Isolation of fungus from the cloves of Allium sativum, and evaluation of the antioxidant, antimicrobial, and cytotoxic potential of the ethyl acetate extract of this fungus

Mohaimenul Hoque¹, Md. Mobarak Hossain¹, Tarekul Islam¹, Mahmuda Ferdous¹, Sarah Sarker ², and Prodip Kumar Baral^1*

¹Department of Pharmacy, Faculty of Science, Noakhali Science and Technology University, Noakhali-3814, Bangladesh.

²National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka-1349, Bangladesh

Author of correspondence. email: pkb.phar@nstu.edu.bd

ORCID ID: https://0000-0003-0569-1763

DOI: 10.70373/RB/2025.10.01.1

Abstract

Endophytic fungi have drawn an intense attraction to drug discovery as endophytes undergo a mutualism; they can mimic the chemical potentials of the host. Allium sativum L. (garlic) is well-reputed for its medicinal values, but no study about endophytic fungi has been reported from its cloves till now. Therefore, the study aimed to isolate garlic fungi and investigate their biological potential. Having structural barriers against microfungal penetration, several attempts successfully isolated a single fungus species. The morphology and DNA sequence confirmed the fungus as Fusarium oxysporum (OR062574) which mainly resides at plant roots and is recognized as a notorious organism for its pathogenicity.  1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging test showed 50% free radical inhibition concentration (IC50) around 20 % of ascorbic acid’s potential. The disc diffusion method detected antimicrobial capacity against gram-negative bacteria (E. coli and P. aeruginosa) and the brine shrimp 50 % Lethal Concentration (LC50) was 2,56 µg/ml was also 20 % activity compared to reference vincristine sulfate. Those findings uncovered the prospect of having pharmacologically valuable molecules in the fungal metabolite.

Keywords: Allium sativum; endophyte; Fusarium; antioxidant; cytotoxicity

Introduction

Endophytic fungi live in host plant tissues in a symbiotic manner and carry their entire or partial life cycle without any evidence of adverse effects or disease, which is considered a natural system of plant’s microenvironment.^1,2 As endophytes live on plants’ nutrition and mimic their natures, plants having medicinal values might host fungi with the same propensity.³ These fungi influence their host to produce hormones and adaptor metabolites to cope with stressful situations.⁴ They earn the capability to synthesize a wide array of bioactive molecules, which are sometimes identical to plants in terms of pharmacology. They are rich candidates for potential chemicals, classified as terpenoids, alkaloids, steroids, flavonoids, and phenolics that exhibit antioxidant, antimicrobial, antimalarial, antitumor, and anti-diabetic properties ⁵.  As a result, the endophytes have drawn an intense attraction of research due to efficient and less tedious ways of drug discovery in recent times.⁶

Allium sativum, the popular spice garlic, belongs to the Amaryllidaceae family, which is globally used for enriching cuisines and is also known for its therapeutic role as a home remedy, Ayurvedic, Homeopathic, and Unani system of medicine, worldwide.^7–10 The major useful part is cloves which have anti-inflammatory, antihypertensive, antimicrobial, antilipemic (cholesterol-lowering), hepatoprotective, and anticancer properties.¹¹ The Garlic clove contains a high amount of sulfur compounds, including allicin, diallyl disulfide, diallyl trisulfide, and, S-allylcysteine, which are considered the troupers for therapeutic activity.^12,13 The clove serves a multiplicity of treatments; the more frequent remedies are for arthritis, back pain, bronchitis, malaria, tuberculosis, chronic fever, rhinitis, skin wounds, hemorrhoid, urinary tract and kidney illnesses, and diabetes.¹⁴

Although the medicinal properties and chemical diversity of A. sativum are explored tremendously, the investigation into its endophytic fungi is very negligible. Nevertheless, few works on endophytic bacteria isolation are available on garlic.^15,16 The literature review shows only one evidence of isolation of one fungus Trichoderma brevicompactum from garlic by the Institute of Plant Protection and Microorganisms, Zhejiang Academy of Agricultural Sciences, however, it does not clarify the plant parts and geolocation from where it was isolated.¹⁷ Therefore, garlic might be a unique source of fungus isolation and its chemical potential exploration. The study targeted to explore the range of the garlic clove’s endophytic fungi and their biological perspectives specifically antioxidant, antimicrobial, and cytotoxic activity as an approach toward drug discovery.

Materials and Methods

Ethical considerations

This study was approved at the Faculty of Science by Noakhali Science and Technology University Ethical Committee, Bangladesh, with the ethical code: NSTU/SCI/EC/2020/55.

Chemicals

Potato dextrose agar medium (HiMedia, Germany); Water agar medium (HiMedia, Germany); Chloroform (S D Fine Chem, India); Dimethyl sulfoxide (Rx Chemicals, India); 2,2- Diphenyl-1-Picrylhydrazyl (Merck, Germany) were used in the study.

Collection of A. stivum bulb

The matured garlic was collected from the farmland of Noakhali, Chittagong, Bangladesh (22.70ºN 91.10ºE) in February 2021. The sample was placed in sterile bags and processed in the laboratory within a few hours after collection.

Isolation of endophytic fungi

The cloves were peeled out and washed with sterile distilled water, which was followed by a sequence of washing for clove’s surface sterilization; immersion in 70% ethanol for 1 min, 1.3 M sodium hypochlorite (3-5% available chlorine) for 3 min, and 70% ethanol for the 30s. After that, the whole cloves were sliced into thin pieces in a sterile environment and placed on an antibiotic (1 mg/ml ciprofloxacin) mixed with potato dextrose agar media for 21 days. The same method was followed for a portion of the sample except for surface sterilization to maintain control. Next, subsequent steps of isolation and purification were described by Kusari et al. with a little bit of modification.¹⁸

Molecular identification

A portion of fungal hyphae was taken to lyophilized in an Eppendorf tube subjected to DNA isolation based on the manufacturer’s protocol using the DNeasy Plant Mini Package (QIAgen, USA). ITS region was amplified by PCR and sequenced as described earlier ¹⁹. The sequences were aligned using Chromas software (V 2.6.2) and matched against the nucleotide-nucleotide database BLASTn of NCBI (National Center for Biotechnology Information) for final Identification ²⁰.

Morphological study of endophytic fungi

The morphological characterization was performed on a few fungal growth parameters: pattern of growth, the colony color at the top and bottom of Petri dishes, surface texture, Shape at the margin, nature of hyphae- vegetative or aerial, and production of acervuli using standard manuals for fungus identification.²¹ Light microscopic (40X) identification described by Sadananda et al. was followed to observe the shape of spores of the endophyte.²²

Fungal cultivation and metabolite extraction

To determine the biological activity of fungal secondary metabolites, small-scale cultivation was performed on 40 pairs of Petri dishes and incubated at 28°C for 21 days. After that, the fugal cultures were frozen and then, water-free contents at normal temperature were taken under solvent extraction procedure using ethyl acetate. Sequential filtration, rotary evaporation at 45°C, and final drying by normal evaporation obtained crude extract of the fungi.²³

Determination of bioactivity of fungal metabolites

Antioxidant Activity

2.0 mL of methanol solution of the extract at ten different concentrations (200.0 to 0.78125µg/ml) was mixed with 2.0 mL of a 1,1-diphenyl-2-picrylhydrazyl (DPPH) methanol solution (20 μg/mL). Then the mixtures were kept dark at room temperature for 30 min for a reaction which was subjected to 50% Inhibitory Concentration (IC₅₀) determination by UV spectrophotometric absorbance at 517 nm against methanol (control)²⁴ Ascorbic acid was used as a positive control to compare the sample values.

Inhibition of free radicals (I%) was calculated as follows:

Antimicrobial activity

Disc diffusion method was followed to determine antimicrobial activity as suggested by Toma & Barriault (1995) with subtle modification against bacteria: two Gr+ bacteria- Staphylococcus aureus (ATCC 25923), Bacillus megaterium (ATCC 13578); two Gr- bacteria- Escherichia coli (ATCC 28739), Pseudomonas aeruginosa (ATCC 27833), and two fungi: two fungi –Aspergillus niger, and Aspergillus flavus. Ciprofloxacin (antibacterial agent) and ketoconazole (antifungal agent) standard discs were used to compare the sample activity.²⁵ The concentrations per disc were 30 µg for standards, and three concentrations: 80 µg, 40 µg, and 20 µg for fungal extracts.

Cytotoxicity test

The brine shrimp nauplii (Artemia salina) eggs were collected from a commercial supplier for the brine shrimp lethality bioassay and hatched for 24 hours. 24-hour aged shrimps were transferred to fungal extract of ten concentrations (200–0.39 μg/mL) and calculation of deaths at different time durations following the methods mentioned by Hannan et al.²⁶ All tests were triplicated. Vincristine sulfate was used as a standard to relate the 50% Lethal Concentration (LC₅₀).

Lethality (L%) was calculated as follows:

Results

The fungal isolates

One endophytic fungus was isolated from the investigated cloves of A. sativum denoted by ASE. DNA sequence, morphology, and microscopic shape of spores confirmed the fungi as Fusarium oxysporum. Subsequently, their metabolites from small-scale cultivation showed significant biological activity.

Information on DNA sequencing

BLAST result revealed a higher possibility of the sample being F. oxysporum. The accession number of the fungus is OR062574 provided by National Center for Biotechnology Information (NCBI).

Morphology of the isolate

The fungus covered approximately the entire area (9 cm plate diameter) on the seventh day of inoculation although vegetative growth was observable on the eleventh day (Table 1). The primary white top view turned grayish, whereas the grayish bottom showed brownish after a week of inoculation. Colony surface texture, side view, and margin were characterized by Wooly, flat, and filamentous, respectively, for the entire period mentioned in Table 1 and Fig. 1(a-c). Microscopic observation revealed separated and two shapes of fungal micro chlamydospore: some were oval tapering and some were narrowly amygdaloid. The mycelium was characterized as filiform and septate in nature (Fig 1d).

Table 1: Morphology of ASE on the culture plate            

Fig. 1: Isolated endophytic fungal strain ASE after 3 days (a), 7 days (b), and 11 days (c). Microscopic view (40x) is the picture (d). The left and right pictures of a-c indicate the culture’s top and bottom views, respectively. The d is mycelium and spores.

Bioactivity of fungal extract

Antioxidant activity

ASE metabolites showed an excellent linear correlation between the percentage of radicals’ inhibition and concentration. The IC₅₀ of standard ascorbic acid was 12.65 µg/ml, whereas this value for ASE was higher at 67.46 µg/ml (fig. 2). So, the free radicals reducing the capacity of F. oxysporum was approximately five times less than that for ascorbic acid.

Fig. 2: Dose-response relationship on free radical scavenging activity. a and b represent linear regression between the percentage of free radicle inhibition and concentration of fungal extract and ascorbic acid, respectively.

Antimicrobial activity

Standards ciprofloxacin and ketoconazole showed a zone of inhibition within 35-45 mm against all of the respective organisms. However, ASE exposed activity against two gram-negative bacteria, E. coli (8 mm) and P. aurigenosa (9 mm) at 80 µg/disc dose (table 2). Therefore, the antibacterial activity of F. oxysporum was one-fifth of ciprofloxacin against gram-negative bacteria at a given concentration.

Table 2: Zone of inhibition of ASE and standards against test organisms

‘…’ means no activity

Cytotoxic activity

The percentage of mortality and logarithmic concentration of ASE extract was almost linear (R²≈1). The reference fifty percent lethal dose (LC₅₀) of vincristine sulfate was 0. 57 µg/ml, where the value for ASE was determined at 2.55 µg/ml (Fig. 3). It ascertains that the cytotoxicity revealed the same extent of potential as showed against gram-negative bacteria in antimicrobial test by one-fifth of references.

Fig. 3: Lethality of ASE extract on brine shrimp nupali after at sixth hour of sample exposure

Discussion

The present study isolated one endophytic fungus that was undertaken for characterization and identification. DNA sequence as well as microscopic structures confirmed the fungus as F. oxysporum. The species was evaluated by its pharmacological potential by three in vitro assays: antioxidant, antimicrobial, and cytotoxicity parameters, and every test exhibited a moderate level of effects compared with their respective standards.

Generally, the investigations on endophytic fungi succeed to explore a good number of the fungi at a time. Nevertheless, this study took several attempts to isolate a single one. The first two attempts did failure to observe the growth of any fungus on A. sativum cloves’ cultivated plates; it was sterile after 21 days. On the third try, the study found only one fungus F. oxysporum. Most probably, some issues addressed the lower scope for residing fungi in garlic cloves. Firstly, a clove of garlic is protected by a few layers of peels, some outer peels, and one inner peel, which provide a barrier to penetrate any fungal entity into cloves. Secondly, garlic itself has resistive potential to grow fungus, and lastly, the clove’s surface is covered by a non-porous waxy layer (cuticle) that does not provide any scope for fungal invasion.^27–29 However, the garlic clove is connected to roots that offer the chance of fungi from the roots as well as soil. Exactly, the idea is proved by the isolated F. oxysporum as it is recognized as a notorious root fungus of³⁰

The study conveyed the first evidence of F. oxysporum isolation from the garlic clove. The fungus is an asexual soilborne fungus found in agricultural soil all over the world.^31,32 The species are saprophytic and can grow and survive in the rhizosphere of many plants and organic matter of soil. It penetrates the vascular system through the roots inducing either root rots or tracheomycosis.³³ However, the fungus has several strains that provide both pathogenic and nonpathogenic characteristics. A study included 38 strains of both types with morphologically indistinguishable features.³⁴ Moreover, diverse morphotypes produce asexual septate spore macroconidia in dissimilar lengths and wide.³⁵ In this study. the isolated fungus looked white or gray in color. However, colors might be varied due to the presence of pigments on several strains.³⁶

In-vitro bioactivity testing on the fungal species showed some extent of activity with respect to the references. Both antioxidant and cytotoxicity tests found the roughly one-fifth potential of ascorbic acid and vincristine sulfate, respectively. Moreover, the gram-negative bacteria (E. coli and P. aeruginosa) were susceptible to the fungal metabolites (80 µg/ml) and it was also nearly one-fifth of reference antibiotics (20 µg/ml). A study reported a polysaccharide from F. oxysporum that exhibited a 20 % free radical scavenging capacity that met the finding of this study.³⁷ However, in another report, F. oxysporum showed 51,5 % activity at 5 min free radical scavenging reaction time.³⁸ Probably, the host of the fungus as well as the geolocation is responsible for the variation. The presence of antibacterial and cytotoxic compounds in F. oxysporum metabolite has been previously reported.^39–41 While the sensitivity tests performed here resemble previous studies, further experiments are needed to confirm its potential for agrochemical and pharmaceutical applications.

Conclusion

The aim to explore endophytic fungi from an untouchable sample garlic clove was successful through repeated cultivation. Only one fungus was isolated and identified by morphological and molecular information. The in-vitro biological potential was also found by antioxidant, antimicrobial, and cytotoxicity tests and fungal metabolites exhibited a moderate level of activity in every aspect. As extracts contain tons of compounds and all are not reliable for a particular effect, further study should discover the prospective molecules to treasure pharmacological entities.

Declaration of interest

The authors declare that there is no conflict of interest. The authors alone are responsible for the content of the paper.

Acknowledgements

We would like to acknowledge Plant Biotechnology Department, National Institute of Biotechnology, Bangladesh for permitting us to conduct a part of the project at their laboratory.

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Abbreviations

ASE: Allium Sativum endophytic fungus; DCM: dichloromethane; DMSO; dimethyl sulfoxide; PDA: potato dextrose agar; DPPH: 2,2-diphenyl-1- picrylhydrazyl; IC₅₀: 50% inhibitory concentration; LC₅₀: 50% lethal concentration

| Received: [12 diciembre 2024] | Accepted: [4 febrero 2025] | Published: [15 marzo 2025]   |

Citation: Hoque, M. Hossain^, M. Islam, T. Ferdous^, M. Sarker, S. Kumar-Baral, P. Isolation of fungus from the cloves of Allium sativum, and evaluation of the antioxidant, antimicrobial, and cytotoxic potential of the ethyl acetate extract of this fungus. 2025.Volumen 10, (No 1). DOI: 10.70373/RB/2025.10.01.1

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