Sixty-nine A. cristatus strains from dark tea samples were collected across various regions of China (Hunan, Hubei, Zhejiang, Shaanxi, Guangxi, and Guizhou Province) and isolated using a gradient dilution (Figures 1A,B). Three standard strains provided by Hunan City University (A. cristatus JH1805, A. pseudoglaucus HL1801, and A. chevalieri XW1803) were also included as controls for the isolated strains to effectively analyze the variation of wild-type strains. The sources of the experimental strains are listed in Supplementary Table S1.

Potato dextrose agar (PDA), modified PDA, 20% Czapek-Dox medium (20% CDA), 60% CDA, czapek yeast extract agar (CYA), potato glucose liquid medium (PDL), and dark tea liquid medium (DTL) were used as culture media. Among them, PDA, modified PDA, 20% CDA, and 60% CDA were used for morphological observations. Whereas PDL and DTL were used for the cultivation of mycelium for DNA extraction and detection of extracellular enzyme activity of the strains, respectively. The formulas for the different culture media are shown in Supplementary Table S2.

Strains were inoculated onto PDA plates and cultured at 28°C for 6 d (Figure 1C). Their characteristics were observed and recorded (for methodology, see Supplementary Figure S1). The assessment and clustering of the morphological characteristics of the colony were performed according to the method described by Yang and Song (2014). The average colony size was determined based on the crossover method. Edge shape was assessed based on the orderliness of colony edges, pigment secretion was evaluated based on the sizes and depths of melanin-containing areas in the colony center; surface characteristics were evaluated to determine colony smoothness; cluster analysis was determined using SPSS AU (Xu et al., 2023a) based on four physiological traits of the tested strains.

The RNA was extracted from sexual and asexual mycelia of A. cristatus JH1805 cultured on PDA medium for 6 d (Hu et al., 2023). The extracted RNA was then used for library construction and sequencing using the methods described by Hu et al. (2023).

MISA software (Liu et al., 2023a; https://webblast.ipk-gatersleben.de/misa/) was used to identify SSR loci within the A. cristatus transcriptome (Figure 1D). The criteria were as follows: single nucleotide repeats >10; dinucleotide repeats >5; trinucleotide repeats >4; and tetranucleotide, pentanucleotide, and hexanucleotide repeats >3.

Primers for SSR-containing sequences ≥12 base pairs (bp) were designed in Primer Premier 5.0. The design criteria were as follows: (1) flanking sequences ≥50 bp; (2) annealing temperature (Tm) of 58–65°C, and a ≤2°C difference between the Tm of positive and negative primers; (3) expected amplicon size, 80–250 bp; (4) primer length, 18–28 bp; and (5) GC content, 40–60%. Hairpins, dimers, false priming, and cross-dimer formation were avoided as much as possible during the primer design. Primer verification was done using BLAST on the unigene library, which resulted in 30 pairs selected for subsequent primer synthesis (Supplementary Table S3).

Representative strains, such as JH1805, A1, A11, A13, A12, A27, A30, B1, B8, C5, C6, D1, E1, E13, and F3 were selected from different provinces as test samples for DNA extraction (Alcaide et al., 2019; Xu et al., 2023b), and A. chevalieri XW1803 was used as the control. The 16 strains were cultured at 28°C in PDL liquid medium for 6 d, then centrifuged at 2550 × g for 5 min. Subsequently, mycelia were collected for DNA extraction.

Extracted DNA was amplified with the 30 EST-SSR primer pairs. The reaction mix comprised 34 μL T3 Super PCR Mix, 2 μL each of upstream and downstream primer, and 2 μL of the DNA template. The parameter settings for PCR amplification of EST-SSR sequences are shown in Supplementary Table S4. Subsequently, PCR products were electrophoresed for 120 min on a 10% polyacrylamide gel at 100 V. Bands were visualized using an EB-stained gel imaging system (Vidya et al., 2021).

To assess the suitability (presence) of SSR markers, their degree of polymorphism (based on length) was used (Zhao et al., 2023b). High, intermediate, and low polymorphism is typically defined as SSR length ≥ 20 bp, 12–19 bp, and <12 bp, respectively (Zhang et al., 2022b).

The analysis of EST-SSR genetic diversity was conducted as follows: the images captured by the gel imaging system in step 2.2.3 were analyzed, and each pair of EST-SSR primer corresponds to a locus. If a polymorphic band is observed, it is considered an allelic variation. The electrophoretic bands were analyzed statistically using the “0,1” system, where no bands were recorded as “0” and bands were recorded as “1.” The statistical results were summarized in an MS Excel table to establish the original database (Li et al., 2023b), which was used for the comparison and clustering analysis of EST-SSR amplified bands. The genetic similarity coefficients among 16 strains were analyzed using NTSYS-pc 2.2, and unweighted pair group method with arithmetic mean (UPGMA) hierarchical clustering analysis was performed based on the similarity coefficient to construct a system tree. The positions of different strains on the system tree can be used to evaluate their EST-SSR genetic diversity (Figure 1E) (Chen et al., 2022). In addition, the conjugate values and matrix correlation coefficients of the UPGMA clustering results were calculated using NTSYS-pc 2.2 to evaluate the clustering results (Yildiz et al., 2021).

The 15 A. cristatus phenotypic traits obtained from the PDA culture were standardized in NTSYS-pc 2.2 to obtain Euclidean distance and genetic distance matrices of EST-SSR markers. The two matrices were subjected to a Mantel correlation analysis (Yang et al., 2022).

Using the three-point method, strains were inoculated onto plates containing PDA, modified PDA, 20% CDA, 60% CDA, and CYA (Figure 1G). Since culture temperature and osmotic pressure affect the reproductive pattern of A. cristatus (Tan et al., 2018; Shao et al., 2022), to observe the different reproductive structures of A. cristatus, all plates were incubated at 28°C except for 60% CDA, which was incubated at 32°C. After culturing for 6 d, colony morphology was recorded by photographing.

Sexual and asexual mycelia cultured in 60% CDA medium for 6 d were selected and prepared as fixed specimens (Figure 1H). Mycelium structure was observed under a scanning electron microscope (SEM). Different regions from the electron microscope images were selected for length and diameter measurements in ImageJ Software (Wainaina and Taherzadeh, 2023). Twenty samples per structure were measured randomly.

Spore suspensions (1.0 mL) for each strain were pipetted into a dark tea liquid medium and then cultivated at 28°C (Figure 1I). After fermentation for 3–8 d, suspensions were centrifuged at 8263 × g for 10 min to obtain the supernatant crude enzyme solution. The enzyme activity per unit volume was calculated by adding the volume of the sample (U/mL).

Cellulase activity was measured using the dinitrosalicylic acid (DNS) method (Sudeep et al., 2020): 0.5 mL of crude enzyme solution was added to 2.0 mL of 1.0% CMC-Na solution, and the solution was placed in a water bath at 40°C for 30 min. Subsequently, 2.0 mL of DNS solution was added to terminate the reaction immediately. The solution was then heated in boiling water for 5 min, after which it was removed and cooled. The volume was maintained at 20 mL. The optical density value was measured at 540 nm using an ultraviolet spectrophotometer, with glucose as the standard curve. One unit of cellulase activity was defined as the amount of enzyme required to produce glucose (1 μg/min) under the above reaction conditions.

During the determination of pectinase activity, 1% CMC-Na solution was replaced by 1% pectin solution, the other methods were similar to those used for cellulase determination. One unit of pectinase activity was defined as the amount of enzyme required to produce glucose (1 μg/min) under the above reaction conditions.

The protease activity was determined using the Folin–Ciocalteu method (Sharma et al., 2021): 1 mL of crude enzyme solution was added to 1 mL of casein. Both the crude enzyme solution and casein substrate were pre-incubated at 40°C in a water bath for 3 min before mixing. Once combined, the reaction mixture was incubated further for 10 min at 40°C in a water bath. Afterward, 2 mL of 0.4 mol/L trichloroacetic acid solution, which is used to inhibit the enzyme, was removed and allowed to sit for 10 min at 20°C and then the solution was centrifuged at 1632 × g for 5 min. After centrifugation, 1 mL of supernatant was added to 5 mL of 0.4 mol/L sodium carbonate solution and then 1 mL of Folin reagent was added to the solution. This was then shaken well and kept in a 40°C water bath for 20 min. An ultraviolet spectrophotometer was used to measure the optical density of the solution at 660 nm, with tyrosine used to plot the standard curve. One unit of protease activity was defined as the amount of enzyme required to hydrolyze casein to produce 1 μg/min tyrosine under the above reaction conditions.

Polyphenol oxidase activity was determined using the catechol method (González-Ceballos et al., 2023): 1.0 mL of crude enzyme solution was added to 3 mL of reaction mixture (citrate buffer: 0.1% proline: 1%, catechol = 10:2:3). The solution was then kept in a thermostatic water bath at 37°C for 10 min, and 3 mL of trichloroacetic acid (1 mL) was added immediately to terminate the reaction. The optical density was measured at 460 nm using an ultraviolet spectrophotometer, and the catechol in the blank control group was replaced by 0.05 mol/L PBS Buffer Solution. One unit of polyphenol oxidase activity was defined as the amount of enzyme required to increase the absorbance by 1 within 1 min under the above reaction conditions.

All seven tested strains grew successfully on the five cultured media (PDA, modified PDA, 20% CDA, 60% CDA, and CYA), forming colonies with different morphologies (Figure 6A). In general, the growth rate of the seven fungi strains was higher on the modified PDA and 60% CDA media, and lower on the PDA and 20% CDA media, the growth rate of the colony was positively correlated with the concentrations of nutrients, such as carbon source. Moreover, the fungal colonies from the seven strains predominantly displayed ascocarps (structure related to sexual reproduction) grown on the PDA, modified PDA and CYA medium. Conversely, asexual reproduction (conidia head) structures were rarely observed, whereas asexual reproduction was predominantly observed in the colonies grown on the 60% CDA medium. Notably, although the growth rate and pigment secretion of the five strains of A. cristatus were affected greatly by the type of medium, the strains showed similar physiological characteristics on different media: for example, B1 grew faster and produced fewer pigments on all the media; A11 grew at a moderate rate and produced more pigments, exhibiting stability of certain physiological characteristics in different strains.

Strain structural SEM images of seven tested strains were quantified in ImageJ (Figure 6B) and analyzed (Table 2). Sexual A. cristatus has a spherical ascocarp (diameter: 50.0–180.0 μm) surrounded by numerous mycelia. As growth progressed, the ascocarp wall became transparent and eventually ruptured, releasing ascospores upon maturation. Ascospores were 3.4–4.7 μm × 4.4–6.4 μm in size and presented a double-convex shape. Their surfaces were rough, with two prominent corona protrusions at the equator and grooves between the protrusions.

Asexual A. cristatus possessed a conidial head of 35.0–80.0 μm in length. At the immature stage, the conidial head exhibited multiple conidial chains, each containing approximately three to seven conidia, which increased in a time-dependent manner. The mature conidia detached from conidial chains and were dispersed within the matrix. The most mature conidia were observed to be ellipsoidal, with small spiny protrusions on the outer wall and 3.2–3.8 μm × 4.2–5.2 μm in size.

The related A. pseudoglaucus is mainly distinguished from A. cristatus by a smooth ascospore surface, less obvious coronal protrusions at the equator, and shallower grooves. In A. chevalieri, ascospores were flat, with a depression in the middle of the bearing-like equator, while conidia were thicker and shorter.

In all seven strains, cellulase activity increased rapidly during fermentation, peaking on days 6 and 7 and then leveling off (Figure 7A). Cellulase activity was the highest among A. cristatus B1 and A11 (1.779 U/mL and 1.786 U/mL on day 7, respectively). Strong cellulase activity was also observed in A. chevalieri XW1803, with a value of 1.607 U/mL on day 6. At the peak of cellulase activity (7 d), the cellulase activity values of each strain were compared in pairs, and there were no significant differences among XW1803, JH1805, B1, A11, and A1 (p > 0.05); there were no significant differences among XW1803, JH1805, D1, and A1 (p > 0.05); and there were significant differences among the other strains (p < 0.05). Among the 5 A. cristatus, D1 had the lowest cellulase activity.

The pectinase activity of the seven test strains is shown in Figure 7B. Activity was the highest in JH1805 and B1, peaking on day 7 at 12.071 U/mL and 12.242 U/mL, respectively. In contrast, pectinase activity was low for A. pseudoglaucus HL1801 and A. chevalieri XW1803, peaking on day 8 (6.029 U/mL) and 7 (6.547 U/mL), respectively. On the 8th day of fermentation, the pectinase activity among the strains was varied. Notably, there were no significant differences observed between strains JH1805, B1, and A11, and A1, as well as between A1 and D1, and between HL1801 and XW1803 (p > 0.05). However, the remaining strains exhibited statistically significant differences (p < 0.05), demonstrating their diverse methods of hydrolyzing pectin. Notably, most strains maintained high pectinase activity during late fermentation.

Protease activity in all seven strains increased initially before decreasing (Figure 7C). Notably, A. cristatus JH1805 and B1 demonstrated robust protease activity, reaching 9.382 U/mL and 8.951 U/mL on day 6, respectively. In contrast, A. chevalieri XW1803 and A. cristatus D1 had low protease activity, peaking at 5.960 U/mL and 6.568 U/mL on fermentation days 6 and 7, respectively. On the 6th day of the peak period of protease activity, each strain displayed a normal distribution, with significant differences between different strains (p < 0.05).

Polyphenol oxidase activity was robust on days 6 and 7 of fermentation, before decreasing (Figure 7D). Activity was particularly high on day 6 in A. cristatus B1 and JH1805, peaking at 0.403 U/mL and 0.388 U/mL, respectively. Activity was lower in A. pseudoglaucus HL1801 and A. chevalieri XW1803, with peak values being 0.107 and 0.114 U/mL, respectively, on day 7. On the 7th day of fermentation, there was no significant difference in polyphenol oxidase activity between B1 and A11, D1 and A1, and HL1801 and XW1803. However, there were significant differences between the other remaining strains (p < 0.05).