Conceived and designed the experiments: SEH WdB JvV. Performed the experiments: SEH. Analyzed the data: SEH WdB JvV. Wrote the paper: SEH WdB JvV.
The authors have declared that no competing interests exist.
In this three year field study the impact of different potato (
Genetic engineering of plants has been used to improve the quality and quantity of crop production in a cost-effective way (e.g. by enhancing resistance to pests and diseases or introducing tolerance to herbicides)
The rhizosphere is a hot-spot of microbial abundance and metabolic activity due to the resources released by plants
The structure and functioning of soil microbial communities is affected by soil type
Most of the studies on soil fungal communities have shown that GM-crops affect soil fungi in a similar way as its isoline
Identifying the normal variation in fungal community structure and function in the soil is very important when aiming to evaluate the possible effects of GM-crops on soil communities
Two agricultural sites VMD and BUI were selected for this experiment
Soil samples were collected from bulk soil before and after harvest whereas both rhizosphere and bulk soil were collected at the growth stages EC30 (seedling/young), EC60 (flowering) and EC90 (senescence)
Quantification of ergosterol, via the alkaline extraction method, was used as an estimate of fungal biomass
DNA was extracted from soil (0.5 g wet weight) with a Power Soil DNA isolation kit (MOBIO Laboratories, Inc. Carlsbad, CA, USA) using a bead beating system. Yields of genomic DNA were checked on 1% agarose gel and visualized under UV after ethidium bromide staining.
Terminal restriction fragment length polymorphism (T-RFLP) combined with the construction of a small library of the most dominant operational taxonomical units (OTUs) was used to determine the fungal community compositions over years. The structures of the three fungal phyla studied, ascomycetes, basidiomycetes and glomeromycetes, were assessed separately. For the analysis of ascomycete and basidiomycete communities, internal transcribed spacer (ITS) regions were used as target regions and the large subunit of ribosomal genes (LSU) was used as a target region for AMF (
Clone libraries were constructed as described in Hannula
Analyses of variance (ANOVA) with a linear mixed effect model was used to compare the ergosterol and enzymatic data as well as number of TRFs using SPSS for windows (Release 17.0.). The assumption of normality was tested with Shapiro-Wilk statistics and homogeneity of variances was assessed with Levene’s test. The field site, growth stage, year of sampling, cultivar and GM-variety were used as fixed factors and block was set as the random factor. Differences between treatments were compared by a post hoc Tukey’s honestly significant difference (HSD) test. Log transformation was used when data were not normally distributed. To estimate the possible effects of GM variety ‘Modena’ to its parental variety over years, a mixed model with repeated measure (growth stage) and block as a random factor was built separately for both fields.
The quality of T-RFLP data was first visually inspected in GeneMapper Software v4.1 (Applied Biosystems) and then transferred to T-Rex
The diversity was calculated from the matched samples with both Shannon-H’ and Simpson diversity indexes and compared with ANOVA as explained above.
Fungal-related parameters in plots cropped with the GM-variety seemed to fall within normal variation among potato cultivars observed in time (
Boxplots of fungal biomass in the rhizosphere as measured by ergosterol concentrations during 3 years in different growth stages and in both field locations. The baseline (all other cultivars combined, n = 16) is marked with green boxplots, the GM-variety (n = 4) with purple and the parental variety ‘Karnico’ (n = 4) with blue markers. The star indicates a significant cultivar effect at the indicated time point. The values under the graphs are the cultivars with highest and lowest values (on average) colored the same as in the boxplots where ‘D’ = ‘Désirée’, ‘Avk’ = ’Aveka’, ‘Avn’ = ’Aventra’, ‘P’ = ‘Premiere’, ‘K’ = ‘Karnico’ (parental cultivar) and ‘M’ = ’Modena’ (modified cultivar).
Field (df. 1) | Year (df.2) | Growth stage (df. 3) | Cultivar (df. 5) | GM- parent (df. 1) | Year×cultivar | Field×cultivar | Growth stage×cultivar | Year×Growth stage×field×cultivar | ||||||||||
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Ergosterol (mg/g) | 0.13 | 0.72 | 48.17 |
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19.38 |
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1.47 | 0.20 | 0.12 | 0.73 | 1.40 | 0.18 | 1.00 | 0.42 | 0.97 | 0.49 | 1.72 | 0.071 |
Laccases (µmol/g) | 0.63 | 0.43 | 14.39 |
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21.19 |
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1.05 | 0.39 | 0.36 | 0.55 | 1.84 | 0.052 | 1.27 | 0.28 | 2.39 |
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1.72 | 0.052 |
Mn-Peroxidases (µmol/g) | 1.06 | 0.10 | 1.96 | 0.14 | 9.81 |
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3.31 | 0.06 | 0.67 | 0.42 | 1.02 | 0.43 | 1.07 | 0.38 | 1.86 |
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1.69 |
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Cellulases (µmol/g) | 17.74 |
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23.94 |
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19.01 |
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1.08 | 0.37 | 0.04 | 0.83 | 4.03 |
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3.96 |
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3.12 |
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1.29 | 0.35 |
# of Ascomycetes | 0.41 | 0.52 | 6.28 |
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25.15 |
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1.51 | 0.19 | 2.73 | 0.11 | 0.72 | 0.69 | 0.48 | 0.79 | 2.67 |
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1.38 | 0.16 |
# of Basidiomycetes | 1.65 | 0.20 | 51.60 |
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20.14 |
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0.72 | 0.61 | 0.16 | 0.69 | 0.52 | 0.88 | 0.08 | 1.00 | 1.12 | 0.34 | 0.39 | 0.97 |
# of AMF | 0.61 | 0.44 | 15.29 |
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6.09 |
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0.66 | 0.65 | 0.35 | 0.55 | 0.49 | 0.88 | 0.89 | 0.49 | 0.34 | 0.98 | 0.50 | 0.91 |
Significant P-values are marked with bold. Only samples from rhizosphere were included in analyses of growth stage, cultivar and GM-parent comparison. # indicates richness of the fungi.
Ergosterol (mg/g) | Laccases (µmol/g) | Mn-Peroxidases (µmol/g) | Cellulases (µmol/g) | # of Ascomycetes | # of Basidiomycetes | # of AMF | |||||||||||
Cultivar | GM-Parent | Cultivar | GM-Parent | Cultivar | GM-Parent | Cultivar | GM-Parent | Cultivar | GM-Parent | Cultivar | GM-Parent | Cultivar | GM-Parent | ||||
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Young |
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0.86 | 0.24 | 0.89 | 2.99 | 1.02 | 1.32 | 0.48 | 0.92 | 0.22 | 0.06 | 0.90 | 0.05 | 1.09 | 0.10 |
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0.53 | 0.65 | 0.51 | 0.15 | 0.44 | 0.30 | 0.79 | 0.38 | 0.95 | 0.82 | 0.50 | 0.83 | 0.42 | 0.78 | |||
Flowering |
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2.13 | 1.00 | 0.59 | 0.76 | 0.42 | 0.79 | 0.00 | 0.38 | 0.25 | 0.48 | 0.02 | 1.89 | 0.47 | ||
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0.20 | 0.44 | 0.47 | 0.59 | 0.84 | 0.57 | 0.96 | 0.86 | 0.63 | 0.79 | 0.89 | 0.15 | 0.53 | |||
Senescence |
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2.07 | 0.15 | 2.61 | 0.20 |
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0.85 | 1.25 | 0.51 |
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0.66 | 0.38 | 1.93 | 0.28 | ||
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0.12 | 0.72 | 0.06 | 0.67 |
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0.40 | 0.33 | 0.50 |
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0.66 | 0.57 | 0.15 | 0.62 | |||
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Young |
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1.22 | 1.13 | 1.24 | 0.50 | 0.73 | 0.61 | 0.94 | 0.61 | 0.13 | 0.07 | 0.47 | 0.19 | 1.12 | 2.42 | |
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0.36 | 0.33 | 0.35 | 0.51 | 0.59 | 0.46 | 0.47 | 0.46 | 0.97 | 0.80 | 0.75 | 0.69 | 0.40 | 0.17 | |||
Flowering |
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1.04 | 0.72 | 0.45 | 2.63 | 1.00 | 0.34 | 0.69 | 5.53 | 0.17 | 3.00 | 0.87 | 0.00 | 0.04 | 5.44 | ||
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0.41 | 0.45 | 0.77 | 0.16 | 0.45 | 0.58 | 0.62 | 0.06 | 0.85 | 0.33 | 0.49 | 0.96 | 0.96 | 0.26 | |||
Senescence |
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1.32 | 0.20 | 1.68 | 1.32 | 1.42 | 1.36 | 1.80 | 0.71 | 0.32 | 1.93 | 1.60 | 2.10 | 1.24 | 1.15 | ||
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0.32 | 0.68 | 0.26 | 0.33 | 0.33 | 0.33 | 0.23 | 0.46 | 0.81 | 0.21 | 0.27 | 0.24 | 0.37 | 0.40 | |||
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Young |
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0.01 | 1.09 | 0.11 | 0.11 | 0.26 | 1.32 | 0.49 | 0.92 | 1.94 | 0.84 | 1.26 | 0.34 | 0.35 | |
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0.94 | 0.41 | 0.75 | 0.99 | 0.63 | 0.31 | 0.52 | 0.49 | 0.21 | 0.54 | 0.31 | 0.88 | 0.58 | |||
Flowering |
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0.85 | 0.77 | 1.06 | 0.64 | 0.84 | 0.69 | 0.47 | 0.13 | 0.19 | 0.02 | 2.52 | 0.06 | 0.34 | 1.08 | ||
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0.54 | 0.42 | 0.41 | 0.46 | 0.54 | 0.80 | 0.79 | 0.73 | 0.96 | 0.89 | 0.31 | 0.81 | 0.88 | 0.49 | |||
Senescence |
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4.73 | 1.42 | 0.65 | 0.22 | 1.89 | 0.30 | 1.55 | 1.51 | 0.33 | 1.04 | 0.68 | 0.00 | 1.53 |
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0.11 | 0.30 | 0.63 | 0.66 | 0.17 | 0.61 | 0.24 | 0.27 | 0.86 | 0.38 | 0.62 | 0.95 | 0.24 |
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Young |
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1.96 | 1.49 | 1.20 | 1.21 | 0.76 | 0.09 | 1.03 | 0.34 | 2.13 | 2.50 | 0.34 | 0.10 | 0.55 | 1.54 |
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0.13 | 0.27 | 0.35 | 0.31 | 0.59 | 0.78 | 0.43 | 0.58 | 0.11 | 0.17 | 0.89 | 0.77 | 0.74 | 0.26 | |||
Flowering |
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0.67 | 0.33 | 1.43 | 1.19 | 0.73 | 0.65 | 1.23 | 1.93 | 0.56 | 0.33 | 0.46 | 1.47 | 2.10 | 1.03 | ||
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0.65 | 0.59 | 0.27 | 0.32 | 0.61 | 0.46 | 0.35 | 0.21 | 0.73 | 0.59 | 0.72 | 0.44 | 0.12 | 0.35 | |||
Senescence |
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1.43 |
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1.24 | 0.51 | 2.43 | 0.18 | 0.81 | 0.10 | 0.66 | 1.93 | 0.56 | 0.26 | 0.66 | 0.93 | ||
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0.26 |
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0.33 | 0.50 | 0.08 | 0.69 | 0.56 | 0.76 | 0.66 | 0.21 | 0.73 | 0.65 | 0.66 | 0.38 | |||
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Young |
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2.91 | 0.54 | 0.66 | 0.22 | 0.27 | 0.06 | 0.32 | 0.15 | 0.57 | 0.24 | 0.58 | 0.36 | 1.06 | 0.62 | |
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0.17 | 0.50 | 0.59 | 0.66 | 0.85 | 0.82 | 0.81 | 0.72 | 0.65 | 0.64 | 0.64 | 0.57 | 0.40 | 0.46 | |||
Flowering |
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7.03 | 0.62 | 0.98 | 0.18 |
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1.18 | 1.22 | 2.83 | 0.67 | 2.00 | 0.25 | 0.85 | 1.58 | 0.29 | ||
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0.23 | 0.47 | 0.44 | 0.69 |
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0.32 | 0.35 | 0.14 | 0.54 | 0.22 | 0.90 | 0.41 | 0.29 | 0.63 | |||
Senescence |
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0.77 |
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1.57 | 1.18 | 1.35 | 1.24 | 0.36 | 0.01 | 1.35 | 1.04 |
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4.09 | 2.38 | 5.89 | ||
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0.68 |
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0.23 | 0.32 | 0.30 | 0.31 | 0.84 | 0.92 | 0.36 | 0.38 |
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0.11 | 0.13 | 0.06 | |||
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Young |
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0.86 | 0.30 | 0.83 | 1.05 | 0.65 | 2.97 | 0.68 | 0.93 | 0.53 | 0.18 | 0.83 | 1.67 |
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4.17 | |
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0.53 | 0.61 | 0.55 | 0.34 | 0.66 | 0.14 | 0.65 | 0.37 | 0.75 | 0.68 | 0.55 | 0.24 |
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0.11 | |||
Flowering |
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0.75 | 0.99 | 1.76 | 1.00 | 2.13 | 1.00 | 0.78 | 0.86 | 1.08 | 0.74 | 0.25 | 0.09 | 2.02 | 8.17 | ||
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0.60 | 0.36 | 0.17 | 0.36 | 0.11 | 3.56 | 0.58 | 0.39 | 0.41 | 0.42 | 0.90 | 0.78 | 0.18 | 0.07 | |||
Senescence |
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0.27 |
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1.54 | 1.17 | 0.18 | 0.13 | 0.30 | 0.06 | 0.95 | 0.46 | 0.47 | 1.02 | 0.12 | 0.52 | ||
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0.84 |
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0.30 | 0.54 | 0.90 | 0.74 | 0.82 | 0.82 | 0.45 | 0.53 | 0.71 | 0.35 | 0.95 | 0.51 |
Significant P-values are marked with bold.
Correlations revealed that all the extracellular enzymes measured in this study (laccases, cellulases and Mn-peroxidases) were positively correlated with the fungal biomass indicator ergosterol (n = 702, R2 between 0.23–0.29 and p<0.001). Further, there were strong positive correlations among all enzyme activities measured. The richness of both ascomycetes and basidiomycetes was positively correlated with the amount of ergosterol (for basidiomycetes R2 = 0.27 and P<0.001 and ascomycetes R2 = 0.08 and P<0.05). AMF richness was negatively correlated with the amount of ergosterol (R2 = .11 and P<0.05). Furthermore, the amount of Mn-Peroxidases in the soil was positively correlated with the ascomycete diversity (R2 = 0.16, P<0.001) while the AMF richness was negatively correlated with production of cellulases (R2 = 0.11 and P<0.005).
The measured extracellular enzymes (laccases, Mn-peroxidases and cellulases) were all affected by plant growth stage; highest activities were measured during senescence (
When looking at individual time points and fields the ascomycete, basidiomycete and glomeromycete richness was only once significantly different between cultivars (
Data on community function, as based on activities of enzymes involved in decomposition of lignocellulose-rich organic matter, and richness were analysed by principal component analyses (PCA). The PCA analyses revealed that the growth stage was the strongest explanatory factor of differences in the community function (
For clarity, the years and field sites are combined. Pre-cropping samples are represented by black circles, young plant stage samples with diamonds, flowering plants stage samples with triangles and senescence stage samples with squares. Green markers and error bars represent baseline cultivars (n = 96), purple markers the GM-variety (n = 24) and blue markers the parental variety ‘Karnico’ (n = 24). The explanatory parameters are mentioned next to the axis. The enzymes measured as functional parameters were laccases, Mn-peroxidases and cellulases.
According to the ANOSIM, the community fingerprints of all TRF peaks as well as identified OTUs of
The PCA analysis was done both at the level of individual OTUs and of orders for total fungi (A & E),
Field | Year | Growth stage | Cultivar |
GM-parent |
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R | P | R | P | R | P | R | P | R | P | |
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0.07 |
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0.29 |
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0.10 |
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0.013 | 0.131 | −0.006 | 1 |
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0.04 |
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0.25 |
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0.19 |
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0.008 | 0.188 | 0.015 | 0.915 |
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0.11 |
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0.22 |
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0.09 |
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−0.005 | 0.689 | −0.011 | 0.863 |
Only samples where plant was present are included in the analyses.
Significant P-values are marked with bold.
The diversity of all fungal phyla was expressed both by the Shannon-Wiener index (H’) and Simpson diversity index. The ascomycete diversity was significantly correlated with ascomycete richness (R2 = 0.55 for total diversity, R2 = 0.45 for orders and R2 = 0.36 for classes, P<0.001 for all) and basidiomycete diversity with basidiomycete richness (R2 = 0.51 for total diversity and R2 = 0.41 for orders, P<0.001 for both). Further, the ascomycete diversity was negatively correlated with basidiomycete diversity (R2 = −0.15, P<0.005). Ascomycete richness was correlated with the amount of Mn-peroxidases in the soil (R2 = 0.15, P<0.05) and basidiomycete richness with ergosterol (R2 = 0.18, P<0.001). The AMF diversity was positively correlated with soil moisture content (R2 = 0.15, P<0.001), AMF richness (R2 = 0.58, P<0.001) and ascomycete diversity (R2 = 0.10, P<0.05).
The diversity of ascomycetes or basidiomycetes at the level of OTUs or orders was not significantly affected by field site. However, AMF diversity was. There was no significant difference in diversity of ascomycetes at the level of OTUs and orders from year to year, although diversity between years 2009 and 2010 was significantly different. However, at the level of classes also 2008 and 2009 were different and year was a more pronounced factor explaining the diversity. For basidiomycetes and AMF, year had a strong influence on diversity both at the level of OTUs and orders (
Boxplots of changes in diversity of
Field | Year | Growth stage | Cultivar |
GM-parent |
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F | P | F | P | F | P | F | P | F | P | ||
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OTUs | 0.005/0.004 | 0.94/0.94 | 7.80/3.89 |
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12.76/9.16 |
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0.65/0.32 | 0.66/0.91 | 2.67/0.49 | 0.11/0.49 |
Orders | 0.33/0.009 | 0.57/0.92 | 7.44/3.56 |
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10.8/13.22 |
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0.59/0.52 | 0.64/0.76 | 2.74/1.58 | 0.10/0.21 | |
Classes | 9.30/9.50 |
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10.80/9.64 |
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6.78/5.76 |
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15.58/34.61 |
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2.97/2.31 | 0.09/0.31 | |
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OTUs | 1.803/0.523 | 0.18/0.47 | 9.49/6.64 |
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13.84/9.37 |
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1.24/1.41 | 0.29/0.23 | 0.03/0.02 | 0.87/0.90 |
Orders | 0.04/0.002 | 0.85/0.97 | 21.85/17.86 |
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8.99/6.48 |
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1.85/2.08 | 0.13/0.09 | 0.19/0.37 | 0.67/0.54 | |
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OTUs | 14.67/15.04 |
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24.48/20.72 |
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3.01/2.76 |
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1.91/1.63 | 0.09/0.15 | 1.91/1.40 | 0.17/0.24 |
Orders | 38.22/35.98 |
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12.50/9.99 |
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2.29/2.13 | 0.08/0.09 | 1.91/1.89 | 0.09/0.10 | 1.17/1.59 | 0.19/0.21 |
Only samples where plant was present are included in the analyses.
All diversities were calculated using both Shannon H’ and Simpson diversity indexes and presented in the table as Shannon H’/Simpson diversity. If both P-values are the same, only one value is presented. Diversity index for classes was not calculated for basidiomycetes and glomeromycetes due to low numbers or unevenness of classes. Significant P-values are marked with bold.
Cultivar-type had no overall effect on basidiomycete, ascomycete and AMF diversity at the level of OTUs or orders. However, at the level of classes cultivar ‘Désirée’ had a significantly less diverse community of ascomycetes in its rhizosphere than all the other cultivars causing a general cultivar effect (
Diversity of Ascomycota (Shannon H) | Diversity of Basidiomycota (Shannon H) | Diversity of Glomeromycota (Shannon H) | ||||||||||
Cultivar | GM-Parent | Order level | Cultivar | GM-Parent | Order level | Cultivar | GM-Parent | Order level | ||||
df. 5 | df. 1 | df. 5 | df. 1 | df. 5 | df. 1 | |||||||
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Young |
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0.12 | 0.12 | 0.21 | 0.60 | 0.38 | 0.38 | |||
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0.89 | 0.75 | n.s. | 0.95 | 0.48 | n.s. | 0.86 | 0.85 | n.s. | |||
Flowering |
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0.92 | 0.00 | 0.79 | 0.56 | 0.62 | 2.34 | |||||
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0.91 | 1.00 | n.s. | 0.58 | 0.50 | n.s. | 0.69 | 0.22 | n.s. | |||
Senescence |
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2.93 | 0.05 | 0.99 | 0.30 | 0.93 | 0.60 | |||||
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0.11 | 0.83 | n.s. | 0.47 | 0.61 | n.s. | 0.50 | 0.50 | n.s. | |||
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Young |
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0.88 | 1.22 | 0.23 | 0.40 | 0.99 | 0.88 | ||||
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0.51 | 0.52 | n.s. | 0.92 | 0.55 | n.s. | 0.46 | 0.38 | n.s. | |||
Flowering |
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2.86 | 1.14 | 3.28 | 0.21 | 0.64 | 0.71 | |||||
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0.14 | 0.35 | n.s. | 0.10 | 0.63 | n.s. | 0.56 | 0.44 | n.s. | |||
Senescence |
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1.35 | 3.29 | 4.88 | 1.19 | nd | nd | |||||
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0.35 | 0.21 | n.s. | 0.77 | 0.29 | n.s. | nd | nd | n.s. | |||
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Young |
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0.79 | 5.98 | 0.54 | 0.60 | |||
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0.58 | 0.07 | n.s. | 0.74 | 0.50 | n.s. | |||
Flowering |
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0.46 | 0.00 | 2.86 | 2.24 | 0.43 | 0.13 | |||||
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0.80 | 0.98 | n.s. | 0.14 | 0.38 | n.s. | 0.82 | 0.74 | n.s. | |||
Senescence |
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1.37 | 1.14 | 0.31 | 0.29 | 4.41 | 0.82 | |||||
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0.30 | 0.35 | n.s. | 0.86 | 0.82 | n.s. | 0.99 | 0.40 | n.s. | |||
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Young |
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1.73 | 1.89 | 0.45 | 0.19 |
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0.19 | 0.49 | n.s. | 0.80 | 0.69 | n.s. |
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Flowering |
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0.85 | 4.35 | 0.43 | 0.37 | 2.23 | 0.41 | |||||
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0.52 | 0.17 | n.s. | 0.79 | 0.58 | n.s. | 0.15 | 0.59 | n.s. | |||
Senescence |
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0.99 | 0.04 | 0.57 | 0.34 | 0.13 | 0.32 | |||||
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0.48 | 0.86 | n.s. | 0.73 | 0.59 | n.s. | 0.93 | 0.62 | n.s. | |||
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Young |
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0.64 | 1.79 | 0.45 | 0.26 | 0.51 | 0.20 | ||||
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0.62 | 0.41 | n.s. | 0.73 | 0.64 | n.s. | 0.69 | 0.67 | n.s. | |||
Flowering |
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0.64 | 1.08 |
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0.90 | 0.47 | 0.71 | |||||
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0.61 | 0.41 | n.s. |
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0.39 | n.s. | 0.76 | 0.44 | n.s. | |||
Senescence |
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0.35 | 1.21 |
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0.93 | 2.31 | 23.78 | |||||
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0.84 | 0.44 | n.s. |
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0.38 | 0.15 | 0.16 | n.s. | ||||
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Young |
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0.49 | 0.42 | 0.62 | 2.92 | 1.12 | 1.89 | ||||
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0.78 | 0.84 | n.s. | 0.66 | 0.15 | n.s. | 0.56 | 0.49 | n.s. | |||
Flowering |
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2.66 |
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1.43 |
2.72 | 2.34 | 0.46 | 0.33 | ||||
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0.08 |
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0.28 |
0.13 | 0.22 | n.s. | 0.77 | 0.67 | n.s. | |||
Senescence |
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0.84 | 0.91 | 0.54 | 2.87 | 0.49 | 0.42 | |||||
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0.50 | 0.38 | n.s. | 0.67 | 0.17 | n.s. | 0.75 | 0.86 | n.s. |
The diversities were estimated using Shannon-H’. The first two columns of each fungal group are performed at the level of OTUs and the third column indicates significance at the level of orders. Significant P-values are marked with bold.
The fields were sampled after the growth seasons 2008 and 2009 and, in addition, rhizosphere of barley was sampled in June 2009 in the field where potatoes were grown in 2008. There were no significant differences in ergosterol content, enzymatic activities, fungal richness or fungal diversity between soils where ‘Modena’ and ‘Karnico’ had been grown (
NMDS of effects of GM-variety in the next crop (barley) rhizosphere in field BUI on ascomycetes (A), basidiomycetes (B) and in fields BUI and VMD on glomeromycetes (C). The GM-variety ‘Modena’ is marked with purple markers, the parental cultivar ‘Karnico’ with blue markers, and baseline (all other cultivars combined) green markers. Details on statistical analysis are given in
Field BUI | Field VMD | Barley rhizosphere | |||||||||||
Cultivar | GM-parent | Cultivar | GM-parent | Cultivar | GM-parent | ||||||||
F/R | P | F/R | P | F/R | P | F/R | P | F/R | P | F/R | P | ||
ANOVA | Ergosterol | 0.14 | 0.89 | 0.00 | 0.95 | 0.81 | 0.47 | 1.04 | 0.35 | 0.03 | 0.98 | 0.00 | 0.98 |
Laccases | 0.70 | 0.63 | 0.28 | 0.62 | 3.63 |
|
0.06 | 0.82 | 0.56 | 0.58 | 1.03 | 0.36 | |
Mn-Peroxidases | 1.77 | 0.19 | 2.76 | 0.15 | 1.30 | 0.31 | 0.00 | 0.98 | 0.00 | 0.99 | 0.00 | 0.99 | |
Cellulases | 1.00 | 0.43 | 0.35 | 0.58 | 1.56 | 0.22 | 5.53 | 0.06 | 0.06 | 0.95 | 0.05 | 0.84 | |
Diversity of basidiomycetes | 1.37 | 0.29 | 0.06 | 0.82 | |||||||||
Diversityof ascomycetes | 0.87 | 0.72 | 0.33 | 0.64 | 0.34 | 0.72 | 0.58 | 0.48 | 0.72 | 0.51 | 0.60 | 0.48 | |
Diversity of AMF | 0.61 | 0.50 | 0.02 | 0.89 | 0.53 | 0.68 | 1.00 | 0.36 | 0.55 | 0.60 | 1.14 | 0.35 | |
ANOSIM | Community of ascomycetes | −0.22 | 0.88 | −0.32 | 1.00 | −0.05 | 0.72 | 0.07 | 0.24 | 0.01 | 0.42 | 0.56 | 0.33 |
Community of basidiomycetes | 0.03 | 0.65 | −0.12 | 0.95 | −0.13 | 0.73 | −0.38 | 0.97 | −0.24 | 0.91 | −0.58 | 1.00 | |
Community of AMF | −0.04 | 0.67 | −0.16 | 0.80 | 0.02 | 0.37 | −0.13 | 0.81 | −0.28 | 1.00 | −0.38 | 1.00 |
ANOVA was used as a similarity measure for fungal biomass, enzymatic measurements and diversity and F values are presented in the table. ANOSIM was used for the community data derived from T-REX and R-values are presented in the table. Significant P-values for both ANOVA and ANOSIM are marked with bold.
The composition and function of fungal communities in the rhizosphere was shown to be highly dynamic and influenced by plant growth stage, soil type, year and, to a smaller extent, also cultivar-type. The largest explaining factor for most of the measured parameters was plant phenological growth stage, followed by year and the soil type. In addition, results confirmed our previous observations that fungal composition and abundance is strongly influenced by the presence of potato roots (i.e. a strong rhizosphere effect)
The succession of microbial communities during plant growing season can be explained by two possible mechanisms
Surprisingly, despite the strong differences in soil organic matter content, field location did not affect the community function or diversity of the higher fungi much and results from the two fields could be even combined for baseline purposes. Earlier studies have found soil type as one of the most explanatory factor
We detected interesting differences between the years. In the first years, mineral fertilizer was used and only from the beginning of 2010 pig manure was used as a fertilizer. This might explain differences in fungal communities observed between 2008 and 2010. Previously, it has been shown that different types of fertilizer treatments contribute to different microbial communities
Community structure and diversity of soil fungi are important determinants of key soil ecosystems functions such as decomposition of organic matter. Indeed, we could detect a correlation between community structure of fungi and decomposition-related enzyme activities. Moreover, the combination of phylogenetic analyses with functional assays proved highly useful, providing a more complete picture of fungal community dynamics. We found a correlation between Mn-peroxidases produced and the ascomycete diversity (and richness). Mn-peroxidases can be produced primarily by basidiomycetes as well as some ascomycetal groups
Only few studies have evaluated the potential impacts of GM-plants in the context of impacts of multiple cultivars on fungal rhizosphere communities. Most of them have found some degree of cultivar dependence of soil fungal community composition
In this study the GM-variety ‘Modena’ was not significantly different from its parental variety ‘Karnico’ in any measured parameter and it seemed that these cultivars had a very similar effect on both the structure and function of soil fungal communities. The only significant effect was the difference in the amount of fungi in the rhizosphere of the two cultivars in the field VMD during senescence, in all years of the study. This was, however, seen only in one of the two soils studied and can, thus, be ruled out as a cultivar-soil interaction effect. There was no overall trend of multiple parameters being consistently changed by any of the cultivars while the other factors (i.e. growth stage and season) had consistent effect on multiple parameters measured.
The growth stage can also affect the outcome of the comparison between the cultivars. Other authors have found differences in microbial communities associated with GM-potatoes mostly at the senescent growth stage
In conclusion, plant growth stage, year and field site were the factors contributing most to variation in the potato-associated fungal communities. Despite some differences in fungal-related parameters between individual cultivars, there were no directional effects and most of the differences observed were not consistent between fields and years. Even at the level of individual OTUs, there were no consistent significant differences between cultivars in community structure and no differences in community function were found during and after the growth of the plant. However, as was seen from conflicting evidence between different studies, we acknowledge that potential effects of GM-crops on soil fungal communities vary between crop species and types of modifications done to the plant making a case-by–case evaluation strategy advisable. We hypothesized that this modification would have no direct but rather indirect unintended effects of the modification on the plant physiology through production of different exudates. Data presented in this study allowed us to conclude that the modification studied here has no long-lasting effects on soil fungal communities and that the potato plant growth stage, season and field location affect the soil fungal community structure and function more than the cultivar-type or starch modification of tubers.
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The authors would like to thank Wiecher Smant, Özgül Ineoglu and Nadia Marttin for technical assistance and collaboration during field sampling and Peter Bruinenberg and Paul Heeres from AVEBE/AVERIS seeds for valuable assistance in the field experiment. This is publication 5227 of the Netherlands Institute of Ecology (NIOO-KNAW).