INTRODUCTION:

Lichen is a self-sustaining ecosystem formed by the interaction of an exhabitant fungus and an extracellular arrangement of one or more photosynthetic partners and an indeterminate number of other microscopic organisms¹. Lichen requires sufficient moisture and temperature to grow and these parameters also provide suitable conditions for the growth of some fungi which lives inside the lichen tissues².Those fungi are called as associative fungi. Associative fungi can be symptomatic or asymptomatic in nature i.e., symptomatic fungi can cause disease symptoms to the lichen host while asymptomatic fungi do not cause any disease to the host.

Thus asymptomatic fungi can also be called as beneficial fungi. One such asymptomatic fungi are endolichenic fungi. Endolichenic fungi is a diverse group of microfungi which resides within the lichen tissues²⁻¹⁰. They mainly belongs to Ascomycetes and Pezizomycetes. Endolichenic fungi produces various kinds of secondary metabolites such as quinones, terpenoids, benzopyranoids, etc.^5,11–17. These secondary metabolites exhibits antifungal, antibacterial, anticancerous, antioxidant and anti-inflammatory properties¹⁶⁻¹⁸. Many works on antimicrobial activity of endolichenic fungi have been reported, similar to that of endophytic fungi and also from medicinal plants^19–28. It has been hypothesized that the medicinal properties of lichens might be due to the presence of endolichenic fungi. The isolation of bioactive metabolites from endolichenic fungi harbouring lichens might result in conserving the lichen population from its natural environment as lichens have been exploited in a tremendous rate and their existence is under danger.

Cryptothecia sp. is a crustose lichen which mainly grows on bark of trees, rocks, etc. Triterpenoids along with many important compounds have been extracted from Cryptothecia sp. which showed strong antimicrobial activity²⁹. The present investigation was undertaken to isolate and identify the endolichenic fungi of Cryptothecia sp. and to screen the isolates for antimicrobial activity against a few selected human test pathogens.

MATERIALS AND METHODS:

Sample Collection and identification of Lichen sample:

Healthy lichen thallus was collected from Tipi region (93.61°N and 27.14°E) of Western Arunachal Pradesh which is a part of Indo-Burma belt. External morphology of collected lichen sample was studied under Leica EZ4 and Leica S9i stereozoom microscopes. Anatomical details were examined under Leica DM2500 compound microscope. Identification of lichen was done by following relevant literature³⁰.

Isolation and identification of endolichenic fungi:

The lichen thallus was firstly surface sterilized following standard protocol³¹. The surface sterilized lichen thallus was cut into smaller fractions and were air dried. The dried surface sterilized lichen fragments were placed onto three media namely PDA (Potato Dextrose Agar), MEA (Malt extract Agar) and WA (water Agar) which were supplemented with 0.01% Streptomycin sulphate. The plates were incubated at 28°C in BOD incubator until the growth of endolichenic fungi appeared. The isolated endolichenic fungi were inoculated in PDA slants and stored at 4°C.

The endolichenic fungi were identified on the basis of colony morphology and reproductive structures referring standard identification manuals^32,33.

Colonization frequency of Endolichenic fungi:

The relative colonization frequency (CF%) of endolichenic species was calculated using the formula:

CF % = (N_col / N_t) × 100

Where, N_col stands for the number of segments colonized by each endophytic fungal species, and N_t stands for the total number of segments plated^34,35which is also applicable for calculating colonization frequency of endolichenic fungal isolates colonization frequency.

Fungal cultivation and secondary metabolites extraction:

Pure endolichenic fungal isolates were cultivated in 100 ml of Potato dextrose broth (PDB).The endolichenic fungal isolates were incubated in BOD shaking incubator at 28°C with periodic shaking at 120rpm for 7 days. After subsequent period of incubation, the culture was filtered to remove the mycelia mats. The liquid broth was collected and extracted with 100ml of Ethyl acetate (EtOAc) in a separating funnel by vigorously shaking for about 15–20 minutes. The collected solvent was then allowed to evaporate in rotary evaporator. The crude extract after the complete evaporation of the EtOAc was dissolved in Dimethyl sulphoxide (DMSO) and stored at 4℃ for the determination of antimicrobial activity³⁵.

Determination of Antimicrobial activity:

The antimicrobial activities of the endolichenic fungi were determined with the help of agar cup diffusion method. The endolichenic fungi were tested against three human pathogens Staphylococcus aureus (MTCC 737), Escherichia coli (MTCC 443) and Candida albicans (MTCC 227). The test pathogens were procured from Institute of Microbial Technology (IMTECH), Chandigarh. The pathogenic bacteria suspension were inoculated in Muller Hinton Agar (MHA) plates and fungal suspension were inoculated in Sabouraud Dextrose Agar (SDA). The pathogens were evenly streaked with the help of sterile cotton swabs. Sterile corkborer (7 mm in diameter) used to scoop out the media to form agar cups. The agar cups were filled with crude metabolites of the endolichenic fungal isolates which were dissolved in DMSO to get the concentration of 1 mg/ml. The plates were incubated at 36±1°C for 24 hr in case of bacterial pathogens and 28±1℃ for 48 hr in case fungal pathogens. Antimicrobial activity of the crude metabolites was determined by the appearance of clear zones around the human test pathogens^35.

RESULTS:

Identification of lichen species:

In the present study, the lichen species was identified as Cryptothecia sp. (Fig 1). Identification was done on basis of its morphological and anatomical features and chemistry of the thallus.

Description:

Thallus corticolous, epiphloeodal, greenish-grey, ecorticate; prothallus white, well developed; photobiont Trentepohlia. Ascigerous tissue scattered in the thallus, slightly elevated fertile areas; paraphysoids densely branched and interwoven, enclosing the asci; asci aggregated in ascigerous areas, globose to broadly clavate thick walled muriform ascospores with wavy septa.

Fig 1: Lichen thallus of Cryptothecia sp.

Isolation of endolichenic fungi:

A total of 19 isolates of endolichenic fungi were isolated from the surface sterilized lichen fragments of Cryptothecia sp. (Table 1). Identification of all the endolichenic fungi was carried out referring standard fungal identification manuals. The fungal isolates that do not sporulate were categorised as Mycelia sterilia. Further, different colony characters and distinct morphological features of non-sporulating isolates were referred as morphotypes. The endolichenic isolates were identified as fungi belonging to the genera Trichoderma, Dreschlera, Penicillium, Mycelia sterilia and Monilinia. Among the endolichenic fungal isolates, colonization frequency of Monilinia sp. was found to be highest (26.66%) with maximum number of isolates (Fig 2).

Fig 2: Isolation of endolichenic fungi from Cryptothecia sp.

Table 1: Endolichenic fungi isolated from Cryptothecia sp. with rate of colonization

Endolichenic Fungi	No. of Isolates	Colonizing Frequency (CF%)
Morphotype 1	01	3.33
Morphotype 2	02	6.66
Morphotype 3	01	3.33
Trichoderma sp.1	02	6.66
Trichoderma sp.2	01	3.33
Penicillium sp.1	01	3.33
Penicillium sp.2	01	3.33
Dreschlera sp.	02	6.66
Monilinia sp.	08	26.66
Total	19

Determination of antimicrobial activity:

All the isolates were determined for antimicrobial activity against three human pathogens namely Escherichia coli (MTCC 443), Candida albicans (MTCC 227) and Staphylococcus aureus (MTCC 737). The results indicated that all the isolates showed antimicrobial activity by inhibiting at least one of the test pathogens (Table 2). Among the endolichenic fungi, Penicillium sp. (isolate CRP 3a) showed considerable antimicrobial activity against all the test pathogens. However, maximum zone of inhibition was observed against Escherichia coli and Candida albicans (Fig 3).

Table 2: Antimicrobial activity of the endolichenic fungal isolates against test pathogens

Endolichenic fungi	Isolate number	*Staphylococcus aureus* (MTCC 737)	*Candida albicans* (MTCC 227)	*Escherichia coli* (MTCC 443)
Morphotype 1	CRP 4	++	+	_
Morphotype 2	CRP 10	++	+	_
Morphotype 3	CRP7	_	++	_
Trichoderma sp.1	CRP 6a	_	+	+
Trichoderma sp.2	CRP 6b	++	_	+
Penicillium sp.1	CRP 3a	++	+++	+++
Penicillium sp.2	CRP 4a	++	+	_
Dreschlera sp.	CRP 2	+	++	_
Monilinia sp.	CRP 8	+	+	+

*Zone of inhibition(mm): >10 <15 = +, >15<20 = ++, >20 = +++, >10 = −

Fig 3: Antimicrobial activity of endolichenic fungal isolates against Staphylococcus aureus (MTCC 737), Escherichia coli (MTCC 443) and Candida albicans (MTCC 227)

DISCUSSION:

Endolichenic fungi have been isolated from many lichen species indicating that they are ubiquitous in nature. In the present study endolichenic fungi were isolated from Cryptothecia sp., a lichen known for its varied medicinal properties. Isolation of endolichenic fungi from medicinal lichen species have also been reported by many workers^2,8,18. The result indicated that Cryptothecia sp. harbours several endolichenic fungi and some of the isolates showed promising antimicrobial activity. This showed that lichen species with medicinal properties can be explored for endolichenic fungi for exploitation as antimicrobial agents. To our knowledge, our study is the first report on the isolation of endolichenic fungi from Cryptothecia sp., from North east India, as no further work has been reported till yet. In this present study Monilinia sp. (23.33%) was found to be the dominant endolichenic fungus followed by Mycelia sterilia (16.66%). It has also been reported that Mycelia sterilia is the dominant endolichenic fungal isolate from Parmelia³⁶.

One important aspect of endolichenic fungi is their ability to produce bioactive metabolites which show strong antimicrobial, antioxidant, anti-inflammatory and cytotoxic activity^37,38. In our present study, secondary metabolites were extracted from the endolichenic fungal isolates, which were then tested against pathogens to determine their antimicrobial activity. It has been found that most of the isolates showed antimicrobial activity by inhibiting atleast one of the pathogens. A total of about 88.8% of all the isolates showed antifungal activity against Candida albicans, while 77.7% and 44.4% of all the isolates showed antibacterial activity against Gram positive bacterium, Staphylococcus aureus and Gram negative bacterium Escherichia coli respectively. Out of all the isolates, Penicillium sp. (CRP 3a) showed highest antimicrobial activity against all the human test pathogens. Thus, our result indicated that most of the endolichenic fungi possess antimicrobial activity because of the presence of biologically active secondary metabolites. Further studies are now needed to identify the active compounds produced in order to discover novel drugs which may be helpful to the mankind.

ACKNOWLEDGEMENT:

First author is highly thankful to UGC National fellowship for OBC students in providing financial assistance to carry out the research work. The authors also thankfully acknowledge the financial support of Department of Biotechnology (DBT, Govt. of India) through Institutional Biotech Hub for establishment of Lichenology Laboratory at Nowgong College, Assam to avail the laboratory facilities for the identification of lichen.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 05.03.2021 Modified on 05.06.2021

Research J. Pharm. and Tech. 2022; 15(5):2193-2197.

DOI: 10.52711/0974-360X.2022.00364