Introduction

The use of genetically modified or transgenic plants has great potential for future agriculture, but asks for a well-defined risk assessment (Bruinsma et al. 2003). The Bacillus thuringiensis (Bt) gene-expressing product of transgenic Bt crops, B. thuringiensis insecticidal crystal protein (Bt toxin), could be introduced into soil through root exudation or through decomposition of the specific crop residues (Palm et al. 1996; Sims and Holden 1996; Saxena et al. 1999; Saxena and Stotzky 2000). Once in the soil, the toxin could be adsorbed or bound on clay particles, humic components, or organic-mineral complexes and then be protected against degradation by soil microorganisms; in this way it could accumulate to a certain concentration that might affect the composition and activity of soil microbial communities (Tapp and Stotzky 1995; Crecchio and Stotzky 1998; Tapp and Stotzky 1998; Stotzky 2000; Crecchio and Stotzky 2001).

The treatment of soil with Bt maize tissues showed that the bioactivity of the Bt toxin decreased quickly after tissue introduction into soil (Sims and Holden 1996), and then the decreasing rate lowered and even became zero when the Bt concentration reached a low value after several weeks (Palm et al. 1996). The free or bound Bt toxin had no effect on soil bacteria, fungi, algae, and protozoa and nematodes (Donegan et al. 1996; Saxena and Stotzky 2001).

Soil urease, acid phosphatase, arylsulfatase, invertase, and cellulase activities play an important role in soil microbial activity, being related to some important N, P, S, and C reactions, respectively (Nannipieri et al. 1990; Deng and Tabatabai 1997; Kandeler et al. 1999; Nannipieri et al. 2002). Jepson et al. (1994) pointed out that soil urease, dehydrogenase, and phosphatase activities could be used as indicators of the impact of toxins on soil microbiological activity. Wu et al. (2004) found that the phosphatase activity of paddy soils was not affected by the incorporation of Bt-transgenic rice straw into soil, whereas the dehydrogenase activity was increased. However, little is known about the effects of Bt toxin of transgenic Bt cotton tissues on soil enzyme activities. Therefore, we have studied the effect of Bt toxin from the decomposing transgenic Bt cotton tissues on soil urease, acid phosphomonoesterase, arylsulfatase, invertase, and cellulase activities.

Materials and methods

Materials and treatments

We used leaves and stems of transgenic Bt cottons Guo-Kang 12 (Bt-GK) and Zhong-Kang 30 (Bt-ZK), which were given by the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences, respectively; non-Bt cotton Zhong-Mian 30 (Non Bt-ZM) was used as the control. The tissues were air-dried, ground, and sieved (<2-mm screen). The average Bt toxin contents in ZK and GK tissues were 208.14 and 169.35 ng g−1, respectively.

The silty loam soil, sampled from the Shenyang Agricultural University of China, had pH 5.72 (1:2.5 water), organic C 14.62 g kg−1, total N 1.22 g kg−1, total P 0.49 g kg−1, total K 20.12 g kg−1, alkali-hydrolyzable N (1 N NaOH, 40°C, 24 h) 106.37 mg kg−1, available P (0.5 N NaHCO3, pH 8.5) 15.92 mg kg−1, and available K (1 N CH3COONH4) 106.37 mg kg−1. All chemical parameters of soil were determined as described by Lu (2000).

The tissues were moistened with distilled water, mixed with soil in a proportion of 1:3 (W/W), and incubated in 10-ml ventilated tubes at 26°C and at a soil moisture content of 20%. Twenty-one replicates were carried out for each treatment, and three tubes of each treatment were sampled after 1, 3, 7, 14, 21, 28, and 56 days, respectively.

Determination of Bt toxin concentration and enzyme activities

Buffer solution (EnviroLogix, Portland, ME, USA) (1 ml) was added to 0.5 g soil sample, then the soil mixture was mixed and centrifuged (13,000×g) at 4°C for 10 min; the Bt toxin concentration in the supernatant was determined by ELISA, using the EnviroLogix Cry1Ab/Cry1Ac Plate Kit (detection limit<0.25 parts per million) (Saxena et al. 1999).

Urease (EC 3.5.1.5), acid phosphomonoesterase (EC 3.1.3.2), and arylsulfatase (EC 3.1.6.1) activities were determined as described by Tabatabai (1994). Briefly, urease activity was determined using urea as substrate, and the soil mixture was incubated at 37°C for 5 h; the residual urea was determined by a colorimetric method. Acid phosphomonoesterase activity was determined with p-nitrophenyl phosphate as substrate, with incubation at pH 6.5 (modified universal buffer) and 37°C. After 1 h, 0.5 M CaCl2 and 0.5 M NaOH were added to precipitate humic molecules responsible for brown coloration and extract p-nitrophenol, respectively. Arylsulfatase activity was determined with p-nitrophenyl sulfate as substrate, with incubation at pH 5.8 (acetate buffer 0.5 M) and 37°C. The enzyme reaction was stopped as reported above.

Both invertase (EC 3.2.1.26) and cellulase (EC 3.2.1.4) activities were determined as described by Schinner et al. (1996). Briefly, invertase activity was determined using sucrose as a substrate and incubation at 50°C for 3 h, before measuring the produced glucose with a colorimetric method. Cellulase activity was determined with carboxymethyl cellulose sodium as a substrate with incubation at 50°C for 24 h, and measuring the produced glucose with a colorimetric method.

Statistical analysis

Comparisons among means were made using the least significant difference test, calculated at P < 0.05. Statistical procedures were carried out with software package SPSS 10.0 for windows (SPSS 2000).

Results and discussion

After amendment with cotton tissues, there was no Bt toxin detected in the non-Bt-ZM treatment. The soils treatment with Bt cotton tissues had a significantly higher content of Bt toxin, which rapidly decreased in the first 7 days of incubation, but then the relative decrease rate lowered and the Bt content remained almost unchanged after 28 days (Fig. 1). By the end of incubation (the 56th day), the Bt toxin content was still high (14.19–22.69 ng g−1 soil), representing 40.79% (Bt-ZK) and 59.98% (Bt-GK) of its initial introduced amounts. Throughout the whole period of incubation, the Bt toxin content was higher in Bt-ZK than in Bt-GK treatment (Fig. 1). Our study confirms that by Sims and Holden (1996), who showed that Bt toxin from amended Bt maize biomass decreased quickly after its introduction into soil.

Fig. 1
figure 1

Persistence of Bt toxin from amended Bt cotton tissues in soil and its influences on soil enzyme activities. For each enzyme activity, values sharing the same letter are not significantly different (P < 0.05) according to the least significant difference test

Full size image

The transgenic Bt cotton tissues and their degradation product had different effects on the tested enzyme activities of soil, with a positive effect on urease, acid phosphomonoesterase, invertase, and cellulase activities, and a negative effect on arylsulfatase activity (Fig. 1). Soil microorganisms are one of the main sources of soil enzymes (Nannipieri et al. 1983). The amendment with Bt-cotton materials supplied a higher content of organic product than the treatment with non-Bt-cotton materials, and this difference was probably responsible for the increase in urease, acid phosphomonoesterase, invertase, and cellulase activities of soil.

Acid phosphomonoesterase activity was significantly stimulated by Bt cotton tissue amendment, probably because these enzymes originate from bacteria, fungi, yeasts, and plant (Nannipieri et al. 1983; Nakas et al. 1987). Wu et al. (2004) showed that there were no significant differences in the soil neutral phosphatase activity between non-Bt-straw and Bt-straw amended soils. The difference between our results and those by Wu et al. (2004) might depend on the fact that the amount of Bt cotton tissues (1: 3 w/w) added to our soil was 10-fold higher than that (3% w/w) of Bt rice straw added to soil by Wu et al. (2004).

The inhibition of arylsulfatase activity by Bt toxin may be partly attributed to the increase in the soil phosphate due to the stimulation of soil acid phosphomonoesterase activity. Lou and Warman (1992) showed that phosphate inhibited the enzymatic hydrolysis of ester sulfate.

According to Falih and Wainwright (1996), soil urease activity was stimulated by plant material amendment probably because of the increase in microbial activity. The same probably occurred in this work where soil urease activity was stimulated by the Bt cotton tissue amendment.

The addition of plant straw to soil strongly increased invertase and cellulase activities (Stemmer et al. 1999; Glissmann and Conrad 2002), probably because this addition increase microbial activity. The same explanation can be suggested to explain the increase in invertase and cellulase activities observed here by the addition of Bt toxin in soil. Therefore, the stimulation of some soil enzyme activities after amendment with Bt cotton tissues depended on the stimulation by the addition of cotton tissue on microbial activity of soil. This effect can mask any negative direct effect of the Bt toxin on enzyme activities.