Soil metagenomics: Practical applications

Phytoremediation, which is the use of plants to clean up environmental pollution, has received much attention as a promising method for the removal of metal pollutants in soils (Cherian and Oliveira, 2005; Van Aken, 2008). Phytoremediation is a cost-effective and environmentally friendly approach compared to other environmentally invasive, expensive and inefficient clean-up technologies (Van Aken, 2008). A number of plant species are capable of high-level organic compound degradation or heavy metal hyperaccumulation. However, slow rates of removal and incomplete metabolism have restricted the application of phytoremediation in the field (Van Aken, 2008). Thus, genetically engineered plants that exhibit enhanced performance with respect to the metabolism of toxic compounds have been developed by the over-expression and/or introduction of genes from other organisms (Doty et al., 2007; French et al., 1999). Engineered poplars have greatly increased the possibility of the practical application of phytoremediation. However, this technology is still in the developmental stage, with the field testing of transgenic plants for phytoremediation being very limited. The major obstacle is biosafety concerns, because the potential unwanted effects of genetically modified organisms are not fully understood.

One of the most postulated potential unwanted effects of genetically modified (GM) plants is alteration to the structure of indigenous microbial communities. Micro-organisms have an important role in regulating soil conditions (Wolfenbarger and Phifer, 2000). Soil micro-organisms are in charge of the global cycling of organic and inorganic matter. A number of microbes decompose organic matter into forms useful to the rest of the organisms in the soil food web, and can break down pesticides and pollutants in soil. Soil microbes perform important services related to water dynamics, nutrient cycling and disease suppression. They also produce substances that constitute the soil structure (Conrad, 1996). Thus, alteration in the diversity or activity of microbial communities may have adverse effects on soil ecology (Kennedy and Smith, 1995), and understanding how GM plants, and plants in general, might alter the soil microbial community is of great interest.

The effect of GM plants on soil microbial communities remains highly controversial. Several studies have reported that microbial communities are clearly altered by engineered plants (Bruce et al., 2007; Donegan et al., 1999; Gyamfi et al., 2002; LeBlanc et al., 2007; Lee et al., 2011; Siciliano and Germida, 1999; Smalla et al., 2001). In contrast, other studies have shown that the associated changes in microbial communities with engineered plants are statistically insignificant (Dunfield and Germida, 2004; Heuer et al., 2002; Kim et al., 2008; Lottmann et al., 2000) or very minor (Di Giovanni et al., 1999; Donegan et al., 1995, 1999; Dunfield and Germida, 2003;

Griffiths et al., 2000; Gyamfi et al., 2002; Jain et al., 2010; Lukow et al., 2000; Schmalenberger and Tebbe, 2002). Most of these studies have used non-sequencing based methods, such as community-level physiological profiles (CLPPs), fatty acid methyl ester (FAME), DGGE and T-RFLP. These techniques are useful for evaluating differences in overall community structure, but these fingerprinting methods are limited in their capacity to detect minor changes and the components of these changes. In addition, the number of clone sequences (<100 sequences per sample) surveyed in a few studies (Kim et al., 2008; LeBlanc et al., 2007; Lee et al., 2011) is insufficient to determine overall community profiles.

Thus, to evaluate the effect of GM plant use on soil microbial communities, extensive sequencing-based community analysis was conducted, while controlling the influence of plant clonality, plant age, soil condition and harvesting season (Hur et al., 2011). The rhizosphere soils of GM and wild type (WT) poplars at a range of growth stages (i.e. rhizosphere of 1.5-, 2.5- and 3-year-old poplars) were sampled together with non-planted contaminated soil, and the microbial community structure was investigated by pyrosequencing the V3 region of prokaryotic 16S rRNA gene. Based on the results of DNA pyrosequencing, poplar type and growth stages were associated with directional changes in the structure of the microbial community. In detail, for both GM and WT poplars, the microbial community of poplars started separating from that of the control soil in the early stage of poplar cultivation (1.5 years), advanced to the middle-stage group (2.5 years), and finally reached the late-stage group (3 years), the composition of which was very different from that of the contaminated soil community. However, the rate of microbial community change was slower in WT poplars than in GM poplars. This phenomenon possibly occurs because of the more active metal uptake ability of GM poplars compared to WT poplars, which resulted in faster changes in the soil environment, and hence the microbial habitat. In conclusion, the shift in the microbial community structure to the late stage was driven faster by the effect of GM phytoremediation than WT phytoremediation. The results of the study demonstrated the superiority of NGS-based technique over traditional risk assessment approaches in the aspect of capacity to detect minor changes and the components of these changes. The next-generation sequencing-enabled metagenomics should be useful and can be widely applied to modern microbiology and bio-technology.

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