Abstract

Background

   Despite significant limitations of growth medium reuse, a large amount
   of organic substrate is reused in soilless cultivation of horticultural
   crops in China. Arbuscular mycorrhizal fungi (AMF) can promote nutrient
   absorption and improve plant tolerance to biotic and abiotic stresses.
   However, the mechanisms governing the effects of AMF on crop growth in
   organic continuous cropping substrates have not been elucidated.

Results

   In this study, we showed that the inoculation of AMF in continuous
   cropping substrates promoted growth and root development, and increased
   the root and NADP-malic enzyme (NADP-ME) activity of tomato seedlings.
   Root transcriptome analysis demonstrated that the plant hormone signal
   transduction pathway was highly enriched, and 109 genes that positively
   correlated with the AMF-inoculated plant phenotype were obtained by
   gene set enrichment analysis (GSEA), which identified 9 genes related
   to indole acetic acid (IAA). Importantly, the levels of endogenous IAA
   in tomato seedlings significantly increased after AMF inoculation.
   Furthermore, the application of AMF significantly increased the
   expression levels of NADP-ME1 and NADP-ME2, as well as the activity of
   NADP-ME, and enhanced the root activity of tomato seedlings in
   comparison to that observed without inoculation of AMF. However, these
   effects were blocked in plants treated with 2,3,5-triiodobenzoic acid
   (TIBA), a polar transport inhibitor of IAA.

Conclusions

   These results suggest that IAA mediates the AMF-promoted tomato growth
   and expression of NADP-MEs in continuous cropping substrates. The study
   provides convincing evidence for the reuse of continuous cropping
   substrates by adding AMF as an amendment.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12870-020-02817-2.

   Keywords: Tomato, Arbuscular mycorrhizal fungi, Continuous cropping
   substrate, IAA, Root activity

Background

   Soilless cultivation promotes crop growth and yield by providing the
   most suitable environment for root growth and development [[39]1]. Due
   to the numerous advantages of organic substrate cultivation, such as
   improved crop yield and quality, reduced pesticide, water and
   fertilizer use, simple operation and management, reduced investment
   cost for equipment, avoidance of soil continuous cropping obstacles,
   and suitability for green organic cultivation, this type of soilless
   cultivation has been widely practiced and currently accounts for over
   75% of the total area of soilless cultivation in China [[40]2]. The
   annual production and sales volume of organic substrates in China is
   approximately 10 million m^3. At the same time, the same volume of the
   substrates used is produced every year. However, the physical and
   chemical properties of the used substrates deteriorate when reused,
   resulting in larger bulk density and lower pH, which are not conducive
   to crop growth and development [[41]3]. Studies have shown that the
   numbers of beneficial fungi decrease and those of harmful fungi
   increase in continuous cropping substrates [[42]4–[43]6]. In addition,
   the effective nutrient content, substrate enzyme activity, and root
   index in the rhizosphere environment significantly decrease with the
   increase in continuous cropping years [[44]4, [45]5]. However, it is
   imperative to improve and reuse substrates to reduce the labor input
   and production costs. At present, few methods have been used to improve
   continuous cropping substrates, such as substrates disinfection and
   crop rotation [[46]4, [47]6]. Interestingly, the inoculation with
   beneficial microorganisms in the continuous cropping soil has been
   demonstrated to be an effective way to overcome continuous cropping
   obstacles [[48]7]. However, the role of beneficial microorganisms in
   continuous cropping substrates is largely unknown.

   Arbuscular mycorrhizal fungi (AMF) are ubiquitous soil microorganisms
   and obligate symbionts that are important components of terrestrial
   ecosystems and can form a mutually beneficial symbiosis with
   approximately 80% of vascular plant roots [[49]8]. The glycoproteins
   produced by AMF hyphae can create a suitable rhizosphere environment
   and better conditions for the growth of almost all vascular plants
   [[50]9]. AMF symbiosis not only increases the input of root carbon but
   also enhances carbon fixation in soil by reducing the decomposition of
   soil organic matter and the rhizosphere priming effect [[51]10]. AMF
   endow soil and roots with a direct connection, enhance plant mineral
   nutrition, water acquisition and photosynthesis, and alleviate the
   adverse effects of abiotic stresses, such as heavy metals and drought
   stress [[52]11–[53]13]. Our previous study showed that AMF inoculation
   on seedling substrates can reduce the harm of saline-alkali land
   exerted on processing tomato [[54]14]. In addition, the application of
   AMF can effectively overcome the continuous cropping obstacles in
   continuous cropping soil, to promote growth and development of many
   crops, such as pepper [[55]15], soybean [[56]16], and cucumber
   [[57]17]. However, the associated mechanism of AMF-promoted growth in
   continuous cropping systems has not been elucidated.

   In this study, we investigated the effect and mechanism of AMF on the
   cultivation of tomato in continuous cropping organic substrates. The
   results showed that the inoculation of AMF in the continuous cropping
   substrates promoted the growth and increased the activity of root and
   NADP-malic enzyme (NADP-ME), and the level of indole acetic acid (IAA)
   in tomato seedlings. However, these effects were compromised when
   plants were treated with IAA polar transport inhibitor, indicating that
   IAA played a critical role in AMF-promoted plant growth. The results
   provide a theoretical and practical basis for the cultivation of tomato
   in continuous cropping substrates.

Results

Effects of AMF on root morphology, plant growth and net photosynthetic rate
in tomato seedlings

   To test the role of AMF in continuous cropping substrates, we first
   compared the growth of tomato in continuous cropping substrates with or
   without inoculation of AMF. AMF successfully colonized tomato roots,
   and the colonization rate was approximately 32% in tomato seedlings
   cultivated in the continuous cropping substrate inoculation with AMF
   (AM) (Additional file [58]1: Figure S1). The inoculation of AMF
   significantly promoted the root growth compared with the tomato
   seedlings cultivated in the continuous cropping substrate (NM)
   (Fig. [59]1a). The root length, total root surface area, total root
   volume, average root diameter, and the number of root tips of AM
   seedlings increased by 32, 30, 23, 6, and 27%, respectively, and the
   fresh weight and dry weight of roots increased by 92 and 54%,
   respectively, compared with those of the NM seedlings
   (Additional file [60]2: Table S1). The plant height, stem diameter,
   fresh weight and dry weight of AM seedlings increased by 29, 14, 30,
   and 23%, respectively, at 40 d in comparison to those observed for the
   NM seedlings (Fig. [61]1b). Furthermore, the net photosynthetic rate
   (Pn) and yield of AM seedlings were higher than those observed for the
   NM seedlings (Fig. [62]1c; Additional file [63]3: Figure S2). The
   results showed that the inoculation of AMF significantly promoted the
   growth of tomato seedlings in continuous cropping substrates.

Fig. 1.

   [64]Fig. 1
   [65]Open in a new tab

   Arbuscular mycorrhizal fungi (AMF) promoted growth of tomato seedlings
   in the continuous cropping substrates. a The root morphology of NM and
   AM seedlings at 40 d. b The plant height, stem diameter, fresh and dry
   weight of NM and AM seedlings. c The net photosynthetic rate (Pn) of NM
   and AM seedlings at 40 d. The results represent the means ± SE. Three
   independent experiments were performed, with similar results. *
   represent significant difference. NM, tomato seedlings cultivated in
   continuous cropping substrate. AM, tomato seedlings cultivated in
   continuous cropping substrate inoculation with AMF

Effects of AMF on root activity and NADP-dehydrogenase activity

   AMF have been shown to improve root development in plants under adverse
   conditions [[66]18, [67]19]. Furthermore, the activity of
   NADP-dehydrogenase is closely related to the root activity of plants
   [[68]20]. Therefore, we measured the root activity of NM and AM
   seedlings at 40 d and the NADP-dehydrogenase activity of tomato roots
   at 20, 25, 30, 35, and 40 d after treatment. The root activity of AM
   seedlings was 90% higher than that of NM seedlings (Fig. [69]2a).
   Furthermore, the NADP-ME activity of AM seedlings was significantly
   higher than that of NM seedlings (Fig. [70]2b). However, there was no
   significant difference in the activity of NADP-isocitrate dehydrogenase
   (NADP-IDCH), glucose-6-phosphate dehydrogenase (G6PDH), or 6-phosphate
   gluconate dehydrogenase (6PGDH), between NM and AM seedlings (Fig.
   [71]2c-e).

Fig. 2.

   [72]Fig. 2
   [73]Open in a new tab

   Effects of arbuscular mycorrhizal fungi (AMF) inoculation on the root
   and NADP-dehydrogenase activity of tomato seedlings. a The root
   activity of NM and AM seedlings at 40 d. b The activity of NADP-ME in
   roots of NM and AM seedlings. c The activity of NADP-ICDH in roots of
   NM and AM seedlings. d The activity of G6PDH in roots of NM and AM
   seedlings. e The activity of 6PGDH in roots of NM and AM seedlings. The
   results represent the means ± SE. Three independent experiments were
   performed, with similar results. * represent significant difference.
   NM, tomato seedlings cultivated in continuous cropping substrate. AM,
   tomato seedlings cultivated in continuous cropping substrate
   inoculation with AMF

Transcriptome analysis

   To further investigate how AMF increased root activity, we used
   transcriptome analysis to identify the differentially expressed genes
   (DEGs) in the roots between NM and AM seedlings. Illumina sequencing
   was performed on 6 root cDNA libraries (NM and AM treatments, each with
   3 biological replicates) after cultivation of tomato seedlings for 30
   d. After removing the low-quality reads, we obtained 397,526,276 clean
   reads (Additional file [74]4: Table S2). The average values of Q20,
   Q30, and clean read rate of the three NM libraries were 98, 92, and
   92%, and these indexes of AM libraries were 98, 92, and 93%,
   respectively (Additional file [75]4: Table S2). Over 92% of the reads
   matched the tomato genome, and more than 90% of the reads were uniquely
   mapped (Additional file [76]5: Table S3). The clustering heat map
   intuitively showed the gene expression of NM and AM with relatively
   consistent and good repeatability (Additional file [77]6: Figure S3).
   Moreover, there were 4522 genes with significant differences between NM
   and AM, including 2566 downregulated genes and 1956 upregulated genes
   (Additional file [78]7: Table S4).

Gene ontology (GO) enrichment analysis

   The DEGs of AM seedlings were significantly enriched for 24 GO items in
   biological processes, 16 GO items in cellular components, and 11 GO
   items in molecular function (Fig. [79]3a; Additional file [80]8: Table
   S5). The GO enrichment analysis results for biological processes showed
   that GO terms, such as hydrogen peroxide decomposition process
   (GO:0042744), defense response (GO:0006952), response to oxidative
   stress (GO:0006979), and response to biotic stimulus (GO:0009607) were
   enriched after AMF inoculation (Fig. [81]3b; Additional file [82]8:
   Table S5). The GO enrichment analysis results for cell components
   indicated that more significantly different genes were concentrated in
   the GO terms related to the cell membrane, such as extracellular region
   (GO:0005576), integral component of membrane (GO:0016021), plasma
   membrane (GO:0005886), and plant cell wall (GO:0009505) (Fig. [83]3c;
   Additional file [84]8: Table S5). The GO enrichment analysis results
   for molecular functions showed that GO items such as DNA-binding
   transcription factor activity (GO:0003700), peroxidase activity
   (GO:0004601), oxidoreductase activity (GO:0016491), iron ion binding
   (GO:0005506), and monooxygenase activity (GO:0004497) were enriched
   (Fig. [85]3d; Additional file [86]8: Table S5).

Fig. 3.

   [87]Fig. 3
   [88]Open in a new tab

   GO enrichment analysis the differentially expressed genes (DEGs)
   between NM and AM seedlings. a The significantly enriched 24 GO terms
   in the biological processes, 16 GO terms in the cellular components,
   and 11 GO terms in the molecular functions. b The highly enriched in
   the GO term of biological processes. c The highly enriched in the GO
   term of the cellular components. d The highly enriched in the GO term
   of the molecular functions

Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis

   The DEGs were mainly enriched in 33 KEGG pathways, including cellular
   processes, environmental information processing, genetic information
   processing, metabolism, and organismal systems (Fig. [89]4a;
   Additional file [90]9: Table S6). The top 20 enriched KEGG pathways (Q
   value is arranged in ascending order) were used to draw a bubble chart
   (Fig. [91]4b; Additional file [92]9: Table S6). The results showed that
   the DEGs were enriched in phenylpropane biosynthesis (00940),
   biosynthesis of secondary metabolites (01110), metabolic pathways
   (01100), MAPK signaling pathways (04016), and plant hormone signal
   transduction (04075). Next, we performed gene set enrichment analysis
   (GSEA) using the genes related to plant signal transduction from the
   results of KEGG enrichment analysis (Fig. [93]4c;
   Additional file [94]10: Table S7) and obtained 109 genes positively
   related to the phenotype of AM seedlings, of which 9 genes were related
   to IAA (Fig. [95]4d). Interestingly, the GO enrichment results showed
   that all 9 genes were enriched in the nucleus (GO: 0005634), binding
   domain containing DNA (GO: 0003677), and were all enriched in the auxin
   activation signal pathway (GO: 0009734) and transcriptional regulation
   pathways (GO: 0006355). In addition, ARF5, an auxin regulator, was
   enriched in the biological process of hormone response (GO:0009725).
   Interestingly, the promoters of NADP-ME1 and NADP-ME2 contained the
   specific binding sites for ARF5 (Fig. [96]4e), indicating that ARF5
   might directly regulate the expression of NADP-ME1 and NADP-ME2.

Fig. 4.

   [97]Fig. 4
   [98]Open in a new tab

   KEGG pathway and GSEA analysis the differentially expressed genes
   (DEGs) between NM and AM seedlings and ARF5 binding sites in NADP-MEs
   promoters. a KEGG pathway analysis the DEGs between NM and AM
   seedlings. b The top 20 enriched KEGG pathways. c GSEA enrichment
   analysis of genes related to plant hormone signal transduction pathway.
   d Gene heat map analysis the core enrichment genes related to IAA. e
   ARF5 binding sites in the promoters of NADP-ME1 and NADP-ME2 of tomato.
   The numbering comes from the predicted transcription starting point

Effects of AMF on endogenous root hormone

   Transcriptome analysis showed that genes related to plant hormone
   signal transduction responded to AMF colonization. Therefore, we
   analyzed the concentrations of IAA, gibberellin (GA[3]), abscisic acid
   (ABA), cytokinin (CTK), and strigolactone (SL) in the roots of tomato
   seedlings. The results showed that the concentrations of IAA and CTK in
   the roots of AM seedlings significantly increased, while the
   concentrations of ABA significantly decreased, and the concentrations
   of GA[3] and SL did not change compared with those in the NM seedlings
   (Fig. [99]5). These results indicated that the AMF colonization of the
   roots of tomato seedlings modulated endogenous hormone levels, which
   presumably further affected other physiological and metabolic
   processes.

Fig. 5.

   [100]Fig. 5
   [101]Open in a new tab

   The contents of IAA, CTK, ABA, GA[3], and SL in roots of NM and AM
   tomato seedlings. The results represent the means ± SE. Three
   independent experiments were performed, with similar results. *
   represent significant difference. NM, tomato seedlings cultivated in
   continuous cropping substrate. AM, tomato seedlings cultivated in
   continuous cropping substrate inoculation with AMF. FW: fresh weight

Response of NADP-MEs to hormones

   To verify whether hormones mediated root activity, tomato seedlings
   were treated with IAA, GA[3], ABA, CTK, and SL, and qPCR was used to
   test the dynamic expression patterns of NADP-ME1 and NADP-ME2. The
   results showed that these 2 genes responded to all 5 hormones
   (Additional file [102]11: Figure S4). The expression of NADP-ME1 and
   NADP-ME2 was upregulated by IAA, and the gene expression remained
   highly upregulated from 1 h to 12 h after treatment, and the expression
   characteristics of these 2 genes were consistent
   (Additional file [103]11: Figure S4a). However, ABA significantly
   reduced the expression of NADP-ME1 and NADP-ME2
   (Additional file [104]11: Figure S4c).

Effects of IAA on AMF-induced root activity

   To clarify the role of IAA in AMF-induced root activity, we
   investigated the effects of IAA and 2,3,5-triiodobenzoic acid (TIBA, an
   auxin polar transport inhibitor) on root activity, NADP-ME activity,
   and NADP-ME1 and NADP-ME2 expression levels in tomato seedlings
   cultivated in continuous cropping substrate with or without AMF
   inoculation. The application of IAA significantly promoted tomato
   seedlings growth after 15 d of treatment, while the application of TIBA
   significantly inhibited the growth of tomato seedlings compared with
   that in the NM seedlings (Fig. [105]6a, b). Furthermore, the root
   activity of AM and tomato seedlings cultivated in the continuous
   cropping substrate irrigated with IAA (NI) increased by 65 and 27%,
   respectively, while the root activity of tomato seedlings cultivated in
   the continuous cropping substrate irrigated with TIBA (NT) decreased by
   54% compared with that in NM seedlings (Fig. [106]6c). Strikingly, the
   root activity of tomato seedlings cultivated in the continuous cropping
   substrate inoculation with AMF and irrigated with TIBA (AT) was
   significantly lower than that in the AM seedlings (Fig. [107]6c). As
   shown in Fig. [108]6d, the NADP-ME activity of AT seedlings markedly
   decreased compared with that in the AM seedlings. The NADP-ME activity
   of NI seedlings significantly increased compared with that in the NM
   seedlings (Fig. [109]6d). In contrast, the NADP-ME activity of NT
   seedlings decreased compared with that of NM seedlings (Fig. [110]6d).
   However, the inhibitory effects of TIBA were ameliorated by the
   application of IAA (Fig. [111]6d).

Fig. 6.

   [112]Fig. 6
   [113]Open in a new tab

   IAA mediated AMF-induced growth, root and NADP-ME activity in tomato
   seedlings. a Fresh weight. b Dry weight. c The root activity of tomato
   seedlings. d The NADP-ME activity of tomato seedlings. e The expression
   of NADP-ME1. f The expression of NADP-ME2. After inoculation with AMF
   for 15 d, IAA and TIBA were irrigated every 5 d, and the data were
   determined at 30 d. The results represent the means ± SE. Means with
   the same letter did not significantly differ at P< 0.05, according to
   Tukey’s test. Three independent experiments were performed, with
   similar results. NM: tomato seedlings cultivated in the continuous
   cropping substrate; AM: tomato seedlings cultivated in the continuous
   cropping substrate inoculation with AMF; AT: tomato seedlings
   cultivated in the continuous cropping substrate inoculation with AMF
   and irrigated with TIBA; NT: tomato seedlings cultivated in the
   continuous cropping substrate irrigated with TIBA; NI: tomato seedlings
   cultivated in the continuous cropping substrate irrigated with IAA;
   NTI: tomato seedlings cultivated in the continuous cropping substrate
   irrigated with TIBA, and then irrigated with IAA after 24 h

   Gene expression results showed that the expression of NADP-ME1 and
   NADP-ME2 in AM plants was higher than that in NM plants (Fig. [114]6e,
   f). Compared with NM and AM, TIBA significantly reduced the expression
   of NADP-ME1 and NADP-ME2 in NT and AT plants; in contrast, the
   application of IAA significantly increased the expression of NADP-ME1
   and NADP-ME2 (Fig. [115]6e, f). The gene expression of NADP-ME1 and
   NADP-ME2 in tomato seedlings cultivated in the continuous cropping
   substrate irrigated with TIBA, and then irrigated with IAA after 24 h
   (NTI) was consistent with the trend of NADP-ME enzyme activity (Fig.
   [116]6d-f). The above results indicated that IAA mediated the
   expression of NADP-ME1 and NADP-ME2 and root activity of tomato
   seedlings in continuous cropping substrates.

Discussion

AMF promoted tomato seedling growth in continuous cropping substrate

   Compared with fresh substrates, continuous cropping substrates have
   many limitations, such as lower pH value, decreased nutrient content,
   reduced permeability, and imbalanced microbial community structure
   [[117]3]. Recently, the use of beneficial microorganisms has become
   increasingly popular to maintain soil fertility and crop productivity
   [[118]21]. AMF play a critical role in plant response to biotic and
   abiotic stresses [[119]15, [120]22–[121]24]. AMF can reduce the risk of
   ionic toxicity and cell membrane damage by decreasing Na^+ absorption
   and increasing antioxidant enzyme activity in cucumber [[122]25].
   Studies have shown that AMF significantly improve the physicochemical
   properties and enzyme activities of continuous cropping systems and
   promote the absorption of nutrients, resulting in promoted plant growth
   and increased yield [[123]15, [124]16, [125]26]. Consistent with the
   previous studies, AMF incorporation into the continuous cropping
   substrate remarkably improved the growth of tomato seedlings and
   enhanced the yield of tomato (Fig. [126]1; Additional file [127]3:
   Figure S2). These results indicate that the inoculation of AMF in
   continuous cropping substrates is an effective way to relieve
   continuous cropping obstacles.

AMF induced an increase in endogenous IAA in tomato roots

   It has been shown that the inoculation of AMF can regulate hormone
   levels in plants [[128]27, [129]28]. In this study, the inoculation of
   AMF significantly increased IAA and CTK concentrations in tomato roots
   and significantly reduced ABA concentrations (Fig. [130]5). Similarly,
   AMF increased the levels of IAA and CTK, decreased ABA levels, and
   enhanced hormone homeostasis in the damaged roots of maize [[131]29].
   Furthermore, AMF inoculation significantly increased the IAA content in
   the root system of trifoliate orange and decreased IAA loss in the root
   system [[132]30]. In this study, the enrichment analysis of the KEGG
   pathway showed that the plant hormone signal transduction pathway was
   relatively enriched (Fig. [133]4b). Through GSEA, 109 genes were
   positively correlated with the AM phenotype, among which 9 genes were
   correlated with IAA (Fig. [134]4c, d). Similarly, AMF can increase the
   IAA level and promote the growth and development of host plants under
   stress by upregulating the expression of IAA transporter genes and
   downregulating the expression of auxin efflux genes [[135]31]. In
   addition, AMF have a positive effect on the regulation of IAA levels in
   plants under drought [[136]32], salt stress [[137]33] and biotic stress
   [[138]34]. These results suggested that the increase in IAA levels in
   tomato roots induced by AMF might be an important reason to promote the
   growth and development of tomato seedlings in continuous cropping
   substrates.

IAA mediated AMF-induced NADP-MEs expression and NADP-ME activity

   NADP-ME is a cytoplasmic protein that is expressed during the
   development of tomato roots, stems, leaves, and fruits [[139]35]. In
   particular, NADP-ME1 is involved in the regulation of malic acid
   content in the root apex, and the absence of NADP-ME1 results in an
   increase in malic acid content. Furthermore, NADP-ME1 also affects the
   signal transmission process [[140]36]. However, NADP-ME2 plays an
   important role in the defense mechanism of plants and seems to be
   involved in the production of reactive oxygen species [[141]37]. Our
   results showed that NADP-ME1 and NADP-ME2 responded to IAA, GA[3], ABA,
   CTK, and SL (Additional file [142]11: Figure S4). Among them, IAA
   significantly increased the expression of NADP-ME1 and NADP-ME2
   (Additional file [143]11: Figure S4a), which indicated that the
   induction of NADP-ME1 and NADP-ME2 by IAA might be a response to the
   stress of tomato under continuous cropping substrates. Notably,
   hormones play a crucial role in regulating complex signaling networks
   in different plant growth and development processes and in plant
   responses to environmental stresses [[144]38]. Likewise, IAA is
   involved in regulating the defense response to various biotrophic and
   necrotrophic pathogens [[145]38]. Our results showed that AMF
   inoculation significantly increased the IAA concentration in tomato
   roots (Fig. [146]5). Furthermore, IAA application significantly
   increased the expression levels of NADP-ME1 and NADP-ME2, NADP-ME
   activity, root activity, and the growth of tomato seedlings cultivated
   in continuous cropping substrates (Fig. [147]6). However, these effects
   were compromised when plants were treated with TIBA (Fig. [148]6).
   Similarly, AMF inoculation promotes the accumulation of endogenous IAA
   to improve the root system and nutrient absorption under stress
   conditions [[149]39]. Exogenous IAA significantly increases the
   activity of NADP-ME in Malus baccata (L.) Borkh. [[150]40]. Thus, AMF
   inoculation improved the endogenous IAA level in plant roots to
   increase the expression of NADP-ME1 and NADP-ME2 and NADP-ME activity.

Conclusions

   In conclusion, our study revealed that IAA mediates the AMF-promoted
   tomato growth and NADP-MEs expression in continuous cropping
   substrates. Our study suggests that the use of AMF can effectively
   improve the growth of tomato cultivated in continuous cropping
   substrates, thereby showing a promising solution for the reuse of
   continuous cropping substrates with AMF.

Methods

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 [[151]41].

   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 [152]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.

Hormone treatments

   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 GA[3] (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 [[153]42].

Morphological index and Pn measurement

   The plant height and stem diameter were measured at 40 d according to
   previously described methods [[154]43, [155]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
   [[156]45].

   The activities of 6PGDH, G6PDH, NADP-IDCH and NADP-ME were measured by
   spectrophotometry, recording the reduction of NADP at 340 nm [[157]46,
   [158]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 MgCl[2] (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, GA[3], CTK, ABA, and SL in tomato roots

   The hormone contents were determined at 30 d as previously described
   [[159]48]. 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).

qPCR analysis

   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 [160]13: Table S9. The relative gene
   expression was calculated as described by Livak and Schmittgen
   [[161]49].

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 [[162]50]. The transcript abundance was normalized
   by the fragments per kilobase of exon per million fragments mapped
   using Cufflinks [[163]51].

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 |log[2](fold
   change)| ≥ 2. After normalization, hierarchical clustering and k-means
   clustering analysis of the expression patterns were performed using
   Mutiexperimental Viewer v4.7 [[164]52].

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 [[165]53]. 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 [[166]54, [167]55].

Statistical analysis

   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.

Supplementary Information

   [168]12870_2020_2817_MOESM1_ESM.pdf^ (223KB, pdf)

   Additional file 1: Figure S1. The colonization of AMF in tomato
   seedlings.
   [169]12870_2020_2817_MOESM2_ESM.docx^ (15.4KB, docx)

   Additional file 2: Table S1. Effects of AMF inoculation on tomato root
   growth.
   [170]12870_2020_2817_MOESM3_ESM.pdf^ (182.6KB, pdf)

   Additional file 3: Figure S2. Effects of arbuscular mycorrhizal fungi
   (AMF) inoculation on the yield of tomato.
   [171]12870_2020_2817_MOESM4_ESM.docx^ (15.5KB, docx)

   Additional file 4: Table S2. Base statistics after filtering from NM1,
   NM2, NM3, AM1, AM2 and AM3 libraries.
   [172]12870_2020_2817_MOESM5_ESM.docx^ (15.5KB, docx)

   Additional file 5: Table S3. Mapping results of clean reads against the
   tomato genome and gene.
   [173]12870_2020_2817_MOESM6_ESM.pdf^ (72.6KB, pdf)

   Additional file 6: Figure S3. Gene heat map showing the gene expression
   differences between NM and AM seedling roots.
   [174]12870_2020_2817_MOESM7_ESM.xlsx^ (1.9MB, xlsx)

   Additional file 7: Table S4. The differentially expressed genes between
   NM and AM.
   [175]12870_2020_2817_MOESM8_ESM.xlsx^ (573.7KB, xlsx)

   Additional file 8: Table S5. GO enrichment analysis the differentially
   expressed genes between NM and AM.
   [176]12870_2020_2817_MOESM9_ESM.xlsx^ (77KB, xlsx)

   Additional file 9: Table S6. KEGG pathway analysis the differentially
   expressed genes between NM and AM.
   [177]12870_2020_2817_MOESM10_ESM.xlsx^ (25.6KB, xlsx)

   Additional file 10: Table S7. GSEA enrichment analysis of genes related
   to plant hormone signal transduction pathway.
   [178]12870_2020_2817_MOESM11_ESM.pdf^ (69.5KB, pdf)

   Additional file 11: Figure S4. Responses of NADP-ME1 and NADP-ME2 to
   hormones.
   [179]12870_2020_2817_MOESM12_ESM.docx^ (15.2KB, docx)

   Additional file 12: Table S8. Comparison of physical and chemical
   properties of continuous cropping substrate and fresh substrate.
   [180]12870_2020_2817_MOESM13_ESM.docx^ (17.3KB, docx)

   Additional file 13: Table S9. Primers used for qPCR assays.

Acknowledgements