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Use of Microbial Inoculants and Organic
Fertilizers for Improving the Growth of
Some Economical Crops of Pakistan
RABIA BADAR
Department of Botany,
Jinnah University for Women,
Nazimabad, Karachi-74600, Pakistan
2012
Use of Microbial Inoculants and Organic
Fertilizers for Improving the Growth of
Some Economical Crops of Pakistan
By
RABIA BADAR
A thesis submitted for the fulfillment of the requirement of
the degree for the Doctor of Philosophy (PhD) in the field
of Botany
Department of Botany,
Jinnah University for Women,
Nazimabad, Karachi-74600, Pakistan
Certificate
This is to certify that Ms. Rabia Badar d/o Mr. Badarul Hasan is a Ph.D
student having enrollment No. 2005/Bot/M.Phil/Ph.D/7 of Jinnah University
for Women. She has completed her research dissertation entitled “Use of
microbial inoculants and organic fertilizers for improving the growth of
some economical crops of Pakistan” under my supervision for the
fulfillment of the requirement for the degree of Doctor of Philosophy (PhD)
in the field of Botany. Without any hesitation I confirm that this is the
original work carried out by the candidate in Department of Botany of same
university.
___________________________ Dr. Shamim Akhter Qureshi
Research Supervisor & Assistant Professor
Department of Biochemistry
University of Karachi, Karachi-75270
Pakistan
Dated: ____________
Declaration
I, Ms Rabia Badar hereby declare that the contents of the thesis entitled “Use
of microbial inoculants and organic fertilizers for improving the growth
of some economical crops of Pakistan”, is my own PhD research work. To
the best of my knowledge and belief, it contains no material previously
published or written by any other person nor material which has been
accepted for the award of any other degree of the University or other
institute of higher studies except where due acknowledgment has been made
in the text.
Research Student
________________________ Ms. Rabia Badar
Department of Botany
Jinnah University for Women, Karachi
Pakistan
Dated: ____________
Dedication
To
My beloved and kind hearted
Parents
who supported me till their last breath of life
May Allah Rest their Souls with Peace
(Ameen)
Use of microbial inoculants and organic fertilizers for
improving the growth of some economical crops of
Pakistan
Table of Contents
S. No. Contents Page No. Acknowledgements
Summary
1. Introduction 1
1.1. Microbial Inoculants / Biofertilizers 4
1.1.1. Rhizobia 7
1.1.2. Fungi 10
1.1.2.1. Trichoderma species 11
1.1.2.1.1. Trichoderma hamatum 12
1.2. Organic fertilizers 14
1.3. Composting 16
1.4. Economical crops of Pakistan 18
1.4.1. Helianthus annus L. 18
1.4.2. Brassica nigra L. 19
1.4.3. Cicer arietinum L 20
1.4.4. Vigna mungo L. Hepper 21
1. 5. Aim of the present study 22
2. Material and Methods 23
2.1. Collection of root samples 23
2.2. Isolation and identification of fungi from rhizoplane 23
2.3. Isolation of rhizobial isolates from root nodule 23
2.4. Characterization of rhizobial isolates 24
2.4.1. Morphological and cultural characteristics 24
2.4.1.1. Hanging drop technique to test motility of rhizobial isolates 25
2.4.1.2. Gram staining 25
2.4.1.3. Differentiation of rhizobial isolates by using YEMA supplemented
with bromothymol blue 25
2.4.2. Biochemical characteristics 26
2.4.2.1. Indole production test 26
2.4.2.2. Methyl red and Voge’s Proskauer (MRVP) tests 26
2.4.2.3. Gelatin liquefaction test 27
2.4.2.4. Starch hydrolysis test 27
2.4.2.5. Hydrogen sulphide formation test 27
2.4.2.6. Nitrate reduction test 28
2.4.2.7. Oxidase test 28
2.4.2.8. Catalase test 28
2.4.2.9. Utilization of carbohydrates 29
2.5. Check the nodulation ability of test Rhizobium 29
2.6. In vitro antifungal activity of test fungus and rhizobial isolates 29
2.7. Legume and non-legume crops used in present experimental
work 30
2.8. Fertilizer and fungicide used in present experimental work 30
2.9. Treatments of microbial inoculants used in present experimental
work 31
2.10. Preparation of conidial and cell inoculums of T.hamatum and
rhizobial isolates 31
2.11. Organic food wastes 34
2.12. Procedure for preparing composted organic fertilizer 34
2.13. Experimental pot design and procedure 34
2.14. Effect of treatments on growth performance of experimental plants 35
2.14.1. Measurement of root and shoot lengths (cm) 35
2.14.2. Estimation of fresh weight (gram) 35
2.15. Effect of treatments on photosynthetic pigment 36
2.15.1. Estimation of chlorophyll content (mg/gm) 36
2.16. Effect of treatments on nutritive values in term of biochemical
parameters of experimental plants 37
2.16.1. Determination of total carbohydrate (mg/g) 37
2.16.2. Determination of crude protein (%) 38
2.17. Effect of treatments on mineral contents of experimental plants 38
2.17.1. Estimation of nitrogen (%) 38
2.17.2. Estimation of phosphorus (%) 42
2.18. Analysis of Data 43
3. Results 45
3.1. Isolation and identification of fungi from rhizoplane 45
3.2. Isolation of rhizobial isolates from root nodules 45
3.3. Characterization of rhizobial isolates 49
3.4. Nodulation ability of test rhizobial isolates 49
3.5. In vitro antifungal activity of T.hamatum and rhizobial isolates
against plant fungal pathogens 53
3.6. Pot experiments (1st Phase) 55
3.6.1. Effect of microbial inoculants on non-legume plants 55
3.6.1.1. Helianthus annuus L. 55
3.6.1.1.1. Growth performance 55
3.6.1.1.2. Photosynthetic pigment 60
3.6.1.1.3. Biochemical parameters 66
3.6.1.1.4. Mineral content 66
3.6.1.2. Brassica nigra L. 76
3.6.1.2.1. Growth performance 76
3.6.1.2.2. Photosynthetic pigment 85
3.6.1.2.3. Biochemical parameters 85
3.6.1.2.4. Mineral content 90
3.6.2. Effect of microbial inoculants on legume plants 96
3.6.2.1. Vigna mungo L. Hepper 96
3.6.2.1.1. Growth performance 96
3.6.2.1.2. Photosynthetic pigment 107
3.6.2.1.3. Biochemical parameters 107
3.6.2.1.4. Mineral content 113
3.6.2.2. Cicer arietinum L. (Chickpea) 124
3.6.2.2.1. Growth performance 124
3.6.2.2.2. Photosynthetic pigment 130
3.6.2.2.3. Biochemical parameters 136
3.6.2.2.4. Mineral content 136
3.7. Composting of rice husk and wheat bran 143
3.7.1. Effect of treatments on total carbohydrate and protein of
composted rice husk and wheat bran 143
3.8. Pot experiments (2nd
Phase) 149
3.8.1. Effect of composted rice husk on non- legume and legume plants 149
3.8.1.1. Helianthus annuus (Sunflower) 149
3.8.1.1.1. Growth performance 149
3.8.1.1.2. Photosynthetic pigment 156
3.8.1.1.3. Biochemical parameters 156
3.8.1.1.4. Mineral content 167
3.8.1.2. Cicer arietinum L. 167
3.8.1.2.1. Growth performance 167
3.8.1.2.2. Photosynthetic pigment 176
3.8.1.2.3. Biochemical parameters 176
3.8.1.2.4. Mineral content 187
3.9. Effect of composted wheat bran on non- legume and legume plants 187
3.9.1. H. annuus L.(sunflower) 187 3.9.1.1. Growth performance 187
3.9.1.2. Photosynthetic pigment 194
3.9.1.3. Biochemical parameters 205
3.9.1.4. Mineral content 205
3.9.2. Cicer arietinum L. 216
3.9.2.1. Growth performance 216
3.9.2.2. Photosynthetic pigment 216
3.9.2.3. Biochemical parameters 225
3.9.2.4. Mineral content 225
4. Discussion 239
4.1. Isolation of T.hamatum from rhizoplane and rhizobial isolates from
root nodules 240
4.2. In vitro antifungal activity of T.hamatum and rhizobial isolates 241
4.3. Pot experiments 243
4.3.1. The effect of microbial inoculants on non-legume plants including
H. annuus and B. nigra 243
4.3.2. The effect of microbial inoculants on legume plants including
V. mungo and C. arietinum. 249
4.4. Composting 255
4.4.1. Effect of microbial treatment on total carbohydrate and protein of
composted rice husk and wheat bran 255
4.4.2. Pot experiments 256
4.4.2.1. The effect of composted rice husk and wheat bran on H. annuus
(non-legume) and C. arietinum (legume) plants 256
5. Conclusion and future prospects 260
6. References 262
7. Publications 310
List of Tables
Titles Page No.
Table 1: Treatments of test microorganism alone and in combination used
in pot experiment 32
Table 2: Treatments of test microorganism alone and in combination used
to prepare composted organic fertilizer 33
Table 3: Absorbance of glucose (µg/ml) 39
Table 4: Absorbance of nitrogen 41
Table 5: Absorbance of phosphorus 44
Table 6: Cultivated and wild plants with their sites of collection 46
Table 7: Test fungal pathogens with their host plants and sites of
collection 47
Table 8: Test microorganisms with host plants, site of collection and
code no. 48
Table 9: Cultural, morphological and staining characteristics of rhizobial
isolates 50
Table 10: Biochemical characteristics of rhizobial isolates 51
Table 11: Utilization of carbohydrates by rhizobial isolates 52
Table 12: In vitro antifungal activity of test microorganisms against
fungal pathogen 54
Table 13: Effect of treatments on growth performance of H.annuus
(sunflower) plants 56
Table 14: Effect of treatments on photosynthetic pigment of H.annuus
(sunflower) plants 61
Table 15: Effect of treatments on biochemical parameters of H.annuus
(sunflower) plants 67
Table 16: Effect of treatments on mineral content of H.annuus
(sunflower) plants 77
Table 17: Effect of treatments on growth performance of B.nigra (black
mustard) plants 79
Table 18: Effect of treatments on photosynthetic pigment of B.nigra
(black mustard) plants 86
Table 19: Effect of treatments on biochemical parameters of B.nigra
(black mustard) plants 91
Table 20: Effect of treatments on mineral content of B.nigra (black
mustard) plants 97
Table 21: Effect of treatments on growth performance of V.mungo (black
gram) plants 102
Table 22: Effect of treatments on photosynthetic pigment of V.mungo
(black gram) plants 110
Table 23: Effect of treatments on biochemical parameters of V.mungo
(black gram) plants 114
Table 24: Effect of treatments on mineral content of V.mungo (black
gram) plants 119
Table 25: Effect of treatments on growth performance of C. arietinum
(chickpea) plants 125
Table 26: Effect of treatments on photosynthetic pigment of C. arietinum
(chickpea) plants 133
Table 27: Effect of treatments on biochemical parameters of C. arietinum
(chickpea) plants 137
Table 28: Effect of treatments on mineral content of C. arietinum
(chickpea) plants 144
Table 29: Total carbohydrate and protein contents of composted rice husk
and wheat bran after 15 days of incubation. 148
Table 30: Effect of composted rice husk on root lengths of H.annuus
(sunflower) plants 150
Table 31: Effect of composted rice husk on shoot lengths of H.annuus
(sunflower) plants 152
Table 32: Effect of composted rice husk on fresh weight of H.annuus
(sunflower) plants 154
Table 33: Effect of composted rice husk on chlorophyll a of H.annuus
(sunflower) plants 157
Table 34: Effect of composted rice husk on chlorophyll b of H.annuus
(sunflower) plants 159
Table 35: Effect of composted rice husk on total chlorophyll of H.annuus
(sunflower) plants 161
Table 36: Effect of composted rice husk on carbohydrate content of
H.annuus (sunflower) plants 163
Table 37: Effect of composted rice husk on crude protein content of
H.annuus (sunflower) plants 165
Table 38: Effect of composted rice husk on percent nitrogen of H.annuus
(sunflower) plants 168
Table 39: Effect of composted rice husk on percent phosphorus of H.annuus
(sunflower) plants 170
Table 40: Effect of composted rice husk on root lengths of C. arietinum
(chickpea) plants 172
Table 41: Effect of composted rice husk on shoot lengths of C. arietinum
(chickpea) plants 174
Table 42: Effect of composted rice husk on fresh weight of C. arietinum
(chickpea) plants 177
Table 43: Effect of composted rice husk on chlorophyll a of C. arietinum
(chickpea) plants 179
Table 44: Effect of composted rice husk on chlorophyll b of C.
arietinum (chickpea) plants 181
Table 45: Effect of composted rice husk on total chlorophyll of C.
arietinum (chickpea) plants 183
Table 46: Effect of composted rice husk on carbohydrate content of
C. arietinum (chickpea) plants 185
Table 47: Effect of composted rice husk on crude protein content of
C. arietinum (chickpea) plants 188
Table 48: Effect of composted rice husk on percent nitrogen of C.
arietinum (chickpea) plants 190
Table 49: Effect of composted rice husk on percent phosphorus of
C.arietinum (chickpea) plants 192
Table 50: Effect of composted wheat bran on root lengths of H.annuus
(sunflower) plants 195
Table 51: Effect of composted wheat bran on shoot lengths of H.annuus
(sunflower) plants 197
Table 52: Effect of composted wheat bran on fresh weight of H.annuus
(sunflower) plants 199
Table 53: Effect of composted wheat bran on chlorophyll a of H.annuus
(sunflower) plants 201
Table 54: Effect of composted wheat bran on chlorophyll b of H.annuus
(sunflower) plants 203
Table 55: Effect of composted wheat bran on total chlorophyll of
H.annuus (sunflower) plants 206
Table 56: Effect of composted wheat bran on carbohydrate content of
H.annuus (sunflower) plants 208
Table 57: Effect of composted wheat bran on crude protein content of
H.annuus (sunflower) plants 210
Table 58: Effect of composted wheat bran on percent nitrogen of
H.annuus (sunflower) plants 212
Table 59: Effect of composted wheat bran on percent phosphorus of
H.annuus (sunflower) plants 214
Table 60: Effect of composted wheat bran on root lengths of C.
arietinum (chickpea) plant 217
Table 61: Effect of composted wheat bran on shoot lengths of C.
arietinum (chickpea) plants 219
Table 62: Effect of composted wheat bran on fresh weight of C.
arietinum (chickpea) plants 221
Table 63: Effect of composted wheat bran on chlorophyll a of C.
arietinum (chickpea) plants 223
Table 64: Effect of composted wheat bran on chlorophyll b of C.
arietinum (chickpea) plants 226
Table 65: Effect of composted wheat bran on total chlorophyll of C.
arietinum (chickpea) plants 228
Table 66: Effect of composted wheat bran on carbohydrate content of
C. arietinum (chickpea) plants 230
Table 67: Effect of composted wheat bran on crude protein content of
C. arietinum (chickpea) plants 232
Table 68: Effect of composted wheat bran on percent nitrogen of C.
arietinum (chickpea) plants 234
Table 69: Effect of composted wheat bran on percent phosphorus of
C. arietinum (chickpea) plants 237
List of Figures
Titles Page No. Figure 1: Standard curve of glucose 39
Figure 2: Standard curve of nitrogen 41
Figure 3: Standard curve of phosphorus 44
Figure 4: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of H.annuus plants 57
Figure 5: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on root length of H.annuus plants 58
Figure 6: Effect of T.hamatum alone and in combination with rhizobial
isolates on shoot length of H.annuus plants 59
Figure 7: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on shoot length of H.annuus plants 62
Figure 8: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of H.annuus plants 63
Figure 9: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on fresh weight of H.annuus plants 64
Figure 10: Effect of T.hamatum alone and in combination with rhizobial
isolates on total chlorophyll of H.annuus plants 65
Figure 11: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total chlorophyll of H.annuus plants 68
Figure 12: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of H.annuus plants 69
Figure 13: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total carbohydrate of H.annuus
plants 70
Figure 14:Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of H.annuus plants 71
Figure 15: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of H.annuus
plants 72
Figure 16: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent nitrogen of H.annuus plants 73
Figure 17: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent nitrogen of H.annuus plants 74
Figure 18: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent phosphorus of H.annuus plants 75
Figure 19: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent phosphorus of H.annuus
plants 78
Figure 20: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of B.nigra plants. 80
Figure 21: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on root length of B.nigra plants. 81
Figure 22: Effect of T.hamatum alone and in combination with rhizobial
isolates on shoot length of B.nigra plants. 82
Figure 23: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on shoot length of B.nigra plants. 83
Figure 24: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of B.nigra plants. 84
Figure 25: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on fresh weight of B.nigra plants. 87
Figure 26: Effect of T.hamatum alone and in combination with rhizobial
isolates on total chlorophyll of B.nigra plants. 88
Figure 27: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total chlorophyll of B.nigra plants. 89
Figure 28: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of B.nigra plants. 92
Figure 29: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total carbohydrate of B.nigra plants. 93
Figure 30: Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of B.nigra plants. 94
Figure 31: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of B.nigra plants. 95
Figure 32: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent nitrogen of B.nigra plants. 98
Figure 33: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent nitrogen of B.nigra plants. 99
Figure 34: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent phosphorus of B.nigra plants. 100
Figure 35: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent phosphorus of B.nigra plants. 101
Figure 36: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of V.mungo plants. 103
Figure 37: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on root length of V.mungo plants. 104
Figure 38: Effect of T.hamatum alone and in combination with rhizobial
isolates on shoot length of V.mungo plants. 105
Figure 39: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on shoot length of V.mungo plants. 106
Figure 40: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of V.mungo plants. 108
Figure 41: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on fresh weight of V.mungo plants. 109
Figure 42: Effect of T.hamatum alone and in combination with rhizobial
isolates on total chlorophyll of V.mungo plants. 111
Figure 43: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total chlorophyll of V.mungo plants. 112
Figure 44: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of V.mungo plants. 115
Figure 45: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total carbohydrate of V.mungo plants. 116
Figure 46: Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of V.mungo plants. 117
Figure 47: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of V.mungo plants. 118
Figure 48: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent nitrogen of V.mungo plants. 120
Figure 49: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent nitrogen of V.mungo plants. 121
Figure 50: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent phosphorus of V.mungo plants. 122
Figure 51: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent phosphorus of V.mungo plant. 123
Figure 52: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of C.arietinum plants. 126
Figure 53: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on root length of C.arietinum plants. 127
Figure 54: Effect of T.hamatum alone and in combination with rhizobial
isolates on shoot length of C.arietinum plants. 128
Figure 55: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on shoot length of C.arietinum plants. 129
Figure 56: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of C.arietinum plants. 131
Figure 57: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on fresh weight of C.arietinum plants. 132
Figure 58: Effect of T.hamatum alone and in combination with rhizobial
isolates on total chlorophyll of C.arietinum plants. 134
Figure 59: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total chlorophyll of C.arietinum plants. 135
Figure 60: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of C.arietinum plants. 138
Figure 61: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total carbohydrate of C.arietinum plants. 139
Figure 62: Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of C.arietinum plants. 140
Figure 63: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of C.arietinum plant. 141
Figure 64: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent nitrogen of C.arietinum plant. 142
Figure 65: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent nitrogen of C.arietinum plants. 145
Figure 66: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent phosphorus of C.arietinum plants. 146
Figure 67: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent phosphorus of C.arietinu plants. 147
Figure 68: Effect of composted rice husk @ 5 and 10 gm on root length of
H.annuus plants. 151
Figure 69: Effect of composted rice husk @ 5 and 10 gm on shoot length
of H.annuus plants. 153
Figure 70: Effect of composted rice husk @ 5 and 10 gm on fresh weight
of H.annuus plants. 155
Figure 71: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a
of H.annuus plants. 158
Figure 72: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-b
of H.annuus plants. 160
Figure 73: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll
of H.annuus plants. 162
Figure 74: Effect of composted rice husk @ 5 and 10 gm on total
carbohydrate of H.annuus plants. 164
Figure 75: Effect of composted rice husk @ 5 and 10 gm on crude protein (%)
of H.annuus plants. 166
Figure 76: Effect of composted rice husk @ 5 and 10 gm on percent nitrogen
of H.annuus plants. 169
Figure 77: Effect of composted rice husk @ 5 and 10 gm on percent phosphorus
of H.annuus plants. 171
Figure 78: Effect of composted rice husk @ 5 and 10 gm on root length of
C.arietinum plants. 173
Figure 79: Effect of composted rice husk @ 5 and 10 gm on shoot length
of C.arietinum plants. 175
Figure 80: Effect of composted rice husk @ 5 and 10 gm on fresh weight
of C.arietinum plants. 178
Figure 81: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a
of C.arietinum plants. 180
Figure 82: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-b
of C.arietinum plants. 182
Figure 83: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll
of C.arietinum plants. 184
Figure 84: Effect of composted rice husk @ 5 and 10 gm on total carbohydrate
of C.arietinum plants. 186
Figure 85: Effect of composted rice husk @ 5 and 10 gm on crude protein (%)
of C.arietinum plants. 189
Figure 86: Effect of composted rice husk @ 5 and 10 gm on percent
nitrogen of C.arietinum plants. 191
Figure 87: Effect of composted rice husk @ 5 and 10 gm on percent
phosphorus of C.arietinum plants. 193
Figure 88: Effect of composted wheat bran @ 5 and 10 gm on root
length of H.annuus plants. 196
Figure 89: Effect of composted wheat bran @ 5 and 10 gm on shoot
length of H.annuus plants. 198
Figure 90: Effect of composted wheat bran @ 5 and 10 gm on fresh
weight of H.annuus plants. 200
Figure 91: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-a
of H.annuus plants. 202
Figure 92: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b
of H.annuus plants. 204
Figure 93: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll
of H.annuus plants. 207
Figure 94: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate
of H.annuus plants. 209
Figure 95: Effect of composted wheat bran @ 5 and 10 gm on crude protein (%)
of H.annuus plants. 211
Figure 96: Effect of composted wheat bran @ 5 and 10 gm on percent nitrogen
of H.annuus plants. 213
Figure 97: Effect of composted wheat bran @ 5 and 10 gm on percent phosphorus
of H.annuus plants. 215
Figure 98: Effect of composted wheat bran @ 5 and 10 gm on root length
of C.arietinum plants. 218
Figure 99: Effect of composted wheat bran @ 5 and 10 gm on shoot length
of C.arietinum plants. 220
Figure 100: Effect of composted wheat bran @ 5 and 10 gm on fresh weight
of C.arietinum plants. 222
Figure 101: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-a
of C.arietinum plants. 224
Figure 102: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b
of C.arietinum plants. 227
Figure 103: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll
of C.arietinum plants. 229
Figure 104: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate
of C.arietinum plants. 231
Figure 105: Effect of composted wheat bran @ 5 and 10 gm on crude
protein (%) of C.arietinum plants. 233
Figure 106: Effect of composted wheat bran @ 5 and 10 gm on percent
nitrogen of C.arietinum plants. 235
Figure 107: Effect of composted wheat bran @ 5 and 10 gm on percent
phosphorus of C.arietinum plants. 238
Acknowledgment
First I humbly offer thanks to my Allah for providing me chance and courage to
successfully completed my doctoral research work. My salutation is always to the Holy
Prophet of Islam Hazrat Muhammad (S.A.W.W.), the most perfect and respectable;
whose sirat-e-tayyaba (way of living) is always an example for the whole Muslim
community.
My heartiest thanks and sincere gratitude is always for my most respected supervisor Dr.
Shamim A. Qureshi, Assistant Professor of Biochemistry Department, University of
Karachi for her continuous, creative, informative and valuable support. Without her entire
supervision, this work would not have been effectively completed.
I offer my sincere thanks to the former Vice Chancellor Prof. Dr Riaz Ahmed Hashmi
who helped me in conducting my research work and his ever lasting cooperation which
always motivates me. I am also grateful to Prof. Dr. Yasmeen Akhter, Dean, Faculty of
Science, Jinnah University for Women, Karachi.
My special gratitude goes to Prof. Dr. Syed Ehteshamul Haque, Dr. Ifran Aziz, Ms.
Mehwish Hussain for supporting me. I express my indebted gratitude to Ms. Saima
Ibrahim, Ms. Shaheen Perveen, Ms. Shabnum Shabbir for their constant help and support
during this whole research work.
Next, I am thankful to Ms. Tooba Lateef, Mr. M.Bilal Azmi, Mr. Ali Zia and other lab
fellows for their appreciation and facilitation provided to me.
I would like to convey my thanks to lab assistants including Mr. Amjad Wazir, Ms. Rabia
Ahmed and Mr Fiasal Jan Baloch for providing technical support. My best regards are
also for Ms. Qaryah Malik, Ms. Amna Shamim, Ms. Yumna Mehmood and Ms. Nosheen
Ghulam.
I feel highly privileged to express my heartiest thanks to my all family members, my
brothers, sisters and all kids for their love and good wishes. I also extend my kind
feelings for my beloved innocent nephew Abdul Rehman Siddiqui whose smile always
energized me throughout this work.
Lastly I refer this work to all scientific personals who are interested in providing healthy
food by using organic fertilizer worldwide.
Ms. Rabia Badar M.Sc (Botany)
Summary
Soil not only serves as a natural habitat for countless living microorganisms but
also an excellent medium for agriculture due to the presence of valuable nutrients
including organic matter, minerals, etc, with favorable chemical and physical properties,
all these components together contribute the soil fertility which is essential for cropping
system. However, the soil flora and fauna is influenced directly or indirectly by farming
practices especially the inappropriate use and frequent application of inorganic fertilizers.
It has been recognized that excessive use of agrochemicals, on one hand, induces
decrease in biodiversity, increase erosion by reducing the soil organic matter and, on the
other hand, produces harmful effects on human health and enviroment. The agricultural
fields of Pakistan are also facing the same threat in order to fulfill the demand of growing
population. Diseases are an additional threat of agriculture which are caused by many
plant pathogenic microorganisms, of which fungi ranks first for crop damage. To provide
defense against these fungal pathogens, excessive use of fungicides have been practiced
globally and due to the presence of harmful ingredients these again produced some
deleterious effects like accumulation of toxic compounds that are potentially dangerous
to whole ecosystem, buildup resistance in pathogens, etc.
From last few decades, biotechnologists have been introducing and motivating
farmers to use alternate biological ways to preserve the soil fertility and increase crop
yield with none or minimal health and environment hazard via microbial inoculants as
biofertilizers and biocontrol agents. Therefore, the present research work was designed to
isolate, inoculate and investigate the effects of Trichoderma hamatum and rhizobial
isolates alone and in combination on physical and biochemical parameters of each of two
non-legumes viz., Helianthus annuus (sunflower) and Brassica nigra (black mustrad)
plants and legumes viz., Vigna mungo (mashbean) and Cicer arietinum (chickpea) plants
in first phase of study and in second phase of study T. hamatum and selected rhizobial
isolates alone and in combination were used to prepare composted rice husk and wheat
bran. Later the effect of these composted food waste based organic fertilizers at 5 and 10g
each were investigated on physical and biochemical parameters of one each of non-
legume (sunflower) and legume (chickpea) plants.
In the present study, test microorganisms including fungus T. hamatum (JUF1)
was isolated from rhizoplane of Amaranthus viridis and nitrogen fixing rhizobial isolates
viz., fast-growing rhizobium sp. (JUR1) isolated from nodules of Trigonella foenum-
graecum (fenugreek) and three slow-growing bradyrizobium species JUR2, JUR3 and
JUR4 isolated from nodules of Phaseolus ungiculata (cowpea) , Vigna radiata
(mungbean) and V.mungo (mashbean) respectively. All these test microorganisms were
screened in vitro by using dual-culture plate method against four plant pathogenic fungi
including Fusarium oxysporum, F.solani, Macrophomina phaseolina and Rhizoctonia
solani in order to evaluate their biocontrol potential where T.hamatum (JUF1) inhibited
the growth of F.oxysporum, two strains each of M.phaseolina (strain-2 & 3) and R.solani
(strains-1 & 2) by mycelial coiling whereas the same JUF1 produced zones of inhibition
ranging from 2- 5.5 mm against F.solani (strain-2), M.phaseolina (strain-1 & 4) and
inhibited the growth of three strains of F.solani (strain-1, 3, & 4) without any zone and
proved another one of its well-reported mode of inhibition, called antibiosis. However,
strong antibiosis was observed by all tested rhizobial isolates including JUR1, JUR2,
JUR3 & JUR4 against F.oxysporum, F.solani, M. phaseolina and R. solani and inhibited
their growth by producing zones ranging from 1.5-11.5 mm.
In the present study, the randomized complete block designed pot experiment
was conducted to investigate the effects of microbial inoculants (test microorganisms)
alone and in different combinations on growth and biochemical parameters of two each
of non-legume and legume experimental plants. In which seeds of experimental plants
were sown in 2 kg soil / pot and at 5th
day of germination, developing seedlings in each
pot of each block were inoculated with 25 milliliters of its respective treatment (5
replicates /treatment). Five plants of each treatment (1 plant/replicate/treatment) were
uprooted at 30th
and 60th
day of growth to measure the selected physical and biochemical
parameters. Similarly five pots treated with each of NPK (fertilizer) and carbendazim
(fungicide) @ 2500 ppm were used as positive controls while five pots of experimental
plants without any treatment were used as control. The results obtained from non-legume
plants viz., sunflower and black mustard plants revealed that T.hamatum (JUF1) alone
and in combination with rhizobial isolates (JUR1, JUR2, JUR3 & JUR4) & fertilizer was
found effective in promoting the root and shoot lengths, total chlorophyll, total
carbohydrate and crude protein contents of test plants as compared to control (untreated)
plants and plants treated with fertilizer and fungicide alone. In addition, T.hamatum
alone and in combination with bradyrhizobium species (JUR3 & JUR4) also improved
mineral content of sunflower plants while JUF1 alone and in combination with JUR1,
JUR2 & JUR3 was found effective on same aspect in black mustard plants. Similarly, all
rhizobial isolates (JUR1, JUR2, JUR3 & JUR4) alone and in combination with each of
fertilizer & fungicide were found effective in improving the growth and biochemical
parameters of sunflower and black mustard plants in one way or another. Whereas only
JUR3 and JUR4 individually in their respective group were found helpful in improving
the mineral content of sunflower and black mustard plants especially the nitrogen
content.
The obtained results of legume plant V.mungo described that out of all tested
rhizobial isolates, host-specific bradyrhizobium sp (JUR4) of same plant found most
effective in improving the growth parameters of test plants followed by JUR2, JUR1 and
JUR3. T.hamatum and host-specific JUR4 were well-matched with each other and their
combination was found effective not only in improving the growth but also total
chlorophyll, total carbohydrate, crude protein and mineral including both nitrogen and
phosphorus contents of test V.mungo plants as compared to control plants. Similarly,
results obtained from C.arietinum plants (another legume), T.hamatum (JUF1) and
rhizobial isolates (JUR1, JUR2 and JUR3) alone were found effective in improving the
growth parameters and total chlorophyll content in their respective groups of test plants.
However, all rhizobial isolates (JUR1, JUR2, JUR3 and JUR4) in combination with
T.hamatum, fertilizer (NPK) and fungicide (carbendazim) were effective in improving the
total carbohydrate, crude protein and mineral contents of chickpea plants.
In the second phase of present study, two food wastes viz., rice husk and wheat
bran were composted with the help of T.hamatum (JUF1), rhizobium (JUR1) and
bradyrhizobium (JUR2) species alone and in combination to form biodegradable value
added product or organic fertilizer. This procedure increased the total carbohydrate and
total protein contents in composted rice husk and wheat bran as compared to
uncomposted and only grinded same organic food wastes. Each of these composted
organic fertilizers @ 5 & 10g /2 kg soil/pot was used to investigate their effects on
growth and biochemical parameters of sunflower (non-legume) and chickpea (legume)
plants. The application of composted organic fertilizer (COF) resulted in significant
improvement in growth and biochemical parameters of both non-legume and legume
plants as compared to control plants treated with uncomposted organic fertilizer (UCOF)
and it was clearly indicated that addition of COF may increase the organic content of soil.
However, effects vary with the microbial treatments involved in composting like rice
husk (RH) composted with T. hamatum (JUF1) and in combination with rhizobium sp
(JUR1) were found effective in improving the shoot & root lengths of plants,
photosynthetic pigments especially chlorophyll-a & total chlorophyll, biochemical
parameters especially crude protein and mineral (nitrogen & phosphorus) content of
sunflower (non-legume) plants. Whereas, RH composted with all treatments including
JUF1, JUR1, JUR2 (bradyrhizobium sp) alone and in combination (JUR1+ JUF1 &
JUR2+ JUF1) at 5 and 10g were found to produce significant effects on growth,
photosynthetic pigments especially chlorophyll-b & total chlorophyll, biochemical
parameters including both total carbohydrate & crude protein and mineral (nitrogen &
phosphorus) content of chickpea (legume) plants. Similarly, wheat bran (WB) composted
with all treatments especially JUF1 was found effective in improving the growth,
photosynthetic pigment and nutritional status of sunflower plants, however, percent
nitrogen content was much improved as compared to phosphorus of same test plants.
While WB composted with all treatments at 5 and 10 g was found efficient only in
improving all growth parameters including root, shoot lengths & fresh weight,
biochemical parameter especially total carbohydrate and phosphorus content of chickpea
plants.
Finally, the conclusion has been achieved that test microorganisms including
T.hamatum (JUF1) and rhizobial (JUR1, JUR2, JUR3 & JUR4) isolates alone and in
combination have shown an excellent growth promoting potential in pot experiments by
not only enhancing the growth but also improving the total carbohydrate, crude protein,
nitrogen and phosphorus contents of plants including sunflower, black mustard, mash
bean and chickpea plants. Hence, these microorganisms can be used as biofertilizers
which may possibly serve as a good substitute of chemical fertilizer in farming practices
in our country and worldwide to enhance the growth and nutritional status of both non-
legume and legume plants. In addition, the same test micoorganisms also proved their
biocontrol potential invitro against M.phaseolina, R.solani and Fusarium species, one of
the frequent fungal pathogens found in agriculture fields of Pakistan. Similarly,
T.hamatum alone and in combination with rhizobial isolates (JUR1 & JUR2) would be
beneficial in the preparation of composted organic fertilizer as the composting procedure
by using these microorganisms converted organic food wastes (rice husk and wheat bran)
into nutritionally rich biodegradable product that was also found effective in improving
the growth and biochemical parameters of sunflower (non-legume) and chickpea
(legume) plants when applied at 5 and 10 gm each /2 kg soil / pot by possibly improving
the organic content of soil. The composting of organic wastes was focused on recycling
of organic waste into biodegradable value added product which can be beneficial as
organic fertilizer for sustainable agriculture and environment. Therefore, the study clearly
indicates that the utilization of biofertilizers (microbial inoculants) and organic fertilizers
(especially composted) are much better than sole application of inorganic fertilizers and
fungicides.
1
Use of microbial inoculants and organic fertilizers for
improving the growth of some economical crops of
Pakistan
1. Introduction
Universally soil is a living matrix and an essential part of terrestrial ecosystem
that contains organic matter, minerals, water, air and living organisms including fungi,
bacteria, protozoa, etc (Raaijmakers and Paulitz, 2009). Hence it serves as an important
source for farming production and maintenance of most life processes including human
(Mishra, 1996). Nutrient availability by decomposing organic materials is a main
function of soil micro-flora (Mishra, 1996; Vikram et al., 2007). However, the organic
matter in soil is severely depleted due to rapid cop production with inappropriate farming
practices which results in decreased microbial activity that eventually affect physical,
chemical and biological conditions of soil (Haynes and Tregurtha, 1999). For this, a
major problem which facing our farmers is the declining of land productivity and reduced
crop yields that creates a gap between production and consumers’ demand. In order to fill
this gap, farming practices related to rapid cropping with no addition of sufficient amount
of mineral fertilizers and manures is the one of the causes of reduced land productivity
(Hornick and Parr, 1987; Parr et al., 1992). Consequently, sustainable agriculture is an
important way to maintain the life of soil and people (Brodt et al., 2011). Though, there
are many imperative reasons that restrict the sustainable crop growth including soil
salinity, alkalinity, erosion, reduction in soil fertility, depletion of water resources,
negligence of irrigation systems, deprived of agricultural land and practices (Zia et al.,
2003; Bhutto and Bazmi, 2007). Among these, decline of fertility because of negative soil
nutrient balance severely affects our cropping system. High-quality crop production can
be possible once these harmful balances are addressed.
Inorganic or chemical fertilizers are meant to provide vital plant nutrients such
as nitrogen, phosphorus, potassium, boron, zinc individually or in combination of two or
2
three of these according to the plant growth requirement (Stewart et al., 2005).
Application of suitable fertilizers increases the level of crop production which can fulfill
adequate and healthy food requirement of world’s increasing population. Thus plant
nutrients are essential elements of sustainable farming practices. Studies described that in
addition of nitrogen & phosphorus, micronutrients (zinc, boron, etc) and sulfur are the
most concern nutrients in the grain-production regions (Ali et al., 2011; Saeed et al.,
2012).
Unfortunately in Pakistan, soils are deficient of 100, 90, 70 and 55% respectively
in nitrogen, phosphorus, zinc and boron (Arain et al ., 2000). Though quantity of
potassium is enough but its shortage is appearing rapidly (Tariq et al., 2011). On the
other hand deficiencies of micronutrients including iron, boron, copper, etc, are reported
for specific crops and areas (Imtiaz et al., 2010; Aref, 2011). Application of fertilizers
become necessary when level of soil nutrients is not enough for ample plant growth.
Various factors including non-availability of right fertilizer on time, their escalating
prices, inappropriate application methods due to short of knowledge among farmers
regarding the need for balanced fertilizer application, fraud and insufficient funding of
soft loans mainly for the small farmers, etc, again put some limitation in the use of
fertilizers (Patil, 2010). Beside these, excessive use of chemical fertilizers which need
for increasing agriculture productivity, also create serious environmental and health risks
such as nitrogen fertilizers increases de-nitrification and release of gases viz., nitric oxide
or ammonia in atmosphere that not only contribute greenhouse warming but also become
the reason of global warming by affecting ozone layer (Smith et al., 2008). In addition,
nitric oxide is a major pollutant that affects the entire ecosystem including the agriculture
and human health (Azam et al., 2002). Nitric acid is reported to produce from nitric
oxide, it along with ammonia severely affect terrestrial and marine life by shifting the pH
towards acidic side (Kennedy, 1992; Reeves et al., 2002). It has been reported that
nitrate leaching induce toxicity in groundwater (Shrestha and Ladha, 1998). Addition of
nitrate in food and drinking water may convert hemoglobin (Fe+2
) into methemoglobin
(Fe+3
) in infants creates a condition called methemoglobinemia which affects lungs
3
efficiency and hepatic content of vitamin A (Phupaibul et al., 2002). Similarly,
application of nitrogen fertilizers for long time may also depletes soil organic carbon
content which originally serves as a versatile resource for soil fertility (Khan et al.,
2007). Hence, the agriculture set-up and practices in the world are changed due to the use
of chemical fertilizer and pesticides.
On one hand, studies reported that soil becomes unfit for cultivation because of
many reasons like inappropriate methods of cultivation, excess use of quick release
chemical fertilizers and extra irrigation (Singh, 1995; Tekle, 1999). On the other hand,
higher expenses in production due to increase application of chemical fertilizers and
transportation to bring crops to the market for consumers not only results in erosion of
natural organic resources but also increases expenditures at farmers’end that contributes
poverty (Moges and Holden, 2007). This increases the migration of youth from rural
areas to urban for earning purpose and leaving their native profession. In addition, rapidly
increasing urbanization also occupied farming areas so in coming years, Pakistan has to
fulfill the demands of growing population from less agriculture land and manpower
(Bhutto and Bazmi, 2007). It is also reported that the yield of most of the crops in our
country is also affected due to the availability of low quality seeds with high prices,
traditional sowing methods, improper utilization of fertilizers, poor management
practices and low rank of farm mechanization (Bhutto et al., 2007). Beside these, plant
diseases cause negative effect on agriculture by decreasing crop yield like fungi are one
of the pathogens accountable for plant diseases and considered as destructive agents for
critical losses of yield, quality and profit in agriculture (Keane, 2012; Gonz´alez-
Fern´andez et al., 2010; Mukhtar, 2009; Shenoy et al., 2007; Than et al., 2008). The
application of chemical fungicides is the most frequent technique to prevent crop yield
losses caused by fungal diseases (Rosslenbroich and Stuebler, 2000; Dias, 2012).
Chemical fungicides act via two modes of action including contact and systemic.
In first mode, fungicide usually kills pathogenic fungus through direct contact whereas in
systemic mode, fungicide one must take up by the affected living being (Dias, 2012). The
fungicides are commercially available in liquid and powdered forms that can be sprayed
4
on affected plants. Sulfur is the most essential active constituent present in all of
fungicides from low to high quantities like moderate fungicides contains 0.08% sulfur,
potent fungicides contain 0.5% and toxic fungicides contain 90% sulfur (Dias, 2012).
Studies proved that effects of chemical fungicides are injurious for individual’s health
and for atmosphere as these are reported to produce skin and eye irritation and also affect
other tissues like lungs, kidneys and heart (Ekpo et al., 2008). Other ingredients of
fungicides such as mercury and cadmium are also harmful to nervous tissues (Mckinney
and Rogers, 1992). One more drawback of frequent and long-term use of chemical
fungicides induced resistance in plant pathogenic fungi which turns fungicides into
useless substance and for effective disease control additional and more potent fungicides
are then required (Dekker, 1976; Georgopoulos, 1977). However, conventional use of
chemical fertilizers and fungicides in increasing the yield and productivity of agriculture
can not be over looked. Therefore, there is an immense need to add or replace chemical
fertilizers by organic fertilizers and to adopt biological ways to have better soil fertility
which in turn improves the crop productivity beside providing biocontrol against fungal
pathogens.
The fertility of soil depends not simply on its chemical composition but also on
number and types of microorganisms inhibiting it. The most abundant group of
microorganisms is bacteria and generally in normal fertile soils, 10 to 100 million
bacteria are present per gram of soil (Nannipieri et al., 2003). Modern technologies have
been continuously in practice to make an additional constructive soil status for excellent
crop production and protection through controlling and manipulating the soil micro flora
by using the microbial inoculants, organic amendments and cultural & management
practices (Lynch et al., 1991).
1.1. Microbial inoculants / Biofertilizers
Microbial inoculants are also referred as biofertilizers or agricultural amendments
(Boraste et al., 2009; Laditi et al., 2012). These are beneficial substitute of chemical
fertilizers to enhance the crop yield by enhancing the availability of soil nutrients like
5
nitrogen, phosphorus, potassium, iron, providing growth-support factors like phyto-
hormones, fixing atmospheric nitrogen or solubilizing phosphorus, oxidizing sulfur,
decomposing (decay) and recycling of solid wastes or organic material (Kaewchai et al.,
2009; Pandya and Saraf, 2010; Saharan and Nehra, 2011). Biofertilizers are actually
mixture of potentially active live microbes (bacteria or fungi) which produced their
effects either directly or indirectly on plant development and crop productivity through
number of mechanisms (Ahemad and Khan, 2009; Pandya and Saraf, 2010).These
microbes are used to treat seeds and roots or applied in rhizosphere (soil adjacent to roots
of plant) as a result of which they enhanced the availability of nutrients due to their innate
activity which in turn improves the soil fertility and produce positive impacts on plant
health and agriculture (Malik et al., 2005). In addition, application of biofertilizers
decreases the environmental contamination (Mia and Shamsuddin, 2010). Studies
described that the use of chemical fertilizers and pesticides could be decreased by using
biofertilizers as these can maintain or restore the physical, chemical and biological
aspects of soil fertility and provide help in sustaining agriculture without harming the
environment (Mahdi et al., 2010; Ahmed et al., 2011). Hence, biofertilizers are
environmental-affable agro-input and cost effective than chemical fertilizers (Gupta et
al., 2003)
Mycorrhizae (arbuscular mycorrhizal fungi), one of the types of biofertilizers are
reported to recover soil fertility and crop yield (Hart and Trevors, 2005; Marin, 2006).
Microbial inoculants, beside being biofertilizer, also act as biocontrol agents and restrict
the occurrence of many plant pathogens in rhizosphere by showing their competitive
nature which protect plants from diseases in a potentially dominant unusual way
(Kulkarni et al., 2007). Microbes are remarkable source of biological actions due to their
vast diversity, complex interactions and various metabolic pathways (Alabouvette et al.,
2006; Pandya and Saraf, 2010; Saharan and Nehra, 2011). Microbial inoculants induce
disease protection, plant development and productivity due to their either free living
presence in rhizosphere or endophytic relation with plant tissues (Nihorimbere et al.,
2011). It has been reported that the use of living entities for biological control is an
6
effective non-chemical way to reduce the harmful effects of phyto-pathogens (Compant
et al., 2005).
Widespread environmental conditions alter the texture of soil by influencing its
three basic components (Buscot, 2005). The physical factors of environment like
moisture, aeration, reaction and temperature affect not only the plant health but also the
soil quality by altering microbial population in rhizosphere (Mishra, 1996). Therefore,
beneficial microorganisms contribute the stability of soil ecosystem. The greater the
diversity and number of microbial population, the higher is the order of their interaction
with plant roots and the more stable the ecosystem (Nihorimbere et al., 2011). The
application of microbial inoculants is basically an attempt to improve the microbiological
balance in agricultural soils to have better plant development, protection and production
by keeping the safety of human health and environment (Higa, 1991; 1994; Parr et al.,
1994).
Biofertilizers are reported to involve efficiently in carry out practices for
maintaining the sustainable agriculture (Pandya and Saraf, 2010). According to the
Agriculture Research Services of United State Department of Agriculture (USDA),
sustainable agriculture is an agriculture which will be productive, profitable, preserve
natural sources, protective for environment & human health and enhance the quality of
food in coming future (Higa and Parr, 1994). In this context, studies reported that
addition of microbial inoculants to enhance crop yield is economic which help to lower
chemical fertilizer doses and significant through which added nutrients can be harvested
from the soil (Somers et al., 2004). It has also been reported that microbial inoculants
may play an important role in low-input agricultural systems of developing countries
(Davison, 1988; Nihorimbere et al., 2011). Therefore farmers of low-income countries
must train, educate and adopt alternatives or modern agriculture technologies as
compared to traditional ones in order to have high yield from low agro-input. However
this objective could be achieved by having skilled authorities (Foster and Rosenzweig,
1995; Tilman et al., 2002; Yesuf and Bluffstoen, 2009).
7
Endophytic microbes both bacteria and fungi serve as both growth stimulators and
biocontrol agents (Sturz and Novak, 2000; Surette et al., 2003; Sessitsch et al., 2004;
Kaewchai et al., 2009). In this respect, many species of bacterial genera including
symbiotic nitrogen fixers such as Rhizobium, Bradyrhizobium and non-symbiotic
nitrogen fixers such as Alcaligenes, Arthrobacter, Azospirillum, Azotobacter, Bacillus,
Burkhoderia, Enterobacter, Klebsiella, Pseudomonas, Serratia, etc, are extensively
studied and reported as plant growth promoters and biocontrol agents (Hayat et al.,
2010). An ideal scientific term used for such helpful bacteria is plant-growth promoting
rhizobacteria (PGPR) because they perform number of roles in promoting plant growth
and yield (Gholami et al., 2009; Saharan and Nehra, 2011). Similarly many fungal
species belong to genera Aspergillus, Cheatomium, Trichoderma, Ectomycorrhizae
(ECM) and Arbuscular mycorrhizae (AM) are collectively reported as mycofungicides
and fungal biofertilizers (Kaewchai et al., 2009).
1.1.1. Rhizobia
Rhizobia (singular: Rhizobium) are strictly aerobic gram-negative rod shaped
bacteria belong to a family Rhizobiaceae, order Rhizobiales and class
alphaproteobacteria (Lee et al., 2005). Rhizobium species are reported to colonize the
plant roots and produce constructive effects both directly and indirectly which in turn
enhanced the growth of crop plants (Akhtar et al., 2012). After establishing themselves
within root nodules of legumes rhizobia fix atmospheric nitrogen or produce ammonia
from nitrogen and organic compound glutamine on one hand and on the other hand the
second party legumes provide organic compounds synthesized through photosynthesis to
rhizobia (Waters and Emerich, 2000; Simms and Taylor, 2002). In this connection both
partners form fruitful association with each other. Rhizobial species are reported to be
responsible for fixing the world largest portion of atmospheric nitrogen which
approximately constitutes 65% of the biosphere available nitrogen (Singh et al., 2010).
8
Species of different genera of rhizobiaceae including Rhizobium,
Mesorhizobium, Bradyrhizobium, Azorhizobium, Allorhizobium and Sinorhizobium
produce close symbiotic association with legume plants by attracting towards flavonoids
that secreted as chemotactic stimuli by the root cells of legumes and helped bacteria to
identify its specific host by attaching with root hairs. These flavonoids stimulate the nod
genes responsible for nodulation in rhizobia that generate lipo-chito-saccharide (LCO)
signals which activate cells to divide mitotically in roots, essential for formation of
nodules (Dakora, 1995; Lhuissier et al., 2001). Legume inoculation is a well-known
technique in agriculture which has been used for more than a century to establish
rhizobial species in soil to obtain its beneficial effect in crop growth promotion and yield
(Brockwell and Bottomley, 1995; Stephens and Rask, 2000; Deaker et al., 2004). The
most widely used way is the seed inoculation by these species alone and in combination
prior to sowing which can direct the establishment of huge rhizobial inhabitants in the
rhizosphere that enhanced nodule formation and nitrogen fixation (Hungria et al., 2003).
On the basis of mechanisms of actions to enhance growth of plants, PGPR are
termed as bioprotectants: by providing biological control against plant diseases,
biofertilzers: by improving nutrient and water uptake by plant, and biostimulants: by
producing phytohormones (Nihorimbere et al., 2011). Many rhizobacteria provide
biological control by different mechanisms that include inducing systemic resistance in
plant by activating certain genes, producing siderophores and toxic extracellular
compounds called antibiotics which are static or cidal in dilute concentration against
pathogenic microorganisms (Seuk Bae et al., 2000). Siderophores are meant to scavenge
heavy metals such as iron in rhizosphere which is one of the nutrients necessary for
pathogen growth thus inhibits its ability to attack crop (Miethke and Marahiel, 2007).
PGPR are well-reported to inhibit the growth of variety of soil-borne pathogens of
legumes and non-legumes such as Rhizoctonia & Fusarium species, sclerotium rolfsii,
Macrophomina phaseolina and Pythium spp. (Ehteshamul-Haque and Ghaffar, 1993;
Nautiyal, 1997; Estevez de Jensen et al., 2002; Baraka et al., 2009). Rhizobium species
are reported to activate plant defense mechanisms against pathogens by indirectly
9
stimulating the synthesis of plant defense compounds like phenols, flavonoids or phyto-
alexins (Ramamoorthy et al., 2001). The PGPR are also reported to oxidize soil elemental
sulfur into sulphate and rock phosphorus to make them easily available for plants
(Salimpour et al., 2010). Studies described that many species of endophytic rhizobium
and bradyrhizobium are capable to solubilize inorganic phosphate (Halder and
Chakrabartty, 1993).
An asymbiotic association with roots of non-legume plants without producing
nodules is another potential of rhizobium and bradyrhizobium (Dawar et al., 2008;
Antoun et al., 1998; Mia et al., 2012). Studies proved that inoculation of rhizobia
enhanced the productivity of many non-legume crops including rice, cereals, etc by
making associative communications with roots of these plants (Hoflich et al., 1994;
Yanni et al., 1997). The rhizobial species are reported to produce direct changes in plant
growth of non-legumes by different ways such as i. by synthesizing various metabolites
such as phytohormones (auxins, cytokynins, gibberellins, abscisic acid, indolacetic acid,
etc), riboflavin and other vitamins, ii. by inhibiting ethylene production in plants, iii. by
enhancing nutrient uptake, iv. by solubilizing the unavailable phosphate or mineralization
of organic phosphate, v. by inducing better stress resistance against plant pathogens, etc
(Biswas et al., 2000a,b; Vessey, 2003; Hafeez et al., 2004; Matiru and Dakora, 2005;
Chandra et al., 2007; Humphary et al., 2007; Pena and Reyes, 2007). Similarly, reduction
in many plant diseases like sheath blight disease of rice produced by Rhizoctonia solani
was observed through rhizobium-mediated production of phenolic compounds generally
gallic, ferulic, tannic and cinnamic acids (Mishra et al., 2006). An innate beneficial
endophytic association between rhizobia and roots help bacteria in root colonization so
many strains of rhizobium and bradyrhizobium could easily colonize and survive in the
rhizosphere of non-legume plants as PGPR and also localized themselves inside the
tissues like xylum (Kloepper and Beauchamp, 1992; Wiehe and Höflich, 1995). Due to
their endophtic nature rhizobial strain have also been isolated from the roots of sweet
corn, cotton (McInroy and Kloepper, 1995), maize (Antoun et al.,1998), wheat
10
(Biederveck et al., 2000), canola (Lupwayi et al., 2000) and other non-legumes species.
Therefore Rhizobia are greatly significant in sustainable agriculture and provide an
alternative way to substitute inorganic fertilizer, pesticide and supplement by inducing
significant increase in overall growth parameters of crop plants and their yield.
1.1.2 . Fungi
Fungi are frequent soil inhabitants beside bacteria, actinomycetes and protozoa,
some are beneficial for plant growth and others are destructive or pathogenic (Anderson
and Caimey, 2004). According to biotechnologists, fungi play important roles in
agriculture, including
i. Act as biocontrol agent: to provide biological control for various plant pathogenic
fungi, insects, nematodes, etc (Burge, 1988; Gillespie, 1988).
ii. Act as biofertilizer or inoculants: to improve crop growth and yield by applying
beneficial fungi in rhizosphere of crop plants (Whips and Lumsden, 1990).
Fungi have many properties for making them active biocontrol agents and
replacement of chemical pesticides like, i. many saprophytic fungal species inhibit the
growth of all pathogenic microorganisms including plant-pathogenic fungi, weeds,
insects, etc., ii. Fungal species can easily grown in culture in order to have large
quantities of spores or mycelia fragments of a particular species that can be used for
inoculation in rhizosphere and these are economically sound. These inoculants then
develop into active mycelium and attack the pathogen with out producing harmful effect
on non-target microorganisms. iii. Fungi can stay alive for long time as resting bodies
which later germinate, grow and kill target population thereby minimizing repeated
inoculation (Wainwright, 1992).
Use of fungi as biofertilizers is another potential impact of beneficial fungi in
agriculture (Kaewchai et al, 2009). The intention behind this approach is the inoculation
of fungal species in soil where they involve in cycling of plant nutrients and minerals to
make them more available for plants, thus improves the crop growth and yield (El-
Azouni, 2008). Many fungal species are reported to increase soil fertility and crop
11
production such as mycorrhizal fungi produce mutualistic relation with roots of 80 - 90 %
plants and other fungi which used as biofertilizers including species of Aspergilllus,
Chaetomium, Penicillium, and Trichoderma are well-reported (Pandya and Saraf, 2010).
The concept of using fungal species as mycofungicides and biofertilizers is not
new though it has been practiced recently. There are number of reports that describe the
importance of fungi to restrict plant diseases and stimulate growth of plants as
biofertilizers which helped to decrease the use of inorganic fungicides and fertilizers
(Hyakumachi & Kubota, 2004). In coming future, the use of biofungicides and
biofertilizers would be increased to produce safe and organic food in order to fulfill the
demand of growing population of the world and to maintain the safety of environment,
human health and the whole ecosystem.
1.1.2.1. Trichoderma species
The genus Trichoderma consists of filamentous, non-pathogenic, saprophytic
fungi and its species including T.viride, T.harzianum, T.hamatum, etc, are common
resident of soil especially rhizosphere of many plants (Harman et al., 2004; Thormann
and Rice, 2007; Kodsueb et al.,2008). These can frequently isolate from soil, decaying
wood and other crude materials (Howell, 2003). Trichoderma species are well-reported
for not only to restrict the growth of root-infecting pathogens and prohibit the occurrence
of plant diseases by acting as mycofungicide but also for promoting the growth of plants
and yield thereby acting as biofertilizer (Kaewchai et al, 2009). These species are known
to control many plant pathogenic fungi including Rhizoctonia, Pythium and Fusarium
species, Botrytis cinerea, Phytophthora palmivora, P. parasitica, etc, by number of
mechanisms, the most important include antibiosis and mycoparasitism (Tang et al.,
2001; Howell, 2003; Harman et al., 2004; Benítez et al., 2004; Harman, 2006; Vinale et
al., 2008). The mycoparasitism induced by these species is depends on detection,
attachment and lyses of host fungal cell wall due to the production of enzymes (Woo and
Lorito, 2007). In addition, these have variety of excellent abilities to control the
pathogens as these are fast growing, efficient in utilizing soil nutrients and make
12
pathogen starving, can stay alive in unpleasant environments or resist abiotic conditions
and show resistance towards many fungicides (Khan and Shahzad, 2007; Mastouri et al.,
2010).
Trichoderma species are included in group of plant growth promoting fungi
(PGPF) due to their abilities to improve photosynthetic rate in plants and their growth
(Harman & Shoresh, 2007; Kaewchai et al, 2009; Cumagun, 2012). The plant growth
promotion in the presence of Trichoderma inoculants are reported due to the
improvement in mineral uptake, decomposing organic matter, production of plant
hormones, enzymes and antibiotics (Daniel and Filho, 2007; Harman et al., 2004; Reino
et al., 2008) and modulation of proteome and transcriptome in plants (Marra et al., 2006;
Alfano et al., 2007; Shoresh and Harman, 2008). The Trichoderma species were found
equally effective in stimulating the growth of both legume and non-legume plants like
rhizosphere competent and endophytic strains of this genus involved in growth
stimulation of Phaseolus vulgaris (bean) (Hoyos-Carvajal et al., 2009) and mustard
plants (Haque et al., 2010).Similarly T. viride induced growth promotion in cotton plants
(Shanmugaiah et al., 2009). Hence, many Trichoderma species are available
commercially as fungicides, biofertilizers and soil amendments and are successfully used
not only in greenhouse but also in field experiments (Kaewchai et al, 2009; Badar and
Qureshi, 2012).
1.1.2.1.1. Trichoderma hamatum
T. hamatum is one of the commonly found species of genus Trichoderma and its
occurence has been reported in forest, cultivated and heathland soils of many countries
including Pakistan (Domsch et al., 1980; Ha, 2010; Mulaw et al., 2010). The same
fungus was also found on roots of pines, rhizosphere of wheat, lettuce, rotting wood, cast
of some insects like earthworm and sewage sludge (Domsch et al., 1980; Mishra, 1996).
T. hamatum, like other Trichoderma species, acts as a biocontrol agent for many
plant pathogenic fungi particularly by showing mycellial coiling (Papavizas, 1985;
Howell, 2003). It has been proved by study which reported that T. hamatum and T.
13
harzianum produce lytic enzymes including chitinases and glucanases that attack the
hyphae and sclerotia of rice sheath blight pathogen (Harman et al., 1981). Antifungal
activity of same biocontrol agants was also reported due to the production of volatile and
non-volatile metabolites that constitutes the mechanism called antibiosis (Dennis and
Webster, 1971 ab). In vitro inhibition of the growth of candida albicans (Marchisio,
1972), Heterobasidion annosum (Gibbs, 1967) and Lententinus edodes (Komatsu, 1976)
was also reported by T.hamatum. Study proved that T. hamatum and other Trichoderma
species not only inhibited the growth of R. solani from 60-78 % in vitro but also soil
treatment with T. hamatum, T. harzianum and T. viride provided highest protection
against damping off disease and improvement in plant heights, fresh & dry weights and
dry seed yield of bean plants (El-Kafrawy, 2002). The study showed that T.hamatum was
effective with other Trichoderma species in controlling of powdery mildew of cluster
beans where culture filtrates of T.hamatum, T.harzianum and T.viride were found
significant and induced 76% increase in yeild of same beans by improving the plant
heights, number of leaves and flowers besides controlling disease upto 78-79% (Deore et
al., 2004).
A study described the antagonistic effects of Trichoderma species against
R.solani and Fusarium solani, causative agents of damping off disease of Phaseolus
vulgaris L., of which T. hamatum gave the highest protection against the disease among
other species of same genus beside giving maximum plant survival and improvement in
growth & yield parameters (Abd-El-Khsir et al., 2010). Similarly, an in vitro study
described that T.hamatum and T.longibrachiatum were effective in inhibiting the growth
of F.verticillioides, isolated from decaying maize stem (Sobowale et al., 2010).
T.hamatum alongwith T.virde and Rhizobium meliloti alone and in combination by using
seed dressing and soil drench methods found effective in reducing infection caused by
Macrophomina phaseolina on fenugreek seedlings on its 30th
day of germination
(Ehteshamul-Haque and Ghaffar, 1992). However, only 7% inhibition of root-rot disease
caused by M.phaseolina on Eggplant was observed by T. hamatum (Ramezani, 2008).
T.hamatum and Trichoderma species were known to control infections caused by
14
F.oxysporum, Pythium ultimum and Sclerotina sclerotium (Harman et al., 1980;
Papavizas, 1985; Manezinger et al., 2002; Sharma and Trivedi, 2010). T. hamatum strain
382 was found to induce systemic resistance against bacterial leaf spot of radish (Han et
al., 2000) and tomato by altering the expression of genes involved in stress and protein
metabolism (Alfano et al., 2007). The same strain 382 of this biocontrol agent was also
observed effective in reducing the risk of Botrytis Blight of Begonia plants in green
house experiments (Horst et al., 2005). Therefore, T.hamatum not only serves as
biocontrol agent but also plant growth promoting agent.
1.2. Organic fertilizers
Naturally occurring organic fertilizers (both untreated and composted) including
animal and plant manures, fall residues, seaweeds, humic acid, food and urban wastes
like industrial wastes, etc are better replacement of synthetic or inorganic fertilizers
(Niemi et al., 2008; Klaus et al., 2008). Traditionally for crop production, animal manure
has always been considered as a significant contribution to the soil fertility (Karmakar et
al., 2007). However, the suitable use of animal manure for enhancing soil quality, crop
nutrition and farm profits depends on farm’s facility and goals that actually related to the
manure management (Nowak et al., 1998). Manure management defines as an
administrative goal to increases the agricultural production with minimum nutrient losses
from manure, not only today but also in coming future (Brandjes et al., 1996). It has been
reported that adding too much nitrogen fertilizer to sunflower not only produces
environmental risk but it may also affect the quality of grain by reducing its oil content
and yield (Steer and Seiler, 1990). Integration of organic manures with inorganic
fertilizers has been traditionally important input in crop yield for the security of soil
fertility and production stability (Xin et al., 2005; Sabiiti, 2011). Therefore, integrated
use of fertilizer, organic manures and biological sources help in maintaining sustainable
agriculture by correcting the deficiencies of macro- and micro nutrients and providing
favorable soil physical condition (Kaur et al., 2005). Interestingly, harmful effects of
different contaminations induced by air, soil and ground water can be positively decrease
15
due to the organic farming (Mokolabate and Hoynes, 2002). Reports proved that organic
farmaing improves soil structure, fertility and soil fauna which eventually affect the crop
production in a constructive manner (Ghosh et al., 2003).
Currently two types of organic fertilizers have been used in agriculture, first is the
synthetic type named urea it is organic compound formed artificially and second is
organic fertilizers that 100% come from nature such as animal & plant manures including
guano, food & industrial wastes, seaweed, sewage sludge and compost materials (Helsel,
1987). Many researchers have well-reported that organic wastes and residues rich in
native inhabitants of microorganisms are actively participate in many biological activities
like to suppress the soil-borne plant pathogens and act as biocontrol agents (Parr et al.,
1994). It has also been reported that variety of off-farm sources of organic waste like
industrial processing wastes can maintain or re-establish the fertility and productivity of
agriculture soils which have exposed to wind and water erosion, nutrient reduction and
loss of organic matter (Espiritu, 2011). Municipal waste water has been in use for tree
plantation or for making lush green areas in industrial compounds and amusement parks
from many years and it has been reported as a good source of organic matter and trace
elements, however the presence of heavy metal in it may affect the ecosystem (Gupta et
al., 1998; Al-Jamal et al., 2000; Sharma et al., 2007; Singh and Agrawal, 2010). Blood
meal is a rich source of nitrogen and phosphorus and organic manures like farmyard,
chicken manure, blood meal and their combination produced positive effects on gowth
and yield of Brassica oleracea var. capitata as compared to inorganic fertilizer (Citak
and Sonmez, 2010).
In spite of many advantages, organic fertilizers have certain disadvantages such
as organic wastes contain small amount of plant nutrients and their large quantity is
required as compared to inorganic ones which also increases the labor and money
expenses, in addition improperly treated organic fertilizers may serve as carrier of
pathogens from plant and animal sources that could harm human and plants, so proper
composting should be required (Westerman and Bicudo, 2005).
16
1.3. Composting
Composting is a technique of recycling the organic waste matters in more
digestible form with improved nutrient and mineral content by using micoorganisms
including fungi and bacteria under specificed conditions of temperature and aeration that
could be used as compost or organic fertilizer or soil amendment or soil conditioner
which generally used to improve soil fertility and helps to ameliorates the crop grwoth
and production (Panda and Hota, 2007). Waste materials of animal and plant origin
including fallen leaves, weeds, straw, water hyacinth, domestic wastes comes from food,
fruit and vegetable, urban trash, saw dust, rice husk, sugar cane bagasse, wastes from
leather factory etc. are normally used to create compost (Inckel et al., 2005). Few wastes
derived from municipal and leather must be refined to make them clear from heavy
metals and other injurious substances (Igwe and Abia, 2006; Akinola et al., 2011).
Globally, there are two types of composting procedures used depends on the
nature of decomposition of organic wastes including anaerobic and aerobic. An anaerobic
composting involves the decomposition of organic wastes with help of anaerobic bacteria
at low temperature and in the absence of oxygen which require more time than aerobic
one results in the formation of compounds with unpleasent odour like methane, hydrogen
sulphide, etc which are not further metabolized and produced toxicity to plants (Voca et
al., 2005). Where as aerobic composting involves the decomposition of organic wastes
with the help of aerobic bacteria at high temperature in the presence of sufficient oxygen,
require less time to produce fully metabolized compounds including humus, carbon
dioxide (CO2), ammonia, heat, water. Heat helps to convert complex protein, fats,
carbohydrate into amino acids, fatty acids, cellulase and hemi-cellulose (Panda and Hota,
2007). Therefore, composting is an easy technique that integrates nutrient-rich humus in
soil which fuels plant development and restores the vigor of uncultivated soil.
Compost is a key component of organic farming and an accelerator of sustainable
agriculture and addition of appropriate effective microbes into composts can reduce the
peroid of its maturity and improves its quality (Panda and Hota, 2007). Greater than or
equals to 80 naturally occuring microorganisms like phosphate solubilizing bacteria,
17
yeast, cellulytic bacteria, actinomycetes, fungi (Aspergilus oryzea), etc are well-
renowned to improve soil fertility and crop yield, these are collectively documented as
effective microorganisms (Ndona et al.,2011; Higa, 2004) and have been frequently used
in the composting of organic waste materials (Yamada and Xu, 2000). Similarly,
cellulytic fungi like Trichoderma species may also play good role in decomposition of
organic wastes during the process of composting. Studies reported that phosphate and
nitrogen concentrations in soil are improved by adding livestock and mushroom wastes
which were composted with phosphate-solubilizing Klebsiella pneumoniae subsp. and
nitrogen-fixing Azospirillum brasilense that promote the growth of many vegetables like
kale, cabbage and corn (Bashan et al., 2004; Hayat et al., 2010). Similarly,
decomposition of rice straw-chicken manure mixture accelerated when inoculated with
Trichoderma harzianum SS33 and Azotobacter sp. H1BFA4b enhanced the process of
nitrogen fixation to improve the nitrogen amount, so Trichoderma-Azotobacter together
stimulated the decomposting and nitrogen fixation rates, hence the biofertilizer made with
these inoculum increased the soil contents of nitrogen, phosphate, potassium and organic
matter which in turn increased the yield of rice, mungbean and pechay (Espiritu and dela
Torre, 2001; Espiritu, 2011).
In different parts of the world along with chemical fertilizer, soil amendments
with organic manure, compost, and composted tea residues are frequently used to
improve crop productivity and yield (Adesemoye and Kloepper 2009). Organic
amendment of soil with rice husk, the natural sheath or productive cover of rice grains,
was found effective in yield of many crops like cowpea, rice, etc (Anonymous, 1979;
Aliyu et al., 2011) and decreased 31-70% occurrence of wilting caused by Fusarium
solani in Parkia biglobosa (Muhammad et al., 2001). Rice husk under different irrigation
intervals can give good rice stand, better grain yield and higher water use efficiency
(Ebaid and El-Refaee, 2007). Soil conditioners based on food waste composted with
suitable microorganism were found effective as same as inorganic fertilizer in promoting
the growth of melon and maize (Means et al., 2005; Ahmad et al., 2006). Wheat bran
another food based waste composted with T.harzianum found effective in reducing the
18
risk of dumping off disease caused by Phythium aphanidermatum in tomato, pea,
cucumber, etc (Sivan et al., 1984).
Therefore, organic waste materials of different sources including animal, plant
and human composted with effective microorganisms used as an excellent biocontrol
agent or soil conditioner or soil amendment or organic fertilizer to improve plant growth
either by protecting from plant diseases or enhancing the availability of nutrient in soil or
improving the physical properties of soil or combination of all these three.
1.4. Economical crops of Pakistan
1.4.1. Helianthus annus L.
Helianthus annuus L. (sunflower) belongs to the family Compositae and one of
most important oilseed plants in the world and ranked second than soybean (Weiss, 2000;
Kaya and Kolsarici, 2011). Its oil generally used for preparation of food, margarine and
biodiesel (Balat and Balat, 2008; De Marco et al., 2007). A number of sunflower varieties
available and their oils are differ in fatty acid composition, some of which contain high
content of oleic acid (a monounsaturated fatty acid) which is significant for health, than
olive oil,sunflower oil is also cheaper than olive oil (Abramovic and Klofutar, 1998). The
sunflower is a biannual crop which give yield in spring and autum, having a big
flowering head (inflorescence) that grows to height from 1.5 to 3.5 meters and
contributes approximately 14% of total world seed oil production (Kaleem and Hassan,
2010). During the last 3 decades, its cultivation is gradually increases globally and
interestingly the climatic conditions of our country are friendly compactable to the
growth of these oil producing plants (Badar and Qureshi, 2012). Therefore, Pakistan
Oilseeds Development Board (PODB) has decided to reserve 80% more area for
sunflower cultivation than its existing under cultivated area which was about 1.2 million
acres that has expected to increase over 1.5 million acres (Imran et al., 2011).
Sunbutter is an alternative of peanut butter processed from sunflower seeds and
after processing seeds for oil, the surplus cake is used as cattle feed (Gibb et al., 2004;
Lima and Guraya, 2005). Sunflower plant like all other angiosperms also produced latex,
19
a type of rubber used for manufacturing gloves, clothes, etc (Pearsona et al., 2010).
Sunflower is also a biotechnologically important plant as it is involved in
phytoremediation and used to extract toxic ingredients such as lead, arsenic and uranium
from soil (Jadia and Fulekar, 2009; Mudgal et al., 2010). Nutritionally, the quality of
crude protein in sunflower silage is superior than corn plus sunflower food contains high
fiber content with small amount of lysine amino acid but higher in methionine than
soybean food (Leite et al., 2002). Studies reported that non-dehulled seeds of sunflower
food contain 28 % protein as compared to dehulled seeds that contains 42% protein
(Teangpook et al., 2011). Generally, crude protein content of sunflower decreases and
lignin content increases after the reproductive stage (Myers and Minor, 1993).
Sunflower oil is commercially used in manufacturing of soaps, detergents,
surfactants, adhesives, plastics, fabric softeners, lubricants and also used as a pesticide
carrier (Erhan, 2005). It is also reported to use in certain paints and varnishes without
color modification because of fine semidrying properties which is associated with the
presence of linolenic acid, another unsaturated fatty acid present in sunflower oil (Erhan,
2005).
1.4.2. Brassica nigra L.
Brassica nigra L. (black mustard) belongs to the family Brassicaceae, is a fast
growing annual herb, in favorable conditions of moisture and temperature, this plants
cover the farm within 4 to 5 weeks and at maturity plant height varies from 30 to 45 inch
depending on nature, variety and environmental conditions while in dry condition, the tap
roots will grow 5 ft into the soil for water absorption (Shekhawat, 2012). Mustard is one
of the economical crops of winter season (Oplinger et al., 1991). After salt & pepper, the
third most important spice is mustard (Downey, 2003). Many species of mustard have
been reported including Brassica juncea (Indian mustard), B. rapa var. yellow sarson
(yellow mustard), B. campestris (brown sarson), B. nigra (black mustard), B. carinata
(karan rai), B. napus (gobhi sarson), B. synapis or Synapis hirta (white mustard), etc so
far (Shekhawat, 2012). The oil of black mustard is used for cooking food in India (Piri,
20
2012). Medicinally its ground seeds are used with honey to relief cough and respiratory
infections while also uses as appetizer, digestive, diuretic, emetic, etc (Hassan, 2006).
The plant residues mainly consist of leaves, stem, pods and husk were reported to use as
feed of livestock, this legume also used as cover crop and green manure and serve as
source of biodiesel (Walt and Breyer-Brandwijk, 1962; Boydston and Al-Khatib, 2005
Bannikov, 2011).
1.4.3. Cicer arietinum L.
Cicer arietinum L. (chickpea or white chana) belongs to the family Fabaceae and
subfamily Faboideae (Al-Mekhlafi et al., 2012). In ancient time, it was the first grain
cultivated and most important legume crop for human being diet (Karasu et al., 2009).
The International Crops Research Institute (ICAI) reported the calculated amount of
different components of chickpea seeds like protein (23%), total carbohydrates (64%)
which chiefly consist of starch (47%) and soluble sugar (6%), fat (5%), crude fiber (6%)
and ash (3%) (Hassan and Khan, 2007). It also has high mineral content such as
phosphorus (340 mg), calcium (190 mg), magnesium (140 mg), iron (7 mg), zinc (3 mg)
per 100 gram of seeds (Daur et al., 2008). In addition chickpeas are low in fat content,
the majority is polyunsaturated which are beneficial for health (Pittaway et al., 2007,
2008). Its varieties include Desi and Kabuli chickpea (Maheri-Sis et al., 2008).
Among pulse production in agriculture, chickpea is the chief winter legume crop
in Pakistan and occupies 73% of area reserved for total pulses while provide more than
75% contribution in total pulse production (Rani et al., 2012). In Pakistan and worldwide
there are number of factors including planting without schedule, inadequate seed price,
too much planting depth, non-uniform seed distribution, unsatisfactory weed control,
inadequate fertilizer, drought, wilt or moisture stress and most important fungal disease
called Ascochyta blight (causative fungus: Ascochyta rabiei) affect the production of
chickpea (Akbar et al., 2011). Several methods adopted globally to increase the yield of
chickpea are, i. use of appropriate sowing method, ii. use of disease resistant varieties, iii.
proper use of nitrogen and phosphorus fertilizers, iv. rhizobial inoculation with
21
fungicide, but it has been recommended to have best result apply fungicide first, dried
and then apply rhizobia (Reddy et al., 2003). Similarly chickpea respond positive when
grown in soils that contain its native Rhizobium species (Sharma et al., 1983). Therefore
rhizobial inoculation normally increased plant growth, yield and nitrogen fixation in
chickpea (Fatima et al., 2008; Aslam et al., 2010).
1.4.4. Vigna mungo L. Hepper
Vigna mungo L. (blackgram) belongs to the family Fabaceae and subfamily
Faboideae, and a member of the Asian Vigna crop group (Ali and Nasir, 1970). It is an
annual pulse crop native to central Asia and staple food of people living in central and
South-East Asia (Delic et al., 2009). It is a short period (90-120 days) and summer season
pulse crop with high nutritive value (El Karamany, 2006). It can digest easily and prevent
flatulence effect (Fery, 2002). Nutritionally, seeds of V.mungo contain crude protein (24-
26%) rich in essential amino acids, crude lipid content (3-4%) that chiefly contains
linoleic and linolenic acids (unsaturated fatty acids), total fiber (4-5%), ash (3%),
carbohydrates (61-64%) which contains Raffinose as the principle oligosaccharide (Soris
et al., 2010; Selvakumar et al., 2012; Shaheen et al., 2012). The seeds also contain
minerals such as Na, K, Mg and P (Suneja et al., 2011) and it is an excellent source of
plant protein that chiefly contain albumin and globulin (Imrie, 2005; Kulsum et al.,
2007).
Blackgram is utilized for many purposes like for human food (vegetable diets),
green manure, a cover crop, forage, silage, hay and chicken pasture (Delic et al., 2009).
Studies reported that blackgram potential used for dual purposes like first as an early
season forage production and later seed production for human consumption (Imrie, 2005;
El Karamany, 2006). Blackgram is sown on most soil but it can grow on heavier soils
having pH 5.5-7.5 (Delic et al., 2009). Seed inoculations with Bradyrhizobium bacteria
earlier to sowing allow a decline in nitrogen mineral fertilization, susceptibility to
environmental stress and production cost (Hussain et al., 2011).
22
1.5. Aim of the present study
By considering the significance of microbial inoculants or biofertilizers and
composted organic fertilizers in promoting plant growth and yield of legume and non-
legume crops, the prersent research work has been designed for conducting the following,
as
1. Isolation of rhizobia from root nodules of different legume plants.
2. Isolation of Trichoderma hamatum from rhizoplane of host plant.
3. Characterization of rhizobial isolates on the basis of cultural, morphological and
biochemical characteristics.
4. Determination of nodulation ability of rhizobial isolates on their respective hosts.
5. To determine in vitro antifungal activity of rhizobial isolates and T.hamatum against
root-infecting fungi to prove their biocontrol potential.
6. To investigate the effect of T. hamatum and rhizobial isolates alone and in combination
on physical (root & shoot length and fresh weight of plants) and biochemical
(chlorophyll, total carbohydrate, crude protein, nitrogen & phosphorus) parameters of
two each of non-legumes viz., Halianthus annuus (sunflower), Brassica nigra (black
mustard) and legumes viz., Vigna mungo (mashbean), Cicer arietinum (chickpea) plants.
7. Preparation of composted rice husk and wheat bran by using T. hamatum and selected
rhizobial isolates in second phase of study.
8. Determination of total carbohydrate and protein contents in uncomposted and
composted rice husk and wheat bran.
9. To investigate the effect of composted rice husk and wheat bran @ 5 & 10 g/ 2 kg
soil/ pot on physical and biochemical parameters of one each of non-legume (sunflower)
and legume (chickpea) plants.
23
2. Material and Methods
2.1. Collection of root samples
The root samples of plants covered with thin rim of rhizopheric soil were
collected from localities of Karachi and Malir district by carefully dugging out kept in
polyethylene bags and brought to laboratory to store them in refrigerator. Fungi and
rhizobia were isolated within 24 hours of collection.
2.2. Isolation and identification of fungi from rhizoplane
Collected root samples of plants were used to isolate fungi including test fungal
pathogens and test fungus from rhizoplane by using standard method (Aneja, 1993). In
which roots were washed in running tap water, 1cm long root pieces were cut from roots
(both tap and lateral) and washed in sterilized distilled water which were then transferred
on plate containing potato dextrose agar (PDA) that also contained anitibiotics viz.,
penicillin (100,000 unit/liter) and streptomycin (0.2g/liter) to inhibit the growth of gram-
positive and gram negative bacteria. Petri plates were kept at 28C for 5 days and finally
grown fungi were identified by expert of Botany Department, University of Karachi,
Karachi, Pakistan with taking reference to manuals of different genera of fungi (Barnett
& Hunter, 1998). Identified test fugal pathogens and test fungus were separated, isolated
pure, coded and preserved on PDA slants for further use.
2.3. Isolation of rhizobial isolates from root nodule
Collected root samples of legume plants were used to isolate rhizobial cultures by
crushed-nodule method (Aneja, 1993). In which, roots were washed in running tap water
to remove adhering rhizospheric soil particles. This helped to select healthy pink,
unbroken and firm nodules. Washed the selected and detached nodules first with
sterilized distilled water and then placed in HgCl2 (0.1%) for 5 minutes for surface
24
sterilization or to remove contamination. Nodules were washed with sterilized distilled
water thrice to remove the effect of sterilizing agent. Dipped the nodules in ethyl alcohol
(70%) for 3 minutes and washed them again with sterilized distilled water. Nodules were
crushed in sterilized distilled water (1 ml) to make uniform suspension of rhizobia that
referred as nodule extract and considered as 1:10 dilution. Serial dilutions of nodule
extract were made from 1:10 to 1:10,000. Spread 0.5 ml of each of the last two highest
dilutions on yeast extract manitol agar (YEMA) plates having 2.5 ml of Congo red (1.0
%) per medium (L) and kept at 28C for 8 -10 days. Large white gummy colonies of
rhizobia were appeared within 3 -7 days. The Rhizobial isolates were picked and
transferred to YEMA plates. The transfering of rhizobia to freshly prepared YEMA
plates has been practiced for 3 to 4 times to obtain pure culture. The rhizobial isolates
were coded and stored on YEMA slants at 4 to 8C. These isolates were further subjected
for characterization and nodulation test to determine their purity and host specificity
respectively.
2.4. Characterization of rhizobial isolates
The study of rhizobial isolates was based on their morphological, cultural and
biochemical characteristics and done by using standard methods (Aneja, 1996; Vincent,
1970). After the confirmation of rhizobium genus, the isolates were maintained on
YEMA slants.
2.4.1. Morphological and cultural characteristics
Morphological characteristics including the size, shape, motility and Gram-stain
reaction of rhizobial isolates were observed under microscope. Whereas colony
characteristics including the configuration, margin, elevation and colour of the colonies
of test rhizobial isolates were observed on standard YEMA containing petri plates.
25
2.4.1.1. Hanging drop technique to test motility of rhizobial isolates
Put a drop of rhizobial suspension on a cover slip of the hanging-drop slide. The
cavity of same slide was upturned on cover slip by making sure that the drop of bacterial
suspension was in its core. Lift the hanging-drop slide gently to face the cavity upward in
such a way that the drop can easily suspended in the cavity. The motility of rhizobium
was examined under microscope by using low power objective with dim light (Aneja,
1996).
2.4.1.2. Gram staining
Gram staining is not only helpful to ensure the purity of rhizobial isolates but also
dividing them into gram-positive and gram-negative groups by observing the morphology
including size, shape, color and arrangement of rhizobial cells. A thin smear of rhizobial
isolate was prepared on a glass slide and air-dried. Slightly heat the slide on a small flame
to fix smear and stained it first through crystal violet (primary dye) for less than 1 minute
and washed with distilled water by using squeezer. Blotted dry and flooded the smear
with iodine solution (secondary dye) for 30 seconds, washed with distilled water and
blotted dry again. After air-dried, rinse the smear with 95% ethanol (decolourizer) drop
by drop until no color was leaked out. After washing with distilled water, the air-dried
and decolorized rhizobial smear was then stained with safranin solution for 20 seconds,
washed, dried and observed with the help of microscope. Gram-negative bacteria become
pink or red by losing the color of primary dye with help of decolourizer and retained the
color of secondary dye where as gram-positive appeared in dark purple color by retaining
the color of primary dye (Aneja, 1996).
2.4.1.3. Differentiation of rhizobial isolates by using YEMA
supplemented with bromothymol blue
The YEMA medium supplemented with bromothymol blue (pH 6.8) was not only
helped to identify fast and slow growing bacterial isolates but also used to make
26
difference in acid and alkali producers such as rhizobium and bradyrhizobium
respectively. Each rhizobial isolate was streaked on petri plates containing YEMA with
0.5% bromothymol blue (5ml/ litre) and incubated at 28oC, finally observations were
recorded (Noris, 1965; Talukder et al., 2008).
2.4.2. Biochemical characteristics
Biochemical characteristics of the rhizobial isolates were studied by conducting
different tests like indole production, methyl red & Voge’s Proskauer, gelatin
liquefaction, starch hydrolysis, nitrate reduction , etc.
2.4.2.1. Indole production test
Production of indole ring was observed in term of deep red color appeared in the
top layer of trptophane broth that was inoculated by test bacterium, incubated for 5 -7
days at 28C when Kovac’s reagent was added. Tryptophane, an essential amino acid, is
oxidized by rhizobial isolates due to the production of tryptophanase enzyme which
results in formation of indole ring (Aneja, 1996).
2.4.2.2. Methyl red and Voge’s Proskauer (MRVP) tests
Glucose phosphate or (methyl red and Voge’s Proskauer; MRVP) broth
inoculated with test bacterium and incubated for 48 - 96 hour. After incubation period, 4 -
5 drops of methyl red (pH indicator) was added and change in color observed. The broth
remained red indicated that test was positive and the test bacterium was acid producer
where as it turned yellow indicated test was negative (Aneja, 1996).
The same MRVP broth was inoculated with test bacterium and incubated for same
period, then it was supplemented with few drops of VP reagent I (α-naphthol solution)
and II (40% potassium hydroxide). The change in color was observed, no change
occurred indicated the test was negative or crimson to ruby-pink color appeared indicated
the test was positive (Aneja, 1996).
27
2.4.2.3. Gelatin liquefaction test
Hydrolysis of gelatin takes place in the presence of gelatinase enzyme produced
by test bacterium. Test bacterium was inoculated on agar slant containing freshly
prepared gelatin (10%) and incubated at 28C for 7 days. After incubation, placed the
tubes in refrigerator at 4C for 15 - 30 minutes then examined. The refrigerated gelatin
tubes appared as melted medium indicates positive test and solidified medium reflects
negative test (Dickey and Kelman, 1988).
2.4.2.4. Starch hydrolysis test
This test was used to determine the ability of test bacterium to hydrolysis the
starch due to the production of amylase enzyme. It was done by streaking the test
bacterium in the center of petri plates containing solidified starch agar medium. After
incubation of 2 - 5 days at 28C, plates were flooded with iodine solution that used as an
indicator for 30 seconds. Discarded the excess iodine solution and observed the clear
zone around the bacterial growth which indicated starch hydrolysis (Aneja, 1996).
2.4.2.5. Hydrogen sulphide formation test
Hydrogen sulphide formation test was performed on slunts containing SIM
(Sulphide Indole Motality) agar. These SIM slunts were stab inoculated with test
bacterium, incubated for 48 - 96 hours and examined the slunt for the appearence of black
color along the line of stab inoculation which reflected test was positive. It was due to the
liberation of H2S from the reduction of sodium thiosulphate, one of the ingredients of
medium by test bacterium. The librated gas then reacted with ferrous ammonium
sulphate, another ingredient present in medium that resulted in the formation of insoluble
black colored precipitates of ferrous sulphide (Aneja, 1996).
28
2.4.2.6. Nitrate reduction test
It was done to asses the ability of test bacterium to convert nitrate into nitrite or to
other nitrogen containing compunds including ammunia, nitric oxide, etc. The test
bacterium was inoculated in nitrate reduction broth that contained large amount of KNO3
and incubated at 28C for 3 - 5 days. After incubation, alpha-naphtylamine and sulfanilic
acid were added, nitrite was in the medium, then it reacted with both the added
compounds and turned the medium red in color that indicated the positive nitrate
reduction test. However no change in color was observed then small amount of zinc
added and again no change observed which confirmed the absence of nitrate indicated
that bacterium is capable to reduce nitrate to ammonia, nitic oxide, etc means positive
nitrate reduction test (Graham and Parker, 1964; Smibert and Krieg, 1981).
2.4.2.7. Oxidase test
It was conducted to investigate the production of enzyme oxidase by test
bacterium. Kovac’s reagent was kpet in brown bottle by dissolving N, N, N, N-tetra
methyl-p-phenylene diamine (1%) in lukewarm water. A filter paper strip was dipped in
Kovac’s reagent and air-dried. Transfered 3-4 days old rhizobial colonies from agar
plates by using sterile wire loop on the same dipped filter paper strip and observed the
change in color. Colonies appeared violet and immediately turned dark purple to black
within 4-5 min reflected positive test (Steel, 1961).
2.4.2.8. Catalase test
It was performed to examine the production of enzyme catalase by test bacterium.
For this the colonies of test rhizobium (3-4 days old) were placed on the surface of glass
slides with the help of wire loop and flood with 3-4 drops of H2O2 (3 %). Gas bubble
formation indicated the presence of enzyme (MacFaddin, 1980).
2.4.2.9. Utilization of carbohydrates
29
Utilization of different carbohydrates including fructose, glucose, lactose,
maltose, sucrose and xylose by test bacterium was done in fermentation tube
anaerobically. The tube containing nutrient broth, specific carbohydrate (particular sugar)
and phenol red (pH indicator) was inoculated with test bacterium and observed the
change in color. The pH indicator appeared red at pH 7 and turned yellow below pH 6.8
due to the formation of organic acid (lactic acid) that indicated positive test
(Somasegaran and Hoben, 1994).
2.5. Check the nodulation ability of test rhizobium
Nodulation ability of test bacterium on its specific host plant roots was checked
by using nitrogen-free medium (modified Jenson’s agar medium). Placed seedling of 3-4
days old legume plant on slope of slunt containing nitrogen free medium in such a way
that root system lied on slope and shoot system came out the tube. Covered these tubes to
prevent dryness of seedlings and allowed these to settle in agar slope. The lower portion
of test tubes was covered with black paper to prevent the enterance of light. A suspention
of test rhizobium from 4 to 5 day old slunts, mixed with ¼ strenght of nitrogen free
nutrient broth and its 5-10 ml was poured in each tube containing seedlings. Inoculated
tubes were incubated in sterilized growth chamber for 3-4 weeks. Removed the seedling
from inoculated tubes at each week interval and observed the process of nodulation on
roots. After nodulation, cut off a nodule from legume and prepared rhizobial extract by
using crushed nodule method (Aneja, 1996), diluted it up to 1:10,000 and streaked the
highest dilution of rhizobial extract on YEMA supplemented with Congo red to match
the bacterial growth with its original plate (Vincent, 1970; Rigaud et al., 1973; Aneja,
1996).
2.6. In vitro antifungal activity of test fungus and rhizobial isolates
Trichoderma hamatum and rhizobial isolates were screened in vitro against root-
infecting fungi viz., F.oxysporum, F. solani, M.phaseolina and R. solani by dual-culture
plate method (Ehteshamul-Haque and Ghaffar, 1993; Siddiqui et al., 2000). For
30
determining antifungal activity of T.hamatum, a 5 mm agar disc of test fungus inoculated
on one side of 90 mm petri plate having solidified potato dextrose agar (PDA)
supplemented with two antibiotics viz., penicillin (100,000 unit/liter) & streptomycin
(0.2g/liter) and opposite side of the same petri plate was inoculated with the same size of
an agar disc of test pathogen and incubated for 4-5 days at 28C. Inhibition zone was
measured on each day, averaged and recorded in mm (Tsuneda and Skoropad, 1980;
Prince et al., 2011).
Simialrly for determining antifungal activity of rhizobial isolates, test rhizobium
was inoculated by streaking on one side of petri plate against a 5mm disc of test fungus
that was placed on other side of same petri plate containing PDA and kept at 28oC for 6-7
days. Inhibition zone was measured daily, averaged and recorded in mm (Subba Rao,
1977). There were 3 replicates of each test (Ehtshamul and Ghaffar, 1993; Siddiqui and
Shaukat, 2002).
2.7. Legume and non-legume crops used in present experimental
work
Seeds of experimental plants including legume viz., Vigna mungo (black
gram), Cicer arietinum (chickpea) and non-legume viz., Helianthus annuus (sunflower),
Brassica nigra (black mustard) plants were purchased from Old vegetable market,
Hyderabad, Pakistan.
2.8. Fertilizer and Fungicide used in present experimental work
Fertilizer named NPK and fungicide named carbendazim were purchased from
dealer of Agrochemical, Old vegetative market, Karachi, Pakistan and were used as
positive controls in pot experiments @ 2500 ppm of each.
2.9. Treatments of microbial inoculants used in present
experimental work
31
Treatments of microbial inoculants (test microorganisms) including T.hamatum,
rhizobium and bradyrhizobium isolates alone and in combination (Table 1) were used to
investigate their effects on growth and biochemical parameters of experimental plants.
Whereas selected treatments of same test microorganisms alone and in combination were
used to prepare composted organic fertilizer (Table 2).
2.10. Preparation of conidial and cell inoculums of T.hamatum
and rhizobial isolates
Conidial and cell inoculums of T.hamatum and rhizobial isolates were prepared
for conducting pot experiments to investigate the effect of microbial inoculants on growth
and biochemical parameters of four experimantal plants. For this four petri plates
containing five day old cultures of same T.hamatum on PDA were blended with 40 mL of
distilled water (10 ml/petri plate) and made its volume up to 50 ml with the help of
sterilized distilled water and considered it as 1:10 dilution. Its serial dilutions from 1:100
to 1:10,000 were made. Twenty five milliliters of highest dilution was used as inoculum
after calculating number of conidia per ml or colony forming unit (cfu) per ml and
adjusted the concentration about 1.2 x 10 6 cfu/ml with the help of SMIC
haemocytometer ART. No.1280. Similar procedure was used to prepare cell inoculum of
rhizobial isolates, calculated and adjusted to 1.9 x 108 cfu/ml (Tuite, 1969; Bader and
Qureshi, 2012).
Condial and cell inoculums were prepared in the same above mentioned manner
for composting organic food wastes including rise husk and wheat bran by using
haemocytometer. Only the concentration of each test microorganism was adjusted to
1011
-1012
cfu per ml.
32
Table 1: Treatments of test microorganism alone and in combination used in pot experiment
S.No. Treatment Code
1. Control Control
2. Trichoderma hamatum (1.2 x 10 6 cfu/ml) JUF1
3. Rhizobium sp- I (1.9 x 108 cfu/ml) JUR1
4. Bradyrhizobium sp-II (1.9 x 108 cfu/ml) JUR2
5. Bradyrhizobium sp-III (1.9 x 108 cfu/ml) JUR3
6. Bradyrhizobium sp-IV(1.9 x 108 cfu/ml) JUR4
7. Fertilizer (NPK @ 2500 ppm) FTZ
8. Fungicide (Carbendazim @ 2500 ppm) FGD
9. Rhizobium sp- I (1.9 x 108 cfu/ml) + T. hamatum (1.2 x 10
6 cfu/ml) JUR1 + JUF1
10. Bradyrhizobium sp-II (1.9 x 108 cfu/ml) + T. hamatum (1.2 x 10
6 cfu/ml) JUR2 + JUF1
11. Bradyrhizobium sp-III (1.9 x 108 cfu/ml) + T. hamatum (1.2 x 10
6 cfu/ml) JUR3 + JUF1
12. Bradyrhizobium sp-IV (1.9 x 108 cfu/ml) + T. hamatum (1.2 x 10
6 cfu/ml) JUR4 + JUF1
13. Rhizobium sp-I (1.9 x 108 cfu/ml) + NPK (2500 ppm) JUR1 + FTZ
14. Bradyrhizobium sp- II (1.9 x 108 cfu/ml) + NPK (2500 ppm) JUR2 + FTZ
15. Bradyrhizobium sp-III (1.9 x 108 cfu/ml) + NPK (2500 ppm) JUR3 + FTZ
16. Bradyrhizobium sp-IV (1.9 x 108 cfu/ml) + NPK (2500 ppm) JUR4 + FTZ
17. Rhizobium sp-I (1.9 x 108 cfu/ml) + carbendazim (2500 ppm) JUR1 + FGD
18. Bradyrhizobium sp-II (1.9 x 108 cfu/ml) + carbendazim (2500 ppm) JUR2 + FGD
19. Bradyrhizobium sp-III (1.9 x 108 cfu/ml) + carbendazim (2500 ppm) JUR3 + FGD
20. Bradyrhizobium sp-IV (1.9 x 108 cfu/ml) + carbendazim (2500 ppm) JUR4 + FGD
21. T.hamatum (1.2 x 10 6 cfu/ml) + NPK (2500 ppm) JUF1 + FTZ
22. Carbendazim (2500 ppm) + NPK (2500 ppm) FGD + FTZ
33
Table 2: Treatments of test microorganism alone and in combination used to prepare
composted organic fertilizer
S.No.
Treatments Code
1. Control Control
2. Rhizobium sp- I (1011
-1012
cfu per ml) JUR1
3. Bradyrhizobium sp-II (1011
-1012
cfu per ml) JUR2
4. Trichoderma hamatum (1011
-1012
cfu per ml) JUF1
5. Rhizobium sp-I (1011
-1012
cfu per ml) + T. hamatum (1011
-1012
cfu per ml) JUR1+ JUF1
6. Bradyrhizobium sp-II (1011
-1012
cfu per ml) + T. hamatum (1011
-1012
cfu per ml) JUR2 + JUF1
2.11. Organic food wastes
Organic food wastes including rice husk and wheat bran were purchased from
local market, Saddar, Karachi, Pakistan and used to prepare composted organic fertilizer.
2.12. Procedure for preparing composted organic fertilizer
Each rice husk or wheat bran (160g) took in conical flask (500 ml) and inoculated
with each of test microorganism (1011
-1012
cfu/ml) alone and in different combinations
(Table 2) under sterilized condition. Three replicates were made for each treatment and
incubated for 15 days at room temperature. After incubation period, oven dried the
composted rice husk or wheat bran at 80C for 2 hour and grinded the dried samples to
use as composted organic fertilizer in pot experiment after estimating its total
carbohydrate and total protein by Anthrone (Yemm and Willis, 1954) and Lowry’s
(Lowry et al., 1951) methods respectively.
2.13. Experimental pot design and procedure
The randomized complete block designed pot experiment was conducted in net
house of Department of Botany, Jinnah University for Women, Nazimabad, Karachi,
Pakistan to investigate the effects of microbial inoculants including T.hamatum and
rhizobial isolates alone and in different combinations (Table 1) on growth and
biochemical parameters of two of each legume and non-legume experimental plants.
Seeds of experimental plants were sown in pots filled with 2 kg soil in each. On 5th
day of
germination, developing seedlings in each pot of each block were inoculated with twenty
five milliliters of its respective treatment. Five replicates were used for each treatment.
During the first few days after inoculation, care was taken in watering the plants to avoid
the washing the inoculums out of the soil and it was done on alternate days. Five plants of
each treatment (1 plant/replicate/treatment) were uprooted at 30th
and 60th
day of growth
to measure the selected physical and biochemical parameters. Similarly five pots treated
with each of NPK and carbendazim @ 2500 ppm and were used as positive controls
while other five pots of experimental plants without any treatment were used as control.
In the second phase of study, same design of pot experiments were conducted to
investigate the effect of composted rice husk and wheat bran @ 5 and 10 g on physical
and biochemical parameters of sunflower (non- legume) and chickpea (legume) plants.
Seeds of experimental plants were sown in pots filled with 2 kg soil in each. At 7 day of
germination of developing seedlings, each sample of composted organic fertilizer @ 5
and 10 g per pot was applied and irrigated by tap water. All the treatments were
maintained for 60 days. The experimental plants were harvested at 30th
and 60th
day of
their germination by uprooting one plant from each pot of each treatment. Finally the
uprooted plants were subjected for physical, biochemical and mineral analysis. Five pots
were used as replicates for each treatment along with control (untreated) plants.
2.14. Effect of treatments on growth performance of experimental
plants
The effect of each treatment on growth performance of experimental plants was
investigated by measuring the lengths of root & shoot and fresh weights of plants at 30th
and 60th
day.
2.14.1. Measurement of root and shoot lengths (cm)
The measurement of root length was done from the point of attachment of the
stem base to the apex of the adventitious/tap root. Where as shoot length was measured
from the base of the stem to the tip of the longest leaf stretched .
2.14.2. Estimation of fresh weight (gram)
Fresh weight (biomass) in grams of both legume and non-legume plants was
recorded at 30th
and 60th
day of each treatment through digital weighing balance.
2.15. Effect of treatments on photosynthetic pigment
2.15.1. Estimation of chlorophyll content (mg/g)
Total chlorophyll and its fractions (a & b) were determined by using 80% acetone
(Arnon, 1949). Chlorophyll concentration related to the photosynthetic potential of plant
and subsequently to its physiological and metabolic status.
Chl- a: C55H72O5N4Mg
Chl- b: C55H70O6N4Mg
Reagents:
Extracting solvent: 80% acetone
Plant material: Green leaves
Procedure
Green leaves (0.25gm) were crushed with 5ml of acetone (80%). The extract was
centrifuged at 2000 rpm for about 5 minutes, the supernatant was transferred to test tube
and remaining debris washed again with 5ml of same acetone. This washing has been
repeated atleast thrice. The volume of collected supernatant was adjusted to 25 ml with
80% acetone and subjected to read its absorbance at 645 and 663 nm on
spectrophotometer.
Calculation
Chlorophyll-a = 12.7 x (Abs 663nm) – 2.69 x (Abs 645nm) x V
_______
1000 x W
Chlorophyll-b = 22.9 x (Abs 645nm) – 4.68 x (Abs 663nm) x V
_______
1000 x W
Total Chlorophyll = 20.2 x (Abs 645nm) + 8.02 x (Abs 663nm) x V
_______
1000 x W
Where:
V = Volume of chlorophyll extract in acetone (80%) = 25ml
W = Weight of fresh plant sample (green leaves) = 0.25gm
2.16. Effect of treatments on nutritive values in term of bio-
chemical parameters of experimental plants
2.16.1. Determination of total carbohydrate (mg/g)
Total carbohydrate (mg/g) was determined by using Anthrone methods (Yemm
and Willis, 1954).
Sample extraction
Leaf sample (0.2 gm) of each plant of each experimental crop was separately
crushed in distilled water by using morter and pestle, transferred in centrifuge tubes and
subjected to centrifugation at 3000 rpm for 15 minutes. Supernatent was collected,
adjusted to 10 ml with distilled water and marked as leaf extract.
Reagents
Anthrone reagent: Anthrone (0.4gm) was dissolved in 200 ml H2SO4 (conc.)
with constant shaking, cooled and transferred drop by drop with constant shaking
in conical flask (500 ml) containing 60 ml distilled water and 15 ml chilled
ethanol (95%).
Working standard solution of glucose (1.0 mg/ml): Glucose (0.25gm) was
Dissolved in 250 ml distilled water (1 mg /ml or 1000 µg/ml).
Procedure for preparation of test, standards and blank
Test: 0.5 ml leaf extract and 5 ml anthrone reagent were added in test tube, mixed
by shaking and kept in boiling water bath for atleast 15 minutes. Later kept in ice
bath and read absorbance spectrophotometerically at 620 nm against blank.
Standards: Different standards having concentrations from 100-1000 µg/ml were
made to prepare standard curve (Table 3 & Figure 1).
Blank: 0.5 ml distilled water + 5ml anthrone were added in test tubes and
proceeded as test.
Calculation
Total carbohydrate (mg/g of tissue) = A 1000
Where:
A = value (µg) from standard curve x total dilution factor (T.D.F)
2.16.2. Estimation of crude protein (%)
The percent of crude protein was calculated by multiplying the value of nitrogen
(%) through 6.25 (Sriperm et al., 2011).
2.17. Effect of treatments on mineral content of experimental plants
2.17.1. Estimation of nitrogen (%)
The nitrogen content (%) was estimated by Nessler’s method (Singh, 1982).
Sample (wet) digestion
Oven dried, powdered plant (leaf) material (0.2 gm) was taken in a conical flask
(250 ml). Add 2 ml H2SO4 (conc.). Heated gently in fume hood over hot plate, slowly
raised the temperature until a black solution was appeared. H2O2 (30%) was added drop
wise until a colourless solution appeared at the bottom of the flask. Sample was digested
Table 3: Absorbance of glucose (µg/ml)
S. No. Standard
(S)
Concentration (µg /ml) Absorbance at 620 nm
1. S1 100 0.8
2. S2 200 1.6
3. S3 300 2.5
4. S4 400 3.3
5. S5 500 3.8
6. S6 600 4.9
7. S7 700 5.6
8. S8 800 6.7
9. S9 900 7.4
10. S10 1000 8.5
Figure 1: Standard curve of glucose
y = 0.0084x - 0.12 R² = 0.9969
0
1
2
3
4
5
6
7
8
9
0 200 400 600 800 1000 1200
Ab
sorb
an
ce a
t 620 n
m
Glucose (µg/ml)
until its 1-2 ml remained. Digested flask was removed from hot plate, cooled, and
adjusted the volume upto 100 ml with distilled water. Filtered it and stored carefully.
Reagents
Nessler’s Reagent: Solution A was prepared by mixing 70 gm of potassium
iodide (KI) and 100gm of murcuric iodide (HgI2) in 300ml distilled water.
Whereas solution B was prepared by dissolving 160 gm sodium hydroxide in
500ml distilled water. Later solution A was completely added to solution B and
adjusted the volume upto 1L.
Sodiumhydroxide (10%): NaOH (10 gm) dissolved in 100 ml distilled water.
Sodium silicate (10%): sodium silicate (10 gm) dissolved in 100 ml distilled
water.
Stock standard solution of nitrogen: NH4NO3 (0.286 gm) was added in 100 ml
distilled water, that was equaled to 100 ppm N (1gm nitrogen in 1L distilled
water).
Working standard solution of nitrogen: Stock standard solution of nitrogen (10
ml) was diluted with distilled water (10 ml) that was equaled to 50 ppm N.
Procedure for preparation of test, standards and blank
Test: An aliquot (1 ml) of digested sample was added in conical flask (50 ml) +
1ml of NaOH (10%) and 1ml of Na-silicate (10%) + 15 ml of Nessler’s reagent and
increased the volume upto 50 ml with distilled water. Incubated for 20 minutes at
room temperature and read the absorbance spectrophotometerically at 410 nm
against the blank.
Standards: Different standards having concentrations from 1-10 ppm N /ml were
made and proceeded as test (Table 4 & Figure 2).
Blank: 1 ml distilled water + 1ml of NaOH (10%) + 1ml of Na-silicate (10%) and
proceeded as test.
Table 4: Absorbance of nitrogen
S. No. Standard
(S)
Concentration (ppm) Absorbance at 410 nm
1. S1 1 0.03
2. S2 2 0.06
3. S3 3 0.09
4. S4 4 0.14
5. S5 5 0.16
6. S6 6 0.195
7. S7 7 0.235
8. S8 8 0.28
9. S9 9 0.305
10. S10 10 0.34
Figure 2: Standard curve of nitrogen
y = 0.035x - 0.009 R² = 0.9971
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 2 4 6 8 10 12
Ab
sorb
an
ce a
t 410 n
m
Nitrogen (ppm)
Calculation
Nitrogen (%) = A /10000
Where:
A (ppm Nitrogen) = nitrogen value (ppm) from standard curve x Total dilution
factor (T.D.F)
2.17.2. Estimation of phosphorus (%)
The percent phosphorus was estimated by Braton reagent (Ashraf et al., 1992).
Sample (wet) digestion
It was done as same as described in 2.16.1.
Reagents
Barton reagent (Ammonium vanadate-molybdate): Solution A contained
ammonium molybdate (25 g) in distilled water (400 ml) and solution B contained
ammonium metavanadate (1.25g) dissolved in hot distilled water (300 ml). Add
solution B to solution A in volumetric flask (1L) and cooled it at room temperature.
Concentrated nitric acid (250 ml) was gradually added, cooled the solution at room
temperature and made the volume up to 1L with distilled water.
Standard stock solution of phosphorus: KH2PO4 (0.2197g) dissolved in 1L of
distilled water, which was equals to 50 ppm phopsphorus.
Procedure for preparation of test, standards and blank
Test: 10 ml of filtrate of digested sample + 20 ml distilled water + 10ml Barton
reagent and made the volume upto 50 ml with distilled water and incubated at room
temperature for 10 min. During which yellow color of phospho-vando-molybdate
complex was appeared. Read the absorbance spectrophotometerically at 420 nm
against blank.
Standards: Different standards having concentrations from 1-10 ppm phosphorus
/ml were made and proceeded as test (Table 5 & Figure 3).
Blank: Barton reagent (10 ml) was taken and adjusted the volume up to 50 ml with
distilled water.
Calculation
Phosphorous (%) = A /10000
Where:
A (ppm Phosphorus) = Phosphorus value (ppm) from standard curve x total dilution
factor (T.D.F)
2.18. Analysis of Data
Results of present pot experiments are expressed as mean standard deviation
(S.D.). The data was analyzed by using One-way ANOVA followed by LSD (least
significant difference) test through SPSS 16 . The differences were considered significant
at p<0.05 when treatments’ mean compared with control.
Table 5: Absorbance of phosphorus
S. No. Standard
(S)
Concentration (ppm) Absorbance at 420 nm
1. S1 1 0.033
2. S2 2 0.061
3. S3 3 0.088
4. S4 4 0.121
5. S5 5 0.146
6. S6 6 0.173
7. S7 7 0.211
8. S8 8 0.259
9. S9 9 0.27
10. S10 10 0.286
Figure 3: Standard curve of phosphorus
y = 0.0296x + 0.0017 R² = 0.9914
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 2 4 6 8 10 12
Ab
sorb
an
ce a
t 420 n
m
Phosphorus (ppm)
3. Results
3.1. Isolation and identification of fungi from rhizoplane
In order to isolate test fungal pathogens and test fungus, root samples
(rhizoplanes) of wild and cultivated plants including Amaranthus viridis L., Brassica
compestris L., Cassia holoceratia Fresen, Gynandropsis gynandra (Linn) Merr.,
Trigonella foenum-graecum L., Triticum astivum L., Phaseolus unguiculata (L.) Walp.,
Sesbania sesban (L.) Merr. and Solonum nigram L. were collected from different areas of
Karachi viz., North Nazimabad, Jinnah University for Women , University of Karachi and
Malir district viz., Maroo Goth and Memon Goth (Table 6). Five replicates of each plant
root sample were collected and processed according to standard method described by
Aneja, 1993. Developed fungi were identified by expert of Botany Department,
University of Karachi, Karachi, Pakistan with taking reference to manual of different
genera of fungi (Barnett & Hunter, 1998) as Fusarium oxysporum Schlecht emend. Snyd.
& Hans, F. solani (Mart) Appel & Wollenw. emend Snyd. & Hans (4 strains),
Macrophomina phaseolina (Tassi) Goid (4 strains) and Rhizoctonia solani Kuhn (2
strains) collectively took as test fungal pathogens and Trichoderma hamatum as test
fungus. Identified test fungal pathogens and test fungus were isolated pure and coded
(Table 7, 8).
3.2. Isolation of rhizobial isolates from root nodules
Root samples of cultivated legume crop plants including Trigonella foenum-
graecum, Phaseolus unguiculata, Vigna radiata and V.mungo were collected from fields
of University of Karachi, Memon Goth (Malir) and net house of Department of Botany,
Jinnah University for Women to obtain healthy pink, unbroken and firm nodules to
isolate rhizobia. The rhizobial isolates were made purified and coded (Table 8).
Table 6: Cultivated and wild plants with their sites of collection
S.No. Plants Location
1. Amaranthus viridis L. North Nazimabad
2. Brassica compestris L. University of Karachi
3. Cassia holoceratia Fresen. University of Karachi
4. Gynandropsis gynandra (Linn.) Merr. North Nazimabad
5. Phaseolus unguiculata (L.) Walp. Memon Goth
6. Solonum nigram L. Jinnah University for Women
7. Susbania susban (L.) Merr. University of Karachi
8. Trigonella foenum graecum L. Maroo Goth
9. Trigonella foenum graecum L. University of Karachi
10. Triticum astivum L. Memon Goth
11. Vigna mungo (L.) Hepper Net house of Jinnah University for Women
12. Vigna radiata (L.) R. Wilczek Net house of Jinnah University for Women
46
Table 7: Test fungal pathogens with their host plants and sites of collection
S.No. Test fungal pathogen Host plant Location
1. Fusarium solani (strain-1) Brassica compestris L. University of Karachi
2. F. solani (strain-2) Solonum nigram L. Jinnah University for Women
3. F.solani (strain-3) Cassia holoceratia Fresen. University of Karachi
4. F.solani (strain-4) Cassia holoceratia Fresen. University of Karachi
5. F. oxysporum - B.R.C. University of Karachi
6. Macrophamina phaseolina (strain-1) Trigonella foenum graecum L. Maroo Goth
7. M. phaseolina (strain-2) Amaranthus viridis L. North Nazimabad
8. M. phaseolina (strain-3) Gynandropsis gynandra (Linn.) Merr North Nazimabad
9. M. phaseolina (starin-4) Triticum astivum L. Memon Goth
10. Rhizocotina solani (strain-1) Phaseolus unguiculata (L.) Walp. Memon Goth
11. R. solani (strain-2) Sesbania sesban (L.) Merr. University of Karachi
B.R.C = M. A. H. Q. Biological Research Center, University of Karachi
47
Table 8: Test microorganisms with host plants, site of collection and code no.
JUW = Jinnah University for Women
S. No. Test microorganism Code No. Host plant Location
1. Trichoderma hamatum JUF1 Amaranthus viridis North Nazimabad
2. Rhizobium sp-I JUR1 Trigonella foenum-graecum University of Karachi
3. Bradyrhizobium sp-II JUR2 Vigna unguiculata Memon Goth
4. Bradyrhizobium sp-III JUR3 Vigna radiata Net house of JUW
5. Bradyrhizobium sp-IV JUR4 V.mungo Net house of JUW
48
3.3. Characterization of rhizobial isolates
On the basis of cultural and morphological characteristics, colonies of JUR1,
JUR2, JUR3 and JUR4 were found round, ranging from 2-6 mm in size, white in color
having entire margin, their elevation include raised for JUR1 and JUR4 while convex for
JUR2 and JUR3. All rhizobial isolates were rod, aerobic and non-spore forming Gram-
negative bacteria which formed white gummy colonies on YEMA medium incorporated
with Congo red (1.0 %). According to observed growth rate of rhizobial isolates on
YEMA medium containing bromothymol blue (0.5%), JUR1 was found fast-growing and
acid producer while rest of three including JUR2, JUR3, and JUR4 were found slow-
growing and alkali producing bacteria (Table 9).
On the basis of biochemical characteristics, all four rhizobial isolates were not
involved in indole production and also gave negative ketolactose, MRVP, starch
hydrolysis, liquafication of gelatin and oxidase tests. Where as all rhizobial isolates were
found actively involved in nitrate reduction and production of hydrogen sulphide. In
addition, gave positive catalase test (Table 10). All four rhizobial isolates were found to
utilize fructose, galactose and glucose as source of carbon while did not utilize lactose.
On the other hand, maltose was only utilized by JUR1, sucrose by JUR1, JUR3 and JUR4
and xylose by only two isolates including JUR1 and JUR4 (Table 11).
3.4. Nodulation ability of test rhizobial isolates
All four rhizobial isolates were found to produce pink nodules on their
respective hosts like roots of Trigonella foenum-graecum, Phaseolus unguiculata, Vigna
radiata and V.mungo were 100% nodulated by JUR1, JUR2, JUR3 and JUR4
respectively and confirmed their host-specificity.
Table 9: Cultural, morphological and staining characteristics of rhizobial isolates
S. No. Characters JUR1 JUR2 JUR3 JUR4
1. Shape Round Round Round Round
2. Size of colony 3-6 mm 2-4 mm 2 mm 2 mm
3. Color/pigmentation White, gummy White, gummy White, gummy White, gummy
4. Elevation Raised Convex Convex Raised
5. Margin Entire Entire Entire Entire
6. Motility Motile Motile Motile Motile
7. Bacterium shape Rod Rod Rod Rod
8. Oxygen demand Aerobic Aerobic Aerobic Aerobic
9. Spore formation -ve -ve -ve -ve
10. Gram’s reaction -ve -ve -ve -ve
11. Growth on YEMA with Congo red White White White White
12. Growth on YEMA with Bromothymol blue
a. Growth rate Fast Slow Fast Slow
b. Acid / alkali Acid Alkali Acid Alkali
50
Table 10: Biochemical characteristics of rhizobial isolates
S.No. Test JUR1 JUR2 JUR3 JUR4
1. Ketolactose test -ve -ve -ve -ve
2. Production of indole -ve -ve -ve -ve
3. Methyl red test -ve -ve -ve -ve
4. Voges-Proskaur test -ve -ve -ve -ve
5. Nitrate reduction test +ve +ve +ve +ve
6. Starch hydrolysis -ve -ve -ve -ve
7. Liquafication of gelation -ve -ve -ve -ve
8. Oxidase test +ve +ve +ve +ve
9. Catalase test +ve +ve +ve +ve
10. Production of H2S +ve +ve +ve +ve
51
Table 11: Utilization of carbohydrates by rhizobial isolates
S.No. Carbohydrates JUR1 JUR2 JUR3 JUR4
1. Furctose + + + ++
2. Glucose + + + ++
3. Lactose - - - -
4. Maltose + - - -
5. Sucrose + - + ++
6. Xylose + - - ++
52
3.5. In vitro antifungal activity of T.hamatum and
rhizobial isolates against plant fungal pathogens
T.hamatum (JUF1) inhibited the growth of Fusarium oxysporum, two strains of
each Rhizoctonia solani (strain 1 & 2) and Macrophomina phaseolina (strain2&3) by
producing mycelial coiling while inhibited the growth of three strains (1, 3 & 4) of
F.solani without any zone. However, the same test fungus produced inhibition zones of
3.0, 5.3 and 2.3 mm against F.solani (strain-2), strain-1 and 4 of M.phaseolina
respectively (Table 12).
Out of test rhizobial isolates, JUR1 produced inhibition zones of 12 mm against
F. oxysporum, from 2.0 - 2.5 mm against three strains (1, 2 & 3) of F.solani while
inhibited the growth of fourth strain of same fungal pathogen without any zone.
Similarly, the same rhizobium sp. produced inhibition zones ranging from 6.5-10 mm
against all strains (1, 2, 3 & 4) of M.phaseolina and from 1.5-2.2 mm against both strains
(1 & 2) of R.solani (Table 12). The Bradyrhizobium sp. (JUR2) inhibited the grwoth of F.
oxysporum with zone of 8.0 mm, from 3.4 -8.3 mm against all four strains (1, 2, 3 & 4) of
F.solani. Likewise the same Bradyrhizobium sp. was found to inhibit all strains (1, 2, 3 &
4) of M.phaseolina with zones ranging from 4 -8.5 mm and 2-5 mm against two strains (1
& 2) of R.solani (Table 12).
The second Bradyrhizobium sp. (JUR3) produced inhibition zone of 6.5 mm
against F. oxysporum. The same Bradyrhizobium also produced zones of inhibition
ranging from 3.5-5.0 mm against all strains (1, 2, 3, & 4) of F.solani, from 8.0 -11.5 mm
against M.phaseolina (strain- 1, 2, 3 & 4) and from 5 -5.5 mm zones against strains (1 &
2) of R.solani (Table 12). The third Bradyrhizobium sp. (JUR4) inhibited the grwoth of
F. oxysporum without any zone while produced zones of inhibition measuring from 5.3-
7.2 mm against F.solani (strain-1, 2, 3 & 4), 8.0 -10.5 mm against M.phaseolina (strain-
1, 2, 3, & 4) and 2.0 mm against each of two strains of R.solani (Table 12).
Table 12: In vitro antifungal activity of test microorganisms against fungal pathogens
S.No. Test fungal pathogens
Zone of inhibition (mm) produced by
test microorganisms
JUF1 JUR1 JUR2 JUR3 JUR4
1. Fusarium oxysporum B 12 8 6.5 A
2. F. solani (strain-1) A 2.5 3.4 5 7.2
3. F. solani (strain-2) 3.0 2.0 6.1 4.4 5.3
4. F. solani (strain-3) A 2.2 8.3 3.5 5.5
5. F.solani (strain-4) A A 5.2 4.2 6.9
6. Macrophomina phaseolina (strain-1) 5.3 10 6 11.5 8.0
7. M. phaseolina (strain-2) B 8.5 4 10.2 10.5
8. M. phaseolina (strain-3) B 8.0 4.5 8.4 9.2
9. M. phaseolina (strain-4) 2.3 6.5 8.5 8.0 9.5
10. Rhizoctonia solani (strain-1) B 1.5 2 5 2.0
11. R.solani (strain-2) B 2.2 5 5.5 2.0
A = Colonies of test microorganism and fungal pathogen inhibited by each other without any zone
B = Test fungus produced mycelial coiling with fungal pathogen
54
3.6. Pot experiments (1st Phase)
3.6.1. Effcet of microbial inoculants on non-legume
plants
3.6.1.1. Helianthus annuus L. (sunflower)
3.6.1.1.1. Growth performance
The percent increase in root length of sunflower plants treated with T.hamatum
(JUF1) alone and combination of T.hamatum with rhizobial isolates including
JUR1+JUF1, JUR2+JUF1, JUR3+JUF1 and JUR4+JUF1 was observed as 44.7, 40.2,
31.7, 18.0 and 8.1% respectively at 30th
day as compared to untreated (control) plants
while T.hamatum with fertilizer (JUF1+ FTZ) induced 28.5% percent increase in same
parameter at 30th
day. Similarly, the treatments include JUF1, JUR1+JUF1, JUR2+JUF1,
JUR3+JUF1, JUR4+JUF1 and JUF1+FTZ induced a significant percent increase in root
length as 135, 66.25, 55.3, 271.47, 21.82 and 250.7% respectively as compared to control
plants at 60th
day (Table 13; Figure 4). The root length of sunflower plants treated with
rhizobial isolates alone including JUR1, JUR2, JUR3 and JUR4 was increased upto 47.6,
39.0, 15.8 and 34.8 % respectively at 30th
day. Whereas fertilizer (FTZ), fungicide (FGD)
and their combination (FTZ+FGD) induced percent increase in root length as 88.1, 23.4
and 37.71% respectively at same day. Likewise, treatments include JUR1, JUR2, JUR3,
JUR4, FTZ, FGD and FGD+FTZ have promoted root length 39.5, 64.38, 170.14, 14.5,
135, 227.3 and 201.3% respectively at 60th
day (Table 13; Figure 5). On the other hand,
the combination of different rhizobial isolates with fertilizer and fungicide include
JUR1+FTZ, JUR2+FTZ, JUR3+FTZ, JUR4+FTZ, JUR1+FGD, JUR2+FGD,
JUR3+FGD and JUR4+FGD induced percent increase in root length of sunflower plants
from 9 -78 % at 30th
day and 15 - 300 % at 60th
day (Table 13; Figure 5).
Sunflower test plants showed only 17.58% increase in their shoot lengths when
treated with T.hamatum (JUF1) alone as compared to test plants which were co-
inoculated with T.hamatum and rhizobial isolates (JUR3 and JUR4) showed significant
percent increase in their shoot lengths about 35 % at 30th
day while same groups of plants
Table 13: Effect of treatments on growth performance of H.annuus (sunflower) plants
Growth performance
30th
day 60th
day
S.No. Treatment Root length* Shoot length* Fresh weight** Root length* Shoot length* Fresh weight**
1. Control 10.5 0.86 26.5 0.5 2.23 0.06 12.831.89 37.4 2.47 2.4 0.24
2. JUR1 15.5 0.86a (47.61) 37.5 0.5
a (41.05) 3.67 0.37 (64.57) 17.9 2.26 (39.51) 48.63 5.59
a (30.62) 3.67 0.37 (52.91)
3. JUR2 14.6 1.27c (39.04) 31.83 2.02
a (20.11) 3.63 0.66 (62.78) 21.06 6.46
d (64.14) 50.33 3.21
a (34.57) 3.63 0.66 (51.25)
4. JUR3 12.16 0.76 (15.80) 32 2.64a (20.75) 3.46 1.05 (55.15) 34.661.15
a (170.14) 53 3.60
a (41.71) 4.54 0.67
c (89.16)
5. JUR4 14.16 0.76c (34.85) 31 2.0
b (16.98) 3.41 0.84 (52.91) 14.7 4.35 (14.57) 37.25 3.05 (-0.40) 3.72 0.29 (55)
6. JUF1 15.2 2.69b (44.76) 31.16 2.75
b (17.58) 4.45 0.19
c (99.55) 30.16 2.46
a (135.07) 53.66 2.08
a (43.47) 5.05 1.08
c (110.41)
7. FTZ 19.76 2.80a (88.19) 29.06 0.45
d (9.66) 2.91 0.23 (30.49) 42 6.92
a (227.35) 54.66 0.57
a (46.14) 5.09 0.25
b (112.08)
8. FGD 12.96 4.60 (23.42) 30.36 0.77d (14.56) 2.66 0.13 (19.28) 25.33 5.50
c (141.23) 44.33 1.15
c (18.52) 3.73 1.04 (55.41)
9. JUR1+JUF1 14.73 0.92c (40.28) 26.33 1.25 (-0.75) 5.97 1.18
a (167.71) 21.33 3.21
d (66.25) 52.33 4.93
a (39.91) 4.21 0.10
d (75.41)
10. JUR2+JUF1 13.831.04d (31.71) 28.33 2.64 (6.90) 3.81 1.44
d (70.85) 19.93 2.40 (55.33) 48.16 2.02
a (28.77) 3.41 0.33 (42.08)
11. JUR3+JUF1 12.4 0.85 (18.09) 35.66 1.04a (34.56) 3.62 0.94 (62.33) 47.66 4.93
a (271.47) 48.66 1.0
a (30.10) 4.45 0.19
c (85.41)
12. JUR4+JUF1 11.36 0.77 (8.19) 35.83 1.60a (35.20) 5.05 1.09
b (1.26) 15.63 2.50 (21.82) 48.96 3.04
a (30.90) 3.79 0.98 (57.91)
13. JUR1+FTZ 11.51.80 (9.52) 29.96 6.03d (13.05) 3.77 0.15
d (69.05) 18.83 2.84 (44.76) 57.33 5.68
a (53.28) 6.44 1.15
a (168.33)
14. JUR2+FTZ 14.83 2.25c (41.23) 28.23 1.72 (6.52) 5.09 0.24
b (128.25) 19.16 1.75 (49.33) 50 4.0
a (33.68) 5.16 1.67
b (89.58)
15. JUR3+FTZ 11.83 0.76 (12.66) 30.4 0.17d (14.71) 3.74 1.04 (67.71) 51.33 3.21
a (300) 54.33 9.23
a (45.26) 6.36 1.90
a (165)
16. JUR4+FTZ 13.83 1.25c (31.71) 30.56 0.40
d (15.32) 5.16 1.67
a (131.39) 16.4 1.08 (27.82) 48.36 1.85
a (29.30) 2.74 0.60 (14.16)
17. JUR1+FGD 13.861.85c (32.0) 27.5 0.86 (3.77) 3.95 0.66
d (77.13) 23.16 7.0
c (80.51) 51.66 1.52
a (38.12) 5.97 1.18
a (148.75)
18. JUR2+FGD 17.431.06a (66) 29.33 0.76
d (10.67) 5.48 1.90
a (145.73) 19.23 1.0 (49.88) 43.5 2.50
d (16.26) 3.81 1.44 (58.75)
19. JUR3+FGD 13.431.40d (27.90) 29.8 1.31
d (12.45) 4.54 0.67
c (103.58) 20.63 2.71 (60.79) 45.16 2.73
c (20.74) 4.35 0.25
c (81.25)
20. JUR4+FGD 18.731.25a (78.38) 30.16 1.04
d (13.81) 4.62 0.71
c (99.55) 14.86 2.05 (15.28) 45.4 2.80
c (21.39) 3.06 1.01 (27.5)
21. JUF1+FTZ 13.51.77d (28.57) 29.73 2.18
d (2.18) 2.29 0.33 (2.69) 45 12.76
a (250.74) 51 3.46
a (36.36) 5.161.66
b (115)
22. FGD+FTZ 14.461.45c (37.71) 27.76 1.80 (4.75) 3.1 0.71 (39.01) 38.66 2.51
a (201.32) 48.33 3.21
a (29.22) 5.48 1.91
a (128.33)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase
or decrease (-) with respective control. * = cm, ** = g, JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=Fertilizer, FGD=Fungicide.
56
Figure 4: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of H.annuus plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
2
4
6
8
10
12
14
16
18
20 R
oot
len
gth
(cm
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a c c
a
c d
d c
b
0
5
10
15
20
25
30
35
40
45
50
Root
len
gth
(cm
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
a
c d
a a
a a
a
Figure 5: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on root length of H.annuus plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
2
4
6
8
10
12
14
16
18
20
Root
len
gth
(cm
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a c
d
a
c c c c c
a
0
10
20
30
40
50
60
Root
len
gth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
c
a
c
a
d
a
Figure 6: Effect of T.hamatum alone and in combination with rhizobial
isolates on shoot length of H.annuus plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30
35
40
Sh
oot
len
gth
(cm
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
a a b d
d
a a
d b
0
10
20
30
40
50
60
Sh
oo
t le
ngth
(cm
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
a a a
c
a a
a a a
a a
showed 30-31% increase at 60th
day (Table 13; Figure 6). However, T.hamatum alone
found active and induced significant 43.47% increase in shoot length of test plants at 60th
day. Similarly, T.hamatum alongwith rhizobial isolates (JUR1, JUR2, JUR3, JUR4) and
NPK (FTZ) induced 28.77-36.36 % increase in same parameter in their respective plant
groups at 60th
day (Table 13; Figure 6). Rhizobial isolates were found more active when
given in combination with fertilizer (FTZ) and fungicide (FGD) at 60th
day and induced
significant increase in shoot length from 16.26 - 53.28 % as compared to its 30th
day
interval. However rhizobial isolates alone were found active at both day except JUR4
which did not increase shoot length at 60th
day (Table13; Figure 7).
T.hamatum (JUF1) alone induced remarkable increase about 99% at 30th
day
and 110.41% at 60th
day in fresh weights of whole sunflower plants where as plants co-
inoculated with T.hamatum and three rhizobial isolates including JUR1, JUR2 and JUR3
showed significant 62-167.7 % increase in fresh weight of plants at 30th
day and 42-85%
at 60th
day. Similarly, T.hamatum alongwith FTZ also induced 115% increase in fresh
weight of plant at 60th
day (Table 13; Figure 8). All four rhizobial isolates in their
respective groups of test plants induced percent increase > 50% in fresh weight at both
days though it was not statistically significant except JUR3 induced significant (89%)
increase in fresh weight of test plants at 60th
day (Table 13; Figure 9).
3.6.1.1.2. Photosynthetic pigment
T.hamatum alone induced 40% increase in total chlorophyll content of
sunflower plants at 30th
day whereas when co-inoculated with JUR3 and JUR4 induced
noteworthy increase in both fractions of chlorophyll (chl-a and chl-b) and total
chlorophyll from 18-150% at 30th
day. Even JUR4+JUF1 induced 94% significant
increase in total chlorophyll content at 60th
day (Table 14; Figure 10).
Out of isolated rhizobia, JUR1 alone enhanced significant production of
chlorophyll content and its fractions at both days of harvesting from 56-140%. The same
isolate in combination with FTZ and FGD also produced significant increase in same
Table 14: Effect of treatments on photosynthetic pigment of H.annuus (sunflower) plants
Photosynthetic pigment
30th
day 60th
day
S.No. Treatment Chl-a* Chl-b* Total Chl* Chl-a* Chl-b* Total Chl*
1. Control 0.48 0.14 0.2 0.01 0.75 0.20 0.98 0.10 0.5 0.20 1.47 0.19
2. JUR1 0.82 0.03d (70.83) 0.48 0.05
a (140) 1.31 0.06
a (74.6) 1.64 0.44
b (63.34) 0.78 0.22
c (56) 2.43 0.64
a (65.3)
3. JUR2 0.59 0.21 (22.91) 0.29 0.03 (45) 0.89 0.18 (18.6) 1.18 0.11 (20.4) 0.44 0.23 (-12) 1.62 0.14 (10.2)
4. JUR3 0.81 0.3d (68.75) 0.49 0.1
a (145) 1.34 0.20
a (78.6) 1.27 0.38 (29.5) 0.43 0.05 (-14) 1.7 0.41 (15.6)
5. JUR4 0.54 0.0 (12.5) 0.35 0.03c (75) 0.89 0.03 (18.6) 1.13 0.27 (15.3) 1.13 0.08
a (126) 2.85 0.06
a (93.8)
6. JUF1 0.73 0.27 (52.08) 0.27 0.1 (35) 1.01 0.22d (39.6) 1.03 0.28 (5.10) 0.35 0.04 (-30) 1.38 0.29 (-6.12)
7. FTZ 0.94 0.07c (95.83) 0.35 0.01
d (75) 1.27 0.08
a (69.3) 0.88 0.3 (-10.20) 0.3 0.07
d (-40) 1.18 0.32 (-19.72)
8. FGD 1.07 0.16a (122.91) 0.37 0.02
d (85) 1.34 0.08
a (78.6) 1.04 0.15 (6.12) 0.31 0.03 (-38) 1.36 0.18 (-7.48)
9. JUR1+JUF1 0.41 0.06 (-14.58) 0.33 0.02c (65) 0.75 0.04 (0.0) 0.99 0.26 (1.02) 0.45 0.15 (-10) 1.45 0.40 (-1.36)
10. JUR2+JUF1 0.54 0.09 (12.5) 0.36 0.02c (80) 0.91 0.07 (21.3) 0.98 0.24 (0.0) 0.45 0.10 (-10) 1.43 0.34 (-2.72)
11. JUR3+JUF1 0.86 0.06c (79.16) 0.33 0.09
d (65) 1.19 0.12
b (58) 0.9 0.11 (-8.16) 0.26 0.04
d (-48) 1.16 0.14 (-21.08)
12. JUR4+JUF1 0.86 0.32c (79.16) 0.5 0.11
a (250) 1.36 0.15
a (18) 1.59 0.24
c (62.24) 1.26 0.28
a (152) 2.85 0.40
a (93.87)
13. JUR1+FTZ 0.93 0.05c (93.75) 0.43 0.03
a (115) 1.37 0.03
a (82) 0.92 0.13 (-6.12) 0.38 0.08 (-24) 1.31 0.22 (-10.88)
14. JUR2+FTZ 0.46 0.16 (-4.16) 0.28 0.1 (40) 0.75 0.12 (0.0) 0.92 0.47 (-6.12) 0.48 0.09 (-4) 1.41 0.54 (-4.08)
15. JUR3+FTZ 0.43 0.09 (-10.41) 0.32 0.1 (60) 0.76 0.15 (1.3) 0.93 0.09 (-5.10) 0.36 0.04 (-28) 1.29 0.14 (-12.24)
16. JUR4+FTZ 0.66 0.14 (39.5) 0.35 0.01c (75) 1.01 0.15
d (34.6) 1.23 0.32 (25.5) 0.74 0.15
d (48) 1.98 0.47
d (34.69)
17. JUR1+FGD 0.85 0.27 (77.08) 0.36 0.05c (80) 1.22 0.16
a (62.6) 0.44 0.15
c (55.10) 0.26 0.04
d (-48) 0.71 0.19 (-51.70)
18. JUR2+FGD 0.48 0.06 (0.0) 0.33 0.33d (65) 0.82 0.09 (9.33) 0.64 0.16 (34.69) 0.3 0.02 (-40) 0.94 0.13 (-36.05)
19. JUR3+FGD 0.65 0.23 (35.41) 0.26 0.02 (30) 0.93 0.2 (24) 1.29 0.12 (31.6) 0.47 0.02 (-6) 1.63 0.19 (10.88)
20. JUR4+FGD 0.79 0.24d (64.58) 0.3 0.07 (50) 1.11 0.31
c (48) 1.23 0.17 (25.5) 0.74 0.24
d (48) 1.98 0.42
d (34.69)
21. JUF1+FTZ 0.77 0.19 (60.41) 0.32 0.05d (60) 1.1 0.23
c (46.6) 1.15 0.15 (17.34) 0.43 0.03 (-14) 1.59 0.12 (8.16)
22. FGD+FTZ 0.9 0.20c (87.5) 0.38 0.07
d (90) 1.35 0.2
a (80) 1.08 0.03 (10.20) 0.35 0.06 (-30) 1.44 0.03 (-2.04)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. * = mg/g, chl = chlorophyll, JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=Fertilizer,
FGD=fungicide. 61
Figure 7: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on shoot length of H.annuus plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30
35
40
Sh
oot
len
gth
(cm
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a b d
d d d d d d d d
0
10
20
30
40
50
60
Sh
ooth
len
gth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a a
a a
c
a
a
a
a
a
d c c a
Figure 8: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of H.annuus plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
1
2
3
4
5
6
Fre
sh w
eigh
t (g
m)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
a
c
b
0
1
2
3
4
5
6
Fre
sh w
eigh
t(gm
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c d
c b
c
b a
Figure 9: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on fresh weight of H.annuus plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
1
2
3
4
5
6
Fre
sh w
eigh
t(gm
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
b a
d
a
c c
0
1
2
3
4
5
6
7
Fre
sh w
eig
ht
(gm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
b
a
b
a a
c
a
0
0.2
0.4
0.6
0.8
1
1.2
1.4 T
ota
l ch
loro
ph
yll
(m
g/g
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
a a a
a
b
a
c
a
Figure 10: Effect of T.hamatum alone and in combination with rhizobial
isolates on total chlorophyll of H.annuus plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
Tota
l ch
loro
ph
yll
(m
g/g
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
a a
parameter at 30th
day. JUR3 found effective in increasing chl-b and total chlorophyll
content at 30th
day. On the other hand, JUR4 induced 75% significant increase in chl-b at
30th
day while 94 - 126 % induced significant increase in chl-b and total chlorophyll
content at 60th
day. Similarly JUR4 with FTZ and FGD found effective in increasing total
chlorophyll content in test plants at both 30th
and 60th
days (Table 14; Figure 11).
3.6.1.1.3. Biochemical parameters
T.hamatum (JUF1) alone effeciently increases carbohydrate and crude protein
contents of sunflower plants at 30th
day. Whereas the same test fungus was also found
effective with JUR1 at 60th
day, with JUR3 at 30th
day and with JUR4 at both days of
harvesting in increaseing carbohydrate and crude protein contents (Table 15; Figure 12,
14).
Rhizobial isolates including JUR1 and JUR4 produced significant effects on
carbohydrate and crude protein contents at 30th
day in their respective groups of
sunflower plants. Interestingly rest of the rhizobial isolates including JUR3 and JUR4
found active and produced significant effects on both biochemical parameters of test
plants when inoculated in combination with FTZ and FGD at both days of harvesting.
JUR1 and JUR2 in combination with FGD found effective in increasing carbohydrate and
crude protein contents of test plants at 30th
day (Table 15; Figure 13, 15).
3.6.1.1.4. Mineral content
Significant increase in percent nitrogen at 30th
day and phosphorus at both days
was observed in leaves of sunflower plants treated with T.hamatum (JUF1) alone. Where
as T.hamatum (JUF1) in combination with JUR3 induced a significant increase in
nitrogen and phosphorus contents at 30th
day while JUR4+JUF1 found effective at both
days of harvesting of plants by increasing the amounts of both minerals from 71.8 -
255.5% (Table 16; Figure 16,18).
Table 15: Effect of treatments on biochemical parameters of H.annuus (sunflower) plants
Biochemical parameters
30th
day 60th
day
S.No. Treatment Total carbohydrate (mg/g) Crude proteins (%) Total carbohydrates (mg/g) Cruude proteins (%)
1. Control 178.49 4.98 8.04 0.13 222.35 37.36 10.281.72
2. JUR1 257.33 25.2c (44.17) 11.89 1.16
c (47.88) 292.31 104.67 (31.46) 13..514.83 (31.42)
3. JUR2 182.09 7.88 (2.01) 8.05 0.67 (0.12) 285.97 53.81 (28.61) 13.22 2.48 (28.59)
4. JUR3 204.38 19.08 (14.50) 9.45 0.88 (17.53) 248.13 18.11 (11.59) 11.47 0.84 (11.57)
5. JUR4 331.62 32.55a (85.79) 15.33 1.50
a (90.67) 400.52 94.28
b (80.13) 18.52 4.36
b (80.15)
6. JUF1 242.0 20.39d (35.58) 11.18 0.94
d (39.05) 252.36 13.49 (13.39) 11.66 0.62 (13.42)
7. FTZ 183.30 12.35 (2.69) 8.47 0.56 (5.34) 191.96 28.98 (-13.66) 8.87 1.33 (13.71)
8. FGD 164.75 15.15 (-7.69) 7.61 0.70 (-5.34) 181.34 82.06 (-18.44) 8.38 3.79 (18.48)
9. JUR1+JUF1 218.44 38.8 (22.38) 10.1 1.79 (25.62) 368.61 99.29c (65.77) 17.04 4.61
c (65.75)
10. JUR2+JUF1 200.47 20.46 (12.31) 9.27 0.94 (15.29) 289.56 89.92 (28.42) 13.38 4.16 (30.15)
11. JUR3+JUF1 242.21 25.84d (35.69) 11.2 1.19
d (39.30) 226.78 73.43 (1.99) 10.48 3.39 (1.94)
12. JUR4+JUF1 297.54 40.95a (66.69) 13.75 1.89
a (71.01) 400.10 40.08
b (79.94) 18.5 1.85
b (79.96)
13. JUR1+FTZ 183.61 30.81 (2.86) 8.48 1.42 (5.47) 316.19 13.66 (42.20) 14.62 0.63 (42.21)
14. JUR2+FTZ 221.45 37.48 (24.06) 10.23 1.72 (27.23) 214.95 9.88 (-3.32) 9.93 0.45 (-3.40)
15. JUR3+FTZ 325.01 86.06a (82.08) 15.02 3.98
a (86.81) 332.04131.97
d (39.33) 15.35 6.10
d (49.31)
16. JUR4+FTZ 402.95 53.81a (125.75) 18.63 2.49
a (131.71) 409.61 8.12
a (84.21) 18.94 0.37
b (84.24)
17. JUR1+FGD 243.23 31.13d (36.27) 10.78 1.68
d (34.07) 243.27 69.70 (9.40) 11.24 3.22 (9.33)
18. JUR2+FGD 241.59 42.26d (35.35) 10.7 2.38
d (34) 245.17 93.58 (10.26) 11.33 4.32 (5.15)
19. JUR3+FGD 248.34 49.43d (39.13) 11.48 2.28
c (42.78) 357.39 29.81
c (60.73) 11.12 1.16 (8.17)
20. JUR4+FGD 426.52 54.35a (138.96) 19.72 2.51
a (145.27) 370.56 83.84
c (66.65) 17.13 3.86
c (66.63)
21. JUF1+FTZ 219.23 15.25 (22.82) 10.13 0.70 (25.99) 200.79 26.75 (-9.69) 9.28 1.23 (-9.72)
22. FGD+FTZ 225.99 29.99 (26.61) 10.44 1.38 (29.85) 254.26 71.95 (14.35) 11.75 3.32 (14.29)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=fertilizer,FGD=fungicide
67
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Tota
l ch
loro
ph
yll
(m
g/g
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a a a
a a
d
a a
a
0
0.5
1
1.5
2
2.5
3
Tota
l ch
loro
ph
yll
(m
g/g
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a
d d
Figure 11: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total chlorophyll of H.annuus plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
50
100
150
200
250
300
350
Tota
l ca
rboh
yd
rate
(m
g/g
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a
d
a
0
50
100
150
200
250
300
350
400
450
Tota
l ca
rboh
yd
rate
(m
g/g
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b
c
b
Figure 12: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of H.annuus plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
50
100
150
200
250
300
350
400
450
Tota
l ca
rboh
yd
rate
(m
g /
g)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
a a
a a
a a a
0
50
100
150
200
250
300
350
400
450
Tota
l ca
rboh
yd
rate
(m
g/g
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b
c
b
d a
Figure 13: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total carbohydrate of H.annuus plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
2
4
6
8
10
12
14
16
18
20
Cru
de
pro
tein
(%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b c
b
0
2
4
6
8
10
12
14
16
Cru
de
pro
tein
(%
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a
d
a
Figure 14: Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of H.annuus plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
2
4
6
8
10
12
14
16
18
20
Cru
de
pro
tein
(%
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b a
a
d d c
a
0
2
4
6
8
10
12
14
16
18
20
Cru
de
pro
tein
(%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b
d
b c
Figure 15: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of H.annuus plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
0.5
1
1.5
2
2.5
Nit
rogen
(%
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a
d d
a
0
0.5
1
1.5
2
2.5
3
Nit
rogen
(%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b c
b
Figure 16: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent nitrogen of H.annuus plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
Nit
rogen
(%
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
a a
a
d d c
a
0
0.5
1
1.5
2
2.5
3
3.5
Nit
rogen
(%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b
d
b c
Figure 17: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent nitrogen of H.annuus plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Ph
osp
horu
s (%
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c d
b
d d
c
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Ph
osp
horu
s (%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
c
b
a
Figure 18: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent phosphorus of H.annuus plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
Out of four rhizobial isolates, JUR4 found active in increasing percent nitrogen
and phosphorus contents in test plants at both 30th
and 60th
days, followed by JUR1 at
30th
day. In combination with FTZ, JUR3 and JUR4 produced significant effect in
increasing the amounts of both minerals in test plants at 30th
and 60th
day. Where as
JUR2 in combination with FTZ induced significant increase in percent phosphorus of
sunflower plants at 30th
day. In combination with FGD, JUR4 found most effective in
promoting the contents of both nitrogen and phosphorus at 30th
and 60th
days, followed
by JUR3 + FGD found effective on both parameters at 30th
day, JUR1+ FGD and JUR2 +
FGD produced significant increase in nitrogen content only at 30th
day (Table 16; Figure
17, 19).
3.6.1.2. Brassica nigra Koch (black mustard)
3.6.1.2.1. Growth performance
T.hamatum (JUF1) alone induced increase in root and shoots lengths of mustard
plants less than 50% at both 30th
and 60th
days. The same positive effects were observed
in groups of plants treated with JUR2+JUF1 and JUR3+JUF1 at both days of harvesting.
JUR1+JUF1 promoted an increase in root length at both days from 22-39% while
JUR4+JUF1 promoted shoot length at 30th
day and root length at 60th
day with 31%
increase in each (Table 17; Figure 20, 22). All four rhizobial isolates encourage the
increase in root length from 16-39% of test plants at both days while shoot length from
27-58% at 30th
day. In combination with FTZ, JUR1 significantly increase root and shoot
lengths from 21-37% at 30th
and 60th
day. JUR2+FTZ and JUR3+FTZ increased root
length from 21-45% at both days and shoot length 43-51% at 30th
day while JUR4+FTZ
induced increase in both of these physical parameters only at 60th
day. Alongwith FGD,
JUR4 found more efficient in increasing root length from 19-22% at both days of
uprooting of plants as compared to JUR1+ FGD, JUR2+ FGD and JUR3 + FGD as all of
these treatments increased root length of test plants from14-24% at 60th
day (Table 17;
Figure 21, 23).
T.hamatum alone and co-inoculated with JUR1 improved fresh weight of
mustard plants at 60th
day (Table 17; Figure 24). Rhizobial isolates include JUR1, JUR2,
Table 16: Effect of treatments on mineral content of H.annuus (sunflower) plants
Mineral content
30th
day 60th
day
S. No. Treatment Nitrogen (%) Phosphorus (%) Nitrogen (%) Phosphorus (%)
1. Control 1.28 0.02 0.08 0.00 1.64 0.27 0.09 0.00
2. JUR1 1.9 0.18c (48.43) 0.15 0.07
d (87.5) 2.16 0.77 (31.70) 0.17 0.06 (88.88)
3. JUR2 1.28 0.11 (0) 0.1 0.00 (25) 2.11 0.40 (28.65) 0.19 0.13c (111.11)
4. JUR3 1.51 0.14 (17.96) 0.12 0.03 (50) 1.83 0.13 (11.58) 0.13 0.03 (44.44)
5. JUR4 2.45 0.24a (91.40) 0.18 0.08
b (125) 2.96 0.69
b (80.48) 0.23 0.11
b (155.55)
6. JUF1 1.79 0.15d (39.84) 0.16 0.08
c (100) 1.86 0.10 (13.41) 0.27 0.07
a (200)
7. FTZ 1.35 0.08 (5.46) 0.12 0.03 (50) 1.42 0.21 (-13.41) 0.1 0.03 (11.11)
8. FGD 1.21 0.11 (-5.46) 0.08 0.00 (0) 1.34 0.60 (-18.29) 0.09 0.00 (0)
9. JUR1+JUF1 1.61 0.28 (25.7) 0.14 0.01d (75) 2.72 0.73
c (65.8) 0.16 0.01 (77.77)
10. JUR2+JUF1 1.48 0.15 (15.62) 0.09 0.00 (12.5) 2.14 0.66 (30.48) 0.12 0.02 (33.33)
11. JUR3+JUF1 1.79 0.19d (39.84) 0.14 0.00
d (75) 1.67 0.54 (1.82) 0.15 0.00 (66.66)
12. JUR4+JUF1 2.2 0.30a (71.8) 0.17 0.00
c (112.5) 2.96 0.29
b (80.48) 0.32 0.02
a (255.55)
13. JUR1+FTZ 1.35 0.22 (5.46) 0.13 0.00 (62.5) 2.33 0.10 (42.07) 0.14 0.02 (55.55)
14. JUR2+FTZ 1.63 0.27 (27.34) 0.14 0.02d (75) 1.80 0.07 (9.75) 0.17 0.02 (88.88)
15. JUR3+FTZ 2.4 0.36a (87.5) 0.18 0.01
b (125) 2.45 0.97
d (49.3) 0.19 0.03
d (111.11)
16. JUR4+FTZ 2.98 0.40a (132.8) 0.19 0.00
a (137.5) 3.03 0.06
b (84.75) 0.21 0.01
c (133.33)
17. JUR1+FGD 1.72 0.26d (34.37) 0.12 0.03 (50) 1.79 0.51 (9.14) 0.14 0.03 (55.55)
18. JUR2+FGD 1.71 0.37d (33.59) 0.11 0.01 (37.5) 1.81 0.68 (10.36) 0.13 0.02 (44.44)
19. JUR3+FGD 1.83 0.36c (42.9) 0.14 0.00
d (75) 1.77 0.25 (7.92) 0.16 0.05 (77.77)
20. JUR4+FGD 3.15 0.40a (146.09) 0.21 0.02
a (162.5) 2.74 0.62
c (67.07) 0.23 0.03
c (155.55)
21. JUF1+FTZ 1.62 0.11 (26.56) 0.1 0.3 (25) 1.48 0.19 (-9.75) 0.12 0.04 (33.33)
22. FGD+FTZ 1.67 0.21 (30.46) 0.13 0.02 (62.5) 1.88 0.53 (14.63) 0.1 0.03 (11.11)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp IV, JUF1=T.hamatum, FTZ=Fertilizer,
FGD=Fungicide.
77
0
0.05
0.1
0.15
0.2
0.25
Ph
osp
horu
s (%
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
b
d
b a
d
a
0
0.05
0.1
0.15
0.2
0.25
Ph
osp
horu
s (%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c b
a
d c
c
Figure 19: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent phosphorus of H.annuus plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
Table 17: Effect of treatments on growth performance of B.nigra (black mustard) plants
Growth performance
30th
days 60th
days
S. No Treatment Root length* Shoot length* Fresh weight** Root length* Shoot length* Fresh weight**
1. Control 9.83 0.50 13.73 0.90 0.42 0.04 23.66 0.57 31.66 0.57 4.72 0.24
2. JUR1 12.23 0.87c (24.41) 21.66 2.51
a (57.75) 0.89 0.24
a (111.90) 27.66 3.40
d (16.90) 34.00 1.00 (7.39) 5.67 0.13 (20.12)
3. JUR2 13.03 1.45a (32.55) 19.83 2.75
a (44.42) 0.72 0.41
d (71.42) 32.93 2.80
a (39.18) 34.66 3.05 (9.47) 5.23 0.84 (10.80)
4. JUR3 12.06 1.47c (22.68) 22.83 1.15
a (66.27) 1.17 0.14
a (178.57) 29.10 1.38b (22.99) 35.66 2.51 (12.63) 6.15 1.77
d (30.29)
5. JUR4 12.00 1.44c (22.07) 17.50 0.90
d (27.45) 0.78 0.13
c (85.71) 29.83 1.15
a (26.07) 33.46 1.17 (5.68) 5.36 0.12 (13.55)
6. JUF1 11.66 0.28d (18.61) 18.00 1.35
c (31.09) 0.53 0.08 (26.19) 31.50 3.12
a (33.13) 38.33 4.04
c (21.06) 6.82 0.46
b (44.49)
7. FTZ 12.2 1.25b (24.10) 16.90 1.21
d (23.08) 0.54 0.10 (28.57) 28.43 1.70
c (20.16) 37.00 5.56
d (16.86) 5.05 1.01 (6.99)
8. FGD 11.13 0.96 (13.22) 15.03 0.80 (9.46) 0.53 0.83 (26.19) 28.40 1.73c (20.03) 29.33 0.57 (-7.35) 2.77 1.05 (-41.31)
9. JUR1+JUF1 12.20 1.57c (22.38) 15.50 1.24 (12.89) 0.46 0.05 (9.52) 32.96 2.45
a (39.30) 40.66 1.15
a (28.42) 6.48 1.59
c (58.68)
10. JUR2+JUF1 11.96 1.40d (23.09) 16.70 1.76
d (21.63) 0.47 0.09 (11.90) 30.60 1.35
a (29.33) 37.00 2.00
d (16.86) 5.51 0.10 (16.73)
11. JUR3+JUF1 11.36 1.50d (15.56) 17.13 0.65
d (24.76) 0.47 0.04 (11.90) 30.50 3.12
a (28.90) 36.00 2.64
d (13.70) 5.18 0.57 (9.74)
12. JUR4+JUF1 11.16 0.61 (17.29) 17.93 3.53c (30.58) 0.44 0.03 (4.76) 31.06 0.75
a (31.27) 35.56 0.77 (12.31) 5.24 0.04 (11.01)
13. JUR1+FTZ 11.86 1.38d (20.65) 17.46 0.92
d (27.16) 0.55 0.13 (30.95) 32.53 2.12
a (37.48) 38.66 1.15
b (22.10) 5.11 0.61 (8.26)
14. JUR2+FTZ 11.96 0.78d (21.66) 19.66 2.08
a (43.19) 0.75 0.15
c (78.57) 30.50 2.35
a (28.90) 35.66 2.30 (12.63) 4.8 0.21 (1.69)
15. JUR3+FTZ 12.6 0.55b (28.17) 20.83 1.21
a (51.71) 0.68 0.15
d (61.90) 34.40 2.15
a (45.39) 28.33 3.21 (-10.51) 6.46 0.51
c (36.86)
16. JUR4+FTZ 11.46 0.60 (16.58) 16.30 3.35 (34.23) 0.56 0.20 (33.33) 30.53 0.83a (29.03) 36.8 1.05
d (16.23) 4.99 0.52 (5.72)
17. JUR1+FGD 10.46 1.26 (6.40) 13.20 1.05 (-3.86) 0.36 0.45 (-14.28) 27.00 1.32d (14.11) 28.66 1.52 (-9.47) 3.36 0.23 (-28.81)
18. JUR2+FGD 10.5 0.36 (6.81) 14.26 0.36 (3.86) 0.28 0.45 (-33.33) 29.53 2.79a (24.80) 34.00 5.19 (7.39) 4.73 0.19 (0.21)
19. JUR3+FGD 11.33 0.70 (15.25) 14.76 0.30 (7.50) 0.36 0.02 (-14.28) 28.46 0.96c (20.28) 34.66 0.57 (9.47) 4.79 0.65 (1.48)
20. JUR4+FGD 11.73 0.37d (19.32) 15.73 0.98 (14.56) 0.45 0.12 (7.14) 28.90 1.15
b (22.14) 32.23 2.63 (1.80) 4.61 0.57 (-2.32)
21. JUF1+FTZ 10.3 0.40 (4.78) 10.56 2.69 (-23.08) 0.29 0.10 (-30.95) 29.66 0.35a (25.35) 34.66 1.52 (9.47) 4.81 0.69 (1.90)
22. FGD+FTZ 13.76 0.64a (39.97) 15.56 1.40 (13.32) 0.52 0.08 (23.80) 22.16 2.02 (-6.33) 28.00 2.64 (-11.56) 4.43 0.44 (-6.14)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. * = cm, ** = gm, JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=Fertilizer,FGD= Fungicide.
79
Figure 20: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of B.nigra plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
2
4
6
8
10
12
14
Root
len
gth
(cm
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a c c b c d
d
0
5
10
15
20
25
30
35
Root
len
gth
(cm
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
d
a
b a c c
a a a a a
0
5
10
15
20
25
30
35
Root
len
gth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
a
b a c c
a a
a
a d
a c b
Figure 21: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on root length of B.nigra plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
2
4
6
8
10
12
14
Root
len
gth
(cm
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
a c c b d d
b d
a
Figure 22: Effect of T.hamatum alone and in combination with rhizobial
isolates on shoot length of B.nigra plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
Sh
oo
t le
ng
th (
cm)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c
a a
a
d d d d c
0
5
10
15
20
25
30
35
40
45
Sh
oot
len
gth
(cm
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c d
a
d d
Figure 23: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on shoot length of B.nigra plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
Sh
oot
len
gth
(cm
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a a
a
d d d
a a
0
5
10
15
20
25
30
35
40
Sh
oo
t le
ngth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d b d
Figure 24: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of B.nigra plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
0.2
0.4
0.6
0.8
1
1.2
Fre
sh w
eigh
t (g
m)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
d
a
c
0
1
2
3
4
5
6
7
Fre
sh w
eigh
t (g
m)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b
d c
JUR3 and JUR4 induced increase in fresh weight of test plants with 112, 71, 179 and
86% respectively at 30th
day while JUR3 also increased the same parameter with 30% at
60th
day. In combination with FTZ, JUR2 and JUR3 improved the fresh weight of test
plants at 30th
day with 62-79% while JUR3+FTZ also produced 37% improvement in
fresh weight at 60th
day (Table 17; Figure 25).
3.6.1.2.2. Photosynthetic pigment
T.hamatum (JUF1) alone and co-inoculated with JUR1 increased total
cholorophyll and its fractions from 48-125% in leaves of mustard plants. JUR2+JUF1
and JUR3+JUF1 significantly promoted the same parameters from 58-105% in test plants
at 30th
day while the same first treatment promoted the chl-b (100%) and the second
treatment has promoted both chl-b (132%) & total chlorophyll (60%) at 60th
day. The
same significant effects were also produced by JUF1+FTZ on test plants (Table 18;
Figure 26, 27).
Out of rhizobial isolates, JUR3 found better in promoting the synthesis of total
chlorophyll (66%) and its fractions (102-131%) in test plants at 30th
day and while chl-b
(112%) at 60th
day, followed by JUR4 promoted the chl-a (78%) at 30th
day and chl-b
(84%) at 60th
day and JUR2 has promoted the chl-a & b from 74-112% at 60th
day. In
combination with FTZ, JUR1 and JUR2 become active and enhanced the synthesis of
total chlorophyll with its fractions from 66-158% at 60th
day. Whereas fraction a & b
were increased by JUR3+FTZ at 60th
day in test plants. In case with FGD, JUR2 found
effective and promoted the total chlorophyll with its fractions from 56-105% in test
plants at 60th
day (Table 18; Figure 26, 27).
3.6.1.2.3. Biochemical parameters
T.hamatum (JUF1) alone increased carbohydrate content with 215% in mustard
plants at 30th
day. The same fungus co-inoculated with JUR1, JUR2, JUR3 and JUR4
significantly stimulated the carbohydrate synthesis in leaves of test plants from 101-277%
Table 18: Effect of treatments on photosynthetic pigment of B.nigra (black mustard) plants
Photosynthetic pigment
30th
days 60th
days
S. No. Treatment Chl-a* Chl-b* Total Chl * Chl-a* Chl-b* Total Chl*
1. Control 0.73 0.03 0.57 0.07 1.67 0.05 0.77 0.03 0.43 0.04 0.93 0.08
2. JUR1 0.87 0.10 (19.17) 0.84 0.07 (47.36) 1.45 0.10 (-13.17) 0.91 0.06 (18.18) 0.70 0.08d (62.79) 1.23 0.07 (32.25)
3. JUR2 0.99 0.36 (35.61) 0.88 0.11 (54.38) 1.87 0.48 (11.97) 1.34 0.09c (74.02) 0.91 0.13
a (111.62) 2.38 0.22
a (155.91)
4. JUR3 1.69 0.49a (131.50) 1.15 0.12
c (101.75) 2.78 0.52
b (66.46) 0.78 0.10 (1.29) 0.91 0.21
a (111.62) 1.23 0.39 (59.13)
5. JUR4 1.30 0.38c (78.08) 0.96 0.04 (68.42) 2.17 0.44 (29.94) 0.71 0.05 (-7.79) 0.79 0.03
c (83.72) 0.80 0.20 (32.25)
6. JUF1 1.45 0.13b (98.63) 0.93 0.02 (63.15) 2.55 0.20
c (52.69) 1.36 0.23
c (76.62) 0.74 0.48
c (72.09) 2.09 0.44
a (124.73)
7. FTZ 0.92 0.13 (26.02) 1.14 0.47c (100) 1.17 0.89 (-29.94) 0.95 0.03 (23.37) 0.74 0.12
d (72.09) 1.05 0.34 (12.90)
8. FGD 0.72 0.09 (-1.36) 0.84 0.12 (47.36) 0.93 0.04 (-44.31) 1.28 0.24c (66.23) 0.90 0.09
a (109.30) 2.05 0.27
a (120.43)
9. JUR1+JUF1 1.29 0.13c (76.71) 1.24 0.00
c (117.54) 2.48 0.31
d (48.50) 1.16 0.22
d (50.64) 0.89 0.07
a (106.30) 1.65 0.54
c (77.41)
10. JUR2+JUF1 1.42 0.15b (94.52) 1.62 0.72
a (184.21) 3.05 0.71
a (82.63) 0.9 0.07 (16.88) 0.89 0.04
a (100) 1.05 0.17 (12.90)
11. JUR3+JUF1 1.46 0.34b (100) 1.17 0.21
c (105.26) 2.64 0.56
c (58.08) 1.04 0.40 (35.06) 1.00 0.15
a (132.55) 1.49 0.05
d (60.21)
12. JUR4+JUF1 0.85 0.14 (16.43) 0.82 0.06 (43.85) 1.68 0.20 (0.59) 0.77 0.97 (0) 0.87 0.06b (102.32) 0.87 0.23 (-6.45)
13. JUR1+FTZ 0.99 0.24 (35.61) 0.92 0.20 (61.40) 1.91 0.40 (14.37) 1.28 0.07c (66.83) 1.02 0.07
a (137.20) 2.40 0.12
a (158.06)
14. JUR2+FTZ 1.35 0.38c (84.93) 0.9 0.32 (57.89) 1.79 0.10 (7.18) 1.3 0.67
c (68.83) 0.83 0.11
c (93.02) 1.8 0.77
b (93.54)
15. JUR3+FTZ 1.03 0.18 (41.09) 0.93 0.06 (63.15) 2.1 0.19 (25.74) 1.19 0.40d (54.54) 0.84 0.13
b (95.34) 1.13 0.35 (21.05)
16. JUR4+FTZ 1.65 0.41a (126.02) 0.93 0.04 (63.15) 2.82 0.61
b (68.86) 0.62 0.02 (-19.48) 0.71 0.05
d (65.11) 0.77 0.06 (-17.20)
17. JUR1+FGD 0.98 0.04 (34.24) 0.86 0.04 (50.87) 1.91 0.18 (14.37) 0.71 0.14 (-7.79) 0.6 0.07 (39.53) 0.98 0.24 (5.37)
18. JUR2+FGD 0.89 0.32 (21.91) 0.8 0.22 (40.35) 1.83 0.63 (9.58) 1.20 0.10d (55.84) 0.7 0.30
d (62.79) 1.91 0.30
a (105.37)
19. JUR3+FGD 0.84 0.06 (15.06) 0.69 0.13 (21.05) 1.44 0.29 (-13.77) 0.98 0.21 (27.27) 0.54 0.08 (25.58) 1.53 0.28d (64.51)
20. JUR4+FGD 1.21 0.02d (65.75) 0.94 0.33 (64.91) 2.02 0.24 (20.95) 0.66 0.01 (-14.28) 0.68 0.60
d (58.13) 0.75 0.22 (-19.35)
21. JUF1+FTZ 0.67 0.04 (-8.21) 0.47 0.19 (-17.54) 0.98 0.4 (-41.31) 1.04 0.50 (35.06) 0.68 0.17d (58.13) 1.50 0.61
d (61.29)
22. FGD+FTZ 0.86 0.12 (17.80) 1.23 0.24c (115.78) 1.42 0.12 (-14.97) 0.72 0.20 (-6.49) 0.35 0.06 (-18.60) 1.08 1.25 (16.12)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control.* = mg/g, , chl = chlorophyll , JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=Fertilizer,FGD=fungicide.
86
0
0.2
0.4
0.6
0.8
1
1.2
Fre
sh w
eigh
t (g
m)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
d
a
c
c
d
Figure 25: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on fresh weight of B.nigra plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
1
2
3
4
5
6
7
Fre
sh w
eigh
t (g
m)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d c
Figure 26: Effect of T.hamatum alone and in combination with rhizobial
isolates on total chlorophyll of B.nigra plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
Tota
l ch
loro
ph
yll
(m
g/g
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c b
d
a
c
0
0.5
1
1.5
2
2.5
Tota
l ch
loro
ph
yll
(m
g/g
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
a
a
c d d
Figure 27: Effect of rhizobial isolates alone and their combination with fertilizer and
fungicide on total chlorophyll of B.nigra plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
Tota
l ch
loro
ph
yll
(m
g/g
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b b
0
0.5
1
1.5
2
2.5
Tota
l ch
loro
ph
yll
(mg/g
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a
a
b a
d
at both days of harvesting of plants. Similarly JUR2 (122%), JUR3 (119-129%) and
JUR4 (146-240%) individually in their respective group improved the carbohydrate
amount at 30th
and 60th
day. In combination with FTZ, JUR3 found most effective in
increasing the carbohydrate content from 120-140% at both days, followed by
JUR2+FTZ and JUR4+FTZ with 297% and 150% increase respectively at 60th
day.
Alongwith FGD, JUR3 and JUR4 found effective in increasing the amount of total
carbohydrate from 150-210% at 30 day and JUR4+FGD increased the same parameter
with 165% and JUR1+FGD induced 153% increase only at 30th
day.
(Table 19; Figure 28, 29).
T.hamatum alone improved crude protein content with 87% in test plants and co-
inoculated with JUR4 via 78% at 30th
day while JUR1+JUF1 and JUR3+JUF1 improved
the same content from 128-183% at both days. However JUR2+JUF1 increased crude
protein content with 277% in test plants at 60th
day. JUR4 found effective in enhancing
the crude protein content with 136-241% in test plants at both days, followed by JUR3
with 109% at 30th
day and JUR2 with 122% at 60th
day. JUR3 and JUR4 with FTZ
improved the same parameter from140-150% at 60th
day. In case with fungicide (FGD),
JUR3 and JUR4 increased crude protein content with 112 and 165% respectively at 30th
and 60th
day (Table 19; Figure 30, 31).
3.6.1.2.4. Mineral content
T.hamatum (JUF1) alone increased percent nitrogen in mustard plants at 30th
day. The same fungus co-inoculated with JUR1 and JUR3 promoted the nitrogen content
from 129-183% and 130-186% respectively at 30th
and 60th
day. JUR2+JUF1 (278%) and
JUR4 + JUF1 (80%) increased the same mineral content only at 60th
day and 30th
day
respectively. Out of four rhizobial isolates, JUR4 found most efficient in increasing the
nitrogen content of test plants from 139-242% at both days, followed by JUR3 with
111% at 30th
day and JUR2 with 123% increase in same parameter at 60th
day. In
combination with FTZ, JUR2, JUR3 and JUR4 induced increase from 141-152 % in
Table 19: Effect of treatments on biochemical parameters of B.nigra (black mustard) plants
Biochemical parameters
30th
days 60th
days
S. No. Treatment Total carbohydrate (mg/g) Crude protein (%) Total carbohydrate (mg/g) Crude proteins (%)
1. Control 83.91 3.98 4.05 0.30 112.91 91.00 5.21 0.13
2. JUR1 112.12 3.48 (33.61) 5.18 0.16 (27.90) 216.38 87.02 (91.63) 9.44 4.37 (81.19)
3. JUR2 185.89 11.28a (121.53) 6.74 1.65 (66.41) 250.99 149.13
d (122.29) 11.6 6.89
d (122.64)
4. JUR3 183.56 46.10b (118.75) 8.48 2.13
c (109.38) 258.75 18.14
d (129.16) 8.41 2.76 (61.42)
5. JUR4 206.86 14.97a (146.52) 9.56 0.69
a (136.04) 384.20 100.11
a (240.27) 17.76 4.63
a (240.88)
6. JUF1 264.87 31.84a (215.65) 7.57 3.05
d (86.91) 188.11 11.94 (66.40) 8.69 0.55 (66.79)
7. FTZ 107.31 10.14 (27.88) 4.96 1.47 (22.46) 247.82 126.30d (119.48) 11.45 5.83 (119.76)
8. FGD 153.39 48.32d (82.80) 7.09 2.23
d (75.06) 172.99 54.55 (53.21) 7.99 2.52 (53.35)
9. JUR1+JUF1 199.78 34.39a (138.08) 9.23 1.58
b (127.90) 318.83 130.48
c (182.37) 14.74 6.03
c (182.91)
10. JUR2+JUF1 169.12 15.98c (101.54) 6.58 0.45 (62.46) 425.46 59.34
a (276.81) 19.67 2.74
a (277.54)
11. JUR3+JUF1 248.24 6.40a (195.84) 11.48 0.29
a (183.45) 258.44 27.38
d (128.89) 11.95 1.26
d (129.36)
12. JUR4+JUF1 179.3118.48b (113.69) 7.21 1.28
d (78.02) 243.96 30.86
d (116.06) 11.28 1.42 (116.50)
13. JUR1+FTZ 126.86 77.13 (51.18) 5.86 3.56 (44.69) 225.83 89.26 (100.00) 10.44 4.13 (100.38)
14. JUR2+FTZ 72.39 28.09 (-13.72) 3.34 1.29 (-17.53) 448.40 66.75a (297.13) 20.73 3.09
a (297.88)
15. JUR3+FTZ 184.96 7.00a (120.42) 5.93 1.13 (46.41) 270.64 203.33
d (139.69) 12.51 9.39
d (140.11)
16. JUR4+FTZ 111.96 3.38 (33.42) 5.17 0.14 (27.65) 282.64 40.02d (150.32) 13.06 1.85
d (150.67)
17. JUR1+FGD 211.99 23.84a (152.63) 9.8 1.10
a (141.97) 220.71 48.34 (95.47) 10.2 2.23 (95.77)
18. JUR2+FGD 116.14 3.93 (38.41) 5.36 0.18 (32.34) 131.62 21.91 (16.57) 6.08 1.01 (16.69)
19. JUR3+FGD 260.27 22.13a (210.17) 8.59 2.20
c (112.09) 202.27 33.10 (79.14) 9.35 1.52 (79.46)
20. JUR4+FGD 210.15 17.72a (150.44) 6.79 0.73 (67.65) 299.44 54.73
c (165.20) 13.84 2.53
c (165.64)
21. JUF1+FTZ 108.95 15.74 (29.84) 5.03 0.72 (24.19) 130.62 83.84 (15.68) 6.03 3.87 (15.73)
22. FGD+FTZ 269.16 17.41a (220.77) 6.89 2.13 (70.12) 166.18 66.81 (47.17) 7.68 3.08 (47.40)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum ,FTZ=Fertilizer,FGD=Fungicide.
91
Figure 28: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of B.nigra plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
50
100
150
200
250
300 T
ota
l ca
reb
oh
yd
rate
(m
g/g
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
a b
a
d
a
c
a
b
a
0
50
100
150
200
250
300
350
400
450
Tota
l ca
rboh
yd
rate
(m
g/g
) 60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d d
a
d
c
a
d
Figure 29: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total carbohydrate of B.nigra plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
50
100
150
200
250
300
Tota
l ca
rboh
yd
rate
(m
g/g
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a b a
d
a a
a
a
a
0
50
100
150
200
250
300
350
400
450
Tota
l ca
rboh
yd
rate
(m
g/g
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d d
a
d
a
d d c
Figure 30: Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of B.nigra plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
2
4
6
8
10
12
Cru
de
pro
tein
(%
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a
d
b
a
d
0
2
4
6
8
10
12
14
16
18
20
Cru
de
pro
tein
(%
)
60th day
Control JUF1 JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+JUF1 JUR2+JUF1 JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
a
c
a
d
Figure 31: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of B.nigra plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
1
2
3
4
5
6
7
8
9
10
Cru
de
pro
tein
(%
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
a
d
a
c
0
5
10
15
20
25
Cru
de
pro
tein
(%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
a a
d d c
nitrogen content of test plants. Where as JUR1 and JUR3 with FGD increased the same
parameter from 114-144% at 30th
day and JUR1+ FGD with 166% at 60th
day (Table 20;
Figure 32, 33).
T.hamatum co-inoculated with JUR3 and JUR4 improved the phosphorus
content in mustard plants respectively 106% at 30th
day and 114% at 60th
day. Rhizobial
isolates include JUR3 and JUR4 significantly increased phopshorus content in test plants
from 75-114% at both days of uprooting of plants (Table 20; Figure 34, 35).
3.6.2. Effect of microbial inoculants on legume plants
3.6.2.1. Vigna mungo L. (black gram)
3.6.2.1.1. Growth performance
T.hamatum alone induced percent increase in shoot and root lengths upto 53 and
23% of V.mungo plants respectively at 30th
and 60th
day. T.hamatum co-inoculated with
JUR1, JUR3 and JUR4 stimulated the length of roots and shoots of test plants from 18 -
81 % at 30th
and 60th
day while JUR2+ JUF1 produced significant results on shoot length
of test plants with 91% increase at 30th
day and on both root (19%) and shoot (39%)
lengths at 60th
day. JUR4 found most effective in increasing the lengths of both roots
(25-33%) and shoots (43-51%) of test plants at both 30th
and 60th
day. Whereas JUR1
induced 17% increase in root length at 30th
day and in roots & shoots both from 15 -24%
at 60th
day. JUR2 supported the root length of test plants with 24% and 16% respectively
on 30th
and 60th
day while promoted the shoot length with 54% increase only on 30th
day.
Opposite results obtained by JUR3 that improved the shoot length of test plants with 32-
38% at both days while roots on 60th
day. In combination with fertilizer (FTZ), JUR3 and
JUR4 accelerated the root and shoot lengths at both days while JUR1+FTZ and
JUR2+FTZ increased the both physical parameters from 17-33% at 60th
day. Alongwith
fungicide (FGD), again JUR4 become active and showed positive effects on both root
and shoot lengths at both days, followed by JUR1 and JUR2 produced significant results
on root and shoot lengths at 30th
day and on roots at 60th
day (Table 21; Figure 36, 37, 38,
39).
Table 20: Effect of treatments on mineral content of B.nigra (black mustard) plants
Mineral content
30th
days 60th
days
S. No. Treatment Nitrogen (%) Phosphorus (%) Nitrogen (%) Phosphorus (%)
1. Control 0.64 0.05 0.16 0.01 0.83 0.02 0.21 0.03
2. JUR1 0.82 0.02 (28.12) 0.12 0.03 (-25) 1.51 0.69 (81.92) 0.22 0.04 (4.76)
3. JUR2 1.07 0.26 (67.18) 0.25 0.05 (56.25) 1.85 1.10d (122.89) 0.36 0.12 (71.42)
4. JUR3 1.35 0.34c (110.93) 0.28 0.06
d (75) 1.34 0.44 (61.44) 0.38 0.11
d (80.95)
5. JUR4 1.53 0.11a (139.06) 0.30 0.08
d (87.5) 2.84 0.74
a (242.16) 0.45 0.08
c (114.28)
6. JUF1 1.21 0.49d (89.06) 0.16 0.02 (0) 1.39 0.90 (67.46) 0.19 0.23 (-9.52)
7. FTZ 0.79 0.07 (23.43) 0.11 0.01 (-31.25) 1.83 0.93 (120.48) 0.14 0.95 (-33.33)
8. FGD 1.13 0.35d (76.56) 0.15 0.05 (-6.25) 1.27 0.40 (53.01) 0.15 0.00 (-28.57)
9. JUR1+JUF1 1.47 0.25b (129.68) 0.18 0.04 (12.5) 2.35 0.96
c (183.13) 0.21 0.05 (0)
10. JUR2+JUF1 1.05 0.07 (64.06) 0.11 0.02 (-31.25) 3.14 0.43a (248.31) 0.32 0.12 (52.38)
11. JUR3+JUF1 1.83 0.04a (185.93) 0.33 0.13
c (106.25) 1.91 0.19
d (130.12) 0.24 0.22 (14.28)
12. JUR4+JUF1 1.15 0.20d (79.68) 0.11 0.03 (-31.25) 1.80 0.22 (116.86) 0.45 0.20
c (114.28)
13. JUR1+FTZ 0.93 0.56 (45.31) 0.08 0.03 (-50) 1.67 0.66 (101.20) 0.28 0.11 (33.33)
14. JUR2+FTZ 0.53 0.21 (-17.18) 0.12 0.03 (-25) 3.31 0.49a (298.79) 0.12 0.03 (-42.85)
15. JUR3+FTZ 0.94 0.17 (46.87) 0.20 0.02 (25) 2.00 1.50d (140.96) 0.25 0.10 (19.04)
16. JUR4+FTZ 0.82 0.02 (28.12) 0.14 0.05 (-12.5) 2.09 0.29d (151.80) 0.20 0.13 (-4.76)
17. JUR1+FGD 1.56 0.17a (143.75) 0.18 0.02 (12.5) 1.63 0.35 (96.38) 0.08 0.03 (-61.90)
18. JUR2+FGD 0.85 0.02 (32.81) 0.15 0.00 (-6.25) 0.97 0.16 (16.86) 0.26 0.15 (23.80)
19. JUR3+FGD 1.37 0.35c (114.06) 0.12 0.73 (-25) 1.49 0.24 (79.51) 0.21 0.11 (0)
20. JUR4+FGD 1.08 0.11 (68.75) 0.10 0.17 (-37.5) 2.21 0.40c (166.26) 0.12 0.04 (-42.85)
21. JUF1+FTZ 0.8 0.12 (25) 0.16 0.76 (0) 0.96 0.62 (15.66) 0.28 0.11 (33.33)
22. FGD+FTZ 1.1 0.34 (71.87) 0.19 0.05 (18.75) 1.22 0.49 (46.98) 0.23 0.07 (9.52)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=Fertilizer, FGD=Fungicide
97
Figure 32: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent nitrogen of B.nigra plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2 N
itro
gen
(%
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a
d
b
a
d
0
0.5
1
1.5
2
2.5
3
3.5
Nit
rogen
(%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
a
c
a
d
Figure 33: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent nitrogen of B.nigra plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Nit
rogen
(%
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c a
d
a
c
0
0.5
1
1.5
2
2.5
3
3.5
Nit
rogen
(%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
a
a
d d c
Figure 34: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent phosphorus of B.nigra plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Ph
osp
horu
s (%
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d d
c
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Ph
osp
horu
s (%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
c c
Figure 35: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent phosphorus of B.nigra plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
0.05
0.1
0.15
0.2
0.25
0.3
Ph
osp
horu
s (%
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d d
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Ph
osp
horu
s (%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
c
Table 21: Effect of treatments on growth performance of V. mungo (black gram) plants
Growth performance
30th
days 60th
days
S.No. Treatment Root length* Shoot length* Fresh weight** Root length* Shoot length* Fresh weight**
1. Control 27.5 2.29 13.33 0.76 0.67 0.09 30.4 0.17 18.83 1.04 1.18 0.39
2. JUR1 32.16 1.82d (16.94) 17.60 2.08 (32.03) 1.18 0.44 (76.11) 34.5 0.88
c (14.80) 23.33 1.52
d (23.89) 1.35 0.10 (14.40)
3. JUR2 34.20 3.12c (24.36) 20.56 1.20
c (54.58) 1.39 0.42
d (107.46) 35.33 1.25
b (16.21) 21.50 0.50 (14.17) 2.01 1.34 (70.33)
4. JUR3 31.16 0.85 (13.30) 18.43 1.40d (38.57) 1.13 0.38 (68.65) 35.76 0.81
b (17.63) 24.83 1.75
c (31.86) 2.02 0.51 (71.18)
5. JUR4 36.73 1.36a (33.56) 24.06 2.53
a (50.82) 1.68 0.42
b (150.74) 38.1 2.02
a (25.32) 27.03 3.28
a (43.54) 3.49 0.19
a (231)
6. JUF1 30.30 1.35 (10.18) 20.33 4.72c (52.85) 1.21 0.50 (80.59) 37.4 1.01
a (23.02) 22.00 2.00 (16.83) 1.37 0.43 (16.10)
7. FTZ 30.06 1.50 (9.30) 20.90 2.28b (57.54) 2.14 0.13
a (219.40) 34.9 1.65
c (14.80) 25.33 0.57
c (34.36) 2.19 0.42 (85.59)
8. FGD 31.86 1.77d (15.85) 21.70 0.64
a (63.15) 1.66 0.16
c (147.76) 32.33 2.51 (6.34) 23.66 3.78
d (25.65) 1.69 0.08 (43.22)
9. JUR1+JUF1 32.70 2.45c (18.90) 23.66 4.01
a (77.89) 1.13 0.45 (68.65) 36.56 1.91
a (20.26) 24.23 1.75
c (28.67) 2.02 0.74 (71.18)
10. JUR2+JUF1 30.46 0.85 (10.76) 25.43 1.40a (91.20) 1.54 0.68
c (129.85) 36.10 1.40
a (18.75) 26.16 1.75
a (38.92) 2.29 0.42 (94.06)
11. JUR3+JUF1 33.70 1.27c (22.54) 24.16 3.68
a (81.65) 1.48 0.26
c (120.89) 35.60 0.85
b (17.10) 25.00 1.00
c (32.76) 2.06 0.31 (74.57)
12. JUR4+JUF1 36.46 2.60a (32.58) 22.33 2.46
a (67.14) 1.73 0.35
b (158.20) 40.16 4.25
a (32.10) 24.70 1.05
c (31.17) 3.09 0.41
c (161.86)
13. JUR1+FTZ 30.06 1.48 (9.30) 19.56 2.20c (47.06) 1.33 0.11
d (98.50) 35.73 1.86
b (17.53) 25.00 2.64
c (32.76) 2.69 1.09
d (127.11)
14. JUR2+FTZ 28.23 0.87 (2.65) 20.66 5.00c (55.33) 1.31 0.30
d (95.52) 38.33 2.25
a (26.08) 24.16 2.02
c (28.30) 2.43 0.22
d (105.93)
15. JUR3+FTZ 34.33 5.39c (24.83) 22.83 2.36
a (71.65) 1.22 0.40 (82.08) 37.30 2.65
a (22.69) 23.66 2.30
d (25.65) 2.41 1.24
d (104.23)
16. JUR4+FTZ 36.63 1.48a (32.21) 25.9 1.65
a (94.73) 1.64 0.03
c (144.77) 40.13 1.18
a (9.53) 27.06 3.27
a (43.70) 2.98 0.56
c (152.54)
17. JUR1+FGD 32.20 1.65d (17.09) 20.33 1.89
c (52.85) 1.07 0.18 (59.70) 37.83 1.05
a (24.44) 22.00 1.73 (16.83) 2.15 0.34 (82.82)
18. JUR2+FGD 33.20 2.60c (20.72) 20.50 1.32
c (54.13) 1.17 0.30 (74.62) 38.06 1.90
a (25.19) 22.66 4.04 (20.33) 2.37 0.42
d (101.84)
19. JUR3+FGD 29.26 0.75 (6.4) 17.16 4.61 (29.02) 1.23 0.44 (83.58) 34.13 1.72d (12.26) 21.66 4.50 (15.02) 1.60 0.35 (35.59)
20. JUR4+FGD 35.40 6.02a (28.72) 21.50 1.32
b (61.65) 1.41 0.24
d (110.44) 36.03 0.45
a (18.51) 24.83 2.01
c (31.86) 2.10 0.10 (77.96)
21. JUF1+FTZ 18.40 5.71 (-33.09) 15.96 1.45 (20) 1.07 0.08 (59.70) 37.5 1.70a (23.35) 24.33 3.21
c (29.20) 2.61 0.32
d (121.18)
22. FGD+FTZ 33.53 3.22c (21.92) 21.86 0.32
a (64.36) 2.7 0.53
a (302.98) 34.76 1.12
c (14.34) 23.66 3.51
d (25.65) 2.74 0.15
c (132.20)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. * = cm, ** = gm, JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum ,FTZ=Fertilizer,FGD=Fungicide.
102
0
5
10
15
20
25
30
35
40
45
Root
len
gth
(cm
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a c b b
a c
a a b a
a c
0
5
10
15
20
25
30
35
40 R
oot
len
gth
(cm
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a
d c c a
c
Figure 36: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of V.mungo plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
Figure 37: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on root length of V.mungo plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30
35
40
Root
len
gth
(cm
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a c
d c
a d c a
c d
0
5
10
15
20
25
30
35
40
45
Root
len
gth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b c b a
c b a a
a a a
d a c
Figure 38: Effect of T.hamatum alone and in combination with rhizobial isolates
on shoot length of V.mungo plants. Columns bearing superscript are statistically
significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30 S
hoot
len
gth
(cm
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c c d
a
b a a
a a
a a
0
5
10
15
20
25
30
Sh
oot
len
gth
(cm
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d c
a c
d c a
c c c d
Figure 39: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on shoot length of V.mungo plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30
Sh
oot
len
gth
(cm
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c d
a b a
c c a
a
c c b a
0
5
10
15
20
25
30
Sh
oot
len
gth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d c a
c d
c c d
a c
d
Fresh weight of V.mungo plants was improved after treated with JUR4+JUF1 at
30th
(158%) and 60th
(162%) day while JUR2+JUF1 and JUR3+JUF1 showed
improvement in same parameter with 121-130% increase at 30th
day. JUR4 alone found
most effective in improving the fresh weight of test plants at 30th
(151%) and 60th
(231%)
day as compared to control plants while JUR2 improved same parameter (107%) at 30th
day. In combination with FTZ, JUR1, JUR2 and JUR4 significantly improved fresh
weights of plants more than 100% at both days in their respective groups while
JUR3+FTZ produced same significant effect on same parameter at 60th
day. In
combination with FGD, JUR2 and JUR4 produced better effects on fresh weights of test
plants respectively at 60th
and 30th
day (Table 21; Figure 40, 41).
3.6.2.1.2. Photosynthetic pigment
T.hamatum induced significant increase in total chlorophyll and its fractions
from 241-278% in leaves of V.mungo plants at 30th
day. T.hamatum co-inoculated with
JUR1, JUR3 and JUR4 stimulated the synthesis of same parameter and its fractions in
their respective groups as compared to control plants at both days (Table 22; Figure 42).
JUR1 produced significant results on chlorophyll content and its fractions from 69-111%
at 30th
and 60th
day while JUR4 at 60th
day. In combination with FTZ, all four rhizobial
isolates found effective in increasing the amount of photosynthetic pigment and its
fractions at both days of uprooting of plants. Similary JUR3 and JUR4 also found active
in combination with FGD in promoting the content of chlorophyll and its fractions in test
plants at both 30 and 60th
day. Whereas JUR2+FGD promoted the total chlorophyll in test
plants at 30th
day only (Table 22; Figure 43).
3.6.2.1.3. Biochemical parameters
T.hamatum (JUF1) alone induced 70% signifiicant increase in each of
carbohydrate and crude protein contents of V.mungo plants at 60th
day. Whereas the same
fungus co-inoculated with JUR1 induced 42-43% increase in both parameters at 30th
day
and with JUR4 it become active and gave prominent results from 74 -77% on both bio-
Figure 40: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of V.mungo plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
Fre
sh w
eigh
t (g
m)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d b
a
c c c
b
a
0
0.5
1
1.5
2
2.5
3
3.5
Fre
sh w
eigh
t (g
m)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
c
c d
Figure 41: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on fresh weight of V.mungo plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3 F
resh
wei
gh
t (g
m)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
b
a
c
d d
c d
a
0
0.5
1
1.5
2
2.5
3
3.5
Fre
sh w
eigh
t (g
m)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
d d d
c
d
c
Table 22: Effect of treatments on photosynthtic pigment of V. mungo (black gram) plant
Photosynthetic pigment
30th
days 60th
days
S. No Treatment Chl-a* Chl-b* Total Chl* Chl-a* Chl-b* Total Chl*
1. Control 0.36 0.02 0.65 0.05 0.81 0.01 0.44 0.04 0.80 0.07 0.95 0.07
2. JUR1 0.76 0.06a (111.11) 1.31 0.00
b (101.53) 1.69 0.08
a (108.64) 0.81 0.08
d (84.09) 1.35 0.10
d (68.75) 1.83 0.04
c (92.63)
3. JUR2 0.54 0.06 (50) 0.99 0.11 (52.30) 1.25 0.08d (54.72) 0.70 0.02 (59.09) 1.27 0.05 (58.75) 1.39 0.04 (46.31)
4. JUR3 0.53 0.02 (47.22) 0.93 0.00 (43.07) 1.14 0.02 (40.74) 0.54 0.12 (22.72) 0.99 0.34 (23.75) 1.55 0.53d (63.15)
5. JUR4 0.52 0.14 (44.44) 1.00 0.36 (53.84) 1.17 0.40d (44.44) 0.86 0.35
d (95.45) 1.33 0.13
d (66.25) 2.2 0.44
a (131.57)
6. JUF1 1.36 0.23a (277.77) 2.46 0.41
a (278.46) 2.76 0.45
a (240.74) 0.58 0.11 (31.81) 1.05 0.21 (31.25) 1.2 0.23 (26.31)
7. FTZ 1.55 0.08a (330.55) 1.70 0.15
a (161.53) 2.84 0.29
a (250.61) 0.54 0.09 (22.72) 1.43 0.46
d (78.75) 1.55 0.52
d (63.15)
8. FGD 1.55 0.11a (330.55) 1.45 0.13
a (123.07) 1.68 0.13
a (107.40) 0.74 0.08 (68.18) 1.34 0.15
d (67.50) 1.27 0.55 (33.68)
9. JUR1+JUF1 0.65 0.09c (80.55) 1.07 0.22
d (64.61) 1.34 0.16
c (65.43) 0.91 0.08
c (106.81) 1.60 0.10
c (100) 1.81 0.16
c (90.52)
10. JUR2+JUF1 0.83 0.02a (130.55) 1.51 0.04
a (132.30) 1.83 0.12
a (125.92) 0.80 0.14 (81.81) 1.18 0.33 (47.5) 1.65 0.11
d (73.68)
11. JUR3+JUF1 0.74 0.03a (105.55) 1.34 0.05
b (106.15) 1.57 0.05
a (93.82) 0.94 0.04
c (113.63) 1.62 0.17
b (102.50) 1.93 0.05
b (103.15)
12. JUR4+JUF1 1.39 0.22a (286.11) 2.35 0.50
a (261.53) 2.84 0.30
a (250.61) 0.85 0.47
d (93.18) 1.46 0.14
c (82.50) 1.70 0.52
c (78.94)
13. JUR1+FTZ 1.48 0.06a (311.11) 2.77 0.05
a (326.15) 2.91 0.23
a (259.25) 1.37 0.08
a (211.36) 2.59 0.63
a (223.75) 2.87 0.06
a (202.10)
14. JUR2+FTZ 1.28 0.30a (255.55) 2.10 0.44
a (223.07) 2.28 0.43
a (181.48) 1.23 0.48
a (179.54) 2.00 0.59
a (150) 2.64 0.22
a (177.89)
15. JUR3+FTZ 1.38 0.22a (263.33) 2.07 0.04
a (218.46) 2.23 0.09
a (175.30) 1.24 0.04
a (181.81) 1.76 0.32
a (120) 2.53 0.09
a (166.31)
16. JUR4+FTZ 0.99 0.13a (175) 1.70 0.11
a (161.53) 2.02 0.25
a (149.38) 1.40 0.04
a (218.18) 0.49 0.14 (-38.75) 2.06 0.26
a (116.84)
17. JUR1+FGD 0.55 0.01 (52.77) 0.89 0.11 (36.92) 1.04 0.19 (28.39) 0.69 0.07 (56.81) 0.83 0.02 (3.75) 1.33 0.55 (40)
18. JUR2+FGD 0.73 0.08b (102.77) 1.32 0.14
b (103.07) 1.48 0.18
a (82.71) 0.76 0.11 (72.72) 1.24 0.43 (55) 1.41 0.40 (48.42)
19. JUR3+FGD 0.77 0.02a (113.88) 1.39 0.04
a (113.84) 1.57 0.09
a (93.82) 0.91 0.02
c (106.81) 1.42 0.18
d (77.5) 1.77 0.07
c (86.31)
20. JUR4+FGD 0.83 0.06a (130.55) 1.52 0.14
a (133.84) 1.69 0.14
a (108.64) 1.01 0.24
b (129.54) 1.54 0.01
c (92.5) 1.95 0.44
b (105.26)
21. JUF1+FTZ 0.20 0.05 (-44.44) 0.37 0.09 (-43.07) 0.42 0.10 (-48.14) 1.13 0.31a (156.81) 1.77 0.13
a (121.25) 1.71 0.58
c (80)
22. FGD+FTZ 0.98 0.10a (172.22) 1.66 0.32
a (155.38) 1.38 0.11
c (70.37) 0.71 0.11 (61.36) 1.38 0.52
d (72.5) 1.28 0.10 (34.73)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represents percent increase or decrease (-) with respective control. * = mg/g, , chl = chlorophyll, JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=Fertilizer, FGD=Fungicide.
110
Figure 42: Effect of T.hamatum alone and in combination with rhizobial isolates
on total chlorophyll of V.mungo plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
Tota
l ch
loro
ph
yll
(m
g/g
)
30th day
Control JUF1 JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+JUF1 JUR2+JUF1 JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
a
a
a
c
a
a
a
c d d
0
0.5
1
1.5
2
2.5
Tota
l ch
loro
ph
yll
(m
g/g
)
60th day
Control JUF1 JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+JUF1 JUR2+JUF1 JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c
d
a
d
c c
b
d c
Figure 43: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on total chlorophyll of V.mungo plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3 T
ota
l ch
loro
ph
yll
(m
g/g
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a
a
a
a a a
b a a
a
0
0.5
1
1.5
2
2.5
3
Tota
l ch
loro
ph
yll
(mg/g
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
d
a
d
a a a
a
c b
chemical parameters at both days of uprooting of plants. Rhizobial isolates include JUR3
and JUR4 improved 67 and 80% respectively the synthesis of total carbohydrate and
crude protein contents at 60th
day. JUR3 and JUR4 alongwith FTZ induced significant
percent increase in carbohydrate and crude protein contents in test plants from 78 - 193%
at both days of harvesting. In combination with FGD, JUR2 and JUR4 enhanced the
synthesis of both biochemical parameters significantly from 45 - 96% at 30th
and 60th
day
(Table 23; Figure 44, 45, 46, 47).
3.6.2.1.4. Mineral content
T.hamatum alone improved the nitrogen content (71%) at 60th
day and the same
fungus when co-inoculated with JUR4 induced significant increase in percent nitrogen
content in V.mungo plants from 76 - 128% at 30th
and 60th
day (Table 24; Figure 48).
Out of rhizobial isolate, JUR3 and JUR4 produced significant promotion in nitrogen
content of test plants from 68-81% at 60th
day. The same two rhizobial isolates in
combination with FTZ also produced significant effect by increasing the nitrogen content
of V.mungo plants from 77-191% at 30th
and 60th
day while JUR2+ FTZ produced 109%
increase in same parameter on 30th
day. In combination with FGD, JUR2 and JUR4
became helpful in enhancing the nitrogen amount in test plants from 45 - 94 % at both
days of uprooting of plants (Table 24; Figure 49).
T.hamatum co-inoculated with JUR4 induced 128% increase in phosphorus
content of V.mungo plants at 60th
day (Table 24; Figure 50). Rhizobial isolates include
JUR1 and JUR4 increased 107 and 114% nitrogen content respectively in test plants at
60th
day. Alongwith FTZ, JUR2 and JUR4 induced increase from 193-282% in the same
mineral in test plant at 30th
and 60th
day while JUR3+ FTZ enhanced 107% phosphorus
content in test plants at 60th
day. All four rhizobial isolates in combination with FGD
stimulated the increase in phosphorus content in their respective groups of test plants
from 86 - 164 % at 60th
day (Table 24; Figure 51).
Table 23: Effect of treatments on biochemical parameters of V. mungo (black gram) plants
Biochemical parameters
30
th
days 60th
days
S. No. Treatment Total carbohydrate (mg/g) Crude proteins (%) Total carbohydrates(mg/g) Crude proteins (%)
1. Control 175.32 11.89 9.65 0.70 192.91 14.55 10.54 0.79
2. JUR1 202.37 6.81 (15.42) 11.06 0.36 (14.61) 236.98 47.22 (22.84) 12.95 2.58 (22.86)
3. JUR2 186.26 7.27 (6.24) 10.17 0.40 (5.38) 228.26 35.67 (18.32) 12.47 1.95 (18.31)
4. JUR3 202.90 7.32 (15.73) 11.08 0.39 (14.81) 322.90 65.13c (67.38) 17.64 3.56
c (20.26)
5. JUR4 241.47 63.31 (37.73) 13.19 3.45 (36.68) 347.68 18.21b (80.22) 19.00 0.97
a (80.20)
6. JUF1 229.53 19.90 (30.92) 12.54 1.08 (29.94) 328.29 105.27c (70.17) 17.94 5.75
b (70.20)
7. FTZ 195.29 50.19 (11.39) 10.67 2.74 (10.56) 215.90 9.88 (11.91) 11.79 0.53 (11.85)
8. FGD 170.56 5.81 (-2.71) 9.32 0.31 (-3.41) 207.66 50.45 (7.64) 11.34 2.75 (7.59)
9. JUR1+JUF1 250.99 11.49d (43.16) 13.71 0.63
d (42.38) 264.09 34.45 (36.89) 14.43 1.88 (36.90)
10. JUR2+JUF1 243.17 27.63 (38.70) 13.28 1.51 (37.61) 255.69 32.59 (32.54) 13.97 1.78 (32.54)
11. JUR3+JUF1 180.71 5.29 (3.07) 9.87 0.28 (2.27) 212.73 25.26 (10.27) 11.62 1.37 (10.72)
12. JUR4+JUF1 310.43 39.55b (77.06) 16.96 2.16
b (75.75) 336.74 17.38
b (74.55) 18.4 0.95
b (74.57)
13. JUR1+FTZ 201.47 36.85 (14.91) 11.00 2.01 (13.98) 223.51 24.94 (15.86) 12.21 1.36 (15.84)
14. JUR2+FTZ 361.42 87.40a (106.14) 19.75 4.78
a (104.66) 415.78 15.53
a (115.53) 13.17 3.6 (24.95)
15. JUR3+FTZ 419.76 77.42a (139.42) 22.94 4.23
a (137.72) 343.03 46.67
b (77.81) 18.74 2.54
a (77.79)
16. JUR4+FTZ 514.23 72.99a (193.30) 28.10 3.98
a (191.19) 367.81 81.56
a (90.66) 20.10 4.45
a (90.70)
17. JUR1+FGD 209.24 59.30 (19.34) 11.43 3.23 (18.44) 233.07 52.62 (20.81) 12.73 2.87 (20.77)
18. JUR2+FGD 256.96 79.80d (46.56) 14.04 4.36
d (45.49) 279.89 62.41
d (45.08) 15.29 3.41
d (45.06)
19. JUR3+FGD 170.40 12.90 (-2.80) 9.31 0.70 (-3.52) 207.18 21.51 (7.39) 11.32 1.17 (7.40)
20. JUR4+FGD 343.61 49.98a (95.99) 18.77 2.73
a (94.50) 350.16 20.41
a (81.51) 19.13 1.11
a (81.49)
21. JUF1+FTZ 200.63 52.75 (14.43) 10.96 2.88 (13.37) 219.86 32.27 (13.97) 12.01 1.76 (13.94)
22. FGD+FTZ 188.74 16.10 (7.65) 10.31 0.87 (6.83) 203.38 5.51 (5.42) 11.11 0.30 (5.40)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum , FTZ=Fertilizer,
FGD=Fungicide.
114
Figure 44: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of V.mungo plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
50
100
150
200
250
300
350
Tota
l ca
rboh
yd
rate
(m
g/g
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b
d
0
50
100
150
200
250
300
350
Tota
l ca
rboh
yd
rate
(m
g/g
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c c b b
Figure 45: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on total carbohydrate of V.mungo plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
100
200
300
400
500
600
Tota
l ca
rboh
yd
rate
(m
g/g
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a
a
d
a
0
50
100
150
200
250
300
350
400
450
Tota
l ca
rboh
yd
rate
(m
g/g
) 60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b
a
b c
a
d
a
Figure 46: Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of V.mungo plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
2
4
6
8
10
12
14
16
18
Cru
de
pro
tein
(%
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
b
0
2
4
6
8
10
12
14
16
18
20
Cru
de
pro
tein
(%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b c a b
Figure 47: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of V.mungo plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
5
10
15
20
25
30
Cru
de
pro
tein
(%
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a
a
d
a
0
5
10
15
20
25
Cru
de
pro
tein
(%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c a a
a
d
a
Table 24: Effect of treatments on percent nitrogen and phosphorus of V. mungo (black gram) plants
Mineral content
30th
days 60th
days
S. No. Treatment Nitrogen (%) Phosphorus (%) Nitrogen (%) Phosphorus (%)
1. Control 1.54 0.11 0.11 0.02 1.68 0.12 0.14 0.09
2. JUR1 1.76 0.05 (14.28) 0.28 0.32 (154.54) 2.07 0.40 (23.21) 0.29 0.08d (107.14)
3. JUR2 1.62 0.06 (5.19) 0.09 0.05 (-18.18) 1.99 0.22 (18.45) 0.18 0.05 (28.57)
4. JUR3 1.77 0.06 (14.93) 0.15 0.16 (36.36) 2.82 0.56c (67.85) 0.19 0.04 (35.71)
5. JUR4 2.11 0.79 (37.01) 0.16 0.08 (45.45) 3.04 0.16a (80.95) 0.30 0.15
d (114.28)
6. JUF1 2.00 0.17 (29.87) 0.18 0.05 (63.63) 2.87 0.91b (70.83) 0.19 0.05 (35.71)
7. FTZ 1.70 0.44 (10.38) 0.44 0.61d (300) 1.88 0.08 (11.90) 0.46 0.07
a (228.57)
8. FGD 1.49 0.05 (-3.24) 0.14 0.09 (27.27) 1.81 0.44 (7.73) 0.15 0.01 (-67.33)
9. JUR1+JUF1 2.19 0.09 (42.20) 0.2 0.07 (81.81) 2.30 0.29 (36.90) 0.23 0.08 (64.28)
10. JUR2+JUF1 2.12 0.24 (37.66) 0.15 0.09 (36.36) 2.23 0.28 (32.73) 0.17 0.07 (21.42)
11. JUR3+JUF1 1.58 0.04 (2.59) 0.21 0.11 (90.90) 1.85 0.21 (10.11) 0.23 0.08 (64.28)
12. JUR4+JUF1 2.71 0.34b (75.97) 0.25 0.17 (127.27) 2.94 0.15
b (75) 0.32 0.12
c (128.57)
13. JUR1+FTZ 1.76 0.32 (14.28) 0.16 0.08 (45.45) 1.95 0.21 (16.07) 0.17 0.07 (21.42)
14. JUR2+FTZ 3.16 0.76a (105.19) 0.42 0.22
d (281.81) 2.10 0.57 (25) 0.43 0.10
a (207.14)
15. JUR3+FTZ 3.67 0.67a (138.31) 0.35 0.98 (218.18) 2.99 0.40
a (77.97) 0.29 0.08
d (107.14)
16. JUR4+FTZ 4.49 0.63a (191.55) 0.38 0.07
d (245.45) 4.68 0.23
a (178.57) 0.41 0.05
a (192.85)
17. JUR1+FGD 1.82 0.51 (18.18) 0.30 0.05 (172.72) 2.03 0.46 (20.83) 0.32 0.09c (128.57)
18. JUR2+FGD 2.24 0.69d (45.45) 0.20 0.05 (81.81) 2.44 0.54
d (45.23) 0.26 0.02
d (85.71)
19. JUR3+FGD 1.48 0.11 (-3.89) 0.30 0.05 (172.72) 1.81 0.18 (7.73) 0.35 0.10b (150)
20. JUR4+FGD 3.00 0.43a (94.80) 0.25 0.00 (127.27) 3.06 0.18
a (82.14) 0.37 0.10
a (164.28)
21. JUF1+FTZ 1.75 0.45 (13.63) 0.16 0.08 (45.45) 1.92 0.28 (14.28) 0.18 0.05 (28.57)
22. FGD+FTZ 1.65 0.14 (7.14) 0.05 0.02 (-54.54) 1.77 0.04 (5.35) 0.10 0.3 (-28.57)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum,
FTZ=Fertilizer,FGD=Fungicide.
119
Figure 48: Effect of T.hamatum alone and in combination with rhizobial isolates on
percent nitrogen of V.mungo plants. Columns bearing superscript are statistically
significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
Nit
rogen
(%
)
30th day
Control JUF1 JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+JUF1 JUR2+JUF1 JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b
0
0.5
1
1.5
2
2.5
3
3.5
Nit
rog
en (
%)
60th day
Control JUF1 JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+JUF1 JUR2+JUF1 JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b c a b
Figure 49: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on percent nitrogen of V.mungo plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5 N
itro
gen
(%
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a
a
d
c
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Nit
rogen
(%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c a a
a
d a
Figure 50: Effect of T.hamatum alone and in combination with rhizobial isolates
on percent phosphorus of V.mungo plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Ph
osp
horu
s (%
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Ph
osp
horu
s (%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d d
a
c
Figure 51: Effect of rhizobial isolates alone and their combination with fertilizer
and fungicide on percent phosphorus of V.mungo plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Ph
osp
horu
s (%
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d d
d
0
0.1
0.2
0.3
0.4
0.5
Ph
osp
horu
s (%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d d
a a
d
a
c
d
b a
3.6.2.2. Cicer arietinum L. (Chickpea)
3.6.2.2.1. Growth performance
Not only T.hamatum (JUF1) alone but also the same test fungus co-inoculated
with rhizobial isolates including JUR1, JUR3, JUR4 found effective in boosting the root
length of chickpea plants at 30th
and 60th
day by inducing the increase from 37-154% .
Where as JUR2+JUF1 was only found effective in same aspect at 30th
day. The group of
test plants treated with JUF1+FTZ also showed increase with 51% in root length at 60th
day (Table 25; Figure 52). Out of rhizobial isolates, JUR3 found most efficient in
increasing root length of chickpea plants by 47-52% at 30th
and 60th
day. JUR4 induced
prominent percent increase (126 %) in same parameter at 30th
day while JUR1 and JUR2
showed their positive performance at 60th
day. In combination with fertilizer (FTZ),
JUR1, JUR3 and JUR4 found successful in increasing the length of roots from 38 -145%
in their respective groups of plants at 30th
and 60th
day. On the other hand, in
combinatiom with fungicide (FGD), JUR2 gave prominent results (63 - 122%) on same
parameter at both 30th
and 60th
day while JUR1+ FGD, JUR3+ FGD and JUR4 + FGD
induced significant increase from 38 - 63% in root length of test plants at 60th
day (Table
25; Figure 53).
T.hamatum alone and co-inoculated with JUR1, JUR3 and JUR4 found to
stimulate the shoot length of chickpea plants from 24-81% at 30th
and 60th
day of
uprooting of plants. Whereas JUR2+JUF1 induced percent increase (32%) in shoot length
only at 30th
day (Table 25; Figure 54). All four rhizobial isolates including JUR1, JUR2,
JUR3 and JUR4 significantly increased the shoot lengths from 21 - 43 % of test plants in
their respective groups at both 30th
and 60th
day. Similarly, JUR1, JUR3 and JUR4 in
combination with FTZ promoted the shoot length from 29 - 48% at both days of
harvesting while JUR2 + FTZ only showed promotion with 41% increase in same
parameter at 30th
day. However, in combination with FGD, JUR2, JUR3 and JUR4 found
efficient in improving the shoot length from 17- 80 % at 30th
and 60th
day (Table 25;
Figure 55).
Table 25: Effect of treatments on growth performance of C. arietinum (chickpea) plants
Growth performance
30th
days 60th
days
S. No. Treatment Root length* Shoot length* Fresh weight** Root length* Shoot length* Fresh weight**
1. Control 11.66 0.57 38.83 2.46 2.00 0.00 21.33 4.64 52.83 3.21 3.65 0.82
2. JUR1 17.16 1.60 (47.16) 47.26 1.77c (21.71) 4.76 0.65
b (138) 32.33 12.26
c (51.51) 70.90 3.51
c (34.20) 7.94 2.36
d (117.53)
3. JUR2 18.8 5.48 (61.23) 47.06 4.36c (21.19) 5.20 0.69
a (160) 32.73 5.03
c (53.44) 75.66 2.41
a (43.21) 9.39 4.39
c (157.26)
4. JUR3 23.0 3.60d (97.25) 47.00 1.00
c (21.04) 6.11 1.76
a (205.05) 30.90 2.30
c (44.86) 71.33 2.12
c (35.01) 6.31 0.24 (72.87)
5. JUR4 26.33 14.28c (125.81) 52.33 0.76
a (34.76) 6.02 0.74
a (201) 27.56 12.56 (29.20) 69.30 2.04
c (31.17) 6.34 0.4 (73.69)
6. JUF1 27.16 9.92c (132.93) 53.66 1.58
a (38.19) 6.96 0.44
a (248) 36.96 5.50
a (73.27) 95.66 3.37
a (81.07) 6.26 0.22 (71.50)
7. FTZ 21.63 7.85d (85.50) 52.46 1.12
a (35.10) 6.00 0.98
a (200) 30.30 9.29
d (42.05) 74.66 0.17
b (41.32) 5.60 0.43 (53.42)
8. FGD 20.06 1.65 (72.04) 56.10 4.82a (44.47) 7.18 0.44
a (259) 22.36 11.71 (4.82) 62.33 0.63 (17.98) 5.36 1.29 (46.84)
9. JUR1+JUF1 22.50 9.34d (92.96) 52.00 3.60
a (33.91) 3.66 0.53
d (83) 34.03 3.32
b (59.54) 82.16 0.50
a (55.51) 8.05 0.64
d (120.54)
10. JUR2+JUF1 24.43 3.57d (109.51) 51.40 2.70
a (32.37) 4.60 0.07
b (130) 26.00 4.64 (21.89) 63.66 7.81 (20.49) 5.49 1.53 (50.41)
11. JUR3+JUF1 22.06 6.21d (89.19) 52.40 8.32
a (34.94) 5.17 0.52
a (158.5) 29.33 3.00
d (37.50) 74.00 1.04
b (40.07) 6.34 2.12 (73.69)
12. JUR4+JUF1 29.66 9.45b (154.37) 54.00 1.90
a (39.06) 5.30 0.67
a (165) 29.83 0.50
d (39.84) 65.76 2.36
d (24.47) 7.18 0.44
d (96.71)
13. JUR1+FTZ 22.66 4.50d (94.33) 50.66 2.88
a (30.46) 3.64 0.71
d (82) 32.33 1.80
c (51.57) 78.50 3.88
a (48.58) 5.74 1.25 (57.26)
14. JUR2+FTZ 19.66 4.61 (68.61) 54.66 1.52a (40.76) 5.44 0.41
a (172) 30.90 9.04
c (44.86) 60.50 8.26 (14.51) 8.13 3.46
d (122.73)
15. JUR3+FTZ 24.33 1.15d (108.66) 55.16 4.25
a (42.05) 4.71 1.63
b (135.5) 30.50 2.64
d (42.99) 68.00 1.50
d (28.71) 6.00 0.75 (64.38)
16. JUR4+FTZ 28.66 4.93b (145.79) 51.00 3.46
a (31.34) 4.50 1.27
c (125) 29.56 3.89
d (38.54) 73.13 2.06
b (38.42) 5.87 1.21 (60.82)
17. JUR1+FGD 16.66 6.02 (42.88) 45.00 3.60 (15.88) 6.12 1.04a (200) 34.50 6.08
a (61.74) 92.00 3.96
a (74.14) 5.92 0.52 (62.19)
18. JUR2+FGD 26.00 1.00c (122.98) 47.16 1.60
d (21.45) 8.80 1.77
a (340) 34.83 6.35
a (63.29) 95.33 3.54
a (80.44) 8.59 0.42
c (135.34)
19. JUR3+FGD 13.00 1.73 (11.49) 45.66 4.04d (17.58) 9.00 0.95
a (350) 31.00 10.50
c (45.33) 67.33 9.64
d (27.44) 9.59 5.71
b (162.73)
20. JUR4+FGD 20.16 3.32 (72.89) 55.50 2.29a (42.93) 6.43 1.11
a (221.5) 29.46 0.81
d (38.11) 64.8 0.90
d (22.65) 5.75 0.24 (57.53)
21. JUF1+FTZ 17.16 3.88 (47.16) 50.50 8.52b (30.05) 5.68 0.27
a (184) 32.2 5.29
c (50.96) 80.00 2.05
a (51.42) 7.15 1.94 (15.89)
22. FGD+FTZ 17.23 0.37 (47.77) 56.43 2.62a (45.32) 4.74 0.22
b (137) 29.06 19.97
d (36.24) 83.00 3.63
a (57.10) 5.69 1.75 (55.89)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. * = cm, ** = gm. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum JUF1=T.hamatum, FTZ=Fertilizer, FGD=Fungicide.
125
Figure 52: Effect of T.hamatum alone and in combination with rhizobial
isolates on root length of C.arietinum plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30 R
oot
len
gth
(cm
) 30 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c
d
c
d d d
d
b
0
5
10
15
20
25
30
35
40
Ro
ot
len
gth
(cm
)
60 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
c c c d
b
d d c
d
Figure 53: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on root length of C.arietinum plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30
Root
len
gth
(cm
) 30 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
c
d d
d
b c
0
5
10
15
20
25
30
35
Root
len
gth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c c c d
c c d d
a a
c d d
Figure 54: Effect of T.hamatum alone and in combination with rhizobial
isolates on shoot length of C.arietinum plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
10
20
30
40
50
60
Sh
ooth
le
ngth
(cm
)
30 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
c c c
a a a
a a a a b
a
0
10
20
30
40
50
60
70
80
90
100
Sh
ooth
len
gth
(cm
)
60 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
c a
c c b
a
b d
a a
Figure 55: Effect of rhizobial isolates alone and their combination with fertilizer and
fungicide on shoot length of C.arietinum plants. Columns bearing superscript are
statistically significant (p< 0.05 LSD) with respective control.
0
10
20
30
40
50
60
Sh
oot
len
gth
(cm
)
30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c c c
a a a
a a a
a d d
a a
0
10
20
30
40
50
60
70
80
90
100
Sh
ooth
len
gth
(cm
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c a
c c b a
d b
a a
d d
a
T.hamatum alone (248%) and co-inoculated with JUR2 (130 %) and JUR3 (156
%) were observed to improve the fresh weight of chickpea plants at 30th
day while
JUR1+JUF1 and JUR4+JUF1 showed improvement in fresh weight of test plants from
24-165% at both 30th
and 60th
day (Table 25; Figure 56). Out of rhizobial isolates, JUR1
and JUR2 found active in improving the fresh weight of test plants from 117-160 % at
both 30th
and 60th
day whereas JUR3 (205%) and JUR4 (201%) found helpful only at 30th
day. In combination with FGD, JUR2 (135 - 340%) and JUR3 (163 - 350%) found
effective in improving the fresh weights of whole test plants at both 30th
and 60th
day.
However, fresh weight of chickpea plants found better when treated with JUR2 + FTZ
from 123-172% at both days as compared to JUR1+FTZ, JUR3+ FTZ and JUR4+FTZ
that showed good results as 82, 135 and 125 % respectively only at 30th
day (Table 25;
Figure 57).
3.6.2.2.2. Photosynthetic pigment
T.hamatum (JUF1) alone and co-inoculated with JUR4 stimulated the production
of total chlorophyll and its fraction in leaves of chickpea plants at 30th
and 60th
day. JUR2
+ JUF1 induced significant increase (68-85%) in same parameters only at 30th
day. All
four rhizobial isolates JUR1 to JUR4 produced positive effects on total chlorophyll and
its fractions (a, b) at 30th
day where as JUR1 to JUR3 stimulated the synthesis of fraction
b and total chlorophyll at 60th
day of test plants. JUR3 and JUR4 in combination with
FTZ induced better improvement in total chlorophyll and its fractions at 30th
day while
JUR1+ FTZ treatment induced improvement in total chlorophyll and its fraction in test
plants at 60th
day. On the contrary, JUR1, JUR2 and JUR3 in combination with fungicide
(FGD) found efficient in improving the same parameters at 30th
and 60th
day. Likewise,
JUR4+FGD only found effective at 30th
day as compared to control plants (Table 26;
Figure 58, 59).
0
1
2
3
4
5
6
7
8
Fre
sh w
eigh
t (g
m)
30 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
b a
a a a
a
d
b a a
a
b
0
1
2
3
4
5
6
7
8
9
10
Fre
sh
wei
gh
t (g
m)
60 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
c
d
d
Figure 56: Effect of T.hamatum alone and in combination with rhizobial
isolates on fresh weight of C.arietinum plants. Columns bearing superscript
are statistically significant (p< 0.05 LSD) with respective control.
0
1
2
3
4
5
6
7
8
9
Fre
sh w
eigth
(gm
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b a
a a a
a
d
a b c
a
a a
a
b
0
1
2
3
4
5
6
7
8
9
10
Fre
sh w
eigh
t (g
m)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
c
d c
b
Figure 57: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on fresh weight of C.arietinum plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
Table 26: Effect of treatments on photosynthetic pigment of C. arietinum (chickpea) plnats
Photosynthetic pigment
30th
days 60th
days
S. No. Treatment Chl-a* Chl-b* Total Chl * Chl-a* Chl-b* Total Chl *
1. Control 0.47 0.02 0.76 0.17 1.03 0.23 0.95 0.08 1.16 0.11 1.97 0.11
2. JUR1 1.04 0.03a (121.27) 1.89 0.05
a (148.68) 2.24 0.17
a (117.47) 1.38 0.37 (45.26) 2.51 0.66
c (116.37) 3.01 0.97
d (52.79)
3. JUR2 0.86 0.24a (82.97) 1.56 0.44
a (105.26) 1.88 0.43
c (82.52) 1.40 0.14 (47.36) 2.54 0.26
c (118.96) 3.21 0.27
d (62.94)
4. JUR3 0.99 0.11a (110.63) 1.92 0.27
a (152.63) 2.17 0.24
b (110.67) 1.50 0.45 (57.89) 2.78 0.77
a (139.65) 3.30 0.85
d (67.51)
5. JUR4 0.80 0.20a (70.21) 1.45 0.38
c (90.78) 1.70 0.40
d (65.04) 1.73 0.06
d (82.10) 1.30 0.25 (12.06) 2.86 0.09 (45.17)
6. JUF1 0.88 0.13a (87.23) 1.60 0.24
a (110.52) 1.81 0.20
d (75.72) 1.38 0.30 (45.26) 2.51 0.55
c (116.37) 3.17 0.65
d (60.91)
7. FTZ 1.68 0.06a (257.44) 1.21 0.65
d (59.21) 2.77 0.24
a (168.93) 1.59 0.44
d (67.36) 2.89 0.80
a (149.13) 3.58 0.77
c (81.72)
8. FGD 1.46 0.14a (210.63) 1.48 0.25
b (94.73) 2.84 0.12
a (175.72) 1.42 0.81 (49.47) 2.91 1.04
a (150.88) 3.37 1.04
c (71.06)
9. JUR1+JUF1 0.96 0.00a (104.25) 1.75 0.00
a (130.26) 1.42 1.22 (37.68) 1.47 0.36 (54.33) 2.76 0.61
a (137.93) 3.35 0.84
c (70.05)
10. JUR2+JUF1 0.87 0.04a (85.10) 1.41 0.36
c (85.52) 1.73 0.44
d (67.96) 1.26 0.25 (32.63) 2.01 0.01
d (73.27) 2.41 0.16 (22.33)
11. JUR3+JUF1 0.84 0.13a (78.72) 1.16 0.26 (52.63) 1.38 0.19 (33.98) 1.27 0.33 (33.68) 2.64 0.03
b (127.58) 3.10 0.14
d (57.36)
12. JUR4+JUF1 1.00 0.12a (112.76) 1.65 0.29
a (117.10) 1.80 0.17
d (74.75) 1.76 0.49
c (85.26) 1.51 0.35 (30.17) 3.04 0.11
d (54.31)
13. JUR1+FTZ 0.49 0.03 (4.25) 0.96 0.46 (26.31) 1.19 0.46 (15.53) 1.92 0.24c (102.10) 3.48 0.43
a (200) 4.28 0.63
a (117.25)
14. JUR2+FTZ 1.42 0.07a (202.12) 2.55 0.77
a (235.52) 3.18 0.19
a (208.73) 1.25 0.28 (31.57) 2.27 0.51
d (95.68) 2.76 0.68 (40.10)
15. JUR3+FTZ 1.38 0.01a (193.61) 2.50 0.34
a (229) 2.72 0.10
a (164.17) 1.12 0.30 (17.89) 2.04 0.54
d (75.86) 2.42 0.62 (22.84)
16. JUR4+FTZ 0.70 0.20d (48.93) 1.08 0.70 (42.10) 1.84 0.11
d (78.64) 1.75 0.31
c (84.21) 1.82 0.10 (56.89) 1.98 0.82 (0.50)
17. JUR1+FGD 1.13 0.04a (140.42) 2.07 0.92
a (172.36) 2.36 0.25
a (129.12) 1.83 0.31
c (92.63) 3.32 0.57
a (186.20) 3.77 0.19
b (91.37)
18. JUR2+FGD 0.91 0.03a (93.61) 2.26 0.51
a (197.36) 2.53 0.54
a (145.63) 1.57 0.41
d (65.26) 2.84 0.74
a (144.82) 3.53 0.85
c (79.18)
19. JUR3+FGD 0.93 0.15a (97.87) 1.58 0.19
a (107.89) 1.97 0.39
c (91.26) 1.37 0.29 (44.21) 2.82 0.10
a (143.10) 2.97 0.59
d (50.76)
20. JUR4+FGD 1.07 0.02a (127.65) 1.88 0.72
a (147.36) 2.27 0.09
a (120.38) 1.77 0.40
c (86.31) 1.39 0.25 (198.20) 2.76 0.60 (40.10)
21. JUF1+FTZ 0.81 0.16a (72.34) 1.38 0.27
c (81.51) 1.53 0.44 (48.54) 1.50 0.29 (57.89) 2.73 0.54
a (135.34) 3.13 0.60
d (58.88)
22. FGD+FTZ 1.60 0.03a (240.42) 0.71 0.26 (-6.57) 2.32 0.31
a (125.24) 1.33 0.22 (40) 2.4 0.40
c (106.89) 2.88 0.53 (46.19)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. * = mg/g, , chl = chlorophyll, JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum ,FTZ=Fertilizer, FGD=Fungicide.
133
Figure 58: Effect of T.hamatum alone and in combination with rhizobial
isolates on total chlorophyll of C.arietinum plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
Tota
l ch
loro
ph
yll
(m
g/g
) 30 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
a
a
a
a
a
a a
d d
a
0
0.5
1
1.5
2
2.5
3
3.5
4
Tota
l ch
loro
ph
yll
(m
g/g
)
60 th day
Control JUF1 JUR1 JUR2 JUR3 JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1 JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d d
d d c
c c d d d
Figure 59: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total chlorophyll of C.arietinum plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
0.5
1
1.5
2
2.5
3
3.5
Tota
l ch
loro
ph
yll
(m
g/g
) 30 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
c
b
d
a a
a
a
d
a a
c
a a
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Tota
l ch
loro
ph
yll
(m
g/g
)
60 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d d d
c c
a
b c
d
3.6.2.2.3. Biochemical parameters
T.hamatum co-inoculated with JUR3 and FTZ found to increase carbohydrate
content significantly from 57-106% in chickpea plants at 30th
and 60th
day. The same
fungus co-inoculated with JUR1, JUR2 and JUR4 found to increase the production of
same parameter from 106-188% at 60th
day. Out of all rhizobial isolates, JUR1 and JUR4
have promoted the carbohydrate production from 56-74% at 30th
day whereas JUR2 and
JUR3 induced significant increase in same biochemical parameter in test plants at 30th
and 60th
. JUR2, JUR3 and JUR4 in combination with FTZ produced remarkable incraese
in carbohydrate content in test plants from 77- 215 % at 60th
day while JUR1 + FTZ (56
%) showed significant change in same parameter at 30th
day. In combination with FGD,
JUR1 induced 69% increase in carbohydrate content of test plants at both 30th
and 60th
day (Table 27; Figure 60, 61).
T.hamatum alone and alongwith JUR2, JUR3 and FTZ promoted the crude
protein contents of chichpea plants from 51-85% at 60th
day while JUR1+JUF1 (42-
106%) and JUR4+JUF1 (67-75%) produced significant increase in same parameter in test
plants both at 30th
and 60th
day. JUR1 and JUR2 found better than JUR3 and JUR4 in
improving the crude protein content of test plants at 60th
day. However the efficiency of
JUR2, JUR3, JUR4 have improved the same aspect when given with FTZ at 30th
and 60th
day. In combination with FGD, JUR2 and JUR4 produced significant improvement in
crude protein content at 30th
and 60th
day (Table 27; Figure 62, 63).
3.6.2.2.4. Mineral content
T.hamatum (JUF1) alone and co-inoculated with JUR2 and JUR4 increased the
percent nitrogen content in chickpea plants from 51-85 % at 60th
day while the same
fungus co-inoculated with JUR1 (51-106%) and FTZ (71-169 %) in their respective
groups at both days (Table 28; Figure 64). JUR1 (73-89 %) and JUR2 (81- 92%) have
improved the nitrogen content significantly at 30th
and 60th
day while JUR3 and JUR4
induced improvement from 69 - 88% only at 30th
day. In combination with
Table 27: Effect of treatments on biochemical parameters of C. arietinum (chickpea) plants
Biochemical parameters
30th
days 60th
days
S. No. Treatment Total carbohydrate (mg/g) Crude protein (%) Total carbohydrate (mg/g) Crude protein (%)
1. Control 239.89 16.65 9.65 0.70 266.52 28.90 14.56 1.58
2. JUR1 417.22 115.43c (73.92) 11.06 0.36 (14.61) 460.87 82.94
d (72.92) 25.18 4.53
c (72.93)
3. JUR2 401.05 50.81c (67.18) 10.17 0.40 (5.38) 607.39 156.94
a (127.89) 27.98 3.21
a (92.17)
4. JUR3 416.16 31.74c (73.47) 11.08 0.39 (14.81) 471.86 8.78
d (77.04) 14.85 0.47 (1.99)
5. JUR4 373.36 120.37d (55.63) 13.19 03.45 (36.68) 315.77 73.71 (18.47) 17.25 4.03 (18.47)
6. JUF1 301.50 29.26 (25.68) 12.53 1.08 (61.03) 403.48 68.45 (51.38) 22.05 3.73d (51.44)
7. FTZ 236.52 14.1 (-1.40) 10.67 2.74 (10.36) 313.87 89.56 (17.76) 17.15 4.89 (17.78)
8. FGD 216.69 33.23 (-9.67) 9.32 0.31 (-3.41) 352.86 112.26 (32.39) 19.28 6.08 (32.41)
9. JUR1+JUF1 335.42 56.00 (39.82) 13.71 0.63d (42.07) 549.06 94.45
b (106.01) 30.00 5.16
a (106.04)
10. JUR2+JUF1 325.60 90.34 (35.72) 13.28 1.51 (37.61) 767.81 47.40a (188.08) 27.00 2.06
b (85.43)
11. JUR3+JUF1 377.17 51.12d (57.22) 9.80 0.28 (1.55) 435.98 155.22
d (63.58) 23.82 8.48
d (63.59)
12. JUR4+JUF1 300.18 7.98 (25.13) 16.96 2.16b (75.75) 634.6 189.74
a (138.10) 24.26 4.17
c (66.62)
13. JUR1+FTZ 375.26 72.71d (56.43) 11.00 2.01 (13.98) 390.85 16.77 (46.64) 21.36 0.91 (46.70)
14. JUR2+FTZ 296.27 44.12 (23.50) 19.75 4.78a (104.66) 470.75 117.87
d (76.62) 25.72 6.44
c (76.64)
15. JUR3+FTZ 287.45 16.49 (19.82) 22.94 4.23a (137.72) 838.57 83.58
a (214.62) 24.99 3.51
c (71.63)
16. JUR4+FTZ 242.06 24.37 (90) 28.10 3.98a (191.19) 620.87 207.30
a (132.95) 24.68 3.27
c (69.50)
17. JUR1+FGD 406.34 134.72c (69.38) 11.43 3.23 (18.44) 452.04 7.19
d (69.60) 24.70 0.39
c (69.64)
18. JUR2+FGD 354.67 29.89d (47.84) 14.04 4.36
d (45.49) 431.28 127.54 (61.81) 23.57 6.96
d (61.88)
19. JUR3+FGD 335.85 20.72 (40.00) 9.31 0.70 (-3.52) 284.28 13.57 (6.66) 11.74 0.59 (19.36)
20. JUR4+FGD 323.59 18.07 (34.89) 18.77 2.73a (94.50) 448.71 68.41
d (68.35) 24.51 3.73
c (95.87)
21. JUF1+FTZ 377.91 41.69d (57.53) 10.96 2.88 (13.57) 549.43 91.37
b (106.15) 23.77 1.67
d (63.25)
22. FGD+FTZ 282.80 79.70 (17.88) 10.31 0.87 (6.83) 272.61 42.94 (2.28) 12.63 4.30 (13.25)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum, FTZ=Fertilizer, FGD=Fungicide.
137
Figure 60: Effect of T.hamatum alone and in combination with rhizobial
isolates on total carbohydrate of C.arietinum plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
50
100
150
200
250
300
350
400
450 T
ota
l ca
rbo
hy
dra
te (
mg
/g)
30 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
c c c
d d d
0
100
200
300
400
500
600
700
800
Tota
l ca
rboh
yd
rate
(m
g/g
)
60 th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
a
d
b
a
d
a
b
Figure 61: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on total carbohydrate of C.arietinum plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
50
100
150
200
250
300
350
400
450 T
ota
l ca
rboh
yd
rate
(m
g/g
) 30 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c c c
d d
c
d
0
100
200
300
400
500
600
700
800
900
Tota
l ca
rboh
yd
rate
(m
g/g
)
60 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
a
d d
a
a
d d
Figure 62: Effect of T.hamatum alone and in combination with rhizobial
isolates on crude protein content of C.arietinum plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
2
4
6
8
10
12
14
16
18 C
rud
e p
rote
in (
%)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b
d
0
5
10
15
20
25
30
Cru
de
pro
tein
(%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
c
a a b
d c d
Figure 63: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on crude protein content of C.arietinum plants.
Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control.
0
5
10
15
20
25
30 C
rud
e p
rote
in (
%)
30 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
a
a
a
d
a
0
5
10
15
20
25
30
Cru
de
pro
tein
(%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
a c c c c
a c c c c
a c c c c
a c c c c d c
Figure 64: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent nitrogen of C.arietinum plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
4 N
itro
gen
(%
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
b c b
c d
c c
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Nit
rogen
(%
)
60th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
c
a a
b
c c
FTZ, JUR2, JUR3 and JUR4 improved the same parameter in test plants from 69 - 76 %
at 60th
day and JUR1 55% at 30th
day. However in combination with FGD, JUR1 and
JUR2 become active and increased the nitrogen content from 55-84% at both days while
JUR3 induced increase 52% in same parameter at 30th
day and JUR4 68% at 60th
day
(Table 28; Figure 65).
T.hamatum (JUF1) alongwith JUR2 and FTZ increased the percent phosphorus
upto 242 and 169% respectively in chickpea plants at 30th
and 60th
day. Out of rhizobial
isolates, JUR3 (157-253%) and JUR4 (153-200%) found efficient in improving the
phosphorus content in their respective test plants at both days while JUR1 (192%) and
JUR2 (231%) at 60th
day (Table 28; Figure 66, 67).
3.7. Composting of rice husk and wheatbran
One hundred and sixty (160 g) gram of each of rice husk and wheat bran was
composted with each of T.hamatum (JUF1), rhizobial isolates include JUR1 & JUR2
alone and combination of both rhizobial isolates with T.hamatum for 15 days in aerobic
condition (Table 2).
3.7.1. Effect of treatments on total carbohydrate and protein
of composted rice husk and wheat bran
Few treatments including JUR1, JUF1 and JUR2+JUF1 increased total
carbohydrate and protein in composted rice husk and wheat bran as compared to
uncomposted (control) wastes. Similar treatments were found effective in same aspect in
case of composted wheat bran while JUR2 was also found efficient in increasing the total
protein content in composted wheat bran after 15 days of incubation (Table 29).
Table 28: Effect of treatments on mineral content of C. arietinum (chicpea) plants
Mineral content
30th
days 60th
days
S. No. Treatment Nitrogen (%) Phosphorus Nitrogen (%) Phosphorus (%)
1. Control 1.93 0.17 0.07 0.00 2.33 0.25 0.13 0.46
2. JUR1 3.64 1.00b (88.60) 0.10 0.03 (42.85) 4.03 0.72
c (72.96) 0.38 0.23
c (192.3)
3. JUR2 3.50 0.44c (81.34) 0.12 0.04 (71.42) 4.47 0.51
a (91.84) 0.43 0.16
c (230.76)
4. JUR3 3.63 0.27b (88.08) 0.18 0.05
d (157.14) 2.37 0.07 (1.71) 0.46 0.10
b (253.84)
5. JUR4 3.26 1.05c (68.91) 0.21 0.05
d (200) 2.76 0.64 (18.45) 0.33 0.12
d (153.84)
6. JUF1 2.63 0.25 (36.26) 0.12 0.03 (71.42) 3.52 0.59d (51.07) 0.19 0.14 (46.15)
7. FTZ 1.98 0.27 (2.59) 0.12 0.03 (71.42) 2.74 0.78 (17.59) 0.25 0.00 (92.3)
8. FGD 1.89 0.29 (-2.07) 0.21 0.05d (200) 3.08 0.98 (32.18) 0.25 0.10 (92.3)
9. JUR1+JUF1 2.93 0.49d (51.81) 0.13 0.10 (85.71) 4.80 0.82
a (106) 0.16 0.06 (23.07)
10. JUR2+JUF1 2.84 0.79 (47.15) 0.31 0.05a (342.85) 4.32 0.33
b (85.4) 0.21 0.16 (61.53)
11. JUR3+JUF1 3.29 0.44c (70.46) 0.12 0.03 (71.42) 3.81 1.15 (63.57) 0.24 0.11 (84.61)
12. JUR4+JUF1 2.62 0.07 (35.75) 0.12 0.03 (71.42) 3.88 0.66c (66.52) 0.14 0.01 (7.69)
13. JUR1+FTZ 2.99 0.96d (54.92) 0.12 0.03 (71.42) 3.41 0.15 (46.35) 0.18 0.00 (38.46)
14. JUR2+FTZ 2.59 0.38 (34.19) 0.10 0.03 (42.85) 4.11 1.03c (76.39) 0.12 0.03 (-7.96)
15. JUR3+FTZ 2.51 0.14 (30.05) 0.12 0.03 (71.42) 3.99 0.56c (71.24) 0.26 0.12 (100)
16. JUR4+FTZ 2.08 0.25 (7.77) 0.10 0.03 (42.85) 3.94 0.52c (69.09) 0.21 0.05 (61.53)
17. JUR1+FGD 3.55 1.18c (83.93) 0.10 0.03 (42.85) 3.95 0.06
c (69.52) 0.21 0.06 (61.53)
18. JUR2+FGD 3.00 0.42d (55.44) 0.08 0.00 (14.28) 3.77 1.11
d (61.8) 0.12 0.3 (-7.96)
19. JUR3+FGD 2.93 0.18d (51.81) 0.10 0.03 (42.85) 1.87 0.09 (-19.74) 0.19 0.09 (46.15)
20. JUR4+FGD 2.82 0.15 (46.11) 0.08 0.00 (14.28) 3.92 0.59c (68.24) 0.16 0.08 (23.07)
21. JUF1+FTZ 3.3 0.36c (70.98) 0.12 0.03 (71.42) 3.80 0.26
c (63.09) 0.35 0.10
d (169.23)
22. FGD+FTZ 2.47 0.69 (27.97) 0.15 0.00 (114.28) 2.02 0.69 (-13.3) 0.16 0.02 (23.07)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within parenthesis
represent percent increase or decrease (-) with respective control. JUR1=Rhizobium sp-I, JUR2=Bradyrhizobium sp-II, JUR3=B. sp-III, JUR4=B. sp-IV, JUF1=T.hamatum ,FTZ=Fertilizer, FGD=Fungicide.
144
Figure 65: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent nitrogen of C.arietinum plants. Columns
bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
0.5
1
1.5
2
2.5
3
3.5
4 N
itro
gen
(%
) 30 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
b c
b
c d
c
d d
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Nit
rogen
(%
)
60 th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
a c c c c
d c
Figure 66: Effect of T.hamatum alone and in combination with rhizobial
isolates on percent phosphorus of C.arietinum plants. Columns bearing
superscript are statistically significant (p< 0.05 LSD) with respective control.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35 P
hosp
horu
s (%
) 30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
d d
a
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Ph
osp
horu
s (%
)
30th day
Control JUF1 JUR1 JUR2 JUR3
JUR4 FTZ FGD JUR1+JUF1 JUR2+JUF1
JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ
d
d d
a
Figure 67: Effect of rhizobial isolates alone and their combination with
fertilizer and fungicide on percent phosphorus of C.arietinum plants.
Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control.
0
0.05
0.1
0.15
0.2
0.25 P
hosp
horu
s (%
) 30th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
d
d d
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Ph
osp
horu
s (%
)
60th day
Control JUR1 JUR2 JUR3 JUR4 FTZ
FGD JUR1+FTZ JUR2+FTZ JUR3+FTZ JUR4+FTZ JUR1+FGD
JUR2+FGD JUR3+FGD JUR4+FGD FGD+FTZ
c
c b
d
Table 29: Total carbohydrate and protein contents of composted rice husk and wheat bran
after 15 days of incubation
S.No.
Treatments
rice husk
wheat bran
Total protein
(µg/g)
Total
carbohydrate
(µg/g)
Total protein
(µg/g)
Total
carbohydrate
(µg/g)
1 Control
(uncomposted) 10.59 ±1.63 524.98 ± 12.04
10.83 ± 1.30
550.6 ±73.34
2 JUR1 18.45 ± 2.60d 598.97 ± 15.05
c
20.16 ± 2.21c
581.5 ± 71.19
3 JUR2 14.74 ± 2.95 558.03 ± 46.60
19.65 ± 2.01d
575.4 ± 56.62
4 JUF1 16.93 ± 5.12 974.79 ± 43.87a
19.37 ± 4.66d
957.8 ± 23.13b
5 JUR1+JUF1 15.84 ± 2.85 550.69 ± 53.81
13.38 ± 4.5
557.8 ± 53.81
6 JUR2+JUF1 20.91± 6.47c 795.77 ± 47.96
a
23.35 ± 5.46b
822.4 ± 213.22d
Each value is a mean ± S.D (standard deviation) of 3 replicates. Means bearing superscripts in each column are significantly different with
respective control at p< 0.05
148
3.8. Pot experiments (2nd Phase)
3.8.1. Effect of composted rice husk on non- legume and
legume plants
Rice husk composted with each treatment was used in two amounts including 5
and 10g /2kg soil per pot to investigate its effect on physical and biochemical parameters
of one each of non-legume and legume crops.
3.8.1.1. H. annuus (sunflower)
3.8.1.1.1. Growth performance
Rice husk (RH) composted with each of JUR1, JUR2, JUF1 and JUR1+JUF1 @
5g/2kg soil/pot found effective in increasing the root length of sunflower plants from 21-
36% at 30th
day as compared to control plants. Interestingly, RH composted with each of
JUF1 and JUR1+JUF1 @10g was also found effective in increasing the root length of test
palnts from 22-27% (Table 30; Figure 68).
RH composted with all treatments @ 5 and 10g produced significant effects on
shoot length of sunflower plants at both days as compared to control plants except RH
composted with JUR1+JUF1 @ 5g found active on 30th
day and composted with JUR2 @
10g on 60th
day in increasing the shoot lengths of test plants (Table 31; Figure 69).
RH composted with JUR2, JUF1 and JUR2+JUF1 @ 5g improved the fresh
weight of sunflower plants from 120-131%, 130-150% and 125-131% respectively at
both 30th
and 60th
day as compared to control plants while at same amount rice husk
composted with JUR1 and JUR1+JUF1 found effective in improving the fresh weight of
test plants with 124 and 169% respectively at 60th
day. On contrary, RH composted with
JUR1+JUF1 and JUR2+JUF1 @ 10g found active in improving same parameter only at
60th
day (Table 32; Figure 70).
Table 30: Effect of composted rice husk on root lengths of H.annuus (sunflower) plants
Root length (cm)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 20.5 0.50
23.96 1.09
25.03 0.70
26.73 2.95
2 JUR1 24.9 1.47d (21.46) 29.66 5.00 (23.78) 28.1 7.36 (12.26) 30.16 2.84 (12.83)
3 JUR2 25.5 2.76 d (24.39) 28.6 1.55 (19.36) 26.23 3.62 (4.79) 30.63 0.40 (14.59)
4 JUF1 28.0 2.29 b (36.58) 28.63 0.77 (19.49) 27.4 1.47 (9.46) 32.60 5.25 d (21.96)
5 JUR1+JUF1 25.43 2.47 d (24.04) 25.7 5.00 (7.26) 26.03 3.69 (3.99) 33.96 1.65
d (27.04)
6 JUR2+JUF1 24.23 1.25 (18.19) 27.13 7.64 (13.23) 27.93 2.20 (11.58) 30.86 4.57 (15.54)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1= T.hamatum
150
0
5
10
15
20
25
30
35
40
45
Ro
ot
len
gth
(cm
)
5 gm
d d
1st column = 30th day, 2nd column = 60th day
b d
a
Figure 68: Effect of composted rice husk @ 5 and 10 gm on root length of H.annuus
plants. Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control.
0
10
20
30
40
50
Root
len
gth
(cm
)
10 gm
d d
a
1st column = 30th day, 2nd column = 60th day
Table 31: Effect of composted rice husk on shoot lengths of H.annuus (sunflower) plants
Shoot length (cm)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 28.03 1.35 31.76 0.66
25.33 0.58 32.06 2.10
2 JUR1 34.86 1.91a (24.36) 47.36 3.68
a (49.11) 40.86 6.21
a (61.31) 39.70 1.77
c (28.83)
3 JUR2 34.03 3.18b (21.40) 44.13 3.09
a (38.94) 34.73 0.64
a (37.11) 35.0 2.16 (9.17)
4 JUF1 36.96 1.86a (31.85) 46.6 1.70
a (46.72) 36.0 0.26
a (42.12) 44.43 3.44
a (38.58)
5 JUR1+JUF1 25.43 2.47 (-9.27) 44.56 2.79a (40.30) 43.66 1.52
a (72.36) 48.53 5.8
a (51.37)
6 JUR2+JUF1 35.93 1.96a (28.18) 43.23 0.20
a (36.11) 40.83 3.30
a (61.19) 46.2 1.3
a (44.10)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1= T.hamatum
152
Figure 69: Effect of composted rice husk @ 5 and 10 gm on shoot length of H.annuus
plants. Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control.
0
10
20
30
40
50
60 S
ho
ot
len
gth
(cm
) 5 gm
a
a
b
a
a
a a
a
a
a
a
Ist column = 30th day,2nd column = 60th day
0
10
20
30
40
50
60
Sh
oo
t le
ngth
(cm
)
10 gm
a
a a
a a
d
c a
a a
a
a
1st column = 30th day, 2nd column = 60th day
Table 32: Effect of composted rice husk on fresh weight of H.annuus (sunflower) plants
Fresh weight (gm)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.41 0.21
1.46 0.13
1.57 0.28
1.38 0.44
2 JUR1 2.27 0.29 (60.99) 3.27 1.15d (123.97) 3.12 2.32 (98.72) 2.73 0.50 (97.82)
3 JUR2 3.11 0.81b (120.56) 3.37 0.56
c (130.82) 1.93 0.21 (22.92) 2.05 0.15 (48.55)
4 JUF1 3.24 0.65a (129.78) 3.66 0.57
c (150.68) 2.51 0.19 (59.87) 3.13 0.81 (126.81)
6 JUR1+JUF1 2.13 0.78 (51.06) 3.93 0.37b (169.17) 3.29 1.00 (109.55) 6.19 3.76
a (348.55)
7 JUR2+JUF1 3.18 0.40a (125.53) 3.37 0.44
c (130.82) 3.01 0.25 (91.71) 3.65 0.45
d (164.49)
______________________________________________________________________________________________________________________
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
154
Figure 70: Effect of composted rice husk @ 5 and 10 gm on fresh weight of H.annuus
plants. Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control.
0
1
2
3
4
5
6
Fre
sh w
eigh
t (g
m)
5 gm
b a a
c c
d c c
b c
a
0
1
2
3
4
5
6
7
Fre
sh w
eigh
t (g
m)
10 gm a
d
c
3.8.1.1.2. Photosynthetic pigment
Rice husk (RH) composted with JUF1 @ 5 and 10g increased chl-a in leaves of
sunflower plants from 60-94% at 60th
day while RH composted with JUR2+JUF1
induced 90% increase in chl-a at 60th
day in its respective group of test plants. The same
organic material composted with JUR1+JUF1 @ 5g induced 50% increase at 30th
day and
@10g from 66-89% increase in chl-a content of test plants (Table 33; Figure 71).
RH composted with JUF1 @ 5g improved the chl-b content with almost 57% at
both days. RH composted with JUF1, JUR2, JUR1+JUF1 and JUR2+JUF1 @ 10g found
effective at 60th
day by improving the same fraction of chlorophyll from 137-261% in test
plants (Table 34; Figure 72).
RH composted with JUR2 @5g produced significant improvement in total
chlorophyll content from 36-41% in leaves of test plants at both days.Whereas RH
composted with JUF1 @ 5 and 10g found to improve total chlorophyll content from 40-
88% at 60th
day. RH composted with JUR1+JUF1 @ 10g found effective in boosting the
total chlorophyll content from 76-102% at both days (Table 35; Figure 73).
3.8.1.1.3. Biochemical parameters
Rice husk (RH) composted with all treatments @ 5g produced significant increase
in carbohydrate content from 46-243% in sunflower plants at 30th
day. Similarly RH
composted with JUF1 and JUR2 @ 10 g also found effective at 30th
and 60th
day in same
aspect (Table 36; Figure 74).
RH composted with JUF1 @ 5g was found more effective at 30th
day in
improving the crude protein content with 239% in test plants than 10 g of same
composted organic material that increased only 43% crude protein at same 30th
day. RH
composted with JUR2 @10g and with JUR1+JUF1 @ 5 g improved crude protein
content at 60th
day (61%) and 30th
day (173%) respectively (Table 37; Figure 75).
Table 33: Effect of composted rice husk on chlorophyll a of H.annuus (sunflower) plants
Chlorophyll a (mg /g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.21 0.04 1.06 0.07 1.14 0.14 1.32 0.58
2 JUR1 1.41 0.19 (16.52) 1.47 0.58 (38.67) 1.27 0.13 (12.26) 1.62 0.53 (22.72)
3 JUR2 1.7 0.10 (40.49) 1.63 0.08 (53.77) 0.67 0.18 (-44.34) 1.19 0.85 (-9.84)
4 JUF1 1.66 0.30 (37.19) 1.7 0.05d (60.37) 1.21 0.21 (6.60) 2.57 0.66
d (94.69)
5 JUR1+JUF1 1.82 0.38d (50.41) 1.5 0.21 (41.50) 1.84 0.22
d (66.03) 2.5 0.93
d (89.39)
6 JUR2+JUF1 1.51 0.58 (24.79) 2.02 0.29c (90.56) 1.17 0.40 (2.83) 2.23 0.14 (68.93)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
157
Figure 71: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a of H.annuus
plants. Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control
0
0.5
1
1.5
2
2.5
Ch
loro
ph
yll
-a (
mg/g
)
5 gm
d d
c
0
0.5
1
1.5
2
2.5
3
Ch
loro
ph
yll
-a (
mg/g
)
10 gm
d
d d
Table 34: Effect of composted rice husk on chlorophyll b of H.annuus (sunflower) plants
Chlorophyll b (mg /g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.14 0.05
1.16 0.38
0.41 0.15
0.56 0.15
2 JUR1 1.26 0.23 (10.52) 1.01 0.25 (-12.93) 0.80 0.27 (95.12) 0.73 0.65 (30.35)
3 JUR2 1.69 0.59 (48.24) 1.49 0.29 (28.44) 0.91 0.49 (121.95) 1.33 0.19c (137.5)
4 JUF1 1.8 0.20d (57.89) 1.82 0.25
c (56.89) 0.96 0.07 (134.14) 1.48 0.29
c (164.28)
5 JUR1+JUF1 1.23 0.22 (7.89) 1.19 0.15 (2.58) 1.24 0.29 (202.43) 2.02 0.08a (260.71)
6 JUR2+JUF1 1.40 0.56 (22.80) 1.49 0.22 (28.44) 0.90 0.24 (119.51) 1.63 0.32b (191.07)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
159
Figure 72: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-b of H.annuus
plants. Columns bearing superscript are statistically significant (p< 0.05 LSD with respective
control.
0
0.5
1
1.5
2
Ch
loro
ph
yll
-b (
mg/g
) 5 gm d c
0
0.5
1
1.5
2
2.5
Ch
loro
ph
yll
-b (
mg/g
)
10 gm
c c
a
b
Table 35: Effect of composted rice husk on total chlorophyll of H.annuus (sunflower) plants
Total chlorophyll (mg/g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 2.41 0.06
2.19 0.08
1.75 0.34
2.22 0.49
2 JUR1 2.68 0.30 (11.20) 2.26 0.12 (3.19) 1.77 0.38 (1.14) 2.35 1.17 (5.85)
3 JUR2 3.40 0.50d (41.07) 2.99 0.14
d (36.52) 1.22 0.31 (-30.28) 3.13 0.44 (40.99)
4 JUF1 3.03 0.81 (25.72) 3.07 0.30c (40.18) 2.06 0.38 (17.71) 4.17 1.16
d (87.63)
5 JUR1+JUF1 3.03 0.48 (25.72) 2.38 0.22 (8.67) 3.09 0.51d (76.57) 4.48 1.11
d (101.80)
6 JUR2+JUF1 2.60 0.53 (7.88) 2.68 0.36 (22.37) 2.19 0.81 (25.14) 3.86 0.27 (73.87)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
161
Figure 73: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll of H.annuus
plants. Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control.
0
0.5
1
1.5
2
2.5
3
3.5
Tota
l ch
loro
ph
yll
(m
g/g
)
5 gm
d
d c
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Tota
l ch
loro
ph
yll
(m
g/g
)
10 gm
d
d d
Table 36: Effect of composted rice husk on carbohydrate content of H.annuus (sunflower) plants
Total carbohydrate (mg/g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 173.55 25.28
184.02 14.76
218.86 20.68
266.31 22.86
2 JUR1 253.94 37.42c (46.32) 204.17 27.03 (10.94) 248.3 20.38 (13.45) 277.62 43.02 (4.24)
3 JUR2 261.87 40.85c (50.89) 208.08 13.67 (13.07) 243.8 51.20 (11.39) 369.35 31.54
c (38.69)
4 JUF1 595.71 48.16a (243.25) 197.62 17.46 (7.39) 299.92 46.02
c (37.03) 253.58 62.75 (-4.78)
5 JUR1+JUF1 443.06 46.63a (155.29) 202.06 4.92 (9.89) 225.20 47.55 (2.89) 348.42 58.96
d (30.83)
6 JUR2+JUF1 270.75 33.89c (56.00) 205.75 3.85 (11.80) 179.44 4.4 (-18.01) 275.51 26.06 (3.45)
______________________________________________________________________________________________________________________________________________
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
163
Figure 74: Effect of composted rice husk @ 5 and 10 gm on total carbohydrate of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05 LSD)
with respective control
0
100
200
300
400
500
600 T
ota
l ca
rboh
yd
rate
(m
g/g
) 5 gm
c c
a
a
c
0
50
100
150
200
250
300
350
400
Tota
l ca
rboh
yd
rate
(m
g/g
) 10 gm
d
d
Table 37: Effect of composted rice husk on crude protein content of H. annuus (sunflower) plants
Crude protein (%)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 7.51 0.76
7.96 0.35
9.71 0.95
10.63 1.92
2 JUR1 11.73 1.74 (56.19) 9.42 1.24 (18.34) 11.64 4.66 (19.87) 13.52 4.08 (27.18)
3 JUR2 12.12 1.89 (61.38) 9.63 0.62 (20.97) 11.25 2.35 (15.85) 17.09 5.97d (60.77)
4 JUF1 25.46 5.22a (239.01) 9.15 0.82 (14.94) 13.87 2.13
d (42.84) 12.57 5.24 (18.25)
5 JUR1+JUF1 20.49 6.78a (172.83) 9.34 0.20 (17.33) 10.42 2.21 (7.31) 16.1 2.71 (51.45)
6 JUR2+JUF1 12.51 1.57 (66.57) 9.53 0.16 (19.72) 8.29 0.20 (-14.62) 12.73 3.98 (19.75)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
165
Figure 75: Effect of composted rice husk @ 5 and 10 gm on crude protein (%) of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05
LSD)with respective control.
0
5
10
15
20
25
30
Cru
de
pro
tein
(%
)
5 gm a
c
0
2
4
6
8
10
12
14
16
18
Cru
de
pro
tein
(%
)
10 gm
d
d
3.8.1.1.4. Mineral content
Rice husk (RH) composted with JUF1 @ 5g (236%) and 10 g (40%) improved the
nitrogen content in sunflower plants at 30th
day. Whereas RH composted with JUR2 @
10g found effective in same aspect at 60th
day. RH composted with JUR1+JUF1 has
accelerated the nitrogen content with 170% increase in test plant at 30th
day (Table 38;
Figure 76).
RH composted with JUF1 @ 5 and 10g increased the phosphorus content in test
plants by 400% at 30th
day and 89% at 60th
day respectively. Similarly RH composted
with JUR2 induced 186% increase in phosphorus content of test plants at 30th
day (Table
39; Figure 77).
3.8.1.2. C.arietinum L. (chickpea)
3.8.1.2.1. Growth performance
Rice husk (RH) composted with JUF1, JUR1+JUF1 and JUR2+JUF1 @ 5 and
10g found efficient in improving the root length of chickpea plants from 40-117% at both
days of harvesting of plants. RH composted with JUR1 @ 5g induced increase (120-
131%) in root length of test plants on both days and @ 10g (33%) on 30th
day while RH
composted with JUR2 @ 5g found effective on both days from 70-78% (Table 40; Figure
78).
RH composted with JUR1, JUF1 and JUR2+JUF1 have promoted the growth of
shoots of test plants by inducing 19-52% increase in their length at 30th
and 60th
days.
Similarly shoot length was also promoted by RH composted with JUR2 @ 5g on both
days while RH composted with JUR1+JUF1 @ 5g on 60th
day and @ 10g on both days
from 31-43% (Table 41; Figure 79).
RH composted with JUR2 and JUF1 @ 5g improved the fresh weight of test
plants from 90-183% at 30th
day while RH composted with JUR1 @ 5 and 10g improved
the same parameter from 72-137% in test plants at 60th
day. RH composted with
Table 38: Effect of composted rice husk on percent nitrogen of H.annuus (sunflower) plants
Nitrogen (%)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.21 0.12
1.27 0.05
1.58 0.1
1.7 0.31
2 JUR1 1.87 0.28 (54.54) 1.50 0.20 (18.11) 1.86 0.74 (17.72) 2.16 0.65 (27.05)
3 JUR2 1.94 0.30 (60.33) 1.54 0.10 (21.25) 1.80 0.37 (13.92) 2.73 0.95d (60.58)
4 JUF1 4.07 0.84a (236.36) 1.46 0.13 (14.96) 2.21 0.34
d (39.87) 2.01 0.84 (18.23)
5 JUR1+JUF1 3.27 1.08c (170.24) 1.49 0.03 (17.32) 1.66 0.35 (5.06) 2.57 0.43 (51.17)
6 JUR2+JUF1 2.0 0.25 (65.28) 1.52 0.02 (19.68) 1.32 0.02 (-16.45) 2.03 0.63 (19.41)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
168
Figure 76: Effect of composted rice husk @ 5 and 10 gm on percent nitrogen of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Nit
rogen
(%
)
5 gm
d
c
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Nit
rogen
(%
)
10 gm
d
d
Table 39: Effect of composted rice husk on percent phosphorus of H.annuus (sunflower) plants
Phosphorus (%)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 0.07 0.01
0.08 0.00
0.07 0.01
0.09 0.05
2 JUR1 0.18 0.05 (157.14) 0.09 0.00 (12.5) 0.07 0.01 (0) 0.12 0.03 (33.33)
3 JUR2 0.2 0.13d (185.71) 0.12 0.10 (50) 0.05 0.02 (-28.57) 0.15 0.00 (66.66)
4 JUF1 0.35 0.42c (400) 0.09 0.00 (12.5) 0.10 0.04 (42.85) 0.17 0.06
d (88.88)
5 JUR1+JUF1 0.12 0.03 (71.42) 0.09 0.00 (12.5) 0.15 0.00 (114.28) 0.12 0.03 (33.33)
6 JUR2+JUF1 0.15 0.09 (114.28) 0.11 0.02 (37.5) 0.10 0.04 (42.85) 0.12 0.03 (33.33)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD).
Values within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
170
Figure 77: Effect of composted rice husk @ 5 and 10 gm on percent phosphorus of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05
LSD)with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Ph
osp
horu
s (%
)
5 gm a
c
0
0.5
1
1.5
2
2.5
3
Ph
osp
horu
s (%
)
10 gm
d
d
Table 40: Effect of composted rice husk on root lengths of C. arietinum (chickpea) plants
Root length (cm)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 17.13 ± 0.76
18.20 ± 0.30
16.56 ± 3.57
18.46 ± 1.85
2 JUR1 39.56 ± 2.42a (130.94) 40.16 ± 11.80
a (120.65) 22.03 ± 4.50
d (33.03) 24.56 ± 3.20 (33.04)
3 JUR2 30.5 ± 2.86a (78.05) 30.93 ± 4.42
c (69.94) 18.56 ± 1.40 (12.07) 22.66 ± 2.33 (22.75)
4 JUF1 35.06 ± 2.20a (104.67) 39.46 ± 2.17
a (116.81) 25.56 ± 1.85
c (54.34) 27.53 ± 2.86
c (49.13)
6 JUR1+JUF1 27.93 ± 2.76b (63.04) 33.3 ± 4.87
a (82.96) 23.86 ± 1.51
c (44.08) 27.5 ± 2.60
c (48.97)
7 JUR2+JUF1 30.56 ± 6.12a (78.40) 39.1 ± 2.47
a (114.83) 23.26 ± 2.93
d (40.45) 28.4 ± 1.38
c (53.84)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values
within parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum..
172
Figure 78: Effect of composted rice husk @ 5 and 10 gm on root length of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control
0
5
10
15
20
25
30
35
40
45
Root
len
gth
(cm
)
5 gm a
a a
b a
a
c
a
a
a
0
5
10
15
20
25
30
35
Root
len
gth
(cm
)
10 gm
d c
c d
c c c
a
d
Table 41: Effect of composted rice husk on shoot lengths of C. arietinum (chickpea) plants
Shoot length (cm)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 35.83 ± 0.80
41.36 ± 1.58
39.8 ± 4.20
43.93 ± 0.47
2 JUR1 54.36 ± 1.35a (51.71) 49.35 ± 2.75
b (19.31) 55.06 ± 4.95
b (38.40) 60.8 ± 0.80
a (38.40)
3 JUR2 49.23 ± 0.28c (37.39) 50.3 ± 2.20
a (21.61) 40.9 ± 4.40 (2.76) 43.05 ± 2.05 (-2.00)
4 JUF1 52.13 ± 0.70b (45.49) 61.55 ± 4.35
a (48.81) 50.25 ± 4.25
d (26.25) 56.45 ± 2.05
a (28.49)
5 JUR1+JUF1 36.56 ± 15.51 (2.03) 57.6 ± 2.40a (39.26) 57.15 ± 1.86
a (43.59) 57.6 ± 2.05
a (31.11)
6 JUR2+JUF1 53.13 ± 2.65a (48.28) 54.43 ± 3.02
a (31.60) 52.75 ± 2.35
c (32.53) 53.06 ± 0.41
a (20.78)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
174
Figure 79: Effect of composted rice husk @ 5 and 10 gm on shoot length of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control
0
10
20
30
40
50
60
70
80
Sh
oot
len
gth
(cm
)
5 gm
a c b a
b a
a a
a
a
c b a
0
10
20
30
40
50
60
70
80
Sh
oot
len
gth
(cm
)
10 gm
b d
a c b
a a
a a a
a
b
JUR1+JUF1 @ 10g on both days and @5g improved the same parameter by 69% on
60th
day (Table 42; Figure 80).
3.8.1.2.2. Photosynthetic pigment
RH composted with all treatment @ 10g enhanced the synthesis of chl-a in test
plants from 24-52% at 30th
day. Similarly RH composted with all treatments @ 5 g
increased the synthesis of chl-a from 48-66% in test plants at 60th
day except JUR1
(Table 43; Figure 81).
RH composted with JUR1 @ 10g and with JUR2 @ 5 g produced significant
effect on chl-b content of test plants on both days respectively from 44-80% and 59-
71%. RH composted with JUF1 @ 5 g induced 60% increase in the synthesis of chl-b
at 60th
day and composted with JUR1+JUF1@ 10g increased the same fraction 54%
in test plants at 30th
day (Table 44; Figure 82).
RH composted with JUR2 and JUR1+JUF1 @ 10g promoted the synthesis of
total chlorophyll from 22-41% in test plants on both days while same each of
composted material @ 5 g produced good enhancing effect on chlorophyll content on
60th
day. RH composted with JUF1 and JUR2+JUF1 @ 5 and 10 g produced positive
effects on same photosynthetic pigment respectively on 60th
and 30th
day (Table 45;
Figure 83).
3.8.1.2.3. Biochemical parameters
Rice husk (RH) composted with JUR1, JUR2 and JUF1 @ 5 and 10 g
promoted the carbohydrate synthesis in leaves of chickpea plants from 38-145% at
30th
and 60th
day. Whereas RH composted with JUR1+JUF1 and JUR2+JUF1 @ 10 g
found more efficient on both days in enhancing the carbohydrate content in test plants
as compared to their 5g amounts that only found active at 60th
day (Table 46; Figure
84).
RH composted with JUR1, JUR1+JUF1 and JUR2+JUF1 @ 10g promoted the
crude protein content in test plants from 36-104% at both days.
Table 42: Effect of composted rice husk on fresh weights of C. arietinum (chickpea) plants
Fresh weight (gm)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.58 ± 0.41
2.36 ± 0.85
1.77 ± 0.37
1.94 ± 0.56
2 JUR1 3.05 ± 0.80 (93.03) 4.07 ± 1.18c (72.45) 2.73 ± 0.16 (54.23) 4.6 ± 2.04
d (137.11)
3 JUR2 4.21 ± 0.56d (166.45) 4.48 ± 0.55
a (89.83) 2.16 ± 1.19 (22.03) 2.2 ± 0.42 (13.40)
4 JUF1 4.48 ± 0.34c (183.54) 4.71 ± 0.11
a (99.57) 2.35 ± 0.32 (32.76) 3.36 ± 1.59 (73.19)
6 JUR1+JUF1 3.25 ± 0.69 (105.69) 3.99 ± 0.75c (69.06) 4.39 ± 0.39
a (148.02) 4.84 ± 1.16
c (149.48)
7 JUR2+JUF1 3.51 ± 0.92 (122.15) 4.53 ± 0.53a (91.94) 2.20 ± 0.69 (24.29) 2.37 ± 0.25 (22.16)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
17
7
Figure 80: Effect of composted rice husk @ 5 and 10 gm on fresh weight of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control
0
1
2
3
4
5
6
7
8
Fre
sh w
eigh
t (g
m)
5 gm
d c
a
a
c a a
c a
a a
0
1
2
3
4
5
6
7
8
Fre
sh w
eigh
t(gm
)
10 gm
a
a
a
d c b c
Table 43: Effect composted rice husk on chlorophyll a of C. arietinum (chickpea) plants
Chlorophyll a (mg/g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.37 ± 0.08 1.65 ± 0.10 1.69 ± 0.18 1.98 ± 0.88
2 JUR1 1.73 ± 0.29 (26.27) 2.08 ± 0.06 (26.06) 2.57 ± 0.09a (52.07) 2.61 ± 0.22 (31.81)
3 JUR2 2.1 ± 0.05 (53.28) 2.61 ± 0.22c (58.18) 2.37 ± 0.33
c (40.23) 2.44 ± 0.20 (23.23)
4 JUF1 1.53 ± 0.21 (11.67) 2.55 ± 0.74d (54.54) 2.34 ± 0.03
c (38.46) 2.52 ± 0.33 (27.27)
5 JUR1+JUF1 1.36 ± 0.35 (-0.72) 2.44 ± 0.32d (47.87) 2.11 ± 0.31
d (24.85) 2.52 ± 0.23 (27.27)
6 JUR2+JUF1 1.71 ± 0.41 (24.81) 2.74 ± 0.57c (66.06) 2.19 ± 0.08
d (29.58) 1.75 ± 0.14 (-11.61)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
179
Figure 81: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
0.5
1
1.5
2
2.5
3 C
hlo
rop
hyll
-a (
mg/g
) 5 gm c d
d
c
0
0.5
1
1.5
2
2.5
3
Ch
loro
ph
yll
-a (
mg/g
)
10 gm a c c
d d
Table 44: Effect of composted rice husk on chlorophyll b of C. arietinum (chickpea) plants
Chlorophyll b (mg /g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 0.83 ± 0.02 1.1 ± 0.10 1.08 ± 0.17 1.4 ± 0.41
2 JUR1 0.97 ± 0.15 (16.86) 1.6 ± 0.26 (45.45) 1.95 ± 0.23c (80.55) 2.02 ± 0.06
d (44.28)
3 JUR2 1.42 ± 0.09c (71.08) 1.75 ± 0.13
d (59.09) 1.53 ± 0.47 (41.66) 1.64 ± 0.08 (17.14)
4 JUF1 0.84 ± 0.16 (1.20) 1.77 ± 0.62d (60.90) 1.53 ± 0.12 (41.66) 1.2 ± 0.08 (-14.28)
5 JUR1+JUF1 1.13 ± 0.12 (36.14) 1.69 ± 0.26 (53.63) 1.67 ± 0.40d (54.62) 1.72 ± 0.23 (22.85)
6 JUR2+JUF1 1.24 ± 0.28 (49.39) 1.67 ± 0.32 (51.81) 1.44 ± 0.17 (33.33) 1.37 ± 0.34 (-2.14)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
181
Figure 82: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-b of C.arietinum
plants. Columns bearing superscript are statistically significant (p< 0.05 LSD) with respective
control.
0
0.5
1
1.5
2
2.5
3 C
hlo
rop
hyll
-b (
mg/g
) 5 gm
c a
d d
d d
b b
0
0.5
1
1.5
2
2.5
3
Ch
loro
ph
yll
-b (
mg/g
)
10 gm
c
d
d
c c
Table 45: Effect of composted rice husk on total chlorophyll of C. arietinum (chickpea) plants
Total chlorophyll (mg/g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 2.05 ± 0.28
2.73 ± 0.19
2.78 ± 0.34
3.29 ± 0.15
2 JUR1 2.7 ± 0.44 (31.70) 3.59 ± 0.17 (31.50) 3.71 ± 0.12d (33.45) 3.98 ± 0.26 (20.97)
3 JUR2 3.52 ± 0.07 (71.70) 4.37 ± 0.35d (60.70) 3.91 ± 0.80
c (40.64) 4.03 ± 0.22
d (22.49)
4 JUF1 2.38 ± 0.38 (16.09) 4.32 ± 1.29d (58.24) 3.87 ± 0.13
d (39.20) 3.59 ± 0.31 (9.11)
5 JUR1+JUF1 2.49 ± 0.24 (21.46) 4.14 ± 0.40d (51.64) 3.77 ± 0.29
d (35.61) 4.25 ± 0.33
c (29.17)
6 JUR2+JUF1 2.95 ± 0.68 (43.90) 4.41 ± 0.83c (61.53) 3.81 ± 0.32
d (37.05) 3.12 ± 0.37 (-5.16)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
183
Figure 83: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5 T
ota
l ch
loro
ph
yll
(m
g/g
) 5 gm
d d d
c
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Tota
l ch
loro
ph
yll
(m
g/g
)
10 gm
d c d d d
d c
Table 46: Effect of composted rice husk on carbohydrate content of C. arietinum (chickpea) plants
Total carbohydrate (mg /g)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 208.75 ± 3.30
355.05 ± 18.82
297.00 ± 10.22
409.69 ± 4.89
2 JUR1 482.69 ± 15.96a (131.22) 515.19 ± 13.28
a (45.10) 697.75 ± 38.31
a (131.27) 831.49 ± 80.07
a (102.95)
3 JUR2 511.91 ± 66.88a (145.22) 633.28 ± 60.43
a (78.36) 557.03 ± 27.59
a (145.22) 660.50 ± 39.53
a (61.21)
4 JUF1 323.96 ± 17.86b (55.19) 673.44 ± 49.19
a (89.67) 594.71 ± 48.47
a (55.19) 566.18 ± 36.31
b (38.19)
5 JUR1+JUF1 238.67 ± 24.81 (14.33) 453.79 ± 44.66c (27.81) 847.55 ± 7.71
a (14.33) 870.80 ± 69.16
a (112.55)
6 JUR2+JUF1 240.42 ± 52.58 (15.17) 556.64 ± 53.63a (56.77) 501.45 ± 56.92
a (15.17) 862.34 ± 98.95
a (110.48)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
185
Figure 84: Effect of composted rice husk @ 5 and 10 gm on total carbohydrate of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05 LSD)
with respective control.
0
100
200
300
400
500
600
700 T
ota
l ca
rboh
yd
rate
(m
g/g
) 5 gm
a a
b
a
a a
c
a
0
100
200
300
400
500
600
700
800
900
Tota
l ca
rboh
yd
rate
(m
g/g
)
10 gm
a
a a
a
a
a
a
b
a a
However RH composted with JUR1 and JUR2 @ 5g also promoted the content of
same parameter from 131-145% at 30th
day. Whereas RH composted with JUF1 @ 5
g produced 48% significant result in crude protein content of test plants at 60th
day
(Table 47; Figure 85).
3.8.1.2.4. Mineral content
Rice husk (RH) composted with JUR1, JUR1+JUF1 and JUR2+JUF1 @ 10 g
have found effective in increasing the nitrogen content of chichpea plants from 36-
105% at 30th
and 60th
day. However RH composted with JUR1 and JUR2 @ 5g
produced significant effect on same parameter in their respective group at 30th
day
and RH composted with JUF1 @ 5 g produced 48% increase in nitrogen content at
60th
day (Table 48; Figure 86).
RH composted with JUR1 @ 5 and 10g found most effective in increasing the
phosphorus content from 154 - 400% in test plants at both days. Similarly RH
composted with JUR2 @ 5 g produced 330 - 475% significant incraese in phosphorus
content at 30th
and 60th
day whereas same composted material @ 10g only produced
145% increase in same parameter at 60th
day. RH composted with JUR1+JUF1 and
JUR2+JUF1 @ 10g produced significant effect on phosphorus content of test plants
from 160 - 410% and @ 5g from 212-237% at 60th
day. RH composted with JUF1 @
5 and 10 g induced positive effect on same parameter respectively at 60th
and 30th
day
(Table 49; Figure 87).
3.9. Effect of composted wheat bran on non- legume and
legume plants
Wheat bran was composted as same as rice husk and then composted wheat
bran with each treatment (Table 2) was used in two amounts (5 and 10g) to
investigate its effects on physical and biochemical parameters of one each of non-
legume and legume crops.
3.9.1. H. annuus L. (sunflower)
3.9.1.1. Growth performance
Table 47: Effect of composted rice husk on crude protein content of C. arietinum (chickpea) plants
Crude protein (%)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 11.39 ± 2.73 24.87 ± 1.00 14.54 ± 2.54 21.13 ± 1.33
2 JUR1 26.36 ± 5.16c (131.43) 28.14 ± 8.97 (13.14) 21.44 ± 2.44
d (47.45) 28.76 ± 3.04
c (36.10)
3 JUR2 27.96 ± 8.29c (145.47) 34.62 ± 7.59 (39.20) 20.20 ± 0.17 (38.92) 23.58 ± 4.63 (11.59)
4 JUF1 17.68 ± 4.97 (55.22) 36.78 ± 2.67d (47.88) 17.88 ± 5.42 (22.97) 22.58 ± 2.86 (6.86)
5 JUR1+JUF1 13.04 ± 1.36 (14.48) 24.81 ± 2.38 (-0.24) 29.65 ± 5.34a (103.92) 33.01 ± 1.5
a (56.22)
6 JUR2+JUF1 13.13 ± 2.83 (15.27) 30.39 ± 9.28 (22.19) 21.14 ± 3.08d (45.39) 30.45 ± 4.46
b (44.10)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
188
Figure 85: Effect of composted rice husk @ 5 and 10 gm on crude protein (%) of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
5
10
15
20
25
30
35
40 C
rud
e p
rote
in (
%)
5 gm
c c
d
0
5
10
15
20
25
30
35
Cru
de
pro
tein
(%
)
10 gm
d
a
d
c
a b
Table 48: Effect of composted rice husk on percent nitrogen of C. arietinum (chickpea) plants
Nitrogen (%)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.81 ± 0.44
3.97 ± 0.16
2.32 ± 0.39
3.38 ± 0.21
2 JUR1 4.21 ± 0.82c (132.59) 4.5 ± 1.43 (13.35) 3.43 ± 0.39
d (47.84) 4.60 ± 0.48
c (36.09)
3 JUR2 4.47 ± 1.33b (146.96) 5.53 ± 1.21 (39.29) 3.23 ± 0.02 (39.22) 3.77 ± 0.74 (11.53)
4 JUF1 2.82 ± 0.79 (55.80) 5.88 ± 0.42d (48.11) 2.86 ± 0.86 (23.27) 3.61 ± 0.45 (6.80)
5 JUR1+JUF1 2.08 ± 0.22 (14.91) 3.97 ± 0.39 (0) 4.75 ± 0.85a (104.74) 5.28 ± 0.23
a (56.21)
6 JUR2+JUF1 2.1 ± 0.45 (16.02) 4.86 ± 1.48 (22.41) 3.38 ± 0.49d (45.68) 4.87 ± 0.71
b (44.08)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
190
Figure 86: Effect of composted rice husk @ 5 and 10 gm on percent nitrogen of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
1
2
3
4
5
6 N
itro
gen
(%
) 5 gm
c b
d
0
1
2
3
4
5
6
Nit
rogen
(%
)
10 gm
d
a
d
c
a b
Table 49: Effect of composted rice husk on percent phosphorus of C. arietinum (chickpea) plants
Phosphorus (%)
Rice husk (5 gm) Rice husk (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 0.10 ± 0.03
0.08 ± 0.00
0.1 ± 0.03
0.11 ± 0.03
2 JUR1 0.36 ± 0.15b (260) 0.4 ± 0.15
b (400) 0.3 ± 0.08
c (200) 0.28 ± 0.15
d (154.54)
3 JUR2 0.43 ± 0.17a (330) 0.46 ± 0.10
a (475) 0.21 ± 0.02 (110) 0.27 ± 0.02
d (145.45)
4 JUF1 0.1 ± 0.04 (0) 0.47 ± 0.12a (487.5) 0.28 ± 0.02
c (180) 0.15 ± 0.00 (36.36)
6 JUR1+JUF1 0.11 ± 0.3 (0.008) 0.25 ± 0.08d (212.5) 0.50 ± 0.05
a (400) 0.56 ± 0.02
a (409.09)
7 JUR2+JUF1 0.1 ± 0.03 (0) 0.27 ± 0.07d (237.5) 0.26 ± 0.15
d (160) 0.31 ± 0.11
d (181.81)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
192
Figure 87: Effect of composted rice husk @ 5 and 10 gm on percent phosphorus
of C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05 LSD) with respective control.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5 P
ho
sph
oru
s (%
) 5 gm
b
a b
a a
d d d d
0
0.1
0.2
0.3
0.4
0.5
0.6
Ph
osp
horu
s (%
) 10 gm
c c
a
d d d
a
d
d d
Wheat bran (WB) composted with each of JUR1, JUF1 and JUR2+JUF1 @ 10
g produced sufficient increase in root length of sunflower plants from 23-40% and 37-
46% respectively at 30th
and 60th
day. WB composted with JUR2 @ 5 and 10 g both
found effective and accelerating the root lengths of test plants at 60th
day. While WB
composted with JUR1+JUF1 @ 5g became active at 30th
day and @ 10g at 60th
day
for promoting the root length (Table 50; Figure 88).
WB composted with all treatments @ 5 and 10 g both found efficient and
observed to increase shoot lengths of sunflower plants significantly from 16-59% as
compared to control plants (Table 51; Figure 89).
WB composted with JUR1 and JUR2 @ 5 g improved the fresh weights of test
plants from 123-128% at 60th
day. However varied results were obtained from WB
composted with JUR1+JUF1 like same WB @ 5g improved the fresh weight (149%)
of test plants at 30th
day and @ 10g (125%) at 60th
day. WB composted with
JUR2+JUF1 @ 5g was only active at 30th
day and improved the fresh weight by
109% (Table 52; Figure 90).
3.9.1.2. Photosynthetic pigment
The amount of chl-a was increased in sunflower plants from 80-117% by WB
composted with all treatments @ 5 g at 30th
day. However positive results were also
obtained on chl-a content of test plants by WB composted with JUR2 @ 10g on both
days from 52-99%. On the other hand 61% increase was also observed in same
parameter by WB composted with JUR1+JUF1@ 10 g on 30th
day (Table 53; Figure
91).
Chl-b content of test plants was significantly increased from 39-69% by WB
composted with JUR2 @ 5 and 10g on both days of uprooting of plants, followed by
WB composted with JUR1 @ 5g produced 41-58% increase in chl-b on both days and
@ 10 g induced 57% increase at 30th
day in same fraction. Similarly WB composted
with JUF1 @ 5g increased the chl-b content (43%) in test plants at 30th
day (Table 54;
Figure 92).
Table 50: Effect of composted wheat bran on root lengths of H.annuus (sunflower) plants
Root length (cm)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 20.16 0.41
22.46 2.20
19.8 1.41
22.23 2.1
2 JUR1 21.2 1.12 (5.15) 24.86 3.84 (10.68) 24.8 3.29d (25.25) 30.43 2.40
c (36.88)
3 JUR2 22.3 0.75 (10.61) 28.66 5.51d (27.60) 20.33 0.28 (2.67) 30.63 0.40
c (37.78)
4 JUF1 20.93 0.63 (3.81) 24.43 2.31 (8.77) 24.3 3.20d (22.72) 32.6 5.25
b (46.64)
5 JUR1+JUF1 24.3 3.7d (20.53) 25.3 2.8 (12.64) 22.0 2.33 (11.11) 33.96 1.65
a (52.76)
6 JUR2+JUF1 24.63 2.4d (22.17) 27.43 1.28 (22.12) 27.66 2.25
a (39.69) 30.86 4.57
c (38.82)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
195
Figure 88: Effect of composted wheat bran @ 5 and 10 gm on root length of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
5
10
15
20
25
30
35
40
45 R
oot
len
gth
(cm
) 5 gm
d
1st column = 30th day, 2nd column = 60th day 1st column = 30th day, 2nd column = 60th day 1st column = 30th day, 2nd column = 60th day
d d
a
0
5
10
15
20
25
30
35
40
45
Roo
t le
ngth
(cm
)
10 gm
d d a
c c b a
c
a
Table 51: Effect of composted wheat bran on shoot lengths of H.annuus (sunflower) plants
Shoot length (cm)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 30.0 0.43
33.83 0.25
31.56 1.05
32.06 2.10
2 JUR1 35.0 3.29d (16.66) 49.8 4.92
a (47.20) 49.1 6.12
a (55.57) 51.13 3.09
a (59.48)
3 JUR2 45.6 3.25a (52) 51.36 7.18
a (51.81) 45.7 3.37
a (44.80) 49.5 1.95
a (54.39)
4 JUF1 42.1 3.40a (40.33) 47.13 1.97
a (39.31) 46.86 4.12
a (48.47) 47.16 2.83
a (47.09)
5 JUR1+JUF1 45.85 2.87a (52.83) 43.5 3.53
b (28.58) 46.46 3.23
a (47.21) 49.26 04.35
a (53.64)
6 JUR2+JUF1 46.66 2.63a (55.53) 50.53 7.33
a (49.36) 43.66 1.45
b (38.33) 46.2 1.30
a (44.10)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
197
Figure 89: Effect of composted wheat bran @ 5 and 10 gm on shoot length of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
10
20
30
40
50
60 S
hoot
len
gth
(cm
) 5 gm
d
a a
a a a a
a b
a
c
a
0
10
20
30
40
50
60
Sh
oot
len
gth
(cm
)
10 gm a a a a b
a a a
a a
a
a
Table 52: Effect of composted wheat bran on fresh weights of H.annuus (sunflower) plants
Fresh weight (gm)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 2.2070.33 2.550.06 2.860.09 2.740.76
2 JUR1 3.9072.09 (77.02) 5.693.21d (123.13) 4.512.43 (57.69) 5.943.67 (116.78)
3 JUR2 4.7631.38 (115.81) 5.813.18d (127.84) 4.080.46 (42.65) 3.680.47 (34.30)
4 JUF1 4.261.03 (93.02) 4.980.56 (95.29) 5.182.09 (81.11) 4.481.12 (63.50)
5 JUR1+JUF1 5.491.04c (148.75) 5.582.50 (118.82) 6.343.65 (121.67) 6.173.78
d (125.81)
6 JUR2+JUF1 4.621.54d (109.33) 4.052.65 (58.82) 4.671.07 (63.28) 3.650.44 (33.21)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
199
Figure 90: Effect of composted wheat bran @ 5 and 10 gm on fresh weight of H.annuus
plants Columns bearing superscript are statistically significant (p< 0.05 LSD) with
respective control
0
1
2
3
4
5
6 F
resh
wei
gh
t (g
m)
5 gm c
d
d d
0
1
2
3
4
5
6
7
Fre
sh w
eigh
t (g
m)
10 gm d
Table 53: Effect of composted wheat bran on chlorophyll a of H.annuus (sunflower) plants
Chlorophyll a (mg/g)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.47 0.05 1.0 0.08 1.15 0.23 1.57 0.10
2 JUR1 1.83 0.41 (24.48) 1.85 0.47b (85) 1.52 0.17 (32.17) 1.71 0.55 (8.91)
3 JUR2 2.09 0.31 (42.17) 2.17 0.30a (117) 2.29 0.69
b (99.13) 2.39 0.31
d (52.22)
4 JUF1 1.84 0.21 (25.17) 1.94 0.03b (94) 1.76 0.17 (53.04) 1.81 0.22 (15.28)
5 JUR1+JUF1 1.84 0.47 (25.17) 1.8 0.06c (80) 1.85 0.17
d (60.86) 1.9 0.77 (21.01)
6 JUR2+JUF1 1.74 0.11 (18.36) 2.03 0.29a (103) 1.39 0.31 (20.86) 2.33 0.74 (48.40)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
201
Figure 91: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-a of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
0.5
1
1.5
2
2.5 C
hlo
rop
hyll
-a (
mg/g
) 5 gm
b
a b
c a
0
0.5
1
1.5
2
2.5
Ch
loro
ph
yll
-a (
mg/g
)
10 gm b
d
d
Table 54: Effect of composted wheat bran on chlorophyll b of H.annuus (sunflower) plants
Chlorophyll b (mg/gm)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.12 0.03 1.02 0.25 1.12 0.39 1.35 0.16
2 JUR1 1.59 0.09d (41.96) 1.62 0.10
c (58.82) 1.76 0.51
d (57.14) 1.42 0.55 (5.18)
3 JUR2 1.68 0.26c (50) 1.73 0.18
b (69.60) 1.86 0.28
d (66.07) 1.88 0.01
d (39.25)
4 JUF1 1.60 0.19d (42.85) 1.76 0.43
a (72.54) 1.56 0.42 (39.28) 1.43 0.13 (5.92)
5 JUR1+JUF1 1.31 0.20 (16.96) 1.36 0.03 (33.33) 1.36 0 .23 (21.42) 0.85 0.21 (-37.03)
6 JUR2+JUF1 1.13 0.21 (.89) 1.4 0.30d (37.25) 1.45 0.13 (29.46) 1.79 0.36
d (32.59)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
203
Figure 92: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8 C
hlo
rop
hyll
-b (
mg/g
)
5 gm
d c d c
b a
0
0.5
1
1.5
2
Ch
loro
ph
yll
-b (
mg/g
)
10 gm d
d d d
In case of increasing total chlorophyll content in test plants again WB
composted with JUR2 @ 5 and 10g found effective at both days from 62-99%,
followed by WB composted with JUR1@ 5 and 10g have promoted the increase in
same parameter on 30th
day. WB composted with JUF1 @ 5g produced better results
on total chlorophyll content at both days as compared to its 10g amount that produced
46% increase in same content only at 30th
day. WB composted with JUR1+JUF1 @
5g produced significant increase in chlorophyll content at both days and WB
composted with JUR2+JUF1@ 5g at 60th
day (Table 55; Figure 93).
3.9.1.3. Biochemical parameters
Wheat bran (WB) composted with all treatments @ 10g found better than its
5g amount and observed to enhance the synthesis of carbohydrate from103-205% in
sunflower plants at 30th
day with few exceptions (Table 56; Figure 94). Similarly WB
composted with all treatments @ 10g produced remarkable effect on crude protein
content in test plants from 104-206% at 30th
day. Whereas WB composted with JUR1
and JUR1+JUF1 @ 5g found effective on 30th
day by producing 97% increase in
crude protein and on 60th
day by 41% increase in same parameter (Table 57; Figure
95).
3.9.1.4. Mineral content
Both amounts (5 and 10g) of WB composted with JUR1 improved the
nitrogen content of sunflower plants from 98-159% at 30th
day. WB composted with
each of JUR2, JUF1 and JUR2+JUF1 @ 10 g was found active at 30th
day on same
aspect with 104-119%. However, WB composted with JUR1+JUF1 @ 5 and 10g was
observed to increase nitrogen content with 41% on 60th
and 206% on 30th
day (Table
58; Figure 96).
The phosphorus content of sunflower plants, WB composted with JUR1 and
JUF1 @ 10g produced positive effects from 100-154% on 60th
day while WB
composted with JUR2 @ 5 g found active (140%) on same day. WB composted with
JUR1+JUF1 @ 10 g increased the phosphorus content (210%) of test plants on 30th
day (Table 59; Figure 97).
Table 55: Effect of composted wheat bran on total chlorophyll of H.annuus (sunflower) plants
Total chlorophyll (mg/g)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 2.310.16 2.030.19 2.280.61 2.750.23
2 JUR1 3.160.22d (36.79) 3.330.81 (64.03) 3.290.39
d (44.29) 3.470.65 (26.18)
3 JUR2 3.970.55a (71.86) 4.050.28
a (99.50) 4.340.95
a (90.35) 4.450.74
b (61..81)
4 JUF1 3.690.64b (59.74) 3.970.33
a (95.56) 3.330.54
d (46.05) 3.510.03 (27.63)
5 JUR1+JUF1 3.150.56d (36.36) 3.430.11
a (68.96) 3.220.41 (41.22) 3.080.37 (12)
6 JUR2+JUF1 2.880.22 (24.67) 3.430.60a (68.96) 2.850.41 (25) 4.130.38
c (50.18)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
206
Figure 93: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05 LSD)
with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5 T
ota
l ch
loro
ph
yll
(m
g/g
) 5gm
d
a b
d
a a
a a
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Tota
l ch
loro
ph
yll
(m
g/g
)
10 gm
d
a
d
b c
Table 56: Effect of composted wheat bran on total carbohydrate of H.annuus (sunflower) plants
Total carbohydrate (mg/g)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 190.27 ± 13.77 324.33 ± 45.38 199.73 ± 34.76 350.96 ± 33.02
2 JUR1 433.90 ± 66.99a (128.04) 326.26 ± 18.70 (0.59) 515.40 ± 15.32
a (158.04) 439.94 ± 25.88
d (25.35)
3 JUR2 195.13 ± 8.10 (2.55) 348.32 ± 22.65 (7.39) 423.24 ± 17.87a (111.90) 354.02 ± 31.31 (0.87)
4 JUF1 199.73 ± 34.76 (4.97) 354.96 ± 6.15 (9.44) 436.45 ± 9.01a (118.52) 416.25 ± 43.63 (18.60)
5 JUR1+JUF1 234.88 ± 19.07 (23.44) 368.08 ± 52.44 (13.48) 609.80 ± 27.39a (205.31) 377.48 ± 36.55 (7.55)
6 JUR2+JUF1 180.87 ± 18.46 (-4.94) 263.76 ± 53.73d (-18.67) 406.60 ± 62.62
a (103.57) 355.93 ± 67.90 (1.41)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
208
Figure 94: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05 LSD)
with respective control.
0
50
100
150
200
250
300
350
400
450 T
ota
l ca
rbo
hy
dra
te (
mg
/gm
) 5 gm a d
0
100
200
300
400
500
600
700
Tota
l ca
rboh
yd
rate
(m
g/g
m)
10 gm
a
a a
a
a d
Table 57: Effect of composted wheat bran on crude protein of H.annuus (sunflower) plants
Crude protein (%)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 8.59 ± 0.55 12.03 ± 3.11 9.21 ± 1.62 16.21 ± 3.23
2 JUR1 16.97 ± 4.45a (97.55) 15.23 ± 3.11 (26.60) 23.82 ± 5.33
a (158.63) 20.35 ± 1.22 (25.53)
3 JUR2 9.02 ± 0.35 (5.00) 16.12 ± 1.06 (33.99) 19.56 ± 4.67c (112.31) 16.37 ± 1.44 (0.98)
4 JUF1 9.21 ± 1.62 (7.21) 16.41 ± 2.96 (36.40) 20.18 ± 0.41c (119.11) 20.29 ± 4.46 (25.16)
5 JUR1+JUF1 10.86 ± 2.49 (26.42) 17.03 ± 2.41d (41.56) 28.17 ± 6.29
a (205.86) 17.46 ± 5.30 (7.71)
6 JUR2+JUF1 8.79 ± 0.20 (2.32) 12.18 ± 3.46 (1.24) 18.79 ± 6.05d (104.01) 16.45 ± 3.12 (1.48)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
210
Figure 95: Effect of composted wheat bran @ 5 and 10 gm on crude protein (%) of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05 LSD)
with respective control.
0
2
4
6
8
10
12
14
16
18 C
rud
e p
rote
in (
%)
5 gm a d
0
5
10
15
20
25
30
Cru
de
pro
tein
(%
)
10 gm
a
c c
a
d
Table 58: Effect of composted wheat bran on percent nitrogen of H.annuus (sunflower) plants
Nitrogen (%)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.37 ± 0.08 1.92 ± 0.49 1.47 ± 0.25 2.59 ± 0.51
2 JUR1 2.71 ± 0.70a (97.81) 2.43 ± 0.49 (26.56) 3.81 ± 0.85
c (159.18) 3.25 ± 0.19 (25.48)
3 JUR2 1.44 ± 0.05 (5.10) 2.57 ± 0.17 (33.85) 3.13 ± 0.74d (112.92) 2.61 ± 0.23 (0.77)
4 JUF1 1.47 ± 0.25 (7.29) 2.62 ± 0.47 (36.45) 3.22 ± 0.06d (119.04) 3.24 ± 0.71 (25.09)
5 JUR1+JUF1 1.73 ± 0.39 (26.27) 2.72 ± 0.38d (41.66) 4.50 ± 1.00
a (206.12) 2.79 ± 0.85 (7.72)
6 JUR2+JUF1 1.40 ± 0.21 (2.81) 1.95 ± 0.55 (1.56) 3.00 ± 0.97d (104.08) 2.63 ± 0.49 (1.54)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
212
Figure 96: Effect of composted wheat bran @ 5 and 10 gm on percent nitrogen of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05 LSD)
with respective control.
0
0.5
1
1.5
2
2.5
3 N
itro
gen
(%
) 5 gm a d
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Nit
rogen
(%
)
10 gm
c
d d
a
d
Table 59: Effect of composted wheat bran on percent phosphorus of H.annuus (sunflower) plants
Phosphorus (%)
Wheat bran (5 gm) Wheat bran (10 gm)
S.No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 0.09±0.05 0.1±0.03 0.1±0.00 0.11±0.04
2 JUR1 0.14±0.09 (55.55) 0.15±0.01 (50) 0.25±0.10 (150) 0.22±0.13d (100)
3 JUR2 0.21±0.08 (133.33) 0.24±0.01c (140) 0.17±0.15 (70) 0.18±0.05 (63.63)
4 JUF1 0.14±0.01 (55.55) 0.17±0.15 (70) 0.25±0.10 (150) 0.28±0.05c (154.54)
5 JUR1+JUF1 0.16±0.02 (77.77) 0.15±0.02 (50) 0.31±0.15d (210) 0.12±0.03 (9.09)
6 JUR2+JUF1 0.15±0.00 (66.66) 0.18±0.01 (80) 0.16±0.08 (60) 0.17±0.02 (54.54)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
214
Figure 97: Effect of composted wheat bran @ 5 and 10 gm on percent phosphorus of
H.annuus plants. Columns bearing superscript are statistically significant (p< 0.05 LSD)
with respective control.
0
0.05
0.1
0.15
0.2
0.25
Ph
osp
ho
rus
(%)
5 gm
c
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Ph
osp
horu
s (%
)
10 gm d
d
c
3.9.2. C. arietinum L. (chickpea)
3.9.2.1. Growth performance
Wheat bran composted with JUR1 and JUF1 @ 5 and 10g found effective in
promoting the root length of chickpea plants from 25-44% on both days of uprooting.
Whereas WB composted with JUR2 @ 5 and 10g significantly elongated the root
length of test plants from 21-42% at 60th
day. JUR1+JUF1 composted WB @ 5g gave
better root length only on 60th
day whereas the same composted WB @ 10g found
efficient in same aspect from 41-50% on both days. Similarly WB composted with
JUR2+JUF1 @ 5g only found active and produced 30% increase in root length of test
plant on 30th
day (Table 60; Figure 98).
WB composted with JUR1, JUR2 and JUF1 @ 5 and 10g each produced
significant effects on shoots of test plants by promoting their length from 15-39%.
WB composted with JUR1+JUF1 @ 5 and 10g produced respectively 35% significant
promotion in root length of test plants on 60th
day and 30-44% on both days. However
21% significant elongation was also observed in shoots of test plants only on 30th
day
by JUR2+JUF1 composted WB (Table 61; Figure 99).
WB composted with JUR2, JUF1 and JUR1+JUF1 @ 5 and 10 g each found
effective in improving the fresh weights of chickpea plants from 110-301% on both
days as compared to control plants while WB composted with JUR2+JUF1 @ 5 g
produced significant effects on fresh weights of test plants from 132-198% on both
days. WB composted with JUR1 @ 5 g was only found effective in same aspect on
30th
day (Table 62; Figure 100).
3.9.2.2. Photosynthetic pigment
Wheat bran (WB) composted with JUR1, JUR2 and JUR2+JUF1 @ 10 g each
efficiently improved the content of chl-a in leaves of chickpea plants from 32-71% on
both 30th
and 60th
day. Whereas WB composted with JUF1 and JUR1+JUF1 @ 10 g
improved the same fraction from 34-36% in test plants on 60th
day. JUR2 composted
WB @ 5 g was only found effective on 30th
day (Table 63; Figure 101).
Table 60: Effect of composted wheat bran on root lengths of C. arietinum (chickpea) plants
Root length (cm)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 17.93 ± 0.73 20.26 ± 5.02 19.63 ± 0.98 23.33 ± 3.81
2 JUR1 25.9 ± 1.58a (44.45) 26.86 ± 1.33
d (32.57) 26.86 ± 2.28
d (36.83) 29.16 ± 1.25
c (24.98)
3 JUR2 20.63 ± 2.11 (15.05) 28.73 ± 2.99c (41.80) 24.63 ± 4.70 (25.47) 28.4 ± 1.93
d (21.73)
4 JUF1 25.10 ± 2.52a (39.98) 28.70 ± 1.25
c (41.65) 27.33 ± 0.90
d (39.22) 30.6 ± 1.87
b (31.16)
5 JUR1+JUF1 20.20 ± 0.20 (12.66) 33.66 ± 2.19a (66.14) 27.7 ± 6.59
d (41.11) 34.93 ± 4.30
a (49.72)
6 JUR2+JUF1 23.36 ± 2.15c (30.28) 25.83 ± 1.04 (27.49) 22.73 ± 2.51 (15.79) 24.33 ± 2.36 (4.28)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
217
Figure 98: Effect of composted wheat bran @ 5 and 10 gm on root length of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
5
10
15
20
25
30
35
Root
len
gth
(cm
) 5 gm
a a c
d c c
a a
0
5
10
15
20
25
30
35
Root
len
gth
(cm
)
10 gm
d d d c d b
a
c
Table 61: Effect of composted wheat bran on shoot lengths of C. arietinum (chickpea) plants
Shoot length (cm)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 37.20 ± 6.93
41.36 ± 6.93
36.3 ± 3.15
40.73 ± 2.83
2 JUR1 47.43 ± 1.15a (27.5) 48.26 ± 4.16
d (16.68) 50.53 ± 0.05
b (39.20) 50.63 ± 0.51
d (24.30)
3 JUR2 44.36 ± 0.80c (19.25) 51.30 ± 3.20
c (24.03) 53.80 ± 0.90
a (48.20) 56.33 ± 12.41
a (38.30)
4 JUF1 42.90 ± 2.60d (15.32) 49.96 ± 3.36
d (20.79) 54.30 ± 5.30
a (49.58) 55.3 ± 0.70
b (35.77)
5 JUR1+JUF1 39.43 ± 1.20 (5.99) 55.75 ± 1.05a (34.79) 52.46 ± 6.41
a (44.51) 53.1 ± 1.01
c (30.37)
6 JUR2+JUF1 45.26 ± 1.62c (21.66) 46.46 ± 1.86 (12.33) 43.53 ± 1.72 (19.91) 46.80 ± 4.32 (14.90)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
219
Figure 99: Effect of composted wheat bran @ 5 and 10 gm on shoot length of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
10
20
30
40
50
60
70
80 S
hoot
len
gth
(cm
) 5 gm
a c d
c
a a d
c d a
a
b
0
10
20
30
40
50
60
70
80
Sh
ooth
len
gth
(cm
)
10 gm
b a a a a a
d a b c
a
c
Table 62: Effect of composted wheat bran on fresh weights of C. arietinum (chickpea) plants
Fresh weight (gm)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.43 ± 0.53 2.07 ± 1.70 1.47 ± 0.90 2.19 ± 0.23
2 JUR1 3.24 ± 0.73d (126.57) 3.52 ± 0.13 (70.04) 3.17 ± 0.12 (115.64) 3.19 ± 0.50 (45.66)
3 JUR2 3.59 ± 0.29c (151.04) 4.36 ± 1.20
d (110.62) 4.92 ± 1.09
c (234.69) 5.46 ± 1.73
c (149.31)
4 JUF1 5.52 ± 0.53a (286.01) 5.87 ± 0.03
c (183.57) 5.90 ± 0.73
b (301.36) 5.56 ± 0.30
c (153.88)
5 JUR1+JUF1 4.02 ± 1.28c (181.11) 6.22 ± 2.50
b (200.48) 4.81 ± 1.51
c (227.21) 4.79 ± 1.45
d (118.72)
6 JUR2+JUF1 4.27 ± 1.20b (198.60) 4.80 ± 0.15
d (131.88) 3.59 ± 1.39 (144.21) 3.94 ± 1.04 (79.90)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
221
Figure 100: Effect of composted wheat bran @ 5 and 10 gm on fresh weight of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
1
2
3
4
5
6
7
8
Fre
sh w
eigh
t (g
m)
5 gm
d c
a
c b
a
a
d
c b
d
c c
0
1
2
3
4
5
6
7
8
Fre
sh w
eigh
t (g
m)
10 gm
c
b
c
a
a
c c d
b c
Table 63: Effect of composted wheat bran on chlorophyll a of C. arietinum (chickpea) plants
Chlorophyll a (mg/g)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 2.27 ± 0.30
2.37 ± 0.51
1.80 ± 0.14
1.97 ± 0.33
2 JUR1 2.56 ± 0.54 (12.77) 2.69 ± 0.22 (13.50) 2.72 ± 0.23d (51.11) 2.60 ± 0.08
d (31.97)
3 JUR2 3.22 ± 0.84d (41.85) 2.30 ± 0.32 (-2.95) 3.08 ± 1.46
c (71.11) 3.23 ± 0.04
a (63.95)
4 JUF1 2.08 ± 0.15 (-8.37) 2.13 ± 0.17 (-10.12) 2.58 ± 0.13 (43.33) 2.68 ± 0.18d (36.04)
5 JUR1+JUF1 2.48 ± 0.11 (9.25) 2.74 ± 0.44 (15.61) 2.51 ± 0.37 (39.44) 2.65 ± 0.11d (34.51)
6 JUR2+JUF1 2.28 ± 0.28 (0.44) 2.44 ± 0.15 (2.95) 2.75 ± 0.32d (52.77) 2.84 ± 0.67
c (44.16)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
223
Figure 101: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-a of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
Ch
loro
ph
yll
-a (
mg/g
) 5 gm
d
0
0.5
1
1.5
2
2.5
3
3.5
Ch
loro
ph
yll
-a(m
g/g
)
10 gm
d
c
d d
a
d d c
WB composted with JUR2 @ 5 g promoted the synthesis of chl-b with 21-62%
on both days and 10g of same composted WB was found 33% effective for the
synthesis of same fraction of chlorophyll on 60th
day while WB composted with JUF1
@ 5g enhanced the synthesis of chl-b on 30th
day (Table 64; Figure 102).
WB composted with JUR2 @ 5 g increased the total chlorophyll content only
at 30th
day while its 10g induced the synthesis of same parameter from 31-53% in test
plants on both days. WB composted with JUF1 @ 5 and 10g promoted the total
chlorophyll content in test plants on 30th
day from 30-35%. JUR2 composted WB @
10g gave better results as compared to its 5g amount. WB composted with
JUR2+JUF1 @ 5g produced 25% increase on 60th
day and @ 10g produecd 43% on
30th
day. In addition, WB composted with JUR1+JUF1 @ 5g induced 35% increase in
total chlorophyll content on 60th
day (Table 65; Figure 103).
3.9.2.3. Biochemical parameters
Among all treatments, wheat bran (WB) composted with JUR1 @ 5 and 10 g
each found efficient and produced significant increased in carbohydrate content in
chickpea plants on both days, followed by WB composted with JUF1 @ 5 and 10 g
produced better results in same parameter on 60th
day. WB composted with JUR2 @ 5
g induced 56% increase in carbohydrate content of test plants on 60th
day while WB
composted with JUR1+JUF1 @ 5 g produced significant effects in carbohydrate
content on both days. However WB composted with JUR2+JUF1 @ 5 and 10 g
produced positive effects on same parameter respectively on 30th
and 60th
day (Table
66; Figure 104). WB composted with JUR1 and JUR2 @ 5g found to increase the
crude protein content from 40-51% in test plants on 60th
day of uprooting of plants
(Table 67; Figure 105).
3.9.2.4. Mineral content
Wheat bran (WB) composted with JUR1 and JUR2 @ 5g found effective in
increasing the total nitrogen content from 40-51% in chickpea plants on 60th
day
(Table 68; Figure 106).
Table 64: Effect of composted wheat bran on chlorophyll b of C. arietinum (cickpea) plants
Chlorophyll b (mg/g)
Wheat bran (5 gm) Wheat bran (10 gm)
S.No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.50 ± 0.10
1.31 ± 0.09
1.41 ± 0.07
1.45 ± 0.05
2 JUR1 1.97 ± 0.40 (31.33) 1.33 ± 0.11 (1.52) 1.68 ± 0.16 (19.14) 1.62 ± 0.28 (11.72)
3 JUR2 2.43 ± 0.42d (62) 1.59 ± 0.03
d (21.37) 1.42 ± 0.46 (0.70) 1.93 ± 0.15
d (33.10)
4 JUF1 2.34 ± 0.70d (56) 1.37 ± 0.12 (4.58) 1.87 ± 0.07 (32.62) 0.93 ± 0.19 (-35.86)
5 JUR1+JUF1 1.50 ± 0.06 (0) 1.53 ± 0.13 (16.79) 1.57 ± 0.12 (11.34) 1.66 ± 0.30 (14.48)
6 JUR2+JUF1 1.48 ± 0.15 (-1.33) 1.52 ± 0.11 (16.03) 2.15 ± 0.33c (52.48) 1.69 ± 0.38 (16.55)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
226
Figure 102: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
Ch
loro
ph
yll
-b (
mg/g
) 5 gm
d d
d
b b
0
0.5
1
1.5
2
2.5
3
Ch
loro
ph
yll
-b (
mg/g
)
10 gm
c d
c c
Table 65: Effect of composted wheat bran on total chlorophyll of C. arietinum (chickpea) plants
Total chlorophyll (mg/g)
Wheat bran (5 gm) Wheat bran (10 gm)
S.No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 3.29 ± 0.11 3.16 ± 0.39 3.42 ± 0.33 3.37 ± 0.67
2 JUR1 4.54 ± 0.84d (37.99) 3.57 ± 0.08 (12.97) 4.37 ± 0.15 (27.77) 3.89 ± 0.26 (15.34)
3 JUR2 4.66 ± 0.47d (41.64) 3.15 ± 0.33 (-0.31) 4.50 ± 1.17
d (31.57) 5.17 ± 0.10
c (53.41)
4 JUF1 4.43 ± 0.69d (34.65) 3.49 ± 0.14 (10.44) 4.46 ± 0.17
d (30.40) 3.01 ± 0.55 (-10.68)
5 JUR1+JUF1 3.99 ± 0.08 (21.27) 4.29 ± 0.56a (35.75) 3.75 ± 0.38 (9.64) 3.66 ± 1.03 (8.60)
6 JUR2+JUF1 3.93 ± 0.56 (19.45) 3.97 ± 0.15c (25.63) 4.91 ± 0.65
c (43.56) 4.54 ± 0.04 (34.71)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
228
Figure 103: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Tota
l ch
loro
ph
yll
(m
g/g
) 5 gm d d d a
c
0
1
2
3
4
5
6
Tota
l ch
loro
ph
yll
(m
g/g
)
10 gm
d d c
c
Table 66: Effect of composted wheat bran on carbohydrate content of C. arietinum (chickpea) plants
Total carbohydrate (mg /g)
Wheat bran (5 gm) Wheat bran (10 gm)
S. No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 203.52 ± 9.42
335.41 ± 8.85
294.19 ± 18.66
348.85 ± 40.43
2 JUR1 292.74 ± 7.76c (43.83) 561.73 ± 29.89
a (67.47) 361.95 ± 52.12
d (23.03) 492.36 ± 57.98
a (41.13)
3 JUR2 255.21 ± 19.21 (25.39) 523.42 ± 14.58a (56.05) 354.02 ± 29.57 (20.33) 372.83 ± 17.09 (6.87)
4 JUF1 250.46 ± 23.35 (23.06) 397.35 ± 46.11d (18.46) 316.51 ± 16.49 (7.58) 442.05 ± 36.39
d (26.71)
5 JUR1+JUF1 263.46 ± 51.54d (29.45) 401.58 ± 49.14
d (19.72) 327.60 ± 56.80 (11.35) 423.88 ± 18.20
d (21.50)
6 JUR2+JUF1 287.02 ± 52.81c (41.02) 320.47 ± 8.83 (-4.45) 333.68 ± 36.15 (13.42) 473.76 ± 50.72
b (35.80)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
230
Figure 104: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
100
200
300
400
500
600
Tota
l ca
rboh
yd
rate
(m
g/g
) 5 gm
c d
c
a a
d d
0
50
100
150
200
250
300
350
400
450
500
Tota
l ca
rboh
yd
rate
(m
g/g
)
10 gm
d
a
d d
b
Table 67: Effect of composted wheat bran on crude protein content of C. arietinum (chickpea) plants
Crude protein (%)
Wheat bran (5 gm) Wheat bran (10 gm)
S.No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 11.83±1.75
19.04±1.06
16.77±2.02
20.30±1.58
2 JUR1 16.01±3.71 (35.33) 28.84±4.77c (51.47) 19.75±2.86 (17.76) 21.88±2.99 (7.78)
3 JUR2 13.96±1.03 (18.00) 26.77±3.94d (40.59) 19.33±4.02 (15.26) 19.51±0.51 (-3.89)
4 JUF1 13.68±0.26 (15.63) 21.68±2.49 (13.86) 17.28±0.92 (3.04) 24.17±6.66 (19.06)
5 JUR1+JUF1 14.41±2.83 (21.80) 21.93±2.70 (15.17) 17.90±3.10 (6.73) 16.87±1.0 (-16.89)
6 JUR2+JUF1 15.66±2.86 (32.37) 17.48±0.50 (-8.19) 18.21±4.36 (8.58) 17.54±2.74 (-13.59)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05(LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
232
Figure 105: Effect of composted wheat bran @ 5 and 10 gm on crude protein (%) of
C.arietinum plants. Columns bearing superscript are statistically significant (p< 0.05
LSD) with respective control.
0
5
10
15
20
25
30
Cru
de
pro
tein
(%
)
5 gm c
d
0
5
10
15
20
25
Cru
de
pro
tein
(%
)
10 gm
Table 68: Effect of composted wheat bran on percent nitrogen of C. arietinum (chickpea) plants
Nitrogen (%)
Wheat bran (5 gm) Wheat bran (10 gm)
S.No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 1.88 ± 0.28 3.04 ± 0.17 2.68 ± 0.32 3.24 ± 0.25
2 JUR1 2.55 ± 0.59 (35.63) 4.61 ± 0.76c (51.64) 3.15 ± 0.45 (17.53) 3.50 ± 0.47 (8.02)
3 JUR2 1.89 ± 0.62 (0.53) 4.27 ± 0.62d (40.46) 3.08 ± 0.64 (14.92) 3.12 ± 0.07 (-3.70)
4 JUF1 2.18 ± 0.19 (15.95) 3.46 ± 0.39 (13.81) 2.76 ± 0.15 (2.98) 3.86 ± 1.06 (19.13)
5 JUR1+JUF1 2.30 ± 0.45 (22.34) 3.5 ± 0.43 (15.13) 2.86 ± 0.49 (6.71) 2.70 ± 0.16 (-16.66)
6 JUR2+JUF1 2.50 ± 0.46 (32.94) 2.79 ± 0.08 (-8.22) 2.91 ± 0.69 (8.58) 2.81 ± 0.43 (-13.37)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
234
Figure 106: Effect of composted wheat bran @ 5 and 10 gm on percent nitrogen
of C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5 N
itro
gen
(%
) 5 gm c
d
0
0.5
1
1.5
2
2.5
3
3.5
4
Nit
rogen
(%
)
10 gm
WB composted with all treatments @ 5 and 10 g each produced significant increased
in phosphorus content of test plants from 110-310% and 287-350% respectively at
60th
day as compared to control plants (Table 69; Figure 107).
Table 69: Effect of composted wheat bran on percent phosphorus of C. arietinum (chickpea) plants
Phosphorus (%)
Wheat bran (5 gm) Wheat bran (10 gm)
S.No. Treatment 30th
day 60th
day 30th
day 60th
day
1 CONTROL 0.11 ± 0.12
0.1 ± 0.03
0.1 ± 0.04
0.08 ± 0.00
2 JUR1 0.21 ± 0.02 (90.90) 0.33 ± 0.02a (230) 0.15 ± 0.00 (50) 0.31 ± 0.07
c (287.5)
3 JUR2 0.19 ± 0.08 (72.72) 0.28 ± 0.02a (180) 0.12 ± 0.03 (20) 0.36 ± 0.02
b (350)
4 JUF1 0.18 ± 0.05 (63.63) 0.21 ± 0.02d (110) 0.14 ± 0.01 (40) 0.34 ± 0.25
c (325)
5 JUR1+JUF1 0.15 ± 0.00 (36.36) 0.41 ± 0.07a (310) 0.16 ± 0.02 (40) 0.35 ± 0.00
c (337.5)
6 JUR2+JUF1 0.23 ± 0.07 (109.09) 0.25 ± 0.00c (150) 0.20 ± 0.08 (100) 0.43 ± 0.05
a (437.5)
Each value is the mean S.D (standard deviation) of 5 replicates. Means bearing superscripts in each column are significantly different with respective control at p< 0.05 (LSD). Values within
parenthesis represent percent increase or decrease (-) with respective control. JUR1= Rhizobium sp-I, JUR2= Bradyrhizobium sp-II, JUF1=T.hamatum.
237
Figure 107: Effect of composted wheat bran @ 5 and 10 gm on percent phosphorus of
C.arietinum plants. Columns bearing superscript are statistically significant
(p< 0.05 LSD) with respective control.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Ph
osp
horu
s (%
)
5 gm
a
a
d
a
c c c
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Ph
osp
horu
s (%
)
10 gm
c
b c c
a
4. Discussion
Soil scientifically is classified as the naturally available ecosystem for
numerous biological existences due to the presence of valuable nutrients with
favorable chemical and physical properties. Accessibility and consistency in pH,
organic matter, moisture and mineral contents including nitrogen, phosphorus, etc
along with various essential living elements that serves as the powerful symbols and
which together enhance the soil fertility or the capacity through which cropping
system is robust. Unfortunately from last few decades lack of attention from the
concerned domains and gradual global climatic changes like greenhouse effects
causes decrement in the land productivity which declines the high crop yield and
capacity.
Pakistan is one of the countries of world whose economy is mainly depends
on agriculture, its more than 67% population is living in rural areas and farming is
their native profession (Jamali et al., 2011; Rahman et al., 2011). However, there has
been reported a gap all the time between farming production and consumers’ demand.
In order to fullfil this space, inorganic or synthetic fertilizers always are the first
choice of our farmers. These synthetic fertilizers are meant to supply essential
nutrients both macro- and micro alone and in combination of two or more than two
like ammonium nitrate, ammonium chloride, patassium sulphate, urea ammonium
phosphate, nitrogen phosphorus potassium (NPK), etc, according to the crop
requirement (Stewart et al., 2005). Inorganic fertilizers though enhanced the crop
production and provide maximum yield to farmers but also produced number of
deleterious effects on whole ecosystem including the enviroment, soil biota, human,
etc, which affects the soil fertility as well (Khan et al., 2007; Smith et al., 2008).
Beside this, day by day increasing prices of fertilizers, lack of knowledge for their
proper use and unavailabilty of specific fertilizer on time are few other drawbacks of
our society (Bhutto and Bazmi, 2007).
In addition, fungal diseases such as root-rot, root-knot, stem-rot, foliar
blights, wilt, damping off, etc, are another big threat for farmers to reduce the crop
productivity. To provide protection against these destructive fungal diseases, chemical
fungicides have prime importance globally. However, the prolong use of these
fungicides again become detrimental to environment and human health as these are
reported to contain different concentrations of harmful ingrediants such as sulfur,
mercury, cadmium, etc, which not only have cidal effect on target pathogens but also
reported to produce certain adverse effects such as inflammation of different body
tissues of persons who are continously in contact with these chemicals (Phupaibul et
al., 2002; Khan and Shahzad, 2007). Now-a-days, modern biotechnologists provide
biological ways by using microbial inoculants as biofertilizers and biocontrol agents
which are reported as good substitutes of synthetic fertilizers and fungicides that not
only preserve the soil fertility but also produce positive effect on crop production.
Therefore, by keeping this concept in mind, the prersent research work has been
designed to isolate, inoculate and investigate the effect of Trichoderma hamatum and
rhizobial isolates alone and in combination on physical and biochemical parameters
of two each of non-legume viz., H.annuus (sunflower) and B. nigra (black mustard)
and legume viz., V.mungo (mashbean) and C. arietinum (chickpea) plants in first
phase of study where as in second phase of study T. hamatum and selected rhizobial
isolates alone and in combination were used to prepare composted rice husk and
wheat bran. Later the effects of these composted food based organic fertilizers have
been investigated on physical and biochemical parameters of one each of non-legume
(sunflower) and legume (chickpea) plants.
4.1. Isolation of T.hamatum from rhizoplane and rhizobial
isolates from root nodules
In the present study, one of the Trichoderma species named T. hamatum
(JUF1) was isolated from rhizoplane of Amaranthus viridis. The species constitute the
genus Trichoderma viz., T.viride, T.harzianum, T.hamatum, T. koningi,
T.pseudokoningi, etc, are widespread inhabitants of rhizosphere (soil form thin film
on root surface) and rhizoplane (the outer surface of root) of plants (Mishra, 1996)
and are reported as nonpathogenic endophytic saprophytes due to their innate ability
to colonies the root surface (cortex) of host plant (Harman et al., 2004). The
rhizoplane has been recognized as the region of highest microbial activity (Clark,
1949). It is the underground region of plant where the most competitive fungal and
bacterial species can be found that could be pathogenic or beneficial for plant growth.
Beside providing mechanical support to plant and serve as a source of nutrient and
water uptake, roots also release root exudates in the form of variety of low molecular
weight compounds that in turn influence the microbial flora in the soil region around
the roots especially in rhizosphere (Bais et al., 2006). Researchers considered young
roots as a “pure echological forte” for soil microorganism (Mishra, 1996). It has been
observed that secreting substances like amino acids, etc, from primary roots after
emergence actually accelerate the germination of fungal spores and attract fungi on
root surface, at first all types of fungi can colonized on root surface but their
competentness make them true root surface fungi (Mishra, 1996). Therefore root-
microbe interaction can be later categories into beneficial which is associated with the
activities of fungi like Mycorhizza, Trichoderma, Aspergillus, Penicillium, etc or
parasitic related to root infecting fungi like Fusarium, Macrophomina, Rhizoctonia,
etc (Badri et al., 2009; Nihorimbere et al., 2011). Studies showed that soybean roots
secrete a group of organic compounds called isoflavones that chemotactically attract
on one hand a nitrogen fixer Bradyrhizobium japonicum and on other hand a pathogen
Phytopthora sojae (Morris et al., 1998). Another positive plant-microbe interaction
observed by rhizobia that form symbiotic association by inducing nodulation in roots
of legume plants and fixed atmospheric nitrogen, this type of interaction is profitable
for both partners (Simms and Taylor, 2002; Masson-Boivin et al., 2009). In the
present study, fast-growing rhizobium sp. (JUR1) isolated from Trigonella foenum-
graecum (fenugreek) and three slow-growing bradyrhizobium species viz., JUR2,
JUR3 and JUR4 isolated from Phaseolus ungiculata, Vigna radiata (mungbean) and
V.mungo (mashbean) respectively. These were identified on the basis of cultural,
morphological and staining characteristics and confirmed their host specificity by
observing nodulation abilities on their respective host.
4.2. In vitro antifungal activity of T.hamatum and rhizobial
isolates
The isolated fungal (JUF1) and rhizobial isolates (JUR1, JUR2, JUR3, and
JUR4) were screened in vitro against four plant pathogenic fungi including Fusarium
oxysporum, F.solani, Macrophomina phaseolina and Rhizoctonia solani in order to
evaluate their potential as biocontrol agents. The Fusarium species are well-reported
phytopathogens globally (Stenglein, 2009; Chehri et al., 2011; Burgess and Bryden,
2012) and considered as most frequent destructive fungi in agriculure fields of
Pakistan that causes number of diseases including root-rot, stem-rot and wilt on
variety of plants (Ehteshamul-Haque and Ghaffar, 1994; Hernandez-Hernandez et al.,
2010; Kawuri et al., 2012). A deuteromycetes M. phaseolina is largely distributed in
tropical and subtropical countries however it is well-suited in temperate regions like
Pakistan and reported as an infecting agent of seedling blight, charcoal rot, root-rot,
stem-rot and pod-rot on many species of plants (Iqbal et al., 2010; Mishra et al., 2011;
Rayatpanah and Dalili, 2012). Similarly, basidomycetes R.solani is known to attack
its host when it is in initial stage and cause diseases like seed-rot, damping off of
seedlings, wilt, root-rot, etc (Brenneman, 1997; Gonzalez-Garcia et al., 2006). In the
present in vitro antifungal study, T.hamatum (JUF1) showed its characteristic ability
of mycelial coiling named mycoparasitisism (Elad et al., 1983; Mohiddin et al., 2010)
and inhibited the growth of F.oxysporum, two strains each of M.phaseolina (strain-2
& 3) and R.solani (strain-1 & 2) whereas the same JUF1 produced zones of inhibition
ranging from 2- 5.5 mm against F.solani (strain-2), M.phaseolina (strain-1 & 4) and
inhibited the growth of three strains of F.solani (strain-1, 3, & 4) without any zone
and proved its another well-reported mode of inhibition known as antibiosis means
release of extracellular metabolites (antibiotics) that could be cidal or static in dilute
concentration and inhibit the growth of pathogens (Harmen et al., 2004; Morgan et
al., 2005; Ryder et al., 2012). However, strong antibiosis was observed by all tested
rhizobial isolates including rhizobium sp. (JUR1) and bradyrhizobium spieces (JUR2,
JUR3 & JUR4) against F. oxysporum, F.solani, M. phaseolina and R. solani and
inhibited their growth by producing zones ranging from 1.5-11.5 mm. Nitrogen fixing
rhizobial strains are well-reported to provide biocontrol against plant pathogenic fungi
through antibiosis like rhizobitoxine producing strains of B.japonicum protect
soybean from infection caused by M.phaseolina, inoculation of seeds of bean with
R.leguminosarum found antagonistic against F.solani (Deshwal et al., 2003). R.
meliloti, a host-specific rhizobium of fenugreek and T. hamatum alone and in
combination were found effective in reducing the infections caused by Fusarium spp.,
M. phaseolina and R. solani in fenugreek plants (Ehteshamul-haque and Ghaffar,
1992). Similar efficacy of Bradyrhizobium sp. of V.radiata was observed in
controlling the same three phytopathogens on sunflower and chickpea plants (Siddiqui
et al., 1998). Therefore the test fungal and rhizobial isolates have proved their
biocontrol potential in vitro against selected fungal pathogens.
4.3. Pot experiments
4.3.1. The effect of microbial inoculants on non-legume plants
including H. annuus and B. nigra
Sunflower (H.annuus) is one of the important kharif oilseed crops of
Pakistan and has 40% oil content which is free from any harmful compound (Weiss,
2000; Kaya and Kolsarici, 2011). In our country, from last few years its cultivation
got fame to increase the production of edible oil and to reduce the foreign import. Its
prominent attributes include high yield in short-growing period, more than one season
suited for its growth such as autum, spring and winter, requires low water for
irrigation and has an extensive flexibility to soil & moisture content and different
croping pattrens (Kazemeini et al., 2009; Yawson et al., 2011; Kirkova and
Stoimenov, 2012). In the present study, T. hamatum (JUF1) alone and in combination
with rhizobium (JUR1) & bradyrhizobium (JUR2) species found effective in
increasing the root length of sunflower plants at both 30th
and 60th
day as compared to
untreated (control) plants and plants treated with other bradyrhizobium species (JUR3
& JUR4). Three rhizobial isolates (JUR1, JUR2 & JUR4) alone in their respective
groups of test plants were found effective in improving the root length at 30th
day
whereas bradyrhizobium species (JUR2 & JUR3) became effective in same aspect at
60th
day. In combination with fertilizer (NPK), only bradyrhizobium species (JUR2,
JUR3 & JUR4) showed significant results and in combination with fungicide
(carbendazim), rhizobium sp. (JUR1) showed promoting effect on root length at both
days, however bradyrhizobium species were only found effective on 30th
day in
promoting the root length of sunflower plants. Similarly, T. hamatum (JUF1) alone
and in combination with rhizobial isolates (JUR1, JUR2) involved in improving the
shoot length of sunflower plants at 60th
day while bradyrhizobium species (JUR3 &
JUR4) were found effective at both days. Rhizobial isolates viz., JUR1, JUR2 & JUR3
individually in their respective groups have promoted the shoot length of sunflower
plants whereas all rhizobial isolates in combination with fertilizer and fungicide found
active on same aspect slightly at 30th
day and prominently at 60th
day. Rhizobial
isolates not alone but in combination with fertilizer and fungicide found efficient in
improving the fresh weight of test plants. Interestingly, T. hamatum alone and in
combination with fertilizer have significantly promoted all three growth parameters
including root & shoot lengths and fresh weight of sunflower plants.
The growth prmoting effect of rhizobium and bradyrhizobium species alone
and in combination with T.hamatum of present work is comparable to previous
studies which described that bradyrhizobium sp. isolated from V.radiata alone and in
combination with fungal anatagonist such as Pacilomyces lilacinus, Memnoniella
echinata and T. harzianum was reported to increase the 40% growth of sunflower
plants besides inhibiting the infections caused by Fusarium sp., M.phaseolina and
R.solani (Siddique et al., 1998). Another researcher reported that R. meliloti, isolated
from fenugreek, alone and alongwith T.hamatum & T.harzianum improved the growth
and vigor index of sunflower plants by controlling root-rot infection caused by
M.phaseolina (Ehteshamul-Haque and Ghaffar, 1998; Anis et al., 2010). Similarly,
the same rhizobium sp. alone and in combination with T.harzianum found effective in
increasing the root & shoot lengths plus their dry weights and overall heights of okra
and sunflower plants (Dawar et al., 2008).
In the present study, T.hamatum alone and in combination with
bradyrhizobium species (JUR3 & JUR4) improved the total chlorophyll content of
sunflower plants. The improvement in this same parameter was also observed by
rhizobial isolates (JUR1, JUR3 & JUR4) alone and in combination with fertilizer and
fungicide in their respective group. Again T.hamatum in combination with fertilizer
found effective in improving the total chlorophyll content of test plants at 30th
day.
T.hamatum (JUF1) and bradyrhizobium species viz., JUR3 & JUR4 alone in their
respective groups at 60th
day, T.hamatum in combination with JUR1on same day and
with JUR4 at both days effectively improved the total carbohydrate and crude protein
contents in sunflower plants. The same bradyrhizobium species (JUR3 & JUR4) with
fertilizer (NPK) found effective at 60th
day whereas JUR2 & JUR4 with fungicide
(carbendazim) found active in improving the same biochemical parameters of
sunflower plants. Mineral content of sunflower plants was also improved by JUF1,
JUR3 & JUR4 alone and in combination of JUF1+JUR4 at 60th
day. The same JUR3
& JUR4 were also found effective in combination with fertilizer and fungicide,
moreover, JUR2 gave significant result in combination with fungicide in increasing
the mineral content of sunflower.
Black mustard (B.nigra) is one of the oilseed crops of Brassicaceae family,
its seeds approximately contains 30-40% oil (Peter, 2004; Shekhawat, 2012). In the
present study, inoculation of T.hamatum (JUF1) and rhizobial isolates (JUR1, JUR2,
JUR3 & JUR4) alone and in combination significantly improved the growth
parameters especially root and shoot lengths of test plants. Similarly, all rhizobial
isolates (JUR1, JUR2, JUR3 & JUR4) in combination with fertilizer (NPK) again
found effective in boosting the same growth parameters of black mustard plants as
compared to untreated (control) plants. However, the same four rhizobial isolates in
combination with fungicide (carbendazem) found not very effective on same aspect
though these produced significant effects on root length at 60th
day as same as the
treatment coded by JUF1+FTZ. T.hamatum, rhizobium sp. (JUR1) and
bradyrhizobium spp. (JUR2 & JUR3) alone in their respective groups again found
efficient in increasing the total chlorophyll content of test mustard plants. The same
positive effect was also observed on total chlorophyll content of test plants which
were treated with T.hamatum (JUF1) in combination with JUR1, JUR2 and JUR3.
Treatment of T.hamatum with fertilizer (JUF1+FTZ) was found effective at 60th
day
as well. In combination with fertilizer, JUR1 & JUR2 and in combination with
fungicide, JUR2 and JUR3 produced significant effects at 60th
day not only on total
chlorophyll content but also its fractions (a & b). T.hamatum alone at 30th
day,
bradyrhizobium species (JUR2, JUR3 & JUR4) alone in their individual groups and
dual inoculation of all rhizobial isolates with T.hamatum as JUR1+JUF1,
JUR2+JUF1, JUR3+JUF1 & JUR4+JUF1 produced significant effects on total
carbohydrate and crude protein contents at both days. In combination with fertilizer,
all bradyrhzibium species (JUR2, JUR3 & JUR4) found effective on same aspect.
Variable results was observed in case of mineral content (nitrogen & phosphorus) of
balck mustard plants treated with JUF1, JUR2, JUR3 & JUR4 alone but overall
nitrogen content was more significantly increased as compared to phosphorus content
in test plants compared with control plants.
The obtained positive effects of rhizobial isolates alone and in combination
with T.hamatum on growth parameters of black mustard plants in pressent study
support the previous reports that described the abilities of rhizobium strains in
producing phytohormones in response to their inoculation via seed dressing or root
drench which helped to accelerate the growth and production of non-legumes
(Sessitsch et al., 2002). A study proved that the direct stimulatory effect of
R.leguminosarum inoculation on roots of B.campestris (another species of Brassica)
and lettuce was found by producing indole-3-acetic acid and cytokinin, the growth
regulators or phytohormones (Noel et al., 1996). Our study also proved that
inoculation of T.hamatum with fertilizer or rhizobial isolates with each of fertilizer
and fungicide produced beneficial effects on growth and biochemical parameters of
non-legume plants. It has also been strengthen by evidences that described the
bacterial inoculation promoted the plant growth by increasing N uptake and reducing
the amount of nitrogen fertilizer that normally used (Mia and Shamsuddin, 2010).
Likewise increased in nitrogen contents of seeds, number of nodules and yield of
different crops have been observed by using rhizobium inoculants with and without
fertilizer in many experiments (Mia and Shamsuddin, 2010). Chlorophyll content
indicates the normal photosynthetic function of plant tissues which results in the
formation of high energy-producing compounds in the presence of sunlight which are
needed by plant for its regular metabolism. It has been reported that increased in
chlorophyll content also linked to increase in total carbohydrate in plant tissues
(Densilin et al., 2010), the same theme was achieved in our present study. On the
other hand, increased protein content in growing parts of plant reflects the metabolic
regulation associated with enhanced enzyme activity which helps plant to withstand
environmental conditions and to promote their growth (Patil, 2010).
In conclusion, the growth promoting effects observed by T.hamatum alone and
in combination with rhizobial isolates was confirmed its ability to produce antibiotics
in rhizosphere that restrict the growth of microorganisms which have detrimental
effects on plant growth (Kaewchai et al., 2009; Mohiddin et al., 2010). It was evident
that seed treatments with T.hamatum have protected both seeds and seedlings of
radish and pea from infections of R. solani and Pythium spp (Harman et al., 1980).
Besides showing antibiosis, Trichoderma species are also reported to control root-
infecting fungi through mycoparasitism, induce struggle for space and nutrients
among microorganism and produce fungal cell wall lytic enzymes such as β-1,3-
glucanase, chitinase, cellulase, protease, etc, and induced resistance in host plant
against diseases by altering plant gene expression, results in formation of enyzmes
and defensive proteins (Pandya and Saraf, 2010; Alfano et al., 2007). Recently strain
382 of T. hamatum 382 reported to reduce the occurrence of foliar diseases of several
vegetable crops including tomato by altering genes involved in stress and protein
metabolism (Al-Dahmani et al., 2005; Khan et al., 2004; Horst et al., 2005). In
addition these cellulytic fungi are reported to have plant growth stimulating effects by
enhancing the availability of nutrients and minerals (Fe, N, P) for plants, producing
plant growth hormones such as alamenthecins, gliotoxin, harzianic acid, trichotoxin,
trichoviridin, viridin, viridiol, etc, and decomposing organic material to improve the
soil fertility which produced positive impact on farming production (Kaewchai et al.,
2009). Trichoderma isolates are reported to improve the nitrogen and phosphorus
contents of crops like tomato seedling, sugarcane, etc by enhancing the nitrogen
uptake and phosphate solubilization (Altomare et al., 1999; Singh et al., 2010; Azarmi
et al., 2011). The same significant improving effect of T.hamatum on mineral content
especially on percent nitrogen of both non-legume plants was also observed in our
study. In addition Trichoderma species helped plants to withstand against abiotic
stresses such as by increasing the length of secondary roots deep in the ground or soil
and improving the water holding capacity to provide protection against drought
(Mastouri et al., 2010). In this regard, T.hamatum was recently reported to induce
tolerance in cocoa plants against water deficit through increasing root growth (Bae et
al., 2009). Therefore, this stress tolerant disease-free environment and improvement
in soil fertility provided by T.hamatum may be found effective for rhizobial isolates to
promote growth and improving the nutritional status of both non-legume plants
asymbiotically or through associative nitrogen fixation in the present study.
Simialrly many researchers proved that rhizobium and bradyrhizobium species
are quite competent in survivng and colonizing the rhizospheres of non-legume crops
(Saharan and Nehra, 2011; Jarak et al., 2012). Studies showed the presence and
duplication of R. legeminosarum bv. trifolii (strain R39) in rhizosphere of many non-
legume crops including barley, corn, raddish, rape and wheat (Wiehe and Hoflick,
1999), the saprophytic and endophytic presence of R. etli in maize roots (Gutierrez-
Zamora and Martinez-Romero, 2001) and bradyrhizobium species in rice roots
(Chaintreuil et al., 2000, 2001). It was also reported that rhizobial inoculation
improved the seed germination, seedling emergence and growth of lowland rice
variety MR219, another non-legume plant (Mia et al., 2012). Other studies provided
evidences that rhizobium species can induced not only increase in germination and
seedling emergence but also improved the growth and output of many cereal and non-
cereal plants (Mia and Shamsuddin, 2010; Saharan and Nehra, 2011). Quite a lot of
studies have been reported that rhizobium and bradyrhizobium species have
prominent plant growth improving effects on non-legume plants by several direct and
indirect mechanisms. Direct mechanisms include 1. production of phytohormones
such as auxin, indole acetic acid, gibberllins, etc, (Humphry et al., 2007; Martinez-
Viveros et al., 2010), 2. Increased nutrients uptake (Biswas et al., 2000a; Biari et al.,
2008), 3. synthesized siderophores which chelate iron (Robin et al., 2008; Avis et al.,
2008), 4. increased phosphate solubilization to make phosphate available for plants
(Richardson et al., 2009; Yazdani et al., 2009), 5. improved root respiration of
inoculated plants (Volpin and Phillips, 1998), 6. induced enzyme generation in
inoculated plants (Glick, 2005; Ahemad and Khan, 2011). Whereas the same two
genera of nitrogen fixing bacteria also promotes plant growth indirectly by acting as
biocontrol agents for plant pathogenic microorganisms through 1. antibiosis by
secreting extra-cellular metabolites (antibiotics) against plant pathogenic bacteria and
fungi, 2. by producing siderophores to make pathogen starving, 3. by producing
hydrogen cyanide (HCN) and 4. by inducting systemic resistance (Antoun et al.,
1998; Gracia-Fraile et al., 2012).
The obtained results of present study clearly concluded that T.hamatum,
rhizobium and bradyrhizobium species alone and in combination are beneficial for the
growth and nutritional status of non-legume plants, thereby improving their
productivity and most important, they showed synergism by not interferring the
natural abilities of one another. This is in accordance to the study that described that
seed treament of pea with Rhizobium and T.hamatum did not effect the nodulating and
protective abilities of each other (Harman et al., 1981).
4.3.2. The effect of microbial inoculants on legume plants
including V. mungo and C. arietinum
Beans are considered as high protein with low fat diet and are widely used in
developing countries especially Asian countries as staple food or as a substitute of
animal protein (Tresina et al., 2010). However legumes are subjected to many fungal
pathogens that severely affect the roots & leaves and become as a major limiting
factor for the yield of these crops in many countries (Puglia and Aragona, 1997).
Again in order to minimize the use of chemical fertilizers and fungicides due to
economical, environmental and health reasons that are normally the first preference of
farmers to increase the yield of legumes (Shaban and El-Bramawy, 2011). Now-a-
days, microbial inoculants have been using as an alternative of commercially
available chemicals to control diseases and promote growth of both legume and non-
legume plants (Nakkeeran et al., 2002).
The biocontrol and growth promoting potentials of Trichoderma species
have been extensively studied and also found that their beneficial effects are not
restricted to non-legumes (Pandya and Saraf, 2010). There are several Trichoderma
products commercially available in market as fungal biofungicides and biofertilizers
which are successfully used not only to control plant diseases but also to promote the
growth of plants in greenhouses and agriculture fields (Kaewchai et al., 2009).
Similarly Rhizobium species are well-documented for their abilities to fix atmospheric
nitrogen in roots of legume plants through nodulation which induced significant
growth promoting and yielding effects in their specific host plants (Mia and
Shamsuddin, 2010). These are also reported to restrict the growth of many soil-borne
pathogens of legume plants such as R. solani, F. oxysporum, F.solani, M. phaseolina,
etc, which also in turn produce health improving effects on plants (Sessitsch et al.,
2002).
In the present study, V. mungo (mashbean) and C. arietinum (chickpea) were
two legume crops seleceted to investigate the effect of microbial inoculants on their
growth and biochemical parameters. The results of V. mungo revealed that host-
specific bradyrhizobium sp (JUR4) of same V.mungo produced accelerating effects on
all growth parameters including root & shoot lengths and fresh weight of same plants
at both days, followed by bradyrhizobium sp. (JUR2) isolated from P. unguiculata on
all three growth parameters at 30th
day as compared to rhizobium (JUR1) and
bradyrhizobium (JUR3) species isolated from T.foenum-graecum and V.radiata
respectively that produced significant effects on root and shoot lengths only at 60th
day. Rhizobial isolates in combination with T.hamatum and fertilizer induced
significant increase in two out of three growth parameters especially on root and shoot
lengths. However, T.hamatum alone did not show any effect but in combination with
fertilizer became active on growth parameters at 60th
day. Almost same treatments
were found effective in increasing the total chlorophyll content and its fractions in test
plants either at 30th
or 60th
or both days but again JUR4 alone and in combination with
T.hamatum or fertilizer produced significant effect on same aspect at both days as
compared to all others. In case of improving the total carbohydrate and crude protein
contents of V.mungo plants, T.hamatum and bradyrhizobium species (JUR3 & JUR4)
alone found efficient in their respective groups and in combination with T.hamatum,
JUR1 at 30th
day and JUR4 at both days found active. JUR3 & JUR4 in combination
with fertilizer produced significant effects on both biochemical parameters including
total carbohydrate and crude protein contents in test plants at both days but
JUR2+FTZ produced same positive effect on 30th
day while in combination with
fungicide, JUR2 and JUR4 found efficient. Interestingly, host-specific
bradyrhizobium sp. (JUR4) alone and in combination with T.hamatum, fertilizer, and
fungicide produced significant increase in nitrogen and phosphorus contents of test
plants.
According to the obtained results of V.mungo plants, it has been observed that
out of all tested rhizobial isolates, host-specific bradyrhizobium sp. (JUR4) of same
plant found effective in improving the growth parameters of these plants followed by
JUR2, JUR1 and JUR3. T.hamatum and host-specific JUR4 were well-matched with
each other and their combination was found effective to improve the growth
parameters and total chlorophyll, carbohydrate and crude protein contents of
experimental crop as compared to control plants which did not treat with any of the
tested microbial inoculant. Similarly, mineral including nitrogen and phosphorus was
significantly increased in test plants treated with the combination of T.hamatum and
JUR4. The plant growth stimulating effect of host specific bradyrhizobium sp. was
reported, besides nodulating the roots of its specific host, which resides in producing
indole acetic acid (IAA), an active auxin and phosphate solubilization while
anatagonistic activity in producing siderophores and enzyme such as chitinase, 1-
aminocyclopropane-1-carboxylate (ACC) deaminase, etc, (Dobey et al., 2012). It has
also been confirmed by another study that root nodules of V.mungo contained high
amount of IAA as compared to non-nodulated roots (Mandal et al., 2007). The same
significant results were also obtained on nodulation, growth and yield of V.mungo by
inoculating B. japonicum alongwith L-tryptophane, a precursor of IAA and author
stated that this type of approach could be beneficial for sustainable production of
legumes (Hussain et al., 2011). Auxin, is one of the plant hormones, reported to
enhance the growth of shoot and root, promote processes of flowering and fruiting,
not confirmed but may be involved in nodulation (Taiz and Zeiger, 2010). There are
scientific evidences which proved the involvement of rhizobial species in the
production of IAA that inturn improved the root growth and uptake of nutrients by
plants (Kavin, 2003). Beside this, inoculation of different strains of bradyrhizobium
spp was also reported to stimulate the growth of V.mungo in Serbian soils by
improving their shoot dry weight yeild, nitrogen and protein contents (Delic et al.,
2009). Other rhizobium species like R. japonicum was found effective in increasing
height, fresh weight, number of roots, nodules, leaves, length of pods and seed weight
of V.mungo and V.radiata (Ravikumar, 2012). The same was observed in our study
that bradyrhizobium (JUR2 & JUR3) and rhizobium (JUR1) species were also found
effective in enhancing almost all three growth parameters either at 30th
or 60th
day
though these were isolated from different hosts. A study showed that Bradyrhizobium
alone and in combination with Pacilimyces lilacinus not only found to control root-
knot nematode but also improved the nitrogen content of root & shoot and nitrogenase
activity in V.mungo plants (Bhat et al., 2012). Similarly, species of genus
Trichoderma including T.hamatum produced number of hydrolytic enzymes that are
involve in inhibiting the growth of infection producing microorganisms via a process
of mycoparasitism (Harman et al., 1981) so it may function in improving the growth
of test plants like T.viride, an abiotic stress tolerant strain was reoprted to increase the
maximum yeild of V.mungo by controling root-rot infection of M.phaseolina (Leo et
al., 2011). On the other hand, a study showed that combined inoculation of rhizobium
and phosphate solubilizing bacteria improved the growth parameters including plant
height, dry matter, number and weight of nodules per plant with yield in terms of
branches, pods per plant, seeds per pod in V.mungo plants under temperate regions of
Kashmir (Hussain et al., 2011). It could be one of the alternate ways for improving
the growth, biochemical parameters and mineral content of V. mungo in the present
study as the three of the tested microbial inoculants including Rhizobium,
Bradyrhizobium and Trichoderma species are reported to have phosphate solubilizing
activities (Barea et al., 2005; Kaewchai et al., 2009; Pandya and Saraf, 2010).
However, little work has been done on investigating the effect of dual inoculation of
either rhizobium and Trichoderma spp. or bradyrhizobium and Trichoderma spp. on
uptake of nutrient, plant growth and yield of legume plants including V.mungo.
Similarly, the results of C. arietinum (chickpea) plants, another legume,
demostrated that treatments including JUR1, JUR2, JUR3 & JUF1 at both days and
JUR4 at 30th
day produced significant effects on almost all growth parameters of these
plants. In combination with T.hamatum (JUF1) and fertilizer, three rhizobial isolates
viz., JUR1, JUR3 & JUR4 at 60th
day and alongwith fungicide, all four rhizobial
isolates produced promoting effect on growth parameters. The total chlorophyll and
its a or b or both fractions were significantly increased in chickpea plants treated with
JUF1, JUR1, JUR2, JUR3 at both days and JUR4 at 30th
day. In combination with
T.hamatum (JUF1), JUR1 & JUR3 at 60th
day, JUR2 at 30th
day and JUR4 at both
days found efficient on same aspect. In combination with fertilizer, JUR1 at 60th
day,
JUR2, JUR3 & JUR4 at 30th
day while in combination with fungicide, JUR1, JUR2,
JUR3 at 60th
day and JUR4 at 30th
day found effective in improving the total
chlorophyll and its fractions (a & b) in test plants. The treatments include JUR1 &
JUR2 improved biochemical parameters including total carbohydrate and crude
protein contents at 60th
day while only total carbohydrate content of test plants was
improved by JUR3 & JUR4 at 30th
day. Likewise rhizobial isolates in combination
with JUF1 found more effective at 60th
day on same aspect. In combination with
fertilizer, JUR2, JUR3 & JUR4 at 60th
day and with fungicide, JUR1 & JUR4 at 60th
day found active in improving the total carbohydrate and crude protein contents of
chickpea plants. Mineral content of chickpea plants were found improved by JUR1 &
JUR2 especially nitrogen at both days and phosphorus at 60th
day whereas by JUR3 &
JUR4 improved phosphorus at both day and nitrogen at 30th
day. Most of the
treatments including T.hamatum in combination with rhizobial isolates and these in
combination with fertilizer or fungicide improved nitrogen content at either 30th
or
60th
day or both days.
On the basis of results obtained from C.arietinum plants, T.hamatum, JUR1,
JUR2 and JUR3 alone found effective in improving the growth parameters and total
chlorophyll of test plants. However, all rhizobial isolates in combination with
T.hamatum, fertilizer (NPK) and fungicide (carbendazim) found efficient in
improving the total carbohydrate, crude protein and mineral contents of chickpea
plants. Our findings are also comparable to the study that described the improvement
in growth, nutrient uptake and production of chickpea under glasshouse and field
experiments due to the combined inoculation of Rhizobium sp. with Trichoderma spp.
(Rudresh et al., 2005). Similarly, another study showed that dual inoculation of
Rhizobium sp. with T.harzianum not only provide biological control against damping
off and root-rot diseases of legume crops including Vicia faba, C. arietinum and
Lupines terms but also the same combination was found effective in increasing
growth parameters of same crops in greenhouse experiments (Shaban and El-
Bramawy, 2011). The growth promotion of both legume plants may also be associated
with the improvement in mineral content of test plants due to the action of microbial
inoculants by increasing their availability and uptake (Biswas et al., 2000a; Biari et
al., 2008), the same was also observed in the present study. Nitrogen and phosphorus
are the essential macronutrients for plant growth as these are required for the
synthesis of many important components of plant like enzymes, hormones, proteins,
DNA, RNA , etc, and their availabilty in soil enhanced the production of food and
feed (Richardson et al., 2009; Hayat et al., 2010). Three of the tested microbial
inoculants included bradyrhizobium, rhizobium and Trichoderma species are actively
involved in nitrogen uptake and phosphate solubilization (Pandya and Saraf, 2010;
Hayat et al., 2010). Rhizobial strains have ability to solubilize inorganic phosphate
compounds viz., dicalcium & tricalcium phosphate, hydroxyl apatite and rock
phosphate by secreting organic acids, of which tricalcium phosphate and hydroxyl
apatite are the most degradable substrates as compared to rock phosphate (Goldstein
1986; Halder and Chakrabarty, 1993; Rodríguez and Fraga 1999; Rodríguez et al.,
2006; Banerjee et al., 2006). There are organic compunds also available in soil which
can serve as a source of phosphorus for plant growth and these must be hydrolyzed to
inorganic phosphate due to the action of enzymes like phosphatase, phytase,
phosphonoacetate hydrolase, D-α-glycerophosphatase, C-P lyase, etc, to make it
available for plant nutrition (Ohtake et al., 1996; Richardson and Hadobas, 1997;
McGrath et al., 1998; Skrary and Cameron 1998; Rodríguez and Fraga, 1999).
Intersetingly, the genus Rhizobium is also reported to express acid phosphatases
which are helpful in mineralization of organic phosphate (Abd-Alla 1994a, b). The
efficiency of rhizobial strains used as biofertilizer in organic farming should not be
depend only on their potential for fixing atmospheric nitrogen but also promote plant
growth by mechanisms like phosphate solubilization (Peix et al., 2001). The
phosphate-solubilizing activity of rhizobium and bradyrhizobium is assumed to be
related with the production of 2-ketogluconic acid (organic acid) which was
neutralized by adding NaOH in a study that indicates these bacteria are involve in
decreasing pH towards acidic side (Halder and Chakrabarty, 1993).
Therefore, T.hamatum alone and in combination with rhizobial isolates have a
great potential value in organic agriculture and can be act as a good replacement of
chemical fertilizer and fungicide for producing positive effect on growth of V.mungo
and C. arietinum by improving the soil fertility and providing disease free
environment by residing in roots tissues and showing rhizosphere competence
especially by T.hamatum, this condition would more strengthen by rhizobium and
bradyrhizobium spp. that could enhanced their natural ability of competition through
antimicrobial substances, siderophores, lytic enzymes, etc and particularly creating
symbiosis or associative interaction with roots of its specific host or non-host legume
plants which in turn promote the growth of legume and improve the mineral (N, P)
content of test plants.
4.4. Composting
Composting is a technique utilized to convert organic matter into value-
added product or nutrient-rich humus with the help of effective microorganisms
which can be used as organic fertilizer that accelerate the growth and nutritional status
of both legume and non-legume plants by improving the soil fertility and water-
holding capacity (Panda and Holta, 2007; Buyukgungor and Gurel, 2009). Most
important it is environment friendly as compared to other synthetic fertilizers. By
keeping this concept in mind, in the present study, two food waste materials including
rice husk and wheat bran were composted with the help of T.hamatum (JUF1),
rhizobium (JUR1) and bradyrhizobium (JUR2) species alone and in combination, later
the effect of each composted organic fertilizer (5g & 10g /2 kg soil/pot) was
investigated on growth and biochemical parameters of sunflower (non-legume) and
chick pea (legume) plants.
4.4.1. Effect of microbial treatment on total carbohydrate and
protein of composted rice husk and wheat bran
In the present study, the microbial treatments (Table 2), used to involve in
composting of food wastes, were efficiently found to increase the total carbohydrate
and total protein contents in composted rice husk and wheat bran as compared to
uncomposted and only grinded same organic food wastes. It is as same as many
studies confirmed that treatment with effective microorganisms (EM) increased the
mineral content of composted waste materials (Shalaby, 2011). Therefore, in the
present study, composted rice husk and wheat bran with increased amount of total
carbohydrate and total protein content used as a good source of carbon and nitrogen
respectively, two of these are important elements for plant growth. These nutritionally
rich composted organic fertilizers may improve the soil texture and fertility which in
turn could produce positive impact on growth and nutritional status of both non-
legume and legume plants.
4.4.2. Pot experiments
4.4.2.1. The effect of composted rice husk and wheat bran on
H. annuus (non-legume) and C. arietinum (legume)
plants
Organic matter is soil normally serves to maintain nutrients, structure, porosity
and water holding capacity which all together improves the fertility of soil, an
essential component for plant growth (Golabi et al., 2004). The amount of this natural
reserve always fluctuates due to the changes in environmental conditions and
agricultural practices. In developing countries like India, Pakistan, etc, the rapidly
increasing population year by year also increases the demand of food which usually
fulfilled with the rigorous cropping system that produce sever depletion in soil
organic matter. Traditional systems of farming, improper and excessive use of
chemical fertilizers and pesticides not only have negative impact on environment and
produce many health problems in human and live stock but also on food safety and
quality. Therefore, biotechnologists have been convincing and motivating farmers and
consumers both that organic farming is the best substitute for inorganic fertilizer and
fungicide based agriculture. Though idea is not innovative but it has been receiving a
huge attention in both developed and developing countries now-a-days (Higa and
Parr, 1994).
Soil organic matter can be improved by adding uncomposted and composted
organic wastes or biodegradable products. This meet the aims of alternative
agriculture practices and provide harmony with all personnel who are anxious about
the environment and human health by creating awareness about waste management
and its use in sustainable agriculture in place of chemical fertilizers. A lot of research
has been done to describe the benefits of organic amendments in improving the three
important aspects of soil including physical, chemical and biological but depend on
amount and composition (Reeves, 1997; Tejada et al., 2008; Badalucco et al., 2010).
However, the physical and chemical parameters are subject to change slowly and
gradually to show noteworthy differences but biological and biochemical parameters
are more quick to respond and can act prompt indicators of changes induced by soil
amendments (Ndiaye et al., 2000; Madejon et al., 2001; Melero et al., 2007; Courtney
and Mullen, 2008; Chitravadivu et al., 2009; Martinez-Salgado et al., 2010).
In the present study, composted organic fertilizer (COF) application provoked
a significant improvement in growth and biochemical parameters of both non-legume
and legume plants as compared to control plants treated with uncomposted organic
fertilizer (UCOF) and it was clearly indicated that addition of COF may increase the
organic content of soil. This possibility was also supported by a study that described
the application of rice straw compost with or without the addition of mineral fertilizer
induced marked increase in organic content of soil which in turn produced positive
effects on its physical properties and microbial activity (Rashad et al., 2011).
Similarly, addition of industrial orange wastes found efficient in improving the soil
characteristics, growth and productivity of durum wheat (Belligno et al., 2005). Study
showed that organic wastes are highly rich in macro- and micro-nutrients (Shah and
Anwar, 2003). Application of organic wastes from different sources including derived
from food in agriculture fields is one of the traditional methods to improve the crop
yield (Parr et al., 1986; Sabiiti, 2011). However, studies proved that direct
application of organic waste without any treatment or processing in agriculture fields
or planting has different negative impact like unprocessed or un-composted organic
materials have heavy metals which produced harmful effect on plants (Gupta et al.,
1998; Singh and Agarwal, 2010) or have wider carbon nitrogen ratio as compared to
ratio which plants really need that would in turn inhibit the availability of nitrogen to
plants by being incarcerated in soil biomass through micro-flora (Ahmad et al., 2006).
Now-a-days, composting is one of the popular methods to produce degradable or
digestible products of organic wastes with improved nutritional and mineral contents
which when applied to soil are easily available to plants (Inckel et al., 1996). It was
also observed in present study, that composting of food wastes (rice husk and wheat
bran) with selected microbial treatments increased their total carbohydrate and total
protein contents which could serve as good source of carbon and nitrogen respectively
and may help to re-establish or improve the fertility of degraded soil or soil. Studies
proved that properly processed organic matter or compost can provide excellent
supply of food and energy for natural microflora especially rhizosphere competent
one (Bunemann et al., 2006; Fuchs et al., 2008).
A preliminary study was conducted as a part of present research work by
applying COF with improved content of total carbohydrate and protein at two
quantities (5 & 10 g / 2 kg soil /pot) on seven day of germination of developing
seedlings of test plants in net house pot experiments where COF have proved its
potential for growth promotion and enhancement in biochemical and mineral contents
of both non-legume and legume test plants but effects vary with the microbial
treatments involved in composting like rice husk (RH) composted with T. hamatum
(JUF1) and in combination with rhizobium sp. (JUR1+JUF1) found effective in
improving the shoot & root lengths of plants, photosynthetic pigment especially
chlorophyll-a & total chlorophyll, biochemical parameters including crude protein and
mineral (nitrogen & phosphorus) contents of sunflower (non-legume) plants. Whereas
RH composted with all treatments including JUF1, JUR1, JUR2 (bradyrhizobium sp)
alone and in combination with JUR1+ JUF1 & JUR2+ JUF1 at 5 and 10 g found to
produce significant effects on growth, photosynthetic pigment especially chlorophyll-
b & total chlorophyll, biochemical parameters including total carbohydrate, crude
protein and mineral (nitrogen & phosphorus) contents of chickpea (legume) plants.
Similarly, wheat bran (WB) composted with all treatments especially JUF1 found
effective in improving the growth, photosynthetic pigment and nutritional status of
sunflower plants, however, percent nitrogen content was much improved as compared
to phosphorus of same test plants. While WB composted with all treatments at both
amounts was found efficient only in improving all growth parameters including root,
shoot lengths and fresh weight, total carbohydrate and phosphorus content of chickpea
plants. These findings of present work strengthen the idea given by researchers that
properly prepared biodegradable product or compost without the addition of effective
microorganisms (EM) is beneficial for plant growth, however, addition of EM may
also increase other characteristics of composts like biocontrol against certain
pathogens and improve its productivity (Shalaby, 2011). Another study also proved
that properly processed compost is usually much better than un-composted materials
which are rich in nutrient, contains appropriate C: N proportion and free from
pathogens or other potential contaminants that could cause pollution (Zia et al., 2003).
Rice husk is a natural productive sheath that covers the rice grains during their
growth and separates from rice grains during winnowing or refining processes. It
constitutes 20% of total weight of rice harvested and about 80% by weight it contains
organic components such as lignin beside silicon dioxide (Anonymous, 1979). Rice
husk has been used as soil amendment to improve crop yield and also reported to
control plant pathogens including fungi, bacteria, nematodes and cowpea mottle virus
(Aliyu et al., 2011). In this regard, study reported that addition of rice husk in soil not
only improved the soil properties but also the yield of many crops (Sharma et al.,
1988). Another study provides evidence that soil composted with rice husks decreased
the occurrence of wilting caused by F. solani on Parkia biglobosa from 31 to 70 %
(Muhammed et al., 2001). The second food waste used in the present study was wheat
bran which is one of the wheat byproducts. It is actually a rough hard covering of
wheat kernel that separates during the milling processes and on the basis of chemical
composition it consists of protein (16.7%), fat (4.6%), crude fiber (11.3%), starch
(11.7%), total sugar (5.5) beside other cell wall material (Sramkovaa et al., 2009). It
has also been reported as a good source of protein and mineral (Kumar et al., 2011).
Wheat bran reported as a medium for the growth of T.harzianum and carrier for the
same fungus which on application found effective in decreasing the Phythium sp
causing damping off disease in pea, tomato, cucumber, etc (Sivan et al., 1984).
The test microorganisms used in the present study for composting of organic
food wastes were T.hamatum, rhizobium and bradyrhizobium species, are well-
famous producers of lytic enzymes including β-1,3-glucanase, chitinase, cellulose,
etc, (Harman et al., 1981; Hayat et al., 2010) found efficient in producing
biodegradable product and improving organic matter of soil on its application. This is
the first study which describes the utilization of T. hamatum in composting of organic
food wastes besides T.harzianum belongs to same genus, a well-reported fungus
utilized in composting procedure and found effective both as alone or in combination
with rhizobium, in improving physical properties, organic content of soil and yield of
many non-legume and legume crops (Rahman et al., 2011; Lopaz-Mondejar et al.,
2010). However, this is the initial study and much work has been required to prove
the claim that biodegradable or digested product of T.hamatum alone and in
combination with rhizobium & bradyrhizobium species can be used on commercial
scale. Study showed that the quality of the compost vary which actually depends on
composting feed material that make difficult to predict its application rates and
investigate its beneficial effects on soil nutrient content, soil conditioning and bio-
control properties (Rashad et al., 2011). Lastly, the use of properly composted organic
nutrient sources in agriculture fields not only helped to recycles organic wastes that
cause pollution to ecosystem but also preserve the nutrients resources which can
minimize the use of chemical fertilizers upto the certain level (Heluf, 2002) though a
study showed that integrated use of synthetic fertilizer with composted material
improves its efficiency and reduces losses (Guar & Geeta, 1993). Keeping all the
points in view, the present study was focused on recycling organic waste into
biodegradable value added product which could be beneficial for sustainable
agriculture and environment.
5. Conclusion and future prospects
The results conclude that test microorganisms of present study including
T.hamatum (JUF1) and rhizobial isolates (JUR1, JUR2, JUR3, & JUR4) alone and in
combination have shown an excellent growth promoting potential in pot experiments
by not only enhancing the growth but also improving the total carbohydrate, crude
protein, nitrogen and phosphorus contents of plants including sunflower, black
mustard, mash bean and chickpea plants. Hence, these microorganisms can be used as
bioinoculants or biofertilizers which may possibly serve as a good substitute for
chemical fertilizers in farming practices in our country and worldwide to enhance the
growth and nutritional status of both non-legume and legume plants. In addition, the
same test microorganisms also proved their biocontrol potential in vitro against
M.phaseolina, R.solani and Fusarium species, one of the frequent fungal pathogens
found in agriculture fields of Pakistan. Similarly, T.hamatum alone and in
combination with rhizobial isolates (JUR1 & JUR2) would be beneficial in the
preparation of composted organic fertilizer as the composting procedure by using
these microorganisms converted organic food wastes including rice husk and wheat
bran into nutritionally rich biodegradable products that were also found effective in
improving the growth and biochemical parameters of sunflower (non-legume) and
chickpea (legume) plants when applied at 5 and 10 g each /2 kg soil / pot by possibly
improving the organic content of soil. The composting of organic wastes with the help
of microbial inoculants not only help in recycling of wastes but also results in the
preparation of economical and environmental friendly organic fertilizer that could
provide benefits to agriculture. Therefore, the study clearly indicates that the
utilization of biofertilizers (microbial inoculants) and organic fertilizers (especially
composted) is beneficial over sole application of inorganic fertilizers and fungicides.
The present research work can be further elaborated and evaluated in future on
the following aspects, as;
1. The test microorganisms used as microbial inoculants or biofertilizers in pot
experiments of present study must be inoculated and evaluated for their effects on
growth and yield of both non-legume and legume plants in field experiments under
different environmental conditions and or with different soil types.
2. To evaluate the rate and relative impact of integration of composted organic
fertilizer (used in the present study) and inorganic fertilizer on growth and yield of
non-legume and legume plants in both pot and field experiments to validate the claim
that biodegradable products prepared through composting of organic wastes enrich
soil fertility on long-term basis for sustainable crop production.
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