Search Results (21)

Mating-type identification is fundamental to studies of genetic diversity and genetic breeding in fungi, especially for tetrapolar basidiomycetes, whose mating types are determined by two multiallelic loci, A and B. Traditional mating-type identification of monokaryons relies on manual inference based on hybridization experiments; however, this process is highly complex, time-consuming, and error-prone when applied to large-scale studies. In this study, we isolated 30 monokaryons from protoplasts derived from 15 dikaryons of Flammulina velutipes and developed a software tool, Mating-Type Imputation (MTI), to automatically, rapidly, and accurately infer monokaryon mating types in tetrapolar fungi using a combinatorial pruning traversal algorithm. Using a compatibility matrix derived from 435 hybridization experiments involving these 30 monokaryons, MTI required only a few minutes to accurately infer the mating types of all monokaryons-a task that typically takes several days for manual inference by experienced investigators. Furthermore, MTI enabled us to investigate how false-positive and false-negative interactions influence mating-type inference results. Using a simulated compatibility matrix, we found that MTI could accurately detect potential false negatives in compatibility and successfully infer the true mating-type combinations even in the presence of limited false negatives; conversely, the tool was easily misled by any false positives, resulting in incorrect mating-type combinations. This indicates that false-positive records in hybridization experiments must be strictly eliminated during mating-type inference. In summary, MTI provides an efficient tool for inferring the mating types of tetrapolar fungi, offering technical support for mating-type studies of edible and medicinal fungi, and holds significant theoretical value and broad application potential in the fields of fungal genetic diversity and breeding research. Full article

Currently, most research on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing in edible fungi focuses on monokaryotic strains. However, the biological mechanisms in a monokaryotic state often do not accurately reflect the actual physiological and metabolic conditions of dikaryotic strains. Therefore, this study used two mating-type-compatible monokaryotic strains, L1 and L2, isolated from Ganoderma lucidum ‘Hunong No.1’ G0119, and employed an RNP (ribonucleoprotein)-based CRISPR/Cas9 system to successfully knock out the cyp512a3 gene in strain L2, resulting in the edited strain L2-KO-cyp512a3. The strain was single-crossed with the previously edited L1 strain L1-KO-cyp512a3 in our laboratory to obtain a dikaryotic editing strain that was homozygous at the cyp512a3 locus, named G0119-KO-cyp512a3. UPLC-MS (Ultra Performance Liquid Chromatography–Mass Spectrometry) analysis showed that compared to the starting strain G0119, the dikaryotic editing strain exhibited varying degrees of reduction in the content of eight types of ganoderic acids, including ganoderic acid Me, ganoderic acid P, ganoderic acid T1, etc., with the reduction ranging from 30.5% to 80.1%. To further validate the function of cyp512a3, we overexpressed this gene in the L1 strain. The results showed that the contents of ganoderic acid Mk, ganoderic acid S, ganoderic acid T, and ganoderic acid R in the mycelium were 0.548 ± 0.020, 1.780 ± 0.028, 2.416 ± 0.148, and 0.281 ± 0.016 mg/g (dry weight), which were 1.5 times, 1.3 times, 1.3 times, and 1.3 times that of G0119, respectively. By integrating the results of gene knockout and overexpression, it can be clearly established that cyp512a3 is a key cytochrome P450 gene regulating the biosynthesis of ganoderic triterpenoids in Ganoderma lucidum. This study not only establishes, for the first time, a homologous recombination-based gene editing system in dikaryotic strains of Ganoderma lucidum, but also provides a research paradigm based on a dikaryon-editing tool for investigating key life traits of other edible fungi. Full article

A recent study challenges a fundamental principle of eukaryotic biology that each nucleus houses a complete genome. Two plant pathogenic fungi, Sclerotinia sclerotiorum and Botrytis cinerea, exhibit a segregated pattern of haploid chromosome distribution across two or more nuclei within each cell. The unequal distribution of the genome between nuclei suggests a coordinated system of internuclear recognition and regulation of cellular functions, a phenomenon previously associated with communication between nuclei of opposite mating type in both ascomycetes and basidiomycetes. Thus, the new study not only shatters expectations about genome biology but also opens new research avenues for understanding fungal adaptation and nuclear behavior. Full article

Auricularia heimuer is a valuable traditional Chinese fungus used as food and medicine. Hei29 is a strain derived from wild A. heimuer through systematic domestication and selection. It has been the primary A. heimuer variety in Northeast China for 30 years and offers the advantages of high yield, good commercial property, and stable traits. This study used protoplast nucleation on Hei29 to produce two amiable and paired monokaryons, Hei29-D1 and Hei29-D2. The genome of Hei29 was sequenced utilizing the Illumina PE150 and PacBio Sequel sequencing platforms. Hei29-D1 and Hei29-D2 had genomic sizes of 47.54 Mb and 47.49 Mb, GC contents of 56.95% and 56.99%, and an N50 of 2.37 Mb and 4.28 Mb, respectively. Hei29’s genome possessed two phytoene synthase (PSY) protein genes, one of which—PSY encoded by g894—has a transmembrane domain. The phylogenetic tree showed that Hei29 shared the closest evolutionary relationship with Auricularia subglabra TFB-10046 SS5. Collinearity analysis showed that the correlation between the two monokaryons was as high as 90.81%. Cluster analysis revealed that Hei29 contains 12,362 core genes, 223 unique genes in Hei29-D1, and 228 unique genes in Hei29-D2. This study is the first to sequence two related and paired monokaryons from A. heimuer, which is critical for fully understanding the genetic composition and information of the characteristic strain of A. heimuer in Northeast China. It establishes the data and theoretical foundation for gene mining, usage, and molecular breeding. It further promotes the genetic breeding and active substance utilization of A. heimuer. Full article

Phallus echinovolvatus is a well-known edible and medicinal fungus with significant economic value. However, the available whole-genome information is lacking for this species. The chromosome-scale reference genome (Monop) and two haploid genomes (Hap1 and Hap2) of P. echinovolvatus, each assembled into 11 pseudochromosomes, were constructed using Illumina, PacBio-HiFi long-read sequencing, and Hi-C technology. The Monop had a size of 36.54 Mb, with 10,251 predicted protein-coding genes and including 433 carbohydrate-active enzyme genes, 385 cytochrome P450 enzyme genes, and 42 gene clusters related to secondary metabolite synthesis. Phylogenetic and collinearity analysis revealed a close evolutionary relationship between P. echinovolvatus and Clathrus columnatus in the core Phallales clade. Hap1 and Hap2 had sizes of 35.46 Mb and 36.11 Mb, respectively. Collinear relationships were not observed for 15.38% of the genes in the two haplotypes. Hap1 had 256 unique genes, and Hap2 had 370 unique genes. Our analysis of the P. echinovolvatus genome provides insights into the genetic basis of the mechanisms underlying the metabolic effects of bioactive substances and will aid ongoing breeding efforts and studies of genetic mechanisms. Full article

The fungal genus Omphalotus is noted for its bioluminescence and the production of biologically active secondary metabolites. We isolated 47 fungal strains of Omphalotus olivascens germinated from spores of a single mushroom. We first noted a high degree of variation in the outward appearances in radial growth and pigmentation among the cultures. Radial growth rates fell into at least five distinct categories, with only slower-growing isolates obtained compared with the parental dikaryon. Scanning UV-vis spectroscopy of liquid-grown cultures showed variation in pigmentation in both the absorption intensity and peak absorption wavelengths, indicating that some isolates vary from the parental strain in both pigment concentration and composition. Bioluminescence intensity was observed to have isolates with both greater and lesser intensities, while the increased emission in response to caffeic acid was inversely proportional to the unstimulated output. Under UV illumination, the media of the parental strain was observed to be brightly fluorescent, which was not due to the pigment, while the isolates also varied from greater to lesser intensity and in their peak emission. At least three separate fluorescent bands were observed by gel electrophoresis from one of the cultures, while only one was observed in others. In a subset of the cultures, fluorescence intensity varied significantly in response to casamino acids. None of this subset produced an antibiotic effective against Staphylococcus aureus, and only the haploids, but not the parental heterokaryon, produced an antibiotic consistent with illudin M effective against Mycobacterium smegmatis. This same subset produced an anticancer agent that was highly potent against MDA-MB-468 breast cancer tumor cells. We interpret these variations in haploids as significant in altering Omphalotus physiology and its production of secondary metabolites, which may in turn alter their ecology and life cycle, and could be further applied to studying fungal physiologies and facilitate linking them to their genetic underpinnings. Full article

Ganoderma is the most important genus in the family Ganodermataceae; many species have attracted much attention and widely cultivated because of their medicinal values, but so far, not a sequenced mitogenome derived from dikaryon strains has been explicitly recorded. Herein, four novel mitogenomes of commonly cultivated Ganoderma (G. leucocontextum H4, G. lucidum G6, G. sinense MZ96 and G. tsugae SS) were de novo assembled and given detail functional annotations. Collinearity analysis revealed that the four mitogenomes shared 82.93–92.02% similarity with their corresponding reference mitogenomes at the nucleotide level. A total of 15 core protein-coding genes (PCGs), along with rrnL and rrnS (mtLSU and mtSSU) were chosen as potential candidates for constructing their individual phylogenetic trees. These trees were compared with those derived from the concatenated sequences of 15 core PCGs. And finally, we found that the atp9 and nad4L were the most reliable markers for the phylogenetic analysis of Ganoderma and chosen as standard sequences to generate new DNA barcodes. This finding was further verified by comparing it against almost all available Ganoderma mitogenomes in the NCBI, with Trametes versicolor (Polyporaceae) and Rigidoporus microporus (Meripilaceae) as two outgroups. A total of 52 mitogenomes from three families were highly conserved, with identical gene lengths for atp9 (222 bp) and nad4L (267 bp). These genes were capable of distinguish distinctly different various species, which are grouped into separate clades within the phylogenetic trees. The closest related clades (I and II), including at least 30 samples of the three classical taxonomic species (G. lingzhi, G. sichuanense and G. lucidum), differed in only one SNP. The single base mutation rate increased with the evolutionary divergence of the phylogenetic clades, from two to three SNPs in earlier clades (e.g., clade IV containing G. leucocontextum) to five to six SNPs in later clades (e.g., clade X containing G. sinense). Despite these variations between species, the atp9 and nad4L genes of Ganoderma mitogenomes consistently encoded the same ATP synthase F0 subunit c (73 aa) and NADH dehydrogenase subunit 4L (88 aa). These two genes have been identified as reliable markers of new DNA barcodes, offering valuable insights and contributing significantly to understanding the evolutionary relationships and phylogeny of the Ganoderma genus and even the Ganodermataceae family. Full article

Plant pathogenic nematodes play an important role in crop production and are difficult to control. One of them is Heterodera schachtii—a cyst nematode, pathogenic to sugar beet. Nature suggests a natural way to resolve this problem by using nematode-trapping fungi, one of which is Pleurotus ostreatus. It is one of the most widespread mushrooms in the world. It is a valuable and widely cultivated edible mushroom with nematocidal properties. The mycelium of this mushroom produces toxin droplets that paralyze nematodes, after which the nematodes become infected with the hyphae filament, resulting in their death. This feature can be put to practical use as a natural nematocidal agent. In this paper, we have described studies on the variability of the nematocidal properties in the progeny of three wild strains of P. ostreatus (Po1, Po2, Po4), obtained by crossing dikaryons with monokaryons (Buller phenomenon). The toxicity of mycelium was tested against the model organism Caenorhabditis elegans and against sugar beet pest—H. schachtii. The results of the study allowed the selection of the progeny heterokaryotic mycelia with the best parameters useful for in vitro control of both nematodes. They differed in the activity against C. elegans and H. schachtii, and in the toxic knobs production. The results indicated that the progeny of the Po1 strain presented a good ability to produce hyphal knobs in the presence of C. elegans, and progenies of the Po4 strain presented good quality of growth in preferred temperatures (10–25 °C). Three strains Po1 5dix27, Po2 20dix21, and Po4 2dix1, as well as a maternal strain Po4 controlled H. schachtii by entwining cysts better than other strains. These strains were moderately effective against C. elegans. Strains Po2 15dix17, Po4 1dix18, and Po4 1dix30 may be considered good controlling isolates against both tested organisms. The results of the research also clearly suggest that the killing properties of P. ostreatus mycelia should not be assessed only by their ability to produce toxic hyphal knobs. Their nematocidal properties also depend on other mechanisms developed by mycelia, which is observed as the lethality of nematodes. The results of this research will lead to a natural way to protect plants against nematodes. The research also proved the nematocidal properties of the wild strains to reduce the H. schachtii population in the soil. Full article

Culture collections (CCs) play an important role in the ex situ conservation of biological material and maintaining species and strains, which can be used for scientific and practical purposes. The Komarov Botanical Institute Basidiomycetes Culture Collection (LE-BIN) preserves a large number of original dikaryon strains of various taxonomical and ecological groups of fungi from different geographical regions. Started in the late 1950s for the investigation of Basidiomycetes’ biological activity, today, in Russia, it has become a unique specialized macromycetes collection, preserving 3680 strains from 776 species of fungi. The Collection’s development is aimed at ex situ conservation of fungal diversity, with an emphasis on preserving rare and endangered species, ectomycorrhizal fungi, and strains useful for biotechnology and medicine. The main methods applied in the collection for maintaining and working with cultures are described, and the results are presented. Some problems for the isolation and cultivation of species are discussed. The taxonomical structure and variety of the strains in the collection fund are analyzed, and they show that the taxonomical diversity of fungi in the LE-BIN is commensurable with the largest CCs in the world. The achievements from the ex situ conservation of the diversity of macromycetes and the main results from the screening and investigation of the collection’s strains demonstrate that a number of strains can be prospective producers of enzymes (oxidoreductases and proteases), lipids, and biologically active compounds (terpenoids, phthalides, etc.) for biotechnology and medicine. Full article

With the advancement of “targeted poverty alleviation”, the main problems we face at this stage are analyzing the causes of poverty and preventing poverty-stricken villages from falling back into poverty. Using the last batch of poverty-stricken villages that have been lifted out of poverty, which were announced by the Henan Provincial poverty alleviation and development office, as the research object, this research discusses the spatial distribution characteristics of 835 poverty-stricken villages in Henan Province from the perspective of the macro province by combining kernel density analysis and spatial autocorrelation analysis, analyzes their influencing factors using the least squares linear regression model, and performs a quantitative analysis of their influencing factors. The results revealed the following: (1) The spatial distribution characteristics of poverty-stricken villages in Henan Province are characterized by significant agglomeration. A banded high-density area is formed in the east of Henan Province, with a sub-density area in the west of Henan Province, and it radiates outward with the “dikaryon” as its center. (2) High-density and sub-density areas of poverty-stricken villages are mainly located in flat areas, with spatial dependence on each other. The high-high clustering areas are mainly areas dominated by agricultural economies. The low-low clustering areas mainly have limited agricultural development and rapid urban–rural integration. (3) Geographical locations and socio-economic and basic public services are the main factors affecting poverty in poverty-stricken villages. At the same time, environmental livability is an advantageous condition in areas where poverty-stricken villages are located, of which the Temperature Humidity Index factor is most closely related to the distribution density of poverty-stricken areas. The research results are intended to provide information for the policies and program intervention strategies for poverty reduction and sustainable development in poverty-stricken villages in Henan Province, and this is conducive to consolidating the results of targeted poverty alleviation and preventing a return to poverty. Full article

Karyotyping in Agaricus bisporus is crucial for both the isolation of homokaryotic strains and the confirmation of dikaryon establishment. For the verification of the karyotype, the A mating type loci of two homokaryotic strains, H39 and H97, were analyzed through comparative sequence analysis. The two loci showed major differences in two sequence regions designated as Region 1 and Region 2. H97 had a putative DNA transposon in Region 1 that had target site duplications (TSDs), terminal inverted repeats (TIRs), and a loop sequence, in contrast to H39, which only had the insertional target sequence. Homologous sequences of the transposon were discovered in the two different chromosomes of H97 and in one of H39, all of which have different TSDs but share high sequence homology in TIR. Region 2 shared three consensus sequences between H97 and H39. However, it was only from H97 that a large insertional sequence of unknown origin was discovered between the first and second consensus sequences. The difference in length in Region 1, employed for the verification of the A mating type, resulted in the successful verification of mating types in the heterokaryotic and homokaryotic strains. This length difference enables the discrimination between homo- and heterokaryotic spores by PCR. The present study suggests that the A mating type locus in A. bisporus H97 has evolved through transposon insertion, allowing the discrimination of the mating type, and thus the nuclear type, between A. bisporus H97 and H39. Full article

Oudemansiella raphanipes, considered as a well-known culinary edible mushroom with a high content of natural bioactive substances, is widely cultivated in China with the commercial name Changgengu. However, due to the lack of genomic data, molecular and genetic study on O. raphanipes is rare. To obtain a comprehensive overview of genetic characteristics and enhance the value of O. raphanipes, two mating-compatible monokaryons isolated from the dikaryon were applied for de novo genome sequencing and assembly using Nanopore and /or Illumina sequencing platforms. One of the monokaryons, O. raphanipes CGG-A-s1, was annotated with 21,308 protein-coding genes, of which 56 were predicted to be involved in the biosynthesis of secondary metabolites such as terpene, type I PKS, NRPS, and siderophore. Phylogenetic and comparative analysis of multiple fungi genomes revealed a close evolutionary relationship between O. raphanipes and Mucidula mucid based on single-copy orthologous protein genes. Significant collinearity was detected between O. raphanipes and Flammulina velutipes on the synteny of inter-species genomes. 664 CAZyme genes in CGG-A-s1 were identified with GHs and AAs families significantly elevated when compared with the other 25 sequenced fungi, indicating a strong wood degradation ability. Furthermore, the mating type locus analysis revealed that CGG-A-s1 and CGG-A-s2 were conserved in the gene organization of the mating A locus but various in that of the mating B locus. The genome resource of O. raphanipes will provide new insights into its development of genetic studies and commercial production of high-quality varieties. Full article

Ustilago esculenta is a fungus of two morphological forms, among the filamentous dikaryon that can induce the plant stem to expand to form fleshy stem. In order to establish biotrophy with Zizania latifolia which belongs to the tribe Oryzeae (Poaceae), U. esculenta firstly needs to secrete a bunch of effectors, among them being cell wall degrading enzymes (CWDEs). We have isolated a gene, UeEgl1, which was differentially expressed in MT-type and T-type U. esculenta at an early stage of infection, and specifically induced in the filamentous growth of the T-type. Bioinformatics analysis and enzyme activity assay indicated that UeEgl1 functions outside the cell as a β-1,4-endoglucanase with a conserved domain of the glycosyl hydrolase family 45 (GH45) which targets the main component of the plant cell wall β-1,4 linked glycosidic bonds. The phenotype analysis of UeEgl1 deletion mutants and UeEgl1 over-expression transformants showed that UeEgl1 had no significant effect on the budding, cell fusion, and filamentous growth of U. esculenta in vitro. Further study found that over-expression of UeEgl1 promoted the proliferation of mycelia inside Z. latifolia, and raised plant defense responses. The above results show that the UeEgl1 gene may play an important role in the early stage of infection through the decomposition of the plant cell wall. Full article

Lentinula edodes is a tetrapolar basidiomycete with two haploid nuclei in each cell during most of their life cycle. Understanding the two haploid nuclei genome structures and their interactions on growth and fruiting body development has significant practical implications, especially for commercial cultivars. In this study, we isolated and assembled the two haploid genomes from a commercial strain of L. edodes using Illumina, HiFi, and Hi-C technologies. The total genome lengths were 50.93 Mb and 49.80 Mb for the two monokaryons SP3 and SP30, respectively, with each assembled into 10 chromosomes with 99.63% and 98.91% anchoring rates, respectively, for contigs more than 100 Kb. Genome comparisons suggest that two haploid nuclei likely derived from distinct genetic ancestries, with ~30% of their genomes being unique or non-syntenic. Consistent with a tetrapolar mating system, the two mating-type loci A (matA) and B (matB) of L. edodes were found located on two different chromosomes. However, we identified a new but incomplete homeodomain (HD) sublocus at ~2.8 Mb from matA in both monokaryons. Our study provides a solid foundation for investigating the relationships among cultivars and between cultivars and wild strains and for studying how two genetically divergent nuclei coordinate to regulate fruiting body formation in L. edodes. Full article

The emergence of new fungal pathogens threatens sustainable crop production worldwide. One mechanism by which new pathogens may arise is hybridization. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected because they both infect Zea mays, can hybridize, and tools are available for their analysis. The hybrid dikaryons of these fungi grew as filaments on plates but their colonization and virulence in Z. mays were reduced compared to the parental dikaryons. The anthocyanin induction caused by the hybrid dikaryon infections was distinct, suggesting its interaction with the host was different from that of the parental dikaryons. Selected virulence genes previously characterized in U. maydis and their predicted S. reilianum orthologs had altered transcript levels during hybrid infection of Z. mays. The downregulated U. maydis effectors, tin2, pit2, and cce1, and transcription factors, rbf1, hdp2, and nlt1, were constitutively expressed in the hybrid. Little impact was observed with increased effector expression; however, increased expression of rbf1 and hdp2, which regulate early pathogenic development by U. maydis, increased the hybrid’s capacity to induce symptoms including the rare induction of small leaf tumors. These results establish a base for investigating molecular aspects of smut fungal hybrid pathogen emergence. Full article