Isolation and Characterization of Denitrifier Strains

Denitrifier strains were isolated from patches in the presence/absence of AMF at the second harvest in pot expt 2 to examine the enriched denitrifier community in the hyphosphere. Fresh soil was vortexed and suspended in ddH₂O. Then, 10⁵-fold dilutions of the soil suspension were spread on bromothymol blue (BTB) agar plates to isolate the denitrifiers [31 (link)]. Each sample was prepared in triplicate. The plates were incubated at 30 °C for 1–3 days. Separate blue colonies were isolated and purified by repeated streaking on BTB plates. The total bacterial DNA of each isolate was extracted from 1 mL culture suspension with a genomic DNA extraction kit (Tiangen Biotech, Beijing, China). The bacterial primers 27F/1492R were used for 16S rDNA amplification, and sequencing was performed by Tsingke Biotech, Beijing, China. The PCR thermal conditions are shown in Table S2. Following dereplication with a cut off value of 99% sequence similarity, the sequences were aligned with reference sequences in the National Center for Biotechnology Information (NCBI) GenBank database. A phylogenetic tree was then constructed by the neighbor-joining method [32 (link)] with bootstrap analysis of 1000 replicates using MEGA version 5 [33 (link)].
The bacterial primers nosZ1527F/nosZ1773R were used for nosZ gene amplification to examine whether the Pseudomonas isolates possessed the nosZ gene (Table S2). The target band was detected, sequenced and then identified using a BLAST search in GenBank in NCBI. Three Pseudomonas fluorescens isolates (JL1, JL2, and JL3) possessing the nosZ gene were screened. The draft genomes of the three strains were sequenced. Details are shown in the Supplementary Information.