Fungal and Bacterial Diversity Profiling

The genomic DNA was extracted with an AxyPrep DNAGel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s instructions, and quantified using the NanoDrop 2000 UV–Vis spectrophotometer (Thermo Scientific, Wilmington, USA). The endophytic and epiphytic fungal 18ITS1 gene was amplified using primers ITS1F-5′-CTTGGTCATTTAGAGGAAGTAA-3′ and ITS2R-5′-GCTGCGTTCTTCATCGATGC-3′ (Gardes and Bruns, 1993 (link)). The amplicons were purified, quantified, and sequenced using the Illumina Miseq PE250 platform. Primers 338F-5′-ACTCCTACGGGAGGCAGCA-3′ and 806R-5′-GGACTACHVGGGTWTCTAAT-3′ (Mori et al., 2014 (link)) were used to amplify the V3-V4 region of the epiphytic bacterial 16s rRNA gene. Primers 779F-5′- AACMGGATTAGATACCCKG -3′ and 1392R-5′- ACGGGCGGTGTGTRC -3′ were used to amplify the endophytic bacterial gene. The amplicons were purified and sequencing using Illumina Miseq PE 300. The 25 μl PCR system contained 12.5 μl Premix Taq Polymerase (Takara, China), 0.5 μl of each primer, 10 ng genomic DNA. PCR was carried out under the following conditions: 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final extension step at 72°C for 10 min. Sequencing was performed by Shanghai Majorbio Biopharm Technology Co., Ltd (China).