Radhajeyalakshmi Raju-APPP

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In vitro evaluation of solvent extracted
compounds from edible macromycetes
against phytopathogenic fungi
R. Radhajeyalakshmi
a
, R. Velazhahan
a
& V. Prakasam
a
a
Department of Plant Pathology, Centre for Plant Protection
Studies, Tamil Nadu Agricultural University, Coimbatore, 641 003,
India
Available online: 03 Nov 2011
To cite this article: R. Radhajeyalakshmi, R. Velazhahan & V. Prakasam (2011): In vitro evaluation
of solvent extracted compounds from edible macromycetes against phytopathogenic fungi, Archives
Of Phytopathology And Plant Protection, DOI:10.1080/03235408.2011.559048
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In vitro evaluation of solvent extracted compounds from edible
macromycetes against phytopathogenic fungi
R. Radhajeyalakshmi*, R. Velazhahan and V. Prakasam
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural
University, Coimbatore 641 003, India
(Received 26 November 2010; final version received 11 December 2010)
Extraction of antimicrobial compounds from medicinal mushrooms, namely,
Auricularia polytricha (Jew’s ear), Lentinulla edodes (Shiitake) and Volvariella
volvacea (paddy straw), has been done with different solvent systems (chloroform,
ethyl acetate, ether and methanol) and tested against a wide range of
phytopathogens by filter paper disk assay. All the three basidiomycetes inhibited
the phytopathogens tested so far. This enumeration was based on the number of
organisms inhibited and the diameter of inhibitory zones produced. From the results
obtained, it could be observed that ethyl acetate was the best solvent for extracting
antimicrobial substances from these medicinal mushrooms. Thin layer chromato-
graphy revealed the presence of several compounds with different Retention Factor
(RF) values. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis profiling
of V. volvacea showed the presence of several antimicrobial proteins with various
molecular weights. Western blotting revealed the presence of thaumatin-like
glycoproteins of molecular weight more than 45 kDa.
Keywords: Auricularia polytricha; Lentinulla edodes; Volvariella volvacea;
basidiomycetes; phytopathogens; TLC; SDS-PAGE
Introduction
Many antibiotics in clinical use were developed from fungal and actinomycetes
metabolites. The first investigations on the potential of basidiomycetes as sources of
antibiotics were performed by Anchel, Hervey and Wilkins in 1941 (Sandven 2000)
by examining the extracts of fruiting bodies and mycelial culture from over 2000
species. In the last decades, the wide range of pharmaceutically interesting
metabolites from basidiomycetes, a large group of terrestrial fungi of the phylum
basidiomycota, has been one of the most attractive groups of natural products
studied (Boh et al. 2007). In fact, several compounds that inhibit the growth of a
large spectrum of saprophytic and phytopathogenic fungi were isolated from
basidiomycetes (Anke 1997). Furthermore, these organisms are able to inhibit the
development of bacteria, actinomycetes and other fungi from their microhabitat,
indicating that the antimicrobial substances produced by them have important
ecological implications (Sidorova and Velikanov 2000). Despite their potential and
enormous diversity in tropical ecosystems, few studies aiming at the discovery of
*Corresponding author. Email: radhajeyalakshmi@hotmail.com
Archives of Phytopathology and Plant Protection
iFirst article 2011, 1–8
ISSN 0323-5408 print/ISSN 1477-2906 online
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bioactive compounds from basidiomycetes were conducted, and most of the
investigations were directed to edible mushrooms (Ishikawa et al. 2001; Paccola
et al. 2001; Oliveira et al. 2002). Pleuromutilin, a diterpene that is especially useful
for the treatment of mycoplasm infections in animals, was one of the first
commercial antibiotics developed from basidiomycete origin (Rosa et al. 2003).
Since it usually takes several months to cultivate basidiomycetes using traditional
basidiome (fruiting body) cultivation method, liquid culture is viewed as a promising
alternative. Submerged cultivation of macrofungi such as basidiomycetes presents
some interesting challenges. Unlike filamentous fungi, with which it is quite easy to
produce spores and use them as inoculum, it is not practical to use spore inocula of
basidiomycetes for fermentation since spores are only produced in the basidiome.
Therefore, the approach used in this study was to prepare mycelium-based inocula
grown on agar. The ability to manipulate culture conditions in submerged
cultivation may lead to the production of a wider range of bioactive compounds
of therapeutic importance by basidiomycetes (Lindequist et al. 2005). The main goals
of this study were to cultivate the basidiomycetes of medicinal importance in
submerged cultures and to evaluate their ability to produce antimicrobial substances
of pharmaceutical interest.
Materials and methods
Macromycetes
Edible macromycetes, namely, Auricularia polytricha (Jew’s ear), Lentinulla edodes
(Shiitake) and Volvariella volvacea (paddy straw), are obtained from ‘‘Culture
collection of wild mushroom species’’ Department of Plant Pathology, Tamil Nadu
Agricultural University (TNAU), Coimbatore 641003 and are used in this study. The
macromycetes strains were maintained in potato dextrose agar (PDA) medium at
258C for further study.
Fungal cultures and growth conditions
The phytopathogenic fungi Alternaria solani, Colletotrichum capsici, Rhizoctonia
solai and Pythium aphanidermatum were collected from the Department of Plant
Pathology, TNAU, Coimbatore. Cultures were maintained on PDA medium. The
mushroom cultures (A. polytricha, L. edodes and V. volvacea) were prepared by
plating the young sporocarp tissue (8-day-old) in the centre of the PDA, and well-
grown mycelium was subcultured to fresh plates of PDA for every 15 days after
culturing.
Extraction of antimicrobial compounds by solvent extraction systems
The inoculum of A. polytricha, L. edodes and V. volvacea for submerged culture
consisted of a 5-mm-diameter plug of each isolate taken from 7-day-old cultures on
PDA plates. Mycelial plugs were transferred into Petri dishes and Erlenmayar flasks
containing 20 ml of liquid substrate based on potato dextrose. The culture filtrates
were collected from 25 days after inoculation, separated with various solvent
systems, namely, ethyl acetate, ether, methanol and chloroform for separating the
non-protein fraction. The fractions were evaporated in vacuo at 408C using Buchi EL
141 Rotavapor, and residues were dissolved in 2 ml of each solvent. These condensed
2 R. Radhajeyalakshmi et al.

extracts were filtered through 0.2-m syringe filter (Millipore, Bedford) and assayed
for antifungal activity.
Fungal growth inhibition assay
For testing antifungal activity, mycelial disk (7 mm) cut from 4-day-old culture of
test pathogen was placed in the centre of the Petri dish (90 mm in diameter)
containing PDA medium. The plates were incubated at room temperature (288C) for
24 h. Then sterile filter paper disks (6 mm in diameter) were placed on the agar
surface at 1 cm away from the periphery of the Petri dish, and 50 ml of the
supernatant fractions were applied to each disk, and plates were further incubated at
room temperature and observed for inhibition of mycelial growth.
Thin layer chromatography
Thin layer chromatography (TLC) analysis was carried out on silica gel (Silica gel 60
F254, Merck, Germany). The plates were activated at 1008C for 30 min, cooled,
spotted with 15 ml of extract from the V. volvacea, L. edodes and A. polytricha.
Samples were loaded at 1.5-cm intervals and 2 cm from the bottom of the plate.
Plates were developed with different solvent systems, namely, isopropanol:
ammonia: water, chloroform: ethyl acetate: acetone: methanol (ethyl acetate
fractions) and chloroform: acetone: water (chloroform fractions), and the
chromatogram was viewed under UV light (365 nm).
Extraction of Pathogenisis Related (PR) proteins
Mycelial mats were harvested from 25-day-old grown cultures, and proteins were
extracted from 1-g fresh weight tissues in 2 ml of 0.2 M citrate phosphate buffer (pH
2.8) in a pre-chilled pestle and mortar at 48C. The homogenate was squeezed through
four layers of cheese cloth and centrifuged at 12,000g for 30 min. The supernatant
was subsequently dialysed against ultra pure water at 48C for 24 h and lyophilised.
The dried residues were redissolved in 200 ml of 0.2 M citrate phosphate buffer (pH
2.8) and stored at 7208C in sample storage eppendorf tubes until use.
Western blotting
Proteins in the samples of the extract obtained above were precipitated overnight
with five volumes of 80% acetone at 7208C. The precipitate was then dissolved in 40
ml of a sample buffer containing 0.06 ml of 25 mM Tris, 2% sodium dodecyl
sulphate (SDS), 10% glycerol, 5% 2-mercaptoethanol and 0.01% bromophenol blue
at pH 6.8 by vigorous vortexing and boiled for 5 min after polyacrylamide gel
electrophoresis (PAGE) (Laemmli 1970). Protein concentrations were determined by
Bradford assay (1976) using bovine serum albumin as a standard. Proteins (100 mg)
were separated by SDS-PAGE in a Mighty Small II gel electrophoresis unit (Hoefer
Scientific Instruments, San Francisco, CA) with 12% acrylamide resolving gel and
4% acrylamide stacking gel. The gels were electrophoresed for 2 h at a constant
current of 20 mA. After electrophoresis, the proteins were electrotransferred to a
polyvinylene difluoride membrane (BioRad, Hercules, CA) for 30 min at 140 mA in
a BioRad semi-dry transblot apparatus in accordance with the manufacturer’s
Archives of Phytopathology and Plant Protection 3

instructions. The membrane was then blocked with Tris-buffered saline (10 mmol l71
Tris-HCl, pH 7.9, 140 mmol l71
NaCl) containing 0.05% (v/v) Tween-20
supplemented with 2.5% (w/v) gelatin. Antiserum raised against maize zeamatin
(a gift of Dr CP Selitrennikoff, University of Colorado Health Sciences Center,
Denver, CO) was used as primary antibody at 1:1000 dilution. Detection of
thaumatin-like protein on the membrane was performed according to Winston et al.
(1987) using a 1:1000 dilution of horseradish peroxidase-conjugated goat-anti rabbit
IgG (BioRad). Colour development was with 4-chloro-1-naphthol (BioRad).
Figure 1. TLC showing the spots with different RF values from A. polytricha, L. edodes and
V. volvacea (methanol, ethyl acetate and chloroform) solvent extracts at 10% (v/v) concentration
with different mobile phases (A) isoproponal: ammonia: water, (B) chloroform: ethyl acetate:
acetone: methanol and (C) chloroform: acetone: water under UV light.
Figure 2. Western blot showing expression of thaumatin-like glycoproteins from
A. polytricha, L. edodes and V. volvacea. Aliquots (100 mg) of proteins from the mycelial
mat were analysed by western blotting after SDS-PAGE, hybridised with zeamatin antiserum.

Results and discussion
The mushroom genome stands out as a virtually untapped resource for novel
antimicrobial metabolites. However, over 6000 metabolites were already identified
from these imperfect fungi, making it more and more difficult to isolate novel
bioactive metabolites from them (Abraham 2001). With the development of new
fermentation and purification technologies, basidiomycetes with medicinal properties
are again receiving attention as potential sources of new classes of antibiotics. In fact,
several compounds that inhibit the growth of a large spectrum of saprophytic and
phytopathogenic fungi were isolated from basidiomycetes. Therefore, the objective of
this work is to provide information on the antimicrobial activities and compounds in
medicinal mushrooms, namely, A. polytricha (Jew’s ear), L. edodes (Shiitake) and
Figure 3. Inhibition of mycelial growth of plant pathogenic fungi by A. polytricha solvent
extract. Antifungal activity of A. polytricha solvent extract at 10% (v/v) concentration was
tested by filter paper disk assay. Mycelial growth inhibition was measured after 72 h at
28 + 28C. Values are the means + SE of six replications.
Figure 4. Inhibition of mycelial growth of plant pathogenic fungi by V. volvacea solvent
extract. Antifungal activity of V. volvacea solvent extract at 10% (v/v) concentration was
tested by filter paper disk assay. Mycelial growth inhibition was measured after 72 h at
28 + 28C. Values are the means + SE of six replications.

V. volvacea (Paddy straw). Among the media tested for the production of antifungal
metabolites, potato dextrose broth promoted the synthesis of antimicrobial
compounds from A. ploytricha, L. edodes and V. volvacea (data not shown).
Extraction of antimicrobial compounds from these species has been done with
different solvent systems and tested against a wide range of phytopathogens by filter
paper disk assay. All the three basidiomycetes inhibited the phytopathogens tested so
far. The protein fractions of these three mushroom fungi V. volvacea, L. edodes and
A. polytricha inhibited the mycelial growth of the foliar pathogens when tested by
filter paper disk assay with a minimum inhibition zone of 45 mm (Figures 3, 4 and 5).
This suggestion was based on the number of organisms inhibited and the
diameter of inhibitory zones produced. The frequency of the resultant antimicrobial
activities was considered as an indicator of the ability of the different genera of
basidiomycetes to produce bioactive secondary metabolites with potential ther-
apeutic interest. From the results obtained, it could be observed that ethyl acetate
was the best solvent for extracting antimicrobial substances from these medicinal
mushrooms. TLC revealed the presence of several compounds with different RF
values. Western blotting revealed the presence of thaumatin-like glycoproteins of
molecular weight 45 kDa (Figure 2). The results of the present study are similar with
the research reports of Rosa et al. (2003) from a total of 103 isolates of
basidiomycetes, representing 84 species from different Brazilion ecosystems were
shown antimicrobial activity. Culture extracts of Mycena leptocephala showed
antifungal activity against Candida lipolylica (Vahidi et al. 2004). Antimicrobial
activity has also been found in liquid cultures of L. edodes and chloroform, ethyl
acetate or water extracts of dried mushrooms (Hirasawa et al. 1999; Ishikawa et al.
2002). Antimicrobial compounds isolated from L. edodes liquid cultures include
lentinamicin (octa-2, 3-diene-5, 7 diyne-1-ol) (Komemushi et al. 1996), b-ethyl
phenyl alcohol (Komemushi et al. 1996) and lentin, an antifungal protein with
molecular mass of 27.5 kDa (Ngai 2003). These findings related to the reasonability
to expect that the observed activities were due to the presence of more than one
active compound in these extracts.
Figure 5. Inhibition of mycelial growth of plant pathogenic fungi by L. edodes solvent
extract. Antifungal activity of L. edodes solvent extract at 10% (v/v) concentration was tested
by filter paper disk assay. Mycelial growth inhibition was measured after 72 h at 28 + 28C.
Values are the means + SE of six replications.

Small changes in the composition of the culture medium for ascomycetes and
basidiomycetes can cause changes in the production of volatiles. Studies on
Lentinellus cochleatus have investigated the fruiting bodies of L. cochleatus are
having volatile compounds p-anisaldehyde, methyl p-anisate, methyl (z)-p-methox-
ycinnamate and methyl (E)-p-methoxy cinnamate and when it has been grown in
liquid culture containing glucose, the amino acid asparagines and mineral salts. The
L. cochleatus distillate are rich in trans-nerolidol, fokienol and 6-formyl-2, 2-
dimethyl chromene (Rapior et al. 2002). Dichloromethane extracts of this fungus
were shown to be antibacterial to Bacillus subtilis and Escherichia coli, fungicidal
towards Candida albicans and Cladosporium cucumerinum (Keller et al. 2002). Both
the culture liquid and the mycelial biomass of V. bombycina were shown to have
good antioxidative activity (Badalyan et al. 2003). Suay et al. (2000) showed that
45% of extracts from a total of 317 isolates of basidiomycetes collected in Spain
showed antimicrobial activity. The results of this study can serve to stimulate the
investigation of tropical, temperate basidiomycetes as a potential source of bioactive
secondary metabolites. It can, therefore, be concluded that while basidiomycetes
may not be as easy to cultivate as bacteria, they may be worth screening as their
diverse biosynthetic abilities may be harnessed in the search and discovery of new
compounds active against new drug targets. The studies highlighted that the rich
fungal genome is an essential component of our natural heritage and may be
society’s greatest protection against microbial diseases.
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