Eight years of throughfall exclusion reshaped bacterial, fungal, and arbuscular mycorrhizal fungal communities in managed forest soil

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Forest soils support tree productivity and resilience.However, long-term manipulations of drought and substrate inputs are rare.This resource reports 16S rRNA gene, ITS2, and long-amplicon (SSU-ITS-LSU) sequencing datasets of bacterial, fungal, and arbuscular mycorrhizal fungal communities from Japanese forest soil following 8 years of throughfall exclusion.KEYWORDS amplicon sequencing, arbuscular mycorrhizal fungi, drought, forest soil, soil bacteria, soil fungi, throughfall exclusion D rought alters soil microbial communities and associated carbon and nutrient dynamics (1).Although short-term throughfall exclusion experiments are common (2), long-term manipulations are limited.We sequenced bacterial, fungal, and arbuscu lar mycorrhizal fungal (AMF) communities in soil from an 8-year throughfall exclusion experiment in a managed forest.These data sets support studies of drought-induced shifts in forest soil microbiomes.Soil was sampled in October 2025 at the Chiyoda Experimental Station of the Forestry and Forest Products Research Institute, Japan (36°10′57.4″N, 140°13′01.2″E).This site has been roofed continuously since 2017 (2 m aboveground) to exclude rainfall and litterfall (3).Surface mineral soils (0-10 cm) were collected from four points per treatment (rainfall/litter exclusion and adjacent control), transported on ice, and stored at -80°C.For bacterial and fungal community profiling, frozen soil samples were freezedried, bead-homogenized, and used for DNA extraction with a Lab-Aid 824s Kit (ZEESAN, Xiamen, China).DNA was quantified fluorometrically.The V4 region of the 16S rRNA gene was amplified with primers 515F/806R (5′-GTGCCAGCMGCCGCGG TAA-3′/5′-GGACTACHVGGGTWTCTAAT-3′) (4) and the fungal ITS2 region with gITS7/ITS4 (5′-GTGARTCATCGARTCTTTG-3′/5′-TCCTCCGCTTATTGATATGC-3′) (5).Amplicon libraries were prepared by two-step tailed PCR using KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan); first-round PCR comprised 94°C for 2 min; 30 cycles of 98°C for 10 s, 50°C for 30 s, and 68°C for 30 s for 16S, or 94°C for 15 s, 50°C for 30 s, and 68°C for 1 min for ITS2; and 68°C for 7 min.Libraries were sequenced on the Illumina NextSeq 1000 platform using 2 × 300 bp paired-end reads (Seibutsu Giken Inc., Kanagawa, Japan) (Table 1).Sequence processing followed previously described methods (6), using DADA2 v1.38.0 (7) in R v4.5.2 with truncLen = c(240, 220) for bacteria.Bacterial and fungal taxonomy was assigned against SILVA v138.1 and UNITE v10.0, respectively.For AMF, genomic DNA was extracted from frozen soils using a NucleoSpin Soil Kit (Macherey-Nagel, Düren, Germany).Long-amplicon sequencing targeting the SSU-ITS-LSU region was performed using nested PCR (8) with primer sets NS31/ LSUmAr13 + LSUmAr24 and AML1/LSUmAr13 + LSUmAr24 (9), followed by final amplification with NS31_Glo3/wLSUmBr (5′-TTGYTGCRGTTAAAAAGCTCG-3′/5′-AACACTC

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