The effect of short-term fallowing on the microbial communities in forest soil cultivated with ginseng: Preliminary research

Experimental site

Three samples were collected in Erdaogang (41°35′56.99″N, 128°2′42.55″E), which is a village in a mountainous region in Malugou, Changbai County, Baishan City, Jilin Province, China. The average annual temperature is 2.0 °C, and the mean annual precipitation is 691 mm. The frost-free period is approximately 90–135 d, and the average yearly sun exposure is 2,466 h. The lowest temperature period from December to February of the next year is −17 °C on average, and the lowest temperature is −20 °C in January. Frost appears at night and thaws during the day in the middle of October. In early November, the frost layer deepens gradually, and the thickness of the frost layer reaches between 150 cm and 200 cm in February. The ground begins to thaw in the middle of March, and thaws completely by the middle of May. The depth of frozen soil is directly related to humidity, and snow depth. The soil is classified as dark brown forest soil, a typical subcategory of dark brown soil with six typical characteristic layers: AOO (leaf litter layer), AO (dark semi-decomposed organic matter layer), A1 (humus layer), Ab (transition boundary layer), B (illuvial layer), and C (parent material layer).

Experimental design

The field experiment was conducted in October 2014. Three kinds of sampling fields were chosen: the field identified as “fallow field” (Hx2) was the field where ginseng had been cultivated for two to four years, harvested, and then the field was left fallow for two years. Another sampling field was designated the “ginseng cultivated field after fallow” (Clsd); this was the field where ginseng had been cultivated for two to four years, was harvested, and ginseng was cultivated for two years. A forest field (Hx0ks) with trees (Betula platyphylla, Larix gmelinii, etc.) was chosen as the control.

Each plot was 5 × 10 m², and ginseng sampling fields were managed using the same agronomic practices. Hx2 and Clsd were old ginseng fields. The distance between the two treatments plot was 50 m, and the control forest soil plots were located next to the Hx2 and Clsd plots.

Short-term fallow procedure of cultivated ginseng field

When ginseng roots had been harvested in the fall of 2014, chosen field plots (Hx2) were irrigated and the soil water content was increased up to about 50–60% of the maximum field water capacity. The soil was tilled and furrowed with a rotary tiller and 30 g of dazomet fine granules (98% C₅H₁₀N₂S₂) per square metre was spread. The soil was sealed with plastic film for more than 20 days, then opened for 15 days, so that the dazomet agent could completely evaporate. A total of 500 g per square meter of forest leaf fertilizer was broadcast into the soil, which marked the start of two years of fallow.

Ginseng variety and soil samples collecting

We used the ginseng variety, Damaya (Panax ginseng strains Damaya). Ginseng seedlings were replanted in 2014 in the plot (Clsd) and were managed using the same conditions mentioned above. This marked the start of two years of ginseng growth.

Soil samples (depth: 0–20 cm) were collected with a four-point sampling method to analyze soil nutrients and microbial community structure. Different treatment soils were randomly collected from five sampling sites in each plot in October 2016. Soil samples were sealed in plastic bags and stored in dry ice before being transported to the laboratory. All soil samples were divided into two fractions: the soil samples, mixed from five sampling sites in each sample plot with stones and large roots removed, were ground through a two mm sieve to measure soil nutrients; another fraction of the soil samples were stored at −80 °C for high-throughput sequencing to analyze the soil bacterial and fungal community structure and diversity.

Soil nutrient determination

The amount of organic matter was determined by quantifying the amount of oxidized soil carbon based on its reaction to acidic dichromate (Cr₂${\mathrm{O}}_7^{2-}$) (Zhang et al., 2012). Soil available nitrogen was determined by alkaline hydrolysis diffusion method (Zhou et al., 2020). Total nitrogen was determined using the Kjeldahl determination (Lu & Foo, 2000). Available phosphate (AP) was extracted with NaHCO₃, and was then determined by molybdenum antimony spectrophotometry (UV-1800 spectrophotometer; Agilent, Santa Clara, CA, USA) at 880 nm, based on the National Environmental Protection Standards of the People’s Republic of China (HJ 704-2014). The C:N was calculated based on the ratio of soil organic carbon (SOC) of total nitrogen, and SOC was determined by the K₂Cr₂O₇ redox titration method (Nelson & Sommers, 1983).

DNA extraction and high-throughput sequencing

Approximately 0.5 g of the chilled soil samples were used to extract the total genomic DNA using a Fast DNA SPIN Kit (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. Data were collected as previously described in Miao et al. (2022). Specifically, in PCR amplification, the primers (5′-ACTCCTACGGGAGGCAGCA-3′) and (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3–V4 of the 16S rRNA gene for bacterial community analysis. Primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA- 3′) and ITS1R (5′-GCTGCGTTCTTCATCGATGC-3) were used to amplify the ITS region for fungal community analysis. The PCR reaction was run using Eppendorf Mastercycler^® Pro S thermal cycler as follows: initial denaturation at 98 °C for 2 min, 10 s denaturation at 98 °C for 40 cycles, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min, and finally at extend at 72 °C for 10 min. PCR products were purified using a Qiagen PCR Purification Kit (Qiagen, Inc., Shanghai, China) and were pooled in equimolar concentrations. High-throughput sequencing was performed using an Illumina MiSeq platform (Biomarker Technologies Co. Ltd., Beijing, China).

Data analysis

The Quantitative Insights into Microbial Ecology (QIIME 1.8.0) toolkit was used to analyze the sequences. After removing low-quality or ambiguous sequences, FLASH (Fast Length Adjustment of Short reads) (v1.2.7) was used to select high-quality, paired-end reads (Magoč & Salzberg, 2011).

OTUs were defined as similarity between sequences higher than 97%. Based on the results of OTU analysis, taxonomic analysis was performed on the samples at each classification level to obtain the community structure diagram of each sample at the taxonomic level of phylum, class, order, family, genus and species. QIIME was used to calculate the α diversity index, including the Simpson index, Chao1 index, ACE index and the Shannon index, and to draw the sample dilution curve. PICRUSt software (Yin & Wang, 2021) was used to predict functional gene composition from 16S rRNA sequences, and metabolic pathways of functional genes were observed through the KEGG database (Palù et al., 2022), leading to heat map using origin software (Biomarker Technologies Co. Ltd., Beijing, China).

Soil nutrient content

Soil organic matter (SOM), total N, available N, available P, and C:N ratios were higher in the sample collected from the fallow plot (Hx2) and the forest plot (Hx0ks) as compared to the sample collected from the plot that was returned to cultivation (Clsd) (Table 1). Soil nutrient content were similar for the fallow and forest plots.

Basic information on the sequencing data

A total of 390,753 bacterial 16S rRNA and 320,897 fungal ITS gene sequences passed the quality control tests. 16S rRNA and ITS gene sequences of each sample ranged from 64,542–65,311 and 52,718–58,334, respectively. After optimized sequence clustering, the number of OTUs of bacteria and fungi in the soil were 1,013–1,150 and 326–612, respectively (Table 2). The number of bacteria unique to libraries generated from the fallow or the forest plot was more than the number of bacteria unique to the plot (Clsd) that was returned to cultivation (Fig. 1A). The number of fungi unique to a specific plot, was highest for the fallow plot (Hx2) (Fig. 1B).

The slope of the rarefaction curve was flat under different similar cut-off values, indicating that this sample of sequences were sufficient for data analysis (Fig. 2).

Microbial community diversity analysis

The library generated from a soil sample collected after two years of fallow period showed higher alpha diversity of bacteria and fungi communities in soil relative to plots that remained in cultivation (Table 2).

Changes in the relative abundance of bacterial communities

Cultivation did not significantly change the structure of bacterial communities, at the phylum level, in soil (Fig. 3A). The main phyla detected (Acidobacteria, Proteobacteria, Actinobacteria, Verrucomicrobia and Gemmatimonadetes) were the same in the three samples (Fig. 3A). Among these, Acidobacteria and Proteobacteria accounted for the majority of bacteria observed. Similarly, the relative abundance of bacterial genera was similar between the three libraries but these genera were not well characterized. About 80% of OTUs were either unknown genera, uncultured or others (Fig. 3B).

Changes in the relative abundance of fungal communities

The main fungal phyla detected in the three samples were Basidiomycota, Ascomycota, and Zygomycota; however, each sample had a partially unknown sequence at the phyla and genus level due to the limited resolution of ITS sequencing (Fig. 4A). Basidiomycota and Ascomycota accounted for >70% of the fungal phyla detected. In the library generated from the forest sample (Hx0ks), Russula (44%) was the dominant fungal genera identified (Fig. 4B). In the library generated from the fallow and cultivated field samples, Mortierella and Leucoagaricus respectively were the dominant fungal genera identified.

Function prediction

Differential analysis of KEGG metabolic pathways allowed for the identification of approximately 27 intensive metabolic pathways (Fig. 5). The sample from a forest plot showed many pathways for signaling molecules and interaction, glycan biosynthesis and metabolism, cell motility, drug resistance, folding, sorting and degradation, and energy metabolism. The sample a fallow plot showed many pathways for substance dependence, sensory system, immune diseases, circulatory system, and cellular community. The sample from the cultivated plot showed pathways for xenobiotics biodegradation and metabolism, transport and catabolism, signal transduction, nucleotide metabolism, metabolism of terpenoids and polyketides, environmental adaptation, drug resistance, cell growth and death, amino acid metabolism. Compared to the sample from forest soil, pathways of signaling molecules and interaction, glycan biosynthesis and metabolism, and cell motility were reduced.

Changes in the relative abundance of pathogens

Fallow periods appeared to reduce the relative abundance of fungal pathogens of ginseng. Pathogens were relatively rare in the library generated from the forest soil sample (Fig. 6). The relative abundance of Armillaria, Rhizoctonia, and Cylindrocarpon, were lower in the fallow plot (Hx2) than the cultivated plot; however, Fusarium (0.70%) and Ilyonectria (1.22%) were relatively higher in the library generated from the fallow plot (Fig. 6).

Compared to the fallow field, the relative abundance of Armillaria (0.04%), Rhizoctonia (0.22%), Cylindrocarpon (0.15%), and Pseudomonas (0.39%) was higher in the library generated from the cultivated plot.