In Vitro Bioactivities and Characterization of Mycelial Extracts from Different Strains of Phellinus igniarius (Agaricomycetes).

To fully utilize Phellinus igniarius fermentation mycelia, the present study investigated the in vitro antioxidant and α-amylase inhibitory properties of four Ph. igniarius strains. Organic solvents were used to extract fatty acids, phenolics, and flavonoids from the selected mushrooms. The composition and bioactivity of the extracts were evaluated. The lipid yield obtained using petroleum ether (7.1%) was higher than that obtained using 1:1 n-hex-ane+methanol (5.5%) or 2:1 dichloromethane+methanol (3.3%). The composition and relative content of saturated and unsaturated fatty acids in the petroleum ether extract were higher than those in other solvent extracts. Furthermore, ethyl acetate extracts had higher flavonoid and phenolic content and better antioxidant activity than other extracts; however, the 70% ethanol extracts had the best α-amylase inhibitory activity. The supernatant from the ethanol precipitation of aqueous and 1% (NH4)2C2O4 extracts could also be biocompound sources. This comparative study is the first highlighting the in vitro antioxidant and α-amylase inhibitory properties of the four strains of Ph. igniarius extracts prepared using different organic solvents, which makes the investigated species and extracts promising for biological application.

Enhanced Heat Resistance in Morchella eximia by Atmospheric and Room Temperature Plasma.

This study focuses on optimizing the mutagenesis process for Morchella eximia (Mel-7) mycelia through atmospheric and room temperature plasma (ARTP) mutation and explores the resultant thermal adaptability and physiological responses of mutant strains. This research demonstrated a clear relationship between ARTP mutagenesis exposure duration and lethality rate, indicating that an exposure time of 40 s resulted in the optimal balance of inducing mutations without causing excessive mortality. Additionally, this study established 43 °C as the ideal screening temperature for identifying mutant strains with enhanced heat resistance, as this temperature significantly challenges the mycelia while allowing thermotolerant strains to be distinguishable. Among the screened mutants, strains L21, L23, L44, and L47 exhibited superior growth and high-temperature tolerance, with notable resilience at 30 °C, highlighting their enhanced adaptability to above-optimal temperatures. Furthermore, this research delved into biochemical responses, including lipid peroxidation and non-enzymatic antioxidant content, highlighting the diverse mechanisms, such as enhanced lipid peroxidation resistance and increased antioxidant content, employed by mutant strains to adapt to temperature fluctuations. The activities of antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), were shown to be significantly influenced by temperature elevations, illustrating their critical roles in the thermal adaptation of mutant strains. These findings shed light on the importance of considering mutation duration and temperature screening in the development of thermotolerant fungal strains with potential applications in various industries. This study's breakthrough lies in its comprehensive understanding of the thermal adaptability of Mel-7 mycelia and the identification of promising mutant strains, offering valuable insights for both academic and industrial purposes.

Reference gene selection for quantitative real-time PCR analysis of Hymenopellis radicata under abiotic stress.

Hymenopellis radicata (H. radicata) is an edible fungus rich in protein and mineral elements, with high edible and medical value. And reference genes suitable for normalization of qRT-PCR data from this species have not been investigated. In this study, therefore, we selected 11 housekeeping genes common in biology. The expression levels of these housekeeping genes were measured in three different tissues and six different abiotic stress treatments in mycelium. They were evaluated for expression stability using online tools. The results showed that gene ACT could be stable expressed in all samples. The expressions of genes TUB and UBQ10 are the most stable under heat stress, ACT and EF are the most stable genes under salt stress, ACT and TUB are the most stable genes under oxidation stress, RPL6 and EF are the most stable genes under pH condition, ACT and RPB2 are the most stable genes under cadmium stress, and RPB2 and UBC are the most stable genes under drought condition. ACT and PP2A are the most stable genes at different tissue sites. This study is of great help to explore the gene expression pattern of H. radicata, and also provides reference for internal reference gene screening under other conditions.

Antioxidant activity of Phellinus igniarius fermentation mycelia contributions of different solvent extractions and their inhibitory effect on α-amylase.

Phellinus spp. have historically been used as traditional medicines to treat various diseases owing to their antioxidant, antitumor, and antidiabetic activities. Polysaccharides exhibit antidiabetic activity. In the present study, the polysaccharide contents of four Phellinus strains were compared. Phellinus igniarius QB72 possessed higher polysaccharide production, stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and α-amylase inhibitory activity. The three polysaccharides were sequentially extracted and partially purified from the fermentation mycelia using hot water, 1 % (NH4)2C2O4, and 1.25 M NaOH. Hot water extract polysaccharides exhibited higher DPPH radical scavenging and strong inhibitory activity against α-amylase with an IC50 value of 6.84 ± 0.37 mg/mL. The carbohydrate content of A1 (approximately 17457 Da) was approximately 88.28 %. The α-amylase inhibitory activity IC50 was decreased (3.178 ± 0.187 mg/mL) after DEAE water elution. P. igniarius QB72 hot-water extracts of partially purified polysaccharides have great potential as α-amylase inhibitors in food and medication-assisted additives.

Application of Atmospheric and Room-Temperature Plasma (ARTP) to Microbial Breeding.

Atmospheric and room-temperature plasma (ARTP) is an efficient microbial mutagenesis method with broad application prospects. Compared to traditional methods, ARTP technology can more effectively induce DNA damage and generate stable mutant strains. It is characterized by its simplicity, cost-effectiveness, and avoidance of hazardous chemicals, presenting a vast potential for application. The ARTP technology is widely used in bacterial, fungal, and microalgal mutagenesis for increasing productivity and improving characteristics. In conclusion, ARTP technology holds significant promise in the field of microbial breeding. Through ARTP technology, we can create mutant strains with specific genetic traits and improved performance, thereby increasing yield, improving quality, and meeting market demands. The field of microbial breeding will witness further innovation and progress with continuous refinement and optimization of ARTP technology.

Polysaccharides Derived from Mushrooms in Immune and Antitumor Activity: A Review.

Mushrooms are full of nutrition and have beneficial properties for human health. Polysaccharides are the main component of edible and medicinal mushrooms, especially ß-glucans, which have attracted much more attention for their complex structure and diverse biological activities. Among all the diverse medicinal activities of mushroom polysaccharides, antitumor and immune-enhancing activities are two excellent bioactivities that have much more potential and deserve application. Their bioactivities are highly dependent on their structural features, including molecular weight, monosaccharide composition, degree of branching, type and configuration of glycosidic bonds, substituent pattern, and chain conformation. This review summarizes the current method for obtaining polysaccharides from mushrooms, chemical characterizations of the structures and their roles in immune and antitumor activities. In addition, the methods for preparation of the polysaccharide derivatives and the potential medicinal clinical application are also discussed in this review, which may provide new guidance for mushroom polysaccharide development.

Combined transcriptomics and metabolomics analysis reveals the molecular mechanism of heat tolerance of Le023M, a mutant in Lentinulaedodes.

Lentinula edodes, one of the most highly regarded edible mushrooms in China, is susceptible to damage from high temperatures. However, a mutant strain derived from L. edodes, known as Le023M, has shown exceptional thermotolerance. Compared to the original strain Le023, Le023M exhibited accelerated mycelial recovery following heat stress. Through RNA-seq analysis, the majority of differentially expressed genes (DEGs) were found to be associated with functions such as "protein refolding", "protein unfolding", "protein folding", and "response to heat", all of which are closely linked to heat shock proteins. Furthermore, qRT-PCR results revealed significant accumulation of heat shock-related genes in Le023M under heat stress. GC-MS analysis indicated elevated levels of trehalose, aspartate, and glutamate in Le023M when subjected to heat stress. The highly expressed genes involved in these metabolic pathways were predominantly found in Le023M. Collectively, these findings highlight the following: (i) the crucial role of heat shock proteins (HSPs) in the thermo-resistant mechanisms of Le023M; (ii) the potential of trehalose accumulation in Le023M to enhance mycelium resistance to heat stress; and (iii) the induction of aspartate and glutamate accumulation in response to heat stress. These results shed light on the molecular mechanisms underlying the thermotolerance of Le023M, providing valuable insights for further understanding and improving heat stress response in L. edodes. The findings also highlight the potential applications of Le023M in the cultivation and production of L. edodes under high-temperature conditions.

Application of omics technology in the research on edible fungi.

Edible fungus is a large fungus distributed all over the world and used as food and medicine. But people's understanding of edible fungi is not as much as that of ordinary crops, so people have started a number of research on edible fungi in recent years. With the development of science and technology, omics technology has gradually walked into people's vision. Omics technology has high sensitivity and wide application range, which is favored by researchers. The application of omics technology to edible fungus research is a major breakthrough, which has transferred edible fungus research from artificial cultivation to basic research. Now omics technology in edible fungi has been flexibly combined with other research methods, involving multiple studies of edible fungus, such as genetic breeding, growth and development, stress resistance, and the use of special components in edible fungus as pharmaceutical additives. It is believed that in the future, the research of edible fungi will also be brought to a deeper level with the help of omics technology. This paper introduces the application progress of modern omics technology to the study on edible fungi and mentions the application prospect of edible fungi research with the constant development of omics technology, thereby providing ideas for the follow-up in-depth research on edible fungi.

Genome-Wide Identification and Analysis of the Heat-Shock Protein Gene in L. edodes and Expression Pattern Analysis under Heat Shock.

Lentinula edodes (L. edodes), one of the most popular edible mushrooms in China, is adversely affected by high temperature. Heat shock proteins (HSPs) play a crucial role in regulating the defense responses against the abiotic stresses in L. edodes. Some HSPs in L. edodes have been described previously, but a genome-wide analysis of these proteins is still lacking. Here, the HSP genes across the entire genome of the L. edodes mushroom were identified. The 34 LeHSP genes were subsequently classified into six subfamilies according to their molecular weights and the phylogenetic analysis. Sequence analysis showed that LeHSP proteins from the same subfamily have conserved domains and one to five similar motifs. Except for Chr 5 and 9, 34 LeHSPs genes were distributed on the other eight chromosomes. Three pairs of paralogs were identified because of sequence alignment and were confirmed as arising from segmental duplication. In LeHSPs' promoters, different numbers of heat shock elements (HSEs) were predicted. The expression profiles of LeHSPs in 18N44 and 18 suggested that the thermo-tolerance of strain 18N44 might be related to high levels of LeHSPs transcript in response to heat stress. The quantitative real-time PCR (qRT-PCR) analysis of the 16 LeHSP genes in strains Le015 and Le027 verified their stress-inducible expression patterns under heat stress. Therefore, these comprehensive findings provide useful in-depth information on the evolution and function of LeHSPs and lay a theoretical foundation in breeding thermotolerant L. edodes varieties.

Pulsed Light Mutagenesis of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), for Enhanced Production of Flavonoids, Laccase, and Fermentation Biomass.

Phellinus igniarius is a medicinal fungus possessing potent therapeutic activity due to the polysaccharides, polyphenols, flavonoids, and other secondary metabolites they contain. Laccases are crucial enzymes involved in lignin degradation in Ph. igniarius and offer great potential to accomplish several bioprocesses. To generate Ph. igniarius strains with high biomass, flavonoid, and laccase activity, we used pulsed light (PL) technology for mutagenesis of Ph. igniarius protoplasts and screened for mutants with high biomass, flavonoid, and laccase activity. At the irradiation power of 100 J, treated distance 8.5 cm, irradiation frequency was 0.5 s/time, three times treatments, after five generations of selection, three mutants were obtained with higher biomass production. Compared with control, the mycelium biomass and the flavonoid production of the screened mutant strain QB72 were increased 20.87% and 53.51%, respectively. The total amount of the accumulated extracellular laccase of the QB72 in the first 6 and 8 days increased 23.38% and 22.37% respectively, and over the total 16 days it increased 9.62%. In addition, RAPD analysis results indicated that the genetic materials of the mutant QB72 were altered. PL mutagenesis method has great potential for developing strains, especially Phellinus.

Edible and medicinal fungi breeding techniques, a review: Current status and future prospects.

Mushrooms of the edible and medicinal which are highly nutritious and environmentally friendly crops carry numerous medicinal benefits. For the abundant and high diversity of bioactive metabolites they possess, which are considered to be an important pool of bioresources. The efficient breeding technique is always a challenging task in mushrooms for obtaining better character strains, which are essential for developing healthy products and even consumption. This review comprehensively summarizes the breeding techniques applied to the edible and medicinal mushrooms. Including the traditional mutagenesis method, and even modern gene-editing breeding techniques, the effects of each method, and the comparison of each breeding technique are systematic illustrations. Strategies for mushroom breeding techniques in the future are also discussed in this review paper. With the ongoing sequencing of the mushroom genome, knowledge of the gene background of the strains and functions can be available for developing better markers for gene-editing breeding as CRISPR/Cas9 systems. Combine the metabolism engineering and in-silico tools analysis was the rational design of the novel strains. Modern physical mutagenesis techniques such as the ARTP and the combination of the other physical, and chemical breeding mutagens with cross-breeding techniques or the protoplasts fusion will also lead to superior strains for cultivation and pave the way for higher quality and yield.

Impacts of size reduction and alkaline-soaking pretreatments on microbial community and organic matter decomposition during wheat straw composting.

This study assessed the impacts of size reduction and alkaline-soaking pretreatments on microbial community shifts and organic matter decomposition in wheat straw composting. Bacterial communities were altered by alkaline soaking rather than size reduction, while fungal communities were altered by both pretreatments. Alkaline-soaking pretreatment promoted lignocellulosic saccharification and humification. A combination of both pretreatments increased the proportion of the fungal genus Coprinopsis (39%) at the early stage and promoted the proliferation of Ornithincoccus (15%) at the late stage. This facilitated the mineralization of ammonium N from amino acids; decreased the total lipids, free fatty acids, and nitrate N contents; and greatly improved the germination index of the final composting product to a high level of 149% as tested with radish seeds. The findings demonstrate that the combined application of size reduction and alkaline-soaking pretreatments is an effective strategy for improving the product quality of wheat straw compost.

Build Your Own Mushroom Soil: Microbiota Succession and Nutritional Accumulation in Semi-Synthetic Substratum Drive the Fructification of a Soil-Saprotrophic Morel.

Black morel, a widely prized culinary delicacy, was once an uncultivable soil-saprotrophic ascomycete mushroom that can now be cultivated routinely in farmland soils. It acquires carbon nutrients from an aboveground nutritional supplementation, while it remains unknown how the morel mycelium together with associated microbiota in the substratum metabolizes and accumulates specific nutrients to support the fructification. In this study, a semi-synthetic substratum of quartz particles mixed with compost was used as a replacement and mimic of the soil. Two types of composts (C1 and C2) were used, respectively, plus a bare-quartz substratum (NC) as a blank reference. Microbiota succession, substrate transformation as well as the activity level of key enzymes were compared between the three types of substrata that produced quite divergent yields of morel fruiting bodies. The C1 substratum, with the highest yield, possessed higher abundances of Actinobacteria and Chloroflexi. In comparison with C2 and NC, the microbiota in C1 could limit over-expansion of microorganisms harboring N-fixing genes, such as Cyanobacteria, during the fructification period. Driven by the microbiota, the C1 substratum had advantages in accumulating lipids to supply morel fructification and maintaining appropriate forms of nitrogenous substances. Our findings contribute to an increasingly detailed portrait of microbial ecological mechanisms triggering morel fructification.

Multi-omic analyses of exogenous nutrient bag decomposition by the black morel Morchella importuna reveal sustained carbon acquisition and transferring.

The black morel (Morchella importuna Kuo, O'Donnell and Volk) was once an uncultivable wild mushroom, until the development of exogenous nutrient bag (ENB), making its agricultural production quite feasible and stable. To date, how the nutritional acquisition of the morel mycelium is fulfilled to trigger its fruiting remains unknown. To investigate the mechanisms involved in ENB decomposition, the genome of a cultivable morel strain (M. importuna SCYDJ1-A1) was sequenced and the genes coding for the decay apparatus were identified. Expression of the encoded carbohydrate-active enzymes (CAZymes) was then analyzed by metatranscriptomics and metaproteomics in combination with biochemical assays. The results show that a diverse set of hydrolytic and redox CAZymes secreted by the morel mycelium is the main force driving the substrate decomposition. Plant polysaccharides such as starch and cellulose present in ENB substrate (wheat grains plus rice husks) were rapidly degraded, whereas triglycerides were accumulated initially and consumed later. ENB decomposition led to a rapid increase in the organic carbon content in the surface soil of the mushroom bed, which was thereafter consumed during morel fruiting. In contrast to the high carbon consumption, no significant acquisition of nitrogen was observed. Our findings contribute to an increasingly detailed portrait of molecular features triggering morel fruiting.

Transcriptional profiling of Auricularia cornea in selenium accumulation.

Auricularia cornea is a widely cultivated edible fungus with substantial nutritive value. This study aimed to enrich the multifunctional bionutrient element selenium in A. cornea to improve its quality and explore the accumulation of selenium in the fungus using high-throughput RNA-Seq technology. In general, the treatment group with a 100 µg/g supply of selenium outperformed the other treatment groups in terms of high yield, rich crude polysaccharides and a high total selenium concentration. Additional evidences demonstrated the budding and mature phases were two typical growth stages of A. cornea and were important for the accumulation of selenium. Therefore, the budding and mature phase tissues of A. cornea in the treatment group with a 100 µg/g supply of selenium were used for transcriptome analysis and compared to those of a control group that lacked additional selenium. A total of 2.56 × 105 unigenes from A. cornea transcriptome were assembled and annotated to five frequently used databases including NR, GO, KEGG, eggNOG and SwissProt. GO and KEGG pathway analysis revealed that genes involved in metabolic process and translation were up-expressed at the budding stage in response to selenium supplementation, including amino acid metabolism, lipid metabolism, ribosome. In addition, the differential gene expression patterns of A. cornea suggested that the up-expressed genes were more likely to be detected at the budding stage than at the mature stage. These results provide insights into the transcriptional response of A. cornea to selenium accumulation.

Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable Morchella importuna provides insights into plant-litter decomposition.

The aim of this study is to determine the key laccase-encoding gene in the life cycle of Morchella importuna SCYDJ1-A1, and to characterize the biochemical properties of the laccase. Two laccase-like multicopper oxidase (LMCO) genes were identified in the genome of M. importuna SCYDJ1-A1 as putative laccase-encoding genes. The two genes, belonging to Auxiliary Activity family 1 subfamily 3, were named as MiLacA and MiLacB. Phylogenetic analysis of deduced amino acid sequences showed that MiLacA is closest to a LMCO of M. importuna 22J1, while MiLacB had low similarity with known Morchella LMCOs. Real-time quantitative PCR results showed that MiLacA was expressed at much higher levels than MiLacB throughout the entire course of artificial cultivation. MiLacA was overexpressed in Pichia pastoris as a recombinant protein. Biochemical characterization of the purified enzyme showed that MiLacA simultaneously possessed laccase and polyphenol-oxidase activities. MiLacA could be strongly inhibited by Fe2+, which is unusual. The optimum pH was four and optimum temperature was 60 °C. The enzyme retained over 74% of the laccase activity after 16-h incubation at 60 °C, which means that its thermostability is at the forefront among the currently known laccases. Our findings may help to elucidate how the laccase of M. importuna is involved in decaying lignin in plant litter, and could also provide a candidate thermostable laccase for potential industrial application.

Influence of Temperature on the Bacterial Community in Substrate and Extracellular Enzyme Activity of Auricularia cornea.

Temperature is an important environmental factor that can greatly influence the cultivation of Auricularia cornea. In this study, lignin peroxidase, laccase, manganese peroxidase, and cellulose in A. cornea fruiting bodies were tested under five different temperatures (20 °C, 25 °C, 30 °C, 35 °C, and 40 °C) in three different culture periods (10 days, 20 days and 30 days). In addition, the V4 region of bacterial 16S rRNA genes in the substrate of A. cornea cultivated for 30 days at different temperatures were sequenced using next-generation sequencing technology to explore the structure and diversity of bacterial communities in the substrate. Temperature and culture days had a significant effect on the activities of the four enzymes, and changes in activity were not synchronized with changes in temperature and culture days. Overall, we obtained 487,694 sequences from 15 samples and assigned them to 16 bacterial phyla. Bacterial community composition and structure in the substrate changed when the temperature was above 35 °C. The relative abundances of some bacteria were significantly affected by temperature. A total of 35 genera at five temperatures in the substrate were correlated, and 41 functional pathways were predicted in the study. Bacterial genes associated with the membrane transport pathway had the highest average abundance (16.16%), and this increased at 35 °C and 40 °C. Generally, different temperatures had impacts on the physiological activity of A. cornea and the bacterial community in the substrate; therefore, the data presented herein should facilitate cultivation of A. cornea.

A bifunctional cellulase-xylanase of a new Chryseobacterium strain isolated from the dung of a straw-fed cattle.

A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase-xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 µmol min-1  mg-1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 µmol min-1  mg-1 at pH 8, 90 °C. The activity level and thermophilicity are in the front rank of all the known cellulases and xylanases. Core hydrophobicity had a positive effect on the thermophilicity of this enzyme. When similar quantity of enzymatic activity units was applied on the straws of wheat, rice, corn and oilseed rape, CbGH5 could obtain 3.5-5.0× glucose and 1.2-1.8× xylose than a mixed commercial cellulase plus xylanase of Novozymes. When applied on spent mushroom substrates made from the four straws, CbGH5 could obtain 9.2-15.7× glucose and 3.5-4.3× xylose than the mixed Novozymes cellulase+xylanase. The results suggest that CbGH5 could be a promising candidate for industrial lignocellulosic biomass conversion.

Biochemical Characterization of a Psychrophilic Phytase from an Artificially Cultivable Morel Morchella importuna.

Psychrophilic phytases suitable for aquaculture are rare. In this study, a phytase of the histidine acid phosphatase (HAP) family was identified in Morchella importuna, a psychrophilic mushroom. The phytase showed 38% identity with Aspergillus niger PhyB, which was the closest hit. The M. importuna phytase was overexpressed in Pichia pastoris, purified, and characterized. The phytase had an optimum temperature at 25°C, which is the lowest among all the known phytases to our best knowledge. The optimum pH (6.5) is higher than most of the known HAP phytases, which is fit for the weak acidic condition in fish gut. At the optimum pH and temperature, MiPhyA showed the maximum activity level (2,384.6 ± 90.4 µmol·min⁻¹·mg⁻¹, suggesting that the enzyme possesses a higher activity level over many known phytases at low temperatures. The phytate-degrading efficacy was tested on three common feed materials (soybean meal/rapeseed meal/corn meal) and was compared with the well-known phytases of Escherichia coli and A. niger. When using the same amount of activity units, MiPhyA could yield at least 3× more inorganic phosphate than the two reference phytases. When using the same weight of protein, MiPhyA could yield at least 5× more inorganic phosphate than the other two. Since it could degrade phytate in feed materials efficiently under low temperature and weak acidic conditions, which are common for aquacultural application, MiPhyA might be a promising candidate as a feed additive enzyme.

White mold on cultivated morels caused by Paecilomyces penicillatus.

Morchella (morel) includes prized edible and medical mushrooms in the world. Since 2012, commercial cultivation of morels in the field has developed rapidly in China. However, coupled with the rapid expansion of morel cultivation, diseases have been become serious threats to morel production. White mold is one of the most serious diseases on cultivated morels. This study aimed to confirm this pathogen by following Koch's postulates, and to identify it using molecular evidence. Our results indicated that healthy Morchella fruiting bodies inoculated with Paecilomyces sp. isolates produced typical white mold symptoms, and the internal transcribed spacer sequences of the Paecilomyces sp. were 99% similar to that recovered from an epitype of Paecilomyces penicillatus. Therefore, P. penicillatus was considered to be the causative agent of white mold. White mold occurred from the initial harvest to the storage and preservation process, and it produced white mold-like symptoms on the caps and stripes of Morchella. This is the first time that white mold has been reported on cultivated Morchella.