4.3.1 Experimental site and sources of pathogens and plant aqueous extracts
The experiment was carried out at the University of Zimbabwe, Crop Science Department pathology laboratory as an in-vitro study. Pure cultures of fungal strains, R. solani, P. ultimum, P.
infestans, and bacterial strains of P. atrosepticum, P. carotovorum subsp. brasiliensis and D.
dadantii were obtained from the pathology laboratory. Fresh disease free Moringa leaves and the disease-free seeds were obtained from the Forestry Commission Head Office in Harare, Zimbabwe.
The Moringa leaf, bark and seed aqueous extracts were analyzed using the GC/MS method to identify the bioactive and phytochemical compounds of the Zimbabwean Moringa accessions as described by Dhen et al. (2014). The analysis was carried out at the Central Analytical Facilities, Stellenbosch University in the Mass spectrometry Unit, at Matieland, South Africa. Three different solvents were used for the plant extract phytochemical analysis: Dichloromethane (DCM), Methanol (MeOH) and Solid Phase Micro Extraction (SPME) (Appendix A).
51 4.3.2 Experimental design and treatments
The experiment was a 2 x 4 factorial experiment laid out in Randomized Block Design with 3 replications. Factor A was Moringa plant aqueous extracts at two levels: a) leaf (MLE) and b) seed (MSE); Factor B were the Moringa aqueous concentration levels at four levels: a) 0%, b) 5%, c) 10%, and d) 15%. The test subjects were the pathogens at six levels: fungi with three levels namely a) P. ultimum, b) R. solani and c) P. infestans, and bacterial also at three levels namely a) Pectobacterium carotovorum subsp. brasiliensis, b) P. atrosepticum and c) D. dadantii. At 0%
aqueous concentration level, distilled water only was used. The positive control factor of copper oxychloride was set up separately to avoid and prevent any chance of cross contamination of the Moringa treatments during the duration of trial. Contamination of any kind would have introduced biases into the study.
4.3.3 Media preparation and sub-culturing
Media was prepared by mixing 39 g nutrient agar (NA) and 54 g Potato dextrose agar (PDA) to 1000 ml distilled water, separately in sterilized conical flasks. P. infestans is hard to culture on the general standard media, hence the need to modify the quantities of media used in this study by slightly increasing from the standardized levels (Sopee et al., 2012). The modification to the quantities of distilled water used was to improve spread, growth and sporulation of the test organism and the dissolution of the Moringa aqueous extracts (Sabat and Gupta, 2009). The media was later autoclaved at 121oC and 15psi for 20 minutes and was left to cool before pouring into 9 cm petri dishes. Pouring was done under aseptic conditions using the lamina flow cabinet and 10 ml of media was poured in each petri-dish and stored for future use. The pathogen culture were sourced from the Crop Science Pathology laboratory, the department provided pure pathogens cultures in quantities as per calculations in consultation with the department.
Bacterial strains were sub-cultured by streaking in nutrient agar (NA) and fungal strains by cutting a 5 mm square disc from pure culture of the strain using a new, sterilized stainless steel surgical blade and placed upside down in PDA and incubated at 35oC and 25oC, respectively.
52 4.3.4 Preparation of the Moringa aqueous extracts
The Moringa leaves and seeds were both first thoroughly washed with distilled water to remove any dust, dirt and debris from their surfaces and allowed to air dry at room temperature under dark conditions for seven days. After drying, both the leaves and seeds were ground to a fine powder using mortar and pestle as described by Suleiman and Emua (2009). The aqueous extracts were then prepared by separately mixing ground seed and leaf powders with distilled water. Leaf and seed powder weighing 5, 10 and 15 grams respectively for each aqueous extract were placed in sterilized beakers and 100 ml of distilled water was added to these beakers to make 5%, 10% and 15% aqueous extracts respectively as described by (Hussain et al., 2011; Hossain et al., 2012).
The mixtures were then stirred continuously for 30 minutes using a sterile glass rod and allowed to stand at room temperature for 24 hours. After 24 hours, the aqueous extracts were separately filtered through a 3 layered cheesecloth and finally, through a sterile Whatman number 1 filter paper placed in a funnel as described by Zahir et al., (2009); Preethi et al., (2010); Chollom, (2012). The aqueous extracts were further filtered through 45 micrometer Nitrocellulose micro- filters for further purification to avoid contamination and to achieve a more purified aqueous solution.
The three concentrations for each plant part and one control (distilled water) were then mixed with prepared PDA and NA in separate sterile petri dishes and later inoculated with fungal and bacterial pathogens respectively. 1ml of Moringa leaf and seed aqueous extract for each concentration level (5%, 10% and 15%), were added into each petri-dish containing molten potato dextrose agar (PDA) and nutrient agar (NA). Bacterial pathogen inoculation was done by streaking each of the different strains of bacteria individually onto the NA media where a 10mm line had been drawn at the center base of the petri dish thus streaking was along the drawn line. Fungal strains were inoculated by cutting a square piece from a pure culture and placing it upside down in the PDA.
The plates were later incubated at 25oC and 35oC for fungi and bacteria respectively after which they were examined daily for the presence or absence of the fungal or bacterial growth for 7 days.
53 4.3.5 Pathogen growth measurements and data analysis
The data required to ascertain and monitor pathogen growth were obtained by measuring the width of the colony growth upon the streaked bacteria line in the petri-dish for the bacteria pathogens, and the diameter of the growth of fungal pathogens. Data was collected daily for seven days. The measurements for the fungal growth on diameter were inclusive of five-millimeter disc used during plating. The growth data for the fungal and bacterial pathogens was then analyzed using Genstat Version 14 statistical analysis package and the means were separated using the Fishers’ Protected least significant difference (LSD) at 5% significant level.