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.

First Report of Sphingobacterium multivorum Causing Bacterial Rot of Cauliflower mushroom (Sparassis latifolia) in China.

Cauliflower mushroom (Sparassis latifolia), is widely distributed in Australia, North America, Europe, and East Asia (Bashir et al., 2020). It is known for its medicinal significance due to the availability of various pharmacological substances and their use in health supplements (Bashir et al., 2017). In recent years, with the development of artificial cultivation technology, S. latifolia has been industrialized in China, with an annual output value 50 million dollars. In March 2023, approximately 15% of S. latifolia showed obvious bacterial rot in mushroom hothouse (about 0.05 ha), located in Shuangliu county, Sichuan province, China (104°7'51"N, 30°25'2"E). The affected parts appear water-soaked, and become sunken and softened as the disease progresses. In the finally, all the fruiting body tissues turn into paste, with colors pale yellow, and have a foul smell. The pathogen was isolated from the margin of the lesions by dilution and streaking techniques onto Nutrient Agar, and incubated at 28℃ in the dark for 2-3 days. A single colony was re-streak for purification. Eight isolates were obtained from five samples collected randomly. The representative three isolates were selected for further characterization. For pathogenicity testing, ten health fruit bodies of S. latifolia were selected (for per isolate). Bacterial suspensions (1 × 107 CFU/ml) of the three isolates were applied to the fruiting body until wet, sterile water was used as controls. All the S. latifolia were maintained at 19±1℃, 85-100% relative humidity, and 18 h of light in the mushroom hothouse. Three days later, the inoculated fruiting bodies developed yellow color, and appear water-soaked, five days later, fruiting body gradually turn to soft and part turn to rot, seven days later, the fruiting body tissues completely turn into paste with a foul smell. The symptoms exhibited were similar to those of the original diseased fruiting bodies, while the control group remained healthy. The same bacterial were re-isolated from the infected fruiting bodies and subsequently identified by morphological characteristics and DNA sequenced. The pathogenicity test was conducted three times, each yielding similar results. The colonies of the pathogen are gram-negative rods, medium sized, convex, smooth, opaque, turning yellow after several days at a temperature 28℃. For molecular identification, the DNA of the representative three isolates was extracted using a Bacterial Genomic DNA Extraction Kit (Solarbio, Beijing). The 16S rRNA genes were amplified and sequenced with the primer 27F/1492R (Lane et al., 1985). Finally, the sequences were identical. The generated representative sequence was deposited in GenBank with accession number OR399122. BLASTn analysis showed 100% identity (1404/1404 bp) with previously deposited sequence (accession number CP068224) of S. multivorum FDAARGOS in GenBank. Based on the maximum likelihood method, phylogenetic analysis revealed 100% bootstrap support values with S. multivorum. Finally, the bacterium was identified as S. multivorum. This is the first report of S. multivorum causing bacterial rot of mushroom. The fruiting body of S. multivorum consists of multiple folded flat lobes, which are thin and have large surface area, may facilitate the infection of S. multivorum. Sphingobacterium sp. are named for their synthesize sphingolipids, which play an important role in bacterial infection (Kunz et al., 2019). These results will contribute to developing control strategies for this disease.

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.

Comparative Transcriptome Analysis of Candidate Genes Associated with Mycelia Growth from a He-Ne Laser with Pulsed Light Mutant of Phellinus igniarius (Agaricomycetes).

A mutant Phellinus igniarius JQ9 with higher mycelial production was screened out by He-Ne laser with pulsed light irradiation, the mechanism underlying the higher mycelial production is still unknown. This study aims to obtain a comprehensive transcriptome assembly during the Ph. igniarius liquid fermentation and characterize the key genes associated with the mycelial growth and metabolism in Ph. igniarius JQ9. Our transcriptome data of Ph. iniarius JQ9 and the wild strain were obtained with the Illumina platform comparative transcriptome sequencing technology. The results showed that among all the 346 differentially expressed genes (DEGs), 245 were upregulated and 101 were downregulated. Candidate genes encoding endoglucanase, beta-glucosidase, cellulose 1,4-beta-cellobiosidase, glycoside hydrolase family 61 protein, were proposed to participate in the carbohydrate utilization from KEGG enrichment of the starch and sucrose metabolism pathways were upregulated in Ph. igniarius JQ9. In addition, three candidate genes encoding the laccase and another two candidate genes related with the cell growth were higher expressed in Ph. igniarius JQ9 than in the wild type of strain (CK). Analysis of these data revealed that increased these related carbohydrate metabolism candidate genes underlying one crucial way may cause the higher mycelia production.

First Report of Lecanicillium aphanocladii Causing Rot of Sparassis crispa in China.

Sparassis crispa, also known as cauliflower mushroom, is a new popularly edible mushroom in China, also a medicinal mushroom, which possesses various biological activities, such as immunopotentiation, anti-diabetes, anti-cancer, and anti-inflammatory effects. (Han et al., 2018). In recent years, the artificial cultivation of S. crispa has gained considerable public attention in China. In 2023, approximately 20% of S. crispa (about 0.05 ha of the planting area) showed obvious rot with white molds symptoms in mushroom hothouse, located in Shuangliu county, Sichuan province, China (GPS, 104°7'51"N, 30°25'2"E). Infected fruiting bodies were covered by white mycelia that later turned red or fuchsia. In the final stages of infection, the S. crispa fruiting bodies turned dark red or brown before rotting. The pathogen was isolated from the margin of the lesions by plating onto potato dextrose agar (PDA), and incubated at 25℃ in the dark for a week. Five pure culture fungal isolates were obtained. Collected isolates with similar morphology were described as Lecanicillium spp. (Zare et al., 2001). The colonies were raised, covered with white, the reverse side were violet brown, produced diffusing reddish-purple pigment. Conidiogenous cells produced singly, in pairs, verticillate or in dense irregular clusters on prostrate hyphae, at first flask-shaped, tapering into threadlike neck, with a size of 3.0-6.2×0.8-2.2 µm. Conidia were solitary, oval to subglobose, and 2.3-4.0×1.1-2.1 µm in size, similar to L. aphanocladii (Higo et al., 2021). For pathogenicity testing, ten fruiting bodies of S. crispa (planted in the bottles) were selected. Fungal cake of the isolate Bx-Ljb of L. aphanocladii were applied to the fruiting body of S. crispa, whereas pieces of sterile PDA medium were used as controls. All the bottles were incubated at 19±1℃, 85-100% relative humidity, and 18 h of light in the mushroom hothouse. A week later, the inoculated fruiting bodies developed brown spots and gradually expanding, with symptoms similar to the original diseased fruiting bodies. The controls remained healthy. The same fungus was reisolated from the infected fruiting bodies and subsequently identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. For molecular identification, the DNA of the isolates was extracted using a Fungi Genomic DNA Extraction kit (Solarbio, Beijing). The SSU, LSU, and TEF1-α genes were amplified with the primer as previously described (Zhou et al., 2018). The generated sequences were deposited in GenBank with accession numbers OR206377, OR206378, and OR204702, respectively. BLASTn analyses showed >99.2% identity with previously deposited sequences of L. aphanocladii. Based on the maximum likelihood method, phylogenetic analysis revealed 99% bootstrap support values with L. aphanocladii. The fungus was identified as L. aphanocladii based on morphological and multilocus phylogenetic analyses. To our knowledge, there are two reports of L. aphanocladii on fruiting bodies of Tremella fuciformis and Morchella sextelata in China, and this is the first report of this fungus causing rot of S. crispa in China. It may be a reminder that the risk of L. aphanocladii in mushroom production in China is gradually increasing. These results will contribute to developing managemental strategies for this disease in S. crispa.

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.

Ultrasound Intensify the Flavonoid Production of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), Fermentation Mycelia.

This research aimed to use a novel and effective ultrasound (US) approach for obtaining high bio-compound production, hence proposing strategies for boosting active ingredient biosynthesis. Furthermore, the US promotes several physiological effects on the relevant organelles in the cell, morphological effects on the structure of Phellinus igniarius mycelium, and increases the transfer of nutrients and metabolites. One suitable US condition for flavonoid fermentation was determined as once per day for 7-9 days at a frequency 22 + 40 kHz, power density 120 W/L, treated 10 min, treatment off time 7 s. The flavonoid content and production increased about 47.51% and 101.81%, respectively, compared with the untreated fermentation (P < 0.05). SEM showed that sonication changes the morphology and structure of Ph. igniarius mycelium; TEM reveals the ultrasonic treatment causes organelle aggregation. The ultrasound could affect the metabolism of the biosynthesis of the active ingredients.

Evaluation of the nutritional value, umami taste, and volatile organic compounds of Hypsizygus marmoreus by simulated salivary digestion in vitro.

Hypsizygus marmoreus is an edible medicinal mushroom species with a high dietary value. The main purpose of this study was to evaluate the nutritional value, umami taste, and volatile organic compounds (VOCs) of H. marmoreus treated with hot water combined with simulated salivary digestion in vitro. Seafood mushroom (Hm3) had the highest content of moisture, soluble polysaccharides, soluble proteins, and total flavonoids while white Hypsizygus marmoreus (Hm1) had the highest total phenolic content. Moreover, Hm1 had a more noticeable equivalent umami concentration (EUC) value, indicating the umami properties of Hm1 as a food or processing ingredient. Results from E-nose and HS-SPME-GC-MS revealed that the VOCs of Hm1 and brown Hypsizygus marmoreus (Hm2) were relatively similar, which differed substantially from Hm3. Among the 134 VOCs, 24 differential metabolites were identified by OPLS-DA analysis, characterized by VIP > 1, p-value < 0.05, and FC > 2 (pairwise comparisons). Furthermore, 10 biomarkers with VIP > 1 and p-value < 0.05 were identified by PLS-DA analysis based on the total differential metabolites to distinguish different strains of H. marmoreus. These results will benefit future research on the chemistry of H. marmoreus and serve as a guide for breeding, introducing, and using the species more effectively.

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.

First Report of Leaf blight Caused by Colletotrichum karstii on Banana Shrub (Michelia figo) in Sichuan, China.

Banana Shrub (Michelia figo (Lour.) Spreng.) is widely cultivated in most of southern China (Wu et al, 2008). It can be used to make essential oil and flower tea（Ma et al, 2012; Li et al, 2010）.The first symptoms were observed in Sept. 2020 at a grower's field in Banana shrub seedlings (0.6 ha), Ya'an city (29°30'N, 102°38'E), Hanyuan county. The symptoms re-occurred in May-June of 2021 and became prevalent from August to September. the incidence rate and the disease index were 40% and 22%, respectively. Initially, purplish-brown necrotic lesions appeared at the leaf tip with dark-brown edges. Progressively, necrosis spread, to the middle of the leaves, and the older area turned gray-white. Dark sunken lesions appeared in the necrotic areas and orange conidial masses were visible under humid conditions. Ten isolates were obtained on potato dextrose agar (PDA) from 10 leaf samples using previously described tissue isolation method (Fang et al. 1998). All the 10 isolates exhibited similar morphological characteristics. Grey to white aerial mycelium at the center and in dispersed tufts, with numerous dark conidiomata scattered over the surface, reverse was pale orange with numerous dark flecks corresponding to the ascomata, orange conidial masses were formed from mature conidiomata. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, apex round, the contents appearing granular 14.8 to 17.2 × 4.2 to 6.4 µm (average: 16.26 × 4.84 µm, n=30) as Colletotrichum spp. (Damm et al. 2012). For molecular identification, DNA was extracted from a representative isolate HXcjA using a plant genomic DNA extraction kit (Solarbio, Beijing). and the partial sequences of internal transcribed spacer region (ITS, OQ641677), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, OL614009), actin (ACT, OL614007), beta-tubulin (TUB2, OL614011), histone3 (HIS3, OL614010), and calmodulin (CAL, OL614008) were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT-512F/ACT-783R, CAL 228F/CAL 737R (Carbone et al. 1999), TUB1F/Bt2bR, CYLH3F/CYLH3R (Crous et al. 2004), respectively. BLASTn analysis for ITS, GAPDH, CAL, ACT, TUB2 and HIS3 sequences showed ≥99.7% identity to C. Karstii, namely, NR_144790 (532/532 bp), MK963048 (252/252 bp), MK390726 (431/431 bp), MG602039 (761/763 bp), (KJ954424, 294/294 bp), (KJ813519, 389/389 bp), respectively. The fungus was identified as C. karstii based on morphology and a multigene phylogeny. The conidial suspension (1 × 107 conidia/mL) with 0.05% Tween 80 buffer was used for pathogenicity test, by spraying 2-year-old Banana Shrub plants. Ten plants were inoculated with spore suspensions (approximately 2ml per plant). An equal number of plants were sprayed with 0.05% Tween 80 buffer to serve as a control. Fifteen days later, the inoculated plants showed similar symptoms as the original diseased plants but the controls remained asymptomatic. C. karstii was re-isolated from the infected leaves and identified by morphology and a multigene phylogeny. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of Banana Shrub leaf blight caused by C. karstii in China. This disease reduces the ornamental and economic value of Banana Shrub, and this work will provide a basis for the prevention and treatment of the disease in the future.

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.

Co-Fermentation of Edible Mushroom By-Products with Soybeans Enhances Nutritional Values, Isoflavone Aglycones, and Antioxidant Capacity of Douchi Koji.

Douchi is a traditional salt-fermented soybean food with various bioactivities, such as anti-oxidation, anti-diabetes, and anti-hypertension, which are greatly affected by the activities of protease and ß-glucosidase during koji production. Edible mushroom by-products are ideal ingredients for enhancing food flavor and nutritional quality due to their unique nutritional characteristics of high protein, rich amino acids, and low calories. However, there is no research on the preparation of Douchi by the mixed fermentation of edible mushroom by-products and soybeans. In this study, response surface methodology (RSM) was used to optimize the fermentation conditions of edible mushroom by-product Douchi koji (EMDK) with protease and ß-glucosidase activities as indicators, and the changes in the main bioactive compounds and antioxidant activities of unfermented raw samples (URS), Douchi koji without edible mushroom by-product (DKWE), and EMDK were compared. The results of single-factor tests and RSM showed that the optimal fermentation conditions of EMDK were the Aspergillus oryzae to Mucor racemosus ratio of 1:1, inoculation amount of 6%, edible mushroom amount of 21%, and fermentation time of 63 h, and the activities of protease and ß-glucosidase under these conditions were 796.03 ± 15.01 U/g and 1175.40 ± 36.98 U/g, respectively. Additionally, compared with URS and DKWE, the contents of total isoflavones and ß-glucoside isoflavones in EMDK were notably decreased, while the contents of amino nitrogen, total phenolics, total flavonoids, and aglycone isoflavone, as well as the antioxidant capacity were significantly increased. Furthermore, significant correlations were found between the above components and antioxidant capacity. These results showed that edible mushroom by-product could be incorporated into soybeans for co-fermentation, conferring higher nutritional value to and antioxidant capacity of Douchi koji.

Effects of ß-1,6-Glucan Synthase Gene (FfGS6) Overexpression on Stress Response and Fruit Body Development in Flammulina filiformis.

ß-1, 6-glucan synthase is a key enzyme of ß-1, 6-glucan synthesis, which plays a vital role in the cell wall cross-linking of fungi. However, the role of the ß-1, 6-glucan synthase gene in the development of the fruiting body and the stress response of macrofungi is largely unknown. In this study, four overexpression transformants of the ß-1, 6-glucan synthase gene (FfGS6) were successfully obtained, and gene function was studied in Flammulina filiformis. The overexpression of FfGS6 can increase the width of mycelium cells and improve the tolerance ability under mechanical injury and oxidative stress. Moreover, FfGS6 gene expression fluctuated in up-regulation during the recovery process of mycelium injury but showed a negative correlation with H2O2 concentration. Fruiting body phenotype tests showed that mycelia's recovery ability after scratching improved when the FfGS6 gene was overexpressed. However, primordia formation and the stipe elongation ability were significantly inhibited. Our findings indicate that FfGS6 is involved in regulating mycelial cell morphology, the mycelial stress response, and fruit body development in F. filiformis.

Reactive Oxygen Species Distribution Involved in Stipe Gradient Elongation in the Mushroom Flammulina filiformis.

The mushroom stipe raises the pileus above the substrate into a suitable position for dispersing spores. The stipe elongates at different speeds along its length, with the rate of elongation decreasing in a gradient from the top to the base. However, the molecular mechanisms underlying stipe gradient elongation are largely unknown. Here, we used the model basidiomycete mushroom Flammulina filiformis to investigate the mechanism of mushroom stipe elongation and the role of reactive oxygen species (ROS) signaling in this process. Our results show that O2- and H2O2 exhibit opposite gradient distributions in the stipe, with higher O2- levels in the elongation region (ER), and higher H2O2 levels in the stable region (SR). Moreover, NADPH-oxidase-encoding genes are up-regulated in the ER, have a function in producing O2-, and positively regulate stipe elongation. Genes encoding manganese superoxide dismutase (MnSOD) are up-regulated in the SR, have a function in producing H2O2, and negatively regulate stipe elongation. Altogether, our data demonstrate that ROS (O2-/H2O2) redistribution mediated by NADPH oxidase and MnSODs is linked to the gradient elongation of the F. filiformis stipe.

Transcriptome Profiling Reveals Candidate Genes Related to Stipe Gradient Elongation of Flammulina filiformis.

Stipe gradient elongation is an important and remarkable feature in the development of most mushroom fruiting bodies. However, its molecular mechanism has rarely been described. Here, the decreasing trend of stipe elongation and increasing trend of cell length in a gradient from the top to the base of the stipe were determined in a model basidiomycete mushroom: Flammulina filiformis. According to RNA-seq results, 1409 differentially expressed genes (DEGs) were identified among elongation region (ER), transition region (TR), and stable region (SR) samples, including 26 transcription factors (TFs). Based on Short Time-series Expression Miner (STEM) clustering of DEGs, clusters 1 and 3, with obvious expression trends that were consistent with or in contrast to the elongation rate, were screened. The cluster 1 DEGs were mainly involved in the GO cellular component category and KEGG genetic information processing class; however, the cluster 3 DEGs were mainly involved in metabolic processes. Furthermore, qRT-PCR confirmed that key genes of the long-chain fatty acid synthesis pathway were involved in stipe gradient elongation and regulated by NADPH oxidase-derived ROS signaling molecules. These findings provide an essential basis for understanding the molecular mechanism of stipe gradient elongation.

Morchella importuna Flavones Improve Intestinal Integrity in Dextran Sulfate Sodium-Challenged Mice.

Morchella importuna, as an edible fungus, has various health benefits. However, the effects of M. importuna on intestinal health are rarely investigated. Hence, this study aims to ascertain the influences of flavones from the fruiting bodies of M. importuna (hereinafter abbreviated as MIF) on dextran sulfate sodium (DSS)-induced damage to intestinal epithelial barrier in C57BL/6J mice. In this (14-day) study, 144 C57BL/6J mice were divided into four groups: (1) Control; (2) DSS treatment; (3) DSS treatment + 100 mg/kg MIF (LMIF); (4) DSS treatment + 200 mg/kg MIF (HMIF). On days 8-14, mice in the challenged groups were challenged with 3.5% DSS, while the control group received an equal volume of normal saline. Then, serum and intestinal samples were obtained from all mice. The results showed that MIF ingestion enhanced intestinal integrity in DSS-challenged mice, as evinced by the elevated (p < 0.05) abundances of occludin, claudin-1, and zonula occludens-1 proteins. Meanwhile, MIF ingestion reduced (p < 0.05) the colonic interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) concentrations and increased the superoxide dismutase and catalase activities and Shannon and Simpson indices in DSS-challenged mice. Moreover, MIF ingestion reduced (p < 0.05) the abundance of phospho-nuclear factor (NF)-κB and increased the abundance of phospho-Nrf2 in DSS-challenged mice. Taken together, MIF protects against intestinal barrier injury in C57BL/6J mice via a mechanism that involves inhibiting NF-κB activation and promoting Nrf2 activation, as well as regulating intestinal microbiota.

Morchella importuna Polysaccharides Alleviate Carbon Tetrachloride-Induced Hepatic Oxidative Injury in Mice.

This study aimed to investigate the effects of Morchella importuna polysaccharides (MIPs) on carbon tetrachloride (CCl4)-induced hepatic damage in mice. A total of 144 female mice were randomly assigned to four treatment groups, namely, control, CCl4, low-dose MIP (LMIP) group, and high-dose MIP (HMIP) group. After the 10-day experiment, serum and liver were sampled for biochemical and metabolomic analyses. The HMIPs markedly decreased the liver weight under CCl4 intoxication. Furthermore, the significantly elevated concentrations of five serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, triglyceride, total cholesterol, and total bile acid under CCl4 treatment were subverted by MIP administration in a dose-dependent manner. Moreover, MIPs relieved the increased hepatic malonaldehyde and protein carbonyl content and the decreased superoxide dismutase and catalase contents caused by CCl4 intoxication. There was also a dose-dependent decrease in the CCl4-induced inflammatory indices, such as the levels of interleukin-1, interleukin-6, tumor necrosis factor-alpha, and myeloperoxidase, with MIP administration. Subsequent ultra-high performance liquid chromatography-tandem mass spectrometry-based serum metabolomics identified nine metabolites between the control and CCl4 groups and 10 metabolites between the HMIP and CCl4 groups, including some critical metabolites involved in flavonoid biosynthesis, amino acid metabolism, energy metabolism, and toxicant degradation. These novel findings indicate that MIPs may be of therapeutic value in alleviating the oxidative stress and inflammation caused by CCl4. Liquid chromatography-mass spectrometry-based metabolomics provides a valuable opportunity for identifying potential biomarkers and elucidating the protective mechanisms of medicinal mushrooms against hepatic oxidative injury.

Temperature affects substrate-associated bacterial composition during Ganoderma lucidum hyphal growth.

The growth of the well-known fungus Ganoderma lucidum is influenced by temperature, which has an impact on the associated microbial structure in the substrate. In this study, we analyzed the bacterial diversity of the substrate at different temperatures using next-generation sequencing technology. A total of 513 733 sequences from 15 samples were assigned to 19 bacterial phyla. The samples were dominated by Proteobacteria, followed by Firmicutes; the 2 phyla exhibited opposite changes with elevated temperature. Bacterial genera showed different abundances at different temperatures, in which Sediminibacterium maintained a stable abundance below 40 °C, while Ochrobactrum and Rhodococcus were enriched with elevated temperature and both showed their highest abundances at 40 °C. Functional prediction uncovered 39 identified KEGG pathways, and bacterial genes involved in the membrane transport pathway exhibited the highest abundance subject to heat (40 °C) during the growth of G. lucidum. In general, our findings illustrated the influence of temperatures on G. lucidum mycelial morphology and the bacterial community in the substrate, and the results will facilitate cultivation of this fungus.