EFFECT OF Bt COTTON ON SOIL BIOTA

The Bt is a short form of ubiquitous soil bacterioum Bacillus thuringiensis. This bacterium is gram positive and spore forming that forms parasporal crystals during stationary phase of its growth cycle. The synthesized crystalline proteins called ‘endotoxins’ are highly toxic to certain insects. They kill the insect by acting on the epithelium tissues of midgut of caterpillars. These proteins are characterized by their insecticidal activity and are therefore grouped into four classes i.e. Lepidoptera-specific (Cry I), Lepidoptera and Diptera-specific (Cry II), Coleoptera-specific (Cry III) and Diptera-specific (Cry IV). Cotton bollworms belong to the order Lepidoptera and therefore are sensitive to Bt Cry I and Cry II proteins, which are specific to them. Other beneficial insects are unaffected by these proteins. popularly and effectively utilized are Cry 1 Ac, Cry 1 Ab in different crops.

1. Seminar Topic     “ EFFECT OF Bt COTTON ON SOIL BIOTA”   Submitted by   GADAMBE ARUN GUNAJI Reg No: – 2018A/105M   Research guide Dr.R.N.Khandare   Assistant Professor AICRP on LTFE, Dept . of SSAC , VNMKV , Parbhani .   Seminar In-charge Dr. Syed Ismail Head of Department of Soil Science And Agril.Chemistry , VNMKV, Parbhani .

2. Introduction:- History:- 1901:- Shigetane Ishiwatari first isolated the bacterium Bacillus thuringiensis as the cause of the sotto disease. 1915:- Berliner reported the existence of a crystal within Bt,but the activity of this crystal was not discovered until much later. 1956:- Researchers Hannay,Fitz -James and Angus found that the main insecticidal activity against moth insect was due to crystal. 1958:- In the US,Bt was used commercially. 1961:- Bt was registered as a pesticide. 1996:- Bt cotton was introduced into US agriculture .

3. Why Bt Cotton ? The sucking pest complex comprising of aphids, jassids , thrips and whitefly are widespread and fairly serious . The bollworms are most important tissue feeders and highly damaging. Three types of bollworms viz. American bollworm ( Helicoverpa armigera ), Pink bollworm ( Pectionphora gossypiella ) and Spotted bollworm ( Earias vitella ), normally referred as bollworm complex are by far the most damaging and loss inducing pests of cotton. Decrease the damage rate of bollworm complex develop Bt cotton. (Source:- Mayee C.D. et al. CICR TECHNICAL BULLETIN NO :22)

4. What is Bt ? The Bt is a short form of ubiquitous soil bacterioum Bacillus thuringiensis . This bacterium is gram positive and spore forming that forms parasporal crystals during stationary phase of its growth cycle. The synthesized crystalline proteins called ‘endotoxins’ are highly toxic to certain insects. They kill the insect by acting on the epithelium tissues of midgut of caterpillars. These proteins are characterized by their insecticidal activity and are therefore grouped into four classes i.e. Lepidoptera-specific (Cry I), Lepidoptera and Diptera -specific (Cry II), Coleoptera -specific (Cry III) and Diptera -specific (Cry IV ). Cotton bollworms belong to the order Lepidoptera and therefore are sensitive to Bt Cry I and Cry II proteins, which are specific to them. Other beneficial insects are unaffected by these proteins. popularly and effectively utilized are Cry 1 Ac, Cry 1 Ab in different crops. ( Source:- Mayee C.D. et al. CICR TECHNICAL BULLETIN NO :22)

5. What is Bt cotton? A genotype or individual which is developed by the techniques of genetic engineering is referred to as transgenic . In other words, genetically engineered organisms are called transgenics . A transgenic may be a plant, an animal or a microbe . . Transgenic plants contain foreign gene or genetically modified gene of the same species. The foreign gene may be from a distantly related species, closely related species or unrelated species or from micro-organisms such as fungi, bacteria and viruses. Bt cotton refers to transgenic cotton which contains endotoxin protein inducing gene from soil bacterium Bacillus thuringiensis .   ( Source:- Mayee C.D. et al. CICR TECHNICAL BULLETIN NO :22)

6. How Bt cotton is developed ? For development of transgenic of any crop, there are five important steps: - Identification of effective gene or genes. Gene transfer technology. Regeneration ability from protoplasts, callus or tissues. Gene expression of the product at desired level. Proper integration of genes so that are carried for generations by usual means of reproduction. ( Source:- Mayee C.D. et al. CICR TECHNICAL BULLETIN NO :22)

7. The major advantage of Bt cotton are summarized below : 1.The Bt cotton has inbuilt genetic resistance to bollworms and is very effective in controlling the yield losses caused by bollworms to a considerable extent.The resistance is governed by a single dominant gene. 2.Use of Bt cotton reduces the use of pesticides resulting in reducing the cost of cultivation. 3.It results in improvement of yield levels and also improves margin of profit to the farmers. 4.It provides opportunities to grow cotton in areas of severe bollworm incidence. 5.It promotes ecofriendly cultivation of cotton and allows multiplication of beneficial insects i.e.parasites and predators of bollworms. 6.It also reduces environmental pollution and risk of health hazards associated with use of insecticides because in Bt cotton the insecticides are rarely used. An average reduction of 3.6 sprays per crop season has been reported in Bt varieties as compared to non-Bt . ( Source:- Mayee C.D. et al. CICR TECHNICAL BULLETIN NO :22)

8. Disadvantages of Bt cotton:- Several advantages with some limitations as well High cost of Bt cotton seeds makes not afforded by small and marginal farmers. Effectiveness up to 120 days. Advers effect on insecticide manufacturing companies. Unemployment. Ineffective against sucking pests like aphids,whitefly etc. Promote malpractices.

9. Soil biota:- Living component present in soil is called soil biota. Types of soil biota:- 1.Macrofauna (>2mm) 2.Mesofauna (0.1-2mm) 3.Microfauna(<0.1mm) Functions of macrofauna :- 1.Pulverize,mix and granulate soil and incorporate o.m.into the lower horizons. 2.Provide large channels through which air and water can move freely. 3.Partially digest organic residues and leave their excrement for microbial degradation.

10. Functions of mesofauna :- 1.First line of attack on litter on the forest floor. 2.Important regulators of some microfaunal biomass and composition. Functions of microfauna :- 1.Soil microfauna is important for the cycling of nutrient in ecosystem. 2.Control populations of microflora .

11. Beneficial Activities of Microorganisms in the Soil:- 1. Humus synthesis. 2. Mineralization of organic nitrogen, sulphur and phosphorus. 3. Increasing Plant Nutrients Availability (P, S, Mn , Fe, Zn and Cu ). 4. Symbiotic mycorrhizal associations. 5. Production of organic chelating agents. 6. Increase oxidation-reduction reactions. 7. Increase phosphorus solubilisation. 8. Increase b iological Nitrogen Fixation (BNF ). 9. Increase free-living bacteria and Cynobacteria . 10.Increase associative microorganisms. 11.Increase symbiotic bacteria with legume and non-legume. 12.Promoting Plant Growth. 13.Production of plant growth hormones. 14.Protection against root pathogens and pseudopathogens . 15.Enhanced nutrient use efficiency. 16.Controlling Deleterious Microorganisms and Plants.  

12. Direct and Indirect Effects of Transgenic Plants on soil microbiota:- FIGURE 1. Direct and indirect effects of transgenic plants on soil microbiota . (source:- Singh et al.(2016)Current Developments in Biotechnology and Bioengineering: Crop Modification, Nutrition, and Food Production)

13. With regard to the nature of genes incorporated into the transgenic plant genome, use of an antibiotic-resistant gene as a marker has been criticized by the World Health Organization , because antibiotic-resistant genes may transferred to the rhizosphere and soil microbes, and then to linking groups by horizontal gene transfer . Deployment of transgenic crops has raised the following scientific concerns: 1. possible adverse effects on nontarget organisms. 2. gene flow into wild plant communities or soil microbiota through horizontal gene transfer. 3. persistence of gene products or crop residues in the environment. 4. development of resistance in target microorganisms.

14. Case studies

15. Table 1: Average value of microbial counts (N = 25) in Bt and non- Bt cotton soils ( vidharbha,MH .) CFU- Colony forming unit; NS- non significant,*p<0.05;**p<0.01 (source-Tarafdar. et al Applied biological research 14 (1):00-00,2012) Microorganisms (CFU g-1) Non- Bt Cotton soil Bt ctton Soil Increase(+) or decrease(-) from non- Bt cotton(%) Level of significance No-crop soil Actinobacteria ( x10 5 ) 52.5±9.7 43.6±7.3 -17.0 ** 31.8±5.9 Bacteria ( x 10 6 ) 85.9±8.9 73.7±8.5 -14.2 * 59.1±6.2 Fungi (x 10 4 ) 31.1±6.9 31.3±5.2 +0.3 NS 19.8±3.4 Nitrifiers (x10 2 ) 19.7±2.5 18.9±2.4 -4.1 NS 12.9±1.9

16. Table 2 (a)Bacterial isolates identified in rhizosphere soils of non- Bt cotton from four regions in Mahabubnagar District,Andhra Pradesh. ( Achampet , Balnagar , Nagarkurnool and Kalwakurthy ) ( Source :- Pindi et al (2013), Bulgarian Journal of Agricultural Science, 19 (No 6) 2013, 1306-1310)   Bacteria Non Bt cotton Total number of isolates Frequency of isolates, % Kaveri Super nova Super seeds N-32 Rhizobium 26 - 26 20 72 14.06 Azospirillum Sp 20 28 22 25 95 18.55 Azotobacter 22 30 - 28 80 15.62 Actinomycetes - 22 27 29 78 15.23 Bacillus Sp 30 27 22 16 95 18.55 Pseudomonas fluorescence 18 24 21 29 92 17.96 Total         512 100

17. Table 2(b)Bacterial isolates identified in rhizosphere soils of Bt cotton from four regions in Mahabubnagar District Andhra Pradesh. ( Achampet , Balnagar , Nagarkurnool and Kalwakurthy ) ( Source :- Pindi et al (2013), Bulgarian Journal of Agricultural Science, 19 (No 6) 2013, 1306-1310)   Bacteria Bt cotton Total number of isolates Frequency of isolates, % Mahyco Marvel Diana Rashi Rhizobium 18 - 18 19 55 16.17 Azospirillum Sp 15 12 18 18 63 18.57 Azotobacter 14 17 20 - 51 15 Actinomycetes 17 19 - 16 52 15.29 Bacillus Sp 13 11 16 18 58 17.05 Pseudomonas fluorescence 18 15 14 14 61 17.94 Total         340 100

18. Table 3(a) Fungal isolates identified in rhizosphere soils of non Bt cotton from four regions in Mahabubnagar District Andhra Pradesh.( Achampet , Balnagar , Nagarkurnool and Kalwakurthy ) (Source :- Pindi et al (2013), Bulgarian Journal of Agricultural Science, 19 (No 6) 2013, 1306-1310)   Fungi Non Bt cotton Total number of isolates Frequency of isolates, % Kaveri Super nova Super seeds N-32 Aspergillus Sp 22 18 22 16 78 15.11 Penicillium 23 - 25 17 65 12.59 Rhizopus 14 20 23 15 72 13.95 Fusarium 19 26 25 21 91 17.63 Trichoderma 25 24 - 17 66 12.79 Alternaria 20 19 16 18 73 14.14 Rhizoctonia 18 15 18 20 71 13.75 Total         516 100

19. Table 3(b) Fungal isolates identified in rhizosphere soils of Bt cotton from four regions in Mahabubnagar District Andhra Pradesh.( Achampet , Balnagar , Nagarkurnool and Kalwakurthy ) (Source :- Pindi et al (2013), Bulgarian Journal of Agricultural Science, 19 (No 6) 2013, 1306-1310)   Fungi Bt cotton Total number of isolates Frequency of isolates, % Mahyco Marvel Diana Rashi Aspergillus Sp 10 11 12 6 39 12.95 Penicillium 10 12 16 - 38 12.62 Rhizopus 13 14 9 11 47 15.61 Fusarium 15 13 16 13 57 18.93 Trichoderma 12 17 8 9 46 15.28 Alternaria 10 10 - 8 28 9.3 Rhizoctonia 15 10 10 11 46 15.28 Total         301 100

20. Table No.4 Dehydrogenase activity ( l g TPF g - 1 h - 1 ) in the rhizosphere soils under Bt ( Bt ) and non- Bt ( NBt ) cotton crops. NC indicates no crop ( IARI,New Delhi) (Source:- Sarkar et al.(2008) Journal of Agronomy & Crop Science ( 2008) 194 ,289-296). Treatments Crop growth period (days)     60 90 120 Mean NBt 20.7 33.0 43.4 28.0 31.3 Bt 19.4 33.0 22.8 28.6 26.0 NC 20.5 27.8 21.1 25.3 23.7 Mean 20.2 31.3 29.1 27.3   LSD (P = 0.05): treatment (T) = 1.90; days (D) = 2.20; T · D = 3.81

21. Table No.5 Number (CFU’s) of total soil bacteria observed in cultivation of Bt and non- Bt cotton by replicate and mean(Brazil) (source:- Farnandes et al .(2019) Journal of Agricultural Science ; Vol. 11, No. 4; 2019)   Soil Collection Time Rep 1 Rep 2 Rep 3 Mean 1 Mean 2   CFU/mL Bt 0 D.A.S 2.75 × 10 -5 3.0 × 10 -5 2.5 × 10 -5 2.75 × 10 -5 Aa   30 D.A.S 2.5 × 10 -5 2.0 × 10 -5 1.8 × 10 -5 2.10 × 10 -5 Aab   60 D.A.S 2.9 × 10 -5 2.9 × 10 -5 2.0 × 10 -5 2.60 × 10 -5 Aab   90 D.A.S 1.7 × 10 -5 1.9 × 10 -5 1.9 × 10 -5 1.83 × 10 -5 Ab   120 D.A.S 2.0 × 10 -5 2.1 × 10 -5 2.1 × 10 -5 2.07 × 10 -5 Aab   150 D.A.S 2.7× 10 -5 2.9 × 10 -5 2.9 × 10 -5 2.83× 10 -5 Aa 2.36 × 10 -5 a Non- Bt 0 D.A.S 2.7 × 10 -5 1.3 × 10 -5 2.5 × 10 -5 2.17× 10 -5 Aa   30 D.A.S 2.6 × 10 -5 1.7 × 10 -5 3.0 × 10 -5 2..43 × 10 -5 Aa   60 D.A.S 2.4× 10 -5 1.7 × 10 -5 1.5 × 10 -5 1.87 × 10 -5 Aa   90 D.A.S 1.4 × 10 -5 2.2 × 10 -5 2.1 × 10 -5 1.90 × 10 -5 Aa   120 D.A.S 2.3 × 10 -5 2.1× 10 -5 1.5 × 10 -5 1.97 × 10 -5 Aa   150 D.A.S 2.6 × 10 -5 2.9 × 10 -5 2.2 × 10 -5 2.75 × 10 -5 Aa 2.15 × 10 -5 a  

22. Table no.6 Effects of Transgenic cotton on Soil Microbiota(Varanasi). ( source:- Singh et al.(2016)Current Developments in Biotechnology and Bioengineering: Crop Modification, Nutrition, and Food Production) Protein/gene plant Method Organisms Impact Cry1Ac Cotton Plate count,enzymatic activities Soil microbes Negative effect Cry1Ac Cotton Plate count Soil bacterial and fungal communities Transient effect Cry1Ac Cotton RCR-RFLP Soil bacterial and fungal communities Transient effect Cry1Ac Cotton Soil microbial biomass, enzymatic activities Soil bacterial, fungal and actinomycetal communities Cry1Ac negatively affects soil microbial and biochemical properties Cry1Ac Cotton AMF colonization Mycorrizal fungi No effect on AMF colonization Cry1Ac Cotton Plate count Rhizosperic microbial community Transient effect Cry1Ab Cotton Plate count Soil microbial community Transient effect

23. Fig. 2 Activities of phosphatase, urease, dehydrogenase, phenol oxidase and protease in rhizosphere soil of a Bt transgenic cotton and its non- Bt near-isogenic counterpart following different growth periods and in a control soil incubated without a growing cotton plant. P1: vegetative period; P2: reproductive period; P3: senescing period; P4: whole life cycle. Vertical bars represent – SD (n = 3) (source:- Shen et al.(2 006) Plant soil (2006)285:149-159 ) (China) non- Bt Bt CK-no straw

24. Fig.3 Enzyme activities in soil incubated with different amounts of biomass from Bt and non Bt cotton and in soil without biomass addition.Vertical bars represent ± SD (n=3 ). (China). ( source:- Shen et al.(2 006) Plant soil ( 2006)285:149-159) non- Bt Bt CK-no straw

25. Table No.7 Aerobic, Heterotrophic Microbial population density of Bt Rhizosphere soil ( Tirupati ) ( source :- Audiseshamma et al .( 2014) International Journal of Current Microbiology and Applied Sciences (2014)3(5):289-294) S. No Sample Microbial population Density (Cfu / gm soil) Bacteria Fungi Actinomycetes Yeasts 1. Bt Rhizosphere soil 27(±3)×10 4 29×10 4 27×10 2 48×10 4 2. Non Bt Rhizosphere Soil 41×10 4 18×10 4 32×10 2 18×10 4

26. Table no.8 Beneficial Microbial population Density in Rhizosphere soils of Bt and Non Bt cotton( Tirupati ) (source:- Audiseshamma et al .(2014) International Journal of Current Microbiology and Applied Sciences (2014)3(5):289-294) S. No Types of organisms Bt Rhizosphere ( Cfu / gm soil) Non Bt Rhizosphere ( Cfu / gm soil) 1 Symbiotic Nitrogen fixing bacteria 7×10 6 11×10 6 2 Asymbiotic nitrogen fixing bacteria Azospirillum Azatobacter 15×10 3 78×104 28×10 3 20×10 4 3 Phosphate Solubilizing bacteria 35×10 4 43×10 4 4 Cellulase degrading bacteria 28×10 4 27×10 4

27. Figure.4a Distribution of Microbial populations in Bt cotton soils( Tirupati ) Figure.4b Distribution of Microbial populations in Non Bt cotton soils ( source:- Audiseshamma et al .(2014) International Journal of Current Microbiology and Applied Sciences (2014)3(5 ): 289294)

28. Fig.5A Distribution of beneficial microbial population in Bt Cotton soils( Tirupati ) Fig.5B Distribution of beneficial microbial population in Non Bt Cotton soils (source:- Audiseshamma et al .(2014) International Journal of Current Microbiology and Applied Sciences (2014)3(5):289294)

29. Table no. 9. Effect of cultivation of Bt cotton on soil microbial population (log 10 CFU g -1 ) ( Warangal,TS .) ( source :- Surapaneni et al. (2015) Green Farming Vol . 6 (6) : 1297-130 Microbial population (log 10 CFU g -1) Soil under non- Bt cotton Soils under Bt cotton for 2-5 years Soils under Bt cotton for >8 years Significance Range Mean±Std dev Range Mean ± Std dev Range Mean ± Std dev Total microbial population 3.2-7.3 4.8-±1.3 3.2-8.8 6.2±1.9 3.0-9.3 6.25±1.8 NS Pseudomonas (PSB) 3.3-5.3 4.1±0.7 3.1-5.6 3.6±0.7 3.1-7.4 4.4±1.5 NS Azotobacter 4.0-5.4 4.6±0.6 3.0-5.3 4.1±0.5 4.0-6.7 4.8±0.8 NS MBC(mg kg -1) 74.8-186.0 141.4±34.8 125.1-250.9 150.9±34.3 96.7-385.2 173.3±68.6 NS

30. Table 10: Effect of Bt and non Bt cotton on soil microbial population in Perambalur district,Tamilnadu (Mean values of ten villages in eachtalukas ). (Source:- Sherene et al .(2018) Biological Forum – An International Journal 11(1): 18-23(2019) Sr. No Talukas General microflora in Bt cotton grown soils (CFU /g) General microflora in non Bt cotton grown soils (CFU /g) Bacteria × 10 6 Fungi × 10 3 Actinomycetes × 10 3 Bacteria × 10 6 Fungi × 10 3 Actinomycetes × 10 3 1. Veppanthattai 42 15.0 4.8 29 14.7 3.8 2. Perambalur 58 14.3 4.0 33 13.8 2.8 3. Alathur 30 14.8 5.2 25 12.0 2.9 4. Veppur 35 16.5 5.7 25 14.3 3.1   Range values 30-58 14.3-16.5 4.0-5.7 25-33 12.0-14.7 2.8-3.8 SD 8.034 1.491 0.56 4.877 1.913 0.814

31. Table 11: Effect of Bt and non Bt cotton on soil microbial respiration and Dehydrogenase activity in soils of Perambalur district,Tamilnadu (Mean values of ten villages in eachtaluks ). ( Source:- Sherene et al .(2018) Biological Forum – An International Journal 11(1): 18-23(2019 ). ss S.No . Talukas Bt cotton grown soils Non Bt cotton grown soils DHA (µg TPF/ g / h Soil respiration µg of CO 2 / g / h DHA (µg TPF/ g / h Soil respiration µg of CO 2 / g / h 1. Veppanthattai 0.2137 224 0.071 164 2. Perambalur 0.2281 264 0.068 181 3. Alathur 0.1983 308 0.075 202 4. Veppur 0.1739 286 0.079 201   Rangevalues 0.174 -0.228 224-308 0.068-0.079 168-202 SD 0.024 26.464 0.006 16.494

32. Table no. 12 Impact of GM cotton on soil biochemical and biological characteristics ( IARI,New Delhi) MBC microbial biomass C, MBN microbial biomass N, MBP microbial biomass P, TOC total organic carbon, MQ microbial quotient, PNM potential nitrogen mineralization, NS not significant. (source:- Sarkar et al .( 2008) Environment Monit Asses (2009)156:595-604) Biological and biochemical Percent increase (+) characteristics or decrease (−) MBC +44.6 MBN +53.6 MBP +104 TOC NS MQ +37 PNM +21.5 Nitrification +11.4 Nitrate reductase +35.3 Alk -phosphatase +24.1 Acid-phosphatase +16.3

33. Figure 6 a&b . Effects of Bt and non- Bt cotton on soil enzyme activities (a) soil urease; (b) soil dehydrogenase. ** P < 0.01. Error bars (± SD) with the same letters within the cotton genotypes do not differ significantly. ( CICR,Nagpur ) (source:- Velmourougane et al .(2013) Plant soil Environment Vol.59,2013,No.3:108-114 ).  

34. Table No. Bacterial populations in soil rhizosphere of 06Z604D expressing both cry1Ac and cry2Ac genes at five different crop growth stages at ( source: Swilla et al .(2015)African Journal of Biotechnology Vol.15(21) pp.930-939) ( source: Swilla et al .( 2015)African Journal of Biotechnology Vol.15(21) pp.930-939)   Mean rhizosphere culturable bacterial population (Cells/g dry Soil)   Treatment     Sampling time (DAS)     64 110 154 175 06Z604D ( Bt ) 1.85x10 5 3.32x10 5 4.32x10 5 5.74x10 5 5.46x10 5 99M03 ( Isoline ) 1.97x10 5 2.92x10 6 8.12x10 4 9.58x10 5 1.06x10 6 HART89M (Conventional) 1.78x10 5 3.56x10 5 8.91x10 4 5.21x10 5 6.23x10 5 Table no.13. Bacterial populations in soil rhizosphere of 06Z604D expressing both cry1Ac and cry2Ac genes at five different crop growth stages at Thika CFT site( Thika ,Kenya).

35. Table 14. Comparisons of colony counts on soils from the rhizosphere of the three different cotton lines (Miles and Misra drop plate method). ( Thika,Kenya ) (source: Swilla et al .(2015 ) African Journal of Biotechnology Vol.15(21) pp.930-939)   Treatment Bacteria Actinomycetes Fungi 06Z604D ( Bt ) 2.38x10 5 4.81x10 5 5.61x10 5 99M03( Isoline ) 4.48x10 5 5.98x10 5 4.91x10 5 HART 89M (Conventional) CD (p=0.05) 3.77x10 5 5.72x10 5 5.43x10 5

36. Table no.15.Effect of cry proteins on soil microbial population and diversity( CICR,Nagpur ) ( source:- Velmourougane et al .(2017) CAB Reviews 2017 12,No.046)   Microorganisms Cry protein Experimentation Conclusions Culturable bacteria and fungi Cry1Ac Bt and non-Bt cotton A significant, increase in numbers in soil with Bt cotton Microbial population and diversity Cry1Ac Bt and non-Bt cotton Higher microbial population and diversity in Bt cotton Total microbial population Cry1Ac Bt and non-Bt cotton No adverse effects Microbial functional diversity Cry1Ac Bt and non- Bt cotton No adverse effects Culturable functional bacteria Cry1Ac Bt and non- Bt cotton No significant differences in numbers after the growing season Methylobacteria Cry1Ac Bt and non- Bt cotton No adverse effects Microbial diversity Cry1Ac Bt and non- Bt cotton No adverse effects Culturable functional bacteria Cry1Ac Multiple-year cultivation of Bt cotton No adverse effects Composition of soil microbiota Cry1Ac Bt and non- Bt cotton More extensive fungal colonization, higher ratios of fungi to bacteria, and different types of fungal spores in soil with Bt cotton

37. Table no.16. Estimation of Microbial Population by Viable Plate Count method and MPN method (Nagpur) ( source:- Ekta et al.( 2016 ) International Journal of Agriculture Sciences Vol.8,Issue 53,2016,pp-2708-2710) Sr.No . Soil sample with different treatment/ variety MPN method/gm of soil (dilution factor) Viable Plate Count /gm of soil (dilution factor) Total Associative Nitrogen Fixers (10 5 ) Heterotrophic population (10 7 ) Phosphate Solubilizers (10 2 ) 1 Bt cotton V1 3.9 x 10 5 38 x 10 7 15 x 10 2 2 Non Bt cotton V1 2.0 x 10 5 28 x 10 7 11 x 10 2 3 Bt cotton V2 6.3 x 10 5 86 x 10 7 7 x 10 2 4 Non Bt cotton V2 3.3 x 10 5 19 x 10 7 4.3 x 10 2 5 Bt cotton V3 16.0 x 10 5 55 x 10 7 12 x 10 2

38. Figure 7 . Bacterial population ( cFu log 10 g −1 of soil) at four locations (Punjab) Non Bt cotton varieties –CIM-591 and CIM-573 (source:- Yasin et al.(2016) plant production science ,2016) Bt cotton varieties –CIM-602 and CIM-599

39. Photos 1 A-D : Scanning electron micrographs of conventional or non- Bt (A and C) and Bt cotton stubble residues (B and D) retrieved from field incubation after 4 weeks (A and B) and 12 weeks (C and B) at Narrabri (Kenya) . Fig. A (non- Bt ) Fig.B ( Bt ) s (source:-Watson et al .(2004) Ecological Impacts of Genetically Modified Organisms)

40. Fig.C (non Bt ) (source:-Watson et al .(2004) Ecological Impacts of Genetically Modified Organisms) Fig.D ( Bt )

41. Photo 2C. Close-up SEM picture of Bt and non- Bt cotton stubble showing fungal spores (Kenya) Fig.E (non- Bt ) Fig.D ( Bt ) (source :-Watson et al .(2004) Ecological Impacts of Genetically Modified Organisms)

42. Conclusion:- Some studies indicate that Bt cotton has no negative effect on soil biota and may even have beneficial effects,while some have reported adverse effect. Some soil specific microbial populations were affected by Bt cotton and some specific microbial populations were unaffected. Cultivation of Bt cotton expressing Cry1Ac gene had no adverse effect on soil biological activities such as microbial population,soil respiration,dehydrogenase activity. Bt toxins from Bt and transgenic crops had no apparent effect on soil microorganisms such as bacteria,fungi,algae,nematode and protozoa. Bt cotton had no harmfull effects on soil enzyme activities. Growth of Bt cotton has a positive effect on most of the microbial,biochemical indicators and enzyme activities. Some soil specific microbial populations were affected by Bt cotton and some specific microbial populations were unaffected.

43. Studies based on Cry1Ac –expressing proteins in the laboratory and field based studies reported “ no” , “significant” and a “ transient effect” of Bt crop on soil microbial communities. Cultivation of Bt cotton either for prolonged period (> 8 years ) or for short terms (2-5 years) did not bring out any significant change in the population of total microbes, Azatobacter , Psuedomonas and soil enzyme activity in rhizosphere soil, when compared to non Bt cotton cultivation. Cultivation of Bt cotton did not affect the diversity of the soil bacterial population.