High-Throughput Isolation of Nucleic Acids from Soil

Chiodi, Claudia; Moro, Matteo; Squartini, Andrea; Concheri, Giuseppe; Occhi, Francesco; Fornasier, Flavio; Cagnin, Massimo; Bertoldo, Giovanni; Broccanello, Chiara; Stevanato, Piergiorgio

doi:10.3390/soilsystems4010003

Cited by 10 publications

(8 citation statements)

References 28 publications

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“…It is worth noting the multi-compound properties of lysis solution during extraction of DNA from belowground bulked plant tissue, related to both plant and soil origin. Among the soil particles HS are often described as contaminants of DNA extraction [ 72 ]. In the case of plants, the direct effects of HS on their physiology are a consequence of the flow of HS into the apoplast, the changes in proteins transport and hormone-like effects [ 73 ], therefore contamination of plant tissue with HS is unavoidable.…”

Section: Discussionmentioning

confidence: 99%

Looking for Hidden Enemies of Metabarcoding: Species Composition, Habitat and Management Can Strongly Influence DNA Extraction while Examining Grassland Communities

et al. 2021

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Despite the raising preoccupation, the critical question of how the plant community is composed belowground still remains unresolved, particularly for the conservation priority types of vegetation. The usefulness of metabarcoding analysis of the belowground parts of the plant community is subjected to a considerable bias, that often impedes detection of all species in a sample due to insufficient DNA quality or quantity. In the presented study we have attempted to find environmental factors that determine the amount and quality of DNA extracted from total plant tissue from above- and belowground samples (1,000 and 10,000 cm2). We analyzed the influence of land use intensity, soil properties, species composition, and season on DNA extraction. The most important factors for DNA quality were vegetation type, soil conductometry (EC), and soil pH for the belowground samples. The species that significantly decreased the DNA quality were Calamagrostis epigejos, Coronilla varia, and Holcus lanatus. For the aboveground part of the vegetation, the season, management intensity, and certain species — with the most prominent being Centaurea rhenana and Cirsium canum — have the highest influence. Additionally, we found that sample size, soil granulation, MgO, organic C, K2O, and total soil N content are important for DNA extraction effectiveness. Both low EC and pH reduce significantly the yield and quality of DNA. Identifying the potential inhibitors of DNA isolation and predicting difficulties of sampling the vegetation plots for metabarcoding analysis will help to optimize the universal, low-cost multi-stage DNA extraction procedure in molecular ecology studies.

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Section: Discussionmentioning

confidence: 99%

Looking for Hidden Enemies of Metabarcoding: Species Composition, Habitat and Management Can Strongly Influence DNA Extraction while Examining Grassland Communities

et al. 2021

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“…The 5 μL reaction mix was composed of 2.5 µL PowerUp SYBR Green Master Mix (Thermo Fisher Scientific), 0.15 µL each of forward and reverse primer (Table 1), 1.2 µL PCR-grade water, and 1 µL template DNA. A standard curve using known amounts of the target genes cloned in plasmids of known length (Chiodi et al, 2020) was obtained and data were used to calculate the copy number of the gene targets based on the Ct value.…”

Section: Soil Chemical Analysesmentioning

confidence: 99%

“…Amann et al (1995) observed that the culturability of bacteria from environmental samples ranged between 0.001 and 0.3% depending on the characteristics of the matrix. High-throughput cultureunrelated techniques, like Next Generation Sequencing (NGS), have been established over time to bypass the underestimation of soil microbial diversity problem (Chiodi et al, 2020). 16S rDNA multi-amplicon metabarcoding, sequencing at the same time several hypervariable regions, can generate a substantial amount of sequences, providing crucial information for a deep characterization of the microbiome even of extremely complex natural matrices such as soils (Young et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Microbial Diversity of Reconstituted, Degraded, and Agricultural Soils Assessed by 16S rDNA Multi-Amplicon Sequencing

Maretto

Deb

Ravi

et al. 2022

Front. Environ. Sci.

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The microbial diversity is, among soil key factors, responsible for soil fertility and nutrient biogeochemical cycles, and can be modified upon changes in main soil physicochemical properties and soil pollution. Over the years, many restoration techniques have been applied to restore degraded soils. However, the effect of these approaches on soil microbial diversity is less understood and thus requires more investigation. In this study, we analyzed the impact, on soil microbial diversity of a patented novel technology, used to restore degraded soils. Soil samples were collected from three nearby sites located in Borgotrebbia, Piacenza, Italy, and categorized as reconstituted, degraded, and agricultural soils. After total soil DNA extraction, 16S rDNA multi-amplicon sequencing was carried out using an Ion GeneStudio S5 System to compare soils’ bacterial community profiles. Sequenced reads were processed to assign taxonomy and then key microbial community differences were identified across the sampling sites. Species diversity featured significant abatement at all rank levels in the degraded soil when compared to the agricultural control. The 5 year restoration technique showed full recovery of this index at the genus level but not at the phylum level, displaying a rank-dependent gradient of restored richness. In parallel, the abundance of genes involved in the nitrogen (N) biogeochemical cycle was assessed using quantitative Real-Time PCR (qPCR). Total DNA content was significantly higher (p < 0.05) in degraded (μ = 12.69 ± 2.58 μg g−1) and reconstituted (μ = 11.73 ± 1.65 μg g−1) soil samples when compared to the agricultural soil samples (μ = 2.39 ± 0.50 μg g−1). The taxonomic diversity of each soil site was significantly different, with some instances unique of the agricultural soil even at the phylum level. The analysis of N functional genes showed that the relative abundance of bacterial amoA (p < 0.05) and nosZ (p < 0.01) genes were significantly lower in the agricultural than in the reconstituted and degraded soils. We concluded that the application of the soil reconstitution technique appears to enhance the active microbial community, with distinct diversity and functionality towards genes involved in N biogeochemical cycle, as compared to both the degraded and the agricultural soil.

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“…For the DNA extraction from soil samples to ascertain the presence of the fungus Cercospora, the method published by Chiodi et al 2020 [25] was used.…”

Section: Dna Extractionmentioning

confidence: 99%

Bacterial Endophytes As Indicators of Susceptibility To Cercospora Leaf Spot (CLS) Disease In Beta vulgaris L.

Broccanello

Ravi

Deb

et al. 2021

Preprint

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Background: The fungus Cercospora beticola causes Cercospora Leaf Spot (CLS) of sugar beet (Beta vulgaris L.). Despite the global importance of this disease, durable resistance to CLS has still not been obtained. Therefore, the development of tolerant hybrids is still a major goal for sugar beet breeding. Although recent studies have suggested that the leaf microbiome composition can offer useful predictors to assist plant breeders, this is an untapped resource in sugar beet breeding efforts. Methods: Using Ion GeneStudio S5 technology to sequence amplicons from seven 16S rRNA hypervariable regions, the most recurring endophytes discriminating CLS-symptomatic and symptomless sea beets (Beta vulgaris L.ssp. maritima) were identified. This allowed the design of taxon-specific primer pairs to quantify the abundance of the most representative endophytic species in large naturally occurring populations of sea beet and subsequently in sugar beet breeding genotypes under either CLS symptomless or infection stages using qPCR. Results: Among the screened bacterial genera, Methylobacterium and Mucilaginibacter were found to be significantly (p<0.05) more abundant in symptomatic sea beets with respect to symptomless. In cultivated sugar beet material under CLS infection, the comparison between resistant and susceptible genotypes confirmed that the susceptible genotypes hosted higher contents of the above-mentioned bacterial genera. Conclusions: These results suggest that the abundance of these species can be correlated with increased sensitivity to CLS disease. This evidence can further prompt novel protocols to assist plant breeding of sugar beet in the pursuit of improved pathogen resistance.

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High-Throughput Isolation of Nucleic Acids from Soil

Cited by 10 publications

References 28 publications

Looking for Hidden Enemies of Metabarcoding: Species Composition, Habitat and Management Can Strongly Influence DNA Extraction while Examining Grassland Communities

Looking for Hidden Enemies of Metabarcoding: Species Composition, Habitat and Management Can Strongly Influence DNA Extraction while Examining Grassland Communities

Microbial Diversity of Reconstituted, Degraded, and Agricultural Soils Assessed by 16S rDNA Multi-Amplicon Sequencing

Bacterial Endophytes As Indicators of Susceptibility To Cercospora Leaf Spot (CLS) Disease In Beta vulgaris L.

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