American Journal of Plant Sciences, 2011, 2, 841-846
doi:10.4236/ajps.2011.26099 Published Online December 2011 (http://www.SciRP.org/journal/ajps)
841
Assessment of Antifungal Activity of Some
Himalayan Foliose Lichens against Plant
Pathogenic Fungi
Priti Tiwari1,2, Himanshu Rai1, Dalip Kumar Upreti1*, Suman Trivedi2, Preeti Shukla1
1Lichenology Laboratory, CSIR—National Botanical Research Institute, Lucknow, India; 2Motilal Vigyan Mahavidyalaya, Barkatul-
lah University, Bhopal, India.
Received July 13th, 2011; revised October 7th, 2011; accepted October 21st, 2011.
ABSTRACT
In vitro antifungal activity of the acetone, methanol and chloroform extracts of four lichen species viz, Bulbothirx set-
schwanensis, Everniastrum nepalense, Heterodermia diademata, Parmelaria thomsonii were investigated against seven
plant pathogenic fungi (Aspergillus flavu s, A. fumigatus, Alternaria alternata, Fusarium oxysporum, F. solani, F. rose-
um and Penicillium citrinum) with reference to commercially available synthetic antifungal drug Ketoconazole (positive
control). Lichen secondary metabolites were extracted using Soxhlet extractor and were further recovered through gen-
tle evaporation of solvents in rotary evaporator. Antifungal activity was analysed employing Bauer-Kirby disc diffu-
sion assay. Acetone and methanol extracts of lichenized fungi were found more effective against tested plant pathogenic
fungi. Principal component analysis concluded that though, Ketoconazole was effective against four of the tested plant
pathogenic fungi, acetone and methanol extracts of lichens were comparatively more effective against some broad
spectrum plant pathogenic fungi (Fusarium oxysporum, F. solani, F. roseum).
Keywords: Acetone Extract, Antifungal Activity, Bauer-Kirby Disc Diffusion Assay, Fo liose, Himalayan Lichen,
Methanol Extract, Rotary Evaporator, Secondary Metabolites, Soxhlet Extractor
1. Introduction
The utilization of lichen in medicine has been cited in
different pharmacopoeias of the world. During the middle-
ages lichens figured prominently among the herbs used
by medicinal practitioners [1]. Lobaria pulmonaria, Ce-
traria islandica, and Cladonia species are reported to
be effective in the treatment of pulmonary tuberculosis
[2].
Lichens synthesize a wide range of primary (polysac-
charides) and secondary organic compounds that show
manifold bioactivities from nematocidal, antimicrobial,
cytotoxic, antimutagenic and antiproliferative to immu-
nostimulatory effects [3-5]. Out of 700 secondary meta-
bolites so far known from lichens 550 are unique to them
[6]. The lichen extracts and their components have a dis-
tinguished antimicrobial activity [2,5,7-9]. On the other
hand, it is well known that microorganisms have well
developed resistance to many antibiotics. This creates
enormous problems in the treatment of infectious disease,
and investigators therefore seek new antimicrobial sub-
stances from different sources so new sources of bioac-
tive substances have been searched for, such as medici-
nal herbs, fungi and lichens [10,11].
India being a mega diversity country exhibit rich di-
versity of different plant groups.The medicinal properties
of higher group of plants is well known from the country.
However, despite of the manifold medicinal uses of lower
plants by traditional and ethnic groups the medicinal po-
tential of these plants are not studied upto a greater ex-
tent. Thus, present study was done to evaluate the in vi-
tro anti- fungal activity of four foliose lichens Bulbothrix
setschwanensis (Zahlbr.) Hale, Parmelaria thomsonii (Stir-
ton) D.D. Awasthi, Everniastrum nepalense (Taylor) Hale,
Heterodermia diademata (Taylor) D.D. Awasthi, against
some common plant pathogenic fungi.
2. Materials and Methods
2.1. Collection and Identification of Lichen
Samples
The lichen specimens of Bulbothrix setschwanensis (Zahlbr.)

Assessment of Antifungal Activity of Some Himalayan Foliose Lichens against Plant Pathogenic Fungi
842
Hale, Parmelaria thomsonii (Stirton) D.D. Awasthi, Ev-
erniastrum nepalense (Taylor) Hale, Heterodermia dia-
demata (Taylor) D.D. Awasthi, growing luxuriantly in
temperate regions of India were collected from different
locations of Pithoragarh district, Uttarakhand. The iden-
tification was done morpho-anatomically using a La-
bomedTM stereomicroscope and LeicaTM DM 500 optical
microscope and chemically with the help of thin-layer
chromatography [12,13]. Identification was done using
relevant key and monographs [14,15]. The voucher spe-
cimens were deposited at the lichen herbarium (LWG),
National Botanical Research Institute (NBRI), Lucknow,
India.
2.2. Extraction from Lichen Sample
Lichen samples were sorted, cleaned of substratum and
dried for extraction. Three different solvent systems i.e.
acetone, methanol and chloroform were used for extrac-
tion. Lichen substances were extracted using Soxhlet ex-
tractor equipped with a reflux condenser [16,17] in se-
lected solvents (acetone, methanol and chloroform) and
further recovered through gentle removal of solvents
from lichen samples by evaporation using rotary evapo-
rator (Büchi Rotavapor R-200TM). 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 h for complete extraction of secondary
compounds.
2.3. Microorganisms and Media
Seven fungal strains were procured from the mycological
collection maintained by the Mycological Laboratory wi-
thin the Department of microbiology Kanpur University.
The fungi used as test organisms were: Aspergillus fla-
vus, Aspergillus fumigatus, Alternaria alternata, Fusa-
rium oxysporum, Fusarium solani, Fusarium roseum
and Penicillium citrinum. Fungal cultures were main-
tained on Potato Dextrose agar (PDA) and were trans-
ferred to Sabourad dextrose agar for experimental pur-
poses.
2.4. Determination of Antimicrobial Activity
The antimicrobial activity of lichen extracts against test
fungi was determined employing disk diffusion method
[18-21]. Fungi strains were inoculated on potato dextrose
agar plate (108 spores/ml) in triplicate.
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 sol-
vent. 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 an-
tifungal 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). The antimicrobial activity was evalu-
ated by measuring the inhibition zone diameter (in mil-
limeter) observed (National Committee for Clincal Labo-
ratory Standards Nccls Document [22]).
2.5. Data Analysis
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 [23,
24]. 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 [25,26].
3. Results
3.1. General Patterns of Antifungal Activity
Disc diffusion assay of the crude extracts of all the four
lichens Bulbothrix setschwanensis, Parmelaria thomsonii,
Heterodermia diademata and Everniastrum nepalense
showed antifungal activity against most of the tested
fungi (Figures 1 and 2). A differential activity of the three
extracts (i.e. Acetone, Methanol and Chloroform) was
observed. Among the three acetone and methanol ex-
tracts were more effective than the chloroform extract.
Lichen extract in acetone was found more effective in
Bulbothrix setschwanensis, Parmelaria thomsonii and
Heterodermia diademata, whereas in Everniastrum ne-
palense methanol extract was found more effective than
other solvents. Positive control Ketoconazole was inef-
fective in case of Fusariu m ro se um , F usa riu m sola n i and
Fusarium oxysporum whereas lichen extracts were found
active against these pathogens. The chloroform extracts
of all the four lichens though were sporadically more
effective than some extracts but overall Chloroform ex-
tracts were not as effective as acetone and methanol ex-
tracts.
The acetone and methanol extracts of Bulbothrix set-
schwanensis showed activity against Fusarium roseum
and Fusarium solani while the chloroform extract and
the positive control Ketoconazole exhibited no activity.
The methanol extract of Bulbot hrix setschwanensis showed
maximum zone of inhibition of 15 mm while the Keto-
conazole exhibited 20 mm zone of inhibition against
Penicillium citrinum. The chloroform extract exhibited
no activity against Fusarium solani, Fusarium roseum
and Alternaria alternata.

Assessment of Antifungal Activity of Some Himalayan Foliose Lichens against Plant Pathogenic Fungi 843
Fig ure 1. Result s of co mparat ive a ntifung al scr eening of the dif ferent solve nt ex tracts (A = Acetone, M = M ethanol, a nd Cl =
Chloroform) of Himalayan foliose lichens and commercially available fungicide Ketoconazole(K), against selected plant
pathog enic fu ngi (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 Arit hmetic mean
± Standar d error.
3.2. Principal Component Analysis (PCA)
The acetone extracts of Parmelaria thomsonii were
active against all the seven tested fungi. Though all the
three lichen extracts in acetone methanol and chloroform
exhibited inhibition zones of 19 mm, 7 mm and 6 mm
respectively, the positive control showed no antifungal
activity against Fusarium oxysporum. The maximum
zone of inhibition 24 mm exhibited by methanol extract
of Parmelaria thomsonii against Fusarium roseum was
better than Ketoconazole. No activity was recorded by
Methanol and chloroform extracts against Aspergillus
flavus, Alternaria a lternata and Penicillium citrinum.
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 (Bulbothrix setschwanensis-72%; Par-
melaria thomsonii-83%; Heterodermia diademata-70%;
Everniastrum nepalense-90%) and were taken into ac-
count in the study (Figure 3). PCA biplots concluded that
though positive control Ketoconazole showed higher
degree of antifungal activity against some of the plant
pathogenic fungi (Alternaria alternata, Aspergillus fla-
vus, Asp ergillus fumiga tus and Penicillium citrinum), the
lichen extracts were more effective against broad spec-
trum plant pathogenic fungi (Fusarium oxysporum, F.
roseum, F. soloni (Figure 3).
Acetone and methanol extracts of Heterodermia dia-
demata were active against all the seven tested fungi
while the chloroform extract showed activity against only
two pathogenic fungi Alternaria alternata and Penicil-
lium citrinum. The maximum zone of inhibition 19 mm
was exhibited by methanol extract. 4. Discussion
The methanol extracts of Everniastrum nepalense sho-
wed antifungal activity against all the tested fungi, while
acetone extract exhibited activity against only four pa-
thogenic fungi. Maximum zone of inhibition 20 mm was
showed by methanol extract which is better than the in-
hibitory zone of Ketoconazole 15 mm against Penicil-
lium citrinum.
Bioactive compounds in recent past are gaining edge
over traditionally known drugs because of their improved
effectiveness against pathogens, the lichen compounds
are not an exception in this field [27]. Extracts of lichen
thalli proved to have strong antifungal activity against
various plant pathogenic fungi [9,28].

Assessment of Antifungal Activity of Some Himalayan Foliose Lichens against Plant Pathogenic Fungi
844
Figure 2. The inhibition zones of tested lichen extracts (A = Acetone, M = Methanol and Cl = Chloroform) against selected
plant pathogenic fungi and commercially availa ble fungicide Ketoconazole (K ). Bar = 10 mm.

Assessment of Antifungal Activity of Some Himalayan Foliose Lichens against Plant Pathogenic Fungi845
Figure 3. P CA Biplots of selected li chen ((a) = Bulbothrix setschwanensis, (b) = Parm ela ria t hom son ii, (c) = Heterodermia dia-
demata (d) = Everniastrum nepalense) extracts (A = Acetone, M = Methanol, Cl = Chloroform) and commercially available
fungicide Ketoc onazole(K) on different pla nt pathogenic fungi .
The present study with different solvent extracts of
Bulbothrix setschwanensis, Parmelaria thomsonii, Hete-
rodermia diademata and Everniastrum nepalense showed
promising results against some well known plant patho-
genic fungi. 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 [28,29].
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 supe-
rior potentials as fungicides.
5. Acknowledgements
Authors are thankful to the Director, National Botanical
Research Institute (CSIR), Lucknow for providing neces-
sary laboratory facilities. This research work is dedicated
to Late Dr. D.D. Awasthi (28 Sept. 1922-21 Aug. 2011)
pioneer Lichenologist, whose work laid foundation for
systematic lichenological research in Indian subcontinent.
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