Evaluation of the Effects of the Extract of Vernonia amygdalina on Fungi Associated with Infected Tomatoes (Lycopersicon esculentum) in Jos North Local Government Area, Plateau State, Nigeria

Aim: To evaluate the effects of different concentrations of ethanolic extract of Vernonia amygdalina on fungi associated with infected tomatoes (Lycopersicon esculentum) obtain from Jos North Local Government Area. Place and Duration of Study: Jos Local Government Area markets; Microbiology Laboratory, Federal College of Forestry between May and August, 2015. Materials and Methods: A total of thirty (30) infected tomatoes were collected. Three species of fungi were isolated, identified and observed in relation to their percentage of distribution. Isolates Original Research Article John et al.; ARRB, 9(4): 1-8, 2016; Article no.ARRB.23698 2 obtained were subjected to Vernonia amygdalina ethanolic leaf extract to determine antifungal sensitivity at varying concentrations. 70% Ethanolic extract of Vernonia amygdalina at 12.5, 25, 50 and 100 mg/ml were used for sensitivity test with positive control using fluconazole (50mg/ml) and negative control using distilled water. The mean effects of the treatments on the isolates were determined at 24hours and 48hours, respectively. Results: The percentage of distribution showed Geotrichum candidum (45.16%), Rhizopus stolonifer (22.58%) and Fusarium oxysporum (32.26%). After 24 hours, no inhibition was recorded on Fusarium oxysporum at all levels of concentration of the test plant, except the positive control which showed the highest inhibitory effect. However after 48 hours, there was inhibitory effect across all the isolates at all concentration levels of the test plant with significant differences between each level. 100 mg/ml of the extract had high inhibitory effect of 13.00 mm in comparison with other concentration levels. The lowest inhibitory effect of 1.00 mm was observed at 12.5 mg/ml concentration. The positive control showed no inhibitory effect on Rhizopus stolonifer within the duration of the observation. The inhibitory effects of the extract results shows significant difference among the three fungi tested (P<001). Conclusion: This study revealed that natural products from higher plants are relatively broad spectrum, bio-efficacious, economical, and biodegradable and can be ideal for use as agrochemicals. Among plants that are known to possess those qualities is Vernonia amygdalina.


INTRODUCTION
Vernonia amygdalina is a valuable medical plant that is widespread in East and West Africa [1], it is known as bitter leaf, due to its characteristics bitter taste and flavor, and may be used as an active anticancer [2], antibacterial, antimalarial and antiparasitic agent [3].
The leaves are used for human consumption and washed before eating to get rid of the bitter taste. They are used as vegetable and stimulate the digestive system, as well as they reduce fever. They are also used as local medicine against leech, which are transmitting bilharzias. Free living chimpanzees eat the leaves, if they had been attacked by parasites. V. amygdalina is also used instead of hops to make beer in Nigeria [4]. Furthermore, it is found in homes in villages as fence post and pot-herb [5].
Ghassan et al. [6] evaluated eco-friendly botanicals (natural plant extracts) as alternatives to synthetic fungicides and reported that fungicides are widely used in conventional agriculture to control plant diseases. Prolonged usage often poses health problems as modern society is becoming more health-conscious. Penicillium digitatum, the cause of citrus green mould, is an important postharvest pathogen which causes serious losses annually. The disease is currently managed with synthetic fungicides. There is, however, a growing concern globally about the continuous use of synthetic chemicals on food crops because of their potential effects on human health and the environment.
The problems caused by synthetic pesticides and their residues have increased the need for effective biodegradable pesticides with greater selectivity. Alternative strategies have included the search for new types of pesticides, which are often effective against a limited number of specific target species, m [7]. Applied chemical pesticides are one of the effective and fast means for reducing the loss of post-harvest diseases. Nevertheless, the excessive use of these chemicals for controlling mould fungi in fruit has been counterproductive, causing damage to the environment and humans, with increased demands to reduce the use of these chemicals that accumulate in fruits and vegetables [8].
The use of these chemicals has increased significantly with improper use and have being left to grow globally, leading to the use of these chemicals that accumulate in fruits and vegetables in circulation. It is claimed that these fruits were major health problems after they became a crop of major commodities exported to different countries worldwide. As persistent hazardous chemicals, the use of these pollutants, such as Thiabendazole (TBZ) and Imazalil to control a wide range of fungi has led to an imbalance in the natural enemies in the environment [9,10]. Natural plant products derived from plants effectively have enormous potential to influence modern agrochemical research. When extracted from plants, these chemicals are referred to as botanicals. The use of botanical pesticides is now emerging as one of the prime means to protect crops and their products and the environment from pesticides [11]. Botanicals degrade more rapidly than most chemical pesticides, and therefore are considered to be eco-friendly and less likely to kill beneficial pests than synthetic pesticides with longer environmental retention. Most of the botanical pesticides generally degrade within a few days, and sometimes even within a few hours [12].
Tomato is one of the widely consumed fresh fruit worldwide since it contributes to a healthy well balanced diet which is rich in vitamins such as vitamin A, B, C and E. Carbohydrates such as fructose and glucose; Minerals which include phosphorus, sodium, potassium, calcium, magnesium and trace elements like iron, copper, zinc and dietary fibres [13]. The deep red colouration of ripened tomato is due to the presence of lycopene, a form of B-carotenoid pigment and a powerful antioxidant that help to protect against prostate cancer, cardiovascular disease and diabetes [14], thus there is an appeal and demands of the fruits by consumers as a result of their knowledge that they are healthy, tasty, convenient and fresh [15].
Vegetable crops including tomatoes are widely cultivated in most parts of Sub Sahara Africa, particularly by small scale farmers in most states of Nigeria [16,17]. Generally, global production of fruits and vegetables tripled from 396 million metric tonnes in 1961 to 1.34 billion metric tonnes in 2003 [18]. Global production of tomatoes is about 89.8 million metric tonnes from an area of about 3,170.000 ha [19]. Adegbola et al. [20] stated that Nigeria is undeniably the 14 th largest producer of tomatoes, second to Egypt in Africa at 1.51 million metric tonnes valued at N87.0 billion with a cultivated area of 254,430 ha being the biggest producer in Sub-Sahara Africa. The aim of the research is to evaluate the effects of different concentrations of ethanolic extract of Vernonia amygdalina on fungi associated with infected tomatoes (Lycopersicon esculentum) obtain from Jos North Local Government Area.

Study Area
The experiment was carried out at Jos North local government area, Plateau state. Located on latitude 9°55 ΄N and longitude 8°54 ΄E, at an altitude of 1200 m above sea level. The area falls under Natural Region II of Nigeria's agroecological zones, the climate of the area is humid with an average annual rainfall and temperature between 140-1480 mm and 10°-32°C respectively.

Samples Collection
Infected tomato fruits with symptoms of softness were randomly procured locally from Farin gada market, Terminus market and Jarawa Tomato market in Jos between May and August 2015. Ten (10) samples were collected randomly from three different sellers at the different markets (a total of thirty), placed in sterile polythene bags and conveyed into the laboratory for fungal isolation and subsequent identification. 100 g of Vernonia amygdalina leaf were collected from Bauchi Ring Road, Jos, placed in a polythene bag and taken to the Pharmacognosy laboratory of pharmaceutical department, University of Jos for plant extraction and concentration preparation.

Isolation of Fungal Organisms
Potato Dextrose Agar (PDA) was prepared according to the manufacturer's instructions, following the techniques described by Arora and Arora [21]. 80 mg of Gentamycin, an antibiotic was added to each 500 ml preparation of the agar to inhibit probable bacteria growth.
Diseased portion of the tomato fruits were cut under aseptic conditions into small bits into a sterile dish with the aid of sterile scissors (flamed over a Bunsen burner flame) and dipped inside methylated spirit [22]. The bits were sterilized with 70% ethanol and placed on Petri dishes containing already prepared solidified potato dextrose agar (PDA). The solidified plates were incubated at room temperature (28±2°C) in the dark until visible growths were seen on the plates. The fungal colonies grown from the incubated plates were sub-cultured into fresh medium until pure cultures were obtained.

Identification of Fungal Organisms
Microscopic examination was used for examining the colony characteristics. A sterile needle was used in taking a little portion of the colony, placed on the sterile glass slide, stained with lactophenol cotton blue and examined under the microscope for morphology and culture characteristics of fungal structures.

Preparation of Plant Extracts
Vernonia amygdalina leaves were air dried and pulverized into powder using blender. 40g of the plant powder was weighed into 500ml conical flasks and was soaked in 70% ethanol. This was left to stand overnight (24hrs), then shook for 3hrs on a mechanical shaker. The content was filtered using a non-absorbent cotton wool on a Buchner funnel-flask using a vacuum pump. The residue was subjected to several parts of rinsing and filtration with fresh solvents to attain some level of maceration (extraction). The collective filtrate was evaporated to dryness using a rotary evaporator and a drying cabinet. The percentage yield of the extract was determined and the extract was transferred into a stirrer sample container and preserved in the refrigerator. % Yield = (Weight of extract / Weight of crude powder) x 100

Antifungal Sensitivity Testing
The Broth dilution method was adopted for the antifungal sensitivity by subjecting the isolate to a fresh culture in a nutrient broth for 24hours, and then a sterile swab stick was used to inoculate the isolate from the broth on an already prepared potato dextrose agar plate. Six holes were bored on the plate afterwards and 0.2mls of the ethanolic extracts and controls were introduced separately in each hole. The set up was incubated at room temperature for 48hours and daily measurements of the fungi growth of the cultures were determined by measuring culture along two diameters with plain metre rule. Fungi growth inhibition was taken and recorded. Four concentrations level of the ethanolic extract (12.5 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml) were used. Fluconazole (50 mg/ml) was used as positive control while sterilized distilled water served as negative control. Three replicates were done for each of the organisms including the concentration levels and controls.

Statistical Analysis
One  Table 1 illustrates the culture and morphological characteristics of the identified fungi. Implicated isolates were Geotrichum candidum, Rhizopus stolonifer and Fusarium oxysporum.  10.00 mm at 100 mg/ml and 4.50 mm at 12.5 mg/ml, a considerably significant difference (P<0.001). However after 48 hours, the three organisms showed increasing inhibitory effect. The positive control inhibited Geotrichum candidum and Fusarium oxysporum while the in the negative control there was no zone of inhibition. Tables 5 and 6 showed the mean effect of concentrations across all isolates. At concentration 100 mg, the isolates showed more susceptibility than other concentrations use after 24 hours and 48 hours respectively. Tables 7  and 8 showed the mean effect of extract on Isolates across all concentrations. Rhizopus stolonifer shows to be more sensitivity to the extract at all concentrations after 24 hours and 48 hours. The Rhizopus stolonifer was more susceptible to the treatment (plant extract) in all concentration level with mean effect of 4.75 mm after 24 hours and 7.25 mm after 48 hours, followed by Geotrichum candidum and then Fusarium oxysporum which was least susceptible. They all showed significant differences (P<0.001).

DISCUSSION
Deductions from the frequency of occurrence of these organisms (Geotrichum candidum, Rhizopus stolonifer and Fusarium oxysporum) that were isolated and identified revealed that Geotrichum candidum had higher prevalence of contamination, followed by Fuarium oxysporum, then Rhizopus stolonifer had least prevalence. Sensitivity test using different concentrations of extract of Vernonia amygdalina showed they significantly affected all the identified organisms with R. stolonifer showing the highest inhibitory effect (P<0.001).
This study resulted into a mixed culture of fungal species, after several stages of sub culturing, three fungi were isolated and identified, then subjected to further experimentation. The identified isolates were Geotrichum candidum, Rhizopus stolonifer and Fusarium oxysporum.
This finding is similar with the reports of previous investigations carried out by Chuku et al. [23] and Ugwu et al. [24]. Chuku   Antifungal effectiveness of some tropical plants extracts in controlling several plant pathogens have been reported by several researchers [25,26,27,28,29,30].    This study observed inhibition after 24 and 48 hours using 70% ethanolic extract of Vernonia amygdalina, the work showed that the extract is effective against fungi associated with tomato infection and this is similar with Ijato et al. [31] who reported that Chromolaena odorata (leaf), Tridax procumbens (leaf), Venonia amygdalina (leaf) and Azadirachta indica (leaf) had inhibitory effect on the radial growth of rot fungi; after 72 hours observation with 1 ml of 15% ethanolic extract of Venonia amygdalina achieved an inhibitory effect of 54.58% on the radial growth of rot fungi. Report by Ugwuoke et al. [32] shows that Venonia amygdalina was use to control Fusarium solani causing tuber rot on cassava. He also reported the use of Azadirachta indica in the control of Fusarium oxysporum. Onyeani et al. [33] showed the inhibitory effect of Venonia amygdalina against Rhizopus stonolifer in his work.
At 24 hours, there was no measurable effect of the extract on Fusarium oxysporum until after 48hours. After 48 hours, there was increase in the effect of the extract but the positive control (Fluconazole at 50 mg/ml) had the same inhibitory effect as it did at 24 hours. However, Rhizopus stonolifer showed resistance to the positive control. The mean effect of the levels of concentration (12.5 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml) and the controls (fluconazole and distilled water) were significantly different (P≤0.001) at both 24 hours and 48 hours.

CONCLUSION
The effect of extract of Venonia amygdalina on fungi isolated from tomatoes at Jos North LGA markets shows that the plant leaves have significant inhibitory effects on the growth of the fungi. The effect of the extract increases with increasing concentrations and time of exposure to the plant leaf powder. This suggests that the plant extract contains some chemical component that has fungicidal activity.

RECOMMENDATIONS
i.
Other parts of the plant (stem and root) can also be subjected to study to check for probable higher fungicidal activities than the leaf part. ii.
Further research work should explore the botanicals to determine a higher effective extracts that can control fungi rot on tomatoes. iii.
Further research work may also seek to do a comparative study on the effect of more than one plant extract.