Deciphering the in vitro Antimicrobial Potential of Some Macrolichens from Burachapori Wildlife Sanctuary, Assam, India

INTRODUCTION

Lichens are complex organisms consisting of two microorganisms, an algae and a fungus, as well as several kinds of bacteria, fungi, and non-obligate viruses that form a microecosystem. Lichenicolous fungi are those that grow on the lichen’s exterior, and endolichenic fungi are those that reside inside the lichen (Pant et al, 2025). These are fascinating biological systems that result from a symbiotic relationship, usually between an algal or cyanobacterial photobiont and a fungal mycobiont. Their distinct biology enables them to synthesize a wide range of secondary metabolites that are unique in other species and to colonize harsh environments, such as exposed rock faces in deserts and polar tundra (Gopal 2024). There are over 3,200 distinct species of lichens in India alone, out of the approximately 20,000 species that are known to exist worldwide (Huneck, 1999). These microorganisms produce a variety of potent secondary metabolites including antibiotics. The different bioactive substances that may be isolated from lichens show promise for biopharmaceutical uses for developing novel formulations or methods that include cytotoxic, antioxidant, and antimicrobial agents (Pradhan, 2023). The antimicrobial active components were found to be primarily concentrated in the organic solvent extract phases, particularly in the solvent extract phases such as methanol, ethanol, ethyl acetate, and acetone, while investigating the antibacterial potential of lichen extract (Tian et al, 2025). Several workers had investigated the antimicrobial effects of lichen species against significant pathogenic microorganisms in recent days showing the antibiotic potential of the lichen species (Prashith et al, 2015,Yadav et al, 2021, Sahoo et al, 2021, Essadki et al, 2024, Piñeiro, 2025). Matić et al. discovered noteworthy results when they examined the antibacterial and antioxidant activities of the methanol extracts of the lichens Cladonia rangiferina and Lobaria pulmonaria against Staphylococcus aureus in vitro. Natural endolichenic fungal products are a substantial source of new metabolites with potential uses in agriculture and medicine (Mansour et al, 2025).In traditional medicine, several lichens have been used to cure a variety of illnesses, such as diabetes, stomach problems, coughs, pulmonary TB, wound healing, and skin ailments (Srivastava, 2013). In today’s era, the fundamental problem of persistent antibiotic resistance in microorganisms is posing a threat to public health around the world. This constant concern has made the development of new antimicrobial agents and drugs to fight infectious diseases necessary and the lichens being the interesting organisms possessing bioactive compounds are being thoroughly screened for antimicrobial agents and properties.

MATERIALS AND METHODS

Lichen collection and identification

The lichen samples used in the study were collected from Burachapori Wildlife Sanctuary of Sonitpur district, Assam (India) during the field work from October 2021 to May 2023.They were identified at Lichenology Laboratory of CSIR-NBRI,Lucknow. The voucher specimens of the identified and used lichen samples in this study- Ramalina hossei no. 65470(LWG), Vain. Ramalina nervulosa (Müll.Arg.) Abbayes no. 65471(LWG), Parmotrema praesorediosum (Nyl.) Hale no. 65456(LWG) and Parmotrema tinctorum (Despr. ex Nyl.) Hale no. 65455(LWG) (as seen in the Figure 1) were deposited in the herbarium LWG of CSIR-National Botanical Research Institute, Lucknow, India.

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Preparation of lichen extracts

The dried samples were finely ground using an electric grinder and pulverized material (10 g) was extracted twice by soaking with 100 mL of acetone for 48 hr at room temperature. Further the separated extracts were filtered through Whatman No. 1 filter paper and the acetone filtrate was condensed to dryness using rotary evaporator at 40˚C. The same method was followed separately with methanol and chloroform. The extracts prepared in this way were used for antimicrobial studies (Tian, 2025).

Test microorganisms

The bioprospection was performed by taking three gram positive three gram negative pathogenic bacterial and three fungal strains were procured as the test organisms from recognized collection centre in India. They are the selected gram-positive bacterial strains such as Enterococcus faecalis MTCC-439, Bacillus cereus MTCC-5521, Clostridium botulinum MTCC-1349 and gram-negative strains Salmonella enteritidis MTCC-734, Pseudomonas aeruginosa MTCC-1688, Acinetobacter baumannii MTCC-1425.Whereas Aspergillus clavatus MTCC-1344, Aspergillus fumigatus and Candida glabrata MTCC-6507 are the fungal specimens.

Determination of Antimicrobial Activity

Preparation of Acetone, Methanol and Chloroform extracts done separately and performed the Antimicrobial studies using agar well diffusion methods.

Agar Well Diffusion method for antibacterial studies

The agar well diffusion method was employed to determine the antibacterial activity of the lichen samples against the selected gram-positive bacterial strains such as Enterococcus faecalis MTCC-439, Bacillus cereus MTCC-5521, Clostridium botulinum MTCC-1349 and gram-negative strains Salmonella enteritidis MTCC-734, Pseudomonas aeruginosa MTCC-1688, Acinetobacter baumannii MTCC-1425. A subculture of each bacterial strain at a volume of 200 µL, equivalent to 10⁶ CFU/mL, was uniformly spread onto the surface of a Petri dish containing 20mL of nutrient agar, using a sterile cotton swab and wells were punched using a sterile gel borer. On the Nutrient agar, five wells with a diameter of 8 mm each were created for the bacterial strains. The first well was designated as the negative control and was loaded with 100 μL of DMSO, using which all the extracts were dissolved in the concentrations of 1mg/mL and 2mg/mL. While second well served as the positive control and contained 100 μL of Ampicillin (an antibiotic). Rest of the wells in all the plates contained 100 μL of test drug concentration 1mg/mL. Another same set of petri dishes were prepared for drug concentration 2mg/mL. The Petri dishes were prepared in triplicates and incubated at 22^oC for 3-4 days, following which the measurement of the zone of inhibition surrounding the wells was performed after the incubation period. The concentration of positive control used was Ampicillin (0.1 mg/mL). The antibacterial activity of each compound was analysed by calculating the diameter of the inhibition zone and the potency of each compound was correlated with Ampicillin.

Agar Well Diffusion method for antifungal studies

The agar well diffusion method was employed to determine the antifungal activity of lichen samples against the selected fungal strains Aspergillus clavatus MTCC-1344, Aspergillus fumigatus MTCC-343 and Candida glabrata MTCC-6507. A standardized suspension of the test fungi were inoculated and incubated for 16-18 hr. at 37^oC to obtain fresh cultures. This was uniformly spread onto the surface of a Petri dish containing 20mL of potato dextrose agar, using a sterile cotton swab and wells were punched using a sterile gel borer. On the agar, five wells with a diameter of 8 mm each were created for the fungal strains. The extracts were dissolved in solvent DMSO with different concentrations such as 1mg and 2 mg/mL. The second well was designated as the negative control and was loaded with 100 μL of DMSO, using which all the extracts were dissolved in the concentrations of 1mg/mL and 2mg/mL. While second well served as the positive control and contained 100 μL of Fluconazole (an antifungal tablet). Rest of the wells in all the plates contained 100 μL of test drug concentration 1mg/mL. Another same set of petri dishes were prepared for drug concentration 2mg/mL. The Petri dishes were prepared in triplicates and incubated at 22^oC for 3-4 days. The antifungal activity of each compound was analysed by calculating the diameter of the inhibition zone and the potency of each compound was correlated with Fluconazole (Zambare, 2012).

RESULTS

Each of the four lichen samples that were examined showed a dose-dependent inhibitory ability against the tested organisms. When comparison to 1 mg/mL, zones are observed to be higher at 2 mg/mL which can be seen in Table 1. Additionally, it is noted that whereas methanol extracts had a moderate effect, acetone extracts of every sample showed excellent inhibition. Chloroform extracts follow next. Every triple experiment showed identical findings.

Anti-bacterial studies Antifungal studies

The screening of the lichen species against fungal specimens can be observed in Table 2:

DISCUSSION

Antibacterial activity

All the 4 lichen samples has shown to be effective against 6 tested organisms having dose dependent inhibitory capacity. Zones are found to be higher at 2 mg/mL when compared to 1mg/ mL. The zone of inhibition range from 16.7±0.17 to 28.2±0.15 (in m.m.). The lowest was recorded for Chloroform extract of Parmotrema praesorediosum against Acinetobacter baumannii in 1 mg concentration. Whereas the highest zone of inhibition was recorded for Acetone extract of Ramalina nervulosa against Enterococcus faecalis in 2 mg concentration. Furthermore, it is noted that whereas methanol extracts had a moderate effect, acetone extracts of every sample shown excellent inhibition and least effect was shown by the Chloroform extracts. Every triple experiment yielded identical findings.

Antifungal activity

In case of antifungal activity also all the 4 lichen samples tested has shown to be effective against 3 tested organisms having the dose dependent inhibitory capacity. Zones are found to be higher at 2 mg/mL when compared to 1 mg/mL. The zones of inhibition were seen in the range 17.7±0.55 to 26.4±0.46 (in m.m.). Chloroform extract of Parmotrema praesorediosum has shown the lowest zone of inhibition against Aspergillus fumigatus in 1mg concentration. On the other hand Acetone extract of Parmotrema praesorediosum has shown the highest zone of inhibition against Aspergillus clavatus in 2 mg concentration. Also it is observed that acetone extracts of all the samples have shown excellent inhibition, whereas methanol extracts have showed moderate effect followed by the chloroform extracts. The results obtained are same in all the triplicate experiments.

CONCLUSION

From the experiment it was confirmed that lichens are interesting organisms having the antibacterial and antifungal activities. The pathogenic microorganisms were screened in vitro and found to have antimicrobial potential with zones of inhibition of various ranges. This study is really helpful to identify, isolate and characterize the principal compounds exhibiting the antimicrobial activity from the potential and medicinally important lichen species.