A New Species Agrocybe striatipes, also a Newly Commercially Cultivated Mushroom with Highly Nutritional and Healthy Values

The species of Agrocybe (Strophariaceae, Agaricales, Agaricomycetes) are saprophytic and widely distributed in temperate regions. In this study, a new species named Agrocybe striatipes from China is described, which has been successfully cultivated in China recently. The phenotypic characteristics examination and molecular phylogenetic analyses using multilocus data (ITS and nrLSU) both support it as a new species in the genus Agrocybe. Moreover, nutritional ingredient analysis showed that the fruiting body of A. striatipes was rich in seventeen amino acids, including eight essential amino acids, in addition to high levels of calcium (78.5 mg/kg) and vitamin D (44.1 μg/100g). The following analysis of the heavy metal contents of the fruiting bodies show that it does not contain lead, cadmium, arsenic, mercury, and other heavy metal elements. In the crude extract of the mushroom, the nutrients in the aqueous phase are amino acids and oligosaccharides, and the active substances in the ethyl acetate layer are sterols, which have a variety of pharmacological effects. In conclusion, A. striatipes is not only a new species but also has highly application values as a cultivated edible mushroom in nutrition and health.


Introduction
The genus Agrocybe was first named by Fayod in 1889 with the type species A. praecox (Pers.) Fayod. Agrocybe species are highly adaptable and widely distributed in temperate regions of Asia, Europe, and North America [1]. In China, sixteen species/varieties were reported from the Guizhou, Yunnan, Fujian, Sichuan, Jiangsu, Zhejiang provinces, etc. [2,3]. Recently, the outline of Basidiomycota documented c. 100 species in this genus [4]. Members of Agrocybe are saprophytic, usually grow in the forest or grassland, and are characterized by small-to-medium-sized basidiomata, most of them have a membranous ring, brown spore prints, pileipellis a hymeniderm or ixohymeniderm, basidiospores ovoid, ellipsoid or fusiform, yellow to light brown, smooth, usually with a broad germ-pore [5,6].
Singer conducted a comprehensive morphological classification study on Agrocybe, and placed this genus in the family Bolbitiaceae based on characteristics of the pileipellis cell consisting of pear-shaped, subspherical, sphaerocytes cells; the presence or absence of hymenial cystidia; and brown spore prints [7]. Furthermore, he divided this genus into two subgenera, subg. Agrocybe and Aporus; subg. Agrocybe was further divided into five sections (sections Agrocybe, Pediades, Microsporae, Allocystides, and Evelatae), and subg. Aporus was separated into two sections (sections Aporus, Velatae) [7]. Later, the section

Morphological Study
Mushroom specimens were collected in the field after taking photographs. Odor and color changes on bruising were recorded at the same time. Macromorphological features and chemical reactions of fresh specimens were recorded. Specimens were dried completely with a food drier under a temperature of 55 • C overnight. Anatomical and cytological features including lamellae, pileipellis, basidiospores, basidia, and cystidia were observed from dried specimens and following the protocols [17][18][19]. A total of 5% KOH were used for a staining reaction. More than twenty measurements of microscopic features (spores, basidia, and cystidia) were recorded, which included tx, the mean of the length by the width ± SD; Q, the quotient of the basidiospore length to width; and Qm, the mean of the Q-values ± SD [17,20].

Molecular Phylogenetic Study
DNA was extracted from the dried specimens using a Broad-spectrum plant Rapid Genomic DNA Kit (Biomed) according to the manufacturer's protocol. Primers ITS4 and ITS5 were used for internal transcribed spacer (ITS) and LROR and LR5 for large ribosomal subunit (nrLSU) PCR reactions [21]. The PCR programs followed previous studies [19,20,22]. The PCR products were sent to a Biomed Biotechnology commercial company for sequencing.
The sequences produced from this study and some from the NCBI GenBank database were used in phylogenetic analyses [3,8,12,13,23,24] (Table 1). Sequences of multigene data were aligned by Muscle version 3.6 separately [25], then manually adjusted to remove ambiguous regions in BioEdit version 7.0.4 [26]. Maximum likelihood (ML) analysis was performed by RAxmlGUI 1.3 under a GTRGAMMA model with one thousand rapid bootstrap (BS) replicates [27]. Bayesian Inference (BI) analysis was performed by MrBayes v3.2.6 [28]. Six Markov chains were run for 2,000,000 generations and trees were sampled every 100th generation. Burn-ins were determined in Tracer version 1.6 with an ESS value higher than 200, and the remaining trees were used to calculate Bayesian posterior probabilities (PP). The trees were displayed in FigTree version 1.4.0 [29]. Nutrient composition analysis was performed using a completely dried artificially cultivated fruiting body. The amino acid content, polysaccharide content, protein content, ash content, mineral content, fat content, energy, and carbohydrate were determined by sending it to the Analysis and Testing Center of Sichuan Academy of Agricultural Sciences. An automatic amino acid analyzer (L-8800) and atomic-fluorescence photometer (AFS3000) were used to assay the amino acid and mineral contents. All determinations are under China's National Food Safety Standard System, respectively [30][31][32][33][34][35]. A comparative analysis of the nutrient compositions of this proposed new species and eight reported cultivated species, namely A. praecox, C. cylindracea, C. salicacicola, Pleurotus placentodes (Rumph. ex Fr.) Boedijn, P. ostreatus (Jacq.) P. Kumm., Lentinus edode (Berk.) Pegler, Flammulina velutipes (Curtis) Singer, and Agaricus bisporus (J.E. Lange) Imbach, was conducted.

Heavy Metal Content Analysis
The artificially cultivated fruiting body was detected for heavy metal content. Pb (Lead), Cd (Cadmium), Hg (Mercury), and As (Arsenic) were determined by sending them to the Analysis and Testing Center of Sichuan Academy of Agricultural Sciences. An atomic fluorescence photometer (AFS3000) was used in the analysis based on the atomic fluorescence spectrometric method, and all determinations were performed according to the corresponding China National Food Safety Standard [36].

Metabolites Analysis
The target compound was purified by HPLC (Agilent 1200), which was performed on C 18 column (4.6 mm × 250 mm, 5 µm) with a flow rate of 1.0 mL/min. The mobile phase consisted of methanol (A) and water containing 0.01% formic acid (B). A gradient elution program was set as follows: 0-5 min, 5% A; 5-40 min, 5-100% A. The sample injection volume was 10 µL and the column temperature was set at 25 • C. Its structure was determined by 1 H and 13 C NMR data analysis.

Phylogenetic Results
Ninety-five samples were included in this multigene phylogenetic analysis, and it contained 50 Agrocybe samples representing 23 species; the genus Cyclocybe was set as the outgroup. The overall topologies of the ML and BI trees did not show a difference, and the ML tree is shown in Figure 1. Among them, six ITS sequences and six nrLSU sequences were newly generated for this study. It is noticeable that Agrocybe was not a monophy-letic group, and all Agrocybe species split into four clades, which correspond with a previous study by Kiyashko [8]. The proposed new species A. striatipes was nested in the Clade I, which comprised the type species of this genus, and it also showed its sister relationship with the known species A. letic group, and all Agrocybe species split into four clades, which correspond with ous study by Kiyashko [8]. The proposed new species A. striatipes was nested in th I, which comprised the type species of this genus, and it also showed its sister rela with the known species A.    There are pure-white fruiting bodies that occurred in the cultivated yard and mixed with those in brown. Due to the fact that they have the same morphological characters except for the color and identical sequences, they were identified as the same species. In Agaricus and some other mushroom genera, these white variants are very frequent in many species, such as Ag. bisporus, Ag. subrufescens [40,41].

Evaluations of Nutrition and Food Security of A. striatipes
The nutrient content of A. striatipes was analyzed from its dry materials, and the tails are shown in Table 2. The protein content of A. striatipes was 5.66 g/100 g, and content was nearly three times as much as those of A. praecox. The richness of calcium (7 mg/kg) and zinc (6.87 mg/100 g) is beneficial to the elderly and growing children; in dition, trace amounts of selenium have been detected, which is beneficial to the norm physiological activities of the human body. Furthermore, the cultivated specimens w also rich in vitamins, with a vitamin D content of 44.1 µg/100 g. Vitamin C and other f soluble vitamins were not detected within the limit of quantification.
As shown in Table 3, a comparative analysis of amino acid content with another eig common edible mushrooms was conducted. Our analysis revealed that this species is r in 17 amino acids; the total amino acid is 19.24 g/100 g, including the eight essential am acids required by the human body. In addition, we have detected gamma-aminobuty acid, which was not reported in others. The ratios of essential and non-essential am acids indicated that these two were in equal amounts.  Notes: The new species A. striatipes is characterized by its yellowish ochraceous-brown pileus, stipe deeply striate-sulcate with somewhat fibril, relatively larger pleurocystidia and cheilocystidia, and smaller basidiospores than other species. In the phylogenetic tree (Figure 1), A. striatipes and A. smithii formed a distinct lineage and separated from the other Agrocybe species. However, A. smithii can be easily distinguished by a fresh cap with olive shades and having bigger basidiospores (11-13.5 × 6.5-8 µm) [37]. A. allocystis and A. striatipes are very similar in the field in terms of morphological characteristics; however, the former has much bigger basidiospores (10-16 × 7-10.5 µm) and is different in pleurocystidia and cheilocystidia shapes, which were ventricose to lageniform, and apically usually sub capitate to capitate [6]. Another morphologically similar species is A. broadwayi, which differs from A. striatipes by its bigger basidiospores (12-15 × 8-9 µm) and margin of pileus usually striated, occasionally covered with small concolorous squamules [38]. A. retigera resembles A. striatipes too because they both have umbonate, yellow-cream colored to pale brownish pileus surfaces; however, this known species' heavy lacunose-rugose will disappear in older ones, and it possesses bigger basidiospores (11.5-18 × 7-10 µm) with a double wall [39].
There are pure-white fruiting bodies that occurred in the cultivated yard and mixed with those in brown. Due to the fact that they have the same morphological characters except for the color and identical sequences, they were identified as the same species. In Agaricus and some other mushroom genera, these white variants are very frequent in many species, such as A. bisporus, A. subrufescens [40,41].

Evaluations of Nutrition and Food Security of A. striatipes
The nutrient content of A. striatipes was analyzed from its dry materials, and the details are shown in Table 2. The protein content of A. striatipes was 5.66 g/100 g, and its content was nearly three times as much as those of A. praecox. The richness of calcium (78.5 mg/kg) and zinc (6.87 mg/100 g) is beneficial to the elderly and growing children; in addition, trace amounts of selenium have been detected, which is beneficial to the normal physiological activities of the human body. Furthermore, the cultivated specimens were also rich in vitamins, with a vitamin D content of 44.1 µg/100 g. Vitamin C and other fat-soluble vitamins were not detected within the limit of quantification.
As shown in Table 3, a comparative analysis of amino acid content with another eight common edible mushrooms was conducted. Our analysis revealed that this species is rich in 17 amino acids; the total amino acid is 19.24 g/100 g, including the eight essential amino acids required by the human body. In addition, we have detected gamma-aminobutyric acid, which was not reported in others. The ratios of essential and non-essential amino acids indicated that these two were in equal amounts.  The analysis results of heavy metal contents are detailed in Table 4, and the results show that the artificially cultivated fruiting body did not contain lead, cadmium, arsenic, mercury, or other heavy metal elements, and their contents were within the standard limits.

The Bioactive Ingredients of A. striatipes
The dry encarpium (200 g) was extracted repeatedly with 95% ethanol 500 mL three times, and the organic solvent was evaporated to dryness under a vacuum to afford the crude extract (3.2 g), which was distributed between water and EtOAc to afford the two fractions. They were analyzed by HPLC. The water fraction was dissolved in DMSO-d6 and analyzed by 1 H NMR, which revealed that the water fraction mainly contained oligosaccharides and amino acids (Appendix A Figure A1). We found that there was a main compound with a retention time of 40.0 min; other peaks with similar retention times have the same UV absorption, which were as shown in Figure 4. The main compound (3.0 mg) was purified by the same HPLC condition. It was identified as ergosterol by an analysis of 1 H and 13 C NMR contrast the reference (Appendices B and C Figure A3) [42]. Ergosterol, the major product of mycosterol biosynthesis, is an important component of fungal cell membranes that maintain membrane structural integrity, permeability, and fluidity, which can promote the absorption of calcium and phosphorus in the human body. It is one of the good sources of exogenous vitamin D2 in the human body and is often used to improve rickets, osteomalacia, and osteoporosis caused by vitamin D deficiency in infants and elderly people [43].
J. Fungi 2023, 9, x FOR PEER REVIEW 11 of 15 mercury, or other heavy metal elements, and their contents were within the standard limits.

The Bioactive Ingredients of A. striatipes
The dry encarpium (200 g) was extracted repeatedly with 95% ethanol 500 mL three times, and the organic solvent was evaporated to dryness under a vacuum to afford the crude extract (3.2 g), which was distributed between water and EtOAc to afford the two fractions. They were analyzed by HPLC. The water fraction was dissolved in DMSO-d6 and analyzed by 1 H NMR, which revealed that the water fraction mainly contained oligosaccharides and amino acids (Appendix A Figure A1). We found that there was a main compound with a retention time of 40.0 min; other peaks with similar retention times have the same UV absorption, which were as shown in Figure 4. The main compound (3.0 mg) was purified by the same HPLC condition. It was identified as ergosterol by an analysis of 1 H and 13 C NMR contrast the reference (Appendix B Figure A2 and Appendix C Figure  A3) [42]. Ergosterol, the major product of mycosterol biosynthesis, is an important component of fungal cell membranes that maintain membrane structural integrity, permeability, and fluidity, which can promote the absorption of calcium and phosphorus in the human body. It is one of the good sources of exogenous vitamin D2 in the human body and is often used to improve rickets, osteomalacia, and osteoporosis caused by vitamin D deficiency in infants and elderly people [43].

Discussion
Although species of the generas Agrocybe and Cyclocybe are morphologically similar, the molecular phylogenetic analysis revealed that Cyclocybe, which is represented by the widely cultivated C. cylindracea complex (=A. cylindracea), belongs to Tubariaceae. However, the phylogenetic study that used multigene sequences of Strophariaceae, Tubariaceae, and other selected Hymenogastraceae species showed that Agrocybe are in fact phylogenetically distant from Cyclocybe and belong to Strophariaceae [24]. In morphology,

Discussion
Although species of the generas Agrocybe and Cyclocybe are morphologically similar, the molecular phylogenetic analysis revealed that Cyclocybe, which is represented by the widely cultivated C. cylindracea complex (=A. cylindracea), belongs to Tubariaceae. However, the phylogenetic study that used multigene sequences of Strophariaceae, Tubariaceae, and other selected Hymenogastraceae species showed that Agrocybe are in fact phylogenetically distant from Cyclocybe and belong to Strophariaceae [24]. In morphology, species of Agrocybe possess a broad germ-pore; however, Cyclocybe species rarely possess germ-pore [6,12].
In this study, a multi-gene phylogenetic analysis was carried out based on 95 specimens, including 50 specimens of 23 Agrocybe species. Phylogenetic results enable the division of the current Agrocybe into four main clades, which coincided with previous research [8,12,13]. Clade I contains the proposed new species and the type species A. praecox, as well as most of the Agrocybe species. Clade II contains only species A. arvalis, which is nested at the sister position with Clade I without statistics supporting the values. The Clade IV is located at the base of the phylogenetic tree and comprises five strongly supported species (97 BS/1 PP). Regardless, the molecular analysis supports A. striatipes as a new species, and this is also supported by its morphological characteristics. To resolve the taxonomic problem of polyphyletic Agrocybe, the taxonomic system for those related taxa may change in the future. However, no matter how the taxonomic system changes, A. striatipes as belonging to Agrocybe will not change due to it being closely clustered with type species A. praecox within clade I in the phylogeny (Figure 1) [8].
Currently, eight edible species of Agrocybe were reported in China [44], and only A. praecox was successfully domesticated and cultivated. The new species A. striatipes introduced from this study is the second species of Agrocybe that can be capable of cultivation. The results of the nutrient analysis revealed that the cultivated A. striatipes can be used as a nutritious food high in proteins. As shown in Table 2, the protein content of A. striatipes is about 5.66 g/100 g, which is significantly higher than that of A. praecox. In addition, it is rich in polysaccharides. Further analysis of polysaccharide extraction and identification of components can be carried out to develop the medicinal value of its polysaccharide active substances and other components. A. striatipes is also rich in mineral elements, with a calcium content (78.5 mg/kg) significantly higher than that of A. praecox. Meanwhile, A. striatipes substrates are rich in Vitamin B2 (0.33 mg/100 g), and B vitamins are mainly involved in bio-oxidation and metabolism in the form of coenzymes, which have very important physiological functions. From the results of the amino acid content, A. striatipes is rich in amino acids, with a total amino acid content of 4.14 g/100 g, which is significantly higher than that of A. praecox, and GLU is the highest amino acid content, accounting for 18%. However, compared with other common edible mushrooms, the total amino acids of A. striatipes are not high, but the ratio of essential amino acids to total amino acids is comparable to other edible mushroom species.
In the crude extract of the mushroom, the nutrients in the aqueous phase are amino acids and oligosaccharides, and the active substances in the ethyl acetate layer are sterols. Ergosterol is the most common class of active substances in fungi. Ergosterol has a variety of pharmacological effects, such as strengthening the immune system, anti-inflammatory and pain-relief properties, lowering cholesterol, anti-fibrosis, anti-oxidant, and delaying aging [45,46].

Conclusions
This study, which involved multiple DNA gene-fragment analyses in combination with morphological analysis, revealed that A. striatipes is highly supported as a new species. The success of its artificial cultivation proved that it was the second species in the genus that can be successfully cultivated. Furthermore, the evaluations of its nutrition, food security, and bioactive ingredients indicated that it could be a healthy food for human beings. Compared with A. praecox, A. striatipes produces higher levels of proteins and amino acids and is richer in calcium, zinc, and vitamin D contents, which especially make it more suitable for the elderly and growing children. In the daily diet structure, we recommend combining A. striatipes with other edible mushrooms, vegetables, or meats, which can provide a complementary balance of amino acids and mineral elements.  Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.