Evaluation Of the Antifungal Activity of Cassia fistula (Linn) On Phytopathogenic Fungi Isolated from Citrus sinensis

Citrus sinensis is a prominent tropical fruit with nutritional and economic benefits for humans. However, it can also be plagued by fungal diseases, causing global economic and production reduction. The extensive application of synthetic fungicides in agricultural systems has posed increased toxicological risks to humans and ecosystems. However, the varying phytochemicals found in medicinal plants have enabled them to be regarded as safe and effective alternatives. However, few studies have evaluated the use of Cassia fistula extracts as potential bio-fungicides. This research purposed to evaluate the antifungal activity of Cassia fistula leaflet extracts against phytopathogenic fungi. Ethanol as well as aqueous extracts of Cassia fistula leaves were assessed for antifungal activity. The agar well-diffusion technique was implemented for in vitro screening, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of all different extracts against isolated fungal species of Aspergillus, Penicillium and Geotrichum. All fungal isolates identified from the spoiled oranges were Aspergillus flavus, Aspergillus fumigatus, Geotrichum sp. and Fusarium sp. Preliminary phytochemical screening exhibited the presence of carbohydrates, terpenoids, quinones and saponins in both ethanolic and aqueous extracts. At 1000 mg/ml, the aqueous and ethanolic extracts had the highest antifungal activity against A. flavus (40 mm) and Fusarium sp. (38 mm), respectively. Both extracts had no antifungal activity against A. fumigatus at 62.5 mg/ml but zones of inhibition were observed from the positive and negative controls of ketoconazole and DMSO, respectively. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were evaluated at 62.5 mg/ml and 250 mg/ml, respectively. This study’s findings demonstrated the antifungal potential of C. fistula leaf extracts as a bio-fungicidal agent against fungal invasion in plants.


Introduction
Oranges (Citrus sinensis) are one of the most important horticultural crops worldwide, with nutritional and economic value because of their use in the production of fruit juices, salads and essential oils [1][2].However, fungal infection of oranges spans from pre-to post-harvest processes, leading to economic losses that vary between 30 and 50% or sometimes complete product loss [3][4].Sources of fungal contamination include irrigation water, insect and animal bitings and human handling [5][6].Although synthetic fungicides are used to reduce phytopathogenic fungi in fruits and vegetables, they pose a toxicological risk to human health and the environment, resulting in the development of resistant fungal strains [7].Therefore, the use of medicinal plants to inhibit phytopathogenic microorganisms should be considered an alternative strategy.The antifungal effects of Cassia fistula extracts have been reported by Bhalodia and Shukla [8]; however, there is a paucity of studies on the potential of C. fistula extracts to inhibit phytopathogenic fungi in oranges.Therefore, this research purposed to evaluate the activity of Cassia fistula extracts on fungal pathogens causing spoilage of oranges.The knowledge gained from this study will help in formulating fungicides that are effective against phytopathogenic fungi in oranges.

Collection and Processing of Samples
Samples that comprised of 24 oranges were obtained from Agbara, Iju and Oja-Ota Markets at Ado-Odo Ota LGA, Ogun State, Nigeria, and were analyzed at the Microbiology Laboratory, Covenant University, Ota, Ogun State, Nigeria.The oranges were surface-sterilized with a 1% hypochlorite solution, rinsed in sterile distilled water three times and aseptically positioned on sterilized filter papers to remove residual moisture.The samples were comminuted using a sterile mortar and pestle, and one gram of every sample was introduced directly into a test tube filled with 10 ml of sterile distilled water from which further dilutions were performed up to the ninth dilution.

Media Preparation
The media used was Potato Dextrose Agar (PDA) which was constituted following the manufacturer's instructions.

Potato Dextrose agar
Potato Dextrose agar (PDA) was made by weighing 39.5 grams of the medium into 1000 ml of distilled water.This solution was then homogenized inside a water bath and sterilized at 121℃ for the stipulated time of 15 minutes.

Fungal Isolation
One milliliter of the inoculum suspension (required dilution of each sample) was dispensed into a Petri plate and immediately followed by the addition of 20 ml of cooled molten PDA.The mixtures in the plates were gently swirled, set and kept in the incubator at 25 °C for 7 days.All mycoflora colonies were sub-cultured onto freshly constituted PDA Petri plates and incubated at 25 °C for 5 days to obtain pure cultures [9].

Identification of Isolates
Pure fungal cultures were phenotypically identified based on morphological and microscopic features.Morphological characterisation was performed by observing the texture and pigmentation of the cultures.The microscopic characterisation was performed using lactophenol cotton blue stain and observing the hyphal type, presence or absence of hyphal septation (hyphal wall type), characteristic spore descriptors (colour, shape and arrangement) as well as rhizoids at X40 microscopy.Barnett and Hunter's Atlas of Mycology and Compendium of Fungi [10] were used for identification.Pure cultures were stored inside a refrigerator at 4 °C for further bioassays.

Collection of plant materials
Mature disease-free leaves of C. fistula were harvested from the Covenant University environs in Ogun State and identified by a botanist in the biology programme of the Biological Sciences Department of the afore-named institution.The specimen was preserved in the herbarium and the voucher specimen number was designated as Cf/Bio/H848.

Crude Extraction
The collected leaves were air-dried for three weeks and blended into a fine powder by using a sterile blender.Then, extraction was carried out according to Oyejide [11].Powdered leaves (300 g) were separately extracted with 1500 ml of ethanol and water for 3 d and then filtered through the Whatman No. 1 filter paper.Further drying was conducted at 50 °C utilizing a rotary evaporator and all the semi-solid extracts were stored in the refridgerator at 4 °C.

In vitro Antifungal Activity of Leaf Extracts
The antifungal activities of both aqueous and ethanolic leaf extracts of C. fistula were evaluated employing the agar well diffusion technique according to [12] and [13].The stock concentration was constituted by dispensing 1000 mg of crude extract into 1 ml of DMSO (ratio 1:1) for each of the aqueous and ethanolic extracts (stock concentrations).These stock concentrations were reconstituted into other concentrations using the doubling dilution method.All fungal isolates were standardized according to the McFarland standard (0.5M) and used to seed freshly prepared PDA plates for incubation at room temperature for 5 days.Sterile cork borers with 10 mm diameter were used in boring wells and 0.2 ml of each plant extract concentration was introduced into the agar wells.Both the positive and negative control plates contained ketoconazole and DMSO, respectively.The plates were placed on sterile work table for an hour to ensure the extracts were completely diffused inside the agar pores.Following this, the Petri dishes were immediately placed inside the incubator for 3 days at 25 °C.Post incubation, the zones of inhibition were immediately assessed and recorded.Every assay was conducted in duplicates and the average zones of inhibition were recorded accordingly.Minimum inhibitory concentration (MIC) was described as the lowest extract concentration that allowed minimal microbial growth, whereas minimum fungicidal concentration (MFC) was established as the lowest extract concentration that disallowed any visible microbial growth post incubation.

Analysis of Data
All diameters of the regions of inhibition for in vitro antifungal assay were situated as the mean of the duplicates.All statistical analyses were conducted implementing the SPSS version 20 software package.

Fungal isolation and identification
The fungal pathogens isolated in this study included Aspergillus flavus, Aspergillus fumigatus, Geotrichum sp. and Fusarium sp.(Table 1).Aspergillus species were the most frequently recorded, with Aspergillus flavus having the highest frequency of occurrence (Figure 1).

O3
Smoky grey cottony strands with a white or tan on the reverse side of the plate.
Hyaline septated hyphae bearing brown conidiospores arranged in chains borne in a conidial sac.

O1, O2 and O3
The lemon green powder grew on a plate with the periphery and yellow coloration on the reverse side of the plate.
Bluish-green conidiospores are contained in a sac suspended by hyaline hyphae.

O3 and O2
White wide sparsely floccose mycelial growth restricted to surface of plate with orange fruiting bodies at the centre.

Oualitative phytochemical screening of the crude extracts
Qualitative phytochemical screening showed the presence of carbohydrates, quinones, terpenoids and saponins in both the aqueous and ethanolic leaf extracts.Coumarins were present only in the aqueous extracts, whereas tannins, glycosides as well as phenols were detected only in every reconstituted ethanolic extract (Table 2).Alkaloids, flavonoids, cardiac glycosides, ninhydrin, anthraquinones, steroids as well as phlobatannins were void in both extracts.

Antifungal activity of the crude extracts
The detailed results of the antifungal assessment of all the aqueous and ethanolic extracts at the concentrations of 1000, 500, 250, 125, and 62.5 mg/ml are presented in Figures ( 2 and 3).At 1000 mg/ml concentration, all the aqueous and ethanolic extracts exhibited highest antifungal activity against A. flavus (40 mm) and Fusarium sp.(38 mm), respectively.Neither extract showed antifungal activity against A. fumigatus at 62.5 mg/ml.Overall, the crude extracts had higher antifungal activity than ketoconazole and DMSO, which were the positive and negative controls, respectively

Discussion
Fungal contamination of oranges generally affects their economic value and public health.In this study, qualitative phytochemical screening of Cassia fistula leaf extracts and evaluation of all the extracts of isolated phytopathogenic fungi were conducted.The fungal pathogens isolated from spoiled oranges in this study were also published by Bali et al. [5], Chukwuka et al. [14] and Mailafia et al. [15].Environmental factors that could have aided the dissemination of these phytopathogenic fungi include cross-contamination during handling, storage and transportation to the markets.Currently, due to their potent, economical and easy accessibility, diverse plants and herbages are been researched and assayed using the biotechnological approach for human application Akinduti et al. [16].Extracts of medicinal plant are alternative or complementary control means which is premised on their antimicrobial activity, non-phytotoxicity, systemicity and biodegradability potentials.Abundant secondary metabolites that possess antimicrobial and antifungal activity are produced by plants Popoola et al. [17].In this study, the ethanolic extracts revealed more phytochemicals than the aqueous extracts but this was not reflected in their antifungal activity.The results of the phytochemical screening in this study partly agreed with the findings of Raji and Samrot [18] and Wijaya et al. [19].Our findings portrayed that the aqueous and ethanolic types of extracts possessed varying inhibitory activities against mycoflora isolates at diverse concentrations.While the extracts had limited antifungal activity on A. fumigatus, they had higher antifungal activity against the other isolated fungal species compared to ketoconazole and DMSO.The results of antifungal activity from this study revealed higher inhibition by the aqueous extract than by the ethanolic extracts, which is in contrast to the study of Panda et al. [20].
The variations in the findings across studies could be as a result of the type of solvents or protocols employed in the extraction and screening processes.All minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values were evaluated at 62.5 mg/ml and 250 mg/ml respectively.Therefore, this study has shown great potential of the aqueous as well as ethanolic extracts of Cassia fistula as a bio-fungicide to inhibit certain phytopathogenic fungal infections.

Conclusion and Recommendation
Plants, particularly medicinal plants, have antifungal potential for the prevention of fungal diseases, thus reducing spoilage in oranges and forestalling the development of resistant fungal strains.The antifungal activity observed in the extracts of C. fistula against phytopathogenic fungi indicated that it can be employed as a natural fungicide for the treatment of phytopathogenic fungal infections.More studies are needed to test the antifungal activities of C. fistula extracts on phytopathogenic fungal isolates.
of Ado-Odo LGA in Ogun State

Figure 1 :
Figure 1: Map of the study area showing the sampled location

7 Figure 2 :
Figure 2: Frequency of occurrence of mycoflora isolates cultured from Citrus sinensis

Figure 3 :
Figure 3: Mean zones of inhibition of C. fistula aqueous leaf extracts against the isolated fungal pathogens.

Figure 4 :
Figure 4: Mean zones of inhibition of C. fistula ethanolic leaf extracts against the isolated fungal pathogens.

Table 1 :
The Preliminary Identification of Mycofloral Pathogens

Table 2 :
Phytochemical Screening of C. fistula