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2023 Volume 3
Article Contents
Abstract
Introduction
Structure and distribution of main bioactive constituents of Lonicera
Biosynthesis and regulation of the main bioactive constituents of Lonicera
Biosynthesis of chlorogenic acids and flavonoids
Biosynthesis of iridoids and triterpene saponins
Metabolic engineering of the main bioactive compounds of the Lonicera genus
Metabolic engineering of chlorogenic acid, luteolin and their glycosides
Metabolic engineering of iridoids and triterpene saponins
Pharmacological activities of the main bioactive compounds of Lonicera
Anti-inflammatory effects
Antimicrobial effects
Anti-oxidative stress effect
Hepatoprotection
Other pharmacological effects
Chloroplast genomic and phylogenetic relationships of Lonicera species
Conclusions and future perspectives
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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The addition of spent oyster mushroom substrates has positive effects on alfalfa growth and soil available nutrients

  • College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China

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  • This study aimed to investigate the effects of spent mushroom substrate (SMS, 0%, 10%, 20%, and 30%, w/w) addition to degraded grassland soil on the growth and nutrient uptake of alfalfa through a greenhouse pot experiment. Meanwhile, we compared with the inorganic fertilizer (CF, 200 N mg/kg and 30 P mg/kg) application treatment, and explored the most suitable SMS addition amount for alfalfa yield. Our results showed, that compared with the control treatment (CK), 10% SMS, 20% SMS, 30% SMS, and CF treatments increased alfalfa shoot biomass by 1.19, 1.67, 1.77, and 1.77 times, respectively. Total carbon content in leaves and total nitrogen content in stems of 20% SMS treatment were significantly higher than other treatments. Adding SMS increased the nodule number, especially the 20% SMS treatment. In addition, the concentrations of dissolved organic carbon, available phosphorus, and microbial biomass carbon and nitrogen were significantly enhanced with increasing SMS addition, and there was no significant difference between CF and CK treatments except for available phosphorus. Shoot biomass was significantly correlated with available phosphorus. In summary, adding SMS (20% and 30%) to degraded grassland soil can significantly improve soil nutrients and microenvironment to increase alfalfa yield, but considering economic benefits, 20% SMS is the most suitable application amount. This study provides the theoretical basis and technical support for the large-scale application of SMS in the field.

  • Introduction

    The Lonicera Linn. genus is a constituent member of the Caprifoliaceae family[1]. It is the largest genus in this family and comprises at least 200 species with a notable presence in North Africa, North America, Asia, and Europe[1]. Members of the Lonicera genus possess a wide range of economic benefits from their use as ornamental plants to food and as plants credited with numerous health benefits. Conspicuous among the numerous members of this genus with known medicinal uses are L. japonica, L. macranthoides, L. hypoglauca, L. confusa, and L. fulvotomentosa[2]. Though these species feature prominently in the Chinese Pharmacopoeia, other species such as L. acuminata, L. buchananii, and L. similis are recognized as medicinal resources in certain parts of China[1]. Among the aforementioned species, L. japonica takes precedence over the rest due to its high medicinal and nutritional value[3,4]. For instance, the microRNA MIR2911, an isolate from L. japonica, has been reported to inhibit the replication of viruses[5−7]. Also, the water extract of L. japonica has been used to produce various beverages and health products[8]. The Lonicera genus therefore possesses huge prospects in the pharmaceutical, food, and cosmetic industries as an invaluable raw material[9].

    The main active constituents of the Lonicera genus include organic acids, flavonoids, iridoids, and triterpene saponins. Chlorogenic acids, iridoids, and flavones are mainly credited with the anti-inflammatory, antiviral, anticancer, and antioxidant effects of the various Lonicera species[10−13]. Their hepatoprotective, immune modulatory, anti-tumor and anti-Alzheimer’s effects are for the most part ascribed to the triterpene saponins[14−16]. As stated in the Chinese Pharmacopoeia and backed by the findings of diverse research groups, the plants of the Lonicera genus are known to possess high amounts of organic acids (specifically chlorogenic acid) and pentacyclic triterpenoid saponins[2,17−19]. The flower and flower bud have traditionally served as the main medicinal parts of the Lonicera genus even though there is ample evidence that the leaves possess the same chemical composition[20]. A perusal of the current scientific literature reveals the fact that little attention has been devoted to exploring the biosynthesis of the chemical constituents of the Lonicera genus with the view to finding alternative means of obtaining higher yields. It is therefore imperative that priority is given to the exploration of the biosynthesis of these bioactive compounds as a possible means of resource protection. There is also the need for further research on ways to fully tap the medicinal benefits of other plant parts in the Lonicera genus.

    Here, we provide a comprehensive review of relevant scientific literature covering the structure, pharmacology, multi-omics analyses, phylogenetic analysis, biosynthesis, and metabolic engineering of the main bioactive constituents of the Lonicera genus. Finally, we proffer suggestions on the prospects of fully exploiting and utilizing plants of the Lonicera genus as useful medicinal plant resources.

    Structure and distribution of main bioactive constituents of Lonicera

    A total of at least 400 secondary metabolites have been reported for the Lonicera genus. These metabolites are categorized into four main groups (Fig. 1a), including not less than 50 organic acids, 80 flavonoids, 80 iridoids, and 80 triterpene saponins[21−23]. Organic acids are mainly derivatives of p-hydroxycinnamic acid and quinic acid. Among the organic acids, chlorogenic acids are reported to be the main bioactive compounds in L. japonica[24−26]. The organic acids are most abundant in the leaves, while the least amounts are found in the stem of L. japonica. The flowers of the plant are known to contain moderately high amounts of organic acids[27]. The basic core structure of the flavonoids is 2-phenylchromogen. Luteolin and its glycoside which are characteristic flavonoids of the Lonicera genus are most abundant in L. japonica[28]. On the whole, the flavonoid contents in L. japonica are also highest in the leaves, available in moderate amounts in the flowers, and in lowest amounts in the stem[21]. The core structures of the iridoids are iridoid alcohols, the chemical properties of which are similar to hemiacetal. The iridoids often exist in the form of iridoid glycosides in plants. Secoiridoids glycosides are predominant in the Lonicera genus[25]. In L. japonica, the contents of the iridoids are most abundant in the flowers, moderate in leaves, and lowest in the stem[21]. The characteristic saponins of the Lonicera genus are mainly pentacyclic triterpenoids, including the hederin-, oleanane-, lupane-, ursulane- and fernane-types, etc[22]. The hederin-type saponins are reported in the highest amounts in L. macranthoides[17] (Fig. 1b).

    Figure 1.  Core structures of main secondary metabolites and their distribution in five species of Lonicera. (a) 1 and 2, the main core structures of organic acids; 3, the main core structures of flavonoids; 4, the main core structures of iridoids; 5, the main core structures of triterpene saponins. (b) Comparison of dry weight of four kinds of substances in five species of Lonicera[17,28].
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    Biosynthesis and regulation of the main bioactive constituents of Lonicera

    Biosynthesis of chlorogenic acids and flavonoids

    The similarities between chlorogenic acid (CGA) and flavonoids can be traced back to their biosynthesis since p-coumaroyl CoA serves as the common precursor for these compounds[29]. p-coumaroyl CoA is obtained through sequential catalysis of phenylalanine and its biosynthetic intermediates by phenylalanine-ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase (4CL)[30−33].

    CGA is a phenolic acid composed of caffeic acid and quinic acid and is the most important bioactive compound among the organic acids. Its biosynthesis has been relatively well-established; three main biosynthetic routes have been propounded (Fig. 2a). One route relates to the catalysis of caffeoyl-CoA and quinic acid by hydroxycinnamoyl-CoA quinate transferase (HQT)/hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) to produce CGA[34−37]. The HQT-mediated pathway has been deemed the major route for CGA synthesis in in different plant species[38,39]. The second biosynthetic route stems from the biosynthesis of p-coumaroyl quinate through the catalytic effect of HCT/HQT and subsequent hydroxylation of p-coumaroyl quinate under the catalysis of p-coumarate 3'-hydroxylase (C3’H)[34,36,37]. For the third route, caffeoyl glucoside serves as the intermediate to form CGA, a process that is catalyzed by hydroxycinnamyl D-glucose: quinic acid hydroxycinnamyl transferase (HCGQT)[40,41].

    Figure 2.  Biosynthetic pathways of main bioactive constituents of Lonicera. (a) Biosynthetic pathways of chlorogenic acid. (b) Biosynthetic pathways of luteoloside. (c) Biosynthetic pathways of secologanin. (d) Biosynthetic pathways of hederin-type triterpene saponins. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; C3’H, p-coumaroyl 3-hydroxylase; HQT, hydroxycinnamoyl-CoA quinate transferase; UGCT, UDP glucose: cinnamate glucosyl transferase; CGH, p-coumaroyl-D-glucose hydroxylase; HCGQT, hydroxycinnamoyl D-glucose: quinate hydroxycinnamoyl transferase; CHS, Chalcone synthase; CHI, Chalcone isomerase; FNS, Flavone synthase; F3H, flavonoid 30-monooxygenase/flavonoid 30-hydroxylase; UF7GT, flavone 7-O-β-glucosyltransferase; GPS, Geranyl pyrophosphatase; GES, geraniol synthase; G8O, geraniol 10-hydroxylase/8-oxidase; 8HO, 8-hydroxygeraniol oxidoreductase; IS, iridoid synthase; IO, iridoid oxidase; 7DLGT, 7-deoxyloganetic acid glucosyltransferase; 7DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferase; SLS, secologanin synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; β-AS, β-amyrin synthase; OAS, oleanolic acid synthetase.
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    The key enzymes in the biosynthesis of p-coumaroyl CoA, and invariably CGA, thus, PAL, C4H, and 4CL have been established in diverse studies such as enzyme gene overexpression/knockdown[42], enzyme activity analysis[33] and transcriptomics[18]. However, the centrality of HQT in the biosynthesis of CGA remains disputable. While some studies have reported a strong correlation between HQT expression level with CGA content and distribution[18,35,39,43,44], others found no such link[45], bringing into question the role of HQT as a key enzyme in CGA biosynthesis.

    Few studies have been conducted on the regulation of CGA biosynthesis in the Lonicera genus. It was found that overexpression of the transcription factor, LmMYB15 in Nicotiana benthamiana can promote CGA accumulation by directly activating 4CL or indirectly binding to MYB3 and MYB4 promoters[46]. LjbZIP8 can specifically bind to PAL2 and act as a transcriptional repressor to reduce PAL2 expression levels and CGA content[47]. Under NaCl stress, increased PAL expression promoted the accumulation of phenolic substances in leaves without oxidative damage, a condition that was conducive to the accumulation of the bioactive compounds in leaves[48].

    Luteolin and its derivative luteolin 7-O- glucoside (luteoloside) are representative flavonoids of the Lonicera genus. Similar to CGA, luteolin is biosynthesized from p-coumaroyl CoA but via a different route. The transition from p-coumaroyl CoA to luteolin is underpinned by sequential catalysis by chalcone synthetase (CHS), chalcone isomerase (CHI), flavonoid synthetase (FNS), and flavonoid 3'-monooxygenase/flavonoid 3'-hydroxylase (F3'H)[45,49,50] (Fig. 2b). Luteoloside is synthesized from luteolin by UDP glucose-flavonoid 7-O-β-glucosyltransferase (UF7GT)[51]. Similar to CGA biosynthesis, the key enzymes of luteolin synthesis include PAL, C4H, and 4CL in addition to FNS[33,45,52]. The content of luteoloside was found to be highly abundant in senescing leaves relative to other tissues such as stem, flowers, and even young leaves[52]. Through transcriptomic analysis, luteoloside biosynthesis-related differentially expressed unigenes (DEGs) such as PAL2, C4H2, flavone 7-O-β-glucosyltransferase (UFGT), 4CL, C4H, chalcone synthase 2 and flavonoid 3'-monooxygenase (F3'H) genes were found to be upregulated in the senescing leaves. Biosynthesis-related transcription factors such as MYB, bHLH, and WD40 were also differentially expressed during leaf senescence[52], while bHLH, ERF, MYB, bZIP, and NAC were differentially expressed during flower growth[53]. Further analysis of the transcription factors revealed that MYB12, MYB44, MYB75, MYB114, MYC12, bHLH113, and TTG1 are crucial in luteoloside biosynthesis[52,53].

    Biosynthesis of iridoids and triterpene saponins

    The biosynthesis of terpenoids mainly involves three stages; formation of intermediates, formation of basic structural skeleton, and modification of basic skeleton[54]. The intermediates of terpenoids are mainly formed through the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, and eventually converted to the universal isoprenoid precursors, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) through a series of enzyme-catalyzed reactions. Under the catalysis of geranyl pyrophosphatase (GPS), IPP is then converted to geranyl pyrophosphate (GPP). Different terpenoids are subsequently derived from GPP as the intermediate product. For instance, in the formation of secoiridoid, GPP first removes the phosphoric acid group to obtain geraniol, second through a series of reactions such as oxidation and cyclization, the skeleton of iridoid, namely iridodial, can be obtained. Finally, through a series of reactions, the basic carbon skeleton of the secoiridoid, namely secologanin, is obtained[55−61] (Fig. 2c). In the formation of triterpene saponins, the key step lies in the formation of the precursor, 2,3-oxidosqualene, a reaction that is catalyzed by squalene epoxidase (SE). There are many pentacyclic triterpenes in the Lonicera genus, the most important type being the hederin-type saponins with hederagenin as aglycones. Hederin-type saponins are produced after the synthesis of oleanolic acid from β-amyrin and catalyzed by β-starch synthetase (β-AS) and Oleanolic acid synthase (OAS)[62,63]. The skeletal modification of the triterpenoid saponins is mainly achieved via the activities of the CYP450 enzymes and UDP-glycosyltransferase (UGT). Hence, the corresponding aglycones are first obtained via oxidation by the CYP450 enzymes (e.g., CYP72A), and further subjected to glycosylation by the appropriate UGT enzyme[63−65] (Fig. 2d). Skeletal formations of the iridoids and triterpene saponins in general have been well researched, but the same cannot be said about the enzymes involved in biosynthesis of these groups of compounds in the Lonicera genus. To fully utilize the iridoids and triterpene saponins in the Lonicera genus, it is necessary to further explore their biosyntheses with the view to enhancing and optimizing the process.

    Metabolic engineering of the main bioactive compounds of the Lonicera genus

    Given the importance of the bioactive compounds in the Lonicera genus, continual isolation of these compounds using the traditional methods are not only tedious and time-consuming, but also unsustainable. With the development and application of microbial metabolic engineering, different strategies have been introduced to produce these bioactive compounds by heterologous synthesis (Table 1).

    Table 1.  Biosynthesis of Lonicera-specialized metabolites using metabolic engineering.
    Engineering bacteriaOperational methodsProductsYieldRefs
    S. cerevisiaeEliminate the tyrosine-induced feedback inhibition, delete genes involved in competing pathways and overexpress rate-limiting enzymesCaffeic acid569.0 mg/L[69]
    S. cerevisiaeEmploye a heterologous tyrosine ammonia lyase and a 4HPA3H complex composed of HpaB and HpaC derived from different speciesCaffeic acid289.4 mg/L[73]
    S. cerevisiaeSupply and recycle of three cofactors: FADH2, S-adenosyl-L-methion, NADPHCaffeic acid
    Ferulic acid    		Caffeic acid: 5.5 g/L;
    Ferulic acid: 3.8 g/L    		[117]
    E. coliKnocking out competing pathwaysCaffeic acid7,922 mg/L[118]
    E. coliArtificial microbial community, a polyculture of three recombinant Escherichia coli strainsChlorogenic acid250 μM[68]
    Cell-free biosynthesisExtract and purify spy-cyclized enzymes (CFBS-mixture)Chlorogenic acid711.26 mg/L[70]
    S. cerevisiaeThree metabolic engineering modules were systematically optimized: shikimate pathway and carbon distribution, branch pathways, CGA pathway genesChlorogenic acidFlask fermentation: 234.8 mg/L;
    Fed-batch fermentation:
    806.8 mg/L    		[119]
    E. coliUsing modular coculture engineering: construction of the defective strain improves the production and utilization of precursor substancesChlorogenic acid131.31 mg/L[122]
    E. coliIntroduce heterologous UDP-glucose biosynthetic genesLuteolin34 mg/L[120]
    Y. lipolyticaOverexpression of the key genes involved in the mevalonate pathway, the gene encoding cytochrome P450 (CYP716A12) to that encoding NADPH-P450 reductaseOleanolic acid129.9 mg/L[85]
    S. cerevisiaeImprove the pairing efficiency between Cytochrome P450 monooxygenase and reductase and the expression level of key genesOleanolic acid606.9 mg/L[121]
    S. cerevisiaeHeterologous expression and optimization of CrAS, CrAO, and AtCPR1, and regulation of ERG1 and NADPH regeneration systemOleanolic acid433.9 mg/L[123]
     | Show Table
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    Metabolic engineering of chlorogenic acid, luteolin and their glycosides

    Due to the demand for CGA in the food, pharmaceutical, chemical, and cosmetic industries, the traditional means of obtaining the same requires a relatively longer period for plant maturation to obtain low yields of the desired product. This therefore brings into question the sustainability and efficiency of this approach. The alternative and sustainable approach has been to produce CGA using synthetic biology and metabolic engineering.

    Current research has sought to utilize Escherichia coli (and its mutant strain) and Saccharomyces cerevisiae to synthetically generate CGA and other flavonoids[66−73]. For instance, Cha et al. employed two strains of E. coli to produce a relatively good yield of CGA (78 mg/L). Their approach was based on the ability of one strain to generate caffeic acid from glucose and the other strain to use the caffeic acid produced and quinic acid as starting materials to synthesize CGA[66]. Using a bioengineered mutant of E. coli (aroD mutant), Kim et al. increased the yield of CGA to as high as 450 mg/L[67]. Others have sought to increase the yield of CGA by employing a polyculture of three E. coli strains that act as specific modules for the de novo biosynthesis of caffeic acid, quinic acid and CGA. This strategy eliminates the competition posed by the precursor of CGA (i.e., caffeic acid and quinic acid) and generally results in improved production of CGA[68]. Saccharomyces cerevisiae is a chassis widely used for the production of natural substances from plants with an intimal structure that can be used for the expression of cytochrome P450 enzymes that cannot be expressed in E. coli. Researchers have used yeast to increase the production of organic acids[69]. A de novo biosynthetic pathway for the construction of CGA in yeast has been reported new cell-free biosynthetic system based on a mixture of chassis cell extracts and purified Spy cyclized enzymes were adopted by Niu et al. to a produce the highest yield of CGA reported so far up to 711.26 ± 15.63 mg/L[70].

    There are many studies on the metabolic engineering for the synthesis of flavonoids, but few on luteolin and its glycosides. Strains of E. coli have been engineered with specific uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) to synthesize three novel flavonoid glycosides. These glycosides were quercetin 3-O-(N-acetyl) quinovosamine (158.3 mg/L), luteolin 7-O-(N-acetyl) glucosaminuronic acid (172.5 mg/L) and quercetin 3-O-(N-acetyl)-xylosamine (160.8 mg/L)[71]. Since most of the flavonoid glycosides synthesized in E. coli are glucosylated, Kim et al. in their bid to synthesize luteolin-7-O-glucuronide, deleted the araA gene that encodes UDP-4-deoxy-4-formamido-L-arabinose formyltransferase/UDP-glucuronic acid C-4'' decarboxylase in E.coli and were able to obtain a yield of 300 mg/L of the desired product[72].

    Metabolic engineering of iridoids and triterpene saponins

    Terpenoidal saponins are mostly derived from slow-growing plants and usually possess multiple chiral centers[74]. Traditional isolation and even chemical synthesis of the terpenoidal saponins are both tedious and uneconomical for large-scale production. Therefore, it is necessary to find other ways to synthesize these compounds known to have diverse pharmacological functions.

    Heterologous synthesis has become an important way to improve the target products. With the development of synthetic biology, heterologous synthesis of triterpene saponins involves chassis of both plant and microbial origin. In this regard, Nicotiana benthamiana is a model plant species for the reconstruction of the biosynthetic pathways of different bioactive compounds including monoterpenes, hemiterpenes, and diterpenes[59,75−77]. Aside from Nicotiana benthamiana, other plants have also been used as heterologous hosts[78]. Heterologous synthesis using microbial hosts mainly involves Saccharomyces cerevisiae and Escherichia coli[79−81], and other microorganisms[82,83]. Comparatively, plants as biosynthetic hosts have the advantages of an established photosynthetic system, abundant supply of relevant enzymes, and presence of cell compartments, etc. They are however not as fast growing as the microorganisms, and it is also difficult to extract and separate the desired synthesized compounds from them as hosts.

    Although heterologous synthesis has many advantages, the premise of successful construction of synthetic pathway in host is to elucidate the unique structure of the compound and the key enzyme reaction mechanism in the biosynthetic pathway. There is little research on metabolic engineering of the hederin-type pentacyclic triterpene saponins in Lonicera, but there are studies on the heterologous synthesis of its aglycone precursor, oleanolic acid[84,85]. There is a dearth of scientific literature on key enzymes in the biosynthesis of pentacyclic triterpenoid saponins in the Lonicera genus.

    Pharmacological activities of the main bioactive compounds of Lonicera

    Scientific evidence by diverse research groups has linked members of the Lonicera genus to a wide range of pharmacological effects (Fig. 3). These pharmacological effects are elicited by different chemical constituents, much of the underlying mechanisms of which have been elucidated by the omics techniques. Here, we summarize the pharmacological effects and pharmacodynamics of the Lonicera genus in the last 6 years.

    Figure 3.  Schematic summary of four main pharmacological effects (anti-inflammatory, antimicrobial, anti-oxidative and hepatoprotective effects) of the Lonicera genus and the underlying mechanisms of actions.
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    Anti-inflammatory effects

    Bioactive compounds of plants in the Lonicera genus have demonstrated varying degrees of anti-inflammatory actions. In a recent study, Lv et al. showed that lonicerin inhibits the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) through regulating EZH2/AtG5-mediated autophagy in bone marrow-derived macrophages of C57BL/6 mice[86]. The polysaccharide extract of L. japonica reduces atopic dermatitis in mice by promoting Nrf2 activation and NLRP3 degradation through p62[87]. Several products of Lonicera have been reported to have ameliorative effects on DSS-induced colitis. Among them, flavonoids of L. rupicola can improve the ulcerative colitis of C57BL/6 mice by inhibiting PI3K/AKT, and pomace of L. japonica can improve the ulcerative colitis of C57BL/6 mice by improving the intestinal barrier and intestinal flora[88,89]. The flavonoids can also ameliorate ulcerative colitis induced by local enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in Wistar rats by inhibiting NF-κB pathway[90]. Ethanol extract from L. Japonica has demonstrated the potential to inhibit the expressions of inflammatory cytokines in serum and macrophages of LPS-induced ICR mice[91]. The water extract of L. japonica and luteolin were found to exhibit their anti-inflammatory effects via the inhibition of the JAK/STAT1/3-dependent NF-κB pathway and induction of HO-1 expression in RAW263.7 cells induced by pseudorabies virus (PRV)[92].

    Antimicrobial effects

    Existing scientific evidence indicates that the extracts of plants in the Lonicera genus exhibit strong inhibition against different pathogenic microorganisms. Phenolic compounds from L. japonica demonstrated a particularly significant inhibitory effect against Staphylococcus aureus and Escherichia coli, in vitro, making these compounds potential food preservatives[93]. Influenza A virus is a serious threat to human health. Recent research has found the ethanol extract of L. japonica to possess a strong inhibitory effect against H1N1 influenza virus-infected MDCK cells and ICR mice[94]. The incidence of the COVID-19 pandemic called to action various scientists in a bid to find safe and efficacious treatment[95]. Traditional Chinese medicines became an attractive alternative in this search. The water extract of the flower bud of L. japonica which has traditionally served as a good antipyretic and antitussive agent attracted the attention of researchers. Scientific evidences have confirmed that the water extract of L. japonica can induce let-7a expression in human rhabdomyosarcoma cells or neuronal cells and blood of lactating mice, inhibiting the entry and replication of the virus in vitro and in vivo[96]. In addition, the water extract of L. japonica also inhibits the fusion of human lung cancer cells Calu-3 expressing ACE2 receptor and BGK-21 cells transfected with SARS-CoV-2 spike protein, and up-regulates the expression of miR-148b and miR-146a[97].

    Anti-oxidative stress effect

    Oxidative stress has been implicated in the pathophysiology of many diseases, hence, amelioration of the same could be a good therapeutic approach[98,99]. In keeping with this therapeutic strategy, various compounds from the Lonicera genus have demonstrated the ability to relieve oxidative stress due to their pronounced antioxidant effects. For instance, the polyphenolic extract of L. caerulea berry was found to activate the expression of AMPK-PGC1α-NRF1-TFAM proteins in the skeletal muscle mitochondria, improve the activity of SOD, CAT and GSH-Px enzymes in blood and skeletal muscle, relieve exercise fatigue in mice by reducing oxidative stress in skeletal muscle, and enhance mitochondrial biosynthesis and cell proliferation[100]. The diverse health benefits of the anthocyanins from L. japonica have been mainly credited to their antioxidant and anti-inflammatory effects. The anthocyanin and cyanidin-3-o-glucoside have been reported to possess the potential to prolong life and delay senescence of Drosophila through the activation of the KEAP1/NRF2 signaling pathway[101].

    Hepatoprotection

    The liver is an essential organ that contributes to food digestion and detoxification of the body. These functions expose the liver to diverse toxins and metabolites. The Lonicera genus is rich in phytochemicals that confer protection on the liver against various toxins. The phenolic compound, 4, 5-di-O-Caffeoylquinic acid methyl ester was shown to be able to improve H2O2-induced liver oxidative damage in HepG2 cells by targeting the Keap1/Nrf2 pathway[102]. Hepatic fibrosis is a complex dynamic process, with the propensity to progress to liver cancer in severe cases. The L. japonicae flos water extract solution increased the cell viability of FL83B cells treated with thioacetamide (TAA), decreased the levels of serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP), inhibited the transformation growth factor β1 (TGF-β1) and liver collagen deposition[103]. Sweroside, a secoiridoid glucoside isolate of L. japonica is known to protect the C57BL/6 mice liver from hepatic fibrosis by up-regulating miR-29a and inhibiting COL1 and TIMP1[104].

    Other pharmacological effects

    Aside from the aforementioned, other pharmacological effects have been ascribed to the Lonicera genus. The ethanolic extract of L. caerulea has been reported to inhibit the proliferation of SMMC-7721 and H22 hepatoma cells, while its anthocyanins induced the apoptosis of tumor cells via the release of cytochrome C and activation of caspase[105]. AMPK/PPARα axes play an important role in lipid metabolism. A chlorogenic acid-rich extract of L. Japonica was found to significantly decrease the early onset of high-fat diet-induced diabetes in Sprague-Dawley rats via the CTRPs-AdipoRs-AMPK/PPARα axes[106]. In a high-fat diet-induced non-alcoholic fatty liver disease in C57BL/6 mice, treatment with L. caerulea polyphenol extract decreased serum inflammatory factors and endotoxin levels and the Firmicutes/Bacteroidetes ratio, an indication of its modulatory effect on the gut microbiota[107]. The iridoid-anthocyanin extract from L. caerulea berry contributed to alleviating the symptoms of intestinal infection with spirochaeta in mice[108].

    Chloroplast genomic and phylogenetic relationships of Lonicera species

    The traditional classification of the Lonicera genus based on the morphology of member plants is further categorized into two subgenera, Chamaecerasus and Periclymenum. The Chamaecerasus includes four categories, Coeloxylosteum, Isika, Isoxylosteum and Nintooa. The Periclymenum includes two categories, Subsect. Lonicera and Subsect. Phenianthi (Supplemental Table S1).

    High-throughput chloroplast genome sequencing of L. japonica found its length to be 155078 bp, which is similar to the structure of the typical angiosperm chloroplast genome. It contains a pair of inverted repeat regions (IRa and IRb, 23774 bp), a large single copy region (LSC, 88858 bp) and a small single copy area (SSC, 18672 bp)[109,110]. However, compared with chloroplast genomes of other plants, the chloroplast genome of L. japonica has a unique rearrangement between trnI-CAU and trnN-GUU[110]. Based on the phylogenetic analysis of the plastid genomes of seven plants in the Lonicera genus, 16 diverging hot spots were identified as potential molecular markers for the development of the Lonicera plants[111]. The phylogeny of Lonicera is rarely researched at the molecular level and the pattern of repetitive variation and adaptive evolution of the genome sequence is still unknown. Chloroplast genome sequences are highly conserved, but insertions and deletions, inversions, substitutions, genome rearrangements, and translocations also occur and have become powerful tools for studying plant phylogeny[112,113].

    We present here the phylogenetic tree of the Lonicera genus based on the published complete chloroplast genome sequences downloaded from the National Center for Biotechnology Information (NCBI) database using the Maximum likelihood method (Fig. 4). Based on our chloroplast phylogenies, we propose to merge L. harae into Sect. Isika and L. insularis into Chamaecerasus, but whether L. insularis belongs to Sect. Isika or Sect. Coeloxylosteum is uncertain. Based on protein-coding regions (CDS) of the chloroplast genome or complete chloroplast genomes, Liu et al. and Chen et al. supported the classification of the two subgenera in Lonicera[111,114]. Sun et al. and Srivastav et al. demonstrated a classification between the two subgenera with more species by using sequences of nuclear loci generated, chloroplast genome, and restriction site-associated DNA sequencing (RADSeq)[115,116]. However, our phylogenetic analysis and that of Sun et al. show relations within the subgenus Chamaecerasus are tanglesome in some respects[116]. Plant traits are affected by the environment to varying degrees. Since evidence of plant speciation is implicit in its genome sequence, comparative analysis at the molecular level provides a relatively accurate depiction of inherent changes that might have occurred over time. These findings suggest the need for more species of the Lonicera genus to be sequenced to provide a more accurate theoretical basis for the evolution of the Lonicera plants and a more effective revision in the classification of the Lonicera genus.

    Figure 4.  Phylogenetic tree of 42 species of the Lonicera genus based on complete chloroplast genome sequence data. The phylogenetic tree was constructed by the maximum likelihood method. Coeloxylosteum, Isika, Isoxylosteum, and Nintooa belong to Chamaecerasus and Subsect. Lonicera belongs to the Periclymenum. Chamaecerasus and Periclymenum are the two subgenera of Lonicera. 'Not retrieved' indicates that the species failed to retrieve a subordinate taxon in the Lonicera.
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    Conclusions and future perspectives

    The Lonicera genus is rich in diverse bioactive compounds with immeasurable prospects in many fields. Members of this genus have been used for thousands of years in traditional Chinese medicine for heat-clearing and detoxification. These plants generally have a good taste and form part of the ingredients of various fruit juices. In cosmetics, they are known to possess anti-aging and moisturizing functions. Plants of the Lonicera genus are also known for their good ecological adaptability and can be used to improve soil and ecological environment. Based on the value of the Lonicera genus, besides researching their use through molecular biological means, their efficient utilization can also be promoted in the following ways: (1) The stems and leaves of the plants could be developed for consumption and use since the chemical profiles of these parts do not differ significantly from the flowers. This way, the wastage of this scarce resource could be minimized or avoided. (2) Most of the Lonicera plants are vines or shrubs and their natural regeneration speed is slow, so the introduction and domestication of species could be strengthened to avoid overexploitation of wild resources.

    At present, only the research on the biosynthesis and efficacy of chlorogenic acid is quite comprehensive and has been used widely in various fields. There is limited research on various aspects of other bioactive compounds and should therefore be given priority in future research goals. Currently, the multi-omics analytical approach has gradually evolved as a reliable and helpful analytical platform. Hence, multi-omics research on the Lonicera genus could lead to discoveries in drug discovery and human health.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design, draft manuscript preparation: Yin X, Chen X, Li W, Tran LSP, Lu X; manuscript revision: Yin X, Chen X, Li W, Tran LSP, Lu X, Chen X, Yin X, Alolga RN; data/literature collection: Chen X, Yin X; figure preparation: Chen X, Yin X; figure revision: Alolga RN, Yin X, Chen X, Li W, Tran LSP, Lu X. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article and its supplementary information file.

    Acknowledgments

    This work was partially supported by the National Natural Science Foundation of China (NSFC, Nos 82173918 and 82373983).

  • The authors declare that they have no conflict of interest. Xiaojian Yin is the Editorial Board member of Medicinal Plant Biology who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

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  • Cite this article

    Shi Y, Cui X, Zhang Y, Liu M. 2023. The addition of spent oyster mushroom substrates has positive effects on alfalfa growth and soil available nutrients. Grass Research 3:19 doi: 10.48130/GR-2023-0019
    Shi Y, Cui X, Zhang Y, Liu M. 2023. The addition of spent oyster mushroom substrates has positive effects on alfalfa growth and soil available nutrients. Grass Research 3:19 doi: 10.48130/GR-2023-0019
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The addition of spent oyster mushroom substrates has positive effects on alfalfa growth and soil available nutrients

  • College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China

  • Received: 08 May 2023
  • Accepted: 21 August 2023
  • Published online: 08 October 2023
Grass Research  3,  Article number: 19  (2023)  |  Cite this article

Abstract: This study aimed to investigate the effects of spent mushroom substrate (SMS, 0%, 10%, 20%, and 30%, w/w) addition to degraded grassland soil on the growth and nutrient uptake of alfalfa through a greenhouse pot experiment. Meanwhile, we compared with the inorganic fertilizer (CF, 200 N mg/kg and 30 P mg/kg) application treatment, and explored the most suitable SMS addition amount for alfalfa yield. Our results showed, that compared with the control treatment (CK), 10% SMS, 20% SMS, 30% SMS, and CF treatments increased alfalfa shoot biomass by 1.19, 1.67, 1.77, and 1.77 times, respectively. Total carbon content in leaves and total nitrogen content in stems of 20% SMS treatment were significantly higher than other treatments. Adding SMS increased the nodule number, especially the 20% SMS treatment. In addition, the concentrations of dissolved organic carbon, available phosphorus, and microbial biomass carbon and nitrogen were significantly enhanced with increasing SMS addition, and there was no significant difference between CF and CK treatments except for available phosphorus. Shoot biomass was significantly correlated with available phosphorus. In summary, adding SMS (20% and 30%) to degraded grassland soil can significantly improve soil nutrients and microenvironment to increase alfalfa yield, but considering economic benefits, 20% SMS is the most suitable application amount. This study provides the theoretical basis and technical support for the large-scale application of SMS in the field.

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    Introduction

    • According to the survey by the China Edible Fungi Association, China is the largest country in the production and export of edible mushrooms[1,2]. Edible mushrooms are rich in minerals and are often considered an alternative source of meat, fish, and vegetables[3,4]. In particular, the demand for Pleurotus mushrooms is increasing year by year because of its high protein, dietary fiber, as well as essential and non-essential amino acids[5,6]. It has been known that each kilogram of mushroom can produce about 2.5−5 kg of the spent mushroom substrate (SMS)[7,8]. Currently, SMS is usually treated as agricultural waste and is landfilled, burned in the open on land, or composted with animal manure. These disposals may lead to environmental issues such as soil, air, and water pollution[9].

      SMS is the lignocellulosic by-product of mushroom cultivation mainly referring to biomass waste which is not completely degraded as the degradation efficiency of edible fungi species only reached 40%−80%[10,11]. The term 'spent mushroom compost' (SMC) can be used interchangeably with SMS describing the agro-residues and fungal mycelium left after mushroom harvest[12]. SMS mainly consisted of residual fungal mycelium, various disintegrated lignocellulosic biomass (such as corn cob, sawdust, livestock litter and manure, cottonseed hull, wood chip, straw), amendments (such as lime, peat, gypsum), nutrients, as well as a high level of enzymes and organic matter[13−15]. A previous study has found that SMS has the characteristics of loose texture, small bulk density, rich particle structure, good air permeability, high nutrient, and water retention rate, which can effectively improve soil physical structure and soil microbial ecological environment[16].

      After harvesting mushrooms, the SMS contains nutrients that are useful for crop, vegetable, fruit tree production and soil improvement[17]. Nowadays, there have been various studies using SMS as a biofertilizer to cultivate plants, including tomato, barley, pineapple, and so on. Lopes et al.[18] concluded that the use of SMS (Agaricus subrufescens) for seedling production had a higher total tomato production compared to previously reported production levels. Some experiments have also shown that cultivating pineapple in soil amended with SMS of oyster mushroom (Pleurotus ostreatus) increased its fruit number and optimized soil properties[19]. Field experiment results showed that the application of SMS at 50 and 100 t/ha significantly increased barley grain yield and soil nutrients, especially soil nitrogen and potassium content[20]. Vahid Afagh et al.[21] revealed that 10%–15% of SMS addition in the growing substrate significantly increased plant growth, flower yield, essential macronutrient uptake and soluble sugar content as well as essential oil percentages on German chamomile (Matricaria recursta L.) compared to the control treatment (no SMS). In addition, a previous report showed that SMS improved soil physical and chemical properties, and the speed of lawn establishment in turf production[22]. However, research on using SMS to cultivate forage has not yet been in-depth. Improving the nutrient content of grasslands in grassland management is one of the important measures to promote forage production. Therefore, exploring the impact mechanism of SMS on forage in degraded grassland soil can help the editable mushroom industry to achieve green development, transformation, and upgrading[23].

      Alfalfa has been grown on approximately 30 million hectares for hay, silage processing and grazing globally[24]. With its long cultivation history and adaptability to a wide range of territories, alfalfa has been commonly used for animal feed[25]. It has also been utilized as a medicinal herb since it is a good source of vitamins (A, C, E, and K), protein, and minerals such as calcium, iron, potassium, and phosphorus[26]. Apart from these uses, alfalfa is an ideal natural resource and model plant for the remediation of contaminated soils, offering a variety of elite characteristics, including a highly productive biomass, drought tolerance, a fast-growing and prosperous root system, and availability in large amounts over several months of the year[27]. Based on the current cultivation status and application prospects of alfalfa mentioned above, it is quite important to study the application of SMS in alfalfa cultivation. Therefore, this research is based on the idea that SMS is expected to be used for alfalfa cultivation, filling the gap in this research field. Using SMS and soil mixed in proportion as a nutrient substrate. This research aims to explore the effect of SMS on the growth of alfalfa in degraded grassland soil, while improving the reuse efficiency of mushroom substrate.

    Materials and methods

      Soil and spent mushroom substrate properties

    • The topsoil (0−20 cm) used in the experiment was taken from Duolun Restoration Ecology Research Station(42°20'N, 116°17'E), Inner Mongolian, China[28]. Soil properties were: pH 6.51, 84,066.46 mg/kg available nitrogen, 48.55 mg/kg dissolved organic carbon (DOC), and 232.79 mg/kg microbial biomass carbon (MBC). The type of experimental soil was classified as Haplic calisols according to the Food and Agriculture Organization of the United Nation (FAO) classification.

      The experimental spent mushroom substrate (SMS) was the waste residue from 4−5 generations of Pleurotus ostreatus from Beijing Academy of Agriculture and Forestry Sciences, and its main components include 50% cottonseed hull, 40% corncob, a small amount of bran, and soybean meal (about 2%−5%). The spent oyster mushroom substrates properties were: pH 6.04, 90,650.61 mg/kg available nitrogen, 50,426.34 mg/kg DOC, and 50,626.21 mg/kg MBC. The spent mushroom substrate was thoroughly air-dried, crushed, and mixed before use.

      Experimental design

    • The experiment included four substrate ratio treatments and one inorganic fertilizer with four replications. Therefore, these five treatments were 100% soil (CK), 90% soil and 10% SMS spent oyster mushroom substrates (10%SMS), 80% soil and 20% SMS spent oyster mushroom substrates (20%SMS), 70% soil and 30% SMS spent oyster mushroom substrates (30%SMS), and 100% soil with adding inorganic fertilizers (CF). Mix the crushed SMS and soil passing through a 2 mm sieve in proportion to each other and put them into disinfected pots one by one. Each plastic pot was filled with 0.4 kg dry weight of mixed substrate. According to previous studies, 200 mg/kg N and 30 mg/kg P (the actual fertilizer substances were (NH4)2SO4 and KH2PO4) were applied in the inorganic fertilizer treatment[29,30].

      Use a 10% H2O2 (SCRC, Shanghai, China) solution to sterilize the surface of alfalfa (Medicago sativa L.) seeds with uniform size and full particles. Rinse them with sterile water several times and then sow them in plastic pots, with a few seeds per pot. Water them every two days. After the emergence of forage grass, eight plants per pot were established, cultivated in an artificial climate chamber with 16 h light, 8 h darkness, and a constant temperature of 25 °C. Under these conditions, alfalfa can have suitable growth conditions and good cultivation effects[31]. The experiment was conducted from April 2022 to December 2022 and harvested 40 d later.

      Experimental method

    • This experiment measured the plants and soil of five treatments of alfalfa, including plant physiological indicators such as shoot biomass, root biomass, alfalfa nodule number, and total carbon (TC) and nitrogen (TN) of plants (including roots, stems, and leaves respectively), as well as soil microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), dissolved organic carbon (DOC), and ammonium nitrogen (NH+4-N), nitrate nitrogen (NO−3-N), soil pH and available phosphorus (P).

      We used scissors to cut aboveground alfalfa from the substrate surface, and then, the belowground was thoroughly washed with deionized water to remove soil particles. Shoots and roots were dried to constant weight at 65 °C and respectively weighed to measure the dry biomass of stems, leaves, and roots. Scan and calculate the number of alfalfa nodules using Epson Perfection V700 Photo (flat panel color image scanner) and WinRHIZO software. To measure TC and TN values of alfalfa components concluding roots, stems, and leaves by using Vario MACRO Cube Elemental.

      We used the chloroform fumigation extraction method to determine MBC and MBN[32]. Five grams of two soil subsamples and incubate the non-fumigated group in a sealed cardboard box for 24 h (with water and NaOH solution inside). The fumigated group is vacuum filtered for 24 h (with water, CCl3, and NaOH solution inside) in a suction filter. After removal, add 20 ml of K2SO4 solution (0.5 M), shake and filter. Take 10 mL of the leaching solution and measure the data using an Analytik Jena Multi N/C3100 instrument to calculate the DOC, MBC, and MBN values.

      Soil NH+4-N and NO−3-N were extracted from 10 g subsamples, added 50 mL KCl solution (1 M), shaken for 30 min, and then analyzed the filtrate with a continuous flow analyzer (AutoAnalyser 3, Analytical, Norderstedt, Germany)[33]. 1:2.5 soil/water suspension was measured substrates pH using Sartorius PB-10.

      We used the molybdate-ascorbic acid method for the colorimetric measurement of available phosphorus[30]. Specifically, a 2.5 g air-dried substrate sample was added to 25 mL 0.5 mol/L NaHCO3 solution and about 1 g dephosphorized activated carbon, shaken at 20–25 °C for 30 min, and the suspension was filtered by double filter paper. Add 2.5 mL of molybdenum-antimony anti-mixed color reagent to 5 mL soil suspension, constant volume to 25 mL, shake and place at room temperature for 30 min. Available phosphorus concentration was determined using a multimode microplate reader (Varioskan LUX; Thermo Scientific, USA) at 880 nm wavelength.

      Data analysis

    • Alfalfa growth data and soil indicator data were analyzed using one-way analysis of variance (ANOVA) by Duncan's multiple rang test and SPSS 20.0 software for processing; the significance level was p < 0.05. Before analysis, the Shapiro-Wilk test was used to evaluate the normality of the data. Levene test was used for analysis of variance to determine homogeneity of variance. Where necessary, the parameters were square root transformed to achieve normality and homogeneity. To explore the relationship between variables, the redundancy analysis (RDA) and Pearson correlation analysis were used for evaluation. All the figures were performed in R 4.2.1.

    Results

      Plant biomass and nutrient uptake

    • The shoot and root biomass of alfalfa cultured with 20% SMS, 30% SMS and CF treatments were significantly higher than those of CK treatment (Fig. 1, p < 0.05), but there was no significant difference between 10% SMS and CK treatments (p > 0.05). And there was no significant difference between the shoot and root biomass of 20% SMS and 30% SMS treatments compared with CF treatment (p < 0.05). CF treatment had the largest number of root nodules number, followed by 20% SMS, 30% SMS, and 10% SMS treatments, and CK treatment had the least number (Fig. 2). The number of nodules with 20% SMS and 30% SMS treatments was higher than CK treatment (Fig. 2, p < 0.05).

      Figure 1. 

      (a) Shoot biomass, (b) root biomass and (c) picture of plant pot experiments of alfalfa under different treatments. CK: 100% soil; 10%: 10% SMS; 20%: 20% SMS; 30%: 30% SMS; CF: 100% soil with chemical fertilizers added. Different lowercase letters above the columns represent significant differences among these treatments according to Duncan tests.

      Figure 2. 

      Number of alfalfa nodules under different treatments. CK: 100% soil; 10%: 10% SMS; 20%: 20% SMS; 30%: 30% SMS; CF: 100% soil with chemical fertilizers added. Different lowercase letters above the columns represent significant differences among these treatments according to Duncan tests.

      TN and TC content of forage grass reflects its excellent quality. Our data showed that 20% SMS treatment had the highest TN and TC contents of alfalfa leaf (Table 1). The TN and TC contents of alfalfa root and stem were the highest with CF treatment, and there was no significant difference compared with 20% SMS and 30% SMS treatments (p > 0.05).

      Table 1.  Total carbon, nitrogen content, and carbon and nitrogen ratio (TC, TN, C/N) of alfalfa harvested in different treatments.

      TreatmentRoot-TN (g)Root-TC (g)Root-C/N (%)Stem-TN (g)Stem-TC (g)Stem-C/N (%)Leaf-TN (g)Leaf-TC (g)Leaf-C/N (%)
      CK0.53 ± 0.26b8.05 ± 3.69b15.44 ± 0.86b0.27 ± 0.11b7.27 ± 2.73c27.17 ± 1.27a0.73 ± 0.49a12.51 ± 8.20b23.32 ± 12.55ab
      10% SMS0.83 ± 0.28ab14.13 ± 4.18ab17.13 ± 1.09a0.28 ± 0.09b8.49 ± 2.96bc29.99 ± 3.39a0.98 ± 0.47a23.49 ± 9.58ab24.8 ± 3.63a
      20% SMS1.00 ± 0.30ab17.71 ± 5.83a17.56 ± 0.79a0.52 ± 0.16a14.58 ± 4.21ab28.23 ± 2.71a2.50 ± 2.16a29.74 ± 7.79a15.7 ± 6.43ab
      30% SMS1.04 ± 0.52ab17.92 ± 8.80a17.28 ± 0.57a0.44 ± 0.19ab13.16 ± 5.88abc29.82 ± 2.14a1.60 ± 1.09a25.07 ± 17.19ab11.76 ± 7.87b
      CF1.16 ± 0.15a19.77 ± 2.30a17.13 ± 0.45a0.58 ± 0.13a15.53 ± 2.61a27.78 ± 6.58a1.93 ± 0.21a29.06 ± 1.85ab15.14 ± 1.13ab
      Different lowercase letters in the table represent significant differences among these treatments according to Duncan tests.

      Substrate physicochemical properties

    • The concentrations of DOC and available P significantly increased with the increase of SMS addition in the substrate (Fig. 3a & b, p < 0.05), and the concentrations were the lowest in the CK treatment. There was no significant difference between the 10%SMS and CF treatments (Fig. 3b, p > 0.05).

      Figure 3. 

      Effects of different treatments on (a) soil dissolved organic carbon (DOC), (b) available P, (c) microbial biomass nitrogen (MBN), (d) microbial biomass carbon (MBC), (e) nitrate nitrogen (NO−3) and (f) ammonium nitrogen (NH+4). Means ± S.E. CK: 100% soil; 10%: 10% SMS; 20%: 20% SMS; 30%: 30% SMS; CF: 100% soil with chemical fertilizers added. Different lowercase letters above the columns represent significant differences among these treatments according to Duncan tests.

      The MBN and MBC concentrations of 10% SMS, 20% SMS, and 30% SMS treatments were significantly higher than those of CF and CK treatments (Fig. 3c, p < 0.05). There was no difference between CF and CK treatments (p > 0.05).

      NO−3 concentrations of 10% SMS, 20% SMS, and 30% SMS treatments were significantly lower than that of CK and CF treatments (Fig. 3e, p < 0.05). NH4+ concentration was the lowest in 20% SMS treatment, while other treatments had no significant difference with CK treatment (Fig. 3d, p < 0.05). There was no significant difference in soil pH among all treatments (Table 2, p > 0.05).

      Table 2.  The pH of substrate with different treatments after harvesting.

      Experimental treatmentpH
      CK7.12 ± 0.18a
      10% SMS7.08 ± 0.09a
      20% SMS7.06 ± 0.08a
      30% SMS7.06 ± 0.15a
      CF7.10 ± 0.06a
      Different lowercase letters in the table represent significant differences among these treatments according to Duncan tests.

      Correlation analysis

    • RDA was used to assess how the physicochemical properties of the substrates influenced alfalfa growth indicators. It can be seen that there are significant differences in the substrate environment among different treatments, which have a significant impact on plant physiological indicators (Fig. 4). Plant physiological indicators respond strongly to the physicochemical characteristics of the substrate. The results show that the variance contribution rate of principal component 1 (PC1) and principal component 2 (PC2) respectively are 42.72% and 26.10%, and the cumulative variance contribution rate of PC1 and PC2 is 68.82%. Obtained through RDA diagram and calculation of Pearson correlation coefficient (PCC), available P had the strongest correlation with the PC1 and treatments distribution (r2 = 0.4035, p = 0.008), followed by root nodule (r2 = 0.2829, p = 0.047).

      Figure 4. 

      The Redundancy Analysis (RDA) shows the effects of five experimental treatments on physical and chemical properties in substrate including pH, dissolved organic carbon (DOC), microbial biomass nitrogen (MBN), microbial biomass carbon (MBC), NH+4 and NO−3, available P (P) and plant indicators including shoot biomass, root biomass, plant total nitrogen (TN), total carbon (TC), and carbon nitrogen ratio (CN). The red arrow in the figure represents plant growth indicators, and the blue arrow in the figure represents physical and chemical properties in substrate.

      The Pearson correlation analysis showed that the shoot biomass value of alfalfa was significantly correlated with the available P in the substrate (Fig. 5, p < 0.05). MBC and MBN were strongly correlated with DOC and available P (p < 0.05). Meanwhile, there is a strong correlation between DOC and P (p < 0.001).

      Figure 5. 

      Correlation heatmaps of alfalfa growth indicators (shoot biomass and root biomass) and of nutrient concentration indicators in substrates (NO−3, NH+4, MBC, MBN, DOC, pH and P). NO−3: nitrate nitrogen; NH+4: ammonium nitrogen; MBC: microbial biomass carbon; MBN: microbial biomass nitrogen; DOC: dissolved organic carbon; pH: acidity and alkalinity of substrates; P: available P. The color intensity in each panel indicates the relative correlation between read numbers of two groups. Blue represents positive correlation, and red represents negative correlation. Statistically significant correlations are indicated with *p < 0.05, **p < 0.01, and ***p < 0.001.

    Discussion

    • SMS has been increasingly used as soil amendment either freshly or after composting processes in recent years[34]. The current study found that adding appropriate amounts of SMS to degraded grassland soil can promote the growth and quality of alfalfa. The shoot biomass of alfalfa in 20% SMS and 30% SMS treatment significantly increased respectively by 1.67 and 1.77 times compared to CK treatment (Fig. 1). 20% SMS treatment had the highest TN contents of alfalfa stem and TC contents of alfalfa leaf (Table 1). This result supports previous studies, which illustrated that sometimes organic manure amendment promoted plant growth more efficiently byproviding a higher nutrient value compared with inorganic fertilizer[35].

      We found that our SMS materials contain far more nutrients needed by plants than soil, which means that adding SMS to degraded grasslands with poor soil is beneficial for plant growth. As an important factor affecting soil quality, soil nutrients typically can maintain soil quality and promote plant productivity[36]. During the growth process of Pleurotus ostreatus, due to the need for autotrophic nutrients, enzymes are secreted to mineralize the nutrients in the substrate, such as laccases, cellulases, hemicellulases, and xylanases[37]. Some of these nutrients are absorbed by the mushrooms themselves, while others are left in the substrate, such as carbohydrates, protein, and fat[38]. So, SMS contains different levels of carbon and nitrogen higher than soil, which can improve the storage capacity of soil nutrients when applied as substitutes for fertilizers[39]. The results of our research concluded that the soil application of SMS improved soil fertility since the DOC and available P were increased significantly (Fig. 3). When applied as an alternative fertilizer, the nitrogen in SMS can be slowly released into the soil, which is beneficial for plants to assimilate nitrogen[40]. And SMS is rich in phosphorus, which can be used as a phosphorus additive in soil to increase soil organic matter and nutrient content[41,42]. Besides, the rich organic matter in SMS improves soil structure, soil aeration, and water retention, and even increases soil microbial activity[43]. The increased organic C and available N in soils amended with organic manure would provide more organic resources that benefit the microbial activity in soils[44]. Based on the previous research, four months after the addition of Agaricus bisporus SMS to the soil, organic N and available P content of the soil increased, while the impact on pH is not significant[7], which is consistent with our experimental results. The effectiveness of SMS as an organic soil amendment has also been positively evaluated by Courtney & Mullen[20]. The effect of applying SMS was equivalent to fertilization, where the C, N and P contents in the soil respectively increased by 40%, 28%, and 230%, meanwhile, the calcium, potassium, and magnesium content increased by three times.

      In addition, SMS increased the beneficial microbial biomass in the substrate, which may be one of the reasons why SMS promotes plant growth. A previous study has found that Rhizobia is beneficial to leguminous plants and can effectively fix nitrogen in the air for plant use[45]. Our data showed that the number of nodules increased with the increase of SMS addition, which can be further inferred to increase the effect of nitrogen fixation (Fig. 2). The mutually beneficial relationship between symbionts and host plants is established of based on an 'investment-return' strategy balance, which means that alfalfa uses rhizobia to obtain nutrients while correspondingly spending energy to maintain microbial life[46]. Although the number of nodules in CF treatment was significantly higher than in other treatments (Fig. 2), there was no significant increase in biomass compared with 20% SMS and 30% SMS. It is possible because higher levels of nitrogen or phosphorus would break potential nutrient limitations, allowing host plants to easily obtain essential nutrients for growth through their roots, and plants would invest fewer resources in rhizobia at this point[47]. The MBN and MBC concentrations of adding SMS treatments were significantly higher than no SMS treatments (CK and CF), which indicated that SMS addition promotes microbial biomass, while inorganic fertilizer addition does not have such effects (Fig. 3c & d). Research has shown that the different microbial populations of bacteria and fungi carried by SMS also can degrade organic foreign compounds in soil[48], which may promote plant absorption of chemical fertilizers. Additional research instructed that microorganisms play an important role in soil fertility and nutrient cycling[49]. Therefore, SMS can promote the formation of root nodules, and increase the N content and the biomass of alfalfa.

      Correlation analysis indicated that the soil available P concentration and the number of alfalfa nodules significantly affect the growth of alfalfa (Fig. 4). The shoot biomass of alfalfa was significantly correlated with the soil available P concentration, and MBC and MBN have a strong correlation with DOC content, and DOC is also related to P content (Fig. 5). Alfalfa has high requirements for phosphorus, sulfur, potassium, and some micronutrients, such as boron, and if one nutrient is deficient in soil, crop growth will be poor even if other nutrients are abundant[50]. Therefore, it is inferred that the rich organic carbon and high microbial activity in the SMS improve nutrient cycling within the substrate, and the high P content in the substrate promotes an increase in alfalfa biomass.

    Conclusions

    • The experimental results showed that the shoot and root biomass, DOC, available P, MBC, and MBN concentrations of alfalfa enhanced with the increase of SMS addition in degraded grassland soil. Among them, 20% SMS and 30% SMS treatments had no significant difference in alfalfa biomass compared with CF treatment. 20% SMS treatment had the second highest number of alfalfa nodules only after the CF treatment. Total carbon content in leaves and total nitrogen content in stems of 20% SMS treatment were significantly higher than other treatments. The available P was significantly correlated with shoot biomass, MBC, MBN, and DOC. In conclusion, the effects of 20% SMS treatment on alfalfa growth and soil improvement were superior to other treatments. Therefore, adding 20% SMS to the degraded grassland soil to cultivate alfalfa has a good economic and application prospect, which can improve the yield and quality of alfalfa, promote the recycling of waste and reduce the waste of resources. This study provides a theoretical basis and support for the large-scale application and popularization of this technology in the field.

    Acknowledgments

    • Thanks to the College of Grassland Science and Technology at China Agricultural University for their guidance and assistance in this experiment. Also thanks to the Beijing Academy of Agriculture and Forest Sciences and Duolun Restoration Ecology Research Station, Institute of Botany, and Chinese Academy of Sciences for their material support in this experiment. Thanks to Hengkang Xu, Lu Lian and Bin Wei for their support in this project.

      Conflict of interest

      • The authors declare that they have no conflicts of interest. Yingjun Zhang is the Editorial Board member of Grass Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer review handled independently of this Editorial Board member and his research groups.

      Rights and permissions
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (2) References (50)
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    Shi Y, Cui X, Zhang Y, Liu M. 2023. The addition of spent oyster mushroom substrates has positive effects on alfalfa growth and soil available nutrients. Grass Research 3:19 doi: 10.48130/GR-2023-0019
    Shi Y, Cui X, Zhang Y, Liu M. 2023. The addition of spent oyster mushroom substrates has positive effects on alfalfa growth and soil available nutrients. Grass Research 3:19 doi: 10.48130/GR-2023-0019
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  • Introduction
  • Materials and methods

  • Soil and spent mushroom substrate properties

  • Experimental design

  • Experimental method

  • Data analysis

  • Results

  • Plant biomass and nutrient uptake

  • Substrate physicochemical properties

  • Correlation analysis

  • Discussion
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