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ROLE OF AUTOCHTHONOUS FUNGI IN

PHYTOEXTRACTION OF HEAVY METALS

FROM TOXIC TANNERY SOLID WASTE

AISHA NAZIR

DEPARTMENT OF BOTANY

UNIVERSITY OF THE PUNJAB

LAHORE, PAKISTAN.

ROLE OF AUTOCHTHONOUS FUNGI IN

PHYTOEXTRACTION OF HEAVY METALS

FROM TOXIC TANNERY SOLID WASTE

A Thesis Submitted to the University of the Punjab in

Partial Fulfillment to the Requirements for the Degree

of Doctor of Philosophy in Botany

By

AISHA NAZIR

DEPARTMENT OF BOTANY

UNIVERSITY OF THE PUNJAB

LAHORE, PAKISTAN.

August, 2013

Dedicated to my supervisor

Prof. Dr. Firdaus-e-Bareen

DECLARATION CERTIFICATE

I, Ms. AISHA NAZIR, under the supervision of DR. FIRDAUS E BAREEN

Professor, College of Earth and Environmental Science, University of the

Punjab, Lahore, admit that the data presented in the PhD dissertation entitled

“ROLE OF AUTOCHTHONOUS FUNGI IN PHYTOEXTRACTION OF HEAVY

METALS FROM TOXIC TANNERY SOLID WASTE” is original. All the

sections, sub-sections and formatting are performed by me. This work has not

been used in part or full in a manuscript already submitted or in the process of

submission in partial or complete fulfilment of the award of any other degree

from any institution.

(AISHA NAZIR)

PhD Scholar

Department of Botany,

University of the Punjab,

Lahore.

Dated:

ABSTRACT

The TSW generated by the Leather industry has been a hazardous

entity for agricultural soils in the vicinity of KTWMA landfill site, Kasur,

Pakistan. The presence of enormous amounts of toxic metals like Cr, Cd, Cu,

Fe, Na, K in the TSW has been a major hindrance in converting its organic

and combustible components into products like manure, compost and

fertilizer, etc. Finding solution for the decontamination of heavy metals present

in TSW has been one the primary concerns of environmental biotechnologists

in Pakistan. The current study is focused at phytoextraction of heavy metals

from TSW through phytoextraction bioreinforced with autochthanous saprobic

fungi isolated from TSW.

After repetitive analyses, the TSW was observed to have high pH (8.9),

ECe (2.89 dS cm-1), NaCl (421 %), bicarbonates and chlorides (359.9 and

3118 mgL-1 respectively), considerable amount (4.5 %) of organic matter and

very low bulk density (0.66 g cm-3). The multi-metal contaminated TSW had

high levels of both essential and trace metals. The total metal fraction of

Category-I and II metals was much higher than the in the upper part of

permissible limits of USEPA (1999) with concentrations of Ca, Mg and Na

(6320, 4210 and 9440 mg kg-1 respectively) as well as Cd, Cr, Cu, Fe, Mg, Ni,

Pb and Zn (10097, 25534, 10554, 2250, 3840, 590, BDL and 7590 mg kg-1

respectively).

Screening of hyperaccumulator fungi isolated from TSW on different

fungal nutrient media and selection of ornamental plants for phytoextraction

on the basis of germination response (%) on TSW-Soil mixtures short listed

the upper level of TSW (%) in TSW-Soil mixtures on the basis of toxicity

contribution in soil. The total thirteen autochthonous fungal species were

isolated from TSW and four of them viz. Alternaria alternata, Aspergillus niger,

Fusarium sp. and Trichoderma pseudokoningii were shortlisted for in vitro

mutual growth interaction studies. The four shortlisted fungi were also tested

for their in situ mutual interaction studies in soil by applying their inoculations

in different combinations to marigold (Tagetes patula). On the basis of plant

vegetative biomass production incurred by the fungal inoculations, the isolates

of Trichoderma pseudokoningii and Aspergillus niger were ultimately selected

for actual phytoextraction trials on marigold (Tagetes patula) and sunflower

(Helianthus annuus) in greenhouse and field conditions. The TSW-Soil

mixtures for these trials were 0 (only soil), 5, 10 and 20 % (TSW-Soil w:w).

The plants cultivated on 20% TSW-soil mixture showed less significant

growth as compared to 5 and 10 % lower TSW-soil Mixtures, with lower

values of all biochemical parameters in terms of chlorophyll content, total

protein, SOD and CAT activity. The metal extraction efficiency was found to

be the highest in F1 + F2 treatment. The metal extraction efficiency from

higher to lower order was in the order: F1 + F2 > F2 > F1 > C.

Both the tested plants were found to be effective accumulators of metals.

The plants given inoculation of both fungi (F1 + F2) showed a significantly

higher growth in all types of soil. Plants given only fungus (F1 or F2) also

showed significant growth rate as compared with control treatment. The

statistical analyses of the results showed increase in all growth parameters in

lower TSW-soil mixtures at all exposures followed by a decrease at the

highest TSW-soil ratio i.e. 20%.

According to Tolerance Index (TI) and translocation Index, H. annuus and

T. patula proved to be the suitable for phytoextraction of multimetal

contaminated TSW and showed the ability to serve as phytostabilizing plants

for metals in the phytoremediation process. Tolerance Index (TI) values more

than 1.0 for Cr and Zn suggested the hyperaccumulative potential of both

plants for these metals. Greater SEY (%) values also suggested the efficiency

of both these plants to remove metals from TSW.

Keeping in view the growth parameters and metal accumulation in the

plant, it was observed that lower percentage (5 and 10%) of tannery solid

waste was suitable for the phytoremediation of most of the studied metals.

The better growth, elevated levels of antioxidants (SOD and CAT), high

accumulation of metals and significant statistical data showed that there is

synergistic effect of both fungal inocula (F1 + F2). Thus autochthonous fungi

along with tolerant plants can be exploited for phytoremediation of tannery

waste by products.

Acknowledgements

Without encouragement and support of many people, this dissertation

would never have been accomplished. My personal very deep appreciation

goes to each of the contributors, right from my very induction into PhD till

submission of the final manuscript.

Firstly and fore mostly, I would like to express my deep gratitude to

learned and dignified teacher and supervisor, Prof. Dr. Firdaus-e-Bareen,

Principal College of Earth and Environmental Science, University of the

Punjab, Lahore, for her keen interest in supervision, valuable guidance

sympathetic attitude and stimulating ideas, which were real source of

inspiration for me throughout the course of this research work. She has the

attitude and the substance of a genius; she continually and convincingly

conveyed a spirit of adventure in regard to research. Without her persistent

help and guidance this synergistic product would not have been possible.

I am particularly indebted to Dr. Janice E. Thies, International Professor

of Soil Ecology at Cornell University, for always extending her expert, sincere,

valuable guidance and encouragement to me. Thanks Dr. J for being so kind

and helpful during my stay in your laboratory and giving us opportunity to

learn very advanced techniques in such a simple way.

Very special thanks to Prof. Dr. Khan Raas Masood, Chairman of

Department of Botany, for allowing me to conduct this research work and for

providing me all the necessary facilities for the accomplishment of the present

work.

I gratefully acknowledge the Project Director of KTWMA, who always kept

the door of sampling site open for me. Special thanks to Higher Education

Commission (HEC) for the financial support.

I feel a deep sense of gratitude for my father and late mother who formed

part of my vision and taught me the good things that really matter in life. The

happy memory of my mother still provides a persistent inspiration for my

journey in this life. I am grateful to my sisters who gave me love, cared for me

and encouraged me to pursue my education.

I cannot find words to express my gratitude to my brother Syed Abid

Shah, who took me on the process of learning, taught me the importance of

hard work and instilled in me the perseverance and strength to overcome all

hardships and made himself always available to complete this work, even

though it was an irrelevant subject for him. Thank you doesn’t seem sufficient

but it is said with appreciation and respect.

I owe my deepest gratitude to my dear husband Mr. Muhammad Shafiq,

for supporting me with his constant love, concern, interest, excellent

suggestions, and creative ideas and for helping to create an environment that

gave the peace of mind to enable me to really focus on research work.

Without his practical and emotional support this dissertation might not have

been accomplished.

I owe my deepest gratitude to my friends and colleagues. I share the credit

of my work with all my laboratory members.

Whenever my steps tried to lose balance and my perseverance during

the laborious journey of PhD become fainted, a glimpse of my wonderful son,

Muhammad Asad Shafiq, always made me forget the worries and instilled

new energy, passion and determination to complete this uphill task as at the

earliest possible. He is who proved to be the light of my life and the hope of

my heart.

I also place on record my deep sense of gratitude to one and all who,

directly or indirectly, have lent their helping hand in this venture from the

people who first persuaded and got me interested into the study of Botany to

those who with the gift of their company made my days more enjoyable and

worth living.

I would like to say more; however, “word are but empty thanks.”

(Aisha Nazir)

LIST OF ABBREVIATIONS

AAS Atomic Absroption Spectrophotometer AM Arbuscular Mycorrhizal AN Aspergillus niger AS Autoclaved Soil BDL Below Detection Limit Ca Calcium CAT Catalase Cd Cadmium CFUs Colony Forming Units Cr Chromium Cu Copper DMRT Duncan’s Multiple Range Test DTPA Diethylene Triamine Pentaacetic Acid ECe Electrical Conductivity of extract EPA Environment Protection Agency Fe Iron HEC Higher Education Commission HM Heavy Metals K Potassium KTWMA Kasur Tannery Waste Management Agency LSD Least Significant Difference MEA Malt Extract Agar Mg Magnesium MMN Modified Melin Norken Na Sodium NAS Non Autoclaved Soil Ni Nickel PDA Potato Dextrose Agar RCBD Randomized Complete Block Design ROS Reactive Oxygen Species SEY (%) Specific Extraction Yield percentage SOD Superoxide Dismutase SOM Soil Organic Matter SPAD Soil-Plant Analysis Development TH Trichoderma harzianum TI Tolerance Index TS Trichoderma pseudokoningii TSW Tannery Solid Waste Zn Zinc

TABLE OF CONTENTS

Sr. No. Title Page No.

Abstract

Acknowledgements

List of Abbreviations

List of Tables ............................................................................................................ i

List of Figures ........................................................................................................... xi

1. INTRODUCTION ...................................................................................................... 1

2. MATERIAL AND METHODS ................................................................................... 12

2.1 Sampling site, surveys and sampling of tannery solid waste ..................... 12

2.2 TSW sample processing for spiking garden soil ......................................... 15

2.3 Screening of fungi and plants tolerant for TSW based toxic metals ........... 19

2.4 Pot trial phytoextraction studies based on ultimate screened fungi and plants 26

2.5 Evaluation of pot-trial findings at field level ................................................. 31

2.6 Phytoextraction studies assisted with Trichoderma harzianum (TH) and TS in

a pot trial at Cornell University, New York USA .......................................... 35

2.7 Statistical analyses ................................................................... 35

3. RESULTS ....................................................................................................... 36

3.1 Physico-chemical properties of TSW and garden soil ................................ 36

3.2 Isolation and identification of TSW representative fungi ............................. 41

3.3 Screening and selection of heavy metal resistant autochthonous fungi ..... 43

3.4 In vitro fungal mutual growth interaction studies for Category-II metal

tolerance............................................. ........................................................ 44

3.5 In situ mutual growth interaction studies for screened Category-II metal tolerant fungal isolates with Tagetes patula in soil ..................................... 49

3.6 Screening of heavy metal tolerant ornamental plant species for phytoextraction of TSW-Soil mixtures ......................................................... 51

3.7 Pot experiments with Marigold on autoclaved (AS) and non-autoclaved TSW-

Soil mixtures (NAS) to verify bio-reinforcing role of fungi ........................... 53

3.8 Experiment with saprobic and AM fungi ..................................................... 78

3.9 Experiments with saprobic fungi ................................................................. 99

3.9A Experiments with Tagetes patula inoculated with saprobic fungi ............... 99

3.9B Experiments with Helianthus annuus inoculated with saprobic fungi ......... 121

3.10 Experiment with French marigold ............................................................... 141

3.11 Field experiments ................................................................... 161

3.11A Experiment with Helianthus annuus .............................................. 161

3.11B Experiment with Tagetes patula .................................................... 184

4. DISCUSSION ....................................................................................................... 209

4.1 Physico-chemical properties of TSW and garden soil ................................ 209

4.2 Isolation and identification of TSW representative fungi ............................. 215

4.3 Screening and selection of heavy metal resistant autochthonous saprobic

fungi........................................ ................................................................... 216

4.4 In vitro fungal mutual interaction studies for Category-II metal tolerance... 218

4.5 In situ mutual growth interaction studies for screened Category-II metals tolerant fungal isolates with Tagetes patula in soil ..................................... 219

4.6 Screening of heavy metal tolerant ornamental plant species for phytoextraction of TSW-Soil mixtures ........................................................ 220

4.7 Experiments with marigold cultivated on autoclaved and non autoclaved

TSW-Soil mixtures and inoculated with selective autochthonous saprobic

fungi..................................................................................................... ....... 221

4.8 Experiments with marigold cultivated on TSW-Soil mixtures and inoculated with selective autochthonous saprobic fungi and AM fungi under greenhouse conditions....................................................... ............................................. 227

4.9 Experiments with marigold and sunflower inoculated with selective autochthonous saprobic fungi under greenhouse and field conditions....... 229

5. REFERENCES ....................................................................................................... 239

6. PUBLISHED RESEARCH PAPERS....................................................................... 264

********************

i

LIST OF TABLES

Table

No. Title

Page

No.

2.1.1

Layout for pot experiments showing selected mixtures of tannery solid

waste with soil (TSW-Soil) either autoclaved (AS) or non-autoclaved

(NAS) and fungi (F1: Aspergillus niger and F2: Trichoderma

pseudokoningii) used for inoculation in soil

25

3.1.1 The physico-chemical properties of TSW, garden soil and their various

(% w:w TSW-Soil) mixtures 37

3.1.2

The total, water soluble and DTPA-extractable fraction of Category-I

metals (mgkg-1

) in TSW, garden soil and their various (% w:w TSW-

Soil) mixtures

38

3.1.3

The total, water soluble and DTPA-extractable fraction of Category-II

metals (mgkg-1

) in TSW, garden soil and their various (% w:w TSW-

Soil) mixtures

39

3.3.1

The tolerance index (TI) of various fungi cultivated on 2 % MEA

prepared in autoclaved extract of TSW along with control of each of

the fungus cultivated on 2 % MEA prepared in distilled autoclaved

water.

43

3.4.1

Comparison of six pairs of mutually interacting fungi for growth

competition on the basis of various morphological parameters

observed after 10 days of fungal inoculation.

47

3.5.1

Morphological and biochemical parameters for 50-days old pot

cultivated plants of Tagetes patula cultivated in soil and applied with

individual and combined fungal inoculations. The mean values ± SD

with different letters are significantly different according to Duncan’s

multiple range test (n = 3; P = 0.05).

49

3.6.1

Screening of plants for their phytoextraction potential on the basis of

percentage germination observed in different TSW-Soil (% w:w)

mixtures. The values ± S.D. are mean of three replicates.

51

3.7.1

The biochemical parameters observed in 50-days old Tagetes patula

cultivated on TSW-Soil mixtures. The mean values S.D. with

common letters (small along the row & capital within a column) are not

significantly different according to Duncan’s multiple range test (P =

0.05; n = 3).

55

3.7.2

Various morphological parameters observed in 50-days old Tagetes

patula cultivated on TSW-Soil mixtures. The mean values S.D. with



0.05; n = 3).

58

3.7.3 The concentration of Category-I Metals (mgkg-1

) observed in SHOOT

of 50-days old Tagetes patula cultivated on TSW-Soil mixtures. The 60

ii

mean values S.D. with common letters (small along the row & capital

within a column) are not significantly different according to Duncan’s

multiple range test (P = 0.05; n = 3).

3.7.4

The concentration of Category-I Metals (mgkg-1

) observed in ROOT of

50-days old Tagetes patula cultivated on TSW-Soil mixtures. The




61

3.7.5

The concentration of Category-II Metals (mgkg-1

) observed in SHOOT

of 50-days old Tagetes patula cultivated on TSW-Soil mixtures. The




65

3.7.6







68

3.7.7

The post-harvest fungal analyses (× 105 c.f.u. g

-1 soil) of 50-days old

Tagetes patula cultivated on TSW-Soil mixtures. The mean values

S.D. with common letters (small along the row & capital within a

column) are not significantly different according to Duncan’s multiple

range test (P = 0.05; n = 3).

70

3.7.8 The Category-I metals translocation index (%) analyzed in 50-days old

Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 71

3.7.9 The Category-II metals translocation index (%) analyzed in 50-days old


3.7.10 The tolerance index (TI) analyzed in shoot and root of 50-days old


3.7.11 The Category-I metals specific extraction yield (SEY %) analyzed in

50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 74

3.7.12 The Category-II metals tolerance index (TI) analyzed in 50-days old


3.8.1


cultivated on TSW-Soil mixtures applied with different fungi. The mean

values S.D. with common letters (small along the row & capital within

a column) are not significantly different according to Duncan’s multiple

range test (P = 0.05; n = 3).

78

3.8.2


patula cultivated on TSW-Soil mixtures applied with different fungi. The




82

iii

3.8.3


) observed in SHOOT

of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied

with different fungi. The mean values S.D. with common letters

(small along the row & capital within a column) are not significantly

different according to Duncan’s multiple range test (P = 0.05; n = 3).

83

3.8.4



50-days old Tagetes patula cultivated on TSW-Soil mixtures and

applied with different fungi. The mean values S.D. with common

letters (small along the row & capital within a column) are not


0.05; n = 3).

85

3.8.5


) observed in SHOOT

of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied




87

3.8.6



50-days old Tagetes patula cultivated on TSW-Soil mixtures applied




90

3.8.7



Tagetes patula cultivated on TSW-Soil mixtures applied with different

fungi. The mean values S.D. with common letters (small along the

row & capital within a column) are not significantly different according

to Duncan’s multiple range test (P = 0.05; n = 3).

93

3.8.8 The Category-I metals translocation index (%) analyzed in 50-days old


3.8.9 The Category-II metals translocation index (%) analyzed in 50-days old


3.8.10 The root and shoot tolerance index (TI) analyzed in 50-days old


3.8.11 The Category-I metals specific extraction yield (SEY %) analyzed in


3.8.12 The Category-II metals specific extraction yield (SEY %) analyzed in


3.9A.1





0.05; n = 3).

100

3.9A.2 Various morphological parameters observed in 55-days old Tagetes


103

iv



0.05; n = 3).

3.9A.3


) observed in SHOOT





105

3.9A.4







107

3.9A.5


) observed in SHOOT





108

3.9A.6







112

3.9A.7






range test (P = 0.05; n = 3).

115

3.9A.8 The Category-I metals translocation index (%) analyzed in 50-days old


3.9A.9 The Category-II metals translocation index (%) analyzed in 50-days old


3.9A.10 The tolerance index (TI) analyzed in 50-days old Tagetes patula

cultivated on TSW-Soil (% w:w) mixtures. 117

3.9A.11 The Category-I specific extraction yield (SEY %) analyzed in 50-days

old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures. 118

3.9A.12 The Category-II metals specific extraction yield (SEY %) analyzed in


3.9B.1

The biochemical parameters observed in 52-days old Helianthus

annuus cultivated on TSW-Soil mixtures. The mean values S.D. with



0.05; n = 3).

122

v

3.9B.2

Various morphological parameters observed in 52-days old Helianthus

annuus cultivated on TSW-Soil mixtures. The mean values S.D. with



0.05; n = 3).

125

3.9B.3


) observed in SHOOT

of 52-days old Helianthus annuus cultivated on TSW-Soil mixtures.

The mean values S.D. with common letters (small along the row &

capital within a column) are not significantly different according to

Duncan’s multiple range test (P = 0.05; n = 3).

127

3.9B.4



52-days old Helianthus annuus cultivated on TSW-Soil mixtures. The




128

3.9B.5


) observed in SHOOT

of 55-days old Helianthus annuus cultivated on TSW-Soil mixtures.

The mean values S.D. with common letters (small along the row &

capital within a column) are not significantly different according to

Duncan’s multiple range test (P = 0.05; n = 3).

130

3.9B.6



52-days old Helianthus annuus cultivated on TSW-Soil mixtures. The




133

3.9B.7



Helianthus annuus cultivated on TSW-Soil mixtures. The mean values



range test (P = 0.05; n = 3).

135

3.9B.8 The Category-I metals translocation index (%) analyzed in 52-days old

Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 136

3.9B.9 The Category-II metals translocation index (%) analyzed in 52-days old

Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 137

3.9B.10 The tolerance index (TI) analyzed in 52-days old Helianthus annuus

cultivated on TSW-Soil (% w:w) mixtures. 138

3.9B.11 The Category-I specific extraction yield (SEY %) analyzed in 52-days

old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 138

3.9B.12 The Category-II specific extraction yield (SEY %) analyzed in 52-days

old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures. 139

3.10.1

The physico-chemical properties, Category-I and Category-II metals

determined in TSW, Caldwell field soil and their various (% w:w TSW-

Soil) mixtures; The mean values with common letters (small along the

row & capital within a column) are not significantly different according

141

vi


3.10.2





0.05; n = 4).

142

3.10.3





0.05; n = 4).

145

3.10.4


) observed in SHOOT





147

3.10.5







148

3.10.6


) observed in SHOOT





150

3.10.7







154

3.10.8






range test (P = 0.05; n = 4).

156

3.10.9

The Category-I metals translocation index (%) analyzed in 45-days old

Tagetes patula cultivated on Caldwell field mixed with different

percentages of TSW (% w:w).

157

3.10.10

The Category-II metals translocation index (%) analyzed in 45-days old

Tagetes patula cultivated on Caldwell field mixed with different

percentages of TSW (% w:w).

157

3.10.11 The tolerance index (TI) analyzed in 45-days old Tagetes patula

cultivated on Caldwell field mixed with different percentages of TSW 158

vii

(% w:w).

3.10.12

The Category-I metals specific extraction yield (SEY %) analyzed in

45-days old Tagetes patula cultivated on Caldwell field mixed with

different percentages of TSW (% w:w).

159

3.10.13

The Category-II metals specific extraction yield (SEY %) analyzed in

45-days old Tagetes patula cultivated on Caldwell field mixed with

different percentages of TSW (% w:w).

159

3.11A.1

The biochemical parameters observed in 78-days old Helianthus

annuus cultivated on field soil mixed with different percentages of

tannery solid waste (TSW-Soil mixtures). The mean values S.D. with



0.05; n = 6).

162

3.11A.2

Various morphological parameters observed in 78-days old Helianthus

annuus cultivated on field soil mixed with different percentages of

tannery solid waste (TSW-Soil mixtures). The mean values S.D. with



0.05; n = 6).

164

3.11A.3


) observed in SHOOT

of 78-days old Helianthus annuus cultivated on field soil mixed with

different percentages of tannery solid waste (TSW-Soil mixtures). The




167

3.11A.4



78-days old Helianthus annuus cultivated on field soil mixed with





169

3.11A.5


) observed in SHOOT

of 78-days old Helianthus annuus cultivated on field soil mixed with





171

3.11A.6



78-days old Helianthus annuus cultivated on field soil mixed with





175

3.11A.7 The post-harvest fungal analyses (× 105 c.f.u. g

-1 soil) of soil used for the

cultivation of 78-days old sunflower (Helianthus annuus) on field soil mixed

178

viii

with different percentages of tannery solid waste (TSW-Soil mixtures). The

mean values S.D. with common letters (small along the row & capital within a

column) are not significantly different according to Duncan’s multiple range test

(P = 0.05; n = 6).

3.11A.8

Meta-analytical phytoextraction indices of sunflower: the Category-I

translocation index (%) analyzed for sunflower cultivated on field soil mixed

with different percentages of tanner solid waste (TSW) and inoculated with

different fungi.

179

3.11A.9

Meta-analytical phytoextraction indices of sunflower: the Category-II

translocation index (%) analyzed for sunflower cultivated on field soil mixed


different fungi.

180

3.11A.10

Meta-analytical phytoextraction indices of sunflower: the tolerance index (TI)

analyzed for shoot and root of sunflower cultivated on field soil mixed with

different percentages of tanner solid waste (TSW) and inoculated with different

fungi.

181

3.11A.11

Meta-analytical phytoextraction indices of sunflower: the specific extraction

yield (SEY %) for Category-I metals in sunflower cultivated on field soil mixed


different fungi.

181

3.11A.12

Meta-analytical phytoextraction indices of sunflower: the specific extraction

yield (SEY %) for Category-II metals in sunflower cultivated on field soil mixed


different fungi.

182

3.11B.1

The concentration of category-I category-II metals (mgkg-1

) observed

in soil amended with different concentration of tannery solid waste

(TSW-Soil % w:w) determined after the harvesting sunflower

(Helianthus annuus) and prior the sowing French marigold (Tagetes

patula). The mean values S.D. with common letters are not


0.05; n = 6).

185

3.11B.2





0.05; n = 6).

187

3.11B.3

Various morphological parameters observed in 82-days old Tagetes patula

cultivated on TSW-Soil mixtures. The mean values S.D. with common letters

(small along the row & capital within a column) are not significantly different

according to Duncan’s multiple range test (P = 0.05; n = 6).

189

3.11B.4


) observed in SHOOT





193

3.11B.5 The concentration of Category-I Metals (mgkg-1


82-days old Tagetes patula cultivated on TSW-Soil mixtures. The 195

ix




3.11B.6


) observed in SHOOT





197

3.11B.7







200

3.11B.8






range test (P = 0.05; n = 6).

203

3.11B.9 The Category-I metals translocation index (%) analyzed in Tagetes

patula cultivated on TSW-Soil (% w:w) mixtures. 204

3.11B.10 The Category-II metals translocation index (%) analyzed in Tagetes

patula cultivated on TSW-Soil (% w:w) mixtures 205

3.11B.11 The translocation index (%) analyzed in Tagetes patula cultivated on

TSW-Soil (% w:w) mixtures. 206

3.11B.12 The Category-I metals specific extraction yield (SEY %) analyzed in


3.11B.13 The Category-II metals specific extraction yield (SEY %) analyzed in


4.7.1 Pearson correlation among selected biochemical properties, dry

weight, Cr and Na uptake observed in Tagetes patula cultivated on

non-autoclaved soil (NAS) mixed with tannery solid waste (TSW).

223

4.7.2 Pearson correlation among selected biochemical properties, dry

weight, Cr and Na uptake in Tagetes patula cultivated in autoclaved

soil (AS) mixed with tannery solid waste (TSW)

225

4.8.1 Pearson correlation among selected biochemical properties and

metals for Tagetes patula cultivated in soil mixed with tannery solid

waste (TSW) and inoculated with AM (arbuscular mycorrhizal) and

saprobic fungi.

228

4.9.1 Pearson correlation among selected biochemical properties, CAT,

SOD, dry weight production, Cr and Na uptake in Tagetes patula

cultivated on TSW:Soil taken in pots and inoculated with saprobic

fungi.

230

4.9.2 Pearson correlation among selected biochemical properties and 232

x

metals for Tagetes patula cultivated in Caldwell field soil mixed with

tannery solid waste (TSW) and inoculated with saprobic fungi (incl.

Trichoderma harzianum).

4.9.3 Pearson correlation among selected biochemical properties and

metals for Helianthus annuus cultivated in soil mixed with tannery solid

waste (TSW) and inoculated with saprobic fungi (FIELD TRIAL).

234

xi

LIST OF FIGURES

Fig. No. Title Page No.

2.1.1 Map showing location of KTWMA plant in Kasur city of Pakistan (upper)

and detailed layout of the effluent treatment plant and landfill of KTWMA

(lower) from where TSW was sampled for the study.

13

2.1.2 View of the KTWMA landfill with open dumping of tannery solid waste

(TSW) selected as sampling site for TSW. (upper) The agricultural fields

can be seen in the vicinity of the landfill with no protective linings; (lower)

the diversity of the components of TSW can be observed.

14

2.2.1 Procurement steps of tannery solid waste (TSW) from landfill to the mixing

in soil at experimental station: (A) scrapping top 15 cm layer of TSW

before sampling; (B) filling sack with TSW dug with hoe; (C) TSW sample

filled sacks; (D) carriage of samples to the experimental station; (E) TSW

spread on polythene sheet for drying; (F) air dried TSW.

16

2.3.1 A view of the experimental station where pot trials were carried out; the

partially controlled conditions can be noted from the available type of wire

house applied with glass roof and airy fairy sides being open to natural

ambient temperature and moisture regimes.

23

2.5.1 The statistical layout of the experimental units showing distribution of field

plots lined with polythene sheet. The vertical grey lines show the

separation between experimental plots (25 × 3 feet each) arranged in split

plot design with no replication having soil mixed with tannery solid waste

(TSW-Soil % w:w) while the horizontal dark black lines show division of

each strip plot into sub-plots (5 × 3 feet each) applied with fungal

inoculations in a randomized complete block design.

32

2.5.2 Preparation of field plots in order to apply pot trial findings at field level: (A)

excavation of soil from strip plots (25 × 3 feet each) up to 2.5 feet depth

and lining with polythene sheet; (B) mixing of soil with 2 mm sieved

tannery solid waste (TSW); (C) refilling of excavated strip plots with soil

mixed with different percentages of TSW; (D) a strip plot refilled with TSW

mixed soil; (E) the vertical orientation of 5 strip plots; (F) the sub-plotting of

each of the strip plot into four experimental units each for fungal

inoculations in a randomized complete block design.

33

3.2.1 Alternaria alternata on Czapek’s medium at 250C isolated from TSW. 41

3.2.2 Aspergillus flavipus on 2% MEA medium at 250C. 41

3.2.3 Aspergillus fumigatus on Czapek’s medium at 250C 41

3.2.4 Aspergillus parasiticus on Czapek’s medium at 250C 41

3.2.5 Aspergillus terreus on MMN medium at 250C isolated from TSW 41

3.2.6 Fusarium sp. on Czapek’s medium at 250C isolated from TSW 41

3.2.7 Rhizopus arrhizus on 2% MEA basal medium at 250C isolated from TSW 42

3.2.8 Trichoderma pseudokoningii on Czapek’s medium at 250C isolated from

TSW

42

3.2.9 Aspergillus flavus on Czapek’s medium at 250C isolated from TSW. 42

3.2.10 Aspergillus japonicus on 2% MEA basal medium at 250C isolated from 42

xii

TSW.

3.2.11 Aspergillus niger on MMN medium at 250C isolated from TSW. 42

3.2.12 Aspergillus penicilloides on Czapek’s medium at 250C isolated from TSW 42

3.2.13 Aspergillus versicolor on 2% MEA medium at 250C isolated from TSW 42

3.4.1 In vitro mutual growth interaction (center) between screened heavy metal

tolerant Aspergillus niger (right) vs. Trichoderma pseudokoningii (left) 44


tolerant Alternaria alternata (left) vs. Trichoderma pseudokoningii (right)

45


tolerant Fusarium sp. (left) vs. Alternaria alternata (right)

45


tolerant Aspergillus niger (left) vs. Alternaria alternata (right)

46


tolerant Fusarium sp. (left) vs. Trichoderma pseudokoningii (right)

46


tolerant Fusarium sp. (left) vs. Aspergillus niger (right)

47

3.5.1 Pot cultivated 50-days old plants of Tagetes patula with vegetative growth

variation in response to individual and combined fungal inoculations in soil.

50

3.6.1 Screening of plants for their phytoextraction potential on the basis of

percentage germination on different TSW-Soil (% w:w) mixtures. A)

Tagetes patula B) Patunia xybrid C) Dahlia coccinea D) Zinnia elegans

52

3.7.1 The vegetative growth variation caN be observed in Marigold (Tagetes

patula) in response to autoclaved soil (AS on right) and non-autoclaved

soil (NAS on left) mixed with different percentages of TSW (% w:w)

ranging from the maximum in plants from 5 % (TSW:Soil) NAS inoculated

with F1 + F2 to the minimum in plants from 20 % (TSW:Soil) AS inoculated

with C i.e. no fungi.

57

3.8.1 The vegetative growth variation in Marigold (Tagetes patula) in response

to soil mixed with different percentages of TSW (% w:w) and inoculated

with different fungi.

81

3.9A.1 The vegetative growth variation in Marigold (Tagetes patula) in response

to soil mixed with different percentages of TSW (% w:w) and inoculated

with different fungi.

104

3.9B.1 The vegetative growth variation in sunflower (Helianthus annuus) in

response to soil mixed with different percentages of TSW (% w:w) and

inoculated with different fungi.

124

3.10.1 The vegetative growth variation in marigold (Tagetes patula) in response

to Caldwell field soil mixed with different percentages of TSW (% w:w) and

inoculated with different fungi; (upper) the representative pots of each of

the treatments with best growth of marigold; (lower) all of the experimental

units with replicates of all the treatments.

146

3.11A.1 Phytoextraction field trials with sunflower (Helianthus annuus) cultivated

on soil amended with different levels of tannery solid waste (TSW:Soil

w:w); 0 % the only treatment without geothermal membrane allowing

165

xiii

leaching (A), 0 % with geothermal membrane to avoid leaching (B), 5 %

(C), 10 % (D) and 20 % (E). The white lines across the strip plots (25 × 3

ft) indicate soil barriers (1.25 ft) subdividing each strip plot into four

subplots (5 × 3 ft each) for fungal inoculations viz. C: No fungal inoculum;

F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger +

T. pseudokoningii, applied in a randomized complete block design.

3.11A.2 The sunflower stem girth variation in response to soil amended with

different levels of tannery solid waste (% w:w) inoculated with fungi;

(clockwise from upper left) 10 % with F2, 20 % with F1 + F2; 5 % with F1 +

F2, 10 % with F1 + F2, 20 % with C, 5 % with F1 + F2.

166

3.11B.1 Phytoextraction field trials with French Marigold (Tagetes patula)

cultivated on soil amended with different levels of tannery solid

waste (TSW-Soil % w:w); upper: (A) 0* % the only treatment

without geothermal membrane allowing leaching, (B) 0 % with

geothermal membrane to avoid leaching, (C) 5 %, (D) 10 % and (E)

20 %. The white lines across the strip plots separated by bricked

walk ways (horizontal in above and vertical in lower; 25 × 3 ft) in

upper and the white arrows in the lower picture indicate soil

separations (1.25 ft) subdividing each strip plot into four subplots (5

× 3 ft each) for fungal inoculations viz. C: No fungal inoculum; F1:

Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A.

niger + T. pseudokoningii, applied in a randomized complete block

design.

190

3.11B.2 The Tagetes patula stem girth variation in response to soil mixed

with different levels of tannery solid waste (TSW : soil w:w)

inoculated with fungi (A- 0*% with F1+F2; B- 0% with F1+F2; C- 5%

with F2; D- 5% with F1+F2 ; E- 5% with C ;F-10% with F1+F2; G-

10% with F2; H- 20% with F2; I- 20% with F1+F2.

191

4.1.1 Simple non-linear regression between bulk density (gcm-3

) of TSW-soil

mixture and different fractions of Category-I metals (mgkg-1

): (A) between

bulk density and water-soluble fraction, (B) between bulk density and

DTPA-extractable fraction.

211

4.1.2 Simple linear regression between bulk density (gcm-3

) of TSW-soil mixture

and different fractions of Category-II metals (mgkg-1

): (A) between bulk

density and water-soluble fraction, (B) between bulk density and DTPA-

extractable fraction.

212

4.1.3 Correlation between TSW (% w/w) of TSW-soil mixture and different

fractions of Category-II metals (mgkg-1

): (A) between bulk density and

water-soluble fraction, (B) between bulk density and DTPA-extractable

fraction.

213

4.5.1 The growth variation observed in Tagetes patula in response to different

fungal inoculations in soil

219

Chapter 1

Introduction

1

CHAPTER 1

INTRODUCTION

Land and water are valuable natural resources that sustain agriculture

and ultimately civilization of mankind. For the last couple of decades they

have not only been subjected to the maximum exploitation and severe

degradation but also been polluted due to anthropogenic activities. The

escalating rate of human activities in the form of industrialization has been

posing unprecedented threats towards the biosphere. Both the number and

diversity of pollution sources have been increasing day by day. Burning of

fossil fuels, mining, smelting of metalliferous ores, electroplating, finishing of

metals and plastics, municipal wastes, fertilizers, pesticides, the use of

pigments, chemical works, wood preservation, vehicle service stations, metal

fabrication shops, paper mills, textile plants, waste disposal sites, and above

all tanning of leather industry have been particularly guilty of polluting the

environment (Wong, 2003; Freitas et al., 2004). Each of the pollution sources

have been variably contaminating the environment and exerting damaging

effects on plants, animals and ultimately human health by adding heavy

metals to the soils and waters. Such a scenario has been a serious concern

due to the persistence of heavy metals in the environment and potent

carcinogenicity to human beings. The contamination of the environment with

heavy metals has become a worldwide problem that affects crop yields, soil

biomass and fertility, and leads to the bioaccumulation of metals in the food

chain (Gratao et al., 2005; Rajkumar et al., 2009).

A heavy metal is a member of an ill-defined subset of elements that

exhibits metallic properties. They mainly include the transition metals, some

metalloids, lanthanides and actinides (Babula et al., 2008). These are metallic

chemical elements that have a relatively high density and are toxic even at

low concentrations. They cannot be destroyed biologically but are only

transformed from one oxidation state or organic complex to another (Garbisu

and Alkorta, 2001; Gisbert et al., 2003). Therefore, heavy metal pollution

poses a great potential threat to the environment and human health.

The leather processing has been one of the top most culpable

industries that cause heavy metal pollution. Among all the industrial wastes

2

tannery effluents have been ranked the greatest pollutants. Industrialized

countries have regulated the emission of toxic substances, but in developing

countries like Pakistan, rapid industrial development and population

explosion, coupled with lack of pollution control has caused an enormous

increase in heavy metal contamination of agricultural soils (Ji et al., 2000).

Because of the relatively inexpensive cost of labor and materials, over half of

the world’s tanning activities have been occurring in low and middle income

countries as in the case of Pakistan. Between 1970 and 1995, the percentage

of low to middle income countries contributing to the global production of light

leather increased from 35 % to 56 % and from 26 % to 56 % for the

production of heavy leather materials (Jenkins et al., 2004). According to a

study conducted by the Blacksmith Institute (2010), about 75% of Cr sites are

located in South Asia and of these, nearly a third have been associated with

tannery operations, with mining and metallurgy sites also contributing

significantly. The high concentration of chromium sites in South Asia has been

primarily due to the abundance of tanneries in the region. Majority of the

tanneries have poor environmental controls (Azom et al., 2012).

The process of turning raw leather into manufactured hide uses a lot of

chemicals, auxiliary materials and water. Various chemicals are used during

the soaking, tanning and post tanning processing of hides and skins (Inamul

Haq, 1998). The main chemicals used include sodium sulphite and basic

chromium sulphate including non-ionic wetting agents, bactericides, soda ash,

CaO, ammonium sulphide, ammonium chloride and enzymes. Others include

sodium bisulphate, sodium chlorite, NaCl, H2SO4, formic acid, sodium

formate, sodium bicarbonate, vegetable tannins, syntans, resins,

polyurethane, dyes, fat emulsions, pigments, binders, waxes, lacquers and

formaldehyde. Various types of processes and finishing solvents and

auxiliaries are also used, as well. It has been reported that only about 20 % of

the large number of chemicals used in the tanning process are absorbed by

leather while the rest are released as waste (Azom et al., 2012).

It has been reported that 90 % of all global production of tanned

leathers is tanned using chromium sulfates (Aslan et al., 2007). The

remainders are tanned using other metal sulfates, mostly aluminum,

vegetable tannins or a combination of both (Aslan, 2009). In the event of

3

leather being released to the environment as waste, the heavy metals within

the leathers might harm the ecosystem and threaten human health by

transferring directly or indirectly into the food chain (Aslan et al., 2006; Aslan,

2009). Chromium salts have been used commonly as tanning agents due to

their excellent tanning property, low cost, easy application and wide supply.

As a result, a world total of 540.000 tons of chrome tanned solid waste is

being produced each year (Afsar et al., 2011). A significant amount of

chromium (60 %) applied in tanning is taken up by the leather and the

remaining is discharged in the effluent (Feed International, 2003).

The major environmental issues of tanneries have been solid wastes,

sludge and wastewater. While processing of animal hides into leather, about

20 % of the materials have been produced as solid wastes, consisting of

leather scraps, hair, soluble proteins, curing salts and fleshing (animal fats,

collagen fibers, meat etc). The collagen based waste has been consisting of

4.5 % three valance chrome on an average. During the preservation of animal

hides, huge loads of pollution have been discharged as total dissolved solids

(TDS) and chlorides as a result of use of lime, sodium sulphide, ammonium

salts, sulphuric acid and chromium salts (Kanagaraj et al., 2006). The toxic

metallic compounds, chemicals, biologically oxidizable materials and large

quantities of putrefying suspended matter solid wastes of tanneries are

usually dumped improperly inside and around the factory area (Khuwaja et al.,

1995). Such solid waste has been observed to augment obnoxious smell

because of the degradation of proteins and fats in the skin and generation of

compounds such as NH3, H2S and CO2. Interest on this waste has increased

in recent years due to its volume and oxidation risk of Cr (III) to Cr (VI) which

is carcinogenic for human and more toxic for environment. Discharging of the

solid waste to restricted areas has been a problem for tanners due to the high

cost.

Tanning industry in Pakistan has not been a new industry like other

South Asian countries. It has existed in its indigenous form since long. It had

started in Pakistan about 100 years ago but received a big boost in 1971. The

province of the Punjab has been playing a major role in the development of

tanning industry in Pakistan. Presently, most of the cities where the industry

has been installed in the Province are located in Sialkot, Lahore, Multan,

4

Gujranwala and Kasur. There have been as many as 600 tanneries in the

formal sector of the country and 50% of these have been located in Kasur, a

district of Punjab province, with a long standing tradition of leather tanning

(Augustus, 1996).

The leather sector has been representing one of the most important

industrial sectors in Pakistan in terms of significant contribution to the national

economy. According to a review on Pakistan exports by Trade Development

Authority of Pakistan (TDAP, 2007-08), leather and its products showed an

increase of 21% with export reaching upto US$ 1220.12 Million from US$

1088.20 Million. However, concerns over the expansion of the leather sector

have been resulting in considerable pressures on the environment in the form

of pollution and occupational hazards. Leather industry has been one of the

key culprits of such pollution in Pakistan (Khan, 2001).

By far, proper solid waste disposal systems have been completely

lacking in the design of tanneries of Pakistan. Even in case of the treatment

plant constructed by Kasur Tannery Waste Management Agency (KTWMA) in

association with United Nations Organization (UNO) at Kasur, Pakistan; the

solid waste produced by the cluster of tanneries has been simply thrown away

at the designated spot with extreme violation of standard recommendations.

The solid waste from tanneries is being removed and disposed off in the solid

waste landfill site indiscriminately without any environmental consideration.

Such approach of solid waste disposal has so far been considered a low-cost-

solution although its actual cost may be many folds especially for small and

medium business enterprises. The presence of toxic compounds, especially

compounds of Cr, has been representing a real danger to the health of the

nearby population (TOOL, 2003). Improper and unguarded use of chemicals

has exposed the workers and residents of the adjoining areas to health

hazards of varied types. Respiratory disorders, lung infection and related

diseases, diarrhea/dysentery and typhoid have been common among the

community population (EMRO, 2003).

Conventional methods for removing metals from aqueous solutions or

extracts taken from solid wastes include chemical and physical methods

(chemical precipitation, chemical oxidation or reduction, ion exchange,

filtration, electrochemical treatment, reverse osmosis, membrane

5

technologies, evaporation recovery, etc.) and activated sludge biological

treatment (Ahluwalia and Goyal, 2007). These processes have been generally

efficient in removing the bulk of metals from solution at high or moderate

concentrations. These techniques might have been ineffective or extremely

expensive especially when the metals in solution are at low concentration i.e.

in the range of 1–100 mg L-1 (Ahluwalia and Goyal, 2007). As a consequence,

their limits (high cost, high reagent requirements, etc.) become more

pronounced when voluminous effluents containing complex organic matter

and low metal contamination have to be treated. Various expensive cleaner

technologies cannot eliminate Cr completely from tannery waste, because

some Cr remains in sludge and solid waste derived from tanning (IULTCS,

2008).

In such a scenario, technologies are badly needed that should not only

be able to remove small to large fractions of heavy metals in the polluted

entities like soil and water but also be sustainable in terms of cost and

efficiency. With regard to this, phytotechnologies involving the use of plants

for pollutant removal has gained importance during the last two decades

(Dhir, 2010). Such technologies have been very commonly used because of

their cost effectiveness. However, one of the major drawbacks is their

tremendously slow efficiency. In order to cover efficiency cons, such

technologies are incorporating various chemical, biological and/or

combination of both as phytoextraction enhancers. For example, application

of crude and commercial plant growth regulators (PGRs) along with the

saprobic fungus Trichoderma pseudokoningii have been found to enhance

both multi-metal accumulation and biomass production in Pennisetum

glaucum cultivated in tannery solid waste amended soils (Bareen et al., 2012).

Application of PGRs along with saprobic fungi help the plants to mitigate toxic

effects of HM by secretion of acids, proteins, phytoantibiotics and other

chemicals (Denton, 2007). Other microorganisms such as, bacteria,

filamentous fungi and yeasts have also been found to be capable of efficiently

accumulating heavy metals (Ahluwalia and Goyal, 2007; Mungasavalli et al.,

2007). When efficiency of plants used for phytoremediation is accelerated by

applying either synthetic chemicals or living microbes in the soil, it is termed

as bioaugmentation-assisted phytoextraction (Lebeau et al., 2008) or simply

6

assisted phytoremediation. The reinforced application of plants and

associated microbes to remove HM from the contaminated bodies has offered

an effective, low cost and sustainable means to achieve sustainable cleaning

(Terry and LeDuc, 2005). Researchers have found that microorganisms

associated with plants are able to degrade a number of contaminants (Suresh

and Ravishankar, 2004; Macek et al., 2004). Such microorganisms increase

the efficiency of plants to decontaminate the toxic environment.

Biotechnological approaches consisting of combined application of plants and

microbes could succeed in such areas (Malik, 2004). Therefore, the use of

biological resources such as those of autochthonous fungi has been explored

worldwide for treatment of toxic wastes and accepted owing to several

advantages including environmental safety and cost effectiveness (Dhir et al.,

2010). El-Kassas and El-Taher (2009) isolated Trichoderma viride from heavy

metal polluted area found to be a strong potent for removal of Cr (VI).

Whereas Ezzouhri et al. (2009) isolated highly tolerant fungi and yeasts from

multi-metal contaminated sites. There has also been work on extracellular

enzymatic activities in the rhizosphere of stress tolerant plants that could

potentially contribute to phytoremediation of HM (Reboreda and Cacador,

2008). Besides autochthonous fungi, people have been advocating the

application of allochthonous fungi to increase microbial diversity in the

historically contaminated soils that could cause fungal augmentation leading

to qualitative differences in the soil (Federici et al., 2007). The expanded and

multi-variate tendency of mycelial fungi could be attributed to their more

varied array of strategies for reducing HM toxicity (Baldrian, 2010). There

have been suggestions about applicability of mycelial fungi in both biosorption

(Prigione et al., 2009) as well as bioremediation (Hatvani and Mecs, 2003)

technologies to treat complex wastes contaminated with HM. One of the key

advances offered in the current research is expanding list of stress tolerant

autochthonous filamentous fungi isolated from the contaminated sites like that

of tannery solid waste dumping sites and putting them back in the

phytoextraction process as enhancers of heavy metal tolerance and uptake.

The introduction of heavy metal compounds into the environment

generally induces morphological and physiological changes in the microbial

communities (Vadkertiova and Slavikova, 2006), hence exerting a selective

7

pressure on the microbiota (Verma et al., 2001). Generally, the contaminated

sites are the sources of metal resistant micro-organisms (Gadd, 1993).

Therefore, it is important to explore autochthonous micro-organisms from

such contaminated niches for the bioremediation of heavy metals.

Besides autochthonous filamentous fungi, there has been extensive

work on isolation from polluted soils, application of arbuscular mycorrhizal

(AM) fungi in enhancing biomass as well as heavy metal uptake of plants

growing on heavy metal contaminated soils. The AM fungi have been

reported in tannery pollutants and affected soils (Raman and Sambandan,

1998). They also provide an attractive system to advance plant-based

environmental clean-up (Gohre and Paszkowski, 2006). The AM fungi have

acquired salient functions that make them thrive in stressed soils, enhance

plant growth and mobilize HM from soil. Such functions comprise nutrient

acquisition, cell elongation, metal detoxification, and alleviation of biotic /

abiotic stress (Rajkumar et al., 2012). It has been suggested that isolation of

the indigenous heavy metal stress-adapted AM fungi could be the potential

biotechnological tool for inoculation of plants for successful restoration of

degraded ecosystems (Gaur and Adholeya, 2004). Vivas et al. (2003) isolated

AM fungus namely G. mosseae from cadmium polluted soil and found its

mycorrhizal colonization potential on Trifolium to yield high growth and multi-

metal tolerance. Audet and Charest (2007) have presented models that

illustrate important compromise between plant growth, plant HM uptake and

HM tolerance, and importance of AM fungi in buffering the soil environment

for plants under stressed plant cultivation conditions. Similarly, the incidence

of AM fungi in HM polluted sites, their role in imparting HM tolerance in plants,

factors affecting AM fungi in HM polluted sites and their mechanism of HM

tolerance have been extensively reviewed by Khade and Adholeya (2009). It

has been reported that mycorrhizal associations increase the absorptive

surface area of the plants and the protection provided to roots and enhanced

capability for greater uptake of minerals results in greater biomass production,

a prerequisite for successful remediation (Khan 2001, 2005). The chemical,

biochemical, structural and genomic aspect of AM fungi colonized on roots of

plants in heavy metal soils have been extensively elaborated as a matter of

support for repeated application of AM fungi to alleviate heavy metal stress of

8

plants (Hildebrandt et al., 2007). A multi-scale application comprising pot

lysimeter and field plot experimental approach for studying the

biogeochemical role of AM fungi has been developed (Neagoe et al., 2013).

To a wider perspective, the AM fungi application in phytoextraction has

resulted in greater effectiveness with respect to increasing the tolerance of

lettuce because of induction of antioxidant enzymes (Kohler et al., 2009;

Azcon et al., 2009). Combined application of AM and filamentous fungi

isolated from multi-contaminated soil resulted in enhanced bioaccumulation of

HM (Azcon et al., 2010). The application of AM fungi in phytoremediation with

plants that cannot translocate HM from root to shoot could be of great interest

since such plants produce high biomass but show less bioaccumulation index

(Citterio et al., 2005).

Several heavy metal-tolerant autochthonous fungi have been isolated

from polluted soils, which can be useful for reclamation of degraded soils as

they are found to be associated with a large number of plant species in heavy

metal-polluted soil. Korda et al. (2004) reported that reintroduction of

indigenous microorganisms isolated from the contaminated sites after

culturing provide a highly effective bioremediation tool. Fungi employed in this

effort include many species that have been commonly found in soil, such as

species of Aspergillus, Fusarium, Rhizopus, Mucor and Penicillium (Ammarati

and Michelle, 2005).

Saprobic fungi are important and a common component of rhizosphere

soil, where they obtain enhanced nutritional benefit from organic and

inorganic compounds released from living roots, together with sloughed cells

(Alexander, 1977; Dix and Webster, 1995). Their importance lies in the large

microbial biomass they supply to the soil and in their capacity to degrade toxic

substances (Madrid et al., 1996). Some experimental results confirmed the

existence of synergism in the saprobic fungi, Fusarium concolor and

Trichoderma koningii, on plant roots colonized by AMF (Garcia-Romera et al.,

1998, Arriagada et al., 2005; Arriagada et al., 2007). Similar synergistic

interaction has been observed in Trichoderma pseudokoningii and the AM

fungus Glomus fasciculatum (Bareen and Nazir, 2010). So the cultivation of

heavily mycorrhizal trees that produce large amounts of biomass on

contaminated soil are recommended for phytoremediation practice to prevent

9

food chain contamination (Arriagada et al., 2009). The current work does

comprise isolation of AM fungi from stressed soils and their application in HM

alleviation endeavors with selected ornamental plants.

Increase in the level of heavy metals poses a pervasive threat to the

natural ecosystem. Although many heavy metals when in trace amounts are

essential for various metabolic processes in organisms, they create

physiological stress leading to generation of free radicals when in high

concentration. Stress in turn induces the production of reactive oxygen

species (ROS). Therefore, a mechanism to interrupt such an autocatalytic

process is required. Under normal circumstances, concentration of oxygen

radicals remains low because of the activity of protective enzymes, including

superoxide dismutase (SOD), catalase, and ascorbate peroxidase (Asada,

1984). In resistant forms, stress condition may enhance protective processes

such as accumulation of compatible solutes and increase in the activities of

detoxifying enzymes. Malondialdehyde (MDA) is a cytotoxic product of lipid

peroxidation and an indicator of free radical production and consequent tissue

damage (Ohkawa et al., 1979). Superoxide dismutase (SOD) is a

metalloenzyme that catalyzes dismutation of superoxide anion into oxygen

and hydrogen peroxide. Such enzymes provide a defense system for the

survival of aerobic organisms (Beyer et al., 1991). Proline accumulates

heavily in several plants under stress, providing the plants protection against

damage by ROS. Proline also plays important roles in osmoregulation

(Ahmad and Hellebust, 1988; Laliberte and Hellebust, 1989), protection of

enzymes (Nikolopoulos and Manetas, 1991; Laliberte and Hellebust, 1989;

Paleg et al., 1984), stabilization of the machinery of protein synthesis (Kadpal

and Rao, 1985), regulation of cytosolic acidity (Venekemp, 1989), and

scavenging of free radicals (Smirnoff and Cumbes, 1989). It also acts as an

effective singlet oxygen quencher (Alia et al., 2001).

Wise selection of hyperaccumulators has played a key role in

designing and successful implementation of an assisted phytoextraction plan.

The key factors involved in making such plant selection include HM tolerance

index of plant, continuous rotational frequency with respect to seasonality and

climatology, to let phytoextraction enhancers effectively integrate into the

rhizosphere of the plant, and be able to propagate under multi-metal

10

contaminated conditions. No plant has yet been found to fulfill all of the

desired pre-requisites of an ideal hyperaccumulator, however, some plants

show many of them. Sunflower (Helianthus annuus L.) has been found to

produce sufficient plant biomass and metal shoot translocation for efficient

phytoextraction (Nehnevajova et al., 2009). It (sunflower) has also been able

to show increased concentration of HM from tannery sludge amended soils

under synergistic effects of AM fungi and Trichoderma pseudokoningii (Nazir

and Bareen, 2011). As a matter of plant rotational frequency in the HM

contaminated site, it has been found to be suitable with its other floriculture

members such as, Marigold, Tagetes patula and T. erecta, while propagating

in soils ameliorated with tannery effluents (Chatterjee et al., 2010). Since the

integration of the phytoextraction enhancers is necessary for the ideal

hyperaccumulator, the Marigold (Tagetes patula) has been found to produce

maximum biomass and HM uptake when accompanied with combined

application of saprobic and mycorrhizal fungi while being cultivated in tannery

solid waste amended multi-metal contaminated soil (Bareen and Nazir, 2010).

Such studies with T. patula on tannery waste amended soils have been

repeated by other workers in India (Singh et al., 2011); with T. erecta in China

(Miao and Yan, 2013) and India (Sivasankar et al., 2012). There have also

been findings about bioremediation of sludge through soil amendments using

hyperaccumulator plant species (Singh and Sinha, 2004). On the basis of

their excellent qualities, Sunflower (H. annuus L.) and Marigold (T. patula)

have been selected for current research work.

Heavy metal exposure stimulates formation of toxic oxygen species

(TOS) or reactive oxygen species (ROS) in plants such as O2− and H2O2

(Halliwell and Gutteridge, 1984). Such studies in Sunflower have also been

reported (Singh et al., 2004). Toxic oxygen species are highly reactive and

damage lipids, proteins and nucleic acids (Foyer et al., 1994). Plants possess

certain defensive mechanisms that either prevent the formation of TOS/ROS

or scavenge free radicals. These include antioxidant enzymes and specific

compounds, the production of which is triggered during stress (Devi and

Prasad, 1998). The synchronous action of various antioxidant enzymes, viz.,

catalase (CAT) and ascorbate peroxidase (APX) with the thiol-regulated

enzymes dehydroascorbate reductase (DHAR), Monodehydroascorbate

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WDM-4W68DYJ-1&_user=2248840&_coverDate=09%2F30%2F2009&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1188142481&_rerunOrigin=google&_acct=C000056732&_version=1&_urlVersion=0&_userid=2248840&md5=b025baf9968ae57a386037269fd1f783#bib14


11

reductase (MDHAR) and glutathione reductase (GR) of the ascorbate

glutathione (GSH) pathway is a predominant mechanism of ROS quenching

under heavy metal stress (Weckx and Clijsters, 1996; Hall, 2002). Apart from

these enzymes low molecular weight antioxidant metabolites like ascorbic

acid (Asc) and glutathione play an important role in protecting plants from

oxidative stress damage (Alscher, 1989; Dhir et. al., 2010). The assessment

of enzymology associated with plants in their defense mechanism while being

cultivated on tannery solid waste multi-metal contaminated soils have been

other key advances of the current research work.

Generation of free radicals under heavy metal stresses and induction

of a defense mechanism in plants inoculated with autochthonous fungi

(saprobic and/or mycorrhizal) have not been studied thoroughly so far. This

situation prompted to investigate whether free radicals are generated under

the stress imposed by tannery solid waste containing heavy metals such as

Pb, Cu, Cr and Zn. The other key purpose of the current work is to determine

the response of selected hyperaccumulator plants to heavy metals in terms of

growth and tolerance reflected by the parameters of plant defense mechanism

based on SOD, CAT, and proline under the influence of autochthonous fungal

inoculated in soils with different toxicity levels of tannery solid waste.

In the light of the given text, the current research work is targeted at

finding phytoextraction potential of sunflower and marigold under the influence

of AM and filamentous fungi isolated from the tannery solid waste in terms of

HM tolerance index and biomass production of selected plants, assessment of

biochemical plant enzymes involved in providing hyperaccumulation and

shoot translocation tendencies, as well as overall cleaning efficiency of the

proposed technology for targeted tannery solid waste (collected from dumping

site of Kasur Tannery Waste Management Agency (KTWMA) located at

Kasur, Pakistan) amended multi-metal contaminated soil both in pilot scale

greenhouse experiment and in the field.


Chapter 2

Materials and

Methods

12

CHAPTER 2

MATERIALS AND METHODS

2.1 Sampling site, surveys and sampling of tannery solid waste

2.1.1 Sampling site

The tannery solid waste (TSW) landfill site constructed and managed

by Kasur Tannery Waste Management Agency (KTWMA) is located at

Depalpur Road, Kasur (31005’16.29’N, 74028’36.16’E), Pakistan (Fig. 2.1.1).

The landfill is surrounded with agricultural fields from three sides while

on the fourth side (towards north) effluent treatment plant of KTWMA is

located (Fig. 2.1.1). The landfill has been receiving TSW from about more

than one hundred local tanneries of Kasur since its establishment in 1994.

The landfill has been ill-managed, lacking of regular leveling of TSW and

application of clay over improperly pressed TSW. As a result, localized heaps

of TSW with variable sizes are randomly distributed in the landfill area. Few of

the TSW heaps are high enough so that during rainy (monsoon) season in the

area, the runoff from TSW slopes flows down the adjacent agricultural fields,

severely affecting the cultivated crops.

The TSW comprises about more than thirty different kinds of

components, mainly being chrome shavings, pieces of partially or completely

tanned leather, wet shavings, bulk amount of salts, hairs, etc. Most of the

precipitation water is retained in the TSW igniting the putrification process of

TSW. As a result, obnoxious odors are produced that contaminate the air as

well.

The KTWMA landfill makes the Kasur city an acute example of

environmental degradation, not only surface water, fertile land and ground

water, but also a direct health hazard to the people living and working in the

affected area (Fig. 2.1.2). The putrification of TSW involving degradation of

proteins and fats from the leather further makes composition of TSW more

complex.

13

Figure 2.1.1. Map showing location of KTWMA plant in Kasur city of Pakistan (upper) and detailed layout of the effluent treatment plant and landfill of KTWMA (lower) from where TSW was sampled for the study.

14

Figure 2.1.2. View of the KTWMA landfill with open dumping of tannery solid waste (TSW) selected as sampling site for TSW. (upper) The agricultural fields can be seen in the vicinity of the landfill with no protective linings; (lower) the diversity of the components of TSW can be observed.

2.1.2 Surveys and sampling

The sampling site was extensively surveyed in order to assess the

variation of TSW and collection of representative sample of the whole lot.

N

15

During the course of study, the TSW landfill was surveyed on: May 05, 2007;

Jul 19, 2008; Sep 20, 2008; Dec 20, 2009; Feb 16, 2009; Apr 15, 2010; Nov

10, 2010; Feb 15, 2011; and Sep 20, 2011.

For sampling of TSW, a completely randomized design was followed.

After scrapping off top 15 cm layer of the surface debris at 1 m2 area, the top

10-inches of TSW was thoroughly mixed with the help of a shovel and placed

approximately 25 kg of TSW in a polypropylene sac. There were as many as

50 random spots from where TSW bulk samples were collected and carried to

the experimental site at Quid-e-Azam Campus, University of the Punjab,

Lahore, Pakistan.

2.2 TSW sample processing for spiking garden soil

A uniform layer of TSW sample from each sack was spread on

polythene sheet in sunlight for drying (Fig. 2.2.1) and turned every other day

for two weeks. Any aggregates were hammered before sieving through a

mesh size of 2.5 mm2. In addition, field soil from Botanical Garden near the

experimental site was collected, dried and sieved through 2.5 mm2 mesh size.

The sieved TSW and soil were further dried to bring them at the same

moisture content prior to mixing. To obtain 5 % TSW-Soil (w : w) mixture, 250

g of TSW and 4650 g of soil were taken in a tumbler. The resulting 5000 g of

mixture were homogenized for 2 minutes by rotating the tumbler and then

1500 g of the mixture were placed in each of the three regular 1.5 liter plastic

pots with no bottom holes taken as replicates while the remaining replicative

500 g of the mixture were placed in labeled zip-loc bag for physic-chemical

analysis in the laboratory. Similarly, 10, 20, 30 through 100 % TSW: Soil

mixtures were procured with three replicates each and replicative 500 g of

each of the mixture ratios in zip-loc bags for pre-sowing analysis in the

laboratory.

The tumbler was cleaned between mixing every other type of TSW-Soil

mixture ratio. The mixtures were allowed to stay by leaving pots in the

greenhouse for 30 days.

16

Figure 2.2.1. Procurement steps of tannery solid waste (TSW) from landfill to the mixing in soil at experimental station: (A) scrapping top 15 cm layer of TSW before sampling; (B) filling sack with TSW dug with hoe; (C) TSW sample filled sacks; (D) carriage of samples to the experimental station; (E) TSW spread on polythene sheet for drying; (F) air dried TSW.

2.2.1 Preparation of soil saturation extract

According to the method given by Rhoads (1982), 500 g of soil sample

were taken and mixed with adequate water to form a supersaturated paste

and left overnight until it glistened under the light and the spatula did not stick

with the paste. A soil saturation extract was obtained under suction pressure

exerted from a vacuum pump.

A B

C D

E F

17

2.2.2 Determination of pH

The soil saturation extract obtained as given in 2.2.1 was used to

determine the pH of the soil samples with the help of a bench type pH meter

(Model Inolab pH 720/Set WTW Germany).

2.2.3 Determination of Electrical Conductivity (ECe) and Sodium

Chloride (%)

The soil saturation extract was used to determine the ECe and NaCl

content of the soil sample using auto ranging portable water proof

microprocessor EC/ TDS/NaCl/oC meter (Model, Hanna 9835).

2.2.4 Determination of carbonates and bicarbonates

For the estimation of carbonates and bicarbonates, the soil saturation

extract was titrated against a standard solution of sulfuric acid using

phenolphthalein as an indicator for carbonates and methyl orange as an

indicator for bicarbonates (Saeed, 1980).

2.2.5 Determination of soil organic matter (SOM %)

The SOM in TSW-Soil mixtures was assessed by loss-on-ignition

method given by Wright et al. (2008).

2.2.6 Digestion of Soil-TSW mixtures

The TSW-Soil mixtures with three replicates each were digested on a

heating digester (Model DK 6, TMD 6 Velp, Italy) and mineralized in a

microwave oven with aqua regia (ISO 11466, 1995) to bring their volume up

to 50 ml final volume of soil saturation extract.

2.2.7 Determination of water soluble metals

The soil saturation extract obtained from the extraction process was

used to determine the water soluble fraction of metals. On the basis of their

method of detection, the metals were grouped into two categories i.e. the

Category-I and Category-II metals.

The Category-I metals were detected by using flame photometer

(Model: PFP7&PFP7/C JENWAY) such as, Ca, K, and Na. In order to

determine the water soluble fraction of Category-I metals, the soil saturation

extracts obtained were run on flame photometer. The quantification of metals

18

was carried out by plotting the absorption values of unknown samples against

the standards solutions of each of the Category-I metal.

The Category-II metals were detected by using flame atomic

absorption spectrophotometer (AAS Model: GBC SAVANT AA, Australia)

such as, Cd, Cr, Cu, Fe, Mg, Ni, Pb and Zn. The water-soluble fraction of

Category-II metals was determined from the soil saturation extracts of

unknown samples by running them on AAS after running the blank and

standard solution for each of the metal and then blank again. After running

every ten samples, the process of AAS calibration with respective standards

was repeated for each of the metal, following the recommendations given in

user manual by the manufacturer.

2.2.8 Determination of DTPA extractable metals

In order to determine diethylene triamine pentaacetic acid or DTPA-

extractable fraction of Category-I as well as Category-II metals, the TSW-Soil

mixtures, 10 g of the TSW-Soil mixtures were solubilized in 20 ml of

extracting solution (0.005M DTPA, 0.01M CaCl2 and 0.1M EDTEA adjusted

to pH 7.3) as given by Lindsay and Norvell (1978). The extracts thus obtained

were run on flame photometer and AAS, as illustrated in 2.2.7.

19

2.3 Screening of fungi and plants tolerant for TSW based toxic

metals

2.3.1 Isolation and screening of TSW-representative autochthonous

fungi

2.3.1.1 Preparation of culture media

The isolation of fungi from TSW was performed on three types of

fungal nutrient media namely, Malt Extract Agar (MEA), Czapek’s medium

and Modified Melin Norken (MMN) Medium.

The 2% MEA medium was prepared by solubilizing 20 gm each of Malt

Extract and Agar in one liter of distilled water as given by Tuite (1969).

For Czapek’s medium (Dox, 1910), 3 gm sodium nitrate, 1gm

potassium hydrogen phosphate, 0.5gm magnesium sulphate, 0.5gm

potassium chloride, 0.01gm ferrous sulphate, 30gm sucrose and 20gm agar

were taken per liter of distilled water.

In order to prepare MMN Medium (Morx, 1969), 3 gm malt extract, 10

gm d-glucose, 0.25 gm (NH4)2HPO4, 0.50 gm MgSO4.7H2O, 0.15 gm

CaCl2.2H2O 0.05 gm, 0.02/1.2ml (1% solution) NaCl, 100 µg thiamin HCl and

12 gm agar were taken per liter of distilled water.

For each of the fungal nutrient medium, their respective ingredients

were thoroughly mixed using a magnetic stirrer in individual volumetric flasks

and autoclaved at 1210C temperature and 15 lb/inch2 pressure for 15 minutes.

After autoclaving, the flasks were allowed to cool down to 650C. The nutrient

medium from flasks was poured into the petri plates with a uniform layer after

adding the antibacterial powder (Chloromycetin chloramphenicol) under

aseptic conditions maintained in a laminar air flow cabinet.

2.3.1.2 Isolating TSW-representative fungi by direct and spread plate

The TSW powder was sprinkled on the fungal nutrient medium taken in

petri plates by the direct and spread plate methods.

20

In the direct plate method, the sieved powder of TSW was sprinkled

directly on top of the medium under aseptic conditions. The petri plates were

sealed with parafilm and incubated at room temperature for 3 days.

In the spread plate method, 1gm powdered TSW was dissolved in

100ml distilled sterilized water by agitating for 20 minutes at room

temperature and then diluting (10 - 10,000 folds) in large beakers. Aliquots of

100 µl of different dilutions were plated onto all of the three fungal nutrient

media with three replicates each to ensure the growth of fungi present in

samples. The petri plates were sealed with parafilm and incubated.

After at least 3 days of incubation at 25°C, the developed fungal

colonies were randomly picked for isolation from MEA and sub-cultured on

petri plates contained with new flush of same nutrient medium. Similarly, the

picking and sub-culturing of fungal colonies was carried out for both Czapek’s

and MMN fungal nutrient media. The process of sub-culturing was kept

continuous until pure isolates for all of the TSW representative fungal colonies

on three fungal nutrient media were obtained.

2.3.1.3 Identification of the TSW-representative fungal species

The pure isolates from TSW were characterized to the genus level on the

basis of macroscopic characteristics (colonial morphology i.e. color,

appearance and shape) and microscopic characteristics (septation of

mycelium, shape, diameter and texture of conidia). The identification at

species level was authenticated at “The First Fungal Bank of Pakistan”,

University of the Punjab, Lahore Pakistan. The pure fungal isolates comprised

of 13 fungal strains.

2.3.1.4 Screening of TSW-representative autochthonous fungi for HM

tolerance

The purified isolates of TSW-representative fungi resulting from 2.3.1.3

were counter-screened for HM tolerance. For this purpose, 2% MEA medium

was prepared by solubilizing 20 gm each of malt extract and agar per liter of

pre-autoclaved extract taken from TSW with distilled autoclaved water. For

each of the pure isolates from 2.3.3.1, the picking and sub-culturing on 2%

MEA meant for counter-screening was performed as described earlier in

21

2.3.1.2. The inoculated plates were incubated at 25°C for at least 7 days. As a

matter of HM tolerance, the growth of the sub-cultured fungal isolates was

estimated by measuring the radius of the colony extension (mm) against the

control (2% MEA medium prepared in distilled and autoclaved water). While

the tolerance index (TI) of TSW representative fungal isolates was determined

by obtaining the ratio of the extension radius of the treated colony

(supplemented with TSW extract) to that of the untreated colony

(supplemented with distilled autoclaved water). The fungal isolates resulting

from the counter-screening process were termed ‘autochthonous saprobic

fungi’ and selected for further experimentation. Four of the 13 pure fungal

isolates were screened for in vitro and in situ mutual interaction studies on the

basis of HM tolerance during the screening process.

2.3.2 In vitro and in situ mutual interaction studies for the

autochthonous saprobic fungi

2.3.2.1 In vitro mutual interaction studies for the autochthonous

saprobic fungi

The four selected isolates of autochthonous saprobic fungi isolated

from TSW were studied for their mutual competitive growth response in petri

plates using 2% MEA medium. For interactions study, petri plates were

labeled as control and experimental on either side of the vertical line crossing

at the center of the petri dish. Two different fungi were introduced opposite to

the vertical line crossing at the center of petri plate. They were incubated at

room temperature and observed every day until the experiment was over. The

combination of fungi competing for mutual growth was as under:

Trichoderma pseudokoningii vs Aspergillus niger (TS vs AN)

Aspergillus niger vs Fusarium sp. (AN vs F)

Aspergillus niger vs Alternaria alternata (AN vs AA)

Trichoderma pseudokoningii vs Fusaium sp. (TS vs F)

Trichoderma pseudokoningii vs Alternaria alternata (TS vs AA)

Fusarium sp. vs Alternaria alternata (F vs AA)

22

2.3.2.2 In situ mutual growth competition studies for autochthonous

saprobic fungi

Parallel to in vitro, the in situ study of the aforementioned combinations

of the autochthonous saprobic fungi was performed for their mutual interaction

with respect to influence on biomass production tendency of Tagetes patula L.

(a reported hyperaccumulator of HM) cultivated on garden soil (mentioned as

control in 2.2) in a pot trial.

2.3.2.2.1 Preparation of Fungal inoculum

The autochthonous saprobic fungal strains namely, Trichoderma

pseudokoningii Persoon ex Gray, Aspergillus niger Van Tieghem, Fusarium

sp. Link ex Gray, and Alternaria alternata Nees ex Wallroth isolated from the

tannery solid waste were used as inoculum in pot experiments. The

autochthonous saprobic fungal strains were cultured on 2% MEA medium.

Then a suspension solution of each fungus was prepared by taking 2-3 loops

full of actively growing young fungal colonies were taken in autoclaved

distilled water under aseptic conditions. After vigorous shaking at least for 15

minutes the inoculum was given through injection to the roots of plants.

Approximately 1.5ml (having colony forming units (CFU) 95,000/ml of

Trichoderma pseudokoningii, 75,000/ml of Aspergillus niger, 10,000/ml of

Fusarium sp and 26,000/ml of Alternaria alternata) was given to each

replicate of each treatment 3 times a week, after germination.

2.3.2.2.2 Pot trial set up

The experiment was set in a wire house having a transparent glass

roof (Fig. 2.3.1) in the Department of Botany, University of the Punjab, Lahore

Pakistan, in a “Completely Randomized Design”.

The experiment was set in medium regular 1.5 liter plastic pots each

filled with 1.5kg soil. The experiment comprised of 12 treatments of fungi with

four replicates each. The pots were watered regularly after every 2 days and

maintained at pot capacity. The combinations of different fungal isolates were

as under:

C = Control (without any inoculum)

23

F1 = Aspergillus niger

F2 = Trichoderma pseudokoningii

F3 = Fusarium sp.

F4 = Alternaria alternata

F1+F2 = Trichoderma pseudokoningii + Aspergillus niger

F1+F3 = Trichoderma pseudokoningii + Fusarium sp.

F1+F4 = Trichoderma pseudokoningii + Alternaria alternata

F2+F3 = Aspergillus niger + Fusarium sp.

F2+F4= Aspergillus niger + Alternaria alternata

F3+F4 = Fusarium sp. + Alternaria alternata

F1+F2+F3+F4 = Trichoderma pseudokoningii + Aspergillus niger + Fusarium

sp. + Alternaria alternata

Figure 2.3.1. A view of the experimental station where pot trials were carried out; the partially controlled conditions can be noted from the available type of wire house applied with glass roof and airy fairy sides being open to natural ambient temperature and moisture regimes.

24

Based on the performance of four pure autochthonous fungal isolates

during their in vitro and in situ interaction studies, two of them namely

Trichoderma pseudokoningii (TS) and Aspergillus niger (AN) were selected

for further experiments.

2.3.3 Verification of TS and AN isolates for HM tolerance by inducting in

phytoextraction with Tagetes patula

Prior to taking TS and AN as ‘ultimate screened fungi’, they were

tested in a phytoextraction pot trial with Tagetes patula for counter verification.

For this purpose, 0, 5, 10 and 20 % (w: w) TSW-Soil mixtures were used as

described in 2.2. Each pot filled with 1.5 kg of respective TSW-Soil mixture.

With three replicates each, one set of pots with said TSW-Soil ratio contained

autoclaved culturing media while the other set of pots was filled with non-

autoclaved mixture.

2.3.3.1 Fungal inoculation

In order to inoculate pots with the selected fungal isolates,

approximately 1.5 ml solution with colony forming units (CFU) 95,000/ml of

Trichoderma pseudokoningii and CFU 75,000/ml of Aspergillus niger was

applied to the rhizosphere of plants by syringe injection through plant

rhizosphere.

The experiment was set up in a wire house with a transparent glass

roof situated at the Department of Botany, University of the Punjab, Lahore

following a randomized complete block design with 96 experimental units (32

× 3). The experimental layout showing combinations of fungi (F) and TSW-

Soil are described in Table 2.1.

The pot capacity was maintained with tap water after every two days

and until the plants were harvested at seeding stage. The activity of the

inoculated fungi was evaluated by estimating their CFU from the soil.

25

Table 2.1.1. Layout for pot experiments showing selected mixtures of tannery solid waste with soil (TSW-Soil) either autoclaved (AS) or non-autoclaved (NAS) and fungi (F1: Aspergillus niger and F2: Trichoderma pseudokoningii) used for inoculation in soil.

Fungal Inoculation

TSW-Soil (% w:w) mixtures

Autoclaved soil (AS) Non-autoclaved soil (NAS)

0 5 10 20 0 5 10 20

C (Control with no

fungus)

0 % AS

with C

5 % AS

with C

10 % AS

with C

20 % AS

with C

0 % NAS

with C

5 % NAS

with C

10 % NAS

with C

20 % NAS

with C

F1 (Aspergillus

niger)

0 % AS with F1

5 % AS with F1

10 % AS with F1

20 % AS with F1

0 % NAS with F1

5 % NAS with F1

10 % NAS with F1

20 % NAS with F1

F2 (Trichoderma

pseudokoningii)

0 % AS with F2

5 % AS with F2

10 % AS with F2

20 % AS with F2

0 % NAS with F2

5 % NAS with F2

10 % NAS with F2

20 % NAS with F2

F1 + F2

0 % AS with

F1+F2

5 % AS with

F1+F2

10 % AS with

F1+F2

20 % AS with

F1+F2

0 % NAS with

F1+F2

5 % NAS with

F1+F2

10 % NAS with

F1+F2

20 % NAS with

F1+F2

2.3.4 Screening of TSW-tolerant plants for HM phytoextraction

For this purpose, a pot trial was set where plants of Helianthus annuus

L., Tagetes patula L., Petunia xhybrida (Hook) Vilm, Zinnia elegans (Jacq.)

and Dahlia coccinea (CAV.) were cultivated on 0, 5, 10, 20, 50 and 100% (w :

w) TSW-Soil mixtures with three replicates each. Each pot had been filled with

1.5 kg of TSW-Soil mixture. For this purpose, certified seeds of the plants

were sterilized with 10 % mercuric chloride solution.

The tolerant plant species Helianthus annuus and Tagetes patula were

selected for actual experiments on the basis of percentage germination. No

germination was observed beyond 20% TSW-soil mixture, due to which 5, 10

and 20% TSW were selected and all other TSW-Soil mixture ratios were

dropped for further experiments.

http://en.wikipedia.org/wiki/Jacq.

26

2.4 Pot trial phytoextraction studies based on ultimate

screened fungi and plants

The ultimate screened fungi and plants resulting from the extensive

screening process were procured together for their HM remediation potential.

The pot trials were conducted with Helianthus annuus and Tagetes patula

cultivated on screened TSW-Soil mixtures along with different combinations of

fungi such as, saprobic and arbuscular fungi as well as two saprobic fungi (TS

and AN). Further details are as under:

2.4.1 Pot trial I: Experiments with saprobic fungi

2.4.1.1 Experiments with saprobic TS and AN fungi

The effect of autochthonous fungi TS and AN on phytoextraction

potential of Tagetes patula as well as Helianthus annuus was studied by

setting a pot trial. The details of the combinations of two fungi TS and AN

were similar to the non-autoclaved part of what is described in 2.3.3.1. The

experiment was set in a randomized complete block design with 48

experimental units (16 × 3) for both Tagetes and Helianthus. Approximately

1.5ml solution with CFU 86,900/ml of Trichoderma pseudokoningii and CFU

69,800/ml of Aspergillus niger of the inoculum was applied in the rhizosphere

of the plants. The experiment was set in large sized earthen pots with glazed

inner wall and filled with 10 kg of the soil. The pots were watered after every

two days in order to maintain pot capacity until the plants were harvested. The

experiment was repeated thrice.

2.4.1.2 Live plant analyses-biochemical assays

For live plant analyses based on biochemical assessment of proline

and enzyme assays like superoxide dismutase (SOD) and catalase (CAT), the

plant leaves were plucked, washed with distilled water and immediately

placed in small cylinders filled with liquid nitrogen.

2.4.1.2.1Chlorophyll contents-SPAD values

Chlorophyll content of the plants was determined using a chlorophyll

meter (Model SPAD-502 Singapore). For this purpose, SPAD readings were

taken by selecting 15 plants at random per treatment.

27

2.4.1.2.2 Estimation of protein

The Biuret method of Racusen and Johnstone (1961) was adopted for

the estimation of soluble protein content. The reaction mixture consisted of

2.0 mL of Biuret reagent (3.8 g CuSO4⋅5H2O, 1.0 g KI, 6.7 g Na-EDTA, 200

mL 5 N NaOH in 1,000 mL of solution) and 0.1 mL of supernatant. The control

consisted of 0.1 mL of distilled water instead of supernatant. The optical

density was measured at 545 nm using a Hitachi U-1100 spectrophotometer.

The amount of protein was calculated from a standard curve of known protein

concentrations, which was prepared from bovine serum albumin.

2.4.1.2.3 Estimation of catalase

The catalase (E.C 1.11.1.6) activity was assayed according to Beers

and Sizer (1952) with certain modifications. The reaction was carried out

using two buffer solutions (A and B). Buffer A consisted of 50 mM potassium

phosphate (pH7.0), while buffer B was 0.036% H2O2 solution in 50 mM

potassium phosphate buffer (pH7.0). The reaction mixture consisted of 2.9 mL

buffer B and 0.1 mL of enzyme extract, while control consisted of only 3.0 mL

of buffer A. The enzyme activity was measured by time required for the

absorbance (at 240 nm) to decrease from 0.45 to 0.40 and expressed as units

per milliliter of enzyme.

2.4.1.2.4 Estimation of superoxide dismutase

The superoxide dismutase (SOD; E.C 1.15.1.1) activity was assayed

spectrophotometrically by measuring its ability to inhibit photochemical

reduction of nitroblue tetrazolium (NBT) according to Maral et al. (1977). Two

tubes were taken, each containing 2.0 mL of 1.0 mM sodium cyanide (NaCN),

13 mM methionine, 75 μM NBT, 0.1 mM EDTA, and 2.0 μM riboflavin as a

substrate. One tube was used as sample containing reaction mixture + 5.0 μL

enzyme extract, placed approximately 30 cm below the bank of two 30-W

fluorescent tubes for 15 min. The other tube containing reaction mixture

without enzyme extract was covered with black cloth at the same time. The

absorbance of the illuminated tube was compared to non-illuminated mixture

at 560 nm. SOD activity was expressed as units per milligram of protein.

28

2.4.1.3 Post-harvest analyses

The TSW-Soil mixtures and plants after harvesting were analyzed in

order to compare pre- and post-harvest trends on the basis of selected

parameters.

2.4.1.3.1 Post-harvest soil analyses

The TSW-Soil mixture from the pots after harvesting plants was

digested by the method given in 2.2.6 to assess the level of category-I as well

as category-II metals.

2.4.1.3.2 Post-harvest plant analyses

2.4.1.3.2.1 Morphological parameters and biomass yield

Different morphological parameters like, shoot length (cm), root length

(cm), seedling length (cm), no. of leaves and roots, fresh and dry weight (g)

as well as the plants were harvested, washed with deionized water and

wrapped in blotting paper for oven drying.

2.4.1.3.2.2 Digestion of plant material and HM estimation

The harvested biomass from pot trial was separated into root and

shoot; oven dried and digested after the method given by Huang and Schulte

(1985). The level of (mgkg-1) category-I as well as category-II metals

accumulation in different plant parts was assessed.

2.4.1.3.3 Post-harvest fungal analyses

Parallel to post-harvest physico-chemical analyses of the TSW-Soil

mixtures, the inoculated fungal CFUs (× 105 cfu g-1 soil) as well as no. of

spores (50 gm-1 soil) were determined.

2.4.1.3.4 Meta-analytical assays

The meta-analytical perspective of the proposed phytoextraction

technology assisted with fungi was developed on the basis of the following

parameters.

29

2.4.1.3.4.1 HM translocation index (%)

The HM translocation index (%) as given by Mattina et al., 2003 is used

to determine the efficiency of a plant to translocate HM from root to aerial

parts. The formula used is as under:

2.4.1.3.4.2 HM tolerance index (TI)

The TI value for different HM as given by Wilkins (1978) gives the ratio

of biomass yielded in TSW-Soil mixture to biomass yielded in soil (Control). Its

formula is given below:

2.4.1.3.4.3 Specific HM extraction yield percentage (SEY %)

The SEY (%) as explained by Audet and Charest (2006) represents

percent ratio of plant HM content to soil HM concentration and its formula is

given as under:

2.4.2 Pot trial II: Experiments with both saprobic and arbuscular

mycorrhizal (AM) fungi

The details of this pot experiment were similar to pot trial mentioned in

2.4.1 including live plant analyses, post-harvest and meta-analytical analyses

except for post-harvest fungal analyses in 2.4.1.2.3 where a new parameter of

percentage infection of AM fungi in roots (%) was incorporated. The AM fungi

were inoculated as described below:

2.4.2.1 Fungal inoculum

In order to inoculate TSW-Soil mixtures with AM fungi, a uniform layer

of about 15 gm fresh roots of Cynodon dactylon Pers., naturally infected with

AM fungi, collected from Botanical Garden, Dept. of Botany University of the

Punjab; were applied in pots 3 inches below the soil surface. The applied

grass roots were confirmed microscopically for AM infection prior to

inoculation in pots. The saprobic TS was applied in the form of conidial

30

suspension. For this purpose, TS was grown in 500-ml conical flasks

containing potato dextrose broth for 8 days. The cultures were filtered through

Whatman no. 1 filter paper and the mycelial mat was macerated using a

waring blender for 1 min and mixed with 250 ml of 0.1 M MgSO4.7H2O

solution. Ten ml of this inoculum containing 5 x 104 CFU per ml was used for

inoculating each pot.

2.4.2.2 Mycorrhizal and fungal assessment

The mycorrhizal growth was expressed in terms of extent of infection

while the activity of the saprobic fungus was estimated by the CFU from the

soil. For this purpose, fresh root samples were stained using 0.05% Trypan

Blue as described by Phillips and Hayman (1970) and the percent root

colonization was estimated by adopting the grid-line intersect method of

Giovanetti and Mosse (1980). Extramatrical chlamydospores in root-zone soil

samples were enumerated using the wet sieving and decanting method of

Gerdemann and Nicolson (1963). The population of TS in the root zone soil

was determined by using 2% MEA.

31

2.5 Evaluation of pot-trial findings at field level

For field trials, the selected site was the same from where garden soil

was taken for preparing TSW-Soil mixtures for pot trials. For this purpose, Plot

# 129 of the Botanical Garden, Dept. of Botany University of the Punjab

Lahore, Pakistan was selected (31O49’96.34’’ N, 74O30’08.53’’E).

2.5.1 Field plotting

2.5.1.1 Strip field plotting

Five strip plots (25 × 3 feet each) were prepared by digging and removing

soil up to 2.5 feet depth. There was a soil barrier of 2 feet width between each

of the strip plot (Figure 2.5.1 and 2.5.2). Four of the 5 ditches resulting after

removal of soil were lined with polythene sheet. The soil removed from all of

the ditches was air-dried by spreading it on plastic sheets separately to bring

its moisture level equal to the powdered TSW. One of the ditches with lining

and one without lining were filled back with relevant soil removed from them

without incorporating any TSW taken as Control. One of the ditches was not

lined with geothermal membrane in order to find any effect of lining of the plot

on plant growth. The remaining three ditches were filled back with their

respective soil after mixing appropriate ratios of TSW i.e. 5, 10 and 20% (w :

w) in order to prepare TSW-Soil mixtures similar to the soil mixtures prepared

for pot trials as described earlier in 2.2.

2.5.1.2 Sub-division of the strip plots for fungal inoculation

In order to apply fungal inoculations in strip plots following randomized

complete block design (RCBD), each of the strip plots was sub-divided into

four sub-plots (5 × 3 feet each separated by a soil barrier of 1.25 feet)

categorized as Control with no fungal inoculation, F1, F2 and F1 + F2, as

shown in Figure 2.5.1 and 2.5.2. An individual network of drip irrigation pipes

was applied in each of the sub-plots at about 3 inches depth of the soil

surface for injecting solution of fungal CFU.

The soil mixtures in all field plots were allowed to equate for 30 days.

During that period, there was neither any precipitation nor any pest attack

observed on plants growing in the vicinity of strip plots.

32

Figure 2.5.1. The statistical layout of the experimental units showing distribution of field plots lined with polythene sheet (upper). The vertical grey lines show the separation between experimental plots (25 × 3 feet each) arranged in split plot design with no replication having soil mixed with tannery solid waste (TSW-Soil % w:w) while the horizontal dark black lines show division of each strip plot into sub-plots (5 × 3 feet each) applied with fungal inoculations in a randomized complete block design. The aerial view of experimental station (lower).

Fun

gal i

no

cula

tio

ns

20 10 5 0 0*

TSW-Soil (% w:w) mixtures

0* = experimental plot with only soil as well as without polythene sheet while 0 is similar except being applied with polythene sheet as in the case of other TSW-Soil mixtures.

C = neither of the fungal inoculations applied

F1 = Aspergillus niger

F2 = Trichoderma pseudokoningii

F1 + F2 = one applied with both fungi together

The physical separation between experimental plots

N

33

Figure 2.5.2: Preparation of field plots in order to apply pot trial findings at field level: (A) excavation of soil from strip plots (25 × 3 feet each) up to 2.5 feet depth and lining with polythene sheet; (B) mixing of soil with 2 mm sieved tannery solid waste (TSW); (C) refilling of excavated strip plots with soil mixed with different percentages of TSW; (D) a strip plot refilled with TSW mixed soil; (E) the vertical orientation of 5 strip plots; (F) the sub-plotting of each of the strip plot into four experimental units each for fungal inoculations in a randomized complete block design.

A

E

B

C D

E F

34

2.5.2 Field Trial I: Experiments with Helianthus annuus

After 30 days of equating the field soils, certified seeds of Helianthus

annuus were sown during local peak crop season i.e. last week of Jan 2011.

The field capacity was maintained by irrigating the field with tap water

according to the agricultural recommendations about sunflower crop.

2.5.2.1 Fungal inoculations

When the seedlings were 2 weeks older, approximately 25 liter solution

containing CFU 76,800/ml of TS and CFU 73,800/ml of AN of the inoculum

were applied in each of the sub-plots by injecting through network of drip

irrigation pipes lined at about 3 inches depth of the soil surface.

2.5.2.2 Live plant analysis-Biochemical assays

The detail of live plant analyses based on biochemical assays SPAD

values were similar to the methods given in 2.4.1.2.

2.5.2.3 Harvesting

The sunflower plants were harvested after the sunflower petals started to

wither off and the seeds were mature. The harvested aerial biomass i.e.

leaves, shoots and roots were collected separately in zip-loc bags for lab

analysis after observing morphological parameters and fresh weight. All of the

roots were collected carefully in a 2 mm sieve and washed with distilled water

before blotting and placing in zip-loc bags. The residual water resulting from

washing was returned back to their respective sub-plot altogether in order to

remove experimental error.

2.5.2.4 Post-harvest analyses

The detail of post-harvest analyses was similar to the descriptions

given in 2.4.1.3.

35

2.5.3 Field Trial II: Experiments with Tagetes patula

The certified seeds of Tagetes were sown during its local peak crop

season i.e. last week of Oct 2011. The rest of the details with regard to pre-

and post-harvest analyses was similar to experiment with sunflower as

described in 2.5.2.

2.6 Phytoextraction studies assisted with Trichoderma

harzianum (TH) and TS in a pot trial at Cornell University,

New York USA

The experiment was set in greenhouse at Cornell University in 2012 as

part of PhD research work. The visit was funded by Higher Education

Commission (HEC) Pakistan under International Research Support Initiative

Program (IRSIP). The greenhouse pot trial was aimed at studying effect of

saprobic Trichoderma harzianum (TH) and Trichoderma pseudokoningii (TS)

on phytoextraction potential of Tagetes patula using three TSW-soil mixtures

i-e 0, 5, and 10%. The TH was provided by Dr. Gary E. Harman, Professor at

Dept. of Horticulture, Cornell University Geneva campus; while TS isolates

were similar to those used in the research work done in Pakistan.

2.7 Statistical analyses

The level of significance among treatments was determined using two-

way analysis of variance (ANOVA). In order to compare treatment means,

least significant difference (LSD) test was applied, while to pin-point exact

difference among group of means, Duncan’s multiple range test (DMRT) was

applied. The SPSS software version 11.3 and statistical power package of the

Microsoft Office Excel 2010 was used for all the statistical analyses.

Chapter 3

Results

36

CHAPTER 3

RESULTS

3.1 Soil and TSW analyses

3.1.1 Physico-chemical properties of TSW and garden soil

The physico-chemical properties of TSW, garden soil and their

selected mixtures are given in Table 3.1. The pH of the TSW was significantly

higher (8.9) as compared to garden soil (7.1). On spiking with TSW, the pH of

garden soil start increasing and it reached as high as 8.1 at 20 %. The TSW-

Soil mixtures beyond 20 % were not carried along for further experimentation

and one of the reasons was such a high pH that didn’t let the seeds of

selected plants to germinate. The values of ECe (dScm-1), bicarbonates (mgL-

1) as well as chlorides (mgL-1) were found to be the maximum in TSW and

were significantly higher as compared to garden soil. The ECe increased

significantly in each of the TSW-Soil mixture with increasing amount of TSW

and showing a increasing trend from 0 (garden soil only) to 5, 10 and 20%

mixtures. Similar trends were found for both NaCl (%) and bicarbonates (mgL-

1). The carbonates (mgL-1) were observed to be BDL in both garden soil and

TSW. The bulk density (gcm-3) of TSW was extremely low as compared to soil

and spiking soil with TSW decreased its bulk density. However, organic

matter (OM %) in TSW was significantly higher as compared to garden soil

and blending of soil added OM increments to the soils, as given in Table

3.1.1.

37

Table 3.1.1. The physico-chemical properties of TSW, garden soil and their various (% w:w TSW-Soil) mixtures

Parameters

TSW-Soil mixtures (% w:w)

LSD0.05 0

(Soil only)

5 10 20 50 100

(TSW only)

pH 7.1bc

7.4c 8.0

b 8.1

ab 8.3

ab 8.9

a 0.515

ECe (dScm-1

) 0.02f 0.12

e 0.31

d 0.81

c 1.42

b 2.89

a 0.053

NaCl (%) 2.9e 3.5

e 52.3

d 125.7

c 255.1

b 421.5

a 0.68

Bicarbonates (mgL-

1)

103.7f 125.3

e 152.5

d 189.1

c 262.3

b 359.9

a 1.279

Carbonates (mgL-1

) ND ND ND ND ND ND -

Chlorides (mgL-1

) 62.1ef 76.8

e 255

d 812

c 2,233

b 3,118

a 17.79

Organic matter (%) 2.61c 2.90

bc 3.1

b 3.4

b 3.8

ab 4.5

a 0.657

Bulk Density (gcm-

3)

1.06a 1.04

a 1.02

ab 0.92

b 0.73

c 0.66

d 0.10

The mean values S.D. with common letters (along the row) are not significantly different according to Duncan’s multiple range test (P = 0.05). LSD = Least Significant Difference (P = 0.05)

3.1.2 Category-I metals in TSW and garden soil

The TSW exhibited the maximum concentration of Category-I metals

(detected with flame photometer) i.e. Ca, K and Na; and it was significantly

higher as compared to garden soil, as shown in Table 3.1.2. Blending soil with

TSW elevated the concentration of all the three metals in different TSW-Soil

mixtures. Such a trend of variation was observed for not only the total metal

fraction of Ca, K and Na, but also for the water soluble and DTPA-extractable

part. The Ca found to be the maximum in TSW, being significantly highest

than soil and its mixtures with TSW. Similarly, K and Na were also found to be

the maximum in TSW and significantly highest than any of the soil treatments.

38

Table 3.1.2. The total, water soluble and DTPA-extractable fraction of Category-I metals (mgkg-1

) in TSW, garden soil and their various (% w:w TSW-Soil) mixtures

Category-I metal fraction


LSD0.05 0

(Soil only)

5 10 20 50 100

(TSW only)

Water soluble (mgkg

-1)

Ca 25

e ±

4.08 135

de ±

10.61 190

d ±

16.33 255

c ±

36.74 425

b ±

36.74 940

a ±

40.83 64.84

K 90

ef ±

16.33 125

e ±

12.25 235

d ±

20.41 410

c ±

40.83 690

b ±

40.83 1,105

a ±

85.73 100.83

Na 15

f ±

4.1 35

e ± 4.1 90

d ± 9.8

280b ±

7.4 120

c ± 7.4 890

a ± 13 18.98

DTPA-extractable

(mgkg-1

)

Ca 55

f ±

8.17 450

e ±

32.66 660

d ±

65.32 830

c ±

32.66 990

b ±

65.32 1,240

a ±

81.65 123.94

K 210

e ±

16.33 380

d ±

40.83 545

cd ±

44.9 675

c ±

28.6 1,040

b ±

106.14 1,780

a ±

73.5 137.55

Na 250

f ±

12.2 995

e ±

11.4 1,270

d ±

114.3 1,920

c ±

40.9 2,130

b ±

42 2,685

a ±

61.3 134.3

Total (mgkg

-1)

Ca 95

f ±

8.17 1,625

e ±

20.41 2,345

d ±

110.23 2,910

c ±

89.82 3,420

b ±

179.83 6,320

a ±

97.98 235.02

K 664

e ±

11.4 890

e ±

65.3 1,210

d ±

16.3 1,980

c ±

57.2 2,340

b ±

81.7 4,210

a ±

49 122.5

Na 995

f ±

81.7 1,355

e ±

61.2 2,515

d ±

20.4 4,645

c ±

53.1 6,350

b ±

45 9,440

a ±

57.2 129.29

The mean values S.D. with common letters (along the row) are not significantly different according to Duncan’s multiple range test (P = 0.05). LSD = Least Significant Difference (P =

0.05)

3.1.3 Category-II metals in TSW and garden soil

Likewise, the concentration of Category-II (AAS detected) metals in

TSW was found to be the maximum and significantly highest than soil and its

selected mixtures with TSW. Being a true representative of the cumulative

chrome tanning waste, the TSW showed extremely high concentration of Cr

and it was highly significantly greater than Cr in garden soil, as shown in

Table 3.1.3.

39

Table 3.1.3. The total, water soluble and DTPA-extractable fraction of Category-II metals (mgkg

-1) in TSW, garden soil and their various (% w:w TSW-Soil) mixtures

Category-II metal fraction


LSD0.05 0

(Soil only)

5 10 20 50 100

(TSW only)

Water soluble (mgkg

-1)

Cd 15

f ±

2.5 490

e ±

16.3 610

d ±

12.3 790

c ±

16.3 935

b ±

19.6 2,750

a ±

49 55.4

Cr BDL 1,350

e ±

120 1,970

d ±

52.3 2,250

c ±

50.6 3,450

b ±

65.3 4,570

a ±

106 176.4

Cu 110

e

± 7.3 235

d ±

11.4 580

c ±

21.2 965

c ± 13

1,160b ±

21.2 2,260

a ±

55.5 62

Fe 15

e ±

2.5 70

d ±

10.6 95

cd ±

11.4 115

c ± 13

225b ±

19.6 570

a ±

23.7 34.7

Mg 10

ef ±

3 35

e ±

3.1 65

d ± 7.4 95

c ± 14

270b ±

15.5 630

a ±

19.6 28.1

Ni BDL BDL BDL BDL BDL BDL -

Pb BDL BDL BDL BDL BDL BDL -

Zn 15

f ±

3.8 45

e ±

5.6 85

d ± 7

130c ±

7.3 190

b ± 4

335a ±

11.4 16.2

DTPA-Extractable

(mgkg-1)

Cd 35

f ±

3.3 1,460

e ±

13.9 3,780

d ±

69.4 3,920

c ±

18 4,230

b ±

33.5 5,030

a ±

81.7 107.7

Cr 55

f ±

4.9 3,560

e ±

62.1 4,350

d ±

41.7 5,450

c ±

44.9 6,750

b ±

68.6 9,050

a ±

49.1 114.4

Cu 235

f ±

12.2 780

e ±

22.8 1,350

d ±

49.8 1,790

c ±

59.6 2,750

b ±

53 5,640

a ±

196 205.7

Fe 40

f ±

6.5 120

e ±

6.9 230

d ±

13.9 640

c ±

7.4 870

b ±

14.7 1,030

a ±

25.3 32.5

Mg 30

f ±

7.2 150

e ±

8.7 340

d ±

22.8 540

c ±

23.3 1,240

b ±

24 1,930

a ±

25.9 46.4

Ni BDL BDL 25d ± 1.4 55

c ± 8.3

110b ±

11.2 370

a ±

13.6 18.7


Zn 110

e

± 10.6 270

de ±

9 325

d ±

18.9 450

c ±

29.4 960

b ±

33.5 1,035

a ±

37.6 59

Total (mgkg

-1)

Cd 55

f ±

5.7 2,650

e ±

53.1 6,580

d ±

81.7 8,750

c ±

57.2 9,720

b ±

49 10,097

a ±

87.4 142

Cr 110

e

± 7.4 8,250

d ±

48.9 10,250

cd ±

163 15,510

c ±

155 19,520

b ±

139 25,534

a ±

191 310.2

Cu 600

f ±

44 1,350

e ±

57.1 2,100

d ±

8.2 5,250

c ±

57.2 7,510

b ±

81.6 10,554

a ±

125.7 165.5

Fe 50

f ±

6.7 250

e ±

13 510

d ±

28.6 910

c ± 18

1,230b ±

25.3 2,250

a ±

40 56.4

Mg 45

f ±

6.1 310

e ±

21 620

d ± 79

1,020c ±

89 2,870

b ±

101 3,840

a ±

118 196

Ni BDL 35

de ±

1.3 55

d ± 10.7

110c ±

9.2 310

b ±

19.6 590

a ±

20.4 39.8


Zn 218

e

± 16.3 1460

d ±

17.1 1890

c ±

19.6 2025

b ±

25.3 5,150

ab ±

57.2 7,590

a ±

15.5 67


0.05)

Similarly, the concentration of Cd and Cu were found to be the maximum in

for the water soluble, DTPA-extractable as well as total metal fraction of the

Category-II metals present in the TSW. The mixing of garden soil with TSW

40

had incremented Cr, Cd and Cu in accordance with increasing percentage of

TSW. The water soluble fraction of Fe and Mg found to be the lowest in TSW,

soil and their various mixtures, followed by DTPA-extractable; however, their

total metal fraction being the highest. The Pb found to be BDL in all the TSW-

Soil mixtures. The water soluble fraction of Ni was also BDL. However,

considerable concentrations of DTPA-extractable and total fraction of Ni were

noticed to be present. As far as Zn is concerned, its total metal fraction was

highest, followed by DTPA-extractable and water soluble fraction, respectively

for TSW, soil and their various mixtures.

41

3.2 Isolation and identification of TSW representative fungi

Using three different fungal nutrient media i.e. 2% MEA, Czapek’s and

MMN medium, 13 autochthonous isolates were purified and confirmed on the

basis of the colony morphology after verification from “The First Fungal Bank

of Pakistan”, University of the Punjab, Lahore Pakistan. There were as many

as nine species of Aspergillus, one species of each of Alternaria, Fusarium,

Rhizopus and Trichoderma. The figure 3.2.1 – 3.2.13 are given to show the

colony morphology of the pure isolates used for further experimentation:

Figure 3.2.1. Alternaria alternata on Czapek’s medium at

250C isolated from TSW.

Figure 3.2.2. Aspergillus flavipus on 2% MEA medium at 25

0C.

Figure 3.2.3. Aspergillus fumigatus on Czapek’s medium

at 250C

Figure 3.2.4. Aspergillus parasiticus on Czapek’s medium at 25

0C

Figure 3.2.5. Aspergillus terreus on MMN medium at 25

0C isolated from TSW

Figure 3.2.6. Fusarium sp. on Czapek’s medium at 25


42

Figure 3.2.7. Rhizopus arrhizus on 2% MEA basal

medium at 250C isolated from TSW.

Figure 3.2.8. Trichoderma pseudokoningii on Czapek’s medium at 25


Figure 3.2.9. Aspergillus flavus on Czapek’s medium at


Figure 3.2.10. Aspergillus japonicus on 2% MEA basal medium at 25


Figure 3.2.11. Aspergillus niger on MMN medium at 25

0C

isolated from TSW. Figure 3.2.12. Aspergillus penicilloides on Czapek’s

medium at 250C isolated from TSW.

Figure 3.2.13. Aspergillus versicolor on 2% MEA medium at 25


43

3.3 Screening and selection of heavy metal resistant autochthonous

fungi

During screening process of autochthonous fungi on 2 % MEA

prepared in autoclaved extract of TSW, the isolates of Trichoderma

pseudokoningii and Aspergillus niger found to be the best in terms of

tolerance index (TI) as compared to their respective control i.e. isolates of

Trichoderma and Aspergillus cultivated on 2 % MEA prepared in distilled

autoclaved water respectively, as given in Table 3.3.1. The TI value for

Alternaria alternata and Fusarium sp. found to be third and fourth respectively,

among all of the 13 tested fungi. The TI gave an indication of a fungus’

tendency to resist colony radius delimitation caused by the very high level of

heavy metals incorporated in 2 % MEA through autoclaved MSW extract. The

order of heavy metal tolerance of fungi on the basis of TI values observed to

be, Trichoderma pseudokoningii > Aspergillus niger > Alternaria alternata >

Fusarium sp.

Table 3.3.1. The tolerance index (TI) of various fungi cultivated on 2 % MEA prepared in autoclaved extract of TSW along with control of each of the fungus cultivated on 2 % MEA prepared in distilled autoclaved water.

Sr # Fungi Tolerance Index (TI)

1 Alternaria alternata 0.93ab

2 Aspergillus flavipus 0.32

g

3 Aspergillus fumigatus 0.78c

4 Aspergillus Parasiticus 0.21h

5 Aspergillus terreus 0.28gh

6 Fusarium spp 0.89

b

7 Rhizopus arrhizus 0.49ef

8 Trichoderma pseudokoningii 0.97a

9 Aspergillus flavus 0.46ef

10 Aspergillus japonicas 0.69d

11 Aspergillus niger 0.96a

12 Aspergillus penicilloides 0.42f

13 Aspergillus versicolor 0.51e

The values with different letters are significantly different as per Duncan’s multiple range test (n = 3; P = 0.05). The screening experiment repeated thrice.

From the 13 fungi used in screening process, four of them with highest

TI value were selected for further mutual fungal interaction study while

withdrawing rest of them for further experimentation.

44

3.4 In vitro fungal mutual growth interaction studies for Category-II metal

tolerance

Analogous to a tug of war, the two fungi inoculated on opposite sides of

the vertical line crossing the petri plat at its center, tried to overwhelm each

other in terms of radial growth of their respective colony on from point of

origin, across the central vertical line to the opposite fungus’ point of origin.

The relative superseding by one fungus over its counterpart was evaluated on

the basis of radial growth in mutual combination with respect to individual

growth rate (taken as control), maximum colony diameter (grown along with

partner and individually), and mycelial growth towards and away from the

other colony, as given in Table 3.4.1.

3.4.1 Aspergillus niger vs. Trichoderma pseudokoningii

The radial growth area covered by A. niger receded as compared to

radial colony expansion exhibited by T. pseudokoningii across the vertical line

at the center of petriplate to quite near the point of origin of A. niger as can be

observed in Figure 3.4.1. The T. pseudokoningii growth was approximately 65

% while for A. niger it observed to be approximately 35 % of the total area. As

compared to their respective controls, both of the competitor fungi displayed

relatively less growth in mutual interaction plate. Conclusively, T.

pseudokoningii was selected over its counterpart for further study.

Figure 3.4.1. In vitro mutual growth interaction (center) between screened heavy metal tolerant Aspergillus niger (right) vs. Trichoderma pseudokoningii (left)

A. niger (Control) A. niger vs. T. pseudokoningii T. pseudokoningii (Control)

45

3.4.2 Alternaria alternata vs. Trichoderma pseudokoningii

The growth area covered by A. alternata and T. pseudokoningii was

not equal with respect to one another. The T. pseudokoningii overlapped its

opponent A. alternata and suppressed its growth as much as approximately

70 % while limiting A. alternata to as low as approximately 30% of the total

area of the petriplate, as shown in Figure 3.4.2. Conclusively, T.

pseudokoningii found to be better over A. alternata.

Figure 3.4.2. In vitro mutual growth interaction (center) between screened heavy metal tolerant Alternaria alternata (left) vs. Trichoderma pseudokoningii (right)

A. alternata (Control) A. alternata vs. T. pseudokoningii T. pseudokoningii (Control)

3.4.3 Fusarium sp. vs. Alternaria alternata

During this paired mutual fungal interaction study, the Fusarium sp.

displayed growth recession in terms of radial growth as compared to

Alternaria alternata, as given in Figure 3.4.3. The latter autochthonous

saprobic fungal strain overlapped its counterpart up to 80 % and supressed

growth of Fusarium sp. limiting it to only 20 % of the total petriplate area.

Conclusively, A. alternata prevailed over Fusarium sp.

Figure 3.4.3. In vitro mutual growth interaction (center) between screened heavy metal tolerant Fusarium sp. (left) vs. Alternaria alternata (right)

Fusarium sp. (Control) Fusarium sp. vs. A. alternata A. alternata (Control)

46

3.4.4 Aspergillus niger vs. Alternaria alternata

The growth area covered by A. niger was overwhelmingly greater than

A. alternata i.e. the whole petriplate was overlaid by A. niger growth,

suppressing underlying A. alternata, as can be seen in Figure 3.4.4. In the

control of each autochthonous saprobic fungal strain, the growth area was

nearly equal.

Figure 3.4.4. In vitro mutual growth interaction (center) between screened heavy metal tolerant Aspergillus niger (left) vs. Alternaria alternata (right)

A. niger (Control) A. niger vs. A. alternata A. alternata (Control)

3.4.5 Fusarium sp. vs. Trichoderma pseudokoningii

During the mutual growth interaction of Fusarium sp. and T.

pseudokoningii, it was observed T. pseudokoningii overlapped Fusarium sp.

The growth area covered by T. pseudokoningii was approximately 80 % as

compared to Fusarium sp. which covered approximately 20 % of the total

growth area of petriplate as shown in Figure 3.4.5. The rate of colony

expansion resulting from radial growth observed to be extremely quick in

Trichoderma pseudokoningii as compared to Fusarium sp.

Figure 3.4.5. In vitro mutual growth interaction (center) between screened heavy metal tolerant Fusarium sp. (left) vs. Trichoderma pseudokoningii (right)

Fusarium sp. (Control) Fusarium vs. T. pseudokoningii

T. pseudokoningii (Control)

47

In case of Control, both of the mutually interacting fungi displayed

nearly equal growth, as can be seen in the Figure 3.4.5. Conclusively, T.

pseudokoningii observed to be dominant over Fusarium sp. in terms of in vitro

mutual competitive growth.

3.4.6 Fusarium sp. vs. Aspergillus niger

In case of interaction between Fusarium sp. and A. niger, growth area

covered by the latter fungus was about 80 % of the total petri plate area while

the former receding itself to an area of as low as 20 %, as can be observed in

Figure 3.4.6. In case of Control; however, both of the fungi displayed healthy

radial growth. Conclusively, A. niger found to be far better than Fusarium sp.

in terms of in vitro competitive growth.

Figure 3.4.6. In vitro mutual growth interaction (center) between screened heavy metal tolerant Fusarium sp. (left) vs. Aspergillus niger (right)

Fusarium sp. (Control) Fusarium sp. vs. A. niger A. niger (Control)

3.4.7 Comparison of the six paired mutual interactions of screened fungi

The comparison of the six pairs of fungi mutually interacting for

competitive growth under in vitro conditions is given in Table 3.4.1.

Table 3.4.1. Comparison of six pairs of mutually interacting fungi for growth competition on the basis of various morphological parameters observed after 10 days of fungal inoculation.

Pair # Mutually

interacting fungi

Parameters

Colony diameter

(cm)

Relative growth area

(%)

Direction of mutual mycelial growth

(proceed/recede)

Colony morphology (color)

Pair I A. niger vs.

T. pseudokoningii 4 35 Proceed black 4 65 Proceed green

Pair II A. alternata vs. T.

pseudokoningii 3 30 Recede black 5 70 Proceed green

Pair III Fusarium sp. vs.

A. alternata 1 20 Recede white 3 80 Proceed black

Pair IV A. niger vs. A.

alternata 5 100 Proceed black 0 0 Recede black

Pair V Fusarium sp. vs. T.

pseudokoningii 1 20 Recede white 4 80 Proceed green

Pair VI Fusarium sp. vs.

A. niger 1 20 Recede white 4 80 Proceed balck

48

The order of dominance displayed by fungi on the basis of relative

growth area (%) was as: T. pseudokoningii = A. niger > A. alternata >

Fusarium sp.

Consequently, T. pseudokoningii and A. niger were found to be the

best fungi for Category-II metal tolerance after the screening process of metal

tolerance and mutual fungal interaction. Both of them were selected for the

forthcoming pot and field trial aimed at using these fungi as bio-reinforcers for

increasing the biomass production of marigold plant.

49

3.5 In situ mutual growth interaction studies for screened Category-II

metal tolerant fungal isolates with Tagetes patula in soil

On inoculating soil with four fungal isolates either individually or in

combinations, the T. patula responded variably in relation to the type and

number of fungi applied in the pot soil. The plants with F1 + F2 inoculations

yielded maximum root-, shoot- and seedling length; fresh and dry weight; as

well as chlorophyll content (SPAD value), as given in Table 3.5.1. The relative

variation in vegetative growth can also be observed in Figure 3.5.1.

The F1 treatment gave the second highest values for all the vegetative

growth parameters, while F2 treatment being the third among all the fungal

Table 3.5.1. Morphological and biochemical parameters for 50-days old pot cultivated plants of Tagetes

patula cultivated in soil and applied with individual and combined fungal inoculations. The mean values ± SD with different letters are significantly different according to Duncan’s multiple range test (n = 3; P =

0.05).

Treatments Root

length (cm)

Shoot length (cm)

Seedling length (cm)

Chlorophyll content

Fresh weight (g)

Dry weight (g)

Control 14.9

bc ±

1.36 15.7

b ± 2.04 31.5

bc ± 2.04 13.1

b ± 2.53 4.9

b ± 1.14

1.47b ±

0.14

F1 17.2b ± 0.49 16.1

b ± 0.98 33.6

b ± 2.12 15.9

ab ± 2.44 5.6

ab ± 1.31 1.74

ab ± 0.2

F2 16.1b ± 0.9 14.8

b ± 1.47 31.2

bc ± 1.8 14.5

ab ± 2.85 4.7

b ± 1.39

1.41b ±

0.25

F3 6.2cd

± 0.65 4.1e ± 0.65 10.7

ef ± 1.39 9.9

bc ± 2.37 2.1

c ± 0.33

0.63cd

± 0.11

F4 5.5ef

± 0.89 6.4de

± 1.41 12.1e ± 0.89 10.7

b ± 2.20 2.2

c ± 0.49

0.68cd

± 0.15

F1+F2 34.6a ± 1.88 23.3

a ± 1.96 58.1

a ± 1.71 19.5

a ± 2.86 7.2

a ± 1.39

2.16a ±

0.13

F1+F3 10.4d ± 1.31 12.4

c ± 1.14 23

cd ± 2.44 17.3

a ± 1.04 3.4

bc ± 0.32

1.05bc

± 0.12

F1+ F4 14.1c ± 2.45 11.1

c ± 1.72 25.3

c ± 2.69 16.6

ab ± 2.12 4.6

b ± 1.3

1.43b ±

0.26

F2+ F3 9.8d ± 1.63 10.9

c ± 1.55 21.3

cd ± 1.88 9.3

bc ± 1.88 3.2

bc ± 0.57

0.96c ±

0.32

F2+ F4 7.1e ± 0.98

10.6cd

± 1.31

18d ± 1.63 10.2

bc ± 1.79 2.3

c ± 0.73

0.69cd

± 0.16

F3+F4 5.3ef

± 0.73 7.5d ± 1.22 13

e ± 2.44 7.1

c ± 1.71 2.1

c ± 0.41

0.65cd

± 0.12

F1+F2+F3+F4 7.4e ± 1.22 8.2

d ± 0.98 15.9

d ± 2.37 11.9

b ± 2.37 2.4

c ± 0.33 0.74

cd ± 0.2

LSD0.05 3.04 3.13 4.42 4.92 2.02 0.43


0.05)

50

inoculations. Surprisingly, increasing number of isolates in the fungal

inoculant decreased the values of all the parameters, as can be seen in case

of F1 + F2 + F3 + F4 treatment, given in Table 3.5.1.

On the basis of the maximum biomass yield incurred by individual as

well as combined inoculation, both F1 and F2 were selected for further

experimentation.

Figure 3.5.1. Pot cultivated 50-days old plants of Tagetes patula with vegetative growth variation in response to individual and combined fungal inoculations in soil.

51

3.6 Screening of heavy metal tolerant ornamental plant species for

phytoextraction of TSW-Soil mixtures

During screening process, the plants’ suitability for phytoextraction of

TSW-Soil mixtures was judged on the basis of seed germination (%).

3.6.1 Seed germination (%)

The plants of Tagetes patula, Petunia xybrid, Dahlia coccinia, Zinnia

elegans and Helianthus annuus were tested for their germination response on

selected range of TSW-Soil (% w:w) mixtures. It was observed that T. patula

and H. annuus displayed the maximum germination in 20 % TSW:Soil, while

Petunia and Dahlia could come up only in 10 % of the TSW:Soil mixtures.

The Zinnia, however, was able to survive only in simple soil i.e. control. The

relative germination on different TSW-Soil mixtures is given in Table 3.6.1.

Table 3.6.1. Screening of plants for their phytoextraction potential on the basis of percentage germination observed in different TSW-Soil (% w:w) mixtures. The values

± S.D. are mean of three replicates.


Plants considered for screening

Tagetes Patula Helianthus

annuus Petunia xybrid

Dahlia coccinea

Xinia elegans

Soil 100 ± 0 100 ± 0 100 ± 0 100 ± 0.19

100 ± 0

5 88 ± 0.82 88 ± 0.67 55 ± 0.92

55 ± 1.3 NG

10 77 ± 1.73 80 ± 0.65 22 ± 0.38

44 ± 0.98

NG

15 - 80 ± 0.15 NG NG NG 20 77 ± 0.92 75 ± 0.98 NG NG NG 50 NG NG NG NG NG

100 NG NG NG NG NG

The relative germination rate of selected plants in response to the

TSW-Soil mixture is given in Figure 3.6.1.

On the basis of germination (%) results, T. patula and H. annuus were

selected for further experimentation while neglecting rest of the two plants.

52

Figure 3.6.1. Screening of plants for their phytoextraction potential on the basis of percentage germination on different TSW-Soil (% w:w) mixtures. A) Tagetes patula B) Patunia xybrid C)

Dahlia coccinea D) Zinnia elegans

B

C

D

A

E

53

3.7 Pot experiments with Marigold on autoclaved (AS) and non-

autoclaved TSW-Soil mixtures (NAS) to verify bio-reinforcing role of

fungi

3.7.1 Pre-sowing analysis of TSW-Soil mixtures

The physico-chemical properties, concentration of Category-I & II

metals are given in Table 3.1, 3.2 and 3.3 respectively and the details are

described in Chapter 3.1.

3.7.2 Biochemical analyses of Tagetes patula

Overall, inoculating soil and its TSW mixtures with selected fungi

increased the stress alleviation tendency of the plants by increasing its

tendency to produce more chlorophyll, soluble protein and different enzymes

as compared to plants where soil was not applied with fungi. The plants from

NAS treatments performed relatively better as compared to respective AS for

all the TSW-Soil mixtures.

The comparisons for influence of fungal inoculations on phytoextraction

parameters are described along the row of a table while the comparisons for

influence of TSW percentages in soil on phytoextraction parameters are

compared down the columns of the same table. This method of description

has been followed for all the forthcoming data tables.

The specific details of each of the biochemical parameters are as

under:

3.7.2.1 Chlorophyll content

After 50 days of cultivation, the plant chlorophyll content observed to

increase with the application of fungal inoculations to soil, as can be seen

along the row of Table 3.7.1. The maximum quantity within row for soil and

rest of the TSW-Soil mixtures observed where combined fungal inoculations

i.e. the F1 + F2 were applied, as given in Table 3.7.1. The F1 + F2 cause the

greatest (29.1 SPAD value) increase in plants from 5 % NAS while influencing

to the least (11.9 SPAD value) in 20 % AS as compared to any of the

treatments within a row. The AS from C gave the least (8.76 SPAD value)

54

performance in terms of chlorophyll contents. Overall, the autoclaving of soil

and its TSW mixtures significantly decreased plant chlorophyll contents than

their respective non-autoclaved treatments.

Within column, the general trend was decrease in plant chlorophyll

contents with the increase of TSW percentage in soil for all the fungal

treatments. The F1 + F2 from the 5 % NAS while 20 % AS with C gave the

best and the poor (performances respectively like the way it was found in

within-a-row comparison. The application of fungus either individually or in

combination help plants perform better as compared to Control in terms of

chlorophyll contents, as given in Table 3.7.1.

3.7.2.2 Soluble protein contents

Parallel to chlorophyll contents, the values within a row for

soluble protein contents also increased with the application of fungal

inoculations for all the treatments except for the AS from Soil where it was

below the detection limits, as given in Table 3.7.1. The F1 + F2 from 5 % NAS

and the C from 20 % AS exhibited the maximum (21 mgg-1) and the minimum

(4 mgg-1) soluble protein contents as compare to any of the soil or fungal

treatments. The non-autoclaved treatments caused increased in plant soluble

protein contents as compared to their respective autoclaved ones except for

Soil where AS didn’t give any values at all.

Within a column, overall there was decrease in soluble protein contents

with the increasing level of TSW in soil treatments. However, addition of fungi

helped plants to reduce stress by increasing soluble protein contents. The F1

+ F2 overwhelm F1, F2 and C for all of the soil treatments. The 5 % NAS with

F1 + F2 and the 20 % AS with C inoculations observed to have the maximum

and the minimum values respectively. The plants cultivated in pots with F1

inoculations performed better than those with F2 for all the TSW-Soil

mixtures.

3.7.2.3 Superoxide dismutase (SOD) contents

Parallel to the trends found for chlorophyll and soluble protein contents,

the SOD values increased with fungal inoculations within a row for all the

55

treatments, as given in Table 3.7.1. The fungal treatments having both of the

F1 and F2 performed better than control and those applied with either F1 or

F2. The plants in 5 % NAS with F1 + F2 gave maximum SOD values (44 Umg-

1 of protein) while being the least (6 Umg-1 of protein) in case of plants from

Soil AS with no fungal inoculations. The plants from NAS leaded the AS for all

of the treatments by giving significantly large SOD values over their respective

counterparts except for Soil-C treatment.

Within a column, the trend of SOD increase or decrease was similar to

that of chlorophyll and soluble protein contents. There was decrease in SOD

with the increasing fraction of TSW in soil for NAS and AS of all of the

treatments. The F1 + F2 plants performed better than control, F1 as well as

Table 3.7.1. The biochemical parameters observed in 50-days old Tagetes patula cultivated

on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).

Parameters TSW-Soil (% w:w)

Type

Fungal treatments

LSD0.05 C F1 F2

F1+F2

Chlorophyll content (SPAD value)

Soil NAS 16.8

abB 0.11 19.21

bB 0.80 18.98

bB 0.55

26.49aAB

0.22

4.71

AS 15.8abBC

0.20 16.5bBC

0.20 17.1bC

0.24 20.4aC

0.12 2.7

5 NAS 20.9

bA 0.17 26.1

abA 0.22 25.4

abA 0.14 29.1

aA 0.28 4.2

AS 17.2abB

0.17 18.3abBC

0.19 19.5abB

0.14 21.7aC

0.22 3.98

10

NAS 18.40bAB

0.45 20.1bB

0.14 20.6bB

0.27 25.3aB

0.13 4.54

AS 10.40cC

0.25 12bC

0.22 13.2bD

0.17 16.83aD

0.13 2.28

20 NAS

15.39bBC

0.28

16.98abBC

0.75 16.68

abC

0.28 20.20

aC 0.20 3.59

AS 8.76cCD

0.65 9.8bD

0.87 10.1bE

0.28 11.9aE

0.72 1.21

LSD0.05 3.04 2.99 3.17 3.85

Soluble Protein content

(mgg-1

)

Soil NAS 0.5

aE 0.67 0.4

abE 0.38 0.5

aE 0.03 0.6

aE 0.87 0.22

AS BDL BDL BDL BDL -

5 NAS 16

bA 0.35 18

abA 0.34 17

bA 0.15 21

aA 0.87 3.90

AS 10bcBC

0.98 13abBC

0.91 12bB

0.19 15aB

0.76 3.06

10 NAS 12

bcB 0.71 15

bB 0.61 14

bAB 0.81 19

aAB 0.10 3.33

AS 9abC

0.73 7bCD

0.23 6bcCD

0.82 10aC

0.71 1.18

20 NAS 8

bC 0.53 9

abC 0.45 8

bC 0.19 10

aC 0.72 1.21

AS 4bD

0.91 4.5bD

0.28 5abD

0.45 6aD

0.26 1.13

LSD0.05 2.79 2.18 2.05 2.25

SOD (Umg

-1 of protein)

Soil NAS 7

cD 0.14 11

bcE 0.33 13

bD 0.19 16

aD 1.39 2.47

AS 6cD

0.16 8bF

0.16 7bcE

0.56 11aE

0.19 1.71

5 NAS 32

bcA 0.57 35

bA 0.39 37

bA 0.78 44

aA 1.2 5.19

AS 25bC

0.12 21bcDE

0.67 20cC

0.98 39aB

0.91 4.28

10 NAS 28

cB 0.17 31

bB 0.23 33

bAB 0.45 38

aB 0.19 4.2

AS 24bC

0.45 23bcD

0.87 25bBC

0.34 35aBC

0.75 3.99

20 NAS 30

bAB 0.34 27

bcC 0.45 28

bcB 0.23 34

aBC 0.45 3.83

AS 27bB

0.55 25bcCD

0.89 24bBC

0.78 31aC

0.37 3.11

LSD0.05 2.88 2.43 4.19 4.98

CAT (Uml

-1)

Soil NAS 0.3

bcDE 0.19 0.4

abCD 0.56 0.32

bDE 0.23 0.5

aD 0.56 0.19


5 NAS 18

bA 0.45 14

cA 0.45 15

cA 0.12 21

aA 0.34 2.35

AS 15abB

0.45 13bAB

0.45 12bB

0.23 17aB

0.45 2.19

10 NAS 11

aC 0.56 7

bcB 0.76 8

bC 0.11 10

abC 0.34 1.17

AS 9.8aCD

0.67 6.5bcB

0.98 7bcCD

0.12 8bCD

0.34 1.04

20 NAS 1.3

abD 0.34 1.4

abC 0.23 1.5

aD 0.12 1.8

aD 0.23 0.71

AS 0.12cDE

0.12 0.14bCD

0.12 0.15abDE

0.11 0.16aDE

0.1 0.02

LSD0.05 1.88 1.81 1.97 2.11

C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil

56

F2. The plants from NAS performed better than their respective AS treatment

even within a column comparison.

3.7.2.4 Catalase (CAT) contents

The fungal inoculations of TSW-Soil mixtures helped plants improve

their defense mechanism by increasing CAT values. Within a row, there was

an increase in plant CAT value with the individual or combined fungal

inoculations, as compared to control. The plants from 5 % NAS with F1 + F2

and 20 % AS with C had the maximum (21 Uml-1) and the minimum (0.12 Uml-

1) SOD values as compared to any of the treatments except Soil AS where the

values were BDL. The AS prevailed less feasible conditions for plants survival

than NAS for all of the TSW-Soil mixtures by significantly decreasing SOD

values as given in Table 3.7.1.

Down the column, there was decrease in SOD contents with the

increasing percentage of TSW in soil mixtures. The SOD contents of NAS of

every fungal treatment were lower than those from the corresponding AS.

Among the columns for all the fungal treatments where soil were mixed with

TSW, the SOD in plant from NAS of 5 % was highest and being the lowest in

20 % (TSW-Soil). Between the columns, plants from all of the soil treatments

with F1 + F2 inoculations had the maximum SOD and those from the C (with

no fungus applied) had the minimum values.

3.7.3 Post-harvest analysis

3.7.3.1 Growth performance of Tagetes patula

Overall, it was noticed that plants cultivated in soil and its TSW

mixtures inoculated with fungal isolates yielded greater shoot, root and

seedling length, no. of leaves and roots, as well as, fresh and dry weight; as

compared to control. The NAS treatments yielded plants with less vigor and

biomass as compared to those from AS treatments. The exact details of each

of the morphological parameters is given in Table 3.7.2 and described as

under:

57

3.7.3.1.1 Shoot, root and seedling length (cm)

Along the row, the maximum plant shoot (41.76 cm), root (20.43 cm)

and seedling length (62.30 cm) was observed in 5 % NAS with F1 + F2

inoculation while being minimum in 20 % AS with no fungal inoculation i.e. the

C. There was increase in length of all the three vegetative parameters with the

application of fungal inoculations and the order of increase observed to be F1

+ F2 > F2 > F1 > C along the row. In NAS, the plant gave better response

than in AS for all the treatments.

Within column, there was decrease in plant shoot, root and seedling

length with the increasing proportion of TSW in the soil. The different TSW-

Soil mixtures under F1 + F2 column gave best results while those in C column

attained the least height as seen in Figure 3.7.1.

Figure 3.7.1. The vegetative growth variation cab be observed in Marigold (Tagetes patula) in response

to autoclaved soil (AS on right) and non-autoclaved soil (NAS on left) mixed with different percentages of TSW (% w:w) ranging from the maximum in plants from 5 % (TSW:Soil) NAS inoculated with F1 + F2 to the minimum in plants from 20 % (TSW:Soil) AS inoculated with C i.e. no fungi.

3.7.3.1.2 No. of leaves and roots

Along the row, the plants in 5 % NAS with F1 + F2 inoculation

observed to have maximum no. of leaves (18) and roots (29) while being the

minimum (4 and 7 respectively) in 20 % AS without any of the inoculation. The

TSW-Soil mixtures with F2 performed better than those inoculated with F1

and no fungal incorporations.

Within a column except, the increasing ratio of TSW decreased the no.

of leaves and roots. The TSW-Soil mixtures under F1 + F2 gave the best

58

vegetative growth than any of the fungal treatments. The worst growth

response observed to be in C column.

Table 3.7.2. Various morphological parameters observed in 50-days old Tagetes patula

cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


Type

Fungal treatments

LSD0.05 C F1 F2

F1+F2

Shoot Length (cm)

Soil NAS 18.38

cAB 0.11 22.12

bcB 0.80 25.47

bAB 0.55 36.31

aB 0.22 4.02

AS 16.12dB

0.20 19.89cC

0.20 22.56bB

0.24 26.20aDE

0.12 3.13

5

NAS 21.76bcA

0.17 26.54bcA

0.22 28.41bA

0.14 41.76aA

0.28 4.79 AS 18.29

cdAB 0.19 21.18

cB 0.16 29.61

bA 0.23 32.30

aC 0.22 3.28

10

NAS 19.11bcAB

0.45 23.33bAB

0.14 24.39bAB

0.27 34.26aBC

0.93 4.41 AS 14.44

cC 0.25 17.34

bcCD 0.22 19.34

bBC 0.17 28.15

aD 0.13 3.97

20 NAS 15.45

cBC 0.89 18.32

bC 0.34 19.34

bBC 0.62 25.23

aDE 0.48 3.13

AS 11.53bcD

1.0 13.54bcD

0.12 14.74bC

0.19 20.82aE

0.74 3.91

LSD0.05 2.95 3.22 4.62 3.71

Root Length (cm)

Soil NAS 11.50

bA 0.23 12.36

bAB 0.17 14.45

abAB 0.14 16.63

aB 0.17 3.22

AS 7.50bB

0.27 10.36abB

0.42 11.36abB

0.21 14.33aBC

0.24 3.83

5

NAS 11.30cA

0.25 16.60bA

0.26 18.43abA

0.12 20.43aA

0.15 3.49

AS 8.30cAB

0.25 12.60bAB

0.16 13.43bB

0.12 16.43aB

0.15 2.88

10

NAS 8.40bAB

0.27 9.46bB

0.13 7.97abC

0.1 8 14.40aBC

0.26 3.09 AS 6.40

bB 0.22 7.46

bB 0.23 8.40

abC 0.18 10.40

aCD 0.26 2.75

20 NAS 7.45

bB 0.23 8.12

bB 0.87 7.51

bC 0.14 12.47

aC 0.16 3.9

AS 5.67abBC

0.65 7.35bBC

0.17 6.29bCD

0.13 10.63aCD

0.67 2.6

LSD0.05 3.33 3.68 4.11 2.38

Seedling Length (cm)

Soil NAS 30.85

dCD 0.15 34.96

cCD 0.56 40.10

bB + 0.95 52.98

aA 0.84 5.85

AS 23.97bC

0.25 30.26abBC

0.26 34.16aB

0.15 40.93aA

0.14 4.89

5

NAS 33.50cC

0.12 43.32bcBC

0.14 46.82bB

0.10 62.30aA

0.12 6.46

AS 26.94dC

0.22 33.96cB

0.24 43.23bAB

0.10 48.98aA

0.12 5.43

10

NAS 27.80bcC

0.12 33.43bB

0.21 32.63bB

0.27 48.97aA

0.19 5.55 AS 21.17

cC 0.12 24.99

bcBC 0.21 27.93

bB 0.07 38.86

aA 0.09 3.82

20 NAS 23.10

bcBC 0.82 27.06

bB 0.32 26.95

bB 0.15 37.96

aA 0.19 4.88

AS 17.11bcBC

0.12 20.98bB

0.92 21.10bB

0.12 32.12aA

0.34 4.25

LSD0.05 4.45 5.61 3.32 6.44

No. Of Leaves

Soil NAS 9

bB 0.22 12

abB 0.17 11

abB 0.23 14

aB 0.29 4.75

AS 6bC

0.2 7bC

0.1 8abC

0.2 10aC

0.2 2.82

5

NAS 13bA

0.26 14abA

0.18 15abA

0.21 18aA

0.13 4.43

AS 7bcBC

0.26 8bC

0.18 9bBC

0.21 13aB

0.13 3.34

10

NAS 9bB

0.16 15aA

0.33 16aA

0.07 17aA

0.51 4.16 AS 5

aC 0.16 4

abD 0.33 3

bD 0.07 6

aD 0.11 2.75

20 NAS 9

abB 0.16 7

bC 0.16 8

bC 0.16 11

aBC 0.16 2.63

AS 4bCD

0.16 5bD

0.16 6abCD

0.16 8aCD

0.16 2.71

LSD0.05 2.77 2.97 2.89 3.09

No. Of Roots

Soil NAS 16

cAB 0.23 19

bAB 0.30 20

bA 0.09 24

aB 0.16 3.63

AS 12bcB

0.23 14bBC

0.30 16bAB

0.09 21aBC

0.16 4.4

5

NAS 18bcA

0.15 21bA

0.19 19bA

0.30 29aA

0.14 4.27

AS 13bcB

0.16 15bB

0.19 18abA

0.10 21aBC

0.14 3.24

10

NAS 10cBC

0.23 16bB

0.25 17bAB

0.15 26aAB

0.16 4.12 AS 9

bcC 0.23 11

bC 0.25 10

bBC 0.15 17

aC 0.16 3.28

20 NAS 9

cC 0.65 12

bC 0.15 13

bB 0.98 16

aC 0.14 2.57

AS 7cCD

0.12 10bCD

0.16 11bB

0.76 15aCD

0.10 2.81

LSD0.05 3.46 3.62 4.31 3.28

Fresh weight (g)

Soil NAS 4.21

bAB 0.96 5.37

ab 0.13 5.51

a 0.66 6.44

aC 0.19 1.21

AS 3.98bB

0.26 4.95abB

0.13 4.72abBC

0.16 5.89aC

0.19 1.88

5 NAS 6.50

cA 0.27 9.40

bA 0.27 10.70

bA 0.18 14.40

aA 0.55 2.93

AS 4.50bAB

0.17 5.40bB

0.17 5.70bB

0.18 9.40aB

0.15 3.61

10 NAS 3.70

bcB 0.12 5.91

bB 0.15 5.30

bB 0.17 9.22

aB 0.19 2.99

AS 2.70cBC

0.12 4.40bB

0.52 5.30abB

0.71 6.22aC

0.96 1.59

20 NAS 3.52

bcB 0.18 4.23

bBC 0.82 4.76

bBC 0.18 6.78

aC 0.32 1.74

AS 2.76bBC

0.29 2.98bC

0.64 3.12abC

0.76 4.20aD

0.98 1.12

LSD0.05 2.71 2.44 1.86 2.58

Dry weight (g)

Soil NAS 1.96

bAB 0.96 2.30

abB 0.13 2.50

abB 0.66 3.10

aB 0.19 0.88

AS 0.97cD

0.14 1.34bD

0.82 1.41bCD

0.51 2.28aC

0.92 0.24

5 NAS 2.26

bcA 0.18 2.98

bA 0.13 3.20

bA 0.16 5.40

aA 0.19 1.09

AS 1.95bAB

0.17 1.93bBC

0.17 1.70bC

0.18 3.40aB

0.15 0.79

10 NAS 1.70

aB 0.12 1.23

abD 0.15 1.30

abCD 0.17 2.22

aC 0.19 1.17

AS 1.01bCD

0.12 1.12bDE

0.54 1.09bD

0.79 1.52aCD

0.9 0.24

20 NAS 1.34

cC 0.61 1.72

abC 0.27 1.64

bC 0.71 1.96

aC 0.23 0.30

AS 0.67bDE

0.02 0.76abE

0.94 0.78abDE

0.62 0.92aD

0.54 0.21

LSD0.05 0.34 0.38 0.47 1.12


59

3.7.3.1.3 Fresh and dry weight (g)

The fresh and dry weight while being inter-dependent observed to be

the maximum and the minimum in accordance with the maximum and the

minimum no. of leaves and roots for both along the row as well as within

column comparisons. In other words, along the row the maximum weight

(14.40 g fresh, 5.40 g dry) observed to be in 5 % NAS with F1 + F2 and the

minimum (2.76 g fresh, 0.67 g dry) in 20 % AS without any fungus.

Within column, the increasing TSW ratio affected the biomass production

negatively except for 5 % TSW-Soil mixture. The TSW-Soil mixtures under F1

+ F2 yielded maximum fresh and dry weight while those in C column yielded

the least.

3.7.3.2 Category-I metals in plant SHOOT

The Category-I metals i.e. the flame photometer detected metals in

shoot were variable with respect to fungal inoculations as well as increasing

ratio of TSW in soil, as given in Table 3.7.3.

3.7.3.2.1 Calcium (Ca) in shoot

Along the row, the Ca concentration in shoot increased with inoculation

of fungi as compared to C. The NAS of all the treatments had more shoot Ca

contents with respect to that of AS. The maximum (1120 mgkg-1) shoot Ca

observed to be in 10 % NAS with F1 + F2 while being the minimum (5.5 mgkg-

1) in plants from Soil AS with C. The NAS of 10 % observed to have the

maximum Ca in shoot than any of the TSW-Soil mixtures for C, F1, F2 and F1

+ F2 inoculations within row.

Within column, the maximum Ca concentrations in shoot of plants from

TSW-Soil mixtures inoculated with F1 + F2 while being the minimum in those

where no fungi was applied. The plants with F2 inoculation had more Ca in

shoot than those with F1. It was observed that shoot Ca increased with the

increasing TSW ratio in soil mixtures for both NAS and AS.

60

3.7.3.2.2 Potassium (K) in shoot

Along the row, the shoot K uptake increased with fungal applications

for all the TSW-Soil mixtures. The maximum K level (550 mgkg-1) was

observed in 10 % NAS with F1 + F2 while being the minimum (12 mgkg-1) in

soil NAS with no fungi. The values for each AS observed to be lesser than its

respective NAS.

Within column, the K shoot uptake increased with the increasing

concentration of TSW in soil mixtures for all the treatments. For all TSW-Soil

mixtures, the F2 plants showed more K uptake than those with F1 and being

the least where no fungus was applied. The maximum values observed under

F1 + F2 column.

Table 3.7.3. The concentration of Category-I Metals (mgkg-1

) observed in SHOOT of 50-days

old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).

Category-I Metal

TSW-Soil (% w:w)

Type

Fungal treatments

LSD0.05 C F1 F2

F1+F2

Ca

Soil NAS 10.12

cD 0.34 12.22

bcD 0.9 15.9

bE 0.87 22.34

aE 1.1 3.44

AS 5.5cD

0.65 7.6bD

1.1 8.5bE

0.56 12.5aE

1.8 2.19

5

NAS 379cAB

1.2 480bAB

3.5 570bBC

3.1 990aB

2.8 189

AS 230cC

1.4 290bC

2.7 310bDE

2.7 450aDE

2.7 75

10

NAS 475bcAB

1.3 610bA 3.9 660

bA 3.4 1120

aA 4.2 225.6

AS 310bcB

1.6 425bB 2.8 415

bC 2.7 780

aC 2.7 164.2

20 NAS 520

bcA 1.3 590

bA 3.7 610

bB 2.8 960

aB 3.8 178.7

AS 210bC

1.7 390aBC

3.1 345abD

2.9 550aD

2.6 155.1

LSD0.05 59.8 51.2 48.7 116.4

K

Soil NAS 12

bD 0.87 15

abE 0.56 21

bD 1.1 40

aE 1.4 7.52

AS BDL BDL BDL 13E 1.1 -

5

NAS 210cB

1.6 340bB 2.8 310

bB 1.3 490

aAB 2.9 101.8

AS 120cC 1.7 280

bC 2.1 240

bcC 1.6 380

aC 3.7 61.3

10

NAS 325cA 2.1 410

bA 3.6 425

bA 1.8 550

aA 3.3 123.3

AS 225bcB

1.4 290bBC

2.5 310bB 1.7 460

aB 3.2 108.7

20 NAS 310

bcA 2.4 340

bB 2.2 370

abAB 1.8 410

aBC 2.7 54.5

AS 110cC 1.4 190

bcD 1.5 210

bCD 2.1 290

aD 2.6 32.6

LSD0.05 31.7 50.7 56.8 74.4

Na

Soil NAS 240

bcC 1.3 310

bBC 3.3 340

bC 2.1 600

aBC 3.7 154.6

AS 125bcD

2.2 225bC

2.1 210bD

2.7 480aC

2.8 140.5

5

NAS 450bcA

2.6 590bA 2.7 610

bA 2.8 720

aB 3.1 92.4

AS 210bcC

2.2 320bBC

2.5 340abC

2.4 580aBC

2.7 148.7

10

NAS 340cB 1.8 440

bB 1.1 510

bB 3.4 880

aA 3.7 163.5

AS 210cC 1.9 280

bcC 1.4 310

bC 2.7 390

aCD 1.8 41.8

20 NAS 110

cD 1.5 120

bcD 1.6 150

bDE 2.6 210

aCD 1.7 35.5

AS 40cDE

0.99 90bD

1.3 85bE 1.7 125

aD 1.5 31.8

LSD0.05 86.7 141.6 94.8 154.3


3.7.3.2.3 Sodium (Na) in shoot

The Na concentration in shoot observed to increase along the row and

it was because of fungal inoculations. The pots with F1 + F2 showed the

greatest Na shoot uptake, the F2 being greater than F1, while those with no

61

fungi being the least. The plants in 10 % NAS with F1 + F2 had the highest

value while those in 20 % AS with no fungi exhibited the lowest Na shoot

contents.

Within column, the increasing ratio of TSW in soil mixtures enhanced

the shoot Na uptake except for 20 % for all the fungal treatments. In case of

20 %, the concentration of shoot Na observed to be the least for all the fungal

treatments.

3.7.3.3 Category-I metals in plant ROOT

The bioavailability of Category-I metals was variable with different fungi

in root also however, it was directly related to the increasing ratio of TSW in

soil mixture, as given in Table 3.7.4.

3.7.3.3.1 Calcium (Ca) in root

The root Ca in both NAS and AS of Soil was BDL for all the fungal

treatments except F1 + F2 of NAS. The application of fungi to the soil helps

increase Ca uptake along the row. The plants in F1 + F2 pots observed to


) observed in ROOT of 50-days

old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according


Category-I Metals

TSW-Soil (% w:w)

Type

Fungal treatments

LSD0.05 C F1 F2

F1+F2

Ca

Soil NAS BDL BDL BDL 12

CD 0.56 -


5 NAS 225

dC 1.4 310

cB 2.9 350

bB 1.8 370

aC 2.6 19.6

AS 125cD 1.7 270

bBC 2.8 250

bBC 1.6 310

aC 2.7 57.8

10 NAS 360

bcA 2.7 590

bA 3.6 550

bA 2.6 980

aA 3.4 310.4

AS 230bcC

2.6 350bB 3.1 380

bB 2.5 690

aB 2.8 275.8

20 NAS 310

bB 2.8 380

bB 2.9 360

bB 2.4 775

aB 3.1 388.8

AS 125cD 1.8 250

bBC 2.7 270

bBC 2.3 320

aC 2.6 47.6

LSD0.05 49.1 206.7 165.9 284.2

K

Soil NAS BDL BDL BDL BDL - AS BDL BDL BDL BDL -

5 NAS 125

cC 1.3 230

bcC 2.6 280

bB 1.6 345

aB 2.2 63.5

AS 55bcD

1.4 125bD

1.2 150bC

1.4 290aBC

2.1 137.4

10 NAS 290

bA 2.5 310

bA 2.4 325

bA 1.8 430

aA 2.9 103.2

AS 220bB 2.7 270

bB 1.4 225

bBC 1.6 355

aB 3.1 128.6

20 NAS 130

bcC 1.8 240

bBC 1.3 260

bB 1.9 310

aBC 2.8 45.9

AS 25cD 0.9 90

bDE 1.1 110

bCD 1.7 155

a C 2.6 42.3

LSD0.05 68.8 39.1 43.3 73.3

Na

Soil NAS 120

bC 0.87 225

bC 2.1 280

bBC 2.1 425

aC 2.7 141.2

AS BDL BDL BDL 25G 0.67 -

5 NAS 380

dA 2.6 420

cA 3.2 470

bA 2.5 490

aB 3.1 18.9

AS 225c 2.1 325

aB 3.1 290

bBC 2.6 330

aD 2.1 15.6

10 NAS 290

bB 2.7 380

bA 2.6 350

bB 3.1 550

aA 1.8 198.8

AS 230bBC

2.6 280bBC

1.5 255bBC

3.5 390aCD

1.6 103.4

20 NAS 95

bC 0.98 110

bD 1.1 120

bC 1.1 180

aE 1.2 58.7

AS 30cD 0.79 55

bDE 0.78 60

bCD 0.87 90

aF 0.99 28.1

LSD0.05 85.5 87.8 116.6 56.7

C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least

significant difference; NAS: Non-autoclaved soil; AS: Autoclaved soil

62

have maximum while those with no fungi having the minimum root Ca than

any of the fungal treatments for all of the TSW-Soil mixtures. The highest root

Ca was in 10 % NAS with F1 + F2 while being the minimum in 20 % AS with

no fungal inoculation.

3.7.3.3.2 Potassium (K) in root

The K contents in both NAS and AS of Soil were BDL for all the fungal

inoculations. However, the 10 % NAS with F1 + F2 exhibited the maximum

root uptake than any of the soil treatments while being the minimum in 20 %

AS with no fungal application. The application of fungus as individual

inoculant i.e. F1 and F2 showed better K uptake than TSW-Soil mixtures

where no fungi has been applied.

Within column, the metal uptake in root increased with increasing ratio

of TSW in 5 and 10 %, however, it decreased in 20 %. The NAS treatments

displayed better root metal uptake than those AS for all of the TSW-Soil-fugal

combinations.

3.7.3.3.3 Sodium (Na) in root

Along the row as observed in case of Ca and K, the application of fungi

helps increase Na uptake in roots. The maximum Na in root was observed in

10 % NAS with F1 + F2 while being minimum in 20 % AS despite of the fact

that it was BDL in few of the fungal treatments from Soil AS. Those with F1

and F2 applications also performed better than C i.e. treatment with no fungal

inoculation.

Within column, trend of Na root uptake was also similar to what

observed in case of Ca and K. The increasing ratio of TSW in soil displayed

increased root Na uptake as compared to Soil with C for all the fungal

treatments. However, in case of 20 % the Na root uptake trend reversed and

metal uptake dropped even lower than what observed in case of NAS of Soil

with C. The plants in NAS accumulated more Na than those from AS for all of

the fungal treatments.

63

3.7.3.4 Category-II metals in plant SHOOT

The Category-II metals i.e. the AAS detected metals in shoot were

variable with respect to fungal inoculations as well as increasing ratio of TSW

in soil, as given in Table 3.7.5. The application of fungi enhanced trace metal

uptake tendency of plant for all the TSW-Soil mixtures and both NAS and AS.

However, the increasing level of Category-II metals in shoot was in

accordance with the increasing ratio of TSW in soil mixtures for all the fungal

treatments except for 20 % where it dropped comparatively.

3.7.3.4.1 Cd in shoot

Along the row, the Cd shoot concentration increased with application of

fungi and found to be the maximum in TSW-Soil mixtures with combined and

F2 for both NAS and AS of all treatments. In AS the metal was found to be

BDL except for F1 + F2 treatment where the metal accumulation was recoded

30 mgkg-1. Maximum amount of metal was observed in 10% TSW-Soil mixture

in NAS having combined inoculation of fungi i.e. F1 + F2.

3.7.3.4.2 Cr in shoot

As compared to control with no fungal inoculation, the Cr concentration

in shoot increased in accordance with the application of fungal inoculations

along the row except NAS of soil with F1 inoculation showing low shoot

extraction than the C. The maximum Cr accumulation in shoot was observed

in treatments applied with combined inoculants i.e. F1 + F2, being significantly

higher than any of the treatments along the row.

Within a column for C, the Cr in 5 % NAS plant shoots significantly

increased than any of the treatments. The values found in NAS and AS of Soil

was significantly least. The plants from AS of every TSW-Soil mixture

displayed lower Cr uptake than every corresponding NAS. Similar trend was

observed in plant shoots harvested from F1, F2 and F1 + F2 treatments.

3.7.3.4.3 Cu in shoot

Along the row, the plants harvested from F1 + F2 exhibited maximum

Cu accumulation as compared to any of the treatments with single or no

64

fungal application. Such a trend was observed in all of the TSW-Soil mixtures.

The F2 proved to be better enhancer of Cu uptake in plant shoot as compared

to the F1. The plants from treatments with no fungal inoculations i.e. C

showed the significantly least Cu accumulation as compared to any of the

fungal treatments.

Within a column, the plants from every AS had significantly less Cu

uptake in shoot than those form NAS for all the TSW-Soil mixtures i.e. soil, 5,

10 and 20 %.

3.7.3.4.4 Fe in shoot

The Fe uptake in plant shoots observed to be BDL in AS of Soil with all

the fungal inoculations except F1 + F2. The plants from all the NAS with F1 +

F2 inoculation displayed the maximum Fe uptake in shoot as compare to

plants from the treatments with single or no fungal inoculations. The values of

plant shoot Fe in C i.e. with no fungal inoculation(s) were the minimum as well

as significantly least as compared to plants from treatments with fungal

inoculation(s).

Within a column, in case of C, the value of shoot Fe found to be the

maximum in 5 % TSW-Soil while being the minimum and significantly least in

plant shoots harvested from AS Soil. Similar trends were observed in F1, F2

as well as F1 + F2.

3.7.3.4.5 Mg in shoot

Along the row, the Mg uptake in plant shoot increased with fungal

application in the pots and found to be the maximum in plants harvested from

pots applied with combined fungi i.e. F1 + F2, while being the minimum as

well as significantly least in shoot so of plants cultivated in soil with no fungus.

Such a pattern was observed for all the TSW-Soil mixtures.

Inside column, the value of plant shoot Mg increased with increasing

level of TSW percentage in soil up to 10 % (TSW-Soil). At 20 %, the shoot

uptake decreased drastically and significantly as compared to the treatments

with preceding lower doses of TSW in soil. Such a pattern of shoot Mg

65

variation was observed in C, F1, F2, as well as F1 + F2. The plants from NAS

displayed less Mg uptake in shoot than corresponding AS for all the

treatments.

Table 3.7.5. The concentration of Category-II Metals (mgkg-1


old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according


Category-II Metals

TSW-Soil (% w:w)

Type

Fungal treatments

LSD0.05 C F1 F2

F1+F2

Cd

Soil NAS 10

cD 0.34 25

bcE 0.86 45

bD 1.6 80

aD 1.4 23.6

AS BDL BDL BDL 30DE

1.7 -

5

NAS 320cA 1.4 410

bA 2.5 450

bA 2.6 590

aA 2.7 129.5

AS 210dBC

2.1 350cB 1.9 390

bAB 2.8 425

aBC 4.3 32.8

10

NAS 250bcB

2.2 325bB 1.4 360

bB 2.6 480

aB 4.1 115.5

AS 210cBC

2.5 290bcC

1.6 325bB 2.8 390

aBC 3.3 58.9

20 NAS 110

cC 1.7 190

bcD 1.8 225

bC 2.2 280

aC 2.9 49.9

AS 20bcD

0.67 45bE 1.4 55

bD 1.9 110

aD 2.1 54.4

LSD0.05 68.7 55.4 79.9 118.6

Cr

Soil NAS 45

bcE 0.98 40

bcF 0.99 80

bE 1.8 215

aD 2.1 68.6

AS 15bcE

0.56 30bF 1.5 50

bE 1.1 190

aD 1.7 62.5

5

NAS 990bcA

1.4 1,100bA 3.7 1,550

aA 2.9 1,540

aA 5.1 125.8

AS 420cC

2.3 650bcC

2.2 720bC

2.7 910aBC

3.3 105.5

10

NAS 810cB 2.7 970

bcB 3.1 1,030

bB 2.6 1,120

aB 3.6 89.4

AS 780cB 2.8 855

bcBC 2.4 945

bB 2.3 1,045

aB 3.8 98.2

20 NAS 415

bcC 3.1 480

bD 2.8 510

bCD 2.5 625

aC 2.8 101.7

AS 220cD

3.5 320bE 2.6 345

bD 1.7 510

aCD 4.2 61.9

LSD0.05 96.7 121.1 265.4 215.8

Cu

Soil NAS 60

bcE 1.1 120

bBC 1.6 155

bC 1.3 290

aB 2.4 125.5

AS 45cE 1.3 110

bC 1.5 125

bD 1.4 190

aCD 2.1 60.6

5

NAS 190bB 1.6 210

bB 2.3 225

bA 1.8 350

aA 2.5 120.2

AS 285bA 2.1 410

aA 2.4 160

cC 1.1 190

cCD 2.4 122.8

10

NAS 155cC

1.9 195bcB

2.1 210bA 3.1 230

aC 2.6 18.8

AS 95bcD

0.98 110bC

1,9 115bE 1.1 150

a 1.6 33.3

20 NAS 125

cCD 1.6 175

bB 1,8 180

bB 1.8 210

aC 2.2 27.6

AS 75bDE

1.1 90bC

1.7 85bE 3.9 115

aD 2.7 38.8

LSD0.05 34.8 90.4 24.7 57.4

Fe

Soil NAS 12

cC 0.98 19

bcC 1.1 25

bD 0.85 90

aB 2.5 11.1

AS BDL BDL BDL 25E 1.1 -

5

NAS 45cA 0.68 70

bA 0.98 90

abA 0.91 105

aA 2.1 23.9

AS 30bB 1.2 20

bcC 1.8 35

bCD 0.83 70

aCD 2.6 25.7

10

NAS 30cB 1.8 50

bB 1.6 75

abB 1.2 80

aBC 2.8 5.8

AS 15bcC

0.98 20bC

1.4 25bD

0.57 45aD

1.9 18.4

20 NAS 20

cBC 1.4 35

bcBC 1.2 45

bC 1.8 60

aC 2.1 14.3

AS 15dC

0.49 25cC

0.89 35bCD

1.2 40aD

1.9 4.99 LSD0.05 14.4 18.9 13.3 14.8

Mg

Soil NAS 15

bcD 0.78 25

bD 1.94 35

bDE 1.6 70

aC 1.7 32.4

AS BDL 10bE 0.92 13

bE 1.8 35

aD 1.8 5.3

5

NAS 55cAB

1.1 70bcB

1.2 80bB 1.7 110

aB 1.6 17.8

AS 30bcCD

1.4 50bC

1.5 45bD

1.6 75aC

1.5 18.8

10

NAS 60cA 1.3 90

bA 1.9 125

abA 1.2 135

aA 2.4 18.1

AS 45cB 1.2 70

bB 1.2 65

bC 0.89 110

aB 2.1 11.7

20 NAS 35

cC 1.3 45

bcC 1.7 60

bC 0.93 95

aBC 1.9 14.8

AS 15cD

0.98 40bCD

1.2 35bDE

0.91 65aC

1.2 13.9

LSD0.05 6.7 13.2 14.3 24.4

Ni


5

NAS 15bcA

0.93 17bA 1.2 20

abA 0.87 22

aA 1.2 2.98

AS 9cB 0.23 12

bB 1.5 10

bcB 0.86 15

aC 1.5 2.11

10

NAS 8bBC

0.12 15abAB

1.6 10bB 0.91 20

aAB 0.93 5.5

AS 7bcC

0.15 5cD

0.23 9bBC

0.92 16aBC

0.95 2.89

20 NAS 5

cD 0.11 9

bC 0.12 8

bcC 0.84 18

aB 0.98 1.92

AS BDL 8abC

0.15 7bCD

0.96 11aD

0.94 1.3 LSD0.05 1.5 2.8 1.6 2.9

Zn

Soil NAS 10

cCD 0.13 15

bcC 0.19 18

bF 0.93 25

aC 0.90 4.1

AS BDL BDL BDL 15aC

0.76 -

5

NAS 160cA 1.1 190

bcAB 1.1 210

bB 1.6 345

aA 3.1 30.4

AS 135bcAB

1.6 160bB 1.2 170

bC 1.8 280

aAB 2.8 34.8

10

NAS 150cA 2.1 215

bA 1.7 235

bA 1.4 360

aA 2.9 64.6

AS 80bcB

1.5 160bB 1.9 140

bD 1.5 240

aB 2.6 78.8

20 NAS 75

cB 1.6 195

bA 1.8 210

bB 1.1 290

aAB 2.1 76.1

AS 40cC

1.3 120bBC

1.2 95bcE

0.98 190aBC

2.1 44.7 LSD0.05 33.3 45.2 24.3 78.6


66

3.7.3.4.6 Ni in shoot

The plant shoot Ni observed in BDL for NAS as well as AS of Soil.

However, its value found to increase with application of fungus and observed

to be the maximum in case of plants cultivated in pots with F1 + F2. The

plants from 5, 10 and 20 % (TSW-Soil) observed to follow the same

sequence.

In a column comparison, the value of plant shoot Ni concentration

decreased with the increasing percentage of TSW in soil and found to be the

maximum in plants harvested from 5 % NAS while being the minimum in

plants from 20 % AS. The plants from NAS showed enhanced shoot Ni uptake

than corresponding AS for all the treatments.

3.7.3.4.7 Zn in shoot

Alongside row, the shoot Zn concentration increased in plants harvest

from pots with fungal inoculations than those harvested from pots applied with

no fungi. Like other metals, the shoot Zn observed to be the maximum in

plants representing soil treatments applied with F1 + F2, those harvested from

the pots filled with autoclaved soil applied with no fungi displayed the

minimum values.

Inside a column, the value of shoot Zn concentration decreased with

increasing fraction of TSW in soil mixture for all the fungal treatments. The

plants from every NAS had significantly greater Zn shoot concentration than

corresponding AS and such a variation observed in C, F1, F2 as well as F1 +

F2.

3.7.3.5 Category-II metals in plant ROOT

The Category-II metals i.e. the AAS detected metals in root were

observed to differ with varying levels of TSW in the soil as well in response to

fungal inoculations, as given in Table 3.7.6.

67

3.7.3.5.1 Cd in root

Along the row, the Cd level in plant roots harvest from Soil observed to

be BDL for NAS and AS of C, F1, F2 and F1 + F2. However, for 5, 10 and 20

% TSW-Soil mixtures, the plant root Cd concentration increased with fungal

inoculations and found to be maximum as well as significantly higher than

those applied with single or no fungal treatments, while being the minimum

and significantly least in C.

Down the columns, the root Cd concentration increased with increasing

percentage of TSW in soil up to 10 % but dropped at 20 % for C, F1, F2 as

well as F1 + F2. The plants from all of the NAS exhibited enhanced Cd uptake

than those harvested from corresponding AS and such a trend was observed

in all of the fungal treatments.

3.7.3.5.2 Cr in root

Alongside the row, the plants from F1 + F1 fungal inoculations showed

the maximum root Cr concentration with C treatment plants having the

minimum uptake for both NAS and AS of Soil, 5, 10 and 20 % TSW-Soil

mixtures. The F2 inoculation enhanced the root Cd concentration better than

F1 as well as C.

Within the columns, the root Cr concentration increased with increasing

percentage of TSW in soil up to 10 % but significantly decreased at 20 % as

compared to 0 %. For C, F1, F2 and F1 + F2, the plants from NAS showed

better root Cr uptake than corresponding AS.

3.7.3.5.3 Cu in root

Alongside the row, the fungal inoculations improvised the root Cu

uptake as compared to C. The plants from F1 + F2 observed to have the

maximum uptake than those cultivated in pots with single or no fungal

inoculations and found to be the minimum in C. The F2 observed to be better

enhancer of root Cu uptake than F1.

Under the columns, the plants from 5 and 10 % TSW-Soil exhibited

better root Cu accumulation than Soil. The 20 % plants had the lower root Cu

68




Category-II Metals

TSW-Soil (% w:w)

Type

Fungal treatments

LSD0.05 C F1 F2

F1+F2

Cd


5

NAS 290cA 1.1 390

bA 1.8 410

bA 2.1 550

aA 2.5 105.3

AS 190cB 1.6 325

bcB 2.8 350

bB 2.5 390

aB 3.1 38.7

10

NAS 240cAB

2.1 310bBC

2.6 340bB 2.9 410

aB 2.7 68.7

AS 180cB 1.7 270

bcBC 2.5 290

bC 1.9 320

aBC 2.6 29.8

20 NAS 90

cC 0.99 155

bcC 1.9 190

bD 1.6 230

aC 2.1 36.6

AS BDL 15bcD

0.94 25bE 0.69 90

aD 1.1 15.5

LSD0.05 88.5 64.3 56.2 134.7

Cr

Soil NAS 20

bcF 0.23 35

bE 0.67 55

bE 0.56 185

aE 1.6 18.8

AS 10bcF

0.27 25bE 0.93 35

bE 0.94 95

aEF .9 21.2

5

NAS 910cA 1.7 990

bA 2.7 1010

bA 1.8 1320

aA 4.1 77.1

AS 410cD 2.4 620

bC 2.9 695

bC 1.6 890

aBC 3.8 193.5

10

NAS 585cdC

3.1 615cC 3.1 655

bC 2.6 795

aC 2.9 36.4

AS 755bcB

2.7 850bB 2.8 835

bB 3.1 990

aB 3.1 93.7

20 NAS 210

cE 2.9 325

bD 2.1 380

bD 1.6 550

aD 2.8 114.4

AS 185bcEF

1.7 295bD

1.9 270bDE

2.1 480aDE

2.5 107.9

LSD0.05 69.9 92.5 105.2 108.3

Cu

Soil NAS 55

bcC 1.1 95

bC 1.8 100

bD 0.99 255

aA 2.6 75.6

AS 25cD 0.94 75

bD 1.5 80

b 1.0 145

aC 2.7 48.8

5

NAS 130bcA

1.3 155bAB

1.7 160bB 0.98 270

aA 2.9 53.4

AS 95cdB

1.6 110cBC

1.1 130bC

1.7 155aC

1.9 17.5

10

NAS 115dAB

1.2 170cA 0.96 190

bA 1.5 210

aB 1.7 19.8

AS 80bcB

0.94 95bC

0.69 110aCD

1.1 120aD

2.6 23.3

20 NAS 90

cB 0.91 120

bB 0.78 135

abBC 1.7 160

aC 2.8 27.9

AS 55bC

0.56 75abD

0.56 70bE 0.94 110

aD 1.3 38.2

LSD0.05 22.5 17.8 25.2 32.5

Fe

Soil NAS BDL BDL BDL 55

AB 1.3 -

AS BDL BDL BDL 20BC

1.8 -

5

NAS 40cdA

0.93 45cA 0.82 55

bA 0.78 75

aA 1.9 9.5

AS 25bB 0.96 15

cBC 0.29 30

bB 0.58 60

aAB 2.1 13.5

10

NAS 15bcC

0.23 30abAB

0.49 25bB 0.49 40

aB 2.8 11.9

AS 10cD 0.87 15

bcBC 0.67 20

bBC 0.39 35

aB 1.9 8.7

20 NAS 10

cD 0.94 25

bB 0.89 20

bBC 0.56 45

aB 2.6 14.6

AS 5bcE

0.45 10bC

0.93 15bC

0.87 35aB 2.6 12.2

LSD0.05 4.89 17.5 14.9 29.4

Mg

Soil NAS 25

cCD 0.29 50

abBC 1.4 65

bC 0.93 90

aD 1.7 24.3

AS 20cD 0.12 45

bBC 1.4 60

abC 1.1 70

aE 1.2 16.9

5

NAS 70cA 0.96 90

bcA 1.5 115

bA 1.5 165

aA 1.8 34.6

AS 35cC 0.59 80

bA 1.9 110

abA 1.7 140

aB 1.6 30.9

10

NAS 55cB 0.83 75

bcAB 1.1 90

bAB 1.5 120

aC 1.5 25.6

AS 50bB 0.39 65

abB 1.2 45

bD 1.3 95

aD 1.1 32.2

20 NAS 45

cBC 0.31 70

bcAB 0.94 85

bB 0.99 130

aBC 1.4 29.9

AS 15cD 0.12 30

bcC 0.95 50

bCD 1.5 125

aBC 1.3 21.3

LSD0.05 12.4 22.8 18.2 19.8

Ni


5

NAS 5cB 0.11 7

bC 0.26 9

abD 0.94 10

aC 0.95 1.7

AS BDL 5bD

0.19 8abDE

0.78 9aC

0.67 1.4

10

NAS 10cA 0.18 12

bcA 0.67 13

bB 1.1 15

aAB 0.95 1.9

AS 8cAB

0.67 10bcB

0.89 11bC

1.6 13aB 0.19 1.8

20 NAS 6

cB 0.18 10

bB 0.28 15

abA 1.8 17

aA 0.95 2.1

AS 5dB 0.31 7

cC 0.83 9

bD 1.6 12

aB 0.78 1.6

LSD0.05 2.1 2 1.5 2.15

Zn

Soil NAS BDL 10

cD 0.94 15

bD 0.78 20

aF 0.29 4.6

AS BDL BDL BDL 12F 1.1 -

5

NAS 125bcA

0.91 150bAB

1.4 195abA

2.6 320aA 2.5 34.6

AS 90cB 1.1 110

bcB 1.9 125

bB 2.1 260

aC 2.6 19.9

10

NAS 130cA

1.8 175bA 1.3 180

bAB 1.7 280

aB 2.9 22.5

AS 70cBC

1.3 120bB 1.7 110

bBC 1.5 210

aDE 2.7 30.1

20 NAS 60

cC 1.2 155

bcAB 1.2 180

bAB 1.8 235

aD 3.1 41.8

AS 25cC 0.92 95

bC 0.91 80

bC 0.93 155

aE 2.7 42.5

LSD0.05 26.5 36.1 28.1 17.2


uptake than those harvested from Soil but less than any of the 5 and 10 %.

Such a trend was observed for C, F1, F2 as well as F1 + F2. The plants from

69

NAS showed enhanced root Cu uptake than corresponding AS for all the

treatments.

3.7.3.5.4 Fe in root

Moving alongside the row, the root Fe observed to be BDL in Soil for

NAS and AS of C, F1, F2 but not in F1 + F2. The plants from C had the lowest

root Fe as compared to plants from any of the single or combined application

of fungi. The root Fe accumulation observed to be the maximum in F1 + F2

and significantly higher than those harvested from any of the treatments with

single or no fungal inoculations.

Inside columns, the root Fe concentration decreased with increasing

percentage of TSW in soil for all the fungal treatments. The plants from NAS

showed enhanced root Fe uptake than corresponding AS.

3.7.3.5.5 Mg in root

Beside the rows, the Mg root concentration increased in NAS and AS

with fungal application as compared to C i.e. with no fungal inoculation. The

F1 + F2 plants displayed the maximum root Mg concentration than those from

C as well as F1 and F2. The F2 inoculations gave better results than F1.

Down the columns, except 20 % the root Mg accumulation increased

with increasing percentage of TSW in soil. The plants from every AS

treatment had lower root Mg concentration than corresponding NAS for all the

fungal treatments.

3.7.3.5.6 Ni in root

The root Ni concentration increased along the row with the application

of fungal inoculations and found to be the maximum in plants applied with

combined application of both of the fungi while being the minimum in C with

no fungus added. The F2 application incurred better root Ni uptake effects

than F1. The plants from NAS and AS of Soil had the root Ni accumulation

below the detection limit.

70

Within columns, the plants from 10 % TSW-Soil mixtures had the

maximum root Ni level than those from both of the 5 and 20 % for all of the

fungal treatments. The NAS plants had the better root Ni concentration than

corresponding AS for C, F1, F2 as well as F1 + F2.

3.7.3.5.7 Zn in root

Alongside the rows, the root Zn accumulation observed to increase in

pots applied with fungal inoculations than C and found to be the maximum in

treatments applied with both of the fungi and being the minimum with no

fungal applications.

Down the columns, the root Zn concentration decreased with

increasing percentage of TSW in soil with every AS treatment showing

comparatively less values than corresponding NAS soils. Such a pattern was

observed in all the fungal treatments.

3.7.4 Fungal analyses

Alongside the row, the c.f.u. increased with fungal application than C

and observed to be the maximum in treatments with combined application of

both of the fungi. The order of c.f.u. abundance was F1 + F2 > F2 > F1 > C.

Within a column, the c.f.u. abundance was observed in NAS of all the TSW-

Soil in varying ratio; however, it was altogether absent in all of the

corresponding AS treatments.

Table 3.7.7. The post-harvest fungal analyses (× 105 c.f.u. g

-1 soil) of 50-

days old Tagetes patula cultivated on TSW-Soil mixtures. The mean

values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).

TSW-Soil (% w:w) Type Fungal Treatment

LSD0.05 C F1 F2 F1+F2

0 NAS 0.12

cdD 0.22 1.1

cB 0.17 2.3

bAB 0.23 3.9

aAB 0.29 0.99

AS - 0.9bBC

0.67 1.8abB

0.71 2.7aB 0.43 0.91

5

NAS 0.22cA

0.26 2.4bcA

0.18 2.9bA

0.21 4.6aA

0.13 1.3

AS - 1.4bB 0.12 1.7

bB 0.31 3.2

aB 0.19 0.95

10

NAS 0.19cB

0.16 2.3bA

0.33 2.5bA

0.07 4.2aA

0.51 1.1

AS - 1.2bB 0.76 1.7

abB 0.67 2.1

aBC 0.09 0.84

20 NAS 0.16

cC 0.23 1.6

bAB 0.34 1.8

bB 0.45 2.7

aB 0.76 0.90

AS - 0.7abC

0.82 1.01bBC

0.23 1.8aC

0.16 0.73

LSD0.05 0.2 0.87 0.96 1.2


71

3.7.5 Meta-analytical perspective

The meta-analytical indices of plant-metal-TSW interactions for

Category-I and Category-II metals are as under:

3.7.5.1 Category-I metals translocation index (%)

The plant translocation index values were also recorded for category-I

metals those detected by flame photometer i.e. Ca, K and Na.

In case of Ca, maximum value was observed in 5% NAS with F1 + F2

i.e. 267.56 % while least value was recorded (103.38 %) in 10% NAS with F1.

For K, 5% AS showed greater values as compared to NAS, similar trend was

seen in 10% TWS-Soil mixture except with F2 and F 1+ F2. The maximum

translocation index value was calculated in 20% AS with C i.e. 440% being

the minimum in 10% AS (102.06 %).

Table 3.7.8. The Category-I metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.

Category I Metals

TSW-Soil (% w:w)

Fungal treatments

Plants C F1 F2

F1+F2

Ca

5 NAS 168.44 154.83 162.85 267.56

AS 184 107.40 124 145.16

10 NAS 131.94 103.38 120 114.28 AS 134.78 121.42 109.21 113.04

20 NAS 167.74 155.26 169.44 123.87 AS 168 156 90.74 171.87

K

5 NAS 168 147.82 110.71 142.02

AS 218.18 224 160 131.03

10 NAS 112.06 132.25 130.76 127.90

AS 102.27 107.40 137.77 129.57

20 NAS 238.46 141.66 142.30 132.25

AS 440 211.11 190.90 187.09

Na

5 NAS 118.42 140.47 129.78 146.78

AS 93.33 98.46 117.24 175.75

10 NAS 117.24 115.78 145.71 160

AS 91.30 100 121.56 100

20 NAS 115.78 109.09 125 116.66

AS 133.33 163.63 141.66 138.88

C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; NAS: Non-autoclaved soil; AS: Autoclaved soil

Similar trend was seen in case of Na in 20% concentration, while NAS values

showed greater values as compared to AS in 5% except in F1 + F2 and in

10%.

3.7.5.2 Category-II metals translocation index (%)

The plant translocation index observed to be greater in treatments

applied with fungus than that of C and found to increase along the row, being

72

the maximum where F1 and F2 applied together for all metals as given in

Table 3.7.9. As compared to different TWS-Soil concentrations there was

increasing trend observed with increasing TWS-Soil mixture as maximum

values for both AS and NAS soil was observed for 20% then 10% and

minimum values were found for 5% for all metals.

Such a pattern of increase was observed for Cd in almost all the TSW-

Soil mixtures except for F1 treatment in 20% AS soil where the translocation

index value was recorded 300 %.

In case of Cr plants showed better efficiency in NAS and F2 treatment

with 5% TWS-Soil mixture than AS and in all treatments. Similarly in 10% F1

and F2 performed better than C and F1 + F2. However for 20% the plants in C

showed maximum values for translocation index 197.1 % as compared to all

other treatments. For Cr all values were found to be highest for NAS as

compared to AS for all fungal treatment and TWS-soil concentration.

For Cu, the values were found to be greater in AS as compared to NAS

in 5% with C and F1. However the translocation index values were found to

be greater in NAS than AS for all treatments and TWS-Soil mixture The

maximum value was recorded in 5% AS with F1 treatment i.e. 372.72 %.

For Fe, the maximum translocation index values was recorded to be

300 % for 10% NAS with F2 and 20% AS with C treatment, while minimum

value was recorded in 5% NAS with C treatment i.e. 112.5 %.

In case of Mg the plants showed least value in 5% AS with F2 (40.90

%), while the maximum values for this metal was recorded in 10% AS with F2

i.e. 144.44 %.

For Ni, there was 300% translocation index value recorded in 5% NAS

with C treatment then decrease in values was observed in F1 then in F2 and

least value were recorded in combined fungal treatment i.e. 220% for the

same TWS-Soil mixture while for 10% NAS there was maximum value

recorded for F1 + F2 i.e. 123.07 %.

73

As far as the Zn is concerned there was maximum translocation index

recorded in 20% AS with C treatment i.e. 160 %. For 5% plants showed

maximum values with C as compared to F1, F2 or combined treatment i.e. F1

+ F2 for both NAS and AS. Similar kind of trend was seen in 20% TWS-Soil

mixture

Overall NAS performed well as compared to AS with respect to

translocation index values (%) except for some values.

3.7.5.3 Tolerance index (TI)

In shoots TI values were found to be higher in NAS as compared to AS

in 5% with C and F1, and case was reverse with F2 and F1 + F2. Similarly in

Table 3.7.9. The Category-II metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.

Metals

TSW-Soil (% w:w)

Fungal treatments

Plants C F1 F2

F1+F2

Cd

5 NAS 110.34 105.12 109.75 107.27

AS 110.52 107.69 111.42 108.97

10 NAS 104.16 104.83 105.88 117.07 AS 116.66 107.40 112.06 121.87

20 NAS 122.22 122.58 118.42 121.73 AS - 300 220 122

Cr

5 NAS 108.79 111.11 153.46 116.66

AS 102.43 104.83 103.59 102.24

10 NAS 138.46 157.72 157.25 140.88

AS 103.31 100.58 113.17 105.55

20 NAS 197.61 147.69 134.21 113.63 AS 118.91 108.47 127.77 106.25

Cu

5 NAS 146.15 135.48 140.62 129.62

AS 300 372.72 123.07 122.58

10 NAS 134.78 114.70 110.52 109.52

AS 118.75 115.78 104.54 125

20 NAS 138.88 145.83 133.33 131.25 AS 136.36 120 121.42 104.54

Fe

5 NAS 112.5 155.55 163.63 140

AS 120 133.33 116.66 116.66

10 NAS 200 166.66 300 200

AS 150 133.33 125 128.57

20 NAS 200 140 225 133.33 AS 300 250 233.33 114.28

Mg

5 NAS 78.57 77.77 69.56 66.66

AS 85.71 62.5 40.90 53.57

10 NAS 109.09 120 138.88 112.5

AS 90 107 144.44 115.78

20 NAS 77.77 64.28 70.58 73.07 AS 100 133.33 70 52

Ni

5 NAS 300 242.85 222.22 220

AS 0 240 125 166.66

10 NAS 80 125 76.92 133.33

AS 87.5 50 81.81 123.07

20 NAS 83.33 90 53.33 105.88 AS 0 114.28 77.77 91.66

Zn

5 NAS 128 126.66 107.69 107.81 AS 150 145.45 136 107.69

10 NAS 115.38 122.85 130.55 128.57 AS 114.28 133.33 127.27 114.28

20 NAS 125 125.80 116.66 123.40 AS 160 126.31 118.75 122.58


74

10% and 20%, plants showed better TI values in NAS than AS except in F1 +

F2. The highest TI value for shoot (1.31) was recorded in 5% AS with F2

treatment while minimum value (0.65) was observed for 20% AS with F2.

In case of TI in roots 1.34 was recorded for plants grown in 5% NAS

with F1, while 0.51 was recorded as minimum value in 20% NAS with F2.

Table 3.7.10. The tolerance index (TI) analyzed in shoot and root of 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.

TSW-Soil % (w:w)

Fungal treatments

Plants C F1 F2

F1+F2

TI Shoot

5 NAS 1.18 1.19 1.11 1.15 AS 1.13 1.06 1.31 1.23

10 NAS 1.03 1.05 0.95 0.94 AS 0.89 0.87 0.85 1.07

20 NAS 0.84 0.82 0.75 0.69 AS 0.71 0.68 0.65 0.79

TI Root

5 NAS 0.98 1.34 1.27 1.22 AS 1.10 1.21 1.18 1.14

10 NAS 0.73 0.76 0.55 0.86 AS 0.85 0.72 0.73 0.72

20 NAS 0.64 0.65 0.51 0.74 AS 0.75 0.70 0.55 0.74


3.7.5.4 Category-I metals Specific extraction yield percentage (SEY %)

Overall SEY % for all category-I metals i.e. Ca, K and Na showed a

maximum value in 5% NAS with F1 + F2 in all TWS-Soil mixture while

minimum in 20% AS with C ( having no fungal inoculum), as shown in Table

3.7.11.

Table 3.7.11. The Category-I metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w)

mixtures.

Category I Metals

TSW-Soil (% w:w)

Fungal treatments

Plants C F1 F2

F1+F2

Ca

5 NAS 37.16 31.41 19.80 83.69 AS 21.84 34.46 34.46 46.76

10 NAS 33.20 47.71 48.11 83.49 AS 21.47 30.81 31.61 58.44

20 NAS 1786 20.88 20.88 37.35 AS 7.21 13.77 11.08 18.72

K

5 NAS 37.64 47.10 29.79 93.82 AS 19.66 45.50 43.82 75.28

10 NAS 50.82 59.50 61.98 80.99 AS 36.77 46.28 44.21 67.35

20 NAS 22.22 29.29 31.81 36.36 AS 6.81 14.14 16.16 22.47

Na

5 NAS 61.25 40.15 23.25 89.29 AS 32.10 47.60 46.49 67.15

10 NAS 25.04 32.60 34.19 56.85 AS 17.49 22.26 22.46 31.01

20 NAS 4.41 4.95 5.81 8.39 AS 1.50 3.12 3.12 4.62


75

In case of Ca, for all TWS-Soil TWS-Soil mixture i.e. 5, 10 and 20% the

plants showed higher values of SEY % in NAS as compared to AS with all

fungal treatments except in 5% with F1 and F2 where the case was found to

be reversed.

In case of K, plants cultivated in NAS showed higher values of SEY%

as compared to AS in all TWS-Soil mixture and fungal treatments except in

5% NAS with F2 having 29.79 % in NAS and 43.82% in AS.

For Na, the highest value (89.29%) was recorded in 5% NAS with F1 +

F2 while minimum values was found to be 1.50% in case of 20% AS with no

fungal inoculum.

3.7.5.5 Category-II metals Specific extraction yield percentage (SEY %)

The SEY (%) was calculated in category-I metals that were detected by

AAS. Overall a similar kind of trend was seen for all metals in various fungal

treatments along the row, that the SEY % values increased with the

application of fungal inoculums and highest value was observed for F1 + F2

treatment.

In case of Cd the maximum value for SEY % was recorded in 5% NAS

with F1 + F2 treatment i.e. 43.01 %, While minimum (0.22%) was observed in

20% AS with C.

Similarly as in case of Cd, the SEY % value for Cr was found to be

highest (34.66) in 5% NAS with F1 + F2 and minimum (2.61) was recorded in

20% AS with no fungal inoculum i.e. C. Moreover, in 5% and 20% the NAS

have greater values for all fungal treatments as compared to AS but the case

is reverse for 10% where the plants in AS showed better results as compared

to NAS.

For Cu again the maximum value was observed for 5% NAS (45.95%)

with combined fungal inoculum F1 + F2 and least value (2.47%) was recorded

in 20% AS with C.

In case of Fe, the plants grown in NAS showed higher SEY % values

as compared to AS in all fungal treatments and TWS-Soil concentrations i.e.

76

5%, 10% and 20%. As in case of above mentioned metals again the highest

(72%) and lowest (2.19%) values were recorded in 5% NAS with F1 + F2 and

20% AS with C respectively.

As far as the highest and lowest values are concerned there was a

same trend seen in case of Mg, where plants in 5% NAS showed highest

SEY% value (88.70) with F1 + F2 and being minimum (2.94) in 20% AS with

C having no fungal inoculum.

There was maximum value for Ni was calculated (91.42 %) in 5% NAS

with combined fungal inoculum and minimum value (4.54 %) was recorded in

20% AS for C.

Table 3.7.12. The Category-II metals tolerance index (TI) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.

Metals

TSW-Soil (% w:w)

Fungal treatments

Plants C F1 F2

F1+F2

Cd

5 NAS 23.01 30.01 32.45 43.01

AS 15.09 25.47 27.92 30.75

10 NAS 7.4 9.6 10.63 13.52 AS 5.92 8.51 9.34 10.79

20 NAS 2.28 3.94 4.74 5.82 AS 0.22 0.68 0.91 2.28

Cr

5 NAS 23.03 25.33 31.03 34.66

AS 10.06 15.39 17.15 21.81

10 NAS 13.60 15.46 16.43 18.68

AS 14.97 16.63 17.36 19.85

20 NAS 4.02 5.19 5.73 7.57 AS 2.61 3.96 3.96 6.38

Cu

5 NAS 23.70 27.03 28.51 45.95

AS 15.18 18.51 21.48 25.55

10 NAS 14.76 17.38 19.04 20.95

AS 8.33 9.76 10.71 12.85

20 NAS 4.09 5.61 6.00 7.04 AS 2.47 3.14 2.95 4.28

Fe

5 NAS 34.00 22.54 15.93 72.00

AS 22 14 26 52

10 NAS 8.82 15.68 19.60 23.52

AS 4.90 6.86 8.82 15.68

20 NAS 3.29 6.59 7.14 11.53 AS 2.19 3.84 5.49 8.24

Mg

5 NAS 40.32 25.80 19.11 88.70

AS 20.96 41.93 50 69.35

10 NAS 18.54 26.61 34.67 41.12

AS 15.32 21.77 17.74 33.06

20 NAS 7.84 11.27 14.21 22.05 AS 2.94 6.86 8.33 18.62

Ni

5 NAS 57.14 43.63 26.36 91.42

AS 25.71 48.57 51.42 68.57

10 NAS 32.72 49.09 41.81 63.63

AS 27.27 27.27 36.36 52.72

20 NAS 10 17.27 20.90 31.81 AS 4.54 13.63 14.54 20.90

Zn

5 NAS 19.52 17.98 20 45.54 AS 15.41 18.49 20.20 36.98

10 NAS 14.81 20.63 21.95 33.86 AS 7.93 14.81 13.22 23.80

20 NAS 6.66 17.28 19.25 25.92 AS 3.20 10.61 8.64 17.03


77

For Zn, plants in NAS showed greater SEY% values as compared to

AS for all TWS-Soil mixture and treatments except in 5% with F1 and F2

where AS showed greater values as compared to NAS. However as far as the

maximum and minimum values of SEY% are concerned again there was

similar trend seen i.e. maximum value (45.54 %) in 5% NAS with F1 + F2 and

minimum (3.20 %) in 20% AS with C.

78

3.8 Experiment with saprobic and AM fungi

3.8.1 Pre-sowing analysis


metals are given Table 3.1, 3.2 and 3.3 and the details are described in

Chapter 3.1.

3.8.2 Biochemical analyses of 50-days old Tagetes patula

The biochemical parameters like chlorophyll contents, soluble protein

CAT and SOD were observed. There was increased production of all these

parameters in combined inoculation of fungi as compared to C and single

fungi. The specific details of each of the biochemical parameters are as

under:


After 50 days of cultivation, the plant chlorophyll contents within a

fungal treatment were observed to increased by applying combined fungal

inoculation i.e. the F1 + F2, as given in Table 3.8.1.


on TSW-Soil mixtures applied with different fungi. The mean values S.D. with common

letters (small along the row & capital within a column) are not significantly different according



Fungal treatments LSD0.05

C F1 F2 F1+F2

Chlorophyll contents (SPAD value)

0 14.4cB

0.98 16.1bAB

0.89 16.9aAB

0.91 17.2aB

0.34 0.9

10 15.2cA

0.67 16.8bcA

1.09 17.4bA

0.56 19.7aA

0.45 1.6

20 13.8cBC

0.23 15.3bcB

1.1 16. 3bB

0.45 18.5aAB

0.78 1.8

LSD0.05 0.7 0.8 0.6 1.3

Protein content

(mgg-1

)

0 1.2abBC

0.67 0.9bC

0.87 1.4aBC

0.34 1.7aB

0.98 0.6

10 8.9dA

1.1 10.5cA

0.89 12.4bA

0.99 15aA

0.55 1.6

20 4.2bB

1.09 5.6abB

0.14 6.4aB

1.01 3.7bcB

0.66 0.9

LSD0.05 3.6 3.8 5.1 7.8

SOD (Umg

-1 of protein)

0 BDL BDL BDL BDL -

10 18bA

1.78 14cB

1.1 16bcB

1.78 21aA

0.99 2.9

20 17cA

1.79 19.7bA

2.0 20bA

1.89 22aA

1.78 1.8

LSD0.05 0.6 2.5 1.8 0.7

CAT (Uml

-1)

0 0.13bBC

1.09 0.17bC

0.33 0.16bC

0.12 2.3aB

1.5 0.9

10 12.2cA

1.2 13.5bcA

0.78 16bA

1.9 22aA

1.45 2.7

20 4.4bB

1.05 5.9aB

0.93 6.3aB

1.4 3.5bcB

1.09 1.3

LSD0.05 4.8 4.6 5.9 6.7

C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii; LSD: least significant difference

79

The F1 + F2 cause the greatest (19.7 SPAD value) increase in plants

from 10 % while influencing to the least (13.8 SPAD value) in 20 % in C

treatment as compared to any of the treatments within a row. However F2

showed better values with respect to chlorophyll content as compared to F1

values.



treatments. The F1 + F2 from the 10 % NAS while 20 % AS with C gave the

best and the poor (performances respectively like the way it was found in

within-a-row comparison. The application of fungus either individually or in

combination help plants perform better as compared to Control in terms of

chlorophyll contents, as given in Table 3.8.1.




inoculations for all the treatment, as given in Table 3.8.1. The F1 + F2 from 10

% and the F1 from 0 % exhibited the maximum (15 mgg-1) and the minimum

(0.9 mgg-1) soluble protein contents as compare to any of the soil or fungal

treatments.

Within a column, overall there was increase in soluble protein contents

with the increasing level of TSW in soil treatments i.e. in 10 % and then

decrease in 20 %. However, addition of fungi helped plants to alleviate stress

by increasing soluble protein contents. The F1 + F2 superseded F1, F2 and C

for all of the soil treatments. The 10 % with F1 + F2 and the 0 % with F1

inoculations observed to have the maximum and the minimum values

respectively. The plants cultivated in pots with F2 inoculations performed

better than those with F1 for all the TSW-Soil mixtures.


Parallel to the trends found for chlorophyll and soluble protein contents,


treatments, as given in Table 3.8.1. The SOD values were found to be below

80

detection limits (BDL) in all treatments of 0 (% TSW-Soil). For rest of the

concentrations the fungal treatments having both of the F1 and F2 performed

better than control and those applied with either F1 or F2. The plants in 20 %

with F1 + F2 gave maximum SOD values (22 Umg-1 of protein) while being

the least (14 Umg-1 of protein) in case of plants from 10% with F1 treatment.

Within a column, the plants cultivated in F1 + F2 performed better than

control, F1 as well as F2. The plants from 20% performed better than 10% in

all treatments except control.


The fungal inoculations of TSW-Soil mixtures helped plants improve

their defense mechanism by increasing CAT values. Within treatments, there

was an increase in plant CAT value with the individual or combined fungal

inoculations, as compared to control. The plants from 10 % with F1 + F2 and

0 % with C had the maximum (22 Uml-1) and the minimum (0.13 Uml-1) CAT

values as compared to any of the treatments as given in Table 3.8.1.

For different TSW-Soil concentration comparison, there was minimum

CAT contents was observed in 0% concentration. The highest values were

observed for 10% then in 20% TWS-Soil mixture The order from lowest to

highest values for CAT content can be written as 0% >20% >10%.

Cumulatively, the values for plant under F1 + F2 inoculations column had

significantly higher values as compared to values under either of the C, F1 or

F2 columns.



The maximum growth of 50-day-old plants of T. patula was observed in

case of soil, being relatively less in 10 % and 20 % as indicated by growth

parameters (Table 3.8.2). It was noticed that plants cultivated in soil and its

TSW mixtures inoculated with fungal isolates yielded greater shoot, root and

seedling length, no. of leaves and roots, as well as, fresh and dry weight; as

compared to control. The statistical analysis of the data showed significant

81

growth in all parameters in lower TSW concentration in soil followed by a

decrease at higher (20 %) concentration. However, the maximum increase in

values was found in F + M treatment over their controls F1 and F2, for each of

the corresponding soil treatments. The details of each of the morphological

parameters is given in Table 3.8.2 and described as under:


Along the row, the maximum plant shoot (68.0 cm), root (4.1 cm) and

seedling length (108.2 cm) respectively were observed in 0 % with F1 + F2

inoculation; while being the minimum for plant shoot and seedling length in 20

% with no fungal inoculation i.e. the C. The minimum values for plant root

length was recorded in 20 % with F1 (mycorrhizal inoculation). There was

increase in length of all the three vegetative parameters with the application of

fungal inoculations and the order of increase observed to be F1 + F2 > F2 >

F1 > C along the row, as can be seen in Figure 3.8.1.

Figure 3.8.1. The vegetative growth variation in Marigold (Tagetes patula) in response to soil mixed with

different percentages of TSW (% w:w) and inoculated with different fungi.

Within column, there was decrease in plant shoot, root and seedling

length with the increasing proportion of TSW in the soil. The different TSW-

Soil mixtures under F1 + F2 column gave best results while those in C column

attained the least height.


Along the row, the plants in 0% with F1 + F2 inoculation observed to

have maximum no. of roots (27) and leaves (21) while being the minimum (6

82

and 6 respectively) in 20 % without any of the inoculation i.e. C. The TSW-Soil

mixtures with F2 performed better than those inoculated with F1 and no fungal

incorporations.


cultivated on TSW-Soil mixtures applied with different fungi. The mean values S.D. with

common letters (small along the row & capital within a column) are not significantly different

according to Duncan’s multiple range test (P = 0.05; n = 3).



C F1 F2 F1+F2

Shoot Length (cm)

0 45.1cdA

0.11 50.5cA

0.21 60.2bA

0.20 68.0aA

0.21 6.9

10 44.2bA

0.17 42.5bB

0.45 43.9bB

0.52 65.10aA

0.35 5.7

20 25.9cA

0.45 31.2bcC

0.12 34.5bBC

0.41 40.3aB

0.30 4.4

LSD0.05 6.2 6.7 9.8 9.5

Root Length (cm)

0 30.1cA

0.23 35.2bA

0.55 39.4aA

0.44 40.1aA

0.21 3.1

10 29.0abA

0.24 21.5cB

0.25 25.4bB

0.27 31.6aB

0.28 2.9

20 18.9bB

0.36 15.2cC

0.25 17.5bcC

0.16 21.4aC

0.27 1.9

LSD0.05 4.1 6.8 7.5 6.5


0 75.4cA

0.28 86.0cA

0.16 99.7bA

0.34 108.2aA

0.66 8.5

10

73.4bA

0.34 64.2cB

0.52 69.4bcB

0.36 97.2aAB

0.17 6.3

20 44.8cB

0.21 46.5cC

0.47 52.1bC

0.23 61.9aB

0.23 4.7

LSD0.05 10.7 13.4 16.8 15.9

No. of roots

0 21cA

O.12 20cA

0.22 24bA

0.43 27aA

0.32 2.9

10 13bcB

0.12 11cB

0.56 14bB

0.31 16aB

0.45 1.7

20 6dC

0.16 8cBC

0.13 10bBC

0.48 12aBC

0.27 1.9

LSD0.05 5.6 4.8 4.9 5.8

No. of leaves

0 14cA

0.11 16bcA

0.21 18bA

0.42 21aA

0.33 2.1

10 7cB

0.34 9bB

0.23 10bB

0.63 13aB

0.41 1.8

20 6cB

0.09 8bB

0.14 9abB

0.12 10aBC

0.54 1.2

LSD0.05 2.8 2.9 3.2 3.6

Fresh wt. (g)

0 26.5cA

0.17 29.1bA

0.16 30.7bA

0.08 35.2aA

0.36 3.5

10 19.2bB

0.36 18.4bcB

0.16 19.1bB

0.46 24.4aB

0.24 2.1

20 12.4bC

0.34 11.3bcC

0.29 12.1bC

0.34 14.2aC

0.22 1.4

LSD0.05 4.9 6.1 6.5 7.1

Dry wt. (g)

0 10.4bA

0.12 9.5bcA

0.13 10.1bA

0.65 13.5aA

0.34 0.98

10 8.1bcB

0.22 8.5bcAB

0.52 8.9bAB

0.38 9.7aB

0.27 0.80

20 5.2cC

0.41 6.1bB

0.12 6.7abB

0.37 7.1aC

0.26 0.56

LSD0.05 1.9 1.6 1.3 2.5


Within different TWS-Soil mixture of TWS-Soil, the increasing ratio of

TSW decreased the no. of leaves and roots. The TSW-Soil mixtures under F1

+ F2 gave the best vegetative growth than any of the fungal treatments. The

worst growth response observed to be in C column.


The fresh and dry weight was observed to be the maximum and the

minimum in accordance with the maximum and the minimum no. of leaves

83

and roots for both along the row as well as within column comparisons. In

other words, along the row the maximum weight (35.2 g fresh, 13.5 g dry)

observed to be in 0 % with F1 + F2 and the minimum (11.3 g fresh, 5.2 g dry)

in 20 % with F1 and C, without any fungus respectively.

Within column, the increasing TSW ratio affected the biomass

production negatively. The TSW-Soil mixtures under F1 + F2 yielded

maximum fresh and dry weight while those in C column yielded the least.






) observed in SHOOT of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied with different fungi. The mean


Parameters TSW-Soil (%

w:w)


C F1 F2 F1+F2

Ca

0 110cB

0.34 190bB

1.23 210bB

0.98 325aB

0.23 56

10 220cA

0.67 280bcA

2.2 305bA

2.6 390aA

0.25 45

20 95cB

0.56 145bC

1.09 170bBC

0.98 385aA

0.67 75

LSD0.05 46 49 48 25

K

0 55cC

0.12 120bcBC

0.23 150bC

0.55 225aB

0.17 55

10 125cB

1.4 165bcB

0. 210bB

0.33 355aAB

0.83 62

20 250cA

0.12 295bcA

0.94 310bA

0.97 390aA

0.45 38

LSD0.05 45 60 56 57

Na

0 440cBC

1.4 510bBC

1.8 525bC

1.9 690aC

1.1 65

10 550cB

2.3 610bcB

0.9 650bB

1.5 760aB

0.23 55

20 810dA

1.6 850cA

1.09 910bA

1.3 950aA

1.6 38

LSD0.05 128 118 130 90




of fungi as compared to C. The maximum (390 mgkg-1) shoot Ca observed to

be in 10 % with F1 + F2 while being the minimum (95 mgkg-1) in 20% with C.

The plants with F2 inoculation had more Ca in shoot than those with F1.

Within column, the maximum shoot concentrations were in TSW-Soil

mixtures with F1 + F2 while being the minimum in those where no fungi was

applied. It was observed that shoot Ca increased with the increasing TSW

ratio in soil mixtures for 10% and then decreased for 20%.

84




observed in 0 % with F1 + F2 while being the minimum (55 mgkg-1) in soil with

no fungi i.e. C.


concentration of TSW in soil mixtures for all the concentrations. For all TSW-

Soil mixtures, the F2 plants showed more K uptake than those with F1 and

being the least where no fungus was applied. The maximum values observed

under F1 + F2 column.





fungi being the least. The plants in 20 % with F1 + F2 had the highest value

(950 mgkg-1) while those in 0 % with no fungi exhibited the lowest Na shoot

contents (440 mgkg-1).


the shoot Na uptake for all the fungal treatments. In case of 0 %, the

concentration of shoot Na observed to be the least for all the fungal

treatments.






The application of fungal inoculum to the soil helps to increase Ca uptake

along the row i.e. various fungal treatments. The plants in F1 + F2 pots

observed to have maximum while those with no fungi having the minimum

root Ca than any of the fungal treatments for all of the TSW-Soil mixtures. The

85

highest root Ca (365 mgkg-1) was in 10 % with F1 + F2 while being the

minimum (55 mgkg-1) in 0 % with no fungal inoculation.


) observed in ROOT of 50-days old Tagetes patula cultivated on TSW-Soil mixtures and applied with different fungi. The

mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2

Ca

0 55cC

0.2 150bD

0.23 170aC

1.09 295aB

0.87 62

10 200cA

0.97 240bcA

0.23 275bA

1.1 365aA

1.8 45

20 70cC

0.46 130bcD

0.35 150bC

1.5 275aBC

1.5 54

LSD0.05 50 40 43 35

K

0 30cDE

1.12 95bcD

1.3 110bD

1.7 210aD

0.12 48

10 90cD

1.05 160bcC

1.45 190bBC

2.01 310aAB

0.98 60

20 230cdA

0.23 255cA

0.09 290bA

1.6 345aA

0.12 35

LSD0.05 71 56 65 52

Na

0 710cD

0.65 850bC

1.67 890bD

1.51 995aD

1.7 85

10 750cCD

0.12 950bBC

1.12 1000bBC

1.71 1240aB

1.9 130

20 940dA

0.67 1150bcA

2.12 1210bA

1.01 1310aA

2.01 98

LSD0.05 82 115 120 120



The K contents in the 20 % with F1 + F2 exhibited the maximum root

uptake (345 mgkg-1) than any of the soil treatments while being the minimum

(30 mgkg-1) in 0 % with no fungal application. The application of fungus as

individual inoculant i.e. F1 and F2 showed better K uptake than TSW-Soil

mixtures where no fungi has been applied. However, F2 showed better uptake

as compared to F1 treatment.


of TSW in 10 and 20 %, however, it was found to be minimum in 0 %.



helps increase Na uptake in roots. The maximum Na in root (1310 mgkg-1)

was observed in 20 % with F1 + F2 while being the minimum in 0 %

amounting 710 mgkg-1. Those with F1 and F2 applications also performed

better than C i.e. treatment with no fungal inoculation.

86




treatments.

3.8.3.4 Category-II metals in plant shoot




uptake tendency of plant for all the TSW-Soil mixtures. However, the

increasing level of Category-II metals in shoot was in accordance with the

increasing ratio of TSW in soil mixtures for all the fungal treatments.



fungi and found to be the maximum in TSW-Soil mixtures with combined

fungal treatments. Maximum amount of metal (850 mgkg-1) was observed in

20% TSW-Soil mixture in combined inoculation of fungi i.e. F1 + F2, while

being minimum in 0% amounting 110 mgkg-1.

Within different concentrations there is increasing trend of metal

accumulation with TSW-Soil TWS-Soil mixture


With different fungal treatments the maximum Cr accumulation in shoot

was observed in treatments applied with combined inoculants i.e. F1 + F2,

being significantly higher than any of the treatments along the rows. Cr was

found to be as low as 1 mgkg-1 in 0% with C and maximum 1,450 mgkg-1 were

recorded in 20% with F1 + F2.

Within different TWS-Soil mixture of TWS-soil there was increasing

trend of accumulation of metal as the TWS-Soil mixture increased i.e.

maximum accumulation was observed in 20%, while minimum accumulation

of metal was observed in 0%.

87

Statistical analysis showed the significant difference of accumulation of

Cr in 0 % and 20 %.



old Tagetes patula cultivated on TSW-Soil mixtures applied with different fungi. The mean

values S.D. with common letters (small along the row & capital within a column) are not

significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2

Cd

0 110bD

1.5 100bcC

1.6 120abCD

1.09 140aD

1.1 25

10 410cAB

0.98 590bA

0.91 620bA 1.04 710

aAB 0.76 80

20 520efA

0.34 590eA

0.47 610dA

1.1 850aA

0.23 85

LSD0.05 145 165 170 205

Cr

0 1cCD

0.01 3bC

0.02 4aBC

0.01 5aC

0.01 1.5

10 1,050cA

2.03 990cdAB

1.8 1,120bA

2.1 1,210aA

2.1 65

20 1,110dA

1.2 1,190cdA

1.3 1,345bA

3.2 1,450aA

2.6 95

LSD0.05 375 398 455 485

Cu

0 85cD

0.43 90cCD

0.59 110abCD

0.23 125aC

0.98 25

10 310dB

0.23 425bcAB

0.12 490bcA

0.18 610aAB

1.1 85

20 450dA

0.68 510cA

0.89 550bcA

1.1 690aA

3.1 75

LSD0.05 105 115 120 140

Fe

0 BDL 12bcCD

0.23 18bC

0.45 30aCD

0.86 12

10 75cdB

0.89 90cA

0.12 110bcAB

0.24 275aA

0.98 55

20 85deA

0.65 115dA

0.75 145cdA

0.89 325aA

0.23 75

LSD0.05 8.8 45 50 110

Mg

0 20cCD

0.11 30cD

0.19 45bcCD

0.89 85aD

0.99 25

10 125cdA

0.89 190cA

0.23 220bcA

1.1 345aA

0.19 70

20 130dA

0.56 155cAB

0.49 170cB

0.89 275aB

0.45 45

LSD0.05 40 45 48 72

Ni

0 BDL BDL BDL BDL -

10 10cB

0.13 15bcB

0.19 20bB

0.99 30aBC

0.33 8

20 15cA

0.15 25bA

0.45 30bA

0.28 50aA

0.73 12

LSD0.05 4 6 9 12

Zn

0 15dDE

0.13 65bcBC

0.72 90bCD

0.89 135aC

1.2 38

10 120cA

0.39 230bA

1.3 250aA

1.5 280aAB

0.99 49

20 70dC

0.38 170cB

0.89 245bA

0.38 320aA

1.5 70

LSD0.05 25 43 69 75






The F2 proved to be better enhancer of Cu uptake in plant shoot as compared

to the F1. The plants from treatments with no fungal inoculations i.e. C

showed the significantly least Cu accumulation as compared to any of the

fungal treatments as shown in Table 3.8.5.

88

Within different TWS-Soil TWS-Soil mixture 0 % showed significantly less Cu

uptake in shoots than those form 10 and 20 % as minimum accumulation (85

mgkg-1) was seen in 0 % with C and maximum accumulation (690 mgkg-1) in

20 % with F1 + F2.


The Fe uptake in plant shoots observed to be BDL in Soil (0 %) with C,

but F1 + F2 inoculation displayed the maximum Fe uptake in shoot as

compare to plants from the treatments with single or no fungal inoculations.

The values of plant shoot Fe was minimum 12 mgkg-1 in 0 % with F1 and

maximum 325 mgkg-1 in 20 % with F1 + F2.

Within a column, a similar kind of trend for metal accumulation was

observed as with Cu that there was increasing trend of metal accumulation by

the plants as the TWS-Soil TWS-Soil mixture increased as shown in Table

3.8.5.


Along various fungal treatments, the Mg uptake in plant shoot

increased with fungal application in the pots and found to be the maximum

345 mgkg-1 in plants harvested from pots applied with combined fungi i.e. F1

+ F2 in 10 % TWS-Soil mixture, while being the minimum as well as

significantly least in shoot of plants cultivated in soil with no fungus i.e. 20

mgkg-1 as shown in Table 3.8.5. Such a pattern was observed for all the TSW-

Soil mixtures.

For different TWS-Soil mixture, the value of plant shoot Mg increased

with increasing level of TSW percentage in soil up to 10 % (TSW-Soil). At 20

%, the shoot uptake decreased drastically and significantly as compared to

the treatments with preceding lower dose of TSW in soil


The plant shoot Ni observed to be BDL in 0 % i.e. Soil. However, its

value found to increase with application of fungus and observed to be the

maximum in case of plants cultivated in pots with F1 + F2. The plants from 10

89

and 20 % (TSW-Soil) observed to follow the same sequence. The maximum

accumulation was observed in 20 % for F1 + F2 while minimum value 10

mgkg-1 in 10 % with C having no fungal application.

In a TWS-Soil mixture comparison, the value of plant shoot Ni

concentration increased with the increasing percentage of TSW in soil and

found to be the maximum in plants harvested from 20 % being the minimum in

plants from 10 %.


The Zn concentration in shoots increased in plants harvest from pots

with fungal inoculations than those harvested from pots applied with no fungi.

Like other metals, the shoot Zn observed to be the maximum in plants

inoculated with F1 + F2 while those harvested from the pots with no fungi

displayed the minimum values. There was increasing trend of accumulation of

metal as the application of fungi. However F2 showed better results as

compared to F1 in terms of accumulation of metal as shown in Table 3.8.5.

For different TWS-Soil mixtures the value of shoot Zn concentration

was found to be maximum 320 mgkg-1 in 20 % with F1 + F2 being minimum

15 mgkg-1 in 0 % with C as shown in Table 3.8.5. The order of accumulation

of metal accumulation within different TWS-Soil mixture from maximum to

minimum was as 10 % >20 % > 0 %.

3.8.3.5 Category-II metals in plant root




3.8.3.5.1 Cd in root

For different fungal treatments within the rows, the Cd level in plant

roots harvest from Soil observed to be minimum 150 mgkg-1 in 0% with C and

found to be maximum 1,190 mgkg-1 in 20 % with F1 + F2 as shown in Table

3.8.6. The plant root Cd concentration increased with fungal inoculations and

found to be maximum as well as significantly higher than those applied with

90

single or no fungal treatments, while being the minimum and significantly least

in C.

Inside the columns, for different TWS-Soil TWS-Soil mixture the root

Cd concentration increased with increasing percentage of TSW in soil being

maximum in 20 % for C, F1, F2 as well as F1 + F2.


) observed in ROOT of 50-days old Tagetes patula cultivated on TSW-Soil mixtures applied with different fungi. The mean



w:w)


C F1 F2 F1+F2

Cd

0 150cdCD

0.89 190cCD

0.12 210bcD

0.67 290aCD

0.98 42

10 710dAB

0.99 850cA

1.2 890bcA

1.1 1,050aA

1.4 90

20 840dA

0.36 950cA

1.5 990cA

0.89 1,190aA

1.3 95

LSD0.05 238 265 280 320

Cr

0 3abCD

0.01 2bC

0.03 4aC

0.05 3abBC

0.23 1.2

10 1,500cdB

1.5 2,550bAB

0.88 2,910aA

0.67 3,100aAB

0.28 425

20 2,500eA

2.5 3,150cdA

0.91 3,410bcA

0.19 3,950aA

0.20 375

LSD0.05 835 1025 1145 1330

Cu

0 110dDE

0.89 90cdCD

0.91 125bCD

0.92 140aC

0.82 12

10 510dC

1.1 640cB

0.29 590cdBC

2.4 890aAB

0.81 115

20 910deA

2.9 1,100cA

2.3 1,250bA

0.89 1,390aA

1.62 135

LSD0.05 280 355 390 440

Fe

0 BDL BDL BDL BDL -

10 45dB

0.92 70cAB

0.29 85cAB

0.89 155aC

0.91 35

20 65dA

0.29 90dA

0.39 120cdA

0.40 270aA

0.23 59

LSD0.05 10 25 45 55

Mg

0 BDL BDL 30bCD

0.29 55aCD

0.39 8

10 75dA

0.39 135bA

0.12 170aA

0.19 110cB

0.1 25

20 65eB

0.49 110dB

0.90 165cA

0.38 260aA

1.1 45

LSD0.05 5 12 55 75

Ni

0 BDL. BDL BDL BDL -

10 6bB

0.22 8abAB

0.23 10abB

0.49 15aC

0.1.3 8

20 10cA

0.34 15bcA

0.95 20bcA

0.48 45aA

2.6 14

LSD0.05 2.5 11 4 15

Zn

0 10cdDE

0.45 30cCD

0.39 45bC

0.59 80aC

0.92 25

10 90dA

0.29 185abA

0.32 210aA

0.38 220aA

2.6 48

20 65dB

0.92 125cBC

0.99 215aA

0.45 245aA

1.1 55

LSD0.05 22 35 70 80


3.8.3.5.2 Cr in root

For different fungal treatments i.e. along the row, the plants from F1 +

F1 fungal inoculations showed the maximum 3,950 mgkg-1 root Cd

concentration in 20 %, while with F1 treatment plants having the minimum

uptake 2 mgkg-1 in 0%. The F2 inoculation enhanced the root Cd

concentration better than F1 as well as C as shown in Table 3.8.6.

91

Within the columns, the root Cd concentration increased with

increasing percentage of TSW in soil having the maximum accumulation of

metal in 20 % for all fungal treatments like C, F1, F2 and F1 + F2.

3.8.3.5.3 Cu in root

The fungal inoculations enhanced the root Cu uptake as compared to

C. The plants from F1 + F2 observed to have the maximum (1,390 mgkg-1)

uptake in 20 % than those cultivated in pots with single or no fungal

inoculations and found to be the minimum 90 mgkg-1 in 0% with F1. The F2

observed to be better enhancer of root Cu uptake than F1.

Under the columns, the plants from 10 and 20 % TSW-Soil exhibited

better root Cu accumulation than Soil. The 20 % plants had the maximum root

Cu uptake than those harvested from Soil and 10%.

3.8.3.5.4 Fe in root

Moving alongside the row while analyzing effect of different fungal

treatments, the root Fe observed to be BDL in Soil with all fungal treatments.

The plants from C had the lowest root Fe as compared to plants from any of

the single or combined application of fungi. The root Fe accumulation

observed to be the maximum in F1 + F2 and significantly higher than those

harvested from any of the treatments with single or no fungal inoculations.

Inside columns, the root Fe concentration decreased with increasing

percentage of TSW in soil for all the fungal treatments. The maximum Fe

uptake (270 mgkg-1) by roots was observed in 20% with F1 + F2 treatment

while the least value of metal uptake (45 mgkg-1) was observed in 10 % with C

as shown in Table 3.8.6.

3.8.3.5.5 Mg in root

While analyzing the fungal application effect on plants, it was observed

that the Mg root concentration increased in plant roots with fungal application

as compared to C i.e. with no fungal inoculation. The F1 + F2 plants displayed

the maximum root Mg concentration than those from C as well as F1 and F2.

92

For 0% the values for metal uptake were found to be BDL with C and F1. The

F2 inoculations gave better results than F1.

For the columns, the root Mg accumulation decreased with increasing

percentage of TSW in soil for all fungal treatments except for F1 + F2 where

the metal accumulation was increased i.e. 260 mgkg-1 in 20%, as compared to

110 mgkg-1 in 10% as shown in Table 3.8.6.

3.8.3.5.6 Ni in root

The root Ni concentration was found to be BDL for all fungal treatments

in 0% i.e. soil. However there was increased metal accumulation along the

row with the application of fungal inoculations and found to be the maximum

in plants applied with combined application of both of the fungi while being the

minimum in C with no fungus added. The F2 application incurred better root

Ni uptake effects than F1.


maximum root Ni level than 10 % for all of the fungal treatments. There was

maximum (45 mgkg-1) accumulation of metal was noted in 20% with F1 + F2

while minimum uptake (6 mgkg-1) was observed in 10% with C.

3.8.3.5.7 Zn in root

The root Zn accumulation observed to increase in pots applied with

fungal inoculations than C and found to be the maximum in treatments applied

with both of the fungi and being the minimum with no fungal applications.

Again as with most of the above discussed metals F2 performed better than

F1 as far as metal accumulation efficiency of the plant is concerned.

For different TWS-Soil TWS-Soil mixture, the root Zn concentration

increased with increasing percentage of TSW in soil with every fungal

treatment except F1 where the uptake was reduced in 20% (125 mgkg-1) as

compared to TWS-Soil mixture in 10 % (185 mgkg-1) with the same fungal

treatment i.e. F1 as shown in Table 3.8.6.

93


The results of assessment of fungal inoculum both in soil and in plants

are shown in Table 3.8.7. For analysis of various fungal treatments it is

observed that for the C treatment (without any inoculum) plants showed 0 %

AM infection and no c.f.u for T. pseudokoningii and no spores in soil. In 10 %

concentration of TWS-Soil, F1 + F2 showed maximum percentage of infection

(95 %) while the minimum infection (35 %) was observed in 20 %

concentration in F2 treatment. Similarly, maximum c.f.u. per gram of soil was

observed in 20 % TWS-Soil mixture in F1 + F2 treatment 3.5 X 105 c.f.u. of T.

pseudokoningii per gram of soil and the minimum value, 0.1 X 105 was

observed for F2 treatment. The maximum spore number of 256 spores per 50

g of soil was observed in F1 + F treatment, while the minimum number of 50

spores per 50 g of soil was observed for 20 % TWS-Soil mixture in F2

treatment.


-1 soil) of 50-days old Tagetes

patula cultivated on TSW-Soil mixtures applied with different fungi. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2

Percentage infection of AM fungi (in roots)

0 - 86.0aA

23 60cA

0.91 90aA

0.33 10.6

10 - 88abA

1.4 64cA

0.67 95aA

1.6 8.3

20 - 55aCD

1.6 35dCD

2.3 59aCD

1.2 7.4

LSD0.05 - 11.9 10.2 12.8

T. pseudokoningii (× 10

5 c.f.u. g

-1 soil)

0 - 0.30bcA

0.34 0.96aCD

0.78 1.20aC

0.23 0.87

10 - 0.16bcC

0.50 1.1aB

0.23 1.40aC

0.56 0.67

20 - 0.20deAB

0.33 1.6bA

0.98 3.5aA

0.93 1.3

LSD0.05 - 0.12 0.43 0.98

Spore No. 50 g-1

soil

0 - 190bcA

0.29 64dA

0.92 256aA

2.1 55

10 - 185bAB

0.23 60dAB

1.2 250aA

1.6 50

20 - 170aD

0.93 50dCD

1.6 188aCD

2.3 42

LSD0.05 8.9 5.8 25






94

The plant translocation index (%) values were recorded for Category-I

metals those detected by flame photometer i.e. Ca, K and Na shown in Table

3.8.8.

Table 3.8.8. The Category-I metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.

Parameters TSW-Soil (% w:w) Fungal treatments

C F1 (M) F2 F1+F2

Ca 10 110 116.66 110.90 106.84

20 135.71 111.53 113.33 140

K 10 138.88 103.12 110.52 114.51

20 108.69 115.68 106.89 113.04

Na 10 73.33 64.21 65 61.29

20 86.17 73.91 75.20 72.51

C: No fungal inoculum; F1: Mycorrhizal fungus F2: Trichoderma pseudokoningii; F1 + F2: Mycorrhizal fungus and T. pseudokoningii;

In case of Ca, maximum value was observed in 20 % with F1 + F2 i.e.

140 % while the minimum value was recorded (110 %) in 10% with C.

For K, the maximum translocation index value was calculated in 10 %

with C i.e. 138.88 % being the minimum in 10 % with F1 (103.12%).

In case of Na in for both 10 and 20 % (TSW-Soil) with C treatment

showed maximum values as compared to any other treatment. Within row, in

10 % (TSW-Soil) the maximum values were noted with C i.e. 73.33 % and for

also for 20 % (TSW-Soil) with C the maximum values (86.17 %) along the row

were noted while the minimum values were recorded for corresponding F1 +

F2.


The plant translocation index found to be greater in treatments applied

with fungus than that of C, being the maximum where F1 and F2 applied

together for all metals as given in Table 3.8.9. As compared to different TWS-

Soil concentrations there was decreasing trend observed with increasing

TWS-Soil mixture of TWS-Soil with few exceptions.

For Cd, the maximum Translocation index (71.42 %) was noted for F1

+ F2 in 20 % (TSW-Soil) while minimum value was recorded 57.74 % in 10%

with C having no fungal application.

95

In case of Cr plants showed better metal translocation efficiency (70 %)

in 10 % with C. similarly for 20 % the plants in C showed maximum values for

translocation index 44.4 % as compared to all other treatments.

For Cu, the maximum translocation index values was recorded to be

83.05 % for 10 % (TSW-Soil) with F2, while minimum value was recorded in

20 % (TSW-Soil) with F2 treatment i.e. 44 %.

Table 3.8.9. The Category-II metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 (M) F2 F1+F2

Cd 10 57.74 69.41 69.66 67.61

20 61.90 62.10 61.61 71.42

Cr 10 70 38.82 38.48 39.39

20 44.4 37.77 39.44 36.70

Cu 10 60.78 66.40 83.05 68.53

20 49.45 46.36 44 49.64

Fe 10 166.66 128.57 129.41 177.41

20 130.76 127.77 120.83 313.63

Mg 10 166.66 140.74 129.41 118.96

20 200 140.90 103.03 105.76

Ni 10 166.66 187.5 200 200

20 150 166.66 150 111.11

Zn 10 133.33 124.32 119.04 127.27

20 107.69 136 113.95 130.61


For Fe, the maximum translocation index values were recorded to be

313.63 % for 20 % (TSW-Soil) with F1 + F2, while the minimum value was

recorded in 20 % (TSW-Soil) with F2 treatment i.e. 120.83 %. For both TWS-

Soil mixtures, the F1 + F2 inoculations showed greater values than any other

fungal treatment.

In case of Mg the plants showed least value in 20 % (TSW-Soil) with

F2 (103.03 %), while the maximum values for this metal was recorded in 20 %

(TSW-Soil) with C i.e. 200 %. For Mg plants showed the maximum values in C

(without fungal treatment) in both 10 and 20 % (TSW-Soil) mixtures.

For Ni, there was as much as 200 % translocation index value

recorded in 10 % (TSW-Soil) with F2 and F1 + F2 treatment and it decreased

in F1 then in C, being the least in combined fungal treatment of 20 % (TSW-

Soil) i.e. 111.11 %.

96

As far as the Zn is concerned, the maximum translocation index

recorded in 20 % (TSW-Soil) with F1 treatment i.e. 136 %. For plants from 10

% (TSW-Soil), the maximum value was with C (133.3 %) as compared to F1

(124.32 %), F2 (119.04 %) or F1 + F2 (127.27 %).


In shoot, the TI values were found to be the highest in 10 % (TSW-Soil)

with C (0.98). In case of 20 % (TWS-Soil), the maximum value was 0.61

noted in F1 treatment as shown in Table 3.8.10.

Table 3.8.10. The root and shoot tolerance index (TI) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 (M) F2 F1+F2

TI Shoot 10 0.98 0.84 0.72 0.95

20 0.57 0.61 0.57 0.59

TI Root 10 0.96 0.61 0.64 0.78

20 0.62 0.43 0.44 0.53


In case of TI in roots, 0.96 was recorded as the maximum TI for plants

grown in 10 % (TSW-Soil) with C, while 0.43 was recorded as the minimum

value in 20 % (TSW-Soil) with F2.

3.8.5.4 Category-I metals specific extraction yield (SEY %)

The SEY (%) for Category-I metals i.e. Ca, K and Na is given in Table

3.8.11.

Table 3.8.11. The Category-I metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 (M) F2 F1+F2

Ca 10 17.91 22.17 24.73 32.19

20 5.6 9.4 10.99 22.68

K 10 17.76 26.85 33.05 54.95

20 24.24 27.77 30.30 37.12

Na 10 51.68 62.02 65.60 79.52

20 37.67 43.05 45.64 48.65


97

In case of Ca, the maximum value (32.19 %) was recorded for plants

grown in 10 % (TSW-Soil) with F1 + F2, and the minimum (5.6 %) in 20 %

(TSW-Soil) with C.

In case of K, plants cultivated in 10 % (TWS-Soil) with F1 + F2 showed

the highest value of SEY (54.95 %) and the minimum (17.76 %)

corresponding C treatment.

For Na, the highest value (679.52 %) was recorded in 10 % (TSW-Soil)

with F1 + F2 while the minimum values (37.67 %) were found to be in case of

20% (TSW-Soil) with C i.e. no fungal inoculums.

3.8.5.5 Category-II metals Specific extraction yield (SEY %)

The SEY (%) was calculated for the Category-II metals is given in

Table 3.8.12. A trend of SEY (%) variation for Category-II similar to Category-

I was observed for all the fungal treatments along the row i.e. the SEY (%)

values increased with the application of fungal inoculums and the highest

values were observed for F1 + F2 treatments.

In case of Cd, the maximum value for SEY (%) was recorded in 10 %

(TSW-Soil) with F1 + F2 treatment being 26.74 %, while the minimum (15.54

%) was observed in 20% (TSW-Soil) with C.

Similarly the SEY % value for Cr was found to be the highest (42.04 %)

in 10 % (TSW-Soil) with F1 + F2 and the minimum (23.27 %) was recorded in

20 % with no fungal inoculums i.e. C. However, plants with F2 inoculums

showed greater SEY values as compared to those from F1.

98

Table 3.8.12. The Category-II metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 (M) F2 F1+F2

Cd 10 17.02 21.88 22.94 26.74

20 15.54 17.6 18.28 23.31

Cr 10 24.87 34.53 39.31 42.04

20 23.27 27.98 30.65 34.81

Cu 10 39.04 50.71 51.42 71.42

20 25.90 30.66 34.28 39.61

Fe 10 23.52 31.37 38.23 84.31

20 16.48 22.52 29.12 65.38

Mg 10 32.25 52.41 62.90 73.38

20 19.11 25.98 32.84 52.45

Ni 10 29.18 41.81 54.54 81.81

20 22.72 36.36 45.45 86.36

Zn 10 11.11 21.95 24.33 26.45

20 6.66 14.56 22.71 27.90


For Cu, again the maximum value (71.42 %) was observed for 10 %

(TSW-Soil) with combined fungal inoculums i.e. F1 + F2 and the minimum

value (25.90 %) was recorded in 20 % (TSW-Soil) with C.

For Fe, the highest (84.31 %) and the lowest (16.48 %) SEY values

were recorded in 10 % (TSW-Soil) with F1 + F2 and 20 % (TSW-Soil) with C

respectively.

The SEY for Mg observed to be highest (73.38 %) in plants from 10 %

(TSW-Soil) with F1 + F2 and being the minimum (10.11 %) in 20 % (TSW-

Soil) with C i.e. having no fungal inoculums.

The maximum value (86.36 %) of SEY (%) for Ni was calculated in 20

% (TSW-Soil) with combined fungal inoculums and the minimum value (22.72

%) was recorded in 20 % (TSW-Soil) for C. The 10 % (TSW-Soil) treatments

showed higher SEY (%) values as compared to 20 % (TSW-Soil) mixtures.

A similar kind of SEY (%) variation was seen for Zn and Ni. The

maximum value (27.90 %) was noted for 20 % (TSW-Soil) with F1 + F2 and

the minimum value (6.66 %) was found in 20 % (TSW-Soil) with C.

99

3.9 Experiments with saprobic fungi

3.9A. Experiment with Tagetes patula inoculated with saprobic fungi

3.9A.1 Pre-sowing analysis


metals are given Table 3.1, 3.2 and 3.3 respectively and their details are

described in Chapter 3.1.

3.9A.2 Biochemical analyses of 55-days old Tagetes patula


CAT and SOD were observed in 55-days old marigold. There was increased

production of all these parameters in plants from soil mixtures applied with

combined inoculation of fungi as compared to those from applied with no or

single fungus. The specific details of each of the biochemical parameters are

as under:

3.9A.2.1 Chlorophyll content

The plant chlorophyll content within a fungal treatment increased by

applying combined fungal inoculation i.e. the F1 + F2, as given in Table

3.9A.1. The F1 + F2 incurred the greatest (19.3 SPAD value) increase in

plants from 10 % (TSW-Soil) while influencing to the least (13.5 SPAD value)

in 20 % in C treatment as compared to any of the treatments within a row.

However F2 showed better values with respect to chlorophyll content as

compared to F1 values for all TWS-soil TWS-Soil mixture except for 10%

where F1 showed greater value (17.3) as compared to F2 (16.8) as shown in

Table 3.9A.1.

Within column, there was increase in plant chlorophyll contents with the

increase of TSW percentage in soil for all the fungal treatments but the values

were dropped being the minimum in 20% TWS-soil mixture. The application of

fungus either individually or in combination help plants perform better as

compared to C in terms of chlorophyll contents, as given in Table 3.9A.1.

100

3.9A.2.2 Soluble protein contents

Parallel to chlorophyll contents, the values within a row for soluble

protein contents also increased with the application of fungal inoculations for

all the treatment, as given in Table 3.9A.1.

Table 3.9A.1. The biochemical parameters observed in 55-days old Tagetes patula cultivated on TSW-Soil mixtures.

The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2


0 14.3abB

0.38 14.9abAB

0.37 15.4aAB

0.12 15.8aB

0.39 2.1

5 15.5bA

0.71 16.6abA

0.22 17.2abA

0.17 19.3aA

0.28 3.2

10 16.4abA

0.88 17.3aA

0.11 16.8abA

0.28 18.7aA

0.19 3.6

20 13.5abCd

0.38 13.9abAB

0.18 14.3aB

0.91 15.4aB

0.11 3.8

LSD0.05 1.9 2.8 2.4 2.8


(mgg-1

)

0 0.3cdD

0.03 0.6bE

0.04 0.8aF

0.12 0.9aE

1.02 0.23

5 12cA

0.38 9deB

0.18 14bB

0.91 17aB

1.04 2.1

10 13dA

0.48 14cA

0.14 18abA

0.49 21aA

0.10 3.5

20 8bB

0.38 6bcC

0.04 7bD

0.13 11aC

0.13 2.8

LSD0.05 2.9 3.2 2.9 3.8

SOD (Umg

-1 of protein)

0 02bF

0.01 01cdD

0.02 03aC

0.21 01cdD

0.02 0.78

5 33deA

0.38 36cdA

0.38 38cA

0.32 45aA

0.88 3.4

10 31cdA

0.59 29dAB

o.45 30cAB

0.76 36aAB

0.16 2.9

20 22cdC

0.18 26bcB

0.12 28bAB

0.98 32aB

0.55 3.7

LSD0.05 4.7 10.2 12.4 9.6

CAT (Uml

-1)

0 0.1cD

0.02 0.3abE

0.12 0.3abF

0.12 0.4aE

0.17 0.13

5 16deA

0.07 18cdA

0.33 21bcA

0.38 26aA

0.38 2.8

10 12cdAB

0.23 15bcAB

0.08 14cBC

0.78 20aB

0.37 2.6

20 09cB

0.38 11bcB

0.45 10cD

0.38 16aBC

0.67 1.9

LSD0.05 4.1 5.1 3.9 4.5

C: No fungal inoculum; F1: Aspergillus niger; F2 Trichoderma pseudokoningii:; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference

The F1 + F2 from 10 % and the C from 0 % exhibited the maximum (21

mgg-1) and the minimum (0.3 mgg-1) soluble protein content as compare to

any of the soil or fungal treatments. Within Fungal treatments C showed

minimum values as compared to other fungal applications like F1, F2 and F1

+ F2 except in 5% (TSW-Soil) where the value is higher (12) than F1 (9).

However in F2 the value found to be 14 and 21 in F1 + F2.


with the increasing level of TSW in soil i.e. in 10 % and then decrease in 20%

(TSW-Soil). However, addition of fungi helped plants to assuage stress by

increasing soluble protein contents. The F1 + F2 superseded F1, F2 and C for

all of the soil treatments. The 10 % with F1 + F2 and the 0 % with F1

inoculations observed to have the maximum and the minimum values

101

respectively. The plants cultivated in pots with F2 inoculations performed

better than those with F1 for all the TSW-Soil mixtures.

3.9A.2.3 Superoxide dismutase (SOD) contents

Following the trends found for chlorophyll and soluble protein contents,


treatments, as given in Table 3.9A.1. The SOD values were found to be 01

mgg-1 in F1 and F1 + F2 treatments of 0% (TSW-Soil). For the rest of the

concentrations the fungal treatments having both of the F1 and F2 performed


with F1 + F2 gave maximum SOD values.

While analyzing the response of plant cultivated in different TWS-Soil

mixtures it was observed that the 0% showed least values, while 5% showed

maximum SOD values in all fungal treatments, then decrease in 10% and

20%. The order from highest to lowest SOD values can be written as 5%

>10% > 20% > 0%.

3.9A.2.4 Catalase (CAT) contents

Within different treatments, there was an increase in plant CAT value

with the individual or combined fungal inoculations, as compared to control.

The plants from 5 % with F1 + F2 and 0 % with C had the maximum (26 Uml-

1) and the minimum (0.1 Uml-1) CAT values as compared to any of the

treatments as given in Table 3.9A.1.

For different TSW-Soil mixtures comparison, there was minimum CAT

contents was observed in 0% (TSW-Soil). The highest values were observed

for 5% then in 10 and 20 %. Cumulatively, the values for plant under F1 + F2

inoculations column had significantly higher values as compared to values

under either of the C, F1 or F2 columns.

3.9A.3 Post-harvest analysis

3.9A.3.1 Growth performance of Tagetes patula

Better growth of 55-day-old plants of T. patula was observed in case of

lower TWS-soil mixture i.e. 5% and 10%, being relatively less in soil (0%) and

102

20% as indicated by growth parameters (Table 3.9A.2). It was noticed that

plants cultivated in soil and its TSW mixtures inoculated with fungal isolates

yielded greater shoot, root and seedling length, no. of leaves and roots, as

well as, fresh and dry weight; as compared to control. The statistical analysis

of the data showed significant growth in all parameters in lower TSW mixtures

in soil followed by a decrease at higher (20%) level. However, the maximum

increase in values was found in F + M treatment over their controls F1 and F2,

for each of the corresponding soil treatments. The details of each of the

morphological parameters is given in Table 3.8.2 and described as under:

3.9A.3.1.1 Shoot, root and seedling length (cm)

The shoot, root and seedling length (cm) of the marigold are given in

Table 3.9A.2. Along the row in comparison with different fungal treatments,

the maximum plant shoot (32.1 cm), root (40.1 cm) and seedling length (35.2

cm) was observed in 5 % with F1 + F2 inoculation while being minimum

values for plant shoot (16.3 cm) and seedling length (28.5 cm) in 20 % with

no fungal inoculation i.e. the C. The minimum value for plant root length (11.7

cm) was recorded in 20% with control having no fungal inoculation. There was



F1 > C along the row.

While in comparison with different TWS-Soil mixture there was

increase in plant shoot, root and seedling length with the increasing proportion

of TSW in the soil. The different TSW-Soil mixtures under F1 + F2 column

gave best results while those in C column attained the least height. However

the highest TWS-Soil TWS-Soil mixture i.e. 20% showed least growth as

shown in Table 3.9A.2.

3.9A.3.1.2 No. of leaves and roots



and 5 respectively) in 20 % without any of the inoculation i.e. C.

Within different TWS-Soil mixture of TWS-Soil, the increasing ratio of TSW

increased the no. of leaves and roots upto 10% but there was decreased for

103

these parameters in 20% TWS-Soil mixture The TSW-Soil mixtures under F1

+ F2 gave the best vegetative growth than any of the fungal treatments. The

least growth response observed to be in C column as shown in Figure 3.9A.1.

Table 3.9A.2. Various morphological parameters observed in 55-days old Tagetes patula



w:w)


C F1 F2 F1+F2

Shoot Length (cm)

0 18.9cAB

0.67 20.4bBC

0.12 21.1bAB

1.1 24.7aC

1.03 2.8

5 19.7dA

0.99 22.5cB

0.98 23.3cA

1.05 32.1aA

1.4 4.7

10 17.6cB

0.78 23.8bA

0.23 21.9bAB

0.33 29.3aAB

0.34 5.2

20 16.3cC

0.56 18.3bCD

0.45 19.0bBC

0.36 23.7aC

0.65 2.9

LSD0.05 1.3 2.2 1.5 3.8

Root Length (cm)

0 12.4cB

0.39 18.1bC

1.02 20.3bBC

1.2 26.7aBC

0.87 4.2

5 15.2dA

0.59 24.7bA

0.29 26.1bA

1.04 35.2aA

0.91 2.9

10 14.9cA

1.4 19.2bBC

0.34 20.7bcBC

1.4 27.1aBC

0.39 4.3

20 11.7dB

1.04 14.5bcC

0.99 15.1bCD

1.2 19.4aCD

1.22 2.8

LSD0.05 2.2 3.2 3.9 4.5


0 31.6dC

1.3 38.8cBC

0.78 41.9bcAB

0.36 51.8aC

0.47 6.2

5 35.3eA

1.5 47.5cdA

1.54 49.7cdA

0.28 67.6aA

0.94 9.3

10 32.9deBC

0.98 43.4bcAB

1.9 42.9bcAB

0.19 56.8aBC

1.5 10.2

20 28.5dD

0.38 33.1cCD

1.02 34.5cC

1.03 43.6aDE

1.39 5.8

LSD0.05 3.1 4.2 4.8 6.9

No. of roots

0 13bcCD

0.27 15bBC

1.07 18abB

0.27 21aCD

0.23 2.9

5 16cdA

0.56 19cA

1.6 20bcA

0.45 27aA

0.99 3.1

10 15cAB

0.98 14cdC

1.89 18bcB

1.2 24aB

1.8 3.9

20 12cdD

0.27 14bcC

2.6 17abB

1.7 19aD

1.46 2.7

LSD0.05 1.4 1.9 1.2 2.9

No. of leaves

0 6cBC

0.27 8bcB

0.23 10bAB

0.56 13aBC

0.78 2.1

5 8cdA

0,21 10cA

1.06 11cA

0.22 19aA

0.27 2.8

10 7cdA

0.21 9cA

0.11 12bcA

0.20 17aAB

0.33 3.8

20 5cdC

0.23 7cB

0.56 9bcBC

0.08 14aB

0.31 3.5

LSD0.05 1.2 1.7 1.5 2.1

Fresh wt. (g)

0 2.1bBC

0.27 2.7abC

0.91 3.0aB

0.12 3.5aB

0.27 1.6

5 3.4bA

0.38 3.9abA

0.34 4.1aA

0.87 4.3aA

1.09 0.7

10 2.3bBC

0.20 2.9abBC

0.56 3.2aB

0.67 3.8aA

1.45 1.4

20 1.9abC

0.99 2.4aCD

0.77 2.6aBC

0.29 2.9aCD

0.27 1.1

LSD0.05 0.53 0.39 0.7 0.28

Dry wt. (g)

0 0.9bBC

0.27 1.1bCD

0.28 1.4aC

0.27 1.9aBC

0.12 0.7

5 1.6abA

1.78 2.0aA

0.34 2.3aA

0.20 2.4aA

0.18 0.8

10 1.0bBC

1.09 1.2bCD

0.27 1.5abC

0.44 2.1aA

0.13 0.7

20 0.7bC

0.99 0.92abD

1.11 1.02aCD

0.02 1.2aC

0.73 0.4

LSD0.05 0.17 0.21 0.24 0.41


3.9A.3.1.3 Fresh and dry weight (g)

The fresh and dry weight were observed to be the maximum and the



104


observed to be in 5 % with F1 + F2 and the minimum (1.9 g fresh, 0.7 g dry) in

20 % with C, without any fungus respectively.

Within column, the increasing TSW ratio affected the biomass production

positively for lower TWS-Soil mixture i.e. 5% and 10 % and negatively for 20

% (TSW-Soil). The TSW-Soil mixtures under F1 + F2 yielded maximum fresh

and dry weight while those in C column yielded the least.

Figure 3.9A.1. The vegetative growth variation in Marigold (Tagetes patula) in response to soil mixed

with different percentages of TSW (% w:w) and inoculated with different fungi.

3.9A.3.2 Category-I metals in plant SHOOT



ratio of TSW in soil, as given in Table 3.9A.3.

3.9A.3.2.1 Calcium (Ca) in shoot




Within different concentrations of TSW in soil, the maximum shoot

concentrations were observed with F1 + F2 while being the minimum in those

where no fungi was applied. It was observed that shoot Ca increased with the

105

increasing TSW ratio in soil mixtures for 5 and 10 % and then decreased for

20 %.

Table 3.9A.3. The concentration of Category-I Metals (mgkg-1

) observed in SHOOT of

55-days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


LSD0.05 C F1 F2 F1+F2

Ca

0 13bcD

0.38 16

bDE

0.11 11

bcE 0.09 25

aDE 0.16 4.8

5 110dB

0.12 390

abA

0.22 360

bA 0.41 425

aA 0.33 85

10 145cdA

0.17 410

abA

0.29 380

bA 0.38 455

aA 0.66 75

20 95cdB

0.88 335

bBC

0.56 340

bAB

0.61 395

aAB 0.91 80

LSD0.05 35 110 98 125

K

0 14cD

0.38 20

bDE

0.87 22

bD 0.18 35

aD 1.09 6.2

5 220cdA

0.78 310

bA

0.38 325

bA 0.68 380

aAB 0.56 49

10 245dA

0.18 295

cdA

0.18 345

bA 0.12 410

aA 0.97 45

20 195dB

0.99 305

bcA

0.67 330

bA 1.09 400

aA 0.38 55

LSD0.05 62 75 85 110

Na

0 5cdDE

0.09 12

bcDE

0.05 18

aE 0.1 22

aDE 0.56 4.8

5 140dA

0.18 260

bA

1.09 275

bA 0.49 355

aB 0.67 55

10 90eBC

0.44 280

cdA

0.67 295

cdA 0.38 425

aA 0.98 45

20 55deC

0.12 160

cCD

0.39 190

bcBC

0.56 270

aC 1.6 60

LSD0.05 38 75 78 109


3.9A.3.2.2 Potassium (K) in shoot



observed in 10 % with F1 + F2 while being the minimum (14 mgkg-1) in soil

with no fungi i.e. C.


concentration of TSW in soil mixtures for all the concentrations except for

20% where the values were observed to be decreased. For all TSW-Soil

mixtures, the F2 plants showed more K uptake than those with F1 and being

the least where no fungus was applied. The maximum values observed in

plants cultivated in 10% TWS-Soil mixture.

3.9A.3.2.3 Sodium (Na) in shoot

The Na concentration in shoot observed to be increased along the row

and it was because of fungal applications. The pots with F1 + F2 showed the

106






the shoot Na uptake for all the fungal treatments except 20 %. In case of 0 %,

the concentration of shoot Na observed to be the least for all the fungal

treatments and maximum for 10% as shown in Table 3.9A.3.

3.9A.3.3 Category-I metals in plant ROOT



soil mixture, as given in Table 3.9A.4.

3.9A.3.3.1 Calcium (Ca) in root

The application of fungal inoculum to the soil helps to increase Ca

uptake along the row i.e. various fungal treatments. The plants in F1 + F2 pots



highest root Ca (410 mgkg-1) was in 5 and 10 % with F1 + F2 while being the


3.9A.3.3.2 Potassium (K) in root




individual i.e. F1 and F2 showed better K uptake than TSW-Soil mixtures

where no fungi has been applied. However, F2 showed better uptake as

compared to F1 treatment except for 0 % where F1 showed slightly better

value (12 mgkg-1) as compared to F2 (11 mgkg-1).

Within column, the metal uptake in root was found to be maximum in

10% while being minimum in 0 %.

107





w:w)


C F1 F2 F1+F2

Ca

0 6cdDE

0.28 12bE

0.77 9bcE

0.33 18aDE

0.16 3.9

5 55dBC

0.38 355bcA

0.49 335bcA

0.28 410aA

0.89 92

10 125deA

0.45 390bA

1.6 340bcA

0.36 410aA

0.34 110

20 40deC

0.81 180cC

1.4 210bcCD

0.19 305aBC

0.29 95

LSD0.05 35 99 105 85

K

0 8cDE

0.12 12bD

0.58 11bDE

0.91 20aD

0.17 4.2

5 110deAB

0.38 210bA

0.87 225abA

0.45 290aA

0.92 55

10 160dA

1.6 230bA

0.13 260bA

0.38 315aA

0.45 60

20 75cdC

0.38 195bAB

0.099 220aAB

0.11 270aAB

0.19 58

LSD0.05 40 65 80 75

Na

0 3cDE

0.67 6bcDE

0.098 9aE

0.34 12aE

0.96 8.3

5 90deA

0.95 220cA

0.25 245bcA

0.38 320aAB

1.3 90

10 70dAB

0.38 190bcA

0.76 210bcAB

0.96 380aA

1.9 80

20 45cdC

0.45 140bcBC

1.08 155bcCD

2.5 245aCD

2.7 65

LSD0.05 28 65 75 90


3.9A.3.3.3 Sodium (Na) in root


helps increase Na uptake in roots. The maximum Na in root (380 mgkg-1) was

observed in 10 % with F1 + F2 while being minimum in 0 % amounting 3

mgkg-1. Those with F1 and F2 applications also performed better than C i.e.

treatment with no fungal inoculation.




treatments.

3.9A.3.4 Category-II metals in plant shoot



in soil, as given in Table 3.9A.5. The application of fungi enhanced trace

metal uptake tendency of plant for all the TSW-Soil mixtures. However, the



108

Table 3.9A.5. The concentration of Category-II Metals (mgkg-1

) observed in SHOOT of 55-

days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2

Cd

0 2deD

0.23 8cDe

0.12 12cE

0.34 25aDE

0.78 8.9

5 210efAB

0.22 390dBC

0.99 425cdCD

1.1 970aAB

1.3 205

10 290eA

1.03 510cA

1.03 590cA

1.5 1,020aA

2.3 190

20 280deA

1.6 490cA

0.12 530cA

1.9 1,050aA

2.6 220

LSD0.05 78 127 155 265

Cr

0 2cdDE

0.04 5cDE

0.3 8bcD

0.11 15aE

0.91 3.8

5 245efB

0.12 560dAB

0.82 625cA

1.4 1,415aCD

1.3 280

10 315eA

1.12 610dA

0.72 670cdA

1.32 1,380aA

2.1 275

20 295efA

0.12 590dA

0.38 600dB

1.91 1,265aB

1.9 255

LSD0.05 83 168 178 365

Cu

0 20cdE

0.29 60bD

0.022 55bDE

0.38 90aE

1.23 19.2

5 450cB

0.19 630aBC

0.11 670aA

1.1 690aC

1.4 55

10 510deA

0.28 760bcA

0.38 710bcA

1.3 990aA

2.9 125

20 325dCD

0.28 410cCD

1.1 485cC

1.9 990aA

1.2 175

LSD0.05 128 185 168 235

Fe

0 BDL BDL BDL BDL -

5 40cB

0.31 70bcC

0.23 110aB

0.12 220aB

1.5 48

10 60cA

0.22 105aAB

0.34 110aB

1.4 120aCD

0.91 25

20 55eA

0.11 130cA

0.31 145cA

1.2 325aA

0.94 72

LSD0.05 6.3 18 9 53 13

Mg

0 BDL BDL BDL BDL -

5 45cC

0.23 90bcBC

0.29 115bB

0.23 170aC

0.321 35

10 80cdA

0.78 130bA

0.56 155bA

0.12 290aA

0.38 55

20 70dA

0.99 145cA

0.38 165cA

0.19 310aA

0.29 65

LSD0.05 4 15 15 38

Ni

0 BDL BDL BDL BDL -

5 8bC

1.2 10bCD

0.11 12aD

0.38 15aC

0.31 3

10 11bcC

0.38 12bcCD

0.20 15bD

0.91 20aC

1.51 4

20 35bA

0.32 40bA

0.98 55aA

0.93 60aA

1.44 8

LSD0.05 8 9 12 13

Zn

0 70bD

0.11 90bD

0.37 110abDE

0.22 145aD

0.74 20

5 220deBC

0.38 425cdB

1.5 470cdB

0.11 875aA

0.19 165

10 340dA

0.18 510cA

1.3 550cA

0.45 920aA

0.29 148

20 290eB

0.36 465cdA

1.2 490cdAB

0.78 895aA

0.75 154

LSD0.05 69 107 114 195


3.9A.3.4.1 Cd in shoot



fungal treatments. Maximum amount of metal (1,020 mgkg-1) was observed in


being minimum in 0% amounting 2 mgkg-1.

109

Within different concentrations there is increasing trend of metal

accumulation with TSW-Soil TWS-Soil mixture

3.9A.3.4.2 Cr in shoot




found to be as low as 2 mgkg-1 in 0% with C and maximum 1,415 mgkg-1 in

5% with F1 + F2.

Within different TWS-soil mixtures there was maximum accumulation of

metal was observed in 10% for all fungal treatments except F1 + F2, as for

this treatment the maximum accumulation was observed in 5% as shown in

Table 3.9A.5.

3.9A.3.4.3 Cu in shoot




The plants from treatments with no fungal inoculations i.e. C showed the

significantly least Cu accumulation as compared to any of the fungal

treatments as shown in Table 3.9A.5.

Within different TWS-Soil mixtures, 0% showed significantly less Cu

uptake in shoots than those form 5, 10 and 20 % as minimum accumulation

20 mgkg-1 was seen in 0% with C and maximum accumulation 990 mgkg-1 in

10 and 20% with F1 + F2.

3.9A.3.4.4 Fe in shoot

The Fe uptake in plant shoots observed to be BDL in Soil (0%) with C,



The values of plant shoot Fe was minimum 40 mgkg-1 in 5% with C and


110

Within a column, maximum accumulation was observed in 10 % as


3.9A.3.4.5 Mg in shoot




+ F2 in 20 % (TSW-Soil), while being the minimum as well as significantly

least in shoot of plants cultivated in soil with no fungus i.e. 45 mgkg-1 as

shown in Table 3.9A.5. Such a pattern was observed for all the TSW-Soil

mixtures.

For different TSW-Soil mixtures the value of plant shoot Mg was found

to be BDL in 0%. For 10 %, the shoot uptake found to be highest as

compared to 5 and 20 % TSW-Soil mixture.

3.9A.3.4.6 Ni in shoot

The Ni concentration observed to be BDL in 0% for all the fungal

treatments along the row. However, for all the TSW-Soil mixtures the shoot Ni

concentration increased with the application of fungus and observed to be the

maximum in plants inoculated with F1 + F2 and being the least in those from

C i.e. where no fungus was applied.

Within the columns comparison, the value of plant shoot Ni



plants from 5 %.

3.9A.3.4.7 Zn in shoot






111


compared to F1 in terms of accumulation of metal as shown in Table 3.9A.5.

For different TWS-Soil mixtures, the value of Zn concentration in shoot


70 mgkg-1 in 0% with C as shown in Table 3.9A.5. The order of accumulation

of metal accumulation within different TWS-Soil mixtures from maximum to

minimum was as 10 % >20 % > 5% > 0%.

3.9A.3.5 Category-II metals in plant root



fungal inoculations, as given in Table 3.9A.6.

3.9A.3.5.1 Cd in root


roots harvest from Soil observed to be minimum 5 mgkg-1 in 0% with F1 and

found to be maximum 990 mgkg-1 in 20 % with F1 + F2 as shown in Table

3.9A.6. The plant root Cd concentration increased with fungal inoculations

and found to be maximum as well as significantly higher than those applied

with single or no fungal treatments, while being the BDL in 0 % with C.

Inside the columns, for different TWS-Soil mixtures the root Cd

concentration was found to be highest in 10% with C, F1 and F2 except F1 +

F2. The highest concentration was noted in 20% with F1 + F2 i.e. 990 mgkg-1.

3.9A.3.5.2 Cr in root


F1 fungal inoculations showed the maximum 1,320 mgkg-1 root Cr

concentration in 5 % (TSW-Soil), while with F2 treatment plants having the

minimum uptake 2 mgkg-1 in 0%. The F2 inoculation enhanced the root Cr

concentration better than F1. However for C and F1 fungal treatments the

values found to be BDL as shown in Table 3.9A.6.

112

Within the columns, the root Cr concentration increased with increasing

percentage of TSW in soil having maximum accumulation of metal in 10 % for

all fungal treatments like C, F1, F2 except F1 + F2.





w:w)


C F1 F2 F1+F2

Cd

0 BDL 5cdD

0.02 7cdE

0.09 21aDE

0.34 5.6

5 190efB

0.89 355dAB

0.38 390dC

0.56 910aAB

0.38 140.8

10 250eA

0.69 485dA

0.86 510cdA

0.88 980aA

0.78 185

20 210dAB

0.45 455cA

0.12 490cA

0.99 990aA

0.98 179

LSD0.05 18 124 128 245

Cr

0 BDL. BDL 2cD

0.01 8aE

0.2 1.3

5 225deB

0.38 390dA

0.99 420cdA

1.23 1,320aA

0.38 205

10 275dA

1.28 425cA

1.2 455cA

0.38 1,290aA

1.23 195

20 235cdA

1.37 370cA

0.38 445bcA

1.11 1,050aB

1.4 215

LSD0.05 14 15 115 330

Cu

0 BDL 15deDE

0.78 20dD

0.36 45aE

0.26 4.9

5 380bcA

1.03 410abA

0.96 425aA

1.2 430aC

1.33 13.4

10 295efBC

1.05 525cA

0.78 505dA

1.07 685aA

1.36 79

20 210eCD

1.3 390dB

1.2 455cdA

0.97 860aA

1.22 165

LSD0.05 45 128 124 205

Fe

0 BDL BDL BDL BDL -

5 35dA

0.45 55cdCD

1.8 85bcBC

0.38 120aC

0.13 24

10 45eA

0.23 80dBC

0.07 110cdA

0.34 235aA

0.32 49

20 30dB

0.35 115bA

0.38 125bA

1.2 210aA

0.29 68

LSD0.05 5 18 12 30

Mg

0 BDL BDL BDL BDL -

5 30cBC

0.12 55abC

0.73 60aC

0.44 65aBC

0.31 7.4

10 45dAB

1.2 95bAB

0.94 120aA

0.51 130aAB

0.22 23

20 55deA

0.99 115cA

0.12 135cA

0.88 290aA

0.44 60

LSD0.05 8 18 20 58

Ni

0 BDL BDL BDL BDL -

5 5cdDE

0.12 5cdCD

0.12 8bC

0.19 10aB

0.16 1.53

10 12cdC

0.38 10dC

0.76 13cdBC

0.31 18aAB

0.35 1.6

20 30bA

0.43 32abA

0.87 35aA

0.44 30bA

1.5 1.3

LSD0.05 6.8 7 9 8

Zn

0 55cdC

0.23 75bcD

0.25 90bCD

0.87 115aC

1.3 18

5 190cA

0.56 380aA

0.31 410aA

0.34 425aAB

1.7 58.8

10 230eA

0.98 415cA

0.23 435cA

0.11 670aA

1.2 112

20 210dA

0.48 395bcA

0.46 455bcA

0.38 690aA

0.99 155

LSD0.05 46 90 95 145


3.9A.3.5.3 Cu in root

Along the row for various fungal inoculations the root Cu uptake was

enhanced as compared to C. The value was found to be BDL in 0% for C

treatment. The plants from F1 + F2 observed to have the maximum (860

113

mgkg-1) uptake in 20 % than those cultivated in pots with single or no fungal

inoculations and found to be the minimum 15 mgkg-1 in 0% with F1.

For different columns, the plants from 5 and 10 % TSW-Soil exhibited

better root Cu accumulation than soil (0 %) and 20 %.

3.9A.3.5.4 Fe in root


treatments, the root Fe observed to be BDL in soil (0 %) with all fungal

treatments. The plants from C had the lowest root Fe as compared to plants

from any of the single or combined application of fungi. The root Fe

accumulation observed to be the maximum (235 mgkg-1) in 10 % with F1 + F2



Inside columns, the maximum Fe uptake by roots was observed in 10

% while the least value of metal uptake was observed in 5 % except the C

treatment as shown in Table 3.9A.6.

3.9A.3.5.5 Mg in root

While analyzing the fungal application effect on plants along the rows,

it was observed that the Mg root concentration increased in plant roots with

fungal application as compared to C i.e. with no fungal inoculation. The F1 +

F2 plants displayed the maximum root Mg concentration than those from C as

well as F1 and F2. For 0% the values for metal uptake were found to be BDL

for all fungal treatments. However the F2 inoculations gave better results than

F1.

For the columns, the root Mg accumulation increased with increasing

percentage of TSW in soil for all fungal treatments being maximum in 20%

with F1 + F2 where the metal accumulation was observed to be 290 mgkg-1

while minimum concentration 30 mgkg-1 in 5% with C treatment was noted as


3.9A.3.5.6 Ni in root

114





minimum in C with no fungus added.



maximum (35 mgkg-1) accumulation of metal was noted in 20% with F2 while

minimum uptake (5 mgkg-1) was observed in 5% with C and F1.

3.9A.3.5.7 Zn in root






For different TWS-Soil mixtures, the root Zn concentration increased

with increasing percentage of TSW in soil with every fungal treatment except

in 20% where the metal uptake was reduced. However the maximum

accumulation was noted in 20 % with F1 + F2 i.e. 690 mgkg-1 and minimum

(55 mgkg-1) in 0 % with C treatment as shown in Table 3.9A.6.

3.9A.4 Fungal analyses

The results of estimation of the post-harvest fungal analyses (× 105

c.f.u. g-1 soil) of 55-days old Tagetes patula cultivated on TSW-Soil mixtures

are shown in Table 3.9A.7. Alongside the row, the c.f.u. increased with fungal

application than C and observed to be the maximum in treatments with

combined application of both of the fungi. The order of c.f.u. abundance was

F1 + F2 > F2 > F1 > C.

Within a column, the c.f.u. abundance was observed in 5% with 11.1 ×

105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2. Statistical

115

analysis indicated that there is significant difference of 5% with other TWS-

Soil mixtures.

Table 3.9A.7. The post-harvest fungal analyses (× 105 c.f.u. g


patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).

TSW-Soil (% w:w) mixture and its type

Treatment LSD0.05

C F1 F2 F1+F2

Soil 1.5deBC

0.22 3.6dAB

0.17 4.2cdA

0.23 9.8aB

0.29 2.3

5 1.8cdA

0.26 4.1bcA

0.18 4.5bcA

0.21 11.1aA

0.13 4.6

10 1.6dA

0.16 3.2cB

0.33 3.9bcAB

0.07 7.5aCD

0.51 1.98

20 1.4cdBC

0.24 2.8bCD

1.2 2.9bBC

0.38 3.8aD

0.98 0.57

LSD0.05 0.19 0.36 1.8 2.9


3.9A.5 Meta-analytical perspective



3.9A.5.1 Category-I metals translocation index (%)

The plant translocation index values were also recorded for Category-I


3.9A.8.

Table 3.9A.8. The Category-I metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Ca

5 200 109.85 101.40 103.65

10 116 105.12 111.76 110.97

20 237.5 186.11 161.90 129.50

K

5 200 147.61 144.44 131.03

10 153.12 128.28 132.69 130.15

20 260 156.41 150 148.14

Na

5 155.55 118.18 112.24 110.93

10 128.57 147.36 140.47 111.84

20 122.22 114.28 122.58 110.20


In case of Ca, maximum value was observed in 20% (TSW-Soil) with C

i.e. 237.5 % while the minimum value was recorded (101.40 %) in 5 % (TSW-

Soil) with F2.

116

For K, the maximum translocation index value was calculated in 20%

with C i.e. 260 % being the minimum 138.28 % in 10% with F1.

In case of Na, the maximum value 155.55 % was observed in 5% with

C treatment and least value was recorded in 20 % (TSW-Soil) for F1 + F2

(110.20 %).

3.9A.5.2 Category-II metals translocation index (%)


with fungal inoculum than that of C, being the maximum where F1 and F2

applied together for all metals as given in Table 3.9A.9. As compared to

different TWS-Soil concentrations there was decreasing trend observed with

increasing percentage of TWS-Soil with few exceptions.

Table 3.9A.9. The Category-II metals translocation index (%) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Cd

5 110.52 109.86 108.97 106.59

10 116 105.15 115.69 104.08

20 133.33 107.69 108.16 106.06

Cr

5 108.88 141.02 148.8 107.2

10 114.55 143.53 147.25 106.98

20 125.53 159.45 134.83 120.47

Cu

5 118.42 153.65 157.64 160.47

10 172.88 144.76 140.59 144.52

20 154.54 141.37 106.59 115.11

Fe

5 114.28 127.27 129.41 183.33

10 133.33 131.25 100 51.06

20 183.33 113.04 116 154.76

Mg

5 150 163.63 191.66 261.53

10 177.77 136.84 129.16 223.07

20 127.27 126.08 122.22 106.89

Ni

5 160 200 150 150

10 91.66 120 115.38 111.11

20 116.66 125 157.14 200

Zn

5 115.78 111.84 114.63 205.88

10 147.82 122.89 126.43 137.31

20 138.09 117.72 107.69 129.71


For Cd, the maximum Translocation index (133.33 %) was noted in

20% (TSW-Soil) with C, while minimum value was recorded 104.08 % in 10%

with F1 + F2.

117

In case of Cr plants showed maximum metal translocation efficiency

(159.45 %) in 20% with F1 and minimum 106.98 % in 10 % with F1 + F2.


172.88 % for 10% with C, while minimum value was recorded in 5 % with C

treatment i.e. 118.42 %.


183.33 % for 5 % with F1 + F2 and for 20 % with C, while minimum value was

recorded in 10% with F1 + F2 treatment i.e. 51.06 %.

In case of Mg the plants showed least value in 20% with F1 + F2

(106.89 %), while the maximum values for this metal was recorded in 10%

with F1 + F2 i.e. 223.07 %.

For Ni, there was 200% translocation index value recorded in 5% with

F1 and in 20% with F1 + F2 treatment then least value was observed in 10%

with C i.e. 91.66 %.


recorded in 5 % with F1 + F2 treatment i.e. 205.88 % while minimum value

was recorded in 20% with F2 i.e. 107.69 % as shown in Table 3.9A.9.

3.9A.5.3 Tolerance index (TI)

In shoots TI values were found to be highest in 5 % with F1 + F2

(1.29) while minimum value was recorded to be 0.86 in 20% with C as shown

in Table 3.9A.10.

Table 3.9A.10. The tolerance index (TI) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

TI Shoot

5 1.04 1.10 1.10 1.29

10 0.93 1.16 1.03 1.18

20 0.86 0.89 0.90 0.95

TI Root

5 1.22 1.36 1.28 1.31

10 1.60 1.06 1.01 1.01

20 0.94 0.80 0.74 0.72


118

In case of TI in roots 1.60 was recorded as maximum for plants grown

in 10% with C, while 0.72 were recorded as minimum value in 20% with F1 +

F2.

3.9A.5.4 Category-I metals Specific extraction yield percentage (SEY %)

SEY % for Category-I metals i.e. Ca, K and Na showed in Table

3.9A.11.

In case of Ca, there was maximum value 51.38 % was recorded for

plants grown in 5 % (TSW-Soil) with F1 + F2, and minimum 4.63 % in 20 %

(TSW-Soil) with C.

In case of K, plants cultivated in 5 % TWS-soil showed highest value of

SEY% (75.28 %) with F1 + F2 and minimum in 20 % with C i.e. 13.63 %.

As in case of Ca and K, the highest value for Na was recorded in 5 %

with F1 + F2 (49.81 %) while minimum values was found to be 2.15 % in case

of 20% with no fungal inoculum i.e. C.

Table 3.9A.11. The Category-I specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Ca

5 10.15 45.84 44.0 51.38

10 11.51 34.11 30.70 36.88

20 4.63 17.69 18.90 24.05

K

5 37.07 58.42 61.79 75.28

10 33.47 43.33 50 59.91

20 13.63 25.25 27.77 33.83

Na

5 16.97 35.42 38.37 49.81

10 6.36 18.68 20.07 32.00

20 2.15 6.45 7.42 11.08


3.9A.5.5 Category-II metals Specific extraction yield percentage (SEY %)

The SEY (%) was calculated in Category-II metals that were detected

by AAS as shown in Table 3.9A.12. Overall a similar kind of trend was seen

for all metals in various fungal treatments along the row, that the SEY %

values increased with the application of fungal inoculum and highest value

was observed for F1 + F2 treatment.

119

In case of Cd the maximum value for SEY % was recorded in 5 %

(TSW-Soil) with F1 + F2 treatment i.e. 70.94 %, while minimum (5.6 %) was

observed in 20% (TSW-Soil) with C.

Similarly the SEY % value for Cr was found to be highest (33.15 %) in

5 % (TSW-Soil) with F1 + F2 and minimum (3.41 %) was recorded in 20%

with no fungal inoculum i.e. C. However F2 showed greater values as

compared to F1.

For Cu again the maximum value was observed for 5 % (79.76 %) with

combined fungal inoculum F1 + F2 and least value (10.19 %) was recorded in

20% (TSW-Soil) with C.

Table 3.9A.12. The Category-II metals specific extraction yield (SEY %) analyzed in 50-days old Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Cd

5 15.09 28.11 30.75 70.94

10 8.20 15.12 16.71 30.39

20 5.6 10.8 11.65 23.31

Cr

5 5.69 11.39 12.66 33.15

10 5.75 10.09 10.97 26.04

20 3.41 6.18 6.73 14.92

Cu

5 61.48 77.03 81.11 82.96

10 38.33 61.19 57.85 79.76

20 10.19 13.33 17.90 35.23

Fe

5 30 50 78 136

10 20.58 36.27 43.13 69.60

20 9.34 26.92 29.67 58.79

Mg

5 24.19 46.77 56.45 75.80

10 20.16 36.29 44.35 67.74

20 12.25 25.49 29.41 58.82

Ni

5 37.14 42.85 57.14 71.42

10 41.81 40 50.90 69.09

20 59.09 65.45 81.81 81.81

Zn

5 28.08 55.13 60.27 89.04

10 30.15 48.94 52.11 84.12

20 24.69 42.46 46.66 78.27


As in case of above mentioned metals again the highest (136 %) and

lowest (9.34 %) values were recorded in 5% with F1 + F2 and 20 % with C

respectively for Fe. As far as the highest and lowest values are concerned

there was a same trend seen in case of Mg, where plants in 5 % (TSW-Soil)

120

showed highest SEY% value (75.80 %) with F1 + F2 and being minimum

(12.25 %) in 20% (TSW-Soil) with C having no fungal inoculum.

There was maximum value for Ni was calculated (81.81 %) in 20%

(TSW-Soil) with combined fungal inoculum and F2, while minimum value

(37.14 %) was recorded in 5 % (TSW-Soil) for C.

In case of Zn the maximum value was noted (89.04 %) for 5 % with F1

+ F2 and minimum value (24.69 %) in 20 % (TSW-Soil) with C.

121

3.9B. Experiments with Helianthus annuus inoculated with saprobic

fungi

3.9B.1 Pre-sowing analysis



Chapter 3.1.

3.9B.2 Biochemical analyses of 52-days old Helianthus annuus





under:

3.9B.2.1 Chlorophyll content

After 52 days of cultivation, the plant chlorophyll content within fungal

treatments increased by applying combined fungal inoculation i.e. the F1 +

F2, as given in Table 3.9B.1. The greatest (28.7 SPAD value) was observed

in 10 % with F1 + F2 while influencing to the least (14.1 SPAD value) in 20 %

in C treatment as compared to any of the treatments within a row. However

F2 showed better values with respect to chlorophyll content as compared to

F1 values for all TWS-soil mixtures.

Within column, there was increase in plant chlorophyll contents with the

increase of TSW percentage in soil i.e. 10 % for all the fungal treatments but

the values were dropped being the minimum in 20%. The application of

fungus either individually or in combination help plants perform better as

compared to C in terms of chlorophyll contents, as given in Table 3.9B.1.

3.9B.2.2 Soluble protein contents



inoculations for all the treatment, as given in Table 3.9B.1. The F1 + F2 from

10 % and the F1 from 0 % exhibited the maximum (21.8 mgg-1) and the

122

minimum (0.8 mgg-1) soluble protein contents as compare to any of the soil or

fungal treatments. Within Fungal treatments C showed minimum values as

compared to other fungal applications like F1, F2 and F1 + F2.

Within a column, there was increase in soluble protein contents with

the increasing level of TSW in soil treatments i.e. in 10 % and then decrease

in 20%. However, addition of fungi helped plants to lessen stress by

increasing soluble protein contents. The plants cultivated in pots with F2

inoculations performed better than those with F1 for all the TSW-Soil

mixtures.

3.9B.2.3 Superoxide dismutase (SOD) contents



treatments, as given in Table 3.9B.1. The SOD values were found to be 0.5

mgg-1 in 0% with C bring minimum. For the rest of the concentrations the

Table 3.9B.1. The biochemical parameters observed in 52-days old Helianthus annuus



w:w)


C F1 F2 F1+F2

Chlorophyll content

(SPAD value)

0 17.7bcBC

1.1 18.5abC

2.1 19.1abCD

0.97 21.9aD

1.1 1.2

10 19.9dA

2.1 22.5cA

1.5 23.3cA

0.22 28.7aA

1.3 2.2

20 14.1bcDE

1.4 15.4aE

0.94 15.9aE

0.34 16.1aEF

1.2 1.5

LSD0.05 0.85 1.9 0.83 1.8


(mgg-1

)

0 0.9bcF

1.5 0.8bcF

0.43 1.1abEF

0.22 1.6aF

0.22 0.19

10 16.1cA

0.66 17.2bcA

1.5 17.8bcA

2.1 21.8aA

0.32 1.4

20 12.7dC

0.34 14.3bcBC

1.7 14.5bcC

0.37 15.9aCD

0.33 0.27

LSD0.05 3.2 4.9 4.3 5.2

SOD (Umg

-1 of protein)

0 0.5cDE

0.97 0.8abE

0.34 1.9abDE

1.3 1.1aCD

0.12 0.15

10 22deA

0.54 37bcA

0.97 39bcA

0.24 44aA

0.34 5.1

20 24cdA

1.2 35bA

0.33 37bA

0.29 41aA

0.97 4.2

LSD0.05 6.8 11.1 14 15

CAT (Uml

-1)

0 BDL BDL BDL BDL -

10 18eA

0.99 24deA

1.4 28cdA

0.92 39aA

0.66 3.4

20 12deBC

1.3 21bcCD

0.12 26aB

0.38 28aDE

0.13 4.3

LSD0.05 1.1 0.98 0.89 3.9

C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference

123

fungal treatments having both of the F1 and F2 performed better than control

and those applied with either F1 or F2. The plants in 10 % with F1 + F2 gave

maximum SOD values (44 Umg-1 of protein).

While analyzing the response of plant cultivated in different TWS-Soil

mixtures, it was observed that the 0% showed least values, while 10 %

showed maximum SOD values in all fungal treatments except C treatment

where highest value was recorded for 20 %.

3.9B.2.4 Catalase (CAT) contents

Within different treatments along the row, there was an increase in

plant CAT value with the individual or combined fungal inoculations, as

compared to control. The plants from 0 % showed CAT value to be BDL for all

fungal treatments. The maximum value was noted (39 Uml-1) in 10 % with F1

+ F2 while the minimum (12 Uml-1) CAT value was noted in 20 % with C

treatment as given in Table 3.9B.1.

For different TSW-Soil mixtures comparison, greater CAT content was

observed in 10% as compared to 20 % for all fungal treatments.

3.9B.3 Post-harvest analysis

3.9B.3.1 Growth performance of Helianthus annuus

Better growth of 52-day-old plants of H. annuus was observed in case

of lower TWS-soil TWS-Soil mixture i.e. 10%, being relatively less in soil (0%)

and 20% as indicated by growth parameters (Table 3.9B.2). It was noticed

that plants cultivated in soil and its TSW mixtures inoculated with fungal

isolates yielded greater shoot, root and seedling length, no. of leaves and

roots, as well as, fresh and dry weight; as compared to control. The statistical

analysis of the data showed significant growth in all parameters in lower TSW

concentration in soil followed by a decrease at higher (20%) concentration.

However, the maximum increase in values was found in F + M treatment over

their controls F1 and F2, for each of the corresponding soil treatments. The

details of each of the morphological parameters is given in Table 3.9B.2 and

described as under:

124

3.9B.3.1.1 Shoot, root and seedling length (cm)

Along the row in comparison with different fungal treatments, the

maximum plant shoot (70.2 cm), root (61.1 cm) and seedling length (131.7

cm) was observed in 10 % with F1 + F2 inoculation while being minimum

values for plant shoot (33.1 cm) and seedling length (71.3 cm) in 20 % with no

fungal inoculation i.e. the C. The minimum value for plant root length (32.5

cm) was recorded in 0% with control having no fungal inoculation. There was



F1 > C along the row as shown in Figure 3.9B.1.

Figure 3.9B.1. The vegetative growth variation in sunflower (Helianthus annuus) in response to soil

mixed with different percentages of TSW (% w:w) and inoculated with different fungi.

While in comparison with different TWS-Soil mixture there was increase in

plant shoot, root and seedling length with the increasing proportion of TSW in

the soil. The different TSW-Soil mixtures under F1 + F2 column gave best

results while those in C column attained the least height.

3.9B.3.1.2 No. of leaves and roots

Along the row, the plants in 10 % with F1 + F2 inoculation observed to


and 8 respectively) in 20 % without any of the inoculation i.e. C and 0% with

C.

125

Within different percentage of TWS-Soil, the increasing ratio of TSW

increased the no. of leaves and roots upto 10% but there was decreased for

these parameters in 20%. The TSW-Soil mixtures under F1 + F2 gave the

best vegetative growth than any of the fungal treatments. The least growth

response observed to be in C column.

Table 3.9B.2. Various morphological parameters observed in 52-days old Helianthus annuus




C F1 F2 F1+F2

Shoot Length (cm)

0 44.1efB

2.9 51.2dB

1.2 56.3bcA

1.1 67.8aA

1.3 5.68

10 48.1eA

1.4 59.5cA

1.3 61.2cA

1.05 70.2aA

1.22 5.1

20 33.1efCD

1.1 41.2cdDE

0.99 43.8cdC

1.03 54.1aD

0.99 4.97

LSD0.05 3.8 4.7 5.7 4.2

Root Length (cm)

0 32.5dD

0.98 38.7cC

1.1 41.1bcBC

1.5 49.7aCD

1.4 4.49

10 43.3deA

2.8 50.3bcA

1.4 52.3bA

1.1 61.1aA

1.07 5.3

20 36.8dC

2.5 38.3cdC

0.88 39.5cdC

1.09 56.1aB

1.02 5

LSD0.05 3.1 3.9 4.7 3.4


0 76.9dBC

0.99 90.2cC

1.4 97.7bcB

1.3 117.9aB

1.4 11.5

10 91.4cdA

1.9 110.1bA

1.2 113.8bA

1.2 131.7aA

1.31 11.1

20 71.3cdC

2.3 79.8cDE

0.98 83.6cCD

1.6 110.6aBC

0.93 10.7

LSD0.05 5.72 7.82 11.3 12.6

No. of roots

0 18dAB

2.1 23bcB

3.3 26bcA

2.1 33aB

1.6 4.2

10 21cdA

3.1 28bcA

2.2 31bA

2.2 39aA

1.9 3.7

20 13cCD

0.45 16bD

0.89 18bC

2.4 22aCD

2.1 2.5

LSD0.05 2.63 3.7 5.1 5.6

No. of leaves

0 8dC

0.99 12bBC

1.3 13bAB

2.1 16aB

2.1 2.1

10 11dA

0.45 14bcA

1.1 15bcA

1.2 18aA

2.5 1.93

20 9cdC

0.33 11cdC

1.9 14bA

0.22 17aA

1.9 1.88

LSD0.05 0.44 0.59 0.62 0.9

Fresh wt. (g)

0 9.5cAB

1.01 11.4bcA

2.09 13.2aA

1.1 15.1aBC

2.09 1.8

10 11.5cA

1.1 12.1cA

2.2 14.3bcA

2.3 19.9aA

0.99 2.9

20 9.1cdAB

1.09 10.4cB

0.11 11.6cAB

2.2 16.7aBC

0.22 2.1

LSD0.05 1.2 1.1 2.1 2.6

Dry wt. (g)

0 2.3cdAB

1.1 2.9cA

0.23 3.2abA

1.2 3.6aC

1.9 0.38

10 2.9bcA

1.2 3.1bcA

0.99 3.6bcA

2.1 4.8aA

0.76 0.72

20 2.1cAB

0.98 2.4bcAB

0.34 2.6bcBC

2.09 3.8aC

0.33 0.49

LSD0.05 0.8 0.9 1.2 2.1


3.9B.3.1.3 Fresh and dry weight (g)





observed to be in 10 % with F1 + F2 and the minimum (9.1 g fresh, 2.1 g dry)

in 20 % with C, without any fungus respectively.

126


production positively for lower percentage i.e. 10 % and negatively for 20 %

TWS-Soil mixture The TSW-Soil mixtures under F1 + F2 yielded maximum

fresh and dry weight while those in C column yielded the least.

3.9B.3.2 Category-I metals in plant SHOOT



ratio of TSW in soil, as given in Table 3.9B.3.

3.9B.3.2.1 Calcium (Ca) in shoot




Within different concentrations of TSW in soil, the maximum shoot



increasing TSW ratio in soil mixture for 10 % and then decreased for 20 %.

3.9B.3.2.2 Potassium (K) in shoot


for all the TSW-Soil mixtures as in case with Ca. The maximum K level (765

mgkg-1) was observed in 20 % with F1 + F2 while being the minimum (30

mgkg-1) in 0% with no fungi i.e. C.


concentration of TSW in soil mixtures for all TSW-Soil mixtures, the F2 plants

showed more K uptake than those with F1 and being the least where no

fungus was applied i.e. the C treatment. The maximum values observed in

plants cultivated in 20% TWS-Soil mixture.

3.9B.3.2.3 Sodium (Na) in shoot



127

highest Na shoot uptake, the F2 being greater than F1, while those with no

fungi being the least. The value for Na in 0 % with C treatment found to be

BDL. The plants in 20 % with F1 + F2 had the highest value (520 mgkg-1)

while those in 0 % with F1 exhibited the lowest Na shoot contents (35 mgkg-1).

Table 3.9B.3. The concentration of Category-I Metals (mgkg-1


days old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


LSD0.05 C F1 F2 F1+F2

Ca

0 16dE

0.12 22cDE

0.76 28bcD

0.17 44aDE

0.23 8.4

10 125cdBC

0.34 230cA

1.3 245cA

0.19 380aB

0.44 65

20 190dA

0.67 250cdA

1.1 270cdA

0.99 430aA

1.6 63

LSD0.05 64 78 86 135

K

0 30deD

0.65 60cE

0.76 75cDE

1.01 145aD

1.1 32

10 210eA

1.2 390cA

2.1 410cAB

1.6 670aA

1.7 125

20 270eA

1.3 455cdA

2.4 480cdA

1.7 765aA

2.1 140

LSD0.05 82 132 143 225

Na

0 BDL 35eDE

0.99 55bcD

0.78 75aDE

1.1 11

10 155dA

2.3 180cA

1.5 225cA

1.1 430aAB

2.7 72

20 145deA

3.1 170dA

1.4 210dA

1.02 520aA

2.4 98

LSD0.05 5 54 68 155



the shoot Na uptake for all the fungal treatments except 20 % where the

values for Na were decreased as compared to 10 %. However in 20% with F1

+ F2 the Na accumulation was found to be greater than in 10 %. In case of 0

%, the concentration of shoot Na observed to be the least for all the fungal

treatments as shown in Table 3.9B.3.

3.9B.3.3 Category-I metals in plant ROOT



soil mixture, as given in Table 3.9B.4.

3.9B.3.3.1 Calcium (Ca) in root





128



3.9B.3.3.2 Potassium (K) in root




individual i.e. F1 and F2 showed better K uptake than TSW-Soil mixtures

where no fungi has been applied. However, F2 showed better uptake as

compared to F1 treatment.

Within column, the metal uptake in root was found to be increased with

increasing TWS-Soil mixtures i.e. being the maximum values in 20% than in

10 % and least values were observed in 0 %.



old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2

Ca

0 10cdD

0.1 15cDE

0.11 20abD

1.1 35aE

1.6 8.4

10 55eBC

0.23 120cdC

1.1 135cdBC

1.12 310aB

2.6 67

20 95deA

0.37 210cA

1.12 225cA

1.5 390aA

1.12 78

LSD0.05 35 74 81 128

K

0 15dDE

0.23 40cDE

0.99 55bcE

0.18 90aE

0.95 22

10 135deBC

0.26 255bcB

1.31 290bA

2.6 380aAB

1.12 68

20 180A 1.1 310

bcA 1.4 325

bcA 1.5 455

aA 2.2 76

LSD0.05 64 78 105 134

Na

0 BDL 15cdDE

0.41 20bcCD

0.12 45aD

1.2 9.4

10 90eB

1.4 125dA

1.12 145dAB

1.5 325aA

1.12 65

20 110dA

1.12 110dB

0.23 190cdA

0.98 355aA

1.05 72

LSD0.05 7.6 6.9 64 115


3.9B.3.3.3 Sodium (Na) in root



observed in 20% with F1 + F2 while being least in 0 % amounting 15 mgkg-1

with F1. Those with F1 and F2 applications also performed better than C i.e.

treatment with no fungal inoculation. For 0% with C treatment the values was

found to be BDL.

129




treatments, however the value was found to be decreased in F1 for 20 % (110

mgkg-1) as compared to 10% (125 mgkg-1).

3.9B.3.4 Category-II metals in plant shoot



in soil, as given in Table 3.9B.5. The application of fungi enhanced trace

metal uptake tendency of plant for all the TSW-Soil mixtures. However, the



3.9B.3.4.1 Cd in shoot



fungal treatments. Maximum amount of metal (1,030 mgkg-1) was observed in


being minimum in 0% amounting 35 mgkg-1 for C treatment.

Within different TWS-Soil mixtures there is increasing trend of metal

accumulation with 10 % and then the values was decreased in 20 % for all

fungal treatments.

3.9B.3.4.2 Cr in shoot




found to be as low as 3 mgkg-1 in 0% with C and maximum 1,135 mgkg-1 in

10% with F1 + F2.

Within different columns, there was maximum accumulation of metal

was observed in 10% for all fungal treatments as shown in Table 3.9B.5.

130

Table 3.9B.5. The concentration of Category-II Metals (mgkg-1


days old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2

Cd

0 35dDE

0.94 70cE

0.23 95bcDE

2.1 145aE

1.4 32

10 380eA

1.4 665cA

3.4 790cA

4.1 1,030aA

2.8 176

20 220eC

1.7 540cB

3.7 610cA

2.4 980aA

2.1 215

LSD0.05 128 205 237 310

Cr

0 3dD

0.1 6cE

0.34 9abDE

0.34 12aE

0.12 2.6

10 415deA

1.8 645cA

2.3 685cAB

2.7 1,135aA

2.5 210

20 395eA

2.1 525cdA

2.6 595cdA

2.3 985aBC

2.1 155

LSD0.05 143 228 232 390

Cu

0 BDL 15dE

0.21 10dDE

0.13 45aE

0.86 10.7

10 210eA

1.6 355cdA

1.6 335cdA

1.71 875aA

1.1 168

20 115efBC

1.41 230dCD

2.1 265dA

1.35 660aBC

0.23 145

LSD0.05 35 122 130 155

Fe

0 6dD

0.94 15cE

0.93 20bcDE

0.43 45aDE

0.87 12.7

10 55deBC

0.91 110cAB

1.01 145bA

1.1 170aBC

1.1 32

20 80efA

0.23 155cdA

1.1 170cdA

1.01 270aA

1.6 48

LSD0.05 28 45 65 72

Mg

0 20eDE

0.78 45dD

0.14 65cDE

0.45 110aD

1.12 27

10 160bcA

1.2 190abA

0.99 220aA

1.5 240aB

1.65 38

20 180dA

1.54 225bcA

1.4 265bA

1.2 310aA

2.1 42

LSD0.05 22 65 72 78

Ni

0 BDL BDL BDL BDL -

10 10cBC

0.12 15bcB

1.6 25aA

0.12 30aA

0.34 7.8

20 15cA

0.41 20abA

0.33 15cBC

0.21 25aA

0.76 3.2

LSD0.05 3.4 2.9 3.1 3.5

Zn

0 25dD

0.12 45bcDE

0.88 55bcD

0.67 80aDE

0.98 16.3

10 435deA

0.13 660bcA

2.6 710bcA

2.6 990aA

1.65 142

20 390eA

0.45 480cBC

2.9 510cB

2.1 860aBC

2.1 128

LSD0.05 142 218 227 327


3.9B.3.4.3 Cu in shoot






treatments as shown in Table 3.9B.5.

Within different TWS-Soil mixtures, 0% showed significantly less Cu

uptake in shoots than those form 10 and 20 % as minimum accumulation 15

mgkg-1 was seen in 0% with F1 and maximum accumulation 875 mgkg-1 in 10

% with F1 + F2. The value was found to be BDL in 0% with C treatment.

131

3.9B.3.4.4 Fe in shoot

The Fe uptake in plant shoots observed to be least in Soil (0%) with C

i.e. 6 mgkg-1, but F1 + F2 inoculation displayed the maximum Fe uptake in

shoot as compared to plants from the treatments with single or no fungal

inoculations. The values of plant shoot Fe was maximum 270 mgkg-1 in 20 %

with F1 + F2.


compared to 10 % and found to be least in 0% for all fungal applications as

shown in Table 3.9B.5.

3.9B.3.4.5 Mg in shoot




+ F2 in 20 % TWS-Soil mixture, while being the minimum as well as

significantly least in shoot of plants cultivated in soil with no fungus i.e. 20

mgkg-1 as shown in Table 3.9B.5.

Along the columns, the value of plant shoot Mg was found to be least in

0%. For 20 %, the shoot uptake found to be highest as compared to 10 %

TSW-Soil mixture.

3.9B.3.4.6 Ni in shoot







concentration increased in 20% with C and F1 treatment while decreased with

F2 and F1 + F2.

3.9B.3.4.7 Zn in shoot



132





compared to F1 in terms of accumulation of metal as shown in Table 3.9B.5.



25 mgkg-1 in 0% with C as shown in Table 3.9B.5. The order of accumulation

of metal accumulation within different TWS-Soil mixtures from maximum to

minimum was as 10 % >20 % > 0% for all fungal treatments.

3.9B.3.5 Category-II metals in plant root



fungal inoculations, as given in Table 3.9B.6.

3.9B.3.5.1 Cd in root




3.9B.6. The plant root Cd concentration increased with fungal inoculations

and found to be maximum as well as significantly higher than those applied

with single or no fungal treatments.


concentration was found to be highest in 10% and decreased values were

noted in 20%.

3.9B.3.5.2 Cr in root


F1 fungal inoculations showed the maximum 875 mgkg-1 root Cr concentration

in 10 %, while with C treatment plants having the minimum uptake 290 mgkg-1

in 20%. The F2 inoculation enhanced the root Cd concentration better than

F1. However in 0 % the values were found to be BDL for all fungal treatments

as shown in Table 3.9B.6.

133



old Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).


w:w)


C F1 F2 F1+F2

Cd

0 20deEF

0.23 60bcDE

1.12 90aE

1.33 110aE

1.24 28

10 310eA

1.1 520cdA

2.45 550cdA

1.23 930aA

2.53 163

20 210deC

1.34 490cA

1.23 510cA

2.1 745aBC

2.81 143

LSD0.05 65 158 162 284

Cr

0 BDL BDL BDL BDL -

10 325eA

1.65 430cdA

2.11 480cdA

2.4 875aA

2.3 145

20 290efAB

1.87 365dAB

2.3 390dBC

2.6 770aC

2.6 129

LSD0.05 13.2 28 33 39.3

Cu

0 BDL 8cEF

0.86 5cE

0.1 25aEF

0.23 5.5

10 90efA

1.5 245cdA

1.32 260cdA

2.1 670aA

1.3 164

20 65eCD

1.4 195cdC

1.3 210cdAB

2.7 455aC

1.4 111

LSD0.05 9.3 82 88 238

Fe

0 BDL 10cdF

0.45 13cdDE

0.95 30aD

0.94 6.5

10 20deD

0.44 90adCD

1.01 135bcA

1.1 210aA

1.66 98

20 60dA

0.98 120bcA

1.1 155aA

1.6 180aA

1.6 45

LSD0.05 17 39.2 55 62

Mg

0 15dDE

0.45 30cDE

0.54 45bcD

0.94 75aE

0.99 18

10 110cA

0.56 145abAB

1.54 150aBC

1.02 170aC

1.3 23

20 75dBC

0.87 190bcA

1.1 215bcA

1.01 290aA

1.1 78

LSD0.05 34 58.9 64 76

Ni

0 BDL BDL BDL BDL -

10 5bcBC

0.1 10aA

0.66 9aA

0.11 10aBC

0.2 2.9

20 8cA

0.13 12bcA

0.65 8cA

0.23 15aA

0.03 2.7

LSD0.05 1.8 2.5 1.4 3.1

Zn

0 15deDE

0.33 30cD

1.1 35cDE

0.34 45aD

1.1 3.1

10 225cdAB

1.1 265cA

1.6 280cA

0.99 480aAB

2.7 67.8

20 295dA

1.4 310cdA

1.4 325cdA

1.66 535aA

2.5 62

LSD0.05 98 102 112 118


Within the columns, the root Cr concentration was found to be greater

in 10 % as compared to 20 % for all fungal treatments.

3.9B.3.5.3 Cu in root


enhanced as compared to C. The value was found to be BDL in 0% for C

treatment. The plants from F1 + F2 observed to have the maximum (670

mgkg-1) uptake in 10 % than those cultivated in pots with single or no fungal

inoculations and found to be the minimum 5 mgkg-1 in 0% with F2.

For different columns, the plants from 10 % TSW-Soil exhibited better

root Cu accumulation than 20 %.

134

3.9B.3.5.4 Fe in root


treatments, the root Fe observed to be BDL in soil (0 %) with C treatment. The

plants from C had the lowest root Fe as compared to plants from any of the

single or combined application of fungi for the rest of TWS-Soil mixtures i.e.

10 % and 20 %. The root Fe accumulation observed to be the maximum (210

mgkg-1) in 10 % with F1 + F2 and significantly higher than those harvested

from any of the treatments with single or no fungal inoculations.


% with F1 + F2 while the least value of metal uptake was observed in 0 % as


3.9B.3.5.5 Mg in root





well as F1 and F2. However the F2 inoculations gave better results than F1.

For the columns, the root Mg accumulation was found to be highest in

20% with F1 + F2 where the metal accumulation was observed to be 290

mgkg-1 while minimum concentration 15 mgkg-1 in 0 % with C treatment was

noted as shown in Table 3.9B.6.

3.9B.3.5.6 Ni in root








135


while minimum uptake (5 mgkg-1) was observed in 10 % with C.

3.9B.3.5.7 Zn in root






For different TWS-Soil mixtures along the columns, the root Zn

concentration increased with increasing percentage of TSW in soil with every

fungal treatment. The maximum accumulation was noted in 20 % with F1 + F2

i.e. 535 mgkg-1 and minimum (15 mgkg-1) in 0 % with C treatment as shown in

Table 3.9B.6.

3.9B.4 Fungal analyses


c.f.u. g-1 soil) of 52-days old Helianthus annuus cultivated on TSW-Soil

mixtures are shown in Table 3.9B.7. Alongside the row, the c.f.u. increased

with fungal application than C and observed to be the maximum in treatments

with combined application of both of the fungi. The order of c.f.u. abundance

was F1 + F2 > F2 > F1 > C.

Within a column, the c.f.u. abundance was observed to be highest in %

with 10.9 × 105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2.

Table 3.9B.7. The post-harvest fungal analyses (× 105 c.f.u. g


Helianthus annuus cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 3).

TSW-Soil (% w:w) Treatment

LSD0.05 C F1 F2 F1+F2

0 2.7deBC

0.22 4.8cA

0.17 5.5cB

0.23 10.9aBC

0.29 3.1

10 3.1cdAB

0.16 4.2cAB

0.33 4.9cB

0.07 9.5aC

0.51 1.8

20 2.4bcC

0.92 2.9bcDE

0.52 3.1bcCD

0.83 4.9aE

0.20 1.54

LSD0.05 0.38 0.64 0.97 2.5


136

3.9B.5 Meta-analytical perspective



3.9B.5.1 Category-I metals translocation index (%)



3.9B.8.

In case of Ca, maximum value was observed in 10% with C i.e. 227.27

% while the minimum value was recorded (110.25 %) in 20 % with F1 + F2.


with F1 + F2 i.e. 176.31 % being the minimum 141.37 % in 10% with F2.

Table 3.9B.8. The Category-I metals translocation index (%) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Ca 10 227.27 191.66 181.48 122.58

20 200 119.04 120 110.25

K 10 155.55 152.94 141.37 176.31

20 150 146.77 147.69 168.13

Na 10 172.22 144 155.17 132.30

20 131.81 154.54 110.52 146.47

C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii

In case of Na, the maximum value 172.22 % was observed in 10% with

C treatment and least value was recorded in 20 % for F2 (110.52 %).

3.9B.5.2 Category-II metals translocation index (%)



applied together for all metals as given in Table 3.9B.9. As compared to

different TWS-Soil mixtures there was decreasing trend observed with

increasing percentage of TWS-Soil with few exceptions.

For Cd, the maximum Translocation index (143.63 %) was noted in

10% with F2, while minimum value was recorded 104.76 % in 20% with C.

137


(152.56 %) in 20% with F2 and minimum 127.69 % in 10 % with C.


233.33 % for 10% with C, while minimum value was recorded in 20 % with F1



275 % for 10 % with C, while minimum value was recorded in 10 % with F1 +

F2 treatment i.e. 80.95 %.

In case of Mg the plants showed least value in 20% with F1 + F2

(106.89 %), while the maximum values for this metal was recorded in 20%

with C i.e. 240 %.


F1 + F2 and least value was observed in 10% with F1 i.e. 150 %.


recorded in 10 % with F2 treatment i.e. 253.57 % while minimum value was

recorded in 20% with C i.e. 132.20 % as shown in Table 3.9B.9.

Table 3.9B.9. The Category-II metals translocation index (%) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Cd 10 122.58 127.88 143.63 110.75

20 104.76 110.20 119.60 131.54

Cr 10 127.69 150 142.70 129.71

20 136.20 143.83 152.56 127.92

Cu 10 233.33 144.89 136.53 130.59

20 176.92 117.94 126.19 145.05

Fe 10 275 122.22 107.40 80.95

20 133.33 129.16 109.67 150

Mg 10 145.45 131.03 146.66 141.17

20 240 118.42 123.25 106.89

Ni 10 200 150 277.77 300

20 187.5 166.66 187.5 166.66

Zn 10 193.33 249.05 253.57 206.25

20 132.20 154.83 156.92 160.74


138

3.9B.5.3 Tolerance index (TI)

In shoots TI values were found to be highest in 10 % with F1 (1.16)

while minimum value was recorded to be 0.75 in 20% with C as shown in

Table 3.9B.10.

Table 3.9B.10. The tolerance index (TI) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

TI Shoot 10 1.09 1.16 1.08 1.03

20 0.75 0.80 0.77 0.79

TI Root 10 1.33 1.29 1.27 1.22

20 1.13 0.98 0.96 1.12



in 10% with C, while 0.96 was recorded as minimum value in 20% with F2.

3.9B.5.4 Category-I metals specific extraction yield percentage (SEY %)

The SEY % for Category-I metals i.e. Ca, K and Na showed in Table

3.9B.11.

Table 3.9B.11. The Category-I specific extraction yield (SEY %) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Ca 10 17.91 22.17 24.73 32.19

20 5.6 9.4 10.99 22.68

K 10 17.76 26.85 33.05 54.95

20 24.24 27.77 30.30 37.12

Na 10 51.68 62.02 65.60 79.52

20 37.67 43.05 45.64 48.65



plants grown in 10 % with F1 + F2, and minimum 5.6 % in 20 % with C.

In case of K, plants cultivated in 10 % TWS-soil showed highest value

of SEY% (54.95 %) with F1 + F2 and minimum in 10 % with C i.e. 17.76 %.

The highest value for Na was recorded in 10 % with F1 + F2 (79.52 %)

while minimum values was found to be 37.67 % in case of 20% with no

fungal inoculum i.e. C.

139

3.9B.5.5 Category-II metals specific extraction yield percentage (SEY %)


by AAS as shown in Table 3.9B.12. Overall a similar kind of trend was seen




Table 3.9B.12. The Category-II specific extraction yield (SEY %) analyzed in 52-days old Helianthus annuus cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Cd 10 17.02 21.88 22.94 26.74

20 15.54 17.6 18.28 23.31

Cr 10 24.87 34.53 39.31 42.04

20 23.27 27.98 30.65 34.81

Cu 10 39.04 50.71 51.42 71.42

20 25.90 30.66 34.28 39.61

Fe 10 23.52 31.37 38.23 84.31

20 16.48 22.52 29.12 65.38

Mg 10 32.25 52.41 62.90 73.38

20 19.11 25.98 32.84 52.45

Ni 10 29.18 41.81 54.54 81.81

20 22.72 36.36 45.45 86.36

Zn 10 11.11 21.95 24.33 26.45

20 6.66 14.56 22.71 27.90


In case of Cd the maximum value for SEY % was recorded in 10 %

with F1 + F2 treatment i.e. 26.74 %, while minimum (15.54 %) was observed

in 20% with C.


10 % with F1 + F2 and minimum (23.27 %) was recorded in 20% with no

fungal inoculum i.e. C. However F2 showed greater values as compared to

F1.

For Cu again the maximum value was observed for 10 % (71.42 %)

with combined fungal inoculum F1 + F2 and least value (25.90 %) was

recorded in 20% with C.

140

As in case of above mentioned metals again the highest (84.31 %) and


respectively for Fe.


same trend seen in case of Mg, where plants in 10 % showed highest SEY%

value (73.38 %) with F1 + F2 and being minimum (19.11 %) in 20% with C

having no fungal inoculum.

There was maximum value for Ni was calculated (86.36 %) in 20% with

combined fungal inoculum, while minimum value (22.72 %) was recorded in

20 % for C.

In case of Zn the maximum value was noted (27.90 %) for 20 % with

F1 + F2 and minimum value (6.66 %) in 20 % with C.

141

3.10 Experiment with French marigold

3.10.1 Pre-sowing analysis

The physico-chemical properties of TSW, Caldwell field soil and their

various (% w:w TSW-Soil) mixtures is shown in Table 3.10.1. The pH of TSW

was extremely high as compared to the Caldwell soil and mixing soil with

TSW increased its pH. Likewise, the OM of soil increased with increasing the

percentage of TSW. The concentration of Category-I as well as Category-II

metals were also extremely in TSW as compare to the soil. However, mixing

of TSW in soil added doses of both of the categories of the metals to the soil

with maximum fractions observed in 10 %.

Table 3.10.1. The physico-chemical properties, Category-I and Category-II metals determined

in TSW, Caldwell field soil and their various (% w:w TSW-Soil) mixtures; The mean values

with common letters (small along the row & capital within a column) are not significantly


Parameters 0

(Soil only) 5 10

100

(TSW only) LSD0.05

pH 5.7bc

6.1b 6.5

b 8.9

a 2.4

Organic matter (OM %) 3.65ab

3.91ab

4.2a 4.5

a 1.2

Ca 699.5f 1,800

e 2,450

cd 6,320

a 1,525

Na 23.92gh

880f 1,550

e 9,440

a 2,398

K 118.5e 290

de 1,330

c 4,210

a 1,088

Cd BDL 2,320e 4,230

cd 10,097

a 2,545

Cr 13.3fg 7,560

de 9,910

d 25,534

a 6,378

Cu 9.32ef 1,210

d 1,930

cd 10,554

a 2,672

Fe 18.62f 280

de 550

cd 2,250

a 678

Mg 75.44f 530

de 945

d 3,840

a 789

Ni 16.21f 80

ef 125

de 590

a 165

Pb 16.5a 13.3

b 11.8

bc BDL 2.1

Zn 7.29f 280

ef 430

ef 7,590

a 1,810

LSD: Least significance difference

3.10.2 Biochemical analyses of 45-days old Tagetes patula





under:

142


The plant chlorophyll content within a fungal treatment increased by

applying combined fungal inoculation i.e the F1 + F2, as given in Table 3.10.2.

The F1 + F2 cause the greatest (22.3 SPAD value) increase in plants from 5

% while having the least 12.2 SPAD value in 0 % with C treatment as

compared to any of the treatments within a row. However F2 showed better

values with respect to chlorophyll content as compared to F1 values.

Within column, there was increase in plant chlorophyll contents in 5 %

for all the fungal treatments but the values were dropped in 10%. The

application of fungus either individually or in combination help plants perform

better as compared to C in terms of chlorophyll contents, as given in Table

3.10.2.


on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range

test (P = 0.05; n = 4).


w:w)


C F1 F2 F1+F2


0 12.2cC

0.67 13.5abC

0.99 13.9aCD

0.67 14.8aBC

1.2 0.9

5 16.5dA

0.11 18.1cA

1.4 19.2bcA

0.69 22.3aA

1.87 2.2

10 14.3abAB

1.3 14.9abBC

0.67 15.4aC

0.23 15.9aBC

0.62 2.8

LSD0.05 1.5 3.2 3.7 4.2


(mgg-1

)

0 0.6bDE

1.5 0.9abCD

0.78 1.1abD

0.60 1.9aDE

0.45 1.7

5 11cdA

1.8 13bcA

0.66 14bcA

0.77 18aA

0.38 1.6

10 12cdA

0.67 15bA

0.34 16bA

0.67 19aA

0.92 2.8

LSD0.05 4.2 4.7 4.5 4.9

SOD (Umg

-1 of protein)

0 BDL BDL BDL BDL -

5 16cA

0.68 18cA

0.56 21bcA

0.45 28aA

0.33 4.1

10 17cA

0.44 19cA

0.61 23abA

0.98 27aA

0.67 2.9

LSD0.05 2.1 2.5 2.6 2.8

CAT (Uml

-1)

0 BDL BDL BDL BDL -

5 13deA

0.6 16cA

0.55 18cA

0.24 27aA

0.23 3.7

10 9cBC

0.63 11bcC

0.89 12bcBC

0.45 16aC

0.45 2.7

LSD0.05 2.3 1.9 2.8 3.8

C: No fungal inoculum; F1: Trichoderma harzianum; F2: Trichoderma pseudokoningii; F1 + F2: T. harzianum and T. pseudokoningii; LSD: least significant difference


Same as the case with chlorophyll contents, the values within a

row for soluble protein contents also increased with the application of fungal

inoculations for all the treatment, as given in Table 3.10.2. The F1 + F2 from

10 % and the C from 0 % exhibited the maximum (19 mgg-1) and the minimum

(0.6 mgg-1) soluble protein contents respectively, as compare to any of the soil

143

or fungal treatments. Within Fungal treatments C showed minimum values as

compared to other fungal applications like F1, F2 and F1 + F2.


with the increasing level of TSW in soil. However, addition of fungi helped

plants to lessen stress by increasing soluble protein contents. The 10 % with

F1 + F2 and the 0 % with C observed to have the maximum and the minimum

values respectively. The plants cultivated in pots with F2 inoculations

performed better than those with F1 for all the TSW-Soil mixtures.




treatments, as given in Table 3.10.2. The SOD values were found to be BDL

for all fungal treatments of 0%. For the rest of the two TWS-Soil mixtures i.e. 5

and 10 % the fungal treatments having both of the F1 and F2 performed


with F1 + F2 gave greater SOD values except with F1 + F2 where 5% showed

slightly better value i.e. 28 Umg-1 of protein.


Within different treatments along the row, there was an increase in

plant CAT value with the individual or combined fungal inoculations, as

compared to control. CAT values for 0 % were found to be BDL for all fungal

treatments. The plants from 5 % with F1 + F2 had the maximum (27 Uml-1)

CAT values. The minimum (9 Uml-1) CAT value was observed in 10 % in C

treatment as given in Table 3.10.1.

For different TSW-Soil mixture comparison, there was minimum CAT

contents was observed in 10% as compared to 5% for all fungal treatments.



Better growth of 45-day-old plants of T. patula was observed in case of

lower TWS-soil percentage i.e. 5% being relatively less in 10% as indicated

by growth parameters (Table 3.10.3). It was noticed that plants cultivated in

144

soil and its TSW mixtures inoculated with fungal isolates yielded greater

shoot, root and seedling length, no. of leaves and roots, as well as, fresh and

dry weight; as compared to control. The statistical analysis of the data showed

significant growth in all parameters in lower TWS-Soil mixtures in soil followed

by a decrease at higher (10%) level. However, the maximum increase in

values was found in F + M treatment over their controls F1 and F2, for each of

the corresponding soil treatments. The details of each of the morphological

parameters is given in Table 3.10.3 and described as under:


Along the row in comparison with different fungal treatments, the

maximum plant shoot (22.3 cm), root (18.1 cm) and seedling length (40.8 cm)

was observed in 0 % with F1 + F2 inoculation while being minimum values for

plant shoot (9.7 cm) and seedling length (18.6 cm) in 10 % with no fungal

inoculation i.e. the C. The minimum value for plant root length (9.7 cm) was

recorded in 10% with control having no fungal inoculation. There was increase

in length of all the three vegetative parameters with the application of fungal

inoculations and the order of increase observed to be F1 + F2 > F2 > F1 > C

along the row as shown in Figure 3.10.1.

While in comparison with columns, there was decrease in plant shoot,

root and seedling length with the increasing proportion of TSW in the soil. The

different TSW-Soil mixtures under F1 + F2 column gave best results while

those in C column attained the least height. However the highest TWS-Soil

mixture i.e. 10% showed least growth as shown in Table 3.10.3.




and 04 respectively) in 10 % without any of the fungal inoculation i.e. C.

Within columns, the increasing ratio of TSW increased the no. of roots

upto 5 % but there was decreased for these parameters in 10%. The TSW-

Soil mixtures under F1 + F2 gave the best vegetative growth than any of the

fungal treatments. The least growth response observed to be in C column.

145



Parameters TSW-Soil

(% w:w)


C F1 F2 F1+F2

Shoot Length (cm)

0 12.1dA

0.34 16.4bcA

1.2 17.8bcA

0.78 22.3aA

0.67 2.7

5 11.7dA

1.33 14.9bcAB

1.4 15.5bcB

0.45 19.3aBC

1.5 3.2

10 9.7cdBC

1.45 12.3bC

1.1 13.7bBC

0.11 17.1aC

0.79 3.8

LSD0.05 0.7 1.5 1.4 1.8

Root Length (cm)

0 11.3bcA

2.4 13.8bA

0.14 14.5bcA

0.14 18.1aA

0.77 1.8

5 9.6bAB

1.5 10.3aBC

0.44 10.9aBC

0.87 12.1aC

.1 2.7

10 8.5abB

0.99 8.1abC

0.57 8.7abC

0.33 10.4aC

1.07 3.2

LSD0.05 0.9 1.7 1.9 2.6

Seedling Length

(cm)

0 23.7dA

1.93 30.5bcA

0.66 32.6bcA

1.2 40.8aA

1.88 1.5

5 21.6dA

2.38 25.5bcC

0.32 26.7bBC

1.5 31.7aC

1.34 1.6

10 18.6dBC

1.66 20.8bcD

0.24 22.7bcC

1.7 27.8aD

1.23 2.5

LSD0.05 1.7 1.8 3.5 4.8

No. of roots

0 09cdB

1.22 13bA

0.98 15aA

0.84 17aC

0.18 2.2

5 12dA

1.54 15bcA

0.54 16bcA

0.38 21aA

0.34 2.7

10 07cdBC

0.95 09cBC

1.3 11cBC

0.32 19aAB

0.56 3.3

LSD0.05 1.8 1.7 1.9 2.2

No. of leaves

0 07dA

0.32 10bcA

0.34 09bA

0.78 13aA

0.45 3.1

5 08bcA

0.11 07cB

0.89 06cA

0.67 15aA

0.86 2.8

10 04cdBC

0.20 06cB

1.3 05cdAB

0.45 12aB

N0.46 4.2

LSD0.05 0.9 1.5 1.7 2.6

Fresh wt. (g)

0 5.6abA

0.12 6.4aB

1.1 6.9aA

1.3 7.2aBC

0.43 2.4

5 6.4cA

0.32 8.1abA

0.26 7.8bA

1.2 9.7aA

0.64 2.9

10 4.2abB

0.67 4.9aCD

0.78 5.1aC

0.92 5.9aC

0.89 1.5

LSD0.05 0.8 1.1 2.6 2.9

Dry wt. (g)

0 2.1abA

0.23 2.4aAB

1.1 2.5aA

1.3 2.9aA

0.44 1.7

5 2.5abA

0.75 3.1aA

0.24 2.8abA

1.1 3.5aA

0.65 1.9

10 1.2bBC

0.61 1.5abBC

0.87 1.8abB

0.95 2.3aAB

0.91 2.6

LSD0.05 1.4 2.2 2.7 3.1







observed to be in 5 % with F1 + F2 and the minimum (4.2 g fresh, 1.2 g dry) in

10 % with C, respectively.


production positively for lower TWS-Soil mixture i.e. 5%. The TSW-Soil

mixtures under F1 + F2 yielded maximum fresh and dry weight while those in

C column yielded the least.

146

Figure 3.10.1. The vegetative growth variation in marigold (Tagetes patula) in response to Caldwell field soil mixed with different percentages of TSW (% w:w) and inoculated with different fungi; (upper) the representative pots of each of the treatments with best growth of marigold; (lower) all of the experimental units with replicates of all the treatments.









Within different mixtures of TSW in soil, the maximum shoot


147


increasing TSW ratio in soil mixtures for 5 % and then decreased for 10 %.





LSD0.05 C F1 F2 F1+F2

Ca

0 07deC

0.23 10dCD

0.66 15cD

0.22 25aDE

0.99 4

5 45efA

0.45 70dA

0.45 110cdA

0.84 225aA

2.3 48

10 30cAB

0.45 55bcAB

0.98 80bB

1.1 145aC

1.2 32

LSD0.05 29 23 34 78

K

0 20dD

0.96 35cCD

0.94 45bcC

1.0 65aCD

1.1 17

5 70cBC

0.09 90abAB

1.5 95aA

1.04 110aAB

2.5 16

10 105cA

0.45 120bcA

1.3 125bcA

2.4 155aA

2.03 18

LSD0.05 36 47 52 65

Na

0 BDL BDL BDL BDL -

5 80deA

0.99 170cA

1.3 210bcA

1.4 290aA

2.5 54

10 60deBC

2.5 135cC

0.98 155cCD

1.2 260aAB

2.1 49

LSD0.05 8 16 27 18





observed in 10 % with F1 + F2 while being the minimum (20 mgkg-1) in soil

with no fungi i.e. C.


percentage of TSW in soil mixtures for all the TWS-Soil mixtures. For all TSW-

Soil mixtures, the F2 plants showed more K uptake than those with F1 and

being the least where no fungus was applied. The maximum values observed

in plants cultivated in 10% TWS-Soil mixture.








148

Within column, 0% showed Na values BDL for all fungal treatments.

For the rest of two TWS-Soil mixtures i.e. 5 and 10 %, the concentration of

shoot Na observed to be higher in 5% as compared to 10% as shown in Table

3.10.4.











minimum (4 mgkg-1) in 0 % with F1. The value was found to be BDL in 0%

with C.



45-days old Tagetes patula cultivated on TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 4).


w:w)


C F1 F2 F1+F2

Ca

0 BDL 4eD

0.23 10cdE

0.34 20aE

0.99 7

5 35cA

1.1 55cA

0.55 90bcA

0.43 160aA

1.5 64

10 20dBC

1.2 35cdB

0.99 60cCD

0.54 110aBC

1.4 32

LSD0.05 8 18 33 48

K

0 10cC

0.2 15cCD

1.5 25bcD

0.65 45aC

1.4 9

5 55bcA

0.34 80aA

1.2 85aA

0.77 90aA

2.5 15

10 50cA

0.99 70bcA

1.1 65bcB

0.35 80aA

1.5 17

LSD0.05 19 26 18 22

Na

0 BDL BDL BDL BDL -

5 35deA

0.44 125bcA

0.76 145bcA

1.1 220aA

2.5 35

10 20efBC

0.56 90dB

0.65 125bcAB

1.5 185aB

2.3 42

LSD0.05 6 17 16 27


149




(10 mgkg-1) in 0 % with no fungal application i.e. C.

Within column, the metal uptake in root was found to be highest in 5 %

while being least in 0 % for all fungal treatments.




observed in 5 % with F1 + F2 while being least in 10 % amounting 20 mgkg-1

with C. Those with F1 and F2 applications also performed better than C i.e.

treatment with no fungal inoculation.

Within column, in 0% the values for Na were found to be BDL in all

fungal treatments. The increasing ratio of TSW in soil displayed increased

root Na uptake for 5 % as compared to 10%.

3.10.3.4 Category-II metals in plant shoot




uptake tendency of plant for all the TSW-Soil mixtures. However, the






fungal treatments. Maximum amount of metal (785 mgkg-1) was observed in


being minimum in 0% amounting 12 mgkg-1 with C.

150

Within columns comparison, there is increasing trend of metal

accumulation with TSW-Soil mixtures for 5 % then decrease in values were

observed for 10%.





LSD0.05 C F1 F2 F1+F2

Cd

0 12cdD

0.12 18cDe

0.91 20cDE

0.83 45aE

0.97 12.2

5 110efA

0.23 230dA

1.4 260cdA

1.4 785aA

2.7 170

10 90deA

0.56 185dAB

1.1 165dBC

1.7 625aC

2.2 124

LSD0.05 38 76 28 235

Cr

0 BDL 3cEF

0.94 5cE

0.78 15aEF

0.11 4.4

5 340eA

0.56 625bcA

3.4 655bcA

2.4 930aA

2.6 132

10 280efBC

0.88 515cdC

2.3 545cdB

2.5 765aCD

1.4 127

LSD0.05 25 210 225 275

Cu

0 10cDE

0.78 12cE

0.93 20bcDE

0.99 45aE

1.02 9.7

5 120cdA

1.3 290bA

1.3 315bA

1.5 450aAB

1.5 78

10 145deA

1.4 310cA

1.03 345cA

2.1 520aA

1.3 98

LSD0.05 55 95 105 170

Fe

0 3dE

0.55 5bcDE

0.39 9bE

0.94 14aE

0.92 7.6

5 30cdCD

0.87 90abAB

0.92 110aA

1.04 115aD

1.6 22

10 65deA

0.99 125cA

1.02 145cA

1.5 255aA

1.4 51

LSD0.05 32 45 65 122

Mg

0 10eDE

0.76 20cdD

0.98 25cdDE

0.92 45aE

0.99 9

5 90cA

0.85 130abA

1.32 145aA

1.3 160aA

2.1 67

10 70bcAB

0.83 110bA

1.2 155abA

1.4 190aA

1.4 22

LSD0.05 30 38 55 63

Ni

0 BDL BDL BDL BDL -

5 10abC

0.45 12aBC

1.3 13aB

0.93 15aB

1.2 6

10 17abA

0.64 19aA

1.1 18aA

0.82 22aA

1.3 9.8

LSD0.05 4.7 3.1 5.5 3.8

Zn

0 25dB

0.87 55bcCD

0.98 80aBC

0.91 95aA

1.1 23

5 35cA

2.2 90abA

2.54 105aA

2.2 120aA

2.1 34

10 20bcBC

2.6 55aCD

2.45 60aC

3.1 70aBC

3.2 17

LSD0.05 8 33 40 80



With different fungal treatments along the rows, the maximum Cr

accumulation in shoot was observed in treatments applied with combined

inoculants i.e. F1 + F2, being significantly higher than any of the fungal

treatments. Cr was found to be as low as 3 mgkg-1 in 0% with F1 and

maximum 930 mgkg-1 in 5% with F1 + F2.

151

Within different mixtures of TWS-soil there was maximum accumulation

of metal was observed in 5 % for all fungal treatments. the value for Cr was

found to be BDL in 0% with C.







treatments as shown in Table 3.10.6.

Within different TWS-Soil mixtures along the column, 0% showed

significantly less Cu uptake in shoots than those form 5 and 10 %. The

minimum accumulation 10 mgkg-1 was seen in 0% with C and maximum

accumulation 520 mgkg-1 in 10 % with F1 + F2.


The Fe uptake in plant shoots observed to be least in Soil (0%) with C,



The values of plant shoot Fe was minimum 3 mgkg-1 in 0 % with C and



shown in Table 3.10.5.





+ F2 in 10 %, while being the minimum as well as significantly least in shoot of

plants cultivated in soil with no fungus i.e. 10 mgkg-1 as shown in Table

3.10.6.

152

For different TWS-Soil mixtures, the value of plant shoot Mg was

found to be least in 0%. For 10 %, the shoot uptake found to be highest as

compared to 5 and 0 % TSW-Soil mixture.










plants from 5 %.








compared to F1 in terms of accumulation of metal as shown in Table 3.10.6.


was found to be maximum 120 mgkg-1 in 5 % with F1 + F2 being minimum 20

mgkg-1 in 10% with C as shown in Table 3.10.6.

3.10.3.5 Category-II metals in plant root




153

3.10.3.5.1 Cd in root




3.10.7. The plant root Cd concentration increased with fungal inoculations and

found to be maximum as well as significantly higher than those applied with

single or no fungal treatments.


concentration was found to be highest in 10% with C and F1 as compared

with 5 and 0 %. However the 5 % showed greater values with F2 and F1 + F2

as compared to 10 and 0%.

3.10.3.5.2 Cr in root


F1 fungal inoculations showed the maximum 875 mgkg-1 root Cd

concentration in 5 %, while with C treatment plants having the minimum

uptake 190 mgkg-1 in 10%. The F2 inoculation enhanced the root Cd

concentration better than F1. However in 0% for all the fungal treatments the

values found to be BDL as shown in Table 3.10.7.

Within the columns, the root Cr concentration decreased with

increasing percentage of TSW in soil having maximum accumulation of metal

in 5 % for all fungal treatments like C, F1, F2 except F1 + F2 than 10%.

3.10.3.5.3 Cu in root


enhanced as compared to C. The plants from F1 + F2 observed to have the

maximum (435 mgkg-1) uptake in 10 % than those cultivated in pots with

single or no fungal inoculations and found to be the minimum 5 mgkg-1 in 0%

with C.

For different columns, the plants from 5 and 10 % TSW-Soil exhibited better

root Cu accumulation than soil (0 %).

154





w:w)


C F1 F2 F1+F2

Cd

0 10dCD

0.44 15bcC

0.56 18bcDE

0.98 35aE

0.67 6.6

5 65deA

0.91 155dA

1.34 190dA

1.34 670aA

1.6 155

10 70deA

0.98 160cA

1.45 110cdBC

1.67 520aC

1.8 128

LSD0.05 22.5 54 67 232

Cr

0 BDL BDL BDL BDL -

5 255deA

1.2 425cA

1.7 550cA

1.34 875aA

1.9 162

10 190eBC

1.1 410cA

1.1 505abAB

2.1 630aC

2.3 115

LSD0.05 23 9 18 88

Cu

0 5aD

0.87 8cD

0.89 15bcDE

1.1 30aD

1.02 6.9

5 90deA

0.81 180dA

0.99 245cA

1.6 315aBC

2.05 59

10 55efBC

0.67 215dA

1.01 265cdA

1.2 435aA

2.1 98

LSD0.05 32 209 85 138

Fe

0 BDL BDL BDL BDL -

5 20cBC

0.98 65abA

1.07 70aC

1.15 80aBC

1.04 19

10 45cdA

1.1 70bA

0.99 95aA

1.12 110aA

1.02 21

LSD0.05 9.4 2.4 9.1 11

Mg

0 5cdDE

0.91 9cE

0.99 13cDE

0.43 30aCD

0.82 7

5 85bA

0.98 95abA

1.2 110aA

0.92 125aA

0.29 13

10 40cC

0.91 65bcBC

0.99 70bcBC

0.94 125aA

0.39 22

LSD0.05 28 32 35 43

Ni

0 BDL. BDL BDL BDL -

5 8aA

0.78 4bCD

0.98 5aC

0.9 7aBC

0.8 2

10 10cdA

1.1 15bcA

0.56 14bcA

1.2 18aA

1.4 3.4

LSD0.05 1.1 2.5 3.4 4.6

Zn

0 20dA

0.99 35cAB

0.98 55bcA

1.3 75aA

2.1 17

5 15cdBC

1.23 40cA

2.4 55bA

3.4 70aA

1.5 15

10 10cdCD

2.4 25bC

2.7 30aBC

1.5 35aC

1.1 6.8

LSD0.05 3.2 5.7 7.6 9.2


3.10.3.5.4 Fe in root


treatments, the root Fe observed to be BDL in soil (0 %) with all fungal

treatments. The plants from C had the lowest root Fe as compared to plants

from any of the single or combined application of fungi. The root Fe

accumulation observed to be the maximum (110 mgkg-1) in 10 % with F1 + F2




% while the least value of metal uptake was observed in 5 % as shown in

Table 3.10.7.

155

3.10.3.5.5 Mg in root





well as F1 and F2. However the F2 inoculations gave better results than F1.

For the columns, the root Mg accumulation increased with increasing

percentage of TSW in soil for all fungal treatments being maximum in 5 and


mgkg-1 while minimum concentration 5 mgkg-1 in 0 % with C treatment was

noted as shown in Table 3.10.6.

3.10.3.5.6 Ni in root






Within columns, the plants from 10 % TSW-Soil mixture had the



while minimum uptake (4 mgkg-1) was observed in 5% with F1.

3.10.3.5.7 Zn in root





F1 as far as metal accumulation efficiency of the plant is concerned. The

maximum accumulation of metal was observed in 0 % with F1 + F2 i.e. 75

mgkg-1 while minimum value for the metal accumulation was noted in 10 %

with C i.e. 10 mgkg-1.

156



c.f.u. g-1 soil) of 45-days old Tagetes patula cultivated on TSW-Soil mixtures

are shown in Table 3.10.8. Alongside the row, the c.f.u. increased with fungal

application than C and observed to be the maximum in treatments with

combined application of both of the fungi. The order of c.f.u. abundance was

F1 + F2 > F2 > F1 > C.





Treatment LSD0.05

C F1 F2 F1+F2

0 0.8cdC

0.22 1.7cD

0.17 2.8bcC

0.23 5.9aCD

0.29 1.5

5 2.7deA

0.26 3.8cdA

0.18 4.9cdA

0.21 9.1aA

0.13 1.9

10 2.4cdA

0.16 3.2cdA

0.33 4.2cA

0.07 7.5aB

0.51 2.6

LSD0.05 0.9 0.8 0.7 1.3


Within a column, the c.f.u. abundance was observed in 5% with 9.1 ×

105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2. Statistical

analysis indicated that there is significant difference of 5% with other TWS-

Soil mixtures.







3.10.9.

In case of Ca, maximum value was observed in 10% with F1 i.e. 157.1

% while the minimum value was recorded (122.2 %) in 5 % with F2.


with C i.e. 210 % being the minimum 111.7 % in 5 % with F2.

157

In case of Na, the maximum value 300 % was observed in 10 % TWS-

Soil mixture with C treatment and least value was recorded in 10 % for F2

(124 %).




applied together for all metals as given in Table 3.10.10.

Table 3.10.10. The Category-II metals translocation index (%) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).


C F1 F2 F1+F2

Cd 5 169.2 148.3 136.8 117.1

10 128.5 115.6 150 120.1

Cr 5 133.3 147.0 119.0 106.2

10 147.3 125.6 107.9 121.4

Cu 5 133.3 161.1 128.5 142.8

10 263.6 144.1 130.1 119.5

Fe 5 150 138.4 157.1 143.7

10 144.4 178.5 152.6 231.8

Mg 5 105.8 136.8 131.8 128

10 175 169.2 221.4 152

Ni 5 125 300 260 214.2

10 170 126.6 128.5 122.2

Zn 5 233.3 225.0 190.9 171.4

10 200.0 220.0 200.0 200.0


For Cd, the maximum Translocation index (169.2 %) was noted in 5 %

with C, while minimum value was recorded 115.6 % in 10% with F1.


(147.3 %) in 10% with C and minimum 106.2 % in 5 % with F1 + F2.

Table 3.10.9. The Category-I metals translocation index (%) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).


C F1 F2 F1+F2

Ca 5 128.5 127.2 122.2 140.6

10 150 157.1 133.3 131.8

K 5 127.2 112.5 111.7 122.2

10 210 171.4 192.3 193.7

Na 5 228.5 136 144.8 131.8

10 300 150 124 140.5


158


263.6 % for 10% with C, while minimum value was recorded in 10 % with F1 +

F2 treatment i.e. 119.5 %.


231.8 % for 10 % with F1 + F2, while minimum value was recorded in 5 %

with F1 treatment i.e. 138.4 %.

In case of Mg the plants showed least value in 5 % with C (105.8 %),

while the maximum value for this metal was recorded in 10% with F2 i.e.

221.4 %.


F1 to be the highest and least value was observed in 10% with F1 + F2 i.e.

122.2 %.


recorded in 5 % with C treatment i.e. 233.3 % while minimum value was

recorded in 5 % with F1 + F2 i.e. 171.4 % as shown in Table 3.10.9.


In shoots TI values were found to be highest in 5 % with C

(0.966) while minimum value was recorded to be 0.75 in 10% with F1 as

shown in Table 3.10.11.

Table 3.10.11. The tolerance index (TI) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).


C F1 F2 F1+F2

TI Shoot 5 0.966 0.908 0.870 0.865

10 0.801 0.75 0.76 0.76

TI Root 5 0.84 0.74 0.75 0.66

10 0.75 0.58 0.60 0.57


In case of TI in roots 0.84 was recorded as highest for plants grown in

5 % with C, while 0.57 was recorded as minimum value in 10% with F1 + F2.

3.10.5.4 Category-I metals specific extraction yield (SEY %)


3.10.12. In case of Ca, there was maximum value 21.3 % was recorded for


159

Table 3.10.12. The Category-I metals specific extraction yield (SEY %) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).


C F1 F2 F1+F2

Ca 5 4.4 6.9 11.1 21.3

10 2.0 3.6 5.7 10.4

K 5 43.1 58.6 62.0 68.9

10 11.6 14.2 14.2 17.6

Na 5 13.0 33.5 40.3 57.9

10 5.1 14.5 18.0 28.7




As in case of Ca and K, the highest value for Na was recorded in 5 %

with F1 + F2 (57.9 %) while minimum values was found to be 5.1 % in case of

10% with no fungal inoculum i.e. C.

3.10.5.5 Category-II metals specific extraction yield (SEY %)


by AAS as shown in Table 3.10.13. Overall a similar kind of trend was seen




Table 3.10.13. The Category-II metals specific extraction yield (SEY %) analyzed in 45-days old Tagetes patula cultivated on Caldwell field mixed with different percentages of TSW (% w:w).


C F1 F2 F1+F2

Cd 5 7.5 16.5 19.3 62.7

10 3.7 8.1 6.5 27.0

Cr 5 7.8 13.8 15.9 23.8

10 4.74 9.3 10.5 14.0

Cu 5 17.3 38.8 46.2 63.2

10 10.3 27.2 31.6 49.4

Fe 5 17.8 55.3 64.2 69.6

10 20.0 35.4 43.6 66.3

Mg 5 33.0 42.45 44.3 46.2

10 11.6 18.5 23.8 33.3

Ni 5 22.5 20 22.5 27.5

10 21.6 27.2 25.6 32.0

Zn 5 17.8 46.4 57.1 67.8

10 6.9 18.6 20.9 24.4


160

In case of Cd the maximum value for SEY % was recorded in 5 % with

F1 + F2 treatment i.e. 62.7 %, while minimum (3.7 %) was observed in 10%

with C.

Similarly the SEY % value for Cr was found to be highest (23.8 %) in 5

% with F1 + F2 and minimum (4.74 %) was recorded in 10% with no fungal

inoculum i.e. C. However F2 showed greater values as compared to F1.



10% with C.

In case of Fe the highest (69.6 %) and lowest (17.8 %) values were

recorded in 5% with F1 + F2 and 5 % with C respectively.



value (46.2 %) with F1 + F2 and being minimum (11.6 %) in 10% with C

having no fungal inoculum.

There was maximum value for Ni was calculated (32.0 %) in 10% with

combined fungal inoculum, while minimum value (20 %) was recorded in 5 %

for F1.

In case of Zn the maximum value was noted (67.8 %) for 5 % with F1 +

F2 and minimum value (6.9 %) in 10 % with C.

161

3.11 Field experiments

3.11A: Experiment with Helianthus annuus

3.11A.1 Pre-sowing analysis



Chapter 3.1.

3.11A.2 Biochemical analyses of Helianthus annuus

The chlorophyll contents, soluble protein, CAT and SOD were

observed in H. annuus while being cultivated in field soil amended with

different levels of TSW. The specific details of each of the biochemical

parameters are as under:

3.11A.2.1 Chlorophyll content

Along the row, the plant chlorophyll contents based on SPAD value

observed in 50-days old sunflower increased in soil treatments applied with

fungus as compared to the C (with no fungus). The plants from F2 showed

enhanced chlorophyll contents than F1, being the maximum in F1 + F2

(applied with combined fungal inoculation), as given in Table 3.11A.1. Such a

trend was observed in all of the TSW-Soil mixtures.



treatments. In all fungal treatments, the plants from all the TSW-Soil 0 (%

w:w) had more chlorophyll contents than every corresponding 0* (% w:w). The

plants from 5 % (% w:w) of all the fungal treatments showed the maximum

SPAD value within a column while those from 20 (% w:w) had the least

chlorophyll contents.

3.11A.2.2 Soluble protein contents

Likewise chlorophyll contents, the soluble protein contents along the

row increased with the application of fungal inoculations for all the fungal

treatments, as given in Table 3.11A.1. The plants from C (with no fungal

162

treatment) had the least protein contents than any of the treatment with single

or combine fungal inoculations, being the maximum in F1 + F2.

Table 3.11A.1. The biochemical parameters observed in 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery solid waste (TSW-Soil

mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).


w:w)


C F1 F2 F1+F2

Chlorophyll contents (SPAD value)

0* 23.5aBC

0.98 23.7aC

1.2 24.1aBC

2.5 24.8aC

2.1 1.1

0 23.6abBC

1.5 22.7abC

2.1 23.3abBC

0.92 25.2aC

3.3 2.1

5 29.7abA

1.4 31.6abA

0.98 32.1aA

2.6 34.5aA

3.9 2.9

10 24.3aBC

2.7 20.4bcCD

3.1 24.5aBC

2.9 23.9aC

1.7 1.3

20 18.8abD

1.8 19.4aD

3.2 19.7aD

3.5 20.2aCD

1.98 0.9

LSD0.05 2.2 3.2 3.9 3.6

Protein content

(mgg-1

)

0* 2.4deCD

0.98 13.6bcC

2.1 14.2bcCD

2.2 25.1aD

3.2 7.4

0 3.2deB

2.9 14.1cC

0.99 13.7cCD

1.9 34.9aBC

0.98 9.1

5 6.7dA

2.7 27.9bA

1.8 24.7bcA

1,5 38.9aA

1.99 10.1

10 4.1cB

3.2 9.7abD

1.7 8.5abE

0.34 12.6aE

2.5 4.4

20 2.1bcCD

3.6 7.6aDE

2.5 6.1aEF

1.2 8.1aE

3.1 3.3

LSD0.05 1.9 6.2 4.3 7.6

SOD

(umg-1

of protein)

0* 4.3aDE

3.6 3.7aD

2.8 4.6aE

2.7 2.8cDE

2.1 1.6

0 1.9aE

2.9 1.8aD

2.1 1.6aE

0.98 1.2bDE

1.5 0.8

5 25.6dAB

0.98 34.7cA

3.2 44. 3abA

2.5 48.6aA

1.9 7,9

10 29.6bcA

2.7 31.1bcA

3.3 33.2bcBC

3.0 45.7aA

3.7 6.3

20 12.3cC

2.2 15.9bBC

2.1 14.9bD

2.6 18.4aC

0.97 3.4

LSD0.05 7.6 8.6 10.6 11.5

CAT (Uml

-1)

0* 1.2bE

0.95 1.5abDE

1.6 1.9aE

0.87 1.1bDE

4.2 0.6

0 1.1aE

2.6. 0.9abDE

0.34 1.0aE

2.1 1.2aDE

3.8 0.2

5 22.1cA

2.4 28.2abA

1.4 32.2aA

0.98 29.7aA

3.2 4.7

10 18.1bcB

2.7 21.3aAB

2.1 22.1aBC

1.7 19.3bcBC

4.1 2.2

20 11.1abCD

3.1 10.3bC

2.7 12.4abD

1.5 14.5aC

0.95 1.2

LSD0.05 7.4 7.5 9.1 7.5

0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant difference

The soluble protein contents increased down the column up to 5 (%

w:w); however, it decreased in 10 and 20 %. Such a variation was observed

for all the fungal treatments’ down the column analyses. The plants from 5 %

had the maximum protein contents in all the columns.

3.11A.2.3 Superoxide dismutase (SOD) contents

Parallel to the trends found for soluble protein contents, the SOD

contents in plants increased with fungal inoculations within a row for all the

treatments; however, it decreased in case of combined fungal application in

both 0* and 0 (% TSW-Soil) mixtures, as given in Table 3.11A.1. For 5, 10

and 20 % (TSW-Soil) mixture, the SOD values increased with the application

163

of single or combined fungal inoculation, being the maximum in plants applied

with F1 + F2.

Within a column, the plants from 0* (control with no geothermal

membrane lining) had shown enhanced SOD contents than 0 (with lining) for

all the fungal treatments. However, it decreased with increasing level of TSW

percentage in soil, being the maximum in 5 % and the minimum in 20 %

except 5 % C.

3.11A.2.4 Catalase (CAT) contents

The CAT contents of the plant exhibited variable pattern along the row.

Other than the 0 (% TSW-Soil) where plants with F1 had lesser values than

the C, the CAT values increased in plants applied with individual fungal

inoculations for F1 and F2 but decreased in treatments inoculated with F1 +

F2. For 5, 10 and 20 % (TSW-Soil), the plants inoculated with F2 had the

maximum CAT contents while those applied with no fungus had the least

values.

Down the column, the plants from 0* showed enhanced CAT contents

than corresponding 0 (% TSW: Soil) for all the fungal treatments except F1 +

F2. However it decreased down the column with increasing percentage of

TSW in soil for all of the fungal treatments, being the maximum in 5 %

(TSW:Soil) and the minimum in 0 for all the treatments.

3.11A.3 Post-harvest analysis

3.11A.3.1 Growth performance of Tagetes patula

The growth of 78-days old sunflower varied in response to different

TSW percentages as well as fungal inoculations, as given in Table 3.11A.2.

The details are as under:

3.11A.3.1.1 Shoot, root and seedling length (cm)

Along the row, the sunflower shoot, root and seedling length increased

in treatments applied with fungus than those applied with no fungus i.e. C. It

164

was the maximum in F1 + F2 being the minimum in C for all the TSW-Soil

mixtures. The plants with F2 inoculation performed better than those from F1.

Table 3.11A.2. Various morphological parameters observed in 78-days old Helianthus annuus cultivated

on field soil mixed with different percentages of tannery solid waste (TSW-Soil mixtures). The mean



w:w)


C F1 F2 F1+F2

Shoot Length (cm)

0* 54.86cC

2.3 57.91cCD

3.1 64.0bcC

2.9 85.34aBC

2.4 7.9

0 57.9dC

1.6 51.82dCD

2.8 67.05cdC

2.5 88.39aBC

1.5 8.7

5 97.53bcA

1.8 112.77abA

2.5 118.87abA

2.7 146.30aA

2.3 13.1

10 81.44cAB

2.2 100.58bcA

3.2 106.68bcA

4.1 140.20aA

2.7 16.8

20 45.72cdCD

2.7 51.81bcCD

3.1 57.91bCD

3.3 67.05aD

3.4 5.6

LSD0.05 12.2 17.5 21 19

Root Length (cm)

0* 40.34abAB

2.5 43.92aA

4.3 42.21aA

1.7 45.24aA

3.2 3.2

0 47.45aA

3.1 40.92bAB

4.6 35.78bcAB

2.4 46.12aA

3.1 5.4

5 50.12aA

2.8 50.78aA

3.8 47.20abA

3.2 52.67aA

4.1 6.7

10 49.29aA

2.4 45.98abA

2.2 39.22cAB

2.6 53.23aA

3.3 6.5

20 25.98cdD

3.5 35.37bC

1.7 32.38aB

2.1 37.27aBC

2.6 8.5

LSD0.05 6.9 7.8 9.2 7.3


0* 95.5cdCD

3.3 101.86cC

4.1 106.4cCD

2.8 130.8aC

2.5 6.8

0 105.5cdC

2.4 92.8dCD

3.8 102.9cdCD

4.1 134.7aC

2.2 9.2

5 147.8dA

1.7 163.7bcA

4.5 166.12bcA

3.3 199.1aA

2.7 11.3

10 130.9dBC

1.9 146.7cdAB

3.9 146.1cdB

3.6 193.5aA

3.4 12.2

20 71.9bcDE

2.2 87.21abCD

4.1 90.31abD

3.8 104.5aDE

3.1 10.9

LSD0.05 5.5 8.3 5.8 3.4

No. of roots/plant

0* 44bcC

3.4 54aB

3.1 58aAB

2.3 61aBC

1.2 5.5

0 42bcC

3.1 48bcBC

1.6 57aAB

3.1 62aBC

2.2 6.2

5 65abA

2.1 68abA

3.7 71aA

2.6 78aA

1.5 4.5

10 72aA

2.3 64abA

1.9 68aA

3.7 71aA

2.7 3.2

20 40aD

2.7 38aCD

2.9 36abC

2.8 41aD

1.8 1.9

LSD0.05 3.5 6.5 8 8.6

No. of leaves/plant

0* 8abAB

1.4 9abBC

1.6 8abC

1.1 11aC

2.1 3.5

0 9aAB

2.1 8aBC

1.5 9aC

2.2 10aC

2.8 4.9

5 14bA

2.2 18abA

2.5 21aA

2.9 24aA

3.1 7.7

10 12aA

3.2 11abB

1.2 10abC

3..7 16aBC

1.7 6.5

20 6abC

1.1 7aBC

1.4 8aC

1.6 9aC

1.2 2.1

LSD0.05 2.7 3.2 2.9 3.8

Fresh wt./plant (g)

0* 305.1aE

2.2 295.6abC

3.1 303.6aCD

1.6 312.3aC

2.4 4.3

0 290.5bcCD

3.3 310.4abC

2.7 314.5abCD

4.6 333.5aC

3.8 5.4

5 470.7bcA

2.4 495.1abA

1.9 505.09aA

3.7 525.8aA

4.5 13.6

10 255.3bD

1.7 263.6abCD

1.8 271.1abD

3.3 291.1aC

3.9 11.6

20 122.9bcF

1.9 131.8aE

2.5 135.2aEF

2.9 149.7aDE

4.1 16.5

LSD0.05 12.6 13.5 17.8 21.3

Dry wt. (g)

0* 65.5aA

2.3 72.1aA

2.6 74.2aA

2.8 76.7aA

3.1 8.9

0 61.9abAB

1.3 68.9aAB

4.1 71.2aA

3.7 73.6aAB

2.6 14.3

5 78.9aA

4.3 81.5aA

3.3 82.7aA

3.1 85.6aA

1.1 16.5

10 57.2abAB

2.2 62.2aAB

2.1 61.9aBC

2.5 67.3aAB

3.3 18.7

20 48.2aC

3.2 52.2aCD

1.1 53.7aC

2.2 54.9aCD

4.2 12.2

LSD0.05 7.6 8.7 7.5 9.2


165

Down the column, the plants from 0* had less efficient vegetative

growth in terms of shoot, root and seedling length than the 0 (TSW-Soil)

except those applied with F1. For 5, 10 and 20 % (TSW-Soil), the plant height

decreased down the column with increasing percentage of TSW, being the

maximum in 5 % and the minimum in 20 %. Such a variability trend was

observed for all the fungal treatments as shown in Figure 3.11A.1.

Figure 3.11A.1. Phytoextraction field trials with sunflower (Helianthus annuus) cultivated on soil

amended with different levels of tannery solid waste (TSW:Soil w:w); 0 % the only treatment without geothermal membrane allowing leaching (A), 0 % with geothermal membrane to avoid leaching (B), 5 % (C), 10 % (D) and 20 % (E). The white lines across the strip plots (25 × 3 ft) indicate soil barriers (1.25 ft) subdividing each strip plot into four subplots (5 × 3 ft each) for fungal inoculations viz. C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger + T. pseudokoningii, applied in a randomized complete block design.

3.11A.3.1.2 No. of leaves and roots

Parallel to the vegetative growth components of plant height, the

number of leaves and roots observed to increase along the row with fungal

inoculation than C (with no fungus applied), being the maximum in F1 + F2

and the minimum in C. The plants with F2 showed more number of leaves

and roots than plants applied with F1.

Within a column, the root and leaf number were lesser for plants

harvested from 0* than 0 (% TSW-Soil) for all the fungal treatments except F1

and F1 + F2 treatments. For 5, 10 and 20 % TSW-Soil mixtures, the trend of

root and leaf number increase was from 5 % towards 20 % being the

A

B

C

D

E

N

166

maximum in 5 % and the minimum in 20 %. Such a variation pattern was

observed for all the fungal treatments.

3.11A.3.1.3 Fresh and dry weight (g)

Similar to the plant vegetative growth parameters, the fresh and dry

plant biomass found the increase along the row in plants applied with fungi

than those applied with no fungi. The maximum sunflower biomass production

was observed in F1 + F2 treatments and the minimum in C. The plants

inoculated with F2 showed enhanced biomass production than F1. Such a

variability pattern was observed for all the TSW-Soil mixtures.

Down the column, the biomass production decreased with increasing

TSW percentage in soil, being the maximum in 5 % and the minimum in 20 %.

The plants from 0* had yielded less biomass than those from the 0 (% TSW-

Soil) for all the fungal treatments.

3.11A.3.2 Category-I metals in plant SHOOT


shoot observed to vary in response to the variability of TSW percentage in soil

as well as fungal inoculations, as given in Table 3.11A.3.

3.11A.3.2.1 Calcium (Ca) in shoot

Along the row, the Ca concentration in sunflower shoot increased with

inoculation of fungi as compared to C, being the maximum in plants from F1 +

Figure 3.11A.2. The sunflower stem girth variation in response to soil amended with different levels of tannery solid waste (% w:w) inoculated with fungi; (clockwise from upper left) 10 % with F2, 20 % with F1 + F2; 5 % with F1 + F2, 10 % with F1 + F2, 20 % with C, 5 % with F1 + F2.

167

F2 treatments while being the minimum in C. The F2 plants showed enhanced

Ca shoot uptake than F1. Such a variability trend was observed for all the

TSW-Soil mixtures.

Down the column, the shoot Ca observed to increase with increasing

percentage of TSW in soil and found to be the maximum in plants harvested

from 10 % except F2. The minimum shoot Ca concentration within a column

observed to be in plants harvested from those mixed with no TSW. The 0*

plants showed lesser shoot Ca uptake than corresponding 0 (% TSW-Soil) for

all the fungal treatments except F2.


) observed in SHOOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery

solid waste (TSW-Soil mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).



C F1 F2 F1+F2

Ca

0* 25bE

3.9 30aDE

1.3 28aE

2.6 32aEF

3.2 2.5

0 29aE

1.7 31aDE

3.9 27bE

2.3 35aEF

3.3 4.7

5 355cB

3.2 370cAB

1.7 410bcA

1.7 550aBC

2.1 119

10 405dA

3.3 445cdA

3.1 390dA

2.1 625aA

3.9 155

20 290cdC

2.1 310cdB

3.2 295cdC

2.7 440aD

1.7 95

LSD0.05 83 95 78 128

K

0* 20dE

3.8 45bE

3.2 35bcE

2.4 65aE

3.9 18

0 22cE

1.4 33bE

3.3 23cE

3.9 55aE

1.7 15

5 210dA

2.1 330aA

2.6 290bcA

3.7 355aA

3.2 94

10 190bcA

2.7 205bcBC

3.9 210bcB

4.1 280aB

3.3 79

20 125abCD

1.9 145aC

1.7 130aCD

1.7 155aCD

2.1 48

LSD0.05 48 54 47 58

Na

0* 25bcE

3.2 35bE

3.2 40aE

3.1 55aDE

3.9 18

0 20bE

3.3 30abE

3.9 35aE

3.2 45aDE

1.7 13

5 210dA

2.1 325bcA

1.7 300bcA

3.3 445aA

1.4 117

10 110bcC

2.7 145aCD

2.5 165aC

2.1 170aC

2.1 48

20 70cD

1.8 95bcD

1.6 110bcCD

2.7 190aC

2.7 52

LSD0.05 43 46 51 56


3.11A.3.2.2 Potassium (K) in shoot

Alongside row, the shoot K uptake increased with fungal inoculations

as compared to C (with no fungus applied), being the maximum in F1 + F2

and the minimum in C. The plants harvest from F1 showed enhanced K

uptake than F2. Such a variability pattern was observed for all the TSW-Soil

mixtures.

168

Within column, the K shoot uptake decreased with the increasing

percentage of TSW in soil mixtures for all TSW-Soil mixtures (5, 10 and 20

%). However, the 5 % plants showed significantly higher shoot K uptake than

soil mixed with no TSW. The 0* plants showed enhanced shoot k uptake than

corresponding 0 (% TSW-Soil) except for C.

3.11A.3.2.3 Sodium (Na) in shoot

Alongside row, the shoot Na concentration increased with fungal

inoculation as compared to C (with no fungus applied) and found to be the

maximum in F1 + F2 being the minimum in C. The plants from F2 exhibited

enhanced shoot Na uptake than F1. Such a variability trend was observed for

all of the TSW-Soil mixtures except 5 %, where F1 plants had greater shoot

Na than F2.

Within columns, the shoot Na concentration decreased with increasing

TSW percentage in soil, being the maximum in 5 % and the minimum in soil

treatments mixed with no TSW. The 0* plants showed greater shoot Na

concentration than corresponding 0 (% TSW-Soil).

3.11A.3.3 Category-I metals in plant ROOT

The sunflower root uptake of Category-I metals was variable with

respect to different fungal inoculations as well as TSW percentage in soil. The

details are as under:

3.11A.3.3.1 Calcium (Ca) in root

Like the Ca variability trend observed plant shoot, the root Ca

concentration increased in plants inoculated with fungi than those applied with

no fungi, being the maximum in F1 + F2 treatments and the minimum in C

(with no fungus applied). Such a variability trend was observed for all the

TSW-Soil mixtures. The F2 inoculations incurred enhanced root Ca uptake

than those inoculated with F1.

Down the column, the root Ca concentration observed to be lesser in

0* than corresponding 0 (TSW-Soil) of all the fungal treatments. Among 5, 10

and 20 % TSW-Soil, the plant from 10 % showed the maximum root Ca while

169

those from 20 % exhibited the least root Ca uptake for all of the fungal

treatments.

3.11A.3.3.2 Potassium (K) in root

Alongside row, the K contents in plant roots increased with fungal

inoculation as compared to those applied with no fungi i.e. C. The plants from

F1 + F2 exhibited the maximum while those from the C showed the minimum

K root concentration. The F2 inoculation caused greater root K accumulation

than corresponding F1 for all the TSW-Soil treatments except 5 % where F1

plants had more root K than those from F2.

Down the column, the 0* plants showed lesser root K concentration

than those from the corresponding 0 (% TSW-Soil) and such a trend was

observed for all the fungal treatments. Among 5, 10 and 20 (% TSW-Soil), the

root K concentration decreased down the column with increasing percentage

of TSW in soil, being the maximum in 5 % and the minimum in 20 %. Such a

variability tendency was observed for all the fungal treatments.


) observed in ROOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery



LSD0.05 C F1 F2 F1+F2

Ca

0* 5cE

1.1 8bcE

1.2 10bcEF

1.0 15aE

1.8 1.2

0 5deE

1.9 10cdE

2.9 15bEF

1.9 20aE

1.8 3.7

5 110cCD

1.2 155abBC

1.9 160aC

2.1 180aC

2.9 47

10 245abA

2.2 260aA

1.3 265aA

2.4 280aA

1.2 73

20 95bcCD

1.6 110bcCD

1.5 115bcD

2.3 175aC

2.4 48

LSD0.05 48 52 51 46

K

0* 5bcD

0.9 8bE

2.1 10aDE

2.7 12aD

2.2 6.5

0 10bD

2.2 12bE

2.9 14abDE

2.2 18aD

1.6 5.9

5 90bcA

2.3 110bA

2.1 105bA

3.5 125aA

2.9 32

10 80cA

1.2 95bcA

1.9 110bA

2.4 130aA

1.8 37

20 45cdC

2.1 55cCD

1.2. 65bcC

3.2 85aB

2.1 29

LSD0.05 18 22 21 25

Na

0* 7cdD

0.6 6cdDE

3.2 10bcE

1.2 15a 2.2 7.6

0 8cD

1.5 10bcDE

0.9 12bcE

2.9 20aE

2.8 5.9

5 65cdA

2.8 90bA

2.2 110aA

2.5 125aA

1.9 32

10

35cBC

2.6 40cC

1.1 50bcCD

0.7 75aBC

1.5 28

20 20bcC

3.1 25bcCD

1.5 30bD

1.3 45aD

2.1 18

LSD0.05 11 15 16 25


170

3.11A.3.3.3 Sodium (Na) in root


enhanced the Na uptake in roots. The maximum Na in root observed to be in

plants harvested from F1 + F2 while the minimum in plants cultivated on soil

inoculated with no fungi. The F2 incurred better root Na uptake effects than

F1. The plants from all of the TSW-Soil treatments followed the above

mentioned variability pattern.

Down the column, root Na concentration in 0* observed to be lesser

than the corresponding 0 (% TSW-Soil). Among 5, 10 and 20 % (TSW-Soil),

the root Na concentration decreased with increasing percentage of TSW in

soil and found to be the maximum in 5 % and the minimum in 20 %. The

plants from all of the fungal treatments followed the similar variability pattern.

3.11A.3.4 Category-II metals in plant shoot

The Category-II metals i.e. the AAS detected metals in shoot varied

with respect to fungal inoculations as well as increasing percentage of TSW in

soil, as given in Table 3.11A.5. The details are as under:

3.11A.3.4.1 Cd in shoot


fungi and found to be the maximum in F1 + F2 treatments and being the

minimum in C i.e. where no fungi was applied. The F2 inoculations incurred

enhanced shoot Cd concentration effects than F1. The plants from all of the

TSW-Soil treatment observed to follow the same variability pattern.

Down the column, the shoot Cd concentration observed to be lesser in

0* than those from the 0 (% TSW-Soil) for all of the fungal treatments. Among

5, 10 and 20 % (TSW-Soil), the plant shoot Cd concentration decreased with

increasing percentage of TSW in soil and found to be the maximum in 5 %

and the minimum in 20 % of all the fungal treatments.

3.11A.3.4.2 Cr in shoot

Alongside row similar to the trend observed for shoot Cd concentration,

the shoot Cr concentration increased with fungal inoculation being the

171

maximum in F1 + F2 while being the minimum in C i.e. with no fungal

inoculation.


) observed in SHOOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery



w:w)


C F1 F2 F1+F2

Cd

0* 5cE

0.8 10bcEF

2.1 15abE

2.2 20aEF

3.2 4.3

0 8bcE

2.9 12abEF

0.9 14aE

1.9 16aEF

0.98 4.1

5 110dA

2.7 290abA

1.8 310aA

1.5 345aA

1.99 55

10 90bAB

3.2 110abCD

1.7 125aCD

0.34 140aD

2.5 34

20 40bcCD

3.6 55bDE

2.5 60aDE

1.2 75aE

3.1 28

LSD0.05 29 62 68 71

Cr

0* 10cEF

0.93 15bcF

3.4 20aE

1.9 22a 2.4 5.6

0 8bcEF

2.2 10bcF

3.79 12bcE

3.5 23a 2.8 7.9

5 280eA

2.9 455dA

3.8 390dA

2.3 880aA

3.9 159

10 190eBC

2.1 395cdAB

3.5 410cdA

2.3 990aA

3.5 174

20 110efD

4.1 290dD

2.1 325dB

3.2 855aA

2.8 143

LSD0.05 58 98 78 85

Cu

0* 15cF

3.9 20abF

2.8 22aE

1.3 25aF

2.7 7

0 10cdF

1.9 15bcF

3.9 18bcE

3.2 30aF

3.8 12

5 285dA

2.4 455bcA

2.8 430bcA

2.9 550aA

2.9 47

10 245cdB

2.4 390abAB

1.9 355bAB

3.4 425aB

3.9 51

20 110deDE

3.1 215bcD

1.3 245bCD

2.4 310aCD

2.4 39

LSD0.05 58 88 76 115

Fe

0* 3cCD

0.12 6aDE

0.9 5abD

1.2 7aD

1.4 5.4

0 5bcCD

2.1 6aDE

1.8 6aD

1.5 8aD

0.32 8.2

5 45dA

3.7 70bcA

1.5 65bcA

1.2 80aA

1.9 23

10 40cA

3.5 50bcB

2.7 40cBC

0.3 70aA

2.5 37

20 10cC

3.1 20bCD

3.5 25abC

2.5 35aBC

2.1 24

LSD0.05 8.7 9.7 10.2 13.8

Mg

0* 8bcC

1.2 10abBC

2.4 12aB

0.8 15aBC

1.5 9

0 5cCD

1.1 7bBC

1.9 8bBC

2.1 12aC

0.9 6.8

5 15bAB

0.9 17bAB

2.8 20aA

3.2 25aA

2.9 5.6

10 20bcA

2.3 22bcA

1.4 18bcA

2.1 30aA

1.2 7.2

20 10bC

2.1 12abB

1.1 11bB

1.2 15aBC

1.5 6.4

LSD0.05 3.2 9.6 11.1 6.5

Ni

0* BDL BDL BDL BDL -

0 BDL BDL BDL BDL -

5 5bcA

1.9 7bAB

2.8 8aB

2.5 10aB

2.7 4.3

10 8cA

1.5 10bcA

2.7 13aA

2.4 15aA

2.2 7.6

20 4cAB

1.1 7bAB

1.5 5bcBC

1.2 10aB

1.6 5.6

LSD0.05 0.8 1.1 1.7 3,1

Zn

0* 15dD

0.8 35bcE

2.3 30bcF

2.7 55aG

3.2 12

0 18cD

2.6 22bcE

1.2 28bcF

1.1 45aG

1.9 22

5 210dA

2.1 490bcA

1.8 510bcA

1.5 620aA

1.9 156

10 235cdA

1.2 310cBC

1.7 290cC

1.4 425aCD

1.3 134

20 190bB

2.6 225aC

1.5 210aCD

1.2 230aE

1.3 127

LSD0.05 46 98 115 165


172

The plants from F2 showed enhanced shoot Cr uptake than

corresponding F1 for all of the TSW-Soil treatments except 5 % where F1

plants performed better than those from F2. The plants from all of the TSW-

Soil mixtures followed the same shoot Cr variability pattern.

Within columns, the shoot Cr concentration observed to be greater in

plants harvested from 0* than corresponding 0 (% TSW-Soil) for all the fungal

treatments apart from F1 + F2. For 5, 10 and 20 %, the shoot Cd

concentration decreased with increasing percentage of TSW in soil for all the

fungal treatments; being the maximum in sunflower shoots harvested from 5

% and the minimum in 20 % except F2 and F1 + F2.

3.11A.3.4.3 Cu in shoot

Along the row, the variability pattern of shoot Cu uptake followed the

same pattern as observed for Cd. The shoot Cu concentration increased with

fungal inoculations as compared to those where no fungus was applied; being

the maximum in plants harvested from F1 + F2 and the minimum C. Such a

shoot Cu variability trend was observed for all of the TSW-Soil treatments.

Down the column, the plants from 0* showed enhanced shoot Cu

uptake than those from the corresponding 0 (% TSW-Soil). Such a variability

pattern was observed for all the fungal treatments except F1 + F2 where 0*

plants showed lesser shoot Cu uptake than the corresponding 0. Among 5, 10

and 15 % (TSW-Soil), the shoot Cu concentration decreased with increasing

percentage of TSW in soil; being the maximum in 5 % and the minimum in 20

%. All of the fungal treatments observed to show the similar shoot Cu uptake

variability.

3.11A.3.4.4 Fe in shoot

Along the row, the sunflower shoot Fe concentration observed to

increase with fungal inoculations as compared to treatments where no fungus

was applied. The maximum value of shoot Fe was observed to be in plants

harvested from F1 + F2 while being the minimum in C i.e. with not fungus

applied. Such a variability of shoot Fe uptake was observed for all of the

173

TSW-Soil mixtures. The F2 incurred lesser shoot Fe uptake than

corresponding F1 for all the TSW-Soil mixtures except 0 and 20 %.

Within columns, the plants from 0* had lesser shoot Fe uptake than

corresponding 0 (% TSW-Soil) for all of the fungal treatments except for F1. In

case of 5, 10 and 20 (% TSW-Soil), the shoot Fe uptake decreased with

increasing percentage of TSW in soil; being the maximum in plants harvested

from 5 % and the minimum in 20 % for all the fungal treatments.

3.11A.3.4.5 Mg in shoot

Along the row, the shoot Mg concentration increased with fungal

inoculations as compared to those where no fungus was applied and such a

trend was observed for all of the TSW-Soil mixtures. The maximum shoot Mg

concentration was observed for plants harvest from F1 + F2 while being the

minimum in C i.e. where no fungus was applied. The plants with F2

inoculations showed enhanced shoot Mg uptake than corresponding F1 plants

except 10 and 20 (% TSW-Soil) mixtures.

Down the column, the value of plant shoot Mg was greater in 0* than

corresponding 0 (% TSW-Soil). Among 5, 10 and 15 %, the maximum shoot

Mg concentration was observed in plants harvested from 10 % while being the

minimum in those harvest from 20 % for all of the fungal treatments.

3.11A.3.4.6 Ni in shoot

Moving along the row, the sunflower shoot Ni observed to be BDL in 0*

as well as 0 %. i.e. Soil. In case of 5, 10 and 20 %, it was observed to

increase where fungus was inoculated than where no fungus was applied;

being the maximum in F1 + F2 and the minimum in C.

Down the column, the plants from 10 % observed to have the

maximum value of shoot Ni concentration while being the minimum in those

harvested from 20 %.

3.11A.3.4.7 Zn in shoot

Alongside row, the shoot Zn concentration increased with fungal

inoculations than those applied with no fungi. The maximum shoot Zn

174

concentration was observed in plants inoculated with F1 + F2 while being the

minimum in C where no fungus was applied. The F2 inoculations incurred

enhanced shoot Zn concentration than F1 for all of the TSW-Soil mixtures

except 0*, 10 and 20 %.

Down the column, the shoot Zn concentration was greater in 0* % than

corresponding 0 % for all the fungal treatments except C. The 10 % plants

showed the maximum shoot Zn concentration than plants from any of the

TSW-Soil mixtures for all the fungal treatments.

3.11A.3.5 Category-II metals in plant root


observed to differ with varying percentage of TSW in the soil as well in

response to different fungal inoculations, as given in Table 3.11A.6.

3.11A.3.5.1 Cd in root

Along the row, the Cd root concentration increased with application of

fungi and found to be the maximum in F1 + F2 treatments and being the

minimum in C i.e. where no fungi was applied. The F2 inoculations incurred

enhanced root Cd concentration than F1. The plants from all of the TSW-Soil

treatment observed to follow the same variability pattern.

Down the column, the root Cd concentration observed to be lesser in

plants from 0* than those from the 0 (% TSW-Soil) for all of the fungal

treatments except F1. Among 5, 10 and 20 % (TSW-Soil), the plant root Cd

concentration decreased with increasing percentage of TSW in soil and found

to be the maximum in 5 % and the minimum in 20 % of all the fungal

treatments.

3.11A.3.5.2 Cr in root

Alongside row similar, the root Cr concentration increased with fungal

inoculation being the maximum in F1 + F2 while being the minimum in C i.e.

with no fungal inoculation. The plants from F2 showed enhanced root Cr

uptake than corresponding F1 for all of the TSW-Soil treatments except 10 %

where F1 plants performed better than those from F2.

175


) observed in ROOT of 78-days old Helianthus annuus cultivated on field soil mixed with different percentages of tannery



LSD0.05 C F1 F2 F1+F2

Cd

0* BDL 5bD

3.1 3bcDE

1.2 8aCD

2.9 0.8

0 BDL 4bcD

1.9 5bcDE

1.1 10aCD

1.8 0.7

5 35cdA

1.8 45cA

2.8 60bA

1.9 80aA

4.4 12.4

10 20dBC

4.2 35cB

3.7 40cBC

2.7 70aA

3.1 11.6

20 10cC

2.8 12cDE

3.2 15bcD

2.7 25aC

3.6 3.3

LSD0.05 4.5 6.5 5.7 9.8

Cr

0* 3cE

4.1 5bD

2.7 6bE

3.2 10aE

2.9 1.1

0 BDL 7bD

1.6 8abE

2.1 11aE

3.8 0.9

5 180deA

2.2 225cA

2.8 255cA

2.4 340aC

2.9 34.2

10 95fC

3.7 210deA

3.2 190deB

3.3 640aA

3.5 94.5

20 55deD

2.9 90dC

3.5 110dCD

1.8 310aC

3.5 35.2

LSD0.05 35 47 52 68

Cu

0* BDL 5bcE

2.1 8aDE

3.2 10aDE

2.1 3.1

0 BDL 8cE

2.9 10bcDE

2.5 15aDE

3.8 1.8

5 155bcA

1.3 210aA

1.8 190aA

1.6 210aA

2.9 30.5

10 110bcB

3.5 125abC

2.7 130abB

3.3 155aBC

2.3 5.9

20 40cdD

2.1 80bCD

3.5 90bC

1.2 115aC

1.1 39.7

LSD0.05 22 27 32 44

Fe

0* BDL 3aE

1.4 2abD

1.2 4aD

1.1 0.5

0 2bD

2.9 3abE

1.12 2bD

1.1 5aD

2.8 0.7

5 20bcA

2.4 25bcA

3.8 30aA

1.3 35aA

2.9 7.6

10 15cAB

3.1 20bcB

2.7 18cBC

1.4 35aA

2.2 7.3

20 8cBC

1.9 12abCD

1.5 13aC

1.1 15aC

1.3 3.5

LSD0.05 4.3 5.7 6.3 8.7

Mg

0* 3cC

0.8 5bcD

1.2 6aD

1.3 8aCD

2.3 1.1

0 4bcBC

1.9 5bD

1.9 6bD

1.7 10aCD

2.9 1.3

5 13cA

2.1 20bcA

1.5 18bcA

1.5 28aA

2.9 2.3

10 10cA

2.5 15bB

1.8 20aA

1.4 22aAB

2.8 2.7

20 5cdBC

1.4 10bcC

1.7 12bcBC

2.2 17aC

3.1 3.8

LSD0.05 1.5 1.7 2.1 3.9

Ni


0 BDL BDL BDL BDL -

5 2bcAB

2.7 4bA

2.4 5bA

1.5 8aA

1.9 0.8

10 4bcA

3.4 5bcA

2.7 3cAB

2.4 10aA

2.5 1.2

20 2cAB

2.9 3bcAB

2.5 2cB

1.2 6aB

1.1 1.1

LSD0.05 0.6 0.4 0.2 0.7

Zn

0* 10dD

1.3 25bDE

1.8 28bD

1.2 35aE

1.2 1.5

0 12dD

1.9 15cdDE

1.3 17cdDE

2.9 35aE

1.8 4.5

5 125dA

1.7 210abA

1.2 190bA

1.3 230aA

1.99 23.2

10 130cdA

1.2 145cBC

1.9 155cB

1.3 225aA

2.5 17.7

20 110bcB

1.6 125bC

2.1 135abBC

2.2 160aC

2.1 19.4

LSD0.05 22.2 24.1 27.3 28.2

0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger and T. pseudokoningii; LSD: least significant

difference

Within columns, the root Cr concentration observed to be lesser in

plants harvested from 0* than those from corresponding 0 (% TSW-Soil) for all

the fungal treatments. Among all the fungal treatments apart from soil with no

176

TSW, the root Cr concentration decreased with increasing percentage of TSW

in soil for all of the TSW-Soil mixtures; being the maximum in sunflower roots

harvested from 5 % and the minimum in 20 % except F1 + F2 where it was

the maximum in 10 %.

3.11A.3.5.3 Cu in root

Along the row, the variability pattern of root Cu uptake followed the

same pattern as observed for root Cd. The root Cu concentration increased

with fungal inoculations as compared to those where no fungus was applied;

being the maximum in plants harvested from F1 + F2 and the minimum in

plants belonging to C. Such a root Cu variability trend was observed for all of

the TSW-Soil mixtures.

Down the column for all of the fungal treatmetns, the plants from 0*

showed reduced root Cu uptake than those from the corresponding 0 (%

TSW-Soil) C where it was BDL. For all of the fungal treatments for soils mixed

with TSW, the root Cu concentration decreased with increasing percentage of

TSW in soil; being the maximum in 5 % and the minimum in 20 %.

3.11A.3.5.4 Fe in root

Along the row, the sunflower root Fe concentration was increased with

fungal inoculations as compared to treatments where no fungus was applied.

The maximum value of root Fe was observed to be in plants harvested from

F1 + F2 while being the minimum in C i.e. with not fungus applied for all of the

TSW-Soil mixtures.

Within columns for all of the fungal treatments, the plants from 0* had

lesser root Fe uptake than corresponding 0 (% TSW-Soil) for all of the fungal

treatments except for F1 and F2. For all the fungal treatments where soils

were mixed with TSW, the root Fe uptake decreased with increasing

percentage of TSW in soil; being the maximum in plants harvested from 5 %

and the minimum in 20 %.

3.11A.3.5.5 Mg in root

Along the row, the root Mg concentration increased with fungal

inoculations as compared to those where no fungus was applied and such a

177

trend was observed for all of the TSW-Soil mixtures. The maximum root Mg

concentration was observed for plants harvest from F1 + F2 while being the

minimum in C i.e. where no fungus was applied. The plants with F2

inoculations showed enhanced root Mg uptake than corresponding F1 plants

except 5 (% TSW-Soil) mixtures.

Down the column, the value of plant root Mg in each of 0* was either

equal to or greater than the corresponding 0 (% TSW-Soil). For all of the

fungal treatments where soil was mixed with TSW, the shoot Mg

concentration decreased with increasing percentage of TSW down the

column; being the maximum in plants harvested from 5 % while being the

minimum in those harvest from 20 % for all of the fungal treatments.

3.11A.3.5.6 Ni in root

While comparing along the row, the sunflower root Ni was observed to

increase where fungus was inoculated than where no fungus was applied;

being the maximum in F1 + F2 and the minimum in C. The plant from both F1

and F2 had almost similar Ni concentration in root.

Down the column for all of the fungal treatments, the root Ni was BDL

in plants harvested from 0* as well as 0 (% TSW-Soil). However, the plants

from 10 % observed to have the maximum value of root Ni concentration

while being the minimum in those harvested from 20 %.

3.11A.3.5.7 Zn in root

Alongside row, the root Zn concentration increased with fungal

inoculations than those applied with no fungus. The maximum root Zn

concentration was observed in plants inoculated with F1 + F2 while being the

minimum in C where no fungus was applied. The F2 inoculations incurred

enhanced root Zn concentration than F1 for all of the TSW-Soil mixtures

except 5 % where F1 plants had more root Zn concentration than

corresponding F2.

Down the column for all of the fungal treatments, the root Zn

concentration was greater in 0* % than corresponding 0 % except C. For all

178

fungal treatments where soil was mixed with TSW, the root Zn concentration

decreased down the column with increasing percentage of TSW in soil; being

the maximum in 5 % and the minimum in 20 % except C treatment where the

maximum root Zn was observed in 10 % while being the minimum in 20 %.

3.11A.4 Fungal analyses

The c.f.u. (× 105 c.f.u. g-1 soil) counts of the soils after the harvest of

78-days old plants of sunflower are given in Table 3.11A.7.

Along the row, the c.f.u. number increased in soil treatments inoculated

with fungus than those applied with no fungus; being the maximum in F1 + F2

treatments and the minimum in C i.e. where no fungus was applied. This trend

of variability was observed for all of the TSW-Soil mixtures. The soil

treatments applied with F2 fungus had greater number of c.f.u. than

corresponding F1 for all the TSW-Soil mixtures except 5 %.

Down the column for all the fungal treatments, the soils from 0* had

lesser number of c.f.u. than corresponding 0 (% TSW-Soil). For all the fungal

treatments where soils were mixed with TSW, the c.f.u. in soil decreased

down the column with increasing percentage of TSW in soil; being the

maximum in 5 % and the minimum in 20 %.

3.11A.5 Meta-analytical perspective



Table 3.11A.7. The post-harvest fungal analyses (× 105 c.f.u. g

-1 soil) of soil used for the cultivation of

78-days old sunflower (Helianthus annuus) on field soil mixed with different percentages of tannery solid

waste (TSW-Soil mixtures). The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n =

6). TSW-Soil (% w:w)

mixture and its type

Treatment LSD0.05

C F1 F2 F1+F2

0* 0.2dBC

0.6 0.5cdC

1.5 0.7cdCD

1.1 2.5aCD

1.6 0.9

0 0.3bcBC

1.2 0.8bC

1.8 1.1aCD

1.2 1.5aD

2.8 0.5

5

2.9dA

2.3 4.1cA

1.6 3.1cdAB

1.3 6.3aA

2.7 1.5

10

2.8cA

2.6 3.2bcAB

1.4 3.9bcA

2.0 5.7aA

2.4 1.2

20 2.1bcA

2.8 2.6bBC

2.4 2.7bBC

2.5 3.8aC

2.3 1.1

LSD0.05 1.2 0.8 0.6 0.7


179

3.11A.5.1 Category-I metals translocation index (%)

The plant translocation index values analyzed for Category-I metals

varied in response to the fungal inoculations as well as with different

percentages of TSW, as given in Table 3.11A.8.

Table 3.11A.8. Meta-analytical phytoextraction indices of sunflower: the Category-I translocation index

(%) analyzed for sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.


C F1 F2 F1+F2

Ca

5 322.7 238.7 256.2 305.5

10 165.3 171.1 147.1 223.2

20 305.2 281.8 256.5 251.4

K

5 233.3 300.0 276.1 284.0

10 237.5 215.7 190.9 215.3

20 277.7 263.3 200.0 182.3

Na

5 323.0 361.1 272.7 356.0

10 314.2 362.5 330.0 226.6

20 350.0 380.0 366.6 422.2


In case of Ca, in terms of influence incurred by mixing of TSW in soil

and fungal inoculations, the 5 % C treatment found to be having the highest

translocation index value (322.7 %) and that of 10 % F2 being the least (147.1

%).

For K, the maximum translocation index value (300 %) was found to be

in 5 % F1 while being the least (182.3 %) in 20 % F1 + F2.

In case of Na, the 20 % F1 + F2 had the maximum translocation index

value (422.2 %) while being the least (226.6 %) in 10 % F1 + F2.

3.11A.5.2 Category-II metals translocation index (%)

The Category-II metals translocation index values showed greater

variability in response to the influence of TSW mixing in soil and inoculations

with different fungi, as given in Table 3.11A.9. For Cd, the maximum

translocation index value (644.4 %) was noted for 5 % F1 while the minimum

value (200 %) was recorded in 10% F1 + F2.

In case of Cr, the highest translocation efficiency (322.2 %) was

observed in 20 % F1 and the minimum (152.9 %) was observed in 5 % F2.

180

For Cu, the maximum translocation index value (312 %) was calculated

for 10 % F1 and the minimum (183.8 %) was recorded in 5 % C.

Table 3.11A.9. Meta-analytical phytoextraction indices of sunflower: the Category-II translocation index

(%) analyzed for sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.


C F1 F2 F1+F2

Cd

5 314.2 644.4 516.6 431.2

10 450.0 314.2 312.5 200.0

20 400.0 458.3 400.0 300.0

Cr

5 155.5 202.2 152.9 258.8

10 200.0 188.0 215.7 154.6

20 200.0 322.2 295.4 275.8

Cu

5 183.8 216.6 226.3 261.9

10 222.7 312.0 273.0 274.1

20 275.0 268.7 272.2 269.5

Fe

5 225.0 280.0 216.6 228.5

10 266.6 250.0 222.2 200.0

20 125.0 166.6 192.3 233.3

Mg

5 115.3 85.0 111.1 89.2

10 200.0 146.6 90.0 136.3

20 200.0 120.0 91.6 88.2

Ni

5 250.0 175.0 160.0 125.0

10 200.0 200.0 433.3 150.0

20 200.0 233.3 250.0 166.6

Zn

5 168.0 233.3 268.4 269.5

10 180.7 213.7 187.0 188.8

20 172.7 180.0 155.5 143.7


For Fe, the maximum translocation index value (280 %) was found in 5

% F1, while the minimum value (125 %) was observed in 20 % C.

In case of Mg, the highest (200 % each) translocation efficiency was

observed in C of both 10 and 20 % while the lowest value (85 %) was

recorded for 5 % F1.

For Ni, the translocation index recorded to be the highest (433 %) in 10

% F2 and the least (125 %) of it was observed to be in 5 % F1 + F2.

As far as the Zn is concerned, there was the maximum (269.5 %)

translocation index in 5 % F1 + F2 while the lowest value (143.7 %) was

recorded in 20 % F1 + F2.

181

3.11A.5.3 Tolerance index (TI) for shoot and root

The tolerance index values for both root and shoot and their variation in

response to TSW mixing in soil and inoculation with different fungi are given in

Table 3.11A.10.

Table 3.11A.10: Meta-analytical phytoextraction indices of sunflower: the tolerance index (TI) analyzed

for shoot and root of sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.


C F1 F2 F1+F2

TI Shoot

5 1.68 2.17 1.77 1.65

10 1.40 1.94 1.59 1.58

20 0.78 0.99 0.86 0.75

TI Root

5 1.05 1.24 1.31 1.14

10 1.03 1.12 1.09 1.15

20 0.54 0.86 0.90 0.80


For shoot, the TI found to be the highest (2.17) in 5 % F1 while being

the lowest (0.75) in 20 % F1 + F2.

In case of root, the highest value (1.31) of TI observed to be in 5 % F2

while being the lowest (0.54) in 20 % C.

3.11A.5.4 Category-I metals specific extraction yield (SEY %)

The SEY (%) for Category-I metals and its variability with respect to

TSW mixing in soil and inoculation with different fungi is given in Table

3.11A.11.

Table 3.11A.11: Meta-analytical phytoextraction indices of sunflower: the specific extraction yield (SEY

%) for Category-I metals in sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.


C F1 F2 F1+F2

Ca

5 28.6 32.3 35.0 44.9

10 27.7 30.6 27.9 38.5

20 13.2 14.4 14.0 21.1

K

5 33.7 49.4 44.3 53.9

10 22.3 24.7 26.4 33.8

20 8.5 10.1 9.8 12.1

Na

5 20.2 30.6 30.2 42.0

10 5.7 7.3 8.5 9.7

20 1.9 2.5 3.0 5.0


182

In case of Ca, the maximum SEY (44.9 %) was found to be in 5 % F1 +

F2 while being the minimum (13.2 %) in 20 % C.

The SEY value for K found to be the highest (53.9 %) in 5 % F1 + F2

and the lowest (8.5 %) of it was recorded in 20 % C.

For Na, the highest value of SEY (42 %) was calculated for 5 % F1 +

F2 and the lowest of it (1.9 %) was found to be in 20 % C.

3.11A.5.6 Category-II metals specific extraction yield (SEY %)

The SEY percentages calculated for Category-II metals and their

variability in response to spiking of soils with variable levels of TSW and

inoculations with different fungi, are given in Table 3.11A.12.

Table 3.11A.12. Meta-analytical phytoextraction indices of sunflower: the specific extraction yield (SEY

%) for Category-II metals in sunflower cultivated on field soil mixed with different percentages of tanner solid waste (TSW) and inoculated with different fungi.


C F1 F2 F1+F2

Cd

5 5.4 12.6 13.9 16.0

10 1.6 2.2 2.5 3.1

20 0.5 0.7 0.8 1.1

Cr

5 5.5 8.2 7.8 14.7

10 2.7 5.9 5.8 15.9

20 1.0 2.4 2.8 7.5

Cu

5 32.5 49.2 45.9 56.2

10 16.9 24.5 23.0 27.0

20 2.8 5.6 6.3 8.0

Fe

5 26.0 38.0 38.0 46.0

10 10.7 13.7 11.3 20.5

20 1.9 3.5 4.1 5.4

Mg

5 9.0 11.9 12.2 17.0

10 4.8 5.9 6.1 8.3

20 1.4 2.1 2.2 3.1

Ni

5 20.0 31.4 37.1 51.4

10 21.8 27.2 29.0 45.4

20 5.4 9.0 6.3 14.5

Zn

5 22.9 47.9 47.9 58.2

10 19.3 24.0 23.5 34.3

20 14.8 17.2 17.0 19.2


For Cd, the highest SEY percentage was found to be 16 in case of 5 %

F1 + F2 while the lowest of it was observed to be 0.5 % for 20 % C.

183

In case of Cr, the maximum SEY value 15.9 % was recorded 10 % F1

+ F2 and the minimum of it being 1 % in 20 % C.

Likewise for Cu, 5 % F1 + F2 exhibited the maximum SEY value (56 %)

with corresponding minimum value (2.8 %) in 20 % C.

Similarly for Fe, the highest SEY value (46 %) was recorded in 5 % F1

+ F2 and that of lowest was found to be 1.9 % in 20 % C i.e. with no fungal

inoculum.

Following the similar trends, the SEY calculated for Mg found to be the

highest (17 %) in 5 % F1 + F2 while the lowest of it (1.4 %) found to be in 20

% C.

As well as, the SEY for Ni calculated to be the highest (51.4 %) in case

of 5 % F1 + F2 while the lowest of it (5.4 %) being in 20 % C.

Also for Zn, the maximum SEY percentage was calculated to be 58.2 in

5 % F1 + F2 while the minimum of it was observed to be 14.8 % in 20 % C.

184

3.11B. Experiment with Tagetes patula

3.11B.1 Pre-sowing analysis

The analyses of the soil in field plots mixed with different percentages

of TSW and inoculated with different fungi after harvesting sunflower

(Helianthus annuus) and before cultivating French marigold (Tagetes patula)

are given in Table 3.11B.1.

3.11B.1.1 Pre-sowing Category-I metals in field soil

For the Category-I metals among the fungal treatments, the

concentration of Ca was significantly greater in all of the TSW-Soil mixtures

than corresponding soil treatments mixed with no TSW i.e. 0* and 0 (% TSW-

Soil). It increased with increasing percentage of TSW in soil for all the fungal

treatments; being the maximum in 20 % and the least in 5 %. Similar trends of

soil Ca variability observed for K as well as Na.

For the Category-I metals between the fungal treatments, the Ca

concentration in 0* of all the fungal treatments found to the maximum in case

of C and the minimum in corresponding F1 + F2. Similar trend of variation was

observed for K as well as Na in 0* of all the fungal treatments. Likewise, the 0,

5, 10 and 20 % (TSW-Soil) of all the fungal treatments had the minimum

values of Category-I metals in C treatment and the corresponding minimum

values in F1 + F2, as given in Table 3.11B.1.

3.11B.1.2 Pre-sowing Category-II metals in field soil

For the Category-II metals among the fungal treatments, the

concentration of Cd was significantly greater in all of the TSW-Soil mixtures

than corresponding soil treatments mixed with no TSW i.e. 0* and 0 (% TSW-

Soil). It increased with increasing percentage of TSW in soil for all the fungal

treatments; being the maximum in 20 % and the least in 5 %. Similar trends of

soil Cd variability observed for rest of the Category-II metals.

For the Category-II metals between the fungal treatments, the Cd

concentration in 0* of all the fungal treatments was found to be the maximum

in case of C and the minimum in corresponding F1 + F2. Similar trends of

variation were observed for Cd in 0* of all the fungal treatments.

185

Table 3.11B.1. The concentration of category-I category-II metals (mgkg-1

) observed in soil amended with different concentration of tannery solid waste

(TSW-Soil % w:w) determined after the harvesting sunflower (Helianthus annuus) and prior the sowing French marigold (Tagetes patula). The mean values

S.D. with common letters are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).

Type of

metals

TSW-Soil (% w:w) mixtures with different fungal inoculations

0* 0 5 10 20 0* 0 5 10 20 0* 0 5 10 20 0* 0 5 10 20

LSD0.05

C F1 F2 F1+F2

Cate

go

ry-I

(mg

kg

-1)

Ca 50

+ 2.4 80

+ 2.3 1,570 + 4.1

1,811 + 2.1

2,700 + 2.6

45 + 2.4

75 + 3.2

1,450 + 3.7

1,640 + 3.7

2,610 + 2.4

38 + 3.4

70 + 2.6

1,410 + 2.3

1,750 + 1.3

2,600 + 3.2

35 + 3.1

65 + 1.8

1,190 + 2.1

14,40 + 3.4

2,290 + 3.2

K 550 + 3.1

640 + 3.1

810 + 3.5

940 + 1.8

1,850 + 2.7

525 + 3.7

610 + 3.3

720 + 2.5

820 + 3.8

1,750 + 2.1

510 + 3.1

590 + 2.4

755 + 3.1

910 + 2.5

1,790 + 2.8

490 + 3.1

550 + 2.6

670 + 3.1

720 + 2.1

1,680 + 3.0

Na 670 + 3.2

950 + 2.4

1,290 + 2.4

2,410 + 2.6

4,600 + 3.3

640 + 3.1

890 + 3.1

1,255 + 2.6

2,320 + 4.4

4,450 + 4.1

630 + 2.5

895 + 2.8

1,235 + 2.9

2,290 + 3.3

4,440 + 3.1

590 + 2.3

740 + 2.1

1,125 + 2.5

2,195 + 3.5

4,350 + 4.0

Cat

ego

ry-I

I

(mg

kg

-1)

Cd 40

+ 2.1 45

+ 1.9 2,530 + 3.1

6,490 + 3.9

8,740 + 3.8

35 + 2.0

40 + 2.5

2,410 + 2.8

6,450 + 4.1

8,735 + 4.4

30 + 1.7

40 + 1.9

2,390 + 3.9

6,430 + 4.4

8,720 + 3.6

25 + 1.8

35 + 2.1

2,200 + 3.5

6,370 + 3.3

8,670 + 4.1

Cr 55

+ 2.3 90

+ 1.8 7,790 + 4.1

10,050 + 4.8

15,410 + 4.6

45 + 2.1

80 + 2.3

7,550 + 2.8

9,610 + 3.4

15,270 + 4.7

50 + 1.4

85 + 2.1

7,610 + 2.2

9,620 + 4.1

15,110 + 4.1

35 + 3.2

70 + 1.8

7,030 + 3.7

8,650 + 3.1

13,400 + 4.4

Cu 510 + 3.1

560 + 2.7

1,025 + 3.7

1,810 + 2.1

5,110 + 3.8

450 + 2.2

490 + 2.5

970 + 2.9

1,730 + 3.5

4,950 + 4.6

430 + 1.4

480 + 2.4

990 + 2.1

1780 + 4.8

4.890 + 3.6

410 + 2.1

450 + 1.9

780 + 3.1

1,690 + 2.1

4,810 + 3.4

Fe 30

+ 2.0 50

+ 1.1 210

+ 2.3 480

+ 2.7 890

+ 2.7 35

+ 1.0 45

+ 2.1 190

+ 1.6 485

+ 2.5 840

+ 3.2 38

+ 1.1 45

+ 2.1 185

+ 2.4 490

+ 3.1 830

+ 3.3 25

+ 1.1 30

+ 1.5 170

+ 1.9 470

+ 1.8 810

+ 2.7

Mg 25

+ 1.9 40

+ 1.5 290

+ 2.6 590

+ 2.1 920

+ 3.6 20

+ 0.89 35

+ 1.4 270

+ 1.7 580

+ 2.1 870

+ 2.2 22

+ 2.2 38

+ 2.1 260

+ 2.6 565

+ 2.8 865

+ 2.9 15

+ 2.2 30

+ 2.1 250

+ 2.3 530

+ 3.1 830

+ 2.1

Ni BDL BDL 28

+ 1.4 45

+ 1.8 95

+ 2.1 BDL BDL

25 + 1.1

40 + 1.1

70 + 2.7

BDL BDL 22

+ 1.1 38

+ 1.3 85

+ 1.4 BDL BDL

19 + 1.6

30 + 1.5

65 + 1.5

Zn 140 + 2.1

190 + 1.8

1230 + 2.4

1620 + 2.5

1,890 + 1.1

135 + 1.1

170 + 2.1

960 + 2.7

1,560 + 3.3

1,720 + 2.5

130 + 1.2

185 + 2.2

980 + 2.1

1,590 + 3.1

1,725 + 2.7

120 + 1.1

160 + 2.4

890 + 3.1

1,210 + 3.3

950 + 2.2

0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least

significant difference

186

Likewise, the 0, 5, 10 and 20 % (TSW-Soil) of all the fungal treatments

had the minimum values of Category-II metals in C treatment and the

corresponding minimum values in F1 + F2, as given in Table 3.11B.1.

3.11B.2 Biochemical analyses of Tagetes patula in field

The biochemical parameters observed in live T. patula and its variation

in response to mixing of soil with different TSW percentages and inoculations

with different fungi are given in 3.11B.2. The specific details of each of the

biochemical parameters are as under:

3.11B.2.1 Chlorophyll content

Along the row, the plant chlorophyll contents based on SPAD value

observed in 50-days old French marigold increased in soil treatments applied

with fungus as compared to the C (with no fungus). The plants from F2

showed enhanced chlorophyll contents than F1, being the maximum in F1 +

F2 (applied with combined fungal inoculation) and the least in C, as given in

Table 3.11A.1. Such a trend of variability was observed in all of the TSW-Soil

mixtures.



treatments. In all fungal treatments, the plants from all the TSW-Soil 0 (%

w:w) had more chlorophyll contents than every corresponding 0* (% w:w)

except for F1 and F2 where plants from 0* exhibited the higher value than

those from corresponding 0 (% TSW-Soil). The plants from 5 % (% w:w) of all

the fungal treatments showed the maximum SPAD value within a column

while those from 20 (% w:w) had the least chlorophyll contents.

3.11B.2.2 Soluble protein contents

Likewise chlorophyll contents, the soluble protein contents along the

row increased with the application of fungal inoculations for all the fungal

treatments, as given in Table 3.11B.2. The plants from C (with no fungal

treatment) had the least protein contents than any of the treatment with single

or combine fungal inoculations, being the maximum in F1 + F2. Such a trend

of variability was observed for all of the TSW-Soil mixtures.

187

Table 3.11B.2. The biochemical parameters observed in 82-days old Tagetes patula



w:w)


C F1 F2 F1+F2

Chlorophyll contents

0* 19.5bB

1.8 21.2aC

2.1 22.4aAB

2.3 22.3aC

2.6 0.8

0 20.6bB

1.2 20.4bC

2.4 19.3bB

1.3 23.1aC

2.3 1.1

5 24.1bcA

1.3 26.6bcA

2.8 25.3bcA

2.7 32.5aA

2.9 2.3

10 22.3abA

1.7 23.4aAB

1.1 23.5aA

1.9 24.9aC

1.6 2.8

20 13.8cD

1.8 14.4cDE

2.2 13.7cCD

2.5 19.2aD

1.8 3.1

LSD0.05 1.2 1.5 2.5 2.8

Protein content

0* 2.6dC

1.9 9.6bBC

2.4 9.2bC

3.2 12.1aC

2.2 2.4

0 3.1dC

2.3 10.1abBC

1.9 9.7bC

2.9 12.9aC

1.8 2.1

5 7.4cdA

1.7 17.1aA

2.8 16.7aA

2,5 18.9aA

1.9 3.4

10 3.8dC

2.2 6.5bcC

2.3 7.2bcCD

1.4 11.6aC

2.9 4.1

20 1.9cdCD

2.1 2.9cDE

1.5 3.1cE

1.5 6.1aDE

3.1 4.7

LSD0.05 3.1 2.8 3.2 4.1

SOD

0* 2.1bcD

1.9 3.6aDE

1.8 2.9aEF

1.4 3.1aE

2.1 1.2

0 1.3cD

1.2 2.3aDE

1.4 2.5aEF

1.8 1.1cE

0.89 0.9

5 12.6abA

1.8 13. 2aA

1.2 14.63aA

1.5 14.8aA

2.2 2.6

10 11.1cA

2.7 13.7abA

2.3 13.2abA

2.3 15.1aA

2.7 3.5

20 9.3bcB

1.2 8.9aBC

1.1 7.8aCD

1.6 8.4aCD

1.7 1.6

LSD0.05 1.4 2.3 2.5 3.6

CAT


0 0.1bcDE

0.6 0.4aE

0.1 0.3aEF

0.2 0.2bEF

0.8 0.2

5 12.2deA

1.4 16.2cA

1.3 19.2abA

1.8 21.4aA

2.2 2.1

10 10.2cA

1.3 11.3cC

1.4 12.3cC

1.7 17.4aB

2.1 4.2

20 9.4bcB

1.3 10.6bC

1.7 11.2bC

1.2 13.2aC

1.5 4.7

LSD0.05 2.1 3.2 3.4 5.4

0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant difference

The soluble protein contents was increased down the column observed

to be greater in 0 than corresponding 0* (% w:w) for all the fungal treatments.

For all the fungal treatments and soils mixed with TSW, the soluble protein

contents decreased down the column being the least in 5 % and the highest in

20 % (TSW-Soil). The plants from 5 % had the maximum protein contents in

all the columns.

3.11B.2.3 Superoxide dismutase (SOD) contents

Parallel to the trends found for soluble protein contents, the SOD

contents in plants increased with fungal inoculations within a row for all the

treatments; however, it decreased in case of combined fungal application in

both 0 (% TSW-Soil) mixture, as given in Table 3.11A.2. For 5, 10 and 20 %

(TSW-Soil) mixture, the SOD values increased with the application of single or

188

combined fungal inoculation, being the maximum in plants applied with F1 +

F2.

Within a column, the plants from 0* (control with no geothermal

membrane lining) had shown enhanced SOD contents than 0 (with lining) for

all the fungal treatments. However, it decreased with increasing level of TSW

percentage in soil, being the maximum in 5 % and the minimum in 20 %

except 5 % F1.

3.11B.2.4 Catalase (CAT) contents

The CAT contents of the French marigold exhibited variable pattern

along the row. Other than the 0* (% TSW-Soil) where observed values were

BDL, the CAT values increased along the row with fungal inoculations and

being the maximum in plants applied with F1 + F2 and being the minimum in

TSW-Soil mixtures applied with no fungus.

Down the column, the plants from 0 showed enhanced CAT contents

than corresponding 0* (% TSW: Soil) for all the fungal treatments. However it

decreased down the column with increasing percentage of TSW in soil for all

of the fungal treatments, being the maximum in 5 % (TSW:Soil) and the

minimum in 0 for all the treatments.

3.11B.3 Post-harvest analysis

The growth of 50-days old French marigold cultivated in field varied in

response to different TSW percentages as well as fungal inoculations, as

given in Table 3.11B.3. The details are as under:

3.11B.3.1 Growth performance of Tagetes patula

The details of each of the morphological parameters observed in

French marigold are given in Table 3.11B.3 and described as under:

3.11B.3.1.1 Shoot, root and seedling length (cm)

Along the row, the sunflower shoot, root and seedling length increased

in treatments applied with fungus than those applied with no fungus i.e. C. It

was observed to be the maximum in F1 + F2 and being the minimum in C for

all the TSW-Soil mixtures.

189

Table 3.11B.3. Various morphological parameters observed in 82-days old Tagetes patula cultivated on

TSW-Soil mixtures. The mean values S.D. with common letters (small along the row & capital within a column) are not significantly different according to Duncan’s multiple range test (P = 0.05; n = 6).


w:w)


C F1 F2 F1+F2

Shoot Length (cm)

0* 24.2bA

2.4 27.3aBC

2.1 24.0bBC

1.9 28.6aBC

2.7 2.1

0 23.5bcAB

1.7 21.8bcBC

2.3 22.0bcC

2.9 28.9aBC

2.5 3.4

5 27.5dA

2.8 38.7bcA

2.6 33.7cA

2.8 46.3aA

3.3 7.2

10 25.4cA

2.9 23.8cbC

2.5 26.8cBC

3.1 40.2aA

2.3 5.7

20 21.2bcAB

2.2 22.8bcC

2.8 21.9bcC

2.1 27.5aBC

2.4 3.4

LSD0.05 2.6 4.7 5.2 5.8

Root Length (cm)

0* 20.4bB

2.8 23.2abB

2.3 22.1abB

2.7 25.4aB

2.2 2.6

0 20.5bcB

2.1 21.9bcBC

2.6 21.8bcB

2.4 26.1aB

2.8 3.8

5 27.2bA

1.7 28.8bA

2.8 27.9bA

2.1 32.6aA

3.1 2.9

10 22.2bcB

2.1 25.9bB

2.6 23.2bAB

2.1 27.3aB

2.3 3.4

20 19.1bB

2.5 15.7cdCD

1.7 18.5aB

2.2 19.7aC

2.7 3.1

LSD0.05 3.7 4.8 2.9 4.8


0* 44.9bcB

2.3 50.7abBC

2.2 46.4bcbC

2.4 54.2aC

2.8 4.6

0 44.3bcB

2.8 44.0bcC

2.8 44.1bcBC

3.1 55.2aC

2.7 3.4

5 55.0cdA

2.5 67.8bcA

3.5 65.9bcA

3.1 79.1aA

2.4 9.1

10 47.9bcB

1.6 50.0bcBC

2.8 50.3bcB

2.6 67.9aB

. 3.1 7

20 40.6bBC

2.8 38.8bC

2.1 40.7bC

2.3 47.5aC

2.8 6.1

LSD0.05 4.1 6.1 8.1 7.8

No. of roots/plant

0* 24aA

2.4 22aB

2.1 20abBC

2.6 21aBC

2.2 4

0 22abA

2.1 23aB

2.7 24aA

2.4 25aB

2.4 8.3

5 25abA

2.4 28aA

2.7 26abA

2.9 30aA

2.8 3.1

10 21abAB

2.4 23aB

2.5 22aB

2.7 24aB

2.3 1.9

20 20bAB

2.1 18bC

2.6 16bcCD

2.3 21aBC

2.8 3.5

LSD0.05 4 3 6 3.1

No. of leaves/plant

0* 7bcC

1.5 8aC

1.8 9abC

1.3 11aBC

2.1 1.4

0 8abC

2.1 9aC

1.5 8abC

2.2 10aBC

2.8 2.1

5 15bcA

2.9 17bA

2.7 16bA

2.6 21aA

2.5 3.4

10 9bC

1.2 10bbC

1.7 9bC

2.6 13aBC

1.9 2.9

20 5cCD

1.5 7bC

1.6 8aC

1.9 9aC

1.8 2.6

LSD0.05 4 6 3.3 4.2

Fresh wt./plant (g)

0* 97.1bB

2.5 115.1aB

3.4 123.8aAB

2.2 112.7aBC

2.5 3.7

0 120.1aA

3.5 110.3aB

2.9 114.1aB

4.1 113.5aBC

3.8 3.6

5 125.3abA

2.9 138.3aA

2.8 135.9aA

3.3 145.2aA

2.5 4.5

10 115.1aA

2.2 113.6aB

2.8 111.7aB

3.7 121.1aB

3.5 2.9

20 102.3aB

2.4 109.8aB

2.5 105.2aB

3.1 114.7aBC

4.2 3.2

LSD0.05 5.4 7.9 9.2 8.8

Dry wt. (g)

0* 30.5bA

2.9 32.3aA

2.7 34.1aA

3.6 36.7aA

3.6 2.2

0 31.4aA

2.3 33.1aA

3.1 31.2aA

3.9 33.9aA

2.8 2.8

5 34.9abA

4.1 36.5aA

3.8 33.7abA

3.3 37.8aA

2.1 5.9

10 27.6aB

2.4 22.8bBC

3.2 21.7bBC

3.4 27.5aBC

3.1 7.9

20 18.8bC

3.7 21.2abBC

2.1 23.7aBC

2.7 24.9aBC

3.2 9.2

LSD0.05 3.4 4.1 4.8 4.2

No. of flowers /plant

0* 10bcB

3.1 12bB

2.7 14aA

2.2 16aAB

2.4 4.8

0 11bB

2.8 13aB

3.3 11bAB

2.8 14aB

2.1 5.3

5 14bcA

4.6 16bA

3.7 13bcA

2.5 20aA

2.9 8.6

10 13bA

2.6 11bBC

3.5 12bA

2.7 17aAB

3.7 5.2

20 9bcB

3.2 12abB

2.8 13aA

2.6 14aB

3.9 4.1

LSD0.05 4.2 3.6 3.1 3.7


190

The plants from 0*, 5 and 20 % with F1 inoculation performed better than

those from corresponding F2 treatments. However; from 0 and 10 % with F2

performed better than those from corresponding F1 treatments as shown in

figure 3.11B.1.

Figure 3.11B.1: Phytoextraction field trials with French Marigold (Tagetes patula) cultivated on soil amended with different levels of tannery solid waste (TSW-Soil % w:w); upper: (A) 0* % the only treatment without geothermal membrane allowing leaching, (B) 0 % with geothermal membrane to avoid leaching, (C) 5 %, (D) 10 % and (E) 20 %. The white lines across the strip plots separated by bricked walk ways (horizontal in above and vertical in lower; 25 × 3 ft) in upper and the white arrows in the lower picture indicate soil separations (1.25 ft) subdividing each strip plot into four subplots (5 × 3 ft each) for fungal inoculations viz. C: No fungal inoculum; F1: Aspergillus niger; F2: Trichoderma pseudokoningii; F1 + F2: A. niger + T. pseudokoningii, applied in a randomized complete block design.

A

B

C

D E

N

N

191

Down the column, the plants from 0* had more efficient vegetative

growth in terms of shoot, root and seedling length than corresponding 0

(TSW-Soil) except those applied with F1 + F2. For all the fungal treatments

where soil was mixed with TSW, the plant height decreased down the column

with increasing percentage of TSW, being the maximum in 5 % and the

minimum in 20 %.

The stem girth variations in different TSW-Soil mixtures and fungal

treatment can be observed in Figure 3.11B.2

Figure 3.11.2B: The Tagetes patula stem girth variation in response to soil mixed with different levels of tannery solid waste (TSW : soil w:w) inoculated with fungi (A- 0*% with F1+F2; B- 0% with F1+F2; C- 5% with F2; D- 5% with F1+F2 ; E- 5% with C ;F-10% with F1+F2; G- 10% with F2; H- 20% with F2; I- 20% with F1+F2.

3.11B.3.1.2 No. of leaves, roots and flowers per plant

Along the row, the plants from pots inoculated with fungi showed better

vegetative growth than those applied with no fungi. Except 0*, the maximum

no. of root, leaves as well as flowers observed in plants inoculated with F1 +

F2 and the minimum of which being in C. The F2 inoculation incurred better

vegetative growth than F1.

A B

D

G H

E

I

F

C

192

Down the columns, plants from 0* of all the fungal treatments exhibited

better growth than those from corresponding 0 (5 TSW-Soil). For fungal

treatments where soil was mixed with TSW, the increasing percentage of

TSW decreased the no. of leaves, roots as well as flowers per plant; being the

maximum in 5 % and the minimum in 20 % as shown in Figure 3.11B.1.

3.11B.3.1.3 Fresh and dry weight (g)

The fresh and dry weight was observed to be the maximum and the


and roots for both along the row as well as within column comparisons.

3.11B.3.2 Category-I metals in plant SHOOT



ratio of TSW in soil, as given in Table 3.11B.4.

193





LSD0.05 C F1 F2 F1+F2

Ca

0* 15bcDE

1.3 20aD

1.1 18abD

2.2 22aDE

2.1 2.2

0 18bDE

1.7 22abD

2.5 19bD

2.3 25aDE

1.3 2.6

5 145cBC

3.4 240aA

2.4 210bA

1.7 250aA

2.9 32

10 210bcA

1.9 245abA

2.2 210bcA

2.6 275aA

2.3 49

20 130cBC

2.6 220aA

2.2 235aA

2.3 240aA

2.5 55

LSD0.05 36 42 51 58

K

0* 10bcD

1.8 15aDE

1.2 12bE

1.4 15aDE

2.9 3.4

0 12bcD

1.2 13bDE

1.1 14bE

1.4 17aDE

1.3 2.6

5 110cA

2.3 130cA

2.5 170bA

2.7 215aA

2.2 43

10 90bcA

2.3 115bA

2.9 130aB

2.1 150aBC

3.3 34

20 65bcBC

1.6 75bcC

1.7 60bcCD

1.3 115aC

2.4 28

LSD0.05 45 56 38 47

Na

0* 5cD

1.2 10bD

1.3 8bcDE

1.1 15aDE

1.9 4.1

0 7cD

1.2 10bcD

1.9 11bcDE

2.4 18aDE

1.5 3.8

5 105bA

1.1 125abA

1.7 110bA

2.3 145aA

1.7 26

10 105bcA

1.8 115bA

2.5 105bcA

2.6 155aA

3.1 31

20 40cdC

1.8 55cCD

1.2 50cC

2.1 90aBC

2.2 46

LSD0.05 22 28 26 31


3.11B.3.2.1 Calcium (Ca) in shoot


of fungi as compared to C. For 0*, the maximum (22 mgkg-1) shoot Ca

observed to be in F1 + F2 while being the minimum (15 mgkg-1) in C. Similar

trends were observed for 0, 5, 10 and 20 %.

Within columns, the plants from 0* had the lower shoot Ca than those

from corresponding 0 (% TSW-Soil) for all the fungal treatments. For those

fungal treatments where soil was mixed with TSW, the shoot Ca increased

(10 %) and then decreased (20 %) down the column as compared to the

shoot Ca observed in plants from 5 %.

3.11B.3.2.2 Potassium (K) in shoot

The trend of variation of shoot K was similar to that of shoot Ca i.e. it

increased along the row with application of fungi and was greater in plants

with fungal inoculations than those without fungi. Plant shoot K observed to

follow the same trend along row for all the TSW-Soil mixtures.

194

Likewise, the K shoot concentration increased down the column with

the increasing concentration of TSW in soil mixtures for all the fungal

treatments.

3.11B.3.2.3 Sodium (Na) in shoot





(155 mgkg-1) while those in 0* % with no fungi exhibited the lowest Na shoot



the shoot Na uptake for 5 and 10 % but decreased for 20 %. In case of 0 %,

the concentration of shoot Na observed to be the least for all the fungal

treatments.

3.11B.3.3 Category-I metals in plant ROOT



soil mixture, as given in Table 3.11B.5.

3.11B.3.3.1 Calcium (Ca) in root

The application of fungal inoculums to the soil helped increase Ca root

uptake along the row. The plants from F1 + F2 pots observed to have

maximum while those from where no fungi was applied having the minimum

root Ca for all the TSW-Soil mixtures. The highest root Ca (110 mgkg-1) was in

5 % with F1 + F2 while being the minimum (4 mgkg-1) in 0* % with C i.e. no

fungal inoculation.

Down the columns, the root Ca decreased with increasing percentage

of TSW in soil being the maximum in plants from 5 % and minimum in those

from 20 %. Such variation was observed for all the fungal treatments.

195

3.11B.3.3.2 Potassium (K) in root

The K root contents in the 5 % with F1 + F2 exhibited the maximum

value (65 mgkg-1) than any of the soil treatments while being the minimum (2

mgkg-1) in 0 % with C i.e. no fungal application. The application of fungus as

individual inoculant i.e. F1 and F2 showed better K uptake than TSW-Soil

mixtures where no fungi has been applied. However, F2 showed better uptake

as compared to F1 for all the soil treatments except 5 %.


of TSW in 5 and 10 %, however, it was decreased in 20 %.


) observed in ROOT of 82-



LSD0.05 C F1 F2 F1+F2

Ca

0* 4cE

0.1 5cE

1.1 7bcDE

1.4 11aE

1.5 1.2

0 5bcE

1.4 8bE

2.9 6bcDE

1.3 10aE

1.8 1.7

5 70cA

1.3 95abA

1.4 90bA

1.7 110aA

2.7 32

10

45cC

2.3 60bcBC

1.3 55bcC

2.1 80aB

2.7 25

20 35cC

1.8 40bcD

1.5 45bC

2.1 55aCD

2.2 38

LSD0.05 12 17 21 26

K

0* 2dD

0.9 5bcD

2.6 7bD

2.3 10aDE

1.2 2.4

0 4cD

1.2 6bcD

1.4 8bD

1.2 12aDE

1.3 3.2

5 40cA

1.4 50bcA

1.6 45bcA

1.5 65aA

2.1 16

10 20dBC

1.5 35bcBC

1.7 40bcA

1.2 50aB

1.4 27

20 15cdC

1.1 20cC 1.5 35

abB 2.7 45

aB 1.6 32

LSD0.05 7 10 9 14

Na

0* 5bcCD

0.4 7bcD

1.2 5cD

0.2 10aD

1.2 1.6

0 6cCD

0.5 8bD

0.6 9bD

1.5 13aD

1.4 1.9

5 45bA

1.2 50abA

1.2 45bA

1.5 55aA

1.3 23

10

30bcB

1.2 35bcB

1.3 32bcB

0.9 45aAB

1.5 34

20 16cC

1.1 19bcCD

1.8 22bC

1.6 30aC

1.7 29

LSD0.05 13 14 8 11


3.11B.3.3.3 Sodium (Na) in root

Along the row, as observed in case of Ca and K, the application of

fungi helped increase Na uptake in roots. The maximum Na in root (55 mgkg-

1) was observed in 5 % with F1 + F2 while being the minimum in 0 % with no

fungi (5 mgkg-1). Those with F1 and F2 applications also performed better

than C i.e. treatment with no fungal inoculation.

196

Within column, the increasing ratio of TSW in soil decreased root Na

uptake as compared to 5 %. And this trend was observed for all the fungal

treatments.

3.11B.3.4 Category-II metals in plant shoot



in soil, as given in Table 3.11B.6.

3.11B.3.4.1 Cd in shoot



fungal treatments while being minimum where no fungi was applied.

Maximum amount of metal (85 mgkg-1) was observed in 5 % TSW-Soil

mixture with combined inoculation of fungi i.e. F1 + F2 and the minimum of

which was observed in 0 % with C (BDL).

Down the columns, for fungal treatments where soil was mixed with

TSW, the shoot Cd significantly decreased as compared to 5 % with

increasing percentage of TSW in soil.

3.11B.3.4.2 Cr in shoot

Like Cd in shoot, the plant Cr also exhibited the same variation pattern

i.e. along the row it increased with fungal application and found to be the

maximum in plants where both of the fungi were inoculated. The minimum of it

was observed in plants where no fungi were applied. This trend was observed

for all of the TSW-Soil mixtures.

Alongside columns, the increasing percentage of TSW in soil

decreased the shoot Cr and such variation was observed in plants from all of

the fungal treatments.

3.11B.3.4.3 Cu in shoot

The shoot Cu concentration increased along the row in plants with the

application of fungal inoculations and the observed increase was with respect

to the C treatments where no fungi were applied. Down the column, the shoot

197

Cu decreased with increasing fraction of TSW in soil and was the most in

plants cultivated on 5 % while being the least in those cultivated on 20 %.





LSD0.05 C F1 F2 F1+F2

Cd

0* BDL 5bDE

1.1 7aDE

1.2 8aDE

1.2 4.2

0 BDL 6aDE

0.7 6aDE

1.4 7aDE

0.8 3.7

5 60bA

1.7 70abA

1.9 65bA

2.5 85aA

1.9 26

10 15cdCD

1.2 30bC

1.9 25bcCD

1.4 40aC

2.1 11

20 10dCD

1.2 25bC

2.8 30aCD

1.8 35aC

2.1 9

LSD0.05 11 18 21 28

Cr

0* 5cDE

1.3 7bDE

1.4 10aE

1.1 12aE

1.1 2.6

0 4cdDE

1.1 8bDE

1.7 12aE

1.5 13aE

1.2 3.1

5 180dA

2.9 230cB

2.3 280cA

2.9 580aA

2.4 115

10 115dBC

2.2 295cA

2.5 270cA

2.9 490aAB

2.5 98

20 70dCD

1.7 190bcBC

2.9 165bcC

2.2 375aBC

2.3 85

LSD0.05 45 57 59 95

Cu

0* 5cDE

0.9 8bcE

1.1 10bE

0.8 15aDE

1.7 2.2

0 3cdDE

0.7 5cE

0.8 8bE

0.5 10aDE

1.2 3.1

5 180dB

2.8 225cBC

2.3 210cBC

2.7 350aAB

2.5 87

10 245cdA

2.4 390aA

1.9 355bA

3.4 425aA

3.9 105

20 70cCD

1.7 115bD

1.3 95bD

2.8 140aCD

2.6 78

LSD0.05 55 62 58 67

Fe

0* BDL 2bDE

0.7 3bDE

0.4 5aD

1.1 2.9

0 BDL 3bcDE

0.6 4bcDE

0.5 8aD

0.9 1.9

5 20dA

1.7 40cB

1.8 35cAB

1.2 70aA

1.8 21

10 15cdB

1.5 55aA

2.2 40bcA

2.3 60aA

2.2 18

20 10dC

1.1 30bcC

2.5 25bcC

1.9 55aAB

2.7 11

LSD0.05 2.6 11 18 21

Mg

0* BDL 8bcCD

1.9 10aC

1.5 12aCD

1.8 3.2

0 BDL 7cCD

1.6 8cC

1.2 15aCD

1.3 4.1

5 20dA

0.9 30cdA

1.8 25cdA

1.2 70aA

1.9 27

10 15dB

2.3 20cdB

1.1 15cdB

1.5 65aA

1.9 19

20 10dC

1.4 25cAB

1.2 20cAB

1.6 45aBC

1.4 16

LSD0.05 2.5 4.6 5.9 8.2

Ni


0 BDL BDL BDL BDL -

5 BDL 5bcAB

0.8 7aA

1.5 8aB

1.7 1.8

10 5bcAB

0.5 7bA

0.7 8abA

0.4 10aA

1.2 1.9

20 7cA

0.8 5cAB

0.6 6cAB

0.5 12a 1.3 3.5

LSD0.05 0.9 0.6 0.8 1.7

Zn

0* 5dCD

0.8 15cD

2.3 10cCD

2.7 25aD

3.2 3.5

0 10cdCD

1.4 20bcD

1.7 15cCD

1.4 30aD

1.3 4.1

5 90dB

2.3 170cA

1.2 160cAB

1.3 310aA

1.7 78

10 125cdA

1.4 150cdA

1.5 190cA

1.5 320aA

1.1 85

20 80cdB

2.1 125cB

1.9 170cA

1.6 280aAB

1.7 89

LSD0.05 28 32 38 42


198

3.11B.3.4.4 Fe in shoot

The application of fungal inoculations to the soil cause increased

uptake of Fe in plant shoot and such an increase was observed along the row

in Table 3.11b.6. The maximum shoot Fe uptake effects was incurred by the

F1 + F2 than any of the individual fungal inoculations.

Down the columns for fungal treatments where soil was mixed with

TSW, the shoot Fe decreased with increasing percentage of TSW in soil.

3.11B.3.4.5 Mg in shoot

The shoot Mg concentration observed in plants after they were

harvested and it appeared that application of fungi in soil cause an increased

uptake as compared to those plant harvested form soil with no fungi. The

combined application of fungi incurring maximum uptake effect and those with

no fungi had the least tendency for phytoextraction.

Moving along the column, the shoot Mg observed to decrease with

increasing percentage of TSW in soil and thus it was accumulated to the most

in plants from 5 % and the least being extracted by plants from 20 %. Such

variation was observed for all the fungal treatments.

3.11B.3.4.6 Ni in shoot

The shoot Ni was BDL in plants from 0* and 0 (% TSW-Soil) with any

of the fungi inoculated in soil. However, for 5, 10 and 15 %, its concentration

increased in plant shoots along the row of table with the application of fungal

inoculums.

Down the column of Table 3.11B.6, the shoot Ni uptake decreased with

increasing percentage of TSW in soil, although negligible amounts of uptake

were observed.

3.11B.3.4.7 Zn in shoot

The Zn shoot concentration increased in plants applied with fungal

inoculums and this trend could be observed along the row for all the TSW-Soil

mixtures.

199

The increasing percentage of TSW in soil caused increase in plant

shoot Zn uptake in 10 % but a decrease was observed in 20 % as compared

to plants from 5 %. Such variations were observed for all the fungal

treatments down the column of Table 3.11B.6.

3.11B.3.5 Category-II metals in plant ROOT

The Category-II metals i.e. the AAS detected metals in plant root

exhibited variation in response to the fungal inoculations as well as to the

increasing percentages of TSW in soil, as given in Table 3.11B.7.

200


) observed in ROOT of 82-



LSD0.05 C F1 F2 F1+F2

Cd

0* BDL BDL 2bD

0.6 4aD

1.2 0.5

0 BDL BDL 2bD

0.8 5aD

0.8 0.3

5 30bcA

1.3 40abA

2.4 25cA

1.5 50aA

2.2 11

10 10bcC

1.2 15bCD

1.7 20abB

1.3 25aC

1.4 18

20 8bC

1.8 10bD

1.2 12aBC

1.3 15aCD

1.4 9

LSD0.05 3.8 3.9 5,7 9.6

Cr

0* BDL 2cD

0.7 4bcD

1.2 8aE

1.3 1.6

0 BDL 3cdD

0.6 5cD

1.1 10aE

1.1 2.2

5 110cA

1.8 125cA

1.4 115cA

1.9 240aA

2.2 78

10 50dC

1.3 110bcA

2.4 90cAB

2.3 210aAB

2.3 56

20 25dD

1.9 60cC

1.5 55cC

1.3 110aCD

2.5 49

LSD0.05 21 27 31 54

Cu

0* BDL 2cdD

0.4 4cDE

0.2 8aDE

0.6 3.2

0 BDL 4abD

0.5 5aDE

0.5 6aDE

0.8 2.8

5 85bAB

1.7 110abA

1.3 90bB

1.9 120aBC

2.1 34

10 110bA

3.5 125abA

2.7 130abA

3.3 155aA

2.3 49

20 30cdCD

2.8 70bBC

2.5 65bBC

1.8 85aC

1.9 28

LSD0.05 22 29 38 43

Fe

0* BDL BDL BDL 2DE

0.6 -

0 BDL BDL BDL 4DE

0.8 -

5 15cdA

2.4 25cA

2.8 20cAB

1.6 45aA

2.9 8

10 10cB

3.1 30aA

2.7 25bA

1.4 35aB

2.2 19

20 5dC

1.9 20bB

1.5 15bcBC

1.1 25aC

1.3 11

LSD0.05 2.9 3.8 5.2 9.3

Mg

0* BDL 2bcCD

0.6 4aC

0.7 5aCD

0.8 2.1

0 BDL 3bcCD

0.9 5bC

0.6 8aC

0.9 1.8

5 15cA

1.1 15cA

1.1 13cA

1.3 30aA

2.5 8

10 10cdB

1.5 12cdAB

1.6 10cdA

1.2 35aA

1.8 6

20 5dC

0.8 10cB

1.7 12bcAB

1.2 20aB

1.1 5.7

LSD0.05 2.1 3.1 5.3 7.6

Ni


0 BDL BDL BDL BDL -

5 BDL 2cB

0.4 2cA

0.9 5aA

0.8 2.2

10 3cA

0.4 4bcA

0.7 3cA

0.8 6aA

0.5 3.8

20 2cdA

1.2 3cA

1.5 2cdA

1.8 6aA

1.3 1.8

LSD0.05 0.4 0.8 0.5 0.6

Zn

0* BDL 10bcD

0.8 8cD

1.1 15aE

1.2 1.7

0 2dDE

0.8 10aD

1.2 12aD

1.2 10aE

1.1 1.5

5 55cdBC

1.5 110abA

1.1 90bcA

1.4 130aC

1.9 37

10 70dA

1.3 115cdA

1.8 95cdA

1.6 275aA

2.7 75

20 50cdBC

1.7 80bcBC

2.5 75bcB

2.3 140aC

2.2 33

LSD0.05 5.4 4.2 17.2 19.2

0*Control with infiltration having no lining; 0 Control with no infiltration applied with lining; C: No fungal inoculum; F1: Trichoderma pseudokoningii; F2: Aspergillus niger; F1 + F2: T. pseudokoningii and A. niger; LSD: least significant

difference

201

3.11B.3.5.1 Cd in root

In 0* and 0 (% TSW-Soil) with C and F1 inoculations, the plant root Cd

observed to BDL; however, detected in soil treatments of F2 and F1 + F2. For

5, 10 and 20 %, the root Cd was higher in plants applied with fungi than those

applied with no fungi. The F 1 + F2 showed the best assistance in root Cd

uptake than any individual or no fungal application.

The increasing percentage of TSW in soil lowered down the root Cd

concentration resulting maximum accumulation in plants from 5 % and

minimum uptake in plants from 20 %.

3.11B.3.5.2 Cr in root

Like root Cd, the concentration of Cr in root increased along the row of

Table 3.11B.7.and it was due to application of fungi in rhizoshpere of plants.

Those applied with no fungi, showed the least Cr accumulation in root.

The increasing level of TSW in soil added to the soil Cr; however,

plants were able to uptake the maximum concentration from 5 % as compared

to 10 and 20 % treatments of soil. Such variation in plant root was observed

for all of the fungal treatments.

3.11B.3.5.3 Cu in root

An increase in the root Cu was observed in plants harvested from soil

inoculated with fungi as compared to those harvested from soil with no fungi.

The F1 + F2 incurred the maximum root Cu accumulation than any of the

individual fungi as well as C.

The root Cu observed to decrease in 20 % treatments but increased in

10 % as compared to 5 %; although its level in plant roots from all the TSW-

Soil mixtures was significantly higher than those harvested from C.

3.11B.3.5.4 Fe in root


treatments, the root Fe observed to be BDL in soil (0 %) with C, F1 and F2

treatments. The plants from 0* % with F1 + F2 had the lowest root Fe as

compared to plants from any of the single or combined application of fungi for

202

the rest of TWS-Soil mixtures i.e. 10 % and 20 %. The root Fe accumulation

observed to be the maximum (45 mgkg-1) in 10 % with F1 + F2 and

significantly higher than those harvested from any of the treatments with



% with F1 + F2 while the least value of metal uptake was observed in 0 % as


3.11B.3.5.5 Mg in root



fungal application as compared to C. for both of 0 % i.e. in soil, the value was

found to be BDL with C. The F1 + F2 plants displayed the maximum root Mg

concentration than those from C as well as F1 and F2. However the F2

inoculations gave better results than F1.

For the columns, the root Mg accumulation was found to be highest in


mgkg-1 while minimum concentration 2 mgkg-1 in 0* % with F1 treatment was

noted as shown in Table 3.11B.7.

3.11B.3.5.6 Ni in root


in 0* and 0 % i.e. soil. However there was increased metal accumulation

along the row with the application of fungal inoculations and found to be the

maximum in plants applied with combined application of both of the fungi

while being the minimum in C with no fungus added.

Within columns, the plants from 10 and 20 % TSW-Soil mixtures had

the maximum root Ni level than 5 % for all of the fungal treatments. There was

maximum (6 mgkg-1) accumulation of metal was noted in 10 and 20% with F1

+ F2 while minimum uptake (2 mgkg-1) was observed in 5 % with F1.

203

3.11B.3.5.7 Zn in root






For different TWS-Soil mixtures along the columns, the root Zn

concentration increased with increasing percentage of TSW in soil with every

fungal treatment. The maximum accumulation was noted in 10 % with F1 + F2

i.e. 275 mgkg-1 and minimum (8 mgkg-1) in 0* % with F2 treatment as shown

in Table 3.11B.7.

3.11B.4 Fungal analyses


c.f.u. g-1 soil) of 82-days old Helianthus annuus cultivated on TSW-Soil

mixtures are shown in Table 3.11B.8. Alongside the row, the c.f.u. increased

with fungal application than C and observed to be the maximum in treatments

with combined application of both of the fungi. The order of c.f.u. abundance

was F1 + F2 > F2 > F1 > C.

Within a column, the c.f.u. abundance was observed to be highest in 5

% with 8.4 × 105 c.f.u. g-1 soil in combined inoculum of fungi i.e. F1 + F2.

Table 3.11B.8. The post-harvest fungal analyses (× 105 c.f.u. g




Treatment LSD0.05

C F1 F2 F1+F2

0* 0.3cdD

0.27 0.7cdDE

1.1 0.9cdD

1.3 2.9aDE

1.9 0.9

0 0.4cdD

1.5 1.0bDE

1.9 1.2bD

1.6 1.8aDE

2.2 0.8

5 3.2cA

2.2 4.8bcA

1.8 5.1bcA

1.21 8.4aA

2.13 1.9

10 3.4cA

2.4 3.8cB

2.3 4.9cA

2.07 7.8aA

2.51 1.5

20 2.4cBC

2.3 3.1bBC

3.2 3.7aBC

2.83 4.7aCD

3.20 0.7

LSD0.05 0.8 1.1 1.4 1.8


204

3.11B.5 Meta-analytical perspective



3.11B.5.1 Category-I metals translocation index (%)

The plant translocation index values were also recorded for Category-I


3.11B.9.

In case of Ca, maximum value was observed in 10% with C i.e. 446.6

% while the minimum value was recorded (207.1 %) in 5 % with C.


with C i.e. 450 % being the minimum 171.4 % in 20% with F2.

Table 3.11B.9. The Category-I metals translocation index (%) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Ca

5 207.1 252.6 233.3 227.2

10 446.6 408.3 381.8 343.7

20 371.4 550 522.2 436.3

K

5 275 260 377.7 330.7

10 450 328.5 325 300

20 433.3 375 171.4 255.5

Na

5 233.3 250 244.4 263.6

10 350 328.5 328.1 344.4

20 250 289.4 227.2 300


In case of Na, the maximum value 350 % was observed in 10% with C

treatment and least value was recorded in 20 % for F2 (227.2 %).

3.11B.5.2 Category-II metals translocation index (%)

The plant translocation index is given in Table 3.11B.10. For Cd, the

maximum Translocation index (260 %) was noted in 5% with F2, while

minimum value was recorded 125 % in 20% with C and in 10% with F2.


(340.9 %) in 20% with F1 + F2 and minimum 163.9 % in 5 % with C.

205


312 % for 10% with F1, while minimum value was recorded in 20 % with F2



220 % for 20 % with F1 + F2, while minimum value was recorded in 5 % with

C treatment i.e. 133.3 %.

Table 3.11B.10. The Category-II metals translocation index (%) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Cd

5 200 175 260 170

10 150 200 125 160

20 125 250 250 233.3

Cr

5 163.6 184 243.4 241.6

10 230 268.1 300 233.3

20 280 316.6 300 340.9

Cu

5 211.7 204.5 233.3 291.6

10 222.7 312 273 274.1

20 233.3 164.2 146.1 164.7

Fe

5 133.3 160 175 155.5

10 150 183.3 160 171.4

20 200 150 166.6 220

Mg

5 133.3 200 192.3 233.3

10 150 100 150 185.7

20 200 250 166.6 225

Ni

5 0 250 350 160

10 166.6 175 266.6 166.6

20 350 166.6 300 200

Zn

5 163.6 154.5 177.7 238.4

10 178.5 130.4 200 116.3

20 160 156.2 226.6 200


In case of Mg the plants showed highest value in 20% with F1 (250 %),

while the least value for this metal was recorded in 10% with F1 i.e. 100 %.


C and 0 value was observed in 5 % with C.


recorded in 5 % with F1 + F2 treatment i.e. 238.4 % while minimum value was

recorded in 10% with F1 + F2 i.e. 116.3 % as shown in Table 3.11B.10.

206

3.11B.5.3 Tolerance index (TI)

In shoots TI values were found to be highest in 5 % with F1 + F2 (1.61)

while minimum value was recorded to be 0.83 in 20% with F1 as shown in

Table 3.11B.11.

Table 3.11B.11. The translocation index (%) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

TI Shoot*

5 1.13 1.41 1.40 1.61

10 1.04 0.87 1.11 1.40

20 0.87 0.83 0.91 0.96

TI Root*

5 1.33 1.24 1.26 1.28

10 1.08 1.18 1.06 1.04

20 0.93 0.67 0.83 0.77



in 5 % with C, while 0.67 was recorded as minimum value in 20% with F1.

3.11B.5.4 Category-I metals specific extraction yield (SEY %)


3.11B.12.

Table 3.11B.12. The Category-I metals specific extraction yield (SEY %) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Ca

5 13.69 23.10 21.27 30.25

10 14.08 18.59 15.14 24.65

20 6.11 9.96 10.76 12.88

K

5 18.51 25 28.47 41.79

10 11.70 18.29 18.68 27.77

20 4.32 5.42 5.30 9.52

Na

5 11.62 13.94 12.55 17.77

10 5.60 6.46 5.98 9.11

20 1.21 1.66 1.62 2.75






207

The highest value for Na was recorded in 5 % with F1 + F2 (17.77 %)

while minimum values was found to be 1.21 % in case of 20% with no fungal

inoculum i.e. C.

3.11B.5.5 Category-II metals specific extraction yield (SEY %)


by AAS as shown in Table 3.11B.13. Overall a similar kind of trend was seen




Table 3.11B.13. The Category-II metals specific extraction yield (SEY %) analyzed in Tagetes patula cultivated on TSW-Soil (% w:w) mixtures.


C F1 F2 F1+F2

Cd

5 3.55 4.56 3.76 6.13

10 0.38 0.69 0.69 1.02

20 0.20 0.40 0.48 0.57

Cr

5 3.72 4.70 5.19 11.66

10 1.64 4.21 3.74 8.09

20 0.61 1.63 1.45 3.61

Cu

5 25.85 34.53 30.30 60.25

10 19.61 29.76 27.24 34.31

20 1.95 3.73 3.27 4.67

Fe

5 16.66 34.21 29.72 67.64

10 5.20 17.52 13.26 20.21

20 1.68 5.95 4.81 9.87

Mg

5 12.06 16.66 14.61 40

10 4.23 6.89 4.42 18.86

20 1.63 4.02 3.69 7.83

Ni

5 0 28 40.90 68.42

10 17.77 27.5 28.94 53.33

20 9.47 11.42 9.41 27.69

Zn

5 11.78 29.16 25.51 49.43

10 12.03 16.98 17.92 49.17

20 6.87 11.91 14.20 44.21


In case of Cd the maximum value for SEY % was recorded in 5 % with

F1 + F2 treatment i.e. 6.13 %, while minimum (0.20 %) was observed in 20%

with C.

208


5 % with F1 + F2 and minimum (0.61 %) was recorded in 20% with no fungal

inoculum i.e. C.



20% with C.

As in case of above mentioned metals again the highest (67.64 %) and


respectively for Fe.



value (40 %) with F1 + F2 and being minimum (1.63 %) in 20% with C having

no fungal inoculum.

There was maximum value for Ni was calculated (68.42 %) in 5 % with

combined fungal inoculum, while minimum value (9.47 %) was recorded in 20

% for C. The value was found to be 0 in 5 % with C.

In case of Zn the maximum value was noted (49.43 %) for 5 % with F1

+ F2 and minimum value (6.87 %) in 20 % with C.

Chapter 4

Discussion

209

CHAPTER 4

DISCUSSION

4.1 Physico-chemical properties of TSW and garden soil

Leather processing with several salts contained with Category-I and II

metals have been used to convert raw leather into tanned leather. Pre-tanning

has been involved with numerous operations, such as curing by dehydration

of skins, soaking for rehydration of skins, liming of skins for swelling, deliming

for deswelling, pickling involving acidification of skins and depickling carried

out by basification (Aravindhan et al., 2007). Furthermore, the skins or hides

are subjected to wide variations of pH. A huge amount of salts with and

without metals, used during different steps of tanning process, become part of

TSW. The TSW originating from Cr tanning plants has mainly been composed

of tanned residue, hair, lime, and salts of Cr (Amir et al., 2008). All of the solid

wastes resulting from tanneries of Kasur have predominantly been contained

with such salts and metals. The lack of sophistication and environmental

consideration in handling of TSW from tanning factories has been

accelerating discharge of untreated heavily polluted TSW on to the

mismanaged dumping sites. Even tannery wastewater when accidently get

spilled into TSW heaped within a tanning factory, high concentrations of

chlorides, sulfates, Cr or tannins and other minerals can be carried to the

TSW (Thanikaivelan et al., 2000).

The high valued physico-chemical properties of TSW such as, pH,

ECe, bicabonates, and chlorides has been resulting due to the incorporation

of salts in the TSW and its untreated disposal at the dumping site. The mixing

of TSW in soil aggravates the values of physic-chemical properties of soil and

putting it to stressed situation. The increasing quantity of TSW in soil

intensified the soil stress and further additions of TSW beyond 20 % (TSW-

Soil) made the soil completely unfit for germination of selected plant seeds.

The high metal (Category-I and II) contents of the TSW has also been

alarming in terms of polluting soil. The higher values of Cr and other heavy

metals in representative samples of TSW, collected from the dumping site,

indicate that majority of tanneries working in Kasur have been the chrome

tanning methods due to its high speed and cost effectiveness. But the

210

environmental hazards associated with heavy metals released from tanning

have been left unnoticed. The high content of heavy metals in TSW has been

reported to make their treatment by biological processes difficult (Amir et al.,

2008).

Plants cultivated on heavy metal polluted soils cannot usually access

the total pool of a metal present in the growth substrate. The fraction of a

metal which plants can absorb is known as the available or bioavailable

fraction (Fageria et al., 1991; Marschner, 1995; Whitehead, 2000). The metals

soluble in the soil solution are the metals directly available for plant uptake

and other soil metal pools are less available (del Castilho et al., 1993). The

metals present in soil are in dynamic equilibrium and have been divided into a

number of fractions including; the soluble metal in the soil solution, metal-

precipitates, metal sorbed to clays, hydrous oxides and organic matter, and

metals within the matrix of soil minerals (Norvell, 1991).

Metal speciation, on the basis of kind of solvent used to check the

metal availability, has been helpful in determining the fraction of plant

available metals (Iwegbue et al., 2007). In the current study, the different

pools of metal in TSW have been carried out to find out the plant available

fraction of different metals. There are several factors that can affect the

concentration and speciation of metals in the soil solution including the plant

available fraction of metals such as, the total metal present in the soil, pH,

clay and hydrous oxide content, bulk density, organic matter and redox

conditions. In the current study, the water soluble and DTPA-extractable

fractions of metals have been found to increase in accordance with increase

in pH, ECe, NaCl (%), bicarbonates as well as chlorides. Parallel to that, a

shift in total metal contents of the TSW as well as its mixtures with soil has

been reported (Reichman, 2002). However, the increasing percentage of

TSW in soil significantly reduces the bulk density of the resulting TSW-Soil

mixtures. The water soluble, DTPA-extractable and total fraction of both

Category-I and II metals have been observed to increase with decreasing bulk

density. Perhaps, the increase in metal fractions could have been due to

increasing volume of air spaces in the soil substratum as well as presence of

inert fibrous leather hide ingredients of crushed TSW.

211

The simple linear regression between bulk densities (gcm-3) and

Category-I metals’ water-soluble as well as DTPA-extractable fractions

showed that both of the fractions of Category-I metals were strongly

dependent on variations in bulk density, as given in Figure 4.1.1. The high

Figure 4.1.1: Simple non-linear regression between bulk density (gcm-3

) of TSW-soil mixture and

different fractions of Category-I metals (mgkg-1

): (A) between bulk density and water-soluble fraction, (B) between bulk density and DTPA-extractable fraction.

Co

nce

ntr

atio

n (

mgk

g-1)

of

wat

er-s

olu

ble

fra

ctio

n

Co

nce

ntr

atio

n (

mgk

g-1)

of

DTP

A-e

xtra

ctab

le f

ract

ion

Variation in bulk density (gcm-3

) of soil mixed with TSW

B

A

212

values of regression co-efficient R2 for both fractions of Category-I metals

indicates that they were highly dependent on the bulk density variations.

Similarly for Category-II metals, both of the fractions were strongly

dependent on bulk density variations, as shown in Figure 4.1.2. The values of

regression co-efficient R2 for both fractions (i.e. water-soluble and DTPA-

extractable) of Category-I were highly dependent on variations on bulk density

variations.

Figure 4.1.2: Simple linear regression between bulk density (gcm-3

) of TSW-soil mixture and different fractions of Category-II metals (mgkg

-1): (A) between bulk density and water-soluble fraction, (B)

between bulk density and DTPA-extractable fraction.

Co

nce

ntr

atio

n (

mgk

g-1)

of

wat

er-s

olu

ble

fra

ctio

n

Co

nce

ntr

atio

n (

mgk

g-1)

of

DTP

A-e

xtra

ctab

le f

ract

ion

Variation in bulk density (gcm-3

) of soil mixed with TSW

A

B

213

The total fraction of Category-I and II metals was directly dependent on

percentage of TSW in soil mixture, as given in Figure 4.1.3. The correlation

between TSW percentage (% w:w) in soil and total fraction of Category-I and

II metals indicated that the total fraction of both metals increased in

accordance with the increase in TSW percentage, as is obvious from the high

values of coefficient of correlation (r2).

Figure 4.1.3: Correlation between TSW (% w/w) of TSW-soil mixture and different fractions of Category-II metals (mgkg

-1): (A) between bulk density and water-soluble fraction, (B) between bulk

density and DTPA-extractable fraction.

Co

nce

ntr

atio

n (

mgk

g-1)

of

tata

l fra

ctio

n o

f m

etal

s

TSW (% w:w) mixed in soil

The total metal has been reported to indicate the maximum pool of

metal in the soil with other factors being important in determining how much of

214

this soil pool will be available to plants (Wolt, 1994). In addition, researchers

have found that the total metal may correlate with the bioavailable soil pools

of metal but is still inadequate by itself to reflect bioavailability (Lexmond,

1980; Sauve et al., 1996; McBride et al., 1997; Sauve et al., 1997;

Peijnenburg et al., 2000).

Such studies of tannery effluents in relation to soil and water have

been carried out in Pakistan (Tariq et al., 2005; Tariq et al., 2006) but the

research work has been carried out for the first time for TSW. Overall, the

average concentrations of Category-II metals were within the above range of

the permissible limits of EPA (USEPA, 1999; Bosnic et al., 2000). Any shift in

the heavy metals carried as TSW by natural hazard or anthropogenic

activities can expand the exposure spectrum heavy metals to the biotic life.

215

4.2 Isolation and identification of TSW representative fungi

The isolation of a fungus on PDA from an industrial waste

contaminated with heavy metals (Ezzouhri et al., 2009) and application of

such isolated fungi back into the same system (autochthonous fungi) for

fungal bioaccumulation purposes has been carried out (Zapotoczny et al.,

2007). The isolation of fungi such as Trichoderma from TSW of KTWMA,

Kasur, has been done (Bareen and Nazir, 2010). Earlier, isolation from other

KTWMA tannery wastes such as, tannery effluent has been carried out

(Bareen et al., 2011). There have been different criteria to define a newly

isolated fungal species. The most common of which has been phenotypic,

being the most classic approach describing a fungus species on the basis of

the morphological characteristics only (Inui et al., 1965). In the current study,

the identification and characterization of fungi isolated from TSW was carried

out according the selected (only of the morphological) formal requirements

and best practices for the publication of descriptions of new fungal species

(Seifert and Rossman, 2010); while bringing legitimacy and validity at the First

Fungal Bank of Pakistan, University of the Punjab, Lahore, Pakistan. The

isolated fungi were obtained after rigorous procurement of powdered TSW on

selected fungal nutrient media. The identifications were mainly based on

color, physical growth attributes of fungal colonies as well as morphological

structures following Schipper and Stalpers (1984).

The TSW representative fungal species, among genera of Aspergillus

and Fusarium, isolated during the current study have also been isolated from

heavy metal polluted soils by Iram et al., (2009) while using similar fungal

nutrient media. As the microbiological and molecular marker requirements for

the identification of isolated fungi were not fulfilled, the rigorous isolation with

repeated appearance of the same 13 species of fungi, during the isolation

process, was taken as the criteria for claiming isolated fungi as the

representative fungal flora of TSW from KTWMA, Kasur. The described

number of fungal species during current work is not been the true proportion

of actually existing fungal flora, as the fungal flora of a contaminated site is

never limited to a few species. (Hawksworth, 2001). However, future research

will be focused on expanding the process of isolation on the bases of testing

216

further fungal nutrient media, identifications based on microbiological features

as well as validations based on DNA markers.

4.3 Screening and selection of heavy metal resistant autochthonous

saprobic fungi

The fungi have known to be heavy metal tolerant (Baldrian, 2003;

Gavrilesca, 2004). The screening process of fungi during mutual interaction

studies have been more appropriate on selected nutrient media taken in petri

plates (Zapotoczny et al., 2006). During the current study, the screening

process of fungi on the basis of mutual interaction has been carried on 2 %

MEA taken in petri plates.

The variability of fungal micoflora in the contaminated growth media

and their adaptation to the heavy metal stress has mainly been driven by

availability of nutrients, pH, concentration of metal ions as well as physical

conditions of the contaminated media (Okoronkwo et al., 2005). During the

current study, the TSW representative fungi tested for their heavy metal stress

tolerance i.e. Trichoderma pseudokoningii, Aspergillus niger, Alternaria

alternata and Fusarium sp. were found to be the best adapted to the multi-

metal contaminated growth media on the basis of their highest tolerance index

(TI), while Aspergillus parasiticus were found the least tolerant to the provided

nutrient conditions. The most reported fungi from the heavy metal pollutes

sites that seems to have adapted themselves to such situation include

Aspegillus, Fusarium and Penicillium, and thus may be called heavy-metal-

stress-adapted fungal flora of Pakistan (Zafar et al., 2007).

The origin, type, source and duration of exposure of fungi are the main

factors that regulate the adaptation of fungi to the heavy metal stressed media

(Anand et al., 2006). The fungi isolated during the current study had taken

their origin from TSW. The highest acclimatization of the four fungal species

to the heavy metals on the basis of their origin enabled them to show high TI

values throughout their recurrent cultivation on growth media impregnated

with TSW extracts taken with water. The species of Aspergillus (Valix et al.,

2001; Anahid et al., 2011); Aspergillus and Fusarium (Iram et al., 2009);

Aspergillus and Trichoderma (Parameswari, et al., 2010); Aspergillus,

Fusarium and Alternaria (Ezzouhri et al., 2009); Aspergillus, Fusarium,

217

Alternaria and Trichoderma (Zafar et al., 2007) have been reported to show

high TI when cultivated on PDA impregnated with multi-metal stress. The

least reduction in colony size of tolerant fungi on TSW impregnated growth

media as compared to their corresponding replicates on growth media with no

TSW extract (i.e. control) indicated that A. niger was highly tolerant to multi-

metal stress including Cu and Cd. Similar findings have been reported by

Saleh and Al-Sohaibani (2011) where A. niger was identified as tolerant to Cu

and Cd stress; and by Shugaba et al. (2010) where A. niger and A. flavus, A.

parasiticus has been shown as tolerant to Cr (VI).

The screened autochthonous saprobic fungi during current study were

able to show maximum fungal biomass under heavy metal stress. Therefore,

the procurement of final selected fungi during the screening process would

show a high potential for the bio-reinforcing tendency on their inoculation in

the phytoextraction trials to be conducted with multi-metal (from TSW mixing)

contaminated soils.

218

4.4 In vitro fungal mutual interaction studies for Category-II metal

tolerance

Although based on hyphal length variation and not on radial colony

distribution, the in vitro interaction studies have been conducted by Arriagada

et al. (2009). During in vitro interaction studies, the prevalence of Aspergillus

niger, Trichoderma pseudokoningii, Alternaria alternata and Fusarium sp. over

rest of the 9 fungal sp. could be attributed to several features. Aspergillus

niger and T. pseudokoningii had acquired a better tendency to utilize the

available nutrients in growth media, more readily and efficiently, than their

respective competitors fungi under the prevalent culturing conditions. Thus,

their mycelial growth occupied greater area of the petri plate during the

observation period. Similarly, Alternaria alternata and Fusarium sp., although

not as efficient as A. niger and T. pseudokoningii, were able to supersede

their competitor fungi and consumed the nutrients from the growth media at

faster pace.

The antagonistic potential of a fungus could also be helpful in

suppressing the growth of competitor fungi. It has been reported that

Trichoderma can be a strong antagonist and completely suppresses the

growth of Fusarium sp. (Dubey et al., 2007; Gupta and Mishra, 2009),

Aspergillus (Lone et al., 2012; Usha et al., 2012) and Alternaria alternata

(Gveroska and Ziberoski, 2012). The fungi that showed radial colony

expansion than their competitor fungi could also be more sensitive to the

growth media and incubation conditions than those which thrived extensively

under the same conditions. Thus it could be a matter of conduciveness of

conditions under the same culturing conditions making these fungi become

overlap or become overlapped by the other.

Conclusively, Trichoderma and Aspergillus proved to be the best

antagonists in suppressing the growth of other fungi (Usha et al., 2012). Thus,

both of them were selected for their assistance potential in phytoextraction

studies with Tagetes patula.

219

4.5 In situ mutual growth interaction studies for screened Category-II

metals tolerant fungal isolates with Tagetes patula in soil

The autochthonous saprobic fungi have been found to show significant

in situ mutual interaction studies with Eucalyptus globulus compared to their

competitor fungi (Arriagada et al., 2009). During the current study, the

interaction of Trichoderma pseudokoningii and Aspergillus niger (F1 + F2)

showed highly significant results in terms of mutual interaction by enhancing

plant biomass and chlorophyll content in Tagetes patula encircled red (Figure

4.5.1). Synergistic effects have been shown by saprobic fungi through plant

dry biomass increase when applied in situ to the soil (Fracchia et al., 1998;

Arriagada et al., 2004). In the present study, the production of higher biomass

in marigold due to inoculation with F1 + F2 on repetitive basis in soil provided

strong grounds for selecting both the fungi for phytoextraction trials in

greenhouse and in the field.

Figure 4.5.1: The growth variation observed in Tagetes patula in response to different fungal

inoculations in soil

220

4.6 Screening of heavy metal tolerant ornamental plant species for

phytoextraction of TSW-Soil mixtures

Over 500 Angiosperms have found to be hyperaccumulators

constituting 0.2 % of the total heavy metal (Sarma, 2011), after passing

through the screening process for heavy metal tolerance. Such screening of

plants for heavy metal tolerance has been performed by several scientists

(Hakmaoui et al., 2006; Kachenko et al., 2007). The adaptation of plants to

the heavy metal polluted soil starts from a healthy germination response,

contained in the inherited, physiological, molecular, genetic and ecological

traits. In the present study, marigold and sunflower showed the maximum

germination as compared to the rest of the test plants. According to Sarma

(2011), there have been several important criteria in selecting any plant as a

suitable candidate for phytoremediation such as high tolerance for targeted

heavy metals, tendency for adequate uptake, accumulation and translocation,

high biomass yield, tolerance for abiotic stress, preferred habitat

acclimatization and tolerance for high pH and salinity. During this study,

results have indicated that both marigold and sunflower had the requirements

for being multi-metal hyperaccumulators, as has been given by Cutright et al.

(2010) for sunflower and Sun et al. (2011) for marigold. Both sunflower and

marigold were selected as hyperaccumulator and taken along for further

experimentations.

221

4.7 Experiments with marigold cultivated on autoclaved and non

autoclaved TSW-Soil mixtures and inoculated with selective

autochthonous saprobic fungi

The experiments with autoclaved soil (AS) were conducted to find

whether inoculation of TSW-Soil mixtures with screened autochthonous

saprobic fungi i.e. F1 (Aspergillus niger) and F2 (Trichoderma pseudokoningii)

have been responsible for bio-reinforcing of marigold phytoextraction or not.

The autoclaving of TSW-Soil mixtures killed all of the soil microbes in the

experimental pots and had only two of the inoculated fungi in plant

rhizosphere.

A trend of increase in all selected biochemical parameters was

observed in plants harvested from AS applied with fungal inoculations as

compared to those harvested from AS with no inoculations and such

variability was observed in all the soil treatments. The increase in production

of antioxidant enzymes SOD and CAT in plants with inoculation was due to

the application of autochthonous saprobic fungi. These findings have been

parallel to the activation of the antioxidant machinery in plants under stress

when inoculated with Trichoderma sp. (Brotman et al., 2013). The plants

applied with Trichoderma in their rhizosphere undergo stimulation of growth

and resistance to a wide range of adverse environmental conditions (Mastouri

et al., 2010; Soresh et al., 2010). The application of combined inoculation of

Trichoderma pseudokoningii and Aspergillus niger enabled the marigold

plants to become tolerant to heavy metals by activating its antioxidant enzyme

system. This is why, the increase in SOD and CAT was observed in plants

from pots with elevated levels of Category-I and II metals. However, those

provided with F1 + F2 inoculations exhibited the highest levels of both of the

antioxidant activities than any of the AS with single or no fungal inoculations.

The experiments with non-autoclaved soil (NAS) were conducted to

find i) validity of both of the screened fungi for inoculation under field

conditions with possible mutual synergism, ii) involvement of other

(unreported) soil microbes other than the inoculated fungi in enhancing the

phytoextraction tendency of marigold, iii) hyperaccumulator tendencies of the

screened fungi for the screening process while being in TSW-Soil mixtures in

222

natural conditions as in in-vitro conditions, and iv) the implications of

application of screened fungi under field conditions. The plants from NAS pots

inoculated with fungi exhibited elevated levels of all the biochemical

parameters than those harvested from NAS with no fungi. Such results have

been reported by Song et al. (2011) where inoculation of saprobic fungus

(Aspergillus niger) enhanced the plant SOD and CAT production as compared

to the un-inoculated control plants. In the present study, chlorophyll contents

observed to decrease with increasing level of metals in TSW-Soil mixture. The

plants from NAS with or without fungal inoculations performed better than

those from AS with or without fungi, respectively in terms of chlorophyll,

soluble protein, SOD and CAT. In this study, the combined application of F1 +

F2 in both NAS and AS pots showed elevated levels of all the biochemical

parameters than those from both NAS and AS with either of the single or no

fungal treatments for of all the TSW-Soil mixtures. The SOD and CAT

production in marigold has been reported to increase by increasing the level

of abiotic stress (Tian et al., 2012). Increase in SOD activity under metal

stress (Cho and Sohn 2004; Zhang et al., 2006) as well as of both SOD and

CAT (Wang et al., 2009) has been reported.

In the present study, the plant chlorophyll content decrease with the

increase in heavy metals concentration in soil. These results are parallel with

decrease in chlorophyll production with increasing concentration of metal in

soil (Zengin and Munzuroglu, 2006; Elloumi et al., 2007). The soluble protein

contents were also noticed to decrease parallel to the decrease in chlorophyll

contents due to increase in metal concentration in soil. It has been reported

that the soluble protein contents decrease with increasing concentration of

metals in soil (Ahmad and John, 2005; Ahmad et al., 2006).

It has also been reported that the free radical species (forms of active

oxygen) may be increased in stress condition, which will enhance the

activities of these detoxifying enzymes (Bhattacharjee, 1997–98; Gallego et

al., 1999). Also, the activities of SOD, CAT, and POD are induced in plants

due to heavy metal exposure (Pereira et al., 2002; Fornazier et al., 2002; Lee

and Shin, 2003; Sk´orzy´nska-Polit et al., 2003–2004; Li et al., 2006).

223

Correlation is the statistical measurement of the relationship among

two variables (Aslam et al., 2012). The substantial mutual variability

relationships between biochemical parameters, dry weight, Na and Cr based

on Pearson correlation coefficient values for T. patula cultivated on non-

autoclaved (NAS) TSW:Soil mixtures was found (Table 4.7.1).

Table 4.7.1. Pearson correlation among selected biochemical properties, dry weight, Cr and Na uptake observed in Tagetes patula cultivated on non-autoclaved soil (NAS) mixed with tannery solid waste (TSW).

Chlorophyll Soluble Protein

SOD CAT Dry

weight Cr Na

Chlorophyll

Pearson correlation

Sig. (2-tailed)

N

1 - 8

0.643*

0.043

8

0.523*

0.046 8

0.524*

0.041 8

0.745**

0

8

0.781**

0 8

0.682*

0.038 8

Soluble Protein

Pearson correlation

Sig. (2-tailed)

N

0.643*

0.043

8

1 -

8

0.213

0.543 8

0.734** 0 8

0.435

0.61

8

0.802** 0 8

0.784**

0.01 8

SOD

Pearson correlation

Sig. (2-tailed)

N

0.523*

0.046 8

0.213

0.543

8

1 - 8

-0.023

0.861 8

0.451

0.54

8

0.572*

0.042 8

0.631*

0.027 8

CAT

Pearson correlation

Sig. (2-tailed)

N

0.524*

0.041

8

0.734**

0

8

-0.023

0.861 8

1 - 8

0.341

0.124

8

0.623*

0.015 8

0.567*

0.031 8

Dry weight

Pearson correlation

Sig. (2-tailed)

N

0.745** 0 8

0.435

0.61

8

0.451

0.54 8

0.341

0.124 8

1 -

8

0.871** 0 8

0.785** 0 8

Cr

Pearson correlation

Sig. (2-tailed)

N

0.781** 0 8

0.802**

0

8

0.572*

0.489 8

0.623*

0.015 8

0.871**

0

8

1 - 8

0.789** 0 8

Na

Pearson correlation

Sig. (2-tailed)

N

0.682*

0.038 8

0.784**

0.01

8

0.631*

0.027 8

0.567*

0.031 8

0.785**

0

8

0.789** 0 8

1 - 8

**Correlation is significant at the 0.01 level (2-tailed); * Correlation is significant at the 0.05 level (2-tailed)

The variation in chlorophyll contents with respect to soluble protein

contents, dry weight, Cr and Na uptake showed considerably stronger

224

correlation; however, the chlorophyll contents found have weak correlation

with SOD and CAT values. A relatively strong correlation was found between

soluble protein contents and uptake of Cr and Na. The SOD contents found to

have little bit strong mutual variability relationship with Na while showing weak

correlation with rest of the selected variables. The CAT contents showed

stronger correlation with soluble proteins only, while dry weight of the plant

showed strong correlation with chlorophyll contents, Cr and Na uptake. The

uptake of Cr showed stronger relationship with chlorophyll production of plant,

CAT, dry weight and Na uptake while giving small correlation value (0.572)

with SOD. Likewise, the Na uptake exhibited relatively strong correlation with

all of the selected variables except CAT.

The mutual variability between selected variables on the basis of

Pearson correlation values for T. patula cultivated on autoclaved (AS)

TSW:Soil mixtures is given in Table 4.7.2.

225

Table 4.7.2. Pearson correlation among selected biochemical properties, dry weight, Cr and Na uptake in Tagetes patula cultivated in autoclaved soil (AS) mixed with tannery solid waste (TSW)


SOD CAT Dry

weight Cr Na

Chlorophyll

Pearson correlation

Sig. (2-tailed)

N

1 - 8

0.546*

0.043 8

0.456*

0.039 8

0.786** 0 8

0.451*

0.045

8

0.534

0.12 8

0.623*

0.035 8

Soluble Protein

Pearson correlation

Sig. (2-tailed)

N

0.546*

0.043 8

1 - 8

0.523*

0.042 8

0.342

0.32 8

0.651**

0.012

8

0.451*

0.038 8

0.410*

0.048 8

SOD

Pearson correlation

Sig. (2-tailed)

N

0.456*

0.039 8

0.523*

0.042 8

1 - 8

-0.012

0.67 8

0.234

0.56

8

0.452

0.23

8

0.523*

0.032 8

CAT

Pearson correlation

Sig. (2-tailed)

N

0.786** 0 8

0.342

0.32 8

-0.012

0.67 8

1 - 8

0.456

0.14

8

0.523*

0.031 8

0.512*

0.041 8

Dry weight

Pearson correlation

Sig. (2-tailed)

N

0.451*

0.045

8

0.651**

0.012 8

0.234

0.56 8

0.456

0.14 8

1 -

8

0.561*

0.021 8

0.612** 0 8

Cr

Pearson correlation

Sig. (2-tailed)

N

0.534

0.12

8

0.451*

0.038 8

0.452

0.23 8

0.523*

0.031 8

0.561*

0.021

8

1 - 8

0.578*

0.023 8

Na

Pearson correlation

Sig. (2-tailed)

N

0.623*

0.036 8

0.410*

0.048 8

0.523*

0.032 8

0.512

0.041 8

0.612*

0.041

8

0.578*

0.023 8

1 - 8


The chlorophyll contents were observed to vary with strong correlation

with CAT and Na uptake, while soluble protein contents displayed weak

correlation with all the variables except dry weight production. The SOD found

226

weakly correlated with all of the selected variables, likewise CAT except its

relation with chlorophyll production. The dry weight production showed

relatively strong correlation with soluble protein and Na uptake, while Cr

uptake had weak correlation with all the variables. The Na uptake found to

vary with mutual correlation with chlorophyll content and dry weigh production.

Correlation between different plants was also studied by Aslam et al. (2012).

227

4.8 Experiments with marigold cultivated on TSW-Soil mixtures and

inoculated with selective autochthonous saprobic fungi and AM fungi

under greenhouse conditions

The AM fungal associations of the plants have been reported to

decrease their sensitivity to the heavy metal stress by increasing production of

antioxidant defense enzymes (Blaudez et al., 2000; Frey et al., 2000;

Jentschke and Godbold, 2000; Auge, 2001; Schützendübel et al., 2001;

Schützendübel and Polle, 2002; Ehsanpour and Amini, 2003; Hernandez et

al., 2003; Cho et al., 2006; Kohler et al., 2009). In the present study, the

application of AM inoculum in trial 3.8 has been observed to show significant

increase in the production of SOD and CAT, enabling marigold plants to

withstand the stress exerted in soil due to mixing of TSW. It has been

reported that the antioxidant defense system of plants is strengthened by the

elevation of SOD and CAT production in soils where AM fungi are applied

either individually (Azcon et al., 2009) or in combination with saprobic fungi

(Medina et al, 2006). Thus in this study (Experiment 3.8), the application of

both AM and saprobic fungi in the same soil were responsible for maximum

production of SOD and CAT in marigold. Other than the antioxidant defense

system of the plant, several other defense strategies against heavy metals

may be there in AM inoculated plants (Vivas et al., 2006). However the

autochthonous saprobic fungi (Trichoderma pseudokoningii) performed better

as compared to AM fungi when inoculated to the marigold individually. This

finding prompted the use of autochthonous saprobic fungi for experimentation

with marigold and sunflower in greenhouse and field trials (3.9-3.11).

On finding Pearson correlation between chlorophyll contents, soluble

protein, SOD, CAT, dry weight production as well as Cr and Na uptake in

plants (Table 4.8.1); it was found that chlorophyll contents had strong

correlation with soluble protein contents while having weak variability

relationship with rest of the variables. The soluble protein showed relatively

strong correlation with chlorophyll; however its variation with respect to other

variables was weakly correlated.

228

Table 4.8.1. Pearson correlation among selected biochemical properties and metals for Tagetes patula cultivated in soil mixed with tannery solid waste (TSW) and inoculated with AM (arbuscular mycorrhizal) and saprobic fungi.


SOD CAT Dry

weight Cr Na

Chlorophyll

Pearson correlation

Sig. (2-tailed)

N

1 - 8

0.674*

0.011

8

0.563

0.431

8

0.546

0.23 8

0.129

0.78

8

0.023

0.91 8

0.034

0.67 8

Soluble Protein

Pearson correlation

Sig. (2-tailed)

N

0.674*

0.011 8

1 -

8

0.453

0.21

8

0.512*

0.023 8

0.220

0.42

8

0.124

0.34 8

0.151

0.62 8

SOD

Pearson correlation

Sig. (2-tailed)

N

0.563

0.431

8

0.453

0.21

8

1 -

8

0.324

0.41 8

0.435

0.71

8

0.241

0.56 8

0.034

0.70 8

CAT

Pearson correlation

Sig. (2-tailed)

N

0.546

0.23 8

0.512*

0.023

8

0.324

0.41

8

1 - 8

0.134

0.391

8

0.142

0.56 8

0.120

0.67 8

Dry weight

Pearson correlation

Sig. (2-tailed)

N

0.129

0.78 8

0.220

0.42

8

0.435

0.71

8

0.134

0.391 8

1 -

8

0.651** 0 8

0.578*

0.034 8

Cr

Pearson correlation

Sig. (2-tailed)

N

0.023

0.91 8

0.124

0.34

8

0.241

0.56

8

0.142

0.56 8

0.651**

0

8

1 - 8

0.789** 0 8

Na

Pearson correlation

Sig. (2-tailed)

N

0.034

0.67 8

0.151

0.62

8

0.034

0.70

8

0.120

0.67 8

0.578*

0..034

8

0.789** 0 8

1 - 8


The SOD, CAT and dry weight found to have weak correlation between

them and with all other variables. The Cr uptake in plant exhibited strong

mutual correlation with Na uptake.

229

4.9 Experiments with marigold and sunflower inoculated with selective

autochthonous saprobic fungi under greenhouse and field conditions

During the current study, the SOD and CAT in sunflower and marigold

was found to increase with increasing level of heavy metal concentration in

soil, due to increasing percentage of TSW. Our studies are parallel to the

elevation in production of antioxidant defense enzymes on increasing the

concentration of tannery waste in soil (Halliwell and Gutteridge, 2004; Singh

et al., 2004; Ahmad et al., 2009; Gupta and Sinha, 2009). On the contrary,

reduced CAT production in sunflower with increasing stress has also been

reported (Quartacci and Navari-Izzo, 1992). However, due to increasing level

of tannery sludge, a decrease in soluble protein content and chlorophyll

content (Singh et al., 2004) and total chlorophyll content (Gupta and Sinha,

2009) has been reported. In the current study, the soluble protein and

chlorophyll content was found to decrease down the column in all the cases

where fungal inoculations were not applied and it was because of increasing

level of multi-metal stress in soil.

The Pearson correlation between selected biochemical parameters, dry

weight, Cr and Na uptake in plant showed that the chlorophyll showed strong

correlation with Cr and Na uptake. The soluble protein contents showed

strong correlation with CAT and dry weight production, while SOD had strong

mutual variation with soluble protein, CAT and dry weight production. The

SOD was strongly variable with soluble protein, CAT and dry weight. The CAT

production in plant strongly correlated with chlorophyll contents, soluble

protein, CAT and dry weight. The CAT in plant was strongly variable with

respect to the soluble protein, SOD, Cr and Na uptake. The dry weight was

found to have strong correlation with soluble protein, SOD, Cr and Na uptake.

The Cr uptake varied strongly with respect to chlorophyll contents, CAT, dry

weight production; however very strongly correlated with Na uptake in plant.

The Na uptake in plant showed strong correlation with chlorophyll contents,

CAT dry weight and Cr uptake in plant, as given in Table 4.9.1.

230

Table 4.9.1. Pearson correlation among selected biochemical properties, CAT, SOD, dry weight production, Cr and Na uptake in Tagetes patula cultivated on TSW:Soil taken in pots and inoculated with saprobic fungi.


SOD CAT Dry

weight Cr Na

Chlorophyll

Pearson correlation

Sig. (2-tailed)

N

1

8

0.453

0.67

8

0.567

0.34

8

0.613*

0.042 8

0.571

0.56

8

0.614

0.067 8

0.678*

0.023 8

Soluble Protein

Pearson correlation

Sig. (2-tailed)

N

0.453

0.67

8

1 -

8

0.651*

0.042

8

0.645*

0.032 8

0.713**

0

8

0.561*

0.043 8

0.589*

0.039 8

SOD

Pearson correlation

Sig. (2-tailed)

N

0.567

0.34

8

0.651*

0.042

8

1 -

8

0.671*

0.023 8

0.651*

0.035

8

0.456

0.142 8

0.478

0.23 8

CAT

Pearson correlation

Sig. (2-tailed)

N

0.613*

0.042

8

0.645*

0.032

8

0.671*

0.023

8

1 - 8

0.569*

0.034

8

0.761** 0 8

0.691*

0.042 8

Dry weight

Pearson correlation

Sig. (2-tailed)

N

0.571

0.56

8

0.713**

0

8

0.651*

0.035

8

0.569*

0.034 8

1 -

8

0.681*

0.041 8

0.718** 0 8

Cr

Pearson correlation

Sig. (2-tailed)

N

0.614

0.067

8

0.561*

0.043

8

0.456

0.142

8

0.761** 0 8

0.681*

0.041

8

1 - 8

0.812** 0 8

Na

Pearson correlation

Sig. (2-tailed)

N

0.678*

0.023

8

0.589*

0.039

8

0.478

0.23

8

0.691*

0.042 8

0.718**

0

8

0.812** 0 8

1 - 8


Thus, in the present study, the inoculation of marigold and sunflower

with saprobic fungi in pot and field trials has led both of the plants to withstand

the stress exerted by the Category-I and II metals. The sparobic Trichoderma

sp. has also been reported to involve in improving the phytoextraction

231

efficiency of Brassica juncea (Cao et al., 2008) and in clover (Medina et al.,

2006). The saprobic fungus Trichoderma harzianum has been reported to

involve in Cd removal from contaminated soils (Lima et al., 2011). In this

work, the application of T. harzianum in marigold phytoextraction trials with

Caldwell field soil has also manifested the similar results.

The Pearson correlation values for chlorophyll with respect to soluble

protein contents, dry weight and Cr uptake in plant showed strong correlation,

as given in Table 4.9.2. The soluble protein contents varied strongly with

respect to chlorophyll contents; however, SOD and CAT didn’t show strong

correlation with other variables. The dry weight production showed strong

mutual variation with chlorophyll production, SOD and Cr uptake in plant. The

Cr uptake in plant showed strong correlation with chlorophyll contents, dry

weight production and Na uptake in plant. The Na uptake in plant observed to

vary strongly with respect to CAT production and Na uptake in plant.

232

Table 4.9.2. Pearson correlation among selected biochemical properties and metals for Tagetes patula cultivated in Caldwell field soil mixed with tannery solid waste (TSW) and inoculated with saprobic fungi (incl. Trichoderma harzianum).


SOD CAT Dry

weight Cr Na

Chlorophyll

Pearson correlation

Sig. (2-tailed)

N

1 - 8

0.647*

0.038 8

0.645*

0.046

8

0.478

0.34

8

0.610*

0.032

8

0.637*

0.046 8

0.563

0.31 8

Soluble Protein

Pearson correlation

Sig. (2-tailed)

N

0.647*

0.038 8

1 - 8

0.564*

0.039

8

0.453

0.56

8

0.356

0.76

8

0.467

0.56 8

0.574

0.24 8

SOD

Pearson correlation

Sig. (2-tailed)

N

0.645*

0.046 8

0.564*

0.039 8

1 -

8

0.235

0.78

8

0.645*

0.034

8

0.545

0.34 8

0.564

0.54 8

CAT

Pearson correlation

Sig. (2-tailed)

N

0.478

0.34 8

0.453

0.56 8

0.235

0.78

8

1 -

8

0.572

0.23

8

0.578

0.32 8

0.610*

0.041 8

Dry weight

Pearson correlation

Sig. (2-tailed)

N

0.610*

0.032 8

0.356

0.76 8

0.645*

0.034

8

0.572

0.23

8

1 -

8

0.671*

0.031 8

0.571

0.43 8

Cr

Pearson correlation

Sig. (2-tailed)

N

0.637*

0.046 8

0.467

0.56 8

0.545

0.34

8

0.578

0.32

8

0.671*

0.031

8

1 - 8

0.678*

0.029 8

Na

Pearson correlation

Sig. (2-tailed)

N

0.563

0.31 8

0.574

0.24 8

0.564

0.54

8

0.610*

0.041

8

0.571

0.43

8

0.678*

0.029 8

1 - 8


Both sunflower and marigold were able to accumulate significant

concentrations of metals in their root and shoot. Such reports for sunflower

have shown effective accumulation of multi-metals in both shoot and root

233

while grown on tannery waste amended soils (Singh et al., 2004). It was

observed that the metal accumulation ability of both of the plants increased

with increasing level of heavy metal concentration in soil i.e. due to increasing

percentage of TSW in soil. Similar results have been reported where metal

accumulation increased with increasing level of tannery waste in soil (Barman

et al., 2000; Armienta et al., 2001). It was also noted during this study that the

metal uptake in both the selected plants increased with increasing

concentration of multi-metals present in soil. Such studies have been reported

by AlHamdani and Blair, (2004); Hoffman et al., (2004) and Sune et al., (2007)

where plant exposure to the increasing concentration of metals such as, Cr,

Cd, and Pb has been reported to elevate metal uptake in plants.

On finding Pearson correlation between chlorophyll and rest of the

selected variables, it was found that chlorophyll strongly correlated with Na

uptake in plant. The soluble protein contents of the plant strongly varied with

respect to dry weight production in plant and Na uptake. The SOD contents

observed to have strong correlation with Cr uptake in plant, while CAT

production observed to correlate weakly with all of the selected variables. The

dry weight production in plant exhibited strong correlation with Cr and Na

uptake in plant. The Cr uptake in plant strongly varied with respect to Na

uptake, while Na uptake found to have strong correlation with chlorophyll

contents, soluble protein, dry weight and Cr uptake in plant, as given in Table

4.9.3. Increased level of SOD activity as a result of oxidative stress caused by

surfeit of heavy metals is well documented (Dey et al., 2007; Zhang et al.,

2007).

234

Table 4.9.3. Pearson correlation among selected biochemical properties and metals for Helianthus annuus cultivated in soil mixed with tannery solid waste (TSW) and inoculated with saprobic fungi (FIELD TRIAL).


SOD CAT Dry

weight Cr Na

Chlorophyll

Pearson correlation

Sig. (2-tailed)

N

1 - 8

0..536

0.43

8

0.456

0.56 8

0.563

0.61

8

0.568

0.32

8

0.563

0.41 8

0.673*

0.038 8

Soluble Protein

Pearson correlation

Sig. (2-tailed)

N

0.536

0.43 8

1 -

8

0.562

0.45 8

0.462

0.67

8

0.615*

0.023

8

0.525

0.56 8

0.681*

0.034 8

SOD

Pearson correlation

Sig. (2-tailed)

N

0.456

0.56 8

0.562

0.45

8

1 - 8

0.013

0.89

8

0.456

0.56

8

0.615*

0.021 8

0.578

0.46 8

CAT

Pearson correlation

Sig. (2-tailed)

N

0.563

0.61 8

0.462

0.67

8

0.013

0.89 8

1 -

8

0.561

0.23

8

0.518

0.34 8

0.591

0.43 8

Dry weight

Pearson correlation

Sig. (2-tailed)

N

0.568

0.32 8

0.615*

0.023

8

0.456

0.56 8

0.561

0.23

8

1 -

8

0.781** 0 8

0.699*

0.031 8

Cr

Pearson correlation

Sig. (2-tailed)

N

0..563

0.41 8

0.525

0.56

8

0.615*

0.021 8

0.518

0.34

8

0.781**

0

8

1 - 8

0.767** 0 8

Na

Pearson correlation

Sig. (2-tailed)

N

0.673*

0.038 8

0.681*

0.034

8

0.578

0.046 8

0.591

0.43

8

0.699*

0.031

8

0.767** 0 8

1 - 8


Some studies have also reported that increasing the level of tannery

waste to a certain limit adds the necessary nutrients to soil (Singh and Sinha,

2004). As a result, the production of biomolecules such as chlorophyll is

235

increased in plants. In the current study, the 5 % TSW:Soil mixture in all the

cases not only improved the chlorophyll content of both marigold and

sunflower but also the biomass production. Such results have also been found

by Singh et al. (2004) where increase in the chlorophyll content of H. annuus

have been reported due to mixing of soil with tannery waste.

The distribution and variation of heavy metals in soil due to amendment

with tannery wastes have been reported (Vankar and Bajpai, 2008; Aceves et

al., 2009). In this study, the plants from TSW-Soil mixtures with fungi where

soil had high levels of Category-I and II metals, showed elevated levels of

biochemical parameters as compared to those harvested from the

corresponding soil treatments without fungal inoculation.

Literature suggests increased plant antioxidant enzymes in response

to increasing level of heavy metals in soils (Pietrini et al., 2003; Al-Hamdani

and Blair, 2004; Ederli et al., 2004; Hu et al., 2007; Dhir et al., 2009).The level

of toxicity caused by heavy metals has been found to affect several processes

in plants (Siedlecka et al., 2001). In the current study, an increase in CAT

production was observed relative to the increase in heavy metal concentration

in TSW-Soil mixture. An increase in CAT with increase in stress level has

been reported (Zhang et al., 2006). In contrast, however, CAT activity has

been reported to decrease on exposing plants to high levels of Cr (Aravind

and Prasad, 2005; Shankar et al., 2005). As the biochemical parameters in

this study were noticed for leaves of both marigold and sunflower, the

assessment of the antioxidant defense system was validated. In leaves of

heavy metal-stressed plants (SOD) activity fluctuated in different stress levels

compared to the control, while CAT activity increased with stress levels

(Zhang et al., 2007).

The decrease in plant growth with increasing level of heavy metals has

been reported by John et al., (2009). During this study, the growth of

marigold was observed to decrease with increasing concentration of

Category-I and II metals, being the minimum in 20 % TSW-Soil mixtures in

most of the cases. The increasing toxicity due to heavy metals has been

found to lower plant growth and tolerance (Mahmood et al., 2007). Thus in

236

this study, the elevated level of Category-I and II concentration levels in soils

for both pot and field trials has been found to lower down the plant growth and

biomass yield. The elevated level of metals in the growing medium has been

reported to cause a decrease in chlorophyll production (Prasad et al., 2001;

Pietrini et al., 2003; Panda and Chaudhary, 2005; Hu et al., 2007; John et al.,

2009). The soluble protein content has also been reported to decrease with

increasing concentration of metal in soil (John et al., 2009). The excess of

heavy metals in soils minimize the tendency of plants to produce the key

biochemical molecules such as, chlorophyll and soluble proteins, which

ultimately result in increasing sensitivity to stress environments. Thus in this

study, the growth variation of marigold was subjected to the variation in

concentration of Category-I and II metals in the soil.

The meta-analytical perspective of Category-I and II in TSW-Soil

mixtures helped analyze the trend of metals in soil, plant and plant parts. In

the literature, the metal analytical parameters have been used to analyze the

metal distribution pattern in soil to plant during different phytoextraction

studies such as tolerance index (TI %) (Bauddh and Singh, 2011); relative

growth rate (RGR) and TI (Umebese and Motajo 2008) and only TI (Mahmood

et al., 2007), translocation index (Zehra et al., 2009); translocation index in

field experiments (Liu et al., 2009) and translocation index and mobility index

of heavy metals (Tukura et al., 2012). In this study, the metal-analytical

comparisons for circulation of Category-I and II metals from TSW to soil and

from soil to sunflower and marigold were carried out.

Conclusion

The present study demonstrates the potential of Helianthus annuus

and Tagetes patula in phytoremediation of multimetal contaminated TSW.

Exposure to metals with fungal inoculation individually or in combinations

resulted in enhanced metal accumulation in the roots and shoots of these

plants, with comparatively high biomass production indicating no toxicity

symptoms, shows that these plants can tolerate high metal concentrations.

On the basis of high values of SEY (%), Tolerance index and translocation

index, it can be concluded that H. annuus and T. patula have a great ability to

237

phytoextract the metals from TSW. Moreover, results suggests that both the

plants have a high capability to detoxify the ROS produced in response to

metal stress throughout their growth and developmental stages, indicated by

high activities of antioxidant enzymes like SOD and CAT. Hence, H. annuus

and T. patula along with autochthonous fungi, proved their phytoremediational

efficiency for novel phytoremediation strategies for metals.

In general, the benefits and disadvantages of phytoremediation must

be assessed by finding the most appropriate organism for the task.

Combining technologies like phytoremediation and mycoremediation offers

the greatest potential to decontaminate the multimetal contaminated TSW.

The application of latest innovations in biotechnology has made it easy to find

a workable and cost effective method of solving a problem. Disposal of

tannery solid waste is not a problem if it is converted into a useful by-product

or fertilizer free of heavy metals. Phytoremediation is a miracle and can be

used to transform byproduct of leather industry into a non toxic or non toxic

material.

Future perspective

A considerable biotechnological approach for increasing the potential

for metal phytoextraction may be to improve the hyperaccumulator growth

rate through selective plants, or by the transfer of metal hyperaccumulation

genes to the high biomass species. The transgenic plant approach has shown

to be promising, but only very few studies have proved authentic till now

under field conditions. Genetic manipulations will involve to change the

expression levels in a number of genes, and also to determine the number of

genes involved and their characteristics. Functions and regulations of genes

involved in metal hyperaccumulation, uptake, root-to-shoot translocation,

detoxification, sequestration mechanisms need to be fully understood to

provide transgenic approach less competent to solve the problem.

In spite of progress made in recent years by numerous studies, the

complexity of hyperaccumulation is far from being understood and several

aspects of this astonishing feature still await explanation. The recent idea that

heavy metals would provide an elemental defense to the plant through joint

effects with organic defense compounds requires much experimental

238

evidence. More elements and a larger number of hyperaccumulator species

need to be examined to validate the hypothesis of defensive effects of heavy

metals.

Furthermore, it is of pivotal importance to increase the understanding

of hyperaccumulator-based remedial mechanisms because they will be able

to provide clues for optimizing the effectiveness of phytoextraction with

appropriate agronomic practices. In addition, knowledge acquired on genes

involved in hyperaccumulation mechanisms will open the opportunity to use

biotechnology to transfer specific genes to high-biomass promising species.

Much research is still needed on rhizosphere and soil microbial composition

under field conditions, in order to identify micro-organisms associated with

metal solubility or precipitation.

There is also an urgent need to find and characterize other

hyperaccumulators, to cultivate them and investigate agronomic practices and

management to enhance plant growth and metal uptake. Even then, metal

uptake might create environmental risks, unless the biomass produced during

the phytoremediation process could be rendered economical by burning it to

produce bio-ore or converting it into bioenergy. Before the commercialization

of phytoextraction using high-biomass hyperaccumulator plants it needs to be

determined that they will not only remediate contaminated sites but also

generate income from agricultural lands not utilized otherwise.

References

239

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