Under Egyptian conditions tomato plants are vulnerable to infection early blight
disease caused by Alternaria solani (Ellis and Martin) Sorauer (Abada
et al., 2008) which causes great reduction in the quantity and quality
of fruit yield. The Alternaria fungus can cause disease on all parts
of the plant (leaf blight, stem collar rot and fruit lesions) and result in
severe damage during all stages of plant development (Abada
et al., 2008).
This disease is controlled mainly with agro chemicals. However, the world wide
trend towards environmentally-safe methods of plant disease control in sustainable
agriculture calls for reducing the use of these synthetic chemical fungicides.
In an attempt to modify this condition some alternative methods of control have
been adopted. Recent efforts have focused on developing environmentally safe,
long lasting and effective biocontrol methods for the management of plant diseases.
Natural plant products are important sources of new agrochemicals for the control
of plant diseases (Kagale et al., 2004). Furthermore,
biocides of plant origin are non-phytotoxic, systemic and easily biodegradable
(Qasem and Aau-Blan, 1996). It is now known that various
natural plant products can reduce populations of foliar pathogens and control
disease development and then these plant extracts have potential as environmentally
safe alternatives and as components in integrated pest management programs (Bowers
and Locke, 2004). A number of plant species have been reported to possess
natural substances that are toxic to several plant pathogenic fungi (Goussous
et al., 2010). Dushyent and Bohra (1997)
studied the effect of 11 different plant extracts on mycelial growth of A.
solani and found that leaf extracts of some plants i.e. Tamarix
aphylla and Salsola baryosma totally inhibited the growth of the
pathogen in vivo. Also, Wszelaki and Miller (2005)
reported that garlic extracts significantly reduced the early blight disease
on tomato. Additionally, several plant extracts have shown antimicrobial activity
against fungal pathogens under in vitro and in vivo conditions
(Kagale et al., 2004). Therefore, our present
study investigated the efficacy of various Egyptian plants leaf extracts, Ocimum
basilicum, Azadirachta indica, Eucalyptus chamadulonsis, Datura
stramonium, Nerium oleander and Allium sativum for control
of early blight of tomato under greenhouse and field conditions. The treatments
were compared with a commonly used fungicide (Ridomil Plus).
MATERIALS AND METHODS
Plant materials: Seeds of tomato (Solanum lycopericum L.) cultivar
Super Strain B were obtained from the Ministry of Agriculture, Egypt and used
in this study. Seeds were sown in plastic pots, each of 30 cm diameter and containing
a soil mixture consisting of sand 3 kg pot and 10 g slow-release fertilizer
per kg (N.P.K 12: 4: 6). All pots were placed on a benchtop in a climate controlled
greenhouse at 30±5°C with 68-80% RH and watered as required.
Isolation and pathogenicity tests of the causal pathogen: Six fungal
isolates were isolated from naturally diseased tomato leaves and fruits showing
blight symptoms. Pathogenicity tests of Alternaria sp. isolates were
carried out under greenhouse conditions in 2007-2008 experiments in greenhouse
of Plant Pathology Department, Faculty of Agriculture, Assiut University, Assiut,
Egypt. The inoculum was prepared by growing each of the tested isolates on PDA
medium at 27°C for 15 days. Then 10 mL of sterile distilled water was added
to each plate and colonies were carefully scraped with a sterile needle. The
resulting conidial suspension from each isolate was adjusted to 5x106
spores mL-1 and used for inoculation of 20 tomato plants (cv. Super
Strain B), using an atomizer. After inoculation, plants were covered with polyethylene
bags for 48 h to maintain a high humidity conditions. After 48 h, bags were
removed and plants were kept under greenhouse conditions. Pots were maintained
in completely randomized design under glasshouse conditions. Two weeks after
inoculation, disease severity was recorded. The trial was repeated twice. The
intensity of disease was recorded in each treatment following the score chart
0-9 scale (0-Healthy; 1 = 1 to 5%; 2 = 6 to 10%; 3 = 11 to 25%; 5 = 26 to 50%
and 7 = 51-75% 9 = >76% leaf area infected) proposed by Latha
et al. (2009).
Preparation of extracts: Extracts from leaves of six plants namely,
O. basilicum, A. indica, E. chamadulonsis, D. stramonium,
N. oleander and A. sativum were collected from different parts
of Assiut, Egypt and tested for their efficacy in reducing the mycelial growth
of A. solani in vitro using the poisoned food technique (Schmitz,
1930). Ten grams of fresh leaf material of each plant species was collected,
washed with water and crushed in a mortar and pestle by adding sterile distilled
water at the rate of 10 mL g-1 of plant tissue and the homogenates
were centrifuged at 10000xg for 15 min at 4°C and the supernatant solutions
were collected. The plant extract was diluted further to have 1 and 5% concentration
(v/v). These fractions were sterilized using 0.2 m disposable syringe filters
and used for assay of antimicrobial activity as described below.
The PDA media amended with five milliliters of aqueous leaf extract, 1 and 5%, of each plant extracts individually were inoculated with mycelial discs (9 mm diameter) taken from the advancing edges of 7 day-old pure cultures of A. solani. The control experiments had distilled water instead of plant extracts. The inoculated media were incubated at temperature 27±1°C. Four plates were each treatment was used as a replicates. The diameter of the fungal colony was measured using a meter rule along two diagonal lines drawn on the reverse side of each Petri plate 7 days after inoculation. Each treatment was replicated three times with four plates per replication.
Testing of plant extracts against early blight of tomato under greenhouse conditions: Fungicide (Ridomil Plus 50% WP, 15% metalaxyl+35% Copper oxychloride, at 2 g L-1) and plant extracts treatments at 1 and 5% were applied as foliar application, 30 mL on tomato plants, seven week olds and every 15 days up to 60 days of planting after two days from second spraying tomato plants were inoculated with 20 mL of A. solani suspension containing 5x106 cfu mL-1. After inoculation, plants were kept in a climate chamber with 28°C day temperature and 85% relative humidity. Disease development was recorded 15 days after inoculation. Disease severity was recorded as described before. Greenhouse experiments were repeated twice.
Testing of plant extracts on early blight of tomato under field conditions: The field trials were conducted at the Experimental Farm of Faculty of Agriculture, Assiut University, Assiut, Egypt in 2008 and 2009 growing seasons. Field plots (3x3.5 m) comprised two rows and 5 plants/row arranged in a completely randomized block design. Three plots were used as replicates for each treatment as well as for the untreated control treatment. Application of plant extracts was carried out as in greenhouse experiments. Disease development was recorded 15 days after inoculation. Disease severity was recorded as described before. Field experiments were repeated twice. At harvest time, the average accumulated yield was calculated for each treatments including untreated control. Ten plants from each replicate were harvested to assess the total yield of each treatment (ton ha-1).
Statistical analysis: All experiments were performed twice. Analyses
showed no significant interaction between the two tests run for any of the treatment.
Therefore, results from duplicate tests were combined for final analysis. Analyses
of variance were carried out using MSTAT-C program version 2.10 (MSTAT-C
1991). Least Significant Difference (LSD) was employed to test for significant
difference between treatments at p = 0.05 (Gomez and Gomez,
Identification of the causal pathogen: Six fungal isolates were obtained
from naturally diseased tomato leaves and fruits showing blight symptoms and
identified as A. solani, based on the morphological characteristics (Ellis,
Pathogenicity tests: Results in Fig. 1 indicate that all the tested isolates of A. solani were able to infect tomato plants causing typical early blight symptoms with different degrees of disease severity. Data indicate that isolates 1, 3 and 5 were highly pathogenic and caused the highest disease severity. Isolates 2 and 4 exhibited the lowest disease severity on tomato plants followed by isolate 6. On the basis of this result, isolate 1 was used in the following experiments.
Effect of plant extracts on radial growth of A. solani: Six plant species were selected and evaluated for the antimicrobial activity against A. solani. All the leaf extracts of tested plants at 1 and 5% concentration were effective in inhibiting the radial growth of A. solani, compared to control. The leaf extract of D. stramonium, A. indica and A. sativum at 5% concentration caused highest reduction of mycelial growth of A. solani (44.4, 43.3 and 42.2%, respectively), followed by E. chamadulonsis and D. stramonium at 1% concentration. O. basilicium at 1 and 5% and N. oleander at 1% caused the lowest inhibition of mycelial growth of the pathogen. Overall the Ridomil Plus at 2 g L-1 caused the highest reduction of the pathogen by 77.8% (Table 1).
Effect of plant extracts on early blight incidence of tomato under artificial infection in greenhouse conditions: The different concentrations of six plant extracts, O. basilicum, A. indica, E. chamadulonsis, D. stramonium, N. oleander and A. sativum, significantly reduced the early blight diseases (Table 2). The greatest reduction 82.8% of disease severity was achieved by Ridomil Plus at 2 g L-1. The most effective treatments from plant extracts were A. sativum at 1 and 5% followed by D. stramonium at 1 and 5% concentration. The least reduction of disease severity was achieved by O. basilicum at 1% (35.2%). Other plant extracts treatments were moderately effective.
Effect of some plant extracts on early blight incidence of tomato under
field conditions: All treatments, plant extracts and fungicide (Ridomil
Plus at 2 g L-1), significantly reduced the early blight disease
under field conditions (Table 3).
||Pathogenicity tests of six isolates of Alternaria solani
on tomato plants (cv. Super Strain B) under greenhouse conditions. Different
letters indicate significant differences among treatments according to least
significant difference test (p = 0.05). Means of standard deviation for
twenty plants per treatment are shown
||In vitro effect of six plants extracts on the linear
growth of Alternaria solani
|A five milliliters of aqueous leaf extracts prepared from
each of the plant sample was mixed with 45 mL of PDA medium (1 and 5%).
b The percent inhibition of radial growth of A. solani
was calculated. Each treatment was replicated three times with four plates
per replication. Values in the column followed by the same letter are not
significantly different at (p = 0.05)
||Influence of six plants extracts on early blight disease of
tomato plants under greenhouse conditions
|The intensity of the disease was recorded in each treatment
as proposed by Latha et al. (2009). Values
in the column followed by different letters indicate significant differences
among treatments according to least significant difference test (p = 0.05)
|| Influence of six plant extracts on early blight disease and
yield of tomato under field conditions
|Values in the column followed by different letters indicate
significant differences among treatments according to least significant
difference test (p = 0.05)
The greatest reduction of disease severity at 74.2% was achieved by Ridomil
Plus at 2 g L-1 followed by A. sativum at 5% and the least
reduction was obtained when tomato plant were treated with O. basilicum
at 1 and 5% (46.1 and 45.2 %, respectively). The other treatments were moderately
Effect of treatments on fruit yield: Data in Table 3 indicate that the efficacy of the Ridomil Plus and plant extracts was reflected in the fruit yield produced. Plants sprayed with fungicide, D. stramonium and A. sativum at 5% increased the fruit yield 85.7, 76.2 and 66.7% respectively, compared to nontreated control. In contrast, O. basilicum, A. indica, E. chamadulonsis and N. oleander treatments increased the fruit yield moderately, ranged between 28.6 to 38.1% compared to infected control.
Our results indicated that all tested plant extracts, Ocimum basilicum,
Azadirachta indica, Eucalyptus chamadulonsis, Datura stramonium,
Nerium oleander and Allium sativum and fungicide, Ridomil Plus
50% WP, caused significant reduction in the linear growth of A. solani.
This reduction was gradually increased by increasing the concentration of extracts
in the growth medium. Ridomil Plus was more effective than the plant extracts.
Similar effect of various other plant products effective against Alternaria
spp. have been reported by several workers (Latha et
al., 2009; Goussous et al., 2010). Vijayan
(1989) reported that the bulb extract of A. sativum, leaf extract
of Aegle marmelos and flower extract of Catharanthus roseus inhibited
the spore germination and mycelial growth of A. solani. The inhibitory
effect of the fungicide on the growth of A. solani was reported by many
researchers (Patil et al., 2001; Abada
et al., 2008). The inhibitory effect of the tested plant extracts
may be due to their direct toxic effect to the pathogen as reported by Vijayan
(1989). Investigations on mechanisms of disease suppression by plant products
have suggested that the active principles present in plant extracts may either
act on the pathogen directly (Amadioha, 2000), or induce
systemic resistance in host plants resulting in reduction of disease development
(Kagale et al., 2004).
The greenhouse and field experiments indicated that foliar spray of tomato
plants by plant extracts and fungicide resulted in significant reduction in
early blight infection. However, the tested fungicide was more efficient than
plant extracts. These results were similar to previous work on the role of plant
extracts in fungal disease control. Several authors including Krebs
et al. (2006), Curtis et al. (2004)
and Latha et al. (2009) reported that plant extracts
from 20 non-host plant species caused reduction of early blight disease and
suppressed the mycelial growth of A. solani. All tested plant extracts
treatments improved the yield of tomato plants compared to infected control.
In conclusion, present study demonstrated that many plant extracts, O. basilicum, A. indica, E. chamadulonsis, D. stramonium, N. oleander and A. sativum, can be used for the bio-control of early blight disease. Thus, this method of control can contribute to minimizing the risks and hazards of toxic fungicides, especially on vegetables produced for fresh consumption. Further research into these extracts will identify the active compounds responsible for their fungicidal activity.