Efficacy of Neem ( Azadirachta indica ) Leaf Extract Against Fungi Pathogen Associated with Watermelon ( Citrullus lanatus ) Spoilage

Background and Objective: Watermelon ( Citrullus lanatus ) is a member of the Cucurbitaceae family native to Africa and has been cultivated since ancient times. Reports of chemo-resistance pathogens and health implications have stalled the application of agrochemicals for improving fruit production. This led to the isolation, purification, identification of pathogens and use of Azadirachta indica leaf extract to inhibit pathogenic growth responsible for post-harvest deterioration of watermelon ( Citrullus lanatus ). The study aims to isolate, purify and determine growth rate, zone of inhibition and colony count of fungi pathogen of watermelon using standard mycological techniques to ascertain the effectiveness of Azadirachta indica leaf extract on the pathogen. Materials and Methods: Sample collection and media preparation, culture and purification of isolates, pathogenicity test, as well as neem leaf aqueous extract, were conducted using standard pathological methods in this research, after which five fungi pathogens such as Aspergillus flavus , Fusarium oxysporum , Aspergillus niger , Rhizopus sp. and Saccharomyces sp. They were isolated from infested watermelon fruits and treated with 5, 10, 20 and 40 g concentrations of neem extracts where 5.4, 5.8, 6.1 and 6.6 cm inhibition rates were observed. Results: Aspergillus species appeared undeterred by the extract at these concentrations, but the overall inhibitory potential of neem leaf extracted was observed at 40 g inhibitory concentration at 50% concentration (IC 50 ). This means that the higher the concentration of the neem extract, the more effective in inhibiting pathogenic diseases of watermelon. Findings revealed that at controlled doses, Azadirachta indica leaf extracts may be able to suppress post-harvest pathogenic fungus in watermelon. Conclusion: To get compounds with significant resistance to post-harvest rotting, bioactive components should be assessed via bioassay-guided isolation.


INTRODUCTION
Watermelon (Citrullus lanatus) is a member of the Cucurbitaceae family native to Africa and has been cultivated since ancient times.The fruit flesh of watermelons is watery, but typically hard-textured, pale-colored and bland or bitter 1 .The watermelon fruit has a deep green smooth thick exterior rind with grey or light green vertical stripes.There are now eight designated flesh colors in watermelon: White, salmon yellow, orange, crimson red, scarlet red, pale yellow, canary yellow and green 2 .Watermelon contains more than 91% water and up to 7% carbohydrates.It is rich in some of the major antioxidants, vitamin C and carotenoids such as lycopene and β-carotene that are responsible for the red, orange and colors of the watermelon, the lycopene content of watermelon has become very important for consumers, as recently lycopene has stimulated attention as a health-promoting antioxidant.The sweetness of watermelon is mainly due to a combination of sucrose, glucose and fructose.Sucrose and glucose account for 20-40% and fructose for 30-50% of total sugars in a ripe watermelon.
Watermelon cultivation is not unconnected with losses due to post-harvest diseases especially those of fungi origin 3 .Synthetic fungicides are typically used to control phytopathogenic fungi.Since fungicides constitute a threat to human health and the environment, their use has been strictly regulated.However, after years of use, these pathogenic fungi have become highly resistant in plants, necessitating the implementation of a control mechanism to overcome this, microbiologists and plant scientists are paying more attention to phytochemicals because they are derived from plant components and have been shown to be safe for human health and biodegradable against microorganisms 4,5 .
As a result, it is inevitable to isolate, purify and identify the pathogenic fungus linked to the post-harvest degradation of watermelon fruits and to employ various plant leaf extraction techniques and disease management strategies to treat the phytopathogens.Therefore, this study aims to Isolate, purify and determine the growth rate, zone of inhibition and colony count of the fungi pathogen of watermelon using standard mycological techniques to ascertain the effectiveness of Azadirachta indica leaf extract on the fungi pathogen.

Sample collection:
Fresh skinned watermelon fruits were obtained from Anyigba's main market.Samples were collected for 6 months (March to August, during the wet season of 2022) during the research and transported immediately to the Plant Science and Biotechnology Research Laboratory of Prince Abubakar Audu University, Anyigba, Kogi State.The sampled fruits were rinsed and air-dried at room temperature.The leaves of neem (Azardirachta indica) were obtained from Prince Abubakar Audu University agricultural farm.The leaves were rinsed with double distilled water (ddw) and were air-dried, pulverized and sieved (mesh size of 20 µm), 100 g of the dried leaves were measured and stored in an air-tight sample container at 4EC until ready for use.

Isolation of pathogen:
About 39 g of potato dextrose agar (PDA) was weighed and dissolved in 1000 mL of distilled water, 1 mL of Tetracycline was prepared and transferred to a set of petri dishes, followed by sterilization at 121EC for 15 min.The infected fruits portion was sterilized using 70% ethanol, after which the infested fruits were scrapped off using a sterile scalpel and then transferred aseptically onto the agar media prepared 6 .

Identification of pathogen:
The Identification and quantification of the pure isolates were investigated and observed at the end of incubation.Then selection of representative colonies and sub-cultured, until pure isolate culture is obtained and maintained on the PDA slant and stored.Pure culture was sub-cultured from the isolate obtained from the watermelon and maintained on a petri dish and daily readings were taken to identify the representative colonies and growth rate of fungal mycelia after 7 days 7  Pathogenicity test: The 70% of ethanol was used to disinfect fresh watermelon after it had been cleaned with distilled water.With the use of a sterile cork borer and the fungus mycelia, 4 mm diameter columns were extracted from the fruits (pure culture of the isolates).With the help of a sterile inoculating loop, the discs were taken out and put in fruit columns (a fruit column for a disc column).To stop other organisms from getting contaminated, the fruit skin columns were covered with vaseline gel and sealed.The inoculated watermelon fruit was kept on the laboratory bench for 21 days at a room temperature of 25EC.After the inoculation period, the watermelon fruit was cut across using a sterilized knife along the plane of inoculation and the rot depth was accessed.

Preparation of plant aqueous extract:
The cold water extraction method was used for the preparation of the plant extracts.Fresh neem leaves were washed thoroughly with double distilled water and dried on the laboratory desk, it was pulverized and sieved to get a powder form, before being weighed at 5, 10, 20 and 40 g, respectively.The soluble powdered ingredient of the plant material was then extracted by adding 100 mL of distilled water to the weighed plant material.This was left for 24 hrs and then subsequently filtered through filter paper placed in the funnel under aseptic condition.This preparation gave 5, 10, 20 and 40% crude aqueous extract 8 .

Bioassay of plant extracts on the fungi isolates:
Potato Dextrose agar medium was used; this was carried out in a sterile petri dish of 8.6 cm diameter.Containing solidified PDA medium.The 1 mL of 5, 10, 20 and 40% of the extract preparation was separately spread thinly on the surface of the PDA in the Petri dishes.A disc of 4 mm diameter (using a sterile cork borer) from each pure cultured fungi isolate was aseptically transferred and inoculated on the thin film of extract formed on the PDA.The organism was swabbed evenly on the extract of different concentrations (5, 10, 20 and 40%/disc).A replica was set for each treatment and control disc was set up with sterile distilled water.The treatment and control were both incubated at room temperature using the method of Amadioha and Uchendu 9 .

Conc. (%) -
From the study, pure phytopathogenic fungi (Aspergillus Niger, Aspergillus flavus, Fusarium oxysporum, Rhizopus ssp, Saccharomyces ssp) were isolated and identified in deteriorated watermelon fruits according to Koch postulate (Table 5).The retro assessment and authenticity of these pathogenic organisms were carried out using a pathogenicity test.Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Rhizopus sp and Saccharomyces spp.

DISCUSSION
The result of the pathogenicity test (Table 2) revealed that the fungi isolated from the spoilt watermelon fruits in this study were able to induce the same disease symptoms present in healthy fruit with the same fungi re-isolated from the inoculated healthy fruit.Thus, it shows that fungi were responsible for the spoilage of fruits.
There appeared no significant inhibition of Rhizopus throughout the treatment, this could be due to various factors including pathogen virulence as well as environmental interactions at the time of the experiment.This was also noted by Ibrahim et al. 11  temperatures, ranging from 15 to 40°C, with the optimal growth temperature of about 30°C.There doesn't seem to be much significant inhibition on the pathogen tested except for Aspergillus flavus (Fig. 1) with an inhibition percentage of 58.14% as compared to others while Rhizopus indicated no inhibition whatsoever.This was in conformity with Bashir et al. 12 who worked on the effect of sodium carbonate and sodium chloride for the control of black rot disease of Mangifera indica caused by Aspergillus niger, much resistance was also observed by the pathogen even though conidia germination is hindered.Fulton, et al. 13 also worked on the fungi diseases of watermelon and found Alternaria leaf blight (Alternaria cucumerina), Anthracnose (Colletotrichum orbiculare), Cercospora leafspot (Cercospora citrullina), downy mildew (Pseudoperonospora cubensis), Fusarium wilt (Fusarium oxysporum), Gummy stem blight (Didymella bryoniae) and powdery mildew (Podosphaera xanthii) affecting the growth of watermelon.He also opined that disease severity is high in areas where there is frequent rainfall and temperatures are high, a characteristic nature of Anyigba where this research was carried out.Alhaji et al. 14 isolated the fungi associated with the rot of watermelon fruit in Sokoto metropolitan, Sokoto State, Nigeria and were able to isolate and identified Aspergillus flavus and Aspergillus niger.Rhizopus stolonifer and Mucor spp.and concluded that these pathogens confer great concerns to public health.
Fusarium oxysporum has also been implicated as the major causal organism of vascular wilt as well as the most important pathogen that causes serious yield loss yearly 15,16 .From this research, the treatment with 5 g concentration of neem doesn't show any form of inhibition throughout the pathogens tested, however, the highest percentage inhibition of 58.14% was observed with 40 g treatment of the pathogen especially Aspergillus flavus (Fig. 1).This indicated that the higher the concentration of the neem extract, the more likely its ability to inhibit pathogenicity of a pathogen given that other environmental effects are within a controlled limit.This had also been suggested by Kutama et al. 7 , who worked on the in vitro inhibitory potential of Trichoderma species on Fusarium oxysporum f. sp vasinfectum the causal organism of vascular wilt of cotton (Gossypium hirsutum L.) in the Nigerian Sudan Savanna.Aspergillus niger appeared to have the least radial growth while Rhizopus solani had the highest mean radial growth (Table 3).
Fusarium diseases have been implicated to be a significant hindrance to food and plant production and are very difficult to manage, especially soil-borne diseases caused by F. oxysporum 17 .Fusariums are mycotoxin-producing fungi of great economic importance as they cause many diseases in man and other animals when infected food products are consumed.Some of the mycotoxins produced by Fusarium include FB 1 and trichothecenes 18 .The effects of these mycotoxins on animals depend on the quantity of mycotoxin intake according to Bouhet and Oswald 19 and after intake, they arrive at the gastrointestinal epithelial cell layer which is covered by the mucous secreted from goblet cells.
Fusarium species according to Sharma and Marques 20 , have diverse ecological functions ranging from saprophytes, endophytes and animal and plant pathogens.The major losses of watermelon postharvest in Kogi State had been a major setback to many farmers and traders alike in this axis, it is, therefore, imperative to employ several control measures for the management of these fungi pathogens.Furthermore, growing disease-resistant species to combat this disease is a more appropriate strategy because synthetic fungicides are not a true way to prevent fungi-related diseases due to their harmful effects on the ecosystem and the environment 21 .This study was carried out to determine the efficacy of neem (Azadirachta indica) leaf extract associated with fungi pathogen affecting watermelon obtained from Anyigba market.The neem (Azadirachta indica) tree has gained global recognition due to its numerous medical benefits.Antifungal, immunomodulatory, anti-inflammatory, antihyperglycemic, antiulcer, antimalarial, antibacterial, antioxidant, antimutagenic and anticarcinogenic qualities have been shown for neem leaf and its compounds 22  Agricultural and chemical precautions cannot be overly successful in the management of fungi-related diseases in plants.The most effective way to address fungal-induced plant diseases is through the application of biological control techniques.such as the extracts of neem that have been proven by the findings of this research work to be of promising, efficient, cheap and eco-friendly means of protecting plants and plant products.

CONCLUSION
To overcome the alarming problem of fungal infections or contaminations on fruits, the discovery of phytochemicals against new targets is a matter of urgency.Thus, A. indica could become promising natural antifungal agents with potential applications in the pharmaceutical industry for controlling pathogenic fungi.The plant extracts in the study (A.indica) could be used to inhibit rot fungi of watermelon fruit at controlled concentrations, thereby increasing the shelf life and high market value of watermelon fruit.

SIGNIFICANCE STATEMENT
Watermelon (Citrullus lanatus) is a large annual plant with long, trailing or climbing stems and belongs to Cucurbitaceae.The mineral and nutritional compositions revealed bioactive constituents such as phenol, crude fiber, flavonoids, phosphorus, sodium, manganese, potassium, copper, calcium, zinc, iron, fat content, lipid, crude ash and moisture content.However, fungi pathogens are a major deterrent to the yield, productivity, shelf life and market value of watermelon especially in this central part of Nigeria.This research has unveiled a new, cheap and environmentally friendly way of controlling the diseases of watermelon via green and friendly sources.This will boost the economy of this region and the entire country and the world at large once these findings are duly accepted and harnessed.

C
dc = Average diameter of fungal colony in the control C dt = Average diameter of fungal colony in the treatment Table . https://doi.org/10.3923/asb.2024.249.257| Page 255 .

Table 1 :
Pathogenicity of fungi on watermelon Aspergillus nigerFruits appeared water-soaked and soft, disease developed rapidly through fruit tissue resulting in total rot and exudation of liquidsAspergillus flavusA uses soft rot spoilage and the infected area turns brown and water-soaked Fusarium sp.Pathogen causes soft rot in the affected area Rrhizopus sp.Pathogen causes soft rot and water-soaked in the infected area, resulting in total rot in whole fruit Saccharomyces sp.A pathogen causes watery and soft rot in the infected area

Table 2 :
Pathogenicity test showing the mean rot depth caused by pathogenic fungi.

Table 3 :
Mean radial growth (cm) of isolated fungi from rotted watermelon on PDA

Table 2
below indicates the pathogenicity test showing the measured depth of the rot caused by the pathogen on watermelon fruit where Rhizopus sp. had the highest rot depth recorded while Fusarium oxysporum recorded the least.

Table 4 :
Percentage Inhibition rate of Azadrachta indica leaf extract on fungi pathogen