N.MANIVANNAN (2008).STUD!

ES ON USE OF BIO-FERTILIZERS ENRICHED VERMICOMPOST FOR ENHANCED CROP PRODUCTION

3
MATERIALS AND METHODS

PREPARATION OF VERMICOMPOST

Vermibed preparation

For the present study an epigeic earthworm, Eudrilus

eugeniae were collected from the breeding stock of the Department of

Biology, Gandhigram Rural University, Gandhigram, Tamilnadu, India

and leaf litter of Tephrosia purpurea Pers.,(Plate 1) Cassia auriculata

Linn.(Plate 2) and Gliricidia sepium Jacq.(Plate 3) were collected from

the Gandhigram Rural University campus. The leaf litters were

separately subjected to predigestion by sprinkling water on the heap and

covering it with gunny bag in order to let out the initial heat produced

during decomposition of organic material (15 days). The vermibeds were

56
prepared in plastic containers of 45x35x15 cm size (Plate 4) and the

substrate was moistened to hold 60-80 percent moisture content and

kept for 24 hours stabilization.

Inoculation of earthworms

20 numbers of healthy clitellate E.eugeniae (Plate 5) were

introduced in each bed. The vermicomposting trials were carried out in

the rearing room with the relative humidity and the temperature of 75-85

percent and 27+2°C respectively. The substrate was turned (mixed)

once in a week and maintained up to 90 days. The experiment was

carried out with three replicates for each substrate with proper control as

indicated in the experimental design. (Daniel and Karmegam 2000 and

Karmegam and Daniel, 2000 -c, 2000-d and 2000-e).

The details regarding the combination of leaf litter, cowdung

etc for each treatment and its control are indicated in the following

experimental design itself.

57
Experimental design showing the details of vermicomposting trials.

SI.No Vermibed substrate Number of Set up

worms
introduced
1 Leaf litter of T. purpurea + 20 T reatment 1
Cowdung (1:1)

2 Leaf litter of T. purpurea + 0 Control 1

Cowdung (1:1)

3 Leaf litter of C. auriculata + 20 Treatment 2

Cowdung (1:1)

4 Leaf litter of C.auriculata + 0 Control 2

Cowdung (1:1)

5 Leaf litter of G.sepium + 20 Treatment 3

Cowdung (1:1)

6 Leaf litter of G.sepium + 0 Control 3

Cowdung (1:1)

7 Leaf litter of T. purpurea + 20 T reatment 4

C.auriculata + Cowdung (1:1:2)

8 Leaf litter of T. purpurea + 0 Control 4

C.auriculata + Cowdung (1:1:2)

9 Leaf litter of C. auriculata + 20 Treatment 5

G.sepium + Cowdung (1:1:2)

10 Leaf litter of C. auriculata + 0 Control 5

G.sepium + Cowdung (1:1:2)

11 Leaf litter of T. purpurea + 20 T reatment 6

G.sepium + Cowdung (1:1:2)

12 Leaf litter of T. purpurea + 0 Control 6

G.sepium + Cowdung (1:1:2)

58
 The total number and total biomass of earthworms in the

vermibed substrates of all the treatments (Experiment 1,2,3,4,5 and 6)

were observed after 30,60 and 90 days and the results were statistically

analyzed using Microsoft excel computer software.

After 90 days the vermibed substrates were removed from

all the treatments and all the controls and sieved and stored for further

study (Plate 6).

PHYSICO-CHEMICAL ANALYSIS OF VERMIBED SUBSTRATES

The vermibed substrates i.e., the worms-worked (W W) and

the worms-unworked (WUW) substrates were analyzed for various

physico-chemical parameters such as pH, Electrical

conductivity(EC),organic carbon, total nitrogen, total phosphorus, total

potassium, total sodium, total calcium, total magnesium zinc, copper,

iron and manganese using standard procedures as given in the following

table.
Methods used for analyzing various physico-chemical parameters of the

vermibed substrates after 90 days.

Parameter analyzed Methodology Reference

Followed

pH Digital pH meter (Elico) Jackson (1973)

Electrical Conductivity bridge (Elico) Jackson (1973)

conductivity (E.C.)

Organic carbon Potassium-dichromate Walkley and Black

oxidation method (1947)

Total nitrogen MicroKjeldhal method Tandon (1993)

Total phosphorous Spectrophotometric Tandon (1993)

method

Total potassium Flame photometric Tandon (1993)

method
*
The sodium Flame photometric Tandon (1993)
method

Total calcium Acid digestion method Tandon (1993)

Total magnesium Acid digestion method Tandon (1993)

Zinc Atomic absorption method Tandon (1993)

Copper Spectrophotometric Tandon (1993)

method

Iron Spectrophotometric Jackson (1973)

method

Manganese Spectrophotometric Chopra and Kan war

method (1991)

60
 The C/N and C/P ratio were also found out for the worm-

worked and for the worm-unworked vermibed substrates. The

percentage increase/decrease of various physico-chemical parameters

in the worm-worked substrates over the worm-unworked substrates were

calculated as hereunder (Ramalingam and Ranganathan, 2001);

A- B
Percentage increase/decrease =------------X 100
A
Where,

A = Values in the worm- worked substrate

B = Values in the worm- unworked substrate

The percentage of increase/decrease of physico-chemical parameters in

the worm-worked over the worm-unworked substrates were subjected to

student T test using the computer software.

ENUMERATION OF MICROBIAL POPULATION

The total colony forming units (CFU) of bacteria, fungus and

actinomycetes in vermibed substrates at the beginning of the experiment

(Initial) and at the end the experiment (Worm-worked and Worm-

unworked substrates) were enumerated using standard plate count

method (Subbarao, 1995; and Kannan, 1996).

61
 Ten gram of each sample was taken in a 250 ml sterile

conical flask containing 100 ml of distilled water and shaken in a

Vortex mixture for 30 minutes. From this stock, various dilutions

were prepared from 10~1 to 10~6 with sterile distilled water. The

diluted samples were used for the analysis of total colony forming

units.

Procedure followed for enumeration of total colony forming units

(CFU) of bacteria, fungus and actinomycetes:

The numbers of colony forming units were counted

after incubation i.e., 2 days for bacteria, 5 days for fungi and 12 days

for actinomycetes as given in the above table.

62
ISOLATION, IDENTIFICATION AND MASS MULTIPLICATION OF

MICROORGANISMS FOR ENRICHING VERMICOMPOST.

Isolation of Rhizobium sp.

Rhizobium sp. were isolated from root nodules of the

leguminous plant, Vigna unguiculata (L.) collected from different

localities in and around the Gandhigram Rural University campus,

(Plate 7) following the technique of Vincent (1970) and maintained

in the Yeast extract mannitol agar medium (Vincent, 1980). The

purity of the culture was periodically ascertained using Congo red

reaction (Hahn, 1966).

Authentication of Rhizobium sp.

The Rhizobium sp. isolated from the root nodules were

authenticated by root nodulation tests such as tube test, pouch test

and Leonard’s jar assembly (Leonard, 1944) method and also by

biochemical tests and molecular studies.

Tube method

The efficiency of newly isolated Rhizobium sp. was

tested using plant infection test in nitrogen free nutrient solution

63
(Broughton and Dilworth, 1971). The test was performed in

germination paper by roll tube method (24 d).

Symbiotic effectiveness (%) = Dry weight of inoculated / Dry weight

of controls x 100.

Pouch method

The efficiency of the newly isolated Rhizobium sp. was

tested by pouch method (21 d) of Udaiyasuriyan et al (1985).

Folded germination paper was inserted in Polyethylene bags to

create pouches giving provision for keeping the sprouted seeds of

green gram, Phaseolus radiatus in its top fold. The entire setup was

sterilized by radiation (UV) for 30 minutes. Nitrogen-free Jensen’s

nutrient solution (Broughton and Dilworth, 1971) was added initially,

and pre-germinated seeds were transferred aseptically to the top

fold of germination paper of the pouches and 1 ml of 7 days old

rhizobial culture was inoculated on the seeds. Two replications

were kept for each isolate. Uninoculated controls were also

maintained.

After 21 days the plants were removed from the pouches

and the root system was observed for the presence or absence of

64
nodules. Suitable control was also maintained. The isolates that

produced nodules were used for the further study.

Jensen’s nutrient solution

Composition (Gm/lt)

CaHP04 1.00
K2HP04 0.20
MgS04 0.20
NaCI 0.20
FeCb 0.10
Distilled water 1000 ml
pH 6.8

Leonard jar assembly method (Leonard, 1944)

Leonard’s jar assembly method was conducted to find

out the efficiency of the newly isolated Rhizobium sp. A wide

mouth (Beer bottle) of 500ml capacity with its bottom cut and neatly

ground was inverted in a glass bottle (20 x 5 cm) of suitable

dimensions in such a way that the neck of the bottle snugly fits in it

(Plate 8). Cotton lamp wick was used to pass through the narrow

mouth of the bottle so as to touch the upper most part of the

vermiculite in the bottle. Vermiculite as the rooting medium and

Broughton and Dilworth solution as the nutrient solution was used

in this experiment. The Leonard jar assembly was sterilized at 15

lbs pressure for 20 minutes. After sterilization the assembly was

covered with brown paper. Three well spaced holes were made in

the rooting medium to a depth that will accommodate the pre­

germinated seeds of cowpea, Vigna unguiculata below the surface.

Well germinated seeds were picked out with sterile forceps and

placed into the holes, one seed on each hole with the radical facing

downwards. Suspension containing 109 cells / ml of the rhizobial

culture was added @ 1 ml per seed around the radical. Each

isolate was tested with two replications. Treatment with N control,

and uninoculated control were also maintained.

Identification of Rhizobium sp.

The bacterial cultures were identified up to genus level by

cultural, morphological and biochemical characteristics in accordance to

the Bergey’s manual of determinative bacteriology, Ed No. II, 2001.

BIOCHEMICAL TESTS

Growth in Congo red medium

A loop full of rhizobial isolates were streaked on Yeast

extract mannitol agar (Vincent, 1970) plates containing 2.5 ml of

one percent Congo red solution per liter and the growth of

rhizobium in the plate was checked (Subbarao, 1999).

66
Growth in Hofler’s Alkaline Broth (Hofler, 1935)
A loop full of the exponential phase culture was inoculated

into 25 ml of Hofler’s alkaline broth (Hofler, 1935) and incubated at

30°C for 4 days and observed the growth. Growth was measured in

a spectrophotometer by recording the turbidity of cultures at 600

nm (Subbarao, 1999). Uninoculated broth served as control. The

positive reaction for Rhizobium sp. was indicated by the absence of

growth in the medium.

Ketolactose production

Production of 3-Ketolactose was determined by the methods of

Bernaerts and De lay (1963). A loop full of bacteria from YEMA slants

was deposited on a lactose agar medium in a petridish as a small heap,

above 0.5 cm in diameter and incubated at 28°C for 5 days. The

petriplate was flooded with a shallow layer of Benedict reagent and left at

room temperature. The formation of yellow ring of Cu20 around the cell

mass was indicative of 3-Ketolactose production. Agrobacterium

tumefaciens was used as positive control for this test.

Growth in Glucose- peptone broth

A loopful of isolate was transferred to 25 ml of glucose

peptone broth (Subbarao, 1999). After 24 hrs of incubation the

67
growth in the glucose peptone broth was observed. Poor growth is

the indication of positive for rhizobial isolates.

Production of Acid or Alkali

Bromothymolblue was incorporated in 5 ml of a 0.5 percent

alcoholic solution per liter of YEM medium, giving a final

concentration of 25 ppm and shows green color (pH 6.8). A loopful

of the isolate was streaked into sterile petriplates containing the

above medium and incubated at 26°C for 4 - 6 days (Cappucino

and Sherman, 2004). Change of color of the medium to deep blue

as the colonies developed indicated the production of alkali by slow

growing Rhizobia; whereas the development of yellow coloration

indicated acid production by the fast growers of Rhizobium sp.

MOLECULAR STUDIES

DNA Extraction

Total genomic DNA was extracted from Rhizobium sp. and

Bacillus sp. by a modified method of Smoker and Barnum, (1988).

16S rDNA sequencing

The PCR products were purified using Microcon PGR

centrifugal filter device (Millipore Crop. Bedford, Mass) and sequencing

was carried out using the facility at Macrogene Inc (Seoul, Korea).

68
Nucleotide sequence accession

The 16S rDNA sequences for the Rhizobium sp. and Bacillus

sp. has been deposited in Gene Bank http://www.ncbi.nlm.nih.gov/qene

bank and the sequences accession numbers are EF420109 and

EF601985 respectively.

Phylogenetic analysis

The nucleotide sequences obtained from the sequencing of

the PCR product were compared with the Rhizobium sp. and Bacillus sp.

sequences available in the NCBI database using the bioinformatics tool

BLAST. All the sequences were aligned using the multiple sequence

alignment program CLUSTAL V developed by Higgins et al. (1992). The

pair wise evolutionary distances were computed using the Kimura

(1980). The multiple distance matrix obtained was then used to construct

phylogenetic trees using Neighbour-Joining method of Saitou and Nei

(1987). A phylogenetic tree was constructed by the program PHYLIP

using the bioinformatics Genebee tool available in on-line i.e.,

www.qenebee.msu.su.

Prediction of RNA secondary structure

The secondary structure of 16S rDNA of Rhizobium sp. and

Bacillus sp. were predicted using the bioinformatics Genebee tool

available in on-line i.e., www.qenebee.msu.su.

69
Restriction site analysis in 16S rDNA

The restriction sites in DNA of Rhizobium sp. and Bacillus sp.

were analyzed using the NEBCutter program version 2.0 tools available

in on-line i.e., www.tools.neb.com/NEBCutter2/index.phD.

Growth characteristics of Rhizobium sp.

An Erlenmeyer flask (250ml) with of YEM broth was

inoculated with 0.1 ml of late log phase culture. The absorbance of the

resulting culture at zero time was 0.01 at 540 nm. The culture was grown

in a rotary shaker with a speed of 150 rpm at room temperature (27°

±3°C). Growth was monitored by recording the absorbance of the culture

at 540 nm in a Spectronic 20.

Mass multiplication of Rhizobium sp.

The selected rhizobial colony was grown in YEMA slant

for 3-9 days. Depending on the growth, the culture was checked for

purity and transferred to conical flask containing sterile liquid

medium and maintained for 4-9 days. This was called as the

“Starter culture”. Later the starter culture was transferred to

production medium (Vincent, 1970) with pH 6.5- 7.0 and incubated

at 30°-32°C for 4-9 days (Plate 9).

70
 After incubation period the cell count was done by

normal plate count method to determine the viable count at 28+ 2°C

incubation. Serial dilutions of the broth were prepared, so as to

provide a dilution at which 30-300 viable bacteria appeared on the

petriplate. Similarly the cell count was determined from fresh broth

after suitable dilution in a haemocytometer (Vincent, 1970).

ISOLATION, SCREENING AND IDENTIFICATION OF

PHOSPHATE SOLUBILIZING MICROORGANISMS FROM THE

RHIZOSPHERE SOIL SAMPLES OF LEGUMINOUS PLANTS

Isolation of phosphate solubilizing microorganisms

Phosphate solubilizing microorganisms namely bacteria

and fungi were isolated from the sites of the rhizosphere of

leguminous plants from in and around Gandhigram Rural University

campus. Soil adhering on the root was gently shaken and collected

to the isolation of rhizosphere bacteria and fungi. Serial dilutions of

the rhizosphere soil samples were individually plated on Nutrient

agar (NA), Rose bengal agar (RBA) and Pikovskaya agar medium

(PVK) as described by Guar (1990).

71
72
 accordance to the Bergey’s manual of determinative bacteriology,

Ed No. II, 2001 and also by molecular sequencing (Plate 10).

Maintenance of pure culture.

The predominant bacterial colonies were isolated from the

master plate and streaked in the Nutrient agar slants, incubated and then

stored in the refrigerator.

Tests for identification of bacterial isolates

The following tests were used for the identification of

bacterial strains.

Gram’s staining

The slides subjected to gram’s staining were examined under

phase contrast microscope. The bacteria that retained the color of

crystal violet Iodine complex were said to be gram positive and the

bacteria that retained the safranin color were said to be gram negative.

This was done for all the bacteria which were isolated from rhizosphere

soil of the leguminous plants.

Motility test by hanging drop method

A ring of petroleum jelly was placed around the cavity of a

depression slide. A loop full of 18hrs culture was placed in the center of

73
clean cover slip and then placed over the depression slide with the

concave surface facing the cover slip and then focused in the phase

contrast microscope to see the motility.

BIOCHEMICAL TESTS FOR BACTERIAL ISOLATES

Indole production test

Bacterial culture was inoculated in nutrient broth and

incubated at 37°C for 24hrs and 0.5ml of Kovac’s reagent was added

and incubated again. Formation of red color ring at the top of the culture

tube indicated the indole positive reactions.

Methyl red test

Bacterial cultures were inoculated in MR broth and incubated

at 37°C for 24hrs. After incubation 5 drops of methyl red were added and

the result was observed. The color formed in the tube indicated the

positive result. The tubes which remained yellow were negative.

Voges Proskauer test

Bacterial cultures were inoculated in VP broth and incubated

at 37°C for 24 hrs. After incubation 10 drops of VP-1 reagent were added

and then 2-3 drops of VP-2 were added, shaken well and the results

74
were observed. Appearance of red color indicated the positive result and

the tubes that remained yellow were noted as negative.

Citrate utilization test

The test bacterial culture was inoculated in Simmon’s citrate

agar slant and incubated at 37°C for 24-48 hours. Following incubation,

citrate positive cultures were identified by the presence of growth on the

surface of the slant, which was accompanied by blue coloration. Citrate

negative culture showed no growth, and the medium remained green.

Catalase test

The isolated culture was aseptically picked up using sterilized

loop and smear was prepared on a clean slide. 2-3 drops of H202 was

added on the slide. The effervescence (bubble) formed on the slide

indicated a positive result, and the slide which did not show the

effervescence indicated the negative result.

Nitrate reduction test

Bacterial culture was inoculated in nitrate broth and

incubated at 37°C for 24-48 hours. After incubation 2-3 drops of reagent

A (0.8 percent solution of sulfanilic acid in 5N acetic acid) and 2-3 drops

of reagent B (0.5 percent solution of dimethyl alpha-naphthol in 5N acetic

75
acid) were added to each tube and observed. The red color formed

immediately indicated the positive result and the tubes which did not

show red color were said to be negative.

Mass multiplication of Bacillus sp.

The selected bacterial colony was grown in Nutrient agar

slant for 48 hours. Depending on the growth, the culture was

checked for purity and transferred to conical flask containing sterile

liquid medium for 48 hours. This was called as the starter culture.

Later starter culture was transferred to the production medium with

pH 6.5-7.0 and incubated for 2-5 days (Plate 9).

Cell count was done by normal,plate count method to

determine the viable,count at 37+2°C incubation. Serial dilution of

the broth was prepared, so as to provide a dilution at which 30-300

viable bacteria appeared on the petriplate. Likewise the cell count

was determined from fresh broth after suitable dilution in a

haemocytometer (Vincent 1970).

Identification of phosphate solubilizing fungi

All the fungal isolates were subjected to macroscopic

observation by plating on Rose bengal agar medium and their colony

characteristics were observed and recorded (Plate 11).

76
Microscopic examination

The fungal isolates were stained with Lacto phenol cotton

blue and observed, for their cultural characteristics and the structure of

their conidiospore in microscopy (Domsch et al., 1980). The Aspergillus

sp. was identified based on their morphological features (Plate 12).

Mass multiplication of Aspergillus sp.

25gms of barley grains were washed well with distilled

water, drained and transferred to a 250 ml conical flask and

sterilized intermittently for two days. Then 3 ml of spore suspension

with 3 ml of sterile water was inoculated aseptically and kept in the

room temperature for 14 days (Plate 13).

Spore count was done by normal plate count method to

determine the viable count on 7th, 9th, 12th and 14th day at 25+2°C

incubation. Serial dilution of the broth was prepared, so as to

provide a dilution at which 0-50 viable fugal colonies appeared on

the petriplate. Similarly the spore count was determined from fresh

broth after suitable dilution in a haemocytometer (Vincent, 1970).

77
POT CULTURE STUDIES

The pot culture studies were conducted in the

Department of Biology, Gandhigram Rural University, Gandhigram.

Certified seeds of Vigna unguiculata (L.) Walp. were procured from

Tamilnadu Agricultural University, Periyakulam, Tamilnadu. Soil

mixture containing sand, red soil, vermicompost and mixed

biofertilizers (Rhizobium sp., Bacillus sp. and Aspergillus sp.) was

used to grow the plants following the given design.

Since Treatment 4, 5 and 6 containing the mixed

combination of leaf litters (T.purpurea + C.auriculata, C.auriculata +

G.sepium and T.purpurea + G.sepium) produced higher number and

biomass of E.eugeniae and also higher NPK and other minerals, only the

vermicompost prepared from these combinations were considered for

further pot culture studies after mixing with biofertilizers i.e., rhizobium for

nitrogen fixation, bacillus and aspergillus for phosphate solubilization.

78
Experimental design for pot culture studies of Vigna unguiculata (L.)
Walp.

Ml - Microbial Inoculants; Rhi - Rhizobium; PSB - Phosphate solubilizing

bacteria; PSF - Phosphate solubilizing fungi; VC - Vermicompost;
TP - Tephrosia purpurea; CA - Cassia auriculata; GS - Gliricidia sepium

79
 Vermicompost enriched with mixed inoculation of

Rhizobium, phosphate solubilizing bacteria (PSB) and phosphate

solubilizing fungi (PSF) of different microbial load (1X104 and 1X108)

in soil was used as substrate for the pot culture studies. Ten

uniform size seeds of cowpea were surface sterilized with 0.5

percent sodium hypochlorite solution for 45 min, rinsed in sterilized

water and then sown in the control (soil only) and in the treated

soil (soil + vermicompost + microbial inoculum). After germination,

seedlings were thinned to four per pot. Treatments were arranged

in a randomized complete block design (RBD) with triplicate for

each treatment. Plants were watered subsequently (Plate 14). The

mean annual rainfall at Gandhigram is 765 mm distributed over 48 rainy

days with minimum and maximum temperature ranging between 12°C

and 39°C respectively.

Physical parameters observed

Three plants were selected at random from each replication

for each treatment. The observations were recorded and the mean

values were statistically analyzed and expressed in their respective units.

80
Root length

The root length was measured from a fixed point just below

the surface of the soil to the end of the root at the vegetative phase on

35th day after sowing (DAS) and at the harvest stage (on 70 DAS) and

expressed in centimeter (cm). The long root which develops from the

seed itself was considered for this experiment.

Shoot length

The height of the plant itself was measured from a fixed point

just above the surface of the soil to the growing tip of the main stem at

two stages i.e., vegetative stage (on 35 DAS) and at harvest stage (on

70 DAS) and was expressed in centimeter (cm) for each treatment.

Number of root nodules

On 35 DAS and 70 DAS the plants were carefully

removed from the pots and gently washed with water, blotted on

a filter paper and the number of root nodules per plant were

recorded. The average number of nodules was expressed in

numbers per plant for each treatment (Plate 15).

81
Number of root branches

The plants were carefully removed from the pots and

gently washed with water, blotted on a filter paper and the

number of root branches per plant were recorded on 35 DAS and

on 70 DAS. The average number of root branches was

expressed in numbers per plant for each treatment.

Shoot wet weight

On 35 DAS and on 70 DAS the plants were carefully

removed from the pots, gently washed with water, blotted on a filter

paper, the shoot portion alone was removed from the whole plant and

then weighed. The average fresh weight was expressed in grams (g) for

each treatment.

Shoot dry weight

After measuring the fresh weight, the plants were dried in hot

air oven at 80°C for 24 h and weighed. The average dry weight of the

shoot on 35 DAS and on 70 DAS were measured and expressed in

grams (g) for each treatment.

82
Root wet weight

The root weight was measured on 35 DAS and on 70 DAS.

The plants were carefully removed from the pots, gently washed with

water, blotted on a filter paper; the root region alone was removed and

weighed. The average fresh weight was expressed in grams (g) for each

treatment.

Root dry weight

After measuring the fresh weight, the root portions were dried

in hot air oven at 80°C for 24 h and then weighed. The average dry

weight of the root on 35 DAS and on 70 DAS were measured and

expressed in grams (g) for each treatment.

Number of internodes

The number of internodes present in the plant was counted

from a fixed point just above the surface of the soil to the growing tip of

the main stem at harvest stage (on 70 DAS) and was expressed in

numbers for each treatment.

83
Number of internodes per feet

The number of internodes per foot in the plant was counted

from the surface of the soil in the main stem at harvest stage (on 70

DAS) and was expressed in numbers for each treatment.

Leaf area index

The total leaf area index was calculated in the harvest stage

(70 DAS) using the formula described by Williams and Steinberg (1958)

as given below (Plate 16):

Total area of plant

Leaf area index = ----------------------------------------------

Land area occupied by the plant

Total number of flowers appeared

This data was collected during the flowering season of

the plant growth (Plate 17). The total number flowers present in

each plant were counted and the average was expressed as

number of flowers per plant on 70 DAS for each treatment.

84
Number of pods

The number of pods harvested from three plants

was summed up and the mean was worked out and expressed as

number of pods per plant for each treatment (Plate 18).

Pod length

The pods were collected from each plant on 70 DAS and

the length of the pod from each treatment was measured and their

mean was calculated and expressed in centimeter (Plate 19).

Pod wet weight

The pods were collected and gently washed with water,

blotted on a filter paper, weighed and the mean wet weight of the

pod from each treatment was calculated and expressed in grams

(g).

Pod dry weight

After measuring the wet weight, the pods were dried in hot air

oven at 80°C for 24 h and then weighed and the average dry weight of

the pod is expressed in grams (g) for each treatment.

85
Number of seeds per pod

The seeds were separated from the pods and the

number of seeds per pod was counted and the average number of

seeds per pod was calculated for each treatment (Plate 20).

Seed dry weight

The seeds were dried in the hot air oven at 80°C for 24 h and

the dry weight of the seeds were calculated and expressed in grams (g)

for each treatment (Plate 21).

Grain I Cotyledon thickness

Three grains from each experimental setup were

randomly selected and the grain thickness of each seed was

measured and expressed in mm for each treatment.

Peeling thickness

Three peelings from each experimental setup were

randomly selected. Peeling thickness per pod was measured and

expressed in mm for each treatment.

86
Leaf chlorophyll content

The leaves of the plant supplied with various substrates and

microbial inoculants were collected on 70 DAS and chlorophyll ‘a’,

chlorophyll ‘b\ total chlorophyll content of the leaves were measured

using the method of Arnon (1949) and expressed as mg/g of fresh tissue

for all the treatments.

Carbohydrate, Protein, Fat contents of seeds

The seeds of each experimental set up were dried, powdered

and analyzed for total carbohydrate (Anthrone’s method), protein

(Lowry’s method) and fat (Soxhlet method) contents.

Enumeration of microbial load

The microorganisms present in the rhizosphere region of

cowpea plant in each treatment were separately collected and

enumerated by serial dilution technique. The total colony forming units

(CFU) of Rhizobium sp. Bacillus sp. and Aspergillus sp. at the initial and

on 35 DAS and on 70 DAS were enumerated using standard plate count

method (Subbarao, 1999) and the results were subjected to one-way

AN OVA using the Computer software, SPSS.

87
Procedure followed for the enumeration of different colony forming

microorganisms

PLANT SAMPLE ANALYSIS FOR NUTRIENT CONTENT

Fully expanded fifth leaf from the tip of the shoot was

utilized for the analysis as recommended by Ward (1967).

Leaf samples were collected in each experimental pot on

the 35 and on the 70 DAS. The dust particles on the samples were

gently removed by using brush and then washed thoroughly with

0.1 N Hcl followed by double distilled water. The plant samples

were dried under shade and then in a stainless steel hot air oven at

60°C. The dried samples were chopped with stainless steel scissors

and ground in a Wiley mill and used for the analysis of nutrients

88
i.e., nitrogen (MicroKjeldhal method), phosphorous

(Spectrophotometric method) and potassium (Flame photometer).

Nitrogen content

The total N was estimated by MicroKjeldhal Method

(Piper, 1966). The total N uptake was calculated by multiplying

estimated N content with total dry matter produced and expressed

in percentage.

Phosphorous content

The total P content of the samples was estimated by the

Vanadomolybdophosphate yellow color method of Piper (1966).

P uptake of the plants was calculated and expressed in percentage.

Potassium content

The total K content of the plant samples was estimated

using digital flame photometer as described by Jackson (1973) and

the total uptake of K was expressed in percentage.

ANALYSIS OF SOIL SAMPLES

Soil samples were collected from the experiment pots at a

depth of 15-30 cm after harvest of the crop, dried under shade,

powdered and sieved through a 2 mm sieve for analysis of the following.

89
Available nitrogen

Available nitrogen content in the soil was estimated by

alkaline permanganate method as given by Subbiah and Asija (1956)

and expressed in mg/kg.

Available phosphorous

Available phosphorous content of the soil was estimated by

calorimetric method as suggested by Olsen et al. (1954) and expressed

in mg/kg.

Available potassium

Available potassium content of the soil was estimated by

flame photometry method as given by Stanfold and English (1949) and

expressed in mg/kg.

Statistical Analysis

The results of the experiments were statistically

analyzed by one-way and Two-way ANOVA with the help of MS-

DOS soft ware SPSS and the critical difference were worked out at

five and one percent level and the results were tabulated.

90