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Effects of three transgenic cotton hybrids (RCH-2 Bt, Bunny Bt and NHH 44 Bt) carrying cry1Ac gene on selected soil biological properties was studied under field condition in deep vertisol. Bt-cotton recorded significantly higher soil respiration rate (1.82 mg of CO2) than non-Bt cotton (1.77 mg) at 1 and 5% significance level. There was significant difference in FDA activities between the Bt-cotton (16.76 at 0-15 cm and 10.65 Fluorescin g/dwt at 15-30 cm) and non-Bt cotton (16.09 at 0-15 cm and 10.21 Fluorescin g/dwt at 15-30 cm). Bt cotton soil recorded significantly higher MBC (168.8 Î¼g/g) as compared to Non-Bt cotton (161.8Î¼g/g). Bacterial, yeast and actinomycetes population was found to be significantly higher in Bt cotton grown soil as compared to Non-Bt soil. The results of this study suggest that growing transgenic Bt cotton may not adversely affect the soil biological properties and pose no environmental risk.
Additional keywords: Transgenic Bt cotton; Soil respiration; Soil microbial biomass; Soil enzymes; Soil microbial population
Over the last decade considerable research effort has been directed at the study and assessment of the use of genetically modified microorganisms and plants in agriculture (Sharma and Ortiz, 2000). An important aspect of the bio-safety assessment of genetically engineered (Transgenic) plants is to study their impact on soil ecosystem including changes in the plant-associated microflora (Kowalchuk et al., 2003). Whilst considerable benefits have been identified for the control of disease, increase in crop yield, and the reduction in the use of herbicides and insecticides, concerns remain of the wider ecological impact that transgenic organisms may have on soil ecosystem function and biodiversity (Icoz and Stotzky, 2008). Most of the studies on impact of transgenic crops on soil properties carried world over were restricted to contained or controlled conditions (Biao Liu et al., 2005) Although some research has examined the environmental impacts of the "aboveground" portion of transgenic crops, relatively less research effort has examined the effects of these crops on soil microbially mediated processes and functions in soils (O'Callaghan and Glare, 2001; Bruinsma et al., 2003).
Bt cotton was approved for commercial cultivation in India in March 2002 after stringent assessment for biosafety and profitability (Morse, 2005) and the country now has 8 years of experience with the crop. In India, Bt cotton occupies more than 80% of area, and there has been wide health concern about its effect on non-target organism in soil and general soil health. Several workers have studied the effects of transgene products and transgenic cotton on the soil biological properties ( Ream et al., 1994; Donegan et al., 1995; Sims and Ream, 1997; Yu et al., 1997; Donegan and Seidler, 1999; Valasubramanian Ramaiah, 2001; USEPA, 2001; Al-Deeb et al., 2003; Schloter et al., 2003; Yu-Kui Rui et al., 2005; Ren Fang Shen et al., Sun et al., 2007; Head et al., 2007; Balachander et al., 2008; Sarkar et al., 2008, 2009). Though many scientific studies from different countries claimed that there is no harmful impact on soil health by transgenic cotton, the issues is not settled. Though India has maximum area under transgenic Bt cotton cultivation, there has been no comprehensive/conclusive field study to respond the problem. To our knowledge, no comprehensive field studies related to impact of transgenic Bt cotton growing on soil biological and microbiological properties have been reported from India.
Based on the facts, the present study was undertaken with an objective to assess the impact of growing transgenic cotton expressing cry1AC on selected soil biological and microbiological properties as assessed by soil respiration, microbial biomass carbon (MBC), Fluorescein diacetate hydrolysis (FDA), soil enzymes and soil microbial populations under field condition. The findings of the present work will be useful in acquisition of scientific data to produce an environmental impact and risk assessment report on effect on transgenic Bt cotton on soil health and identification of a monitorable key soil indicators for quick assessment of impact of transgenic Bt cotton on soil functions and ecosystem.
Materials and methods
Field design, sampling and soil baseline properties
Three popular cotton hybrids comprising of Gossypium hirsutum transgenic (expressing Cry1AC) and non-transgenic (RCH-2 Bt, Bunny Bt and NHH 44 Bt), were assessed with a randomized block design in triplicates for each cultivar under field condition. A control treatment was also included along with the main treatments in form of bulk soil to assess the soil quality changes with no crop. The experimental field was at the Central Institute for Cotton Research, Nagpur, India. The soil was deep black vertisols and the crop was raised under rainfed conditions during the rainy season (June-Dec) with 90 x 45 cm spacing. Normal agronomic practices were followed for raising the crop (basal fertilizer N:P:K: 90:45:45 kg/ha). Rhizosphere soils samples were collected just before the harvest of crop at two depths (0-15 and 15-30 cm) from three plants in the same experimental plot and mixed thoroughly to form a representative samples. The soil samples were labeled and transported back to the laboratory in polyethylene bags and stored at 4°C before analysis.
The baseline data on soil biological properties of the experimental block before experimentation are as follows: soil respiration- 0.93 and 0.52 CO2 (mg)/3d; Fluorescein diacetate hydrolysis (FDA activity)- 9.45 and 6.45 Fluorescin g/dwt; microbial biomass carbon (MBC)-98.5 and 65.4 Î¼g/g of soil; Microbial quotient (MQ)-0.003 and 0.001 qCo2; soil urease 0.61 and 0.42Î¼g NH4-N/g dwt/2h; soil dehydrogenase (DHA) 0.59 and 0.21µg TPF g-1 24h; bacterial count-15.4 and 9.6 cfu x 103/g; fungi-1.2 and 1.0 cfu x 103/g; yeast-6.8 and 2.3 cfu x 103/g and actinomycetes- 5.6 and 4.2 cfu x 103/g at 0-15 and 15-30 cm soil depth respectively.
Soil biological analysis
The selected soil biological parameters viz., soil respiration, microbial biomass carbon (MBC), soil dehydrogenase activity (DHA), soil urease activity and Fluorescein Diacetate Hydrolysis (FDA) were studied according to standard methods. Soil dehydrogenase activity was determined by colorimetric measurement of the reduction of 2,3,5-triphenyltetrazolium chloride to triphenylformazan according to the method of Tabatabai (1994). Soil respiration was measured according to a modification of the soda lime method originally developed by Edwards (1982). Microbial biomass C (MBC) was estimated by the chloroform-fumigation-extraction method (Jenkinson and Ladd, 1981; Bremner and Kesssel, 1990) and the microbial quotient (MQ) was calculated as the ratio of MBC to soil respiration (Anderson and Domsch, 1989). Urease activity was determined according to method described by Tabatabai and Bremner (1972). Fluorescein diacetate hydrolysis was measured following the method of Schnürer and Rosswall (1982) using 3,6- diacetyl fluorescein as substrate and measuring the absorbance of released fluorescein at 490 nm.
Soil microbiological analysis
Samples (10 g, fresh weight) were serially diluted in 90 ml Ringers solution up to 10-3 dilution and 1 ml of the aliquot was pour plated into selective media (Nutrient agar for bacteria) (Allen, 1959), Martin's rose Bengal agar for fungi (Martin, 1950), Ken-knights and Munaier's agar (Allen, 1959) for actinomycetes and buffered yeast agar for yeast. The plates were incubated at optimum temperature (250C±10C) for 3-7 days in triplicates. The functional/physiological groups of microbes were enumerated by following standard microbiological methods (Wollum, 1982). The functional groups from the soil samples were enumerated using Pikovskaya agar (Pikovskaya, 1948) for phosphorus solubilizing microbes (PSM), Waksmann No.77 media for Azotobacter, Beckings media for Beijerinckia (Becking, 1959) and Kings-B for fluorescent pseudomonads (King et al., 1954). The microbial colonies appearing after the stipulated time period of incubation were counted as Colony Forming Units (Cfus)/g fresh weight of the sample. The colony characteristics were observed and representative single colonies were isolated and sub cultured on respective media. Cell morphology was observed microscopically after staining. All the bacterial cultures were identified according to Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). Identification of yeast and fungi were done as per the available standard manual (Barnett, 1960; Watanabe, 1994).
Soil property measurements were statistically analyzed using the procedure suggested by Gomez and Gomez (1984) for randomized block design, using Microsoft Excel and SPSS packages. Least significance difference (LSD) at P=0:05 were tested to determine the significant difference between treatment means.
Soil under Bt-cotton recorded significantly higher soil respiration rate (mean value =1.82 for 0-15 cm and 0.79 for 15-30 cm soil depth) than non-Bt cotton (mean value =1.77 for 0-15 cm and 0.75 for 15-30 cm) at 1% and 5% significance level (Table 1). There found to be significant differences in FDA activities between the Bt-cotton (16.76 at 0-15 cm and 10.65 Fluorescin g/dwt at 15-30 cm soil depth) and non-Bt cotton (16.09 at 0-15 cm and 10.21 Fluorescin g/dwt at 15-30 cm) treatments at both 1% and 5% level (Table 1). Higher FDA value was recorded with NHH 44 Bt followed by RCH-2 Bt.
There found to be significant differences in soil microbial biomass C (MBC) values between Bt and Non-Bt cotton hybrids. Bt cotton soil recorded significantly higher value at both the depths (168.8 and 134.9 Î¼g/g at 0-15 and 15-30 cm soil depth respectively) as compared to Non-Bt cotton (161.8 and 131.6 Î¼g/g) (Table 1). Microbial quotient (MQ) was also found to be higher in Bt cotton as compared to Non-Bt cotton at both the depths.
The soils under Bt-cotton recorded significantly higher soil urease and soil dehydrogenase activities than under non-Bt and no-crop. Bt cotton soil recorded significantly higher mean value of soil urease activity at both the depths (1.55 and 0.77 42Î¼g NH4-N/g dwt/2h at 0-15 and 15-30 cm respectively) as compared to Non-Bt cotton (1.36 and 0.70 42Î¼g) (Figure 1). Bt cotton soil had significantly higher mean value of soil dehydrogenase activity (1.38 and 1.0621µg TPF g-1 24h at 0-15 and 15-30 cm respectively) as compared to Non-Bt cotton (0.91and 0.7421µg TPF g-1 24h) (Figure 2).
Bacterial population was found to be significantly higher in Bt cotton grown soil as compared to Non-Bt soil, its population was 36.8 and 28.78 cfu x 103/g at 0-15 and 15-30 cm soil depth respectively in Bt cotton, while Non-Bt cotton recorded 27.6 and 24.5 cfu respectively (Table 2). Yeast and actinomycetes population followed the same trend as that of bacteria. Bt cotton soil recorded significantly higher fungal counts at 5% level, while 1% level produced non-significant value at 0-15 cm depth. There found to be no significant differences in fungal populations between Bt and Non-Bt cotton at 15-30 cm soil depth. The data on differences in functional microflora population in Bt and Non-Bt cotton is presented in Table 3. There found to be no significant differences in population of functional microflora viz., Azotobacter spp., Beijerinckia spp and Phosphorus solubilising microbes between Bt and Non-Bt cotton soil at both the depths at 1% level. However, Bt cotton recorded significantly higher Beijerinckia spp (15-30 cm at 5% significance) and Fluorescent Pseudomonad's counts (0-15 cm at 1 and 5% significance level) as compared to Non-Bt cotton. Soil respiration found to highly correlate with soil urease activity and MQ, while urease activity was found to positively correlate with MQ at 0-15 cm soil depth (Table 4). Soil DHA showed positive correlation with all the parameters, while FDA showed significant correlation with MBC. Higher positive correlation for all the soil biological parameters was observed at 15-30 cm soil depth as compared to 0-15 cm (Table 5).
An important aspect of the bio-safety assessment of genetically engineered (Transgenic) plants is to study their impact on soil ecosystem including changes in the plant-associated microflora (Kowalchuk et al., 2003). Respiration activity was frequently used to evaluate soil quality, soil fertility or soil contamination with organic pollutants or heavy metals (Brookes, 1995, Kubát et al., 2002) and for the evaluation of the effect of the change in land use (VoÅ™íÅ¡ek et al., 2002). Soil respiration, a common measure of soil biological activity, represents the amount of CO2 evolved from roots, soil microbes, and to a lesser extent by oxidation of root exudates, plant detritus and humified organic matter (Raich and Schlesinger, 1992). Measurements of soil CO2 emissions therefore provide useful insights into soil C cycling, and provide a basis for evaluating soil C dynamics and potential C sequestration under different crop management systems (Paustian et al., 1990; Paul et al., 1999). Several studies provide evidence that seasonal changes in soil respiration rates correlate with plant growth processes (Kuzyakov and Cheng, 2001; Franzluebbers et al., 2002). The increased soil respiration in soil grown with Bt cotton in the present study may be the consequence of both microbial growth and stimulation of microbial activity by enhanced resource availability, as well as of changes in microbial community composition.
Nayak et al. (2007) suggested FDA hydrolysis as a sensitive indicator of soil microbial activity changes. Generally more than 90% of the energy flow in a soil system passes through microbial decomposers; therefore an assay which measures microbial decomposer activity will provide a good estimate of total microbial activity. Non-specific esterases, proteases and lipases, which have been shown to hydrolyse FDA, are involved in the decomposition of many types of tissue. The ability to hydrolyse FDA thus seems widespread, especially among the major decomposers, bacteria and fungi (Schnurer and Rosswall, 1982). Sánchez-Monedero et al. (2008) found an increase of both FDA hydrolysis and respiration in soils freshly amended with compost. The higher FDA activity in Bt cotton soil in this study directly indicates the healthy soil condition and total microbial activity.
The microbial biomass is an essential component of nutrient cycling in the agro-ecosystems. Soil management practices strongly affect the size of the microbial biomass pool. Possibly a greater amount of root exudates and readily metabolisable C are perhaps the most influential factors contributing to as much as increase in MBC in soils under Bt-cotton. Lynch and Panting (1980) reported that soil MBC increased with root growth and rooting density of the crop. The metabolic quotient, also known as the respiratory quotient (qCO2), is frequently used to determine stress in the microbial population (Anderson and Domsch, 1993) and measures the ratio of respiration to the soil microbial biomass (SMB). The qCO2 index is an important ecosystem index contributing to the understanding of the SOM recycling rate (Parker et al., 1983).It is assumed that the soil microorganisms produce more CO2-C per unit microbial biomass per unit time as stress increases and hence results in an increase in qCO2 (Anderson and Domsch, 1993). Earlier, Sarkar et al. (2008, 2009) reported significantly higher root volume, MBC, MBN, MBP and MQ in Bt cotton than non-Bt cotton isoline.
Urease is an important enzyme in soil mediating the conversion of organic nitrogen to inorganic nitrogen by the hydrolysis of urea to ammonia. Increase in soil urease activity with increasing organic matter content has been already reported (Tabatabai and Bremner, 1972; Ladd, 1985). Urease activity was significantly correlated with organic carbon (OC) and total nitrogen (TN). Soil dehydrogenase is an indicator of overall microbial activity, because it occurs intracellulary in all living microbial cells and is linked with microbial oxydoreduction processes (electron transport chain). Dehydrogenase activity reflects the oxidative activity or intensity of metabolism of soil microflora and can be used as an indicator of microbial activity or populations in soils (Nannipieri et al., 1990). Earlier, Sun et al. (2007) reported higher soil urease, acid phosphomonoesterase, invertase, and cellulase by the addition of Bt cotton tissues, supporting similar observations on a typic Haplustept soil (Mandal et al., 2007). Higher nitrate reductase, acid and alkaline phosphatase activities were also reported by Sarkar et al. (2009) in the soil under Bt-cotton.
Higher microbial counts in transgenic cotton grown soil have also been reported by several other workers in cotton (Yu-Kui Rui et al., 2005; Ren Fang Shen et al., 2006; Balachandar et al., 2008).The difference in the numbers of the functional bacteria in the rhizosphere may be due to different crop cultivars with different root exudates and root characteristics. Root exudates have a profound qualitative and quantitative effect on the rhizosphere microflora (Schenck, 1976; Donegan et al., 1995; Rengel et al., 1998). Genetic manipulation or tissue culturing of the plants had produced a change in plant characteristics aside from Bt toxin production that could influence the growth and species composition of the soil microorganisms (Donegan et al., 1995). Moreover, significantly greater populations of different microbial groups have been reported in field plots under transgenic alfalfa (Medicago sativa L.) (Donegan and Seidler, 1999), cotton (Donegan et al., 1995), papaya (Wei et al., 2006), and maize (Griffiths et al., 2006).
Ren Fang Shen et al. (2006) suggested that there was no evidence to indicate any adverse effects of Bt cotton on the soil ecosystem in their study. Head et al. (2002) reported that Cry1Ac protein was undetectable from soil samples of six fields that are under cultivation of Bt cotton from last 3-6 yrs and have incorporated Bt cotton plant residues by postharvest tillage. Binoy Sarkar et al. (2009) studied the impact of Bt-cotton over the non-Bt isoline on soil properties and concluded that there were some positive or no negative effects of Bt-cotton on the studied indicators, and therefore cultivation of Bt cotton appears to be no risk to soil ecosystem functions. Ream et al. (1994) in a lab study on cotton carrying Cry 1Ab, Cry1Ac, Cry3Aa proteins reported no persistence of proteins in soil; proteins degraded in soil with a half-life of 20 days. Purified proteins and Cry proteins (Cry 1Ab, Cry1Ac) from cotton tissue decreased rapidly, with a half-life of approximately 4 and 7 days, respectively, by ELISA (Palm et al., 1996). Half-life of bioactivity Bt cotton expressing Cry 2A was estimated at 15.5 days by insect assay (Lab) and Half-life of bioactivity was estimated at 31.7 days by insect assay (Field) (Sims and Ream, 1997). No detectable level of protein (Cry 1Ac) in soil for 3-6 consecutive years by Bt cotton cultivation was reported by Head et al. (2002). Valasubramanian Ramaiah (2001) in a lab study concluded that transgenic cotton expressing Cry 1Ac has no adverse effect on earthworms (Eisenia fetida). Cultivation of Bt cotton expressing Cry1Ab, Cry1Ac has no effects on Collembola (Folsomia candida) population USEPA (2001) and Yu et al. (1997).
Our study clearly demonstrated that growing transgenic Bt cotton has no negative impacts on soil properties, but significant positive effect on soil biological indicators with different magnitudes on soil respiration, MBC, microbial quotient, FDA hydrolysis, soil urease activity, dehydrogenase activity and microbial populations has been observed with growing Bt cotton. These results suggest that cultivation of Bt cotton probably not pose any ecological or environmental risk.