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.AppliedMicrobiology
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 tthhaann iinn cclluummppss tthhaatt aarree
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 aass ppoossssiibbllee ttoo tthhee ssooiill
Actively aerate the soil
Re-emphasize biology
Rediscover the potentials of synergy and symbiosis
2

AIM :DifferentMicrobial loads under system of rice intensification.
Objective :
Rice cultivation by SRI technique.
Sample collection from SRI and conventional rice ccuullttiivvaattiioonn ffiieelldd..
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)
7. Molecular Characterization pH, NPK value
1. East Field (Giridih)
2. Rice cultivation in SRI field
3. Sample collection
4
Identification of isolated soil
microbes
4. Physicochemical
characterization of soil
5. Isolation of microbes
from soil
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
SSuubb PPlloott SSiizzee :: 11 ×× 77 mm²
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 ffeerrttiilliizzeerr ((ccoonnttrrooll))
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
Phosphorus
Exchangeable
Potassium
9

Isolation of Microbes from collected Soil samples
Soil Sample
10
Serial Dilution
Potato Dextrose Agar Nutrient Agar

Characterization of Isolates
MACROSCOPIC
Colony Morphology
CFU Count
Antibiotic Susceptibility Test
Antifungal Activity Test
MICROSCOPIC BIOCHEMICAL TEST
11
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 mmeetthhoodd..
PCR (Polymerase Chain Reaction).
16srDNA Sequencing.
ITS Sequencing for Fungus.
12

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
13
Average(%) 6.43 6.52 6.32 6.58 6.50
Fungus Bacteria Algae
Table 1. pH of collected soils

50
40
30
20
10
0
D1S1
D1S2
D1S3
D1S4
D1S5
D2S1
D2S2
D2S3
D2S4
D2S5
D3S1
D3S2
D3S3
D3S4
D3S5
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 soils
14

800
700
600
500
400
300
200
100
0
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 soils
15

600
500
400
300
200
100
0
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
16

250
200
150
100
50
0
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
17

Fields 1st 2nd 3dr MEAN CFU/Gm/ML
D2SI 24 10 14 16 2.66*10⁶
D1S4 31 10 19 20 3.33*10⁶
D2S5 3 14 4 7 1.16*10⁶
D2S2 12 7 5 8 1.33*10⁶
D1S3 26 17 20 21 3.5*10⁶
D3S4 5 12 10 9 1.5*10⁶
D3S1 4 2 12 6 1*10⁶
D2S3 16 5 12 11 1.83*10⁶
D3S5 8 6 7 7 1.16*10⁶⁶
D1S5 15 13 8 12 2*10⁶
D2S4 2 1 6 3 O.5*10⁶
D3S5 6 2 1 3 0.5*10⁶
D1S1 31 26 18 25 4.16*10⁶
D3S2 10 11 6 9 1.5*10 ⁶
D3S3 17 9 13 13 2.16*10 ⁶
Table. 6. Fungal population in different soils
18

Fields 1st 2nd 3dr MEAN CFU/Gm/ML
D2SI 24 10 14 16 2.66*10⁶
D1S4 31 10 19 20 3.33*10⁶
D2S5 3 14 4 7 1.16*10⁶
D2S2 12 7 5 8 1.33*10⁶
D1S3 26 17 20 21 3.5*10⁶
D3S4 5 12 10 9 1.5*10⁶
D3S1 4 2 12 6 1*10⁶
D2S3 16 5 12 11 1.83*10⁶
DD33SS55 88 66 77 77 11..1166**110⁶⁶
D1S5 15 13 8 12 2*10⁶
D2S4 2 1 6 3 O.5*10⁶
D3S5 6 2 1 3 0.5*10⁶
D1S1 31 26 18 25 4.16*10⁶
D3S2 10 11 6 9 1.5*10 ⁶
D3S3 17 9 13 13 2.16*10 ⁶
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
ssuurrrroouunnddiinngg
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 fields
21

Figure. 3. Lactophenol cotton blue staining for fungal isolates
22

Figure.4. Antifungal assay of fungal isolates
23

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
FFlluuccoonnaazzoollee -- -- ++
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
27

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
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 NNoo zzoonnee NNoo zzoonnee
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
d
e f
g
h
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.
180
160
140
120
100
36
80
60
40
20
0
SRI
Conventional
Direct Seedling
Figure.9. Comparative studies of fungal population using different techniques.

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 oorr iinnddiirreecctt mmeeaannss..
• 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 SSRRII ffiieellddss ((WWeesstteerrnn
Plateau vs. Eastern Plateau).
SEM Analysis.
FAME Analysis.
38

AKNOWLEDGEMENT
I am very thankful to the management of VIT University and
ISI(Indian Statistical Institute) for providing well furnished lab and
facilities 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. Bhaskara
Rao, Programme chair, Environmental BBiiootteecchhnnoollooggyy
Division, VIT, for granting the permission to carry out the project at
ISI, Kolkata.
I also thankful to Dr. Godwin Christopher J. and Dr. Pabitra Banik for
his guidance.
I would like to thank my parents and my friends for their
encouragement, moral support and everlasting love and affection.
39

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Systematics 36: 563–595.
• Van den Tweel, W. J. J., J. P. Smits, and J. A. M. de Bont. 1986. Microbial metabolism of
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41

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