Effect of Ocimum gratissimum (L.) and Aframomum melegueta (K. Schum.) Extracts on the Growth of Sclerotium rolfsii (Sacc.)
Southern blight disease of tomato induced by Sclerotium rolfsii Sacc.
is known to cause about 60% reduction in tomato yield under rain fed condition.
The use of resistant varieties and chemicals has not provided adequate control
due to the persistent nature of the pathogen, poor adaptation of cultivars,
high cost and scarcity of the chemicals. An alternative method of control was
examined by evaluating the inhibitory effect of some selected spices on Southern
blight disease of tomato in vitro and in vivo. Water and ethanol
extraction of the seeds of Aframomum melegueta K. Schum. and leaves of
Ocimum gratissimum L. was done at 1-5% v/v. Sclerotium rolfsii was
isolated from infected plants. The in vivo experiment was laid out in
triplicates in completely randomised design using the same extracts. The synthetic
fungicide served as the control. All data collected were subjected to Analysis
of Variance (ANOVA) and significant means were separated using fishers
LSD. The results showed that the spice extracts differed significantly (p<0.05)
in their potential to inhibit the growth of S. rolfsii. In all, the extracts
inhibition decreases with increase in concentration. Ethanol extract of the
two spices at 3-5% concentration showed total inhibition (0.00 mm) on the mycelial
growth. The in vivo experiment showed that plants treated with 5% O.
gratissimum extract performed better in reducing Southern blight severity,
gave highest fruit weight at 5% extract concentration. It was also observed.
The two spice extracts are potential options for the management of S. rolfsii
as they compared favourably with fungus force, a synthetic pesticide recommended
for the control of S. rolfsii.
to cite this article:
E.A. Adesegun, E.O. Ajayi, O.S. Adebayo, A.K. Akintokun and O.A. Enikuomehin, 2012. Effect of Ocimum gratissimum (L.) and Aframomum melegueta (K. Schum.) Extracts on the Growth of Sclerotium rolfsii (Sacc.). International Journal of Plant Pathology, 3: 74-81.
Received: November 04, 2011;
Accepted: February 15, 2012;
Published: June 05, 2012
Sclerotium rolfsii Sacc. is an economically important soil borne fungal
pathogen which causes disease on a wide range of agricultural and horticultural
crops (Farr et al., 1989) including the diseases
known as Southern blight especially on tomato. Sclerotium rolfsii forms
brownish sclerotia that can survive in soil for long periods frequently tolerating
biological and chemical degradation due to the presence of melanin in the outer
membrane (Chet, 1975). Although no statistical data
are available, the disease caused by this pathogen lead to heavy losses in vegetable
crop yield especially during wet season when weather conditions are favourable
for both crop production and for the growth and dissemination of the sclerotia
of the pathogen (Okabe et al., 2000). Among over
500 plants attacked by Sclerotium rolfsii particularly in the tropical,
sub-tropical and warm temperate areas (Okereke and Wokocha,
2007) is tomato (Lycopersicon esculentum Mill.) which is an important
vegetable in Nigeria for domestic purpose, accounting for about 18% of the daily
consumption of vegetables which averages out at 50.6% per person (Kateria
and Mittal, 1984). One medium-sized tomato fruit of about 150 g provides
half the recommended dietary allowance of vitamin C for an adult (Yusuf
and Okusanya, 2007). Egharevba (1991) and Yusuf
and Okusanya (2007) stressed that fresh tomato fruits and other vegetables
are essential components of human diet which have necessitated the increased
production and commerce of commodities.
Major methods employed to manage S. rolfsii are fungicide applications,
solarisation, use of antagonistic microorganisms, deep ploughing, crop rotation
and incorporation of organic and inorganic residues (Punja,
1985). Fungicides such as Captan and Calixin have been used for seed dressing
and other chemicals such as methyl bromide and Chloropicrin are used as soil
fumigants to the control of this pathogen. These chemicals are environmentally
hazardous, recently banned, not readily available and difficult therefore to
adopt in subsistence agriculture in West Africa (Okereke
and Wokocha, 2007). There is therefore the need to search for the use of
cheaper environmentally friendly and readily available alternatives such as
plant extracts for the control of Sclerotium rolfsii. The purpose of
this trial was to evaluate the efficacy of A. melegueta and O. gratissimum
for the in vitro inhibition of the S. rolfsii.
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
Fishers Least Significant Difference (Fishers 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 Fishers 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
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.
|| Inhibition of mycelial growth of S. rolfsii by O.
gratissimum extracts after 5 days incubation at 28°C
|| 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,
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.
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