Chapter – 3

Materials and Methods 3.1. Introduction

The present investigations were carried out in two phases. The first

phase of the investigations was to identify the AM fungal flora naturally

associated with green gram in their field conditions in South India. Extensive

field surveys were carried out at this stage in the traditional green gram fields

of Tamil Nadu and Karnataka States. Plant specimens and soil samples were

collected from the fields and brought to laboratory for further investigations.

During the second phase of the investigation, experimental trials of certain

important AM fungi, Piriformospora indica and Rhizobium on green gram

were carried out in pots. Experiments of both individual and combined

applications of these microbes were done to understand the influence of them

on the crop. Among the AM fungi tested, the better isolates were further

tested as consortium to learn the impact on the crop at different levels of

fertilizer supply. All the details of these investigations including soil and plant

tissue analyses are given below.

3.2. Field Survey

3.2.1. Study area

Studies were conducted in the major pulse growing regions of

Tamilnadu and Karnataka. The sampling locations were selected at random

with a minimum distance of ten kilometer in between. The sampling sites S1

to S15 were in four Districts of Tamilnadu (Tirunelveli, Teni, Dindigal and

Coimbatore) and S16 to S21 were in one District of Karnataka

(Chamarajnagar) [Fig.1].

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S1. Ambalavanapuram S12. Sukkumanaikampetty S2. Valliyur S13. Oddanchathram S3. Ambasamudram S14. Thalayathu S4. Alangulam S15. Udumelpetta S5. Thevaram S16. Gundalpet I S6. Dwarasamipuram I S17. Gundalpet II S7. Dwarasamipuram II S18. Gundalpet III S8. Puthupetty S19. Gundalpet IV S9. Periyakulam I S20. Gundalpet V S10. Allinagaram S21. Gundalpet VI S11. Periyakulam II

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3.2.2. Sampling procedure

Soil samples from the rhizosphere zone of green gram at a depth of 20-

30 cm were collected from 21 locations during October-November 2005. Two

samples were collected from each sampling location (field) spaced within ten

meters. All the samples were collected when the plants were either in blossom

or bearing fruit. Each plant, along with a cylinder of soil was dug out without

damaging the root system. The plant was then removed and the feeder roots

were fixed in 10% potassium hydroxide (KOH) for assessment of percentage

colonization.

The soil left was mixed to form a composite sample and assigned

sample number 1 to 21 to represent samples from S1 to S21 respectively.

Those composite soil samples were used for isolation of AM fungal spores

and for studying soil chemistry. The soil was stored in plastic bags and

brought to the laboratory. The samples were kept open for air drying in clean

undisturbed open shelves separately. They were then used for extracting

spores as well as for studying soil parameters, within one week from the date

of sampling.

3.2.3. Spore identification and counting

The soil samples were analyzed for AM fungal spores, following wet

sieving and decanting procedure (Gerdemann and Nicolson, 1963) and the

spore count was carried out by the plate method described by Smith and

Skipper (1979). One gram of moist soil was added to 9 ml of distilled water in

a test tube capped with a rubber stopper. The tube was then vigorously

agitated by hand and 1 ml was immediately pipetted in parallel lines on to a 9

cm filter paper disc in a petridish. The filter paper was then scanned wet

under a dissecting microscope and the spores were counted. The spore count

was multiplied upward to represent 100g soil. Ten plates were prepared from

one soil sample and the data were represented as average. Identification of

AM fungi was based on the system proposed by Hall and Fish (1979). The

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spores were identified from the plate for spore count and the proportion of

each species was determined. It was then multiplied upward to represent 100

g soil.

3.2.4. Study of Percent colonization

Estimation of mycorrhizal colonization of the root system was done

after clearing and staining the roots (Philips and Hayman, 1970). The roots

were cut into pieces of 1cm length and boiled in 10% KOH and washed with

water. After washing, the root segments were neutralized with 10%

hydrochloric acid (HCl). The clear root segments were stained with 0.05%

trypan blue in lactophenol. The root segments were destained after 2 hours

using lactophenol and examined for AM fungal colonization. 100 root bits

were examined for mycorrhizal infection. The results were expressed as

percent colonization.

3.2.5. Study of soil parameters

The soil from different sampling sites was analyzed for soil fertility

parameters such as pH, N, P and K following standard methodology; following

the methodology described by Jackson (1973). Flame photometry (Flame

photometer Model 128 Systronics) was used for potassium estimation.

3.3. Experimental Materials

3.3.1. Plant for experiments

Green gram (Phaseolus aureus Roxb., Syn. Phaseolus radiatus L.,

Vigna radiata (L.) Wilczek.) belongs to family Leguminosae, sub family

Fabaceae. The plant is an erect or sub erect deep-rooted, branched, hairy

annual herb. Leaves are trifoliate with ovate leaflets and alternate phyllotaxy.

Inflorescence racemose, flowers yellow coloured, pods grey or brownish

when mature. Seeds are small and green. Various cultivars of green gram are

available, differing in habit, height, period of maturity, color of pod, size and

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color of seeds. Selection, hybridization and mutation are the methods adopted

for crop improvement in green gram.

The seeds of green gram (P. aureus) used for the experiment were

procured from the Tamilnadu Agriculture University, Coimbatore. The

variety used was ‘Co6’, which was a hybrid between ‘WGG 37’ and ‘Co5’. It

was released in 1999. The duration of the crop is 62-67 days with 50%

flowering in 26-30 days. The plant height is 35-55 cm. The leaves are broad

and the fruits are pubescent, turning brownish black when dry. The grains are

green with smooth surface. Average seed weight is 3.4 g/100seeds.

3.3.2. Rhizobial isolates

The Rhizobium isolates used for screening the most effective strain in

green gram were taken from six different host plants belonging to Fabaceae.

The criteria for the selection of host plants were (i) the plants belong to the

same family as that of green gram, so that the Rhizobium isolates would

nodulate green gram easily (ii) the number of healthy effective nodules on the

respective hosts. The host plants and the numbers given to these isolates are

shown in the Table 1.

Table 1. The host plants and the Rhizobium isolates used in the screening test

Sl. No. Name of host plant Rhizobium isolate

1. Crotalaria striata R1

2. Vigna wightii R2

3. Pueraria phaseoloides R3

4. Phaseolus aureus R4

5. Flemingia bracteata R5

6. Phaseolus multiflorus R6

The percentage of effective nodules (determined on the basis of nodule

size and presence of leghemoglobin) on the hosts ranged from 40 to 55. The

nodules were separated from the root system and surface sterilized with

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PLAT 1

Root nodules isolated from different host plants

alcohol (Plate1). They were crushed in sterile water to form the stock

inoculum. The nodule bacteria from each host were multiplied in nutrient

medium as per Dye (1979).

The flasks containing nutrient medium were autoclaved at a

temperature 1200 C and 15 lbs pressure for 30 minutes. 10ml each of the

Rhizobium suspension was added to the flasks after proper cooling. The

Rhizobium cultures were incubated at 350 C for 48 hrs and used for

inoculating the seedlings.

3.3.3. AM fungal cultures

The pure cultures of AM fungi used for the screening test were

obtained from the AM fungal collections of Microbiology Division of Central

Tuber Crops Research Institute (CTCRI), Thiruvananthapuram, Kerala. The

selection of the AM fungi for screening test was made on the basis of field

observation. The most common AM fungal associates with green gram under

field conditions were Glomus mosseae (M1), Glomus microcarpum (M2) and

Gigaspora margarita (M3). Hence pure cultures of G. mosseae, G.

microcarpum, and G. margarita were used to assess the individual

performance on green gram. The inocula of the AM fungi were multiplied on

Italian millet (Setaria italica Beauv.) root in pot condition. The soil along

with spores and root segments were used for inoculating the test plants.

3.3.4. Culture of Piriformospora indica (P. indica)

P. indica is an axenically culturable fungus that mimics AM fungi in

plant growth promoting effect. In order to test the possibility of P. indica as a

phytopromoter in green gram, an evaluation of the performance of the fungus

on the crop was done. The stock culture of P. indica was obtained from

Microbiology Division of CTCRI, Thiruvananthapuram. It was then

multiplied in sterilized Potato Dextrose Broth (PDB). The hyphae recovered

from the broth were used for inoculation.

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3.4. Experimental trials

3.4.1. Experiments with Rhizobial isolates

3.4.1.(a). Experimental design and details of experiment

The Rhizobium isolates were screened for their effectiveness on green

gram under pot culture conditions. 56 small pots of 10cm diameter were used

for the experiment. The pots were filled with double sterilized (using

autoclave) pot mixture consisting of top soil (Gravelly Clay Loam on

Laterites with a mechanical composition of about 40.1% coarse sand, 28.5%

fine sand, 8.8% silt, 22.6 % clay) coarse sand and dried and powdered cow

dung in the ratio 3:1:1 by weight. The nitrogen content of the soil for the

experiment was 196 kg ha-1. The pots were arranged in 7 sets having 8

replications each. In each pot one surface sterilized and pre soaked seed was

laid in a small pit made for the seed. The Rhizobium inoculation was made on

the second day after sowing the seeds. 10ml of 48 hr old bacterial culture

(containing approximately 109 cells per ml) was added to the root zone of

each seedling using a pipette. The treatments were designated as follows:

R0 R2 R4 R6

R1 R3 R5

R0- without Rhizobium; R1 to R6- with respective Rhizobium isolate

The pots were arranged separately to avoid contamination. Sampling

was done on 20, 40 and 60 days after inoculation (DAI).

3.4.1.(b). Sampling

Two plants each from a treatment were sampled at a time. Growth

parameters such as root length, shoot length, total fresh weight, total dry

weight, percentage moisture, total chlorophyll, total leaf area and leaf area

index were analyzed. Total number of nodules, total weight of nodules,

weight per nodule and percentage of effective nodules were estimated for

each plant. Tissue nitrogen (%) was also determined for each treatment. The

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harvest was made after 68 days of sowing. The harvest data relating to each

plant include total number of pods, total weight of pods, total number of

seeds, total weight of seeds and weight of 100 seeds.

3.4.2. Experiments with AM fungal type alone and in combination of

Rhizobium

3.4.2.(a). Experimental design and details of experiment

To evaluate the effectiveness of AM fungal types and P. indica, pot

culture experiments were conducted. 100 pots of 10cm diameter were filled

with double sterilized (autoclaved) pot mixture consisting of top soil

(Gravelly Clay Loam on Laterites with a mechanical composition of about

40.1% coarse sand, 28.5% fine sand, 8.8% silt, 22.6 % clay) coarse sand and

dried and powdered cow dung in the ratio 3:1:1 by weight. The NPK content

of the soil was 50.76, 10.8 and 142 kg ha-1 respectively. The pots were

arranged in 10 sets with 10 replications each. The treatment combinations

were:

M0 M2 M4 M1R M3R

M1 M3 R M2R M4R

M0- without mycorrhiza; M1- G. mosseae; M2- G. microcarpum; M3-

Gigaspora margarita; M4- P. indica, R- Rhizobium alone; M1R to M3R-

respective AM fungi and Rhizobium; M4R- P. indica and Rhizobium

The pots were arranged separately to avoid contamination of

inoculated and non inoculated treatments. The pots assigned for mycorrhizal

treatments (M1 to M3) were inoculated with AM fungal inoculum consisting

of about 400 spores / 100g soil. The inoculum was mixed with the topsoil.

One surface sterilized and pre soaked seed of green gram was placed in each

pot, about one centimeter below the soil surface. The pot assigned for P.

indica (M4) was inoculated with hyphal mass recovered from the culture

broth. Each pot was inoculated with 1g of hyphae of P. indica.

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The pots assigned for the study of interaction between AM fungi and

Rhizobium, and P. indica and Rhizobium were also prepared in the same

manner as for the evaluation of AM fungi and P. indica. Rhizobium culture

raised from the most effective Rhizobium isolate (R6) was used for

inoculation. The procedure of Rhizobium culturing and inoculation was the

same as that for screening of the Rhizobium isolates.

3.4.2.(b). Sampling

Samples were taken on 20th, 40th and 60th day after inoculation (DAI).

Two pots from each treatment were selected and the plants were up rooted,

without damaging the root system. The root system was cleaned with water

and the fine fragments of root were recovered. Growth parameters such as

root length, shoot length, total fresh weight, total dry weight, % moisture,

total leaf area and leaf area index were analyzed. Total number of nodules,

total weight of nodules, weight per nodule and percentage of effective nodules

were estimated for each plant treated with Rhizobium. Root samples were

taken for estimation of AM fungal and P. indica colonization according to the

procedure developed by Philips and Hayman (1970). Soil samples from the

rhizosphere zone were taken for estimation of N, P and K. Leaf samples (fully

expanded third leaf from the tip of the plant) were taken for estimating total

chlorophyll, tissue N, P and K. The harvest data was taken on 68th day after

sowing.

3.4.3. Experiments with AM fungi and Rhizobium under different

nutritional levels

The interaction between the AMF consortium individually and in

combination with Rhizobium at different dosages of N and P on the crop was

carried out as the third major set of experiments.

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3.4.3.(a). AM fungal selection for further evaluation

Based on the performance in the screening test, Glomus microcarpum

was found to be better performer when applied alone. But along with

Rhizobium, Glomus mosseae was found to be effective in promoting growth

parameters and nodulation. Hence a study of interactive effect of these two

AMF and Rhizobium, either alone or in combination, on green gram at

different fertilizer doses was carried out. The two AMF were developed

separately in pot condition on Italian millet (Setaria italica Beauv.) root for

inoculum production. The spore load of G. mosseae was 200/100g soil, while

that of G. microcarpum was 734/100g soil. The total spore load of the

inoculum was adjusted to 400-spores/100g soil, with approximately equal

number of spores of each AM fungus.

3.4.3.(b). Rhizobial isolate selection for evaluation

The Rhizobium isolate selected for the final evaluation was R6 (from

Phaseolus multiflorus). It was multiplied in Yeast extract-Manitol medium as

described earlier. 48 hr old Rhizobium culture was used for inoculation.

3.4.3(c). Fertilizer doses

Urea was used as the source of N, while Rock phosphate (Raj Phos)

was used as the source of P. The fertilizers were applied at the rate of 0, 50

and 100% of the recommended dose (RD). The nutrient K was kept constant

at 100% of the recommended dose to all the treatment combinations in the

form of muriate of potash (MOP). The recommended doses of N, P and K for

green gram were; N – 20kg ha-1; P2O5 – 30kg ha-1; K2O – 30kg ha-1 (Kerala

Agriculture University, 2002).

3.4.3.(d). Preparation of pots

Since plastic pots are recommended for soil fertility experiments

(Allen et al, 1976), the present study was done with 114 plastic pots of 23cm

diameter in the completely randomized block (CRB) design. The soil used for

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the experiment showed pH- 6.33, N- 46.65 kg/ha, P- 9.86 kg/ha and K-

132.16 kg/ha. Each pot was filled with 5 kg potting mixture. The pots were

filled to ¾ of its capacity. The pot mixture consisted of soil, sand and

farmyard manure in the ratio 3:1:1 and was sterilized with 10% formaldehyde

for 48 hrs. The AMF inoculum was added and mixed with the topsoil in each

pot. The pots were arranged in 19 sets of six replications each (Plate 2A &B).

The treatment combinations were as given in the Table 2.

Table 2. Treatment combinations involving AM fungal consortium,

Rhizobium, N and P

Sl. No. Treatment combinations Sl. No. Treatment combinations

1 M0 R0 P2N2 11 M1 R1 P0N0

2 M1 R0 P0N0 12 M1 R1 P0N1

3 M1 R0 P0N1 13 M1 R1 P0N2

4 M1 R0 P0N2 14 M1 R1 P1N0

5 M1 R0 P1N0 15 M1 R1 P1N1

6 M1 R0 P1N1 16 M1 R1 P1N2

7 M1 R0 P1N2 17 M1 R1 P2N0

8 M1 R0 P2N0 18 M1 R1 P2N1

9 M1 R0 P2N1 19 M1 R1 P2N2

10 M1 R0 P2N2

M0- without mycorrhiza P0- without additional P

M1 – with AM fungal consortium P1 – with 50% RD of P

N0- without additional N P2 – with 100% RD of P

N1 – with 50% RD of N R0 – without Rhizobium

N2 – with 100% RD of N R1 – with Rhizobium

The pots were carefully arranged to avoid contamination between the

treatments. Two surface sterilized and pre soaked seeds of green gram were

placed in each pot with a distance of 15 cm in between. The fertilizer

application was done on the 3rd day after sowing. The plants were watered

once in a day up to 30 days and then twice in a day until harvest so as to avoid

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PLATE 2 Experiment with AM fungal consortium, Rhizobium and NPK

A. Experiments after 4 days of inoculation

B. Experiments after 15 days of inoculation

PLATE 3 Experiment with AM fungal consortium, Rhizobiurn and NPK

A. Experiments after 30 da.ys of inoculation

I

R Experiments after 60 days of inoculation

any chance of water stress (Borse et al., 2002). Well water with poor nutrient

content was used for watering all the test plants through out the experimental

period.

3.4.3.(e). Sampling

Sampling was done on 30th (Plate 3A) and 60th (Plate 3B) day after

inoculation. The sampling procedure was the same as that described for

evaluation of effective AM fungal type and interaction with Rhizobium. The

harvest data was taken on 68th day after sowing. The soil samples were taken

for estimation of nitrogen, phosphorus and potassium left in the soil after

harvest. The soil sampling was also done after harvest for estimating the total

number of spores. The growth parameters were analyzed for each plant after

each sampling. The leaf tissues were collected for various analyses. One gram

of fresh leaf was taken from each plant for estimating total chlorophyll

content.

3.4.4. Procedure for analyses

3.4.4.1. Study of soil parameters

The soil from different sampling was analyzed for soil fertility

parameters such as pH, N, P and K following standard methodology.

3.4.4.1.(a). Measurement of soil pH

Ten gram fine soil was taken in a 50ml beaker. 25 ml distilled water

(1:2.5) was added to it and stirred well. It was allowed to stand for 30 minutes,

with occasional stirring using a glass rod. Measurement of pH was done using

pH meter (Micro pH system 362 Systronics).

3.4.4.1. (b). Estimation of soil Nitrogen

Micro-diffusion method (Sparks, 1996) was employed for quantifying

soil nitrogen. The soil samples collected from the root zone of plants were

52

dried, powdered and sieved. 2g of powered soil was taken in an injection

bottle and 2ml of 0.32% KMNO4 and 2ml of 40% NaOH solutions were

added. After mixing it well the bottle was kept inside a plastic bottle, which

contained 10ml boric acid - mixed indicator. The plastic bottles were tightly

closed and kept in an incubator for 18 hrs at 38o C. The injection bottles were

removed after the release of nitrogen as ammonia from the soil. The bottles

were rinsed from outside with distilled water into the same plastic bottle. The

ammonia absorbed by boric acid resulted in bluish colour. It was titrated

against 0.01N H2SO4. From the titre value the amount of N was estimated.

3.4.4.1.(c). Estimation of soil Phosphorous

Estimation of soil phosphorus was carried out as per Bray and Kurtz

(1945). The soil samples collected from the root zone of plants were dried,

powdered and sieved. 5 grams of soil was taken to extract the phosphate using

Bray and Kurtz reagent. Soil samples were shaken well with Bray and Kurtz

No. I extracting solution for ten minutes in a reciprocating shaker and filtered

through Whatman No. 40 filter paper. The filtrate was used for estimation

with Ammonium molybdate reagent and ascorbic acid. 5ml of the filtered

extract was pipetted into a 25ml volumetric flask. After dilution with distilled

water 4ml of reagent B was added. This was made up to 25ml. After ten

minutes a blue colour was developed, which was read at 730-840nm in a

spectrophotometer (Visible spectrometer-Visican 167 Systronics). The

absorbancy was plotted against standard graph prepared with KH2PO4.

3.4.4.1.(d). Estimation of Potassium

Estimation of soil K was carried out as per Jackson (1973). Soil

samples collected from the root zone of plants were dried, powdered and

sieved. 5 g of soil was taken in a shaking bottle to estimate available

potassium. 50 ml neutral 1N ammonium acetate solution was added and

shaken in a reciprocating shaker for 5 minutes. The sample was then filtered

through Whatman No. 42 filter paper. 5 ml of the filtrate was pipetted into a

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50 ml volumetric flask and made up to 50 ml with 1N ammonium acetate and

K was estimated using a flame photometer (Flame photometer Model 128

Systronics) using K filter.

3.4.4.2. Plant analysis

3.4.4.2.(a) Estimation of total chlorophyll

Chlorophyll content of the leaves indicates photosynthetic ability. For

estimating total chlorophyll of the leaves 1g of fresh leaf was taken and

ground with 80% acetone in a mortar using pestle. The extract was filtered

through Whatman No.1 filter paper. The residue was washed repeatedly with

80% acetone till the residue on the filter paper became colourless. The

volume of the filtrate was made up to 100ml using 80% acetone. The

absorbancy of the chlorophyll extract was read at 645 and 663nm in a

spectrophotometer with 80% acetone as blank. The amount of total

chlorophyll was calculated following Arnon (1949).

3.4.4.2.(b). Total leaf area and leaf area index

Total leaf area of a plant is an index of its photosynthetic ability. It was

measured using computer with the help of MATLAB software, employing the

provision for image processing. The leaves were scanned and the images were

stored in the computer. The measurement of the total leaf area was done using

the computer software. The steps involved in the measurement of leaf area

were:

Scaned the image of a leaf into the computer and saved it as ‘leaf.tif’. When

the program functioned, MATLAB first displayed the original image on the

monitor.Then it showed the black and white equivalent image on the screen.

Then it displayed the complemented image. And finally it calculated the area

and displayed it.

In the graphical method of calculating leaf area, the probable error per

unit boundary length was . In the image processing method, the error 25mm±

54

was practically zero, since the area calculation involved only the counting of

pixels. Leaf area index (LAI) is an indication of the ability of utilizing light. It

was calculated from the equation of Watson (1958).

3.4.4.2.(c) Tissue preparation for chemical analysis

The leaves were dried in hot air oven at 650C after recording the fresh

weight, till it showed constant weight. It was powdered in a Cyclotec mill and

kept in a desiccator. The dried and powdered leaf samples were used for

estimating tissue N, P and K.

3.4.4. 2 (d). Estimation of tissue Nitrogen

Micro-Kjeldahl method (AOAC, Washington, 1978) was employed for

estimating tissue N. Well ground leaf sample (0.5g) was taken in a digestion

tube of the digestor and mixed with 10 ml concentrated sulphuric acid. A

tablet of potassium sulphate- copper sulphate mixture was added as catalyst

and was placed in the digestor. The temperature of digestion was set to 410oC

by adjusting the thermostat. The digestion tube containing the digested

sample was distilled automatically in the Kjeltec 1026 distilling unit. The

receiver flask containing 20 ml of 4% boric acid along with 3-4 drops of

mixed indicator (cherry red colour) was kept for collecting the distillate. The

ammonia liberated from the plant sample during distillation collected in the

receiver flask changed the colour to blue from cherry red. The distillate

collected was titrated with 0.1 N H2SO4 changing the colour back to cherry

red. The titre value was noted and calculations were made.

3.4.4. 2.(e). Tissue Phosphorous

For estimating the tissue P, Vanado-molybdate yellow- colour method

(Jackson, 1973) was followed. 0.5gm of the dried plant tissue was weighed

accurately in a digestion tube. Cold digestion done by adding triple acid

mixture and it was kept overnight. Triple acid was made of concentrated nitric

55

acid, perchloric acid and sulphuric acid in the ratio 10:4:1. After the precold

digestion the digestion mixture was heated at 180 to 200 degree Celsius for 2

hrs or till the digestion mixture became a clear solution. The digest was made

up to 100ml. 5ml of made up digest was taken in a standard flask and 5ml of

Vanado-molybdate reagent was added. The volume was made to 25ml with

distilled water. The yellow colour developed was read after 10 minutes at 490

nm in a spectrophotometer. Standard graph was prepared and calculations

were done.

3.4.4.2.(f). Estimation of tissue Potassium

The triple acid digest was used for estimation of K in the sample. The

made up extract was directly read in the flame photometer by changing the

filter for K. Percentage of K in the tissue was calculated from the photometer

readings.

3.4.4.(g). Estimation of crude protein content

The crude protein content of the plants was estimated from the

percentage of N in the tissue as per Horowitz (1960).

3.5. Statistical analysis The data obtained from each analysis was subjected to statistical analysis.

Analysis of variance (ANOVA), correlation and critical difference were analyzed

using GENSTAT-6 software.

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