Fungicidal effect of three plants extracts in control of four phytopathogenic fungi of tomato (Lycopersicum esculentum L.) fruit rot.

Fungicidal effect of leaf aqueous extracts of Azadirachta indica, Tithonia diversifolia and Chromolaena odorata were determined on rot causing fungi. In the study, the phytopathogenic fungi isolated from the infected tomato fruit parts and identified based on morphological and cultural characters were: Aspergillus Niger Van Tiegh, Fusarium oxysporum Schlecht, Geotrichum candidium Link and Rhizopus stolonifer Ehrenb. ex. Fr. as confirmed by pathogenicity tests. Leaf aqueous extracts of different concentrations (20, 40, 80, 60 and 100 % w/v) of A. indica, T. diversifolia and C. odorata were added to growth media prior to inoculation. All aqueous extracts of the tested plants significantly (p < 0.05) reduced mycelial growth of the fungal pathogens and this effect gradually increased with increasing concentration. Fungicidal activity was strongly exhibited by A. indica extract at 100% w/v against all the pathogenic fungi. In the case of T. diversifolia extracts inhibitiory effects at 20, 40, 60, 80 and 100% w/v were greater than those of C. odorata on A. Niger , F. oxysporum and G. candidium while for R. stolonifer inhibition, C. odorata produced the highest in the all five concentrations than T. diversifolia extracts. It could be emphatically concluded that the tested plant extracts can effectively control rot causing fungi disease of tomato. This makes them potential biocide in diseases management in that they are cheap and environmentally safe as they showed fungicidal and fungitoxic ability.

INTRODUCTION Tomato (Lycopersicum esculentum L.) is the second most important vegetable crop in the world because of its special nutritive value. The edible fruit of the tomato plant has a series of usages in different forms. The crop is nutritious and contain high amount of dietary source of vitamins A, B, C, E and nicotinic acid (Kanneh et al., 2015;Godia, 2014). In Sierra Leone and other parts of the world, it is consumed as fresh fruit, salads, soup and stew and often used in other dishes (Osei et al., 2014). Its cultivation provides source of employment to many and continue to play a key horticultural role in the sub-region in terms of reducing poverty and food insecurity (Osei et al., 2014). Recently, according to Kanneh et al. (2015), the Global production of fresh fruit tomato is about 100 million tons cultivated on 3.7 million hectares. In Sierra Leone as well as other countries in Africa, the average yield on farm is between 7.5-10t/ha (Godia, 2014) which is far below the potential yield of the crop 45-50 M t/ha. Tomato production is seriously affected by over 200 diseases caused by pathogenic fungi, bacteria, viruses and nematodes (Abad Z. G. and Abad J. A., 1997; Koch et al. 1992;Lukyanenko, 1991; Moriones and Navas-Castillo, 2000). Major fungal diseases affecting tomato production are late blight, early blight, septoria leaf spot, fusarium wilt, and verticillium wilt, corky root rot, damping-off, leaf mould and powdery mildew (Panthee and Chen, 2010). Most farmers control these diseases using fungicides. However, the negative environmental impacts, mammalian toxicity and high costs are making their use unattractive thereby searching for alternatives such as natural plant-based chemicals (Asawalam, 2006). Plants have ability to synthesize aromatic secondary metabolites, like phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins and coumarins (Cowan, 1991). These groups of compounds show antimicrobial effect and serves as plant defense mechanisms against pathogenic microorganisms (Das et al., 2010). Many research workers have tried to find out safe and economical control of plant diseases by using extracts of different plant parts (Hasan et al., 2005;Bdliya and Alkali, 2008). Hence the objective of the study was to determine to efficacy of aqueous leaf extracts for controlling some important postharvest tomato fruit rot diseases in in-vitro.

Preparation of culture media
Potato Dextrose Agar (PDA) (BAM Media M127) was prepared by dissolving 39 grams in 1 litre Erlenmeyer flask and then made up to 1 litre using sterile distilled water. The medium was autoclaved at 121 o C for 15 minutes at 15 lb. The sterilized medium was allowed to cool to 45°C, before supplemented with streptomycin sulphate (3 grams) and aseptically dispensed into sterilized 9 cm diameter glass Petri dishes.

Isolation and Identification of Fungal Pathogens
Diseased tomato fruits were randomly collected from the two different Markets. The Chiejina (2008) isolation method was used. Thin sections (2 mm diameter) were cut from the Periphery of diseased tomato fruits and surface sterilized in 0.1% mercuric chloride for 2-3 min, after which they were rinsed in three changes of sterile distilled water. The sections were plated in water agar and mycelium was transferred into clean PDA plates. The plates were incubated at room temperature (27 ± 2° C) for 6-7 days. Subcultures were made ascetically from the plates into similar clean PDA plates and were incubated under similar conditions until pure cultures were obtained. The identification of the isolated fungi was done macroscopically and microscopically. Macroscopic identification was based on observed culture growth patterns and mycelial colour. Small portions of the fungal cultures were teased and mounted in lactophonel in cotton blue on clean slides, covered with clean cover slips and then viewed microscopically. Fungal identification was confirmed with the aid of books by Barnett

Pathogenicity Test
Each of the fungal isolates obtained from the diseased tomato fruits were tested for their ability to cause the same disease condition previously observed in healthy tomato fruits. Health tomato fruits were washed in sterile distilled water and surface sterilized by dipping into 0.1% mercuric chloride and with the aid of a sterile cork borer, cylindrical cores were removed from each of the tomato fruits. Pure cultures of the isolates of the isolates were introduced into the cores and the cores were replaced and sealed with sterile petroleum jelly. The fruits were kept at room temperature for 7-10 days. On establishment of disease condition, inocula were taken from the infected tomato fruits and cultured. The organisms were reisolated and identified as previously isolated organisms. This was taken as evidence that they incited the disease.

Sources of plant materials and Preparation of Extracts.
Leaves of three plant species namely Azadirachta indica (A. Juss) (Neem), Tithonia diversifolia (Hemsley) A. Gray (Mexican sunflower) and Chromolaena odorata (Linn) were used in the experiment. These were obtained within the premises of the Federal University of Agriculture, Abeokuta. Fresh leaves of A. indica, T. diversifolia and C. odorata were washed in tap water then surfaced-sterilized with (1% NaOCl for 5min and rinsed in five changes of sterile distilled water) and air dried at (28 ± 2 °C) for 1h. 20grams, 40grams, 60grams, 80grams and 100 grams of each plant material were grounded using sterilized Brabantia 5-speed blender (Model BBEK 1051) in 100 ml distilled water, and then filtered through a Whatman ® No. 9 filter paper separately into a 250 ml Erlenmeyer flask to produce 20 %, 40 %, 80%, 60% and 100% extract concentrations.

Effect of plant extracts on mycelial growth
inhibition of fungal pathogens. Extract-media mixtures were prepared by mixing 1 ml extract with 9 ml molten PDA prior to solidification for each extract concentration. Media amended with mycelial disc of a 5-day-old cultures of each fungus were placed in the centre of the petri dishes. The control plates consisted of PDA mixed with 1 ml sterile distilled water. Benlate a standard fungicide, at a concentration of 20 mg/ml was used to assess the efficacy of the plant extracts Owolade and Oskkanlu (1999) modified method. All treatments were in three replicates and incubated at 28 ± 2°C. Radial growth in treatments and control were measured at 24 h interval for seven days. This was expressed as the mean growth along two axes on two predraw perpendicular lines on the reverse side of each plate. The percentage inhibition of mycelial growth by each extract was computed using formula. III. STATISTICAL ANALYSIS Data were subjected to analysis of variance (ANOVA) and significant means were separated using Duncan's Multiple Range Test (DMRT) at 5% level of probability.

Inhibition of mycelial growth of Aspergillus niger by three aqueous plant extracts
The aqueous extracts of the tested plants reduced mycelial growth and inhibition of A. niger.
However, the inhibitory effect of the plant extracts and benlate solution on the mycelial growth A. niger was significantly different (P <0.05) at all the various concentrations. A. indica exerted the highest inhibitory effect of (92.0 %) at 100 % (w/v) followed by T. diversifolia and C. odorata at the same concentration. Fungitoxicity of tested plant extracts against A. niger increased as the concentration increased (Table 1).

Inhibition mycelial growth of Fusarium oxysporum with three aqueous plant extracts
The effect of aqueous tested plant extracts mycelial growth F. oxysporum revealed that three extracts produced significant (P<0.05) levels of inhibition of mycelial growth of F. oxysporum at various concentrations. The highest inhibitory of mycelial growth was manifested by A. indica (87.4%) at 100 % (w/v) concentration while the least (15.5%) inhibition was recorded for A. indica at the concentration of 20 % (w/v). However, benlate solution was superior in mycelial growth inhibition (Table 2)  Table 3)
However, the highest inhibition was exerted by A. indica at 100 % (w/v) while the least inhibition (15.9 %) was exhibited by T. diversifolia at 20 % (w/v). As the concentration increased mycelial inhibition growth increased (Table 4)

IV. DISCUSSION
The mycelial growth inhibition and of the pathogens by the leaf aqueous extracts of A. indica T. diversifolia, and C. odorata investigated in this study indicated that, antifungal activity showed by the tested plant extracts had inhibitory effects on the growth of A. niger, F. oxysporum, G. candidium and R. stolonifer. These results further revealed that antifungal activities of the extracts were enhanced by increasing the concentration from 20 to 100 % (w/v); hence the inhibition activities of the extracts were concentration dependent. This is in agreement with the report of Ilondu (2012) and Chiejina and Ukeh (2013) who indicated that increase in the antifungal activities had corresponding increase in concentration of plant extracts.  Olufolaji, 1999 andOnifade, 2002). However, the differences in the efficacy of the extracts could be attributed to the differences their active ingredients (Onifade, 2002;Okigbo et al. 2009). Major compounds of plant extracts are phenols, flavonoids, alkaloids, quinones, saponines, tannins and sterols (Halama and Van Haluwin, 2004) and their fungicidal or fungistatic properties against various plant pathogens have been established (Scheuerella and Mahaffee, 2002). These products might either have direct inhibitory effects on pathogens, exhibiting fungicidal or fungistatic properties. They could help in the establishment of favorable conditions for antagonistic microbes (Scheuerella and Mahaffee, 2002). Benlate solution at 20mg/ml was found to be more effective than aqueous plant extracts in inhibiting the mycelial growth of the pathogens. This may be as result of the refined nature of benlate and its active ingredients being more concentrated than of the plant extracts.

V.
CONCLUSION This study demonstrated that A. indica, T. diversifolia, and C. odorata should be used as a potential biocide in plant disease management, as they showed fungicidal and fungitoxic ability (at 100% w/v). The utilization of plant extracts to control disease in vegetable filed minimizes or eliminates the risks and hazards of toxic fungicides, especially on freshly consumed vegetables. It is anticipated that further research into these extracts would identify the active compounds responsible for their fungicidal activity.