project on woodely land

DECLARATION

I do herby declare that this project titled “Detection of Enzyme Amylase From Woolly Aphids”. Submitted by me to the department of Chemistry New Arts, Commerce and Science College, Ahmednagar is the product of my own efforts. To the best of my knowledge and belief that this report is based on reliable scientific measurements. This dissertation has not been submitted to any university for the award of the any degree.









Date:- Chopde R.M.
M.Sc.-II
(Bio Chemistry)
New Arts, Commerce
& Science College,
Ahmednagar.

Place:-Ahmednagar












ACKNOWLEDGMENT



It is indeed a great pleasure and moment of immense satisfaction for me to express my deep sense of gratitude towards project guide Dr.M.V.Padul for ken inspiring interest and Dr. A.D. Chaugale invaluable guidance at each stage, during the entire course the entire course of my project.

I am grateful to Prof. Humbe A.M.head of chemistry department, for providing the requisite laboratory facilities without which this project could not have taken the present shape.
I am grateful to Prof. Mr.Bladye & Prof. Mrs. Shaikh R.R. my classmates and laboratory assistants for their timely help and suggestions.

Last but not the least I thank my parents my friends for their everlasting support in all my endeavors.






Mr. Chopde Ramakant Maruti.













INDEX



SR.NO. TOPIC PAGE NO.
1 Abstract
2 Introduction
3 Life Stages of C. lanigera
4 Material and Method
a) Apparatus
b) Chemical
c) Method
1. Agar diffusion assay technique
2. Comparative study between woolly aphide. Fungal, salivary, amylase at different pH tem. By DNS method
3. Biogel chromatography
4. Pricipitation Polyacrylamide gel electophoresis & Agrose gel Electrophoresis.
5 Result and Discussion
6 References









ABSTRACT

The amylase activity of woolly acid aphids was determine by agar plate diffusion assay method. Amylase activity was also determined different pH by agarplate diffusion method. Comparative study between woolly aphids, salivary, fungal amylase was the different pH-temp. by chemical method that is by DNSA method.
Protein concentration determined by Lawry method. substrate concentration also determine. Biogel chromatography was also done precipitation was carry out higher activity shown by fraction was used to determine further study. Pattern of protein was determine by poly acrylamide gel electrophoreses and agarosse gel electrophoresis.









NOT AFFECTED LESS AFFECTED HIGHLY AFFECTED






INTRODUCTION


Sugar woolly afide has been recently reported inout break proportion from westerm and southern India. Through the pest was first reported from west bangol in 1958 and later from other part of India. Resent survey in part of past affected area of maharashtra and Karnataka.
Sugar is a universal sweetening agent and sugarcane (Saccharum affleinarum L.) is the primary age old source of it.It is mainly cultivated in tropics in the world, but in India it is grown even under subtropical areas.
Sugarcane occupies an area of 42.97 lakhs hectares with a production of 278.56 mt(Anonymous, 2003a) the area in Maharashtra was 5.9 lakhs hectares and annual production of 45.14 mt (Anonymous, 2003 a.) Thus Maharashtra account for 15.4 % of the total production in India.
It is a prerennial crop and occupies the land for 12-18 month. It is propagated by setts. Planting of sugarcane is done in 3 seasons, viz. Suru ( Dec-jan plating), adsali ( June-July planting) and preseason ( Oct.-Nov.planting) in The crop requires a long warm growing season with adequate amount of rainfall, Maharashtra. a fairly sunny and cool conditions, nut frost free condition during ripening and haresting freedom from stormy and severe winds.
Sugarcane is damaged by about 288 species of insects and non insets (David and Nandagopal 1986) and tissue borers, whte grubs,white flies, rodends, mealy bugs, pyrilla, scale insects etc. are of an economic
Important. Avasthy (1977) reported losses to the extent of 20 and 15 percent in cane yield and sugar recovery, repectively, due to the ravages of the pests. Recently,in Maharashtra.state during July-2002,an epidemic of sugarcane wooly aphid, Ceratovacuna Lanigera Zehnter was noticed in sangli, kolhapur and Satara districts and later on spread in parts of Solapur, pune and Ahmednagar districts. By September, 2002 it spread to neighboring Karnataka state. (Anonymous,2003b0 upto February 203, 1.32 lakh hectares of sugarcane crop was found affected by this pest in maharashtra.
In other countries, ceretovacuna lanigera (Takara and Auma, 1968, hill, 19930 C japonica (Anonymous, 202 and C. graminum (David and nandagopal, 1986) are the common species of white woolly sugarcane aphids damaging the sugarcane. In Indonesia,Farina (19940 observed the loss due to heavy incidence of C. lanigera by 26% in sugarcane yield and 24% in sugar content.

NATURE OF DAMAGE:-
Initial aphid infestation was seen on the under surface of leaves along the midrib and then over the entire under surface, covering it with flocculent, waxy secretion. Co-pious honeydew excretion often covers the entire upper surface of the leaves, leading to growth of sooty molded. (lopez A.W.) and continuous infestation leads to reduction in the length. Circumference, weight and sugar content of the stalk, and in susceptible varieties in vienam,( Vietnam 1963, P 266) loss in tonnage as well as sugar recovery (Tripathi G.M.) Gupta and Goswami assessed the effect of 25 and 100% alphid infested leaves on some yield and quality parameters of sugarcane and found that 100% infested had detrimental effects on the length (11% reduction), girth (3.5% reduction) weight (16.6 % Reduction) length of internodes (18.4% reduction) and width of leaf (4.9% reduction) juice quality parameters also exhibited considerable reduction. The percent reduction in sucrose, brix, glucose, purity and commercial cane sugar (CSS) was 53.3. 32.3, 25.3, 31.7, and 64.0 respectively. Preliminary studies on loss estimation being conducted at University of agricultural Sciences, Dharwad indicate adverse effects of aphid infestation on yield and quality parameters(Patil R.K. Lingappa S.)

LIFE STAGES OF C. LANIGERA

Systematic position:
Scientific name of species : Ceratovacuna Lanigera zehnt.
Family : Pomphigidae.
Superfamily : Aphidoidea
Order : Homoptera
English (Common) name : White Woolly
Marathi (Local) Name : Pandhara Lokari Oosmava
Obeservation on duration of different life stages of C.I. lanigera are follows

First instar nymph

The first instar nymphs are yellowish or greenish yellow in colour. It is very active and moves fast on the surface of leaf when exposed to sunlight. A pair of hollow tube is present at the posterior end of the abdomen oat the firth segment and is mainly used to secrete the defensie fuluid. The average length of the 1st instar was 0.87 mm and the width was 0.36 mm. ghosh (1974) reported that early instar nymph length ranges from 0.82-096 mm and width ranges from 0.3-0.45 mm.

Table duration of nymphal instars of C lanigera
Nymphal instars (Days)
Total
Duration
(Days)

I
II
III
IV
6 7 7 9 29

Patil (*20020 reported that first instar nymph length averaged 0.77 mm and width between 0.27-0.38 mm. the average duration of the first instar nymph was found to be 6.33 days.

Second instar nymph

The second instar nymphs are found to be less active and brownish in colour with black marking on the body. The white wooly filaments are absent. The average length of the 2nd instar was 1.17 mm and was 0.57 mm.
Patil (2002) reported that second instar nymph length as 1.23 mm and width ranged between 0.3-0.46 mm.
The duration of the second instar was found to be 6.66 days.




Third instar nymph

The third instar nymph is partially convered by white woolly filaments. These filaments are dense at the posterior end of the abdomen and also covered thorax portion but not head. The white wooly filaments are very dry, soft, nonpolar and dydrophobic. The average length length of the third instar nymph was 1.81 m and width was 1.05mm.
Patil(2002) reported the average length of this instar nymph as 1.83 mm and width as 0.46-1.07 mm.
The duration of this instar was found to be 7.99 days.


Fourth instar nymph

The fourth instar is covered with dense wooly filaments. They are sedentary on the leaf. A four segmented antenna is seen which was very short and measured 0.15 mm length. The length of fourth instar nymph was 1.98 mm and width was 1.02 mm.
Patil (2002) reported that length of fourth instar nymph as 2.09 mm and width as 0.47-0.78 mm.
The duration of this instar nymph was found to be 8.22 days.

Adult

The alate form adult emerged after fourth moult is blackish in colour, two pairs of transparent wings with clear wing venation are observed. Wings expanse of female is 2.7 mm in length and 1.2 mm in width. Adult possesses a pair of compound eyes. The winged adult was 2.00 in length and 1.0 mm in width.
Apterous forms lived for 30 days and alate forms lived for 10 days. The alate forms were responsible for the spreads for colony to new areas.
Pan and yang (1989) reported the longevity of adult from 32 to 57 days. Cheng et al. (2000) reported the longevity of adults as 20.5-24.1 days.

Life span

The nymphs undergo 4 instars and average of 29 days was required to complete the four instars on potted sugarcane plants. Total adult longevity was 30 days in case of apeterous aphids and 10 days in case of alate forms.
However, change et. Al. (20000 reported that a period of 15.8 to 16.5 days was required to complete nymphal stages.
Patil (20000 reported that life cycle is completed in one month period.


Sex Ratio

A number of aphids were critically observed and it was found that no sexual were recorded in the colony. The colony consists of females only and this was responsible for rapid increase in number.
Ghosh (1974) reported that no sexual were reported and the results were found to be in conformity with it.
In view of the above, it became indispensable to have a through knowledge of the pest so as to frame effective control measures against it. The available literature showed that so far into much work has been reported on this pest in India. It was therefore felt necessary to enzymatic study of woolly aphid










MATERIALS AND METHODOLOGY

Materials:-
A) Apparatus:-
1. mortar & pestle
2. Knife or Sissor
3. Petri Plates
4. Measuring Cylinder
5. Conical Flask
6. Beakers
7. Test Tube
8. Colorimeter
9. Spectrophotometer
10. Cold Centrifuge
11. Refrigerator
12. Ependrop tube
13. pH meter
14. Micropipett
B) Chemical:-
1. Soluble starch powder
2. agar Powder
3. Dintiro Salicylic Acid
4. Naoh
5. Glycine
6. Sodium Acetate
7. acetic Acid
8. Monobasic Sodium Phosphate
9. Dibasic Sodium Phosphate
10. Biogelp100
11. Disterase

METHODS:-
Collection of woolly aphids
Woolly aphids were collected from the rahuri sugar cane fields which were affected by woolly aphids. After collection of the woolly aphids were separated from the infected leaf and taken into separate dish. Then woolly aphids are preserved deepfreeze.

PREPARATION OF DIFFERENT PH BUFFERS:-
1. acetate buffer ( pH 4,5)
2. Phosphate buffer (pH 6,7,8)
3. Glycine NaOH buffer (pH 9,10)

EXTRACTION OF ENZYME FROM WOOLLY APHIDS:-
For this experient the 2 gm of woolly aphids is taken in seprate dish and 10 to 15 ml of buffer solution is added then the content is crushed in mortar pastle and the pest is prepared. Then after complete homogenation the sample is cold centrifuged the supernantant obtained is used for the further study of enzyme analysis. If some turbidity was observed then supernatant again centrifuge at high speed about 15000 rpm and time 30 min. the super natant filter by whatman filter paper.


CHECKING AMYLASE ACTIVITY BY DIFFUSION ASSAY TECHNIQUE:-
Starch and agar (3gm + 1.5 gm ) was taken in 100 ml distil water. Heat the solution and pour in to the petriplates. When the medium was solidified. After solidification the wells were prepared with the help of borer. After same time sample was added different wells with different pH. Enzyme extract then incubate 1 hour. The iodine solution was added in the petridish for staining after 4-5 min. the petridish is washed with D/W 3-4 times. Then incubating the petridish for 1 hour. The activity of aylase was seen at alkaline pH.



EXTIMATION OF AMYLASE ACTIVITY BY DINTRO SALICYLIC ACID METHOD REAGENT:-
a) Dinitro salicylic acid reagent (DNSA)
Dissolved 1 gram DNSA + 200 mg crystal phenol & 50 mg Sodium sulphate in 100 ml 1 % NaOH stored at 40 C.

Method:-
For estimation of amylase activity by DNSA method the starch is taken as substrate.
For the enzyme amylase, the activity of enzyme is estimated by checking optical density on the colorimeter.
REAGENTS TEST CONTROL
Enzyme extract 0.5 ml 0.00ml
Substrate (Sttarch) 1.00 ml 1.00 ml
Distilled water 0.00 ml 05 ml
Incubate at 37 c for 30 minutes
DNSA 1.00 ml 1.00 ml
Keep in boiling water bath for 10 minutes
After all addition ckecked the optical density on the colorimeter at 530 mm.

BIOGEL CHROMATOGRAPHY:-
Take 5 gm of biogel in 100 ml distil water and kept for one hour for the purpose of swelling. The coloum was properly cleaned with distil water the glass wool was wetted. And pour into the coloum with the help of long glass rod. The biogel was added to the coloum. The distil water was slowely added setting of gel at the bottom of coloum. The coloum was equillibriated with equilibration buffer pH-8 (phosphate buffer)
The one ml of crude sample is loaded on coloum with the help of pipette. The flow rate was adjested to 2 ml per 5 min. phosphate buffer was added help of pipette when fraction was collected. Each ependrop tube 2 ml fraction collected. Activity of each fraction was measured by DNSA method and protein concentration also measured.
Some number of fraction get highest activity they get separated from other and acetone preptation was carried out.
ACCETONE PRICIPITATION:-
The equal volume of sample and chilled acetone was mixed by drop by drop. After mixing the sample were placed in freeze for over night. The next day the sample were centrifugal at 12000 rpm for 15 min at 100 C. the supernatant was discarded and pellet was collected by dissolving 1 ml buffer and sample used as electophorotic study.

Reagents:

1. Stock buffer for resolving gel (1.5 M Tris HCL pH 8.8) :-
Tris-hydrozymethyl aminomethane (23.64g) was dissolved in 50 ml distilled water and the pH adjusted to 8.8 by adding conc. HCL drop wise the final volume was made up to 100 ml and the solution was stored in refrigerator.
2. Stock buffer for stacking gel (0.5 M Tris-HCL, pH 6.8)
Tris (7.88g0 was dissolved in 50 mlo of distilled water and the pH was adjusted to 6.8 by adding conc. HCL drop. Final volume was made to 100 ml using distilled water.
3. Electrode (tank) buffers (pH8.3):-
9.0g Tris, 42.3 g glycine were dissolved in distilled water to make 3 liters (electrode buffer was used for 2-3 subsequent runs)
4. Staining Solution:-
10 ml of 1 per cent coomassie blue prepared in methanol solution starining solution was filtered two to three times before use.
5. 30% acryl amide for stacking gel:-
75g acryl amideand 2 g bis-acryl amides were dissolved in distilled water. Volume was made to 250 mlo by addition of istilled water.

Apparatus & equipments :-
1. The vertical slab gel electrophoresis apparatus of atto with 16*16 cm glass plates.
2. Gasket.
3. Well comb & power supply.


Chemicals:-
The following chemicals (analytical grade) ware used for preparation of different solution
1. Acryl amide ”Electrophoresis grade “
2. Bisacreylamide”Electrophoresis grade “(N’N’ methylene bisacrlymide)
3. Tris ( hydroxymethyl aminomethane)
4. Glycine
5. HCL
6. Glycerol
7. Ammonium persulphate
8. TEMED (N, N, N’N’ tetranmethylmethylene diamine)
9. glycial acetic acid
10. Coomassie brilliant blue
11. bromophenol blue

Preparation of Gel
The gel plates ware cleaned and dried and cassettes ware assembled as per the instruction.
1. Separating Gel
The following solution ware mixed (10%)
Buffer pH 8.8 :7.5ml
Water : 12.3ml
30% running gel acrylamide : 9.9ml
5% ammonium persulphate : 0.15ml
TEMED : 0.02

(TEMED was added just before pouring gel)
All the reagents were mixed well and poured between the plates of the cassette.care was taken, so that no air bubble was trapped in the gel solution. The cassettes was filled ¾ with 3-4 cm left from the top the polymerized gel solution was overlayed with butanol and the gel was allowed to polymerize (it took about 30-60 min)
2. Stacking gel (4%)
After the polymerization of the separating gel the butanol layer was removed using filter paper and the cassette was blotted. The following stacking gel mixture was carefully poured into it.

Tris buffer 6.8 : 1.25 ml
Water : 3.075 ml
30% stacking gel acryl amide : 0.67 ml
10% ammonium per sulphate : 0.025 ml
TEMED : 0.005 ml

(TEMED was added just before pouring the gel)
After puring the stacking gel solution an acrylic comb having 11 teeth or wells was set without trapping any bubble. The gel was allowed to polymerize (it look about 10-15 min,)
After polymerization of gel the comb was carefully removed from the gel without disturbing the shape of wells. The sample wells were washed with tank buffer to remove the unpolymerised material. The tank filled with an appropriate volume of tank buffer solution.

ELECTROPHORESIS:-
The gel casstte was fixed into the electrophoresis unit. An aliquot soluble protein (enuivalent of 100 mg) was loaded to each well w8ith help of micro syringes. The gel plates were fixed over cathode chamber cum heat exchange with clamps and screws. The cathode chamber was placed into the4 tank. The upper tank was filled in with power supply with the anode connected to the lower reservoir and cathode to upper reservoir. The electrophoresis was conducted at 35 mA till the samples migrate into the running gel and subsequently at 60 mA.
The unit was disconnected from the power supply and cassette was removed from the electrophoresis tank as soon as the tracking dye reached the bottom of gel.




Fixing and staining the gel:-
The cassette was removed from the unit and the gel was taken out gently. Gel was placed in a staining tray and sufficient staining solution was poured of cover the gel uniformly. It was incubated for 6 hrs. And the stain was decanted, rinsed with water. To clear the gel back ground detaining in water was followed for a day or two. The pattern of staining drawn as well as photographed.

Agarose gel electropheresi:
Principle:- Agarose gel electophoresis refers to movement of charged particle/ molecules under the influence of electric field.
Agrose gel is more porous and used to separation of large particles. In this method charge molecules moves to the charged when electric field supplied. This movment is depend on the size of molecules and molecular weight smaller size protein molecules move faster than larger size molecules.
Procedure:-
1. Phosphate buffer (0.2 m) pH-8 is prepared about 200 ml with distil water.
2. 0.5 gm of agarose was dissolved in 50 ml distill water.1% agrose gel from it dissolved by heating to form clear solution.
3. Above containt cooled at 600 C and pored in to tray and comb was inserted in to it. Precautions were taken that no air bubble should enter.
4. Gel thickness were made up to 4 to 5 mm. and agarose solution were poure and allowed to cool comb was removed gentle when agar solidified.
5. phosphate buffer poured into tank to immerse the gel by about 5 ml
6. Power set was connected.
7. sample is loaded in well in derived order
8. Voltage was set to 50 V and switch was start of the power supply.
9. Electrophoresis was run until dye migrate to the other end of the gel ( approx.1 hr)
10. Power supply was turn off. The agrose gel was removed from the electro phorsis tank.
11. Kept in to the staining solution for 1 to 2 hr.
12. It was distained by water or solution.
13. the band pattern was observed

RESULT AND DISCUSSION:-
1. Amylase activity

pH7 pH8 pH9
The enzyme amylase present in woolly aphides activity is checked at pH 4, 5,6,7,8,9,10 woolly aphids amylase shown activity in alkaline pH 7,8,9 but pH-8 show maximum activity. pH-8 get maximum zone of activity.

2. Protein Concentration

NO ENZYME(ml) WATER(ml) LAWARYREAG. F.P. O.D.
Reg. constant one 0.0 1 3 0.5 0.0
Enzyme const. 2 0.1 4.4 0.0 0.0 0.2
Test 3 0.1 0.9 3 0.5 0.34
Test 4 0.1 0.9 3 0.5 0.34

O.D. = Test – Enzyme control
= 34-2
O.D. = 32
O.D. plotted on the standard graph
1 ml sample = 124mg/ml
But sample is diluted with distil water up to 10 ml.
1 ml = 124 µg X 10
I.e. 1 ml = 1240 µg/ml
I.e. 1 ml = 1.245 mg/ml
1 ml = 0.00124 gm/ml
i.e. 1 gm woolly aphide contain = 0.00124 X 10
i.e. 1 gm contain = 0.0124 gm/ml

3. AMYLASE ACTIVITY BY DNSA METHOD:-
Standard Graph for Activity by using DNS method.




O.D.



Constration of Maltose (µgm)

A) Substrate Concentration:-



O.D.




Substrate Cons.

Substrate conc in % O.D Activity(µgm/ml/min)
2 0.73 96
1.8 0.73 96
1.6 0.73 96
1.4 0.73 96
1.2 0.66 176
1 0.54 144
0.8 0.48 128
0.6 0.34 89.33
0.4 0.27 70.33
0.2 0.18 48

Maximum substrate concentration for woolly aphids amylase is 1.2%
Graph shown increase any activity with increase in substrate const. but further steady state observed.
B) Different pH with comparative study between salivary, fungal, woolly aphides amylase


pH Woolly Aphid Activity(µgm/ml/min) Fungel Activity(µgm/ml/min) Salivary Activity(µgm/ml/min)
4 0.18 48 0.64 171.66 0.65 173.33
5 0.41 109.33 0.79 212 0.7 186.60
6 0.58 154.66 0.8 213 0.8 213
7 0.64 171.66 0.61 162.66 0.65 173.33
8 0.71 189.33 0.43 154.66 0.6 160
9 0.42 105.33 0.16 41.33 0.55 146.66
10 0.36 96 0.15 40 0.51 122.66

In above graph shows woolly aphids amylase show maximum activity at pH-8 salivary, fungal amylase shown maximum activity at pH-6.
The graph shown the woolly aphides amylase is different from salivary and fungal amylase.

C) Different temperature with comparative study between woolly aphids salivary, fungal amylase.

Temp Woolly Aphid Activity(µgm/ml/min) Salivary Activity(µgm/ml/min) Fungel Activity(µgm/ml/min)
20 0.16 41.33 0.22 60 0.24 64
30 0.2 53.33 0.24 64 0.24 64
40 0.22 60 0.28 74.66 0.28 74.66
50 0.18 48 0.26 69.33 0.25 66.66
60 0.16 41.33 0.19 50.66 0.21 56
70 0.11 28 0.16 41.33 0.18 48

The woolly aphids, salivary, fungal amylase shown maximum activity at 40o C temperature but at higher temperature suddenly decrease the activity. It clear that enzyme is not thermos table..



4) BIOGEL CHROMATOGRAPHY:-

The above graph peak has shown maximum activity. These fractions is collected and carry out acetone precipitation.

5) ELECTROPHORESIS STUDY:-

Crude sample
Band Accetone Preciption Sample
band







a) Electrophoresis pattern of protein is woolly aphide shown only one band in chromatography fraction but crude sample get diffused pattern of band.


b) Agarosegel Electrophoresis:-


Crude Sample Band with
Diff Concentration




in agarosegel Electrophoresis load crude sample but not get clear band some diffused band observed

6) Molecular weight determination:-













REFERANCE

1. Ghosh A.K, Homoptera: Aphidoidea, 4, Subfamily phloemyzinae, Anoeciinae and Hormaphidinae. In the fauna of India and adjacent countries, Zoological survey of India, kolkata, 1988 p 429
2. Lingappa, S. Kulkarni. K.A. Patil R.K. Thippanavar, P.S. and shekhappa, status of woolly aphid ceratovacuna lanigera zehntner on sugarcane in Karnataka. Paper presented at brain storming session on sugarcane woolly aphid, 10 june 2003.
3. Gupta, M.K. and Goswami, P.K. incidence of sugarcane woollyu aphid and its effect on yield attributes and juice quality. Indian sugar, 1995, 44, 883-885. Lopez, A.W.Ann. Rep. Res. Bur. Philipp. Sugar assoc, Entomology department report, 1931, p 273
4. Anonymous, annual work progress report on crop improvement program of rice, sugarcane, vegetable and field crops, directorate of rural Affairs, Vietnam, 1963, p, 263
5. Tripathi, G.M. Record of parasite and predator complex of sugarcane woolly aphide, ceratovacuna lanigera Zehnter in Nagaland India sugar, 1995, 44, 883-885.
6. Raychaudhari. D. N. food plant catalogue of India aphidede, aphidological socity of India, kolkata, 1984, p 188.
7. Mote U.N. & Puri.S.N., present status of sugarcane woolly aphid, ceratovacuna lanigera, a new pest problem on sugarcane in maharashtra. Paper presented at brain stroming.
8. David & Nandagopal, study of pest problem on sugarcane in India 1986
9. Avasthy, Study on losses percent in cane yield and sugar recovery due to the ravage of the pests.