Collection and isolation of associated fungi from rotted yam tuber samples
Rotted tuber samples of D. rotundata were collected from different locations across three agroecological zones (AEZ): Humid rainforest, Derived savanna and southern Guinea savanna. Fifteen tubers showing rot symptoms were collected from fourteen locations across the three AEZ. A total of 210 rotten tubers were collected and evaluated for fungal rot pathogens. Each rotten sample was washed in running tap water and cut to expose the fresh necrotic tissues. From the areas in advance of the necrosis, tissue pieces were surface-sterilized with 10% sodium hypochlorite for 2 minutes, and rinsed in five changes of sterile distilled water (SDW) before plating on potato dextrose agar (PDA). The inoculated plates were incubated in the dark in a Gallenkamp incubator operating at a temperature of 28°C for 3 to 4 days and examined for fungal growth from the tissue pieces. Identification of the isolates was carried out using standard procedure (Barnett and Hunter 1998).
Preparation of inoculum
Pure cultures of the pathogens were subcultured from different stock cultures. These were placed on PDA for 7–10 days depending on the pathogen, to allow full sporulation. This was then scooped out into a Warring blender containing one litre of SDW; a drop of Tween 20 (Polyoxyethylene sorbitan mono-oleat) detergent was added to aid the dislodgment of the spores from the medium. The spores were then strained off the mycelia fragments using a double layer of cheese cloth placed inside a sterile funnel. An aliquot of 0.1 mL spore concentration was then placed on an hemacytometer slide and the spores were quantified under a compound microscope. The final spore count and concentration was determined by using this method:
Where: C1 = Initial inoculum concentration, V1 = Initial volume of water used in streaking the culture plate
C2 = Final inoculum concentration desired, V2 = Final volume of water added to obtain desired concentration.
Preparation of cold water extracts
Fresh Oryza sativa husks were washed thoroughly under running tap water and air-dried at a temperature of 28°C for 7–10 days until they became crisp. The dried plant husks were pulverized by blending using a high speed blender (Waring Commercial, Springfield, MO, USA) to form a fine powder while the bark of Quercus phillyraeodes (Oak plant) was dried in a Gallenkamp oven at 80°C for two days and crushed to a coarse powder in a mortar. Ultraviolet light (UV) was used to surface-sterilize the botanicals to prevent fungal and bacterial contaminants. Cold water extract was prepared by adding the powder to 100 mL of sterile distilled water in a 250- mL beaker. This was stirred vigorously and allowed to stand for 24 hours before filtering the extract through folds of sterile cheese cloth. Five different extract concentrations were prepared by blending 0.5 g, 1.0 g, 1.5 g, 2.5 g, and 3.5 g of each plant part in 100 mL of sterile distilled water to produce 0.5%, 1.0%, 1.5%, 2.5%, and 3.5% extract concentrations, respectively.
Measurement of mycelial growth inhibition
This involved creating a four equidistant section on each Petri dish by drawing two perpendicular lines at the reverse bottom of the plate, the point of intersection indicating the center of the plate. This was done before dispensing PDA into each of the plates. An aliquot of 1 mL each of the extracts was separately introduced into the Petri dish containing 15mL PDA. A 5-mm cork borer was used to inoculate a disc of the pathogen culture and placed on the medium containing extract just at the point of intersection of the two previously drawn lines at the bottom of the Petri dish in three replicates. Control experiments were set up without the addition of any plant material. Inhibitory effect of the extract was expressed as percentage inhibition and calculated using the formula:
Where: XC = Average diameter of control
YT = Average diameter of fungal colony with treatment
Determination of the efficacy of extracts for control of tuber rot
The experiment was a 2 × 3 × 6 factorial in a randomized complete block design with three replicates. Both extracts that had earlier proved effective in the in vitro inhibition of the pathogens were used for the trials against the virulent fungal pathogens. Clean, healthy tubers of yam genotype TDr 95–18544 were washed under running tap water to remove adhering soil and surface-borne microflora. Eighteen tubers were inoculated per pathogen with three replicates in two batches. The first batch of nine tubers consisting of three sets of tubers in each treatment, were separately scratched, wounded, and bored, respectively. The tubers were inoculated with the test pathogens, the surface area treated with 1 ml of the extract and then exposed. Similarly, the second batch of nine tubers was scratched, wounded, bored, prior to inoculation and treated with the extract and covered with black polyethylene sheets. For scratched treatments, the tuber periderm was removed with a scapel up to 0.1 cm; wounded tubers were cut to a depth of 0.5 cm. Bored treatments were inoculated to a depth of 1 cm, using a 5-mm cork borer. The tubers were inoculated with the test pathogens 48 h before the application of the extracts and stored at room temperature (28 ± 2°C) in the yam barn for 6 months. Tubers were inoculated with a spore concentration of 106 conidia/mL of the test pathogen. Control tubers were scratched, bored, wounded, and dipped in SDW and inoculated with test pathogen only. Percent rot inhibition was determined using the method:
Phytochemical screening of plant extracts
Fresh samples of O. sativa husk and Q. phillyraeoides bark extracts were prepared as previously described and were concentrated using a rotary evaporator. Chemical tests were carried out on the extracts for the qualitative determination of phytochemical constituents using standard procedures (Sofowora 1993). Mayer’s and Draggendoff’s reagents were used for test and confirmation of the presence of alkaloids in the extracts. The formation of a cream colour and reddish brown precipitate with Mayer’s and Draggendoff’s reagent respectively was regarded as the confirmatory test for the presence of alkaloids. The amount of total phenolics in extracts was determined with the Folin-Ciocalteu reagent. Gallic acid was used as a standard and the total phenolics were expressed as mg/g gallic acid equivalents (GAE). The formation of blue colour upon reaction with Folin-Ciocalteu reagent was the confirmatory test for the presence of phenols in the plant extract samples. Similarly, the formation of a red precipitate when the extract was boiled with 1% aqueous hydrochloric acid indicated the presence of phlobatannins. Other bioactive constituents such as saponins, tanins, flavonoids, steroids, terpenoides, anthraquinones etc. were also determined using standard procedures.
All numerical data were statistically analysed using generalized linear model (GLM) of SAS. Means were separated using Least Significant Difference Test (LSD) and standard error was determined where applicable at 5% level of significance.