Sensitivity evaluations were conducted for sclerotia and soil samples. Soil samples, which were autoclaved at 121 °C for 45 min, were artificially inoculated with a representative isolate of each of three target Sclerotinia species (41065 for S. sclerotiorum, 41067 for S. minor, and 45150 for S. nivalis) separately and together. Inoculation was carried out by depositing a mycelium plug (5-mm diameter) taken from 3-d-old PDA cultures per pot and a sclerotium per pot on the top of the soil, then incubating in growth chamber at 25 °C under a 12/12 h (day/night) cycle for 7 d. In addition, mycelium plugs of the three species were inoculated together in a pot, and the sclerotia of the three species were inoculated in a pot. Three replicates (pots) were inoculated for each fungal isolate. As a control, an agar plug was transferred to the soil surface. Soil DNA was extracted using the SPINeasy DNA Kit for Soil (MP Biomedicals, Santa Ana, CA). A total of 300–400 mg of the inoculated soil was harvested from the surface of the soil, placed in a lysis tube, and homogenized at 30 Hz/s for 10 min using an MM400 mixer mill (Retsch GmbH, Haan, Germany). Sclerotia were harvested from colonies formed on the PDA medium, crushed with a pellet pestle, and homogenized at 30 Hz/s for 30 min using an MM400 mixer mill (Retsch GmbH). DNA was extracted using a NucleoSpin Plant II Kit (Macherey-Nagel).
Spineasy dna kit for soil
Manufactured by MP Biomedicals
Sourced in Germany, United States
The SPINeasy DNA Kit for Soil is a laboratory equipment product designed for the extraction and purification of DNA from soil samples. The kit provides the necessary reagents and tools to efficiently isolate high-quality genomic DNA from a variety of soil types.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt fragmentation enzyme, by Illumina (2 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Ampure magnetic beads, by Beckman Coulter (2 mentions)
Miseq platform, by Illumina (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nucleospin plant 2 kit, by Macherey-Nagel (1 mentions)
Dsdna hs assay kit, by Illumina (1 mentions)
Nextseq 2000 platform, by Illumina (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Eb buffer, by Beckman Coulter (1 mentions)
Hiseq x platform, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Miseq pe300 platform, by Illumina (1 mentions)
8 protocols using spineasy dna kit for soil
1
Sclerotinia species detection in soil
2
Whole Genome Sequencing of Bacterial Samples
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Initially, a bacterial cell pellet was obtained by centrifugation of 1 mL sample for 5 min at 9000× g. DNA was extracted via the SPINeasy DNA Kit for Soil (MP Biomedicals, Eschwege, Germany), according to manufacturer’s instructions. Subsequently, DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and IDT Unique Dual Indexes with total DNA input of 1 ng. Genomic DNA was fragmented using a proportional amount of Illumina Nextera XT fragmentation enzyme. Unique dual indexes were added to each sample followed by 12 cycles of PCR to construct libraries. DNA libraries were purified using AMpure magnetic Beads (Beckman Coulter, Brea, CA, USA), eluted in QIAGEN EB buffer, quantified using a Qubit 4 fluorometer and a Qubit dsDNA HS Assay Kit, and sequenced on an Illumina Nextseq 2000 platform 2 × 150 bp. Unassembled sequencing reads were converted to relative abundances (%) using the CosmosID-HUB Microbiome Platform (CosmosID Inc., Germantown, MD, USA) [66 (link),67 (link)].
3
16S rRNA Gene Amplicon Sequencing
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Quantitative data were obtained by correcting abundances (%; 16S rRNA gene profiling) with total cell counts for each sample (cells/mL; flow cytometry), resulting in estimated cell counts/mL of different taxonomic groups. Initially, a bacterial cell pellet was obtained by centrifugation of 1 mL sample for 5 min at 9000× g. DNA was extracted via the SPINeasy DNA Kit for Soil (MP Biomedicals, Eschwege, Germany), according to manufacturer’s instructions. Upon DNA extraction, library preparation and sequencing were performed on an Illumina MiSeq platform with v3 chemistry. The 16S rRNA gene V3-V4 hypervariable regions were amplified using primers 341F (50 -CCT ACG GGN GGC WGC AG-30) and 785Rmod (50 -GAC TAC HVG GGT ATC TAA KCC-30). Results were analyzed at different taxonomic levels (phylum, family and OTU (operational taxonomic unit) level). For total cell count analysis, liquid samples were diluted in anaerobic phosphate-buffered saline (PBS), after which cells were stained with SYTO 16 at a final concentration of 1 µM and counted via a BD FACS Verse flow cytometer (BD, Erembodegem, Belgium). Data were analyzed using FlowJo, version 10.8.1.
4
Estimating Absolute Microbial Cell Counts
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Quantitative data were obtained by correcting abundances (%; 16S rRNA gene profiling) with total cell counts (cells/mL; flow cytometry), resulting in the estimated absolute cell counts per mL of different taxonomic groups. Initially, a bacterial cell pellet was obtained by the centrifugation of 1 mL samples for 5 min at 9000× g. DNA was extracted via the SPINeasy DNA Kit for Soil (MP Biomedicals, Eschwege, Germany), according to the manufacturer’s instructions. Subsequently, library preparation and sequencing were performed on an Illumina MiSeq platform with v3 chemistry. The 16S rRNA gene V3–V4 hypervariable regions were amplified using primers 341F (5′-CCT ACG GGN GGC WGC AG-3′) and 785Rmod (5′-GAC TAC HVG GGT ATC TAA KCC-3′). The results were analysed at different taxonomic levels (phylum, family, and operational taxonomic unit (OTU) level).
For the total cell count analysis, liquid samples were diluted in anaerobic phosphate-buffered saline (PBS), after which cells were stained with SYTO 16 at a final concentration of 1 µM and counted via a BD FACS Verse flow cytometer (BD, Erembodegem, Belgium). Data were analysed using FlowJo, version 10.8.1.
For the total cell count analysis, liquid samples were diluted in anaerobic phosphate-buffered saline (PBS), after which cells were stained with SYTO 16 at a final concentration of 1 µM and counted via a BD FACS Verse flow cytometer (BD, Erembodegem, Belgium). Data were analysed using FlowJo, version 10.8.1.
5
High-quality DNA Extraction and Sequencing
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DNA was isolated from harvested samples using SPINeasy DNA kit for Soil (MP Biomedicals, Eschwege, Germany), according to the manufacturer’s protocol. Extracted DNA samples were quantified using Qubit 4 fluorometer and Qubit™ dSDNA HS Assay Kit (Thermofisher Scientific, Waltham, MA). DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and IDT Unique Dual Indexes with total DNA input of 1 ng. Genomic DNA was fragmented using a proportional amount of Illumina Nextera XT fragmentation enzyme. Unique dual indexes were added to each sample followed by 12 cycles of PCR to construct libraries. DNA libraries were purified using AMpure magnetic Beads (Beckman Coulter, Brea, CA) and eluted in QIAGEN EB buffer (Hilden, Germany). DNA libraries were quantified using Qubit 4 fluorometer and Qubit™ dsDNA HS Assay Kit. Libraries were then sequenced on an Illumina HiSeq X platform with a 2x150bp chemistry yielding an average of 1.8M read pairs per sample (S1 Table ).
6
Microbiome Analysis of Fusarium solani Soil
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Microbiome analysis was performed to investigate the change in the comprehensive microbial community according to cultivation time in the three fields with differing incidence rates. In addition, microbiome analysis was performed on both inoculated and uninoculated F. solani soils. Microbial communities were analyzed using 16S and 18S rDNA sequences. Microbial DNA was extracted from samples using the SPINeasy DNA kit for soil (MP Biomedicals, Germany) according to the manufacturer’s protocols. 16S and 18S rRNA amplicon libraries were prepared using the LoopSeq™ 16S and 18S Long Read Kit (Loop Genomics, California, United States). Each of the 24 genomic DNA samples was fragmented and assigned a barcode adapter according to the 16S and 18S rRNA genes for sequencing on an Illumina platform. The libraries were read on an Illumina NovaSeq6000 (Illumina Inc., San Diego, CA, United States) using a paired-end 2 × 150 bp reading system. Sequencing data were analyzed using the QIIME2 classifier (Caporaso et al., 2010 (link)) and Silva database (Quast et al., 2013 (link)).
7
Soil Microbiome Dynamics with Fungal Agents
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Both FN and IM-FN were added to plastic pots (9 × 9 cm) filled with soil samples, respectively. The soil samples were collected after 3, 5, 7, 14, and 21 days and stored in the refrigerator at 4°C. Soil DNA extraction kit (SPINeasy DNA Kit for Soil, MP Biomedicals, LLC, Solon, OH, United States) was used to extract total soil DNA based on 1 g of soil sample from different treatments. The Zn2+-dependent hydrolase gene PydC (Zhang J. P. et al., 2021 (link)) and the esterase gene estD (Dong, 2019 ) were cloned from strains FH-1 and NJ-1, respectively.
The standard curve was plotted using the quantitative real-time PCR (qRT-PCR) method for absolute quantification. The total DNA of the soil samples treated with FN and IM-FN was used to perform the qRT-PCR (10 μl reaction) as described inSupplementary material . The total copy numbers of PydC and estD genes were calculated by the standard curves, with the dynamics of colonization of strains FH-1 and NJ-1 in soil as the functional coordinate and IM-FN expressed in time as horizontal coordinate. The primers used in the PCR are provided in Supplementary Table 1 .
The standard curve was plotted using the quantitative real-time PCR (qRT-PCR) method for absolute quantification. The total DNA of the soil samples treated with FN and IM-FN was used to perform the qRT-PCR (10 μl reaction) as described in
8
Salinity Effect on Archaeal Community
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To explore the effect of salinity on the archaeal community, high-throughput sequencing of archaeal 16S rRNA genes was performed on the CK and S4 treated samples of each habitat soil after the incubation. First, DNA in soil (0.5 g) was extracted strictly according to the instruction manual of SPINeasy DNA Kit for Soil (MP Biomedicals, Santa Ana, CA, USA). The concentration of DNA was determined with a NanoDrop (NanoDropOneC, Thermo Scientific, USA). The qualified DNA samples were stored in a -20°C refrigerator. PCR amplification on V4-V5 regions of archaeal 16S rRNA gene used 524F10extF (TGYCAGCCGCCGCGGTAA) and Arch958RmodR (YCCGGCGTTGAVTCCAATT) primer pair (Liu et al., 2016) (link). The amplification reaction system (20 µL) included 10 µL 2×Pro Taq, 0.8 µL each for upstream and downstream primers (5 µmol L -1 ), and 10 ng µL -1 DNA template. PCR reaction parameters were 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 45 s, and then 72°C for 10 min. PCR amplification and sequencing were performed on the Illumina MiSeq PE300 platform (Shanghai Majorbio Bio-pharm Technology Co., Ltd).
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