Doctoral Degrees (Plant Pathology)
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Browsing Doctoral Degrees (Plant Pathology) by browse.metadata.advisor "Fourie, P. H."
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- ItemOptimisation of imazalil application and green mould control in South African citrus packhouses(Stellenbosch : Stellenbosch University, 2014-04) Erasmus, Arno; Fourie, P. H.; Lennox, C. L.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.ENGLISH ABSTRACT: South Africa is the largest exporter of shipped fresh citrus fruit worldwide. One of the major factors that can lead to substantial losses is postharvest decay. Penicillium digitatum (PD) and P. italicum (PI) are the main wound pathogens, respectively causing green and blue mould decay. PD is more prevalent than PI and therefore also the focus in the majority of research in this field. Imazalil (IMZ) is applied by the majority of citrus packhouses through an aqueous dip treatment, and provides good curative and protective control, as well as sporulation inhibition activity. Two IMZ formulations are in use: the sulphate salt applied in aqueous treatments and the emulsifiable concentrate (EC) applied with wax coatings. The majority of research on IMZ has been done using the EC formulation. The maximum residue limit (MRL) for IMZ on citrus fruit is 5 μg.g-1, whereas 2-3 μg.g-1 is regarded as a biologically effective residue level that should at least inhibit green mould sporulation. A study was conducted to assess the current status of IMZ application in South African packhouses, to determine the adequate residue levels needed to control green mould and inhibit sporulation using IMZ sensitive and resistant isolates, and to study optimisation of modes of IMZ application in citrus packhouses. Factors studied were IMZ concentration, application type (spray vs. dip and drench), exposure time, solution temperature and pH, as well as curative and protective control of PD. The packhouse survey showed that the majority of packhouses applied IMZ in a sulphate salt formulation through a fungicide dip tank, and loaded an IMZ residue of ≈1 μg.g-1. In dip applications, IMZ had excellent curative and protective activity against Penicillium isolates sensitive to IMZ. However, curative control of IMZ resistant isolates was substantially reduced and protective control was lost, even at twice the recommended concentration, nor was sporulation inhibited. The use of sodium bicarbonate (2%) buffered imazalil sulphate solutions at pH ±8, compared with pH ±3 of the unbuffered solutions, markedly increased IMZ residue loading on navel and Valencia oranges and improved curative and protective control of IMZ resistant isolates. Exposure time did not affect IMZ residue loading in IMZ sulphate solutions at pH 3, although the MRL was exceeded after 45 s exposure in pH 8 solutions. Imazalil applied through spray or drench application improved residue loading, but green mould control was less effective than after dip application. IMZ formulation (IMZ sulphate and EC), solution pH (IMZ sulphate at 500 μg.mL-1 buffered with NaHCO3 or NaOH to pH 6 and 8) and exposure time (15 to 540 s) were subsequently investigated in order to improve IMZ residue loading and green mould control on Clementine mandarin, lemon, and navel and Valencia orange fruit. As seen previously, exposure time had no significant effect on residue loading in the unbuffered IMZ sulphate solution (pH 3). No differences were observed between the pH buffers used, but residue loading improved with increase in pH. The MRL was exceeded following dip treatment in IMZ EC (after 75 s exposure time), and IMZ sulphate at pH 8 using NaHCO3 (77 s) or NaOH (89 s) as buffer. The MRL was exceeded after 161 s in IMZ sulphate solutions buffered at pH 6 with either NaHCO3 or NaOH. Green mould control as influenced by residue data was modelled to predict control of IMZ-sensitive and IMZ-resistant PD isolates. From this model the effective residue levels for 95% control of an IMZ-sensitive isolate and of an IMZ-resistant isolate were predicted to be 0.81 and 2.64 ug g-1, respectively. The effects of incubation time (infection age), exposure time, solution pH, wounds size and fruit brushing after dip treatments on residue loading and curative green mould control were also investigated. Exposure time did not have a significant effect on residue loading on fruit dipped in pH 3 solutions of IMZ (< 2.00 μg.g-1). Increasing the pH to 6 resulted in significantly increased residue loading, which increased with longer exposure time, but mostly to levels below the MRL after 180 s. Post-dip treatment brushing reduced residue levels obtained in IMZ pH 3 solutions by up to 90% to levels < 0.5 μg.g-1; however, curative control of the IMZ sensitive isolate was mostly unaffected, but with poor sporulation inhibition. At pH 6, post-dip brushing reduced residues to ≈ 60%; again curative control of the sensitive isolate was unaffected, but with improved sporulation inhibition. Wounded rind sections loaded higher residue levels compared to intact rind sections and large wounds loaded higher levels than small wounds (≈ 10.19, ≈ 9.06 and ≈ 7.91 μg.g-1 for large, small and no wound, respectively). Curative control of infections originating from large wounds was significantly better than those from small wounds. The ability of IMZ to control sensitive green mould infections declined from 6 and 12 h after inoculation on Clementine mandarin fruit of infections induced by small and large wounds, respectively; on navel orange fruit, curative control declined 18 and 36 h after inoculation for the respective wound size treatments. Effective IMZ concentrations that inhibit 50% (EC50) growth of nine PD and five PI isolates were determined in vitro and the IMZ sensitivity of the various isolates categorized according to their EC50 values and resistance (R) factors. Effective residue levels that predicted 50% curative (ER50C) and protective (ER50P) control of these isolates were determined in vivo. All the PI isolates had sensitive EC50 values of 0.005 - 0.050 μg.mL-1. Three PD isolates were sensitive (0.027 – 0.038 μg.mL-1), while one resistant isolate was categorized as low resistant (R-factor of 19), one as moderately resistant (R-factor of 33.2), three as resistant (R-factor of 50 - 57.6) and one as highly resistant (R-factor of 70.7). Sensitive PD isolates had mean ER50C and ER50P values on Valencia orange fruit of 0.29 and 0.20 μg.g-1, and 0.33 and 0.32 μg.g-1 on navel fruit, respectively. ER50 values for resistant isolates did not always correlate with EC50 values and ranged from 1.22 – 4.56 μg.g-1 for ER50C and 1.00 – 6.62 μg.g-1 for ER50P values. ER50P values for resistant isolates could not be obtained on navel orange fruit, but ER50C values (1.42 – 1.65 μg.g-1) were similar to those obtained on Valencia fruit. The PI isolates all behaved similar to the sensitive PD isolates with ER50C and ER50P values on navel and Valencia fruit < 0.38 μg.g-1. Alternative fungicides were assessed for the control of an IMZ sensitive, resistant and highly resistant PD isolates; these included sodium ortho-phenylpenate (SOPP), thiabendazole (TBZ), guazatine (GZT), imazalil (IMZ), pyrimethanil (PYR) and Philabuster® (PLB; a combination of IMZ and PYR), fludioxonil (FLU), azoxystrobin (AZO), Graduate®A+ (a combination of FLU and AZO) and propiconazole (PPZ). Multiple resistance was shown against IMZ, GZT, TBZ and PPZ in both resistant PD isolates. For the sensitive isolates, IMZ, SOPP, TBZ, GZT and PLB provided best curative control, while IMZ, GZT and PLB provided best protective control. For the IMZ-resistant isolates, SOPP, PYR and PLB gave the best curative control, while none of the fungicides provided adequate protective control. Globally, this is the first in-depth study of green and blue mould control with the sulphate formulation of IMZ. Findings from this study are already being implemented by industry. Solution pH is monitored, exposure time is measured and residue loading specific to application method is assessed and interpreted by means of the ER50 values. Aqueous dip applications performed best in terms of curative control, and IMZ residue loading in wound sites was most important for curative control. Other studies confirmed this and showed that IMZ is better protectively applied with wax coatings. The practical impact of IMZ resistance has been highlighted as resistant isolates infections could never be adequately controlled. IMZ alternative fungicides were assessed and SOPP, TBZ, GZT, PYR and/or PLB could be used to reduce the development and impact of IMZ resistance.