African Journal of Microbiology Research Vol. 6(22), pp.

4655 -4661, 14 June, 2012

Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR11.979
ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Assessment of the antifungal activity of Nicotiana

glauca Graham aqueous and organic extracts against
some pathogenic and antagonistic fungi
Asma Rinez1*, Mejda Daami-Remadi2, Faten Omezzine1, Afef Ladhari1, Imen Rinez1 and
Rabiaa Haouala3
1
Department of Biology, Faculty of Sciences of Bizerte, University of Carthage, Tunisia (UR03AGR04).
2
Laboratory of Phytopathology, Regional Center of Research in Horticulture and Organic Agriculture, University of
Sousse, Chott-Mariem, 4042, Tunisia.
3
Department of Biology and Plant Protection and Environment, Higher Agronomic Institute of Chott-Mariem, University of
Sousse, Chott-Mariem, 4042, Tunisia. (UR03AGR04).

Accepted 17 November, 2011

In the present research work, Nicotiana glauca Graham was used as a potential source of biologically
active compounds. The antifungal activity of leaf and flower aqueous and organic extracts (petroleum
ether, chloroforme and methanol) was assessed in vitro against three phytopathogenic fungi and two
antagonistic agents. These target fungi were subjected to the different types of extracts already
incorporated into the Potato Dextrose Agar (PDA) medium at various concentrations. Results revealed
an important antifungal activity of N. glauca leaf and flower aqueous extracts at all concentrations
tested (1, 2, 3 and 4%). However, a relative difference in the extent of the response of the same fungal
agent to the extracts tested was observed. In fact, Trichoderma viride was found to be more sensitive
than the other target species, where the radial growth inhibition noted varied from 37.4 to 63.14%
depending on aqueous extracts concentrations and the maximum inhibition was obtained with leaf
aqueous extracts applied at 1 and 2% concentrations. Moreover, T. viride and Fusarium oxysporum f.
sp. melonis were found to be the most sensitive to leaf and flower organic extracts as compared to the
other agents. Growth of T. viride was inhibited by 33.7% in the presence of chloroforme leaf extract and
petroleum ether flower extracts tested at 3000 and 9000 ppm, respectively. Furthermore, the radial
growth of F. oxysporum f. sp. melonis was reduced by more than 31 and 20% with leaf petroleum ether
and flower chloroform extracts, respectively, applied at 9000 ppm. T. harzianum, F. oxysporum f. sp.
tuberosi and F. oxysporum f. sp. lycopersici were found to be less sensitive to N. glauca organic
extracts as compared to aqueous extracts.

Key words: Antifungal activity, aqueous extracts, allelopathy, Nicotiana glauca Graham, Fusarium,
Trichoderma and radial growth.

INTRODUCTION
primary concern of agricultural scientists (Guleria and
Fungal diseases of cultivated crops remain the principal Kumar, 2006). Since the very beginning of their
limitation to increased agriculture production every year. appearance, researchers have succeeded in controlling
Therefore, protection of plants from pathogens remains a some devastating diseases by synthetic fungicides. As
several synthetic fungicides are highly effective in
controlling plant diseases, their negative effect on human
and animal health and also on the agro-ecosystem was
*Corresponding author. E-mail: [email protected]. gradually realized which entailed serious research in developing
4656 Afr. J. Microbiol. Res.

alternative environmentally acceptable (environment- MATERIALS AND METHODS

friendly) methods (Sahni et al., 2005). These efforts Fungal agents
included biological control, genetic engineering, use of
systemic acquired resistance (SAR) with the help of biotic Pure culture of two Trichoderma species (Trichoderma harzianum
and abiotic agents (Lyon et al., 1995), and biodegradable and T. viride) and three formae speciales of Fusarium oxysporum
natural products especially from medicinal plants that is F. oxysporum f. sp. melonis (FOM), F. oxysporum f. sp.
lycopersici (FOL) and F. oxysporum f. sp. tuberosi (FOT) infecting
(Prithiviraj et al., 1996). The use of pesticides of plant melon, tomato and potato, respectively, were obtained from the
origin has been suggested by some workers as Laboratory of Phytopathology of the Regional Center of Research
alternatives to synthetic chemicals (Amadioha and Obi, in Horticulture and Organic Agriculture, Chott-Mariem (Tunisia). The
1999; Amadioha, 2000, 2002). Recent studies has isolates were obtained from the diseased samples on Potato
confirmed the efficacy of plant extracts in the control of Dextrose Agar (PDA), purified and maintained at 4°C un til use.
fungal diseases (Amadioha and Uchendu, 2003; Singh et
al., 2010), with the view to countering obvious pollution Collection of plant material
problems in the environment and avoiding the toxic
effects of synthetic chemicals on non-target organisms. Fresh and healthy leaves and flowers of N. glauca were collected
Organic (Shafique and Shafique, 2008; Bajwa et al., from Tunisian littoral (Monastir, Tunisia). Fresh materials were
washed thoroughly with detergent to remove any dust. Washed
2008) and aqueous extracts (Bajwa et al., 2001) of many leaves and flowers were dried in an electric oven at 30°C for 72 h
allelopathic plants are known to exhibit antifungal and crushed to make powder.
properties. Allelochemicals reduce the germination of
spores and the mycelial growth of pathogenic fungi
(Bajwa et al., 2003). Extracts preparation
The quest for development of new antifungal agents Aqueous extract was prepared by soaking thirty grams of dried leaf
with powerful and wide range of fungicidal activity led us and flower powder of the test species in 100 ml of sterilized distilled
to investigate Nicotiana glauca Graham for potentially water for 24 h. Extract was filtered through a double layered muslin
important antifungal compounds. N. glauca, a fast cloth followed by Whatman No. 1 filter paper and then passed
growing shrub or small tree, belongs to the family through 0.22 µm micro-filter pore size to remove bacteria. Filtrates
Solanaceae (Mizrachi et al., 2000). It branches profusely, were preserved at 4°C. To avoid any prospective chemi cal
alterations, the extracts were generally used within a week.
and can grow vigorously to 3 m, particularly after good Sequential extraction was carried out in organic solvents with
rainfall events (Florentine and Westbrooke, 2005). The rising polarity: petroleum ether, chloroform and methanol. Eighty
leaves are stalked, alternate, elliptical to lanceolate or grams of powder were immersed in the organic solvent for 7 days at
oval, pointed, bluish or greyish-green. The flowers are room temperature. Organic extracts were evaporated to dryness
greenish-yellow, 30 to 40 mm long; many are borne in a under reduced pressure in a rotary evaporator at 45 to 50°C,
lax panicle (Bogdanovic et al., 2006). Studies have respectively, to remove the petroleum ether, chloroforme and
methanol. Samples of 15, 30, and 45 mg were individually
demonstrated that N. glauca is highly toxic to humans dissolved in 2 ml of methanol and then diluted by adding 3 ml of
(Mizrachi et al., 2000) and animals (Panter et al., 2000). sterilized distilled water, to make final volume of 5 ml, to give three
The first investigation on toxic effects of secondary extract concentration (3000, 6000 and 9000 ppm). The stock
metabolites extracted from this plant was initiated as extract was stored at 4°C and used within four days.
early as the 1930’s (Steyn, 1934). In the late seventies,
the four major pyridine alkaloids, that is, nicotine, Antifungal bioassays
anabasine, anatabine and nornicortine were produced
from this species (Hawley, 1977; Waller and Edmund, The antifungal activity against the test pathogens was determined
1978). Several researchers have reported biological according to the poisoned food technique of Grover and Moore
(1962). In fact, PDA medium was prepared and autoclaved at
activities of N. glauca extracts, but a few comprehensive
150°C for 30 min. Appropriate quantities of aqueous extracts (1.5,
antifungal activities of N. glauca have been reported, 3, 5, and 6.25 ml) and distilled water were added to this medium
although it is widely used by traditional healers. Mdee et (40 ml), cooled to 45 to 50°C, to get 1, 2, 3 and 4% (w/v)
al. (2009) reported antifungal activity of acetone extracts concentrations of leaf and flower aqueous extracts. The control
of N. glauca against ten fungal phytopathogens. Soberon medium received the same quantity (1.5, 3, 5, and 6.25 ml) of
et al. (2007) have also reported its antibacterial and sterile distilled water. Stock solution of organic extracts (5 ml)
prepared above at 3000, 6000 and 9000 ppm was added to PDA
cytotoxic effects. Allelopathic activity of N. glauca extracts medium. Control received the same quantity (5 ml) of diluted
was reported by Heisey and Delwiche (1983), Florentine methanol used as control for all bioassays with organic extracts.
and Westbrooke (2005) and Alshahrani (2008). The plant extracts were thoroughly mixed with the medium. Ten
The aim of this investigation is, therefore, is to assess ml of each medium was poured in each 9 cm diameter sterilized
the in vitro antifungal activity of aqueous and organic Petri plate. After solidification, mycelial discs of 5 mm diameter
extracts of N. glauca leaves and flowers. This allelopathic were taken with a pre-sterilized cork borer from 5 to 7 days old
culture of each tested fungus and were placed in each Petri plate.
plant species was tested, as potential source of natural Each treatment was replicated thrice. Plates were incubated in an
substances biologically active against three phytopatho- incubator at 25 ± 2°C for 3 to 7 days. Fungal growth was measured
genic and two antagonistic fungi. by averaging the two diameters taken from each colony.
 Rinez et al. 4657

Table 1. Percentage of inhibition of the mycelial growth of fungal agents induced by Nicotiana glauca leaf and flower aqueous
extracts tested at different concentrations.

Leaf aqueous extract Flower aqueous extract

Fungal agents 1% 2% 3% 4% 1% 2% 3% 4%
a a b ab a a a a
TH 23.37 30.99 46.63 33.74 11.57 23.62 21.76 19.78
b b ab a a b b ab
TV 58.84 63.14 50.65 44.87 37.40 51.17 56.38 46.60
a a a a a b b b
FOM 22.13 14.23 14.74 18.70 1.36 25.66 24.72 18.20
b a a a a ab ab b
FOL 25.63 16.45 19.65 19.22 8.31 14.41 12.76 19.89
b a b a a b b b
FOT 22.78 6.21 18.66 5.38 2.88 15.63 19.08 24.40

For each fungus tested and each extract type, values (indicating concentrations) affected by the same letters are significantly similar
according to Duncan’s test at the 0.05 level. Incubation temperature: 25 ± 2ºC; Incubation period: 3-4 days. TH: Trichoderma
harzianum; TV: T. viride; FOM: Fusarium oxysporum f. sp. melonis; FOL: F. oxysporum f. sp. lycopersici; FOT: F. oxysporum f. sp.
tuberosi..

Percentage growth inhibition of the fungal colonies was calculated to Trichoderma species. In fact, the maximum allelopathic
by applying the following formula (Khanh et al., 2005): stress (25.63%) induced by leaf aqueous extract at 1%
Growth/inhibition (%) = [(Growth in control – Growth in treatment)/
concentration was recorded in F. oxysporum f. sp.
Growth in control] * 100 lycopersici. In contrast, flower aqueous extracts exhibited
less inhibitory effects (of about 21.14%) on the mycelial
growth of F. oxysporum f. sp. lycopersici and F.
Statistical analysis oxysporum f. sp. tuberosi at the highest concentration
tested (4%).
The SPSS statistical methods [predictive analytics software
(PASW) statistics 18] were used to calculate the means, standard
errors and standard deviations. Statistical analysis one-way ANOVA
was applied to the data to determine differences in the three factors Effect of Nicotiana glauca organic extracts on fungal
tested (Extracts, concentrations and fungi tested, and their
interactions) according to a completely randomized factorial design.
mycelial growth
To check significant differences between the levels of the main
factor, Duncan multiple comparison tests at 5% significance were The effects of N. glauca leaf and flower organic extracts
applied. on the radial growth of the three phytopathogenic and the
two antagonistic fungi tested are presented in Table 2. In
fact, the mycelial growth of T. harzianum was inhibited on
RESULTS PDA medium amended with the leaf and flower organic
N. glauca extracts at all concentrations tested. The
Effect of Nicotiana glauca aqueous extracts on fungal highest inhibition of about 33.7% was recorded in the
mycelial growth presence of chloroforme leaf extract and petroleum ether
flower extract applied at 3000 and 9000 ppm,
Results presented in Table 1 revealed an important respectively. The response of T. viride to leaf organic
antifungal activity exhibited by N. glauca leaf and flower extracts of N. gluaca was slightly different as compared
aqueous extracts at all concentrations tested. However, to T. harzianum. The mycelial growth of T. viride was
the response to extracts seems to be different depending slightly inhibited or even stimulated in the presence of
on target agents used. Indeed, T. viride was the more flower organic extracts. The addition of the petroleum
affected by the extracts tested than the other fungal ether fraction at 9000 ppm induced increased by 6.46%
agents; the recorded radial growth inhibition varied from the radial growth of this fungus.
37.4 to 63.14%, depending on extracts and A relatively higher inhibitory activity was exerted by N.
concentrations used. Moreover, T. harzianum exhibited glauca leaf and flower organic extracts against F.
more sensitivity to leaf than to N. glauca flower extracts; oxysporum f. sp. melonis in all the concentrations used.
the highest radial growth inhibition of 46.63% was The pathogen radial growth was reduced by more than
obtained with leaf aqueous extracts applied at 3%. These 31 and 20%, with leaf petroleum ether extract and flower
results revealed the inhibitory effect of aqueous extracts chloroform extract, respectively, applied at 9000 ppm.
of this allelopathic plant exhibited against these both However, F. oxysporum f. sp. lycopersici and F.
antagonistic agents. oxysporum f. sp. tuberosi were found to be less sensitive
Also, as shown in Table 1, the mycelial growth of the to N. glauca organic extracts as compared to F.
three F. oxysporum formae speciales tested seems to be oxysporum f. sp. melonis.
less affected by N. glauca aqueous extracts as compared The addition of relatively increasing or decreasing
 4658 Afr. J. Microbiol. Res.

Table 2 Percentage of inhibition of the mycelial growth of fungal agents induced by Nicotiana glauca leaf and flower organic
extracts tested at different concentrations.

Fungal agents Organic extract (ppm) Leaf organic extract Flower organic extract
3000 6000 9000 3000 6000 9000
b a c a a b
Petroleum ether 8.65 5.48 19.01 14.13 16.65 33.98
c a b b a ab
TH Chloroform 33.42 12.96 27.39 24.79 8.02 14.11
b a a b ab a
Methanol 24.46 8.07 10.64 19.88 16.15 11.79
a a a a a a
Petroleum ether 10.00 16.79 17.47 -5.46 -1.50 -6.46
b a a a b a
TV Chloroform 14.62 2.38 4.37 12.73 -0.27 9.42
a a a a b ab
Methanol 0.76 2.39 2.91 -3.37 7.88 6.87
a b c ab a b
Petroleum ether 8.53 15.88 31.38 12.78 6.23 19.00
b ab a a a b
FOM Chloroform 14.33 10.10 5.39 11.62 8.92 20.56
a b ab b a a
Methanol -0.01 14.30 2.28 11.62 1.95 4.67
a a a a b a
Petroleum ether 6.83 -0.84 2.94 4.95 -7.48 -0.06
a a a a b b
FOL Chloroform 3.70 -5.88 0.47 -7.15 7.74 9.21
a a a a a a
Methanol 1.40 -5.89 -1.50 -1.90 -14.96 0.57
a b a a a b
Petroleum ether 7.49 -4.08 11.49 1.60 8.62 -14.16
a b a a a a
FOT Chloroform 10.27 -4.92 11.36 6.96 8.87 18.20
a b b a a a
Methanol -10.92 14.04 21.02 8.01 -4.93 7.14
For each fungus tested and each extract type, values (indicating concentrations) affected by the same letters are significantly
similar according to Duncan’s test at the 0.05 level. TH: Trichoderma harzianum; TV: T. viride; FOM: Fusarium oxysporum f.
sp. melonis; FOL: F. oxysporum f. sp. lycopersici; FOT: F. oxysporum f. sp. Tuberosi

concentrations of N. glauca leaf and flower organic The relative intensity of this effect, however, varies with
extracts caused depression or stimulation of fungal the target fungus, as well as the origin, types and
growth of F. oxysporum f. sp. lycopersici and F. concentrations of the extracts used. The differences
oxysporum f. sp. tuberosi. In fact, as clearly recorded in the fungitoxic activity of the extracts tested is
demonstrated in the present screening, leaf methanol likely due to the solubility of the active compound(s) in
extracts applied at 9000 ppm exhibited the highest water or the presence of inhibitors to the fungitoxic
inhibitory (of about 21%) effect against F. oxysporum f. principle as noted by Qasem and Abu-Blan (1966),
sp. tuberosi. However, the radial growth of this fungus Amadioha (2001), and Okigbo and Ogbonnaya (2006).
was enhanced by 10.92% with this same extract when According to the previously mentioned results, a strong
used at the lowest concentration tested (3000 ppm). F. toxicity of N. glauca leaf and flower aqueous extracts
oxysporum f. sp. lycopersici was found to be the less against all the tested fungi at all concentrations (1, 2, 3
sensitive to all N. glauca organic extracts as compared to and 4%) was shown. However, the response to extracts
the other F. oxysporum formae speciales tested; the seems to be different depending on target agents used.
recorded percentage of inhibition did not exceed 6.83 and In fact, T. viride was more affected by the aqueous
9.21%, respectively with leaf petroleum ether used at extracts tested than the other fungal agents; the recorded
3000 ppm and the flower chloroform extract applied at radial growth inhibition varied from 37 to 63%, depending
9000 ppm. However, with the flower methanol extracts on extracts and concentrations used. The inhibition of the
tested at 6000 ppm, the growth of this pathogen was mycelial growth of the sensitive agent may be attributed
14.96% higher than the untreated control. to the presence and detrimental effects of allelochemicals
on cell division, cell elongation and nutrient uptake
(Blake, 1985). In contrast, mycelial growth of T. viride
DISCUSSION was slightly inhibited or even stimulated in the presence
of flower organic extracts as observed with the petroleum
Plants are a repository of various biomolecules involved ether fraction used at 9000 ppm which enhanced by 6%
in their different biological activities (Kiran et al., 2010). In of the radial growth of this fungus. Similar phenomena
fact, various plant extracts have been examined by were observed by Mughal et al. (1996) who found that
different investigators for their antifungal activity with the some allelochemicals can enhance fungal growth at
objective of exploring environmentally safe alternatives of different concentrations. The differences in the toxicity of
plant disease control (Bajwa et al., 2006). different extracts could be attributed to the presence of
The results of this conceptual study clearly reflect that the active principles that are extracted by different
N. glauca has inherent ability to induce allelopathic solvents, which may be influenced by several factors
effects on the in vitro growth of the tested fungal species. such as method of extraction, type of extracting solvent
 Rinez et al. 4659

and time of harvesting plant materials (Nicolls, 1969; differences in chemical nature of those solvents. It is
Qasem et al., 1996). In fact, as shown in our study, likely that various types of chemicals were dissolved in
organic extracts were found to be relatively more different solvents resulting in variable biological activity
effective in decreasing the mycelial growth of T. even in the same plant part or organ extracts when
harzianum whereas T. viride exhibited greater resistance distinct solvents were used. Many examples in the
against allelopathic compounds of N. glauca. This literature support these findings. Indeed, Jabeen et al.
difference in Trichoderma species response may be (2008) observed differences in the inhibitory effects of
attributed to their genetic or physiological differences aqueous and organic extracts of different Melia
(Shaukat et al., 1983). Previous studies support also azedarach parts when tested against Ascochyta rabiei.
these results as reported by Martinez-Lozano et al. Alkhail (2005) studied the effect of aqueous and ethanolic
(2000) that Sargassum filipendula extracts also exhibited extracts of Allium sativum, Carum carvi, A. indica and
variable inhibitory effects against Aspergillus species Eugenia caryophyllus against F. oxysporum, Botrytis
including Aspergillus niger, A. flavus and A. parasiticus. cinerea and Rhizoctonia solani and found that aqueous
The mycelial growth of the three F. oxysporum formae extracts exhibited more inhibitory activity to fungal growth
speciales tested seems to be less affected, precisely at than ethanolic extract. Similarly, Mokbel and Hashinaga
the concentrations tested, by N. glauca extracts as (2005) found variable antimicrobial activity of n-hexane,
compared to Trichoderma species. Differential sensitivity ethyl acetate, butanol and methanol extracts of Citrus
of fungi to various bio-pesticides may be due, among grandis against five species of bacteria and three fungal
other factors, to the chemical structure of the active species (Botrytis cinerea, Rhizopus stolonifer and
ingredient and or metabolic activity of the target fungus Penicillium expansum). Zafar et al. (2002) reported that
(Viyas, 1984). As demonstrated in our study, the chloroform extract of leaves of M. azedarach was active
maximum allelopathic stress (inhibition by about 23.5%) against F. chlamdosporum while hexane, ethanol and
induced by N. glauca leaf aqueous extract was recorded water extracts were not.
at 1% concentration. In contrast, at this same In our study, the inhibitory effect recorded with N.
concentration, flower aqueous extracts exhibited the glauca extracts may be attributed, in part, to the presence
lowest (4%) inhibitory effects against all the three of some alkaloids. In fact, Saunders (1979) signaled that
phytopathogenic species tested. Pandey et al. (2010) the genus Nicotiana has been reported as containing
observed a similar phenomenon with Cinnamomum alkaloids in the vacuole. Several alkaloids are able to
zeylanicum extracts. These authors concluded that affect biological functions even at very low concentrations
antifungal substances seem to be more prominently and thus, they exhibit antimicrobial activity (Mahajan et
present in the bark as compared to leaves. This al., 1982; McCarthy et al., 1992; Srivastava et al., 1994;
difference could be attributed to the presence of variable Atta-Ur et al., 1997; Singh et al., 1994, 1999, 2000).
amounts of bioactive secondary metabolites in different Antifungal activity of alkaloids was already reported in
parts of the plant. The composition of these secondary several other works including different plants (Maurya et
metabolites in turn varies from species to species, al., 2001; 2002; Ahmed et al., 2004; Annapurna et al.,
climatic conditions, and the physiological stage of plant 2004; Chung et al., 2004). For example, Olugbade et al.
development (Pandey, 2007). (1992) showed that alkaloids present in the bulb of Crinus
The addition of relatively increasing or decreasing jagus possessed antifungal activity against Candida and
concentrations of N. glauca leaf and flower organic Aspergillus spp.
extracts caused depression or stimulation of F. It is concluded from our study that aqueous and organic
oxysporum f. sp. lycopersici and F. oxysporum f. sp. extracts of various parts (leaf and flower) of N. glauca
tuberosi growth. These results are also supported by the may be used as biofungicides against some pathogenic
fact that some allelopathic substances, as previously fungi. In fact, among the tested aqueous extracts, N.
reported by Puruis et al. (1985), have variable effects, glauca leaf extract was proved to be the most effective
either inhibitory or stimulatory, when applied at different against F. oxysporum formae speciales at the lowest
concentrations. Similarly, Fabry et al. (1996) reported that concentration. However, their use, at a given dose, may
extracts of Entada abyssinica, Terminalia spinosa, negatively affect growth of some antagonistic fungi as
Harrisonia abyssinica, Ximenia caffra, Azadirachta indica, shown with Trichoderma species. Thus, further
Zanha africana and Spilanthes mauritiana, at different researches are needed concerning target pathogens and
concentrations had different effects on the radial growth the adverse effects on the antagonistic microorganisms,
of Candida spp. and Aspergillus spp. The findings of the and on the concentrations that may effectively inhibit
current study are consistent with those of Farooq (2002) plant pathogens without harming biocontrol agents.
on the effects of different concentrations of Achillea
millefolium extracts on the linear growth of
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