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Ischaemic stroke is a common neurological disease and a leading cause of severe disability and death in developed countries. In most cases, stroke is thought to be a multifactorial disorder or complex trait for which classic patterns of inheritance cannot be shown. Xuesaitong is one of the most commonly used medicines for treating ischemic stroke in China. However, compared to the conventional therapy, the effectiveness and safety of Xuesaitong for ischemic stroke needs to be further systematically reviewed and determined.Relevant randomized controlled trials (RCTs) examining the use of the Xuesaitong soft capsule in the treatment of patients with ischemic stroke were identified from databases, including the China National Knowledge Infrastructure, Wanfang, PubMed, Embase, and Web of Science databases. Next, 2 researchers independently extracted information from the included studies, analyzed the data using STATA 15.0 software, and evaluated the quality of the included studies using RevMan 5.3.A total of 17 RCTs (comprising 1,942 patients with ischemic stroke) were included in the meta-analysis. The meta-analysis results showed that the Xuesaitong soft capsule treatment increased patients' total effective rate compared to conventional or other drug treatments, and improved patients' Clinical Severity Score (CSS scores) or Barthel index (BI) score. A further subgroup analysis stratified by different treatment times showed that Xuesaitong soft capsule treatment at 4 and 8 weeks improved CSS scores more than treatment at 2 weeks in patients with ischemic stroke. Additionally, the Xuesaitong soft capsule also significantly improved plasma viscosity, whole-blood viscosity at high and low shear rates, fibrinogen, hematocrit, and the effect on traditional Chinese medicine (TCM) single symptoms or signs in patients with ischemic stroke.In summary, compared to conventional or other drug treatments, the Xuesaitong soft capsule treatment was beneficial in improving patients' TCM symptoms (e.g., crooked mouth and tongue, and dizziness) and various indicators. Further, Xuesaitong soft capsule may be a safe and effective drug for the treatment of ischemic stroke. And large-scale randomized clinical trials are needed to further confirm our findings.

This paper examines the impact of cross-sectoral climate policy on forest carbon sinks. Due to the complexity of the climate change issue and the professional division of labor among government departments, cross-sectoral cooperation in formulating climate policy is a desirable strategy. Forest carbon sinks play an important role in addressing climate change, but there are few studies focusing on forest carbon sinks and cross-sectoral climate policies. Thus, based on the panel data of 30 provinces and cities in China from 2007 to 2020, this paper establishes a benchmark regression model and a spatial panel model to analyze the impact of cross-sectoral climate policies on forest carbon sinks. We find that cross-sectoral climate policies positively impact forest carbon sinks. Under the influence of the “demonstration effect”, we find that cross-sectoral climate policies have a positive impact not only on the forest carbon sinks in the region but also on those in the neighboring region. Further analysis shows that for provinces with less developed forestry industry and small forest areas, the positive effect of cross-sectoral climate policies on forest carbon sinks is more obvious. Overall, this paper can serve as an important reference for local governments to formulate climate policies and increase the capacity of forest carbon sinks.

Metabolite detection from complex biological samples faces challenges due to interference from endogenous substrates and the inherent limitation of multiple subsequent tandem scanning rates of instruments. Here, a new integrated approach based on gas-phase fractionation with a staggered mass range (sGPF) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) molecular network was developed to accelerate the data processing of the targeted and untargeted constituents absorbed in rats after oral administration of the traditional Chinese medicine (TCM) prescription Gui Ling Ji (GLJ). Compared with three conventional acquisition methods, sGPF at 3, 5, and 7 mass fractions could enhance MS/MS coverage with an increased MS/MS triggering rate of 29.4-206.2% over data-dependent acquisition (DDA), fast DDA and gas-phase fractionation. A mass range fraction setting of five optimized the performance. Based on the similar diagnostic fragment ions and characteristic neutral loss behaviors in the DDA-MS/MS spectrum, an initial molecular network of GLJ was created with the help of the global natural products social molecular networking (GNPS) platform. Furthermore, to remove the endogenous interference nodes, Cytoscape software was adopted to produce a clean and concise molecular network of prototype compounds and their corresponding metabolites. Using this strategy, a total of 210 compounds, including 59 prototype constituents and 151 metabolites, was unambiguously or tentatively identified in GLJ. This first systematic metabolic study of GLJ in vivo elucidated the potential pharmacodynamic basis of GLJ in clinical treatment. More importantly, this work can serve as a practical example and establish a guide for rapidly identifying TCM metabolites in biological matrices.

Background/Aims: Heat stress could cause huge losses for Lentinula edodes in China and other Asian cultivation areas. Yet our understanding of mechanism how to defend to heat stress is incomplete. Methods: Using heat-tolerant and heat-sensitive strains of L. edodes, we reported a combined proteome and transcriptome analysis of L. edodes response to 40 °C heat stress for 24 h. Meanwhile, the effect of LeDnaJ on the thermotolerance and IAA (indoleacetic acid) biosynthesis in L. edodes was analyzed via the over-expression method. Results: The proteome results revealed that HSPs (heat shock proteins) such as Hsp40 (DnaJ), Hsp70, Hsp90 and key enzymes involved in tryptophan and IAA metabolism process LeTrpE, LeTrpD, LeTam-1, LeYUCCA were more highly expressed in S606 than in YS3357, demonstrating that HSPs and tryptophan as well as IAA metabolism pathway should play an important role in thermotolerance. Over-expression of LeDnaJ gene in S606 strains showed better tolerance to heat stress. It was also documented that intracellular IAA accumulation of S606 (8-fold up) was more than YS3357 (2-fold up), and exogenous IAA enhanced L. edodes tolerance to heat stress. Conclusion: Our data support the interest of LeTrpE, LeDnaJ, tryptophan and IAA could play a pivotal role in enhancing organism thermotolerance.

The prominent problem of "uncertainty" has been frustrating morel farming since the commercial production of Morchella mushrooms was realized in 2012 in China. Spawn aging may be the main underlying reason. In this paper, aging in cultivated strains of M. importuna T4 and M. sextelata T6 was achieved by successive subculturing. Oxidative stress, mycelial growth rate, biomass, sclerotial formation, pigmentation, and yield of different subcultures were determined. The results suggested that M. importuna T4 and M. sextelata T6 exhibited systemic senescence manifested as 24 and 17 subcultures and lifespan of 3048 and 2040 h, respectively. Aging showed a close relevance to oxidative stress. Regression analysis revealed a strong positive correlation between time of subculturing (aging) and lipid peroxidation (oxidative stress) in both morels. In addition, pigmentation tended to increase, while the number of sclerotia tended to decrease, with the rise of subculturing times in both morels. Moreover, the mycelial growth rate and biomass of the last two subcultures were significantly lower than those of others, indicating that cultural characteristics may be used as signs of seriously aging culture. Finally, the yield of subcultures was significantly lower than that of the original strains in artificial cultivation. Regression analysis showed a strong negative correlation between time of subculturing and yield in two morels. This work will improve the understanding of "uncertainty" and thus be beneficial for stable development of morel farming.

The commercial production of Morchella mushrooms calls for urgent breeding of excellent varieties or strains with appropriate tools, such as protoplast fusion. However, the protoplast fusion in morels has not been studied. In this paper, interspecific hybridization between cultivated morels of M. importuna and M. sextelata by PEG-induced protoplast fusion was conducted. Apart from functional complementation of double inactivated protoplasts, the fusants were characterized by cultural and cultivated characters and molecular markers of random amplified polymorphic DNA (RAPD). The results suggested that the hybrids and their parents showed significant difference in their inoculum recovery time, mycelial growth rate, yield of cultivation and total amino acid content of ascocarps. Moreover, positive barrage reactions were observed between parental strains as well as between each parent and a hybrid line. A dendrogram created on the basis of RAPD fingerprints exhibited three major clusters, in which morel hybrids showed intra-cluster variations, M. sextelata #6 formed an out group, while M. importuna #4 was phylogenetically closer to morel hybrids. All the results demonstrated that real fusants were obtained in our study. Protoplast fusion may provide an ideal alternative for new strain selection, and thus will promote the healthy development of morel industry.

Morchella crassipes (Vent.) Pers., a typical yellow morel species with high economic value, is mainly distributed in the low altitude plains of Eurasia. However, rare research has been performed on its genomics and polarity, thus limiting its research and development. Here, we reported a fine physical map of the nuclear genome at the subchromosomal-scale and the complete mitochondrial genome of M. crassipes. The complete size of the nuclear genome was 56.7 Mb, and 23 scaffolds were assembled, with eight of them being complete chromosomes. A total of 11,565 encoding proteins were predicted. The divergence time analysis showed that M. crassipes representing yellow morels differentiated with black morels at ~33.98 Mya (million years), with 150 gene families contracted and expanded in M. crassipes versus the two black morels (M. snyderi and M. importuna). Furthermore, 409 CAZYme genes were annotated in M. crassipes, containing almost all plant cell wall degrading enzymes compared with the mycorrhizal fungi (truffles). Genomic annotation of mating type loci and amplification of the mating genes in the monospore population was conducted, the results indicated that M. crassipes is a heterothallic fungus. Additionally, a complete circular mitochondrial genome of M. crassipes was assembled, the size reached as large as 531,195 bp. It can be observed that the strikingly large size was the biggest up till now, coupled with 14 core conserved mitochondrial protein-coding genes, two rRNAs, 31 tRNAs, 51 introns, and 412 ncORFs. The total length of intron sequences accounted for 53.67% of the mitochondrial genome, with 19 introns having a length over 5 kb. Particularly, 221 of 412 ncORFs were distributed within 51 introns, and the total length of the ncORFs sequence accounted for 40.83% of the mitochondrial genome, and 297 ncORFs had expression activity in the mycelium stage, suggesting their potential functions in M. crassipes. Meanwhile, there was a high degree of repetition (51.31%) in the mitochondria of M. crassipes. Thus, the large number of introns, ncORFs and internal repeat sequences may contribute jointly to the largest fungal mitochondrial genome to date. The fine physical maps of nuclear genome and mitochondrial genome obtained in this study will open a new door for better understanding of the mysterious species of M. crassipes.

Our previous study found that isolates from morel stipes showed a large proportion of mating type deletion, which implied the nuclear evolution and spatial heterogeneity. In this paper, the specific mating type genes of Mat1-1-1 and Mat1-2-1 in Morchella importuna were analyzed using the relative quantitative method of qRT-PCR to study the uneven nuclear distribution in morel life cycle. The results demonstrated obvious unbalanced distribution of two karyotypes in tested commercially cultivated strains and stipe issue isolates from cultivated ascocarps. The ratio of two mating type genes in tissue isolates from different parts of the ascocarps (base soil, inside and surface of stipe, inside and outside of cap pit) was also different. The disequilibrium also existed in primordium, young ascocarp and mature ascocarp. Moreover, the imbalance in different positions of mature ascocarp (base of stipe, middle of stipe, junction of stipe and cap, middle of cap, and top of cap) gradually converged from bottom to top with the largest difference in base of stipe and almost equal distribution of two mating types in morel caps, which indicated that the deviation of two kinds of nuclei in sexual reproduction stage slowed down. More than half of the protoplast regenerated strains from the heterokaryotic M04 strain were homokaryotic, with the dominant Mat1-1-1 genotype accounted for 85.7%. Overall, the Mat1-1-1 genotype nucleus dominated in all samples from different stage of the life cycle. The imbalance of nuclear distribution during the life cycle of M. importuna may imply the evolutionary-driven nuclear-level selection. This is the first study of spatial and temporal disequilibrium of nuclear distribution in macro-ascomycetes. For scale cultivation, it is necessary to perform genotype integrity testing in potential cultivated strains to ensure the existence of both mating type genes.

In March 2013, pileus rot disease was first observed on cultivated Morchella importuna. Infected ascomata were covered by white, velvety mycelia mainly on the pileus, and the infection resulted in malformed fruiting bodies. The causal pathogen was identified as Diploospora longispora based on its morphology and the internal transcribed spacer of its ribosomal DNA sequences. After inoculation of young ascomata with the isolates, the original symptoms were reproduced, and the same pathogen was reisolated from diseased ascomata. This is the first report of pileus rot disease on morels caused by D. longisporain China.

The commercial production of Morchella mushrooms, especially M. importuna , has been realized since 2012 in China, which facilitates the fundamental studies of Morchella spp. In this paper, the cytological characteristics at three stages of ascocarpic development and the nuclear behavior during meiosis and ascosporogenesis in cultivated strain M. importuna 1 # was visualized by confocal laser scanning microscopy. The results suggested that the strain sporulated at sporulation stage of ascocarpic development. A total of six nuclear divisions typically took place during ascosporogenesis. The first and second divisions were meiotic in which the single diploid nucleus divided into four haploid nuclei. The subsequent mitosis gave rise to eight nuclei, and eight incipient ascospores with one nucleus in each spore were formed after spore delimitation in the clavate ascus. Then, the nucleus in most of the young ascospore underwent three successive mitoses producing 6–8 haploid nuclei in each mature spore, and thus the multinucleate ascospores in each ascus were all homokaryons. To the best of our knowledge, this is the first dynamic tracing study of nuclear behavior during meiosis and ascosporogenesis in cultivated morels, and the spore delimitation time is also the first report. The study will be beneficial for the genetics study and strain breeding of Morchella mushrooms.

Lentinula edodes has the potential to degrade woody and nonwoody lignocellulosic biomass. However, the mechanism of lignocellulose degradation by L. edodes is unclear. The aim of this work is to explore the profiling of soluble secreted proteins involved in lignocellulose degradation in L. edodes. For that, we compared the secretomes of L. edodes grown on microcrystalline cellulose, cellulose with lignosulfonate and glucose. Based on nanoliquid chromatography coupled with tandem mass spectrometry of whole-protein hydrolysate, 230 proteins were identified. Label-free proteomic analysis showed that the most abundant carbohydrate-active enzymes involved in polysaccharide hydrolysis were endo-β-1,4-glucanase, α-galactosidase, polygalacturonase and glucoamylase in both cellulosic secretomes. In contrast, enzymes involved in lignin degradation were most abundant in glucose culture, with laccase 1 being the predominant protein (13.13%). When the cellulose and cellulose with lignosulfonate secretomes were compared, the abundance of cellulases and hemicellulases was higher in cellulose with lignosulfonate cultures, which was confirmed by enzyme activity assays. In addition, qRT-PCR analysis demonstrated that the expression levels of genes encoding cellulases and hemicellulases were significantly increased (by 32.2- to 1166.7-fold) when L. edodes was grown in cellulose with lignosulfonate medium. Biological significance In this article, the secretomes of L. edodes grown on three different carbon sources were compared. The presented results revealed the profiling of extracellular enzymes involved in lignocellulose degradation, which is helpful to further explore the mechanism of biomass bioconversion by L. edodes.

The complete mitochondrial genome of Morchella importuna, the famous edible and medicinal mushroom, was assembled as a 272,238 bp single circular dsDNA. As the largest mitogenome among fungi, it exhibits several distinct characteristics. The mitogenome of M. importuna encoded 14 core conserved mitochondrial protein-coding genes and 151 mitochondrial non-conserved open reading frames (ncORFs) were predicted, of which 61 were annotated as homing endonuclease genes, and 108 were confirmed to be expressed during the vegetative growth stages of M. importuna. In addition, 34 introns were identified in seven core genes (cob, cox1, cox2, cox3, nad1, nad4 and nad5) and two rRNA genes (rrnS and rrnL) with a length from 383 bp to 7453 bp, and eight large introns with a length range of 2340 bp to 7453 bp contained multiple intronic mtORFs. Moreover, 34 group I (IA, IB, IC1, IC2, ID and derived group I introns) and four group II intron domains were identified for the 34 introns, including five hybrid ones. Furthermore, the M. importuna mitogenome showed the presence of about 18.7% mitogenomic interspersed repeats. These and the aforementioned ncORFs and introns, contributed to the enlarged size of the mitogenome.

True morels (Morchella spp.) are edible, medicinal mushrooms which have recently been artificially cultivated in China but stable production remains a problem. Here, we describe complete and comprehensive transcriptome of Morchella importuna at the stages of vegetative mycelium (VM), initial sclerotium (IS) and mature sclerotium (MS) by deep transcriptional sequencing and de novo assembly for the first time and which will potentially provide useful information for improving its cultivation. A total of 26,496 genes were identified with a contig N50 length of 1763 bp and an average length of over 1064 bp. Additionally, 11,957 open reading frames (ORFs) were predicted and 9676 of them (80.9%) were annotated. The 2605 differentially expressed genes (DEGs) identified by gene expression clustering were mainly involved with energy metabolism and could be divided into three broad clusters, of which genes in cluster_1 and cluster_2 were involved in the metabolic process of carbohydrate, polysaccharide, hydrolase, caprolactam, beta-galactosidase, and disaccharide, respectively. Genes in cluster_3 were the largest category, mainly identified with the catalytic activity and transporter activity. Overall, the enzymes involved in the carbohydrate metabolism were highly expressed, and the CAZyme (carbohydrate-active enzyme) genes were significantly expressed within cluster_3. For expression verification, 16 CAZYme genes were selected for qRT-PCR, and the results suggested that the catabolism of carbohydrates occurs mainly in the vegetative mycelium stage, and the anabolism of the energy-rich substances is the main event of mycelial growth and sclerotial morphogenesis of M. importuna.

True morels (Morchella spp.) are edible, medicinal mushrooms which have recently been artificially cultivated in China but stable production remains a problem. Here, we describe complete and comprehensive transcriptome of Morchella importuna at the stages of vegetative mycelium (VM), initial sclerotium (IS) and mature sclerotium (MS) by deep transcriptional sequencing and de novo assembly for the first time and which will potentially provide useful information for improving its cultivation. A total of 26,496 genes were identified with a contig N50 length of 1763 bp and an average length of over 1064 bp. Additionally, 11,957 open reading frames (ORFs) were predicted and 9676 of them (80.9%) were annotated. The 2605 differentially expressed genes (DEGs) identified by gene expression clustering were mainly involved with energy metabolism and could be divided into three broad clusters, of which genes in cluster_1 and cluster_2 were involved in the metabolic process of carbohydrate, polysaccharide, hydrolase, caprolactam, beta-galactosidase, and disaccharide, respectively. Genes in cluster_3 were the largest category, mainly identified with the catalytic activity and transporter activity. Overall, the enzymes involved in the carbohydrate metabolism were highly expressed, and the CAZyme (carbohydrate-active enzyme) genes were significantly expressed within cluster_3. For expression verification, 16 CAZYme genes were selected for qRT-PCR, and the results suggested that the catabolism of carbohydrates occurs mainly in the vegetative mycelium stage, and the anabolism of the energy-rich substances is the main event of mycelial growth and sclerotial morphogenesis of M. importuna. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-1614-y) contains supplementary material, which is available to authorized users.

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya), the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution.

Sclerotial formation is a key phase of the morel life cycle and lipids have been recorded as the main cytoplasmic reserves in sclerotia of Morchella fungi without any experimental verification. In this study, the ultrastructural features of the undifferentiated mycelia stage (MS) and three main sclerotial differentiation states (sclerotial initial [SI], sclerotial development [SD] and sclerotial maturation [SM]) were compared by transmission electron microscopy. The nature of the energy-rich substance in hypha and sclerotium of Morchella importuna was qualitatively investigated by confocal laser scanning microscopy and quantitatively studied by extraction of lipids. Sclerotia were observed to form from the repeated branching and enlargement of either terminal hyphae or subordinate hyphal branches, indicating a complex type of sclerotial development. Autophagy and apoptosis were involved in the sclerotial metamorphosis of the cultivated strain of M. importuna. During the SI phase, the characteristic features of autophagy (vacuolation, coalescence of small vacuoles, existence of autophagosomes and engulfment of autophagosomes by vacuoles) were observed. At the SD phase, apoptotic characteristics (condensation of the cytoplasm and nucleus, shrinkage of plasma membrane, extensive plasma membrane blebbing and existence of phagosomes) could be seen in some developing sclerotial cells. In the final stage of sclerotial morphogensis, the sclerotial cells showed a necrotic mode of cell death. In addition, confocal laser imaging studies of live cells indicated that the energy-rich substance in morel hyphae and sclerotia was lipid. The lipid content in sclerotia was significantly more than that in hyphal cells. To the best of our knowledge, this is the first detailed ultrastructural description highlighting the involvement of autophagy and apoptosis in sclerotial metamorphosis of Morchella species and lipid accumulation during morel sclerotial development was also first experimentally verified. This work will promote a better understanding of the mechanism of morel sclerotial metamorphosis.

Lentinula edodes, one of the most popular, edible mushroom species with a high content of proteins and polysaccharides as well as unique aroma, is widely cultivated in many Asian countries, especially in China, Japan and Korea. As a white rot fungus with lignocellulose degradation ability, L. edodes has the potential for application in the utilization of agriculture straw resources. Here, we report its 41.8-Mb genome, encoding 14,889 predicted genes. Through a phylogenetic analysis with model species of fungi, the evolutionary divergence time of L. edodes and Gymnopus luxurians was estimated to be 39 MYA. The carbohydrate-active enzyme genes in L. edodes were compared with those of the other 25 fungal species, and 101 lignocellulolytic enzymes were identified in L. edodes, similar to other white rot fungi. Transcriptome analysis showed that the expression of genes encoding two cellulases and 16 transcription factor was up-regulated when mycelia were cultivated for 120 minutes in cellulose medium versus glucose medium. Our results will foster a better understanding of the molecular mechanism of lignocellulose degradation and provide the basis for partial replacement of wood sawdust with agricultural wastes in L. edodes cultivation.

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

In recent years, the artificial cultivation of Morchella mushrooms that belong to Elata Clade, including Morchella elata, has been developed rapidly in China. However, the prominent problem of spawn aging has been frustrating the morel farming. In this paper, aging in M. elata was achieved from 12 to 17 subcultures and lifespan of 1536-2256 h by successive subculturing. The lifespan can be roughly divided into juvenile phase and senescent phase with respect to the mycelia linear growth rate. After a certain period of rapid growth with almost constant rate at the juvenile phase, the isolate entered the senescent phase characterized by slow down of mycelia growth, producing pigments ahead of time and final death of the apical hyphae. The period of the senescent phase was definitely 240-288 h; while that of the juvenile phase was diverse relying on different isolates. Moreover, microscopic study showed that angles between the leading and primary hyphae increased constantly with aging. In senesced hyphal cells of M. elata, the typical characteristics of autophagy (enlargement of vacuoles and existence of organelles sequestrated lysosomes) and apoptosis (condensation of the cytoplasm and nuclear and plasmolysis) were observed. In addition, in the final stage of senescence, the apical hyphae collapsed with the plasma membrane and all the cellular organelles disrupted, indicated a necrotic mode of cell death. Taken together, these data revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The characterization and molecular mechanism of autophagy, apoptosis and necrosis need further study and the systematic study of morel aging will be beneficial for the healthy development of morel farming.