Courtship Ritual of Male and Female Nuclei during Fertilization in Neurospora crassa.

Sexual reproduction is a key process influencing the evolution and adaptation of animals, plants, and many eukaryotic microorganisms, such as fungi. However, the sequential cell biology of fertilization and the associated nuclear dynamics after plasmogamy are poorly understood in filamentous fungi. Using histone-fluorescent parental isolates, we tracked male and female nuclei during fertilization in the model ascomycete Neurospora crassa using live-cell imaging. This study unravels the behavior of trichogyne resident female nuclei and the extraordinary manner in which male nuclei migrate up the trichogyne to the protoperithecium. Our observations raise new fundamental questions about the modus operandi of nucleus movements during sexual reproduction, male and female nuclear identity, guidance of nuclei within the trichogyne and, unexpectedly, the avoidance of "polyspermy" in fungi. The spatiotemporal dynamics of male nuclei within the trichogyne following plasmogamy are also described, where the speed and the deformation of male nuclei are of the most dramatic observed to date in a living organism. IMPORTANCE Using live-cell fluorescence imaging, for the first time we have observed live male and female nuclei during sexual reproduction in the model fungus Neurospora crassa. This study reveals the specific behavior of resident female nuclei within the trichogyne (the female organ) after fertilization and the extraordinary manner in which male nuclei migrate across the trichogyne toward their final destination, the protoperithecium, where karyogamy takes place. Importantly, the speed and deformation of male nuclei were found to be among the most dramatic ever observed in a living organism. Furthermore, we observed that entry of male nuclei into protoperithecia may block the entry of other male nuclei, suggesting that a process analogous to polyspermy avoidance could exist in fungi. Our live-cell imaging approach opens new opportunities for novel research on cell-signaling during sexual reproduction in fungi and, on a broader scale, nuclear dynamics in eukaryotes.

Apoptotic and Nonapoptotic Activities of Pterostilbene against Cancer.

Cancer is a major cause of death. The outcomes of current therapeutic strategies against cancer often ironically lead to even increased mortality due to the subsequent drug resistance and to metastatic recurrence. Alternative medicines are thus urgently needed. Cumulative evidence has pointed out that pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, PS) has excellent pharmacological benefits for the prevention and treatment for various types of cancer in their different stages of progression by evoking apoptotic or nonapoptotic anti-cancer activities. In this review article, we first update current knowledge regarding tumor progression toward accomplishment of metastasis. Subsequently, we review current literature regarding the anti-cancer activities of PS. Finally, we provide future perspectives to clinically utilize PS as novel cancer therapeutic remedies. We, therefore, conclude and propose that PS is one ideal alternative medicine to be administered in the diet as a nutritional supplement.

Comparative analysis of genome sequences of the conifer tree pathogen, Heterobasidion annosum s.s.

The causal agent of root and butt rot of conifer trees, Heterobasidion annosum, is widespread in boreal forests and economically responsible for annual loss of approximately 50 million euros to forest industries in Finland alone and much more at European level. In order to further understand the pathobiology of this fungus at the genome level, a Finnish isolate of H. annosum sensu stricto (isolate 03012) was sequenced and analyzed with the genome sequences of 23 white-rot and 13 brown-rot fungi. The draft genome assembly of H. annosum has a size of 31.01 Mb, containing 11,453 predicted genes. Whole genome alignment showed that 84.38% of H. annosum genome sequences were aligned with those of previously sequenced H. irregulare TC 32-1 counterparts. The result is further supported by the protein sequence clustering analysis which revealed that the two genomes share 6719 out of 8647 clusters. When sequencing reads of H. annosum were aligned against the genome sequences of H. irregulare, six single nucleotide polymorphisms were found in every 1 kb, on average. In addition, 98.68% of SNPs were found to be homo-variants, suggesting that the two species have long evolved from different niches. Gene family analysis revealed that most of the white-rot fungi investigated had more gene families involved in lignin degradation or modification, including laccases and peroxidase. Comparative analysis of the two Heterobasidion spp. as well as white-/brown-rot fungi would provide new insights for understanding the pathobiology of the conifer tree pathogen.

Trichoderma reesei meiosis generates segmentally aneuploid progeny with higher xylanase-producing capability.

BACKGROUND: Hypocrea jecorina is the sexual form of the industrial workhorse fungus Trichoderma reesei that secretes cellulases and hemicellulases to degrade lignocellulosic biomass into simple sugars, such as glucose and xylose. H. jecorina CBS999.97 is the only T. reesei wild isolate strain that is sexually competent in laboratory conditions. It undergoes a heterothallic reproductive cycle and generates CBS999.97(1-1) and CBS999.97(1-2) haploids with MAT1-1 and MAT1-2 mating-type loci, respectively. T. reesei QM6a and its derivatives (RUT-C30 and QM9414) all have a MAT1-2 mating type locus, but they are female sterile. Sexual crossing of CBS999.97(1-1) with either CBS999.97(1-2) or QM6a produces fruiting bodies containing asci with 16 linearly arranged ascospores (the sexual spores specific to ascomycetes). This sexual crossing approach has created new opportunities for these biotechnologically important fungi. RESULTS: Through genetic and genomic analyses, we show that the 16 ascospores are generated via meiosis followed by two rounds of postmeiotic mitosis. We also found that the haploid genomes of CBS999.97(1-2) and QM6a are similar to that of the ancestral T. reesei strain, whereas the CBS999.97(1-1) haploid genome contains a reciprocal arrangement between two scaffolds of the CBS999.97(1-2) genome. Due to sequence heterozygosity, most 16-spore asci (>90%) contain four or eight inviable ascospores and an equal number of segmentally aneuploid (SAN) ascospores. The viable SAN progeny produced higher levels of xylanases and white conidia due to segmental duplication and deletion, respectively. Moreover, they readily lost the duplicated segment approximately two weeks after germination. With better lignocellulosic biomass degradation capability, these SAN progeny gain adaptive advantages to the natural environment, especially in the early phase of colonization. CONCLUSIONS: Our results have not only further elucidated T. reesei evolution and sexual development, but also provided new perspectives for improving T. reesei industrial strains.

Potential roles of laccases on virulence of Heterobasidion annosum s.s.

Laccases, multi-copper-containing proteins, can catalyze the oxidation of phenolic substrates and have diverse functions such as a virulence factor in fungi. However, limited information can be found on the role of laccases in the interaction of Heterobasidion annosum s.s. to its host plant. Due to genome availability of the close-related species Heterobasidion irregulare, which contains 18 predicted laccase-encoding genes, phylogenetic analysis and gene expression profiling were performed. Eighteen laccase genes could be classified into 4 groups based on protein domains and phylogenetic analysis. However, there is no clear indication between phylogeny and domain compositions in laccases, and lifestyles of fungal species. The results of qRT-PCR showed that the expression of 8 laccase genes was highly up-regulated in Scots pine seedlings at 1 wpi. These data suggested that they might be involved in early stage of host infection. In addition, up-regulation of gene expression under glucose condition as a sole carbon source suggests that those laccases are not under carbon catabolite repression. Higher activities of laccase were observed in culture media containing cellulose, sucrose, or glucose compared to that of cellobiose as a sole carbon source. The highest mortality of Scots pine seedlings was observed when infected by H. annosum s.s. on extra carbon source as glucose. This was supported by the facts that glucose plays significant roles on up-regulation of laccase genes in planta and higher activity of laccase in H. annosum s.s.. Taking all together, laccases in H. annosum s.s. have diverse functions and a group of laccases may play a role during interactions with Scots pine seedlings.

Genome sequence of Trichoderma virens FT-333 from tropical marine climate.

Environmental factors can cause changes in the content of the fungal genome during evolution. In this study, a fungus used as a biocontrol agent, Trichoderma virens FT-333 (from a tropical marine climate) has been isolated. The genome (38.6 Mbp; GC content, 49.43%) has a total of 12,751 proteins. Gene ontology terms (cellular component and molecular function) and KEGG analyses demonstrated the importance of the secretion function in FT-333. Compared to the other Trichoderma species, copy number of genes related to defense and nutrient utilization was variable.

Cordyceps s.l. (Ascomycetes) Species Used as Medicinal Mushrooms are Closely Related with Higher Ability to Produce Cordycepin.

Cordyceps s.l. (sensu lato) species have been used as herbal medicines; one of their main constituents is cordycepin. As genome sequencing techniques have become more cost-effective and more popular, more entomogenous fungal genomes have been sequenced and published. Here, we constructed a phylogenetic tree based on 18S rRNA sequences from Cordyceps species and analyzed the copy number of the key enzymes involved in biosynthesis of cordycepin from related fungal genomes that have been published. The sequences of the 18S rRNA gene were examined, and seven single nucleotides were found that could represent the evolutionary history of Cordyceps s.l. and which perfectly fit the phylogenetic tree. Their evolution was influenced mainly by host factors, rather than geographical location. The Cordyceps s.l. species that are used as herbal medicines are closely related in the phylogenetic tree. The major species for Chinese pharmaceutical markets, such as C. militaris and C. sinensis, have higher copy numbers of 5'-nucleotidase and adenylate kinase, and ribonucleotide reductases (RNRs), respectively. Moreover, absence of an RNR inhibitor may cause cordycepin accumulation. Presence of an RNR inhibitor may lead to lower cordycepin levels in fungal species in which no medicinal applications have been described. Cordycepin is not only an important secondary metabolite that is used as an herbal medicine, but it also has significance for understanding the evolution of these entomogenous species.

In silico sequence analysis reveals new characteristics of fungal NADPH oxidase genes.

NADPH oxidases (Noxes), transmembrane proteins found in most eukaryotic species, generate reactive oxygen species and are thereby involved in essential biological processes. However, the fact that genes encoding ferric reductases and ferric-chelate reductases share high sequence similarities and domains with Nox genes represents a challenge for bioinformatic approaches used to identify Nox-encoding genes. Further, most studies on fungal Nox genes have focused mainly on functionality, rather than sequence properties, and consequently clear differentiation among the various Nox isoforms has not been achieved. We conducted an extensive sequence analysis to identify putative Nox genes among 34 eukaryotes, including 28 fungal genomes and one Oomycota genome. Analyses were performed with respect to phylogeny, transmembrane helices, di-histidine distance and glycosylation. Our analyses indicate that the sequence properties of fungal Nox genes are different from those of human and plant Nox genes, thus providing novel insight that will enable more accurate identification and characterization of fungal Nox genes.

Secret lifestyles of Neurospora crassa.

Neurospora crassa has a long history as an excellent model for genetic, cellular, and biochemical research. Although this fungus is known as a saprotroph, it normally appears on burned vegetations or trees after forest fires. However, due to a lack of experimental evidence, the nature of its association with living plants remains enigmatic. Here we report that Scots pine (Pinus sylvestris) is a host plant for N. crassa. The endophytic lifestyle of N. crassa was found in its interaction with Scots pine. Moreover, the fungus can switch to a pathogenic state when its balanced interaction with the host is disrupted. Our data reveal previously unknown lifestyles of N. crassa, which are likely controlled by both environmental and host factors. Switching among the endophytic, pathogenic, and saprotrophic lifestyles confers upon fungi phenotypic plasticity in adapting to changing environments and drives the evolution of fungi and associated plants.

Development of pedigree classification using microsatellite and mitochondrial markers for Giant grouper broodstock (Epinephelus lanceolatus) management in Taiwan.

Most giant groupers in the market are derived from inbred stock. Inbreeding can cause trait depression, compromising the animals' fitness and disease resistance, obligating farmers to apply increased amounts of drugs. In order to solve this problem, a pedigree classification method is needed. Here, microsatellite and mitochondrial DNA were used as genetic markers to analyze the genetic relationships among giant grouper broodstocks. The 776-bp fragment of high polymorphic mitochondrial D-loop sequence was selected for measuring sibling relatedness. In a sample of 118 giant groupers, 42 haplotypes were categorized, with nucleotide diversity (π) of 0.00773 and haplotype diversity (HD) of 0.983. Furthermore, microsatellites were used for investigation of parentage. Six out of 33 microsatellite loci were selected as markers based on having a high number of alleles and compliance with Hardy-Weinberg equilibrium. Microsatellite profiles based on these loci provide high variability with low combined non-exclusion probability, permitting practical use in aquaculture. The method described here could be used to improve grouper broodstock management and lower the chances of inbreeding. This approach is expected to lead to production of higher quality groupers with higher disease resistance, thereby reducing the need for drug application.

Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems.

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

Blue light acts as a double-edged sword in regulating sexual development of Hypocrea jecorina (Trichoderma reesei).

The industrially important cellulolytic filamentous fungus Trichoderma reesei is the anamorph of the pantropical ascomycete Hypocrea jecorina. H. jecorina CBS999.97 strain undergoes a heterothallic reproductive cycle, and the mating yields fertilized perithecia imbedded in stromata. Asci in the perithecia contain 16 linearly arranged ascospores. Here, we investigated H. jecorina sexual development under different light regimes, and found that visible light was dispensable for sexual development (stroma formation and ascospore discharge). By contrast, constant illumination inhibited stroma formation, and an interruption of the darkness facilitated timely stroma formation in a 12 h/12 h light-dark photoperiod. The results of genetic analyses further revealed that H. jecorina blue-light photoreceptors (BLR1, BLR2) and the photoadaptation protein ENV1 were not essential for sexual development in general. BLR1, BLR2 and ENV1 are orthologues of the conserved Neurospora crassa WC-1, WC-2 and VVD, respectively. Moreover, BLR1 and BLR2 mediate both positive and negative light-dependent regulation on sexual development, whereas ENV1 is required for dampening the light-dependent inhibitory effect in response to changes in illumination. Comparative genome-wide microarray analysis demonstrated an overview of light-dependent gene expression versus sexual potency in CBS999.97 (MAT1-2) haploid cells. Constant illumination promotes abundant asexual conidiation and high levels of hpp1 transcripts. hpp1 encodes a h (hybrid)-type propheromone that exhibits features of both yeast a and a pheromone precursors. Deletion of hpp1 could rescue stroma formation but not ascospore generation under constant illumination. We inferred that the HPP1-dependent pheromone signaling system might directly prevent stroma formation or simply disallow the haploid cells to acquire sexual potency due to abundant asexual conidiation upon constant illumination.

An automated microfluidic chip system for detection of piscine nodavirus and characterization of its potential carrier in grouper farms.

Groupers of the Epinephelus spp. are an important aquaculture species of high economic value in the Asia Pacific region. They are susceptible to piscine nodavirus infection, which results in viral nervous necrosis disease. In this study, a rapid and sensitive automated microfluidic chip system was implemented for the detection of piscine nodavirus; this technology has the advantage of requiring small amounts of sample and has been developed and applied for managing grouper fish farms. Epidemiological investigations revealed an extremely high detection rate of piscine nodavirus (89% of fish samples) from 5 different locations in southern Taiwan. In addition, positive samples from the feces of fish-feeding birds indicated that the birds could be carrying the virus between fish farms. In the present study, we successfully introduced this advanced technology that combines engineering and biological approaches to aquaculture. In the future, we believe that this approach will improve fish farm management and aid in reducing the economic loss experienced by fish farmers due to widespread disease outbreaks.

Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper.

Infection of virus (such as nodavirus and iridovirus) and bacteria (such as Vibrio anguillarum) in farmed grouper has been widely reported and caused large economic losses to Taiwanese fish aquaculture industry since 1979. The multiplex assay was used to detect dual viral infection and showed that only nervous necrosis virus (NNV) can be detected till the end of experiments (100% mortality) once it appeared. In addition, iridovirus can be detected in a certain period of rearing. The results of real-time PCR and in situ PCR indicated that NNV, in fact, was not on the surface of the eggs but present in the embryo, which can continue to replicate during the embryo development. The virus may be vertically transmitted by packing into eggs during egg development (formation) or delivering into eggs by sperm during fertilization. The ozone treatment of eggs may fail to remove the virus, so a new strategy to prevent NNV is needed.

Grouper (Epinephelus coioides) CXCR4 is expressed in response to pathogens infection and early stage of development.

Chemokine (C-X-C motif) receptor 4 (CXCR4) from orange-spotted grouper (Epinephelus coioides) was identified and characterized in this study. gCXCR4 shared common features in protein sequence and predicted structure of CXCR4 family. This suggested that gCXCR4 is a member of G protein-coupled receptors with seven transmembrane domains. The expression patterns revealed that gCXCR4 may play a key role in early development of grouper. Furthermore, overexpression of gCXCR4-GFP for 48 h had significant effects on the GF-1 cell viability. gCXCR4 protein was mainly expressed in the marginal zone of head kidney and on the surface of intestinal villi. gCXCR4 expression can be detected in all the examined tissues and significantly up-regulated in eye and brain, which are the main targets for nervous necrosis virus (NNV) infection and replication. gCXCR4 gene expression can be induced in the spleen and eye by lipopolysaccharide and NNV, respectively. Our data suggested that gCXCR4 may not only play a role in the early immune response to microbial infection but also restrain to the immune system and central nervous system.

A gene delivery system based on the N-terminal domain of human topoisomerase I.

The N-terminal 200 amino acid residues of topoisomerase I (TopoN) is highly positive in charge and has DNA binding activity, without DNA sequence and topological specificity. Here, a fusion protein (6 x His-PTD-TopoN) containing a hexahistidine (6 x His) tag, a membrane penetration domain and TopoN (amino acid 3-200) was designed and developed. The protein can bind to different sizes (3.0-8.0 kb) and forms (circular and linear) of DNA and translocates the bound DNA to the nucleus. The protein also showed low cytotoxicity to GF-1 grouper fish fin cells that were previously very sensitive and difficult to transfect in vitro. Maintaining the hexahistidine tag increased the protein's transfection efficiency in COS7 African green monkey kidney cells and simplified the purification process. The plasmid pEGFP-N1 was delivered into COS7 cells by the protein in ATP- and temperature-dependent manners. The results indicate that the binding ability of TopoN is very useful for DNA delivery and the carrier protein can be expressed in Escherichia coli without removal of the hexahistidine tag.

Real-time quantitative PCR assay for monitoring of nervous necrosis virus infection in grouper aquaculture.

Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Nuclear dynamics, mitosis, and the cytoskeleton during the early stages of colony initiation in Neurospora crassa.

Neurospora crassa macroconidia form germ tubes that are involved in colony establishment and conidial anastomosis tubes (CATs) that fuse to form interconnected networks of conidial germlings. Nuclear and cytoskeletal behaviors were analyzed in macroconidia, germ tubes, and CATs in strains that expressed fluorescently labeled proteins. Heterokaryons formed by CAT fusion provided a rapid method for the imaging of multiple labeled fusion proteins and minimized the potential risk of overexpression artifacts. Mitosis occurred more slowly in nongerminated macroconidia (1.0 to 1.5 h) than in germ tubes (16 to 20 min). The nucleoporin SON-1 was not released from the nuclear envelope during mitosis, which suggests that N. crassa exhibits a form of "closed mitosis." During CAT homing, nuclei did not enter CATs, and mitosis was arrested. Benomyl treatment showed that CAT induction, homing, fusion, as well as nuclear migration through fused CATs do not require microtubules or mitosis. Three ropy mutants (ro-1, ro-3, and ro-11) defective in the dynein/dynactin microtubule motor were impaired in nuclear positioning, but nuclei still migrated through fused CATs. Latrunculin B treatment, imaging of F-actin in living cells using Lifeact-red fluorescent protein (RFP), and analysis of mutants defective in the Arp2/3 complex demonstrated that actin plays important roles in CAT fusion.

Identification of Chinese medicinal fungus Cordyceps sinensis by PCR-single-stranded conformation polymorphism and phylogenetic relationship.

Fungi belonging to the Cordyceps species have long been used as food and herbal medicines in Asia and are especially popular as commercially available powdered supplements. Despite this acceptance and use, little is known of the phylogenetic relationships of the genus. Presently, the neighbor-joining method based on the ITS1, 5.8S rRNA, and ITS2 regions was used to construct a phylogenetic tree of 17 Cordyceps isolates. Five major groups were evident. Cordyceps sinensis was less closely related to 15 Cordyceps species but shared a closer relationship with Cordyceps agriota. PCR-single-stranded conformational polymorphism was applied to differentiate seven Cordyceps isolates: five were different from those used to construct the phylogenetic tree, based on differences in the internal spacer 2 (ITS2). The length of ITS2, amplified by primers 5.8SR and ITS4, vary between 334 and 400 bp. This segment could be used for intraspecies classification or detection of mutations and represents potential novel means of identification of this fungal genus in herbal medicines and in quality control applications in the fermentation industry.