4. IN VITRO AND IN VIVO DISINFECTION AND BIOCONTROL TREATMENTS
4.3 Materials and Methods
4.3.1 Sample fruit production
Nagami (Fortunella margarita) was identified as the sample fruit, being the main kumquat variety exported from South Africa. Kumquat fruit samples were obtained from the Letsitele region, just outside Tzaneen, and Levubu near the Kruger National Park, Limpopo Province, South Africa. The kumquat orchards are registered with the Department of Agriculture, Forestry and Fisheries and are clear of citrus black spot and fruit fly. Rooister Boerdery and Premier Fruit Exports (Pty) Ltd provided the necessary samples for testing. After harvest commercially mature kumquat fruit were couriered overnight to the UKZN laboratories. This was to ensure minimal fruit exposure to temperature fluctuations between harvesting and sampling. A total of 3 kg were used for this experiment.
4.3.2 Fungal cultures
The fungal cultures used in this study were P. digitatum and P. italicum isolated from citrus. Pure cultures of P. digitatum and P. italicum were prepared by and purchased from the Agricultural Research Council - Plant Protection Research Institute, Pretoria, South Africa and delivered in sealed potato dextrose agar (PDA) Petri dishes.
4.3.3 Preparation of inoculum
All laboratory utensils and apparatus were sterilized for 15 minutes at 121C using a
Africa). Procedures were carried out aseptically next to a flame within a laminar flow unit. Each of the Petri dishes containing the fungal culture were flooded with 20 mL of sterile distilled water (Smilanick et al., 1999). The conidia were then loosened with the aid of a laboratory glass rod. The conidial suspensions were passed through muslin and collected in a sterilized glass jar with Tween 20 (Uni Laboratory, South Africa) added as a surfactant (0.05 mL per 50 mL). Conidia suspension concentrations were quantified using a Neubauer Improved Haemocytometer (Hirschmann, Eberstadt, Germany) and then diluted to the desired concentration of 1 104 conidia.mL-1 using sterilized distilled water (Abraham et al., 2010).
4.3.4 Treatment preparation
The treatments for this study included two disinfection treatments of anolyte water and chlorinated water and a yeast biocontrol agent, which was a strain of Candida fermentati (B13). The treatments were as follows:
1. Anolyte water at a concentration of 100 mg.kg-1 (A).
2. Chlorinated water (calcium hypochlorite) at a concentration of 100 mg.kg-1 (B).
3. C. fermentati yeast isolate - B13 biocontrol agent (C).
4. Anolyte water and B13 (D).
5. Chlorinated water and B13 (E).
6. Tap water (F).
Commercially available anolyte water was obtained from Radical Waters (Johannesburg, South Africa) delivered in plastic containers to avoid loss of the ionized properties of the solution. The 100 mg.kg-1 chlorinated water was prepared by adding 22.06 g of calcium hypochlorite granules (Frexus CH, Arch Chemicals, Bloemfontein, South Africa) per 100 litres of tap water. This quantity was adjusted to 10 litres for this experiment. The presence of freely available chlorine in the tap water is discounted as negligible at < 5 mg.kg-1. The freely available chlorine concentrations and pH of the treatment solutions was measured using Hydrion chlorine test strips and a Hydrion pH and sanitizer test kit (MicroEssential Laboratory, Inc., Brooklyn, USA), respectively.
Yeast B13, a strain of C. fermentati was supplied by Plant Health Products (Pty) Ltd (Nottingham Road, South Africa), on a grain substrate and packaged in a porous fabric.
The recommended concentration of B13 is 100 g per 100 litres of warm water (25-27°C), which was adjusted to accommodate 10 litres of water for this experiment.
4.3.5 Sample preparation
Untreated kumquat fruit were inspected based on uniformity of size, colour and damage (Hong et al., 2007). Fruit that showed signs of damage or deformity were discarded. The fruit were then thoroughly rinsed in a plastic strainer under running tap water to remove any dirt, debris or soil prior to treatments. After rinsing the fruit were dried using laboratory paper towels. The fruit were sorted in to 6 batches of 18 fruit and labelled at the base of the fruit using a white marker. Of these batches, 3 batches were inoculated with P. digitatum and the remaining 3 batches were inoculated with P. italicum.
4.3.6 In vitro experiment
Rose Bengal (with chloramphenicol) agar (Oxoid, Basingstoke, England) was used to culture the fungi. The agar was prepared by adding 16 g per 500 mL distilled water and autoclaved (121°C/ 15 minutes). Three replications per treatment were performed for each fungal inoculum. Diffusion disks of 0.65 mm diameter were prepared from Whatman® filter paper and autoclaved. 0.1 mL of each of the prepared inoculum was transferred aseptically onto the agar and evenly spread. Forceps were aseptically used to transfer the diffusion disks into the required treatment solution/s before being evenly positioned onto the plates to form a triangular shape. Three disks per plate were used. The plates were incubated at 25°C for five days. The diameters for the zones of growth inhibition around the disks were measured in mm (Espina et al., 2011).
4.3.7 In vivo experiment
4.3.7.1 Inoculation of kumquat fruit
Three replications, each comprising of three fruit per treatment, were performed for each fungal culture. A portion of each of the sample kumquat surfaces, near the pedicel, were disinfected with 70% ethanol. This area was selected for uniformity and for easy detection
mm deep. Care was taken to avoid piercing the fruit albedo (Abraham et al., 2010). The wounds were allowed to dry before the fruit were divided into two batches. Each fruit of the first batch was inoculated with 10 uL of P. digitatum inoculant at a concentration of 1 104 conidia.mL-1. The second batch of kumquat fruit were inoculated with P. italicum at the same concentration. The fruit were stored at ambient conditions for two weeks to observe mould growth each day.
4.3.7.2 Isolation of fungi from infected fruits
After a period of 14 days the microorganisms on the surface of the fruits were isolated on rose Bengal agar following the method used by Sivakumar et al. (2012). The plates were then incubated at 28°C and mould formation was observed after 3 days.