Pleurotus tuoliensis, also known as P. ferulae, P. eryngii var. tuoliensis or P. nebrodensis, is a valuable and rare edible fungus (Zhao et al., 2016). It has extremely high nutritional and medicinal value. Modern pharmacological studies have shown that fungal polysaccharides contained in P. tuoliensis have many biological properties such as improving human immunity (Li et al., 2016), anti-tumor (Wang et al., 2014), antioxidant (Alam et al., 2012) and cholesterol-lowering activities (Alam et al., 2011). Wild P. tuoliensis usually grows together with the roots of Ferula plants in the Gobi desert, which is mainly situated in Xinjiang, China (Mou, 1987). Although large-scale commercial cultivation of P. tuoliensis began as early as 1997, the technology used in its production remains imperfect, leading to unstable yield and quality. It is reported that total yield of P. tuoliensis in 2012 reached 300 thousand tons, while in 2016 it was only 100 thousand tons (data from the China Edible Fungi Association). This is mainly ascribed to estimates on mycelium maturity. P. tuoliensis must undergo a mycelium physiological maturation period (MPMP) of 30–60 days, which is far longer than that of 7 days required by P. ostreatus and P. eryngii (Duan et al., 2013). This period is the process time of nutrients accumulation by mycelium metabolism. Mycelia can reach physiological maturation at the end of MPMP and the level of maturity directly affects the differentiation of primordia and the growth of fruiting bodies. Immature mycelia greatly reduce the growth rate and quality of mushrooms, while overmature mycelia have reduced viability and over-utilize nutrients in the cultivation substrate, resulting in a relative lack of nutrients during the development of fruiting body. At present, in the commercial production of P. tuoliensis, estimate on mycelium maturity was only based on the days of MPMP (>30 days) and mycelial growth (such as dense and thick, white in color) (Gou et al., 2003). The lack of measurable indicators that can clearly characterize the physiological maturation of P. tuoliensis mycelia leads to subjective evaluation of mycelium physiological maturation, resulting in fluctuations in yield and quality of P. tuoliensis, as well as differences in production efficiency among producers. Therefore, a clear indicator that can be used to estimate the physiological maturity of mycelia and thus provide a basis for optimal cultivation of P. tuoliensis is urgently needed. In addition, the uncertainty of the ambient temperature during MPMP also limited the yield of P. tuoliensis.

Metabolomics is a new discipline that has developed rapidly following genomics, transcriptomics and proteomics (Trivedi et al., 2017). It aims to detect the overall trajectory of endogenous metabolites in organisms or cells under specific conditions to reflect the pathological and physiological processes of organisms, and some differential metabolites detected have become potential markers that characterize the pathological and physiological states of organisms (Bracewellmilnes et al., 2017; Kaushik and Deberardinis, 2018). Metabolomics has been widely used in many fields such as disease diagnosis (Lin et al., 2017), drug development (Webhofer et al., 2011), microbial metabolism (Guo et al., 2018), and animal and plant metabolism (Pandey et al., 2016).

In recent years, metabolomics technology has been gradually applied in the field of edible fungi to study their metabolic mechanisms in response to certain environmental conditions. Zhang et al. (2008) used GC-MS-based metabolomics to study changes in volatile substances during the ripening process of Volvariella volvacea and P. ostreatus, and to evaluate the antioxidant activity of these edible fungi related to the non-volatiles and volatiles. Agaricus bisporus is highly susceptible to mechanical damage during storage or transportation. GC-MS technology has been used to identify metabolites that can be used as markers for A. bisporus injury (O’Gorman et al., 2012). Qiu et al. (2018) used GC-MS and LC-MS to analyze changes in extracellular metabolites in the mycelium of P. ostreatus under high temperature conditions, as well as to study the effect of exogenous addition of some significantly increased metabolites on the growth of Trichoderma asperellum, with the goal of determining why P. ostreatus was easily infected by T. asperellum. These reports above also serve as a guide to explore metabolic profiles during mycelium physiological maturation in P. tuoliensis by using a metabolomics approach.

In this study, GC-MS-based metabolomics was performed to study metabolic changes in mycelia collected at MPMP day 0, and day 35 under different temperatures (17 and 29°C). The primary goal of this study was to provide metabolomic information to assist in the identification of metabolites that can be used as potential indicators of mycelium physiological maturation, which could improve P. tuoliensis cultivation practices and thus maximize yield and quality.

Mycelium physiological maturation period is an extremely important stage before primordia formation. Mycelia reach physiological maturity during the process of MPMP, and their level of maturity is positively correlated with their fruiting rate. In industrial production, thick and white mycelia and a compact cultivation bag are usually used as appearance indicators for judging the physiological maturity of mycelia, but no measurable signals have been found. In addition, we have confirmed that P. tuoliensis MPMP lasting 35 days at 17°C resulted in larger cap diameters, higher fruiting rates, increased yields and improved biological efficiency, in comparison with an MPMP of 35 days at 29°C, but the reason lower temperatures during MPMP facilitate fruiting body development remains unclear.

Some metabolites in mycelia change greatly during physiological maturation. Thus they may be important signals of entry into primordia differentiation stage. Metabolomics provides an effective approach for studying the metabolic profiles of mycelia before and after MPMP, as well as during temperature stress (Zhang et al., 2014). In our study, GC-MS-based metabolomics was performed to screen for significantly altered metabolites in mycelia at MPMP day 0 (Mycelia A samples), MPMP day 35 at 17°C (Mycelia B samples) and MPMP day 35 at 29°C (Mycelia C samples), with the goal of identifying potential biomarkers for mycelial maturation. Multivariate PCA and OPLS-DA analyses, which could confirm the intrinsic variation of metabolites and the stability of the entire analysis process (Song et al., 2013; Brusco, 2014), showed that there were changes in the composition and concentrations of extracellular metabolites among three tested groups of P. tuoliensis mycelia, which indicated that metabolic profiles were influenced by the growth of the mycelia as well as by temperature stress.

Using statistical analysis and VIP values obtained from the OPLS-DA analysis, 72 differential metabolites were found to be greatly altered among three tested groups of mycelium samples (p < 0.01 and VIP > 1.0). A heat map (Figure 4A) showed that the abundance of 37.8% of identified extracellular metabolites was significantly increased, while that of 62.8% of identified extracellular metabolites decreased at MPMP day 35. The results of metabolic pathway enrichment analysis indicated that many of the identified metabolites were involved in sugar metabolism, amino acid metabolism, and tricarboxylic acid (TCA) cycle.

Tricarboxylic acid cycle is a key metabolic pathway that unifies protein, fat and carbohydrate metabolism (Wahyuni et al., 2013). The reactions of TCA cycle are carried out by eight enzymes that completely oxidize acetyl-CoA into two molecules of carbon dioxide. Through catabolism of proteins, fats and sugars, acetate is produced in the form of acetyl-CoA, which enters the citric acid cycle (Vagelos et al., 1963). A decrease of citrate, succinate, fumarate, malate, and ribose in physiologically mature mycelia suggested that TCA cycle was depressed and mycelium metabolism was slowed down. We speculate that the slowing of metabolism is to prevent mycelia from excessive decomposition of cultivation substrate, and thus avoid insufficient nutrients during the formation of primordia and fruiting bodies.

The concentrations of all measured sugars were decreased in mature mycelia. This phenomenon was in accordance with growth profiles of fungal mycelia. Basidiomycetes, such as the oyster mushroom, king oyster mushroom, winter mushroom, and shiitake, can enzymatically degrade cultivation substrates containing lignin, hemicellulose, and cellulose into low molecular weight sugars, which are then absorbed by fungal hyphae to support growth and maturation in a process called nutritive absorption (Lim et al., 2012, 2013; Chen et al., 2016). In the late stage of mycelial maturation, the slowing of metabolism causes a decrease in the levels of extracellular enzymes, so the abundance of degradation products is correspondingly reduced. In our study, an extremely low concentration of ribose, a key intermediate of pentose phosphate pathway, was observed in mature mycelia, suggesting that this pathway was also depressed. The pentose phosphate pathway is a biochemical pathway that functions in parallel with glycolysis and generates nicotinamide adenine dinucleotide phosphate (NADPH) and pentoses (5-carbon sugars) and involves oxidation of glucose. Therefore, its primary role is anabolic rather than catabolic (Kruger and Von, 2003; Keller et al., 2014). The pentose phosphate pathway is a major source of reductants for biosynthetic processes such as fatty-acid synthesis and assimilation of inorganic nitrogen (Emes and Neuhaus, 1995). In our study, the abundance of octanoic acid, dodecanoic acid, octadecanoic acid, and octadecenoic acid in fatty acid synthesis pathway were sharply decreased at MPMP day 35. The reductant used for fatty acid synthesis originated mainly from pentose phosphate pathway, which was found to be depressed. Therefore down-regulation of these fatty acids was in good agreement with that of pentose phosphate pathway intermediates.

Most amino acids identified in this study were evidently abundant in mycelia at MPMP day 35, suggesting that amino acid metabolism was essential during mycelial maturation. The abundance of glycine, a glucogenic amino acid, which could be converted into glucose via gluconeogenesis (Brosnan, 2003), was significantly elevated at MPMP day 35 (p < 0.05). Aspartate is mainly from oxaloacetate, which is involved in TCA cycle (Scandiani et al., 2015). The abundance of aspartate showed insignificant changes (p < 0.05) after maturation. But aspartate-derived amino acids (asparagine and CA-asp) accumulated significantly in mature mycelia (1.62- and 2.02-fold for asparagine at 17 and 29°C, respectively, and 14.6- and 40.5-fold increases for CA-asp at 17 and 29°C, respectively). The increases in the abundance of asparagine and CA-asp were attributed to active participation of other metabolic pathways, and not to the presence of alanine, aspartate, and glutamate metabolism in which aspartate is involved.

Our observations indicate that pyrimidine synthesis, which is related to CA-asp, was significantly activated, suggesting that physiological maturation of mycelia increased the demand for nucleotides for the rapid synthesis of cellular materials, which was fulfilled by de novo synthesis and/or nucleotide salvage of pyrimidines. Figure 8 shows the entire pyrimidine metabolic pathway in mycelia after physiological maturation (intermediates with significantly increased abundance are shown in red). Mature mycelia showed elevated concentrations of nucleotides, including cytidine monophosphate (CMP) and cytosine, which were indicative of the high demand for rapid nucleotide synthesis during physiological maturation. Methylmalonate is a degradation product that is catalyzed by methylbarbiturate and is associated with valine, leucine and isoleucine metabolism. The increase in the abundance of methylmalonate in mature mycelia might mean that the corresponding metabolic network was more active than that in immature mycelia. Moreover, extracellular nucleotides stimulate mycelial survival via purinergic signaling (Schneider et al., 2015).

As stated above, MPMP temperature influences the yield and quality of fruiting bodies, which is also reflected in differences in the abundance of metabolites. The concentrations of citric acid, maleic acid and L-malic acid in Mycelia C samples (at 29°C) were significantly higher than those in Mycelia B samples (at 17°C) at MPMP day 35 (Figure 4B). These differential metabolites were involved in TCA cycle, which is related to energy metabolism. Therefore, the metabolism of Mycelia C samples was likely more active than that of Mycelia C samples, indicating that Mycelia C samples consumed more cultivation substrate, which led to insufficient concentrations of nutrients for the formation of primordia and fruiting bodies. This difference also explains why mycelia that were exposed to low temperatures during physiological maturation may have larger cap diameters, higher fruiting rates, increased yield and improved biological efficiency.

Among the identified metabolites, CA-asp attracted our attention because of high fold-change difference in its abundance in Mycelia B and Mycelia C samples in comparison with its abundance in Mycelia A samples. Interestingly, almost no CA-asp was detected in mycelia collected at day 0 of the MPMP, but its content was extremely high in physiologically mature mycelia. We deduced that CA-asp may be used as a potential indicator to identify whether mycelia have reached physiological maturity. Therefore, we determined the content of CA-asp in mycelia at different MPMP stages, which revealed that CA-asp content continued to rise until day 50 of the MPMP. Therefore, in our study, the content of CA-asp in mycelia showed a regular change during physiological maturation. However, the utility of CA-asp content as an indicator of the physiological maturation of mycelia was not determined, and this question merits additional fruiting experiments.

Our cultivation results demonstrated that a lower MPMP temperature (17°C) is beneficial for the growth and development of P. tuoliensis. A GC-MS-based metabolic approach was employed to compare the abundance of metabolites in mycelia sampled at MPMP day 0, MPMP day 35 (17°C) and MPMP day 35 (29°C). 236 differential metabolites were identified. For the purpose of analysis, 72 differential metabolites (37.8% up-regulated and 62.2% down-regulated) were selected based on the criteria of VIP greater than 1 and p < 0.01. These metabolites are mainly involved in glycolysis, organic acid metabolism, amino acid metabolism, TCA cycle, sugar metabolism, nicotinate and nicotinamide metabolism, and oxidative phosphorylation. The pyrimidine metabolic pathway may be related to the physiological maturation of mycelia, and metabolite CA-asp which participated in this pathway can be a potential indicator for mycelial maturation, because its abundance was significantly enhanced at MPMP day 35. Subsequently, we proved that CA-asp content in mycelia collected at different MPMP stages showed a regular change, which indicated the feasibility of CA-asp used as indicator for mycelial maturation, but whether this indicator can be applied for the commercial production of P. tuoliensis requires the verification of large-scale fruiting experiences.

FD, YZ, YJ, and XY conceived, designed, and performed the experiments, analyzed the data, and wrote and revised the manuscript. QH conceived and designed the experiments.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.