Research Article

Effect of the Leaf Litter of Transgenic Populus Simonii P. nigra on the Soil Microbial Community and Horizontal Transfer Possibility of the Foreign Gene  

Mu Peng1 , Syed Sadaqat Shah 2 , Qiuyu Wang1 , Fanjuan  Meng1
1 School of Life Science, Northeast Forestry University, Harbin, 150040, China
2 Key Laboratory of Vegetation Ecology, Ministry of Education, Institute of Grassland Science, Northeast Normal University, Changchun 130024, China
Author    Correspondence author
Molecular Microbiology Research, 2017, Vol. 7, No. 1   doi: 10.5376/mmr.2017.07.0001
Received: 26 Jun., 2017    Accepted: 20 Jul., 2017    Published: 27 Jul., 2017
© 2017 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Peng M., Shah S.S., Wang Q.Y., and Meng F.J., 2017, Effect of the leaf litter of transgenic Populus simonii×P. nigra on the soil microbial community and horizontal transfer possibility of the foreign gene, Molecular Microbiology Research, 7(1): 1-9 (doi: 10.5376/mmr.2017.07.0001)

Abstract

To study the potential influence of transgenic Populus simonii×P. nigra on the soil microbial community composition and horizontal exogenous gene transfer, fresh transgenic leaves were treated in broadleaf and coniferous forest soil for 6 months in the laboratory. The results showed that DNA fragments of the target and marker genes were completely degraded before 120 days. The dominant colony is the bacterium whose number is much more than that of other microbes under the same treatment. The number of cultivable bacteria treated by transgenic litters was significantly higher than the number treated by non-transgenic litters in selective medium with kanamycin. The target gene in the entophytic and soil bacteria, with 1.92% and 3.08% PCR-positive results, was found only in broadleaf forest soil. This result showed a certain possibility of horizontal gene transfer from the transgenic poplar to soil bacteria.

Keywords
Transgenic poplar; Bt-the spider insecticidal peptide gene; NPTII gene; Soil microbe; Horizontal gene transfer

Introduction

Transgenic plants have great economic and social benefits for the pharmaceutical sector, the food industry, and agriculture and animal husbandry, but they are also associated with some level of risk. One identified risk is their effect on soil environments, microbial communities, and horizontal gene transfer in particular. Transgenes may escape and enter wild gene pools when transgenic plants are released into the field. In addition, the instability of transgene expression may cause unforeseen negative effects on plant fitness after genetic transformation (Stewart et al., 2000).

 

The horizontal transfer of hereditary substances is ubiquitous in natural ecosystems. Horizontal transfer is a fundamental evolution and adaptation mechanism of bacteria (Garcia-Vallvé et al., 2000; Rensing et al., 2002). Traditionally, it has been believed that microorganisms propagate asexually, with little or no exchange of hereditary substance among diverse species (Jain et al., 2002). However, some soil bacteria can undergo natural or passive genetic transformation under certain circumstances (Paget and Simonet, 1994; Demanèche et al., 2001). In soil ecosystems, there is an interdependent association among plants, microbes, and the environment (Wheatley, 2009). Plant inputs from plant detritus and root exudates are the major drivers of soil ecosystem functioning (Wheatley, 2009). The quantity and chemical constituents of these inputs vary by plant species; thus, different plant species can have differing effects on soil. Microbes are essential in the mineralization and decomposition of plant inputs. Horizontal gene transfer may occur when microbes are in contact with plant inputs. Thus, the possible impacts of transgenic plants on soil ecosystems and horizontal gene transfer from transgenic plants to soil microorganisms are worth consideration.

 

The influence of transgenic plants on natural ecosystems has been widely reported. Some researchers have stated that the impacts of transgenic plants on microbial community and horizontal gene transfer between transgenic plants and soil bacteria were negligible (Devare et al., 2004; Riglietti et al., 2008). However, Hur et al. (2012) reported that transgenic poplar can affect the diversity of indigenous fungal communities during metal phytoremediation. In addition, Axelsson et al. (2011) revealed that transgenic plants can affect adjacent waterways in unanticipated ways, which could change the composition of the aquatic insect colonies on the leaf litter and increased the average insect abundance. One recent laboratory feeding trial showed that transgenic corn by-products reduced the growth and increased the mortality of nontarget stream insects (Rosi-Marshall et al., 2007).

 

In recent years, an increasing number of transgenic crops have been released into the field. The USA is a leader in planting transgenic crops, with 54.6 million hectares planted as of 2006, accounting for 53% of the global transgenic crop area (Icoz and Stotzky, 2008). Herbicide-tolerant transgenic crops accounted for 68% of the global transgenic crop area, while insect-resistant Bt crops accounted for 19%. Combined insect-resistant and herbicide-tolerant transgenic crops accounted for 13% (James, 2006). Relative to transgenic crops, only a few transgenic tree species have been tested in the field. A search of publicly accessible databases worldwide yields approximately 700 field trials of transgenic woody plants (Walter et al., 2010). However, little information is available regarding what was actually investigated in these studies. Because transgenic trees are perennial and anemophilous, they will have stronger and longer-lasting effects on the natural ecosystem than transgenic crops, especially on the soil ecosystem (Kang et al., 2004). The release of transgenic trees may create problems in the soil ecosystem, potentially changing the soil physical and chemical properties, the structure of the soil microbial community, and the entire soil microenvironment (Hawes, 1990; Maloney et al., 1997; Sørensen, 1997). All of these problems require further study.

 

The objective of this study is to assess the effect of transgenic Populus simonii×P. nigra on the soil microbial community and the possibility of horizontal gene transfer from transgenic poplar to soil microbes. We hope to contribute to the basic data on the potential ecological effects of releasing transgenic trees on the forest environment.

 

1 Results and Analysis

1.1 Decomposition of the leaves and foreign genes in the soil

The total DNA of the treated leaves was extracted after 10, 20, 30, 60, 90, and 120 days, with the remaining target and marker genes being detected using PCR. The target gene could be detected clearly after 10, 20, 30, and 60 days (Figure 1c). The marker gene could be observed until 90 days after the leaf decomposition began (Figure 1b). Neither the target nor marker genes were observed after 120 days (Figure 1a).

 

 

Figure 1 Poplar leaf decomposition in two soil types and DNA degradation of the transgenic genes

Note: a): 1-B: the broad-leaf forest soil treated by non-transgenic leaves after 30 days degradation, 1-C: the coniferous forest soil treated by non-transgenic leaves after 30 days degradation, 2-B: the broad-leaf forest soil treated by TT1 transgenic leaves after 30 days degradation, 2-C: the coniferous forest soil treated by TT1 transgenic leaves after 30 days degradation, 3-B: the broad-leaf forest soil treated by TT1 transgenic leaves after 60 days degradation, 3-C: the coniferous forest soil treated by TT1 transgenic leaves after 60 days degradation.b): NTPII gene; c): Bt, the spider insecticidal peptide gene; M: marker; P: positive control; N: negative control; band 4-band 8: transgenic samples treated after different decomposition durations

 

1.2 Changes in the number of soil microbial communities

Three types of microbes from two types of forest soil were cultivated and counted, including bacteria, actinomycetes, and fungi (Figure 2). Similar changes could be seen in the numbers of all three microbial types, with increasing and decreasing trends occurring over the course of the leaf decomposition. The highest values were generally observed on the 20th day of leaf degradation: 4.43×105~5.27×105 CFU/mL for bacteria, 8.20×104~11.40×104 CFU/mL for actinomycetes, and 6.50×104~8.17×104 CFU/mL for fungi. For the control soil, the number of all microbes decreased over the course of leaf decomposition, and the numbers were much lower than those in the other three treatments. This was true for both broadleaf and coniferous soils.

 

 

Figure 2 The number of cultivable soil microbes by treatment day

Note: CKs is the soil without leaves; CKp is the soil treated by non-transgenic ramet; TT1 is the soil treated by TT1 transgenic ramet; TT3 is the soil treated by TT3 transgenic ramet. Error bars represent one standard deviation with 5 replicates per sample

 

The microbial number differs significantly by microbial type, treatment, and decomposition period. Variance analysis reveals strong variation among all treatments in bacterial number for broadleaf forest soil (F=158.39, P<0.001) and coniferous soil (F=10.484, P<0.001), fungal number for broadleaf soil (F=6.532, P<0.01) and coniferous soil (F=32.214, P<0.001), and actinomycetic number for broadleaf soil (F=20.26, P<0.001) and coniferous soil (F=45.553, P<0.001). There are no significant differences in fungal or actinomycetic number between broadleaf soil and coniferous soil, but the bacterial numbers differ greatly between broadleaf and coniferous soil for the same treatment (F=6.804–58.23, P<0.05). Bacteria are the dominant microbial group in the horizontal transfer study of the exogenous gene, as the bacterial number is much higher than the fungal and actinomycetic numbers.

 

1.3 The number of bacterial KanR clones

The number of bacterial clones cultured in selective medium is significantly less than that in non-selective medium. In non-selective medium, there is no significant difference in the clone number between the transgenic ramets (TT1 and TT3) and non-transgenic ramet (CKp). In selective medium, the bacterial number differs significantly among treatments (F=15.468, p<0.05), as does the percentage (F=56.3025, p<0.01). The number of KanR bacterial clones treated by transgenic litter was approximately 3 times higher than the number in the non-transgenic control in selective medium (Table 1). This finding indicates that the transgenic litter could greatly increase the number of soil bacterial clones with antibiotic resistance.

 

 

Table 1 Clone number and percentage of cultivable soil bacteria among different treatments

Note: a: soil without leaves; b: soil treated by non-transgenic ramet; c: soil treated by TT1 transgenic ramet; d: soil treated by TT3 transgenic ramet

 

1.4 Horizontal transfer of the target and marker genes

Two types of bacteria from transgenic treatments were cultured: soil bacteria and entophytic bacteria (Table 2). The bacterial DNA were extracted from different treatments, and PCR reaction was performed using the primers designed by sequences of the target and marker genes of transgenic poplar to ensure the horizontal transfer of transgenic genes from the plants to soil bacteria (Figure 3). All percentages of PCR-positive results for the NPTII gene in the bacteria treated by transgenic litter exceed 40%, and those from broadleaf forest soil are higher than those from coniferous forest soil. The percentages of PCR-positive results for the NPTII gene for the bacteria from CKs and CKp are only 9.38% and 12.5% in broadleaf forest soil and 6.67% and 11.43% in coniferous forest soil, respectively. Variance analysis shows that the percentages of PCR-positive results for the NPTII gene differ significantly among treatments (F=102.513, P<0.01). NPTII-positive results are slightly more common for the entophytic bacteria than the soil bacteria. The target gene in bacterial clones from coniferous forest soil is not detected, and only 3 bacterial clones from broadleaf forest soil are detected, with percentages of 1.92% for soil bacteria and 3.08% for entophytic bacteria.

 

 

Table 2  PCR-positive results of the target and marker genes from cultivable bacteria for different treatments

 

 

Figure 3 PCR results for the marker and target genes in cultivable bacteria

Note: (a): NTPII gene; (b): Bt, the spider insecticidal peptide gene; (c): PCR-Southern blotting of the NTPII gene; (d): PCR-Southern blotting of Bt, the spider insecticidal peptide gene; M: marker; P: positive control; N: negative control; band 4-band 11: PCR-positive results of marker and target genes in soil bacteria

 

In a laboratory study, Donegan et al. (1995) found that a transient increase in the cultivable population of soil bacteria and fungi was caused by two-thirds of the transgenic cotton lines tested. In a field experiment, in the first growing season, transgenic potato lines expressing the cysteine proteinase inhibitors appeared to have no effect on the abundance and metabolic activity of the soil microbial community. However, in the second growing season, compared with the control, the microbial abundance was decreased by 23% in the transgenic treatment (Cowgill et al., 2002). Our study found that there was a large difference in microbial number among different treatments, indicating that transgenic poplar might affect the microbial composition and abundance.

 

The release of transgenic plants in a soil environment may increase the risk of contamination by transgenic DNA. Douville et al. (2007) reported that the exogenous gene from Bt maize was found in nearby streams and rivers, occasionally even several kilometers downstream, indicating contamination by agricultural transgenic DNA. Some studies have detected the recombinant DNA of transgenic plants in soil bacteria, although some controlled studies have demonstrated natural transformation (De-Vries et al., 2004). (Tepfer et al., 2003) reported foreign gene transfer from different transgenic plants to soil bacteria when neomycin phosphotransferase (NPTII) was used as a marker gene. Gene transfer was detected in the presence but not in the absence of homologous NPTII receptor bacteria. Similar results were found in the present study. The number of KanR bacterial clones treated by transgenic litters was significantly higher than the number of non-transgenic controls in selective medium. Thus, it appears that the transgenic litters affected the antibiotic resistance of the soil bacteria.

 

In the present study, the percentage of PCR-positive results for the NPTII gene was much higher for the transgenic treatments compared to the non-transgenic treatments. This rapid change in the number of resistant clones in soil bacteria might be caused by the horizontal transfer of foreign genes, but this is difficult to prove. In general, many resistant genes, including the KanR gene, naturally exist in many environmental microorganisms that can multi-replicate along with rapid bacterial propagation. Additionally, natural transformation has been shown to occur in approximately 90 different species of prokaryotes, including the soil microorganisms Bacillus subtilis and Acinetobacter spp. (De-Vries et al., 2004). However, 3 bacterial clones were detected with PCR-positive results for target genes from the entophytic and soil bacteria only in broadleaf forest soil (Table 2), which could be the first evidence of horizontal transfer of the exogenous genes. The PCR-Southern blotting results are consistent with the PCR results. This horizontal gene transfer might also be related to the flora and soil environment.

 

2 Materials and Methods

2.1 Materials

The strain and vector were donated from the Faculty of Life Sciences of Beijing University. The vector pYHY was transformed into Populus simonii×P. nigra by Agrobacerium tumefaciens (Smith and Townsend) LBA4404 in the National Key Laboratory of Northeast Forestry University. pYHY is 1.39 Kb in size, including a fusion gene composed of the C-terminus of the toxin gene from Bacillus thuringiensis and the spider insecticidal peptide gene as the target gene (800 bp) as well as NPTII and GUS gene as the marker gene (500 bp).

 

A three-year field trial of insect-resistant transgenic Populus simonii×P. nigra expressing both insecticidal crystal and spider insecticidal peptide protein was conducted in the Tree Breeding Base of Northeast Forestry University near Harbin in Northern China. All sample leaves were collected from the same poplar clone, including two transgenic ramets named TT1 and TT3 and one non-transgenic ramet named CKp as the plant control.

 

Soil from broadleaf and coniferous forests of the Maoershan forest station of Northeast Forestry University, 90 km from Harbin, were used as the media to treat the leaves.

 

2.2 Treatment

The soil was collected in a 30 cm×40 cm plastic bucket. Fresh leaves (1-3 g) were collected from one-year-old branches, sealed in a 10 cm×10 cm nylon net bag, and buried in the soil at a depth of 14-16 cm for 6 months in the laboratory. Four treatments were prepared for each soil type: the soil without leaves as the soil control (CKs), the soil treated by non-transgenic leaves (CKp), the soil treated by TT1 transgenic leaves (TT1), and the soil treated by TT3 transgenic leaves (TT3). Five replications were conducted for each treatment. Because the leaf decomposition was fast in the beginning of treatment, the soil and degraded leaves from each treatment were sampled every 10 days in the first month and then once per month thereafter.

 

2.2.1 Soil microbial separation and count

First, 10 g of freshly sieved soil was added to 90 mL of sterile saline water, after which 10-1 soil dilutions were obtained. For the plate count test, 1-mL aliquots of different dilutions were transferred to plates with different solid media as follows. The cultivable soil microbes were separated in the Luria-Bertani medium for bacteria, Martin medium for fungi, and Gause’s synthetic agar medium for actinomycetes and then counted by the plate dilution method (Yao and Huang, 2006).

 

After 120 days of leaf degradation, two types of soil bacterium were separated by LB medium with 50 mg/L kanamycin. One type of bacterium was from the treated soil, and the other was from the decomposed leaves treated by rinsing, sterilizing in alcohol, and rinsing again before the entophytic bacteria were cultivated. Bacteria cultivated in the medium without kanamycin were also measured as the control.

 

2.2.2 DNA extraction and PCR reaction

The genomic DNA of the decomposed leaves was extracted by the CTAB method (Clark, 1998). The total DNA of the cultivable entophytic and soil bacteria was extracted by the CTAB/NaCl method (Li and Jin, 2006). The DNA concentrations were determined using a UV-1800 UV-Vis spectrophotometer (Shanghai Lorderan Scientific Instrument Co., Ltd.).

 

The primers were designed according to sequences of the target and marker gene. The target gene was amplified by primer 1 (5`-ATGTCTCCAACTTGCATTCC-3`) and primer 2 (5`-GTGGATCCTTAATCAATSTG-3` S=G/C.). The marker gene was amplified by primer 3 (5`-CCTGTCCGGTGCCCTGAATGAAC-3`) and primer 4 (5`-CGGCCACAGTCGATGAATCCAGAAAA-3`) on an MJ/PTC-100 PCR instrument (MJ Research, Reno, NV). The PCR reaction contained 2 µL of 1 × PCR buffer, 2 µL of dNTP (0.2 mmol/L), 10 pM of each primer, 1.5 units of Taq polymerase, and 10 ng of template DNA, which was raised to a constant volume of 20 μL using ddH2O. The cycling parameters were 94ºC for 5 min, followed by 40 cycles of 94ºC for 1 min, 56ºC for 1 min, and 72ºC for 2 min, with a final extension at 72ºC for 7 min. The reactions were performed in duplicate, and negative controls were included in each reaction. The amplification products were tested by electrophoresis in 0.8% (w/v) agarose gels, stained with ethidium bromide, and visualized using the GeneGenius Gel BioImaging system (Syngene, Ltd). PCR-Southern blotting (Li, 2007) was also conducted to avoid false positive results.

 

2.2.3 Probe synthesis and southern blot detection

pYHY DNA was amplifed by the probes labeled with DIG by PCR primer method, where the primers were used as the same as above. The reaction system (50 μL) included 5 μL 10×PCR Buffer, 1 μL DIG-dNTPS (40 mmol/μL), each of F-primer and R-primer (20 μmol/L), 0.5 μL LA Tagalog enzyme (5 U/μL), 10 ng DNA template. The probes were purified by the high pure PCR product purification kit (REF: 11732 668 001 Version 15.0 Roche) and saved in the mini tubes under -20ºC.

 

The genomic DNA extracted from the individual bacterial clone with positive result by PCR was used as the template, the southern blot analysis was performed as described (Sambrook and Russell, 2001), with pYHY DNA as a positive control and the non-temple system as a negative control.

 

2.2.4 Statistical analyses

The differences between the broadleaf and coniferous soil types and the treatments between transgenic and non-transgenic leaves on microbial number, the bacterial number between selective and non-selective media, and the percentage of PCR-positive results among different treatments were analyzed by one-way ANOVA using the Statistical Package for the Social Sciences statistical software (SPSS 16.0). A p value of ≤0.05 was considered to be statistically significant.

 

Acknowledgments

The authors are grateful to the State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University, China) for providing transgenic poplar materials, the National Natural Science Foundation of China (31901142, 31170568) and the Key Project of Science and Technology in Heilongjiang Province for financial support (GA06B301).

 

Authors' contributions

Qiuyu Wang conceived and designed the experiments; Mu Peng performed the experiments; Mu Peng and Syed Sadaqat Shah analyzed the data; Qiuyu Wang contributed reagents/materials/analysis tools; Fanjuan Meng wrote the paper.

 

Conflict of interests

The authors declare no conflict of interest.

 

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