New insights into the stipitate hydnoid fungi Sarcodon, Hydnellum, and the formerly informally defined Neosarcodon, with emphasis on the edible species marketed in Southwest China.

Sarcodon and Hydnellum are two ectomycorrhizal genera of important ecological and economic value in Southwest China, and they are common in the free markets in this region. It was estimated that more than 1,500 tonnes of them were sold as edible per year, but there was little information about the taxonomic placements of these edible mushrooms sold in the markets. Traditional concepts of the two genera have also been challenged recently, and circumscription of Sarcodon and the informally defined clade "Neosarcodon" remained unresolved. In the present study, specimens collected in the field and purchased from the markets in Southwest China were analyzed based on morphological characters and DNA sequences. Phylogeny of the traditional Sarcodon s. lat. and Hydnellum s. lat. was reconstructed from the combined internal transcribed spacer (ITS), nuclear large ribosomal subunit (nLSU) and RNA polymerase II second largest subunit (RPB2) dataset based on expanded samples to reevaluate the taxonomic placements of the two genera. In the present molecular analyses, four distinct clades were recovered and strongly supported: Hydnellum, Neosarcodon, Phellodon and Sarcodon. Neosarcodon is formally introduced as a generic name to include nine species previously placed in Sarcodon, and the delimitation of Sarcodon is revised based on phylogenetic and morphological studies. Phylogenetic analyses also revealed an unexpected species diversity (17 phylogenetic species) of Sarcodon and Hydnellum in the markets; nine phylogenetic species of Sarcodon and eight of Hydnellum were uncovered from the samples collected in the markets. Eight species were resolved in the traditional S. imbricatus complex, with S. imbricatus s.str. being the most common edible stipitate hydnoid fungal species. Three of the edible Hydnellum species (H. edulium, H. subalpinum, and H. subscabrosellum), and five separated from the S. imbricatus complex (Sarcodon flavidus, S. giganteus, S. neosquamosus, S. nigrosquamosus, and S. pseudoimbricatus), are described as new. Three new Chinese records (H. illudens, H. martioflavum, and H. versipelle), and the notable S. imbricatus and S. leucopus are also reported.

Comparative analyses of Pleurotus pulmonarius mitochondrial genomes reveal two major lineages of mini oyster mushroom cultivars.

Pleurotus pulmonarius, commonly known as the mini oyster mushroom, is highly esteemed for its crisp texture and umami flavor. Limited genetic diversity among P. pulmonarius cultivars raises concerns regarding its sustainable industrial production. To delve into the maternal genetic diversity of the principal P. pulmonarius cultivars, 36 cultivars and five wild isolates were subjected to de novo sequencing and assembly to generate high-quality mitogenome sequences. The P. pulmonarius mitogenomes had lengths ranging from 69,096 to 72,905 base pairs. The mitogenome sizes of P. pulmonarius and those of other mushroom species in the Pleurotus genus showed a significant positive correlation with the counts of LAGLIDAG and GIY-YIG homing endonucleases encoded by intronic open reading frames. A comparison of gene arrangements revealed an inversion of a fragment containing atp9-nad3-nad2 between P. pulmonarius and P. ostreatus. The mitogenomes of P. pulmonarius were clustered into three distinct clades, two of which were crowded with commercial cultivars. Clade I, all of which possess an inserted dpo gene, shared a maternal origin linked to an ancestral cultivar from Taiwan. Primers were designed to target the dpo gene, potentially safeguarding intellectual property rights. The wild isolates in Clade III exhibited more divergent mitogenomes, rendering them valuable for breeding.

Comparative analyses of Flammulina filiformis mitochondrial genomes reveal high length polymorphism in intergenic regions and multiple intron gain/loss in cox1.

The golden-needle mushroom Flammulina filiformis is one of the bulk mushroom products in the world. This study obtained complete mitogenomes of 44 wild isolates collected from nine provinces and two artificially bred cultivars of F. filiformis, together with three Flammulina rossica isolates and one Flammulina fennae isolate for comparison. The mitogenome of F. filiformis ranged from 83,540 bp to 90,938 bp, consisting of 14 conserved protein-coding genes (PCGs), two rRNA genes, and 25 tRNA genes. To the best of our knowledge, it contained the highest proportion of intergenic regions compared to the other known Basidiomycota mitogenomes. Introns and intergenic regions were two major contributing factors to the total size of the F. filiformis mitogenome. The conserved PCG cox3 is located in an intron of another conserved PCG, nad5. This is a unique phenomenon in all known fungal mitogenomes. Gain/loss of introns was observed in cox1, nad5, and rnl. Length polymorphism was widely observed in intergenic regions. Accordingly, primers were designed as useful markers for rapid identification of F. filiformis isolates with differentiated mitogenomes. Our findings provide a basis for further studies related to variety identification and population genetics of this economically important mushroom.

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.

Application of the mushroom volatile 1-octen-3-ol to suppress a morel disease caused by Paecilomyces penicillatus.

Morels (Morchella spp.) are of great economic and scientific value. Paecilomyces penicillatus can cause white mold disease (WMD) widely emerging on morel ascocarps and is also a potential factor causing morel fructification failure. 1-octen-3-ol is a mushroom volatile compound with broad-spectrum antimicrobial activities. This study aimed to control the morel disease caused by P. penicillatus through suppressing P. penicillatus in the soil cultivated with Morchella sextelata using 1-octen-3-ol. Safe concentration of 1-octen-3-ol was estimated by comparing its inhibitory effect against P. penicillatus and M. sextelata, respectively, with mycelium-growth experiments on agar plates. The results showed that M. sextelata possesses a higher tolerance to 1-octen-3-ol than P. penicillatus with a 1-octen-3-ol concentration between 0 and 200 µL/L. Based on that, a sandy soil was supplemented with low (50 µL/L) or high concentration (200 µL/L) of 1-octen-3-ol. The effects of 1-octen-3-ol on soil microbial communities, WMD incidence, and morel yield were investigated. Compared to the non-supplemented control group, the incidence of WMD and the proportion of Paecilomyces in the soils of low- and high-concentration treatment groups were significantly decreased, corresponding to a significant increase in morel ascocarp yield. It suggests that 1-octen-3-ol effectively suppressed P. penicillatus in the soil, thereby reducing the severity of WMD and improving the morel yield. The diversity of soil bacterial communities was also altered by 1-octen-3-ol supplement. The proportion of Rhodococcus spp. in the soil was positively correlated with the 1-octen-3-ol concentration and ascocarp yield, suggesting its potential role in improving morel yield. KEY POINTS: • A novel method for morel disease suppression was proposed. • Paecilomyces in soil affects white mold disease and fructification yield of morel. • 1-Octen-3-ol suppresses Paecilomyces and alters bacterial community in soil.

Dual RNA-Seq Analysis of the Interaction Between Edible Fungus Morchella sextelata and Its Pathogenic Fungus Paecilomyces penicillatus Uncovers the Candidate Defense and Pathogenic Factors.

Morels (Morchella spp.) are economically important mushrooms cultivated in many countries. However, their production and quality are hindered by white mold disease because of Paecilomyces penicillatus infection. In this study, we aimed to understand the genetic mechanisms of interactions between P. penicillatus and Morchella. M. sextelata, the most prevalent species of Morchella in China, was inoculated with P. penicillatus; then, the expression profiles of both fungi were determined simultaneously at 3 and 6 days post-inoculation (dpi) using a dual RNA-Seq approach. A total of 460 and 313 differentially expressed genes (DEGs) were identified in P. penicillatus and M. sextelata, respectively. The CAZymes of ß-glucanases and mannanases, as well as subtilase family, were upregulated in P. penicillatus, which might be involved in the degradation of M. sextelata cell walls. Chitin recognition protein, caffeine-induced death protein, and putative apoptosis-inducing protein were upregulated, while cyclin was downregulated in infected M. sextelata. This indicates that P. penicillatus could trigger programmed cell death in M. sextelata after infection. Laccase-2, tyrosinases, and cytochrome P450s were also upregulated in M. sextelata. The increased expression levels of these genes suggest that M. sextelata could detoxify the P. penicillatus toxins and also form a melanin barrier against P. penicillatus invasion. The potential pathogenic mechanisms of P. penicillatus on M. sextelata and the defense mechanisms of M. sextelata against P. penicillatus were well described.

Morel Production Related to Soil Microbial Diversity and Evenness.

Black morel is a widely prized ascomycetous mushroom with culinary value. It was once uncultivable but can now be cultivated routinely in ordinary farmland soils. Large-scale morel farming sometimes encounters nonfructification for unknown reasons. In spring 2020, many morel farms in the area of Chengdu-Plain, China, exhibited no fructification at all, causing disastrous economic loss to the farmers. To determine potential ecological factors associated with the different performance of morel production in these farms, 21 affected sites versus 11 sites with normal fructification performance were analyzed to compare soil microbiota and physiochemical characteristics during fructification. The results indicated that soil physiochemical characteristics were unlikely to be a major reason for the difference between successful fructification and nonfructification. The soils with successful fructification had significantly higher diversity in both the fungal and bacterial communities than those with nonfructification. Morel yield was positively correlated with the α-diversity of fungal communities. The higher diversity of the successfully fructified soils was contributed by community evenness rather than taxonomic richness. In contrast, most nonfructification soils were dominated by a high proportion of a certain fungal genus, typically Acremonium or Mortierella, in the fungal communities. Our findings demonstrate the importance of microbial ecology to the large-scale agroindustry of soil-cultivated mushrooms. IMPORTANCE Saprotrophic mushrooms cultivated in soils are subject to complex influences from soil microbial communities. Research on growing edible mushrooms has revealed connections between fungi and a few species of growth-promoting bacteria colonizing the mycosphere. The composition and diversity of the whole microbial community may also have an influence on the growth and production of soil-saprotrophic mushrooms. Morel mushrooms (Morchella spp.) are economically and culturally important and are widely prized throughout the world. This study used the large-scale farming of morels as an example of an agroecosystem for soil-saprotrophic mushroom cultivation. It demonstrated a typical pattern of how the microbial ecology in soil agroecosystems, especially the α-diversity level and community evenness among soil fungal taxa, could affect the production of high-value cash crops and the income of farmers.

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.

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.

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.

Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges.

A novel phytase of Acidobacteria was identified from a soil metagenome, cloned, overexpressed, and purified. It has low sequence similarity (<44%) to all the known phytases. At the optimum pH (2.5), the phytase shows an activity level of 1,792 µmol/min/mg at physiological temperature (37°C) and could retain 92% residual activity after 30 min, indicating the phytase is acidophilic and acidostable. However the phytase shows poor stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time of the engineered phytase at 60°C and 80°C, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.