Potential antifungal activity of Himalayan lichens: a methodological

assessment.

1Department of Botany

Pt. L.M.S. Government Post Graduate College,

Rishikesh(Dehradun),

Uttarakhand-249201, India

Himanshu Rai1, 2*, Roshni Khare1, 2, R.K. Gupta1, D.K. Upreti2, Priti Tiwari2

2Lichenology laboratory;

Plant Diversity, Systematics and Herbarium Division;,

CSIR-National Botanical research Institute,

Lucknow-226001, Uttar Pradesh, India

Lichen: Introduction

• Lichens, a symbiotic composite

of a fungus and a green or/ and

blue green algae

• Lichens are named based on the

fungal component, which plays

the primary role in determining

the lichens form.

• Lichens are among the most

tolerant organisms, capable of

inhabiting most of the terrestrial

habitats ranging from hot deserts

to alpine Antarctic habitats.

Parmelia sulcata

Lichens produce a wide array of both

Primary (intracellular) metabolites : amino acids,

polyols, carotenoids, polysaccharides, and

vitamins.

Secondary(extracellular) metabolites : Lichen

acids

Lichen Chemistry: Introduction

Lichen Secondary metabolites

Lichen secondary compounds are produced primarily by

the mycobiont.

Lichen secondary compounds are extracellular products

of relatively low molecular weight crystallized on the

hyphal cell walls of the cortex and/or medulla. .

Lichen secondary compounds are usually insoluble in

water and can be extracted into organic solvents.

Out of 800 secondary metabolites so far known from

lichens 650 are unique to them (82%).

Lichen Secondary metabolites: Major pathways

Lichen secondary metabolites are

derived from three chemical

pathways:

Shikimic acid pathway

•pulvinic acid derivatives (yellow

pigments)

Mevalonic acid pathway

•terpenes

Acetate-polymalonate pathway

•depsides, depsidones, usnic acid,

anthraquinones, xanthones,

aliphatic acids (majority of lichen

compounds)

Lichen Secondary compounds: Activities

LICHENS

Pulvinic acid

derivatives

Depsides,

Depsidones, Usnic

acid,

Anthraquinones,

Xanthones,

Aiphatic acids

Terpenes

Ecophysiological,

Immunostimulatory

and Enzyme

inhibitory

Objectives

Lichens from Uttarakhand have been studied taxonomically for past five decades (> 700 species known)

Lichens from Uttarakhand not have been screened for their biological activities (few reports).

A study was undertaken : To assess the antifungal activity of Himalayan lichens /

Lichens from Uttarakhand against some broad-spectrum plant pathogenic fungi.

To device an extraction protocol with maximum efficiency in terms of yield of secondary metabolites and screening assay for antifungal activity of lichen substances.

Experiment: Components

Lichens:

Four foliose lichen species:

Bulbothirx setschwanensis,

Everniastrum nepalense,

Heterodermia diademata,

Parmelaria thomsonii

collected from temperate

habitats of Pithoragarh

(1350-1760m), Uttarakhand

FUNGI:

Seven plant pathogenic

fungi :

Aspergillus flavus,

Aspergillus fumigatus

Alternaria alternata

Fusarium oxysporum

Fusarium solani

Fusarium roseum

Penicillium citrinum

Ketoconazole (positive control).

SOLVEN SYSTEM: .

Acetone,

Methanol Chloroform

Methodology: Procedure

2 Recovery

(Rotary evaporator )

3 Antifungal Assay

(Bauer-Kirby disc diffusion assay)

1 Extraction

(Soxhlet extractor)

Methodology: Soxhlet extraction

The solvent extraction was carried out at the specific boiling temperature of the solvents (acetone-56˚C, methanol-65˚C and chloroform -61.2˚C) for 48 hr for complete extraction of secondary compounds.

Methodology: Recovery

Rotary evaporation is a technique used to remove large volumes of volatile solvents from solutions. The rotary evaporator

rotates a flask containing the solution under a reduced pressure. The rotation spreads the solution out to form a thin film with a

greatly increased surface area speeding up the evaporation.

Lichen secondary metabolites were further recovered through gentle removal of solvents from lichen samples by evaporation using rotary evaporator (Büchi Rotavapor R-200TM).

Antifungal activity: Bauer-Kirby disc diffusion assay

Test solutions of lichen substances were prepared by dissolving recovered lichen substances in 10 ml of their respective solvents.

Experimental diffusion discs were prepared by loading five milliliters of lichen extract, 1 ml in each load on filter paper disks (6 mm in diameter), allowing the solvent to evaporate between each loading and leaving the lichen extracts on disk without the solvent.

All the three lichen extracts (i.e. acetone, methanol and chloroform) were loaded in this manner.

Loaded discs were planted on test plant pathogenic fungi culture plate in triplicate.

Commercially available synthetic antifungal drug Ketoconazole was used as positive control.

The plates were incubated for 5 days at 20°C to 25°C.

Growth was evaluated visually by comparing a particular plate with the negative control plates (having only plant pathogenic fungi).

Data Analysis: Multivariate study-PCA

Indirect gradient ordination method, principal component analysis (PCA) was used to summarise the effect of three solvent extracts of test lichens on test plant pathogenic fungi with reference to positive control Ketoconazole .

PCA was done on the basis of inhibition zone (mm) produced on test fungi colonies, utilizing correlation ma-trix, using multivar option in PAST 2.09.

Results: Extraction efficiency

15%

12%

15%

12%

The extraction process resulted in 10-15% efficiency in yield of secondary metabolites.

Results: Differential Antifungal activity

Preeti Tiwari, Himanshu Rai, Dalip Kumar Upreti, Suman Trivedi, Preeti Shukla

(2011).Assessment of antifungal activity of some Himalayan foliose lichens against

plant pathogenic fungi. American Journal of Plant Sciences, 2:841-846.

Preeti Tiwari, Himanshu Rai, Dalip Kumar Upreti, Suman Trivedi, Preeti Shukla

(2011).Assessment of antifungal activity of some Himalayan foliose lichens against plant

pathogenic fungi. American Journal of Plant Sciences, 2:841-846.

Results of comparative antifungal screening of the different solvent extracts (A = Acetone, M = Methanol, and Cl = Chloroform) of Himalayan foliose lichens and commercially available fungicide Ketoconazole(K), against selected plant pathogenic fungi (AF = Aspergillus flavus, Alt = Alternaria alternata, AFU = Aspergillus fumigatus, FUR = Fusarium roseum, FUS = Fusarium soloni, FOX = Fusarium oxysporum, and PC = Penicilium citrinum). Reported values are in Arithmetic mean ± Standard error.

Results: Principal Component Analysis

PCA Biplots of selected lichen

(a) = Bulbothrix setschwanensis,

(b) = Parmelaria thomsonii,

(c) = Heterodermia dia-demata

(d) = Everniastrum nepalense)

extracts (A = Acetone,

M = Methanol,

Cl = Chloroform)

and commercially available fungicide Ketoconazole(K) on different plant pathogenic fungi.

Preeti Tiwari, Himanshu Rai, Dalip Kumar Upreti, Suman Trivedi, Preeti Shukla (2011).Assessment of antifungal activity of some Himalayan

foliose lichens against plant pathogenic fungi. American Journal of Plant Sciences, 2:841-846.

PCA analysis required four components (axis) to account for 100% variance in dataset for all the four lichenized fungi. The first two components (axis) of PCA explained maximum variation

PCA biplots concluded that though positive control Ketoconazole showed higher degree of antifungal activity against some of the plant pathogenic fungi (Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus and Penicillium citrinum), the lichen extracts acetone and methanol extracts of lichens were comparatively more effective against some broad spectrum plant pathogenic fungi (Fusarium oxysporum, F. solani, F. roseum)

Conclusions

The better performance of lichenic extracts against commercially available antifungal Ketoconazole against some (Fusarium roseum, Fusarium solani and Fusarium oxysporum) plant pathogenic fungi suggests their superior potentials as fungicides.

The selective antifungal effect of acetone and methanol extracts of test lichens over chloroform extracts can be attributed to the presence of different constituent secondary metabolites in lichen thalli .