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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
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 method.Spectrophotometric method.
� 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
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
Fields S1 S2 S3 S4 S5 Avg.
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
Fields S1 S2 S3 S4 S5 Avg.
0
100
200
300
400
500
600
S1 S2 S3 S4 S5
SRI
Dierect Seedling
Conventional
Fields S1 S2 S3 S4 S5 Avg.
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
Fields S1 S2 S3 S4 S5 Avg.
0
50
100
150
200
250
S1 S2 S3 S4 S5
SRI
Direct Seedling
Conventional
Fields S1 S2 S3 S4 S5 Avg.
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*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 +++ - -
Clotrimazole No zone No zone No zone
Fluconazole No zone No zone No zone
Fungus c Grisefulvin - - +
Ketoconazole +++ - -
Micronazole No zone No zone No zone
Terbinafine - - +
24
Clotrimazole No zone No zone No zone
Fluconazole No zone No zone No zone
Fungus d Grisefulvin No zone No zone No zone
Ketoconazole No zone No zone No zone
Micronazole +++ - -
Terbinafine - - +
Clotrimazole No zone No zone No zone
Fluconazole No zone No zone No zone
Fungus e Grisefulvin No zone No zone No zone
Ketoconazole No zone No zone No zone
Micronazole No zone No zone No zone
Terbinafine No zone No zone No zone
Clotrimazole +++ - -
Fluconazole +++ - -
Fungus f Grisefulvin - - +
Ketoconazole - - +
Micronazole - ++ -
Terbinafine - - +
Clotrimazole No zone No zone No zone
Fluconazole No zone No zone No zone
Fungus g Grisefulvin No zone No zone No zone
Ketoconazole No zone No zone No zone
Micronazole No zone No zone No zone
Terbinafine +++ - - 25
Clotrimazole +++ - -
Fluconazole - ++ -
Fungus h Grisefulvin No zone No zone No zone
Ketoconazole No zone No zone No zone
Micronazole No zone No zone No zone
Terbinafine - - +
Clotrimazole +++ - -
Fluconazole - - +
Fungus i Grisefulvin - - +
Ketoconazole - - +
Micronazole - ++ -
Terbinafine - - +
Clotrimazole No zone No zone No zone
Fluconazole No zone No zone No zone
Fungus j Grisefulvin No zone No zone No zone
Ketoconazole +++ - -
Micronazole - ++ -
Terbinafine - - +
26
Clotrimazole +++ - -
Fluconazole +++ - -
Fungus m Grisefulvin +++ - -
Ketoconazole - - +
Micronazole - - +
Terbinafine - - +
Clotrimazole No zone No zone No zone
Fluconazole No zone No zone No zone
Fungus n Grisefulvin No zone No zone No zone
Ketoconazole No zone No zone No zone
Micronazole No zone No zone No zone
Terbinafine No zone No zone No zone
Clotrimazole +++ - -
Fluconazole +++ - -
Fungus o Grisefulvin - ++ -
Ketoconazole - - +
Micronazole No zone No zone No zone
Terbinafine 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.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
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
Ciprofloxacin No zone No zone No zone
Methicillin No zone No zone No zone
Bacteria c Gentamycin - - +
Streptomycin - - +
Erythomycin No zone No zone No zone
Penicillin - - +
Ciprofloxacin No zone No zone No zone
Methicillin No zone No zone No zone
Bacteria d Gentamycin No zone No zone No zone
Streptomycin No zone No zone No zone
Erythomycin +++ - -
Penicillin - ++ -
Ciprofloxacin No zone No zone No zone
Methicillin No zone No zone No zone
Bacteria e Gentamycin No zone No zone No zone
Streptomycin 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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