phyllosphere fungi
After quality evaluation of the extracted sample DNA, the ITS1 region of fungal 18S rRNA was amplified using 2Hieff Robust PCR Master Mix (Yeasen,10105ES03, China). Two universal fungal rRNA gene amplicon PCR primers (PAGE purified) were used: the primers were as follows: ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2 (5'-GCTGCGTTCTTCATCGATGC-3'). Each PCR mixture consisted of 15 l Hieff  Robust PCR Master Mix (2); 3 l Bar-PCR primer F (2 mol/l); 10 l DNA (1 ng/l); and 2 l ddH2O. The plate was sealed and PCR performed in a thermal instrument (Applied Biosystems 9700, USA) using the following program: 1 cycle of denaturing at 95 C for 3 min, first 5 cycles of denaturing at 95 C for 30 s, annealing at 45 C for 30 s, elongation at 72 C for 30 s, then 20 cycles of denaturing at 95 C for 30 s, annealing at 55 C for 30 s, elongation at 72 C for 30 s and a final extension at 72 C for 5 min. The PCR products were separated by electrophoresis on 2% agarose gels in 1  TAE buffer, purified, and then quantified. A test library was constructed using Qubit 3.0, and then subjected to high-throughput amplicon sequencing on the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA). Sequencing was performed by the Sangon Bioengineering Co., Ltd. According to the manufacturers instructions.