Effect of Bt Cotton on Enzymes Activity and Microorganisms in Rhizosphere

For three consecutive years Bacillus thuringiensis (Bt) transgenic cotton (var. Mech 162) and its isogenic non Bt counterpart were assessed for the risks of transgenic crop on the soil ecosystem under Indian subtropical conditions. To observe effect of Bt cotton on soil biochemical properties, activities of dehydrogenase, alkaline phosphatase, nitrate reductase and urease soil enzymes were assayed at its different growth stages i.e., seedling, vegetative, flowering, bolling and harvesting stages. To observe effect of Bt cotton on soil microorganisms, number of nematodes, collembola and ants representing micro, meso and macrofauna, respectively were observed in Bt and nonBt cotton plants rhizosphere at different growth stages.


Introduction
In India nine million hectares (m ha) area was under cotton cultivation and it is one fourth of the global area under cotton cultivation (35 m ha).Bacillus thuringiensis (Bt) cotton was introduced in India in 2002.Following its success, during the last 7 years (2002)(2003)(2004)(2005)(2006)(2007)(2008), the area under Bt cotton has increased by 7.6 m ha from 0.029 m ha (James, 2008).
Although there are diverse benefits of Bt cotton, public concern also exist because both in vitro and in vivo studies on Bt cotton showed that Bt toxin produced in leaves, stems and roots of Bt cotton plants is introduced in soil.Bt-toxin from Bt cotton plants introduced into the soil through two pathways, i.e., biomass incorporation and root exudates (Saxena and Stotzky 2001;Mina et al., 2008;Liu 2009).Bt toxin released in soil get adsorbed or bound on clay particles, humic components, or organic mineral complexes and then be protected against degradation by soil microorganisms (Tapp et al., 1995).Although Bt toxin also found naturally in many soils, but continuous growing of Bt crops on same location enhance its existing levels to a certain concentration that might affect the composition and activity of soil microbial communities (Donegan and Seidler 1999;Stotzky 2004;Wei et al., 2006;Griffiths et al., 2007;Rui et al., 2005) and the soil biochemical properties (Rui et al., 2005;Fang et al., 2007;Sun et al., 2007;Sarkar et al., 2009).
Several experiments were conducted to assess risk of Bt cotton on flora and fauna in diverse agroecosystems (Zhang et al., 2000;Li et al., 2002;Liu et al., 2002;Men et al., 2003;Bai et al., 2003).Some studies indicate that Bt cotton has no negative effects on soil flora and fauna and may even have beneficial effects (Saxena and Stotzky 2001;Sarkar et al., 2009), while some have reported adverse effects (Cui and Xia 2000;Tan et al., 2002).However, similar experimental studies on risk assessment of Bt cotton with respect to soil ecosystem in India are very limited.Climatically India is a subtropical country, thus as compared to temperate and sub temperate countries, biological and biochemical response of Indian soil to increasing acreage of Bt cotton may vary.Therefore, we have studied the effects of Bt cotton (var.Mech 162, approved by Genetic Engineering Approval Committee for commercial cultivation in North India) on activity of soil enzymes and microorganisms at Indian Agriculture Research Institute farms, New Delhi, India.An evaluation of the ecological risks of Bt cotton was made on the basis of changes in enzymes activity and number of nematodes, collembola and ants as compared to non Bt cotton in the respective rhizospheres.

Field preparation and cultural practices
Field experiments were conducted at the research farm of the Indian Agricultural Research Institute, New Delhi, India.The soil of the experimental site was slightly alkaline with silty clay loamy texture and has low organic matter content.Experiments were conducted for three consecutive years (2004, 2005 and 2006) in a large plot (450 m 2 ) where Bt and non Bt cotton had ever planted.This plot was subdivided into 24 subplots (3.75m x 5m).Treated seeds of non Bt cotton (Mech 162-Bt) and Bt cotton (Mech 162+Bt), procured from MAHYCO (Mahyco seed limited, Mumbai) were sown in plots at the rate of 15 kg/ha.Each subplot was sown with a different cotton line, under irrigated conditions.Distance between row to row and plant to plant was 60 cm and 45 cm respectively.To ensure uniform germination and better stand, line sowing with seed drill was carried out.Seeds were sown uniformly at a depth of 4-5 cm.A 6 m buffer zone was also established surrounding the main plot.Recommended dose of NPK fertilizer (660 g N as Urea, 250 g P as SSP, 350 g K as MOP) were applied in the field.Recommended dose of phosphorus and Potash was applied at time of sowing, whereas nitrogen was given in split dose, half at the time of thinning and other half at flowering stage.First irrigation to cotton was given one week after sowing and subsequent irrigations at an interval of two weeks.Cotton crop at maturity was harvested in the first week of December in all the three years.Soil samples of experimental field for physicochemical characterization (Table 1) were collected from the top layer (0-20 cm).The soil was air-dried at room temperature, passed through a 2 mm sieve, homogenized by mixing three times with a shovel and stored at room temperature before use.

Soil Sampling
Soil samples from the rhizosphere of Bt cotton and non Bt cotton plants were collected at regular interval (30 days interval, coinciding with different growth stage of cotton) since the date of sowings till harvesting in all the three years.First Sampling was done before seed sowing from prepared field, second sampling 30 Days After Sowing (DAS) (at seedling stage, growth stage1), third sampling 60 DAS (at vegetative stage, growth stage 2), fourth sampling 90 DAS (at Flowering stage growth stage3), fifth sampling 120 DAS (at Boll formation stage, growth stage 4) and last sixth sampling 150 DAS (at mature stage, growth stage 5).Ten rhizosphere samples (0-15cm) were taken along two transects across each plot and were mixed to make a representative sample for analysis.Collected rhizospheric soil samples were processed by air drying, grounding and sieving (passed through a 1 mm sieve) and analysed for all enzymes activities except dehydrogenase and nitrate reductase.For observing dehydrogenase as well as nitrate reductase activity, the soil samples were kept moist.

Dehydrogenase activity
Dehydrogenase activity was analyzed as described by Min et al. (2001).Five grams of soil was incubated for 12 h at 37 0 C in 5 ml of a TTC solution (5 g TTC in 0.2M Tris-HCl buffer, pH 7.4).Two drops of concentrated sulfuric acid were added immediately after the incubation to end the reaction.The sample was then blended with

Alkaline phosphatase activity
Alkaline phosphatase activity was measured spectrophotometrically as described by Tabatabai and Bremner (1969). 1 g soil was placed in a 50 ml erlenmeyer flask and treated with 0.25 ml of toluene and 4 ml MUB buffer (P H 11) and 1 ml of p-nitrophenolphosphate solution made in same buffer.After that the flask contents were mixed and incubated for 1 h at 37 0 C.After 1 h of incubation 1 ml of CaCl 2 (0.5 M) and 4 ml of NaOH (0.5 M) were added to the flask.The colored soil suspension was filtered through Watmann no. 2 filter paper and absorbance of filtrate was measured at 400 nm.The phosphatase activity was expressed as µg p-nitrophenol g _1 h _1

Urease activity
Urease enzyme activity in soil sample was estimated according to "determination of urea remaining" methodology (Tabatabi, 1994).This methodology estimate urea hydrolysis in soils on account of urease enzyme activity.For this 5 g of soil mixed with 5 ml of urea solution (10 mg urea/ml) in a 50-ml erlenmeyer flask, and incubated for 5 h at 37°C.After 5 h, 50 ml 2M KC1-PMA was added to flasks and kept for 1h shaking.After 1 h shaking soil suspension was filtered under suction through whatmann no 42 filter paper.Out of filtrate 2 ml of aliquot was taken and mixed with 10 ml 2M KC1-PMA and 30 ml colouring reagent ( 25 ml 2.5% DAM + 10 ml of 0.25% TSC in 500 ml acid reagent ).This mixture was first kept in water bath for 30 minutes and than kept in ice coldwater for 15 minutes for colour development.Absorbance of coloured end product was measured at 527nm.Activity of Urease enzyme in soil was expressed as μg urea N hydrolysed g −1 h −1

Nitrate reductase activity
Nitrate reductase activity was determined using methodology of Abdellmagid and Tabatabai (1987).For this 5 gm field-moist soil was mixed with 4 ml of 0.19 mM 2,4 dinitrophenol, 1 ml of (25 mM) substrate solution (KNO 3 ) and 5 ml distilled water into screw cap test tubes.These tubes were incubated at 37°C for 24 h and the control was frozen immediately at -20°C.After incubation 10 ml of 4M KCL solution was added in the samples and control and filtered immediately.5 ml of the filtrate was used for color development with 3 ml of 0.19 M ammonium chloride buffer and 2 ml of color reagent.Absorbance of coloured end product was measured at 520nm.Activity of nitrate reductase enzyme in soil was expressed as μg NO 2 -Ng -1 h -1

Nematodes
Nematodes were extracted from Bt and non Bt cotton plants rhizosphere (250 gm) by the Baermann technique (Van Gundy 1982) in water.Nematodes samples from Bt and non Bt cotton plants in water suspension were uniformly placed over tissue paper supported by screen in a petriplates, which were filled with water.After placing the sample, petriplates were covered with respective cap and incubated at 24 ± 2 0 C for 36 h.To estimate the total number of nematodes, the content of the petriplates were transferred to a flat bottom partitioned counting dish (36 squares), and the number of nematodes were counted in 10 random squares using stereoscopic dissecting microscope (Nikon, 10-100 X zoom).Total nematodes in the sample were calculated by multiplying the average count in 10 squares by 36.

Collembola and Ants
Collembola and ants were extracted from soil cores (5 cm dia x 20 cm deep; collected from Bt and non Bt cotton plants rhizosphere) using Tullgreen funnel ("Berlese funnels").Reagent grade ethylene glycol was used to capture the Collembola and ants (moving out from the soil core as the soil heated up from bulbs light energy) in 275 ml plastic cups.Collected collembola and ants were counted using stereoscopic dissecting microscope (Nikon, 10 -100 X zoom)

Experimental design and statistics
This study was designed as a randomized complete block.The block treatment was the month of sampling and the two cotton lines were the main source of variation.The interaction of month and cotton lines was used for testing significance of the sources of variation.These treatments were tested with analysis of variance (ANOVAs) using SPSS software.

Results and discussion
Seven ANOVAs were calculated to test significant differences among the variables due to Bt cotton.ANOVA results for the dehydrogenase enzyme is given in Table 2. Cotton line was a significant source of variation in one of seven ANOVA's.Year block term was non significant in all seven ANOVA's, whereas month by cotton variety interaction source of variation being non significant for three variables.Therefore the interaction term was used to test the variation on account of cotton variety.
On average dehydrogenase, alkaline phosphatase, nitrate reductaase and urease enzymes activity was high in Bt cotton rhizosphere as compared to non Bt rhizosphere (Table 3).Except dehydrogenase enzyme, differences in activity of alkaline phosphatase, nitrate reductase and urease enzymes between Bt and non Bt plants rhizosphere were statistically non significant.Over the crop growth period (of 150 days) the activity of Phosphatase, nitrate reductaase and urease enzymes activity was high in the rhizosphere at early vegetative and flowering stage, whereas activity of dehydrogenase enzyme was high at boll formation stage (Figure 1).Activity of all the four enzymes declined at crop harvesting stage (Figure 1).On comparing enzymes activity for the interaction of crop variety (Bt and non Bt trait) over a monthly period, activity of dehydrogenase and urease enzymes were non significant and activity of Phosphates and nitrate reductase enzymes were significant (Table 3).
Significant differences (P<0.05) were observed in the number of nematodes, collembola and ants between the Bt and non Bt cotton rhizosphere (Table 4).Number of nematodes, collembola and ants were more in Bt plants rhizosphere as compared to non Bt plants rhizospere.Number of nematodes, collembola and ants were highest in Bt and non Bt cotton rhizosphere at flowering stage.
The biochemical properties of soil have often been proposed as early and sensitive indicators of soil ecosystem health (Oliveira and Pampulha, 2006).Activities of soil enzymes indicate the direction and strength of all kinds of biochemical processes in soil and act as key biological indicator of soil.Soil enzymes play an essential role in energy transfer, environmental quality, organic matter decomposition, nutrient cycling and crop productivity (Tabatabai, 1969;Kumar et al., 1992).Measurement of enzymes activity in combination with count of number of key microorganisms provides sensitive information of the changes occurring in soil (Brookees 1995).The results are consistent with an experiment conducted with Cry 1A Bt cotton (Sukang-103) and non Bt cotton (Sumian-12) (Shen et al., 2006).Their study demonstrated enhanced phosphatase, nitrate reductase and urease activity in the rhizosphere of Bt cotton, but most of the time differences in enzymes activity between Bt and non-Bt cotton rhizosphere were non significant.Similarly, Sun et al., (2007) also observed stimulated activities of urease, phosphatase, invertase and cellulase enzymes by the addition of Bt cotton tissues in soil.In present study variation in dehydrogenase enzyme activity between Bt and non-Bt cotton rhizosphere was significant (P<0.05) and activity of dehydrogenase enzyme was high in Bt cotton rhizosphere.This observation of the present study is consistent with the observation of Wu et al., (2004) observed enhanced dehydrogenase activity in soil incubated with Bt transgenic rice straw (during 7-14 days of incubation, later on it declined) compared to soil without straw.Results of the present study with respect to dehydrogenase enzyme were contrary to research performed by Liu et al. (2008) and Oliveira et al. (2008).They observed no significant differences in dehydrogenase activity in Bt maize and Bt rice rhizosphere.The enhancement in activity of dehydrogenase in Bt cotton rhizosphere may be due to altered composition of its root exudates.Root exudates have a profound qualitative and quantitative effect on the rhizospheric microorganisms (Schenck, 1976).
In the present study temporal variation was observed in number of nematodes, collembola and ants in Bt and non Bt plants rhizopshere.Temporal variation in number of nematodes, collembola and ants is a complicating factor in impact assessment of Bt transgenic cotton, because the impact of the genetic modification may be transient and minor compared to the variation caused by soil condition and growth stage of crop (Rasche et al., 2006).Number of nematodes, collembola and ants in Bt plants rhizosphere were significantly more than non Bt plants rhizopshere.This observation of the present study is contrary to Saxena and Stotzky (2001) observation.They observed that transgenic corn crops had no apparent effect on nematodes.However these results should be considered as preliminary, as nematodes, collembola and ants are mobile and slight change in their niche environment may affect their mobility.In general, we speculate that effects of Bt cotton on the number of nematodes, collembola and ants in rhizosphere might mainly depend on the extent of change in root exudates (quality and/or quantity) that may related to variety differences.More detailed studies on the composition and diversity of these groups of microorganisms using different molecular and biochemical techniques are necessary.
In conclusion, based on three year field experiments it was found that Mech 162 variety of Bt cotton has no adverse effect on soil biochemical and microbial indicators.The differences caused by growing Bt cotton were not as large as those resulting from seasonal changes indicating that effect of Bt cotton on soil ecology was within normal variation expected in conventional agriculture.

Figure 1 .
Figure 1.Variations in activity of (a) Dehydrogenase (b) Alkaline phosphatase (c) Nitrate reductase and (d) Urease enzymes at different growth stages in Bt and Non Bt cotton rhizosphere

Table 2 .
Example of ANOVA for dehydrogenase enzyme

Table 3 .
Enzymes activity in Bt and non Bt cotton rhizosphere during crop growth period

Table 4 .
Number of nematodes, collembola, ants in Bt and non Bt cotton rhizosphere