The experiment was conducted at Council of Scientific and Industrial Research – Oil Palm Research Institute (OPRI), Plant Pathology Laboratory, Kusi. Kusi is located in the Dekyeambour District in the Eastern Region of Ghana. The rainfall pattern in Kusi is bimodal with an annual rainfall of 1,621.2 mm and temperature ranging from 28.9 to 35.4 oC (Oil Palm Research Institute Meteorological Weather Station, 2020).
Collection of diseased samples, isolation and purification of the pathogens
Symptomatic disease samples were collected from the OPRI nursery, kept in tablet envelopes, labeled and brought to the laboratory. Diseased leaves were washed under running tap water for one minute, they were surface sterilized in 10 % (v/v) sodium hypochlorite solution and washed in two changes of sterile distilled water. They were blotted dry and plated on chloramphenicol amended water agar. Emerging fungal growth from peripheries of cultured leaves was sub-cultured onto a half strength cPDA and pure cultures were established for purification purposes.
Ten leaves from healthy oil palm seedling leaves were collected from the OPRI nursery, washed under running tap water, surface sterilized with 70 % (v/v) ethanol, washed in two changes of sterile distilled water. The leaves were wounded with an inoculating needle and inoculated with the Curvularia isolate (KOP-21-5) and Pestalotiopsis isolate (KOP-21-20) separately. Five leaves were inoculated with each pathogen to prove Koch’s postulates.
Media preparation and fungicide amendments
Two percent (w/v) of 250 ml water agar was prepared and amended with 125 mg of chloramphenicol for isolation of disease pathogens. 39 g of Oxoid potato dextrose agar (PDA) was weighed (using Mettler PM 600 electronic balance) into a medium bottle, one litre distilled water was added for the bioassay of the pathogens. 100 ml of PDA was measured separately into seven 250 ml Erlenmeyer flasks and sterilized at 121oC, 15 psi for 15 minutes in a Tuttnauer Autoclave. Upon cooling, the medium was adjusted to the required volume and amended with different concentrations of wood vinegar (Control (0), 0.67 %, 1.34 %, 2.01%, 2.68 % and 3.35 % v/v) and the manufacturer’s recommended rate of carbendazim (0.1% v/v).
In vitro assay of foliar fertilizer against the pathogens
Food poisoning technique23 was employed to screen the organic product against pathogens. Five mm of actively growing mycelial plug of a seven-day old Curvularia species (KOP-21-5) and Pestalotiopsis species (KOP-21-20) were centrally placed inversely on each plate medium (five plates per concentration) and incubated at 31 ± 1 oC in a humidified transparent container under diffused sunlight during the day and darkness during the night (Five plates per treatment). Colony diameters were measured daily from the reverse side of plates with a ruler (average of two diagonal measurements per plate). Sclerotia production was scored qualitatively from day 7 to day 14 after incubation. Percentage inhibition was calculated using the formula by Chaurasia et al.24 expressed as:
I = Inhibition percentage
C = Average growth of control plate
T = Average growth of fungicide treated plate
Also, average daily growth rate was calculated with the formula below;
A = Average colony diameter for current day
B = Average colony diameter for previous day
Effect of wood vinegar on different developmental stages of Curvularia and Pestalotiopsis species
After colony diameters were measured for the respective pathogens, six mycelial plugs (5 mm) of Curvularia species per plate (three plates per concentration) were excised along a radius centrally to the periphery of the culture25. Acervuli produced by Pestalotiopsis species after 10 days of culturing were harvested from three plates per concentration into 2 ml of sterilized distilled water in Bijoux bottle. The mycelial plugs of Curvularia were teased with a sterilized inoculating needle and shaken for two minutes on a Heidolph REAX 2000 vortex. 20µl of spores were pipetted onto Weber Scientific International haemocytometer to determine spore count of both pathogens. Spore production was expressed as the number of spores per mm2 of colony area.
Spore germination was determined using a Riddell mount26. Three agar blocks per concentration (0, 0 .67, 1.34, 2.01, 2.68, 3.35 and 0.1 Carbendazim) were used. 60 µl of spore suspension prepared from unamended PDA plates were spread at different locations on each setup. Germination of spore was measured after incubating the setup for 6 hours at room temperature in the dark by examining 30 and 100 spores for Curvularia and Pestalotiopsis species, respectively at 100X magnification of an Amscope Compound Microscope (T490B). A spore was scored as germinated if the germ tube had reached at least the full length of the spore (Fig. 5).
Completely Randomized Design (CRD) was used for the in-vitro assay. Both transformed (log transformation) and untransformed data were analyzed using GenStat statistical package 12th edition and the means were compared with Fisher’s protected Highest Significant Differences at 1 %.