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The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application Mareli Kellermana,b , Jéanine Joubertc , Arno Erasmusb , Paul H. Fouriea,b,* a b c
Department of Plant Pathology, Stellenbosch University, Private bag X1, Stellenbosch 7602, South Africa Citrus Research International, PO Box 28, Nelspruit, 1200, South Africa Department of Horticulture, Stellenbosch University, Private bag X1, Stellenbosch 7602, South Africa
A R T I C L E I N F O
Article history: Received 15 February 2016 Received in revised form 16 May 2016 Accepted 17 June 2016 Available online xxx Keywords: Fungicide Postharvest Citrus
A B S T R A C T
Imazalil (IMZ) is the most relied upon fungicide for use against citrus green mould, caused by Penicillium digitatum. In South Africa, the IMZ sulphate formulation is used as dip application in packhouses. Previous studies showed that IMZ efficacy and residue loading of this formulation is highly affected by pH in the application solution. This study investigated the effect of pH, exposure time and temperature on residue loading, green mould control and sporulation inhibition when citrus fruit were dipped in IMZ sulphate. Clementine mandarins, lemons and navel oranges were dipped in a 500 mg L 1 solution of IMZ sulphate for 15, 45 or 90 s, at a temperature of 23, 35 or 45 C, and at pH levels of 3 or 6. At pH 3, similar residues were loaded on fruit regardless of fruit type, temperature or exposure time (1.18–1.51 mg kg 1). At pH 6, both temperature and exposure time influenced residue loading: at 23 C, higher residue levels were observed than at pH 3 (2.03–2.73 mg kg 1, increasing with longer exposure time); at 35 C and 45 C, residue levels increased significantly with 15 s, 30 s and 45 s exposure time (3.34, 5.11, 7.24 mg kg 1 and 5.31, 9.65 and 13.10 mg kg 1, respectively). Clementine mandarin fruit loaded higher residues at pH 6 (6.96 mg kg 1) than lemons and navels (5.20 and 4.82 mg kg 1, respectively). The MRL was frequently exceeded at longer exposure times. Green mould control was lower at pH 3 and was influenced by temperature and exposure time (92.9–97.4% and 42.8–45.1% for lemons and Clementine mandarins, respectively). Better control was observed at pH 6 regardless of temperature and exposure time (96.9–98.7% and 39.3–57.9% for lemons and Clementine mandarins, respectively). On navel oranges, increased exposure time led to increased green mould control (84.7–92.5%). Sporulation incidence was lower at pH 6 (12.5–28.8%, 19.1–39.2% and 4.6% for lemons, Clementine mandarins and navel oranges, respectively) than at pH 3 treatments (72.7–89.9%, 35.8–91.4% and 77.2% for lemons, Clementine mandarins and navel oranges, respectively). The results show that temperature, pH and exposure time are important parameters in dip application using IMZ sulphate formulation with significant effects on green mould control, more so than residue loading alone. ã 2016 Published by Elsevier B.V.
1. Introduction South Africa exported 1,460,633 cartons (15 kg) of fresh fruit in the 2013/14 season (CGA, 2015), making this country the second largest exporter of citrus in the world, exceeded only by Spain (DAFF, 2010). Postharvest decay is frequently a problem, of which 90% is due to green mould, caused by Penicillium digitatum (Pers.:Fr) Sacc. (Kavanagh and Wood, 1967; Eckert and Eaks, 1989).
* Corresponding author at: Citrus Research International, PO Box 28, Nelspruit, 1200, South Africa. E-mail address: [email protected] (P.H. Fourie).
Penicillium digitatum is an ascomycete that survives on orchard debris and produce airborne spores (asexual conidia) that infect wounded and split fruit in the orchard (Brown et al., 1988). Being exclusively a wound pathogen P. digitatum enters through wounds on the fruit made by insects or during harvest (Smilanick et al., 2005). Moisture and nutrients in the wound facilitates conidial germination (Pelser and Eckert, 1977). Sporulating fruit may cause soilage of healthy neighboring fruit, which makes cleaning and repacking necessary, and leads to extra expenses (Brown et al., 1988). Practices such as sanitation and careful handling of fruit during harvesting are essential to control this disease. In addition, fungicides are used for control, of which imazalil (IMZ) is currently
http://dx.doi.org/10.1016/j.postharvbio.2016.06.014 0925-5214/ã 2016 Published by Elsevier B.V.
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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the most reliable (Altieri and Renzo, 2005; Zhang, 2007; Liebenberg, 2011; Erasmus, 2014). Imazalil is a sterol demethylation inhibitor (DMI) (Siegel et al., 1977), with anti-sporulant activity. The maximum residue limit (MRL) for IMZ in South Africa, Japan and most European countries are 5.0 mg kg 1 for citrus on whole fruit (Brown and Dezman, 1990). Residue levels of 2.0–3.5 mg kg 1 are required for green mould control and to inhibit sporulation (Kaplan and Dave, 1979; Brown and Dezman, 1990; Smilanick et al., 1997). It has been reported through recent studies that the sensitive strain of P. digitatum can be effectively controlled in the fungicide bath by IMZ residue of 0.97 mg kg 1, while higher residue levels were required for sporulation inhibition (Erasmus et al., 2011; Njombolwana et al., 2013a). IMZ applications in packhouses in South Africa generally loaded lower residue levels (Erasmus et al., 2011). For effective disease control and compliance with food safety standards, IMZ applications must be done as accurately and effectively as possible. Different application methods are commonly used: dip, drench or wax applications (Kaplan and Dave, 1979; Erasmus et al., 2011). The majority of South-African packhouses apply IMZ either as a single application in the fungicide bath, or as a dip application followed by IMZ incorporated in wax coating application. This additional application in wax improved IMZ residue loading, protective green mould control and sporulation inhibition (Njombolwana et al., 2013a). IMZ application in fungicide baths or dip tanks was the most effective application method for curative control (Erasmus et al., 2011; Njombolwana et al., 2013a). However, there was a lot of variation in specification of fungicide baths used by different packhouses, including the pH and temperature of the solution. Erasmus et al. (2011, 2013, 2015a) studied the effects of pH on IMZ residue loading and green mould control, specifically using the IMZ sulphate salt formulation, which appeared to be very sensitive to pH effects. Several studies reported on the application of the IMZ emulsifiable concentrate (EC) formulation in dip tanks (Smilanick et al., 1997; Schirra et al., 1998; Cabras et al., 1999; Smilanick et al., 2005), but not much on the IMZ sulphate formulation, which is almost exclusively used in South Africa. Erasmus et al. (2011) showed the pH of a IMZ sulphate solution prepared at the registered 500 mg kg 1 in pH neutral water declined to pH3. At this low pH, fruit exposure time and solution temperatures at 25 and 35 C had no effect on IMZ residue loading when fruit was dip treated, but the MRL was exceeded after 45 s in a pH 8 solution. It was highlighted that IMZ sulphate has a pKa level of 6.5 and dissociated at higher pH solutions (Siegel et al., 1977; Erasmus et al., 2011). Erasmus et al. (2013) showed that residue loading increased at longer exposure times when fruit was dipped in an IMZ sulphate solution at pH 6; however, exposure times did not affect residue loading at pH 3, although improved green mould control was observed. One of the factors that have not been investigated fully in these studies with the IMZ sulphate solution was solution temperature. As temperature also influenced residue loading and green mould control (Cabras et al., 1999; Erasmus et al., 2011), the influence of temperature and pH needed to be studied further. Therefore, the aims of this study were to determine combined effects of temperature, exposure time and pH of IMZ sulphate dip applications on curative control of green mould, sporulation inhibition and residue loading. 2. Material and methods 2.1. Penicillium digitatum isolates One isolate of P. digitatum were used in all the trials: isolate STEU 6560 that is sensitive to TBZ and IMZ (Erasmus et al., 2011;
Kellerman et al., 2014). The isolate was plated out from 80 C storage culture onto potato dextrose agar (PDA; DIFCO, Becton, Dickinson and Company, USA) and grown for 7–14 days at 25 C before each trial. Spore suspensions were freshly prepared each day of inoculation by filtering the culture grown on PDA through two layers of cheesecloth with deionised water amended with Tween 20 (Sigma–Aldrich, St Louis, MO, USA) at a concentration of 0.01 mL L 1. Spores were counted with a haemocytometer and final spore concentration adjusted to 106 spores mL 1. Viability of spores was verified after each trial by plating out the used spore suspension on PDA. 2.2. Fruit Untreated export quality citrus fruit were collected from various citrus packhouses in the Western Cape province of South Africa. This was done during the 2013 harvest season as the specific citrus type, lemons (cv. Eureka), mandarin (cv. Nules Clementine) and early navels (cv. Washington), became available for trials. Before the trials commenced, fruit was washed with chlorine (70 mg L 1), air dried and stored at 3.5–7 C for 3 days. Fruit was transferred from cold storage to ambient ( 23 C) a day before each trial. 2.3. Inoculation Inoculations for curative treatments were done 24 h before IMZ dip treatment. Wounding and inoculation were conducted simultaneously by dipping a wounding tool into a spore suspension of P. digitatum (106 spores mL 1) immediately prior to wounding (Erasmus et al., 2011). Wounding tools consisted of a 7 mm diameter cylindrical stainless steel rod with a protruding tip 2 mm long and 1 mm in diameter. With equal distances apart, four wounds were induced on each fruit around the calyx. Three replications were done for every treatment, with 12 fruit being inoculated per treatment combination. Untreated fruit served as controls. 2.4. Temperature and pH trial A 3 3 2 factorial experiment was done for each fruit type with three different temperatures (23 C, 35 C and 45 C), three exposure times (15, 45 and 90 s) and two pH levels (3 and 6). For every citrus type the experiment was done twice. Temperaturecontrolled stainless steel warm water baths (25 L capacity; Unitemp, Baird and Tatlock Ltd., Essex, UK) were used as IMZ dip tanks at 23 C, 35 C and 45 C. Fruit from the various treatment combinations were dipped in a 500 mg L 1 solution of IMZ sulphate (Imazacure, 750 g kg 1 SG, ICA International Chemicals, Stellenbosch, South Africa). The pH of the IMZ sulphate solutions was adjusted to a pH of 6 by using sodium bicarbonate (NaHCO3; Alkalinity Plus, Pool Perfect, Bellville, South Africa) and pH was measured using a pH meter with temperature probe (HI 991002; Hanna Instruments, Woonsocket, Rhode Island). Fruit were left to dry at ambient temperature (23 C) after treatment. On count 13 soft fibreboard trays (SFT) nectarine trays (Huhtamaki South Africa (Pty) Ltd, Atlantis, South Africa), 12 fruit from each treatment combination were packed in lock back table grape cartons (APL Cartons, Worcester, South Africa). Each carton was covered with a transparent polyethylene bag. This created a humid incubation chamber, and also excluded cross-contamination. The cartons were incubated at ambient temperature (22– 25 C). Infection was rated with a UV light (UV-A at 365 nm, Labino Mid-light; www.labino.com) after 4 5 days’ incubation. The number of infected wounds was recorded using the UV light to
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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indicate yellow fluorescent lesions caused by P. digitatum that could not yet be seen with the naked eye (Erasmus et al., 2011). Sporulation was rated after 10–14 days on a scale of 0–6 (0 = no sign of disease; 1 = visible lesion but no sporulation; 2 = sporulating area on lesion smaller than a quarter of the fruit; 3 = sporulating area larger than a quarter of the fruit, but smaller than half of the fruit; 4 = sporulating area larger than half of the fruit, but smaller than three quarters of the fruit; 5 = sporulating area larger than three quarters of the fruit, but smaller than the whole fruit; 6 = sporulating area covering the whole fruit). Sporulation incidence was calculated as a percentage of infected fruit per treatment combination, with a sporulation index of 1–3 regarded as inhibition of sporulation and 4–6 regarded as sporulating. Sporulation incidence (%) was used in further data analysis.
IMZ residue loading at pH 3 with residues ranging from means of 1.18–1.51 mg kg 1 (Table 1). However, at pH 6 both these factors had significant effects: at 23 C, IMZ residue levels were marginally higher than those at pH 3 (2.03–2.73 mg kg 1, increasing with longer exposure time); at 35 C and 45 C, residue levels increased significantly with 15 s, 30 s and 45 s exposure time (3.34, 5.11, 7.24 mg kg 1 and 5.31, 9.65 and 13.10 mg kg 1, respectively). Clementine, lemon and navel fruit loaded similar residues at pH 3 (1.27, 1.35 and 1.29 mg kg 1, respectively; Table 2), compared with significantly higher residues loaded at pH 6, with Clementine loading higher levels (6.96 mg kg 1) than lemons and navels (5.20 and 4.82 mg kg 1, respectively).
2.5. Imazalil residue analysis
Analysis of variance indicated no significant interaction between temperature, pH, exposure time and fruit type (P = 0.702; Supplementary data Table 5), but significant 3-factor interactions with fruit type was observed. Hence the fruit types were analyzed separately.
For each treatment combination, six non-inoculated fruit from two replications were sampled for residue analysis, and stored at 20 C until they were prepared for residue analysis. Fruit were chopped, then weighed and pulped for 2 min using a blender (Salton Elite, Almalgamated Appliance Holdings Limited, Reuven, South Africa). Pulped fruit samples were stored at 20 C. Samples from the first and last replications of each treatment were sent for IMZ residue analyses by an accredited analytical laboratory (Hearshaw and Kinnes Analytical Laboratory, Westlake, Cape Town, South Africa). Samples were extracted using acetonitrile followed by a matrix solid phase dispersion extraction. Analysis of the extracts for chloramizol (IMZ) was conducted by liquid chromatography mass spectrometry (LCMS/MS; Agilent 6410, Agilent Technologies Inc., Santa Clara, CA, USA).
3.2. Green mould control
3.2.1. Lemons Analysis of variance of green mould control data indicated a significant interaction between temperature and pH (P = 0.004; Supplementary data Table 6) and exposure time was significant as main effect (P = 0.001). Very high levels of control were observed (up to 98.7%; Table 3), and generally significantly higher at pH 6 (96.9–98.7%) compared with pH 3 (92.7–97.4%), except at pH 3 at 23 C (97.4%), which was comparable to control achieved at pH 6 at the same temperature. Exposure time of 45 and 90 s gave significantly better control (96.7 and 97.9%, respectively; results not shown) than 15 s (94.3%).
2.6. Statistical analysis For green mould control data, wound infection data were normalized by calculating the percentage control relative to the untreated control. For the sporulation data, the percentage incidence was used in analysis. Data were subjected to analysis of variance, means were separated using Fisher’s least significant difference test at P < 0.05 in XLSTAT version 2011.4.02 (www.xlstat. com). 3. Results 3.1. IMZ residue loading Analysis of variance of the IMZ residue data showed a significant temperature pH exposure time and pH citrus type interactions (P = 0.003 and 0.005, respectively; Supplementary data Table 1). Exposure time and temperature did not influence Table 1 Mean IMZ residues loaded on citrus in different trials where different fruit types (Clementine, navel and lemon fruit) were dipped in 500 mg L 1 IMZ for 15, 45 or 90 s, at temperatures of 23 C, 35 C or 45 C and a pH level of 3 or 6. pH
Temperature ( C)
IMZ residues (mg L
pH 6
a
23 35 45 23 35 45
)
15 s
45 s
90 s
1.23f 1.18f 1.31f
1.21f 1.23f 1.51f
1.23f 1.34f 1.47f
2.03ef 3.34e 5.31d
3.2.3. Navels Analysis of variance indicated no significant interactions, but exposure time had a significant effect on green mould control (P < 0.0001; supplementary data Table 8). Exposure time of 90 s (92.5%) and 45 s (90.9%) gave significantly better control compared with exposure time of 15 s (84.7%). Contrasting with other fruit types, pH and temperature did not have significant effects on control (P = 0.237 and 0.433, respectively).
1 a
Exposure time
pH 3
3.2.2. Clementines Analysis of variance indicated a significant interaction between temperature and exposure time, as well as temperature and pH (P = 0.0013 and P = 0.0003, respectively; Supplementary data Table 7). In general lower levels of green mould control (31.7– 65.6%) was observed than with lemons. Similar control levels were obtained following the various pH and temperature treatments (39.3–45.1%), but with 45 C at a pH 6 resulting in significantly better curative green mould control (57.9%) and pH 3 at a temperature of 35 C significantly poorer (31.7%). Levels of control significantly increased with longer exposure times at all temperatures, except at 35 C where no difference was observed between 15 and 45 s (Table 4).
2.45ef 5.11d 9.65b
Means with the same letter do not differ significantly (p 0.05).
2.73ef 7.24c 13.10a
Table 2 Mean IMZ residues loaded on citrus in different trials where different fruit types (Clementine, navel and lemon fruit) were dipped in 500 mg L 1 IMZ for 15, 45 or 90 s, at temperatures of 23 C, 35 C or 45 C and a pH level of 3 or 6. IMZ residues (mg L
pH 3 pH 6 a
1 a
)
Clementine
Lemon
Navel
1.27c 6.96a
1.35c 5.20b
1.29c 4.82b
Means with the same letter do not differ significantly (p 0.05).
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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Table 3 Mean curative green mould control (%) on lemon fruit after dip treatment with 500 mg L 1 IMZ with exposure times of 15, 45 or 90 s at temperatures of 23, 35 or 45 C. pH
Temperature ( C)
Green mould control (%)a
pH 3
23 35 45
97.4a 94.2bc 92.9c
6.5 12.5 12.5
pH 6
23 35 45
96.9ab 97.8a 98.7a
5.1 6.9 3.7
a
Infected fruit (%)
Means with the same letter do not differ significantly (p 0.05).
3.3. Sporulation incidence There was no interaction between temperature, pH, exposure time and fruit type (P = 0.128; results not shown), but significant 3factor interactions with fruit kind was observed. Since experiments were not designed specifically for studying sporulation incidence, results are not shown but briefly discussed. 3.3.1. Lemons Analysis of variance of sporulation incidence data showed a significant temperature pH interaction (P = 0.012), where much lower sporulation incidence was observed at pH 6 treatments (12.5–28.8%) than pH 3 treatments (72.7–89.9%). Sporulation incidence increased significantly with increasing temperature at pH 3, but not at pH 6. Sporulation tended to decrease in treatments with longer exposure time (effect significant at P = 0.088). 3.3.2. Clementines A significant temperature pH exposure time interaction was observed (P = 0.047). Sporulation incidence was generally significantly higher at pH 3 (35.8 91.4%) compared with pH 6 (19.1– 39.2%). At pH 3, sporulation levels were generally lower at increased temperatures and longer exposure times. The highest level of sporulation was observed at 23 C with 15 s exposure time (91.4%), and the lowest level was observed at 45 C at 45 s (51.5%). At pH 6, temperature and exposure time generally did not have a significant effect on sporulation. 3.3.3. Navels Analysis of variance showed significant effects for exposure time (P = 0.022) and pH (P < 0.0001), but not for temperature (P = 0.335). Sporulation incidence following 15 s dips (47.4%) differed significantly from 45 s (39.2%) and 90 s (36.2%). Sporulation incidence at pH 6 (4.6%) was significantly lower compared to pH 3 (77.2%). 4. Discussion Solution pH, solution temperature and fruit exposure time had significant effects on IMZ residue loading and curative green mould Table 4 Mean curative green mould control (%) on Clementine fruit after dip treatment with 500 mg L 1 IMZ with exposure times of 15, 45 or 90 s at temperatures of 23, 35 or 45 C. Temperature ( C)
23 35 45 a
Green mould control (%)a Exposure time (s)
Infected fruit (%) Exposure time (s)
15 s
45 s
90 s
15 s
45 s
90 s
34.8ef 32.9f 34.9ef
43.6dc 32.7f 50.6bc
52.7b 40.9de 65.6a
91.7 92.4 93.8
85.4 93.1 83.3
79.9 86.8 68.1
Means with the same letter do not differ significantly (p 0.05).
control when using the IMZ sulphate formulation. Managing these factors is important to ensure adherence to MRLs while still loading effective residues for disease control. Three different citrus fruit types, lemon, Clementine mandarin and navel orange fruit were used in this study. Similar trends in IMZ residue loading and green mould control were evident among different citrus types. Eureka lemons appeared to be more resistant to green mould infection than Clementine mandarins and navel oranges, as was evident from decay levels on untreated fruit (results not shown) and control levels obtained in trials using the same methodology. For all three citrus types, a solution pH of 6 loaded higher IMZ residue levels than pH 3 solutions. For the unbuffered (pH 3) IMZ sulphate solutions exposure time and temperature did not significantly influence residue loading. This corroborates similar findings by Erasmus et al. (2011, 2013, 2015a). In an IMZ sulphate solution buffered at pH 6, IMZ residue loading increased on all citrus types when the temperature was increased from 23 C to 45 C. As demonstrated by Erasmus et al. (2011, 2013, 2015a) and confirmed in this study, exposure times at higher pH should be limited to prevent exceedance of the 5 mg kg 1 MRL. Our study did not consider the effect of fruit brushing after dip treatment. Citrus packlines generally have brushes after dip treatment to aid in fruit drying. South African packhouses have at least 8 roller brushes after the dip tank and can be as much as 52 (Erasmus et al., 2011). These brushes reduce the potential residue levels with as much as 90% and 60% after dip treatment in a pH 3 and pH 6 solution, respectively (Erasmus et al., 2015a), and would have prevented MRL exceedance in many of our treatments. Whilst clear differences in residue loading were observed between treatments, it did not correlate with curative green mould control. Imazalil residue loading on lemons at a pH of 3 was similar regardless of temperature, but control levels decreased from 97.4% at 23 C to 92.9% at 45 C. It is possible that prolonged exposure to the heated fungicide solution caused higher green mould incidence, as this was observed with hot air treatment of citrus (Lay-Yee and Rose, 1994). At pH 6, higher residue levels were loaded at higher temperature, but no effect was observed with regards to green mould control; very high levels of control (>96.9%) might have masked any subtle differences that might have been expected. Despite these high levels of control, it was clear that longer exposure times led to better curative control. For all citrus types, better control was observed at pH 6 than pH 3. Clementine mandarin fruit had poor levels of green mould control in general; the best results were seen at pH 6 at the highest temperature, regardless of exposure time. The lower level of control observed on Clementine mandarin fruit might be attributed to inherent fruit susceptibility of the citrus type (BenYehoshua et al., 1992; Palou et al., 2002) as well as the fruit quality of the Clementine batches. Variable green mould control levels between fruit batches have been reported previously (Njombolwana et al., 2013a; Kellerman et al., 2014). Erasmus et al. (2015a) showed that curative control on the more green mould sensitive Clementine mandarin fruit started to steeply decline on infections older than 6 h that was induced by means of a small wound. Control levels were reduced to 65% on 24 h old infections; this was opposed to very good levels of control (>95%) on Navel orange fruit. In our trials, green mould control on navel oranges was only influenced by exposure time (i.e. better control with longer exposure time), although residue loading was influenced by pH and temperature. The same trend applied for sporulation inhibition; for Clementines and lemons lower incidence was observed at pH 6, but temperature and exposure time only had an effect at pH 3, except for navel oranges where exposure time had a main effect. The addition of sodium bicarbonate to thiabendazole (TBZ), azoxystrobin, fludioxonil and pyrimethanil (PYR) also improved
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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their performance to manage postharvest green mould (Smilanick et al., 2005; Smilanick et al., 2006a; Smilanick et al., 2006b; Kanetis et al., 2008). Using sodium bicarbonate on its own to control green mould is effective (Larrigaudiere et al., 2002; Palou et al., 2002). However, this was at much higher levels (>0.5–3%) than what was applied in this study ( 0.03%). At these low levels, the higher efficacy of a sodium bicarbonate buffered IMZ solution (pH 6) can be ascribed to more biologically active molecules of unprotonated IMZ being available than in an unbuffered solution at pH 3 (Erasmus et al., 2013). Improved green mould control at pH 6 is therefore unlikely as a result of sodium bicarbonate’s fungistatic abilities (Smilanick et al., 1999; Palou et al., 2001), but rather to its buffering effect of the IMZ sulphate solution therewith improving IMZ residue loading and efficacy. Heating TBZ, fludioxonil and PYR increased their effectiveness without causing excessive residues on citrus fruit (Smilanick et al., 2008). Smilanick et al. (1997) found that IMZ [notably using the emulsifiable concentrate (EC) formulation] applied in heated solutions for 30 s or longer, resulted in adequate residue deposition to make a second application of IMZ in wax for sporulation control unnecessary. This improvement is probably due to more effective infiltration of the fungicide into the wound infection courts that are exploited by P. digitatum on the rind of citrus fruit (Brown, 1984). In Smilanick et al.’s 1997 study, increasing the solution temperature from 21.1 to 40.6 C had an increasing effect on residue loading of IMZ (EC formulation) at solution pH 7.2, as well as on green mould control. Our study showed similar residue loading effects when using the IMZ sulphate formulation at pH 6. This effect was, however, not so spectacular on green mould control where marginal increases of control was observed with increased solution temperature with the Clementine fruit showing the only exception. Very high levels of control obtained with lemon and navel fruit (>84.7%) might have masked these effects, or 45 C studied as the maximum temperature might have been too low and that higher temperatures should be investigated. In general, the SC formulation seems to lead to higher levels of green mould control than the EC formulation, but that its efficacy lasts for a limited time after application (Sepulveda et al., 2015). Understanding the interactions between solution temperature, solution pH and fruit exposure time will enable packhouses to correctly manage IMZ application to ensure that they adhere to maximum residue limits (MRL), but still ensure biologically effective residue loading. Improved residue loading should lead to more sporulation inhibition, which would also reduce P. digitatum population build-up in packhouses and reduce the risk of fungicide resistance development. IMZ application in aqueous solutions was found to be superior in terms of curative control (Brown et al., 1983; Erasmus et al., 2011; Njombolwana et al., 2013b). Previous studies clearly demonstrated improved protective control and sporulation inhibition following IMZ application in wax coating (Waks et al., 1985; Smilanick et al., 1997; Njombolwana et al., 2013b), whilst aqueous IMZ applications were found to be inferior. In this study, only curative control was evaluated. Further work should be done to determine the effects of temperature and exposure time in IMZ aqueous applications on protective green mould control. This study confirms the work previously done by Erasmus et al. (2013), which indicated that IMZ residue levels should not be used as sole predictor of the effectiveness of a specific application for green mould control. Relatively low levels of IMZ can be highly effective for the curative control of green mould in dip applications. Erasmus et al. (2015b) showed that the effective residue level for 50% curative control of IMZ sensitive isolates of P. digitatum was 0.30 mg kg 1 on Valencia and navel orange fruit. In another study it was also shown that low levels of IMZ residues (<0.5 mg kg 1) can still be highly effective for curative control
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(>90%) (Erasmus et al., 2015a). Whilst low IMZ residue levels might be sufficient to cure relatively fresh green mould infections (<24 hold), higher residue levels are required for protective control (Dore et al., 2009; Erasmus et al., 2011), sporulation control (Smilanick et al., 1997; Njombolwana et al., 2013a) and for the control of IMZ resistant isolates (Smilanick et al., 1997; Erasmus et al., 2011, 2015b). The success of using a fungicide dip tank does not come without challenges. Previous studies have proven that for curative control IMZ is best applied in the dip tank, but IMZ in wax coating is needed for protective control and sporulation inhibition (Njombolwana et al., 2013a). Thus, packhouses that do not apply IMZ in the wax coating, or using excessive brushing after the dip tank, should consider using IMZ at pH of 6 and at a higher temperature for effective sporulation inhibition. Since it is so difficult to restrict exposure time in commercial packhouses, a pH of 3, which gives good curative control, is much safer to use to decrease the risk of exceeding the MRL. IMZ stripping from the fungicide bath solution will also happen faster at a pH of 6 (Erasmus et al., 2013), possibly even more so at higher temperatures. If the IMZ concentration in the dip tank is not carefully maintained, this might lead to inadequate residue loading. The combination of increased solution temperatures and solution pH levels will require strict management to ensure residue loading below the MRL. Brushing after dip will reduce this risk (Erasmus et al., 2015a). Increasing the temperature of the fungicide dip tank will definitely increase energy cost for packhouses. It is evident that with careful management of pH and exposure time, green mould can be effectively controlled at lower temperatures. However, it is not evident how lower temperatures will influence control of other postharvest diseases Acknowledgements The authors thank Citrus Research International, Citrus Academy and Technology and Human Resources for Industry Programme (THRIP) for financial support; personnel at the Department of Plant Pathology, University of Stellenbosch for assistance; Hearshaw and Kinnes Analytical Laboratory (Pty) Ltd. for residue analyses, and ICA International Chemicals (Pty) Ltd. for chemicals. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. postharvbio.2016.06.014. References Altieri, G., Renzo, G.C., 2005. Imazalil on-line control in postharvest treatments of citrus fruit. Acta Hortic. 682, 1773–1777. Ben-Yehoshua, S., Rodov, V., Kim, J.J., Carmeli, S., 1992. Preformed and induced antifungal materials of citrus fruits in relation to the enhancement of decay resistance by heat and ultraviolet treatments. J. Agric. Food Chem. 40, 1217–1221. 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. Brown, G.E., Nagy, S., Maraulja, M., 1983. Residues from postharvest non-recovery spray applications of imazalil to oranges and effects on green mould caused by Penicillium digitatum. Plant Dis. 67, 954–957. Brown, G., Eckert, J., Whiteside, J., Garnsey, S., Timmer, L., 1988. Green mold, pages 35–36. Compendium of Citrus Diseases. APS Press, St.Paul, MN, USA. Brown, G.E., 1984. Efficacy of citrus postharvest fungicides applied in water or resin solution water wax. Plant Dis. 68, 415–418. CGA, 2015. Key Industry Statistics. Citrus Growers Association of Southern Africa (pp. 12). Cabras, P., Schirra, M., Pirisi, F.M., Garau, V.L., Angioni, A., 1999. Factors affecting imazalil and thiabendazole uptake and persistence in citrus fruits following dip treatments. J. Agric. Food Chem. 47, 3352–3354.
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Department of Agriculture, Forestry and Fisheries (DAFF). 2010. A profile of the South African citrus market value chain. http://www.daff.gov.za/docs/AMCP/ Citrusmvcp2011-12.pdf (accessed 2.12.13). Dore, A., Molinu, M.G., Venditti, T., D’Hallewin, G., 2009. Immersion of lemons into imazalil mixtures heated at 50 C alters the cuticle and promotes permeation of imazalil into rind wounds. J. Agric. Food Chem. 57, 623–631. Eckert, J.W., Eaks, I.L., 1989. Postharvest disorders and diseases of citrus fruits, pp. 179–259. In: Reuther, W., Calavan, E., Clair Carman, G.E., Jeppson, L.R. (Eds.), The Citrus Industry. University of California, Division of Agriculture and Natural Resources, Oakland, Calif. 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. Erasmus, A., Lennox, C.L., Smilanick, J.L., Lesar, K., Fourie, P.H., 2013. Imazalil residue loading and green mould control on citrus fruit as affected by formulation: solution pH and exposure time in aqueous dip treatments. Postharvest Biol. Technol. 77, 43–49. Erasmus, A., Lennox, C.L., Njombolwana, N.S., Lesar, K., Fourie, P.H., 2015a. Curative control of citrus green mould by imazalil as influenced by infection age wound size, fruit exposure time, solution pH and fruit brushing after treatment. Postharvest Biol. Technol. 101, 26–36. Erasmus, A., Lennox, C.L., Korsten, L., Lesar, K., Fourie, P.H., 2015b. Imazalil resistance in Penicillium digitatum and P: italicum causing citrus postharvest green and blue mould: impact and options. Postharvest Biol. Technol. 107, 66–76. Erasmus, A., 2014. Optimization of imazalil application and green mould control in South African packhouses. PhD Dissertation. Department Plant Pathology, University of Stellenbosch, Stellenbosch. Kanetis, L., Förster, H., Adaskaveg, J.E., 2008. Optimizing efficacy of new postharvest fungicides and evaluation of sanitizing agents for managing citrus green mold. Plant Dis. 92, 261–269. Kaplan, H.J., Dave, B.A., 1979. The current state of imazalil: a postharvest fungicide for citrus. Proc. Florida State Hortic. Soc. 92, 37–43. Kavanagh, J.H., Wood, R.K.S., 1967. The role of wounds in the infection of oranges by Penicillium digitatum Sacc. Annu. Appl. Biol. 60, 375–383. Kellerman, M., Erasmus, A., Cronjé, P.J., Fourie, P.H., 2014. Thiabendazole residue loading in dip, drench and wax coating applications to control green mould and chilling injury on citrus fruit. Postharvest Biol. Technol. 96, 78–87. Larrigaudiere, C., Pons, J., Torres, R., Usall, J., 2002. Storage performance of Clementines treated with hot water: sodium carbonate and sodium bicarbonate dips. J. Hortic. Sci. Biotechnol. 77, 314–319. Lay-Yee, M., Rose, K.J., 1994. Quality of ‘Fantasia’ nectarines following forced-air heat treatments for insect disinfestation. Hortscience 29, 663–666. Liebenberg, E.C., 2011. Optimal use of pyrimethanil, a new postharvest fungicide, for the control of green mould on citrus in South Africa. BSc(Hons) Report. Department Microbiology: University of Stellenbosch, Stellenbosch. Njombolwana, N.S., Erasmus, A., Fourie, P.H., 2013a. Evaluation of curative and protective control of Penicillium digitatum following imazalil application in wax coating. Postharvest Biol. Technol. 77, 102–110.
Njombolwana, N.S., Erasmus, A., Van Zyl, J.G., du Plooy, W., Cronje, P.J.R., Fourie, P.H., 2013b. Effects of citrus wax coating and brush type on imazalil residue loading: green mould control and fruit quality retention of sweet oranges. Postharvest Biol. Technol. 86, 362–371. Palou, L., Smilanick, J.L., Usall, J., Viñas, I., 2001. Control of postharvest blue and green molds of oranges by hot water sodium carbonate, and sodium bicarbonate. Plant Dis. 85, 371–376. Palou, L., Usall, J., Muñoz, J.A., Smilanick, J.L., Viñas, I., 2002. Hot water sodium carbonate, and sodium bicarbonate for the control of postharvest green and blue molds of Clementine mandarins. Postharvest Biol. Technol. 24, 93–96. Pelser, P.D.T., Eckert, J.W., 1977. Constituents of orange juice that stimulate germination of conidia of Penicillium digitatum. Phytopathology 67, 747–754. Schirra, M., Cabras, P., Angioni, A., Ruggiu, R., Minelli, E., 1998. Synergistic actions of 50 C water and imazalil dip treatments to preserve quality of late-season ‘Marsh’ grapefruit. Adv. Hortic. Sci. 12, 63–66. Sepulveda, M., Cuevas, I.I., Smilanick, J.L., Cerioni, L., Rapisarda, V.A., Ramallo, J., 2015. Improvement in imazalil treatments in commercial packinglines to control green mold on lemon fruit. Sci. Hortic. 192, 387–390. Siegel, M.R., Kerkenaar, A., Kaars-Sijpesteijn, A., 1977. Antifungal activity of the systemic fungicide imazalil. Plant Pathol. 83, 121–133. 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 mould of citrus with imazalil in warm water compared with its use in wax. Plant Dis. 81, 1299–1304. Smilanick, J.L., Margosan, D.A., Mlikota, F., Usall, J., Michael, I.F., 1999. Control of citrus green mold by carbonate and bicarbonate salts and the influence of commercial postharvest practices on their efficacy. Plant Dis. 83, 139–145. Smilanick, J.L., Mansour, M.F., Margosan, D.A., Gabler, F.M., Goodwin, W.R., 2005. Influence of pH and NaHCO3 on effectiveness of imazalil to inhibit germination of Penicillium digitatum and to control postharvest green mould on citrus fruit. Plant Dis. 89, 640–648. Smilanick, J.L., Mansour, M.F., Gabler, F.M., Goodwin, W.R., 2006a. The effectiveness of pyramethanil to inhibit germination of Penicillium digitatum and to control citrus green mould after harvest. Postharvest Biol. Technol. 42, 75–85. Smilanick, J.L., Mansour, M.F., Sorenson, D., 2006b. Pre- and postharvest treatments to control green mold of citrus fruit during ethylene degreening. Plant Dis. 90, 89–96. Smilanick, J.L., Mansour, M.F., Gabler, F.M., Soenson, D., 2008. Control of citrus postharvest green mould and sour rot by potassium sorbate combined with heat and fungicides. Postharvest Biol. Technol. 47, 226–238. 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. Zhang, J., 2007. The potential of a new fungicide fludioxonil for stem-end rot and green mold control on Florida citrus fruit. Postharvest Biol. Technol. 46, 262–270.
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The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application Mareli Kellermana,b , Jéanine Joubertc , Arno Erasmusb , Paul H. Fouriea,b,* a b c
Department of Plant Pathology, Stellenbosch University, Private bag X1, Stellenbosch 7602, South Africa Citrus Research International, PO Box 28, Nelspruit, 1200, South Africa Department of Horticulture, Stellenbosch University, Private bag X1, Stellenbosch 7602, South Africa
A R T I C L E I N F O
Article history: Received 15 February 2016 Received in revised form 16 May 2016 Accepted 17 June 2016 Available online xxx Keywords: Fungicide Postharvest Citrus
A B S T R A C T
Imazalil (IMZ) is the most relied upon fungicide for use against citrus green mould, caused by Penicillium digitatum. In South Africa, the IMZ sulphate formulation is used as dip application in packhouses. Previous studies showed that IMZ efficacy and residue loading of this formulation is highly affected by pH in the application solution. This study investigated the effect of pH, exposure time and temperature on residue loading, green mould control and sporulation inhibition when citrus fruit were dipped in IMZ sulphate. Clementine mandarins, lemons and navel oranges were dipped in a 500 mg L 1 solution of IMZ sulphate for 15, 45 or 90 s, at a temperature of 23, 35 or 45 C, and at pH levels of 3 or 6. At pH 3, similar residues were loaded on fruit regardless of fruit type, temperature or exposure time (1.18–1.51 mg kg 1). At pH 6, both temperature and exposure time influenced residue loading: at 23 C, higher residue levels were observed than at pH 3 (2.03–2.73 mg kg 1, increasing with longer exposure time); at 35 C and 45 C, residue levels increased significantly with 15 s, 30 s and 45 s exposure time (3.34, 5.11, 7.24 mg kg 1 and 5.31, 9.65 and 13.10 mg kg 1, respectively). Clementine mandarin fruit loaded higher residues at pH 6 (6.96 mg kg 1) than lemons and navels (5.20 and 4.82 mg kg 1, respectively). The MRL was frequently exceeded at longer exposure times. Green mould control was lower at pH 3 and was influenced by temperature and exposure time (92.9–97.4% and 42.8–45.1% for lemons and Clementine mandarins, respectively). Better control was observed at pH 6 regardless of temperature and exposure time (96.9–98.7% and 39.3–57.9% for lemons and Clementine mandarins, respectively). On navel oranges, increased exposure time led to increased green mould control (84.7–92.5%). Sporulation incidence was lower at pH 6 (12.5–28.8%, 19.1–39.2% and 4.6% for lemons, Clementine mandarins and navel oranges, respectively) than at pH 3 treatments (72.7–89.9%, 35.8–91.4% and 77.2% for lemons, Clementine mandarins and navel oranges, respectively). The results show that temperature, pH and exposure time are important parameters in dip application using IMZ sulphate formulation with significant effects on green mould control, more so than residue loading alone. ã 2016 Published by Elsevier B.V.
1. Introduction South Africa exported 1,460,633 cartons (15 kg) of fresh fruit in the 2013/14 season (CGA, 2015), making this country the second largest exporter of citrus in the world, exceeded only by Spain (DAFF, 2010). Postharvest decay is frequently a problem, of which 90% is due to green mould, caused by Penicillium digitatum (Pers.:Fr) Sacc. (Kavanagh and Wood, 1967; Eckert and Eaks, 1989).
* Corresponding author at: Citrus Research International, PO Box 28, Nelspruit, 1200, South Africa. E-mail address: [email protected] (P.H. Fourie).
Penicillium digitatum is an ascomycete that survives on orchard debris and produce airborne spores (asexual conidia) that infect wounded and split fruit in the orchard (Brown et al., 1988). Being exclusively a wound pathogen P. digitatum enters through wounds on the fruit made by insects or during harvest (Smilanick et al., 2005). Moisture and nutrients in the wound facilitates conidial germination (Pelser and Eckert, 1977). Sporulating fruit may cause soilage of healthy neighboring fruit, which makes cleaning and repacking necessary, and leads to extra expenses (Brown et al., 1988). Practices such as sanitation and careful handling of fruit during harvesting are essential to control this disease. In addition, fungicides are used for control, of which imazalil (IMZ) is currently
http://dx.doi.org/10.1016/j.postharvbio.2016.06.014 0925-5214/ã 2016 Published by Elsevier B.V.
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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the most reliable (Altieri and Renzo, 2005; Zhang, 2007; Liebenberg, 2011; Erasmus, 2014). Imazalil is a sterol demethylation inhibitor (DMI) (Siegel et al., 1977), with anti-sporulant activity. The maximum residue limit (MRL) for IMZ in South Africa, Japan and most European countries are 5.0 mg kg 1 for citrus on whole fruit (Brown and Dezman, 1990). Residue levels of 2.0–3.5 mg kg 1 are required for green mould control and to inhibit sporulation (Kaplan and Dave, 1979; Brown and Dezman, 1990; Smilanick et al., 1997). It has been reported through recent studies that the sensitive strain of P. digitatum can be effectively controlled in the fungicide bath by IMZ residue of 0.97 mg kg 1, while higher residue levels were required for sporulation inhibition (Erasmus et al., 2011; Njombolwana et al., 2013a). IMZ applications in packhouses in South Africa generally loaded lower residue levels (Erasmus et al., 2011). For effective disease control and compliance with food safety standards, IMZ applications must be done as accurately and effectively as possible. Different application methods are commonly used: dip, drench or wax applications (Kaplan and Dave, 1979; Erasmus et al., 2011). The majority of South-African packhouses apply IMZ either as a single application in the fungicide bath, or as a dip application followed by IMZ incorporated in wax coating application. This additional application in wax improved IMZ residue loading, protective green mould control and sporulation inhibition (Njombolwana et al., 2013a). IMZ application in fungicide baths or dip tanks was the most effective application method for curative control (Erasmus et al., 2011; Njombolwana et al., 2013a). However, there was a lot of variation in specification of fungicide baths used by different packhouses, including the pH and temperature of the solution. Erasmus et al. (2011, 2013, 2015a) studied the effects of pH on IMZ residue loading and green mould control, specifically using the IMZ sulphate salt formulation, which appeared to be very sensitive to pH effects. Several studies reported on the application of the IMZ emulsifiable concentrate (EC) formulation in dip tanks (Smilanick et al., 1997; Schirra et al., 1998; Cabras et al., 1999; Smilanick et al., 2005), but not much on the IMZ sulphate formulation, which is almost exclusively used in South Africa. Erasmus et al. (2011) showed the pH of a IMZ sulphate solution prepared at the registered 500 mg kg 1 in pH neutral water declined to pH3. At this low pH, fruit exposure time and solution temperatures at 25 and 35 C had no effect on IMZ residue loading when fruit was dip treated, but the MRL was exceeded after 45 s in a pH 8 solution. It was highlighted that IMZ sulphate has a pKa level of 6.5 and dissociated at higher pH solutions (Siegel et al., 1977; Erasmus et al., 2011). Erasmus et al. (2013) showed that residue loading increased at longer exposure times when fruit was dipped in an IMZ sulphate solution at pH 6; however, exposure times did not affect residue loading at pH 3, although improved green mould control was observed. One of the factors that have not been investigated fully in these studies with the IMZ sulphate solution was solution temperature. As temperature also influenced residue loading and green mould control (Cabras et al., 1999; Erasmus et al., 2011), the influence of temperature and pH needed to be studied further. Therefore, the aims of this study were to determine combined effects of temperature, exposure time and pH of IMZ sulphate dip applications on curative control of green mould, sporulation inhibition and residue loading. 2. Material and methods 2.1. Penicillium digitatum isolates One isolate of P. digitatum were used in all the trials: isolate STEU 6560 that is sensitive to TBZ and IMZ (Erasmus et al., 2011;
Kellerman et al., 2014). The isolate was plated out from 80 C storage culture onto potato dextrose agar (PDA; DIFCO, Becton, Dickinson and Company, USA) and grown for 7–14 days at 25 C before each trial. Spore suspensions were freshly prepared each day of inoculation by filtering the culture grown on PDA through two layers of cheesecloth with deionised water amended with Tween 20 (Sigma–Aldrich, St Louis, MO, USA) at a concentration of 0.01 mL L 1. Spores were counted with a haemocytometer and final spore concentration adjusted to 106 spores mL 1. Viability of spores was verified after each trial by plating out the used spore suspension on PDA. 2.2. Fruit Untreated export quality citrus fruit were collected from various citrus packhouses in the Western Cape province of South Africa. This was done during the 2013 harvest season as the specific citrus type, lemons (cv. Eureka), mandarin (cv. Nules Clementine) and early navels (cv. Washington), became available for trials. Before the trials commenced, fruit was washed with chlorine (70 mg L 1), air dried and stored at 3.5–7 C for 3 days. Fruit was transferred from cold storage to ambient ( 23 C) a day before each trial. 2.3. Inoculation Inoculations for curative treatments were done 24 h before IMZ dip treatment. Wounding and inoculation were conducted simultaneously by dipping a wounding tool into a spore suspension of P. digitatum (106 spores mL 1) immediately prior to wounding (Erasmus et al., 2011). Wounding tools consisted of a 7 mm diameter cylindrical stainless steel rod with a protruding tip 2 mm long and 1 mm in diameter. With equal distances apart, four wounds were induced on each fruit around the calyx. Three replications were done for every treatment, with 12 fruit being inoculated per treatment combination. Untreated fruit served as controls. 2.4. Temperature and pH trial A 3 3 2 factorial experiment was done for each fruit type with three different temperatures (23 C, 35 C and 45 C), three exposure times (15, 45 and 90 s) and two pH levels (3 and 6). For every citrus type the experiment was done twice. Temperaturecontrolled stainless steel warm water baths (25 L capacity; Unitemp, Baird and Tatlock Ltd., Essex, UK) were used as IMZ dip tanks at 23 C, 35 C and 45 C. Fruit from the various treatment combinations were dipped in a 500 mg L 1 solution of IMZ sulphate (Imazacure, 750 g kg 1 SG, ICA International Chemicals, Stellenbosch, South Africa). The pH of the IMZ sulphate solutions was adjusted to a pH of 6 by using sodium bicarbonate (NaHCO3; Alkalinity Plus, Pool Perfect, Bellville, South Africa) and pH was measured using a pH meter with temperature probe (HI 991002; Hanna Instruments, Woonsocket, Rhode Island). Fruit were left to dry at ambient temperature (23 C) after treatment. On count 13 soft fibreboard trays (SFT) nectarine trays (Huhtamaki South Africa (Pty) Ltd, Atlantis, South Africa), 12 fruit from each treatment combination were packed in lock back table grape cartons (APL Cartons, Worcester, South Africa). Each carton was covered with a transparent polyethylene bag. This created a humid incubation chamber, and also excluded cross-contamination. The cartons were incubated at ambient temperature (22– 25 C). Infection was rated with a UV light (UV-A at 365 nm, Labino Mid-light; www.labino.com) after 4 5 days’ incubation. The number of infected wounds was recorded using the UV light to
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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indicate yellow fluorescent lesions caused by P. digitatum that could not yet be seen with the naked eye (Erasmus et al., 2011). Sporulation was rated after 10–14 days on a scale of 0–6 (0 = no sign of disease; 1 = visible lesion but no sporulation; 2 = sporulating area on lesion smaller than a quarter of the fruit; 3 = sporulating area larger than a quarter of the fruit, but smaller than half of the fruit; 4 = sporulating area larger than half of the fruit, but smaller than three quarters of the fruit; 5 = sporulating area larger than three quarters of the fruit, but smaller than the whole fruit; 6 = sporulating area covering the whole fruit). Sporulation incidence was calculated as a percentage of infected fruit per treatment combination, with a sporulation index of 1–3 regarded as inhibition of sporulation and 4–6 regarded as sporulating. Sporulation incidence (%) was used in further data analysis.
IMZ residue loading at pH 3 with residues ranging from means of 1.18–1.51 mg kg 1 (Table 1). However, at pH 6 both these factors had significant effects: at 23 C, IMZ residue levels were marginally higher than those at pH 3 (2.03–2.73 mg kg 1, increasing with longer exposure time); at 35 C and 45 C, residue levels increased significantly with 15 s, 30 s and 45 s exposure time (3.34, 5.11, 7.24 mg kg 1 and 5.31, 9.65 and 13.10 mg kg 1, respectively). Clementine, lemon and navel fruit loaded similar residues at pH 3 (1.27, 1.35 and 1.29 mg kg 1, respectively; Table 2), compared with significantly higher residues loaded at pH 6, with Clementine loading higher levels (6.96 mg kg 1) than lemons and navels (5.20 and 4.82 mg kg 1, respectively).
2.5. Imazalil residue analysis
Analysis of variance indicated no significant interaction between temperature, pH, exposure time and fruit type (P = 0.702; Supplementary data Table 5), but significant 3-factor interactions with fruit type was observed. Hence the fruit types were analyzed separately.
For each treatment combination, six non-inoculated fruit from two replications were sampled for residue analysis, and stored at 20 C until they were prepared for residue analysis. Fruit were chopped, then weighed and pulped for 2 min using a blender (Salton Elite, Almalgamated Appliance Holdings Limited, Reuven, South Africa). Pulped fruit samples were stored at 20 C. Samples from the first and last replications of each treatment were sent for IMZ residue analyses by an accredited analytical laboratory (Hearshaw and Kinnes Analytical Laboratory, Westlake, Cape Town, South Africa). Samples were extracted using acetonitrile followed by a matrix solid phase dispersion extraction. Analysis of the extracts for chloramizol (IMZ) was conducted by liquid chromatography mass spectrometry (LCMS/MS; Agilent 6410, Agilent Technologies Inc., Santa Clara, CA, USA).
3.2. Green mould control
3.2.1. Lemons Analysis of variance of green mould control data indicated a significant interaction between temperature and pH (P = 0.004; Supplementary data Table 6) and exposure time was significant as main effect (P = 0.001). Very high levels of control were observed (up to 98.7%; Table 3), and generally significantly higher at pH 6 (96.9–98.7%) compared with pH 3 (92.7–97.4%), except at pH 3 at 23 C (97.4%), which was comparable to control achieved at pH 6 at the same temperature. Exposure time of 45 and 90 s gave significantly better control (96.7 and 97.9%, respectively; results not shown) than 15 s (94.3%).
2.6. Statistical analysis For green mould control data, wound infection data were normalized by calculating the percentage control relative to the untreated control. For the sporulation data, the percentage incidence was used in analysis. Data were subjected to analysis of variance, means were separated using Fisher’s least significant difference test at P < 0.05 in XLSTAT version 2011.4.02 (www.xlstat. com). 3. Results 3.1. IMZ residue loading Analysis of variance of the IMZ residue data showed a significant temperature pH exposure time and pH citrus type interactions (P = 0.003 and 0.005, respectively; Supplementary data Table 1). Exposure time and temperature did not influence Table 1 Mean IMZ residues loaded on citrus in different trials where different fruit types (Clementine, navel and lemon fruit) were dipped in 500 mg L 1 IMZ for 15, 45 or 90 s, at temperatures of 23 C, 35 C or 45 C and a pH level of 3 or 6. pH
Temperature ( C)
IMZ residues (mg L
pH 6
a
23 35 45 23 35 45
)
15 s
45 s
90 s
1.23f 1.18f 1.31f
1.21f 1.23f 1.51f
1.23f 1.34f 1.47f
2.03ef 3.34e 5.31d
3.2.3. Navels Analysis of variance indicated no significant interactions, but exposure time had a significant effect on green mould control (P < 0.0001; supplementary data Table 8). Exposure time of 90 s (92.5%) and 45 s (90.9%) gave significantly better control compared with exposure time of 15 s (84.7%). Contrasting with other fruit types, pH and temperature did not have significant effects on control (P = 0.237 and 0.433, respectively).
1 a
Exposure time
pH 3
3.2.2. Clementines Analysis of variance indicated a significant interaction between temperature and exposure time, as well as temperature and pH (P = 0.0013 and P = 0.0003, respectively; Supplementary data Table 7). In general lower levels of green mould control (31.7– 65.6%) was observed than with lemons. Similar control levels were obtained following the various pH and temperature treatments (39.3–45.1%), but with 45 C at a pH 6 resulting in significantly better curative green mould control (57.9%) and pH 3 at a temperature of 35 C significantly poorer (31.7%). Levels of control significantly increased with longer exposure times at all temperatures, except at 35 C where no difference was observed between 15 and 45 s (Table 4).
2.45ef 5.11d 9.65b
Means with the same letter do not differ significantly (p 0.05).
2.73ef 7.24c 13.10a
Table 2 Mean IMZ residues loaded on citrus in different trials where different fruit types (Clementine, navel and lemon fruit) were dipped in 500 mg L 1 IMZ for 15, 45 or 90 s, at temperatures of 23 C, 35 C or 45 C and a pH level of 3 or 6. IMZ residues (mg L
pH 3 pH 6 a
1 a
)
Clementine
Lemon
Navel
1.27c 6.96a
1.35c 5.20b
1.29c 4.82b
Means with the same letter do not differ significantly (p 0.05).
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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Table 3 Mean curative green mould control (%) on lemon fruit after dip treatment with 500 mg L 1 IMZ with exposure times of 15, 45 or 90 s at temperatures of 23, 35 or 45 C. pH
Temperature ( C)
Green mould control (%)a
pH 3
23 35 45
97.4a 94.2bc 92.9c
6.5 12.5 12.5
pH 6
23 35 45
96.9ab 97.8a 98.7a
5.1 6.9 3.7
a
Infected fruit (%)
Means with the same letter do not differ significantly (p 0.05).
3.3. Sporulation incidence There was no interaction between temperature, pH, exposure time and fruit type (P = 0.128; results not shown), but significant 3factor interactions with fruit kind was observed. Since experiments were not designed specifically for studying sporulation incidence, results are not shown but briefly discussed. 3.3.1. Lemons Analysis of variance of sporulation incidence data showed a significant temperature pH interaction (P = 0.012), where much lower sporulation incidence was observed at pH 6 treatments (12.5–28.8%) than pH 3 treatments (72.7–89.9%). Sporulation incidence increased significantly with increasing temperature at pH 3, but not at pH 6. Sporulation tended to decrease in treatments with longer exposure time (effect significant at P = 0.088). 3.3.2. Clementines A significant temperature pH exposure time interaction was observed (P = 0.047). Sporulation incidence was generally significantly higher at pH 3 (35.8 91.4%) compared with pH 6 (19.1– 39.2%). At pH 3, sporulation levels were generally lower at increased temperatures and longer exposure times. The highest level of sporulation was observed at 23 C with 15 s exposure time (91.4%), and the lowest level was observed at 45 C at 45 s (51.5%). At pH 6, temperature and exposure time generally did not have a significant effect on sporulation. 3.3.3. Navels Analysis of variance showed significant effects for exposure time (P = 0.022) and pH (P < 0.0001), but not for temperature (P = 0.335). Sporulation incidence following 15 s dips (47.4%) differed significantly from 45 s (39.2%) and 90 s (36.2%). Sporulation incidence at pH 6 (4.6%) was significantly lower compared to pH 3 (77.2%). 4. Discussion Solution pH, solution temperature and fruit exposure time had significant effects on IMZ residue loading and curative green mould Table 4 Mean curative green mould control (%) on Clementine fruit after dip treatment with 500 mg L 1 IMZ with exposure times of 15, 45 or 90 s at temperatures of 23, 35 or 45 C. Temperature ( C)
23 35 45 a
Green mould control (%)a Exposure time (s)
Infected fruit (%) Exposure time (s)
15 s
45 s
90 s
15 s
45 s
90 s
34.8ef 32.9f 34.9ef
43.6dc 32.7f 50.6bc
52.7b 40.9de 65.6a
91.7 92.4 93.8
85.4 93.1 83.3
79.9 86.8 68.1
Means with the same letter do not differ significantly (p 0.05).
control when using the IMZ sulphate formulation. Managing these factors is important to ensure adherence to MRLs while still loading effective residues for disease control. Three different citrus fruit types, lemon, Clementine mandarin and navel orange fruit were used in this study. Similar trends in IMZ residue loading and green mould control were evident among different citrus types. Eureka lemons appeared to be more resistant to green mould infection than Clementine mandarins and navel oranges, as was evident from decay levels on untreated fruit (results not shown) and control levels obtained in trials using the same methodology. For all three citrus types, a solution pH of 6 loaded higher IMZ residue levels than pH 3 solutions. For the unbuffered (pH 3) IMZ sulphate solutions exposure time and temperature did not significantly influence residue loading. This corroborates similar findings by Erasmus et al. (2011, 2013, 2015a). In an IMZ sulphate solution buffered at pH 6, IMZ residue loading increased on all citrus types when the temperature was increased from 23 C to 45 C. As demonstrated by Erasmus et al. (2011, 2013, 2015a) and confirmed in this study, exposure times at higher pH should be limited to prevent exceedance of the 5 mg kg 1 MRL. Our study did not consider the effect of fruit brushing after dip treatment. Citrus packlines generally have brushes after dip treatment to aid in fruit drying. South African packhouses have at least 8 roller brushes after the dip tank and can be as much as 52 (Erasmus et al., 2011). These brushes reduce the potential residue levels with as much as 90% and 60% after dip treatment in a pH 3 and pH 6 solution, respectively (Erasmus et al., 2015a), and would have prevented MRL exceedance in many of our treatments. Whilst clear differences in residue loading were observed between treatments, it did not correlate with curative green mould control. Imazalil residue loading on lemons at a pH of 3 was similar regardless of temperature, but control levels decreased from 97.4% at 23 C to 92.9% at 45 C. It is possible that prolonged exposure to the heated fungicide solution caused higher green mould incidence, as this was observed with hot air treatment of citrus (Lay-Yee and Rose, 1994). At pH 6, higher residue levels were loaded at higher temperature, but no effect was observed with regards to green mould control; very high levels of control (>96.9%) might have masked any subtle differences that might have been expected. Despite these high levels of control, it was clear that longer exposure times led to better curative control. For all citrus types, better control was observed at pH 6 than pH 3. Clementine mandarin fruit had poor levels of green mould control in general; the best results were seen at pH 6 at the highest temperature, regardless of exposure time. The lower level of control observed on Clementine mandarin fruit might be attributed to inherent fruit susceptibility of the citrus type (BenYehoshua et al., 1992; Palou et al., 2002) as well as the fruit quality of the Clementine batches. Variable green mould control levels between fruit batches have been reported previously (Njombolwana et al., 2013a; Kellerman et al., 2014). Erasmus et al. (2015a) showed that curative control on the more green mould sensitive Clementine mandarin fruit started to steeply decline on infections older than 6 h that was induced by means of a small wound. Control levels were reduced to 65% on 24 h old infections; this was opposed to very good levels of control (>95%) on Navel orange fruit. In our trials, green mould control on navel oranges was only influenced by exposure time (i.e. better control with longer exposure time), although residue loading was influenced by pH and temperature. The same trend applied for sporulation inhibition; for Clementines and lemons lower incidence was observed at pH 6, but temperature and exposure time only had an effect at pH 3, except for navel oranges where exposure time had a main effect. The addition of sodium bicarbonate to thiabendazole (TBZ), azoxystrobin, fludioxonil and pyrimethanil (PYR) also improved
Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014
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their performance to manage postharvest green mould (Smilanick et al., 2005; Smilanick et al., 2006a; Smilanick et al., 2006b; Kanetis et al., 2008). Using sodium bicarbonate on its own to control green mould is effective (Larrigaudiere et al., 2002; Palou et al., 2002). However, this was at much higher levels (>0.5–3%) than what was applied in this study ( 0.03%). At these low levels, the higher efficacy of a sodium bicarbonate buffered IMZ solution (pH 6) can be ascribed to more biologically active molecules of unprotonated IMZ being available than in an unbuffered solution at pH 3 (Erasmus et al., 2013). Improved green mould control at pH 6 is therefore unlikely as a result of sodium bicarbonate’s fungistatic abilities (Smilanick et al., 1999; Palou et al., 2001), but rather to its buffering effect of the IMZ sulphate solution therewith improving IMZ residue loading and efficacy. Heating TBZ, fludioxonil and PYR increased their effectiveness without causing excessive residues on citrus fruit (Smilanick et al., 2008). Smilanick et al. (1997) found that IMZ [notably using the emulsifiable concentrate (EC) formulation] applied in heated solutions for 30 s or longer, resulted in adequate residue deposition to make a second application of IMZ in wax for sporulation control unnecessary. This improvement is probably due to more effective infiltration of the fungicide into the wound infection courts that are exploited by P. digitatum on the rind of citrus fruit (Brown, 1984). In Smilanick et al.’s 1997 study, increasing the solution temperature from 21.1 to 40.6 C had an increasing effect on residue loading of IMZ (EC formulation) at solution pH 7.2, as well as on green mould control. Our study showed similar residue loading effects when using the IMZ sulphate formulation at pH 6. This effect was, however, not so spectacular on green mould control where marginal increases of control was observed with increased solution temperature with the Clementine fruit showing the only exception. Very high levels of control obtained with lemon and navel fruit (>84.7%) might have masked these effects, or 45 C studied as the maximum temperature might have been too low and that higher temperatures should be investigated. In general, the SC formulation seems to lead to higher levels of green mould control than the EC formulation, but that its efficacy lasts for a limited time after application (Sepulveda et al., 2015). Understanding the interactions between solution temperature, solution pH and fruit exposure time will enable packhouses to correctly manage IMZ application to ensure that they adhere to maximum residue limits (MRL), but still ensure biologically effective residue loading. Improved residue loading should lead to more sporulation inhibition, which would also reduce P. digitatum population build-up in packhouses and reduce the risk of fungicide resistance development. IMZ application in aqueous solutions was found to be superior in terms of curative control (Brown et al., 1983; Erasmus et al., 2011; Njombolwana et al., 2013b). Previous studies clearly demonstrated improved protective control and sporulation inhibition following IMZ application in wax coating (Waks et al., 1985; Smilanick et al., 1997; Njombolwana et al., 2013b), whilst aqueous IMZ applications were found to be inferior. In this study, only curative control was evaluated. Further work should be done to determine the effects of temperature and exposure time in IMZ aqueous applications on protective green mould control. This study confirms the work previously done by Erasmus et al. (2013), which indicated that IMZ residue levels should not be used as sole predictor of the effectiveness of a specific application for green mould control. Relatively low levels of IMZ can be highly effective for the curative control of green mould in dip applications. Erasmus et al. (2015b) showed that the effective residue level for 50% curative control of IMZ sensitive isolates of P. digitatum was 0.30 mg kg 1 on Valencia and navel orange fruit. In another study it was also shown that low levels of IMZ residues (<0.5 mg kg 1) can still be highly effective for curative control
5
(>90%) (Erasmus et al., 2015a). Whilst low IMZ residue levels might be sufficient to cure relatively fresh green mould infections (<24 hold), higher residue levels are required for protective control (Dore et al., 2009; Erasmus et al., 2011), sporulation control (Smilanick et al., 1997; Njombolwana et al., 2013a) and for the control of IMZ resistant isolates (Smilanick et al., 1997; Erasmus et al., 2011, 2015b). The success of using a fungicide dip tank does not come without challenges. Previous studies have proven that for curative control IMZ is best applied in the dip tank, but IMZ in wax coating is needed for protective control and sporulation inhibition (Njombolwana et al., 2013a). Thus, packhouses that do not apply IMZ in the wax coating, or using excessive brushing after the dip tank, should consider using IMZ at pH of 6 and at a higher temperature for effective sporulation inhibition. Since it is so difficult to restrict exposure time in commercial packhouses, a pH of 3, which gives good curative control, is much safer to use to decrease the risk of exceeding the MRL. IMZ stripping from the fungicide bath solution will also happen faster at a pH of 6 (Erasmus et al., 2013), possibly even more so at higher temperatures. If the IMZ concentration in the dip tank is not carefully maintained, this might lead to inadequate residue loading. The combination of increased solution temperatures and solution pH levels will require strict management to ensure residue loading below the MRL. Brushing after dip will reduce this risk (Erasmus et al., 2015a). Increasing the temperature of the fungicide dip tank will definitely increase energy cost for packhouses. It is evident that with careful management of pH and exposure time, green mould can be effectively controlled at lower temperatures. However, it is not evident how lower temperatures will influence control of other postharvest diseases Acknowledgements The authors thank Citrus Research International, Citrus Academy and Technology and Human Resources for Industry Programme (THRIP) for financial support; personnel at the Department of Plant Pathology, University of Stellenbosch for assistance; Hearshaw and Kinnes Analytical Laboratory (Pty) Ltd. for residue analyses, and ICA International Chemicals (Pty) Ltd. for chemicals. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. postharvbio.2016.06.014. References Altieri, G., Renzo, G.C., 2005. Imazalil on-line control in postharvest treatments of citrus fruit. Acta Hortic. 682, 1773–1777. Ben-Yehoshua, S., Rodov, V., Kim, J.J., Carmeli, S., 1992. Preformed and induced antifungal materials of citrus fruits in relation to the enhancement of decay resistance by heat and ultraviolet treatments. J. Agric. Food Chem. 40, 1217–1221. 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. Brown, G.E., Nagy, S., Maraulja, M., 1983. Residues from postharvest non-recovery spray applications of imazalil to oranges and effects on green mould caused by Penicillium digitatum. Plant Dis. 67, 954–957. Brown, G., Eckert, J., Whiteside, J., Garnsey, S., Timmer, L., 1988. Green mold, pages 35–36. Compendium of Citrus Diseases. APS Press, St.Paul, MN, USA. Brown, G.E., 1984. Efficacy of citrus postharvest fungicides applied in water or resin solution water wax. Plant Dis. 68, 415–418. CGA, 2015. Key Industry Statistics. Citrus Growers Association of Southern Africa (pp. 12). Cabras, P., Schirra, M., Pirisi, F.M., Garau, V.L., Angioni, A., 1999. Factors affecting imazalil and thiabendazole uptake and persistence in citrus fruits following dip treatments. J. Agric. Food Chem. 47, 3352–3354.
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Please cite this article in press as: M. Kellerman, et al., The effect of temperature, exposure time and pH on imazalil residue loading and green mould control on citrus through dip application, Postharvest Biol. Technol. (2016), http://dx.doi.org/10.1016/j.postharvbio.2016.06.014