The development of genetically modified (GM) crops is a big bang in the history of agriculture which transformed the conventional agricultural system into an advanced one using genetic tools and molecular approaches ( Udriste & Badulescu, 2017 ). We have witnessed a revolution in traditional agriculture just after the successful advent of transgenic varieties in late 1970s. One of the leading GM traits is insect resistance, where Bacillus thuringiensis (Bt) genes transformed into the plant, express a toxic protein ( Cry ) which targets the gut epithelium of attacking insect and eventually protects the host plant from pathogens ( Lee et al. , 2016 ). Transgenic crops with Bt insecticidal gene can specifically protect itself from Lepidopterans, i.e. cotton bollworm, corn borer, tobacco beetle, pink bollworm and budworms ( Dutton et al. , 2002 ). The economical benefits of Bt planta­tion include the decreased pesticidal sprays and increased crop productivity. The global hectarage of Bt crop cultivation is reported to be greatly increased from 1.68 million hectares in 1995 to 181.5 million hec­tares in 2014 ( Liang et al. , 2018 ).

Despite the popularity of Bt crops, their regular cultivation, however, could result in unintentional effects on soil organisms. In view of this, a number of risk assessment studies have been conducted in order to evaluate the potential unintended effect of Bt crops on non-target organisms (NTOs). However, most of the outcomes of such studies were controversial Tsatsakis et al. , 2017 ). As such, the need for further safety assessment of Bt crops on NTOs, particularly the beneficial soil microbes cannot be ignored because Bt toxin, when expressed by plant cells, could influence the soil organisms by entering the soil either through plant residues or via root exudates ( Strain & Lydy, 2015 ). But, contrarily, further studies ruled out this threat by reporting a rapid degradation of Bt protein soon after its release into the soil ( Li et al. , 2016 ). Although it was declared that Bt protein does not persist for long term in soil, the risks are still associated with their short-term existence in rhizosphere, as the Bt proteins or even genetic material can possibly be taken up by soil microbes through natural phenomena of trans­formation. Hence, a critical investigation is needed to assess the potential impacts of Bt crop rhizosphere on soil bacteria specially the plant growth promoting rhizobacteria (PGPR) which are the most important and least studied group of microbiotas ( Yaqoob et al. , 2016 ).

PGPR are significant elements of plant rhizosphere, promoting plant growth and improving the soil structure through aggregate formation, nitrogen fixation, nitrate/nitrite reduction, mineral solubilization, increasing the organic contents of rhizosphere and phytohormone production ( Yaqoob et al. , 2013 ). Among PGPR, Pseudomonas and Bacillus sp. are widely distributed in soil but least studied for GM risk assessment. Therefore, these bacterial rods were selected specifically for the present study to assess the potential risks of Bt toxin on these soil bacteria.

Proteins Cry1AC and Cry2A were detected in all the collected soil samples. However, a significant decrease in concentration of Cry proteins was noticed when root pieces were soaked in water as compared with direct protein extraction method (Fig. 2).

Fig. 3 shows the bacterial colony count from both control and experimental groups during the two years. There were no significant differences ( p >0.05) in bacterial count at both experimental periods.

More than 90% of all the strains were found to be positive for nitrate reduction and nitrogen fixation tests. No noticeable difference was observed between the control and experimental strains by comparing their test results. However, significant decrease ( p <0.05) in phosphate solubilization ability was observed in experimental strains at maturity stages when compared with control (Fig. 4).

Significant potential for auxin biosynthesis was revealed by all bacterial strains upon the stimulation of L-tryptophan and a reduced auxin production was observed in experimental strains at maturity stages when compared with that of control isolates (Fig. 5).

The 16S rDNA sequences of experimental strains 03-N4, 03-N9, 01-I and 08-H were deposited in the NCBI under accession nos. JQ912576.1, AB920753.1, KP698780.1, and KF732997.1 respectively, and se­quences of control strains 04-P5 and 06-N5 under no. JN861778.1 and KP685409.1, respectively.

Analysis of a 450-650 bp sequence of 16S rDNA from six representative bacterial strains indicated that three strains (03-N9, 03-N4 and 04-P5) exhibited ≥90% similarity to Pseudomonas spp., while the other three (01-1, 08-H and 06-N5) exhibited ≥90% similarity to Bacillus spp. (Table 3).

Soil is the basic medium of growth for all plants. The soil ecosystem plays a significant role in food chain, housing not only the macroorganisms but also the plant growth promoting microorganisms. Besides this ecological role, the contribution of soil components in nutrient cycling is also worth mentioning: a primary role in nutrient cycling is played by soil microbes due to their diverse ability of solubilizing complex organic/inorganic compounds into simpler molecules or ions. These molecules or ions when dissolved in soil particles become easily available to plant roots for their proper growth and metabolism ( Prasad et al. , 2015 ). Therefore, plant rhizosphere is considered to be most important for risk assessment studies against Bt crop cultivation. The present study was planned to specifically assess the potential effects of Bt Cotton (VH-289) on soil bacterial count during two consecutive years (2017-2018). It was observed that both plant growth stage and planting date impacted soil microflora but the Bt trait was the weediest clarifying factor to impart any significant change. Our results were in agreement with those of Liang et al. (2015) , who found that cultivation of GM plants did not affect the soil microbial community structure.

Along with the biosafety assessment, flow rate of Cry proteins from roots into the external medium was also assessed to confirm previous contradictory studies on the release of Bt protein into the soil. For this purpose, both experimental and control plant roots were soaked in distilled water for two months and the water was then used for protein extraction and Cry1AC / Cry2A estimation through ELISA. It was revealed that very minute quantities of Cry proteins were released into the water as compared to total extracted protein (as shown in Fig. 5). Our findings are in accordance with Yaqoob et al. (2016) , who found a decreased persistence of Bt proteins in soil.

The biochemical activities of the bacterial isolates were found to be non-significantly affected. However, a reduced rate of phosphate solubilization and auxin biosynthesis was observed during maturity stages of both years when compared with that of early stages. Potentially this reduced auxin biosynthesis might be occurred as per the need of plant growth, i.e. more auxin is produced at early growth stages of plants for excessive root and shoot growth as compared to maturity stages. Similarly, environmental factors and weather conditions must also be kept in consideration while assessing the bacterial activities.

The impact of transgenic plants on bacterial communities in soil could be either temporary or have no effect at all ( Rashid et al. , 2016 ). According to Hannula et al. (2012) and Liang et al. (2018) minor changes in microbial populations that occur due to transgenic plants are mostly due to change in season and field and these changes do not persist in next field season. The findings of this research is in line with Li & Liu (2013) , Wu et al. (2014) and Zhang et al. (2015) , who found that Bt toxins released from transgenic plants had no short-span lethal effects on soil bacterial populations, but conclude that long-term impact due to toxin accumulation on biodiversity of soil needs to be evaluated.

Differences of temperature, rainfall and soil charac­teristics were observed in both planting years with significant differences in total soil pH ( p <0.01). These differences could also be the reason of effects on biochemical activities of bacteria as rhizobacterial activities are moderately affected by change in soil pH ( Simonin et al. , 2016 ). Different soil properties and other environmental parameters in different seasons significantly affect soil microbial population.

The present study concluded that presence of minute quantities of Bt toxin in soil rhizosphere is non-toxic to soil bacteria. No harmful effect is noticed on biochemical or molecular characteristics of isolated soil bacteria from Bt cotton rhizosphere except a decreased phosphatase activity in some of the strains. The in­formation presented in this study is useful for Bt crop development and its biosafety. However, more critical investigation is required to assess the risks of Bt crops on other soil micro and macrorganisms.