UNIVERSITY OF AGRICULTURE FAISALABAD
Faculty of Basic Sciences
Department of Botany
(SYNOPSIS FOR M. Phil. IN BOTANY)
TITLE: ROLE OF SODIUM 4-CPA AND FUNGICIDES TO CONTROL
POST HARVEST ISSUES OF KINNOW
Name of the Student: Lubna Shehzadi
Registration No: 2019-Ag-6572
ABSTRACT
The Kinnow is the most widely grown mandarin in Pakistan. It is a King x Willow leaf hybrid,
developed in 1915 by H.B. Frost of the Riverside University Research Centre, California,
classified and released in 1935. The fruit is medium-sized, with a smooth, glossy rind, yellowish
orange when ripe. The flesh is very juicy with a rich, fragrant, distinctive flavour, and has
numerous seeds. The tree is vigorous with dense foliage, thornless branches and lanceolate
leaves. The Kinnow mandarin ripens between January, March, and holds well on the tree. The
most prominent citrus variety produced in Pakistan is the Kinnow mandarin, which accounts for
about 80% of the total citrus production. Other varieties include sweet oranges, grapefruit,
lemons, and limes. The Kinnow is particularly valued for its juiciness, ease of peeling, and high
nutritional content. In 2022, Pakistan produced approximately 2.6 million tons of citrus, with
Punjab province being the primary production area, especially the districts of Sargodha. In this
study, sodium 4-CPA and different fungicide treatments will be used to check their effect on the
storage quality of Kinnow Mandarin. Treatment will include control T3(Wax only) T4(Wax+
TBZ+ IMZ) T5(Wax+ TBZ+ IMZ+ Beijia) T6(Wax+ Beijia and Bixian). All fruits will be placed
in modified atmosphere packaging and stored in cold storage for maximum 90 days. Change in
fruit quality will be determined every 15 days until eatable ripening stage. Data will be checked
from physical parameters (produce quality, colour content, fruit firmness, weight loss, softness,
and overall visual qualities), organoleptic parameters and biochemical parameters (TSS, TA,
Vitamin C, and pH). Using Statistic 8.1 software, the analysis of variance technique will be used
for statistical analysis. The study will employ a completely randomized design.
UNIVERSITY OF AGRICULTURE FAISLABAD
Department of Botany
Faculty of Sciences
(SYNOPSIS FOR M. Phil. IN BOTANY)
TITLE: EFFECT OF 1-METHYLECYCLOPROPENE ON STRORAGE
QUALITY OF KINNOW MANDARIN
II. a) Date of Admission
b) Date of Initiation (Research) 14-03-2024
c) Probable (Duration Research) (Three Months)
III. PERSONNEL
a) Name of the student Lubna Shahzadi
b) Registration Number 2019-ag-6572
IV. SUPERVISORY COMMITTEE
i) Supervisor Dr Raheel Anwar
raheelanwar@uaf.edu.pk
ii) Member Dr Muhammad Shahbaz
shahbazmuaf@uaf.edu.pk
iii) Member Mr. Karim Yar Abbasi
karimabbasi@uaf.edu.pk
Introduction
Citrus belongs to the family Rutaceae, a mainly cultivated crop of subtropical and tropical
regions in the world (Shireen et al., 2018). Citrus is a highly nutritious fruit and considered a rich
source of vitamin C (Malik et al., 2021), dietary components and antioxidant activity (Goulas
and Manganaris, 2012). In Pakistan, citrus ranks top of the list in area (0.18 million ha) and
production (2.47 million tons) among fruits (MNFSR, 2020). ‘Kinnow’ mandarin is considered
as the prime cultivar of Pakistan, retain highest position in production (>2.1 million tons) and
export (300,000 metric tons, worth > USD 180 million).
Kinnow’ mandarin is a prime cultivar which has dominated Pakistan’s citrus industry (Khan et
al., 2020). Nevertheless, ‘Kinnow’ mandarin fruit is fetching low prices in the international
market; thus, the national economy faces a loss of millions of dollars annually . The reason
behind the lower price is low fruit quality production, mostly B grade. The basic reason for its
downgraded quality is the higher extent of blemishes on the skin of fruit. Blemished fruit
production has not only affected our export to international markets but also caused huge losses
to ‘Kinnow’ growers.
A comprehensive report in 2014 has been released on ‘Kinnow’ quality issues with their causes
and provides a way out for possible management strategies. Poor rind quality has been reported
as the major cause for farmgate rejection of fruits by the processors and traders. However, the
rejection rate varies between 20 to 50%, sometimes rejecting the complete orchard due to poor
management and quality (Malik and Khan, 2014). Such a high farm gate rejection and low price
make our ‘Kinnow’ production and export unsustainable.
Earlier studies revealed that citrus groves had been found susceptible to different diseases
markedly damaging the plant health and downgrading the external and internal fruit quality.
Nevertheless, fungal infections, including citrus scab and melanose known as viruses’ among the
local community, have been reported as the most devastating issue in citrus quality, end up in
high rejection at farmgate (Malik and Khan, 2014; Malik et al., 2021). In addition, bacterial
diseases such as citrus canker and greening are also reported in citrus groves, badly affecting the
cosmetic quality of fruits (Ahmed, 2005).
Currently, crop losses have been reported prevented by the regular use of fungicides employed
worldwide. However, the application of fungicides is extremely needed to be optimized to reduce
pesticide residues, environmental pollution, fungicide efficacy, risk of resistance development
and negative impact on beneficial organisms (Rebollar-Alviter and Nita, 2011). Multiple
chemicals have been formulated according to a specific disease.
There have been several attempts, with differing degrees of success, to increase the storage life
of Kinnow Mandarin, such as cold storage (Singh and Jain, 2004), use of sodium bicarbonate
(2%) dip for 2 mints (Singla et al., 2018), applying Nipro Fresh SS40T and SS50 coatings, and
packing with altered atmosphere. The main causes of postharvest losses, which drastically reduce
the shelf life of the Kinnow, are fungus-related illnesses and physiological processes like
respiration, ethylene release, and enzyme activity (Singh and Mandal, 2006). Pre-harvest tree
treatments or post-harvest pre-storage actions can prolong the storage life. For mandarins, to
increase their shelf-life antifungal agent sodium 4-CPA and fungicides such as IMZ and TBZ and
also wax coating are frequently used postharvest pre storage treatment.
4-Chlorophenoxyacetic acid or parachlorophenoxyacetate (pCPA) is a synthetic pesticide similar
to chemicals in a group of plant hormones called auxins.4-Chlorophenoxyacetic acid or
parachlorophenoxyacetate (pCPA) is a synthetic pesticide similar to chemicals in a group of
plant hormones called auxins.
Fungicide application in citrus packinghouses is one of the most important steps for the effective
control of postharvest decay. To date, the most efficient method of fungicide application utilises a
warm aqueous suspension of the fungicide where water temperature, duration of fruit contact
with the fungicide solution and fungicide concentration in the solution affect fungicide efficacy.
However, the application of IMZ mixed with wax shows low efficacy ( Erasmus et al., 2011).
Imazalil (IMZ) is a postharvest fungicide that often gets incorporated in wax coatings (Kaplan
and Dave, 1979) and applied to control infections caused by Penicillium digitatum Sacc. and
Penicillium italicum Wehmer. According to previous studies, the incorporation of IMZ in the
wax coating reduces its effectiveness to control green mould as the residues remain bound to the
surface of the fruit with only small amounts penetrating wounds infected with P. digitatum
(Njombolwana et al, 2013) confirmed that IMZ in the wax coating provided better green mould
control in protective treatments than in curative treatments and improved sporulation inhibition.
Thiabendazole can also be applied in a wax coating, but it was shown to be less effective in
controlling disease than other application methods (Brown, 1984). Fungicides applied in a wax
coating led to better protective control (Njombolwana et al.,2013).
VI. Objective
The aim of this research was to evaluate the effects of fungicide, wax coating and
various combinations of above treatments which are more effective on shelf life and quality of
Kinnow mandarin cultivars during cold storage.
VII. Review of Literature
Citrus fruits have a relatively longer post-harvest life in comparison with other tropical and
subtropical fruits. Proper storage of citrus fruits for an extended period is very essential for their
proper utilization in the glut season (Sonkar et al. 2008). Kinnow is highly cherished as fresh and
in processed form as well due to its quality attributes, particularly its tangy taste. While at its
peak production during winter, it is processed into juices by the industry and fruit vendors (Rafiq
et al. 2018). The fruit is abundantly rich in vitamin C, vitamin B, β-carotene, calcium and
phosphorous. Its startling colour, distinguishing flavour and nutritional content create an impulse
for beverages preparation (Sogi and Singh 2001). Despite its commercial prominence and
nutritional benefits, the availability of fruit during off-season is very limited due to its poor shelf
life of 8–10 days.
The most common and most serious postharvest diseases for citrus fruits are green and blue
moulds, which are caused by Penicillium digitatutm and P. italicum, respectively ( Ismail &
Zhang, 2004). Both pathogens predominantly attack blood oranges and lemons picked during the
period late winter to early spring. Decay control actions are based on both chemical and
technological treatments. The first step in pathogen control is temperature and humidity
management during handling, storage and transport operations (Lanza, Strano, & Calandra,
2006). An improvement in the postharvest lifetime of citrus fruit is contingent on the chemical
fungicide treatment coupled with temperature and humidity control (Lanza & Strano, 2009).
Fungicide application in citrus packinghouses is one of the most important steps for the effective
control of postharvest decay. To date, the most efficient method of fungicide application utilises
a warm aqueous suspension of the fungicide where water temperature, duration of fruit contact
with the fungicide solution and fungicide concentration in the solution affect fungicide efficacy.
However, the application of IMZ mixed with wax shows low efficacy (Erasmus et al., 2011)
fungicides applied in a wax coating led to better protective control (Njombolwana et al., 2013).
Wounds occur on fruit due to poor harvesting practices, and this is an entry point for P.
digitatum spores from the orchard (Smilanick et al., 2005).
Imazalil (IMZ) is the most widely used postharvest fungicide in citrus (Smilanick, Brown, &
Eckert, 2006). However, it is ineffective in the treatment of other diseases such as stem rot, sour
rot and brown rot. At 20 °C, the recommended dose of IMZ is 1000 mg/kg when used in a
water-based mixture and 2000 mg/kg when used as a wax-based mixture (Angioni, et.al. 2011).
Citrus fruits are perishable products and are not only prone to postharvest decay, but also to a
reduction in quality due to aspects such as postharvest water loss through transpiration and
respiration (Mannheim and Soffer, 1996). Fruit has natural wax layers on the surface that
reportedly get removed or disturbed as the fruit go through a long washing process in the
packhouse, and therefore need to be replaced in the packhouse to avoid dehydration (Ahmed et
al., 2007).
Waxing is one of the most important postharvest applications to fruit to prevent undesirable
changes and extend shelf-life. Important attributes of wax coatings on citrus fruit include
imparting a good shiny attractive appearance that will last through the whole marketing process,
reducing fruit weight loss and shrinkage, and preservation of fruit quality. Additionally, it is
expected to be useful as a carrier of fungicides (Hagenmaier, 2002). A good coating also
provides more protection against postharvest physiological disorders such as stem-end rind
breakdown and chilling injury (Dou, 2004). Fungicides are usually added to the wax or wax
emulsion. Waxing in combination with some chemicals viz. sesame oil, captafol, potassium
permanganate, and mustard oil helped in improving the shelf life and reducing the physiological
loss in weight (PLW) of kinnow (Sharma et al. 1991). Waxol™ is a commercial paraffin wax
emulsion and its 12% concentration helped in the retention of highest quality kinnow for 35
days.
In packhouses, IMZ is usually mixed with wax which is typically sprayed onto citrus fruit over
rotating brushes. This method simplifies packinghouse operations because the upgrade needed in
the packing line is inexpensive and straightforward, and, unlike dipping, there are no problems
due to drainage and water treatment. Moreover, various problems arise when spraying IMZ
mixed with wax. The nozzles may become blocked, which adversely affects treatment efficacy
and uniformity of distribution. There may also be dispersion and loss of fungicide and wax onto
the brushes and along the packing line as well as an accumulation of fungicide on the brushes
leading to waste problems. Spores may also accumulate in the brushes, requiring regular
cleaning (Taverner & Cunningham, 1999).
The wax contains several alkaline soluble materials that can cause more IMZ to be partitioned
into the wax, which results in reduced amounts of IMZ in the aqueous phase available to
penetrate the fruit peels (Brown, Nagy, & Maraulja, 1983). These problems all require an
increase in fungicide concentration (Eckert and Eichom,1977).
Thiabendazole (4-(1H-1,3-benzodiazol-2-yl)-1,3-thiazole; TBZ) is a benzimidazole fungicide
and can be used curatively (Schirra et al., 2008) and protectively against Penicillium digitatum,
which causes green mould on citrus, and may lead to 90% of post-harvest losses and also
controls its sporulation (Ladaniya, 2008).
TBZ fungicide with wax are also used to control the post-harvest disease of Kinnow mandarin.
There are several methods of applying TBZ to fruit, and different methods influence the efficacy
of TBZ to control disease. Common methods are drenching, dipping and wax coating with TBZ.
Degreening conditions favour the development of green mould and effective fungicide
application in the postharvest drench is crucial (Smilanick et al., 2006).
(Schirra et al. 2008) did extensive studies with TBZ in the dip tank and stated that the location of
the fungicide is more important than the amount of residue present on the fruit for control of P.
digitatum. Their study found that different application parameters led to TBZ being loaded at
different depths into the fruit skin, and this influenced the fungicide's bio-efficacy, even in
controlling TBZ-resistant isolates. It is therefore important that the application of TBZ is optimal
to ensure efficacy and prevent the development of resistance. Thiabendazole can also be applied
in a wax coating, but it was shown to be less effective in controlling disease than other
application methods (Brown, 1984).
VII. Material and methods
Fruit collection and layout of the experiment
The Kinnow mandarin fruit will be purchased from selected processing factories (National and
Al-Rafique Kinnow Factory, Sargodha). Fruits having qualifying A grade (intact pedicle, no sign
of any damage, clean <20%, blemished, especially from the upper-half side, without canker signs
and recently harvested and processed for export (washed and waxed within 24h) will be
purchased and transferred to the post-harvest lab, Institute of Horticulture Sciences, University of
Agriculture, Faisalabad. Upon arrival, fruit will be stored at the 20°C and then treated these fruits
with different treatments and packed them. Packed fruit will be stored at low temperature
regimes for a maximum of 91 days. First fruit quality assessments will be conducted after 21
days and then remaining removal conducted every fifteen days. At every removal, six fruit boxes
will be removed from cold storage and kept at 20 °C for 5 days to simulate handling in retail and
permit post-shipment quality assessment. Each experimental unit will have 80 fruits from each
box that will be divided into 6 applications. The experiment will be conducted using a
completely randomized design under factorial arrangements.
Treatments
T1= Water only
T2= Water+ Beijia+ Bixian
T3= Wax (control)
T4= Wax + TBZ + IMZ
T5= Wax + TBZ + IMZ + Beijia
T6= Wax + Beijia + Bixian
Parameters
Four types of parameters will be studied in this experiment.
A) Physical parameters
B) Physiological parameter
C) Biochemical parameters
D) Phytochemical parameter
A) Physical parameters
We will study the following physical parameters in this experiment
a) Peel colour of fruit
Peel colour of fruit will be checked by using portable colorimeter. We will check it from two side
right and left.
b) Fruit Weight Loss
Fruit weight will be weighed by digital weight balance (Setra BL-4100S) and then weight loss
percentage will be assessed by formula.
c) Disease Incidence
Disease score will be given to fruit depending upon the percentage of disease on fruit surface
(Amin et al., 2008).
1- No symptoms of any disease (Normal)
2- Less than 5% surface of fruit is affected with disease (trace)
3- 5 to 10% surface of fruit is affected with disease (slight)
4- 10 to 25% surface of fruit is affected with disease (moderate)
5- More than 25 % surface of fruit is affected with disease (severe)
d) Blemishes
Blemishes Score will be given to fruit depending upon the percentage of blemishes on fruit
(Amin et al., 2008)
1- No Blemishes is present on surface of fruit (Normal)
2- Less than 5% blemishes are present on surface of fruit (trace)
3- 5 to 10% blemishes are present on surface of fruit (slight)
4- 10 to 25% blemishes are present on surface of fruit (moderate)
5- More than 25 % blemishes are present on surface of fruit (severe)
e) Shrivelling
Shrivelling Score will also be given to fruit depending upon the percentage of shrivelling on fruit
(Amin et al., 2008).
1- No sign of shrivelling (Normal)
2- Less than 5% of surface are shrivelled (trace)
3- 5 to 10% of surface are shrivelled (slight)
4- 10 to 25 % of surface are shrivelled (moderate)
5- More than 25 % of surface are shrivelled (severe)
B) Physiological Parameters
Physiological parameters which will we check in this experiment are following.
a) Peel weight
The fruit were peeled off manually and the peel weight was measured using digital weight
balance.
b) Peel thickness
Peel thickness was measured using Vernier Calliper.
c) Electrical Conductivity
The electrical conductivity (EC) of fruit will be checked by using EC meter (Arwa AD 310).
EC of distilled water will be recorded, and then uniform cubes of fruit (5mm 3) will be placed in
graduated cylinder and will fill it up to 50 ml with distilled water and place it for 30 min and will
check the EC after 30 min. After solution will be boiled for 5 min and cooled down for 30 min
before recording final EC and EC will be calculated by using following formula.
Initial EC
EC (%) = × 100
Final EC
d) Fruit Firmness
Fruit firmness will be determined by using penetrometer (Lutron FR-5120) and firmness will be
presented in Newton (N) unit.
C) Biochemical Parameters
Biochemical parameters which will be studied in this experiment are following.
a) Total Soluble Solids
Total soluble solids will be calculated by using refractometer (ATAGO Hybrid PAL-BXI ACID
F5).
b) Titratable Acidity
Titratable acidity will be measured by using Auto titrator (HI84532:H0089885: ROMANIA:
11/2022: Titratable Acidity & pH meter for juice).
c) pH
Auto titrator will also calculates the pH of fruit. (HI84532:H0089885: ROMANIA: 11/2022:
Titratable Acidity & pH meter for juice).
d) Vitamin C
Vitamin C will be calculated by using Ruck (1961) method and will present it in mg/100g.
e) Dye
The Kinnow juice was extracted with a manual extractor, 10mL juice add into 90ml oxalic acid
and then filtered through Whatman filter paper into separate beaker almost 5ml solution were
filtered through Whatman filter paper and then few drops of Ascorbic dye added until its color
were changed.
f) Juice weight
The Kinnow juice was extracted with a manual extractor, and juice weight was measured using
digital weight balance.
VIII. Statistical Analysis
The collected data will be evaluated statistically through Analysis of Variance (ANOVA). The
differences of means will be compared by using Least Significant Difference (LSD) test (P ≤
0.05) by using Statistic 8.1 software.
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