Plant materials and treatments
Tomato (Solanum lycopersicum L. cv hezuo 903, obtained from Shanghai Changzhong Tomato Seed Industry Co., Ltd.) was used in this study. The AMF used in this study was an isolate of Funneliformis mosseae (BGC HEB07B, 1511C0001BGCAM0049, obtained from Huaian Chaimihe Agriculture Science and Technology Co., Ltd.), which was obtained through propagation with maize (Zea mays L.) as previously described .
The continuous cropping substrate, which had been used for the cultivation (complete growth cycle) of pepper and eggplant, was sterilized and then used to cultivate tomato in this study. The physical properties of the continuous cropping substrate deteriorated, the bulk density (BD) significantly increased, and the values of pH and electrical conductivity (EC) decreased (Additional file 12: Table S8).
The tomato seeds were placed on moist filter paper and germinated at 28 °C for 30 h in the dark. After germination, the seeds were sown in plastic pots containing continuous cropping substrate or continuous cropping substrate inoculated with F. mosseae at a dose of 600 spores per plant. Seedlings were cultivated in an artificial growth chamber. The growth conditions were as follows: 25 ± 2 °C/18 ± 2 °C (day/night), 12 h/12 h (day/night) photoperiod, 60–75% relative humidity, and 300 μmol m− 2 s− 1 light density. The growth parameters, root morphology, and Pn of tomato seedlings were determined at 40 d. The enzyme activity related to root activity was measured at 20, 25, 30, 35, and 40 d. The transcriptome sequencing was performed at 30 d with 3 biological replicates.
Tomato seedlings were cultured in fresh substrate and grown under in the same conditions as described above. The tomato seedlings were watered with 50 ml of 100 μM GA3 (Solarbio, Beijing, China), IAA (Solarbio, Beijing, China), ABA (Solarbio, Beijing, China), CTK (Solarbio, Beijing, China), and SL (Solarbio, Beijing, China) solution per plant, respectively, and the control plants were watered with the same volume of deionized water. Root samples were taken at 0, 1, 6, 12, and 24 h, respectively, to determine the gene expression of NADP-MEs.
IAA and TIBA treatment
To clarify the relationship between IAA and tomato root activity and NADP-MEs, 15-d-old NM and AM tomato seedlings were watered with 50 ml of 100 μM IAA or TIBA (Aladdin, Shanghai, China), every 5 d, and the control plants were watered with the same volume of deionized water. There were 6 treatments in the experiment, including NM, AM, AT, NT, NI, and NTI. Root samples were taken at 30 d to determine the root activity, enzyme activities, and NADP-MEs expression.
Mycorrhizal colonization rate measurement
For measurement of the rate of mycorrhizal colonization, fresh roots were collected at 30 d, cleaned and cut to 1–2 cm and then incubated in 10% (w/v) KOH (Sinopharm Chemical Reagent, Shanghai, China) at 90 °C for 40 min. The roots were rinsed with distilled water and then soaked in 2% lactic acid (Solarbio, Beijing, China) at 90 °C for 20 min. Then, the roots were stained with 0.05% Trypan Blue dye (Solarbio, Beijing, China) at 90 °C for 30 min. After cooling, the root samples were decolored with destaining solution (distilled water: lactic acid: glycerin [Sinopharm Chemical Reagent, Shanghai, China] =1:2:2, v/v) for 2–4 d at room temperature. Thirty pieces of randomly selected stained root fragments were observed with a Leica DM1000 microscope (Leica Microsystems, Wetzlar, Hesse, Germany) to confirm the presence of fungal structures, including intraradical mycelia, vesicles, and arbuscules. The root colonization rate was measured as previously described .
Morphological index and Pn measurement
The plant height and stem diameter were measured at 40 d according to previously described methods [43, 44]. After removing the plants from the cultivation pot, the roots were washed with distilled water and the fresh weight was measured with an electronic balance (OHAUS, Parsippany, NJ, USA). The plant materials were enclosed in envelopes and placed in an oven (Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China) at 105 °C for 30 min. Then, the oven temperature was adjusted to 75 °C for 2 d to obtain the dry weight. A WinRHIZO LA2400 root scanner system (Regent Instruments Inc., Québec, QC, Canada) was used to collect root morphological indexes.
The Pn was measured with a portable photosynthesis measurement system (Li-6400; Li-COR, Lincoln, NE, USA) after 1 h of light in the morning.
Root activity and related enzyme activities
The root activity of tomato was determined with the triphenyltetrazolium chloride (TTC) method as previously described .
The activities of 6PGDH, G6PDH, NADP-IDCH and NADP-ME were measured by spectrophotometry, recording the reduction of NADP at 340 nm [46, 47]. The experiment was conducted at 25 °C, and the reaction system volume was 1 ml, including 50 mM HEPES (pH 7.6, Solarbio, Beijing, China), 2 mM MgCl2 (Sinopharm Chemical Reagent, Shanghai, China), 0.8 mM NADP (Solarbio, Beijing, China) and plant samples. The reaction was initiated by adding 5 mM 6-phosphate gluconate (Aladdin, Shanghai, China), 5 mM glucose 6-phosphate (Solarbio, Beijing, China), 10 mM 2R,3S-isocitrate (Aladdin, Shanghai, China) and 10 mM malic acid (Solarbio, Beijing, China), respectively.
Analysis of the contents of IAA, GA3, CTK, ABA, and SL in tomato roots
The hormone contents were determined at 30 d as previously described . Tomato root tissue was fully ground in liquid nitrogen, and transferred to a precooled 50-ml centrifuge tube that contained 4 ml of precooled 80% chromatographic methanol (Aladdin, Shanghai, China). The mixture was placed on ice in the dark for 12 h. The tubes were centrifuged at 10000 g and 4 °C for 15 min, and the supernatant was transferred to another 50-ml tube and stored in a refrigerator at 4 °C. Afterward, 3 ml of 80% methanol was added to the remaining precipitate 3 times, and the supernatants were combined. Then, 1.0 g of polyvinylpyrrolidone (Sinopharm Chemical Reagent, Shanghai, China) was added to the supernatant and shaken in a shaker at 4 °C in the dark for 1 h. Subsequently, the mixture was centrifuged at 10000 g and 4 °C for 15 min, and the supernatant was passed through a C18 extraction cartridge (Waters, Milford, MA, USA) that had already been rinsed in the dark. The liquid was stored in a 50-ml centrifuge tube and freeze-dried in vacuum for 3 d under dark conditions. Then, 1 ml of precooled chromatographic methanol was added to the tube to completely dissolve the hormone, and the samples were filtered with a 0.45-μm organic microfiltration membrane before loading. The samples were detected with a high-performance liquid chromatography 1525 system (Waters, Milford, MA, USA).
Total RNA was isolated from tomato roots with the RNA Simple Total RNA Kit (Tiangen, Beijing, China). Total RNA (1 μg) was reverse transcribed into cDNA using HiScript® II Q RT SuperMix (+ gDNA-wiper) (Vazyme, Nanjing, China) for qPCR. qPCR assays were performed using ChamQ Universal SYBR-qPCR Master Mix (Vazyme, Nanjing, China) in a StepOne (TM) real-time PCR system (Applied Biosystems, Foster, CA, USA). The tomato Ubi3 gene was used as an internal control. The primer sequences are shown in Additional file 13: Table S9. The relative gene expression was calculated as described by Livak and Schmittgen .
RNA extraction, cDNA library construction and Illumina sequencing
Total RNA was extracted from the roots of tomato seedlings using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The RNA quality and purity were verified by a Nanodrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA) and electrophoresis in a 1.0% agarose gel. The mRNAs were purified from total RNA using poly-T oligo-attached magnetic beads (Invitrogen, Carlsbad, CA, USA). Subsequently, the mRNAs were fragmented, and cDNA was synthesized using random hexamers, DNA polymerase I (Thermo Fisher Scientific, Rockford, IL, USA) and RNase H (Thermo Fisher Scientific, Rockford, IL, USA). The purified double-stranded cDNAs were ligated to adaptors for Illumina paired-end sequencing. An Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and ABI real-time RT-PCR (Applied Biosystems, Foster, CA, USA) were used to verify the quality and quantity of the library, respectively. The cDNA libraries were sequenced with the Illumina HiSeq2000 platform (Illumina, San Diego, CA, USA) by the Beijing Genomics Institute.
Sequence data analysis and annotation
After the raw reads, adaptor sequences and low-quality reads were removed, all the clean reads were mapped to the tomato reference genome using TopHat v1.4.0 . The transcript abundance was normalized by the fragments per kilobase of exon per million fragments mapped using Cufflinks .
Identification of DEGs
The significance of the gene expression difference was recognized based on the false discovery rate (FDR) value less than 0.01 and |log2(fold change)| ≥ 2. After normalization, hierarchical clustering and k-means clustering analysis of the expression patterns were performed using Mutiexperimental Viewer v4.7 .
GO and KEGG enrichment analysis
For identification of putative biological functions and pathways of the DEGs, the GO and KEGG database were searched for annotation. GO classification was performed by WEGO . The AgriGO and KOBAS2.0 packages were used to analyze the enrichment of GO and KEGG at a significance cutoff of 0.05 FDR, respectively [54, 55].
The experiment was carried out in a completely randomized design with three independent replicates, and each replicate contained 12 plants. Significant differences (P< 0.05) between treatments were determined using Tukey’s test.