Artificial cultivation of true morels: current state,issues and perspectives

Morels (Morchella, Ascomycota), which are some of the most highly prized edible and medicinal mushrooms, are of great economic and scientific value. Morel cultivation has been a research focus worldwide for more than 100 years, and the outdoor cultivation of morels has succeeded and expanded to a large scale in China in recent years. In this study, we review the progress in recent research regarding the life cycle and reproductive systems in the genus Morchella and the current state of outdoor cultivation. Sclerotia formation and conidia production are two important phases during the life cycle. The morel species cultivated commercially in America is M. rufobrunnea based on molecular phylogenetic analysis. The species currently cultivated in China are black morels, including M. importuna, M. sextalata and M. eximia. The field cultivation of morels expanded in the majority of the provinces in China with a yield of fresh morels of 0–7620?kg per ha. The key techniques include spawn production, land preparation and spawning, the addition of exogenous nutrition, fruiting management and harvesting. The application of exogenous nutrition is the most important breakthrough in the field of morel cultivation, but the mechanism remains unclear. It was estimated that the total amount of field cultivated fresh morels was ～500 t in 2015–2016. We also discuss the potential issues remaining in the current literature and suggest directions for future studies.     

Molecular characterization of Morchella species from the Western Himalayan region of India

The molecular diversity of thirty-two different Morchella cultures/fruiting bodies, collected from the Western Himalayan region was studied in this investigation. Considerable taxonomic confusion exists regarding many species of Morchella. Although classical taxonomy is helpful in identification for many ascomycetes, morels exhibit considerable morphological diversity and there is disagreement in the identification of morel species. Phylogenetic analyses based on DNA sequences could help in sorting out morel taxonomy which is essential to better define the morel diversity. In this study, sequence analysis revealed that in the Western Himalayan region of India, both yellow (M. crassipes, M. spongiola) and black morels (M. elata, M. angusticeps, and M. gigas) were prominent along with two Verpa species. Phylogenetic analysis by maximum parsimony, maximum likelihood and Bayesian inference revealed two different clades and a clear distinction between yellow and black morels.     

Observations on post-fire black morel ascocarp development in an Israeli burnt forest site and their preferred micro-sites

Morchella species ascocarps (morels) are sought-after edible mushrooms that exhibit pyrophilous behavior, proliferating in fire-affected soils of certain types of forests. The factors governing fruiting in this habitat are poorly understood. An observational approach was used to determine the spatial distribution on preferred micro-sites of black morel fruiting in a forest after a summer fire, subjected to different post-fire forestry management activities. Clearing the burnt tree stumps from the site, compaction of the burnt soil by heavy machinery (bulldozers) and covering the soil with chopped wood created preferred micro-sites for black morel fruiting. Fewer fruit bodies developed on untouched burnt soil, and almost none on non-burnt soil at the same site. These observations enhance understanding the ecological principles underlying the distribution and abundance of morel ascocarp development in natural habitats; such an understanding could contribute to conservation and management of morel fruiting in the wild.     

Molecular and cultural characterization of Morchella spp. from disturbed environments of central-southern Chile

In Northwestern Patagonia (Chile), three species of Morchella from undisturbed environments have been identified to date: Morchella tridentina, Morchella andinensis and Morchella aysenina, all belonging to the Elata clade and associated mainly with Nothofagus forests. In this study, the search for Morchella specimens was extended to disturbed environments in Central-Southern Chile, to further explore Morchella species diversity in the country, which is still very limited. The Morchella specimens were identified through multilocus sequences analysis, and the mycelial cultures were characterized, establishing comparisons with specimens from undisturbed environments. To the best of our knowledge, these results reveal for the first time in Chile the presence of the species Morchella eximia and Morchella importuna, and in the case of the last one also the first record in South America. These species were found associated almost exclusively with harvested or burned coniferous plantations. The in vitro mycelial characterization revealed certain inter- and intra-specific patterns of the morphology, such as pigmentation, mycelium type, and development and formation of sclerotia, which varied according to growth media and incubation temperature. The growth rates (mm/day) and mycelial biomass (mg) were significantly influenced by the temperature (p < 0.05), with maximum rates (>10 mm/day) and biomass (approx. 20 mg) between 20 and 24 °C, while a significant growth reduction (70–90%) was observed at 28 °C, mainly in the species from undisturbed environments. Potato-dextrose (PDA) medium stimulated the greatest mycelial density and sclerotia formation in most of the isolates, mainly in M. eximia (UDEC-LAF 236 isolate) which recorded the best mycelial growth performance. Among isolates, UDEC-LAF 236 also showed the best performance in sclerotia production (>350 sclerotia/dish) in 10 days of growth. This study contributes to the knowledge of the diversity of Morchella species in Chile by broadening the species range to those from disturbed environments. It also provides molecular and morphological characterization of the in vitro cultures of different Morchella species. The report on M. eximia and M. importuna, species known as cultivable, adapted to local climatic and edaphic conditions could represent the first step to developing artificial Morchella cultivation methods in Chile.     

Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61–63 % and 51–55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59–60 % identity.Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.     

Hybridization,characterization and transferability of SSRs in the genus Morchella

Recently, Morchella importuna, M. sextelata, M. eximia, M. exuberans, Mel-13, and Mel-21 have been successfully cultivated in China and planting areas rapidly expanded because of their economic importance. Effective molecular markers are urgently needed for accurately identifying morel cultivars. Microsatellites are widely utilized for strain authentication in many fungal groups. To our knowledge, for the first time we characterized the distribution of microsatellites (simple sequence repeats, SSRs) in the M. importuna genome with 12902 SSRs and reported the first set of SSRs developed for Morchella species. Mono-nucleotides (66.2 %) were the most frequent motifs, followed by tri- (15.4 %), di- (12.1 %), tetra- (3.7 %), penta- (1.3 %) and hexa-nucleotides (1.3 %). We tested the cross-species amplification of 180 SSRs on 24 samples from the six species and high cross-species transferability of SSRs (87.7 %) was found. Among twenty-two microsatellites selected for genetic diversity analysis on 127 samples from the six species, fifteen to twenty polymorphic loci were identified in M. importuna, M. sextelata, M. eximia, M. exuberans, Mel-13 and Mel-21. Interspecific hybridization events were detected among morel species, indicating the potential application of morel crossbreeding. Ninety-one cultivated samples were characterized as new cultivars with different genotypes, but cultivar names used for these by farmers was confusing, with misnaming, synonyms and homonyms. Our results are not only helpful for cultivar identification and morel breeding programs in China, but also provide molecular tools for genetic studies in morels.     

Optimization of a solid culture medium based on Monochamus alternatus for Cordyceps militaris fruiting body formation

Monochamus alternatus (Coleoptera: Cerambycidae; M. alternatus), popularly known as the Japanese pine sawyer, is a vector of pinewood nematode (Bursaphelenchus xylophilus) that causes pine wilt disease. A solid medium culture with M. alternatus produced Cordyceps militaris fruiting bodies with the longest strips and the highest biological efficiency. Supplementing the original form of M. alternatus with oats resulted in slightly enhanced fruiting body production. The original form of M. alternatus showed higher production than its powder form. The solid culture medium was optimized using a response surface methodology, and the optimal medium contained the following: 8·5 g per bottle of M. alternatus and 11·5 g per bottle of oats mixed with 22·4 ml of water in a 300-ml cylindrical plastic bottle. The optimal culturing period for the fruiting body formation was 37·1 days. Under these conditions, a fruiting body dry weight of 38·0 g per bottle (actual value) was attained. The fruiting body produced using a solid culture medium based on M. alternatus had a cordycepin content of about 25 µg g⁻¹. The solid culture medium containing M. alternatus is highly efficient and eco-friendly, and its effectiveness in large-scale fruiting body production from C. militaris has been demonstrated.     

羊肚菌分子鉴定及功能基因研究进展

羊肚菌作为珍贵的食药用真菌,具有很高的经济价值.就近年来羊肚菌分子生物学研究的进展从分子鉴定、系统学研究、功能基因等方面进行了分析总结.结果显示,目前利用分子标记已实现羊肚菌属快捷的分子鉴定,较全面揭示了属内种群的遗传多样性和亲缘关系;利用组学技术初步探索了羊肚菌菌核形成及子实体发育机制;此外还克隆表达了与羊肚菌抗逆响...     

Using Local Ecological Knowledge to Assess Morel Decline in the U.S. Mid–Atlantic Region

Using Local Ecological Knowledge to Assess Morel Decline in the U.S. Mid–Atlantic Region. Morels (Morchella spp.) are prized wild edible mushrooms. In the United States, morels are the focus of family traditions, local festivals, mycological society forays, and social media, as well as substantial commercial trade. A majority of the anglophone research on morels has been conducted in Europe and in the U.S. Pacific Northwest and Midwest. This literature provides insights into a diverse and plastic genus, but much of its biology and ecology remains a mystery. In 2004, we initiated a study of morel mushroom harvesting in the U.S. Mid–Atlantic region in response to concerns that morels might be in decline in the national parks in that area. This paper presents results from that research with an emphasis on morel hunters’ local ecological knowledge of morel types, phenology, habitat, vegetative associations, and responses to disturbance. We conclude that experienced morel harvesters possess local ecological knowledge that complements scientific knowledge and can increase our understanding of the complex and regionally variable ecology of Morchella and inform conservation efforts.     

Ecological characterization of Morel (Morchella spp.) habitats: A multivariate comparison from three forest types of district Swat,Pakistan

Morchella spp. are wild edible mushrooms growing ephemerally under particular environmental conditions in temperate regions of the world. The present study was aimed to outline some principal eco-edaphic characteristics influencing distribution of these mushrooms from three different forest types of Swat, Pakistan. A randomized circular plots were laid down along the transect line in 14 stands in study areas. Environmental variables were recorded and soil characteristics were determined, analyzed and compared. Five species were collected from N to NW slope at elevation range of 1100-2695m asl. Mean relative humidity of 58.7%, range of air temperature 13 °C-27 °C and soil temperature 6-26 °C were recorded at morel growing sites. A higher canopy cover (average 57%) favours sporocarp formation in morels. Soil analysis showed that morel preferably appeared in sandy loam to loamy textured soil at slightly acidic to neutral pH (mean=6.4) and with consistently higher organic matter (1.5%) and salt activities (mean = 38.8 μS) in the soil. IVI calculation showed that Pinus wallichiana A. B. Jackson, Juglans regia L., Abies pindrow Royle, Cedrus deodara (Roxb. ex D. Don) G. Don were dominant trees and Viburnum grandiflorum Wall. ex DC, Hedra nepalensis K. Koch, Hert and Sarcococca saligna (D. Don) Muell. Arg. were shrub species associated with morels. Whilst Fragaria nubicola (Hook.f.) Lindl., Viola canescens Wall ex Roxb. and Podophyllum hexandrum Roylae were the predominant herbs in morel habitat. This study will help to understand the natural habitat and to provide a base line information for the future conservation and management planning in the area.     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by regulation of hyphal development

The morphological effects of biotin and L-arginine on fruiting body formation of the ascomycete Sordaria macrospora are investigated by scanning electron and light microscopy. Biotin is recognized as an elongation factor and arginine as a branching factor in vegetative and reproductive hyphae. In the absence of exogenous biotin, development is blocked after the ascogonium-core hypha stage of protoperithecial morphogenesis, whereas linear growth of the myceliar front is maintained. The addition of exogenous arginine to a biotin deficient culture induces the formation of numerous side branches even in the older mycelium. Fruiting body formation, however, remains blocked at the protoperithecial stage as before, because of the inability of the side branches to elongate. When biotin and arginine are administered simultaneously, a most vigorous branching and growth are induced in the older mycelium, accompanied by a rapid and maximal formation of fruiting bodies. The results are summarized in a model of the exogenous control of hyphal morphogenesis. The model is designed to explain the relationship between fruiting and hyphal density as well as the edge effect on fruiting body formation.     

Kin Discrimination in Dictyostelium Social Amoebae

Evolved cooperation is stable only when the benefactor is compensated, either directly or through its relatives. Social amoebae cooperate by forming a mobile multicellular body in which, about 20% of participants ultimately die to form a stalk. This benefits the remaining individuals that become hardy spores at the top of the stalk, together making up the fruiting body. In studied species with stalked migration, P. violaceum, D. purpureum, and D. giganteum, sorting based on clone identity occurs in laboratory mixes, maintaining high relatedness within the fruiting bodies. D. discoideum has unstalked migration, where cell fate is not fixed until the slug forms a fruiting body. Laboratory mixes show some degree of both spatial and genotype‐based sorting, yet most laboratory fruiting bodies remain chimeric. However, wild fruiting bodies are made up mostly of clonemates. A genetic mechanism for sorting is likely to be cell adhesion genes tgrB1 and tgrC1, which bind to each other. They are highly variable, as expected for a kin discrimination gene. It is a puzzle that these genes do not cause stronger discrimination between mixed wild clones, but laboratory conditions or strong sorting early in the social stage diminished by later slug fusion could be explanations.     

Cell‐fate determination in Myxococcus xanthus development: Network dynamics and novel predictions

Myxococcus xanthus is a myxobacterium that exhibits aggregation and cellular differentiation during the formation of fruiting bodies. Therefore, it has become a valuable model system to study the transition to multicellularity via cell aggregation. Although there is a vast set of experimental information for the development on M. xanthus, the dynamics behind cell‐fate determination in this organism's development remain unclear. We integrate the currently available evidence in a mathematical network model that allows to test the set of molecular elements and regulatory interactions that are sufficient to account for the specification of the cell types that are observed in fruiting body formation. Besides providing a dynamic mechanism for cell‐fate determination in the transition to multicellular aggregates of M. xanthus, this model enables the postulation of specific mechanisms behind some experimental observations for which no explanations have been provided, as well as new regulatory interactions that can be experimentally tested. Finally, this model constitutes a formal basis on which the continuously emerging data for this system can be integrated and interpreted.     

Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies

Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators.     

Spatial and temporal patterns of morel fruiting

The biotic and abiotic factors conditioning morel fruit body production are incompletely known. We examined spatial and temporal patterns of Morchella esculenta fruiting over five years in a wooded site in Missouri, USA. Fruiting onset was inversely correlated with spring air and soil temperatures, whereas abundance was positively correlated with rain events (>10 mm) during the 30 d preceding fruiting. The two years with the greatest fruiting had the shortest fruiting seasons (6–7 d). Fruiting season length was positively correlated with soil warming, suggesting that a narrow range of optimum soil temperatures favour the explosive production of fruit bodies. All woody stems of at least 1 cm diam were mapped and stem diameter and crown condition were noted. Morel fruit bodies were significantly closer to stems of Carya spp., Tilia americana and Ulmus americana than predicted by the frequencies of these woody species or their contribution to the total basal area on the site. Although intra-annual clustering of fruit bodies was often observed, inter-annual clustering was not. The spatial pattern of M. esculenta fruiting appears to be associated with vegetation pattern, whereas the onset and abundance of fruiting are determined by the interaction of spring temperatures with availability of supporting precipitation.     

Protein expression during Flammulina velutipes fruiting body formation

Formation of the Flammulina velutipes fruiting body can be induced by lowering the ambient temperature (first treatment) in complete darkness. Fruiting bodies formed under these conditions elongate without pileus formation (pinhead fruiting body), suggesting that they cannot mature in complete darkness. However, after light treatment of the pinhead fruiting body (second treatment), a pileus develops immediately, and the stipe also thickens and becomes increasingly pigmented. The apical region swells as a result of cell division starting 2 days after light treatment, the pileus–stipe junction fracture and hymenium primordia form on day 4, and gills appear at day 6. Pf1 and Pf3 are specifically expressed after exposure to low temperature without light. The cell wall-associated protein [pileus-specific hydrophobin-like protein (PSH)] is specifically induced in the pileus, but not in the stipe, following the second light treatment to the pinhead fruiting body. These results suggest that Pf1 and Pf3 would be involved in fruiting body induction and that PSH would be involved in pileus formation. These phenomena will aid further histological and molecular biological investigations into the mechanisms behind fruiting body development in F. velutipes.     

Growth performance of Ganoderma lucidum using billet method in Garhwal Himalaya,India

Medicinal mushrooms have been used in various treatments from a very long time, among which, Ganoderma lucidum is one of the most important medicinal mushroom. It is cultivated worldwide to meet its ever-increasing demand in the market. It is generally cultivated by bed log (Sawdust) and wood log (billet) method. This study was an attempt to observe the growth performance of G. lucidum on poplar billets (Populus deltoides) in the Sherpur Village (Dehradun) and Manjgaun village (Tehri Garhwal) of Garhwal Himalaya, India. The farmers’ field with empty house/ rooms having proper growing conditions especially humidity and light were used for the cultivation of G. lucidum. The G. lucidum spawn was inoculated in poplar wood billets and these billets were installed in well prepared soil. The results demonstrated that cropping cycle of G. lucidum was shorter (132–136 days) in Sherpur Village (Dehradun) as compared to Manjgaun village (141–145 days) in Tehri Garhwal. Further the results also revealed that yield was decreased in the subsequent flushes. In Village Sherpur, the fruiting bodies of G. lucidum were harvested between 64-66 days, 100-101  days and 135-136  days during first, second and third flush after the installation of billets, respectively. However; in village Manjgaun, the fruiting bodies of G. lucidum were harvested between 69 and 71 days, 107-108  days and 144-145 days in first, second and third after the installation of billets respectively. Warmer temperature in Village Sherpur resulted in the early emergence and development of the fruiting bodies as compared to village Manjgaun where pinhead and fruiting body development was delayed due to the lower temperature during cropping cycle.     

Phylogenetic diversity of true morels (Morchella), the main edible non-timber product from native Patagonian forests of Argentina

Morchella species are edible fungi in high demand and therefore command high prices in world markets. Phenotypic-based identification at the species-level remains inadequate because of their complex life cycles, minor differences and plasticity of morphological characteristics between species, and the lack of agreement between scientific and common names. In Patagonia–Argentina, morels are associated with native forests of Austrocedrus chilensis (Cordilleran or Chilean cypress) and Nothofagus antarctica (ñire) and several exotic conifers that were introduced from western North America. Little is known about their taxonomy and phylogenetic relationships with other species in the genus. This work focused on the identification of collections of Morchella from Patagonia and their phylogenetic relationships with other species from the Northern Hemisphere. The comparison was made by analysis of DNA sequences obtained from four loci: the nuclear ribosomal internal transcribed spacer region (ITS) and the partial RNA polymerase I gene (RPB1) for the complete collection; and ITS, RPB1, RNA polymerase II gene (RPB2), and translation elongation factor (EF1-α) for the species-rich Elata Subclade. Analyses of individual and combined data sets revealed that Patagonian morels belong to the Elata Clade and comprised three strongly supported species-level lineages from both Patagonian native forest, and exotic trees introduced from western North America. One lineage was identified as Morchella frustrata phylogenetic species Mel-2, which is known from the USA and Canada. The second lineage, which appeared to be ‘fire-adapted’, was identified as Morchella septimelata phylogenetic species (Mel-7), which is also known from the USA. This species was collected from burned native forests mainly composed of A. chilensis and N. antarctica but also Pseudotsuga menziesii (Mirb.) Blanco, which is native to western North America. The phylogenetic analyses suggested that the third species from Patagonia was nested within the species-rich Elata Subclade and represents a new species-level lineage (informally designated Mel-37) within Elata Clade. The present collections from Patagonia constitute the southernmost latitude from which Morchella has been reported to date. The identification of two Argentine morels as North American taxa is therefore a remarkable biogeographic pattern. In view of the hypothesis that the Elata Clade originated in western North America, we speculate that at least two of the lineages colonized South America from North America via long distance dispersal, migration or, more likely, they were introduced with the exotic tree species that they were collected near.