Imazalil and wax coating application in citrus packhouses to control green mould and preserve fruit quality

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.

Masters