Different microbial loads under system of rice intensification (sri) copy pdf copy

Different Microbial Loads Under System of Rice

Intensification (SRI)

Project works submitted to the VIT University in partial fulfillment of the

requirement for the degree of Master of Science in Applied Microbiology

Guide :- Dr. Godwin Christopher J. (Associate Professor, VIT

University)

Dr. Pabitra Banik (Associate Professor, A.E.R.U, ISI, Kolkata)

Presented by,

Ishita Das (12MSM0041)

MSc.Applied Microbiology

VIT University, Vellore, Tamilnadu

Agriculture is the noblest of all alchemy; for it turns earth, and even

manure, into gold……….

Conferring upon its cultivator the additional reward of health………

Paul Chatfield.

Introduction

System of Rice Intensification (SRI) is a cultivation practice for Rice that is

taken up in a different and more biologically enriched environment for growth.

SRI is based on the following principles:

�Young seedlings between 8-12 days old (2-3 leaf stage) are transplanted

to preserve potential for tillering and rooting ability;

� Careful planting of single seedlings rather than in clumps that are� Careful planting of single seedlings rather than in clumps that are

often plunged in the soil;

�Wider spacing at 25 cm x 25 cm. in square planting rather than in

rows;

� Use of cono-weeder/ rotary hoe/power weeder to aerate the soil as

well as controlling weeds;

1

�Alternate wetting and dry method rather than continuous flooding in the field;

� Use of organic manure or vermicompost / FYM.

KEY FEATURES OF SRI:

� Transplant young seedlings

�Reduce plant population

�Maintain aerated soil conditions

� Provide as much organic matter as possible to the soil� Provide as much organic matter as possible to the soil

�Actively aerate the soil

�Re-emphasize biology

�Rediscover the potentials of synergy and symbiosis

2

AIM :Different Microbial loads under system of rice intensification.

Objective :

� Rice cultivation by SRI technique.

� Sample collection from SRI and conventional rice cultivation field.� Sample collection from SRI and conventional rice cultivation field.

� Physicochemical characterization of collected soil samples.

� Isolation of microorganisms from collected soil samples.

�Macroscopic and microscopic characterization of isolates.

�Molecular characterization and identification of microorganisms isolated

from SRI soil samples ( DNA isolation, PCR, 16srDNA& ITS sequencing ).

3

METHODOLOGY(OVERVIEW)

pH, NPK value7. Molecular Characterization

1. East Field (Giridih)

2. Rice cultivation in SRI field

3. Sample collection

4

pH, NPK value7. Molecular Characterization

& Identification of isolated soil

microbes 4. Physicochemical

characterization of soil

5. Isolation of microbes

from soil

5. Isolation of microbes

from soil

6. Microscopic & Macroscopic

microbes

6. Microscopic & Macroscopic

Identification of isolated soil

microbes

7a. Genomic DNA isolation

7b. Polymer chain reaction (PCR)

7c. 16s rDNA& ITS Sequencing

Rice Cultivation by SRI Technique

Field Details :

� Place : Giridhi, Jharkhand

� Latitude : 23º 5’N to 24º 7’N

� Longitude : 86º 18’E to 86º19’E

� Design : Split Plot Design

�Main Plot Size : 7×7 m²

� Sub Plot Size : 1 × 7 m²� Sub Plot Size : 1 × 7 m²

�Main Plot bund Size : 0.75m

� Sub Plot bund Size : 0.5m

� Replication : 4

� Bund Size between two plots : 0.75m

� Season :Winter (Dec-Feb’2014)

� Normal RF : 33mm

5

Figure 1. Rice cultivation by SRI method

7

SRI Soil Sample

Total soil Sample = 15

Sample code details :

D1 = SRI methods

D2 = Direct Seedling Main Plot Treatment

D3 = Normal Transplanting

S1 = 100% of recommended inorganic fertilizers (120 : 60: 40 kg NPK/ha)

S2 = 50% inorganic + 50% organic (equivalent of N dose)

S3 = 100% recommended dose through organic source ( equivalent of N dose ) Sub Plot Treatment

S4 = 150% recommended fertilizer dose

S5 = No fertilizer (control)S5 = No fertilizer (control)

S1: UREA = 182g, SSP = 262g, MOP = 47g

S2: UREA = 91g, SSP = 131g, MOP = 24g, VC = 3.23kg/ COWDUNG = 8.4 kg Fertilizer Requirement per plot

S3: VC = 6.64kg/COWDUNG = 1608kg

S4: UREA = 273g, SSP = 393g, MOP = 71g

S5: = No fertilizer (control)

8

Physicochemical Characterization of soil samples

pH

Organic

Carbon

Available

Nitrogen

Available

12

Available

Phosphorus

Exchangeable

Potassium

9

Isolation of Microbes from collected Soil samples

Soil Sample

10Potato Dextrose Agar Nutrient Agar

Serial Dilution

Characterization of Isolates

MACROSCOPIC

MICROSCOPIC BIOCHEMICAL TEST

�Colony Morphology

�CFU Count

�Antibiotic Susceptibility Test

�Antifungal Activity Test

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�Lactophenol Cotton

Blue Statining

�Gram Statining

�Indol Test

�Catalase Test

�Citrate Utilization Test

�MR-VP Test

Molecular Characterization and

Identification of Isolates

� Genomic DNA Isolation.

�Agarose Gel Electrophoresis.

� Determination of the purity and quantity of DNA by

Spectrophotometric method.Spectrophotometric method.

� PCR (Polymerase Chain Reaction).

� 16srDNA Sequencing.

� ITS Sequencing for Fungus.

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Results & Discussion

Fields S1 S2 S3 S4 S5 Avg.

D1 6.40 6.64 6.43 6.50 6.51 6.49

D2 6.39 6.53 6.10 6.62 6.60 6.44

D3 6.51 6.40 6.44 6.63 6.40 6.41

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Average(%) 6.43 6.52 6.32 6.58 6.50

Fungus Bacteria Algae

Table 1. pH of collected soils

0

10

20

30

40

50

D1

S1

D1

S2

D1

S3

D1

S4

D1

S5

D2

S1

D2

S2

D2

S3

D2

S4

D2

S5

D3

S1

D3

S2

D3

S3

D3

S4

D3

S5

SRI

Direct Seedling

Conventional


D1 0.890 0.909 0.894 0.897 0.916 0.901

D2 0.881 0.905 0.912 0.911 0.912 0.904

D3 0.905 0.887 0.885 0.915 0.890 0.896

Average(%) 0.892 0.900 0.897 0.907 0.906

Table. 2. Organic carbon content in collected soils14

Fields S1 S2 S3 S4 S5 Avg

0

100

200

300

400

500

600

700

800

S1 S2 S3 S4 S5

SRI

Direct Seedling

Conventional

Fields S1 S2 S3 S4 S5 Avg

D1 188.16 169.34 181.88 169.34 188.16 179.3

D2 175.61 194.43 344.96 194.43 75.26 196.9

D3 194.43 181.88 175.61 206.97 206.97 193.1

Average(kg/ha) 186.06 181.88 234.15 190.24 156.79

Table.3. Available Nitrogen (kg/ha) in collected soils15


0

100

200

300

400

500

600

S1 S2 S3 S4 S5

SRI

Dierect Seedling

Conventional


D1 144.48 147.61 145.15 145.60 148.73 146.31

D2 143.13 146.94 148.06 147.84 148.06 146.80

D3 146.94 144.03 143.58 148.51 144.48 145.50

Average(kg/ha) 144.85 146.19 145.59 147.31 147.09

Table.4. Available Phosphorus (kg/ha) in collected soils

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0

50

100

150

200

250

S1 S2 S3 S4 S5

SRI

Direct Seedling

Conventional


D1 63.95 73.58 78.51 64.51 52.64 66.63

D2 57.23 71.68 74.92 50.28 70.67 64.95

D3 64.40 72.46 64.62 76.83 85.68 72.79

Average(kg/ha) 61.86 72.57 72.68 63.87 69.66

Table. 5. Exchangeable Potassium (kg/ha) in collected soils

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Fields 1st 2nd 3dr MEAN CFU/Gm/ML

D2SI 24 10 14 16 2.66*10⁶⁶⁶⁶

D1S4 31 10 19 20 3.33*10⁶⁶⁶⁶

D2S5 3 14 4 7 1.16*10⁶⁶⁶⁶

D2S2 12 7 5 8 1.33*10⁶⁶⁶⁶

D1S3 26 17 20 21 3.5*10⁶⁶⁶⁶

D3S4 5 12 10 9 1.5*10⁶⁶⁶⁶

D3S1 4 2 12 6 1*10⁶⁶⁶⁶

D2S3 16 5 12 11 1.83*10⁶⁶⁶⁶

⁶⁶⁶⁶D3S5 8 6 7 7 1.16*10⁶⁶⁶⁶

D1S5 15 13 8 12 2*10⁶⁶⁶⁶

D2S4 2 1 6 3 O.5*10⁶⁶⁶⁶

D3S5 6 2 1 3 0.5*10⁶⁶⁶⁶

D1S1 31 26 18 25 4.16*10⁶⁶⁶⁶

D3S2 10 11 6 9 1.5*10 ⁶⁶⁶⁶

D3S3 17 9 13 13 2.16*10 ⁶⁶⁶⁶

Table. 6. Fungal population in different soils

18

Fields 1st 2nd 3dr MEAN CFU/Gm/ML

D2SI 24 10 14 16 2.66*10⁶⁶⁶⁶

D1S4 31 10 19 20 3.33*10⁶⁶⁶⁶

D2S5 3 14 4 7 1.16*10⁶⁶⁶⁶

D2S2 12 7 5 8 1.33*10⁶⁶⁶⁶

D1S3 26 17 20 21 3.5*10⁶⁶⁶⁶

D3S4 5 12 10 9 1.5*10⁶⁶⁶⁶

D3S1 4 2 12 6 1*10⁶⁶⁶⁶

D2S3 16 5 12 11 1.83*10⁶⁶⁶⁶

D3S5 8 6 7 7 1.16*10⁶⁶⁶⁶D3S5 8 6 7 7 1.16*10⁶⁶⁶⁶

D1S5 15 13 8 12 2*10⁶⁶⁶⁶

D2S4 2 1 6 3 O.5*10⁶⁶⁶⁶

D3S5 6 2 1 3 0.5*10⁶⁶⁶⁶

D1S1 31 26 18 25 4.16*10⁶⁶⁶⁶

D3S2 10 11 6 9 1.5*10 ⁶⁶⁶⁶

D3S3 17 9 13 13 2.16*10 ⁶⁶⁶⁶

Table.7. Bacterial population in different soils

19

Fungus Size Shape Margin Surface Color

a. 6mm Irregular Lobate Wrinkled Milky white

b. 4mm Round Wavy Smooth White center, clear

surrounding

c. 13mm Irregular Lobate Smooth White center,

milky white

surrounding

d. 5mm Irregular Wavy Smooth Yellow, gold, clear

surrounding

e. 9.5mm Irregular Wavy Smooth, contoured

edges

Tan center, white

ring, clear ring

f. 6mm Irregular Lobate Wrinkled Black

g. 6mm Round Wavy Smooth Pink center, clear

surrounding surrounding

h. 20mm Irregular Lobate Smooth Dusty brown

i. 7mm Regular Wavy Smooth Yellow, gold, clear

surrounding

j. 9.5mm Irregular Wavy Smooth, contoured

edges

Red center, white

ring, clear ring

k. Punctiform one. Round Smooth Smooth Slightly white

l. 7mm Irregular Wavy Smooth Creamy white

m. 8.5mm Irregular Lobate Wrinkled Black

n. 9mm Round smooth Smooth Pink center, clear

surrounding

o. 10mm Irregular Lobate Smooth Blakish green

Table.8. Fungal colony morphology in PDA medium 20

Figure. 2. Different types of fungal isolates from treated soil fields21

Figure. 3. Lactophenol cotton blue staining for fungal isolates

22

Figure.4. Antifungal assay of fungal isolates23

Antifungal discs Zone diameter in mm

Sensitive Intermediate Resistance

Clotrimazole +++ - -

Fluconazole - ++ -

Fungus a Grisefulvin - - +

Ketoconazole - ++ -

Micronazole - - +

Terbinafine No zone No zone No zone

Clotrimazole No zone No zone No zone

Fluconazole - - +Fluconazole - - +

Fungus b Grisefulvin - - +

Ketoconazole No zone No zone No zone

Micronazole - - +

Terbinafine +++ - -


Fluconazole No zone No zone No zone

Fungus c Grisefulvin - - +

Ketoconazole +++ - -

Micronazole No zone No zone No zone

Terbinafine - - +

24



Fungus d Grisefulvin No zone No zone No zone


Micronazole +++ - -

Terbinafine - - +



Fungus e Grisefulvin No zone No zone No zone





Fluconazole +++ - -

Fungus f Grisefulvin - - +

Ketoconazole - - +

Micronazole - ++ -

Terbinafine - - +



Fungus g Grisefulvin No zone No zone No zone



Terbinafine +++ - - 25


Fluconazole - ++ -

Fungus h Grisefulvin No zone No zone No zone



Terbinafine - - +


Fluconazole - - +

Fungus i Grisefulvin - - +

Ketoconazole - - +

Micronazole - ++ -

Terbinafine - - +



Fungus j Grisefulvin No zone No zone No zone

Ketoconazole +++ - -

Micronazole - ++ -

Terbinafine - - +

26


Fluconazole +++ - -

Fungus m Grisefulvin +++ - -

Ketoconazole - - +

Micronazole - - +

Terbinafine - - +



Fungus n Grisefulvin No zone No zone No zone





Fluconazole +++ - -

Fungus o Grisefulvin - ++ -

Ketoconazole - - +



** “+++” indicate that > 35 mm zone of inhibition (sensitive)

“++” indicate that 15-25mm zone of inhibition (intermediate)

“+” indicate that < 10 mm zone of inhibition (resistance)

Table.9. Antifungal assay of fungal isolates

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Bacteria Size Shape Margin Surface Color

a. 8mm Round Smooth Smooth Yellow

b. 8mm Irregular Lobate Smooth Clear to

creamy

white

c. 3mm Star Smooth Concentric White

d. 2mm Round Lobate Smooth Clear to

off white

e. 3mm Round Lobate Wrinkled,

smooth

Clear

Table.10. Bacterial colony morphology in Nutrient Agar medium

28

Figure.5. Different bacterial strains isolated from treated soils

29

Bacterial Isolates Positive Negative

a. ***

b. ***

c. ***

d. ***

e. ***

Sr. no Bacterial isolates Catalase

Test

Citrate

Test

Indol

Test

Voges-

Proskauer

Test

Methyl

Red Test

Table. 11. Gram staining of bacterial isolates

Test

1 a. + + - - -

2 b. + + - + +

3 c. - + + + +

4 d. - + + + +

5 e. + - + + +

Table. 12. Biochemical test for bacterial isolates

30

Figure .6. Biochemical test for SRI bacterial isolates; a) Citrate test, b) Catalase

test, c) Methyl Red test, d) Voges Proskauer test.

31

Figure. 7. Antibiotic Susceptibility Test for bacterial isolates

32

Antibiotic discs Zone diameter in mm

Sensitive Intermediate Resistance

Ciprofloxacin +++ - -

Methicillin - - +

Bacteria a Gentamycin - - +

Streptomycin - - +

Erythomycin - - +

Penicillin No zone No zonePenicillin No zone No zone

Ciprofloxacin No zone No zone No zone

Methicillin +++ - -

Bacteria b Gentamycin - - +

Streptomycin No zone No zone No zone

Erythomycin - - +

Penicillin - ++ -

33


Methicillin No zone No zone No zone

Bacteria c Gentamycin - - +

Streptomycin - - +

Erythomycin No zone No zone No zone

Penicillin - - +



Bacteria d Gentamycin No zone No zone No zone


Erythomycin +++ - -

Penicillin - ++ -



Bacteria e Gentamycin No zone No zone No zone


Erythomycin No zone No zone No zone

Penicillin No zone No zone No zone

** “+++” indicate that > 35 mm zone of inhibition (sensitive)

“++” indicate that 15-25mm zone of inhibition (intermediate)

“+” indicate that < 10 mm zone of inhibition (resistance)

Table.13. Antibiotic Susceptibility Test for Bacterial isolates

34

Partial Identification of Microorganisms

.

a

.

b c

de f

gh

i

Figure. 8. Macroscopic and microscopic identification of fungal isolates;

a, b, and c showing the fungal growth on PDA (Potato Dextrose Agar); d)

Aspergillus sp., e) Alternaria sp., f) Fusarium sp, g) Rhizopus sp.,. h)

Cunninghamella i) Trichoderma sp. 35

• All bacterial isolates were obtained in pure cultures by using

standard techniques. The photomicrographs of all the

bacterial isolates were taken helps in identification of

bacterial isolates. Five genera were identified as, Bacillus sp.,

Flavobacterium sp. and Pseudomonas sp.

100

120

140

160

180

36

Figure.9. Comparative studies of fungal population using different techniques.

0

20

40

60

80

100

SRI

Conventional

Direct Seedling

Conclusion• Fungus are dominating the bacterial growth in East SRI field.

• Among the isolates Aspergillus sp. and Rhizopus sp. were dominating in all

agricultural fields due to high sporelation capacity and the Aspergillus sp. producing

different kinds of toxins such as aflotoxins, achrotoxins etc.

• Fungus are enhancing the rice plant growth in East SRI field.

• Bacterial and fungal diversity increase soil quality by affecting soil agglomeration

and increasing soil fertility.

• They are both important in nutrient cycling and in enhancing plant health through

direct or indirect means.direct or indirect means.

• Soil pH, organic carbon, available nitrogen, potassium, phosphorus are enhancing

the microbial growth in East SRI fields.

• Pathogenic fungus are negligible in SRI fields as compare to conventional and direct

seedling fields.

• It can also be concluded that yields of SRI are increased by 50-100 % or more with

less water (by 25-50%), without using new improved varieties or using chemical

fertilizer(compost+soil), with usually lowered costs of production and thus

considerably increased net economic returns per hectare.

37

Future Works

� Heavy metal quantity in SRI fields.

� Species level identification of isolated microbes

(16srDNA & ITS Sequencing).

�Enzymatic activity of microbes in SRI fields.

� Comparative studies of different SRI fields (Western� Comparative studies of different SRI fields (Western

Plateau vs. Eastern Plateau).

� SEM Analysis.

� FAME Analysis.

38

AKNOWLEDGEMENT

I am very thankful to the management of VIT University andISI(Indian Statistical Institute) for providing well furnished lab andfacilities to carry out of our project work.

I am grateful to Dr. G. Viswanathan, Chancellor, VIT, TN and Dr. C.Ramalingam, Dean, SBST, VIT, TN and Dr. K.V. BhaskaraRao, Programme chair, Environmental & BiotechnologyRao, Programme chair, Environmental & BiotechnologyDivision, VIT, for granting the permission to carry out the project atISI, Kolkata.

I also thankful to Dr. Godwin Christopher J. and Dr. Pabitra Banik forhis guidance.

I would like to thank my parents and my friends for theirencouragement, moral support and everlasting love and affection.

39

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