Screening and Characterization of Wild Sarcomyxa edulis Strains from Heilongjiang, China, for Strain Development

Abstract

Sarcomyxa edulis is a characteristic low-temperature, edible mushroom in Northeast China. It has a delicious taste and rich nutritional and medicinal value. The artificial cultivation of S. edulis has been increasing in recent years. However, the number of S. edulis varieties is scarce, and strain degradation is a serious issue, affecting the yield and quality of S. edulis. Therefore, we collected 21 wild strains of S. edulis (Y1–21) in this study, aiming to develop strains of S. edulis. Five strains without antagonistic reaction were eliminated via the antagonism test, and the remaining sixteen strains were identified as S. edulis using internal transcribed spacer (ITS) marker identification. The mycelial growth rate, mildew resistance, fruiting body yield, agronomic traits, and nutrient content of the 16 strains were determined. The results demonstrate that Y12, Y13, Y14, and Y15 exhibited a rapid mycelial growth rate (6.43–6.8 mm·day⁻¹). Their colony density was moderate; their edges were neat; and their colonies were leathery and had obvious pigmentation. Moreover, they exhibited strong mildew resistance and a low Trichoderma contamination rate (<40%). Their fruiting body yield (281.15–342.03 g) and biological efficiency (56.23–68.40%) were high. Their fruiting body shape was good. Their polysaccharide and crude protein contents were higher, while their crude fiber, ash, and crude fat contents were lower. Overall, these four S. edulis strains exhibited high yield, excellent traits, and good quality for commercial production and food production with high nutrient contents. This study provides a foundation for the further cross-breeding and matrix improvement of S. edulis.

1. Introduction

Sarcomyxa edulis (also called delicious mushroom, frozen mushroom, “huanglinggu”, “huangmo”, etc.) belongs to the order Agaricales, family Mycenaceae, and genus Sarcomayxa. It is a characteristic low-temperature, edible mushroom in Northeast China [1,2,3]. S. edulis is delicate; fragrant; delicious [4,5]; and rich in nutrients such as proteins, polysaccharides, and amino acids [6]. Moreover, it exhibits therapeutic properties and can be used to strengthen muscles and bones, treat gas problems, and promote blood circulation [4,5,6]. S. edulis polysaccharides have antitumor, antiradiation, immunity-improving, and other effects that give this species a high medicinal value [7,8,9].

Most research on S. edulis is focused on the mechanisms of growth and development and the biological effects of polysaccharides. For example, Tian et al. [3] sequenced the whole genome of S. edulis. Many annotated protein-coding genes in S. edulis’s genome are associated with its pharmacodynamic activity. Phylogenetic and comparative analyses of Basidiomycota based on single-copy homologous proteins demonstrated that Sarcomyxa is an independent group evolved from the family Pleurotacae. Duan et al. [5] conducted transcriptome analysis during the six developmental stages of S. edulis, clarified its genetic background, and screened the key genes at various developmental stages. Some studies reported that polysaccharides from S. edulis exhibit various biological activities, such as intestinal health protection, antioxidation, and blood sugar reduction [10,11,12].

In China, S. edulis is artificially cultivated on a large scale in bags containing a sterile substrate (oak sawdust, wheat bran, and gypsum) in greenhouses. The cultivation area is concentrated in the northeast region. With the continuous increase in market demand in China in recent years, the yield of S. edulis is increasing, and the industrial scale is rapidly expanding. For example, according to incomplete statistics, S. edulis cultivation increased from approximately 200,000 bags to 300,000 bags in Heilongjiang Province between 2021 and 2023. Moreover, the total fresh weight of S. edulis was estimated to be from approximately 5200 kg to 7800 kg during this period. The average fresh weight of S. edulis in each bag was 260 g, each containing a dry substrate weight of 500 g. S. edulis is very popular in China. The market price of fresh S. edulis is 20–26 RMB/kg, and the cost per bag is RMB 2–2.5, with considerable economic benefits. S. edulis is not currently exported, and no S. edulis species have been reported to be artificially cultivated outside China. However, due to the scarcity of existing S. edulis strains and long-term cultivation [13], strain degradation (Figure 1) in the subculture process has been seriously affecting the yield and quality of S. edulis and causing huge losses to farmers. In this study, we aimed to screen S. edulis strains exhibiting high yield, excellent traits, and good quality. In total, 21 wild S. edulis strains were collected from a field and artificially domesticated. S. edulis strains suitable for artificial cultivation and exhibiting excellent traits at this stage were screened using ITS marker identification, agronomic trait evaluation, and nutrient content analysis [14]. This study provides a reference for S. edulis strain development and food production by screening S. edulis strains with high nutrient contents.

2. Materials and Methods

2.1. Sample Collection, Taxonomic Identification, Mycelial Culture, and Antagonism Test

In total, 21 wild strains of S. edulis were collected in Heilongjiang Province, China (121°11′–135°05′ E, 43°26′–53°33′ N) from 2013 to 2020 and artificially domesticated. They were stored at the Institute of Edible Fungi, Mudanjiang Branch, the Heilongjiang Academy of Agricultural Sciences (Mudanjiang, China), and named Y1–21.

The fruiting bodies of the wild strains (the clustered morphology of the fruiting body, the pileus color, and the pileus length) were observed and recorded, alongside the morphology of the hyphae (diameter and presence of clamps), via macroscopic observations. The tissue blocks between pileus and fold of the fruiting bodies of the wild strains were collected and quickly transferred to potato dextrose agar (PDA; 200 g L⁻¹ diced potatoes, 20 g L⁻¹ glucose, and 15 g L⁻¹ agar) medium at 25 °C under sterile conditions.

The pure culture was inoculated on a medium consisting of a filtrate collected after boiling 40 g of oak sawdust, 200 g of potato, 0.5 g of peptone, 20 g of glucose, 20 g of agar, and distilled water at a volume of 1000 mL. The antagonism test was performed as described in [15]. The antagonism of one strain was tested against six other strains on one culture dish. One strain was inoculated at the center of each plate, and an equal amount of the other six strains was inoculated at the corresponding position on the culture dish. The plates were incubated at 25 °C. The experiment was performed in triplicate. The growth was observed and recorded for approximately 10 days.

2.2. Molecular Identification of Sarcomyxa edulis Strains

The strains that did not exhibit antagonistic reactions were eliminated. DNA was extracted from the mycelium of the remaining strains using the Fungi Genomic DNA Extraction Kit (Beijing Solarbio Science, Beijing, China) as per the manufacturer’s instructions. Two universal primers—ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′)—were used to amplify the internal transcribed spacer (ITS) region [16,17]. The extracted samples were sent to Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) for ITS sequencing. The spliced sequences were compared in NCBI using BLAST search.

2.3. Cultivation of S. edulis Strains on Spawn Medium

Fresh raw material without mildew was selected, weighed, and evenly mixed to prepare the spawn medium (86% oak sawdust, 13% wheat bran, and 1% gypsum). Its water content was 50–55%. An amount of 500 g of the dry spawn medium was placed in each polypropylene bag (17 cm diameter × 35 cm height × 0.05 mm thickness) and subjected to high-pressure sterilization at 125 °C for 3 h. Furthermore, the bags were cooled to a temperature below 30 °C, inoculated (10–15 g of spawn per bag) with the mycelia of the strains under sterile conditions, and immediately transferred to an incubation room with a temperature of 22–25 °C. In total, 60 bags were used for each strain. The experiment was repeated 3 times.

The bags with the mycelia were transferred to a greenhouse and kept on the ground. The cotton plug at the top of the bag was removed while the mushrooms were growing, and the bag was opened using a knife when mushroom buds were formed. The incubation temperature was maintained at 10–20 °C during the fruiting period, and the air relative humidity was 85–90%.

2.4. Mycelial Morphology

The strains identified using ITS marker identification were inoculated on the mycelia culture medium plates and cultured at 25 °C for 14 days. The mycelial morphology was recorded, including the colony density (high, moderate, or low), degree of aerial mycelial development (strong, moderately strong, or weak), uniformity of the colony edge (neat or uneven), presence of leathery colony, and colony pigmentation.

2.5. Determination of Mycelial Growth Rate and Resistance to Trichoderma Contamination

The tested strains were inoculated in a sterile spawn medium. Each strain was inoculated in 10 bags. Mycelial germination was observed after 5 days of inoculation. The mycelial growth rate was recorded every 3 days. The time required for the mycelia to fill the bags was recorded.

Ten bags were used for each strain. The culture conditions were the same as described in Section 2.4. Trichoderma was inoculated when the mycelia grew to fill up half of the bag. The incubation was continued, and the number of bags with Trichoderma growth was recorded.

2.6. Agronomic Characteristics of Various S. edulis Strains

Nine agronomic characteristics of the fruiting bodies of the various strains were assessed (

Table 1. Assessment of nine agronomic traits of Sarcomyxa edulis.

Agronomic Traits	Observation
Clustered morphology of fruiting body	Stacked-up; overturned tile
Shape of pileus	Sector; mytiliform
Uniformity of color of pileus	Nonuniform; uniform
Color of pileus	Yellow; yellowish-brown; yellowish-green
Pileus edge	Smooth; wavy; incision
Number of fruiting bodies	Small; medium; large
Length–width ratio of pileus	Small; middle; big
Pileus thickness	Thin; middle; thick
Time from inoculation to primordium formation	Short; moderate; long

). The phenotypic traits included the clustered morphology of the fruiting body, pileus traits, pileus color uniformity, pileus color, pileus edge, fruiting body number, pileus length–width ratio, pileus thickness, and time from inoculation to primordium formation [18].

2.7. Determination of Biological Efficiency

S. edulis strains were cultivated as described in Section 2.3. The fruiting bodies were harvested once at the mature stage. The fresh weight (g) of the fruiting bodies in each bag was calculated to assess the average yield and biological efficiency (BE).

The biological efficiency (BE) was calculated as shown in Equation (1):

BE (%) = Ffw/Sdw × 100%

(1)

where Ffw and Sdw are the average fresh mushroom weight and the weight of the substrate dry matter per bag (g), respectively [19].

2.8. Nutrient Contents in Fruiting Bodies

The contents of polysaccharides, crude protein, crude fiber, ash, and crude fat in the fruiting bodies were determined. The polysaccharides were extracted using water extraction and alcohol precipitation [20], and their content was determined using the phenol–sulfuric acid method [21]. The crude protein content was determined using the Kjeldahl method [22]. The crude fiber and ash contents were determined according to the method described by the official Association of Analytical Chemists (AOAC 2000). The crude fat content was determined via continuous extraction using a Soxhlet apparatus [23].

2.9. Statistical Analysis

Data were expressed as the averages of three replicates and collated using Microsoft Excel 2021. SPSS 20 was used to analyze data such as the mycelial growth rate and the yield and agronomic traits of the fruiting bodies. A phylogenetic tree was constructed using MAGA 5.0. GraphPad Prism10 was used to analyze the nutrient content, BE, and uniformity of the fruiting bodies with heatmap clustering analysis. A test was performed to assess the significance of differences between groups. p < 0.05 was considered significant.

3. Results

3.1. Taxonomic Identification and Antagonism Test

The characterization of the wild strains included the analysis of the clustered morphology of the fruiting bodies (stacked-up or overturned tile, no or short), and the pileus shape (sector or mytiliform). The pileus length was approximately 3–20 cm. The hyphae length was approximately 4–13 µm. There were obvious clamps with branches, and some of the middle part was slightly thick (Figure 2).

Somatic incompatibility in basidiomycetes refers to ineffective fusion (allorejection) due to allogeneic recognition between basidiomycetes with different genetic characteristics. When the mycelia are in contact, an incompatible reaction occurs in the contact area, such as pigment formation, dense hyphae uplift, or sparse hyphae, known as antagonism [24,25,26]. The results of the antagonism test of the 21 strains are shown in

Table 2. Antagonism test results of the strains.

Strain	Y1	Y2	Y3	Y4	Y5	Y6	Y7	Y8	Y9	Y10	Y11	Y12	Y13	Y14	Y15	Y16	Y17	Y18	Y19	Y20	Y21
Y1		0	0	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++
Y2			0	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++
Y3				++	+	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	++
Y4					++	++	++	++	++	++	++	++	++	++	++	++	++	++	++	0	++
Y5						++	++	++	++	++	+	++	++	+	++	++	++	++	++	++	++
Y6							0	++	++	++	++	++	++	+	++	++	++	++	++	++	++
Y7								++	++	++	++	++	++	++	++	++	++	++	++	++	++
Y8									+	++	++	++	0	++	++	++	++	++	++	++	++
Y9										++	++	+	+	+	++	++	++	++	++	++	++
Y10											++	++	++	++	++	++	++	++	++	++	++
Y11												++	++	++	++	++	++	++	++	++	++
Y12													++	++	++	++	++	++	++	++	++
Y13														++	++	++	++	++	++	++	++
Y14															++	++	++	++	++	++	++
Y15																++	++	++	++	++	++
Y16																	++	++	++	++	++
Y17																		++	++	++	++
Y18																			++	++	++
Y19																				++	++
Y20																					++
Y21

Note: ++—the antagonism was obvious; +—the antagonism was not obvious; 0—no antagonism.

. If two strains do not possess antagonism, this may indicate that they are the same strain. Therefore, the strains with antagonistic reactions were screened for further study. No antagonistic reaction was observed between Y1, Y2, and Y3; Y4 and Y20; Y6 and Y7; and Y8 and Y13. Obscure antagonistic reactions were observed between Y5 and Y11; Y5 and Y14; Y6 and Y14; Y9 and Y12; Y9 and Y13; and Y9 and Y14. Antagonistic reactions were observed between other strains. A total of 16 strains were screened for further experiments, comprising Y3, Y5, Y6, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, and Y21 (Figure 3).

3.2. ITS Marker Identification

The ITS fragments obtained by sequencing and splicing the 16 strains screened after the antagonism test were compared with those in the NCBI Genbank. A phylogenetic tree was constructed with Lentinula edodes as the outgroup and S. edulis (LC150017.1, AB819088.1, MZ158699.1, MH747088.1, and AB819095.1) and Passiflora edulis (GQ219730.1) as the reference strain (Figure 4). The 16 ITS sequences obtained in this experiment were clustered into one branch with the reference strain, and the homology was more than 99%. An outgroup was clustered into one branch, and these strains were determined to be S. edulis.

3.3. Mycelial Morphologies of Various S. edulis Strains

As shown in Figure 5 and

Table 3. Mycelial morphologies of various S. edulis strains.

Strain	Colony Density	Degree of Aerial Mycelial Development	Colony Edge Uniformity	Leathery Colony	Obvious Colony Pigmentation
Y3	High	Moderately strong	Neat	No	No
Y5	Medium	Moderately strong	Neat	Yes	Yes
Y6	High	Strong	Uneven	No	No
Y9	High	Strong	Neat	No	No
Y10	Medium	Weak	Uneven	Yes	Yes
Y11	Low	Weak	Uneven	Yes	Yes
Y12	Medium	Weak	Neat	Yes	Yes
Y13	Medium	Moderately strong	Neat	Yes	Yes
Y14	Medium	Weak	Neat	Yes	Yes
Y15	Medium	Weak	Neat	Yes	Yes
Y16	Medium	Weak	Uneven	Yes	Yes
Y17	High	Strong	Uneven	No	No
Y18	High	Moderately strong	Neat	No	No
Y19	Medium	Weak	Uneven	Yes	Yes
Y20	Medium	Weak	Neat	Yes	Yes
Y21	High	Weak	Neat	Yes	Yes

, the mycelial morphology of the various strains differed. Overall, 6, 9, and 1 strain had high, medium, and low colony densities, respectively; 3, 4, and 9 strains exhibited strong, moderate, and weak aerial mycelial development, respectively; 10 and 6 strains had neat and uneven colony edges, respectively; 11 and 5 strains did and did not exhibit leathery colonies, respectively; and 11 and 5 strains did and did not exhibit obvious colony pigmentation, respectively.

3.4. Average Daily Growth Rates of Mycelia of Various S. edulis Strains

The mycelial growth rates of the various strains in the sawdust medium differed [(5.6 ± 0.10) to (6.8 ± 0.10) mm·day⁻¹] (Figure 6). Y12, Y15, and Y13 exhibited significantly faster mycelial growth (6.6–6.8 mm·day⁻¹) than the other strains, but no significant difference was observed between these three strains. Y3 exhibited the lowest mycelial growth rate (5.6 mm·day⁻¹).

3.5. Correlation Analysis between Trichoderma Contamination Rate and Mycelial Growth Rate

A correlation was observed between the Trichoderma contamination rate and the mycelial growth rate of the various S. edulis strains (Figure 7). The 16 strains could be divided into three groups according to the Trichoderma contamination rate. The first group contained six strains (Y13, Y17, Y6, Y10, Y14, and Y18) with a Trichoderma contamination rate of <20% and an average mycelial growth rate of 6.27 mm·day⁻¹, which was 1.62% and 5.03% faster than the second and third groups, respectively. The second group contained eight strains (Y5, Y9, Y12, Y15, Y16, Y19, Y20, and Y21) with a Trichoderma contamination rate of 30–40% and an average mycelial growth rate of 6.17 mm·day⁻¹. The third group contained two strains (Y11 and Y3) with a Trichoderma contamination rate of 60–70% and an average mycelial growth rate of 5.97 mm·day⁻¹.

3.6. Analysis of Fruiting Body Yield

The fruiting body yields of the various strains were 180.57–342.02 (average 230.62) g per bag (

Table 4. Fruiting body yields and biological efficiencies of various S. edulis strains.

Strain	Yield (g)	Biological Efficiency (%)
Y3	180.57 ± 5.76 e	36.11 ± 1.21 e
Y5	186.32 ± 6.18 cde	37.26 ± 1.37 cde
Y6	205.07 ± 4.96 cde	41.01 ± 0.99 cde
Y9	215.13 ± 5.17 cde	44.03 ± 1.03 cde
Y10	195.05 ± 6.81 cde	39.01 ± 0.78 cde
Y11	221.22 ± 6.34 cde	44.24 ± 1.86 cde
Y12	306.03 ± 3.70 ab	61.21 ± 1.11 ab
Y13	342.02 ± 4.43 a	68.40 ± 0.74 a
Y14	281.15 ± 5.56 b	56.23 ± 1.12 b
Y15	294.12 ± 5.62 b	58.82 ± 1.88 b
Y16	230.93 ± 7.13 cd	46.19 ± 1.42 cd
Y17	197.98 ± 6.46 cde	39.60 ± 1.69 cde
Y18	207.03 ± 3.56 cde	41.41 ± 0.71 cde
Y19	236.32 ± 4.04 c	46.32 ± 1.03 c
Y20	181.90 ± 5.32 de	36.38 ± 1.06 de
Y21	209.07 ± 3.92 cde	41.81 ± 0.78 cde

Note: Various lowercase letters indicate significant differences (p < 0.05).

). Y13 and Y12 exhibited the highest yields (342.03 and 306.03 g, respectively, 32.70–48.30% higher than the average yield), exhibiting no significant difference between them. Y15 and Y14 exhibited yields of 294.12 and 281.15 g, respectively, which were 21.91–27.53% higher than the average yield. Y3 exhibited the lowest yield (180.57 g). The BE of all strains was 36.11–68.40% (average: 46.12%). Y13 and Y12 exhibited the highest BE values (68.40% and 61.21%, respectively, 15.09–22.28% higher than the average BE), with no significant difference between them. The BE values of Y15 and Y14 were 58.82% and 56.23%, respectively, which were 10.11–12.70% higher than the average BE. Y3 exhibited the lowest BE (36.11%).

3.7. Cluster Analysis of Agronomic Traits of Fruiting Bodies

A cluster analysis of nine agronomic traits of the various S. edulis strains could more intuitively assess the agronomic differences (Figure 8 and Figure 9). The 16 strains of S. edulis could be divided into two categories at a Euclidean distance of five. The first type included nine strains (Y13, Y14, Y18, Y19, Y5, Y20, Y11, Y12, and Y16). Combined with the fruiting body morphological observations, the common characteristics of the first type of fruiting body were a wavy pileus edge; a mostly yellow pileus color; a large pileus size; hypertrophic flesh; and a short time from inoculation to primordium formation. The second type included seven strains of S. edulis (Y10, Y15, Y3, Y6, Y9, Y17, and Y21). The common characteristics of the second type of fruiting body were a smooth pileus edge; a mostly yellowish-brown pileus color; a large number of fruiting bodies; a moderate pileus size; and a long time from inoculation to primordium formation.

3.8. Heatmap of the BE, Uniformity, and Nutrient Contents of Fruiting Bodies

After data standardization, the BE, uniformity, and nutrient contents of the fruiting bodies (

Table 5. The results of the nutrient contents in the fruiting bodies.

Strain	Polysaccharide (g/100 g)	Crude Protein (g/100 g)	Crude Fiber (%)	Ash (%)	Crude Fat (g/100 g)
Y3	6.12 ± 0.04 e	15.33 ± 0.06 g	3.65 ± 0.02 e	7.65 ± 0.08 a	24.10 ± 0.1 a
Y5	7.55 ± 0.07 cd	13.41 ± 0.03 i	5.62 ± 0.04 b	6.32 ± 0.06 ab	23.20 ± 0.2 b
Y6	6.98 ± 0.04 de	14.32 ± 0.09 h	2.41 ± 0.01 g	5.83 ± 0.04 ab	20.30 ± 0.3 cd
Y9	6.35 ± 0.03 e	16.32 ± 1.01 fg	6.65 ± 0.04 a	6.01 ± 0.06 ab	20.30 ± 0.1 cd
Y10	6.88 ± 0.08 de	15.78 ± 0.05 fg	4.12 ± 0.05 d	5.78 ± 0.05 bc	19.70 ± 0.3 de
Y11	7.42 ± 0.06 cd	16.95 ± 0.06 ef	3.67 ± 0.06 e	7.03 ± 0.08 ab	19.50 ± 0.2 de
Y12	8.03 ± 0.05 c	18.02 ± 0.07 d	5.32 ± 0.03 bc	5.66 ± 0.02 bc	22.00 ± 0.2 cd
Y13	7.96 ± 0.04 cd	20.65 ± 0.04 b	4.12 ± 0.06 d	5.34 ± 0.06 c	15.50 ± 0.3 f
Y14	8.86 ± 0.07 a	19.24 ± 0.09 c	3.65 ± 0.03 e	5.44 ± 0.08 bc	15.60 ± 0.1 f
Y15	8.45 ± 0.04 b	22.64 ± 0.06 a	2.98 ± 0.07 f	5.81 ± 0.05 bc	17.40 ± 0.4 e
Y16	6.07 ± 0.05 e	17.02 ± 0.04 ef	4.66 ± 0.04 c	6.98 ± 0.04 ab	20.10 ± 0.2 cd
Y17	7.46 ± 0.06 cd	16.36 ± 0.06 ef	5.13 ± 0.12 bc	7.05 ± 1.11 c	19.80 ± 0.3 de
Y18	7.32 ± 0.03 cd	15.46 ± 0.02 fg	3.48 ± 0.05 ef	7.22 ± 0.08 ab	17.90 ± 0.1 e
Y19	7.69 ± 0.07 cd	16.48 ± 0.07 ef	6.54 ± 0.03 a	7.43 ± 0.06 ab	22.40 ± 0.2 c
Y20	7.77 ± 0.05 cd	17.23 ± 1.02 de	2.87 ± 0.04 f	6.64 ± 0.05 ab	21.30 ± 0.3 c
Y21	7.04 ± 0.06 de	16.45 ± 0.06 ef	4.35 ± 0.11 cd	7.15 ± 0.04 ab	20.60 ± 0.2 cd

Note: Various lowercase letters indicate significant differences (p < 0.05).

) of the various strains were analyzed using a heatmap cluster analysis, which could more intuitively compare the differences between the various S. edulis strains (Figure 10). The 16 strains could be divided into three categories. The first category included Y12, Y13, Y14, and Y15; the second included Y6, Y11, Y16, and Y21; and the third included Y3, Y5, Y9, Y10, Y17, Y18, Y19, and Y20. The first category exhibited a high BE; a neat uniformity of the fruiting body edge; high polysaccharide and crude protein contents; and low crude fiber, ash, and crude fat contents. The second category exhibited a moderate BE; a neat uniformity of fruiting body edge; low polysaccharide, crude protein, and crude fiber contents; and high ash and crude fat contents. The third class generally exhibited a low BE; an irregular edge of the fruiting body; and a low crude protein content.

4. Discussion

The wild resources of S. edulis are distributed in the eastern mountainous areas of Northeast China; the regional distribution is relatively narrow. The wild resources of S. edulis are scarce [1,27]. Therefore, the sustainable development of germplasm resources of S. edulis has great significance. All S. edulis strains in this study were domesticated from wild strains and are vital germplasm resources. Germplasm resources are the basic and original material for breeding excellent varieties and are crucial to the production and scientific research of edible fungi [28].

Antagonism tests are usually based on observing antagonism between two strains on the same plate by observing hyphal growth to preliminarily determine the genetic relationship between various strains. However, S. edulis is tetrapolar heterothallic [29]. The results of antagonistic experiments can vary per the culture time duration and whether the strain is degraded. Molecular marker technology is used to identify strains more accurately [24,25,26]. The ITS region of the ribosomal cistron can be used to identify an extensive range of fungi with the highest probability; it has the most clearly defined barcode gap between inter- and intraspecific variations [30]. Various ribosomal DNA regions, including ITS1, ITS2, and ITS4, have been used to determine the genetic variations of fungi at the species and subspecies levels [31,32,33]. Therefore, 21 wild strains collected from 2013 to 2020 were subjected to antagonism tests in this study to eliminate strains without antagonistic reactions, and 16 strains were screened. Furthermore, ITS marker identification was used to confirm that all test strains were S. edulis.

Agronomic traits are a key component of variety protection, new variety breeding, and DUS (distinct, uniform, and stable) tests [34]. For edible mushroom breeders, most genetic traits of wild strains are unknown [35,36]. Therefore, we evaluated the agronomic traits of various S. edulis strains in this study. The cultivation steps included substrate preparation, inoculation, mycelial culture, and fruiting body harvest after maturation. According to the characteristics of various edible mushrooms, the investigation indexes of the agronomic traits differed. Gao et al. [37] investigated six agronomic and quality traits, including earliness (first harvesting day), firmness, distribution, pileus color, compost colonization, and scaling (smoothness of the pileus skin) of Agaricus bisporus (button mushrooms), using multi-trait QTL analyses in a mixed model. Yan et al. [34] tested the fruiting body, pileus, pileus diameter, pileus thickness, gill width, stipe length, and stipe thickness of Pleurotus giganteus. The results suggested that a high degree of difference was observed in terms of some morphological traits in some varieties. In our study, we tested the clustered morphology of the fruiting body, pileus shape, pileus color, number of fruiting bodies, pileus length–width ratio, pileus thickness, and time from inoculation to primordium formation of the fruiting bodies of S. edulis. This was based on the DUS test guidelines [38]. The 16 strains were divided into two categories using cluster analysis combined with fruiting body morphological analysis. The first category included nine strains with common characteristics, including a wavy pileus edge, a mostly yellow pileus color, a large pileus, thick flesh, and a short time from inoculation to primordium formation. The second category included seven strains with common characteristics, including a smooth pileus edge and a mostly yellowish-brown pileus color. Compared with the first category, the number of fruiting bodies was high, the size of the pileus was moderate, and the time of inoculation to primordium formation was long.

Improving yield and disease resistance is the main goal of edible mushroom breeding [39]. The green mold Trichoderma is one of the most common pathogens in edible fungi cultivation, seriously affecting the yield and quality of edible mushrooms [40,41]. In this study, the rate of Trichoderma contamination in strains Y13, Y17, Y6, Y10, Y14, and Y18 was <20%, indicating strong resistance to Trichoderma. The fruiting body yield and BE of edible fungi result from the interaction between multiple factors, including variety characteristics, cultivation substrates, and environmental factors [42]. In this study, the fruiting body yields and BE values of the various strains significantly differed in the same cultivation substrate and cultivation environment (p < 0.05). Y13 and Y12 exhibited the highest average fruiting body yields and BE values.

Edible mushrooms are rich in protein and cellulose but low in fat [43]. The edible mushrooms A. bisporus and L. edodes are extensively artificially cultivated globally. A. bisporus contains 14.1% protein, 2.2% fat, 9.7% ash, and 325 kcal/kg of energy. L. edodes contains 12.8% protein, 1.0% fat, 4.3% ash, and 388 kcal/kg of energy [44,45,46]. Both are standard examples of high-protein and low-fat food, in line with people’s current demand for a low-fat diet. Polysaccharides, one of the main active substances of edible fungi, have immunomodulatory [47,48], antitumor [49], anti-inflammatory [50], and hypoglycemic [51,52] effects. In our study, the polysaccharide, crude protein, crude fiber, ash, and crude fat contents in the fruiting bodies of the various strains differed (6.07–8.86 g/100 g, 13.41–22.64 g/100 g, 2.41–6.65%, 5.34–7.65%, and 1.56–2.41 g/100 g, respectively). Studies have demonstrated that the yield and quality of edible fungi are greatly correlated with the cultivation substrate [53,54]. In a follow-up study, the screened strains with excellent traits will be applied in breeding experiments to improve the yield and quality of the fruiting bodies via matrix improvement. We assessed the appropriate medium composition, matrix composition, and cultivation methods for S. edulis in previous studies [55,56,57]. The four S. edulis strains screened in this study exhibited 8.13–31.55% higher yields than current S. edulis strains in production. Moreover, the four strains exhibited stable traits, which is significant for promoting the development of the S. edulis industry. However, this study had some limitations. This was only a screening study, and the genetic material was not changed. Furthermore, these four strains still require further development for commercial production.

5. Conclusions

This study lays a foundation for the strain development of S. edulis in China. In this study, 21 wild strains of S. edulis were collected from a field in Heilongjiang Province, China. Sixteen strains were identified as S. edulis using the antagonism test and ITS marker identification. The mycelial growth rate, resistance to Trichoderma, yield, agronomic traits, and nutrient contents of the fruiting bodies of the 16 strains were analyzed. The results demonstrate that Y12, Y13, Y14, and Y15 exhibited high fruiting body yields, excellent traits, and good quality that can be applied in production. Moreover, they can be used as the basic materials for the genetic and matrix improvement of S. edulis. However, further studies are needed for the highly efficient cultivation and breeding of S. edulis for commercial production.