Total soil DNA was extracted from 500 mg of each soil sample using ISOIL for Beads Beating kit (Nippon Gene Co., Ltd., Tokyo, Japan) following the protocol of the manufacturer. For root DNA extraction, root tissue up to 1 cm from the root tip was separated from root sample using a sterile pair of scissors after washing the roots three times with sterile deionized water in order to remove adhering soil particles. Total root DNA was extracted from the 30 roots tips with two laboratory replicates using ISOIL (Nippon Gene Co., Ltd., Tokyo, Japan) without bead beating to avoid extracting root DNA (Toju and Sato, 2018 (link)). The DNA was eluted in 100 μl of TE buffer.
Bacterial and fungal gene copy numbers were quantified by real-time SYBR Green PCR assays in a StepOne Real-Time PCR System (Life Technologies Japan, Tokyo, Japan) with the 16S rRNA gene primer pair Eub338 and Eub518 and the 18S rRNA gene primer pair 5.8 s and ITS1f (Rousk et al., 2010 (link)) as previously described (Sawada et al., 2021 (link)). Standard curves were obtained using a 10-fold serial dilution of a plasmid containing either the Escherichia coli 16S rRNA gene or the Saccharomyces cerevisiae 18S rRNA gene. The measurements were expressed as copy numbers on an oven-dry soil weight basis (105°C, 24 h).
Bacterial and fungal gene copy numbers were quantified by real-time SYBR Green PCR assays in a StepOne Real-Time PCR System (Life Technologies Japan, Tokyo, Japan) with the 16S rRNA gene primer pair Eub338 and Eub518 and the 18S rRNA gene primer pair 5.8 s and ITS1f (Rousk et al., 2010 (link)) as previously described (Sawada et al., 2021 (link)). Standard curves were obtained using a 10-fold serial dilution of a plasmid containing either the Escherichia coli 16S rRNA gene or the Saccharomyces cerevisiae 18S rRNA gene. The measurements were expressed as copy numbers on an oven-dry soil weight basis (105°C, 24 h).
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