Summary 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.

SummaryA 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.

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.

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.

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.

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.

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.

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 Fe 2+ , 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.