Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer

Guerra, Victor; Beule, Lukas; Lehtsaar, Ena; Liao, Hui‐Ling; Karlovský, Petr

doi:10.3390/microorganisms8040532

Cited by 37 publications

(32 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The extracts were stored at −20 °C. The effect of co-extracted PCR-inhibiting substances on the amplification of DNA was determined using a qPCR inhibition test [ 27 ]. The extracts were diluted 1:50 ( v/v ) in 0.5 × TE prior to amplification to overcome PCR inhibition.…”

Section: Methodsmentioning

confidence: 99%

Relative Abundances of Species or Sequence Variants Can Be Misleading: Soil Fungal Communities as an Example

2021

Self Cite

View full text Add to dashboard Cite

Plant production systems that are more sustainable than conventional monoculture croplands are the vision of future agriculture. With numerous environmental benefits, agroforestry is among the most promising alternatives. Although soil fungi are key drivers of plant productivity and ecosystem processes, investigations of these microorganisms in temperate agroforestry systems are scarce, leaving our understanding of agricultural systems under agroforestry practice incomplete. Here, we assessed the composition and diversity of the soil fungal community as well as the frequency (relative abundance) of fungal groups in three paired temperate poplar-based alley cropping (agroforestry) and monoculture cropland systems by amplicon sequencing. Analysis of microbiomes using relative abundances of species or sequence variants obtained from amplicon sequencing ignores microbial population size, which results in several problems. For example, species stimulated by environmental parameters may appear unaffected or suppressed in amplicon counts. Therefore, we determined absolute abundances of selected fungal groups as well as total fungal population size by real-time polymerase chain reaction (PCR). Tree rows strongly affected the community composition and increased the population size and species richness of soil fungi. Furthermore, ectomycorrhiza were strongly promoted by the tree rows. We speculate that mycorrhiza improved the nutrient acquisition in unfertilized tree rows, thereby contributing to the total productivity of the system. Comparison of relative and absolute abundances revealed dramatic discrepancies, highlighting that amplicon sequencing alone cannot adequately assess population size and dynamics. The results of our study highlight the necessity of combining frequency data based on amplicon sequencing with absolute quantification.

show abstract

Section: Methodsmentioning

confidence: 99%

Relative Abundances of Species or Sequence Variants Can Be Misleading: Soil Fungal Communities as an Example

2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…Soil DNA extracts were stored at -20˚C. The extracts were tested for the absence of PCR inhibitors as described previously [32].…”

Section: Plos Onementioning

confidence: 99%

Tree rows in temperate agroforestry croplands alter the composition of soil bacterial communities

Beule

Karlovský

2021

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Background Tree-based intercropping (agroforestry) has been advocated to reduce adverse environmental impacts of conventional arable cropping. Modern agroforestry systems in the temperate zone are alley-cropping systems that combine rows of fast-growing trees with rows of arable crops. Soil microbial communities in these systems have been investigated intensively; however, molecular studies with high taxonomical resolution are scarce. Methods Here, we assessed the effect of temperate agroforestry on the abundance, diversity and composition of soil bacterial communities at three paired poplar-based alley cropping and conventional monoculture cropland systems using real-time PCR and Illumina sequencing of bacterial 16S rRNA genes. Two of the three systems grew summer barley (Hordeum vulgare); one system grew maize (Zea mays) in the sampling year. To capture the spatial heterogeneity induced by the tree rows, soil samples in the agroforestry systems were collected along transects spanning from the centre of the tree rows to the centre of the agroforestry crop rows. Results Tree rows of temperate agroforestry systems increased the abundance of soil bacteria while their alpha diversity remained largely unaffected. The composition of the bacterial communities in tree rows differed from those in arable land (crop rows of the agroforestry systems and conventional monoculture croplands). Several bacterial groups in soil showed strong association with either tree rows or arable land, revealing that the introduction of trees into arable land through agroforestry is accompanied by the introduction of a tree row-associated microbiome. Conclusion The presence of tree row-associated bacteria in agroforestry increases the overall microbial diversity of the system. We speculate that the increase in biodiversity is accompanied by functional diversification. Differences in plant-derived nutrients (root exudates and tree litter) and management practices (fertilization and tillage) likely account for the differences between bacterial communities of tree rows and arable land in agroforestry systems.

show abstract

“…Following DNA extraction, the quality and quantity of DNA were examined on 0.8% (w/v) agarose gels stained with ethidium bromide. The extracts were tested for PCR inhibitors as described previously (Guerra, Beule, Lehtsaar, Liao, & Karlovsky, 2020) and diluted 1:50 (v/v) in double-distilled water (ddH 2 O) prior to qPCR analysis. We quantified bacterial 16S rRNA and fungal 18S rRNA genes, as well as genes involved in denitrification, namely narG and napA for NO 3 − reduction, nirK and nirS for NO 2 − reduction, and nosZ clade I and II for N 2 O reduction.…”

Section: Dna Extraction From Soil and Qpcrmentioning

confidence: 99%

The potential of ryegrass as cover crop to reduce soil N₂O emissions and increase the population size of denitrifying bacteria

Wang

Beule

Zang

et al. 2020

European J Soil Science

Self Cite

View full text Add to dashboard Cite

Nitrogen (N) fertilization is the major contributor to nitrous oxide (N 2 O) emissions from agricultural soil, especially in post-harvest seasons. This study was carried out to investigate whether ryegrass serving as cover crop affects soil N 2 O emissions and denitrifier community size. A microcosm experiment was conducted with soil planted with perennial ryegrass (Lolium perenne L.) and bare soil, each with four levels of N fertilizer (0, 5, 10 and 20 g N m −2 ; applied as calcium ammonium nitrate). The closed-chamber approach was used to measure soil N 2 O fluxes. Real-time PCR was used to estimate the biomass of bacteria and fungi and the abundance of genes involved in denitrification in soil. The results showed that the presence of ryegrass decreased the nitrate content in soil. Cumulative N 2 O emissions of soil with grass were lower than in bare soil at 5 and 10 g N m −2 . Fertilization levels did not affect the abundance of soil bacteria and fungi. Soil with grass showed greater abundances of bacteria and fungi, as well as microorganisms carrying narG, napA, nirK, nirS and nosZ clade I genes. It is concluded that ryegrass serving as a cover crop holds the potential to mitigate soil N 2 O emissions in soils with moderate or high NO 3 − concentrations. This highlights the importance of cover crops for the reduction of N 2 O emissions from soil, particularly following N fertilization. Future research should explore the full potential of ryegrass to reduce soil N 2 O emissions under field conditions as well as in different soils. Highlights

show abstract

Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer

Cited by 37 publications

References 71 publications

Relative Abundances of Species or Sequence Variants Can Be Misleading: Soil Fungal Communities as an Example

Relative Abundances of Species or Sequence Variants Can Be Misleading: Soil Fungal Communities as an Example

Tree rows in temperate agroforestry croplands alter the composition of soil bacterial communities

The potential of ryegrass as cover crop to reduce soil N₂O emissions and increase the population size of denitrifying bacteria

Contact Info

Product

Resources

About

Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer

Cited by 37 publications

References 71 publications

Relative Abundances of Species or Sequence Variants Can Be Misleading: Soil Fungal Communities as an Example

Relative Abundances of Species or Sequence Variants Can Be Misleading: Soil Fungal Communities as an Example

Tree rows in temperate agroforestry croplands alter the composition of soil bacterial communities

The potential of ryegrass as cover crop to reduce soil N2O emissions and increase the population size of denitrifying bacteria

Contact Info

Product

Resources

About

The potential of ryegrass as cover crop to reduce soil N₂O emissions and increase the population size of denitrifying bacteria