I

APPENDIX – I

Composition of Media

Sr. No. Name of the medium Composition Quantity (g) 1. Potato Dextrose Agar Peeled potatoes 200.0

Dextrose 20.0

Agar 20.0

DW 1 Litre

2. Cellulose Basic Medium C4H12N2O6 5.0

KH2PO4 1.0

MgSO4.7H2O 0.5

Yeast Extract 0.1

CaCl2.2H2O 0.001

DW 1 Litre

3. Lignin Basic Medium KH2P04 1.0

Yeast Extract 0.01

C4HI2N206 0.5

CuS04.5H20 0.001

MgS04·7H20 0.5

Fe(S04)3 0.001

CaCl2·2H20 0.01

MnS04.H20 0.001

DW 1 Litre

4. Phosphate Saline Buffer Na2HPO4 1.02

NaH2PO4 0.437

NaCl 0.85

DW 100 ml

pH 7.2

5. Alsver's Solution Glucose 2.05

Sodium citrate 0.8

NaCl 0.42

DW 100 ml

pH 6.1

II

APPENDIX – II

Composition List of Abbreviations

HPLC High Performance Liquid Chromatography

GC Gas Chromatography

Ca Calcium

Mg Magnesium

Na Sodium

K Potassium

Cu Copper

Zn Zinc

Fe Iron

Pb Lead

Ag Silver

As Arsenic

Hg Mercury

Sb Antimony

µg Microgram

µm Micrometer

g Grams

N Nitrogen

D.W. Distilled Water

Min. Minutes

mg Milligrams

SFA Saturated fatty acids

MUFA Monounsaturated Fatty acids

PUFA Polyunsaturated Fatty acids

HCl Hydrochloric acid

HNO3 Nitric acid

SLM Spatial Light Modulator

ELSD Evaporative Light Scattering Detector

ACN Acetonitrile

N2 Nitrogen gas

III

H3PO4 Phosphoric acid

MTBE Methyl Tertiary-Butyl Ether

GAEs Gallic acid equivalents

m Meters

nd Not detected

ANOVA Analysis of variance

rpm Rotation per minute

vol. Volume

mL Milliliters

nm Nanometers

C Carbon

ºC Degree centigrade

NaOH Sodium Hydroxide

Na2HPO4 Disodium hydrogen phosphate

NaH2PO4 Sodium dihydrogen phosphate

μg Microgram

% Percent

UPLC Ultra Performance Liquid Chromatography

RT Retention time

IV

APPENDIX – III

List of Published Research Papers

N.S. Atri, Sapan Kumar and B.M. Sharma. 2010. Lentinus torulosus (Pers.:Fr.)

Llyod- A new fungus record from India. Mushroom Research. 19 (1): 33 – 35.

N.S. Atri, Dimple Kumari and Sapan Kumar Sharma. 2010. Effect of vitamins and

growth regulators on the vegetative growth of Lentinus connatus Berk- Indian

Journal of Mushroom. 28 (1&2) 63 – 69.

Arvind Gulati, N.S.Atri, Sapan Kumar Sharma and B.M. Sharma. 2011. Nutritional

studies on five wild Lentinus species from North West India. World Journal of

Dairy and Food Science. 6(2): 140 -145.

N.S.Atri, Dimple Kumari and Sapan Kumar Sharma. 2011. Studies on the effect of

media and substrates on growth and yield of Lentinus connatus. Mushroom

Research. 20 (1): 55 – 62.

N.S.Atri, Sapan Kumar Sharma, Robin Joshi, Ashu Gulati and Arvind Gulati. 2012.

Amino acid composition of five wild Pleurotus species from North West

India. European Journal of Biological Sciences. 4 (1): 31 - 34.

N.S. Atri, Sapan Kumar Sharma, Navdeep Kaur, Praveen Rahi and Arvind Gulati.

2012. Morpho-cultural, molecular and nutraceutical studies on

coremiopleurotus from India. World Applied Sciences Journal. 17 (6): 759 –

763.

MAP – 1

Map – 1 : Surveyed area during 2008 – 2011.

PLATE – 1

Figs. (A – F) : Pine forest in Solan, B. Quercus forest in Mcleodganj, C. Mixed forest

with Deodar as dominant component in Jhatingari (Mandi), D. Sal forest in Sirmour

(H.P.), E. Dense mixed Oak and Deodar forest in Shimla, F. Pine forest in Bhadrol

(H.P.)

PLATE - 2

Figs. (A-F) Lentinus squarrosulus var. squarrosulus: A. Deeply infundibuliform

basidiocarps, B. Gill trama, C. Basidiospores (Scale bar=10μm), D. Hyphal pegs

(Scale bar=10μm) E. Bovista type hyphae, F. Mature mycelium with dark extending

patches.

PLATE – 3

Figs. (A-G) Lentinus squarrosulus var. squarrosulus: A. Carpophore, B. Basidia, C.

Basidiopspores, D. Pleurocystidia, E. Cystidiform hairs, F. Generative hyphae, G.

Bovista type hyphae.

PLATE - 4

Figs (A-G) Lentinus squarrosulus var. mangoensis var. nov. : A. Basidiocarps, B.

Hymenophore showing basidia (Scale bar=10μm), C. Basidiospores (Scale

bar=10μm), D. Hyphal pegs (Scale bar=10μm), E. Cheilocystidia on gill edge (Scale

bar=10μm), F. Stipe hyphae, G. Mature culture.

PLATE - 5

Figs (A-G) Lentinus squarrosulus var. mangoensis var. nov.: A. Basidiocarp, B.

Basidia, C. Basidiospores, D. Hyphal pegs, E. Pleurocystidia, F. Caulocystidia on

stpe surface, G. Bovista type hyphae.

PLATE - 6

Figs. (A-F). Lentinus cladopus : A. Caespitose fruit bodies, B. Hymenophore showing

basidia (Scale bar=10μm), C. Basidiospores (Scale bar=10μm), D. Cheilocystidia on

the gill edge (Scale bar=10μm), E. Caulocystidia on the stipe surface (Scale

bar=10μm), F. Mature mycelium.

PLATE – 7

Figs. (A-F) Lentinus cladopus: A. Basidiocarps, B. Basidiospores, C. Basidia, D.

Cystidial hairs, E. Skeletal hyphae, F. Generative hyphae.

PLATE - 8

Figs. (A-F) Lentinus sajor-caju: A. Basidiocarps on host, B. Undersurface of pileus

showing decurrent lamellae, C. Spore (Scale bar=10μm), D. Hyphal pegs (Scale

bar=10μm), E. Seven days old mycelium, F. Eleven days old mycelium

PLATE – 9

Figs. (A-F) Lentinus sajor-caju: A. Carpophore, B. Basidiospores, C. Basidia, D.

Cheilocystidia, E. Hyphal pegs, F. Generative and skeletal hyphae.

PLATE - 10

Figs. (A-G) Lentinus torulosus: A. Lobed pileus of basidiocarps, B. Hymenophore

showing pleurocystidia (Scale bar=10μm), C. Basidiospores (Scale bar=10μm), D.

Clamped cheilocystidia (Scale bar=10μm), E. Cheilocystidia on gill edge (Scale

bar=10μm), F. Six days old culture, G. Fruit bodies on medium.

PLATE – 11

Figs. (A-G) Lentinus torulosus : A. Basidiocarp, B. Basidiospores, C. Basidia, D.

Pleurocystidia, E. Cheilocystidia, F. Skeletal hypha, G. Generative hyphae.

PLATE – 12

Fig. (A-F) Lentinus connatus: A. Carpophore of Lentinus connatus (upper surface)

with excentric stipe, B. Basidia, C. Basidiospores (Scale bar=10μm), D. Generative

hyphae with clamp connection, E. Thick walled skeletal hyphae, F. Mature mycelium.

PLATE - 13

Fig. (A-G). Lentinus connatus : A. Basidiocarps, B. Basidiospores, C. Basidia, D.

Pileocystidia, E. Cheilocystidia, F. Skeletal hyphae, G. Generative hyphae

PLATE - 14

Figs. (A-F) Pleurotus sapidus; A. Basidiocarps in the caespitose clusters, B.

Basidiospore (Scale bar=10μm), C. Cheilocystidia (Scale bar=10μm), D.

Hymenophore showing Basidia (Scale bar=10μm), E. Generative hypha with clamp

connection, F. Eight days old culture.

PLATE - 15

Figs. (A-F) Pleurotus sapidus: A. Basidiocarps, B. Basidiospores, C. Basidia, D.

Generative hyphae, E. Cheilocystidia, F. Pileus surface.

PLATE - 16

Figs. (A-F) Pleurotus cystidiosus subsp. abalonus : A Basidiocarp of

Pleurotus cystidiosus subsp. abalonus, B. Coremia formation on the tissue, C.

Toxocyst on the mycelium, D. Coremial spores (Scale bar=10μm), E. Basidiospore

(Scale bar=10μm), F. Clamp connection on hypha.

PLATE - 17

Fig. (A-G) Pleurotus cystidiosus sp. abalonus : A. Carpophore, B. Basidiospores, C.

Basidia, D. Pleurocystidia, E. Pileocystidia, F. Cheilocystidia, G. Hypha with clamp

connection.

PLATE–18

Figs. (A-F) Pleurotus floridanus : A. Basidiocarp, B. Undersurface of basidiocarp

showing decurrent gills, C Basidiospores (Scale bar=10μm), D Hymenobasidia (Scale

bar=10μm), E Clamp connection in pileus hypha, F. Mature mycelium.

Figs. (A-F) Pleurotus floridanus: A. Carpophore, B. Hymenobasidia, C.

Basidiospores, D. Chielocystidia, E. Hyphae on pileus surface, F. Hyphae on stipe

surface.

PLATE - 19

PLATE – 20

Figs. (A-E) Pleurotus pulmonarius var indica var. nov. : A. Basidiocarps, B. Basidia

in hymenium (Scale bar=10μm), C. Basidiospores (Scale bar=10μm), D. Gill trama,

E. Mature culture after ten days of inoculation.

PLATE - 21

Figs. (A-E) Pleurotus pulmonarius var. indica var. nov. : A. Basidiocarp, B.

Basidiospores, C. Hymenophore bearing Basidia, D. Pleurocystidia, E. Pileus

surface, F. Stipe surface.

PLATE – 22

Figs. (A-F) Pleurotus sajor-caju : A. Basidiocarp on Host, B. Hymenophore showing

basidia (Scale bar=10μm), C. Basidiospores (Scale bar=10μm), D. Cheilocystidia,

(Scale bar=10μm), E. Gill trama, F. Fully grown culture.

PLATE - 23

Figs. (A-E). Pleurotus sajor-caju : A. Basidiocarp, B. Basidiospores, C. Basidia, D.

Pileus surface, E. Stipe surface.

Isolated DNA and PCR amplification

A B

C D

Figs. (A-D) : A. Isolated DNA lane M. Marker, lane 1- 6 fungal DNA, 1. L. sajor-

caju, 2. L. connatus, 3. L. torulosus, 4. L. cladopus, 5. Lentinus squarrosulus, 6. L.

squarrosulus var. mangoensis, B. Amplification of ITS region, lane M-100bp plus

ladder, lane 1-6 fungus DNA, C. Isolated DNA lane M. Marker, lane 1- 5 fungal

DNA, 1. Pleurotus cystidiosus, 2. P. floridanus, 3. P. pulmonarius, 4. P. sapidus, 5. P.

sajor-caju, D. Amplification of ITS region, lane M-100bp plus ladder, lane 1-5

fungus DNA.

PLATE - 24

Fig. Phylogenetic tree showing the relationships among six Lentinus species and

representatives of related taxa, based on the ITS region sequences. The numbers on

the nodes indicate how often (no. of times, %) the species to the right are grouped

together in 100 bootstrap samples. Bar 0.2 substitutions per site.

Lentinus crinitus GU207300

Lentinus bertieri GU207303

Lentinus bertieri GU207304

Lentinus swartzii GU207278

Lentinus tigrinus GU207267

Lentinus glabratus GU207250

Lentinus badius GU207275

Lentinus squarrosulus var. mangoensis JQ868747

Lentinus cladopus (S3004) JQ868754

Lentinus cladopus GU849477

Lentinus scleropus GU207310

Lentinus striatulus GU207311

Lentinus sajor-caju JQ428820

Lentinus squarrosulus GU001951

Lentinus tigrinus JQ428822

Lentinus squarrosulus (S3005) JQ868748

Lentinus connatus (S3002) JQ868752

Lentinus squarrosulus GQ 849475

Lentinus cyathiformis EF524038

Lentinus torulosus M98650

Lentinus tuber-regium AB115045

Lentinus sajor caju (S3001) JQ868751

Lentinus sajor-caju iJN8493900

Lentinus torulosus (S3003) JQ868753

Lentinus torulosus 175098 100

98

89

100

42

49

100

64

98

96

96

92

63

86

93

98

82

70

48

35

57

0.2

Phylogenetic analysis of investigated Lentinus species

PLATE - 25

Pleurotus floridanus strain (S2002) JQ868739

Pleurotus eryngii FJ904749

Pleurotus floridanus (strain dd08070) FJ810170

Pleurotus sapidus (strain S047) AY540327

Pleurotus ostreatus FJ501549.1

Pleurotus sapidus (strain S2004) JQ868741

Pleurotus pulmonarius JF908604

Pleurotus floridanus AY265832

Pleurotus pulmonarius JN043318

Pleurotus sajor-caju EF458652

Pleurotus sajor-caju (S2005) JQ868742

Pleurotus pulmonarius voucher HMAS86396

Pleurotus pulmonarius strain (S2003) JQ868740

Pleurotus sajor-caju (strain S049)AY540129

Pleurotus albidus AF345661

Pleurotus cystidiosus GU947713

Pleurotus citrinopileatus JN234853

Pleurotus cornucopiae AB102785

Pleurotus eryngii FJ904779

Pleurotus eryngii isolate FJ904777

Pleurotus nebrodensis FJ904773

Pleurotus nebrodensis FJ904771

Pleurotus cystidiosus ACCC 51280

Pleurotus ostreatus JN977548

53

100

100

63

88

99

37

86

19

17

22

27

42

51

35

13

34

0.2

PLATE - 26

Fig. Phylogenetic tree showing the relationships among five Pleurotus species and

representatives of related taxa, based on the ITS region sequences. The numbers on the

nodes indicate how often (no. of times, %) the species to the right are grouped together in

100 bootstrap samples. Bar 0.2 substitutions per site.

Phylogenetic analysis of investigated Pleurotus species

PLATE – 41

Figs. (A-E) : A. Button formation in Lentinus sajor-caju, B. Button formation in L.

connatus species, C. Button formation in L. torulosus,D. Button formation in L.

cladopus, E. Mat formation in L. squarrosulus var. squarrosulus.

PLATE - 42

Figs. (A-E) : A. Button formation in Pleurotus cystidiosus, B. Button formation in P.

floridanus, C. Button formation in P. sapidus, D. Button formation in P. pulmonarius

var. indica, E. Button formation in P. sajor-caju .

PLATE - 43

Figs. (A-E): Cellulase activity of Lentinus species grown at 28 ± 1°C for eight days

on Cellulose Basic Medium (CBM) and stained with Congo-red dye : A. Lentinus

sajor-caju, B. Lentinus connatus, C. Lentinus torulosus, D. Lentinus cladopus, E.

Lentinus squarrosulus var. squarrosulus.

PLATE - 44

Figs. (A-E): Cellulase activity of Pleurotus species grown at 28 ± 1°C for eight days

on Cellulose Basic Medium (CBM) and stained with Congo-red dye : A. Pleurotus

cystidiosus ssp. abalonus, B. Pleurotus floridanus, C. Pleurotus sapidus, D.

Pleurotus pulmonarius var. indica, E. Pleurotus sajor-caju.

PLATE – 45

Figs. (A-E): Lignin modifying enzyme activity of Lentinus species grown at 28 ± 1°C

for eight days on Lignin Basic Medium (LBM): A. Lentinus sajor-caju, B. Lentinus

connatus, C. Lentinus torulosus, D. Lentinus cladopus, E. Lentinus squarrosulus var.

squarrosulus.

PLATE - 46

Figs. (A-E): Lignin modifying enzyme activity of Pleurotus species grown at 28 ±

1°C for eight days on Lignin Basic Medium (LBM): A. Pleurotus cystidiosus ssp.

abalonus, B. Pleurotus floridanus, C. Pleurotus sapidus, D. Pleurotus pulmonarius

var. indica, E. Pleurotus sajor-caju.

Title of the Thesis: Diversity, Taxonomy and Nutritional Aspects of Wild Species of

Lentinus and Pleurotus from North West India

Name of the candidate – Sapan Kumar

Supervisor Name- Dr. N.S. Atri,

Date of Submission: 19.6.2012

Keywords: Lentinus, Pleurotus, taxonomy, wild mushrooms, nutraceuticals

Abstract

Genus Lentinus is represented by 40 species and genus Pleurotus is represented

by 20 species the world over. However, there are 24 valid species of genus Lentinus and

12 valid species of genus Pleurotus documented from India. As a consequence of various

exploratory tours undertaken during 2008-2011 to various localities of North west India,

collections belonging to six taxa of Lentinus, namely L. sajor-caju, L. connatus, L.

torulosus, L. cladopus, L. squarrosulus var. squarrosulus, L. squarrosulus var.

mangoensis var. nov. and five taxa of genus Pleurotus, namely P. cystidiosus subsp.

abalonus, P. floridanus, P. pulmonarius var. indica, P. sapidus and P. sajor-caju were

made and subsequently investigated for taxonomic, nutritional and nutraceutical

attributes.Out of eleven taxa of Lentinus and Pleurotus taxonomically investigated,

Lentinus torulosus and Pleurotus cystidiosus ssp. abalonus are new records for India. L.

squarrosulus var. mangoensis var. nov. and P. pulmonarius var. indica are two new

varieties proposed. In the investigated wild Lentinus and Pleurotus species, carbohydrates

were found to be in higher amount followed by protein, fibres, ash content and fat. In all

mushroom species seven minerals including four macro (Ca,Mg,Na,K) and three micro

minerals (Cu,Zn,Fe) were documented. All the minerals were found to be in substantial

amount. No heavy metal was detected in any of the investigated species. Nutraceutical

components evaluated include sugars, fatty acids, ascorbic acid, β-carotene, lycopene,

phenolic compounds, amino acids and alkaloids. Out of all the investigated species the

assay for the presence of lectin was positive only in Lentinus squarrosulus var.

squarrosulus. On qualitative basis all the species of both the genera investigated

exhibited the formation of inhibition zones during enzyme assay demonstrating the

production of cellulose and lignin modifying enzymes.