Optimisation of imazalil application and green mould control in South African citrus packhouses

Erasmus, Arno (2014-04)

Thesis (PhD(Agric))--Stellenbosch University, 2014.

Thesis

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.

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