Study area and sample design
The study site was located in Maoershan Forest Research Station (127°30′–127°34′E, 45°21′–45°25′N) of Northeast Forestry University, Heilongjiang province, China. This area is characterized by a continental monsoon climate of a windy spring, a warm and humid summer, and a dry and cold winter. Mean annual temperature is 2.8℃, with the minimum temperature in January (-40.9℃) and maximum temperature in July (34.2℃). The frost-free period fluctuates between 120 and 140 days. Annual precipitation ranges from 600 to 800 mm. Soils are Hap-Boric Luvisols [58] with high organic matter content and well-developed horizons, and are well drained.
In spring 1987, one-year-old seedlings of Juglans mandshurica and Larix gmelinii were obtained from the Maoershan Forest Farm and transplanted to the experimental station for establishing the monoculture (Juglans mandshurica, JM) and mixed plantation (Juglans mandshurica × Larix gmelinii, J × L) of Manchurian walnut. The seedlings were designed by 1.5 m × 1.5 m in each plantation, and mixed by lining (three rows of JM × five rows of larch) for mixed plantation. These plantations have similar site conditions with an average gradient of 7°. Detailed tree growth for each plantation were shown in Table S1. Voucher specimen of J. mandshurica and L. gmelinii were not deposited in this study since they are the most common trees in Northeastern China.
Sample Collection
In April 2016, three random sampling plots (20 m × 30 m 0.06 ha) were respectively selected for pure and mixed plantation described above, which were identified to serve as replicates. Rhizosphere soil and plant root samples were collected in July 2016. In each of these experimental plots, root samples were collected in 0–10 soil layers from nine individuals of Juglans mandshurica and mixed as a composite sample of each plot. The rhizosphere soils were sampled adjacent to the roots and brushed off from the plant root systems. The soil and root samples were packed in an ice box and transported to laboratory. Soil samples were sieved (1 mm mesh) to remove roots and debris, and subsamples were stored at -80℃ for DNA extraction. The first three root orders of the roots of Juglans mandshurica are infected by mycorrhiza [59]. Root samples were washed using distilled water to remove soil particles and were stored at -80℃ for DNA extraction.
Dna Extraction And Pcr Amplification
Microbial DNA was extracted from root and soil samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA), and DNA quality was checked by 1% agarose gel electrophoresis. The fungal 18S rRNA genes were amplified by a nested PCR reaction. According to the study of Lumini et al. 2010, AML1 (5’-ATCAACTTTCGATGGTAGGATAGA-3’) and AML2 (5’-GAACCCAAACA CTTTGGTTTCC-3’) where barcode is an eight-base sequence unique to each sample, were used in the first round PCR, and AMV4-5NF (5’-AAGCTCGTAGTTGAATTTCG-3’) and AMDGR (5’-CCCAACTATCCCTATTAATCAT-3’) were used in the second round PCR by thermocycler PCR system (GeneAmp 9700, ABI, USA). The two rounds of PCR yielded amplicons of approximately 800 bp and 300 bp, respectively. PCR reactions were performed in triplicate 20 µL mixture containing 4 µL of 5 × FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of FastPfu Polymerase and 10 ng of template DNA. The PCR reactions were conducted using the following program: 3 min of denaturation at 95 °C, 30 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, and 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min. The procedure of the second round PCR reaction was same as the first round, except for the cycle number, which was 35 [60].
Illumina Miseq Sequencing
The resulted PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s protocol. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform (Illumina, San Diego,USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP227587).
Processing Of Sequencing Data
Raw fastq files were demultiplexed, quality filtered by Trimmomatic and merged by FLASH with the following criteria: (i) The reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window. (ii) Primers were exactly matched allowing two nucleotide mismatching and reads containing ambiguous bases were removed. (iii) Sequences whose overlap longer than 10 bp were merged according to their overlap sequence.
Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE(version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 18S rRNA gene sequence was analyzed by blast against the MaarjAM database using confidence threshold of 97%.
Soil Physicochemical Analyses
Total carbon (C) and total nitrogen (N) were measured by a Macro Elemental Analyzer (vario MACRO, Elementar Co., Germany), and total phosphorus (P) was determined colometrically with a UV spectrophotometer (TU-1901, Puxi Ltd., Beijing, China) after wet digestion with HClO4–H2SO4. Soil pH was measured using a pH meter (MT-5000, Shanghai). Soil nitrate-N (NO3−-N) and ammonium-N (NH4+-N) were extracted in 2 M KCl and measured using a continuous-flow ion auto-analyzer (Scalar SANplus segmented flow analyzer, The Netherlands). Soil total phenol content was measured by the ultraviolet spectrophotometer method [61]. The Folin reagent colorimetric method was used to determine soil water-soluble phenol and complex phenol content [61]. Soil microbial biomass carbon (MBC) and nitrogen (MBN) was measured using a chloroform fumigation extraction method [62]. The mycorrhizal colonization rate of fine roots was determined according to method of Guo [59].
Statistical analysis
For the Illumina MiSeq sequencing data, the Alpha diversity indices (OTU number of observed, Chao1, ACE, Faith’s PD, Shannon and Simpson diversity indices) were generated using QIIME [63]. For beta diversity analysis, Bray-Curtis distances were calculated and Principal coordinates analysis (PCoA) was conducted to visualize the community similarity using by ‘vegan’ package in ‘R’ (Version 3.6.1) [64]. ANOSIM (Analysis of similarities) and PERMANOVA (Permutation multivariate analysis of variance) were carried out to test the differences in microbial communities with the Bray-Curtis distances and 999 permutations. Heat map analysis was used to compare the relative abundances of the top 50 most abundant classified AM fungal genera among treatments with ‘pheatmap’ package in ‘R’ (Version 3.6.1). The shared and unique OTUs among treatments were counted, and their distributions shown in a Venn diagram with the ‘VennDiagram’ package in ‘R’ (Version 3.6.1). Differences in relative abundances of microbial taxa between treatments were analyzed using Welch’s t test and Tukey’s honestly significant difference (HSD) test with Bonferroni correction in ‘STAMP’ [65]. The differences were considered statistically significant if P < 0.05. Redundancy analysis (RDA) was used to identify soil properties that predict the variation of AM fungal communities. Mantel test with a Monte Carlo simulation with 999 randomizations was used to assess the relationships between the Euclidean distance of AM fungal community and soil characteristic. RDA and Mantel test analyses were performed with the rda function in the ‘vegan’ package and the mantel.rtest function in the ‘ade4’ package in ‘R’ (Version 3.6.1), respectively.
Supplementary information
Please see the supplementary files.