MATERIALS AND METHODS

Isolation of S. rolfsii-Infected tomato stems were obtained from the experimental plot of National Horticultural Research Institute, Ibadan Nigeria. The stems were washed with sterile distilled water, cut into 5 mm segments which were surface sterilized in 0.5% sodium hypochlorite (NaOCl) solution for 5 min and rinsed thrice in sterilized distilled water. The segments were then air dried between sterile filter paper and plated on Potato dextrose agar (DIFCO) amended with chloramphenicol (60 mg mL^-1). The plates were incubated at 28±2°C and examined for 5 days. Cultural and morphological identification of the organism was done with the use of microscope. The plates were sub-cultured to obtain pure cultures of isolate which were kept in slants and stored at room temperature.

Preparation of plant extracts: Dried seeds of A. melegueta (Alligator pepper) and fresh leaves of O. gratissimum (African basil) were the plant materials used. A. melegueta seeds were purchased from Bode market in Nigeria while O. gratissimum leaves were obtained from the progeny garden of the National Horticulural Research Institute (NIHORT) Ibadan, Nigeria. The plant materials were rinsed in sterile distilled water and dried at 60°C with the O. gratissimum leaves also rinsed as above and dried for 10 days at room temperature, after which they were milled with Marlex blender separately into powder. The powder were sieved and packed into glass bottles and sterilized in a hot air oven at 160°C for 5 h (Enikuomehin, 1995).

In vitro control of S. rolfsii: One milligram, 2, 3, 4 and 5 g of each sterilized sample were suspended in 100 mL of sterilized distilled water to obtain concentrations of 1, 2, 3, 4 and 5%, respectively. Each mixture was agitated manually to obtain even particle distribution. Solution of ethanol extracts of the same plant materials were made by dissolving the same quantity as described for water 100 mL absolute ethanol to obtain 1, 2, 3, 4 and 5% concentrations, respectively. Each of the extract was evaluated for antifungal properties against the fungal pathogen by agar diffusion plate method. One millilitre of each extract concentration was poured into sterile 9 cm-diameter Petri dishes, 9 mL of cooled (about 45°C) molten chloramphenicol amended (60 mg mL^-1) PDA was aseptically poured into each Petri-dish and rotated gently to ensure even dispersion of extract. A 6 mm mycelial disc obtained from the margin of a 5-day-old culture of S. rolfsii was placed at the centre of each petri-dish containing each spice extract samples. Control plates had either 1 mL of sterile distilled water of 1 mL ethanol plus 9 mL distilled water mixed with cooled molten Chloramphenicol modified PDA and inoculated as described above. There were three replicates for each plant extract concentration. Ethanol extracts of the samples were also prepared as above. All plates were incubated at 28±2°C for 5 days. Measurement was taken as the mean growth along the two axes on two pre-drawn perpendicular lines on the reverse side of plates. Data were analysed using Fisher’s Least Significant Difference (Fisher’s LSD) at 5%.

In vivo control of S. rolfsii: The experiment was complete randomised design replicated three times. Tomato seedlings UC82B were raised in steam sterilized soil for four weeks in the nursery. S. rolfsii inoculum was prepared by adding 10 sclerotia of S. rolfsii to 100 g of moist autoclaved wheat seeds in 500 mL conical flasks. Incubation was done for two weeks at 28°C. Soil infestation was carried out after watering the soil for one week by mixing 10 g of inoculum in 10 kg sterilized soil. (Enikuomehin et al., 1998). The four weeks old seedlings were transplanted into the inoculated soil at one seedling per pot. The extracts and the synthetic fungicide were applied at the base line of the stem at the rate of 10 mL per pot. Plants were watered every other day throughout the period of the experiment. The data collected include plant height, leaf number, disease severity and fruit weight. Data were collected weekly and disease severity was rated according to the method of De Cal et al. (1995).

Statistical analysis: The data collected were subjected to analysis of variance (ANOVA) (Steel and Torrie, 1984) using SAS software. Means of significant treatments were separated by Fisher’s Least Significant Difference (LSD).

RESULTS AND DISCUSSION

The effect of A. melegueta and O. gratissimum extract concentrations at various time on the growth of Sclerotium rolfsii is shown in Table 1 and 2.

The efficacy of two spice extracts against Southern blight fungus (S. rolfsii) of tomato was tested in vitro and in vivo. The results showed that the two spice extracts significantly (p<0.05) inhibited the radial growth of the organism with inhibition varying from one extract to another. It was observed that the antifungal effectiveness of these spices in culture depends on the concentration used and the solvent of extraction. This is in agreement with the work of Udo et al. (2001) who reported that methanol and ethanol extracts of spices have high potency for the control of pathogenic fungi of potato and yam tubers. They also reported that the potency of aqueous extracts was low compared to the methanol and ethanol extracts. According to Zaker and Mosallanejad (2010), methanol extracts of peppermint (15%), Lavandula (15%), peppermint (10%) and eucalyptus (15%) demonstrated promising ability in inhibiting the mycelial growth of Alternaria alternata causal organism of Alternaria leafspot of potato. Derbalah et al. (2011) also reported the antifungal activity of crude extracts of seven plant species (Cassia senna, Caesalpinia gilliesii, Thespesia populnea var. acutiloba, Chrysanthemum frutescens, Euonymus japonicus, Bauhinia purpurea and Cassia fistula) against Alternaria solani the causal organism of early blight disease in tomato at 150 ppm and 200 p. As concentration increases it was observed that O. gratissimum aqueous extract gave comparable reduction in mycelial growth this is in agreement with the work of Awuah (1989) who found that extracts of O. gratissimum led to 24.6% reduction in radial growth of Rhizopus species and a 60% reduction of Ustilaginoidea virens. Ijeh et al. (2005) reported that aqueous and ethanolic extracts of O. gratissimum and Xylopia aethiopica showed their antimicrobial activities against five pathogenic organisms; Staphylococcus aureus, Escherichia coli, Streptococcus faecalis, Pseudomonas aeruginosa and Lactobacillius specie.

Okigbo and Ogbonnanya (2006) reported that O. gratissimum and A. melegueta ethanol extracts inhibited the mycelia growth and spore germination of many rot causing microorganisms. O. gratissimum leaf extracts controlled spore germination and mycelia growth of Rhizopus oryzae (Amadioha, 2000). Enikuomehin et al. (1998) also proved the inhibitory effect of O. gratissimum leaf ash on the mycelial growth of S. rolfsii in vitro. Generally, radial growth increased as the hours increases. Twenty four hours after incubation, the radial growth of S. rolfsii on plates impregnated with water extract of O. gratissimum decreased with increasing extract concentration. At 120 h after incubation, plates with no extract had the highest radial growth of 4.3 mm while the least of 0.5 mm was on plates with 5% extract concentration. Plates impregnated with ethanol extracts of O. gratissimum had highest mycelia growth at 1% concentration and the least at 3% after 120 h of incubation. Also, at 120 h plates impregnated with water extract of A. melegueta had highest radial growth of 4.6 mm while the least was 2.5 mm. Highest radial growth of 4.5 mm was observed at 120 h while the least of 0 was observed for ethanol extract of A. melegueta. The aqueous and ethanol extracts of the two plant materials used in this trial differed significantly in their potential to inhibit the mycelium growth of S. rolfsii. Though, non of the aqueous extracts had total inhibition on the growth of the pathogen at (p<0.05). However, concentration had significant effect on the mycelium growth which decreases with increasing concentrations for the two extracts as the number of hours increased. Highest significant effect was observed at 5% for the water extracts while the least was recorded at 1%. The ethanol extracts of the two samples had significant effect on the mycelium growth. Highest inhibition was observed at 3% in the two samples with no growth on the plates.

Results of analysis of variance showed that different concentrations of A. melegueta and O. gratissimum had significant effect on plant height (6 WAT). Southern blight severity and fruit yield per plant but plant height at 8 WAT, number of leaf/plant and shoot dry weight were not significantly affected by spice extract application (Table 1). O. gratissimum extract at 5% concentration gave the highest plant (85.26) though this was not significantly different from O. gratissimum at 2% (80.83 cm) and A. melegueta at 1% (76.43 cm) (Table 2). At 6 WAT disease severity was significantly higher on plants treated with A. melegueta at 1 and 2% concentrations than those treated with A. melegueta at 3 and 4%, O. gratissimum at 1 -4% concentrations. Disease severity recorded on plants treated with A. melegueta extract at 5% and fungus force were significantly lower than those mentioned above while the least disease severity was observed on plants treated with O. gratissimum extract at 5% concentration (Table 2). At 8 WAT, disease severity observed on plants treated with A. melegueta extract at 1 and 2% and O. gratissimum at 1% concentrations was significantly higher than other treatments, this was followed by A. melegueta at 3, 4%, O. gratissimum at 2, 3 and 4% concentrations which were significantly different from each other.

Table 1:	Inhibition of mycelial growth of S. rolfsii by O. gratissimum extracts after 5 days incubation at 28°C

Table 2:	Inhibition of mycelial growth of S. rolfsii by A. melegueta extracts after 5 days incubation at 28°C

Disease severity recorded on plants treated with 5% A. melegueta was comparable with that of the fungus force, this was lower than what was observed at lower concentrations of A. melegueta and O. gratissimum extracts. Among all the treatments, the lowest disease severity was recorded on plants treated with O. gratissimum extract (Table 2). Significant difference was observed in the fruit yield of plants treated with the spice extracts at different concentrations (p<0.05). The highest fruit yield of 154.9 g was recorded in plants treated with 5% concentration of O. gratissimum extracts but this was comparable with 108.9 and 116.2 g obtained from plants treated with 4% concentration of X. aethiopica extract and fungus force, respectively.

Maximum reduction in disease severity to (1.67) was achieved at the highest concentration (5%) of O. gratissimum and this was comparable to (2.3) obtained with synthetic fungus force (Mancozeb 63 + 12.5% WP). This agrees with the work of Afroz et al. (2008) who affirmed that the occurrence of late blight attained an epipytotic momentum when the plants entered into their reproductive phase, that is, between 40 and 55 days after transplanting. The results obtained from this study on the effectiveness of the two spice extracts on Southern blight disease severity revealed that the extracts vary in their ability to reduce disease severity. This is in consonance with the earlier reports that many plant products contain fungitoxic constituents that have potential to control plant diseases (Amadioha and Obi, 1999; Enikuomehin, 2005).

Disease severity varied with each spice extract at different concentrations. Okigbo and Ogbonnanya (2006) had reported the antifungal effectiveness of O. gratissimum and A. melegueta in vitro and in vivo on rot pathogens of yam tubers and that the two extracts did not show significant difference in their potency against rot pathogens.

The results obtained from this study on the effectiveness of the two spice extracts on Southern blight disease severity revealed that the extracts vary in their ability to reduce disease severity. It was observed that increase in concentration of the extracts enhanced the effectiveness of individual extract to reduce disease severity. Plants treated with O. gratissimum extract had better growth in terms of plant height. This result agrees with the study of Okigbo and Ogbonnanya (2006) who reported that O. gratissimum and A. melegueta extracts proved effective against mycelial inhibition and spore germination of many rot causing microorganisms. Amadioha (2000) ascertained that O. gratissimum leaf extracts controlled spore germination and mycelial growth of Rhizopus oryzae. Enikuomehin (2005) also reported the inhibitory effect O. gratissimum leaf ash on the mycelial growth of S. rolfsii of wheat on agar. The antifungal effectiveness of these spice extracts in culture depends on the concentration and the solvent of extraction. Sallam (2011) concluded that extract of Ocimum basilicum (Sweat Basil), Azadirachta indica (Neem), Eucalyptus chamadulensis (Eucalyptus), Datura stramonium (Jimsonweed), Nerium oleander (Oleander) and Allium sativum (Garlic) against Alternaria solani at 5% concentration caused reduction in mycelial growth of A. solani causal organism of Early blight of tomato in vitro and increased the fruit yield in vivo. According to Abera et al. (2011) extracts of Allium sativum and Croton macrostachyus on Colletotrichum kahawae radial growth and disease development vary depending on the concentration of the extracts applied. Oyelana et al. (2011) reported that the leaf extracts of Ficus species at 75 and 100 mg mL^-1 concentrations had profound antimicrobial properties on the fungal and bacterial pathogens of Dioscorea rotundata. These extracts contained alkaloids, flavonoids and cardiac glycosides and may have conferred the antimicrobial properties on this group of plants. Neem leaf extract at 15% concentration has also been reported to inhibit radial growth in Fusarium oxysporum f. sp. psidii (Derbalah et al., 2011). From this trial, ethanol extracts performed better in inhibition of the mycelial growth of S. rolfsii. The inhibitory activities of these extracts suggest their fungitoxic ability on S. rolfsii. This observation agrees with the study of Iwalokun et al. (2003), Onwuliri and Wonang (2005), Udo et al. (2001), Eweis and Amber (2011), who concluded that the antifungal properties of these plants are due to their phytochemical contents and the concentration of the extracts and essential oil used. This investigation demonstrates the potential of A. melegueta and O. gratissimum as potential alternatives to synthetic chemical in the management of Sclerotium rolfsii which causes stem rot disease of tomato.