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“Wood Ear Mushrooms Of West

Dissertation submitted for the partial fulfilment of project (Molecular and

applied Mycology and Plant Pathology) for the degree of Master of Science
in Botany

Susmita Sett
Roll Number: 91/BOT/171004
Registration Number: 613─1221─1246─14

Under the supervision of

Prof. Krishnendu Acharya
Department Of Botany, University Of Calcutta
Genus: Auricularia

The genus Auricularia, belonging to the family Auriculariaceae, was established by

Bulliard in 1787.

Auricularia is a cosmopolitan saprophytic basidiomycetious jelly group of fungi

usually found in association with decaying wood & it popularly named as wood ear
Kingdom Fungi
 It has gelatin like consistency.
Auricularia is a phragmobasidious group Division Basidiomycota

of fungus with three transverse septation.

Class Agaricomycetes,
It is fourth most edible cultivated
mushroom worldwide and popular as low Order Auriculariales
calorie dietary food with good source of
Family Auriculariaceae
various amino acids, crude fibres and
Type species: Auricularia mesenterica
biologically active metabolites. (Dicks) Pers.
Literature Review

 World scenario:
MycoBank database (http://www.mycobank.org) has 175 specific or infraspecific records in Auricularia, while the
number in Index Fungorum (http://www.indexfungorum.org) is 165.

Table:1 distribution of different species across the world

 Indian scenario:
 According to the earliest report four species reported in “Fungi of India” viz A. epitricha, A. mesenterica, A. rugosissima, A.
vespertilis (E. J. Buttler & G. R. Bisby 1931).
 Seven species (A. delicata, A fuscosuccinea, A mesenterica, A peltata, A polytricha, A tenuis, A auriculae judae) reported in
“Manual of Indian Edible Mushrooms” from West Bengal (R. P. Purakayashtha 1985).
 Four species viz. A. delicata, A. polytricha, A. mesenterica, A. auricula from Manipur (Singh S.M. 2008).
 Six species viz A. polytricha, A. auricular-judae, A. cornea, A. mesenterica, A. delicata, and A. auricula repoted from different
parts of India (Sohi & Upadhyay 1990, Singh 2008, Mukherji & Manoharachary 2010).
 A. olivaceous a new species was reported from North India ( Kumari et al. 2013).
Auricularia spp. Mushrooms used as food:

 Several species of Auricularia cultivate world

wide and consumed.

A.auricula-judae , A. fuscosuccinea, A. delicata, A.

nigricans, A. tenuis, A. mesenterica (Boya

• A. auricula-judae known as “Black food of

china” due to high melanin content.

Beneficial property:
 Low fat high calorie diet with high amount of
fibre. A. mesenterica contain 76%
carbohydrate, 15% protein, 0.80% crude fat,
3.92% fibre, 3% ash content.

 Rich source of amino acids viz.glutamic acid,

lysine,alanine,serine, threonine (Sekara et al

 Novel functional food AHP Auricularia mushrooms

(polyasaccharide of A. auricula, polyphenols of used in soups, salads,
Hawthorn & Puneria radix) active against various Chinese dishes.
dyslipidemia (Luo et al. 2009).
Medicinal properties of Auricularia sp.

 Antioxidant
• Ethanolic extract of A. auricula activate nitric oxide synthase enzyme
(Acharya et al. 2004).
• Poolysaccharide of A.auricula increased SOD & glutathione activity (Lin et
al. 2013).
 Antimicrobial
• Melanin of A. auricula showing antibiofim activity against E.Coli (Bin et al.
• Inhibit Violecein production which effect the quorum sensing of
Chromobacterium violeceum (Zhu et al. 2011)
 Antitumour
• β – D glucan of A. auricula induces apoptosis (Ma et al. 2010).
 Prebiotics
• ethanolic extract of A. auricula-judae promote activity of Lactobacillus and
inhibit pathogenic strain (Swagnagn et al. 2018).
 Anti-coagulant
• A. polytricha suppress platelet aggregation (Hokma & Hokma 1981)
 Anti-inflammatory
• Polysacharide of A. auricula-judae having anti-inflammatory effect (Ukai et
al. 1983).
 Immunomodulatory
• Proteins isolated from A. polytricha shows immunodulatory property (Sheu
et al 2004)
Study area: The state West Bengal

 Located at the eastern part of the

India, in between 27°13'15"‒
21°25'24"N latitudes and 85°48'20"‒
89°53'04" E longitudes.
 It is the only state in India where
Himalayas are in the north and Sea is
at the south, with both plains and
plateaus covering the remaining
region and seems to be a miniature of
 The climate varies from tropical
savannah in humid subtropical.
Barring the mountainous parts of
Darjeeling and Jalpaiguri, the entire
states experience a warm tropical
monsoon climate.
 The summer temperatures in the
states ranges between 26˚c to 43˚c
and the winter temperatures ranges
from 10˚c to 19˚c.
 North Bengal receives the highest
rainfall, 200–400 cm. In the coastal
area rainfall is about 200 cm, in the
Ganga plain and in the central part of

Main objective of the study is to explore diversity of macro-fungal

genera Auricularia of West Bengal based on different macro and
micro morphological characters.

Identification and characterization of different species of

Auricularia collected from various parts of West Bengal & provide a
artificial key of the studied genus.

Phytochemical profiling of two specimens measuring the

bioactive metabolites and antioxidant potential.
1.Collection of sample from
the field with proper
photograph & field notes
2.Properly dry the sample with
3.Documentation of
macroscopic characterization
of the macrofungi
4.Study the microscopic
characters of the specimens
Sample.ressurect after 5.Preparation of complete
soaking into water description sheet of the
studied macrofungi
6. Identifiy the macrofungi
with the help of literatures &
7.Prservation of the studied

Data sheet for the characterization of

Auricularia species using the internal
anatomy of the basidiocarp (created by
Paola González-Colón). Type A refers to
species that have a medulla and eight
zones. Type B refers to species that
lack a medulla and have only six
Dried & preserved sample zones, where the Superior and Inferior
Laxa are absent.
Identifying Characters of Auricularia species


• Cross section through ZC

basidiomata showing eight
hyphal zones, i.e., Zona Pilosa
(ZP), Zona Compacta (ZC), Zona
Subcompacta Superior (ZSS),
Zoma Laxa Superior (ZLS),
Medulla layer, Zona Laxa Inferior
(ZLI), Zona Subcompacta Inferior
(ZSI), Hymenium (Scale bar=100

• Abhymenial hairs (A) •Monomitic type of hyphal system; generative
(scale bar 100 µm) hyphae thin walled, hyaline, highly branched &
clamped (B-C) ) (scale bar 5 µm)

•Smooth, thin
walled, hyaline,
(D) (scale bar 5

a with three
septations (E-F)
(scale bar 10
D µm) E F
Identification of six species of Auricularia with detailed macro-
microscopic features based on standard keys and literatures
(Lowy 1951, Montoya- Alvarez et al. 2011 ,Looney et al. 2013).
1 2

Fig:1 Auricularia fuscosuccinea Fig:2 Auricularia cornea Ehrenb. (Scale

(Mont.) Henn. ( Scale Bar 10mm). Bar 20mm)
Macro-microscopic features: Macro-microscopic features:
•Basidiomata, gregarious, sessile, orbicular, •Basidiomata, gregarious, substipitate,
3.2−4.5 cm wide and 2−3 mm thick; orbicular, 1.7–2.9 cm wide and 1.5–2 mm
abhymenial surface reddish orange (7B6); thick; abhymenial surface pilose reddish
hymenium smooth brownosh orange (6C3), brown (9D4) ; hymenium smooth greyish
smooth. violet (19D4).
•Presence of disticnt eight zones; zona pilosa •Presence of distinct eight zones; zona pilosa
7.74−25.8 µm wide, hair 21.52−34.4 × 181.83−314.07 µm wide, hair 60.34−349.11 ×
3.44−5.17 µm. 4.31−8.18 µm.
•Medulla distinctly present. •Medulla distinctly present.
•Basidium cylindrical, 30.17-56.03 × 4.3-5.1 •Basidium cylindrical, 30.14-51.72 × 4.3-5.1
µm in diam. With three transverse septations. µm in diam. With three transverse septations.
•Basidiospores measuring 8.6-11.32-13.76 × •Basidiospores measuring 10.6-13.93-17.24 ×
3.87-4.9-6.02 µm with mean Q value of 2.26. 4.31-5.77-7.75 µm with mean Q value of 2.46.
Fig:3 Auricularia fuscosuccinea (a)
Fig:4 Auricularia cornea (a) Cross
Cross section of basidiomata
section of basidiomata showing
showing different zones, (b)
different zones, (b) Abhymenial hairs,
Abhymenial hairs, (c) Basidia, (d)
(c) Basidia, (d) Basidiospores, (e)
Basidiospores, (e) Clamp connections
Clamp connections of hyphae. (Bars:
of hyphae. (Bars: (a) 100 µm, (b˗e) 5
(a) 100 µm, (b˗e) 5 µm).
3 4

Fig:5 Auricularia delicata (Mont. ex. Fr.) Fig: 6. Auricularia mesenterica

Henn. (Scale Bar 20mm) (Dicks.:Fr.) Pers. ( Scale Bar 10mm)
Macro-microscopic features: Macro-microscopic features:

•Basidiomata solitary, sessile, orbicular, 2.6−5.6 •Basidiomata gregarious, sessile, effuse-

cm wide and 3−4 mm thick; abhymenial surface reflexed, 2.2−2.6 cm wide and 0.7−1.5 mm
reddish brown (8D7); hymenium reticulate, thick; abhymenial surface densely villose with
greyish ruby (12C6). concentric zones alternate with light brown
(7D4) and orange grey (5B2); hymenium with
• Presence of disticnt six zones; zona pilosa folded ridge greyish red (7B4), smooth.
25.86−47.41 µm wide, hair 25.86−47.41 ×
4.3−6.4 µm. • Presence of disticnt six zones; zona pilosa
• Medulla absent. 528.96−1520.79 µm wide, hair
•Basidium cylindrical, 34.48-40.94 × 4.3-6.46 396.72−1405.5 × 1.79−3.58 µm.
µm in diam. With three transverse septations. • Medulla absent.
•Basidiospores measuring 8.62-11.32-13.79 × •Basidium & basidiospores not observed.
4.31-4.97-6.46 µm with mean Q value of 2.
Fig: 7. Auricularia delicata (a) Cross Fig:8 Auricularia mesenterica (a)Cross
section of basidiomata showing section of basidiomata showing different
different zones, (b) Abhymenial hairs, zones, (b) Abhymenial hairs, (c) Clamp
(c) Basidia, (d) Basidiospores, (e) Clamp connections of hyphae. (Bars: (a) 100 µm,
connections of hyphae. (Bars: (a) 100 (b) 10 µm, (e) 5 µm.
µm, (b˗e) 5 µm).
5 6

Fig: 9. Auricularia nigricans (Sw.) Birkebak Fig:10 Auricularia Sp. (Scale Bar 20 mm).
Looney & Sánchez- Garcia (Scale Bar 10mm).
Macro-microscopic features: Macro-microscopic features:

•Basidiomata solitary, sub-stipitate, orbicular, •Basidiomata gregarious, sessile, orbicular,

2.2−3.5 cm wide and 0.8−1 mm thick; 1.7−3.5 cm wide and 0.5−1 mm thick;
abhymenial surface violet brown (10E6) densely abhymenial surface greyish violet (16E7) pilose;
pilose; hymenium smooth brownish red (10D6), hymenium smooth reddish brown (9E8), smooth.
smooth. •Presence of disticnt eight zones; zona pilosa
•Presence of disticnt eight zones; zona pilosa 11.42−57.12 µm wide, hair 40.94−97.3 ×
146.54−318.94 µm wide, hair 107.25−474.1 × 4.31−6.46 µm.
4.31−6.46 µm. •Medulla distinctly present.
•Medulla distinctly present. •Basidium cylindrical, 51.72-64.65 × 4.3-5.6 µm
•Basidium cylindrical, 30.17-34.48 × 4.3-5.1 µm in diam. With three transverse septations.
in diam. With three transverse septations. •Basidiospores measuring 8.62-9.57-12.93 ×
•Basidiospores measuring 9.48-12.54-13.79 × 4.31-4.69-6.89 µm with mean Q value of 2.04.
4.31-4.43-4.74 µm with mean Q value of 2.82.
Fig: 11. Auricularia nigricans, (a) Cross section of Fig:12 Auricularia sp. (a) Cross section of
basidiomata showing different zones, (b) basidiomata showing different zones, (b)
Abhymenial hairs, (c) Basidia, (d) Basidiospores, (e) Abhymenial hairs, (c) Basidia, (d)
Clamp connections of hyphae. (Bars: (a) 100 µm, Basidiospores, (e) Clamp connections of
(b˗e) 5 µm). hyphae. (Bars: (a) 100 µm, (b˗e) 5 µm).
Artificial key to the species of Auricularia:

1.Hymenium smooth.………………………............................................................2

1a. Hymenium reticulate…………………………………………………............ A.delicata

2. Medulla present .……………………………………………………..........………………. 3

2a. Mesulla absent ……………………………………………….......……… A. mesenterica

3. Abhymenial hairs up to 40 µm long …………………….......……. A. fuscosuccinea

3a. Abhymenial hairs more than 40 µm long ……………........…………………………4

4. Basidiospores up to 13 µm long………………………..........……………………….… 5

4a. Basidiospores more than 13 µm long …………….........………..……….. A. cornea

5. Basidia <50 µm long and hairs > 100 µm long …...........……………. A. nigricans

5a. Basidia >50 µm long and hairs <100 µm long ……….......…….... Auricularia sp.

2. Phytochemical profiling of two specimens A. delicata
& A. mesenterica using infusion, decoction &
hydroalcoholic extraction method measuring the
bioactive metabolites and antioxidant potential.

Dried specimen Powder made from dried

Bioactive profiling of different
(Martins et
al. 2015)

(Khatua et
Quantification of Bioactive Metabolites

Fig: 13. Determination of phenol, flavonoid and ascorbic acid content of infusion, decoction
and hydroalcoholic extract obtained from A. delicata & A mesenterica

Abbreviations: GAE: Gallic Acid Equivalent; QE: Quercetin Equivalent

• ẞ carotene, lycopene were detectecd in very scanty amount.

Determination of DPPH radical scavenging
DPPH radical scavenging activity

10 µl
20 µl

30 µl
40 µl
Infusion Decoction Hydroalcoholic

Fig:14 Graph representing the working principle of DPPH assay(A),
Microtitre plate showing gradual discolouration of DPPH withincreasing
concentration of extract reflecting the radical scavenging activity (B)

Fig:15. Determination of DPPH radical

scavenging activity of infusion, decoction
Table 2: EC50value of DPPH radical scavenging activity of three and hydroalcoholic extract obtained from
different extracts of A. delicata and A. mesenterica. the values have A. delicata (a) infusion, (b) decoction, (c)
been expressed as mean±standard deviation. hydroalcoholic] & A. mesenterica (d)
infusion, (e) decoction, (f) hydroalcoholic].
Determination of ABTS radical scavenging
(A) (B)
Control ABTS radical
scavenging activity

20 µl
50 µl

70 µl

100 µ l
Infusion Decoctio
lic extract (a)

Fig16: Working principle of ABTS radical scavenging assay (A),

Microtitre plate showing gradual discolouretaion with increasing
concentration of extract depict the increase radical scavenging
activity (B).

45.03 ±0.022 35.10 ±0.034 56.69 ±0.061

A. delicata

A. 109.6 ±0.003 106.7 ±0.001 116.2 ±0.007

Fig:17. Determination of ABTS scavenging
Table 3: EC50 values of three different extracts of A. delicata and activity of infusion, decoction and
A. mesenterica. In ABTS radical scavenging activity. the values hydroalcoholic extract obtained from A.
has been expressed as mean±standard deviation. delicata (a) & A mesenterica (b),
Determination of Total
Antioxidant Activity

Fig: 19. Mechanism of total antioxidant activity

Total Infusion Decoction Hydroalcoho
Antioxida Fig:20. Determination of total antioxidant
lic Extract
nt Activity activity of infusion, decoction and hydroalcoholic
(µg/mg of
ascorbic extract obtained from A. delicata & A.
acid) mesenterica.

A delicata 0.00165 ± 0.000403 ± 0.00742 ±

0.002 0.003 0.003  This is a direct method to evaluate
reducing efficiency of antioxidant. It
A 0.00125 ± 0.00132 ± 0.00 0.00742 ±
mesenteri 0.001 0.003
is different from other method as
ca phosphomolybdenum complex is
formed without generations o free
Table 4: Quantitative estimation of total radicals.
antioxidant activity of three different extracts of A.
delicata and A. mesenterica. The values has been
expressed as mean ± standard deviation.
 The proposed work has been described Six taxa of the
genus Auricularia of which five of them are identified upto
species level and one upto genus level.
 Auricularia cornea is reported first time from West
 Quantitative estimation shows that A. delicata & A.
mesenteica both contain high amount of phenol, flavonoid
& ascorbic acids and showing promising antioxidant
 EC50 value of hydroalcoholic extract of A. delicata
showing highest DPPH & ABTS radical scavenging activity
although all the three extracts of both the specimens
showing positive results in antioxidant screening.
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I would like to express my sincerest gratitude to my respected supervisor in this project, Prof.
Krishnendu Acharya, Department Of Botany, University Of Calcutta for his constant guidance
and encouragement which kept me inspired throughout the course of my work.

I would like to express my heartfelt gratitude to the Department of Botany, University of

Calcutta, for giving me the opportunity to finish my final semester project from this esteemed

I would like to convey my thanks to Miss Rituparna saha and Miss Sandipta Ghosh, research
scholar, Department of Botany, University of Calcutta, for their constant support and immense

I would also like to thank Dr. Khushi Mukherjee, Dr. Surojit Sarkar, Mr. Adhiraj Dasgupta, Mr.
Pranab Samanta, Miss Somanjana Khatua, Miss Sudeshna Nandi, Mr. Anik Sarkar, Mr. Entaj
Tarafdar, Mr. Diptosh Das, Mr. Mangal Biswas, Miss Juna Tamang, Miss Alisha Thapa
Research Scholars of Department of Botany, University of Calcutta, for supporting me and
helping me with this work.

I would like to take this opportunity to thank my classmate Gouri Basak, Sreeparna Sarkar,
Ipsita Bandopadhyay and my seniors Tribeni Chatterjee, Anirban Sardar for their help and

Finally, I express my heartfelt gratitude to my parents and other family members, without
whose blessings and help this work could not have been furnished within the stipulated time
Thank you