APPENDIX I - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10214/17/17_appendix.pdf · SLM...
Transcript of APPENDIX I - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/10214/17/17_appendix.pdf · SLM...
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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
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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
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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
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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.
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MAP – 1
Map – 1 : Surveyed area during 2008 – 2011.
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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.)
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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.
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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.
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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.
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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.
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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.
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PLATE – 7
Figs. (A-F) Lentinus cladopus: A. Basidiocarps, B. Basidiospores, C. Basidia, D.
Cystidial hairs, E. Skeletal hyphae, F. Generative hyphae.
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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
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PLATE – 9
Figs. (A-F) Lentinus sajor-caju: A. Carpophore, B. Basidiospores, C. Basidia, D.
Cheilocystidia, E. Hyphal pegs, F. Generative and skeletal hyphae.
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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.
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PLATE – 11
Figs. (A-G) Lentinus torulosus : A. Basidiocarp, B. Basidiospores, C. Basidia, D.
Pleurocystidia, E. Cheilocystidia, F. Skeletal hypha, G. Generative hyphae.
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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.
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PLATE - 13
Fig. (A-G). Lentinus connatus : A. Basidiocarps, B. Basidiospores, C. Basidia, D.
Pileocystidia, E. Cheilocystidia, F. Skeletal hyphae, G. Generative hyphae
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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.
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PLATE - 15
Figs. (A-F) Pleurotus sapidus: A. Basidiocarps, B. Basidiospores, C. Basidia, D.
Generative hyphae, E. Cheilocystidia, F. Pileus surface.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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PLATE - 23
Figs. (A-E). Pleurotus sajor-caju : A. Basidiocarp, B. Basidiospores, C. Basidia, D.
Pileus surface, E. Stipe surface.
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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
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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
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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
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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.
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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 .
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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.
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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.
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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.
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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.
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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.