EFFECT OF RHIZOBIUM AND PSB IN MYCORRHIZAL...

Chapter - 5

5.1 Introduction with review of literature

With the introduction of green revolution, the modern agriculture is getting

more and more dependent on the steady supply of synthetic inputs i.e.

Chemical fertilizers. Indiscriminate and injudicious use of chemical fertilizers

for the crop production has compounded the problem of environmental

pollution, deterioration of soil health and residue problems. Adverse effects

of the chemical fertilizers have compelled the scientific fraternity to look for

alternatives in the form of Biofertilizers.The term biofertilizer refers to all

organisms, which add, conserve and mobilize the plant nutrients in the soil.

Such microorganisms have come to be called as “biofertilizers” a term, which

is a misnomer, compared to commercial fertilizers manufactured on large

scales in factories. Biofertilizers are based on renewable energy sources and

ecofriendly compared to commercial fertilizers (Yawalkar et al, 1996).

Biofertilizers play a very significant role in improving soil fertility by fixing

atmospheric nitrogen, both symbiotic and non-symbiotic, solubilise insoluble

soil phosphate and produce plant growth substances in the soil. Depending

up on the nutrient produced, Verma and Battacharya (Yawalkar et al, 1996)

has broadly classified biofertilizers as follows.

EFFECT OF RHIZOBIUM AND PSB IN MYCORRHIZAL LEGUMINOUS PLANTS

216 Chapter 5Chapter 5Chapter 5Chapter 5

Biofertilizers can be generally defined as preparations containing

live or latent cells of efficient strains of nitrogen fixing, phosphate

solubilising or cellulolytic microorganisms, used for application to seed, soil

or composting areas with the objective of increasing the number of such

microorganisms and accelerate certain microbial processes to augment

the extent of the availability of nutrients in the form which can be easily

assimilated by plants. Nitrogen is an essential element for plants’ normal

growth and development. Even though nitrogen is present in the

atmosphere about 78%, it is not readily available to plants. As nitrogen is

diatomic, strong bond is held in between, to break this bond to form

nitrogenous compounds, it requires high energy and temperature. But

nitrogen-fixing bacteria can break the bond to produce nitrogenous

compounds. Two kinds of nitrogen fixing bacterias are there (1) Symbiotic

nitrogen fixing bacterias and (2) Asymbiotic (free-living) nitrogen fixing

bacterias. In natural environment, the leguminous plants had symbiotic

Biofertilisers

Nitrogen fixing Biofertilisers (NBF)

Phosphate Mobilizing Biofertilisers

NBF for Legumes

Rhizobium

NBF for Cereals Azospirillum Azotobacter Azolla BGA

Phosphate Solubiliser Bacillus Pseudomonas

Aspergillus

Phosphate Absorber Vesicular Arbuscular

Mycorrhiza

Effect of Rhizobium and Effect of Rhizobium and Effect of Rhizobium and Effect of Rhizobium and PSBPSBPSBPSB In Mycorrhizal Leg In Mycorrhizal Leg In Mycorrhizal Leg In Mycorrhizal Leguminous Plantsuminous Plantsuminous Plantsuminous Plants 217

association of nitrogen fixing bacteria- Rhizobium in the root nodules.

Phosphorus is an important plant nutrient, which is referred to as the

‘master key’ element in crop production (Pierre,1938). The most vital

functions of phosphorus in organisms are for cell division, photosynthesis;

break down of sugar, energy storage, transfer and as a component of

DNA, essential for the passing on of heredity traits from one generation to

another. Plants cannot absorb organic phosphorus unless converted to

inorganic forms. Certain microbial enzymes could cleave organic

phosphorus to inorganic forms available to plants. The enzymes, which

cleave phosphorus from organic compounds, are known as phosphatases.

Two basic types of phosphatases exist in soils- acid phosphatases which

exhibit optimum activity in acidic PH range; and alkaline phosphatases

exhibiting optimum activity in the alkaline PH range. The present study is

designed to evaluate the effect of Rhizobium and PSB inoculation on

mycorrhizal and non-mycorrhizal leguminous plants.

5.2 Materials and Methods

5.2.1 Isolation and maintenance of Rhizobium

The Nitrogen fixing bacteria were isolated from a naturally growing

leguminous plant- Mimosa pudica.L, in (yeast extract maltose agar)

“YEMA” medium (The composition of the medium is given in the appendix)

Surface sterilized(using 0.01% mercuric chloride solution) root nodules

washed several times in distilled water were crushed in a mortar and

pestle. It was serially diluted and at 10¯ 6 dilution pure colony of the

rhizobium was obtained.


Plate 1 Nitrogen fixing bacteria, Rhizobium leguminosarum L colony developed on YEMA medium.

Plate 2 Clearing zone of P solubilising bacteria, Bacillus sp on Apatite Agar medium.


From that pure colony, sub culturing was done. It was maintained in

nutrient broth, whose composition is given in the appendix.1ml of the

nutrient broth containing about 106 bacterial cells, was given to each plant.

5.2.2 Isolation and maintenance of Phosphate Solubilising Bacteria (PSB)

Inoculation of crop plants with phosphate solubilising

microorganisms could enhance the utilization of sparingly soluble phosphate

by plants. Use of efficient phosphate solubilising microorganisms as seed

inoculants or direct use in soil when crops are raised greatly help in

phosphate solubilisation and mobilization for crop use.

5.2.2.1 Collection of samples-: Soil samples were collected from the

rhizosphere of crop plants from 3 different points from a plot. They were

mixed and homogenized. 10gms of soil were taken from the mixed

samples of soil. They were labeled and stored in polythene bags, at 4˚C

until they were processed. Pikovskay’s medium (1948) was used for the

isolation, cultivation and maintenance of Phosphate solubilising Bacteria.

5.2.2.2 Enrichment culture technique-: 100ml of pikovskaya’s broth

sterilized at 10lbs pressure for 30minutes, was dispersed along with 10 gm

of rhizosphere soil which was added aseptically in 250ml flask and were

incubated at 30˚C for one week.

5.2.2.3 Phosphatase activity-: The isolated strain of bacteria was

cultured in nutrient broth for 72hrs. 1ml of the broth was then transferred to

a test tube containing 5ml pre-sterilized basal medium and 5ml of sterilized

tris buffer of pH 8.5. To this, 0.5ml of sterilized solution of the substrate, P-

nitro phenyl phosphate (0.005m) was added; and 0.5ml of distilled water

instead of the substrate served as control. The test tubes were incubated

at room temperature for 72hrs. To this added 1ml of 0.2N sodium

carbonate and centrifuged at 3000rpm for 15 minutes. The absorbance of

supernatant was measured at 418nm. The values of the control tubes were

subtracted from the experimental ones for calculating the nitro phenol


liberated. The maximum intensity of color developed showed the maximum

phosphatase activity.

The isolated P solubilising bacteria were grown in nutrient broth in

a shaker for 72 hrs. 1ml of the nutrient broth containing 106 bacterial cells

was given to each plant.

Pot culture experiments were conducted to study the effect of co-

inoculation of PSB and Rhizobium along with VAM fungus in leguminous

plants. The soil used in this study was an acidic sandy loam with organic

matter- 2%, PH-5.3, total nitrogen- 6.8mg/Kg soil, phosphorus-2.5 mg/Kg

soil and potassium- 20mg/ Kg soil. The soil was air dried, sieved to less

than 2mm and mixed with sand at a ratio of soil: sand 4:1 (v/v) and

sterilized at 15 lbs/cm for 30 minutes in an autoclave to eliminate native

arbuscular mycorrhizal propagules. The soil was taken in polythene bags,

each holding 5 Kg dry soil. The isolation and maintenance of mycorrhizal

inoculum of Glomus mosseae was described elsewhere in chapter 3. 10

gm of the soil containing spores and propagules of Glomus. mosseae from

the stock culture were laid 1cm below the surface soil. Seeds of four

leguminous plants namely Vigna unguiculata and Arachis hypogeae,

provided by Kerala Agricultural university farm Kozha, and Agicultural

University Tamil Nadu, were grown separately and studied. Seeds were

put in water for about 2-4 hrs before sowing. The seeds were surface

sterilized in 0.05% mercuric chloride solution and after that thorough

washing in glass distilled water. The surface sterilized seeds 3-5 in number

were sowed in each bag. 1 ml each of the nutrient broth containing N2

fixing bacteria- Rhizobium and Phosphate Solubilising Bacteria-Bacillus

sp from the stock cultures were also added to the plants as described

below


Groups Treatments

I Control

II VAM

III Rhizobium

IV PSB

V VAM+ Rhizobium

VI VAM+ PSB

VII VAM+Rhizobium+ PSB

The plants were kept in green house with adequate sunlight and

ventilation. Daily watering (sterilized and de-ionized water) and

observations were done.20ml/pot of modified Hogland’s nutrient

solution were given once in three days to all the plants under study. The

composition of the modified Hogland’s solution was given in the

appendix. The plants were uprooted after 30, 60 and 120 days of

growth. The plants’ growth rates were assessed by standard

parameters like shoot length; shoot weight (fresh & dry), root length,

root weight (fresh & dry), nodule number, nodule weight etc. The

nutrients NPK uptake and the biochemical constituents like total

chlorophyll, total carbohydrates, total reducing sugars and total proteins

etc were estimated. Nitrogenase, Acid and alkaline Phosphatases were

also studied as per the procedures given in chapter 2.


5.3 Results

5.3.1 Growth rate and nutrient levels

Growth rate and NPK levels of Vigna unguiculata and Arachis

hypogeae inoculated with Rhizobium, PSB and VAM fungus, after 30, 60

and 120 days of growth were studied and the results are given in tables

LCIV to CI. It was found that the co-inoculation of two bacterias with VAM

fungus enhanced the growth rate and NPK levels in leguminous plants

significantly than the single inoculums and uninoculated plants.

Table LCIV: Growth rate of Vigna unguiculata inoculated with Rhizobium, PSB and VAM fungus, after 30 days of growth

Shoot weight Plant-1 gm

Root weight Plant-1 (gm)

Groups

Shoot length Plant-1

cm fresh dry

Root Length Plant-1

cm fresh dry

No. of Nodules Plant-1

Weight of Nodule-1

Gm

% of mycorrhizal infection Plant-1

I 20±

0.641

1.83±

0.054

0.21±

0.011

8.31±

0.171

0.63±

0.023

0.08±

0.007 - - -

II 32±

0.912

3.85±

0.116

0.78±

0.034

12.4±

0.268

1.34±

0.031

0.20±

0.014 - - 28±1.45

III 28.3±

0.833

2.64±

0.071

0.64±

0.025

11.2±

0.215

1.08±

0.024

0.18±

0.015

16±2.43

0.03±

0.0006 -

IV

27.6±

0.814

3.42±

0.118

0.72±

0.029

11.4±

0.234

1.12±

0.026

0.19±

0.014 - - -

V

48.4±

1.431

4.56±

0.142

0.87±

0.038

13.4±

0.259

1.48±

0.027

0.26±

0.011 24±3.11

0.08±

0.009 4 0.4±2.31

VI 45.1±

1.162

4.43±

0.135

0.85±

0.037

12.8±

0.263

1.58±

0.034

0.23±

0.016 - - 34.2±1.70

VII 54.3±

2.614

5.30±

0.192

0.97±

0.041

14.5±

0.271

1.94±

0.041

0.26±

0.015 28±3.52

0.9±

0.007

4 6.7±2.71

Average of six values in each case ±SD


Table LCV: Growth rate of Vigna unguiculata inoculated with Rhizobium, PSB and VAM fungus, after60 days of growth.



Groups


cm fresh dry

Root

Length Plant-1

cm fresh dry


Weight of Nodule-1

gm


I 28±

1.051

3.83±

0.074

0.79±

0.025

9.10±

0.241

1.02±

0.023

0.18±

0.009 - - -

II 60±

1.341

8.60±

0.176

2.80±

0.081

13.43±

0.273

2.87±

0.051

0.83±

0.021 - - 50±2.74

III 54.3±

2.314

7.32±

0.148

2.06±

0.072

12.81±

0.254

2.08±

0.044

0.76±

0.019 34±3

0.04±

0.006 -

IV 52.8±

2.182

7.29±

0.157

2.01±

0.076

12.52±

0.261

2.03±

0.049

0.76±

0.016 - - -

V 78.0±

3.651

11.51±

0.232

4.80±

0.113

15.24±

0.330

3.21±

0.065

0.99±

0.027 61±4

0.11±

0.009

80±3.57

VI 74.7±

3.145

11.34±

0.247

4.68±

0.109

14.76±

0.278

3.16±

0.068

0.98±

0.023 - -

40±2.18

VII 86.2±

3.873

13.25±

0.281

5.89±

0.121

15.91±

0.346

3.89±

0.072

1.06±

0.038 54±2

0.124±

0.008 76±3.14

Average of six values in each case ± SD


Table LCVI: Growth rate of Vigna unguiculata, inoculated with Rhizobium, PSB and VAM fungus after 120 days of growth



Groups


cm fresh dry

Root Length Plant-1

cm fresh dry


Weight of Nodule-1

gm


I 32.0±

1.021

4.21±

0.109

0.86±

0.046

9.801±

0.368

1.48±

0.081

0.21±

0.010 - - -

II 88.0±

3.461

17.61±

0.741

5.62±

0.147

15.62±

0.523

4.27±

0.108

1.34±

0.071 - - 70.0±1.67

III 76.02±

2.873

15.89±

0.548

4.49±

0.118

14.24±

0.369

3.86±

0.097

1.13±

0.066

61.0±

1.071

0.05±

0.007 -

IV 75.09±

2.746

15.85±

0.574

3.47±

0.096

13.28±

0.381

2.95±

0.078

0.92±

0.049 - - -

V 110.3±

4.211

21.64±

0.884

9.07±

0.345

16.83±

0.714

5.34±

0.136

3.44±

0.092

120±

4.089

0.07±

0.008 85.20±2.86

VI 105.7±

3.982

20.93±

0.869

8.87±

0.283

15.64±

0.611

4.86±

0.084

3.08±

0.055 - - 86.03±2.45

VII 126.4±

4.367

24.91±

0.972

10.42±

0.348

18.43±

0.889

6.96±

0.135

3.98±

0.087

132±

5.121

0.133±

0.021 94.28±4.39


The growth rate of Vigna unguiculata inoculated with rhizobium, PSB

and VAM fungus, after 30, 60 and 120 days of growth was studied and the

results are given in tables LCIV to LCVI. The no: of nodules and

percentage of mycorrhizal infection were increased in the tetrapartiate

inoculations than the single ones. As a result of co-inoculation, the number

and weight of the nodule and the percentage of VAM infection were

increased and that might have resulted in increased growth. Gradual

increase in growth rate in all the groups was observed. In vigna plants


inoculated with Rhizobium, PSB and VAM fungi showed three fold

increase over control plants after 120 days of growth. Among the different

single inoculations, the VAM fungal inoculation showed significant increase

than the other single inoculations.

Table LCVII: Nutrient uptake levels of Vigna unguiculata, inoculated with Rhizobium, PSB and VAM fungus, after 30, 60 &120 Days of growth

% of N % of P % of K Groups

30 DAP

60 DAP

120 DAP

30 DAP

60 DAP

120 DAP

30 DAP

60 DAP

120 DAP

I 2.18±

0.063

3.08±

0.091

3.48±

0.092

0.09±

0.0027

0.12±

0.0031

0.14±

0.044

1.23±

0.035

1.36±

0.056

1.82±

0.042

II 4.64±

0.097

5.86±

0.124

6.92±

0.147

0.32±

0.0064

0.73±

0.0224

0.81±

0.0247

2.26±

0.054

4.31±

0.095

4.58±

0.094

III 4.71±

0.094

6.03±

0.125

7.04±

0.140

0.18±

0.0053

0.29±

0.0083

0.31±

0.0096

1.72±

0.037

2.86±

0.064

3.12±

0.062

IV 3.76±

0.087

3.98±

0.087

4.48±

0.092

0.28±

0.0091

0.43±

0.0138

0.58±

0.0173

1.29±

0.0261

3.72±

0.073

2.87±

0.067

V 5.38±

0.102

6.42±

0.134

7.36±

0.151

0.43±

0.0115

0.78±

0.0232

0.89±

0.0271

3.38±

0.062

4.53±

0.095

5.24±

0.103

VI 4.86±

0.086

5.78±

0.123

7.28±

0.156

0.48±

0.0142

0.81±

0.0242

0.98±

0.037

3.21±

0.067

4.23±

0.084

5.12±

0.101

VII 5.62±

0.114

6.96±

0.134

7.98±

0.162

0.62±

0.0197

0.92±

0.0281

1.21±

0.0362

3.69±

0.076

4.87±

0.102

5.48±

0.115

Average of six values in each case ±SD

The nutrients uptake (NPK) levels of Vigna unguiculata inoculated

with Rhizobium, PSB and VAM fungus, after 30, 60 and 120 days of

growth were studied and the results are shown in table LCVII. The VAM


inoculated plants showed significant increase in NPK levels than any other

single inoculums, except nitrogen. With other single inoculums, VAM

showed increased NPK levels. The highest level was observed in

tetrapartite association. The rhizobium inoculated plants showed more

nitrogen than the other single inoculums.

Table LCVIII: Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungus after 30 days of growth

Shoot weight Plant-1 gm Root weight Plant-1

(gm) Groups


cm Fresh Dry

Root Length Plant-1

cm Fresh Dry


Weight of

Nodule-1 gm


I 15.04±

0.510

1.10±

0.031

0.20±

0.006

8.31±

0.204

0.72±

0.018

0.091±

0.002 - - -

II 28.03±

0.826

2.68±

0.079

0.72±

0.029

12.34±

0.267

1.52±

0.046

0.232±

0.008 - -

34.0±

1.140

III 23.51±

0.724

2.30±

0.071

0.61±

0.018

10.25±

0.213

1.25±

0.034

0.206±

0.006

13.0±

1.004

0.02±

0.003 -

IV 24.43±

0.721

2.53±

0.078

0.65±

0.021

12.14±

0.238

1.38±

0.037

0.215±

0.007 - - -

V 38.81±

1.142

3.40±

0.106

0.82±

0.036

13.42±

0.277

1.61±

0.041

0.261±

0.008

22.0±

1.671

0.06±

0.004

42.0±

2.113

VI 36.40±

1.071

3.29±

0.097

0.78±

0.027

13.23±

0.258

1.56±

0.039

0.246±

0.006 - -

39.0±

1.841

VII 43.62±

1.448

4.40±

0.118

0.97±

0.044

14.38±

0.279

1.92±

0.062

0.295±

0.007

29.0±

1.074

0.07±

0.005

49.0±

2.014



Table LCIX: Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungus after 60 days of growth



Groups


cm fresh dry

Root Length Plant-1

cm fresh dry


Weight of Nodule-1

gm


I 22.03±

0.681

3.51±

0.071

0.78±

0.006

10.12±

0.514

1.16±

0.031

0.19±

0.002 - - -

II 54.01±

1.841

7.42±

0.201

2.16±

0.078

13.64±

0.341

2.93±

0.083

0.84±

0.005 - - 62.1±2.411

III 45.16±

1.162

6.84±

0.186

1.97±

0.051

12.18±

0.285

2.01±

0.059

0.75±

0.007

28.0±

1.10

0.040±

0.006 -

IV 40.33±

1.024

5.98±

0.143

1.62±

0.049

12.36±

0.276

2.04±

0.071

0.78±

0.007 - - -

V 65.41±

2.411

9.23±

0.372

3.41±

0.095

14.23±

0.293

3.38±

0.064

0.97±

0.009

48.0±

2.24

0.120±

0.007 72.4±2.820

VI 63.83±

2.503

8.96±

0.311

2.95±

0.084

13.91±

0.371

3.29±

0.089

0.91±

0.008 - - 63.6±2.571

VII 77.2±

2.815

10.1±

0.410

4.18±

0.115

14.80±

0.388

3.84±

0.097

1.14±

0.021

54.0±

2.03

0.140±

0.009 80.7±3.875



Table C: Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungus after 120 days of growth



Groups


cm fresh dry

Root

Length Plant-1

Cm fresh dry


Weight of

Nodule-1

gm


I 28.16±

0.681

4.89±

0.122

1.12±

0.037

10.6±

0.311

2.08±

0.068

0.78±

0.022 - - -

II 61.24±

1.731

15.64±

0.471

4.28±

0.116

15.12±

0.447

4.43±

0.129

1.89±

0.078 - - 82±3.11

III 50.09±

1.082

14.08±

0.271

3.48±

0.089

13.84±

0.412

3.72±

0.107

1.34±

0.063 59±2.11

0.05±

0.006 -

IV 51.71±

1.065

14.32±

0.351

3.62±

0.097

13.56±

0.385

3.65±

0.084

1.09±

0.031 - - -

V 63.42±

1.338

16.93±

0.410

6.21±

0.133

16.08±

0.467

5.54±

0.146

2.98±

0.081 110±2.15

0.07±

0.008 83.9±4.12

VI 65.21±

1.285

16.46±

0.511

6.05±

0.172

15.91±

0.408

5.24±

0.092

2.73±

0.043 - - 79.3±3.80

VII 76.73±

2.163

19.82±

0.684

7.12±

0.236

17.63±

0.523

6.69±

0.161

3.49±

0.094 126±3.24

0.121±

0.012 92.1±4.61


Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB

and VAM fungi after 30, 60 and 120 days of growth were studied and the

results are given in tables LCVIII to C. All the groups showed significant

increase in growth rate than the control. The co-inoculation of VAM fungus

with either Rhizobium or both bacteria showed significant increase and the

plants with both Rhizobium and PSB along with VAM fungus showed

highest growth rate.


Table CI: Nutrient uptake levels of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungi, after 30, 60 &120 Days of growth

% of N % of P % of K Groups

30

DAP

60

DAP

120

DAP

30

DAP

60

DAP

120

DAP

30

DAP

60

DAP

120

DAP

I 1.94±

0.042

2.62±

0.051

3.13±

0.067

0.06±

0.0011

0.09±

0.0033

0.14±

0.0047

1.62±

0.031

1.86±

0.045

2.18±

0.046

II 4.19±

0.086

4.94±

0.097

6.92±

0.143

0.29±

0.0082

0.51±

0.0154

0.81±

0.0241

2.86±

0.065

4.23±

0.083

5.28±

0.112

III 4.38±

0.084

5.11±

0.101

7.06±

0.142

0.16±

0.0037

0.24±

0.0042

0.42±

0.0126

2.28±

0.054

3.87±

0.073

4.08±

0.084

IV 2.92±

0.048

3.74±

0.055

5.28±

0.114

0.18±

0.0053

0.39±

0.0114

0.53±

0.0147

1.29±

0.036

3.92±

0.085

3.96±

0.082

V 4.96±

0.097

6.34±

0.133

7.63±

0.164

0.36±

0.0102

0.62±

0.0196

0.89±

0.0274

3.16±

0.067

4.35±

0.095

5.32±

0.112

VI 4.86±

0.087

6.28±

0.132

6.94±

0.141

0.40±

0.0135

0.68±

0.0203

0.83±

0.0252

3.21±

0.067

4.28±

0.093

5.28±

0.115

VII 5.28±

0.102

6.97±

0.144

8.31±

0.173

0.48±

0.0145

0.92±

0.0284

1.16±

0.0356

3.69±

0.073

4.85±

0.104

6.21±

0.127

Average of six values in each case ±SD, DAP- days after planting

NPK levels of Arachis hypogeae inoculated with Rhizobium, PSB

and VAM fungus, after 30, 60 and 120 days of growth were studied and

the results are given in table CI. All the treated plants showed two to three

fold increase in nutrient contents except the plants inoculated with PSB,

when compared to controls. The phosphorus content in Rhizobium

inoculated plants showed the least value among the different treatments.

Gradual increase in the level of NPK was observed in all plants compared

to controls when they were uprooted after 30 to 120 days of growth. The


most significant increase in the NPK level was observed in Arachis

hypogeae when it was inoculated with Rhizobium, PSB and VAM fungus.

The percentage of infection of VAM fungus was greater in this combination

and the no: of nodules were also higher than other treatments.

5.3.2 Bio chemical constituents

Biochemical constituents like total chlorophyll, carbohydrates,

reducing sugars and proteins in mycorrhizal and non-mycorrhizal

leguminous plants, inoculated with PSB and Rhizobium separately and in

mixture were studied in two leguminous plants and the results are given in

tables CII to CIX.

Table CII: Total chlorophyll in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth

Total chlorophyll (mg/gm fresh tissue Groups

30 DAP 60 DAP 120 DAP Significance between groups

I 1.270±

0.034

1.680±

0.0411

2.890±

0.082

II 2.760±

0.062

4.560±

0.0941

8.270±

0.171

III 2.061±

0.054

3.894±

0.0971

6.218±

0.137

IV 2.014±

0.049

3.786±

0.0891

6.011±

0.128

V 2.972±

0.061

5.093±

0.162

9.107±

0.186

VI 2.901±

0.064

4.984±

0.118

8.909±

0.169

VII 3.218±

0.073

5.892±

0.161

10.316±

0.307

I&II-*

I&III-**

I&IV-**

I&V-**

I&VI-**

I&VII-**

Other interactions-NS

Average of six values in each case ±SD, * P<0.05, **P<0.01,NS- not significant


The total chlorophyll in mycorrhizal and non-mycorrhizal Vigna

unguiculata inoculated with PSB and Rhizobium, after 30,60 & 120 days of

growth was studied and the results are given in table CII. It was found that

among the different inoculations, VAM fungal inoculation was better than

the other single inoculations. PSB or Rhizobium inoculations along with

VAM showed increased chlorophyll content. Highest chlorophyll content

was observed in the tetra partite association of VAM, Rhizobium and PSB

in Vigna plants. A progressive increase in the chlorophyll content was

observed in all the plants, when they were uprooted after 30 to 120 days of

growth when compared to controls.

Table CIII: Total Chlorophyll in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth.

Total chlorophyll (mg/gm fresh tissue) Groups

30 DAP 60 DAP 120 DAP

Significance between groups

I 1.760±

0.0322

2.980±

0.064

3.260±

0.112

II 3.310±

0.0962

5.840±

0.113

9.630±

0.177

III 3.065±

0.0842

5.621±

0.109

8.916±

0.168

IV 2.867±

0.0691

5.218±

0.119

8.519±

0.173

V 3.932±

0.118

6.817±

0.137

9.879±

0.197

VI 3.549±

0.096

6.611±

0.128

9.481±

0.186

VII 4.118±

0.153

8.116±

0.173

11.942±

0.314

I & II- *

I & III- *

I & IV- *

I & V- **

I & VI- **

I & VII- ***

other interactions - NS

Average of six values in each case± SD,* P<0.05, ** P<0.01, *** P<0.001, NS- not significant


Total chlorophyll in mycorrhizal and non-mycorrhizal Arachis

hypogeae inoculated with PSB and rhizobium after 30, 60 and 120 days of

growth was studied and the results are given in table CVI. Total chlorophyll

content in mycorrhizal plants inoculated with both PSB and rhizobium was

found to be increased very significantly, after 120 days of growth than any

other treatments. Chlorophyll content was more in VAM infected plants

than PSB or rhizobium inoculated plants, but not significant. In Arachis,

the dual inoculation did not have significant change in the level of

chlorophyll than VAM treatment alone. Increase in chlorophyll was

observed in all the groups, from 30 to 120 days of growth.

Table CIV: Total Carbohydrates in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth.

Total carbohydrate (mg/gm fresh tissue

Groups 30

DAP

60

DAP 120 DAP


I 3.630±

0.104

4.690±

0.137

5.830±

0.143

II 7.630±

0.153

10.700±

0.214

14.700±

0.271

III 6.116±

0.132

8.646±

0.167

13.163±

0.241

IV 6.021±

0.124

8.219±

0.153

13.098±

0.259

V 8.693±

0.185

11.493±

0.239

16.162±

0.331

VI 8.187±

0.1730

11.118±

0.216

16.018±

0.316

VII 10.240±

0.2241

14.118±

0.293

18.816±

0.372

I&II- ***

I&III- **

I&IV- **

I&V- ***

I&VI- ***

I&VII- ***

II&VII-*

III&VIII- **

IV&VII- ***

Other interactions –NS

Average of six values in each case± SD, * P<0.05, ** P<0.01, *** P<0.001, NS- not significant


Total carbohydrates in mycorrhizal and non-mycorrhizal Vigna

unguiculata inoculated with PSB & Rhizobium after 30, 60 and 120 days of

growth were studied and the results are given in table CIII. Carbohydrate

content was increased significantly in mycorrhizal plants inoculated with

rhizobium and PSB when compared to other inoculums. VAM fungal

inoculum showed high levels of carbohydrates than PSB or Rhizobium

inoculations. The PSB and Rhizobium inoculations did not show significant

change in carbohydrates content, when compared.

Table CV: Total Carbohydrates in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth.

Total carbohydrates (mg/gm fresh tissue)

Groups 30 DAP 60 DAP 120 DAP


I 3.820±

0.0864

4.980±

0.103

5.460±

0.106

II 7.830±

0.163

10.400±

0.211

14.620±

0.244

III 7.120±

0.1461

9.206±

0.193

12.116±

0.235

IV 6.693±

0.1267

9.018±

0.187

11.907±

0.216

V 8.392±

0.206

11.811±

0.234

16.118±

0.311

VI 8.063±

0.186

11.492±

0.241

15.943±

0.261

VII 10.319±

0.326

14.821±

0.298

17.371±

0.294

I & II- ***

I &III- **

I & IV- **

I & V- ***

I & VI- ***

I & VII- ***

III & VII- **

IV & VII- **



Total carbohydrates in Arachis hypogeae inoculated with

Rhizobium, PSB and VAM fungi, after 30, 60 and 120 days of growth were

studied and are given in table CVII. Increased levels of carbohydrates

were found in all groups, when compared to control plants. But the


Rhizobium or PSB inoculated plants showed no significant difference in

carbohydrate contents. The co-inoculation of rhizobium or PSB along with

VAM fungus also showed no significant change. VAM plants inoculated

with Rhizobium and PSB showed significant increase in the carbohydrates,

when compared to any other groups.

Table CVI: Total Reducing Sugars in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth.

Total reducing sugars (mg/gm fresh tissue Groups

30 DAP 60DAP 120 DAP Significance between groups

I 1.280±

0.0312

2.030±

0.0432

2.120±

0.0641

II 4.310±

0.0942

6.060±

0.135

7.240±

0.146

III 3.924±

0.0781

4.924±

0.107

6.711±

0.135

IV 3.615±

0.0664

4.739±

0.113

6.583±

0.142

V 5.244±

0.113

7.103±

0.162

9.183±

0.192

VI 5.019±

0.108

6.916±

0.143

9.007±

0.169

VII 6.114±

0.119

8.853±

0.185

11.321±

0.234

I &II- ***

I &III- **

I &IV- **

I &V- ***

I &VI- ***

I &VII- ***

II &VII- ***

III &VII- ***

IV &V- *

IV &VI- *

IV &VII- ***

Other interactions- NS


The total reducing sugars in mycorrhizal and non-mycorrhizal Vigna

unguiculata, inoculated with PSB and Rhizobium, after 30, 60 and 120

days of growth were studied and the results are given in table CIV. The

mycorrhizal plants showed more reducing sugars than the other bacterial

inoculations and controls. The dual inoculated plants also showed

increased sugar content than the single inoculums. The mycorrhizal


inoculation along with both PSB and Rhizobium showed significant

increase in sugar contents than any other inoculations.

Table CVII: Total Reducing Sugars in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth.

Total reducing sugars (mg/gm fresh tissue) Significance between groups

Groups 30 DAP 60DAP 120 DAP

I 1.030±

0.0241

2.180±

0.063

3.080±

0.091

II 5.620±

0.114

7.790±

0.163

9.460±

0.194

III 3.716±

0.113

7.426±

0.157

8.831±

0.179

IV 3.618±

0.133

7.014±

0.148

8.118±

0.167

V 6.118±

0.167

8.618±

0.173

10.864±

0.218

VI 6.089±

0.154

8.129±

0.165

10.293±

0.207

VII 7.513±

0.169

9.627±

0.198

12.467±

0.253

I & II- ***

I & III- ***

I & IV- ***

I & V- ***

I & VI- ***

I & VII- ***

II & VII- *

III & V- *

III & VII- ***

IV & V- **

IV & VI- *

IV & VII- ***


Average of six values in each case± SD, * P<0.05, ** P<0.01, *** P<0.001, NS-

not significant

Total reducing sugars in Arachis hypogeae, inoculated with

Rhizobium, PSB and VAM fungi, after 30,60 and 120 days of growth

were studied and the results are given in table CVIII. The reducing

sugars were significantly high in all groups compared to controls. Plants

in-group II showed increased sugar content when compared to group III

and IV. Plants in group V showed no change in sugars when compared

to group VI, but both showed significant increase when compared to

control and single inoculations. Plants in-group VII showed significantly

high sugar content when compared to other groups.


Table CVIII: Total Proteins in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth.

Total proteins (mg/gm fresh tissue) Groups



I 4.810±

0.0941

4.837±

0.095

5.630±

0.127

II 8.320±

0.181

11.410±

0.234

13.140±

0.284

III 6.812±

0.192

8.162±

0.183

11.614±

0.231

IV 5.181±

0.152

7.493±

0.162

11.495±

0.218

V 10.784±

0.231

12.897±

0.271

14.867±

0.291

VI 10.618±

0.211

12.091±

0.283

14.743±

0.279

VII 12.812±

0.256

14.581±

0.316

16.870±

0.316

I &II- ***

I &III- **

I &IV- *

I & V- ***

I & VI- ***

I & VII- ***

II & IV- *

II &VII- **

III & V- **

III &VI- **

III &VII- ***

IV & V- ***

IV &VI- ***

IV &VII- ***

Other interactions- NS


Total proteins in mycorrhizal and non- mycorrhizal Vigna unguiculata

inoculated with PSB and rhizobium, after 30, 60 and 120 days of growth were

studied and the results are given in table CV. It was found that the proteins

increased in all plants, when they were uprooted after 30 to 120 days of

growth. Mycorrhizal plants had significant increase in the protein content over

the plants inoculated with either PSB or rhizobium, but less than the dual

inoculums. The tetra partite inoculations of all the microbes showed very

significant increase in proteins than any other treatments.


Table CIX: Total Proteins in mycorrhizal and non-mycorrhizal Arachis

hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth

Total proteins (mg/gm fresh tissue) Groups



I 5.140±

0.102

5.260±

0.131

6.140±

0.153

II 10.820±

0.214

13.310±

0.271

14.860±

0.294

III 7.632±

0.1631

9.718±

0.192

12.384±

0.239

IV 6.918±

0.1434

8.694±

0.184

11.938±

0.226

V 12.736±

0.2718

14.542±

0.316

16.842±

0.318

VI 11.425±

0.2361

14.091±

0.293

16.139±

0.326

VII 14.311±

0.2681

16.863±

0.351

18.428±

0.349

I & II- ***

I & III- ***

I & IV- ***

I & V- ***

I & VI- ***

I & VII- ***

II & III- **

II & IV- **

II & VII- ***

III & V- ***

III & VI- ***

III & VII- ***

IV & V- ***

IV & VI- ***

IV & VII- ***

VI & VII- *



Total proteins in Arachis hypogeae inoculated with Rhizobium, PSB

and VAM fungus, after 30, 60 and 120 days of growth were studied and

the results are given in table CIX. Plants with single inoculations showed

significant increase in protein content than the control plants. Plants in

group V showed no significant change when compared to group VI, but

both showed significant increase when compared to other single

inoculations. Plants in tetrapartiate associations of VAM, Rhizobium and

PSB showed maximum protein content when compared to other groups.


5.3.3 Nitrogenase and phosphatases (ACP & ALP) activity

The experiment was conducted as mentioned earlier. The two

leguminous plants were uprooted after 30,60 and 120 days of their growth.

The enzymes nitrogenase, acid and alkaline phosphatases responsible for

nitrogen and phosphorus metabolism in plants were estimated as

mentioned in chapter: 2 and the results are given below.

Table CX: Nitrogenase activity in Mycorrhizal and non-Mycorrhizal Vigna unguiculata inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth.

Nitrogenase activity in n moles of ethylene produced hr-1 plant-1 at Groups

30 days 60 days 120 days

Average

I 214±4 240±6 281±4 245

II 421±7 480±5 520±4 473.667

III 436±4 520±11 552±9 502.667

IV 320±6 380±6 416±6 372

V 520±6 615±4 668±4 601

VI 485±9 580±6 624±7 563

VII 680±6 760±7 818±6

Average 439.429 510.714 554.143 752.667

Average of 6 values in each case ±S.D,

C D values (P=0.05)

Between Groups Between Days after planting Between

Groups x DAP

6.084 3.983 10.539


Nitrogenase activity in mycorrhizal and non-mycorrhizal Vigna

unguiculata inoculated with Rhizobium and Phosphate solubilizing

bacteria, after 30, 60 and 120 days of growth was studied and the results

are given in table CX. It was found that the enzyme activity was more in

mycorrhizal plants inoculated with both Rhizobium and P.S.B than single

or un-inoculated controls. Group VII plants showed three-fold increase

over controls. Increased activity was observed in all groups from 30 to 120

days of their growth.

Table CXI: Nitrogenase activity in Mycorrhizal and non-Mycorrhizal Arachis hypogeae inoculated with Rhizobium and Phosphate Solubilizing Bacteria , after 30, 60,120 days of growth.

Nitrogenase activity in n moles of ethylene produced hr-1 plant-1 at Groups


Average

I 196±7 220±6 260±9 225.333

II 432±8 489.33±8.67 510±9 477.111

III 476±6 510±9 525±6 503.667

IV 324±7 432±8 496±9 417.333

V 534±10 596±11 639±12 589.667

VI 502±10 565±11 607±12 558.00

VII 642±13 723±14 806±14

Average 443.714 505.048 549.00 723.667


C D values (P=0.05)

Between Groups Between Days after planting Between

Groups x DAP

9.453 6.188 16.373


Nitrogenase activity in mycorrhizal and non-mycorrhizal Arachis

hypogeae inoculated with Rhizobium and Phosphate solubilizing bacteria,

after 30, 60 and 120 days of growth was studied and the results are given

in table CXI. The P.S.B inoculated plants had less nitrogenase activity than

other groups, but showed higher activity than controls. Plants in-group III

showed more nitrogenase activity than group II. Group V plants showed

increased nitrogenase activity when compared to all groups except VII.

Table CXII: Acid Phosphatase activity in Mycorrhizal and non-Mycorrhizal Vigna unguiculata inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth.

ACP activity in KA units/100 ml sample at. Groups


Average

I 1.260±0.08 1.443±0.09 1.480±0.08 1.394

II 2.180±0.08 4.130±0.05 4.210±0.08 3.507

III 6.800±0.15 8.370±0.09 10.190±0.05 8.453

IV 4.620±0.09 6.790±0.05 8.160±0.07 6.523

V 10.760±0.08 12.810±0.07 15.680±0.07 13.083

VI 12.320±0.09 14.740±0.06 16.390±0.06 14.483

VII 13.673±0.24 16.450±0.26 19.723±0.18

Average 7.373 9.248 10.833

16.616


C D values (P=0.05)

Between Groups Between Days after planting Between Groups x DAP

0.113 0.074 0.195


The level of acid phosphatase (ACP) activity in mycorrhizal and

non-mycorrhizal Vigna unguiculata inoculated with P.S.B and Rhizobium,

after 30, 60 and 120 days of growth was studied and the results are given

in table CXII. It was found that the enzyme activity was maximum in

mycorrhizal plants inoculated with Rhizobium and P.S.B and the least in

single culture of Rhizaobium. Even though P.S.B was responsible for

phosphate metabolism, more acid phosphatase activity was observed in

VAM plants. Dual inoculation of VAM and P.S.B showed two fold increase

in the enzyme activity than single inoculated ones.

Table CXIII: Acid Phosphatase activity in Mycorrhizal and non-Mycorrhizal Arachis hypogeae inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth.

ACP activity in KA units/100 ml sample at. Groups


Average

I 1.210±0.03 1.460±0.04 1.490±0.04 1.387

II 5.810±0.08 6.920±0.14 7.460±0.15 6.730

III 2.890±0.06 3.147±0.08 3.280±0.07 3.106

IV 3.920±0.09 4.890±0.10 5.630±0.11 4.813

V 6.340±0.12 7.210±0.14 8.730±0.17 7.427

VI 6.720±0.13 7.520±0.15 9.050±0.18 7.763

VII 6.960±0.13 7.890±0.16 9.670±0.19

Average 4.836 5.577 6.473

8.173



C D values (P=0.05)

Between Groups

Between Days after planting

Between Groups x DAP

0.116 0.076 0.201

Acid phosphatase activity in mycorrhizal and non-mycorrhizal

Arachis hypogeae inoculated with P.S.B and Rhizobium, after 30, 60 and

120 days of growth was studied and the results are given in table CXIII.

The enzyme activity was found to be increased maximally in-group VII.

Plants in group V and VI also showed increased activity, but not significant

when compared. Group II showed an increase in the activity when

compared to group III and IV. All the groups showed increased enzyme

activity when compared to controls.

Table CXIV: Alkaline Phosphatase (ALP) activity in Mycorrhizal and non-Mycorrhizal Vigna unguiculata inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth.

ALP activity in KA units/100 ml sample at. Groups


Average

I 0.830±0.03 0.910±0.04 0.900±0.04 0.880

II 2.927±0.06 3.820±0.09 4.647±0.12 3.798

III 1.420±0.11 2.373±0.08 2.360±0.06 2.051

IV 2.890±0.05 3.530±0.06 4.020±0.09 3.480

V 4.210±0.06 5.360±0.06 7.240±0.05 5.603

VI 6.280±0.07 7.840±0.07 8.920±0.07 7.680

VII 7.260±0.08 8.320±0.16 10.190±0.20

Average 3.688 4.593 5.468

8.590



C D values (P=0.05)


0.084 0.055 0.146

Alkaline phosphatase activity in mycorrhizal and non-mycorrhizal

Vigna unguiculata inoculated with Rhizobium and PSB, after 30, 60 and

120 days of growth was studied and the results are given in table CXIV.

Plants in-group VII showed maximum enzyme activity when compared to

other groups. Plants in-group II showed increased enzyme activity than

group III and IV. No significant increase was observed in-group V when

compared to group VI, but both showed significant increase when

compared to single inoculations or controls.

Table CXV: Alkaline Phosphatase (ALP) activity in Mycorrhizal and non-Mycorrhizal Arachis hypogeae inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth.

ALP activity in KA units/100 ml sample at.

Groups 30 days 60 days 120 days

Average

I 0.780±0.02 0.840±0.03 0.890±0.04 0.837

II 2.860±0.06 3.760±0.08 4.180±0.08 3.600

III 1.080±0.02 1.890±0.04 1.910±0.04 1.627

IV 1.290±0.03 2.060±0.04 2.920±0.06 2.090

V 3.280±0.07 3.810±0.08 4.680±0.09 3.923

VI 3.410±0.07 4.120±0.08 5.290±0.11 4.273

VII 3.780±0.08 4.460±0.09 5.717±0.11

Average 2.354 2.991 3.655 4.652



C D values (P=0.05)


0.065 0.043 0.113

Alkaline phosphatase activity in mycorrhizal and non-mycorrhizal

Arachis hypogeae inoculated with P.S.B and Rhizobium, after 30, 60 and

120 days of growth was studied and the results are given in table CXV.

Dual and tripartite inoculations in these plants showed more enzyme

activities than single inoculums. Among the different groups studied, the

group VII showed maximum enzyme activity after 120 days of growth.

Group VI also showed increased enzyme activity when compared to other

groups except VII. Plants in-group II showed significant increase in

enzyme activities when compared to other single inoculations.

5.4 Discussion

Results indicated that the percentage of VAM infection was

increased in tetrapartiate association. The percentage of VAM infection in

Vigna unguiculata and Arachis hypogeae in different groups after 30, 60

and 120 days of growth were graphically shown in figure XVIII and XIX.

Both leguminous plants showed same kind of VAM infection. Among the

bacterial inoculums, Rhizobium enhanced the VAM infection than PSB

when inoculated dually.


Figure XVIII: Percentage of VAM infection in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with Rhizobium and PSB, after 30, 60 and 120 days of growth

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Figure: XIX. Percentage of VAM infection in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with Rhizobium and PSB, after 30, 60 and 120 days of growth

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A few reports are available on the effect of tetrapartiate association

of VAM, PSB and Rhizobium in leguminous plants. Numerous reports

available on dual or tripartite associations of VAM, Rhizobium or PSB in

leguminous plants. The results obtained in this study revealed that the

single inoculation of mycorrhizal fungus is better than the other single

inoculations in leguminous plants. The growth rate and nutrient uptake

were more in mycorrhizal plants. Any combination containing VAM fungus

showed significant increase in growth and nutrient level. The biochemical

constituents like total chlorophyll, carbohydrates, reducing sugars and

proteins were increased significantly in tetrapartiate associations in

leguminous plants. Green house studies conducted by Cabello et al,

(2005) reported that the dual inoculation of a VAM fungus Glomus

mosseae and a phosphorus solubilizing microorganism Penicillium thomii

in Mentha piperita with or with out rock phosphate, showed a positive

effect of these microbes on host plant’s growth when compared to control.

Rodruez et al, (2005) reported that the combined application of mycorrhizal

fungus and rhizobacteria in banana plants, showed significant increase in

total fresh weight, aerial dry weight, shoot length, leaf area and leaf

mineral contents N P& K than non-treated control bananas. Wu et al,

(2005) studied the effects of biofertilizer containing N-fixer, P & K

solubilizers and AM fungi on Maize growth, in green house trials reported

that the application of the combined inoculums showed significant increase

in plant height and biomass and improved assimilation of nutrients by

improving soil properties, organic matter content and total N in soil.

Artusson et al, (2006) reported that Arbuscular mycorrhizal (AM) fungi and

rhizobacteria could interact synergically to stimulate plant growth through a

range of mechanisms that include improved nutrient acquisition (Nitrogen

and phosphorus bioavailability) and inhibition of fungal plant pathogens.

Weber et al (2005) reported that the dual inoculation with Glomus

intraradices and Bradyrhizobium in Acacia mangium under aeroponic

culture, showed increased growth rate than single or non-inoculated ones.


The higher ‘P’ concentrations reduced the AM frequencies while lower

concentrations of ‘P’ stimulated the development of AM with out affecting

plant development. Schenck and Hinson, (1973) reported that the

mycorrhizal infection improved ‘N’ content in the shoots and seeds of

nodulated plants than the non-nodulating ones. Thus nodule function is

enhanced by mycorrhizal infection. The plants require relatively large

amount of phosphorus for optimum growth, nodulation and nitrogen

fixation (Carling et al, 1978). Co-inoculation of Rhizobium sps and VAM

fungi had a positive effect on growth and nutrient uptake in nodulating

plants. Dual inoculation of leguminous plants with Rhizobium and VAM

was found to enhance chlorophyll content and photosynthetic rates in

Cyamopsis sp (Neeraj and Ajit Verma, 1995). Rice bean (Vigna umbellate)

inoculated with G. fasciculatum and Rhizobium sp in a ‘p’ deficient soil

significantly increased VAM colonization, nodulation and yield of plants

(Kaur and Singh, 1985). Joshi et al, (1991) reported that a significantly

positive correlation exists between percent VAM infection and nodule

number in 60 days old groundnut. Gaur (1990), Kucey (1988) reported

that the inoculation of plants with phosphate solubilizing fungi was

encouraging the proliferation of other phosphate solubilizing fungi. ‘P’ and

‘N’ are the two major plant nutrients and co- inoculation of Nitrogen fixers

and Phosphate solubilizers may benefit the plant better than either group

of organisms alone (Saxena and Tilak, 1994). Manjunath et al, (1981)

reported that mixed inoculation of bacteria and fungi increased the yield of

onion to a greater extent than the individual microbial content. Smith and

Draft, (1977) reported that mycorrhiza- induced, increase in nitrogen fixing

rates in Medicago sativa preceded any effect on plant growth. This

suggested the idea that nodules demand phosphate. When phosphate

was added, the non-mycorrhizal plants showed higher value of % N as that

of mycorrhizal ones.


Results in the enzymes assays revealed that the enzymes

nitrogenase, alkaline phosphatases and acid phosphatases were found to

be increased in tetrapartiate associations. When plants were inoculated

with single inoculums, VAM inoculation showed maximum activity of these

enzymes when compared to others. In dual inoculums also VAM

association showed maximum enzyme activity. This ability suggests VAM

as a prominent recommendable microbe for soil fertility. Higher amount of

‘P’ uptake may also be the result of higher activity of acid phosphatase as

the total and specific activity of this enzyme in root extracts from the

inoculated seedlings was found to be significantly higher than the

uninoculated control (Choudhury et al, 2002) Nitrogenase activity in

Pueraria sps increased when the growth phosphate response curve

become asymptotic (Waidyanatha et al, 1979). It supports the suggestion

that nodule function may be preferentially stimulated by mycorrhizal

infection, which makes phosphate directly available to the nodules

(Bagyaraj et al, 1979, Dixon, et al, 1993, Mehrotra, 1995 and Priyarani et

al, 1999). Bagyaraj and Menge, (1978) reported that the dual inoculation of

VAM and Nitrogen fixing bacteria enhance the plant growth, as the

nitrogen fixing bacteria produce growth regulators and thus enhancing the

root production. The increased root growth might have resulted in more

root area for the VAM fungi and Nitrogen fixing bacteria to colonize and

thus enhancing the growth. Mosse et al, (1976) found that plants did not

nodulate unless their ‘P’ concentrations were at least 0.15%, mycorrhizal

infection helped the plants to reach this required level and nodulation then

occurred. Carling et al (1978) found that soluble phosphate can replace

VAM in increasing nitrogenase and nitrate reductase activities in nodulated

soybeans. Mycorrhizas markedly increase ‘P’ up take, growth and

nodulation in clover at low and intermediate rates of applied ‘P’, although

plant growth depressions may occur at high levels of available Phosphorus

(Crush, 1976). Large P additions to the soil are known to decrease

mycorrhizal infection in several legumes. Anguilar and Barea, (1978)


reported that a satisfactory nodulation was greatly dependent on the

mycorrhizal symbiosis. Rhizobium and Phosphobacteria cultures improved

plant growth, nodulation and mycorrhiza formation. The important benefit

of VAM association with leguminous plants is the absorption of phosphates

from soil. Legumes generally have less extensive root system as

compared to other plants and many of them are poor foragers of soil

phosphates. Phosphate is an important macronutrient for leguminous

plants due to its role in energy transfer during the process of nitrogen

fixation. A good supply of phosphorus is essential for effective nodulation,

which could be accomplished by VAM association. Nodules are generally

known to possess higher concentration of phosphorus than root tissues.

The requirement of P is high in legumes (Bagyaraj, 1991) and there fore

leguminous plants respond more to mycorrhizal infection than cereals,

which indirectly enhances the biological nitrogen fixation through increased

P availability especially in soils with low P content (Sieverding, 1983).

Legumes can form two types of associations with microorganisms. One

with Rhizobium sp involved in the fixation of atmospheric nitrogen and the

other with fungi, that form vesicular arbuscular endomycorrhizas which is

concerned with the up take of phosphorus and other nutrients (Crush,

1974). Inoculation of plants with VA mycorrhizal fungi can stimulate

nodulation and nitrogen fixation by legumes (Mosse, 1981, Timmer and

Leyden, 1980, Giahmi, 1976).

Carling (1979) reported that the mycorrhizal nodulated soybean

plants exhibited higher levels of nitrogenase and nitrate reductase as

compared to non-mycorrhizal plants. This increase in the nitrate reductase

system might have a role in increasing the symbiotic effectiveness of VAM.

A close association of fungal hyphae with the plant root changes

the structure and morphology of the root. It is clear now that the root

environment is very complex, where soil and rhizosphere microorganisms

and mycorrhizal fungi live and interact with each other. Interactions


between mycorrhizal fungi and microorganisms in the microenvironment

are of a very complex type. Mineral nutrition and Carbon allocation of host

plants alter the chemical and physical environment, which undoubtedly has

its effect on root, associated microbes in the soil. Bagyaraj, (1984)

observed that in most of the studies on tripartite association of leguminous

plants, VAM fungus and Rhizobium, mycorrhizas generally improve

nodulation and nitrogen fixation. Smith et al, (1985) noted that mycorrhizal

fungus encourages the production of glutamine synthatase in VAM root,

which accounts for the increased ammonium up take from the soil.

Pacovsky et al, (1986) observed an improvement in the shoot and root dry

weight of Sorghum when VAM fungus and Azospirillum were inoculated.

Dual inoculation with both organisms had an additive effect on shoot dry

weight. Phosphate Solubilizing Bacteria in the rhizosphere of mycorrhizal

plants have a special significance. Reports are available showing that such

bacteria interact with VAM for a better exploitation of a poorly soluble

phosphorus source. Better growth, increased dry matter and enhanced up

take of phosphorus from the soil were observed when VAM fungi and

phosphate solubilizing bacteria were inoculated together (Raj et al, 1981,

Piccini and Azcon, 1987). Bartlett and Lewis (1973) noted that the surface

acid phosphatases present in ECM and VAM are useful in utilizing

complex phosphates. Higher phosphatase activity in the germ tube and

hyphae of VAM fungus, Glomus mosseae has also been reported Dodd et

al, (1987). Huang-Ruey-shyong, et al (1983) and Manjunath, et al (1984)

were reported that the inoculation of Leucaena (Leucaena leucocephala, a

tropical leguminous tree) with mycorrhizal fungi was found to improve

nodulation, nitrogen fixation, plant growth and phosphorus uptake. Studies

of Singh and Subba Rao, (1987) reported that the yield and P content of

wheat increased with the co-inoculation of Azospirillum brasiliensis and

Glomus fasciculatum. Increased activity of acid phosphatase in roots of

trigonella has been reported due to inoculation with VAM fungus (Kapoor

et al, 1988).

EFFECT OF RHIZOBIUM AND PSB IN MYCORRHIZAL...

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