Effects of citrus wax coating and brush type on imazalil residue loading, green mould control and fruit quality retention of sweet oranges

Postharvest Biology and Technology 86 (2013) 362–371

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Effects of citrus wax coating and brush type on imazalil residue loading, green mould control and fruit quality retention of sweet oranges Ncumisa S. Njombolwana a,b , Arno Erasmus b , J. Gideon van Zyl a,b , Wilma du Plooy c , Paul J.R. Cronje b , Paul H. Fourie a,b,∗ a

Department of Plant Pathology, University of Stellenbosch, Stellenbosch, South Africa Citrus Research International, Disease Management, 2 Baker Street Nelspruit, Mpumalanga, Nelspruit 1200, South Africa c John Bean Technologies, Brackenfell, Cape Town 7560, South Africa b

a r t i c l e

i n f o

Article history: Received 26 March 2013 Accepted 13 July 2013 Keywords: Penicillium digitatum Carnauba Polyethylene Morpholine-free Synthetic Horsehair

a b s t r a c t Wax application plays an important role in prolonging fruit quality, and the addition of imazalil (IMZ) furthermore protects fruit against green mould caused by Penicillium digitatum. The objectives of this study were to evaluate green mould control and quality preservation effects of carnauba or polyethylene citrus coatings supplemented with IMZ, as well as the effect of synthetic or horsehair brush types used on sweet orange fruit. Single applications of IMZ at 3000 ␮g mL−1 at rates of 0.6, 1.2 and 1.8 L t−1 resulted in residues that increased with increasing coating loads on navel oranges (1.31 to 3.32 ␮g g−1 ) and Valencia oranges (3.22 to 6.00 ␮g g−1 ). Coating with IMZ generally provided poorer curative control (≈14%) than protective control (≈58%), with less sporulation in treatments using horsehair (≈59%) than synthetic brushes (≈64%). More fruit weight and firmness losses were found in fruit treated with the polyethylene coating (≈1.18 and ≈0.93 ratios of treated vs. untreated, respectively) and lower in carnauba treated fruit (≈0.76 and ≈0.74 ratios, respectively). However, polyethylene coatings resulted in shinier fruit before (≈10.85 shine ratio) and after storage (11.60), whereas carnauba coatings resulted in lower shine ratios (≈7.45 and 10.15, respectively). Gas (CO2 ) exchange ratios remained similar for both waxes (≈0.67). Higher polyethylene coating loads (1.8 L t−1 ) resulted in off-tastes similar to uncoated control fruit (≈2.21 rating on a 5-point scale) and higher than the rating for carnauba coated fruit (≈1.82) at this rate. Scanning electron micrographs showed an amorphous crystallised natural wax layer with uncovered stomatal pores on the surface of uncoated fruit. The thickness of the applied wax layer increased with increasing coating load. A single application of IMZ in wax provided good protective green mould control and sporulation inhibition, with differing effects on some fruit quality parameters due to coating and brush types. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Citrus fruit 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 have natural wax layers on the surface that reportedly get removed or disturbed as the fruit go through a long washing process in the packline, and therefore need to be replaced in the packline to avoid dehydration (Gillepsie, 1986; 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

∗ Corresponding author. Tel.: +27 832902048; fax: +27 865717273. E-mail addresses: [email protected], [email protected] (P.H. Fourie). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.07.017

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 (Hall, 1981; Mannheim and Soffer, 1996; Petracek et al., 2000; Hagenmaier, 2002). A good coating also provides more protection against postharvest physiological disorders such as stem-end rind breakdown (Gillepsie, 1986; Dou et al., 2001) and chilling injury (Dou, 2004). Emulsion waxes, mainly carnauba and polyethylene coatings, are most commonly used, with shellac and wood rosin being included in smaller amounts (Hall, 1981; Hagenmaier, 1993). These wax coatings contribute to shine of the fruit as well as maintaining gaseous exchange and water retention. The specific formulation varies depending on fruit type and market preference.

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Despite the importance of meeting consumer needs, the composition of the coating is extremely important, as it could negatively affect the quality of the fruit if the formulation does not allow for the fruit physiological processes to continue. Fruit continue to respire after harvest and although the content and composition of coatings provide high levels of wax gloss, they tend to negatively affect the permeation of gases through the peel, which might lead to development of off-flavours (Hagenmaier and Shaw, 2002; Porat et al., 2005). Typical increased off-flavour volatiles associated with anaerobic respiration in fruit include ethanol and acetaldehyde (Chen and Nussinovitch, 2000; Porat et al., 2005). Polyethylene is a synthetic wax coating with high shine and highly permeability to gases but has also been reported to affect fruit quality and increase development of off-flavours (Cohen et al., 1990; Mannheim and Soffer, 1996). It is manufactured from ethylene, which is generally produced from gas and either oxidised or co-polymerised with acrylic acid to give the polyethylene chemical functionality that allows it to be emulsified (Hagenmaier, 1998; Dou, 2004). It is used as a protective coating in various fruit types, e.g. oranges, avocados, bananas, mango, pineapples, and vegetables (Hagenmaier and Shaw, 1991). Carnauba wax is a natural vegetable wax that occurs as a protective coating on the leaves of Copernica cerifera, the Brazilian palm tree (Dou, 2004). Carnauba wax is cultivated, harvested and processed in Brazil. It is hard, brittle and melts at 86 ◦ C (Hagenmaier, 1998). Shellac, on the other hand, is obtained from India and Thailand by the refinement of raw lac which is secreted by the insect Laccifer lacca (Bourtoom, 2008; Dou, 2004). The formulation of coating consists of solids, mainly of shellac, wood resin, polyethylene and carnauba, and bases, like morpholine or ammonia, which are mainly used for drying (Petracek et al., 2000). However, the use of morpholine in emulsions applied on citrus fruit and apples has been totally banned in the European Union (EU) (De Boer, 2010). Shellac is included in both polyethylene and carnauba coatings to enhance shine; however, reduction of off-flavour development and gas permeability can be avoided by reducing the total amount of soluble solids and the amount of shellac added; this emulsion results in a shiny fruit appearance with reduced weight loss (Porat et al., 2005). The performance of coatings depends on the quality of the application (Tugwell, 1980; Hall, 1981). Polyethylene and carnauba coatings give good results when applied to fruit that is thoroughly cleaned and dried (Hall, 1981). There are different types of coating applicators used and they mainly assist in delivering a uniform amount of coating to each fruit. Coatings are applied by means of a variety of nozzle types over a set of rotating brushes, which are either made of polyethylene or horsehair (Hall, 1981; Hagenmaier, 1993). Brush types vary not only in hair types, but also in the configuration of the tufts on the brush shaft. Grierson et al. (1978) and Tugwell (1980) stated that brushes consisting of 50% horsehair resulted in a good spread of coating over the fruit. During the application of the emulsion to the fruit surface, the coating becomes incorporated onto the fruit surface as a film consisting of the applied wax and the natural fruit wax; this film can only be removed again by physical interference (Du Plooy, 2006). However, some researchers have claimed that solvents like hexane or dichloromethane completely removed the natural wax from the fruit peel (Albrigo, 1971; Freeman et al., 1979; Cajuste et al., 2010). 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 (Kavanagh and Wood, 1967; Eckert and Eaks, 1989). 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 into wounds infected with

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P. digitatum (Brown, 1984; Smilanick et al., 1997). 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. Green mould control was improved with a single application of IMZ in the wax coating at 3000 ␮g mL−1 at higher coating loads (1.8 L t−1 ), which is higher than coating loads generally recommended for use on citrus fruit (1.2 L t−1 ). At these rates the maximum residue levels allowed in certain export markets (5 ␮g g−1 ) can be exceeded. From these studies, questions arose over the effects of higher coating loads on fruit quality and whether the type of coating or applicator brush type could affect coating application, IMZ residue loading, green mould control and/or fruit quality. The objective of the present study was therefore to evaluate these effects for both a carnauba and a polyethylene citrus coating applied with either horsehair or synthetic brush types to sweet orange fruit. 2. Materials and methods 2.1. Fruit Export quality Valencia (‘Midknight’) and navel (‘Robyn’) orange fruit (Citrus sinensis (L.) Osbeck) were collected from Citrusdal in Western Cape immediately upon the arrival at the packhouse from the orchard, before they were subjected to any fungicide in the packhouse. The fruit were washed with 1 mL L−1 didecyl dimethyl ammonium chloride (Sporekill, ICA International Chemicals, Stellenbosch, South Africa) and allowed to dry overnight at ambient temperature prior to commencement of the trials. 2.2. Inoculation Two-week old cultures of IMZ sensitive isolates of P. digitatum (STE-U 6559 and 6560) cultured on potato dextrose agar (PDA) (DIFCO, Becton, Dickinson and Company, USA and Le Pont de Claix, France) were used to prepare spore suspensions. Conidia were gently dislodged from a culture plate using 5 mL of sterile deionised water [containing 0.001 mL L−1 Tween 20 (Sigma–Aldrich, St. Louis, MO, USA)], which was then transferred to a 500 mL Scott’s bottle, to obtain a 200 mL volume of spore suspension. The spore concentration was adjusted to 1 × 106 spores mL−1 using a haemocytometer. Twelve fruit of similar size was wound inoculated through the flavedo into the top albedo layer at four sites surrounding the stem end, using a wound inducer. This instrument was made by milling a stainless steel rod to form a narrow, concave tip able to produce a 0.2 mm2 lesion. The wound inducers were dipped into the spore suspension prior to making a small wound 2 mm deep. The inoculations were done curatively and protectively, i.e. before and after treatment, respectively. For curative treatments, the fruit were incubated for 24 h before treatment. 2.3. IMZ and coating application Commercial application of IMZ in wax coatings as a fruit treatment was simulated using an experimental pack-line. This line is custom-built (Dormas, Johannesburg, South Africa) and represents a scaled-down, but comparable version of packlines used in commercial packhouses in South Africa. Imazalil EC (Imazacure, 750 g kg−1 EC, ICA International Chemicals, Stellenbosch, South Africa) as per registered concentration of 3000 ␮g mL−1 , was incorporated into two types of morpholine-free coatings (Endura-fresh® , John Bean Technologies, Foodtech, Brackenfell, South Africa). Both formulations contained 18% solids, one being a carnauba-based coating (Carnauba 6100) and the other a polyethylene-based coating (Quick-Dry Poly). Imazalil and coating emulsion were constantly agitated on a magnetic stirrer. Each

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coating was applied over either a set of seven rotating synthetic brushes (made of polyethylene), or seven horsehair brushes (made of horsehair); (RKF Brushes (Pty) Ltd, RKF Distributors, Johannesburg, South Africa). Fruit exposure time on the applicator was 10, 20 and 30 s, which resulted in a coating load of 0.6, 1.2 and 1.8 L t−1 of fruit, respectively. The coating applicator was calibrated using one pulsating nozzle (0.5 s on, 2 s off) at 22 mL min−1 for the two navel orange batches. Thereafter, for two Valencia orange batches the nozzles reconfigured and two nozzles were calibrated to each deliver 11 mL min−1 . Untreated fruit without coating and IMZ served as controls. 2.4. Storage and evaluation of green mould control Twelve treated and inoculated fruit from each treatment combination were placed in new lock-back table grape boxes (APL Cartons, Worcester, South Africa) on count SFT13 nectarine trays (Huhtamaki South Africa (Pty) Ltd., Atlantis, South Africa). Each box was covered with a transparent polyethylene bag and sealed. Treatments were divided into two batches. One batch was incubated at 20 ◦ C and after 4 days the number of infected wounds per fruit were evaluated using a ultra-violet light source (UV-A at 365 nm, Labino Mid-light; www.labino.com). Under UV light, infected wounds was clearly visible as yellow fluorescence on the surface of fruit, even though the infection might not have been visible with the naked eye. Wound infection data was normalised to percentage control relative to untreated control fruit: (average percentage wound infection of untreated fruit–percentage wound infection of treated fruit)/average infection of untreated fruit × 100. Green mould sporulation was rated after 10–12 days. A sporulation index of 0 to 6 was used, which described the percentage of the fruit surface covered with green spores, where 0 = no sign of disease, 1 = lesion but no sporulation, 2 = sporulating area covering a quarter of the lesion, 3 = sporulating area larger than a quarter but smaller than the half of the lesion, 4 = sporulating area larger than half but smaller than three quarters of the lesion, 5 = sporulating area larger than three quarters but smaller than the whole lesion, and 6 = sporulating area covering the whole fruit (Erasmus et al., 2011). Sporulation incidence (%) was determined from infected fruit with a sporulation index of 3 and higher. 2.5. Residue analysis Following the various treatments, IMZ residues were determined from fruit sampled from two of the three repetitions. From each treatment combination, six fruit were sampled and stored at −20 ◦ C until prepared for IMZ residue analysis. Fruit were defrosted, measured, weighed and macerated to a fine pulp, using a fruit blender (Salton Elite, Amalgamated Appliance Holdings Limited, Reuven, South Africa) where after it was re-frozen. Similar to Erasmus et al. (2011), sub-samples of the macerated fruit were submitted for IMZ (chloramizol) residue analyses by an accredited analytical laboratory (Hearshaw and Kinnes Analytical Laboratory, Westlake, Cape Town, South Africa). Imazalil was extracted from the samples by using acetonitrile followed by a matrix solid phase dispersion extraction. Extracts were analysed using liquid chromatography mass spectrometry (LCMS/MS; Agilent 6410, Agilent Technologies Inc., Santa Clara, CA, USA). 2.6. Fruit quality 2.6.1. Weight loss and fruit firmness A total of 432 un-inoculated fruit was treated along with the curative and protective treatment to evaluate the quality parameters including: weight loss, fruit firmness, appearance and respiration. Coated, un-inoculated fruit and uncoated controls were

stored under conditions simulating export and marketing, i.e. 26 days at −0.5 ◦ C and 7 days at ambient temperature simulating shelf life. Prior to and after cold storage, 12 fruit of each replication were weighed individually using a weighing scale (Centrotec, Zeiss West, Germany). For each fruit, weight loss was calculated as a percentage relative to its weight prior to cold storage. Fruit firmness was analysed using a texture analyser (TA.XT.PLUS, Stable Microsystems) fitted with P/35 probe (35 mm diameter cylinder aluminium supplied by Microsystems Limited). The texture analyser settings were calibrated to produce a compression force of 10 g and compression distance of 7 mm. The force (kg) was measured as an indication of firmness. Six fruit from each replication in all treatments was measured on two perpendicular sides of the fruit; the average of the two measurements was then calculated. Similar for weight loss, firmness loss was calculated for each fruit as a percentage relative to its firmness prior to cold storage. 2.6.2. Shine Shine was determined from twelve fruit per treatment combination. A single fruit was positioned in the middle of a backilluminated white Perspex box (300 × 210 × 110 mm; L × W × H) to reduce any shadowing and to enhance edging of fruit in captured images during analysis. Digital photos of the side of the fruit were taken in Canon RAW file format (img.CR2 ≈ 10 MB) using a Canon EOS 40D camera equipped with a 60 mm macro lens and the builtin flash switched on. The camera was attached to a tripod in a fixed position directly above the fruit. Images were captured before and after storage. RAW image files were converted to 8-bit Exif-TIFF files (img.TIF ≈ 30 MB) with Digital Photo Professional version 3.1.0.0 (CANON INC., www.canon.com) for digital image analysis (Image Pro Plus software version 6.2, Media Cybernetics) to determine the amount of shine as reflected light from the fruit surface. The amount of light reflected from each fruit surface was determined as the total count of white (pre-selected histogram value) pixels reflected from the fruit surface expressed as a percentage of the total fruit area. This was done by extracting the blue colour channel, for better contrast enhancement and more sensitive expression of white pixels on the fruit surface and to determine total fruit area (sum of all pixels in area of interest). 2.6.3. Respiration Respiration was measured on three replications of each treatment including the untreated fruit as controls. Twelve fruit of each treatment were sealed in an airtight 10 L jar for 2.5 h at ambient temperature (20 ◦ C) before a headspace gas sample was taken from each, using an airtight syringe. Carbon dioxide levels were determined by injecting into a gas chromatograph (Model N6980, Agilent Inc., Wilmington, USA) fitted with a PorapakQ (Hayes Separation Inc. Analytical Polymers, Bandera, Texas) packed column and thermal conductivity detector (Dries van Vuuren Consulting, Somerset West, South Africa). The volume of free space in the jar as well as the mass of the fruit was used to calculate CO2 production rate in mg CO2 kg−1 fruit h−1 . Evaluations were done before and after cold storage. 2.6.4. Taste Six fruit per treatment combination were randomly selected and juiced using a juice extractor (Sunkist Growers Inc., Fontana, CA, USA). The various treatments were blind-tasted by 10 tasters, rating each of the levels of sweetness, acidity and off-tastes on a 5-point scale. 2.7. Scanning electron microscopy (SEM) of waxed rind surfaces One fruit was randomly selected from the first and third replication of each treatment and processed within 36 h after treatment.

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Table 1 Imazalil residues loaded on navel and Valencia orange fruit, treated with imazalil (IMZ) (EC) and coating at 3000 ␮g mL−1 using carnauba or polyethylene coating on horsehair or synthetic brushes at 0.6, 1.2 and 1.8 L t−1 . Fruit/coating load

Carnauba wax

Polyethylene wax

Horsehair brushes

Synthetic brushes

Horsehair brushes

Synthetic brushes

1.48c 2.74b 3.65a

1.55c 3.05ab 3.00ab

1.09c 1.39c 2.81b

1.09c 1.70c 3.71a

3.69cd 4.37bc 7.56a

2.68cd 3.74cd 4.22bcd

4.17bcd 3.69cd 6.33a

2.32d 3.43cd 5.90ab

*

Navel 0.6 1.2 1.8 Valencia* 0.6 1.2 1.8 *

For each fruit type, means followed by the same letter do not differ significantly (P > 0.05).

Three sections of orange rind were dissected from a blemish-free central area of the fruit, and processed by plunge freezing in liquid propane (AFROX, Mobil, Cape Town, South Africa) at −180 ◦ C (Du Plooy, 2006). Samples were then transferred to liquid nitrogen for temporary storage (5 to 10 days) in a 1-L Dewar container and kept at −80 ◦ C. Before the sample viewing, each were subjected to liquid CO2 in a critical point drying apparatus (Quorum Technologies Limited, ASHFORD, UK) for 90 min, allowing the complete displacement of N2 by CO2 in the plant tissue, with subsequent drying when the liquid CO2 has evaporated from the sample. Samples were mounted on a stub with double sided carbon tape and coated with a thin layer of gold in order to make the sample surface electrically conducting. Imaging of the samples was done using a scanning electron microscope (SEM; Leo® 1430VP Scanning Electron Microscope, Oxford Instruments) at high resolution (3072 × 2304 pixels). Digital SEM images were captured at magnifications ranging between 100× and 500×, visualising the rind surface with natural wax and applied coatings. Beam conditions during surface analysis were 7 kV and approximately 1.5 nA, with a working distance of 13 mm. 2.8. Experimental layout and statistical analysis Experimental layout was a 2 (curative and protective treatment) × 2 (polyethylene and carnauba wax) × 2 (synthetic and horsehair brushes) × 3 (IMZ and wax loads) factorial design with 12 fruit per treatment combination and three replications; the trial was conducted four times: twice on navel and twice on Valencia orange fruit. Fungicide residues on the fruit, wound infection, green mould and sporulation incidence data were analysed using appropriate analysis of variance, and Fisher’s test was used to observe the significant differences between means at a 95% confidence interval. The fruit quality data (percentage weight loss, firmness loss, shine and respiration) were analysed by determining the ratio of the treatment relative to the untreated control, which was then submitted to analysis of variance and Fisher’s tests to compare means. 3. Results

this rate. A significant interaction between the navel batch, coating type and coating load was also observed (P = 0.038). Although the reasons for this observation are unclear, it was ascribed to generally higher residue loading in the first navel batch; residue loading trends were nonetheless similar (results not shown). 3.1.2. Valencia orange fruit Analysis of variance for the residue data on Valencia oranges also indicated a meaningful 3-factor interaction between coating type, brush type and coating load (P = 0.150). Significant effects for coating load (P < 0.0001) and brush type (P = 0.003) were observed. Compared to navel fruit, generally higher residues were recorded from Valencia fruit (2.32 to 7.56 ␮g g−1 ; Table 1). Residue loading increased significantly with increasing carnauba coating load when horsehair brushes were used (3.60 to 7.56 ␮g g−1 ), while lower residues were recorded when carnauba coating was applied using synthetic brushes at higher coating load (2.68 to 4.22 ␮g g−1 ). Similarly, for polyethylene coating, higher residue levels were recorded when it was applied with horsehair brushes (4.17 to 6.33 ␮g g−1 ) in comparison to application with synthetic brushes (2.32 to 5.90 ␮g g−1 ). Residue levels generally increased with increasing coating load. 3.2. Green mould control 3.2.1. Navel orange fruit Analysis of variance for green mould control on navel oranges indicated a 3 factor interaction (P = 0.002) involving action (curative and protective), brush type (synthetic and horsehair) and coating load (0.6, 1.2 and 1.8 L t−1 ). A significant 3-factor interaction (P = 0.005) was also observed between action, coating type (polyethylene and carnauba) and coating load. Curative control was poor, but improved with increasing coating load on both brushes, although the use of horsehair brushes resulted in better control (30% to 46%) than synthetic brushes (14% to 31%; Table 2). Protectively, there was a similar trend and both brushes exhibited successful green mould control (80% to 97%), especially at higher coating loads. Similar levels of curative control were observed between coating types (17% to 40%); however, polyethylene coating demonstrated significantly better control at low coating load (0.6 L t−1 ) (27% and 17% for polyethylene and carnauba, respectively). Conversely, the application of carnauba coating resulted in significantly better protective control at 0.6 and 1.2 L t−1 (82% and 94%, respectively) than polyethylene (78% and 83%, respectively).

Table 2 Mean percentage control on navel oranges that were wound inoculated with a imazalil (IMZ) sensitive P. digitatum isolate and curatively or protectively treated with IMZ at a concentration of 3000 ␮g mL−1 in carnauba or polyethylene coating and horsehair or synthetic brushes at a coating load of 0.6, 1.2, and 1.8 L t−1 incubated for 4 days at ambient temperature (20 ◦ C). Coating load (L t−1 )

3.1. Residue loading 3.1.1. Navel orange fruit Analysis of variance for the residue data in navel oranges indicated a meaningful interaction between coating type, brush type and coating load (P = 0.081). For application of carnauba-based coating using horsehair brushes, IMZ residue loading increased significantly with increasing coating load from 1.48 to 3.65 ␮g g−1 (Table 1). When synthetic brushes were used, similar residues were recorded (1.55 to 3.05 ␮g g−1 ), with no significant difference between coating loads of 1.2 and 1.8 L t−1 . The application of polyethylene-type coating using horsehair brushes loaded lower residues levels (1.09 to 2.81 ␮g g−1 ), which were significantly lower than levels loaded with carnauba coating using horsehair brushes at 1.2 and 1.8 L t−1 . For synthetic brushes, residue levels varied from 1.09 to 3.71 ␮g g−1 at 1.8 L t−1 , which was significantly higher than for polyethylene coating applied with horsehair brushes at

Curative 0 0.6 1.2 1.8 Protective 0 0.6 1.2 1.8

Brush type*

Coating type*

Horsehair

Synthetic

Carnauba

Polyethylene

0.00h 29.79ef 24.72f 46.26d

0.00h 14.29g 26.35f 31.45ef

0.00g 16.71f 23.89e 37.76d

0.00g 27.38e 27.18e 39.95d

0.00h 80.24c 87.58b 95.95a

0.00h 80.38c 90.26b 96.63a

0.00g 82.16bc 94.02a 95.09a

0.00g 78.46c 83.82b 97.49a

* For brush and coating type separately, means followed by the same letter do not differ significantly (P > 0.05).

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Table 3 Sporulation incidence (%) on navel oranges that were wound inoculated with a imazalil (IMZ) sensitive P. digitatum isolate and curatively or protectively treated with IMZ at a concentration of 3000 ␮g mL−1 in carnauba or polyethylene coating on horsehair or synthetic brushes at a coating load of 0.6, 1.2, and 1.8 L t−1 incubated for 4 days at ambient temperature (20 ◦ C). Coating load (L t−1 )

Carnauba coating*

0 0.6 1.2 1.8 *

Polyethylene coating*

Horsehair brushes

Synthetic brushes

Horsehair brushes

Synthetic brushes

100.0a 56.0bc 34.9de 25.6ef

99.3a 51.7c 25.0ef 29.2ef

93.1a 35.7de 29.8ef 25.4ef

92.1a 65.2b 46.1cd 20.9f

Means followed by the same letter do not differ significantly (P > 0.05).

Analysis of variance for sporulation data indicated a significant interaction (P = 0.0001) between coating, brush and load. For the carnauba coating, recorded sporulation levels following application with horsehair and synthetic brushes were similar (26% to 56%; Table 3), and decreased with increasing coating load. The application of polyethylene coating with horsehair brushes resulted in significantly less sporulation at 0.6 and 1.2 L t−1 (36% and 30%, respectively) than synthetic brushes (65% and 46%, respectively) at the same rates, while levels recorded at 1.8 L t−1 were similar (21% to 25%). When considering the significant interaction (P = 0.023; Table 3) between action, coating and brush type, mean sporulation levels were generally lower for curative treatments (48% to 53%; results not shown) than protective treatments (42% to 61%). For protective treatments, significantly lower levels (42%) were recorded when polyethylene coating was applied using horsehair brushes. 3.2.2. Valencia orange fruit Analysis of variance of green mould control data indicated a significant interaction between brush and coating load (P = 0.002), action and coating load (P < 0.0001) and action and brush (P = 0.008); a meaningful effect for coating was also observed (P = 0.166). Horsehair and synthetic brushes resulted in similar mean levels of control with increasing coating load (24% to 44%; Table 4), but coating applied at 0.6 L t−1 with horsehair brushes resulted in significantly better control (32%) in comparison to application with synthetic brushes (24%). Curative control in all coating loads (3% to 10%) was significantly poorer than protective control levels, which increased with increasing coating load (53% to 78%; Table 4). Brush type did not influence the level of curative control, but horsehair brushes resulted in significantly better protective control (52%) than synthetic brushes (46%). No significant difference was observed between the two coatings, but carnauba coating resulted in a slightly improved level of control (28%) against that of polyethylene coating (26%). Analysis of variance for sporulation data indicated a significant interaction between action, coating type and brush type (P = 0.004), as well as between action and coating load (P = 0.0001). For the action, coating type and brush type interaction, high levels of sporulation were recorded in curative treatments (97% to 99%) with protective treatments resulting in significantly less sporulation; carnauba coating applied on synthetic brushes resulted in a significantly higher level of sporulation (57%) than the other protective treatments (38% to 42%). In terms of coating load, there was also no sporulation reduction observed in curative treatments even at higher coating load; however, protective treatments resulted in decrease of sporulation with increasing coating load (from 33% to 24%) (results not shown). 3.3. Quality 3.3.1. Weight loss and firmness 3.3.1.1. Navel orange fruit. Analysis of variance for the weight loss ratio data of navel fruit indicated a significant 3-factor interaction for coating type, brush type and coating load (P = 0.001). Carnauba coating applied with horsehair brushes showed no significant difference among the three coating loads with ratios indicating less weight loss than the uncoated controls (0.85 to 0.90; Table 5). However, when applied with synthetic brushes at 0.6 L t−1 , carnauba coating resulted in weight loss

Table 4 Mean percentage green mould control on Valencia oranges that were wound inoculated with a imazalil (IMZ) sensitive P. digitatum isolate and curatively or protectively treated with IMZ at a concentration of 3000 ␮g mL−1 in carnauba or polyethylene coating on horsehair or synthetic brushes and a coating load of 0.6, 1.2, and 1.8 L t−1 incubated for 4 days at ambient temperature (20 ◦ C). Coating load (L t−1 )

0 0.6 1.2 1.8

Action*

ratio significantly higher (1.31) than with 1.2 or 1.8 L t−1 (0.83 to 0.78). Polyethylene coating resulted in increased weight loss ratios with both horsehair (1.09 to 1.34) and synthetic brushes (0.96 to 1.19), but no apparent trend with regards to amount of coating loaded. According to the analysis of variance for firmness loss ratio data in navel oranges, there is a significant interaction between coating type and load (P = 0.0001) and between coating type and brush type (P = 0.005). Mean firmness loss ratio was significantly lower for carnauba coating applied at 1.8 L t−1 (0.77) when compared to the other carnauba and all polyethylene coating loads showing similar ratios of firmness loss (0.91 to 0.97). Similar to weight loss ratios, carnauba coating applied on horsehair brushes resulted in significantly lower firmness ratios (0.81), when compared to the polyethylene coating treatments (0.94) or carnauba coating × synthetic brush treatments (0.95). 3.3.1.2. Valencia orange fruit. For weight loss ratio data in Valencia orange fruit, analysis of variance indicated a significant interaction between brush type and coating load (P = 0.041), and a meaningful effect for coating type and load (P = 0.099). Irrespective of the brush type and the coating load, weight loss ratio was ≈0.76, except for the 1.8 L t−1 coating load applied with synthetic brushes, which had a significantly higher ratio (0.90), indicating more weight loss. Carnauba coating resulted in significantly reduced weight loss at all three coating loads (≈0.60) when compared to polyethylene coating with weight loss ratios of 0.90, 0.93 and 1.05 for 0.6, 1.2 and 1.8 L t−1 , respectively. Analysis of variance for firmness loss ratio data in Valencia oranges indicated a significant interaction between brush type and coating load (P = 0.041), as well as between coating type and brush type (P = 0.024). Firmness loss ratios remained fairly similar for fruit coated using horsehair (0.71 to 0.78; results not shown) and synthetic (0.74 to 0.79) brushes, resulting in an insignificant ratio decrease with increasing coating load; however, the firmness loss ratio at 1.8 L t−1 was significantly higher for synthetic brushes when compared to horsehair brushes (0.79 vs. 0.71). Further observations showed that carnauba coating applied with horsehair brushes resulted in significantly lower firmness loss ratio (0.57) than synthetic brushes (0.63), with polyethylene coating resulting in significantly higher firmness loss ratios for both brush types (0.90 to 0.93). 3.3.2. Respiration 3.3.2.1. Navel orange fruit. Analysis of variance for pre-storage and post-storage respiration data indicated no significant interaction between treatments (P > 0.214 and P > 0.170, respectively). Coating type had a significant effect on postharvest respiration (P = 0.001), whereas fruit batch differed significantly at pre- and poststorage evaluation (P < 0.0001 and P = 0.021). The second batch of navels resulted in higher CO2 ratios relative to the control treatments when measured at pre- and post-storage evaluations (0.98 and 0.94, respectively) compared with the first batch (0.69 and 0.80, respectively). For the post-storage evaluation of navel oranges, polyethylene coating resulted in higher CO2 ratios (0.98) than carnauba coating (0.76). 3.3.2.2. Valencia orange fruit. The analysis of variance for pre-storage and poststorage respiration data of Valencia orange fruit indicated no significant interactions for the 4 factors (fruit batch, coating, brush and coating load; P > 0.320 and P > 0.238, respectively. A significant effect (P < 0.0001) for fruit kind was observed in both evaluation periods, but no significant effect was observed for any of the other factors (P > 0.761). The second Valencia orange fruit batch had significantly higher CO2 ratios relative to the control treatments when measured at pre- and post-storage evaluations (0.78 and 0.91, respectively; results not shown) and compared to the first batch (0.66 and 0.54, respectively).

Brush type*

Curative

Protective

Horsehair

Synthetic

0.0f 2.6ef 6.7de 10.4d

0.0f 53.5c 65.0b 78.5a

0.0e 32.0c 37.7b 44.4a

0.0e 24.1d 34.0bc 44.4a

* For action and brush type separately, means followed by the same letter do not differ significantly (P > 0.05).

3.3.3. Shine Navel orange fruit. An incomplete shine test dataset was obtained for the first batch of navel oranges, and only the second batch was analysed. Analysis of variance indicated a meaningful interaction involving coating type, brush type, and coating load for pre- and post-storage data (P = 0.077and 0.108, respectively). From prestorage evaluations of navel oranges, for carnauba coating applied with horsehair and synthetic brushes, increased levels of shine with increasing coating load (0.6 to 1.8 L t−1 ) on horsehair (shine ratios of 3.82 to 7.69) and synthetic brushes (4.48 to 7.24) was observed, with significantly higher ratios on horsehair brushes at 1.2 L t−1

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Table 5 Weight loss ratio of navel orange fruit treated with imazalil (IMZ) (EC) at 3000 mg L−1 in either carnauba or polyethylene coating on synthetic or horsehair brushes at loads of 0.6, 1.2 and 1.8 L t−1 , evaluated at pre-storage and post-storage of 26 days at −0.5 ◦ C and 7 days of shelf life at 20 ◦ C. Coating load (L t- 1 )

Carnauba coating*

0.6 1.2 1.8 *

Polyethylene coating*

Horsehair brushes

Synthetic brushes

Horsehair Brushes

Synthetic brushes

0.85ef 0.90def 0.87def

1.31ab 0.83ef 0.78f

1.34a 1.08bcde 1.27ab

0.96cdef 1.09bcd 1.19abc

For coating type, means followed by the same letter do not differ significantly (P > 0.05).

Table 6 Shine ratio results of navel and Valencia orange fruit treated with imazalil (IMZ) (EC) at 3000 mg.L−1 in either carnauba or polyethylene coating on synthetic or horsehair brushes at loads of 0.6, 1.2 and 1.8 L t−1 , evaluated at pre-storage and post-storage of 26 days at −0.5 ◦ C and 7 days of shelf-life at 20 ◦ C. Coating load (L t- 1 )

Navel*

Valencia*

Carnauba coating

Pre-storage 0.6 1.2 1.8 Post-storage 0.6 1.2 1.8 *

Polyethylene coating

Carnauba coating

Horsehair brushes

Synthetic brushes

Horsehair brushes

Synthetic brushes

Horsehair brushes

Synthetic brushes

Polyethylene coating Horsehair brushes

Synthetic brushes

3.82f 6.16bcd 7.69ab

4.48ef 4.55ef 7.24abc

5.90cde 6.32abc 6.82abcd

5.31def 6.99abc 7.82a

7.22f 9.27cde 11.65a

8.41e 9.34cde 8.73de

11.34a 11.34a 11.24a

9.54cd 10.10bc 10.19bc

2.42f 4.37bcd 5.62a

3.12ef 3.37def 5.41ab

3.16ef 3.83cde 4.88abc

2.93ef 4.30bcd 4.93abc

13.71e 16.71cd 21.66a

13.98e 15.63de 14.63de

18.63bc 18.84bc 18.96b

15.32de 18.53bc 15.64de

For each fruit type separately, means followed by the same letter do not differ significantly P > 0.05).

(6.16). Polyethylene coating also resulted in similar effects, but with higher ratios at 0.6 L t−1 and no apparent difference between horsehair (5.90 to 6.82) and synthetic (5.31 to 7.82) brushes. Post-storage evaluations generally resulted in lower shine ratios but following the same trend as pre-storage. Carnauba coating had similar shine with horsehair (2.42 to 5.62) than with synthetic brushes (3.12 to 5.41), as was the case for polyethylene coating (2.93 to 4.93 and 3.16 to 4.88 for synthetic and horsehair brushes, respectively). In general, polyethylene coating resulted in increased shine ratios before storage (6.51) compared to carnauba coating (5.61); however, no significant difference was observed in the after storage evaluations as both coatings resulted in ≈4.01 ratios (Table 6). 3.3.3.1. Valencia orange fruit. The analysis of variance for shine test data in Valencia oranges indicated a significant 3-factor interaction involving coating type, brush type, and coating load in both pre-storage (P < 0.0001) and post-storage data (P = 0.0003). A significant effect was observed for Valencia batches (P < 0.0001), which was mostly attributed to significantly lower levels of shine when comparing the first batch to the second batch. Trends for the effects of coating, brush and coating load were nonetheless similar, and the batch effect was ignored. In pre-storage evaluations, carnauba coating applied with horsehair brushes resulted in increasing shine ratios (7.22 to 11.65) with increasing coating load from 0.6 to 1.8 L t−1 , while similar shine ratios (8.41 to 9.34) were observed when applied with synthetic brushes. Application of polyethylene coating with horsehair brushes resulted in higher shine ratios (11.24 to 11.34) at all three coating loads, while this coating type on synthetic brushes yielded significantly lower shine ratios (9.54 to 10.09). As for navels, similar trends were observed in the post storage evaluation, but shine ratios were generally higher, given the poorer shine of the control treatment. Carnauba coating applied using horsehair brushes resulted in higher shine ratios with increasing coating load (13.71 to 21.66), while carnauba coating applied using synthetic brushes resulted in lower shine ratios (13.98 to 15.73). Post-storage, polyethylene coating had high shine ratios, while results seemed better with horsehair brushes (18.63 to 18.96) than synthetic brushes (15.32 to 18.64) (Table 6). 3.3.4. Taste 3.3.4.1. Navel orange fruit. Analysis of variance for taste data indicated no significant interaction for sweetness data between any of the factors. According to the off-taste data, a significant interaction between coating type, brush type and coating load (P < 0.001) and a significant effect for kind was observed (P < 0.001). Carnauba coating resulted in similar level of off-taste ratings (1.65 to 1.94; results not shown) which did not differ significantly with the control fruit (2.16) for all three coating loads, except for fruit coated with carnauba coating on synthetic brush at 1.8 L t−1 (1.65). Polyethylene coating resulted in higher off-taste levels at higher coating loads on horsehair (2.38) and synthetic brushes (2.65). Lower coating loads (0.6 and 1.2 L t−1 ) resulted in significantly lower off-taste levels with both horsehair (≈1.65) and synthetic brushes (≈1.81). The first batch of navel oranges resulted in higher off-taste level (2.04) than the first batch (1.81).

3.3.4.2. Valencia orange fruit. Analysis of variance of sweetness taste data indicated a significant 4-factor interaction involving fruit batch, coating type, brush type and coating load (P < 0.0001). This interaction was ascribed to the fruit batch effect as there were significant differences in level of sweetness between batches; trends for treatments were nonetheless similar and the batch interactions were therefore ignored and not included in further interpretations of the data. A 3-factor interaction between coating type, brush type and load was also observed for sweetness and off-taste (P < 0.0001 and P = 0.043, respectively). Carnauba coating on horsehair brushes resulted in similar levels of sweetness in all coating loads, but on synthetic brushes an increasing level of sweetness with increasing coating load (2.74 to 3.29) was tasted. Sweetness level was higher in polyethylene coating (≈3.19) but 1.2 L t−1 loads resulted in lower levels both on horsehair (2.70) and synthetic brushes (2.58). Compared with the uncoated control treatment (2.09), carnauba coating showed similar off-taste levels in all coating loads on horsehair or synthetic brushes (1.71 to 2.18), except for lower levels on synthetic brushes at 1.2 L t−1 (1.61). Polyethylene coating resulted in higher off-taste levels with horsehair at 0.6 L t−1 (1.91) and with synthetic brushes at 1.8 L t−1 (2.25), while the other polyethylene coating treatments were similar (1.50 to 1.70). 3.4. Scanning electron microscopy Due to processing, Valencia orange rind samples on SEM had a lot of fracturing of the applied wax on the fruit surface as can be observed at 100× magnification (Fig. 1B, C and D), irrespective of coating type, brush type or coating load. The surface of the untreated fruit, which served as control, showed a natural wax layer with visible stomatal pores and consisted of a crystallised undisturbed amorphous structure (Fig. 1A and Fig. 2A). Coated fruit clearly had a much smoother wax coating on the fruit and some differences could be observed. At 500× magnification, polyethylene coating applied using synthetic brushes had a smooth, thin layer of wax at 0.6 L t−1 (Fig. 2B), but at these lower wax loads, incomplete coverage of the fruit surface was often observed. Higher coating loads (1.2 and 1.8 L t−1 ) had thicker and smoother wax layers on the surface (Fig. 2C and D). Although it could not be quantified, horsehair brushes appeared to yield a thinner but smoother layer of polyethylene coating (Fig. 2E) and carnauba coating (Fig. 2F) at 1.2 L t−1 .

4. Discussion This study contributes to our current understanding of IMZ application in coating and its role in the protection of the fruit against green mould, sporulation inhibition and preservation of fruit quality by the wax coatings (Hall, 1981; Waks et al., 1985; Smilanick et al., 1997; Porat et al., 2005; Du Plooy et al., 2009). The comparison of the carnauba and polyethylene coating formulations applied using horsehair or synthetic brushes pointed towards little

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Fig. 1. Scanning electron microscope micrographs (100×) of Valencia orange fruit surface, showing an un-waxed fruit as a control (A), fruit treated with imazalil (IMZ) in carnauba and polyethylene coatings at 3000 mg L−1 using horsehair brushes at a rate of 1.2 L t−1 (B and C, respectively) and polyethylene coating using synthetic brushes at 1.2 L t−1 (D).

Fig. 2. Scanning electron microscope micrographs (500×) of Valencia orange fruit surface, showing an uncoated fruit as a control (A), fruit treated with IMZ in polyethylene coating at 3000 mg L−1 using synthetic brushes at rates of 0.6 L t−1 (B), 1.2 L t−1 (C) and 1.8 L t−1 (D), polyethylene and carnauba coating applied with horsehair brushes at 1.2 L t−1 (E and F, respectively).

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difference between the two coatings and brushes in terms of bioefficacy. However, differences in IMZ residue loading and certain fruit quality parameters were observed, which possibly indicate improved distribution of wax coating on the fruit surface. Imazalil residue loading increased with increasing coating load, and similar to findings in Njombolwana et al. (2013), single application of IMZ at 3000 ␮g mL−1 in coating demonstrated the risk of exceeding the maximum residue limit (5 ␮g g−1 ) at coating loads exceeding the recommended dosage. Higher concentrations of IMZ in coating increased the amount of residues on Hamlin orange fruit (Brown et al., 1983). The recommended concentration for IMZ application in wax coating in South Africa is 3000 ␮g mL−1 , but at a recommended dosage of 1.2 L t−1 fruit. These findings therefore clearly indicate the importance of adhering to the recommended dosage (i.e. coating load). Interestingly, higher residue levels were recorded on Valencia fruit than on navel fruit. This is contrary to a previous study (Njombolwana et al., 2013), according to which significantly lower residue levels on Valencia oranges (≈1.58 ␮g g−1 ) were found when compared to navel oranges (≈7.09 ␮g g−1 ). One of the reasons for this increased level of residue on Valencia fruit in the present study could be the reconfiguration of the coating applicator. For navel oranges, as well as the experiments in Njombolwana et al. (2013), only one coating application nozzle was used. In order to improve coating application, two nozzles delivering the same total amount of wax coating as with one nozzle were used for the two batches of Valencia oranges. These settings therefore conformed to the recommendation made by Tugwell (1980) that at least two nozzles should be used in order to obtain an even spread of coating on the surface of the fruit. Furthermore, this re-calibration resulted in a more even coverage of the brushes with coating and possibly improved coating and resultant IMZ application. Imazalil residue loading was influenced by coating type and brush type. This was particularly evident on Valencia fruit where improved residue levels were achieved following IMZ application in carnauba or polyethylene coating using horsehair brushes. On navel oranges, this difference was not evident for carnauba coating. However, polyethylene coating loaded IMZ better using synthetic brushes. Imazalil residue levels could be indicative of the quantity of coating applied, and since fruit were treated with similar coating loads, these differences in IMZ residue levels might indicate improved quality of coverage of the fruit surface. Our findings therefore indicate that carnauba coating is better applied using horsehair brushes, which was also the case for polyethylene coating on Valencia fruit. However, further work needs to be conducted to support specific coating type × brush type recommendations for specific citrus fruit cultivars. Although IMZ residue levels were significantly lower on navel fruit, the residue levels obtained at the recommended and higher coating loads (1.2 to 1.8 L t−1 ) were higher or similar to the required level (2 to 3.5 ␮g g−1 ) reported to successfully control green mould and inhibit sporulation (Kaplan and Dave, 1979; Brown and Dezman, 1990; Smilanick et al., 1997). Results from this study confirmed the previous reports that IMZ in coating gives better protective control than curative control of green mould (Waks et al., 1985; Gillepsie, 1986; Du Plooy et al., 2009; Njombolwana et al., 2013). This was clearly observed and could be attributed to the immobility of IMZ in coating as it becomes bound on the surface of the fruit therefore failing to penetrate into the albedo where the developing green mould infection is taking place (Brown, 1984). It should be noted that there was no marked difference in level of sporulation between the carnauba coating and polyethylene coatings after application with horsehair and synthetic brushes. However, as far as sporulation was concerned, some differences in brush type were observed as horsehair brushes reduced levels of sporulation even at lower coating loads when polyethylene coating was used. On navel fruit, similar residue levels were recorded and

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improved sporulation inhibition should therefore be attributed to improved quality of deposition, i.e. coverage of fruit. Further work is, however, needed to conclusively support this hypothesis. The horsehair brushes used in this study contained 100% horsehairs. It was reported that, for better results, brushes should consist of at least 50% horsehair as these combined brushes held the emulsion better and spread it more evenly over the surface of the fruit instead of throwing it off due to the centrifugal force of the rotating brush (Grierson et al., 1978; Tugwell, 1980). IMZ application in coating provided better sporulation inhibition than aqueous dip application (Smilanick et al., 1997; Erasmus et al., 2011, Njombolwana et al., 2013). In cases where control was inadequate in the IMZ + coating treatments, such as the curative treatments, white mycelia grew abundantly on the surface of the fruit without producing olive-green green mould spores, proving a successful inhibition of the sporulation (Eckert and Kolbezen, 1977; Brown et al., 1983). An anti-sporulant activity is essential for the control of spoilage, a cosmetic defect resulting from the dusting of healthy fruit in the cartons by spores from the nearby infected fruit (Eckert and Brown, 1986). Superior green mould sporulation inhibition following coating application was also observed for other fungicides, notably thiabendazole (Eckert and Kolbezen, 1977; Kellerman, unpublished results). Apart from being a carrier for postharvest fungicides, and the disease control attributes thereof, wax coating is primarily applied to citrus fruit to maintain fruit quality and improve appearance. Generally, untreated fruit that served as control treatments exhibited a significant decline in firmness and weight when compared to coated fruit; as was also reported elsewhere (Cohen et al., 1990; Banks et al., 1997; Hu et al., 2011). However, better reduction of weight and firmness loss was achieved on fruit treated with carnauba coating, with improved results at increased coating loads. This finding concurred with studies conducted on grapefruit and “Murcot” tangerine fruit to evaluate coating application using shellac, carnauba and polyethylene waxes. Weight loss was best limited by carnauba coating, while polyethylene coating resulted in moderate protection from weight loss (Cohen et al., 1990; Hagenmaier and Baker, 1997; Dou, 2004). Formulation of coatings differ between types, and polyethylene-based coatings are formulated to consist of minimal levels of fatty acids, which might affect the ability of the wax to reduce fruit weight loss as some of them often cause brittleness in the wax (Hagenmaier and Baker, 2006). Additionally, as similar to the observation with sporulation inhibition, horsehair brushes performed better than synthetic brushes in terms of weight loss reduction after the application of carnauba coating. Grierson et al. (1978) recommended horsehair brushes for better quality of coating application. Despite the relatively poor performance in terms of reducing weight loss and firmness loss, the polyethylene coating exhibited a better shine at all coating loads, with carnauba coating resulting in improved shine at higher coating loads. Hagenmaier (2000) reported similar findings using candililla wax coating, a plant extract obtained from the leaves of a small shrub called Euphobia antisyphilitica, which provided less shiny fruit. In this case, shine was improved when polyethylene wax was added to the mixture. Valencia oranges had high shine ratios relative to the uncoated fruit before storage and even higher after storage. For navel oranges it seemed that the level of shine decreased less dramatically during the storage period. Despite consumer preference for shiny fruit given its fresh appearance (Hagenmaier, 1993), previous studies indicated that high levels of fruit gloss, which are often obtained with the use of shellac-based coatings, resulted in the development of off-flavours (Cohen et al., 1990; Mannheim and Soffer, 1996). In our study, this was evident in stored fruit that were treated with polyethylene coating at higher loads. The development of off-flavours in coated

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fruit is due to ethanol and acetaldehyde build-up, which are the products of anaerobic respiration (Mannheim and Soffer, 1996). This was reported to occur when fruit that were coated with shellac or wood rosin develop low internal oxygen levels and increased internal carbon dioxide levels (Hagenmaier and Baker, 1994; Sun and Petracek, 1999). Coatings that rely on ammonia as a base are more permeable to oxygen and carbon dioxide as this base dissolve and evaporate when the coating dries (Hagenmaier and Shaw, 1991). Hagenmaier and Baker (1994) stated that the rate at which the coating is applied had no negative impact on gaseous exchange but that gaseous exchange is rather influenced by the type of wax. However, when comparing carnauba and polyethylene coatings, we only observed off-tastes at higher polyethylene coating loads. As our study did not conduct formal sensory analyses, this aspect should be studied further. Simulated cold-sterilisation treatment of citrus fruit (−0.5 ◦ C for 26 days) to combat the occurrence of false codling moth (Thaumatotibia leucotreta) during export shipping was used (Boardman et al., 2011). The first batch of navel oranges exhibited a physiological rind disorder called chilling injury after storage, which was visible in both coated and uncoated fruit. Chilling injury is considered as cellular collapse in distinct area of the rind that eventually becomes dark and depressed and often appears in circle form (Dou, 2004; Cronje, 2009). This was attributed to an inadequate amount of coating due to poor coverage on the surface of the fruit. However, storage temperature did not appear problematic to the fruit in further experiments as no rind disorders were observed. The coating application experiments were conducted under controlled laboratory conditions but we endeavoured to simulate commercial packhouse application. However, due to scale differences between the laboratory and commercial coating application and limitations of the scanning electron microscopy methodology used, it is possible that certain conspicuous differences could not be discerned between coating type and brush type treatments from the SEM micrographs. For instance, in commercial packhouses typically use 10 to 18 brushes for thorough coating application (Kaplan, 1986). In South African packhouses the number of brushes varies from 6 to 13. Additionally, the width of packlines and the amount of fruit that passes through varies and therewith calibration of the coating applicator and the number of brushes utilised. It was anticipated that the rougher-textured synthetic brushes would produce a poorer quality wax layer on the fruit surface than the finer-textured horsehair brushes. In our study, only seven brushes were used with a low fruit load and no obvious difference in the quality of wax layer could be discerned regardless of coating or brush type. During sample preparation, fracturing of the applied wax layer inevitably occurred, but it was nonetheless obvious that the fruit surface and stomatal pores were covered. Increased coating load, in our case, resulted in increased exposure time in the wax applicator, and consequently, in improved quality of wax deposition. At the lowest coating loads, uncoated surface areas were clearly visible. The surface of uncoated fruit had a crystalline structure with irregular shapes and bare stoma as was also reported elsewhere (Ben-Yehoshua et al., 1985; Vercher et al., 1994; Chen and Nussinovitch, 2000). Micrographs of the recommended industry dosage of 1.2 L t−1 revealed that a thicker layer of carnauba or polyethylene coating is applied with synthetic brushes than horsehair brushes. However, these observations will have to be studied further for conclusivity, as well as to elucidate the practical relevance. Our work gives some indications on effect of brush type on the distribution and polishing of the coating on the surface of the fruit, which is not documented in scientific literature. Coating companies have been encouraging the use of horsehair brushes as they apparently provide a better distribution of wax on the surface. Horsehair brushes are made of a natural product and therefore more expensive compared to synthetic brushes; as a result packhouses

are still resistant to their use. Although not conclusively, our study supports this recommendation, particularly in the case of carnauba coating, where green mould control, sporulation inhibition and quality parameters were often improved when applied with horsehair brushes. However, these findings are by no means exhaustive and warrant further investigation. The South African citrus export industry is challenged by long distances to export markets. Inevitably, fruit gets exposed to different storage regimes during transportation and marketing, leading to the deterioration of fruit quality. Coatings therefore protect the fruit against weight loss and firmness loss, as was particularly evident in the carnauba coating treatments in this study. Additionally, wax coating improved the fruit’s appearance following simulated shipping conditions, which is vitally important to improve marketability of fresh fruit. In this case, the polyethylene coating provided superior shine levels in comparison to the carnauba coating formulation used in this study. The choice of coating should, however, be governed by market preference as well as citrus type. Despite marked differences between coating types as observed in our study, especially in terms of quality parameters, we obtained no conclusive evidence disqualifying a particular coating for use on Valencia or navel oranges. Notably, the inferiority of the polyethylene coating used in this study in terms of weight and firmness loss prevention already resulted in reformulation of the product (Wilma du Plooy, pers. comm.). Diligent application of any coating should be stressed to packhouse operators, as was clear from relatively poor results that were obtained following under- or over-coating. The recommended coating load of 1.2 L t−1 consistently provided good results for both green mould control and quality preservation. Sub-optimal coating loads (0.6 L t−1 in this study) provided non-satisfactory results, while higher coating loads (1.8 L t−1 ) provided higher levels of green mould control, but the MRL was exceeded in some cases and indications of off-flavours were observed in the case of fruit with polyethylene coating. Cautious application is therefore advised to ensure thorough coverage of the fruit surfaces with a smooth, thin wax layer without exceeding optimally recommended coating loads. Acknowledgements The researchers acknowledge Citrus Research International, Citrus Academy, National Research Foundation and THRIP for financial assistance; Hearshaw and Kinnes Analytical Laboratory (Pty)Ltd for residue analysis; John Bean Technologies for supplying wax coatings, ICA International Chemicals (Pty)Ltd for supplying IMZ; Department of Horticultural Science, Stellenbosch University for the fruit quality measuring equipment; Central Analytical Facilities, Stellenbosch University for SEM study and technical assistance. References Ahmed, D.M., El-Shami, S.M., El-Mallah, M.H., 2007. Jojoba oil as a novel coating for exported Valencia orange fruit, part 1, the use of trans (isomerized) jojoba oil. Agric. Environ. Sci. 2, 173–181. Albrigo, L.G., 1971. Distribution of stomata and epicuticular wax on oranges as related to stem end rind breakdown and water loss. Hortic. Sci. 97, 220–223. Banks, N.H., Johnston, J.W., Watson, R.A., Kingsley, A.M., Mackay, B.R., 1997. Coating to enhance fruit life. In: Cutting, J.G. (Ed.), Proceeding from Conference ‘97, Searching for Quality. Joint Meeting of the Australian Avocado Growers Federation, Inc. and New Zealand Avocado Growers Association, Inc., 23–26 September, pp. 46–54. Ben-Yehoshua, Burg, S.P., Young, R., 1985. Resistance of citrus fruit to mass transport of water vapour and other gases. Plant Physiol. 79, 1048–1053. Boardman, L., Grout, T.G., Terblanche, J.S., 2011. False codling moth Thaumatotibia leucotreta (Lepidoptera,Tortricidae) larvae are chill-susceptible. Insect Sci. 00, 1–14.

N.S. Njombolwana et al. / Postharvest Biology and Technology 86 (2013) 362–371 Bourtoom, T., 2008. Edible films and coatings, characteristics and properties. Int. Food Res. 15, 1–12. Brown, G.E., Nagy, S., Maraulja, M., 1983. Residues from postharvest non-recovery spray applications of imazalil to oranges and effects on green moulds caused by Penicillium digitatum. Plant Dis. 67, 954–957. Brown, G.E., 1984. Efficacy of citrus postharvest fungicides applied in water or resin solution water wax. Plant Dis. 68, 954–957. Brown, G.E., Dezman, D.J., 1990. Uptake of imazalil by citrus fruit after postharvest application and the effect of residue distribution on sporulation of Penicillium digitatum. Plant Dis. 74, 927–930. Cajuste, J.F., Gonzalez-Candelas, L., Veyrat, A., Garcia-Beijio, F.J., Reig-Aminana, J., Lafuente, M.T., 2010. Epicuticular wax content and morphology as related to ethylene and storage performance of ‘Navelate’ orange fruit. Postharvest Biol. Technol. 55, 29–35. Cohen, E., Shalom, Y., Rosenberger, I., 1990. Postharvest ethanol buildup and offflavor in ‘Murcott’ tangerine fruit. J. Amer. Soc. Hortic. Sci. 115, 775–778. Chen, S., Nussinovitch, A., 2000. Permeability and roughness determinations of waxhydrocolloid coating, and their limitations in determining citrus fruit overall quality. Food Hydrocolloids 15, 127–137. Cronje, P.J.R., 2009. Summary of Known Management Practices to Reduce Common Citrus Fruit Rind Disorders. Citrus Research International Department of Horticultural Science University of Stellenbosch, South Africa. De Boer, A., 2010. Unapproved Food Additives Discovered in Wax on Apples. Report Food. Standards Agency. Dou, H., Zhang, J., Ismail, M.A., 2001. Postharvest factors influencing stem-end rind break down (SERB) of Valencia oranges. Proc. Fla. State Hortic. Soc. 114, 164–169. Dou, H., 2004. Effect of coating application on chilling injury of grapefruit cultivars. Postharvest Biol. Technol. 39, 558–561. Du Plooy, W., 2006. Aspects of Mango Magnifera indica L. Fruit Rind Morphology and Chemistry and Their Implication for Postharvest Quality. Doctoral Thesis. University of Pretoria, Pretoria, South Africa. Du Plooy, W., Regnier, T., Combrinck, S., 2009. Essential oils amended coatings as alternatives to synthetic fungicides in citrus postharvest management. Postharvest Biol. Technol. 53, 117–122. Eckert, J.W., Kolbezen, M.J., 1977. Influence of formulation and application method on the effectiveness of fungicides for controlling postharvest diseases of citrus fruits. Plant Pathol. 83, 343–352. Eckert, J.W., Brown, G.E., 1986. Postharvest citrus diseases and their control. In: Wardowski, W.F., Nagy, S., Grierson W. (Eds.), Fresh Citrus Fruits, Avi Publishing Westport, pp. 315–360. Eckert, J.W., Eaks, I.L., 1989. Postharvest disorders citrus diseases and their control. In: Reuther, W., Calavan, E.C., Carman, G.E. (Eds.), The Citrus Industry, Division of Agriculture and Natural Resources, University of California, CA, pp. 175–255. Erasmus, A., Lennox, C.L., Jordaan, H., Smilanick, J.L., Lesar, K., Fourie, P.H., 2011. Imazalil residue loading and green mould control in citrus packhouses. Postharvest Biol. Technol. 62, 193–203. Freeman, B., Albrigo, L.G., Biggs, R.H., 1979. Ultrastructure and chemistry of cuticular waxes of developing citrus leaves and fruits. Hortic. Sci. 104, 801–808. Gillepsie, K., 1986. Stem end dehydration of Valencias. In: Packingshed Newsletter. Department of Agriculture, Australia. Grierson, W., Smith, A.F.G., Thornton, F., Felsenstein, G., 1978. Packingline machinery. In: Wardowski, W.F., Nagy, S., Grierson, W. (Eds.), Fresh Citrus Fruit. Van Nostrand Reinhold Company, FL, USA, pp. 287–313. Hall, D., 1981. Innovation in citrus waxing—an overview. Proc. Fla. State Hortic. Soc. 94, 258–263.

371

Hagenmaier, R.D., Shaw, P.E., 1991. Permeability of coating made with emulsified polyethylene wax. Food Chem. 39, 1705–1709. Hagenmaier, R.D., 1993. Selection of citrus ‘wax’ coatings on criteria other than short-term gloss. In: Wardowski, W.F. (Ed.), Packinghouse Newsletter. University of Florida, FL, USA. Hagenmaier, R.D., Baker, R.A., 1994. Internal gases, ethanol content and gloss of citrus fruit coated with polyethylene wax, carnauba wax, shellac or resin at different application levels. Proc. Fla. State Hortic. Soc. 107, 261–265. Hagenmaier, R.D., Baker, R.A., 1997. Edible coatings from morpholine free wax microemulsions. Food Chem. 45, 349–352. Hagenmaier, R.D., 1998. Wax microemulsion formulations used as fruit on coatings. Proc. Fla. State Hortic. Soc. 111, 251–255. Hagenmaier, R.D., 2000. Evaluation of a polyethylene-candelilla coating for ‘Valencia’ oranges. Postharvest Biol. Technol. 19, 147–154. Hagenmaier, R.D., 2002. The flavor of mandarin hybrids with different coatings. Postharvest Biol. Technol. 24, 79–87. Hagenmaier, R.D., Shaw, P.E., 2002. Changes in volatile components of stored tangerines and other specialty citrus fruit with different coatings. Food Chem. Toxicol. 67, 1742–1745. Hagenmaier, R.D., Baker, R.A., 2006. Edible coatings from candelilla wax microemulsions. Food Sci. 61, 552–565. Hu, H., Li, X., Dong, C., Chen, W., 2011. Effects of wax treatment on quality and postharvest physiology of pineapple fruit in cold storage. Biotechnology 39, 7592–7603. Kaplan, H.J., Dave, B.A., 1979. The current state of imazalil: a postharvest fungicide for citrus. Proc. Fla. State Hortic. Soc. 92, 37–43. Kaplan, H.J., 1986. Washing, waxing and color-adding. In: Wardowski, W.F., Nagy, S., Grierson, W. (Eds.), Fresh Citrus Fruit. Van Nostrand Reinhold Company, FL, USA, pp. 379–395. Kavanagh, J.H., Wood, R.K.S., 1967. The role of wounds in the infection of oranges by Penicillium digitatum Sacc. Ann. Appl. Biol. 60, 375–383. Mannheim, C., Soffer, T., 1996. Permeability of different wax coatings and their effect on citrus fruit quality. Food Chem. 44, 919–923. Njombolwana, N.S., Erasmus, A., Fourie, P.H., 2013. Evaluation of curative and protective control of Penicillium digitatum following imazalil application in wax coating. Postharvest Biol. Technol. 77, 102–110. Petracek, P.D., Dou, H., Mourer, J., Davis, C., 2000. Measurement of gas exchange characteristics of waxes applied to citrus fruit. In: Janoudi, A. (Ed.), Proceedings from International Symposium on Growth and Development of Fruit Crops. Porat, R., Weiss, B., Cohen, L., Daus, A., Biton, A., 2005. Effects of polyethylene wax content and composition on taste, quality, and emission of off-flavor volatiles in ‘Mor’ mandarins. Postharvest Biol. Technol. 38, 262–268. Smilanick, J.L., Michael, I.F., Mansour, M.F., Mackey, B.E., Morgosan, D.A., Flores, D., Weist, C.F., 1997. Improved control of green mold of citrus with imazalil in warm water compared with its use in wax. Plant Dis. 81, 1299–1304. Sun, D., Petracek, P.D., 1999. Grapefruit gland oil composition is affected by wax application, storage temperature, and storage time. Agric. Food Chem. 47, 2067–2069. Tugwell, B., 1980. Citrus waxing can be improved. In: Gillespie, K. (Ed.), Packingshed Newsletter, No. 3. Vercher, R., Tadeo, F.R., Almela, V., 1994. Rind structure, epicuticular wax morphology and water permeability of ‘Fortune’ mandarin fruits affected by peel pitting. Ann. Bot. 74, 619–625. Waks, J., Schiffmann-Nadel, M., Lomaniec, E., Chalutz, E., 1985. Relation between fruit waxing and development of rots in citrus fruit during storage. Plant Dis. 69, 869–870.