Therefore, this study was designed to evaluate the physico-chemical properties and growth response of
Zea mays
in
mycoremediated crude oil polluted soil using
Lentinus squarrosulus.
2. MATERIALS AND METHODS
2.1 Collection of materials: Soil samples used for this experiment were collected (1-45cm depth) from University of
Port Harcourt Botanical Garden, River State, Nigeria. The soil samples were air dried to constant weight and sieved
with 2mm Mesh. Pure cultures of
Lentinus squarrosulus
were obtained from African Centre for Mushroom Research
and Innovation, University of Benin, Benin City, Edo State, Nigeria. Crude oil was collected from Nigerian National
Petroleum Corporation, River State, Nigeria.
2.2 Method of spawn preparation: Fresh cultures of
L. squarrosulus
were by flaming an inoculating loop to be red
hot, after which, it was used to cut a portion of the fresh mushroom into freshly prepared PDA. The cultures were
then incubated for about seven days in an incubator. The centre of each PDA plate containing each of the mushroom
isolates was punched with a cork borer with a diameter of 0.5 mm to form a fungal mycelia disc. This was done
according to the method of [11]. 40g of saw dust was measured in a weighing balance and then transferred to a
clean bowl where it was filtered to remove chaff and other particles. The saw dust was then transferred again into a
clean bowl and mixed with water to make it moist. The moist saw dust was then put into a spawn flask. After which it
was autoclaved at 121
0
C for 30 minutes for 3 days. The saw dust in the bottles was inoculated with four 0.5mm
mycelial discs of
L. squarrosulus
under aseptic conditions [12]. These were then incubated at room temperature
(28±2
0
C) for three months.
2.3 Preparation of the crude oil contaminated substrate: The technique used for the screening of the
bioremediative properties of
L. squarrosulus
was done by modifying the method of [13]. Two hundred grams (200g)
of soil were measured into locally available bottles and mixed thoroughly with the crude oil based on the
concentration. The concentration levels of 5, 10, 15, 20, and 30mls were used alongside a control treatment
(0ml).Thirty grams (30g) of sawdust were laid on the contaminated soil in each bottle separated with wire gauze. The
bottles containing the soil, saw dust and crude oil were then sterilized in an autoclaved at 115
0
C for 30 minutes. Ten
grams (10g) of fungal inocula of
L. squarrosulus
were aseptically weighed and transferred into the already sterilized
bottles containing the soil and sawdust substrate for the mushroom. The cultures were incubated at 25
0
C for three
months. The experimental set up consisted of the following treatment: Control (soil only), Pollution treatment (Soil +
each concentration level of crude oil: 5, 10, 15, 20, and 30mls, respectively), and Bioremediation treatment (Soil +
each concentration of crude oil + spawns of
L. squarrosulus
). The experimental set up was replicated five times, and
further used for assessment of test crop growth response in the bioremediated crude oil polluted soil.
2.4 Germination studies: Viable seeds of
Zea mays
were sorted out, sterilized with 0.01% mercuric chloride
solution for 30 seconds, washed several times with distilled water and air dried. Five (5) seeds of the test crop were
sown in plastic containers containing one-quarter level of spawn of crude oil polluted soil colonized by
L. squarrosulus
based on treatment: Control (soil only), Pollution treatment (Soil + each concentration level of crude oil - 5, 10, 15,
20, and 30mls, respectively), Bioremediation treatment (Soil + each concentration of crude oil + spawns of
L.
squarrosulus
). The seedlings were thinned to three (3) per container. Five replicates were used for each treatment
using randomized complete block design. The experimental set up was maintained at a mean minimum temperature
of 22.32
o
C and mean maximum temperature of 34.18
o
C, under natural light condition for four (4) months.
2.5 Analysis of experimental soils: The physico-chemical properties (pH, organic carbon, calcium, magnesium,
sodium, nitrogen, potassium, and phosphorus) of the experimental soils were analyzed using standard procedures
[14]. Experimental soils (control, pollution, and bioremediation treatments) were analyze before and after harvest.
2.6 Growth Studies: Plant height, leaf number, stem diameter, moisture content, and grain yield were measured at
the end of the experiment.
2.7 Statistical Analysis: Data analysis was carried out using analysis of variance (ANOVA) (P < 0.05) using the
method of [15].
3. RESULTS
In Table 1, the pH values, and mineral nutrient contents of the experimental soil are presented. The pH values of the
soil decreased with increase in the concentration of crude oil in all treatments (Table 1). The calcium, magnesium,
phosphorus, sodium, and potassium contents in crude oil pollution and bioremediation treatments were relatively
higher (P < 0.05) than those of the control treatment. The contents of calcium, sodium, magnesium, nitrogen,