Soil sampling and amplicon analysis of soil fungi
36 soil cores, one from each treatment combination (i.e., one column, consisting of three 40×40 cm quadrats; see Table 1b), were collected in June 2018 (near the end of the three-year experiment) for analyzing the fungal composition. Soil samples were collected from the interstices between the transplanted seedlings in each column (bulk soils instead of rhizosphere soils were collected because it was not feasible to trace the roots of the seedlings that died in the first two years). After removal of loose surface debris, surface soil samples (0–10 cm in depth) were collected using a soil corer with a 5-cm inner diameter. The soil corer was rinsed thoroughly using double distilled water after each sampling.
The collected soil samples were immediately frozen and shipped on dry ice to Magigene Technology Ltd. (Guangzhou, China) laboratory and were stored at -80 °C prior to DNA extraction for Illumina amplicon sequencing. The sequencing steps are described as follows:
In total, 11407 OTUs (Data S1) were observed. Among these OTUs, 88.73% (9802) belonged to fungi (Data S2), while the remaining OTUs belonged to kingdom Animalia (0.05%), Chromista (2.10%), Plantae (1.92%), and Protista (0.71%), or showed no blast hit (5.51%). According to the previously published criteria for grouping functional guilds based on the FUNGuild database (Nguyen et al. 2016; Vetrovsky et al. 2019; Delgado-Baquerizo et al. 2020), the fungal OTUs identified to the genus level were further grouped as saprotrophs, plant pathogens, EcM fungi, etc. An OTU identified to the genus level was assigned to a guild if it could unequivocally match a guild, otherwise its trophic strategy was considered unknown (Nguyen et al. 2016; Hannula et al. 2017; Mommer et al. 2018; Vetrovskyet al. 2019). During analyses of the response of pathogenic fungi and EcM fungi to experimental treatments, we only chose the fungal OTUs as plant-pathogenic fungi or EcM fungi with “probable” or “highly probable” confidence levels (Data S3).
Because the ITS primers used in this study were invalid to classify oomycete pathogens (belonging to Chromista), Chrmista OTUs were excluded from the community analysis. Even though oomycete-specific primers are currently available, they can merely be used to qualitatively analyze oomycete community and diversity monitoring (Legeay et al. 2019), infeasible to quantitatively assess relative abundance of oomycete pathogens.