Abstract

   Ganoderma lucidum has been used as a rare medical mushroom for
   centuries in China, due to its health-promoting properties. Successive
   cropping obstacles are common in the cultivation of G. lucidum,
   although the remaining nutrients in the germ substrate are sufficient
   for a second fruiting. Here, we aimed to study the metabolite profile
   of G. lucidum via nontargeted metabonomic technology. Metabonomic data
   revealed that organic acids played an important role in the cropping
   obstacles of G. lucidum, which is accordance with the pH decrease in
   the germ substrate. A Kyoto encyclopedia of genes and genomes (KEGG)
   enrichment analysis indicated that most differential acids participated
   in the metabolic pathways. Five acids were all significantly
   upregulated by two MS with high energy (MS^E) modes in two cultivars,
   among which
   5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylic acid is
   also involved in purine metabolism regulation and microbial metabolism
   in diverse environments. Taken together, this work illustrated the
   organic acid stress generated by G. lucidum, which formed the
   autotoxicity feedback, and resulted in cropping obstacles. Determining
   the cause of the cropping obstacles in G. lucidum will promote the
   utilization rate of fungus substrate to realize the sustainable use of
   this resource.

   Keywords: Ganoderma lucidum, organic acids, autotoxicity, cropping
   obstacle

1. Introduction

   Most edible mushrooms can produce a qualified fruiting body at least
   two consecutive times under proper artificial conditions, such as
   Pleurotus ostreatus, Flammulina velutipes, Coprinus comatu, etc. Other
   edible fungi only produce qualified fruit the first time, and then tend
   to consecutively produce defective fruits [[38]1]. This is known as a
   successive cropping obstacle, which is common during the culture of G.
   lucidum. G. lucidum is one of the most typical varieties, with abundant
   bioactive components [[39]2,[40]3], including triterpenoids,
   polysaccharides, nucleosides, alkaloids, ganoderic acid, etc. The
   functional compounds’ immunoregulation, antitumor, neuroprotection,
   hepatoprotection, antioxidation, anti-radiation, and anti-mutation
   properties have been proven [[41]4,[42]5,[43]6] since the discovery of
   wall-breaking technology. The medical mushroom has shown promising
   potential for pharmacological and economic applications. However,
   successive cropping obstacles limit its cultivation promotion, as only
   the first fruit of G. lucidum has large output and contains plentiful
   bioactive substances; the fruiting body, output, and bioactive
   substances from the second fruiting are unsatisfactory [[44]7]. It has
   been considered that the successive cropping obstacles in G. lucidum
   are mainly related to environmental conditions and nutrients in
   cultivation materials. Although the remaining nutrients in the germ
   substrate are enough for continuous fruiting, the remaining cultivated
   substances cannot be applied to growing homologous edible fungi again,
   unless the germ substrate or covering soil are exposed to sun, rain,
   and decay over two years. This is a passive way to change the
   cultivation environment and supplementation of germ nutrients, which
   will also increase the cost, instead of enhancing the utilization of
   cultivation substances as an alternative.

   The continuous cropping obstacle in edible fungi is similar to that in
   crops and vegetables, which is closely related to root exudate
   dispersion and accumulation in soil [[45]8,[46]9,[47]10,[48]11]. Fungi
   metabolites inhibit its second fruiting when exudates accumulate to a
   certain amount. Recently, we found that the germ substrate pH
   significantly decreased from 7.50 to 4.74 after the first fruiting,
   indicating that the mycelium of G. lucidum secreted organic acids or
   other metabolites into cultivated substances, and resulted in the
   subsequent fruiting body becoming defective. Moreover, the ethanol
   extract and water extract of the germ substrate after the first
   fruiting had an inhibitory effect on the growth of homologous G.
   lucidum, and the inhibition was positively correlated with extract
   doses (unpublished data). The germ substrate contains almost
   mycelium-secreted organic acids; meanwhile, the accumulated organic
   acid may inhibit feedback on its own growth. Continuous cropping
   increases the accumulation of inhibitors and changes the soil
   microenvironment. Therefore, we assumed that the organic acid exudate
   may be the main reason for the failure of continuous cropping.

   To the best of our knowledge, there have been few reports on the
   feedback inhibition of G. lucidum. In this study, we compared the
   changes in organic acids at different G. lucidum growth stages using
   non-targeted metabonomic technology. Then, we determined the
   differential organic acids and related pathways, which were used to
   analyze the promising autotoxicity mechanism. The metabolite feedback
   inhibition study on edible mushrooms is helpful when screening
   inhibiting components and inhibition scavengers, which will overcome
   successive cropping obstacles, make full use of cultivation substances,
   and improve the biological efficiency of fungi culture.

2. Results and Discussion

2.1. pH Variation of Germ Substrates during Cultivation

   [49]Figure 1 shows the pH changes in substrates from inoculation to a
   mature fruiting body. Arrows point to important growth stages of G.
   lucidum, including primordium at Day 8, pileus at Day 18, fruiting
   initiation at Day 28, and spore ejection at Day 42. As seen in
   [50]Figure 1, pH decreases can be observed before every stage of growth
   point, especially before spore ejection (Day 42). The pH of L09-1
   (5.36) decreased to 4.63 from the inoculation to the end of first
   fruiting, and the pH of L09-2 showed little change from 4.46 to 4.3.
   The transcriptional dynamics of genes during sporulation in G. lucidum
   revealed that the differential expressed genes involved in the
   carbohydrate metabolic process were upregulated at the later stage of
   sporulation, and the accumulation of specific carbohydrates may serve
   as energy sources for further development [[51]12]. The decreased pH of
   the germ substrates indicated the increase in some organic acid
   compounds. Organic acids can modify the physical and chemical
   properties of substrates, and influence nutrient absorption; therefore,
   organic acids play an important ecological role in the growth of G.
   lucidum. In addition, organic acids in medical mushrooms are potential
   candidates as antifungal agents, resulting in autotoxicity in the
   second fruiting [[52]1,[53]13,[54]14]. Therefore, we focused on the
   organic acid and derivative changes in germ substrate affected by G.
   lucidum through non-targeted metabolomics.

Figure 1.

   [55]Figure 1
   [56]Open in a new tab

   pH values of germ substrates of two G. lucidum from inoculation to
   fruiting. Arrows point to several important growth stages. The pH
   decreased before every stage of growth point, especially before spore
   ejection. The pH of L09-1 (5.36) decreased to 4.63 from the inoculation
   to the end of first fruiting, and the pH of L09-2 showed little change
   from 4.46 to 4.3.

2.2. Acquisition of Mass Spectrum Data

   Since quality control (QC) samples are prepared with mixed samples, the
   differences in the varieties and abundances of metabolites in QC
   samples should be within the random error. Therefore, the consistency
   of QC sample results can be used as an important method to evaluate
   laboratory automation and quality management. In this study, samples
   from different groups were arranged in a random order, and a QC sample
   was inserted every ten samples. In [57]Figure 2, the heat map of the QC
   samples’ correlation was entirely above 0.9, indicating that the data
   obtained from HPLC-QTOF-MS were valid.

Figure 2.

   [58]Figure 2
   [59]Open in a new tab

   Correlation analysis of QC samples. QC samples were a mixture of all
   tested samples, used to evaluate instrument stability. The correlation
   was entirely above 0.9, indicating that the data were valid.

2.3. Comparison of Organic Acids and Derivatives between L09-1 and L09-2

   The separation and identification of organic acids from the germ
   substrate was performed using UPLC-QTOF-MS^E. MS^E is an MS with high
   energy that can collect ion fragments ranging from low to high energy.
   [60]Figure 3 shows an example of chromatograms with marked retention
   times of peaks obtained in positive and negative modes. According to
   the positive MS^E mode by UPLC-QTOF-MS, 3521 metabolites in the two
   varieties were screened through qualitative and quantitative analysis,
   among which 1007 metabolites were significantly different. A total of
   724 compounds out of 3521 metabolites belonged to organic acids and
   derivatives. [61]Supplementary Table S1 lists 194 significantly
   upregulated and downregulated organic acids and derivatives in both
   species. There were 119 upregulated compounds, while the others were
   downregulated.

Figure 3.

   [62]Figure 3

   [63]Figure 3
   [64]Open in a new tab

   Example of total obtained ion chromatogram of L09-1 germ substrate
   sample using UPLC-QTOF-MS^E in positive mode (a) and negative mode (b).

   For the negative MS^E mode, there were 43 differential metabolites in
   total, among which there were 17 organic acids and derivatives. As
   shown in [65]Table S2, nine organic acids were all upregulated in two
   G. lucidum, and only the content of C19:1 (CIS-10) acid was
   downregulated, compared to that of the inoculation. The reference
   showed that organic acids are closely related to the synthesis and
   catabolism of the cell wall [[66]15,[67]16]. The cell wall is the first
   barrier for external ions entering the protoplasm, which plays an
   important role in the resistance to external ion stress [[68]17]. The
   feedback of organic acids may inhibit the synthesis of the cell wall
   during the second fruiting, thus producing defective G. lucidum.

   In [69]Figure 4, the KEGG pathway enrichment analysis indicates that
   most of the differential acids participate in the metabolic pathways,
   which is in keeping with the finding of Cai and colleagues [[70]12].
   There were 22 acids in positive mode and 1 in negative mode (map01100,
   seen in [71]Figure S2). [72]Table 1 lists five acids, which appeared in
   two MS^E modes, and all were significantly upregulated in the two
   cultivars. The differential compound
   5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylic
   acid—which occurs in both MS^E modes and cultivars—is also involved in
   both purine metabolism regulation (map00230, seen in [73]Figure S3a)
   and microbial metabolisms in diverse environments (map01120, seen in
   [74]Figure S3b). However, other compounds in [75]Table 1 were not found
   in the related KEGG pathway. Moreover, 13 differential organic acids
   were involved in the biosynthesis of secondary metabolites (map01110,
   in [76]Figure S4a), 5 organic acids were involved in the biosynthesis
   of amino acids (map01230, in [77]Figure S4b) and 3 organic acids were
   involved in lysine degradation (map00310, in [78]Figure S4c) in
   positive ion mode. Other than that, either two or one differential
   compounds in positive ion mode appeared in the corresponding KEGG
   pathway (data not shown). The KEGG pathways suggest that these
   metabolic processes may be important in connecting edible mushrooms’
   secondary metabolic networks [[79]18].

Figure 4.

   [80]Figure 4
   [81]Open in a new tab

   KEGG pathway enrichment analysis of differential metabolites of two
   cultivars.

Table 1.

   Organic acids and derivatives in the germ substrates of two cultivars
   using two MS^E modes.
   No. Differential Compounds Content Change
   1 5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylic acid
   UP
   2 malonic acid UP
   3 2-(2-Carboxyethyl)-5-hydroxyphenyl hexopyranosiduronic acid UP
   4
   3-C-Carboxy-2-deoxy-4-O-[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]pentaric
   acid UP
   5 2-Deoxy-4-O-[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]pentaric acid UP
   [82]Open in a new tab

   G. lucidum is an important edible fungus with medical value [[83]19].
   Improvements in the yield and utilization rate of the germ substrate is
   helpful to lower the planting cost and to avoid wasting resources.
   However, most reports have been concerned with evaluations of its
   medical activity and analyses of its functional ingredients, but paid
   little attention to the metabolite-autotoxicity-induced successive
   cropping obstacles [[84]20]. In our study, we found that organic acids
   and derivatives account for the majority of all differential
   metabolites. Most differential organic acids were upregulated in two G.
   lucidum cultivars, which is in accordance with the decrease in the pH
   of the germ substrate observed during cultivation (seen in [85]Figure
   1). In the study of Xie et al., most triterpenoids, including ganoderic
   acid, guanidylic acid, and lucidenic acid, were upregulated in the
   fruiting body stage, and this may be positively correlated with the
   increase in the organic acid content in the substrate [[86]5]. Organic
   acids can preserve the quality of fruit and defend against abiotic
   stress [[87]21,[88]22]. The increased organic acid content may offer a
   reasonable explanation for the fact that the remaining nutrients of the
   germ substrate cannot be reapplied to grow qualified G. lucidum. The
   content of organic acids represent the metabolic status, and a higher
   level of organic acids reflect an organism’s ability to survive and
   maintain its basal metabolism [[89]23]. The accumulation of organic
   acids increases energy production and amino acid content and better
   maintains the osmoregulation of cells, thus enhancing the tolerance to
   stress [[90]22,[91]24]. Mycelia secrete organic acids into the germ
   substrate and change the environmental state. Organic acids may
   negatively regulate the growth in the fruiting body by participating in
   metabolic pathways, purine metabolism regulation, and microbial
   metabolism in diverse environments or other pathways, thus forming the
   autotoxicity feedback. In addition, the substrate bacterial community
   structure may be affected by the pH change [[92]25,[93]26], and may in
   turn influence the growth of G. lucidum, which will be considered in a
   further study of ours.

3. Materials and Methods

3.1. Strains and Inoculation

   Strains of G. lucidum were bred by our lab and were named L09-1 and
   L09-2. The germ substrate consisted of 78% wood chips, 20% wheat bran,
   1% lime, and 1% gypsum; they were then mixed with equivalent water
   [[94]27,[95]28,[96]29]. Polypropylene bags containing 2 kg of the
   substrate were ready for mycelium inoculation after autoclaving at 121
   ℃ for 40 min. Next, L09 and L09-2 were planted on solid substrates.
   After inoculation, germ barns were collected every 48 h until the end
   of first fruiting. A total of seven samples were collected during the
   assay. The substrate samples were dissolved in hot water and filtered.
   Supernatants were ready for subsequent analysis. The pH values of each
   sample were determined. All other reagents were of the highest
   available quality (Sigma-Aldrich Co. Ltd., Burlington, MA, USA).

3.2. Sample Preparations

   A total of 100 mg germ substrate sample was ground with liquid
   nitrogen, and vortexed with 300 μL of 80% methanol (Sigma-Aldrich Co.
   Ltd., Burlington, MA, USA). The mixture was ground by a tissue crusher
   (Zhengqiao Instrument Company, Shanghai, China) at 45 Hz for 4 min, and
   then treated with ultrasonication in an ice water bath for 5 min.
   Samples were kept at −20 ℃ for 30 min, and centrifuged at 14,000× g for
   30 min. The supernatant was filtered ready for further measurement. The
   quality control (QC) sample was prepared by drawing 10 μL of
   supernatants from all abovementioned samples and then mixing them up.
   Samples from different groups were arranged in a random order, and a QC
   sample was inserted every ten samples.

3.3. UPLC-MS/MS Data Acquisition

   Samples were determined by Waters Acqquity UPLC equipped with Waters
   Vion IMS QTOF (Waters Co. Ltd., Taunton, MA, USA). A 5 μL sample was
   loaded and separated on a Waters ACQUITY UPLC HSS T3 C18 column (2.1 ×
   100 mm and particle size of 1.8 µm) with 0.1% formic acid aqueous
   solution (eluent A) and 0.1% formic acid acetonitrile solution (eluent
   B) in gradient elution mode at a rate of 0.25 mL/min. The Elution
   program was set as follows: from 98% A at 0 min to 70% A at 5 min;
   constant 50% A from 7 to 16 min; from 10% A at 16 min to 2% A at 17
   min; constant 2% A from 17 min to 20 min. The column temperature was
   kept at 25 ℃.

   The MS detection conditions were as follows: the desolvation gas N[2]
   flow was 800 L/h at 500 ℃, and the flow rate of cone gas is 50 L/h. The
   capillary voltages were set at 2.00 KV and 1.20 KV for positive and
   negative MS^E modes, respectively, and separately. Scan range was from
   100 to 1200 m/z, and scan time was 0.3 s.

   Samples were analyzed in random sequence to avoid the influence caused
   by fluctuations in instrumental signals. QC samples were inserted every
   ten samples to evaluate the stability of the system and the data
   reliability.

3.4. Processing and Statistical Analysis of MS Spectra Data

   The primary data were imported from QTOF-MS to Progenesis QI 2.3 (PQI)
   software (Waters Co. Ltd., Taunton, MA, USA), and the data were aligned
   within 0.2 min of retention time deviation with 5 ppm of quality
   deviation. Peaks were selected according to a coefficient of variance
   (CV) of 30%, a signal-to-noise ratio of 3, and a minimum signal of 10^5
   in order to provide more accurate identification. Meanwhile, peak area
   was quantified and target ions were integrated. Organic acids and
   derivates were predicted by the exact mass of potential difference m/z.
   Possible molecular formulas were predicted by comparing ion peaks and
   fragment ions with the use of databases, which included METLIN, the
   Human Metabolome database, and ChemSpider. The quantitative results
   were normalized, and a labeled matrix was produced and recognized
   [[97]30]. The mass spectra with variable importance in projection (VIP)
   above 1 were selected in this study, and a non-parametric t test with a
   critical p value of 0.05 was applied to further determine whether a
   significant difference existed between at least two groups for each
   organic acid and derivate.

4. Conclusions

   This study is the first exploratory work to determine the cropping
   obstacle of G. lucidum. The metabolic data were analyzed using
   UPLC-QTOF-MS^E. Most of the differential metabolites were organic acids
   and derivatives. Moreover, 119 out of 194 and 9 out of 17 differential
   organic acids were upregulated in both positive and negative ion modes,
   respectively. The increased contents of organic acids were consistent
   with the pH decrease during the whole cultivation. KEGG pathway
   enrichment analysis revealed that differential compounds were mainly
   involved in the metabolic pathways. Additionally, we found that the
   organic acid-induced stress may inhibit the second fruiting of G.
   lucidum. In the next step, we will focus on studying the relationship
   between successive cropping obstacles and differentially expressed
   genes and protein in order to find reasonable solutions, or provide
   novel breeding ideas, for G. lucidum. Moreover, the functional
   nutrients in G. lucidum are also important parameters for evaluating
   its quality, which may be affected by organic acid stress, and will be
   explored in the future.

Supplementary Materials

   The following supporting information can be downloaded at:
   [98]https://www.mdpi.com/article/10.3390/molecules27196734/s1, Figure
   S1: Total ion chromatogram (TIC) of germ substrate sample of G. lucidum
   by LC-QTOF-MS in positive ion mode and negative ion mode. a and c are
   TICs of L09-1 germ substrate at the beginning and the end of whole
   cultivation in positive ion mode; b and d are TICs of L09-1 germ
   substrate at the beginning and the end of whole cultivation in negative
   ion mode; e and g are TICs of L09-2 germ substrate at the beginning and
   the end of whole cultivation in positive ion mode; f and h are TICs of
   L09-2 germ substrate at the beginning and the end of whole cultivation
   in negative ion mode.; Figure S2: Metabolic pathway is the main pathway
   in two G. lucidum by KEGG pathway enrichment analysis. There were 22
   acid compounds involved in positive ion mode and one acid in negative
   ion mode. The red points in metabolic pathways indicate that the acids
   were upregulated, and the green points perform the downregulated
   compounds; Figure S3:
   5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylic acid,
   occurring in both modes and both cultivars, participates in purine
   metabolism (a) and microbial metabolism in diverse environment (b). The
   red points in pathways indicate the compound was upregulated
   significantly; Figure S4: Related KEGG pathway of differential
   compounds in germ substrates of G. lucidum. a. 13 organic acids were
   involved in the biosynthesis of secondary metabolites. b. There were
   five organic acids participating in the biosynthesis of amino acids. c.
   There were three organic acids involved in Lysine degradation; Table
   S1: Organic acids and derivatives in germ substrates of two cultivars
   by positive MS^E continuum; Table S2: Organic acids and derivatives in
   germ substrates of two cultivars by negative MS^E continuum.
   [99]Click here for additional data file.^ (1.5MB, zip)

Author Contributions

   P.Z. drafted the manuscript. Y.G. collected test data. S.D. and Z.S.
   conducted the cultivation of G. lucidum. H.C. and Y.Z. collected
   samples for analysis. Z.Z. and X.C. designed the study. All authors
   have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The data used to support the findings of this study are available from
   the corresponding author (Xiaoming Chen, chxm001@126.com) upon request.

Conflicts of Interest

   The authors declare no conflict of interest.

Sample Availability

   Samples of the G. lucidum used in this study are available from the
   authors.

Funding Statement

   The study was financially supported by the Innovative Research Fund of
   Young Researchers of Tianjin Academy of Agricultural Sciences
   (No.2020003), Special Fund for Scientific Research of Public Welfare
   Industry (Agriculture, No.201503142-09) and Innovative Research Fund of
   Young Researchers of Institute of Agricultural Quality Standard and
   Testing Technology (No.ZBS-2020001).

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References