Evaluation of curative and protective control of Penicillium digitatum following imazalil application in wax coating

Postharvest Biology and Technology 77 (2013) 102–110

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Evaluation of curative and protective control of Penicillium digitatum following imazalil application in wax coating Ncumisa S. Njombolwana a,b , Arno Erasmus b , Paul H. Fourie a,b,∗ a b

Department of Plant Pathology, University of Stellenbosch, Stellenbosch, South Africa Citrus Research International, Nelspruit, South Africa

a r t i c l e

i n f o

Article history: Received 11 September 2012 Accepted 24 November 2012 Keywords: Citrus green mould Residue loading Sporulation inhibition

a b s t r a c t Imazalil (IMZ) is widely used in citrus packhouses to manage green mould, caused by Penicillium digitatum. The aim of this study was to investigate green mould control efficacy of IMZ applied in a wax coating, and the combination of aqueous dip and coating IMZ applications. Single application of IMZ at 3000 ␮g mL−1 in carnauba wax coating at rates of 0.6, 1.2 and 1.8 L tonne−1 of fruit gave better protective (mean 13% infection) than curative (mean 70% infection) control of the sensitive isolate. Imazalil residue levels increased (0.85 to 1.75 ␮g g−1 ) with increasing coating load. However, the resistant isolate could not be controlled (>74% infection). Dip only treatment (IMZ sulphate at 500 ␮g mL−1 for 45 s and 90 s) gave good curative control (≈77%) of the sensitive isolate at residue loading of 0.12–0.73 ␮g g−1 . Wax coating only treatment (IMZ at 3000 ␮g mL−1 at 1.8 L wax tonne−1 ) gave good protective control and improved sporulation inhibition (≈80%) at residue loading of 1.32–7.09 ␮g g−1 . The MRL of 5 ␮g g−1 was exceeded at higher wax loads on navels and clementines. Double application with dip (45 s in IMZ sulphate at 500 ␮g mL−1 ) followed by 2000 ␮g mL−1 IMZ in wax coating at 0.6, 1.2 and 1.8 L wax tonne−1 resulted in residue loading of 1.42 to 2.83 ␮g g−1 , increased protective control (≈69%) as well as curative control (≈83%). In all treatments, poor curative and protective control of the resistant isolate was observed (<46% and <55%, respectively). Double application demonstrated superior green mould control by giving good curative and protective control and sporulation inhibition. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Green mould is a well-known citrus postharvest disease worldwide and it often results in considerable loss of citrus fruit each year (Kaplan and Dave, 1979). The causal agent of green mould is Penicillium digitatum Sacc. (Kavanagh and Wood, 1967). The pathogen penetrates the fruit through wounds that are formed during harvesting, improper handling and by insects (Smilanick et al., 2005). The availability of nutrients and moisture in the wounds favour the germination of P. digitatum spores (Kavanagh and Wood, 1971; Pelser and Eckert, 1977). In addition, some physiologically induced injuries such as chilling injuries and stem-end rind breakdown can also provide entry for the pathogen into the fruit (Brown, 2003). Therefore, disease management strategies employed to control the occurrence of green mould encompass sanitation and application of fungicides (Smilanick et al., 2006). One of the most effective fungicides registered for citrus green mould control is imazalil (IMZ), which has been used for over two

∗ Corresponding author at: Citrus Research International, Nelspruit, South Africa. Tel.: +27 832902048; fax: +27 865717273. E-mail address: [email protected] (P.H. Fourie). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.11.009

decades (Brown et al., 1983; Zhang, 2007). Imazalil is a systemic fungicide inhibiting the demethylation of ergosterol biosynthesis with an ability to inhibit sporulation of P. digitatum (Siegel et al., 1977; Eckert and Brown, 1986). Imazalil can be applied in a number of ways: by means of aqueous dip tank applications, as a fungicide spray and with incorporation in wax (Kaplan and Dave, 1979). The maximum residue limits (MRLs) for IMZ in most European countries, South Africa and Japan are regulated to be 5 ␮g g for citrus on whole fruit (Brown et al., 1983; Brown and Dezman, 1990). According to the toxicological tests conducted on IMZ to illustrate safety to humans and the environment, the data clearly showed that IMZ should be regarded as a safe product to utilise (Kaplan and Dave, 1979). In South Africa, IMZ is available in two forms (Erasmus et al., 2011). The first is IMZ sulphate, which consists of a soluble granule formulation that is highly soluble in water. It is mainly used in dip tanks in an aqueous solution at a recommended concentration of 500 ␮g mL−1 . Recent studies showed that dip application of IMZ sulphate was very effective in IMZ residue loading and control of green mould caused by the sensitive isolate of P. digitatum; however, there was poor sporulation inhibition (Erasmus et al., 2011). The other form is IMZ emulsifiable concentrate (EC), which is an oily emulsion of IMZ and is mainly applied incorporated

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in postharvest wax coatings at a recommended concentration of 3000 ␮g mL−1 . Wax coating is applied to citrus fruit over rotating brushes in the packline to give it a shiny attractive appearance that will last through the whole marketing process, to reduce postharvest weight loss, to preserve fruit quality and to also provide a carrier for fungicides (Hall, 1981; Porat et al., 2005). Previous studies showed that IMZ applied through wax coating was less effective against green mould compared to when applied as an aqueous solution; the poorer efficacy was ascribed to the viscosity of the coating (Brown, 1984; Smilanick et al., 1997). The effectiveness of IMZ in coating becomes reduced due to encapsulation or binding of the fungicide by coating hence it is required to double the concentration of IMZ when applied through coating to boost the availability of residues at the site of infection (Brown, 1984; Smilanick et al., 1997). It has been reported that commercial packhouses in Florida apply IMZ in dip tanks and also in coating at a concentration of 1000 to 2000 ␮g mL−1 resulting in 4 ␮g g−1 residues on fruit, which is still within the tolerance level of 10 ␮g g−1 in the United States and 5 ␮g g−1 of other countries, including South Africa (Dezman et al., 1986). Applying IMZ in coating had an additional effect of inhibiting sporulation of P. digitatum. Eckert and Brown (1986) stated that the anti-sporulant activity is vital as it prevents soilage, which occurs when decayed fruit rubs off spores to healthy fruit in the same box, which then becomes unappealing to the buyer. Also, reducing sporulation helps to reduce the inoculum in citrus packhouses, and is beneficial to fungicide resistance management strategies as sporulating IMZ treated fruit often contains resistant isolates (Eckert et al., 1994). A survey conducted by Erasmus et al. (2011) showed that the application of IMZ in South African citrus packhouses differed distinctly. About 65% of packhouses surveyed applied IMZ with the wax coating and the majority of these packhouses had less than 15 s exposure time under the coating applicator; the mean IMZ residue level was 2.31 ␮g g−1 (Erasmus, unpublished results). It was also found that 38% of the packhouses applied IMZ as a double application, i.e. first in the fungicide dip tank and then in the wax. Application of IMZ through dip tanks has been thoroughly studied by Erasmus et al. (2011), specifically with regard to residue loading and curative and protective control. However, the application of IMZ with wax coating has not been fully documented and as a result there is limited knowledge on residue loading and concomitant bio-efficacy. The objective of this study was to evaluate the protective and curative control of sensitive and resistant isolates of P. digitatum as well as sporulation inhibition following IMZ application in wax coating, focusing on single and double application on different citrus fruit kinds. 2. Materials and methods 2.1. Fruit Untreated Valencia and navel oranges (Citrus sinensis (L.) Osbeck), satsuma (Citrus unshiu Marc) and clementine (Citrus reticulata Blanco) mandarins of export quality were collected from Citrusdal and Franchoek areas in the Western Cape province of South Africa. The fruit were washed in a 1 mL L−1 didecyl dimethyl ammonium chloride solution (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 (STE-U 6560) and IMZ resistant (STE-U 6590) isolates of P. digitatum on potato dextrose

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agar (PDA) were used to prepare spore suspensions. Conidia was gently dislodged from cultures in 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 volume of 200 mL spore suspension. The spore concentration was adjusted to 1 × 106 spores mL−1 using a haemocytometer. Twelve fruit of similar size were wound inoculated through the flavedo into the top albedo layer using a wound inducer at four sites surrounding the stem end. Wound inducers consisted of three insect needles placed in a needle clamp to create three small wounds of 0.5 mm wide and 2 mm deep at a triangular distance of 1.5 mm apart. The inoculations were done curatively and protectively, i.e. prior to treatment and after treatment, respectively. For curative treatments, the fruit were incubated for 24 h before treatment. 2.3. Packline A custom-built experimental packline (Dormas, Johannesburg, South Africa) similar to a packline at commercial packhouses was used. It consisted of four modular units: an elevator feeding fruit into the line, a re-cycling spray-on washing system over 8 brushes, a coating applicator with 7 rotating synthetic brushes and coating applicator (John Bean Technology Foodtech, Brackenfell, South Africa); The coating applicator was calibrated using one pulsating nozzle (0.5 s on, 2 s off) at 22 mL min−1 (3 bar), and a drying tunnel that uses high volume air at low speeds (ambient temperature) to dry the fruit. The whole packline is speed-controlled and the wash and coating units have brush-sweep paddles to move fruit across the unit at the set speed. 2.4. Single IMZ application in coating The fruit was treated with a medium solids (18%) carnaubashellac based coating (875 High Shine, John Bean Technologies, Brackenfell, South Africa) incorporated with IMZ EC (Imazacure, 750 g kg−1 EC, ICA International Chemicals, Stellenbosch, South Africa) at a concentration of 3000 ␮g mL−1 ; the registered concentration of IMZ applied through coating in South African citrus industry. Imazalil and the wax coating were kept agitated on a magnetic stirrer. The fruit exposure time on the coating applicator was 10 s, 20 s and 30 s, which affected a coating load of 0.6, 1.2 and 1.8 L tonne−1 of fruit, respectively. Fruit treated with wax coating not amended with imazalil (wax coating only), and untreated fruit (without coating or imazalil) served as controls. 2.4.1. Storage and evaluation Twelve treated and inoculated fruit from each treatment combination were placed in 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 were evaluated using a light source (UV-A at 365 nm, Labino Mid-light; www.labino.com). Under the light, the infected wound was visible in a form of yellow fluorescence on the surface of the fruit that was not visible with the naked eye at the period of evaluation. The other batch simulated the export conditions of cold sterilisation for false codling moth (Myburgh, 1965 in Boardman et al., 2011) and was stored at −0.5 ◦ C for 30 days and 7 days at ambient temperature before evaluation. Sporulation was rated 10–12 days after treatment. A sporulation index of 0–6 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

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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. 2.4.2. Residue analysis IMZ residues following the various treatments were determined from fruit sampled from 2 of the 3 replicates. From each treatment combination, 6 fruit were sampled and were frozen (20 ◦ C) until prepared for IMZ residue analysis. Fruit were defrosted, measured and weighed and macerated to a fine pulp by using a fruit blender (Salton Elite, Amalgamated Appliance Holdings Limited, Reuven, South Africa) and 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). Samples were extracted by using acetonitrile followed by a matrix solid phase dispersion extraction. Analysis of the extracts was conducted in liquid chromatography mass spectrometry (LCMS/MS; Agilent 6410, Agilent Technologies Inc., Santa Clara, CA, USA). 2.5. Double IMZ application in dip and wax coating Inoculation, treatment and evaluation procedures were similar to those described above; however, a number of different treatments were included using different fruit kinds, including Valencia and navel oranges, clementine and satsuma soft citrus. For dip only treatment, fruit were dipped for 45 s and 90 s in an aqueous solution of IMZ sulphate (Imazacure, 750 g kg−1 SG, ICA International Chemicals, Stellenbosch, South Africa) at a concentration of 500 ␮g mL−1 as is registered for commercial use. Following dip treatment, fruit were moved over IMZ saturated rotating brushes and donuts (simulating commercial packline treatment following the dip tank fungicide application) to the drying tunnel. For double application treatments, fruit were dipped for 45 s in 500 ␮g mL−1 IMZ sulphate, moved over IMZ saturated rotating brushes and donuts to the drying tunnel, and then treated with IMZ (EC) at a concentration of 2000 ␮g mL−1 in the coating. For the latter, fruit were exposed for 10 s, 20 s and 30 s on the coating applicator, which realised a coating load of 0.6, 1.2 and 1.8 L tonne−1 of fruit, respectively. For wax coating only treatment, the fruit was exposed to IMZ (EC) in wax coating at a concentration of 3000 ␮g mL−1 for 30 s (1.8 L tonne−1 fruit) on the coating applicator. Untreated fruit served as controls. The fruit were packed into boxes and evaluated after 4 days for wound infection and after 10–12 days for sporulation, as described previously. 2.6. Experimental layout and statistical analysis The single application trial was a 2 (curative and protective treatment) × 2 (sensitive and resistant isolate) × 3 (IMZ and the coating loads) factorial design with 12 fruit per treatment combination and 3 replications; the trial was conducted four times on Valencia oranges. The double application trial was a 2 (curative and protective treatment) × 2 (sensitive and resistant isolate) × 6 (IMZ treatments) factorial design with 12 fruit per treatment combination and 3 replications. The trial was conducted twice on Valencia oranges during the 2010 season and on clementine, satsuma mandarins and navel oranges during the 2011 season. Fruit residue, wound infection, green mould control and sporulation incidence data were analysed using appropriate analysis of variance and

Fisher’s test was used to observe the significance differences among the treatments at a 95% confidence interval. 3. Results 3.1. Single IMZ application in wax coating Analysis of variance of fruit residue data showed no significant interaction between fruit batch and treatment (P = 0.633; Anova not shown), but there was a significant effect for treatment (P < 0.0001). There was no significant difference in IMZ fruit residue following IMZ and the coating loads at 0.6 L tonne−1 (0.85 ␮g g−1 ; results not shown) and 1.2 L tonne−1 (0.82 ␮g g−1 ). IMZ application in a coating load of 1.8 L tonne−1 resulted in significantly higher IMZ residues on the fruit (1.75 ␮g g−1 ). 3.1.1. Fruit stored at 20 ◦ C for 4 days Analysis of variance of wound infection data (excluding data for cold-stored incubation treatment) showed a significant 4factor interaction (P < 0.0001; Anova not shown) between the four Valencia oranges fruit batches, curative and protective actions, sensitive and resistant isolate, and the treatment applied. This was largely ascribed to variable effects between fruit batches, but general trends across treatments were similar. Data could not be normalised (to percentage control relative to an untreated control) as a separate control treatment for the cold-stored fruit was not included in the experimental layout. The significant action × isolate × treatment interaction (P < 0.011) for wound infection data will be described further. The treated and untreated control resulted in 58–68% infected wounds in the curative treatments, regardless of isolate (Fig. 1). There was no significant difference between the two types of control treatments. There was poor curative control of the resistant isolate (63–59% wound infection), but an increasing decline of infections by the sensitive isolate with increasing coating load from 0.6 to 1.8 L tonne−1 (48–28%). For protective treatments, untreated (no coating) and treated controls (coating only) of the resistant isolate resulted in ≈60% of infected wounds. Treated control of the sensitive isolate for protective treatments showed significantly less infected wounds (49%) than the untreated controls (59%). Infection by the sensitive isolate significantly decreased from 14% for lower coating load (0.6 L tonne−1 ) to 3% to the higher coating load (1.8 L tonne−1 ), while the resistant isolate was reduced to 38% wound infection only at the highest coating load. 3.1.2. Cold stored fruit (−0.5 ◦ C for 30 days and 7 days at 20 ◦ C) and fruit stored at ambient (20 ◦ C) for 4 days Similar to the wound infection data, a significant fruit batch interaction (P < 0.0001; Anova not shown) was observed for green mould and sporulation incidence data. The interaction was largely ascribed to significant but non-informative differences in treatment effects between fruit batches. Hence the significant action × isolate × treatment interactions (P < 0.0001 and 0.018, respectively) will be described further. For curative treatments, regardless of isolate, both treated and untreated controls resulted in more than 95% infected fruit (Table 3) with no significant differences. Imazalil and coating treatment showed poor curative control of both sensitive and resistant isolates resulting in an infection incidence that exceeded 70% for the fruit that was incubated at 20 ◦ C (Table 1). However, the fruit stored at −0.5 ◦ C for 30 days and 7 days at 20 ◦ C exhibited significantly lower infection levels (52%) by the sensitive isolate for the 1.8 L tonne−1 treatments, but with no improved control of the resistant isolate where none of the treatments differed from the control. The controls for the protective treatments also showed very high levels of green mould incidence (>89%), with no significant difference between the treated

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Fig. 1. The percentage wound infection of green mould on Valencia oranges caused by sensitive (white bars) and resistant (black bars) isolates of P. digitatum after 4 days’ incubation at 20 ◦ C, following curative and protective imazalil treatment in wax coating at 3000 ␮g mL−1 at a rate of 0.6, 1.2 and 1.8 L tonne−1 fruit treated with clean wax coating (treated control) and untreated controls (control). Means for each fruit batch followed by the same letter do no differ significantly (P = 0.05).

and untreated control regardless of isolate. The infection by the sensitive isolate was increasingly reduced from 50% to 13% with increasing the coating load to 1.8 L tonne−1 . At the highest coating load, green mould incidence by the resistant isolate was significantly lower than the controls (74%). The infection in cold-stored fruit was reduced to 4% for the sensitive isolate and to 50% for the resistant isolate. For sporulation incidence, both coated and uncoated controls resulted in 80% sporulating fruit for both isolates, except for the uncoated control (protective treatment) for the resistant isolate (68%). Imazalil in wax coating in curative treatments resulted in a significant reduction in sporulation incidence of the sensitive isolate, progressively with increased coating load (45–10%); cold storage did not appear to influence sporulation incidence (44–5%). Similar trends were observed for the protective treatments against the sensitive isolate with higher coating load treatments reducing the sporulation to 0% in both ambient and cold stored fruit. Despite some significant reductions in sporulation incidence of the resistant

isolate, it could not be reduced below 50% in any of the treatments (Table 1). 3.2. Double IMZ application in dip and wax coating. There was a significant interaction (P < 0.0001; Anova not shown) between the fruit type and the treatments applied. Citrus types exhibited varying levels of IMZ residues loaded. Clementine, satsuma and navel generally showed a similar trend in terms of residue loading, while Valencia oranges loaded distinctly lower residue levels at higher coating loads. This difference was, however, not evident for dip only treatments (Table 2), where residue levels varied from 0.12 to 0.73 ␮g g−1 , with no significant difference between 45 s and 90 s or fruit batches. Improved residue loading was evident from double application yielding up to 2.83 ␮g g−1 residues on fruit. Although not significant, increasing coating load increased the residues for the dip and coating application, with 0.6 L tonne−1 loading 1.42–2.31 ␮g g−1 and 1.8 L tonne−1 loaded

Table 1 Mean percentage fruit infection (green mould incidence) and sporulation incidence on Valencia oranges that were wound inoculated with a IMZ sensitive and a resistant P. digitatum isolate and curatively or protectively treated with imazalil (IMZ) at a concentration of 3000 ␮g mL−1 in the coating and incubated for 4 days at ambient (20 ◦ C) and 30 days at −0.5 ◦ C and 7 days at 20 ◦ C. Treatment

Curative Control (no coating) Control (coating) Coating 0.6 L tonne−1 Coating 1.2 L tonne−1 Coating 1. 8 L tonne−1 Coating 0.6 L tonne−1 + cold Coating1.2 L tonne−1 + cold Coating 1.8 L tonne−1 + cold Protective Control (no coating) Control (coating) Coating 0.6 L tonne−1 Coating 1.2 L tonne−1 Coating 1.8 L tonne−1 Coating 0.6 L tonne−1 + cold Coating 1.2 L tonne−1 + cold Coating 1.8 L tonne−1 + cold x y z

Green mould incidence (%)x

Sporulating fruit (%)x

Sensitivey

Resistantz

Sensitive

Resistant

95.6a 96.5a 84.6c 84.7c 70.1de 76.1d 62.2ef 51.8gh

95.5a 97.9a 91.7abc 95.8a 92.3abc 91.7abc 87.5bc 90.1abc

90.1bc 96.4ab 44.3hi 45.6hi 29.6j 38.8ij 9.5k 4.8k

87.5cd 97.9a 89.7bcd 79.1e 69.1fg 56.6h 89.1bcd 74.8ef

96.9a 90.1abc 50.0h 28.2i 13.2j 15.1j 25.0i 4.2k

89.2bc 93.8ab 84.7c 86.1bc 74.3d 64.6ef 59.4fg 50.0h

95.4ab 81.1de 38.1ij 50.8hi 40.3hij 37.6ij 0k 0k

66.1g 81.7de 75.3ef 58.1gh 65.8g 56.9gh 80.2de 49.1hi

Means followed by the same letter do no differ significantly (P = 0.05). Fruit were inoculated with a sensitive isolate of P. digitatum. Fruit were inoculated with a resistant isolate of P. digitatum.

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Table 2 Imazalil (IMZ) residues loaded on different citrus fruit batches (clementine, satsuma, navel and Valencia) that were dip-treated with IMZ sulphate at 500 ␮g mL−1 for 45 s and 90 s exposure time, treated with IMZ sulphate at 500 ␮g mL−1 for 45 s in the dip followed by IMZ (EC) and coating at 2000 ␮g ml−1 at rates of 0.6 L tonne−1 , 1.2 L tonne−1 and 1.8 L tonne−1 , and treated with only a IMZ (EC) and coating at 3000 ␮g mL−1 at a rate of 1.8 L tonne−1 . Treatment

Dip (45 s) Dip (90 s) Dip (45 s) + coating 0.6 L tonne−1 Dip (45s) + coating 1.2 L tonne−1 Dip (45s) + coating 1.8 L tonne−1 Coating 1.8 L tonne−1 x y

IMZ Concentration (␮g mL−1 )

500 500 500 + 2000 500 + 2000 500 + 2000 3000

Imazalil residues (␮g g−1 )x Clementine

Satsuma

Navel

Valencia 1y

Valencia 2y

0.22hi 0.25hi 2.31cde 2.30cde 2.83c 6.04a

0.16i 0.12i 1.42defg 2.01cdef 2.26cde 4.14b

0.28hi 0.29hi 1.46defg 1.41defg 2.40 cd 7.09a

0.14i 0.73ghi 0.48ghi 0.64ghi 1.29efgh 1.32defgh

0.23hi 0.18i 0.38ghi 0.70ghi 1.08fghi 1.84cdef

Means followed by the same letter do not differ significantly (P = 0.05). Two batches of Valencia oranges (Valencia 1 and 2).

2.26–2.83 ␮g g−1 for clementine, satsuma and navel. On Valencia oranges, lower residue levels were recorded 0.38–0.48 ␮g g−1 at 0.6 L tonne−1 and 1.08–1.29 ␮g g−1 at 1.8 L tonne−1 . Single application of IMZ in coating at the higher concentration of 3000 ␮g mL−1 resulted in significantly higher residues loaded on clementine (6.04 ␮g g−1 ), satsuma (4.14 ␮g g−1 ) and navel (7.09 ␮g g−1 ) fruit, but not significantly higher for Valencia fruit (1.32–1.84 ␮g g−1 ). Since mean infection levels on control treatments differed between fruit types, data were normalised by determining percentage control in treatments relative to the untreated control. Analysis of variance resulted in a significant 4-factor fruit type × action × isolate × treatment interaction (P < 0.0001; Anova not shown). Data for each fruit type was therefore analysed separately. For each fruit type, there was a 3-factor action × isolate × treatment interaction (P < 0.0001; Anova not shown) for percentage control data, as well as for sporulation incidence (P < 0.0001). For sporulation data, this interaction was ascribed to the effect of treatment action together with difference in level of sporulation in sensitive and resistant isolates. There was a significant interaction between isolate and treatment (P < 0.05) and a significant effect for action (P < 0.05). For the latter, curative treatments generally resulted in better sporulation inhibition than protective treatments (results not shown). Except for the clementine which resulted in 79% sporulation in the curative and 82% in the protective treatments, sporulation incidence was higher in protective treatments than in curative treatments, varying from 48% to 73% in the protective and 42% to 62% in the curative treatments (results not shown). For navel oranges, dip only treatments (45 s and 90 s) curatively controlled the sensitive isolate up to 82% and 93%, respectively (Fig. 2A), but poorly controlled the resistant isolate (40% and 38%, respectively). Dip and coating treatments resulted in marginally better curative control of the sensitive isolate than 45 s dip only treatments, and the level of control improved with coating load from 86% (0.6 L tonne−1 ) to 95% (1.8 L tonne−1 ). The resistant isolate was poorly controlled (30%), even at the highest coating load of 1.8 L tonne−1 . Wax coating only treatment resulted in poor curative control of both sensitive (47%) and resistant (27%) isolates. For protective treatments, dip only treatments resulted in poorer control of the sensitive isolate (38% and 43% for 45 and 90 s, respectively) than curative treatments, while the resistant isolate was poorly controlled (<18%). Dip and coating significantly improved protective control of the sensitive isolate and treatments from 86% (0.6 L tonne−1 ) to 95% (1.8 L tonne−1 ). Wax coating only treatments yielded similar protective control (93%) of the sensitive isolate, while the resistant isolate was also marginally controlled (53%). Dip treatments (45 and 90 s) resulted in significantly less sporulation of the sensitive isolate (68% to 54%, respectively; Table 3) than the untreated control (100%). Following double application, increasing coating load resulted in decreasing levels of sporulation from 38% (0.6 L tonne−1 ) to 7.3% (1.8 L tonne−1 ). Coating only also resulted

in reduced level of sporulation (21%). For the resistant isolate, all treatments resulted in poor or lack of sporulation inhibition (>80%). Lower levels of curative and protective control of the sensitive isolate were observed on clementine fruit (Fig. 2B) than was observed for navel fruit, while the resistant isolate was poorly controlled (<20%). Trends for the different treatments were similar. For sporulation incidence, significant inhibition was observed for the sensitive isolate only and only for treatments that included application of IMZ in wax coating (Table 3). On satsuma fruit (Fig. 2C), curative control of the sensitive isolate by the various treatments was similar to levels observed for clementine fruit, but higher levels of control were observed for the resistant isolate with treatments that included an IMZ dip application (20–40%). Protective control of the sensitive isolate also followed similar trends as for clementine, albeit at lower levels; 52–61% for treatments that included IMZ in coating application and significantly lower for the dip only applications (40–42%). Protective control of the resistant isolate varied from 22% to 35%, with no significant differences between the various treatments. Similarly to the other fruit types, sporulation incidence levels of the sensitive isolate were significantly lower than the control treatments only for treatments that included IMZ application in wax coating, while sporulation of the resistant isolate was not inhibited (Table 3). For control and sporulation data obtained from Valencia oranges, a significant fruit batch interaction was observed, but trends were nonetheless similar. Hence the significant 3-factor action × isolate × treatment interaction (P < 0.0001; Anova not shown) was described further to simplify results. Curative control of the sensitive isolate was significantly higher in 45 s and 90 s dip treatments (82% and 93%, respectively; Fig. 3), with an improved level of control of the resistant isolate at longer exposure time (28–40%, respectively). Similar trends to the other fruit types with double application and wax coating only treatments were observed with good curative control of the sensitive isolates (92–94%) and poorer control of the resistant isolate (34–38%). Protective control of the sensitive isolate improved significantly following double application of IMZ in dip and wax coating treatments, varying from 66% to 76%. Wax coating only treatments resulted in significantly superior protective control (90%), while dip only treatments yielded moderate protection against the sensitive isolate (49%). The resistant isolate was poorly controlled in all treatments (<40%). Dip treatments resulted in significantly lower sporulation levels for the sensitive (42% and 31% for 45 and 90 s, respectively (Table 3) relative to the control (75%), but for the resistant isolate reduced sporulation relative to the control (77%) was only observed following the 90 s dip treatment (34%). Dip and wax coating resulted in successful reduction of sporulation incidence with increasing coating loads for sensitive isolate (19% to 6%), but not for the resistant isolate (>56%). Likewise, wax coating only treatment reduced sporulation of the sensitive isolate (25%), but not of the resistant isolate (74%).

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Fig. 2. The percentage curative and protective control of green mould on navel (A), clementine (B) and satsuma (C) fruit caused by sensitive (white bars) and resistant (black bars) isolates of P. digitatum, following 45 s and 90 s dip treatments in imazalil (IMZ) sulphate at 500 ␮g mL−1 (Dip 45 s and Dip 90 s), double IMZ application of 45 s dip in IMZ sulphate at 500 ␮g mL−1 followed by IMZ (EC) and wax coating at 2000 ␮g mL−1 at a rate of 0.6 L tonne−1 (DW 0.6), 1.2 L tonne−1 (DW 1.2) and 1.8 L tonne−1 (DW 1.8), wax coating only treatment of IMZ (EC) and coating at 3000 ␮g mL−1 at a rate of 1.8 L tonne−1 (W 1.8), and untreated controls (control). Means for each fruit batch followed by the same letter do no differ significantly (P = 0.05). Table 3 Mean percentage of sporulation incidence after 10 days on citrus fruit that were wound inoculated with P. digitatum imazalil (IMZ) sensitive and resistant isolates and curatively or protectively treated with IMZ sulphate dip application at 500 ␮g mL−1 for 45 s and 90 s, IMZ sulphate at 500 ␮g mL−1 for 45 s in the dip and exposed in IMZ emulsifiable concentrate (EC) and coating at 2000 ␮g mL−1 , IMZ (EC) at a concentration of 3000 ␮g mL−1 and untreated controls (control). Sporulation incidencex Treatment Sensitivey Dip (45 s) Dip (90 s) Dip (45 s) + Coating (0.6 L tonne−1 ) Dip (45 s) + Coating (1.2 L tonne−1 ) Dip (45 s) + Coating (1.8 L tonne−1 ) Coating (1.8 L tonne−1 ) Control Resistantz Dip (45 s) Dip (90 s) Dip (45 s) + Coating (0.6 L tonne−1 ) Dip (45 s) + Coating (1.2 L tonne−1 ) Dip (45 s) + Coating (1.8 L tonne−1 ) Coating (1.8 L tonne−1 ) Control x y z

Clementine

Satsuma

Navel

Valencia

95.8a 97.2a 41.7b 45.8b 42.5b 19.4c 100.0a

91.7a 91.7a 30.7c 12.9a 30.2c 20.0d 100.0a

68.1e 54.3f 37.9 g 20.8 h 7.3i 20.8 h 99.5a

41.7c 30.7de 18.8f 17.4f 6.3 g 25.0ef 75.3a

98.6a 100.0a 95.8a 98.6a 98.6a 100.0a 99.1a

100.0a 97.2ab 98.6ab 98.6ab 96.9ab 100.0a 100.0a

95.8abc 88.7bcd 91.7abcd 87.5 cd 88.9bcd 86.1d 96.3ab

76.4a 34.0 cd 55.6b 79.2a 76.4a 73.6a 77.4a

Means for each fruit batch followed by the same letter do no differ significantly (P = 0.05). Fruit were inoculated with a sensitive isolate of P. digitatum. Fruit were inoculated with a resistant isolate of P. digitatum.

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Fig. 3. The percentage curative and protective control of green mould on two batches of Valencia, caused by sensitive (white bars) and resistant (black bars) isolates of P. digitatum, following 45 s and 90 s dip treatments in imazalil (IMZ) sulphate at 500 ␮g mL−1 (Dip 45 s and Dip 90 s), IMZ sulphate at 500 ␮g mL−1 45 s in the dip and exposed in IMZ (EC) and wax coating at 2000 ␮g mL−1 at a rate of 0.6 L tonne−1 (DW 0.6), 1.2 L tonne−1 (DW 1.2) and 1.8 L tonne−1 (DW 1.8), IMZ (EC) and wax coating at 3000 ␮g mL−1 at a rate of 1.8 L tonne−1 (W 1.8), and untreated controls (control). Means for each fruit batch followed by the same letter do no differ significantly (P = 0.05).

4. Discussion Results from this study clearly demonstrated the benefits of IMZ application in wax coating, specifically with regards to improved protective control (Waks et al., 1985) and sporulation inhibition (Smilanick et al., 1997) compared with aqueous applications, which was found to be superior in terms of curative control (Brown et al., 1983; Erasmus et al., 2011). Moreover, the study provides better insight of IMZ residue loading through coating application, specifically demonstrating residue levels required for effective control and sporulation inhibition and indications of coating loads that could lead to the exceedance of the 5 ␮g g−1 MRL (maximum residue level). The low IMZ residue levels (0.8 ␮g g−1 ) obtained with single application of IMZ at 3000 ␮g mL−1 in coating at 0.6 L tonne−1 and 1.2 L tonne−1 were higher than those obtained by Kaplan and Dave (1979) with 2000 ␮g mL−1 on navel oranges and grapefruit (0.32 and 0.18 ␮g g−1 , respectively). At 1.8 L tonne−1 , residue loading improved (1.75 ␮g g−1 ), but still remained less than 2–3.5 ␮g g−1 , the level that is regarded as the ideal in terms of sustained green mould control and sporulation inhibition (Kaplan and Dave, 1979; Brown and Dezman, 1990; Smilanick et al., 1997). Adequate residues on the fruit are required for the disease control and hinder the development of resistance. Nonetheless, satisfactory protective control and sporulation inhibition was obtained by residue levels obtained following a coating load of 1.2 L tonne−1 and 1.8 L tonne−1 . These results strongly concur with what has been reported previously (Waks et al., 1985; DuPlooy et al., 2009), that IMZ in wax coating is better protective than curative when controlling the sensitive isolate of P. digitatum. Because wax acts as a barrier on the surface of the fruit against fungal pathogens, it is better for protection than to heal the already established infection on the albedo layer of the peel (Brown, 1984). Improved sporulation inhibition would also reduce P. digitatum population build-up in packhouses and therewith the risk of fungicide resistance development. For curative treatments, the sensitive isolate was poorly controlled and no control was observed for the resistant isolate. It was also observed that coating alone (coated controls) could not limit decay since the infection was as severe as with the uncoated controls. This was also the case when the effect of essential oils and carnauba coating was investigated for green mould control (DuPlooy et al., 2009). In cases where control was inadequate in the IMZ + wax coating treatments, such as the curative treatments, white mycelia grew abundantly on the surface of the fruit without

turning into green olive spores showing 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 that takes place when healthy fruit in the cartons are affected by spores from the nearby infected fruit (Eckert and Brown, 1986). Previous studies showed that wax coatings particularly shellac and wood rosin, have a negative impact on the internal quality of the fruit whereby there is development of off-flavours which subsequently form because of less permeance for gaseous exchange (oxygen and carbon dioxide) as the natural openings get clogged by a wax coating and lead to disruption of movement of these gases (Mannheim and Soffer, 1996). The coating used in this study is a medium solid carnauba-shellac based coating, which was suitable for late season citrus. Quality-related aspects were not evaluated, but there were no obvious signs of rind disorders or quality defects on the fruit stored in cold temperatures (−0.5 ◦ C). Benschoter (1984) showed that Valencia oranges were more tolerant than grapefruit to lower temperatures (1.7 ◦ C) when stored for a period of 19 days to control larvae of the Caribbean fruit fly. In transit to the export market, Valencia oranges can withstand lower temperatures of 0.5 ◦ C without developing chilling injury (CI). The low temperatures and the duration of the storage used in this study was an aggravated condition given that commonly used transit temperature is 3.5 ◦ C for 24 days during transportation. P. digitatum grows optimally at ambient temperatures ranging from 20 ◦ C to 25 ◦ C and temperatures below that or above deteriorated the growth and development of the pathogen (Kassim and Khan, 1996; Plaza et al., 2003). In comparison with green mould control at ambient temperatures, we observed a clear additive effect of cold storage (−0.5 ◦ C) on curative and protective green mould control by IMZ. Infection levels of the sensitive isolate were greatly reduced and control of the resistant isolate was improved, especially at the higher IMZ and coating loads. Following 45 s and 90 s dip-treatments in 500 ␮g mL−1 IMZ sulphate solutions (pH 3), similar residue loading trends was observed for clementine, satsuma, navel and Valencia oranges with all the citrus types loading ≈0.3 ␮g g−1 . For similar treatments, Erasmus et al. (2011) reported markedly higher residues on navel and Valencia oranges (0.97 ␮g g−1 ). Lower residue levels following dip application in our study can be attributed to the post-dip brushing of fruit followed by drying. These treatments removed excess fungicide solution from the fruit, thereby limiting IMZ residue loading

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to the exposure time in the dip tank and on the wet brushes only. Nonetheless, the aqueous IMZ dip treatment successfully controlled green mould caused by the sensitive isolate of P. digitatum in all fruit kinds, particularly at longer exposure time (90 s), in curative treatments. Erasmus et al. (2011) reported similar findings, but found superior curative treatments of 6-hour-old infections compared with the 24 h-old infections in the present study. In a commercial packhouse, the harvested fruit can easily stand for 2–4 days before fungicide treatment. A 24 h incubation period therefore better simulates such situations. The curative action of IMZ on soft citrus was shown to be most effective in infections that were less than 24 h old; control and sporulation inhibition were progressively diminished in more established infection (N. Njombolwana, unpublished data). Sporulation could not be inhibited on clementine and satsuma fruit following dip treatments, while some sporulation inhibition was evident on navel and Valencia oranges, but for the sensitive isolate only. Lack of sporulation inhibition was attributed to residues that were significantly lower than the ideal level recommended to adequately control green mould and inhibit sporulation (Kaplan and Dave, 1979; Brown and Dezman, 1990; Smilanick et al., 1997). Brown and Dezman (1990) reported that control of sporulation following aqueous application depended solely on the deposition of IMZ on the natural wax of Valencia oranges. Post-dip brushing of fruit, which is a common practice in commercial packlines, most probably contributed to reduced IMZ residue loading and possibly to reduced sporulation inhibition. Imazalil residues loading following double application of IMZ in dip (500 ␮g mL−1 for 45 s) and wax coating (2000 ␮g mL−1 ) improved quite significantly for all citrus types compared to the dip only application. Depending on citrus type and coating load used, residues ranged from 0.4 to 1.8 ␮g g−1 on Valencia oranges and from 1.4 to 2.8 ␮g g−1 on clementine, satsuma and navel fruit. Double application resulted in both curative and protective control of the sensitive isolate, especially at the recommended 1.2 L tonne−1 and higher 1.8 L tonne−1 coating loads. Therefore, double application appears to be an ideal application system by integrating the superior curative control by IMZ following dip application with the superior protective control and sporulation inhibition following IMZ application in coating. This is supported by Smilanick et al. (1997), who also found that a second application of IMZ in coating after an aqueous application will ensure the inhibition of sporulation, although in their work the IMZ EC formulation was used for both applications. The encapsulation or binding of the lipophilic IMZ in the coating affects its movement into the rind and this immobility of IMZ led to ineffectiveness at low concentration in the coating (Brown, 1984). This was also observed for thiabendazole and benomyl, which were less effective when applied in wax coating compared with aqueous application (Cho et al., 1977; Eckert and Kolbezen, 1977; Brown, 1984). In this study, it was evident that the application of coating following dip application played an important role in sporulation inhibition as opposed to dip application only (Erasmus et al., 2011). The ideal residue level for sporulation inhibition was often reached (>2 ␮g g−1 ) with the double application of IMZ in dip and wax coating application. Loss of control of resistant isolate following dip, wax coating or double application was clearly evident in this study. It might be attributed to the severe inoculation methods and high inoculum dosage used, which might represent a worst case scenario in comparison with the situation at a commercial packhouses (Erasmus et al., 2011). However, it is clear that practical level of IMZ resistance in P. digitatum occurs and packhouses should implement antiresistance strategies to limit its development and effects (Holmes, 1995; Holmes and Eckert, 1999; Karaoglanidis et al., 2001; Lado et al., 2011).

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A survey of coating application in commercial packhouses indicated a huge variation in terms of the fruit exposure time on the coating applicator: it ranged from 7 s (probably less than the lower loads used in this study, 0.6 L tonne−1 ) to 41 s (probably more than the higher loads used in this study, 1.8 L tonne−1 ). This irregularity in coating application is cause for concern, as residue data obtained from this study indicated that the MRL of 5 ␮g g−1 was often exceeded at higher coating loads (1.8 L tonne−1 ), particularly in clementine and navel fruit. Brown et al. (1983) stated that higher treatment concentrations of IMZ in coating enhanced the resulting residue loading. Interestingly, this was not observed on Valencia oranges, which showed a markedly reduced propensity to load IMZ residues following dip and coating application. Higher residue loading on navel oranges might be attributed to its rougher rind (Brown et al., 1983) surface texture as well as the open navel end. Similar behaviour substantiating this phenomenon was also noticed with thiabendazole (TBZ), where more residues were obtained from navel oranges than in Valencia oranges (M. Kellerman, unpublished results). These observations warrant further investigation. Most South African packhouses have adopted the practice of double application of imazalil, but care should be taken when applying IMZ in the wax coating, as MRL levels can be exceeded at coating loads higher than the recommended 1.2 L tonne−1 . This study successfully showed the additive benefits of double application of IMZ in dip and wax coating. However, despite improved control of the sensitive isolate, the resistant isolate could not be controlled. This highlights the importance of fungicide resistance management strategies and alternative chemistry or methods of green mould control.

Ackowledgements The study was financially supported by Citrus Research International, Citrus Academy, National Research Foundation and THRIP. The authors thank the personnel at Department of Plant Pathology, Stellenbosch University for technical support; Hearshaw and Kinnes Analytical Laboratory (Pty)Ltd for residue analysis; Dr Wilma du Plooy and John Bean Technologies for supplying wax coatings and technically assisting in coating application; ICA International Chemicals (Pty)Ltd for supplying IMZ chemicals.

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