Transcriptome analysis and development of EST-SSR markers in the mushroom Auricularia heimuer.

Auricularia heimuer, the third most frequently cultivated edible mushroom species worldwide, has high medicinal value. However, a shortage of molecular marker hinders the efficiency and accuracy of genetic breeding efforts for A. heimuer. High-throughput transcriptome sequencing data are essential for gene discovery and molecular markers development. This study aimed to clarify the distribution of SSR loci across the A. heimuer transcriptome and to develop highly informative EST-SSR markers. These tools can be used for phylogenetic analysis, functional gene mining, and molecular marker-assisted breeding of A. heimuer. This study used Illumina high-throughput sequencing technology to obtain A. heimuer transcriptome data. The results revealed 37,538 unigenes in the A. heimuer transcriptome. Of these unigenes, 24,777 (66.01%) were annotated via comparison with the COG, Pfam, and NR databases. Overall, 2510 SSRs were identified from the unigenes, including 6 types of SSRs. The most abundant type of repeats were trinucleotides (1425, 56.77%), followed by mononucleotides (391, 15.58%) and dinucleotides (456, 18.17%). Primer pairs for 102 SSR loci were randomly designed for validity confirmation and polymorphism identification; this process yielded 53 polymorphic EST-SSR markers. Finally, 13 pairs of highly polymorphic EST-SSR primers were used to analyze the genetic diversity and population structure of 52 wild A. heimuer germplasms, revealing that the 52 germplasms could be divided into three categories. These results indicated that SSR loci were abundant in types, numbers, and frequencies, providing a potential basis for germplasm resource identification, genetic diversity analysis, and molecular marker-assisted breeding of A. heimuer.

Evaluation of Rice Straw, Corncob, and Soybean Straw as Substrates for the Cultivation of Lepista sordida.

Lepista sordida is a type of high-quality rare edible and medicinal mushroom, and its research boom is just beginning. More than 80 million tons of grain crop residues are produced each year in Heilongjiang Province. To realize the exploration and utilization of wild L. sordida mushrooms and also provide a theoretical support for the high-value utilization of these resources in Heilongjiang Province, we evaluated the cultivation of L. sordida mushrooms using rice straw, corncob, and soybean straw as substrates. L. sordida grew on all three substrates, and the biological efficiency and yield of the mushrooms grown on soybean straw and corncob were 32.33 ± 1.78% and 4.20 ± 0.23 kg m-2, and 30.15 ± 0.93% and 3.92 ± 0.12 kg m-2, respectively, which increased by 9.38% and 2.08% compared with that on the rice straw substrate with 3.84 ± 0.12 kg m-2 and 29.56 ± 0.89%. The time it took for the mycelia to colonize and initiate primordia on the soybean straw substrate was 22.33 ± 0.58 d and 19.67 ± 0.58 d, respectively, which was delayed by 2 d and 3 d compared with that on the rice straw substrate with 20.67 ± 2.08 d and 16.33 ± 0.58 d, respectively. The fruiting bodies grown on corncob and soybean straw substrates were relatively larger than those on the rice straw substrate. The highest amount of crude protein was 57.38 ± 0.08 g 100 g-1, and the lowest amount of crude polysaccharide was 6.03 ± 0.01 g 100 g-1. They were observed on mushrooms collected from the corncob substrate. The contents of the heavy metal mercury, lead, arsenic, and cadmium in the fruiting bodies grown on each substrate were within the national safety range.

Extraction, physicochemical properties, and antioxidant activity of natural melanin from Auricularia heimuer fermentation.

Introduction: Natural melanin from Auricularia heimuer have numerous beneficial biological properties, which were used as a safe and healthy colorant in several industries. Methods: In this study, single-factor experiments, Box-Behnken design (BBD), and response surface methodology (RSM) were employed to investigate the effects of alkali-soluble pH, acid precipitation pH, and microwave time on the extraction yield of Auricularia heimuer melanin (AHM) from fermentation. Ultraviolet-visible spectrum (UV-Vis), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscope (SEM), and high-performance liquid chromatography (HPLC) were used to analyze the extracted AHM. The solubility, stability, and antioxidant activities of AHM were also measured. Results: The results showed that alkali-soluble pH, acid precipitation pH, and microwave time significantly affected the AHM yield, with the following optimized microwave-assisted extraction conditions: alkali-soluble pH of 12.3, acid precipitation pH of 3.1, and microwave time of 53 min, resulting in an AHM extraction yield of 0.4042%. AHM exhibited a strong absorption at 210 nm, similar to melanin from other sources. FT-IR spectroscopy also revealed that AHM exhibited the three characteristic absorption peaks of natural melanin. The HPLC chromatogram profile of AHM showed a single symmetrical elution peak with a 2.435 min retention time. AHM was highly soluble in alkali solution, insoluble in distilled water and organic solvents, and demonstrated strong DPPH, OH, and ABTS free radical scavenging activities. Discussion: This study provides technical support to optimize AHM extraction for use in the medical and food industries.

Research progress of Auricularia heimuer on cultivation physiology and molecular biology.

Auricularia heimuer (A. heimuer F. Wu, B. K. Cui, Y. C. Dai), a well-known gelatinous fungus used for both food and medicine, is a major edible fungus with a more than 1000-year history of cultivation in China. The nutrients of A. heimuer are abundant, including polysaccharides, melanin, mineral elements, etc. The A. heimuer polysaccharides exhibit antioxidant, immunomodulatory, and anticancer properties. A. heimuer is a completely different species grown in China, unlike Auricularia auricula-judae (Bull.) Quel, which was used to characterize it. The cultivated strain varies based on the local climatic factors and cultivation practices. Hardwood chips are the primary material utilized in the cultivation of substitute materials, which is the principal cultivation technique. However, in actual production, straw is frequently replaced for some wood chips to address the issue of a lack of wood. There are three different types of growing techniques: open-air ground cultivation, arch cultivation, and shed-type hanging substitute cultivation of these three, the quality of A. heimuer grown in a shed is superior to that grown in an open-air environment. The A. heimuer genome sequencing project started later than expected, and the entire genome sequencing was not finished until 2019. A. heimuer's molecular biology studies have mostly concentrated on analyzing genetic diversity and identifying cultivars using molecular markers including RAPD, ISSR, and ITS. There have only been a small number of studies on the function of A. heimuer genes, which have only focused on the preliminary cloning and expression study of a few genes, including the laccase gene and the triterpene compound production gene, among others. However, there is still a lack of comprehensive information concerning A. heimuer, necessitating a synopsis. To our knowledge, this is the first published review of A. heimuer, and it summarizes the most recent studies on its molecular biology and cultivation. This review can serve as a guide for future research on the fungus.

Development of a Strain-Specific Quantification Method for Monitoring Bacillus amyloliquefaciens TF28 in the Rhizospheric Soil of Soybean.

Bacillus amyloliquefaciens TF28 can be used to control soybean root disease. To assess its commercial potential as a biocontrol agent, it is necessary to develop a strain-specific quantification method to monitor its colonization dynamics in the rhizospheric soil of soybean under field conditions. Based on genomic comparison with the same species in NCBI databases, a strain-unique gene ukfpg was used as molecular marker to develop strain-specific PCR assay. Among three primer pairs, only primer pairs (F2/R2) could specifically differentiate TF28 from other strains of B. amyloliquefaciens with the detection limit of 10 fg and 100 CFU/g for DNA extracted from pure culture and dry soil, respectively. Then, a colony count coupled with PCR assay was used to monitor the population of TF28 in the rhizospheric soil of soybean in the field. The results indicated that TF28 successfully colonized in the rhizospheric soil of soybean. The colonization population of TF28 changed dynamically within the 120-day growth period with high population at the branching (V6) and flowering stages (R2). This study provides an efficient method to quantitatively monitor the colonization dynamics of TF28 in the rhizospheric soil of soybean in the field and demonstrates the potential of TF28 as a biocontrol agent for commercial development.

Optimization of Melanin Extraction from the Wood Ear Medicinal Mushroom, Auricularia auricula-judae (Agaricomycetes), by Response Surface Methodology and Its Antioxidant Activities In Vitro.

The optimal conditions for melanin extraction from Auricularia auricula-judae (Hei 29) fruiting bodies was determined on the basis of the extract yield of melanin, calculated by using a single-factor experiment and response surface methodology. Its antioxidant activities were also studied in vitro. Various optimal process conditions for melanin extraction were determined by using Design-Expert software: incubation temperature, 69.11°C; incubation time, 58.66 minutes; and incubation pH, 12.81. Under these conditions, the melanin yield was 2.59%. We found that the antioxidant activities of A. auricula-judae melanin in vitro were strong against DPPH radicals and superoxide anions. The rate of DPPH radical scavenging was 63.04% when the A. auricula-judae melanin concentration was 0.36 mg/mL; the rate of superoxide anion scavenging reached 39.79% when the concentration was 0.375 mg/mL. However, the antioxidant activity against hydroxyl radicals was somewhat weak; the rate of scavenging reached only 7.47% when the A. auricula-judae melanin concentration was 0.06 mg/mL.

Identification of a New Fungal Pathogen Causing White Villous Disease on the Fruiting Body of the Culinary-Medicinal Mushroom Auricularia auricula-judae (Agaricomycetes) in China.

Auricularia auricula-judae is an edible and medicinal fungus ranking fourth in production among the edible fungi cultivated worldwide. White villous disease is rampant in Northeast China; it infects the fruiting bodies of A. auricula-judae by forming a white mycelial layer on its ventral side. The disease not only causes an unacceptable morphological appearance and a poor-quality product, but it also significantly reduces the yield. In this study, based on fungal morphology, ribosomal DNA internal transcribed spacer sequences, identification of species-specific primers, and the pathogenicity of the mycelia and spores, 2 fungal pathogens were isolated and identified as Fusarium equiseti and F. sporotrichioides.