African Journal of Biotechnology Vol. 10(42), pp.

8291-8295, 8 August, 2011

Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB11.241
ISSN 1684–5315 © 2011 Academic Journals

Full Length Research Paper

In vitro antifungal evaluation of various plant extracts

against early blight disease (Alternaria solani) of potato
Yusuf Yanar1*, Ayhan Gökçe1, Izzet Kadioglu1, Halit Çam1 and Mark Whalon2
1
Department of Plant Protection, Agriculture Faculty, Gaziosmanpaşa University, Taşliçiftlik Yerleşkesi, Tokat, Turkey.
2
Department of Entomology, Michigan State University, East Lansing, MI, USA.
Accepted 23 May, 2011

Antifungal activities of 27 plant extracts were tested against Alternaria solani (E. & M.) Jones and Grout
using radial growth technique. While all tested plant extracts produced some antifungal activities, the
results revealed that Circium arvense, Humulus lupulus, Lauris nobilis and Salvia officinalis showed
significant antifungal activities. The leaf extract of L. nobilis was most effective in inhibiting the
mycelial growth of A. solani (79.35%) at 4% concentration, followed by S. officinalis, H. lupulus, and C.
arvense with 76.50, 61.50 and 55.83% inhibition, respectively. The other tested plant extracts exhibited
modarate activity and average mycelial growth inhibition of fungus varied from 9.15 to 50.58%. The
lowest antifungal activity was observed on Hypericum perforatum extract. The antifungal activity of
extracts of C. arvense, H. lupulus, L. nobilis and S. officinalis were further evaluated at different
concentrations (0.2, 0.4, 2, 4 and 8% (w/v)) against A. solani. Inhibitory effects of these extracts were
increased with increasing concentration. The minimum inhibitory concentration (MIC) of H. lupulus, L.
nobilis and S. officinalis were 8% (w/v). Further studies on isolation and characterization of the active
(antifungal) compound is needed before the possible use of the tested extracts in control strategies of
this fungus.

Key words: Plant extracts, Alternaria solani, antifungal, minimum inhibitory concentration (MIC).

INTRODUCTION

Potato is one of the most important crop in the world and susceptibility. It has been reported that severe epidemics
is planted in 18.2 million ha and total yield reached 314.1 can reduce yields by up to 30% (Christ and Maczuga,
million ton (FAO, 2010). In Turkey, it is grown in an area 1989; Shtienberg et al., 1990). Control of early blight
of 154000 ha with an annual production of 4.3 million ton disease has been accomplished primarily by the appli-
(FAO, 2010). Potato plants are subjected to attack by cation of chemical fungicides (Jones et al., 1991).
numerous diseases wherever the crop is planted. Fungal Several effective pesticides have been recommended
pathogens, Alternaria solani (E. & M.) Jones and Grout for use against this pathogen, but they are not considered
causes early blight disease of potato and occurs annually to be long-term solutions, due to concerns of expense,
to some degree in most production areas. The timing of exposure risks, fungicide residues and other health and
appearance and rate of disease progression help to environmental hazards. In an attempt to modify this
determine the impact of the disease on the potato crop. condition, some alternative methods of control have been
The disease occurs over a wide range of climatic adopted. Natural products isolated from plant appear to
conditions and depends in a large part, on the frequency be one of the alternatives as they are known to have
of foliage wetting from rainfall, fog, dew, or irrigation, and minimal environmental impact and danger to consumers
on the nutritional status of foliage as well as cultivar in contrast to synthetic pesticides (Varma and Dubey,
1999). Control of microorganism linked plant disease with
plant extracts as components in integrated pest manage-
ment strategy has been tested by many researchers.
*Corresponding author. E-mail: yyanar@gop.edu.tr. Fax: +90- Chapagain et al. (2007) reported that saponin rich-
356-2521488. extracts (4%) from Balanites aegyptiaca fruit mesocarp,
8292 Afr. J. Biotechnol.

showed 34.7% growth inhibition against A. solani. Also placed in the centre of each Petri plate. The position of the disc was
Muto et al. (2005) tested the extracts derived from fresh marked on the base of the dish with a marker pen and two
orthogonal axes passing through the centre of the disc were
and dry tissues of 14 plant species against A. solani.
marked to be used as references for recording growth. Plates were
Mohana and Raveesha (2007) reported that the aqueous incubated at temperature of 28 ± 2°C for 7 days. Radial growth
extract from Decalepis hamiltonii at 30% concentration along each line was recorded at exactly 24 h intervals using
caused 84.83% mycelial growth inhibition on A. alternata callipers (Mitutoyo). The inhibitory activity of each treatment was
and increase in extract concentration up to 50% resulted expressed as the percent growth inhibition as compared to the
in 100% inhibition. Report also showed that all tested negative control (0%) using the following formula (Pandey et al.,
1982):
plant extracts lowered the disease severity of early blight
in summer growing season, especially the extracts of Growth inhibition (%) = DC – DT / DC x 100
lemon grass leaves, garlic bulbs, basil leaves and marjo-
ram leaves, respectively. Where, DC = Diameter of control and DT = diameter of fungal
The objectives of the present study is to evaluate the colony with treatment. Each treatment was replicated four times
with five plates per replication.
antifungal activity of methanolic extracts of 27 plant
species against A. solani under in vitro conditions.
Determination of minimum inhibitory concentration (MIC)

MATERIALS AND METHODS Based on effects of plant extracts on radial growth experiment,
Circium arvense, Humulus lupulus, Lauris nobilis and Salvia
The pathogenic isolates of A. solani (E. & M.) Jones and Grout (AS- officinalis extracts were further tested for the determination of MIC.
5) was isolated from the potato leaves showing typical symptoms of Various concentrations (0.2, 0.4, 2, 4 and 8% (w/v)) of the extracts
early blight by using potato dextrose agar (PDA) medium and of plant species were prepared by adding appropriate quantities of
identified as A. solani according to Simmsons (2007). stock solution of each extract and distilled water to PDA medium
and thoroughly mixed with the medium. Twenty milliliters of each
medium was poured into each 90 mm diameter sterilized Petri
Plant materials and extract preparation plates. Plates were inoculated, incubated and evaluated as des-
cribed earlier. Each treatment was replicated four times with five
The extract of twenty seven naturally growing plant species (Table plates per replication. Antracol WP 70 (Propineb %70) (Bayer) was
1) were used in the present study. The plants were collected during used as a standard, synthetic fungicide for comparison of results
spring and summers of 2002 to 2003 from different localities of under identical conditions. The MIC value was defined as the
Taşlıçiftlik, Tokat, a temperate region of Turkey, where the altitude lowest extract concentration required for complete suppression of
is 640 m and the soil is sandy lime soil, except fruits of Styrax mycelial growth of the tested fungus (Barbour et al., 2004).
officinalis and Hedera helix which were collected from Mersin. The
plant parts (leaves and fruits) were air dried at room temperature for
three weeks in dark conditions. The dried plant parts were milled to Statistical analysis
a fine powder in a mill (Model M 20 IKA Universal Mill, IKA Group),
and stored at room temperature in closed 2000 ml glass jars in the The data on effect of the treatments on the growth of pathogens
dark, at 20°C until used. Fifty grams each of the dried, powdered was analyzed by analysis of variance (ANOVA), and treatment
plant sample were weighed and placed into 1000 ml Erlenmeyers means were compared by Fishers least significant difference test
flasks and then 500 ml of absolute methanol (Sigma-Aldrich) was (LSD) at P = 0.05.
added to the flask. The flasks were closed with cotton balls and
covered with aluminium foil then placed on a horizontal shaker (HS
260 Basic, IKA Group) and shaked at 120 rpm for 24 h in the dark
RESULTS
and then the suspension were filtered through two layers of cheese
cloth into different 250 ml evaporating flasks. Excess alcohol was
evaporated to dryness using a rotary evaporator (RV 05 Basic 1B, Effect of plant extracts on radial growth of A. solani
IKA Group) at 32 ± 2°C and the remaining residue was diluted by
adding appropriate quantity of sterilized distilled water containing Twenty seven plant species, belonging to the various
10% acetone (v/v) to prepare 40% (w/v) stock suspension (Gokce families were selected and evaluated for antifungal
et al., 2006). These stock suspensions were stored at 4°C and used
within four days.
activity. Twenty two out of twenty seven plant extracts at
4% concentration were effective in inhibiting the radial
growth of A. solani (E. & M.) Jones and Grout isolate
Screening of plant extracts against A. solani (AS-5). The leaf extract of L. nobilis was most effective in
The antifungal properties of the extracts were tested using the
inhibiting the mycelial growth of A. solani (79.35%) at 4%
radial growth method as described by Banso et al. (1999). PDA concentration, followed by S. officinalis, H. lupulus and C.
medium was prepared by autoclaving at 121°C and cooled to 45°C. arvense with 76.50, 61.50 and 55.83% inhibition, respec-
Afterwards, appropriate quantities of stock solution of each extract tively (Table 1). Inhibitory activities of L. nobilis and S.
was added to PDA medium to get 4% (w/v) concentrations of the officinalis extracts were significantly different when com-
extracts in the medium and mixed. In the control, 10% acetone (v/v)
pared with the rest of the tested plant extracts. Not much
water mixture was added to PDA. Twenty milliters of each medium
was poured into 90 mm diameter sterilized Petri plates and left to difference was observed in the activities of H. lupulus and
solidify over night. Mycelial discs of 5 mm diameter were taken from C. arvense extracts in the period of test. On the other
7 days old A. solani cultures with a sterilized cork borer and were hand, activities in the extracts of E. elaterium, H.
 Yanar et al. 8293

Table 1. Effect of plant extracts on the mycelial growth of A. solani.

Family Plant species Plant part Per cent reduction*

Lauraceae Laurus nobilis L. Leaves 79.35a

Lamiaceae Salvia officinalis L. Leaves 76.50a

Cannabinaceae Humulus lupulus L. Flower bud 61.50b

Asteraceae Circium arvense (L.) Scop. Leaves 55.83bc

Styracaceae Styrax officinalis L. Fruit 50.58cd

Apocynaceae Nerium oleander L. Leaf 49.75cd

Rubiaceae Galium aperina L. Leaves 44.10de

Solanaceae Solanum nigrum L. Fruit 40.88e

Chenopodiacea Chenopodium album L. Leaves 36.80e

Caprifoliaceae Sambacus nigra L. Fruit 27.90f

Poaceae Sorghum halepense (L.) Pers. Fruit 27.03f

Asteraceae Arctium lapa L. Leaves 26.28f

Araliaceae Hedera helix L. Leaves 25.95f

Fabaceae Glycrrhiza glabra L. Fruit 22.30fg

Solanaceae Datura stramonium L. Fruit 22.20fg

Asteraceae Artemisia vulgaris L. Leaves 21.38 fg

Poaceae Cynodon dactylon L. Leaves 16.63gh

Scrophulariaceae Verbascum songaricum L. Leaves 15.20gh

Poaceae Lolium temulentum L. Leaves 12.10hi

Asteraceae Xanthium strumarium L. Fruit 11.53hi

Urticaceae Urtica urens L. Leaves 9.85hi

Rubiaceae Rubia tinctoria L. Leaves 9.15hi

Cucurbitaceae Ecballium elaterium (L.) A. Rich. Fruit 5.60ij

Guttiferae Hypericum perforatum L. Flowers 4.50ij

Ranunculaceae Delphinium consolida L. Leaves 0.10j

Asteraceae Chrysanthemum segetum L. Leaves 0.00j

Apiaceae Conium maculatum L. Leaves 0.00j

Control (PDA with 10% acetone ) 0.00j

*Percentage (%) growth inhibition was calculated with comparison with the growth of the control. In
column, means followed by the same letter are not significantly different at 5% level by LSD (7.90).

perforatum and Delphinium consolida was not signifi- Conium maculatum did not show any inhibitory activity
cant, whereas extracts of Chrysanthemum segetum and against A. solani (Table 1).
8294 Afr. J. Biotechnol.

Table 2. Minimum inhibitory concentration of four plant extracts extract produced similar inhibition at high concentration.
against A. solani. Inhibition reached 100% at 8% concentration (w/v) for L.
nobilis, S. officinalis and H. lupulus, but for C. Arvense, it
Extract Concentration (% w/v) Growth inhibition (%) was higher than 8% (Table 2). The MIC of L. nobilis, S.
C. arvense (LSD=6.7) officinalis and H. lupulus was 8% (w/v).
0.2 35.8a
0.4 42.9b
2.0 46.2b DISCUSSION
4.0 55.8c
8.0 76.8d In the present study, we evaluated the antifungal activity
of the extracts of 27 plant species against early blight
L. nobilis (LSD=11.0) pathogen (A. solani). Leaf extract of L. nobilis (4%) was
0.2 24.7a
highly effective in reducing the radial growth of A. Solani.
At some concentrations, extracts from S. officinalis (leaf),
0.4 26.0a
H. lupulus (flower bud), C. arvense (leaf) and S. officinalis
2.0 58.6b
also inhibited the mycelial growth of the fungus over 50%.
4.0 79.4c Similar effect of other various plant extracts effective
8.0 100.0d against Alternaria spp. have been reported by several
workers (Hassanein et al., 2008; Abd-El-Khair and
S. officinalis (LSD=1.6) Haggag, 2007; Muto et al., 2005; Patil et al., 2001;
0.2 63.8a Srivastava et al., 1997). The aqueous neem leaf extracts
0.4 65.9b inhibited the mycelial growth of A. solani (Hassanein, et
2.0 69.6c al., 2008). Muto et al. (2005) showed that the extracts
4.0 76.5d derived from potato sprouts and Solanum nigrum roots
8.0 100.0e showed complete inhibition of conidial germination of
Alternaria brassicicola at a concentration of 10% (w/v)
H. lupulus (LSD=5.9) from fresh tissues, and 1% (w/v) from dry tissues. Also,
the ethanol extracts from dry fruit tissues of S. nigrum
0.2 42.4a
completely inhibited conidial germination of the fungus.
0.4 45.0a
Vijayan (1989) reported that the bulb extract of Allium
2.0 51.5b sativum, leaf extract of Aegle marmelos and flower
4.0 61.5c extract of Catharanthus roseus inhibited the spore germi-
8.0 100.0d nation and mycelial growth of A. solani. The bulb extract
of A. sativum inhibited the mycelial growth of Alternaria
Propineb 70% (Antracol WP 70) 100.0 helianthi (Sivagami, 2003). Similarly, Mishra et al. (2009)
Control (10% acetone) 0.0 reported that the complete inhibition (100%) of spore
*Means followed by same letter in each column are not germination in A. solani was observed with chloroform
significantly different at p = 0.05 by Fisher’s least significant and acetone extract of Cinnamomum zeylanicum bark as
difference value. Percentage (%) growth inhibition was calculated well as with petroleum ether and ethanol extracts of C.
with comparison with the growth of the control (0%). zeylanicum leaf at the lowest concentration (50 µg/ml).
Nineteen out of twenty seven plant extracts accounted
for less than 50% inhibition of mycelial growth of A. solani
Determination of MIC at a concentration of 4% (w/v). In contrast, extracts from
leaves of D. consolida, C. segetum, and C. maculatum
Based on effects of plant extracts on radial growth were ineffective in inhibiting mycelial growth of A. solani
experiment, C. arvense, H. lupulus, L. nobilis and S. isolate AS-5. Evaluation of L. nobilis, S. officinalis, H.
officinalis extracts were further tested for determination of lupulus and C. arvense extracts further with increasing
MIC. Various concentrations (0.2, 0.4, 2, 4 and 8% (w/v)) concentration of the extracts up to 8% (w/v) exhibited
of these plants extracts were tested against A. solani. increased inhibitory properties of the extracts up to 100%.
Results indicate that inhibition values of the extracts from This is in conformity with the finding of Abd-El-Khair and
C. arvense, L. nobilis, S. officinalis leaves and H. lupulus Haggag (2007) who observed that higher concentration
flower bud were higher in comparison to the control, and of plant extracts induced maximun inhibition in fungal
this effect gradually increased with concentration (Table growth. Similar observations have been reported by
2). The C. arvense leaf extract inhibited mycelial growth Farcasanu and Oprea (2006) who found that doubling the
of A. solani at 35.8% and even at 0.2% (w/v), reaching S. officinalis leaf extract concentration (100 µl/ml),
76.8% at 8% concentration. On the other hand, L. nobilis, inhibited cell growth of yeast almost completely. The
S. officinalis leaves extracts and H. Lupulus flower bud effective antifungal nature of L. nobilis, S. officinalis, and
 Yanar et al. 8295

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