Genetically modified Bt maize lines containing cry3Bb1, cry1A105 or cry1Ab2 do not affect the structure and functioning of root-associated endophyte communities
Highlights
► We investigate potential effects of Bt-maize on bacterial endophytes ► We study endophyte diversity by cultivation-based and cultivation-independent analysis ► We investigate the plant growth-promoting potential of endophytic isolates ► Genetic modification did not affect the diversity and functional potential of endophytes.
Introduction
The major advantage of genetically modified (GM) maize expressing Bacillus thuringiensis (Bt) toxins is the reduced need for broad spectrum pesticides, providing benefits for human health and the environment. However, public concern has been raised regarding the release of transgenic crops because of potential environmental implications. GM maize varieties expressing various insecticidal crystal (Cry) proteins, derived from the soil bacterium Bt, are among the most important transgenic crops today. Since the introduction of genetically modified plants in 1996 the area of Bt maize cultivation has continuously increased up to an area of 39 million hectares worldwide and 91 thousand hectares in the EU in 2010 (James 2010).
Microbial Bt products specified as “bio-pesticides” have a long history of safe use (Glare and O’Callaghan, 2000). To enhance their insecticidal effect, genes encoding Bt toxins have been integrated into plant genomes. The “first generation” Cry1Ab protein confers to the plants resistance against lepidopteran insects (European corn borer, Mediterranean corn borer). Increased protection against lepidopteran pests was achieved by the introduction of a Bt expression cassette coding for cry1A.105 and the cry2Ab2, resulting in the transformation event MON88034, conferring resistance to the European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera ssp.), black cutworm (Agrotis ipsilon) and corn earworm (Helicoverpa zea). The chimeric CryA.105 protein contains sequences of the naturally occurring Cry1Ab, Cry1F, and Cry1Ac proteins, whereas Cry2Ab2 is a variant of the wild-type Cry2Ab protein isolated from Bacillus thuringiensis subsp. kurstaki. Resistance to coleopteran insect pests (Diabrotica spp.) was obtained by the expression of modified B. thuringiensis (subsp. kumamotoensis) cry3Bb1 by the transformation event MON88017. Conventional crossing of MON89034 and MON88017 generated the stacked maize line MON89034 × MON88017, comprising combined expression of insecticidal genes from the parental lines (EFSA, 2010).
Environmental risk assessment of GM plants includes the investigation of unintended effects on plant fitness due to genetic modifications (EFSA, 2010). Beneficial plant-microbe interactions are of particular importance for soil nutrient cycling, plant nutrition and resistance to biotic and abiotic stresses (Compant et al., 2010a, Domey and Lippmann, 1989, Glick, 2005, Lethbridge and Davidson, 1983, Rodriguez and Fraga, 1999, Yang et al., 2009).
Endophytes, living inside the plants tissue, are particularly known for their plant beneficial properties (Berg et al., 2005, Lottmann et al., 1999, Sessitsch et al., 2004). Changes in communities of plant-associated microorganisms, both in the rhizosphere or in the endosphere, may have adverse effects on the plants’ fitness and should be considered in a risk assessment of transgenic crops. Nonetheless, studies addressing plant-associated microbiota such as endophytes of transgenic crops are rare (Heuer et al., 2002, Rasche et al., 2006a).
Studies on soil microbes of Bt maize (event Bt176) producing Cry1Ab proteins have demonstrated consistent differences in fungal and bacterial communities compared to the conventional counterpart or another Bt maize (event Bt11) also expressing cry1Ab (Castaldini et al., 2005, Turrini et al., 2005). However, these effects could be related to possible changes in plant physiology and composition of root exudates of the specific transformation event Bt176, which affected rhizosphere microbial communities. Other studies have found only minor changes in the soil microbial community structure (Blackwood and Buyer, 2004, Brusetti et al., 2005, Castaldini et al., 2005, Griffiths et al., 2006, Mulder et al., 2006) or did not reveal negative effects on soil microbial communities due to the expression of cry1Ab (Flores et al., 2005, Hönemann et al., 2008, Saxena and Stotzky, 2001). Reported effects of the Cry1Ab protein on microbial communities were transient and were exceeded by other environmental factors (Fang et al., 2005, Filion, 2008, Griffiths et al., 2006, Griffiths et al., 2005, Lilley et al., 2006). Few studies have so far considered the expression of Cry3Bb1 proteins (Devare et al., 2007, Devare et al., 2004, Icoz and Stotzky, 2008), and they could not show any significant effects on soil microbial colonization. However, no information on potential effects of Bt maize MON88017 (cry1A.105 and cry2Ab2) and the stacked event MON88017xMON89034 (cry1A.105 and cry2Ab2, cry3Bb1) on plant-associated microorganisms is presently available.
The expression of yet poorly investigated cry genes might lead to subtle changes in plant metabolite composition and thereby cause alterations of associated endophyte communities as compared to the nearly isogenic wildtype. Therefore, the aim of the present study was to assess whether the expression of different novel cry gene combinations in the maize lines MON88017, MON89034 and MON88017xMON89034 leads to different endophytic communities as compared to the wildtype. To have some additional information on the baseline variation we included several additional non-transgenic maize lines in our study and cultivated plants on two different soils. Endophytic communities were characterized by cultivation and cultivation-independent analysis. Isolated strains were identified by 16S rRNA gene sequencing and tested for plant growth-promoting functions. Potential effects due to the genetic modification or other parameters on the endophytic community structure were assessed by 16S rRNA gene-based community analysis.
Section snippets
Containment experiment and sampling
We used seven different maize cultivars, including three transgenic cultivars (MON89034, MON88017, MON88017 × 89034), their near isogenic line DK315, and three conventional maize cultivars (DKC3420, DKC5143, Antares). MON88017 carries the cry3Bb1 gene mediating resistance towards Coleoptera, whereas MON89034 carries cry1A105 and cry2Ab2 conferring resistance towards Lepidoptera. The stacked event MON89034xMON88017 carries the combination of cry3Bb1, cry1A105 and cry2Ab2. Maize plants were grown
Identification of cultivable bacterial isolates
Seven hundred bacterial isolates were obtained from the root tissue of seven different maize lines grown on two different soil types. Based on RFLP analyses of the intergenic spacer region between 16S and 23S rRNA genes isolates could be grouped according to their banding pattern into 143 IGS types. Isolates from plants grown on Fischamend soil showed 102 distinct IGS types, whereas isolates obtained from plants grown on Tulln soil grouped in 81 distinct IGS types. Based on partial 16S rRNA
Discussion
Genetically modified plants are of high agricultural and economic importance in many countries. Despite their agricultural benefits their environmental safety needs to be considered. Hence, risk assessment has to warrant on a case-by-case basis that the genetic modification does not pose any unintended negative effects on the environment, including any alteration of important biogeochemical cycles (EFSA, 2010). Plant growth and health greatly depend on the surrounding soil environment, and the
Conclusions
In conclusion, we could identify highly diverse bacterial communities in the maize endosphere with various isolates showing high potential to promote plant growth by the production of ACC deaminase and IAA. These results emphasize the suitability and importance of endophytes as a part of risk assessment of transgenic crops. While an influence of maize cultivar and soil environment was detected, we could not find an evidence for the influence of the Bt constructs used in the transgenic maize
Acknowledgements
We are grateful to Anton Grahsl for help with the containment experiment and technical advice. This study was financed by the Federal Ministry of Agriculture, Forestry, Environment and Water Management and the Federal Ministry of Health.
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