use of microbial inoculants and organic fertilizers for

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





3.6.2. Effect of microbial inoculants on legume plants 96

3.6.2.1. Vigna mungo L. Hepper 96





3.6.2.2. Cicer arietinum L. (Chickpea) 124





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





3.8.1.2. Cicer arietinum L. 167





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


Table 15: Effect of treatments on biochemical parameters of H.annuus


Table 16: Effect of treatments on mineral content of H.annuus


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


Table 27: Effect of treatments on biochemical parameters of C. arietinum


Table 28: Effect of treatments on mineral content of C. arietinum


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


Table 31: Effect of composted rice husk on shoot lengths of H.annuus


Table 32: Effect of composted rice husk on fresh weight of H.annuus


Table 33: Effect of composted rice husk on chlorophyll a of H.annuus


Table 34: Effect of composted rice husk on chlorophyll b of H.annuus


Table 35: Effect of composted rice husk on total chlorophyll of H.annuus


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


Table 38: Effect of composted rice husk on percent nitrogen of H.annuus


Table 39: Effect of composted rice husk on percent phosphorus of H.annuus


Table 40: Effect of composted rice husk on root lengths of C. arietinum


Table 41: Effect of composted rice husk on shoot lengths of C. arietinum


Table 42: Effect of composted rice husk on fresh weight of C. arietinum


Table 43: Effect of composted rice husk on chlorophyll a of C. arietinum


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.


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


Table 48: Effect of composted rice husk on percent nitrogen of C.


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


Table 51: Effect of composted wheat bran on shoot lengths of H.annuus


Table 52: Effect of composted wheat bran on fresh weight of H.annuus


Table 53: Effect of composted wheat bran on chlorophyll a of H.annuus


Table 54: Effect of composted wheat bran on chlorophyll b of H.annuus


Table 55: Effect of composted wheat bran on total chlorophyll of


Table 56: Effect of composted wheat bran on carbohydrate content of


Table 57: Effect of composted wheat bran on crude protein content of


Table 58: Effect of composted wheat bran on percent nitrogen of


Table 59: Effect of composted wheat bran on percent phosphorus of


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.


Table 62: Effect of composted wheat bran on fresh weight of C.


Table 63: Effect of composted wheat bran on chlorophyll a of C.


Table 64: Effect of composted wheat bran on chlorophyll b of C.


Table 65: Effect of composted wheat bran on total chlorophyll of C.


Table 66: Effect of composted wheat bran on carbohydrate content of


Table 67: Effect of composted wheat bran on crude protein content of


Table 68: Effect of composted wheat bran on percent nitrogen of C.


Table 69: Effect of composted wheat bran on percent phosphorus of


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


isolates on shoot length of H.annuus plants 59


fertilizer and fungicide on shoot length of H.annuus plants 62


isolates on fresh weight of H.annuus plants 63


fertilizer and fungicide on fresh weight of H.annuus plants 64


isolates on total chlorophyll of H.annuus plants 65


fertilizer and fungicide on total chlorophyll of H.annuus plants 68


isolates on total carbohydrate of H.annuus plants 69


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


fertilizer and fungicide on crude protein content of H.annuus

plants 72


isolates on percent nitrogen of H.annuus plants 73


fertilizer and fungicide on percent nitrogen of H.annuus plants 74


isolates on percent phosphorus of H.annuus plants 75


fertilizer and fungicide on percent phosphorus of H.annuus

plants 78


isolates on root length of B.nigra plants. 80


fertilizer and fungicide on root length of B.nigra plants. 81


isolates on shoot length of B.nigra plants. 82


fertilizer and fungicide on shoot length of B.nigra plants. 83


isolates on fresh weight of B.nigra plants. 84


fertilizer and fungicide on fresh weight of B.nigra plants. 87


isolates on total chlorophyll of B.nigra plants. 88


fertilizer and fungicide on total chlorophyll of B.nigra plants. 89


isolates on total carbohydrate of B.nigra plants. 92


fertilizer and fungicide on total carbohydrate of B.nigra plants. 93


isolates on crude protein content of B.nigra plants. 94


fertilizer and fungicide on crude protein content of B.nigra plants. 95


isolates on percent nitrogen of B.nigra plants. 98


fertilizer and fungicide on percent nitrogen of B.nigra plants. 99


isolates on percent phosphorus of B.nigra plants. 100


fertilizer and fungicide on percent phosphorus of B.nigra plants. 101


isolates on root length of V.mungo plants. 103


fertilizer and fungicide on root length of V.mungo plants. 104


isolates on shoot length of V.mungo plants. 105


fertilizer and fungicide on shoot length of V.mungo plants. 106


isolates on fresh weight of V.mungo plants. 108


fertilizer and fungicide on fresh weight of V.mungo plants. 109


isolates on total chlorophyll of V.mungo plants. 111


fertilizer and fungicide on total chlorophyll of V.mungo plants. 112


isolates on total carbohydrate of V.mungo plants. 115


fertilizer and fungicide on total carbohydrate of V.mungo plants. 116


isolates on crude protein content of V.mungo plants. 117


fertilizer and fungicide on crude protein content of V.mungo plants. 118


isolates on percent nitrogen of V.mungo plants. 120


fertilizer and fungicide on percent nitrogen of V.mungo plants. 121


isolates on percent phosphorus of V.mungo plants. 122


fertilizer and fungicide on percent phosphorus of V.mungo plant. 123


isolates on root length of C.arietinum plants. 126


fertilizer and fungicide on root length of C.arietinum plants. 127


isolates on shoot length of C.arietinum plants. 128


fertilizer and fungicide on shoot length of C.arietinum plants. 129


isolates on fresh weight of C.arietinum plants. 131


fertilizer and fungicide on fresh weight of C.arietinum plants. 132


isolates on total chlorophyll of C.arietinum plants. 134


fertilizer and fungicide on total chlorophyll of C.arietinum plants. 135


isolates on total carbohydrate of C.arietinum plants. 138


fertilizer and fungicide on total carbohydrate of C.arietinum plants. 139


isolates on crude protein content of C.arietinum plants. 140


fertilizer and fungicide on crude protein content of C.arietinum plant. 141


isolates on percent nitrogen of C.arietinum plant. 142


fertilizer and fungicide on percent nitrogen of C.arietinum plants. 145


isolates on percent phosphorus of C.arietinum plants. 146


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


Figure 71: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a


Figure 72: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-b


Figure 73: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll


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 (%)


Figure 76: Effect of composted rice husk @ 5 and 10 gm on percent nitrogen


Figure 77: Effect of composted rice husk @ 5 and 10 gm on percent phosphorus



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


Figure 81: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a


Figure 82: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-b


Figure 83: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll


Figure 84: Effect of composted rice husk @ 5 and 10 gm on total carbohydrate


Figure 85: Effect of composted rice husk @ 5 and 10 gm on crude protein (%)


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


Figure 92: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b


Figure 93: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll


Figure 94: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate


Figure 95: Effect of composted wheat bran @ 5 and 10 gm on crude protein (%)


Figure 96: Effect of composted wheat bran @ 5 and 10 gm on percent nitrogen


Figure 97: Effect of composted wheat bran @ 5 and 10 gm on percent phosphorus


Figure 98: Effect of composted wheat bran @ 5 and 10 gm on root length


Figure 99: Effect of composted wheat bran @ 5 and 10 gm on shoot length


Figure 100: Effect of composted wheat bran @ 5 and 10 gm on fresh weight


Figure 101: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-a


Figure 102: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b


Figure 103: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll


Figure 104: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate


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

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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

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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

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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).

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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,

http://en.wikipedia.org/wiki/Organic_farming

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


11. Bradyrhizobium sp-III (1.9 x 108 cfu/ml) + T. hamatum (1.2 x 10


12. Bradyrhizobium sp-IV (1.9 x 108 cfu/ml) + T. hamatum (1.2 x 10


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.


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.


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


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




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




a

c

a

c

a

d

a


isolates on shoot length of H.annuus plants. Columns bearing superscript are


0

5

10

15

20

25

30

35

40

Sh

oot

len

gth

(cm

)

30th day




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




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


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


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


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




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




a a

a a

c

a

a

a

a

a

d c c a


isolates on fresh weight of H.annuus plants. Columns bearing superscript are


0

1

2

3

4

5

6

Fre

sh w

eigh

t (g

m)

30th day




d

a

c

b

0

1

2

3

4

5

6

Fre

sh w

eigh

t(gm

)

60th day




c d

c b

c

b a


and fungicide on fresh weight of H.annuus plants. Columns bearing


0

1

2

3

4

5

6

Fre

sh w

eigh

t(gm

) 30th day




d

b a

d

a

c c

0

1

2

3

4

5

6

7

Fre

sh w

eig

ht

(gm

)

60th day




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




d

a a a

a

b

a

c

a


isolates on total chlorophyll of H.annuus plants. Columns bearing superscript


0

0.5

1

1.5

2

2.5

3

Tota

l ch

loro

ph

yll

(m

g/g

)

60th day




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




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




a

a

d d


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




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




b

c

b


isolates on total carbohydrate of H.annuus plants. Columns bearing


0

50

100

150

200

250

300

350

400

450

Tota

l ca

rboh

yd

rate

(m

g /

g)

30th day




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




b

c

b

d a


fertilizer and fungicide on total carbohydrate of H.annuus plants. Columns


control.

0

2

4

6

8

10

12

14

16

18

20

Cru

de

pro

tein

(%

)

60th day




b c

b

0

2

4

6

8

10

12

14

16

Cru

de

pro

tein

(%

)

30th day




d c

a

d

a


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




b a

a

d d c

a

0

2

4

6

8

10

12

14

16

18

20

Cru

de

pro

tein

(%

)

60th day




b

d

b c


fertilizer and fungicide on crude protein content of H.annuus plants. Columns


control.

0

0.5

1

1.5

2

2.5

Nit

rogen

(%

)

30th day




d c

a

d d

a

0

0.5

1

1.5

2

2.5

3

Nit

rogen

(%

)

60th day




b c

b


isolates on percent nitrogen of H.annuus plants. Columns bearing superscript


0

0.5

1

1.5

2

2.5

3

3.5

Nit

rogen

(%

)

30th day




c

a a

a

d d c

a

0

0.5

1

1.5

2

2.5

3

3.5

Nit

rogen

(%

)

60th day




b

d

b c


fertilizer and fungicide on percent nitrogen of H.annuus plants. Columns


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




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




a

c

b

a


isolates on percent phosphorus of H.annuus plants. Columns bearing


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


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)


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




d

b

d

b a

d

a

0

0.05

0.1

0.15

0.2

0.25

Ph

osp

horu

s (%

)

60th day




c b

a

d c

c


fertilizer and fungicide on percent phosphorus of H.annuus plants. Columns


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


isolates on root length of B.nigra plants. Columns bearing superscript are


0

2

4

6

8

10

12

14

Root

len

gth

(cm

)

30th day




d c

a c c b c d

d

0

5

10

15

20

25

30

35

Root

len

gth

(cm

)

60th day




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




d

a

b a c c

a a

a

a d

a c b


fertilizer and fungicide on root length of B.nigra plants. Columns bearing


0

2

4

6

8

10

12

14

Root

len

gth

(cm

)

30th day




c

a c c b d d

b d

a


isolates on shoot length of B.nigra plants. Columns bearing superscript are


0

5

10

15

20

25

Sh

oo

t le

ng

th (

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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



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



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


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

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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


0

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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



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


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


isolates on total carbohydrate of B.nigra plants. Columns bearing superscript


0

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) 30th day




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control.

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Control JUF1 JUR1 JUR2 JUR3 JUR4 FTZ

FGD JUR1+JUF1 JUR2+JUF1 JUR3+JUF1 JUR4+JUF1 JUF1+FTZ FGD+FTZ

d

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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)


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


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


isolates on percent nitrogen of B.nigra plants. Columns bearing superscript


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2 N

itro

gen

(%

)

30th day




d c

a

d

b

a

d

0

0.5

1

1.5

2

2.5

3

3.5

Nit

rogen

(%

)

60th day




d

a

c

a

d


fertilizer and fungicide on percent nitrogen of B.nigra plants. Columns


control.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Nit

rogen

(%

)

30th day




c a

d

a

c

0

0.5

1

1.5

2

2.5

3

3.5

Nit

rogen

(%

)

60th day




d

a

a

d d c


isolates on percent phosphorus of B.nigra plants. Columns bearing


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Ph

osp

horu

s (%

)

30th day




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




d

c c


fertilizer and fungicide on percent phosphorus of B.nigra plants. Columns


control.

0

0.05

0.1

0.15

0.2

0.25

0.3

Ph

osp

horu

s (%

)

30th day




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




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




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




d c

a

d c c a

c


isolates on root length of V.mungo plants. Columns bearing superscript are



and fungicide on root length of V.mungo plants. Columns bearing superscript are


0

5

10

15

20

25

30

35

40

Root

len

gth

(cm

)

30th day




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




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




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




d c

a c

d c a

c c c d


and fungicide on shoot length of V.mungo plants. Columns bearing superscript


0

5

10

15

20

25

30

Sh

oot

len

gth

(cm

)

30th day




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




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



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).


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-


isolates on fresh weight of V.mungo plants. Columns bearing superscript are


0

0.5

1

1.5

2

2.5

3

Fre

sh w

eigh

t (g

m)

30th day




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




a

c

c d


and fungicide on fresh weight of V.mungo plants. Columns bearing superscript are


0

0.5

1

1.5

2

2.5

3 F

resh

wei

gh

t (g

m)

30th day




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




a

d d d

c

d

c

Table 22: Effect of treatments on photosynthtic pigment of V. mungo (black gram) plant


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


on total chlorophyll of V.mungo plants. Columns bearing superscript are


0

0.5

1

1.5

2

2.5

3

Tota

l ch

loro

ph

yll

(m

g/g

)

30th day



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



c

d

a

d

c c

b

d c


and fungicide on total chlorophyll of V.mungo plants. Columns bearing


0

0.5

1

1.5

2

2.5

3 T

ota

l ch

loro

ph

yll

(m

g/g

) 30th day




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




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).


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


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


Table 23: Effect of treatments on biochemical parameters of V. mungo (black gram) plants


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


isolates on total carbohydrate of V.mungo plants. Columns bearing superscript


0

50

100

150

200

250

300

350

Tota

l ca

rboh

yd

rate

(m

g/g

) 30th day




b

d

0

50

100

150

200

250

300

350

Tota

l ca

rboh

yd

rate

(m

g/g

)

60th day




c c b b


and fungicide on total carbohydrate of V.mungo plants. Columns bearing


0

100

200

300

400

500

600

Tota

l ca

rboh

yd

rate

(m

g/g

) 30th day




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




b

a

b c

a

d

a


isolates on crude protein content of V.mungo plants. Columns bearing


0

2

4

6

8

10

12

14

16

18

Cru

de

pro

tein

(%

) 30th day




d

b

0

2

4

6

8

10

12

14

16

18

20

Cru

de

pro

tein

(%

)

60th day




b c a b


fertilizer and fungicide on crude protein content of V.mungo plants. Columns


control.

0

5

10

15

20

25

30

Cru

de

pro

tein

(%

) 30th day




a

a

a

d

a

0

5

10

15

20

25

Cru

de

pro

tein

(%

)

60th day




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


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



b

0

0.5

1

1.5

2

2.5

3

3.5

Nit

rog

en (

%)

60th day



b c a b


and fungicide on percent nitrogen of V.mungo plants. Columns bearing


0

0.5

1

1.5

2

2.5

3

3.5

4

4.5 N

itro

gen

(%

) 30th day




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




c a a

a

d a


on percent phosphorus of V.mungo plants. Columns bearing superscript are


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




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




d d

a

c


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




d d

d

0

0.1

0.2

0.3

0.4

0.5

Ph

osp

horu

s (%

)

60th day




d d

a a

d

a

c

d

b a

3.6.2.2. Cicer arietinum L. (Chickpea)


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


isolates on root length of C.arietinum plants. Columns bearing superscript are


0

5

10

15

20

25

30 R

oot

len

gth

(cm

) 30 th day




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




a

c c c d

b

d d c

d


fertilizer and fungicide on root length of C.arietinum plants. Columns bearing


0

5

10

15

20

25

30

Root

len

gth

(cm

) 30 th day




d

c

d d

d

b c

0

5

10

15

20

25

30

35

Root

len

gth

(cm

)

60th day




c c c d

c c d d

a a

c d d


isolates on shoot length of C.arietinum plants. Columns bearing superscript


0

10

20

30

40

50

60

Sh

ooth

le

ngth

(cm

)

30 th day




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




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


0

10

20

30

40

50

60

Sh

oot

len

gth

(cm

)

30th day




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




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).


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




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




d

c

d

d


isolates on fresh weight of C.arietinum plants. Columns bearing superscript


0

1

2

3

4

5

6

7

8

9

Fre

sh w

eigth

(gm

) 30th day




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




d

c

d c

b


fertilizer and fungicide on fresh weight of C.arietinum plants. Columns


control.

Table 26: Effect of treatments on photosynthetic pigment of C. arietinum (chickpea) plnats


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


isolates on total chlorophyll of C.arietinum plants. Columns bearing


0

0.5

1

1.5

2

2.5

3

Tota

l ch

loro

ph

yll

(m

g/g

) 30 th day




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


fertilizer and fungicide on total chlorophyll of C.arietinum plants. Columns


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




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




d d d

c c

a

b c

d


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


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



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


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


isolates on total carbohydrate of C.arietinum plants. Columns bearing


0

50

100

150

200

250

300

350

400

450 T

ota

l ca

rbo

hy

dra

te (

mg

/g)

30 th day




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




d

a

d

b

a

d

a

b


fertilizer and fungicide on total carbohydrate of C.arietinum plants. Columns


control.

0

50

100

150

200

250

300

350

400

450 T

ota

l ca

rboh

yd

rate

(m

g/g

) 30 th day




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




d

a

d d

a

a

d d


isolates on crude protein content of C.arietinum plants. Columns bearing


0

2

4

6

8

10

12

14

16

18 C

rud

e p

rote

in (

%)

30th day




b

d

0

5

10

15

20

25

30

Cru

de

pro

tein

(%

)

60th day




d

c

a a b

d c d


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




a

a

a

d

a

0

5

10

15

20

25

30

Cru

de

pro

tein

(%

)

60th day




c

a c c c c

a c c c c

a c c c c

a c c c c d c


isolates on percent nitrogen of C.arietinum plants. Columns bearing


0

0.5

1

1.5

2

2.5

3

3.5

4 N

itro

gen

(%

) 30th day




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




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


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


fertilizer and fungicide on percent nitrogen of C.arietinum plants. Columns


control.

0

0.5

1

1.5

2

2.5

3

3.5

4 N

itro

gen

(%

) 30 th day




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




c

a c c c c

d c


isolates on percent phosphorus of C.arietinum plants. Columns bearing


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35 P

hosp

horu

s (%

) 30th day




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




d

d d

a


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




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




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)


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


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


Table 31: Effect of composted rice husk on shoot lengths of H.annuus (sunflower) plants

Shoot length (cm)



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)


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


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


Table 32: Effect of composted rice husk on fresh weight of H.annuus (sunflower) plants

Fresh weight (gm)



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)

______________________________________________________________________________________________________________________


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


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


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).


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)



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)



157

Figure 71: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a of H.annuus


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)



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)



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)



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)



161

Figure 73: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll of H.annuus


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)



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)

______________________________________________________________________________________________________________________________________________



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 (%)



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)



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


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)


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 (%)



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)



168

Figure 76: Effect of composted rice husk @ 5 and 10 gm on percent nitrogen of


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 (%)



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)



170

Figure 77: Effect of composted rice husk @ 5 and 10 gm on percent phosphorus of


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)



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


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)



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



RH composted with all treatment @ 10g enhanced the synthesis of chl-a in test


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


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


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).


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


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)



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


17

7

Figure 80: Effect of composted rice husk @ 5 and 10 gm on fresh weight of


(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)



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)



179

Figure 81: Effect of composted rice husk @ 5 and 10 gm on chlorophyll-a of



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)



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)



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




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)



183

Figure 83: Effect of composted rice husk @ 5 and 10 gm on total chlorophyll of


(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)



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)



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



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


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


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 (%)



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)



188

Figure 85: Effect of composted rice husk @ 5 and 10 gm on crude protein (%) of



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 (%)



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)



190

Figure 86: Effect of composted rice husk @ 5 and 10 gm on percent nitrogen of



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 (%)



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)



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


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


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)


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)



195

Figure 88: Effect of composted wheat bran @ 5 and 10 gm on root length of



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)



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)



197

Figure 89: Effect of composted wheat bran @ 5 and 10 gm on shoot length of



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)



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)



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




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)



201

Figure 91: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-a of



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)



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)



203

Figure 92: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b of



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


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


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


Table 55: Effect of composted wheat bran on total chlorophyll of H.annuus (sunflower) plants




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)



206

Figure 93: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll of



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)



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)



208

Figure 94: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate of



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 (%)



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)



210

Figure 95: Effect of composted wheat bran @ 5 and 10 gm on crude protein (%) of



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 (%)



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)



212

Figure 96: Effect of composted wheat bran @ 5 and 10 gm on percent nitrogen of



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 (%)


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)



214

Figure 97: Effect of composted wheat bran @ 5 and 10 gm on percent phosphorus of



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


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


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


Table 60: Effect of composted wheat bran on root lengths of C. arietinum (chickpea) plants

Root length (cm)



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)



217

Figure 98: Effect of composted wheat bran @ 5 and 10 gm on root length of



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)



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)



219

Figure 99: Effect of composted wheat bran @ 5 and 10 gm on shoot length of



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)



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)



221

Figure 100: Effect of composted wheat bran @ 5 and 10 gm on fresh weight of



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




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)



223

Figure 101: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-a of



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


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


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


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 64: Effect of composted wheat bran on chlorophyll b of C. arietinum (cickpea) plants

Chlorophyll b (mg/g)



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)



226

Figure 102: Effect of composted wheat bran @ 5 and 10 gm on chlorophyll-b of



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




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)



228

Figure 103: Effect of composted wheat bran @ 5 and 10 gm on total chlorophyll of



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)



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)



230

Figure 104: Effect of composted wheat bran @ 5 and 10 gm on total carbohydrate of



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 (%)



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)



232

Figure 105: Effect of composted wheat bran @ 5 and 10 gm on crude protein (%) of



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 (%)



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)



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


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 (%)



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)



237

Figure 107: Effect of composted wheat bran @ 5 and 10 gm on percent phosphorus of



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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