0.6 K 0.6
Citric acid (%)

0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 4.16 Effect of foliar B application on postharvest quality of fruit during storage
at 10°C and relative humidity of >90%: extractable colour (A and B); firmness (C
and D); water content (E and F); TSSC (G and H) and TA (K and I) for cv. Aries
and cv. Papri Queen, respectively in the second trial (higher B concentrations). Fruit
were harvested from plants that received a foliar application of 0, 2 or 7 mM H₃BO₃
from flowering to harvest of red fruit at weekly intervals by hand-held sprayer pump
at an average rate of 200 mL m⁻² (equivalent to 2000 L ha⁻¹). Data are means ± SE
from n = 3. LSDs (P ≤ 0.05) are shown for among individual B concentration for
each time-point during storage

55
When considering grey mould development on fruit derived from young fruit that
had been inoculated preharvest, lesion area on fruit from plants sprayed with 7.0 mM
H₃BO₃ was significantly smaller (P = 0.037) from 18 DAH to 22 DAH than that
from plants sprayed with 2.0 mM H₃BO₃ for cv. Aries (Fig 4.17A). For cv. Papri
Queen, there was no significant difference for lesion area on fruit, regardless of the B
concentration used on plants (Fig 4.17C). The percentage of fruit exhibiting rot from
plants that were sprayed with 7.0 mM H₃BO₃ appeared to be lower than those that
were not sprayed or sprayed with lower B concentration, regardless of cultivar (Fig
4.17B, D).

Time to reach 50% of maximum lesion area on fruit derived from young fruit that
had been inoculated preharvest was not significantly different for cv. Aries,
regardless of B concentration used on plants (Fig 4.18A). In contrast, for cv. Papri
Queen, the time to reach 50% of maximum lesion area on fruit from control plants
was significantly shorter (P = 0.013) than that for fruit from plants sprayed with 2.0
or 7.0 mM H₃BO₃ (Fig 4.18B). However, time to reach 50% of maximum lesion area
was similar on fruit of plants sprayed with either B concentration.

The lesion area on fruit inoculated postharvest was not significantly different,
regardless of B application on plants or cultivar (Fig 4.19A, B).

56
 B
A
9000 0 mM H₃BO₃ 100
8000 2 mM H₃BO₃
80

Fruit exhibing rot (%)

7000
Lesion area (mm²)

7 mM H₃BO₃
6000
60
5000
4000
40
3000
2000
20
1000
0 0
14 16 18 20 22 24 26 28 0 2 7

9000 C D
100
8000 90
7000

Fruit exhibiting rot (%)

80
Lesion area (mm²)

6000 70
5000 60
50
4000
40
3000
30
2000 20
1000 10
0 0
14 16 18 20 22 24 26 28 0 2 7
Foliar boron application
Time during storage (days) (mM H₃BO₃)

Fig. 4.17 Effect of foliar B application on grey mould development and percentage
of fruit exhibiting rot of capsicum cv. Aries (A and B), cv. Papri Queen (C and D)
during storage at 10°C and relative humidity of >90% in the second trial (higher B
concentrations). Fruit were derived from young fruit inoculated with 100 µL
suspension of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri
Queen at 3 DAA when plants received a foliar application 0, 2 or 7 mM H₃BO₃.
Plants were sprayed foliar boron from flowering to harvest of red fruit at weekly
intervals by hand-held sprayer pump at an average rate of 200 mL m⁻² (equivalent to
2000 L ha⁻¹). Data are means ± SE from n = 10. LSDs (P ≤ 0.05) are shown for
among individual boron concentration for each time-point during storage

57
 35
A
30

Time (days) to reach 50% of

maximum lesion area
25

20

15

10

0
0 2 7
35

30
Time (days) to reach 50% of

B
maximum lesion area

25

20

15

10

0
0 2 7
Foliar B application (mM H₃BO₃)

Fig. 4.18 Time after harvest for lesions to reach 50% of maximum size on fruit of
capsicum cv. Aries (A and B) and cv. Papri Queen (C and D) during storage at 10°C
and relative humidity of >90% for 28 days in the second trial (higher B
concentrations) when plants received a foliar application of 0, 2 or 7 mM H₃BO₃.
Plants were sprayed foliar boron from flowering to harvest of red fruit at weekly
intervals by hand-held sprayer pump at an average rate of 200 mL m⁻² (equivalent to
2000 L ha⁻¹). Fruit were derived from young fruit inoculated with 100 µL suspension
of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3
DAA. Data are means ± SE from n = 10. LSDs (P ≤ 0.05) for among individual
boron concentrations at 28 DAH are shown

58
 6000
A

5000
0 mM H₃BO₃
2 mM H₃BO₃

Lesion area (mm²)

4000
7 mM H₃BO₃

3000

2000

1000

0
4 6 8 10 12

6000
B
5000
Lesion area (mm²)

4000

3000

2000

1000

0
4 6 8 10 12
Time post-inoculation (days)

Fig. 4.19 Effect of foliar B application on grey mould development on postharvest-

inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at
10°C and relative humidity of >90% in the second trial (higher B concentrations)
when plants received a foliar application of 0, 2 or 7 mM H₃BO₃. Plants were
sprayed foliar boron from flowering to harvest of red fruit at weekly intervals by
hand-held sprayer pump at an average rate of 200 mL m⁻² (equivalent to 2000 L
ha⁻¹). Fruit were wounded both sides and inoculated postharvest with 40 µL
suspension of 10⁵ conidia mL¯¹. Data are means ± SE from n = 10. LSDs (P ≤ 0.05)
are shown for among individual boron concentrations for each time-point post-
inoculation

59
4.3.3 Negative correlation between lesion area on fruit and boron concentration
on leaf and fruit tissues

The correlation between grey mould development on fruit derived from young fruit
that had been inoculated preharvest and B concentration in leaves or fruit of
capsicum cv. Aries and cv. Papri Queen was determined from 12 individual
measurements of lesion area on fruit at 28 DAH and B concentration in leaves (Fig
4.20A) or in fruit (Fig 4.20B). Data from soil and foliar B application were used to
build the trendline. Higher concentrations of B in the leaves and the fruit were
associated with less severe disease, regardless of cultivar. However, there was only a
significant negative correlation between B concentration in the leaves (R² =0.95) or
in the fruit (R² =0.68) from cv. Aries and grey mould development on fruit.

When grey mould development was assessed on fruit inoculated postharvest, there
was also a negative relationship between grey mould development on fruit and B
concentration in leaf or fruit tissue (Fig 4.21A, B). However, there was only a
significant correlation between B concentration in the fruit (R² =0.85) from cv. Papri
Queen and grey mould development on fruit.

4.4 Discussion

In the present research, shelf life of fruit from B-treated plants from cv. Aries was
longer than that from control plants, but preharvest B application did not affect yield
or postharvest quality of fruit. Boron concentration in leaves and fruit from plants
that received a soil B application was significantly higher than that from plants that
received a foliar B application. Increasing B concentration in plant tissues resulted in
restricting grey mould development on fruit, regardless of whether inoculation
occurred before or after harvest.

60
 A
6000 Aries
Papri Queen
5000

Lesion area (mm²/fruit)

4000 y = -10.742x + 5393.7
R² =0.9488
3000

2000
y = -4.2459x + 1739.7
1000 R² =0.3509

0
0 100 200 300 400
Boron concentration in leaves (mg kg¯¹ DW)

6000
B
5000
Lesion area (mm²/fruit)

y = -196.56x + 6368.9
4000 R² =0.6833

3000

2000
y = -99.729x + 2379
1000 R² =0.3821

0
0 5 10 15 20
Boron concentration in fruit (mg kg¯¹ DW)

Fig. 4.20 The negative correlation between grey mould development on fruit from
latent infection and B concentration in leaves (A) and fruit (B) of capsicum cv. Aries
and cv. Papri Queen. Lesion area at 28 DAH on fruit (at 10°C and relative humidity
of >90%) derived from young fruit inoculated with 100 µL suspension of 10⁴
conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3 DAA.
Young mature leaves and red fruit for nutrient analysis were collected at harvest.
Data was collected from twelve individual measurements of grey mould
development on fruit and B concentration in leaves or fruit in the experiments
involving soil and foliar B application

61
 A
1800 Aries
1600
y = -1.802x + 1401.7 Papri Queen

Lesion area (mm²/fruit)

1400 R² =0.4161
1200
1000
800
600
400
200 y = -2.7584x + 1084.6
R² =0.4865
0
10 110 210 310 410
Boron concentration in leaves (mg kg¯¹ DW)

1800
B

1600
1400
Leasion area (mm²/fruit)

y = -39.56x + 1648.2
1200 R² =0.4192

1000
800
600
400
y = -82.034x + 1695.3
200 R² =0.8492
0
5 10 15 20
Boron concentration in fruit (mg kg¯¹ DW)

Fig. 4.21 The negative correlation between grey mould development on postharvest-
inoculated fruit and B concentration in leaves (A) or fruit (B) of capsicum cv. Aries
and cv. Papri Queen. Lesion area at day 10 on fruit (at 10°C and relative humidity of
>90%) that were inoculated postharvest. Fruit were wounded on opposite sides and
inoculated with 40 µL suspension of 10⁵ conidia mL¯¹. Young mature leaves and red
fruit for nutrient analysis were collected at harvest. Data was collected from 12
individual measurements of grey mould development on fruit and B concentration in
leaves or fruit in the experiments involving soil and foliar B application

62
The shelf life of fruit was related to water loss during storage which depends on
surface area, epicuticular wax content and initial water content of fruit (Lownds et al.
1993; Kissinger et al. 2005). Water loss of pepper fruit has been shown to be
positively correlated with initial water content and surface area, but negatively
correlated with the amount and type of epicuticular wax (Lownds et al. 1993). In the
present research, small fruit and low initial water content in fruit from cv. Papri
Queen prevented water loss during storage. As a result, shelf life of fruit from cv.
Papri Queen was not significantly different among B treatments. In contrast, fruit
from cv. Aries of plants treated with boron generally had a longer shelf life than
those from control plants, particularly when B was applied to the soil during growth
of plants. Fruit from B-treated plants were observed to see less shrivelled during
storage than those from control plants. However, water loss of fruit from B-treated
plants (18.48%) was not significantly different compared with those from control
plants (21.65%) after 23 days of storage. Foliar application of B with the highest rate
of B (300 mg L¯¹) was reported to lower the rate of defect formation in tomato fruit
compared to a control (Huang and Snapp 2004). Boron may possibly decrease the
cuticular micro-cracking on the fruit surface because B is believed to affect
physiology of fruit when B binds apoplastic proteins to the cis-hydroxyl groups of
membranes and cell walls (Dugger 1973) or when B interferes with manganese-
dependent enzymatic reactions (Blevins and Lukaszewski 1998). In the present
research, B-treated fruit may have less cracks and smaller cracks than control fruit.
After harvest, softening in fruit that occurs due to a change of cell-wall carbohydrate
metabolism and TSSC and TA of capsicum fruit increased during storage resulting
from increasing activity of the degrading enzyme PG (Sethu et al. 1996). In this
research, TSSC and TA from plants of the cv. Aries treated with B were generally
lower than those from control plants after storage which means that postharvest
quality of fruit from B-treated plants was better than those from control plants.
Boron sprays before full bloom lowered TSSC in pear fruit cv. Conference than
control plants due to reducing membrane permeability significantly after ripening
(Wojcik and Wojcik 2003). Moreover, pear fruit cv. Conference from B-treated
plants was also reported to have a lower respiration rate at harvest and during storage
than those from control plants (Xuan et al. 2002). Ethylene is known as a ripening

63
hormone that can affect cell wall structure. Increasing ethylene production in fruit
was associated with higher membrane permeability (Brady 1987). Lower ethylene
production may have contributed to reducing membrane permeability in fruit from
B-treated plants although ethylene production by capsicum fruit is low during
storage due to its non-climacteric nature (Pham 2007). Therefore, more research is
necessary to examine the effect of preharvest B application on membrane
permeability of capsicum fruit.

In the present research, preharvest application of B, regardless of whether B was

applied to the soil or as a foliar spray, did not affect extractable colour or firmness of
fruit at harvest or during storage. The results were in agreement with previous
research that showed quality parameters of fruit from control strawberry plants
(Singh et al. 2007) or control pear plants did not differ with fruit from B-treated
plants (Wojcik and Wojcik 2003).

Boron concentration in the leaf, regardless of whether B was applied to the soil or
foliage, was significantly higher than B concentration in fruit. Boron uptake by roots
of plants in the form of boric acid is believed to be a passive process (Brown et al.
2002). After uptake by roots, B is loaded in to the xylem for transporting to the
growing point of stems and leaves, with this process being driven by the
transpiration stream (Brown and Shelp 1997). Thus, nutrients in the xylem are
mainly accumulated in the sites which have the highest transpiration rate (such as
large leaves). In addition, B has been considered to be immobile in the phloem, with
phloem transport being independent of transpiration. Nutrient movement in the
phloem mainly supplies requirements of fruit and seeds, which do not have water
loss (Brown and Shelp 1997). The results from the present research about nutrient
status of capsicum plants confirmed the lack of mobility of B in phloem. In the
present study, preharvest B application as a soil fertiliser was more effective than
foliar B application for increasing B status in leaves and fruit. Similarly, spraying B
before full bloom did not increase leaf and fruit B concentration of ‘Conference’
pear (Wojcik and Wojcik 2003). Boron concentration in leaves and in fruit from the
first trial was significantly higher than that from the second trial. Leaf B

64
concentration from control plants was deficient in the second trial, but was adequate
in the first trial, even though B concentration in the nutrient solution for control
plants was the same in both trials. In addition, foliar spraying with 2.0 and 7.0 mM
H₃BO₃ in the second trial increased leaf B concentration significantly but it was still
significantly lower than leaf B concentration in the first trial, regardless of cultivar.
Many species of plants maintain B concentration in the old leaves, while young
leaves do not receive sufficient B for growth (Tanaka and Fujiwara 2008). In
addition, B leaf concentration is not only related to transpiration rate but also to
uptake rate by roots (Wojcik et al. 2008). The observation that B concentration in
leaves from control plants was adequate in the first trial, but was deficient in the
second trial probably reflects lower B uptake by roots as plants aged.

Boron is an important micronutrient for normal growth, yield and fruit quality.
However, there is a narrow range between B deficiency and toxicity. In this study,
capsicum plants that received a soil application at 0.1 mM H₃BO₃ showed symptoms
of B toxicity in leaves, but this did not affect yield or quality of fruit as evidenced by
the non-significant effect of length, width and weight of fruit as well as the number
of fruit. This result was in agreement with research conducted by Eraslan et al.
(2007) that examined the effect of a range of B concentrations regarded as toxic in
capsicum plants. The fresh weight and dry weight were only significantly reduced at
B concentration of 2483 mg kg¯¹. In the present research, the highest B concentration
(287.50 mg kg¯¹ for cv. Aries and 305 mg kg¯¹ for cv. Papri Queen) was much less
than 2483 mg kg¯¹. In addition, leaf B concentration from control plants in the
second trial of B foliar application (14.98 mg kg¯¹ for cv. Aries and 18.38 mg kg¯¹
for Papri Queen) was deficient, based on the critical value of 20 mg kg¯¹ suggested
by Reuter and Robinson (1997), but no obvious effect of deficiency on yield or
quality of fruit was observed.

Preharvest application of B significantly decreased grey mould development

following inoculation with B. cinerea preharvest and less fruit from B-treated plants
exhibiting rot harvested than those harvested from control plants. The results of the
present research showed a negative correlation between lesion area on fruit and B

65
concentration in leaves and fruit. Boron treatment was reported to also reduce
infection of peaches by Monilinia laxa, which causes brown rot (Thomidis and
Exadaktylou 2010) and to decrease the rate of defects on tomato fruit (Huang and
Snapp 2004). Preharvest foliar application of B reduced blossom end rot, caused by
Ca deficiency, on “Anna” apple fruit significantly (Khalifa et al. 2009). The higher
concentration of B in fruit might maintain cell wall structure and reduce membrane
permeability (Wojcik and Wojcik 2003). Given boron’s role in reducing cuticle
degradation (Blevins and Lukaszewski 1998), cell wall synthesis and maintaining
cell wall structure by cross-linking of cell wall poly-saccharides (Hansch and
Mendel 2009), infection by fungi may therefore be minimised by having adequate
levels of B in the tissues, even though B concentration in the fruit was lower than in
the leaves.

4.5 Conclusion

Preharvest soil application of B could be recommended for capsicum plants as an

environmentally friendly method to reduce grey mould growth on fruit during
storage as well as to improve the shelf life of fruit. Applying B to the soil was more
effective than foliar sprays in terms of increasing B concentration in the leaves and
the fruit at the early stages of plant development. However, foliar application at
appropriate concentrations may be necessary when plants have a larger shoot mass to
ensure B remains in the plant tissues at adequate levels.

66
 CHAPTER FIVE

Effect of preharvest calcium applications

5.1 Introduction

Calcium (Ca) is an essential component of the plant cell wall that produces structural
rigidity by forming cross-bridges between adjacent pectic acids or between pectic
acids and other polysaccharides (Maas 1998; Easterwood 2002). However, Ca
concentrations in fruit are often low because of the immobility of Ca in the phloem
sap (Marschner 1995; White 2001). Calcium deficiency may cause physiological
disorders of fruit including blossom-end rot in tomato and pepper (Geraldson 1957),
splitting and spoilage in cherry (Meheriuk et al. 1991) or increased susceptibility to
Botrytis cinerea (Schwab et al. 1993).

Botrytis cinerea, which causes serious loss in more than 200 crops worldwide
(Williamson et al. 2007), may infect flowers or young developing fruit during
preharvest and remain latent until environmental conditions are suitable for
resumption of growth and ripening causes physiochemical and biochemical changes
in the fruit, especially after harvest, leading to the development of grey mould
(Droby and Lichter 2004). Ensuring that Ca is present at a high enough level in plant
tissue, especially fruit, may therefore be necessary to reduce pathogen infection.
Calcium ions appear to play a key role in the induction of the plant defence
responses (Benhamou 1996). For example, phytoalexins thought to be antifungal, are
produced by cultured soybean cells when treated with Ca2+ in vitro (Stäb and Ebel
1987) while intracellular Ca2+ appears necessary for β-1,3-glucan synthase to form
callose, which re-enforces plant cell walls (Kauss et al. 1989). Moreover, increased
Ca concentration in fruit may enhance cell wall resistance to the action of the
pathogen’s pectolytic enzymes including polygalacturonase (PG) and pectinesterase
(PE) (Preston 1979; Conway et al. 1994a). Therefore, many studies have been
focussed on increasing Ca concentration in the skin and flesh of fruit, including
strawberry (Chéour et al. 1990; Naradisorn et al. 2006), peach (Manganaris et al.
2005; Elmer et al. 2007), sweet cherry (Ippolito et al. 2005) and table grape (Amiri et
al. 2009), to improve postharvest quality of fruit and reduce fungal infection.

67
Fungicides are the most common way of reducing grey mould but alternative
methods are necessary to minimise chemical use in fresh food crops (Sharma et al.
2009). Preharvest Ca application, by soil amendment or foliar spray, is one potential
strategy to improve the quality of fruit and reduce disease incidence in fruit. Calcium
chloride (CaCl₂) is not commonly used in the fruit industry because high chloride ion
concentrations in plant tissues can be toxic to the plant (Xu et al. 1999). Calcium
nitrate [Ca(NO₃)₂] is therefore preferred for use in research as a source of Ca
because it is more soluble than other Ca-containing salts and not toxic at high
concentrations. Calcium applied to soil at concentrations ranging from 1.0 to 8.0 mM
Ca(NO₃)₂ was an effective method to increase Ca in fruit such as tomato (Elad and
Volpin 1993), strawberry (Nam et al. 2006) and sweet pepper (Rubio et al. 2010).
However, high temperature and relative humidity may affect Ca uptake by plants
(Tadesse et al. 2002) and immobility of Ca within the plant may cause inefficient
distribution of Ca in fruit (Conway et al. 1994a). Thus, soil fertilisation with Ca may
not result in a sufficient increase of Ca in fruit. Application of Ca as a foliar spray
may be an alternative way to increase Ca concentration in fruit. Spraying Ca during
fruit development increased Ca in some fresh fruit, such as peach (Manganaris et al.
2005), strawberry (Singh et al. 2007), table grape (Amiri et al. 2009) and apple
(Khalifa et al. 2009). However, the process of Ca penetration in fruit is not well
understood (Serrano et al. 2004). Lenticels, cracks, stomata or surface discontinuities
could allow Ca penetration (Glenn et al. 1985; Christensen 1995).

In previous studies, capsicum plants have been shown to respond positively to

increased Ca concentration, regardless of whether Ca was applied to the soil or as a
foliar spray. Increasing Ca in the root medium from 1.5 to 4 mM increased the
marketable yield of sweet pepper (cv. Orlando) from 1.67 to 2.38 kg plant¯¹ (Rubio
et al. 2010). Weekly foliar sprays with 1% w/v Ca(NO₃)₂ for sweet peppers (cv.
Hungarian Wax) increased Ca concentration in leaves as well as fruit, and were the
most effective method in reducing blossom-end rot (Schon 1993), a physiological
disorder commonly associated with Ca deficiency (Marcelis and Ho 1999).
Increasing Ca in nutrient solution was effective against B. cinerea of sweet paper
plants (Yoon et al. 2010). However, the role of Ca in postharvest quality of capsicum

68
fruit from other cultivars and grey mould development in capsicum has not been
reported. Therefore, the research reported in this chapter aimed to examine the effect
of preharvest soil or foliar Ca application on postharvest quality of fruit and grey
mould development in capsicum fruit from cv. Aries and cv. Papri Queen.

5.2 Methods and materials

5.2.1 Plants and growth conditions

Capsicum plants of cv. Aries and cv. Papri Queen were used in these experiments.
All plants were grown in a greenhouse at the Waite Campus of the University of
Adelaide as per Section 4.2.1.

5.2.2 Nutrient solution for Ca application

5.2.2.1 Soil application

Initially, the basal nutrient solution was modified from Hoagland’s solution
(Hoagland and Arnon 1938) as described by Rubio et al. (2010) to obtain four Ca
treatments: 0.0, 1.5, 4.0 or 8.0 mM Ca (Table 5.1). Calcium nitrate [Ca(NO₃)₂] was
used as the source of Ca. Micronutrient solutions were also prepared and applied to
plants in all the treatments as described in Section 4.2.2.1. Hoagland’s solution (200
mL) in the form of four Ca concentrations, plus micronutrients, was applied
manually to each plant by pouring into the soil every 2 days, while reverse osmosis
(RO) water was used to maintain soil moisture, particularly for hot days during
summer.

A pilot experiment showed that application of nutrient solution containing 0 or 1.5

mM Ca from when seedlings had been transplanted to individual pots was not
appropriate for growth of either cultivar, due to Ca deficiency (Fig 5.1).
Consequently, there were not enough red fruit for subsequent experiments.
Therefore, in the first trial, plants received 4.0 mM Ca in Hoagland’s solution until
the plants had flowers, after which one of the three concentrations (1.5, 4.0 or 8.0
mM Ca) was applied until fruit were harvested at the red stage (100% with low
intensity) (60 - 65 days after anthesis). However, nutrient analysis from the first trial

69
showed that the Ca concentration in leaf and fruit tissues was not significantly
different among the three Ca concentration treatments and all were in the adequate
range (Reuter and Robinson 1997) (Table 5.2). Therefore, a new set of plants was
not used, but based on continued growth of the plants used in the first experiment
Plants received 4.0 mM Ca in Hoagland’s solution until the plants had flowers and
then they were treated with one of the three Ca concentrations: 0.0, 1.5 or 4.0 mM
Ca until fruit were harvested at the red stage.

For each cultivar, there were twenty plants per treatment divided into five replicate
groups. For each group, there were four plants per treatment arranged randomly in
the greenhouse.

5.2.2.2 Foliar application

Plants that were treated with a foliar Ca application continued to be watered with
basal nutrient solution containing no Ca and in which 4.0 Ca(NO₃)₂ was replaced by
6.0 mM NH₄NO₃ (Section 5.2.2.1). Capsicum plants were sprayed from flowering to
harvest of red fruit (five - seven sprays) as described in Section 4.2.2.2 but with
Ca(NO₃)₂. Three Ca concentrations: 0.5, 0.75 or 1.0% w/v Ca(NO₃)₂ were initially
trialled. Results showed that Ca concentration in leaf or fruit tissue was not
significantly different among the three Ca treatments and Ca concentration in leaves
was within the critical range (Table 5.4). Calcium concentrations: control, 0.5 or
1.5% Ca(NO₃)₂ were therefore selected for the second foliar application trial (Table
5.5). However, spraying plants with 1.5% w/v Ca(NO₃)₂ caused leaf necrosis, so Ca
concentration treatment at 1.5% w/v Ca(NO₃)₂ was adjusted to 1.0% w/v Ca(NO₃)₂
after the first spray. For each cultivar, there were twenty plants per treatment divided
into three treatment groups which were arranged randomly in the greenhouse.

70
Table 5.1 Concentration of nutrients (mM) in Hoagland’s solution to form four Ca
treatments which were applied to the soil preharvest

Nutrient source 0.0 mM Ca 1.5 mM Ca 4.0 mM Ca 8.0 mM Ca

KNO₃ 6 5 6 0
Ca(NO₃)₂ 0 1.5 4 7
NH₄NO₃ 6 6 0 0
K₂SO4 0 0 0 3
MgSO₄.7H₂O 1 1 1 1
NaH₂PO4.2H₂O 0 0 0 1
KH₂PO4 1 1 1 0
KCl 0.05 0.05 0.05 0.05
CaCl₂.6H₂O 0 0 0 1

71
5.2.3 Nutrient analysis

Youngest mature leaves (~ 20 g) were randomly collected at harvest from a total of

20 treated plants and three replicate red fruit per treatment for each cultivar were
randomly collected from a total of 20 treated plants for nutrient analysis. Nutrient
analysis was conducted as described in Section 4.2.3.

5.2.4 Inoculation of fruit preharvest or postharvest

5.2.4.1 Isolation, maintenance and culture of B. cinerea

The isolation, maintenance and culture of B. cinerea were as described in Chapter 3.

Production of conidia for use as inoculum (at 10⁴,10⁵, 10⁶ conidia mL¯¹) was as per
Chapter 3.

5.2.4.2 Preharvest inoculation of young developing fruit

Fifteen young developing fruit [3 days after anthesis (DAA)] on multiple flowering
nodes on each plant were randomly selected, tagged and inoculated as described as
in Section 4.2.4.2.

5.2.4.3 Postharvest inoculation of fruit

Ten symptomless, uniform red fruit (100% red with low intensity) were harvested
per treatment for each cultivar from a total of 20 treated plants for postharvest
inoculation as described in Section 4.2.4.3.

5.2.5 Assessment of grey mould development and postharvest quality in fruit

5.2.5.1 Grey mould development on fruit

Assessment of development of grey mould during storage used the protocols

described in Section ‘Assessment of grey mould development’ in Chapter 3. The
fruit were monitored daily and the number of fruit exhibiting rot was recorded. Rot
development was quantified as lesion area on individual fruit by measuring the

72
length and width of decayed areas using digital callipers (digiMax, Switzerland), and
multiplying them.

The correlation between lesion area on fruit and Ca concentration in leaves and fruit
of capsicum cv. Aries and cv. Papri Queen was determined based on 12 individual
measurements of grey mould development on fruit and Ca concentration in leaves
and fruit from experiments involving soil or foliar Ca application. Lesion area on
fruit derived from young developing fruit inoculated preharvest was measured at 28
days after harvest (DAH), while lesion area on fruit inoculated postharvest were
measured at 10 days post-inoculation (DPI).

5.2.5.2 Postharvest quality of fruit

To assess postharvest quality of fruit, fruit were assessed at harvest and then at 5, 10,
15 and 23 days after harvest. Three fruit at each time-point per treatment (a total of
15 fruit) were used to assess shelf life, water content, extractable colour, firmness,
total soluble solid content (TSSC) and titratable acidity (TA) as per Section 4.2.5.1.
Soil application of Ca in both trials and foliar application of Ca in the first trial
showed that extractable colour and TA in fruit were not affected by Ca treatment.
Therefore, extractable colour and TA were not measured in the second trial of foliar
application.

5.2.6 Statistical analysis and photography

Experiments were designed randomly using the factors of cultivar and Ca

concentration. Data were subjected to repeated measurements analysis of variance
(ANOVA) using GenStat and means of the treatments compared using the LSD at a
significance level of ≤0.05. Means and standard errors were determined using
Microsoft Excel. Photography was by a digital camera (Canon D500, Japan).

73
5.3 Results

5.3.1 Symptoms of Ca deficiency in plants treated with low Ca concentration

Symptoms of Ca deficiency such as yellow leaves and dwarfing were observed for
plants that received a soil application of 0.0 or 1.5 mM Ca, regardless of cultivar (Fig
5.1A, B). No mature fruit survived on plants treated with 0.0 mM Ca from transplant
to fruiting. Fruit died at an early stage from 7 to 10 DAA. Most fruit (80 - 85%) also
died and very few fruit survived until red stage when plants were treated with 1.5
mM Ca. Symptoms of Ca deficiency on dying fruit from both cultivars were
observed as likely blossom-end rot (Fig 5.1C, D).

5.3.2 Effect of Ca applied to soil on nutrient status of plant tissues, postharvest

quality and grey mould development on capsicum fruit

5.3.2.1 Soil application of Ca in the first trial (1.5, 4.0 or 8.0 mM Ca)

Regardless of cultivar, Ca concentration in leaf tissues increased with increasing Ca

concentrations applied to the soil (Table 5.2). When plants were treated with 4.0 or
8.0 mM Ca, Ca concentration in the leaves from cv. Aries was greater than that in
leaves from cv. Papri Queen. For cv. Aries, Ca concentration in leaves from plants
treated with 8.0 mM Ca was significantly higher (P<0.001) than that in leaves from
plants treated with the two lower Ca concentrations and Ca concentration in leaves
from plants treated with 4.0 mM Ca was significantly greater than that in leaves from
plants treated with 1.5 mM Ca. For cv. Papri Queen, Ca concentration in leaves from
plants treated with 1.5 mM Ca was significantly lower (P<0.001) than that in leaves
from plants treated with 4.0 or 8.0 mM Ca, which were similar to each other.
However, Ca concentration in fruit, regardless of cultivar, was not significantly
different among plants treated with any of the three Ca concentrations (Table 5.2).
There were no significant differences in other nutrients in leaf or fruit tissues,
regardless of cultivar or Ca concentration (Appendix A.5). However, manganese
concentration in leaves from both cultivars appeared deficient compared to that
considered to be adequate (26 - 100 mg kgˉ¹), regardless of cultivar or Ca treatment
(Reuter and Robinson 1997).

74
 A 0.0 mM 1.5 mM B 0.0 mM 1.5 mM
Ca Ca Ca Ca

C D

Fig. 5.1 Symptoms of Ca deficiency in capsicum plants and fruit from cv. Aries (A,
C) and cv. Papri Queen (B, D) in the pilot experiment. Plants received a soil
application of 0.0 or 1.5 mM Ca from transplant to fruiting (A, B). Fruit from cv.
Aries (C) and cv. Papri Queen (D) when capsicum plants received 1.5 mM Ca from
transplant to fruiting. Representative images are shown

75
Table 5.2 Effect of soil Ca application on nutrient status in capsicum plant tissues in
the first trial. Other nutrients are detailed in table in Appendix A.5. The nutrient
concentrations were determined using ICP-OES analysis. Youngest mature leaves
and red fruit for nutrient analysis were collected at harvest from plants that received
a soil application of 1.5, 4.0 or 8.0 mM Ca in Hoagland’s solution (200 mL per plant
every 2 days) from flowering to harvest of red fruit. Data are presented as means ±
SE from n = 3. For each cultivar, means with the same letters in each column were
not significantly different as determined using the LSD (P<0.05)

Ca concentration
Ca treatment
Cultivar (mg kg¯¹ DW)
[mM Ca(NO₃)₂]
Leaf Fruit
1.5 10250.00 ± 50.00a 993.33 ± 150.26a
4.0 25000.00 ± 0.00b 1120.67 ± 153.67a
Aries
8.0 34000.00 ± 1000.00c 1096.67 ± 8.82a
LSD (P value) 2601.70 (<0.001) (0.75)
1.5 7850.00 ± 550.00a 836.67 ± 92.80a
4.0 20500.00 ± 500.00b 1053.33 ± 48.07a
Papri Queen
8.0 21000.00 ± 0.00b 1226.67 ± 221.69a
LSD (P value) 1931.40 (<0.001) (0.23)

76
Calcium treatment did not significantly affect shelf life of fruit, regardless of cultivar
(Fig 5.2A, B). The shelf life of fruit cv. Aries was similar to those from cv. Papri
Queen, regardless of Ca treatment

Extractable colour in fruit generally increased during storage and was significantly
higher at the end of storage than that in fruit at harvest (Fig 3A, B). For cv. Aries,
application of Ca to the soil did not significantly affect extractable colour of fruit
during storage (Fig 5.3A). In contrast, extractable colour of fruit from cv. Papri
Queen plants treated with 8.0 mM Ca was significantly higher (P = 0.004) at 5 DAH,
but significantly lower at 23 DAH than that in fruit from plants treated with lower Ca
concentrations (Fig 5.3B). At the end of storage, extractable colour in fruit from cv.
Papri Queen was significantly greater (P = 0.01) than that in fruit from cv. Aries.

Firmness of fruit was not significantly affected by soil application of Ca, regardless
of cultivar (Fig 5.3C, D). Firmness of fruit from cv. Aries significantly decreased
(P<0.001) at 5 DAH but significantly increased at 10 DAH and then no change was
observed at 23 DAH. Firmness of fruit from cv. Papri Queen increased significantly
(P<0.001) from 5 DAH to 10 DAH but then remained constant from 15 to 23 DAH.

TSSC in fruit from cv. Aries increased during storage and was significantly higher
(P<0.001) at 23 DAH than that in fruit at harvest (Fig 5.3E). Soil application of Ca
did not affect the TSSC in fruit from cv. Aries at harvest or during storage. In
contrast, soil application of Ca significantly affected TSSC in fruit from cv. Papri
Queen (Fig 5.3F). TSSC in fruit from plants treated with 4.0 or 8.0 mM Ca increased
steadily during storage and was significantly higher (P<0.001) at 23 DAH than that
at harvest. TSSC in fruit from plants treated with 4.0 mM Ca was significantly
higher than that in fruit from plants treated with 8.0 mM Ca at harvest, but was the
same during storage. TSSC in fruit from plants treated with 1.5 mM Ca fluctuated
and was significantly lower (P = 0.011) than that in fruit from plants treated with
higher Ca concentrations at 0 DAH, 10 DAH and 23 DAH. TSSC in fruit from cv.
Papri Queen was significantly greater (P = 0.001) than that in fruit from cv. Aries,
regardless of Ca treatment.

77
 30 A

25

Predicted shelf life (days)

20

15

10

0
1.5 4 8
30
B
25
Predicted shelf life (days)

20

15

10

0
1.5 4 8
Soil Ca application [mM Ca(NO₃)₂]

Fig. 5.2 Effect of soil Ca application on shelf life of fruit from cv. Aries (A) and cv.
Papri Queen (B) during storage at 10°C and relative humidity of >90% in the first
trial. Fruit were harvested from plants treated by watering with 1.5, 4 or 8 mM
Ca(NO₃)₂ in Hoagland’s solution at a rate of 200 mL per plant every 2 days from
flowering to harvest of red fruit. Shelf life of fruit was predicted at a general
appearance (GA) of 5.5 by using a third degree polynomial in GenStat (Section
4.2.4.2). Data are means ± SE from n = 3. LSDs (P ≤ 0.05) for among individual Ca
concentrations are shown

78
TA in fruit from cv. Aries fluctuated during storage, but was significantly higher
(P<0.001) at the end of storage than that in fruit at harvest, regardless of Ca
concentration treatment (Fig 5.3G). For cv. Aries, TA in fruit was not significantly
different at harvest or at the end of storage among Ca concentration treatments.
However, TA in fruit from plants treated with 8.0 mM Ca was significantly higher
(P<0.001) at 10 DAH, but significantly lower at 15 DAH than that in fruit from
plants treated with lower Ca concentrations. At 15 DAH, TA in fruit from plants
treated with 1.5 mM Ca was significantly higher than that in fruit from plants treated
with higher Ca concentrations. For cv. Papri Queen, TA in fruit from plants treated
with 1.5 mM Ca was significantly (P<0.001) lower than that in fruit from plants
treated with higher Ca concentrations from 0 DAH to 10 DAH and 23 DAH (Fig
5.3H). TA in fruit from cv. Papri Queen was significantly greater (P<0.001) than that
in fruit from cv. Aries, regardless of Ca treatment.

When considering grey mould development on fruit derived from young developing
fruit that had been inoculated preharvest, lesion area on fruit from plants treated with
4.0 mM Ca was significantly smaller (P = 0.021) by the end of the experiment than
on fruit from plants treated with 1.5 mM or 8.0 mM Ca for cv. Aries (Fig 5.4A). The
percentage of fruit exhibiting rot from plants that were treated with 4.0 mM Ca
appeared to be lower than those from plants that were treated with 1.5 mM or 8.0
mM Ca (Fig 5.4B). For Papri Queen, lesion area on fruit was not significantly
different among fruit from plants treated with different Ca concentrations (Fig 5.4C).
The percentage of fruit exhibiting rot from plants that were treated with 8.0 mM Ca
appeared to be lower than those from plants that were treated with 1.5 mM or 4.0
mM Ca (Fig 5.4D). Lesions on fruit from cv. Aries were significantly larger than
those on fruit from cv. Papri Queen, regardless of Ca treatment.

Although time to reach 50% of maximum lesion area on fruit appeared to be one day
different, soil application did not delay the time for grey mould to reach 50% of
maximum lesion, regardless of cultivar (Fig 5.5A, B).

The lesion area on fruit inoculated postharvest was not significantly different,
regardless of Ca application to plants or cultivar (Fig 5.6A, B).

79
 350 1.5 mM Ca(NO₃)₂ 350
A B
Extractable colour (ASTA) 300 4 mM Ca(NO₃)₂ 300
250 8 mM Ca(NO₃)₂ 250
200 200
150 150
100 100
50 50
0 0
0 5 10 15 20 25 0 5 10 15 20 25
100 100 D
C
80 80
Firmness (N cm⁻²)

60 60

40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
18 E 18 F
16 16
14 14
12 12
TSSC (⁰Bx)

10 10
8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 0.7
G H
0.6 0.6
0.5
Citric acid (%)

0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after harvest (days) Time after harvest (days)

Fig. 5.3 Effect of soil Ca application on postharvest quality of fruit during storage at
10°C and relative humidity of >90%: extractable colour (A and B); firmness (C and
D); TSS (E and F) and acidity (G and H) for cv. Aries and cv. Papri Queen,
respectively in the first trial. Fruit were harvested from plants treated by watering
with 1.5, 4 or 8 mM Ca(NO₃)₂ in Hoagland’s solution at a rate of 200 mL per plant
every 2 days from flowering to harvest of red fruit. Data are means ± SE from n = 3.
LSDs (P ≤ 0.05) are shown for among individual Ca concentrations for each time-
point during storage

80
 B
A
8000 100
1.5 mM Ca(NO₃)₂
7000 90
80

Fruit exhibiting rot (%)

Lesion area (mm²) 4 mM Ca(NO₃)₂
6000
70
5000 8 mM Ca(NO₃)₂ 60
4000 50
40
3000
30
2000 20
1000 10
0
0
14 16 18 20 22 24 26 28 1.5 4 8

8000 100
C D
7000 90
80

Fruit exhibiting rot (%)

6000
70
Lesion area (mm²)

5000 60
4000 50
40
3000
30
2000 20
1000 10
0
0
1.5 4 8
14 16 18 20 22 24 26 28
Soil Ca application
Time after harvest (days)
[mM Ca(NO₃)₂]

Fig. 5.4 Effect of soil Ca application on grey mould development and the number
of fruit of capsicum cv. Aries (A and B) and cv. Papri Queen (C and D) exhibiting
rot during storage at 10°C and relative humidity of >90% in the first trial. Fruit
were derived from young developing fruit inoculated with 100 µL suspension of
10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3
DAA when plants received a soil application of 1.5, 4 or 8 mM Ca(NO₃)₂ in
Hoagland’s solution at a rate of 200 mL per plant every 2 days from flowering to
harvest red fruit. Data are means ± SE from n = 10. LSDs (P ≤ 0.05) are shown for
among individual Ca concentrations for each time-point during storage

81
 30 A

25

Time (days) to reach 50% of

maximum lesion area
20

15

10

0
1.5 4 8

30 B
Time (days) to reach 50% of

25
maximum lesion area

20

15

10

0
1.5 4 8
Soil Ca application [mM Ca(NO₃)₂]

Fig. 5.5 Time after harvest for grey mould lesions to reach 50% of maximum size
on fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at 10°C
and relative humidity of >90% for 28 days in the first trial. Fruit were derived
from young developing fruit inoculated with 100 µL suspension of 10⁴ conidia
mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3 DAA when
plants received a soil application of 1.5, 4 or 8 mM Ca(NO₃)₂ in Hoagland’s
solution at a rate of 200 mL per plant every 2 days from flowering to harvest of
red fruit. Data are means ± SE from n = 10. LSDs (P ≤ 0.05) for among individual
Ca concentrations at 28 days after harvest are shown

82
 3000
A
2500 1.5 mM Ca(NO₃)₂
4 mM Ca(NO₃)₂

Lesion area (mm²)

2000
8 mM Ca(NO₃)₂
1500

1000

500

0
5 6 7 8
3000 B
2500
Lesion area (mm²)

2000

1500

1000

500

0
5 6 7 8
Time post-inoculation (days)

Fig. 5.6 Effect of soil Ca application on grey mould development on postharvest-

inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage
at 10°C and relative humidity of >90% in the first trial when plants received a soil
application of 1.5, 4 or 8 mM Ca(NO₃)₂ in Hoagland’s solution at a rate of 200
mL per plant every 2 days from flowering to harvest red fruit. Fruit were wounded
on opposite sides and inoculated postharvest with 40 µL suspension of 10⁵ conidia
mL¯¹. Data are means ± SE from n = 10. LSDs (P ≤ 0.05) are shown for among
individual Ca concentrations for each time-point post-inoculation

83
5.3.2.2 Soil application of Ca in the second trial (0.0, 1.5 or 4.0 mM Ca)

Ca concentration in leaf tissues was significantly different among plants treated

with the three Ca concentrations: 0.0, 1.5 or 4.0 mM Ca, from flowering to
harvest of red fruit (Table 5.3). The Ca concentration in leaf tissues from plants
treated with 4.0 mM Ca was significantly greater (P = 0.001) than that from plants
treated with 1.5 mM Ca or control plants and Ca concentration in leaf tissues from
control plants was significantly lower than that from plants treated with 1.5 mM
Ca, regardless of cultivar. Calcium concentration in leaf tissue from control
plants, regardless of cultivar, appeared deficient compared to levels considered to
be adequate (100000 - 37000 mg kg¯¹ for cv. Aries and 4000 - 10000 mg kg¯¹ for
cv. Papri Queen) (Reuter and Robinson 1997). However, symptoms of Ca
deficiency were not observed. Calcium concentration in fruit, regardless of
cultivar, was not significantly different among plants treated with any of the three
Ca concentrations. There were no significant differences in other nutrients in leaf
or fruit tissues, regardless of Ca concentration (Appendix A.6). However,
concentration of copper in leaf tissues from both cultivars appeared deficient
compared to that considered to be adequate (6 - 25 mg kg¯¹) (Reuter and
Robinson 1997).

For cv. Aries, length and width of fruit were not significantly different among
fruit from plants treated with the three Ca concentrations (Fig 5.7A). Weight of
fruit from plants treated with Ca was similar but was significantly heavier (P =
0.02) than those from control plants. For cv. Papri Queen, length, width and
weight were not significantly different among fruit from plants treated with the
three Ca concentrations (Fig 5.7A, B). The number of fruit was similar on plants
treated with different Ca concentrations, regardless of cultivar (data not shown).

The shelf life of fruit was not significantly different, regardless of cultivar (Fig
5.8A, B). The shelf life of fruit from cv. Aries was similar to that for fruit from cv.
Papri Queen.

84
Table 5.3 Effect of soil Ca application on nutrient status in capsicum plant tissues
in the second trial. Other nutrients are detailed in Appendix A.6. The nutrient
concentrations were determined using ICP-OES analysis. Youngest mature leaves
and red fruit for nutrient analysis were collected at harvest from plants that
received a soil application: 0.0, 1.5 or 4.0 mM Ca in Hoagland’s solution (200 mL
per plant every 2 days) from flowering to harvest of red fruit. Data are presented
as means ± SE from n = 3. For each cultivar, means with the same letters in each
column were not significantly different as determined using the LSD (P<0.05)

Ca concentration
Ca treatment
Cultivar (mg kg¯¹ DW)
[mM Ca(NO₃)₂]
Leaf Fruit
Control *9450.00 ± 450.00a 726.67 ± 43.72a
1.5 24500.00 ± 500.00b 903.33 ± 103.33a
Aries
4.0 48000.00 ± 2000.00c 983.33 ± 48.42a
LSD (P value) 5483.00 (<0.001) (0.1)
a
Control *3600.00 ± 1000.00 966.67 ± 179.01a
1.5 18350.00 ± 950.00b 943.33 ± 117.24a
Papri Queen
4.0 28500.00 ± 1500.00c 1456.67 ± 79.65a
LSD (P value) 5295.10 (0.001) (0.056)

*deficient as stated by Reuter and Robinson (1997)

85
 A
180.00 180.00
0 mM Ca(NO₃)₂ 4 mM Ca(NO₃)₂
160.00 160.00
1.5 mM Ca(NO₃)₂
140.00 140.00
120.00 120.00

mm per fruit

g per fruit
100.00 100.00
80.00 80.00
60.00 60.00
40.00 40.00
20.00 20.00
0.00 0.00
length width weight
180.00 180.00
B
160.00 160.00
140.00 140.00
120.00 120.00
mm per fruit

g per fruit
100.00 100.00
80.00 80.00
60.00 60.00
40.00 40.00
20.00 20.00
0.00 0.00
length width weight
Size of fruit

Fig. 5.7 Length, width and weight of fruit from cv. Aries (A) and cv. Papri Queen
(B) when plants received a soil application of 0, 1.5 or 4 mM Ca(NO₃)₂ in the
Hoagland’s solution at a rate of 200 mL per plant every 2 days from transplant to
harvest of red fruit. Data are means ± SE from n = 10

86
 25
A

20

Predicted shelf life (days)

15

10

0
0 1.5 4

25
B

20
Predicted shelf life (days)

15

10

0
0 1.5 4
Soil Ca application [mM Ca(NO₃)₂]

Fig. 5.8 Effect of soil Ca application on shelf life of fruit from cv. Aries (A) and
cv. Papri Queen (B) during storage at 10°C and relative humidity of >90% in the
second trial. Fruit were harvested from plants that received a soil application of 0,
1.5 or 4 mM Ca(NO₃)₂ in Hoagland’s solution at a rate of 200 mL per plant every
2 days from flowering to harvest of red fruit. Shelf life of fruit was predicted at a
general appearance (GA) of 5.5 by using a third degree polynomial in GenStat.
Data are means ± SE from n = 3. LSDs (P ≤ 0.05) for among individual Ca
concentrations are shown

87
Extractable colour in fruit increased steadily during storage and was significantly
higher at the end of storage than that in fruit at harvest, regardless of Ca
concentration treatment or cultivar (Fig 5.9A, B). Soil application of Ca did not
affect extractable colour in fruit at harvest or during storage, regardless of
cultivar. At the end of storage, extractable colour in fruit from cv. Papri Queen
was significantly greater (P = 0.01) than that in fruit from cv. Aries, regardless of
Ca treatment.

Firmness of fruit at harvest or during storage was not affected by soil application
of Ca, regardless of cultivar (Fig 5.9C, D). Firmness of fruit from cv. Aries did
not significantly change during storage, regardless of the Ca concentration applied
to soil. In contrast, firmness of fruit from cv. Papri Queen increased steadily
during storage and was significantly higher at 23 DAH than at harvest.

Water content in fruit decreased steadily during storage and was significantly
lower (P = 0.02) at 23 DAH than that in fruit at harvest, regardless of cultivar or
Ca treatment (Fig 5.9E, F). Soil application of Ca did not affect water content
during storage in both cultivars.

TSSC increased steadily during storage and was significantly higher at 23 DAH
than at harvest, regardless of Ca concentration treatment or cultivar (Fig 5.9G, H).
Soil application of Ca did not affect TSSC in fruit at harvest or during storage,
regardless of cultivar. TSSC in fruit from cv. Papri Queen was significantly
greater (P = 0.01) than that in fruit from cv. Aries, regardless of Ca treatment.

For cv. Aries, TA in fruit from plants treated with 4.0 mM Ca was significantly
lower (P = 0.007) at 10 DAH than that in fruit from plants treated with lower Ca
concentrations (Fig 5.9I). For cv. Papri Queen, TA in fruit increased significantly
(P<0.001) from 5 DAH to 15 DAH but remained constant from 15 to 23 DAH,
regardless of which Ca concentration was applied to soil (Fig 5.9K). TA in fruit
from cv. Papri Queen was significantly greater (P<0.001) than that in fruit from
cv. Aries, regardless of Ca treatment.

88
 350 350
A B

Extractable colour(ASTA)
300 300
250 250
200 200
150 0 mM Ca(NO₃)₂ 150
100 1.5 mM Ca(NO₃)₂ 100
50 4 mM Ca(NO₃)₂ 50
0 0
0 5 10 15 20 25 0 5 10 15 20 25
120 120 D
Firmness (N cm⁻²)

100 C 100
80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
80 E 80
Water content (%)

F
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
16 16
14 14 H
12 12
G
TSSC (°Bx)

10 10
8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 0.7
0.6 0.6
Citric acid (%)

0.5 I 0.5 K
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 5.9 Effect of preharvest soil Ca application on postharvest quality of fruit

during storage at 10°C and relative humidity of >90%: extractable colour (A and
B); firmness (C and D); water content (E and F); TSSC (G and H) and TA (I and
K) for cv. Aries and cv. Papri Queen, respectively in the second trial. Fruit were
harvested from plants that received a soil application of 0, 1.5 or 4 mM Ca(NO₃)₂
in Hoagland’s solution at a rate of 200 mL per plant every 2 days from flowering
to harvest of red fruit. Data are means ± SE from n = 3. LSDs (P ≤ 0.05) are
shown for among individual Ca concentrations for each time-point during storage

89
When considering grey mould development on fruit derived from young
developing fruit that had been inoculated preharvest, for cv. Aries, lesion area on
fruit from plants treated with 4.0 mM Ca via soil application was significantly
smaller (P = 0.02) than lesion area on fruit from plants treated with 1.5 mM Ca at
18 DAH and lesion area on fruit from control plants at 20 DAH (Fig 5.10A).
Lesion area on fruit from plants treated with 1.5 mM Ca application to the soil
was similar to lesion area on fruit from control plants. For cv. Papri Queen, lesion
area on fruit from plants treated with the 4.0 mM Ca soil application was
significantly smaller (P = 0.01) than those on fruit from control plants at 22 DAH
and from 20 DAH to 24 DAH for plants treated with 1.5 mM Ca (Fig 5.10C). The
percentage of fruit exhibiting rot from plants that were treated with 4.0 mM Ca
appeared to be lower than control plants or those that were treated with the lower
Ca concentration, regardless of cultivar (Fig 5.10B, D).

Time to reach 50% of maximum lesion area on fruit derived from young fruit that
had been inoculated preharvest was not significantly different among the three Ca
treatments, regardless of cultivar (Fig 5.11).

The lesion area on fruit inoculated postharvest was not significantly different for
cv. Aries, regardless of Ca application to plants (Fig 5.12A). For cv. Papri Queen,
lesion area on fruit from control plants was significantly larger (P<0.001) than
that on fruit from plants treated with Ca from 8 DPI to 10 DPI (Fig 5.12B). Lesion
area on fruit from plants treated with 1.5 mM Ca was also significantly larger
(P<0.001) than that on fruit from plants treated with 4.0 mM Ca at 10 DPI.

90
 9000 A 100 B
8000 0 mM Ca(NO₃)₂ 90

7000 1.5 mM Ca(NO₃)₂ 80

Fruit exhibiting rot (%)

70
6000 4 mM Ca(NO₃)₂
Lesion (mm²)

60
5000
50
4000
40
3000
30
2000 20
1000 10
0 0
14 16 18 20 22 24 26 28 0 1.5 4

9000 100 D
C
8000 90
7000 80
Fruit exhibiting rot (%) 70
6000
Lesion (mm²)

60
5000
50
4000
40
3000
30
2000 20
1000 10
0 0
14 16 18 20 22 24 26 28 0 1.5 4
Time after harvest (days) Soil Ca application
[mM Ca(NO₃)₂]

Fig. 5.10 Effect of soil Ca application on grey mould development and the
number of fruit of capsicum cv. Aries (A and B) and cv. Papri Queen (C and D)
exhibiting rot during storage at 10°C and relative humidity of >90% in the second
trial. Fruit were derived from young developing fruit inoculated with 100 µL
suspension of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri
Queen at 3 DAA when plants received a soil application of 0, 1.5 or 4 mM
Ca(NO₃)₂ in Hoagland’s solution at a rate of 200 mL per plant every 2 days from
flowering to harvest red fruit. Data are means ± SE from n = 10. LSDs (P ≤ 0.05)
are shown for among individual Ca concentrations for each time-point during
storage

91
 30 A

Time (days) to reach 50% of maximum

25

20

lesion area
15

10

0
0 1.5 4

30 B
Time (days) to reach 50% of maximum

25

20
lesion area

15

10

0
0 1.5 4
Soil Ca application [mM Ca(NO₃)₂]

Fig. 5.11 Time after harvest for grey mould lesions to reach 50% of maximum
size on fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at
10°C and relative humidity of >90% for 28 days in the second trial. Fruit were
derived from young fruit inoculated with 100 µL suspension of 10⁴ conidia mL¯¹
for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3 DAA when plants
received a soil application of 0, 1.5 or 4 mM Ca(NO₃)₂ in Hoagland’s solution at a
rate of 200 mL per plant every 2 days from flowering to harvest red fruit. Data are
means ± SE from n = 10. LSDs (P ≤ 0.05) for among individual Ca concentrations
at 28 days after harvest are shown

92
 8000 A
7000 0 mM Ca(NO₃)₂
6000 1.5 mM Ca(NO₃)₂

Lesion area (mm²)

5000 4 mM Ca(NO₃)₂
4000
3000
2000
1000
0
4 5 6 7 8 10 12
8000
7000
B

6000
Lesion area (mm²)

5000
4000
3000
2000
1000
0
4 5 6 7 8 10 12
Time post-inoculation (days)

Fig. 5.12 Effect of soil Ca application on grey mould development on postharvest-

inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage
at 10°C and relative humidity of >90% in the second trial when plants received a
soil application of 0, 1.5 or 4 mM Ca(NO₃)₂ in Hoagland’s solution at a rate of
200 mL per plant every 2 days from flowering to harvest red fruit. Fruit were
wounded on opposite sides and inoculated with 40 µL suspension of 10⁵ conidia
mL¯¹. Data are means ± SE from n = 10. LSDs (P ≤ 0.05) are shown for among
individual Ca concentrations for each time-point post-inoculation

93
5.3.3 Effect of Ca as a foliar application on nutrient status of plant tissues,
postharvest quality and grey mould development on capsicum fruit

5.3.3.1 Foliar application of Ca in the first trial [0.5, 0.75 or 1% Ca(NO₃)₂]

Ca concentration in leaf and fruit tissues was statistically similar among plants
sprayed with the three Ca concentrations: 0.5, 0.75 or 1.0% w/v Ca(NO₃)₂,
regardless of cultivar (Table 5.4). There were no significant differences in other
nutrients in leaf or fruit tissue, regardless of cultivar or Ca concentration
(Appendix A.7). No symptoms of Ca deficiency were observed and they appeared
all the same (data not shown). However, leaf concentration of copper (6.15 - 8.50
mg kg¯¹) and zinc (14.23 - 20.00) from cv. Papri Queen, regardless of Ca
concentration applied, appeared to be lower than the range considered adequate (8
- 15 mg kg¯¹ for copper and 20 - 60 mg kg¯¹ for zinc, respectively) (Reuter and
Robinson 1997).

The shelf life of fruit was not significantly different among fruit from plants
sprayed with the three Ca concentrations, regardless of cultivar (Fig 5.13A, B).

Extractable colour in fruit from cv. Aries was not significantly different among
the three Ca applications at harvest or during storage. Extractable colour
significantly increased (P<0.001) at 10 DAH then remained constant to the end of
storage, regardless of Ca application (Fig 5.14A, B). For cv. Papri Queen,
extractable colour in fruit from plants sprayed with 1.0 % w/v Ca(NO₃)₂ was
significantly lower (P = 0.032) than that from plants sprayed with 0.5% w/v
Ca(NO₃)₂ at 23 DAH. At the end of storage, extractable colour in fruit from cv.
Papri Queen was significantly greater (P = 0.01) than that in fruit from cv. Aries,
regardless of Ca treatment.

94
Table 5.4 Effect of foliar Ca application on nutrient status in capsicum plant
tissues in the first trial. Other nutrients are detailed in Appendix A.7. The nutrient
concentrations were determined using ICP-OES analysis. Youngest mature leaves
and red fruit for nutrient analysis were collected at harvest from plants that
received a foliar application: 0.5, 0.75 or 1.0% w/v Ca(NO₃)₂ from flowering to
harvest of red fruit. Plants were watered with Hoagland’s solution without Ca
(200 mL per plant for every 2 days). Data are presented as means ± SE from n =
3. There were no significant difference between Ca treatments within tissue types
(P values are shown)

Ca concentration
Ca treatment
Cultivar (mg kg¯¹ DW)
[% w/v Ca(NO₃)₂]
Leaf Fruit
0.5 16050.00 ± 1150.00 700.00 ± 90.74
0.75 17750.00 ± 2250.00 753.33 ± 89.69
Aries
1.0 22500.00 ± 1500.00 613.33 ± 44.10
P value 0.15 0.48
0.5 12450.00 ± 1550.00 586.67 ± 33.33
0.75 16450.00 ± 1650.00 446.67 ± 38.44
Papri Queen
1.0 17100.00 ± 1100.00 593.33 ± 35.28
P value 0.19 0.16

95
 30
A
25

Predicted shelf life (days)

20

15

10

0
0.5 0.75 1

30
B
25
Predicted shelf life (days)

20

15

10

0
0.5 0.75 1
Foliar Ca application [% w/v Ca(NO₃)₂]

Fig. 5.13 Effect of foliar Ca application on shelf life of fruit of capsicum cv. Aries
(A) and cv. Papri Queen (B) during storage at 10°C and relative humidity of
>90% in the first trial. Fruit were harvested from plants that received a foliar
application of 0.5, 0.75 or 1% w/v Ca(NO₃)₂. Plants were sprayed from flowering
to harvest of red fruit at weekly intervals by hand-held sprayer pump at an average
rate of 200 mL m¯² (equivalent to 2000 L ha¯¹). Shelf life of fruit was predicted at
a general appearance (GA) of 5.5 by using a third degree polynomial in GenStat.
Data are means ± SE from n = 10. LSDs (P ≤ 0.05) for among individual Ca
concentrations are shown

96
Firmness of fruit during storage was not affected by foliar application of Ca,
regardless of cultivar (Fig 5.14C, D). For cv. Papri Queen, firmness of fruit
increased during storage and was significantly higher (P<0.001) at 23 DAH than
that at harvest.

TSSC in fruit was not significantly different among the three Ca concentrations
applied as a foliar spray, regardless of cultivar (Fig 5.14E, F). For cv. Aries, TSSC
in fruit significantly (P = 0.002) decreased at 23 DAH compared to harvest, while
TSSC in fruit from cv. Papri Queen increased steadily during storage and was
significantly higher (P<0.001) at 23 DAH than at harvest, regardless of Ca
concentration. TSSC in fruit from cv. Papri Queen was significantly greater (P =
0.001) than that in fruit from cv. Aries, regardless of Ca treatment.

For cv. Aries, TA in fruit was also not significantly different among the three Ca
concentrations applied as a foliar spray (Fig 5.14G). TA in fruit significantly
increased (P<0.001) at 5 DAH then decreased steadily to 23 DAH. For cv. Papri
Queen, TA in fruit from plants sprayed with 0.5% w/v Ca(NO₃)₂ was significantly
higher (P<0.001) than that in fruit from plants sprayed with higher Ca
concentrations at 5 DAH (Fig 5.14H). At 15 DAH, TA in fruit from plants
sprayed with 1.0% w/v Ca(NO₃)₂ was significantly lower than that in fruit from
plants sprayed with lower Ca concentrations. At 23 DAH, TA in fruit from plants
sprayed with 0.75% was significantly lower than that from plants sprayed with
0.5% or 1.0% w/v Ca(NO₃)₂. TA in fruit from cv. Papri Queen was significantly
greater (P<0.001) than that in fruit from cv. Aries, regardless of Ca treatment.

97
 350 350
A 0.5% Ca(NO₃)₂ B
Extractable colour (ASTA)
300 300
0.75% Ca(NO₃)₂
250 250
1% Ca(NO₃)₂
200 200
150 150
100 100
50 50
0 0
0 5 10 15 20 25 0 10 20 30
120 120
C D
100 100
Firmness (N cm⁻²)

80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
16 16
14 E 14 F
12 12
TSSC (°Bx)

10 10
8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 0.7
0.6 G H
0.6
0.5
Citric acid (%)

0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 5.14 Effect of foliar Ca application on postharvest quality of fruit during

storage at 10°C and relative humidity of >90%: extractable colour (A and B);
firmness (C and D); TSS (E and F) and acidity (G and H) for cv. Aries and cv.
Papri Queen, respectively in the first trial. Fruit were harvested from plants that
received a foliar application of 0.5, 0.75 or 1% w/v Ca(NO₃)₂ from flowering to
harvest of red fruit at weekly intervals by hand-held sprayer pump at an average
rate of 200 mL m¯² (equivalent to 2000 L ha¯¹). Data are means ± SE from n = 3.
LSDs (P ≤ 0.05) are shown for among individual Ca concentrations for each time-
point during storage

98
When considering grey mould development on fruit derived from young fruit that
had been inoculated preharvest, lesion area on fruit from plants sprayed with
0.75% w/v Ca(NO₃)₂ was significantly smaller (P = 0.001) from 24 DAH to 28
DAH than that on fruit from plants sprayed with 0.5 or 1.0% w/v Ca(NO₃)₂ for cv.
Aries (Fig 5.15A). Lesion area was similar on fruit from plants sprayed with 0.5
and 1.0% w/v Ca(NO₃)₂. The percentage of fruit exhibiting rot from plants
sprayed with 0.75% w/v Ca(NO₃)₂ appeared to be lower than those from plants
sprayed with 0.5 or 1.0% w/v Ca(NO₃)₂ (Fig 5.15B). Foliar application of Ca did
not significantly affect lesion area on fruit from cv. Papri Queen (Fig 5.15C). The
percentage of fruit exhibiting rot appeared to be similar for plants sprayed with
any of the three Ca concentrations (Fig 5.15D). Lesion area on fruit from cv.
Aries was significantly larger (P = 0.001) than lesion area on fruit from cv. Papri
Queen, regardless of Ca concentration applied.

Time to reach 50% of maximum lesion area was not significantly different for
fruit from plants sprayed with any of the three Ca concentrations, regardless of
cultivar (Fig 5.16A, B).

The lesion area on fruit inoculated postharvest was not significantly different
during storage, regardless of Ca concentration and whether applied as a foliar
spray or cultivar (Fig 5.17A, B).

99
 8000 B
A 100
7000 0.5% Ca(NO₃)₂ 90
6000 80

Fruit exhibiting rot (%)

0.75% Ca(NO₃)₂
Lesion area (mm²)

70
5000 1% Ca(NO₃)₂
60
4000 50
3000 40
30
2000
20
1000 10
0 0
14 16 18 20 22 24 26 28 0.5 0.75 1
8000 100
7000 90 D

Fruit exhibiting rot (%)

C 80
6000
Lesion area (mm²)

70
5000 60
4000 50
3000 40
30
2000
20
1000 10
0 0
14 16 18 20 22 24 26 28 0.5 0.75 1
Time after harvest (days) Foliar Ca application
[% w/v Ca(NO₃)₂]

Fig. 5.15 Effect of foliar Ca application on grey mould development and

percentage of fruit exhibiting rot of capsicum cv. Aries (A and B), cv. Papri
Queen (C and D) during storage at 10°C and relative humidity of >90% in the first
trial. Fruit were derived from young developing fruit inoculated with 100 µL
suspension of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri
Queen at 3 DAA when plants received a foliar application of 0.5, 0.75 or 1% w/v
Ca(NO₃)₂. Plants were sprayed with foliar Ca from flowering to harvest of red
fruit at weekly intervals by hand-held sprayer pump at an average rate of 200 mL
m¯² (equivalent to 2000 L ha¯¹). Data are means ± SE from n = 10. LSDs (P ≤
0.05) are shown for among individual Ca concentrations for each time-point
during storage

100
 30 A

Time (days) to reach 50% maximum

25

20

lesion area
15

10

0
0.5 0.75 1

30 B
Time (days) to reach 50% maximum

25

20
lesion aera

15

10

0
0.5 0.75 1
Foliar Ca application [% w/v Ca(NO₃)₂]

Fig. 5.16 Time after harvest for grey mould lesions to reach 50% of maximum
size on fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at
10°C and relative humidity of >90% for 28 days in the first trial. Fruit were
derived from young fruit inoculated with 100 µL suspension of 10⁴ conidia mL¯¹
for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3 DAA when plants
received a foliar application of 0.5, 0.75 or 1% w/v Ca(NO₃)₂. Plants were
sprayed with foliar Ca from flowering to harvest at weekly intervals by hand-held
sprayer pump at an average rate of 200 mL m¯² (equivalent to 2000 L ha¯¹). Data
are means ± SE from n = 10. LSDs (P ≤ 0.05) for among individual Ca
concentrations at 28 days after harvest are shown

101
 2500 A

2000 0.5% Ca(NO₃)₂

0.75% Ca(NO₃)₂

Lesion area (mm²)

1500 1% Ca(NO₃)₂

1000

500

0
5 6 7 8
2500
B

2000
Lesion area (mm²)

1500

1000

500

0
5 6 7 8
Time post inoculation (days)

Fig. 5.17 Effect of foliar Ca application on grey mould development on

postharvest-inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B)
during storage at 10°C and relative humidity of >90% in the first trial when plants
received a foliar application of 0.5, 0.75 or 1% w/v Ca(NO₃)₂. Plants were
sprayed with foliar Ca from flowering to harvest of red fruit at weekly intervals by
hand-held sprayer pump at an average rate of 200 mL m¯² (equivalent to 2000 L
ha¯¹). Fruit were wounded on opposite sides and inoculated postharvest with 40
µL suspension of 10⁵ conidia mL¯¹. Data are means ± SE from n = 10. LSDs (P ≤
0.05) are shown for among individual Ca concentrations for each time-point post-
inoculation

102
5.3.3.2 Foliar application of calcium in the second trial [0.0, 0.5 or 1% w/v
Ca(NO₃)₂]

Ca concentration in leaf tissues was significantly different among plants sprayed

with three Ca concentrations: 0.0, 0.5 or 1.0% w/v Ca(NO₃)₂ from flowering to
harvest of red fruit (Table 5.5). Calcium concentration in fruit, regardless of
cultivar, was not significantly different among plants sprayed with any of the
three Ca concentrations.

Regardless of cultivar, Ca concentration in leaf tissues from plants sprayed with

1.0% w/v Ca(NO₃)₂ was significantly greater (P<0.001) than that in leaf tissues
from plants sprayed with 0.5% w/v Ca(NO₃)₂ or control plants. Calcium
concentration in leaf tissues from control plants was significantly lower than that
in leaf tissues from plants sprayed with 0.5% w/v Ca(NO₃)₂. Calcium
concentration in leaf tissue from control plants, regardless of cultivar, appeared
deficient compared to that considered to be adequate (100000 - 37000 mg kg¯¹ for
cv. Aries and 4000 – 10000 mg kg¯¹ for cv. Papri Queen) (Reuter and Robinson
1997). However, symptoms of Ca deficiency on leaves and fruit were not
observed. There were no significant differences in other nutrients in leaf or fruit
tissues, regardless of cultivar or Ca concentration (Appendix 5.5). However,
concentration of copper in leaf tissues from both cultivars appeared deficient
compared to that considered to be adequate (6 - 25 mg kg¯¹) (Reuter and
Robinson 1997).

The shelf life of fruit was not significantly different among the three Ca
applications, regardless of cultivar (Fig 5.18A, B).

103
Table 5.5 Effect of foliar Ca application on nutrient status in capsicum plant
tissues in the second trial. Other nutrients are detailed in Appendix A.8. The
nutrient concentrations were determined using ICP-OES analysis. Youngest
mature leaves and red fruit for nutrient analysis were collected at harvest from
plants that received a foliar application: 0.0, 0.5 or 1.0% w/v Ca(NO₃)₂ from
flowering to harvest of red fruit. Plants were watered with Hoagland’s solution
without Ca (200 mL per plant for every 2 days). Data are presented as means ± SE
from n = 3. For each cultivar, means with the same letters in each column were
not significantly different as determined using the LSD (P<0.05)

Ca concentration
Ca treatment
Cultivar (mg kg¯¹ DW)
[% w/v Ca(NO₃)₂]
Leaf Fruit
Control *5150.00 ± 50.00a 485.00 ± 25.00a
0.5 12600.00 ± 200.00b 470.00 ± 70.00a
Aries
1.0 19250.00 ± 50.00c 640.00 ± 80.00a
LSD (P value) 551.20 (<0.001) (0.26)
Control *3550.00 ± 50.00a 485 ± 45.00a
0.5 8350.00 ± 50.00b 675.00 ± 35.00a
Papri Queen
1.0 13250.00 ± 250.00c 785.00 ± 2.5.00a
LSD (P value) 373.20 (<0.001) (0.35)

*deficient as stated by Reuter and Robinson (1997)

104
 25 A

20

Predicted shelf life (days)

15

10

0
0 0.5 1

25 B

20
Predicted shelf life (days)

15

10

0
0 0.5 1
Foliar Ca application [% w/v Ca(NO₃)₂]

Fig. 5.18 Effect of foliar Ca application on shelf life of fruit of capsicum cv. Aries
(A) and cv. Papri Queen (B) during storage at 10°C and relative humidity of
>90% in the second trial. Fruit were harvested from plants that received a foliar
application of 0, 0.5 or 1% w/v Ca(NO₃)₂ in the second trial. Plants were sprayed
from flowering to harvest of red fruit at weekly intervals by hand-held sprayer
pump at an average rate of 200 mL m¯² (equivalent to 2000 L ha¯¹). Shelf life of
fruit was predicted at a general appearance (GA) of 5.5 by using a third degree
polynomial in GenStat. Data are means ± SE from n = 10. LSDs (P ≤ 0.05) for
among individual Ca concentrations are shown

105
Firmness of fruit during storage was not affected by Ca application as a foliar
spray, regardless of cultivar (Fig 5.19A, B). For cv. Aries, firmness of fruit from
plants sprayed with 1.0% w/v Ca(NO₃)₂ remained constant from 0 DAH to 10
DAH, while firmness of fruit from control plants or plants sprayed with 0.5% w/v
Ca(NO₃)₂ significantly decreased (P<0.001) at 5 DAH to 10 DAH compared to
that at harvest. Firmness of fruit then significantly increased at 15 DAH then
decreased at 23 DAH, regardless of Ca concentration applied to plants. For cv.
Papri Queen, firmness of fruit remained constant from 0 DAH to 15 DAH but
then significantly increased at 23 DAH, regardless of Ca concentration.

TSSC in fruit was not significantly different among the three Ca concentrations
applied as a foliar spray, regardless of cultivar (Fig 5.19C, D). For cv. Aries,
TSSC in fruit significantly (P<0.001) decreased by 10 DAH compared to at
harvest but significantly increased at 15 DAH and remained constant at 23 DAH,
regardless of Ca concentration. For cv. Papri Queen, TSSC in fruit remained
constant during storage, regardless of Ca concentration. TSSC in fruit from cv.
Papri Queen was significantly greater (P<0.001) than that in fruit from cv. Aries,
regardless of Ca treatment.

When considering grey mould development on fruit derived from young

developing fruit that had been inoculated preharvest, for cv. Aries, lesion area on
fruit from plants sprayed with 0.5 or 1.0% w/v Ca(NO₃)₂ was significantly smaller
(P<0.001) than that on fruit from control plants from 22 DAH to 28 DAH (Fig
5.20A). Lesion area on fruit from plants sprayed with 0.5% w/v Ca(NO₃)₂ was
significantly larger than that on fruit from plants sprayed with 1.0% w/v Ca(NO₃)₂
from 26 DAH to 23 DAH. However, foliar application of Ca did not significantly
affect lesion area on fruit from cv. Papri Queen (Fig 5.20C). The percentage of
fruit exhibiting rot from plants that were sprayed with 1.0% w/v Ca(NO₃)₂
appeared to be lower than those that were not sprayed or sprayed with lower Ca
concentration, regardless of cultivar (Fig 5.20B, D).

106
 140 A 0% Ca(NO₃)₂ 140 B
120 0.5% Ca(NO₃)₂ 120
100 1% Ca(NO₃)₂
Firmness (N cm⁻²)
100
80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25

18 18 D
C
16 16
14 14
12 12
TSSC (°Bx)

10 10
8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 5.19 Effect of foliar Ca application on postharvest quality of fruit during

storage at 10°C and relative humidity of >90%: firmness (A and B); TSSC (C and
D) for cv. Aries and cv. Papri Queen, respectively in the second trial. Fruit were
harvested from plants that received a foliar application of 0, 0.5 or 1% w/v
Ca(NO₃)₂ from flowering to harvest of red fruit at weekly intervals by hand-held
sprayer pump at an average rate of 200 mL m¯² (equivalent to 2000 L ha¯¹). Data
are means ± SE from n = 3. LSDs (P ≤ 0.05) are shown for among individual Ca
concentration for each time-point during storage

107
Foliar sprays of Ca delayed the time for grey mould to reach 50% of maximum
lesion area on fruit compared to control for cv. Aries (Fig 5.21A), but did not
affect time to reach 50% of maximum lesion area on fruit from cv. Papri Queen
(Fig 5.21B).

The lesion area on fruit inoculated postharvest was not statistically significant
during storage, regardless of cultivar or Ca concentration applied as a spray (Fig
5.22A, B).

5.3.4 Correlation between lesion area on fruit and calcium concentration on

leaf and fruit tissues

The correlation between grey mould development on fruit inoculated preharvest

and Ca concentration in leaves or fruit from cv. Aries and cv. Papri Queen was
determined from 12 individual measurements of lesion area on fruit at 28 DAH
and Ca concentration in leaves (Fig 5.23A) or in fruit (Fig 5.23B). Data from soil
and foliar application of Ca were used to build the trend line. The correlation was
not significant between Ca concentration in the leaves or in the fruit and lesion
area on fruit, regardless of cultivar.

When grey mould development was assessed at 10 DPI on fruit inoculated

postharvest, there was no significant correlation between Ca concentration in
leaves and lesion area on fruit from cv. Aries or from cv. Papri Queen (Fig 24A).
Similarly, higher Ca concentration in fruit was not significantly correlated with
smaller lesion area on fruit from cv. Aries or from cv. Papri Queen (Fig 24B).

108
 A B
8000 0% Ca(NO₃)₂ 100

7000 0.5% Ca(NO₃)₂ 90

80

Fruit exhibiting rot (%)

6000 1% Ca(NO₃)₂
70
Lesion area (mm²)

5000 60
4000 50
40
3000
30
2000 20
1000 10
0
0
0 0.5 1
14 16 18 20 22 24 26 28
8000 100 D
C
90
7000
80

Fruit exhibiting rot (%)

6000
70
Lesion area (mm²)

5000 60
4000 50

3000 40
30
2000
20
1000 10
0 0
14 16 18 20 22 24 26 28 0 0.5 1
Time after harvest (days) Foliar Ca application
[% w/v Ca(NO₃)₂]

Fig. 5.20 Effect of foliar Ca application on grey mould development and

percentage of fruit exhibiting rot of capsicum cv. Aries (A and B), cv. Papri
Queen (C and D) during storage at 10°C and relative humidity of >90% in the
second trial. Fruit were derived from young developing fruit inoculated with 100
µL suspension of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶ conidia mL¯¹ for cv.
Papri Queen at 3 DAA when plants received a foliar application of 0, 0.5 or 1%
w/v Ca(NO₃)₂. Plants were sprayed with foliar Ca from flowering to harvest of red
fruit at weekly intervals by hand-held sprayer pump at an average rate of 200 mL
m¯² (equivalent to 2000 L ha¯¹). Data are means ± SE from n = 10. LSDs (P ≤
0.05) are shown for among individual Ca concentration for each time-point during
storage

109
 30
A
25

Time (days) to reach 50% of

maximum lesion area
20

15

10

0
0 0.5 1

30 B
Time (days) to reach 50% of

25
maximum lesion area

20

15

10

0
0 0.5 1
Foliar Ca application [% w/v Ca(NO₃)₂]

Fig. 5.21 Time after harvest for grey mould lesions to reach 50% of maximum
size on fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at
10°C and relative humidity of >90% for 28 days in the second trial when plants
received a foliar application of 0, 0.5 or 1% w/v Ca(NO₃)₂. Plants were sprayed
with foliar Ca from flowering to harvest at weekly intervals by hand-held sprayer
pump at an average rate of 200 mL m¯² (equivalent to 2000 L ha¯¹). Fruit were
derived from young fruit inoculated with 100 µL suspension of 10⁴ conidia mL¯¹
for cv. Aries and 10⁶ conidia mL¯¹ for cv. Papri Queen at 3 DAA. Data are means
± SE from n = 10. LSDs (P ≤ 0.05) for among individual Ca concentrations at 28
days after harvest are shown

110
 6000 A
0% Ca(NO₃)₂
5000
0.5% Ca(NO₃)₂
1% Ca(NO₃)₂

Lesion area (mm²)

4000

3000

2000

1000

0
4 6 8 10 12
6000
B
5000
Lesion area (mm²)

4000

3000

2000

1000

0
4 6 8 10 12
Time post inoculation (days)

Fig. 5.22 Effect of foliar Ca application on grey mould development on

postharvest-inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B)
during storage at 10°C and relative humidity of >90% in the second trial when
plants received a foliar application of 0, 0.5 or 1% w/v Ca(NO₃)₂. Plants were
sprayed foliar Ca from flowering to harvest of red fruit at weekly intervals by
hand-held sprayer pump at an average rate of 200 mL m¯² (equivalent to 2000 L
ha¯¹). Fruit were wounded on opposite sides and inoculated postharvest with 40
µL suspension of 10⁵ conidia mL¯¹. Data are means ± SE from n = 10. LSDs (P ≤
0.05) are shown for among individual Ca concentrations for each time-point post-
inoculation

111
 6000 A Aries Papri Queen

5000

Lesion area (mm²)

4000

3000
y = -0.0544x + 3990.3
R² =0.1283
2000

1000
y = -0.01x + 1248.8
R² =0.0117
0
0 10000 20000 30000 40000 50000
Calcium concentration in leaves (mg kg¯¹ DW)
6000
B
5000

4000 y = -2.0854x + 4530.9

Lesion area (mm²)

R² =0.0686
3000

2000
y = 0.4047x + 767.27
1000 R² =0.0319

0
0 300 600 900 1200 1500
Calcium concentration in fruit (mg kg¯¹ DW)

Fig. 5.23 The correlation between grey mould development on fruit inoculated
preharvest and Ca concentration in leaves (A) and in fruit (B) of capsicum cv.
Aries and cv. Papri Queen. Lesion area was determined at 28 DAH on fruit (at
10°C and relative humidity of >90%) derived from young developing fruit
inoculated with 100 µL suspension of 10⁴ conidia mL¯¹ for cv. Aries and 10⁶
conidia mL¯¹ for cv. Papri Queen at 3 DAA. Data was collected from twelve
individual measurements of grey mould development on fruit and Ca
concentration in leaves or fruit in the experiments involving soil and foliar
application of Ca

112
 1800 Aries Papri Queen
A
1600

1400
y = -0.0036x + 1258.9
R² =0.02
Lesion area (mm²)
1200

1000

800

600

400
y = -0.0164x + 793.32
200 R² =0.4853
0
0 10000 20000 30000 40000 50000
Calcium concentration in leaves (mg kg¯¹ DW)
1800 B
1600
1400
y = 0.3466x + 910.82
1200 R² =0.0659
Lesion area (mm²)

1000
800
600
400
y = -0.2331x + 754.31
200 R² =0.1618
0
0 300 600 900 1200 1500
Calcium concentration in fruit (mg kg¯¹ DW)

Fig. 5.24 The correlation between grey mould development on fruit inoculated
postharvest and Ca concentration in leaves (A) or fruit (B) of capsicum cv. Aries
and cv. Papri Queen. Lesion area was determined at 10 days post incoulation on
fruit (at 10°C and relative humidity of >90%) that were inoculated postharvest.
Fruit were wounded on opposite sides and then inoculated with 40 µL suspension
of 10⁵ conidia mL¯¹. Data was collected from 12 individual measurements of grey
mould development on fruit and Ca concentration in leaves or fruit in the
experiment involving soil and foliar application of Ca

113
5.4 Discussion

Preharvest application of Ca did increase Ca concentration in leaf tissues

significantly, but did not increase Ca concentration in fruit tissues, regardless of
cultivar or application method. For both cultivars, preharvest application of Ca did
not affect quality parameters of fruit at harvest or during storage. Lesion area on
fruit from Ca-treated plants was significantly smaller than that on fruit from
control plants when fruit were derived from young developing fruit inoculated
with B. cinerea preharvest. However, when fruit were wounded and inoculated
with B. cinerea postharvest, lesion area on fruit from Ca-treated plants was similar
to that on fruit from the control plants.

Ca concentration did not change in fruit tissue, regardless of application method

or Ca concentration treatments. Calcium has been demonstrated to be xylem
mobile, but phloem immobile (Mengel and Kirkby 1987; White and Broadley
2003). Therefore, Ca uptake into the plant from the soil is reliant on transpiration
rate but, because fruit have a very low transpiration rate, very little Ca translocates
to fruit (White and Broadley 2003). The finding in this research confirms phloem
immobility of Ca in capsicum plants and/or the low level of transpiration by fruit
that may not have many stomata or if they do, stomata are unlikely to be open
(Peschel et al. 2003). Given the inability to increase fruit Ca via soil fertilisation
as observed in this study and by others (Rubio et al. 2010), foliar application is
thought to have potential because Ca can penetrate into fruit due to natural
openings including stomata, lenticels and cracks (Harker and Ferguson 1988).
Spraying peach with 0.12% w/v Ca in the form of Ca-EDTA at weekly intervals
from 6 or 12 weeks before harvest until commercial ripening increased Ca
concentration in peach fruit significantly (Manganaris et al. 2005). Calcium
concentration in tomato fruit increased significantly when plants were sprayed
with 0.5 CaCl₂ + 20 mg L¯¹ NAA at 4 days-intervals from 18 days after
transplanting to harvest (Dong et al. 2005). When surfactants such as Tween 20
are added to solutions used to spray plants, Ca uptake into fruit is increased
significantly due to altered permeability of the cuticles (Harker and Ferguson
1991). Calcium concentration in capsicum fruit did not increase significantly after
foliar application in this study perhaps because the natural waxy structure of the

114
skin (Govindarajan and Salzer 1985) did not allow Ca penetration. Adding
surfactants to the solution before spraying onto capsicum plants may therefore
increase Ca concentration in to the fruit and should be examined in further
research.

Ca concentration in leaf tissues from capsicum cv. Aries was significantly greater
than that from capsicum cv. Papri Queen, while Ca concentration in fruit appeared
to be similar. Calcium uptake is dependent on transpiration rate as noted earlier
and this is related to the total of leaf area per plant. Capsicum cv. Aries was
observed to have larger leaves than those from cv. Papri Queen, while leaves of
capsicum cv. Papri Queen were observed to be waxier than those from cv. Aries.
These reasons may result in better Ca uptake by cv. Aries than cv. Papri Queen. In
addition, Ca uptake ability of each cultivar could rely on the number and type of
Ca transporter in the root (Demarty et al. 1984). Calcium transporter in the root of
different cultivars of capsicum has not been reported and needs further research.

Length and width of fruit from both cultivars as well as weight of fruit from cv.
Papri Queen were not affected by Ca treatments via soil. Therefore, the size of
fruit could not be used to predict Ca deficiency in capsicum plants, but poor
development of flowers and fruit was caused by Ca deficiency as observed in this
research. For cv. Aries, weight of fruit from Ca-treated plants was significantly
greater than that from the control. Higher Ca concentration in the root medium
could have caused greater vegetative growth which affected the fruit weight
(Rubio et al. 2010). Calcium may be linked to an increase of leaf photosynthetic
efficiency which possibly increased number and weight of fruit per plant in
tomato (Lopez and Satti 1996).

Preharvest application of Ca did not affect quality parameters of fruit including

shelf life, extractable colour, firmness and TSSC at harvest or during storage,
regardless of cultivar. The results in this research were in agreement with a
previous report that quality of sweet pepper fruit (cv. Orlando) at harvest was not
affected when plants were fertilised with different Ca concentrations (Rubio et al.
2010). Preharvest Ca spray did not affect TSSC and TA in ‘Asgari’ table grapes
(Amiri et al. 2009). Quality of fruit is lost during storage due to cell-wall

115
breakdown and increasing membrane permeability (Brady 1987). Preharvest
application of Ca was expected to maintain postharvest quality of capsicum fruit
by increasing Ca concentration in the flesh and therefore stabilising the cell-wall
membrane and reducing tissue break down by the linkage of the pectic substances
of the cell wall (Demarty et al. 1984). Indeed, preharvest application of Ca has
been shown to increase Ca concentration in fruit leading to increased firmness and
shelf life of kiwifruit (Gerasopoulos et al. 1996) and strawberry fruit (Singh et al.
2007). In the present research, when plants received enough Ca to grow and
produce red fruit, postharvest quality of fruit was similar because Ca
concentration in fruit was not significantly different among the different Ca
concentration treatments preharvest, regardless of cultivar.

Most of the observations in this research showed that when applying higher
concentration of Ca to capsicum plants appeared to reduce grey mould
development on fruit, regardless of cultivar. Preharvest Ca treatment was also
reported to reduce grey mould in crops such as tomato (Elad and Volpin 1993)
and strawberry (Chéour et al. 1990; Naradisorn et al. 2006). Firstly, Ca is known
to be essential for the induction of plant defence (Benhamou 1996). Antifungal
substances including phytoalexins and phenolics have been found in cell walls
when Ca was applied to cultured soybean cells (Stäb and Ebel 1987). Increased
Ca2+ intracellular concentration is closely linked to the establishment of lignin-
like compounds at sites of potential fungal penetration to provide a further barrier
preventing pathogen spread and enzyme-catalysed degradation (Benhamou 1996).
In the present research, lower disease incidence on fruit from Ca-treated plants
may be due to the role of Ca in plant defence responses to reduce infection of
capsicum fruit by B. cinerea preharvest. However, there was no significant
relationship between the highest Ca concentration in plant tissues and the lowest
amount of disease. In the present research, Ca(NO₃)₂ was used as source of Ca,
increased nitrate concentration in nutrient solution may effect on disease
incidence on fruit. The high nitrate concentration in nutrient solution may increase
susceptibility of plant to disease (Snoeijers et al. 2000). This may be linked to
defence-related compounds in plants treated with different nitrate concentrations.
Soluble phenols, rutin and α-tomatine in tomato have been shown to be lower at

116
higher N availability (Hoffland et al. 1999). For cv. Aries in the first trial of soil
Ca application, fruit from plants treated with the highest Ca(NO₃)₂ showed greater
grey mould incidence than fruit from plants treated with moderate Ca(NO₃)₂, but
grey mould development was similar to that on fruit from plants treated with low
Ca(NO₃)₂. This could be due to the negative effect of nitrate overlapping the
positive effect of Ca on plant defence responses leading to greater grey mould on
fruit. This could be due to the negative effect of nitrate overlapping the positive
effect of Ca on plant defence responses leading to greater grey mould on fruit
(Snoeijers et al., 2000).

For Papri Queen, increasing Ca concentration in the leaf tissues appeared to not
affect grey mould development on fruit. Also lesion area on fruit from cv. Papri
Queen was significantly smaller than that on fruit from cv. Aries, regardless of Ca
concentration in leaf or fruit tissues. Although Ca deficiency developed in cv.
Papri Queen, grey mould lesions on fruit were still smaller than those on fruit
from cv. Aries at adequate Ca levels which reflects that Ca is not the only factor
contributing to plant defence response to pathogen infection. Others factors
including pathogenesis-related proteins in different cultivars of capsicum, as noted
in Chapter 3, may play a role in restricting fungal growth.

Lesion area on fruit inoculated postharvest was not significantly different among
Ca concentration treatments, regardless of cultivar or application method.
Wounding fruit probably caused cell death and failure of natural defence by the
fruit allowing grey mould to develop rapidly. Antifungal substances may be
present in fruit from Ca-treated plants, but rapid growth of B. cinerea on wounded
fruit may have overcome any inhibitory effect.

5.5 Conclusion

An increase of Ca concentration applied to plants via soil amendment or foliar

spray resulted in reducing grey mould development on fruit derived from young
developing fruit inoculated with B. cinerea preharvest. Therefore, preharvest
application of Ca might be recommended for capsicum industry to reduce grey
mould caused by B. cinerea postharvest and not affect the quality of fruit.

117
However, preharvest application of Ca could not protect fruit from growth of B.
cinerea when fruit were wounded and inoculated postharvest.

118
 CHAPTER SIX

Effect of postharvest calcium dips or vacuum infiltration

6.1 Introduction

Postharvest losses of capsicum fruit are mainly due to shrivelling and infection by
Botrytis cinerea. Even though fruit may be infected by B. cinerea during
development, grey mould does not develop until after harvest, when storage
conditions and the changes due to ripening are suitable for its growth (Droby and
Lichter 2004). Fungicide application is the most commonly-used method to
control fungal pathogens preharvest and postharvest. However, fungicides may be
toxic for human health and damage the environment. Alternative means of disease
control, such as nutrient application, may be a safer solution that can also improve
fruit quality (Fallahi et al. 1997; Hansch and Mendel 2009).

Calcium (Ca) is known to have an important role in reducing softening and fungal
growth in fruit because Ca strengthens cell walls by cross-linking the carboxyl
groups of polyuronide chains in the pectins found in the middle lamella (Sams
1999). Calcium also stabilises cell membranes and increases cell turgor pressure
(Mignani et al. 1995; Picchioni et al. 1995). Therefore, increasing Ca content in
fruit, via preharvest application of Ca, may be an effective way to extend the
postharvest shelf life in a wide range of soft fruit and vegetables, such as
capsicum. However, Ca is immobile in plant tissues and only small amounts are
translocated from leaves to fruit, so that Ca content in fruit is often low (Chamel
1989) (Chapter 5). Alternative methods are therefore needed to increase Ca
content and these may include the postharvest application of Ca-containing salts,
such as calcium chloride (CaCl₂), using dips and vacuum or pressure infiltration.
Calcium content increased 270% in peel and 74% in flesh when peach fruit were
dipped in 62.5 mM CaCl₂ (Manganaris et al. 2007). Dipping strawberry fruit in
1% calcium chloride solution increased Ca content of fruit, maintaining firmness
and controlling postharvest decay caused by B. cinerea (García et al. 1996;
Hernandez-Munoz et al. 2008). Even though the chloride anion from CaCl₂ could
potentially affect fungal growth, the Ca cation has been shown to be responsible
for the inhibition of growth of B. cinerea and Penicillium expansum in vitro

119
(Wisniewski et al. 1995). CaCl₂ has been mostly used as a dipping solution to
increase firmness and as a preservative agent for whole fruit and fresh-cut
commodities (Martín-Diana et al. 2007).

Although the Ca concentration of the skin and outer layers of the fruit is increased
with dipping, active infiltration appears to be a more effective way to ensure Ca
concentration increases in flesh (Conway et al. 1994a; Klein et al. 1997).
However, tissue damage prevents the use of active infiltration of Ca in some soft
fruit and so dips are the preferred method to improve postharvest quality of soft
fruit and control decay. Dipping whole kiwifruit with 1, 2 or 3% w/v CaCl₂
solution for 25 min maintained firmness of fruit slices compared to the control
(Beirão-da-Costa et al. 2008). In addition, when diced Roma tomato fruit were
dipped in 1% CaCl₂ for 1 min, the Ca concentration in fruit increased more than
300%, while firmness was enhanced and soluble pectin was decreased (Magee et
al. 2003). Dipping fruit in 1% CaCl₂ solution for 2 min reduced postharvest rots of
sweet cherries significantly compared to the control (Ippolito et al. 2005).

However, the effect of postharvest treatment with Ca on grey mould development

and the storage life of capsicum has not been reported. Thus, this research
examined the effect of postharvest Ca application on quality and grey mould
development in capsicum fruit. Both dips and vacuum infiltration were
investigated.

6.2 Materials and methods

6.2.1 Plant material and growth conditions

Capsicum plants of cv. Aries and cv. Papri Queen were used in this research and
all plants were grown in UC potting mix at the Waite Campus of the University of
Adelaide as previously described in Chapter 3. Pests were controlled as per
Section 4.2.1.

6.2.2 Isolation, maintenance and culture of B. cinerea

The isolate of B. cinerea described in Chapter 3 was used to produce conidia for
use as inoculum (at 10⁵ conidia mL¯¹) as per Chapter 3.

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6.2.3 Effect of calcium chloride on growth of B. cinerea in vitro

To examine the effect of CaCl₂ on fungal growth in vitro, B. cinerea was cultured
on potato dextrose agar (PDA, Difco, USA) amended with 100 mg L¯¹
streptomycin (Sigma, St Louis, MO) and various concentrations of calcium
chloride (CaCl₂.2H₂O): 0, 2, 4, 6 or 8% w/v. Briefly, a disc (1 cm diameter) from
the edge of a 2-week old culture of B. cinerea was placed in the centre of a 8.5-cm
Petri dish containing the amended PDA before incubation at 22 - 24°C under a 12
h light/12 h dark cycle. Colonies in PDA were monitored daily and diameter was
measured by using a digital calliper (digiMax, Switzerland). Five replicate plates
were prepared for each concentration of CaCl₂ and the experiment was conducted
twice. The daily rate of hyphal growth was also calculated.

6.2.4 Postharvest calcium dips

To study the effect of postharvest Ca dips on grey mould development and

postharvest quality of fruit, CaCl₂.2H₂O was used as the Ca-containing compound
because it has been used most often by other authors (see Section 6.1).
Symptomless, uniform-sized red fruit were harvested from plants of capsicum cv.
Aries and cv. Papri Queen. Before treatment, fruit were surface-sterilised by
dipping in 2% sodium hypochlorite for 1 min, rinsing with reverse osmosis (RO)
water and air-drying at room temperature in a laminar flow. Fruit were then
treated with four concentrations: 0, 2, 5 or 8% w/v CaCl₂.2H₂O. Dipping was
conducted by placing fruit in the respective Ca solution for 5 min. Only one
length of time for dipping was used due to time limitations. As such, 5 min was
chosen, as it has been used previously by Hernández-Muñoz et al. (2006) in
strawberry and Manganaris et al. (2007) in peach. Each treatment for each cultivar
had 25 fruit: 10 treated fruit for postharvest inoculation and 15 for the assessment
of postharvest quality of fruit during storage (see Section 6.2.7).

6.2.5 Postharvest calcium vacuum infiltration

An initial experiment was conducted to test if the Ca solution would infiltrate into
fruit. Fruit immersed in Ca solution were placed in a glass desiccator and then a
vacuum applied using a vacuum pump (BÜCHI Vac® V-500, Switzerland) for

121
times ranging between 10 sec and 5 min before holding the fruit in solution for 2
min and then releasing the vacuum. Holding fruit in solution for 2 min was
necessary for Ca absorted into fruit tissue, particularly at short time of vacuum.
Only one length of time for holding fruit in solution was used due to time
limitations. Results showed that if the vacuum was applied for more than 10 sec,
the fruit changed colour because of water soaking and had tissue damage while
the solution remained inside the fruit after the vacuum was released. Therefore, it
was decided fruit would be treated by applying the vacuum for 10 sec followed by
holding the fruit for 2 min in solution before the vacuum was released.

Symptomless, uniform-sized red fruit were harvested from plants of capsicum cv.
Aries and cv. Papri Queen and surface-sterilised before vacuum infiltration as per
Section 6.2.4. Vacuum infiltration was applied as above. Twenty five fruit of each
cultivar for one treatment were used: 10 treated fruit for postharvest inoculation
and 15 for the assessment of postharvest quality of fruit during storage (see
Section 6.2.7).

6.2.6 Effect of infiltration method on absorption of Ca into flesh of fruit

Visual assessment and tissue analysis of Ca concentration were used to examine

whether Ca was absorbed into the flesh of fruit. Symptomless, uniform-sized fruit
were harvested from plants of capsicum cv. Aries and cv. Papri Queen to examine
whether Ca solution directly penetrated into the flesh. Three fruit for each cultivar
were either dipped or vacuum infiltrated with Ca solution at a concentration of 8%
w/v CaCl₂.2H₂O amended with aniline blue 0.01% w/v as described earlier
(Sections 6.2.4 and 6.2.5). Treated fruit were then cut in half and images were
recorded using a digital camera (Canon D500, Japan). Fruit were then peeled with
a knife to about 1 mm deep before collecting for nutrient analysis to ensure that
Ca concentration was measured only in the flesh. Samples for ICP-OES were then
prepared as per Section 4.2.3.

In a separate experiment, to determine whether Ca was absorbed into flesh after

storage, three fruit of cv. Aries were dipped or vacuum infiltrated with Ca solution
at a concentration of 8% w/v CaCl₂.2H₂O as described earlier (Section 6.2.4 and
6.2.5) before storage at 10°C for 10 days. Fruit of cv. Papri Queen was not used
122
due to its thin flesh making it too soft to peel after 10 days of storage. RO water
was used to treat fruit as a control. After 10 days of storage, peel and flesh were
collected for nutrient analysis as per Section 4.2.3.

6.2.7 Postharvest inoculation and assessment of postharvest quality of fruit

Ten treated fruit (as per Section 6.2.4 and Section 6.2.5) were wounded on
opposite sides and inoculated with B. cinerea as per Section 4.2.4.3. After
inoculation, fruit were placed in plastic container lined with paper towel and
stored at 10°C and a relative humidity (RH) of >90%. Rot development was
quantified daily by measuring lesion area on individual fruit as per Section
4.2.5.1.

To assess postharvest quality, fruit were assessed at harvest and then after storage
for 5, 10, 15 or 23 days. Three fruit at each time-point per treatment (a total of 15
fruit) were used to assess shelf life, extractable colour, firmness, total soluble
solid content (TSSC) and titratable acidity (TA) as per Section 4.2.5.1.

6.2.8 Statistical analysis and photography

Experiments were designed using the factors of cultivar and Ca concentration.

Data were subjected to repeated measurements analysis of variance (ANOVA)
using GenStat and means of the treatments compared using the LSD at a
significance level of P ≤ 0.05. Means and standard errors were determined using
Microsoft Excel.

6.3 Results

6.3.1 Effect of calcium chloride on fungal growth of B. cinerea

The diameter of fungal colonies on PDA from day 2 to day 4 was significantly
smaller with increasing Ca concentration from 4% to 8% w/v CaCl₂ 2H₂O (Fig
6.1A). The effect of 0% and 2% w/v CaCl₂ 2H₂O on fungal colonies was similar
from day 1 to day 4. Calcium chloride initially inhibited the rate of hyphal growth
from day 1 to day 2 or day 2 to day 3, but the rate of hyphal growth was not
significantly different from day 3 to day 4 (Fig 6.1B).

123
Colony morphology showed that mycelial growth on PDA amended with calcium
chloride concentration of 4, 6 or 8 % w/v CaCl₂ 2H₂O was woollier with white
colour after 4 days, while mycelial growth on PDA amended with lower calcium
chloride was almost smooth on the surface (Fig 6.1C).

6.3.2 Effect of postharvest calcium dips on Ca uptake and nutrient status of

fruit

Nutrient analysis showed that Ca concentration in fruit was not significantly

different among the four Ca concentrations tested for either cv. Aries or cv. Papri
Queen (Table 6.1). After dipping in 8% w/v CaCl₂.2H₂O amended with aniline
blue, dye was observed on the skin but not in the flesh of fruit from either cultivar
(Fig 6.2A, B). No significant differences in the concentration of other nutrients
were evident and all nutrients were sufficient (Reuter and Robinson 1997)
(Appendix A.9).

After 10 days of storage, Ca concentration in flesh from Ca-treated fruit was not
statistically significant (Table 6.2). No significant differences in the concentration
of other nutrients were evident and all nutrients were sufficient (Reuter and
Robinson 1997) (Appendix A.10).

124
 85
80 A B
35
75
Diameter of colony (mm)
70

Rate of hyphal growth (mm)

65 30
60
55 25
50
45 0% CaCl₂.2H₂O 20
40
35 2% CaCl₂.2H₂O 15
30
25 4% CaCl₂.2H₂O 10
20
15 6% CaCl₂.2H₂O 5
10
5 8% CaCl₂.2H₂O
0 0
Day 1 to 2 Day 2 to 3 Day 3 to 4
1 2 3 4
Days Time

Fig. 6.1 Effect of CaCl₂.2H₂O on mycelial growth of B. cinerea; (A) Diameter of

colony on PDA; (B) Rate of hyphal growth on PDA. Data are means ± SE from n
= 10 across two experiments. LSDs (P ≤ 0.05) for among individual CaCl₂.2H₂O
concentrations for each time-point are shown. (C) Prepresentative image shown
for fungal colonies on PDA amended with 0, 2, 4, 6 or 8% w/v CaCl₂.2H₂O after
4 days

125
Table 6.1 Effect of postharvest Ca dips on Ca concentration in fruit tissues. Other
nutrients are in a table in Appendix A.9. The nutrient concentrations were
determined using ICP-OES analysis. Fruit for nutrient analysis were collected at 0
days after harvest and storage. Fruit were treated at four concentrations: 0, 2, 5
and 8% w/v CaCl₂.2H₂O. Dipping was conducted by placing fruit in the
respective calcium solution for 5 min. Data are presented as means ± SE from n =
3.Calcium concentration in fruit tissue was not significantly different between Ca
treatments (P values are shown)

CaCl₂.2H₂O concentration Ca concentration in fruit

Cultivar
(%) tissue (mg kg¯¹ DW)
0 845.00 ± 185.00
2 765.00 ± 135.00
Aries 5 690.00 ± 160.00
8 780.00 ± 20.00
P value P = 0.89
0 915.00 ± 25.00
2 1070.00 ± 460.00
Papri Queen 5 725.00 ± 155.00
8 1020.00 ± 250.00
P value P = 0.82

126
Table 6.2 Effect of storage time and postharvest Ca dips on Ca concentration in
fresh of fruit from cv. Aries. Other nutrients are in a table in Appendix A.10. Fruit
were treated with 8% w/v CaCl₂.2H₂O and analysed after 10 days of storage.
Control fruit were treated with RO water and nutrient analysed at harvest. Dipping
was conducted by placing fruit in calcium solution for 5 min. Fruit were peeled
with a knife to about 1 mm deep to collect peel and flesh for nutrient analysis.
Data are presented as means ± SE from n = 3. Means with the same letters in
column were not significantly different as determined using the LSD (P<0.05)

Ca concentration in fruit
Sample Fruit tissue
tissue (mg kg⁻¹ DW)
Control Flesh 485.00 ± 25.00a
Flesh 800.00 ± 10.00a
Dip (8% w/v CaCl₂.2H₂O) Peel 1390.00 ± 190.00b
LSD (P value) 498.60 (P = 0.023)

127
 A B

Fig. 6.2 Effect of postharvest Ca dips on penetration of Ca solution into fruit.

Capsicum fruit from cv. Aries (A) and cv. Papri Queen (B) were dipped in Ca
solution at a concentration of 8.0% w/v CaCl₂.2H₂O amended with 0.01% w/v
aniline blue for 5 min. No stain in placenta was observed. Representative images
are shown

128
6.3.3 Effect of postharvest calcium dips on postharvest quality and grey
mould development

General appearance (GA) (9 = the best condition and 1 = the worst condition) (as
in Section 4.2.5.2), regardless of cultivar or Ca treatment, decreased significantly
during storage while GA was not significantly different among fruit treated with
any of the four Ca concentrations (Fig 6.3A, B). Shelf life of fruit was not
significantly different among fruit, regardless of Ca concentration applied or
cultivar (Fig 6.3C, D).

Extractable colour generally appeared to increase during storage in fruit,

regardless of Ca concentration applied and cultivar (Fig 6.4A, B). Extractable
colour of fruit was significantly higher at 23 DAH than at harvest. However, there
was no significant difference for extractable colour among fruit treated with any
of the four Ca concentrations, regardless of cultivar.

Firmness of fruit from both cultivars was not significantly different among fruit
treated with any of the four Ca concentrations (Fig 6.4C, D). For cv. Aries,
regardless of Ca concentration, firmness of fruit was significantly lower at 5 DAH
compared with those at harvest but then significantly increased at 10 DAH, with
no change from 15 DAH to 23 DAH (Fig 6.4C). For cv. Papri Queen, firmness of
fruit at harvest and at 5 DAH was not significantly different but then firmness
significantly increased at 10 DAH (Fig 6.4D).

TSSC of fruit from both cultivars was not significantly different among fruit,
regardless of Ca concentration applied and cultivar (Fig 6.4E, F). TSSC of fruit
from cv. Aries and treated with any of the four Ca concentrations was not
significantly different during storage from that observed at harvest (Fig 6.4E). For
cv. Papri Queen, TSSC of fruit treated with four Ca concentrations was similar
from harvest to 15 DAH and then significantly increased at 23 DAH (Fig 6.4F).
TSSC in fruit from cv. Papri Queen was significantly higher than in fruit from cv.
Aries, regardless of Ca concentration.

129
 A
B
9 9
8 8
General appearance (GA)

7 7
6 6
5 5
4 0% CaCl₂.2H₂O 4
3 2% CaCl₂.2H₂O 3
2 5% CaCl₂.2H₂O 2
1 8% CaCl₂.2H₂O 1
0 0
0 10 20 30 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

25 C 25 D

20
Predicted shelflife (days)

20

15 15

10 10

5 5

0 0
0 2 5 8 0 2 5 8
Ca concentration treament Ca concentration treatment
(% w/v CaCl₂.2H₂O) (% w/v CaCl₂.2H₂O)

Fig. 6.3 Effect of postharvest calcium dips on general appearance (A and B) and
shelf life (C and D) of fruit from cv. Aries and cv. Papri Queen, respectively
during storage at 10°C and relative humidity of >90%. Fruit were treated at four
concentrations: 0, 2, 5 or 8% w/v CaCl₂.2H₂O. Dipping was conducted by placing
fruit in the respective calcium solution for 5 min. Shelf life of fruit was predicted
at a general appearance (GA) of 5.5 by using a third degree polynomial in
GenStat. Data are means ± SE from n = 3. LSDs (P ≤ 0.05) for among individual
calcium concentrations are shown

130
TA of fruit from cv. Aries was significantly different among fruit treated with
different Ca concentrations (Fig 6.4G). Initial TA of fruit treated with 0% and 2%
CaCl₂.2H₂O was similar and significantly lower than for those treated with 5%
and 8% CaCl₂.2H₂O. TA of fruit treated with the two higher Ca was significantly
lower than those treated with two lower Ca concentrations at 15 DAH, but was the
same at 23 DAH. For cv. Papri Queen, TA of fruit treated with any of the four Ca
concentrations was not significantly different for all time points of storage (Fig
6.4H). TA in fruit from cv. Papri Queen was significantly higher than in fruit from
cv. Aries, regardless of Ca concentration.

Grey mould developed significantly more slowly in fruit dipped in high Ca

concentrations in cv. Aries (Fig 6.5A). Lesion area on fruit treated with 8%
CaCl₂.2H₂O was significantly smaller than on those treated with 0% or 2% w/v
CaCl₂.2H₂O at 10 DPI, whereas lesion area on fruit treated with 5% w/v
CaCl₂.2H₂O was not significantly different from fruit treated with 0%, 2% or 8%
w/v CaCl₂.2H₂O. At 12 DPI, lesion area on fruit treated with 8% w/v
CaCl₂.2H₂O was significantly smaller than on those treated with the three lower
concentrations, while lesions on fruit treated with 5% CaCl₂.2H₂O was also
significantly smaller than on fruit treated with 0% or 2% w/v CaCl₂.2H₂O. Lesion
area was the same on fruit treated with 2% CaCl₂.2H₂O and control fruit. For cv.
Papri Queen, lesion area on fruit from cv. Papri Queen was significantly smaller
than that on fruit from cv. Aries, regardless of Ca concentration, and lesion area
was not affected by Ca concentrations (Fig 6.5B).

131
 350 350 B
A

Extractable colour (ASTA)

300 300
250 250
200 200
150 0% CaCl₂.2H₂O 150
100 2% CaCl₂.2H₂O 100
5% CaCl₂.2H₂O
50 50
8% CaCl₂.2H₂O
0 0
0 5 10 15 20 25 0 5 10 15 20 25
120 C 120 D
100 100
Firmness (N cm⁻²)

80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
16 16 F
14 E 14
12 12
TSSC (°Bx)

10 10
8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 G 0.7 H
0.6 0.6
0.5 0.5
Citric acid (%)

0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 6.4 Postharvest quality of capsicum fruit during storage at 10°C and relative
humidity of >90%: extractable colour (A and B); firmness (C and D); TSSC (E
and F) and acidity (G and H) for fruit from cv. Aries and fruit from cv. Papri
Queen, respectively. Fruit were dipped in Ca solution at 0, 2, 5 or 8% w/v
CaCl₂.2H₂O for 5 min before storage. Data are means ± SE from n = 3. LSDs (P
≤ 0.05) are shown for among individual Ca concentrations for each time-point
during storage

132
 3500 A 0% CaCl₂.2H₂O

2% CaCl₂.2H₂O
3000
5% CaCl₂.2H₂O

Lesion area (mm²)

2500
8% CaCl₂.2H₂O

2000

1500

1000

500

0
4 6 8 10 12

3500
B
3000

2500
Lesion area (mm²)

2000

1500

1000

500

0
4 6 8 10 12
Time post inoculation (days)

Fig. 6.5 Effect of Ca dips on grey mould development on postharvest-inoculated

fruit of capsicum cv. Aries (A) and cv. Papri Queen (B) during storage at 10°C
and relative humidity of >90%. Fruit were dipped in Ca solution at 0, 2, 5 or 8%
w/v CaCl₂.2H₂O for 5 min. Fruit were wounded on opposite sides and inoculated
with 40 µL suspension of 10⁵ conidia mL⁻¹. Data are means ± SE from n = 10.
LSDs (P ≤ 0.05) are shown for among individual Ca concentrations for each time-
point post inoculation

133
6.3.4 Effect of postharvest Ca vacuum infiltration on Ca uptake and nutrient
status of fruit

Nutrient analysis showed that Ca concentration in fruit was not significantly

different among the four Ca concentration treatments for either cv. Aries or cv.
Papri Queen (Table 6.3). No significant differences in concentration of other
nutrients were evident and all nutrients were sufficient (Reuter and Robinson
1997) (Appendix A.11). Dye was observed in the placenta of fruit from both
cultivars, but not in flesh (Fig 6.6A and B).

After 10 days of storage, the Ca concentration in flesh from Ca-treated fruit was
greater (P = 0.009) than that in control fruit (Table 6.4). Calcium concentration in
flesh of fruit treated by using vacuum infiltration was significantly greater (P =
0.009) than that in flesh of fruit treated by using dips. No significant differences in
concentration of other nutrients were evident and all nutrients were sufficient
(Reuter and Robinson 1997) (Appendix A.12).

6.3.5 Effect of postharvest Ca vacuum infiltration on postharvest quality and

grey mould development

GA, regardless of cultivar and Ca concentration applied, decreased significantly

after storage but was not significantly different among fruit treated with any of the
four Ca concentrations (Fig 6.7A, B). Shelf life of fruit was not significantly
different among fruit, regardless of Ca concentration applied and cultivar (Fig
6.7C, D).

Extractable colour generally appeared to increase during storage of fruit,

regardless of Ca concentration applied and cultivar (Fig 6.8A, B). Extractable
colour of fruit was significantly higher at 23 DAH than at harvest. However, there
was no significant difference for extractable colour among fruit treated with any
of the four Ca concentrations, regardless of cultivar.

134
Table 6.3 Effect of postharvest Ca vacuum infiltration on Ca concentration in
fruit tissues. Other nutrients are in a table in Appendix A.11. The nutrient
concentrations were determined using ICP-OES analysis. Fruit for nutrient
analysis were collected at harvest. Fruit were treated at four concentrations: 0, 2, 5
or 8% w/v CaCl₂.2H₂O. Vacuum infiltration was conducted by placing fruit in the
respective calcium solution, drawing a vacuum for 10 sec and then holding fruit in
solution for 2 min before vacuum release. Data are presented as means ± SE from
n = 3. Calcium concentration in fruit tissue was not significantly different between
Ca treatments (P values are shown)

CaCl₂.2H₂O concentration Ca concentration in fruit

Cultivar
(%) tissue (mg kg¯¹ DW)
0 710.00 ± 160.00
2 900.00 ± 160.00
Aries 5 730.00 ± 160.00
8 890.00 ± 300.00
P value 0.83
0 765.00 ± 95.00
2 1715.00 ± 65.00
Papri Queen 5 845.00 ± 345.00
8 825.00 ± 95.00
P value 0.06

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Table 6.4 Effect of storage time and postharvest Ca vacuum infiltration on Ca
concentration in fruit from cv. Aries. Other nutrients are in a table in Appendix
A.12. Fruit were vacuum infiltrated with 8% w/v CaCl₂.2H₂O and nutrient
analysed after 10 days of storage while control fruit were vacuum infiltrated with
RO water and nutrient analysed at harvest. Vacuum infiltration was conducted by
placing fruit in Ca solution, drawing a vacuum for 10 sec and then holding fruit in
solution for 2 min before vacuum release. Fruit were peeled with a knife to about
1 mm deep to collect peel and flesh for nutrient analysis. The nutrient
concentrations were determined using ICP-OES analysis. Data are presented as
means ± SE from n = 3. Means with the same letters in column were not
significantly different as determined using the LSD (P<0.05)

Ca concentration in fruit tissue

Sample Fruit tissue
(mg kg¯¹ DW)
Control Flesh 485.00 ± 25.00a
Flesh 2075.00 ± 325.00b
Vacuum infiltration
Peel 3500.00 ± 600.00c
(8% w/v CaCl₂.2H₂O)
LSD(P value) 1274.3(P = 0.028)

136
 A B

Fig. 6.6 Effect of postharvest Ca vacuum infiltration on penetration of Ca solution

into fruit. Capsicum fruit from cv. Aries (A) and cv. Papri Queen (B) were placed
in Ca solution at 8% w/v CaCl₂.2H₂O amended with 0.01% w/v aniline blue,
drawing a vacuum for 10 sec and then holding fruit in solution for 2 min before
vacuum release. Representative images are shown. Arrows show whether the
placenta tissues were stained

137
 A B
9 9
8 8
General appearance (GA) 7 7
6 6
5 5
0% CaCl₂.2H₂O
4 4
3 2% CaCl₂.2H₂O
3
2 5% CaCl₂.2H₂O 2
1 8% CaCl₂.2H₂O 1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

25 25
D
C
Predicted shelflife (days)

20 20

15 15

10 10

5 5

0 0
0 2 5 8 0 2 5 8
Ca concentration treatment Ca concentration treament
(% w/v CaCl₂.2H₂O) (% w/v CaCl₂.2H₂O)

Fig. 6.7 Effect of postharvest calcium vacuum infiltration on general appearance

(A and B) and shelf life (C and D) of fruit cv. Aries and fruit cv. Papri Queen,
respectively during storage at 10°C and relative humidity of >90%. Shelf life was
predicted for fruit treated at four concentrations: 0, 2, 5 or 8% w/v CaCl₂.2H₂O.
Vacuum infiltration was conducted by placing fruit in Ca solution, drawing a
vacuum for 10 sec and then holding fruit in solution for 2 min before vacuum
release. Shelf life of fruit was predicted at a general appearance (GA) of 5.5 by
using a third degree polynomial in GenStat. Data are means ± SE from n = 5.
LSDs (P ≤ 0.05) for among individual Ca concentrations are shown

138
Firmness of fruit from both cultivars was not significantly different among fruit
treated with any of the four Ca concentrations (Fig 6.8C, D). For cv. Aries,
firmness of fruit did not change from harvest to 10 DAH and then significantly
declined at 15 DAH, but did not change at 23 DAH (Fig 6.8C). For cv. Papri
Queen, firmness of fruit treated with any of the four Ca concentrations did not
change at 5 DAH compared with those at harvest but then significantly increased
at 10 DAH but did not change at 15 DAH and 23 DAH (Fig 6.8D).

TSSC of fruit was not significantly different among fruit, regardless of Ca

concentration applied and cultivar (Fig 6.8E, F). TSSC of fruit from cv. Aries
treated with any of the four Ca concentrations was not changed after harvest (Fig
6.8E). For cv. Papri Queen, TSSC of fruit treated with four Ca concentrations was
similar from harvest to 15 DAH and then significantly increased at 23 DAH (Fig
6.8F). TSSC in fruit from cv. Papri Queen was significantly higher than in fruit
from cv. Aries, regardless of Ca concentration.

TA of fruit from cv. Aries was significantly different among fruit vacuum
infiltrated with different Ca concentrations (Fig 6.8G). Initial TA of fruit treated
with 0% CaCl₂.2H₂O was significantly lower than those treated with any of the
three concentrations of CaCl₂.2H₂O. At10 DAH, regardless of Ca concentration,
TA of treated fruit was significantly greater than at harvest. TA of fruit treated
with the two higher Ca concentrations was significantly lower than that treated
with the two lower Ca concentrations at 15 DAH, but the same at 23 DAH. TA in
fruit from cv. Papri Queen was significantly higher than in fruit from cv. Aries,
regardless of Ca concentration.

Lesion area on fruit from cv. Aries treated with 0% CaCl₂.2H₂O was significantly
greater than on those treated with any of the three Ca concentrations from 10 DPI
to 12 DPI. However, there was no significant difference for lesion area on fruit
treated with 2, 5 or 8% w/v CaCl₂.2H₂O (Fig 6.9A). For cv. Papri Queen, lesion
area was not significantly different among fruit treated with any of the four Ca
concentrations (Fig 6.9B). Lesion area on fruit from cv. Papri Queen was
significantly smaller than that on fruit from cv. Aries, regardless of Ca
concentration.

139
 300 A B
300

Extractable colour (ASTA)

250 250
200 200
0% CaCl₂.2H₂O
150 2% CaCl₂.2H₂O 150
100 5% CaCl₂.2H₂O 100

50 8%CaCl₂.2H₂O 50
0
0
0 5 10 15 20 25
0 5 10 15 20 25
120 C 120
D
Firmness (N cm⁻²)

100 100
80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 0 5 10 15 20 25
16 16 F
14 14
12 E 12
TSSC (°Bx)

10 10
8 8
6 6
4 4
2 2
0 0
0 5 10 15 20 25 0 5 10 15 20 25
0.7 0.7 H
0.6 0.6
Citric acid (%)

0.5 0.5
G
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time after storage (days) Time after storage (days)

Fig. 6.8 Postharvest quality of fruit during storage at 10°C and relative humidity
of >90%: extractable colour (A and B); firmness (C and D); TSSC (E and F) and
acidity (G and H) for fruit cv. Aries and fruit cv. Papri Queen, respectively. Fruit
were vacuum infiltrated by placing fruit in Ca solution at 0, 2, 5 or 8% w/v
CaCl₂.2H₂O, drawing a vacuum for 10 sec and then holding fruit in solution for 2
min before vacuum release. Data are means ± SE from n = 3. LSDs (P ≤ 0.05) are
shown for among individual Ca concentrations for each time-point during storage

140
 A
5000
4500 0% CaCl₂.2H₂O

4000 2%CaCl₂.2H₂O

Lesion area (mm²)

3500 5% CaCl₂.2H₂O
3000 8% CaCl₂.2H₂O
2500
2000
1500
1000
500
0
4 6 8 10 12

5000 B
4500
4000
3500
Lesion area (mm²)

3000
2500
2000
1500
1000
500
0
4 6 8 10 12
Time post-inoculation (days)

Fig. 6.9 Effect of Ca vacuum infiltration on grey mould development on

postharvest-inoculated fruit of capsicum cv. Aries (A) and cv. Papri Queen (B)
during storage at 10°C and relative humidity of >90%. Fruit were vacuum
infiltrated by placing fruit in Ca solution at 0, 2, 5 or 8% w/v CaCl₂.2H₂O,
drawing a vacuum for 10 sec and then holding fruit in solution for 2 min before
vacuum release. Fruit were wounded opposite sides and inoculated with 40 µL
suspension of 10⁵ conidia mL⁻¹. Data are means ± SE from n = 10. LSDs (P ≤
0.05) are shown for among individual Ca concentrations for each time-point post
inoculation

141
6.4 Discussion

Calcium concentration in fruit tissue was not significantly different among

calcium treatments, regardless of application method. Postharvest application of
Ca did not affect the shelf life or postharvest quality of capsicum fruit during
storage. However, for cv. Aries, lesion area on Ca-treated fruit inoculated
postharvest was significantly smaller than that on control fruit suggesting that
postharvest Ca treatment may be a successful control method for grey mould of
capsicum.

Postharvest Ca dips or vacuum infiltration, regardless of Ca concentration, did not

increase Ca concentration in flesh of capsicum fruit significantly when sampling
for nutrient analysis was immediately after Ca treatment. Calcium concentration
in flesh of ‘Golden Delicious’ apples was reported to not increase significantly
compared to a control when fruit were dipped with a CaCl₂ solution at
concentrations of 0, 2, 4, 8 or 12%, but to increase significantly when fruit were
vacuum infiltrated with the same Ca concentrations (Conway and Sams 1983).
However, dipping peach fruit in Ca solution for 5 min increased flesh Ca
concentration significantly (Manganaris et al. 2007). Flesh Ca concentration in
strawberry was also higher compared to a control when fruit were dipped with 1%
CaCl₂ for 5 min (Hernández-Muñoz et al. 2006). These observed differences of
the ability of Ca to penetrate into different fruit may be explained by differences
in the surface properties of fruit. The waxiness of capsicum (Govindarajan and
Salzer 1985) could therefore possibly prevent Ca from being taken up easily. The
surface properties of capsicum fruit may also be variable among cultivars with
fruit from cv. Papri Queen appearing to be more waxy than fruit from cv. Aries.
Lenticels are probably the primary pathway for Ca to enter fleshy fruit (Betts and
Bramlage 1977) but Ca may also penetrate through cracks in the cuticle and
epidermis (Glenn et al. 1985). Calcium may also penetrate through stomata given
that there is a positive correlation between stomatal pore area with the rate of
water uptake into fruit as reported in sweet cherry (Simon 2006). The lack of Ca
penetration into capsicum flesh in the present research may therefore be due to a
shortage of cracks and/or stomata and the waxiness of the capsicum peel. Further
research is suggested to examine surface properties of capsicum fruit compared to

142
another fruit. Vacuum infiltration improved Ca uptake but Ca solution entered
fruit through stem-end tissues and went straight to the placenta but not into the
flesh (Fig 6.6A and B). This was probably due to vesicle structure of the placenta
(Broderick and Cooke 2007), Ca was stored in vesicle and not easily transported
to flesh. In order to confirm Ca was only in flesh, the placenta was removed when
sampling for nutrient analysis.

Although Ca concentration was not increased initially by dipping or vacuum

infiltration, the Ca concentration did increase significantly after 10 days of storage
when fruit had been vacuum infiltrated. Calcium concentration increased by more
than 300% in the flesh and 600% in the peel of vacuum-infiltrated fruit after 10
days storage (Table 6.4). The amount of Ca solution that was taken up into
‘Golden Delicious’ apples by using pressure infiltration was greater after 6
months of storage than after shorter storage intervals (Roy et al. 1999) and the
authors revealed that cracks in the epicuticular wax layers and the cuticular layers
of apple fruit extended during cold storage. Calcium in the peel of peach fruit
decreased while there was a corresponding increase of Ca in the flesh after 5 days
of cold storage (Manganaris et al. 2007). In the present research, increasing cracks
or extension of cracks in the cuticular layers of capsicum during cool storage may
create more openings for penetration of Ca. Further research is required to
examine the cracks on the surface of capsicum fruit during cool storage.
Moreover, Ca solution possibly penetrated into the flesh from the placenta during
storage because of cell-wall breakdown in the flesh tissue due to ripening and
senescence (Brady 1987).

The increased Ca concentration in the peel and flesh of fruit treated with Ca
postharvest, has been accompanied by a maintained quality of fruit during storage
in strawberry (García et al. 1996; Hernández-Muñoz et al. 2006), peach
(Manganaris et al. 2007) and apple (Conway et al. 1994b). The common
explanation is that the presence of Ca ion stabilises cell wall structure by forming
cross bridges to link the pectin components and/or to other acidic polysaccharides
(Demarty et al. 1984). In the present research, postharvest Ca application was
expected to maintain the postharvest quality of capsicum fruit during storage.
However, postharvest quality of capsicum fruit was not affected by postharvest

143
application of Ca, regardless of cultivar or Ca concentration. Calcium
concentration in Ca-treated fruit may increase in flesh during storage, but was not
sufficient to affect postharvest quality of fruit and the shelf life. However, there
could be a potential economic impact as the shelf life of Ca-treated fruit may be
significantly longer than the control if capsicum fruit were stored for a longer
period. Symptoms of surface damage were not observed on Ca-treated fruit, but
salt accumulation was observed on the surface of fruit treated with Ca
concentrations of 5% or 8% w/v CaCl₂.2H₂O. This contributes to the penetration
of Ca into the flesh during storage as noted above. A residual amount of CaCl₂ on
the surface of fruit has been linked to detection of bitterness by consumers (Luna-
Guzmán and Barrett 2000). However, dipping fruit with Ca concentration of 4%
w/v CaCl₂ did not affect sensorial quality of strawberry fruit (García et al. 1996).

Lesion areas on Ca-treated fruit from cv. Aries were significantly smaller than
those on the control fruit, regardless of whether Ca was applied as a dip or by
vacuum infiltration. Postharvest treatment with Ca solution at 2% or 4% w/v
CaCl₂ by using pressure infiltration reduced decay areas caused by B. cinerea in
apples by 40% or 60%, respectively (Conway et al. 1994b). Postharvest treatment
with Ca solution at the concentration of 2.0% or 3.0% w/w CaCl₂ was also
reported to delay Botrytis rot on strawberry cv. Selva (Naradisorn 2008). Effect of
increased Ca concentration in fruit on reducing the development of B.cinerea may
be directly by inhibiting conidial germination and germ-tube elongation or
indirectly by strengthening cell wall structure (Demarty et al. 1984; Conway et al.
1991). Direct inhibition of growth of B. cinerea seems most likely in this research
given that adding CaCl₂.2H₂O to PDA on which B. cinerea was grown resulted in
reduced hyphal growth. This result was in agreement with research conducted by
Wisniewski et al. (1995). The inhibitory effect of CaCl₂ or MgCl₂ on B. cinerea
and P. expansum in vitro was examined with a range of concentrations from 0 to
200 mM. The research concluded that the Ca cation rather than chloride anion was
responsible for the inhibition of conidial germination and germ-tube growth. In
higher eukaryotes, high internal Ca concentration has been shown to cause
inhibition of fungal growth and cell death (Carafoli 1987). In the present research,
presence of Ca on the surface of Ca-treated fruit from cv. Aries may have caused

144
fewer conidia to germinate. Even though wounding fruit promotes B. cinerea
growth, Ca concentration at wounded sites on Ca-treated fruit was most likely
higher than those on the control fruit. Therefore, increasing Ca concentration on
the surface of fruit may be a potential way to reduce decay in fruit during storage
although an increase of Ca concentration in flesh did not occur. However, grey
mould development on fruit from cv. Papri Queen was not significantly different
among Ca treatments. Natural resistance of cv. Papri Queen to B. cinerea as
described in Chapter 3 may overcome the positive effect of Ca in reducing the
numbers of conidial germination on surface of fruit. Further research is required
to examine conidial germination on wound sites of capsicum fruit treated with
different Ca concentrations at a commercial scale.

If Ca is able to be taken up into fruit, the stability of the cell walls can be
increased due to Ca binding with polygalacturonate chains, making the cell walls
less accessible to enzymes that cause softening or to degrading enzymes produced
by fungi (Conway et al. 1994b). High concentrations of Ca ions in tissues of
tomato was thought to inhibit the activity of degrading enzymes, including
pectinesterase (PE) and polygalacturonase (PG) (Wills and Rigney 1980), which
were most important for B. cinerea to access more nutrients from cell-wall
breakdown (Williamson et al. 2007). Decay caused by P. expansum on apple fruit
reduced more than 50% when Ca tissue concentration was increased to 1200 -
1500 g Ca kg⁻¹ DW, but a lower concentration may restrict progress of B. cinerea
(Conway et al. 1991). In the present research, increasing Ca concentration reduced
grey mould development significantly in cv. Aries, but was not always associated
with higher levels of Ca in flesh as evidenced by the non-significant effect of
firmness of fruit. Therefore, a direct effect of Ca on inhibition of the fungus itself
than on cell-wall strengthen of fruit was indicated in this research.

6.5 Conclusion

Postharvest application of Ca could be recommended for capsicum cv. Aries fruit

before storage or commercialisation to reduce grey mould growth on fruit during
storage. However, the effect of different Ca concentrations on conidial
germination on wound sites of capsicum fruit should be researched further. The
vacuum infiltration method was more effective than dipping in increasing Ca
145
concentration in the flesh when capsicum fruit were subsequently stored in cool
conditions. Therefore, Ca vacuum infiltration would be recommended for
capsicum fruit which needs a long period of storage. However, the ideal length of
time for vacuum infiltration needs to be determined in further research.

146
 CHAPTER SEVEN

General discussion

7.1 Outcomes of study

Preharvest and postharvest losses of horticultural crops, including capsicum, due

to grey mould disease caused by the fungus Botrytis cinerea are of significant
concern to growers and produce marketers. A reduction of disease in fruit and
improvement of fruit quality by using preharvest application of Ca and/or B via
soil amendment or as a foliar spray has been reported in a wide range of
horticultural crops, including strawberry (Chéour et al. 1990; Naradisorn et al.
2006), peach (Manganaris et al. 2005; Elmer et al. 2007), sweet cherry (Ippolito et
al. 2005) and table grape (Amiri et al. 2009). However, how B. cinerea infects
capsicum fruit has not been previously reported. In addition, knowledge of the
effect of Ca or B on grey mould of capsicum and quality of fruit is limited. This
research established the infection pathway of B. cinerea in capsicum and explored
the effect of Ca or B application on grey mould of capsicum and quality of fruit.

Regardless of cultivar, when B. cinerea infected capsicum fruit preharvest,

symptoms of grey mould were only apparent after harvest, while a greater
concentration of inoculum caused more servere disease on fruit. Flowers were
particularly sensitive to infection by B. cinerea and often died. The infection of
flowers by B. cinerea followed by a latent period while fruit developed prior to
disease symptoms forming has been reported for other fruit, including red
raspberry (Dashwood and Fox 1988), strawberry (Jarvis and Borecka 1968), grape
(Keller et al. 2003) and tomato (Lavy-Meir et al. 1989). This research confirmed
the latency of B. cinerea in capsicum fruit, but infection of flowers during
anthesis was probably not the only way for B. cinerea to infect fruit. Attached
petals and shed pollen lodged on young fruit may supply moisture and nutrients
for subsequent penetration of B. cinerea in fruit and this hypothesis requires
further research.

Wounded fruit that were inoculated with B. cinerea after harvest showed a quicker
development of grey mould than unwounded fruit when B. cinerea was present
but latent before harvest. Wounding breaks the cuticle and epidermis which are

147
the primary defence to protect fruit from pathogen. Wounding is not desirable for
marketability of fruit, but often occurs during harvest and postharvest handling of
capsicum. B. cinerea requires a wound in the epidermis to enter susceptible tissue
and initiate infection (Spotts et al. 1998). Wounding, therefore, probably caused
failure of cuticle and epidermis resistance, leading to cell death and nutrient
release which allowed B. cinerea to colonise tissue rapidly Also, the accumulation
of aldehydic products at wounding sites contributed to cell death associated with
preading lesion into healthy tissues (Deighton et al., 1999). The number of dead
flowers and disease incidence on fruit after harvest was positively correlated with
conidial concentration used to inoculate flowers or young developing fruit.
Development of grey mould on wounded fruit inoculated postharvest was also
positively correlated with conidial concentration of B. cinerea, regardless of
ripening stage. Cultural practices, including ensuring an open canopy and removal
of debris, to reduce moisture and inoculum could be applied to capsicum growing
in glasshouses to minimise infection by B. cinerea. Careful and hygienic
postharvest handling may help to reduce loss during subsequent storage and
marketing of capsicum fruit.

Capsicum cv. Aries was more susceptible to infection by B. cinerea than

capsicum cv. Papri Queen, suggesting the susceptibility of different cultivars to B.
cinerea may be genetically controlled. A genotypic difference in the production of
antifungal compounds has already been described for capsicum fruit (Howard et
al. 2000; Deepa et al. 2007). A greater accumulation of capsidiol, an antifungal
compound, was found a capsicum cultivar resistant to fungal pathogens (Egea at
al., 1996). A long term might therefore be to use plant breeding strategies to
create resistant cultivars. However, the basis of resistance in cv. Papri Queen in
the present research is not known and therefore further research is needed.

Preharvest application of Ca or B to the soil or as a foliar application reduced grey

mould development on fruit after harvest. In this study, Ca or B concentration in
fruit tissues did not increase greatly in response to their application to plants, but
did increase significantly in leaf tissues. There was a negative correlation between
grey mould on fruit from cv. Aries and B concentration in leaves and fruit, while
grey mould on fruit was not correlated with Ca concentration in leaves or fruit.

148
However, applying a higher concentration of Ca [4.0 mM Ca(NO₃)₂ via soil
amendment or 1.0% w/v Ca(NO₃)₂ as a foliar spray] appeared to reduce disease
incidence. The decrease of grey mould on fruit from cv. Aries that was derived
from young developing fruit inoculated preharvest may be due to the role of Ca
and B in the plant defence response, reducing cuticle degradation and maintaining
cell wall structure (Benhamou 1996; Blevins and Lukaszewski 1998; Hansch and
Mendel 2009), limiting latent infection of B. cinerea in fruit. Capsidiol, a
phytoalexin, is an antifungal compound produced by capsicum, but its presence
has not been reported specifically in response to Ca or B treatment in capsicum.
However, Ca and B (0.05 or 0.1 mM B via soil amendment or 2.0 or 7.0 mM B as
a foliar spray) were not important in reducing grey mould development on fruit
from the less susceptible cv. Papri Queen. The encouraging results from this
research suggest that preharvest application of Ca or B might be recommended to
growers as a potential way to reduce grey mould on fruit from susceptible
cultivars. However, preharvest application of Ca or B did not reduce grey mould
development on wounded fruit inoculated with B. cinerea after harvest. In
contrast, postharvest application of Ca, regardless of whether by dipping or
vacuum infiltration, reduced grey mould development on wounded fruit. A direct
inhibitory effect of Ca on conidial germination and germ-tube elongation on
wounded fruit was probably responsible for any effect seen (Wisniewski et al.
1995). Postharvest application of Ca could therefore be used to reduce grey mould
on fruit during storage and marketing in capsicum.

Quality parameters of fruit including extractable colour, firmness, total soluble

solid content (TSSC) and titratable acidity (TA), from plants treated with Ca and
B, regardless of whether by soil or foliar application did not improve compared to
the control. This was probably because application of Ca and B did not change
concentrations of nutrients in fruit due to natural immobility of Ca and B within
the plant (Brown and Shelp 1997; White and Broadley 2003). Calcium and B
increases cell wall strength and maintains structural integrity of the fruit
membrane (Hewett 2006). Increasing Ca and B has been demonstrated to improve
quality of fruit, such as apples (Khalifa et al. 2009), peach (Manganaris et al.
2005; Elmer et al. 2007), sweet cherry (Ippolito et al. 2005), strawberry

149
(Naradisorn et al. 2006) and table grape (Amiri et al. 2009). Therefore, if Ca and
B concentration in fruit can be increased the the quality of capsicum fruit may
also improve. Further research should therefore be focused on ways to increase Ca
or B in fruit tissue.

When capsicum plants received the same amount of Ca or B, regardless of

cultivar or application method, Ca or B concentration in the fruit was significantly
lower than that in the leaves. Even though Ca or B increased significantly in the
leaf tissue, there was no concomitant increase in the fruit tissue. These results
present a challenge to the capsicum industry for increasing Ca or B concentration
in the fruit. In addition, B concentration in the leaves and the fruit from plants that
received a soil application appeared to be greater than those from plants that
received a foliar spray. The natural waxiness of capsicum fruit (Govindarajan and
Salzer 1985) may prevent Ca and B from penetrating directly into the flesh when
applied as a foliar spray. Altering surface properties of capsicum fruit for Ca or B
penetration may be necessary by adding surfactants in nutrient solution before
spraying but this requires further research to determine appropriate levels of
surfactants in the nutrient solution for increasing Ca and B in fruit. Calcium
concentration in flesh after treatment also did not increase significantly when a Ca
solution was applied directly to red fruit postharvest by using a dip or vacuum
infiltration. This study only tested one duration of dipping and the effect of longer
times for Ca penetration may need to be examined.

Using fertigation to supply plants with Ca and B is easier than foliar spray for
growers because equipment use and labour can be saved. However, foliar
application would be necessary to maintain Ca and B nutrition at adequate levels
in fields without irrigation systems and/or when nutrient uptake by plants is low.
Foliar sprays should not be applied close to harvest because the negative effect on
taste of fruit may be detected by the customer due to residual salt on the surface of
fruit even after washing (Wójcik and Lewandowski 2003). Therefore, which
method is used depends on cultural practice and the desired quality of products.
However, care needs to be taken with B application because of the narrow range
between B deficiency and B toxicity and yield losses are possible when B is
applied to the soil because of toxicity. Symptoms of B toxicity were observed on

150
leaves from capsicum plants that received a soil application at 0.1 mM H₃BO₃, but
the yield and quality of fruit were not affected. Moreover, when plants had a
larger shoot mass (at the time of the second harvest), the B uptake appeared to be
decreased. Given that individual capsicum plants normally have more than two
harvests of red fruit, if B is not supplied between harvests then B deficiency is
very likely. Although foliar B spray was less effective than soil amendment with
H₃BO₃ to increase B in the shoot by the first harvest, foliar sprays may be
necessary to ensure B remains in the plant tissues at adequate levels throughout
the lifecycle.

Boron or Ca concentration in leaves from cv. Aries was significantly greater than
that in leaves from cv. Papri Queen. Boron or Ca uptake is dependent on
environmental and genotypic factors. Higher relative humidity was reported to
reduce transpiration rate in leaves leading to reduced Ca concentration in the
leaves, but increased Ca concentration in fruit of tomato (Adams and Holder
1992). At higher temperatures and in moist soil conditions, the rate of Ca and B
uptake by plants was significantly higher than that at lower temperatures and
when soil was dry soil (Gupta 1979; Klein and Ferguson 1987). Capsicum plants
in this research were grown in the greenhouse which differ in humidity and
temperature compared to the field. The effect of these factors on B or Ca status in
capsicum should be examined in further research. Moreover, the effect of
genotypic factors on Ca and B efficiency has been previously reported. For
example, maize inbred with the “Ca-efficient” genotype (‘Oh43’) only required
one quarter of the level of Ca required by maize inbred with the “Ca-inefficient”
(A251’) to produce the same shoot biomass (Clárk 1983). A genotype-related
difference in B uptake and distribution has also been reported in tomato (Bellaloui
and Brown 1998) and wheat (Ahmed et al. 2007). Having Ca and B available in
plant tissues may enable better plant defence responses against the pathogen
(Benhamou 1996; Blevins and Lukaszewski 1998). Therefore, Ca and B
efficiency in capsicum may provide better defence to pathogen attack. The effect
of genotypic factors on Ca and B efficiency in capsicum plant requires more
research.

151
7.2 Further research

From the findings of this research, there are a number of things that need further
research.

1. Determining the inoculum source for infection of B. cinerea preharvest.

Given the possible involvement of floral parts in infection of capsicum fruit
by B. cinerea, further study is required to examine the role of petals and shed
pollen in latent infection of developing fruit.

2. Identifying the basis of resistance against B. cinerea in capsicum.

Characterisation of genes in relation to coding for pathogenesis-related
proteins and defence responses may provide valuable information for
breeding programs to create resistance cultivars for the future. In tomato,
transgenic plants expressing polygalacturonase inhibitor protein (qPGIP)
reduced growth of B. cinerea on fruit (Powell et al. 2000). Absence of the
endo-β-1,4 glucanases Cel1 and Cel2 enhances resistance to B. cinerea (Flors
et al. 2007).

3. Determining factors which influence Ca and B uptake into the fruit.

Further research on the effects of environmental factors on Ca and B uptake
and distribution in the plant may provide better ways to increase Ca and B in
the capsicum fruit. Moreover, in order to increase Ca or B penetration into
fruit by using a foliar application, further studies are required to examine the
role of surfactants in altering permeability of the cuticles in capsicum fruit. In
addition, longer length of time for dipping fruit in Ca solution to provide
more opportunity for Ca to penetrate into flesh is required in further research.

Genetic engineering to increase Ca and B uptake and distribution in capsicum

fruit are required in further research to create Ca and B efficient cultivars for
long-term strategy.

152
7.3 Conclusions

Based on findings of this research and previously proposed questions (Chapter 2),
a model of the infection pathway of B. cinerea in capsicum fruit and the effect of
Ca or B nutrition on grey mould and quality of fruit has been established (Fig
7.1). This model may provide the basis for management of B. cinerea in the
capsicum industry. Moreover, there are some existing questions which need
further research so that better methods for controlling grey mould in capsicum
may be developed in the future.

153
 Fig. 7.1 Model of the infection pathway of B. cinerea in capsicum fruit and the effect of Ca or B on grey mould and quality of capsicum fruit

Postharvest Plants receive soil or

application of Postharvest infection foliar application of Preharvest infection
Ca Ca or B

Wounding Petals and Stem-end Stigma

? shed pollen

No increased Increased Ca or
Green stage Red stage ? Young fruit
Ca or B in fruit B in fruit Flowers
Low inoculum High inoculum (3 or 6 DAA)
?
? Improved quality of
? Grey mould on
Less grey mould Greater grey No change in Death
quality of fruit
fruit and no grey fruit after harvest
mould mould

Inhibitory effect Inhibitory effect of Increased lignification,

of Ca on hyphal Ca on conidial antifungal proteins
germination and Strengthened cell walls
growth germ-tube elongation
Reduced membrane permeability

?
Grey mould development not reduced Grey mould development reduced on fruit
on fruit from both cultivars from cv. Aries, but not fruit from cv.
Papri Queen

Infection pathway of B. cinerea in capsicum

The effect of preharvest application of Ca or B on grey mould development and quality of fruit
The effect of postharvest application of Ca on grey mould development on fruit
?? Unclear and further research is needed
154
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170
Appendix A.1

171
 Appendix A.2

Effect of soil boron application on nutrient concentrations in leaf and fruit of capsicum. The nutrient concentrations were determined using ICP-
OES analysis. Youngest mature leaves and red fruit for nutrient analysis were collected at harvest from plants that received 0, 0.05 or 0.1 mM
H₃BO₃ in Hoagland’s solution (200 mL per plant every 2 days). Data are presented as means ± SE from n = 6
cv. Aries cv. Papri Queen
Nutrient
(mg kg¯¹ 0.00 mM H₃BO₃ 0.05 mM H₃BO₃ 0.1 mM H₃BO₃ 0 mM H₃BO₃ 0.05 mM H₃BO₃ 0.1 mM H₃BO₃
DW) Fruit Fruit Fruit Leaf Fruit Leaf Fruit Leaf Fruit
Leaf tissues Leaf tissues Leaf tissues
tissues tissues tissues tissues tissues tissues tissues tissues tissues
41.22 ±
Iron 82.92 ± 9.21 47.19 ± 3.57 104.39 ± 12.76 48.33 ± 3.09 88.95 ± 3.95 44.87 ± 1.70 84.64 ± 3.64 34.71 ± 0.66 84.65 ± 1.11 44.31 ± 1.78 89.17 ± 5.40
1.88
Manganese 59.21 ± 11.68 9.29 ± 0.33 56.99 ± 6.20 10.85 ± 0.46 49.73 ± 7.99 11.93 ± 0.55 36.88 ± 2.09 8.71 ± 0.28 34.78 ± 5.11 8.51 ± 0.46 35.59 ± 3.61 9.15 ± 0.34
2.19 ±
Copper 5.19 ± 0.57* 4.23 ± 0.24 4.72 ± 0.51* 3.91 ± 0.28 4.33 ± 0.27* 3.69 ± 0.16 2.74 ± 0.23* 3.27 ±0.05
0.046*
2.57 ± 0.06 2.66 ± 0.18* 2.57 ± 0.12

Molybdenum 4.57 ± 1.08 1.05 ± 0.04 3.92 ± 0.72 1.28 ± 0.02 3.81 ± 0.15 1.18 ± 0.02 4.62 ± 0.49 < 1.00 4.54 ± 0.29 < 1.00 3.97 ± 0.22 < 1.00
20.32 ±
Zinc 33.56 ± 2.46 17.77 ± 0.51 37.60 ± 0.35 18.79 ± 0.87 34.43 ± 0.99 19.74 ± 0.36 22.96 ± 1.25 19.65 ± 0.33 24.68 ± 0.92 19.24 ± 0.66 26.00 ± 0.87
1.42
33500.00 ± 29250.00 ± 31000.00 ± 34750.00 ± 1008.33 ± 33000.00 ± 1141.67 ± 33750.00 ± 768.33 ±
Calcium 3500.00
836.67 ± 23.94
4346.93
1013.33 ± 54.28
3511.88
928.33 ± 49.27
2015.56 42.02 2160.25 27.76 478.71 25.63
5175.00 ± 1218.33 ± 5025.00 ± 1286.67± 5250.00 ± 1230.00 ±
Magnesium 4875.00 ± 221.27 1140.00 ± 39.82 4675.00 ± 385.95 1251.67 ± 36.09 4650.00 ± 322.75 1301.67 ± 53.72
85.39 34.62 118.15 30.92 107.78 42.73
35.04 ±
Sodium 0.80 ± 0.00 103.74 ± 2.26 0.78 ± 0.03 97.99 ± 3.52 0.80 ± 0.00 72.99 ± 3.56 < 0.80 51.98 ± 3.31 < 0.90 29.25 ± 1.20 < 0.90
1.70
50500.00 ± 26166.67 ± 48000.00 ± 25166.67 ± 49250.00 ± 24666.67 ± 47000.00 ± 24000.00 ± 49250.00 ± 21683.33 ± 45250.00 ± 25833.33 ±
Potassium 645.50 245.33 1683.25 245.33 1250.00 291.87 816.50 394.41 1547.85 486.53 750 702.51
2733.33 ± 2850.00 ± 2225.00 ± 2000.00 ± 2375.00 ± 2178.33 ± 2350.00 ± 2015.00 ±
Phosphorus 2475.00 ± 232.29
104.70
2800.00 ± 302.77
128.56
2525.00 ± 131.50 2681.67 ± 92.62
75.00 27.91 85.39 62.19 28.87 82.13
5025.00 ± 1591.67 ± 5100.00 ± 1588.33 ± 4675.00 ± 1623.33 ±
Sulphur 6250.00 ± 342.78 1941.67 ± 43.11 5650.00 ± 221.74 1951.67 ± 32.10 5700.00 ± 173.21 1911.67 ± 31.08
201.56 23.34 302.77 10.30 188.75 26.83
Aluminium 2.71 ± 0.63 2.49 ± 0.31 1.61 ± 0.43 1.85 ± 0.26 1.72 ± 0.23 0.81 ± 0.10 4.27 ± 1.39 1.43 ± 0.34 3.07 ± 0.43 0.49 ± 0.04 4.45 ± 1.84 0.57 ± 0.08
*deficient as stated by Reuter and Robinson (1997)

172
 Appendix A.3

Effect of foliar boron application on nutrient concentrations in leaf and fruit tissues of cv. Aries. The nutrient concentrations were determined
using ICP-OES analysis. Youngest mature leaves and red fruit for nutrient analysis were collected at harvest from plants that sprayed 0, 0.025 or
0.075 mM H₃BO₃ in the first trial and 0.0, 2.0 and 7.0 mM in the second trial from flowering to harvest red fruit. Plants were watered with
Hoagland’s solution without boron (200 mL per plant every 2 days). Data are presented as means ± SE from n = 3
The 1st trial The 2nd trial
Nutrient 0.00 mM H₃BO₃ 0.025 mM H₃BO₃ 0.075 mM H₃BO₃ 0.0 mM H₃BO₃ 2.0 mM H₃BO₃ 7.0 mM H₃BO₃
(mg kg¯¹ DW) Leaf Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit
Fruit tissues
tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues
Iron 68.00 ± 0.23 40.56 ± 4.81 60.88 ± 0.4 41.14 ± 2.04 57.24 ± 0.32* 34.06 ± 3.07 58.39 ± 0.01* 41.80 ± 41.12 69.25 ± 0.01 50.13 ± 3.60 56.79 ± 0.19* 47.77 ± 6.41
Manganese 64.10 ± 0.13 12.60 ± 1.03 66.64 ± 0.65 11.74 ± 0.71 72.14 ± 0.3 10.15 ± 1.89 53.31 ± 0.37 13.72 ± 0.68 54.43 ± 1.00 10.45 ± 1.12 62.44 ± 0.75 9.69 ± 0.82
Copper 3.01 ± 0.04* 3.56 ± 0.19 2.86 ± 0.01* 3.34 ±0.14 2.68 ± 0.01* 3.33 ± 0.26 3.05 ± 0.03* 4.63 ± 1.03 3.65 ± 0.03* 2.65 ± 0.09 3.17 ± 0.05* 3.02 ± 0.28
Molybdenu
5.68 ± 0.11 1.56 ± 0.05 5.39 ± 1.12 1.32 ± 0.04 4.65 ± 0.02 1.25 ± 0.09 3.51 ± 0.06 3.57 ± 1.26 3.82 ± 0.00 < 1.00 3.76 ± 0.02 1.00
m
Zinc 40.05 ± 0.18 18.86 ± 1.14 38.13 ± 0.16 18.82 ± 0.72 38.21 ± 0.2 15.58 ± 1.39 29.31 ± 0.11 27.26 ± 4.00 29.80 ± 0.76 17.52 ± 1.01 29.11 ± 0.26 22.76 ± 2.52
49000.00 ± 47000.00 ± 48000.00 ± 29500.00 ± 28000.00 ± 34000.00 ± 816.67 ±
Calcium 973.33 ± 20.28 903.33 ± 65.66 946.67 ± 124.1 823.33 ± 46.31 776.6 ±69.36
1000.00 0.00 0.00 500.00 0.00 0.00 102.69
5650.00 ± 1090.00 ± 3650.00 ± 1120.00 ± 4100.00 ± 1093.33 ± 4250.00 ± 1060.00 ±
Magnesium 5300.00 ± 0.00 1223.33 ± 50.44 5100.00 ± 0.00 1193.33 ± 66.92
50.00 129.00 50.00 78.10 100.00 60.64 50.00 77.67
Sodium 5.01 ± 0.72 45.87 ± 1.75 2.92 ± 0.58 48.01 ± 6.26 6.96 ± 1.09 49.94 ± 6.73 44.22 ± 0.89 74.21 ± 6.71 24.45 ± 1.03 83.16 ± 10.83 20.49 ± 0.87 90.53 ± 4.70
53500.00 ± 27666.67 ± 50000.00 ± 50500.00 ± 24000.00 ± 47000.00 ± 18900.00 ± 51500.00 ± 21100.00 ± 48000.00 ± 20666.67 ±
Potassium 24000.00 ± 0.00
500.00 881.92 0.00 500.00 2000.00 0.00 1050.40 1500.00 900.00 0.00 333.33
2550.00 ± 3650.00 ± 2633.33 ± 3600.00 ± 2800.00 ± 3400.00 ± 2566.67 ±
Phosphorus 2700.00 ± 0.00 3033.33 ± 202.76 2700.00 ± 0.00 2866.67 ± 66.67 2500.00 ± 0.00
453.70 50.00 240.37 100.00 208.17 100.00 176.38
1800.00 ± 1820.00 ± 1866.67 ± 5900.00 ± 1736.67 ±
Sulphur 5700.00 ± 0.00 2133.33 ± 88.12 5100.00 ± 0.00 1936.67 ± 58.41 5000.00 ± 0.00 6000.00 ± 0.00 6300.00 ± 0.00
161.70 140.12 17.64 100.00 78.39
Aluminium 1.36 ± 0.01 < 0.40 1.61 ± 0.17 < 0.30 1.33 ±0.36 < 0.40 3.52 ± 0.15 10.11 ±4.65 3.32 ± 0.33 0.76 ± 0.12 2.88 ± 0.31 1.29 ± 0.60
*deficient as stated by Reuter and Robinson (1997)

173
 Appendix A.4

Effect of foliar boron application on nutrient concentrations in leaf and fruit tissues of cv. Papri Queen. The nutrient concentrations were
determined using ICP-OES analysis. Youngest mature leaves and red fruit for nutrient analysis were collected at harvest from plants that sprayed
0, 0.025 or 0.075 mM H₃BO₃ in the first trial and 0, 2 and 7.0 mM in the second trial from flowering to harvest red fruit. Plants were watered
with Hoagland’s solution without boron (200 mL per plant every 2 days). Data are presented as means ± SE from n = 3
The 1st trial The 2nd trial
Nutrient 0.00 mM H₃BO₃ 0.025 mM H₃BO₃ 0.075 mM H₃BO₃ 0.0 mM H₃BO₃ 2.0 mM H₃BO₃ 7.0 mM H₃BO₃
(mg kg¯¹ DW) Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit
tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues
Iron 67.81± 0.97 36.01 ± 5.39 52.63 ± 1.64* 33.65 ± 2.13 57.14 ± 1.01* 43.26 ± 6.50 70.21 ± 0.89 42.42 ± 1.21 57.08 ± 0.48* 39.34 ± 5.86 57.69 ± 0.62* 35.39 ± 5.18
Manganese 52.38 ± 0.86 11.47 ± 0.50 45.79 ± 1.71 9.32 ± 0.48 58.87 ± 0.43 11.06 ± 1.10 46.28 ± 1.69 10.78 ± 0.26 46.49 ± 0.09 10.32 ± 1.05 48.17 ± 0.04 11.09 ± 0.39
Copper 3.69 ± 0.01* 3.39 ± 0.5 2.51 ± 0.05* 2.89 ± 1.34 2.73 ± 0.05* 3.86 ± 0.48 4.00 ± 0.11* 2.83 ± 0.03 2.87 ± 0.06* 3.35 ±0.19 3.94 ± 0.19* 3.0 ± 0.72
Molybdenum 5.04 ± 0.12 1.34 ± 0.20 5.65 ± 0.07 1.10 ± 0.06 4.78 ± 0.1 1.39 ± 0.31 4.17 ± 0.01 < 1.00 3.57 ± 0.15 < 1.00 3.62 ± 0.01 < 1.00
Zinc 47.04 ± 0.38 20.55 ± 1.93 42.70 ± 1.11 17.41 ± 1.18 40.19 ± 0.24 16.28 ± 1.17 31.53 ± 1.07 15.54 ± 0.56 36.11 ± 0.26 16.74 ± 1.47 30.37 ± 0.35 15.15 ± 1.89
1730.00 ± 43000.00 ± 41500.00 ± 1583.33 ± 23500.00 ± 27500.00 ± 23500.00 ± 800.00 ±
Calcium 36000.00 ± 0.00
191.40 0.00
1073.33 ± 44.1
500.00 257.70 500.00
746.67 ± 93.51
500.00
793.33 ± 23.33
500.00 174.74
5300.00 ± 1293.33 ± 5650.00 ± 4850.00 ± 1173.33 ± 4400.00 ± 1006.67 ±
Magnesium 100.00 23.33 50.00
1003.33 ± 33.83
50.00 121.97 100.00
990.00 ± 23.09 4800.00 ± 0.00 973.33 ± 89.69 4300.00 ± 0.00
38.44
Sodium < 1.00 56.67 ± 7.47 17.91 ± 1.89 42.72 ± 3.78 < 1.00 51.08 ± 6.50 51.62 ± 1.94 95.24 ± 8.78 23.54 ± 1.20 99.54 ± 8.03 28.06 ± 0.37 75.53 ± 7.72
26333.33 ± 54500.00 ± 26666.67 ± 54000.00 ± 25666.67 ± 49500.00 ± 25666.67 ± 43500.00 ± 24666.67 ± 50500.00 ± 23000.00 ±
Potassium 62000.00 ± 0.00
881.92 500.00 333.33 0.00 333.33 1500.00 1333.33 500.00 2333.33 500.00 1527.53
2600.00 ± 3066.67 ± 4200.00 ± 2700.00 ± 2966.67 ± 3950.00 ± 2566.67 ±
Phosphorus 3300.00 ± 0.00 3400.00 ± 0.00 2733.33 ± 145.3 3500.00 ± 0.00 4400.00 ± 0.00
251.66 284.80 100.00 57.74 133.33 50.00 317.98
1966.67 ± 2026.67 1840.00 ± 1976.67 ± 1773.33 ±
Sulphur 7450.00 ± 50.00
185.59
7100.00 ± 0.00 1923.33 ± 62.27 6000.00 ± 0.00
±126.67
7000.00 ± 0.00
85.05
6200.00 ± 0.00
75.35
6600.00 ± 0.00
78.81
Aluminium 4.19 ± 1.83 < 0.4 17.34 ± 13.52 < 0.3 3.00 ± 0.69 < 0.68 4.21 ± 0.44 2.71 ± 0.72 2.42 ± 0.10 3.50 ± 0.9 2.54 ± 0.23 0.82 ± 0.43
*deficient as stated by Reuter and Robinson (1997)

174
 Appendix A.5

Effect of soil Ca application on nutrient status in capsicum plant tissues. The nutrient concentrations were determined using ICP-OES analysis.
Youngest mature leaves and red fruit for nutrient analysis were collected at harvest from plants that received a soil application: 1.5, 4.0 or 8.0
mM Ca in Hoagland’s solution (200 mL each plant every 2 days) from flowering to harvest of red fruit. Data are presented as means ± SE from n
=3
cv. Aries cv. Papri Queen
Nutrient 1.5 mM Ca 4.0 mM Ca 8.0 mM Ca 1.5 mM Ca 4.0 mM Ca 8.0 mM Ca
(mg kg¯¹ DW)
Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit
tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues
Iron 85.50 ± 9.50 43.00 ± 1.53 113.00 ± 2.00 32.00 ± 3.00 93.00 ± 2.00 31.67 ± 1.86 84.50 ± 4.50 37.67 ± 3.76 127.50 ± 2.50 29.67 ± 2.85 104.00 ± 3.00 35.00 ± 2.00
Manganese *4.65 ± 0.65 13.33 ± 1.86 *7.55 ± 0.65 9.70 ± 0.66 *8.70 ± 0.40 10.73 ± 0.82 *4.50 ± 0.70 13.67 ± 0.88 *5.30 ± 0.00 8.73 ± 0.52 *7.50 ± 0.00 9.67 ± 0.33
Boron 43.00 ± 3.00 12.67 ± 0.67 61.00 ± 1.00 12.67 ± 1.20 56.50 ± 2.50 12.67 ± 0.33 48.50 ± 1.50 12.67 ± 0.67 49.00 ± 1.00 12.33 ± 0.33 49.00 ± 0.00 15.67 ± 0.88
Copper 8.10 ± 0.80 6.03 ± 0.38 8.35 ± 0.25 5.47 ± 0.68 7.90 ± 0.20 5.33 ± 0.65 6.15 ± 0.15 3.77 ± 0.18 8.20 ± 0.20 3.50 ± 0.15 8.50 ± 0.10 3.90 ± 0.06
Molybdenu
m 33.00 ± 0.00 1.77 ± 0.18 67.00 ± 0.00 1.90 ± 0.15 17.00 ± 0.00 1.03 ± 0.03 34.00 ± 1.00 0.73 ± 0.00 44.50 ± 0.50 1.43 ± 0.13 18.00 ± 1.00 1.01 ± 0.01
7200.00 ± 1153.33 ± 6000.00 ± 1283.33 ± 6800.00 ± 1050.00 ± 6450.00 ± 1123.33 ± 1206.67 ± 5400.00 ± 1083.33 ±
Magnesium
200.00 91.35 200.00 115.66 100.00 45.09 250.00 63.60 7000.00 ± 0.00 37.56 100.00 8.82
96.33 ±
Sodium 7.80 ± 1.00 183.00 ± 26.85 7.15 ± 0.95 96.00 ± 6.66 19.50 ± 7.50 142.00 ± 9.81 20.00 ± 15.00 170.00 ± 45.35 19.55 ± 11.45 68.67 ± 6.17 14.30 ± 3.00 11.84
82000.00 ± 25333.33 ± 62500.00 ± 25333.33 ± 54000.00 ± 26333.33 ± 75500.00 ± 23333.33 ± 68000.00 ± 24000.00 ± 59000.00 ± 24666.67 ±
Potassium 1000.00 666.67 1500.00 1855.92 0.00 1333.33 2500.00 1452.97 2000.00 577.35 1000.00 1201.85
7000.00 ± 3433.33 ± 5700.00 ± 2033.33 ± 6000.00 ± 2966.67 ± 2533.33 ± 1776.67 ±
Phosphorus
100.00 120.19 100.00 2900.00 ±208.17 3300.00 ± 0.00 66.67 100.00 145.30 5400.00 ± 0.00 66.67 4100.00 ± 0.00 33.33
7550.00 ± 2013.33 ± 1970.00 ± 7850.00 ± 1840.00 ± 6850.00 ± 1536.67 ± 7100.00 ± 1423.33 ± 6600.00 ± 1556.67 ±
Sulphur
450.00 101.71 8250.00 ± 50.00 165.63 250.00 65.06 50.00 35.28 100.00 50.44 100.00 38.44
Selenium < 9.5 <8 <7 <8 <7 <8 < 8.00 < 8.00 < 7.50 < 8.00 < 7.5 < 8.00

*deficient range as stated by Reuter and Robinson (1997)

175
 Appendix A.6

Effect of soil Ca application on nutrient status in capsicum plant tissues. The nutrient concentrations were determined using ICP-OES analysis.
Youngest mature leaves and red fruit for nutrient analysis were collected at harvest from plants that received a soil application: 0.0, 1.5 or 4.0
mM Ca in Hoagland’s solution (200 mL each plant every 2 days) from flowering to harvest of red fruit. Data are presented as means ± SE from n
=3
cv. Aries cv. Papri Queen
Nutrient 0.0 mM Ca 1.5 mM Ca 4.0 mM Ca 0.0 mM Ca 1.5 mM Ca 4.0 mM Ca
(mg kg¯¹ DW) Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit
tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues
Iron 66.30 ± 0.54 47.82 ± 0.66 77.93 ± 2.83 52.03 ± 3.79 *40.94 ± 3.08 37.08 ± 0.18 78.92 ± 1.80 46.55 ± 6.93 79.50 ± 9.71 47.69 ± 4.32 66.40 ± 3.33 41.80 ± 5.02
Manganese 195.46 ± 14.54 10.27 ± 0.21 220.00 ± 0.00 10.37 ± 0.76 57.21 ± 0.41 8.04 ± 0.36 78.88 ± 2.09 13.14 ± 1.12 132.75 ± 8.31 12.08 ± 0.33 47.26 ± 2.32 8.04 ± 1.16
Boron 117.40 ± 6.79 13.05 ± 0.08 105.21 ± 4.64 11.98 ± 0.40 113.69 ± 1.88 11.56 ± 0.51 62.87 ± 7.32 12.06 ± 0.17 86.41 ± 2.36 9.77 ± 0.42 86.79 ± 1.65 12.13 ± 0.26
Copper *3.91 ± 0.03 3.17 ± 0.17 *3.56 ± 0.23 3.54 ± 0.41 *2.57 ± 0.17 3.19 ± 0.10 *4.95 ± 1.05 3.08 ± 0.17 *2.94 ± 0.02 2.63 ± 0.03 *2.84 ± 0.19 2.60 ± 0.17
Molybdenum 2.65 ± 0.12 < 1.00 2.97 ± 0.03 1.12 ± 0.05 2.88 ± 0.21 1.14 ± 0.08 2.74 ± 0.09 1.03 ± 0.01 2.84 ± 0.07 < 1.00 3.49 ± 0.30 < 1.00
6200.00 ± 5300.00 ± 4100.00 ± 4800.00 ± 1033.33 ± 4400.00 ± 3700.00 ± 916.67 ±
Magnesium
300.00 890.00 ± 30.55 200.00 826.67 ± 29.06 100.00 960.00 ± 45.83 100.00 83.53 300.00 840.00 ± 11.55 300.00 143.45
223.12 ±
Sodium
6.74 ± 0.62 184.28 ± 3.16 20.43 ± 0.38 172.77 ± 7.25 19.75 ± 3.66 137.62 ± 18.11 17.93 ± 1.26 224.36 ± 23.34 10.76 ± 0.19 204.05 ± 25.95 11.19 ± 2.20 12.23
44500.00 ± 21000.00 ± 33500.00 ± 19500.00 ± 35500.00 ± 21466.67 ± 42000.00 ± 21333.33 ± 39500.00 ± 19400.00 ± 43000.00 ± 20200.00 ±
Potassium
500.00 577.35 500.00 776.75 1500.00 1348.25 1000.00 1201.85 1500.00 208.17 1000.00 416.33
2166.67 ± 2666.67 ± 1975.00 ± 2850.00 ± 2600.00 ± 2850.00 ± 2566.67 ± 2700.00 ± 2396.67 ±
Phosphorus
2100.00 ± 0.00 88.19 2550.00 ± 50.00 218.58 125.00 2600.00 ± 57.74 250.00 173.21 50.00 185.59 200.00 254.97
6300.00 ± 1823.33 ± 6200.00 ± 1950.00 ± 4750.00 ± 5800.00 ± 1876.67 ± 5550.00 ± 1833.33 ± 1736.67 ±
Sulphur
300.00 27.28 200.00 126.62 150.00 1846.67 ± 60.64 300.00 113.48 250.00 122.52 5300.00 ± 0.00 108.68
Aluminium 25.41 ± 0.07 3.08 ± 0.54 7.96 ± 0.11 3.62 ± 0.86 1.40 ± 0.21 1.84 ± 0.20 18.54 ± 2.41 4.90 ± 1.19 8.38 ± 0.74 3.33 ± 0.33 2.65 ± 0.31 4.69 ± 1.29

*deficient range as stated by Reuter and Robinson (1997)

176
 Appendix A.7

Effect of foliar Ca application on nutrient status in capsicum plant tissues. The nutrient concentrations were determined using ICP-OES analysis.
Youngest mature leaves and red fruit for nutrient analysis were collected at harvest from plants that received a foliar application: 0.5, 0.75 or
1.0% w/v Ca(NO₃)₂ from flowering to harvest of red fruit. Plants were watered with Hoagland’s solution without Ca (200 mL each plant for
every two days). Data are presented as means ± SE from n = 3
cv. Aries cv. Papri Queen
Nutrient 0.5% w/v Ca(NO₃)₂ 0.75% w/v Ca(NO₃)₂ 1.0% w/v Ca(NO₃)₂ 0.5% w/v Ca(NO₃)₂ 0.75% w/v Ca(NO₃)₂ 1.0% w/v Ca(NO₃)₂
(mg kg¯¹ DW)
Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit
tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues tissues
Iron 75.50 ± 0.50 67.00 ± 12.34 72.00 ± 2.00 54.00 ± 1.53 62.50 ± 0.50 37.00 ± 1.53 83.00 ± 1.00 46.00 ± 2.31 74.00 ± 4.00 39.67 ± 0.88 71.00 ± 3.00 42.67 ± 3.71
126.50 ±
Manganese 12.50 18.33 ± 2.60 128.50 ± 7.50 15.33 ± 1.67 104.50 ± 8.50 13.00 ± 0.58 100.00 ± 4.00 16.33 ± 1.20 90.00 ± 3.00 14.33 ± 0.67 77.50 ± 5.50 15.00 ± 0.58
Boron 69.00 ± 7.00 10.63 ± 0.37 72.50 ± 8.50 10.60 ± 0.95 76.00 ± 8.00 9.87 ± 0.66 74.50 ± 2.50 11.00 ± 0.58 66.00 ± 4.00 12.00 ± 0.78 47.00 ± 2.00 12.67 ± 0.33
Copper 10.30 ± 0.70 9.27 ± 0.38 9.75 ± 0.05 8.80 ± 0.40 8.85 ± 0.35 6.97 ± 0.23 *6.65 ± 0.15 7.30 ± 0.12 *5.15 ± 0.15 7.13 ± 0.27 *4.30 ± 0.10 6.40 ± 0.35
Molybdenum 6.95 ± 0.05 1.80 ± 0.25 5.80 ± 0.20 1.77 ± 0.12 7.10 ± 0.35 1.33 ± 0.12 6.00 ± 0.50 1.17 ± 0.07 6.05 ± 0.05 1.00 ± 0.00 4.80 ± 0.00 0.83 ± 0.04
5250.00 ± 1440.00 ± 5250.00 ± 1283.33 ± 4250.00 ± 4000.00 ± 1263.33 ± 3750.00 ± 1330.00 ± 3250.00 ± 1240.00 ±
Magnesium 350.00 87.18 150.00 77.53 50.00 1103.33 ± 3.33 100.00 85.70 150.00 36.06 150.00 45.09
7850.00 ± 118.33 ± 7550.00 ± 6850.00 ± 6600.00 ± 6700.00 ± 5750.00 ±
Sodium 250.00 14.53 150.00 110.33 ± 12.12 200.00 80.33 ± 4.10 250.00 94.67 ± 14.31 650.00 69.67 ± 8.45 350.00 34.00 ± 5.51
44000.00 ± 23666.67 ± 47500.00 ± 23333.33 ± 43500.00 ± 20233.33 ± 48500.00 ± 25666.67 ± 42500.00 ± 24666.67 ± 39500.00 ± 27666.67 ±
Potassium 2000.00 333.33 1500.00 881.92 1500.00 392.99 500.00 881.92 500.00 666.67 1500.00 1666.70
3050.00 ± 3500.00 ± 3050.00 ± 3500.00 ± 2850.00 ± 2933.33 ± 3050.00 ± 3566.67 ± 2450.00 ± 3466.67 ± 2300.00 ± 3500.00 ±
Phosphorus
250.00 152.75 50.00 57.74 150.00 66.67 50.00 66.67 50.00 33.33 100.00 115.47
7850.00 ± 2133.33 ± 7550.00 ± 2103.33 ± 6850.00 ± 1766.67 ± 6600.00 ± 1700.00 ± 6700.00 ± 1550.00 ± 5750.00 ± 1583.33 ±
Sulphur 150.00 33.33 150.00 112.60 50.00 24.04 300.00 46.19 100.00 20.00 150.00 86.67
Aluminium 9.65 ± 1.35 6.03 ± 0.23 9.50 ± 1.50 4.50 ± 0.49 11.50 ± 1.500 2.47 ± 0.58 8.05 ± 0.15 3.80 ± 3.06 9.25 ± 0.75 1.77 ± 1.27 7.40 ± 0.2 0.90 ± 0.01

177
 Appendix A.8

Effect of foliar Ca application on nutrient status in capsicum plant tissues. The nutrient concentrations were determined using ICP-OES analysis.
Youngest mature leaves and red fruit for nutrient analysis were collected at harvest from plants that received a foliar application: 0.0, 0.5 or
1.0% w/v Ca(NO₃)₂ from flowering to harvest of red fruit. Plants were watered with Hoagland’s solution without Ca (200 mL each plant for
every two days). Data are presented as means ± SE from n = 3
cv. Aries cv. Papri Queen
Nutrient 0.0% w/v Ca(NO₃)₂ 0.5% w/v Ca(NO₃)₂ 1.0% w/v Ca(NO₃)₂ 0.0% w/v Ca(NO₃)₂ 0.5% w/v Ca(NO₃)₂ 1.0% w/v Ca(NO₃)₂
(mg kg¯¹ DW)
Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit
Leaf tissues Fruit tissues Leaf tissues Fruit tissues
tissues tissues tissues tissues tissues tissues tissues tissues
44.13207 ±
Iron 92.29 ± 1.23 49.83 ± 9.12 99.13 ± 0.06 38.41 ± 2.55 90.96 ± 0.66 44.46 ± 0.87 109.79 ± 0.64 0.18 121.40 ± 0.99 60.05 ± 3.10 113.88 ± 1.32 56.09 ± 4.14
15.57581 ±
Manganese 146.52 ± 1.67 17.21 ± 0.52 161.76 ± 1.49 15.14 ± 1.45 174.19 ± 1.27 14.24 ± 0.27 112.52 ± 0.25 0.53 115.76 ± 0.04 18.98 ± 0.63 120.45 ± 0.72 20.43 ± 4.74
19.27215 ±
Boron 330.00 ± 0.00 17.14 ± 1.49 285.00 ± 5.00 12.32 ± 0.22 250.00 ± 0.00 12.89 ± 0.52 280.00 ± 0.00 1.86 270.00 ± 0.00 18.83 ± 0.63 250.00 ± 0.00 20.37 ± 2.58
2.547399 ±
Copper 5.69 ± 0.98 4.45 ± 0.75 5.90 ± 0.72 3.93 ± 0.07 5.01 ± 0.04 3.97 ± 0.09 *3.94 ± 0.02 0.25 *4.64 ± 0.16 3.98 ± 0.54 *4.07 ± 0.03 3.67 ± 0.26
Molybdenum 2.22 ± 0.03 1.35 ± 0.02 2.13 ± 0.08 < 2.00 1.62 ± 0.01 < 2.00 2.79 ± 0.03 < 1.03 ± 0.03 3.38 ± 0.05 < 1.00 2.77 ± 0.05 < 1.50
Zinc 30.51 ± 0.79 27.72 ± 3.04 30.97 ± 0.08 21.95 ± 3.82 26.62 ± 0.23 20.37 ± 0.69 24.87 ± 0.06 17.29 ± 0.39 25.47 ± 0.03 26.68 ± 2.41 19.39 ± 0.31 23.58 ± 1.38
7100.00 ± 1285.00 ± 5550.00 ± 6450.00 ± 1810.00 ± 1900.00 ±
Magnesium 100.00 1660.00 ± 60.00 6300.00 ± 0.00 1340.00 ± 40.00 5900.00 ± 0.00 105.00 50.00 1515.00 ± 5.00 50.00 120.00 5200.00 ± 0.00 200.00
189.81 ±
Sodium 44.50 ± 2.30 169.60 ± 16.64 66.32 ± 0.26 176.00 ± 15.78 45.80 ± 0.84 215.00 ± 5.00 29.63 ± 0.66 134.98 ± 21.61 32.43 ± 0.42 205.32 ± 34.68 60.16 ± 0.45 20.19
75000.00 ± 27500.00 ± 77500.00 ± 72500.00 ± 26000.00 ± 74000.00 ± 25500.00 ± 75000.00 ± 28000.00 ± 75500.00 ± 32500.00 ±
Potassium 1000.00 1500.00 1500.00 25000.00 ± 0.00 500.00 1000.00 0.00 500.00 0.00 0.00 1500.00 3500.00
3250.00 ± 3000.00 ± 4050.00 ± 3750.00 ±
Phosphorus 3450.00 ± 50.00 3400.00 ± 400.00 4000.00 ± 0.00 3150.00 ± 150.00 3300.00 ± 0.00 150.00 4800.00 ± 0.00 100.00 4500.00 ± 0.00 3600.00 ± 0.00 50.00 350.00
9500.00 ± 8050.00 ± 2150.00 ± 8050.00 ± 1615.00 ± 1960.00 ± 1985.00 ±
Sulphur 100.00 2300.00 ± 200.00 9600.00 ± 0.00 2075.00 ± 125.00 50.00 50.00 50.00 55.00 8200.00 ± 0.08 140.00 7550.00 ± 0.00 15.00
Aluminium 12.66 ± 0.47 2.13 ± 0.09 13.29 ± 0.01 5.45 ± 0.34 9.84 ± 0.54 4.93 ± 1.76 11.43 ± 0.21 1.77 ± 1.20 7.02 ± 0.00 1.95 ± 1.10 8.29 ± 0.00 2.91 ± 1.04
*deficient range as stated by Reuter and Robinson (1997)

178
Appendix A.9

Effect of postharvest calcium dips on nutrient concentrations in fruit tissues. The nutrient concentrations were determined using ICP-OES
analysis. Fruit for nutrient analysis were collected at 0 day after harvest and 0 day of storage. Fruit were treated at four concentrations: 0, 2, 5
and 8% w/v CaCl₂.2H₂O. Dipping was conducted by placing fruit in the respective calcium solution for 5 min. Data are presented as means ± SE
from n = 3
cv. Aries cv. Papri Queen
Nutrient
(mg kg¯¹ DW) 0.0 % 2.0 % 5.0 % 8.0 % 0.0 % 2.0 % 5.0 % 8.0 %
CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O
Iron 49.05 ± 6.29 39.77 ± 3.12 45.85 ± 2.73 39.21 ± 2.13 37.01 ± 2.20 36.57 ± 2.42 37.28 ± 2.87 35.07 ± 1.18
Manganese 12.59 ± 0.85 13.21 ± 3.12 14.46 ± 1.93 16.53 ± 1.08 14.47 ± 1.15 16.92 ± 3.61 11.81 ± 0.72 16.78 ±0.36
Boron 14.86 ± 1.86 15.07 ± 1.56 16.80 ± 2.02 14.34 ± 0.02 16.89 ± 0.34 18.60 ± 1.85 17.11 ± 0.02 17.91 ± 0.07
Copper 7.85 ± 0.92 8.97 ± 0.67 13.15 ± 4.11 9.15 ± 0.80 8.10 ± 1.31 8.12 ± 1.77 7.39 ± 0.89 6.88 ± 0.96
Nickel < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00
Zinc 30.17 ± 8.87 25.62 ± 0.59 21.33 ± 0.51 22.55 ± 1.88 29.56 ± 2.13 27.16 ± 0.48 22.02 ± 2.64 21.87 ± 0.89
1075.00 ±
Magnesium 970.00 ± 20.00 945.00 ± 175.00 1075.00 ± 45.00 950.00 ± 70.00 1090.00 ± 10.00
105.00
955.00 ± 85.00 1165.00 ± 95.00
Sodium 198.52 ± 41.48 193.93 ± 16.07 138.52 ± 0.28 130.41 ± 15.71 99.61 ± 14.79 123.70 ± 26.53 91.00 ± 3.03 90.62 ± 17.81
20950.00 ± 22000.00 ± 21500.00 ± 24500.00 ± 26000.00 ±
Potassium 1050.00 1000.00
21500.00 ± 500.00
500.00 1500.00
26000.00 ± 0.00 25000.00 ± 0.00
1000.00
3150.00 ± 3000.00 ± 3150.00 ±
Phosphorus 2950.00 ± 50.00 3000.00 ± 300.00 3350.00 ± 50.00 3100.00 ± 100.00 3300.00 ± 100.00
550.00 200.00 150.00
1910.00 ± 1730.00 ± 1860.00 ±
Sulphur 1825.00 ± 15 1840.00 ± 100.00 1955.00 ± 45.00 1735.00 ± 15.00 1940.00 ± 360.00
390.00 250.00 340.00
Aluminium 9.08 ± 4.18 5.96 ± 0.79 3.71 ± 1.58 5.52 ± 0.59 4.52 ± 2.97 4.77 ± 0.24 1.72 ± 0.89 3.75 ± 2.78

179
Appendix A.10

Effect of storage time and postharvest Ca dips on nutrient status of fruit from cv. Aries. The nutrient concentrations were determined using ICP-
OES analysis. Fruit were treated with 8% w/v CaCl₂.2H₂O and analysed after 10 days of storage. Control fruit were treated with RO water and
nutrient analysed at harvest. Dipping was conducted by placing fruit in calcium solution for 5 min. Fruit were peeled with a knife to about 1 mm
deep to collect peel and flesh for nutrient analysis. Data are presented as means ± SE from n = 3
Nutrient Dip (8% w/v CaCl₂.2H₂O)
Control (flesh)
(mg kg¯¹DW) Flesh Peel
Iron 49.83 ± 9.12 36.74 ± 0.86 53.52 ± 1.59
Manganese 17.21 ± 0.52 13.15 ± 0.1 23.73 ± 0.65
Boron 17.14 ± 1.49 15.24 ± 0.9 16.76 ± 0.87
Copper 4.45 ± 0.75 3.65 ± 0.4 4.07 ± 0.00
Molybdenum 1.35 ± 0.02 < 2.00 < 1.00
Zinc 27.72 ± 3.04 17.08 ± 0.58 13.69 ± 1.67
Magnesium 1660.00 ± 60.00 1570.00 ± 70.00 1580.00 ± 180.00
Sodium 169.60 ± 16.64 191.00 ± 29.00 141.77 ± 14.19
Potassium 27500.00 ± 1500.00 24500.00 ± 500.00 18350.00 ± 1650.00
Phosphorus 3400.00 ± 400.00 2500.00 ± 100.00 2500.00 ± 400.00
Sulphur 2300.00 ± 200.00 1850.00 ± 80.00 2085.00 ± 115.00
Aluminium 2.13 ± 0.09 4.07 ± 0.67 0.92 ± 0.13

180
 Appendix A.11

Effect of postharvest Ca vacuum infiltration on nutrient concentrations in fruit tissues. The nutrient concentrations were determined using ICP-
OES analysis. Fruit for nutrient analysis were collected at harvest. Fruit were treated at four concentrations: 0, 2, 5 or 8% w/v CaCl₂.2H₂O.
Vacuum infiltration was conducted by placing fruit in the respective calcium solution, drawing a vacuum for 10 sec and then holding fruit in
solution for 2 min before vacuum release. Data are presented as means ± SE from n = 3
cv. Aries cv. Papri Queen
Nutrient
0.0 % 2.0 % 5.0 % 8.0 % 0.0 % 2.0 % 5.0 % 8.0 %
(mg kg¯¹ DW)
CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O CaCl₂.2H₂O
Iron 43.76 ± 6.50 46.31 ± 5.41 54.31 ± 7.69 51.98 ± 7.36 43.78 ± 2.42 53.94 ± 10.48 43.93 ± 8.19 36.76 ± 2.87
Manganese 13.35 ± 3.44 17.53 ±0.01 13.00 ± 1.08 17.94 ± 1.18 14.34 ± 0.66 19.40 ± 1.91 13.19 ± 4.08 20.35 ± 0.89
Boron 13.77 ± 2.52 15.43 ± 2.80 15.29 ±1.01 16.63 ± 0.53 15.69 ± 2.54 14.94 ± 0.32 13.02 ± 0.24 19.03 ± 2.27
Copper 8.56 ± 1.43 10.58 ± 1.81 9.65 ± 2.80 11.77 ± 1.54 8.89 ± 1.48 9.27 ± 1.05 9.40 ± 1.74 6.55 ± 0.30
Nickel < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.50 < 1.00 < 1.00
Zinc 28.88 ± 4.20 34.49 ± 13.21 42.91 ± 13.21 27.32 ± 6.32 30.94 ± 6.31 35.42 ± 5.92 25.68 ± 1.91 23.93 ± 1.11
1515.00 ±
Magnesium 1010.00 ± 150.00 1090.00 ± 30.00 995.00 ± 30.00 1205.00 ± 35.00 1085.00 ± 75.00
145.00
1115.00 ± 85.00 1285.00 ± 75.00
Sodium 195.38 ± 0.21 172.71 ± 27.29 206.74 ± 27.29 143.96 ± 3.63 82.46 ± 1.75 95.32 ± 6.96 147.49 ± 47.12 71.96 ± 15.88
22300.00 ± 24500.00 ± 24500.00 ± 28000.00 ± 26500.00 ± 28000.00 ±
Potassium 2700.00 4500.00 4500.00 1000.00
24000.00 ± 0.00 26000.00 ± 0.00
1500.00 3000.00
3500.00 ± 3000.00 ±
Phosphorus 3150.00 ± 250.00 3400.00 ± 300.00 3150.00 ± 300.00 3650.00 ± 50.00 3350.00 ± 150.00 3650.00 ± 50.00
300.00 100.00
2255.00 ±
Sulphur 1860.00 ± 240.00 2055.00 ± 245.00 1915.00 ± 245.00 2300.00 ± 0.00 1800.00 ± 300.00 2040.00 ± 60.00
345.00
1645.00 ± 95.00
Aluminium 4.82 ± 1.54 6.16 ± 3.14 7.72 ± 3.14 4.59 ± 3.38 1.58 ± 0.14 7.35 ± 5.50 2.08 ± 1.65 0.97 ± 0.12

181
Appendix A.12

Effect of storage time and postharvest Ca vacuum infiltration on nutrient status of fruit from cv. Aries. Fruit were vacuum infiltrated with 8%
w/v CaCl₂.2H₂O and nutrient analysed after 10 days of storage while control fruit were vacuum infiltrated with RO water and nutrient analysed
at harvest. Vacuum infiltration was conducted by placing fruit in Ca solution, drawing a vacuum for 10 sec and then holding fruit in solution for
2 min before vacuum release. Fruit were peeled with a knife to about 1 mm deep to collect peel and flesh for nutrient analysis. The nutrient
concentrations were determined using ICP-OES analysis. Data are presented as means ± SE from n = 3
Nutrient Vacuum infiltration
Control (flesh)
(mg kg¯¹DW) Flesh Peel
Iron 49.83 ± 9.12 39.09 ± 0.18 59.78 ± 5.13
Manganese 17.21 ± 0.52 15.26 ± 0.32 28.86 ± 0.26
Boron 17.14 ± 1.49 13.16 ± 0.28 17.23 ± 0.68
Copper 4.45 ± 0.75 2.76 ± 0.45 4.07 ± 0.08
Molybdenum 1.35 ± 0.02 < 3.00 < 1.00
Zinc 27.72 ± 3.04 15.23 ± 0.56 19.08 ± 0.34
Magnesium 1660.00 ± 60.00 1725.00 ± 105.00 1580.00 ± 40.00
Sodium 169.60 ± 16.64 250.00 ± 20.00 176.09 ± 33.92
Potassium 27500.00 ± 1500.00 21000.00 ± 1000.00 20150.00 ± 1850.00
Phosphorus 3400.00 ± 400.00 2200.00 ± 0.00 2650.00 ± 250.00
Sulphur 2300.00 ± 200.00 1820.00 ± 0.00 2250.00 ± 150.00
Selenium < 5.00 < 10.00 < 4.00

182