A. lancea plantlets were induced by buds of young stems obtained by the germination of wild A. lancea seeds collected from the Taurus Mountains, Jintan City, Jiangsu Province. Initially, full-grained wild seeds were carefully selected and washed thrice with soap water and then running tap water, respectively. The surface of the seeds was drained with absorbent paper. Thereafter, the remaining steps were carried out under aseptic conditions. The seeds were surface-sterilized by immersing in 75% ethanol for 30 s and then washed four times by soaking in sterile distilled water. They were immersed in 2% sodium hypochlorite for 15 min, then in 0.1% mercury chloride solution for 4 min, and finally rinsed five times in sterile distilled water. Eventually, the sterile surfaces of the seeds were dried with sterilized absorbent paper and placed on MS medium to accelerate germination (Wang et al. 2014).
The buds (approximately 2–3 cm long) of young stems of the germinated seedlings were intercepted and placed on 100 mL MS solid medium containing 100 mL distilled water, 0.02 mg naphthalene acetic acid (NAA), 0.2 mg 6-benzyladenine (6-BA), 3 g sucrose and 0.78% agar, to induce bud differentiation in 750 mL tissue culture flasks. The medium pH was adjusted to 5.8 before autoclaving at 121 °C for 20 min. Four weeks after differentiation, the newborn axillary buds were separated and placed in the rooting medium (1/2 MS) comprising 1.0 mg/L NAA, 30 g/L sucrose, and 7% agar. All in vitro cultures were maintained in a growth chamber (23 ± 2 °C entire day, with a light intensity of 1,500–2,000 lx and a photoperiod of 12 h) and sub-cultured every 4 weeks (Wang et al. 2012; Wang et al. 2014).
Soil microorganisms: Native soil collected from the soil beneath forests of the mountain slopes (31° 36ʹ 18ʺ N, 119° 6ʹ 48ʺ E; the habitat of wild A. lancea) in Lishui District, Jiangsu Province, was used to prepare a soil suspension comprising the entire microbial community; 10 g of soil was dissolved into 100 mL sterile distilled water and mixed on a shaker at a rotating speed of 220 r/min for 10 min.
Sterile nutrient soil: The nutrient soil (thickness, 6 cm) was added into a 750-mL tissue culture flask and then placed in a high-pressure steam sterilization pot at 121 °C for 60 min.
Thereafter, A. lancea plantlets with short roots, grown on rooting medium for four weeks, were transplanted to the sterile nutrient soil. Nine days later, the new roots were observed from the bottom of the bottle. Subsequently, 5 mL soil suspension was inoculated around the roots of the plantlets (see Supplementary Fig. 1). In the present study, we set two temperature conditions of 23 ± 2 °C and 30 ± 2 °C. The 23 °C condition is a common temperature for plant culture in the laboratory. As high temperatures > 30 °C in the laboratory are likely to cause the death of A. lancea plantlets, the critical temperature of 30 °C was selected as the high temperature treatment. Fifteen repetitions of plantlets inoculated with soil suspension and 15 repetitions of plantlets inoculated with sterilized soil suspension were placed in a greenhouse (23 ± 2 °C entire day, with a light intensity of 1,500–2,000 lx and 12 h of daylight). N indicates normal temperature (23 °C) and free-microbe treatment group. N+B depicts inoculated microorganisms at normal temperature. Another 15 repetitions of plantlets inoculated with soil suspension and 15 repetitions of plantlets inoculated with sterilized soil suspension were placed in an artificial climatic incubator (30 ± 2 °C for the entire day, with a light intensity of 1,500–2,000 lx and 12 h of daylight). H indicates high temperature (30 °C) and free-microbe treatment group. H+B indicates inoculated microorganisms at high temperatures. Three repetitions of sterile nutrient soils that were inoculated with sterilized soil suspension were placed in a greenhouse with a 23 °C temperature as the primary soil (CK). Simultaneously, nine repetitions of sterile nutrient soil without transplanting plantlets were placed in the greenhouse (23 ± 2 °C) after inoculation of the soil suspension. NBS indicates control soil inoculated with soil microorganisms at 23 ± 2 °C. Thereafter, another nine repetitions of sterile nutrient soil without transplanting plantlets were placed in the greenhouse (30 ± 2 °C) after inoculating the soil suspension. HBS presents control soil inoculated with soil microorganisms at 30 ± 2 °C. All the aforementioned treatment groups were sampled after 30 days and sample measurements were recorded.
All samples were collected and measured 30 days after inoculation. A total of 3 CK soil samples, each containing a repeat, were used for testing primary soil chemical properties. Nine NBS repetitions were randomly divided into three treatment groups with three repetitions each. Three duplicated soils were blended and used as a NBS for soil chemistry and microbiological testing. Nine HBS were sampled and tested in the same way. Fifteen repetitions were randomly divided into three treatment groups with five plantlets each. First, the plantlets were pulled from the soil and excess loose soil was shaken off. The soil tightly adhered to the roots surface was removed and collected as rhizosphere soil. Five rhizosphere soils from each treatment group were combined as one rhizosphere soil sample. NRS and HBS represent the rhizosphere soil in which plantlets were cultivated at normal temperatures and high temperatures, respectively, without microbial inoculation. NBRS and HBRS indicate the rhizosphere soil in which plantlets were cultivated at normal temperatures and high temperatures, respectively, with microbial inoculation. Thereafter, the roots of these plantlets were cleaned with sterile water, and then surface-dried with sterile absorbent paper for measuring the fresh weight. Second, each plantlet was weighed and a cut was made at the junction of the root and stem. The fresh weight of the whole plant minus the fresh weight of the root was used as the fresh weight of the stems and leaves. The roots were carefully straightened and the length was measured with a vernier caliper. The ratio of fresh weight of stems and leaves to the fresh weight of the roots was used as the root-shoot ratio. Third, two roots were removed from the roots of all plantlets and combined according to the aforementioned random grouping to detect the composition of endophytic bacterial diversity. Mixed root tissues from each grouping were placed in clean 50 mL conical tubes and pre-rinsed three times with sterile distilled water. The washed roots were then treated with 70% ethanol for 10 min, followed by a treatment with 2.5 % sodium hypochlorite and sonication for an additional 10 min. The samples were then drained and rinsed with sterile distilled water for three times. To check for surface sterility, 100 μL of the final rinsed solution was plated in Potato Dextrose Agar (PDA) and Nutrient agar (NA) and this resulted in zero colonies. Similarly, two other roots were removed and combined as above to detect the expression of key enzyme genes. Tissue samples and soil samples used for the detection of endophytic and rhizosphere bacteria were cooled in liquid nitrogen immediately after sampling, and then stored at -80℃ for later use. The remaining roots were mixed accordingly to identify the contents of the four volatile oils including hinesol, β-eudesmol, atractylon, and atractylodin. Eventually, the root dry weight was measured after the roots used to detect the volatile oils had been dried.
Analysis of content of four volatile oil and key enzyme gene expression
The content of hinesol, β-eudesmol, atractylon, and atractylodin was measured via gas chromatography mass spectrometer (GC-MS). The rhizomes of different treatment groups used to detect volatile oil were dried in a freeze-dryer for approximately 72 h until they attained a constant weight, and were then ground into powders. The volatile oil was extracted with a solid–liquid ratio of 1:4, that is, 0.1 g powder was soaked in 0.4 mL n-hexane. After 15 min of sonication (60 Hz), the mixture was centrifuged for 5 min at 5000 × g at 4 °C. The supernatant was separated, filtered through a 0.22 µm microporous filter, and stored in a brown sample vial at 4 °C for GC-MS analysis (Yang et al. 2019; Yuan et al. 2019). The aforementioned components were quantified using a Trace 1310 series GC with a TSQ8000 MS detector (Thermo Fisher Scientific Co. Ltd, Waltham, Massachusetts, USA). A TR-5ms capillary column (30 m 3 0.25 mm i.d., DF = 0.25 mm, Thermo Fisher Scientific) was used for GC-MS analysis. A few adjustments were made to the program, according to the method of Li et al. (2018), as follows: the injected sample (1 μL) was separated with a Helium flow rate of 1 ml/min with a temperature program of 2 min at 120 °C, followed by a gradient from 120 °C to 240 °C at 5 °C/min, and held at 240 °C for 5 min. The injector and detector temperatures were set at 240 °C and 350 °C, respectively. MS operating conditions were as follows: the MS ionization mode indicated the electron impact ion source (EI) at 230 °C, with an acceleration voltage of 70 eV. The interface temperature was 240 °C and the total ion current was recorded for a mass range of 40–500 amu (Li et al. 2018; Yang et al. 2019; Yuan et al. 2019). The contents of four volatile oils in each sample were quantitatively determined by the standard curves (see Supplementary Table 1).
In plants, the precursors of terpenoids can be produced through mevalonate (MVA) and methylerythritol-4-phosphate (MEP) pathways (Vranová et al. 2013). HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and DXS (1-deoxy-D-xylulose 5-phosphate synthase) are the first rate-limiting enzymes in the MVA and MEP pathways, respectively (Zhao et al. 2010). Moreover, sesquiterpene biosynthesis requires a key enzyme, farnesyl diphosphate synthase (FPPS) (Cane 1999; Shakeel et al. 2016). Real time quantitative reverse transcription PCR (real time qRT-PCR) was performed to detect the expression levels of key enzyme genes of plantlets in different groups, including HMGR, FPPS, and DXS (Liu et al. 2007; Deng et al. 2017; Jiang et al. 2017; Lu et al. 2019). Ribosomal protein 18 (18S) was used as an internal reference (Jiang et al. 2017). Primers for the three selected genes are listed in supplementary Table 2; they were synthesized by Beijing Ruibo Biotechnology Co., LTD, Beijing, China. The total RNA extracted from the roots was used to detect the expression of key enzyme genes using a quick RNA isolation kit (Hua Yue Yang biotechnology, Beijing, China). The results of agarose gel electrophoresis revealed high quality total RNA (see Supplementary Fig. 2); therefore, further tests could be carried out. Approximately 2 μg of total RNA was reverse-transcribed into cDNA using a kit (Prime Script One Step RT Reagent Kit; Takara, Dalian, China) (Jin et al. 2019). The reaction system of real time-qPCR was as follows: 10 μL SYBR Premix Ex Taq (2×), 1 μL PCR forward primer (10 pmol/μL), 1 μL PCR reverse primer (10 pmol/μL), 2 μL cDNA template, replenished with ddH2O to 20 μL. Real time-qPCR was performed at 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 40 s (Lu et al. 2019).
Analysis of soil chemical properties
Five physical and chemical indices of soil were detected, namely, pH, nitrate nitrogen, ammonium nitrogen, available phosphorus, and available potassium, respectively. The pH values of soil were measured using the glass electrode method (soil and water suspension v/v 1:2.5) (Iso 1994). Soil nitrate nitrogen, soil ammonium nitrogen, available phosphorus, and available potassium contents were determined via ultraviolet spectrophotometry (GB/T 32737-2016), indophenol blue colorimetry (LY/T 1228-2015), colorimetry (NY/T 1121.7-2014), and the extraction-molybdenum-antimony anticolorimetric method (NY/T 889-2004), respectively.
DNA extraction, 16S amplicon sequencing, and bioinformatics analysis
DNA extraction and 16S amplicon sequencing
The total bacterial DNA extracted from 100 mg root tissues, 150 mg rhizosphere soil, and 150 mg control soil using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), was used for amplification and sequencing of the 16S rRNA, targeting the variable V3–V4 regions (Xu et al. 2016; Perez-Jaramillo et al. 2019), thereby resulting in amplicons of approximately 460 bp. The gene primers were 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Error-correcting barcodes were added to both forward and reverse primers (Hamady et al. 2008). Extracted DNA was detected by 1% agarose gel electrophoresis. PCR reaction was carried out on a GeneAmp 9700 PCR system (Applied Biosystems, Foster City, CA, USA). The total volume of each PCR amplified reaction system was 25 μL, including 1 μL DNA template, 0.5 μL forward primer, 0.5 μL reverse primer, 0.25 μL bovine serum albumin, 12.5 μL 2× DreamTaq Green PCR Master Mix (Thermo Scientific, USA), replenished with ddH2O to 25 μL. Setting three replicates for each reaction, PCR was carried out as follows: 95 °C for 3 min, followed by 27 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 10 min. Three technical repeats of one sample were mixed into a single PCR product. The products were separated via 2% agarose gel electrophoresis and purified using a Qiagen PCR purification kit (Qiagen, Hilden, Germany). Furthermore, the purified products were quantified with Pico Green using a QuantiFluorTM-ST Fluorometer (Promega Biotech, Beijing, China) and were then pooled at equal concentrations. Thereafter, the amplicons were sequenced in an Illumina MiSeq platform (San Diego, CA, USA) at Shanghai Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China.
First, paired-end (PE) reads obtained by MiSeq sequencing were spliced according to their overlap relations using FLASH (Magoč and Salzberg 2011), and quality-filtered using Trimmomatic (Bolger et al. 2014). All sequences were divided into operational taxonomic units (OTUs) with 97% similarity or greater using UPARSE software (version 7.0) (Edgar 2013), and a majority consensus taxonomy was obtained for each OTU. Singletons were removed from the datasets to minimize the impact of sequencing artifacts (Dickie 2010). Chimeric sequences were identified and removed using UCHIME (Edgar et al. 2011). In order to obtain the species classification information corresponding to each OTU, the RDP classifier algorithm (https://sourceforge.net/projects/rdp-classifier/) was applied to compare the OTU representative sequences with the Silva database (SSU138) for taxonomic analysis using confidence threshold of 70%. Among these, chloroplasts and mitochondrial sequences were removed. The bacterial community diversity and richness were demonstrated using the Shannon index, Simpson index，Chao index, and ACE index using Mothur v.1.30.1 (http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity). Student’s t-test was used to compare the significance of the index differences between groups. The significance level to threshold (P value) was set at 0.05. The relative abundance bar of bacteria at the phylum and genus levels was visualized using R language tools (v.3.3.1). Analysis of Principal Coordinates Analysis (PCoA) between root tissue samples was performed using QIIME (version 1.9.1) based on unweighted-Unifrac distance matrix. A non-metric multidimensional scaling (NMDS) ordination to illustrate the clustering of control soil and rhizosphere soil bacterial community composition variation was conducted using the Vegan software based on the Bray-Curtis distance of genus. The Student’s t-test was used to examine differences in bacterial composition between the two groups. The relation between relative abundance of genus and the contents of four volatile oils or soil chemical properties was performed using Spearman’s correlation analyses.
In the present study, biomass, volatile oil, key enzyme gene expression, and soil physicochemical data as aforementioned were recorded and processed by Excel (Office 2019). Thereafter, GraphPad Prism 8.0.1 (GraphPad Software Inc., USA) was used for rendering graphics. One-way ANOVA of plant biomass, the contents of four volatile oils, key enzyme gene expression, and soil chemical properties were determined using IBM SPSS Statistics 19.0 (SPSS, Chicago, IL, USA). Significance was calculated by Tukey’s test (p < 0.05). Results are expressed as mean ± standard deviation (S.D.). P<0.05 and P<0.01 were considered to be statistically significant and extremely significant, respectively.