Ce travail de thèse a porté sur l'élimination d'un pesticide, l'imazalil, en phase aqueuse par trois procédés de dégradation (UV/TiO2, UV/TiO2/K2S2O8 et UV/K2S2O8) en mettant en oeuvre les outils analytiques nécessaires pour identifier les photoproduits de dégradation, suivre leur cinétique de formation et la minéralisation. L'ajout de K2S2O8 est bénéfique pour la dégradation et la minéralisation car il produit des radicaux sulfates qui sont des espèces oxydantes puissantes. Par ailleurs, le rôle des espèces radicalaires a été mis en évidence dans les trois systèmes de dégradation par des inhibiteurs de radicaux tels que les alcools (inhibiteurs des radicaux OH•). Dans une première partie, la dégradation de l'imazalil a été réalisée dans le système UV/TiO2 et les principaux photoproduits ont été identifiés par LC/MS/MS, leurs cinétiques ont été tracées. La minéralisation a été suivie et un mécanisme de dégradation a été proposé. La dégradation se déroule selon deux voies, par l'attaque des radicaux OH• ou par les trous. Dans une deuxième partie, la méthodologie de plans d'expériences a été établie dans le système UV/TiO2/K2S2O8 afin i) d'identifier les paramètres les plus influents et leurs interactions et ii) de déterminer les conditions expérimentales les plus favorables à la dégradation. Enfin, la dégradation de l'imazalil a été comparée dans les trois systèmes de dégradation en utilisant différentes concentrations de persulfate et différents pH. Il est apparu que la dégradation dans UV/TiO2, est plus efficace en milieu basique alors le pH acide est plus favorable à la dégradation de l'imazalil dans le système UV/TiO2/K2S2O8. Par contre, le pH ne joue pas un rôle important dans le système UV/K2S2O8 / The aim of this thesis was the degradation of the pesticide imazalil in water in three different systems of degradation (UV/TiO2, UV/TiO2/K2S2O8 and UV/K2S2O8). Analytical techniques were used to identify photoproducts, to follow their kinetics and mineralization. The addition of K2S2O8 is beneficial for the degradation and mineralization because it produces sulfate radicals that are powerful oxidizing species. In addition, the role of radical species has been highlighted in the three systems of degradation by using scavengers of these radicals such as alcohols (hydroxyl radical scavenger). In the first part, the degradation of imazalil was performed in the system UV/TiO2 and the main photoproducts were identified by LC/MS/MS also their kinetics were plotted. Furthermore, mineralization was followed and degradation mechanism was proposed. The degradation of imazalil could happen in two ways, by the attack of OH• radicals or by holes. In the second part, the methodology of experimental design was established in the system UV/TiO2/K2S2O8 to identify the most influential parameters also their interactions and to determine the experimental conditions that are most favorable to the degradation. Finally, the degradation of imazalil was compared in the three degradation systems using different concentrations of persulfate and different pH. It appears that the degradation in UV/TiO2 is more efficient in alkaline medium while the acidic pH is more favorable to the degradation of imazalil in the system UV/TiO2/K2S2O8. On the other hand, the pH does not play an important role in the system UV/K2S2O8

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These compounds, when reach aquatic environments, can cause serious environmental problems and affect target and non-target organisms such as microorganisms, that play key ecological functions to the ecosystem functioning. So, the objectives of this study were: (i) to evaluate the bactericidal action of oxytetracycline (OTC) and fungicide imazalil (IMZ) on bacterial ecological function of pesticide fipronil degradation; (II) evaluate the effect of oxytetracycline on density and aquatic bacterial diversity; (III) to monitor changes in bacterial dynamics in the presence of fipronil. Water was sampled in Beija-Flor Reservoir for mounting the experiments. Microcosms were prepared in triplicates for six different treatments: water; water and OTC; water and IMZ; water and fipronil; water, fipronil and OTC; water, fipronil and IMZ. The microcosms were kept in the dark at 21oC. Samples were collected at 1, 5, 10, 20, 45 and 70 days of incubation. It was determined the water pH, the dissolved organic carbon (DOC), total nitrogen (TN), OTC and fipronil concentrations. Water samples from the microcosms were also used for bacterial density analysis by epifluorescence microscopy and diversity by DGGE band profiles analysis. The presence of OTC alone and especially mixed with IMZ affects bacterial diversity, but does not cause significant effect on fipronil degradation, suggesting the existence of functional redundancy among the individuals that formed the bacterial community. The oxytetracycline, at certain concentrations, promotes a bacterial density decrease and diversity increase, probably by mediating the coexistence of species as a result of the growth control of the best competitive bacteria. Fipronil may cause bacterial density increase but, concerning its diversity, initially in the experiment, it was reduced, comparing with the control samples. However, after 70 days, the diversity was similar, suggesting that the community was reestablished, leading us to consider that the community was resilient to the impact caused by fipronil. / O atual sistema de produção agropecuário exige a utilização de produtos químicos como os antimicrobianos e agrotóxicos. Estes compostos, ao alcançarem os ambientes aquáticos, podem trazer sérios problemas ambientais e afetar vários organismos, como os microrganismos, que desempenham funções ecológicas fundamentais ao funcionamento ecossistêmico. Desta maneira, os objetivos deste trabalho foram: (I) avaliar a ação do bactericida oxitetraciclina (OTC) e fungicida imazalil (IMZ) na função ecológica bacteriana de degradação do agrotóxico fipronil; (II) avaliar o efeito da oxitetraciclina na densidade e diversidade bacteriana aquática; (III) acompanhar alterações na dinâmica bacteriana na presença do fipronil. Foi coletada água da Represa do Beija-Flor para a montagem dos experimentos. Foram preparados microcosmos em tréplica para seis tratamentos distintos: água; água e OTC; água e IMZ; água e fipronil; água, fipronil e OTC; água, fipronil e IMZ. Os microcosmos foram mantidos a 21 oC, no escuro. Foram feitas amostragens nos tempos 1, 5 10, 20, 45 e 70 dias de incubação. Foram determinados o pH da água, carbono orgânico dissolvido (COD), nitrogênio total (NT) e as concentrações de OTC e fipronil. Amostras de água provenientes dos microcosmos foram utilizadas para análise bacteriana de densidade por microscopia de epifluorescência e análise de diversidade por meio dos perfis de bandas do DGGE. A presença da OTC sozinha e principalmente em mistura com IMZ, afeta a diversidade bacteriana, porém não causa efeito significativo na degradação do fipronil, sugerindo existência de redundância funcional entre os indivíduos que constituíram a comunidade bacteriana. A oxitetraciclina, em determinadas concentrações, causa a diminuição na densidade bacteriana e pode promover o aumento da diversidade, provavelmente por mediar à coexistência de espécies em consequência do controle do crescimento de bactérias mais competitivas. O fipronil pode causar o aumento da densidade bacteriana mas quanto à diversidade, no início do experimento, ela foi menor que nas amostras controle. Porém, após 70 dias, as diversidades ficam semelhantes, sugerindo que a comunidade se reestabeleceu, podendo esta ser considerada resiliente a perturbação causada pelo fipronil.

L’usage excessif des pesticides a entraîné de nombreux effets néfastes sur l’environnement et aussi sur la santé des populations. Il est donc important d’approfondir, nos connaissances sur leur comportement dans toutes les sphères de l’environnement et de mettre en place de nouvelles technologies de dépollution d’effluents chargé en pesticides avant leur rejet dans le milieu naturel. Le travail de cette thèse est situé dans ce contexte. Le premier volet de thèse a été consacré au traitement des solutions aqueuses chargées par deux fongicides (imazalil, thiabendazole), par le procédés d’oxydation électrochimiques directe, afin d’optimiser les différents paramètres expérimentaux de leur dégradation. Ces études confirment l’efficacité du procédé électrochimique dans le traitement des eaux polluées par les pesticides, avec un taux de minéralisation qui dépasse 80%. D’autre part, les résultats obtenus dans ce travail ont montré que la nature du matériel d’anode a une grande influence sur la performance du procédé d’oxydation. Dans la deuxième partie, nous avons étudié la réactivité atmosphérique en phase gazeuse des composés aromatiques hétérocycliques azotés. Ces espèces sont attendues lors de la dégradation des pesticides. Les études cinétiques de ces composés avec l’ozone et les radicaux OH, ont été effectuées dans une chambre de simulation atmosphérique à différentes températures. Les réactions d’ozonolyse de ces composés sont très lentes. Dans le cas de leur réaction avec les radicaux OH, les résultats obtenus montrent que la réactivité atmosphérique de ces composés dépend fortement de leur structure moléculaire. En effet, les composés hétérocycliques aromatiques à 5 atomes présentent une réactivité plus importante vis-à-vis les radicaux OH, par rapport aux composés hétérocycliques aromatiques à six atomes. Par ailleurs, ces études ont montrés que la présence des atomes d’azote dans le cycle aromatique a un effet désactivant sur la réactivité de ces composés avec les radicaux OH. Sur le plan spectroscopique, les spectres d’absorption UV-visibles des composés : 1,2,3 triazole, pyrazole, pyrazine, pyrimidine,et pyridazine, ont été déterminés en phase gazeuse et leurs constantes de photodissociation atmosphérique ont été estimées. Enfin, les données cinétiques et spectroscopiques obtenues ont permis de discuter les implications atmosphériques de ces composés en termes de durée de vie par rapport aux différents processus de dégradation atmosphérique / This work concerns a new insight of treatment of organic pollutants pesticides before their rejection in the natural environment. This phenomenon is increasing dramatically causing many adverse effects on the environment and the health. The first step of this work focused on the treatment of aqueous solution charged with fungicides by direct electrochemical processes. The efficiency of this latter in the treatment of waters polluted by pesticides has been confirmed with a mineralization rate exceeding 80%. In the other hand, the obtained results have ensured that the nature of the anode material is one of the keys and factors having an influence on the oxidation pathway. The second step conducted by absorption spectroscopy and focused on the atmospheric reactivity in the gas phase of nitrogenous heterocyclic aromatic compounds. These species are expected during the degradation of pesticides. The use of an atmospheric simulation chamber at different temperatures allowed us to follow the kinetics of these compounds with ozone and OH radicals. The kinetics of ozonolysis of these compounds is very slow. The molecular structure is the factor, according to the results obtained, controlling the atmospheric reactivity of these compounds in the case of their reaction with OH radicals. Indeed, the 5-atom aromatic heterocyclic compounds have a greater reactivity with respect to the OH radicals, compared with six-membered aromatic heterocyclic compounds. Moreover, according to these studies, the presence of nitrogen atoms in the aromatic ring has a deactivating effect on the reactivity of these compounds with the OH radicals. Spectroscopically, the UV-visible absorption spectra of the compounds 1,2,3-triazole, pyrazole, pyrazine, pyrimidine, and pyridazine were determined in the gas phase and their atmospheric photodissociation constants were estimated. Finally, the kinetic and spectroscopic data obtained made it possible to discuss the atmospheric implications of these compounds in terms of their lifetime with respect to the different processes of atmospheric degradation.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Please refer to full text for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Thesis (PhD(Agric))--Stellenbosch University, 2014. / 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. / AFRIKAANSE OPSOMMING: Suid-Afrika is die grootste uitvoerder van verskeepde vars sitrusvrugte wêreldwyd. Een van die vernaamste faktore wat tot substansiële verliese kan lei, is na-oesverrotting. Penicillium digitatum (PD) en P. italicum (PI) is die hoof wondpatogene, en veroorsaak onderskeidelik groenskimmel- en blouskimmelverval. PD is meer algemeen as PI en daarom ook die fokus in die meerderheid van navorsing in hierdie veld. Imazalil (IMZ) word deur die meerderheid van sitruspakhuise in ‘n waterige doopbehandeling toegedien, en verskaf goeie genesende en beskermende beheer, sowel as sporulasie-inhibisie aktiwiteit. Twee IMZ-formulasies word gebruik: die sulfaatsout wat in waterige behandelings toegedien word, en die emulsifiseerbare konsentraat (EK) wat in wakslaagbehandelings toegedien word. Die meerderheid van navorsing op IMZ is gedoen deur die gebruik van die EK-formulasie. Die maksimum residu limiet (MRL) vir IMZ op sitrusvrugte is 5 μg.g-1, terwyl 2-3 μg.g-1 as ‘n biologies effektiewe residuvlak beskou word wat ten minste groenskimmelsporulasie moet inhibeer. ‘n Studie is uitgevoer ten einde die huidige status van IMZ-toediening in Suid-Afrikaanse pakhuise vas te stel, om die voldoende residuvlakke vas te stel wat nodig is om groenskimmel te beheer en sporulasie te inhibeer deur die gebruik van IMZ-sensitiewe en -weerstandbiedende isolate, en om optimisering van metodes van IMZ-toediening in sitruspakhuise te bestudeer. Faktore wat bestudeer is, was IMZ-konsentrasie, toedieningstipe (spuit vs. doop en stort), blootstellingsperiode, oplossingstemperatuur en pH, asook genesende en beskermende beheer van PD. Die pakhuis-opname het aangedui dat die meerderheid van pakhuise IMZ in ‘n sulfaatsoutformulasie deur ‘n fungisieddooptenk toegedien het, en ‘n IMZ-residu van ≈1 μg.g-1 gelaai het. In dooptoedienings het IMZ uitstekende genesende en beskermende aktiwiteit teen ‘n Penicillium IMZ-sensitiewe isolaat gehad. Genesende beheer van ‘n IMZ-weerstandbiedende isolaat was egter substansiëel minder, en beskermende beheer was verlore, selfs teen twee keer die aanbevole konsentrasie. Sporulasie is ook nie geïnhibeer nie. Die gebruik van natriumbikarbonaat (2%) gebufferde imazalil sulfaat-oplossings by pH ±8, in vergelyking met pH ±3 van die ongebufferde oplossings, het IMZ-residulading op nawel en Valencia lemoene merkbaar verhoog, en genesende en beskermende beheer van IMZ-weerstandbiedende isolaat verbeter. Blootstellingsperiode het nie IMZ-residulading in IMZ-sulfaat-oplossings by pH 3 geaffekteer nie, hoewel die MRL ná 45 s blootstelling in pH 8 oplossings oorskry is. Imazalil wat deur spuit- of drenkhandeling toegedien is, het residulading verbeter, maar groenskimmelbeheer was minder effektief as ná dooptoediening. IMZ-formulasie (IMZ-sulfaat en EK), oplossing pH (IMZ-sulfaat teen 500 μg.mL-1 gebuffer met NaHCO3 of NaOH na pH 6 en 8) en blootstellingsperiode (15 tot 540 s) is daaropvolgend ondersoek ten einde IMZ-residulading en groenskimmelbeheer op Clementine mandaryn, suurlemoen, en nawel en Valencia lemoen vrugte te verbeter. Soos voorheen opgelet, het blootstellingsperiode geen betekenisvolle effek op residulading in die ongebufferde IMZ-sulfaat-oplossing (pH 3) gehad nie. Geen verskille is tussen die pH buffers wat gebruik is, waargeneem nie, maar residulading het met verhoogde pH verbeter. Die MRL is ná die doopbehandeling in IMZ EK (ná 75 s blootstellingsperiode), en IMZ-sulfaat by pH 8 en gebruik van NaHCO3 (77 s) of NaOH (89 s) as buffer, oorskry. Die MRL is ná 161 s in IMZ-sulfaat-oplossings gebuffer by pH 6 met óf NaHCO3 óf NaOH oorskry. Groenskimmelbeheer, soos beïnvloed deur residulading, is gemodelleer ten einde beheer van IMZ-sensitiewe en IMZ-weerstandbiedende PD isolate te voorspel. Vanaf hierdie model is die effektiewe residuvlakke vir 95% beheer van ‘n IMZ-sensitiewe isolaat en van ‘n IMZ-weerstandbiedende isolaat as onderskeidelik 0.81 en 2.64 ug.g-1 voorspel. Die effekte van inkubasieperiode (infeksie-ouderdom), blootstellingsperiode, oplossing pH, wondgrootte en borsel van vrugte ná doopbehandelings, op residulading en genesende groenskimmelbeheer, is ook ondersoek. Blootstellingsperiode het geen betekenisvolle effek op residulading op vrugte wat in pH 3 oplossings van IMZ (< 2.00 μg.g-1) gedoop is, gehad nie. Verhoging van pH tot 6 het tot betekenisvolle verhoogde residulading gelei, wat met verlengde blootstellingsperiode toegeneem het, maar meestal tot vlakke onder die MRL ná 180 s. Ná-doop borsel van vrugte het residuvlakke wat in IMZ pH 3 oplossings verkry is, met tot 90% verminder na vlakke < 0.5 μg.g-1; genesende beheer van die IMZ-sensitiewe isolaat was egter meestal ongeaffekteer, maar met swak sporulasie-inhibisie. By pH 6, het ná-doop borsel van vrugte residue tot ≈ 60% verminder; genesende beheer van die sensitiewe isolaat is weer nie geaffekteer nie, maar met verbeterde sporulasie-inhibisie. Gewonde skilsegmente het hoër residuvlakke gelaai in vergelyking met heel skilsegmente, en groot wonde het hoër vlakke gelaai in vergelyking met klein wonde (≈ 10.19, ≈ 9.06 en ≈ 7.91 μg.g-1 vir groot, klein en geen wond, onderskeidelik). Genesende beheer van infeksies wat vanaf groot wonde ontstaan het, was betekenisvol beter as dié vanaf klein wonde. Die vermoë van IMZ om sensitiewe groenskimmel-infeksies te beheer, het vanaf 6 en 12 h ná inokulasie op Clementine mandaryn vrugte van infeksies wat deur klein en groot wonde onderskeidelik geïnduseer is, afgeneem; op nawel lemoen vrugte, het genesende beheer 18 en 36 h ná inokulasie vir die onderskeie wondgrootte behandelings, afgeneem. Effektiewe IMZ-konsentrasies wat 50% (EK50) groei van nege PD en vyf PI isolate inhibeer, is in vitro vasgestel en die IMZ-sensitiwiteit van die verskillende isolate is volgens hul EK50 waardes en weerstandsfaktore (R) gekatogeriseer. Effektiewe residuvlakke wat 50% genesende (ER50C) en beskermende (ER50P) beheer van hierdie isolate voorspel, is in vivo vasgestel. Al die PI isolate het sensitiewe EK50 waardes van 0.005 - 0.050 μg.mL-1 gehad. Drie PD isolate was sensitief (0.027 – 0.038 μg.mL-1), terwyl een weerstandbiedende isolaat as laag weerstandbiedend (R-faktor van 19) gekatogeriseer is, een as matig weerstandbiedend (R-faktor van 33.2), drie as weerstandbiedend (R-faktor van 50 - 57.6) en een as hoogs weerstandbiedend (R-faktor van 70.7). Sensitiewe PD isolate het gemiddelde ER50C en ER50P waardes op Valencia lemoen vrugte van 0.29 en 0.20 μg.g-1 gehad, en 0.33 en 0.32 μg.g-1 op nawel vrugte, onderskeidelik. ER50 waardes vir weerstandbiedende isolate het nie altyd met EK50 waardes gekorreleer nie en het van 1.22 – 4.56 μg.g-1 vir ER50C en 1.00 – 6.62 μg.g-1 vir ER50P waardes gevariëer. ER50P waardes vir weerstandbiedende isolate kon nie op nawel lemoen vrugte verkry word nie, maar ER50C waardes (1.42 – 1.65 μg.g-1) was soortgelyk aan dié verkry op Valencia vrugte. Die PI isolate het almal soortgelyk aan die sensitiewe PD isolate opgetree, met ER50C en ER50P waardes op nawel en Valencia vrugte < 0.38 μg.g-1. Alternatiewe swamdoders is vir die beheer van ‘n IMZ-sensitiewe, -weerstandbiedende en -hoogs weerstandbiedende PD isolate getoets; hierdie het ingesluit: “sodium ortho-phenylpenate” (SOPP), thiabendazole (TBZ), guazatine (GZT), imazalil (IMZ), pyrimethanil (PYR) en Philabuster® (PLB; ‘n kombinasie van IMZ en PYR), fludioxonil (FLU), azoxystrobin (AZO), Graduate®A+ (‘n kombinasie van FLU en AZO) en propiconazole (PPZ). Veelvoudige weerstand is teen IMZ, GZT, TBZ en PPZ in beide weerstandbiedende PD isolate aangetoon. Vir die sensitiewe isolate, het IMZ, SOPP, TBZ, GZT en PLB die beste genesende beheer verskaf, terwyl IMZ, GZT en PLB die beste beskermende beheer verskaf het. Vir die IMZ-weerstandbiedende isolate, het SOPP, PYR en PLB die beste genesende beheer verskaf, terwyl geen van die swamdoders voldoende beskermende beheer verskaf het nie. Hierdie studie is wêreldwyd die eerste in-diepte studie van groenskimmel- en blouskimmelbeheer met die sulfaatformulasie van IMZ. Bevindinge vanuit hierdie studie word alreeds in die industrie geïmplementeer. Oplossing pH word gemonitor, blootstellingsperiode word gemeet en residulading spesifiek tot toedieningsmetode word bepaal en volgens die ER50 waardes geïnterpreteer. Waterige dooptoedienings het die beste ten opsigte van genesende beheer gevaar, en IMZ-residulading in wond-areas was die belangrikste vir genesende beheer. Ander studies het dit bevestig en getoon dat IMZ beter beskermend is wanneer in ‘n wakslaag toegedien word. Die praktiese impak van IMZ-weerstand is uitgelig aangesien weerstandbiedende isolaat-infeksies nooit voldoende beheer kon word nie. IMZ alternatiewe swamdoders is getoets en SOPP, TBZ, GZT, PYR en/of PLB kon gebruik word om die ontwikkeling en impak van IMZ-weerstand te verminder.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Green mould is a major citrus postharvest disease caused by a wound pathogen called Penicillium digitatum. Imazalil (IMZ) is the most important postharvest fungicide that is currently registered against green mould and blue mould, given its good protective and curative control and sporulation inhibition. Imazalil can be applied in drench, dip, spray and wax coating applications. Wax coatings improve the quality of the fruit during shelf life by limiting moisture loss and providing a shiny appearance. The primary objective of this study was to investigate the application of imazalil in wax coatings in citrus packhouses to control green mould and preserve fruit quality. The first aim of this study was to evaluate the protective and curative control and sporulation inhibition of IMZ sensitive and resistant isolates of P. digitatum following single IMZ application in wax coating and IMZ application in aqueous dip as well as wax coating. For single application, Valencia orange fruit was curatively or protectively treated with a carnauba-based coating into which IMZ at 3000 μg.mL-1 was mixed. For curative treatments, fruit was treated after 24 hour's incubation. Coating was applied at 0.6, 1.2 and 1.8 L.ton-1 of fruit on a commercial coating applicator. Untreated control and fruit treated with coating only showed up to 80% infection for both sensitive and resistant isolates. Imazalil in coating proved to have better protective than curative action against the sensitive isolate, while control of the resistant isolate was poor. Imazalil residue levels increased with increasing coating load (0.85 to 1.75 μg.g-1). For double application, clementine, satsuma, navel and Valencia orange fruit were used. Curative and protective control following dip only treatment (IMZ sulphate at 500 μg.mL-1 for 45 s and 90 s) was compared with coating only treatment (IMZ at 3000 μg.mL-1 at 1.8 L.ton-1), and double application comprising dip (45 s in IMZ sulphate at 500 μg.mL-1) followed by 2000 μg.mL-1 IMZ in coating at 0.6, 1.2 and 1.8 L.ton-1. Double application resulted in improved residue loading (0.85 to 2.06 μg.g-1) compared to dip only treatments (≈ 0.49 μg.g-1). Coating only resulted in the highest residue levels (1.82 to 7.09 μg.g-1), often exceeding the maximum residue limit of 5 μg.g-1. In all treatments, poor curative and protective control of the resistant isolate (<46% and <55%, respectively) and generally no sporulation inhibition was observed. For the sensitive isolate, dip only treatments resulted in better curative control (≈ 77%) than protective control (≈ 38%). Double application showed increased protective control with increasing wax load (≈ 69%) as well as good curative control (≈ 83%). Wax coating only treatment resulted in poor curative control (≈ 26%), but good protective control (≈ 80%). Sporulation inhibition of the sensitive isolate was generally improved in treatments that included IMZ application in the wax coating. This study successfully showed the additive benefits of double application of IMZ in the wax coating following an aqueous dip application. However, despite improved control of the sensitive isolate, the resistant isolate could not be controlled. The second objective of the study was to evaluate green mould control and quality preservation following IMZ application using different coating and brush types. The inoculation and treatment procedure was similar to the single application of wax coating described above but only the sensitive isolate of P. digitatum was used. Carnauba- or polyethylene-based wax coatings, including IMZ at 3000 μg.mL-1, was applied at a dosage of 0.6, 1.2 and 1.8 L.ton-1 using commercial horsehair or synthetic brushes. Fruit was either incubated for 4 days prior to wound infection ratings or stored at -0.5oC for 26 days and 7 days shelf life. Imazalil residues on fruit increased with increasing coating loads on navel oranges (1.31 to 3.32 μg.g-1) and Valencia oranges (3.22 to 6.00 μg.g-1). Better protective (≈ 58%) than curative control (≈ 14%) was observed, with horsehair brushes resulting in lower sporulation levels (≈ 59%) than synthetic brushes (≈ 64%). Fruit weight loss and firmness loss ratios relative to uncoated control fruit were significantly higher in fruit treated with polyethylene coating compared with carnauba coating treated fruit. However, polyethylene coating resulted in shinier fruit before and after storage, whereas carnauba coating resulted in lower shine ratios. Gaseous (CO2) exchange ratios remained similar for both coatings, but higher polyethylene coating loads (1.8 L.ton-1) resulted in off-tastes similar to the uncoated control fruit and higher than the ratings for carnauba. Scanning electron micrographs revealed an amorphous crystallised natural wax layer with uncovered stomatal pores on the surface of uncoated fruit. The thickness of the applied coating layer increased with increasing coating load. Coating layers following application with synthetic brushes at 1.2 L.ton-1 appeared to be thicker than when applied using horsehair brushes. This study indicated the effects of brush type on the distribution and polishing of the wax coating on the surface of the fruit and also the importance of cautious application of coating as under or over application may lead to poor results in terms of bio-efficacy, residue loading and fruit quality. / AFRIKAANSE OPSOMMING: Groenskimmel is 'n belangrike sitrus na-oessiekte, veroorsaak deur 'n wondpatogeen, genaamd Penicillium digitatum. Imazalil (IMZ) is die belangrikste na-oesfungisied wat tans teen groenskimmel geregistreer is, gegewe sy goeie beskermende en genesende beheer en sporulasie-inhibisie. Imazalil kan in bad-, doop-, spuit- en wakslaagbehandelings toegedien word. Wakslae behou die kwaliteit van die vrugte gedurende raklewe deur vogverlies te beperk en 'n glansvoorkoms te verskaf. Die primêre doel van hierdie studie was om die toediening van imazalil in wakslaagbehandelings in sitruspakhuise vir groenskimmel en vrugkwaliteit beheer te ondersoek. Die eerste doelwit van hierdie studie was om die beskermende en genesende beheer en sporulasie-inhibisie van IMZ sensitiewe en weerstandbiedende isolate van P. digitatum, gevolg deur enkel IMZ toediening in 'n wakslaagbehandeling, en IMZ toediening in 'n doop-, asook wakslaagbehandeling, te evalueer. Vir enkelltoediening is Valencia lemoenvrugte genesend of beskermend met 'n carnauba-gebaseerde wakslaag behandel, waarin IMZ teen 3000 μg.mL-1 gemeng is. Vir genesende behandelings is vrugte ná 24 uur inkubasie behandel. Wakslaagbehandeling is teen 0.6, 1.2 en 1.8 L.ton-1 vrugte op 'n semi-kommersiële paklyn toegedien. Onbehandelde kontrole en vrugte met waks behandel, het slegs tot 80% infeksie vir beide sensitiewe en weerstandbiedende isolate getoon. Imazalil in waks het beter beskermende as genesende aksie teen die sensitiewe isolaat getoon, terwyl beheer van die weerstandbiedende isolaat swak was. Imazalil residuvlakke het met toename in wakslading toegeneem (0.85 tot 1.75 μg.g-1). Vir dubbeltoediening is clementine, satsuma, navel en Valencia lemoenvrugte gebruik. Genesende en beskermende beheer van IMZ na enkel doopbehandeling (IMZ sulfaat teen 500 μg.mL-1 vir 45 s en 90 s), is met enkel waksbehandeling (IMZ teen 3000 μg.mL-1 teen 1.8 L.ton-1) vergelyk, asook dubbeltoediening bestaande uit doop (45 s in IMZ sulfaat teen 500 μg.mL-1), gevolg deur 2000 μg.mL-1 IMZ in waksbehandeling teen 0.6, 1.2 en 1.8 L.ton-1. Dubbeltoediening het verbeterde residulading (0.85 tot 2.06 μg.g-1) tot gevolg gehad, in vergelyking met enkel doopbehandelings (≈ 0.49 μg.g-1). Enkel waksbehandeling het die hoogste residuvlakke (1.82 tot 7.09 μg.g-1) tot gevolg gehad, wat dikwels die maksimum residu-limiet van 5 μg.g-1 oorskry het. In alle behandelings is swak genesende en beskermende beheer van die weerstandbiedende isolaat (<46% en <55%, onderskeidelik), en oor die algemeen, geen sporulasie-inhibisie, waargeneem. Vir die sensitiewe isolaat, het die enkel doopbehandelings beter genesende beheer (≈ 77%) in vergelyking met die beskermende beheer (≈ 38%) tot gevolg gehad. Dubbeltoediening het verhoogde beskermende beheer met toenemende wakslading (≈ 69%) getoon, asook goeie genesende beheer (≈ 83%). Enkel wakslaagbehandeling het swak genesende beheer (≈ 26%) tot gevolg gehad, maar goeie beskermende beheer (≈ 80%). Sporulasie-inhibisie van die sensitiewe isolaat was oor die algemeen beter in behandelings wat IMZ toediening in die wakslaag ingesluit het. Hierdie studie het suksesvol die toegevoegde voordele van dubbeltoediening van IMZ in doopbehandeling en wakslaag getoon. Ten spyte van die verbeterde beheer van die sensitiewe isolaat, kon die weerstandbiedende isolaat egter nie beheer word nie. Die tweede doelwit was om groenskimmelbeheer en behoud van kwaliteit te evalueer, volgende op IMZ toediening deur gebruik van verskillende waks- en borseltipes. Inokulasie- en behandelingsprosedure was soortgelyk aan die enkeltoediening van wakslaagbehandeling soos bo beskryf, maar slegs die sensitiewe isolaat van P. digitatum is gebruik. Carnauba- of poli-etileen-gebaseerde wakslae, insluitende IMZ teen 3000 μg.mL-1, is teen 'n dosis van 0.6, 1.2 en 1.8 L.ton-1 toegedien, deur die gebruik van kommersiële perdehaar of sintetiese borsels. Vrugte is óf vir 4 dae vóór wond-infeksie evaluering geïnkubeer, óf teen -0.5oC vir 26 dae en 7 dae raklewe gestoor. Imazalil residuvlakke op vrugte het met toenemende waksladings op navellemoene (1.31 tot 3.32 μg.g-1) en Valencia lemoene (3.22 tot 6.00 μg.g-1) toegeneem. Beter beskermende (≈ 58%) as genesende beheer (≈ 14%) is waargeneem, met perdehaar borsels wat laer sporulasievlakke (≈ 59%) tot gevolg gehad het, in vergelyking met sintetiese borsels (≈ 64%). Vruggewig- en fermheidsverlies verhoudings, relatief tot onbedekte kontrole vrugte, was betekenisvol hoër in vrugte wat met 'n poli-etileenwaks behandel is, in vergelyking met carnauba-waks behandelde vrugte. Poli-etileen waksbehandeling het egter blinker vrugte vóór en ná opberging tot gevolg gehad, terwyl canauba waksbehandeling laer blink verhoudings tot gevolg gehad het. Gas (CO2) uitruilverhoudings het dieselfde vir beide waksbehandelings gebly, maar hoër poli-etileen waksladings (1.8 L.ton-1) het tot afsmake soortgelyk aan die onbedekte kontrole vrugte gelei, en hoër as die evaluerings vir carnauba. Skandeer-elektronmikrograwe het 'n amorfe gekristalliseerde natuurlike wakslaag met onbedekte stomata op die oppervlak van onbedekte vrugte getoon. Die dikte van die toegediende waks het met toename in wakslading toegeneem. Wakslae na toediening met sintetiese borsels teen 1.2 L.ton-1 het dikker vertoon in vergelyking met perdehaar borsels. Hierdie studie het die effekte van borseltipe op die verspreiding en polering van die wakslaag op die oppervlak van vrugte getoon, en versigtige toediening van wakslae onderskryf, aangesien ónder- of óórtoediening swak resultate, in terme van bio-effektiwiteit, residu-lading en vrugkwaliteit, tot gevolg kan hê. / National Research Foundation, THRIP, Citrus Academy & Citrus Research International for bursary and research funding.

Las podredumbres verde y azul causadas por los hongos patógenos Penicillium digitatum (Pers.: Fr.) Sacc y Penicillium italicum Wehmer, respectivamente, son las responsables de grandes pérdidas económicas en poscosecha de cítricos a nivel mundial. En España, las pudriciones de la fruta en poscosecha fluctúan del 3 al 6% durante una estación típica. Sin embargo, bajo condiciones favorables a la enfermedad, las pérdidas pueden alcanzar el 50%. Las podredumbres verde y azul son las enfermedades de poscosecha más importante de los cítricos en España, California y todas las áreas de producción caracterizadas por lluvias escasas en verano (Ecker y Eaks, 1989). Se han logrado avances importantes en estudios de resistencia adquirida sistémica (SAR) y resistencia sistémica inducida (ISR) tanto natural como artificial (Terry y Joyce, 2004; Tavallali et al., 2008). También se está avanzando en la evaluación de sustancias conocidas como GRAS (Generally Regarded as Safe), o en el uso del calor y la luz ultravioleta (Palou et al., 2008; Montesinos et al., 2009). Todos estos enfoques constituyen estrategias de control alternativo no contaminante. Sin embargo, actualmente seguimos dependiendo del uso de fungicidas químicos sintéticos convencionales para controlar las enfermedades de poscosecha de fruta fresca en general y de cítricos en particular. Asimismo, existe un incremento de la demanda de los mercados internacionales de frutas y hortalizas frescas de buena calidad e inocuas. Además, el uso de fungicidas convencionales conlleva el problema del desarrollo de resistencia en cepas de P. digitatum y P. italicum. Por otro lado, los resultados en efectividad de la mayoría de los métodos de control no contaminantes no han sido equivalentes a los obtenidos con métodos de control químico convencional. Por todo ello sigue siendo un reto la búsqueda e implementación comercial de estrategias no contaminantes efectivas para el control de las podredumbres verde y azul de los cítricos. Un importante mediador de la respuesta en la patogénesis de estos hongos es el gas etileno, ya que su biosíntesis se incrementa durante la interacción planta-patógeno o después de la aplicación de químicos inductores de resistencia. Además, la producción del etileno puede ser una señal para activar mecanismos de defensa contra patógenos como hongos, bacterias o virus (Ecker y Davis, 1987). Se han realizado estudios orientados a buscar relaciones entre el etileno y la patogenicidad de hongos causantes de enfermedades en poscosecha (Brown, 1973). Sin embargo, discrepancias en los resultados han sido observadas. El etileno es aplicado durante la desverdización de los frutos cítricos, que es una práctica comercial común realizada en muchas partes del mundo (Smilanick et al., 2006), incluyendo España, y se estima que más de 1,2 millones de toneladas de fruta de mandarina de estación temprana se desverdiza anualmente (Martínez-Jávega et al., 2007). Por lo tanto, es de importancia primordial determinar el efecto de esta práctica en la susceptibilidad y desarrollo de las podredumbres verde y azul de los frutos cítricos. Los objetivos serán los siguientes: 1) Evaluar sustancias químicas con antecedentes de potencial de inducción de resistencia para controlar las podredumbres verde y azul de poscosecha en frutos cítricos, 2) Evaluar sustancias conocidas como GRAS (General Regarded as Safe), solas o combinadas con fungicidas para controlar las podredumbres verde y azul en frutos cítricos y, 3) Determinar el efecto del desverdizado comercial con etileno sobre la incidencia y desarrollo de las podredumbres verde y azul en mandarinas y naranjas de estación temprana; así como el efecto del desverdizado comercial sobre los atributos de calidad del fruto. / Moscoso Ramírez, PA. (2013). Aproximaciones a una estrategia integrada para el control no contaminante de las podredumbres verde y azul en poscosecha de cítricos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34626 / TESIS