Evaluation of curative and protective control of Penicillium digitatum following imazalil...

Postharvest Biology and Technology 77 (2013) 102–110

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Postharvest Biology and Technology

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

Ncumisa S. Njombolwanaa,b, Arno Erasmusb, Paul H. Fouriea,b,!

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

a r t i c l e i n f o

Article history:Received 11 September 2012Accepted 24 November 2012

Keywords:Citrus green mouldResidue loadingSporulation 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, andthe 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 levelsincreased (0.85 to 1.75 !g g"1) with increasing coating load. However, the resistant isolate could notbe controlled (>74% infection). Dip only treatment (IMZ sulphate at 500 !g mL"1 for 45 s and 90 s) gavegood curative control (#77%) of the sensitive isolate at residue loading of 0.12–0.73 !g g"1. Wax coatingonly treatment (IMZ at 3000 !g mL"1 at 1.8 L wax tonne"1) gave good protective control and improvedsporulation inhibition (#80%) at residue loading of 1.32–7.09 !g g"1. The MRL of 5 !g g"1 was exceededat higher wax loads on navels and clementines. Double application with dip (45 s in IMZ sulphate at500 !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 resultedin 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 goodcurative and protective control and sporulation inhibition.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Green mould is a well-known citrus postharvest disease world-wide and it often results in considerable loss of citrus fruit each year(Kaplan and Dave, 1979). The causal agent of green mould is Peni-cillium digitatum Sacc. (Kavanagh and Wood, 1967). The pathogenpenetrates the fruit through wounds that are formed during har-vesting, improper handling and by insects (Smilanick et al., 2005).The availability of nutrients and moisture in the wounds favourthe germination of P. digitatum spores (Kavanagh and Wood, 1971;Pelser and Eckert, 1977). In addition, some physiologically inducedinjuries such as chilling injuries and stem-end rind breakdown canalso provide entry for the pathogen into the fruit (Brown, 2003).Therefore, disease management strategies employed to control theoccurrence of green mould encompass sanitation and applicationof fungicides (Smilanick et al., 2006).

One of the most effective fungicides registered for citrus greenmould 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).

decades (Brown et al., 1983; Zhang, 2007). Imazalil is a systemicfungicide inhibiting the demethylation of ergosterol biosynthesiswith an ability to inhibit sporulation of P. digitatum (Siegel et al.,1977; Eckert and Brown, 1986). Imazalil can be applied in a numberof ways: by means of aqueous dip tank applications, as a fungi-cide spray and with incorporation in wax (Kaplan and Dave, 1979).The maximum residue limits (MRLs) for IMZ in most Europeancountries, South Africa and Japan are regulated to be 5 !g g for cit-rus on whole fruit (Brown et al., 1983; Brown and Dezman, 1990).According to the toxicological tests conducted on IMZ to illustratesafety to humans and the environment, the data clearly showedthat IMZ should be regarded as a safe product to utilise (Kaplanand 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 gran-ule formulation that is highly soluble in water. It is mainly usedin dip tanks in an aqueous solution at a recommended concentra-tion of 500 !g mL"1. Recent studies showed that dip application ofIMZ sulphate was very effective in IMZ residue loading and con-trol 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), whichis an oily emulsion of IMZ and is mainly applied incorporated

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N.S. Njombolwana et al. / Postharvest Biology and Technology 77 (2013) 102–110 103

in postharvest wax coatings at a recommended concentration of3000 !g mL!1.

Wax coating is applied to citrus fruit over rotating brushesin the packline to give it a shiny attractive appearance that willlast through the whole marketing process, to reduce postharvestweight loss, to preserve fruit quality and to also provide a car-rier for fungicides (Hall, 1981; Porat et al., 2005). Previous studiesshowed that IMZ applied through wax coating was less effectiveagainst green mould compared to when applied as an aqueous solu-tion; the poorer efficacy was ascribed to the viscosity of the coating(Brown, 1984; Smilanick et al., 1997). The effectiveness of IMZ incoating becomes reduced due to encapsulation or binding of thefungicide by coating hence it is required to double the concentra-tion of IMZ when applied through coating to boost the availabilityof residues at the site of infection (Brown, 1984; Smilanick et al.,1997). It has been reported that commercial packhouses in Floridaapply IMZ in dip tanks and also in coating at a concentration of1000 to 2000 !g mL!1 resulting in 4 !g g!1 residues on fruit, whichis still within the tolerance level of 10 !g g!1 in the United Statesand 5 !g g!1 of other countries, including South Africa (Dezmanet al., 1986). Applying IMZ in coating had an additional effect ofinhibiting 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 fruitin the same box, which then becomes unappealing to the buyer.Also, reducing sporulation helps to reduce the inoculum in citruspackhouses, and is beneficial to fungicide resistance managementstrategies as sporulating IMZ treated fruit often contains resistantisolates (Eckert et al., 1994).

A survey conducted by Erasmus et al. (2011) showed that theapplication of IMZ in South African citrus packhouses differed dis-tinctly. About 65% of packhouses surveyed applied IMZ with thewax coating and the majority of these packhouses had less than15 s exposure time under the coating applicator; the mean IMZresidue level was 2.31 !g g!1 (Erasmus, unpublished results). Itwas also found that 38% of the packhouses applied IMZ as a doubleapplication, i.e. first in the fungicide dip tank and then in the wax.Application of IMZ through dip tanks has been thoroughly studiedby Erasmus et al. (2011), specifically with regard to residue load-ing and curative and protective control. However, the applicationof IMZ with wax coating has not been fully documented and as aresult there is limited knowledge on residue loading and concomi-tant bio-efficacy. The objective of this study was to evaluate theprotective and curative control of sensitive and resistant isolates ofP. digitatum as well as sporulation inhibition following IMZ appli-cation in wax coating, focusing on single and double application ondifferent 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 retic-ulata Blanco) mandarins of export quality were collected fromCitrusdal and Franchoek areas in the Western Cape province ofSouth Africa. The fruit were washed in a 1 mL L!1 didecyl dimethylammonium chloride solution (Sporekill, ICA International Chem-icals, Stellenbosch, South Africa) and allowed to dry overnight atambient temperature prior to commencement of the trials.

2.2. Inoculation

Two-week old cultures of IMZ sensitive (STE-U 6560) and IMZresistant (STE-U 6590) isolates of P. digitatum on potato dextrose

agar (PDA) were used to prepare spore suspensions. Conidia wasgently 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 toobtain a volume of 200 mL spore suspension. The spore concentra-tion was adjusted to 1 " 106 spores mL!1 using a haemocytometer.Twelve fruit of similar size were wound inoculated through theflavedo into the top albedo layer using a wound inducer at foursites surrounding the stem end. Wound inducers consisted of threeinsect needles placed in a needle clamp to create three smallwounds of 0.5 mm wide and 2 mm deep at a triangular distanceof 1.5 mm apart. The inoculations were done curatively and pro-tectively, i.e. prior to treatment and after treatment, respectively.For curative treatments, the fruit were incubated for 24 h beforetreatment.

2.3. Packline

A custom-built experimental packline (Dormas, Johannesburg,South Africa) similar to a packline at commercial packhouses wasused. It consisted of four modular units: an elevator feeding fruitinto the line, a re-cycling spray-on washing system over 8 brushes,a coating applicator with 7 rotating synthetic brushes and coat-ing applicator (John Bean Technology Foodtech, Brackenfell, SouthAfrica); The coating applicator was calibrated using one pulsatingnozzle (0.5 s on, 2 s off) at 22 mL min!1 (3 bar), and a drying tunnelthat uses high volume air at low speeds (ambient temperature) todry the fruit. The whole packline is speed-controlled and the washand coating units have brush-sweep paddles to move fruit acrossthe unit at the set speed.

2.4. Single IMZ application in coating

The fruit was treated with a medium solids (18%) carnauba-shellac 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, SouthAfrica) at a concentration of 3000 !g mL!1; the registered con-centration of IMZ applied through coating in South African citrusindustry. Imazalil and the wax coating were kept agitated on a mag-netic stirrer. The fruit exposure time on the coating applicator was10 s, 20 s and 30 s, which affected a coating load of 0.6, 1.2 and1.8 L tonne!1 of fruit, respectively. Fruit treated with wax coatingnot amended with imazalil (wax coating only), and untreated fruit(without coating or imazalil) served as controls.

2.4.1. Storage and evaluationTwelve treated and inoculated fruit from each treatment com-

bination were placed in lock back table grape boxes (APL cartons,Worcester, South Africa) on count SFT13 nectarine trays (Huhta-maki South Africa (Pty) Ltd., Atlantis, South Africa). Each box wascovered with a transparent polyethylene bag and sealed. Treat-ments were divided into two batches. One batch was incubatedat 20 #C and after 4 days the number of infected wounds wereevaluated using a light source (UV-A at 365 nm, Labino Mid-light;www.labino.com). Under the light, the infected wound was visiblein a form of yellow fluorescence on the surface of the fruit thatwas not visible with the naked eye at the period of evaluation.The other batch simulated the export conditions of cold sterili-sation 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 ambi-ent temperature before evaluation. Sporulation was rated 10–12days after treatment. A sporulation index of 0–6 was used, whichdescribed the percentage of the fruit surface covered with greenspores, where 0 = no sign of disease, 1 = lesion but no sporulation,2 = sporulating area covering a quarter of the lesion, 3 = sporulating

104 N.S. Njombolwana et al. / Postharvest Biology and Technology 77 (2013) 102–110

area larger than a quarter but smaller than the half of the lesion,4 = sporulating area larger than half but smaller than three quar-ters of the lesion, 5 = sporulating area larger than three quartersbut smaller than the whole lesion, and 6 = sporulating area cover-ing the whole fruit (Erasmus et al., 2011). Sporulation incidence (%)was determined from infected fruit with a sporulation index of 3and higher.

2.4.2. Residue analysisIMZ residues following the various treatments were determined

from fruit sampled from 2 of the 3 replicates. From each treat-ment combination, 6 fruit were sampled and were frozen (20 !C)until prepared for IMZ residue analysis. Fruit were defrosted, mea-sured and weighed and macerated to a fine pulp by using a fruitblender (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 labora-tory (Hearshaw and Kinnes Analytical Laboratory, Westlake, CapeTown, South Africa). Samples were extracted by using acetonitrilefollowed by a matrix solid phase dispersion extraction. Analysis ofthe extracts was conducted in liquid chromatography mass spec-trometry (LCMS/MS; Agilent 6410, Agilent Technologies Inc., SantaClara, CA, USA).

2.5. Double IMZ application in dip and wax coating

Inoculation, treatment and evaluation procedures were similarto those described above; however, a number of different treat-ments were included using different fruit kinds, including Valenciaand navel oranges, clementine and satsuma soft citrus. For diponly treatment, fruit were dipped for 45 s and 90 s in an aqueoussolution of IMZ sulphate (Imazacure, 750 g kg"1 SG, ICA Interna-tional Chemicals, Stellenbosch, South Africa) at a concentrationof 500 !g mL"1 as is registered for commercial use. Following diptreatment, fruit were moved over IMZ saturated rotating brushesand donuts (simulating commercial packline treatment followingthe dip tank fungicide application) to the drying tunnel. For doubleapplication treatments, fruit were dipped for 45 s in 500 !g mL"1

IMZ sulphate, moved over IMZ saturated rotating brushes anddonuts to the drying tunnel, and then treated with IMZ (EC) at aconcentration of 2000 !g mL"1 in the coating. For the latter, fruitwere exposed for 10 s, 20 s and 30 s on the coating applicator, whichrealised a coating load of 0.6, 1.2 and 1.8 L tonne"1 of fruit, respec-tively. For wax coating only treatment, the fruit was exposed toIMZ (EC) in wax coating at a concentration of 3000 !g mL"1 for30 s (1.8 L tonne"1 fruit) on the coating applicator. Untreated fruitserved as controls. The fruit were packed into boxes and evalu-ated after 4 days for wound infection and after 10–12 days forsporulation, as described previously.

2.6. Experimental layout and statistical analysis

The single application trial was a 2 (curative and protectivetreatment) # 2 (sensitive and resistant isolate) # 3 (IMZ and thecoating loads) factorial design with 12 fruit per treatment com-bination and 3 replications; the trial was conducted four times onValencia oranges. The double application trial was a 2 (curative andprotective treatment) # 2 (sensitive and resistant isolate) # 6 (IMZtreatments) factorial design with 12 fruit per treatment combina-tion and 3 replications. The trial was conducted twice on Valenciaoranges during the 2010 season and on clementine, satsuma man-darins and navel oranges during the 2011 season. Fruit residue,wound infection, green mould control and sporulation incidencedata were analysed using appropriate analysis of variance and

Fisher’s test was used to observe the significance differences amongthe 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 significantinteraction between fruit batch and treatment (P = 0.633; Anova notshown), but there was a significant effect for treatment (P < 0.0001).There was no significant difference in IMZ fruit residue followingIMZ and the coating loads at 0.6 L tonne"1 (0.85 !g g"1; results notshown) and 1.2 L tonne"1 (0.82 !g g"1). IMZ application in a coatingload of 1.8 L tonne"1 resulted in significantly higher IMZ residueson the fruit (1.75 !g g"1).

3.1.1. Fruit stored at 20 !C for 4 daysAnalysis of variance of wound infection data (excluding data

for cold-stored incubation treatment) showed a significant 4-factor interaction (P < 0.0001; Anova not shown) between the fourValencia oranges fruit batches, curative and protective actions,sensitive and resistant isolate, and the treatment applied. Thiswas largely ascribed to variable effects between fruit batches,but general trends across treatments were similar. Data couldnot be normalised (to percentage control relative to an untreatedcontrol) as a separate control treatment for the cold-stored fruitwas not included in the experimental layout. The significantaction # isolate # treatment interaction (P < 0.011) for wound infec-tion data will be described further. The treated and untreatedcontrol resulted in 58–68% infected wounds in the curative treat-ments, regardless of isolate (Fig. 1). There was no significantdifference between the two types of control treatments. There waspoor curative control of the resistant isolate (63–59% wound infec-tion), but an increasing decline of infections by the sensitive isolatewith increasing coating load from 0.6 to 1.8 L tonne"1 (48–28%). Forprotective treatments, untreated (no coating) and treated controls(coating only) of the resistant isolate resulted in $60% of infectedwounds. Treated control of the sensitive isolate for protective treat-ments showed significantly less infected wounds (49%) than theuntreated controls (59%). Infection by the sensitive isolate signif-icantly 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 resistantisolate was reduced to 38% wound infection only at the highestcoating 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 batchinteraction (P < 0.0001; Anova not shown) was observed forgreen mould and sporulation incidence data. The interaction waslargely ascribed to significant but non-informative differencesin treatment effects between fruit batches. Hence the signifi-cant 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 resultedin more than 95% infected fruit (Table 3) with no significant differ-ences. Imazalil and coating treatment showed poor curative controlof both sensitive and resistant isolates resulting in an infection inci-dence 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 7days at 20 !C exhibited significantly lower infection levels (52%) bythe sensitive isolate for the 1.8 L tonne"1 treatments, but with noimproved control of the resistant isolate where none of the treat-ments differed from the control. The controls for the protectivetreatments also showed very high levels of green mould inci-dence (>89%), with no significant difference between the treated


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 waxcoating (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 thesensitive isolate was increasingly reduced from 50% to 13% withincreasing the coating load to 1.8 L tonne"1. At the highest coatingload, green mould incidence by the resistant isolate was signifi-cantly lower than the controls (74%). The infection in cold-storedfruit was reduced to 4% for the sensitive isolate and to 50% for theresistant isolate.

For sporulation incidence, both coated and uncoated controlsresulted in 80% sporulating fruit for both isolates, except for theuncoated control (protective treatment) for the resistant isolate(68%). Imazalil in wax coating in curative treatments resulted ina significant reduction in sporulation incidence of the sensitiveisolate, progressively with increased coating load (45–10%); coldstorage did not appear to influence sporulation incidence (44–5%).Similar trends were observed for the protective treatments againstthe sensitive isolate with higher coating load treatments reducingthe sporulation to 0% in both ambient and cold stored fruit. Despitesome 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 notshown) between the fruit type and the treatments applied. Citrustypes exhibited varying levels of IMZ residues loaded. Clementine,satsuma and navel generally showed a similar trend in terms ofresidue loading, while Valencia oranges loaded distinctly lowerresidue levels at higher coating loads. This difference was, how-ever, not evident for dip only treatments (Table 2), where residuelevels varied from 0.12 to 0.73 !g g"1, with no significant differencebetween 45 s and 90 s or fruit batches. Improved residue loadingwas evident from double application yielding up to 2.83 !g g"1

residues on fruit. Although not significant, increasing coating loadincreased the residues for the dip and coating application, with0.6 L tonne"1 loading 1.42–2.31 !g g"1 and 1.8 L tonne"1 loaded

Table 1Mean 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) and30 days at "0.5 !C and 7 days at 20 !C.

Treatment Green mould incidence (%)x Sporulating fruit (%)x

Sensitivey Resistantz Sensitive Resistant

CurativeControl (no coating) 95.6a 95.5a 90.1bc 87.5cdControl (coating) 96.5a 97.9a 96.4ab 97.9aCoating 0.6 L tonne"1 84.6c 91.7abc 44.3hi 89.7bcdCoating 1.2 L tonne"1 84.7c 95.8a 45.6hi 79.1eCoating 1. 8 L tonne"1 70.1de 92.3abc 29.6j 69.1fgCoating 0.6 L tonne"1 + cold 76.1d 91.7abc 38.8ij 56.6hCoating1.2 L tonne"1 + cold 62.2ef 87.5bc 9.5k 89.1bcdCoating 1.8 L tonne"1 + cold 51.8gh 90.1abc 4.8k 74.8efProtectiveControl (no coating) 96.9a 89.2bc 95.4ab 66.1gControl (coating) 90.1abc 93.8ab 81.1de 81.7deCoating 0.6 L tonne"1 50.0h 84.7c 38.1ij 75.3efCoating 1.2 L tonne"1 28.2i 86.1bc 50.8hi 58.1ghCoating 1.8 L tonne"1 13.2j 74.3d 40.3hij 65.8gCoating 0.6 L tonne"1 + cold 15.1j 64.6ef 37.6ij 56.9ghCoating 1.2 L tonne"1 + cold 25.0i 59.4fg 0k 80.2deCoating 1.8 L tonne"1 + cold 4.2k 50.0h 0k 49.1hi

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


Table 2Imazalil (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 sand 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 IMZ Concentration (!g mL!1) Imazalil residues (!g g!1)x

Clementine Satsuma Navel Valencia 1y Valencia 2y

Dip (45 s) 500 0.22hi 0.16i 0.28hi 0.14i 0.23hiDip (90 s) 500 0.25hi 0.12i 0.29hi 0.73ghi 0.18iDip (45 s) + coating 0.6 L tonne!1 500 + 2000 2.31cde 1.42defg 1.46defg 0.48ghi 0.38ghiDip (45s) + coating 1.2 L tonne!1 500 + 2000 2.30cde 2.01cdef 1.41defg 0.64ghi 0.70ghiDip (45s) + coating 1.8 L tonne!1 500 + 2000 2.83c 2.26cde 2.40 cd 1.29efgh 1.08fghiCoating 1.8 L tonne!1 3000 6.04a 4.14b 7.09a 1.32defgh 1.84cdef

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

2.26–2.83 !g g!1 for clementine, satsuma and navel. On Valenciaoranges, lower residue levels were recorded 0.38–0.48 !g g!1 at0.6 L tonne!1 and 1.08–1.29 !g g!1 at 1.8 L tonne!1. Single applica-tion 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 differedbetween fruit types, data were normalised by determiningpercentage control in treatments relative to the untreated con-trol. Analysis of variance resulted in a significant 4-factor fruittype " action " isolate " treatment interaction (P < 0.0001; Anovanot shown). Data for each fruit type was therefore ana-lysed separately. For each fruit type, there was a 3-factoraction " isolate " treatment interaction (P < 0.0001; Anova notshown) for percentage control data, as well as for sporulationincidence (P < 0.0001). For sporulation data, this interaction wasascribed to the effect of treatment action together with differencein level of sporulation in sensitive and resistant isolates. There wasa significant interaction between isolate and treatment (P < 0.05)and a significant effect for action (P < 0.05). For the latter, curativetreatments generally resulted in better sporulation inhibition thanprotective treatments (results not shown). Except for the clemen-tine which resulted in 79% sporulation in the curative and 82% inthe protective treatments, sporulation incidence was higher in pro-tective 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) curativelycontrolled 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 marginallybetter curative control of the sensitive isolate than 45 s dip onlytreatments, and the level of control improved with coating loadfrom 86% (0.6 L tonne!1) to 95% (1.8 L tonne!1). The resistant iso-late was poorly controlled (30%), even at the highest coating loadof 1.8 L tonne!1. Wax coating only treatment resulted in poor cura-tive control of both sensitive (47%) and resistant (27%) isolates.For protective treatments, dip only treatments resulted in poorercontrol of the sensitive isolate (38% and 43% for 45 and 90 s, respec-tively) than curative treatments, while the resistant isolate waspoorly controlled (<18%). Dip and coating significantly improvedprotective control of the sensitive isolate and treatments from 86%(0.6 L tonne!1) to 95% (1.8 L tonne!1). Wax coating only treatmentsyielded similar protective control (93%) of the sensitive isolate,while the resistant isolate was also marginally controlled (53%). Diptreatments (45 and 90 s) resulted in significantly less sporulationof the sensitive isolate (68% to 54%, respectively; Table 3) than theuntreated control (100%). Following double application, increasingcoating 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, alltreatments resulted in poor or lack of sporulation inhibition (>80%).

Lower levels of curative and protective control of the sensi-tive isolate were observed on clementine fruit (Fig. 2B) than wasobserved for navel fruit, while the resistant isolate was poorly con-trolled (<20%). Trends for the different treatments were similar.For sporulation incidence, significant inhibition was observed forthe sensitive isolate only and only for treatments that includedapplication of IMZ in wax coating (Table 3).

On satsuma fruit (Fig. 2C), curative control of the sensitive iso-late by the various treatments was similar to levels observed forclementine fruit, but higher levels of control were observed for theresistant isolate with treatments that included an IMZ dip appli-cation (20–40%). Protective control of the sensitive isolate alsofollowed similar trends as for clementine, albeit at lower levels;52–61% for treatments that included IMZ in coating application andsignificantly lower for the dip only applications (40–42%). Protec-tive control of the resistant isolate varied from 22% to 35%, with nosignificant differences between the various treatments. Similarly tothe other fruit types, sporulation incidence levels of the sensitiveisolate were significantly lower than the control treatments onlyfor treatments that included IMZ application in wax coating, whilesporulation of the resistant isolate was not inhibited (Table 3).

For control and sporulation data obtained from Valenciaoranges, a significant fruit batch interaction was observed, buttrends were nonetheless similar. Hence the significant 3-factoraction " isolate " treatment interaction (P < 0.0001; Anova notshown) was described further to simplify results. Curative controlof the sensitive isolate was significantly higher in 45 s and 90 s diptreatments (82% and 93%, respectively; Fig. 3), with an improvedlevel of control of the resistant isolate at longer exposure time(28–40%, respectively). Similar trends to the other fruit types withdouble application and wax coating only treatments were observedwith good curative control of the sensitive isolates (92–94%) andpoorer control of the resistant isolate (34–38%). Protective con-trol of the sensitive isolate improved significantly following doubleapplication of IMZ in dip and wax coating treatments, varying from66% to 76%. Wax coating only treatments resulted in significantlysuperior protective control (90%), while dip only treatments yieldedmoderate protection against the sensitive isolate (49%). The resis-tant isolate was poorly controlled in all treatments (<40%). Diptreatments resulted in significantly lower sporulation levels forthe sensitive (42% and 31% for 45 and 90 s, respectively (Table 3)relative to the control (75%), but for the resistant isolate reducedsporulation relative to the control (77%) was only observed follow-ing the 90 s dip treatment (34%). Dip and wax coating resultedin successful reduction of sporulation incidence with increasingcoating loads for sensitive isolate (19% to 6%), but not for the resis-tant isolate (>56%). Likewise, wax coating only treatment reducedsporulation of the sensitive isolate (25%), but not of the resistantisolate (74%).


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 (blackbars) 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 inIMZ 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 followedby the same letter do no differ significantly (P = 0.05).

Table 3Mean percentage of sporulation incidence after 10 days on citrus fruit that were wound inoculated with P. digitatum imazalil (IMZ) sensitive and resistant isolates andcuratively 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 IMZemulsifiable 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 Clementine Satsuma Navel Valencia

Sensitivey

Dip (45 s) 95.8a 91.7a 68.1e 41.7cDip (90 s) 97.2a 91.7a 54.3f 30.7deDip (45 s) + Coating (0.6 L tonne!1) 41.7b 30.7c 37.9 g 18.8fDip (45 s) + Coating (1.2 L tonne!1) 45.8b 12.9a 20.8 h 17.4fDip (45 s) + Coating (1.8 L tonne!1) 42.5b 30.2c 7.3i 6.3 gCoating (1.8 L tonne!1) 19.4c 20.0d 20.8 h 25.0efControl 100.0a 100.0a 99.5a 75.3aResistantz

Dip (45 s) 98.6a 100.0a 95.8abc 76.4aDip (90 s) 100.0a 97.2ab 88.7bcd 34.0 cdDip (45 s) + Coating (0.6 L tonne!1) 95.8a 98.6ab 91.7abcd 55.6bDip (45 s) + Coating (1.2 L tonne!1) 98.6a 98.6ab 87.5 cd 79.2aDip (45 s) + Coating (1.8 L tonne!1) 98.6a 96.9ab 88.9bcd 76.4aCoating (1.8 L tonne!1) 100.0a 100.0a 86.1d 73.6aControl 99.1a 100.0a 96.3ab 77.4a

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


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 inIMZ (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 IMZapplication in wax coating, specifically with regards to improvedprotective control (Waks et al., 1985) and sporulation inhibition(Smilanick et al., 1997) compared with aqueous applications, whichwas found to be superior in terms of curative control (Brown et al.,1983; Erasmus et al., 2011). Moreover, the study provides bet-ter insight of IMZ residue loading through coating application,specifically demonstrating residue levels required for effective con-trol and sporulation inhibition and indications of coating loadsthat could lead to the exceedance of the 5 !g g!1 MRL (maximumresidue level).

The low IMZ residue levels (0.8 !g g!1) obtained with singleapplication of IMZ at 3000 !g mL!1 in coating at 0.6 L tonne!1 and1.2 L tonne!1 were higher than those obtained by Kaplan and Dave(1979) with 2000 !g mL!1 on navel oranges and grapefruit (0.32and 0.18 !g g!1, respectively). At 1.8 L tonne!1, residue loadingimproved (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 sustainedgreen mould control and sporulation inhibition (Kaplan and Dave,1979; Brown and Dezman, 1990; Smilanick et al., 1997). Adequateresidues on the fruit are required for the disease control and hinderthe development of resistance. Nonetheless, satisfactory protectivecontrol and sporulation inhibition was obtained by residue levelsobtained 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 pre-viously (Waks et al., 1985; DuPlooy et al., 2009), that IMZ in waxcoating is better protective than curative when controlling the sen-sitive isolate of P. digitatum. Because wax acts as a barrier on thesurface of the fruit against fungal pathogens, it is better for protec-tion than to heal the already established infection on the albedolayer of the peel (Brown, 1984). Improved sporulation inhibitionwould also reduce P. digitatum population build-up in packhousesand therewith the risk of fungicide resistance development.

For curative treatments, the sensitive isolate was poorly con-trolled and no control was observed for the resistant isolate. Itwas also observed that coating alone (coated controls) could notlimit decay since the infection was as severe as with the uncoatedcontrols. This was also the case when the effect of essential oilsand carnauba coating was investigated for green mould control(DuPlooy et al., 2009). In cases where control was inadequate inthe 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 ofthe sporulation (Eckert and Kolbezen, 1977; Brown et al., 1983).An anti-sporulant activity is essential for the control of spoilage, acosmetic defect that takes place when healthy fruit in the cartonsare affected by spores from the nearby infected fruit (Eckert andBrown, 1986).

Previous studies showed that wax coatings particularly shellacand wood rosin, have a negative impact on the internal quality ofthe fruit whereby there is development of off-flavours which sub-sequently form because of less permeance for gaseous exchange(oxygen and carbon dioxide) as the natural openings get cloggedby a wax coating and lead to disruption of movement of thesegases (Mannheim and Soffer, 1996). The coating used in this studyis a medium solid carnauba-shellac based coating, which wassuitable for late season citrus. Quality-related aspects were notevaluated, but there were no obvious signs of rind disorders orquality defects on the fruit stored in cold temperatures (!0.5 "C).Benschoter (1984) showed that Valencia oranges were more tol-erant than grapefruit to lower temperatures (1.7 "C) when storedfor a period of 19 days to control larvae of the Caribbean fruitfly. In transit to the export market, Valencia oranges can with-stand lower temperatures of 0.5 "C without developing chillinginjury (CI). The low temperatures and the duration of the stor-age used in this study was an aggravated condition given thatcommonly used transit temperature is 3.5 "C for 24 days duringtransportation.

P. digitatum grows optimally at ambient temperatures rangingfrom 20 "C to 25 "C and temperatures below that or above deteri-orated the growth and development of the pathogen (Kassim andKhan, 1996; Plaza et al., 2003). In comparison with green mouldcontrol at ambient temperatures, we observed a clear additiveeffect of cold storage (!0.5 "C) on curative and protective greenmould control by IMZ. Infection levels of the sensitive isolate weregreatly 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 sul-phate solutions (pH 3), similar residue loading trends was observedfor clementine, satsuma, navel and Valencia oranges with all the cit-rus types loading #0.3 !g g!1. For similar treatments, Erasmus et al.(2011) reported markedly higher residues on navel and Valenciaoranges (0.97 !g g!1). Lower residue levels following dip appli-cation in our study can be attributed to the post-dip brushing offruit followed by drying. These treatments removed excess fungi-cide solution from the fruit, thereby limiting IMZ residue loading


to the exposure time in the dip tank and on the wet brushesonly. Nonetheless, the aqueous IMZ dip treatment successfully con-trolled green mould caused by the sensitive isolate of P. digitatum inall fruit kinds, particularly at longer exposure time (90 s), in cura-tive treatments. Erasmus et al. (2011) reported similar findings,but found superior curative treatments of 6-hour-old infectionscompared with the 24 h-old infections in the present study. In acommercial packhouse, the harvested fruit can easily stand for 2–4days before fungicide treatment. A 24 h incubation period there-fore better simulates such situations. The curative action of IMZ onsoft citrus was shown to be most effective in infections that wereless than 24 h old; control and sporulation inhibition were progres-sively diminished in more established infection (N. Njombolwana,unpublished data).

Sporulation could not be inhibited on clementine and satsumafruit following dip treatments, while some sporulation inhibitionwas evident on navel and Valencia oranges, but for the sensitive iso-late only. Lack of sporulation inhibition was attributed to residuesthat were significantly lower than the ideal level recommended toadequately control green mould and inhibit sporulation (Kaplanand Dave, 1979; Brown and Dezman, 1990; Smilanick et al., 1997).Brown and Dezman (1990) reported that control of sporulation fol-lowing aqueous application depended solely on the deposition ofIMZ on the natural wax of Valencia oranges. Post-dip brushing offruit, which is a common practice in commercial packlines, mostprobably contributed to reduced IMZ residue loading and possiblyto reduced sporulation inhibition.

Imazalil residues loading following double application of IMZin 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 diponly application. Depending on citrus type and coating load used,residues ranged from 0.4 to 1.8 !g g!1 on Valencia oranges andfrom 1.4 to 2.8 !g g!1 on clementine, satsuma and navel fruit. Dou-ble application resulted in both curative and protective control ofthe sensitive isolate, especially at the recommended 1.2 L tonne!1

and higher 1.8 L tonne!1 coating loads. Therefore, double applica-tion appears to be an ideal application system by integrating thesuperior curative control by IMZ following dip application withthe superior protective control and sporulation inhibition follow-ing IMZ application in coating. This is supported by Smilanick et al.(1997), who also found that a second application of IMZ in coatingafter an aqueous application will ensure the inhibition of sporula-tion, although in their work the IMZ EC formulation was used forboth applications.

The encapsulation or binding of the lipophilic IMZ in the coatingaffects its movement into the rind and this immobility of IMZ led toineffectiveness at low concentration in the coating (Brown, 1984).This was also observed for thiabendazole and benomyl, which wereless effective when applied in wax coating compared with aqueousapplication (Cho et al., 1977; Eckert and Kolbezen, 1977; Brown,1984). In this study, it was evident that the application of coatingfollowing dip application played an important role in sporulationinhibition 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 coat-ing application.

Loss of control of resistant isolate following dip, wax coating ordouble application was clearly evident in this study. It might beattributed to the severe inoculation methods and high inoculumdosage used, which might represent a worst case scenario in com-parison with the situation at a commercial packhouses (Erasmuset al., 2011). However, it is clear that practical level of IMZ resistancein P. digitatum occurs and packhouses should implement anti-resistance strategies to limit its development and effects (Holmes,1995; Holmes and Eckert, 1999; Karaoglanidis et al., 2001; Ladoet al., 2011).

A survey of coating application in commercial packhouses indi-cated a huge variation in terms of the fruit exposure time onthe coating applicator: it ranged from 7 s (probably less than thelower loads used in this study, 0.6 L tonne!1) to 41 s (probablymore than the higher loads used in this study, 1.8 L tonne!1). Thisirregularity in coating application is cause for concern, as residuedata 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), par-ticularly in clementine and navel fruit. Brown et al. (1983) statedthat higher treatment concentrations of IMZ in coating enhancedthe resulting residue loading. Interestingly, this was not observedon Valencia oranges, which showed a markedly reduced propen-sity to load IMZ residues following dip and coating application.Higher residue loading on navel oranges might be attributed toits rougher rind (Brown et al., 1983) surface texture as well as theopen navel end. Similar behaviour substantiating this phenomenonwas also noticed with thiabendazole (TBZ), where more residueswere obtained from navel oranges than in Valencia oranges (M.Kellerman, unpublished results). These observations warrant fur-ther investigation.

Most South African packhouses have adopted the practice ofdouble application of imazalil, but care should be taken whenapplying IMZ in the wax coating, as MRL levels can be exceededat coating loads higher than the recommended 1.2 L tonne!1. Thisstudy successfully showed the additive benefits of double appli-cation of IMZ in dip and wax coating. However, despite improvedcontrol of the sensitive isolate, the resistant isolate could not becontrolled. This highlights the importance of fungicide resistancemanagement strategies and alternative chemistry or methods ofgreen mould control.

Ackowledgements

The study was financially supported by Citrus Research Interna-tional, Citrus Academy, National Research Foundation and THRIP.The authors thank the personnel at Department of Plant Pathol-ogy, Stellenbosch University for technical support; Hearshaw andKinnes Analytical Laboratory (Pty)Ltd for residue analysis; DrWilma du Plooy and John Bean Technologies for supplying waxcoatings and technically assisting in coating application; ICA Inter-national Chemicals (Pty)Ltd for supplying IMZ chemicals.

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