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

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Ishita Das
Ishita Das

SRI, is the well known name in the field of Agriculture. Here I was check the involvement of microbes in the SRI (System rice intensification)

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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.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.
Different microbial loads under system of rice intensification (sri) copy pdf copy
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 : 23? 5¡¯N to 24? 7¡¯N Longitude : 86? 18¡¯E to 86?19¡¯E Design : Split Plot Design Main Plot Size : 7¡Á7 m? SSuubb PPlloott SSiizzee :: 11 ¡Á¡Á 77 mm? 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
Different microbial loads under system of rice intensification (sri) copy pdf copy
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 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 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 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
250 200 150 100 50 0 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*10? D1S4 31 10 19 20 3.33*10? D2S5 3 14 4 7 1.16*10? D2S2 12 7 5 8 1.33*10? D1S3 26 17 20 21 3.5*10? D3S4 5 12 10 9 1.5*10? D3S1 4 2 12 6 1*10? D2S3 16 5 12 11 1.83*10? D3S5 8 6 7 7 1.16*10?? D1S5 15 13 8 12 2*10? D2S4 2 1 6 3 O.5*10? D3S5 6 2 1 3 0.5*10? D1S1 31 26 18 25 4.16*10? D3S2 10 11 6 9 1.5*10 ? D3S3 17 9 13 13 2.16*10 ? Table. 6. Fungal population in different soils 18
Fields 1st 2nd 3dr MEAN CFU/Gm/ML D2SI 24 10 14 16 2.66*10? D1S4 31 10 19 20 3.33*10? D2S5 3 14 4 7 1.16*10? D2S2 12 7 5 8 1.33*10? D1S3 26 17 20 21 3.5*10? D3S4 5 12 10 9 1.5*10? D3S1 4 2 12 6 1*10? D2S3 16 5 12 11 1.83*10? DD33SS55 88 66 77 77 11..1166**110?? D1S5 15 13 8 12 2*10? D2S4 2 1 6 3 O.5*10? D3S5 6 2 1 3 0.5*10? D1S1 31 26 18 25 4.16*10? D3S2 10 11 6 9 1.5*10 ? D3S3 17 9 13 13 2.16*10 ? 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 +++ - - 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 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
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 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
References ?Acinas, S., Rodriguez-Valera, R., Pedros-Alio, C., 1997. Spatial and temporal variation in marine bacteria plankton diversity as shown by RFLP fingerprinting of PCR amplified 16S rDNA. FEMS Microbial. Ecol. 24, 27¨C40. ?Atlas, R.M., Bartha, R., 1993. Microbial Ecology Fundamentals and Applications. 3rd ed. Benjamin Cummings Publishing, New York. ? Aderson, 1982. Soil respiration. Methods of soil analysis. Page, A.L. (Ed), 2 nd edition, American society of Agronomy, Madison, West Indies, 831-872. ?Alexander.M.,1977 Introduction to soil Microbiology, John Wiley Sons, New York. ?Bagwell, C.E., Lovell, C.R., 2000. Persistence of selected Spartina alterniflora rhizoplane diazotrophs exposed to natural and manipulated environmental variability.Appl. Environ. Microbiol. 66, 4625¨C4633. ?Thompson, C. J., N. R. Movva, R. Tizard, R. Crameri, J. E. Davies, M. Lauwereys, and J. Botterman. 1987. Characterization of the herbicide-resistence gene bair from Streptoinvces hygroscopicus. EMBO J. 6:2519-2523. 40
? Umbreit, W. W., R. H. Burris, and J. F. Stauffer (ed.). 1959. Manometric techniques, 2nd ed. Burgess Publishing, Minneapolis. ? Ulrich G. Mueller, Nicole M. Gerardo, Duur K. Aanen, Diana L. Six, and Ted R. Schultz. 2005. The Evolution of Agriculture in Insects. Annual Review of Ecology, Evolution, and Systematics 36: 563¨C595. ? Van den Tweel, W. J. J., J. P. Smits, and J. A. M. de Bont. 1986. Microbial metabolism of D- and L-phenylglycine by PseucdotnonIals piatida LW-4. Arch. Microbiol. 144:169-174. ?Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39:971-974. ?Wild, A., and R. Manderscheid. 1984. The effect of phosphinothricin on the assimilation of ammonia in plants. Z. Naturforsch. 39c:500-504. ?Yu C, Lv DG, Qin SJ. Du GD, Liu GC, 2007. J.Appl. Ecol.,18(10):2277-2281. 41
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Different microbial loads under system of rice intensification (sri) copy pdf copy

  • 1. 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
  • 2. 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.
  • 4. 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
  • 5. 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
  • 6. 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
  • 7. 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
  • 8. Rice Cultivation by SRI Technique Field Details : Place : Giridhi, Jharkhand Latitude : 23? 5¡¯N to 24? 7¡¯N Longitude : 86? 18¡¯E to 86?19¡¯E Design : Split Plot Design Main Plot Size : 7¡Á7 m? SSuubb PPlloott SSiizzee :: 11 ¡Á¡Á 77 mm? 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
  • 10. Figure 1. Rice cultivation by SRI method 7
  • 11. 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
  • 12. Physicochemical Characterization of soil samples pH Organic Carbon Available Nitrogen Available 12 Phosphorus Exchangeable Potassium 9
  • 13. Isolation of Microbes from collected Soil samples Soil Sample 10 Serial Dilution Potato Dextrose Agar Nutrient Agar
  • 14. 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
  • 15. 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
  • 16. 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
  • 17. 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 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 soils 14
  • 18. 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
  • 19. 600 500 400 300 200 100 0 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
  • 20. 250 200 150 100 50 0 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
  • 21. Fields 1st 2nd 3dr MEAN CFU/Gm/ML D2SI 24 10 14 16 2.66*10? D1S4 31 10 19 20 3.33*10? D2S5 3 14 4 7 1.16*10? D2S2 12 7 5 8 1.33*10? D1S3 26 17 20 21 3.5*10? D3S4 5 12 10 9 1.5*10? D3S1 4 2 12 6 1*10? D2S3 16 5 12 11 1.83*10? D3S5 8 6 7 7 1.16*10?? D1S5 15 13 8 12 2*10? D2S4 2 1 6 3 O.5*10? D3S5 6 2 1 3 0.5*10? D1S1 31 26 18 25 4.16*10? D3S2 10 11 6 9 1.5*10 ? D3S3 17 9 13 13 2.16*10 ? Table. 6. Fungal population in different soils 18
  • 22. Fields 1st 2nd 3dr MEAN CFU/Gm/ML D2SI 24 10 14 16 2.66*10? D1S4 31 10 19 20 3.33*10? D2S5 3 14 4 7 1.16*10? D2S2 12 7 5 8 1.33*10? D1S3 26 17 20 21 3.5*10? D3S4 5 12 10 9 1.5*10? D3S1 4 2 12 6 1*10? D2S3 16 5 12 11 1.83*10? DD33SS55 88 66 77 77 11..1166**110?? D1S5 15 13 8 12 2*10? D2S4 2 1 6 3 O.5*10? D3S5 6 2 1 3 0.5*10? D1S1 31 26 18 25 4.16*10? D3S2 10 11 6 9 1.5*10 ? D3S3 17 9 13 13 2.16*10 ? Table.7. Bacterial population in different soils 19
  • 23. 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
  • 24. Figure. 2. Different types of fungal isolates from treated soil fields 21
  • 25. Figure. 3. Lactophenol cotton blue staining for fungal isolates 22
  • 26. Figure.4. Antifungal assay of fungal isolates 23
  • 27. 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 +++ - - 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
  • 28. 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
  • 29. 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
  • 30. 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
  • 31. 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
  • 32. Figure.5. Different bacterial strains isolated from treated soils 29
  • 33. 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
  • 34. Figure .6. Biochemical test for SRI bacterial isolates; a) Citrate test, b) Catalase test, c) Methyl Red test, d) Voges Proskauer test. 31
  • 35. Figure. 7. Antibiotic Susceptibility Test for bacterial isolates 32
  • 36. 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
  • 37. 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
  • 38. 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
  • 39. ? 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.
  • 40. 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
  • 41. 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
  • 42. 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
  • 43. References ?Acinas, S., Rodriguez-Valera, R., Pedros-Alio, C., 1997. Spatial and temporal variation in marine bacteria plankton diversity as shown by RFLP fingerprinting of PCR amplified 16S rDNA. FEMS Microbial. Ecol. 24, 27¨C40. ?Atlas, R.M., Bartha, R., 1993. Microbial Ecology Fundamentals and Applications. 3rd ed. Benjamin Cummings Publishing, New York. ? Aderson, 1982. Soil respiration. Methods of soil analysis. Page, A.L. (Ed), 2 nd edition, American society of Agronomy, Madison, West Indies, 831-872. ?Alexander.M.,1977 Introduction to soil Microbiology, John Wiley Sons, New York. ?Bagwell, C.E., Lovell, C.R., 2000. Persistence of selected Spartina alterniflora rhizoplane diazotrophs exposed to natural and manipulated environmental variability.Appl. Environ. Microbiol. 66, 4625¨C4633. ?Thompson, C. J., N. R. Movva, R. Tizard, R. Crameri, J. E. Davies, M. Lauwereys, and J. Botterman. 1987. Characterization of the herbicide-resistence gene bair from Streptoinvces hygroscopicus. EMBO J. 6:2519-2523. 40
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