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Management of postharvest diseases of apples using essential oils

Mbili, Nokwazi Carol (2015-12)

Thesis (PhDAgric)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Grey mould, blue mould and bull’s eye rot, caused by Botrytis cinerea, Penicillium expansum and Neofabraea alba, respectively, are postharvest diseases which cause significant losses to the apple industry in South Africa and other parts of the world. Current control methods of the diseases raises some concerns, such as development of fungicide resistance, residues harmful to humans, and increasing public health restrictions on the use of certain fungicides. These public concerns over synthetic pesticides in foods and the environment has spurred interest among researchers and the global public to find effective and safe non-fungicide means of controlling postharvest pathogens. The aim of this study was therefore to evaluate the effect of lemongrass and citrus essential oils in combination with cold storage regimes to control B. cinerea, P. expansum and N. alba, both in vitro and in vivo. Volatile composition of essential oils of lemon (Citrus limon), lime (Citrus aurantifolia) and lemongrass (Cymbopogan citratus) were analysed using gas chromatography-mass spectrometry (GC-MS). A total of 44 volatile compounds were detected and identified in C. limon, with limonene (58.52%) and γ-terpinene (19.80%) as the major compounds; C. aurantifolia showed 20 components with limonene (79.00%) and γ-terpinene (11.22%) as the abundant compounds; while C. citratus revealed 26 components with geranial (48.14%) and neral (38.32%) as the major compounds. Direct contact and vapour phase methods were used to test the in vitro antifungal activity of citrus and lemongrass oils against B. cinerea, P. expansum and N. alba. Direct contact phase of lemongrass oil, mixtures of lemon + lemongrass and lime + lemongrass oils exhibited the strongest toxicity and completely inhibited the mycelial growth and spore germination of all three tested pathogens, at concentrations of 1.50-100.00% at 20°C (95% relative humidity), regular atmosphere (-0.5°C, air), and controlled atmosphere (5%O2 + 10% CO2, -0.5°C). Lemongrass oil, mixtures of lemon + lemongrass and lime + lemongrass oils were fungicidal against the three postharvest pathogens both in direct contact and vapour phase of 1.50% and 0.13%, respectively, at 20°C, regular atmosphere and controlled atmosphere. Lemon and lime oils also showed antifungal activity, but were much less effective than lemongrass essential oil. In the vapour phase method, all essential oil treatments significantly (p < 0.05) controlled all three pathogens at concentrations of 0.02-1.00% compared to control treatments. Mycelial growth of fungal pathogens was inhibited by the tested essential oils in a dose-dependent manner. The mechanisms of antifungal action of lemongrass oil against B. cinerea was investigated. Light microscopy and scanning electron microscopy observations indicated that B. cinerea hyphae exposed to lemongrass oil undergo morphological damage such as vesiculation, cytoplasmic disruption and collapsed hyphae. Glucose-induced reduction in external pH of B. cinerea was inhibited by lemongrass oil in a time and concentration dependent manner. Botrytis cinerea spores treated with lemongrass oil showed strong propidium iodide fluorescence in the cytosol. Lemongrass oil significantly altered the plasma membrane, the release of cell constituents, and the total lipid content of B. cinerea. These observations indicate that the antifungal activity of lemongrass oil can be attributed to the disruption of the cell membrane integrity and membrane permeability. Application of lemongrass and citrus essential oils through fumigation, thermal fogging and dipping were tested in vivo for their potential to inhibit postharvest decay caused by B. cinerea, P. expansum and N. alba on ‘Granny Smith’, ‘Golden Delicious’ and ‘Pink Lady’ apples. Treated fruit were stored at controlled atmosphere (‘Granny Smith’ and‘Pink Lady’: 1.5% O2 + 1% CO2,-0.5°C and ‘Golden Delicious’: 1.5% O2 + 2.5% CO2,-0.5°C) for 28 days followed by 7 days at 20°C. After storage, lesion diameter was measured and expressed as percentage inhibition relative to control treatment. Application of citrus and lemongrass essential oils through fumigation, dipping and thermal fogging showed some potential to inhibit B. cinerea, P. expansum and N. alba in vivo. Postharvest control of B. cinerea, P. expansum and N. alba by essential oils through dipping and thermal fogging in combination with controlled atmosphere indicated that a mixture of lime + lemongrass oil was the most effective, followed by a mixture lemon + lemongrass oil, lemongrass oil, lime oil and lemon oil. The effects of citrus and lemongrass essential oil treatments on quality parameters of apples was evaluated after controlled atmosphere storage. Citrus and lemongrass essential oils tested had no significant effect on fruit firmness of ‘Granny Smith’ and ‘Pink Lady’ apples. Significant differences were observed in total soluble solids, titratible acidity, pH and total soluble solids: titratible acidity ratio of both cultivars treated with essential oils compared to control treatments. Citrus and lemongrass essential oil exerted a positive influence in postharvest and quality of ‘Granny Smith’ and ‘Pink Lady’ apples. The results presented in this thesis highlight the potential of essential oils in combination with cold storage regimes as an alternative control strategy against grey mould, blue mould and bull’s eye rot in the apple industry. This suggest the application of essential oils by incorporating the oil into wax or coating treatments that are being used in the packing-line and the possibility of fumigation and thermal fogging during the storage period.

AFRIKAANSE OPSOMMING: Grys-, blou- en teikenvrot word veroorsaak deur die patogene Botrytis cinerea , Penicillium expansum en Neofabraea alba, onderskeidelik, en is na-oes siektes wat beduidende verliese in die appel-bedryf van Suid-Afrika en ander wêrelddele veroorsaak. Huidige beheer metodes van die siektes bring 'n paar bekommernisse; soos weerstand ontwikkeling teen swamdoders, residue nadelig vir die mens en toenemende beperkings as gevolg van openbare gesondheidbewustheid op die gebruik van sekere swamdoders. Die openbare bekommernis oor sintetiese plaagdoders in voedsel en die effek van dit op die omgewing het navorsers aangespoor om meer doeltreffende en veiliger alternatiewe middels vir na-oes pathogene te ontwikkel. Dus was die doel van hierdie studie om die in vitro en in vivo effek van sitroengras en sitrus essensiële olies tesame met koelkamer sisteme op B. cinerea, P. expansum en N. alba te ondersoek. Die vlugtige samestelling van essensiële olies van suurlemoen (Citrus limon), lemmetjie (Citrus aurantifolia) en sitroengras (Cymbopogan citratus) is ontleed met behulp gaschromatografie-massaspektrometrie (GC-MS). Altesaam is 44 vlugtige verbindings opgespoor en geïdentifiseer in C. limon, met limoneen (58,52 %) en γ-terpineen (19,80 %) as die belangrikste verbindings. Citrus aurantifolia het 20 komponente bevat waarvan limoneen (79,00 %) en γ-terpineen (11,22 %) die oorvloedigste verbindings was. Van C. citratus se 26 komponente was geraniaal (48,14 %) en neraal ( 38,32 %) as die belangrikste verbindings beskou. Direkte kontak en dampfase metodes is gebruik om die in vitro antifungale aktiwiteit van sitrus en sitroengras olies teen B. cinerea, P. expansum en N. alba toets. Die direkte kontak van sitroengras olie, mengsels van suurlemoen+ sitroengras en lemmetjie + sitroengras olie was die mees toksiese olies en het miseliumgroei en spoor-ontkieming vir al drie pathogene geïnhibeer. Inhibisie was verkry by konsentrasies van 1,50-100,00% teen 20°C (95% relatiewe humiditeit), normale atmosfeer (-0,5 °C, lug), en gereguleerde atmosfeer (5% O2 + 10% CO2, -0,5 °C). Sitroengras olie, mengsels van suurlemoen + sitroengras en lemmetjie + sitroengras olies het swamdodende aktiwiteit getoon vir al drie na-oes patogene in direkte kontak en damp fase van 1,50% en 0,13%, onderskeidelik, by 20°C in normale asook gereguleerde atmosfeer. Suurlemoen en lemmetjie olies het ook antifungale aktiwiteit getoon, maar was minder doeltreffend as sitroengras essensiële olie. Met die dampfase metode het al die essensiële olie behandelings aansienlike beheer (P < 0.05) getoon vir al drie patogene by konsentrasies van 0,02-1,00% in vergelyking met die kontrole behandeling. Die swamme se miseliumgroei was geïnhibeer deur die essensiële olies in 'n dosis-afhanklike wyse te toets. Die meganisme van sitroengras olie se antifungale werking teenoor B. cinerea was ondersoek met behulp van ligmikroskopie en skandering elektronmikroskopie. Die hifes van B.cinerea was blootgestel aan sitroengras olie en morfologiese skade soos vesikulasie, sitoplasmiese ontwrigting en ineenstorting is waargeneem. Sitroengras olie het glukose-geïnduseerde vermindering in die eksterne pH van B. cinerea geïnhibeer op ‘n tyd- en konsentrasie-afhanklike wyse. Botrytis cinerea spore wat behandel is met sitroengras olie het sterk propidium jodied fluoressensie in die sitosol getoon. Sitroengras olie veroorsaak aansienlike veranderinge in die plasma membraan, uitlating van sel inhoud en die totale lipied inhoud van B. cinerea. Hierdie waarnemings dui daarop dat die antifungale aktiwiteit van sitroengras olie toegeskryf kan word aan die ontwrigting van die selmembraan se integriteit en deurlaatbaarheid. Die potensiaal van sitroengras en sitrus essensiële olies om na-oes bederf, wat veroorsaak word deur B. cinerea, P. expansum en N. alba, is in vivo getoets as beroking, termiese verneweling en doopmiddel op 'Granny Smith', "Golden Delicious" en " Pink Lady’ appels. Behandelde vrugte is gestoor in gereguleerde atmosfeer. Vir 'Granny Smith' en 'Pink Lady': 1,5% O2 + 1% CO2, -0,5°C en vir ‘Golden Delicious': 1,5% O2 + 2,5% CO2, -0,5°C vir 28 dae gevolg deur 7 dae by 20°C. Na opberging was letsel deursnee gemeet en weergee as persentasie inhibisie relatief tot die kontrole behandeling. Toepassing van sitrus en sitroengras essensiële olies in die vorm van beroking, doop en termiese verneweling beskik oor die potensiaal om B. cinerea, P. expansum en N. alba te inhibeer in vivo. Die behandeling van B. cinerea, P. expansum en N. alba met essensiële olies as ‘n doop en termiese verneweling gekombineer met gereguleerde atmosfeer het getoon dat ‘n mengsel van lemmetjie+ sitroengras olie die mees doeltreffende na-oes beheer gehad het. Dit is gevolg met ‘n mengsel van suurlemoen + sitroengras olie, sitroengras olie, lemmetjie olie en suurlemoen olie. Die effek van behandeling met sitrus en sitroengras essensiële olies op die kwaliteit van appels is geëvalueer na opberging in gereguleerde atmosfeer. Sitrus en sitroengras essensiële olies het geen wesenlike uitwerking op die fermheid van 'Granny Smith’ en ‘Pink Lady’ appels gehad nie. Beduidende verskille is waargeneem in die totale oplosbare vastestowwe, titreerbare suur, pH en totale oplosbare vastestowwe. Die titreerbare suurverhouding van beide kultivars behandel met essensiële olies was vergelyk met die kontrole behandelings. Sitrus en sitroengras essensiële olie het 'n positiewe invloed op die na-oes beheer en gehalte van 'Granny Smith’ en ‘Pink Lady' appels. Die resultate wat verkry is in hieride tesis beklemtoon die potensiaal van essensiële olies in kombinasie met koelkamer stelsel as 'n alternatiewe beheerstrategie teen grys vorm Grys-, blou- en teikenvrot in die appelbedryf. essensiële olies kan dus moontlik geinkorporeer word in was- en filmlaag behandelings van paklyne, beroking en en termiese verneweling tydens die opbergingsfase.

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