WO2019040991A1

WO2019040991A1 - A method for the treatment of eukaryotic microorganisms

Info

Publication number: WO2019040991A1
Application number: PCT/AU2018/050939
Authority: WO
Inventors: Kirsty Louise BAYLISS; Giles Edward StJohn HARDY; Shaikh Sharmin SIDDIQUE
Original assignee: Murdoch University
Current assignee: Murdoch University
Priority date: 2017-08-31
Filing date: 2018-08-31
Publication date: 2019-03-07
Anticipated expiration: 2020-02-29
Also published as: AU2018326395A1

Abstract

A method for the treatment of fresh produce, the method comprising the steps of contacting the fresh produce with cold plasma or cold plasma-treated water, wherein where the fresh produce is contacted with cold plasma, the cold plasma source is between 1 and 20 cm from the fresh produce and the fresh produce is contacted for between 1 and 20 minutes; or wherein where the fresh produce is contacted with cold plasma-treated water, the fresh produce is contacted with the cold plasma- treated water for 1 second to 36 hours, such that at least a portion of the eukaryotic microorganisms on or in the fresh produce are inactivated.

Description

A Method for the Treatment of Eukaryotic Microorganisms Field of the Invention

[0001 ] The present invention relates to a method for the treatment of eukaryotic microorganisms.

Background Art

[0002] Fruits and vegetables are susceptible to a large number of postharvest fungal pathogens causing major losses due to postharvest decay of produce. These postharvest fungi mostly develop disease symptoms on fruit after harvest, during storage at cold temperature, shipment, and decrease shelf life prior to consumer consumption. Different genera of fungi can cause postharvest disease of fruit, such as Penicillium, Aspergillus, Botrytis, Monilinia, and Colletotrichum. Many of these fungi have a wide host range and infection ability (pathogenicity), and account for crop yield losses as well as economic losses every year. Even in the absence of appropriate control measures, some genera like Colletotrichum can cause up to 100 % postharvest losses in some produce.

[0003] The use of synthetic fungicides is the current strategy to control postharvest diseases as they are easily applicable, relatively inexpensive, and have both curative as well as preventive action against established and new infections. However, their intense use has given rise to important issues such as human health hazards and environmental pollution. These negative impacts of chemical fungicides has prompted the search for safer control methods.

[0004] The development of alternative chemical-free techniques to control postharvest diseases is increasing in many research programs worldwide including biological control, modified atmospheres, essential oils and physical treatments. These alternative methods often adversely affect the fruit colour, aroma or taste after treatment.

[0005] The preceding discussion of the background art is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was part of the common general knowledge in Australia as at the priority date of the application.

Summary of the Invention

[0006] In accordance with the present invention, there is provided a method for the treatment of eukaryotic microorganisms, the method comprising the steps of: contacting a eukaryotic microorganism with cold plasma or cold plasma-treated water, wherein where the eukaryotic microorganism is contacted with cold plasma, the cold plasma source is between 0.5 cm and 20 cm from the eukaryotic microorganism and the eukaryotic microorganism is contacted for between 1 second and 20 minutes; or wherein where the eukaryotic microorganism is contacted with cold plasma- treated water, the eukaryotic microorganism is contacted with the cold plasma- treated water for 1 second to 36 hours, such that at least a portion of the eukaryotic microorganisms are inactivated.

[0007] In accordance with the present invention, there is provided a method for the treatment of fresh produce, the method comprising the steps of: contacting the fresh produce with cold plasma or cold plasma-treated water, wherein where the fresh produce is contacted with cold plasma, the cold plasma source is between 1 and 20 cm from the fresh produce and the fresh produce is contacted for between 1 and 20 minutes; or wherein where the fresh produce is contacted with cold plasma-treated water, the fresh produce is contacted with the cold plasma-treated water for 1 second to 36 hours, such that at least a portion of the eukaryotic microorganisms on or in the fresh produce are inactivated.

[0008] The fresh produce may be selected from the group comprising fruits, vegetable, grains, nuts and seeds. Preferably, the fresh produce are fruits and vegetables. Where the fresh produce are fruits and vegetables, the fruits and vegetables may include citrus fruit, stone fruit, pome fruit, tropical fruit, melons, leafy vegetables, roots and tubers. More preferably, in highly specific forms of the invention, the fresh produce are avocados and strawberries. The fresh produce may also be provided in the form of fresh cut produce such as shredded lettuce leaves which may be available in packaged salads.

[0009] Preferably, the cold plasma has an intensity of at least 0.1 Jem ². [0010] Preferably, the cold plasma is a gliding arc plasma.

[001 1 ] Without being limited by theory, it is believed that the distance of the cold-plasma source to the eukaryotic microorganism and the period of contact are directly proportional. That is, shorter distances may require briefer contact times.

[0012] Without being limited by theory, it is believed that the distance of the cold-plasma source to the fresh produce and the period of contact are directly proportional. That is, shorter distances may require briefer contact times.

[0013] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma, the cold plasma source is between 1 and 20 cm from the eukaryotic microorganism. Alternatively, the cold plasma source is between 1 and 10 cm from the eukaryotic microorganism. Alternatively, the cold plasma source is between 2 and 8 cm from the eukaryotic microorganism. Alternatively, the cold plasma source is between 2 and 5 cm from the eukaryotic microorganism. Alternatively, the cold plasma source is about 2.5 cm from the eukaryotic microorganism. Alternatively, the cold plasma source is about 5 cm from the eukaryotic microorganism.

[0014] In one form of the invention, where the eukaryotic organism is contacted with cold plasma, the eukaryotic organism is contacted for between 1 0 seconds and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 10 minutes. Alternatively, the eukaryotic microorganism is contacted for between 30 seconds and 5 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 minute and 5 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 minute and 3 minutes.

[0015] In one form of the invention, where the eukaryotic organism is contacted with cold plasma, the eukaryotic microorganism is contacted for between 1 minute and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 minute and 10 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 minute and 5 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 minute and 3 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 minute and 2 minutes.

[0016] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma, the eukaryotic microorganism is contacted for between 5 minutes and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 minutes and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 15 minutes and 20 minutes.

[0017] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma, the eukaryotic microorganism is contacted for between 1 second and 10 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 5 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 4 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 3 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 2 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 1 minute. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 30 seconds. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 20 seconds. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 10 seconds. Alternatively, the eukaryotic microorganism is contacted for between 1 second and 5 seconds. Alternatively, the eukaryotic microorganism is contacted for between 5 seconds and 60 seconds. Alternatively, the eukaryotic microorganism is contacted for between 5 seconds and 30 seconds. Alternatively, the eukaryotic microorganism is contacted for between 5 seconds and 20 seconds. Alternatively, the eukaryotic microorganism is contacted for between 5 seconds and 10 seconds. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 60 seconds. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 50 seconds. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 40 seconds. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 30 seconds. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 20 seconds. Alternatively, the eukaryotic microorganism is contacted for between 20 seconds and 60 seconds. Alternatively, the eukaryotic microorganism is contacted for between 20 seconds and 50 seconds. Alternatively, the eukaryotic microorganism is contacted for between 20 seconds and 40 seconds. Alternatively, the eukaryotic microorganism is contacted for between 20 seconds and 30 seconds. Alternatively, the eukaryotic microorganism is contacted for between 30 seconds and 60 seconds. Alternatively, the eukaryotic microorganism is contacted for between 30 seconds and 50 seconds. Alternatively, the eukaryotic microorganism is contacted for between 30 seconds and 40 seconds.

[0018] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma, the eukaryotic microorganism is contacted for between 10 seconds and 10 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 5 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 4 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 3 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 2 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 1 minute. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 30 seconds. Alternatively, the eukaryotic microorganism is contacted for between 10 seconds and 20 seconds.

[0019] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma, the eukaryotic microorganism is contacted for between 10 seconds and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 1 minute and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 2 minutes and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 15 minutes and 20 minutes. Alternatively, the eukaryotic microorganism is contacted for between 10 minutes and 20 minutes.

[0020] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma, the temperature of the surface of the eukaryotic microorganism is between 0 and 120 °C. Preferably, the temperature is less than 50 °C. [0021 ] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma-treated water, the eukaryotic microorganism is contacted with the cold plasma-treated water for 10 seconds to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 30 seconds to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma- treated water for 1 minute to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 5 minutes to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 10 minutes to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 20 minutes to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 30 minutes to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma- treated water for 1 hour to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 2 hours to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 4 hours to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 8 hours to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 12 hours to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 18 hours to 36 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 24 hours to 36 hours.

[0022] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma-treated water, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 24 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 18 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma- treated water for 1 second to 12 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 8 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 4 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 2 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 1 hour. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 30 minutes. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 20 minutes. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 10 minutes. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 5 minutes. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 1 minute. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 30 seconds. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 20 seconds. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 10 seconds. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 5 seconds.

[0023] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma-treated water, the eukaryotic microorganism is contacted with the cold plasma-treated water for 10 seconds to 24 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 30 seconds to 18 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma- treated water for 1 minute to 12 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 5 minutes to 8 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 10 minutes to 4 hours. Alternatively, the eukaryotic microorganism is contacted with the cold plasma-treated water for 30 minutes to 2 hours.

[0024] Preferably, the eukaryotic microorganism is contacted at a temperature of between 10 and 35 °C.

[0025] In one form of the invention, the cold plasma-treated water is prepared by contacting water with cold plasma for a period of 1 -120 minutes. Alternatively, the water is contacted with the cold plasma for a period of 1 -60 minutes. Alternatively, the water is contacted with the cold plasma for a period of 1 -30 minutes. Alternatively, the water is contacted with the cold plasma for a period of 1 -20 minutes. Alternatively, the water is contacted with the cold plasma for a period of 1 -10 minutes. Alternatively, the water is contacted with the cold plasma for a period of 1 -5 minutes. Alternatively, the water is contacted with the cold plasma for a period of 1 -3 minutes. Alternatively, the water is contacted with the cold plasma for a period of 1 -2 minutes. Alternatively, the water is contacted with the cold plasma for a period of 2-120 minutes. Alternatively, the water is contacted with the cold plasma for a period of 4-120 minutes. Alternatively, the water is contacted with the cold plasma for a period of 10-120 minutes. Alternatively, the water is contacted with the cold plasma for a period of 20-120 minutes. Alternatively, the water is contacted with the cold plasma for a period of 60-120 minutes.

[0026] In one form of the invention, the cold plasma-treated water is prepared by contacting water with cold plasma for a period of 1 and 24 hours. Cold plasma-treated water may be prepared on a continuous basis, 24 hours a day.

[0027] The cold plasma-treated water may be prepared by subjecting the water to cold plasma at a distance of about 1 to 5 cm from the water surface. Alternatively, cold plasma-treated water may be prepared by subjecting the water to cold plasma immersed in the water and bubbled through the water.

[0028] The water may be provided in the form of tap water, deionised water or distilled water.

[0029] The cold plasma-treated water may be used any time up to 30 days from preparation. In one form of the invention, the cold plasma-treated water is used within 15 days of preparation. Preferably, the cold plasma-treated water is used within 24 hours of preparation.

[0030] In one form of the invention, where the fresh produce is contacted with cold plasma, the cold plasma source is between 1 and 20 cm from the fresh produce. Alternatively, the cold plasma source is between 1 and 10 cm from the fresh produce. Alternatively, the cold plasma source is between 2 and 8 cm from the fresh produce. Alternatively, the cold plasma source is between 2 and 5 cm from the fresh produce. Alternatively, the cold plasma source is about 2.5 cm from the fresh produce. Alternatively, the cold plasma source is about 5 cm from the fresh produce.

[0031 ] In one form of the invention, where the fresh produce is contacted with cold plasma, the fresh produce is contacted for between 10 seconds and 20 minutes. Alternatively, the fresh produce is contacted for between 10 seconds and 10 minutes. Alternatively, the fresh produce is contacted for between 30 seconds and 5 minutes. Alternatively, the fresh produce is contacted for between 1 minute and 5 minutes. Alternatively, the fresh produce is contacted for between 1 minute and 3 minutes.

[0032] In one form of the invention, where the fresh produce is contacted with cold plasma, the fresh produce is contacted for between 1 minute and 20 minutes. Alternatively, the fresh produce is contacted for between 1 minute and 10 minutes. Alternatively, the fresh produce is contacted for between 1 minute and 5 minutes. Alternatively, the fresh produce is contacted for between 1 minute and 3 minutes. Alternatively, the fresh produce is contacted for between 1 minute and 2 minutes.

[0033] In one form of the invention, where the fresh produce is contacted with cold plasma, the fresh produce is contacted for between 5 minutes and 20 minutes. Alternatively, the fresh produce is contacted for between 10 minutes and 20 minutes. Alternatively, the fresh produce is contacted for between 15 minutes and 20 minutes.

[0034] In one form of the invention, where the fresh produce is contacted with cold plasma, the fresh produce is contacted for between 1 second and 10 minutes. Alternatively, the fresh produce is contacted for between 1 second and 5 minutes. Alternatively, the fresh produce is contacted for between 1 second and 4 minutes. Alternatively, the fresh produce is contacted for between 1 second and 3 minutes. Alternatively, the fresh produce is contacted for between 1 second and 2 minutes. Alternatively, the fresh produce is contacted for between 1 second and 1 minute. Alternatively, the fresh produce is contacted for between 1 second and 30 seconds. Alternatively, the fresh produce is contacted for between 1 second and 20 seconds. Alternatively, the fresh produce is contacted for between 1 second and 10 seconds. Alternatively, the fresh produce is contacted for between 1 second and 5 seconds. Alternatively, the fresh produce is contacted for between 5 seconds and 60 seconds. Alternatively, the fresh produce is contacted for between 5 seconds and 30 seconds. Alternatively, the fresh produce is contacted for between 5 seconds and 20 seconds. Alternatively, the fresh produce is contacted for between 5 seconds and 10 seconds. Alternatively, the fresh produce is contacted for between 10 seconds and 60 seconds. Alternatively, the fresh produce is contacted for between 10 seconds and 50 seconds. Alternatively, the fresh produce is contacted for between 10 seconds and 40 seconds. Alternatively, the fresh produce is contacted for between 10 seconds and 30 seconds.

Alternatively, the fresh produce is contacted for between 10 seconds and 20 seconds.

Alternatively, the fresh produce is contacted for between 20 seconds and 60 seconds.

Alternatively, the fresh produce is contacted for between 20 seconds and 50 seconds.

Alternatively, the fresh produce is contacted for between 20 seconds and 40 seconds.

Alternatively, the fresh produce is contacted for between 20 seconds and 30 seconds.

Alternatively, the fresh produce is contacted for between 30 seconds and 60 seconds.

Alternatively, the fresh produce is contacted for between 30 seconds and 50 seconds. Alternatively, the fresh produce is contacted for between 30 seconds and 40 seconds.

[0035] In one form of the invention, where the fresh produce is contacted with cold plasma, the fresh produce is contacted for between 10 seconds and 10 minutes. Alternatively, the fresh produce is contacted for between 10 seconds and 5 minutes. Alternatively, the fresh produce is contacted for between 10 seconds and 4 minutes. Alternatively, the fresh produce is contacted for between 10 seconds and 3 minutes. Alternatively, the fresh produce is contacted for between 10 seconds and 2 minutes. Alternatively, the fresh produce is contacted for between 10 seconds and 1 minute. Alternatively, the fresh produce is contacted for between 10 seconds and 30 seconds. Alternatively, the fresh produce is contacted for between 10 seconds and 20 seconds.

[0036] In one form of the invention, where the fresh produce is contacted with cold plasma, the fresh produce is contacted for between 10 seconds and 20 minutes. Alternatively, the fresh produce is contacted for between 1 minute and 20 minutes. Alternatively, the fresh produce is contacted for between 2 minutes and 20 minutes. Alternatively, the fresh produce is contacted for between 15 minutes and 20 minutes. Alternatively, the fresh produce is contacted for between 10 minutes and 20 minutes.

[0037] In one form of the invention, where the eukaryotic microorganism is contacted with cold plasma, the temperature of the surface of the eukaryotic microorganism is between 1 and 120 °C. Preferably, the temperature is less than 50 °C.

[0038] In one form of the invention, where the fresh produce is contacted with cold plasma-treated water, the fresh produce is contacted with the cold plasma-treated water for 10 seconds to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 30 seconds to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 minute to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 5 minutes to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 10 minutes to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma- treated water for 20 minutes to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 30 minutes to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 hour to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 2 hours to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma- treated water for 4 hours to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 8 hours to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 12 hours to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 18 hours to 36 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 24 hours to 36 hours.

[0039] In one form of the invention, where the fresh produce is contacted with cold plasma-treated water, the fresh produce is contacted with the cold plasma-treated water for 1 second to 24 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 18 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 12 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 8 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 4 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 2 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 1 hour. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 30 minutes. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 20 minutes. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 10 minutes. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 5 minutes. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 1 minute. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 30 seconds. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 20 seconds. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 10 seconds. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 second to 5 seconds.

[0040] In one form of the invention, where the fresh produce is contacted with cold plasma-treated water, the fresh produce is contacted with the cold plasma-treated water for 10 seconds to 24 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 30 seconds to 18 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 1 minute to 12 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 5 minutes to 8 hours. Alternatively, the fresh produce is contacted with the cold plasma-treated water for 10 minutes to 4 hours. Alternatively, the fresh produce is contacted with the cold plasma- treated water for 30 minutes to 2 hours.

[0041 ] The fresh produce may be contacted by the cold plasma-treated water by immersing the produce in the water or by introducing the water to the produce, for example, by spraying. Where the produce is immersed in the water, the contact time may be between 1 and 15 minutes.

[0042] Preferably, the temperature of the fresh produce is maintained between 10 and 35 °C while in contact with the cold plasma-treated water.

[0043] Preferably, the eukaryotic microorganisms are fungi.

[0044] Different genera of fungi can cause postharvest disease of fruit, such as Penicillium, Aspergillus , Botrytis, Monilinia, and Colletotrichum. Many of these fungi have a wide host range and infection ability (pathogenicity), and account for crop yield losses as well as economic losses every year. Even in the absence of appropriate control measures, some genera like Colletotrichum spp. can cause up to 100 % postharvest losses in some produce.

[0045] Postharvest fungi that may be treated by the method of the present invention include but not limited to Alternaria (including alternata, citri, mali spp.), Aspergillus (including flavus, niger, parasiticus, ochraceus spp.), Botrytis (including cinerea, fabae spp.), Cercospora (including apii, longissima spp.), Cladosporium (including cladosporioides, herbarum, tenuissimum spp.), Colletrotrichum (including acutatum, alienum, florinae, gloeosporioides, musae spp.), Fusarium (including decemcellulare, oxysporum, proliferatum spp.), Geotrichum (including citri-aurantii spp.), Lasiodiplodia (including theobromae spp.), Monilinia (including fructicola, fructigena, mail, laxa spp.), Mucor (including piriformis, racemosus spp.), Neofabraea (including alba spp.), Nigrospora (including sphaerica, oryzae spp.), PenicHlium (including italica, digitatum, expansum, verrucosum spp.), Phytophthora (including cactorum, palmivora, erythroseptica, infestans, syringae spp.), Phoma (including carica-papayae spp.), Phomopsis (including citri, perseae spp.), Pseudocercospora (including purpurea spp.), Pythium (including ultimum spp.), Rhizopus (including oryzae, stolonifer spp.), Rhizoctonia (including solani spp.), Sclerotinia (including sclerotiorum spp.), Sclerotium (including ro/fe/7^' spp.), Stemphyllium (including lycopersici spp.).

[0046] In one form of the invention, the ratio of plasma-treated water to fungal spores is at least 1 :1 . In one form of the invention, the ratio of plasma-treated water to fungal spores is between 1 :1 and 10:1 . In one form of the invention, the ratio of plasma- treated water to fungal spores is between 1 :1 and 3:1 .

[0047] The method of the present invention is applicable to the treatment of fresh produce such as fruits and vegetables. Preferably, the fruits and vegetables are treated postharvest. Fresh produce may be treated by cold plasma in any number of ways including the use of a hand-held cold plasma device. Preferably, the plasma flame or device does not touch the produce. Alternatively, the fresh produce may pass through a cold plasma on a conveyor or similar apparatus. Alternatively, the fresh produce may be washed or immersed in plasma-treated water.

[0048] The cold plasma may be prepared in an atmosphere of air, compressed air, oxygen, nitrogen or argon either individually or in combination.

[0049] Preferably, the cold plasma is at a temperature of between 25 and 450 °C. More preferably, the temperature is between 25 and 100 °C. More preferably, the temperature is room temperature.

[0050] Brief Description of the Figures

[0051 ] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:

Figure 1 . Mean colony growth of C. alienum on 90 mm half PDA plates after cold plasma treatment of freshly inoculated cultures for 0 to 360 s; A) cold plasma treatment without lids; B) cold plasma treatment with lids. Bars indicate standard errors of the mean (n=10);

Figure 2. Mean colony growth of C. alienum on 90 mm half PDA plates after cold plasma treatment of 2-day old actively-growing cultures for 0 to 360 s; A) cold plasma treatment without lids; B) cold plasma treatment with lids. Bars indicate standard errors of the mean (n=10);

Figure 3. Mean percentage germination of conidia of C. alienum on half PDA following cold plasma treatment. Conidia were plated within 60 s, and 3, 6, 12 or 24 hr after treatment. Germination was counted 12 hr after each plating or 12, 15, 18, 24, and 36 hr after treatment. Bars indicate standard errors of the mean (n=6);

Figure 4. Mean colony growth of Colletotrichum spp. on 90 mm half PDA plates after cold plasma treatment of freshly inoculated cultures for either 180 and 360 s; A and B) strongly pathogenic C. alienum (WAC-13971 , and WAC-13891 ); C and D) weakly pathogenic C. alienum (WAC-13972, and WAC-13973); E and F) strongly pathogenic C. florinae (WAC-13896). Bars indicate standard errors of the mean (n=10);

Figure 5 Mean colony growth of Colletotrichum spp. on 90 mm half PDA plates after cold plasma treatment of 2 day old actively-growing cultures for 180 and 360 s; A and B) strongly pathogenic C. alienum (WAC-13971 , and WAC-13891 ); C and D) weakly pathogenic C. alienum (WAC-13972, and WAC-13973); E and F) strongly pathogenic C. florinae (WAC-13896). Bars indicate standard errors of the mean (n=10);

Figure 6 Mean colony growth of Colletotrichum spp. on 90 mm half PDA plates after cold plasma treatment of freshly inoculated cultures for either 180 and 360 s in a sealed box; A and B) strongly pathogenic C. alienum (WAC-13971 , and WAC-13891 ); C and D) weakly pathogenic C. alienum (WAC-13972, and WAC- 13973); E and F) strongly pathogenic C. florinae (WAC-13896). Bars indicate standard errors of the mean (n=10);

Figure 7 Mean colony growth of Colletotrichum spp. on 90 mm half PDA plates after 180 and 360 s cold plasma treatment of 2 day old actively-growing cultures in a sealed box; A and B) strongly pathogenic C. alienum (WAC-13971 , and WAC-13891 ); C and D) weakly pathogenic C. alienum (WAC-13972, and WAC- 13973); E and F) strongly pathogenic C. florinae (WAC-13896). Bars indicate standard errors of the mean (n=10);

Figure 8. Mean percentage germination of conidia of Colletotrichum alienum and C. florinae isolates on half PDA following a 180 s and 360 s cold plasma treatment of spore suspensions. Conidia were plated immediately (time 0) or at 3, 6, 12 or 24 hr after treatment, and germination measured 12 hr later. A and B) strongly pathogenic C. alienum (WAC-13971 , and WAC-13891 ); C and D) weakly pathogenic C. alienum (WAC-13972, and WAC-13973); E and F) strongly pathogenic C. florinae (WAC-13896). Bars indicate standard errors of the mean (n=3);

Figure 9 Mean percentage germination of conidia of different Colletotrichum spp. mixtures on half PDA following a 180 and 360 s cold plasma treatment of spore suspensions. Conidia were plated immediately (time 0) or at 3, 6, 12 or 24 hr after treatment, and germination measured 12 hr later. A and B) mixture of strongly and weakly pathogenic C. alienum isolates (WAC-13891 , WAC-13972); C and D) mixture of strongly pathogenic C. alienum and C. florinae isolates (WAC-13891 , WAC-13896); Bars indicate standard errors of the mean (n=6);

Figure 10. Average optical emission spectra observed during cold plasma generation in A) an open environment, or B) a sealed environment. OIV= triple ionised oxygen notated, Olll = doubly ionised oxygen, O II = singly ionised oxygen, Ol = non ionised atomic oxygen, O²⁺ = singly ionised molecular oxygen; Arlll = doubly ionised argon, Aril = singly ionised argon, Arl = non ionised atomic argon; N2H2 = diazene, N2O2 = hyponitrite, NO = nitric oxide; NO2 = nitrogen dioxide ? = singly ionised molecular nitrogen, Nil = singly ionised atomic nitrogen, Nl = non ionised atomic nitrogen; Culll = doubly ionised copper, Cull = singly ionised copper; Cul = non ionised atomic copper; 0111 =doubly ionised carbon, CM =singly ionised carbon, CI =neutral carbon;

Figure 1 1 . Temperature increased observed during cold plasma treatment in a fume hood in open or sealed environment;

Figure 12. Mean percentage germination of C. alienum conidia on half PDA following treatment with PAW30 or PAW60 derived from tap, deionised or distilled water at one of three ratios of conidia:PAW (1 :1 , 1 :2, or 1 :3). Conidia were treated with PAW for 30 s, 3, 6, 12 and 24 hr, and then plated on half PDA. Percentage germination was counted 12 hr after plating the conidia. A and B) Plasma activated tap water; C and D) Plasma activated deionised water; E and F) Plasma activated distilled water. The controls were 1 :1 of conidia:untreated sterile water (tap, deionised, or distilled water). Bars indicate standard errors of the mean (n=6);

Figure 13. Mean percentage germination of C. alienum conidia on half PDA following treatment with PAW generated from distances of 2.5, 5 and 10 cm from plasma emission point to the water surface. The ratio of conidia:PAW was 1 :3. Conidia were treated with PAW for 30 s, 3, 6, 12 and 24 hr, and then plated on half PDA. Percentage germination was counted 12 h after plating the conidia. Positive control indicates conidia treated with PAW60 (PAW produced from 60 ml deionised water, 60 min of CP treatment, 2.5 cm distance at 1 :3 ratio for conidia and PAW); Negative control indicates conidia treated with sterile deionised wateri :3 ratio (conidia: sterile deionised water). Bars indicate standard errors of the mean (n=6);

Figure 14. Mean percentage germination of C. alienum conidia on half PDA following treatment with PAW generated from different volumes (100, 500 and 1000 ml) of water at 1 :3 ratio of conidia:PAW. Conidia were treated with PAW for 30 s, 3, 6, 12 and 24 r, and then plated on half PDA. Percentage germination was counted 12 hr after plating the conidia. A) PAW100, PAW500 and PAW1000 produced from different volume of water; B) PAW [1000 (comb)] produced by combining 10 volume of PAW100. Positive control indicates conidia treated with PAW60 (PAW produced from 60 ml deionised water, 60 min of CP treatment, 2.5 cm distance at 1 :3 ratio for conidia and PAW); Negative control indicates conidia treated with sterile deionised wateri :3 ratio (conidia: sterile deionised water). Bars indicate standard errors of the mean (n=6);

Figure 15. Mean percentage germination of C. alienum conidia on half PDA following treatment with PAW generated from different volume of water (at 1 :3 ratio of conidia:PAW) after different storage period. Conidia were treated with PAW for 30 s, 3, 6, 12 and 24 hr, and then plated on half PDA. Percentage germination was counted 12 hr after plating the conidia. A) after 1 d of storage; B) after 3 d of storage; C) after 7 d of storage; and D) after 15 d of storage. Positive control indicates conidia treated with PAW60 (PAW produced from 60 ml deionised water, 60 min of CP treatment, 2.5 cm distance at 1 :3 ratio for conidia and PAW); Negative control indicates conidia treated with sterile deionised wateri :3 ratio (conidia: sterile deionised water). Bars indicate standard errors of the mean (n=9);

Figure 16. pH of different PAW-treated conidia suspensions, in three ratios (1 :1 , 1 :2, and 1 :3) measured up to 24 hr following treatment with PAW. pH of conidia after treatment with A and B) Plasma activated tap water; C and D) Plasma activated deionised water; E and F) Plasma activated distilled water. The controls were 1 :1 of conidia:untreated sterile water (tap, deionised, or distilled water). The initial conidia suspension had pH of 5. Bars indicate standard errors of the mean (n=6);

Figure 17. pH of different PAW. A) PAW produced from 100 ml of water and different cold plasma emission distance; B) PAW produced from 2.5 cm cold plasma emission distance and different volume of water. Bars indicate standard errors of the mean (n=8);

Figure 18. Mean percentage germination of C. alienum conidia on half PDA following exposure to deionised water of varying pH, at three ratios (conidia:pH water; 1 :1 , 1 :2, or 1 :3). Conidia were treated with the pH-adjusted water for 30 s, 3, 6, 12 and 24 hr, and then plated on half PDA. Percentage germination was counted 12 h after plating the conidia. A) pH 1 .8; B) pH 2; C) pH 2.5; D) pH 3. Positive control indicates conidia treated with PAW60 (PAW produced from 60 mL deionised water, 60 min of CP treatment, 2.5 cm distance at 1 :3 ratio for conidia and PAW); Negative control indicates conidia treated with sterile deionised wateri :3 ratio (conidia: sterile deionised water). Bars indicate standard errors of the mean (n=6);

Figure 19. Active nitrogen or nitrogen oxide (NOx= NO3+NO2) concentration of different PAW. A) PAW60 and PAW30 generated from different sources of water, here Dei W, T W, and Dist W indicated deionised water, tap water, and distilled water respectively; B) PAW produced from different cold plasma emission distance; B) PAW produced from different volume of water (and 2.5 cm distance) and stored up to 15 dy. Bars indicate standard errors of the mean (n=4);

Figure 20. Optical emission spectra from both 200 nm and 400 nm fibre optic cables observed during PAW generation. OV= quadruple ionised oxygen, OIV= triple ionised oxygen, Olll = doubly ionised oxygen, O II = singly ionised oxygen, OI= non ionised atomic oxygen, O²⁺= singly ionised molecular oxygen; Arlll= doubly ionised argon, Aril = singly ionised argon, Arl= non ionised atomic argon; NH = nitrogen monohydride N2H2= diazene, N2O5 = dinitrogen pentoxide, NO= nitric oxide; NO2= nitrogen dioxide J = singly ionised molecular nitrogen, Nil = singly ionised atomic nitrogen, Nl = non ionised atomic nitrogen; Cu 111= doubly ionised copper, Cull= singly ionised copper; Cul= non ionised atomic copper; 0111= doubly ionised carbon, Cll=singly ionised carbon, Cl=neutral carbon. CH⁺= carbon-hydrogen positive ion, OH = hydroxide;

Figure 21 . Body rot diseases score. A) score 0 = no external or internal disease spot; B) score 1 = slight fungal growth at stem end point both external and internal spot size less than 10 mm in size or less than 10% area diseased; C) sccore 2= 2 to 5 spots (scattered small spots), total spot size less than 30 mm (both external and internal), moderate dense fungal growth, with presence of conidiomata or 10 to 25% area diseased; D) score 3= large spots, more than 30 mm but less than 50 mm, dense fungal growth, profuse conidiomata or 25 to 50 % area diseased; E) score 4= large spots, more than 50 mm but less than 80 mm, dense fungal growth, profuse conidiomata or 50 to 75 % area diseased and; F) score 5= completely rotted fruit or 100 % area diseased;

Figure 22. Stem end rot score of avocado. A) Stem end rot score 0= no symptoms; B) stem end rot score 4= more than 30 mm spot;

Figure 23. Changes in the firmness of avocado after CP treatment at different ripening stage. Bars indicate standard errors of the mean (n=60);

Figure 24. Number of infected fruit and disease score of body rot symptom at different ripening stage after CP treatment. A) Body rot score at eating ripe stage; B) body rot score at eating ripe +5 days; C) body rot score at eating ripe +10 days; and D) body rot score at eating ripe +15 days. Bars indicate standard errors of the mean (n=60);

Figure 25. Number of infected fruit and disease score of stem end rot symptom at different ripening stage after CP treatment. A) Stem end rot score at eating ripe stage; B) stem end rot score at eating ripe +5 days; C) stem end rot score at eating ripe +10 days; and D) stem end rot score at eating ripe +15 days. Bars indicate standard errors of the mean (n=60);

Figure 26. Changes in length (A), width (B), weight (C), and colour (D) of avocado before and 13 days after PAW treatment. Bars indicate standard errors of the mean (n=8);

Figure 27. Changes in firmness of avocado before and after 13 days PAW treatment. Bars indicate standard errors of the mean (n=8). Here Firmness scale 1 = Hard, 2= Slightly soft and 3= Very soft;

Figure 28. Average number of days before the first appearance of body rot symptoms after PAW treatment of avocado. Bars indicate standard errors of the mean (n=8);

Figure 29. Body rot and stem end rot disease score 13 days after PAW treatment of avocado. A) Body rot score; B) Stem end rot score. Bars indicate standard errors of the mean (n=8);

Figure 30. Reduction in the growth of pure cultures of Botrytis cinema, isolated from strawberry following treatment with cold plasma; and Figure 31 . Spore germination of Botrytis cinerea, isolated from strawberry, following treatment with cold plasma.

Detailed Description of the Preferred Embodiments of the Invention

[0052] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0053] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however be understood to be common general knowledge.

[0054] Manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0055] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0056] The invention described herein may include one or more range of values (e.g. size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. [0057] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0058] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0059] Features of the invention will now be discussed with reference to the following non-limiting description and examples.

[0060] In a general form, the invention relates to a method for the treatment of eukaryotic microorganisms, the method comprising the steps of: contacting a eukaryotic microorganism with cold plasma or cold plasma- treated water, wherein where the eukaryotic microorganism is contacted with cold plasma, the cold plasma source is between 0.5 cm and 20 cm from the eukaryotic microorganism and the eukaryotic microorganism is contacted for between 1 second and 20 minutes; or wherein where the eukaryotic microorganism is contacted with cold plasma- treated water, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 36 hours, such that at least a portion of the eukaryotic microorganisms are inactivated.

[0061 ] In a general form, the invention relates to a method for the treatment of fresh produce, the method comprising the steps of: contacting the fresh produce with cold plasma or cold plasma-treated water, wherein where the fresh produce is contacted with cold plasma, the cold plasma source is between 1 and 20 cm from the fresh produce and the fresh produce is contacted for between 1 and 20 minutes; or wherein where the fresh produce is contacted with cold plasma-treated water, the fresh produce is contacted with the cold plasma-treated water for 1 second to 36 hours, such that at least a portion of the eukaryotic microorganisms on or in the fresh produce are inactivated.

Examples

Preferred Machine

[0062] A Dyne-A-Mite™ HP Surface Treatment Plasma Machine (Enercon, USA) was used in all experiments. This machine produces a gliding arc plasma which is classed as an indirect plasma since the electrodes are separate from the product being treated. The plasma outlet was 6.5 cm in length and 0.9 cm in width and emitted a gliding arc plasma flame of approximately 4.5 cm in length.

Data analysis

[0063] All results were analysed by repeated measures ANOVA (using SPSS software) to determine the statistical difference between the two repeats for each experiment. Where there were no differences between any of the repeated experiments (P>0.005), results were combined and expressed as the mean (of all replicates) for both experiments. Duncan's multiple range test was used to separate the means.

Treatment of fruit with cold plasma

[0064] One hundred untreated fresh avocados {Persea americana variety Hass) were harvested from an orchard 48 hours prior to collection and stored at 4 ^°C. The avocados were washed twice with sterile distilled water and dried with sterile paper towel prior to CP treatment. An area of 8 cm² was marked on every fruit using a felt marker pen, to represent the cold plasma application zone. Six CP exposure times 0, 5, 10, 15, 20, and 25 s and four exposure distances 5, 10, 15, and 20 cm from the CP outlet were tested, to give 24 combinations in total. For each combination, there were four replicate fruit, and all fruit were treated in a fumehood. The plasma flame did not touch the fruit during treatment. After treatment, avocados were transferred to individual sealed transparent plastic boxes (1 L size [Microready CA-CM 650]), and incubated at 25±1 ^°C in the dark for 10 days. This experiment was conducted once. [0065] Colour, length, width, weight, and firmness of each fruit was recorded prior to treatment, and thereafter every 24 h for 8 days. The number of days for fruit to reach the eating ripe stage was recorded. Length and width of each fruit was measured with a slide caliper (Craftright, 150 mm Digital Caliper). Fruit weights were measured with a digital balance (Sartorius Basic B3100P). The peel colour and firmness of the treated zone was rated according to Table 1 .

Table 1 . Avocado colour and firmness scale.

Optimisation of in-vitro cold plasma treatment of mycelia

[0066] A series of experiments were conducted to optimise the duration of CP treatment to reduce colony growth of the C. alienum isolate. In the first experiment, 90 mm half strength Potato Dextrose Agar (PDA [Difco] powder 19.5 g + Agar [Difco] 7.5 g + deionised water 1 L) plates in a laminar flow cabinet were inoculated with a single five mm diameter mycelial disc transferred from the edge of a three day-old fungal culture and immediately treated with CP in a fumehood for one of six time periods 0, 5, 10, 15, 20, 25 s at a distance of 5 cm from the CP outlet. The plates were treated with the lids off. The second experiment was identical, except the plates were treated with the lids on. Both of these experiments were also repeated with 2-day-old fungal cultures. In all experiments, the controls were inoculated, untreated plates. After treatment, all plates were incubated at 25±1 °C in the light and colony growth was measured every 24 hours until the growth of the controls had reached the edge of the plates. Following these experiments, it was observed that the colony growth was not affected by the CP treatment for any of the six treatment times. Therefore, all experiments were repeated, but with treatment times of 0, 30 60, 120, 180, or 360 s. There were five replicate plates per treatment and all experiments were repeated once.

Optimisation of in-vitro cold plasma treatment of conidia

[0067] Conidial suspensions (10 mL) prepared from the PDA cultures were transferred in a laminar flow cabinet to six individual sterile glass crystallising dishes (150 mL) for CP treatment for 0, 30, 60, 120, 180, or 360 s, with a distance of 5 cm between the CP outlet and the base of the crystallising dish. As the temperature of the conidial suspension was observed to increase during treatment, the method was modified by placing each dish in the centre of a plastic container (1 L) containing crushed ice. Within 20 s of treatment, the conidial suspensions were transferred to individual sterile 20 mL glass bottles, sealed and incubated at 25±1 °C in the dark. A 100 μί aliquot of each conidial suspension was transferred in a laminar air flow to the centre of a fresh half strength PDA plate within 60 s and at 3, 6, 12 or 24 hr after treatment. All plates were incubated at 25±1 °C in the light. Germination was counted 12 hr after each plating, so 12, 15, 18, 24, and 36 hr after CP treatment, by assessing a minimum of 100 conidia per plate under a light microscope (Olympus CX31 ) at 10 x magnification. There were two untreated controls, one was a conidial suspension held in the treatment vessel placed on ice for 360 s, and the other was left at room temperature (20±2 °C). There were three replicate plates for each treatment and the experiment was repeated once.

Results

Optimisation of in-vitro cold plasma treatment of mvcelia

[0068] Significant (P = 0.001 ) differences were observed in the colony growth of the freshly-inoculated fungal cultures treated for more than 120 s (Figure 1 ). The colony growth was significantly lower (P = 0.001 ) when the pathogen was exposed to CP with the lid off the Petri-dish compared to the CP treatment with the lid on. With lids off the Petri-dish, the colony growth ceased completely following the 360 s treatment, and for 120 s or longer, colony growth was significantly (P= 0.001 ) reduced compared to the controls (Figure 1 A). With the Petri-dish lids on, only the 360 s CP treatment significantly (P <0.05) reduced colony growth compared to the control (Figure 1 B). [0069] For the 2-day old cultures, there was a significant (P < 0.05) reduction in colony growth compared to the controls, for all treatments of 120 s or longer with lids off (Figure 2A). With lids on, only the 180 s or longer treatments significantly (P < 0.05) reduced the colony growth (Figure 2B).

Optimisation of in-vitro cold plasma treatment of conidia

[0070] CP treatment of the conidial suspensions significantly (P= 0.001 ) reduced the germination of conidia compared to the control. After the 360 s treatment, C. alienum did not germinate at all (Figure 3).

Discussion

[0071 ] Cold plasma treatment of avocado, with a commercially available machine emitting indirect plasma even from the shortest possible distance (5 cm) did not affect any external fruit quality traits measured, or the number of days until the avocado reached the eating ripe stage.

[0072] The present study is the first to report the application of CP to avocado. CP was applied for different distances and durations up to 25 s with a commercially available machine and no negative effects of CP on visible quality traits were observed. With a selected distance, 360 s of CP treatment successfully reduced avocado postharvest disease causing pathogen growth.

[0073] The in vitro cold plasma treatment of Colletotrichum alienum successfully reduced colony growth and conidia germination when treated at a distance of 5 cm.

In-planta pathogenicity test of Colletotrichum isolates

[0074] One hundred fresh avocados {Persea americana variety Hass) were treated with Sportak (a.i. 450 g/L Prochloraz, Bayer CropScience) 15 day prior to harvest and there was no postharvest treatment. The harvested avocados were stored at 4 °C for 3 day (2 day in the grower's store and 1 day at the laboratory).

[0075] The avocados were surface sterilised with 1 % sodium hypochlorite solution for 5 min, rinsed twice with sterile deionised water and air dried in a laminar flow cabinet. The fruit were inoculated by creating a small wound (less than 1 mm deep) with a sterile needle. Within 30 s of wounding, a 5 mm² plug of mycelium from a three-day-old culture of one of the 20 single conidia isolates (grown on half strength PDA) was placed on the wound. After inoculation, each fruit was placed into a one litre plastic box with sterile blotter paper (moistened with sterile deionised water) and a second plastic box was used as a lid. There were four replicate fruit per isolate, and the control consisted of four avocados inoculated with a half strength PDA plug only. The boxes were sealed with masking tape and incubated at 25±1 °C in the dark. Lesion development was measured on the external surface and also under the skin nine days after inoculation, and expressed as the mean lesion diameter. The experiment was repeated once. Five isolates ranging in pathogenicity were selected for further experiments as indicated in Table 2.

WAC number Name of species Pathogenicity level

WAC-13971 Colletotrichum alienum High

WAC-13891 Colletotrichum alienum High

WAC-13972 Colletotrichum alienum Low

WAC-13973 Colletotrichum alienum Low

WAC-13896 Colletotrichum florinae High

WAC = Western Australian Culture Collection

Table 2: Colletotrichum isolates selected for direct cold plasma treatment.

In-vitro cold plasma treatment of selected Colletotrichum isolates

Experiments conducted in an open environment

[0076] Separate experiments were conducted to assess the direct effect of CP on pathogen growth. 90 mm half strength PDA plates were inoculated with a single 5 mm diameter mycelial disc, transferred from the edge of a three-day-old single culture of one of five selected isolates (Table 2), and immediately treated with CP in the fumehood for either 180 s or 360 s. The lids of the PDA plates were removed during treatment, and the plates were treated at a distance of 5 cm from the plasma emission point.

[0077] A second experiment was similar except the plates contained 2-day-old cultures. In both experiments, the controls were untreated cultures. After CP treatment, all cultures were incubated under continuous light at 25±1 °C and the colony growth was measured every 24 hr, as described above. Both experiments had five replicate plates for each treatment and the experiments were repeated once.

Experiments conducted in a sealed environment [0078] The experiments above were replicated in inside a 42 L plastic box covered with a piece of cardboard and weighted with a glass lid (775 cm²). The plasma emission head was inserted via a 7 cm diameter opening and the distance between the plasma emission point and the culture was 20 cm. Growth measurements and replicates were as described above and the experiments were conducted in a fumehood.

Cold plasma treatment of conidia in an open environment

[0079] Conidia suspensions for each of the five isolates were prepared in treatment vessels. Each treatment vessel was placed in the centre of a plastic container (1 L) containing ice and the conidial suspensions were treated in a fumehood with CP for either 180 or 360 s, from a distance of 5 cm from the bottom of the vessel. Within 20 s of treatment the conidial suspensions were transferred to individual 20 imL glass bottles and 100 μΙ_ aliquots of each transferred to fresh half-strength PDA plate as described above. The controls were conidial suspensions held in the treatment vessel on ice for 360 s.

Cold plasma treatment of mixed conidial suspensions in an open environment

[0080] The experiment was identical to that immediately above, except two mixtures of conidia from different isolates were treated. One mixture was prepared by combining WAC-13891 and WAC-13972 (highly and weakly pathogenic C. alienum, respectively) in 1 :1 ratio. A separate mixture was prepared by combining WAC-13891 and WAC- 13896 (highly pathogenic C. alienum and C. florinae, respectively). The controls were untreated conidial suspensions of the individual isolates. This experiment had three replicates for each treatment and was repeated once. Germination was calculated as described above and the was repeated once.

Optical emission spectroscopy (PES)

[0081 ] An AvaSpec-2048-8 Fibre Optic Spectrometer (Avantes, USA) with Avaspec version 8.3 software was used to measure the major excited reactive species during plasma generation. Two fibre optic cables (200 nm and 400 nm) were used to detect the spectra during plasma generation. The fibre optic cables were placed 5 cm underneath the plasma generation point, or 20 cm under the plasma generation point if inside the sealed environment. Five data points were acquired for each cable during CP emission and the average emission spectra for each cable were determined. These were combined to produce the final spectra graph and spectra identified using Plasus Specline (version 2.1 ) software.

Temperature data collection

[0082] The temperature increased during the 360 s CP treatment of the two pathogens in open and sealed environments were assessed on half strength PDA plates. The distance between the CP emission point and bottom of the PDA plate was 5 cm and 20 cm for both open and sealed environment, respectively. One probe was placed adjacent to the CP emission point, and the second just above the surface of the PDA plate and the temperature was monitored every 20 s for up to 360 s.

Cold plasma effect ln-vitro cold plasma treatment of selected Colletotrichum isolates in an open environment

[0083] The colony growth of all freshly inoculated cultures was significantly (P = 0.001 ) reduced following the 360 s CP treatment, compared to the controls (Figure 4). In comparison, the 180 s CP treatment significantly (P <0.05) reduced the colony growth of three isolates of C. alienum WAC-13971 , WAC-13972 and WAC-13973, but the remaining isolate of C. alienum (WAC-13891 ) and the C. florinae (WAC-13896) isolate did not differ from the control (Figure 4).

[0084] Following CP treatment of 2-day old cultures for 360 s, all of the C. alienum isolates had significantly (P < 0.05) reduced colony growth, compared to the controls (Figure 5), but C. florinae was not affected. Treatment for 180 s did not significantly reduce the colony growth of any actively-growing isolates (Figure 5).

In-vitro cold plasma treatment of selected Colletotrichum isolates in sealed environment

[0085] When CP treatment was conducted in a sealed environment for 360 s, the colony growth of all freshly inoculated isolates was significantly (P = 0.001 ) reduced compared to the control (Figure 6).

[0086] Similarly, for 2-day-old active cultures treated in a sealed environment, the colony growth of all isolates of C. alienum was significantly (P = 0.001 ) reduced following the 360 s treatment compared to the control (Figure 7). This was also observed for the 180 s treatment of C. alienum WAC-13891 (strongly pathogenic) and WAC-13973 (weakly pathogenic) but not the other C. alienum isolates or C. florinae.

Cold plasma treatment of conidia in an open environment

[0087] CP treatment of the conidial suspensions significantly (P = 0.001 ) reduced the germination of conidia compared to the control. After the 360 s treatment, C. alienum isolates WAC-13971 and WAC-13973 and C. florinae (WAC- 13896) did not germinate at all, and C. alienum isolates WAC-13891 and WAC-13972 germinated at less than 2 %. The latter two isolates stopped germinating completely within 15 hr of treatment (Figure 8).

[0088] When the conidial suspensions were treated with plasma for 180 s, the initial germination percentage ranged from 60-80% for C. alienum to 20% for C. florinae, but this was reduced significantly (P = 0.001 ) after 15 hr. Conidia stopped germinating 18 hr after the 180 s treatment. No conidia germinated after 15 days of incubation period, following either treatment.

Cold plasma treatment of mixed conidila suspensions in an open environment

[0089] When a mixture of conidia of weakly and highly pathogenic C. alienum, or highly pathogenic C florinae was treated with CP, conidia germination was significantly (P = 0.001 ) reduced compared to the untreated individual conidia of each isolate. After 360 s CP treatment less than 10 % of conidia germinated in the mixture of strong and weakly pathogenic C. alienum. Alternatively, after 180 s of CP treatment, more than 40 % of conidia germinated from the same conidia mixture. For the mixture of C. alienum and C florinae, less than 5 % and 40 % conidia germinated after 360 s and 180 s of CP treatment, respectively. Conidia germination ceased after 15 hr for both 180 s and 360 s CP treatment in both mixtures (Figure 9).

Optical emission spectroscopy

[0090] Six major peaks of different reactive species were identified during CP treatment in an open environment (Figure 10A). The emission spectra were dominated by Argon (Aril) and Nitrogen species (Nl, Nil, N2, NO, N2O2 and N j). There were also strong emissions of atomic oxygen (Ol, Oil, ONI, OIV, OV and Olll). Moreover, C I, C II, Cu I, Cu II and Cu III were also recorded. In a sealed environment, only three major peaks were recorded (Figure 10B) and their intensity was three times higher than the peaks observed in the open environment (Figure 10A and 10B).

Temperature emission during CP treatment

[0091 ] Temperature during CP treatment varied significantly (P=0.001 ) between the open and sealed environments. The initial temperature was 23 °C and in the open environment reached 52 °C within 20 s of CP treatment, peaking at 142 °C after 360 s (Figure 1 1 ). In the sealed environment, the temperature increased slowly to 90 °C after 160 s and peaked at 106 °C after 360 s (Figure 1 1 ).

Discussion

[0092] In this study Colletotrichum alienum and C. florinae colony growth and conidia germination were reduced following CP treatment. To our knowledge this is the first report of either being controlled following application of CP.

[0093] A reduction in colony growth was observed for multiple isolates of Colletotrichum that ranged in pathogenicity, following CP treatment.

[0094] A significant temperature rise was recorded during CP emission in both the open and sealed conditions. CP emission at a distance of 5 cm increased the temperature more when compared to a distance of 20 cm, and this held true even when the CP was delivered in a sealed environment. Therefore, it can be concluded that using a greater CP emission distance within a sealed environment reduces the likelihood of temperature increases during CP treatment, and consequently any detrimental effect of heat on the treated product.

Conclusion

The current investigation was conducted to observe the efficacy of CP for the in vitro control of two Colletrotrichum species. CP significantly reduced both colony growth and conidial germination of multiple isolates of both species.

In vitro treatment of conidia with PAW generated from different water types

[0095] Two volumes (50 and 60 imL) of unsterilised tap water were placed into two individual sterile glass crystallising dishes (150 imL) and treated with cold plasma produced with a Dyne-A-Mite™ HP (Enercon, USA) Surface Treatment Plasma Machine in a fumehood from a distance of 5 cm from the bottom of the dish for either 30 or 60 min, to produce 'PAW30' and 'PAW60'. In a laminar flow, the conidia suspension was mixed with either PAW30 or PAW60 within <60 s of preparation in one of three ratios, 1 :1 , 1 :2 or 1 :3 (conidia :PAW). The mixture was transferred to a 5 ml McCartney bottle and incubated for up to 24 h at 25±1 °C in the dark. The control treatment consisted of a 1 :1 ratio of conidia:tap water. The same experimental procedure was repeated using PAW prepared from unsterilised deionised water and unsterilised distilled water. The entire experiment was repeated once. All subsequent experiments PAW60 was prepared using 60 mL of deionised water treated for 60 min from a distance of 5 cm, and used to treat conidia in a ratio of 1 :3, unless otherwise stated.

In vitro treatment of conidia with PAW generated from different distances

[0096] In this experiment PAW was generated in a fumehood from a distance of 2.5, 5 or 10 cm (from the emission point to water surface) for 60 min and the volume of water was 100 mL. Conidia were mixed with PAW and incubated as described above. There were two controls, a negative control which was 1 :1 conidia:sterile deionised water, and a positive control (PAW60 but generated from 2.5cm). The experiment was repeated once.

In vitro treatment of conidia with PAW generated from different volumes

[0097] Three different volumes of water, 100, 500 or 1000 mL, were treated in a fumehood for 60 min in a glass beaker from a distance of 2.5 cm from the water surface. The two larger volumes were constantly stirred with a magnetic stirrer during preparation, and the pH of the PAW was recorded within 20 s of generation. Conidia were mixed with PAW and incubated as described above. In addition to the 1000 m L treated volume, another volume of 1000 ml was prepared by combining 10 volumes of 100 mL of PAW. The positive and negative controls were as described above. Both experiments were repeated once.

Long term activity of PAW in large volumes

[0098] PAW in volumes of 100, 500 or 1000 mL was prepared as described above and stored in sealed 20 ml polypropylene vials at 25±1 °C in the dark. After 1 , 3, 7 or 15 days an aliquot of each was removed from storage and used to treat conidia in a ratio of 1 :3 as described above. The controls were described as above The experiment was repeated twice.

In vitro treatment of conidia with different pH solutions

[0099] To test the effect of pH on conidia germination, 50 imL of unsterilised deionised water was adjusted to one of four pH levels (1 .8, 2, 2.5, or 3) with HNO3 (70% analytical reagent with density 1 .42 and 15.7 M). In a laminar flow, a 100 μΙ_ conidia suspension was transferred to a five imL glass bottle and then treated with one of the pH solutions in a ratio of either 1 :1 , 1 :2 or 1 :3 (conidia:pH-adjusted water). The controls were as described above. The experiment was repeated once.

Conidia germination assessment

[00100] For assessing germination of conidia, a 100 μΙ_ aliquot of each of the conidiaPAW solutions was plated on to half strength PDA within 30 s of mixing with PAW or pH water, and then again after 3, 6, 12 and 24 hr, and incubated at 25±1 °C in the light. Percentage germination was determined 12 hr after conidia were plated, as previously described above. In every experiment for each treatment, there were three replicates and each replicate was treated with fresh PAW, except the storage experiment where each batch of conidia was treated with PAW after the mentioned storage period. pH and NOx (NO3 + NO2) measurement of PAW

[00101 ] The pH of all solutions was recorded with an Oakton pH 700 Benchtop Meter prior to plasma treatment, within 30 s of PAW generation, and again at 3, 6, 12 and 24 hr after PAW generation. The pH was not measured for the storage experiment because the change of pH was insignificant from 30 s to 24 hr. In addition, NOx concentration (NOx = NO3+NO2) of all PAW was measured within 24 hr of PAW generation or after 3, 7 and 15 d of storage, according to the American Public Health Association standard method 2100 (American Public Health Association, 2012).

Optical emission spectroscopy (PES)

[00102] An AvaSpec-2048-8 Fibre Optic Spectrometer (Avantes, USA) along with Avaspec 8.3 software was used to identify the major excited reactive species during PAW60 generation. Two fibre optic cables (200 nm and 400 nm) were placed 5 mm from the bottom of the glass crystallising dish (contained unsterilised deionised water) to capture the spectra being emitted during PAW generation. Five data points for each cable were acquired and measured as described above.

Data analysis

[00103] The data from long term activity of PAW experiment was analysed through factorial analysis, where 5 treatment was Factor 1 , and 4 storage period was considered as Factor 2. Duncan's multiple range tests was used to separate the means.

Results

In vitro treatment of conidia with PAW generated from different water types

[00104] Both PAW30 and PAW60 significantly reduced the germination of conidia compared to the control (Figure 12). The most effective conidia:PAW ratio was 1 :3.

In vitro treatment of conidia with PAW generated from different distances

[00105] The germination of conidia was significantly (P=0.001 ) reduced after treatment with PAW produced from different distances, however 2.5 cm was the most effective (Figure 13). For PAW produced from 5 and 10 cm, conidial germination was significantly (P= 0.005) lower than the negative control at 15 hr after treatment, and decreased to 10 % by 36 hr.

In vitro treatment of conidia with PAW generated from different volumes

[00106] PAW produced from different volumes of water significantly (P= 0.001 ) reduced the percentage germination of conidia compared to the negative control. However, PAW produced from the smaller volume of water was more effective compared to PAW produced from the larger volume of water. For all PAW, the percentage germination of conidia declined with an increase in exposure time (Figure 14A). Less than 1 % conidia germinated 15 hr after treatment with PAW produced from the smaller volume of water (100 imL). This result was similar to the positive control. Less than 15 % of the conidia germinated 24 hr after treatment with PAW produced from the higher volume of water (500 and 1000 imL), and this was significantly lower than negative control.

The conidial germination was significantly (P= 0.001 ) reduced over negative control when treated with PAW generated from the combination of several small volumes of water (1000 mL comb). After 12 hr of treatment with combined PAW, 80 % of conidia germinated which was significantly higher than positive control. However conidial germination ceased completely after 15 hr of treatment with this combined PAW, and it was similar to the positive control (Figure 14B).

Long term activity of PAW in large volumes

[00107] All three PAW produced from different volumes of water (100, 500 and 1000 mL) significantly (P=0.001 ) reduced the percentage conidial germination compared to the negative control after different storage periods (Figure 15). For the negative control, 88 to 100 % conidia germinated when plated 30 s after preparation. After 1 d of storage, in all PAW treatments and the positive control, up to 80% of conidia germinated 12 hr after treatment. The most effective reduction in germination was observed after treatment of PAW prepared with the 100 mL volume (Figure 15). The conidial germination for this PAW ceased 15 hr after treatment, and this was similar to the positive control. For PAW prepared from the larger volumes conidial germination decreased over time. This trend was observed for all storage periods.

Efficacy of different pH solution on conidia germination reduction in vitro

[00108] Water adjusted to pH 1 .8 and 2 significantly (P = 0.001 ) reduced the germination of conidia compared to the positive control, declining to less than 30 %, 36 hr after treatment. Water at pH 2.5 reduced conidial germination to 55 %, 36 hr after treatment. There was no significant difference observed in conidial germination treated with water at pH 3 even 36 hr after treatment (Figure 18). In contrast, conidia from the positive control stopped germinating 15 hr after treatment . pH measurement of PAW

[00109] The initial pH of the three water sources ranged from 4.6 for distilled water and deionised water to 7.3 for tap water but was reduced to between 1 .8 to 3 after treatment with CP (Figure 16). Addition of the conidial suspension (initial pH 5) to the PAW increased the pH slightly, but the change was insignificant for 24 hr post PAW treatment (Figure 16). The change of initial pH of all PAWs was also insignificant (P>0.05) after 24 hr of incubation (Figure 16). [001 10] Both the distance from CP emission and the volume of deionised water affected the final pH of PAW (Figure 17). The lowest pH of 2 was recorded from PAW produced from 2.5 cm and 100 imL of deionised water. The highest pH of 3.3 was recorded from PAW produced from 10 cm and 1000 imL of deionised water (Figure 17).

The active nitrogen or nitrogen oxide or NOx concentration of PA W

[001 1 1 ] The NOx (MO +NO, ) concentration of all PAW generated from different types of water was significantly (P=0.001 ) higher than the source water. The initial NOx concentrations ranged from 0.00425 mg/L to 0.135 mg/L, which increased to a maximum of 58.5 mg/L in PAW60 derived from tap water (Figure 19). The highest NOx concentration was recorded from tap water and the lowest NOx concentration was recorded from distilled water (Figure 19). Moreover, the NOx concentration of PAW60 was almost double the concentration observed in PAW30 for all three types of water.

[001 12] The NOx concentration was significantly (P= 0.001 ) different between PAW generated from different distances and different volumes of water, and the control. The highest NOx concentration (407.5 mg/L) was recorded from PAW produced from 2.5 cm and 100 ml of water. This was comparable to the positive control (447.5 mg/L). The NOx concentrations of PAW generated from 100 mL and 5 or 10 cm distances were not significantly different to the negative control (Figure 17). PAW produced from 500 or 1000 mL had significantly (P=0.001 ) lower NOx concentration (55.25 and 40.5 mg/L respectively) compared to the PAW produced from 100 mL and the positive control.

[001 13] The NOx concentration changed significantly (P=0.001 ) after the different storage periods (Figure 19). The highest NOx concentration was observed in PAW produced from 100 mL water and stored for 24 hr, whilst the lowest was in the same PAW stored for 15 d.

Optical emission spectroscopy

[001 14] Six major peaks of different reactive species were identified during production of PAW (Figure 20). The emission spectra were dominated by argon and nitrogen species from 357 to 436.69 nm. There were also strong emissions of atomic oxygen recorded.

Discussion [001 15] In the present investigation, it was observed that conidial germination reduced after a certain duration of PAW exposure and germination declined as exposure time to PAW increased.

[001 16] The duration of CP exposure during PAW generation also influenced conidia germination. PAW generated following 60 min of CP exposure was more effective than PAW generated following 30 min exposure, for all three types of water.

[001 17] In the current study, PAW was produced by treating different volumes of water, from varying distances and both factors influenced the efficacy of PAW. Specifically, PAW produced from the shorter distance 2.5 cm was more effective at reducing conidia germination. Similarly, PAW demonstrated greater efficacy when prepared from smaller volumes of water. Indeed, when PAW was produced by combining multiple smaller volumes, it was more effective than the same volume produced in a single batch. However, although PAW produced from the shortest distance and smallest volume of water was most active, others also demonstrated potential.

[001 18] PAW retained its efficacy at least 15 days of storage. The storage temperature was constant (25±1 °C) in the present investigation.

[001 19] In the current study, it was observed that exposure of the conidia to water at acidic pH did not have the same effect as exposure of the conidia to PAW. More than 80% conidia germination in water solutions of pH 3 to 3.5, yet less than 20 % germinated in PAW at the same pH. Further, up to 20 % conidia germinated in extremely acidic solutions (pH 1 .8), yet in PAW at the same pH none germinated. Although pH decreases during PAW generation, it does not appear to be the actual mechanism for reducing conidial germination.

[00120] In the present investigation, significant NOx production was recorded after PAW generation and over different storage period.

Conclusion

[00121 ] It was demonstrated that PAW can successfully reduce or inhibit the conidia germination of an avocado postharvest anthracnose pathogen, C. alienum.

Source of fruit [00122] Untreated, unripe fresh avocados {Persea americana variety Hass) were harvested 48 hours prior to collection and stored at 4 °C .

Effect of CP treatment on avocado firmness at different ripening stages of the avocado

[00123] Five avocados were placed in a 42 L plastic box, covered with a lid comprised of 6 mm thick cardboard and weighted with a glass plate, and sealed with plastic adhesive tape. The plasma head was inserted through a 90 mm diameter opening in the lid, the box was placed in a fume hood, and plasma applied for either 30, 60 or 300 s. The distance between the CP emission point to the base of the box was 20 cm. In order to check for systemic infection of the fruit after treatment, a 2 by 5 cm oblong mark was drawn on each avocado. During treatment, the marked areas of five avocados were directly exposed to the CP emission. Immediately after treatment, the avocados were transferred to cardboard packing trays and stored at 25 ± 1 °C in the dark. The firmness of all the treated fruit was assessed at four different stages and included the eating ripe stage, plus 5, 10 and 15 days after the eating ripe stage. There were 15 pieces of fruit per treatment and two controls. The positive control was a Prochloraz treatment at 0.55 imL/L (450 g ai /L prochloraz) for 5 min followed by air drying, and the negative control was untreated fruit. This experiment was repeated three times.

Fruit characteristics

[00124] Fruit firmness was assessed at each of the four ripeness stages with a penetrometer (Firmness gauge: Handpi model GY-2, Range: 0.5-4kg/cm² (x10 5pa), Head Dim: SR 3.5mm, Resolution: 0.02). For each fruit, five firmness measurements were taken and then the average calculated.

Postharvest body rot and stem end rot data collection

[00125] The number of pieces of fruit exhibiting postharvest body rot or stem end rot symptoms were counted at each of the four ripeness stages. The skin of each piece of fruit was peeled from the 2 x 5 cm marked area to assess the size of the body rot disease lesion with a digital slide caliper (Craftright, 150mm Digital Caliper). The remaining skin from the whole fruit was then removed to measure the extent of body rot disease over the entire fruit. All the body rot data was scored according to a 0 to 5 scale (Figure 21 ). The stem end rot lesions were assessed after cutting the fruit in half. The stem end rot disease symptom was recorded according to a 0 to 4 scale : 0= no symptoms, 1 = 1 -10 mm spot, 2= 1 1 -20 mm spot, 3= 21 - 30 mm spot, 4= more than 30 mm spot (Figure 22).

Results

Effect of CP treatment on avocado firmness at different ripening stage of avocado

[00126] The fruit firmness was significantly affected (P = 0.001 ) after CP treatment. The highest firmness was recorded from the 300 s CP treated fruit at the eating ripe stage (Figure 23). There was no significant difference observed between 30 s and 60 s CP treated fruit compared to the 300 s CP treated fruit although all of them were higher in firmness than both the negative and positive controls. The lowest firmness recorded was from the positive control (Figure 23). At eating ripe + 1 0 days the firmness for all fruit ranged from 0.63 to 0.51 X 10⁵ Pa. The highest firmness was recorded from positive control treated fruit, which was significantly (P= 0.008) higher than CP treated and negative control fruit. There was no significant difference in firmness recorded in any treated fruit at eating ripe + 5 days (P> 0.05), and eating ripe+ 15 days (P > 0.05) after CP treatment (Figure 23).

Number of infected fruit and disease score of body rot and stem end rot symptoms after CP treatment at different ripening stages

[00127] No significant (P > 0.05) difference was observed in number of infected fruit, or for postharvest disease scores (body rot and stem end rot) after CP treatment, at any of the ripeness stages (Figure 24 and 25). At the eating ripe stage, the number of infected fruit and scores for both diseases were the lowest. The highest scores (5 for body rot and 3 for stem end rot) for both diseases were recorded at the eating ripe +15 days when almost all treated fruit showed both disease symptoms (Figure 24 and 25).

Discussion

[00128] CP treatment affected the fruit firmness differently depending on the ripening stage, but did not affect the postharvest disease development in avocado. Fruit quality parameters are important for consumer satisfaction postharvest. For example, colour is an obvious selection parameter for consumers, and plays a key role in food choice, food preference and acceptability, and may also influence taste thresholds, sweetness perception and pleasantness. In the present experiment, it was observed that avocados were more firm at the eating ripe stage after CP treatment. The other quality parameters did not change compared to the controls.

PA W treatment of avocado

[00129] For PAW treatment, two litres of plasma activated water was prepared from 100 mL deionised water following 60 min of CP treatmentas described previously. Twenty-four avocados were surface sterilised and dried with a sterile paper towel as described above. Air dried fruit were individually dipped in 250 mL of plasma activated water contained in a 500 mL glass beaker for 5, 10 or 15 min. There were three control treatments; surface sterilised fruit dipped in sterile distilled water for 5 or 15 min (to assess the dipping effect on fruit) and untreated avocados. The avocados were allowed to dry at room temperature. Immediately after drying, all the avocados were incubated at 25±2 °C in the dark in a sealed box for 13 days. There was four replicate fruit per treatment. The experiment was repeated once. The appearance of postharvest body rot symptoms and the area of fruit affected by body rot and stem end rot disease were recorded 13 days after plasma treatment,. Different fruit characteristics such as colour, length, width, and weight of each fruit were also recorded.

Results

[00130] There was no significant (P= > 0.005) change observed in length, width and weight after PAW treatment of fruit compared to the three controls (Figure 26). The colour of all fruit changed from 2 before treatment to 4, 13 days after treatment. No significant difference was observed in fruit colour (Figure 26). The firmness of 10 and 15 min of PAW treated fruit increased from 1 to 1 .5, whereas for other treatments the fruit firmness increased from 1 to 2.5 after 13 days of treatment (firmness scale 1 = Hard, 2= Slightly soft and 3= Very soft) (Figure 27). Although the change was insignificant between all treatments.

First appearance of postharvest disease symptom and disease scale after PAW treatment of avocado

[00131 ] PAW treatment for 15 min significantly (P=0.012) increased the average number of days before the first disease symptoms appeared on fruit compared to controls. Briefly, the three control treatments took between 8-10 days before first symptoms appeared whilst symptoms first appeared after 12-14 days in the three PAW treatments (Figure 28).

[00132] The scale of both body rot and stem end rot symptoms were significantly reduced after PAW treatment of avocado compared to the control (Figure 29). Both disease scales were less than 1 for 15 min PAW treated fruit, 13 days after treatment. The disease scales remained below 2 for the 5 min PAW treated avocado (Figure 29). By contrast, for body rot and stem end rot, the diseases scale increased up to 2 and 4 respectively in the controls.

Plasma effect on Botrytis cinema

[00133] Cold plasma has been shown to significantly reduce the growth of pure cultures of Botrytis cinema, isolated from strawberry (Figure 30), and prevent the spores from germinating within 18 hours of treatment (Figure 31 ).

Claims

1 . A method for the treatment of fresh produce, the method comprising the steps of: contacting the fresh produce with cold plasma or cold plasma-treated water, wherein where the fresh produce is contacted with cold plasma, the cold plasma source is between 1 and 20 cm from the fresh produce and the fresh produce is contacted for between 1 and 20 minutes; or wherein where the fresh produce is contacted with cold plasma-treated water, the fresh produce is contacted with the cold plasma-treated water for 1 second to 36 hours, such that at least a portion of the eukaryotic microorganisms on or in the fresh produce are inactivated.

2. A method for the treatment of fresh produce according to claim 1 , wherein the cold plasma source is about 2.5 cm from the fresh produce.

3. A method for the treatment of fresh produce according to claim 1 or claim 2, wherein the fresh produce is contacted with the cold plasma for between 1 and 10 minutes.

4. A method for the treatment of fresh produce according to claim 1 or claim 2, wherein the fresh produce is contacted with the cold plasma for less than 1 minute.

5. . A method for the treatment of fresh produce according to any one of the preceding claims, wherein the fresh produce is contacted with cold plasma, the temperature of the surface of the fresh produce is between 0 and 120 °C.

6. . A method for the treatment of fresh produce according to any one of the preceding claims, wherein the fresh produce is contacted with cold plasma, the temperature of the surface of the fresh produce is less than 50 °C.

7. A method for the treatment of fresh produce according to claim 1 , wherein the fresh produce is contacted with the cold plasma-treated water for 10 seconds to 36 hours.

8. A method for the treatment of fresh produce according to claim 1 , wherein the fresh produce is contacted with the cold plasma-treated water for 1 hour to 24 hours.

9. A method for the treatment of fresh produce according to any one of claims 1 or 7 to 8, wherein the fresh produce is contacted by the cold plasma-treated water by immersing the fresh produce in the cold plasma-treated water or by spraying the water on the fresh produce.

10. A method for the treatment of fresh produce according to claim 9, wherein the fresh produce is immersed in the cold plasma-treated water for between 1 and 15 minutes.

1 1 . A method for the treatment of fresh produce according to any one of claims 1 or 7 to 9, wherein the temperature of the fresh produce is maintained between 10 and 35 °C while in contact with the cold plasma-treated water.

12. A method for the treatment of fresh produce according to any one of the preceding claims, wherein the fresh produce are selected from the group comprising fruits, vegetable, grains, nuts and seeds.

13. A method for the treatment of fresh produce according to any one of the preceding claims, wherein the fresh produce include citrus fruit, stone fruit, pome fruit, tropical fruit, melons, leafy vegetables, roots and tubers.

14. A method for the treatment of fresh produce according to any one of the preceding claims, wherein the fresh produce include avocados and strawberries.

15. A method for the treatment of fresh produce according to any one of the preceding claims, wherein the fresh produce are treated post-harvest.

16. A method for the treatment of eukaryotic microorganisms, the method comprising the steps of: contacting a eukaryotic microorganism with cold plasma or cold plasma- treated water, wherein where the eukaryotic microorganism is contacted with cold plasma, the cold plasma source is between 0.5 cm and 20 cm from the eukaryotic microorganism and the eukaryotic microorganism is contacted for between 1 second and 20 minutes; or wherein where the eukaryotic microorganism is contacted with cold plasma- treated water, the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 second to 36 hours, such that at least a portion of the eukaryotic microorganisms are inactivated.

17. A method for the treatment of fresh produce according to claim 16, wherein the eukaryotic microorganism is contacted with the cold plasma for less than 1 minute.

18. A method for the treatment of eukaryotic microorganisms according to claim 16 or claim 17, wherein the eukaryotic microorganism is contacted with cold plasma, the temperature of the surface of the eukaryotic microorganism is between 0 and 120 °C.

19. .A method for the treatment of eukaryotic microorganisms according to any one of claims 16 to 18, wherein the eukaryotic microorganism is contacted with cold plasma, the temperature of the surface of the fresh produce is less than 50 °C.

20. A method for the treatment of eukaryotic microorganisms according to claim 16, wherein the eukaryotic microorganism is contacted with the cold plasma-treated water for 10 seconds to 36 hours.

21 . A method for the treatment of eukaryotic microorganisms according to claim 16, wherein the eukaryotic microorganism is contacted with the cold plasma-treated water for 1 hour to 24 hours.

22. A method for the treatment of eukaryotic microorganisms according to any one of claims 16 to 21 , wherein the eukaryotic microorganism is a fungi.