MATERIALS AND METHODS

Arbuscular Mycorrhizal (AM) fungi is one of the most important microbes of soil that form symbiotic associations with most of the terrestrial plants on the earth. These fungi are chiefly responsible for Phosphorus (P) uptake. Arbuscular mycorrhizal fungi are of considerable interest because of their archaic existence, ability to form symbiotic associations with 85% of plant taxa and their potential use as a biofertilizer to increase yield of crop and tree species. Hence, an attempt was to study the effect of AM fungi on the growth and yield of Abelmoscus esculentus. SOIL SAMPLE COLLECTION The soil samples used for the isolation of Arbuscular mycorrhizal fungi were collected from Soukku, Banana, Sugarcane and Coconut field at Nagercoil, Tirunelveli, Tenkasi and Kanyakumari. Four samples were collected from each sampling site and mixed to form a composite sample. The collected soil samples were stored at 4C for two months for spore development. ISOLATION AND QUANTIFICATION OF SPORES The soil samples were analyzed for AM fungal spores, following a wet sieving and decanting procedure (Gerdemann, 1995). 250gm of soil was mixed in 1000ml of water and allowed the heavier particles to settle down for few seconds. The liquid was poured through coarse sieve (750 mm, 450 mm, 250 mm and 45 mm) to remove large pieces of organic matter. The filtrate was mixed with some more quantity water, well shaked and allowed heavier particles to settle for few more seconds. The suspension was passed through a sieve fine enough to filter the desired spores. The materials retained on the sieve were washed to ensure that all colloidal material pass through the sieve. Small amounts of remaining debris were transferred to a petridish and examined under a dissecting microscope. (Leyva et al., 2008)

CARRIER BASED INOCULUM PRODUCTION

The isolated spores were used to establish pot cultures, since, soil inoculums are considered to be more rapidly infective than spore inoculums. AM fungi are propagated in pot cultures by placing the mycorrhizal inoculums with sterilized soil. Onion (Allium cepa) was used as a host plant to propagate the AM fungal spores for two months. The presence and abundance of AM fungi propagates are constantly monitored. At the end of two months, soil was stopped watering and the cultures were allowed to dry slowly. The mycorrhizal inoculum was harvested and stored, later used it for plant inoculation. For quality control of mycorrhizal inoculum, evaluation of the proportion of root length colonized by AM fungi (percentage of mycorrhizal colonization) isolation and quantification of spores per gram of soil was done (Corkidia et al., 2008).

ROOT SAMPLE COLLECTION For mycorrhizal assay, root was collected carefully, by excavating from soil, assuring sufficient terminal feeder roots attached to lower order roots.

CLEARING AND STAINING SPECIMENS The roots were cut into small pieces (0.5 cm -1.0 cm) and heated/boiled in beaker containing 10% potassium hydroxide (KOH) solution for 20 minutes to 1 hour till it become soft in a well-ventilated exhaust hood. Added KOH solution clears the host cytoplasm and nuclei, which readily allows stain penetration. Pour off the KOH solution and rinse the root sample in a beaker with at least three complete changes of water or until no brown color appears in the rinsed water. Do not agitate the root vigorously. Cover the root in the beaker with alkaline H2O2 (NaoH-3ml; H2O2 (10%) 30 ml; water-567 ml) and keep it room temperature for 10 to 20 min or until roots are bleached. Rinse the root in beaker thoroughly using at least threecomplete changes of tap water to remove H2O2 Cover the root in the beaker with 1% HCL and soak for 3 to 4 min and then pour of the solution. Do not rinse after this soak because the specimens must be acidified for proper staining. Cover the root in the beaker with 0.01% trypanblue lactic acid staining solution and warm it again in beaker for 10 minutes. (Lactic acid solution; lactic acid875 ml, glycerol-63 ml; tap water-63 ml; trypanblue-0.1 g). Remove excess of stain and cover

the root with above solution without trypanblue for destaining and mycorrhizal assay can be done (Khadge., 1992). Mycorrhizal infection can now quantify in the samples. SOIL ANALYSIS The four different soil samples were used for the pot experiment to analyze the effect of AM on the physiological and biochemical changes on plant They were collected from Nagercoil, Tirunelveli, Sivakasi, Thenkasi and the samples were labeled asS1, S2, S3 and S4 respectively.

NITROGEN, PHOSPHOURS AND POTASSIUM ANALYSIS OF SOIL SAMPLES The pH nutrient analysis of soil samples (S1.S2,S3 and S4) such as Nitrogen, phosphorus and potassium were analyzed in Regional Research Institute at Kovilpatti.

DESIGNING OF POT EXPERIMENT To assess the effect of AM fungi the pot experiment was selected. Four different types of soil samples were taken in pots and the seeds were sown in the sterilized soil. An AM fungus was inoculated as the soil containing AMF (soil inoculums) used as an inoculum. The plants were analyzed for various parameters after 45 days of germination.

EFFECT OF ARBUSCULAR MYCORRHIZAL FUNGI ON MAIZE PLANT ON FOUR DIFFERENTSOILS The effect of AM fungi on the physiological and biochemical changes on plant grown in different soil samples were analyzed after forty five days of seedling.

DAY OF EMERGENCE After seedling the day of emergence was noted for all the plants grown in different soil.

ROOT INFECTION The percentage of root infection was calculated according to Kaushik et al ., 2008.

MEASUREMENT OF ROOT LENGTH To measure the root length of plant, the plants were uprooted carefully from soil, washed with water and its length was measured. An average value of the root was taken in to consideration and expressed in centimeter (cm).

MEASUREMENT OF SHOOT LENGTH To measure the shoot length of plant, the plants were uprooted carefully from soil, washed with water and its length was measured. An average value of the shoot was taken in to consideration and expressed in centimeter (cm).

EFFECT OF AM ON FRESH WEIGHT AND DRY WEIGHT OF PLANT

The plant parts are collected and he fresh weight of the plants was calculated. After drying the plants in oven, dry weight of the plants was calculated and expressed in gram (g).

EFFECT OF AM ON CHLOROPHYLL CONTENT OF PLANT 0.5 gm of control and AM treated leaf material were ground separately in 80% acetone and centrifuged at 5000 rpm for 5 minutes. Extraction with 80% acetone was repeated till the pellet becomes non-green color. The supernatants were polled after each centrifugation and the combined supernatant was used for the estimation of chlorophyll. Absorbance was read at 645 nm and 662 nm.

Thimmaiah. S.K., (1999) formulae were used for establishing the content of Chl a, Chl b and total Chlorophyll. Chl a (mg/g) =10.65 xA662 -2.35 x A645 Chl b (mg/g) =17.51 xA645 - 3.96 x A662

Total chlorophyll (mg/g) =6.69 x A662 -16.26 x A645

ESTIMATION OF CAROTENOIDS The carotenoids present in the acetone extract were quantified by measuring absorbance at 470 nm and amount of carotenoids were calculated by the following formula of 1000 x A4702.27x Ca 80.2 x Cb Carotenoids 229

ESTIMATION OF ANTHOCYANIN The estimation of anthocyanin content was determined by Thimmaiah. S.K., (1999). 100 mg fresh leaves were taken and ground with 10ml of ethanol and filtered through Whatman No 1 filter paper. One ml of extract along with Methanolic - Hcl was added to 1 ml of peroxide reagent and kept in dark for 15 minutes and the absorbance was read at 525 nm. Anthocyanin content was determined by O.D value (A525) / gram of leaf tissue.

ESTIMATION OF PROLINE Free proline from plant tissues may be selectively extracted in aqueous Sulphosalicylic acid and its concentration was measured using Ninhydrin method Thimmaiah. S.K., (1999). 200 mg of leaf sample was taken and ground with 10 ml Sulphosalicylic acid and filtered with what man 1 filter paper. 2ml of the extract along with 2ml acid Ninhydrin and 2 ml of glacial acetic acid was taken, mixed well and kept in boiling water bath (100C) for one hour. It was cooled in an ice for 5 minutes and added with 4ml of toluene. The tubes were agitated vigorously. The upper pink chromophore layer was separated and the absorbance was read at 520 nm. The amount of proline was estimated using proline as the standard

ESTIMATION OF PHENOL The phenolic content was estimated by Folin Ciocalteu method (Thimmaiah. S.K., (1999).100 mg fresh leaves were taken and ground with 10 ml of ethanol and filtered through what man no 1 filter paper. One ml of extract was added with 2 ml of 20% sodium carbonate. It

was shaken well and kept in boiling water bath for 1 minute and cooled. The blue solution obtained was diluted to 25 ml with water and the absorbance was read at 650 nm. The amount of phenol was estimated using Catachol as the standard.

ESTIMATION OF PROTEIN BY LOWREY ET AL METHOD (1951) (Thinmmaiah.S. K.,1999) Reagents 1. Alkaline Copper mixture, 2. 0.1N NaOH 3. 10% TCA, 4. Folins Ciocalteu Reagent. 5. Procedure

100 mg of leaf sample of both control and treated plants were taken and homogenized separately with 10 ml distilled water with the help of mortar and pestle. The extract was centrifuged at 3000 rpm for 5 minutes. The supernatant was taken and the pellet was discarded. 1 ml of ice cold TCA was added with the supernatant and kept it in ice for 10 minutes. The supernatant was centrifuged at 50000 rpm for 10 minutes. The pellet was taken and dissolved in 1 ml of 0.1 N NaOH. It was used as test solution. From this test solution 0.5 ml was taken and make up to 1 ml with distilled water. Add solution was mixed thoroughly and boiled for 10 minutes and kept in dark for half an hour. The absorbance was noted at 650 nm. Blank was also prepared with 0.5 ml of distilled water, 0.5 ml of folin reagent and add 5.5 ml of alkaline copper reagent. The amount of protein content was calculated from the standard graph of protein constructed with bovine serum albumin (BSA) as marker protein. (An aliquot of 100 mg BSA showed 0.197 absorbance of 650 nm)

CARBOHYDRATE ESTIMATION BY ANTHRONE METHOD (Thimmaiah. S. K., 1999) The carbohydrate content can be measured by hydrolyzing the polysaccharides into simple sugars using dilute hydrochloric acid and estimating the resultant monosaccharides. In

hot acidic medium glucose was dehydrated to hydroxymethyl furfural. This compound formed with Anthrone a green colored product with absorption maximum at 630 nm. Reagents 1. 2.5 N-HCL, 2. Anthrone reagent,

3. Standard glucose

Procedure Weighed 100 mg of each samples (control and AM treated) in to sampling tubes. It was hydrolyzed by keeping it in a boiling water bath for three hours with 5 ml of 2.5 N-HCL and cooled to room temperature. Then neutralized with sodium carbonate until effervescence cease. The volume was made up to 100 ml and centrifuged. Standard was also prepared by taking 0,0.2,0.4,0.6,0.8,1 ml of the working standard. The volume was made to 1 ml in all the tubes including sample tubes with distilled water. Then added 4 ml of anthrone reagent and heated for 8 minutes in a boiling water bath. Cooled rapidly and read the green to dark green colors at 630 nm. The standard graph was drawn and the amount of carbohydrate present in the sample tubes was calculated. Calculation

Amount of carbohydrate present in 100 mg of sample = mg of glucose / volume of Test sample x100 ESTMATION OF STARCH BY ANTHRONE METHOD (Thimmaiah. S. K., 1999) Reagents 1. Anthrone: Dissolve 200 mg anthrone in 100 ml of ice cold 95% sulphuric acid. 2. 80% ethanol

3. 52% Perchloric acid 4. Standard glucose: stock-100 mg in 100 ml water. 5. Working standard- 10 ml of stock diluted to 100 ml with

water (100 mg/ml)Method

0.1 to 0.5 gm of the sample was homogenized in hot 80% ethanol to remove sugars. It was centrifuged and retained the residue. The residue washed repeatedly with hot 80% ethanol till the washing did not gave color with Anthrone reagent. The residue was dried over a water bath. To the residue, 5 ml of water and 6.5 ml of 52% Perchloric acid were added and extracted at 0 c for 20 minutes. Centrifuged and save the supernatant. The extraction was repeated using fresh Perchloric acid. Centrifuge and pool the supernatant and make up to 100 ml. Pipette out 0.1 or 0.2 ml of the supernatant and make up to the volume to 1 ml with water. The standards were prepared by taking 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the working standard and make up the volume to 1 ml in each tube with water. 4 ml of Anthrone reagent was added to each tube and heated for 8 minutes in a boiling water bath. Cooled rapidly and read the intensity of green to dark green color at 630 nm.

Calculation The glucose content in the sample was found using the standard graph and multiply the value by a factor 0.9 to arrive at starch content. ESTIMATION OF NITROGEN BY MICRO-KJELDHAL METHOD Reagents 1.Conc.H2SO4 2.Mercuric oxide 3.Potassium sulphate 4.Potassium hydroxide- Sodium thiosulphate solution: Dissolve 600g of NaOH and 50 g of Na 2SO3.5H2O in water and make up to 1 liter. 5.0.2N standard HCL or H 2SO4 6.4% Boric acid solution: Dissolve 4g of H3BO3 in warm water and diluted 100 ml distilled water. 7.Mixed indicator solution:

Mix 2 parts of 0.2% methyl red in ethanol with 1 part of 0.2% methylene blue in ethanol. Methods 40-100 mg of finely powdered homogenous sample was taken into 30 ml digestion flask.2g K2SO4 and 90 mg of HGO, 2ml of Conc.H2SO4 Were added and mixed well. Boiling chips/glass beads were added and digested the sample over digestion rack. Cooled and add minimum quantity of water along the sides of the flask to dissolve solids and transferred quantitatively to the distillation apparatus with successive rinsing with water. A 100ml conical flask containing 5ml of boric acid solution with a few drops of mixed indicator in such a way that the tip of the condenser dipped inside the solution.10 ml sodium hydroxide sodium thiosulphate solution to the digest in the apparatus through the funnel and rinse with water. Distilled and collected the ammonia in boric acid. The color change from violet to green was an indication of ammonium absorbed. Rinsed the tip of the condenser with water and titrated the distilled sample against the standard HCl or H2SO4 until the appearance of original violet color as the end point. Run a blank digested similarly with an equal volume of water after washing the distillation apparatus by back suction with excess of water. Calculation %Nitrogen= (ml HCl in sample)-(ml HCl in blank) x Normality of acid x 14.01 x 100 Weight of the sample

EFFECT OF AM ON Ca AND Fe CONTENT The control and AM treated leaf sample were digested with concentrated nitric acid and 30% hydrogen peroxide and Ca, Fe content were determined by using Atomic Absorption Spectrophotometer (AAS) . SDS PAGE Reagents Separating gel (12%) 10 ml: Distilled H2O = 3.3 ml 30% acryl amide, bis acryl amide = 4 ml

1.5 M Tris (pH 8.8) = 2.5 ml 10% SDS 10% APS = 0.1ml = 0.1 ml

TEMED = 0.004 ml Stacking gel (5%)3 ml Distilled H2O = 2.1 ml 30% acryl amide, bis acryl amide = 0.5ml 1 M Tris (pH 6.8) = 0.38 ml 10% SDS 10% APS = 0.03ml = 0.03 ml

TEMED = 0.003 ml Sample loLoading buffer (10 ml) Tris pH (6.8) = 2.5 ml 10% SDS = 4 ml 100% Glycerol = 2ml -mercapto ethanol = 0.8 ml Bromophenol blue (0.1%) = 0.3 ml Distilled H2O = 0.4 ml Tank buffer Glycine = 36g Tris = 7.5g APS 10%: Dissolve 300mg of solid APS in 5ml of Distilled H2O. Staining solution: (200 ml):

Coomasive brilliant blue = 0.3 g Methanol (or) ethanol = 80 ml Glacial acetic acid Distilled H2O = 20 ml

= 100ml

Stock Acryl amide Mix 29.2 g Acryl amide, 0.8 g bisacrylamide and final volume is made upto 100ml. 1.5 M Tris- HCL (pH 8.8) Dissolve 18.15 g of Tris in 50 ml of Dis. H2O. Adjust the pH to 8.8 with HCL; make the volume to 100 ml. 10% SDS Dissolve 18.15 g of Tris in 50 ml of Dis. H2O and make the final volume equal to 10 ml. Procedure Thoroughly clean the glass plates and spacer then assemble them proper by hold the assembly together bulldog clips clamp in an upright position white petroleum gelly or 2% agar (meltated in a boiling water bath) is then applied around the edges of the spacers. To hold them in place and seal the chamber between the glass plates.Prepare the sufficient volume of separating gel mixture. Mix gently and carefully pour the gel solution in the chamber between the glass plates. Layered distilled water on the top of the gel and leave to set for 30 to60 minutes.

Prepare the sufficient volume of stocking gel mixture. Remove the water from the top of the gel and wash with a little stocking solution. Pour the stacking gel mixture, place the comb in the stacking gel and allow the gel to set (30-60 minutes). After the stacking gel has polymerized remove the comb without distorting shape of well carefully install the gel after removing the clips, agar etc in the electrophoresis apparatus. Fill it with electrode buffer and remove and any trapped, air bubbles at thebottom of gel.

Connect the cathode of the top and turn on the DC power briefly to check the electrical circuit. Plate can be kept cooled using a suitable facility. Heat generator during the run is dissipated and does not affect the gel and resolution. Prepare samples for electrophoresis, following suitable extraction procedure. Adjust the protein concentration in each sample using the 5-strength sample buffer and water in such a way that the same account of proteins (50-200 g) in a volume (25-50l) not greater than the sample well. Sample solutions in a boiling water bath for 2-3 minutes to ensure complete interaction between proteins and SDS. Cool the sample solutions and carefully inject it into a sample well through an electrode buffer. Marking the position of wells on the glass plate with a marker pen and the presences of chromo phenol blue in the sample buffer facilitates easy loading of the samples. Similarly load the a few wells with standard marker proteins in the sample buffer. Turn on the circuit to 10-15 mA for initial 10-15 minutes until the samples travel through the stacking gel. The stacking gel helps concentration of the samples. Then continue the run at 30 mA until the Bromo phenol blue reaches the bottom of the gel (about 3 hours). The gel may be run at a high current (60-70 mA) for short period (1 hour) with proper cooling. After the run is complete carefully remove the gel from between the plates &immerse in staining solution (3 hour)&uniform shaking. Transfer the gel to suitable container with at least 200-300 ml ofdestaining solution and shake gently. Destaining process should be stopped at appropriate stage the visualize as many bands as possible. Gel as can be photographed stored in polythene bags or dried in vacuum for permanent record.